Synthesis of sphingomyelin sulfur analogue and its behavior toward sphingomyelinase

Article abstract of DOI:10.1016/j.bmcl.2005.02.020

The sulfur analogue of sphingomyelin was designed and stereoselectively synthesized from S-benzyl-N-Boc-cysteine. The introduction of the phosphoryl choline moiety was successfully achieved by our own method using 2-bromoethyl dimethyl phosphite and carbon tetrabromide followed by a trimethylamine treatment. The synthesized compound proved to be a useful substrate for monitoring the enzyme activity of sphingomyelinase by detecting the liberated thiol group with a thiol-sensitive reagent.

Full text of DOI:10.1016/j.bmcl.2005.02.020

Bioorganic & Medicinal Chemistry Letters 15 (2005) 2141–2144
Synthesis of sphingomyelin sulfur analogue and its
behavior toward sphingomyelinase
Toshikazu Hakogi,a Shinobu Fujii,b Michio Morita,b
Kiyoshi Ikedab and Shigeo Katsumuraa,*
aSchool of Science, Kwansei Gakuin University, Gakuen, Sanda, Hyogo 669-1337, Japan
bDepartment of Biochemistry, Osaka University of Pharmaceutical Sciences, Nasahara, Takatsuki, Osaka 569-1094, Japan
Received 8 January 2005; revised 5 February 2005; accepted 5 February 2005
Available online 19 March 2005
Abstract—The sulfur analogue of sphingomyelin was designed and stereoselectively synthesized from S-benzyl-N-Boc-cysteine. The
introduction of the phosphoryl choline moiety was successfully achieved by our own method using 2-bromoethyl dimethyl phosphite
and carbon tetrabromide followed by a trimethylamine treatment. The synthesized compound proved to be a useful substrate for
monitoring the enzyme activity of sphingomyelinase by detecting the liberated thiol group with a thiol-sensitive reagent.
Ó 2005 Elsevier Ltd. All rights reserved.
1. Introduction
logue 1 (Fig. 2) and its behavior toward B. cereus
SMase.
Previously, we reported the syntheses of the carbon and
nitrogen analogues of sphingomyelin, in which the phos-
phonate oxygen to be hydrolyzed by sphingomyelinase
(SMase) was replaced by carbon1 and nitrogen atoms,2
respectively. These new analogues showed moderate
inhibitory activities toward Bacillus cereus SMase. Gen-
erally, SMase produces phosphoryl choline and cera-
mide resulting from the specifically catalyzed
hydrolysis of the phosphoester linkage of sphingomye-
lin.3 The produced ceramide has been known as a lipid
second messenger in mammalian cell membranes and
plays key roles in the cellular signal transmission path-
way, in particular, as a signal transduction factor in cell
differentiation and apoptosis derivation (Fig. 1).4 To
elucidate the detailed catalytic mechanism of SMase,
development of sphingomyelin analogues that competi-
tively act at the catalytic site and strongly inhibit the
hydrolytic ability of the enzyme, has strongly been
desired.5
A sulfur analogue would behave in a manner similar to
that for the original sphingomyelin, since sulfur and
oxygen atoms possess very similar orbital arrangements
particularly in the s and p orbitals. Therefore, the ana-
logue 1 was expected to act as an inhibitor or as a sub-
strate for SMase.
For example, a sulfur analogue of glycerophospholipids
was reported by MartinÕs group6 to be a strong inhibitor
toward phosphatidylcholine-specific phospholipase C
(PC-PLC). On the other hand, phosphatidylinositol-spe-
cific phospholipase C (PI-PLC)7 and phospholipase A2
(PLA2)8 were shown to be capable of cleaving the S–P
bond of the thiophosphate linkage in addition to the
O–P bond of the phosphoester linkage. Therefore, sulfur
analogues have been used as substrates for continuous
spectrophotometric assays of PI-PLC and PLA2, in
which the release of the free thiol could be followed by
the coupling reaction with a colorimetric thiol reagent.9
A number of methods to measure SMase activity have
been developed, and NBD-labeled sphingomyelin,10 2-
hexadecanoylamino-4-nitrophenylphosphorylcholine,11
In this paper, we describe the highly efficient and
stereocontrolled synthesis of the short chain sulfur ana-
14
and C-labeled sphingomyelin12 have often been used
as substrates. Unlike these compounds, the analogue 1
might have the possibility to be hydrolyzed by SMase,
and might be useful as a substrate for the continuous
spectrophotometric assay.
Keywords: Sphingomyelinase; Substrate analogue.
*
Corresponding author. Tel.: +81795658314; fax: +81795659077;
e-mail: katsumura@ksc.kwansei.ac.jp
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.02.020

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T. Hakogi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2141–2144
Figure 1. Sphingolipid catabolite.
Figure 2. Designed sulfur analogue of sphingomyelin.
2. Results and Discussion
In order to achieve the efficient synthesis of 1, we started
the synthesis from the Weinreb amide 3, which was
easily prepared from S-benzyl-N-Boc-cysteine 2 accord-
ing to the literature13 in 80% yield (Scheme 1).
The monoalkynylation of the amide successfully pro-
ceeded at ꢀ43 °C with 3 equiv of lithium acetylide to af-
ford 4 in 80% yield. The anti-selective reduction of this
ketone 4 was investigated. Reduction with diisobutylalu-
minum 2,6-di-tert-butyl-4-methylphenoxide,14 diisobut-
ylaluminum hydride, and Red-Al gave a 4:1, 3:1, and
5:2 mixture of the anti- and syn-derivatives, respectively.
Reduction with sodium borohydride produced no selec-
tivity. When lithium tri-tert-butyloxyaluminohydride,
however, was employed in ethanol at ꢀ78 °C,15 the
selectivity was improved to 6:1 by NMR, and both iso-
mers were not separable at this stage. The reduction
products were transformed into the corresponding oxazo-
lidinone derivative 6 by a sodium hydride treatment in
THF at 50 °C. The stereochemistry of the major product
was confirmed to be a syn-orientation concerning the
two substituents on the oxazolidinone ring by compar-
ing the J4–5 value of the oxazolidinone derivative 6;
J4–5 of the major product was 7.8 Hz, and that of minor
product was 5.8 Hz.16 The undesired stereoisomer could
be separated by simple column chromatography after
introduction of a tert-butoxycarbonyl group into the
carbamate nitrogen of 6. The pure oxazolidinone deriva-
tive thus obtained was hydrolyzed by treatment with
cesium carbonate in methanol to give 7.17 The treatment
of 7 with lithium in liquid NH3 under reflux produced
the desired thiol 8 in 93% yield resulting from the reduc-
tion of the triple bond and removal of the protecting
group at the primary mercapto group. In this reaction,
the corresponding dimeric disulfide was often produced
Scheme 1. (i) C5H11CCLi, THF, ꢀ43 °C; (ii) LiAl(OBu)3H, EtOH,
ꢀ78 °C; (iii) NaH, THF, 50 °C; (iv) (a) Boc2O, DMAP, Et3N, DMF,
0 °C; (b) separation; (c) Cs2CO3, MeOH, 47% for six steps; (v) Li, liq.
NH3, THF, reflux, 93%; (vi) (a) TBSCl, DMAP, Et3N, DMF, 73%; (b)
TBAF, THF, ꢀ78 °C, quant.
and the yield of the desired compound was lowered, un-
less a careful workup under nitrogen was employed. In
order to introduce a phosphoryl choline group into the
primary mercapto group, protection of the secondary
hydroxy group was necessary. Fortunately, after protec-
tion of both the mercapto and hydroxy groups of 8 as
tert-butyldimethylsilyl ethers, selective deprotection of
the primary mercapto group was successful by treatment
with TBAF in THF at ꢀ78 °C to quantitatively produce 9.
With the desired thiol 9 in hand, the introduction of a
phosphorylcholine group into the mercapto group was
examined. In our previous synthesis of sphingomyelin,

T. Hakogi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2141–2144
2143
Table 1. Phosphorylation
Conditions
Yield
Products
No reaction
or <5%
Unidentified
product
Scheme 2. (i) B, CBr4, pyr, 0 °C, quant.; (ii) 2N HCl, MeOH; (iii)
Ac2O, pyr; (iv) TFA, CH2Cl2, then 1N NaOH, C5H11COCl, 88%; (v)
Me3N, toluene, 95%; (vi) KOH, MeOH, 90%.
57%a
lective synthesis of the sulfur analogue 1 was efficiently
achieved from cysteine.22
Quant.
The inhibitory activity of analogue 1 toward B. cereus
SMase was tested,23 and we found that this compound
seemed to inhibit the enzyme activity much weaker than
the previous carbon and nitrogen analogues of sphingo-
myelin,1,2 but was hydrolyzed by this enzyme to release
a free thiol. This compound 1 appeared to be a useful
substrate for monitoring the enzyme activity of SMase,
because the liberated thiol group easily reacts with a
thiol-sensitive reagent such as 5,50-dithiobis-(2-nitroben-
zoic acid) (DTNB) to form the yellow dianion of 5-thio-
2-nitrobenzoic acid (TNB), which can be continuously
measured spectrophotometrically.9 Figure 3 shows the
time-course of the increases in absorbance after the
addition of B. cereus SMase in the presence of several
concentrations of the mixed micelle of the sulfur ana-
logue 1 with Triton X-100 (the molar ratio of 1:10).
The result showed that the sulfur analogue 1 was hydro-
lyzed by SMase and the initial velocities could be deter-
mined. Thus, the sulfur analogue 1 proved to be a useful
substrate for the SMase assay.
a The reaction conditions were not optimized.
this group was successfully introduced with cyclic chlo-
rophosphate in the presence of triethylamine.18 Under
this condition, however, the phosphorylation of 9 pro-
ceeded only with a low yield. After various attempts as
shown in Table 1,19 the phosphorylation of 9 was
realized by treatment with trimethyl phosphite A and car-
bon tetrabromide20 to afford the corresponding dimeth-
ylester 10 in 57% yield. Based on the obtained re-
sults, we designed 2-bromoethyl dimethyl phosphite B
instead of trimethyl phosphite A and expected to obtain
the desired compound 11, which would be easily trans-
formed into the desired choline moiety. Compound B
was actually prepared by the reaction of methyl dichlo-
rophosphite with 2-bromoethanol and methanol in the
presence of diisopropylethylamine in THF at
ꢀ78 °C,21 and compound 2 was treated with a mixture
of B and carbon tetrabromide in pyridine at 0 °C to suc-
cessfully and quantitatively produce the corresponding
11. Moreover, the obtained compound 11 showed an or-
dinary polarity, and its purification and transformation
were much easier than the compounds containing the
highly polar phosphorylcholine moiety.
The remaining issues from 11 to 1 were the introduction
of an acyl group at the nitrogen group and formation of
the choline moiety (Scheme 2). Transformation of the
secondary hydroxy protecting group into an acetyl
group, subsequent removal of the tert-butoxycarbonyl
group, and introduction of an acyl group onto the
resulting primary amine afforded 14, which was treated
with anhydrous trimethylamine in toluene in a pressure
bottle at 60 °C to quantitatively give the desired choline
compound 15. Finally, deprotection of the secondary
hydroxy group produced the desired compound 1 after
purification by reverse phase HPLC. Thus, the stereose-
Figure 3. Enzymatic hydrolysis of mixed micelle of sulfur analogue 1
with Triton X-100 (the molar concentration of 1:10) by B. cereus
SMase. The analogue concentrations: (1) 1.6; (2) 1.2; (3) 1.0; (4) 0.8; (5)
0.6; (6) 0.4; (7) 0.2 mM.

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T. Hakogi et al. / Bioorg. Med. Chem. Lett. 15 (2005) 2141–2144
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Triton X-100 (the molar ratio of 1:10) at the ionic
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3418, 2928, 1640, 1547, 1229, 1073 cmꢀ1 1H NMR
;
(CD3OD, 400 MHz) d 5.70 (td, J = 6.8, 15.1 Hz, 1H),
5.45 (mdd, J = 7.1, 15.4 Hz, 1H), 4.28–4.30 (m, 2H), 4.02
(dd, J = 7.1, 7.8 Hz, 1H), 3.97 (ddd, J = 3.7, 6.8, 8.8 Hz,
1H), 3.62–3.72 (m, 2H), 3.24 (s, 9H), 3.13 (ddd, J = 3.7,
12.2, 13.7 Hz, 1H), 2.83 (ddd, J = 8.8, 13.7, 14.9 Hz, 1H),
2.19 (t, J = 6.8 Hz, 2H), 2.03 (td, J = 7.1, 7.1 Hz, 2H), 1.61
(tt, J = 7.3, 7.3 Hz, 1H), 1.28–1.42 (m, 10H), 0.92 (t,
J = 6.8 Hz, 3H), 0.90 (t, J = 6.8 Hz, 3H); 13C NMR
(CDCl3, 100 MHz): d 176.2, 134.9, 130.8, 75.1, 67.5 (m),
60.5 (JC–P = 5.0 Hz), 56.1 (JC–P = 2.5 Hz), 54.78, 54.75,
54.7, 49.0, 49.4, 49.2, 49.0, 48.8, 48.6, 48.4, 37.3, 33.4, 32.6
(3C), 30.0, 26.9, 23.6, 23.5, 14.4, 14.3; FAB HRMS m/z
calcd for C21H44N2O5PS (M++H) 467.2708, found
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