Efficient Total Chemical Synthesis of ¹³C=¹⁸O Isotopomers of Human Insulin for Isotope‐Edited FTIR

Abstract

Isotope-edited two-dimensional Fourier transform infrared spectroscopy (2D FTIR) can potentially provide a unique probe of protein structure and dynamics. However, general methods for the site-specific incorporation of stable 13C=18O labels into the polypeptide backbone of the protein molecule have not yet been established. Here we describe, as a prototype for the incorporation of specific arrays of isotope labels, the total chemical synthesis – via a key ester insulin intermediate – of 97% enriched [(1-13C=18O)PheB24]human insulin, stable-isotope labeled at a single backbone amide carbonyl. The amino acid sequence as well as the positions of the disulfide bonds and the correctly folded structure were unambiguously confirmed by the X-ray crystal structure of the synthetic protein molecule. In vitro assays of the isotope labeled [(1-13C=18O)PheB24]human insulin showed that it had full insulin receptor binding activity. Linear and 2D IR spectra revealed a distinct red-shifted amide I carbonyl band peak at 1595 cm−1 resulting from the (1-13C=18O)PheB24 backbone label. This work illustrates the utility of chemical synthesis to enable the application of advanced physical methods for the elucidation of the molecular basis of protein function.

Scope and Limitations of Fmoc Chemistry SPPS‐Based Approaches to the Total Synthesis of Insulin Lispro via Ester Insulin

“…Because of the reported instability of the Thz-moiety to oxidation at reduced pH,[13] which we confirmed in a model study (see SI), we were unable to use our previously published C-to-N segment condensation strategy,[14] so a revised strategy was devised that made use of N-to-C native chemical ligation segment condensation (Scheme 4). This strategy obviated the need for Thz-protection of the N-terminal Cys-residue of the middle segment, while the hydrazide at the C-terminus of that peptide served as a cryptic thioester, facilitating the N → C ligation strategy.…”

“…[14] Formation of the desired folded ester insulin lispro protein was indicated by the formation of a sharp, early eluting peak that had a mass lower by 5.5 ± 0.6 Da which corresponds to the formation of three disulfide bonds) (Figure 5). The earlier elution behavior of the folded ester insulin lispro protein compared with the unfolded polypeptide is consistent with the burying of hydrophobic side chains inside the protein tertiary structure stabilized by three disulfide bonds.…”

“…The ester insulin lispro was then saponified,[14] to give insulin lispro (Figure 6). After prep-HPLC purification, 3.39 mg insulin lispro was obtained (66% yield, 0.58 μmol from 5.1 mg, 0.88 μmol of ester insulin lispro).…”

Crystallization of Enantiomerically Pure Proteins from Quasi‐Racemic Mixtures: Structure Determination by X‐Ray Diffraction of Isotope‐Labeled Ester Insulin and Human Insulin

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To tal chemical synthesis was used to prepare stable isotope labeled [(1-13 C= 18 O)Phe B24 )] human insulin, via [(1-13 C= 18 O)Phe B24 )] ester insulin as ad efined intermediate product (see accompanying article). [1] Human insulin is a5 1-residue protein hormone that consists of two polypeptide chains held together by two interchaind isulfide bonds;t he folded protein also contains one intrachain disulfide bond ( Figure 1). [2] In ab iologicalc ontext, insulin forms hexamersinthe presence of Zn ions that coordinate the His B10 sidec hains from each insulin molecule.

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“…Human insulin was obtainedb ys aponificationo fe ster insulinu sing LiOH at 4 8C. [1] In order to confirm the formation of the correct disulfide bonds and their preservation during base-catalyzed hydrolysis of the ester bond, we set out to crystallize both isotope-labeledp roteins and to determine their high resolution structures by X-ray diffraction.We have previously reported determination of the crystal structure of DKP ester insulin prepared by total chemical synthesis. [6] DKP insulin is am onomeric form of human insulin in which His B10 has been replaced by Asp B10 ,a nd in which the native-Pro B28 -Lys B29 -sequence has been inverted.…”