Peter Jan Leonard Mario Quaedflieg

Click chemistry as a means to functionalize macroporous PolyHIPE

The surface functionalization of macroporous polyHIPE (pHIPE) was achieved by Huisgen-type ‘click’ chemistry. In the first step a 600–800 nm thick layer of poly(glycidyl methacrylate) (pGMA) was grafted from the pHIPE surface by atom transfer radical polymerization (ATRP). Near quantitative azidation of the pGMA layer was achieved by the ring-opening reaction of the epoxide groups with sodium azide. The influence of the reaction conditions on the uniformity of the ‘click’ reaction on the three-dimensional macroporous materials was shown in model reactions with propargyl alcohol. Under optimized conditions, azide conversions of around 80% were estimated from IR-spectra. Visualization of the homogeneous functionalization was achieved by the attachment of a fluorescent molecule. Moreover, the first proof of the versatility for biofunctionalization of pHIPE by this method was provided by the attachment of several protected amino acids. The hydrolytic stability of the triazole ring allows for the successful deprotection of the amino acids on the pHIPE.

Aspartame dipeptide analogues: effect of number of side-chain methylene group spacers and Cα-methylation in the second position

Abstract Our model of the active site of the sweet taste receptor is shown to be consistent with the aspartame analogues in which the L-Phe 2 residue is replaced by L-(αMe)Phg, L-(αMe)Phe or L-(αMe)Hph. The analogues containing either the first or the third C α -methylated, phenyl-containing residue in the second position of the dipeptide were synthesized and found to be approximately as sweet as aspartame itselfand its L-(αMe)Phe 2 analogue.

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.

An Enantioselective Organocatalytic Approach to Both Enantiomers of Lasubine II

A concise stereoselective route providing access to both enantiomers of the bioactive quinolizidine alkaloid lasubine II has been developed. The enantioselectivity was introduced by taking advantage of a proline-catalyzed asymmetric Mannich reaction. Next, the bicyclic system was constructed via a diastereoselective Mannich cyclization and subsequent ring-closing metathesis as the key steps.

Proteolysin, a Novel Highly Thermostable and Cosolvent-Compatible Protease from the Thermophilic Bacterium Coprothermobacter proteolyticus

ABSTRACT Through genome mining, we identified a gene encoding a putative serine protease of the thermitase subgroup of subtilases (EC 3.4.21.66) in the thermophilic bacterium Coprothermobacter proteolyticus. The gene was functionally expressed in Escherichia coli, and the enzyme, which we called proteolysin, was purified to near homogeneity from crude cell lysate by a single heat treatment step. Proteolysin has a broad pH tolerance and is active at temperatures of up to 80°C. In addition, the enzyme shows good activity and stability in the presence of organic solvents, detergents, and dithiothreitol, and it remains active in 6 M guanidinium hydrochloride. Based on its stability and activity profile, proteolysin can be an excellent candidate for applications where resistance to harsh process conditions is required.

Papain-Specific Activating Esters in Aqueous Dipeptide Synthesis

Enzymatic peptide synthesis has the potential to be a viable alternative for chemical peptide synthesis. Because of the increasing commercial interest in peptides, new and improved enzymatic synthesis methods are desirable. In recently developed enzymatic strategies such as substrate mimetic approaches and enzyme‐specific activation, use of the guanidinophenyl ester (OGp) group has been shown to suffer from some drawbacks. OGp esters are sensitive to spontaneous chemical hydrolysis and the group is expensive to synthesize and therefore not suitable for large‐scale applications. On the basis of earlier computational studies, we hypothesized that OGp might be replaceable by simpler ester groups to make the enzyme‐specific activation approach to peptide bond formation more accessible. To this end, a set of potential activating esters (Z‐Gly‐Act) was designed, synthesized, and evaluated. Both the benzyl (OBn) and the dimethylaminophenyl (ODmap) esters gave promising results. For these esters, the scope of a model dipeptide synthesis reaction under aqueous conditions was investigated by varying the amino acid donor. The results were compared with those obtained from a previous study of Z‐XAA‐OGp esters. Computational docking analysis of the set of esters was performed in order to provide insight into the differences in the reactivities of all the potential activating esters. Finally, selected ODmap‐ and OBn‐activated amino acids were applied in the synthesis of two biologically active dipeptides on preparative scales.

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.

Synthesis of (R)-4,4,4-trifluoro-2-mercaptobutyric acid

Abstract Syntheses of (R)-4,4,4-trifluoro-2-mercaptobutyric acid from (S)-malic acid via a Mitsunobu reaction and from (rac)-thiomalic acid on enzymatic resolution, using Pseudomonas cepacia (Amano lipase PS), are described. A new method for direct determination of ees for (R)- and (S)-4,4,4-trifluoro-2-mercaptobutyric acid derivatives by HPLC on a polysaccharide phase is disclosed.

Enzyme-Specific Activation versus Leaving Group Ability.

Enzyme‐specific activation and the substrate mimetics strategy are effective ways to circumvent the limited substrate recognition often encountered in protease‐catalyzed peptide synthesis. A key structural element in both approaches is the guanidinophenyl (OGp) ester, which enables important interactions for affinity and recognition by the enzyme—at least, this is usually the explanation given for its successful application. In this study we show that leaving group ability is of equal or even greater importance. To this end we used both experimental and computational methods: 1) synthesis of close analogues of OGp, and their evaluation in a dipeptide synthesis assay with trypsin, 2) molecular docking studies to provide insights into the binding mode, and 3) ab initio calculations to evaluate their electronic properties.