Herein we report a thiol-labile cysteine protecting group based on an unsaturated pyridazinedione (PD) scaffold. We establish compatibility of the PD in conventional solid phase peptide synthesis (SPPS), showcasing this in the on-resin synthesis of biologically relevant oxytocin. Furthermore, we establish the applicability of the PD protecting group towards both microwave-assisted SPPS and native chemical ligation (NCL) in a model system

Bahou, C

Baker, JR

Chudasama, V

Forte, N

McMahon, C

Rochet, LNC

Shamsabadi, M

Spears, RJ

Thanasi, IA

Thoreau, F

English

UCL Discovery

This journal is © The Royal Society of Chemistry 2021 Chem. Commun.Cite this: DOI: 10.1039/d1cc03802hA novel thiol-labile cysteine protecting groupfor peptide synthesis based on a pyridazinedione(PD) scaffold†Richard J. Spears, Clı́ona McMahon, Monika Shamsabadi, Calise Bahou,Ioanna A. Thanasi, Léa N. C. Rochet, Nafsika Forte, Fabien Thoreau,James R. Baker * and Vijay Chudasama *Herein we report a thiol-labile cysteine protecting group based onan unsaturated pyridazinedione (PD) scaffold. We establish com-patibility of the PD in conventional solid phase peptide synthesis(SPPS), showcasing this in the on-resin synthesis of biologicallyrelevant oxytocin. Furthermore, we establish the applicability of thePD protecting group towards both microwave-assisted SPPS andnative chemical ligation (NCL) in a model system.Protecting group strategies for chemoselective protection anddeprotection, also referred to as ‘‘orthogonal’’ protectinggroups, have proved invaluable in the synthesis of complexbiomolecules.1 In particular, construction of peptides contain-ing multiple disulfide bonds frequently relies on orthogonalcysteine protecting groups to engineer the disulfide linkages ina regioselective manner.2 Examples of these include synthesisof hormones such as human insulin3 and bioactive moleculessuch as conotoxins.4 In addition, orthogonal cysteine protect-ing groups have also been used in protein semisynthesis5 andmore recently in the synthesis of homogeneous antibody drugconjugates (ADCs) bearing multiple payloads.6 The majority ofcommonly used cysteine protecting groups require acidic con-ditions and/or toxic heavy metals such as Hg for deprotection,which has thus resulted in a great deal of literature focusing ondesigning strategies that offer milder deprotection conditions.7,8In particular, thiol labile protecting groups show promise asorthogonal cysteine protecting groups as these can be removedunder mild reducing conditions in peptide synthesis. This hasprimarily been demonstrated with the tert-butylsulphenyl/tert-butylthio (StBu) protecting group.9 Deprotection of this groupis notably sluggish however, requiring deprotection times ofseveral hours or days depending on peptide sequence.10In an effort to address this, the S-dimethoxyphenylthio/S-trimethoxyphenylthio (S-Dmp/S-Tmp) groups11 and the sec-isoamylmercaptan/2-methyloxolane-3-thiol (SIT/MOT) groups12 have beenreported as replacements for StBu, offering significantly fasterdeprotection times albeit with minor instability towards stronglyacidic conditions/piperidine occasionally observed.11,12 As evi-denced by the development of S-Dmp/S-Tmp and SIT/MOT,expansion of the thiol-labile class of cysteine protecting groupswould greatly enhance the peptide chemist’s current toolkit.Over the last decade, we have reported pyridazinediones(PDs) as a class of multifunctional molecules for cysteinefunctionalisation.13 We have found that PDs have provedextremely versatile in organic synthesis and chemicalbiology14–16 and can be readily cleaved via addition of smallmolecule thiols in mild, aqueous conditions i.e. phosphatebuffer (PB) pH 7–8.13,17 Given these properties, we anticipatedthe PD would potentially be well suited to act as a novel, thiol-labile cysteine protecting group in peptide synthesis. We beganby validating the use of PDs as cysteine protecting groups inconventional 9-fluorenylmethoxycarbonyl solid phase peptidesynthesis (Fmoc SPPS). First, we performed a one-step reactionbetween Fmoc-Cys-OH 1 and bromopyridazinedione 2 to pre-pare a suitably protected model Fmoc-Cys(PD)-OH 3 in 71%yield (Fig. 1, see ESI† for full synthesis). Fmoc-Cys(PD)-OH 3was subsequently incorporated into peptides via standardFmoc SPPS, using 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HCTU) and N,N-diisopropylethylamine (DIPEA) coupling on a 2-chlorotritylFig. 1 Synthesis of the protected amino acid Fmoc-Cys(PD)-OH 3.UCL 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 DOI: 10.1039/d1cc03802hReceived 14th July 2021,Accepted 1st October 2021DOI: 10.1039/d1cc03802hrsc.li/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 01 October 2021. Downloaded on 11/18/2021 9:56:38 AM.  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 2021resin with Fmoc deprotections carried out using 20% piper-idine in DMF (5  2 min treatments). We envisaged that, uponcompletion of on-resin synthesis, TFA-mediated cleavage of thepeptide from the resin (TFA : TIS : H2O, 95 : 2.5 : 2.5) would givethe corresponding PD protected peptide, whereas the free thiol-containing peptide would be obtained instead if the resin wastreated with a suitable thiol-containing cocktail prior to resincleavage. Initially, we synthesised on-resin the protected 7-merpeptide Cys(PD)-Ser(tBu)-Leu-Tyr(tBu)-Arg(Pbf)-Ala-Gly, whichwas cleaved with TFA : TIS : H2O (95 : 2.5 : 2.5). The corres-ponding PD protected peptide H-Cys(PD)-Ser-Leu-Tyr-Arg-Ala-Gly-OH (C(PD)SLYRAG) 4 (Fig. 2a) was obtained as judged byliquid-chromatography mass spectrometry (LCMS), suggestingthe PD protecting group was stable to both the acidic and basicconditions used during Fmoc SPPS. Furthermore, crude pep-tide 4 was obtained in 495% purity as judged by high-performance liquid chromatography (HPLC) analysis. Next,we investigated conditions for on-resin deprotection of thePD protecting group to synthesise the corresponding CSLYRAGpeptide CSLYRAG 5 (Fig. 2b). We have previously establishedthat the unsaturated PD motif in protein–PD bioconjugates canbe cleaved by addition of excess thiols.18 With this in mind, weattempted on-resin PD deprotection with 1,4-dithiothreitol(DTT) due to its capacity to remove thiol-labile cysteine protect-ing groups and its comparative lack of toxicity/stench com-pared to other thiols such as b-mercaptoethanol (BME) and 1,2-ethanedithiol (EDT). Taking inspiration from the previouslydescribed S-Dmp/S-Tmp protecting groups,11 we initially trea-ted resin-bound peptide (following the final Fmoc deprotectioncycle) with a 5% DTT in DMF, 0.1 M N-methylmorpholine(NMM) deprotection solution (3 5 min treatments). Followingpeptide cleavage from the resin, we obtained the CSLYRAGpeptide CSLYRAG 5 with minimal deprotection of the PDobserved by LCMS (see ESI†). In an effort to increase thisconversion, we modified the deprotection solution used tocomprise an aqueous component, given that our previous workinvolving deconjugation of protein–PD bioconjugates has primarilybeen performed in aqueous buffer systems. We therefore treatedportions of resin-bound PD protected peptide (3  5 min treat-ments) with deprotection solutions containing either 5% or 10%DTT w/v with differing ratios of DMF and 5 mM PB pH 8.0,followed by peptide cleavage with TFA : TIS : H2O (95 : 2.5 : 2.5, with5% DTT w/v to ensure the liberated thiols were kept in the reducedform). Improved conversions (as judged by LCMS) to the corres-ponding peptide 5 were observed, particularly when using a 5% or10% DTT w/v in DMF : 5 mM phosphate buffer (8 : 2) deprotectionsolution (3  5 min treatments), of which the best results wereobserved when using 10% DTT w/v. Incorporation of Fmoc-Cys(PD)-OH 3 at an internal position within a peptide sequencewas then investigated. In a similar manner to the synthesis of 4 and5, we synthesised peptides SLYC(PD)RAG 6 (Fig. 2c) and SLYCRAG7 (Fig. 2d) which were obtained following resin cleavage andconfirmed by LCMS, with crude purities of 90% and 75% respec-tively as judged by HPLC. Additionally, we found that no racemisa-tion from L-Cys to D-Cys had occurred in the synthesis of SLYCRAG7 when using the Cys(PD) protecting group strategy (see ESI†). As adirect comparison for the lability of PD protecting group vs. theStBu protecting group, we also synthesised SLYC(StBu)RAG 8 inaddition to SLYC(PD)RAG 6 and attempted on-resin synthesis ofSLYCRAG 7 (Fig. 3a), with Cys deprotection performed using a 10%DTT w/v in DMF : 5 mM phosphate buffer (8 : 2) deprotectionsolution (3  5 min treatments) and resin cleavage achieved usingTFA : TIS : H2O (95 : 2.5 : 2.5, forgoing 5% DTT w/v addition in thecocktail for both peptides; this was done to avoid unintended StBudeprotection unrelated to prior DTT treatment). Synthesis ofSLYC(StBu)RAG 8 proceeded similarly to that of SLYC(PD)RAG 6,whereas attempts to synthesise the deprotected peptide 7 using theCys(StBu) protecting group strategy gave a mixture of deprotected7/protected peptide 8 (60% deprotected) as judged by HPLC, asopposed to near quantitative deprotection achieved using theCys(PD) protecting group strategy (490% deprotected, Fig. 3b).Overall, the PD protecting group shows faster deprotection timesthan those reported for the StBu protecting group10 and similar toS-Dmp/S-Tmp11 and SIT/MOT.12Fig. 2 Peptides synthesised (a) C(PD)SLYRAG 4, (b) CSLYRAG 5,(c) SLYC(PD)RAG 6 and (d) SLYCRAG 7.Fig. 3 (a) Outline of synthesis of peptides 6, 7, and 8. (b) HPLC dataobtained for attempted synthesis of peptides 6 and 8, along withattempted synthesis of 7.Communication ChemCommOpen Access Article. Published on 01 October 2021. Downloaded on 11/18/2021 9:56:38 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is © The Royal Society of Chemistry 2021 Chem. Commun.Previously published work featuring PDs reports that the unsa-turated motif typically displays an absorption maximum at ca.l = 300–350 nm, e.g. e345 = 9100 cm1 for a dithiopyridazinedione.19We hypothesised that on-resin PD deprotection would yield thiol–PD adducts that could be monitored by performing UV-vis analysisof wash solutions post incubation with a given resin-bound, PDprotected peptide (Fig. 4a). To test this hypothesis, we collected thewash solutions from resin-bound peptides that had been treatedwith our deprotection solutions and performed UV-vis analysis. Weobserved a large absorbance readout that decreased with eachwashing of the resin; for example, in the synthesis of SLYCRAG7 (Fig. 4b). This decrease in absorbance was also found to coincidewith efficient PD deprotection as judged by LCMS (following resincleavage). We found that UV-vis analysis of wash solutions collectedafter treating the resin with deprotection solution in this mannerproved to be a rapid and reliable way of tracking PD deprotectionduring peptide synthesis without the need to cleave portions of theresin-bound peptide to analyse deprotection by HPLC or LCMS. Wetherefore continued to use this method of analysis throughout theremainder of our studies of the PD protecting group.We next investigated the applicability of the PD protectinggroup towards the synthesis of peptides containing more thanone cysteine. To this end, we focused on synthesising oxytocin,a disulfide containing 9-mer peptide which can be used forvarious therapeutic applications such as induction of labour.20We performed on-resin peptide synthesis on a Rink Amideresin via Fmoc SPPS, with Fmoc-Cys(PD)-OH 3 incorporated atboth positions 1 and 6 of the peptide. Following the finalcoupling/Fmoc deprotection step and subsequent peptide clea-vage from the resin with TFA : TIS : H2O (95 : 2.5 : 2.5), successfulsynthesis of the crude PD protected linear oxytocin 9 wasconfirmed by LCMS analysis and obtained in 87% purity asjudged by HPLC. Next, we performed stepwise PD deprotectionand subsequent thiol oxidation for the on-resin synthesis ofoxytocin. PD deprotection of the resin-bound protected linearoxytocin was performed using a 10% w/v DTT in DMF : 5 mMphosphate buffer (8 : 2) deprotection solution (7  5 mintreatments, deprotection tracked by UV-vis analysis). Followingquantitative PD deprotection (as judged by HPLC/UV-vis analy-sis), on-resin oxidation of the resin-bound, protected, free thiol-containing linear oxytocin peptide was carried out usingN-chlorosuccinimide (NCS) as reported previously by Postmaand Albericio.21 Following thiol oxidation and peptide cleavagefrom the resin with TFA : TIS : H2O (95 : 2.5 : 2.5), the crudematerial was subsequently purified by semi-preparative HPLC,with purified oxytocin 10 confirmed by LCMS (Fig. 5a–d).Finally, we sought to investigate the applicability of the PDprotecting group within two different aspects of peptide synth-esis as a proof of concept. We first focused on the compatibilityof the PD protecting group with microwave-assisted peptidesynthesis. We synthesised SLYC(PD)RAG 6 on-resin usingmicrowave-assisted conditions (see ESI† for details) for Fmoc-amino acid couplings (Fig. 6a). As a test of stability, uponcompletion of on-resin synthesis we also subjected the resin-bound peptide to 20% piperidine in DMF under microwave-assisted conditions. The desired PD protected peptide wasobtained in a crude purity of 82% (Fig. 6b), suggesting the PDprotecting group was compatible with microwave-assistedconditions. Next, we turned our attention to native chemi-cal ligation (NCL),22 a well-documented strategy for thesynthesis of large peptides/proteins through reaction ofan N-terminal cysteine-containing peptide fragment and aFig. 4 (a) Outline monitoring on-resin PD deprotection by UV-vis analysisof wash solutions. (b) Example UV-vis traces of wash solutions in thesynthesis of SLYCRAG 7.Fig. 5 (a) SPPS of protected linear oxytocin 9. (b) SPPS of oxytocin 10. (c) HPLC data for the synthesis of oxytocin 10. (d) LCMS data for 9 and purified 10.ChemComm CommunicationOpen Access Article. Published on 01 October 2021. Downloaded on 11/18/2021 9:56:38 AM.  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 2021C-terminal thioester-containing fragment.23 We envisaged thataddition of thiol would act to deprotect the PD protecting groupto yield the N-terminal cysteine required for NCL and thatthis could be done in a one pot manner along with NCL inthe presence of a suitable thioester fragment. We attempteda one-pot PD deprotection/NCL procedure by incubatingC(PD)SLYRAG 4 with a model N-acetylglycine thioester 11 inthe presence of either DTT or 4-mercaptophenylacetic acid(MPAA) in ammonium acetate (NH4OAc) pH 6.8 for 18 h at37 1C (Fig. 6c). Total consumption of the PD protected peptide 4was observed, along with conversion to the anticipated NCLproduct Ac-GCSLYRAG-COOH 12 as judged by LCMS analysis(Fig. 6d).In conclusion, we have explored the use of pyridazinedionesin peptide synthesis, establishing the motif as a thiol-labileprotecting group compatible with Fmoc SPPS. We found on-resin deprotection of the PD could be achieved through addi-tion of thiols and monitored by UV-vis analysis. We havedemonstrated the applicability of the PD protecting groupwithin on-resin synthesis of disulfide-containing oxytocin,microwave-assisted synthesis and peptide coupling via a onepot deprotection/NCL procedure under aqueous conditions. Weanticipate the ability to track thiol deprotection by UV-visanalysis, as well as the potential to diversify the ethyl chainsof the PD scaffold with moieties such as affinity/solubility tagsand clickable handles will assist in enabling more complexpeptide syntheses. Furthermore, given its compatibility withaqueous conditions, we anticipate the PD protecting groupcould be applicable within research focused on designing moreenvironmentally-friendly, ‘‘greener’’ peptide synthesis.24We gratefully acknowledge the Leverhulme Trust (RPG-2017-288, 176274 and RPG-2020-010) for funding R. J. S. and C. J. B.(respectively), the EU (projects 859458 and 838703 respectively)for funding L. N. C. R. and F. T. (respectively), and the EPSRC(EP/T517793/1 and EP/R034621/1) for funding I. A. T. and N. F.(respectively). We also acknowledge the UCL Chemistry MassSpectrometry (MS) Facility (Dr Kersti Karu).Conflicts of interestV. C. and J. R. B. are Directors of the spin-out ThioLogics, butthere are no competing financial interests to declare.References1 A. Isidro-Llobet, M. Álvarez and F. Albericio, Chem. Rev., 2009, 109,2455–2504.2 R. He, J. Pan, J. P. Mayer and F. Liu, ChemBioChem, 2020, 21,1101–1111.3 K. Akaji, K. Fujino, T. Tatsumi and Y. Kiso, J. Am. Chem. Soc., 1993,115, 11384–11392.4 A. Cuthbertson and B. Indrevoll, Org. Lett., 2003, 5, 2955–2957.5 M. Jbara, S. K. Maity, M. Seenaiah and A. Brik, J. Am. Chem. Soc.,2016, 138, 5069–5075.6 M. R. Levengood, X. Zhang, J. H. Hunter, K. K. Emmerton,J. B. Miyamoto, T. S. Lewis and P. D. Senter, Angew. Chem., Int.Ed., 2017, 56, 733–737.7 R. J. Spears, C. McMahon and V. Chudasama, Chem. Soc. Rev., 2021,50, 11098–11155.8 S. Laps, G. Satish and A. Brik, Chem. Soc. Rev., 2021, 50, 2367–2387.9 R. Eritja, J. P. Ziehler-Martin, P. A. Walker, T. D. Lee, K. Legesse,F. Albericio and B. E. Kaplan, Tetrahedron, 1987, 43, 2675–2680.10 M. Góngora-Benı́tez, J. Tulla-Puche, M. Paradı́s-Bas, O. Werbitzky,M. Giraud and F. Albericio, Biopolymers, 2011, 96, 69–80.11 T. M. Postma, M. Giraud and F. Albericio, Org. Lett., 2012, 14,5468–5471.12 A. Chakraborty, A. Sharma, F. Albericio and B. G. de la Torre,Org. Lett., 2020, 22, 9644–9647.13 V. Chudasama, M. E. B. Smith, F. F. Schumacher, D. Papaioannou,G. Waksman, J. R. Baker and S. Caddick, Chem. Commun., 2011, 47,8781–8783.14 J. C. F. Nogueira, K. Paliashvili, A. Bradford, F. Di Maggio,D. A. Richards, R. M. Day and V. Chudasama, Org. Biomol. Chem.,2020, 18, 2215–2218.15 C. Bahou, R. J. Spears, A. E. Aliev, A. Maruani, M. Fernandez,F. Javaid, P. A. Szijj, J. R. Baker and V. Chudasama, Chem. Commun.,2019, 55, 14829–14832.16 A. Maruani, P. A. Szijj, C. Bahou, J. C. F. Nogueira, S. Caddick,J. R. Baker and V. Chudasama, Bioconjugate Chem., 2020, 31,520–529.17 M. Fernández, A. Shamsabadi and V. Chudasama, Chem. Commun.,2020, 56, 1125–1128.18 C. Bahou, P. A. Szijj, R. J. Spears, A. Wall, F. Javaid, A. Sattikar,E. A. Love, J. R. Baker and V. Chudasama, Bioconjugate Chem., 2021,32, 672–679.19 A. Maruani, M. E. B. Smith, E. Miranda, K. A. Chester,V. Chudasama and S. Caddick, Nat. Commun., 2015, 6, 6645.20 J. M. Shyken and R. H. Petrie, Clin. Obstet. Gynecol., 1995, 38,232–245.21 T. M. Postma and F. Albericio, Org. Lett., 2013, 15, 616–619.22 P. E. Dawson, T. W. Muir, I. Clark-Lewis and S. B. Kent,Science, 1994, 266, 776–779.23 V. Agouridas, O. El Mahdi, V. Diemer, M. Cargoët, J.-C. M. Monbaliuand O. Melnyk, Chem. Rev., 2019, 119, 7328–7443.24 O. Al Musaimi, B. G. de la Torre and F. Albericio, Green Chem., 2020,22, 996–1018.Fig. 6 (a) Outline of microwave-assisted synthesis of SLYC(PD)RAG 6.(b) HPLC data obtained. (c) Outline of Ac-GCSLYRAG 12 synthesis. (d)LCMS data of crude reaction containing peaks corresponding to 12.Communication ChemCommOpen Access Article. Published on 01 October 2021. Downloaded on 11/18/2021 9:56:38 AM.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

A novel thiol-labile cysteine protecting group for peptide synthesis based on a pyridazinedione (PD) scaffold

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A novel thiol-labile cysteine protecting group for peptide synthesis based on a pyridazinedione (PD) scaffold

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UCL Discovery