Timo Nuijens

Enzymatic synthesis of C-terminal arylamides of amino acids and peptides.

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.

Enzyme-mediated ligation technologies for peptides and proteins

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.

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.

Papain-catalyzed peptide bond formation: Enzyme-specific activation with guanidinophenyl esters

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‐XAA‐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.

Enzyme-catalyzed peptide cyclization

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.

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

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.