Dichloro Butenediamides as Irreversible Site-Selective Protein Conjugation Reagent

Article abstract of DOI:10.1002/anie.202108791

We describe maleic-acid derivatives as robust cysteine-selective reagents for protein labelling with comparable kinetics and superior stability relative to maleimides. Diamide and amido-ester derivatives proved to be efficient protein-labelling species with a common mechanism in which a spontaneous cyclization occurs upon addition to cysteine. Introduction of chlorine atoms in their structures triggers ring hydrolysis or further conjugation with adjacent residues, which results in conjugates that are completely resistant to retro-Michael reactions in the presence of biological thiols and human plasma. By controlling the microenvironment of the reactive site, we can control selectivity towards the hydrolytic pathway, forming homogeneous conjugates. The method is applicable to several scaffolds and enables conjugation of different payloads. The synthetic accessibility of these reagents and the mild conditions required for fast and complete conjugation together with the superior stability of the conjugates make this strategy an important alternative to maleimides in bioconjugation.

Full text of DOI:10.1002/anie.202108791

A Journal of the Gesellschaft Deutscher Chemiker
Angewandte
Chemie
International Edition
www.angewandte.org
Accepted Article
Title: Dichloro Butendiamides as Irreversible Site-Selective Protein
Conjugation Reagent
Authors: Victor Laserna, Daniel Abegg, Cláudia Afonso, Esther Martin,
Alexander Adibekian, Peter Ravn, Francisco Corzana, and
Gonçalo J. L. Bernardes
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.202108791
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10.1002/anie.202108791
Angewandte Chemie International Edition
RESEARCH ARTICLE
Dichloro Butendiamides as Irreversible Site-Selective Protein
Conjugation Reagent
Victor Laserna,*[a] Daniel Abegg,[b] Cláudia F. Afonso,[c] Esther M. Martin,[d] Alexander Adibekian,[b]
Peter Ravn,[d, e] Francisco Corzana,[f] and Gonçalo J. L. Bernardes*[a,c]
[a]
Dr. V. Laserna, Dr. G. J. L. Bernardes
Yusuf Hamied Department of Chemistry
University of Cambridge
Lensfield Road, CB2 1EW, Cambridge (UK)
E-mail: gb453@cam.ac.uk; vla28@cam.ac.uk
Dr. D. Abegg, Dr. A Adibekian
Department of Chemistry
The Scripps Research Institute
130 Scripps Way, Jupiter, Fl 33418 (USA)
C. F. Afonso, Dr. G. J. L. Bernardes
Instituto de Medicina Molecular João Lobo Antunes
Faculdade de Medicina Universidade de Lisboa
Avenida Professor Egas Moniz, 1649-028 Lisboa (Portugal)
Dr. E. M. Martin, Dr. P. Ravn
[b]
[c]
[d]
AstraZeneca
R&D BioPharmaceuticals Unit | Antibody Discovery & Protein Engineering (ADPE)
Milstein Building, Granta Park, Cambridge CB21 6GH, UK
Dr. P Ravn
Department of Biotherapeutic Discovery, H. Lundbeck A/S
Ottiliavej 9, 2500 Valby, Denmark
[e]
[f]
Dr. F. Corzana
Departamento de Química. Centro de Investigación en Síntesis Química
Universidad de La Rioja. 26006 Logroño (Spain)
Supporting information for this article is given via a link at the end of the document.
Abstract: We describe maleic-acid derivatives as robust cysteine-
selective reagents for protein labelling with comparable kinetics and
superior stability relative to predominantly used maleimides. Diamide
and amido-ester derivatives proved to be efficient protein-labelling
strategies have been reported for Cys-selective conjugation,
usually based on alkylation[12]/arylation[13] reagents or Michael
acceptors.[14] Michael acceptors, particularly maleimides, remain
the most commonly used reagent for the construction of
conjugates for biological applications[15] because of their
associated fast reaction kinetics. In fact, a number of Food and
Drug Administration approved conjugates, such as antibody-drug
conjugates (ADCs) Brentuximab vedotin[15] and Trastuzumab
emtansine[16] or PEGylated conjugate Certolizumab pegol,[17]
species with
a common mechanism in which a spontaneous
cyclization reaction occurs upon addition to cysteine. Introduction of
chlorine atoms in their structures triggers ring hydrolysis or further
conjugation with adjacent residues, which results in conjugates that
are completely resistant to retro-Michael reactions in the presence of
biological thiols and human plasma. By controlling the
microenvironment of the reactive site, we can control selectivity
towards the hydrolytic pathway, forming homogeneous conjugates.
The method is applicable to several scaffolds and enables conjugation
of different payloads. The synthetic accessibility of these reagents and
the mild conditions required for fast and complete conjugation
together with the superior stability of the conjugates makes this
strategy an important alternative to maleimides in the bioconjugation
toolbox.
contain
a thio-succinimide adduct derived from maleimide
conjugation.[18]
However, it is well known that thio-succinimide adducts can
undergo fast and uncontrolled disruptive cleavage by thiol-
exchange in plasma, which ultimately compromises the safety
and efficacy of the conjugate.[19] Considerable efforts have been
reported on how to increase the stability of maleimide-based
constructs.[20] The most common approach consists of
hydrolyzing the thio-succinimide linkage to form a stable, linear
thio-succinamic acid species.[21] Different strategies to control the
hydrolysis kinetics have been described. For instance, the
Over the last decade protein site-selective conjugation has
become a thriving research field.[1] A plethora of methods have
been reported for the construction of protein conjugates with a
wide array of applications in chemical biology and medicine.[2]
Cysteine[3] (Cys) and lysine[4] (Lys) remain the main target
residues in protein modification although recently, methodologies
that target alternative residues, such as aspartic/glutamic acid,[5]
tryptophan,[6] methionine,[7] serine[8] or tyrosine[9] have been
described. Cys-targeting methods are particularly ubiquitous, as
they yield structurally homogeneous conjugates in proteins that
contain native[10] or genetically engineered free Cys.[11] Multiple
addition of electron-withdrawing groups in the payload,[22] tuning
[21a]
the amino-acid environment near the inserted Cys,
or mild
ultrasonication[23] can accelerate thio-succinimide hydrolysis.
Alternatively, maleimides with different leaving groups attached to
the vinylic bond or similar structures have been reported to form
conjugates with higher stability based on maleimide or maleamic
acid linkages.[24]
There are also acyclic reagents, such as benzoyl acrylic
25]
reagents[11,
or fumarate derivatives.[26] They produce
conjugates with linear linkages, which can present enhanced
1
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10.1002/anie.202108791
Angewandte Chemie International Edition
RESEARCH ARTICLE
25]
stability in plasma relative to maleimides.[11,
Despite these
experiments, we now exposed these reagents to the presence of
a free Cys-containing protein (Figure 1, Conditions B, Figures
S25–S31). As a model protein we chose Ub, which was
engineered with a reactive Cys at position 63 (K63C). Surprisingly,
the reactivity of the different species varied depending on the
setup. Some species showed high reactivity in small-molecule
reactions but no protein conjugation reactivity, whereas others
proved to be very efficient protein conjugation reagents but were
unreactive in a small molecule setup. In successful conjugations,
Cys-selectivity was confirmed after digestion by MS/MS studies
(Figure S82–S83) and by using Ellman’s reagent as a chemical
control (Figure S32).
advances, most of these strategies fail to combine fast
conjugation kinetics along with the formation of stable conjugates,
which is required for biological applications, and where
maleimides remain the first choice. The challenge remains to find
general, simple alternative for Cys bioconjugation with
comparable kinetics and synthetic accessibility to maleimides, but
complete linkage stability.
a
We hereby report on diamide and amido esters derivatives from
maleic acid as new, efficient reagents for Cys-selective protein
conjugation. These reagents undergo fast Michael addition
reactions with free Cys containing proteins in stoichiometric
amounts under physiological conditions. Conjugation occurs
through an unprecedented mechanism in which, upon Cys
addition, spontaneous cyclization is promoted by attack of an
amide moiety to the distal carbonyl group to form a succinimide
link (Scheme 1). By expanding this approach to 2,3-dichloro but-
en-diamides, we can selectively target free Cys residues that form
chloro-maleimide intermediate linkages, which quickly undergo
hydrolysis. The final conjugates are fully resistant to thiol-
exchange and cleavage in the presence of biological thiols and
human plasma. Through this approach an array of free Cys
containing proteins could be irreversibly tagged, such as ubiquitin
(Ub), human serum albumin (HSA) or different antibody
fragments, with different tags including azide or alkyne
functionalities. The favourable kinetics, stability and stoichiometry
of the conjugation makes a flexible platform to access complex
constructs, such as ADCs, which is an advantage over currently
existing platforms.
Figure 1. Small molecule and Ub reaction with different maleic acid derivatives.
DMF = dimethyl formamide.
Mass analysis of the protein conjugates formed revealed a unique
conjugate with a mass of 8754 Da (protein mass + 188 Da) for all
the examples. Interestingly, the mass of the newly formed
conjugate did not correspond to the addition of the full reagent.
Instead, we observed the formation of a thio-succinimide linkage.
The origin of such a linkage is through intramolecular attack of the
amide to the distal carbonyl group (Scheme 2a), which can
happen prior or after Cys conjugation. Similar cyclizations are
known although never triggered by aqueous solution or thiol
conjugation.[27] Furthermore, the cyclization step seems to play a
key role in the viability of the conjugation, because only
compounds with at least one secondary amide, capable of driving
such a cyclization, underwent conjugation. To gain better
mechanistic insight, compounds 2, and 5–10 were stirred for 2 h
at 25 ºC in DMSO/D20 (NaPi pH 8, 50 mM) to determine if the
cyclization was induced as a result of the pH of the solvent or the
thiol addition. Amido esters 2 and 8 cyclized spontaneously in the
Scheme 1. Synthesis of conjugates with linear succinamic acid linkages.
With the goal of finding alternative approaches to access
homogeneous conjugates with fully stable linkages in free Cys-
containing proteins, we set out to investigate maleic acid
derivatives with labile functionalities, which we envisioned could
undergo fast Michael addition with thiols under physiological
conditions and, upon conjugation, would hydrolyze to form
succinamic acid structures or equally stable analogs.
The reactivity of b-mercaptoethanol (BME) was evaluated with
different maleic acid derivatives (1–10, Figure 1, Conditions A,
Figures S3–S6) to assess their reactivity as Michael acceptors.
Having determined the reactivity of 1–10 in small molecule
2
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10.1002/anie.202108791
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RESEARCH ARTICLE
presence of D2O pH 8 to form a maleimide species (Figures S1–
S2). However, this was not the general behaviour for amido
esters; compound 6 together with diamides 5, 7, 9 and 10 were
completely stable under these conditions. This aqueous stability
supports the idea that thiol addition in the protein environment is
responsible for triggering the cyclization. By using diamide 10 and
Ub as a model, the reaction showed great tolerance for pH [5–9,
Figures S33–S37)] and temperature [25–37 ºC (Figures S31
and S36)].
When a reagent with two different secondary amide groups, such
as 9, is conjugated, a regioselectivity issue arises. The presence
of two secondary amide moieties allows for two possible
cyclizations each driven by one of the amides (Scheme 2b), each
pathway leads to a different conjugate: 9a or 9b. The preferred
pathway could be controlled by the pH at which the reaction was
done. Under slightly acidic conditions, pH 6–7, the aryl amide
group directed cyclization was favored, whereas under basic
conditions, pH 8–9, the opposite selectivity was observed
(Figures S38–39).
or desirable with proteins because they can result in unfolding and
loss of functional properties. To address this problem, we focused
on accelerating the hydrolysis of the linkages created by the
conjugation of maleic acid diamides.
Aqueous instability of dichloro maleimides[24c] suggests that
hydrolysis kinetics of maleimides can be exponentially increased
by the introduction of halogens into the ring. This led us to think
that, by introducing chlorine substituents into the alkene scaffold
of maleic acid diamides, the stability of the compounds and their
reactivity would not be affected, although now instead of obtaining
conjugates with succinimide linkages, chloro-maleimide linkages
would be generated, which would quickly hydrolyze to stable
linear chloro maleamic acid species. By following reported
procedures[28]
we
synthesized
symmetrical
di-chloro
butendiamides (11–14, Figure 2a) and, to our delight, their
reactivity still included a cyclization step which yielded a cyclic
chloro-maleimide linkage with enhanced hydrolysis kinetics.
Addition of 11 (3 equiv) to a solution of Ub resulted in complete
conjugation (Figure 2) after 30 min at 25 ºC. The cyclic
intermediate was observed if the reaction mixture was analyzed
shortly after addition of the reagent (Figure S71). No trace of
cyclic maleimide linkage and only linear chloro maleic acid linkage
was noted after 30 min at different temperatures (25–37 ºC) and
pH [pH 5–9 (Figures S67–S70)], which further supports the
hypothesis of rapid hydrolysis rates of the linkage. The
mechanism associated with this conjugation is similar to that of
dibromomaleimides[23] in that the linkages obtained are similar,
but an additional cyclization step and much more favourable
hydrolysis kinetics, makes this approach advantageous to obtain
homogenous, stable maleamic acid linkages.
Another appealing aspect of this conjugation reaction is the
controlled release of small molecules in
a
particular
microenvironment of the protein sequence. Upon cyclization, an
amine (diamides) or alcohol (amido-esters) molecule is released,
which could open new approaches for monitoring reactions
(release of fluorophores), Cys-targeted decaging of payloads or
site-directed bifunctionalization of proteins. We are currently
investigating this directed release in our laboratories and will be
reported in due course.
The generality of the method was demonstrated by conjugation of
7 and 14 to alternative free Cys containing proteins. Under similar
conditions, HSA, the N-terminal domain of phage repressor R434
and a camelian nanobody targeted at amyloid precursors (HET
nanobody)[29] were effectively conjugated (Figures S40–S42) to
7. In all examples Michael addition/cyclization mechanism to yield
a succinimide was observed, which supports the generality of the
mechanism in different protein sequence microenvironments.
Analysis of the circular dichroism (CD) profiles of the conjugates
relative to native proteins confirmed that overall tertiary structure
of the proteins was maintained throughout conjugation (Figures
S85–S86).
An array of di-chloro but-en-diamide analogs with different
functionalities were synthesized (compounds 11–14). Alyphatic
11 and aromatic 12 amide species could be accessed as well as
amides with an incorporated alkyne 13 or azide 14 functionality.
These new functionalities were incorporated into the protein
structure to enable further functionalization through copper(I)-
catalyzed alkyne-azide cycloadditon. Compounds 12–14 showed
similar reactivity to 11 upon reaction with Ub to form stable, linear
chloro maleamic acid linkages (Figures S44–S46). However,
when compound 14 was tested with different proteins, upon
conjugation-cyclization, different reactivity was observed for some
of these proteins. In Ub, C2Am[30] or peptide ASCATN the
expected sequential Michael addition-cyclization-hydrolysis
occurs to give a final conjugate with a chloro-maleamic acid
linkage. When the scope was expanded and proteins like HSA,
H3K4C or a full-length IgG antibody Gemtuzumab, a second
alternative pathway, in which a second Michael addition from an
adjacent residue takes place, occurs instead of the hydrolysis
step (Figure 2b). This alternative pathway forms cyclic maleimide
Scheme 2. Regioselectivity of the cyclization step with compound 9.
The thio-succinimide linkages obtained are known to hydrolyze
over time under physiological conditions to give a linear and
stable N-substituted thio-succinamic acid linkage, but the kinetics
of hydrolysis is extremely slow and often yields mixtures of
conjugates. Harsh hydrolyzing conditions, such as high pH and
temperature, could ultimately lead to the complete hydrolysis to
succinamic acid linkages, but such conditions are not compatible
3
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10.1002/anie.202108791
Angewandte Chemie International Edition
RESEARCH ARTICLE
linkages with two coordination sites to the protein. Both linkages
the distance from all lysine residues to the reactive carbon (C–Cl).
cannot undergo retro-Michael addition and form stable conjugates, According to these calculations, lysine at position 207 had the
which was demonstrated by analyzing the stability of Ub-14; the
conjugate remained stable in buffer, and no degradation was
observed after 48 h at 37 ºC in glutathione (1 mM) or human
plasma (Figures S73–S74).
smallest average distance and was presumably the residue that
directed the second nucleophilic attack to form the cyclic
derivative. The simulations indicated also that the incorporation of
this cyclic derivative into Fab does not significantly disrupt its
structure (Figure 2b). The involvement of K207 in the formation
of the cyclic linkage was further confirmed by MS/MS studies on
Trastuzamab V205C-14 (Figure S84). By mutating lysine 207 into
an alanine, the new Trastuzumab V205C+K207A was conjugated
to 14 and this time complete selectivity to the linear linkage was
obtained (Figure S55). This result supported the idea that nearby
lysines residues were the driving factors for the formation of the
cyclic linkage, allowing us to predict the final linkage formed and
if mixtures were to arise, avoid the formation of the cyclic species
by introducing cysteines in the appropriate place or mutating the
problematic lysine residues. We found, however, that mutation of
lysine 207 in Trastuzumab V205+K207A reduces the
nucleophilicity of cysteine 205 and only ~50% conjugation is
achieved under the previously described conditions (Figure S55).
To find suitable positions to genetically encode a reactive Cys that
allow complete conversion, yet selective formation of the stable
linear linkage, we produced Fc fragments of an IgG with Cys
insertions at different positions in the absence of nearby Lyz
residues (239iC, 268iC, 274iC, 289iC and 442iC). Under identical
reaction conditions with 14, the Fc fragments with 274iC and
289iC reacted completely and selectively formed the linear
linkage (Figures S56–S60). These simple maleic acid derivatives
enable installation of modifications on antibodies at specific sites
to form homogenous and stable antibody-conjugates.
To compare the kinetics of conjugation between maleic acids and
maleimides, an equimolar mixture of 11 and its analogous
maleimide reagent were conjugated to Fc fragment 289iC. Under
the same conditions, 11 and maleimide showed comparable
conjugation kinetics as a 1:1 mixture of Fc-11 and Fc-maleimide
constructs were obtained (Figure S81). However, when we
compared the stability of the constructs in the presence of GSH
(1mM) after 66 h at 37 ºC, the constructs (trastuzumab-11, Fc
274iC-11, Fc 289iC-11) conjugated to 11 showed higher stability
than the corresponding species conjugated with maleimide
(Figures S75–S80). While the conjugates formed with reagent 11
remain intact in the presence of a thiol, constructs built using
Figure 2. a, Conjugation reaction of reagents 11–14 with different Cys-
containing proteins. b, Representative snapshot derived from 0.5 µs MD
simulations on conjugate HER2-14. The RMSD value of the protein, relative to
the first frame, is also shown. c, Conjugation reaction of Trastuzumab V205C,
K207A with 14. d, Conjugation reaction of Fc fragments 274iC and 289iC with
14.
maleimides form
a mixture of hydrolyzed maleimide and
unconjugated antibody. This data shows a key advantage of our
reagents to build stable conjugates and illustrates how enhanced
hydrolysis kinetics of the cyclic linkages allows the formation of
fully stable species.
The enhanced plasma stability displayed by the conjugates
formed from the reaction of Cys with reagents 11–14 has potential
for the construction of stable ADCs. However, because two
alternative linkages can be formed, this could constitute an issue
regarding homogeneity of the final construct. Next, we analysed
the ratio between cyclic and linear conjugates using LCMS. The
conjugate derived from Gemtuzumab V205C showed a mixture of
species, [ratio 2:1 cyclic vs linear (Figure S53)], but all others
formed selectively one of the two potential linkages. Conjugation
of HER2 targeted IgG Trastuzumab V205C with 14 led to a similar
result (ratio 4:1, Figure S54).
In summary, we describe the protein conjugation reactivity of a
series of maleic acid derivatives and identify amido esters and
diamides as viable conjugation reagents to produce succinimide
linkages through a new stepwise mechanism. The strategy was
optimized to use dichloro butendiamides, which promote a further
hydrolysis step to form completely stable linkages to proteins.
This latter species shows comparable conjugation kinetics
relative to maleimides yet benefits from superior stability. The
general mechanism described composes a Michael addition
followed by spontaneous cyclization to form cyclic linkages similar
to those obtained by conjugation to maleimides. The presence of
chlorine atoms in these cyclic linkages promotes subsequent
hydrolysis or to give completely stable linkages. Nearby
nucleophilic residues can promote an alternative mechanism
To explain this result at the atomic level, we performed molecular
dynamics (MD) simulations on the Fab fragment of HER2
conjugated to compound 14 (Figures 2b and S87) and examined
4
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10.1002/anie.202108791
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M. Bouzelha, M. Mével, D. Deniaud, M. Boujtita, S. G. Gouin, J. Am.
Chem. Soc, 2018, 140, 17120–17126; d) S. Leier, S. Richter, R.
Bergmann, M. Wuest, F. Wuest, ACS Omega 2019, 4, 22101–22107.
[10] P. R. Lindstedt, F. A. Aprile, M. J. Matos, M. Perni, J. B. Bertoldo, B.
Bernardim, Q. Peter, G. Jiménez-Osés, T. P. J. Knowles, C. M. Dobson,
F. Corzana, M. Vendruscolo, G. J. L. Bernardes, ACS Cent. Sci. 2019, 5,
1417–1424.
where a second nucleophilic attack leads to the formation of a
cyclic linkage, but this pathway can be controlled and depleted if
undesired conjugate mixtures are obtained. The approach allows
for the creation of conjugates with azide/alkyne moieties and
opens up possibilities to create tailored conjugation for a number
of relevant proteins, including nanobodies or IgGs relevant for the
formation of ADCs.
[11] B. Bernardim, P. M. Cal, M. J. Matos, B. L. Oliveira, N. Martínez-Sáez, I.
S. Albuquerque, E. Perkins, F. Corzana, A. C. Burtoloso, G. Jiménez-
Osés, G. J. Bernardes, Nat. Commun. 2016, 7, 13128.
[12] S. Sechi, B. T. Chait, Anal. Chem. 1998, 70, 5150–5158.
[13] C. Zhang, E. V. Vinogradova, A. M. Spokoyny, S. L. Buchwald, B. L.
Pentelute, Angew. Chem. Int. Ed. 2019, 58, 4810–4839.
Acknowledgements
This project has received funding from the European Union’s
Horizon 2020 research and innovation programme under the
Marie Skłodowska-Curie grant agreement Nº 836698. We also
thank Dr. Ester Jiménez-Moreno for providing Ub, Dr. André
Neves and Prof. Kevin Brindle for supplying C2Am, Dr. Phil
Lindstedt and Prof. Michele Vendruscolo for the HET nanobody,
HSA was provided by Albumedix, and AstraZeneca for producing
and supplying the Gemtuzumab, Trastuzumab and Fc domain
antibodies. The authors thank Dr Vikki Cantrill for her help with
the editing of this manuscript. Funding from the Scripps Research
Institute is also acknowledged (A.A.), FCT Portugal (PhD
scholarship PD/BD/135512/2018 to C.F.A.) and Agencia Estatal
Investigación of Spain (AEI; Grant RTI2018-099592-B-C21 to
F.C.). G.J.L.B. is a Royal Society University Research Fellow
(URF\R\180019) and an FCT Stimulus (CEECIND/00453/2018).
[14] J. Ravasco, H. Faustino, A. Trindade, P. M. P. Gois, Chem. Eur. J. 2019,
25, 43–59.
[15] P. D. Senter, E. L. Sievers, Nat. Biotechnol. 2012, 30, 631–637.
[16] P. M. LoRusso, D. Weiss, E. Guardino, S. Girish, M. X. Sliwkowski, Clin.
Cancer Res. 2011, 17, 6437–6447.
[17] N. Goel, S. Stephens, MAbs 2010, 2, 137–147.
[18] A. Beck, L. Goetsch, C. Dumontet, N. Corvaïa, Nat. Rev. Drug Discov.
2017, 16, 315–337.
[19] A. D. Baldwin, K. L. Kiick, Bioconjugate Chem. 2011, 22, 1946–1953.
[20] P. A. Szijj, C. Bahou, V. Chudasama, Drug Discov. Today Technol. 2018,
30, 27–34.
[21] a) B. Q. Shen, K. Xu, L. Liu, H. Raab, S. Bhakta, M. Kenrick, K. L.
Parsons-Reponte, J. Tien, S. F. Yu, E. Mai, D. Li, J. Tibbitts, J. Baudys,
O. M. Saad, S. J. Scales, P. J. McDonald, P. E. Hass, C. Eigenbrot, T.
Nguyen, W. A. Solis, R. N. Fuji, K. M. Flagella, D. Patel, S. D. Spencer,
L. A. Khawli, A. Ebens, W. L. Wong, R. Vandlen, S. Kaur, M. X.
Sliwkowski, R. H. Scheller, P. Polakis, J. R. Junutula, Nat. Biotechnol.
2012, 30, 184–189; b) L. N. Tumey, M. Charati, T. He, E. Sousa, D. Ma,
X. Han, T. Clark, J. Casavant, F. Loganzo, F. Barletta, J. Lucas, E. I.
Graziani, Bioconjugate Chem. 2014, 25, 1871–1880.
Keywords: bioconjugation • cysteine • irreversible • maleimides
[22] a) R. J. Christie, R. Fleming, B. Bezabeh, R. Woods, S. Mao, J. Harper,
A. Joseph, Q. Wang, Z. Q. Xu, H. Wu, C. Gao, N. Dimasi, J. Control.
Release 2015, 220, 660–670; b) S. D. Fontaine, R. Reid, L. Robinson,
G. W. Ashley, D. V. Santi, Bioconjugate Chem. 2015, 26, 145–152.
[23] W. Huang, X. Wu, X. Gao, Y. Yu, H. Lei, Z. Zhu, Y. Shi, Y. Chen, M. Qin,
W. Wang, Y. Cao, Nat. Chem. 2019, 11, 310–319.
• Michael addition
[1]
a) E. A. Hoyt, P. M. S. D. Cal, B. L. Oliveira, G. J. L. Bernardes, Nat. Rev.
Chem. 2019, 3, 147–171; b) J. N. deGruyter, L. R. Malins, P. S. Baran,
Biochemistry 2017, 56, 3863–3873; c) J. A. Shadish, C. A. DeForest,
Matter 2020, 2, 50–77; d) L. Xu, S. L. Kuan, T. Weil, Angew. Chem. Int.
Ed. 2021,133, 13874–13894.
[24] a) M. E. 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; b) C. Marculescu, H. Kossen, R. E. Morgan, P.
Mayer, S. A. Fletcher, B. Tolner, K. A. Chester, L. H. Jones, J. R. Baker,
Chem. Commun. 2014, 50, 7139–7142; c) N. Forte, M. Livanos, E.
Miranda, M. Morais, X. Yang, V. S. Rajkumar, K. A. Chester, V.
Chudasama, J. R. Baker, Bioconjugate Chem. 2018, 29, 486–492; d) J.
P. M. Nunes, V. Vassileva, E. Robinson, M. Morais, M. E. B. Smith, R. B.
Pedley, S. Caddick, J. R. Baker, V. Chudasama, RSC Adv. 2017, 7,
24828–24832; e) E. De Geyter, E. Antonatou, D. Kalaitzakis, S. Smolen,
A. Iyer, L. Tack, E. Ongenae, G. Vassilikogiannakis, A. Madder, Chem.
Sci. 2021, 12, 5246–5252.
[2]
a) E. M. Milczek, Chem. Rev. 2018, 118, 119–141; b) N. Krall, F. P. da
Cruz, O. Boutureira, G. J. L. Bernardes, Nat. Chem. 2016, 8, 103–113;
c) V. Chudasama, A. Maruani, S. Caddick, Nat. Chem. 2016, 8, 114–119.
J. M. Chalker, G. J. L. Bernardes, Y. A. Lin, B. G. Davis, Chem. Asian J.
2009, 4, 630–640.
[3]
[4]
a) S. Asano, J. T. Patterson, T. Gaj, C. F. Barbas, 3rd, Angew. Chem.
Int. Ed. 2014, 53, 11783–11786; b) M. J. Matos, B. L. Oliveira, N.
Martínez-Sáez, A. Guerreiro, P. M. S. D. Cal, J. Bertoldo, M. Maneiro, E.
Perkins, J. Howard, M. J. Deery, J. M. Chalker, F. Corzana, G. Jiménez-
Osés, G. J. L. Bernardes, J. Am. Chem. Soc, 2018, 140, 4004–4017; c)
S. R. Adusumalli, D. G. Rawale, U. Singh, P. Tripathi, R. Paul, N. Kalra,
R. K. Mishra, S. Shukla, V. Rai, J. Am. Chem. Soc. 2018, 140, 15114–
15123.
[25] B. Bernardim, M. J. Matos, X. Ferhati, I. Compañón, A. Guerreiro, P.
Akkapeddi, A. C. B. Burtoloso, G. Jiménez-Osés, F. Corzana, G. J. L.
Bernardes, Nat. Protoc. 2019, 14, 86–99.
[5]
[6]
[7]
N. Ma, J. Hu, Z. M. Zhang, W. Liu, M. Huang, Y. Fan, X. Yin, J. Wang, K.
Ding, W. Ye, Z. Li, J. Am. Chem. Soc, 2020, 142, 6051–6059.
Y. Seki, T. Ishiyama, D. Sasaki, J. Abe, Y. Sohma, K. Oisaki, M. Kanai,
J. Am. Chem. Soc, 2016, 138, 10798–10801.
[26] a) B. W. Zaro, L. R. Whitby, K. M. Lum, B. F. Cravatt, J. Am. Chem. Soc.
2016, 138, 15841–15844; b) B. W. Zaro, E. V. Vinogradova, D. C. Lazar,
M. M. Blewett, R. M. Suciu, J. Takaya, S. Studer, J. C. de la Torre, J. L.
Casanova, B. F. Cravatt, J. R. Teijaro, J. Immunol. 2019, 202, 2737–
2746; c) M. M. Blewett, J. Xie, B. W. Zaro, K. M. Backus, A. Altman, J. R.
Teijaro, B. F. Cravatt, Sci. Signal. 2016, 9, rs10.
a) S. Lin, X. Yang, S. Jia, A. M. Weeks, M. Hornsby, P. S. Lee, R. V.
Nichiporuk, A. T. Iavarone, J. A. Wells, F. D. Toste, C. J. Chang, Science
2017, 355, 597–602; b) M. T. Taylor, J. E. Nelson, M. G. Suero, M. J.
Gaunt, Nature 2018, 562, 563–568.
[27] M. B. Richardson, K. N. Gabriel, J. A. Garcia, S. N. Ashby, R. P. Dyer, J.
K. Kim, C. J. Lau, J. Hong, R. J. Le Tourneau, S. Sen, D. L. Narel, B. B.
Katz, J. W. Ziller, S. Majumdar, P. G. Collins, G. A. Weiss, Bioconjugate
Chem. 2020, 31, 1449–1462.
[8]
[9]
J. C. Vantourout, S. R. Adusumalli, K. W. Knouse, D. T. Flood, A.
Ramirez, N. M. Padial, A. Istrate, K. Maziarz, J. N. deGruyter, R. R.
Merchant, J. X. Qiao, M. A. Schmidt, M. J. Deery, M. D. Eastgate, P. E.
Dawson, G. J. L. Bernardes, P. S. Baran, J. Am. Chem. Soc, 2020, 142,
17236–17242.
[28] G. Bonse, H. U. Blank, Liebigs Ann. Chem. 1981, 1981, 1658–1664.
[29] P. R. Lindstedt, F. A. Aprile, P. Sormanni, R. Rakoto, C. M. Dobson, G.
J. L. Bernardes, M. Vendruscolo, Cell Chem. Biol. 2021, 28, 70–77 e75.
[30] I. S. Alam, A. A. Neves, T. H. Witney, J. Boren, K. M. Brindle,
Bioconjugate Chem. 2010, 21, 884–891.
a) K. L. Seim, A. C. Obermeyer, M. B. Francis, J. Am. Chem. Soc, 2011,
133, 16970–16976; b) S. Sato, H. Nakamura, Angew. Chem. Int. Ed.
2013, 52, 8681–8684; c) D. Alvarez-Dorta, C. Thobie-Gautier, M. Croyal,
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10.1002/anie.202108791
Angewandte Chemie International Edition
RESEARCH ARTICLE
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Tag It Irreversibly. We describe maleic acid derivatives as cysteine-selective reagents for protein labelling with comparable kinetics
and superior stability to existing reagents. A novel mechanism is described to readily access stable chloro-maleamic acid. The mild
conditions, fast kinetics and irreversible conjugation make this method an appealing alternative to existing cysteine-targeted methods.
Institute and/or researcher Twitter usernames: @gbernardes_chem; @AdibekianG; @vlasernayora
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