Synthesis of a glycolipid for studying mechanisms of mitochondrial uncoupling proteins

Article abstract of DOI:10.1016/j.carres.2005.04.004

Glucose-O-ω-palmitic acid is an amphipathic molecule that is useful as a tool for studying the mechanism of mitochondrial uncoupling proteins. The synthesis of this glycolipid is described herein. The study of the reaction of a series of glycosyl donors with appropriate acceptors derived from 16-hydroxyhexadecanoic acid showed that a glycosyl trichloroacetimidate donor was more efficient than thioglycoside, glycosyl halide and glycosyl acetate donors for synthesis of this glycolipid.

Full text of DOI:10.1016/j.carres.2005.04.004

Carbohydrate
RESEARCH
Carbohydrate Research 340 (2005) 1547–1552
Note
Synthesis of a glycolipid for studying mechanisms of
mitochondrial uncoupling proteins
Sebastien G. Gouin,a Wayne Pilgrim,a Richard K. Porterb,* and Paul V. Murphya,*
aCentre for Synthesis and Chemical Biology, Department of Chemistry, Conway Institute of Biomolecular and Biomedical Research,
University College Dublin, Belfield, Dublin 4, Ireland
bDepartment of Biochemistry, Trinity College Dublin, Dublin 2, Ireland
Received 23 February 2005; received in revised form 7 April 2005; accepted 7 April 2005
Abstract—Glucose-O-x-palmitic acid is an amphipathic molecule that is useful as a tool for studying the mechanism of mitochon-
drial uncoupling proteins. The synthesis of this glycolipid is described herein. The study of the reaction of a series of glycosyl donors
with appropriate acceptors derived from 16-hydroxyhexadecanoic acid showed that a glycosyl trichloroacetimidate donor was more
efficient than thioglycoside, glycosyl halide and glycosyl acetate donors for synthesis of this glycolipid.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Keywords: Mitochondrial uncoupling protein; Glycoconjugates; Glucose-O-x-palmitic acid; Palmitic acid; 16-(b-D-Glucopyranosyloxy)-hexadeca-
noic acid
Mitochondrial uncoupling protein 1 (UCP 1 also known
as UCP and thermogenin) is a 32 kDa protein situated
in the inner membrane of mitochondria from brown adi-
pose tissue (BAT) of mammals and human infants.1 The
protein uncouples ATP synthesis from oxygen consump-
tion by catalysing the dissipation of the proton electro-
chemical gradient across the mitochondrial inner
membrane. UCP 1 is the basis of the non-shivering ther-
mogenesis generated by BAT under conditions of cold
exposure. Long chain free fatty acids, such as palmitic
acid, are known to increase proton leak through UCP
1.2 However, the mechanism by which free fatty acids
are involved in the UCP uncoupling process is a matter
of investigation. The two main models for the mecha-
nism of action of the uncoupling proteins are: (i) that
UCP 1 acts as a proton conduit across the mitochon-
drial inner membrane and importantly that fatty acids
act as cofactors/activators providing an addition car-
boxyl group at a key intra membrane site enhancing
the rate of proton movement3 or: (ii) that protonated
fatty acids freely flip across the mitochondrial inner
membrane and uncouple the mitochondria and that
the uncoupling proteins act as ÔflippasesÕ translocating
the anionic fatty acids back across the bilayer leaflets
of the inner membrane.4 Thus the latter model proposes
that UCP 1 facilitates a cycle of uncoupling by free fatty
acids. There is evidence for both models but crucial
experimental evidence distinguishing both models is
lacking. To that end, we describe the synthesis of glu-
cose-O-x-palmitic acid 1 (Chart 1) an amphipathic mol-
ecule that cannot ÔflipÕ across the mitochondrial inner
membrane but can theoretically provide the carboxyl
group for catalysis of proton translocation. This glyco-
lipid has been cited previously in the literature5 but nei-
ther details regarding the synthesis of this molecule nor
its analytical data nor criteria of purity have been re-
ported to date.
O
OH
O
HO
HO
( )14
O
OH
OH
*
Corresponding authors. Tel.: +353 1 7162504; fax: +353 1 7162127;
e-mail: paul.v.murphy@ucd.ie
Chart 1. Structure of glycolipid 1.
0008-6215/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.04.004

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S. G. Gouin et al. / Carbohydrate Research 340 (2005) 1547–1552
Table 1. Glycosidation reactionsa
Entry
Donor
Acceptor
Promoter
Time (h)
3
Main product (%) a:b
AcO
AcO
O
4
(1.0 equiv)
SnCl4
(1.0 equiv)
12 (20)
<5:95
1
OAc
AcO
OAc
5 (1.1 equiv)
2
3
5 (1.1 equiv)
4
(1.0 equiv)
SnCl4
(1.0 equiv)
45
2
13 (20)
>90:10b
AcO
O
AcO
3
(1.0 equiv)
Ag2CO3
12 (31)
b
AcO
AcO
Br
(3.5 equiv),
AgClO4
(0.12 equiv)
6 (1.5 equiv)
BzO
O
BzO
4
5
3
(1.0 equiv)
AgOTf
2
2
10 (33), 11 (58)
b
SMe
SMe
BzO
(0.1 equiv),
NIS
(1.0 equiv)
BzO
7 (1.0 equiv)
AcO
O
AcO
3
(1.0 equiv)
AgOTf
14 (51), 15 (29)
c
—
AcO
(0.1 equiv),
NIS
(1.0 equiv)
OAc
8 (1.0 equiv)
BzO
O
BzO
BzO
6
3
(1.0 equiv)
TMSOTf
(0.1 equiv)
0.33
10 (55), 11 (5)
b
BzO
O
CCl3
NH
9 (1.0 equiv)
a All glycosidations were carried out in CH2Cl2 and yields correspond to isolated yield after purification by chromatography.
b Higher amounts of a-glycoside 13 were obtained from reactions carried out on >1 g scale after 29 h.
c No glycoside product was isolated.
The glycosidation6 reactions of a series of donors 5–9
(Table 1 and Scheme 2)7–9 in presence of acceptors 3 or 4
were investigated. The acceptors 3 and 4 were obtained
from 16-hydroxyhexadecanoic acid 2; reaction of 2 with
MeOH catalysed by PPTS gave ester 3 and subsequent
silylation gave 4 (Scheme 1).
The results from glycosidation are summarised in Ta-
ble 1. The Schmidt–Michel glycosidation from benzoyl
protected trichloroacetimidate10 donor 9 was found to
be significantly more efficient than reactions of thio-
glucosides 7 and 8, glucosyl bromide (6) or b-D-glucose
penta-acetate (5), giving 10 in 55% yield (Scheme 2) as
well as a minor amount of benzoate ester 11. The ester
11 was a major product from reaction of the thioglyco-
side 7. A similar by-product, 15, was isolated, as well as
orthoester 14 (Chart 2), from reaction of thioglycoside
8. A proposal, which would explain the formation of
11 and 15 during glycosidation is shown in Scheme 3.
This involves coordination by a Lewis acid reagent to
the pyranose 2-O atom followed by formation of the
product facilitated by glycosyl oxocarbenium ion forma-
tion. The stereoselectivity observed from the SnCl4
catalysed glycosidation of 5 was dependent on the reac-
tion time (Table 1). It was possible to obtain pure b-ano-
mer 12 (Chart 2) after 3 h (Table 1, entry 1), however the
a-anomer 13 (>90:10 selectivity) can be obtained if the
experiment is conducted over a longer time (45 h, entry
2). The anomerisation11 reaction of 12 to 13 is catalysed
by SnCl4 and can be monitored by TLC and the yield
was similar in all experiments (ꢀ20%). The Koenigs–
Knorr glycosidation of glucosyl bromide 6 gave 12
(31%), with no anomerisation occurring, even after
24 h. The protecting groups were efficiently removed
from 10 (or 12) using first treatment with NaOMe–
MeOH and subsequent reaction with LiOH to give the
desired glycolipid 1.
O
PPTS
O
( )14
HO
OMe
( )14
HO
OH
MeOH
97%
2
3
TMSCl, Et3N,
CH2Cl2, 88%
O
( )14
TMSO
OMe
4
Scheme 1. Synthesis of 3 and 4.

S. G. Gouin et al. / Carbohydrate Research 340 (2005) 1547–1552
1549
O
O
TMSOTf (0.1 eq)
CH2Cl2
BzO
O
O
BzO
( )14
O
BzO
( )14
OMe
OMe
+
BzO
( )14
HO
OMe
BzO
BzO
O
10 55%
NaOMe, MeOH
then LiOH, THF, MeOH, H2O,
>95%
11 5%
3
BzO
BzO
BzO
O
CCl3
NH
9
O
HO
HO
O
HO
( )14
O
OH
HO
1
Scheme 2. Synthesis of 1 from trichloroacetimidate donor 9.
O
D-glucopyranoside as an internal standard. This seems a
reasonable assumption given the hygroscopic nature of
some carbohydrate compounds. Biochemical experi-
ments with 1 are underway and the insights these provide
to the mechanisms, which operate for mitochondrial
uncoupling proteins will be reported in due course.
OAc
O
OAc
O
AcO
AcO
AcO
AcO
( )14
O
Me
O
OAc
AcO
( )14
O
Me
12
13
OAc
O
AcO
AcO
O
O
O
O
O
O
( )14
( )14
O
Me
1. Experimental
1.1. General
Me
14
15
Chart 2. Structure of 12–15.
Optical rotations were determined with a Perkin–Elmer
1
241 model polarimeter at 23 ꢁC. H NMR spectra were
recorded with a Varian Inova 300 MHz spectrometer;
13C NMR spectra were recorded on the same instrument
at 75 MHz. Chemical shifts are reported relative to inter-
nal Me4Si in CDCl3 (d 0.0) or HOD for D2O (d 4.79) for
1H and (d 77.16) for 13C. 13C signals were assigned with
RO
RO
RO
RO
'A'
O
O
RO
X
RO
O
O
O
O
O
HO
( )14
OMe
R'
R'
1
the aid of DEPT-135. H signals were assigned with the
RO
RO
RO
RO
RO
RO
O
O
aid of COSY. IR spectra were recorded with a Mattson
Galaxy Series FTIR 3000 using either thin film between
NaCl plates or KBr discs, as specified. High resolution
mass spectra were recorded on a Micromass LCT
KC420 or Micromass Quattro. TLC was performed on
aluminium sheets precoated with Silica Gel 60 (HF254,
E. Merck) and spots visualised by UV and charring with
1:20 H2SO4–EtOH. Flash column chromatography was
carried out with Silica Gel 60 (0.040–0.630 mm, E.
Merck) and employed a stepwise solvent polarity gradi-
ent correlated with the TLC mobility. Chromatography
solvents used were MeOH, CH2Cl2 (Riedel-de Haen),
EtOAc and cyclohexane (Fluka). Reaction solvents were
dried and distilled before use.
O
O
O
O
O
O
LA
R'
LA
( )14
O
R'
OMe
O
( )14
OMe
O
O
R'
O
( )14
OMe
Scheme 3. A proposal for formation of 11 and 15.
Biochemists who work in this area are interested in the
stability and purity of 1. We have found that 1 is resistant
to acid catalysed hydrolysis and remained unchanged
when treated with aq HCl and MeOH, aq HCl and
THF or 18% HCl for 24 h at room temp. Hydrolysis or
methanolysis of 1 to expected products was only
1.2. Methyl 16-hydroxyhexadecanoate (3)
1
achieved (detected by TLC, MS and H NMR), after
heating the compound to 60 ꢁC in (i) a 2:1 mixture of
aq HCl (9%) and THF for 6 h or (ii) a 1:1 mixture of
MeOH and aq HCl (18%) for 3 h. The 1H NMR spectra
of 1, prepared from both 10 and 12, have been provided
in the Supplementary information section. The purity of
1 was determined to be 93% by 1H NMR, using methyl a-
16-Hydroxyhexadecanoic acid 2 (3.0 g, 11 mmol) and p-
toluenesulfonic acid monohydrate (669 mg, 3.5 mmol)
were dried under diminished pressure for 2 h. MeOH
(180 mL) was added and the reaction mixture stirred
at rt for 16 h. Solid NaHCO3 (750 mg) was then added
and the mixture stirred for a further 30 min, then filtered

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S. G. Gouin et al. / Carbohydrate Research 340 (2005) 1547–1552
through Celite and the solvent removed under dimin-
ished pressure. Chromatography (1:3 EtOAc–cyclohex-
ane) gave 3 (2.89 g, 92%) as a white powder; mp
53 ꢁC; IR (KBr) cmÀ1: 3313, 2920, 2850, 1741, 1462;
1H NMR (CDCl3): d 3.66 (3H, s, OCH3), 3.64 (2H, t,
J 8.1 Hz, aromatic CH), 7.96 (2H, d, J 8.1 Hz, aromatic
CH), 7.90 (2H, d, J 8.1 Hz, aromatic CH), 7.84 (2H, d, J
8.1 Hz, aromatic CH), 7.56–7.26 (12H, m, aromatic
CH), 5.90 (1H, t, J2,3 9.6 Hz, J3,4 9.6 Hz, H-3), 5.67
(1H, t, J3,4 9.6 Hz, J4,5 9.9 Hz, H-4), 5.51 (1H, dd, J1,2
7.8 Hz, J3,4 9.6 Hz, H-2), 4.83 (1H, d, J1,2 7.8 Hz, H-
1), 4.63 (1H, dd, J6a,6b 12.3 Hz, J5,6a 3.6 Hz, H-6a),
4.50 (1H, dd, J6a,6b 12.3 Hz, J5,6b 5.1 Hz, H-6b), 4.15
(1H, ddd, J4,5 9.9 Hz, J5,6a 3.6 Hz, J5,6b 5.1 Hz, H-5),
3.92 (1H, m, CHHCO2Me), 3.66 (3H, s, OCH3) 3.52
J
6.6 Hz, HOCH2CH2), 2.30 (2H, t, J 7.5 Hz,
CH2CH2CO2Me), 1.64–1.52 (4H, m, HOCH2CH2 and
CH2CH2CO2Me), 1.25 (22H, br s, 11CH2); 13C NMR
(CDCl3): d 174.6 (s, CO2Me), 63.3 (t, CH2OH), 51.6
(q, OCH3), 34.4, 33.1, 29.9 (2s) 29.8 (2s), 29.7 (2s),
29.5, 29.4, 26.0, 25.2 (each t, each CH2); ESI-HRMS
calcd for C17H35O3 287.2586, found m/z 287.2572
[M+H]+. Anal. Calcd for C17H34O3: C, 71.28; H,
11.96. Found: C, 71.30; H, 11.74.
(1H, m, CHHCO2Me), 2.30 (2H, t,
J 7.5 Hz,
OCH2CH2), 1.64–1.42 (4H, m, 2CH2), 1.27–1.07 (24H,
m, 12CH2); 13C NMR (CDCl3, 75 MHz): d 174.5 (s,
CO2Me), 166.2, 166.0, 165.3, 165.2 (each s, each
C@O), 133.5, 133.4, 133.3, 133.2 (each s, each aromatic
C), 130.0, 129.9, 129.8, 129.5, 129.0, 128.5, 128.4 (each
d, each aromatic CH), 101.4 (d, C-1), 73.1, 72.3, 72.0
(each d), 70.5 (t, C-6), 70.0 (d), 63.4 (t, CH2CH2OCHO),
51.5 (q, CH3O), 34.3 (t, CH2CH2CO2Me), 29.8, 29.7,
29.6, 29.5, 29.4, 29.3, 25.9, 25.1 (each t); ESI-HRMS
calcd for C51H60O12Na 887.3982, found m/z 887.3981
[M+Na]+. Analytical data for methyl 16-benzoyloxyhex-
adecanoate 11: 1H NMR (CDCl3, 300 MHz): d 8.04
(2H, d, J 7.8 Hz, aromatic CH), 7.55 (1H, dd, J
7.8 Hz, J 7.2 Hz, aromatic CH), 7.41 (2H, m, aromatic
CH), 4.31 (1H, t, J 6.6 Hz, CH2O), 3.66 (3H, s, CH3O-
CO), 2.30 (2H, t, J 7.5 Hz, CH2CH2CO2Me), 1.77 (2H,
m, CH2CH2OCOAr), 1.63–1.59 (2H, m, CH2CH2CO),
1.25 (22H, m, 11CH2); 13C NMR (CDCl3, 75 MHz): d
(ppm) 174.5, 166.8 (each s, each C@O), 132.9 (d, aro-
matic CH), 130.7 (s, aromatic C), 129.7, 128.5 (each d,
each aromatic CH), 65.3 (t, CH2O), 51.6 (q, OCH3),
34.2 (t, CH2CO2), 29.8, 29.7, 29.6, 29.5, 29.3, 28.9,
26.2, 25.9, 25.1 (each t, each CH2); ESI-HRMS calcd
for C24H39O4 391.2848, found m/z 391.2853 [M+H]+.
1.3. Methyl 16-trimethylsilyloxyhexadecanoate (4)
Methyl 16-hydroxyhexadecanoate 3 (1.12 g, 3.9 mmol)
was dissolved in anhyd CH2Cl2 (15 mL) and the soln
cooled to 0 ꢁC under N2. Et3N (819 lL, 5.9 mmol) and
TMSCl (574 lL, 4.7 mmol) were added dropwise and
the reaction mixture was stirred at rt for 2 h. The mixture
was diluted with CH2Cl2 (50 mL) and washed with satd
aq NaHCO3 (50 mL). The organic layer was dried
(MgSO4), filtered and the solvent was removed under
diminished pressure. Chromatography (1:50 Et3N–cyclo-
hexane) gave 4 as a colourless oil (1.07 g, 88%); IR (film)
cmÀ1: 2926, 2854, 1744, 1463, 1436, 1250, 1098; 1H NMR
(CDCl3): d 3.65 (3H, s, OCH3), 3.55 (2H, t, J 6.6 Hz,
TMSOCH2CH2), 2.29 (2H, t, J 7.5 Hz, CH2CH2-
CO2Me), 1.66–1.48 (4H, m, TMSOCH2CH2 and
CH2CH2CO2Me), 1.25 (22H, s, CH2), 0.1 (9H, s,
Si(CH3)3); 13C NMR (CDCl3) d: 174.5 (s, CO2Me), 63.0
(t, CH2OSi), 51.6 (q, OCH3), 34.4, 33.1, 29.9 (three sig-
nals), 29.8, 29.7, 29.5, 29.4, 26.1, 25.2 (each t, each
CH2), À0.2 (q, Si(CH3)3); Anal. Calcd for C20H42O3Si:
C, 66.98; H, 11.80. Found: C, 67.10; H, 11.68.
1.5. 16-(b-D-Glucopyranosyloxy)-hexadecanoic acid (1)
1.4. Methyl 16-(2,3,4,6-tetra-O-benzoyl-b-D-glucopyr-
anosyloxy)-hexadecanoate (10)
Protected glycoside 10 (32 mg, 37 lmmol) was dissolved
in dry MeOH (1 mL). A catalytic amount (20 lL) of so-
dium methanolate (1 M in MeOH) was added, and the
resulting soln was stirred for 2 h at rt. The solvent was
removed under diminished pressure and water (1 mL)
was added. THF was added dropwise into the cloudy
solution until it became clear and LiOH (10 mg) then
added and the solution was stirred for 1 h at rt. Amber-
lite IR-120 (plus) was added until pH 6.0. The resin was
filtered off and rinsed with THF and then solvent was
evaporated under diminished pressure until the soln be-
came cloudy and the remainder of solvent removed by
freeze drying. Residual benzoic acid was removed by
repeated freeze drying (·3) from water and the title
compound further purified by chromatography
(MeOH–CH2Cl2) to give a white powder (16 mg,
A mixture of trichloroacetamide 99 (129 mg, 174 lmol),
acceptor 3 (50 mg, 174 lmol) and molecular sieves 4 A
˚
(100 mg) were placed under diminished pressure
(<1 mmHg) for 1 h. Anhyd CH2Cl2 (2 mL) was added
and the sol stirred for 2 h at room temp and TMSOTf
(3 lL, 17.4 lmol) then added and stirring continued
for a further 20 min at room temp. Solid NaHCO3
(25 mg) was then added and the mixture stirred for
15 min, then filtered through Celite, which was rinsed
with CH2Cl2 (5 mL). The solvent was removed under
diminished pressure and the residue purified by flash
chromatography (1:9 EtOAc–cyclohexane) to give the
title compound as a colourless syrup (85 mg, 55%) and
11 (3.4 mg, 5%). Analytical data for 10: [a]D À7 (c 0.1,
1
100%); [a]D À7 (c 0.1, MeOH); mp 85 ꢁC; H NMR
1
MeOH); H NMR (CDCl3, 300 MHz): d 8.01 (2H, d,
(CD3OD): d 4.27 (1H, d, J1,2 7.8 Hz, H-1), 3.93–3.68

S. G. Gouin et al. / Carbohydrate Research 340 (2005) 1547–1552
1551
(2H, m), 3.71–3.65 (1H, m), 3.59–3.51 (1H, m,
CHHCO2Me), 3.39–3.27 (3H, m, CHHCO2Me and H-
6a,6b), 3.18 (1H, t, J 8.4 Hz), 2.30 (2H, t, J 7.5 Hz,
OCH2CH2), 1.64–1.56 (4H, m, 2CH2), 1.31 (24H, s,
12CH2); 13C NMR (CD3OD): d 176.1 (s, CO2H), 104.4
(d, C-1), 78.2, 78.0, 75.2, 71.8 (each d, each CH), 71.0
(t, C-6), 62.8 (t, CH2CH2OCO), 34.9 (t, CH2CO2H),
30.9, 30.8, 30.7, 30.6, 30.4, 30.2, 27.2, 26.0 (each t);
ESI-HRMS calcd for C22H42O8Na 457.2770, found m/z
5.06 (1H, d, J1,2 3.9 Hz, H-1), 5.06 (1H, t, J3,4 9.6 Hz,
J4,5 9.6 Hz, H-4), 4.85 (1H, dd, J1,2 3.9 Hz, J2,3
10.0 Hz, H-2), 4.26 (1H, dd, J6a,6b 12.3 Hz, J5,6a 4.5 Hz,
H-6a), 4.08 (1H, dd, J6a,6b 12.3 Hz, J5,6b 2.1 Hz, H-6b),
3.99 (1H, J4,5 10.2 Hz, J5,6a 4.5 Hz, J5,6b 2.1 Hz, ddd,
H-5), 3.70–3.66 (4H, m, CHHCO2Me and OCH3),
3.46–3.40 (1H, m, CHHCO2Me), 2.30 (2H, t, J 7.5 Hz,
OCH2CH2), 2.09, 2.06, 2.03, 2.01 (12H, each s, each
CH3COO), 1.64–1.56 (4H, m, 2CH2), 1.25 (24H, se,
12CH2); 13C NMR (CDCl3, 75 MHz): d 174.6 (s,
CO2Me), 170.9, 170.5, 170.4, 169.9 (each s, each C@O),
95.9 (d, C-1), 71.3, 70.6 (each d), 69.1 (t, C-6), 69.0,
67.5 (each d), 62.3 (t, CH2CH2OCO), 51.7 (q, OCH3),
34.4 (t, CH2CH2CO2Me), 29.9 (5s), 29.7, 29.6 (2s), 29.5
(2s), 26.3, 25.3 (each t), 21.0, 20.9 (2s) (each q); ESI-
HRMS calcd for C31H52O12Na 639.3356, found m/z
639.3340 [M+Na]+. Anal. Calcd for C31H52O12: C,
60.37; H, 8.50. Found: C, 59.97; H, 8.35.
457.2762 [M+Na]+. To determine the purity,
1
(2.997 mg, 6.90 lmol) and methyl a-D-glucopyranoside
as the internal standard (99%, 1.356 mg, 6.99 lmol) were
dissolved in Me2SO-d6 and the 1H NMR spectrum of the
mixture recorded. Integration of the respective anomeric
proton signals indicated that the purity of 1 was 93%.
1.6. Methyl 16-(2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
syloxy)-hexadecanoate (12)
Alcohol 3 (50 mg, 0.17 mmol), silver carbonate (164 mg,
0.60 mmol), silver perchlorate (3.6 mg, 0.02 mmol) and
molecular sieves were dried under diminished pressure
for 3 h. Anhyd CH2Cl2 (0.75 mL) was added and the
mixture was stirred at rt for 2 h. Bromide 6 (109 mg,
0.26 mmol) in dry CH2Cl2 (0.2 mL) was added dropwise
and the mixture was stirred under N2 at rt for 1.5 h. The
mixture was diluted with CH2Cl2 (20 mL) and washed
with water (20 mL). The organic layer was dried
(MgSO4), filtered and solvent removed under dimin-
ished pressure. Chromatography (1:4–1:3 EtOAc–cyclo-
hexane) gave 12 as a white powder (32 mg, 31%); mp
1.8. Analytical data for methyl 16-acetyloxyhexadecano-
ate (15)
1H NMR (CDCl3, 300 MHz): d 4.05 (1H, t, J 7 Hz,
CH2OCOMe), 3.66 (3H, s, CH3OCO), 2.31 (2H, t, J
7.5 Hz, CH2CH2CO2Me), 2.04 (3H, s, CH3CO2), 1.66–
1.59 (4H, m, OCH2CH2 and CH2CH2CO), 1.25 (22H,
m, 11CH2); 13C NMR (CDCl3, 75 MHz): d (ppm)
174.4, 171.3 (s, each C@O), 64.8 (t, CH2O), 51.5 (q,
OCH3), 34.2 (q, CH2CO2), 29.7, 29.6, 29.5, 28.7, 26.5,
25.0, 21.1 (t, 13CH2).
1
55 ꢁC; [a]D À14 (c 0.3, CH2Cl2); H NMR (CDCl3): d
1.9. Methyl 16-O-[2-(3,4,6-tri-O-acetyl-a-D-glucopyra-
nose-1,2-di-O-yl)ethyl]-hexadecanoate (14)
5.20 (1H, t, J2,3 = J3,4 = 9.6 Hz, H-3), 5.09 (1H, t,
J3,4 = J4,5 = 9.6 Hz, H-4), 4.98 (1H, t, J1,2 7.8 Hz, J2,3
9.6 Hz, H-2), 4.49 (1H, d, J1,2 7.8 Hz, H-1), 4.27 (1H,
dd, J6a,6b 12.3 Hz, J5,6a 4.8 Hz, H-6a), 4.13 (1H, dd,
J6a,6b 12.3 Hz, J5,6b 2.4 Hz, H-6b), 3.90–3.83 (1H, m,
CH2CHHO), 3.70–3.66 (1H, ddd, J5,6b 2.4 Hz, J5,6a
4.8 Hz, J4,5 9.6 Hz, H-5), 3.66 (3H, s, OCH3), 3.51–
3.43 (1H, m, CH2CHHO), 2.30 (2H, t, J 7.5 Hz,
CH2CO2Me), 2.09, 2.04, 2.02, 2.01 (12H, each s, each
CH3COO), 1.64–1.56 (4H, m, CH2CH2O and
CH2CH2CO2Me), 1.25 (22H, s, CH2); 13C NMR
(CDCl3): d 174.5 (s, CO2Me), 170.9, 170.5, 169.6,
169.5 (each s, each C@O), 101.1 (d, C-1), 73.2, 72.0,
71.7 (each d), 70.5 (t, C-6), 68.8 (d), 62.3 (t, CH2CH2O-
CO), 51.6 (q, CH3O), 34.3 (t, CH2CO2Me), 29.9, 29.8,
29.7, 29.6, 29.5, 29.4, 26.1, 25.2 (each t), 21.0, 20.9,
20.8 (each q); ESI-HRMS calcd for C31H52O12Na
639.3356, found m/z 639.3326 [M+Na]+.
1H NMR (CDCl3, 300 MHz): d 5.70 (1H, d, J1,2 5 Hz,
H-1), 5.19 (1H, m, H-3), 4.90 (1H, dd, J3,4 10 Hz, J4,5
3 Hz, H-4), 4.31 (1H, m, H-2), 4.20 (2H, m, H-6a,6b),
3.95 (1H, dt, J4,5 3 Hz, J5,6a = J5,6b = 9 Hz, H-5), 3.66
(1H, s, CO2Me), 3.44 (2H, t, J 7 Hz, CH2O), 2.29 (2H,
t, CH2CO2), 2.11, 2.09, 2.08 (9H, each s, each CH3CO),
1.71 (3H, s, CH3C(O)3), 1.63–1.50 (4H, m, CH2CH2O
and CH2CH2CO2Me), 1.25 (22H, m, 11CH2);
13C NMR (CDCl3, 75 MHZ): d (ppm) 174.0, 170.7,
169.7, 169.2 (each s, each C@O), 121.3 (s), 96.9 (s, C-
1), 73.1, 70.2, 67.0 (each d), 63.7, 63.1 (each t, CH2O
and C-6), 51.4 (q, CH3O), 34.1 (t, CH2CH2CO2Me),
29.6, 29.8, 29.4, 29.2, 26.0, 24.0 (each t, each CH2),
20.8 (q); ESIMS: m/z [M+Na]+: calcd 639.3, found
639.3.
1.7. Analytical data for methyl 16-(2,3,4,6-tetra-O-acetyl-
a-D-glucopyranosyloxy)-hexadecanoate (13)
Acknowledgements
The authors thank Enterprise Ireland for Basic
Research funding, the European Commission for a
Marie-Curie Fellowship to S.G., Science Foundation
White solid mp 31 ꢁC; [a]D +87 (c 0.3, CH2Cl2); 1H NMR
(CDCl3): d 5.48 (1H, t, J2,3 10.0 Hz, J3,4 9.6 Hz, H-3),

1552
S. G. Gouin et al. / Carbohydrate Research 340 (2005) 1547–1552
3. For reviews see Ref.
Huang, S.-G. Biochim. Biophys. Acta 1999, 1415, 271–
2
and Klingenberg, M.;
Ireland for a Programme Investigator Award (P.V.M.).
The research was in part funded by the Programme for
Research in Third Level Institutions Cycle 3 adminis-
tered by the HEA (of Ireland).
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4. Garlid, K. D.; Jaburek, M.; Jezˇek, P.; Varecha, M.
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Supplementary data associated with this article can be
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