SOLID-PHASE SYNTHESIS OF PEPTIDE-4-NITROANILIDES

A wide variety of Glu/Asp and Gln containing peptide-4-nitroanilides a nd other chromogenic peptidyl-arylamides could be quickly synthesized by a Fmoc-based solid-phase synthesis strategy employing the side-chai n carboxyl groups for transient anchoring to the resin. Suitable synth ons for this method. Fmoc-Glu-NH-Np and Fmoc-Asp-NH-Np, were prepared using a diphenylphosphinic chloride-mediated coupling reaction. Peptid es of the common structure Suc-Ala-Phe-Pro-Xaa-NH-Np (Xaa=Glu/Asp, Gln ) were synthesized and were shown to be substrates for the protease su btilisin Carlsberg (E.C.3.4.21.14a) and for peptidyl-prolyl cis/trans- isomerases (PPIases E.C. 5.2.1.8). The method was extended to amino ac ids possessing a side chain missing an anchor for binding to the matri x. We synthesized Suc-Ala-Phe-Pro-Gln-Phe-NH-Np anchoring the dipeptid e derivative Fmoc-Glu-Phe-NH-NP with the carboxyl group to Rink amide resin using staadard SPPS procedures. Additionally this procedure allo wed us the preparation of peptidyl-arylamides, utilizing the commercia l available Fmoc-Glu-OAll as building block. A mixture of pentapeptide -4-nitroanilides with the general sequence Ala-Ala-Xaa-Pro-Gln-NH-Np w as synthesized. Electrospray ionization mass spectrometry (ESI-MS) was used to evaluate the hydrolysis of the peptide mixture by the proteas e subtilisin Carlsberg. It could be shown that peptides with the hydro phobic amino acids Phe, Tyr, Leu and Val in the varied P3-position wer e most rapidly cleaved under the chosen conditions. Hydrolysis of the Gln-NH-Np bond in Ala-Ala-Pro-Pro-Gln-NH-Np has not been observed. (C) Munksgaard 1996.