Tetrapeptide-4

The ubiquitous cyclophilins belong to peptidyl-prolyl cis/trans isomerases (PPIases; EC 5.2.1.8). They are able to catalyze the cis/trans isomerization about peptidyl-prolyl amide bonds. The mode of action of human cytosolic cyclophilin (Cyp18cy) has been studied on substrate analog tetrapeptide-4-nitroanilides containing the thioxo peptidyl-prolyl bond. Five peptides of the general structure Ala-Xaa-psi [CS-N]-Pro-Phe-NH-Np (Xaa = Gly, Ala, (S)-2-aminobutyric acid, Phe, and Leu) containing the thioxo peptidyl-prolyl bond were synthesized. The kcat values for the chymotryptic cleavage of 4-nitroanilide bond of the thioxo tetrapeptide-4-nitroanilides ranged from 1.7 to 9.0 s-1 and were sufficiently high to analyze the conformational equilibria by isomer-specific proteolysis. The rate constants of the cis/trans isomerization of the thioxo peptidyl-prolyl bond were found to be 25-100-fold lower due to the O/S substitution. Cyp18cy binds both thioxo peptides and oxo peptides in similar manner in the active center but cannot utilize the sulfur analogs as substrates. Instead, competitive inhibition occurs, which was further characterized for Ala-Gly-psi[CS-N]-Pro-Phe-NH-Np. The inhibition was nearly independent of the pH value in the range of pH 4.5-9, exhibiting apparent Ki values ranging from 200 to 600 microM. In comparison to Ala-Gly-trans-psi[CS-N]-Pro-Phe-NH-Np, the cis thioxo peptide Ala-Gly-cis-psi[CS-N]-Pro-Phe-NH-Np was found to possess an approximately 30-fold higher affinity for the active site of the enzyme. Thus, in the presence of stoichiometric amounts of Cyp18cy, the total amount of Ala-Leu-cis-psi[CS-N]-Pro-Phe-NH-Np in solution, detectable by isomer-specific proteolysis, was considerably enhanced.

The solid-phase synthesis and an ADME investigation with albino and pigmented male rats of the doubly 14C-labelled beta/alpha-tetrapeptide derivative Ac-beta3 hTyr-(D)Trp-beta3 hLys-beta3 hThr-lactone (3; Fig. 3) are described. After intravenous (i.v.) and peroral (p.o.) administration of the peptide, its concentration in blood and plasma, its tissue distribution, and the metabolism and the excretion of the peptide were analyzed over a period of up to 7 days post dose. The tetrapeptide in its ring opened form, 5, has a bioavailability of ca. 25%; radioactivity is distributed in the animals in an organ-specific way, and the compound appears to pass the blood-brain barrier to a very small extent, if at all (Tables 1-3 and Figs. 2-6). Excretion (37% renal, 44% fecal, including biliary) of the tetrapeptide 4 days after i.v. administration is almost complete, with only 4.3% remaining in the carcass; 4 days after p.o. administration 97% of the dose has been excreted in the feces. Radiochromatograms taken of plasma (0.5 and 24 h after i.v. dosing) and of urine and feces extracts (0-48 h collected) reveal the presence of lactone 3 and/or the corresponding hydroxy acid 5 with essentially no or very minor other peaks, respectively, representing possible metabolites (Tables 4-6, and Fig. 7 and 8). A comparison with a previous ADME investigation of a beta-nonapeptide show that--except for the lack of metabolism--all aspects of exposure, distribution, and elimination are different (structure-specific properties). The investigated tetrapeptide 3 is a potent and highly specific agonist of the somatostatin receptor hsst4, rendering the results described herein promising for diagnostic and therapeutic applications of beta-peptides.

Peptide-4-nitroanilides can be quickly synthesised using an Fmoc-based approach on 2-chlorotritylchloride resin. Preformed building blocks Fmoc-Xaa-NH-Np (Xaa = Cit, Cys, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp) can be attached via side chain to the 2-chlorotritylchloride linker of the resin. N-terminal elongation yields the respective peptide-4-nitroanilides after detachment from the solid support. We synthesised a set of tetrapeptide-4-nitroanilides with the general structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa = Asp, Cit, Cys, Glu, Gln, His, Lys, Orn, Ser, Thr, Tyr, Trp). Even peptidyl-arginine-4-nitroanilides are available by a slightly modified procedure. First, the appropriate ornithine-containing peptide was synthesised. After detachment of the peptide from the resin the side-chain primary amino group was transformed to the guanidino function of arginine using 1-guanyl-3,5-dimethylpyrazole. A further application of this method is the convenient synthesis of phosphorylated peptide-4-nitroanilides. Five phosphopeptides with the general structure Ac-Ala-Xaa(PO3H2)-Pro-Yaa-NH-Np (Xaa = Ser, Thr, Tyr; Yaa = Tyr, Lys) and their nonphosphorylated analogues were prepared. Global phosphorylation was carried out on the resin-bound peptides using dibenzyl-N, N-diisopropyl-phosphoramidite/tetrazole followed by oxidation with tert-butyl hydroperoxide.