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

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

Natural Occurring and Engineered Enzymes for Peptide Ligation and Cyclization

The renaissance of peptides as prospective therapeutics has fostered the development of novel strategies for their synthesis and modi\ufb01cation. In this context, besides the developmentofnewchemicalpeptideligationapproaches,especiallytheuseofenzymes as a versatile tool has gained increased attention. Nowadays, due to their inherent properties such as excellent regio- and chemoselectivity, enzymes represent invaluable instruments in both academic and industrial laboratories. This mini-review focuses on natural- and engineered peptide ligases that can form a new peptide (amide) bond between the C-terminal carboxy and N-terminal amino group of a peptide and/or protein. The pro\u2019s and cons of several enzyme classes such as Sortases, Asparaginyl Endoproteases, Trypsin relatedenzymesand as a centralfocus subtilisin-derived variants are summarized. Most recent developments with regards to ligation and cyclization are highlighte

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

Synthesis of Constrained Tetracyclic Peptides by Consecutive CEPS, CLIPS, and Oxime Ligation

In Nature, multicyclic peptides constitute a versatile molecule class with various biological functions. For their pharmaceutical exploitation, chemical methodologies that enable selective consecutive macrocyclizations are required. We disclose a combination of enzymatic macrocyclization, CLIPS alkylation, and oxime ligation to prepare tetracyclic peptides. Five new small molecular scaffolds and differently sized model peptides featuring noncanonical amino acids were synthesized. Enzymatic macrocyclization, followed by one-pot scaffold-assisted cyclizations, yielded 21 tetracyclic peptides in a facile and robust manner