Chemoenzymatic Synthesis of Poly(l-alanine) in Aqueous Environment

Abstract

L-alanine ethyl ester was polymerized into poly(L-alanine) (polyAla), one of the insoluble polypeptides, by papain in aqueous buffer at varying pH. At neutral pH, a maximum chain length of 11 repeats was observed. These polymers were dominated by random coiled structure and demonstrated a lack of patterned macromolecular assembly. Under alkaline conditions, longer polymer chain lengths were achieved, and the maximum chain length was 16 repeats. These longer chains showed distinct β-sheet formation and were capable of fibril assembly. The present study reports on chemoenzymatic synthesis of a hydrophobic homopolypeptide under aqueous conditions as well as demonstrates a chain length dependency of secondary structure formation and macromolecular assembly of chemoenzymatically synthesized polyAla, providing a new insight into material design of polypeptide.

Chemoenzymatic Synthesis of Polypeptides for Use as Functional and Structural Materials

“…In contrast, CPDY is characterized as an exopeptidase that typically cleaves only amide bonds at C-termini. Therefore, CPDY-catalyzed polymerization avoids the hydrolysis of the polypeptide backbones of pre-existing polypeptides, resulting in efficient suppression of the broadening of the molecular weight distribution of the products that Methyl, [49,54] ethyl, [50][51][52][53][54][55][56] benzyl [54] Proteinase I, [49] papain (13)(14)(15)(16)(17)(18) [50][51][52][53][54][55][56] Glycine Methyl, [54] ethyl, [49,51,54,55] benzyl [54] Proteinase I, [49] papain (10)(11)(12)(13)(14)(15)(16)(17)(18)(19) [51,54,55] l-Leucine…”

“…PolyAla was synthesized by the papain-catalyzed polymerization of l-alanine ethyl ester. [50] Papain was selected because it has relatively broad specificity, especially for hydrophobic substrates. [103] The polymerization of l-alanine ethyl ester in two buffer solutions with different pH values (i.e., 1 m sodium phosphate buffer [pH 7.0] and 1 m sodium carbonate buffer [pH 11.0]) was examined.…”

Papain-Catalyzed Synthesis of Polyglutamate Containing a Nylon Monomer Unit

“…Methyl and ethyl esters of monomeric substrates were used in this study because of their relatively higher reactivities in comparison to those of the free amino acids and nylon units [15,28]. Because the pK a of substrate monomer is the main determinant of the reaction efficiency in the chemoenzymatic polymerizatrion, yield and degree of polymerization are largely dependent on the pH of the reaction medium [14].…”

“…The aminolysis can proceed kinetically when excess substrate amino acids are present around the enzyme. Papain, a cysteine protease, shows broad substrate specificity and exhibits endopeptidase, amidase, and esterase activities, which have been widely used in chemoenzymatic synthesis [15][16][17][18][19][20][21]. Beer et al have provided the first demonstration that papain can recognize the nylon 3 monomer β-alanine as a substrate in a dipeptide synthesis [16].…”

Synthesis of peptides with narrow molecular weight distributions via exopeptidase-catalyzed aminolysis of hydrophobic amino-acid alkyl esters

“…14,15 Although these methods have both drawbacks and beneficial aspects, the protease-catalyzed chemoenzymatic synthesis of peptides is an alternative to these conventional methods. 10,[16][17][18][19][20] Importantly, this method offers advantages over other processes because it requires limited or no side-chain protection of amino acids. 21 In addition, this alternative uses mild conditions and retains stereospecificity.…”