Fmoc-(Fmoc-Hmb)-Val-OH

During the Fmoc-protection of H-alpha-Me-Val-OH, an unknown side product was found and isolated. The characterization using various analytical methods led unambiguously to the result that Fmoc-beta-Ala-OH was formed during the reaction. The reagent Fmoc-OSu was proven to be the source of Fmoc-beta-Ala-OH, following a mechanism that involved many deprotonation and elimination steps and a Lossen-type rearrangement as key sequence. The impurity Fmoc-beta-Ala-OH was found in a variety of reactions in which Fmoc-OSu was applied, either in the reaction mixture or as a contamination of the crude product. Purification of the Fmoc-amino acid derivatives from this impurity incurred high costs and significant reductions in yield.

A new method for solid-phase peptide synthesis in which a protein is used as the solid support has been developed. Two aspects of the method have been demonstrated. The peptides H-Phe-Leu-Glu-Glu-Val-OH (1) and H-Leu-Leu-Ala-Gly-Val-OH (2), respectively, were synthesized on the amino groups of BSA via a cleavable linker, using the Fmoc group protecting scheme. The free peptides were obtained by cleavage from the protein with 95% TFA, precipitation in diethyl ether and additional work-up by either dialysis or centrifugation through a membrane followed by gel filtration. The identity of the products was determined by amino acid analysis and HPLC. The peptide-protein conjugates, H-Ser-Met-Asp-Thr-Ser-Asn-Lys-Glu-Glu-Lys-BSA (3) and H-Thr-Val-Leu-BTG (4), were obtained in the same manner, omitting the cleavable linker group. It was found that 35-50 peptide chains were conjugated per molecule BSA and BTG, respectively. Immunization of rabbits with conjugate 3 gave rise to peptide specific antibodies. This method will be useful for generation of sequence specific antibodies, since the peptide is conjugated to the carrier protein exclusively via its C-terminus, and will allow synthesis of highly specific peptide-protein conjugates.

The N(alpha)-Fmoc-peptide isocyanates 3a-q, 4a-c, and 5a-c were prepared by the Curtius rearrangement of N(alpha)-Fmoc-peptide acid azides in toluene under thermal, microwave, and ultrasonic conditions. All the N(alpha)-Fmoc-oligo-peptide isocyanates made were isolated as stable crystalline solids with 71 to 94% yield and were fully characterized by 1H NMR, 13C NMR, and mass spectroscopy. Their utility for the synthesis of oligo-alpha-peptidyl ureas 7a-f and 8a-c by the divergent coupling approach was demonstrated. The coupling of N(alpha)-Fmoc-dipeptide isocyanates with amino acid ester or with N,O-bis(trimethylsilyl)amino acids resulted in N(alpha)-Fmoc-tripeptidyl urea ester and acids containing one each of peptide bond and urea bond. The divergent approach is extended to the synthesis of tetrapeptidyl ureas by the 2 + 2 strategy using bis-TMS-peptide acid as an amino component. To incorporate urea bonds in adjacent positions, N(alpha)-Fmoc-peptidyl urea isocyanates 9a-d were prepared and employed in the synthesis of three tetrapeptidyl ureas 10a-b and 11 containing one peptide bond and two urea bonds in series from the N-terminal end. The protocol was then employed for the synthesis of five urea analogues 13-15, 18, and 21 of [Leu5]enkephalin containing urea bonds at the 2, 3, 4 positions as well as at the 2, 4 and 2, 3, 4 positions. The analogue 2l was made by the convergent synthesis by the N --> C terminal chain extension. Finally, two urea analogues 22 and 23 of repeat units of bioelasto polymers, namely Val-Pro-Gly-Val-Gly-OH and Pro-Gly-Val-Gly-Val-OH, were synthesized incorporating the urea bond by the concomitant isocyanate generation and urea bond formation under thermal conditions.