Baker, JR

Caddick, S

Chudasama, V

Molloy, J

Moody, P

Ryan, CP

Smith, ME

UCL Discovery

DOI: 10.1002/cbic.201100603Bromomaleimide-Linked Bioconjugates Are Cleavable in Mammalian CellsPaul Moody,[a, b] Mark. E. B. Smith,[a] Chris P. Ryan,[a] Vijay Chudasama,[a] James R. Baker,*[a] Justin Molloy,*[b] andStephen Caddick*[a]Bromomaleimides have recently been shown to actas small, versatile scaffolds for the controlled assem-bly of thiolated biomolecules.[1–4] They present threepoints of chemical attachment, thereby allowing themodular construction of multifunctional bioconju-gates. The bromomaleimide scaffold introduces nonew chiral centres to the final construct and, in con-trast to disulfide linkages, bromomaleimides can beused to link two unactivated biomolecules of equalconcentration with minimal formation of homodi-meric side products.[2] Bromomaleimide adductshave been shown to dissociate in vitro in the pres-ence of reducing agents to liberate the compositethiols.[2] The cytoplasm of cells contains 1–10 mm re-duced glutathione,[5] thus raising the possibility thatbromomaleimide conjugates could be cleavable invivo. If this could be demonstrated, then a numberof medical and academic applications can be envis-aged that combine in vivo cleavage with multiplepoints of scaffold attachment. However, a number offactors might inhibit cytoplasmic cleavage, in partic-ular pH-dependant[3] and protease-catalysed amidebond hydrolysis. Using a series of bromomaleimide-linked green fluorescent protein (GFP)–rhodamineconjugates, designed as FRET pairs, we demonstrateherein that bromomaleimide-linked bioconjugatescleave in the cytoplasm of mammalian cells.Rhodamine–maleimide derivatives 4–6 were gen-erated by condensation of the relevant maleic anhy-dride with a common intermediate, 3 (Scheme 1).The two native cysteines in wild-type superfolderGFP,[6] C48 and C70, were shown to be inaccessibleto maleimide functionalisation under our reactionconditions (see Section 4 in the Supporting Informa-tion). A GFP with a free, accessible thiol close to itsfluorophore (GFP-SH) was generated by introducingan S147C mutation into superfolder GFP. The mutat-ed GFP-SH produces a similar emission spectrum towild-type superfolder GFP. The folded protein was shown to beresistant to disulfide-mediated dimerisation, thus allowingGFP-SH to be conjugated to maleimides without the need forreducing agents (see Section 5 in the Supporting Information).Compounds 4–6 were attached to GFP-SH as described inthe Supporting Information, Section 5. Stoichiometric additionof rhodamine–maleimide was confirmed by mass spectrometry.The emission spectra of the resultant constructs 7–10 are illus-trated in Figure 1. In each case, addition of rhodamine–malei-mide was shown to result in efficient quenching of GFP fluo-rescence. Little increase in emission at 590 nm was seen.Cleavage of compounds 7–10 by a physiologically relevantconcentration of reduced glutathione (1 mm) was monitoredin vitro by dual-channel measurement of the GFP and rhoda-Scheme 1. Synthesis of rhodamine-bromomaleimides. a) (COCl)2, 20 8C, 15 h; b) piperidin-4-yl carbamic acid tert-butyl ester (10.4 equiv), CsCO3 (10.4 equiv), CH2Cl2, 20 8C, 24 h,71% (2 steps) ; c) TFA/CH2Cl2 (1:1), 20 8C, 5 h, 100%; d) maleic anhydride (1.4 equiv),AcOH, 120 8C, 5 h, 40%; e) bromomaleic anhydride (1.4 equiv), AcOH, 120 8C, 5 h, 66%;f) dibromomaleic anhydride (1.4 equiv), AcOH, 120 8C, 5 h, 66%.[a] P. Moody, Dr. M. E. B. Smith, Dr. C. P. Ryan, Dr. V. Chudasama,Dr. J. R. Baker, Prof. S. CaddickDepartment of Chemistry, University College LondonGordon Street, London, WC1H 0AJ (UK)E-mail : s.caddick@ucl.ac.uk[b] P. Moody, Dr. J. MolloyMRC National Institute of Medical ResearchThe Ridgeway, Mill Hill, London, NW7 1AA (UK)Supporting information for this article is available on the WWW underhttp://dx.doi.org/10.1002/cbic.201100603.ChemBioChem 2012, 13, 39 – 41  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim 39mine emission intensities upon excitation of GFP at 494 nm(Figure 2).As expected, the GFP emission intensity associated with 7did not change upon addition of glutathione, whereas thatassociated with 8–10 increased. The data fitted well to a singleexponential increase in free GFP concentration over time, sug-gesting first-order kinetics with respect to conjugates 8–10under these conditions. Rate constants of 1.9 s1 for 8, 26 s1for 9 and 2.8 s1 for 10 were calculated.For compounds 7–9, little change in apparent rhodamineemission (590 nm) was seen during the experiments. For com-pound 10, a decrease in rhodamine emission was observed;this suggesting that FRET is disrupted. The efficient quenchingof GFP fluorescence allowed us to use the ratio of GFP/rhoda-mine emission intensities as a quantitative measure of cleav-age during subsequent microinjection experiments. Cleavageof constructs 8–10 was also confirmed by mass spectrometryunder analogous reaction conditions (see Section 7 in the Sup-porting Information).Cleavage of constructs 8–10 in human cells was demonstrat-ed by microinjection into live, cultured HeLa cells, followed bytime-lapse fluorescence microscopy. GFP was excited by usinga 450–490 nm filter, and GFP and rhodamine emission weremeasured simultaneously by using an image splitter that con-tained GFP (500–550 nm) and rhodamine (575–630 nm) emis-sion filters (Figures 3 and 4).In each case, both the absolute GFP intensity post-microin-jection (see Section 8 in the Supporting Information) and ratio-metric data fitted well to a single exponential increase, thusshowing that bromomaleimide-linked constructs are cleaved incells. The order of reactivity is the same as that observed invitro (see Section 9 in the Supporting Information); this sug-gests that the mechanism of cleavage in cells is the same asthe mechanism in vitro. The ratio of GFP/rhodamine emissionintensity at equilibrium is consistent with the ratio observedFigure 1. Emission spectra of superfolder GFP, the mutant GFP-SH and therhodamine conjugates; compounds 7–9 (0.85 mm), compound 10 (0.425 mm ;lex=494 nm).Figure 2. In vitro cleavage of the rhodamine–GFP conjugates. Glutathione(1 mm) was added to compounds A) 7, B) 8, C) 9 (0.85 mm each) and D) 10(0.43 mm). GFP was excited at 494 nm; GFP emission (515 nm, green) andrhodamine emission (590 nm, red) were measured simultaneously.Figure 3. In-cell cleavage of compound 9 following microinjection into HeLacells. The image was cast onto the upper and lower halves of the CCDsensor by using an image splitter (Optical Insights). Top: GFP emission,bottom: rhodamine emission. A) DIC image immediately before injection.B) Fluorescence image immediately after injection. C) Fluorescence image10 min after cleavage. Scale bars=5.0 mm.40 www.chembiochem.org  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim ChemBioChem 2012, 13, 39 – 41for cleaved constructs deposited directly onto glass slides (seeSection 10 in the Supporting Information). This suggests thatcomplete cleavage of the bromomaleimide linkages occurs inHeLa cells. Disassembly of the fully conjugated dibromomalei-mide (10) was also successfully demonstrated in COS-7 cells(see Section 8 in the Supporting Information), thus highlight-ing that observed in cell cleavage is not restricted to HeLacells.In summary, we have demonstrated that bromomaleimide-linked conjugates can be cleaved with first-order kinetics bothin vitro and in cells. Dibromomaleimide derivatives (9 and 10)cleave at a faster rate than monobromomaleimide construct 8.Maleimide construct 7 is stable to thiol-promoted cleavage.We envisage that bromomaleimides have potential as scaffoldsfor the synthesis of a variety of multifunctional reagents. Webelieve that the FRET construct 9 has a specific application inthe screening of attached thiolated cell-penetrating structurescapable of successfully delivering protein cargo to the cyto-plasm. More broadly, we believe that bromomaleimide scaf-folds provide a potential core structure for the facile and versa-tile construction of targeted therapeutics that will cleave to re-lease their cargo once internalised into the cytoplasm. We shallreport on the further development and application of thismethodology in due course.AcknowledgementsWe gratefully acknowledge the Wellcome Trust, MRC, RCUK,EPSRC, BBSRC and UCLB for support of our programme. Wethank Dr. Phillip Behe for helpful advice on microinjection, andthe Registry of Standard Biological Parts for the donation of thesuperfolder GFP gene.Keywords: drug delivery · fluorescent probes · in-cellcleavage · microinjection · protein modifications[1] L. M. Tedaldi, M. E. B. Smith, R. I. Nathani, J. R. Baker, Chem. Commun.2009, 6583–6585.[2] M. E. B. Smith, F. F. Schumacher, C. P. Ryan, L. M. Tedaldi, D. Papaioannou,G. Waksman, S. Caddick, J. R. Baker, J. Am. Chem. Soc. 2010, 132, 1960–1965.[3] C. P. Ryan, M. E. B. Smith, F. F. Schumacher, D. Grohmann, D. Papaioan-nou, G. Waksman, F. Werner, J. R. Baker, S. Caddick, Chem. Commun.2011, 47, 5452–5454.[4] F. F. Schumacher, M. Nobles, C. P. Ryan, M. E. B. Smith, A. Tinker, S. Cad-dick, J. R. Baker, Bioconjugate Chem. 2011, 22, 132–136.[5] C. V. Smith, D. P. Jones, T. M. Guenthner, L. H. Lash, B. H. Lauterburg, Toxi-col. Appl. Pharmacol. 1996, 140, 1–12.[6] J.-D. Pedelacq, S. Cabantous, T. Tran, T. C. Terwilliger, G. S. Waldo, Nat. Bio-technol. 2006, 24, 79–88.Received: September 24, 2011Published online on November 18, 2011Figure 4. In-cell cleavage of compounds 7–10 in HeLa cells. The GFP/rhoda-mine emission ratio and exponential fits were calculated as described in theSupporting Information.ChemBioChem 2012, 13, 39 – 41  2012 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chembiochem.org 41

Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

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Bromomaleimide-linked bioconjugates are cleavable in mammalian cells.

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