Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via a click-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting their applications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids (2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B–N bond. This technology was applied in the selective modification of N-terminal cysteine-containing functional peptides

António, J. P. M.

Cavadas, R. A. N.

Gois, P. M. P.

Merino, P.

Padanha, R.

English

Repositorio Universidad de Zaragoza

N‑Terminal Cysteine Bioconjugation with (2-Cyanamidophenyl)boronic Acids Enables the Direct Formation ofBenzodiazaborines on PeptidesPublished as part of the Organic Letters virtual special issue “Chemoselective Methods for Labeling andModification of Peptides and Proteins”.Rita Padanha, Rafaela A. N. Cavadas, Pedro Merino, Joaõ P. M. António,* and Pedro M. P. Gois*Cite This: Org. Lett. 2023, 25, 5476−5480 Read OnlineACCESS Metrics & More Article Recommendations *sı Supporting InformationABSTRACT: Benzodiazaborines (BDABs) have emerged as a valuable tool to produce stable and functional bioconjugates via aclick-type transformation. However, the current available methods to install them on peptides lack bioorthogonality, limiting theirapplications. Here, we report a strategy to install BDABs directly on peptide chains using (2-cyanamidophenyl)boronic acids(2CyPBAs). The resulting BDAB is stabilized through the formation of a key intramolecular B−N bond. This technology wasapplied in the selective modification of N-terminal cysteine-containing functional peptides.In recent years, boron compounds evolved from usefulsynthetic reagents to central actors of different fields.1 Inchemical biology, the dynamic coordination profile of boron hasbeen extensively explored to prepare biologically activecompounds, to functionalize biomolecules, and to assemblebioconjugates with specific mechanisms to respond to differentstimuli.2−5 Among the different families of boron-containingmolecules, benzodiazaborines (BDABs) are a particularlynoteworthy class of boronic acid derivatives because they elicitan array of biological properties and exhibit a naphthoid isosterstructure that is now emerging as a powerful construction tool offunctional bioconjugates.6Dependent upon the substituent periphery, BDABs canexhibit very high stability in physiological conditions,7responsiveness to reactive oxygen species,8 and a click-typesynthesis using hydrazines and 2-carbonyl phenyl boronicacids.9−11 Despite these very positive features, the installation ofBDABs on peptide chains requires the initial bioconjugation ofhydrazine or 2-carbonyl phenyl boronic acid onto the peptide,followed by the addition of the second component. However,this reaction is poorly orthogonal because 2-carbonyl phenylboronic acids are well-known to react with ε-amino groups oflysine12,13 and N-terminal residues, like cysteine,14,15 which canlimit the post-functionalization with hydrazines. Therefore, thediscovery of a bioconjugation reagent that could generateBDABs directly on the peptide chain, using natural amino acidsas building blocks, would give direct access to this valuablefunctional core.Cyanamides are electrophilic warheads that have been used invarious inhibitors of cysteine proteases.16,17 However, thisfunction has been mostly overlooked in the development ofbioconjugation reagents targeting solvent-exposed cysteineresidues. A likely explanation for this lack of use is the factthat, upon reaction with Cys, cyanamide generates a thiourea-like function that in dilute aqueous conditions rapidlyReceived: June 5, 2023Published: July 19, 2023Letterpubs.acs.org/OrgLett© 2023 The Authors. Published byAmerican Chemical Society5476https://doi.org/10.1021/acs.orglett.3c01835Org. Lett. 2023, 25, 5476−5480Downloaded via UNIV DE ZARAGOZA on August 28, 2023 at 10:12:03 (UTC).See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.hydrolyzes to form urea. Considering this structure, weenvisioned that the incorporation of cyanamide in the orthoposition of a phenyl boronic acid (2CyPBA) would generate acysteine alkylation reagent that directly generates the BDABcore through the formation of an intramolecular B−N bond(Figure 1).To test this idea, we prepared 2CyPBA 1 and tested it in thereaction with 1.0 equiv of cysteine in NH4CH3CO2 (20 mM) atpH 7.0, and the reaction was monitored by electrosprayionization mass spectrometry (ESI−MS). After 24 h, proposedBDAB 2 was the major product observed (Figure 2B).Compound 2 was then isolated by semi-preparative high-performance liquid chromatography (HPLC) in 75% yield andfully characterized by nuclear magnetic resonance (NMR) andhigh-resolution mass spectrometry (HRMS). Differently,thiourea 4 is not stable under these conditions, which clearlyshowcases the importance of the proximal boronic acid functionto favor the bioconjugation reaction (Figure 4A).After our initial hypothesis was confirmed, we proceeded tooptimize the reaction conditions. As shown in Figure 2, BDAB 2was readily formed in a pH range of 5−8, with a preference forneutral and slightly basic pH values. Similarly, the reaction isfavored at higher concentrations, with 20 mM achieving cleanerreaction profiles than 2 and 10 mM (section 4 of the SupportingInformation).Then, to evaluate the kinetics of this reaction, we prepared a50 mM solution of cyanamide 1 in ND4CD3CO2 solution (20mM) at pH 7.0 and added 10 equiv of cysteine (Figure 3A). Thereaction was monitored by 1H NMR over 26 h, at 2 h intervals,Figure 1. Proposed strategy to install BDABs on peptide chains using(2-cyanamidophenyl)boronic acid (2CyPBA).Figure 2. Reaction of cyanamide 1 with cysteine (1.0 equiv) inNH4CH3CO2 (20mM), 25 °C, 20mM, and 24 h at pH (A) 5.0, (B) 7.0,and (C) 8.0.Figure 3. (A) Cyanamide 1 reacts with cysteine (10 equiv, 50 mM) inND4CD3CO2 (20 mM, pH 7.0) at room temperature (rt) to generateBDAB 2. (B) Reaction of compound 1 with cysteine (10 equiv) inND4CD3CO2 (20mM, pH 7.0) was followed by 1HNMR. (C) Stabilityof BDAB 2 (10 mM) was evaluated by HRMS in PBS at pH 7.4.Organic Letters pubs.acs.org/OrgLett Letterhttps://doi.org/10.1021/acs.orglett.3c01835Org. Lett. 2023, 25, 5476−54805477and a clean reaction was observed, with no visible intermediatespecies/side products being formed (section 5 of the SupportingInformation). From this experiment, a pseudo-first-order kobs of3.44 × 10−5 s−1 was calculated (Figure 3B). Moreover, BDAB 2displayed adequate stability in physiological conditions withdegradation half-lives of approximately 40 h in both phosphate-buffered saline (PBS) at pH 7.4 (Figure 3C) and the presence of10 equiv of glutathione (section 6 of the SupportingInformation).Next, we set out to expand the scope of the reaction and tounderstand its mechanism. As previously mentioned, boronicacid plays an important role in the reaction because, without it,the product formed is unstable and quickly hydrolyzed (Figure4A). Likewise, the reaction of compound 2 with acetylcysteineappears to block 2CyPBA reactivity, because BDAB 5 was notdetected by ESI−MS (Figure 4B). This result is very promising,because it unlocks the possibility of using this technology toselectively modify N-terminal cysteines in the presence of in-chain cysteines. Moreover, the presence of a methyl group incyanamide (compound 6) led to a sluggish reaction with a lowapparent conversion after 24 h when compared to the reactionwith cysteine (Figure 4C). Finally, the introduction of an estergroup in position 4 was well-tolerated and may act as aderivatization handle for future applications of this technology(Figure 4D and section 7 of the Supporting Information).Considering these results, we performed a detailed mecha-nistic study using density functional theory (DFT) calculationsto rationalize the experimental findings. Therefore, we studieddiazaborine formation with cyanamides 1 and 6. For the N-methyl derivative 6, the barrier is higher (28.5 kcal/mol versus24.4 kcal/mol for compound 1) as a result of steric hindranceexerted by the methyl group to the S attack, which closes theentry path (Figure 5A). On the other hand, two possibleorientations of the N-cyano group were considered for 2CyPBA1 (section 14 of the Supporting Information). As illustrated inFigure 5B, the most stable orientation model presents anadditional interaction between the NH group and oxygen ofboron ester. Furthermore, the reaction takes place through ahydrogen transfer from the thiol group mediated by the freeamino group of cysteine, which should be basic enough to acceptthe proton and promote the hydrogen transfer to the cyanogroup. The observed α-amine effect may justifiy the absence ofreactivity observed with N-acetyl cysteine, because theacetamido group is not protonated at pH 7 and is unable toundergo the initial hydrogen transfer step. After proton transferand nucleophilic S attack, hydrolysis of the boronate groupprovides the final diazaborine.After establishing the reaction mechanism, we beganevaluating its performance in more complex systems. Variousreports indicate that the neighboring amino acids may have animportant effect in bioconjugation efficiency.18,19 As such, weprepared a small library of cysteine-containing dipeptidesfeaturing hydrophobic, polar uncharged, and charged aminoacids and evaluated their bioconjugation with boronatedcyanamide 1. The reaction proceeds with moderate to highconversion rates with most dipeptides tested, with the exceptionof Cys-Glu, where only 51% conversion is observed (Figure 6).Moreover, no cross-reactivity was observed with othernucleophilic amino acids, such as lysine, histidine, serine,threonine, and tyrosine, which confirmed the proposedselectivity for cysteine (section 8 of the Supporting Informa-tion).Finally, we advanced to the modification of more complexpeptides. Cys-bombesin and C-ovalbumin, both featuring N-terminal cysteines, were incubated for 24 h with 10 equiv of2CyPBA 1 at pH 7.0, and high conversions (99 and 76%,respectively) were observed with both peptides. However, whenusing the GV-1001 peptide, which displays a C-terminalcysteine, no conjugation is observed after 24 h of incubationwith cyanamide 1 (Figure 7 and sections 9, 10, and 12 of theSupporting Information). These results validate the applicationof this technology for the modification of N-terminal cysteines.Then, we investigated the possibility of selectively modifyingN-terminal cysteine in the presence of other cysteines. For this,we designed a modified bombesin that displays bothN-terminalFigure 4. Scope and mechanistic studies by low-resolution massspectrometry (LRMS). (A) Absence of proximal boronic acid rendersthiourea 4 unstable and detected in low concentrations. (B) Compound5 is not detected as a result of the lack of reactivity between N-acetylcysteine and cyanamide 1. (C) Methylation of cyanamide 6 hinders itsreactivity, and compound 7 is detected with low conversion. (D)Introduction of an ester group in the meta position of cyanamide doesnot interfere with the formation of BDAB 9.Figure 5. Transition structures corresponding to BDAB formationusing cysteine and (A) cyanamide 6 and (B) cyanamide 1.Organic Letters pubs.acs.org/OrgLett Letterhttps://doi.org/10.1021/acs.orglett.3c01835Org. Lett. 2023, 25, 5476−54805478and in-chain cysteine (CysCys-bombesin). Upon incubationwith 10 equiv of compound 1 for 24 h at pH 7.0, the majorproduct observed corresponded to the single-modified con-jugate with 67% conversion (section 11 of the SupportingInformation).After confirming the selective modification of the N-terminalcysteine, we envisioned the construction of a dual-modifiedfunctional conjugate. First, we accomplished the selectivemodification of CysCys-bombesin with cyanamide 8, to obtainconjugate 24. Then, the addition of 10 equiv of maleimide 25promoted the alkylation of the in-chain cysteine, generating thedesired dual-modified conjugate 26 (Figure 8 and section 13 ofthe Supporting Information).In summary, in this work, we developed a novel methodologythat enables the direct installation of BDABs in peptides. Thismethodology is grounded on the role of proximal boronic acid instabilizing the addition of a cysteine to cyanamides. Wedemonstrated the BDAB 2 formation in ammonium acetatesolution preferably at neutral or slightly basic pH values and athigher reaction concentrations as well its stability underphysiological conditions. A detailed DFT study was performedto elucidate the mechanism of BDAB formation, which revealedthe importance of the B−N bond formation in the stabilizationof the conjugate and the role of the free amine group of N-terminal cysteines for the conjugation success. This method-ology is compatible with different amino acid side chains,resulting in dipeptide conjugates with moderate to highconversion rates. Particularly, these cysteine alkylation reagentsare selective for N-terminal cysteines, showing reactivity towardCys-bombesin and C-ovalbumin but not toward GV-1001. Withthis strategy, we obtained a dual modification of CysCys-bombesin with 2CyPBA 8 and maleimide 25 as derivatizationhandles. As future work, we intend to apply this strategy toprotein modification. Although natural proteins containing a N-terminal cysteine are rare, several methods for the production ofproteins with N-terminal cysteines are available, empoweringthe practical use of this site-specific bioconjugation strategy.Figure 6. Effect of the neighboring amino acid on the conjugationefficiency with 2CyPBA 1.Figure 7. Reaction of cyanamide 1 with peptides featuring cysteine indifferent positions (N-terminal, in-chain, and both).Figure 8. Sequential site-selective modification of CysCys-bombesinwith boronated cyanamide 8 and maleimide 25 to obtain dual-modifiedconjugate 26.Organic Letters pubs.acs.org/OrgLett Letterhttps://doi.org/10.1021/acs.orglett.3c01835Org. Lett. 2023, 25, 5476−54805479■ ASSOCIATED CONTENTData Availability StatementThe data underlying this study are available in the publishedarticle and its Supporting Information.*sı Supporting InformationThe Supporting Information is available free of charge athttps://pubs.acs.org/doi/10.1021/acs.orglett.3c01835.Experimental details, NMR spectra, ESI−MS spectra, andother materials (PDF)■ AUTHOR INFORMATIONCorresponding AuthorsPedro M. P. Gois − Research Institute for Medicines(iMed.ULisboa), Faculty of Pharmacy, Universidade deLisboa, 1649-003 Lisboa, Portugal; orcid.org/0000-0002-7698-630X; Email: pedrogois@ff.ulisboa.ptJoão P. M. António − Research Institute for Medicines(iMed.ULisboa), Faculty of Pharmacy, Universidade deLisboa, 1649-003 Lisboa, Portugal; Email: jantonio@ff.ulisboa.ptAuthorsRita Padanha − Research Institute for Medicines(iMed.ULisboa), Faculty of Pharmacy, Universidade deLisboa, 1649-003 Lisboa, PortugalRafaela A. N. Cavadas − Research Institute for Medicines(iMed.ULisboa), Faculty of Pharmacy, Universidade deLisboa, 1649-003 Lisboa, PortugalPedro Merino − Instituto de Biocomputación y Física deSistemas Complejos (BIFI), Universidad de Zaragoza, 50009Zaragoza, Spain; orcid.org/0000-0002-2202-3460Complete contact information is available at:https://pubs.acs.org/10.1021/acs.orglett.3c01835NotesThe authors declare no competing financial interest.■ ACKNOWLEDGMENTSThis work was generously supported by the PortugueseFundação para a Cien̂cia e Tecnologia, Ministério da Cien̂ciae da Tecnologia, Portugal [Grants SFRH/BD/150677/2020 (toRita Padanha), 2022.06817.CEECIND (to João P.M. António),PTDC/QUI-OUT/3989/2021 (to Pedro M. P. Gois), andInstitutional Grants UIDB/04138/2020 and UIDP/04138/2020 (iMed)]. The NMR spectrometers are part of thePortuguese National NMR Network (PT NMR) and arepartially supported by Infrastructure Project 022161 (co-financed by FEDER through COMPETE 2020, POCI, PORL,and FCT through PIDDAC). Pedro Merino thanks theMinisterio de Ciencia e Innovacion (Spain, Project PID2019-104090RB-100) and the Government of Aragón (Spain, GruposConsolidados, E34_20R) for financial support. The authorsthankfully acknowledge the resources from the supercomputers“Memento” and “Cierzo”, with technical expertise and assistanceprovided by BIFI−ZCAM (Universidad de Zaragoza, Spain).■ REFERENCES(1) Stubelius, A.; Lee, S.; Almutairi, A. The Chemistry of BoronicAcids in Nanomaterials for Drug Delivery. Acc. Chem. Res. 2019, 52(11), 3108−3119.(2) António, J. P. M.; Russo, R.; Carvalho, C. P.; Cal, P. M. S. D.; Gois,P. M. P. Boronic Acids as Building Blocks for the Construction ofTherapeutically Useful Bioconjugates. Chem. Soc. Rev. 2019, 48 (13),3513−3536.(3) Akgun, B.; Hall, D. G. 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N-terminal cysteine bioconjugation with (2-cyanamidophenyl)boronic acids enables the direct formation of benzodiazaborines on peptides

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N-terminal cysteine bioconjugation with (2-cyanamidophenyl)boronic acids enables the direct formation of benzodiazaborines on peptides

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