A novel biotinylated diazirinyl ceramide analogue for photoaffinity labeling

Article abstract of DOI:10.1016/j.bmcl.2007.11.071

A novel photoreactive ceramide analogue, which contains (3-trifluoromethyl)phenyldiazirinyl lipid and biotinylated sphingosine, was synthesized. The probe was recognized as an antigen by anti-ceramide antibody and as a substrate for sphingolipid ceramide N-deacylase. These results indicate that the probe may be useful as a photoaffinity-biotinylating agent in sphingolipid studies.

Full text of DOI:10.1016/j.bmcl.2007.11.071

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 650–652
A novel biotinylated diazirinyl ceramide analogue
for photoaffinity labeling
Makoto Hashimotoa,* and Yasumaru Hatanakab
aDepartment of Agricultural and Life Science, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho,
Obihiro 080-8555, Hokkaido, Japan
bFaculty of Pharmaceutical Sciences, Toyama University, 2360 Sugitani, Toyama 930-0194, Japan
Received 4 September 2007; revised 30 October 2007; accepted 17 November 2007
Available online 22 November 2007
Abstract—A novel photoreactive ceramide analogue, which contains (3-trifluoromethyl)phenyldiazirinyl lipid and biotinylated
sphingosine, was synthesized. The probe was recognized as an antigen by anti-ceramide antibody and as a substrate for sphingolipid
ceramide N-deacylase. These results indicate that the probe may be useful as a photoaffinity-biotinylating agent in sphingolipid
studies.
Ó 2007 Elsevier Ltd. All rights reserved.
Research into glycosphingolipids has been attracting
increasing attention recently because they are closely in-
volved in cell adhesion, and the regulation of cell growth
and differentiation.1,2 They consist of a hydrophilic su-
gar moiety and hydrophobic ceramide.
to solve these difficulties by using the combinational
introduction of a diazirinyl photophore and an avidin–
biotin system (photoaffinity biotinylation).6–9 In this pa-
per, we describe a novel photoreactive ceramide ana-
logue for use in photoaffinity biotinylation of
glycosphingolipid-related biomolecules.
Although many investigations have reported the biolog-
ical roles of sugar moieties, there is little information on
those of the ceramide moiety in glycosphingolipids.
Several diazirine-based photoreactive ceramide10–13 or
glycosphingolipid14–16 analogues have been reported,
but they have no tag or radiolabeled iodine on the ben-
zene ring to detect photolabeled components. This is not
an easy method to handle radiolabeled compounds for
organic synthesis.
Photoaffinity labeling is a useful biochemical method to
reveal structural and functional relationships between
low molecular weight bioactive compounds and biomol-
ecules.3 The method is suitable for analyzing biological
interactions because it is based on the affinity of bioac-
tive compounds for biomolecules. Various photophores,
such as phenyldiazirine, arylazide, and benzophenone,
are used.4 Although comparative irradiation studies of
these three photophores in living cells indicated that a
carbene precursor (3-trifluoromethyl)phenyldiazirine is
the most promising photophore,4 the rather complicated
synthesis of the (3-trifluoromethyl)phenyldiazirinyl
three-membered ring has resulted in fewer applications
in biomolecular studies than other photophores. Fur-
thermore, the low cross-linking yield of photoaffinity
labeling experiments still hampers the purification and
isolation of labeled components.5 We have attempted
For photoaffinity biotinylation we attempted to intro-
duce a biotinylated diazirinyl fatty acid N-hydroxy-
succinimide ester
3 to lyso GM1 1 to give a
photoreactive GM1 analogue 2 in moderate yield. Com-
pound 2 was assayed using Pseudomonas sp. sphingo-
lipid ceramide N-deacylase (SCDase), which generates
sphingosine and fatty acids from the ceramide moiety
of sphingolipids,17–20 but unfortunately, the photoreac-
tive GM1 analogue 2 was not a substrate recognized
by the enzyme. We therefore had to establish new con-
cepts applying photoaffinity biotinylation to SCDase.
SCDase recognized not only sphingolipids but also cer-
amide, which is composed of fatty acid and D-erythro
sphingosine. We introduced a photoreactive group and
biotin to fatty acid and D-erythro sphingosine,
respectively.
*
Corresponding author. Tel.: +81 155 49 5542; fax: +81 155 49
5577; e-mail: hasimoto@obihiro.ac.jp
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.071

M. Hashimoto, Y. Hatanaka / Bioorg. Med. Chem. Lett. 18 (2008) 650–652
651
D-Erythro sphingosine derivatives were synthesized as
reported by Koskinen, with slight modifications.21 10-
Undecenoyl chloride 4 was converted to tert-butyl ester
5, followed by conversion to aldehyde 6 by oxidation of
olefin with sodium periodate and OsO4 in moderate
yield. Trans-selective double bond formation with com-
pound 6 and b-ketophosphonate 7 was achieved by a
modified Horner–Wadsworth–Emmons reaction with
K2CO3 in CH3CN. Enone reduction of 8 with DI-
BAL-H in toluene gave anti-diastereomer 9 as the main
product. The terminal aldehyde was converted to Schiff
base 10 with biotinhydrazide. After acid deprotection,
diazirinyl fatty acid N-hydroxysuccinimide ester 115
was introduced to give biotinylated diazirinyl ceramide
derivative 12.22 Several sphingolipids have antigenic
activities that are used to detect the localization of these
compounds in the cell.23 We examined whether photore-
active ceramide analogue 12 was recognized by mouse
anti-ceramide antibody. The synthetic ceramide ana-
logue was developed on TLC. The developed sphingo-
lipid was transferred to PVDF membrane (far-Eastern
blotting) as reported in the literature24 (Figs. 1 and 2).
The membrane was subjected to two immunodetection
methods.
The first method was treatment with streptavidin–horse-
radish peroxidase (HRP) conjugate to detect the biotin
component. As the second method, we used mouse
anti-ceramide antibody and subsequently goat anti-
mouse IgM-HRP conjugate to detect the ceramide skel-
eton. Both treatments then used chemiluminescence re-
agents to detect the immune complex (Fig. 3, lanes 1
and 2, respectively). Both detection methods gave a
chemiluminescence signal in the same position on
TLC. The result indicated that the synthetic ceramide
derivative 12 was recognized by both streptavidin and
the mouse anti-ceramide antibody without dissociation
of the Schiff base moiety.
Enzymatic reaction of compound 12 resulted in biotinyl-
ated D-erythro sphingosine 13 and diazirinyl fatty acid
derivative 14 under hydrolyzed conditions by SCDase.
In contrast, compounds 13 and 14 were condensed to
12 under reverse reaction conditions by SCDase
(Fig. 4).17–20 It is therefore evident that SCDase recog-
nized compound 12 as a substrate.
OH OH
O
OH
O
OH
O
HO
OH
O
O
OH
O
HO
HN
R
Photoaffinity labeling of SCDase with compound 12 was
performed under hydrolysis conditions. The enzyme was
incubated briefly with excess photoligand, and then the
mixture was irradiated with a black light lamp (15 W)
CH3COHN
HOOC
O
O
(CH2)14CH3
O
HO HO
HO
HO
HO
O
OH
HO
H3COCHN
HO
1 R = H
N
F3C
O
i)
2 R =
N
HN
NH
O
lane
1
2
H
N
O
O
O
N
H
O
S
Figure 3. Immunochemical detection of compound 12 with streptavi-
din–HRP (for biotin, lane 1) and mouse anti-ceramide antibody (for
ceramide skeleton, lane 2). Compound 12 was developed on silica-TLC
(CHCl3/CH3OH = 9:1), and then the TLC was subjected to far-Eastern
blotting on a PVDF membrane.23 The membrane was treated for
chemiluminescence detection as described previously.24
C
O
O
N
H
Figure 1. Synthesis of biotinylated diazirinyl GM1 derivative 2.
Reagents and conditions: (i) 3 (2 R-OSu), triethylamine, DMF, rt,
8 h, 65%.
O
O
O
i)
ii)
O
H
(CH2)8
O
(CH2)8
(CH2)8
Cl
O
O
6
5
4
O
O
O
P(OCH3)2
iii)
v)
iv)
O
NBoc
NBoc
OH
O
O
8
7
O
S
HN
O
NH
OH
O
H
NNH
O
NBoc
NBoc
O
O
10
9
N
N
F3C
HN
NH
O
OH
HN
NNH
O
S
CF3
N
O(CH2)10COOSu
vi)
11
HO
12
N
O
Figure 2. Synthesis of bifunctional ceramide derivatives 12. Reagents and conditions: (i) tert-butanol, TEA, rt, 2 h (91%); (ii) OsO4, NaIO4, dioxane,
H2O, rt, 1 h (60%); (iii) 6, K2CO3, CH3CN, rt, 12 h (72%, 93% ee); (iv) DIBAL-H, toluene, À70 °C, 1 h (67%, syn/anti = 1:6); (v) biotin hydrazide,
DMF, rt, 12 h (93%); (vi) 50% TFA, CH2Cl2, then, 11, CHCl3 CH3OH, TEA, rt, 3 h (50%).

652
M. Hashimoto, Y. Hatanaka / Bioorg. Med. Chem. Lett. 18 (2008) 650–652
Acknowledgments
O
HN
NH
O
OH
HN
This research was supported partly by Grants-in-Aid for
Scientific Research on a Priority Area, 18032007, and
for Scientific Research (C), 19510210 from the Ministry
of Education, Science, Sports and Culture (to M.H.).
M.H. also thanks the Fugaku Foundation and Research
for Promoting Technological Seeds for financial support
for the study.
NNH
O
S
CF3
N
HO
HO
12
N
O
i)
O
ii)
HN
NH
O
OH
NNH
S
N
N
NH2
F3C
References and notes
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+
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14
O(CH2)10COOH
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Figure 4. Enzymatic reaction for synthetic compounds by SCDase.
Reagents and conditions: (i) 0.8% Triton X-100, 50 mM sodium
acetate buffer, pH 6.0, 37 °C, 12 h, 86%; (ii) 0.1% Triton X-100, 25 mM
sodium phosphate buffer, pH 7, 48 h, 25%.
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at 0°C for 20 min. Competitive inhibition was per-
formed in the presence of an excess amount of ganglio-
side mixtures as the natural substrate. The irradiated
samples were subjected to SDS–PAGE followed by
Western blotting to detect the labeled components by
chemiluminescence as described previously.25 The
chemiluminescence signal was detected in a substance
of the reported molecular weight of SCDase (52 KDa)
(Fig. 5, lane 1). Competitive inhibition of photoaffinity
biotinylation was observed in the presence of the gangli-
oside mixtures (Fig. 5, lane 2); therefore, compound 12
was incorporated in the binding site of the natural
substrates.
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Sphingolipids have been studied recently because they
are highly enriched intracellularly in the cell membrane
of most mammalian cells.1,2 The compartmentalization
of sphingolipids in membranes serves as the starting
pool for sphingolipid metabolism. All metabolites of
sphingolipids presumably function either as intercellular
secondary messengers or as ligand molecules for cell sur-
face receptors. Ceramide biosynthesized from dihydro-
ceramide and sphingomyelin is well known as the
precursor for sphingoshine-1-phosphate, which is a
member of the lysophospholipid growth factor family;
however, there have been few reports on the structure–
activity relationships of fatty acid and sphingosine moi-
eties, due to the difficulty of obtaining their derivatives.
This is the first report, to our knowledge, of the synthe-
sis of a bifunctional ceramide analogue, and will help to
elucidate the functions of sphingolipids.
22. Compound 12 FAB-MS (negative) m/z 864 ([MÀH]+); 1H
NMR (CDCl3) d 7.30 (1H, t, J = 8.3 Hz), 6.92 (1H, d,
J = 8.3 Hz), 6.75 (1H, d, J = 8.3 Hz), 6.66 (1H, s), 5.72
(1H, m), 5.51 (1H, m), 5.20 (1H, m), 4.52 (dd, 2H, J = 7.6,
4.9 Hz), 4.36 (dd, 2H, J = 7.6, 4.4 Hz), 3.90 (2H, t,
J = 6.4 Hz), 3.36 (m, 2H), 3.24 (m, 1H), 2.97 (dd, 1H,
J = 12.9, 4.9 Hz), 2.74 (d, 1H, J = 12.9 Hz), 2.54 (2H, m),
2.33 (m, 2H), 1.80–1.20 (34H, m).
52 kDa
23. Benjamins, J. A.; Callahan, R. E.; Montgomery, I. N.;
Studzinski, D. M.; Dyer, C. A. J. Neuroimmnol. 1987, 14,
325.
24. Ishikawa, D.; Taki, T. Methods Enzymol. 2000, 312,
145.
lane
1
2
Figure 5. Chemiluminescence detection of photoaffinity labeled
SCDase with compound 12. Labeled proteins without (lane 1) or with
(lane 2) excess ganglioside mixtures (Sigma G-2375) were subjected to
SDS–PAGE (10%), followed by transfer to a PVDF membrane to
detect the biotin moiety.
25. Hatanaka, Y.; Hashimoto, M.; Nishihara, S.; Narimatsu,
H.; Kanaoka, Y. Carbohydr. Res. 1996, 294, 95.