Dipeptide synthesis in biphasic medium: evaluating the use of commercial porcine pancreatic lipase preparations and the involvement of contaminant proteases

Abstract

Sínteses bem sucedidas de dipeptídeos a partir de Ac-L-Tyr-OEt ou Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys ou Thr) e glicina amidada em meio reacional bifásico foram realizadas usando dois tipos de preparações comerciais de lipase pancreática suína (PPL) (bruta (cPPL) e purificada (pPPL)). Nas mesmas condições reacionais, a α-quimotripsina, protease pancreática que também apresenta atividade esterásica, catalisou a síntese de Ac-L-Tyr-Gly-NH 2 com alta produtividade. Na maioria das sínteses também ocorreu a hidrólise do produto formado. Eletroforese em gel de poliacrilamida, ensaios enzimáticos com substratos cromogênicos específicos e cromatografia de exclusão molecular demonstraram que cPPL e pPPL apresentam proteases contaminantes e, portanto, atividades esterásica e amidásica. Em conjunto, esses resultados indicam que tais proteases, e não a PPL, possam ser os catalisadores majoritários da síntese da ligação peptídica quando ésteres de N α -acil-L-aminoácidos e preparações comerciais de PPL são usados. Por outro lado, tais dados não contradizem a possibilidade de usar cPPL como fonte barata de catalisadores para a síntese de dipeptídeos em condições reacionais brandas.Dipeptide syntheses starting from Ac-L-Tyr-OEt or Z-L-X-OMe (X: Asp, Tyr, Phe, Arg, Lys or Thr) and glycine amide in biphasic reaction media were achieved using two commercially available porcine pancreatic lipase (PPL) preparations (crude (cPPL) and purified PPL (pPPL)). Under the mild conditions employed, α-chymotrypsin, a pancreatic protease that also presents esterase activity, catalyzed Ac-L-Tyr-Gly-NH 2 synthesis with high productivity. Product hydrolysis also occurred in most of the syntheses studied. Polyacrylamide gel electrophoresis, enzymatic assays employing specific chromogenic substrates and size-exclusion chromatography revealed that cPPL and pPPL contain contaminant proteases and, therefore, exhibit esterase and amidase activities. Overall, these data indicate that those contaminants may be the main catalysts of peptide bond synthesis when N α -blocked-L-amino acid esters and the commercial PPL preparations are used. On the other hand, such data do not contest the possibility of using such enzyme preparations as an inexpensive source of catalysts for dipeptide synthesis under soft conditions. Keywords: biocatalysis, enzymes, peptide bond synthesis, n-hexane IntroductionIn the last decade, interest in environment friendly technologies has increased drastically. As a result, the ability of enzymes to catalyze reactions required for the manufacture of fine chemicals has been explored extensively. 1 Proteases that also present esterase activity have been widely used to catalyze reactions related to peptide synthesis, such as ester hydrolysis, esterification, transesterification and peptide bond formation. 2,3 However, it is well known that the main concern in protease-catalyzed peptide bond synthesis is protease-catalyzed product consumption. [4][5][6] Therefore, the following approaches have been used to minimize such an undesirable r...

Candida tropicalis CE017: a new Brazilian enzymatic source for the bioreduction of aromatic prochiral ketones

“…Bioreduction reactions were performed during 9 days at 28 ºC in potato dextrose broth using 6,8,10,12,14,15,20, 30 and 40 μL of acetophenone 1a to obtain (S)-1-phenylethanol in high to excellent enantiomeric excess (Table 5).…”

“…7 The use of microorganism new strains as biocatalysts may offer an alternative opportunity to investigate the local resources for the effective conduct of key synthetic transformations with significant economic and ecological implications. 8 Our research interest is based on the development of chemoenzymatic methodologies to obtain chiral alcohols using Brazilian local sources of low cost. Herein, we report our current investigations in the study of the Candida tropicalis CE017 strain behavior as a novel stereoselective reducing agent of aromatic prochiral ketones to the corresponding chiral alcohols.…”

Difficult Peptides

“…Since acetylated Hmb (AcHmb) as the amide backbone protecting group led to a substantial improvement in the solubility of the synthetic bA4 amyloid fragment (34)(35)(36)(37)(38)(39)(40)(41)(42) [125], they concluded that the 2-hydroxy moiety was Hmbs essential feature and proposed the 2-hydroxybenzyl (Hbz) group, TFA-stable and trifluoromethanesulfonic acid (TFMSA) labile, for SPPS by the Boc/Bzl strategy. Since acetylated Hmb (AcHmb) as the amide backbone protecting group led to a substantial improvement in the solubility of the synthetic bA4 amyloid fragment (34)(35)(36)(37)(38)(39)(40)(41)(42) [125], they concluded that the 2-hydroxy moiety was Hmbs essential feature and proposed the 2-hydroxybenzyl (Hbz) group, TFA-stable and trifluoromethanesulfonic acid (TFMSA) labile, for SPPS by the Boc/Bzl strategy.…”

A green route for the synthesis of a bitter-taste dipeptide combining biocatalysis, heterogeneous metal catalysis and magnetic nanoparticles

“…[21][22][23][24] Nevertheless, since then, its main limitation has become apparent: it is mostly applicable to the production of peptides containing up to 5 amino-acid residues. 16,25,26 In addition, it is well known that the technology has disadvantages that make it impractical for reuse and recovery from reaction media, including use of puried proteases and expensive autolytic enzymes with short half-lives in solution. To overcome these limitations and reduce costs, many researchers have immobilized the proteases using a large variety of solid supports, [27][28][29][30] including nanoparticles.…”