ABSTRACT
Protein synthesis and semisynthesis offer immense promise for life sciences and have impacted pharmaceutical innovation. The absence of a generally applicable method for traceless peptide conjugation with a flexible choice of junction sites remains a bottleneck for accessing many important synthetic targets, however. Here we introduce the PALME (protein activation and ligation with multiple enzymes) platform designed for sequence-unconstrained synthesis and modification of biomacromolecules. The upstream activating modules accept and process easily accessible synthetic peptides and recombinant proteins, avoiding the challenges associated with preparation and manipulation of activated peptide substrates. Cooperatively, the downstream coupling module provides comprehensive solutions for sequential peptide condensation, cyclization and protein N/C-terminal or internal functionalization. The practical utility of this methodology is demonstrated by synthesizing a series of bioactive targets ranging from pharmaceutical ingredients to synthetically challenging proteins. The modular PALME platform exhibits unprecedentedly broad accessibility for traceless protein synthesis and functionalization, and holds enormous potential to extend the scope of protein chemistry and synthetic biology.
ABSTRACT
Omniligase-1 is a broadly applicable enzyme for peptide bond formation between an activated acyl donor peptide and a non-protected acyl acceptor peptide. The enzyme is derived from an earlier subtilisin variant called peptiligase by several rounds of protein engineering aimed at increasing synthetic yields and substrate range. To examine the contribution of individual mutations on S/H ratio and substrate scope in peptide synthesis, we selected peptiligase variant M222P/L217H as a starting enzyme and introduced successive mutations. Mutation A225N in the S1' pocket and F189W of the S2' pocket increased the synthesis to hydrolysis (S/H) ratio and overall coupling efficiency, whereas the I107V mutation was added to S4 pocket to increase the reaction rate. The final omniligase variants appeared to have a very broad substrate range, coupling more than 250 peptides in a 400-member library of acyl acceptors, as indicated by a high-throughput FRET assay. Crystal structures and computational modelling could rationalize the exceptional properties of omniligase-1 in peptide synthesis.
ABSTRACT
The renaissance of peptides as prospective therapeutics has fostered the development of novel strategies for their synthesis and modification. In this context, besides the development of new chemical peptide ligation approaches, especially the use of enzymes 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's and cons of several enzyme classes such as Sortases, Asparaginyl Endoproteases, Trypsin related enzymes and as a central focus subtilisin-derived variants are summarized. Most recent developments with regards to ligation and cyclization are highlighted.
ABSTRACT
Omniligase-1-catalyzed ligation represents a powerful tool for the efficient intermolecular and intramolecular (head-to-tail cyclization) ligation of peptides. Reactions are irreversible and proceed with unprotected peptides (µM-mM concentration) in aqueous solution at slightly basic pH. Due to its high catalytic efficiency, only very low molar equivalents of omniligase-1 are required. In this chapter, a chemoenzymatic peptide synthesis (CEPS) approach for the assembly of medium-to-long-sized linear peptides as well as for efficient peptide head-to-tail cyclization is described. In particular, we provide protocols for the chemoenzymatic synthesis of the peptide therapeutic exenatide, a GLP-1 (glucagon-like peptide) analogue, and the macrocyclization and oxidative folding of the cyclotide MCoTI-II in a one-pot procedure.
Subject(s)
Ligases/chemistry , Peptides, Cyclic/chemical synthesis , Amino Acid Sequence , Catalysis , Chromatography, High Pressure Liquid , Cyclization , Cyclotides/chemistry , Peptides, Cyclic/analysis , Peptides, Cyclic/chemistryABSTRACT
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.
ABSTRACT
Disulfide-rich macrocyclic peptides-cyclotides, for example-represent a promising class of molecules with potential therapeutic use. Despite their potential their efficient synthesis at large scale still represents a major challenge. Here we report new chemoenzymatic strategies using peptide ligase variants-inter alia, omniligase-1-for the efficient and scalable one-pot cyclization and folding of the native cyclotides MCoTI-II, kalataâ B1 and variants thereof, as well as of the θ-defensin RTD-1. The synthesis of the kB1 variant T20K was successfully demonstrated at multi-gram scale. The existence of several ligation sites for each macrocycle makes this approach highly flexible and facilitates both the larger-scale manufacture and the engineering of bioactive, grafted cyclotide variants, therefore clearly offering a valuable and powerful extension of the existing toolbox of enzymes for peptide head-to-tail cyclization.
Subject(s)
Cyclotides/chemistry , Defensins , Peptide Synthases , Cyclization , Cyclotides/chemical synthesis , Defensins/chemical synthesis , Defensins/chemistry , Peptide Synthases/chemical synthesis , Peptide Synthases/chemistry , Plant Proteins/chemical synthesis , Plant Proteins/chemistryABSTRACT
A broadly applicable one-pot methodology for the facile transformation of linear peptides into tetracyclic peptides through a chemoenzymatic peptide synthesis/chemical ligation of peptides onto scaffolds/copper(I)-catalyzed reaction (CEPS/CLIPS/CuAAC; "triple-C") locking methodology is reported. Linear peptides with varying lengths (≥14 amino acids), comprising two cysteines and two azidohomoalanines (Aha), were efficiently cyclized head-to-tail by using the peptiligase variant omniligase-1 (CEPS). Subsequent ligation-cyclization with tetravalent (T41/2 ) scaffolds containing two bromomethyl groups (CLIPS) and two alkyne functionalities (CuAAC) yielded isomerically pure tetracyclic peptides. Sixteen different functional tetracycles, derived from bicyclic inhibitors against urokinase plasminogen activator (uPA) and coagulation factor XIIa (FXIIa), were successfully synthesized and their bioactivities evaluated. Two of these (FF-T41/2 ) exhibited increased inhibitory activity against FXIIa, compared with a bicyclic control peptide. The corresponding hetero-bifunctional variants (UF/FU-T41/2 ), with a single copy of each inhibitory sequence, exhibited micromolar activities against both uPA and FXIIa; thus illustrating the potential of the "bifunctional tetracyclic peptide" inhibitor concept.
Subject(s)
Peptides, Cyclic/chemical synthesis , Peptides/chemistry , Alanine/analogs & derivatives , Alanine/chemistry , Amino Acid Sequence , Combinatorial Chemistry Techniques , Cyclization , Cysteine/chemistry , Factor XIIa/antagonists & inhibitors , Humans , Models, Molecular , Peptides, Cyclic/chemistry , Peptides, Cyclic/pharmacology , Urokinase-Type Plasminogen Activator/antagonists & inhibitorsABSTRACT
The synthesis of thymosin-α1, an acetylated 28 amino acid long therapeutic peptide, via conventional chemical methods is exceptionally challenging. The enzymatic coupling of unprotected peptide segments in water offers great potential for a more efficient synthesis of peptides that are difficult to synthesize. Based on the design of a highly engineered peptide ligase, we developed a fully convergent chemo-enzymatic peptide synthesis (CEPS) process for the production of thymosin-α1via a 14-mer + 14-mer segment condensation strategy. Using structure-inspired enzyme engineering, the thiol-subtilisin variant peptiligase was tailored to recognize the respective 14-mer thymosin-α1 segments in order to create a clearly improved biocatalyst, termed thymoligase. Thymoligase catalyzes peptide bond formation between both segments with a very high efficiency (>94% yield) and is expected to be well applicable to many other ligations in which residues with similar characteristics (e.g. Arg and Glu) are present in the respective positions P1 and P1'. The crystal structure of thymoligase was determined and shown to be in good agreement with the model used for the engineering studies. The combination of the solid phase peptide synthesis (SPPS) of the 14-mer segments and their thymoligase-catalyzed ligation on a gram scale resulted in a significantly increased, two-fold higher overall yield (55%) of thymosin-α1 compared to those typical of existing industrial processes.
Subject(s)
Peptide Synthases/chemistry , Thymalfasin/chemical synthesis , Amino Acid Sequence , Mutation , Peptide Synthases/genetics , Protein Engineering/methods , Solid-Phase Synthesis Techniques/methodsABSTRACT
The recent advancement of peptide macrocycles as promising therapeutics creates a need for novel methodologies for their efficient synthesis and (large scale) production. Within this context, due to the favorable properties of biocatalysts, enzyme-mediated methodologies have gained great interest. Enzymes such as sortase A, butelase 1, peptiligase and omniligase-1 represent extremely powerful and valuable enzymatic tools for peptide ligation, since they can be applied to generate complex cyclic peptides with exquisite biological activity. Therefore, the use of enzymatic strategies will effectively supplement the scope of existing chemical methodologies and will accelerate the development of future cyclic peptide therapeutics. The advantages and disadvantages of the different enzymatic methodologies will be discussed in this review.
Subject(s)
Peptides/chemistry , Catalysis , Cyclization , Cysteine Endopeptidases/chemistry , Subtilisin/chemistryABSTRACT
With the steadily increasing complexity and quantity requirements for peptides in industry and academia, the efficient and site-selective ligation of peptides and proteins represents a highly desirable goal. Within this context, enzyme-mediated ligation technologies for peptides and proteins have attracted great interest in recent years as they represent an extremely powerful extension to the scope of chemical methodologies (e.g. native chemical ligation) in basic and applied research. Compared to chemical ligation methods, enzymatic strategies using ligases such as sortase, butelase, peptiligase or omniligase generally feature excellent chemoselectivity, therefore making them valuable tools for protein and peptide chemists.
Subject(s)
Enzymes/metabolism , Peptides/chemistry , Peptides/metabolism , Proteins/chemistry , Proteins/metabolism , HumansABSTRACT
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.
Subject(s)
Bacterial Proteins/metabolism , Chemistry Techniques, Synthetic , Deinococcus/enzymology , Peptide Hydrolases/metabolism , Peptides/metabolism , Solvents , Thermus/enzymology , 2-Propanol , Acetone , Acetonitriles , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Biocatalysis , Chemical Precipitation , Deinococcus/genetics , Dimethylformamide , Escherichia coli/metabolism , Genes, Bacterial , Hot Temperature , Peptide Hydrolases/genetics , Peptide Hydrolases/isolation & purification , Protein Stability , Recombinant Fusion Proteins/metabolism , Solubility , Thermus/genetics , tert-Butyl AlcoholABSTRACT
A series of novel glycine esters was evaluated for efficiency in subtilisin A-CLEA-catalysed peptide synthesis. The reactivity of the easily accessible carboxyamidomethyl (Cam) ester was further enhanced by elongating it with an amino acid residue, thereby creating more recognition space for subtilisin A.
Subject(s)
Bacillus subtilis/enzymology , Glycine/analogs & derivatives , Peptides/chemical synthesis , Peptides/metabolism , Subtilisins/metabolism , Catalysis , Esters/chemistry , Peptide Biosynthesis , Peptides/chemistryABSTRACT
The substrate mimetics approach is a versatile method for small-scale enzymatic peptide-bond synthesis in aqueous systems. The protease-recognized amino acid side chain is incorporated in an ester leaving group, the substrate mimetic. This shift of the specific moiety enables the acceptance of amino acids and peptide sequences that are normally not recognized by the enzyme. The guanidinophenyl group (OGp), a known substrate mimetic for the serine proteases trypsin and chymotrypsin, has now been applied for the first time in combination with papain, a cheap and commercially available cysteine protease. To provide insight in the binding mode of various Z-X(AA)-OGp esters, computational docking studies were performed. The results strongly point at enzyme-specific activation of the OGp esters in papain through a novel mode of action, rather than their functioning as mimetics. Furthermore, the scope of a model dipeptide synthesis was investigated with respect to both the amino acid donor and the nucleophile. Molecular dynamics simulations were carried out to prioritize 22 natural and unnatural amino acid donors for synthesis. Experimental results correlate well with the predicted ranking and show that nearly all amino acids are accepted by papain.
Subject(s)
Biocatalysis , Biomimetic Materials/chemistry , Guanidine/chemistry , Papain/metabolism , Peptides/chemistry , Peptides/chemical synthesis , Dipeptides/chemical synthesis , Dipeptides/chemistry , Esters , Molecular Dynamics Simulation , Protein Conformation , Reproducibility of Results , Water/chemistryABSTRACT
A mild and cost-efficient chemo-enzymatic method for the synthesis of C-terminal arylamides of amino acid and peptides is described. Using the industrial serine protease Alcalase under near-anhydrous conditions, C-terminal arylamides of N-Cbz-protected amino acids and peptides could be obtained from the corresponding C-terminal carboxylic acids, methyl (Me) or benzyl (Bn) esters, in high chemical and enantio- and diastereomeric purities. Yields ranged between 50% and 95% depending on the size of the aryl substituents and the presence of electron-withdrawing substituents. Complete alpha-C-terminal selectivity could be obtained even in the presence of various unprotected side-chain functionalities such as beta/gamma-carboxyl, hydroxyl, and guanidino groups. In addition, the use of the cysteine protease papain and the lipase Cal-B gave anilides in high yields. The chemo-enzymatic synthesis of arylamides proved to be completely free of racemization, in contrast to the state-of-the-art chemical methods.
Subject(s)
Amides/chemistry , Amides/metabolism , Amino Acids/chemistry , Peptides/chemistry , Subtilisins/metabolism , Anilides/chemistry , Anilides/metabolism , Bacillus/enzymology , Lipase/metabolism , Molecular Conformation , Papain/metabolism , StereoisomerismABSTRACT
Modification through beta-elimination has proven to be a reliable first step in the approach for enrichment of serine/threonine-phopshorylated (Ser-/Thr) peptides. However, under harsh basic conditions, Ser-/Thr-glycosylated peptides are susceptible to beta-elimination as well. Therefore, we have optimized these conditions to achieve a beta-elimination that is highly selective for phosphorylated peptides. This is the first report of selective beta-elimination and enrichment of phosphorylated peptides in the presence of glycosylated peptides.