Stable and Rapid Thiol Bioconjugation by Light-Triggered Thiomaleimide Ring Hydrolysis

Article abstract of DOI:10.1002/anie.201609733

Maleimide-mediated thiol-specific derivatization of biomolecules is one of the most efficacious bioconjugation approaches currently available. Alarmingly, however, recent work demonstrates that the resulting thiomaleimide conjugates are susceptible to breakdown via thiol exchange reactions. Herein, we report a new class of maleimides, namely o-CH₂NHⁱPr phenyl maleimides, that undergo unprecedentedly rapid ring hydrolysis after thiol conjugation to form stable thiol exchange-resistant conjugates. Furthermore, we overcome the problem of low shelf lives of maleimide reagents owing to their propensity to undergo ring hydrolysis prior to bioconjugation by developing a photocaged version of this scaffold that resists ring hydrolysis. UV irradiation of thiol bioconjugates formed with this photocaged maleimide unleashes rapid thiomaleimide ring hydrolysis to yield the desired stable conjugates within 1 h under gentle, ice-cold conditions.

Full text of DOI:10.1002/anie.201609733

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Communications
Chemie
International Edition: DOI: 10.1002/anie.201609733
German Edition: DOI: 10.1002/ange.201609733
Bioconjugation
Stable and Rapid Thiol Bioconjugation by Light-Triggered
Thiomaleimide Ring Hydrolysis
Dimpy Kalia,* Sharad P. Pawar, and Jyoti S. Thopate
Abstract: Maleimide-mediated thiol-specific derivatization of
biomolecules is one of the most efficacious bioconjugation
approaches currently available. Alarmingly, however, recent
work demonstrates that the resulting thiomaleimide conjugates
are susceptible to breakdown via thiol exchange reactions.
Herein, we report a new class of maleimides, namely o-
CH2NHiPr phenyl maleimides, that undergo unprecedentedly
rapid ring hydrolysis after thiol conjugation to form stable thiol
exchange-resistant conjugates. Furthermore, we overcome the
problem of low shelf lives of maleimide reagents owing to their
propensity to undergo ring hydrolysis prior to bioconjugation
by developing a photocaged version of this scaffold that resists
ring hydrolysis. UV irradiation of thiol bioconjugates formed
with this photocaged maleimide unleashes rapid thiomaleimide
ring hydrolysis to yield the desired stable conjugates within 1 h
under gentle, ice-cold conditions.
C
hemical reactions that enable site-specific protein biocon-
Figure 1. Thiomaleimide bioconjugation via a) the conventional
approach and b) our proposed approach.
jugation have revolutionized both clinical and biological
research.[1] In particular, Michael addition reactions between
cysteinyl thiols and maleimides have been extensively used.
Indeed, both of the antibody-drug conjugates (ADCs) in the
market and 24 out of the 34 ADCs currently undergoing
clinical trials employ this chemistry.[2] Additionally, thiol–
maleimide conjugation is routinely used for a variety of other
applications, including labeling of proteins with fluorescent
dyes and affinity tags,[3] protein PEGylation,[4] and fabrication
of nanoparticles and polymers.[5]
thiol exchange-mediated breakdown is highly desirable. One
strategy for achieving this goal is to allow conventional N-
alkyl thiomaleimide adducts to undergo ring hydrolysis to
yield thiol exchange-resistant thiomaleamic acids (II, Fig-
ure 1a), an extremely slow process that takes about 1 month
at 378C for completion,.[6b,d] Recently, thiol conjugates of N-
aminoethylmaleimide have been reported to undergo faster
ring hydrolysis that proceeds to completion within 1 day at
room temperature and pH 7.4.[6b,d] This method is not ideal, as
day-long room temperature exposure of proteins[9] and
protein conjugates such as ADCs[10] can lead to aggregation-
mediated loss of activity. Therefore, the development of
maleimide derivatives that undergo faster thiomaleimide ring
hydrolysis to rapidly yield stable conjugates at milder temper-
atures (preferably at 48C or under ice-cold conditions) is
urgently needed.
We reasoned that thiol conjugates of N-phenyl malei-
mides appended with an amino group at the ortho position
would undergo much more rapid ring hydrolysis due to the
synergistic effects of the electron withdrawing phenyl group
and the ability of the amino group to orchestrate intra-
molecular acid catalysis (III in Figure 1b). To test this
hypothesis, we synthesized N-acetyl cysteine conjugates of
o-NH2 phenyl maleimide (1) and o-CH2-NH2 phenyl malei-
mide (2) and studied their ring hydrolysis at pH 7 and room
temperature via HPLC. These studies revealed that 1 was
extremely resistant to ring hydrolysis and yielded no new
peak for the hydrolyzed conjugate over the course of 4 h
(Figure 2a; Supporting Information, Figure S3). In contrast, 2
Despite its widespread use, recent work has demonstrated
a major limitation of thiol–maleimide bioconjugation: the
resulting thiosuccinimidyl adducts (I in Figure 1a) are sus-
ceptible to breakdown via thiol exchange reactions with
biological thiols under physiological conditions (Figure 1a).[6]
This drawback has motivated several researchers, including
us, to develop alternate thiol bioconjugation approaches.[7]
Nevertheless, in contrast to maleimides that have a proven
track record of use for a wide variety of both in vitro and
in vivo applications, the suitability of these new linkages for
in vivo applications remains unexplored.[8]
Considering the tremendous potential of thiomaleimide
conjugates, the development of methods that prevent their
[*] Dr. D. Kalia, S. P. Pawar, J. S. Thopate
Department of Chemistry, Savitribai Phule Pune University (SPPU)
Pune, Maharashtra, 411007 (India)
E-mail: dkalia@chem.unipune.ac.in
Supporting information for this article (experimental methods, MS
characterization of HPLC peaks, additional HPLC chromatograms,
NMR spectra) can be found under:
http://dx.doi.org/10.1002/anie.201609733.
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Figure 2. Time course of ring hydrolysis of N-acetyl cysteine maleimide conjugates at pH 7 and room temperature.
hydrolyzed rapidly; the peak for the intact conjugate com-
pletely disappeared within 2 h at room temperature and pH 7,
and was replaced by the peak for the hydrolyzed conjugate (3,
Figure 2b). This observation can be accounted for by the low
pKa (ca. 4.6)[11] of the conjugate acid of the o-NH2 group of
1 due to which it primarily exists in the deprotonated state at
pH 7, and is therefore unable to orchestrate intramolecular
acid catalysis, in contrast to the o-CH2NH2 group of 2 that
possesses a much higher pKa (ca. 9.3),[11] enabling it to exist
primarily in the catalytic protonated state at pH 7. We next
compared the rate of hydrolysis of 2 with those of the N-acetyl
cysteine conjugates of two maleimide derivatives that have
been previously reported to undergo fast thiomaleimide ring
hydrolysis-N-aminoethylmaleimide (S19)[6b,d] and N-p-fluoro-
phenylmaleimide (S20).[12] Whereas S19 progressively hydro-
lyzed over 4.5 h (Supporting Information, Figure S9), S20 did
not demonstrate detectable hydrolysis over 4 h (Supporting
Information, Figure S10). Nevertheless, the extent of hydrol-
ysis of S19 was considerably lower (only 57% at the 4.5 h time
point; Supporting Information, Figure S9) than that of the N-
acetyl cysteine adduct of our new scaffold (2), which proceeds
to 100% completion within 2 h (Figure 2b).
Despite these encouraging results with 2, we were
intrigued by a slowly emerging peak at 14.4 min (marked
with an asterisk in Figure 2b; Supporting Information, Fig-
ure S4) in the HPLC chromatograms obtained with this
compound. Mass spectrometric characterization of this peak
gave a mass of 18 amu lower (corresponding to the loss of
a molecule of H2O) than that of the ring-hydrolyzed
conjugate 3 (Supporting Information, Table S1), leading us
to speculate that the amino group of the o-CH2NH2 sub-
stituent of 3 was undergoing intramolecular condensation
with one of the two COOH groups of the molecule. 1H-COSY,
DEPT, and HMBC experiments convincingly established that
the product formed was the 9-membered macrocycle 4 (for
details, see the Supporting Information).
bioconjugation approach, as the slow rate of macrocyclization
would introduce undesirable heterogeneity in aqueous sol-
utions of thiomaleimide conjugates. We reasoned that the
introduction of N-alkyl substituents on the o-CH2NH2 group
of these compounds could reduce the rate of intramolecular
nucleophilic attack by the amine due to increased steric bulk
without compromising the rate of thiomaleimide ring hydrol-
ysis. Consistent with this hypothesis, we found that the N-
acetyl cysteine conjugate of the o-CH2NHMe derivative (5,
Figure 2c) possessed a much lower macrocyclization rate
without demonstrating any decrease in the rate of ring
hydrolysis as compared to 2. Indeed, whereas the ring
hydrolysis of both 2 and 5 proceeded to completion within
2 h at pH 7 and room temperature (middle panels of Fig-
ure 2b and c, respectively), the rate of macrocyclization of the
ring hydrolyzed conjugate of 5 was much slower: 9 h into the
incubation, 19% of 2 had macrocyclized (Supporting Infor-
mation, Figure S4), as compared to only 4% after 14 h for 5
(Figure 2c; Supporting Information, Figure S6). Encouraged
by this decrease in the propensity for macrocylization upon N-
alkylation, we proceeded to introduce the bulky isopropyl
group on the amino group of the scaffold to form compound
S16 (Supporting Information, Scheme S1c). HPLC studies on
the N-acetyl cysteine adduct of this derivative (6, Figure 2d)
demonstrated that its ring hydrolysis was as fast as that of 2
and 5, proceeding to completion within 2 h, whereas macro-
cyclization was not detectable even after storing the conjugate
in aqueous solutions at pH 7 for 8 d (5 d at 228C followed by
3 d at 378C). Similar results were obtained with the N-acetyl
cysteamine analogue of the o-CH2NHiPr derivative (S18;
Supporting Information, Scheme S2) which demonstrated no
macrocyclization even after 8 d at 378C (Supporting Infor-
mation, Figure S8). We also studied the hydrolysis of 6 in
acidic (pH 6) and basic (pH 9) aqueous solutions and
observed that as expected, the ring hydrolysis is base-
catalyzed (Supporting Information, Figure S7). Additionally,
the ring-hydrolyzed conjugate of 6 was observed to be
completely stable in serum over 5 days (Supporting Informa-
tion, Figure S12).
Although our discovery of spontaneous macrocyclization
via amide bond formation in the absence of coupling agents is
fascinating from a synthetic chemistry perspective,[13] it was
not ideal for our immediate goal of developing an efficacious
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Data obtained from these studies on conjugates 1, 6, S19
the BSA conjugate gave a mass corresponding to the attach-
and S20 yielded kinetic traces depicted in the Supporting
Information, Figure S1 that clearly demonstrate the superi-
ority of 6 over other scaffolds. Indeed, t1/2 for the ring
hydrolysis of 6 was observed to be 20 min at room temper-
ature and pH 7, in comparison to 3.6 h for the recently
reported N-aminoethylmaleimide derivative (S19). These
studies demonstrate that o-CH2NHiPr phenyl maleimide is
a much improved scaffold for stable thiol bioconjugation,
possessing the fastest known rates of thiomaleimide ring
hydrolysis.
ment of one molecule of 7 on the protein (Figure 3b, bottom
right). Attachment of a single fluorophore molecule/protein
molecule was further established from UV absorbance
measurements that yielded a [Fluorophore]/[BSA] value
close to one of the conjugate (see the Supporting Information
for details). Treating BSA with the S29 (Supporting Informa-
tion, Scheme S4) also yielded the mass of its expected ring-
hydrolyzed monoadduct (Figure 3b, top right).
It is well known that maleimide reagents are susceptible to
ring hydrolysis to form thiol-unreactive maleamic acids.[12,14]
Not surprisingly, therefore, maleimide reagents are typically
stored at or below À208C until use. Consistent with this
property, S29 demonstrated progressive formation of its ring-
hydrolyzed maleamic acid derivative over 3 months (Fig-
ure 4c, left panel). To enhance its shelf life, we decided to
mask the ring hydrolysis-facilitating catalytic activity of the
amino group of S29 with the photocleavable 6-nitroveraltry-
loxycarbonyl (NVOC) moiety[15] that can be removed after
bioconjugation upon UV irradiation to trigger thiomaleimide
ring hydrolysis and form the desired stable conjugate (Fig-
ure 4a). The resulting compound (10; Supporting Informa-
tion, Scheme S4) possessed a much improved shelf life: there
was no observable formation of the hydrolyzed maleamic acid
product even after three months at room temperature, in
contrast to S29, which undergoes hydrolysis at a much lower
temperature of À108C over three months (Figure 4c). The
extremely favorable shelf life of o-CH2N(NVOC)iPr phenyl
maleimide, along with its ability to rapidly form stable thiol
conjugates via light-triggered thiomaleimide ring hydrolysis,
renders it ideal for thiol-specific conjugation.
To evaluate the thiol selectivity of our scaffold, we treated
o-CH2NHiPr phenyl maleimide (S29; Supporting Informa-
tion, Scheme S4) with an aqueous solution (at pH 7) contain-
ing N-acetyl cysteine (1 equiv), tyrosine (5 equiv), and N-
acetyl lysine (5 equiv). HPLC analysis demonstrated the
exclusive formation of the N-acetyl cysteine adduct, thereby
convincingly establishing the thiol selectivity of this scaffold
(Supporting Information, Figure S13). We next explored the
scope of o-CH2NHiPr phenyl maleimides for cysteine bio-
conjugation of peptides and proteins. Towards this goal, we
synthesized a fluorescent derivative of this scaffold (7;
Supporting Information, Scheme S3) and treated a model
peptide, glutathione (GSH), with 1 equivalent of 7 at pH 7
and room temperature. After 15 min of reaction time, the
complete disappearance of 7 and the appearance of two
closely spaced peaks for the hydrolyzed (9) and the unhy-
drolyzed (8) GS-maleimide adducts (Figure 3a) were
observed. After 1 h, the peak for 8 had completely disap-
peared and that for the stable ring-hydrolyzed adduct 9
remained. We next tested the suitability of this scaffold for
cysteine bioconjugation on proteins by treating 7 with
a protein containing a single free-cysteine, bovine serum
albumin (BSA), at room temperature and also at 48C. In
a control experiment, we treated 7 with lysozyme, a protein
that has no free cysteines. SDS-PAGE analysis (Figure 3b,
left) showed successful fluorescent labeling of BSA and no
labeling of lysozyme under both these conditions, thereby
further demonstrating thiol selectivity of the o-CH2NHiPr
phenyl maleimide scaffold. MALDI mass spectrometry on
To perform bioconjugation with 10, we treated it with one
equivalent GSH on ice to generate the photocaged biocon-
jugate 11 within 10 min (middle panel, Figure 4b). The
reaction mixture was then irradiated with UV light (lmax
=
306 nm) on ice, and decaging was confirmed by UV spectros-
copy (Supporting Information, Figure S14). HPLC analysis
after 50 min of irradiation (top panel, Figure 4b) revealed
that 11 had completely converted into the desired ring-
hydrolyzed thiol adduct of GSH (13). Experiments with N-
Figure 3. Bioconjugation of a) GSH peptide and b) BSA protein with o-CH2NHiPr phenyl maleimide at pH 7.
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Figure 4. Light-triggered thiomaleimide ring hydrolysis. a) Bioconjugation of GSH with the photocaged o-CH2NHiPr phenyl maleimide 10 followed
by light-triggered ring hydrolysis to yield the stable conjugate at pH 7 on ice. b) HPLC analysis of bioconjugation of GSH with 10. c) Comparison
of the shelf lives of unconjugated o-CH2NHiPr phenyl maleimide with (10, right) and without (S29, left) photocaging. S29 was stored at À108C
and 10 at room temperature (both as neat compounds).
acetyl cysteine instead of GSH gave identical results (Sup-
porting Information, Figure S15). Similar results were
obtained upon irradiating with more biocompatible UV
light (lmax = 352 nm; Supporting Information, Figure S16).
We next employed this photocaged maleimide moiety for
fluorescently labeling a peptide by synthesizing dansyl con-
taining o-CH2N(NVOC)iPr phenyl maleimide, S34, and using
it for the quantitative labeling of GSH followed by light-
triggered ring hydrolysis to rapidly generate the desired stable
conjugate, 9 (Supporting Information, Figure S17).
Finally, we compared the thiol-conjugation rate of o-
CH2NHiPr phenyl maleimides with those of several recently
reported scaffolds. These studies (Supporting Information,
Figures S18–S23) revealed that the o-CH2NHiPr phenyl
maleimides form thiol conjugates much faster than 3-aryl-
propiolonitriles,[7i] phenyloxadiazole sulfones,[7j] and alkynoic
amides,[7k] and at rates similar to those of bromomaleimid-
es.[7e] We envisage that along with protein bioconjugation, o-
CH2NHiPr phenyl maleimides will be employed for a wide
range of other applications, including fabrication of nano-
particles and polymers.
Conflict of interest
The authors declare no conflict of interest.
Keywords: bioconjugation · maleimide · photodecaging ·
ring hydrolysis
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Acknowledgements
This work was supported by the Department of Chemistry,
SPPU and the DST-INSPIRE Faculty Award to D.K.
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Manuscript received: October 4, 2016
Revised: December 6, 2016
Final Article published: && &&, &&&&
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Communications
Bioconjugation
D. Kalia,* S. P. Pawar,
J. S. Thopate
&&& — &&&
Stable and Rapid Thiol Bioconjugation by
Light-Triggered Thiomaleimide Ring
Hydrolysis
Light and water go together! A photo-
activable reagent for stable thiol biocon-
jugation under ice-cold conditions has
been rationally designed. The reagent first
forms a covalent adduct with the bio-
molecule of interest, and then undergoes
extremely rapid ring hydrolysis upon UV
irradiation via intramolecular catalysis
orchestrated by a protonated amino
group to yield the stable bioconjugate.
6
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