Fully Convergent Chemical
Synthesis of Ester Insulin:
Determination of the High Resolution X‑ray Structure by Racemic
Protein Crystallography
- Publication date
- Publisher
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 bondas 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., [Asp<sup>B10</sup>, Lys<sup>B28</sup>, Pro<sup>B29</sup>]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