Engineering a Diverse Ligase Toolbox for Peptide Segment Condensation

The substrate profile of peptiligase, a stable enzyme designed for peptide ligation in aqueous environments, was mapped using six different peptide libraries. The most discriminating substrate binding pocket proved to be the first nucleophile binding subsite (S1), which is crucial for the peptide ligation yield. Two important amino acids shaping the S1 pocket are M213 and L208. A site-saturation library of the M213 position yielded two variants with a significantly broadened substrate profile, i.e., M213G and M213P. Next, examination of two libraries with M213G+L208X and M213P+L208X (with X being any proteinogenic amino acid) resulted in a toolbox of enzymes which can accommodate any proteinogenic amino acid in the S1 pocket, except proline. The applicability of a particular enzyme variant in chemoenzymatic peptide synthesis was demonstrated by coupling at the gram scale of two peptide segments to yield exenatide, a 39-mer therapeutic peptide used in the treatment of diabetes type II. The overall yield of 43% is at least 2-fold higher than yields reported for conventional syntheses of exenatide by full solid-phase peptide synthesis; large-scale production costs are expected to be significantly reduced if the enzymatic coupling process is employed to manufacture this peptide.</p

Enantio- and diastereoselective synthesis of γ-amino alcohols

The γ-amino alcohol structural motif is often encountered in drugs and natural products. We developed two complementary catalytic diastereoselective methods for the synthesis of N-PMP-protected γ-amino alcohols from the corresponding ketones. The anti-products were obtained through Ir-catalyzed asymmetric transfer hydrogenation, the syn-products via Rh-catalyzed asymmetric hydrogenation

Versatile Peptide C-Terminal Functionalization via a Computationally Engineered Peptide Amidase

The properties of synthetic peptides, including potency, stability, and bioavailability, are strongly influenced by modification of the peptide chain termini. Unfortunately, generally applicable methods for selective and mild C-terminal peptide functionalization are lacking. In this work, we explored the peptide amidase from <i>Stenotrophomonas maltophilia</i> as a versatile catalyst for diverse carboxy-terminal peptide modification reactions. Because the scope of application of the enzyme is hampered by its mediocre stability, we used computational protein engineering supported by energy calculations and molecular dynamics simulations to discover a number of stabilizing mutations. Twelve mutations were combined to yield a highly thermostable (Δ<i><i>T</i></i>_m = 23 °C) and solvent-compatible enzyme. Protein crystallography and molecular dynamics simulations revealed the biophysical effects of mutations contributing to the enhanced robustness. The resulting enzyme catalyzed the selective C-terminal modification of synthetic peptides with small nucleophiles such as ammonia, methylamine, and hydroxylamine in various organic (co)­solvents. The use of a nonaqueous environment allowed modification of peptide free acids with >85% product yield under thermodynamic control. On the basis of the crystal structure, further mutagenesis gave a biocatalyst that favors introduction of larger functional groups. Thus, the use of computational and rational protein design provided a tool for diverse enzymatic peptide modification

Peptide synthesis in neat organic solvents with novel thermostable proteases

Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions

Peptiligase, an Enzyme for Efficient Chemoenzymatic Peptide Synthesis and Cyclization in Water

We describe a novel, organic cosolvent-stable and cation-independent engineered enzyme for peptide coupling reactions. The enzyme is a variant of a stable calcium-independent mutant of subtilisin BPN, with the catalytic Ser212 mutated to Cys and Pro216 converted to Ala. The enzyme, called peptiligase, catalyzes exceptionally efficient peptide coupling in water with a surprisingly high synthesis over hydrolysis (S/H) ratio. The S/H ratio of the peptide ligation reaction is correlated to the length of the peptide substrate and proved to be >100 for the synthesis of a 13-mer peptide, which corresponds to >99% conversion to the ligated peptide product an

Omniligase-1: A Powerful Tool for Peptide Head-to-Tail Cyclization

Strategies for the efficient synthesis of peptide macrocycles have been a long-standing goal. In this paper, we demonstrate the use of the peptide ligase termed omniligase-1 as a versatile and broadly applicable enzymatic tool for peptide cyclization. Several head-to-tail (multi) cyclic peptides have been synthesized, including the cyclotide MCoTI-II. Cyclization and oxidative folding of the cyclotide MCoTI-II were efficiently performed in a one-pot reaction on a 1-gram scale. The native cyclotide was isolated and the correct disulfide bonding pattern was confirmed by NMR structure determination. Furthermore, compatibility of chemo-enzymatic peptide synthesis (CEPS) using omniligase-1 with methods such as chemical ligation of peptides onto scaffolds (CLIPS) was successfully demonstrated by synthesizing a kinase-inhibitor derived tricyclic peptide. Our studies indicate that the minimal ring size for omniligase-1 mediated cyclization is 11 amino acids, whereas the cyclization of peptides longer than 12 amino acids proceeds with remarkable efficiency. In addition, several macrocycles containing non-peptidicbackbones (e.g., polyethylene glycol), isopeptide bonds (amino acid sidechain attachment) as well as d-amino acids could be efficiently cyclized

One-Step C-Terminal Deprotection and Activation of Peptides with Peptide Amidase from Stenotrophomonas maltophilia in Neat Organic Solvent

Chemoenzymatic peptide synthesis is a rapidly developing technology for cost effective peptide production on a large scale. As an alternative to the traditional C -> N strategy, which employs expensive N-protected building blocks in each step, we have investigated an N -> C extension route that is based on activation of a peptide C-terminal amide protecting group to the corresponding methyl ester. We found that this conversion is efficiently catalysed by Stenotrophomonas maltophilia peptide amidase in neat organic media. The system excludes the possibility of internal peptide cleavage as the enzyme lacks intrinsic protease activity. The produced peptide methyl ester was used for peptide chain extension in a kinetically controlled reaction by a thermostable protease