Chromogenic compound

Details Chromogenic compound Chromogenic compound

2000-07-03

2002-09-10

Carr, Deborah D. (Department: 1621)

Organic compounds -- part of the class 532-570 series

Organic compounds

Fatty compounds having an acid moiety which contains the...

C554S078000, C558S207000, C558S208000, C558S218000

Reexamination Certificate

active

06448422

ABSTRACT:

This invention is directed to a chromogenic compound, a thiophospholipid enzyme substrate, specifically thiophosphatidyl ethyleneglycol (thioPEG), which is useful as an indicator compound in an analytical test system. In particular, the present invention relates to a novel chromogenic substrate compound, its preparation and use in an assay and for the spectrophotometric detection of enzymes in a liquid test sample.
All biological membranes have the same classes of chemical compounds and a number of properties in common. These membranes are very dynamic structures with a movement that permits the cell as well as subcellular structures to adjust their shape and to move. The chemical components of membranes, which include lipids and protein, are well suited for the dynamic movement of membranes. Further, these membranes control the composition of the space they enclose by their ability to exclude a variety of molecules and via selective transport systems which permit the movement of molecules from one side to another. By controlling the translocation of substrates, cofactors and ions from one compartment to another, membranes modulate the concentration of substances, thereby exerting a strong influence on the body's metabolic pathways.
Lipids are a major component of membranes. The three major lipid components of cell membranes are phosphoglycerides, sphingolipids, and cholesterol. The phosphoglycerides and sphingomyelin, a sphingolipid containing phosphate, are classified as phospholipids.
Phospholipids, which are waxy solids, are found almost exclusively in cellular membranes and in the lipoproteins of blood plasma. Phospholipids thus serve primarily as structural elements and are never stored in large amounts. As their name implies, this group of lipids contains phosphorus in the form of phosphoric acid. The major phospholipids found in cells contain two fatty acid molecules which are esterified to the first and second hydroxyl groups of glycerol. The third hydroxyl group, at carbon atom 3, is esterified with phosphoric acid. Phospholipids contain a second alcohol which is also esterified to the phosphoric acid to form a phosphodiester; the second alcohol group is thus located on the polar head of the phospholipid molecule. The general structural formula of the phospholipids is shown below. RO—denotes the second alcohol group.
Different types of phospholipids are named according to the second alcohol at their polar heads. The most abundant phospholipids are the closely related phosphatidylethanolamine (also called cephalin) and phosphatidylcholine (also called lecithin), which contain ethanolamine and choline, respectively, at their heads. Each of these can occur in different forms depending on the fatty acids they contain.
Phospholipids readily undergo hydrolysis, catalyzed by acids, bases, or enzymes. Dilute base removes the two fatty acid groups of phosphatidylcholine, leaving the rest of the molecule intact. Strong base causes cleavage of both the fatty acids as well as the choline, leaving glycerol 3-phosphate, which can then be cleaved to yield glycerol and phosphoric acid by boiling with hydrochloric acid.
Different types of phospholipases are categorized based on the specific linkage for which they catalyze hydrolysis in the phospholipid molecule. Sites of action of phospholipases A
1
, A
2
, C and D on phosphatidylcholine are shown below.
Via assay, phospholipase enzymatic activity may be measured.
The determination of phospholipase enzymes via assay is important in a variety of fields such as biochemical research, environmental and industrial testing, and medical diagnostics. The quantitation of enzyme levels in body fluids such as serum and plasma provides very useful information to the physician in diagnosing disease states and their treatment. In addition to being analytes of interest in biological fluids, enzymes can also serve as detection reagents in a variety of analytical systems such as immunoassays and nucleic acid hybridization techniques. In such systems, enzymes are useful directly or indirectly as indicators to monitor the extent of antigen-antibody binding or nucleic acid hydridization that occurs.
Accordingly, the desire to detect enzyme analyte and to use enzyme labels as a diagnostic tool in various analytical test systems has given rise to the development of optical indicator compounds for use in the detection and measurement of the activity of such enzymes. Typically, such known optical indicator compounds comprise a detectable chemical group, such as a fluorogen or a chromogen, which has been derivatized with an enzyme cleavable substrate group specific for the enzyme of interest. Such optical indicator. compounds exhibit an optical signal which is different from the optical signal which is provided by the cleaved native form of the fluorogen or chromogen. In principle, the enzyme cleaves the indicator compound to liberate the chromogen in the form of a distinctly fluorescent or colored product to provide a change in fluoroescence or color which is proportional to the amount of enzyme present which, in turn, can be correlated to the amount of analyte present in a liquid test sample.
Currently, there are numerous methods used to measure phospholipase enzymatic activity in an assay. Phospholipases are capable of cleaving thio ester bonds of an unnatural substrate. For example, phospholipase A
2
hydrolyzes and cleaves an sn-2 thio ester. The fact that hydrolysis releases a free thiol group has been utilized as the basis for a spectrophotometric assay shown below in Scheme A. See also, Lin Yu and Edward A. Dennis,
Methods in Enzymology,
Vol. 197, 65-75 (1991). Similarly, phospholipase A
1
hydrolyzes and cleaves an sn-1 thio ester. This sn-1 cleavage has also been utilized as the basis for a spectrophotometric assay. See, Kucera et al.,
Journal of Biological Chemistry,
Vol. 263, 1264-1269 (1988). Further, the enzymatic activity of hepatic lipase, which catalzyes the hydrolysis of phospholipids, has also been measured via assay. See Deckelbaum R. J., et al.,
Biochemistry
, 31, 8544-8551, 8545 (1992).
Phospholipase A
2
cleaves the sn-2 oxy ester of phospholipids; it will also hydrolyze an sn-2 thio ester. As shown in Scheme B below, the liberated thiol is allowed to react with a thiol-sensitive reagent, and the formation is measured continuously by monitoring the increase in absorption associated with its production.
DTNB is used to detect the free thiol group. This reagent is commercially available from Aldrich (Milwaukee, Wis.). DTNB is preferable because it is sufficiently soluble in buffer such that stock solutions are aqueous.
The ability of phospholipases to cleave the thio ester bonds of unnatural substrates has also been utilized to develop continuous spectrophotometric assays for phospholipase A
2
(PLA2), phospholipase A1 (PLA1), phospholipase C (PLC), lysophospholipase and lipase. The detection methods available for phospholipase assays include titrametric, acidimetric, radiometric, nuclear magnetic resonance and others, including the thio assay.
The thio assay possesses many characteristics that recommend it as a general assay for phospholipases. The most important are that it is a continuous, spectrophotometric assay which is very convenient, it directly detects one of the products liberated upon hydrolysis, it is one of the more sensitive assays, and it is also suitable for detailed kinetic studies. The thio assay can be used for phospholipases A
1
and A
2
and with appropriate modification of the substrate would be applicable to other phospholipases. However, owing to the lack of commercial availability of thiophospholipid substrates and their complicated synthesis, the thio assay has not been used extensively.
Although commonly employed in phospholipase assays, natural phospholipid substrates bring disadvantages to an assay due to their long fatty acid chains. Phospholipids with long fatty acid chains solubilize into mixed micelles in the presence of detergents commonly used in an assay. Long-chain phospholipids also f

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