Synthesis of Epidermal Growth Factor by Native Chemical Ligation and Incorporation of Non-Natural Amino Acids
The objective of this study was to develop a method for the efficient and rapid generation of analogs of hEGF and to incorporate unnatural amino acids into EGF and its fragments. Since the analogs share sequences, for optimal versatility and economy, it was decided that hEGF would be assembled from fragments which could be used as building blocks in the synthesis of several analogs. Coupling of amino acids to the conformationally constrained residues Tic and HOTic, is difficult. In model experiments, use of HOAt in combination with either DIC or F1ATU for activation was effective in solving these coupling difficulties. HOTic was then incorporated into the 20-31 fragment of hEGF. [Abu'ui,HOTic ;]hEGF(20-31 )-NH- was shown to be a 'difficult sequence', but replacement of Tyr at position 29 with HOTic facilitates the complete dodecapeptide synthesis. Chemoselective ligation techniques allow the use of unprotected peptide fragments which can eliminate solubility problems associated with fragment condensation. Native chemical ligation results in a peptide bond thus, joining the fragments. Therefore, it was selected as the method for the synthesis of hEGF. To compromise between yield and flexibility, it was decided that the hEGF would be assembled from three fragments. During the synthesis of the thioester fragments, [Cys (Aem)6]hEGF(l-13)-MPAL and [Cys(Acm)20]hEGF(14-30)-MPAL, significant aspartimide formation occurred. To suppress this side reaction, several methods were examined. 'Low-high' TFMSA cleavage, deprotection with 'low' TFMSA cleavage conditions followed by thiolyzis and use of 2-Ada or i-Bu protection on Asp did not solve the problem. cHx protection on Asp, in combination with HF cleavage, supressed the side reaction, but in the case of Msc-[Cys(Acm)20]hEGF(14-30)-MPAL, further variation of HF cleavage conditions is necessary to eliminate other side reactions. Fmoc-Cys(Phacm)-OH was synthesized and side products were identified. The synthesis of [Cys(Phacm)3342]hEGF(3 l-53)-OH required double couplings. The pure peptide decomposed during storage. The decomposed product originated from the reaction of the ^-terminal Cys and formaldehyde. The resulting thiaproline was opened up with O-methyl-hydroxylamine. Ligations proceded in a rather low yield, possibly due to solubility problems. The overall yield of the synthesis of [Cys(Acm)6’20,Cys(Phacm)33 42]hEGF was 0.4%. Addition of ß-dodecyl-D-maltoside to the ligation mixture could increase the overall yield 4-5 fold. [Cys(Acm)6-20,Cys(Phacm)33 4:’]hEGF was not soluble in conditions required to remove Phacm from Cys. To improve yield and obtain correctly folded hEGF analogs, changes in the protection scheme and/or the synthesis of the peptide with optimized step by step solid phase methodology is considered.
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