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

Abstract

Strategies for the efficients ynthesis of peptide macrocyclesh ave been al ong-standing goal. In this paper, we demonstrate the use of the peptide ligase termedo mniligase-1 as av ersatile and broadly applicable enzymatic toolf or peptide cyclization. Severalh ead-to-tail (multi)cyclic peptides have been synthesized, including the cyclotide MCoTI-II. Cyclization ando xidative foldingo ft he cyclotide MCoTI-II were efficiently performed in ao ne-pot reactiono na1 -gram scale.T he native cyclotide was isolated andt he correct disulfide bonding pattern was confirmed by NMRs tructure determination. Furthermore,c ompatibility of chemo-enzymatic peptide synthesis( CEPS) using omniligase1w ith methods such as chemical ligation of peptides onto scaffolds( CLIPS)w as successfully demonstrated by synthesizing ak inase-inhibitor derived tricyclic peptide.O ur studies indicate that the minimal ring size for omniligase-1 mediated cyclization is 11 amino acids,w hereas the cyclization of peptides longer than 12 amino acids proceeds with remarkablee fficiency.I na ddition,s everal macrocycles containing non-peptidic backbones( e.g.,p olyethylene glycol), isopeptide bonds (aminoa cid sidechain attachment) as well as d-amino acids couldbe efficiently cyclized.Keywords: chemo-enzymatic peptide synthesis (CEPS);c yclic peptides; cyclization;c yclotide synthesis;c yclotides;e nzyme catalysis;h ead-to-tail cyclization; ligases;m acrocycles;o mniligase-1;p eptides Peptide macrocyclesr epresent an extremely diverse class of molecules that attracti ncreased attentiona s drug leads and prospective pharmaceuticals,w ith currently over 30 cyclic peptides registered or in clinical trials.[1-3] Togetherw ith linearp eptides, they fill the gap between small-molecule drugs (less than 500 Da) and biologics (over 5000 Da) andh avet he potential to address previously "undruggable" targets. Many cyclicp eptides are characterized by their unique structural and enhancedb iopharmaceutical properties,s uch as an improved metabolic stability due to ar educed sensitivity to proteolytic cleavage.T he increasing number of cyclic peptidesu sed as therapeutics is accompanied by the need for efficient andc osteffectiver outes that enable their synthesis on al arge scale.M oreover, efficients ynthesiso fl arge libraries of cyclic peptides (e.g.,f or screeningp urposes) or of cyclicp eptides containing non-canonical amino acids (e.g., d-o ru nnatural amino acids to increase stability and diversity) is of importance.H owever, efficient cyclization of peptidesu sing traditional synthetic methodsi ss till ac hallenge.[4] Classical coupling reagents are often used to cyclize side-chain protected peptides in anhydrous organic solvents.H owever, heavy dilution to prevent polymerization,r isk of epimerization and ap oors olubility of side-chain protected peptideslimitthis approach, especially for peptides longer than 20 amino acids.[5] Native chemical ligation (NCL) [6] is often used to cyclize unprotected peptides in aqueous solution. However, not all pep...

A One‐Pot “Triple‐C” Multicyclization Methodology for the Synthesis of Highly Constrained Isomerically Pure Tetracyclic Peptides

“…Over the years, a variety of different technologies for constraining peptide structures have been described, including side‐chain‐to‐side‐chain crosslinks and the use of small‐molecule organic scaffolds . We recently established an efficient chemoenzymatic peptide synthesis (CEPS) strategy by using omniligase‐1‐catalyzed head‐to‐tail cyclization for the preparation of various (multi)cyclic peptides, such as the cyclotide MCoTI‐II . Although the cystine‐knotted core of cyclotides represents a promising scaffold for drug design, the majority of disulfide‐rich peptides form isomeric mixtures upon oxidation, which further emphasizes the need for a straightforward, sequence‐independent methodology that enables the manufacture of isomerically pure multicyclic constructs.…”

“…We evaluated the general applicability of triple‐C locking by synthesizing a library of peptides that differed in the length and sequence, which contained both two cysteine and two Aha residues combined with a C‐terminal elongated carboxamidomethyl ester (Cam ester) . Considering the requirement for efficient omniligase‐1‐catalyzed cyclization to be ≥13 amino acids, we first attempted cyclization of the linear 16‐mer peptide 2 3333 (H‐SYCQGA[Aha]KSE[Aha]KFGCK‐ OCam ‐ L ‐OH). Enzymatic cyclization (CEPS) gave the monocyclic peptide c 2 3333 within 30 min (Figure A).…”

A gold mine for drug discovery: Strategies to develop cyclic peptides into therapies

“…The use of enzymes as biocatalysts in peptide cyclization increasingly attracted attention due to their nontoxic nature, cost‐effectiveness, and high chemoselectivity. With the development of protein engineering biotechnology, a variety of enzymes have been exploited for peptide cyclization, such as butelase, trypsin, protease OaAEP1b, prenyl transferase, PatG, sortase, subtiligase, peptiligase, thioesterase, glutathione S‐transferase, transglutaminase, etc. In 2002, Kohli et al discovered an isolated thioesterase, which displayed the capability of catalyzing the cyclization of linear peptides tethered to a carrier protein via a phosphopantetheine linker.…”

General and Facile Route to Isomerically Pure Tricyclic Peptides Based on Templated Tandem CLIPS/CuAAC Cyclizations

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We report aone-pot ligation/cyclization technology for the rapid and clean conversion of linear peptides into tricyclic peptides that is based on using tetravalent scaffolds containing two benzyl bromide and two alkyne moieties.These react via CLIPS/CuAACreactions with cysteines and azides in the peptide.Flexibility in the scaffolds is key to the formation of isomerically pure products as the flexible scaffolds T4 1 and T4 2 mostly promote the formation of single isomeric tricycles while the rigid scaffolds T4 3 and T4 4 do not yield clean products. [12][13][14][15][16][17][18][19][20][21][22][23] In 2005, our group introduced "CLIPS technology" (chemical linkage of peptides onto scaffolds), which involves the tandem ligation/cyclization of tris(cysteine)-containing linear peptides with a,a',a''-tribromomesitylene (T3)t og ive the corresponding bicyclic peptides. We also observed that azides at the peptide termini and cysteine residues in the center gave better results than compounds with the functional groups placed the other wayr ound.

Cyclic peptides have emerged as ap romising class of drug compounds [1][2][3][4] showing aw ide therapeutic window that ranges from antifertility [5] to anticancer [6][7][8] and antiviral [9][10][11] applications.A st he number of cyclic peptides entering clinical trials has drastically increased over the last years, [6] the search for novel synthetic routes to multicyclic peptides has received significant interest.

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“…[25] Currently,c omplex syntheses impede the large-scale production of vancomycin-like drugs,and complicate structural diversification processes aimed at optimizing their biological activities. [26] Examples of synthetic tricyclic peptides have been reported as the groups of both Ruchala [17] and Wu [18] used either a T h -o raD 2h -symmetric scaffold to assemble linear peptides into tricycles,w hile Suga, [19] Nuijens, [20] and our group [21] used various types of backbone cyclization in combination with T3 CLIPS.T hese and other examples emphasize the interest in multicyclic peptides although the formation of multiple isomers,laborious reaction procedures, as well as difficulties in generating structural diversity remain delicate issues.CLIPS reactions of tetrakis(cysteine)-containing peptides with 1,2,4,5-tetrabromodurene (T4)w ould provide as traightforward route to manufacturing tricycles. However,t his reaction yields am ixture of six different regioisomers, [18] which renders this strategy not suitable for atherapeutic development trajectory (Figure 2a).…”