Design and Chemical Synthesis of a Homogeneous Polymer-Modified Erythropoiesis Protein

Abstract

We report the design and total chemical synthesis of "synthetic erythropoiesis protein" (SEP), a 51-kilodalton protein-polymer construct consisting of a 166-amino-acid polypeptide chain and two covalently attached, branched, and monodisperse polymer moieties that are negatively charged. The ability to control the chemistry allowed us to synthesize a macromolecule of precisely defined covalent structure. SEP was homogeneous as shown by high-resolution analytical techniques, with a mass of 50,825 +/-10 daltons by electrospray mass spectrometry, and with a pI of 5.0. In cell and animal assays for erythropoiesis, SEP displayed potent biological activity and had significantly prolonged duration of action in vivo. These chemical methods are a powerful tool in the rational design of protein constructs with potential therapeutic applications.

Total chemical synthesis and electrophysiological characterization of mechanosensitive channels from Escherichia coli and Mycobacterium tuberculosis

“…indicating that these side chains are not critical to channel function. To minimize contributions from nonnative groups in the target sequence, the full-length protein was reacted with bromoacetamide (23), which selectively modifies the two introduced cysteine residues and produces side chain functional groups that are sterically and electronically similar to those of the WT residues. For the synthesis of Tb-MscL, glutamate 102 (E102) and serine 52 (S52) (22) were changed to cysteine and then masked in the final product with appropriate cysteinereactive molecules (Fig.…”

Diels–Alder Ligation of Peptides and Proteins

“…[25][26] A recent application of this ligation method includes the synthesis of a glycoprotein human hormone erythropoietin protein polymer. [27] Because hydrazones are known to hydrolyze rather easily in water, the resulting backbone-engineered peptidyl hydrazone can be reduced with sodium cyanoborohydride to produce the more stable peptidyl hydrazide. Furthermore, the hydrazone ligation concept was explored for the development of a novel bioconjugation system (HydralinK TM ) which is based on the reaction of a 2-hydrazinopyridyl moiety with a benzaldehyde moiety to yield a stable bis-aromatic hydrazone ( Figure 5C).…”

From Chemical Mutagenesis to Post‐Expression Mutagenesis: A 50 Year Odyssey

“…This approach too, has often exploited heteroatom–carbon bond formation to create thia‐Xxx analogues such as thia‐Lys,23 thia‐Arg,24 or even thia‐homo‐Glu,25 thereby allowing sometimes unique mechanistic insight (into for example, p K a or geometry) beyond that offered by the limited palette of classical mutagenesis. In some cases, the trace Cys left from native chemical ligation (NCL; see below) has been advantageously exploited,25b and even the degree of the mimicry has been investigated 26. Notably, however, careful control of reaction conditions may prove necessary to prevent unwanted over‐reaction on non‐Cys residues (e.g., His.…”