(19)
(11)EP 3 004 131 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
22.07.2020 Bulletin 2020/30

(21)Application number: 14808105.2

(22)Date of filing:  05.06.2014
(51)International Patent Classification (IPC): 
C07H 1/02(2006.01)
C07H 19/16(2006.01)
C07H 19/06(2006.01)
C07H 21/00(2006.01)
(86)International application number:
PCT/US2014/041133
(87)International publication number:
WO 2014/197714 (11.12.2014 Gazette  2014/50)

(54)

PHOSPHORAMIDITE BUILDING BLOCKS FOR SUGAR-CONJUGATED OLIGONUCLEOTIDES

PHOSPHORAMIDIT-BAUSTEINE FÜR ZUCKERKONJUGIERTE OLIGONUKLEOTIDE

BLOCS DE CONSTRUCTION À BASE DE PHOSPHORAMIDITE POUR OLIGONUCLÉOTIDES CONJUGUÉS À DES SUCRES


(84)Designated Contracting States:
AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

(30)Priority: 05.06.2013 US 201361831521 P

(43)Date of publication of application:
13.04.2016 Bulletin 2016/15

(73)Proprietor: AM Chemicals Llc
Oceanside, CA 92056-5824 (US)

(72)Inventors:
  • GUZAEV, Andrei, Pavel
    Escondido, CA 92026 (US)
  • VVEDENSKIY, Vladimir, Y.
    San Diego, CA 92121 (US)

(74)Representative: Hutter, Anton et al
Venner Shipley LLP 5 Stirling House Stirling Road The Surrey Research Park
Guildford GU2 7RF
Guildford GU2 7RF (GB)


(56)References cited: : 
US-A- 4 668 777
US-A1- 2002 147 331
US-A1- 2006 178 509
US-A- 5 700 919
US-A1- 2003 229 218
US-B1- 6 610 842
  
      
    Note: Within nine months from the publication of the mention of the grant of the European patent, any person may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. 99(1) European Patent Convention).


    Description

    CROSS-REFERENCE TO RELATED APPLICATIONS



    [0001] This application claims priority from U.S. application Ser. No.61/831,521 filed on Jun 5,2013.

    BACKGROUND OF THE INVENTION


    1. Field of the Invention



    [0002] In one aspect, this invention is directed to methods for the preparation of oligonucleotides conjugated to at least one sugar moiety. In the other aspect, the invention provides methods for preparation of protected forms of oligonucleotides wherein at least one of the internucleosidic phosphate moieties is converted to its phosphate triester analog and to compounds useful in preparation of such protected oligonucleotides. Oligonucleotides prepared by the methods of the invention are useful for therapeutic and diagnostic applications and as research reagents.

    2. Summary of the Related Art



    [0003] Commonly, synthetic oligonucleotides are prepared on solid phase using protected nucleoside phosphoramidites as building blocks by a general approach first disclosed in US 4,668,777. To obtain the desired oligonucleotide, said building blocks are sequentially coupled to the growing oligonucleotide chain in the presence of an acidic catalyst (activator) in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected.

    [0004] Optionally, phosphoramidite compounds comprising two or more nucleoside moieties linked by one or more internucleoside phosphorus atoms may be used as building blocks as disclosed in US 2003/0229218A1.

    [0005] Optionally, the activator may comprise a mixture of an acidic catalyst with N-alkylimidazole as disclosed in US 2006/0178509A1 for mixtures of 5-benzylmercaptotetrazole with N-methylimidazole.

    [0006] As disclosed in US 6,610,842, more cost-efficient phosphoramidite compositions may optionally substitute for highly purified phosphoramidite building blocks that are traditionally used in oligonucleotide synthesis.

    [0007] It is well known that oligonucleotides and their phosphorothioate analogues are of limited stability in blood and tissues. Also, since such compounds are negatively charged they lack the ability to efficiently permeate biological membranes. Thus, both their oral bioavailability and cellular uptake are usually low. To overcome this problem, several types of modified oligonucleotides have been introduced. Among such oligonucleotides, backbone modified neutral oligonucleotides, phosphate triester analogs, have gained wide recognition.

    [0008] To date, the art recognizes simple alkyl triester analogs, methyl phosphotriester analogs disclosed in US 5,955,599, and ethyl phosphotriester analogs disclosed in Pless, R. C.; Ts'o, P. O. P. Biochemistry 1977, 16 (6), 1239-1250; Miller, P. S.; Barrett, J. C.; Ts'o, P. O. P. Biochemistry 1974, 13 (24), 4887-96; Lesser, D. R.; et al. J Biol. Chem. 1992, 267 (34), 24810-18; and Stawinski, J.; et al. Tetrahedron Lett. 1992, 33 (22), 3185-8. More complex, O-acyloxyaryl triester analogs have been disclosed in Iyer, R. P.; et al. Bioorg. Med. Chem. Lett. 1997, 7 (7), 871-876.

    [0009] Further development was presented by 2-aminoethyl analogs prepared using nucleoside 2-aminoethyl phosphoramidites wherein the amino function was protected with trifluoroacetyl or fluorenylmethoxycarbonyl groups as disclosed in US 5,700,919. Similar acylaminoethyl analogs have been disclosed more recently in US 6,121,437, US 6,610,837, US 2001/0044529, WO 2003/048179, and WO 2006/065751.

    [0010] Alternatively, 2-(dialkylamino)ethyl analogs were prepared by a combination of phosphoramidite and H-phosphonate methods as disclosed in US 2002/0147331A1.

    [0011] (Pivaloylthio)ethyl triester analogs have been disclosed in US 6,919,437. The latter modification has been even further developed with the introduction of extended side chains to replace the ethyl fragment as disclosed in WO2010/039543 and in WO2014/031575. Several other, less developed, embodiments of triester analogs have been reported in Sekine, M.; et al. Eur. J. Org. Chem. 2001, (10), 1989-1999; Sekine, M.; et al. J. Org. Chem. 2000, 65 (20), 6515-6524; Sobkowski, M.; et al. Tetrahedron Lett. 1995, 36 (13), 2295-8; Ayukawa, H.; et al. Chemistry Lett. 1995, (1), 81.

    [0012] The common feature of the analogs disclosed in the prior art was that the elimination of negative charge from the backbone of modified oligonucleotides led to the loss of solubility of said oligonucleotides in aqueous media, which is highly desirable for their successful use in biological applications.

    SUMMARY OF THE INVENTION



    [0013] Those skilled in the art will appreciate the fact that natural carbohydrates, particularly mono- and oligosaccharides, are characterized by a high degree of solubility in aqueous media. Should mono- or oligosaccharides be artificially conjugated to compounds of low solubility, this property may be used for the benefit of dissolution of the compounds in aqueous systems.

    [0014] Several processes for the solid phase synthesis of oligonucleotide compounds are known to those skilled in the art and may be employed with the present invention. Exemplary processes are disclosed in US 4,458,066 issued on Jul. 3, 1984, US 4,500,707 issued on Feb. 19, 1985, and US 5,132,418 issued on Nov. 27, 1990.

    [0015] A process for the preparation of phosphoramidite building blocks is disclosed in US 4,415,732 issued on Nov. 15, 1983. Certain nucleoside phosphoramidite compounds are disclosed in US 4,668,777 issued on May 26, 1987.

    [0016] It is an object of this invention to provide novel compounds which may serve as building blocks for the preparation of oligomeric compounds, phosphotriester analogs of natural oligonucleotides, wherein a carbohydrate moiety is linked to the internucleosidic phosphate residue.

    [0017] It is a further object of the present invention to provide novel oligomeric compounds, phosphotriester analogs of natural oligonucleotides with improved physico-chemical properties, wherein a carbohydrate moiety is linked to the internucleosidic phosphate residue.

    [0018] It is a further object of this invention to provide methods for synthetic preparation of said oligomeric compounds.

    [0019] Other objects of this invention will be apparent to those skilled in the art.

    [0020] These objects are satisfied by the present invention which provides novel nucleoside phosphoramidite reagents useful in preparation of oligomeric compounds and methods for making such oligomeric compounds.

    ABBREVIATIONS



    [0021] As used herein:

    Ac is Acetyl;

    Bz is benzoyl;

    DCM is dichloromethane;

    dmf is N,N-dimethylformamidino;

    iPrPac is (4-isopropylphenoxy)acetyl;

    MeCN is acetonitrile;

    Pac is phenoxyacetyl;

    TEA is triethylamine;

    NMI is N-methylimidazol;

    HPLC is high-performance liquid chromatography; and

    ES-MS is mass-spectrometry with electron-spray ionization.


    BRIEF DESCRIPTION OF THE FIGURES



    [0022] 

    FIG. 1 shows structures of compounds 1, 2, and 3 and a synthetic scheme for the preparation of compounds 7a - 7e.

    FIG. 2 shows a synthetic scheme for the preparation of compounds 12, 13a - 13c, 14a - 14d, and 15a - 15d.

    FIG. 3 shows a synthetic scheme for the preparation of compounds 16, 17a - 17c, 18a - 18d, and 19a - 19d.

    FIG. 4 shows a synthetic scheme for the preparation of compounds 20, 21a, 21c, 22a, 22b, 23a, and 23b.

    FIG. 5 shows a synthetic scheme for the preparation of compounds 24, 25a, 25c, 26a, 26b, 27a, and 27b.

    FIG. 6 shows a synthetic scheme for the preparation of compounds 28, 29a, 29c, 30a, 30b, 31a, 31b, 32, 33a, 33c, 34a, 34b, 35a, 35b, 36, 37a - 37c, 38a - 38d, 39a - 39d, 40, 41a, 41c, 42a, 42b, 43a, and 43b.

    FIG. 7 shows a synthetic scheme for the preparation of compounds 52, 53a, 53c, 54a, 54b, 55a, 55b, 56, 57a, 57c, 58a, 58b, 59a, and 59b.

    FIG. 8 shows an alternative synthetic scheme for the preparation of compound 28.

    FIG. 9 shows phosphotriester nucleoside residues 65, 66, 75, 76, 77, 78, 79, and 80 incorporated into synthetic oligonucleotides.

    FIG. 10 shows exemplary 31P NMR spectrum of 5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]thymidine (36, Example 57).


    DETAILED DESCRIPTION OF THE INVENTION



    [0023] In a first aspect, the invention provides novel compounds which may serve as building blocks for preparation of oligomeric compounds, phosphotriester analogs of natural oligonucleotides, wherein a carbohydrate moiety is linked to the internucleosidic phosphate residue according to Formula I:

    wherein:

    R1 is an optionally protected nucleic base selected from adenine, cytosine, guanine, thymine, uracil, 2-aminoadenine, N6-methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 8-aminoadenine, 5-methylcytosine, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-aminoguanine, 7-deazaxanthyne, or hypoxanthine;

    one of R2 and R2a is a protecting group of trityl type including but not limited to (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, and the other is a phosphoramidite moiety:

    wherein:

    each R4 and R5 is, independently, C1 to C6 alkyl, or R4 and R5 together with the nitrogen atom they are attached form a cycle wherein R4 + R5 = (CH2)nX(CH2)m,

    wherein:

    X is an atom of oxygen or CH2 group;

    each n and m is, independently, an integer from 2 to 5;

    L is -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, or -[(CH2)2-O]5-;

    R6 is a substituted monosaccharide residue of general Formula II



    wherein:

    each R10 is, independently, an acyl protecting group including but not limited to acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, or 4-methylbenzoyl groups, a trityl-type protecting group including but not limited to (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, a silyl protecting group including but not limited to triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, or diphenylmethylsilyl, an alkyl group containing from 1 to 18 atoms of carbon, a benzyl group, a 4-methoxybenzyl group, a propargyl group, or another substituted monosacharide residue of Formula II;

    A is a chemical bond or CHOR10;

    Z is a hydrogen, OR10, or N(Q2)Q3 wherein:

    each Q2 and Q3 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group;

    R3 is hydrogen atom, fluorine atom, substituted hydroxy group OR7, or substituted amino group NR8R9,
    wherein:

    R7 is a C1 to C6 alkyl, 2-alkoxyethyl group, or N-methylcarboxamidomethyl group; and

    each R8 and R9 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group.



    [0024] In an embodiment of the present invention, R1 of Formula I is a nucleic base selected from N6-benzoyladenine, N6-phenoxyacetyladenine, N6-(4-isopropylphenoxy)acetyladenine, adenine, N4-benzoylcytosine, N4-acetylcytosine, N4-phenoxyacetylcytosine, N4-(4-isopropylphenoxy)acetylcytosine, N4-benzoyl-4-methylcytosine, N4-acetyl-4-methylcytosine, cytosine, N4-phenoxyacetyl-4-methylcytosine, N4-(4-isopropylphenoxy)acetyl-4-methylcytosine, 4-methylcytosine, N2-isobutyrylguanine, N2-phenoxyacetylguanine, N2-(4-isopropylphenoxy)acetylguanine, N2-(N,N-dimethylformamidino)guanine, guanine, thymine, or uracil.

    [0025] In another embodiment, R2 of Formula I is 4,4'-dimethoxytrityl group and R2a is



    [0026] In yet another embodiment, R2a of Formula I is 4,4'-dimethoxytrityl group and R2 is



    [0027] In yet another embodiment of the present invention, R3 of Formula I is hydrogen.

    [0028] In yet another embodiment of the present invention, R3 of Formula I is OCH3.

    [0029] In yet another embodiment of the present invention, R3 of Formula I is fluorine.

    [0030] In yet another embodiment of the present invention, each R4 and R5 of Formula I is isopropyl group.

    [0031] In yet another embodiment of the present invention, L of Formula I is -(CH2)3-.

    [0032] In yet another embodiment of the present invention, L of Formula I is -(CH2)4-.

    [0033] In yet another embodiment of the present invention, L-O of Formula I is -[(CH2)2-O]2-.

    [0034] In yet another embodiment of the present invention, L-O of Formula I is -[(CH2)2-O]3-.

    [0035] In yet another embodiment of the present invention, L-O of Formula I is -[(CH2)2-O]4-.

    [0036] In yet another embodiment of the present invention, L-O of Formula I is -[(CH2)2-O]5-.

    [0037] In yet another embodiment of the present invention, R6 of Formula I is a protected β-D-glucopyranoside.

    [0038] In yet another embodiment of the present invention, R6 of Formula I is a protected β-D-galactopyranoside.

    [0039] In yet another embodiment of the present invention, R6 of Formula I is a protected 2-amino-2-deoxy-β-D-glucopyranoside.

    [0040] In still another embodiment of the present invention, R6 of Formula I is a protected 2-amino-2-deoxy-β-D-glucopyranoside wherein one of Q2 and Q3 is hydrogen, and the other is an acetyl group.

    [0041] In a further embodiment of the present invention, R6 of Formula I is a protected 2-amino-2-deoxy-β-D-galactopyranoside.

    [0042] In a still further embodiment of the present invention, R6 of Formula I is a protected 2-amino-2-deoxy-β-D-galactopyranoside wherein one of Q2 and Q3 is hydrogen, and the other is an acetyl group.

    [0043] In yet another embodiment of the present invention, each R10 of Formula II is an acetyl group.

    [0044] In yet another embodiment of the present invention, each R10 of Formula II is a benzoyl group.

    [0045] In yet another embodiment of the present invention, each R10 of Formula II is a butyryl group.

    [0046] In yet another embodiment of the present invention, each R10 of Formula II is an isobutyryl group.

    [0047] In yet another embodiment of the present invention, each R10 of Formula II is a propionyl group.

    [0048] In yet another embodiment of the present invention, each R10 of Formula II is a 4-methylbenzoyl group.

    [0049] In yet another preferred embodiment of the present invention, one of R10 of Formula II is another protected monosaccharide residue of Formula II, and each of the other R10 is an acetyl group.

    [0050] In a second aspect, the present invention provides novel oligomeric compounds, phosphotriester analogs of natural oligonucleotides, having the structure according to Formula III wherein a carbohydrate moiety is linked to the internucleosidic phosphate residue:



    wherein:

    each R1 is independently an optionally protected nucleic base selected from adenine, cytosine, guanine, thymine, uracil, 2-aminoadenine, N6-methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 8-aminoadenine, 5-methylcytosine, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-aminoguanine, 7-deazaxanthyne, or hypoxanthine;

    each R3 is, independently, hydrogen atom, fluorine atom, substituted hydroxy group OR7, or substituted amino group NR8R9,

    wherein:

    each R7 is, independently, a C1 to C6 alkyl, 2-alkoxyethyl group, or N-methylcarboxamidomethyl group;

    each R8 and R9 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group;

    L is -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, or -[(CH2)2-O]5-;

    each R6 is, independently, a substituted monosaccharide residue of general Formula II,

    wherein:

    each R10 is, independently, an acyl protecting group including but not limited to acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, and 4-methylbenzoyl groups, a trityl-type protecting group including but not limited to (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(p-methoxyphenyl)xanthen-9-yl, a silyl protecting group including but not limited to triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, and diphenylmethylsilyl, an alkyl group containing from 1 to 18 atoms of carbon, a benzyl group, a 4-methoxybenzyl group, a propargyl group, or another substituted monosacharide residue of Formula II;

    A is a chemical bond or CHOR10;

    Z is a hydrogen, OR10, or N(Q2)Q3 wherein:

    each Q2 and Q3 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group;

    each R11 is independently a negative charge compensated by a cation, a phosphate protecting group, or R6O-L-;

    each R12 and R13 is, independently, hydrogen atom, a protecting group selected from (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl; or a point of attachment to solid phase material with the proviso that R12 and R13 are not both simultaneously a linker connected to a solid support;

    each W1, W2, and W3 is independently oxygen or sulfur; and

    each s and t is, independently, an integer from 0 to 100.



    [0051] In a third aspect, the present invention provides methods for synthetic preparation of oligomeric compounds according to Formula III, said method comprising:

    reacting a compound of Formula I with a compound of Formula IV containing at least one reactive hydroxy group wherein the compound of Formula IV has the structure:

    wherein:

    one of R12 and R13 is a hydrogen atom, and the other is a protecting group or a linker connected to a solid support; and

    k is an integer selected from 0 to 100.



    [0052] In an embodiment, the method further comprises treating the oligomeric compound with a reagent under conditions of time, temperature, and pressure effective to oxidize or sulfurize the oligomeric compound.

    [0053] In an embodiment, the method further comprises treating the oligomeric compound with a reagent under conditions of time, temperature, and pressure effective to deprotect the oligomeric compound.

    [0054] In an embodiment, wherein R13 is a linker connected to a solid support, the method further comprises treating the oligomeric compound with a reagent under conditions of time, temperature, and pressure effective to remove the oligomeric compound from the solid support.

    [0055] Certain of the starting materials used in the practice of the present invention are protected nucleosides 8, 9a - 9c, 10a - 10d, and 11a - 11d, which are readily available from commercial sources (such as, for example, ChemGenes, Inc., Waltham, MA; Rasayan, Inc., Encinitas, CA).

    [0056] Certain other starting materials used in the practice of the present invention are readily prepared following methods and procedures familiar to those skilled in the art and disclosed in the chemical literature. Compounds 1, 2, and 3 (FIG. 1) were prepared as disclosed in Karjala, S. and Link, K. P., J. Amer. Chem. Soc., 1940, 62, 917-922. Compound 4 (FIG. 1) was prepared as disclosed in Fukase, K; et al. Bull. Chem. Soc. Japan 2003, 76, 485-500, and compound 5 (FIG. 1) was prepared as disclosed in Matta, K. L.; et al. Carbohydrate Res. 1973, 26, 215-218.

    [0057] Compounds 6b and 7a - 7e were prepared as disclosed in FIG. 1 from compounds 4 and 5 and ethyleneglycol, di- tri- tetra- and pentaethyleneglycols.

    [0058] As illustrated in FIG. 2, exemplary phosphoramidite building blocks 12, 13a - 13c, 14a - 14d, 15a - 15d comprising a protected β-D-glucopyranose moiety attached to the phosphite function via a C3-spacer arm may be readily synthesized from protected 2'-deoxynucleosides 8, 9a - 9c, 10a - 10d, and 11a - 11d and 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 1.

    [0059] To synthesize compounds 12, 13a - 13c, 14a - 14d, 15a - 15d, the respective protected 2'-deoxynucleoside 8, 9a - 9c, 10a - 10d, and 11a - 11d was first treated with bis(N,N-diisopropylamino) chlorophosphite (R. I. Chemical, Orange, CA) in the presence of excess N-ethyl-N,N-diisopropylamine (Alfa Aesar, Ward Hill, MA) in MeCN at -20°C followed by stirring at room temperature for 1 to 2 h. The obtained solution of protected nucleoside-3'-O-bis(N,N-diisopropylamino) phosphite was further treated with compound 1 and 1H-tetrazole (Glen Research, Sterling, VA) for 14 h at room temperature. Quenching with 5% aqueous NaHCO3 followed by the standard work-up and purification by chromatography on a silica gel column afforded the desired phosphoramidite building blocks 12, 13a - 13c, 14a - 14d, 15a - 15d.

    [0060] Among these compounds, phosphoramidite building blocks 13a, 14a, and 15a feature the standard protecting scheme for exocyclic amino groups of nucleic bases.

    [0061] The method described above is also applicable to a peracetylated β-D-glucopyranosidebearing ethyleneglycol spacer arm. FIG. 3 illustrates a reaction of protected nucleosides 8, 9a - 9c, 10a - 10d, and 11a - 11d with 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside 2. Following the procedure described above gave the phosphoramidite building blocks 16, 17a - 17c, 18a - 18d, 19a - 19d featuring a diethyleneglycol spacer arm.

    [0062] In certain embodiments, it is desirable to introduce monosaccharide residues other than glucose into oligonucleotides. By following the general synthetic method described above, the preparation of phosphoramidite building blocks 20, 21a, 21c, 22a, 22b, 23a, and 23b derivatized with a peracetylated β-D-galactospyranoside residue and ethyleneglycol spacer arm was accomplished by reacting protected nucleosides 8, 9a - 9c, 10a - 10d, 11a - 11d with compound 3 (see FIG. 4).

    [0063] By following the general synthetic method described above, the preparation of phosphoramidite building blocks 24, 25a, 25c, 26a, 26b, 27a, and 27b derivatized with a protected N-acetyl-D-glucosamine residue and C3 spacer arm was accomplished by reacting protected nucleosides 8, 9a - 9c, 10a - 10d, 11a - 11d with compound 6 (see FIG. 5).

    [0064] Analogously, the preparation of phosphoramidite building blocks 28, 29a, 29c, 30a, 30b, 31a, 31b, 32, 33a, 33c, 34a, 34b, 35a, 35b, 36, 37a - 37c, 38a - 38d, 39a - 39d, 40, 41a, 41c, 42a, 42b, 43a, and 43b derivatized with protected N-acetyl-D-galactosamine residues starting from protected nucleosides 8, 9a - 9c, 10a - 10d, and 11a - 11d and compounds 7a - 7e is described in FIG. 6. The phosphoramidite reagents of this series feature oligoethyleneglycol linkers of various lengths to allow the placement of the monosaccharide residue at different distances from polyphosphate backbone of oligonucleotides.

    [0065] In certain embodiments of the invention, synthetic oligonucleotides bearing nucleotide residues derivatized at the 2'- position of the ribose ring are desirable due to their enhanced base-pairing properties. Those skilled in the art recognize that 2'-OMe and 2'-fluoro substitution at the ribose ring results in the enhancement of hybridization affinity of derivatized oligonucleotides to the complementary strands of oligonucleotides or nucleic acids. FIG. 7 describes the preparation of exemplary phosphoramidite building blocks 52, 53a, 53c, 54a, 54b, 55a, and 55b derived from 2'-O-methylnucleosides 44, 45a, 45c, 46a, 46b, 47a, 47b and phosphoramidite building blocks 56, 57a, 57c, 58a, 58b, 59a, and 59b derived from 2'deoxy-2'-fluoronucleosides 48, 49a, 49c, 50a, 50b, 51a, and 51b. All compounds 52 - 59 comprise a peracetylated D-galactosamine residue and tetraethyleneglycol spacer arm.

    [0066] It is well known in the art that the standard conditions of oligonucleotide deprotection comprise treating a solid support-bound, protected oligonucleotide with 28-33% aqueous ammonium hydroxide for 8 h at 55°C. In certain embodiments of the present invention, compounds 13a, 14a, 15a, 17a, 18a, 19a, 21a, 22a, 23a, 25a, 26a, 27a, 29a, 30a, 31a, 33a, 34a, 35a, 41a, 42a, 43a, 53a, 54a, 55a, 57a, 58a, and 59a, are designed to be used within the standard deprotection strategy. However, certain oligonucleotides comprising unnatural nucleosidic residues may demonstrate a rather low stability under the standard conditions.

    [0067] Various protecting schemes have been developed in order to remove the protecting groups under milder conditions. These works have been expertly reviewed in Beaucage, S. L. and Iyer, R. P. Tetrahedron 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P. Tetrahedron 1993, 49, 6123-6194 and references referred to therein. In order to satisfy the requirements of alternative deprotection schemes, the building blocks bearing CAc, Admf, and Gdmf protected bases as, for instance, in compounds 13c, 14d, and 15d, are compatible with mild deprotecting strategies of oligonucleotide synthesis. The building blocks bearing CPac, APac, AiPrPac, GPac, and GiPrPac protected bases as, for instance, in compounds 13b, 14b, 14c, 15b, and 15c were prepared to be compatible with ultra-mild deprotecting strategies.

    [0068] The present disclosure is not limited by the methods described in FIGs. 1-7. For example, an alternative method is exemplified in FIG. 8 by the preparation of phosphoramidite building block 28. Treatment of compound 7b with bis(N,N-diisopropylamino) chlorophosphite in the presence of N-ethyl-N,N-diisopropylamine gave a phosphorodiamidite compound. After purification on a silica gel column, the resulting compound was reacted with protected nucleoside 8 to give the desired phosphoramidite building block 28.

    [0069] Most phosphoramidite building blocks described herein were isolated as solid foams or, in the case of compounds comprising tetraethyleneglycol and pentaethyleneglycol spacer, arms, as colorless oils sufficiently stable to be stored for several months at -18°C.

    [0070] The hydrolysis of phosphoramidite building blocks 12 and 16 in 95% aqueous MeCN at 25°C was followed by reverse-phase HPLC. It was determined that, under the conditions used, the half-life of compounds 12 and 16 was 48 and 33 h, respectively. This demonstrates that solutions of phosphoramidite reagents according to the present invention can be safely stored on an oligonucleotide synthesizer for 2-3 days, which is sufficient for most applications.

    [0071] The use of novel phosphoramidite building blocks described herein in solid phase synthesis of oligonucleotides is illustrated with respect to compound 12:
    d(Tp Tp Tp Tp TT TTT T)
    wherein internucleosidic phosphate linkages p are phosphotriester linkages with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification (SEQ ID NO:6).

    [0072] Oligonucleotides 61-64 were synthesized by the phosphoramidite method. Briefly, a commercial solid support, DMT-T CPG1000 (1.0 µmol) was placed in a synthetic column and installed on ABI394 synthesizer. The standard synthetic cycle useful in assemblying oligonucleotides with phosphate backbone comprised the following steps:
    1. (a) detritylation of the solid phase-bound material with 3% dichloroacetic acid in dichloromethane followed by washing with acetonitrile;
    2. (b) coupling of nucleoside phosphoramidite building block (0.1 M in acetonitrile) required by the sequence to the solid support-bound material in the presence of 1H-tetrazole (0.45 M in acetonitrile);
    3. (c) capping of unreacted solid support-bound hydroxy groups with a mixture of acetic anhydride and N-methyl imidazole and
    4. (d) oxidation of the solid support-bound phosphite triester groups with iodine (0.05 M in a mixture of pyridine, tetrahydrofurane, water).


    [0073] Preparation of oligonucleotide phosphorothioates was conducted as disclosed in US 7,723,528. Accordingly, steps (a) and (b) remained as disclosed above. The iodine solution was replaced with a solution of N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide (0.025 M in pyridine). Upon completion of coupling step (b) and washing the solid phase with acetonitrile, the sulfurization step was conducted. The capping step followed the sulfurization. The cycle appropriate for the assembly of the desired oligonucleotide was repeated as required by the sequence in preparation.

    [0074] The synthetic cycle for incorporation of the phosphoramidite building blocks described herein was different from those disclosed above in that the coupling time for the incorporation of all novel phosphoramidite building blocks was extended to 1 min.

    [0075] Following the general procedures disclosed above, the parent, unmodified oligonucleotide 60, plus oligonucleotides 61-64 wherein Tp denotes the residues of thymidine-3'-[[(β-D-glucopyranosyl)oxy]propyl]phosphate 65, were assembled:

    60: TTT TTT TTT T (SEQ ID NO: 1), parent natural oligonucleotide,

    61: Tp TTT TTT TTT (SEQ ID NO: 2),

    62: (Tp)2 TTT TTT TT (SEQ ID NO: 3),

    63: (Tp)3 TTT TTT T (SEQ ID NO: 4), and

    64: (Tp)4 TTT TTT (SEQ ID NO: 5).



    [0076] To determine the optimal deprotection conditions, solid support-bound oligonucleotides 61-64 thus obtained were treated under the following conditions suitable for removing all phosphate- and base-protecting groups from synthetic oligonucleotides:

    A. Concentrated ammonium hydroxide, room temperature, 18 h;

    B. Concentrated aqueous ammonium hydroxide, 65°C, 8 h;

    C. 0.05 M K2CO3 in anhydrous MeOH, room temperature, 4 h;

    D. 0.5 M Piperidine in anhydrous acetonitrile, room temperature, 15 min, followed by a mixture of 1,2-diaminoethane and toluene (50:50 v/v), room temperature, 4 h;

    E. 25% Aqueous t-butylamine, 65°C, 4 h;

    F. Mixture of concentrated aqueous ammonium hydroxide and 40% aqueous methylamine (50:50 v/v), room temperature, 120 min.



    [0077] Upon completion of the deprotection under the conditions A, B, E, and F, the liquid phase was collected and evaporated in vacuo to dryness. The residue was dissolved in water (1 mL) and analyzed by reverse-phase HPLC and by ES MS.

    [0078] Upon completion of the deprotection under the condition C, the reaction mixtures were neutralized by addition of 0.25 M aqueous NaH2PO4 (250 µL per 1 mL of deprotection solution). The solid phase was spun down; the liquid phase was collected and analyzed as described above.

    [0079] Upon completion of the deprotection under the condition D, the solid phase was spun down, and the liquid phase was discarded. The solid phase was washed with toluene (2×1 mL) and with acetonitrile (2×1 mL). The solid phase was suspended in water (1 mL) and the suspension was centrifugated. The liquid phase was collected and analyzed as described above.

    [0080] The results of HPLC and LC-ES MS analysis showed that deprotection under conditions A, C, D, and F resulted in products containing over 95% of the desired oligonucleotides. In contrast, the use of the conditions B and E led to the formation of the parent oligonucleotide 60 (SEQ ID NO:1) where the (glucopyranosyloxy)propyl modifier was removed from the phosphotriester moiety. A more detailed analysis of the crude oligonucleotide products showed that conditions A and C were the most appropriate for deprotection of phosphotriester-derivatized oligonucleotides.

    [0081] In the next round of experiments, oligonucleotides 67 - 72 were assembled and deprotected under conditions A and C as described above:

    67: Tp Tp Tp Tp TT TTT T (SEQ ID NO: 6) wherein nucleoside phosphate moieties Tp are phosphotriester moieties 75 (FIG. 9) with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification;

    68: Tp Tp Tp Tp T Tp Tp Tp Tp T (SEQ ID NO: 7) wherein nucleoside phosphate moieties Tp are phosphotriester moieties 65 (FIG. 9) with 1-[3-(β-D-glucopyranosyloxy)propyl] modification;

    69: Tp Tp Tp Tp Tp Tp Tp Tp Tp T (SEQ ID NO: 8) wherein all nucleoside phosphate moieties Tp are phosphotriester moieties 65 (FIG. 9) with 1-[3-(β-D-glucopyranosyloxy)propyl] modification;

    70: Tp Tp Tp Tp Tp Tp Tp Tp Tp T (SEQ ID NO: 9) wherein all nucleoside phosphate moieties Tp are phosphotriester moieties 75 (FIG. 9) with 1-[2-[[2-(β-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification;

    71: Tp Tp Tp Tp TT TTT T (SEQ ID NO: 10) wherein nucleoside phosphate moieties Tp are phosphotriester moieties 79 (FIG. 9) with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy) ethyl] oxy] ethyl] modification; and

    72: Tp Tp Tp Tp T Tp Tp Tp Tp T (SEQ ID NO: 11) wherein nucleoside phosphate moieties Tp are phosphotriester moieties 79 (FIG. 9) with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification.



    [0082] Analysis of crude oligonucleotide products by reverse-phase HPLC and LC-ES MS confirmed that both deprotection conditions A and C were appropriate for preparation of highly-modified 10-mer oligonucleotides 68, 69, 70, and 71 comprising 8 to 9 phosphotriester moieties, thus confirming the stability of the novel modification of internucleosidic phosphate groups disclosed in the present invention. Further comparison of HPLC profiles obtained for oligonucleotides 64, 67, and 71 revealed no appreciable difference in hydrolytic stability between nucleoside phosphate residues 65, 75, and 79 under the basic deprotection conditions.

    [0083] Finally, full-length oligonucleotide phosphorothioates 81 - 84 were synthesized and deprotected under the condition A:

    81: d(TGT GAG TAC CAC TGA TTC) (SEQ ID NO: 12) phosphorothioate wherein all internucleoside moieties are phosphorothioate triester moieties with 1-[3-(β-D-glucopyranosyloxy)propyl] modification as exemplified for the triester of thymidine-3'-monophosphorothioate by structure 66 (FIG. 9);

    82: d(TpGpTp GAG TAC CAC TGAp TpTpA) (SEQ ID NO: 13) phosphorothioate wherein internucleoside moieties Tp and Ap are phosphorothioate triester moieties with 1-[3-(N-acetyl-2-deoxy-2-amino-β-D-glucopyranosyloxy)propyl] modification as exemplified for the triester of thymidine-3'-monophosphorothioate by structure 78 (FIG. 9);

    83: d(TpGpTp GAG TAC CAC TGAp TpTpA) phosphorothioate (SEQ ID NO: 14) wherein internucleoside moieties Tp and Ap are phosphorothioate triester moieties with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)ethyl]oxy]ethyl] modification as exemplified for the triester of thymidine-3'-monophosphorothioate by structure 80 (FIG. 9);

    84: dTp dGp dTp GAG UAC CAC UG dAp dTp dTp dA phosphorothioate (SEQ ID NO: 15) wherein internucleoside moieties Tp and Ap are phosphorothioate triester moieties with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-β-D-galactopyranosyloxy)ethyl]oxy]ethyl] modification as exemplified for the triester of thymidine-3'-monophosphorothioate by structure 80 (FIG. 9).



    [0084] Oligonucleotide 81 was characterized by reverse-phase HPLC, and oligonucleotides 82 - 84 were characterized by reverse-phase HPLC, ion exchange HPLC, and LC-MS with electron-spray ionization. Importantly, oligonucleotides 68 and 72 bearing only one negative charge and 69, 70, and 81 wherein all negatively charged internucleosidic phosphates were replaced by neutral phosphotriested moieties were readily soluble in aqueous media.

    [0085] Accordingly, the efficient preparation of monosaccharide-derivatized phosphotriester analogs of oligonucleotides and their phosphorothioate analogs using the novel phosphoramidite building blocks described herein has been demonstrated. Said phosphoramidite building blocks can be readily synthesized by artisans possessing ordinary skills. Conveniently, phosphotriester oligonucleotide analogs synthesized using said phosphoramidite building blocks are stable under the basic deprotection conditions as opposed to methyl triester analogs (US 5,955,599), O-acyloxyaryl triester analogs (Iyer, R. P.; et al. Bioorg. Med. Chem. Lett. 1997, 7 (7), 871-876), acylaminoethyl analogs (US 6,121,437, US 6,610,837, US 2001/0044529, WO 2003/048179, and WO 2006/065751), (pivaloylthio)ethyl triester and similar analogs (US 6,919,437, WO2010/039543 and in WO2014/031575).

    EXAMPLES



    [0086] The following examples are intended to further illustrate certain preferred embodiments of the invention and are not intended to be limiting in their nature.

    GENERAL INFORMATION



    [0087] Protected 2'-deoxynucleoside 2-cyanoethyl phosphoramidites, protected ribonucleoside 2-cyanoethyl phosphoramidites, and all ancillary reagents for oligonucleotide synthesis were purchased from Glen Research (Sterling, VA). Sulfurizing reagent, N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide was prepared as disclosed in US 7,723,582. Anhydrous MeCN was purchased from Honeywell Burdick & Jackson (Muskegon, MI). Protected 2'-deoxynucleosides, 2'-O-methylribonucleosides, and 2'-fluoro-2'-deoxyribonucleosides were purchased from Rasayan, Inc. (Encinitas, CA). Tri-tetra- and pentaethyleneglycols were purchased from Sigma-Aldrich (Milwaukee, WI). All other chemicals were purchased from TCI America (Portland, OR).

    EXAMPLE 1



    [0088] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propyl]phosphino]thymidine (12).

    [0089] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.94 g, 11 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 1, (5.88 g, 10.8 mmol) and N-ethyl-N,N-diisopropylamine (1.44 g, 11.1 mmol) in anhydrous dichloromethane (25 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl- β-D-glucopyranoside (4.47 g, 11 mmol, prepared as disclosed in Karjala, S. and Link, K. P., J. Amer. Chem. Soc., 1940, 62, 917-922) and 1H-tetrazole (0.45 M in MeCN, 7.8 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (200 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient of ethyl acetate (from 20 to 80%) plus triethylamine (5%) in hexane. Collected fractions were evaporated to give the title compound as a mixture of Sp and Rp diastereomers (8.11 g, 69.5%). 31P NMR (CD3CN): δ 147.94, 147.74. 1H NMR (CD3CN): δ 8.941 (1H, br. s); 7.467-7.431 (3H, m); 7.331-7.291 (6H, m); 7.28-7.20 (1H, m); 6.886-6.855 (4H, m) 6.250-6.211 (1H, m); 5.247-5.171 (1H, m); 5.016-4.977 (1H, m); 4.866-4.822 (1H, m); 4.600-4.526 (1H, m); 4.203-4.168 (1H, m); 4.054-4.038 (2H, m); 3.90-3.83 (0.5H, m); 3.83-3.70 (2.5H, m); 3.765 (3H, s); 3.761 (3H, s); 3.68-3.45 (5H, m); 3.329-3.273 (2H, m); 2.45-2.38 (0.5H, m); 2.37-2.28 (1.5H, m); 2.015-1.929 (12H, m); 1.82-1.74 (1H, m); 1.72-1.64 (1H, m); 1.498 (1.5H, d, J = 1.0 Hz); 1.476 (1.5H, d, J = 1.0 Hz); 1.155-1.117 (9H, m); 1.033 (3H, d, J = 6.8 Hz).

    EXAMPLE 2



    [0090] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propyl]phosphino]-2'-deoxycytidine (13a).

    [0091] Solution of bis(N,N-diisopropylamino) chlorophosphite (5.27 g, 19.75 mmol) in anhydrous dichloromethane (20 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (10.01 g, 15.8 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (3.83 g, 29.6 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (7.06 g, 17.38 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:3:87) to ethyl acetate - TEA (94:6). Collected fractions were evaporated to give the title compound (13.30 g, 72.0%). 31P NMR (mixture of diastereomers, CD3CN): δ 147.86, 147.53.

    EXAMPLE 3



    [0092] N4-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxycytidine (13b).

    [0093] Solution of bis(N,N-diisopropylamino) chlorophosphite (4.75 g, 17.81 mmol) in anhydrous dichloromethane (20 mL) was added dropwise to a stirred solution of N4-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9b, (9.46 g, 14.25 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (3.45 g, 26.7 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.37 g, 15.68 mmol) and 1H-tetrazole (0.45 M in MeCN, 12.7 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound (11.60 g, 67.9%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.12, 147.77.

    EXAMPLE 4



    [0094] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxycytidine (13c).

    [0095] Solution of bis(N,N-diisopropylamino) chlorophosphite (4.62 g, 17.32 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (7.83 g, 13.69 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.12 g, 16.42 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.40 g, 15.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (6:94). Collected fractions were evaporated to give the title compound (6.78 g, 44.7%). 31P NMR (mixture of diastereomers, CD3CN): δ 147.97, 147.63. 1H NMR (fast diastereomer, CD3CN): δ 8.805 (1H, br. s); 8.172 (1H, d, J = 7.5 Hz); 7.429-7.412 (2H, m); 7.322-7.280 (6H, m); 7.257-7.228 (1H, m); 7.040 (1H, d, J = 7.5 Hz); 6.878-6.848 (4H, m); 6.087 (1H, dd, J = 6.5 Hz, 4.8 Hz); 5.323 (1H, t, J = 9.6 Hz); 4.997 (1H, t, J = 9.8 Hz); 4.838 (1H, dd, J = 9.8, 8.0 Hz); 4.627 (1H, d, J = 8.1 Hz); 4.50-4.48 (1H, m); 4.230 (1H, dd, J = 12.3, 5.0 Hz); 4.103 (1H, m); 4.042 (1H, dd, J = 12.3, 2.4 Hz); 3.885-3.849 (2H, m); 3.768 (6H, s); 3.625-3.510 (5H, m); 3.41-3.32 (2H, m); 2.573 (1H, d, J = 13.7 Hz, t, J = 6.7 Hz); 2.261 (1H, d, J = 13.7 Hz, d, J = 6.5 Hz, d, J = 4.8 Hz); 2.116 (3H, s); 2.018 (3H, s); 1.975 (3H, s); 1.956-1.784 (6H, m); 1.772 (2H, tt, J = 6.2 Hz); 1.123 (3H, d, J = 6.8 Hz); 1.046 (3H, d, J = 6.8 Hz).

    EXAMPLE 5



    [0096] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (14a).

    [0097] Solution of bis(N,N-diisopropylamino) chlorophosphite (4.12 g, 15.44 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (8.12 g, 12.35 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (1.99 g, 15.44 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.07 g, 14.94 mmol) and 1H-tetrazole (0.45 M in MeCN, 8.2 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (11.49 g, 78.0%). 31P NMR (mixture of diastereomers, CD3CN): δ 147.87, 147.58. 1H NMR (mixture of diastereomers, CD3CN): δ 9.318 (1H, br. s); 8.591 (1H, br. d, J = 3.3 Hz); 8.263 (1H, s); 8.997 (1H, br d, J = 7.2 Hz); 7.646 (1H, t, J = 7.3 Hz); 7.551 (2H, t, J = 7.7 Hz); 7.39-7.34 (2H, m); 7.25-7.16 (7H, m); 6.80-6.74 (4H, m); 6.48-6.42 (1H, m); 5.24-5.16 (1H, m); 5.05-4.96 (1H, m); 4.90-4.78 (2H, m); 4.556 (1H, dd, J = 8.0, 5.1 Hz); 4.26-4.17 (2H, m); 4.08-3.98 (1.5H, m); 3.91-3.78 (1.5H, m); 3.740, 3.736, 3.733, 3.728 (total 6H, singlets); 3.78-3.69 (1H, m); 3.64-3.50 (5H, m); 3.48-3.24 (2H, m); 3.060 (1H, sept, J = 6.4 Hz); 2.68-2.52 (1H, m); 2.00-1.90 (12H, m); 1.795 (1H, pent., J = 6.1 Hz); 1.711 (1H, pent., J = 6.1 Hz); 1.20-1.12 (9H, m); 1.080 (3H, d, J = 6.8 Hz).

    EXAMPLE 6



    [0098] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (14b).

    [0099] Solution of bis(N,N-diisopropylamino) chlorophosphite (3.50 g, 13.13 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (7.22 g, 10.50 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.55 g, 19.74 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (4.69 g, 11.55 mmol) and 1H-tetrazole (0.45 M in MeCN, 9.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound (9.05 g, 70.5%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.54, 148.01.

    EXAMPLE 7



    [0100] N6-(4-i-Propylphenoxy)acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (14c).

    [0101] Solution of bis(N,N-diisopropylamino) chlorophosphite (3.94 g, 14.75 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N6-(4-i-propylphenoxy) acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10c, (8.61 g, 11.80 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.87 g, 22.18 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (5.27 g, 12.98 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.5 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound (10.44 g, 69.9%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.22, 147.72.

    EXAMPLE 8



    [0102] N6-(N,N-Dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (14d).

    [0103] Solution of bis(N,N-diisopropylamino) chlorophosphite (4.67 g, 17.5 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N6-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10d, (8.52 g, 14.0 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (3.40 g, 26.32 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.25 g, 15.4 mmol) and 1H-tetrazole (0.45 M in MeCN, 12.44 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (10:2:87) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound (12.56 g, 78.4%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.65, 148.12.

    EXAMPLE 9



    [0104] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (15a).

    [0105] Solution of bis(N,N-diisopropylamino) chlorophosphite (4.27 g, 16.0 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (8.53 g, 13.33 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.15 g, 16.66 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (6.50 g, 16.0 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.0 mL, 4.5 mmol) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound as an off-white foam (8.54 g, 54.5%).31P NMR (mixture of diastereomers, CD3CN): δ 147.96, 147.62. 1H NMR (mixture of diastereomers, CD3CN): δ 7.817, 7.812 (total 1H, s, s); 7.43-7.36 (2H, m); 7.29-7.18 (7H, m); 6.83-6.72 (4H, m); 6.249-6.214 (1H, m); 5.24-5.14 (1H, m); 5.03-4.94 (1H, m); 4.89-4.81 (1H, m); 4.64-4.52 (2H, m); 4.22-4.12 (2H, m); 4.07-3.98 (1H, m); 3.90-3.88 (1H, m); 3.748, 3.744, 3.740 (total 6H, s, s, s); 3.77-3.69 (1H, m); 3.68-3.49 (5H, m); 3.37-3.28 (1H, m); 3.27-3.19 (1H, m); 2.94-2.83 (1H, m); 2.601 (1H, sept., J = 6.8 Hz); 2.58-2.43 (1H, m); 1.99-1.93 (12H, m); 1.777 (1.5H, pent., J = 6.2 Hz); 1.698 (0.5H, m); 1.186-1.131 (12H, m); 1.063 (3H, d, J = 6.8 Hz).

    EXAMPLE 10



    [0106] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (15b).

    [0107] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.83 g, 10.6 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (5.98 g, 8.5 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.06 g, 16.0 mmol) in anhydrous dichloromethane (25 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (3.97 g, 9.78 mmol) and 1H-tetrazole (0.45 M in MeCN, 7.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound as an off-white foam (6.54 g, 62.1%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.90, 148.48.

    EXAMPLE 11



    [0108] N2-(4-i-Propylphenoxy)acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (15c).

    [0109] Solution of bis(N,N-diisopropylamino) chlorophosphite (3.33 g, 12.5 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-(4-i-propylphenoxy)acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11c, (7.46 g, 10.0 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.43 g, 18.8 mmol) in anhydrous dichloromethane (25 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (4.67 g, 11.5 mmol) and 1H-tetrazole (0.45 M in MeCN, 8.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound as an off-white foam (8.10 g, 63.3%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.88, 148.32.

    EXAMPLE 12



    [0110] N2-(N,N-Dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (15d).

    [0111] Solution of bis(N,N-diisopropylamino) chlorophosphite (3.67 g, 13.75 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11d, (6.87 g, 11.0 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (2.67 g, 20.7 mmol) in anhydrous dichloromethane (25 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 3-hydroxypropyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 1, (2.92 g, 6.7 mmol) and 1H-tetrazole (0.45 M in MeCN, 9.8 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (10:90). Collected fractions were evaporated to give the title compound as an off-white foam (8.10 g, 63.3%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.88, 148.32.

    EXAMPLE 13



    [0112] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]thymidine (16).

    [0113] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.86 g, 6.98 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (3.17 g, 5.82 mmol) and N-ethyl-N,N-diisopropylamine (0.99 g, 7.68 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.92 g, 6.69 mmol, prepared as disclosed in Karjala, S. and Link, K. P., J. Amer. Chem. Soc., 1940, 62, 917-922) and 1H-tetrazole (0.45 M in MeCN, 3.9 mL, 1.76 mmol) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (6:94). Collected fractions were evaporated to give the title compound (4.87 g, 75.4%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.23, 148.12. 1H NMR (mixture of diastereomers, CD3CN): δ 8.910 (1H, br. s); 7.48-7.40 (3H, m); 7.37-7.28 (6H, m); 7.28-7.20 (1H, m); 6.90-6.82 (4H, m); 6.28-6.20 (1H, m); 5.26-5.18 (1H, m); 5.05-4.97 (1H, m); 4.89-4.82 (1H, m); 4.68-4.53 (2H, m); 4.26-4.18 (1H, m); 4.12-4.01 (2H, m); 3.87-3.68 (8H, m, s, s); 3.68-3.41 (9H, m); 3.38-3.23 (2H, m); 2.45-2.29 (2H, m); 2.02-1.92 (12H, m); 1.495 (1.5H, d, J = 1.2 Hz); 1.471 (1.5H, d, J = 1.2 Hz); 1.17-1.11 (9H, m); 1.040 (3H, d, J = 6.8 Hz).

    EXAMPLE 14



    [0114] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxycytidine (17a).

    [0115] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (3.80 g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (5.14 g, 71.4%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.76, 148.39.

    EXAMPLE 15



    [0116] N4-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxycytidine (17b).

    [0117] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9b, (3.98 g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (4.86 g, 65.9%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.05, 147.66.

    EXAMPLE 16



    [0118] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxycytidine (17c).

    [0119] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.00 g, 7.5 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (3.43 g, 6.0 mmol) and N-ethyl-N,N-diisopropylamine (1.47 g, 11.4 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (3.01 g, 6.9 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (5.00 g, 73.3%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.58, 148.14.

    EXAMPLE 17



    [0120] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (18a).

    [0121] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (3.29 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (4.33 g, 70.8%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.32, 147.78.

    EXAMPLE 18



    [0122] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (18b).

    [0123] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (3.44 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl- β-D-glucopyranoside, 2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (3.98 g, 63.5%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.44, 147.63.

    EXAMPLE 19



    [0124] N6-(4-i-Propylphenoxy)acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (18c).

    [0125] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-(4-i-propylphenoxy) acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10c, (3.65 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (4.22 g, 65.1%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.20, 147.69.

    EXAMPLE 20



    [0126] N6-(N,N-Dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (18d).

    [0127] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10d, (3.04 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (1.23 g, 9.5 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.51 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:2:77) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound (4.56 g, 77.7%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.88, 148.27.

    EXAMPLE 21



    [0128] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (19a).

    [0129] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.23 g, 8.4 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (4.29 g, 6.7 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (1.64 g, 12.7 mmol) in anhydrous dichloromethane (30 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (3.36 g, 7.7 mmol) and 1H-tetrazole (0.45 M in MeCN, 10.0 mL, 6.0 mmol) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound as an off-white foam (5.75 g, 71.2%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.91, 148.38.

    EXAMPLE 22



    [0130] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (19b).

    [0131] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.80 g, 6.75 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (3.80 g, 5.4 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (1.33 g, 10.3 mmol) in anhydrous dichloromethane (20 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.71 g, 6.2 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.8 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound as an off-white foam (4.58 g, 66.8%). 31P NMR (mixture of diastereomers, CD3CN): δ 149.04, 148.60.

    EXAMPLE 23



    [0132] N2-(4-i-Propylphenoxy)acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (19c).

    [0133] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.97 g, 7.38 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-(4-i-propylphenoxy) acetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11c, (4.40 g, 5.9 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (1.45 g, 11.2 mmol) in anhydrous dichloromethane (20 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.96 g, 6.8 mmol) and 1H-tetrazole (0.45 M in MeCN, 5.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound as an off-white foam (4.84 g, 62.6%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.75, 148.19.

    EXAMPLE 24



    [0134] N2-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (19d).

    [0135] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.73 g, 6.50 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11d, (3.25 g, 5.20 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (1.28 g, 9.88 mmol) in anhydrous dichloromethane (20 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-glucopyranoside, 2, (2.61 g, 5.98 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column eluted with a step gradient from ethyl acetate - TEA - hexane (20:5:75) to TEA - ethyl acetate (5:95). Collected fractions were evaporated to give the title compound as an off-white foam (4.34 g, 70.1%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.63, 148.08.

    EXAMPLE 25



    [0136] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]thymidine (20).

    [0137] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.28 g, 4.79 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (2.08 g, 3.83 mmol) and N-ethyl-N,N-diisopropylamine (0.94 g, 7.28 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.92 g, 4.40 mmol, prepared as disclosed in Karjala, S. and Link, K. P., J. Amer. Chem. Soc., 1940, 62, 917-922) and 1H-tetrazole (0.45 M in MeCN, 3.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 16 to give the title compound (3.60 g, 84.8%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.66, 148.09.

    EXAMPLE 26



    [0138] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxycytidine (21a).

    [0139] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.28 g, 4.79 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (2.08 g, 3.73 mmol) and N-ethyl-N,N-diisopropylamine (1.01 g, 7.83 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (2.07 g, 4.74 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.7 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 17a give the title compound (3.97 g, 80.3%). 31P NMR (mixture of diastereomers, CD3CN): δ 148.69, 148.33.

    EXAMPLE 27



    [0140] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxycytidine (21c).

    [0141] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.48 g, 5.35 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (2.45 g, 4.28 mmol) and N-ethyl-N,N-diisopropylamine

    [0142] (1.05 g, 8.13 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl- β-D-galactopyranoside, 3, (2.15 g, 4.92 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.8 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 17c to give the title compound (4.05 g, 83.1%). 31P NMR (mixture of diastereomers, CD3CN): δ 149.16, 148.66.

    EXAMPLE 28



    [0143] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (22a).

    [0144] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.32 g, 4.94 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (2.60 g, 3.95 mmol) and N-ethyl-N,N-diisopropylamine (0.97 g, 7.51 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl- β-D-galactopyranoside, 3, (1.98 g, 4.54 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.5 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 18a give the title compound (3.65 g, 75.5%).31P NMR (mixture of diastereomers, CD3CN): δ 148.73, 148.18.

    EXAMPLE 29



    [0145] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyadenosine (22b).

    [0146] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.25 g, 4.68 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (2.57 g, 3.74 mmol) and N-ethyl-N,N-diisopropylamine (0.92 g, 7.1 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside, 3, (1.88 g, 4.30 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.4 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 18b to give the title compound (3.30 g, 70.4%).31P NMR (mixture of diastereomers, CD3CN): δ 148.80, 148.17.

    EXAMPLE 30



    [0147] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (23a).

    [0148] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.33 g, 5.0 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (2.56 g, 4.0 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (098 g, 7.60 mmol) in anhydrous dichloromethane (30 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl- β-D-galactopyranoside, 3, (2.01 g, 4.60 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 19a to give the title compound as an off-white foam (3.56 g, 73.9%). 31P NMR (mixture of diastereomers, CD3CN): δ 149.32, 148.66.

    EXAMPLE 31



    [0149] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [2-[[2-[(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)oxy]ethyl]oxy]ethoxy]phosphinyl]-2'-deoxyguanosine (23b).

    [0150] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.22 g, 4.59 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (2.58 g, 3.67 mmol, Rasayan, Inc., Encinitas, CA) and N-ethyl-N,N-diisopropylamine (0.90 g, 6.97 mmol) in anhydrous dichloromethane (20 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 2-[(2-hydroxyethyl)oxy]ethyl 2,3,4,6-tetra-O-acetyl- β-D-galactopyranoside, 3, (1.84 g, 4.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 3.3 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated chromatographically on a silica gel column as described above for the respective β-D-glucopyranoside analog 19b to give the title compound as an off-white foam (3.23 g, 69.3%). 31P NMR (mixture of diastereomers, CD3CN): δ 149.33, 148.69.

    EXAMPLE 32



    [0151] 1-O-(3-Hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside (6).

    [0152] A mixture of compound 4 (8.80 g, 26.7 mmol, prepared as disclosed in Fukase, K.; Ueno, A.; Fukase, Y; et al., Bull. Chem. Soc. Japan 2003, 76, 485-500), 1,3-propanediol (30.5 g, 0.4 mol), and pyridinium p-toluenesulfonate (5.37 g, 21.4 mmol) was heated at 80 °C for 18 h, then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5% aqueous NaHCO3 (100 mL). The organic phase was separated, dried over Na2SO4, and evaporated in vacuo. The product was isolated by column chromatography on silica gel eluted with a step gradient from DCM to a mixture of MeOH and DCM (8:92). The solid obtained by evaporation of the relevant fractions was re-crystallized from a mixture of methyl t-butyl ether and DCM to give 2.91 g of pure ?? as an off-white solid. Found: C, 49.69; H, 6.77; N, 3.57. C17H27NO10·½H2O. Calculated: C, 49.27; H, 6.81; N, 3.38. 1H NMR (CD3CN): δ 6.395 (1H, br. d, J = 9.5 Hz); 5.134 (1H, dd, J = 10.5, 9.5 Hz); 4.932 (1H, t, J = 9.7 Hz); 4.612 (1H, d, J = 8.5 Hz); 4.215 (1H, dd, J = 12.5, 5.0 Hz); 4.074 (1H, dd, J = 12.5, 2.5 Hz); 3.876-3.835 (1H, m); 3.802-3.759 (2H, m); 3.625-3.560 (1H, m); 3.560-3.523 (2H, m); 2.554 (1H, t, J = 5.2 Hz); 2.015 (3H, s); 1.965 (3H, s); 1.935 (3H, s); 1.820 (3H, s); 1.707 (2H, pent., J = 6.2 Hz). The mother liquor was evaporated, and the residue was re-crystallized from the same mixture to give additional product (2.61 g) in a total yield of 51.3%.

    EXAMPLE 33



    [0153] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]thymidine (24).

    [0154] To the magnetically stirred mixture of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (5.99 g, 11.0 mmol), N-ethyl-N,N-diisopropylamine (2.70 g, 20.90 mmol), and anhydrous CH3CN (30 mL) was added a solution of N,N,N',N'-tetraisopropyl chlorophosphite (3.67 g, 13.75 mmol) in anhydrous DCM (10 mL) at -20 °C. The mixture was allowed to warm up to room temperature over 2 h. A solution of 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (0.405 g, 1 mmol) in anhydrous acetonitrile (5 mL) was added followed by the addition of a solution of 1H-tetrazole in acetonitrile (0.45 M, 9.8 mL). The reaction mixture was stirred for 18 h at room temperature and was quenched by adding 5% aqueous NaHCO3. The product was extracted with ethyl acetate (3×100 mL). Combined extracts were washed with brine (50 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave compound 24 as white solid foam in a yield of 10.37 g (87.4 %). 31P NMR (CD3CN): δ 147.60, 147.57. 1H NMR (CD3CN): δ 8.955 (1H, br. s); 7.45-7.41 (3H, m); 7.35-7.28 (6H, m); 7.28-7.21 (1H, m); 6.90-6.84 (4H, m); 6.590 (0.5H, d, J = 9.0 Hz); 6.342 (0.5H, d, J = 4.5 Hz); 6.230 (0.5H, t, J = 9.0 Hz); 6.195 (0.5H, t, J = 7.0 Hz); 5.184 (0.5H, dd, J = 10.5, 9.5 Hz); 5.135 (0.5H, dd, J = 10.5, 9.5 Hz); 4.920 (1H, q, J = 9.7 Hz); 4.630-4.525 (2H, m); 4.188 (1H, ddd, J = 12.5, 12.5, 5.0 Hz); 4.13-4.09 (0.5H, m); 4.09-3.95 (2H, m); 3.89-3.805 (0.5H, m); 3.771 (3H, s); 3.765 (3H, s); 3.805-3.62 (3H, m); 3.62-3.475 (4H, m); 3.39-3.23 (2H, m); 2.46-230 (2H, m); 1.982, 1.965, 1.962, 1.954, 1.940, 1.935 (total 9H, singlets); 1.789 (1.5H, s); 1.777 (1.5H, s); 1.691 (2H, pent., J = 6.5 Hz); 1.553 (1.5H, d, J = 1.0 Hz); 1.490 (1.5H, d, J = 1.0 Hz); 1.18-1.11 (9H, m); 1.040 (3H, d, J = 7.0 Hz).

    EXAMPLE 34



    [0155] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxycytidine (25a).

    [0156] Solution of bis(N,N-diisopropylamino) chlorophosphite (3.20 g, 12.0 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (6.08 g, 9.6 mmol) and N-ethyl-N,N-diisopropylamine (2.36 g, 18.2 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (4.48 g, 11.0 mmol) and 1H-tetrazole (0.45 M in MeCN, 8.5 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave the target compound as white solid foam in a yield of 9.10 g (81.1 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.63, 147.21.

    EXAMPLE 35



    [0157] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxycytidine (25c).

    [0158] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.67 g, 6.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (2.86 g, 5.0 mmol) and N-ethyl-N,N-diisopropylamine (12.3 g, 9.5 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (2.33 g, 5.75 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.5 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave the title compound as white solid foam in a yield of 4.14 g (74.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.43, 148.77.

    EXAMPLE 36



    [0159] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (26a).

    [0160] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.77 g, 6.63 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (3.49 g, 5.3 mmol) and N-ethyl-N,N-diisopropylamine (1.30 g, 10.1 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (2.47 g, 6.10 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.7 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 4.47 g (70.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.58, 148.11.

    EXAMPLE 37



    [0161] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyadenosine (26b).

    [0162] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.57 g, 5.88 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (3.23 g, 4.70 mmol) and N-ethyl-N,N-diisopropylamine (1.15 g, 8.9 mmol) in anhydrous dichloromethane (15 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (2.19 g, 5.41 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.2 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 3.92 g (68.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.98, 148.29.

    EXAMPLE 38



    [0163] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (27a).

    [0164] Solution of bis(N,N-diisopropylamino) chlorophosphite (2.23 g, 8.38 mmol) in anhydrous dichloromethane (15 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (4.29 g, 6.70 mmol) and N-ethyl-N,N-diisopropylamine (1.64 g, 12.73 mmol) in anhydrous dichloromethane (30 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (3.12 g, 7.71 mmol) and 1H-tetrazole (0.45 M in MeCN, 6.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×100 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 5.68 g (72.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.91, 148.33.

    EXAMPLE 39



    [0165] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[3-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranosyl)oxy]propoxy]phosphinyl]-2'-deoxyguanosine (27b).

    [0166] Solution of bis(N,N-diisopropylamino) chlorophosphite (1.50 g, 5.63 mmol) in anhydrous dichloromethane (10 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (3.17 g, 4.50 mmol) and N-ethyl-N,N-diisopropylamine (1.10 g, 8.55 mmol) in anhydrous dichloromethane (20 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(3-hydroxypropyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-glucopyranoside, 6, (2.10 g, 5.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 4.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (100 mL), and the product was extracted with ethyl acetate (3×70 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 3.66 g (65.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.72, 148.27.

    EXAMPLE 40



    [0167] 1-O-(5-Hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside (7b).

    [0168] A mixture of compound 5 (13.17 g, 40.0 mmol, prepared as disclosed in Fukase, K.; Ueno, A.; Fukase, Y; et al., Bull. Chem. Soc. Japan 2003, 76, 485-500), anhydrous diethyleneglycol (42.5 g, 0.4 mol), and pyridinium p-toluenesulfonate (9.05 g, 36.0 mmol) was heated at 80 °C for 18 h, then distributed between DCM (300 mL) and a 1:1 mixture of brine and 5% aqueous NaHCO3 (100 mL). The organic phase was separated, dried over Na2SO4, and evaporated in vacuo. The product was isolated by column chromatography on silica gel eluted with a step gradient from DCM to a mixture of MeOH and DCM (8:92). Evaporation of the relevant fractions gave the title compound as an off-white solid (7.92 g, 45.4%). 1H NMR (CD3CN): δ 6.572 (1H, d, J = 9.5 Hz); 5.278 (1H, d, J = 3 Hz); 5.010 (1H, dd, J = 11.5 Hz, J = 3Hz); 4.673 (1H, d, J = 8.5 Hz); 4.109 (1H, dd, J = 9 Hz, J = 7 Hz); 4.066 (1H, dd, J = 9 Hz, J = 6 Hz); 3.951 (2H, m); 3.828 (1H, m); 3.703 (1H, m); 3.64-3.54 (4H, m); 3.54-3.45 (2H, m); 3.053 (1H, br. s); 2.098 (3H, s) 1.988 (3H, s); 1.910 (3H, s), 1.845 (3H, s). ES MS: 436.1 (MH+). Calculated for C18H29NO11·H+: 436.2.

    EXAMPLE 41



    [0169] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]thymidine (28).

    [0170] To the magnetically stirred mixture of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (1.142 g, 2.10 mmol), N-ethyl-N,N-diisopropylamine (0.515 g, 3.99 mmol), and anhydrous CH3CN (10 mL) was added a solution of N,N,N',N'-tetraisopropyl chlorophosphite (0.700 g, 2.63 mmol) in anhydrous DCM (5 mL) at -20 °C. The mixture was allowed to warm up to room temperature over 2 h. A solution of 1-O-(5-hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7b, (1.097 g, 2.52 mmol) in anhydrous acetonitrile (5 mL) was added followed by the addition of a solution of 1H-tetrazole in acetonitrile (0.45 M, 1.87 mL). The reaction mixture was stirred for 18 h at room temperature and was quenched by adding 5% aqueous NaHCO3. The product was extracted with ethyl acetate (3×50 mL). Combined extracts were washed with brine (50 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave compound 24 as white solid foam in a yield of 1.760 g (75.6 %). Fast diastereomer: 1H NMR (CD3CN): δ 9.09 (1H, br. s); 7.5-7.4 (3H, m); 7.375-7.30 (6H, m); 7.30-7.23 (1H, m); 6.9-6.8 (4H, m); 6.335 (1H, d, J = 9 Hz); 6.248 (1H, t, J = 7 Hz); 5.31-5.27 (1H, m); 5.025 (1H, dd, J = 11.2 Hz, J = 3.5 Hz); 4.66-4.59 (1H, m); 4.565 (1H, d, J = 8.5 Hz); 4.15-4.11 (1H, m); 4.11-4.03 (2H, m); 3.99-3.89 (2H, m); 3.84-3.70 (2H, m); 3.783 and 3.766 (total 6H, s, s); 3.67-3.53 (5H, m); 3.53-3.43 (4H, m); 3.355 (1H, dd, J = 10.5 Hz, J = 3.0 Hz); (1H, dd, J = 10.5 Hz, J = 3.5 Hz); 2.41-2.33 (2H, m); 2.090 (3H, s); 1.968 (3H, s), 1.913 (3H, s); 1.820 (3H, s); 1.480 (3H, d, J = 0.5 Hz); 1.149 (12H, t, J = 6.5 Hz). 31P NMR (CD3CN): δ 149.99.
    Slow diastereomer: 1H NMR (CD3CN): δ 8.976 (1H, br. s); 7.5-7.4 (3H, m); 7.375-7.30 (6H, m); 7.30-7.23 (1H, m); 6.9-6.8 (4H, m); 6.358 (1H, d, J = 9.5 Hz); 6.242 (1H, t, J = 7 Hz); 5.29-5.27 (1H, m); 5.030 (1H, dd, J = 11.5 Hz, J = 3.5 Hz); 4.64-4.54 (2H, m); 4.15-4.03 (3H, m); 3.99-3.89 (2H, m); 3.85-3.79 (1H, m); 3.814 and 3.764 (total 6H, s, s); 3.78-3.70 (1H, m); 3.70-3.61 (2H, m); 3.61-3.48 (5H, m); 3.35-3.255 (1H, m); 2.46-2.39 (1H, m); 2.37-2.29 (1H, m); 2.094 (3H, s); 1.974 (3H, s), 1.913 (3H, s); 1.832 (3H, s); 1.498 (3H, s); 1.143 (6H, d, J = 6.5 Hz); 1.051 (6H, d, J = 6.5 Hz). 31P NMR (CD3CN): δ 149.67.

    EXAMPLE 42



    [0171] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxycytidine (29a).

    [0172] Solution of bis(N,N-diisopropylamino) chlorophosphite (667 mg, 2.50 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (1267 mg, 2.0 mmol) and N-ethyl-N,N-diisopropylamine (491 mg, 3.80 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1045 mg, 2.40 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.8 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave the target compound as white solid foam in a yield of 1769 mg (73.8 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.91, 148.37.

    EXAMPLE 43



    [0173] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxycytidine (29c).

    [0174] Solution of bis(N,N-diisopropylamino) chlorophosphite (767 mg, 2.88 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (1315 mg, 2.30 mmol) and N-ethyl-N,N-diisopropylamine (564 mg, 4.37 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1202 mg, 2.76 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.1 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave the title compound as white solid foam in a yield of 1835 mg (70.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.78, 148.11.

    EXAMPLE 44



    [0175] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxyadenosine (30a).

    [0176] Solution of bis(N,N-diisopropylamino) chlorophosphite (750 mg, 2.81 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (1480, 2.25 mmol) and N-ethyl-N,N-diisopropylamine (552 mg, 4.28 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1176 mg, 2.70 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 1851 mg (67.3 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.83, 148.23.

    EXAMPLE 45



    [0177] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxyadenosine (30b).

    [0178] Solution of bis(N,N-diisopropylamino) chlorophosphite (700, 2.63 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (1444 mg, 2.10 mmol) and N-ethyl-N,N-diisopropylamine (515 mg, 3.99 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1097 mg, 2.52 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 1681 mg (63.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.94, 147.52.

    EXAMPLE 46



    [0179] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxyguanosine (31a).

    [0180] Solution of bis(N,N-diisopropylamino) chlorophosphite (734 mg, 2.75 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (1407 mg, 2.20 mmol) and N-ethyl-N,N-diisopropylamine (540 mg, 4.18 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1149 mg, 2.64 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 1706 mg (64.4 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.88, 147.43.

    EXAMPLE 47



    [0181] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]-2'-deoxyguanosine (31b).

    [0182] Solution of bis(N,N-diisopropylamino) chlorophosphite (750 mg, 2.81 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (1583 mg, 2.25 mmol) and N-ethyl-N,N-diisopropylamine (552 mg, 4.28 mmol) in anhydrous dichloromethane (10 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7b (1176 mg, 2.70 mmol) and 1H-tetrazole (0.45 M in MeCN, 2.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 1758 mg (61.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.80, 147.39.

    EXAMPLE 48



    [0183] 1-O-(8-Hydroxy-3,6-dioxaoctyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside (7c).

    [0184] A mixture of compound 5 (3.29 g, 10.0 mmol), anhydrous triethyleneglycol (12.0 g, 80 mmol), and pyridinium p-toluenesulfonate (2.26 g, 9.0 mmol) was heated at 80 °C for 18 h, then distributed between DCM (70 mL) and a 1:1 mixture of brine and 5% aqueous NaHCO3 (100 mL). The organic phase was separated, dried over Na2SO4, and evaporated in vacuo. The product was isolated by column chromatography on silica gel eluted with a step gradient from DCM to a mixture of MeOH and DCM (8:92). Evaporation of the relevant fractions gave the title compound as an off-white solid (2.32 g, 48.4%). ES MS: 480.2 (MH+). Calculated for C18H29NO11·H+: 480.2.

    EXAMPLE 49



    [0185] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]thymidine (32).

    [0186] Solution of bis(N,N-diisopropylamino) chlorophosphite (347 mg, 1.30 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (566 mg, 1.04 mmol) and N-ethyl-N,N-diisopropylamine (255 mg, 1.98 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(8-hydroxy-3,6-dioxaoctyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7c, (598 mg, 1.25 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as white solid foam in a yield of 920 mg (76.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.95, 147.41.

    EXAMPLE 50



    [0187] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxycytidine (33a).

    [0188] Solution of bis(N,N-diisopropylamino) chlorophosphite (357 mg, 1.34 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (678 mg, 1.7 mmol) and N-ethyl-N,N-diisopropylamine (263 mg, 2.03 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (616 mg, 1.28 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as white solid foam in a yield of 980 mg (73.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.22, 147.62.

    EXAMPLE 51



    [0189] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxycytidine (33c).

    [0190] Solution of bis(N,N-diisopropylamino) chlorophosphite (333 mg, 1.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (572 mg, 1.0 mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (575 mg, 1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as white solid foam in a yield of 829 mg (70.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.08, 147.72.

    EXAMPLE 52



    [0191] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxyadenosine (34a).

    [0192] Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (671, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (587 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 831 mg (64.3 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.19, 147.47.

    EXAMPLE 53



    [0193] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxyadenosine (34b).

    [0194] Solution of bis(N,N-diisopropylamino) chlorophosphite (370, 1.39 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (763 mg, 1.11 mmol) and N-ethyl-N,N-diisopropylamine (272 mg, 2.11 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (639 mg, 1.33 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 872 mg (60.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.51, 147.90.

    EXAMPLE 54



    [0195] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxyguanosine (35a).

    [0196] Solution of bis(N,N-diisopropylamino) chlorophosphite (320 mg, 1.20 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (614 mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (552 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 784 mg (65.4 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.60, 147.11.

    EXAMPLE 55



    [0197] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[8-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6-dioxaoctyloxy]phosphinyl]-2'-deoxyguanosine (35b).

    [0198] Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (697 mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88 mmol) in anhydrous dichloromethane (10 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7c (570 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as white solid foam in a yield of 803 mg (61.8 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.55, 147.73.

    EXAMPLE 56



    [0199] 1-O-(11-Hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside (7d).

    [0200] A mixture of compound 5 (7.25 g, 22.0 mmol), anhydrous tetraethyleneglycol (34.2 g, 176 mmol), and pyridinium p-toluenesulfonate (4.98 g, 19.8 mmol) was heated at 80 °C for 18 h, then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5% aqueous NaHCO3 (100 mL). The organic phase was separated, dried over Na2SO4, and evaporated in vacuo. The product was isolated by column chromatography on silica gel eluted with a step gradient from DCM to a mixture of MeOH and DCM (8:92). Evaporation of the relevant fractions gave the title compound as an off-white solid (4.92 g, 42.7%). ES MS: 524.4 (MH+). Calculated for C18H29NO11·H+: 524.2.

    EXAMPLE 57



    [0201] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]thymidine (36).

    [0202] Solution of bis(N,N-diisopropylamino) chlorophosphite (370 mg, 1.39 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (604 mg, 1.11 mmol) and N-ethyl-N,N-diisopropylamine (272 mg, 2.11 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7d, (697 mg, 1.33 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1099 mg (82.7 %). 31P NMR (mixture of diastereomers, CD3CN, FIG. 10): δ 152.37, 152.34.

    EXAMPLE 58



    [0203] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxycytidine (37a).

    [0204] Solution of bis(N,N-diisopropylamino) chlorophosphite (313 mg, 1.18 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (596 mg, 0.94 mmol) and N-ethyl-N,N-diisopropylamine (231 mg, 1.79 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (590 mg, 1.13 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 940 mg (77.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 150.06, 149.74.

    EXAMPLE 59



    [0205] N4-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxycytidine (37b).

    [0206] Solution of bis(N,N-diisopropylamino) chlorophosphite (323 mg, 1.21 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9b, (644 mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) in anhydrous dichloromethane (5 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (609 mg, 1.16 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1014 mg (79.4 %). 31P NMR (mixture of diastereomers, CD3CN): δ 151.67, 151.24.

    EXAMPLE 60



    [0207] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino) [11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxycytidine (37c).

    [0208] Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (583 mg, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 998 mg (79.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.82, 149.19.

    EXAMPLE 61



    [0209] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyadenosine (38a).

    [0210] Solution of bis(N,N-diisopropylamino) chlorophosphite (327 mg, 1.23 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (645, 0.98 mmol) and N-ethyl-N,N-diisopropylamine (240 mg, 1.86 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (616 mg, 1.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (50 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 944 mg (73.5 %). 31P NMR (mixture of diastereomers, CD3CN): δ 150.09, 149.36.

    EXAMPLE 62



    [0211] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyadenosine (38b).

    [0212] Solution of bis(N,N-diisopropylamino) chlorophosphite (333, 1.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (688 mg, 1.0 mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (628 mg, 1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 913 mg (68.1 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.84, 149.23.

    EXAMPLE 63



    [0213] N6-(4-i-Propylphenoxyacetyl)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyadenosine (38c).

    [0214] Solution of bis(N,N-diisopropylamino) chlorophosphite (340, 1.28 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-(4-i-propylphenoxy acetyl)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10c, (744 mg, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 982 mg (69.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.91, 149.42.

    EXAMPLE 64



    [0215] N6-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyadenosine (38d).

    [0216] Solution of bis(N,N-diisopropylamino) chlorophosphite (320, 1.20 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N6-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10d, (584 mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (603 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 798 mg (65.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 151.19, 150.55.

    EXAMPLE 65



    [0217] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-triO-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyguanosine (39a).

    [0218] Solution of bis(N,N-diisopropylamino) chlorophosphite (343 mg, 1.29 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (659 mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol) in anhydrous dichloromethane (5 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (40 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 900 mg (67.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 152.01, 151.92.

    EXAMPLE 66



    [0219] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyguanosine (39b).

    [0220] Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (718 mg, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) in anhydrous dichloromethane (10 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 944 mg (68.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 151.95, 151.86.

    EXAMPLE 67



    [0221] N2-(4-i-Propylphenoxyacetyl)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]-3-oxapentyloxy]phosphinyl]-2'-deoxyguanosine (39c).

    [0222] Solution of bis(N,N-diisopropylamino) chlorophosphite (333 mg, 1.25 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-(4-i-propylphenoxy acetyl) -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11c, (746 mg, 1.0 mmol) and N-ethyl-N,N-diisopropylamine (245 mg, 1.90 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (628 mg, 1.20 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 860 mg (61.5 %). 31P NMR (mixture of diastereomers, CD3CN): δ 150.29, 149.95.

    EXAMPLE 68



    [0223] N2-(N,N-Dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-2'-deoxyguanosine (39d).

    [0224] Solution of bis(N,N-diisopropylamino) chlorophosphite (317 mg, 1.19 mmol) in anhydrous dichloromethane (5 mL) was added dropwise to a stirred solution of N2-(N,N-dimethylformamidino)-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11d, (594 mg, 0.95 mmol) and N-ethyl-N,N-diisopropylamine (233 mg, 1.81 mmol) in anhydrous dichloromethane (10 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (597 mg, 1.14 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (30 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 808 mg (66.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 150.03, 149.72.

    EXAMPLE 69



    [0225] 1-O-(14-Hydroxy-3,6,9,12-tetraoxatetradecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside (7e).

    [0226] A mixture of compound 5 (1.64 g, 5.0 mmol), anhydrous pentaethyleneglycol (7.15 g, 30.0 mol), and pyridinium p-toluenesulfonate (1.13 g, 4.5 mmol) was heated at 80 °C for 18 h, then distributed between DCM (50 mL) and a 1:1 mixture of brine and 5% aqueous NaHCO3 (25 mL). The organic phase was separated, dried over Na2SO4, and evaporated in vacuo. The product was isolated by column chromatography on silica gel eluted with a step gradient from DCM to a mixture of MeOH and DCM (8:92). Evaporation of the relevant fractions gave the title compound as an off-white solid (1.08 g, 38.1%). ES MS: 568.0 (MH+). Calculated for C18H29NO11·H+: 568.2.

    EXAMPLE 70



    [0227] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]thymidine (40).

    [0228] Solution of bis(N,N-diisopropylamino) chlorophosphite (213 mg, 0.8 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (348 mg, 0.64 mmol) and N-ethyl-N,N-diisopropylamine (157 mg, 1.22 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(14-hydroxy-3,6,9,12-tetraoxatetradecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7e, (436 mg, 0.77 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 617 mg (77.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.99, 149.66.

    EXAMPLE 71



    [0229] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2'-deoxycytidine (41a).

    [0230] Solution of bis(N,N-diisopropylamino) chlorophosphite (240 mg, 0.90 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9a, (456 mg, 0.72 mmol) and N-ethyl-N,N-diisopropylamine (177 mg, 1.37 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (490 mg, 0.86 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.7 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 692 mg (72.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.17, 147.63.

    EXAMPLE 72



    [0231] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2'-deoxycytidine (41c).

    [0232] Solution of bis(N,N-diisopropylamino) chlorophosphite (220 mg, 0.83 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxycytidine, 9c, (377 mg, 0.66 mmol) and N-ethyl-N,N-diisopropylamine (157 mg, 1.22 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (450 mg, 0.79 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 590 mg (70.5 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.88, 147.21.

    EXAMPLE 73



    [0233] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2' -deoxyadenosine (42a).

    [0234] Solution of bis(N,N-diisopropylamino) chlorophosphite (200 mg, 0.75 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10a, (395, 0.60 mmol) and N-ethyl-N,N-diisopropylamine (147 mg, 1.14 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (409 mg, 0.72 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 563 mg (69.3 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.82, 147.28.

    EXAMPLE 74



    [0235] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2'-deoxyadenosine (42b).

    [0236] Solution of bis(N,N-diisopropylamino) chlorophosphite (230, 0.86 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyadenosine, 10b, (475 mg, 0.69 mmol) and N-ethyl-N,N-diisopropylamine (169 mg, 1.31 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (470 mg, 0.83 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.7 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 610 mg (63.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.17, 147.52.

    EXAMPLE 75



    [0237] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-triO-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyloxy]phosphinyl]-2'-deoxyguanosine (43a).

    [0238] Solution of bis(N,N-diisopropylamino) chlorophosphite (203 mg, 0.76 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11a, (390 mg, 0.61 mmol) and N-ethyl-N,N-diisopropylamine (150 mg, 1.16 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (415 mg, 0.73 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 543 mg (66.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.83, 147.24.

    EXAMPLE 76



    [0239] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[14-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9,12-tetraoxatetradecyl] phosphino]-2'-deoxyguanosine (43b).

    [0240] Solution of bis(N,N-diisopropylamino) chlorophosphite (217 mg, 0.81 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-deoxyguanosine, 11b, (457 mg, 0.65 mmol) and N-ethyl-N,N-diisopropylamine (160 mg, 1.24 mmol) in anhydrous dichloromethane (4 mL) at - 20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7e (443 mg, 0.78 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.6 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 565 mg (62.1 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.29, 147.47.

    EXAMPLE 77



    [0241] 5'-O-(4,4'-Dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]-5-methyluridine (52).

    [0242] Solution of bis(N,N-diisopropylamino) chlorophosphite (313 mg, 1.18 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)-3'-O-methyl-5-methyluridine, 44, (540 mg, 0.94 mmol) and N-ethyl-N,N-diisopropylamine (231 mg, 1.79 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7d, (591 mg, 1.13 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 977 mg (84.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.43, 148.62.

    EXAMPLE 78



    [0243] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]cytidine (53a).

    [0244] Solution of bis(N,N-diisopropylamino) chlorophosphite (323 mg, 1.21 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methylcytidine, 45a, (646 mg, 0.97 mmol) and N-ethyl-N,N-diisopropylamine (238 mg, 1.84 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (609 mg, 1.16 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1038 mg (81.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 149.17, 148.75.

    EXAMPLE 79



    [0245] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]cytidine (53c).

    [0246] Solution of bis(N,N-diisopropylamino) chlorophosphite (340 mg, 1.28 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methylcytidine, 45c, (616 mg, 1.02 mmol) and N-ethyl-N,N-diisopropylamine (250 mg, 1.94 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (641 mg, 1.22 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1061 mg (82.8 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.98, 148.43.

    EXAMPLE 80



    [0247] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]adenosine (54a).

    [0248] Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyladenosine, 46a, (681 mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (622 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 1039 mg (78.3 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.92, 148.37.

    EXAMPLE 81



    [0249] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino) [11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]adenosine (54b).

    [0250] Solution of bis(N,N-diisopropylamino) chlorophosphite (343, 1.29 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyladenosine, 46b, (739 mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 1011 mg (71.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.92, 147.47.

    EXAMPLE 82



    [0251] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyloxy]phosphinyl]guanosine (55a).

    [0252] Solution of bis(N,N-diisopropylamino) chlorophosphite (317 mg, 1.19 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methylguanosine, 47a, (636 mg, 0.95 mmol) and N-ethyl-N,N-diisopropylamine (233 mg, 1.81 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (597 mg, 1.14 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 966 mg (76.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.90, 147.32.

    EXAMPLE 83



    [0253] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-O-methyl-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino]guanosine (55b).

    [0254] Solution of bis(N,N-diisopropylamino) chlorophosphite (337 mg, 1.26 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl -5'-O-(4,4'-dimethoxytrityl)-2'-O-methylguanosine, 47b, (741 mg, 1.01 mmol) and N-ethyl-N,N-diisopropylamine (248 mg, 1.92 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (635 mg, 1.21 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 1011 mg (72.2 %). 31P NMR (mixture of diastereomers, CD3CN): δ 147.51, 146.96.

    EXAMPLE 84



    [0255] 5'-O-(4,4'-Dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl]phosphino]-2'-deoxy-5-methyluridine (56).

    [0256] Solution of bis(N,N-diisopropylamino) chlorophosphite (350 mg, 1.31 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of 5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxy-5-methyluridine, 48, (591 mg, 1.05 mmol) and N-ethyl-N,N-diisopropylamine (258 mg, 2.00 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, 1-O-(11-hydroxy-3,6,9-trioxaundecyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7d, (660 mg, 1.26 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1060 mg (83.1 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.38, 147.87.

    EXAMPLE 85



    [0257] N4-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino]-2'-deoxycytidine (57a).

    [0258] Solution of bis(N,N-diisopropylamino) chlorophosphite (343 mg, 1.31 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-benzoyl-5'-O-(4,4'-dimethoxytrityl)- 2'-fluoro-2'-deoxycytidine, 49a, (673 mg, 1.03 mmol) and N-ethyl-N,N-diisopropylamine (253 mg, 1.96 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (647 mg, 1.24 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 1075 mg (79.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.52, 147.81.

    EXAMPLE 86



    [0259] N4-Acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino]-2'-deoxycytidine (57c).

    [0260] Solution of bis(N,N-diisopropylamino) chlorophosphite (337 mg, 1.26 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N4-acetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxycytidine, 49c, (598 mg, 1.01 mmol) and N-ethyl-N,N-diisopropylamine (248 mg, 1.92 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (635 mg, 1.21 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (5:95). Evaporation of fractions gave the title compound as viscous oil in a yield of 985 mg (78.4 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.23, 147.69.

    EXAMPLE 87



    [0261] N6-Benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino] -2' -deoxyadenosine (58a).

    [0262] Solution of bis(N,N-diisopropylamino) chlorophosphite (347 mg, 1.30 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-benzoyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxyadenosine, 50a, (703 mg, 1.4 mmol) and N-ethyl-N,N-diisopropylamine (255 mg, 1.98 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (653 mg, 1.25 mmol) and 1H-tetrazole (0.45 M in MeCN, 1.0 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 995 mg (72.0 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.23, 147.53.

    EXAMPLE 88



    [0263] N6-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino]-2'-deoxyadenosine (58b).

    [0264] Solution of bis(N,N-diisopropylamino) chlorophosphite (327, 1.23 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N6-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxyadenosine, 50b, (692 mg, 0.98 mmol) and N-ethyl-N,N-diisopropylamine (240 mg, 1.86 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (616 mg, 1.18 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 917 mg (68.9 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.47, 147.88.

    EXAMPLE 89



    [0265] N2-Isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino] -2' -deoxyguanosine (59a).

    [0266] Solution of bis(N,N-diisopropylamino) chlorophosphite (320 mg, 1.20 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxyguanosine, 51a, (631 mg, 0.96 mmol) and N-ethyl-N,N-diisopropylamine (236 mg, 1.82 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (603 mg, 1.15 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 850 mg (67.6 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.34, 147.71.

    EXAMPLE 90



    [0267] N2-Phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-3'-O-[(N,N-diisopropylamino)[11-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3,6,9-trioxaundecyl] phosphino]-2'-deoxyguanosine (59b).

    [0268] Solution of bis(N,N-diisopropylamino) chlorophosphite (330 mg, 1.24 mmol) in anhydrous dichloromethane (3 mL) was added dropwise to a stirred solution of N2-phenoxyacetyl-5'-O-(4,4'-dimethoxytrityl)-2'-fluoro-2'-deoxyguanosine, 51b, (715 mg, 0.99 mmol) and N-ethyl-N,N-diisopropylamine (243 mg, 1.88 mmol) in anhydrous dichloromethane (4 mL) at -20°C. The solution was allowed to stir at room temperature for 2 h. To the obtained mixture, compound 7d (622 mg, 1.19 mmol) and 1H-tetrazole (0.45 M in MeCN, 0.9 mL) were added followed by stirring at room temperature for 14 h. The reaction mixture was quenched by addition of excess 5% aqueous NaHCO3. The emulsion was diluted with 5% aqueous NaHCO3 (10 mL), and the product was extracted with ethyl acetate (3×50 mL). Extracts were washed with brine, dried over anhydrous Na2SO4, and evaporated to oil. Evaporation of fractions gave the title compound as viscous oil in a yield of 840 mg (61.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.59, 147.90.

    EXAMPLE 91



    [0269] 5'-O-(4,4'-Dimethoxytrityl)-3'-O-[(N,N-diisopropylamino)[5-[(3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranosyl)oxy]-3-oxapentyloxy]phosphinyl]thymidine (28).

    [0270] To the magnetically stirred mixture of 1-O-(5-hydroxy-3-oxapentyl)-3,4,6-tri-O-acetyl-2-acetylamino-2-deoxy-β-D-galactopyranoside, 7b, (501 g, 1.15 mmol) N-ethyl-N,N-diisopropylamine (282 mg, 2.19 mmol), and anhydrous CH3CN (10 mL) was added a solution of N,N,N',N'-tetraisopropyl chlorophosphite (364 mg, 1.44 mmol) in anhydrous DCM (5 mL) at - 20 °C. The mixture was allowed to warm up to room temperature over 2 h. A solution of 5'-O-(4,4'-dimethoxytrityl)thymidine, 8, (565 mg, 1.09 mmol), in anhydrous DCM (5 mL) was added followed by the addition of a solution of 1H-tetrazole in acetonitrile (0.45 M, 1.1 mL). The reaction mixture was stirred for 18 h at room temperature and was quenched by adding 5% aqueous NaHCO3. The product was extracted with ethyl acetate (3×50 mL). Combined extracts were washed with brine (50 mL), dried over anhydrous Na2SO4, and evaporated to dryness. The product was isolated by column chromatography on silica gel eluted with a step gradient starting from TEA - ethyl acetate - hexane (5:20:75) to a mixture of TEA in ethyl acetate (10:90). Evaporation of fractions gave compound 24 as white solid foam in a yield of 941 mg (77.7 %). 31P NMR (mixture of diastereomers, CD3CN): δ 148.12, 147.63.

    EXAMPLE 92



    [0271] Oligonucleotides 61-64, 67-72, and 81-84 were synthesized by the phosphoramidite method on an Applied Biosystems DNA/RNA synthesizer 394 on 1 µmol scale starting with a commercial DMT-nucleoside-succinyl-CPG (1000 Å, Glen Research, Sterling, VA). The standard synthetic cycle useful in assemblying oligonucleotides with phosphate backbone comprised the following steps:
    1. (a) detritylation of the solid phase-bound material with 3% dichloroacetic acid in dichloromethane followed by washing with acetonitrile;
    2. (b) coupling of nucleoside phosphoramidite building block (0.1 M in acetonitrile) required by the sequence to the solid support-bound material in the presence of 1H-tetrazole (0.45 M in acetonitrile);
    3. (c) capping of unreacted solid support-bound hydroxy groups with a mixture of acetic anhydride and N-methyl imidazole and
    4. (d) oxidation of the solid support-bound phosphite triester groups with iodine (0.05 M in a mixture of pyridine, tetrahydrofurane, water).
    Preparation of oligonucleotide phosphorothioates was conducted as disclosed in US 7,723,528. Accordingly, steps (a) and (b) remained as disclosed above. The iodine solution was replaced with a solution of N'-(3-thioxo-3H-1,2,4-dithiazol-5-yl)-N,N-dimethylmethanimidamide (0.025 M in pyridine). Upon the completion of the coupling step (b) and washing the solid phase with acetonitrile, the sulfurization step was conducted. The capping step followed the sulfurization. The cycle appropriate for the assembly of the desired oligonucleotide was repeated as required by the sequence in preparation.

    [0272] The synthetic cycle for incorporation of the protected ribonucleoside phosphoramidite building blocks was different from that disclosed above in that the coupling time was extended to 10 min.

    [0273] The synthetic cycle for incorporation of the phosphoramidite building blocks disclosed in the present invention was different from that disclosed above in that the coupling time was extended to 1 min.

    [0274] The final release of oligonucleotides 61-64 from the solid support, deprotection of internucleosidic phosphates, and monosaccharide residues was carried out by treatment under the following conditions:
    1. A. Concentrated ammonium hydroxide, room temperature, 18 h;
    2. B. Concentrated aqueous ammonium hydroxide, 65°C, 8 h;
    3. C. 0.05 M K2CO3 in anhydrous MeOH, room temperature, 4 h;
    4. D. 0.5 M Piperidine in anhydrous acetonitrile, room temperature, 15 min, followed by a mixture of 1,2-diaminoethane and toluene (50:50 v/v), room temperature, 4 h;
    5. E. 25% Aqueous t-butylamine, 65°C, 4 h;
    6. F. Mixture of concentrated aqueous ammonium hydroxide and 40% aqueous methylamine (50:50 v/v), room temperature, 120 min.


    [0275] Upon completion of the deprotection under the conditions A, B, E, and F, the liquid phase was collected and evaporated in vacuo to dryness. The residue was dissolved in water (1 mL) and analyzed by reverse-phase HPLC and by ES MS.

    [0276] Upon completion of the deprotection under the condition C, the reaction mixtures were neutralized by addition of 0.25 M aqueous NaH2PO4 (250 µL per 1 mL of deprotection solution). The solid phase was spun down; the liquid phase was collected and analyzed as described above.

    [0277] Upon completion of the deprotection under the condition D, the solid phase was spun down, and the liquid phase was discarded. The solid phase was washed with toluene (2×1 mL) and with acetonitrile (2×1 mL). The solid phase was suspended in water (1 mL) and the suspension was centrifugated. The liquid phase was collected and analyzed as described above.

    [0278] The final release of oligonucleotides 67-72 and 81-84 from the solid support, deprotection of internucleosidic phosphates, monosaccharide residues, and nucleic bases was carried out by treatment with concentrated aqueous ammonium hydroxide (2 mL) at room temperature for 18 h. The removal of 2'-O-tBDMS groups from oligonucleotide 84 (SEQ. ID 15) was additionally carried out as disclosed in Song, Q. and Jones, R.A. Tetrahedron Lett. 1999, 40, 4653-4654. Upon evaporation in vacuo, the crude deprotection mixtures were dissolved in water and analyzed by ES MS and reverse-phase HPLC. HPLC analysis was carried out on Phenomenex Gemini C18 column (4.6×150 mm) using 0.05 M Tris-HCl as Buffer A, acetonitrile as Buffer B, and a linear gradient from 0 to 100% B in 45 min at a flow rate of 1 mL/min. Peaks were detected by a UV-VIS detector at 264 nm.

    SEQUENCE LISTING



    [0279] 

    <110> AM Chemicals LLC Guzaev, Andrei P. Vvedenskiy, Yladimir Y.

    <120> Phosphoramidite Building Blocks for Sugar-Conjugated Oligonucleotides

    <130> 95111-910083

    <150> US 61/831,521
    <151> 2013-06-05

    <160> 15

    <170> PatentIn version 3.5

    <210> 1
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <400> 1
    tttttttttt   10

    <210> 2
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <400> 2
    tttttttttt   10

    <210> 3
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <400> 3
    tttttttttt   10

    <210> 4
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <400> 4
    tttttttttt   10

    <210> 5
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified to thymidine-3'-[[(beta-D-glucopyranosyl)oxy]propyl]phosphate

    <400> 5
    tttttttttt   10

    <210> 6
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <400> 6
    tttttttttt   10

    <210> 7
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <400> 7
    tttttttttt   10

    <210> 8
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <400> 8
    tttttttttt   10

    <210> 9
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(beta-D-glucopyranosyloxy)ethyl]oxy]ethyl] modification

    <400> 9
    tttttttttt   10

    <210> 10
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <400> 10
    tttttttttt   10

    <210> 11
    <211> 10
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified at phosphate moiety to phosphotriester with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <400> 11
    tttttttttt   10

    <210> 12
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (10)..(10)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (11)..(11)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (12)..(12)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (13)..(13)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (14)..(14)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (15)..(15)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (16)..(16)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <220>
    <221> modified_base
    <222> (17)..(17)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(beta-D-glucopyranosyloxy)propyl] modification

    <400> 12
    tgtgagtacc actgattc   18

    <210> 13
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base (9)..(9) Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (10)..(10)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (11)..(11)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (12)..(12)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (13)..(13)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (14)..(14)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (15)..(15)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <220>
    <221> modified_base
    <222> (16)..(16)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <220>
    <221> modified_base
    <222> (17)..(17)
    <223> Residue modified with phosphorothioate further modified with 1-[3-(N-acetyl-2-deoxy-2-amino-beta-D-glucopyranosyloxy) propyl] modification

    <400> 13
    tgtgagtacc actgatta   18

    <210> 14
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (8)..(8) Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (10)..(10)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (11)..(11)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (12)..(12)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (13)..(13)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (14)..(14)
    <223> Residue modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (15)..(15)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (16)..(16)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (17)..(17)
    <223> Residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <400> 14
    tgtgagtacc actgatta   18
    <210> 15
    <211> 18
    <212> DNA
    <213> Artificial Sequence

    <220>
    <223> Synthetic polynucleotide

    <220>
    <221> modified_base
    <222> (1)..(1)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (2)..(2)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (3)..(3)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (4)..(4)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (5)..(5)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (6)..(6)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (7)..(7)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (8)..(8)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (9)..(9)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (10)..(10)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (11)..(11)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (12)..(12)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (13)..(13)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (14)..(14)
    <223> Residue is ribonucleic acid moiety modified with phosphorothioate

    <220>
    <221> modified_base
    <222> (15)..(15)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (16)..(16)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <220>
    <221> modified_base
    <222> (17)..(17)
    <223> Residue is deoxy residue modified with phosphorothioate further modified with 1-[2-[[2-(N-acetyl-2-deoxy-2-amino-beta-D-galactopyranosyloxy) ethyl]oxy]ethyl] modification

    <400> 15
    tgtgaguacc acugatta   18




    Claims

    1. A compound of Formula I:

    wherein:

    R1 is an optionally protected nucleic base selected from adenine, cytosine, guanine, thymine, uracil, 2-aminoadenine, N6-methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 8-aminoadenine, 5-methylcytosine, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-aminoguanine, 7-deazaxanthyne, or hypoxanthine;

    one of R2 and R2a is a protecting group of trityl type selected from (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, and the other is a phosphoramidite moiety:

    wherein:

    each R4 and R5 is, independently, C1 to C6 alkyl, or R4 and R5 together with the nitrogen atom they are attached to form a cycle, wherein R4 + R5 = (CH2)nX(CH2)m,

    wherein:

    X is an atom of oxygen or CH2 group;

    each n and m is, independently, an integer from 2 to 5;

    L is -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, or -[(CH2)2-O]5-;

    R6 is a substituted monosaccharide residue of Formula II

    wherein:

    each R10 is, independently, an acyl protecting group including but not limited to acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, and 4-methylbenzoyl groups, a trityl-type protecting group including but not limited to (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, and 9-(p-methoxyphenyl)xanthen-9-yl, a silyl protecting group including but not limited to triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, and diphenylmethylsilyl, an alkyl group containing from 1 to 18 atoms of carbon, a benzyl group, a 4-methoxybenzyl group, a propargyl group, or another substituted monosacharide residue of Formula II;

    A is a chemical bond or CHOR10

    Z is a hydrogen, OR10, or N(Q2)Q3 wherein:

    each Q2 and Q3 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group;

    R3 is hydrogen atom, fluorine atom, substituted hydroxy group OR7, or substituted amino group NR8R9 wherein:

    each R7is, independently, a C1 to C6 alkyl, 2-alkoxyethyl group, or N-methylcarboxamidomethyl group; and

    each R8 and R9 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, phenoxyacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group.


     
    2. The compound of claim 1, wherein R1 is a nucleic base selected from N6-benzoyladenine, N6-phenoxyacetyladenine, N6-(4-isopropylphenoxy)acetyladenine, adenine, N4-benzoylcytosine, N4-acetylcytosine, N4-phenoxyacetylcytosine, N4-(4-isopropylphenoxy)acetylcytosine, N4-benzoyl-4-methylcytosine, N4-acetyl-4-methylcytosine, cytosine, N4-phenoxyacetyl-4-methylcytosine, N4-(4-isopropylphenoxy)acetyl-4-methylcytosine, 4-methylcytosine, N2-isobutyrylguanine, N2-phenoxyacetylguanine, N2-(4-isopropylphenoxy)acetylguanine, N2-(N,N-dimethylformamidino)guanine, guanine, thymine, or uracil.
     
    3. The compound of either claim 1 or claim 2, wherein R2 is 4,4'-dimethoxytrityl group and R2a is


     
    4. The compound of either claim 1 or claim 2, wherein R2a is 4,4'-dimethoxytrityl group and R2 is


     
    5. The compound of any preceding claim, wherein R3 is selected from hydrogen, OCH3 or fluorine.
     
    6. The compound of any preceding claim, wherein each R4 and R5 is an isopropyl group.
     
    7. The compound of any preceding claim, wherein R6 is selected from a protected β-D-glucopyranoside, or a protected β-D-galactopyranoside.
     
    8. The compound of any one of claims 1 to 6, wherein R6 is a protected 2-amino-2-deoxy-β-D-glucopyranoside or a protected 2-amino-2-deoxy-β-D-galactopyranoside.
     
    9. The compound of claim 8 wherein one of Q2 and Q3 is hydrogen, and the other is an acetyl group.
     
    10. The compound of any preceding claim, wherein each of R10 is independently selected from an acetyl group, a benzoyl group, a butyryl group, an isobutyryl group, a propionyl group, or a 4-methylbenzoyl group.
     
    11. The compound of any one of claims 1 to 9, wherein one of R10 is a protected monosaccharide residue of Formula II, and each of the other R10 is an acetyl group,
     
    12. An oligomeric compound of Formula III:

    wherein:

    each R1 is, independently, an optionally protected nucleic base selected from adenine, cytosine, guanine, thymine, uracil, 2-aminoadenine, N6-methyladenine, 7-deazaadenine, 7-deaza-8-azaadenine, 8-aminoadenine, 5-methylcytosine, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-azaguanine, 8-aminoguanine, 7-deazaxanthine or hypoxanthine;

    each R3 is, independently, hydrogen atom, fluorine atom, substituted hydroxy group OR7, or substituted amino group NR8R9 wherein:

    R7 is a C1 to C6 alkyl, 2-methoxyethyl group, N-methylcarboxamidomethyl group;

    each R8 and R9 is, independently, hydrogen atom, methyl group, ethyl group, propyl group, isopropyl group, acetyl group, trifluoroacetyl group, benzoyl group, or 9-fluorenylmethyloxycarbonyl group;

    L is -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, or -[(CH2)2-O]5-;

    R6 is a substituted monosaccharide residue of Formula II of claim 1

    wherein:

    each R10 is, independently, an acyl protecting group selected from acetyl, propionyl, butyryl, isobutyryl, pivaloyl, benzoyl, or 4-methylbenzoyl groups, a trityl-type protecting group selected from (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl, 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl, a silyl protecting group selected from triisopropylsilyl, t-butyldimethylsilyl, t-butyldiphenylsilyl, triphenylsilyl, or diphenylmethylsilyl, an alkyl group containing from 1 to 18 atoms of carbon, a benzyl group, a 4-methoxybenzyl group, a propargyl group, or another substituted monosacharide residue of Formula II;

    A is a chemical bond or CHOR10

    Z is a hydrogen, OR10, or N(R8)R9;

    each R11 is, independently, a negative charge compensated by a cation, a phosphate protecting group, or R6O-L-;

    each R12 and R13 is, independently, hydrogen atom, a protecting group selected from (4-methoxyphenyl)diphenylmethyl, bis-(4-methoxyphenyl)phenylmethyl, tris-(methoxyphenyl)methyl; 9-phenylxanthen-9-yl, or 9-(p-methoxyphenyl)xanthen-9-yl; or a point of attachment to solid phase material with the proviso that R12 and R13 are not both simultaneously a linker connected to a solid support;

    each of W1, W2, and W3 is, independently, oxygen or sulfur; and

    each s and t is, independently, an integer from 0 to 100.


     
    13. A method for the preparation of an oligomeric compound of Formula III of claim 12, said method comprising reacting a compound of Formula I of claim 1 with a compound of Formula IV:

    wherein

    said compound of Formula IV contains at least one reactive hydroxy group thereon,

    one of R12 and R13 is a hydrogen atom, and the other is a protecting group or a linker connected to a solid support; and

    k is an integer selected from 0 to 100.


     
    14. The method of claim 13, further comprising treating said oligomeric compound with a reagent under conditions of time, temperature, and pressure effective to oxidize or sulfurize the oligomeric compound.
     
    15. The method of claim 14, further comprising treating said oligomeric compound with a reagent under conditions of time, temperature, and pressure effective to deprotect the oligomeric compound and, optionally, effective to remove said oligomeric compound from the solid support.
     


    Ansprüche

    1. Verbindung der Formel (I):

    wobei:
    R1 eine gegebenenfalls geschützte Nukleinsäurebase ist, ausgewählt aus Adenin, Cytosin, Guanin, Thymin, Uracil, 2-Aminoadenin, N6-Methyladenin, 7-Deazaadenin, 7-Deaza-8-Azaadenin, 8-Aminoadenin, 5-Methylcytosin, N4-Ethylcytosin, 7-Deazaguanin, 7-Deaza-8-Azaguanin, 8-Aminoguanin, 7-Deazaxanthin oder Hypoxanthin; eines von R2 und R2a eine Schutzgruppe vom Trityltyp ist, ausgewählt aus (4-Methoxyphenyl)diphenylmethyl, Bis-(4-methoxyphenyl)phenylmethyl, Tris-(methoxyphenyl)methyl, 9-Phenylxanthen-9-yl oder 9-(p-Methoxyphenyl) xanthen-9-yl, und das andere eine Phosphoramidit-Gruppierung ist:

    wobei:

    jedes R4 und R5 unabhängig C1- bis C6-Alkyl ist oder R4 und R5 zusammen mit dem Stickstoffatom, an das sie gebunden sind, um einen Zyklus zu bilden, wobei R4 + R5 =

    (CH2)nX(CH2)m, wobei:

    X ein Sauerstoffatom oder eine CH2-Gruppe ist;

    jedes n und m unabhängig eine ganze Zahl von 2 bis 5 ist;

    L -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4- oder -[(CH2)2-O]5- ist;

    R6 ein substituierter Monosaccharidrest der Formel II ist



    wobei:

    jedes R10 unabhängig eine Acylschutzgruppe, einschließend unter anderem Acetyl-, Propionyl-, Butyryl-, Isobutyryl-, Pivaloyl-, Benzoyl- und 4-Methylbenzoylgruppen, eine Schutzgruppe vom Trityltyp, einschließend unter anderem (4-Methoxyphenyl)diphenylmethyl, Bis-(4-methoxyphenyl)phenylmethyl, Tris-(methoxyphenyl)methyl, 9-Phenylxanthen-9-yl und 9-(p-Methoxyphenyl)xanthen-9-yl, eine Silylschutzgruppe, einschließend unter anderem Triisopropylsilyl, t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Triphenylsilyl und Diphenylmethylsilyl, eine Alkylgruppe, enthaltend 1 bis 18 Kohlenstoffatome, eine Benzylgruppe, eine 4-Methoxybenzylgruppe, eine Propargylgruppe oder ein anderer substituierter Monosaccharidrest der Formel II ist;

    A eine chemische Bindung oder CHOR10 ist

    Z ein Wasserstoff, OR10 oder N(Q2)Q3 ist, wobei:

    jedes Q2 und Q3 unabhängig ein Wasserstoffatom, eine Methylgruppe, eine Ethylgruppe, eine Propylgruppe, eine Isopropylgruppe, eine Acetylgruppe, eine Trifluoracetylgruppe, eine Phenoxyacetylgruppe, eine Benzoylgruppe oder eine 9-Fluorenylmethyloxycarbonylgruppe ist; R3 ein Wasserstoffatom, ein Fluoratom, eine substituierte Hydroxygruppe OR7 oder eine substituierte Aminogruppe NR8R9 ist, wobei:

    jedes R7 unabhängig ein C1- bis C6-Alkyl, eine 2-Alkoxyethylgruppe oder N-Methylcarboxamidomethylgruppe ist; und

    jedes R8 und R9 unabhängig ein Wasserstoffatom, eine Methylgruppe, eine Ethylgruppe, eine Propylgruppe, eine Isopropylgruppe, eine Acetylgruppe, eine Trifluoracetylgruppe, eine Phenoxyacetylgruppe, eine Benzoylgruppe oder eine 9-Fluorenylmethyloxycarbonylgruppe ist.


     
    2. Verbindung nach Anspruch 1, wobei R1 eine Nukleinsäurebase ist, ausgewählt aus N6-Benzoyladenin, N6-Phenoxyacetyladenin, N6-(4-Isopropylphenoxy)acetyladenin, Adenin, N4-Benzoylcytosin, N4-Acetylcytosin, N4-Phenoxyacetylcytosin, N4-(4-Isopropylphenoxy)acetylcytosin, N4-Benzoyl-4-methylcytosin, N4-Acetyl-4-methylcytosin, Cytosin, N4-Phenoxyacetyl-4-methylcytosin, N4-(4-Isopropylphenoxy)acetyl-4-methylcytosin, 4-Methylcytosin, N2-Isobutyrylguanin, N2-Phenoxyacetylguanin, N2-(4-Isopropylphenoxy)acetylguanin, N2-(N,N-Dimethylformamidino)guanin, Guanin, Thymin oder Uracil.
     
    3. Verbindung nach Anspruch 1 oder Anspruch 2, wobei R2 eine 4,4'-Dimethoxytritylgruppe ist und R2a Folgendes ist:


     
    4. Verbindung nach Anspruch 1 oder Anspruch 2, wobei R2a eine 4,4'-Dimethoxytritylgruppe ist und R2 Folgendes ist:


     
    5. Verbindung nach einem der vorhergehenden Ansprüche, wobei R3 ausgewählt ist aus Wasserstoff, OCH3 oder Fluor.
     
    6. Verbindung nach einem der vorhergehenden Ansprüche, wobei jedes R4 und R5 eine Isopropylgruppe ist.
     
    7. Verbindung nach einem der vorhergehenden Ansprüche, wobei R6 ausgewählt ist aus einem geschützten β-D-Glucopyranosid oder einem geschützten β-D-Galactopyranosid.
     
    8. Verbindung nach einem der Ansprüche 1 bis 6, wobei R6 ein geschütztes 2-Amino-2-desoxy-β-D-glucopyranosid oder ein geschütztes 2-Amino-2-desoxy-β-D-galactopyranosid ist.
     
    9. Verbindung nach Anspruch 8, wobei eines von Q2 und Q3 Wasserstoff ist und das andere eine Acetylgruppe ist.
     
    10. Verbindung nach einem der vorhergehenden Ansprüche, wobei jedes von R10 unabhängig ausgewählt ist aus einer Acetylgruppe, einer Benzoylgruppe, einer Butyrylgruppe, einer Isobutyrylgruppe, einer Propionylgruppe oder einer 4-Methylbenzoylgruppe.
     
    11. Verbindung nach einem der Ansprüche 1 bis 9, wobei eines von R10 ein geschützter Monosaccharidrest der Formel II ist und jedes von dem anderen R10
    eine Acetylgruppe ist.
     
    12. Oligomere Verbindung der Formel III:

    wobei:

    jedes R1 unabhängig eine gegebenenfalls geschützte Nukleinsäurebase ist, ausgewählt aus Adenin, Cytosin, Guanin, Thymin, Uracil, 2-Aminoadenin, N6-Methyladenin, 7-Deazaadenin, 7-Deaza-8-Azaadenin, 8-Aminoadenin, 5-Methylcytosin, N4-Ethylcytosin, 7-Deazaguanin, 7-Deaza-8-Azaguanin, 8-Aminoguanin, 7-Deazaxanthin oder Hypoxanthin;

    jedes R3 unabhängig ein Wasserstoffatom, ein Fluoratom, eine substituierte Hydroxygruppe OR7,

    oder eine substituierte Aminogruppe NR8 R9 ist, wobei:

    R7 ein C1- bis C6-Alkyl, eine 2-Methoxyethylgruppe, N-Methylcarboxamidomethylgruppe ist; jedes R8 und R9 unabhängig ein Wasserstoffatom, eine Methylgruppe, eine Ethylgruppe, eine Propylgruppe, eine Isopropylgruppe, eine Acetylgruppe, eine Trifluoracetylgruppe, eine Benzoylgruppe oder eine 9-Fluorenylmethyloxycarbonylgruppe ist;

    L -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4- oder -[(CH2)2-O]5- ist;

    R6 ein substituierter Monosaccharidrest der Formel II nach Anspruch 1 ist



    wobei:

    jedes R10 unabhängig eine Acylschutzgruppe, ausgewählt aus Acetyl-, Propionyl-, Butyryl-, Isobutyryl-, Pivaloyl-, Benzoyl- oder 4-Methylbenzoylgruppen, eine Schutzgruppe vom Trityltyp, ausgewählt aus (4-Methoxyphenyl)diphenylmethyl, Bis-(4-Methoxyphenyl)phenylmethyl, Tris-(methoxyphenyl)methyl, 9-Phenylxanthen-9-yl oder 9-(p-Methoxyphenyl)xanthen-9-yl, eine Silylschutzgruppe, ausgewählt aus Triisopropylsilyl, t-Butyldimethylsilyl, t-Butyldiphenylsilyl, Triphenylsilyl oder Diphenylmethylsilyl, eine Alkylgruppe, enthaltend 1 bis 18 Kohlenstoffatome, eine Benzylgruppe, eine 4-Methoxybenzylgruppe, eine Propargylgruppe oder ein anderer substituierter Monosaccharidrest der Formel II ist;

    A eine chemische Bindung oder CHOR10 ist

    Z ein Wasserstoff, OR10 oder N(R8)R9 ist;

    jedes R11 unabhängig eine negative Ladung, die durch ein Kation kompensiert wird, eine Phosphatschutzgruppe oder R6O-L- ist;

    jedes R12 und R13 unabhängig ein Wasserstoffatom, eine Schutzgruppe, ausgewählt aus (4-Methoxyphenyl)diphenylmethyl, Bis-(4-methoxyphenyl)phenylmethyl, Tris-(methoxyphenyl)methyl; 9-Phenylxanthen-9-yl oder 9-(p-Methoxyphenyl)xanthen-9-yl; oder eine Bindungsstelle an Festphasenmaterial ist, mit der Maßgabe, dass R12 und R13 nicht beide gleichzeitig ein Linker sind, der mit einem festen Träger verbunden ist;

    jedes von W1, W2 und W3 unabhängig Sauerstoff oder Schwefel ist; und jedes s und t unabhängig eine ganze Zahl von 0 bis 100 ist.


     
    13. Verfahren zur Herstellung einer oligomeren Verbindung der Formel III nach Anspruch 12, wobei das Verfahren das Umsetzen einer Verbindung der Formel I nach Anspruch 1 mit einer Verbindung der Formel IV umfasst:

    wobei
    die Verbindung der Formel IV mindestens eine reaktive Hydroxygruppe daran enthält,
    eines von R12 und R13 ein Wasserstoffatom ist und das andere eine Schutzgruppe oder ein Linker ist, der mit einem festen Träger verbunden ist; und k eine ganze Zahl ist, ausgewählt aus 0 bis 100.
     
    14. Verfahren nach Anspruch 13, ferner umfassend das Behandeln der oligomeren Verbindung mit einem Reagenz unter Zeit-, Temperatur- und Druckbedingungen, die wirksam sind, um die oligomere Verbindung zu oxidieren oder zu schwefeln.
     
    15. Verfahren nach Anspruch 14, ferner umfassend das Behandeln der oligomeren Verbindung mit einem Reagenz unter Zeit-, Temperatur- und Druckbedingungen, die wirksam sind, um die oligomere Verbindung zu entschützen, und gegebenenfalls wirksam sind, um die oligomere Verbindung von dem festen Träger zu entfernen.
     


    Revendications

    1. Composé de Formule I :

    dans lequel :

    R1 est une base nucléique facultativement protégée choisie parmi l'adénine, la cytosine, la guanine, la thymine, l'uracile, la 2-aminoadénine, la N6-méthyladénine, la 7-déazaadénine, la 7-déaza-8-azaadénine, la 8-aminoadénine, la 5-méthylcytosine, la N4-éthylcytosine, la 7-déazaguanine, la 7-déaza-8-azaguanine, la 8-aminoguanine, la 7-déazaxanthyne ou l'hypoxanthine ;

    l'un de R2 et R2a est un groupement protecteur de type trityle choisi parmi le (4-méthoxyphényl)diphénylméthyle, le bis-(4-méthoxyphényl)phénylméthyle, le tris-(méthoxyphényl)méthyle, le 9-phénylxanthén-9-yle ou le 9-(p-méthoxyphényl)xanthén-9-yle, et l'autre est une fraction phosphoramidite :

    dans lequel :

    chaque R4 et chaque R5 sont, indépendamment, un alkyle en C1 à C6, ou R4 et R5 ensemble avec l'atome d'azote auquel ils sont attachés pour former un cycle, où R4 + R5 =

    (CH2)nX(CH2)m, dans lequel :

    X est un atome d'oxygène ou un groupement CH2 ;

    chaque n et chaque m sont, indépendamment, un nombre entier de 2 à 5 ;

    L est -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, ou -[(CH2)2-O]5- ;

    R6 est un résidu monosaccharide substitué de formule II



    dans lequel :

    chaque R10 est, indépendamment, un groupement protecteur acyle incluant, mais sans s'y limiter, les groupements acétyle, propionyle, butyryle, isobutyryle, pivaloyle, benzoyle et 4-méthylbenzoyle, un groupement protecteur de type trityle incluant mais sans s'y limiter le (4-méthoxyphényl)diphénylméthyle, le bis-(4-méthoxyphényl)phénylméthyle, le tris-(méthoxyphényl)méthyle, le 9-phénylxanthén-9-yle et le 9-(p-méthoxyphényl)xanthén-9-yle, un groupement protecteur silyle incluant mais sans s'y limiter le triisopropylsilyle, le t-butyldiméthylsilyle, le t-butyldiphénylsilyle, le triphénylsilyle et le diphénylméthylsilyle, un groupement alkyle contenant de 1 à 18 atomes de carbone, un groupement benzyle, un groupement 4-méthoxybenzyle, un groupement propargyle ou un autre résidu monosacharide substitué de formule II ;

    A est une liaison chimique ou CHOR10

    Z est un hydrogène, OR10, ou N(Q2)Q3 dans lequel :

    chaque Q2 et chaque Q3 sont, indépendamment, un atome d'hydrogène, un groupement méthyle, un groupement éthyle, un groupement propyle, un groupement isopropyle, un groupement acétyle, un groupement trifluoroacétyle, un groupement phénoxyacétyle, un groupement benzoyle, ou un groupement 9-fluorénylméthyloxycarbonyle ;

    R3 est un atome d'hydrogène, un atome de fluor, un groupement hydroxy substitué OR7, ou un groupement amino substitué NR8 R9 dans lequel :

    chaque R7 est, indépendamment, un alkyle en C1 à C6, un groupement 2-alcoxyéthyle ou un groupement N-méthylcarboxamidométhyle ; et

    chaque R8 et chaque R9 sont, indépendamment, un atome d'hydrogène, un groupement méthyle, un groupement éthyle, un groupement propyle, un groupement isopropyle, un groupement acétyle, un groupement trifluoroacétyle, un groupement phénoxyacétyle, un groupement benzoyle, ou un groupement 9-fluorénylméthyloxycarbonyle.


     
    2. Composé selon la revendication 1, dans lequel R1 est une base nucléique choisie parmi la N6-benzoyladénine, la N6-phénoxyacétyladénine, la N6-(4-isopropylphénoxy)acétyladénine, l'adénine, la N4-benzoylcytosine, la N4-acétylcytosine, la N4-phénoxyacétylcytosine, la N4-(4-isopropylphénoxy)acétylcytosine, la N4-benzoyl-4-méthylcytosine, la N4-acétyl-4-méthylcytosine, la cytosine, la N4-phénoxyacétyl-4-méthylcytosine, la N4-(4-isopropylphénoxy)acétyl-4-méthylcytosine, la 4-méthylcytosine, la N2-isobutyrylguanine, la N2-phénoxyacétylguanine, la N2-(4-isopropylphénoxy)acétylguanine, la N2-(NN-diméthylformamidino)guanine, la guanine, la thymine ou l'uracile.
     
    3. Composé selon la revendication 1 ou la revendication 2, dans lequel R2 est un groupement 4,4'-diméthoxytrityle et R2a est


     
    4. Composé selon la revendication 1 ou la revendication 2, dans lequel R2a est un groupement 4,4'-diméthoxytrityle et R2 est


     
    5. Composé selon l'une quelconque des revendications précédentes, dans lequel R3 est choisi parmi l'hydrogène, OCH3 ou le fluor.
     
    6. Composé selon l'une quelconque des revendications précédentes, dans lequel chaque R4 et chaque R5 sont un groupement isopropyle.
     
    7. Composé selon l'une quelconque des revendications précédentes, dans lequel R6 est choisi parmi un β-D-glucopyranoside protégé ou un β-D-galactopyranoside protégé.
     
    8. Composé selon l'une quelconque des revendications 1 à 6, dans lequel R6 est un 2-amino-2-désoxy-β-D-glucopyranoside protégé ou un 2-amino-2-désoxy-β-D-galactopyranoside protégé.
     
    9. Composé selon la revendication 8 dans lequel l'un parmi Q2 et Q3 est l'hydrogène, et l'autre est un groupement acétyle.
     
    10. Composé selon l'une quelconque des revendications précédentes, dans lequel chaque R10 est indépendamment choisi parmi un groupement acétyle, un groupement benzoyle, un groupement butyryle, un groupement isobutyryle, un groupement propionyle ou un groupement 4-méthylbenzoyle.
     
    11. Composé selon l'une quelconque des revendications 1 à 9, dans lequel l'un de R10 est un résidu monosaccharide protégé de formule II, et chacun des autres R10 est un groupement acétyle.
     
    12. Un composé oligomère de formule III :

    dans lequel :

    chaque R1 est, indépendamment, une base nucléique facultativement protégée choisie parmi l'adénine, la cytosine, la guanine, la thymine, l'uracile, la 2-aminoadénine, la N6-méthyladénine, la 7-déazaadénine, la 7-déaza-8-azaadénine, la 8-aminoadénine, la 5-méthylcytosine, la N4-éthylcytosine, la 7-déazaguanine, la 7-déaza-8-azaguanine, la 8-aminoguanine, la 7-déazaxanthine ou l'hypoxanthine ;

    chaque R3 est, indépendamment, un atome d'hydrogène, un atome de fluor, un groupement hydroxy substitué OR7,

    ou un groupement amino substitué NR8 R9 dans lequel :

    R7 est un alkyle en C1 à C6, un groupement 2-méthoxyéthyle, un groupement N-méthylcarboxamidométhyle ;

    chaque R8 et chaque R9 sont, indépendamment, un atome d'hydrogène, un groupement méthyle, un groupement éthyle, un groupement propyle, un groupement isopropyle, un groupement acétyle, un groupement trifluoroacétyle, un groupement benzoyle, ou un groupement 9-fluorénylméthyloxycarbonyle ;

    L est -(CH2)3-, -(CH2)4-, -[(CH2)2-O]2-, -[(CH2)2-O]3-, -[(CH2)2-O]4-, ou -[(CH2)2-O]5- ;

    R6 est un résidu monosaccharide substitué de formule II de la revendication 1



    dans lequel :

    chaque R10 est, indépendamment, un groupement protecteur acyle choisi parmi les groupements acétyle, propionyle, butyryle, isobutyryle, pivaloyle, benzoyle ou 4-méthylbenzoyle, un groupement protecteur de type trityle choisi parmi le (4-méthoxyphényl) diphénylméthyle, le bis-(4-méthoxyphényl)phénylméthyle, le tris-(méthoxyphényl)méthyle, le 9-phénylxanthén-9-yle ou le 9-(p-méthoxyphényl)xanthén-9-yle, un groupement protecteur silyle choisi parmi le triisopropylsilyle, le t-butyldiméthylsilyle, le t-butyldiphénylsilyle, le triphénylsilyle ou le diphénylméthylsilyle, un groupement alkyle contenant de 1 à 18 atomes de carbone, un groupement benzyle, un groupement 4-méthoxybenzyle, un groupement propargyle ou un autre résidu monosacharide substitué de formule II ;

    A est une liaison chimique ou CHOR10

    Z est un hydrogène, OR10 ou N(R8)R9 ;

    chaque R11 est, indépendamment, une charge négative compensée par un cation, un groupement protecteur phosphate ou R6O-L- ;

    chaque R12 et chaque R13 sont, indépendamment, un atome d'hydrogène, un groupement protecteur choisi parmi le (4-méthoxyphényl)diphénylméthyle, le bis-(4-méthoxyphényl) phénylméthyle, le tris-(méthoxyphényl)méthyle ; le 9-phénylxanthén-9-yle, ou le 9-(p-méthoxyphényl)xanthén-9-yle ; ou un point de fixation à un matériau en phase solide à condition que R12 et R13 ne soient pas tous les deux simultanément un segment de liaison connecté à un support solide ;

    chacun de W1, W2 et W3 est, indépendamment, de l'oxygène ou du soufre ; et chaque s et chaque t sont, indépendamment, un nombre entier de 0 à 100.


     
    13. Procédé pour la préparation d'un composé oligomère de formule III de la revendication 12, ledit procédé comprenant la réaction d'un composé de formule I de la revendication 1 avec un composé de formule IV :

    dans lequel
    ledit composé de formule IV contient au moins un groupement hydroxy réactif sur celui-ci,
    l'un de R12 et R13 est un atome d'hydrogène et l'autre est un groupement protecteur ou un segment de liaison connecté à un support solide ; et k est un nombre entier sélectionné de 0 à 100.
     
    14. Procédé selon la revendication 13, comprenant en outre le traitement dudit composé oligomère avec un réactif dans des conditions de temps, de température et de pression efficaces pour oxyder ou sulfurer le composé oligomère.
     
    15. Procédé selon la revendication 14, comprenant en outre le traitement dudit composé oligomère avec un réactif dans des conditions de temps, de température et de pression efficaces pour déprotéger le composé oligomère et, facultativement, efficaces pour éliminer ledit composé oligomère du support solide.
     




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    Cited references

    REFERENCES CITED IN THE DESCRIPTION



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    Patent documents cited in the description




    Non-patent literature cited in the description