THE MERRIFIELD PEPTIDE-SYNTHESIS STUDIED BY NEAR-INFRARED FOURIER-TRANSFORM RAMAN-SPECTROSCOPY

The stepwise solid-phase synthesis of the peptide H-(Ala)6-Lys-OH on a polyacrylamide gel resin was followed by near-infrared (NIR) Fourier- transform (FT) Raman spectroscopy. In particular, this investigation i nvolved the use of Fmoc as the N-alpha-protecting group. The deprotect ion of the Fmoc group by standard methods could be quantitatively foll owed. The deprotection was essentially complete until a number of alan ine residues in multiples of six were reached, with nearly one-third o f the peptide chains left protected after the standard piperidine trea tment. Even a prolonged deprotection time did not result in a complete deprotection of the Fmoc group. This phenomenon could be attributed t o the formation of secondary structures, which were indicated by struc turally sensitive Raman bands, with particular focus on the amide III bands. The Fmoc group was found to have a clear influence upon the sec ondary structure, supporting mainly a beta-sheet conformation, whereas more coiled forms were found for the deprotected samples. Preliminary studies with the use of Boc as the N-alpha-protecting group showed th at this group had essentially no importance for the secondary structur e of the pendent peptide chains. Investigation of peptides containing both D- and L-chiral forms of alanine supported the hypothesis that th e presence of the Fmoc group influences the secondary structure. The s welled forms of the sequence Fmoc-(Ala)6-Lys(Boc)-OR in DMSO or DMF sh owed different secondary structures, indicating different interactions between the peptide chains and the two solvents. Our studies show tha t Raman spectroscopy is a nondestructive analytical tool which allows a recording of spectra while the peptide is directly bound to the soli d support under normal synthetic conditions.