An efficient and convenient synthesis of deuterium-labelled seminolipid isotopomers and their ESI-MS characterization

Article abstract of DOI:10.1016/j.chemphyslip.2008.02.002

Seminolipids 1a and 1b and galactosylalkylacylglycerols 2a and 2b, labelled with deuterium on the alkyl or acyl chain, respectively, were obtained isotopically and chemically pure through a straightforward synthesis from protected glycidyl galactoside 3 i

Full text of DOI:10.1016/j.chemphyslip.2008.02.002

Available online at www.sciencedirect.com
Chemistry and Physics of Lipids 152 (2008) 78–85
An efficient and convenient synthesis of deuterium-labelled seminolipid
isotopomers and their ESI-MS characterization
Laura Franchinia,∗, Luigi Panzab, Kessiri Kongmanasc, Nongnuj Tanphaichitrc,
Kym F. Faulld, Fiamma Ronchettia
a Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina, Universita` di Milano, Via Saldini 50, 20133-Milano, Italy
b Dipartimento di Scienze Chimiche, Alimentari, Farmaceutiche e Farmacologiche, Universita` del Piemonte Orientale,
Via Bovio 6, 28100-Novara, Italy
c Departments of Obstetrics and Gynecology, Biochemistry/Microbiology/Immunology, Ottawa Health Research Institute,
725 Parkdale Avenue, Ottawa, Ontario K1Y 4E9, Canada
d The Pasarow Mass Spectrometry Laboratory, NPI - Semel Institute for Neuroscience and Human Behavior, David Geffen School of Medicine,
University of California Los Angeles, 760 Westwood Plaza, Los Angeles, CA 90024, USA
Received 9 November 2007; received in revised form 6 February 2008; accepted 7 February 2008
Available online 14 February 2008
Abstract
Seminolipids 1a and 1b and galactosylalkylacylglycerols 2a and 2b, labelled with deuterium on the alkyl or acyl chain, respectively, were
obtained isotopically and chemically pure through a straightforward synthesis from protected glycidyl galactoside 3 in an overall 22% yield. The
identity and purity of compounds was ascertained by NMR spectroscopy and ESI mass spectrometry analysis. These labelled compounds are
important as internal standards for quantification of these lipids by mass spectrometry, and they could also be used in metabolic studies in in vitro
and even in vivo systems. Extension of the procedure could provide a route for the preparation of isotopomers of other compounds of the same
general class.
© 2008 Elsevier Ireland Ltd. All rights reserved.
Keywords: Sperm–egg interaction; Seminolipid; Mammalian sulfoglycolipids; Deuterium isotopomers; ESI-MS
1. Introduction
and alkyl chain lengths, and in the degree of unsaturation and
the presence or absence of hydroxylation, SGG from boar, rat
(Kornblatt et al., 1972), human (Ueno et al., 1977) and bovine
(Alvarez et al., 1990) spermatozoa is composed mainly of hex-
adecyl and hexadecanoyl alkyl and acyl chains, respectively.
Like sulfatide (cerebroside sulfate), the other main sulfogly-
colipid in mammals, seminolipid 1 (SGG, Fig. 1) is biosyn-
thesized by the sequential reactions of UDP-galactose:ceramide
galactosyltransferase (CGT) which promotes galactosylation of
alkylacylglycerol (Van der Bijl et al., 1996), and cerebroside
sulfotransferase (CST) which is responsible for the sulfation of
galactosylalkylacylglycerol 2 (GG, Fig. 1) (Honke et al., 1997;
Zhang et al., 1990). Seminolipid is degraded to GG by the action
of arylsulfatase A that catalyzes also the desulfation of sulfatide
(Tanphaichitr et al., 2003).
The sulfated glycerogalactolipid seminolipid is the main gly-
colipid in mammalian spermatozoa and testis (up to 3% of the
total lipids and 90% of total glycolipids in boar testis) (Ishizuka
et al., 1973; Kornblatt et al., 1972; Ueno et al., 1977; Alvarez et
al., 1990).
It was identified as a single molecular species with the
general composition: 1-O-alkyl-2-O-acyl-3-O-␤-d-(3ꢀ-sulfo)-
galactopyranosyl-sn-glycerol. While most naturally occurring
glycolipids exhibit a high degree of heterogeneity in their acyl
Abbreviations: ESI-MS, electrospray ionization mass spectrometry;
MS/MS, tandem mass spectrometry; LC, liquid chromatography; Q1,
first quadrupole; Q3, third quadrupole; THF, tetrahydrofuran; EDCI,
(1-ethyl-3-[3-(dimethylamino)-propyl]carbodiimide hydrochloride; DMAP, 4-
di∗methylaminopyridine; EtOAc, ethyl acetate.
Seminolipid is synthesized in primary spermatocytes and
remains stable during spermatogenesis. It is essential for this
process to occur normally (Fujimoto et al., 2000), although the
precise role of SGG in spermatogenesis and other processes that
Corresponding author. Tel.: +39 02 50316049.
E-mail address: franchini laura@hotmail.com (L. Franchini).
0009-3084/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.chemphyslip.2008.02.002

L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
79
[16,16,16-2H3]-fatty acyl chain. Since in our synthetic sequence
(see Scheme 1) SGG 1a and SGG 1b are obtained by direct sul-
fation of the GG 2a and GG 2b precursors (Fig. 1), this route
provides also a straightforward access to deuterium-labelled iso-
topomers of galactosylalkylacylglycerol.
2. Experimental
2.1. General
Optical rotations were determined on a PerkinElmer 241
polarimeter in a 1 dm cell at 20 ◦C using chloroform solu-
tions. All NMR spectra were recorded at 303 K with a
Bruker FT-NMR Avance DRX500 spectrometer in CDCl3 or
CDCl3/CD3OD solutions with TMS as internal standard; chem-
ical shifts are reported as δ (ppm) relative to CHCl3 fixed at
7.24 ppm for CDCl3 and 7.60 ppm for CDCl3/CD3OD solu-
tions, for 1H NMR spectra and relative to CDCl3 fixed at
77.00 ppm for 13C NMR spectra. All reactions were moni-
tored by thin layer chromatography (TLC) on Silica Gel 60
F-254 plates (Merck) with detection by spraying with 50%
H2SO4 solution or with phosphomolybdate-based reagent and
heating to 110 ◦C. Flash column chromatography was per-
formed on Silica Gel 60 (230–400 mesh, Merck). Dry solvents
and liquid reagents were distilled prior to use: tetrahydrofu-
ran (THF) was distilled from sodium; dichloromethane was
Fig. 1. Structures of SGG, GG and their deuterium-labelled isotopomers.
take place during and following egg fertilization, is still not com-
pletely understood. SGG is exposed on the plasma membrane
of spermatogenic cells and sperm, and like other glycolipids
it contributes to the sperm cell membrane shape and stability
(Attar et al., 2000; Bou Khalil et al., 2006), being also involved
in recognition events (at least 20 proteins with affinity to SGG
have been identified) and cell–cell adhesion. Specifically, SGG
on the mammalian sperm head surface has direct affinity for the
egg zona pellucida (ZP) and is an integral component of sperm
lipid rafts, which are the ZP binding microdomains (White et al.,
2000; Weerachatyanukul et al., 2001; Bou Khalil et al., 2006).
Severe diseases are associated with defective enzymes in
glycolipid metabolism: in humans affected by metachromatic
leukodistrophy the deficit of arylsulfatase A, and less frequently
the deficit of the lipid transporter saposin B (also known as
cerebroside sulfate activator protein), causes the accumulation
of sulfatide and subsequent neurological damage (Norris et al.,
2005), while in male mice lacking the CGT gene, the absence
of GG and SGG may be responsible for apoptosis of germ cells,
which subsequently leads to a spermatogenesis arrest at the pri-
mary spermatocyte level (Zhang et al., 2005). Since sperm SGG
is involved in sperm–egg interaction, it is possible that decreased
levels of sperm SGG are one of the causes of male infertility
and/or subfertility. In this context, characterization, monitoring
and quantitative determination of SGG and its metabolites (e.g.,
GG) in biological samples is important. Mass spectrometry, due
to an unparalleled combination of sensitivity and specificity, is
extensively applied to analyze and quantify lipids and glycol-
ipids; in particular SGG and GG were analyzed by ESI-MS and
their concentration in the total lipid extracts from mice testis
was determined by LC-ESI-MS (Tadano-Aritomi et al., 2003).
The accuracy of the quantitative determination of SGG and GG
from biological extracts could be improved by the availability
of suitable internal standards (Mills et al., 2005) with the same
chemical structure as the analyte and the same fragmentation
patterns. Such internal standards can be generated by incorpo-
ration of stable labels such as deuterium in the lipid chains of
seminolipid.
˚
distilled from calcium hydride; methanol was dried on 4 A
molecular sieves. All solvents and reagents were purchased
from Aldrich; [16,16,16-2H3]-hexadecanoic acid (99.9 atom%
D) was purchased from CDN isotopes. (R)-Glycidyl-2,3,4,6-
tetra-O-benzyl-␤-d-galactopyranoside 3 and 1-O-hexadecyl-3-
O-(2,3,4,6-tetra-O-benzyl-␤-d-galactopyranosyl)-sn-glycerol 7
were synthesized according to Lindberg et al. (2002); the phys-
ical data of the products agreed with those reported by these
authors.
2.2. Electrospray mass spectrometry
A triple quadrupole mass spectrometer (PerkinElmer
Sciex API III+) was tuned and calibrated as previously
described (Glasgow et al., 1998). Samples (20 pmol/␮L based
on dry weight assuming 100% purity) were dissolved in
methanol/chloroform (4/1, v/v, with or without 100 mM lithium
chloride), and injected (10–20 ␮L/injection) in a stream of
methanol/chloroform (4/1, v/v) entering an IonsprayTM ion
source. Mass spectra (positive and negative ion mode) were
obtained by scanning Q1 (m/z 200–1500, 0.3 Da step size, 1 ms
dwell time, 4.56 s/scan, orifice 70 V). Fragment ion spectra (pos-
itive and negative ion mode) of Q1 pre-selected parent ions
were produced by scanning Q3 (m/z 50–800, 0.3 Da step size,
2 ms dwell time, 5.13 s/scan, orifice 150–180 V) under colli-
sionally activated conditions (collision gas: 10% nitrogen in
argon), thickness instrumental setting (CGT) of 200, R0–R2
offset of 30 V). Averaging of all the scans accrued from each
sample injection was achieved with the MacSpecTM computer
program.
With the aim of quantifying SGG and GG in biological sam-
ples, as well as studying their metabolism in vivo, we have
synthesized two deuterium-labelled SGG isotopomers (Fig. 1):
SGG 1a with a [16,16,16-2H3]-ether chain and SGG 1b with a

80
L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
Scheme 1. (a) [16,16,16-2H3]-hexadecanol 4, BF3·OEt2, CH2Cl2, r.t., 60%; (b) see Lindberg et al. (2002); (c) [16,16,16-2H3]-hexadecanoic acid or hexadecanoic
acid, EDCI, DMAP, CH2Cl2, r.t., 72 h, 75%; (d) H2, Pd/C, EtOAc/MeOH 1:1, 24 h, 70%; (e) (i) Bu2SnO, MeOH, reflux, 2 h, (ii) Me3N·SO3, THF, r.t., 4 h, 70%.
2.3. Purification of pig testis SGG and GG
dropwise addition of saturated Na2SO4. CH2Cl2 (40 mL) was
added and the solution was filtered over celite cake, phases
separated and the organic layer was dried over Na2SO4 and con-
centrated. Flash chromatography (n-hexane/EtOAc 8:2) of the
product gave 4 (0.41 g, 85%) as a white solid. 1H NMR (CDCl3)
δ 1.20–1.45 (m, 26H, 13CH2), 1.50–1.65 (m, 2H, CH2), 3.70 (t,
3H, CH2OH). 13C NMR (CDCl3) δ 22.4, 25.7, 29.4–29.7, 31.8,
32.8, 63.1. ESI-MS (positive-ion mode): m/z 268 [M + Na]+.
Total lipids were extracted from pig testes using the method
of Bligh and Dyer (1959), as modified by Kates (1986). SGG
was then purified from total testis lipids via Bio-sil A column
chromatography, followed by preparative TLC (Kates, 1986;
Tupper et al., 1994). To remove endogenous cations, the puri-
fied SGG was converted to the free acid form and then to the
sodium salt form (SGG-Na+) as previously described (Kates,
1986, Tupper et al., 1994). The purity of the final product was
verified by mass spectrometry (see Section 3, Fig. 2) and analyt-
ical HPTLC using chloroform/methanol/water (65:25:4, v/v/v)
as eluent and orcinol and Coomassie blue G-250 staining, which
reveals glycolipids and all lipids bands, respectively. The puri-
fied SGG gave a single band with an Rf value of 0.329 with both
stains. The yield was ∼20 mg, accounting for approximately 1%
of the total testis lipids. GG was further prepared from the puri-
fied SGG by mild acid hydrolysis (Kornblatt et al., 1972); it also
appeared as a single band with an Rf of 0.659 and the identity
was also confirmed by mass spectrometry (see Section 3, Fig. 2).
2.5. 1-O-(16,16,16-2H3)-Hexadecyl-3-O-(2,3,4,
6-tetra-O-benzyl-β-d-galactopyranosyl)-sn-glycerol (5)
To a stirred solution of 3 (0.2 g, 0.34 mmol) and [16,16,16-
H3]-hexadecanol 4 (0.17 g, 0.68 mmol) and 4 A molecular
2
˚
sieves in CH2Cl2 (8 mL) was added BF3·Et2O (0.1 mL, 10% in
CH2Cl2). After 6 h more BF3·Et2O (0.1 mL) was added. After
48 h, the mixture was diluted with CH2Cl2 and washed with
aqueous sat. NaHCO3. The organic layer was filtered over celite
cake, dried over Na2SO4 and concentrated. Flash chromatog-
raphy (petroleum ether/EtOAc 8:2) gave 5 (0.18 g, 60%) as a
1
white solid. [␣]D = −6.0 (c 1). H NMR (CDCl3) δ 1.22–1.39
2.4. [16,16,16-2H3]-Hexadecanol (4)
(m, 26H, 13CH2), 1.53–1.62 (m, 2H, CH2), 3.18 (bs, 1H, OH),
3.40–3.65 (m, 8H, CH2CH2O, H-1a, H-1b, H-6ꢀa, H-6ꢀb, H-5ꢀ,
H-3ꢀ), 3.79 (dd, J3a,3b = 11.0 Hz, J2,3a = 6.7 Hz, 1H, H-3a), 3.86
(dd, J2,3 = 9.7 Hz, J1,2 = 7.7 Hz, 1H, H-2ꢀ), 3.87–3.94 (m, 2H,
To a stirred suspension of LiAlH4 (0.33 g, 8.82 mmol) in
THF (10 mL), a THF (10 mL) solution of [16,16,16-2H3]-
hexadecanoic acid (0.50 g, 1.96 mmol) was added via cannula
at 0 ◦C. The reaction was refluxed for 2 h, then quenched by
H-4ꢀ, H-3b), 3.96–4.02 (m, 1H, H-2), 4.39 (d, J1 ,2 = 7.7 Hz,
ꢀ
ꢀ
1H, H-1ꢀ), 4.42, 4.48, 4.64, 4.73, 4.77, 4.82, 4.91 and 4.96 (8d,

L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
81
8H, CH2Ph), 7.20–7.49 (m, 20H, Ph). 13C NMR (CDCl3) δ
22.4, 26.1, 29.3–29.7, 31.8, 68.7, 69.7, 71.6, 71.7, 73.1, 73.3,
73.4, 73.5, 73.5, 74.6, 75.3, 79.4, 82.2, 104.8, 127.6–128.4,
137.8–138.6. ESI-MS (positive-ion mode): found m/z 865.8,
calculated 865.16 Da for C53H712H3O8Na corresponding to
(M + Na)+.
173.3. ESI-MS (positive-ion mode): found m/z 726.8, calculated
1
726.62 Da for C41 H782H3O9Li corresponding to (M + Li)+.
The physical data of 2a is in agreement with those reported
for the unlabelled compound (Lindberg et al., 2002).
2.8. 1-O-(16,16,16-2H3)-Hexadecyl-2-O-palmitoyl-3-O-[3-
O-(sodium oxysulfonyl)-β-d-galactopyranosyl]-
sn-glycerol (1a)
2.6. 1-O-(16,16,16-2H3)-Hexadecyl-2-O-palmitoyl-3-O-
(2,3,4,6-tetra-O-benzyl-β-d-galactopyranosyl)-
sn-glycerol (6)
Compound 2a (0.02 g, 0.03 mmol), and Bu2SnO (0.01 g,
0.045 mmol) were stirred in MeOH (2 mL) at reflux under Argon
for 2 h. The solvent was removed under reduced pressure and
the dibutylstannylene complex was treated with Me3N·SO3
(0.008 g, 0.06 mmol) in THF (2 mL) for 2 h. The solvent was
removed under reduced pressure, then the residue was dissolved
inCHCl3/MeOH1:1(2 mL), loadedontoacationexchangeresin
column (Dowex 50 × 8 Na+ form, 0.5 cm × 6 cm), eluted with
CHCl3/MeOH (1:1), concentrated under reduced pressure and
subjected to flash chromatography (CHCl3/MeOH 9:1) to give
the target compound 1a (70%, 0.017 g) as a foam. [␣]D = +3.3
Compound 5 (0.10 g, 0.12 mmol) was dissolved in CH2Cl2
(6 mL), then hexadecanoic acid (0.06 g, 0.24 mmol), EDCI
(0.09 g, 0.48 mmol) and DMAP (0.04 g, 0.30 mmol) were added.
The reaction mixture was stirred at room temperature for
96 h, then diluted with CH2Cl2 and washed with aqueous sat.
NaHCO3. The layers were separated and the organic layer was
dried over Na2SO4 and concentrated. The crude product was
purified by flash chromatography (n-hexane/EtOAc 9:1) afford-
1
ing 6 (0.096 g, 75%) as a foam. [␣]D = +1.6 (c 1). H NMR
1
(c 0.4, CHCl3/MeOH, 1:1). H NMR (CDCl3/CD3OD, 1:1)
(CDCl3) δ 0.91 (t, J = 7.0 Hz, 3H, CH3), 1.20–1.45 (m, 50H,
25CH2), 1.53–1.62 (m, 2H, CH2), 1.49–1.66 (m, 4H, 2CH2),
2.23–2.35 (m, 2H, CH2), 3.37 (m, 1H, CH2O), 3.44 (m, 1H,
CH2O), 3.50–3.56 (m, 2H, H-5ꢀ, H-3ꢀ), 3.57–3.67 (m, 4H, H-
δ 0.91 (t, J = 7.0 Hz, 3H, CH3), 1.20–1.35 (m, 50H, 25CH2),
1.50–1.57 (m, 2H, CH2), 1.58–1.66 (m, 2H, CH2), 2.34 (m, 2H,
CH2), 3.39–3.52 (m, 2H, CH2O), 3.55 (t, J = 6.0 Hz, 1H, H-5ꢀ),
3.58–3.67 (m, 2H, H-1a, H-1b), 3.70–3.84 (m, 4H, H-2ꢀ, H-6ꢀa,
H-6ꢀb, H-3a), 3.95 (dd, J3a,3b = 11.0 Hz, J2,3a = 5.5 Hz, 1H, H-
3b), 4.23 (dd, J2,3 = 10.5 Hz, J3,4 = 3.0 Hz, 1H, H-3ꢀ), 4.27 (d,
1a, H-1b, H-6ꢀa, H-6ꢀb), 3.72 (dd, J3a,3b = 10.5 Hz, J2,3a = 4.5 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
1H, H-3a), 3.84 (dd, J2 ,3 = 9.7 Hz, J1 ,2 = 7.5 Hz, 1H, H-2 ),
ꢀ
ꢀ
ꢀ
3.91 (d, J3 ,4 = 3.5 Hz, 1H, H-4 ), 4.03 (dd, J3a,3b = 10.5 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
J3 ,4 = 3.0 Hz, 1H, H-4 ), 4.33 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 5.20
(m, 1H, H-2). 13C NMR (CDCl3/CD3OD, 1:1) δ 13.6, 22.5, 24.8,
25.9, 28.9–29.6, 31.8, 34.2, 61.2, 67.1, 69.2, 69.3, 71.5, 71.6,
74.8, 80.6, 103.6, 174.1.
ꢀ
ꢀ
ꢀ
J2,3b = 4.7 Hz, 1H, H-3b), 4.36 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 4.43,
4.47, 4.64, 4.72, 4.76, 4.78, 4.94 and 4.96 (8d, 8H, CH2Ph),
5.21 (m, 1H, H-2), 7.20–7.49 (m, 20H, Ph). 13C NMR (CDCl3)
δ 14.1, 22.4, 22.7, 24.9, 26.1, 29.1–29.7, 31.8, 31.9, 34.4, 68.2,
68.7, 69.3, 71.3, 71.6, 73.1, 73.5, 73.5, 73.6, 74.6, 75.0, 79.3,
82.1, 104.3, 127.5–128.4, 137.9, 138.5, 138.6, 138.8, 173.3.
ESI-MS (positive-ion mode): m/z 1103.0, calculated 1103.57 Da
for C69H1012H3O9Na corresponding to (M + Na)+.
ESI-MS (negative-ion mode): found m/z 798.5, calculated
1
798.55 Da for C41 H762H3O12S corresponding to (M − H)−.
2.9. 1-O-Hexadecyl-2-O-(16,16,16-2H3)-palmitoyl-3-O-
(2,3,4,6-tetra-O-benzyl-β-d-galactopyranosyl)-sn-glycerol
(8)
2.7. 1-O-(16,16,16-2H3)-Hexadecyl-2-O-palmitoyl-
3-O-(β-d-galactopyranosyl)-
Compound
7 (0.10 g, 0.12 mmol) was dissolved in
sn-glycerol (2a)
CH2Cl2 (6 mL), then [16,16,16-2H3]-hexadecanoic acid (0.06 g,
0.24 mmol), EDCI (0.09 g, 0.48 mmol) and DMAP (0.04 g,
0.30 mmol) were added. The reaction mixture was stirred at
room temperature for 96 h, then diluted with CH2Cl2 and
washed with aqueous sat. NaHCO3. The layers were separated
and the organic layer was dried over Na2SO4 and concen-
trated. The crude product was purified by flash chromatography
(n-hexane/EtOAc 9:1) affording 8 (0.09 g, 70%) as a foam.
[␣]D = +1.1 (c 1). 1H NMR (CDCl3) δ 0.91 (t, J = 7.0 Hz,
3H, CH3), 1.20–1.45 (m, 50H, 25CH2), 1.53–1.62 (m, 2H,
CH2), 1.49–1.66 (m, 4H, 2CH2), 2.23–2.35 (m, 2H, CH2),
3.37 (m, 1H, CH2O), 3.44 (m, 1H, CH2O), 3.50–3.56 (m,
2H, H-5ꢀ, H-3ꢀ), 3.57–3.67 (m, 4H, H-1a, H-1b, H-6ꢀa, H-6ꢀb),
A mixture of 6 (0.06 g, 0.06 mmol) and 10% Pd on acti-
vated charcoal (0.05 g) in EtOAc–MeOH 1:2 (3 mL) was
stirred under hydrogen atmosphere for 96 h, filtered over
Celiteandconcentrated. Flashchromatography(CH2Cl2/MeOH
9:1) afforded the target compound 2a (70%, 0.03 g) as a
white solid. [␣]D = −3.2 (c 0.75, CHCl3/MeOH 1:1). 1H
NMR (CDCl3/CD3OD, 1:1) δ 0.87 (t, J = 7.0 Hz, 3H, CH3),
1.20–1.45 (m, 50H, 25CH2), 1.49–1.70 (m, 4H, 2CH2), 2.34
(t, J = 7.5 Hz, 2H, CH2), 3.40–3.50 (m, 4H, CH2O, H-5ꢀ, H-
3ꢀ), 3.52 (dd, J2 ,3 = 9.7 Hz, J1 ,2 = 7.5 Hz, 1H, H-2 ), 3.57–3.67
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
(m, 2H, H-1a, H-1b), 3.70–3.76 (m, 2H, H-3a, H-6ꢀa), 3.80
ꢀ
ꢀ
ꢀ
ꢀ
(dd, J6a ,6b = 11.5 Hz, J5 ,6b = 6.5 Hz, H-6b), 3.86 (d, J = 3.0 Hz,
3.72 (dd, J3a,3b = 10.5 Hz, J2,3a = 4.5 Hz, 1H, H-3a), 3.84 (dd,
1H, H-4ꢀ), 3.95 (dd, J3a,3b = 11.0 Hz, J2,3b = 5.5 Hz, 1H, H-
J2 ,3 = 9.7 Hz, J1 ,2 = 7.5 Hz, 1H, H-2 ), 3.91 (d, J3 ,4 = 3.5 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3b), 4.22 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 5.20 (m, 1H, H-2). 13C
NMR (CDCl3/CD3OD, 1:1) δ 14.1, 22.7, 24.9, 26.1, 29.1–29.7,
31.2, 34.4, 61.7, 68.7, 69.1, 69.4, 71.3, 71.7, 73.5, 75.0, 104.3,
1H, H-4ꢀ), 4.03 (dd, J3a,3b = 10.5 Hꢀz, J2,3b = 4.7 Hz, 1H, H-
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
3b), 4.36 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 4.43, 4.47, 4.64, 4.72,
4.76, 4.78, 4.94 and 4.96 (8d, 8H, CH2Ph), 5.21 (m, 1H, H-

82
L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
Fig. 2. ESI mass spectra of SGG 1a (panel 1a), SGG 1b (panel 1b), and natural (unlabelled) SGG 1 purified from pig testis (panel 1), GG 2a (panel 2a), GG 2b
(panel 2b), and natural (unlabelled) GG 2 purified from pig testis (panel 2). Spectra 1a, 1b and 1 were obtained from solutions in methanol/chloroform (4/1, v/v),
spectra 2a, 2b and 2 were obtained from solutions in methanol/chloroform (4/1, v/v) containing 100 mM lithium chloride, as described in Section 2.
2H, H-3a, H-6ꢀa), 3.80 (dd, J6a ,6b = 11.5 Hz, J5 ,6b = 6.5 Hz,
2), 7.20–7.49 (m, 20H, Ph). 13C NMR (CDCl3) ␦ 14.1, 22.4,
22.7, 25.0, 26.1, 29.1–29.7 (23C), 31.9, 34.4, 68.3, 68.7, 69.4,
71.3, 71.6, 73.1, 73.5 (2C), 73.6, 74.8, 75.0, 79.3, 82.1, 104.3,
127.3–128.4 (20C), 137.9, 138.5, 138.6, 138.8, 173.3. ESI-
MS (positive-ion mode): m/z 1103.0, calculated 1103.57 Da for
C69H1012H3O9Na corresponding to (M + Na)+.
ꢀ
ꢀ
ꢀ
ꢀ
H-6b), 3.86 (d, J = 3.0 Hz, 1H, H-4ꢀ), 3.95 (dd, J3a,3b = 11.0 Hz,
ꢀ
ꢀ
ꢀ
J2,3b = 5.5 Hz, 1H, H-3b), 4.22 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 5.20
(m, 1H, H-2). 13C NMR (CDCl3/CD3OD, 1:1) δ 14.1, 22.7,
24.9, 26.1, 29.1–29.7, 31.2, 34.4, 61.7, 68.7, 69.1, 69.4, 71.4,
71.8, 73.5, 75.0, 104.3, 174.0. ESI-MS (positive-ion mode):
1
found m/z 726.8, calculated 726.62 Da for C41 H782H3O9Li
corresponding to (M + Li)+. The physical data of 2b is in
agreement with those reported for the unlabelled compound
(Lindberg et al., 2002).
2.10. 1-O-Hexadecyl-2-O-(16,16,16-2H3)-palmitoyl-3-
O-(β-d-galactopyranosyl)-sn-glycerol (2b)
Compound 2b was prepared from 0.08 g of 8 as described
for 2a. Purification of the crude material by flash chromatog-
raphy (CH2Cl2/MeOH 9:1) afforded 2b as a white solid.
(0.04 g, 75%). [␣]D = −2.5 (c 0.5, CHCl3/MeOH 1:1).1H NMR
(CDCl3/CD3OD, 1:1) δ 0.87 (t, J = 7.0 Hz, 3H, CH3), 1.20–1.45
(m, 50H, 25CH2), 1.53–1.62 (m, 2H, CH2), 1.49–1.66 (m,
4H, 2CH2), 2.34 (t, J = 7.5 Hz, 2H, CH2), 3.40–3.50 (m, 4H,
2.11. 1-O-Hexadecyl-2-O-(16,16,16-2H3)-palmitoyl-3-O-
[3-O-(sodium oxysulfonyl)-β-d-galactopyranosyl]-
sn-glycerol (1b)
Compound 1b was prepared from 2b (0.03 g, 0.04 mmol) as
described for 2a; purification of the crude material by flash chro-
matography (CH2Cl2/MeOH, 9:1) gave the target compound
1b (70%, 0.024 g) as a foam. [␣]D = +2.5 (c 1, CHCl3/MeOH,
CH2O, H-5ꢀ, H-3ꢀ), 3.52 (dd, J2 ,3 = 9.7 Hz, J1 ,2 = 7.5 Hz,
ꢀ
ꢀ
ꢀ
ꢀ
1H, H-2ꢀ), 3.57–3.67 (m, 2H, H-1a, H-1b), 3.70–3.76 (m,

L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
83
Fig. 3. ESI tandem mass spectra produced as described in Section 2 under collisionally activated dissociation conditions of designated parent ions (P) from SGG
1a (panel 1a), SGG 1b (panel 1b), natural (unlabelled) SGG 1 purified from pig testis (panel 1), GG 2a (panel 2a), GG 2b (panel 2b), and natural (unlabelled) GG
2 purified from pig testis (panel 2). Spectra 1a, 1b and 1 were obtained from solutions in methanol/chloroform (4/1, v/v), spectra 2a, 2b and 2 were obtained from
solutions in methanol/chloroform (4/1, v/v) containing 100 mM lithium chloride.
1
confirmed the structure of the natural material. This elabo-
rate route exploits 3-O-allyl-l-glycerol as starting material and
requires tedious manipulation of protecting groups both on the
glycerol acceptor and the galactosyl donor.
1:1). H NMR (CDCl3/CD3OD, 1:1) δ 0.91 (t, J = 7.0 Hz, 3H,
CH3), 1.20–1.35 (m, 50H, 25CH2), 1.50–1.57 (m, 2H, CH2),
1.58–1.66 (m, 2H, CH2), 2.34 (m, 2H, CH2), 3.39–3.51 (m,
2H, CH2O), 3.55 (t, J = 6.0 Hz, 1H, H-5ꢀ), 3.58–3.67 (m, 2H,
H-1a, H-1b), 3.70–3.84 (m, 4H, H-2ꢀ, H-6ꢀa, H-6ꢀb, H-3a),
3.95 (dd, J3a,3b = 11.0 Hz, J2,3a = 5.5 Hz, 1H, H-3b), 4.23 (dd,
The glycidyl galactoside 3, derived from S-glycidol, was
recently described (Lindberg et al., 2002) as a precursor in the
synthesis of galactoglycerolipid tumor antigens. Compound 3
was the key intermediate to obtain the deuterated SGG 1a,b and
GG 2a,b.
For the preparation of d3-GG 2a, labelled on the ether chain,
opening of the epoxide in 3 with [16,16,16-2H3]-hexadecanol 4
(obtained by LiAlH4 reduction of [16,16,16-2H3]-hexadecanoic
acid) gave the galactosylglycerol derivative 5 labelled on
the hexadecyl chain (route a, Scheme 1). Then, acylation
with unlabelled hexadecanoic acid following standard proce-
dures afforded protected 6 in 75% yield after purification.
Hydrogenolytic debenzylation of [16,16,16-2H3]-ether 6, fol-
lowedbyflashchromatography, yieldedGG2ain70%. Selective
J2,3 = 10.5 Hz, J3,4 = 3.0 Hz, 1H, H-3ꢀ), 4.2ꢀ6 (d, J3 ,4 = 3.0 Hz,
ꢀ
ꢀ
1H, H-4ꢀ), 4.33 (d, J1 ,2 = 7.5 Hz, 1H, H-1 ), 5.20 (m, 1H, H-
2). 13C NMR (CDCl3/CD3OD, 1:1) δ 13.6, 22.4, 24.8, 25.9,
28.9–29.6, 31.8, 34.2, 61.2, 67.1, 69.2, 69.3, 71.5, 71.6, 74.8,
80.6, 103.6, 174.0. ESI-MS (negative-ion mode): found m/z
ꢀ
ꢀ
1
798.5, calculated 798.55 Da for C41 H762H3O12S correspond-
ing to (M − H)−.
3. Results and discussion
Seminolipid 1 (carrying hexadecyl and hexadecanoyl chains)
was first synthesized by Gigg (Gigg, 1978) and its properties

84
L. Franchini et al. / Chemistry and Physics of Lipids 152 (2008) 78–85
sulfation (Guilbert et al., 1994) finally gave the target SGG 1a in
70% yield. Selective functionalization at position 3 of galactose
was effectively accomplished through reaction of dibutylstan-
nilene acetal with SO3·Me3N, as indicated by the downfield
shift of the H-3ꢀ signal (Franchini et al., 2007) in the 1H NMR
spectrum of SGG 1a.
Vice versa, by opening of the epoxide in 3 with unlabelled
hexadecanol (route b, Scheme 1) we obtained the known alcohol
7 (Lindberg et al., 2002), that was acylated with commercially
available [16,16,16-2H3]-hexadecanoic acid leading to the pro-
tected derivative 8 carrying the [16,16,16-2H3]- label on the
fatty acyl chain. Removal of the benzyl groups afforded GG 2b
in 70% yield after purification. Finally sulfation of GG 2b, con-
ducted in the same conditions as for 2a gave the target SGG 1b
in 70% yield; regioselectivity was once again confirmed by the
downfield shift of the signal of H-3ꢀ in the 1H NMR spectrum
of SGG 1b.
In conclusion we have provided a straightforward synthe-
sis of four glycolipid isotopomers along with their NMR and
mass spectrometric analysis; extension of this procedure could
provide a route for the preparation of isotopomers of other
compounds of the same general class. The use of these com-
pounds, synthesized with high isotopic and chemical purity, for
metabolism studies will be the subject of future research and
hopefully publications.
Acknowledgments
This work was supported by the University of Milan. Kessiri
Kongmanas holds a scholarship from the Development and Pro-
motion of Science and Technology Talented Project, Thailand.
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