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Protecting groups for biological labeling

a technology of biological labeling and protection groups, applied in the direction of phosphorous compound active ingredients, bulk chemical production, instruments, etc., can solve the problems of inability to store for extended periods of time in aqueous solutions, hydrolytic instability, and inapplicability to us

Inactive Publication Date: 2004-10-07
KECZER STEYE DE +4
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the .alpha.-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 .alpha.-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.
.alpha.-Haloketones are often not used because they are generally too reactive, cause cross-linking of the protein, and are otherwise hydrolytically unstable.
Some of the .alpha.-haloketones are also insufficiently soluble for use in such contexts.
Thus, despite their potential usefulness in a number of diagnostic and synthetic applications, the .alpha.-haloketones are often considered too unstable to be used in such applications.
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.
BABA has limited stability when stored above physiological pH and limited solubility at lower pH.
The percent residual sodium chloride can be further reduced by additional cold water washes of the precipitated product with a concomitant loss in product yield.

Method used

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  • Protecting groups for biological labeling
  • Protecting groups for biological labeling
  • Protecting groups for biological labeling

Examples

Experimental program
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Effect test

example 1

Demonstration of Deprotection of the Alkylating Group

[0194] 1. Deprotection of a Phosphate Protecting Group by Alkaline Phosphatase

[0195] A mixture of BABA-phosphate and CABA-phosphate (resulting from preparation of BABA-phosphate), 5 mg in 1 mL pH 8-9 NaOD / D.sub.2O is treated with 1 mg (15 units) of alkaline phosphatase (Sigma, human placenta) for 15 minutes at room temperature. TLC analysis (250 micron SiO.sub.2 (Analtech Uniplate), using 20% H.sub.2O / ACN indicates that the phosphate group of the protected alkylating reagent is removed by treatment with alkaline phosphatase, whereas the nontreated protected alkylating reagent remains intact. This is further corroborated by NMR (D.sub.2O) analysis, which shows the disappearance of the vinyl proton peak (normally found at 6.3 ppm for the enol-phosphate).

[0196] Further demonstration of the efficient removal of the protecting group is shown by TLC (as above) analysis of the reaction of the deprotected alkylating reagent with cysteine....

example 2

Homocysteine Assay Using LOCI (Luminescent Oxygen Channeling Immunoassay)

[0199] A variety of assay methods and compositions are useful in the detection of compounds and compositions in biological samples--e.g., serum samples. One useful assay which uses the compositions and methods described herein is referred to herein as LOCI. (See U.S. Pat. Nos. 5,709,994, 5,340,716 and 5,478,729--the disclosures of which are incorporated by reference herein--for further details regarding luminescent oxygen channeling immunoassays.)

[0200] Hcy exists in numerous forms in plasma--e.g., as a small percentage in the free form, some as disulfides with itself and cysteine, and some as disulfides with albumin (about 70%). Total hcy is the clinically-relevant measure, however, with reference values in fasting subjects of about 5 to 15 .mu.mol / L. Even a moderate increase in plasma hcy, referred to as hyperhomocysteinemia, is considered a risk factor for premature cardiovascular diseases in the general pop...

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Abstract

Alpha-haloketones are useful alkylating agents for coupling to sulfhydryl-containing biomolecules. However, they 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. The present invention provides novel solutions to these problems, however, as novel compounds and compositions comprising protected haloketones are disclosed herein. Methods of preparing and using protected haloketones which are useful in a variety of applications-e.g., in assays and conjugation reactions-are also disclosed herein.

Description

INTRODUCTION AND BACKGROUND[0001] 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 .alpha.-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 .alpha.-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.[0002] .alpha.-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 .alpha....

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): G01N33/532C07F9/113C07H15/10C12P11/00C12Q1/42G01N33/53G01N33/58G01N33/68
CPCC07F9/113C07H15/10C12Q1/42G01N33/6815G01N33/6842Y10S435/975Y10S436/815Y10S436/825Y02P20/55
Inventor KECZER, STEYE DELIU, YEN PINGDAVALIAN, DARIUSHKURN, NURITHULLMAN, EDWIN F.
Owner KECZER STEYE DE
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