Design and Synthesis of Ligand-Tag Exchangeable Photoaffinity Probe Utilizing Nosyl Chemistry

Article abstract of DOI:10.1002/ejoc.201901348

Construction of a fluorophore or high-sensitive mass tag on target molecules would promise the facile analysis in photoaffinity labeling. A novel 2-nitrobenzenesulfonyl (nosyl; Ns) diazirine, which exhibits bifunctional properties for efficient photoaffinity labeling, was designed. This strategy highlights the simplicity of the chemical probe and its ability to install identification tags via Meisenheimer complex by S_NAr after photo-labelling. Herein, in this study, we synthesized new Ns diazirine and demonstrated further studies in terms of photoactivation and S_NAr reaction in aqueous condition.

Full text of DOI:10.1002/ejoc.201901348

A Journal of
Accepted Article
Title: Design and synthesis of ligand-tag exchangeable photoaffinity
probe utilizing nosyl chemistry via Meisenheimer complex
Authors: Aimi Suhaily Saaidin, Yuta Murai, Takuya Ishikawa, and Kenji
Monde
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201901348
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10.1002/ejoc.201901348
European Journal of Organic Chemistry
COMMUNICATION
Design and synthesis of ligand-tag exchangeable photoaffinity
probe utilizing nosyl chemistry
Aimi Suhaily Saaidin,[a] Yuta Murai,*[b] Takuya Ishikawa, [a] and Kenji Monde*[b]
Abstract: Construction of a fluorophore or high-sensitive mass tag on
target molecules would promise the facile analysis in photoaffinity
labeling. A novel 2-nitrobenzenesulfonyl (nosyl; Ns) diazirine, which
exhibits bifunctional properties for efficient photoaffinity labeling, was
designed. This strategy highlights the simplicity of the chemical probe
and its ability to install identification tags via Meisenheimer complex
by SNAr after photo-labelling. Herein, in this study, we synthesized
new Ns diazirine and demonstrated further studies in terms of
photoactivation and SNAr reaction in aqueous condition.
temperature, nucleophiles, reducing agents, acidic and basic
conditions. Besides, diazirine is able to generate an active
species carbene to cross-link in a short time with high wavelength
(∼365 nm) which can avoid the damage of target molecules.
Furthermore, due to this high reactivity, carbenes are often quickly
quenched by water, which could be an advantage as it minimizes
non-specific binding5. Despite, the promising characteristics of
phenyl diazirines, identification of the cross-linked target
molecules often involves complexity of reaction mixtures and
resulting in small quantity of the photo-labelled molecules.
Therefore, the photolabeled molecule requires its enrichment by
using biotin-avidin system, fluorous tag6 and clickable group7. In
addition to them, multi-functional crosslinkers having high-
sensitive functions such as scissile8, fluorescence and stable
isotope showed excellent performance9 with multiple steps.
Ideally, analysis of photolabeled molecules should be more direct
and reliable. With regards to this analysis, we report a novel 2-
nitrobenzenesulfonyl (nosyl; Ns) diazirine with scissile function
which can simultaneously exchange various tags by SNAr with an
effort to provide more direct and reliable analysis.
Ns group has often been used for the protection of
secondary amine groups and its deprotection can be specifically
accomplished with high yield by treatment of thiols nucleophilic
aromatic substitution (SNAr) reaction via Meisenheimer complex10.
This interesting mechanistic feature has been applied to the
development of biosensor11 and imaging fluorescent probes for
cysteine residue on peptides12. Furthermore, Fukuyama and co-
workers also have reported on benzophenone based photoaffinity
label probe with nosyl-type biotin tag linker to cleave from avidin
under physiological environment13.
Introduction
Identification of target biomolecules of a bioactive compound has
been invaluable in the field of life science for deeper
understanding of complex biological mechanisms. For these
purposes, some strategies have been implemented such as
genetics approach, 3D structure determination involving solution-
state NMR and X-ray crystallography. Although these approaches
can provide a detailed structural information of target biomolecule,
an abundant purified molecule is always required. As an
alternative, chemical method using affinity-based chemical probe
has also been used for direct identification and visualization of
target biomolecules in complex systems without having to
undergo purification process. Photoaffinity labeling is one of the
powerful chemical approaches used to identify the interaction,
where it can conduct binding site mapping, protein-protein
interaction and visualizes live-cell imaging by photoinduced
covalent bond between ligand and its target molecule1. The
formation of covalent bond facilitates identification of the binding
partner, even if the binding affinity of the ligand is weak. In
addition, photoaffinity labeling is also able to proceed in a native
cellular environment, therefore it is possible to apply in membrane
protein where X-ray crystallography and solution-state NMR
method are difficult to be applied. In designing a photoaffinity
probe, it is important to select which photoreactive group
(photophore) is used for efficient photoaffinity labeling.
Phenyldiazirines, particularly the trifluoromethyl derivative, have
a number of advantages over other commonly employed photo-
cross-linkers such as phenylazides2, benzophenones3 and the
most recently discovered 2-aryl-5-carboxytetrazole4. Typically,
diazirines are stable toward a number of factors such as high
molecules
O2N
CF3
O
O
Ns group
F3C
S
1. photo-
crosslink
O2N
N
HN
N
O
S
diazirine
HN
O
365 nm
substrate
etc…
O
tag
N
H
H
N
N
N
N
HS
B
=
MeO
O
O
F
F
O
high sensitive
mass tag
coumarin
bodipy
F3C
N
O2N
F3C
N
O2N
N
O
S
O
N
HN
S
S
[a]
[b]
Graduate School of Life Science, Hokkaido University
Kita 11 Nishi 21, Sapporo, 001-0021, Japan,
2. tag exchange
(installation)
NH2
Meisenheimer complex
Faculty of Advanced Life Science, Hokkaido University
Kita 21 Nishi 11, Sapporo, 001-0021, Japan
http://altair.sci.hokudai.ac.jp/infchb/
Figure 1. Structure of the Ns-based photo-crosslinker and its functional view.
E-mail: ymurai@sci.hokudai.ac.jp, kmonde@sci.hokudai.ac.jp
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10.1002/ejoc.201901348
European Journal of Organic Chemistry
COMMUNICATION
Inspired by these works, the photoaffinity probe was
designed based on a Ns scaffold with two functional properties
which 1) forms photo-crosslink and 2) installs different tags as
shown in Figure 1.
with TMS-CF3 and followed by the deprotection of TMS group in
acidic condition to afford alcohol 3 in high yield. Next, alcohol 3
was oxidized by using IBX to obtain ketone 4 in 83% yield. The
trifluoroacetyl
moiety
was
converted
into
the
(trifluoromethyl)diazirinyl moiety 8 using a general method16.
Subsequently, compound 8 was subjected to the introduction of
benzyl mercaptan in the presence of diisopropylethyl amine as
base to give the desired compound 9 with yield of 90%. Finally,
diazirinyl benzene mercaptan 9 was subjected to oxidative
chlorination with 2,4-Dichloro-5,5-dimethylhydantoin (DCDMH)17
to construct 2-nitrobenzenesulfonyl chloride. However, 4-
diazirinyl-2-nitrobenzenesulfonyl chloride 10 was not purified by
silica column chromatography due to its instability. Thus, 4-
diazirinyl-2-nitrobenzenesulfonyl chloride 10 was directly treated
with ethanol amine under basic condition to obtain Ns diazirine 11
in moderate yield (Scheme 1).
Results and Discussions
To evaluate the properties of photoreactivity and exchangeability
on Ns diazirine, a model compound bearing sulfonamide ethanol
Ns diazirine 10 was synthesized. In designing Ns diazirine, we
decided to install the trifluoromethyldiazirine moiety on the meta
position of the nitro group so that the nitro group enhances
photolysis14 and facilitates SNAr reaction15. Our synthesis began
with the introduction of nitro group on 4-chlorobenzaldehyde 1 via
substitution aromatic nucleophilic reaction in the presence of
potassium nitrate and sulphuric acid which gave us 86% yield of
nitro benzaldehyde 2. The desired compound 2 was then treated
Next, Ns diazirine 11 was irradiated under black light to
evaluate its photoreactive properties and kinetic photolysis by
comparing with normal trifluoromethyl diazirine 13. Irradiation of
both compounds under black light (30W) in methanol revealed a
decrease in the characteristic diazirinyl absorbance around 355
nm14,18 as the irradiation time proceeds (Figure 2). Based on these
results, Ns diazirine was found to be photolyzed more rapidly in
A)
H
OMe
CF3
365 nm
(100 W)
0.8
0.6
(min)
O
11
HO
S
0
1
2
3
4
5
6
7
8
9
10
N
O
NO2
MeOH
H
12
0.4
0.2
350
360
370
380
390
400
wavelength [nm]
B)
(min)
0
1
2
3
4
365 nm
(100 W)
N
N
H
OMe
CF3
CF3
0.6
MeOH
14
13
5
6
7
8
9
10
15
0.4
0.2
350
360
370
380
390
400
wavelength [nm]
Figure 2. UV-vis spectra in methanol of A) Ns diazirine 11 (2.00 ×10-3 M), B)
trifluoromethyl phenyldiazirine 13 (2.00 ×10-3 M) under irradiation of 365 nm
light at 0 °C in a sealed quartz cuvette.
Scheme 1. Synthesis of amine ethanol Ns diazirine 11.
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10.1002/ejoc.201901348
European Journal of Organic Chemistry
COMMUNICATION
comparison to normal diazirine as the half-live (t1/2) of these
compounds were calculated as 2.5 and 4.5 min, respectively.
Compound 12 was also confirmed with ESI-MS (m/z 381.2
M+Na+) after the completion of photolysis (Figure S1). These
results indicated that the irradiation effectively promoted
decomposition of the Ns diazirinyl ring and the generation of
highly reactive carbene. This strengthen the fact that Ns diazirine
exhibited suitable characteristics for the photoaffinity reagent.
then reacted with Ns diazirine 11 in the same basic condition as
described above. Expectedly, both 17 and 19 successfully
confirmed the feasibility of tag-exchanged reaction to produce the
tag inserted Ns diazirine 18 and 20 (Scheme 2). Additionally, to
prove the advantage of high sensitive mass tag 19, compound 20
was subjected to measuring ESI-MS after tag-exchanged reaction
without purification. Compound 20 exhibited strong intensity peak
(m/z 349.1), which is 120 times higher than that of the reference
compound, N,N,N-diethylamine type 21 (m/z 363.1) in the same
condition (Figure S2, ESI). Therefore, we envisaged that
photolabeled target molecules with Ns diazirine will simplify its
analysis procedure with the installation of a high sensitive mass
tag by SNAr.
Subsequently, with Ns diazirine 11 in hand, we attempted to
cleave the sulfonamide unit under physiological conditions. Ns
diazirine 11 was treated with 1.5 equiv of coumarin thiol derivative
15 in various conditions. The reaction progress was monitored by
TLC and determined by isolation of compound 16. The SNAr
proceeded smoothly in THF with the presence of Cs2CO3 to afford
16 in excellent yield, whereas the tag-exchanged reaction without
basic condition did not proceed entirely (Table 1. Entries 1–2). In
contrast, this reaction proceeded moderately in ethanol and
acetonitrile with the yield of 70-80% (Table 1. Entries 3–4).
Moreover, the SNAr reaction was also carried out under aqueous
condition, in which photoaffinity labeling is often performed. The
cleavage reaction proceeded smoothly in aqueous media in the
presence of Cs2CO3 which gave 16 in excellent yield 92% (Table
1. Entry 6). However, in the case of using NaHCO3 and
triethylamine (TEA) as weak bases, the reaction required longer
than 24 h reaction time (Table 1. Entries 7–8). According to these
results, although longer time is required for the reaction to be
consumed under aqueous media, the tag-exchanged reaction
proceeded smoothly which means it is further applicable under
physiological condition.
Scheme 2. The utility of the tag-exchanged reaction on Ns diazirine
Finally, we examined the utilities of Ns diazirines by using bovine
serum albumin (BSA) as the model protein (Figure 3). Thus, for
this reason, Ns diazirine bearing biotin was synthesized (Scheme
S1, ESI). After incubating the biotinylated Ns diazirine with BSA
in phosphate buffer (pH 7.0) at rt, photoirradiation was carried out
with 365 nm light (250 W) at 0 °C. Photoproducts were then
further subjected to 10% SDS-PAGE, followed by detection with
chemiluminescence method using horseradish peroxidase
(HRP)-conjugated avidin after blotting on PVDF membrane. The
emission intensity of BSA photolabeled with biotinylated Ns
diazirine increased accordingly with the irradiation time. Based
on the emission intensity, the cross-linking efficiency was
calculated as approximately maximum of 20%, determined by
comparing it with the calibration curve of biotinylated BSA (Figure
S3, ESI).
O
SH
N
N
N N
N
H
MeO
O
O
CF3
CF3
1.5 eq
O
O
O
15
HO
S
S
N
N
O
NO2
NO2
H
H
conditions
O
11
16
a Yield (%)
Entry
Solvent
THF
Base (3eq)
Time (h)
1
2
3
4
5
6
7
8
−
24
1
0
THF
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
Cs2CO3
NaHCO3
TEA
90
EtOH
CH3CN
2
73
78
82
92
47
35
2
5% DMSO in H2O
24
24
24
24
H2O
H2O
H2O
Table 1. Screening SNAr conditions between Ns diazirine 11 and coumarin thiol
15.
[a] Isolated yield after purification using silica gel column chromatography
To demonstrate the generality of the tag-exchanged reaction
between Ns diazirines and thiol linkage tags, we prepared a
bodipy-thiol tag (fluorescence) 17 and N,N,N-trimethylamine
ethanthiol (high sensitive mass tag) 19. These compounds were
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10.1002/ejoc.201901348
European Journal of Organic Chemistry
COMMUNICATION
Conclusions
A)
N
N
O
BSA
CF3
NH
S
O
O
HN
We successfully synthesized the first bifunctional Ns diazirine
based photoaffinity labelling probe with higher photoreactivity in
comparison with the normal phenyldiazirine. Additionally, this
probe also proves the feasibility of the tag-exchangeable reaction
via formation of Meisenheimer complex by installing fluorescent
or high sensitive mass tag in relatively high yield even in aqueous
condition. Interestingly, the product installed with high sensitive
mass tag could be detected without any purification. In the future,
we envisioned that this Ns diazirine methodology will simplify the
procedure for analysis in photoaffinity labeling.
O
S
365 nm
0 °C, time
N
N
2
O
NO2
H
H
F3C
22
O
S
N
linker
O
NO2
H
biotin
BSA
B)
Irradiation time
15
0
1
5
30 (min)
Chemiluminescence
detection
BSA
Acknowledgements
CBB detection
Authors are grateful to Dr. Yuyama Kohei for the biological study.
This work was supported by a grant-in-aid for scientific research
KAKENHI (grant 18K14350, 19H02836 and 17K19188) from the
MEXT of Japan, the MEXT Doctoral pro-gram for Data Related
Innovation Expert Hokkaido University (D-DRIVE-HU) program
and the Photo-excitonix Project in Hokkaido University. This work
was inspired by JSPS Asian CORE Program “Asian Chemical
Biology Initiative" and JSPS A3 Foresight Program. A.A.S. thanks
the International Graduate Program (IGP), Hokkaido University
for the scholarship given.
Figure 3. (A) Photoaffinity labeling in photoprobe 21 with BSA and (B)
chemiluminescence and Coomasie Brilliant Blue (CBB) detection of the BSA
photolabeled with biotinylated Ns diazirine. Photoreaction proceeded under 365
nm light at 0 oC for 0, 1, 5, 15, 30 min
Subsequently, photolabeled BSA was subjected to SNAr
with bodipy-thiol. The emission intensity of bodipy-thioether linked
BSA band at 525 nm increased accordingly in response with time,
and the formation of photolabeled BSA was obtained with
approximately 78% yield after 24 h (Figure 4). This value was
estimated from the relative intensity of the corresponding BODIPY
derivative separately.
Keywords: Photoaffinity probe • Diazirines • Nosyl •
Meisenheimer • SNAr
bodipy-thiol 17
N
H
N
N
B
F
F
. .
SH
O
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COMMUNICATION
Key Topic:
Nosyl, Diazirine
First design and synthesis of nosyl
diazirine based photoaffinity probe was
reported. This efficient approach of
ligand-tag exchangeable photoaffinity
labeling utilizes SNAr via Meisenheimer
complex.
Aimi Suhaily Saaidin, Yuta Murai*,
Takuya Ishikawa, Kenji Monde*
Page No. – Page No.
Title:
Design and synthesis of ligand-tag
exchangeable photoaffinity probe
utilizing nosyl chemistry
Layout 2:
COMMUNICATION
Key Topic*
Author(s), Corresponding Author(s)*
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