Design and synthesis of cysteine-specific labels for photo-crosslinking studies

Article abstract of DOI:10.1039/C8RA10436K

Chemical cross-linking mass-spectrometry (XL-MS) represents a powerful methodology to map ligand/biomacromolecule interactions, particularly where conventional methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (EM) are not feasible. In this manuscript, we describe the design and synthesis of two new photo-crosslinking reagents that can be used to specifically label free thiols through either maleimido or methanethiosulfonate groups and facilitate PXL-MS workflows. Both crosslinkers are based on light sensitive diazirines-precursors of highly reactive carbenes which offer additional advantages over alternative crosslinking groups such as benzophenones and aryl nitrenes given the controlled rapid and more indiscriminate reactivity.

Full text of DOI:10.1039/C8RA10436K

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Design and synthesis of cysteine-specific labels for
photo-crosslinking studies†
Cite this: RSC Adv., 2019, 9, 7610
ab
Martin Walko,ab Eric Hewitt, bc Sheena E. Radford bc and Andrew J. Wilson
*
Chemical cross-linking mass-spectrometry (XL-MS) represents a powerful methodology to map ligand/
biomacromolecule interactions, particularly where conventional methods such as X-ray crystallography,
nuclear magnetic resonance (NMR) spectroscopy or cryo-electron microscopy (EM) are not feasible. In
this manuscript, we describe the design and synthesis of two new photo-crosslinking reagents that can
be used to specifically label free thiols through either maleimido or methanethiosulfonate groups and
facilitate PXL-MS workflows. Both crosslinkers are based on light sensitive diazirines – precursors of
highly reactive carbenes which offer additional advantages over alternative crosslinking groups such as
benzophenones and aryl nitrenes given the controlled rapid and more indiscriminate reactivity.
Received 20th December 2018
Accepted 23rd February 2019
DOI: 10.1039/c8ra10436k
rsc.li/rsc-advances
resultant carbenes insert rapidly into X–H bonds (including O–
H, N–H, S–H and C–H bonds).10 Diazirines have been shown to
Introduction
be advantageous over other common cross-linking groups (e.g.
Chemical crosslinking-mass spectrometry (XL-MS) is an
increasingly important approach to map biomacromolecule
and
small-molecule/biomacromolecule
interactions.1–4
Furthermore, XL-MS is uniquely placed to facilitate analysis of
transient and dynamic interactions and/or conformational
changes. Given that cross-linking “covalently traps” a protein or
its complex, XL-MS has the potential to map changes in
conformation and interactions with time. However, to do so
requires reactive intermediates that react rapidly and indis-
criminately with proximal functionality to ensure supramolec-
ular connectivity is accurately captured upon cross-linking.5 In
this regard photoinduced cross-linking (PXL)6 is advantageous
in that light is used to trigger cross-linking and the suite of
functionalities used for PXL tend to be more indiscriminate in
their reactivity than traditional cross-linking reagents7–9 such as
NHS-esters which require nucleophilic residues to be present at
an interface. We recently synthesized two cysteine specic dia-
zirines as PXL reagents and outlined a workow for mapping
protein interactions that exploits tag-and-transfer (Fig. 1a and
b).10 Using a cysteine selective methanethiosulfonate (MTS) or
maleimido group to attach the reagents to the protein allows
precise positioning of the crosslinking functionality within the
protein. The photosensitive diazirines represent ideal func-
tional groups for PXL-MS workows5,11–17 – upon irradiation, the
aSchool of Chemistry, University of Leeds, Leeds, LS2 9JT, UK. E-mail: a.j.wilson@
leeds.ac.uk
Fig. 1 Overview of tag-transfer PXL for mapping PPIs and diazirine based
PXL reagents, (a) tag-transfer PXL workflow: a thiol-containing “bait” protein
(green) is labelled with the reagent (here MTS-diazirine). After adding
a partner protein (blue), irradiation with 365 nm UV light, reveals a carbene
that can react with the partner protein. Reduction of the disulphide reveals
a thiol which can be further labelled, (b) previously described diazirine based
PXL reagents (c) diazirine based PXL reagents described in this study.
bAstbury Centre for Structural Molecular Biology, University of Leeds, Leeds, LS2 9JT,
UK
cSchool of Molecular and Cellular Biology, Faculty of Biological Sciences, University of
Leeds, Leeds, LS2 9JT, UK
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c8ra10436k
7610 | RSC Adv., 2019, 9, 7610–7614
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
benzophenone and phenyl azide) arising as a consequence of
This reagent could in principle be used to label thiols on
more rapid and indiscriminate reactivity leading to effective proteins and exploited in PXL-MS (Fig. 2b). A difference in using
encoding of supramolecular connectivity.18 However the this reagent in contrast to the tag-and-transfer reagents
synthesis of dizarines is more challenging, limiting their use.
described previously is that following cross-linking, a perma-
In our prior work, we introduced two heterobifunctional nent covalent link between bait and partner proteins will be
reagents (MTS-diazirine 1 and MTS-TFMD 2) (Fig. 1b) bearing generated, which may be advantageous under conditions where
a methanethiosulphonate group to facilitate specic labelling the former reagents are unstable (e.g. the reducing environment
of Cys residues on a “bait” protein, creating a cleavable disulde of the cell).
bond bearing a diazirine.10 Following cross-linking with partner
The second reagent has been designed to be used as a tag-
proteins, this disulde could be reduced leaving a thiol on the and-transfer diazirine, but with an additional biorthogonal
partner protein. In this work we describe the design and alkyne. Consequently, the synthesis was longer, however gram
synthesis of two further reagents; (i) N-maleimido-diazirine 3 quantities could be prepared. Beginning with ethyl acetoacetate
and (ii) MTS-alkynyldiazirine 4 (Fig. 1c). N-Maleimido-diazirine 8, alkylation with 3-bromopropyne 9 and protection of the
3 can be used to label thiols on peptides or proteins via ketone 10 as the cyclic acetal 11, followed by reduction of the
a conventional conjugate addition, used widely in protein ester to alcohol 12 23 and removal of the acetal afforded the
labelling.19 MTS-alkynyldiazirine 4 can be used to label thiols on hydroxyl ketone substrate 13 for diazirine ring formation. As
peptides or proteins, yet bears an additional bio-orthogonal before, the reaction of the ketone with hydroxylamine-O-
group (the alkyne) that could be exploited to introduce further sulfonic acid in liquid ammonia followed by oxidation with
functionality (e.g. uorophore, biotin) by “click” chemistry20,21 iodine gave diazirine 14.24 Subsequent conversion of the alcohol
to support chemical proteomics workows.
14 to an alkyl iodide 15 and reaction with sodium meth-
anethiosulfonate afforded the nal tag-transfer MTS-
alkynyldiazirine 4 (Fig. 3).
Results and discussion
This label could be used in tag and transfer PXL-MS appli-
cations in the same way as our previously described workow.10
Labelling of bait protein followed by photocrosslinking and
reduction, transfers onto a partner protein a thiol group, but
also an alkyne. We envision this second cysteine-specic label
as being of use for the enrichment of cross-linked proteins
where multiple thiols are already present in the partner protein
or other components of the sample under analyses; the
We rst prepared an alkyl diazirine based reagent that could be
used to label proteins via Michael addition of a thiol to a mal-
eimide. This bioconjugation reaction is widely used19 and trivial
to perform for non-specialists whilst avoids the use of more
hazardous and less chemoselective alkylating reagents e.g. a-
halocarbonyl containing or iodoalkyl reagents. N-Maleimido-
diazirine 3 was prepared in two steps (Fig. 2a) from 4-
hydroxybutan-2-one 5. One pot introduction of the diazirine
group to give compound 6 22 was achieved via iodine mediated
oxidation of the diaziridine that results from reaction of the
ketone hydroxylamine-O-sulfonic acid in liquid ammonia. This
was followed by Mitsunobu reaction with N-maleimide 7, to
yield the labelling reagent in 43% over two steps.
Fig. 3 Synthesis and use of MTS-alkynyldiazirine (a) synthesis (b) utility
for PXL workflows; a thiol on a protein can be used to displace the MTS
Fig. 2 Synthesis and use of N-maleimido diazirine (a) synthesis (b) group following which, introduction of partner proteins and excitation
utility for PXL workflows; a thiol on a protein can be used to react with generates a cross-link via the reactive carbene, then the disulphide can
the N-maleimide following which introduction of partner proteins and be reduced resulting in the transfer of a thiol and biorthogonal alkyne
excitation generates a permanent cross-link via the reactive carbene. onto the partner protein.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 7610–7614 | 7611

View Article Online
RSC Advances
Paper
biorthogonal advantages of the alkyne should be readily dH (400 MHz, CDCl3) 1.08 (s, 3H, CH3), 1.45 (brs, 1H, OH), 1.64
exploited. Alternatively, the alkyne could be used to ligate e.g. (t, 2H, J ¼ 6.3 Hz, CH2), 3.54 (t, 2H, J ¼ 6.3 Hz, CH2).
uorophores for the construction of biosensors.25
1-(2-(3-Methyl-3H-diazirin-3-yl)ethyl)-1H-pyrrole-2,5-dione (3)
Diisopropyl azodicarboxylate (2.4 mL, 12 mmol) was added
dropwise to a solution of alcohol 6 (1 g, 10 mmol), Ph3P (2.885 g,
Conclusions
In summary, we have described the design and synthesis of two
new diazirine based cross-linking reagents that can be used to
specically label free thiols. These labels could be applied in
structural analyses of protein–protein interactions using PXL-
MS by conjugating them to bait peptides or proteins and
subsequent crosslinking with partner proteins. In principle,
such a strategy could readily be applied to other ligand/
biomacrolecule interactions as long as a thiol is present in the
bait ligand.
11 mmol) and maleimide 7 (1.067 g, 11 mmol) in THF (30 mL) at
0 ꢁC. Aer stirring the mixture for 16 h at room temperature, the
solvent was evaporated and the residue was triturated with
mixture of 15 mL Et2O and 15 mL of hexane and ltered. The
ltrate was concentrated and puried by column chromatog-
raphy (SiO2, hexane/EtOAc 4/1 to 2/1) to give 1.52 g (85%) of
product 3 as a colourless oil. dH (400 MHz, CDCl3) 1.06 (s, 3H,
CH3CN2), 1.60 (t, 2H, J ¼ 7.1 Hz, CH2CN2), 3.57 (d, 2H, J ¼
7.1 Hz, CH2N), 6.73 (s, 2H, CH ¼ CH); dC (100 MHz, CDCl3)
19.28, 24.04, 33.33, 33.51, 134.41, 170.59; ESI-HRMS found m/z
202.0575 [M + Na]+ C8H9N3NaO2 requires 202.0592. IR: nmax
/
Experimental section
General considerations
cmꢀ1 (oil) ¼ 1699, 1593, 1443, 1406, 1387, 1364, 1310, 825, 693;
UV-vis: lmax(3) (CH3CN) ¼ 216 (14 100), 301 (607), 360sh (60).
All solvents were purchased from Fisher Scientic and all
reagents were purchased from Sigma-Aldrich or Fluorochem
unless otherwise stated and used without further purication.
Purication by column chromatography was carried out using
silica gel. Analytical thin layer chromatography (TLC) was con-
ducted using Merck 0.25 mm silica gel pre-coated aluminium
plates with uorescent indicator active at UV245. 1H and
13CNMR spectra were acquired on Bruker Avance III HD 400
Ethyl 3-oxohept-6-ynoate (10)23
n-BuLi (100 mL of 1.6 M solution in hexanes, 160 mmol) was
added to a solution of N,N-diisoproꢁ pylethylamine (22.5 mL, 160
mmol) in dry THF (150 mL) at 0 C under N2. Aer 30 min of
stirring, ethyl acetoacetate 8 (10.1 mL, 80 mmol) was slowly
added and aer another 30 min propargyl bromide 9 (7.1 mL,
80% solution in toluene, 80 mmol) was added to the reaction
mixture at 0 ꢁC under N2. Aer stirring for additional 1 h under
the same conditions, AcOH (9.2 mL, 160 mmol) was added to
quench the reaction, followed by water (100 mL). The organic
layer was separated and water layer was extracted with ether (2
ꢂ 100 mL). The combined organic layers were washed with
brine (100 mL), dried over MgSO4 and concentrated in vacuo.
Target material was puried by column chromatography (SiO2,
hexane/ethyl acetate 4/1 to 2/1) to give 9.81 g (73%) of product as
a colourless oil. dH (400 MHz, CDCl3) 1.26 (t, 3H, J ¼ 7.2 Hz,
CH3), 1.94 (t, 1H, J ¼ 2.6 Hz, C^CH), 2.45 (dt, 2H, J ¼ 7.2, 2.6 Hz,
CH2C^C), 2.79 (t, 1H, J ¼ 7.2 Hz, CH2CH2CO), 3.44 (s, 2H,
CH2COOEt), 4.18 (q, 2H, J ¼ 7.2 Hz, CH2CH3).
1
series spectrometer at 400 MHz for H, and 100 MHz for 13C.
Chemical shis are expressed as parts per million using solvent
1
as internal standard (CDCl3 7.26 ppm in H and 77.16 ppm in
13C spectra) and coupling constants are expressed in Hz. The
following abbreviations are used: s for singlet, d for doublet, t
for triplet, q for quartet, m for multiplet and br for broad.
2-(3-Methyl-3H-diazirin-3-yl)ethanol (6)22
NH3 (approx. 100 mL) was condensed, using a dry ice/acetone
condenser, into a ask containing 4-hydroxy-2-butanone 5
(15.2 mL, 170 mmol) and cooled to ꢀ78 ꢁC. Aer reuxing
(ꢀ30 ꢁC bath temperature) for 5 h, hydroxylamine-O-sulfonic
acid (21.15 g, 187 mmol) dissolved in MeOH (150 mL) was
added at ꢀ78 ꢁC and the reaction mixture was allowed to heat to
Ethyl 2-(2-(but-3-yn-1-yl)-1,3-dioxolan-2-yl)acetate (11)23
room temperature overnight. The resulting mixture was ltered, Ethyl 3-oxohept-6-ynoate 10 (9.50 g, 56.5 mmol) was dissolved in
the solid residue was washed with MeOH (2 ꢂ 20 mL), and the the mixture of ethylene glycol (12.6 mL, 226 mmol) and triethyl
ltrate was concentrated to about 100 mL. Triethylamine (26 orthoformate (18.8 mL, 113 mmol). p-Toluenesulfonic acid
mL, 187 mmol) was added to the resulting solution followed by monohydrate (1.08 g, 5.7 mmol) was added and the resulting
iodine in several portions while cooling the reaction mixture in solution was stirred at rt for 16 h, then sat. aq. NaHCO3 (50 mL)
ice. Aer adding 29.2 g (115 mmol) of I2, the colour of iodine was added and the mixture was extracted with ether (3 ꢂ 100
persisted, indicating the end of the reaction. The solvents were mL). The combined organic layers were washed with brine (100
carefully removed from the reaction mixture (25 ꢁC and 120 mL), dried over MgSO4 and concentrated in vacuo. Target
mbar) and the residue was partitioned between Et2O (200 mL) material was puried by column chromatography (SiO2, hexane/
and brine (200 mL) containing sat. aq. Na2S2O3 (10 mL). The ethyl acetate 4/1 to 2/1) to give 7.56 g (63%) of product as
organic layer was separated and the aqueous layer was extracted a colourless oil. dH (400 MHz, CDCl3) 1.26 (t, 3H, J ¼ 7.2 Hz,
with Et2O (2 ꢂ 100 mL). The combined organic extracts were CH3), 1.92 (t, 1H, J ¼ 2.6 Hz, C^CH), 2.11 (t, 2H, J ¼ 7.9 Hz,
dried over Na2SO4 and concentrated to give crude diazirine 6, CH2CH2C^C), 2.27–2.32 (m, 2H, CH2C^C), 2.64 (s, 2H, CH2-
which was puried by column chromatography (SiO2, pentane/ COOEt), 3.94–4.03 (m, 4H, OCH2CH2O), 4.15 (q, 2H, J ¼ 7.1 Hz,
Et2O 1/1) to give 8.64 g (51%) of product as a colourless liquid. CH2CH3).
7612 | RSC Adv., 2019, 9, 7610–7614
This journal is © The Royal Society of Chemistry 2019

View Article Online
Paper
RSC Advances
2-(2-(But-3-yn-1-yl)-1,3-dioxolan-2-yl)ethanol (12)23
3-(But-3-yn-1-yl)-3-(2-iodoethyl)-3H-diazirine (15)25
Ethyl 2-(2-(but-3-yn-1-yl)-1,3-dioxolan-2-yl)acetate 11 (7.5 g, 35.3 Iodine (305 mg, 1.2 mmol) was added to a solution of Ph3P
mmol) in dry ether (20 mL) was added to a mixture of LiAlH4 (315 mg, 1.2 mmol) and imidazole (163 mg, 2.4 mmol) in
ꢁ
(1.47 g, 38.8 mmol) in dry ether (200 mL) at 0 C. The reaction dichloromethane (5 mL) at 0 ꢁC. Aer 15 min stirring, the
mixture was stirred for 2 h at room temperature and then slowly alcohol 14 (138 mg, 1.0 mmol) in dichloromethane (1 mL) was
quenched with water (100 mL). The organic layer was separated added and the mixture was stirred for 4 h at room temperature.
and water layer was extracted with ether (2 ꢂ 100 mL). The Water (10 mL) was added to the reaction mixture, organic layer
combined organic layers were washed with brine (100 mL), was separated and aqueous layer was extracted with dichloro-
dried over MgSO4 and concentrated in vacuo. The resulting methane (2 ꢂ 10 mL). The combined organic extracts were
material was puried by column chromatography (SiO2, hexane/ dried over MgSO4 and concentrated. The residue was puried by
ethyl acetate 4/1) to give 5.07 g (84%) of product as a colourless column chromatography (SiO2, hexane/Et2O 10/1) to give
oil. dH (400 MHz, CDCl3) 1.90–1.95 (m, 5H, 2ꢂ CH2, C^CH), 180 mg (73%) of product as a colourless oil. dH (400 MHz,
2.23–2.29 (m, 2H, CH2C^C), 2.66 (brs, 1H, OH), 3.71–3.77 (m, CDCl3) 1.67 (t, 2H, J ¼ 7.2 Hz, CH2CH2C^C), 1.99–2.04 (m, 3H,
2H, CH2OH), 3.94–4.03 (m, 4H, OCH2CH2O).
CH2C^CH), 2.11 (t, 2H, J ¼ 7.6 Hz, CH2CH2I), 2.88 (t, 2H, J ¼
7.6 Hz, CH2I); dC (100 MHz, CDCl3) ꢀ3.87, 13.37, 28.79, 31.94,
37.63, 69.57, 82.53.
1-Hydroxyhept-6-yn-3-one (13)24
2-(2-(But-3-yn-1-yl)-1,3-dioxolan-2-yl)ethanol 12 (5.00 g, 29.4
mmol) was dissolved in acetone (50 mL), p-toluenesulfonic acid
monohydrate (0.288 g, 1.5 mmol) was added and the resulting
solution was stirred at rt for 3 h. Sat. aq. NaHCO3 (50 mL) was
added to the reaction mixture which was subsequently extracted
with ethyl acetate (3 ꢂ 50 mL). The combined organic layers
were washed with brine (50 ml), dried over MgSO4 and
concentrated in vacuo. The target material was puried by
column chromatography (SiO2, hexane/ethyl acetate 1/1 to pure
ethylacetate) to give 2.56 g (69%) of product as a colourless oil;
(400 MHz, CDCl3) 1.95 (t, 1H, J ¼ 2.7 Hz, C^CH), 2.45 (td, 2H, J
¼ 7.4, 2.7 Hz, CH2C^C), 2.46–2.50 (m, 1H, OH), 2.67–2.70 (m,
4H, 2ꢂ CH2), 3.85 (q, 2H, J ¼ 5.7 Hz, CH2OH).
O-Methyl 2-(3-(but-3-yn-1-yl)-3H-diazirin-3-yl)ethanesulfono-
thioate (4)
Sodium methanethiosulfonate (117 mg, 0.87 mmol) was added
to a solution of iodide 15 (180 mg, 0.73 mmol) in DMF (1 mL)
and the resulting solution was heated at 50 ꢁC for 4 h. Aer
evaporation of the solvent, the residue was puried by column
chromatography (SiO2, hexane/ethyl acetate 3/1) to give 140 mg
(83%) of product as colourless oil. dH (400 MHz, CDCl3) 1.66 (t,
2H, J ¼ 7.2 Hz, CH2CH2C^C), 1.92 (t, 2H, J ¼ 7.6 Hz, CH2-
CH2SSO2), 1.99–2.04 (m, 3H, CH2C^CH), 2.95 (t, 2H, J ¼ 7.6 Hz,
CH2SSO2), 3.31 (s, 3H, CH3SO2); dC (100 MHz, CDCl3) 13.25,
27.35, 30.41, 32.03, 33.46, 50.67, 69.71, 82.19. ESI-HRMS found
m/z 255.0226 [M + Na]+ C8H12N2NaO2S2 expected 255.0238; IR:
nmax/cmꢀ1 (solid state) ¼ 3288, 1588, 1434, 1312, 1128, 954, 743;
UV-vis: lmax(3) (CH3CN) ¼ 346 (51), 360sh (41).
2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethanol (14)24
NH3 (approx. 50 mL) was condensed, using dry ice/acetone
condenser, into a ask containing 1-hydroxyhept-6-yn-3-one
13 (2.50 g, 19.8 mmol) and cooled to ꢀ78 ꢁC. Aer reuxing
(ꢀ30 ꢁC bath temperature) for 5 h, hydroxylamine-O-sulfonic
acid (2.47 g, 21.8 mmol) dissolved in MeOH (20 mL) was added
at ꢀ78 ꢁC and the reaction mixture was allowed to heat to room
temperature overnight. Resulting mixture was ltered, solid
residue was washed with MeOH (2 ꢂ 20 mL), and ltrate was
concentrated to about 20 mL. Triethylamine (3 mL, 21.8 mmol)
was added to the resulting solution followed by iodine in several
portions while cooling the reaction mixture in ice. Aer adding
2.06 g (8.1 mmol) of I2, the colour of iodine persisted, indicating
the end of the reaction. The solvents were removed from the
reaction mixture and residue was partitioned between Et2O (50
mL) and brine (50 mL) containing sat. aq. Na2S2O3 (5 mL). The
organic layer was separated and the aqueous layer was extracted
with Et2O (2 ꢂ 50 mL). The combined organic extracts were
dried over MgSO4 and concentrated to give crude diazirine 14,
which was puried by column chromatography (SiO2, pentane/
Et2O 1/1) to give 0.528 g (20%) of product as a colourless liquid.
dH (400 MHz, CDCl3) 1.57 (brs, 1H, OH), 1.68 (t, 2H, J ¼ 7.4 Hz,
CH2CH2C^C), 1.71 (t, 2H, J ¼ 6.2 Hz CH2CH2OH), 2.00 (t, 1H, J
¼ 2.6 Hz, C^CH), 2.04 (td, 2H, J ¼ 7.4; 2.5 Hz, Hz, CH2C^C),
3.49 (t, 2H, J ¼ 6.2 Hz, CH2OH)
Conflicts of interest
There are no conicts to declare.
Acknowledgements
The EPSRC (EP/N035267/1) are acknowledged for their support.
S. E. R. acknowledges funding from the European Research
Council under the European Union's Seventh Framework Pro-
gramme grant FP7.2007-2013/grant agreement number 322408.
References
1 F. J. O'Reilly and J. Rappsilber, Nat. Struct. Mol. Biol., 2018,
25, 1000.
2 A. Sinz, Angew. Chem., Int. Ed. Engl., 2018, 57, 6390.
3 M. Schneider, A. Belsom and J. Rappsilber, Trends Biochem.
Sci., 2018, 43, 157.
4 A. N. Calabrese and S. E. Radford, Methods, 2018, 147, 187.
5 G. W. Preston, S. E. Radford, A. E. Ashcro and A. J. Wilson,
Anal. Chem., 2012, 84, 6790.
6 G. W. Preston and A. J. Wilson, Chem. Soc. Rev., 2013, 42,
3289.
This journal is © The Royal Society of Chemistry 2019
RSC Adv., 2019, 9, 7610–7614 | 7613

View Article Online
RSC Advances
Paper
7 D. Tan, Q. Li, M. J. Zhang, C. Liu, C. Ma, P. Zhang, Y. H. Ding, 16 L. Dubinsky, B. P. Krom and M. M. Meijler, Bioorg. Med.
S. B. Fan, L. Tao, B. Yang, X. Li, S. Ma, J. Liu, B. Feng, X. Liu, Chem., 2012, 20, 554.
H. W. Wang, S. M. He, N. Gao, K. Ye, M. Q. Dong and X. Lei, 17 J. Brunner, H. Senn and F. M. Richards, J. Biol. Chem., 1980,
eLife, 2016, 5, e12509. 255, 3313.
8 S. Madler, C. Bich, D. Touboul and R. Zenobi, J. Mass 18 G. W. Preston, S. E. Radford, A. E. Ashcro and A. J. Wilson,
Spectrom., 2009, 44, 694. ACS Chem. Biol., 2013, 9, 761.
9 S. Kalkhof and A. Sinz, Anal. Bioanal. Chem., 2008, 392, 305. 19 K. Renault, J. W. Fredy, P.-Y. Renard and C. Sabot,
10 J. E. Horne, M. Walko, A. N. Calabrese, M. A. Levenstein, Bioconjugate Chem., 2018, 29, 2497.
D. J. Brockwell, N. Kapur, A. J. Wilson and S. E. Radford, 20 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem.,
Angew. Chem., Int. Ed., 2018, 57, 16688. 2002, 67, 3057.
11 Y. Yang, H. Song, D. He, S. Zhang, S. Dai, X. Xie, S. Lin, 21 H. C. Kolb and K. B. Sharpless, Drug Discov. Today, 2003, 8,
Z. Hao, H. Zheng and P. R. Chen, Nat. Protoc., 2017, 12, 2147.
12 T. Yang, Z. Liu and X. D. Li, Chem. Sci., 2015, 6, 1011.
13 M. Suchanek, A. Radzikowska and C. Thiele, Nat. Methods,
2005, 2, 261.
1128.
´
22 P. Morieux, C. Salome, K. D. Park, J. P. Stables and H. Kohn,
J. Med. Chem., 2010, 53, 5716.
23 K. Hayakawa, M. Yodo, S. Ohsuki and K. Kanematsu, J. Am.
Chem. Soc., 1984, 106, 6735.
14 J. Liang, L. Zhang, X. L. Tan, Y. K. Qi, S. Feng, H. Deng,
Y. Yan, J. S. Zheng, L. Liu and C. L. Tian, Angew. Chem., 24 Z. Li, P. Hao, L. Li, C. Y. J. Tan, X. Cheng, G. Y. J. Chen,
Int. Ed. Engl., 2017, 56, 2744.
15 C. Iacobucci, M. Gotze, C. Piotrowski, C. Arlt, A. Rehkamp,
S. K. Sze, H.-M. Shen and S. Q. Yao, Angew. Chem., Int. Ed.,
2013, 52, 8551.
C. Ihling, C. Hage and A. Sinz, Anal. Chem., 2018, 90, 2805. 25 I. Hamachi, T. Nagase and S. Shinkai, J. Am. Chem. Soc.,
2000, 122, 12065.
7614 | RSC Adv., 2019, 9, 7610–7614
This journal is © The Royal Society of Chemistry 2019