Protein modification, bioconjugation, and disulfide bridging using bromomaleimides

Article abstract of DOI:10.1021/ja908610s

The maleimide motif is widely used for the selective chemical modification of cysteine residues in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible nature of the reaction and, hence, the modification and the number of attachment positions. We conceived of a new class of maleimide which would address some of these limitations and provide new opportunities for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin. Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent somatostatin analogue. These results highlight the significant potential for this new class of reagents in protein modification.

Full text of DOI:10.1021/ja908610s

Published on Web 01/21/2010
Protein Modification, Bioconjugation, and Disulfide Bridging
Using Bromomaleimides
Mark E. B. Smith, Felix F. Schumacher, Chris P. Ryan, Lauren M. Tedaldi,
Danai Papaioannou,† Gabriel Waksman,† Stephen Caddick,* and James R. Baker*
Department of Chemistry, UniVersity College London, 20 Gordon Street, London WC1H OAJ, U.K.,
and Institute of Structural Molecular Biology at UCL/Birkbeck, Malet Street, London WC1E 7HX, U.K.
Received October 9, 2009; E-mail: s.caddick@ucl.ac.uk; j.r.baker@ucl.ac.uk
Abstract: The maleimide motif is widely used for the selective chemical modification of cysteine residues
in proteins. Despite widespread utilization, there are some potential limitations, including the irreversible
nature of the reaction and, hence, the modification and the number of attachment positions. We conceived
of a new class of maleimide which would address some of these limitations and provide new opportunities
for protein modification. We report herein the use of mono- and dibromomaleimides for reversible cysteine
modification and illustrate this on the SH2 domain of the Grb2 adaptor protein (L111C). After initial
modification of a protein with a bromo- or dibromomaleimide, it is possible to add an equivalent of a second
thiol to give further bioconjugation, demonstrating that bromomaleimides offer opportunities for up to three
points of attachment. The resultant protein-maleimide products can be cleaved to regenerate the unmodified
protein by addition of a phosphine or a large excess of a thiol. Furthermore, dibromomaleimide can insert
into a disulfide bond, forming a maleimide bridge, and this is illustrated on the peptide hormone somatostatin.
Fluorescein-labeled dibromomaleimide is synthesized and inserted into the disulfide to construct a fluorescent
somatostatin analogue. These results highlight the significant potential for this new class of reagents in
protein modification.
vivo applications.9 Furthermore, there are only two points of
attachment, thus limiting the number of chemical or biological
Introduction
The selective chemical modification of cysteine residues in
proteins is widely employed to enable a range of fundamental
biological and biophysical studies.1,2 Cysteine is often the most
nucleophilic residue in a protein and as such is generally the
easiest to manipulate with chemical reagents in a selective
manner. Furthermore, the relatively low natural abundance3 of
cysteine makes the introduction of single cysteines via site-
directed mutagenesis and subsequent chemical modification a
very effective method to access modified proteins. A variety of
electrophilic reagents have been developed in order to take
advantage of the nucleophilic characteristics of cysteine, but of
these the maleimide motif remains one of the most widely
employed and there are numerous N-functionalized maleimide
reagents available.4 The reaction of a cysteine residue with a
maleimide is a highly selective process5,6 and is considered to
be irreversible.7,8
entities that can be attached to a system of interest. We report
herein on a new class of maleimides, the bromomaleimides,
which offer reversible protein modification, three points of
attachment for efficient construction of bioconjugates, and the
ability to insert into disulfide bonds, retaining their bridging
character.
Results and Discussion
In prior work we had found that bromomaleimides could be
used for the reversible chemical modification of a model system
of the amino acid cysteine.10 In order to illustrate the applica-
bility of this approach on proteins, we chose to examine a single
point mutant (L111C) of the SH2 domain of the Grb2 adaptor
protein 1, a protein domain which does not otherwise contain
any cysteine residues. We initially treated 1 with Ellman’s
reagent and observed clean formation of the expected mixed
disulfide by LCMS, confirming the presence of an available
cysteine residue. Treatment of 1 with 1 equiv of N-methylbro-
momaleimide at 0 °C (sodium phosphate buffer, pH 8) for 1 h
Despite the successful utilization of maleimide as a reagent
for the chemical modification of proteins, there are limitations.
The irreversible nature of the addition prevents any possibility
for controlled disassembly of the conjugate regenerating the
unmodified protein, which may be desirable for in vitro or in
(5) Schelte, P.; Boeckler, C.; Frisch, B.; Schuber, F. Bioconjugate Chem.
2000, 11, 118–123.
† Institute of Structural Molecular Biology at UCL/Birkbeck.
(1) Lundblad, R. L. Chemical Reagents for Protein Modification, 3rd ed.;
CRC Press: Boca Raton, FL, 2005.
(6) Vanderhooft, J. L.; Mann, B. K.; Prestwich, G. D. Biomacromolecules
2007, 8, 2883–2889.
(7) Gregory, J. D. J. Am. Chem. Soc. 1955, 77, 3922–3923.
(8) Bednar, R. A. Biochemistry 1990, 29, 3684–3690.
(9) Saito, G.; Swanson, J. A.; Lee, K. D. AdV. Drug DeliVer. ReV. 2003,
55, 199–215.
(2) Chalker, J. M.; Bernardes, G. J. L.; Lin, Y. A.; Davis, B. G. Chem.
Asian J. 2009, 4, 630–640.
(3) Fodje, M. N.; Al-Karadaghi, S. Protein Eng. 2002, 15, 353–358.
(4) Hermanson, G. T. Bioconjugate Techniques; Academic Press: London,
1996.
(10) Baker, J. R.; Tedaldi, L. M.; Smith, M. E. B.; Nathani, R. Chem.
Comm. 2009, 6583–6585.
9
1960 J. AM. CHEM. SOC. 2010, 132, 1960–1965
10.1021/ja908610s  2010 American Chemical Society

Protein Modification Using Bromomaleimides
A R T I C L E S
Scheme 1. Modification of the Grb2 SH2 Domain (L111C) with N-Methylbromomaleimide
Scheme 2. Conjugate Addition of Cysteine to Bromomaleimide
Generates the Vicinal Bis-cysteine Adduct
carried out on the single amino acid model system N-Boc-Cys-
OMe. The addition of an excess of N-Boc-Cys-OMe to
bromomaleimide in the buffer (sodium phosphate buffer, pH
8) afforded the vicinal bis-adduct as a mixture of diastereomers
(Scheme 2), as confirmed by NMR spectroscopy.
We have found that dibromomaleimide can also be employed
for protein modification at cysteine. Thus, treatment of the Grb2
SH2 domain 1 with dibromomaleimide led to exclusive forma-
tion of the monobromo adduct 4. Treatment of this adduct with
Ellman’s reagent resulted in no reaction, confirming once again
that the maleimide had added exclusively to the cysteine residue.
Addition of 1 equiv of a second nucleophilic thiol, in the form
of either glutathione or thioglucose, gave the protein-peptide
and protein-sugar conjugates 5 and 6, respectively (Scheme
3). We envisaged that the maleimide-substituted adducts would
still be susceptible to further conjugate addition with nucleo-
philes resulting in cleavage. However, in this instance we found
that TCEP was unsuitable, leading to only a small amount of
cleavage and other unidentifiable products. Instead, we found
that treatment of these adducts with an excess of a thiol (2-
mercaptethanol or glutathione, 100 equiv) led to clean conver-
sion to the free unmodified protein 1.
gave complete conversion to the conjugate 2, as evident by
LCMS. Addition of Ellman’s reagent to 2 led to no reaction,
revealing that the bromomaleimide had reacted exclusively on
the cysteine residue. It should be noted that there are eight lysine
residues present on this protein, and thus, in this case the reagent
is highly selective for thiols over amine nucleophiles. Treatment
of the adduct 2 with 100 equiv of TCEP [tris(2-carboxyethyl)-
phosphine] resulted in 85% conversion back to protein 1,
demonstrating the potential of bromomaleimides for the revers-
ible covalent modification of cysteine residues in proteins.
Further experiments demonstrated that the protein-maleimide
adduct 2 could undergo a second thiol conjugate addition. Thus,
treatment of 2 with glutathione (1 equiv) led to the protein-pep-
tide conjugate 3 in 95% conversion (Scheme 1). In a similar
fashion, treatment of 2 with 2-mercaptoethanol (1 equiv) gave
the analogous bis-thioether adduct.
We are proposing that the mechanism involved in the
cleavage reactions is a conjugate addition-elimination sequence.
In order to provide evidence for our assumption that the
analogous conjugate addition using maleimide is irreversible,
we prepared the succinimide 7 by treatment of 1 with N-
We are proposing that the product 3 is the vicinal and not
geminal bis-thioether by analogy with a related experiment
Scheme 3. Reversible Modification of the Grb2 SH2 Domain (L111C) 1 with Dibromomaleimide
9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1961

A R T I C L E S
Smith et al.
Scheme 4. Irreversible Modification of the Grb2 SH2 Domain (L111C) 1 with NEM
ethylmaleimide (NEM). Treatment of 7 with 100 equiv of TCEP
or mercaptoethanol led to no reaction, even at 37 °C for 4 h,
confirming that NEM irreversibly labels the cysteine residue
(Scheme 4).
glutathione,11 37 °C). We observed after 4 h complete cleavage
to afford the protein 1 (Scheme 5). This result suggests that
such conjugates will cleave in cells and offers many exciting
possibilities which we will be actively pursuing in due course.
We have also investigated the utilization of dibromomale-
imides for the modification of disulfides. Recent work by
Brocchini and co-workers has highlighted the potential of
modifying disulfides via bridging reagents that retain the
structure and function of the protein.12-14 We envisaged that
dibromomaleimide could serve as an alternative reagent for the
selective modification of disulfides without the requirement for
introduction of an additional asymmetric carbon and with the
resulting adduct incorporating a two-carbon bridge between the
two cysteine residues.
The endogenous peptide hormone somatostatin served as an
excellent system to test our hypothesis. It is a 14-amino acid
peptide containing a disulfide bridge, and stable analogues of
somatostatin are clinically employed in the treatment of condi-
tions including acromegaly and gastroenteropancreatic tumors.15,16
Treatment of somatostatin at pH 6.2 with 1 equiv of TCEP
followed by 1.1 equiv of dibromomaleimide led to complete
conversion to the bridged somatostatin 8 (Scheme 6). This
reaction, together with the conditions used for the Grb2 SH2
domain (pH ) 8), demonstrate the broad pH suitability of these
reagents. Treatment of the maleimide bridged peptide 8 with
2-mercaptoethanol led to complete reversion to afford the
reduced somatostatin 9 after 1 h at room temperature. This
demonstrated that dibromomaleimides offer the first effective
reagent for controlled reversible bridging of disulfides.
To gain an appreciation for the relative reactivity of bromo-
maleimides with cysteines, we decided to compare the reaction
times of protein 1 with some commonly employed protein-
modifying reagents. Thus, we treated protein 1 with N-
methylbromomaleimide, dibromomaleimide, N-ethylmaleimide,
and iodoacetamide, respectively, and monitored the reaction
between 5 and 30 min using LCMS (Figure 1). We found that
the order of reactivity was N-methylbromomaleimide ≈ N-
ethylmaleimide > dibromomaleimide . iodoacetamide. Iodoacet-
amide is not shown in Figure 1, as <5% reaction had taken place
after 30 min. The bromomaleimides thus react with the cysteine
residue on a similar time scale to NEM, which is notably far
faster than that of iodoacetamide.
The observed cleavage of the maleimide conjugates by thiols
using excess glutathione suggested the enticing possibility that
thiomaleimides could be used to develop conjugates designed
to cleave in the cytoplasm of cells.9 To test this hypothesis, we
subjected the protein-sugar conjugate 6 to conditions that would
approximate the conditions of the cytoplasm (20 mM HEPES,
100 mM KCl, 1 mM MgCl2, 1 mM EDTA, pH 7.4, 1 mM
Fluorescein-5-maleimide is a commercially available reagent
used widely in protein labeling.4 To demonstrate that bromo-
maleimides provide three possible points of attachment for
protein bioconjugation, we synthesized the fluorescein-di
bromomaleimide 10. The synthesis was carried out by treatment
of dibromomaleic anhydride17 with fluoresceinamine at room
temperature, followed by heating at reflux in AcOH to effect
cyclization to give the maleimide. We chose to treat somatostatin
with this reagent to illustrate the fluorescent labeling of disulfide-
containing biomolecules. Thus, treatment of somatostatin with
N-fluoresceindibromomaleimide 10 afforded the fluorescent
bridged construct 11 in quantitative conversion after 10 min.
In order to demonstrate the reversible nature of this modification,
Figure 1. Relative reactivity of Grb2 SH2 domain (L111C) 1 with
N-methylbromomaleimide, dibromomaleimide, and N-ethylmaleimide.
(11) Schafer, F. Q.; Buettner, G. R. Free Radic. Biol. Med. 2001, 30, 1191–
1212.
Scheme 5. Cleavage of Grb2 SH2 Domain-Thioglucose
Conjugate 6 under Intracellular-like Conditions
(12) Balan, S.; Choi, J. W.; Godwin, A.; Teo, I.; Laborde, C. M.;
Heidelberger, S.; Zloh, M.; Shaunak, S.; Brocchini, S. Bioconjugate
Chem. 2007, 18, 61–76.
(13) Brocchini, S.; Godwin, A.; Balan, S.; Choi, J. W.; Zloh, M.; Shaunak,
S. AdV. Drug DeliVery ReV. 2008, 60, 3–12.
(14) Shaunak, S.; Godwin, A.; Choi, J. W.; Balan, S.; Pedone, E.;
Vijayarangam, D.; Heidelberger, S.; Teo, I.; Zloh, M.; Brocchini, S.
Nat. Chem. Biol. 2006, 2, 312–313.
(15) Freda, P. U. J. Clin. Endocrinol. Metab. 2002, 87, 3013–3018.
(16) Oberg, K. Expert ReV. Anticancer Ther. 2009, 9, 557–566.
(17) Dubernet, M.; Caubert, V.; Guillard, J.; Viaud-Massuard, M. C.
Tetrahedron 2005, 61, 4585–4593.
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Protein Modification Using Bromomaleimides
A R T I C L E S
Scheme 6. Reversible Modification of the Disulfide Bond of Somatostatin by Dibromomaleimide
Scheme 7. Reversible Modification of the Disulfide Bond of Somatostatin by a Fluorescent Dibromomaleimide Reagent
we treated conjugate 11 with 2-mercapoethanol (100 equiv),
which led after 1 h to complete cleavage of the fluorescent
maleimide to regenerate the reduced somatostatin (Scheme 7).
modification.19 In addition to the potential benefits afforded by
this temporary or reversible modification, these reagents also
provide opportunities for further attachment of functional groups.
Thus, a second thiol can also be added to the protein-maleimide
adducts to form maleimide or succinimide conjugates. As with
other maleimide based reagents,4 a functional moiety can also
be attached to the nitrogen, thus offering three points of
attachment for protein bioconjugation and we have illustrated
this by synthesizing a fluorescently labeled somatostatin. We
envisage numerous potential applications for bromomaleimides
incorporating functional groups, including biotin or solid
supports for protein purification and immobilization; fluoro-
phores, radiolabels, and quantum dots for imaging; polymers,
e.g., PEG, for protein stability and others. In the present work,
we have also shown that the dithiomaleimide constructs cleave
under conditions which approximate those encountered in the
cytoplasm, and this introduces the possibility that these male-
imide reagents could serve as a new motif for the design of
prodrugs. We will report on further developments with this
powerful new class of reagent, in due course.
The fluorescent properties of fluoresceindibromomaleimide
10 and somatostatin conjugate 11 were confirmed (Figure 2).
Fluoresceinamine is known to be a poor fluorophore until
conjugated (e.g., to a maleimide)18 and this was observed.
Summary and Implications
In summary, we have shown that mono- and dibromomale-
imides can be used for the selective modification of a cysteine
residue in a protein and that the modification can be reversed.
Thus, bromomaleimides can be employed in temporary cysteine
Experimental Section
Lyophilized somatostatin, 3,4-dibromomaleimide, and fluores-
ceinamine isomer 1 were purchased from Sigma-Aldrich and used
without further purification. Bromomaleimide and N-methylbro-
momaleimide were synthesized as described previously.10 LCMS
(18) Munkholm, C.; Parkinson, D. R.; Walt, D. R. J. Am. Chem. Soc. 1990,
112, 2608–2612.
(19) Baker, J. R.; Caddick, S.; Smith, M. E. B. Patent applications
0913965.0 (reversible covalent linkage of functional moities), 0913967.6
(functionalization of solid substrates), and 0914321.5 (thiol protecting
group), August 2009.
Figure 2. Fluorescence measurements of samples excited at 488 nm.
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A R T I C L E S
Smith et al.
was performed on protein samples using a Waters Acquity uPLC
connected to Waters Acquity Single Quad Detector (SQD) [column,
Acquity uPLC BEH C18 1.7 µm 2.1 × 50 mm; wavelength, 254
nm; mobile phase, 95:5 water (0.1% formic acid):MeCN (0.1%
formic acid), gradient over 4 min to 5:95 water (0.1% formic acid):
MeCN (0.1% formic acid); flow rate, 0.6 mL/min; MS mode, ES+;
scan range, m/z ) 85-2000; scan time, 0.25 s]. Data were obtained
in continuum mode. The electrospray source of the MS was operated
with a capillary voltage of 3.5 kV and a cone voltage of 50 V.
Nitrogen was used as the nebulizer and desolvation gas at a total
flow of 600 L/h. Total mass spectra for protein samples were
reconstructed from the ion series using the MaxEnt 1 algorithm
preinstalled on MassLynx software.
Ellman’s Test. The solution of 4 was treated with Ellman’s
reagent (5 µL, 282 mM solution in H2O) at 0 °C. The mixture was
vortexed for 1 s and maintained at 0 °C for 10 min, after which
the mixture was analyzed by LCMS. Analysis showed that no
reaction with Ellman’s reagent was evident, highlighting that
dibromomaleimide functionalization had occurred at C111.
Mercaptoethanol- or Glutathione-Mediated Cleavage of 4
To Regenerate Protein 1. The solution of 4 was treated with
2-mercaptoethanol or glutathione (5 µL, 282 mM solution in H2O)
at 0 °C. The mixture was vortexed for 1 s and maintained at 0 °C
for 4 h, after which the mixture was analyzed by LCMS. Analysis
showed that the protein-dibromomaleimide adduct had been
cleanly cleaved, yielding the Grb2-SH2 (L111C) 1 (mass ) 14170)
in quantitative conversion.
Formation of the Protein-Glutathione Bioconjugate 5. The
solution of 4 was treated with glutathione (5 µL, 2.82 mM solution
in H2O) at 0 °C. The mixture was vortexed for 1 s and maintained
at 0 °C for 2 h, after which the mixture was analyzed by LCMS.
Analysis showed that the double conjugate 5 had been formed (mass
) 14 573) in near quantitative conversion.
Formation of the Protein-Sugar Bioconjugate 6. The solution
of 4 was treated with ꢀ-1-thioglucose, sodium salt (5 µL, 2.82 mM
solution in H2O) at 0 °C. The mixture was vortexed for 1 s and
maintained at 0 °C for 2 h, after which the mixture was analyzed
by LCMS. Analysis showed that the double conjugate 6 (mass )
14461) was formed in near quantitative conversion.
Modification of Grb2-SH2 (L111C) 1 with Ellman’s Reagent.
To a solution of Grb2-SH2 (L111C) 1 (100 µL, [protein] 2.0 mg/
mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0 °C
was added Ellman’s reagent (5 µL, 282 mM solution in H2O) at 0
°C. The mixture was vortexed for 1 s and maintained at 0 °C for
10 min, after which the mixture was analyzed by LCMS. Analysis
showed a single product with a mass of 14 370, corresponding to
the mixed disulfide, confirming that C111 was available for
functionalization.
Modification of Grb2-SH2 (L111C) 1 with N-Methylbromoma-
leimide. To a solution of Grb2-SH2 (L111C) 1 (100 µL, [protein]
2.0 mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at
0 °C was added N-methylbromomaleimide (5 µL, 2.82 mM solution
in DMF). The mixture was vortexed for 1 s and then maintained at
0 °C for 1 h. Analysis using LCMS showed that the conjugate 2
had been formed in quantitative conversion (mass 14 280). Identical
solutions of 2 formed in this manner were then subjected to the
following experiments.
Ellman’s Test. The solution of 2 was treated with Ellman’s
reagent (5 µL, 282 mM solution in H2O) at 0 °C. The mixture was
vortexed for 1 s and maintained at 0 °C for 10 min, after which
the mixture was analyzed by LCMS. Analysis showed that no
reaction with Ellman’s reagent was evident, highlighting that
N-methylbromomaleimide functionalization had occurred at C111.
TCEP-Mediated Cleavage of 2 To Regenerate Protein 1. The
solution of 2 was treated with TCEP ·HCl (5 µL, 282 mM solution
in H2O) at 0 °C. The mixture was vortexed for 1 s and maintained
at 0 °C for 3 h after which the mixture was analyzed by LCMS.
Analysis showed that the protein-N-methylbromomaleimide adduct
2 had been cleaved, yielding Grb2-SH2 (L111C) 1 (mass ) 14 170)
in 85% conversion.
Mercaptoethanol- or Glutathione-Mediated Cleavage of 5
To Regenerate Protein 1. The solution of 5 was treated with
2-mercaptoethanol or glutathione (5 µL, 282 mM solution in H2O)
at 0 °C. The mixture was vortexed for 1 s and maintained at 0 °C
for 4 h, after which the mixture was analyzed by LCMS. Analysis
showed that the Grb2-SH2 (L111C) 1 (mass ) 14 170) was formed
in quantitative conversion.
Mercaptoethanol- or Glutathione-Mediated Cleavage of 6
To Regenerate Protein 1. The solution of 6 was treated with
2-mercaptoethanol or glutathione (5 µL, 282 mM solution in H2O)
at 0 °C. The mixture was vortexed for 1 s and maintained at 0 °C
for 4 h, after which the mixture was analyzed by LCMS. Analysis
showed that the Grb2-SH2 (L111C) 1 (mass ) 14170) was formed
in quantitative conversion.
Modification of Grb2-SH2 (L111C) 1 with NEM. To a solution
of Grb2-SH2 (L111C) (100 µL, [protein] 2.0 mg/mL, 100 mM
sodium phosphate, 150 mM NaCl, pH 8.0) at 0 °C was added
N-ethylmaleimide (5 µL, 2.82 mM solution in DMF). The mixture
was vortexed for 1 s and then maintained at 0 °C for 1 h. Analysis
using LCMS showed that the desired conjugate 7 had been formed
in quantitative conversion (mass 14 295). The solution of 7 was
treated with either 2-mercaptoethanol (5 µL, 282 mM solution in
H2O) or TCEP ·HCl (5 µL, 282 mM solution in H2O), vortexed
for 1 s, and maintained at 37 °C for 4 h, after which the mixtures
were analyzed by LCMS, showing no reaction had occurred in either
case.
Formation of the Protein-Glutathione Bioconjugate 3. The
solution of 2 was treated with glutathione (5 µL, 2.82 mM solution
in H2O) at 0 °C. The mixture was vortexed for 1 s and maintained
at 0 °C for 2 h, after which the mixture was analyzed by LCMS.
Analysis showed that diconjugate 3 had been formed (mass )
14 588) in 95% conversion. The remaining material was Grb2-SH2
(L111C) 1.
Formation of the Protein-Mercaptoethanol Bioconjugate.
The solution of 2 was treated with 2-mercaptoethanol (5 µL, 2.82
mM solution in H2O) at 0 °C. The mixture was vortexed for 1 s
and maintained at 0 °C for 2 h, after which the mixture was analyzed
by LCMS. Analysis showed that the Grb2-SH2 domain L111C-N-
methylbromomaleimide-2-mercaptoethanol adduct (mass ) 14359)
had been formed in 90% conversion. The remaining material was
Grb2-SH2 domain L111C.
Reaction Profile Experiments of N-Methylbromomaleim-
ide, Dibromomaleimide, N-Ethylmaleimide, and Iodoacet-
amide with Grb2-SH2 (L111C) 1. To a solution of Grb2-SH2
(L111C) (100 µL, [protein] 2.0 mg/mL, 100 mM sodium phosphate,
150 mM NaCl, pH 8.0) at 0 °C was added the relevant electrophile
(5 µL, 2.82 mM solution in DMF). The mixture was vortexed for
1 s and then maintained at 0 °C. The reaction was sampled at regular
time intervals and reaction progress monitored by MS. Percentage
reaction completion was determined using total ion count (TIC)
via the formula TIC adduct/(TIC adduct + TIC unreacted Grb2)
× 100. Iodoacetamide resulted in <5% reaction after 30 min, and
thus no time points are shown for this reaction. Reactions were
performed in triplicate, and percentage reaction completion was
plotted as the mean value. Errors were determined and are reported
both as standard deviations and standard errors in the Supporting
Information.
Modification of Grb2-SH2 (L111C) 1 with Dibromomaleim-
ide. To a solution of Grb2-SH2 (L111C) 1 (100 µL, [protein] 2.0
mg/mL, 100 mM sodium phosphate, 150 mM NaCl, pH 8.0) at 0
°C was added dibromomaleimide (5 µL, 2.82 mM solution in
DMF). The mixture was vortexed for 1 s then maintained at 0 °C
for 2 h. Analysis using LCMS showed that conjugate 4 had been
formed in quantitative conversion (mass 14346). Identical solutions
of 4 formed in this manner were then subjected to the following
experiments.
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Protein Modification Using Bromomaleimides
A R T I C L E S
Cytoplasm Mimicking Cleavage Experiment. A solution of
sugar-protein bioconjugate 6 (made as described above) was
subjected to a buffer swap (Micro Bio-Spin 6 Chromatography
Column, Bio-Rad). To a solution of 6 (95 µL, [adduct] 0.2 mg/
mL, 20 mM HEPES, 100 mM KCl, 1 mM MgCl2, 1 mM EDTA,
pH 7.4) was added glutathione (5 µL, 20 mM solution in 20 mM
HEPES, 100 mM KCl, 1 mM MgCl2, 1 mM EDTA, pH 7.4). The
mixture was vortexed for 1 s then maintained at 37 °C for 4 h.
Analysis showed that Grb2-SH2 (L111C) 1 was formed (mass )
14 170) in quantitative conversion.
an inseparable 1:1 mix of two symmetrical diastereomers: 1H NMR
(400 MHz, CDCl3) δ 8.62 (s, 1H, NH from one diastereomer), 8.66
(s, 1H, NH from one diastereomer), 5.62 (d, 2H, J ) 8.4, 2 × NH
from one diastereomer), 5.51 (d, 2H, J ) 8.0, 2 × NH from one
diastereomer), 4.72-4.58 (m, 4 × R-CH from both diastereomers),
3.80 (s, 6H, 2 × CH3 from one diastereomer), 3.79 (s, 6H, 2 ×
CH3 from one diastereomer), 3.68 (s, 2H, 2 × succinimide CH
from one diastereomer), 3.64 (s, 2H, 2 × succinimide CH from
one diastereomer), 3.46 (dd, 2H, J ) 4.8 and 12.0 Hz, 2 × CHH
from one diastereomer), 3.37 (dd, 2H. J ) 6.0 and 14.4, 2 × CHH
from one diastereomer), 3.21 (dd, 2H, J ) 4.8 and 14.0 Hz, 2 ×
CHH from one diastereomer), 3.11 (dd, 2H, J ) 6.4 and 14.0 Hz,
2 × CHH from one diastereomer), 1.463 (s, 18H, 6 × CH3 from
one diastereomer), 1.460 (s, 18H, 6 × CH3 from one diastereomer);
13C NMR (125 MHz, CDCl3) (three signals missing due to overlap
of diastereomers) δ 174.32 (CdO), 171.25 (CdO), 155.33 (CdO),
80.61 (C), 80.58 (C), 53.51 (CH), 53.18 (CH), 52.91 (CH3), 52.90
(CH3), 48.45 (CH), 47.89 (CH), 34.66 (CH2), 34.59 (CH2), 28.37
Bridging of Somatostatin with Dibromomaleimide. Lyophilized
somatostatin (mass ) 1638) was solubilized in buffer (50 mM
sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF) to yield a
concentration of 152.6 µM (0.25 mg/mL) and reduced with 1.1
equiv of TCEP for 1 h at 20 °C. Completeness of the reduction
was confirmed by LCMS (mass ) 1640); 1.1 equiv of dibromo-
maleimide was added and the reaction maintained at 20 °C for 1 h.
Quantitative insertion of the maleimide into the disulfide bond to
give conjugate 8 was confirmed by LCMS (mass ) 1734).
Mercaptoethanol-Mediated Cleavage of 8 To Regenerate
Reduced Somatostatin 9. The solution of 8 was treated with
2-mercaptoethanol (100 equiv) and the reaction maintained at 4
°C for 1 h. Analysis by LCMS showed complete cleavage of the
conjugate, yielding reduced somatostatin 9 (mass ) 1640).
Bridging of Somatostatin with N-Fluoresceindibromomale-
imide. Lyophilized somatostatin (mass ) 1638) was solubilized in
buffer (50 mM sodium phosphate, pH 6.2, 40% MeCN, 2.5% DMF)
to yield a concentration of 152.6 µM (0.25 mg/mL) and reduced
with 1.1 equiv of TCEP for 1 h at 20 °C. Completeness of the
reduction was confirmed by LCMS (mass ) 1640); 1.1 equiv of
N-fluoresceindibromomaleimide was added and the reaction main-
tained at 20 °C for 10 min. Quantitative insertion of the maleimide
into the disulfide bond to give conjugate 11 was confirmed by
LCMS (mass ) 2066) [M+1 peak of product above spectrometer
threshold, M+2 (m/z ) 1033) and M+3 (m/z ) 689) clearly
visualized].
Mercaptoethanol-Mediated Cleavage of 11 To Regenerate
Reduced Somatostatin 9. The solution of 11 was treated with
2-mercaptoethanol (100 equiv) and the reaction maintained at 4
°C for 1 h. Analysis by LCMS showed complete cleavage of the
conjugate yielding reduced somatostatin 9 (mass ) 1640).
2-tert-Butoxycarbonylamino-3-[4-(2-tert-butoxycarbonylamino-
2-methoxycarbonyl-ethylsulfanyl)-2,5-dioxopyrrolidin-3-ylsulfanyl]-
propionic Acid Methyl Ester (Inseparable Mixture of Diastereo-
mers). N-Boc-Cys-OMe (660 mg, 2.81 mmol) in N,N-dimethyl-
formamide (DMF) (0.25 mL) was added to a stirred solution of
bromomaleimide (50 mg, 0.281 mmol) in aqueous buffer (100 mM
sodium phosphate, 150 mM NaCl, pH 8.0):DMF 95:5 (9.25 mL),
and the reaction was stirred at 25 °C for 5 min. The aqueous reaction
mixture was extracted with ethyl acetate (3 × 25 mL). The
combined organic layers were washed with saturated lithium
chloride solution (aq) (5 × 25 mL), water (25 mL), and brine (25
mL); dried (MgSO4); and filtered, and the solvent was removed in
vacuo. Purification by column chromatography (gradient elution
10-40% ethyl acetate in petroleum ether 40-60 °C) gave the
product as a yellow waxy oil (150 mg, 0.265 mmol, 94% yield),
(CH3), 28.36 (CH3); IR (thin film, neat) 3348, 2978, 1719 cm-1
;
LRMS (EI) 566 (20), 564 (100 [M - H]-); HRMS (EI) calcd for
C22H34N3O10S2 [M - H]- 564.1669; observed 564.1686.
N-Fluoresceindibromomaleimide 10. Dibromomaleic anhy-
dride17 (77.0 mg, 0.30 mmol) was added in one portion to a solution
of fluoresceinamine isomer 1 (105 mg, 0.30 mmol) in AcOH (10
mL) and the reaction mixture was stirred for 6 h at room
temperature. The solid was then filtered off, washed with EtOAc,
and resuspended in AcOH (10 mL). The reaction mixture was then
heated to reflux for 3 h. Upon cooling to room temperature, toluene
(10 mL) was added and the solvent removed in vacuo. This
procedure was repeated twice more to azeotropically remove the
AcOH, affording 10 as an orange solid (148 mg, 0.25 mmol, 84%
1
yield): H NMR (400 MHz, CD3OD) δ 8.07 (d, J ) 1.5 Hz, 1H,
CH), 7.81 (dd, J ) 1.5, 2.0 Hz, 1H, CH), 7.34 (d, J ) 8.5 Hz, 1H,
CH), 6.71 (d, J ) 2.5 Hz, 2H, CH), 6.66 (d, J ) 8.5 Hz, 2H, CH),
6.58 (dd, J ) 1.5, 2.5 Hz, 2H, CH); 13C NMR (150 MHz, CD3OD)
δ 170.23 (C), 164.34 (C), 161.63 (C), 154.18 (C), 152.93 (C),
134.59 (C), 134.19 (CH), 131.01 (C), 130.35 (CH), 129.25 (C),
126.25 (CH), 123.63 (CH), 113.84 (CH), 111.02 (C), 103.55 (CH);
IR (MeOH) 3063, 2924, 1725, 1588 cm-1; HRMS (ES+) calcd
for C24H12NO7Br2 [M+H]+ 583.8980, observed 583.8964; mp >220
°C (dec).
Acknowledgment. We gratefully acknowledge RCUK, EPSRC,
UCL, the Wellcome Trust, and UCLB for support of our program.
We also thank Dr. Lisa Harris for assistance with the mass
spectrometry analyses and Dr. Adam McKay for helpful discussions.
We also thank the referees for useful comments.
Supporting Information Available: Full experimental details
on the cloning and expression of Grb2-SH2 L111C mutant 1,
MS spectra on the protein modification experiments, and NMR
spectra on the small molecule experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA908610S
9
J. AM. CHEM. SOC. VOL. 132, NO. 6, 2010 1965