Tuning the properties of maleimide reagents holds significant promise in expanding the toolbox of available methods for bioconjugation. Herein we describe aryloxymaleimides which represent 'next generation maleimides' of attenuated reactivity, and demonstrate their ability to enable new methods for protein modification at disulfide bonds

Baker, JR

Chester, KA

Fletcher, SA

Jones, LH

Kossen, H

Marculescu, C

Mayer, P

Morgan, RE

Tolner, B

UCL Discovery

This journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 7139--7142 | 7139Cite this:Chem. Commun., 2014,50, 7139Aryloxymaleimides for cysteine modification,disulfide bridging and the dual functionalizationof disulfide bonds†Cristina Marculescu,a Hanno Kossen,a Rachel E. Morgan,a Patrick Mayer,aSally A. Fletcher,a Berend Tolner,b Kerry A. Chester,b Lyn H. Jonesc andJames R. Baker*aTuning the properties of maleimide reagents holds significant promise inexpanding the toolbox of available methods for bioconjugation. Hereinwe describe aryloxymaleimides which represent ‘next generationmaleimides’ of attenuated reactivity, and demonstrate their abilityto enable new methods for protein modification at disulfide bonds.The maleimide motif 1 is a highly reactive conjugate acceptor andcan be employed eﬀectively in selective reactions with thiols, such ascysteine residues in proteins.1 As a result it is one of the most widelyused functional groups in bioconjugation (Scheme 1). We haverecently described bromomaleimides 3 as the parent members ofa new class of reagents, which can be termed ‘next generationmaleimides’ (NGMs). The incorporation of the leaving group leads toa switch in the mechanism that occurs upon reaction with a thiol,from an addition reaction to an addition–elimination reaction.The result is the formation of a thiomaleimide product 4, ratherthan thiosuccinimide 2. Such next generation maleimide reagentsenable new applications including reversible cysteine modification,2the construction of triconjugates,2b,3 and the bridging of disulfidebonds.2b,4We envisaged that the choice of leaving group on the maleimidecore could result in reagents with finely tuned properties. Forexample the reactivity could be controlled, in a manner similarto the use of iodo-, bromo- and chloroacetamides as varyinglyreactive reagents for selective cysteine modification.5 In additionto guiding the reactivity, the choice of leaving group couldalso control other properties of the reagents, such as aqueoussolubility. Herein we report on the aryloxymaleimides, as NGMsof attenuated reactivity, and demonstrate application of thesereagents in cysteinemodification and in newmodes of conjugationof disulfide bonds.Having previously shown that thiophenol served as a suitableleaving group in dithiophenolmaleimide reagents,4b we postulatedthat phenol may do like-wise. Furthermore we considered thatthe increased mesomeric contribution from the oxygen into themaleimide ring would lead to reagents of reduced electrophilicity.The synthesis of aryloxymaleimides 6–8 was carried out by simpleaddition of the corresponding phenol toN-methyl bromomaleimide 5(Scheme 2).Addition of these reagents to a model protein, the singlecysteine mutant (L111C) of the SH2 domain of the Grb2 adaptorprotein 9,2b was monitored over 2 hours and compared to theuse of bromomaleimide 5 (Fig. 1). All three aryloxymaleimidesunderwent the desired addition–elimination reaction to affordthiomaleimide conjugate 10, at a reduced rate compared tobromomaleimide 5.2b As expected, the incorporation of electronScheme 1 Cysteine labelling of proteins using maleimide 1 and nextgeneration maleimide (NGM) 3.Scheme 2 Synthesis of monoaryloxymaleimides 6–8.a Department of Chemistry, University College London, 20 Gordon St, London, UK.E-mail: j.r.baker@ucl.ac.uk; Tel: +44 (0)2076792653b UCL Cancer Institute, 72 Huntley Street, London, WC1E 6BT, UKc Pfizer, Chemical Biology Group, BioTherapeutics Chemistry,WorldWide Medicinal Chemistry, 610 Main Street, Cambridge, MA 02139, USA† Electronic supplementary information (ESI) available: Experimental proceduresand full spectroscopic data are available. See DOI: 10.1039/c4cc02107jReceived 20th March 2014,Accepted 14th May 2014DOI: 10.1039/c4cc02107jwww.rsc.org/chemcommChemCommCOMMUNICATIONOpen Access Article. Published on 15 May 2014. Downloaded on 21/01/2016 17:20:22.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineView Journal  | View Issue7140 | Chem. Commun., 2014, 50, 7139--7142 This journal is©The Royal Society of Chemistry 2014withdrawing groups on the aryloxy ring (7 and 8) led to an accelera-tion in rate compared to the unsubstituted phenoxymaleimide 6. It isnotable that the aryloxymaleimides are still considerably morereactive than iodoacetamide, another commonly employed cysteinereactive reagent, which underwent o5% reaction with this proteinafter 30 min under the same conditions.2b Furthermore the reactioncould be accelerated by increasing the number of equivalents ofphenoxymaleimide 6 to ten, leading to complete conversion tothe product in just 10 min. These results demonstrate thatphenoxymaleimides represent cysteine modification reagentsof attenuated reactivity, a feature we postulated could be usefulin affording improved selectivity in certain bioconjugations.A particular regioselectivity problem had become apparent in ourstudies on disulfide bridging for which we envisaged that thearyloxymaleimides could oﬀer an ideal solution. We were interestedin attempting to use bromomaleimide to bridge a disulfide bond inthe peptide hormone somatostatin 11, in a manner related to ourwork with dibromomaleimide.2b,4b This would aﬀord a saturateddithiosuccinimide 14 (presumed to be as a mixture of diastereo-mers), in contrast to the dithiomaleimides generated by dibromo-maleimide bridging. Such dithiosuccinimides would be expected tohave diﬀerent properties to dithiomaleimides (e.g. reduced reactivitywith thiols)2b,3b and could serve as a complementary approach.However, addition of bromomaleimide 5 to reduced somatostatin12 led to a mixture of the desired bridged adduct 14 and the bis-addition product 15 (Scheme 3). Thus addition of bromomaleimide tothe second thiol in intermediate 13 was competing with the bridgingreaction. What was required was an NGM analogue whichunderwent a slower reaction with cysteine, to ensure that thecyclisation of 13 would take place before the second maleimideaddition. The aryloxymaleimides appeared to fit this specifica-tion. Indeed treatment of reduced somatostatin 12with phenoxy-maleimide 6 led after 1 h to quantitative conversion to thesuccinimide bridged somatostatin conjugate 14. Increasing theamount of phenoxymaleimide to 10 equiv. in this reaction stillaﬀorded none of the bis-labelled product 15, demonstrating theextremely high selectivity for disulfide bridging. To prove thatsuccinimide bridging had reached completion, product 14 wastreated with 1 equiv. ofmaleimide. No reaction was observed by LC-MS analysis after 5 min at room temperature, proving the absenceof detectable amounts of free thiol.We also found that, unlike bromomaleimides,4b aryloxym-aleimides are resistant to cross-reactivity with the reducing agentTCEP and therefore that the succinimide bridging could becarried out in situ. The optimised in situ conditions involveaddition of 1.5 equiv. aryloxymaleimide to the peptide followedby addition of the equimolar amount of the reducing agent TCEPleading to quantitative conversion after 2 h. In situ protocols arevaluable in disulfide bridging as they limit the amount of timethe peptide or protein lacks its bridging functionality and thusreduces the propensity to aggregate or unfold.4b,6A selection of derivatised aryloxymaleimides (Fig. 2) were thensynthesised (see ESI,† Scheme S1) to explore the scope of thissuccinimide bridging. Notably a new, scalable synthesis of bromom-aleimidewas developed to allow convenient access to thesemolecules.A water soluble aryloxy motif was incorporated in 16 to investigate thepossibility of organic solvent free labelling. A ‘miniPEG’ phenoxym-aleimide 17was developed as amodel for PEGylation of proteins, andrhodamine phenoxymaleimide 18 for fluorescent labelling.The use of the water soluble aryloxymaleimide 16 successfullyenabled the labelling of somatostatin under organic solvent freeconditions. It is noteworthy that this is the first example of amaleimide reagent incorporating a water solubilising group whichis released upon conjugation, and further exemplifies the enhancedversatility of NGMs over traditional maleimides.To demonstrate that the succinimide bridging was also applicableto larger proteins, chemical modification of an antibody fragmentFig. 1 Addition of aryloxymaleimides to Grb2 SH2 (L111C), with a graph ofconversion over time measured by LCMS.Scheme 3 Succinimide bridging of the disulfide bond in somatostatin.Communication ChemCommOpen Access Article. Published on 15 May 2014. Downloaded on 21/01/2016 17:20:22.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article OnlineThis journal is©The Royal Society of Chemistry 2014 Chem. Commun., 2014, 50, 7139--7142 | 7141was investigated. A substantial portion of all biotechnology productsunder development are antibodies or antibody fragments,7 andhomogeneous conjugation technologies have a key role to play inrealising the full potential in this area.6,8 Treatment of cystinestabilized anti-carcinoembryonic antigen single chain Fv fragment(anti-CEA ds-scFv) 196 with N-miniPEG phenoxymaleimide 17 andbenzeneselenol as the reducing agent6 led to quantitative conversionto the conjugate 20 (Scheme 4). The same outcomewas observe usingN-methyl phenoxymaleimide 6 to aﬀord conjugate 21.We have previously demonstrated that maleimide bridges aresusceptible to thiol mediated cleavage at high concentration ofthiols, such as the mM concentrations found in the cytoplasm ofcells.2b,6 We wanted to test the stability of the succinimide bridgeto thiols, to investigate if a diﬀerent reactivity profile wasdemonstrated. Treatment of succinimide bridged scFv conjugate21 with 100 equiv. GSH (7 mM, pH 7.4) led to no reaction evenafter 48 h at room temperature. Thus the succinimide bridge inthis scFv is completely resistant to thiol mediated cleavage incontrast to the maleimide bridge.Returning to somatostatin however, it became apparent thatthis thiol stability is variable depending on the exact nature of thedisulfide bridge. A comparison of maleimide and succinimidebridged somatostatin revealed that both were cleaved at similarrates by GSH under cytoplasmmimicking conditions to release thefree peptide 12 (see ESI,† Fig. S1). We propose that the mechanismfor the thiol cleavage of the succinimide bridge starts with a retro-conjugate addition to generate acyclic thiomaleimide 13 (seeScheme 3), and thus 13 and 14 are in fact in equilibrium.Thiomaleimide 13 is then cleaved by GSH in a manner consistentwith our previous results,9 via a conjugate addition–eliminationsequence. The difference in thiol stability of the somatostatin–succinimide 14 to the scFv-succinimide 20 we postulate to beattributable to the extent of intramolecular interactions that arepresent constraining these structures. In the case of somatostatinthe formation of acyclic thiomaleimide intermediate 13 can befollowed by a shift in conformation reducing the rate of rebridgingand allowing a significant amount of this intermediate to form insolution. In contrast in the case of the scFv the extensive inter-actions between the variable regions of the light and heavy chainhold the cysteines in close proximity, precluding the formation ofsignificant amounts the acyclic thiomaleimide intermediaterequired for thiol mediated cleavage.Notably employing N-phenyl phenoxymaleimide to bridgesomatostatin led to the resultant conjugate which underwentrapid hydrolysis of the succinimide bridge (consistent with theequivalent N-phenyl maleimide bridge,4a and see ESI,† Fig. S2),aﬀording a succinamic acid which was completely stable tothiols; this represents a general method of producing completelythiol stable conjugates.Whilst the focus of this study was investigating the use ofphenoxymaleimides in succinimide bridging, we realised that thereversibility of the bridging oﬀered an interesting possibility for duallabelling of disulfide bonds. We envisaged that addition of a secondelectrophile could lead to interception of intermediate 13, attaching asecond tag. Dual labelling of a reduced disulfide bond is extremelydiﬃcult to achieve using traditional reagents as it relies on asignificant diﬀerence in reactivity between the two cysteines.10Methods for the site-selective dual-labelling of peptides are widelysought, to allow controlled introduction of multiple functionalities.11For example, the incorporation of two diﬀerent fluorophores allowsFRET studies while the attachment of an in vivo stabilising group (e.g.a PEG) and a drug can prospectively enable therapeutic applications.Thus treatment of reduced somatostatin 12withphenoxymaleimide6 (R1 = Me) aﬀorded the succinimide bridged somatostatin 14 whichwas then treated, without purification, with monobromomaleimideFig. 2 Aryloxymaleimide derivatives.Scheme 4 Succinimide bridging of ds-scFv shMFE.Scheme 5 Dual modification of somatostatin (conditions: 1. 1 h, r.t.,pH 6.4; 2. 2 h, 37 1C, pH 6.4).ChemComm CommunicationOpen Access Article. Published on 15 May 2014. Downloaded on 21/01/2016 17:20:22.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online7142 | Chem. Commun., 2014, 50, 7139--7142 This journal is©The Royal Society of Chemistry 2014(R2 = H) (Scheme 5). The reaction required warming to 37 1C for 2 h toensure complete conversion to the dual modified peptide 22. Toconfirm the formation of two cleavable thiomaleimides the conjugatewas treated with 2-mercaptoethanol (100 equiv.) to quantitativelyconvert to reduced somatostatin. To demonstrate that this approachcould be employed to access dual functionalised peptides, we con-structed miniPEG–fluorescein and rhodamine–fluorescein bis-conjugates (23 and 24). Notably other electrophiles can also beemployed in the second modification step; for example maleimidewas employed affording a dual conjugate in which one of the tags wascleavable by thiols and the other was not. Trypsin digest of 25confirmed that the reaction led, as expected, to a mixture of the twopossible regioisomers (see ESI,† Fig. S3).In conclusion, by varying the leaving groups on a maleimide core,a range of next generationmaleimides can be producedwith tuneableproperties. Here a selection of aryloxymaleimides have been demon-strated to serve as eﬃcient cysteine labelling reagents of attenuatedreactivity. This enables the aryloxymaleimides to be employed insuccinimide bridging, oﬀering a novel strategy for disulfide labelling.The reversibility of the succinimide bridge in the peptide also enableda one-pot dual labelling strategy to be developed.The authors gratefully acknowledge Pfizer, BBSRC and UCLfor supporting this work.Notes and references1 (a) J. D. Gregory, J. Am. Chem. Soc., 1955, 77, 3922–3923; (b) R. A.Bednar, Biochemistry, 1990, 29, 3684–3690; (c) J. M. Chalker,G. J. L. Bernardes, Y. A. Lin and B. G. Davis, Chem. – Asian J.,2009, 4, 630–640.2 (a) L. M. Tedaldi, M. E. B. Smith, R. Nathani and J. R. Baker, Chem.Commun., 2009, 6583–6585; (b) M. E. B. Smith, F. F. Schumacher,C. P. Ryan, L. M. Tedaldi, D. Papaioannou, G. Waksman, S. Caddickand J. R. Baker, J. Am. Chem. Soc., 2010, 132, 1960–1965.3 (a) J. Youziel, A. R. Akhbar, Q. Aziz, M. E. B. Smith, S. Caddick,A. Tinker and J. R. Baker, Org. Biomol. Chem., 2014, 12, 557;(b) P. Moody, M. E. B. Smith, C. P. Ryan, V. Chudasama,J. R. Baker, J. Molloy and S. Caddick, ChemBioChem, 2012, 13, 39–41.4 (a) C. P. Ryan, M. E. B. Smith, F. F. Schumacher, D. Grohmann,D. Papaioannou, G. Waksman, F. Werner, J. R. Baker and S. Caddick,Chem. Commun., 2011, 47, 5452–5454; (b) F. F. Schumacher, M. Nobles,C. P. Ryan, M. E. B. Smith, A. Tinker, S. Caddick and J. R. Baker,Bioconjugate Chem., 2011, 22, 132–136; (c) M. W. Jones, R. A. Strickland,F. F. Schumacher, S. Caddick, J. R. Baker, M. I. Gibson and D. M.Haddleton, J. Am. Chem. Soc., 2012, 134, 1847–1852; (d) M. W. Jones,R. A. Strickland, F. F. Schumacher, S. Caddick, J. R. Baker, M. I. Gibsonand D. M. Haddleton, Chem. Commun., 2012, 48, 4064–4066.5 R. L. Lundblad, Chemical reagents for protein modification, CRCPress, 2005.6 F. F. Schumacher, V. A. Sanchania, B. Tolner, Z. V. F. Wright,C. P. Ryan, M. E. B. Smith, J. M. Ward, S. Caddick, C. W. M. Kay,G. Aeppli, K. A. Chester and J. R. Baker, Sci. Rep., 2013, 3, 1525.7 S. Aggarwal, Nat. Rev. Drug Discovery, 2010, 9, 427–428.8 M. M. Sun, K. S. Beam, C. G. Cerveny, K. J. Hamblett, R. S.Blackmore, M. Y. Torgov, F. G. Handley, N. C. Ihle, P. D. Senterand S. C. Alley, Bioconjugate Chem., 2005, 16, 1282–1290.9 R. I. Nathani, V. Chudasama, C. P. Ryan, P. R. Moody, R. E. Morgan,R. J. Fitzmaurice, M. E. B. Smith, J. R. Baker and S. Caddick, Org.Biomol. Chem., 2013, 11, 2408–2411.10 Y. Santoso, C. M. Joyce, O. Potapova, L. Le Reste, J. Hohlbein,J. P. Torella, N. D. F. Grindley and A. N. Kapanidis, Proc. Natl. Acad.Sci. U. S. A., 2010, 107, 715–720.11 R. Nathani, P. Moody, V. Chudasama, M. E. B. Smith, R. Fitzmauriceand S. Caddick, Chem. Sci., 2013, 4, 3455.Communication ChemCommOpen Access Article. Published on 15 May 2014. Downloaded on 21/01/2016 17:20:22.  This article is licensed under a Creative Commons Attribution 3.0 Unported Licence.View Article Online

Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

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Aryloxymaleimides for cysteine modification, disulfide bridging and the dual functionalization of disulfide bonds

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