Peptide substrates, method of preparation and use in the determi

Details Peptide substrates, method of preparation and use in the determi Peptide substrates, method of preparation and use in the determi

1991-10-02

1994-11-08

Lee, Lester L.

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

Tripeptides, e.g., tripeptide thyroliberin , etc.

514 18, 514365, 514369, 435 23, A61K 3700, C07K 500, C07K 700, A01N 4378

Patent

active

053628519

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

The present invention relates, by way of novel industrial products, to the peptide substrates of formula I below and their addition salts.
It further relates to the method of preparing these novel products and to their use in the field of the determination of Protein C.


PRIOR ART

It is known that a number of peptide substrates have already been proposed in the past for the identification and assay of numerous substances involved in the cascade mechanism of hemostasis.
It is known in particular that the substrate pyrrolidin-2-one-5-carbonyl) and pNA is the p-nitroanilino radical], described in EP-A-0 004 256 as a chromogenic substrate for the determination of serine proteases and SH-proteases, has recently proved effective in the determination of Protein C.
It is also known that the substrate that its efficacy is inferior to that of the aforementioned compound IIa.
There is therefore a need for specific peptide substrates for the determination of Protein C. In fact, with the exception of the afore-mentioned substrates IIa and IIb, there are no other specific substrates for Protein C which are commercially available.
To meet this need, a novel technical solution is proposed, according to the invention, which uses peptide substrates having a different structure from that of the afore-mentioned products of formulae IIa and IIb known in the prior art.


SUMMARY OF THE INVENTION

According to a first feature of the invention, peptide substrates are provided, as novel industrial products, which are selected from the group consisting of
(i) the tripeptides of the formula
Y is H or an appropriate group blocking the N-terminal end,
A.sub.1 is an amino acid residue selected from the group consisting of (2-oxothiazolidin-4-yl)carbonyl [abbreviated to THC], (2-oxotetrahydro-l,3-thiazin-5-yl)carbonyl [abbreviated to TZC], thiazolidine-4-carbonyl [abbreviated to ATC] and (tetrahydro-1,3-thiazin-5-yl)carbonyl [abbreviated to AZC] residues,
A.sub.2 is an amino acid residue selected from the group consisting of Pro, 3Hyp and 4Hyp residues, where the OH side-group of 3Hyp and 4Hyp is capable of being protected by an ether or ester protecting group,
A.sub.3 is an amino acid residue selected from the group consisting of Arg and Lys residues, and
R is a labeling means; and
(ii) their addition salts.
According to a second feature of the invention, the tripeptide compounds of formula I and their addition salts are used for the determination of Protein C. To this end, a method of determination for the assay or identification of Protein C is provided which comprises bringing a tripeptide of formula I or one of its addition salts into contact with a body fluid (especially plasma, blood serum, or urine) or any other sample of natural or synthetic origin which may contain Protein C.
Finally, according to a third feature of the invention, a method of preparing the tripeptide compounds of formula I and their addition salts is provided.


ABBREVIATIONS

For convenience, the following abbreviations have been used in the present invention:


______________________________________ the amino acid residues: Arg = arginyl ATC = thiazolidine-4-carbonyl (or thioprolyl) AZC = (tetrahydro-1,3-thiazin-5-yl)carbonyl 3Hyp = 3-hydroxypropyl (or 3-hydroxypyrrolidine-2- carbonyl) 4Hyp = 4-hydroxypropyl (or 4-hydroxypyrrolidine-2- carbonyl) Lys = lysyl Pro = prolyl Pyr = pyroglutaminyl (or pyrrolidin-2-one-5- carbonyl) THC = 2-oxothiazolidin-4-yl)carbonyl TZC = (2-oxotetrahydro-1,3-thiazin-5-yl)carbonyl the other abbreviations: Ac = acetyl AcOH = acetic acid Adoc = adamantyloxycarbonyl Aoc = t-amyloxycarbonyl Boc = t-butoxycarbonyl Bop = (benzotriazol-1-yl)oxytris(dimethylamino)- phosphonium hexafluorophosphate (alternative nomenclature: CASTRO's reagent) of the for- mula ##STR1## Bu = n-butyl Bz = benzoyl Bzl = benzyl Cbo = carbobenzoxy o-Cl-pNA = o-chloro-p-nitroanilino [or (2-Cl)pNA] DCCI = dicyclohexylcarbodiimide DIEA = diisopropylethylamine DMF = dimethylfo

REFERENCES:
patent: 4335210 (1982-06-01), Meister et al.
patent: 4508644 (1985-04-01), Heber et al.
patent: 4665016 (1987-05-01), Heber et al.

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