The site specific attachment of the reactive TMTHSI-click handle to the N-terminus of peptides and proteins is described. The resulting molecular constructs can be used in strain-promoted azide alkyne cycloaddition (SPAAC) for reaction with azide containing proteins e.g., antibodies, peptides, nanoparticles, fluorescent dyes, chelators for radioactive isotopes and SPR-chips etc

Dijkgraaf, Ingrid

Hauwert, Niels J

Liskamp, Rob M J

Peeters, Wouter

Rijcken, Cristianne J F

Timmers, Matt

Vermonden, Tina

English

Utrecht University Repository

This journal is © The Royal Society of Chemistry 2023 Chem. Commun., 2023, 59, 11397–11400 |  11397Cite this: Chem. Commun., 2023,59, 11397Specific N-terminal attachmentof TMTHSI linkers to native peptidesand proteins for strain-promoted azidealkyne cycloaddition†Matt Timmers, *ab Wouter Peeters,c Niels J. Hauwert, dCristianne J. F. Rijcken,b Tina Vermonden, a Ingrid Dijkgraaf *c andRob M. J. Liskamp *bceThe site specific attachment of the reactive TMTHSI-click handle tothe N-terminus of peptides and proteins is described. The resultingmolecular constructs can be used in strain-promoted azide alkynecycloaddition (SPAAC) for reaction with azide containing proteinse.g., antibodies, peptides, nanoparticles, fluorescent dyes, chelatorsfor radioactive isotopes and SPR-chips etc.Selective modification of the N-terminus of peptides or proteinsremains a challenge in view of the usually abundant presence offunctional and hence reactive side chains of amino acids.However, in many cases site specific N-terminal modificationis required to circumvent interference with active or bindingdomains. These domains play crucial roles in the biologicalactivity of for example antibodies, cell penetrating peptides,signalling or immunologically active peptides and as a conse-quence should not be modified. Recently, MacDonald et al.1have developed a highly suitable reagent for specific N-terminalmodification of native peptides and proteins. They showed aspecific reaction of the 2-pyridinecarboxaldehyde (2PCA)moiety with the N-terminus of peptides and proteins. Theaim of this research was to combine this versatile 2PCA reagentwith a highly reactive click moiety capable of a fast strainpromoted azide alkyne cycloaddition (SPAAC). After N-terminalspecific introduction of this novel molecular combination theclick moiety can then undergo a mild and efficient reaction withrelatively easy accessible azide containing compounds, not requir-ing the presence of a copper catalyst‡. We realised that it might bechallenging to develop a molecular combination of a reactive clickmoiety and 2PCA followed by its specific N-terminal introduction.However, the relatively easy accessibility of a diversity of azidespresent in antibodies and other proteins, peptides, nucleic acids,nano particles, fluorescent dyes, chelators for radioactive isotopes,SPR surfaces etc. made this the preferred approach instead ofsynthesis of an azide containing 2PCA reagent, which then has toreact with much more difficult accessible click moiety containingantibodies etc.We wished to combine the 2PCA reagent with our recentlypublished tetramethylthiocycloheptyne sulfoximine (TMTHSI)-click handle.2 This click reagent is the fastest reacting SPAACreagent to date. TMTHSI is small, and not sterically crowded toreduce any observer effect, that is a biological response inducedby the steric bulk of the reagent on the formed construct.2,3This molecular combination of TMTHSI and 2PCA should thenspecifically react with the N-terminus of a peptide or proteinafter which a SPAAC reaction is possible. Thus, we describe thesynthesis of TMTHSI-2PCA linker constructs, show the specificN-terminal introduction onto peptides and a protein as well aspurification of the obtained molecular constructs. Finally, SPAACof selected TMTHSI molecular constructs is illustrated especiallyby the successful preparation of a protein construct from an azidecontaining protein component and the N-terminal TMTHSImoiety containing Evasin-3 protein.The first click construct (3) was conveniently obtained in50% yield by combining piperazine-2PCA (2) and TMTHSI in areaction with our earlier described TMTHSI-hydroxy succini-mide derivative 1 (Scheme 1 and ESI†).Octreotide 4, a clinically used peptide for the treatment ofacromegaly, was used for selective coupling to this new TMTHSI-2PCA reagent 3. Since octreotide is a cyclized peptide, containingseveral functionalities including a lysine side chain NH2 and adisulfide bridge, it is a relevant model to evaluate the N-terminalspecificity of reagent 3. After reaction with 15 eq. of TMTHSI-2PCA 3,a 490% conversion was achieved based on the decrease of octreo-tide peak shown by HPLC (Scheme 2, details in ESI,† Section S2).a Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences,Utrecht University, Utrecht 3584 CG, The Netherlandsb Cristal Therapeutics, Maastricht 6229 EV, The Netherlandsc Department of Biochemistry, Cardiovascular Research Institute Maastricht(CARIM), Maastricht University, Maastricht 6229 ER, The Netherlandsd Symeres, Kerkenbos 1013, Nijmegen 6546 BB, The Netherlandse School of Chemistry, University of Glasgow, Glasgow G12 8QQ, UK.E-mail: robert.liskamp@glasgow.ac.uk† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3cc03397jReceived 14th July 2023,Accepted 30th August 2023DOI: 10.1039/d3cc03397jrsc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 30 August 2023. Downloaded on 10/23/2023 10:08:48 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineView Journal  | View Issue11398 |  Chem. Commun., 2023, 59, 11397–11400 This journal is © The Royal Society of Chemistry 2023Octreotide was selectively functionalized on the N-terminus aswas proven by LC-MS (Fig. 1). Not only was the expected mass ofthe modified octreotide observed, but also proof of the occur-rence of cyclization, resulting from the N-terminal specificEdman-degradation-like cyclization reaction4 was apparentfrom the presence of two diastereomers, having the same massand which could be separated by LC-MS.Next, we exposed the oncolytic LTX-315 peptide5,6 6 contain-ing several (five) lysine residues to TMTHSI-2PCA 3 (Scheme 3).Here too, a LTX peptide adduct 7 was formed having only oneTMTHSI-linker moiety (Fig. 2). As expected, no (stable) Schiffbase adducts were formed with the side chains of the lysineresidues. As with octreotide, the N-terminal modification ofLTX-315 was also apparent from the formation of a diastereo-meric mixture (Fig. 2).Thus, the TMTHSI-2PCA 3 reagent is a suitable reagent forN-terminus site specific introduction of a TMTHSI-containinglinker to peptides.For larger peptides and proteins, a TMTHSI derivative (16) witha larger distance between the aldehyde reacting with the N-terminus and the TMTHSI-moiety was envisioned (Scheme 4), toavoid any steric encumbrance in both the N-terminal specificreaction and the subsequent SPAAC. A suitable distance betweenthe SPAAC moiety and the site of attachment of the reagent in asizable peptide or protein will be beneficial for the final biocon-jugate. The larger distance formed by an ethylene glycol spacermay also be favourable for water solubility of the construct. TheTMTHSI-glycol-2PCA derivative 16 was obtained in a straight-forward 7-step synthesis in an overall yield of 25% starting fromethylene glycol derivative 8 and pyridine bismethylene alcohol11 (Scheme 4, details in ESI,† Section S3).This TMTHSI-glycol-2PCA derivative 16 was used in the N-ter-minal modification of the peptide angiopep2 17 (Scheme 5).Angiopep2 is a conjugate peptide to facilitate intracellular deliveryand blood–brain barrier transport of oligonucleotides.7–10 Thecrude reaction mixture 18 required extensive purification to removethe excess of unreacted TMTHSI-glycol-2PCA 16. Fortunately, excessScheme 1 Synthesis of TMTHSI-2PCA 3.Scheme 2 Reaction of TMTHSI-2PCA 3 with octreotide 4.Fig. 1 HPLC-trace of TMTHSI-2PCA reaction with octreotide.Scheme 3 Reaction of TMTHSI-2PCA 3 with LTX 6 containing severallysine residues.Fig. 2 HPLC trace of the TMTHSI-2PCA reaction with LTX-315.Communication ChemCommOpen Access Article. Published on 30 August 2023. Downloaded on 10/23/2023 10:08:48 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article OnlineThis journal is © The Royal Society of Chemistry 2023 Chem. Commun., 2023, 59, 11397–11400 |  11399of TMTHSI-glycol-2PCA can be removed by incubation withhydroxylamine-containing beads (Scheme 5). These beads cap-ture the aldehyde of TMTHSI-glycol-2PCA by formation ofan oxime, which is illustrated in the work-up of the reactionof peptide TP10 (AGYLLGKINLKALAALAKKIL, membrane-transporting peptide11–13) 17 with hydroxyl amine beads(Scheme 5, bottom and ESI,† Section S4).The TMTHSI-moiety integrity and reactivity was monitoredby addition of azido-ethanol to the worked-up product of theN-terminal modified TP10 peptide (Scheme 6). This resulted ina complete shift of the diastereomeric peptides to the left, amore polar region of the HPLC-trace indicating a full reactivityof the TMTHSI-modified peptide (ESI,† Section S5).Finally, TMTHSI-glycol-2PCA was evaluated for its ability tospecifically react with the N-terminus of a protein. Here, Evasin-3 19 was selected, This is a protein which shows a high affinityto CXC-type chemokines, in particular CXCL1 and CXCL8,inflammatory chemokines associated with the developmentof numerous diseases14 such as cancer and atherosclerosis.N-terminal modification of 19 with 16 will enable attachment ofthe bioconjugate to a bacterial chimeric designer protein(CDP)15 to boost the immune recognition (Fig. 3).Thus, 12 equivalents of 16 were reacted with Evasin-3 19 for26 hours. While no full conversion of Evasin-3 19 was observed(ESI,† Section S6), separation on HPLC was possible and there-fore we could purify Evasin-3-TMTHSI 20 by preparative HPLCScheme 4 Synthesis of TMTHSI-glycol-2PCA 16.Scheme 5 Reaction of 17 with TMTHSI-glycol-2PCA (top) and capture ofexcess of TMTHSI-glycol-2PCA 16 using hydroxyl amine beads (bottom).Scheme 6 Click reaction of the TMTHSI-glycol-2PCA peptide constructwith azido ethanol.Fig. 3 Representation of the reaction of Evasin-3 19 with TMTHSI-glycol-2PCA 16 leading to molecular construct 20 (top HPLC trace of purified 20),followed by reaction with CDP-azide 21 leading to Evasin-3-CDP 22(purified 22, bottom HPLC trace).ChemComm CommunicationOpen Access Article. Published on 30 August 2023. Downloaded on 10/23/2023 10:08:48 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online11400 |  Chem. Commun., 2023, 59, 11397–11400 This journal is © The Royal Society of Chemistry 2023in a yield of 40%. Considering that only 12 equivalents of 16were used and preparative HPLC purification was performed, ayield of 40% of pure product was quite satisfactory.Next, conjugation with azide-functionalised CDP 21 wascarried out (Fig. 3). Two equivalents of Evasin-3-TMTHSI 20were added dropwise to 1 equivalent of CDP-azide 21. Afterreacting for 3.5 hours, the reaction mixture was purified usingpreparative HPLC. This resulted in a pure Evasin-CDP conju-gate 22, as was confirmed by MS. The yield of this conjugateafter purification was 60%, which is certainly acceptable con-sidering loss of product during preparative HPLC.We have synthesized clickable, N-terminal site-specific con-structs TMTHSI-2PCA and TMTHSI-glycol-2PCA. The synthesisof both derivatives proceeded without problems, which cer-tainly is not obvious using very reactive click compounds (e.g.,BARAC).16 These reagents have proven to be N-terminal speci-fic, and the reaction via cyclization was demonstrated byobservation of the formation of diastereomers on HPLC andconfirmed by mass spectrometry. Pleasantly, also in theN-terminal reaction no side products have been observeddespite the possible formation of an intermediate diene, inprinciple capable of reacting with the TMTHSI-moiety.4Furthermore, we found that it was possible to use a muchsmaller excess of 2PCA-reagent than was earlier reported1 andthat if necessary, excess can be efficiently removed by captureusing functionalized (hydroxyl amine containing) beads. Thispurification strategy together with HPLC purification has beenused to determine yields which is an improvement compared todetermination of conversion yields solely based on MS.1These clickable N-terminal specific constructs can also beused in larger proteins, as was demonstrated by the clicking ofEvasin-3 with CDP-azide. These combined findings demon-strate the high potential of these TMTHSI-2PCA reagents forin-solution N-terminal specific introduction of a click handle inpeptides and proteins. As such this approach affords pureN-terminally click-reactive peptides and proteins leaving theirinternal functional domains unaffected. It is expected that thisspecific bioconjugation strategy will soon enable a wealth oftherapeutic and diagnostic applications facilitated by the con-venient SPAAC-based possible introduction of a variety of azidecontaining (bio)molecular constructs originating from antibo-dies, nucleic acids, nanoparticles, surfaces etc.Conflicts of interestThere are no conflicts to declare.Notes and references‡ Copper catalysts are usually considered toxic and therefore have to beremoved carefully, before any use of the resulting molecular constructsin vitro or in vivo. In some cases this is even hardly possible, e.g., when ametal chelator has to be introduced for a radio-active isotope which canalso bind copper-ions.1 J. I. MacDonald, H. K. Munch, T. Moore and M. B. Francis, One-stepsite-specific modification of native proteins with 2-pyridinecar-boxyaldehydes, Nat. Chem. Biol., 2015, 11(5), 326–331.2 J. Weterings, C. J. F. Rijcken, H. Veldhuis, T. Meulemans, D. Hadavi,M. Timmers, M. Honing, H. Ippel and R. M. J. Liskamp, TMTHSI, asuperior 7-membered ring alkyne containing reagent for strain-promoted azide–alkyne cycloaddition reactions, Chem. Sci., 2020,11(33), 9011–9016.3 S. B. Engelsma, L. I. Willems, C. E. Van Paaschen, S. I. Van Kasteren,G. A. Van Der Marel, H. S. Overkleeft and D. V. Filippov,Acylazetine as a dienophile in bioorthogonal inverse electron-demand Diels–Alder ligation, Org. Lett., 2014, 16(10), 2744–2747.4 F. P. J. T. Rutjes, How to pick a single amine?, Nat. Chem. Biol., 2015,11(5), 306–307.5 B. Sveinbjørnsson, K. A. Camilio, B. E. Haug and Ø. Rekdal,LTX-315: a first-in-class oncolytic peptide that reprogramsthe tumor microenvironment, Future Med. Chem., 2017, 9(12),1339–1344.6 H. Zhou, S. Forveille, A. Sauvat, T. Yamazaki, L. Senovilla, Y. Ma,P. Liu, H. Yang, L. Bezu and K. Müller, et al., The oncolytic peptideLTX-315 triggers immunogenic cell death, Cell Death Dis., 2016,7(3), e2134.7 H. Gao, S. Zhang, S. Cao, Z. Yang, Z. Pang and X. Jiang, Angiopep-2and activatable cell-penetrating peptide dual-functionalized nano-particles for systemic glioma-targeting delivery, Mol. Pharmaceutics,2014, 11(8), 2755–2763.8 S. Habib and M. Singh, Angiopep-2-Modified Nanoparticles forBrain-Directed Delivery of Therapeutics: A Review, Polymers, 2022,14(4), 712.9 Y. Lei, S. Chen, X. Zeng, Y. Meng, C. Chang and G. Zheng, Angiopep-2 and cyclic RGD dual-targeting ligand modified micelles across theblood–brain barrier for improved anti-tumor activity, J. Appl. Polym.Sci., 2022, 139(24), 52358.10 X. X. Shi, W. M. Miao, D. W. Pang, J. S. Wu, Q. S. Tong, J. X. Li,J. Q. Luo, W. Y. Li, J. Z. Du and J. Wang, Angiopep-2 conjugatednanoparticles loaded with doxorubicin for the treatment of primarycentral nervous system lymphoma, Biomater. Sci., 2020, 8(5),1290–1297.11 M. Z. Islam, H. Ariyama, J. M. Alam and M. Yamazaki, Entry of cell-penetrating peptide transportan 10 into a single vesicle by translo-cating across lipid membrane and its induced pores, Biochemistry,2014, 53(2), 386–396.12 U. Soomets, M. Lindgren, X. Gallet, M. Hällbrink, A. Elmquist,L. Balaspiri, M. Zorko, M. Pooga, R. Brasseur and Ü. Langel, Dele-tion analogues of transportan, Biochem. Biophys. Acta, 2000, 1467(1),165–176.13 C. Zhang, W. Ren, Q. Liu, Z. Tan, J. Li and C. Tong, Transportan-derived cell-penetrating peptide delivers siRNA to inhibit replicationof influenza virus in vivo, Drug Des., Dev. Ther., 2019, 13,1059–1068.14 S. S. Denisov, J. H. Ippel, A. C. A. Heinzmann, R. R. Koenen,A. Ortega-Gomez, O. Soehnlein, T. M. Hackeng and I. Dijkgraaf,Tick saliva protein Evasin-3 modulates chemotaxis by disruptingCXCL8 interactions with glycosaminoglycans and CXCR2, J. Biol.Chem., 2019, 294(33), 12370–12379.15 A. Blanas, H. Karsjens, A. de Ligt, E. J. M. Huijbers, K. van Loon,S. S. Denisov, C. Durukan, D. J. M. Engbersen, J. Groen andS. Hennig, et al., Vaccination with a bacterial peptide conjugatedto SARS-CoV-2 receptor-binding domain accelerates immunity andprotects against COVID-19, Science, 2022, 25(8), 104719.16 M. F. Debets, J. C. M. van Hest and F. P. J. T. Rutjes, Org. Biomol.Chem., 2013, 11, 6439–6455.Communication ChemCommOpen Access Article. Published on 30 August 2023. Downloaded on 10/23/2023 10:08:48 AM.  This article is licensed under a Creative Commons Attribution-NonCommercial 3.0 Unported Licence.View Article Online

Specific N-terminal attachment of TMTHSI linkers to native peptides and proteins for strain-promoted azide alkyne cycloaddition

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Specific N-terminal attachment of TMTHSI linkers to native peptides and proteins for strain-promoted azide alkyne cycloaddition

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