DOI: 10.1002/adsc.201400109

One-Step C-Terminal Deprotection and Activation of Peptideswith Peptide Amidase from Stenotrophomonas maltophilia inNeat Organic Solvent

Muhammad I. Arif,a Ana Toplak,a Wiktor Szymanski,a,b Ben L. Feringa,b

Timo Nuijens,c Peter J. L. M. Quaedflieg,c Bian Wu,a,* and Dick B. Janssena,*a Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen,

Nijenborgh 4, 9747 AG Groningen, The NetherlandsFax: (+31)-50-363-4165; phone; (+ 31)-50-363-4209; e-mail: b.wu@rug.nl or d.b.janssen@rug.nl

b Center for Systems Chemistry, Stratingh Institute for Chemistry, University of Groningen, Nijenborgh 4, 9747 AGGroningen, The Netherlands

c Enzypep B.V., P.O. Box 18, 6160 MD Geleen, The Netherlands

Received: February 7, 2014; Published online: June 20, 2014

Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201400109.

Abstract: Chemoenzymatic peptide synthesis isa rapidly developing technology for cost effectivepeptide production on a large scale. As an alterna-tive to the traditional C!N strategy, which employsexpensive N-protected building blocks in each step,we have investigated an N!C extension route thatis based on activation of a peptide C-terminalamide protecting group to the correspondingmethyl ester. We found that this conversion is effi-ciently catalysed by Stenotrophomonas maltophiliapeptide amidase in neat organic media. The systemexcludes the possibility of internal peptide cleavageas the enzyme lacks intrinsic protease activity. Theproduced peptide methyl ester was used for peptidechain extension in a kinetically controlled reactionby a thermostable protease.

Keywords: chemoenzymatic synthesis; esterifica-tion; organic solvents; peptide amidase; peptidesynthesis; peptides

In the past decades, a large number of peptides hasbeen commercialised for a wide variety of applica-tions, e.g., as therapeutic agents, nutritional supple-ments, and cosmetics.[1] Despite the increasing indus-trial demand for bioactive peptides, cost-efficientlarge-scale peptide synthesis remains challenging.[2] Inrecent years, chemoenzymatic peptide synthesis hasbecome a useful alternative to conventional chemicalsynthesis and recombinant fermentation methods.[3]

Compared to solid phase and solution phase chemicalpeptide synthesis, enzyme-catalysed peptide synthesis

offers notable advantages, including minimal need forprotection of side chains, absence of racemisation,and environmentally friendly reaction conditions.Chemoenzymatic peptide synthesis is usually carriedout with a protease via a kinetically controlled ap-proach.[4] To prevent undesired oligomerisation, theC-terminally activated amino acid or peptide used asthe acyl donor is N-terminally protected and the nu-cleophile is an amino acid or a peptide modified bya C-terminal amide group. After each elongation step,a selective deprotection of one of the termini is re-quired for further chain extension. Elongation of thepeptides in the C!N direction is highly cost-ineffi-cient because it requires an expensive acyl donor ateach step and repeated laborious N-terminal depro-tections. On the other hand, in the direction of N!Cpeptide elongation, much cheaper amino acid or pep-tide amides can be used. This strategy requires depro-tection (amide removal) and re-activation (e.g., asalkyl ester) of the product at its C-terminus. Ideally,the deprotection and reactivation should be per-formed simultaneously.

Recently, such a peptide C-terminal deprotection–reactivation was achieved by alkoxy-deamidation,using the protease subtilisin A.[5] However, the inher-ent nature of the protease always presents the risk ofinternal peptide hydrolysis. To avoid unwanted pro-teolysis of the substrate, such reactions should employa C-terminus selective enzyme. Peptide amidase fromflavedo of oranges (PAF) has been used for the inter-conversion of peptide amides into methyl esters.[6]

However, PAF is not stable enough in neat organicsolvents and requires at least 0.5% (v/v) of water inthe reaction system. Consequently, the yield of pep-tide ester was only moderate due to substantial hy-

Adv. Synth. Catal. 2014, 356, 2197 – 2202 � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 2197

COMMUNICATIONS

drolysis of the peptide amide. In addition, commercialutilisation of PAF is limited by the fact that theenzyme has not been cloned and isolation of theenzyme from orange flavedo is a low-yield process. Adifferent peptide amidase, originating from Stenotro-phomonas maltophilia (PAM), has been cloned, ex-pressed and characterised.[7] PAM is a serine hydro-lase that specifically catalyses the C-terminal deami-dation of peptide amides, without affecting internalpeptide bonds or amide functions in amino acid side-chains.[7] So far, only hydrolytic deamidation activityof this enzyme was reported and the possibility tosuppress hydrolysis by using a neat organic solvent

has not been investigated. Therefore, we decided toexplore the use of PAM as a catalyst for peptide C-terminal alkoxy-deamidation (Scheme 1).

First, we tested the catalytic activity of PAM inaqueous solution. The methoxy-deamidation of themodel substrate Z-Gly-Tyr-NH2 was carried out in50 mM phosphate buffer (pH 8.0) in the presence ofmethanol. With high methanol content (>85%, v/v),the formation of Z-Gly-Tyr-OMe could be detectedby LC/MS, albeit with low conversion rate and veryhigh degree of hydroxy-deamidation (hydrolysis ofthe amide). A further decrease of the water contentresulted in complete inactivation of the enzyme. Nev-ertheless, it was clear that PAM indeed possesses pro-miscuous esterase activity and tolerates considerableamounts of organic solvent.

Encouraged by these initial results, we decided touse methanol in neat organic solvents to prevent thehydrolytic side reaction. Different organic solvents,covering a broad logP range, were tested (Table 1).We performed conversions in neat organic solventcontaining 5% (v/v) methanol and 2% (v/v) DMF,lyophilised PAM, and molecular sieves to retaina very low water activity during the reaction. PAMshowed activity in a variety of organic solvents, withacetonitrile being the best solvent for the model reac-tion (Table 1). Further experiments showed that theoptimal concentration of methanol was 10% (v/v)(see the Supporting Information). Although it wassuggested that the use of apolar solvents is beneficialfor enzyme stability,[8] we did not observe a correlationbetween solvent polarity and PAM in our study.

Since small molecules have been used to protect anenzyme from deactivation during freeze-drying,[9] wefurther optimised the biocatalyst preparation method.Different lyoprotectants, including sugars, polymers,and simple salts were tested (Table 2). Sucrose pro-vided the highest degree of lyoprotection for PAM.Lyophilising the enzyme with sucrose in an optimised1:16.7 weight ratio resulted in over three-fold higherconversion (83% conversion in 24 h, see the Support-ing Information), although a certain degree of hydrol-ysis of the amide substrate was observed (~10%).

Scheme 1. Peptide C-terminal alkoxy-deamidation usingPAM.

Table 1. Effect of organic solvents on the C-terminal me-thoxy-deamidation.[a]

Organic solvent logP Yield [%][b] ofZ-Gly-Tyr-OCH3

DMSO �1.412 <0.5DMF �0.829 <0.5methanol �0.69 <0.5acetonitrile �0.334 40dioxane �0.255 5acetone �0.042 172-propanol 0.173 9THF 0.473 10tert-butyl alcohol 0.584 6MTBE 0.94 28toluene 2.73 <0.5

[a] Reaction conditions: lyophilised PAM (0.35 mg), variousorganic solvents (93%, v/v), methanol (5%, v/v), DMF(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3 � molecular sieves(7 mg), total volume 0.25 mL, 30 8C, 72 h.

[b] Determined by HPLC.

Table 2. Effect of different excipients on PAM esterification activity in the acetonitrile/methanol co-solvent system.[a]

Excipients Total conversion [%] Yield [%][b] of Z-Gly-Tyr-OCH3 Yield [%][b] of Z-Gly-Tyr-OH

polyethylene glycol 19.5 19.5 <0.5sucrose 52.1 51.0 1.1sorbitol <0.5 <0.5 <0.5trehalose 3.9 3.9 <0.5potassium chloride 26.9 26.9 <0.5tris 40.4 33.2 7.2

[a] Reaction conditions: PAM (0.35 mg, excipients:enzyme ratio= 98:1, w/w), acetonitrile (93%, v/v), methanol (5%, v/v),DMF (2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3 � molecular sieves (7 mg), total volume 0.25 mL, 30 8C, 20 h.

[b] Determined by HPLC.

2198 asc.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2014, 356, 2197 – 2202

COMMUNICATIONSMuhammad I. Arif et al.

To determine the scope of the PAM-mediated pep-tide C-terminal alkoxy-deamidation, we testeda range of alcohols. We observed conversion of Z-Gly-Tyr-NH2 only if small, aliphatic, primary alcoholswere used, with methanol giving the best ester yield(Table 3). This observation suggests the nucleophilebinding pocket of PAM is relatively small, which is inagreement with what has been observed in the PAMX-ray structure.[7] In this near-anhydrous organic co-solvent system, the alcohol still needs to competewith the remaining water for access to the enzymeactive site and reaction with the covalent acyl-enzymeintermediate.[10] We observed a higher degree of hy-drolysis upon increasing the length of alkyl chain ofthe alcohol. Likely, steric hindrance accounts for thisphenomenon.

In an aqueous environment, PAM displayeda broad peptide substrate scope in the hydrolytic re-action.[7] To explore the substrate specificity of PAMin a neat organic co-solvent system, we tested sverealamino acids amides and peptide amides. As shown inTable 4, several substrates were efficiently converted.

To investigate the practical applicability of PAM-catalysed peptide C-terminal alkoxy-deamidation, we

performed the reaction on a 50 mg semi-preparativescale under optimised conditions. After 7 days, theconversion of Z-Gly-Tyr-NH2 reached a value of 99%(95% of Z-Gly-Tyr-OMe, 4% of Z-Gly-Tyr-OH).After work-up and purification, the yield of Z-Gly-Tyr-OMe was 78%. The identity and purity of the en-zymatically prepared peptide ester werer confirmedby HPLC and NMR analysis, and compared to thechemically prepared reference compound.

Finally, we tested the applicability of PAM in a che-moenzymatic N!C peptide elongation concept. Forthis two-enzyme reaction, PAM was used first for theconversion of Z-Gly-Tyr-NH2 to the correspondingmethyl ester. Subsequently, two newly described ther-mostable proteases DgSbt and TaqSbt[11] were usedfor coupling of the obtained Z-Gly-Tyr-OMe withPhe-NH2 as the nucleophile (Scheme 2). For a one-pot reaction, PAM was removed by centrifugationafter the first reaction, and the protease DgSbt wasadded directly to the reaction mixture. Although thereaction rate was low, the yield of coupled peptidereached a notable value of 76%. In another experi-ment, we used purified Z-Gly-Tyr-OMe as substratein a subsequent TaqSbt protease-catalysed coupling

Table 3. Alkoxy-de-amidation of Z-Gly-Tyr-NH2 with different alcohols.[a]

Alcohol Reaction time (days) Yield [%][b] of the peptide ester Yield [%][b] of Z-Gly-Tyr-OH

methanol 1 68.5�8.7 1.5�1.5ethanol 11 21.8�2.5 24.8�1.5propanol 11 11.9�0.2 22.8�1.22-propanol 11 <0.5 30.0�5.2butanol 11 8.4�0.5 32.4�9.6tert-butyl alocohol 11 <0.5 21.2�1.2

[a] Reaction conditions: PAM (0.35 mg, lyophilised with 5.85 mg sucrose), acetonitrile (93%, v/v), alcohol (5%, v/v), DMF(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3 � molecular sieves (7 mg), total volume 0.25 mL, 30 8C.

[b] Product identity was confirmed by LC/MS and yields were determined by HPLC.

Table 4. Methoxy-de-amidation of different amino acids and peptides with methanol.[a]

Substrate Yield [%][b] of the peptide ester Yield [%][b] of the hydrolytic product Total conversion [%]

Z-Tyr-NH2 68.6�0.4 7.0�0.2 75.6�0.6Z-Gly-NH2 70.5�0.7 4.1�0.3 74.6�0.4Z-Gly-Tyr-NH2 73.9�4.3 4.1�0.2 78.0�4.2Z-Gly-Leu-NH2 75.4�0.6 5.1�0.2 80.4�0.8Z-Phe-Gly-NH2 43.0�0.3 4.5�0.7 47.1�0.5Z-Gly-Phe-NH2 67.7�4.2 8.1�3.7 75.8�7.9Z-Leu-Phe-NH2 42.1�5.3 9.2�0.8 51.3�6.1Z-Pro-Gly-NH2 65.6�3.6 9.9�3.0 75.5�6.6Z-Phe-Ala-NH2 58.1�1.3 7.0�1.5 65.1�0.3Z-Pro-Leu-Gly-NH2 23.5�4.0 3.7�0.4 27.3�4.4

[a] Reaction conditions: PAM (0.35 mg, lyophilised with 5.85 mg sucrose), acetonitrile (88%, v/v), alcohol (10%, v/v), DMF(2%, v/v), Z-Gly-Tyr-NH2 (5 mM), 3 � molecular sieves (7 mg), total volume 0.25 mL, 30 8C, 16 h.

[b] Product identity was confirmed by LC/MS and yields were determined by HPLC.

Adv. Synth. Catal. 2014, 356, 2197 – 2202 � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim asc.wiley-vch.de 2199

COMMUNICATIONS One-Step C-Terminal Deprotection and Activation of Peptides

reaction, yielding 77% of Z-Gly-Tyr-Phe-NH2 productover 6 days. With the removal of protease from thereaction mixture, the product could in principle beused in another elongation step.

In conclusion, we have established that PAM isa useful catalyst for converting a peptide C-terminalamide to a C-terminal ester, which can readily beused for enzymatic peptide elongation. To the best ofour knowledge, this is the first reported application ofthis class of enzyme in a near-neat organic solventsystem. Compared with the use of an enzyme isolatedfrom a natural source, recombinant production ofpeptide amidase is convenient and more efficient.More importantly, it forms a platform for further pro-tein engineering aimed at developing higher activitytowards the target of interest. Considering the specif-icity for the C-terminus of peptides, the negligible hy-drolysis of internal peptide bonds and side-chainamides, the broad substrate spectrum and the hightolerance towards organic solvents, this enzyme is anattractive addition to the toolbox of enzymes for che-moenzymatic peptide synthesis and may provide newpossibilities for various C-terminal modifications ofpeptides.

Experimental Section

Expression and Purification of PAM

The PAM gene from S. maltophilia was obtained as codon-optimised synthetic construct from DNA 2.0. The PAMgene, without the N-terminal signal sequence, was furthercloned in the pET21a(+) vector (Novagen), containing a C-terminal his-tag sequence, and transformed to E. coli Origa-mi (DE3) for expression. Cultures were grown in 2 L of LBmedium initially at 37 8C and cells were induced at 30 8Cwith 75 mM IPTG at OD600 = 0.6. After 24 h, cells were col-lected by centrifugation and a 20% (w/w) cell suspensionwas prepared in 20 mM potassium phosphate buffer, pH 7.5,containing 0.5 M NaCl and 0.02 M imidazole. The suspensionwas sonicated and the cell-free extract was obtained by cen-trifugation at 15000 rpm for 1 h. The cell-free extract wasapplied on a 5-mL HisTrap column and proteins were col-lected by elution with 20 mM potassium phosphate buffer,pH 7.5, containing 0.5 M NaCl and 0.2 M imidazole. The pu-rified enzyme was desalted in 20 mM potassium phosphatebuffer (pH 7.5) and further concentrated via Amicon filtra-tion (30 kDa). The enzyme was further incubated at 30 8Cfor up to 10 days, since we observed that the purifiedenzyme slowly became more active by an as yet unknownmechanism. After incubation, precipitates were removed by

centrifugation and the clear enzyme solution was stored inaliquots at �20 8C until further use. The yield of purifiedprotein was ~100 mgL�1 culture. For routine purposes, ali-quots (50 mL) containing 0.35 mg enzyme were lyophilisedovernight.

Solvent Optimisation for Methoxy-deamidation withPAM

Samples of 0.35 mg enzyme were lyophilised in a reactionvial. Approximately 7 mg of activated 3 � molecular sieveswere added to each reaction vial followed by addition of or-ganic solvent (93%, v/v), methanol (5%, v/v) and Z-Gly-Tyr-NH2 (5 mM, final concentration) as substrate. All reac-tions contained 2% (v/v) DMF to solubilise the substrate.Total reaction volume was 250 mL. The reaction mixtureswere incubated at 30 8C for 24 h with shaking at 400 rpm ona table top incubator. For analysis, reaction vials were brief-ly centrifuged and 5 mL samples were taken, mixed with50 mL of glacial acetic acid and analysed by HPLC (JascoLC-NetII/ADC) equipped with a reverse phase column(Altech Alltima, C18, 3 mm particle size, 53 mm �7 mm).Elution was carried out in an isocratic mode with 40% ace-tonitrile in water as eluent. The chromatograms were re-corded at 210 nm.

Preparative Scale Conversion of Z-Gly-Tyr-NH2 to Z-Gly-Tyr-OCH3

A 25 mg sample of PAM was lyophilised with 417 mg of su-crose for 48 h. To this, 2 g of 3 � molecular sieves wereadded followed by addition of 50 mg of Z-Gly-Tyr-NH2 dis-solved in acetonitrile:DMF (49:1, v/v). Subsequently, metha-nol was added to a final concentration of 10% (v/v). Thetotal volume of the reaction mixture was 15 mL. All solventsused were pre-incubated with 3 � molecular sieves for30 min. The reaction was performed at 30 8C with shaking at90 rpm. Approximately 50–100 mL samples were withdrawnat 24 h intervals to monitor the progress of the reaction.After 4 days, 1 g of 5 � molecular sieves was added toabsorb the released ammonia and the reaction was furtherincubated for two days. After 6 days of incubation, another1 g of 5 � molecular sieves was added to the system. At theend of the 7th day, the suspension was centrifuged toremove lyophilised enzyme and molecular sieves. The re-maining supernatant was evaporated under vacuum and thepellet was redissolved in 20 mL ethyl acetate. The ethyl ace-tate layer was washed twice with 15 mL NaHCO3, twicewith 15 mL 0.1 N HCl and twice with 15 mL brine. The or-ganic phase was dried (Na2SO4) and concentrated undervacuum after removing salt from the suspension. The driedpellet was redissolved in ethylacetate:pentane (1:1, v/v) andapplied on a 2 mL silica gel column (60 �, 230–400 meshparticle size, Aldrich) and fractions were collected. TLC wasperformed to analyse the purity of fractions. TLC plates

Scheme 2. N!C peptide elongation using a combination of PAM-catalysed deprotection/activation and protease-catalysedtransacylation.

2200 asc.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2014, 356, 2197 – 2202

COMMUNICATIONSMuhammad I. Arif et al.

were visualised by immersion in cerium ammonium molyb-date (CAM) solution followed by drying with hot air. Puri-fied fractions (Rf = 0.2) were pooled and further analysed onHPLC for purity. The material was further dried undervacuum and subjected to NMR analysis. 1H NMR(400 MHz, CDCl3): d= 2.96–3.04 (m, 2 H, CH2CH), 3.73 (s,3 H, CH3O), 3.72–3.90 (m, 2 H, Gly-CH2), 4.81–4.85 (m, 1 H,CH2CH), 5.12 (s, 2 H, CH2O), 5.45 (br s, 1 H, OH), 6.13 (brs, 1 H, CbzNH), 6.53 (br d, 1 H, NHCH), 6.67 (d, 3J= 8.0 Hz,2 H, phenol-H), 6.90 (d, 3J=8.0 Hz, 2 H, phenol-H), 7.31–7.40 (m, 5 H, Ph-H).

Preparation of Z-Gly-Tyr-OCH3 ReferenceCompound

A mixture of tyrosine methyl ester hydrochloride (1.0 mmol,232 mg), Z-Gly (1.0 mmol, 209 mg), HOBt (1.5 mmol,203 mg) and TEA (3.0 mmol, 418 mL) in DCM (10 mL) wascooled in an ice-water bath. EDC (1.1 mmol, 211 mg) wasadded and the cooling was removed. After 20 h, volatileswere evaporated and the residue was redissolved in AcOEt(30 mL). The organic solution was washed with 10% (w/v)aqueous citric acid solution (3� 20 mL), saturated aqueousNaHCO3 (2 �20 mL) and brine (20 mL), dried (MgSO4) andthe solvent was evaporated. The products were purified byflash chromatography (silica gel, 40–63 mm, pentane/AcOEt,1:1, v/v) to give a white powder; yield: 292 mg (75%); Rf =0.15 (pentane/AcOEt, 1:1, v/v). 1H NMR (400 MHz,CDCl3): d=2.96–3.04 (m, 2 H, CH2CH), 3.73 (s, 3 H, CH3O),3.72–3.90 (m, 2 H, Gly-CH2), 4.81–4.85 (m, 1 H, CH2CH),5.12 (s, 2 H, CH2O), 5.42 (br s, 1 H, OH), 5.88 (br s, 1 H,ZNH), 6.48 (br d, 1 H, NHCH), 6.67 (d, 3J=8.0 Hz, 2 H,phenol-H), 6.90 (d, 3J= 8.0 Hz, 2 H, phenol-H), 7.31–7.40(m, 5 H, Ph-H); 13C NMR (75 MHz, CDCl3): d= 37.1, 44.5,52.7, 53.6, 67.5, 115.9, 127.0, 128.3, 128.5, 128.8, 130.5, 136.2,155.8, 157.1, 169.6, 172.3; HR-MS (ESI+): m/z= 409.1366,calcd. for C20H22N2O6Na: 409.1370.

Kinetic Coupling of Z-Gly-Tyr-OMe with H-Phe-NH2

using DgSbt Protease

To the amidase reaction mixture containing N-protectedacyl donor Z-Gly-Tyr-OMe (8 mmol) in acetonitrile(1.5 mL), C-terminally protected Phe-NH2 (10 equiv.,80 mmol) was directly added, as well as 90 mg IPREP (iso-propyl alcohol-rinsed enzyme precipitate) containing 4 mgof DgSbt and activated 3 � crushed molecular sieves(200 mgmL�1). The reaction mixture was shaken at 400 rpmat 60 8C. Samples were taken and quenched with DMSO(1:3, v/v). After 21 days conversion to product was 76%.Conversions were estimated using HPLC by integration ofthe acyl donor starting material and the product peaks as-suming identical response factors. Products were identifiedby LC/MS.

Kinetic Coupling of Z-Gly-Tyr-OMe with H-Phe-NH2

using TaqSbt Protease

In a parallel coupling reaction, to the purified N-protectedacyl donor Z-Gly-Tyr-OMe (28 mmol) in acetonitrile(3 mL), C-terminally protected Phe-NH2 (10 equiv.,280 mmol) was added, followed by 35 mg IPREP enzymepreparation (2.2 mg of TaqSbt enzyme) and activated 3 �

molecular sieves powder (100 mg mL�1). The reaction mix-ture was shaken at 400 rpm at 60 8C. Samples were takenand quenched with DMSO (1:3 v/v ratio). After 6 days, 77%conversion was achieved. Conversions were estimated byHPLC using the same procedure as described above and fur-ther identified by LC/MS.

Acknowledgements

This project is part of Integration of Biosynthesis and Organ-ic Synthesis program (IBOS-2; program number: 053.63.014)funded by The Netherlands Organization for Scientific Re-search (NWO) and Advanced Chemical Technologies forSustainability (ACTS). The authors thank Dr. J.-M. van derLaan from DSM Food Specialties for helpful discussions.

References

[1] a) D. J. Craik, D. P. Fairlie, S. Liras, D. Price, Chem.Biol. Drug. Des. 2013, 81, 136–147; b) N. Sewald, H. D.Jakubke, Peptide Synthesis, Wiley-VCH, Weinheim,2009 ; c) K. Lintner, O. Peschard, Int. J. Cosmet. Sci.2000, 22, 207–218; d) I. Gill, R. L. Fandino, X. Jorba,E. N. Vulfson, Enzyme. Microb. Technol. 1996, 18, 162–183; e) F. Guzman, S. Berberis, A. Illanes, Electron. J.Biotechnol. 2007, 10, 279–314; f) H. Korhonen, A. Pih-lanto, Curr. Pharm. Des. 2003, 9, 1297–1308.

[2] a) D. Hans, P. R. Young, D. P. Fairlie, Med. Chem. 2006,2, 627–646; b) A. M. Thayer, Chem. Eng. News. 2011,89, 21–25.

[3] a) F. Guzm�n, S. Barberis, A. Illanes, Electron. J. Bio-tech. 2007, 10, 279–314; b) T. Nuijens, C. Cusan,T. J. G. M. van Dooren, H. M. Moody, R. Merkx,J. A. W. Kruijtzer, D. T. S. Rijkers, R. M. J. Liskamp,P. J. L. M. Quaedflieg, Adv. Synth. Catal. 2010, 352,2399–2404; c) T. Nuijens, P. J. L. M. Quaedflieg, H. D.Jakubke, Enzyme catalysis in organic synthesis, Wiley-VCH, Weinheim 2012, pp 675–748.

[4] a) J. Bongers, E. P. Heimer, Peptides 1994, 15, 183–193;b) F. Bordusa, Chem. Rev. 2002, 102, 4817–4867; c) R. J.de Beer, B. Zarzycka, M. Mariman, H. I. Amatdjais-Groenen, M. J. Mulders, P. J. L. M. Quaedflieg, F. L.van Delft, S. B. Nabuurs, F. P. Rutjes, ChemBioChem2012, 13, 1319–1326; d) I. Kira, Y. Asano, K. Yokozeki,J. Biosci. Bioeng. 2009, 108, 190–193.

[5] T. Nuijens, E. Piva, J. A. W. Kruijtzer, D. T. S. Rijkers,R. M. J. Liskamp, P. J. L. M. Quaedflieg, Adv. Synth.Catal. 2011, 353, 1039–1044.

[6] P. J. L. M. Quaedflieg, T. Sonke, G. K. M. Verzijl, R. W.Wiertz, Patent Appl. EP 1937826(B1), 2009.

[7] S. Neumann, M. R. Kula, Appl. Microbiol. Biotechnol.2002, 58, 772–780.

[8] a) A. M. Klibanov, Nature 2001, 409, 241–246; b) G.Carrea, S. Riva, Angew. Chem. 2000, 112, 2312–2341;Angew. Chem. Int. Ed. 2000, 39, 2226–2254; c) E. P.Hudson, R. K. Eppler, D. S. Clark, Curr. Opin. Biotech-nol. 2005, 16, 637–643.

[9] A. L. Serdakowski, J. S. Dordick, Trends Biotechnol.2008, 26, 48–54.

Adv. Synth. Catal. 2014, 356, 2197 – 2202 � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim asc.wiley-vch.de 2201

COMMUNICATIONS One-Step C-Terminal Deprotection and Activation of Peptides

[10] a) J. Labahn, S. Neumann, G. B�ldt, M. R. Kula, J.Granzin, J. Mol. Biol. 2002, 322, 1053–1064; b) A. L.ValiÇa, D. Mazumder-Shivakumar, T. C. Bruice, Bio-chemistry 2004, 43,15657–15672.

[11] A. Toplak, B. Wu, F. Fusetti, P. J. L. M. Quaedflieg,D. B. Janssen, Appl. Environ. Microbiol. 2013, 79,5625–5632.

2202 asc.wiley-vch.de � 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim Adv. Synth. Catal. 2014, 356, 2197 – 2202

COMMUNICATIONSMuhammad I. Arif et al.