Azabicyclic vinyl sulfones for residue-specific dual protein labelling

Article abstract of DOI:10.1039/c9sc00125e

We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are moderately resistant to retro Diels-Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1-5 equiv. of reagent in NaP_i pH 7.0, room temperature, 30 min). The resulting thioether-linked conjugates were stable and retained the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein could be fluorescently labelled through an inverse electron demand Diels-Alder reaction in cells to allow selective apoptosis imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.

Full text of DOI:10.1039/c9sc00125e

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E. Jiménez-Moreno, B. Oliveira, C. D. Navo, P. M. S. D. Cal, G. Jiménez-Osés, I. Robina, A. J. Moreno-
Vargas and G. J. L. Bernardes, Chem. Sci., 2019, DOI: 10.1039/C9SC00125E.
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Azabicyclic Vinyl Sulfones for Residue-specific Dual Protein
Labelling
Enrique Gil de Montes,a Ester Jiménez-Moreno,b Bruno L. Oliveira,b,c Claudio D. Navo,d Pedro M. S.
D. Cal,c Gonzalo Jiménez-Osés,d,e,f Inmaculada Robina,a Antonio J. Moreno-Vargas,*a and Gonçalo J.
L. Bernardes*b,c
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
We have developed [2.2.1]azabicyclic vinyl sulfone reagents that simultaneously enable cysteine-selective protein
modification and introduce a handle for further bioorthogonal ligation. The reaction is fast and selective for cysteine relative
to other amino acids that have nucleophilic side-chains, and the formed products are stable in human plasma and are
moderately resistant to retro Diels–Alder degradation reactions. A model biotinylated [2.2.1]azabicyclic vinyl sulfone
reagent was shown to efficiently label two cysteine-tagged proteins, ubiquitin and C2Am, under mild conditions (1–5 equiv.
of reagent in NaPi pH 7.0, room temperature, 30 min.). The resulting thioether-linked conjugates were stable and retained
the native activity of the proteins. Finally, the dienophile present in the azabicyclic moiety on a functionalised C2Am protein
could be fluorescently labelled through an inverse electron demand Diels–Alder reaction in cells to allow selective apoptosis
imaging. The combined advantages of directness, site-specificity and easy preparation mean [2.2.1]azabicyclic vinyl sulfones
can be used for residue-specific dual protein labelling/construction strategies with minimal perturbation of native function
based simply on the attachment of an [2.2.1]azabicyclic moiety to cysteine.
www.rsc.org/
Chudasama,
Caddick
and
co-workers
have
explored
Introduction
For certain biological applications, the installation of two or more
dibromopyridazinedione reagents that are able to site-specifically
rebridge disulfide bonds and — at the same time — leave two click-
reactive handles available for subsequent modifications with
fluorophores, PEG derivatives or drugs through strain-promoted or
copper-mediated azide-alkyne cycloaddition reactions.4, 7, 8 Water-
soluble allyl sulfones were also shown by Weil and co-workers to
rebridge disulfides whilst enabling dual protein functionalisation.9 In
distinct synthetic modifications into
a protein of interest is
desirable.1 To introduce two distinct modifications onto a protein
two options are possible: 1) site-selectively modify two different
amino-acid residues within the protein’s sequence2, or 2) use a
3
scaffold that simultaneously displays two different modifications and
one handle for site-selective protein modification.4, The latter,
another example, Swarts and co-workers developed
a
5
bicyclo[6.1.0]nonyne-based cyclooctyne reagent for the
modification of azide-labelled biomolecules with functional handles
which uses multifunctional scaffolds, is perhaps the most
straightforward because it does not require orthogonal
chemoselective protein reactions.1 Senter and co-workers developed
a double-drug carrier with a hydrophilic tail that reacts with the
protein through a cysteine-reactive handle (maleimide).5 In another
example, Gonçalves and co-workers reported dichlorotetrazine as a
trivalent platform for conjugation to cysteine and showed dual
labelling of albumin with a macrocyclic chelator for simultaneous
nuclear imaging and a fluorescent probe for imaging.6 In addition,
that
allow
both
photo-crosslinking
and
subsequent
detection/enrichment of binders.10 The latter example is the only
one in which the ability to perform dual protein labelling was shown
in cells through bioorthogonal labelling, with the others being used
to prepare dual-labelled conjugates in the test tube. Thus, the
development of simple and robust methodologies that enable the
installation of two specific modifications at a single site on a protein
specially when one handle can be used for bioorthogonal ligation in
cells is an area of prominent interest.
Herein, we present strained [2.2.1]azabicyclic vinyl sulfones as
efficient and versatile reagents for residue-specific dual protein
labelling. Our design includes a highly reactive and selective alkene
handle for bioorthogonal inverse electron demand Diels–Alder
(iEDDA) ligation and one easily accessible functionalisation site
through N-substitution reactions on the azanorbornadiene scaffold.
Vinyl sulfones are useful Michael acceptors and dipolarophiles in
cycloaddition reactions for synthesis and medicinal chemistry
applications,11 and as handles with multi-purpose functions in
protein modification.12 Their first use for protein modification dates
a. Departamento de Química Orgánica, Facultad de Química, Universidad de Sevilla,
C/ Prof. García González, 1, 41012-Sevilla, Spain. E-mail: ajmoreno@us.es
b. Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW
Cambridge, UK. E-mail: gb453@cam.ac.uk
c. Instituto de Medicina Molecular, Faculdade de Medicina da Universidade de
Lisboa, Av. Prof. Egas Moniz, 1649-028 Lisboa, Portugal.
d. Departamento de Química, Centro de Investigación en Síntesis Química,
Universidad de La Rioja, 26006 Logroño, Spain.
e. CIC bioGUNE, Bizkaia Technology Park, Building 801A, 48170 Derio, Spain.
f. Ikerbasque, Basque Foundation for Science, Maria Diaz de Haro 13, 48009 Bilbao,
Spain.
Electronic Supplementary Information (ESI) available: [detailed methods and
additional characterisation]. See DOI: 10.1039/x0xx00000x
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from 1988 in which simple methyl and ethyl vinyl sulfones were used
to alkylate thiol, -amino or imidazole side-chain amino acids.13 Since
that time, there have been a number of reports that describe the
development of allyl9 and vinyl14, 15 sulfones for disulfide rebridging16
and cysteine17 conjugation methodologies, respectively. Under
slightly basic conditions, the sulfhydryl side-chains of the cysteine
residues are typically more nucleophilic than the amino groups of
lysine and imidazole, and the hydroxyl groups of serine and
threonine and thus, selective modification can be achieved.18
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DOI: 10.1039/C9SC00125E
Strained systems offer a unique opportunity for protein labelling
because the significant energy stored in the system is liberated either
in part or fully in a click-like bioconjugation process.19 Among them,
[2.2.1]bicyclic systems, such as norbornenes, exhibit both high
reactivity and high stability, and are synthetically accessible.
(Hetero)norbornenes and (hetero)norbornadienes can be easily
obtained in one step through Diels–Alder reactions between a cyclic
diene (cyclopentadiene/furan/pyrrole derivatives) and the
appropriate dienophile (electron-deficient alkene or alkyne). Of
particular interest are the 7-oxanorbornadiene derivatives
developed by Finn and co-workers, which were previously used for
site-selective modification of cysteine residues in proteins by
Fig. 1 Precedent and proposed linker for cysteine-specific dual protein bioconjugation.
a. 7-Oxanorbornadiene derivatives for cysteine-selective modification and retro Diels–
Alder cleavage.20-22 b. [2.2.1]Azabicyclic vinyl sulfones highlight the site of cysteine-
selective modification, the second reactive handle for biorthogonal labelling in cells and
the explored point-of-attachment.
Results and discussion
Development of [2.2.1]bicyclic vinyl sulfones for cysteine
modification.
21
conjugate addition (Figure 1a).20, After cysteine ligation on the
protein, oxanorbornadiene undergoes a controllable retro-Diels–
Alder (rDA) fragmentation that allowed their use as drug delivery
Based on a reported procedure,26, 27 we first carried out the synthesis
of racemic azanorbornadiene 1 through a Diels–Alder reaction
between inexpensive N-tert-butyloxycarbonyl (Boc)-pyrrole and
ethynyl p-tolyl sulfone. We then tested the reactivity of
azanorbornadiene 1 towards cysteine by using N-Boc-Cys-OMe as a
model system. When a stoichiometric amount of bicyclic system 1
was reacted with the cysteine model in a buffered mixture of NaPi
(100 mM, pH 7.3) and N,N-dimethylformamide (DMF) at room
temperature for 10 min, complete conversion to the corresponding
trans thioether adduct was detected (Scheme 1a). This experimental
data confirmed the high reactivity of this particular vinyl sulfone
embedded in the strained 7-azanorbornadiene skeleton towards
thiol-Michael addition. The exo-facial preference of the nucleophilic
attack is well documented in other [2.2.1]azabicyclic systems.26-30
Furthermore, to attest the chemoselectivity of our strained
[2.2.1]azabicyclic reagent we performed a competition experiment
between N-Boc-Cys and N-Boc-Lys. Notably, under our experimental
conditions [buffered mixture of NaPi (50 mM), pH 7.3,
dimethylsulfoxide (DMSO), room temperature, 10 min] only the Cys
adduct was obtained and no modification of the ε-amino group of
lysine was detected (Scheme 1b and Figure S6). Together our data
show that [2.2.1]azabicyclic vinyl sulfones can react rapidly under
aqueous conditions in a chemo- and stereoselective manner with
cysteine residues.
systems.22
Strained
[2.2.1]bicyclic
systems,
especially
oxanorbornadiene and norbornene systems,19 have also been widely
used in the field of bioorthogonal chemistry as handles, for example,
for fast reaction with tetrazines in iEDDA.23 On the contrary, the use
of [2.2.1]azabicyclic systems for bioconjugation is rare. A single
example showed the use of a 7-azanorbornene as hub molecule for
the incorporation of a fluorophore for imaging and biotin as an
affinity probe for protein recognition.24 This system lacks a handle
that would allow for site-selective covalent protein modification.
In this work, we envisioned N-substituted azanorbornadienes of type
A, which combine a vinyl sulfone functionality, an additional reactive
double bond and a functionalisable bridging nitrogen as an attractive
reagent for cysteine-specific and dual protein labelling (Figure 1b).
The reactive double bond of the resulting azanorbornene upon
cysteine modification offers an attractive handle for further
bioorthogonal modification at the same residue through iEDDA.23, 25
Finally, the bridging nitrogen can be used to incorporate relevant
fragments for protein studies/functions, such as fluorophores,
affinity probes or drugs. Thus, with the designed chemical probe we
can selectively target cysteine within a protein sequence and
functionalise the molecule through simple N-derivatisations of the
azanorbornadiene and further modify it in situ through fast iEDDA
reactions with tetrazine reagents.
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cysteine with [2.2.1]azabicyclic vinyl sulfone reagents, which is
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DOI: 10.1039/C9SC00125E
promising for their potential utility to build conjugates for in vivo
studies.
Entry
Vinyl
Azabicycle-
R
P
solvent
half-
life
sulfonea Cys adduct
1
2
3
4
1
5
7
7
2
6b
8
H
Boc
CDCl3
CDCl3
CDCl3
16.4 h
<5 min
37 hc
59 h
Sch. 1 Reaction engineering on amino acid models. a. Reaction of vinyl sulfone 1 with
N-Boc-Cys methyl ester derivative. b. Competition experiment that shows the
chemoselective nature of the reaction between [2.2.1]azabicyclic vinyl sulfone 1 and
cysteine.
Me Boc
H
H
Ac
Ac
8
DMSO-d6
Table 1 rDA studies of azabicycle-cysteine adducts. aSee ESI for details on the synthesis
of vinyl sulfones 5 and 7. bAzabicycle-cysteine adduct 6 was not observed because it
decomposes directly to give 9 and N-Boc-2,5-dimethylpyrrole.c t1/2 = 40 h; CDCl3 is pre-
neutralized with K2CO3.
Stability of cysteine-[2.2.1]azabicyclic vinyl sulfone towards rDA.
Having shown the chemoselectivity of 1 towards cysteine residues,
we then studied the stability of the formed thioether because we
were concerned by some rDA reports of cleavage with similar bicyclic
systems.22, 31-34 Thus, fragmentation of azanorbornene 2 into N-Boc-
pyrrole and thio-vinyl sulfone 9 was studied by 1H NMR spectroscopy
at 37 ºC in CDCl3 (Table 1, Entry 1; see Figures S8 and S9 for NMR
spectroscopic monitoring). Compound 2 has a half-life of 16 h,
which is nearly half of the longest half-life reported for the
corresponding oxanorbornadiene derivative.22 As reported, the rDA
fragmentation has a tuneable rate depending upon the substituents
of the norbornene so we decided to modulate the stability of our
reagent. To prove the concept, we introduced two bridgehead
methyl groups on the bicyclic system (vinyl sulfone 5, ESI for
synthetic details)), which significantly decreased the half-life (<5 min)
of the corresponding cysteine adduct (Table 1, Entry 2). In fact, in
this case, the corresponding bicyclic adduct was not observed and
afforded directly the rDA products. Next, instead of modifying the
diene structure of the pyrrole ring, we decided to change the
protection of the amino group from Boc in 1 to an acetyl group in 7
(ESI for synthetic details), which doubled the half-life of the cysteine
adduct 8 (37 h; Table 1, Entry 3). The stabilising effect was even more
predominant when we changed to more polar DMSO-d6, which
resulted in a half-life of 59 h, which is nearly 4 times higher than the
initial conditions (Table 1, Entry 4). Moreover, when a mixture of
DMF and buffered aqueous solution was chosen to test the potential
rDA, after 59 h at 37 ºC only 37% of adduct 8 had decomposed into
the rDA products.
[2.2.1]azabicyclic vinyl sulfones for residue-specific protein
labelling.
We decided to evaluate whether our findings on small molecules
could be translated into cysteine-tagged proteins. We started by
adding D-biotin to the bridging nitrogen of the strained
[2.2.1]azabicyclic vinyl sulfone scaffold for potential use in imaging
or enrichment experiments. The bridging nitrogen is ideal as a point
of attachment because it is not expected to influence the reactivity
of the vinyl sulfone towards thiol-Michael addition. Briefly, acidic
Boc-deprotection of 1 followed by acylation with D-biotin acid
chloride 10 afforded biotinylated azanorbornadiene 11 in 75%
overall yield (see Scheme S1 in the ESI). An identical synthetic route
may be used to incorporate other motifs routinely used for protein
modification including PEG, drugs or fluorophores (e.g. dansyl
moiety, see Scheme S2, compound 12). As a model protein for
labelling, we decided to produce a mutant of a multivalent protein –
ubiquitin (Ub-K63C) – that has been engineered to feature a surface-
exposed cysteine residue at position 63 (for further details see the
ESI).36 By using an equimolar amount of biotin-functionalized
[2.2.1]azabicyclic vinyl sulfone 11 in NaPi buffer (20 mM, pH 7.0)
using DMF as co-solvent (10%) at room temperature for 30 min, Ub-
K63C was fully converted into a single and homogenous product
(Figure 2a and 2b) as analysed by LC-MS. The crude reaction was then
subjected to excess Ellman’s reagent and after 1 h at 37 ºC, no change
was noted, which confirms that all cysteine was consumed during the
Michael reaction with vinyl sulfone 11 (see Figure S18), and further
confirmed the chemoselectivity of our method. Importantly, we
showed that the Ub-K63C-biotin conjugate is relatively stable in
plasma media. After 24 hours incubation at 37 ºC with plasma, only
modest degradation of the product (<20%) was observed as a result
of retro-Diels–Alder reaction (see Figure S19). Moreover, the original
thioether linkage remained unaltered and did not exchange with
other biological thiols present in plasma media. Our data show a
cysteine-specific protein modification with a [2.2.1]azabicyclic vinyl
sulfone that bears a synthetic modification inserted at the bridging
nitrogen of the reagent that leaves the remaining alkene unreacted,
which may be further used for iEDDA ligation in a test tube or in cells.
Quantum mechanical calculations on model compounds (Figure S12
in the ESI) reproduced the experimentally observed slight slowing
down of the reaction when replacing the Boc by the Ac group, and
when using a more polar solvent (DMSO vs. CHCl3), reflecting the
high nonpolar character of the rDA transition structures. However,
and unlike in the case of Finn’s oxanorbornadiene thiol and amine
adducts, which have different substitution patterns and exhibit much
more asynchronous transition structures as studied by Houk,35 the
difference in reactivity of the bridgehead-methylated and non-
methylated analogues was not so apparent computationally, and
only using nearly complete models could reproduce the
experimental trend. While the rDA can take place under
physiologically relevant conditions, our data shows that this reaction
is very slow (>59 h) for conjugates formed through modification of
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resonance-detectable tag was used for imaging apoptotic tissues in
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vivo.37 Thus, we reacted this protein mutant with compound 11 by
DOI: 10.1039/C9SC00125E
using similar conditions to those described above (5 equiv., 20 mM
of NaPi buffer/10% DMF at pH 7.0, room temperature, 30 min
reaction) to give a single homogenous product (Figures 3a and 3b)
determined by LC-MS. The efficiency of the biotinylation reaction
was also examined by Western blot. Non-modified C2Am (control)
and biotinylated protein C2Am-11 were resolved by SDS-PAGE.
Biotinylation was confirmed by using a Streptavidin fluorescent
probe (Streptavidin-Alexa555) after transfer to a polyvinylidene
difluoride membrane (Figure 3c). We then tested if this C2Am
derivative retains its inherent functionality of binding to the PS
phospholipid on the surface of apoptotic cells. Therefore, C2Am-11
was used for imaging apoptotic cells by pre-targeting followed by
labelling with Streptavidin-Alexa555.38 Apoptosis in HeLa cells was
induced by treatment with actinomycin D (Act D, 1 μM, 12 h).
Following treatment with Act D, cells were washed and incubated
with C2Am-11 (6 μM, 20 min) at 37 ºC. After pre-incubation,
apoptotic cells were further washed and incubated with the
counterpart of biotin, Streptavidin-Alexa 555 (2.5 μL of 1 mg/mL, 20
min). Blocking studies were performed by incubating apoptotic cells
with a large excess of non-modified C2Am and then with biotinylated
surrogate C2Am-11 and Streptavidin-Alexa555. Fluorescent
microscopy images showed staining of only apoptotic cells by C2Am-
11 + Strep-Alexa 555, whereas healthy control cells remained
unstained (Figure 3d). Moreover, in the blocking experiments, in
which cells were first incubated with the non-labelled C2Am, a
significant decrease of the mean fluorescence intensities (MFI) was
observed (Figure 3e). These results attest the ability of modified
C2Am-11 to bind to PS to enable specific labelling of apoptotic cells.
Fig. 2 Cysteine site-selective bioconjugation with [2.2.1]azabicyclic reagent 11. a.
Reaction between Ub-K63C and 11. General reaction conditions: Ub-K63C reacted with
11 (1 equiv.) in NaPi (20 mM, pH 7.0) at 25 ºC for 30 min. b. Deconvoluted mass spectrum
obtained from the represented reaction. c. Deconvoluted mass spectrum obtained after
incubation of Ub-K63C-11 in plasma for 24 h at 37 °C.
Imaging C2Am-11-labelled apoptotic cells.
Encouraged by our results, we envisioned to apply our biotin-
functionalised reagent for cysteine-selective protein modification
and further labelling with a streptavidin probe for imaging. For these
studies we used the C2A domain of Synaptotagmin-I (C2Am) that
binds to phosphatidylserine (PS), which is localized on the external
leaflet of the plasma membrane during apoptosis. In previous studies
a C2Am mutant that has a single cysteine at the position 78 for
modification with a radionuclide, a fluorescent probe, or a magnetic
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DOI: 10.1039/C9SC00125E
Fig. 3 Imaging of apoptotic HeLa cells with C2Am-11. a. Reaction between C2Am and 3 (5 equiv.) in NaPi (20 mM, pH 7.0) at 25 °C for 30 min. b. Deconvoluted mass spectrum obtained
from a representative reaction. c. Treatment of C2Am-11 with Streptavidin-Alexa 555 after SDS-PAGE and membrane transfer gave a single fluorescent band as detected by Western
blot that confirms biotinylation of C2Am and binding of the fluorescent streptavidin to the biotin. Lanes 1 and 2, Spyro Protein Ruby staining (SDS-PAGE); lanes 3 and 4, fluorescence
at excitation and emission wavelengths for Alexa 555 (Western Blot membrane). d. Epifluorescent images of non-apoptotic (control) and apoptotic HeLa cells after labelling with
C2Am-11 + Streptavidin Alexa 555. Blocking studies were performed by pre-incubating apoptotic cells with an excess of non-fluorescent C2Am before incubation with C2Am-11 +
Streptavidin Alexa 555. Apoptotic cells are shown in green. Blue represents Hoechst-stained nuclei. e. MFI from apoptotic and blocked cells determined with ImageJ (average of 30
regions of interest). Statistical significance was determined by paired t test by using GraphPad Prism (****, P < 0.0001).
Reactivity of cysteine-[2.2.1]azabicyclic reagents towards
tetrazines.
dienophile present in 2 can quickly and easily undergoes iEDDA
reactions with tetrazines.23 Considering the properties of the diene
to perform an iEDDA most electron-deficient tetrazines 13a and 13b
resulted in a faster reaction, whereas more electron-rich tetrazine
13c reacted an order of magnitude slower. (Table 2). As
demonstrated by quantum mechanical calculations (Figure 4 and
S13), and contrary to what is commonly assumed, the superior
reactivity of N-substituted azanorbornenes compared to their non-
bicyclic analogues does not arise from any special strain at the double
bond, as it is the case for other highly strained π-systems such as
trans-cyclooctene or cyclooctyne. This can be deduced from the very
similar double bond lengths and angles in non-bicyclic and bicyclic
analogues. Moreover, such apparent activation of the alkene group
strongly depends on the type of substitution at the bridging atom.
After having studied the reactivity, stability and utility of the cysteine
attachment point of [2.2.1]azabicyclic vinyl sulfones, we explored the
possibility of a second reactive handle for site-specific modification
with the addition of a proper counterpart. In our case, the
subsequent modification would take advantage of the remaining
double bond present in the bicyclic system by its reaction with
tetrazines through iEDDA.
Based on a precedent with reactions between azanorbornene
derivatives and tetrazines,24 we evaluated the reactivity of thioether
bicyclic system 2 with different tetrazines (13a–13c) by monitoring
the decrease of tetrazine absorbance over time.23 We found that the
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the transition state geometry in A1c, while in A1b such distortion is already developed up
While N-alkyl azanorbornenes or norbornenes show nearly the same
(or even slower) calculated iEDDA reaction rates with tetrazine 13a
than their non-bycyclic five-membered analogues (1-alkyl-2,5-
dihydro-1H-pyrroles and cyclopentene, respectively), calculated
activation barriers for N-carbonyl (carbamate or amide)-substituted
azanorbornenes are 5-6 kcal mol–1 smaller than those of their non-
bicyclic counterparts, resulting in a 3-4 orders of magnitude
acceleration. The source of this acceleration is the distortion of the
otherwise planar 1-carbonyl-2,5-dihydro-1H-pyrrole ring into a
puckered conformation closely resembling that at the transition
state, similarly to what described for norbornenes vs. (Z)-but-2-ene
or cyclohexene.39 Figure 4 shows that the iEDDA reaction causes a
puckering of the planar five-membered 2,5-dihydro-1H-pyrrole ring
in model system A1c of ≈13° at the transition state, while for bicyclic
analogue A1b the structural changes at the transition state are
minimal. This reflects the pre-distorted nature of azanorbornenes
and thus their superior ability to achieve transition state geometries
through lower activation barriers, which ultimately translates into
reaction rate acceleration.
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in the dienophile, translating into a reaction rate acceleration of several orders of
DOI: 10.1039/C9SC00125E
magnitude
[2.2.1]azabicyclic vinyl sulfones for residue-specific dual protein
labelling.
With the chemoselectivity towards cysteine residues evaluated (one
attachment point studied with an affinity tag, and the viability of
chemically modifying the second reactive handle in our
[2.2.1]azabicyclic vinyl sulfones with tetrazines appraised), we then
needed to see whether this last achievement was also reproducible
in a physiological environment. This would allow us to demonstrate
the dual-labelling character present in this vinyl sulfone.
We planned to pre-target apoptotic cells with our previously
synthesized bioconjugate with the affinity tag (C2Am-11) and then
use fluorogenic and commercially available 6-methyl-tetrazine-sulfo-
cy3 (Tz-Cy3) to functionalise the remaining double bond (Figure 5a).
The protocol for targeting cells with C2Am-11 was similar to the one
mentioned above. After incubation with the bioconjugate, cells were
washed and further incubated with fluorogenic tetrazine for 90 min.
Tetrazine labelling of pre-targeted C2Am-11 allowed ready
visualization of cells rendered apoptotic by treatment with Act D
(Figure 5a). Similarly, cells previously blocked with “native” C2Am,
and then labelled with C2Am-11 + Tz-Cy3 following the same
protocol, showed a significant decrease of fluorescence (Figure 5c).
These experiments confirm that the specific affinity of C2Am-11 for
apoptotic cells is retained and the double bound is accessible by a
These data highlight the suitability of strained [2.2.1]azabicyclic vinyl
sulfone reagents to site-specifically modify cysteine residues whilst
simultaneously leaving a reactive alkene free for further iEDDA
labelling.
tetrazine fluorophore in
a cellular environment (Figure 5d).
Reactivity of the double bond towards the fluorescent tetrazine was
studied by SDS-PAGE (Figure 5b). After 90 min a fluorescent band
that corresponded to C2Am-11-Tz-Cy3 modified protein was
observed (Figure 5c and 5d). We further tested the versatility of this
system for dual modification of proteins. Reaction with precursor 12
Entry
Tetrazine
13a
13b
k x 10–2 M–1·s–1
2.6 ± 0.1
3.1 ± 0.1
1
2
3
13c
0.143 ± 0.005
(dansylglycine-azanorbornadiene) enabled installation of
a
Table 2. Kinetic studies for the iEDDA reaction between azanorbornene 2 and tetrazines
13a–13c.
fluorescent tag on ubiquitin that could be further modified by
reaction with a tetrazine compound (accessed by MS and SDS-PAGE
in-gel fluorescence; Figures S20, S21). Overall, the azanorbornadiene
handle can be used as a platform for double modification of the same
protein without changing its native properties.
Fig. 4. Kinetic studies for the iEDDA reaction between azanorbornene 2 and tetrazines
13a–13c. Lowest-energy conformations calculated for N-Moc-protected (Moc = methyl
carbamate) cyclic 2,5-dihydro-1H-pyrrole (A1c) and bicyclic azanorbornene (A1b) models
and their iEDDA reactions with tetrazine 13a calculated with PCMDMF/M06-2X/6-
31+G(d,p). Five-membered ring puckering is represented by the torsion angle ϕ and
310
푘
푟푒푙
relative reaction rate constants at 37 ºC (310 K,
) are calculated from activation free
energies (ΔG‡). A significant distortion of the dihydropyrrole ring is required to achieve
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a cytotoxic molecule and a fluorophore for following cancer
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DOI: 10.1039/C9SC00125E
treatment.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
We thank the Universidad de Sevilla (E.G.M. short stay PP2017-
8144), the Spanish Ministry of Education, Culture and Sport
under the FPU program (E.G.M.), the Spanish Ministry of
Economy and Competitiveness (CTQ2016-77270-R to A.J.M.-V,
E.G.M. and I.R and CTQ2015-70524-R and RYC-2013-14706 to
G.J.-O. and C.D.N.), the EU (Marie-Sklodowska Curie ITN Protein
Conjugates to G.J.L.B., GA No. 675007; Marie-Sklodowska Curie
IEF to B.L.O., GA No. 702574 and to E.J.M., GA No. 701473), FCT
Portugal (FCT Investigator to G.J.L.B., IF/00624/2015 and
postdoctoral
fellowship
SFRH/BPD/103172/2014
to
P.M.S.D.C.), and the EPSRC (grant EP/M003647/1 to G.J.L.B.) for
funding. We thank Dr André Neves and Prof. Kevin Brindle for
providing the C2Am protein; Dra. Elena Moreno Clavijo for her
helpful discussions, and Dr Vikki Cantrill for her help with the
preparation and editing of this manuscript. We also thank
CITIUS for NMR and mass spectrometry assistance and BERONIA
(Universidad de La Rioja) for computer support. G.J.L.B. is a
Royal Society University Research Fellow (UF110046 and
URF\R\180019) and the recipient of a European Research
Council Starting Grant (TagIt, GA No. 676832).
Fig. 5 Imaging apoptotic cells with tetrazine-Cy3 after pre-targeting with bioconjugate
C2Am-11. a. Scheme of the general procedure for imaging cells. b. Fluorescent images
of non-apoptotic (control) and apoptotic HeLa cells after labelling with C2Am-11 + Tz-
Cy3. Blocking studies were performed by pre-incubating of apoptotic cells with an excess
of non-fluorescent C2Am before incubation with C2Am-11. c. MFI from apoptotic and 1.
blocked cells determined with ImageJ (average of 30 regions of interest). Statistical
significance was determined by paired t test by using GraphPad Prism (****, P < 0.0001).
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