MAPN: First-in-class reagent for kinetically resolved thiol-to-thiol conjugation

Article abstract of DOI:10.1021/acs.bioconjchem.5b00440

Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no specifi

Full text of DOI:10.1021/acs.bioconjchem.5b00440

Communication
pubs.acs.org/bc
MAPN: First-in-Class Reagent for Kinetically Resolved Thiol-to-Thiol
Conjugation
Oleksandr Koniev,# Sergii Kolodych,
,†,§
§
#,†,§
‡,⊥
Zoljargal Baatarkhuu, Johann Stojko, Jitka Eberova,†
†
‡,⊥
‡,⊥
,†
́
Jean-Yves Bonnefoy, Sarah Cianferani, Alain Van Dorsselaer, and Alain Wagner*
†Laboratory of Functional ChemoSystems (UMR 7199), Labex Medalis, University of Strasbourg, 74 route du Rhin, 67401
Illkirch-Graffenstaden, France
§
Syndivia SAS, 4 Rue Boussingault, 67000 Strasbourg, France
‡
BioOrganic Mass Spectrometry Laboratory (LSMBO), IPHC, University of Strasbourg, 25 rue Becquerel, 67087 Strasbourg, France
⊥IPHC, CNRS, UMR7178, 25 rue Becquerel, 67087 Strasbourg, France
*
S Supporting Information
ABSTRACT: Thiols are among the most frequently used functional groups in the field of bioconjugation. While there exists a
variety of heterobifunctional reagents that allow for coupling thiols to other functions (e.g., amines, carboxylic acids), there is no
specific reagent for creating heteroconjugates using two different thiols. In response to the ever-increasing demand for
bioconjugation tools, we have developed p-(maleimide)-phenylpropionitrile (MAPN)an efficient reagent for kinetically
resolved thiol-to-thiol coupling. In a comparative study with its closest commercially available analogue, p-phenylenedimaleimide,
MAPN has shown substantial advantages for the preparation of thiol−thiol heteroconjugates. Namely, an antibody−drug
conjugate (ADC) with mertansine (DM1), conjugated to the cysteine residues of Trastuzumab, was prepared for the first time.
INTRODUCTION
scope of its application. The drawbacks include the instability of
the conjugate in reducing media and in exchange with other
■
Heterobifunctional reagents are comprehensively applied in
biological research for linking biological molecules to the
10
thiols. The stability issue is, for instance, of crucial importance
in the development of antibody−drug conjugates (ADC), since
the extremely potent cytotoxic payload conjugated to the
antibody should not be lost before the ADC reaches the target
cell. In order to create a stable bond, thiol-containing drugs
such as mertansine are usually conjugated to the lysine residues
of the antibody, using amine-to-thiol heterobifunctional
1
,2
functional entities of interest. Among the functional groups
used for coupling, sulfhydryl groups are of paramount
3
importance for three primary reasons. First, their high
nucleophilicity allows for specific reactions with many reactive
4
5
electrophiles. Second, these groups have low abundance but
omnipresence in biological molecules, which allows for the
preparation of well-defined bioconjugates. Free sulfhydryl
functions on proteins can for instance be obtained through
11
12
coupling reagents such as SMCC or CBTF. However,
these conjugation strategies provide highly heterogeneous
ADCs, due to the presence of more than 60 lysine residues
in the antibody backbone.
6
the reduction of disulfide bonds, naturally present in most
7
proteins, or introduced via lysine modification with SATA or
8
One successful way to decrease heterogeneity is through the
cysteine modification, using the reduction-alkylation strategy.
Indeed, IgG1 antibodies contain only four solvent-exposed
Traut’s reagent. Third, many molecular tags (dyes, linkers,
drugs, DNA, RNA, tracers, lipids) are available as sulfhydryl
derivatives. Therefore, thiol-to-thiol coupling appears as a
highway for the preparation of bioconjugates.
Most commonly, two thiols are coupled through the
formation of disulfide bonds. While this strategy is widely
Received: January 29, 2015
Revised: August 29, 2015
9
applied, it has some significant drawbacks, which limits the
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.bioconjchem.5b00440
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Scheme 1. Synthesis of MAPN
Figure 1. Heteroconjugation of glutathione and cysteine using MAPN (A) and p-phenylenedimaleimide (B). HPLC traces of MAPN and p-
phenylenedimaleimide prior to conjugation are indicated in black; HPLC traces after 2 min of incubation of the reagents with glutathione are
indicated in red; HPLC traces after a subsequent 30 min of incubation with cysteine are shown in green.
interchain disulfide bonds that can be reduced in order to yield
free thiol residues. Selective alkylation of these nucleophilic
residues was shown to yield a much cleaner mixture of
conjugate. It therefore seems appealing to combine this
reduction-alkylation strategy with a thiol-to-thiol heterocou-
pling reagent in order to access well-defined conjugates using
readily available sulfhydryl molecules.
Briefly, 4-iodoaniline reacted with propargylic alcohol under
standard Sonogashira coupling conditions to yield intermediate
2, which was oxidized with MnO in the presence of ammonia
2
14
to form 3-(4-aminophenyl)propionitrile 3. This intermediate
reacted with maleic anhydride to yield product 4. Finally,
MAPN was obtained by dehydrative cyclization of 5 in the
presence of acetic anhydride and sodium acetate.
Herein, we describe the development and application of p-
maleimide)-phenylpropionitrile (MAPN, 5), a ready-to-use
In order to confirm the applicability of MAPN for thiol-to-
thiol heterocoupling, we carried out a reaction between two
model substrates: glutathione and cysteine. The reaction was
conducted in PBS/DMSO (9:1, pH 7.4) in a sequential manner
(
reagent that allows for the covalent heterocoupling of two thiol-
containing entities. MAPN contains two thiol-specific reactive
1
3
groups, an APN and a maleimide, exhibiting distinctive
reaction rates. This feature allows for the selective formation of
monoadduct upon coupling of the more reactive group with the
first thiol, leaving the second reactive group intact for the
subsequent reaction with the second thiol.
(
Figure 1A). First, 1 equiv of MAPN 5 was added to 1 equiv of
the reduced glutathione (1 mM solution). After 2 min of
incubation at 25 °C, HPLC analysis showed full conversion
into the single adduct 6. To the resulting solution, we added 1
equiv of cysteine and incubated the mixture for 30 min to allow
for clean formation of the glutathione-cysteine adduct 7.
Compounds 6 and 7 were isolated and characterized in order to
confirm the order of thiol addition to the maleimide and APN
functions. A side-by-side comparison with the closest
commercially available thiol-to-thiol coupling reagent, p-
phenylenedimaleimide, was carried out under the same
conditions (Figure 1B). As expected, following the addition
of 1 equiv of glutathione, p-phenylenedimaleimide yielded a
mixture with four main products: the adduct, the homocoupling
product, the hydrolysis product, and the starting compound.
RESULTS AND DISCUSSION
■
Recently, we described a 3-arylpropyonitrile (APN) group as an
efficient reactive function for the selective and irreversible
1
3
labeling of thiols. Given the at least 10-fold higher reaction
−1 −1
rate of maleimide (k > 50 M s ) compared to APN (k = 3.1
−
1 −1
M
s ), we hypothesized that combining these two functions
into a heterobifunctional reagent will allow for kinetically
resolved thiol-to-thiol coupling.
We synthesized MAPN 5 with a 29% overall yield from 4-
iodoaniline 1 in a four-step reaction sequence (Scheme 1).
B
DOI: 10.1021/acs.bioconjchem.5b00440
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Figure 2. Conjugation of the antibody with TAMRA-SH using MAPN and p-phenylenedimaleimide.
Subsequent addition of 1 equiv of cysteine resulted in a highly
complex mixture.
Encouraged by these results, we decided to test the MAPN
reagent in the heterocoupling of a biomolecule and a
fluorescent tag (Figure 2). Again, a side-by-side comparison
with p-phenylenedimaleimide was carried out in the coupling of
the Trastuzumab with a thiol-containing fluorophore 11.
Trastuzumab was first reacted with 10 equiv of Traut’s
reagent to form the modified antibody 8 with a number of
solvent-exposed thiol groups. The resulting conjugate was
incubated with 10 equiv of either MAPN or p-phenyl-
enedimaleimide yielding conjugates 9 and 13, respectively.
After 1 h of incubation at 25 °C, 10 equiv of TAMRA-SH 11
was added to the mixtures. Following overnight incubation, the
mixtures were purified by size-exclusion chromatography.
The resulting conjugates 10 and 14 were analyzed by SDS-
PAGE (Figure 3). Gel fluorescence showed that MAPN (lane
6) allowed for the fluorescent labeling of the antibody, in
contrast to p-phenylenedimaleimide (lane 4). We hypothesized
that the lack of antibody labeling in the case of p-
phenylenedimaleimide 5 was due to the lower hydrolytic
stability of maleimides, compared to APN. To verify this
hypothesis, we prepared the p-phenylenedimaleimide-gluta-
thione monoadduct 12a and the MAPN-glutathione mono-
adduct 6 and tested their stability in PBS. Indeed, the
maleimide function of 12a underwent 50% hydrolysis in PBS
(pH 7.4) within 1 h, yielding the unreactive maleic acid
derivative (Supporting Information Figure S1). The APN
function of 6 remained stable in the PBS solution for 24 h.
We performed a series of control experiments to confirm the
absence of false positives that might be related to an unspecific
conjugation process. Thus, the unmodified antibody was
incubated with TAMRA-SH and showed no formation of the
antibody−fluorophore conjugate (lane 1), nor was formation of
the conjugate observed upon incubation of the activated
antibody 8 with TAMRA-SH (lane 2). The unmodified
antibody was also incubated with MAPN and p-phenyl-
enedimaleimide 11 (lanes 3 and 4), followed by reaction with
TAMRA-SH. In this case, gel fluorescence did not reveal any
unspecific conjugation product.
Figure 3. SDS-PAGE analysis of antibody−dye conjugates. Control
experiments were carried out on the unmodified antibody without the
use of a bifunctional reagent (lane 1), with the addition of 12 (lane 3)
and 5 (lane 5). The activated antibody was reacted with TAMRA-SH
directly (lane 2) as a negative control. The reaction of the activated
antibody with 12 and then 11 (lane 4) yielded no fluorescent labeling.
Reaction of the activated antibody with MAPN and then 11 (lane 6)
allowed for efficient fluorescent labeling.
reagent. Random conjugation to nearly 30 surface-exposed
lysines provides a highly heterogeneous mixture of different
coupling species. In contrast, the reduction-alkylation strategy
offered only eight attachment points. This strategy resulted in
more defined ADCs with a narrower distribution of species and
with mostly even drug-to-antibody ratios (Figure 4).
To further demonstrate the utility of the MAPN reagent, we
carried out the preparation of an ADC by coupling mertansine
(DM1) to Trastuzumab via a reduction-alkylation strategy. In
the commercial ADC (T-DM1, trade name: Kadcyla),
mertansine is conjugated to lysine residues using SMCC
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DOI: 10.1021/acs.bioconjchem.5b00440
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Communication
Figure 4. Representative structures and MS spectra of T-DM1 (Kadcyla) and T-MAPN-DM1.
T-MAPN-DM1 was prepared via a two-step process. First,
mertansine was reacted with 1 equiv of MAPN to yield the thiol
reactive payload. The payload was conjugated to the partially
reduced antibody by incubating the components in the PBS
buffer. The conjugate was purified by size exclusion
chromatography and the average DAR was determined by
native ESI-TOF-MS. T-MAPN-DM1 was shown to have the
average DAR of 3.3 ± 0.2, which was comparable with the
average DAR of 3.4 ± 0.1 measured for T-DM1.
comparison with its closest commercial analog showed superior
efficiency on the part of MAPN for bioconjugation applications.
The MAPN reagent was further applied to conjugate
mertansine and the cysteine residues of Trastuzumab, providing
a more homogeneous analog of Trastuzumab emtansine (T-
DM1). Considering the vast use of thiol groups in biological
research, we believe that this reagent will be of great utility in
the area of bioconjugation. Through an agreement with
15
Syndivia, MAPN is now commercially available.
Finally, in vitro cytotoxicity of the T-MAPN-DM1 conjugate
was evaluated on SK-BR-3 (HER2+++) and MDA-MB-231
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bioconj-
chem.5b00440.
■
(
HER2-) cells. Similar to T-DM1, the new conjugate showed
*
S
high toxicity on HER2 positive cells and significantly lower
toxicity on HER2 negative cells (Figure 5).
CONCLUSION
■
Materials and instrumentation, experimental procedures,
analytical data (PDF)
In conclusion, we developed MAPNa first-in-class reagent
for the efficient kinetically resolved coupling of thiols. The
reagent was applied for conjugation of a thiol-activated
antibody and a thiol-containing fluorophore. A side-by-side
AUTHOR INFORMATION
Corresponding Author
E-mail: alwag@unistra.fr.
■
*
Author Contributions
#
Oleksandr Koniev and Sergii Kolodych contributed equally to
this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by SATT Conectus “BioReLiable”
project, CNRS, University of Strasbourg, Reg
́
ion Alsace, and
GIS IBiSA. The International Center for Frontier Research in
Chemistry (icFRC) is also acknowledged for financial support.
J.S. acknowledges the Institut de Recherche Servier for funding
of his PhD fellowship.
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