Convergent Chemical Synthesis of [Lysine^{24, 38, 83}] Human Erythropoietin

Abstract

Ein neuer Weg zu EPO: Mit einer konvergenten chemischen Ligationsstrategie wurde nichtglycosyliertes menschliches Erythropoetin (EPO) hergestellt. Das synthetische [Lysin24, 38, 83]‐EPO‐Analogon, das über HPLC gereinigt wurde und das korrekte Molekulargewicht hat (siehe Bild), hat eine wohldefinierte kovalente Struktur, ist korrekt gefaltet und zeigt biologische Aktivität in einem Assay mit einer faktorabhängigen Zell‐Linie.

Chemical Synthesis of an Erythropoietin Glycoform Containing a Complex‐type Disialyloligosaccharide

“…Chemical syntheses of EPO derivatives have been performed by several groups23, 24, 27, 28 and we also employed the convergent synthetic strategy shown in Figure 5 b. The native EPO sequence contains only four Cys residues (Figure 5 a), but this number and the positions are not sufficient for the use of NCL for the synthesis of the full‐length EPO polypeptide chain.…”

Design, Total Chemical Synthesis, and X‐Ray Structure of a Protein Having a Novel Linear‐Loop Polypeptide Chain Topology

“…[22] Kinetically controlled ligation chemistry allows more flexible, fully convergent synthetic strategies and has already proved to be useful for the total chemical synthesis of a variety of other challenging proteins in our laboratory. [27,35,36] The high-resolution X-ray structure determination reported here for the novel synthetic topological analogue protein is a vivid demonstration of the utility of the quasiracemate crystallization technique. In our hands, quasiracemic protein crystallization is proving to be as effective as racemic crystallization for structure determination of recalcitrant proteins.…”

Towards the Simplification of Protein Synthesis: Iterative Solid‐Supported Ligations with Concomitant Purifications

“…To expand the range of targets accessible by chemical synthesis to larger and morecomplex proteins, sophisticated, iterative or convergent synthetic strategies are required. [7] Thus, ligations of more than three segments, and numerous nontrivial purification steps, would be required; the handling of the ligation product intermediates is often tricky, owing to aggregation or limited solubility.Peptide segments are typically obtained through solidphase peptide synthesis (SPPS), which is generally limited to the synthesis of peptides containing up to 40-50 amino acids. [8] Decades of continuous efforts in optimizing coupling reagents and protective groups have eliminated most prominent side reactions, but incomplete amide couplings are still a significant limitation in Fmoc-based SPPS.…”

“…To expand the range of targets accessible by chemical synthesis to larger and morecomplex proteins, sophisticated, iterative or convergent synthetic strategies are required. [7] Thus, ligations of more than three segments, and numerous nontrivial purification steps, would be required; the handling of the ligation product intermediates is often tricky, owing to aggregation or limited solubility.…”