Design and synthesis of the diazirine-based clickable photo-affinity probe targeting sphingomyelin synthase 2

Article abstract of DOI:10.2174/1570180816666181106154601

Background: SMS family plays a very important role in sphingolipids metabolism and is involved in the membrane mobility and signaling transduction. Methods: SMS2 subtype was related to a variety of diseases and could be regarded as a promising potential d

Full text of DOI:10.2174/1570180816666181106154601

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Letters in Drug Design & Discovery, 2019, 16, 678-684
RESEARCH ARTICLE
ISSN: 1570-1808
eISSN: 1875-628X
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Design and Synthesis of the Diazirine-based Clickable Photo-Affinity
Probe Targeting Sphingomyelin Synthase 2
BENTHAM
SCIENCE
Penghui Wang, Zhining Li, Lulu Jiang, Lu Zhou* and Deyong Ye*
Department of Medicinal Chemistry, School of Pharmacy, Fudan University, Shanghai, P. R. China
Abstract: Background: SMS family plays a very important role in sphingolipids metabolism and is
involved in the membrane mobility and signaling transduction.
Methods: SMS2 subtype was related to a variety of diseases and could be regarded as a promising
potential drug target. However, the uncertainty of the binding sites and the molecular mechanism of
action limited the development of SMS2 inhibitors. Herein, we discovered a photo-affinity probe
A R T I C L E H I S T O R Y
PAL-1 targeting SMS2.
Received: March 18, 2018
Revised: October 19, 2018
Accepted: November 01, 2018
Results: The enzyme inhibitory activity and the photo-affinity labeling experiments showed that
PAL-1 could be mono-labeled on SMS2.
DOI:
Conclusion: In summary, starting from the N-arylbenzamides core structure and the minimalist
10.2174/1570180816666181106154601
terminal alkyne-containing diazirine photo-crosslinker, we designed and synthesized a photo-
affinity probe PAL-1 targeting SMS2. The enzymatic inhibitory activity study showed that PAL-1
exhibited superior selectivities for SMS2 with an IC50 of 0.37 µM over SMS1.
Keywords: Sphingomyelin synthase 2, sphingomyelin, photo-affinity labeling, diazirine, phosphatidylcholine, phosphoethanolamine.
1. INTRODUCTION
inflammation [7] and metabolic disorders such as
atherosclerosis [8, 9], diabetes [10], and obesity [11]. SMS2
deficiency could attenuate LPS-induced lung injury [12],
decrease atherosclerosis in mice [13], and impair insulin
sensitivity [14] and secretion [15] in mice. Besides, SMS2
was also involved in the HIV-1 infection [16] and the
induction of murine colitis-associated colon cancer [17].
These studies suggested that SMS2 could be regarded as a
promising drug target for the diseases mentioned above to
drive the discovery of SMS2 inhibitors (Fig. 1) [18-20].
Sphingomyelin synthase (SMS) is the last step enzyme in
the de novo synthesis of sphingomyelin in mammals,
catalyzing the conversion of phosphatidylcholine (PC) and
ceramide to diacylglycerol (DAG) and sphingomyelin (SM).
SMS has three isoforms with different subcellular localizations.
Sphingomyelin synthase 1 (SMS1) mainly localized in Golgi
membrane, was believed to undertake the majority of SM
production in the cell. Whereas, sphingomyelin synthase 2
(SMS2) is predominantly localized in the plasma membrane,
although
a
minority portion was found in Golgi.
All the three isoforms of SMS share high sequence
identity with each other and are predicted as six-pass
transmembrane proteins, which brought challenges to
understand the molecular mechanism of actions and
characterize the structure of SMSs through crystallography
[21]. Although the 3D structure model of SMS1 has been
generated by homology modeling, the validity of this
constructed model is not experimentally demonstrated and
exposed to the risk of over-interpretation due to the low
sequence similarities between the homologous protein and
the template proteins [22]. The interaction between SMSs
and their small molecular inhibitors still remains unclear that
limits the further development of SMS2 inhibitors as so far.
Sphingomyelin synthase related protein (SMSr) with the
weak function of ceramide phosphoethanolamine (CPE)
production rather than the function of SM production, is
mainly localized in the endoplasmic reticulum. These three
SMSs regulate the levels of sphingomyelin and other
sphingolipids to regulate the membrane mobility, as well as
signaling transduction [1-3].
Recently, more and more investigations showed that the
overexpression or dysfunction of SMSs was related to a
variety of diseases [3]. SMS1 was found to be necessary for
insulin secretion and mitochondrial function [4]. SMS1
deficiency may cause hearing impairments [5], male infertility
[6] and other serious metabolic abnormalities in mice [4].
Interestingly, SMS2 overexpression was involved in
Photo-affinity labeling (PAL) is an effective strategy in
structural biology that uses a chemical probe to generate
photoactive intermediate under the irradiation of the light
with a specific wavelength for subsequent covalently binding
to its target. Cooperating with the activity-based protein
profiling (ABPP) and the mass spectrometry, PAL has been
widely used in chemical biology and drug discovery for
target identification, molecular interactions confirmation and
*Address correspondence to these authors at the Department of Medicinal
Chemistry, School of Pharmacy, Fudan University, Shanghai, P. R. China;
Tel/Fax: +86-21-51980125 (Lu Zhou), +86-21-51980117 (Deyong Ye);
E-mails: zhoulu@fudan.edu.cn (Lu Zhou);
dyye@shmu.edu.cn (Deyong Ye)
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©2019 Bentham Science Publishers

Design and Synthesis of the Diazirine-based Clickable Photo-Affinity Probe
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 6 679
for probing the location and structure of binding sites [23-29].
Herein, we adopted the PAL strategy to design and
synthesize the photo-affinity probe targeting sphingomyelin
synthase 2, thus providing a chemical tool for exploring the
binding sites and the interaction of small molecules with
SMS2.
62%). 1H NMR (400 MHz, Chloroform-d) δ 7.32 – 7.22 (m,
2H), 6.96 (t, J = 7.5 Hz, 1H), 6.80 (d, J = 8.2 Hz, 1H), 4.74
(s, 2H), 3.85 (t, J = 5.9 Hz, 2H), 2.13 – 1.97 (m, 5H), 1.74 (t,
J = 7.4 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ 156.20,
129.37, 129.23, 128.98, 121.09, 110.76, 82.59, 69.43, 62.33,
61.97, 32.53, 32.38, 26.85, 13.26.
2.1.4. 2-((2-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)
benzyl)-oxy)-N-(pyridin-3-yl)benzamide hydrochloride (PAL-
1 HCl)
2. EXPERIMENTAL SECTION
2.1. The Procedures of PAL-1’s Preparation
To a solution of compound 4 (90 mg, 0.368 mmol) in dry
tetrahydrofuran (20 mL), PPh3 (145 mg, 0.553 mmol) and 2-
hydroxy-N-(pyridin-3-yl)benzamide (80 mg, 0.368 mmol)
were added. After stirring for 5 min at 0oC, DEAD (90 µL,
0.553 mmol) was injected into the reaction mixture through a
syringe under a nitrogen atmosphere. The reaction was
completed after another 2 h monitored by TLC. The mixture
was diluted with ethyl acetate (10 mL) and saturated NH4Cl
solution (15 mL). The organic phase was washed with brine,
dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by flash column
chromatography to give a crude product (120 mg) containing
triphenylphosphine oxide.
2.1.1. 2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethan-1-ol (2)
Compound 1 (173 mg, 1.37 mmol) was dissolved in
anhydrous ammonia (10 mL). After stirring for 2 h at -78oC,
a solution of hydroxylamine-O-sulfonic acid (187 mg, 1.65
mmol) in 1 mL dry methanol was added dropwise. The
reaction system was allowed to stirring overnight. The
resulting slurry was diluted with ethyl acetate (5 mL) and
filtered. The filtrate was concentrated under reduced pressure
and the residue was re-dissolved in dry dichloromethane (10
mL) and treated with triethylamine (0.83 mL, 5.94 mmol)
subsequently. A solution of iodine (665 mg, 2.62 mmol) in
dichloromethane (10 mL) was slowly added with stirring
until the reaction solution was turned into orange-brown.
The reaction mixture was continuously stirring for another 2
h and a solution of sodium thiosulfate (1 mol/L, 10 mL) in
water was added. The organic phase was washed with brine,
dried over sodium sulfate and concentrated under reduced
pressure. The residue was purified by flash column
chromatography to give a colorless oil (60 mg, yield: 32%).
1H NMR (400 MHz, Chloroform-d) δ 3.50 (q, J = 5.7 Hz,
2H), 2.05 (td, J = 7.4, 2.5 Hz, 2H), 2.01 (t, J = 2.4 Hz, 1H),
1.71 (dt, J = 11.3, 6.7 Hz, 4H), 1.48 (t, J = 4.5 Hz, 1H). 13C
NMR (151 MHz, CDCl3) δ 82.85, 69.23, 57.42, 35.56,
35.52, 32.68, 26.60, 13.25.
The above crude product was dissolved in ethyl acetate
(2 mL). Hydrogen chloride in ethyl acetate (1 mol/L, 1mL)
was added dropwise under vigorous stirring. The reaction
solution gradually became cloudy. After stirring for 1 h, the
mixture was filtered and the filter cake was recrystallized in
Et2O/EtOAc three times to give a white solid PAL-1
1
hydrochloride (13 mg, yield:12%). H NMR (400 MHz,
DMSO-d6) δ 10.84 (s, 1H), 9.06 (s, 1H), 8.54 (d, J = 5.6 Hz,
1H), 8.31 (s, 1H), 7.81 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H), 7.58
(t, J = 8.0 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.31 (t, J = 7.8
Hz, 2H), 7.14 (t, J = 7.5 Hz, 1H), 7.02 (d, J = 8.2 Hz, 1H),
6.92 (t, J = 7.4 Hz, 1H), 5.33 (s, 2H), 3.86 (t, J = 6.0 Hz,
2H), 2.82 (t, J = 2.8 Hz, 1H), 1.96 (td, J = 7.6, 2.8 Hz, 2H),
1.84 (t, J = 5.9 Hz, 2H), 1.60 (t, J = 7.4 Hz, 2H). 13C NMR
(151 MHz, DMSO) δ 165 .07, 155.85, 155.49, 139.69,
137.05, 135.39, 132.82, 131.04, 129.87, 129.37, 128.63,
125.83, 124.18, 123.71, 120.85, 120.52, 113.31, 111.61,
82.94, 71.57, 65.54, 62.69, 31.67, 31.49, 26.85, 12.45. MS
2.1.2. 3-(2-Bromoethyl)-3-(but-3-yn-1-yl)-3H-diazirine (3)
To a solution of compound 2 (172 mg, 1.24 mmol) in dry
dichloromethane (5 mL), CBr4 (620 mg, 1.87 mmol) and
PPh3 (450 mg, 1.87 mmol) were added at r.t. After stirring
for 4 h, the reaction mixture was concentrated under reduced
pressure. The residue was dissolved in ethyl ether and
filtered to remove the insoluble triphenylphosphine oxide.
The filtrate was concentrated under reduced pressure and the
residue was purified by flash column chromatography to
(ESI+) m/z 441.2 (M + H)+. HRMS (ESI+) m/z: 441.1922 (M
+
+ H)+ (calcd for Chemical Formula: C26H25N4O3
441.1921).
:
1
give a colorless oil (160 mg, 64%). H NMR (400 MHz,
2.2. In Vitro SMS Enzyme Activity Assay
Chloroform-d) δ 3.17 (t, J = 7.2 Hz, 2H), 2.09 – 1.97 (m,
5H), 1.71 (t, J = 7.3 Hz, 2H). 13C NMR (151 MHz, CDCl3) δ
82.45, 69.42, 36.46, 32.05, 27.41, 25.59, 13.25.
2.2.1. SMS1 Inhibitory Activities
5 µL PAL-1 HCl in DMSO with different concentrations
was added to 75 µL SMS1 protein solution (0.041 µg/µL) in
DDM buffer (containing 1mM DDM, 150 mM NaCl, and 20
mM Hepes, pH 7.5) and the mixture was incubated at room
temperature for 5 min. Then, another 20 µL of DDM
solution (containing 1 µL anhydrous ethanol solution of
DMPC (40 mM) and 1 µL DMSO solution of C6-NBD-
ceramide (1.16 mM)) were added. To the mixture, the
sample was added, incubated at 37 oC for 30 min and
quenched with 200 µL anhydrous ethanol. After centrifuged
at 10000 rpm for 10 min, the supernatant was collected for
HPLC test.
2.1.3. (2-(2-(3-(But-3-yn-1-yl)-3H-diazirin-3-yl)ethoxy)phenyl)-
methanol (4)
Compound 3 (160 mg, 0.796 mmol), salicyl alcohol (120
mg, 0.955 mmol) and potassium carbonate (165 mg, 1.19
mmol) were added into acetonitrile (10 mL) and heated to
80oC for 4 h. After cooling to r.t, the reaction mixture was
diluted with ethyl acetate (5 mL) and filtered to remove the
potassium carbonate. The filtrate was concentrated under
reduced pressure and the residue was purified by flash
column chromatography to give a colorless oil (120 mg,

680 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 6
Wang et al.
2.2.2. SMS2 Inhibitory Activities
acetonitriles were discovered as pan SMS inhibitors with low
selectivity through virtual screening and molecular docking
by using the homology modeling structure of SMS1 [18].
Starting optimization from the α-aminophenylacetonitriles, a
series of oxazolopyridines [19] and N-arylbenzamides (our
unpublished results) was obtained with improved activity
and selectivity. The 2-quinolone derivatives were discovered
through a high-throughput mass spectrometry-based screening
system [20]. Considering the preliminary structure and
activity relationship (SAR) of the N-arylbenzamides compounds
(our unpublished data), we selected the N-arylbenzamide as the
core of photo‐affinity probe and installed the minimalist
terminal alkyne-containing diazirine as the photo-crosslinker
[30] to constitute the clickable photo-affinity probe PAL-1
(Fig. 2).
The assay was similar to that of SMS1 using 79 µL of the
SMS2 protein solution (0.0041 µg/µL) in DDM and 1 µL of
a DMSO solution of the compound instead of the above.
2.3. PAL-1 Photolysis Assay
A solution of PAL-1 HCl (100 µM) in methanol was
irradiated by UV lamp (365nm, 40 W) on ice for 0 min, 10
min, 20 min, 40 min and 80 min, respectively. The samples
were kept within 1 cm from the lamp. After irradiation, the
samples were injected into the LC-MS system to measure the
concentration ratios and molecular weight of PAL-1 and its
photolysis product.
2.4. Photo-Affinity Labeling Experiment
The PAL-1 was synthesized according to Scheme 1. The
diazirine alcohol 2 was prepared as described in Li’s work
[30] and was transformed to the corresponding bromide 3 via
Appel reaction. The bromide 3 was nucleophilically
substituted by the salicylic alcohol to yield the substituted
benzyl alcohol 4. And then the alcohol 4 was condensed
with the 2-hydroxy-N-(pyridin-3-yl)benzamide to afford the
target molecule PAL-1 via Mitsunobu reaction, which was
subsequently converted into the corresponding hydrochloride
salt.
To a solution of SMS2 in DDM (with the protein content
of 1.6 µg/µL, 30 µL) 0.5 µL PAL-1 HCl was added in
DMSO (4 mM). The solution was incubated for 30 min and
subsequently irradiated by UV lamp (365nm, 40 W) on ice
for 40 min. The samples were kept within 1 cm from the
lamp. After irradiation, the samples were injected into the
Agilent 6550 iFunnel Q-TOF LC/MS system to measure the
molecular weight of the complex of PAL-1 labeled SMS2.
3. RESULTS AND DISCUSSION
The inhibitory activities of the probe PAL-1 against the
SMS1 and SMS2 were evaluated through the HPLC based
assay [31] by using pure SMS1 and SMS2 proteins as
As shown in Fig. 1, the inhibitors of SMS2 could be
classified into four structural categories. The α-aminophenyl
N
N
O
CN
O
O
N
N
H
N
H
O
O
Cl
SMS2 IC50= 13.5 PM
SMS2 IC50= 4.1 PM
oxazolopyridines
SMS2 IC50= 3.2 PM
N-arylbenzamides
D-aminophenylacetonitriles
Me
CF3
N
O
O
Me
N
Me
N
CF3
O
O
CF3
Me
O
N
CF3
O
SMS2 IC50= 6.5 nM
SMS2 IC50= 16 nM
N
N
O
O
Me
Fig. (1). The structures of representative SMS2 inhibitors.
2-quinolone derivatives

Design and Synthesis of the Diazirine-based Clickable Photo-Affinity Probe
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 6 681
N
O
N
O
N
H
N
install the Minimalist Photo-Crosslinker
H
O
O
N N
O
PAL-1
Fig. (2). The design of the photo‐affinity probe.
N N
N N
b
a
O
c
HN NH
Br
OH
OH
OH
3
2
1
d
•HCl
N
O
OH
N
H
e, f
N N
O
O
O
N N
4
PAL-1 hydrochloride
Scheme 1. Reagents and conditions: a. NH3 (l), then NH2OSO3H in methanol, NH3 (l), -78oC; b. I2, NEt3, MeOH, r.t, 32% (2 steps); c. CBr4,
PPh3, DCM, r.t, 64%; d. 2-(hydroxymethyl)phenol, K2CO3, DMF, 80oC, 62%; e. 2-hydroxy-N-(pyridin-3-yl)benzamide, DEAD, PPh3, THF,
0oC; f. HCl (1 mol/L) in EtOAc, Et2O, r.t, 12% (2 steps).
100
50
SMS1 IC50= 58 uM
SMS2 IC50= 0.37 uM
0
-4
-2
0
2
4
LOG(PAL-1 concentration) uM
Fig. (3). The IC50 curves of PAL-1 against SMS1 and SMS2.
enzyme sources. As shown in Fig. 3, PAL-1 with an IC50 of
58 µM and 0.37 µM against SMS1 and SMS2 respectively
showed 150-fold selectivity on SMS2 compared to on
SMS1. The hydrophobic cross-linker on the C2 of benzyl
group of the N-arylbenzamides could enhance the inhibitory
activity, which was consistent with our previous work [19].
photolysis product was analyzed by LC-MS. As shown in
Table 1, the concentration of the photolysis product was
significantly increased in a time-dependent manner in the
first 20 minutes. However, the photolysis efficiency was
attenuated with the increase of time (40 and 80 minutes). In
addition, the PAL-1 should be activated by UV light
irradiation (365 nm) to generate the carbene intermediate
PAL-C, which was then quenched by the solvent methanol
to yield the intended methoxy product (Fig. 4) [32, 33]. To
our surprise, the mass spectrum data revealed that the
Prior to the labeling experiments with SMS2, the
photochemical reactivity of PAL-1 in methanol was
examined. The concentration ratio of PAL-1 with its

682 Letters in Drug Design & Discovery, 2019, Vol. 16, No. 6
Wang et al.
After the above photochemical reactivity test, the
labeling experiment was conducted on the pure SMS2
enzyme system. The PAL-1 was incubated with SMS2 for
30 minutes followed by UV light (365 nm) irradiation for 40
minutes. The sample was injected into the Agilent 6550
iFunnel Q-TOF LC/MS system to measure the molecular
weight of the PAL-1 labeled SMS2. As shown in Fig. 5, the
mass shift between the SMS2 (42933.2255) and the major
PAL-SMS2 complex (43345.2965) was 412.0710, which
was consistent with the molecular weight of the labeling
fragment of PAL-1 after the loss of N2. This mass spectrum
data proved that PAL-1 could mono-label the SMS2. As for
the specific labeling sites, the further tryptic digestion for
mass spectrometry analysis is still ongoing.
Table 1. The concentration ratio of PAL-1 and its photolysis
product analyzed by LC-MS.
Time(min)*
Concentration Ratio
0
100:0
73:27
32:68
22:78
20:80
10
20
40
80
*The PAL-1 in methanol (100 µM) was irradiated with the UV light (365 nm) for
different time.
CONCLUSION
photolysis product’s m/z value was 412.2 rather than 444.2
in our system. We conjectured that the carbene intermediate
PAL-C has undergone self-quenching before being
quenched by solvent methanol.
In summary, starting from the N-arylbenzamides core
structure and the minimalist terminal alkyne-containing
diazirine photo-crosslinker, we designed and synthesized a
photo-affinity probe PAL-1 targeting SMS2. The enzymatic
N
N
N
O
O
O
O
O
O
O
N
N
H
N
H
H
Methanol
quench
hv
O
365 nm
OMe
N
N
O
m/z: 440.18
PAL-1
m/z: 412.18
PAL-C
m/z: 444.20
PAL-OMe
Fig. (4). The intended photochemical reaction of PAL-1 in methanol.
x106
2.2
+ESI Scan (11.648 min) Frag=175.0V yedeyong5-2.d Deconvoluted (Isotope Width=14.1)
42933.2255
43345.2965
100.00
97.64
2.0
1.8
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
42000 42200 42400 42600 42800 43000 43200 43400 43600 43800 44000 44200 44400 44600 44800 45000
Counts vs. Deconvoluted Mass (amu)
Fig. (5). The mass spectrum of photoaffinity labeling product of PAL-1 with SMS2.

Design and Synthesis of the Diazirine-based Clickable Photo-Affinity Probe
Letters in Drug Design & Discovery, 2019, Vol. 16, No. 6 683
inhibitory activity study showed that PAL-1 exhibited
superior selectivities for SMS2 with an IC50 of 0.37 µM
over SMS1. Besides, the PAL-1’s photolysis product in
methanol was conjectured as the self-quenching product
based on the molecular weight test. Moreover, the
photoactivated labeling experiment of PAL-1 with SMS2
was conducted and the labeled complex was identified as a
mono-labeled product through the mass spectrum. Thus, we
believed that PAL-1 would be a chemical tool for probing
the binding sites and the interaction of the small molecule
inhibitors of SMS2. In addition, with this clickable photo-
affinity probe PAL-1, the activity-based protein profiling
(ABPP), the labeling site analysis, and the cell-based photo-
affinity labeling are ongoing in our lab for further
understanding the molecular mechanism of action of small
molecules that bind to SMS2.
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LIST OF ABBREVIATIONS
ABPP
CPE
PAL
PC
=
=
=
=
=
=
Activity-Based Protein Profiling
Ceramide Phosphoethanolamine
Photo-Affinity Labeling
Phosphatidylcholine
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SM
Sphingomyelin
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Sphingomyelin Synthase
CONSENT FOR PUBLICATION
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FUNDING
This study was financially supported by the Shanghai
Municipal Committee of Science and Technology (No
17431902300), the program for Shanghai Rising Star (No
15QA1400300), and the Open Grant (2015-2017) of the
State Key Laboratory of Bio-organic and Natural Products
Chemistry, Chinese Academy of Sciences and the National
Science & Technology Major Project “Key New Drug
Creation and Manufacturing Program”, China (No
2018ZX09711002).
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CONFLICT OF INTEREST
The authors declare no conflict of interest, financial or
otherwise.
ACKNOWLEDGEMENTS
Lu Zhou & Deyong Ye conceived and designed the
research; Penghui Wang performed the experimental works
and wrote the manuscript, Zhining Li and Lulu Jiang
participated in the experimental works, analyzed the data and
discussed the results. The authors also gratefully acknowledge
Prof. Youhong Hu and Dr. Wenming Ren from the Shanghai
Institute of Materia Medica, Chinese Academy of Sciences, and
Dr. Chao Peng from the National Center for Protein Science
(Shanghai) for providing the 365 nm UV lamp and the analysis
of mass spectrometry of macromolecules respectively.
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