An easy α-glycosylation methodology for the synthesis and stereochemistry of mycoplasma α-glycolipid antigens

Article abstract of DOI:10.3762/bjoc.8.70

Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homologue (C16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I isomers were characterized with 1H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail, while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical stress.

Full text of DOI:10.3762/bjoc.8.70

An easy α-glycosylation methodology for the
synthesis and stereochemistry of mycoplasma
α-glycolipid antigens
Yoshihiro Nishida*1, Yuko Shingu2, Yuan Mengfei1, Kazuo Fukuda1,
Hirofumi Dohi1, Sachie Matsuda2 and Kazuhiro Matsuda2
Full Research Paper
Open Access
Address:
Beilstein J. Org. Chem. 2012, 8, 629–639.
1Chiba University, Graduate School of Advanced Integration Science,
Matsudo 271-8510, Chiba, Japan and 2M. Biotech. Co. Ltd.,
Setagaya-ku, Fukazawa 2-1-3-1103,Tokyo 158-0081, Japan
doi:10.3762/bjoc.8.70
Received: 15 January 2012
Accepted: 28 March 2012
Published: 24 April 2012
Email:
Yoshihiro Nishida* - YNishida@faculty.chiba-u.jp
This article is part of the Thematic Series "Synthesis in the
glycosciences II".
* Corresponding author
Keywords:
Guest Editor: T. K. Lindhorst
cytoplasm membrane; glycolipid antigen; glycosylation; mycoplasma;
stereochemistry
© 2012 Nishida et al; licensee Beilstein-Institut.
License and terms: see end of document.
Abstract
Mycoplasma fermentans possesses unique α-glycolipid antigens (GGPL-I and GGPL-III) at the cytoplasm membrane, which carry a
phosphocholine group at the sugar primary (6-OH) position. This paper describes a practical synthetic pathway to a GGPL-I homo-
logue (C16:0) and its diastereomer, in which our one-pot α-glycosylation method was effectively applied. The synthetic GGPL-I
isomers were characterized with 1H NMR spectroscopy to determine the equilibrium among the three conformers (gg, gt, tg) at the
acyclic glycerol moiety. The natural GGPL-I isomer was found to prefer gt (54%) and gg (39%) conformers around the lipid tail,
while adopting all of the three conformers with equal probability around the sugar position. This property was very close to what
we have observed with respect to the conformation of phosphatidylcholine (DPPC), suggesting that the Mycoplasma glycolipids
GGPLs may constitute the cytoplasm fluid membrane together with ubiquitous phospholipids, without inducing stereochemical
stress.
Introduction
Mycoplasmas constitute a family of gram-positive microbes only as the main antigens but also as probable pathogens. Also
lacking rigid cell walls. They are suspected to be associated in our research team, Matsuda et al. [7-10] isolated a new class
with human immune diseases, in either direct or indirect ways, of α-glycolipid antigens (GGPL-I and GGPL-III, Figure 1) from
although the molecular mechanism is not fully understood [1]. M. fermentans. Another α-glycolipid (MfGL-II), which has a
In recent biochemical studies, Mycoplasma outer-membrane chemical structure very close to GGPL-III, was identified and
lipoproteins [2,3] and glycolipids [4-6] are thought to serve not characterized by other groups [11-14].
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Beilstein J. Org. Chem. 2012, 8, 629–639.
In order to exploit their biological functions in detail, it is
necessary to obtain these α-glycolipids in sufficient amounts.
Thus, both genetic [20-22] and chemical synthetic approaches
[23,24] are being followed, although no practical way has yet
been established. In this paper, we report a chemical access to
both a natural GGPL-I homologue (C16:0) and its diastereomer
I-a and I-b, in which our one-pot α-glycosylation methodology
[25,26] is effectively applied. The two GGPL-I isomers
prepared thereby were characterized with 1H NMR spec-
troscopy, in terms of configuration and conformation at the
asymmetric glycerol moiety.
Results and Discussion
A practical synthetic access to GGPL-I homo-
logues
Figure 1: Absolute chemical structures of M. fermentans
α-glycolipid antigens, GGPL-I and GGPl-III (GGPL: Glycosyl-sn-glyc-
erophospholipid).
GGPL-I provides two key asymmetric centers to be controlled,
literally, in the synthetic pathway. One is the configuration at
the chiral glycerol moiety, and another is the sugar α-glycoside
linkage. In former synthetic works on 3-O-(α-D-glycopyra-
nosyl)-sn-glycerol [27-30], chiral 1,2-O-isopropylidene-sn-gly-
cerol has often been employed [29,30] as the acceptor substrate
for different α-glycosylation reactions. In this case, however,
attention should be paid to the acid-catalyzed migration of the
dimethylketal group [23,29-31]. In our synthetic pathway, chiral
(S)- or (R)-glycidol is employed as an alternative source of the
chiral glycerol to circumvent this problem. In an established
synthetic approach, 6-O-acetyl-2,3,4-tri-O-benzyl protected
sugar 1 [23] is used as the donor and treated with a reagent
combination of CBr4 and Ph3P (Appel–Lee reagent) in either
CH2Cl2 or DMF solvent, or a mixture of the two (Scheme 1).
For the reaction in CH2Cl2, N,N,N’,N’-tetramethylurea (TMU)
is added after in situ formation of α-glycosyl bromide 2, which
Absolute chemical structures of GGPL-I [15] and GGPL–III
[16] have already been established by chemical syntheses of
stereoisomers; these α-glycolipids have a common chemical
backbone of 3-O-(α-D-glucopyranosyl)-sn-glycerol carrying
phosphocholine at the sugar primary (6-OH) position. The fatty
acids at the glycerol moiety are saturated, namely palmitic acid
(C16:0) and stearic acid (C18:0). GGPL-I has a structural feature
analogous to 1,2-di-O-palmitoyl phosphatidylcholine (DPPC) as
a ubiquitous cell membrane phospholipid. Apparently, GGPLs
are amphiphilic compounds that can form certain self-assem-
bled structures under physiological conditions [12,13] and may
give physicochemical stress on the immune system of the host
[17]. In fact, our research team has proven that these
α-glycolipid antigens have certain pathogenic functions [18,19].
Scheme 1: An established synthetic pathway to α-glycosyl-sn-glycerols 4a and 5a. A reagent combination of CBr4 and Ph3P (Appel–Lee reagent) is
utilized in either CH2Cl2 or DMF as solvent.
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Beilstein J. Org. Chem. 2012, 8, 629–639.
equilibrates with a more reactive β-glycosyl bromide species derived after deprotection at the sugar hydroxymethyl position
[32]. In the pathway using DMF, the α-glycosylation is routed and SN2 substitution with cesium palmitate at the glycerol sn-1
via α-glycosyl cationic imidate 3, which was predicted in position. Then, this compound was converted to glycolipid 8a
former studies [33] and evidenced in our preceding NMR and after sequential reaction of the temporary tert-butyl-
MS study [25,26].
dimethylsilyl (TBDMS) -protected sugar, and O-acylation at the
glycerol 2-OH position to give 7a, followed by removal of the
The reaction between 1 and (S)-glycidol in CH2Cl2 (+ TMU) TBDMS protecting group. For introducing the phosphocholine
gave a mixture of epoxy compound 4a (60–70%) and bromide group at the sugar 6-OH position, we employed a phospho-
5a (30–40%). In 5a, the oxirane ring was opened by nucle- roamidite method using 1H-tetrazole as a promoter [34]. First,
ophilic Br− ions produced by Ph3P and CBr4. Also in the DMF- 8a was treated with 2-cyanoethyl-N,N,N’,N’-tetraisopropyl
promoted reaction, a mixture of 4a (70–90%) and 5a (10–30%) phosphorodiamidite in the presence of 1H-tetrazole, and then
was derived. In both reaction pathways, however, the glycosyl- with choline tosylate to give 9a. After removal of the sugar
ation was α-selective (α:β ≥ 90:10, yields >80%) and not O-benzyl group by catalytic hydrogenolysis, the GGPL-I homo-
accompanied by isomerization at the glycerol moiety.
logue I-a was obtained. In the same way, the GGPL-I sn-isomer
I-b was derived from a mixture of 4b and 5b available from the
A mixture of 4a and 5a was used in the following chemical reaction between 1 and (R)-glycidol (Scheme 1 and
transformation (Scheme 2). First, a lyso-glycolipid 6a was Scheme 2b).
Scheme 2: Syntheses of GGPL-I homologue I-a and its isomer I-b. Conditions: (a) K2CO3, CH3OH; (b) cesium palmitate in DMF; (c) TBDMS chlo-
ride then palmitoyl chloride in pyridine + DMAP; (d) TFA in CH3OH; (e) (i) 2-cyanoethyl-N,N,N’,N’-tetraisopropyl phosphorodiamidite, 1H-tetrazole and
MS-4 Å in CH2Cl2; (ii) choline tosylate, 1H-tetrazole, (iii) mCPBA, (iv) aq. NH3 in CH3OH, (f) H2, Pd(OH)2/C in CH3OH.
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Beilstein J. Org. Chem. 2012, 8, 629–639.
1H NMR characterization of I-a, I-b and the
Natural GGPL-I and GGPL-III gave 1H NMR data very close to
related glycerolipids
those of I-a, indicating that both have a common skeleton of
1H NMR spectroscopy provides a useful tool for discriminating 3-O-(α-D-glucopyranosyl)-sn-glycerol [15,16].
between the two GGPL-I isomers as shown in Figure 2. A clear
difference was observed in the chemical shifts of the glycerol The glycerol moiety has two C–C single bonds. By free rota-
methylene protons as designated by “a” and “b”. Conversely, tion, each of them is allowed to have three staggered
little difference was observed between the sn-isomers at the conformers of gg (gauche-gauche), gt (gauche-trans) and tg
sugar H-1 signal as well as at the glycerol H-2 (Table 1). (trans-gauche) (Figure 3). In solution and also in self-
Figure 2: 1H NMR spectra of I-a and I-b (500 MHz, 25 °C, CDCl3/CD3OD 10:1). The assignment of sn-glycerol methylene protons (HproR and HproS)
was performed on the basis of our preceding studies on deuterium-labeled glycerols [35-37] and α(1→6)-linked disaccharides [38-40].
Table 1: 1H NMR data (500 MHz) of I-a, I-b, and their precursors (9a and 9b).
δ [ppm] ( 3J and 2J [Hz] )
Compound
glucose
H-1’
sn-glycerol moiety
H-1
proS
H-2
H-3
proS
proR
proR
I-aa
9a
4.80 (3.5)
4.70 (3.5)
4.80 (3.5)
4.74 (3.5)
4.14 (6.5)
4.17 (6.5)
3.80 (6.0)
3.76 (5.5)
4.40 (3.5, 12.0)
4.38 (3.0, 12.0)
3.57 (5.5,11.0)
3.57 (5.5, 11.0)
5.24
5.22
5.23
5.23
3.76 (5.5)
3.72 (5.5)
4.34 (3.5)
4.37 (3.0)
3.61 (5.5, 11.0)
3.52 (6.0, 11.0)
4.20 (6.5,12.0)
4.19 (6.5, 12.0)
I-ba
9b
aThese α-glycolipids were dissolved in a mixture of CDCl3 and CD3OD (10:1) at 11.2 mM concentration.
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Beilstein J. Org. Chem. 2012, 8, 629–639.
Figure 3: Distributions of gg, gt and tg-conformers in 3-substituted sn-glycerols at 11 mM in solutions of CDCl3 and CD3OD (10:1) at 298 K.
contacting liquid-crystalline states, these conformers are In this equation, a perfect staggering (Φ1 and Φ2 = +60, −60 or
thought to equilibrate with each other. In this study, we calcu- 180 degree) is assumed for every conformer. Figure 3 summa-
lated time-averaged populations of the three conformers by rizes the results for a series of 3-substituted 1,2-di-O-palmitoyl-
means of 1H NMR spectroscopy. As we reported in a preceding sn-glycerols, which involve tripalmitin (Figure 3, entry 1),
paper [41], the Karplus-type equation proposed by Haasnoot et DPPC (1,2-di-O-palmitoylphosphatidylcholine) (Figure 3, entry
al.[42] was adapted as follows:
2), and GGPL-I homologues (Figure 3, entries 3–5). In a solu-
tion state with a mixture of CDCl3 and CD3OD (10:1) as the
solvent and at a concentration of 11.2 mM, tripalmitin adopts
the three conformers in the ratio of gt (45%), gg (37%) and tg
(18%). In comparison with this symmetric lipid, the asym-
metric phospholipid (DPPC) favors the gt-conformer more
strongly around the tail lipid moiety along the sn-1,2 position,
while disfavoring the tg-conformer, in the ratio of gt (59%), gg
2.8gg + 3.1gt + 10.7tg = 3JH2,H1S (or 3JH2,H3R)
0.9gg + 10.7gt + 5.0tg = 3JH2,H1R (or 3JH2,H3S) and
gg + gt + tg = 1.
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Beilstein J. Org. Chem. 2012, 8, 629–639.
Figure 4: Distributions of gg, gt and tg-conformers in 1-substituted sn-glycerols. In these sn-isomers, Φ1 and Φ2 represent the dihedral angles
around the C–C single bond at the glycerol sn-2,3 and 1,2-position, respectively.
(34%) and tg (7%). The head phosphate moiety along the sn-2,3
position adopts the three conformers in equilibrated popula-
tions (gg = gt = tg).
3-O-(α-D-glucopyranosyl)-sn-glycerolipids 10 and 11
(Figure 3, entries 3 and 4) were found to have conformational
properties very similar to DPPC; the lipid tail moiety prefers the
gauche conformations (gt and gg), while the sugar moiety
allows a random conformation. Here, it should be mentioned
that the conformer distribution coincides between I-a (Figure 3,
entry 5) and DPPC (Figure 3, entry 2) at the head moiety [gt
(33%), gg (34%) and tg (33%)].
The above analysis was carried out also for the stereoisomer I-b
and the related glycolipids (Figure 4, entries 6–8). The isomer
(Figure 4, entry 8) showed an overall conformational property
similar to I-a and DPPC (Figure 5), although a small difference
was observed in the conformer distribution at the sugar head
moiety. However, it should be recognized here that the helical
Figure 5: A common conformational property of GGPL-I and DPPC.
The tail lipid moiety favors two gauche-conformers of gt and gg (gt >
direction (helicity) of the gt conformer in I-b is reversed (anti-
gg >> tg), while the head moiety takes three conformers in averaged
populations (gt = gg = tg).
clockwise) from the case of DPPC and GGPL-I (clockwise), as
depicted in Figure 3 and Figure 4.
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Beilstein J. Org. Chem. 2012, 8, 629–639.
Conclusion
gradient modes. For thin-layer chromatography (TLC) analysis,
We have proposed a synthetic pathway to a GGPL-I homo- Merck precoated TLC plates (silica gel 60 F254, layer thickness
logue and its stereoisomer, in which our one-pot α-glycosyl- 0.25 mm) and Merck TLC aluminum roles (silica gel 60 F254,
ation methodology was effectively applied. We envisage that layer thickness 0.2 mm) were used. All other chemicals were
the simple method will allow us to prepare a variety of purchased from Tokyo Kasei Kogyo Co., Ltd., Kishida Chem-
α-glycolipid antigens other than GGPLs and to prove their bio- ical Co., Ltd., and Sigma–Aldrich Chemical Company Co, Int.,
logical significance [43]. By the 1H NMR conformational and were used without further purification.
analysis, which was based on our former studies on deuterium-
labeled sn-glycerols and sugars, we have proven that GGPL-I A typical procedure for the one-pot α-glycosylation: CBr4
and other 3-O-(α-D-glucopyranosyl)-sn-glycerolipids have a (1.6 g, 6.09 mmol) and Ph3P (2.02 g, 6.09 mmol) were added to
common conformational property at the chiral glycerol moiety: a solution of 6-O-acetyl-2,3,4-tri-O-benzyl-D-glucose (1) (1.0
The lipid tail moiety prefers two gauche-conformations (gg and g, 2.03 mmol) in 10 mL of DMF and stirred for 3 h at rt. Then,
gt) in the order gt > gg >> tg, while the sugar head moiety (S)-glycidol (301 mg, 4.06 mmol) was added to the reaction
adopts three conformers in an averaged population (gg = gt = mixture and stirred for 14 h at rt. Products were diluted with a
tg). At the lipid tail position, the gt-conformer with clockwise mixture of toluene and ethyl acetate (10:1), and the solution was
helicity is predominant over the anticlockwise gg-conformer. washed with saturated aq. NaHCO3 and aq. NaCl solution, dried
The observed conformation was very close to what we have and concentrated. The residue was purified by silica gel column
seen in DPPC (Figure 5). Although these results were based on chromatography in toluene and ethyl acetate to give a mixture
the solution state in a solvent mixture of CHCl3 and CH3OH of 4a and 5a (the ratio changed with reaction time) as
(10:1), it may be possible to assume that the mycoplasma colorless syrup. The total yield of 4a and 5a was between 80%
GGPLs and the related 3-O-(α-D-glycopyranosyl)-sn-glycero- and 90%.
lipids can constitute cytoplasm membranes in good cooperation
with ubiquitous phospholipids without inducing stereochemical 3-O-(2,3,4-tri-O-benzyl-α-D-glucopyranosyl)-1,2-di-O-
stress at the membrane.
palmitoyl-sn-glycerols 8a and 8b: K2CO3 (379 mg,
2.74 mmol) was added to the mixture of 4a and 5a (1 g,
The GGPL-I isomer I-b showed an overall conformational 1.83 mmol based on 4a) in CH3OH (20 mL) and stirred for 1 h
property similar to the natural isomer I-a and DPPC. However, at rt. The reaction mixture was neutralized, washed with water,
it should be mentioned here that the chiral helicity of dried, and concentrated. The residue was dried under reduced
gt-conformers in I-b is reversed (anticlockwise) from the clock- pressure and directly subjected to the next reaction. A mixture
wise helicity of DPPC and GGPL-I. The difference in chirality of caesium palmitate (2.7 g, 7.3 mmol) in DMF (40 mL) was
seems critical in biological recognition events and also in heated at 100–110 °C, to which the DMF solution of the above
physicochemical contact with other chiral constituents in cell residue was added slowly. The reaction mixture was stirred for
membranes [44,45].
2 h at 110 °C, cooled to rt, and then filtered through a pad of
Celite powder with ethyl acetate. The filtrate was washed with
saturated aq. NaCl solution, dried, and concentrated. The
residue was purified by silica gel column chromatography to
Experimental
General methods
Infrared (IR) spectra were recorded on a JASCO FT/IR-230 give 6a as a colorless syrup (830 mg, 60% yield). To a solution
Fourier transform infrared spectrometer on KBr disks. All 1H of 6a (300 mg, 0.39 mmol) in pyridine (20 mL), TBDMS chlo-
NMR (500 MHz) spectra were recorded by using a Varian ride (107 mg, 0.71 mmol) and 4-N,N-dimethylaminopyridine
INOVA-500 or Varian Gemini 200. 1H chemical shifts are (cat.) were added. The reaction mixture was stirred for 12 h at
expressed in parts per million (δ ppm) by using an internal stan- rt, treated with methanol (2 mL) for 3 h and concentrated. The
dard of tetramethylsilane (TMS = 0.000 ppm). Mass spectra residue was purified by silica gel column chromatography in a
were recorded with a JEOL JMS 700 spectrometer for fast atom mixture of toluene and ethyl acetate. The main product was
bombardment (FAB) spectra. Silica gel column chromatog- dissolved in pyridine (20 mL) and then reacted with palmitoyl
raphy was performed on silica gel 60 (Merck 0.063–0.200 mm chloride (162 mg, 0.59 mmol) for 3 h at rt. The reaction mix-
and 0.040–0.063 mm) and eluted with a mixture of toluene and ture was treated with methanol (2 mL) and then concentrated
ethyl acetate or a mixture of CHCl3 and CH3OH in gradient with toluene. The residue was dissolved in a mixture of CH3OH
modes (100:0 to 80:20). For purification of phosphocholine- and CH2Cl2 (1:1, 20 mL) and treated with trifluoroacetic acid
containing products, a chromatographic column packed with (1 mL) for 2 h at rt. After concentration, the residue was puri-
Iatrobeads (IATRON LABORATORIES INC., 6RS-8060) was fied by silica gel column chromatography in a mixture of
applied and eluted with a mixture of CH3OH and CHCl3 in toluene and ethyl acetate to give 8a as a white waxy solid
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Beilstein J. Org. Chem. 2012, 8, 629–639.
(0.32 g, 81% yield from 6a). [α]D30 +21.1 (c 1.0, CHCl3); IR and concentrated. The residue was dissolved in a mixture of
(KBr, film): 3413, 2923, 2853, 1736, 1630, 1457, 1361, 1158, CH3OH (10 mL) and 30% aq. NH3 (1 mL) and stirred for
1069, 736 cm−1; 1H NMR (500 MHz, CDCl3): δH 7.40–7.23 15 min at rt. The reaction mixture was concentrated, and the
(m, 5H × 3, -CH2C6H5), 5.23 (m, 1H, glycerol H-2), 4.96–4.64 residue was purified by column chromatography
(d, 2H × 3,-CH2C6H5), 4.70 (d, 1H, J = 3.5 Hz, H-1), 4.40 (dd, (IATROBEADS in a mixture of CHCl3 and CH3OH) to give 9a
1H, J = 4.0 and 12.0 Hz, glycerol H-1proS), 4.19 (dd, 1H, J = (186 mg, 80% yield). [α]D26 +13.0 (c 0.45, CHCl3); IR (KBr,
6.0 and 12.0 Hz, glycerol H-1proR), 3.96 (dd, 1H, J = 9.5 and film): 3301, 2929, 2856, 2537, 1731, 1577, 1419, 1216, 1093,
9.5 Hz, H-3), 3.72 (dd, 1H, J = 5.5 and 10.5 Hz, glycerol 925, 788, 746; 1H NMR (500 MHz, CDCl3): δH 7.35–7.25 (m,
H-3proR), 3.72 and 3.66 (b, 2H, H-6ProR and H-6proS), 3.65 (m, 5H × 3, -CH2C6H5), 5.22 (m, 1H, glycerol H-2), 4.93–4.61 (d,
1H, H-5), 3.54 (dd, 1H, J = 5.5 and 10.5 Hz, glycerol H-3proS), 2H × 3, -CH2C6H5), 4.70 (d, 1H, J = 3.5 Hz, H-1), 4.38 (dd,
3.50 (dd, 1H, J = 9.5 and 10.0 Hz, H-4), 3.49 1H, J = 3.0 and 12.0 Hz, glycerol H-1proS), 4.19 (b, 2H, choline
(dd, 1H, J = 3.5 and 9.5 Hz, H-2), 2.29 (m, 2H × 2, -CH2CH2N+(CH3)3), 4.17 (dd, 1H, J = 6.5 and 12.0 Hz, glycer-
- O C O C H 2 C H 2 ( C H 2 ) 1 2 C H 3 ) , 1 . 5 8 ( b , 2 H
-OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 25 (b, 24H
×
×
2 , ol H-1proR), 4.15 and 4.02 (m, 2H × 2, H-6ProR and H-6proS),
2, 3.93 (dd, 1H, J = 9.0 and 9.5 Hz, H-3), 3.72 (dd, 1H, J = 5.5
-OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz, and 11.0 Hz, glycerol H-3proR), 3.71 (b, 1H, H-5), 3.62 (t, 1H,
-OCOCH2CH2(CH2)12CH3); FABMS m/z: [M + Na]+ 1023.7. H-4), 3.58 (b, 2H, choline -CH2CH2N+(CH3)3), 3.52 (dd, 1H, J
= 6.0 and 11.0 Hz, glycerol H-3proS), 3.46 (dd, 1H, J = 3.5 and
In the same way as derived for the synthesis of 8a, (R)-glycidol 9.5 Hz, H-2), 3.15 (s, 9H, -POCH2CH2N+(CH3)3),
and 6b (0.30 g, 0.39 mmol) was used for the synthesis of 8b 2.28 (m, 2H × 2, -OCOCH2CH2(CH2)12CH3), 1.58 (b, 2H × 2,
(0.30 g, 77% yield). [α]D32 +18.5 (c 1.0, CHCl3); IR (KBr, -OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 25 (b, 24H
× 2,
film): 3452, 2924, 2854, 1739, 1586, 1455, 1296, 1159, 1095, -OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz,
710 cm−1; 1H NMR (500 MHz, CDCl3): δH 7.40–7.23 (m, 5H × -OCOCH2CH2(CH2)12CH3); FABMS m/z: [M + Na]+ 1188.7.
3, -CH2C6H5), 5.23 (b, 1H, glycerol H-2), 4.96–4.63 (d, 2H ×
3,-CH2C6H5), 4.75 (d, 1H, J = 3.5 Hz, H-1), 4.38 (dd, 1H, J = (b) Compound 9a (0.18 g, 0.15 mmol) was hydrogenated with
3.5 and 12.0 Hz, glycerol H-3proR), 4.21 (dd, 1H, J = 6.5 and Pd(OH)2/C (8 mg) under atmospheric pressure in a mixture of
12.0 Hz, glycerol H-3proS), 3.96 (dd, 1H, J = 9.5 and 9.5 Hz, CH3OH (10 mL) and acetic acid (0.1 mL) for 7 h at rt. The
H-3), 3.73 (dd, 1H, J = 6.0 and 11.0 Hz, glycerol H-1proS), 3.76 reaction mixture was neutralized by the addition of Et3N,
and 3.66 (b, 2H, H-6ProR and H-6proS), 3.65 (m, 1H, H-5), 3.57 filtered and concentrated. The residue was purified by column
(dd, 1H, J = 5.5 and 11.0 Hz, glycerol H-1proR), 3.51 (dd, 1H, J chromatography with IATROBEADS (CH3OH and CHCl3) to
= 9.5 and 10.0 Hz, H-4), 3.50 (dd, 1H, J = 3.5 and 9.5 Hz, H-2), give I-a (101 mg, 75% yield). [α]D31 +18.7 (c 1.0, CHCl3/
2.29 (b, 2H × 2, -OCOCH2CH2(CH2)12CH3), 1.59 (b, 2H × 2, CH3OH 10:1); IR (KBr, film): 3372, 2927, 2852, 1731, 1573,
-OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 25 (b, 24H
× 2, 1469, 1112, 975, 727; 1H NMR (500 MHz, CDCl3/CD3OD
-OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz, 10:1): δH 5.23 (m, 1H, glycerol H-2), 4.80 (d, 1H, J = 3.5 Hz,
-OCOCH2CH2(CH2)12CH3); FABMS m/z: [M + Na]+ 1023.7. H-1), 4.39 (dd, 1H, J = 3.5 and 12.0 Hz, glycerol H-1proS), 4.30
(b, 2H, -CH2CH2N+(CH3)3), 4.24 and 3.95 (b, 2H, H-6ProR and
A phosphorodiamidite method for the syn-
thesis of I-a
H-6proS), 4.14 (dd, 1H, J = 6.5 and 12.0 Hz, glycerol H-1proR),
3.75 (dd, 1H, J = 5.5 and 11.0 Hz, glycerol H-3proR), 3.67 (b,
(a) The reaction vessel was kept under anhydrous conditions 2H, choline-CH2CH2N+(CH3)3), 3.65 (b, 1H × 2, H-3 and H-5),
with Ar gas in the presence of molecular sieves (50%w/w), and 3.61 (dd, 1H, J = 5.5 and 11.0 Hz, glycerol H-3proS),
a solution of 8a (0.20 g, 0.20 mmol) and 2-cyanoethyl- 3.56 (b, 1H, H-4), 3.46 (b, 1H, H-2), 3.22 (s, 9H,
N,N,N',N'-tetraisopropyl phosphorodiamidite (90.4 mg, 0.30 - P O C H 2 C H 2 N + ( C H 3 ) 3 ) , 2 . 3 1 ( m , 2 H
mmol) in 10 mL of CH2Cl2 was injected. 1H-tetrazole (28.4 - O C O C H 2 C H 2 ( C H 2 ) 1 2 C H 3 ) , 1 . 6 0 ( b , 2 H
mg, 0.40 mmol) was added and stirred for 2 h at rt. Then -OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 25 (b, 24H
×
2 ,
2 ,
2,
×
×
1H-tetrazole (42.6 mg, 0.60 mmol, 3.0 equiv) and choline tosy- -OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz,
late (220.3 mg, 0.8 mmol: thoroughly dried overnight under -OCOCH2CH2(CH2)12CH3); HRMS–FAB (m/z): [M + Na]+
vacuum) were added to the reaction mixture and stirred for calcd for C46H90NO13PNa, 918.6048; found, 918.6028.
1.5 h at rt. The reaction was quenched by the addition of water
(1 mL), and then m-chloroperbenzoic acid (51.8 mg, 0.3 mmol) In the same way as described above, 9b (180 mg) was derived
was added at 0 °C and stirred for 10 min at rt. The reaction mix- from 8b (200 mg, 0.20 mmol) in 76% yield and converted to the
ture was washed with 10 % aq. Na2SO3 solution, saturated aq. GGPL-I isomer I-b [70 mg, 81% yield from 9b (120 mg)]. 9b:
NaHCO3 solution, water and saturated aq. NaCl solution, dried [α]D26 +8.1 (c 0.62, CHCl3); IR (KBr, film): 3413, 2923, 2857,
636

Beilstein J. Org. Chem. 2012, 8, 629–639.
1735, 1461, 1241, 1097, 744, 495, 445; 1H NMR (500 MHz, -OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz,
CDCl3): δH 7.35–7.23 (m, 5H × 3, -CH2C6H5), 5.23 (b, 1H, -OCOCH2CH2(CH2)12CH3); HRMS–FAB (m/z): [M +
glycerol H-2), 4.94–4.64 (d, 2H × 3,-CH2C6H5), 4.74 (d, 1H, J Na]+calcd for C41H78O10Na, 753.5493;found, 753.5519.
= 3.5 Hz, H-1), 4.37 (dd, 1H, J = 3.0 and 12.0 Hz, glycerol
H-3proR), 4.23 (b, 2H, choline-CH2CH2N+(CH3)3), 4.19 (dd, 3-O-(6-O-palmitoyl-α-D-glucopyranosyl)-1,2-di-O-palmi-
1H, J = 6.5 and 12.0 Hz, glycerol H-3proS), 4.16 and 4.09 (b, toyl-sn-glycerol (11, entry 4): A mixture of 8a (120 mg, 0.12
2H, H-6proR and H-6proS), 3.94 (dd, 1H, J = 9.5 and 9.5 Hz, mmol) and palmitoyl chloride (165 mg, 0.6 mmol) in pyridine
H-3), 3.76 (dd, 1H, J = 5.5 and 11.0 Hz, glycerol H-1proS), 3.72 was stirred at rt for 3 h and then treated with CH3OH (1 mL) for
(m, 1H, H-5), 3.61 (dd, 1H, J = 9.0 and 9.5 Hz, H-4), 3.60 (b, 3 h. After concentration in vacuo, the residue was purified on
2H, choline-CH2CH2N+(CH3)3), 3.57 (dd, 1H, J = 5.5 and 11.0 silica gel (toluene/ethyl acetate). The main product (138 mg)
Hz, glycerol H-1proR), 3.49 (dd, 1H, J = 3.5 and 9.5 Hz, H-2), was dissolved in a mixture of cyclohexene/ethanol 1:4 and
3.20 (s, 9H, choline-CH2CH2N+(CH3)3), 2.28 (dd, 2H × 2, J = subjected to catalytic hydrogenation at atmospheric pressure in
7.5 and 15 Hz, -OCOCH2CH2(CH2)12CH3), 1.58 (b, 2H × 2, the presence of Pd(OH)2/C (50 mg). The product was purified
-OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 25 (b, 24H
× 2, by silica gel column chromatography (CH3OH/CHCl3) to afford
-OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz, 11 (99 mg, 85% yield from 8a). [α]D30 +20.9 (c 1.0, CHCl3/
-OCOCH2CH2(CH2)12CH3); FABMS m/z: [M + Na]+ 1188.7. CH3OH 10:1); IR (KBr, film): 3414, 2919, 2851, 1739, 1605,
I-b: [α]D31= +10.7 (c 1.0, CHCl3/CH3OH 10:1); IR (KBr film): 1465, 1375, 1176, 1054, 720 cm−1; 1H NMR (500 MHz,
3390, 2919, 2856, 1731, 1463, 1228, 1074, 964, 711; 1H NMR CDCl3/CD3OD 10:1): δH 5.25 (m, 1H, glycerol H-2), 4.82 (d,
(500 MHz, CDCl3/CD3OD 10:1): δH 5.24 (m, 1H, glycerol 1H, J = 4.0 Hz, H-1), 4.40 (dd, 1H, J = 3.5 and 12.0 Hz, glycer-
H-2), 4.80 (d, 1H, J = 3.5 Hz, H-1), 4.34 (dd, 1H, J = 3.5 and ol H-1proS), 4.34 and 4.30 (dd × 2, 2H, J = 5.0 and 12.0, 2.5 and
12.0 Hz, glycerol H-3proR), 4.28 (b, 2H, choline- 12.0 Hz, H-6proR and H-6proS), 4.16 (dd, 1H, J = 6.5 and 12.0
CH2CH2N+(CH3)3), 4.25 and 3.97 (b, 2H, H-6ProR and Hz, glycerol H-1proR), 3.82 (dd, 1H, J = 5.0 and 10.5 Hz, gly-
H-6proS), 4.20 (dd, 1H, J = 7.0 and 12.0 Hz, glycerol H-3proS), cerol H-3proR), 3.73 (m, 1H, H-5), 3.65 (dd, 1H, J = 9.0 and 9.5
3.80 (dd, 1H, J = 6.0 and 11.0 Hz, glycerol H-1proS), 3.63 (b, Hz, H-3), 3.61 (dd, 1H, J = 5.5 and 10.5 Hz, glycerol H-3proS),
1H × 2, H-3 and H-5), 3.63 (b, 2H, choline-CH2CH2N+(CH3)3), 3.45 (dd, 1H, J = 4.0 and 9.5 Hz, H-2), 3.33 (dd,
3.58 (dd, 1H, J = 5.5 and 11.0 Hz, glycerol H-1proR), 3.55 (b, 1H, J = 9.0 and 10.0 Hz, H-4), 2.33 (m, 2H × 3,
1H, H-4), 3.46 (dd, 1H, H-2), 3.22 (s, 9H, choline- - O C O C H 2 C H 2 ( C H 2 ) 1 2 C H 3 ) , 1 . 6 1 ( b , 2 H
CH2CH2N+(CH3)3), 2.31 (dd, 2H × 2, J = 7.5 and 15 Hz, -OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 26 (b, 24H
×
×
3 ,
3,
- O C O C H 2 C H 2 ( C H 2 ) 1 2 C H 3 ) , 1 . 6 0 ( b , 2 H
-OCOCH2 CH2 (CH2 )1 2 CH3 ), 1. 26 (b, 24H
×
×
2 , -OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 3, J = 7.0 Hz,
2, -OCOCH2CH2(CH2)12CH3); HRMS–FAB (m/z): [M + Na]+
-OCOCH2CH2(CH2)12CH3), 0.88 (t, 3H × 2, J = 7.0 Hz, calcd for C57H108O11Na, 991.7789; found, 991.7832.
-OCOCH2CH2(CH2)12CH3); HRMS–FAB (m/z): M + Na]+
calcd for C46H90NO13PNa, 918.6048; found, 918.6078.
Acknowledgements
This work was supported by the Industrial Technology
3-O-(α-D-glucopyranosyl)-1,2-di-O-palmitoyl-sn-glycerol Research Grant Program from New Energy and Industrial Tech-
(10, entry 3): Compound 10 was obtained as a waxy solid (73 nology Development Organization (NEDO) of Japan and from
mg, 83% yield) from 8a (120 mg, 0.12 mmol) by catalytic the Science & Technology Project for a Safe & Secure Society
hydrogenation under the same reaction conditions as those from the Ministry of Education, Culture, Sports, Science and
described above for the preparation of I-a. [α]D30 +27.2 (c 1.0, Technology (MEXT) of Japan.
CHCl3/CH3OH 10:1); IR (KBr, film): 3411, 2919, 2851, 1739,
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