Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using Bifunctional NHS-Activated Acrylamides

Article abstract of DOI:10.1002/anie.202016936

Widely used reagents in the peptide functionalization toolbox, Michael acceptors and N-hydroxysuccinimide (NHS) activated esters, are combined in NHS-activated acrylamides for efficient chemoselective amino-sulfhydryl stapling on native peptides and proteins. NHS-activated acrylamides allow for a fast functionalization of N-terminal cysteines (k₂=1.54±0.18×10³ M^?1 s^?1) under dilute aqueous conditions, enabling selectivity over other nucleophilic amino acids. Additionally, the versatility of these new bioconjugation handles was demonstrated in the cross-linking of in-chain or C-terminal cysteines with nearby lysine residues. NHS-activated acrylamides are compatible with the use of other cysteine selective reagents, allowing for orthogonal dual-modifications. This strategy was successfully applied to the late-stage functionalization of peptides and proteins with a PEG unit, fluorescent probe, and cytotoxic agent. The level of molecular control offered by NHS-activated acrylamides is expected to promote amino-sulfhydryl stapling technology as a powerful strategy to design functional bioconjugates.

Full text of DOI:10.1002/anie.202016936

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How to cite: Angew. Chem. Int. Ed. 2021, 60, 10850–10857
International Edition:
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doi.org/10.1002/anie.202016936
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Peptide Functionalization
Efficient Amino-Sulfhydryl Stapling on Peptides and Proteins Using
Bifunctional NHS-Activated Acrylamides
Maria J. S. A. Silva, HØlio Faustino, Jaime A. S. Coelho, Maria V. Pinto, Adelaide Fernandes,
Ismael CompaÇón, Francisco Corzana, Gilles Gasser, and Pedro M. P. Gois*
Abstract: Widely used reagents in the peptide functionaliza-
tion toolbox, Michael acceptors and N-hydroxysuccinimide
efficacy, but as importantly, site-selectivity, orthogonality and
new mechanisms to control the peptide structure.
[
2]
(
NHS) activated esters, are combined in NHS-activated
The low abundance of cysteine (Cys) and the high
reactivity of the sulfhydryl side chain have made this residue
the preferred hotspot to introduce site-selectively modifica-
acrylamides for efficient chemoselective amino-sulfhydryl
stapling on native peptides and proteins. NHS-activated
acrylamides allow for a fast functionalization of N-terminal
cysteines (k = 1.54 Æ 0.18 ” 10 M s ) under dilute aqueous
[
3]
tions on peptide chains. However, apart from a few specific
3
À1 À1
[4]
reagents that modify N-terminal Cys, most of the chem-
2
conditions, enabling selectivity over other nucleophilic amino
acids. Additionally, the versatility of these new bioconjugation
handles was demonstrated in the cross-linking of in-chain or
C-terminal cysteines with nearby lysine residues. NHS-activat-
ed acrylamides are compatible with the use of other cysteine
selective reagents, allowing for orthogonal dual-modifications.
This strategy was successfully applied to the late-stage func-
tionalization of peptides and proteins with a PEG unit,
fluorescent probe, and cytotoxic agent. The level of molecular
control offered by NHS-activated acrylamides is expected to
promote amino-sulfhydryl stapling technology as a powerful
strategy to design functional bioconjugates.
istries available to functionalize this amino acid target the
reactivity of the sulfhydryl side chain and, for that reason,
these methods are often unable to discriminate between
different reactive thiol groups on the biomoleculeꢀs surface.
In addition to this lack of orthogonality, with the notable
exception of bridging reagents used in the stapling of disulfide
[5]
bonds, existing chemical methods to modify Cys typically
offer poor control over the residue chemical environment,
limiting by this way, the manipulation of the peptide chain to
improve binding to biological targets, proteolytic stability,
[5l,6]
among other important pharmacokinetic properties.
Therefore, to further expand the utility of Cys modification
for the construction of more complex and well defined
bioconjugates, it will be fundamental to devise new Cys
modification reactions that offer not only orthogonality but
Introduction
[7]
Novel aqueous chemistries for the selective modification
of native peptide chains have been described in recent years
and become a key instrument for chemical biology and drug
development. It is commonly agreed that these tools will
provide major insight into basic biology and enable the
development of functional bioconjugates with unprecedented
properties. To deliver on these promises, it will be key to
discover new technologies that offer not only bioconjugation
also innovative mechanisms to control the peptide structure.
Motivated by this challenge, we envisioned that a chemo-
selective amino-sulfhydryl stapling reaction would deliver on
such prerequisites. The bridging of these functions would
enable the bioconjugation to proceed orthogonally at
N-terminal Cys sites and promote peptide macrocyclization
between in chain Cys and a lysine (Lys) close by, which could
be explored as a mechanism to control the structure and
properties of the bioconjugate. Despite its potential, the
amino-sulfhydryl stapling reaction remains underdevelo-
[1]
[
5f,l,8]
ped
for bioconjugation applications. Therefore, we set-
[
*] M. J. S. A. Silva, Dr. H. Faustino, Dr. J. A. S. Coelho, M. V. Pinto,
Dr. A. Fernandes, Dr. P. M. P. Gois
out to develop a reagent that is capable of stapling amino-
sulfhydryl functions with high chemoselectivity, fast kinetic
under dilute aqueous conditions and that enables a straight-
forward structural diversification.
Research Institute for Medicines (iMed.ULisboa)
Faculty of Pharmacy, Universidade de Lisboa
Lisbon (Portugal)
E-mail: pedrogois@ff.ulisboa.pt
Michael acceptors like carbonyl acrylic reagents reported
by the Bernardes group and N-Hydroxysuccinimide (NHS)-
[
9]
M. J. S. A. Silva, Dr. G. Gasser
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for
Life and Health Sciences, Laboratory for Inorganic Chemical Biology
activated esters are among the leading warheads for Cys and
[1b]
N-terminus/Lys functionalization, respectively. Therefore,
7
5005 Paris (France)
we envisioned that merging these two functional groups in an
NHS-activated acrylic ester (1) would generate a bifunctional
reagent for the selective stapling of amino-sulfhydryl groups
Dr. I. CompaÇón, Dr. F. Corzana
Departamento de Química, Centro de Investigación en Síntesis
Química, Universidad de La Rioja
(
Scheme 1).
2
6006 LogroÇo, La Rioja (Spain)
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.202016936.
1
0850
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(products 3a, 3c–3 f; Supporting Information,
Section 8, Figures S35–S42).
The formation of this seven-member hetero-
1
cycle was confirmed by H NMR analysis of the
diastereoisomers 6c and 6c’ obtained in the
reaction of Cys methyl ester 4 with NHS-
activated acrylamide 1c (Supporting Informa-
tion, Sections 3.1.6 and 3.1.7, Figures S1–S3).
The diastereoisomer mixture of products was
then evaluated in terms of stability in different
buffers (pD 9.0, 8.0, 7.0 and 4.5) and in the
presence of glutathione. In these conditions, the
Scheme 1. NHS-activated acrylic ester 1 as an amino-sulfhydryl stapling reagent in
peptides and proteins.
Results and Discussion
scaffolds were shown to be stable over one month (Support-
ing Information, Section 4, Figures S4–S8).
N-Terminal cysteine functionalization
Next, we investigated the mechanism of the 1,4-thiazepan-
5
-one formation by means of experimental (NMR and ESI-
To test this hypothesis, we evaluated the reactivity of
several NHS-activated acrylates featuring different substitu-
ents at the b-position (1a–f), against a model peptide, laminin
fragment, that comprises a N-terminal Cys residue. As shown
in Scheme 2, while b-phenyl substituted NHS-acrylate (1b)
offered an incomplete reaction, both unsubstituted NHS-
acrylate (1a) and NHS-acrylates 1c–1 f with different
b-amide substituent profiles, generated very efficiently
a 1,4-thiazepan-5-one unit at the peptide N-terminal residue
MS) and theoretical (DFT calculations) methods (Scheme 3).
For this purpose, we first monitored the reaction of Cys 5 with
1
1c by H NMR in deuterated KPi buffer at pD 7.0, at 19 mM
and 218C and by ESI-MS direct injection in ammonium
acetate 20 mM, pH 7.0, at 25 mM and 378C (Supporting
Information, Sections 5.1 and 5.2, Figures S9–S20). Under
both experiment conditions, the reaction generated the
expected heterocycle 7c as the major product (Scheme 3A).
N-acetyl Cys 8, under the same reaction conditions,
afforded the 1,4-addition product 9c (Scheme 3B) as the
major product along with the thioester product S2 (Support-
ing Information, Section 5.1), while methionine 10 slowly
reacted to produce amide 11c (Scheme 3C). These results
suggest that the heterocycle formation is initiated by the thiol-
Michael addition that is followed by the intramolecular
amidation step. DFT calculations support the formation of
the 1,4-thiazepan-5-one product via a thiol-Michael addition-
intramolecular amidation sequence with energy barriers of 26
À1
and 12 kcalmol , respectively. In comparison to this favored
sequence, the calculated energy barriers associated with an
alternative mechanism involving a thioester formation,
S-to-N transfer and intramolecular thio-Michael addition
À1
sequence are higher (31, 34 and 48 kcalmol , respectively).
Furthermore, this second proposed mechanism was discarded
as it would result in the preferential formation of a six- over
seven-membered heterocyclic ring, which was not observed
experimentally (calculated energy barrier of 30 vs. 48 kcal
À1
mol for a 6-exo-trig vs. 7-endo-trig cyclization, Supporting
Information, Section 5.3, Figures S21 and S22). Considering
these results, we then studied the reaction kinetics by
designing a non-fluorescent coumarin NHS-activated acryl-
amide 1g that turns-on upon saturation of the conjugated
[10]
double bond (Scheme 4). The reaction of Cys with 1g under
second order conditions, resulted in an emission at lmax
4
93 nm of 7g product that enabled to establish a rate constant
3
À1 À1
of k = 1.54 Æ 0.18 ” 10 M
s
for the cyclization (Support-
2
ing Information, Section 6, Figures S23–S33, Table S1).
Scheme 2. Screening of NHS-activated acrylates 1a–1 f in the reaction
with the Laminin fragment and the respective ESI-MS spectra (750–
NHS-activated esters are known to cross react with
+
[1b]
different amino acid nucleophilic side chains.
Therefore,
1
250 m/z) at 5 min reaction time. Conditions: Laminin in ammonium
we performed the evaluation of the chemoselectivity of NHS-
activated acrylamides in a series of experiments. First, 1c was
incubated with an equimolar mixture of Cys and Lys amino
acids to test the cross reactivity of the bifunctional reagent
acetate (20 mM, pH 7.0), TCEP (3 equiv), NHS-activated acrylic deriv-
+
atives (1a–1 f, 3–10 equiv), 10 mM, 378C. [Mconj3a-3f+H] is the proton-
ated molecular ion of the corresponding 7-substituted-3-laminin-1,4-
thiazepan-5-one products 3a–3 f.
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residues using the calcitonin peptide 12. This 32-
amino acid hormone presents a disulfide bond,
bridging the N-terminal Cys and in-chain Cys
(Cys-7). After reducing the disulfide bond with
TCEP and decreasing the buffer pH to 4.5, the
sequential addition of NHS-activated acrylamide
1
c and PEG-maleimide 14 resulted in the dual
site-selective functionalization of calcitonin (prod-
uct 15) (Scheme 5; Supporting Information, Sec-
tion 9.5.2, Figures S56 and S58). The selectivity of
NHS-acrylates towards the N- terminal Cys posi-
tion of calcitonin peptide could be confirmed by
all-ion-fragmentation (AIF, Supporting Informa-
tion, Figures S57 and S59). This observation was
further corroborated in the competition experi-
ment between Cys (5) and N-acetylcysteine (8) in
the presence of 1c, to provide 7c with 98%
selectivity over N-acetylated related products
Scheme 3. Mechanistic investigations of the 1,4-thiazepan-5-one using model sub-
strates. A) Calculated TS and corresponding DG in water (kcalmol ) at the M06-
À1
2
X/Def2TZVPP//M06-2X/6-31G(d) level of theory using Cys-Me ester as substrate.
(Scheme 6; Supporting Information, Section 9.4,
Conditions: 1c (1.2 equiv) in KPi (50 mM), pD 7.0/[D ]DMSO (4:1), 218C and:
6
Figures S50–S53). This selectivity is possibly
caused by a synergistic effect of the proximity of
thiol to the N-terminus amine which presents
A) cysteine, 5:1 ratio (1,4-thiazepan-5-one (7c):thioester S2 (Supporting Information,
Figure S9); B) N-acetyl Cys, 4:1:2 ratio (9c:9c-COOH:9c–thioester); C) methionine,
4
:2:1 ratio (1c:11c:1c hydrolyzed). Products observed by NMR spectroscopy (KPi
À50 mM pD 7.0) at 5 min reaction time (Supporting Information, Section 5.1).
a lower pK , enhancing the reactivity of this
a
[12]
particular position.
In these sets of competitive experiments, NHS-
activated acrylamides demonstrated to be selective
towards the functionalization of the N-terminal
Cys. Based on these results, we questioned if these
reagents could also bridge in-chain or C-terminal
Cys and nearby Lys residues.
Amino-sulfhydryl stapling reaction
To study this macrocyclization, peptides with
a C-terminal Cys and an internal Lys residue
spaced by one, two or three glycine (Gly) residues
(peptides 16–18) were synthesized and reacted
with 1 f. As shown in Scheme 7, the stapling
reaction proceeded as expected and the macro-
cycles were isolated and characterized by
Scheme 4. Kinetic studies for 1,4-thiazepan-5-one formation. A) Coumarin NHS-
activated acrylamide 1g fluorescence turn-on after reacting with Cys. Reactions were
performed in second order conditions (equimolar amounts (10 mM) of coumarin 1g
and Cys) in KPi 50 mM, pH 7.0, 238C. B) Full emission spectra (420–600 nm) traced
1
H NMR, TOCSY-NMR and HRMS (Supporting
Information, Section 10, Figures S60–S77). These
results were further supported by the reaction of
1 f with a peptide with an acetylated Lys residue
every 10 s for 900 s (l =373 nm). C) Emission increase (lmax 493 nm) vs. time (s).
ex
(peptide S1), in which the macrocycle was not
formed (Supporting Information, Section 10.2,
with these two nucleophilic residues. This competition experi-
ment afforded a 99:1 mixture of the 1,4-thiazepane-5-one 7c
and the Lys amidation product S5 (Supporting Information,
Section 9.1, Figures S43–S45). The reaction of NHS-activated
acrylamide 1c with Cys-bombesin 35, that contains a histidine,
or with C-Ovalbumin S7, that exhibits serine and a Lys
residues, generated site-selectively the 1,4-thiazepane-5-one
at the N-terminal Cys (conjugates S6 and S8, Supporting
Information, Sections 9.2 and 9.3, Figures S46–S49, Tables S3
Figures S78–S82). With the isolated macrocyclized products
we could assess the efficiency of macrocyclization reaction by
determining the LC-MS yields under bioconjugation condi-
tions, in ammonium acetate 20 mM, pH 7.0, at diluted
conditions (10 mM) and 258C. As shown in Scheme 7, the
reaction of 1 f with peptide 16, with one Gly between the
amino and sulfhydrylꢀs groups, was slightly less efficient than
the reactions with peptides 17 and 18, featuring two and three
Gly spacers, respectively. This can be explained with stereo
constraints imposed by the shorter peptide spacer, that
difficult the amidation step (Supporting Information, Sec-
tion 10.3, Figures S83–S85, Tables S5–S7).
[1b, 11]
and S4).
Then, the reactivity profile of NHS-activated acrylamides
was tested in the presence of N-terminal and in-chain Cys
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Orthogonal dual-modification of peptides
Once the amino-sulfhydryl stapling at the
N-terminal and in-chain sites was established, we
tested if these reagents could also engage in the
orthogonal functionalization of well-defined pep-
tide 22 featuring a N-terminal Cys, a C-terminal
Cys and an internal Lys residue. This evaluation
was initiated with the sequential addition of 1c
1.2 equiv) and maleimide 23 to the peptide. As
shown in Scheme 9, the preference for the
N-terminal modification over the macrocycliza-
tion enabled the straightforward installation of
the 1,4-thiazepan-5-one unit (product S12) and
the subsequent alkylation of the C-terminal Cys
with the maleimide (conjugate 24, Supporting
Information, Section 11, Figure S86). We then
tested if the macrocyclization could also be
operated in a similar protocol. Considering the
observed preference for the 1,4-thiazepan-5-one
formation, we anticipated that to favor the macro-
cyclization pathway, this mechanism would have
to be inhibited, namely by using a N-terminal
reversible protection group like a boronated
(
Scheme 5. Dual site-selective functionalization of Calcitonin salmon 12 with NHS-
activated acrylamide 1c and maleimide 14. Conditions: A) Calcitonin in ammonium
acetate (20 mM, pH 4.5), TCEP (10 equiv), 1c (5 equiv), 10 mM, 378C, at 5 min;
[4a]
thiazolidine.
Due to the hydrolysis of 1c to
B) maleimide 14 (10 equiv).
the corresponding unreactive acid (t1/2 = 27 min,
Supporting Information, Sections 6.4 and 7, Fig-
ures S30–S34, Table S2) and the reversible nature
of boronated thiazolidine, the addition of malei-
mide 23 allowed to intercept the free N-terminal
Cys and to generate the targeted well-defined
conjugate 28. These complementary strategies
offered control over the selective incorporation of
a maleimide and an NHS-activated acrylamide
orthogonally at different sites.
Scheme 6. Competition assay of Cys versus N-acetyl Cys reaction with NHS-
activated acrylamide 1c. Conditions: Cys (5) (2 equiv) and N-acetyl Cys (8) (2 equiv)
in ammonium acetate (20 mM, pH 7.0), 1c (1 equiv), 50 mM, RT, at 5 min. The
product ratio was calculated based on the extracted ion current (EIC) chromatogram
of LC-HRMS analysis to the reaction crude, showing that 1,4-thiazepane-5-one 7c
was the major product detected by LC-MS. The selectivity is further corroborated by
comparison of HPLC UV traces and TIC of the competition assay with control
experiments (Figures S52 and S53).
As abovementioned, at pH 4.5 we could
selectively functionalize the N-terminal Cys with
1
c in calcitonin salmon peptide 12. However,
conducting the reaction at pH 7.0 and sequen-
tially adding 1c (10 equiv), we obtained the dual
modified product 32 with a 1,4-thiazepan-5-one at
the N-terminal Cys and a macrocycle between
Cys-7 and Lys-11 (Scheme 10, conditions A).
More importantly, taking advantage of the rela-
After confirming the possibility to perform the macro-
cyclization in model peptides, the methodology was applied in
the functionalization of the antinucleolin F3 peptide 29
modified with a C-terminal Cys. Incubation of this 32-amino
acid sequence with 1.5 equiv of NHS-acrylate 1 f resulted in
the formation of a mono-modified conjugate 30 that was
isolated by semi-preparative HPLC in 89% yield (Scheme 8;
Supporting Information, Section 12, Figures S88–S90). The
conjugate digestion with trypsin indicated that the stapling
occurred between the C-terminal Cys and the adjacent Lys
tively fast hydrolysis of NHS-activated acrylamides to gen-
erate the unreactive acid functions, we succeeded in orthog-
onally modifying calcitonin peptide with two distinct NHS-
activated acrylamides (dual-modified conjugate 33). This
stepwise protocol involved the selectively modification of the
N-terminal Cys with 1c at pH 4.5 to generate conjugate 13
(Scheme 10, conditions B), followed by a macrocyclization
reaction at pH 7, between Cys-7 and Lys-11 using a different
NHS-activated acrylamide 1j (Scheme 10, conditions C). The
identification of these modifications was confirmed by trypsin
digestion and by AIF fragmentation (Supporting Informa-
tion, Section 13, Figures S92–S97, Tables S9 and S10).
+
residue (m/z [M+H] = 437.1853) (Supporting Information,
Figure S91, Table S8).
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Applications of NHS-activated acrylamides
Considering the observed level of control offered by the
NHS-activated acrylamides in the modification of peptide
chains, we envisioned that the bis(2,5-dioxopyrrolidin-1-yl)
fumarate 34 could further improve the versatility of this
platform by permitting the sequential derivatization with
nucleophilic amines. To test this protocol, 34 was readily
prepared and functionalized with the cytotoxic drug doxor-
ubicin, a PEG unit and a dansyl fluorescent probe, as
representatives of nucleophilic amines widely used in the
assembly of bioconjugates. These reactions rapidly generated
Scheme 7. Macrocyclization assays in model peptides with 1 to 3 Gly
spacers between in-chain Lys and C-terminal Cys model peptides 16–
1
8. a) yield determined by LC-MS (reactions performed at 10 mM) and
isolated yields from reactions performed at a concentration of 250 mM
21% À19; 7% À20; 35% À21).
(
Scheme 9. Orthogonal modification of model peptide 22. Conditions:
A) selective modification of N-terminal Cys with 1c (1.2 equiv) in
ammonium acetate (20 mM, pH 7.0), 10 mM, RT, TCEP (10 equiv),
followed by the addition of maleimide 23 to the C-terminal Cys to
afford conjugate 24; B) selective modification of N-terminal Cys with
2
5 (10 equiv) in ammonium acetate (20 mM, pH 7.0), 10 mM, RT,
Scheme 8. Selective cyclization of F3 peptide with NHS-activated
acrylamide 1 f and HRMS of the trypsin digested fragment. Conditions:
F3 peptide in ammonium acetate (20 mM, pH 7.0), with 1 f
TCEP (10 equiv), at 5 min; C) formation of the macrocycle by addition
of NHS-activated acrylamide 1c (15 equiv), at 5 min; D) selective
modification of N-terminal Cys with maleimide 23 (10 equiv), at 24 h
to afford conjugate 28.
(1.5 equiv), 10 mM, 378C, 2 h.
1
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examined the distances from all Lys residues to
the reactive ester. The two possible diastereomers
resulting from the attack of Cys-34 to the
acrylamide moiety were also studied. Interesting-
ly, in both conjugates, Lys-136 showed, in average,
the lower distance to the reactive center in good
agreement with the experimental data. (Support-
ing Information, Section 16, Figure S116). Addi-
tional MD simulations were then performed on
BSA 40j conjugate derivative displaying a (R)
configuration at the new stereocenter generated
in the reaction (Scheme 13). According to the
simulations, the average root-mean-square devia-
tion (RMSD) values of the peptide backbone is
.08 Š. The calculations performed on this system
revealed that the installation of 1j scaffold does
not induce any relevant structural modification on
the protein. Therefore, we used this fluorescent
construct in the labelling of microglia cells. These
cells are the resident immune cells of the central
nervous system which, among different surveil-
lance duties, protect the brain from foreign
proteins. BSA is typically not present in a healthy
brain, therefore microglia cells phagocyte this
protein, promoting its degradation in the lyso-
4
[15]
somes.
Based on this mechanism, the BSA-
Scheme 10. N-terminal and dual site-selective functionalization of Calcitonin salmon
2 with NHS-activated acrylamides. Conditions: A) calcitonin in ammonium acetate
20 mM, pH 7.0), TCEP (10 equiv), 1c (10 equiv), 10 mM, 378C, at 5 h. B) Calcitonin
in ammonium acetate (20 mM, pH 4.5), TCEP
10 equiv), 1c (5 equiv), 10 mM, 378C, at 5 min; C) pH
adjustment to 7.0 with NH (10%), 1j (10 equiv),
dansyl conjugate 40j was studied in the presence
of microglia cells, and confocal microscopy
1
(
(
3
1
0 mM, 378C, 1 h.
the targeted NHS-activated acrylamides, which
were then used without any purification step in
the functionalization of Cys-bombesin 35, laminin
2
and F3 peptide 29 to prepare the bioconjugates
depicted in Scheme 11 in high conversions (Sup-
porting Information, Section 14, Figure S98–
S110, Tables S11–S15).
Finally, we performed the amino-sulfhydryl
stapling in BSA 39, a 66 kDa protein, featuring 59
Lys residues and a single free sulfhydryl group of
Cys residue (Cys-34, Scheme 12). Remarkably,
the conjugation of BSA with 4 equiv of 1j or
2
equiv of 1g produced a mixture containing
mostly the mono-modified conjugates 40g and
0j, along with unmodified BSA 39 and di-
4
modified conjugates S21g and S21j, indicating,
to some extent, cross reactivity with a Lys residue.
This solution was then subjected to trypsin
digestion, which indicated that the stapling
occurred between BSA Cys-34 and Lys-136
Scheme 11. Derivatization of bis(2,5-dioxopyrrolidin-1-yl) fumarate 34 platform with
different payloads (doxorubicin S17, PEG-NH S19, dansyl S20) for peptide function-
2
alization. Conditions: first step—nucleophilic amine and 34 in ACN, RT, 5 min at
À1
equimolar amount. Second step—peptide fresh solution (1 mgmL ) in ammonium
(
Supporting Information, Section 15, Fig-
acetate (20 mM, pH 7.0), crude mixture of NHS-activated acrylamides 1h–1j
ures S111–S115).
(1.2 equiv), 10 mM, RT, at 5 min. The peptides’ conversions (35 over 99%; 2 over
To explain this result, we performed molec-
ular dynamics (MD) simulations on BSA carrying
the NHS-activated derivative 1j at Cys-34 and
9
9%; 24–89%) were calculated based on the extracted ion current (EIC) chromato-
gram of LC-HRMS analysis and the conjugates were the major products detected by
LC-MS.
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Scheme 12. A) Modification of BSA with fluorescent NHS-activated acrylamides 1j and 1g in ammonium acetate (20 mM, pH 7.0), 5 mM, RT.
B) Deconvoluted ESI mass spectrum of BSA (left) and deconvoluted ESI mass spectrum of the reaction mixture with derivative 1j (right). The
spectrum of BSA exhibits the characteristic main peaks assigned to its native form (66428 Da) and to its principal physiological post-translational
[
13]
modifications. a) BSA 39, b) mono-modified BSA conjugate 40j, c) di-modified BSA with 1j (S21j). Mass spectral deconvolution was performed
[14]
using a Zscore algorithm in MagTran1.03 software. C) Modification of BSA with dansyl-NHS-activated acrylamide 1j (2 equiv added twice with
h intervals) in ammonium acetate (20 mM, pH 7.0), 5 mM, RT; and modification of BSA with coumarin-NHS-activated acrylamide 1g (2 equiv)
1
in KPi (50 mM, pH 7.0), 5 mM, RT; respective observed conversions.
images were recorded at 1 h and 24 h after incubation. The
experiments clearly support a time dependent uptake of this
conjugate and its accumulation in the lysosomes. These results
indicate that this functionalization strategy can be a valuable
tool for designing well-defined fluorescent BSA
conjugates for lysosome imaging in microglia
cells.
Conclusion
In summary, NHS-activated acrylamides were
shown to be simple and very versatile bioconju-
gation handles that permit the amino-sulfhydryl
stapling in peptides and proteins. The faster
reaction kinetics observed for N-terminal Cys
over other nucleophilic amino acid residues,
including in-chain Cys, allowed the selective
dual-modification of different Cys residues.
Moreover, NHS-activated acrylamides were also
successfully applied on in-chain or C-terminal Cys
stapling to nearby Lys residues. This bioconju-
gation strategy involving sustained product sta-
bility and fast kinetics under biocompatible con-
ditions, good to full conversion to the desired
bioconjugate, is extremely promising in view of
applications in biology or medicine. These bifunc-
tional reagents can be easily prepared with
diverse and complex payloads requiring minimal
Scheme 13. A) Representative snapshot derived from 0.5 ms MD simulations of BSA-
j. Derivative 1j linked to Cys-34 and Lys-136 is shown in blue and the protein as
a white surface. B) Confocal microscopy images of CHME3 microglial cells after
manipulation for late-stage bioconjugation of
peptides. Finally, the same macrocyclization strat-
egy was applied to BSA protein by stapling free
Cys-34 to the nearest Lys-136 as corroborated by
MD simulations. The fluorescent BSA-dansyl
conjugate was used to label lysosomes in micro-
glia cells. Overall, the versatility demonstrated by
NHS-activated acrylamides in the modification of
1
incubation with BSA-dansyl conjugate 40j (5 mM diluted in serum-free culture media
(green)) for 1 h and 24 h. 4’,6-diamidino-2-phenylindole (DAPI (blue)) was used for
nucleus labelling after 3 min of incubation. C) Quantitative analysis of BSA-dansyl
conjugate 40j colocalization with microglial lysosomes. After each time point, cells
were stained with Lysotracker (yellow), for 30 min, at 378C, to observe lysosomes
and assess the respective colocalization.
1
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Angewandte
Research Articles
Chemie
peptide chains by stapling amino-sulfhydryl groups enlarges
the chemical biology toolbox for the construction of func-
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Keywords: Bioconjugation · esters · macrocyclization ·
NHS-Activated-Acrylamides · stapling
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