MONITORING OF A SOLID-PHASE PEPTIDE-SYNTHESIS BY NIR-FT-RAMAN SPECTROSCOPY

Solid phase peptide synthesis (SPPS) of H-(Ala)(n)Lys-OH with n up to 10 is performed on a TentaGel(R) resin. The linker is (p-(hydroxymethy l)phenoxy) acetic acid (HMPA) and 9-fluorenylmethyloxycarbonyl (Fmoc) is used as the N-alpha-protecting group. For each step in the synthesi s near-infrared-(NIR)-Fourier transform-(FT)-Raman spectra with an exc itation wavelength at 1064 nm are obtained of both the protected and d eprotected resin bound peptide. No fluorescence background was observe d. Amide-I and amide-III bands are used to obtain information about th e secondary structure of the growing peptide chain, Bands are observed , which can be attributed to the occurence of beta-sheet structures. T he efficiency of the standard deprotection procedure, which consists o f a treatment with 20% piperidine in dimethylformamide (DMF), v/v, for 10 min depends upon the secondary structure. With six to ten residues attached, standard treatment with piperidine only results in a partia l removal of the Fmoc-group, in accordance with the occurrence of beta -sheet structures. An important conclusion of the present investigatio n is that resins even like TentaGel(R) containing monosubstituted phen yl rings can be used in NIR-FT-Raman studies of SPPS. The Raman spectr oscopic studies were corroborated by HPLC and mass spectrometry analys is.