Development of a fully automated multichannel peptide synthesizer with integrated TFA cleavage capability.

A fully automated multichannel peptide synthesizer has been constructed which performs simultaneous and rapid assembly of peptides on a 20-200 mumol scale. In situ activation of amino acids using BOP or PyBOP was chosen to give an optimized coupling chemistry. Specially designed blocks of valves, with a zero dead volume combined with an original circuitry, permit the distribution of amino acids derivatives and reagents pre-dissolved in DMF. Either Boc or Fmoc chemistry can be adapted on the synthesizer. In Boc synthesis a very rapid protocol involving Boc group deprotection in neat TFA, followed by the concomitant steps of neutralization and coupling, allows the addition of three amino acids per hour on each channel. In Fmoc chemistry we have integrated into the synthesizer an automatic TFA cleavage system that allows the peptides to be cleaved from the resin directly within the reactors used for synthesis. The stability of the Fmoc amino acid derivatives in solution in DMF was investigated, and decomposition was found to be insignificant during the time-span of a synthesis. The satisfactory performance of the instrument was demonstrated by routine synthesis of 15-20 mer peptides.

Multiple autoepitope presentation for specific detection of antibodies in primary biliary cirrhosis.

Antimitochondrial autoantibodies are present in sera from close to 95% of patients with primary biliary cirrhosis. The so-called primary biliary cirrhosis-specific antigen, named M2, was found to be associated with an enzyme complex of the inner mitochondrial membrane and, more precisely, with the E2 component, dihydrolipoamide acetyltransferase, of the pyruvate dehydrogenase complex. We recently established that an immunodominant epitope recognized in direct enzyme-linked immunosorbent assay by primary biliary cirrhosis M2+ sera, but not by non-primary biliary cirrhosis M2+ sera, could be mimicked by a synthetic peptide encompassing residues 167-184 of the E2 component and associated with lipoic acid. This fragment is present in the natural inner lipoyl-binding site of the human enzyme, and the presence of lipoic acid located on lysine 173 was found to be essential to allow IgG antibody binding. In this study we have improved the enzyme-linked immunosorbent assay test based on the synthetic peptide-lipoic acid conjugate by using a multiple antigen peptide system containing eight copies of the peptide as antigen. This approach avoids the use of a peptide conjugated to a carrier protein and was found to be particularly efficient because 23 of 27 primary biliary cirrhosis M2+ sera (85%) could be identified. A multiple antigen peptide without lipoic acid was not recognized by primary biliary cirrhosis antibodies. The peptide used in the multiple antigen peptide construction was a short 13-mer peptide encompassing a highly conserved sequence present in both the outer (residues 40-52) and the inner (residues 167-179) lipoyl-binding sites of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Incomplete TFA deprotection of N-terminal trityl-asparagine residue in fmoc solid-phase peptide chemistry.

We demonstrate that TFA deprotection of trityl-protected N-terminal asparagine is incomplete under normal conditions, resulting in low yields or impure products. This phenomenon does not occur if the asparagine is internal, nor for trityl-protected N-terminal glutamine. Studies on the deprotection of H Asn(Trt)OH show that the incomplete deprotection is due to the extremely slow removal of a trityl group close to an amino group. The use of the new methyl-trityl protecting group overcomes this problem resulting in rapid and complete deprotection.

A home-made solid-phase sequencer operating at the nanomole level.

The building and functioning of a fully automated solid-phase sequencer is described. The peptide is coupled via its alpha-carboxyl end to activated glass beads and successively reacted with Chang's and Edman's reagents. All operations are electronically controlled by the automated programmer. All components necessary to build the machine are commercially available. This sequencer has been used at a nanomole level in the final phase of a protein sequence determination. The overall cost as well as the sensitivity and efficiency of the final product compare favourably to those of commercial machines.