Synthesis of diazirine-based photoreactive saccharin derivatives for the photoaffinity labeling of gustatory receptors

Article abstract of DOI:10.1002/ejoc.201500184

Saccharin is one of the most common artificial sweeteners that has a bitter taste at high concentrations. Currently, there are no detailed functional analyses of these gustatory receptors. Therefore, we designed and synthesized photoreactive saccharin der

Full text of DOI:10.1002/ejoc.201500184

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Date: 31-03-15 16:48:51
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FULL PAPER
DOI: 10.1002/ejoc.201500184
Synthesis of Diazirine-Based Photoreactive Saccharin Derivatives for the
Photoaffinity Labeling of Gustatory Receptors
Lei Wang,[a] Takuma Yoshida,[a] Yasuyuki Muto,[a] Yuta Murai,[a][‡]
Zetryana Puteri Tachrim,[a] Akiko Ishida,[a] Shiori Nakagawa,[a] Yasuko Sakihama,[a]
Yasuyuki Hashidoko,[a] Katsuyoshi Masuda,[b] Yasumaru Hatanaka,[c] and
Makoto Hashimoto*[a]
Keywords: Synthetic methods / Photoaffinity labeling / Receptors / Nitrogen heterocycles / Saccharin
Saccharin is one of the most common artificial sweeteners
that has a bitter taste at high concentrations. Currently, there
are no detailed functional analyses of these gustatory recep-
tors. Therefore, we designed and synthesized photoreactive
saccharin derivatives that contain a (trifluoromethyl)dia-
zirinyl moiety at the 5- or 6-position for use as functional
analysis tools for photoaffinity labeling.
pounds bind to the same sweet taste receptor.[3,4] To study
how the receptor can distinguish between saccharin and
other sweeteners as well as the structural features of
saccharin derivatives that favor the activation of the sweet
taste receptor, approaches such as conformational analysis
by using X-ray crystallography, NMR spectroscopy, and
molecular modeling have been used. However, the receptor-
bound conformations of the sweeteners remain unclear as
a result of limited structural information on the ligands
complexes with the receptor.
Photoaffinity labeling is a useful biochemical method to
explore the structural and functional relationships between
low molecular weight bioactive compounds and biomolec-
ules.[5] This method is suitable for analyzing biological in-
teractions because it is based on the affinity of bioactive
compounds for biomolecules. Various photophores, such as
phenyldiazirine, arylazide, and benzophenone, are used. To
the best of our knowledge, the synthesis of saccharin
derivatives for photoaffinity labeling has not been reported
yet. Arylazide saccharins, which can be utilized as photo-
affinity labeling reagents, have been reported very recently.
However, the arylazide moiety was utilized for click
reaction substrates and photoaffinity labeling was not
employed.[6] Although comparative irradiation studies of
these three photophores in living cells indicates that a carb-
ene precursor, [3-(trifluoromethyl)phenyl]diazirine, is the
most promising photophore,[7] the relatively complicated
synthesis of the [3-(trifluoromethyl)phenyl]diazirinyl ring
has resulted in fewer applications in biomolecular studies
relative to other photophores. To resolve this problem, we
have reported on the post-functional synthesis of a family
of [3-(trifluoromethyl)phenyl]diazirines by using many reac-
tion conditions.[8] Suami et al. reported on several struc-
ture-activity relationships for saccharin, and found that
Introduction
Humans distinguish gustatory sensations as five basic
tastes: bitterness, saltiness, sourness, sweetness, and
savoriness. Sweetness is almost universally regarded as a
pleasurable experience for human beings. Numerous chemi-
cal substances, both natural and artificial, have been re-
ported as sweeteners. Saccharin is one of the most common
artificial sweeteners in the world and is several hundred
times sweeter than sucrose. However, it has a bitter taste at
high concentrations. Sweet and bitter taste receptors are
both G protein-coupled receptors. For saccharin, the bio-
activity underlying its sweetness involves binding with the
sweet taste receptor, which has a heterodimeric structure
with T1R2 and T1R3 subunits. Each subunit has a large
amino-terminal domain linked by a cysteine-rich domain
at the extracellular site to a seven transmembrane helical
domain.[1] The human heterodimeric sweet taste receptor
(hT1R2-hT1R3) responds to a wide variety of chemical
substances, both natural and artificial.[2] Although these
sweeteners have various chemical structures, all of the com-
[a] Division of Applied Science, Graduate School of Agriculture,
Hokkaido University,
Kita 9, Nishi 9, Kita-ku, Sapporo 060-8589, Japan
E-mail: hasimoto@abs.agr.hokudai.ac.jp
http://www.agr.hokudai.ac.jp/en/
[b] Suntory Institute for Bioorganic Research,
1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka
618-8503, Japan
[c] Graduate School of Medicine and Pharmaceutical Sciences,
University of Toyama,
2630 Sugitani, Toyama 930-0194, Japan
[‡] Present address: Faculty of Advanced Life Science, Frontier
Research Center for Post-Genome Science and Technology,
Hokkaido University,
Kita 21, Nishi 11, Kita-ku, Sapporo 001–0021, Japan
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500184.
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substitutions at the 5- and 6-positions in saccharin were
tolerated with regards to its biological activities.[9] In
addition, some sweet compounds also interact with other
taste modalities. For example, saccharin triggers both the
sweet and bitter taste modalities. The mechanisms underly-
ing changes in taste modalities have not yet been eluci-
dated.[10] In this report, we aim to describe the synthesis of
photoreactive (trifluoromethyl)diazirinyl saccharin deriva-
tives, which can be used to elucidate the sweet and bitter
taste mechanisms. Our approach involves post-functional
derivatization[11] for [3-(trifluoromethyl)]phenyldiazirine de-
rivatives.
Results and Discussion
Although several methods for the synthesis of saccharin
have been reported,[12] we were required to choose the
simplest synthetic methods for the diazirine three-
membered ring structure. Each synthetic step for oxidative
cyclization of N-tBu-o-toluenesulfonamide derivatives to
Scheme 1. Improved synthesis of 3-(m- or p-tolyl)-3-(trifluorometh-
yl)-3H-diazirines.
construct saccharin skeleton[13] could be applied without 2 equiv.; experimental conditions are reported in ref.[13]
)
decomposition of the (trifluoromethyl)diazirine moiety. Un- afforded a more complex mixture than that in tBuNH2 only
der previously reported conditions,[14] 2,2,2-trifluoro-1-(p- (4 equiv.). Oxidative cyclization with H5IO6 and CrO3 be-
tolyl)ethanone (1a) was treated with hydroxylamine hydro- tween the methyl group and o-oriented N-tert-butyl sulfon-
chloride in the presence of sodium hydroxide in ethanol at amide synthesized saccharin skeletons 6a and 6b in a mod-
reflux temperatures for 16 h, which resulted in 87% yield of erate yield. Purification for each step generated lower iso-
diazirine. The isolated yield was increased to 95% within lated yields for cyclized compounds 6a and 6b (Ͻ 10%).
3 h when pyridine was used as a solvent at 70 °C.[15] Re- We performed these three steps as one-pot reactions (≈ 30%
gioisomer 1b was also converted into corresponding oxime yields). Deprotection of the tBu group with trifluoroacetic
2b under identical conditions with 1a in 99% yield. The acid (TFA) under reflux conditions afforded diazirinyl sac-
oximes were converted into tosyl oximes 3a and 3b by using charin derivatives 7a and 7b (Scheme 2). The synthesized
Tosyl(Ts)Cl in pyridine in very good yield. Then, we sub- photoreactive saccharin derivatives were insoluble in water.
jected the tosyl oximes to classical stepwise conversions to We performed further purification of the synthesized sac-
give diazirines 5a[14] and 5b[16] through diaziridines 4a and charin derivatives by converting them into the correspond-
4b with up to 70% yield for the two steps (Scheme 1, ing sodium salts with aqueous sodium hydroxide and then
route A). We recently reported on effective one-pot conver- subjecting them to reversed phase HPLC. Both samples
sions into diazirine derivatives from the corresponding tosyl were eluted at 22.5 and 21.0 min on an ODS column with
oximes.[17] The tosyl oximes were subjected to two condi- 30% methanol for sodium salt of 7a and 7b, respectively, at
tions. The first was the treatment of tosyl oximes in liquid 215 nm (Figure 1). These peaks were also detected at
ammonia at 80 °C for 12 h in a stainless pressure-resistant 350 nm. 19F NMR spectra for the (trifluoromethyl)-3H-di-
tube. A detailed product analysis revealed that diaziridine azirines (–65 ppm) revealed the three-membered ring, which
formation occurred within an hour, then slow conversion was identical to those previous reported.[18] These results
–
into diazirine with in situ generated NH2 species at high indicate that the diazirinyl groups are preserved in the final
temperature (Scheme 1, route B). The reaction rate for the compounds.
latter step was further improved by adding lithium amide
Irradiation studies of a methanolic solution (1 mm) of 7a
–
to enhance the concentration of NH2 at lower tempera- and 7b by using 15 W black light indicated that the diaziri-
tures (room temperature; Scheme 1, route C). The chemical nyl moiety of both saccharin derivatives decomposed very
yields of both one-step conversions into diazirine from tosyl rapidly (Scheme 3). Reports have suggested that diazo de-
oxime were almost quantitative.
rivatives, which are one of the main by-products of the irra-
3-(p- or m-Tolyl)-3-(trifluoromethyl)-3H-diazirines (5a diation of diazirines, cannot generate carbenes under irradi-
and 5b) were subjected to aromatic chlorosulfonation at ation at 350 nm at a diazirine concentration over 10 mm.[19]
adjacent positions of the toluene methyl group with chloro- However, a diluted (Ͻ 1 mm) solution can generate carbene
sulfonic acid at –20 °C. Addition of strong acid at higher from diazo compounds when irradiated at 350 nm for
temperatures promoted decomposition of the diazirinyl periods Ͼ 10 min. The absorbance at around 350 nm, which
ring.[18] The sulfonyl chloride was converted into N-alkyl- is characteristic of a diazirinyl three-membered ring,[20]
ated sulfonamide with tBuNH2 at room temperature. The diminished with increasing irradiation time (Figure 1). The
reaction in the triethylamine and tBuNH2 system (both half-life of diazirinyl saccharin derivatives 7a and 7b were
2
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Synthesis of Diazirine Based Photoreactive Saccharin Derivatives
Scheme 2. Post-functional synthesis of (trifluoromethyl)diazirinyl
saccharin derivatives at the 5- or 6-positions.
Figure 1. HPLC profiles of 5- or 6-(trifluoromethyl)diazirinyl-
sodium saccharin. The chromatograms for sodium salts of 7a and
7b are a) and b), respectively. HPLC conditions; Tosoh TSKgel
ODS-80 Ts (4.6ϫ250 mm), 30% MeOH/H2O, flow rate 1 mL/min,
detection at 215 nm.
calculated at 77 and 107 s, respectively. These half-lives are
Scheme 3. Photolysis of diazirine-based saccharin derivatives 7a (a)
and 7b (b) in methanol (1 mm) with black light (15 W). UV spectra
shorter than those of the diazirinyl α-amino acid deriva-
tives[21] that we reported with the photoreactive d-isomer of the photolysis reaction were recorded every 1 min for 10 min.
acting as the sweetener.[22] An electron-donating moiety at
The photoreactive compounds will be subjected to further
detailed biological analyses for their gustatory responses.
the p-position (carbonyl and sulfonamide moieties for 7a
and 7b, respectively) may promote rapid photolysis of
diazirinyl saccharins.
A required characteristic for photoaffinity labeling was
sufficiently met because no influence of irradiation effects
on other biomolecules occurred. The photoreactivities of
the studied compounds were also consistent with previous
19F NMR spectroscopic studies in which the 19F NMR
spectroscopic chemical shifts changed from –65 to –78 ppm
after 10 min of irradiation. No diazo isomer signal
(–58 ppm) was detected in the photoirradiated mixture.[19]
The results indicate that the diazirine moieties generated
carbenes, which were then quenched by solvent.
The synthesized photoreactive saccharin derivatives were
Scheme 4. Sweetness potential represented as normalized responses
subjected to preliminary gustatory receptor assays at
10 mm. Saccharin, 7a, and 7b have 90, 80, and 65% relative
sweetness activity, respectively, against the same concentra-
tion of aspartame for the hT1R2-hT1R3 expressed HEK-
293 T cell.[23] The response was completely inhibited by
addition of lactisole, which is one of the specific inhibitors
in the sweet taste assay. (Scheme 4). The bitter receptor
hT2R31 response to 7a and 7b at 10 mm activity was calcu-
lated as 60 and 80%, respectively. Other bitter receptors,
for 10 mm of aspartame, sucrose and synthetic photoreactive com-
pounds (7a and 7b). Cell responses, which are triggered with 10 mm
aspartame, were set as normalization standard. The degree of cell
response to 10 mm of each chemical was reported as a normalized
ratio. Each column represents the mean standard error of three
independent experiments.
Conclusions
These results indicate that the preparation of the dia-
such as hT2R43,[10] did not respond to 10 mm 7a and 7b. zirinyl saccharin derivatives is effective, and that these
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117.5 (q, JC,F = 283.9 Hz), 21.5, 21.3, 21.2 ppm. 19F NMR
(470 MHz, CDCl3): δ = –61.5, –66.5 ppm. HRMS (ESI): calcd. for
C16H15F3NO3S 358.0725; found 358.0745.
1
photoreactive compounds have enough affinity for the
sweet and bitter taste receptors to elucidate the binding sites
of their ligands in these receptors. This will ultimately allow
an understanding of the underlying molecular mechanisms
of gustatory receptors.
2,2,2-Trifluoro-1-(m-tolyl)ethanone O-Tosyl Oxime (3b): Treatment
of 2b (1.04 g, 5.1 mmol) was carried out as described above to
afford 3b as a colorless amorphous solid (1.68 g, 92%, mixture of
syn- and anti-isomers). 1H NMR (270 MHz, CDCl3): δ = 7.87–7.92
(m, 2 H), 7.30–7.40 (m, 4 H), 7.16–7.23 (m, 2 H), 2.48 (s, 0.7 H),
2.46 (s, 2.3 H), 2.39 (s, 0.7 H), 2.37 (s, 2.3 H) ppm. 13C NMR
Experimental Section
2
(68 MHz, CDCl3): δ = 154.3 (q, JC,F = 32.3 Hz), 146.3 and 146.1,
General Remarks: NMR spectra were measured with JEOL EX-
270 or Bruker AMX500 spectrometers. All solvents were of reagent
grade and distilled by using the appropriate methods. ESI-TOF-
MS data were obtained with a Waters UPLC ESI-TOF mass spec-
trometer.
138.8 and 138.7, 132.6 and 132.5, 131.6 and 131.3, 129.9 and 129.4,
129.3 and 129.1, 128.7 and 128.6, 127.7, 126.0, 125.5, 117.4 (q,
1JC,F = 283.8 Hz), 21.5, 21.2 and 21.1 ppm. 19F NMR (470 MHz,
CDCl3):
δ = –61.5, –66.9 ppm. HRMS (ESI): calcd. for
C16H15F3NO3S 358.0725; found 358.0710.
2,2,2-Trifluoro-1-(p-tolyl)ethanone Oxime (2a). Method (i) in EtOH:
2,2,2-Trifluoro-1-(p-tolyl)ethanone (1a; 0.104 g, 0.55 mmol) in
EtOH (5 mL) was added to hydroxylamine hydrochloride (0.0459 g,
0.66 mmol) and NaOH (0.064 g, 1.6 mmol) in EtOH (5 mL). The
reaction mixture was heated to reflux for 16 h, and then concen-
trated. The residue was partitioned between ether and water. The
organic layer was washed with HCl (0.01 m) and water, dried with
MgSO4, filtered, and concentrated to afford a colorless amorphous
solid (0.0974 g, 87%). Method (ii) in pyridine: 2,2,2-Trifluoro-1-(p-
tolyl)ethanone (1a; 1.13 g, 6.0 mmol) was dissolved in pyridine
(30 mL), then hydroxylamine hydrochloride (0.500 g, 7.2 mmol)
was added. The mixture was stirred at 70 °C for 1 h and was then
subjected to rotary evaporation to remove the pyridine. The residue
was dissolved in ethyl acetate and washed with HCl (1 m), the or-
ganic layer was washed with H2O and brine, dried with MgSO4,
and concentrated to afford a colorless amorphous solid (1.17 g,
96%). The product was mixture of syn- and anti-isomers. 1H NMR
(270 MHz, CDCl3): δ = 9.01 (br. s, 1 H), 7.37–7.44 (m, 2 H), 7.21–
7.29 (m, 2 H), 2.39 (s, 0.8 H), 2.38 (s, 2.2 H) ppm. 13C NMR
3-(p-Tolyl)-3-(trifluoromethyl)diaziridine (4a): To liquid NH3
(20 mL) at –78 °C in a sealed tube, tosyloxime 3a (1.02 g,
2.9 mmol) in Et2O (5 mL) was added. The reaction mixture was
stirred at room temperature for 8 h. After evaporation of NH3 gas,
the reaction mixture was partitioned between ether and water. The
organic layer was washed with brine, dried with MgSO4 and the
solvents evaporated. The residue was subjected to silica-gel column
chromatography (EtOAc/hexane, 1:5) to afford 3-(p-tolyl)-3-(tri-
fluoromethyl)diaziridine (4a) as
a colorless amorphous solid
(0.502 g, 87%). 1H NMR (270 MHz, CDCl3): δ = 7.50 (d, J =
8.0 Hz, 2 H), 7.22 (d, J = 8.0 Hz, 2 H), 2.76 (d, J = 8.0 Hz, 1 H),
2.38 (s, 3 H), 2.19 (d, J = 8.0 Hz, 1 H) ppm. 13C NMR (68 MHz,
CDCl3): δ = 140.3, 129.5, 128.9, 128.1, 123.7 (q, 1JC,F = 278.1 Hz),
57.8 (q, 2JC,F = 35.9 Hz), 21.1 ppm. 19F NMR (470 MHz, CDCl3):
δ = –75.7 ppm. HRMS (ESI): calcd. for C9H10F3N2 203.0796;
found 203.0785.
3-(m-Tolyl)-3-(trifluoromethyl)diaziridine (4b): Treatment of 3b
(1.030 g, 2.9 mmol) was carried out as described above to afford 4b
(68 MHz, CDCl3): δ = 148.2 [q, 2J(C,F) = 30.6 Hz], 141.2 and
as
a
colorless amorphous solid (0.524 g, 90%). 1H NMR
1
141.0, 129.42 and 129.36, 128.7 and 128.3, 123.0, 118.5 (q, JC,F
=
(270 MHz, CDCl3): δ = 7.40–7.42 (m, 2 H), 7.31 (t, J = 7.8 Hz, 1
H), 7.25 (d, J = 7.8 Hz, 1 H), 2.77 (d, J = 8.0 Hz, 1 H), 2.38 (s, 3
283.0 Hz), 21.3 and 21.2 ppm. 19F NMR (470 MHz, CDCl3): δ =
–62.4, –66.6 ppm. HRMS (ESI): calcd. for C9H9F3NO 204.0636;
found 204.0632.
H), 2.21 (d, J = 8.0 Hz, 1 H) ppm. 13C NMR (68 MHz, CDCl3): δ
1
= 138.7, 131.7, 131.0, 128.73, 128.69, 125.3, 123.6 (q, JC,F
=
2
2,2,2-Trifluoro-1-(m-tolyl)ethanone Oxime (2b): Treatment of 2,2,2- 278.2 Hz), 58.0 (q, JC,F
=
35.9 Hz), 21.2 ppm. 19F NMR
trifluoro-1-(m-tolyl)ethanone (1b; 1.13 g, 6.0 mmol) in pyridine was
carried out as described above to afford 2b (1.21 g, 99%) as a color-
less amorphous solid. H NMR (270 MHz, CDCl3): δ = 8.76 (br.
(470 MHz, CDCl3): δ = –76.0 ppm. HRMS (ESI): calcd. for
C9H10F3N2 203.0796; found 203.0796.
1
3-(p-Tolyl)-3-(trifluoromethyl)-3H-diazirine (5a). Route A (stepwise
conversion through the diaziridine): Diaziridine 4a (0.143 g,
0.71 mmol) was dissolved in CH2Cl2 (5 mL) and triethylamine
(0.29 mL), and cooled to 0 °C. Iodine (0.199 g, 0.78 mmol) was
added dropwise. The reaction mixture was stirred for 1 h and
washed with NaOH (1 m), H2O, and brine. The organic layer was
dried with MgSO4, filtered and concentrated. The residue was sub-
jected to silica-gel column chromatography (CHCl3/EtOAc, 19:1)
to afford 5a as a colorless oil (0.114 g, 80%). Route B (one-pot
synthesis): To liquid NH3 (10 mL) at –78 °C in a sealed tube,
tosyloxime 3a (0.711 g, 2.0 mmol) was added. The reaction was
stirred at 80 °C for 11 h. The sealed tube was cooled to –78 °C and
the reaction mixture was diluted with Et2O (50 mL). The sealed
tube was warmed to room temperature to remove the ammonia
gradually. The organic layer was washed by H2O, brine, dried with
MgSO4, and carefully evaporated (0 °C) to afford a colorless oil
(0.392 g, 98%). Route C (one-pot with LiNH2): To liquid NH3
(5 mL) at –78 °C in a sealed tube, tosyloxime 3a (0.711 g,
s, 0.6 H), 8.59 (br. s, 0.4 H), 7.29–7.40 (m, 4 H), 2.40 (s, 1.8 H),
2.39 (s, 1.2 H) ppm. 13C NMR (68 MHz, CDCl3): δ = 148.4 (q,
2JCF = 30.7 Hz), 148.1 (q, 2JC,F = 32.4 Hz), 138.6, 131.6 and 131.4,
129.8 and 129.1, 129.0 and 128.6, 125.9 and 125.7, 125.5, 120.7 (q,
1
1JC,F = 274.8 Hz), 118.4 (q, JC,F = 282.9 Hz), 21.2 and 21.1 ppm.
19F NMR (470 MHz, CDCl3): δ = –62.4, –66.8 ppm. HRMS (ESI):
calcd. for C9H9F3NO 204.0636; found 204.0628.
2,2,2-Trifluoro-1-(p-tolyl)ethanone O-Tosyl Oxime (3a): To
a
solution of oxime 2a (0.910 g, 4.5 mmol) in acetone (30 mL) at
0 °C, triethylamine (1.87 mL, 13.4 mmol) was added. Then, p-
toluenesulfonyl chloride (0.945 g, 5.0 mmol) was added to the reac-
tion mixture that was stirred at room temperature for 1 h. After
evaporation, the residue was purified by silica gel column
chromatography (EtOAc/hexane, 1:3) to afford a colorless amorph-
ous solid (1.50 g, 93%, mixture of syn- and anti-isomers). 1H NMR
(270 MHz, CDCl3): δ = 7.90 (d, J = 8.4 Hz, 2 H), 7.28–7.40 (m, 4
H), 7.22 (d, J = 8.4 Hz, 2 H), 2.48 (s, 0.6 H), 2.46 (s, 2.4 H), 2.40
(s, 0.6 H), 2.39 (s, 2.4 H) ppm. 13C NMR (67 MHz, CDCl3): δ = 2.0 mmol) and LiNH2 (0.230 g, 10 mmol) was added. The reaction
2
154.1 (q, JC,F = 32.1 Hz), 146.2 and 146.1, 142.6 and 142.4, 131.5 was stirred at room temperature for 12 h. The sealed tube was
and 131.3, 129.9, 129.5, 129.2 and 129.1, 128.8 and 128.4, 124.8,
cooled to –78 °C and the reaction mixture was diluted with Et2O
4
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Synthesis of Diazirine Based Photoreactive Saccharin Derivatives
(50 mL). The sealed tube was warmed to room temperature to
remove the ammonia gradually. The organic layer was washed
by H2O (three times), brine, dried with MgSO4, and carefully
evaporated (0 °C) to afford a colorless oil (0.388 g, 97%). 1H NMR
(270 MHz, CDCl3): δ = 7.20 (d, J = 8.2 Hz, 2 H), 7.08 (d, J =
8.2 Hz, 2 H), 2.36 (s, 3 H) ppm. 13C NMR (68 MHz, CDCl3): δ =
140.0, 129.6, 127.8, 126.5, 122.4 (q, 1JC,F = 274.5 Hz), 28.3 (q, 2JC,F
N-tert-Butyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]-1,2-benziso-
thiazole-3-one 1,1-Dioxide (6b): Treatment of 5b (0.136 g,
0.68 mmol) was carried out as described above to afford 2-methyl-
4-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzene-1-sulfonyl chloride.
1H NMR (270 MHz, CDCl3): δ = 8.10 (d, J = 8.6 Hz, 1 H), 7.24
(d, J = 8.6 Hz, 1 H), 7.16 (s, 1 H), 2.80 (s, 3 H) ppm. Treatment of
the residue was carried out as described above to afford N-tert-
butyl-2-methyl-4-[3-(trifluoromethyl)-3H-diazirin-3-yl] benzene-
=
40.4 Hz), 21.0 ppm. 19F NMR (470 MHz, CDCl3):
δ =
–65.4 ppm. HRMS (ESI): calcd. for C9H8F3N2 201.0640; found sulfonamide. 1H NMR (270 MHz, CDCl3): δ = 8.05 (d, J = 8.6 Hz,
201.0632.
1 H), 7.12 (d, J = 8.6 Hz, 1 H), 7.04 (s, 1 H), 4.44 (s, 1 H), 2.66 (s,
3 H), 1.22 (s, 9 H) ppm. Treatment of the residue with H5IO6,
CrO3, and acetic anhydride as described for 6b (0.0781 g, 33%).
UV/Vis (CH3OH): λmax [log (ε/m–1 cm–1)] = 280 (900), 340
3-(m-Tolyl)-3-(trifluoromethyl)-3H-diazirine (5b). Route A (stepwise
conversions through the diaziridine): Treatment of 4b (0.143 g,
0.71 mmol) was carried out as described above to afford 5b
(0.094 g, 66%) as a colorless oil. Route B (one-pot synthesis): Treat-
ment of 3b (0.715 g, 2.0 mmol) was carried out as described above
to afford 5b (0.3960 g, 99%) as a colorless oil. Route C (one-pot
with LiNH2): Treatment of 3b (0.722 g, 2.0 mmol) was carried out
as described to afford 5b (0.400 g, 99%) as a colorless oil. 1H NMR
(270 MHz, CDCl3): δ = 7.28 (t, J = 7.6 Hz, 1 H), 7.22 (d, J =
7.6 Hz, 1 H), 7.01 (d, J = 7.6 Hz, 1 H), 6.97 (s, 1 H), 2.35 (s, 3
H) ppm. 13C NMR (68 MHz, CDCl3): δ = 138.9, 130.5, 129.2,
1
(300) nm. H NMR (270 MHz, CDCl3): δ = 7.89 (d, J = 8.0 Hz, 1
H), 7.82 (s, 1 H), 7.63 (d, J = 8.0 Hz, 1 H), 1.77 (s, 9 H) ppm. 13C
NMR (68 MHz, CDCl3): δ = 158.7, 138.5, 135.8, 132.2, 128.3,
1
2
122.7, 121.4 (q, JC,F = 281.1 Hz), 120.9, 61.8, 28.3 (q, JC,F
=
38.8 Hz), 27.7 ppm. 19F NMR (470 MHz, CDCl3): δ = –64.7 ppm.
HRMS (ESI): calcd. for C13H13F3N3O3S 348.0630; found
348.0626.
6-[3-(Trifluoromethyl)-3H-diazirin-3-yl]-1,2-benzisothiazole-3-one
1,1-Dioxide (7a): Compound 6a (13.5 mg, 39 μmol) was dissolved
in TFA (2 mL). The reaction mixture was heated to reflux for 24 h
and then concentrated. The residue was dissolved in EtOAc and
washed with saturated NaHCO3, HCl (1 m), and brine. The organic
layer was dried with MgSO4 and filtered. The filtrate was concen-
trated, then the residue was recrystallized from EtOAc and hexane
at –20 °C to afford 7a (6.5 mg, 57%) as a colorless amorphous
solid. UV/Vis (CH3OH): λmax [log(ε/m–1 cm–1)] = 290 (920), 337
1
2
128.8, 127.1, 123.7, 122.4 (q, JC,F = 274.5 Hz), 28.3 (q, JC,F
=
40.4 Hz), 21.2 ppm. 19F NMR (470 MHz, CDCl3): δ = –65.2 ppm.
HRMS (ESI): calcd. for C9H8F3N2 201.0640; found 201.0644.
N-tert-Butyl-6-[3-(trifluoromethyl)-3H-diazirin-3-yl]-1,2-benziso-
thiazole-3-one 1,1-Dioxide (6a): Chlorosulfonic acid (0.380 mL,
5.7 mmol) was cooled to –20 °C. Compound 5a (0.115 g,
0.57 mmol) was added dropwise and the reaction mixture was
stirred at the same temperature for 1 h, warmed to room tempera-
ture, and stirred for 4 h, then poured into ether and ice water. The
organic layer was washed with saturated NaHCO3, dried with
MgSO4 and filtered. The filtrate was concentrated to afford crude
2-methyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]benzene-1-sulfonyl
chloride as a pale yellow oil. 1H NMR (270 MHz, CDCl3): δ =
7.80 (d, J = 1.7 Hz, 1 H), 7.53 (dd, J = 1.1, 8.0 Hz, 2 H), 7.50 (d,
J = 8.0 Hz, 2 H), 2.80 (s, 3 H) ppm. The crude residue in CH2Cl2
(1 mL) was added to tBuNH2 (0.130 mL, 1.2 mmol) in CH2Cl2
(1 mL) at 0 °C. The reaction mixture was stirred at the same tem-
perature for 2 h, and then warmed to room temperature for 3 h.
The reaction mixture was washed with HCl (0.1 m) and saturated
NaHCO3, dried with MgSO4 and filtered. The filtrate was concen-
trated to afford N-tert-butyl-2-methyl-5-[3-(trifluoromethyl)-3H-di-
azirin-3-yl]benzenesulfonamide as a pale yellow oil. 1H NMR
(270 MHz, CDCl3): δ = 7.84 (d, J = 2.3 Hz, 1 H), 7.35 (d, J =
8.0 Hz, 1 H), 7.29 (dd, J = 2.3, 8.0 Hz, 1 H), 4.45 (s, 1 H), 2.67 (s,
3 H), 1.23 (s, 9 H) ppm. CrO3 (6.0 mg, 0.06 mmol) and acetic
anhydride (0.430 mL, 4.5 mmol) were added to ortho-periodic acid
(1.06 g, 4.6 mmol) in CH3CN (10 mL). The crude material in a
minimum volume in CH3CN was added at 0 °C. The reaction mix-
ture was stirred at room temperature for 2 d and concentrated. The
residue was dissolved in EtOAc and washed with saturated
NaHCO3, saturated Na2S2O3, and brine. The organic layer was
dried with MgSO4, filtered, and concentrated. The crude oil was
subjected to silica-gel column chromatography (hexane/CH2Cl2,
3:1) to afford 6a as a pale yellow amorphous solid (0.056 g, 28%).
UV/Vis (CH3OH): λmax [log (ε/m–1 cm–1)] = 290 (850), 340
1
(303) nm. H NMR (270 MHz, CD3OD): δ = 8.11 (d, J = 8.0 Hz,
1 H), 7.90 (s, 1 H), 7.84 (d, J = 8.0 Hz, 1 H) ppm. 13C NMR
(68 MHz, CD3OD): δ = 161.3, 142.3, 137.3, 133.8, 130.6, 126.8,
1
2
123.1 (q, JC,F = 275.1 Hz), 120.4 29.7 (q, JC,F = 41.2 Hz) ppm.
19F NMR (470 MHz, CD3OD): δ = –66.7 ppm. HRMS (ESI):
calcd. for C9H5F3N3O3S 292.0004; found 292.0020.
5-[3-(Trifluoromethyl)-3H-diazirin-3-yl]-1,2-benzisothiazole-3-one
1,1-Dioxide (7b): Compound 6b (13.2 mg, 38 μmol) in TFA (2 mL)
was treated in the same manner as described for 6a. The residue
was recrystallized from EtOAc and hexane at –20 °C to afford 7b
as a colorless amorphous solid (6.0 mg, 54%). UV/Vis (CH3OH):
λmax [log(ε/m–1 cm–1)] = 280 (1005), 340 (280) nm. 1H NMR
(270 MHz, CD3OD): δ = 8.11 (d, J = 8.0 Hz, 1 H), 7.86 (s, 1 H),
7.83 (d, J = 8.0 Hz, 1 H) ppm. 13C NMR (68 MHz, CD3OD): δ =
1
161.2, 142.3, 136.5, 134.3, 130.5, 123.9, 123.1, 123.0 (q, JC,F
=
275.1 Hz), 29.5 (q, JC,F = 41.2 Hz) ppm. 19F NMR (470 MHz,
CD3OD): δ = –62.7 ppm. HRMS (ESI): calcd. for C9H5F3N3O3S
292.0004; found 292.0014.
2
HPLC Purification for 7a and 7b: The suspension of 7a and 7b in
aqueous solution were made alkaline with NaOH (1 m). The so-
dium salts were subjected to HPLC [Tosoh TSKgel ODS-80 Ts
(4.6ϫ250 mm), 30% MeOH, 1 mL/min, detection at 215 nm or
350 nm].
Photolysis of a Methanolic Solution of 7a and 7b: Methanolic
solutions of 7a and 7b (1 mm) were irradiated with black light
(15 W) at a distance 3 cm. Spectra were recorded at minute-long
intervals. The decrease in absorbance at around 350 nm was plotted
and used to calculate half-life. 19F NMR (470 MHz, CD3OD): δ =
–78.0 ppm (from 7a and 7b). HRMS (ESI): calcd. for
C10H9F3NO4S 296.0199; found 296.0191 (from 7a), 296.0194 (from
7b).
1
(300) nm. H NMR (270 MHz, CDCl3): δ = 8.03 (d, J = 8.0 Hz, 1
H), 7.64 (s, 1 H), 7.59 (d, J = 8.0 Hz, 1 H), 1.77 (s, 9 H) ppm. 13C
NMR (68 MHz, CDCl3): δ = 158.8, 138.7, 136.2, 131.9, 128.2,
1
2
125.2, 121.4 (q, JC,F = 275.5 Hz), 118.4, 61.8, 28.4 (q, JC,F
=
41.6 Hz), 27.8 ppm. 19F NMR (470 MHz, CDCl3): δ = –64.7 ppm.
HRMS (ESI): calcd. for C13H13F3N3O2S 348.0630; found Gustatory-Tasting Effect Assay: Synthetic compounds 7a and 7b
348.0646.
were tested in a gustatory-tasting effect assay. This test used Ca2+
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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M. Hashimoto et al.
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Acknowledgments
The authors are very grateful to Professor G. D. Holman (Univer-
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M. H. thanks the Japanese Suhara Memorial Foundation for finan-
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Materials and Devices”.
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Published Online: ᭿
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Eur. J. Org. Chem. 0000, 0–0

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Date: 31-03-15 16:48:51
Pages: 7
Synthesis of Diazirine Based Photoreactive Saccharin Derivatives
Nitrogen Heterocycles
L. Wang, T. Yoshida, Y. Muto, Y. Murai,
Z. P. Tachrim, A. Ishida, S. Nakagawa,
Y. Sakihama, Y. Hashidoko, K. Masuda,
Y. Hatanaka, M. Hashimoto* ........... 1–7
Saccharin is one of the most common arti-
ficial sweeteners. Synthesis of photoreactive
saccharin derivatives that contain a (tri-
fluoromethyl)diazirinyl moiety at the 5- or
6-position is reported. These saccharin de-
rivatives could be applied for functional
analysis of gustatory receptors.
Synthesis of Diazirine-Based Photoreactive
Saccharin Derivatives for the Photoaffinity
Labeling of Gustatory Receptors
Keywords: Synthetic methods
/ Photo-
affinity labeling / Receptors / Nitrogen het-
erocycles / Saccharin
Eur. J. Org. Chem. 0000, 0–0
© 0000 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7