DOI: 10.1002/adsc.201000313 Fully Enzymatic Peptide Synthesis using C-Terminal tert-Butyl Ester Interconversion Timo Nuijens, a,b Claudia Cusan, a Theodorus J. G. M. van Dooren, a Harold M. Moody, a Remco Merkx, a John A. W. Kruijtzer, b Dirk T. S. Rijkers, b Rob M. J. Liskamp, b and Peter J. L. M. Quaedflieg a, * a DSM Innovative Synthesis BV, P.O. Box 18, NL-6160 MD Geleen, The Netherlands Fax: (+ 31)-46-476-7604; phone: (+ 31)-46-476-1592; e-mail: peter.quaedflieg@dsm.com b MedicinalChemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, NL-3508 TB Utrecht, The Netherlands Received: April 22, 2010; Revised: August 11, 2010; Published online: October 7, 2010 Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201000313. Abstract: Chemoenzymatic peptide synthesis is po- tentially the most cost-efficient technology for the synthesis of short and medium-sized peptides with some important advantages. For instance, stoichio- metric amounts of expensive coupling reagents are not required and racemisation does not occur, thus rendering purification easier compared to chemical peptide synthesis. The economically most attractive synthesis runs in the N !C terminal direction wherein a cheap C-terminally protected amino acid is employed as the building block for elongation. However, C-terminal deprotection and activation after an elongation step – without cleavage of the side-chain protective groups or the peptide bonds – was hitherto still a challenge. In this paper we de- scribe a novel C-terminal ester interconversion cat- alysed by the serine endopeptidase Alcalase. C-Ter- minally protected peptide tert-butyl esters were en- zymatically converted into primary alkyl esters in quantitative yield and used directly in the next en- zymatic elongation step with another amino acid tert-butyl ester. This fully enzymatic N !C elonga- tion strategy by C-terminal ester interconversion was applied toward the synthesis of biologically active peptides up to the pentamer level. Keywords: cross-linked enzyme aggregates (CLEA); enzyme catalysis; peptides; serine pro- tease alcalase; transesterification Peptides, from simple dipeptides to complex oligopep- tides, are increasingly abundant on the market as (pro)drugs or in clinical development. [1] Additionally, peptides have also gained importance as nutritional and cosmetic ingredients. [2] During the past decades, an increased interest for peptide synthesis has arisen but this is still an expensive and time-consuming pro- cedure up to date. [3] There are four main approaches available for pep- tide synthesis, i.e., fermentation, solid-phase or solu- tion-phase chemical peptide synthesis, and chemoen- zymatic peptide synthesis. [4] Currently, fermentation is only well feasible for long peptides (> 50 amino acids). Solid-phase and solution-phase chemical pep- tide syntheses are the most commonly used methods, but they require full protection of the side-chain func- tionalities, except in the case of fragment ligation techniques where a C-terminal thioester is involved, which is difficult to introduce. Moreover, to suppress racemisation, expensive and environmentally un- friendly coupling reagents are required in stoichio- metric amounts. Chemoenzymatic peptide synthesis, wherein peptides are stepwise elongated enzymatical- ly has been studied in academia during the past de- cades and proved to be suitable for certain short pep- tide sequences (up to 5 amino acids). [5] The amino acid side chains do not require protection and, most importantly, racemisation is completely absent so that purification is potentially much easier. However, (de)- protection of the N- and C-termini still requires harsh reaction conditions. [6] When the peptide is elongated in the C !N direction, expensive N-protected and C- activated amino acid building blocks are employed and after each coupling step the N-protective group should be removed. Elongation in the N !C direction (Scheme 1) would require the less expensive C-pro- tected amino acid building blocks, such as carboxy amides or tert-butyl esters, but a selective C-deprotec- tion should then be at hand. Adv. Synth. Catal. 2010, 352, 2399 – 2404 # 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2399 COMMUNICATIONS