Restrictions to large-scale implementation of activating esters The objective to explore the scope and limitations of the substrate mimetics strategy,

which should ultimately have led to a universal approach to couple amino acids to a peptide chain or effect fragment couplings using cheap enzymes, appeared to be more challenging than expected.

First of all, we showed that in some situations it is better to use the term activating esters instead of substrate mimetics, although both strategies have a similar potential for becoming a universal and versatile approach.

Secondly, for every enzyme the search for suitable activating esters has to be carried out separately, as the properties of each enzyme are different. The S/H ratio is enzyme dependent and is moreover varying for each amino acid. An intrinsic problem is that specificity is the main reason to use an enzyme, but at the same time is also its most limiting characteristic. Possibly, the OCam and OTfe esters have the best chances for being universal activating esters, since they are relatively small and will therefore fit in many active sites. Furthermore, they possess the right electronic properties.

In the third place, an essential condition for activating esters becoming part of an industrial process is to develop an effective system to install the activating ester. At this moment all the advantages of enzymatic peptide coupling are lost, because the ester has to be synthesised by chemical methods. In this situation, it would be more efficient to accomplish the peptide bond directly. Without a doubt, the strategy of substrate mimetics and activating esters is only worthwhile, when the ester itself is synthesised in an enzymatic process.

In conclusion, the development of a universal method for chemoenzymatic peptide synthesis has appeared troublesome. However, the synthesis of a particular peptide product using enzyme-specific activation in my view may still be feasible. In that case, the focus is shifted from general difficulties to finding a solution for specific challenges occurring in that particular sequence.

8.5 Outlook

In my opinion, the most promising strategy for developing enzyme-specific activation procedures would consist of a combination of approaches. Several issues have to be tackled, the main two being 1) the still existing hydrolytic capacity of the enzyme, and 2) the recognition of a broad range of amino acid substrates. The problem of undesired enzymatic hydrolysis resulting in low S/H ratios can be solved by genetic engineering. The conversion of the protease subtilisin into subtiligase (S221C and P225A) is a very successful example of such an approach.[3] _{To prevent secondary hydrolysis of peptide} products containing a specific amino acid residue recognised by the enzyme, another approach is required. Ideally, using directed evolution protocols an enzyme is created, which is specific for an ‘orthogonal’ amino acid, i.e. a residue not likely to be incorporated in any peptide chain. The previously discussed spectrophotometrical assay to demonstrate synthesis instead of hydrolysis is essential at this point to enable high throughput screening of the clones. For the resulting reprogrammed enzyme, a specifically activating group should be developed to achieve more universal substrate acceptance. All in all, a challenging task.

8.6 References

[1] (a) L. Edens, P. Dekker, R. van Hoeven, F. Deen, A. de Roos, R. Floris, J. Agric. Food

Chem. 2005, 53, 7950-7957; (b) M. Lopez, L. Edens, J. Agric. Food Chem. 2005, 53,

7944-7949.

[2] D. V. Besedin, G. N. Rudenskaya, Russ. J. Bioorg. Chem. 2003, 29, 1-17.

[3] T. Chang, D. Y. Jackson, J. P. Burnier, J. A. Wells, Proc. Natl. Acad. Sci. U. S. A. 1994, 91, 12544-12548.

Summary

This thesis describes a section of the chemoenzymatic peptide synthesis project, which has been performed within the framework of the NWO-IBOS programme. The integration of Biosynthesis and Organic Synthesis is the central approach to synthesise peptides in a sustainable and efficient way. This is important, because peptides make up a growing segment of drugs within the pharmaceutical industry, but the development of improved production methods lags behind.

Peptides are composed of amino acids, which can be coupled both in a chemical and enzymatic way. An overview of these procedures is provided in Chapter 1. In addition, special attention is paid to the substrate mimetics strategy, which was taken as a starting point for the research described in this thesis. The substrate mimetics strategy combines the advantages of chemical synthesis (almost unlimited choice of amino acids) with the benefits of enzymatic synthesis (region- and stereospecific, mild reaction conditions). In practice, this is realised by equipping amino acids with an additional universal moiety, the so-called mimetic, resulting in recognition by the enzyme.

Figure 1 Principle of the substrate mimetics strategy

A well-known mimetic from literature is the guanidinophenyl ester (OGp), which, by resembling the amino acid arginine, is specifically recognised by the enzyme trypsin (Figure 1). Until now, OGp has been mainly applied in small-scale academic research. However, to make this concept of substrate mimetics applicable for large-scale industrial processes, a simple and cheap replacement of OGp is required. Preferably, it should be possible to install the mimetic enzymatically in the amino acid of choice.

Our quest for an alternative mimetic commenced in a fairly fundamental way by investigating whether OGp would prove a suitable mimetic for papain, a cysteine protease with a broader specificity than trypsin (Chapter 2). A less specific enzyme may allow more variation in the structure of the mimetic without losing activity. As a test substrate Z-Gly- OGp was synthesised and subjected to a chemoenzymatic reaction with papain in the presence of H-Phe-NH2 as acyl acceptor under aqueous conditions (Scheme 1). The reaction was followed in time and analysed using HPLC.