Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-09
2004-08-31
Celsa, Bennett (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007500, C435S975000, C436S531000, C436S815000, C436S825000
Reexamination Certificate
active
06783947
ABSTRACT:
INTRODUCTION AND BACKGROUND
Alpha-haloketones are alkylating agents that are useful in a variety of contexts, including the coupling of various molecules to other, sulfhydryl-containing molecules. However, the &agr;-haloketones possess characteristics that make them problematical and impractical for use in assays and reactions in which the components of a kit for use in such methods need to be stored together and/or for a period of time. For example, the &agr;-haloketones react spontaneously with water, alkali and organic bases and therefore cannot be stored for extended periods of time in aqueous solutions, particularly in the presence of proteins at physiological pH.
&agr;-Haloketones are often not used because they are generally too reactive, cause cross-linking of the protein, and are otherwise hydrolytically unstable. Were it not for these problems, their higher reactivity would permit the use of lower concentrations of the drug derivative. Some of the &agr;-haloketones are also insufficiently soluble for use in such contexts. Thus, despite their potential usefulness in a number of diagnostic and synthetic applications, the &agr;-haloketones are often considered too unstable to be used in such applications.
Similarly, proteins are often coupled to other proteins by attaching an alkylating group to one protein and a sulfhydryl group to the other. However, it is often difficult to obtain quantitative coupling in this matter without using high concentrations of one or both protein members and/or attaching multiple or single alkylating agents to one of the proteins. Additionally, the protein having alkylating agents attached must either have no sulfhydryl group or its sulfhydryl groups must be protected to avoid polymerization. The problem again could be overcome by using a more reactive &agr;-haloketone if it were not for the difficulty of preparing and storing a protein containing a highly-reactive alkylating agent.
The present invention provides novel solutions to the foregoing problems, however, thereby rendering this class of alkylating agents practical for various diagnostic and synthetic utilities, among others. In conjunction with the disclosure of novel compounds and compositions comprising protected haloketones, the present invention also discloses methods of preparing and using protected haloketones which are useful in a variety of applications—e.g., in assays and conjugation reactions.
As an example of the latter, certain G6PDH drug conjugates are prepared by first coupling the enzyme with an &agr;-haloacid to form an &agr;-haloamide and then coupling this conjugate with a sulfhydryl-labeled drug. The method is particularly useful when the drug has a free amino group that would interfere with coupling by means of an active ester of the drug.
SUMMARY OF THE INVENTION
Therefore, in one embodiment of the invention, protecting groups that permit long-term storage of protected &agr;-haloketones, provide additional water solubility where needed, and offer a simple biocompatible method for removal are disclosed.
Therefore, in one embodiment, the invention discloses a composition comprising a protected alkylating reagent wherein deprotection of the reagent is catalyzed by an enzyme. In various preferred embodiments, the reagent includes a protecting group selected from the group consisting of a phosphate, an ester, a carbohydrate, a nucleic acid, and a lipid. Various preferred embodiments also disclose that the enzyme is selected from the group consisting of glycosidases, nucleases, lipases, esterases, hydroxylases and phosphatases.
In alternative embodiments of the invention, the reagent is a 2-halovinyl ether, a 2-halovinyl ester, a 4-halobutadienyl ether, or a 4-halobutadienyl ester. In various embodiments, the 2-halovinyl ether or ester is a 2-halovinyl monophosphate. In other embodiments, the vinyl group is substituted with one or two alkyl or aryl groups; the alkyl or aryl groups may be substituted or unsubstituted.
In various preferred embodiments of the invention, the haloketone is an &agr;-haloketone. Preferred &agr;-haloketones include &agr;-bromoacetylbenzoic acid (BABA) and &agr;-chloroacetylbenzoic acid (CABA). The compositions of the present invention may further comprise a nucleophilic agent and/or a disulfide reducing agent. Useful disulfide reducing agents include phosphines, such as tris(carboxyethyl)phosphine (TCEP).
The present invention also discloses various kits, such as kits for use in carrying out a coupling reaction. One such kit comprises, in a packaged combination, a first reagent comprising a protected alkylating reagent, in an amount sufficient to conduct at least one reaction.
In one variation, the first reagent includes a protecting group selected from the group consisting of phosphate esters, carboxylate esters, sulfate esters, glycosides and ketals. In another variation, the first reagent includes a protecting group selected from the group consisting of phosphate and carbohydrate. In various embodiments, the first reagent may be a protected haloketone and may comprise a 2-halovinyl ether or ester. An exemplary 2-halovinyl ester is a 2-halovinyl monophosphate.
A protected haloketone of the present invention preferably comprises an &agr;-haloketone, such as BABA or CABA. Alternatively, the first regent may be a 4-halobutadienyl ether or ester. In various embodiments, the vinyl group is substituted with one or two alkyl or aryl groups.
The kit may further comprise a second reagent comprising a catalyst capable of deprotecting the protected alkylating reagent. In various embodiments, the first and second reagents are included in separate containers.
In another variation of the disclosed kits, the catalyst comprises an enzyme. Suitable enzymes for use as disclosed may be selected from the group consisting of glycosidases, nucleases, lipases, esterases, hydroxylases, phosphatases (e.g., alkaline phosphatase) and ribozymes.
The present invention also discloses kits for use in a method for detecting quantitative determination homocysteine in a sample, comprising in a packaged combination: a first reagent comprising a protected alkylating reagent capable of chemically modifying homocysteine to form modified homocysteine when deprotected, a second reagent comprising an activating reagent capable of deprotecting the protected alkylating reagent, and a third reagent capable of specifically binding to the modified homocysteine, each in an amount sufficient to conduct at least one assay.
In various kits of the present invention, the first reagent comprises a protected haloketone having a phosphate protecting group. In various disclosed embodiments, the protected haloketone included in a kit is CABA or BABA. The first reagent may further comprise a homocysteine disulfide reducing agent; it may also further comprise a solid matrix coated with modified homocysteine. The modified homocysteine may be the product formed by deprotecting the protected haloketone and reaction of the deprotected haloketone with homocysteine in the sample. Alternatively, the modified homocysteine may be a 4-carboxyphenacyl thioether of homocysteine (hcy-ABA).
In various alternative embodiments, the solid matrix comprises latex beads, glass beads, a microtiter plate, nitrocellulose, agarose, liposomes, and the like. In embodiments including a homocysteine disulfide reducing agent, that agent may comprise a phosphine. One exemplary phosphine useful in the kits of the present invention is TCEP.
In other embodiments of the kits of the present invention, the second reagent further comprises a phosphatase. In one variation, the phosphatase is alkaline phosphatase. Other variations include kits wherein the second reagent further comprises a solid matrix coated with a receptor capable of specifically binding modified homocysteine. The receptor may comprise an antibody or an immunologically active fragment thereof. When the second reagent further comprises a solid matrix, that matrix may comprise latex beads, glass beads, a microtiter plate, nitrocellulose, agarose, liposomes, and the li
Davalian Dariush
de Keczer Steve
Kurn Nurith
Liu Yen Ping
Ullman Edwin F.
Celsa Bennett
Cullman Louis C.
Dade Behring Marburg GmbH
Tymeson Cynthia G.
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