Herein we report a fundamental discovery on the use of tris(dialkylamino)phosphine reagents for peptide and protein modification. We discovered that C-terminal thiophosphonium species, which are uniquely stable, could be selectively and rapidly generated from their disulfide counterparts. In sharp and direct contrast, internal thiophosphonium species rapidly degrade to dehydroalanine. We demonstrate this remarkable chemoselectivity on a bis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as characterise the species in various small molecule/peptide studies

Aliev, Abil E

Baker, James R

Chrzastek, Alina

Chudasama, Vijay

Karu, Kersti

Spears, Richard J

Yap, Steven Y

English

UCL Discovery

This journal is © The Royal Society of Chemistry 2022 Chem. Commun.Cite this: DOI: 10.1039/d2cc01090aUnearthing the unique stability ofthiophosphonium-C-terminal cysteine adductson peptides and proteins†Richard J. Spears, Alina Chrzastek, Steven Y. Yap, Kersti Karu, Abil E. Aliev,James R. Baker * and Vijay Chudasama *Herein we report a fundamental discovery on the use oftris(dialkylamino)phosphine reagents for peptide and protein mod-ification. We discovered that C-terminal thiophosphonium species,which are uniquely stable, could be selectively and rapidly gener-ated from their disulfide counterparts. In sharp and direct contrast,internal thiophosphonium species rapidly degrade to dehydroala-nine. We demonstrate this remarkable chemoselectivity on abis-cysteine model peptide, and the formation of a stable C-terminal-thiophosphonium adduct on an antibody fragment, as well as charac-terise the species in various small molecule/peptide studies.Protein modification has established itself as a cornerstone ofchemical biology over the past two decades.1,2 Applications ofthis field are wide ranging; examples include design of imagingagents3 and novel therapeutics such as antibody–drug conju-gates (ADCs).4 Typically, protein modification occurs at anamino acid side chain, such as lysine5 or tyrosine.6,7 Cysteinein particular is regularly used due to the suitability of its thiolside chain8–11 (ease of incorporation via conventional site-directed mutagenesis, high nucleophilicity at neutral pH, lownatural abundance which assists in avoiding heterogeneity).12In addition to side chain modification, the N-terminus isfrequently used for site-specific modification due to the uniqueproperties of the a-amino group compared to those of the e-amino groups of lysine residues (e.g. pKa 6–8 vs. pKa ca. 10).13Occasionally, strategies targeting the N-terminus use both thea-amino group and amino acid side chain, as seen for N-terminal cysteine modification using cyanobenzothiazolederivatives.14,15 In contrast to the N-terminus or side chains,examples of chemical modification specific to the C-terminusare comparatively rare; enzymatic-based strategies tend todominate this area of research.16 This is logical given thechallenge of both activating and selectively targeting the car-boxylic acid group of the C-terminus in the presence of gluta-mate and aspartate carboxylic acid side chains. Currently, theonly two examples reported in the literature of chemicalbioconjugation specific for proteins bearing canonical aminoacid residues at the C-terminus include (i) an N - S acyltransfer of C-terminal cysteines to generate thioesters, whichcan be converted to hydrazides using subsequent nativechemical ligation (NCL)17 and (ii) photoredox-mediated decar-boxylative addition of Michael acceptors to the C-terminus,which selectively targets the C-terminus carboxylate over gluta-mate/aspartic acid carboxylates through differences in oxida-tive potentials.18 We therefore hypothesised that investigatingprotein modification strategies focused on the C-terminuswould be of high value. In particular, we envisaged thatpotential modifications specifically on the C-terminus sidechain could be ‘‘locked’’ into place by the adjacent carboxylicacid (akin to that of the a-amino group of the N-terminus for N-terminal modifications). We decided to focus on C-terminalcysteine, given cysteine’s aforementioned favourable proper-ties, anticipating that a modification strategy for a C-terminalcysteine may have a different outcome compared to modifica-tion of cysteine residues at internal positions within an aminoacid sequence. Moreover, several peptides and proteins (e.g. themajority of therapeutic full antibodies)19 contain a naturalC-terminal cysteine residue.During our investigation of chemical modification strategiesfocused at the C-terminus, our attention was turned towardstris(dialkylamino)phosphines. These reagents, specificallytris(dimethylamino)phosphine/hexamethylphosphorous tria-mide, (HMPT 1), have previously been reported for use inactivation of alkyl thiols towards nucleophilic displacementreactions in organic synthesis,20 desulfurization reactions21,22and in formation of dehydroalanine (Dha) on small moleculesand proteins.23 All of the aforementioned transformationsproceed via a thiophosphonium intermediate, which are tradi-tionally regarded as highly reactive and difficult to isolate.24 InUCL Department of Chemistry, 20 Gordon Street, London WC1H 0AJ, UK.E-mail: v.chudasama@ucl.ac.uk, j.r.baker@ucl.ac.uk† Electronic supplementary information (ESI) available. See https://doi.org/10.1039/d2cc01090aReceived 22nd February 2022,Accepted 1st April 2022DOI: 10.1039/d2cc01090arsc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 02 April 2022. Downloaded on 4/12/2022 3:04:58 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView JournalChem. Commun. This journal is © The Royal Society of Chemistry 2022some specific cases, however, the thiophosphonium intermedi-ate has been isolated either as a small molecule salt20 orstabilised through use of rotaxanes,24 or observed by LCMSupon treating a disulfide containing protein with HMPT 1.23We were particularly intrigued how the thiophosphoniummotif might behave when located at the C-terminus near thecarboxylate group, and whether or not the presence of a nearbycarboxylate may (i) impart stabilising interactions between thethiophosphonium cation and carboxylate, (ii) affect propensityof thiophosphonium species towards nucleophilic attack/desul-furisation, and (iii) affect the pKa of the cysteine a proton, andtherefore affect the likelihood of the thiophosphonium decom-position to give Dha.To begin with, we reacted N-acetyl cysteine 2 with 4-nitrophenyl disulfide 3 to synthesise model N-acetyl cysteinedisulfide 4 for use in validation of thiophosphonium for-mation. We chose to use unsymmetrical alkyl–aryl disulfides(S–SAr) as these have previously been reported to assist infacilitating regioselective phosphine attack at the alkyl sulfurof the S–SAr disulfide,25 generating the desired thiophospho-nium species. We initially screened a panel of commerciallyavailable phosphine reagents 1 and 5–11 against disulfide 4 inMeOH, and analysed formation of thiophosphonium by LCMSanalysis (Fig. 1a). Tris(dialkylamino)phosphines 1, 5, 6, and 7 allgave rise to thiophosphonium cations 12–15 (respectively) as judgedby LCMS. Minor thiophosphonium formation was noted in the caseof mixed N-/O-phosphine 8 to give 16, whereas no thiophosphoniumspecies 17–19 was detected when using mixed N-/O-phosphine 9and alkyl phosphines tris(2-carboxyethyl)phosphine (TCEP) 10 andtris(hydroxypropyl)phosphine (THPP) 11. We next sought to see ifone of these thiophosphonium species could both be formed inaqueous conditions and isolated for characterisation. We chosethiophoshonium 12 for this purpose, as HMPT 1 has been reportedbefore for formation of thiophosphonium intermediates. We foundthat thiophosphonium 12 could additionally be formed underaqueous conditions in H2O : MeCN (1 : 1) as judged by LCMS andsubsequently purified by semi-preparative high-pressure liquidchromatography (HPLC) in a 52% yield. 31P NMR of 12 revealed asingle peak at d 65.6 ppm, which was in accordance with previouslyreported 31P NMR data for thiophosphonium salts derived fromHMPT 1.26 Thiophosphonium 12 formation was further confirmedvia tandem mass spectrometry (MSMS), with a fragment corres-ponding to the thiophosphonium motif as the major speciesfollowing MS1 (Fig. 1b). Thiophosphonium 12 proved resistant todecomposition when incubated in 0.2 M PB pH 8 buffer for 18 hand, in buffer, proved inert towards nucleophiles previouslyreported to facilitate nucleophilic displacement of the thiopho-sphonium group (see ESI†).20 Thiophosphonium 12 also provedto be completely resistant to strongly acidic conditions (10 mM HCl,18 h, 37 1C), with only partial decomposition of the thiophospho-nium motif observed under strongly basic conditions (10 mMNaOH, 18 h, 37 1C, see ESI†).Next, thiophosphonium formation on small peptide unitswas investigated using HMPT 1. Peptides FEKGC 20, FCEKG 21and CFEKG 22 (containing cysteine at the C-terminus, at aninternal position and at the N-terminus respectively) weresynthesised via solid phase peptide synthesis (SPPS) and sub-sequently derivatised with Ellman’s reagent 23 to give thecorresponding disulfide-containing peptides 24 (C-terminal),25 (internal) and 26 (N-terminal, Fig. 2, see ESI† for full details).To begin with, peptide 24 was subjected to HMPT 1 in PBpH 8.0 (10% MeCN) at 37 1C, and the results were analysed byLCMS. Full conversion to thiophosphonium peptide species 27(Fig. 3a) was clearly observed at t = 5 min (as judged by LCMS,see ESI†). We additionally saw formation of a previouslyreported SAr thiophosphonium species (see ESI†).23 Thiopho-sphonium species 27 exhibited stability during incubation at37 1C at t = 1 h and t = 18 h (see ESI†) and MSMS analysisfurther confirmed the thiophosphonium as being located onthe C-terminus. We next treated peptide 25 with HMPT 1.Initially, this resulted in thiophosphonium 28 formation att = 5 min (see ESI†); in contrast to thiophosphonium 27,Fig. 1 (a) Formation of thiophosphonium species and detection by LCMS (b) synthesis of thiophosphonium 12 and 31P NMR and MSMS data forisolated 12.Communication ChemCommOpen Access Article. Published on 02 April 2022. Downloaded on 4/12/2022 3:04:58 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is © The Royal Society of Chemistry 2022 Chem. Commun.however, thiophosphonium 28 had almost completelydegraded at t = 1 h (as observed by LCMS, see ESI†). Wehypothesised, in accordance with previous literature, decom-position of the thiophosphonium species to Dha to give peptide29 was occurring following HMPT 1 addition (Fig. 3b). AlthoughDha peptide 29 could not be reliably observed in LCMS analy-sis, Dha formation was confirmed through thiol addition ofn-hexanethiol 30 to the reaction mixture of peptide 28 andHMPT 1 to give thiol-capped peptide 31, suggesting Dha 29 asthe intermediate resulting from thiophosphonium degradation(see ESI†). Finally, we treated peptide 26 with HMPT 1. Thio-phosphonium 32 formation was observed, with some degrada-tion after 18 h of incubation observed via LCMS (see ESI†). Weobserved a peak in the LCMS trace which could havecorresponded to the associated Dha peptide or ketone-containingpeptide 33, which could have formed through degradation ofthiophosphonium 32 to the corresponding Dha/enamine-containing peptide, followed by tautomerisation to an imine andfinally hydrolysis to the corresponding ketone 33. Addition of n-hexanethiol 30 to the reaction mixture gave no reaction as observedvia LCMS, whilst addition of benzyloxyamine 34 under slightlyacidic conditions gave a new peak in the LCMS trace correspondingto oxime product 35 (see ESI†). We thus hypothesised that the peakcorresponded to ketone-containing peptide 33.To further demonstrate this difference in reactivity, we nextturned our attention towards the model peptide FCEKAGC 36,which contains two cysteine residues; Cys2 at an internalposition and Cys7 at the C-terminus of the peptide. Followingderivatisation of the thiol groups with Ellman’s reagent 23 togive peptide 37, the peptide was then treated with HMPT 1 togenerate Dha at position 2 and a thiophosphonium adduct atposition 7 (peptide 38). Further treatment with n-hexanethiol30 subsequently gave dually modified peptide 39 (Fig. 4a),which was isolated as two diastereoisomers by preparative-HPLC and confirmed by LCMS and MSMS analysis (Fig. 4b).Finally, as a proof of concept we tested HMPT 1 within thecontext of modifying a protein containing a C-terminalcysteine. Specifically, we focused on modifying the C-terminalcysteine of the light chain of the herceptin (Trastuzumab) Fabfragment. Initially, reduction of the Fab disulfide bond andcapping of the majority of the heavy chain free thiol only wasperformed (see ESI† for details), i.e. to give the free thiol lightchain and thiol-capped heavy chain as the major products. TheFab light chain free thiol was then derivatised with Ellman’s reagentFig. 2 Cysteine-containing peptides and their disulfide-containingcounterparts.Fig. 3 (a) Reaction of peptide 24 with HMPT 1 to give the stable C-terminal thiophosphonium 27 as confirmed by LCMS and MSMS analysis. (b) Reactionof peptide 25 with HMPT 1 to give internal thiophosphonium 28, which rapidly degrades to Dha 29, which can be modified with n-hexanethiol 30 to givepeptide 31. (c) Reaction of peptide 26 with HMPT 1 to give N-terminal thiophosphonium 32 as a major product and ketone 33 as a minor product. Ketone33 can be functionalised with aminooxy 34 to give oxime product 35.ChemComm CommunicationOpen Access Article. Published on 02 April 2022. Downloaded on 4/12/2022 3:04:58 PM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineChem. Commun. This journal is © The Royal Society of Chemistry 2022to give the corresponding light chain aryl thiol disulfide. This wassubsequently treated with HMPT 1, which generated the thiopho-sphonium species with complete conversion from the Elman’scapped light chain. LCMS analysis confirmed the presence of theFab light chain thiophosphonium, with no decomposition to Dhaobserved (see ESI† for details).To conclude, we have demonstrated the synthesis of a thiopho-sphonium species based on N-acetyl cysteine, along with C-terminalcysteine-containing peptides and trastuzumab Fab light chain.These thiophosphonium species displayed unexpected stabilityand could be subsequently isolated and analysed by NMR and/orMS. In contrast, thiophosphonium formation at a cysteine posi-tioned internally within a peptide sequence resulted in rapiddecomposition to Dha. We hypothesise that the presence of anadjacent carboxylic acid imparts stabilising interactions that preventdecomposition of the C-terminal thiophosphonium, either throughionic interactions between the carboxylic acid and thiophospho-nium, or through a reduced propensity for abstraction of the alphaproton and subsequent collapse of the thiophosphonium species toDha. We intend to explore this difference in reactivity of C-terminaland internal cysteines for further development of protein modifica-tion strategies in future work, but share our current findings here inview of the high impact of this fundamental discovery.We gratefully acknowledge the Leverhulme Trust (RPG-2017-288, RPG-2020-010) and EPSRC (EP/T016043/1) for funding.Conflicts of interestV. C. and J. R. B. are Directors of the spin-out ThioLogics, butthere are no competing financial interests to declare.References1 C. D. Spicer and B. G. Davis, Nat. Commun., 2014, 5, 4740.2 E. A. Hoyt, P. M. S. D. Cal, B. L. Oliveira and G. J. L. Bernardes, Nat.Rev. Chem., 2019, 3, 147–171.3 P. Agarwal, B. J. Beahm, P. Shieh and C. R. Bertozzi, Angew. Chem.,Int. Ed., 2015, 54, 11504–11510.4 S. J. Walsh, J. D. Bargh, F. M. Dannheim, A. R. Hanby, H. Seki,A. J. Counsell, X. Ou, E. Fowler, N. Ashman, Y. Takada, A. Isidro-Llobet, J. S. Parker, J. S. Carroll and D. R. Spring, Chem. Soc. 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Unearthing the unique stability of thiophosphonium-C-terminal cysteine adducts on peptides and proteins

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Unearthing the unique stability of thiophosphonium-C-terminal cysteine adducts on peptides and proteins

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