DOI: 10.1002/cbic.201100434

Simultaneous “One Pot” Expressed Protein Ligation and CuI-CatalyzedAzide/Alkyne Cycloaddition for Protein Immobilization

Max Steinhagen,[a] Kai Holland-Nell,[b] Morten Meldal,[b] and Annette G. Beck-Sickinger*[a]

Proteins, enzymes in particular, are becoming more and moreimportant in synthesis and the creation of stereo- and regiose-lectively novel products with high specificity. They also play animportant role in the development of diagnostic systems, mi-croarrays and biosensors.[1] The anchoring of proteins to solidsupports can facilitate these systems, but to maintain thenative properties and homogeneity, site specific immobilizationis preferred,[2] which in turn calls for site specific introductionof reactive groups into the protein structure. However, modifi-cations can influence protein stability, activity or electrostaticproperties of the protein surface.[3] Alternatively, naturally oc-curring functionalities, for example, the e-amino group oflysine[4] or the thiol group of cysteine,[5] can be employed.However, frequently more than one such reactive group is ex-posed on the protein surface leading to formation of severaldistinct protein species. To circumvent these problems orthog-onal ligation strategies are promising alternatives.

A commonly used method is expressed protein ligation(EPL).[6] The wide scope of combining C-terminal thioesters

with N-terminal cysteine residues of another molecule hasbeen demonstrated in various cases, as recently reviewed.[7]

The formation of native peptide bonds, the selectivity and themild reaction conditions are just some advantages of thistechnique.

Another ligation method is the CuI-catalyzed azide/alkynecycloaddition (CuAAC), which was independently described bythe groups of Meldal and Sharpless.[8] This catalyzed Huisgen[3+2] cycloaddition[9] is accelerated 108-fold, and regioselec-tively forms the 1,4-disubstituted 1,2,3-triazole through CuI cat-alysis. The robustness to side reactions, regio- and chemoselec-tivity, and compatibility with an aqueous environment facili-tates its application to biomolecules. Accordingly, the reactioncan be classified as a “click” reaction.[10]

We have combined expressed protein ligation and CuI-cata-lyzed azide/alkyne cycloaddition in a single simultaneous reac-tion step to circumvent purification of the ligation productafter EPL (Scheme 1). With this combination we have succeed-ed in immobilizing two model proteins, enhanced green fluo-

rescent protein (eGFP) and aldo–keto reductase 1A1 (AKR1A1;EC 1.1.1.2), on PEG-based solid support in high yields.

The proteins, eGFP and AKR1A1, were expressed as proteinthioesters by using the commercially available IMPACT (inteinpurification with an affinity binding tag) vector system (Fig-ure 1 B and C).[11] As a linker peptide (Scheme 1) the short hep-tapeptide 1 labeled with the fluorophore 5(6)-carboxytetrame-

Scheme 1. Illustration of the “one-pot” immobilization process with the linker peptide 1 and the azide PEGA resins 2 and 3. Ahx: 6-aminohexanoic acid;HMBA: 4-(hydroxymethyl)benzoic acid; TCEP: tris(2-carboxyethyl)phosphine.

[a] M. Steinhagen, Prof. Dr. A. G. Beck-SickingerInstitute of Biochemistry, Universit�t LeipzigBr�derstrasse 34, 04103 Leipzig (Germany)E-mail : beck-sickinger@uni-leipzig.de

[b] Dr. K. Holland-Nell, Prof. Dr. M. MeldalCarlsberg LaboratoryGamle Carlsberg Vej 10, 2500 Valby (Denmark)

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thylrhodamine (TAMRA) was synthesized by using solid phasepeptide synthesis (Figure 1 A).

Subsequently, the azide derivatized PEGA resins 2 and 3were synthesized by using standard coupling protocols.[12] Theprotein thioester, peptide 1 and resins 2 or 3 were incubatedwith the reagents CuSO4, tris(2-carboxyethyl)phosphine (TCEP)as the reducing agent and the tridentate CuI ligand tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA). The N-termi-nal cysteine of the linker peptide 1 was used to ligate the pep-tide to the protein thioester by EPL. Simultaneously, the alkynemoiety of 1 was used to ligate the peptide to the azide deriv-

atized PEGA resin by CuAAC. The addition of TCEP as reducingagent was advantageous for both EPL and the CuAAC reac-tion.[13] In EPL it prevents disulfide formation and generatesthe free thiol group of cysteine. In the CuAAC reaction, TCEPreduces CuII to the active CuI species.

To distinguish between unspecific adsorptive binding of theprotein and specifically immobilized protein, control reactionswere performed. To this end, the resin was incubated with theprotein thioesters in the absence of reagents and the linkerpeptide. The controls provided quantification of unspecificallybound protein.

The introduction of a fluorescence labeled linker peptideand eGFP allowed the visual control of the reaction process,and furthermore, indicated the functionality of the immobi-lized eGFP. The successful immobilization of peptide (red) andeGFP (green) could be illustrated by the fluorescence of thebeads upon immobilization (Figure 2). The control beadsshowed no detectable fluorescence, whereas the TAMRA fluo-rescence of the peptide indicated effective peptide ligationwith both proteins. Moreover, the measurement of fluores-cence from immobilized eGFP confirmed the immobilization ofintact protein on the beads. This demonstrated that both reac-tions could be performed simultaneously in one reactionvessel by using common conditions because of their regio-and chemoselectivity.

The protein thioester was selected as the limiting factor withrespect to concentration. We then determined the efficiency ofthe reaction by comparing the amount of immobilized proteinrelative to the amount of protein thioester, which was initiallyadded to the reaction mixture. The immobilized proteinamount was calculated after cleaving the protein from theresin followed by lyophilization, redissolution and Bradfordprotein assay. Therefore, a cleavage site was introduced priorto the azide functionality at the PEGA resin. In resin 3 a baselabile ester was incorporated by using 4-(hydroxymethyl)ben-zoic acid (HMBA). In resin 2 an acid labile cleavage site was in-troduced through the Rink functionality. The immobilized pro-teins were cleaved by using NaOH (1 m) for the HMBA ester or90 % aq. TFA when using the Rink linker.

Figure 1. A) MALDI-ToF-MS of the purified peptide (Mw calcd 1207.9 Da). Puritywas confirmed by RP-HPLC (gradient 20 %!80 % solvent B in 40 min). Asingle peak was detected at 25.7 min, solvent A: 0.1 % TFA, solvent B: 0.08 %TFA in AcN. B) MALDI-ToF-MS of the purified AKR1A1-thioester (Mw calcd

36.719 kDa). Inset : SDS-PAGE showed pure protein; lane 1: solubilized fusionprotein of AKR1A1-intein-chitin binding domain (CBD); lane 2: AKR1A1-thio-ester after cleavage of the fused intein-CBD-tag. C) MALDI-ToF-MS of the pu-rified eGFP-thioester (Mw calcd 27.195 kDa). Inset: SDS-PAGE showed pure pro-tein; lane 1: supernatant after cell lysis ; lane 2: eGFP-thioester after cleavageof the fused intein-CBD-tag.

Figure 2. Fluorescence microscopy of PEGA beads after the immobilizationreaction. Control : PEGA beads incubated without peptide; TAMRA: fluores-cence label of the peptide recorded with 30 ms exposure time; EGFP: eGFPfluorescence recorded with 400 ms of exposure time; Merge: overlay of fluo-rescence channels.

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The cleavage efficiency of the immobilized proteins from thebeads was investigated by ELISA (Figure 3). In case of the Rink-linker modified resin (Figure 3 B), cleavage of the proteins wasnot successful. The HMBA modified resin showed different re-sults for the two proteins (Figure 3 A). While AKR1A1 was quan-titatively cleaved from the resin, residual eGFP remained onthe beads probably due to hydrophobic interactions. In the ex-periments in which the ELISA assay showed quantitative cleav-age, the protein amount was determined by Bradford proteinassay and compared to the amount added to the immobiliza-tion reaction. This indicated 10.3(�2.6) % specific immobiliza-tion of the added thioester versus 1.4(�2.0) % unspecificallybound protein.

To investigate the functionality of the immobilized AKR1A1,we performed an enzyme activity assay. AKR1A1 uses NADPHas cofactor during enzymatic catalysis. NADPH consumptioncan easily be followed by using a photometric assay. NADPHconsumption of specifically immobilized AKR1A1 with the pres-ent approach is significantly higher than that of adsorbedAKR1A1 (Figure 4 A). The final enzyme activity was calculatedas a percentage (Figure 4 B). The slope of the absorbancecurves was determined and compared to the specific activityin solution prior to the immobilization reaction. For specificallyimmobilized AKR1A1 the activity was calculated with38.3(�7.2) %, while for the unspecifically bound AKR1A1 it wasjust 11.3(�3.1) %. This indicates that the immobilized proteinsmaintain their active conformation and are unaffected by thereaction conditions, whereas unspecific binding leads to de-creased activity and/or less immobilization.

It was recently described that stepwise application of EPLand CuAAC reactions can be employed.[13b, 14] Also, other appli-cations using Staudinger ligation,[15] Diels–Alder reaction,[16]

oxime ligation,[17] “click-sulfonamide” reaction[18] or the photo-chemical thiol-ene reaction[19] were successfully applied forprotein immobilization. All these approaches use a stepwisemethodology, in which a modification is introduced into the

protein structure and subsequently followed by the immobili-zation reaction. The present method is a significant improve-ment. We introduce a “one-pot” approach of optimized EPLand CuAAC. The reactions run simultaneously in the samevessel and lead to specifically immobilized proteins retainingtheir native conformation and activity in the polymer-boundstate. This combined approach is time saving and unreactedreactants/protein are readily washed away.

As solid support we selected a PEGA resin.[20] This poly(ethy-lene glycol)-based resin is biocompatible and is able to swell10–20-fold in aqueous environments.[21] These swelling proper-ties allow proteins of up to 50 kDa to enter the PEG network.Thereby, the native conformation is not affected and enzymaticactivity is conserved.[21] The PEGA resin is also compatible withorganic synthesis, whereby specific functionalization of theresin can easily be achieved.[22]

Since copper, as a transition metal, can form reactive oxygenspecies and is known to bind thiol groups, its usage can causeproblems in the presence of biomolecules.[23] Thus, in the pres-ent approach only 1 mol % CuSO4 is used to minimize oxida-tive inactivation or modification of the protein. Furthermore,the tridentate ligand TBTA was used to complex the CuI spe-cies while conserving its catalytic activity. To our knowledge,we could demonstrate for the first time that the used Cu spe-cies have no influence on EPL.

TCEP as phosphine is known to be a good disulfide reducingagent.[24] It is also known to reduce azides in an effectivemanner at room temperature.[25] Nevertheless, it was shownthat TCEP in the presence of CuII and at 4 8C was not able toreduce azides within 48 h.[26] Therefore, we preincubated thecysteine containing peptide and CuSO4 with TCEP and TBTA at37 8C, pH 5.5, to reduce possible disulfide bonds and the CuII

to CuI. The immobilization reaction was performed at pH 8.0and 4 8C to prevent the azides from TCEP reduction. Usingthese conditions we were able to successfully immobilize theproteins on solid support.

The efficiency of immobilization was determined to be10.3(�2.6) % by cleavage of proteins from the resin followed

Figure 3. On-bead ELISA. The measured signal was the absorbance at450 nm. A) Ester modified resin; data are mean � standard error ; &: controlbeads without protein; &: beads after the immobilization reaction; &: beadstreated with NaOH after protein immobilization; results are from two inde-pendent experiments. B) Rink linker modified resin; data are mean � stan-dard error; &: control beads without protein; &: beads with immobilizedeGFP; &: beads treated with TFA after eGFP immobilization; results are fromthree independent experiments.

Figure 4. AKR1A1 enzyme activity. A) Sample measurement of the enzymaticactivity of unspecifically bound AKR1A1 compared to specifically immobi-lized AKR1A1 on PEGA beads; B) remaining activity determined by compari-son of soluble enzyme activity with immobilized enzyme activity ; mean� standard error ; * p<0.05; control was unspecifically retained AKR1A1 bythe azide derivatized resin in the absence of linker peptide; data representthree independent experiments for the control and six independent experi-ments for specific immobilization.

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by Bradford protein assay (data of four experiments in whichcleavage was quantitative according to ELISA). Treatment ofthe resin with NaOH (1 m) as cleavage reagent might have af-fected the protein,[27] and the total amount might even behigher. It is possible that partial hydrolysis takes place wherebythe protein is degraded. The subsequent elution of proteinfragments from the resin might also affect the results of theBradford assay. Lower NaOH concentrations could not be ap-plied because reduced cleavage rates were observed. Surpris-ingly, usage of the acid labile Rink linker was not successful(Figure 3 B). The results of the ELISA assay showed that thecleavage or separation of the immobilized proteins from theresin was not quantitative. Accordingly, the degree of immobi-lization could be significantly better than that determined bythe cleavage of the protein from the resin.

In addition to the cleavage procedure we determined theenzymatic activity of immobilized AKR1A1 in a photometricassay and compare this to the initial enzyme activity in solu-tion. The slope of NADPH consumption of immobilizedenzyme was calculated and compared to the specific enzymeactivity in solution prior to the reaction. This calculation resultsin the amount of immobilized AKR1A1. Compared to the activi-ty in solution the on-bead activity of specifically immobilizedAKR1A1 was 38.3(�7.2) % while the activity of unspecifically re-tained AKR1A1 in the control resin was 11.3(�3.1) %. The com-parison of these activities indicated an immobilization efficien-cy of about 30 %. As recently reported, specifically immobilizedAKR1A1 remains more than 70 % active upon site specific im-mobilization.[13a] Other studies on immobilized enzymes havereported that the kinetics mostly differ from the classical Mi-chealis–Menten kinetics.[28] Furthermore, the enzyme activitycould be reduced through interactions with the polymer or de-formations in the bound protein structure.[29] Such effectscannot be excluded in the present immobilization process andconsequently, the efficiency might be underestimated. Thecleavage procedures caused some protein precipitation, andthe determination of ligation efficiency by the enzymatic activi-ty may be considered more reliable.

Comparison between the reported immobilization reac-tions[13a] and our new approach is not feasible because of thedifference in calculations. While recent reports present their ef-ficiencies to modified protein, we have used the protein thio-ester as reference. Our previous experiments with a stepwiseapproach showed results similar to those of the one-pot reac-tion presented in this article (data not shown).

In conclusion, the presented method of EPL and protein im-mobilization is facile, rapid and reduces the number of re-quired protein purification steps. Herein, we have used pro-teins that need no disulfide bonds for their native structure.Since TCEP also reduces disulfide bridges in proteins, a refold-ing procedure should be applied after the immobilization. Ac-cordingly, the linker structure and length might have to be op-timized for every protein. We believe that the “one-pot” com-bination of EPL and CuAAC is generally applicable for produc-ing immobilized or modified proteins while preserving theiractive conformation.

Experimental Section

Solid-phase peptide synthesis: All Na-Fmoc protected aminoacids, Na-Boc protected cysteine and Fmoc-Rinkamide-AM-polystyr-ene resin were purchased from Iris Biotechnology, Novabiochem orBachem. Peptides were synthesized by using standard Fmoc/tert-butyl strategy. All coupling steps were performed manually byusing fivefold molar excess of the Fmoc l-amino acids, HOBt andDIC. 5(6)-Carboxytetramethylrhodamin (TAMRA; 1.5 equiv) was acti-vated with O-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyluroniumhexafluorophosphate (HATU; 1.5 equiv) and N,N-diisopropylethyl-amine (DIPEA; 1.5 equiv). The peptide was deprotected andcleaved from the resin with a mixture of TFA/thioanisol/ethandi-thiol (90:7:3 v/v/v). After being precipitated and washed in dry di-ethyl ether, the peptide was dissolved in H2O/tBuOH (3:1 v/v) andlyophilized. The crude peptide was purified by RP-HPLC by using aC18 Phenomenex column (90 �, 7.78 mm). A linear gradient ofwater/acetonitrile containing TFA (0.08 %) was applied (6 mL min�1,detection 220 nm, 20–60 % B in 50 min). Fractions were collectedand analyzed by RP-HPLC and MALDI-ToF mass spectrometry. Thepurity of the peptide was >95 %.

Synthesis of PEGA1900-azide resin: Functionalization was per-formed by using the Fmoc/tert-butyl strategy. Manual couplingsteps were carried out with coupling reagent (3 equiv), N-methyl-morpholine (NMM; 4 equiv) and O-(benzotriazol-1-yl)-N,N,N’,N’-tet-ramethyluronium tetrafluoroborate (TBTU; 2.8 equiv). Na-Fmoc-Ne-azido-Lys-OH was coupled to 4-(hydroxymethyl)benzoic amide(HMBA) by using 1-(2-mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole(MSNT; 4 equiv) and 1-methylimidazole (MeIm; 3 equiv). The finalamount of azido groups on the resin was determined by usingFmoc cleavage and measurement of absorption at 300 nm.

Protein expression and purification: The protein thioesters ofeGFP and AKR1A1 were generated as described previously.[30] Theexpression and purification process was monitored by SDS-PAGEand protein concentrations were determined by using the Bradfordprotein assay (Bio-Rad). The identities of protein thioesters were as-sessed by MALDI-ToF mass spectrometry.

Protein immobilization: All reagents were dissolved in reactionbuffer (20 mm HEPES, 0.5 m NaCl, pH 8.0) except the linker peptide,which was dissolved in H2O/tBuOH (3:1 v/v). Protein thioester(1 mL; 10–70 mm, in reaction buffer with 0.2 m MESNa) was addedto a small amount of azide derivatized PEGA resin (fourfold molarexcess of azide functionalities). The linker peptide (2 equiv) waspreincubated with CuSO4 (1 mol %), TBTA (2 mol %) and TCEP(10 equiv; relative to the protein). After incubation for 30 min at37 8C, pH 5.5, this mixture was added to the resin and the pH wasadjusted to 8.0. The immobilization reaction was continued 24 h at4 8C. Afterwards, the resin was washed three times with washingbuffer (1 mL; 20 mm HEPES, 0.5 m NaCl, 1 mm EDTA, 0.1 % Triton X-100, pH 8.0) and a further three times with reaction buffer (1 mL;20 mm HEPES, 0.5 m NaCl, pH 8.0).

Kinetic assay: AKR1A1 activity was determined by using a photo-metric assay. Glucuronic acid (100 mL; 500 mm) was used as sub-strate and NADPH (100 mL; 500 mm) as cofactor. The consumptionof NADPH was measured at 340 nm with a Cary 50 UV/Vis spectro-photometer from Varian. Measurements were carried out for 1 minwhen the soluble protein thioester was used, and for 30 min whenthe immobilized protein was used. The linear slope was deter-mined with the CaryWinUV software (v 3.00(182)).

Fluorescence microscopy of the PEGA beads: Fluorescence mi-croscopy studies were performed by using a Zeiss Axio Observer

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microscope. As software Axio Vision Rel. 4.6 was used. The PEGAbeads were transferred into 8-well m-slides, and fluorescence wasdetected by using the following filter sets: Axio-filter set 38(green), and filter set 31 (red).

On-bead ELISA: All antibodies were obtained from Santa Cruz Bio-technology, Inc. Bead suspension (100 mL) was incubated with a so-lution of BSA (1 mL; 1 mg mL�1) in Tris-buffered saline with Tween-20 (TBS-T) for 1 h at room temperature. After three washing stepswith TBS-T for 5 min each, the beads were incubated with mono-clonal mouse-anti-AKR1A1 antibody or rabbit-anti-GFP antibody(dilutions: 1:500 in TBS-T) for 2 h at room temperature. Again threewashing steps with TBS-T for 5 min each were performed. The sec-ondary antibodies, goat-anti-mouse conjugated with horseradishperoxidase (HRP) and goat-anti-rabbit–HRP were diluted 1:10 000in TBS-T and incubated with the resin for 2 h. Three times washingwith TBS-T for 5 min each was followed by 2.5 min incubation with3,3’,5,5’-tetramethylbenzidine (TMB) as developing reagent(500 mL). This reaction was stopped with HCl (500 mL; 0.25 m). Theabsorption of the solution was measured with TECAN infiniteM200 plate reader at 450 nm.

Cleavage of immobilization products: The proteins immobilizedon HMBA modified PEGA resins were cleaved two times withNaOH (1 mL; 1 m) treatment for 24 h. After elution of the cleavagemixture the pH was adjusted to 8.0 and the cleaved product waslyophilized. The lyophilisate was dissolved in water (200–500 mL)and protein concentration was determined by using the Bradfordprotein assay. Notably, lower NaOH concentrations (0.1 or 0.5 m) aswell as incubation times of 3 or 12 h did not cleave the whole pro-tein amount, as demonstrated by ELISA.

The proteins immobilized on Rink linker modified PEGA resin werecleaved two times with TFA (1 mL; 90 %) for 3–24 h. The cleavedproduct was precipitated in diethyl ether, dissolved in H2O andlyophilized.

After the cleavage procedure, the resin was washed with formicacid (1 mL; 100 %), NaOH (1 mL; 0.1 m) or reaction buffer (1 mL;20 mm HEPES, 0.5 m NaCl, pH 8.0) with different detergents (10 %Triton X-100, 1 % SDS, 1 % methylene glycol). Also, 20 min sonica-tion of the resin was applied. Inclusion of eluate from these wash-ing steps did not increase the recovery of the cleavage product.

Acknowledgements

We gratefully thank R. Reppich-Sacher for mass spectrometryanalysis. Funded in part by the European Union, the Free State ofSaxony and by the DFG (TR67, TPA4 and Graduate School LeipzigSchool of Natural Sciences–Building with Molecules and Nanoob-jects (BuildMoNa)).

Keywords: click chemistry · enzymes · expressed proteinligation · immobilization · multicomponent reactions

[1] S. F. Kingsmore, Nat. Rev. Drug Discovery 2006, 5, 310 – 320.[2] T. Cha, A. Guo, X. Y. Zhu, Proteomics 2005, 5, 416 – 419.[3] C. J. Noren, S. J. Anthony-Cahill, M. C. Griffith, P. G. Schultz, Science

1989, 244, 182 – 188.[4] a) G. MacBeath, S. L. Schreiber, Science 2000, 289, 1760 – 1763; b) C. D.

Hahn, C. Leitner, T. Weinbrenner, R. Schlapak, A. Tinazli, R. Tampe, B.

Lackner, C. Steindl, P. Hinterdorfer, H. J. Gruber, M. Holzl, BioconjugateChem. 2007, 18, 247 – 253.

[5] V. E. Ferrero, L. Andolfi, G. Di Nardo, S. J. Sadeghi, A. Fantuzzi, S. Canni-straro, G. Gilardi, Anal. Chem. 2008, 80, 8438 – 8446.

[6] P. E. Dawson, T. W. Muir, I. Clark-Lewis, S. B. Kent, Science 1994, 266,776 – 779.

[7] T. W. Muir, Annu. Rev. Biochem. 2003, 72, 249 – 289.[8] a) C. W. Tornøe, M. Meldal, Peptides : The Wave of the Future : Proceedings

of the 2nd International and the 17th American Peptide Symposium (Eds. :M. Lebl, R. A. Houghten), American Peptide Society, San Diego, 2001;b) C. W. Tornøe, C. Christensen, M. Meldal, J. Org. Chem. 2002, 67, 3057 –3064; c) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless, Angew.Chem. 2002, 114, 2708 – 2711; Angew. Chem. Int. Ed. 2002, 41, 2596 –2599.

[9] R. Huisgen, G. Szeimies, L. Mobius, Chem. Ber. Recl. 1967, 100, 2494 –2507.

[10] H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. 2001, 113, 2056 –2075; Angew. Chem. Int. Ed. 2001, 40, 2004 – 2021.

[11] S. Chong, F. B. Mersha, D. G. Comb, M. E. Scott, D. Landry, L. M. Vence,F. B. Perler, J. Benner, R. B. Kucera, C. A. Hirvonen, J. J. Pelletier, H.Paulus, M. Q. Xu, Gene 1997, 192, 271 – 281.

[12] M. Meldal, M. A. Juliano, A. M. Jansson, Tetrahedron Lett. 1997, 38,2531 – 2534.

[13] a) K. Holland-Nell, A. G. Beck-Sickinger, ChemBioChem 2007, 8, 1071 –1076; b) P. C. Lin, S. H. Ueng, M. C. Tseng, J. L. Ko, K. T. Huang, S. C. Yu,A. K. Adak, Y. J. Chen, C. C. Lin, Angew. Chem. 2006, 118, 4392 – 4396;Angew. Chem. Int. Ed. 2006, 45, 4286 – 4290.

[14] a) J. Xiao, T. J. Tolbert, Org. Lett. 2009, 11, 4144 – 4147; b) D. R. Elias, Z.Cheng, A. Tsourkas, Small 2010, 6, 2460 – 2468.

[15] A. Watzke, M. Kohn, M. Gutierrez-Rodriguez, R. Wacker, H. Schroder, R.Breinbauer, J. Kuhlmann, K. Alexandrov, C. M. Niemeyer, R. S. Goody, H.Waldmann, Angew. Chem. 2006, 118, 1436 – 1440; Angew. Chem. Int. Ed.2006, 45, 1408 – 1412.

[16] A. D. de Araujo, J. M. Palomo, J. Cramer, O. Seitz, K. Alexandrov, H. Wald-mann, Chem. Eur. J. 2006, 12, 6095 – 6109.

[17] E. H. Lempens, B. A. Helms, M. Merkx, E. W. Meijer, ChemBioChem 2009,10, 658 – 662.

[18] T. Govindaraju, P. Jonkheijm, L. Gogolin, H. Schroeder, C. F. Becker, C. M.Niemeyer, H. Waldmann, Chem. Commun. 2008, 3723 – 3725.

[19] D. Weinrich, P. C. Lin, P. Jonkheijm, U. T. Nguyen, H. Schroder, C. M. Nie-meyer, K. Alexandrov, R. Goody, H. Waldmann, Angew. Chem. 2010,9122, 1274 – 1279; Angew. Chem. Int. Ed. 2010, 49, 1252 – 1257.

[20] M. Renil, M. Ferreras, J. M. Delaisse, N. T. Foged, M. Meldal, J. Pept. Sci.1998, 4, 195 – 210.

[21] M. Meldal, F. I. Auzanneau, O. Hindsgaul, M. M. Palcic, J. Chem. Soc.Chem. Commun. 1994, 1849 – 1850.

[22] F. I. Auzanneau, M. Meldal, K. Bock, J. Pept. Sci. 1995, 1, 31 – 44.[23] M. E. Letelier, S. Sanchez-Jofre, L. Peredo-Silva, J. Cortes-Troncoso, P.

Aracena-Parks, Chem. Biol. Interact. 2010, 188, 220 – 227.[24] J. A. Burns, J. C. Butler, J. Moran, G. M. Whitesides, J. Org. Chem. 1991,

56, 2648 – 2650.[25] A. M. Faucher, C. Grand-Maitre, Synth. Commun. 2003, 33, 3503 – 3511.[26] Q. Wang, T. R. Chan, R. Hilgraf, V. V. Fokin, K. B. Sharpless, M. G. Finn, J.

Am. Chem. Soc. 2003, 125, 3192 – 3193.[27] S. Gulich, M. Linhult, S. Stahl, S. Hober, Protein Eng. 2002, 15, 835 – 842.[28] T. Kobayashi, K. J. Laidler, Biochim. Biophys. Acta Enzymol. 1973, 302, 1 –

12.[29] I. I. Willner, E. Katz, Angew. Chem. 2000, 112, 1230 – 1269; Angew. Chem.

Int. Ed. 2000, 39, 1180 – 1218.[30] a) Z. Machova, C. M�hle, U. Krauss, R. Tr�hin, A. Koch, H. P. Merkle, A. G.

Beck-Sickinger, ChemBioChem 2002, 3, 672 – 677; b) M. P. Richter, A. G.Beck-Sickinger, Protein Pept. Lett. 2005, 12, 777 – 781.

Received: July 7, 2011

Published online on September 8, 2011

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