(19)
(11)EP 0 299 985 B1

(12)EUROPEAN PATENT SPECIFICATION

(45)Mention of the grant of the patent:
16.03.1994 Bulletin 1994/11

(21)Application number: 87907075.3

(22)Date of filing:  05.10.1987
(51)International Patent Classification (IPC)5C12Q 1/00, C12Q 1/34
// C07H15/203
(86)International application number:
PCT/US8702/592
(87)International publication number:
WO 8805/827 (11.08.1988 Gazette  1988/18)

(54)

ENZYME CONTROLLED RELEASE SYSTEM

ENZYMKONTROLLIERTES FREISETZUNGSSYSTEM

SYSTEME DE LIBERATION CONTROLE PAR DES ENZYMES


(84)Designated Contracting States:
DE FR GB IT NL

(30)Priority: 30.01.1987 US 8939

(43)Date of publication of application:
25.01.1989 Bulletin 1989/04

(73)Proprietor: POLAROID CORPORATION
Cambridge, Massachusetts 02139 (US)

(72)Inventors:
  • ARNOST, Michael, J.
    North Andover, MA 01845 (US)
  • MENEGHINI, Frank
    Arlington, MA 02174 (US)
  • PALUMBO, Paul, S.
    West Newton, MA 02165 (US)

(74)Representative: Patentanwälte Dipl.-Ing. R. Splanemann Dr. B. Reitzner Dipl.-Ing. K. Baronetzky 
Tal 13
80331 München
80331 München (DE)


(56)References cited: : 
EP-A- 60 793
US-A- 3 880 934
EP-A- 85 554
  
      
    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

    FIELD OF THE INVENTION



    [0001] The present invention is concerned with systems for the controlled release of a ligand on demand and is particularly concerned with enzyme controlled release systems which employ an intramolecular displacement reaction mechanism for the release of a specific ligand.

    BACKGROUND OF THE INVENTION



    [0002] Compounds and systems for the controlled release of a specific ligand have been the subject of considerable research and product development for a variety of different applications. For example, enzymes and enzyme-conjugates are well recognized reactants in immunoassays for reaction with specific substrates by which a variety of different chromophoric and fluorescent dyes are released. These enzymes and enzyme-conjugates are utilized in mobile and immobilized formats in which the rate of enzymatic reaction and/or the concentration of enzyme reaction product provides qualitative and quantitative data. Illustrative of such enzyme release systems are United States Patent Nos. 4,423,143; 4,501,692; 4,318,846; 4,351,760; 4,439,356; 4,447,527; and 4,481,136.

    [0003] Alternatively, non-enzymatic release systems are also conventionally known, a representative example being displacement reaction mechanisms and release systems used in photographic processes for the release of color-imaging compounds. Such compounds, in the presence of oxidized color developing agents, typically undergo an intramolecular displacement reaction to form a new heterocyclic ring; and as a function of such displacement reaction mechanism and ring formation, split off mobile and diffusible color-imaging material. Such non-enzyme release systems and compositions reflect the pioneer work of Messrs. Raiford and Inman [J. Amer. Chem. Soc. 56:1586 (1934)] and Messrs. Hutchins and Fife [J. Amer. Chem. Soc. 95:7 (1973)]; illustrative examples of the displacement reaction release mechanism as it relates to photographic processes and compositions are described in United States Patent Nos. 3,443,939; 3,443,940; 3,751,406; 3,980,479; and 4,139,379.

    [0004] Despite the ongoing innovations and developments in each of these respective technical areas, there has been no overlap or merger of these individual controlled release techniques. Although the benefits and advantages of such a potential merger between enzymes and compositions able to undergo an intramolecular displacement reaction are substantial, there has not been any innovative development bridging the gap between these technical areas insofar as is presently known.

    SUMMARY OF THE INVENTION



    [0005] An enzyme controlled release system is provided for the release of an identifiable ligand comprising an active enzyme able to a cleave the covalent bond joining the substrate L to the neighbouring group Q from an organic conjugate; and
       the organic conjugate able to react with the enzyme having the formula


    wherein a, b, d, and e individually are 0 or 1;
       W may be omitted entirely but when present comprises the number of atoms necessary to form a saturated or an unsaturated cyclic moiety;
       X and X' individually are hydrogen, a hydrocarbon group or a substituted hydrocarbon group;
       Y may be omitted entirely but when present comprises from 1-5 carbon atoms;
       U and U' individually are hydrogen or a second covalent bond;
       V and V' may be omitted individually or jointly provided that they cannot be omitted jointly when b is 0, and that only one of V and V' can be present when b is 3,
       but when present are


       where p is an integer from 0-3,
       t is an integer from 0-3,
    the sum of p+t is from 0-3,
       J is


    -O-, -S-, or


    and R₂, R₃, R₄, R₅, R₆, R₇, R₈ individually are hydrogen, an alkyl group having from 1-6 carbons or an aryl group;
       Q is O, S, NH or NR' , R' being an organic group.

    [0006] Z is O, S, NH or NR', R' being an organic group;
       L is an enzyme substrate which is bound to Q in a manner so that cleavable the covalent bond joining L to Q is cleavable by said enzyme;
       M is any organic moiety;
       R₁ is hydrogen or one or more substituents affecting the mobility or reactivity of the conjugate composition;
    and wherein Z-M is the displaceable fragment released in anionic, neutral, or cationic form by intramolecular displacement reaction from the organic conjugate after enzymatic cleavage of the covalent bond joining L to Q.

    DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS



    [0007] The present invention is an enzyme controlled release system which uniquely combines at least one enzyme with a prepared organic conjugate able to undergo an intramolecular displacement reaction after enzymatic cleavage of a covalently bonded enzyme substrate from the organic conjugate. The controlled release system thus comprises a series of successive chemical events summarized most generally by Reactions I and II below, wherein the neighboring group Q is seen in its anionic form.


    wherein a, b, d, and e individually are 0 or 1;
       W may be omitted entirely but when present comprises the number of atoms necessary to form a saturated or unsaturated cyclic molecule;
       X and X' individually are hydrogen, a hydrocarbon group or a substituted hydrocarbon group;
       Y may be omitted entirely but when present is a hydrocarbon comprising from 1-5 carbon atoms;
       U and U' individually are hydrogen or a second covalent bond;
       V and V' may be omitted individually or jointly provided that they cannot be omitted jointly when b is 0, and that only one of V and V' can be present when b is 3,
       but when present are


    where p is an integer from 0-3,
       t is an integer from 0-3,
    and the sum p+t is an integer from 0-3, and where J is


    -O-, -S-, or


    and R₂,R₃,R₄,R₅,R₆,R₇,R₈, individually are hydrogen, an alkyl group having 1-6 carbon atoms, or an aryl group;
       Q is O, S, NH, or NR', R' being an organic moiety;
       Z is O, S, NH, or NR', R' being an organic moiety;
       L is an enzyme substrate which is cleaved by said enzyme at the covalent bond joining L to Q;
       M is an organic moiety without limitation;
       R₁ is hydrogen or a substituent affecting the mobility and reactivity of the conjugate composition;
    whereby an identifiable fragment Z-M is released in anionic, neutral, or cationic form by intramolecular displacement reaction from the organic conjugate after enzymatic cleavage of L;
    and whereby a reaction product represented by the formula


    is formed, the heterocyclic ring of the fused ring system being not less than 5 atoms and not more than 9 atoms in size.

    [0008] As stated, Reaction I provides the broadest definition of the reactants and clearly relies upon the selective ability of the active enzyme employed to combine with an enzyme substrate L forming a component part of the organic conjugate and to specifically cleave the L-Q bond. For this reason, enzymes useful for this purpose within the controlled release system will be described in detail.

    The Enzyme



    [0009] Enzymes having the specific activity to selectively cleave the covalent bond joining the substrate L to the neighboring group Q are conventionally recognized and described in the literature as hydrolases, oxo-reductases, and the like. The user is presumed to have sufficient familiarity with and knowledge of enzyme reactions, enzyme kinetics, and enzyme specificity thereby making the individual choice of substrates, co-factors, reaction conditions and resulting products merely one of personal preference or convenience. Specific details and descriptions of enzymes and enzyme reactions are available in Dixon and Webb, Enzymes, Academic Press, 1979; and Dawson, Elliott, and Jones, Data for Biochemical Research, 3rd edition, Clarendon Press, 1986. A representative, but incomplete, listing of individual enzymes suitable for use with specific choices of Q and L which are deemed to be useful when practicing the present invention are given in Table 1 below.
    Table 1
    EnzymeQL
    Alkaline phosphatase O PO₃⁻²
    Acid phosphatase O PO₃⁻²
    β-D-galactosidase O β-D-galactose
    α-D-mannosidase O α-D-mannose
    β-D-glucosidase O β-D-glucose
    β-D-fructofuranosidase O β-D-fructofuran
    thioglucosidase S glycosyl residue
    Bovine trypsin NH CBZ-glycine-arginine
    Leucineaminopeptidase NH leucine
    Snake venom phosphodiesterase O adenosine-5'-phosphate
    Pancreatic ribonuclease O cytidine-3'-phosphate


    [0010] The enzyme chosen for use in the controlled release system may be employed in several formats: as an unjoined enzyme; as an enzyme conjugated to a ligand of known composition or activity; as a mobile molecule in either unjoined or conjugated form; and as an immobilized molecule in either unjoined or conjugated form.

    [0011] As an unjoined entity, the enzyme chosen need only demonstrate a measurable and reproducible degree of specific activity characterized by the ability to combine with an enzyme substrate L and to selectively cleave the covalent bond joining L to the neighboring group Q.

    [0012] In the conjugated enzyme format, the chosen enzyme is joined in a conventional manner to a ligand having specific properties. Common examples of such ligands are: one of a specifically binding pair of immunological reactants, typically antigens and their specific antibodies; specific binding proteins such as biotin and avidin; and hormones and their target organs. Methods for conjugating ligands to enzymes without substantial loss or modification of the enzyme's specific activity are illustrated by United States Patent Nos. 4,423,143 and 4,501,692. Other such useful techniques (in a homogeneous enzyme immunoassay context) are described in United States Patent Nos. 4,376,825; 4,282,325; 4,203,802; 4,067,774; 3,852,157; 4,190,496; and 4,191,613 respectively.

    [0013] As regards the immobilization of either unjoined or conjugated enzymes, the specific manner and chemical reaction means by which the enzyme is attached to a solid phase surface or carrier may be selected from those presently available to meet the user's needs or convenience. Such immobilization techniques and processes by which the unjoined enzyme or conjugated enzyme is attached directly or via the use of "linker-arms" to solids such as plastic and latex, test tubes, discs, agarose and plastic beads, porous glass, and polyacrylamide gels are described in: Methods In Enzymology, Academic Press, 1980; Updike, Antibiotics And Chemotherapeutics 26:67 (1979); and United States Patent Nos. 3,793,445; 3,970,429; and 4,138,474.

    [0014] In its most general form, the organic conjugate composition reactant of Reaction I is


    where V, V', Y, U, U', X, X' Q, L, Z, M, and R₁ are as previously defined herein. As is apparent from this general structure, the prepared reactant comprises three requisite components and provides an optional fourth component. These are: an organic base supporting structure,


    a primary enzyme-sensitive appendage comprising a variable entity (̵V)̵e, a masked neighboring group Q, and a masking enzyme substrate L bound in sequence to the organic base structure; a secondary displacement appendage comprising a variable entity (̵V')̵d, a carbonyl group, and a displaceable fragment Z-M bound in sequence to the base structure; and an optional organic moiety R₁ through which the rate of Reactions I and II may be modulated or through which the prepared reactant may be immobilized or solubilized. Each of these component parts will be described in detail.

    The Organic Base Supporting Structure



    [0015] The supporting structure of the prepared reactant is based upon the presence of an organic base structure having at least two carbon atoms available for the attachment of other appendages and which are not distanced from each other by more than 5 atoms. The base supporting structure may comprise saturated and unsaturated molecules, straight and branched linear chains, single and multiple rings, and include a variety of heterocyclic ring structures. Each of these base structures may also contain substituted hydrocarbons and organic groups known in the art. A representative, but incomplete, listing of organic base structures deemed to be useful in the present invention is provided by Table 2 below.






    The Primary Enzyme-Sensitive Appendage



    [0016] The primary enzyme-sensitive appendage is a linear sequence represented as


    The variable entity


    (e being 1 or 0) when present is


       wherein p is an integer from 0-3,
       t is an integer from 0-3,
    the sum of p+t is an integer from 0-3, and
       wherein J is


    ―O―, ―S―,
       or


    and R₂,R₃,R₄,R₅,R₆,R₇, and R₈, individually are hydrogen, an alkyl group having from 1-6 carbons, or an aryl group. Clearly, the maximum length for V is four carbon atoms in size. In preferred embodiments, however, e is zero and V is omitted entirely from the appendage sequence.

    [0017] Q is selected from the group consisting of oxygen, sulfur, nitrogen and substituted nitrogen groups; in preferred embodiments Q is an oxygen atom. L is an enzyme substrate which is attached by covalent bonds to Q and while in this state serves to mask (or block) the chemical reactivity of Q. L is chosen in accordance with the specific activity of the enzyme to be employed in the release system. A representative listing of substances useful as L has been given by Table 1 previously.

    [0018] It will be appreciated by practitioners ordinarily skilled in this art that the primary enzyme-sensitive appendage sequences comprise some elements which are relatively unrestricted in size and composition; clearly the masking enzyme substrate L is limited only with respect to the existence of a corresponding active enzyme able to react with it. In comparison, the composition of V and Q in combination is limited to a known set of specific entities; moreover the size of V and Q together can not be less than 1 atom and not more than 5 atoms in length.

    [0019] The techniques and reactions by which V and Q and L are joined in sequence to the supporting base structure are conventionally known and need not be described in detail herein. It is required that the masking enzyme substrate L be such that the corresponding active enzyme is able to bind with the substrate L and to cleave those covalent bonds joining the substrate L to the neighboring group Q given suitable conditions for reaction (pH, temperature, necessary co-factors, and the like). The kinetics of Reaction I and the disposition of the cleaved substrate L are of no consequence so long as these factors do not substantially interfere with the consequential intramolecular displacement mechanism of Reaction II or the release of the identifiable ligand for the purposes of the particular application.

    [0020] The event of enzymatically cleaving the masking substrate L from the organic conjugate composition in accordance with Reaction I serves to unmask (deblock) the neighboring group Q. Once unmasked, the presence of the neighboring group Q is the direct initiator of an intramolecular displacement reaction within the secondary displacement appendage, thereby causing the release of an identifiable fragment Z-M and the formation of a heterocyclic ring in accordance with Reaction II. While the particulars of the mechanism by which an anchimerically assisted, intramolecular displacement reaction is initiated (as a result of enzymatically cleaving the substrate L from the organic conjugate) is of theoretical interest to practitioners in this art, the individual details regarding the mechanism of action do not themselves limit or restrict the subject matter as a whole comprising the present invention. Accordingly, so long as at least one enzyme is employed to cleave a masking enzyme substrate L from the linkage sequence


    comprising part of an organic conjugate with the consequence that an intramolecular displacement reaction is initiated and an identifiable fragment is released, that embodiment is deemed to be within the scope of the present invention.

    The Secondary Displacement Appendage



    [0021] A requisite part of the prepared reactant is a secondary linear displacement appendage represented as


       The variable entity (̵V')̵d (d being 1 or 0), can be omitted entirely but when present is


    wherein p is an integer from 0-3,
       t is an integer from 0-3, and the sum of p+t is an integer from 0-3; and wherein
       J is


    -O-, -S-, or


    and
    R₂-R₈ individually are hydrogen, an alkyl group having from 1-6 carbon atoms, or an aryl group. In preferred embodiments V' is -NR₈-.

    [0022] Z is selected from the group consisting of oxygen, sulfur, nitrogen or a substituted nitrogen group NR'; where R' is an organic group. M is any organic moiety without limitation and is chosen in accordance with the desired application.

    [0023] A critical feature of the prepared reactant is that after the occurrence of an initiating enzyme reaction (Reaction I), the secondary displacement appendage sequence becomes subject to an intramolecular displacement reaction such that the Z-M portion of the appendage is actually displaced (i.e. released into the surrounding environment). Furthermore, as a result of this intramolecular displacement reaction, a heterocyclic ring is formed.

    [0024] The selection of the displaceable fragment Z-M will vary directly with the properties and characteristics of the ligand desired to be released, and thus is a function of the intended application. It is expected that the displaceable fragment Z-M will take the form of a chromophoric, fluorescent, or chemiluminescent dye; a dye precursor; a specific amino acid such as serine, cysteine, tyrosine, lysine, glutamic acid; or be a biologically active species-as for example drugs, steroids, polypeptides or proteins which permit them to be attached to the appendage without substantially affecting the biological properties of the molecule as a whole. This last category particularly includes polypeptides of recognized specific activity such as enzymes; polypeptides able to bind specifically to another molecule such as hormones; and polypeptides having defined pharmacological properties such as tissue plasminogen activator (tPA). A representative, but incomplete, listing of groups suitable for use as M in conjunction with various choices of Z is presented in Table 3.














    The Anchimerically Assisted, Intramolecular Displacement Reaction



    [0025] Intramolecular reactions are analogous to bimolecular (intermolecular) reactions in which two discrete reactants combine to form one or more end products, but differ enormously in their individual rates of reaction. The rate of a bimolecular reaction such as



            RCO₂R' + R''NH₂ → RCONHR'' + R'OH



    is expressed as a second order equation whose rate is dependent upon the initial concentration of the two reactants, RCO₂R' and R''NH₂ respectively. However, if the same two reactants comprise component parts of a single large molecule, an intramolecular reaction becomes possible and the rate can be reduced to a first order equation. In addition, if certain electronic and sterochemical parameters are controlled, the driving energy forces favoring such an intramolecular displacement become large and the rate of intramolecular cyclization can far exceed the rate of reaction for the analogous bimolecular (intermolecular) reaction. In those instances where the intramolecular displacement reaction is accelerated in rate via the presence of a neighboring group whose sterochemical and electronic characteristics are well defined (such as neighboring group Q of the present invention), such intramolecular reactions are said to be anchimerically assisted because the displacement and cyclization proceeds through what is, in effect, an internally attached form of the reactants. Such anchimercially assisted reactions often exhibit very rapid rates of reaction with rate factors of 10³-10⁴ being commonly observed.

    [0026] The pertinent literature in this art has recognized the general characteristics of intramolecular reactions and identified many factors influencing intramolecular catalysis, anchimeric assistance, and the formation of ring systems. These include: Hutchins & Fife, J. Am. Chem. Soc. 95:2282 (1973); Morris & Page, J. Chem. Soc., Perkins II:679 (1980); T.H. Fife, Advanced Physical Organic Chemistry, Volume 11 (Gold & Bethell, editors), Academic Press, New York, 1975, pp. 5-19 and 39-60; B. Capon & S.P. McManus, Neighboring Group Participation, Plenum Press New York, 1976, pp. 43-71 and 182-187; and E.L. Eliel, Sterochemistry Of Carbon Compounds, McGraw-Hill Book Co. Inc., New York, 1962, pp. 198-203.

    [0027] Clearly important factors to be considered are the nature of the nucleophilic and displacing groups, ring size, electronic and sterochemical influences, substituent effects, strain energy and entropic effects. The present invention utilizes these factors to effect fast intramolecular displacement of an identifiable fragment from a precursor conjugate reactant under carefully controlled conditions. In simpler terms, the organic conjugate described herein can properly be thought of as means for the cyclic joining together of two functional appendages within the same molecule by way of controlled intramolecular displacement. This is made possible by use of a strong nucleophilic neighboring group Q having sufficient activation energy to initiate displacement and cyclization; by masking (or blocking) the activity of the nucleophilic neighboring group Q with a masking (or blocking) enzyme substrate L which can be cleaved at will by addition of a suitable enzyme; and by prior selection, control and adjustment of the intramolecular reactants and structural geometry such that the two reactive partners are in the appropriate orientation one to the other. Control of the masking (blocking) enzyme substrate L therefore allows for control over the entire anchimerically assisted intramolecular displacement reaction. This is best demonstrated by individually describing some of the preferred embodiments.

    Embodiment A:



    [0028] If the base supporting structure for the organic conjugate takes the form of an aryl base represented generally as


    the prepared reactant will react in conformity with Reactions III and IV below.


    where V, V', Z, M, Q, L and R₁ are as previously defined herein.

    [0029] Clearly, the primary enzyme sensitive appendage


    lies ortho and in a cis orientation with respect to the secondary displacement appendage


    The proximity and steric constraint is provided by the benzene ring structure and the adjacent positioning of the respective appendages on the benzene ring without any intervening atoms between them. Similarly, the available degrees of rotational freedom are reduced such that a reactive sterochemical position is maintained intramolecularly. After cleavage of the masking substrate L by the appropriate enzyme (Reaction III), the neighboring group Q is unmasked and functions as a nucleophile to initiate displacement of the Z-M moiety (Reaction IV). The size of the ring formed by Reaction IV is carefully controlled to be within specified limits and comprises a minimum of 5 atoms and a maximun of only 9 atoms.

    [0030] This size limitation as to the number of atoms in the heterocyclic ring is best visualized by the minimal and maximal forms. Recognizing that both V and V' cannot be omitted concurrently in this embodiment and keeping V (or V') to the minimal one atom in size, the reaction product yielded is the minimal situation where the heterocyclic ring contains 5 atoms. Alternatively, if both V and V' are present and take the largest of the permissible forms, the resulting product is the maximal permissible situation in which 9 atoms comprise the newly formed heterocyclic ring.

    Embodiment B:



    [0031] If the base supporting structure for the organic conjugate takes the form of a bicyclic aromatic ring such as


    the prepared conjugate reactant will act in conformity with Reactions V and VI below.


    where V, V', Q, L, Z, M and R₁ as previously defined.

    [0032] Embodiment B illustrates the situation where there is one carbon atom separating the primary enzyme-sensitive appendage from the secondary displacement appendage at their respective points of attachment to the bicyclic base structure.

    [0033] Reaction V demonstrates the enzymatic control over the masking group L which when cleaved unmasks (deblocks) the nucleophile Q⁻ to initiate Reaction VI. The size of the heterocyclic ring formed is held to be within carefully controlled limits of not less than 5 atoms and not more than 9 atoms in size. The number of carbon atoms (3) in the bicyclic base between the respective appendages permits both V and V' to be entirely omitted and still meet the minimal required ring size of 5 atoms. Alternatively, where both V and V' are present, the reaction products can be the maximal compositional size of 9 atoms.

    Embodiment C:



    [0034] If the base supporting structure for the organic conjugate takes the form of a linear (straight or branched) saturated hydrocarbon such as



            R-CH₂-CH₂-CH₂-CH₂-CH₂-R'



    where R and R' are hydrogen or an organic entity, the prepared conjugate reactant will act in conformity with Reactions VII and VIII below.


    where V, V', Q, L, M, Z, and R₁ are as previously described.

    [0035] Embodiment C illustrates the permissible situation where Y is a 3 carbon atom linkage. Clearly, in comparison with Embodiments A and B reactant C presents the least favorable embodiment for molecular displacement and cyclization. The proximity distance is large; there is no steric constriction provided by the base structure; and the largest number of degrees of rotational freedom are available. These less desirable features will markedly reduce the rate of Reaction VIII to be the slowest among Embodiments A-C. However, there is no doubt whatsoever that Reactions VII and VIII will occur so long as the selection of V, V', Q, conform to the previously defined forms.

    [0036] Accordingly, the number of carbon atoms (5) in the linear base between the respective appendages narrows the possible choices of V and V' to comprise a maximum of 2 atoms in size yielding the maximal 9 atom ring size. On the other hand, if both V and V' are omitted entirely, the minimal 7 atom heterocyclic ring is formed.

    Optional R1 Moiety



    [0037] It is recognized and expected that in many applications, the prepared organic conjugate will be employed either in an immobilized form or a freely mobile format. One provision for immobilizing the organic conjugate is made via the R₁ moiety which is used to join the organic conjugate to a solid surface or carrier. A wide variety of different alkyl and aryl groups and reactions are conventionally known for this purpose. Particularly useful as immobilizing linkages are groups comprising the piperizinyl sulfonyl series represented as


    where each R'' is hydrogen or a lower alkyl radical bonded directly to the solid surface or carrier. Other immobilizing linkages deemed to be useful are described in Example 5 which follows herein. Alternatively, the R₁ moiety will increase the mobility of the organic conjugate as a whole in a fluid. An illustrative embodiment of the R₁ moiety able to increase mobility is the succinylated piperazinyl sulfonyl group described in Example 15 hereinafter. It should be understood that the R₁ moiety can be used to modulate the reactivity of the conjugate composition. For example, when the pKa of Q is important in reactions I and II, those reactions can be accelerated by suitable choice of R₁.

    [0038] To ensure a clear and complete understanding of the enzyme controlled release system, the reactants employed, their manner of use, and the reactions responsible for the release of an identifiable ligand, the following Examples describing the synthesis and use of one preferred embodiment


    are provided. It will be expressly understood, however, that the particulars of the components comprising each of the reactants, the nature of the identifiable ligand released, the specific enzyme employed, and the conditions of use are not limiting or restrictive of the invention in any way or manner. To the contrary, it is expected and intended that the user will freely choose among the many compounds and constituents presently known and available commercially in this art for his reactants, his choice of enzyme, the specific ligand to be released, and the precise conditions for use, all of which will vary with the intended application and the needs of the user.

    Example 1: Synthesis of 2-[N-ethyl-N-carbo (p-nitrophenoxy)]-4-dimethylamino sulfonyl phenyl-β-D-galactopyranoside.



    [0039] The preparation of the aryl conjugate composition, 2-[Nethyl-N-carbo (p-nitrophenoxy)]-4-dimethylamino sulfonylphenyl-β-D-galactopyranoside, proceeds according to the following reaction Scheme I.


       Initially a stirred mixture comprising 10.0 grams (0.028 moles) of compound 1 [Williams, R.T., J. Chem. Soc. 708 (1942)] and 5.3 milliliters (hereinafter "ml") of phosphorous oxychloride in 100 ml of methylene chloride (CH₂Cl₂) was refluxed under a calcium chloride drying tube for 4 hours. The resulting solution was cooled to room temperature, allowed to stand overnight, and then added dropwise with stirring to 100 ml of cold water. The aqueous and organic layers formed in this manner were separated and the organic layer treated with 50 ml of aqueous dimethylamine (25% in water). This mixture was vigorously stirred for 4 hours and the resulting two-phase mixture cooled in ice and then acidified with concentrated HCl to approximately pH 4. A precipitate results which is isolated by suction filtration, washed five times consecutively with water and then dried to yield 3.62 grams (50%) of a fine white solid-compound 2. [¹H-NMR(d-6 DMSO/CDCl₃): δ2.2 (s,3H); 2.65 (s,6H); 6.95 (d, J=9 Hz, 1H); 7.33 (dd, J₁=9 Hz, J₂=2.5, 1H); 8.2 (d, J=2.5 Hz); 9.1 (bs, 1H) 10.6 (bs, 1H); MS (258 M+)].

    [0040] The next step utilizes a prepared mixture comprising 1.0 grams (3.87 millimoles) of compound 2, 0.88 grams (3.87 millimoles) of benzyltriethyl ammonium chloride and 0.77 grams (19.0 millimoles) sodium hydroxide in 15 ml of CH₂Cl₂ and 5.0 ml of water. This prepared mixture was vigorously stirred at room temperature and was treated in a single portion with 1.6 grams (3.87 millimoles) of 2, 3, 4, 6-tetra-0-acetyl-α-D-galactopyranosyl bromide (Sigma Corporation). The resulting two phase mixture was stirred continuously for six hours, followed by the addition of 0.8 grams (1.94 millimoles) of the α-bromo-tetra-acetyl-D-galactose, and stirred continuously overnight. Subsequently, the organic and aqueous phases (layers) were separated and the organic phase dried over sodium sulfate and evaporated in vacuo. The resulting residue was subjected to liquid chromatography using a silica column (Silica Woelm 32-63) and an eluent comprising 1.0% of CH₃OH in CH₂Cl₂ to yield 0.8 grams (35%) of compound 3. [¹H NMR (CDCl₃): δ2 (s, 3H); 2.03 (s, 3H); 2.1 (s, 3H); 2.15 (s, 3H); 2.2 (s, 3H); 2.65 (s, 6H); 4.15 (bs, 3H); 5.1-5.55 (m, 4H); 7.03 (d, J=9 Hz, 1H); 7.35 (dd, J₁=9 Hz, J₂=2.5 Hz, 1H); 7.93 (bs, 1H); 8.73 (d, J=2.5 Hz, 1H); IR (CH₂Cl₂) 3400 (b), 1760, 1750, 1745 cm⁻¹; M.S. (M+ 588)].

    [0041] Subsequently, a solution comprising 0.705 grams (1.2 mmole) of compound 3 in 40 ml of tetrahydrofuran (hereinafter "THF") was made and placed under an atmosphere of nitrogen with constant stirring. To this stirred solution 1.32 ml (2.6 mmole) borane-dimethyl sulfide solution (2M in THF) was added dropwise at room temperature. The resultant mixture was stirred overnight at room temperature and then refluxed for approximately one-half hour. The liquid mixture was then cooled to room temperature, treated with 1.0 ml of acetic acid, and then evaporated to dryness in vacuo. The formed residue was subjected to liquid chromatography using a silica column with an eluent comprising 1.0-3.0% CH₃OH in CH₂Cl₂. 0.27 grams (39%) of compound 4 was obtained as a solid foam upon removal of effluent from the appropriate fractions and high vacuum evaporation [¹H NMR (CDCl₃): δ1.30 (t, J=7 Hz, 3H); 2.05-2.2 (3 singlets, 12H); 2.74 (s, 6H); 3.23 (q, J=7 Hz, 2H); 4.1-4.3 (m, 3H); 5.0-5.7 (m, 4H); 6.9-7.1 (m, 3H)].

    [0042] The synthesis continues by combining 13.0 mg (0.023 mmole) of compound 4 with 3.5 ul (0.025 mmole) of triethylamine in 1.0 ml of THF and treating this mixture in a single portion with 5.0 mg (0.025 mmole) p-nitrophenyl-chloroformate, under an atmosphere of nitrogen (N₂). The reaction mixture was stirred overnight at room temperature, suction filtered, and the filtrate evaporated to dryness. The resulting residue was subjected to liquid chromatography using a silica column with a 1% CH₃OH/CH₂Cl₂ eluent to yield 10.0 mg (60%) of compound 5 [¹H NMR (CDCl₃): δ1.2 (m, 3H); 1.5-2.2 (3 major singlets, 2 minor broad singlets, total of 12H); 3.5-4.0 (m, 2H); 4.0-4.4 (m, 3H); 5.1-5.6 (m, 4H); 7.1-7.6 (m, 3H); 7.65-7.8 (m, 2H); 8.2-8.4 (m, 2H)].

    [0043] Reaction scheme I is completed by preparing a mixture comprising 0.334 grams (0.452 mmol) of compound 5 and 3.1 ml (22.6 mmol) of triethylamine in 35 ml of methanol. This mixture was stirred at room temperature for approximately 8.5 hours and then evaporated to dryness under high vacuum overnight. The resulting residue was subjected to liquid chromatography using a silica column with an eluent comprising 10% CH₃OH in CH₂Cl₂.

    [0044] The effluent after high vacuum evaporation provided 0.165 grams (64%) of compound 6 as a solid foam [¹H NMR (CD₃OD/CDCl₃): δ1.2 (t, J=7 Hz, 3H); 2.75 (s, 6H); 3.3-4.1 (complex multiplet, 12H); 5.0 (m, 1H); 7.2-7.5 (m, 3H); 7.6-7.8 (m, 2H); 8.1-8.4 (m, 2H). Analysis calculated for C₂₃H₂₉N₃SO₁₂:C, 48.33; H, 5.11; N, 7.35; S, 5.61. Found: C, 47.98; H, 5.20; N, 7.01; S, 5.45].

    Example 2: Synthesis of 2-[N-ethyl-N-carbo (resorufinyl)]-4-dimethylamino sulfonylphenyl-β-D-galactopyranoside.



    [0045] The preparation of the aryl conjugate composition, 2-[N-ethyl-N-carbo (resorufinyl)-4-dimethylamino sulfonyl phenyl-β-D-galactopyranoside, is summarized by Reaction Scheme II below.


       Initially, compounds 1-4 are synthesized in the manner described within Example 1 above. Subsequently, a solution comprising 0.27 grams (0.47 mmol) of compound 4 in 10.0 ml of CH₂Cl₂ is added dropwise over 15 minutes to a stirred solution comprising 0.12 ml of phosgene (12.5% solution in benzene) in 10 ml of CH₂Cl₂ at a temperature ranging from 0-5°C. Alternatively, 28 µl (0.24 mmole) of trichloromethyl-chloroformate (a phosgene equivalent) and 67 µl triethylamine (0.48 mmol) may be used. The reaction mixture is slowly warmed to room temperature and then stirred overnight. Subsequently, the liquid is evaporated in vacuo leaving a colorless solid residue. All of this solid residue is then combined with 0.15 grams (0.71 mmol) resorufin dissolved in 3.0 ml of pyridine at 0-5°C. The formed liquid mixture is again slowly warmed to room temperature and then is continuously stirred at room temperature for approximately 4 hours. After evaporation of volatile solvent under high vacuum, a residue is formed with is subjected to liquid chromatography using a silica column and an eluent comprising 1.0% CH₃OH in CH₂Cl₂. 60 mg (16%) of yellow-orange solid foam was recovered as compound 7 [¹H NMR (CDCl₃): δ1.25 (m, 3H); 1.8-2.4 (3 major singlets, 1 minor singlet, 12H); 2.8 (s, 6H); 3.6-4.1 (m, 2H); 4.1-4.4 (m, 3H); 5.1-5.7 (m, 4H); 6.33 (bs, 1H); 6.85 (dd, J₁=10 Hz, J₂=2 Hz, 1H); 7.0-7.6 (m, 4H); 7.7-7.9 (m, 3H)].

    [0046] Compound 7 was finally treated with triethylamine in methanol according to the procedure given for preparation of compound 6. Analysis of Compound 7 revealed: [¹H NMR (CD₃ OD/CDCl₃): δ1.2 (t, J=7 Hz, 3H); 2.75 (s, 6H); 3.5-4.1 (m, 12H); 5.0 (m, 1H); 6.35 (bs, 1H); 6.83 (dd, J₁=10 Hz, J₂=2 Hz, 1H); 7.1-7.5 (m, 4H); 7.6-7.9 (m, 3H). M.S.: (M+ 646).


    457 (ε10,230)].

    Example 3: Synthesis of the trifluoroacetic acid salt of 2-[N-ethyl-N-carboresorufinyl]-4-piperazinosulfonylphenyl-β-D-galacto pyranoside.



    [0047] The preparation of the aryl conjugate composition, the trifluoroacetic acid salt of 2-[N-ethyl-N-carbo-resorufinyl]-4-piperazino-sulfonylphenyl-β-D-galactopyranoside, is summarized by Reaction Scheme III below.


       The preparation begins with compound 1 of Example 1 above in which 5.0 grams (0.014 mol) of compound 1 and 2.65 ml of phosphorous oxychloride are combined in 50 ml of CH₂Cl₂ and refluxed under a calcium chloride drying tube for approximately 4 hours. The resulting solution is cooled to room temperature, allowed to stand at room temperature overnight, and then added dropwise to 100 ml of cold water with continuous stirring. The formed aqueous and organic phases are separated and the organic phase is dried over Na₂SO₄ (anhydrous) and then added dropwise to a cold solution containing 6.03 grams (0.07 mol) piperazine dissolved in a mixture of 60 ml dimethylformamide (hereinafter "DMF") /20 ml CH₂Cl₂. This reaction mixture was slowly warmed to room temperature and stirred for 4 hours. Evaporation of the volatile solvent yielded a solid residue which was triturated with CH₂Cl₂, filtered, and washed with excess CH₂Cl₂. The isolated filtrate was evaporated in vacuo to yield a crude amber syrup. Subsequently, this crude product was dissolved in a mixture comprising 50 ml CH₂Cl₂/10 ml DMF and treated dropwise with stirring at room temperature with 8.0 ml (0.035 mol) of di-t-butyl dicarbonate. After allowing the mixture to react for 1.5 hours, the volatiles were removed by high vacuum evaporation to yield a residue. The formed residue was then combined with ethylacetate; washed successively with water and 1 N HCL; dried over anhydrous MgSO₄; and again subjected to evaporation. The resulting residue was triturated with diethylether; filtered; and again dried to yield 3.0 grams (54%) of solid comprising a mixture of compound 9 and compound 10.

    [0048] Subsequently, a solution comprising 3.0 grams (0.0075 mol) of the white solid comprising compounds 9 and 10 respectively and 45 ml of THF was prepared and treated with a dropwise addition of 11.3 ml (0.023 mol) borane-dimethyl sulfide solution (2 M in THF). This reaction mixture was refluxed under nitrogen for two hours. The resultant mixture was then allowed to stand at room temperature overnight followed by cautious quenching with excess absolute ethanol and subsequent evaporation to dryness. The formed residue is then dissolved in excess ethanol and again evaporated in vacuo to dryness to yield a white solid. This white solid reaction production is then combined with 6.2 grams (0.015 mol) of 2, 3, 4, 6-tetra-0-acetyl-α-D-galactopyranosyl bromide, 1.7 grams (0.0075 mol) benzyl triethylammonium chloride, and 37.5 ml (0.011 mol) of 0.3 M NaOH in 40 ml of CH₂Cl₂. This biphasic mixture is stirred vigorously at room temperature for approximately three hours. The mixture is then allowed to separate into aqueous and organic phases which were isolated. The aqueous phase is then extracted with another 40 ml of CH₂Cl₂. The combined organic phases were then dried over anhydrous Na₂SO₄ followed by evaporation under vacuum. The resulting residue is then subjected to liquid chromatography using a silica column and an eluent comprising 1.0% CH₃OH in CH₂Cl₂. 4.0 grams (75%) of compound 11 is obtained from the collected effluent. [¹H NMR (CDCl₃) : δ; 1.25 (t, J=7 Hz, 3H); 1.45 (s, 9H); 2.0 (s, 3H); 2.07 (bs, 6H); 2.15 (s, 3H); 2.8-3.3 (m, 6H); 3.3-3.6 (m, 4H); 4.0-4.4 (m, 3H); 4.9-5.5 (m, 4H); 6.7-6.9 (m, 3H)].

    [0049] Subsequently, a solution containing 3.33 grams (4.65 mmol) of compound 11 and 0.65 ml (4.65 mmol) of triethylamine in 60 ml THF is added dropwise under nitrogen to a stirred solution of 0.28 ml (2.33 mmol) trichloromethyl chloroformate in 60 ml of THF at a temperature ranging from 0-5°C. After the addition, the reaction mixture is allowed to warm to room temperature and stirred for approximately one hour. The resulting mixture is then suction filtered; washed with 10 ml of THF; and the filtrate evaporated to dryness. The formed residue is then combined with 1.1 grams (4.65 mmol) resorufin-sodium salt dissolved in 30 ml pyridine and stirred overnight at room temperature. After evaporation of the solvent, the residue is dissolved in excess ethylacetate; then washed successively with water, an aqueous saturated NaHCO₃ solution, and brine; and then dried over anhydrous MgSO₄. Evaporation in vacuo to dryness affords a residue which is subjected to liquid chromatography using a silica column and an eluent comprising 2% CH₃OH in CH₂Cl₂. 2.53 grams (57%) of an orange solid is obtained as compound 12. [¹H NMR (CDCl₃): δ1.25 (t, J=7 Hz, 3H); 1.45 (s, 9H); 1.7-2.15 (multiple singlets, 12H); 2.9 (m, 4H); 3.4 (m, 4H); 3.5-3.9 (m, 2H); 4.15 (m, 3H); 4.9-5.5 (m, 4H); 6.25 (bs, 1H); 6.75 (dd, J₁=10 Hz, J₂=2 Hz, 1H); 6.9-7.5 (m, 4H); 7.5-7.8 (m, 3H)].

    [0050] To complete Scheme III, a mixture containing 2.45 grams (2.57 mmol) of compound 12 and 17.9 ml (128.0 mmol) triethylamine dissolved in 70 ml of methanol is prepared and stirred at room temperature for approximately 7.5 hours. This mixture is then allowed to stand overnight in a freezer. The mixture is allowed to warm to room temperature and is stirred for an additional hour before evaporating under vacuum to dryness. The formed residue is dissolved in 70 ml CH₂Cl₂; combined with 30 ml of trifluoroacetic acid; and is stirred at room temperature for approximately 10 minutes. The volatile solvent is removed by evaporation. The formed residue was then combined with CH₂Cl₂ and evaporated in vacuo several times in sequence. Subsequently, the residue is triturated with 100 ml of CH₂Cl₂ containing 10 ml of ethyl ether overnight and then suction filtered to afford 2.0 grams (98%) of an orange powder identified as compound 13 [M.S.: (M⁺ 687: free base)].

    Example 4: Immobilization of Compound 13 onto Sepharose 4B®



    [0051] A useful solid phase carrier for immobilization of prepared aryl conjugate compositions such as composition 13 are Sepharose® gels commercially available from Pharmacia Corporation. A useful immobilization reaction is as follows: 1.0 gram (12 µmol tresyl) of freeze dried tresyl-activated Sepharose 4B® was suspended and allowed to swell for several minutes in 1 mM HCl. The swollen gel is then filtered and washed for one hour's duration on a sintered glass filter using a total volume of 200 ml of 1 mM HCl. The washed gel is then vacuum suctioned into a damp filter cake and then transferred to a solution containing 5.0 milligrams (7.0 µmol) of compound 13 in 5.0 ml of phosphate buffer (pH 7.0). This mixture was allowed to stand at room temperature with occasional swirling for approximately 6 hours. Subsequently, the gel is suction filtered on a sintered glass frit, thoroughly washed with phosphate buffer (pH 7.0), followed by additional washing in water. The washed gel is then again suctioned to a damp filter cake. The orange colored gel obtained in this manner comprises immobilized compound 13 but it is preferred that the prepared gel be dialyzed to remove all remaining contaminants prior to use. Accordingly, the orange gel was transferred to dialysis tubing having a 12-14K molecular weight cutoff (Spectraphor® membrane tubing, Spectrum Medical Industries) and dialyzed against one liter of distilled water for 3 days with the water being changed every 12 hours. The resulting gel demonstrates a measurable release of resorufin after reaction wth Asp. oryzae β-galactosidase.

    Example 5: Useful Immobilization Materials and Reactions



    [0052] To demonstrate the feasibility of immobilizing the aryl conjugate compositions comprising the present invention, a variety of resorufin containing conjugate reactants were prepared in the manner described within Example 3 to yield the aryl conjugate composition whose structural formula is


    wherein R' is chosen from the group identified below and corresponds to compound 13, 14, or 15 respectively.


       Compounds 13, 14, and 15 respectively may individually be immobilized to a wide variety of different solid support materials and a range of immobilizing reactions conventionally known in this art. Merely to illustrate the variety of solid support carriers and reactions which are deemed useful to prepare immobilized embodiments of the present invention, a summary of different formats and the mobilizing reactions are provided by Tables 4 and 4A respectively.









    [0053] It will be appreciated that each of these immobilized embodiments released resorufin in measurable quantities after combination with Asp. oryzae β-galactosidase.

    Example 6: The Enzyme Controlled Release System



    [0054] The release system of the present invention comprising an active enzyme and an aryl conjugate composition comprising a substrate which is open to attack by the enzyme is demonstrated by the interaction between the enzyme Beta-galactosidase in combination with compound 6 described earlier in Example 1; with compound 8 described earlier in Example 2; and with ortho-nitro phenyl galactopyranose (hereinafter "ONPG"), commercially available from Sigma Corporation. The test conditions and kinetic results of enzymatic hydrolysis are summarized in Table 5.


    Example 7: Specificity of the Organic Conjugate Composition



    [0055] A major advantage provided by the organic conjugate compositions comprising the present invention is the selective ability to discriminate not only between different classes of enzymes, but also to distinguish the same enzyme obtained from different sources. To demonstrate this unique ability, beta-galactosidase derived from Asperillus orzyhae, beef liver, and E. coli. were individually evaluated using compounds 6 and 8 respectively as reactants. Each of the individual, separately derived, beta-galactosidases were commercially obtained and empirically evaluated using the test protocol described previously in Example 6. The results of these evaluations are provided by Table 6 below.



    [0056] It is clearly demonstrated that the release of ortho-nitrophenol from compound 6 (and the release of resorufin from compound 8) will vary directly with the individual source of β-galactosidase and that the rate at which the displaceable ligand is released will vary drastically under similar test conditions. The ability of the present invention to identify and discriminate among different sources of the same enzyme is thus unequivocally demonstrated.

    Example 8: Detection of 1.0 x 10⁻¹² M Enzyme



    [0057] Experimental conditions: Ten microliters of 1.0 x 10⁻¹⁰ M Aspergillus oryzhae Beta galactosidase (Cal Biochem) in 100 mM citrate phosphate, pH 5, with 0.05% w/v gelatin (Buffer A) was added to one milliliter of 1.6 mM compound 15 in Buffer A, yielding a final enzyme concentration of 1.0 x 10⁻¹² M. One half milliliter of this reaction mixture was monitored in a spectrofluorimeter in a two millimeter cuvette with instrument settings of 540 nm excitation, 580 emission and 5 nm slit widths. The signal was measured on a chart recorder at a speed of one centimeter per minute and at a sensitivity setting that gave fifteen percent deflection with 1.7 x 10⁻⁷ M Sulforhodamine 101® in ethanol.

    [0058] An increase in signal over time was observed with the enzyme reaction mixture and in a ten minute interval there was a one and one half percent increase in deflection. No increase in the signal generated by the substrate solution alone was seen. Accordingly, the detection of 1.0 x 10⁻¹² M concentrations of enzyme is deemed to be proven unequivocally.

    APPLICATIONS



    [0059] The enzyme controlled release system and the prepared organic conjugate used as reactants in combination with an active specific enzyme may be beneficially employed in a variety of different modes of application. Different features and characteristics of the invention are utilized to advantage in each of these modes of use. It will be recognized also that the described modes which follow are merely representative of the many other applications for the present invention; and are not in any manner or form restrictive of the present invention to the described modes of use.

    Immunoassays for Research or Clinical/Diagnostic Analyses



    [0060] In this mode, the enzyme is covalently bonded to a ligand having known specific binding properties for an analyte of interest. Typically, there is an analyte analogue employed to provide a competition between the analyte of interest and the analyte analogue for the binding sites of the ligand (the specific binding partner) joined to the enzyme. Frequently, the specific binding partner ligand joined to the enzyme will be a protein or a polypeptide and may functionally be classified as an antibody (of monoclonal or polyclonal origin); or an antibody fragment (such as a Fab fragment or Fab' fragment); or a specific binding molecule (such as avidin or biotin). The analyte of interest demonstrates a specific binding affinity for the ligand attached to the enzyme and is typically identified as an antigen or hapten; a specific binding protein; or a molecule having specific receptor sites for the ligand. The analyte analogue typically is a chemical composition identical or similar to the analyte of interest and is also chosen on the basis of its demonstrated ability to selectively bind to the ligand previously joined to the enzyme. After the interaction of the immunological components, the ligand-enzyme complex is brought into reactive contact with the organic conjugate composition whereby the displaceable fragment Z-M is released. In this manner, the presence and concentration of one or more analytes of interest may be detected with precision and accuracy.

    Enzyme Triggered Fluorescence Assays



    [0061] This mode of use is a general one, but is especially applicable to the immunoassay mode described above. The prepared organic conjugate composition brought into reactive contact with an appropriate active enzyme or the immunological ligand-enzyme complex described previously comprises a Z-M fragment which is a fluorescent dye. While attached to the secondary displacement appendage as a component part of the conjugate reactant, the dye moiety does not exhibit any meaningful fluorescence or exhibits a change in its excitation maxima. The attack of the enzyme or ligand-enzyme complex cleaves the attached enzyme substrate, which, in turn initiates the intramolecular displacement reaction and the release of the fluorescent dye as a mobile entity into the reaction medium. Upon release, and the introduction of light at the appropriate wavelength, the mobile dye will fluoresce and be detectable as such. In this manner, the mode may be employed in either a qualitative or quantitative methodology.

    [0062] One aspect of the enzyme triggered fluorescent assay mode described herein deserves special attention: the organic conjugate comprising the attached fluorescent (or chromophoric, or chemiluminescent) dye as a component part may be employed as either a mobile reactant or as an immobilized reactant joined to a solid carrier. The ability to immobilize a prepared organic conjugate comprising a fluorescent dye precursor and subsequently to release from it a mobile fluorescent dye is an important advantage of the present invention.

    Analytical Systems Requiring Enzyme Amplification



    [0063] In many instances where the detection of an enzyme is the goal of the assay, a major drawback of known detection systems is their relative insensitivity and/or inability to provide sufficient quantities of an identifying label with respect to the quantity of enzyme in the test sample. More precisely, although conventional reactants interact with the enzyme in the sample, that quantity of identifying label which is released by the test system as an indication of reactive contact is often insufficient to be measured by the existing instrumentation for the analysis to be accurately made. This is especially true in assays for the detection of specific enzymes which are generally present in nanogram or picogram quantities. The present invention provides for amplification of such detection assays using the release system in series or multiple stages. For example, if the detection of beta-galactosidase were the goal of the assay, a first stage reactant would comprise an immobilized organic conjugate comprising a substrate L specific for beta-galactosidase and a displaceable fragment Z-M which is another active enzyme having a broader substrate range, such as amylase. The amylase enzyme molecule would be joined to the immobilized organic base structure by one of its constituent lysine, tyrosine, serine, or thiol residues via its extending side groups. A second stage conjugate would also be prepared as a second immobilized reactant in which another attached substrate L would be one open to attack by the displaced amylase enzyme molecule after its release from the first stage reactant. The displaceable fragment Z-M of this second prepared reactant typically would be a measurable dye (chromophoric, chemiluminescent or fluorescent).

    [0064] In the analytical protocol, the presence of β-galactosidase in the test sample would initiate an attack on the first immobile conjugate reactant and cause the release of the amylase enzyme as the first displaced Z-M fragment; the released amylase would then be able to react with a known concentration of the second organic conjugate having its own substrate L open to attack by the released amylase which, in turn, will cause the release of an identifiable dye as the second displaced Z-M fragment. The concentration of the second organic conjugate reactant would typically be much greater than the concentration of the first conjugate reactant to achieve the amplification effect. Accordingly, a single molecule of β-galactosidase (through the two-stage enzyme controlled release system) will cause the release of many molecules of identifiable dye in an amount sufficient to be reproducibly and accurately measured by conventional photometric means. In this manner, the presence of minute quantities of β-galactosidase in the test sample can be amplified using the present invention to achieve positive detection and quantitative measurement.

    Release of Specific Agents on Demand



    [0065] This mode of use is employed when the release of a specific agent is required on demand or is desirable in response to a specific triggering event. Such situations include the release of a therapeutic drug, protein, or pharmacologically active agent; or the release of a molecule having specific properties and characteristics such as a photographic dye. In such situations, the prepared organic conjugate will comprise the agent to be released as the displaceable fragment Z-M in the prepared reactant. The addition of an active enzyme able to cleave a substrate L of the conjugate will cause the release of the desired displaceable ligand (agent) directly on demand. In this manner, the desired active agent will be in an attached state subject to immediate release upon the introduction of an appropriate active enzyme.

    [0066] The organic conjugate composition may comprise a saturated or unsaturated cyclic moiety (a = 1) which is substituted with R₁. It will be understood by those skilled in the art that the cyclic moiety may be further appropriately substituted with one or more additional substituents which affect the mobility or reactivity of the conjugate composition.


    Claims

    1. An enzyme controlled release system for the release of a ligand comprising:
    an active enzyme able to a cleave the covalent bond joining the substrate L to the neighbouring group Q in an organic conjugate; and
       the organic conjugate able to react with said enzyme, said organic conjugate having the formula

    wherein a, b, d, and e individually are 0 or 1;
       W may be omitted entirely but when present comprises the number of atoms necessary to form a saturated or unsaturated cyclic molecule;
       X and X' individually are hydrogen, a hydrocarbon group or a substituted hydrocarbon group;
       Y may be omitted entirely but when present comprises from 1-5 carbon atoms;
       U and U' individually are hydrogen or a second covalent bond;
       V and V' may be omitted individually or jointly provided that they cannot be omitted jointly when b is 0 and that only one of V and V' can be present when b is 3,
       but when present are

       wherein p is an integer from 0-3,
       t is an integer from 0-3,
    the sum of p+t is from 0-3, and wherein
       J is

    -O-, -S-, or

    and R₂, R₃, R₄, R₅, R₆, R₇, R₈ individually are hydrogen, an alkyl group having from 1-6 carbons or an aryl group;
       Q and Z individually are O, S, NH or NR', R' being an organic group;
       L is an enzyme substrate which is bound to Q in a manner so that the covalent bond joining L to Q is cleavable by said enzyme;
       M is an organic moiety;
       R₁ is hydrogen or one or more substituents affecting the mobility or reactivity of said organic conjugate;
    and whereby Z-M is a displaceable ligand released by intramolecular displacement reaction from said organic conjugate after said enzymatic cleavage of the covalent bond joining L and Q.
     
    2. The release system as recited in claim 1 wherein said organic conjugate comprises a base supporting structure selected from the group consisting of saturated and unsaturated cyclic organic compounds; or selected from the group consisting of saturated or unsaturated linear organic compounds.
     
    3. The release system as recited in claim 1 or 2 wherein said organic conjugate comprises a base supporting structure selected from the group consisting of phenyl and phenyl derivatives, naphthalene and naphthalene derivatives, quinoline and quinoline derivatives, indole and indole derivatives, and phenanthrene and phenanthrene derivatives.
     
    4. The release system as recited in claims 1-3 wherein said organic conjugate is represented by the formula

    wherein Q and Z individually are O, S, NH or NR', R' being an organic group;
       L is an enzyme substrate which is bound to Q in a manner so that the bond joining L to Q is cleavable by said enzyme;
       M is an organic moiety;
       R₁ is hydrogen or a substituent affecting the mobility or reactivity of said organic conjugate;
       R₈ is an organic substituent; and
    wherein Z-M is a displaceable ligand released by intramolecular displacement reaction from said organic conjugate composition after said enzymatic cleavage of the bond joining L and Q.
     
    5. The release system as recited in any one of claims 1 to 4 wherein said enzyme is a hydrolase.
     
    6. The release system as recited in claim 1 or 2 wherein said displaceable fragment Z-M is a dye or dye precursor, in particular a fluorescent dye or a chemiluminescent dye.
     
    7. The release system as recited in claim 1 or 2 wherein said displaceable fragment Z-M comprises at least one amino acid.
     
    8. The release system as recited in claim 1 or 2 wherein said displaceable fragment Z-M is an active enzyme.
     
    9. The release system as recited in claim 1 or 2 wherein said displaceable fragment Z-M has specific binding properties for another compound.
     
    10. The release system as recited in claim 1 or 2 wherein said active enzyme is attached to a ligand.
     


    Ansprüche

    1. Enzymgesteuertes Freisetzungssystem zur Freisetzung eines Liganden, enthaltend:
    ein aktives Enzym, das in der Lage ist, die kovalente Bindung, die das Substrat L mit der benachbarten Gruppe Q in einem organischen Konjugat verbindet, zu spalten;
    wobei das organische Konjugat in der Lage ist, mit dem Enzym zu reagieren und das organische Konjugat die Formel

    hat, worin bedeuten:
    a, b, d und e bedeuten jeweils 0 oder 1,
    W kann ganz weggelassen werden; wenn es aber vorhanden ist, bedeutet es die Anzahl der Atome, die zur Bildung eines gesättigten oder ungesättigten cyclischen Moleküls erforderlich sind;
    X und X' bedeuten jeweils Wasserstoff, eine Kohlenwasserstoffgruppe oder eine substituierte Kohlenwasserstoffgruppe;
    Y kann ganz weggelassen werden; wenn es aber vorhanden ist, enthält es 1 bis 5 Kohlenstoffatome;
    U und U' bedeutenjeweils Wasserstoff oder eine zweite kovalente Bindung;
    V und V' können einzeln oder gemeinsam weggelassen werden; sie können aber für b = 0 nicht gemeinsam weggelassen werden; es kann für b = 3 nur eines von V und V' vorhanden sein; wenn sie aber vorhanden sind, bedeuten sie

       worin p eine ganze Zahl von 0 bis 3,
       t eine ganze Zahl von 0 bis 3,
       die Summe von p + t 0 bis 3 ist, und worin
       J =

    -O-, -S-, oder

       und R₂, R₃, R₄, R₅, R₆, R₇, R₈ jeweils Wasserstoff, eine Alkylgruppe mit 1 bis 6 Kohlenstoffatomen oder eine Arylgruppe darstellen;
    Q und Z bedeuten jeweils O, S, NH oder NR', wobei R' eine organische Gruppe darstellt;
    L bedeutet ein Enzymsubstrat, das in einer solchen Weise an Q gebunden ist, daß die kovalente Bindung, die L mit Q verbindet, durch das Enzym spaltbar ist;
    M bedeutet eine organische Gruppierung;
    R₁ bedeutet Wasserstoff oder einen oder mehrere Substituenten, die die Mobilität oder Reaktivität des organischen Konjugats beeinflussen;
    und Z-M bedeutet einen entfernbaren Liganden, der nach der enzymatischen Spaltung der kovalenten Bindung, die L mit Q verbindet, durch eine intramolekulare Verdrängungsreaktion aus dem organischen Konjugat freigesetzt wird.
     
    2. Freisetzungssystem nach Anspruch 1, worin das organische Konjugat eine Basis-Trägerstruktur enthält, die aus der Gruppe, bestehend aus gesättigten und ungesättigten cyclischen organischen Verbindungen; oder aus der Gruppe, bestehend aus gesättigten oder ungesättigten linearen organischen Verbindungen, ausgewählt ist.
     
    3. Freisetzungssystem nach Anspruch 1 oder 2, worin das organische Konjugat eine Basis-Trägerstruktur enthält, die aus der Gruppe, bestehend aus Phenyl und Phenylderivaten, Naphthalin und Naphthalinderivaten, Chinolin und Chinolinderivaten, Indol und Indolderivaten und Phenanthren und Phenanthrenderivaten ausgewählt ist.
     
    4. Freisetzungssystem nach einem der Ansprüche 1 bis 3, worin das organische Konjugat durch die Formel

    dargestellt ist, worin bedeuten:
    Q und Z bedeuten jeweils O, S, NH oder NR', wobei R' eine organische Gruppe darstellt;
    L bedeutet ein Enzymsubstrat, das in einer solchen Weise an Q gebunden ist, daß die Bindung, die L mit Q verbindet, durch das Enzym spaltbar ist;
    M bedeutet eine organische Gruppierung;
    R₁ bedeutet Wasserstoff oder einen Substituenten, der die Mobilität oder Reaktivität des organischen Konjugats beeinflußt;
    R₈ bedeutet einen organischen Substituenten; und
    Z-M bedeutet einen entfernbaren Liganden, der nach der enzymatischen Spaltung der Bindung, die L mit Q verbindet, durch eine intramolekulare Verdrängungsreaktion aus dem organischen Konjugat freigesetzt wird.
     
    5. Freisetzungssystem nach einem der Ansprüche 1 bis 4, worin das Enzym eine Hydrolase darstellt.
     
    6. Freisetzungssystem nach Anspruch 1 oder 2, worin das entfernbare Fragment Z-M einen Farbstoff oder eine Farbstoff-Vorstufe, insbesondere einen fluoreszierenden Farbstoff oder einen chemolumineszierenden Farbstoff, darstellt.
     
    7. Freisetzungssystem nach Anspruch 1 oder 2, worin das entfernbare Fragment Z-M mindestens eine Aminosäure enthält.
     
    8. Freisetzungssystem nach Anspruch 1 oder 2, worin das entfernbare Fragment Z-M ein aktives Enzym darstellt.
     
    9. Freisetzungssystem nach Anspruch 1 oder 2, worin das entfernbare Fragment Z-M spezifische Bindeeigenschaften für andere Verbindungen hat.
     
    10. Freisetzungssystem nach Anspruch 1 oder 2, worin das aktive Enzym an einen Liganden gebunden ist.
     


    Revendications

    1. Système de libération controlé par des enzymes pour la libération d'un ligand comprenant :
    une enzyme active capable de couper la liaison covalente reliant le substrat L au groupe voisin Q dans un conjugué organique ; et
    le conjugué organique capable de réagir avec ladite enzyme, ledit conjugué organique ayant la formule :

    dans laquelle a, b, d et e sont individuellement 0 ou 1 ;
       W peut être omis entièrement mais s'il est présent il comprend le nombre d'atomes nécessaire pour former une molécule cyclique saturée ou insaturée ;
       X et X' représentent individuellement l'hydrogène, un groupe hydrocarboné ou un groupe hydrocarboné substitué ;
       Y peut être omis entièrement mais s'il est présent il comprend 1 à 5 atomes de carbone ;
       U et U' représentent individuellement l'hydrogène ou une deuxième liaison covalente ;
       V et V' peuvent être omis individuellement ou conjointement, à condition qu'ils ne puissent être omis conjointement lorsque b vaut 0, et que seul l'un de V et V' peut être présent, lorsque b vaut 3, mais s'ils sont présents, ils représentent :

    dans laquelle
       p est un nombre entier de 0 à 3,
       t est un nombre entier de 0 à 3,
    la somme de p + t allant de 0 à 3 et dans laquelle
    J représente

    - O -, - S -,

    ou et R₂, R₃, R₄, R₅, R₆, R₇ et R₈ représentent individuellement l'hydrogène, un groupe alkyle ayant 1 à 6 atomes de carbone ou un groupe aryle ;
       Q et Z représentent individuellement O, S, NH ou NR', R' désignant un groupe organique ;
       L est un substrat d'enzyme qui est lié à Q de sorte que la liaison covalente reliant L à Q puisse être coupée par ladite enzyme ;
       M est un reste organique ;
       R₁ désigne l'hydrogène ou un ou plusieurs substituants affectant la mobilité ou la réactivité dudit conjugué organique ; et
    dans laquelle Z-M est un ligand labile libéré du conjugué organique par réaction de déplacement intra-moléculaire après ladite coupure enzymatique de la liaison covalente liant L et Q.
     
    2. Système de libération selon la revendication 1, caractérisé en ce que le conjugué organique comprend une base supportant la structure choisie parmi les composés organiques cycliques saturés ou insaturés ; ou choisie parmi les composés organiques linéaires saturés ou insaturés.
     
    3. Système de libération selon la revendication 1 ou 2, caractérisé en ce que ledit conjugué organique comprend une base supportant la structure choisie parmi le phényl et ses dérivés, le naphtalène et ses dérivés, la quinoline et ses dérivés, l'indole et ses dérivés et le phénanthrène et ses dérivés.
     
    4. Système de libération selon l'une quelconque des revendications 1 à 3, caractérisé en ce que le conjugué organique est représenté par la formule :

    dans laquelle
       Q et Z représentent indépendamment O, S, NH ou NR', R' étant un groupe organique ;
       L est un substrat d'enzyme qui est lié à Q de sorte que la liaison liant L à Q peut être coupée par ledit enzyme ;
       M est un reste organique ;
       R₁ représente l'hydrogène ou un substituant affectant la mobilité ou la réactivité dudit conjugué organique ;
       R₈ est un substituant organique ; et
    dans laquelle Z-M est un ligand labile libéré du conjugué organique par réaction de déplacement intra-moléculaire après la coupure enzymatique de la liaison liant L et Q.
     
    5. Le système de libération selon l'une quelconque des revendications 1 à 4, caractérisé en ce que l'enzyme est une hydrolase.
     
    6. Système de libération selon la revendication 1 ou 2, caractérisé en ce que le ligand labile Z-M est un colorant ou un précurseur de colorant, en particulier un colorant fluorescent ou un colorant chemiluminescent.
     
    7. Système de libération selon la revendication 1 ou 2, caractérisé en ce que le ligand labile Z-M comprend au moins un acide aminé.
     
    8. Système de libération selon la revendication 1 ou 2, caractérisé en ce que le ligand labile Z-M est une enzyme active.
     
    9. Système de libération selon la revendication 1 ou 2, caractérisé en ce que le ligand habile Z-M a des propriétés particulières de liaison avec un autre composé.
     
    10. Système de libération selon la revendication 1 ou 2, caractérisé en ce que ladite enzyme active est liée à un ligand.