Versatile synthesis of phenoxydiazirine-based fatty acid analogues and photoreactive galactosylceramide

Article abstract of DOI:10.1016/S0960-894X(01)00669-2

A versatile synthesis of diazirine-based photoreactive fatty acid analogues is reported. The key step is phenoxy alkylation of diazirine with halo alkyl acid esters. The conditions described will be acceptable for the synthesis of various alkyl-length derivatives. The fatty acid derivatives are acceptors for reverse reactions of sphingolipid ceramide N-deacylase (SCDase), which catalyzes the condensation of psychosine and fatty acids to form photoreactive galactosylceramide. The photoreactive galactosylceramide can also be prepared with chemical synthesis, condensation of psychosine and fatty acid succinimidyl ester, and is recognized with anti-GarCer antibody both before and after irradiation.

Full text of DOI:10.1016/S0960-894X(01)00669-2

Bioorganic & Medicinal Chemistry Letters 12 (2002) 89–91
Versatile Synthesis of Phenoxydiazirine-Based Fatty Acid
Analogues and Photoreactive Galactosylceramide
Makoto Hashimoto,a,* Yasumaru Hatanakab and Kensuke Nabetaa
aDepartment of Bioresource Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro 080-8555, Hokkaido, Japan
bFaculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, 2360 Sugitani, Toyama, 930-0194 Japan
Received 23 August 2001; accepted 4 October 2001
Abstract—A versatile synthesis of diazirine-based photoreactive fatty acid analogues is reported. The key step is phenoxy alkylation
of diazirine with halo alkyl acid esters. The conditions described will be acceptable for the synthesis of various alkyl-length deri-
vatives. The fatty acid derivatives are acceptors for reverse reactions of sphingolipid ceramide N-deacylase (SCDase), which cata-
lyzes the condensation of psychosine and fatty acids to form photoreactive galactosylceramide. The photoreactive
galactosylceramide can also be prepared with chemical synthesis, condensation of psychosine and fatty acid succinimidyl ester, and
is recognized with anti-GarCer antibody both before and after irradiation. # 2001 Elsevier Science Ltd. All rights reserved.
Photoaffinity labeling is a well-established method to
elucidate ligand–biomolecule interactions.1À5 Many
investigators consider carbene precursors, especially
3-phenyl-3-trifluoromethyl diazirine, as the photo-
reactive groups of choice, because of the specific char-
acter of the photolabeling reaction.4,6 Photoaffinity
labeling with fatty acid analogues will reveal hydro-
phobic interactions between biomolecule and lipids,7
but the complicated synthesis of the 3-phenyl-3-tri-
fluoromethyl diazirinyl three-membered ring has resul-
ted in fewer applications of it in biomolecular studies
than those of other photophors (azide and benzophe-
none). The synthesis of photoreactive fatty acid analo-
gues presents a similar problem. A few routes for
synthesis of diazirine-based fatty acid analogues were
reported,8,9 but the fatty acid equivalents should be
introduced before construction of diazirinyl three-
membered ring. We have been elucidating approaches
for the post-functionalization of the 3-phenyl-3-tri-
fluoromethyl diazirinyl photophor.10À13 In this paper,
we describe a versatile and easy approach for synthesis
of diazirine-based fatty acid analogues by phenoxy
alkylation, introduction to sphingolipid, and the biolo-
gical properties of the photoreactive compounds.
To archive versatile synthesis of photoreactive fatty
acids, m-methoxy diazirine 1,10 which is available for
large scale preparation (over 0.5 mol), was demethy-
lated with BBr3 to afford 2. The phenolic compound
was subjected to the phenoxy alkylation condition11
with methyl 11-bromoundecanoate. The reaction pro-
ceeded smoothly at 60 ꢀC to afford phenoxydiazirinyl
fatty acid methyl ester 3 with yield of 70%. The reaction
with ethyl 5-bromovalerate afforded the shorter carbon-
length fatty-acid derivative 6 under the same condition.
These esters were easily hydrolyzed to afford the corre-
sponding acids 4 and 7. These synthetic routes will be
more convenient to synthesis variable carbon length
diazirine-based photoreactive fatty acid analogues than
previous routes. The carboxylic acid 4 was converted to
succinimidyl ester 5 with N-hydroxysuccinimide and
water-soluble carbodiimide in CH3CN in moderate
yield (86%). The compound 5 reacted with psychosine
8, lyso-galactosylceramide, in the anhydrous triethyl-
amine and DMF to afford photoreactive galactosylcera-
mide analogues 9 with 74% yield (Fig. 1).14 The
synthetic routes will enable us to condense variable
length photoreactive fatty acid and various N-deacyl-
ated sphingolipids (sphingosine, lysosphingomyelin,
lysoganglioside, etc.) to synthesis of various photo-
reactive sphingolipid derivatives.
It is recently reported that sphingolipid ceramide N-
deacylase (SCDase) catalyze the hydrolytic break down
*Corresponding author. Tel.: +81-155-49-5542; fax: +81-155-49-
5577; e-mail: hasimoto@obihiro.ac.jp
0960-894X/02/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00669-2

90
M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 12 (2002) 89–91
of N-acyl linkage between fatty acids and sphingosine
bases, and its reverse reaction, condensation of sphin-
golipid and fatty acid, under different detergent and pH
conditions.15À18 To elucidate the biological properties of
phenoxy diazirinyl fatty acid, the compound 4 was sub-
jected to enzymatic condensation of psychosine 8 with
reverse reaction of SCDase. The same amount of psy-
chosine and photoreactive fatty acid 4 (10 nmol) were
incubated with SCDase (10 mU) at 37 ꢀC for 48 h. One
fifth of the fatty acid was converted to photoreactive
galactosylceramide 9. The results indicated that the
enzyme recognized phenoxy diazirinyl fatty acid 4 as
substrate. It is first time that the enzyme utilized the
photoreactive fatty acid derivative to produce sphingoli-
pids. The compounds 4 will be useful to elucidate hydro-
phobic interaction between fatty acid and those utilized
enzymes.
The photoreactive galactosylceramide analogue 9 was
applied to irradiation experiments to ensure photo-
activation abilities. The compound 9 in methanol/
chloroform=1:2 (0.7 mM) was placed at distance 2 cm
from black light lamp(15 W) at 0 ꢀC. The spectrogram
at each interval was measured. The diazirine specific
broad adsorption at 355 nm was smoothly decreased
during the irradiation (Fig. 2A). The half-life of the
compound in this condition was calculated as 1.7 min
from semi-log plot (Fig. 2B). Photoreactive fatty acid
derivatives 4 also smoothly decomposed in the same
condition (t1/2 was calculated as 1.2 min in CHCl3).
Several sphingolipids have antigen activities used to
detect localization of these compounds in the cell. Anti-
galactosylceramide antibody is of great interest due to
its usefulness as oligodendroglial markers. We per-
formed an antigenicity test of photoreactive galacto-
sylceramide 9 against rabbit anti-galactosylceramide
antibody.20 The synthetic galactosylceramide analogues,
one is un-irradiated and the other one is irradiated for
30 min, and enzymatic synthesis reaction mixture (Fig. 3
lanes 1, 2 and 3, respectively) were developed on TLC.
The developed sphingolipids were transferred to PVDF
membrane (far-eastern blotting) in a manner identical
with literature.19 The membrane was subjected to
Figure 1. Synthesis of diazirine based fatty acids and galactosylcer-
amide: (i) BBr3, CH2Cl2, 0 ꢀC, 85%; (ii) Br(CH2)10COOCH3, (n-
Bu)4NI, DMF, 60 ꢀC, 19 h, 70%; (iii) NaOH, CH3OH, rt, 2 h, 90%;
(iv) HOSu, EDC–HCl, CH3CN, rt, 3 h, 86%; (v) Br(CH2)4COOC2H5,
(n-Bu)4NI, DMF, 60 ꢀC, 12 h, 60%; (vi) NaOH, C2H5OH, rt, 2 h,
95%; (vii) DMF, TEA, rt, 10 h, 74%; (viii) SCDase, 0.1% Triton X-
100, phosphate buffer pH 7.0, 37 ꢀC, 48 h, 20%.
Figure 3. Immunodetection of photoreactive galactosylceramide ana-
logues, blotted to PVDF membrane, by rabbit anti-galactosylceramide
antibody. TLC plate was developed with CHCl3/methanol=6:1, then
transferred to PVDF membrane in a manner identical with litera-
ture. The membrane was blocked for 1 h in 5% skim milk in 0.1%
Tween 20, phosphate-buffered saline pH 7.4 (T-PBS), washed twice
with T-PBS for 5 min and immersed in rabbit anti-galactosylceramide
antibody (1:50 dilution of the manufacturer’s solution) for 2 h. After
washing six times with T-PBS for 5 min, the membrane was immersed
in goat anti-rabbit IgG peroxidase conjugate (1:25,000 dilution of the
manufacturer’s solution) for 1 h, then washed with T-PBS six times.
The immuno-treated membrane was subjected to chemiluminescence
detection and exposed to film for 0.5 min. Lane 1: chemically synthe-
sized compounds 9; lane 2: photolyzed compound 9 in CHCl3/metha-
nol=2:1; lane 3: enzymatic reaction mixture of psychosine and fatty
acid 4 with SCDase.
Figure 2. Photolysis of a synthetic galactosylceramide analogue 7: (A)
UV spectra of the photolysis reaction mixture at times (in min) indi-
cated with numbers; (B) the decay of the absorbance at 355 nm as a
function of time of photolysis in a semi-log representation.

M. Hashimoto et al. / Bioorg. Med. Chem. Lett. 12 (2002) 89–91
91
immunodetection with rabbit anti-galactosylceramide
antibody and goat anti-rabbit IgG peroxidase conjugate,
then treated with chemiluminescence reagent to detect
galactosylceramide analogues. Both un-irradiated and
irradiated diazirine based galactosylceramide analogue
was recognized by the rabbit anti-galactosylceramide
antibody. It is first time photoactivated galactosylcer-
amide was recognized by anti-galactosylceramide anti-
body. Furthermore, the enzymatic reaction mixture of
fatty acid 4 and psychosine afforded chemiluminescence
signal. We subjected psychosine, lesser antigen for the
antibody, for far-eastern blotting and immunodetection,
but no chemiluminescence signals was afforded. It can
be presumed that psychosine was removed from PVDF
membrane during the immunodetection process. It is
considered that lipophilicity of fatty acid moiety should
be important in this manner. The results indicate that
small amounts of enzymatic reaction are detectable with
this method and without a radioisotope precursor.
5. Dorm, G.; Prestwich, G. D. Trends Biotechnol. 2000, 18,
64.
6. Gillingham, A. K.; Koumanov, F.; Hashimoto, M.; Hol-
man, G. D. Detection and Analysis of Glucose Transporters
Using Photolabelling Techniques. In Membrane Transport: A
Practical Approach; Baldwin S. A. Ed.; Oxford University
Press: Oxford, 2000; p193.
7. Knoll, F.; Kolter, T.; Sandohoff, K. Methods Enzymol.
1999, 311, 568.
8. Weber, T.; Brunner, J. J. Am. Chem. Soc. 1995, 117, 3084.
9. Li, G.; Bittman, R. Tetrahedron Lett. 2000, 41, 6737.
10. Hatanaka, Y.; Hashimoto, M.; Kurihara, H.; Nakayama,
H.; Kanaoka, Y. J. Org. Chem. 1994, 59, 383.
11. Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. Bioorg. Med.
Chem. 1994, 2, 1367.
12. Hashimoto, M.; Kanaoka, Y.; Hatanaka, Y. Heterocycles
1997, 46, 119.
13. Hashimoto, M.; Hatanaka, Y.; Nabeta, K. Bioorg. Med.
Chem. Lett. 2000, 10, 2481.
14. Compound 3: EI-MS m/z 372 (M+ÀN2); 1H NMR
(CDCl3) d 7.43 (1H, dd, J=8.6, 7.6 Hz), 7.07 (1H, d, J=8.6
Hz), 6.90 (1H, d, J=7.6 Hz), 6.82 (1H, s), 4.08 (2H, t, J=6.4
Hz), 3.81 (3H, s), 2.45 (2H, t, J=7.5 Hz), 1.90 (2H, m), 1.78
(2H, m), 1.72–1.45 (12H, m).
Compound 4: EI-MS m/z 358 (M+ÀN2); 1H NMR (CDCl3)
d 7.19 (1H, dd, J=8.3, 7.6 Hz), 6.84 (1H, d, J=8.3 Hz), 6.67
(1H, d, J=7.6 Hz), 6.59 (1H, s), 3.85 (2H, t, J=6.4 Hz), 2.27
(2H, t, J=7.4 Hz), 1.69 (2H, m), 1.56 (2H, m), 1.36–1.23 (12H,
m).
Compound 5: EI-MS m/z 456 (M+ÀN2); 1H NMR (CDCl3)
d 7.21 (1H, dd, J=8.2, 7.9 Hz), 6.86 (1H, d, J=8.2 Hz), 6.68
(1H, d, J=7.9 Hz), 6.61 (1H, s), 3.87 (2H, t, J=6.4 Hz), 2.77
(4H, s), 2.54 (2H, t, J=7.4 Hz), 1.68 (4H, m), 1.42–1.25 (12H,
m).
Specific tags should be needed to manipulate photo-
labeled components from the labeled mixture. We have
previously reported that combination of photoaffinity
labeling and avidin–biotin technology (photoaffinity
biotinylation) is a useful non-radioisotopic method to
detect and retrieve the labeled components from a
complicated mixture.10,13,21À25 Here we show that blot-
ted and irradiated photoreactive galacytosylceramide
was recognized by a specific antibody. This indicates
that the technique of antigen–antibody interaction for
label reagent itself will also be useful to apply to isola-
tion of labeled components. The galactosylceramide is
closely related to Krebbe’s disease26 and metachromatic
leukodystrophy.27 The diazirine-based photoreactive
galactosylceramide should be useful for investigating
the molecular mechanisms in biological functions.
Compound 9: FAB-MS m/z 830 ([M+H]+); 1H NMR
(CD3OD) d 7.40 (1H, dd, J=8.3, 7.9 Hz), 7.06 (1H, d, J=8.3
Hz), 6.82 (1H, d, J=7.9 Hz), 6.72 (1H, s), 5.73 (1H, m), 5.48
(1H, m), 4.26 (1H, t, J=7.3 Hz), 4.00 (2H, t, J=6.4 Hz), 3.90–
3.30 (10H, m), 2.21 (2H, m), 2.06 (2H, m), 1.84 (2H, m), 1.62–
1.31 (12H, m), 0.93 (3H, m).
15. Ito, M.; Kurita, T.; Kita, K. J. Biol. Chem. 1995, 270,
24370.
16. Mitsutake, S.; Kita, K.; Okino, N.; Ito, M. Anal. Biochem.
1997, 247, 52.
Acknowledgements
This research was partially supported by the Ministry of
Education, Science, Sports and Culture, Grant-in-Aid
for Encouragement of Young Scientists, 12780433
(M.H.), for Scientific Research 12470504 and for Scien-
tific Research on Priority Areas (C) ‘Genome Science’,
No. 13202023 (Y.H.). M.H. thanks the Akiyama
Foundation for support of this work.
17. Ito, M.; Mitsutake, S.; Tani, M.; Kita, K. Methods Enzy-
mol. 1999, 311, 682.
18. Kita, K.; Kurita, T.; Ito, M. Eur. J. Biochem. 2001, 268,
592.
19. Benjamins, J. A.; Callahan, R. E.; Montgomery, I. N.;
Studzinski, D. M.; Dyer, C. A. J. Neuroimmunol. 1987, 14, 325.
20. Ishikawa, D.; Taki, T. Methods Enzymol. 2000, 312, 145.
21. Hatanaka, Y.; Hashimoto, M.; Nishihara, S.; Narimatsu,
H.; Kanaoka, Y. Carbohydr. Res. 1996, 294, 95.
22. Hatanaka, Y.; Hashimoto, M.; Kanaoka, Y. J. Am.
Chem. Soc. 1998, 120, 453.
References and Notes
23. Hashimoto, M.; Hatanaka, Y. Chem. Pharm. Bull. 1999,
47, 667.
24. Hashimoto, M.; Yang, J.; Holman, G. D. ChemBioChem.
2001, 2, 52.
25. Hashimoto, M.; Hatanaka, Y.; Yang, J.; Dhesi, J.; Hol-
man, G. D. Carbohydr. Res. 2001, 331, 119.
26. Wenger, D. A.; Rafi, M. A.; Luzi, P.; Datto, J.; Costan-
tino-Ceccarini, E. Mol. Genet. Metab. 2000, 70, 1.
27. Gieselmann, V.; Polten, A.; Kreysing, J.; von Figura, K. J.
Inherit. Metab. Dis. 1994, 17, 500.
1. Bayley, H. In Laboratory Techniques in Biochemistry and
Molecular Biology: Photogenerated Reagents in Biochemistry
and Molecular Biology; Work, T. S., Burdon, R. H., Eds.;
Elsevier: Amsterdam, 1983; p25.
2. Brunner, J. Annu. Rev. Biochem. 1993, 62, 483.
3. Kotzyba-Hilbert, F.; Kapfer, I.; Goeldner, M. Angew.
Chem., Int. Ed. Engl. 1995, 34, 1296.
4. Hatanaka, Y.; Nakayama, H.; Kanaoka, Y. Rev. Heteroatom.
Chem. 1996, 14, 213.