STEFs: Activated Vinylogous Protein-Reactive Electrophiles

Article abstract of DOI:10.1002/anie.201814073

Reported here is the synthesis of a class of semi-oxamide vinylogous thioesters, designated STEFs, and the use of these agents as new electrophilic warheads. This work includes preparation of simple probes that contain this reactive motif as well as its installation on a more complex kinase inhibitor scaffold. A key aspect of STEFs is their reactivity towards both thiol and amine groups. Shown here is that amine conjugations in peptidic and proteinogenic samples can be facilitated by initial, fast conjugation to proximal thiol residues. Evidence that both the selectivity and the reactivity can be tuned by the structure of STEFs is provided, and given the unique ability of this functionality to conjugate by an addition-elimination mechanism, STEFs are electrophilic warheads that could find broad use in chemical biology.

Full text of DOI:10.1002/anie.201814073

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Accepted Article
Title: STEFs - Activated Vinylogous Protein-Reactive Electrophiles
Authors: Bente Kring Hansen, Christopher James Loveridge, Stine
Thyssen, Gustav Julius Wørmer, Andreas Deleuran Nielsen,
Johan Palmfeldt, Mogens Johannsen, and Thomas Bjørnskov
Poulsen
This manuscript has been accepted after peer review and appears as an
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of the final Version of Record (VoR). This work is currently citable by
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content of this Accepted Article.
To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814073
Angew. Chem. 10.1002/ange.201814073
Link to VoR: http://dx.doi.org/10.1002/anie.201814073
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10.1002/anie.201814073
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STEFs - Activated Vinylogous Protein-Reactive Electrophiles
Bente K. Hansen,[a] Christopher J. Loveridge,[a] Stine Thyssen,[b] Gustav J. Wørmer,[a] Andreas D.
Nielsen,[a] Johan Palmfeldt,[c] Mogens Johannsen,*,[b] Thomas B. Poulsen*,[a]
Abstract: We report the synthesis of a class of semi-oxamide
vinylogous thioesters that we have designated STEFs and the use of
these agents as new electrophilic warheads. This includes
preparation of both simple probes that contain this reactive motif as
well as its installation on a more complex kinase inhibitor scaffold. A
key aspect of STEFs is their reactivity towards both thiol- and amine
groups and we substantiate that amine-conjugations in peptidic and
proteinogenic samples can be facilitated by initial, fast conjugation to
proximal thiol residues. We provide evidence that both the selectivity
and the reactivity can be tuned by the structure of STEFs and given
the unique ability of this functionality to conjugate via an addition-
elimination mechanism, STEFs are electrophilic warheads that could
find broad use in chemical biology.
be a promising approach to discover new therapeutically relevant
proteins that can be addressed using small molecules. 6
The warhead functionality dictates the overall properties of an
electrophilic probe.7 Of particular relevance is the reactivity (and
stability), the nucleophile selectivity: S(cys), N(lys/arg/N-term),
O(ser/thr/tyr/asp/glu), C(tyr/trp), and the (ir)reversibility of
conjugation. New types of reactive groups are of strong interest
as they can widen the portion of the proteome that can be
addressed with covalent ligands.7 Here, we report the first
biological studies of
a class of protein reactive acrylate
electrophiles (STEFs – to be further defined below) that function
via an addition-elimination mechanism.
A : Michael-acceptor electrophiles
C : STEF-probe synthesis
e.g. R =
O
R
X
The utility of electrophilic functionalities in chemical biology
ranges from proteomic profiling to structure-based drug design. In
classic activity-based protein profiling (ABPP), hybrid molecules
consisting of an electrophilic warhead and a reporter group are
designed to react irreversibly with nucleophilic amino acid
residues in the active site of defined classes of enzymes.1 This
method principally allows for labelling of functionally active
enzymatic sub-populations and has resulted in a series of
important insights, notably in the protease field but also for other
enzyme-classes.2 Generalization of the ABPP concept to
electrophiles with broad reactivity has been remarkably
productive, e.g. with proteome-wide methods to identify the sub-
population of cysteines with enhanced nucleophilic reactivity
revealing that hyperreactive cysteines are overrepresented
amongst functional and regulatory residues.3 The broad reactivity
required in those studies is enabled by simple thiol-reactive
probes such as the iodoacetamide-derivative IA-alkyne (IA-Alk).
Potent and broadly reactive electrophiles can however be
tempered when placed in molecules with a more complex design,
thus imparting enhanced selectivity to the covalent interaction
with a biomolecule. Complex natural products that include highly
reactive groups are extreme examples of this,4 but even fragment-
sized synthetic molecules with appended electrophiles can
display remarkable selectivity.5 Phenotypic screening using such
covalent-fragments has recently been combined with broadly
reactive probes to assist target-identification and this appears to
NMe2
H
N
R
H
1) (COCl)2
0 o
2)
1
NH2
C
O
O
O
DMAP, CH2Cl2
0 o
C
N
H
S
Het
Ar
N
R
2
3
CN
H
O
O
O
H
N
R
Et
O
N
O
N
H
5
R = Et (6), 56%
R = c-Hexyl (7), 29%
R = CH2CF3 (8), 19%
O
4
O
B : Semi-oxamide vinylogous (thio)esters - STEFs
(trans/cis)
O
H
N
O
R
S
XR
N
H
R = Et (9), 87%, (2.5:1)
R = Ph (10), 72%, (1:1.3)
O
O
STEFs
X = O, S, N
n-BuNH2
CH2Cl2
From cis-9
Advantages:
Straight-forward
synthesis
Questions:
Biological stability
H
n-Bu
O
N
Nucleophile
preference
Reversibility
H
N
Addition-elimination
mechanism
11, 90%
O
Tunable structure
Figure 1. Synthesis and properties of STEFs compared to established Michael-
acceptors.
Acrylamides (1) are among the most used electrophilic warheads
and their reactivity can be modulated by substitution at the b-
carbon (Fig. 1A).8 Similar to other classes of Michael acceptors
such as vinyl sulfones, Knoevenagel adducts, and maleimides (2-
4), these functionalities typically have selectivity for thiol-
conjugation and have found extensive use both as broadly
reactive probes for profiling and as warheads in highly specific
bioactive compounds. In contrast, b-heteroatomsubstituted
acrylamides, such as vinylogous carbamates (exemplified by 5),
undergo mechanistically distinct reactions with nucleophiles
resulting in displacement of the heteroatom via an addition-
elimination pathway – a vinylic substitution reaction9 – but the low
chemical reactivity of these systems limits their biological
applications. We hypothesized that additional activation by an
electron withdrawing group could bring the reactivity into a domain
with potential biological relevance (Fig. 1B). We have chosen to
[a]
B. K. Hansen, C. Loveridge, G. J. Wørmer, A. D. Nielsen,
Prof. Dr. T. B. Poulsen
Department of Chemistry, Aarhus University
Langelandsgade 140, 8000 Aarhus C, Denmark
E-mail: thpou@chem.au.dk
[b]
[c]
S. Thyssen, Prof. Dr. M. Johannsen
Department of Forensic Medicine, Aarhus University
Palle Juul-Jensens Boulevard 99, 8200 Aarhus N, Denmark
E-mail: mj@forensic.au.dk
Prof. Dr. J. Palmfeldt
Department of Clinical Medicine – Research Unit for Molecular
Medicine, Aarhus University hospital
Palle Juul-Jensens Boulevard 82, 8200 Aarhus N, Denmark
Supporting information for this article is given via a link at the end of
the document
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10.1002/anie.201814073
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COMMUNICATION
designate these activated vinylogous compounds as STEF-
electrophiles (STEFs) after Stachel and Effenberger who
provided the first examples of their preparation.10,11 Several
groups, notably Tietze12, have demonstrated the applicability of
STEF-compounds as synthetic intermediates, e.g. as chelating
substrates in metal-catalyzed cycloaddition reactions,13 but their
performance in a biological context has to the best of our
knowledge not been investigated. More than anything, we were
fascinated by the possibility that biological nucleophiles could
undergo either reversible or irreversible conjugation to STEFs
depending on the residue and specific molecular context.
Figure 2. Stability and in vitro reactivity of STEFs. A) Model of proposed STEF-reactivity enabling cysteine-directed conjugation to proximal amines. B) Studies of
the relative reactivity of STEF-probe 9 with thiols and amines in vitro analyzed by HPLC. Probe 9 reacts fast and reversibly with thiols and irreversibly with amines.
All data points represent mean ± s.d. (n=2) and are normalized to the peak area of probe 9 at time zero (left) or the peak area of 11 at time 360 min (right). C-E)
Comparative reactivity of probe 9 towards model hexapeptides that differ by either cysteine or serine at position 5. C) Experimental setup. D) LC-MS traces of the
*
*
*
reaction mixtures at different timepoints. E) MS-Fragmentation spectra from the reaction of probe 9 with the cysteine-containing peptide. bn -ion series (b1 -b4 )
*
*
*
indicate a single STEF-modification present on the N-terminal amine/arginine sidechain (b1 -ion). The doubly-modified peptide show y2 - and y4 -ions, that indicate
additional modification at the cysteine residue. The mass of the STEF-modification (m=150.0555) is observed in both modified peptides which suggest instability to
fragmentation. This could further explain the presence of unmodified cysteine residues (y2#, y2#-NH3) in the doubly-modified peptides.
Starting from either vinyl ethers or vinyl sulfides, we prepared a
small collection of alkyne-tagged STEF-probes (6-10) by initial
reaction with oxalyl chloride14,15 followed by DMAP-catalyzed
trapping with but-3-yn-1-amine to provide a biorthogonal handle
(Fig. 1C). All compounds were formed in reasonable yields
without extensive optimization, however we observed significant
differences in reactivity correlating to the relative electron
deficiency of the starting olefin. In this study, we have focused our
attention on the simplest disubstituted olefin-scaffolds, although
we note that more complex STEFs having additional substituents
in the a and b-positions can also very likely be prepared.14 Probes
6-8 were isolated as single geometric isomers whereas sulfides
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9-10 were formed as mixtures of cis and trans-isomers, albeit
separable by standard flash chromatography. Finally, we
performed an exchange of the thiol moiety in 9 with n-butylamine
under preparative conditions to afford enamide 11 in good yield.
Interestingly, this exchange was found to be stereoconvergent to
afford exclusively the cis-isomer, irrespective of the
stereochemical constitution of the sulfide-precursor (Fig. 1C). To
ascertain the potential of STEFs as biologically relevant
electrophiles, we first studied their stability. Alkoxy substituted
probes (6-8) were found to degrade upon storage even at -20 °C
and we therefore did not further consider their use. In contrast,
vinyl sulfides such as 9 and 10 were stable in neutral buffer as
well as under both acidic and basic conditions. Enamide 11 was
also stable under neutral and basic conditions, however under
acidic conditions (pH < 4) the compound degraded over the
preparation of 11 partially illustrates the latter reactivity albeit in
organic solvent. In the event that thiol-exchange is fast, such
reactivity principally enables cysteine-directed conjugation to
proteinogenic amine nucleophiles and, as consequence of the
addition-elimination mechanism, the thiol-conjugated probe will
remain bound during irreversible amine-conjugation (Fig. 2A). To
investigate these interesting and possibly unique aspects of
STEFs, probe 9 (100 µM) was first treated with an excess of either
n-butylamine or n-butanethiol in 30% CH3CN/PBS (Fig. 2B).
HPLC-analysis demonstrated that while both conditions could
convert 9, this occurred faster with n-butanethiol (Fig. 2B, left).
Similar to the reaction in organic solvent, 11 was formed
selectively from a mixture of cis/trans-isomers of 9 (Fig. S2). Next,
we co-incubated the nucleophiles and monitored formation of the
respective products, 11 and 12. This experiment strongly indicate
course of hours, presumably by acid-catalyzed hydrolysis (Fig. S1, initial formation of 12 which subsequently convert to 11 (Fig. 2B,
Supporting Information).
right). In accord with our initial hypothesis concerning the
irreversibility of amine conjugations, incubation of pure 11 with a
large excess of n-butanethiol did not result in any conversion (Fig.
S1).
We hypothesized that thiol-STEFs (9 and 10) under physiological
conditions would undergo reversible conjugation with thiol
nucleophiles (e.g. cysteine-residues) and that subsequent
reactions with amine nucleophiles could be irreversible. The
Figure 3. A) Proteome labelling by STEFs. MCF7 cell lysates were treated with STEFs (9-11), 5, IA-alkyne (IA-Alk) or DMSO for 2 h, followed by click chemistry
with rhodamine-azide, SDS-PAGE and in-gel fluorescence scanning. Lower panel shows Coomassie blue staining and serves as loading control. B) Proteome
labelling by STEFs following cysteine-blockade with iodoacetamide. MCF7 cell lysate were pre-incubated with iodoacetamide (20 mM) before probe incubation (2
h). C) Affinity isolation of STEF-binding proteins and examples of binding sites identified. See Table S1 and S2, Supporting Information for all data. More examples
of binding sites, see Fig. S9 and Fig. S10.
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Further increasing complexity, we incubated two short peptides
with 9 and used UPLC-MS/MS to monitor formation of potential
conjugates (Fig. 2C-E, Supporting information Fig. S3-7). The two
peptides were designed to differ only by cysteine vs. serine at
position 5 and to contain three different amine nucleophiles (N-
term/Arg/lys). In the presence of 500 µM of 9, we observed full
conversion of the cysteine-containing peptide after 30 min and
formation of new peptides with masses matching single and
double modifications, respectively (Fig. 2C-D). Fragmentation
analysis (Fig. 2E, Supporting Information Fig. S3-5) revealed the
single STEF-modification to be present at the N-terminal
amine/arginine-side chain and the doubly-modified peptide to
conditions throughout the analysis together with pull-down of
peptides instead of entire proteins. To our delight, this resulted in
the identification of a series of direct binding sites for probe 9,
which in all cases were at lysine residues (Table S2, Supporting
Information). Compared with the former experiment, 17 proteins
were in common and an additional 15 were identified. These two
types of experimental approaches have different advantages.
Protein pull-down can give protein hits even if the probed peptide
sequence in question isn’t suitable for MS detection, or if the
probe is unstable during the procedures. Peptide enrichment, on
the other hand, might capture conjugation sites sitting in
inaccessible positions of proteins, and has the clear advantage of
have an additional modification at the cysteine residue. In contrast, offering direct evidence of the modification site. The majority of
the serine-containing peptide barely converted under identical
conditions resulting in low quantities of a peptide carrying a STEF-
modification at the N-terminal amine/arginine sidechain (Fig. 2C,
Supporting Information Fig. S6-7). Collectively, these and the
prior experiments suggest that STEFs have an intrinsic ability for
fast and reversible thiol conjugation to form intermediates that
may be irreversibly intercepted by amine-nucleophiles with
favorable proximity and reactivity.16 Next, we tested the reactivity
and selectivity of STEFs in proteomic samples by gel-based
analysis. MCF7 cell lysates were treated with STEFs (9-11, 1-400
the binding sites, for which high-resolution structural information
is available, did indeed feature a cysteine-residue in close
proximity to the STEF-bound lysine in accordance with the
previous experiments (Fig. 3C, Fig. S9, Supporting Information).
In addition, we identified a series of sites where the STEF-bound
lysine residue was in close association with another lysine-
residue (Fig. 3C, Fig. S10), potentially constituting another
favored binding motif.
µM,
2 h) and subjected to copper-catalyzed azide-alkyne
cycloaddition (CuAAC) with rhodamine-azide. The probe-labelled
proteins were separated by SDS-PAGE and visualized by in-gel
fluorescence scanning (Fig. 3A). We observed
a clear
concentration-dependent labelling of several proteins by probe 9
and 10, with probe 10 displaying markedly enhanced reactivity.
Despite the significant structural similarity of 9 and 10, different
labelling patterns were apparent, e.g. a ~10-kDa protein was
strongly labelled by probe 9 and not by 10. As expected, probe 11
showed no protein labelling and the simple vinylogous carbamate
5 (Fig. 1A) showed 4- to 80-fold reduced reactivity compared to
probe 9 and 10, respectively. The labelling patterns of STEFs
were largely distinct from the thiol-selective probe IA-alkyne (IA-
Alk, Fig. 3A). Next, lysates were pre-treated with iodoacetamide
before exposure to STEFs (Fig. 3B). As expected, this completely
blocked IA-alkyne labelling but, interestingly, also strongly
reduced labelling with STEFs, especially with probe 9. This data
further supports a role for cysteine residues as direct targets and,
as suggested by the previous experiments, as intermediary
conjugation-sites towards irreversible amine-conjugations. In
order to probe these questions, we sought the specific protein
targets of 9. We performed an affinity enrichment and mass
spectrometry workflow using a cleavable azido-azo-biotin tag17
conjugated to probe-treated MCF-7 lysates. Biotinylated proteins
were enriched by streptavidin agarose resin and eluted with
sodium dithionite and resolved with SDS-PAGE (Fig. S8,
Supporting Information). Protein bands were excised, digested
with trypsin and the released peptides were analyzed by nanoLC-
MS/MS. We identified 114 protein targets (≥2 unique peptides of
each target captured in three separate replicates) of probe 9
(Supporting Information Figure S8 and Table S1). DMSO-treated
negative controls did not result in any protein identifications using
these cut-off criteria. Unfortunately, these experiments did not
result in identification of specific conjugation sites of probe 9
which may be due to the observed (Fig. 2A, Fig. S1) instability of
N-linked STEF-conjugates under the strongly acidic conditions
needed for the proteomics analysis. Consequently, we devised an
alternative sample processing scheme using milder acidic
Figure 4. Crystal structure of HSA (pdb: 1BM0) with indicated lysine residues
(purple) targeted by electrophilic probes. STEF-probe 9 preferentially targets
K414 and K199 (Table S3, Supporting Information).
Finally, to assess whether STEFs have potential for use in
bioconjugations, we tested their reactivity in a well-known context,
human serum albumin (HSA). Recently reported probes have
been demonstrated to target specifically K5737b and K647d in this
system (Fig. 4). Interestingly, based on the number of mass
spectral scans, we detect two major conjugation-sites of probe 9,
K414 and K199, that are both located in shallow hydrophobic
groves (Fig. 4B and Table S3, Supporting Information) and not in
proximity to the free cysteine (Cys34) present in HSA. Collectively,
our experiments with STEF-probe 9 demonstrate that while
cysteine-proximity appears to define the major lysine-reactivity
determinant, other favored target-motifs exist suggesting that this
class of reagents have potential to be uniquely tuned for selective
bioconjugations.
In conclusion, we have reported the use of STEFs as new
electrophilic motifs in various scenarios and demonstrated that
thiol-STEFs are both stable in physiological buffer and capable of
conjugation to proteins. We have provided evidence of structure-
dependent selectivity in proteomic labelling with simple STEF-
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[10] H.-D. Stachel, Chem. Ber. 1960, 93, 756-757.
probes and we have laid support for the ability of these
electrophiles to conjugate to proteinogenic amine-nucleophiles
via intermediacy of a reversibly bound thiol-conjugate even in
highly complex samples. In addition to the small probes studied
here, we have also demonstrated that it is chemically feasible to
install STEFs onto more complex molecules, such as analogs of
the EGFR-inhibitor, afatinib (see Supporting Information) which
presents another direction for future studies.
[11] F. Effenberger, Chem Ber. 1965, 98, 2260-2265.
[12] L. F. Tietze, A. Bergman, G. Brill, K. Brüggemann, U. Hartfiel, E.Voss,
Chem. Ber. 1989, 122, 83-94.
[13] See e.g.: G. Dujardin, S. Rossignol, E. Brown, Synthesis 1998, 763-770.
[14] M. Hojo, R. Masuda, H. Sano, M. Saegusa, Synthesis 1986, 137-139.
[15] L. F. Tietze, C. Schneider, M. Pretor, Synthesis 1993,1079-1080.
[16]
A similar effect was recently reported for non-enzymatic lysine
acetylation by AcCoA: A. M. James, K. Hoogewijs, A. Logan, A. R. Hall,
S. Ding, I. M. Fearnley, M. P. Murphy, Cell Rep. 2017, 18, 2105-2112.
[17] (a) Y. Y. Yang, J. M. Ascano, H. C. Hang, J. Am. Chem. Soc. 2010, 132,
3640–3641; (b) C. Sibbersen, A.-M. S. Oxvig, S. B. Olesen, C. B. Nielsen,
J. J. Galligan, K. A. Jørgensen, J. Palmfeldt, M. Johannsen, ACS Chem.
Biol. DOI: 10.1021/acschembio.8b00732.
Acknowledgements
We gratefully acknowledge financial support from the
Independent Research Fund Denmark (Sapere Aude 2 grant
6110-00600A to T.B.P.), the Lundbeck foundation (fellow grant
R105-9308 to T.B.P.), the Lundbeck Foundation (grant R180-
2014-2740 to M.J.) and the Carlsberg Foundation (infrastructure
grant CF15-0431 to T.B.P.)
Keywords: Acrylate • Cysteine • Bioconjugation • Electrophile •
proteomics
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Entry for the Table of Contents
Layout 2:
COMMUNICATION
Bente K. Hansen, Christopher
RPS2
STEF-target
KHRSP
Cys
STEF-target
O
Proximal Cys
Loveridge, Stine Thyssen, Gustav J.
Wørmer, Andreas D. Nielsen, Johan
Palmfeldt, Mogens Johannsen*, Thomas
B. Poulsen*
SR
N
H
O
STEFs
Page No. – Page No.
STEFs - Activated Vinylogous
Protein-Reactive Electrophiles
S-Marks the spot. A new class of activated vinylogous thioesters, called STEFs,
were evaluated for use as biological electrophiles. These compounds e.g. enable
cysteine-directed lysine conjugations via consecutive addition-elimination reactions.
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