Simple synthesis of deuterium and 13C labeled trifluoromethyl phenyldiazirine derivatives as stable isotope tags for mass spectrometry

Article abstract of DOI:10.1248/cpb.52.1385

The synthesis of trifluoromethyl diazirine with a stable isotope tag is reported. We found that both Friedel-Crafts acylation and reduction of aryl carbonyl to methylene, using commercially available stable-isotope reagents, were utilized for the synthesi

Full text of DOI:10.1248/cpb.52.1385

November 2004
Notes
Chem. Pharm. Bull. 52(11) 1385—1386 (2004)
1385
Simple Synthesis of Deuterium and 13C Labeled Trifluoromethyl
Phenyldiazirine Derivatives as Stable Isotope Tags for Mass Spectrometry
b
Makoto HASHIMOTO*,a and Yasumaru HATANAKA
a Department of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine; Inada-cho,
Obihiro, Hokkaido 080–8555, Japan: and b Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical
University; 2360 Sugitani, Toyama 930–0194, Japan.
Received June 29, 2004; accepted September 10, 2004; published online September 10, 2004
The synthesis of trifluoromethyl diazirine with a stable isotope tag is reported. We found that both
Friedel–Crafts acylation and reduction of aryl carbonyl to methylene, using commercially available stable-iso-
tope reagents, were utilized for the synthesis of diazirinyl fatty acid derivatives. The stable isotope labeled
diazirine may be valuable for identifying binding sites by mass spectrometry.
Key words diazirine; photoaffinity label; stable isotope
Photoaffinity labeling is a powerful method in the study of with deuterium labeled triethylsilane in unlabeled TFA, was
biological structures and functions.1,2) It will be suitable for easily achieved for both 13C and D labeled fatty acid deriva-
analysis of biological interactions in vivo because it is based tive 6b.
on the affinity of the ligand moiety. Various photophores,
The photoreactive 4b was converted to succinimide ester,
such as phenyldiazirine, arylazide and benzophenone have then reacted with psychosine to make a photoreactive galac-
been used. Comparative irradiation studies of these three tosylceramide (Gal-Cer) analogue. The synthesized Gal-Cer
photophors in living cells suggested that a carbene precursor analogue was used for Far Eastern blotting8) for transfer onto
(3-trifluoromethyl)phenyldiazirine is the most promising.3)
a PVDF membrane. As it has homology to the natural
MS spectrometry enables us to identify important struc- sphigolipid, it was recognized by the anti Gal-Cer antibody
tures in target biomolecules.4) Elucidation of the different as an antigen. This result indicates that the introduction of an
mass number derived from a mixture of unlabeled and stable- ethyl moiety into the benzene ring of a diazirinyl fatty acid
isotope labeled photophors may be useful in identifying does not affect antigenicity (Fig. 2).
photoligand components on the MS spectrum. However, to
Friedel–Crafts acylation followed by the reduction of aryl
the best of our knowledge, few protocols for the synthesis of ketone to methylene may be useful for the synthesis of stable
a stable-isotope labeled diazirinyl photophor have been isotope-labeled photoaffinity labeling reagents. Stable-iso-
reported. Here, we describe the effective synthesis of stable tope labeled diazirinyl compounds can be used to identify
isotope labeled diazirinyl compounds using commercially labeled regions by MS spectrometry.
available reagents. The construction of diazirinyl skelton
needed at least 5 step reactions. Addition of the stable-
isotope after construction of the phenyl diazirinyl ring (post-
functional) is the recommended synthetic strategy. Recently,
the first example of reduction of an aryl carbonyl group with
trifluoromethyl diazirine to methylene without any photophor
damage was reported.5) This method was easily applied to the
synthesis of stable-isotope labeled diazirinyl derivatives
using commercially available reagents (Fig. 1). Friedel–
Crafts acylation of compound 1a6) with acetyl chloride-1-13C
easily introduced a 13C labeled acetyl group with a moderate
yield. The acetophenone derivatives 2a and 3a were treated
with deuterium labeled triethylsilane in unlabeled trifluo-
Fig. 1. Synthesis of Stable Isotope Labeled Diazirinyl Compounds
roacetic acid (TFA), to introduce two deuteriums on the
methylene moiety for both unlabeled 5a and 13C labeled 6a.
No differences in deuterium introduction were observed
when the reaction proceeded in deuterium-labeled TFA.
These strategies were applied to synthesize photoreactive
fatty acid derivatives.6) The diazirinyl fatty acid derivative
1b7) was subjected to Friedel–Crafts acetylation with acetyl
chloride to afford 2b. NOE studies of 2b revealed that the
isomer of the acetyl group has a concentration of less than
3%. The reduction of 2b with triethylsilane and TFA gave 4b
without damaging the alkoxy moiety. Friedel–Crafts acetyla-
Fig. 2. Immunodetection of Diazirinyl Galactosylceramide Analogue 7
tion with acetyl chloride-1-13C afforded 13C-labelled 3b in a
Lane 1, authentic galactosylceramide (a mixture of a-hydroxy and non-hydroxy fatty
acid); lane 2, compound 7; equal volumes are loaded on silica HPTLC.
manner identical to that of 13C-labeled 3a. Then reduction,
∗ To whom correspondence should be addressed. e-mail: hasimoto@obihiro.ac.jp
© 2004 Pharmaceutical Society of Japan

1386
Vol. 52, No. 11
Experimental
11-[2-[1-CD2]Ethyl-5-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl]oxy
Undecanoic Acid (5b) Compound 2b (0.0226 g, 52.75 mmol) was
dissolved in TFA (0.2 ml). Triethylsilane-D (0.016 ml, 99.32 mmol) was
1
All H-NMR spectra were measured using JEOL JNM-FX270 and ECA-
500 spectrometers. MS spectra were obtained using Hitach M-80B and
JEOL JNM-LA400 spectrometers. All stable isotope reagents were added to the TFA solution. The reaction mixture was treated in the same
purchased from Aldrich. Anti-galactosylceramide was purchased from
Sigma. All solvents were reagent grade and distilled using the appropriate
methods.
manner as that described for 4b to afford a colorless oil (0.0141 g,
1
33.86 mmol, 64%). H-NMR (CDCl3) d: 7.14 (1H, d, Jꢀ7.9 Hz), 6.71 (1H,
d, Jꢀ7.9 Hz), 6.55 (1H, s), 3.93 (2H, t, Jꢀ6.6 Hz), 2.35 (2H, t, Jꢀ7.6 Hz),
1.80—1.30 (16H, m), 1.15 (3H, s, CD2CH3). HR-MS m/z: 416.2268 (Calcd
3-(3-Methoxy-4-[1-13C]acetylphenyl)-3-trifluoromethyl-3H-diazirine
(3a) To a chilled suspension of AlCl3 (0.1300 g, 0.975 mmol) in CH2Cl2 for C21H27D2F3N2O3 (Mꢁ): 416.2254).
(0.1 ml) was added the solution of acetyl chloride-1-13C (0.1 ml,
1.406 mmol) in CH2Cl2 (0.1 ml) and compound 1a (0.0433 g, 0.200 mmol)
in CH2Cl2 (0.25 ml), respectively. The reaction mixture was warmed to room
temperature and stirred for 1 h. The mixture was poured into ice water and
11-[2-[1-13CD2]Ethyl-5-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl]oxy
Undecanoic Acid (6b) Compound 3b (14.0 mg, 32.60 mmol) and triethyl-
silane-D (0.015 ml, 94.25 mmol) were dissolved in TFA (0.2 ml). The reac-
tion mixture was treated in the same manner as that described for 4b to
CH2Cl2 to quench the reaction. The aqueous layer was washed with CH2Cl2. afford a colorless oil (11.2 mg, 26.83 mmol, 82%). 1H-NMR (CDCl3) d: 7.14
The organic layer was washed with saturated NaCl, dried over MgSO4, (1H, dd, JHHꢀ7.9 Hz, J13CCCHꢀ4.6 Hz), 6.71 (1H, d, Jꢀ7.9 Hz), 6.55 (1H, s),
filtered and concentrated. The residue was subjected to silica gel column
3.93 (2H, t, Jꢀ6.3 Hz), 2.35 (2H, t, Jꢀ7.6 Hz), 1.79 (2H, m), 1.64 (2H, m),
chromatography (ethyl acetate : hexaneꢀ1 : 8) to afford a colorless oil 1.79—1.25 (16H, m), 1.15 (3H, d, J13CCHꢀ4.3 Hz). 13C-NMR (CDCl3)
(0.0484 g, 93%). 1H-NMR (CDCl3) d: 7.72 (1H, dd,
JHHꢀ8.3 Hz,
d: 22.54 (J13CDꢀ19.6 Hz), HR-MS m/z: 417.2275 (Calcd for C2013CH27D2
J
13CCCHꢀ4.3 Hz), 6.80 (1H, d, Jꢀ8.3 Hz), 6.66 (1H, s), 3.90 (3H, s), 2.58 F3N2O3 (Mꢁ): 417.2287).
(3H, d,
J
13CCHꢀ6.3 Hz), 13C-NMR (CDCl3) d: 198.76, HR-MS m/z:
Photoreactive Galactosylceramide Derivative (7) Compound 6b
(4.7 mg, 11.38 mmol) was dissolved in CH2Cl2 (0.5 ml). N-Hydroxysuccin-
imide (9.0 mg, 0.078 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)-
259.0635 (Calcd for C1013CH9F3N2O2 (Mꢁ): 259.0650).
3-(4-[1-D2]Ethyl-3-methoxyphenyl)-3-trifluoromethyl-3H-diazirine
(5a) To a stirred solution of compound 2a (15.0 mg, 58.1 mmol) in trifluo- carbodiimide hydrochloride (18.2 mg, 0.095 mmol) in CH3CN (0.25 ml) was
roacetic acid (0.01 ml) at room temperature was added triethylsilane-D added. The reaction mixture was stirred for 10 h and poured into mixed solu-
(0.004 ml, 25.1 mmol). The reaction mixture was stirred for a further 30 min
tion of H2O and hexane. The organic layer was washed with saturated NaCl,
at room temperature and diluted with hexane. The organic layer was washed dried over MgSO4, filtered and concentrated. The residue and psychosine
with saturated sodium bicarbonate and saturated NaCl, dried with MgSO4,
filtered and concentrated. The residue was subjected to silica gel column
(8.2 mg, 17.76 mmol) were dissolved in CHCl3 and methanol (0.5 ml, 2 : 1).
Triethylamine (50 ml) was added at room temperature. The reaction mixture
chromatography (ethyl acetate : hexaneꢀ1 : 8) to afford a colorless oil was stirred at room temperature for 24 h in the dark then subjected to silica
1
(10.5 mg, 73%). H-NMR (CDCl3) d: 7.15 (1H, d, Jꢀ7.9 Hz), 6.74 (1H, d, gel column chromatography (CHCl3 : methanolꢀ6 : 1) to afford a colorless
Jꢀ7.9 Hz), 6.57 (1H, s), 3.82 (3H, s), 1.14 (3H, s), HR-MS m/z: 246.0967 mass (4.2 mg, 43%). 1H-NMR (CD3OD) d: 7.21 (1H, d, Jꢀ7.9 Hz), 6.75
(Calcd for C11H9D2F3N2O (Mꢁ): 246.0947).
(1H, d, Jꢀ7.9 Hz), 6.63 (1H, s), 5.685 (1H, m), 5.45 (1H, m), 4.22 (1H, t,
Jꢀ6.6 Hz), 4.00—3.30 (12H, m), 2.64 (1H, q, Jꢀ7.6 Hz), 2.21 (2H, m),
3-(4-[1-13CD2]Ethyl-3-methoxyphenyl)-3-trifluoromethyl-3H-diazirine
(6a) To a stirred solution of compound 3a (18.5 mg, 71.38 mmol) in triflu- 2.06 (2H, m), 1.84 (2H, m), 1.62—1.31 (12H, m), 1.16 (3H, d, Jꢀ7.6 Hz),
oroacetic acid (0.2 ml) was added triethylsilane-D (0.03 ml, 187.8 mmol) at 0.88 (3H, m), HR-FAB-MS m/z: 858.5477 (Calcd for C45H75F3N3O9
room temperature. The reaction mixture was treated in the same manner as (MꢁHꢁ): 858.5455).
that described for 5a, to obtain a colorless oil (12.8 mg, 73%). 1H-NMR
(CDCl3) d: 7.15 (1H, dd, JHHꢀ7.9 Hz, J13CCCHꢀ4.3 Hz), 6.74 (1H, d,
Immunodetection of the Compound
7 Compound 7 in CHCl3 :
methanolꢀ2 : 1 (3.26 mM, 4 ml) was spotted onto a silica HPTLC plate, then
Jꢀ7.9 Hz), 6.58 (1H, s), 3.82 (3H, s), 1.15 (3H, d, J13CCHꢀ4.3 Hz). 13C- developed with CHCl3 : methanolꢀ6 : 1. The plate was dipped in
NMR (CDCl3) d: 22.37 (J13CDꢀ19.6 Hz), HR-MS m/z: 247.0972 (Calcd for
2-PrOH : 10% CaCl2 : methanolꢀ40 : 20 : 7 for 20 s and overlaid with a
PVDF membrane and glass fiber, then heated at 180 °C for 30 s with an
C1013CH9D2F3N2O2 (Mꢁ): 247.0981).
11-[2-Acetyl-5-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl]oxy Unde- iron.8) The membrane was soaked with the above buffer for 5 s, 0.1% Tween
canoic Acid (2b) To a chilled suspension of AlCl3 (0.8548 g, 6.41 mmol)
in CH2Cl2 (10 ml) was added acetyl chloride (1.0 ml, 14.06 mmol) and
20-PBS (T-PBS) for 5 min, 10% skimmed milk in T-PBS for 1 h, washed
twice with T-PBS for 10 min, incubated in 50 times diluted anti-galactosyl-
compound 1b (0.3023 g, 0.78 mmol) in CH2Cl2 (1 ml), respectively. The ceramide at room temperature for 2 h, washed with T-PBS twice for 10 min,
reaction mixture was warmed to room temperature and stirred for 1h, then then incubated in 21300 times diluted anti-rabbit IgG peroxidase conjugate
poured into ice water and CH2Cl2. The aqueous layer was washed with in 1% BSA in T-PBS at room temperature for 1 h. After washing with T-PBS
CH2Cl2 three times. The organic layer was washed with saturated NaCl, five times for 10 min, the membrane was subjected to chemiluminescence
dried over MgSO4, filtered and concentrated. The residue was subjected to
detection.
silica gel column chromatography (ethyl acetate : hexaneꢀ1 : 3) to afford a
pale yellow oil (0.2433 g, 0.57 mmol, 73%). H-NMR (CDCl3) d: 7.73 (1H,
d, Jꢀ8.3 Hz), 6.80 (1H, d, Jꢀ8.3 Hz), 6.65 (1H, s), 4.034 (2H, t, Jꢀ6.6 Hz),
2.61 (3H, s), 2.35 (4H, m), 1.80—1.20 (14H, m), HR-MS m/z: 428.1918
(Calcd for C21H27F3N2O4 (Mꢁ): 428.1923).
11-[2-Ethyl-5-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl]oxy Unde-
canoic Acid (4b) Compound 2b (0.0496 g, 0.116 mmol) was dissolved in
TFA (1 ml). Triethylsilane (0.1 ml, 0.627 mmol) was added to the TFA solu-
tion. The reaction mixture was treated in the same manner as that described
for 5a and the residue was subjected to silica gel column chromatography
(ethyl acetate : hexaneꢀ1 : 5) to afford a colorless oil (0.0375 g, 78%).
1H-NMR (CDCl3) d: 7.14 (1H, d, Jꢀ7.9 Hz), 6.71 (1H, d, Jꢀ7.9 Hz), 6.55
(1H, s), 3.92 (2H, t, Jꢀ6.3 Hz), 2.62 (2H, q, Jꢀ7.6 Hz), 2.35 (2H, t,
Jꢀ7.6 Hz), 1.80—1.30 (16H, m), 1.16 (3H, t, Jꢀ7.6 Hz), HR-MS m/z:
414.2107 (Calcd for C21H29F3N2O3 (Mꢁ): 414.2130).
1
Acknowledgments This research was partially supported by the
Ministry of Education, Science, Sports and Culture, Grant-in-Aid for
Encouragement of Young Scientists, 16710151 (M.H.), by Exploratory
Research 14658185 and by CLUSTER (Cooperative Link of Unique Science
and Technology for Economy Revitalization) (Y.H.). We also thank Dr. N.
Nakajima (Toyama Prefectural University) for measurement of the MS
spectra.
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11-[2-[1-13C]Acetyl-5-(3-trifluoromethyl-3H-diazirin-3-yl)phenyl]oxy
Undecanoic Acid (3b) To a chilled suspension of AlCl3 (0.0757 g,
0.568 mmol) in CH2Cl2 (0.3 ml) was added the solution of acetyl chloride-1-
13C (0.075 ml, 1.055 mmol) in CH2Cl2 (0.3 ml) and compound 1b (0.0170 g,
0.044 mmol) in CH2Cl2 (0.5 ml), respectively. The reaction mixture was
treated in the same manner as that described for 3a and the residue was
subjected to silica gel column chromatography (ethyl acetate : hexaneꢀ1 : 4)
to afford a colorless oil (0.0140 g, 74%). 1H-NMR (CDCl3) d: 7.74 (1H, dd,
JHHꢀ8.2 Hz, J13CCCHꢀ4.0 Hz), 6.80 (1H, d, Jꢀ8.2 Hz), 6.65 (1H, s), 4.04
(2H, m), 2.61 (3H, d, J13CCHꢀ6.3 Hz), 2.35 (4H, m), 1.80—1.20 (14H, m),
13C-NMR (CDCl3) d: 198.97, HR-MS m/z: 429.1938 (Calcd for
C2013CH27F3N2O4 (Mꢁ): 429.1956).