Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast Cysteine-Selective Protein Modification and Charge Modulation

Article abstract of DOI:10.1002/anie.201901405

Quaternized vinyl- and alkynyl-pyridine reagents were shown to react in an ultrafast and selective manner with several cysteine-tagged proteins at near-stoichiometric quantities. We have demonstrated that this method can effectively create a homogenous an

Full text of DOI:10.1002/anie.201901405

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Accepted Article
Title: Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast
Cysteine-Selective Protein Modification and Charge Modulation
Authors: Maria J. Matos, Claudio D. Navo, Tuuli Hakala, Xhenti
Ferhati, Ana Guerreiro, David Hartmann, Barbara Bernardim,
Kadi L. Saar, Ismael Compañón, Francisco Corzana, Tuomas
P. J. Knowles, Gonzalo Jiménez-Osés, and Gonçalo J. L.
Bernardes
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901405
Angew. Chem. 10.1002/ange.201901405
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Quaternization of Vinyl/Alkynyl Pyridine Enables Ultrafast
Cysteine-Selective Protein Modification and Charge Modulation
[
a],+
[c],+
[a],+
[c],+
[b]
Maria J. Matos,
Claudio D. Navo, Tuuli Hakala,
Xhenti Ferhati, Ana Guerreiro, David
[
a]
[a]
[a]
[c]
*[c]
Hartmann, Barbara Bernardim, Kadi L. Saar, Ismael Compañón, Francisco Corzana, Tuomas
*
[a]
*[c,d]
*[a,b]
P. J. Knowles, Gonzalo Jiménez-Osés,
and Gonçalo J. L. Bernardes
Abstract: Quaternised vinyl- and alkynyl-pyridine reagents were
shown to react in an ultrafast and selective manner with several
cysteine-tagged proteins at near-stoichiometric quantities. We have
drug conjugates (ADCs). This choice is a result of the high
nucleophilicity of the sulfhydryl side-chain combined with the low
abundance of free cysteine residues, since many cysteines are
paired as structural disulfides. Thus, many research groups have
focused on developing efficient methods to chemoselectively
modify cysteine-tagged proteins. For example, electrophiles, such
demonstrated that this method is able to create effectively
a
homogenous antibody-drug conjugate that features a precise drug-to-
antibody ratio of 2 that was stable in human plasma and retained its
specificity towards Her2+ cells. Finally, the developed warhead
introduces a +1 charge to the overall net charge of the protein, which
enabled us to show that a protein’s electrophoretic mobility may be
tuned through the simple attachment of a quaternised vinyl pyridinium
reagent at cysteine residues. We anticipate the generalized use of
quaternised vinyl- and alkynyl-pyridine reagents not only for
bioconjugation, but also as warheads for covalent inhibition and as
tools to profile cysteine reactivity.
[10]
as carbonylacrylic acid reagents for Michael addition, arylation
reactions based on transition metals[11] or a selective amino acid
sequence,[12] or conjugate additions at dehydroalanine[13] formed
from cysteine or thiol-yne reactions using cyclooctynes[14] have
been developed. Each of these methods has relative advantages
and disadvantages, but a method based on the simple attachment
of a “warhead-like” structure whose utility would go beyond
bioconjugation and for example be used to tune protein
pharmacokinetics — through changes in the overall net charge of
a protein — or be used to design cysteine covalent inhibitors is
missing from the current toolbox.
Chemical site-selective modification offers a means to diversify a
protein’s function and properties.[1] For example, by using the
targeting capabilities of an antibody, it is possible to covalently
attach a very potent drug to the antibody through a precise
chemical reaction to shuttle this drug to a specific tissue.[2] The
toolbox of reactions for protein modification has expanded
significantly in the last decade.[3] Of these, reactions that target
proteinogenic amino acids seem particularly suitable to modify
native proteins in the test tube. Lysine,[4] methionine,[5]
tryptophan,[6] the N–[7] and the C–terminus[8] may now be targeted
by using a range of approaches. However, cysteine[9] remains
perhaps the residue of choice to produce functional and, in
particular, clinically useful protein conjugates, namely antibody-
Here, we report the computational chemistry assisted
discovery of quaternization of the nitrogen of vinyl- and alkynyl
pyridines to convert otherwise non-reactive reagents into ultrafast
and chemoselective cysteine-modifying reagents. The utility of
these reagents was demonstrated for bioconjugation and for
modulation of electrophoretic mobility through charge
incorporation by using microfluidics.
Based on computational predictions, we identified promising
alkenes and alkynes through modelling the nucleophilic addition
reaction between a simple thiolate or an amine with various
a
electrophiles. As a result of the lower pK of solvent-exposed
thiols relative to amines, it is expected that at neutral or slightly
basic pH a larger proportion of thiols would be deprotonated.
‡
Activation free energies (ΔG ) for the corresponding addition
[
a]
Dr. M.J. Matos, T. Hakala, Dr. B. Bernardim, D. Hartmann, K. L.
Saar, Prof. T.P.J. Knowles, Dr. G.J.L. Bernardes
Department of Chemistry
reactions were predicted by using quantum mechanical
calculations (Figure 1a and 1b). 2-Vinyl- and 2-ethynyl pyridines
(
compounds 1 and 3) were calculated to be poorly reactive
University of Cambridge
toward thiolates (ΔG‡ > 24 kcal mol ) and unreactive toward
–1
Lensfield Road, CB2 1EW Cambridge (UK)
E-mail: tpjk2@cam.ac.uk; gb453@cam.ac.uk
A. Guerreiro, Dr. G.J.L. Bernardes
primary amines (ΔG‡ 31 kcal mol–1). However, when
>
[
[
b]
c]
quaternised, the resulting N-methylpyridinium derivatives were
calculated to be exceedingly reactive towards nucleophilic
addition, with dramatically decreased activation free energies.
For instance, 2-vinyl- and 2-ethynyl-pyridiniums 2 and 4 were
Instituto de Medicina Molecular
Faculdade de Medicina, Universidade de Lisboa
Avenida Professor Egas Moniz, 1649–028 Lisboa (Portugal)
E-mail: gbernardes@medicina.ulisboa.pt
Dr. C.D. Navo, X. Ferhati, I. Compañón, Prof. F. Corzana, Dr. G.
Jiménez-Osés
calculated to be more than a billion times more reactive with
thiolates (ΔG‡ = 10–12 kcal mol–1) than their non-quaternised
Departamento de Química, Universidad de La Rioja
Centro de Investigación en Síntesis Química
analogues 1 and 3, which shows predicted reactivities similar to
those of maleimide[15] and carbonylacrylic derivatives[10]
previously used for cysteine ligation. In all cases, a complete
selectivity for cysteine over lysine addition was predicted, as
reflected by the nearly 8 kcal mol–1 higher activation barrier
calculated for the latter.
2
6006 Logroño (Spain)
E-mail: francisco.corzana@unirioja.es
Dr. G. Jiménez-Osés
[
[
d]
+]
CIC bioGUNE, Bizkaia Technology Park
Building 801A, 48170 Derio (Spain)
E-mail: gjoses@cicbiogune.es
These authors contributed equally.
We validated our computational predictions by reacting
stoichiometric amounts of minimal cysteine and lysine side-chain
model compounds (i.e. 1-propanethiol and 1-propylamine,
Supporting information for this article is given via a link at the end of
the document.
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respectively) with commercially available 1–3 and synthesized
reagent 4 (Figure 1a and 1b), and monitoring reaction with 1
H
2
NMR spectroscopy (3 mM, phosphate buffer in D O, pH 7.6; see
the Supporting Information for additional data and a detailed
discussion on the effect of using a slightly more acidic pH). In
agreement with theoretical predictions, the reactions with non-
quaternised reagents 1 and 3 were very slow and gave 62 and
6
% conversions, respectively, of addition products after 24 h
(
Figure 1c, Entries 1 and 3). On the contrary, pyridine N-
methylated reagents 2 and 4 gave a conversion of 99% of addition
product after 1 h with only 1 equivalent of electrophile (Figure 1c,
Entries 2 and 4). 2-Alkynyl reagent 4 gave a » 6:4 mixture of
isomers as products, namely the cis and trans alkene adducts
(
See the Supporting Information for their structural
characterization and thermal stability). The kinetic profiles
obtained with a protected cysteine (N-acetylcysteine amide, Ac-
Cys-NH
2
) were similar, albeit slightly faster than with 1-
propanethiol (89% adduct formation in 24 h with non-quaternised
1
, and 93% adduct formation in <4 min with quaternised 2; Figure
1
c, Entries 5 and 6). Also, in agreement with computational
predictions, the fast kinetics observed with quaternised pyridinium
reagents and were comparable to those of N-
ethylmaleimide, for which the reaction with 1-propanethiol and
Ac-Cys-NH were complete within the 4 min necessary to obtain
the first 1H NMR spectroscopic measurements, although the
formation of an undetermined alkene by-product was observed in
these reactions with maleimide (see the Supporting Information).
With lysine side-chain mimic 1-propylamine (Figure 1c, Entry 7)
2
4
[15]
2
Figure 1. Computer-assisted evaluation and experimental validation of a vinyl-
and b alkynyl pyridinium electrophile reagents 1–4 for cysteine modification.
Accurate reaction rate constants could not be obtained for
and N-acetyllysine amide (Ac-Lys-NH
2
; Figure 1c, Entry 8) the
very reactive quaternised pyridiniums 2 and 4, and N-
reactions with quaternised 1-methyl-2-vinylpyridinium (2) were
exceedingly slow (<6% adduct formation after 24 h with 1
equivalent of electrophile). This result agrees with the
computationally predicted orthogonality of these electrophiles
towards cysteines, with no expected reaction on typically solvent-
exposed lysines. It is interesting to note that the terminal alkyne
hydrogen in compounds 3 and 4 quickly exchanged to deuterium
ethylmaleimide as a result of the ultrafast kinetics observed under
1
the reaction conditions used (3 mM was the detection limit for H
NMR spectroscopy in our experiments). Also, the high level of
sensitivity to small reagent concentration changes on the
observed rate for second-order reactions, further complicates the
derivation of the rate constants under stoichiometric conditions.
Furthermore, although alkynyl pyridinium 4 was selective towards
mono-functionalization under strict stoichiometric conditions, it
was found to undergo double thiol addition to the external position
of the triple bond to give a stable dithioacetal when a slight excess
of the thiol was used (see the Supporting Information). Once the
dramatic effect of pyridine quaternization in boosting the
electrophilicity of reagents 1 and 3 was demonstrated and given
the very similar and ultrafast reactivity of quaternised reagents 2
and 4 towards thiols, we decided to use more convenient 1-
methyl-2-vinylpyridinium 2 for cysteine-selective modification on
proteins (i.e. to avoid terminal alkyne deuteration and formation of
mixtures of potentially reactive alkene isomers upon nucleophilic
addition).
2
in D O at pH 7.6, particularly in the case of quaternised 4, for
which deuteration takes place within 4 min. H/D exchange at the
vinyl hydrogens of quaternised 2 was also observed when DMF-
7
d was used as a co-solvent.
Having demonstrated the superiorreactivity of 2 towards thiols
on small molecule models, we decided to test the use of vinyl
pyridinium reagents towards cysteine-tagged proteins (Figure
2
a). We selected four representative proteins, the C2A domain of
Synaptotagmin-I, Annexin-V, ubiquitin and albumin, which display
either natural or engineered single surface-exposed cysteines
(
see the Supporting Information). In all cases, and with
stoichiometric amounts or only a small excess of 2 (1 equiv. for
ubiquitin and albumin and 10 equiv. for annexin-V and C2Am)
i
under buffered (NaP pH 8.0, 50 mM) conditions for 1 h at 37 °C,
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analysis of albumin and albumin–2. g) Surface plasma resonance of binding to
complete conversion into the corresponding thioether adduct was
observed, as confirmed by LC-MS analysis, and in high yield
D
the FcRn receptor. k for rHSA and rHSA-2.
(
>95%), as shown by Bradford protein assay (Figure 2b–2e; see
also the Supporting Information; no reaction was detected with
either 1 or 2 under slightly acidic pH 5.5). Despite the use of 10
equiv. to ensure efficient modification of even sterically crowded
Next, we decided to demonstrate that vinyl pyridinium
reagents may be easily functionalized first through quaternization
of the pyridine nitrogen with an alkyne linker followed by
subsequent derivatization through Cu(I)-catalysed azide–alkyne
cycloaddition reaction with a suitable synthetic motif that displays
an azido group (see the Supporting Information). We chose to
—
and thus poorly deprotonated — cysteine residues, such as in
the case of Annexin-V, no non-specific reactions at lysine were
detected. Other studies that use vinyl-substituted pyridine
derivatives achieved a maximum of only 85% conversion after 24
[16]
build a quaternised vinyl pyridinium reagent that features
h, which demonstrates well the effect of quaternization of the
pyridine on the kinetics of the bioconjugation reaction.
Furthermore, the final conjugates did not react with Ellman’s
reagent [5,5′-dithiobis(2-nitrobenzoic acid)] that showed that all
cysteine had been consumed (see the Supporting Information).
Importantly, we verified that the formed thioether bond is stable
towards potential thiol exchange reactions, and the conjugates
were also fully stable in human plasma in buffered pH 5–8
solutions (see the Supporting Information). Next, we confirmed
that the conditions used are mild and do not induce changes in
both the structure or function of the native proteins. In the case of
albumin, conjugate albumin–2 showed no significant changes in
its secondary structural content as determined by circular
dichroism (CD; Figure 2f) and retained its ability to bind to the
neonatal Fc receptor (FcRn) as determined by Surface Plasma
cytotoxic drug monomethyl auristatin E (MMAE; Figure 3a). By
connecting the cysteine-selective reagent and the drug, we
introduced the dipeptide valine–citrulline (ValCit), which is known
[17]
to cleave on exposure to cathepsin B, and the self-immolating
spacer p-aminobenzyl carbamate (PABC).[18] This reagent 5 was
then conjugated to Thiomab, an HER2 targeting antibody that has
been engineered to contain an additional cysteine residue at
position 205 in each light-chain (Thiomab LC-V205C).[19] To our
delight, the conjugation reaction proceeded to completion with 5
equiv. per light-chain of 5, within 1 h at 37 °C in NaP
i
pH 8.0, 20
single
mM buffer (Figure 3a). LC-MS analysis showed
a
modification in each-light chain and no modifications in the heavy-
chain (Figure 3b) to form a conjugate with a precise drug-to-
antibody ratio of 2. Importantly, Thiomab–5 conjugate retained its
specificity towards Her2 positive cells, as demonstrated by flow
cytometry analysis (Figure 3c and 3d). Our data demonstrate the
use of quaternised vinyl pyridinium reagents to efficiently
generate homogenous and functional antibody conjugates
Resonance (K
albumin–2; Figure 2g). Together, our data demonstrates the
generalization of method for cysteine-selective protein
D D
= 11.8 µM for albumin versus K = 8.88 µM for
a
modification based simply on the use of quaternised vinyl
pyridinium reagents. The products are formed in high conversion
and yield, are stable in human plasma, and both native structure
and function activities are retained.
Figure 3. Cysteine-selective antibody modification. a) Schematic for the
bioconjugation of 5 to Thiomab and chemical structure of 5. b) ES-MS spectra
of the light- and heavy-chain of Thiomab after conjugation with 5. c) Counter
plot indicates the binding affinity of Thiomab-5 to Her2 expressing SKBR3 cells
at 50 nM. d) Percentage of SKBR3 cells bound to Thiomab-5 at 50 nM.
Trastuzumab and Thiomab were used as positive controls and IgG Isotype was
used as negative control.
Figure 2. Cysteine-selective protein modification. a) Schematic of the reaction
of a cysteine-tagged protein with 2. General conditions: a cysteine-tagged
protein (10–20 µM) is reacted with 2 (1–10 equiv.) in NaP
i
(50 mM, pH 8.0) at
3
7 °C for 1 h. b–e) ES-MS spectra of the product of the reaction between b.
C2Am, c. Annexin V, d. Ubiquitin and e. albumin with 2 (deconvoluted spectra;
expected increase of 118 Da in the total protein mass). f) Comparative CD
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A protein’s conjugation can be monitored directly by following
changes in a protein’s electrophoretic mobility because the
quaternised vinyl pyridinium reagent introduces an additional
charge of +1 per cysteine residue to the net charge. For instance,
albumin features one single free cysteine and thus conjugation of
In summary, an efficient and irreversible cysteine-selective
bioconjugation method is reported. This method is enabled by the
discovery that quaternization of the nitrogen of vinyl- and alkynyl
pyridines transforms these molecules into extremely reactive
electrophiles towards thiols. We demonstrate the utility of these
reagents for cysteine-selective bioconjugation of five different
protein scaffolds, including a clinically used antibody. Importantly,
the conjugates formed are resistant towards thiol exchange
reactions and retain their native activity. By using a microfluidic
setup, we show that the electrophoretic mobility of a protein may
be modulated through simple attachment of the quaternised vinyl
pyridinium reagent at cysteine to introduce a +1 charge to the
overall net charge of the protein. This property of the quaternised
vinyl pyridinium may prove useful to enhance ADC’s
hydrophilicity, half-life in circulation or internalization rates. The
simplicity of these reagents combined with their excellent
reactivity and selectivity towards thiols, makes them ideal for
2
adds +1 charge to the overall net charge of the protein (charge
»
–7 at pH 7). We decided to test this hypothesis by using a
[20]
microfluidic free-flow electrophoresis device, which allows the
charge of proteins to be determined under native conditions in
solution and requires only low volumes of sample. The samples
were introduced into the device with a flanking buffer, and the
deflection of the protein samples was observed while a transverse
electric field was applied by increasing the potential between
electrodes in the device from 0–120 V (Figure 4a). When the
measured deflection in the field was converted into drift velocity
(
(
Vdrift) and the electric field inside the devices was calibrated
Figure 4b), a clear difference in the electrophoretic behaviour of
albumin and albumin–2 was observed. Furthermore, the
protein bioconjugation, but also for use as warheads in the design
–
[22]
electrophoretic mobilities were estimated to be: –1.71 ± 0.21 × 10
of cysteine covalent ligands
or to probe hyper-reactive
8
m V s to give a charge »–6.84 ± 0.58 and –1.12 ± 0.10 × 10–
2
–1 –1
cysteines within the human proteome in chemical proteomic
approaches.[23]
8
2
–1 –1
m V s to give a charge »–4.64 ± 0.12 for albumin and
albumin–2, respectively. The decrease in mobility can be
explained by the direct proportionality of the charge (q) to the
B
electrophoretic mobility [µel; q=(µel × k T)/D], which implies that if
Acknowledgements
the charge is decreased, the mobility should also decrease as
confirmed by the data. Moreover, because we are adding positive
charge through conjugation of the quaternised vinyl pyridinium
reagent 2 into a negatively charged albumin, the net charge of
conjugate albumin–2 should be smaller, as observed in the results
obtained with the microfluidic electrophoresis. These data thus
demonstrate the application of site-selective protein modification
to change the overall net charge of a protein in a controlled
manner, and consequently modulate its electrophoretic mobility.
Funded under the EU Horizon 2020 programme, Marie
Skłodowska-Curie ITN GA No. 675007, the Royal Society
(
UF110046 and URF\R\180019 to G.J.L.B.), FCT Portugal (iFCT
IF/00624/2015 to G.J.L.B. and PhD studentship
SFRH/BD/115932/2016 to A.G.), Xunta de Galicia (Galician Plan
of research, innovation and growth 2011–2015, ED481B
2
014/086-0 and ED481B 2018/007 to M.J.M.), D.G.I.
MINECO/FEDER (grants CTQ2015-70524-R and RYC-2013-
4706 to G.J.-O. and C.D.N and CTQ2015-67727-R to F.C.),
Universidad de la Rioja (FPI PhD studentship to I.C.), FAPESP
BEPE 2015/07509-1 and 2017/13168-8 to B.B.) and by an ERC
1
(
StG (GA No. 676832). We also thank Genentech for providing the
Thiomab antibody, Albumedix for providing rHSA, Dr. André
Neves and Prof. Kevin Brindle for providing C2Am, and Dr. Vikki
Cantrill for her help with the editing of this manuscript.
Keywords: bioconjugation • cysteine • protein modification •
antibody-drug conjugates • microfluidics
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Angewandte Chemie International Edition
10.1002/anie.201901405
COMMUNICATION
COMMUNICATION
+
+
Maria J. Matos, Claudio D. Navo Tuuli
+
+
Hakala, Xhenti Ferhati, Ana Guerreiro,
David Hartmann, Barbara Bernardim,
Kadi L. Saar, Ismael Compañón,
Francisco Corzana,* Tuomas P. J.
Knowles,* Gonzalo Jiménez-Osés,* and
Gonçalo J. L. Bernardes*
Quaternise to click: The computation-inspired discovery of quaternisation of the
nitrogen of vinyl and alkynyl pyridine reagents to enable selective and ultrafast
modification of cysteine at near-stoichiometric quantities is presented. With this
strategy, stable and defined antibody-drug conjugates can be prepared.
Furthermore, a protein’s electrophoretic mobility may be modulated through simple
introduction a +1 charge to its overall as shown using microfluidics.
Page No. – Page No.
Quaternization of Vinyl/Alkynyl
Pyridine Enables Ultrafast Cysteine-
Selective Protein Modification and
Charge Modulation
This article is protected by copyright. All rights reserved.