Bioconjugation of superfolder GFP involving the formation of an unusually stable, and unprecedented, cyclic sulfonium species is described. This sulfonium can undergo smooth reaction with a range of nucleophiles to give sulfur-, selenium- and azide-modified GFP derivatives in high conversions

Caddick, S

Fitzmaurice, RJ

Moody, P

Nathani, R

Smith, MEB

English

PubMed

UCL Discovery

DOI: 10.1002/cbic.201200231Bioconjugation of Green Fluorescent Protein via an Unexpectedly StableCyclic Sulfonium IntermediateRamiz Nathani, Paul Moody, Mark E. B. Smith, Richard J. Fitzmaurice, and Stephen Caddick*[a]The development of methods for the modification of peptidesand proteins under benign aqueous conditions has receiveda great deal of attention from both industrial and academiclaboratories.[1] The ability to introduce a range of functionalpost-translational modifications, either natural or synthetic, torecombinantly expressed proteins, such as sugars, lipids, fluo-rophores, affinity tags or radionuclides, has been exploited togain further insight into a plethora of biological process.[2] Anumber of strategies for protein modification through manipu-lation of the amino or carboxy terminus have been devel-oped.[3] However, the vast majority of approaches rely on thereactivity of naturally occurring amino acid side chains such aslysine, tyrosine or cysteine.[4]One bioconjugation approach exploits the elimination ofcysteine to dehydroalanine, which can then be modified byadding suitable nucleophiles. A number of reagents have beendeveloped to convert both peptide- and protein-based cys-teines to dehydroalanine, for example O-mesitylenesulfonyl-hydroxylamine and hexamethylphosphorous triamide.[4b,5]However, the majority of currently used reagents require harshconditions that limit their application. Recently, Davis has de-scribed the elegant use of 1,4-bishaloalkanes for the b-elimina-tion of cysteine to effect protein modification under mild con-ditions.[6] An illustrative example is the treatment of a singlecysteine mutant of subtilisin from Bacillus lentus SBL(S156C) (1)with 2,5-dibromohexanediamide (2) at pH 8, which affordedintermediate dehydroalanine 3, presumably via the transientformation of sulfonium 4 (Scheme 1). Dehydroalanine 3 couldbe subsequently functionalised with a nucleophile to givemodified protein 5.Intrigued by the potential application of the Davis double-al-kylation methodology, we explored its applicability to a rangeof biological systems. Herein, we report preliminary studies onthe modification of a cysteine mutant (S147C) of “superfolder”green fluorescent protein (GFP) bearing a single surface-acces-sible cysteine. We were able to synthesise unusually stablecyclic sulfonium species and to employ these in a novel bio-conjugation that is complementary to current dehydroalanine-based technologies for substrates for which dehydroalanineformation is not facile. Although the presence of sulfoniumions in peptides and proteins derived from the alkylation ofmethionine is well established,[7] to our knowledge this repre-sents the first evidence for the formation of a stable cysteine-sulfonium on a protein.We treated GFP(S147C) (6) with 2 under Davis’ conditions(1500 equiv, 378C, 2 h, pH 8; Scheme 2).[6] Unexpectedly theproduct was not the corresponding GFP dehydroalanine 7,rather electrospray (ES) MS indicated quantitative conversionto a species with a molecular weight equivalent to that of sul-fonium 8 (29486 calcd, 29488 obs.). Indeed, throughout thisstudy, we observed no evidence for the formation of dehy-droalanine 7, or products derived therefrom, even upon incu-bation of sulfonium 8 for prolonged periods (vide infra). Theisolation of this sulfonium species as a stable adduct and itsreluctance to undergo elimination to give the correspondingdehydroalanine product 7 was noteworthy and we embarkedupon further studies to understand and exploit this unexpect-ed mode of reactivity.Scheme 1. Modification of SBL(S156C) by cysteine b-elimination.Scheme 2. Reaction of GFP(S-147C) (100 mL, 1 mgmL1 in H2O, pH 8, 100 mmphosphate) with dibromodiamide (2). a) 2 (1500 equiv; 10 mL, 340 mm inDMF), 378C, 2 h.[a] R. Nathani, P. Moody, Dr. M. E. B. Smith, Dr. R. J. Fitzmaurice,Prof. S. CaddickDepartment of Chemistry, University College LondonLondon, WC1H 0AJ (UK)E-mail: VPEnterprise@ucl.ac.ukSupporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cbic.201200231.Re-use of this article is permitted in accordance with the Terms andConditions set out at http://onlinelibrary.wiley.com/journal/10.1002/(ISSN)1439-4227/homepage/2268_onlineopen.html.ChemBioChem 2012, 13, 1283–1285   2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 1283In order to gain further evidence for the site of reaction be-tween GFP(S147C) and 2,5-dibromohexanediamide, sulfonium8 was treated with N-methylbromomaleimide (1 equiv).[4d] Noreaction was observed with 8, whereas 6 reacted cleanly, thussuggesting that 2 had reacted exclusively on cysteine to gen-erate 8 (see the Supporting Information).Optimisation of the formation of 8 was achieved by varyinga number of parameters including the number of equivalentsof 2, reaction temperature and reaction time (Table 1). Treat-ment of GFP(S147C) with 10 equivalents of 2 afforded no sulfo-nium 8 after 5 h; nevertheless, clear evidence for the formationof thioether 9 (29566 calcd, 29563 obs.) was apparent byLCMS (Table 1, entry 2). However, a prolonged incubation time,20 h, afforded 8 as the only identifiable product in 53% con-version (Table 1, entry 3). Increasing the concentration of 2,5-dibromohexanediamide afforded, as expected, more rapid for-mation of 8, with complete conversion of 6 at 218C with 25 or50 equivalents of 2 within 20 h (Table 1, entries 4–9). At 48C,no sulfonium formation is apparent after 20 h even using25 equivalents of 2,5-dibromohexanediamide (Table 1, entries10 and 11). However, formation of sulfonium 8 at 48Ci sobserved at higher concentrations of 2 (Table 1, entry 12). At378C with 50 equivalents of 2 complete conversion of 6 to 8could be rapidly achieved (Table 1, entry 13).We then sought to establish the stability and reactivity pro-file of sulfonium 8. After preparation of sulfonium 8 under ouroptimised conditions (50 equiv 2,3 7 8C, 2 h) excess 2 could bereadily removed by repeated di-afiltration into fresh buffer. Sulfo-nium 8 was found to be stablein the absence of nucleophilesat 218C for 24 h and at 48C for1 month; although, significantdecomposition was observed at378C after 4 h (vide supra).Treatment of 8 with b-mercapto-ethanol (1000 equiv, 378C, 2.5 h)afforded bisthioether 10a(29564 calcd, 29566 obs.) incomplete conversion, presuma-bly through ring opening of thecyclic sulfonium (Scheme 3).We then evaluated the reactiv-ity of 8 with a range of simplenucleophiles. Treatment of 8with the sodium salt of 1-thio-b-d-glucose (10 equiv) affordedthe expected GFP–thioglucoseconjugate 10b as the sole prod-uct, with complete conversionafter 5 h (Table 2, entry 1). Simi-larly, treatment of 8 with gluta-thione was also successful atboth 21 and at 378C, generatingthe corresponding GFP–gluta-thione adduct 10c in excellentconversion (entries 3–5). We alsoconfirmed that these reactions could be carried out effectivelyat room temperature (218C) in 5 h, or more rapidly at 378Co rwith increased concentrations of the nucleophile (entries 2, 4and 5). Treatment of sulfonium 8 with phenyl selenolTable 1. Optimisation of formation of sulfonium 8.[a]2 [equiv] T [8C] t [h] 6 [%][b] 9 [%][b] 8 [%][b]11 0 2 1 2 >95 0 025 9 1 9 032 0 4 7 0 5 34 25 21 2 81 19 05 5 50 17 3362 0 0 0 >957 50 21 2 56 23 218 5 27 25 4892 0 0 0 >9510 10 4 20 >95 0 011 25 20 80 20 012 50 20 56 24 2013 50 37 2 0 0 >95[a] Conditions: 6 (100 mL, 1 mgmL1 in H2O) at pH 8 (100 mm phosphate),2 (10 mL in DMF). [b] Determined by ratio of peak heights in deconvolut-ed mass spectrum.Table 2. Conjugation of nucleophiles with GFP sulfonium 8.[a]RH Equiv T [8C] t [h] Conv. 8 [%][b] Product1210100372155>95>9510b34510100200372137552>95>95>9510c6 100 37 5 >95 10d7 1000 37 2.5 40 10e8 1000 37 2.5 >95 10 f[a] Conditions: 8 (100 mL, 1 mgmL1 in H2O) at pH 8 (100 mm phosphate), NuH (see the Supporting Informa-tion). [b] Determined by ratio of peak heights in deconvoluted mass spectrum.1284 www.chembiochem.org   2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2012, 13, 1283–1285(100 equiv) afforded the mixed thio/seleno bisether 10d inexcellent conversion (entry 6). Although the conversion fromreaction between 8 and phthalimide to give 10e was onlymodest (entry 7), we were delighted to observe clean reactionin high conversion upon treatment of 8 with sodium azide(1000 equiv) to generate azide-labelled GFP 10 f (entry 8).In conclusion, we have shown that treatment of superfolderGFP with bis-alkylating agents can be used to prepare highlystable, yet synthetically useful cyclic protein sulfonium adducts.These species undergo clean addition reactions to allow the in-troduction of a variety of useful chemical motifs. We envisagethat the stability of the generated cysteine-derived sulfoniumis presumably strongly correlated to the chemical environmentof the alkylated cysteine. This new bioconjugation techniquecould provide a useful entry into multiply functionalised pro-teins and further work will be directed toward extending thesefindings to other systems.AcknowledgementsWe gratefully acknowledge EPSRC (EP/E000754/1), UCL, the Well-come Trust (090145/Z/09/Z), UCLB and GSK for support of ourprogramme.Keywords: alkylation · bioconjugation · cysteine · proteinmodifications · sulfonium[1] a) J. M. Antos, M. B. Francis, Curr. Opin. Chem. Biol. 2006, 10, 253–262;b) L. M. Artner, L. Merkel, N. Bohlke, F. Beceren-Braun, C. Weise, J. Der-nedde, N. Budisa, C. P. R. Hackenberger, Chem. Commun. 2012, 48, 522–524; c) C. R. Behrens, J. M. Hooker, A. C. Obermeyer, D. W. Romanini, E. M.Katz, M. B. Francis, J. Am. Chem. Soc. 2011, 133, 16398–16401; d) L.Cohen, Annu. Rev. Biochem. 1968, 37, 695–726; e) A. N. Glazer, Annu. Rev.Biochem. 1970, 39, 101–130; f) T. J. Holmes, Jr., R. G. Lawton, J. Am.Chem. Soc. 1977, 99, 1984–1986; g) C. T. Walsh, S. Garneau-Tsodikova,G. J. 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Bioconjugation of Green Fluorescent Protein via an Unexpectedly Stable Cyclic Sulfonium Intermediate

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