Fully Convergent Chemical Synthesis of Ester Insulin: Determination of the High Resolution X-ray Structure by Racemic Protein Crystallography

Abstract

Efficient total synthesis of insulin is important to enable the application of medicinal chemistry to the optimization of the properties of this important protein molecule. Recently we described ‘ester insulin’ – a novel form of insulin in which the function of the 35 residue C-peptide of proinsulin is replaced by a single covalent bond – as a key intermediate for the efficient total synthesis of insulin. Here we describe a fully convergent synthetic route to the ester insulin molecule from three unprotected peptide segments of approximately equal size. The synthetic ester insulin polypeptide chain folded much more rapidly than proinsulin, and at physiological pH. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin (i.e. [AspB10, LysB28, ProB29]ester insulin) were prepared by total chemical synthesis. The atomic structure of the synthetic ester insulin molecule was determined by racemic protein X-ray crystallography to a resolution of 1.6 Å. Diffraction quality crystals were readily obtained from the racemic mixture of {D-DKP ester insulin + L-DKP ester insulin}, whereas crystals were not obtained from the L-ester insulin alone even after extensive trials. Both the D-protein and L-protein enantiomers of monomeric DKP ester insulin were assayed for receptor binding and in diabetic rats, before and after conversion by saponification to the corresponding DKP insulin enantiomers. L-DKP ester insulin bound weakly to the insulin receptor, while synthetic L-DKP insulin derived from the L-DKP ester insulin intermediate was fully active in binding to the insulin receptor. The D- and L-DKP ester insulins and D-DKP insulin were inactive in lowering blood glucose in diabetic rats, while synthetic L-DKP insulin was fully active in this biological assay. The structural basis of the lack of biological activity of ester insulin is discussed.

High-resolution structures of a heterochiral coiled coil

“…The recently reported structure of racemic ester insulin exhibits such a heterochiral β-sheet-like interaction between L-and D strands (64), whereas the racemic structures reported here exemplify this inversion relationship between mirror-image α-helices.…”

An oxazetidine amino acid for chemical protein synthesis by rapid, serine-forming ligations

“…The first KAHA ligation between segment 2 α-ketoacid (17) bearing the Fmoc-protected Ozt and segment 3 5-oxaproline 18 furnished the ligated ester, which was kept in this form to improve handling and solubility 24,25 . Fmoc-deprotection revealed N-terminal oxazetidine 20, which was ligated with two equivalents of segment 1 α-ketoacid 21.…”

Racemic and Quasi‐Racemic X‐ray Structures of Cyclic Disulfide‐Rich Peptide Drug Scaffolds

“…A robust method that can be used to facilitate the elucidation of high quality atomic resolution structures would therefore be a valuable tool. Herein we explored the emerging approach of racemic crystallography, which has been used for overcoming the crystallization 'bottleneck' for a range of proteins [17][18][19][20][21][22] but has not yet been applied to cyclic disulfide-rich peptides. Using this approach, we obtained high-resolution structures of a range of cyclic disulfide-rich peptides of varying disulfide bond and amino acid content.…”

“…Data collection and refinement statistics are given in Supplementary As shown in Figure 2a, the X-ray structure of the SFTI-1 true racemate was solved in the centrosymmetric space group P-3. This was initially surprising because theoretical calculations predict that P-1 is the most likely space group for obtaining racemate structures [24][25] and, also, most current examples of protein/peptide racemate structures have been solved in space group P-1, [20][21][26][27][28][29][30][31] with P1 [17,32] , P21/c [33] and I41/a [34] being the other reported examples. The asymmetric unit cell contains a single molecule of the D-form of SFTI-1 (Figure 2b), which adopts a compact tertiary fold that is identical to its inverted L-form.…”