Synthesis of α-O- and α-S-glycosphingolipids related to sphingomonous cell wall antigens using anomerisation

Article abstract of DOI:10.3390/molecules180911198

Analogues of glycolipids from Spingomonadacaece with O- and S- and SO ₂-linkages have been prepared using chelation induced anomerisation promoted by TiCl₄. Included are examples of the anomerisation of intermediates with O- and S-glycosidic linkages as well as isomerisation of β-thioglycuronic acids (β-glycosyl thiols). The β-O-glucuronide and β-O-galacturonide precursors were efficiently prepared using benzoylated trichloroacetimidates. β-Glycosyl thiols were precursors to β-S-derivatives. Triazole containing mimics of the natural glycolipids were prepared using CuI promoted azide-alkyne cycloaddition reactions in THF. The glycolipid antigens are being evaluated currently for their effects on iNKT cells.

Full text of DOI:10.3390/molecules180911198

Molecules 2013, 18, 11198-11218; doi:10.3390/molecules180911198
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of α-O- and α-S-Glycosphingolipids Related to
Sphingomonous cell Wall Antigens Using Anomerisation
Wayne Pilgrim 1,2, Ciaran O’Reilly 1,2 and Paul V. Murphy 2,*
1
School of Chemistry and Chemical Biology, University College Dublin, Dublin 4, Ireland
2
School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland
* Author to whom correspondence should be addressed; E-Mail: paul.v.murphy@nuigalway.ie;
Tel.: +353-91-492460; Fax: +353-91-495576.
Received: 31 July 2013; in revised form: 4 September 2013 / Accepted: 5 September 2013 /
Published: 12 September 2013
Abstract: Analogues of glycolipids from Spingomonadacaece with O- and S- and
SO2-linkages have been prepared using chelation induced anomerisation promoted by
TiCl4. Included are examples of the anomerisation of intermediates with O- and
S-glycosidic linkages as well as isomerisation of β-thioglycuronic acids (β-glycosyl thiols).
The β-O-glucuronide and β-O-galacturonide precursors were efficiently prepared using
benzoylated trichloroacetimidates. β-Glycosyl thiols were precursors to β-S-derivatives.
Triazole containing mimics of the natural glycolipids were prepared using CuI promoted
azide-alkyne cycloaddition reactions in THF. The glycolipid antigens are being evaluated
currently for their effects on iNKT cells.
Keywords: α-GalCer; anomerisation; glucuronic acid; galacturonic acid; glycoside bond;
antigens; NKT cells
1. Introduction
CD1d restricted invariant natural killer T cells (iNKT cells) are a class of lymphocytes activated in
response to specific glycolipid antigens. Stimulation of iNKT cells by glycolipids can cause secretion
of Th1, Th2, Th17 and Treg cytokines and there could be advantages in clinical therapy for glycolipids
to be identified that can induce a bias towards secretion of Th1 or Th2 cytokines. The prototype
antigen α-galactosylceramide (α-GalCer, 1) stimulates iNKT cells to kill tumour cells, release
cytokines and activate other cells of the immune system in mice. Consequently there has been interest

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in evaluating glycolipid antigens with potential as anti-infective agents & vaccine adjuvants [1–10], and
clinical trials of some glycolipids have been undertaken [11] or their preclinical evaluation is
advanced [12]. The cell walls of Sphingomonadacae bacteria present uronic acid containing
glycosphingolipids such 2–4 [1–3,7] which stimulate iNKT cells. Sphingomonadacae are
gram-negative bacteria that can cause infection in humans. While monosaccharide and higher order
saccharide (e.g., 3, Figure 1) antigens are known in these bacteria, it seems that the most potent
stimulators of the iNKT cells are monosaccharides [7]. Such bacterial glycolipids have been shown to
induce septic shock and bacterial clearance in infected mice, demonstrating that glycolipids stimulate
an innate-type immune response to gram negative bacteria [2,3]. The Sphingomonadacae glycolipids
contain glucuronic acid or galacturonic acid residues which are α-O-linked to sphinganine derivatives
(Figure 1), and they are structurally related to α-GalCer 1. The α-glycosidic linkage seems
advantageous in generating highly potent stimulatory properties. In this article we present a
full account of work on the synthesis of α-S-, SO2- and O-linked glycolipids 5–10 which are
based on glucuronic acid and galacturonic acid. This article supplements two preliminary
communications [13,14] where we have outlined how chelation induced anomerisation [15–18] can be
used for the preparation of S- and O- glycolipids relevant to biology and medicine. Herein we provide
additional examples and more detail regarding our initial efforts in this area.
Figure 1. Structures of glycosphingolipids 1–10.

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2. Results and Discussion
The structures of the key building blocks 11–18 used in the synthesis of the glycosphinoglipid
antigens 5–10 are shown in Figure 2. Highly stereoselective glycosidations of uronic acids can be
difficult to achieve and we had observed high selectivity in favour of the α-anomer in anomerisation of
simple α-O and α-S-glucuronic acid derivatives. The synthesis of this type of glycolipid provided a
more challenging biologically relevant target with a view to testing the scope of the Lewis acid
catalysed anomerisation reactions. The overarching aim of this research was thus to establish whether
TiCl4 promoted anomerisation reactions could be utilized in synthesis of these biologically interesting
compounds. The planned strategy involved preparation of potential α-O or α-S glycoside precursors to
the glycolipids and to investigate their subsequent anomerisation and then work out the remaining steps
to give the unprotected glycolipids. The synthesis of 6, 7 and 9 has been previously detailed [13,14] and we
supplement the reports on the preparation of those compounds with details of the preparation of 5, 8
and 10. We have included comparisons of yields and stereoselectivities of the various steps from the
previous reports for completeness. In recent research, a triazole was incorporated into α-GalCer
analogues as an isostere of the sphingosine amide. This conferred interesting and desirable biological
properties, as triazole analogues with a long alkyl chains had potent stimulatory effects on cytokine
production and showed a stronger Th2 cytokine response than found for α-GalCer [19]. Therefore, we
included the preparation of such a triazole analogue containing a uronic acid 10 as part of this research
work. The research therefore began with the synthesis of O-glycosides from the trichloroacetimidates
11 and 12 which were used in conjunction with alcohol 16. In addition we used the thioglycuronic acid
derivatives 13–15 which were used jointly with the bromides 17 and 18 in the generation of
S-linked glycolipids.
Figure 2. Structures of key building blocks.
The preparation of the trichloroacetimidates 11 and 12 is summarized in Scheme 1. The
regioselective protection of D-galactose and D-glucose using trityl chloride in pyridine was followed by
benzoylation in the presence of pyridine. Subsequent hydrolysis of the trityl group using sulfuric acid
in dichloromethane gave the alcohols 19a and 19b. Oxidation of the glucose derivative 19b with
TEMPO-NaOCl proceeded satisfactorily to give the glucuronic acid. However, these conditions were
not successful for the oxidation of the galactose derivative 19a. Nevertheless, the reaction of 19a with

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TEMPO and BAIB as co-oxidant proceeded smoothly to give the galacturonic acid precursor of 20a.
Esterification of the acids via the generation of the carboxylates and their reaction with allyl iodide
gave 20a and 20b. Formation of the ester 20a via the acid chloride, synthesised from reaction of the
acid with oxalyl chloride and DMF in CH2Cl2 followed by addition of allyl alcohol was also
investigated. This route provided the allyl ester 20a in similar yield (40% vs. 44%). Next the glycosyl
bromides were formed by treatment of 20a and 20b with HBr-AcOH in dichloromethane. Reaction of
the isolated bromides with silver carbonate in water and acetone followed by reaction with
trichloroacetonitrile in the presence of DBU gave the glycosyl donors 11 and 12.
Scheme 1. Synthesis of trichloroacetimidates.
With the synthesis of both donors 11 and 12 achieved, the glycoside coupling reactions and
subsequent chelation induced anomerisation were investigated (Scheme 2). The optimal strategy was
to first prepare the β-glycoside 22 by coupling the trichloroacetimidates 11 and 12 with acceptor 16 [13]
using TMSOTf. The benzoylated donor 21b was superior to the related acetylated donor 25.
Glycosidation of 25 with 16 under the same conditions as reaction with 12 led to the isolation of the
corresponding orthoester 26. Evidence for the orthoester was obtained by NMR analysis which, for
1
example, showed an anomeric proton at δ 5.74 ppm (J1,2 5.2 Hz) in the H-NMR spectrum. The next
step was the application of TiCl4 to promote anomerisation. This reaction using 2 equiv of TiCl4 did
proceed in a satisfactory manner to generate the required α-anomers 23a and 23b from the β-glycoside
precursors. The yields and stereoselectivities were high (97:3 or greater) for these reactions. The Lewis
acid conveniently removed the benzyl group from the sphinganine residue under these conditions. It
was found that anomerisation proceeded faster than the cleavage of the benzyl ether. Thus, the benzyl
protected α-anomer could be isolated if required. With 23 in hand, the azide groups were reduced using
the Staudinger reaction and subsequent coupling of the amine with nonadecanoyl chloride in the
presence of triethylamine gave amides 24. Although the benzoates were advantageous in the
glycosidation reaction, they were not easily removed in the final step. Reaction with methoxide in
methanol led to elimination of benzoic acid and formation of an unsaturated product whereas reaction
with K2CO3 in methanol and water led to removal of the allyl ester but under these conditions the
benzoyl protecting groups were found to be stable. Finally the removal of all the protecting groups was

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successfully carried out using hydroperoxide ion generated from hydrogen peroxide in n-propanol
using sodium propoxide as base. These conditions were used in order to maximise solubility of the
glycolipid by using a more hydrophobic solvent than methanol or ethanol. Centrifugation of the
product mixture and precipitation of the insoluble glycolipids and subsequent water washing was used
to isolate the products 5 and 6. Success of the centrifugation method for purification was dependant on
the relative solubility of each glycolipid. Therefore the amount of solvent used in each reaction had to
be adjusted to match the solubility of each glycolipid. The deprotection of 24a was carried out in less
solvent (3 mL for 10 mg precursor) than 24b (5 mL for 10 mg precursor) in order to ensure that
precipitation of the glycolipid would occur. The low yields in the final step are generally attributed to
the product not precipitating and to some of the precipitate being re-dissolved in the water washing step.
Scheme 2. Synthesis of 5 and 6.
Given that the azide 22b was available its conversion into the triazole-containing glycolipid 10 was
also investigated. The synthesis of 1-nonadecyne 28 from 1-nonadecene 27 used a method related to an
established procedure for the synthesis of 1-heptadecyne [20]. Thus, the addition of bromine followed
by didehydrohalogenation with KOH in EtOH gave 28 (73%) as well as 2-bromononadec-1-ene 29

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(24%) (Scheme 3). Initial attempts at promoting the copper catalysed azide-alkyne cycloaddition [21]
were investigated by coupling 23b and 28. However reactions using CuSO4 and sodium ascorbate in
t
various mixtures of THF, H2O, and BuOH were not successful [22,23]. This was attributed to the low
solubility of 28. In contrast the use of CuI in THF [24] led to a successful coupling of 23b and 28 and
gave an 86:14 mixture of the triazoles 30 and 31 (32%). The 1,4 configuration was assigned to the
major product (30) on the basis of both NOESY and ROESY analysis. The 1,4-regioisomer (30)
showed interactions between the triazole proton with a number of protons on the dihydroceramide
chain (see Scheme 3 for NOE correlations) indicating that they are in close proximity. The triazole
proton of the minor regioisomer 31 is more remote from the dihydroceramide chain, and as would be
expected for this isomer there was not any NOE cross-peaks observed between this proton and the
dihydroceramide chain. The regioselectivity of this Cu(I) promoted cycloaddition reaction was not as
high as that reported for the previously synthesised triazole containing glycolipids, were the 1,4 isomer
was not reported [19]. The protecting groups were removed from 30/31 to give a mixture of triazoles
(52%) where 10 was the major component (ratio of 1,4 to 1,5-isomer, 88:12).
Scheme 3. Synthesis of 10.
The synthesis of S-glycoside analogues of natural bioactive O-glycosides has been of interest as
potential glycomimetics [25–27]. S-Glycosides are apparently more stable in vivo than native
O-glycosides and also there is the possibility that they are more immunogenic, which is relevant to

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vaccine development [28]. Kinetic studies on the anomerisation of S-butyl and O-butyl glycosides, as
well as the formation of 23a and 23b, indicated that anomerisation of S-glycolipids would be feasible [14].
As reviewed by Pachamuthu and Schmidt [27], the synthesis of thioglycolipids can be approached by
preparation of a lipid containing thiol [29], which is then reacted with a glycosyl halide. Alternatively
a glycosyl thiol can be generated, which is then reacted with a lipid which has an appropriate leaving
group. We investigated the former approach for the glycosphingolipids, however in our hands this was
not successful. In light of this, the formation of the thioglycoside by direct anomeric alkylation of a
glycosyl thiol with a dihydroceramide moiety containing a suitable leaving group was investigated.
Thiol 13 (79%) was synthesised from the glycosyl bromide 32 through its reaction with KSAc and
subsequent selective deprotection using NaSMe (Scheme 4). Initially the direct anomeric alkylation to
give 35 was attempted using the mesylate of the alcohol 16. However efforts to use this mesylate led to
recovery of both starting materials, with no thioglycoside formation occurring. Reaction of the
mesylate at 90 °C with KBr in DMF gave the bromide 17 (93%). The coupling of this bromide with
the thiol 13 was carried out using NaH as base, and this gave the β-thioglycoside 35. It is worth noting
that this alkylation reaction should not be carried out using excess NaH as this gave rise to the
previously mentioned elimination of benzoic acid and formation of an unsaturated saccharide.
Scheme 4. Synthesis of 7.

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With 35 in hand, the thioglycosides 36–37 were also prepared, and their anomerisations
investigated. Anomerisation of 37 using TiCl4 in CH2Cl2 led to the formation of 40 (36%) with 37 also
being recovered (53%) after 2 h. When longer reaction times were investigated decomposition
occurred. The galactose derivative 36 was prepared in high yield from the bromide 17 (93%) and the
corresponding thiol. However anomerisation, using TiCl4 in CH2Cl2 led to the α-chloride 39 (21%)
being formed, while 36 was also recovered in substantial amounts after 24 h at room temp. A
prolonged reaction with TiCl4 (72 h) or the use of SnCl4 led to the α-chloride 39 as the sole product.
Formation of 39 is consistent with activation of the thioglycoside. It is possible that the presence of the
azide and the OBn group in the lipid is chelating efficiently to the Lewis acid leading to activation of
the thioglycoside which is followed by substitution with a chloride from the Lewis acid. In contrast the
anomerisation of 35 was more successful and is explained by chelation to the C-5 carbonyl group and
the pyranose oxygen atom to the Ti(IV) species, which leads to endocyclic cleavage and anomerisation
rather than thioglycoside activation and glycosyl chloride formation. This was supported by kinetics
studies of simple galacturonic acid and galactose substrates which we have described previously [18].
Hence the anomerisation reaction of 35 carried out with TiCl4 in dichloromethane gave the α-anomer 38 in
55% yield after chromatography. The stereoselectivity (4:1) in the anomerisation reaction was not as
high as for anomerisation of the O-galacturonide. This was presumed to be because of a weaker
anomeric effect for sulfur when compared with oxygen which could be due to the sulfur being less
electron withdrawing than oxygen or due to steric reasons where the larger sulfur atom shows a higher
preference for the equatorial position or due to an equilibrium not being attained. Although a mixture
of anomers was formed they could be separated by column chromatography to give 38 in an isolated
55% yield. Reduction of the azide and coupling with nonadecanoyl chloride was carried out as
described earlier to provide 42 in 40% yield. Treatment of 42 with NaOPr-H2O2-PrOH led to the
removal of the protecting groups with concomitant oxidation of the anomeric sulfur atom to give the
sulfone 7 (56%).
The synthetic strategy to the S-glycolipids was next revised, given that the final deprotection led to
sulfur oxidation. We thus investigated the synthesis of glycosyl thiols 14 and 15 and envisaged they
could be used in conjunction with the bromide 18. The glycosyl thiols 34 and 43 which had the
β-configuration were prepared as described previously [30]. Our earlier work indicated that the
stereoselectivity of the anomerisation reaction of simple O- and S-glycosides could be influenced by
the relative amount of TiCl4 that would be used. The concentration of TiCl4 used was varied from 0.5
equivalents to 4.5 equivalents during an investigation of the anomerisation reactions of 34. The α-thiol
15 was formed and the use of 2.5 equivalents of TiCl4 gave the optimum proportion of 15:34 (8:1).
The formation of the α-thiogalacturonate 14 proceeded from 43 with good selectivity (>9:1) for 15
under the same conditions. The α:β ratio was not just dependent on TiCl4 concentration but also on
temperature, as higher ratios were observed for reactions at 0 °C as compared to reactions at room
temp. The α:β ratio was found to be scale dependant. For the formation of 15 on a 100 mg scale, the
α:β ratio was > 97:3 and was 9:1 when carried out on a one gram scale. This difference may be due to
a greater exotherm on the larger scale which led to warming of the reaction mixture with a consequent
increase in proportion of the β-anomer. An alternative explanation is that this reaction may not have
attained equilibrium on the larger scale. Nevertheless, the transformation was synthetically useful. The
presence of the C-6 carbonyl was found to be critical in order for the anomerisation of the thiol to

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proceed efficiently. The anomerisation reaction of the corresponding 2,3,4,6-tetra-O-acetyl
β-D-thiogalactopyranose, for example, did not proceed under identical conditions, supporting the
notion that chelation by both the C-5 carbonyl and ring oxygen to the Lewis acid is necessary for
efficient anomerisation (Scheme 5).
Scheme 5. Synthesis of α-thioglycopyuranuronic acid derivatives.
TiCl4
CH2Cl2
CO2Me
O
CO2Me
O
AcO
AcO
AcO
AcO
SH
24 h, 0 oC
AcO
AcO
SH
15
34
TiCl4 (no of equiv) : Ratio 15:34
0.5
1.5
2.5
3.5
4.5
60:40
80:20
88:11
83:17
78:22
TiCl4
(2.5 equiv)
AcO
AcO
AcO
CO2Me
O
CO2Me
O
SH
AcO
CH2Cl2
0 oC, 18h
AcO
AcO
SH
43
14
> 90:10
With 14 and 15 available the completion of the synthesis of the S-glycolipid could be achieved
(Scheme 6). The coupling of the thiols 14 and 15 was brought about using NaH (<1 equiv) and 18 to
give the protected lipids 44a and 44b in 36%–39% yield. The use of other bases for this reaction led to
lower yields or only trace amounts of 44a and 44b. Also the addition of additives such as tetra-N-butyl
ammonium iodide did not lead to an improvement in yield. The use of a Mitsunobu condensation
reaction was also investigated. In this case the thiol could be reacted with the alcohol precursor to 18
using 1,1'-(azodicarbonyl)dipiperidine and trimethylphosphine [31] and this approach gave similar
yields to the S-alkylation of 18. The protected thioglycolipids 44a and 44b were then treated with
formic acid [32] for 30 min to remove both the oxazolidine and Boc groups and this gave the
aminoalcohol intermediates in 85% yield. Reaction of this aminoalcohol with the succinate 45 [33] in
dichloromethane gave the amides 46a and 46b (60%); these yields that were better than those from the
corresponding acid chloride. Finally, the removal of the protecting groups from the uronic acid
residues gave 8 and 9. This was achieved in two steps. The methyl ester was removed using LiI in
EtOAc [34], and a subsequent reaction with guanidine and guanidinium nitrate [35] in
methanol-dichloromethane gave 8 and 9.

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Scheme 6. Synthesis of 8 and 9.
3. Experimental
General
Optical rotations were determined with a Perkin-Elmer 343 model polarimeter at the sodium D line
at 20 °C. 1H-NMR spectra and and 13C-NMR were recorded at the frequencies stated. Chemical shifts
are reported relative to internal Me4Si (δ 0.0) in CDCl3 or CDCl3-MeOD, or HOD for D2O (δ 4.84) or
CD2HOD (δ 3.31) for 1H and Me4Si in CDCl3 (δ 0.0) or CDCl3 (δ 77.0) or CD3OD (δ 49.05) for 13C.
1H- NMR signals were assigned with the aid of COSY. 13C-NMR signals were assigned with the aid of
DEPT, HSQC and/or HMBC. Coupling constants are reported in hertz. The IR spectra were recorded
as thin films between NaCl plates or with an ATR attachment. Low and high resolution mass spectra
were measured on either a micromass VG 70/70H or VG ZAB-E or Waters LCT premiere XE
spectrometers and were measured in positive and/or negative mode. Thin layer chromatography (TLC)
was performed on aluminium sheets precoated with silica gel and spots visualized by UV and charring
with H2SO4-EtOH (1:20) or cerium molybdate. Flash chromatography was carried out with silica gel
60 (0.040–0.630 mm). Chromatography solvents were used as obtained from suppliers. CH2Cl2,
MeOH, and THF reaction solvents were dried using a Pure Solv™ Solvent Purification System and
acetonitrile, DMF, pyridine, and toluene were used as purchased from Sigma-Aldrich. The
experimental details for the preparation of 6, 7, 9, 13, 14, 16–18, 19b–24b, 35, 38, 42, 43, 44 and 46b
and their analytical data have been reported previously [13,14].

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1,2,3,4-Tetra-O-benzoyl-α-D-galactopyranuronic acid, allyl ester (20a). D-Galactose (5.4 g, 0.03 mol)
and trityl chloride (17.0 g, 0.04 mol) were dried under diminished pressure for 3 h. The mixture was
taken up in pyridine (80 mL) and heated at 90 °C for 16 h, then The solution was then cooled to 0 °C
and benzoyl chloride (17 mL, 0.15 mol) was added slowlyand he reaction mixture was then allowed to
attain room temp, stirred for 20 h, and the mixture was then diluted with CH2Cl2 and washed with
water, 2 M HCl, satd aq NaHCO3, brine, dried over Na2SO4 and the solvent was removed under
diminished pressure. The resulting syrup was dissolved in CH2Cl2-MeOH (1:2, 150 mL) and conc.
H2SO4 (15 mL) was added. The mixture was stirred for 1 h at room temp and then washed with water,
satd aq NaHCO3, brine, and dried over Na2SO4, and the solvent was removed under diminished
pressure. Flash chromatography (petroleum ether-EtOAc, 4:1) gave 19a as a white solid and a mixture
of anomers (10.9 g, 61%). The NMR data (1H and 13C) for 19a were in agreement with data reported in
the literature [36]. To 19a (10.0 g, 0.017 mol) in CH2Cl2 (100 mL) and H2O (40 mL) were added
TEMPO (0.5 g) and BAIB (16.2 g, 0.05 mol). The mixture was stirred vigorously for 2 h and satd Na2S2O3
(40 mL) was then added and the resulting mixture stirred for 15 min. The layers were separated and the
aq phase acidified with 1M HCl and extracted with CH2Cl2 (×3). The combined organic layers were
dried over Na2SO4, and the solvent was removed under diminished pressure to give the galacturonic
acid intermediate (9.5 g, 91%). The resulting solid was dissolved in THF (100 mL), to this K2CO3 (115 mg,
0.84 mmol), 18-crown-6 (10 mg) and allyl iodide (140 mg, 0.84 mmol, 80 µL) were added. The
reaction was stirred in the dark for 16 h at room temp. The reaction mixture was diluted with Et2O,
washed with water, sodium thiosulfate, water, brine, dried over MgSO4. The solvent was then removed
under reduced pressure and then flash chromatography (EtOAc-cyclohexane 1:4) of the residue gave
20a (484 mg, 44%) as a white solid; Rf 0.53 (EtOAc-cyclohexane 2:5); [α]D +87 (c 4.7, CHCl3); IR
(film) cm−1: 3065, 2930, 1733, 1451, 1263, 1092, 1069; 1H-NMR (CDCl3, 400 MHz ): δ 8.05 (4H, m,
aromatic H), 7.89 (2H, dd, J = 7.2 Hz, J = 1.3 Hz, aromatic H), 7.83 (2H, dd, J = 7.2, J = 1.3 Hz,
aromatic H), 7.61 (1H, tt, J = 7.5 Hz, J = 1.2 Hz, aromatic H), 7.56 (1H, tt, J = 7.4, J = 1.2 Hz,
aromatic H), 7.45 (6H, m, aromatic H), 7.30 (4H, m, aromatic H), 6.28 (2H, m, H-1, H-4), 6.01 (1H, dd,
J = 10.3 Hz, J = 8.3 Hz, H-2), 5.79 (1H, dd, J = 10.3 Hz, J = 3.5 Hz, H-3), 5.72 (1H, ddt, J = 17.2 Hz,
J = 10.3 Hz, J = 6.1 Hz, CH=CH2), 5.22 (1H, dd, J = 17.2 Hz, J = 1.3 Hz, CH=CH2), 5.07 (1H, J = 10.3 Hz,
J = 1.0 Hz, CH=CH2), 4.86 (1H, d, J = 1.5 Hz, H-5), 4.59 (2H, m, OCH2CH=CH2); 13C-NMR (CDCl3,
100 MHz): δ 165.4, 165.2, 165.1, 165.0 164.7 (each C=O), 133.8, 133.6, 133.4 (2s), 130.7, 130.3,
130.1, 129.8, 129.7, 128.8, 128.7, 128.6, 128.5, 128.4 (3s), 128.3 (each C or CH), 120.0 (alkene CH2)
92.6 (C-1), 73.7 (C-5), 71.2 (C-3), 69.0 (C-4), 68.2 (C-2), 66.7 (OCH2CH=CH2); ESI-HRMS calcd for
C37H30O11Na 673.1686, found m/z 673.1714 [M+Na]+.
2,3,4-Tri-O-benzoyl-1-deoxy-1-(2,2,2-trichloro-1-iminoethoxy)-α-D-galactopyranuronic acid,allyl ester (11).
Allyl ester 20a (325 mg, 0.50 mmol) was dissolved in CH2Cl2 (3 mL) and cooled to 0 °C. To this HBr
33% in AcOH (2 mL) was added and the reaction stirred at room temp for 3 h. The reaction was
quenched with satd NaHCO3), washed with water, brine, dried over MgSO4, and the solvent removed
under reduced pressure to give the intermediate bromide (237 mg, 78%); 1H-NMR (CDCl3, 400 MHz): δ
7.99 (4H, d, J = 7.4 Hz, aromatic H), 7.82 (2H, d, J = 7.4 Hz, aromatic H), 7.61 (1H, t, J = 7.4 Hz,
aromatic H), 7.54 (1H, t, J = 7.4 Hz, aromatic H), 7.46 (3H, m, aromatic H), 7.39 (2H, t, J = 7.8 Hz,
aromatic H), 7.28 (2H, t, J = 7.8 Hz, aromatic H), 7.02 (1H, d, J = 3.9 Hz, H-1), 6.32 (1H, dd, J = 3.2 Hz,

Molecules 2013, 18
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J = 1.3 Hz, H-4), 6.05 (1H, dd, J = 10.5 Hz, J = 3.2 Hz, H-3), 5.75 (1H, ddt, J = 17.1 Hz, J = 10.5 Hz,
J = 6.1 Hz, CH=CH2), 5.66 (1H, dd, J = 10.5 Hz, J = 3.9 Hz, H-2), 5,24 (1H, dd, J = 17.1 Hz, J = 0.8 Hz,
J = CH=CH2), 5.17 (1H, d, J = 1.3 Hz, H-5), 5.10 (1H, d, J = 10.3 Hz, CH=CH2), 4.60 (2H, m,
OCH2CH=CH2); 13C-NMR (CDCl3, 100 MHz): δ 165.4, 165.3, 165.0, 164.9 (each C=O), 133.9, 133.7,
133.4, 130.5, 130.0, 129.9, 129.7 (3s), 128.6, 128.5, 128.4, 128.3 (each C or CH), 120.2 (alkene CH2),
87.4 (C-1), 72.9 (C-5), 68.7 (C-4), 68.6 (C-3), 67.9 (C-2), 66.8 (OCH2CH=CH2). This bromide
(237mg, 0.39 mmol) was dissolved in acetone (18 mL) and H2O (2 mL). To this Ag2CO3 (64 mg, 0.23
mmol) was added and the reaction stirred in the dark at room temp for 24 h. The reaction mixture was
filtered through celite, which was rinsed with CH2Cl2. The filtrate was washed with water, brine, dried
over MgSO4, and the solvent removed under reduced pressure. Flash chromatography (EtOAc-
cyclohexane 2:5) gave the hemiacetal (164 mg, 77%) as a colourless oil; Rf 0.18 (EtOAc-cyclohexane
2:5); IR (film) cm−1: 3446, 3069, 2961, 1730, 1264, 1094, 1070, 1026; ES-HRMS calcd for C30H27O10
1
547.1604, found m/z 547.1612 [M+H]+; NMR data for the α anomer: H-NMR (CDCl3, 400 MHz): δ
8.00-7.26 (15H, ms, aromatic H), 6.30 (1H, dd, J = 3.5 Hz, J = 1.6 Hz, H-4), 6.08 (1H, dd, J = 10.7
Hz, J = 3.5 Hz, H-3), 5.96 (1H, t, 3.6 Hz, H-1), 5.70 (2H, overlapping signals, H-2 & CH=CH2), 5.22
(1H, dd, J = 17.2 Hz, J = 1.3 Hz, CH=CH2), 5.18 (1H, d, J = 1.5 Hz, H-5), 5.06 (1H, dd, J = 10.4 Hz,
J = 1.1 Hz, CH=CH2), 4.62 (1H, dd, J = 12.5 Hz, J = 6.5 Hz, OCH2CH=CH2), 4.55 (1H, dd, J = 12.5
Hz, J = 6.3 Hz, OCH2CH=CH2), 4.16 (1H, d, J = 3.6 Hz, OH); 13C-NMR (CDCl3, 100 MHz): δ 167.3,
165.9, 165.6, 165.2 (each C=O), 133.5, 133.4, 133.1, 130.7, 129.9, 129.8 (2s), 129.7, 129.1 (2s), 129.0,
128.5, 128.4, 128.2, 119.9 (alkene CH2), 91.2 (C-1), 69.9 (C-4), 68.8 (2s, C-2 & C-5), 67.7 (C-3), 66.5
1
(OCH2CH=CH2); selected NMR data for the β anomer: H NMR (CDCl3, 400 MHz): δ 6.22 (1H, dd,
J = 3.4 Hz, J = 1.5 Hz, H-4), 5.29 (1H, s), 4.68 (1H, d, J = 1.5 Hz), 4.30 (1 H, d, J = 8.3 Hz, OH); 13C
NMR (CDCl3, 100 MHz): δ 166.6, 165.9, 165.5, 165.1 (each C=O), 120.0 (alkene CH2), 96.1 (C-1),
73.0 (C-5), 71.4 (C-2), 70.7, 69.0, 66.7 (OCH2CH=CH2). This hemiacetal (166 mg, 0.30 mmol) was
dissolved in CH2Cl2 (6 mL) and cooled to 0 °C. To this trichloroacetonitrile (0.3 mL, 3.0 mmol) and
DBU (5 drops) were added, and the mixture was stirred at 0 °C for 4 h. The solvent was removed
under reduced pressure to a volume of 2 mL and flash chromatography (EtOAc-cyclohexane 1:4) then
gave 11 (133 mg, 63%) as a yellow oil; Rf 0.34 (EtOAc-cyclohexane 2:5); IR (film) cm−1: 3328, 3072,
1
2958, 1735, 1677, 1452, 1265, 1106, 1068, 1027, 709; H-NMR (CDCl3, 500 MHz): δ 8.69 (1H, s,
NH), 8.02 (2H, d, J = 7.2 Hz, aromatic H), 7.94 (2H, J = 7.2 Hz, aromatic H), 7.82 (2H, d, J = 7.2 Hz,
aromatic H), 7.60 (1H, t, J = 7.5 Hz, aromatic H), 7.46 (4H, m, aromatic H), 7.34 (2H, t, J = 7.8 Hz,
aromatic H), 7.27 (2H, t, J = 7.8Hz, aromatic H), 7.05 (1H, d, J = 3.5 Hz, H-1), 6.37 (1H, dd, J = 3.3
Hz, J = 1.4 Hz, H-4), 6.10 (1H, dd, J = 10.7 Hz, J = 3.3 Hz, H-3), 5.96 (1H, J = 10.7 Hz, J = 3.5 Hz,
H-2), 5.75 (1H, ddt, J = 17.1 Hz, J = 10.4, J = 6.1 Hz, CH=CH2), 5.22 (1H, dd, J = 17.1 Hz, J = 1.2 Hz,
CH=CH2), 5.11 (1H, d, J = 1.4 Hz, H-5), 5.07 (1H, dd, J = 10.4 Hz, J = 0.7 Hz, CH=CH2), 4.62 (1H,
dd, J = 12.7 Hz, J = 6.1 Hz, OCH2CH=CH2), 4.56 (1H, dd, J = 12.7 Hz, J = 6.1 Hz, OCH2CH=CH2);
13C-NMR (CDCl3, 125 MHz): δ 165.6, 165.4, 165.4, 165.0 (each C=O), 160.2 (C=N), 133.6, 133.5,
133.3, 130.7, 129.9, 129.8, 129.7, 128.8 (2s), 128.6, 128.5, 128.4, 128.3, 120.0 (CH=CH2), 93.6 (C-1),
71.2 (C-5), 69.4 (C-4), 67.8 (C-3), 67.3 (C-2), 66.6 (OCH2CH=CH2); ES-HRMS calcd for
C32H26O10NCl3Na 712.0520, found m/z 712.0545 [M+Na]+.

Molecules 2013, 18
11210
((2S,3R)-2-Azido-3-benzyloxyicosanyl) 2,3,4-tri-O-benzoyl-β-D-galactopyranuronic acid, allyl ester (22a).
Alcohol 16 (78 mg, 0.18 mmol) and imidate 11 (133 mg, 0.19 mmol) were dissolved in CH2Cl2 (6 mL)
and stirred over 4Å MS for 20 min then cooled to 0 °C. To this TMSOTf (0.1M, 0.035 mmol, 0.35 mL)
was added and the reaction stirred at 0 °C for 40 min. Solid NaHCO3 (30 mg) was added and the
mixture stirred for 20 min and then filtered through celite, which was rinsed with CH2Cl2. The solvent
was then removed under reduced pressure and flash chromatography (EtOAc-cyclohexane 1:4) gave
22a (127 mg, 75%) as a yellow oil; Rf 0.50 (EtOAc-cyclohexane 2:5); [α]D +76 (c 6.6 ,CHCl3); IR
(film) cm−1: 3064, 2924, 2853, 2099, 1734, 1261, 1108; 1H-NMR (CDCl3, 500 MHz): δ 7.99 (2H, dd,
J = 8.3 Hz, J = 1.1 Hz, aromatic H), 7.95 (2H, J = 8.3 Hz, J = 1.1 Hz, aromatic H), 7.81 (2H, J = 8.3 Hz,
J = 1.1 Hz, aromatic H), 7.56 (1H, tt, J = 7.5 Hz, J = 1.2 Hz, aromatic H), 7.50 (1H, tt, J = 7.4 Hz,
J = 1.2 Hz, aromatic H), 7.43 (1H, t, J = 7.5 Hz, aromatic H), 7.36 (8H, m, aromatic H), 7.26 (3H, m,
aromatic H), 6.21 (1H, dd, J = 3.5 Hz, J = 1.2 Hz, H-4), 5.82 (1H, dd, J = 10.4 Hz, J = 7.9 Hz, H-2),
5.75 (1H, ddt, J = 17.2 Hz, J = 10.3 Hz, J = 6.0 Hz, CH=CH2), 5.63 (1H, dd, J = 10.4 Hz, J = 3.5 Hz,
H-3), 5.23 (1H, dd, J = 17.2 Hz, J = 1.3 Hz, CH=CH2), 5.07 (1H, dd, J = 10.3 Hz, J = 1.0 Hz,
CH=CH2), 4.91 (1H, d, J = 7.9 Hz, H-1), 4.58–4.64 (3H, overlapping signals, BnCHH, H-5,
OCH2CH=CH2) 4.56 (1H, ddt, J = 13.0 Hz, J = 6.0 Hz, J = 1.0 Hz, OCH2CH=CH2), 4.46 (1H, d, 11.3 Hz,
BnCHH), 4.25 (1H, dd, J = 10.7 Hz, J = 5.6 Hz, CHHO), 3.81 (1H, dd, J = 10.7 Hz, J = 5.0 Hz,
CHHO), 3.70 (1H, dd, J = 10.7 Hz, J = 5.4 Hz, CHN3), 3.50 (1H, ddd, J = 3.5 Hz, J = 5.5 Hz, J = 8.0 Hz,
CHOBn), 1.45 (2H, m, CH2), 1.27 (30H, s, 15 CH2), 0.88 (3H, t, J = 6.9 Hz, CH3); 13C-NMR (CDCl3,
125 MHz): δ 165.5, 165.4, 165.1, 165.0 (each C=O), 138.2, 133.4, 133.3, 133.2, 130.9, 130.0, 129.8,
129.7, 129.2, 128.9, 128.7, 128.5, 128.3 (3s), 128.0, 127.6, 119.7 (CH=CH2), 101.3 (C-1), 78.3
(CHOBn), 72.9 (C-5), 72.7 (BnCH2), 71.3 (C-3), 69.2 (C-2), 69.0 (C-4), 68.9 (CH2O), 66.5
(OCH2CH=CH2), 63.1 (CHN3), 31.9, 30.7 (3s), 29.6 (2s), 29.5 (2s), 29.3, 26.9, 25.0, 22.6 (each CH2),
14.1 (CH3); ES-HRMS calcd for C57H71O11N3Na 996.4986, found m/z 996.4960 [M+Na]+.
((2S,3R)-2-Azido-3-hydroxyicosanyl) 2,3,4-tri-O-benzoyl-α-D-galactopyranuronic acid, allyl ester (23a).
The β-glycoside 22a (125 mg, 0.129 mmol) was dissolved in CHCl3 (6 mL), to this TiCl4 (48 mg,
0.25 mmol, 27 µL) was added and the reaction was stirred at room temp for 3 h. The reaction mixture
was poured onto satd NaHCO3-Et2O (1:9) and stirred for 30 min. The mixture was filtered through
celite, which was rinsed with Et2O, the organic layer was decanted and dried over MgSO4. The solvent
was removed under reduced pressure and then flash chromatography of the residue (EtOAc-
cyclohexane 1:9) gave 23a (114 mg, 99%) as a yellow oil; Rf 0.26 (EtOAc-cyclohexane 1:4);
[α]D +110 (c 5.7, CHCl3); IR (film) cm−1: 3521, 3068, 2924, 2853, 2098, 1732, 1265, 1094, 1026;
1H-NMR (CDCl3, 500 MHz): δ 8.00 (4H, t, J = 7.0 Hz, aromatic H), 7.81 (2H, dd, J = 8.4 Hz, J = 1.2 Hz,
aromatic H), 7.59 (1H, t, J = 7.5 Hz, aromatic H), 7.51 (1H, t, J = 7.5 Hz, aromatic H), 7.45 (3H, m,
aromatic H), 7.37 (2H, t, J = 7.8 Hz, aromatic H), 7.26 (2H, t, J = 7.8 Hz, Hz), 6.28 (1H, dd, J = 3.5 Hz,
J = 1.4 Hz, H-4), 6.01 (1H, dd, J = 10.8 Hz, J = 3.5 Hz, H-3), 5.76 (1H, ddt, J = 17.2 Hz, J = 10.4 Hz,
J = 6.1 Hz, CH=CH2), 5.70 (1H, dd, J = 10.8 Hz, J = 3.6 Hz, H-2), 5.61 (1H, d, J = 3.6 Hz, H-1), 5.23
(1H, dd, J = 17.2 Hz, J = 1.3 Hz, CH=CH2), 5.08 (1H, dd, J = 10.4 Hz, J = 0.9 Hz, CH=CH2), 4.96
(1H, d, J = 1.4 Hz, H-5), 4.62 (1H, dd, J = 12.7 Hz, J = 6.1 Hz, OCH2CH=CH2), 4.57 (1H, dd,
J = 12.7 Hz, J = 6.1 Hz, OCH2CH=CH2), 4.12 (1H, dd, J = 10.6 Hz, J = 2.8 Hz, CHHO), 3.80 (1H,
dd, J = 10.6 Hz, J = 6.8 Hz, CHHO), 3.69 (1H, m, CHOH), 3.48 (1H, td, J = 6.5 Hz, J = 2.8 Hz,

Molecules 2013, 18
11211
CHN3), 1.80 (1H, d, J = 4.6 Hz, OH), 1.38 (4H, m, 2 CH2), 1.26 (s, 28H, 14 CH2), 0.88 (3H, t,
J = 0.88 Hz, CH3); 13C-NMR (CDCl3, 125 MHz): δ 166.4, 165.9, 165.4, 165.1 (each C=O), 133.5 (2s),
133.2, 130.8, 129.9, 129.8, 129.7, 129.0 (2s), 128.9, 128.5, 128.4, 128.2, 119.9 (alkene CH2), 97.6 (C-1),
71.2 (CHOH), 69.8 (C-4), 69.3, 69.2, 68.5 (C-2), 67.8 (C-3), 66.5 (OCH2CH=CH2), 65.3 (CHN3),
33.7, 31.9, 29.7, 29.6, 29.5 (3s), 29.3, 26.9, 25.5, 22.6 (each CH2), 14.1 (CH3); ES-HRMS calcd for
C50H65O11N3Na 906.4517, found m/z 906.4526 [M+Na]+.
((2S,3R)-2-(N-Nonadecanoylamino)-3-hydroxyicosan-1-yl) 2,3,4-tri-O-benzoyl-α-D-galactopyran-uronic
acid, allyl ester (24a). Azide 23a (110 mg, 0.125 mmol) was dissolved in CH2Cl2 (4 mL) and PBu3 (62 μL,
0.25 mmol) was added. The reaction was stirred at room temp for 1.5 h followed by the addition of
H2O (2 mL) and MeOH (0.5 mL) and the reaction stirred for a further 2 h. The reaction was diluted
with Et2O, washed with water, brine, dried over MgSO4, and the solvent removed under reduced
pressure. The crude amine was take up in CH2Cl2 (4 mL) and cooled to 0 °C, to this DIEPA (48 mg,
0.37 mmol, 65 µL) and nonadecanoyl chloride (0.50 mL of 0.25 mM, 0.125 mmol) were respectively
added, and the reaction allowed to attain room temp over 16 h. The reaction mixture was taken up in
Et2O, washed with water, brine, dried over MgSO4 and the solvent was removed under reduced
pressure. Flash chromatography (EtOAc-cyclohexane 1:4) gave 24a (15 mg, 12%) as a white solid; Rf
1
0.18 (EtOAc-cyclohexane 2:5); IR (film) cm−1: 3309, 2917, 2850, 1731, 1650, 1266, 1094; H-NMR
(CDCl3, 500 MHz): δ 8.01 (2H, d, J = 7.2 Hz, aromatic H), 7.96 (2H, d, J = 7.3 Hz, aromatic H), 7.82
(2H, d, J = 7.2 Hz, aromatic H), 7.61 (1H, t, J = 7.5 Hz, aromatic H), 7.54 (1H, t, J = 7.4 Hz, aromatic H),
7.46 (3H, m, aromatic H), 7.40 (2H, t, J = 7.8 Hz, aromatic H), 7.29 (2H, t, J = 7.9 Hz, aromatic H),
6.31 (1H, d, J = 8.1 Hz, NH), 6.26 (1H, dd, J = 3.4 Hz, J = 1.3 Hz, H-4), 5.96 (1H, dd, J = 10.7 Hz,
J = 3.4 Hz, H-3), 5.76 (1H, ddt, J = 17.2 Hz, J = 10.3 Hz, J = 6.2 Hz, CH=CH2), 5.68 (1H, dd,
J = 10.7 Hz, J = 3.7 Hz, H-2), 5.58 (1H, d, J = 3.7 Hz, H-1), 5.24 (1H, dd, J = 17.2 Hz, J = 1.2 Hz,
CH=CH2), 5.10 (1H, dd, J = 10.3 Hz, J = 1.2 Hz, CH=CH2), 4.81 (1H, d, J = 1.3 Hz, H-5), 4.63 (1H,
dd, J = 12.8 Hz, J = 6.2 Hz, OCH2CH=CH2), 4.57 (1H, dd, J = 12.8 Hz, J = 6.2 Hz, OCH2CH=CH2),
4.10 (1H, dd, J = 10.4 Hz, J = 2.2 Hz, CHHO), 4.00 (1H, m, CHOH), 3.83 (1H, dd, J = 10.4 Hz,
J = 3.6 Hz, CHHO), 3.56 (1H, m, CHN), 2.39 (1H, brs, OH), 2.24 (2H, m, COCH2), 1.62 (4H, m, 2
CH2), 1.27 (60H, br s, 30 CH2), 0.89 (6H, t, J = 6.9 Hz, 2 CH3); 13C-NMR (CDCl3, 125 MHz): δ173.2,
166.1, 165.5 (2s), 165.1 (each C=O), 133.8, 133.6, 133.3, 130.8, 130.0, 129.8, 129.7, 128.9 (2s), 128.6
(2s), 128.5, 128.3, 120.0 (alkene CH2), 97.4 (C-1), 73.2 (CHOH), 69.7 (2s), 69.1 (C-5), 68.7 (C-2),
67.8 (C-3), 66.6 (OCH2CH=CH2), 52.1 (CHN), 36.8 (COCH2), 34.9, 31.9, 29.8, 29.7 (4s), 29.6, 29.5
(2s), 29.4 (4s), 26.9, 25.9, 25.8, 22.7 (each CH2), 14.1 (2 × CH3). ES-HRMS calcd for C69H104O12N
1138.7559, found m/z 1138.7515 [M+H]+.
((2S,3R)-2-(N-Nonadecanoylamino)-3-hydroxyicosan-1-yl) α-D-galactopyranuronic acid (5). The
protected lipid 24a (8 mg, 7 µmol) was dissolved in n-PrOH (4 mL) and H2O2 (30%, 0.3 mL), and to
this nPrONa-nPrOH (0.1 M, 60 µmol, 600 µmL) was added at a rate of 100 µL/h. After the addition
was completed, the reaction mixture was then centrifuged at 15000 rpm for 15 min, the supernatant
was removed and the precipitate washed twice with water. The precipitate was then lyophilised to give
5 (1.2 mg, 22%) as a white solid; 1H-NMR (CDCl3, 600 MHz): δ 4.93 (1H, d, J = 2.3 Hz, H-1), 4.26
(1H, s, H-5), 4.22 (1H, m), 3.88 (1H, m, CNH), 3.81 (1H, dd, J = 10.1 Hz, J = 3.0 Hz, H-2), 3.78 (3H,

Molecules 2013, 18
11212
overlapping signals), 3.62 (1H, td, J = 7.2 Hz, J = 1.9 Hz, CHOH), 2.21 (2H, t, J = 7.6 Hz, CH2CO2),
13
1.64-1.53 (4H, m, CH2), 1.27 (60H, br s, CH2), 0.89 (6H, t, J = 7.0 Hz, CH3); C-NMR (CDCl3,
125 MHz): δ 175.1 (C=O), 99.9 (C-1), 72.0 (C-5), 71.0 (C-3), 71.0 (CHOH), 70.8, 68.9, 68.2, 53.7
(CHN), 36.5 (COCH2), 36.7, 32.3, 30.0 (2s), 29.9, 27.8, 29.7 (2s), 26.3, 26.1, 23.0, 14.2.
1-Nonadecyne (28). Nonadec-1-ene 27 (1.96 g, 7.37 mmol) was dissolved in CHCl3 (15 mL) and
bromine (1.44 g, 9.00 mmol) was added, and the reaction mixture was then stirred for 30 min at room
temp. The solution was diluted with cyclohexane, washed with Na2S2O4, water, brine, dried over
MgSO4, and concentrated under reduced pressure to give 1,2-dibromononadecane (3.1 g, 97%) as a
1
white solid; Rf 0.51 (cyclohexane); IR (film) (cm−1): 2923, 2852, 1465, 1143; H-NMR (CDCl3, 400
MHz): δ 4.17 (1H, m, CHBr), 3.85 (1H, dd, J = 10.2 Hz, J = 4.4 Hz, CHHBr), 3.63 (1H, t, J = 10.0 Hz,
CHHBr), 2.14 (1H, m, CHHCHBr), 1.78 (1H, m, CHHCHBr), 1.56 (2H, m), 1.27 (28H, s, CH2), 0.88
(3H, t, J = 6.8 Hz, CH3); 13C-NMR (CDCl3, 100 MHz): δ 53.2 (CHBr), 36.4 (CH2Br), 36.0, 31.9, 29.7
(2 s), 29.6, 29.5, 29.4 (2s), 28.8, 26.8, 22.7, 14.1 (CH3). 1,2-Dibromononadecane (2.00 g, 4.69 mmol)
and KOH (2.67 g, 46.9 mmol) were dissolved in EtOH (40 mL) and heated at reflux for 48 h. The
reaction mixture was then diluted with cyclohexane, washed with water, brine, dried over MgSO4, and
the solvent was removed under reduced pressure. Flash chromatography (cyclohexane) gave 28 (0.91
g, 73%) as a white solid; Rf 0.51 (cyclohexane);IR (film) (cm−1): 3287, 2917, 2849, 1462, 908, 731,
630; 1H-NMR (CDCl3, 400 MHz): δ 2.16 (2H, td, J = 7.1 Hz, J = 2.6 Hz, HCCCH2), 1.89 (1H, t, J =
13
2.6 Hz, CH), 1.52 (2H, m), 1,43 (30H, s), 0.88 (3H, t, J = 6.7, CH3); C-NMR (CDCl3, 100 MHz): δ
84.5 (HCC), 68.0 (CCH), 32.0, 29.8, 29.7 (2s), 29.6, 29.5, 29.2, 28.8, 28.6, 27.0, 22.7, 18.4, 14.1 (CH3).
((2S,3R)-2-(4-Heptadecyl-1H-1,2,3-triazolyl)-3-hydroxyicosan-1-yl) 2,3,4-tri-O-benzoyl-α-D-galacto-
pyranuronic acid, allyl ester (30) and ((2S,3R)-2-(5-heptadecyl-1H-1,2,3-triazolyl)-3-hydroxyicosan-1-
yl) 2,3,4-tri-O-benzoyl-α-D-galactopyranuronic acid, allyl ester (31). Azide 23b (78 mg, 88.3 µmol)
and alkyne 28 (46 mg, 177 µmol) were dried under vacuum. The reagents were taken up in toluene
(1.5 mL), which was followed by the addition of copper(I) iodide (17 mg, 88 µmol), and the reaction
mixture heated at reflux for 24 h. The reaction mixture was purified by flash chromatography to give
(32 mg, 32%) as an inseparable 86:14 mixture of 30:31 (yellow solid); [α]D +32.0 (c 1.0, CHCl3); IR
1
(film) cm−1: 3406, 3070, 2919, 2850, 1733, 1452, 1264, 1108, 1069, 709; H-NMR for major isomer
(CDCl3, 500 MHz): δ 7.93 (2H, dd, J = 8.4 Hz, J = 1.2 Hz, aromatic H), 7.91 (2H, dd, J = 8.4 Hz,
J = 1.2 Hz, aromatic H), 7.88 (2H, dd, J = 8.4 Hz, J = 1.2 Hz, aromatic H), 7.52 (1H, m, aromatic H),
7.44 (2H, m, aromatic H), 7.37 (5H, m, aromatic H), 7.31 (2H, t, J = 7.8 Hz, aromatic H), 6.13 (1H, t,
J = 9.8 Hz, H-3), 5.92 (1H, ddt, J = 17.0 Hz, J = 10.4 Hz, J = 6.1 Hz, CH=CH2) 5.67 (1H, t,
J = 9.8 Hz, H-4), 5.41 (1H, d, J = 3.6 Hz, H-1), 5.30 (1H, dd, J = 9.8 Hz, J = 3.6 Hz, H-2), 5.18 (1H,
dd, J = 17.1 Hz, J = 1.3 Hz, CH=CH2), 5.08 (1H, dd, J = 10.4 Hz, J = 0.9 Hz, CH=CH2), 4.56–4.64
(2H, overlapping signals), 4.48–4.54 (2H, overlapping signals), 4.30 (1H, dd, J = 11.1 Hz, J = 2.8 Hz,
CHHO), 4.15 (1H, dd, J = 11.1 Hz, J = 6.8 Hz, CHHO), 4.05 (1H, br s, CHOH), 2.69 (1H, s, OH),
2.50 (1H, dt, J = 15.0 Hz, J = 7.9 Hz, CHH), 2.42 (1H, dt, J = 15.0 Hz, J = 7.8 Hz, CHH), 1.00–1.75
13
(64H, 32 x CH2), 0.88 (6H, t, J = 6.9 Hz, 2 x CH3); C-NMR (CDCl3, 125 MHz): δ 167.0, 165.6,
165.3, 165.1 (each C=O), 148.4 (N=CH), 133.6, 133.4, 133.3, 130.7, 129.9, 129.8, 129.7, 128.9,
128.8, 128.6 (2s), 128.4 (2s), 128.3, 121.2, 119.6 (alkene CH2), 97.0 (C-1), 71.4 (CHN), 71.2 (C-2),

Molecules 2013, 18
11213
69.8 (C-4), 69.5 (C-5), 69.3 (C-3), 67.8 (CH2O), 66.9 (OCH2CH=CH2), 64.9 (CHOH), 33.8, 31.9, 29.7
(2s), 29.6 (2s), 29.5 (2s), 29.4 (2s), 29.3, 25.6, 25.5, 22.7 (each CH2), 14.1 (CH3); ES-HRMS calcd for
C69H102O11N3 1148.7514, found m/z 1148.7570 [M+H]+.
((2S,3R)-2-(4-Heptadecyl-1H-1,2,3-triazolyl)-3-hydroxyicosan-1-yl) α-D-galactopyranuronic acid (10).
The 86:14 mixture of protected glycolipids 30 and 31 (10 mg, 8.7 µmol) were dissolved in n-PrOH (2 mL)
and H2O2 (30%, 0.2 mL), and to this nPrONa-nPrOH (500 µmL of 0.1 M, 50 µmol) was added at a
rate of 80 µL/h. Water (5 mL) was then added to the reaction mixture and the solution centrifuged at
15000 rpm for 15 min, the supernatant was removed and the precipitate washed twice with water.
The precipitate was then lyophilised to give a mixture of regioisomers (3.6 mg, 52%) where the
1,4-regioisomer 10 was the major product and there was also 12% of the 1,5-regioisomer. Analytical
data for the major isomer 10: 1H-NMR (CDCl3-CD3OD 2:1, 600 MHz): δ 7.64 (1H, s, triazole H), 4.85
(1H, d, J = 2.9 Hz, H-1), 4.59 (1H, br s, CHN), 4.10-4.20 (2H, m, CH2O), 4.03 (1H, td, J = 7.8 Hz,
J = 3,4 Hz, CHOH), 3.88 (4H, br s, OH), 3.63–3.73 (2H, m, H-3, H-5), 3.40–3.45 (2H, m, H-2, H-4),
2.69 (2H, t, J = 7.8 Hz, CH2), 1.62–1.72 (2H, overlapping signals, CH2), 1.1–1.55 (60H, overlapping
13
signals, CH2), 0.89 (6H, t, J = 6.8 Hz, 2 x CH3); C-NMR (CDCl3-CD3OD 2:1, 150 MHz): δ 176.5
(C=O), 130.1, 128.6 (triazole C and CH), 100.1 (C-1), 73.9, 72.6, 72.0, 70.7, 70.3, 68.2, 66.2, 33.8,
32.2, 30.0, 29.7, 29.6, 25.8, 25.7, 22.9, 14.2; ES-HRMS calcd for C45H84O8N3 794.6258, found m/z
794.6276 [M-H]−.
2,3,4-Tri-O-acetyl-α-thio-D-glucopyranosiduronic acid, methyl ester (15). To a stirred solution of the
β-thiol10 34 (100 mg, 0.28 mmol) in CH2Cl2 (3 mL) was added TiCl4 (76 µL, 0.7 mmol) dropwise. The
reaction mixture was stirred for 10 min at room temp and then cooled to 0 °C overnight, and CH2Cl2
and satd NH4Cl were then added, and the phases were separated and the aqueous phase was extracted
with CH2Cl2. The combined organic layers were washed with water, brine, dried over MgSO4 and the
solvent was removed to give an 8:1 mixture of 15 and 34 (65 mg, 65%) as a white foam. Analytical
1
data for 15: IR (film) cm−1: 2955, 2572, 1747, 1438, 1370, 1214, 1077, 1042; H-NMR (CDCl3, 500
MHz): δ 5.98 (1H, t, J = 5.7, H-1), 5.39 (1H, t, J = 9.0, H-3), 5.17 (1H, t, J = 8.9, H-4), 5.02 (1H, dd,
J = 9.3, 5.2, H-2), 4.76 (1H, d, J = 9.2, H-5), 3.76 (3H, s, OCH3), 2.09 (3H, s), 2.06 (3H, s), 2.05 (3H, s)
13
(each OAc), 2.02 (1H, s, SH); C-NMR (126 MHz, CDCl3) δ 169.5, 169.4, 167.7 (each C=O), 76.4
(C-1), 69.8 (C-2), 69.4 (C-5), 68.9 (C-4), 68.6 (C-3), 52.8 (OMe), 20.6, 20.5 (2s) (each OAc);
ESI-HRMS calcd for C13H17O9S 349.0593, found m/z 349.0584 [M-H]−.
Methyl ((4S,5R)-5-heptadecyl-2,2-dimethyl-3-t-butoxycarbonyl-oxazolidin-4-yl)methyl 2,3,4-tri-O-acetyl-
thio-α-D-glucopyranosiduronate (44a). Thiol 15 (155 mg, 0.44 mmol) was dissolved in dry DMF (3 mL)
and cooled to 0 °C. Sodium hydride (16 mg of a 60% dispersion in mineral oil, 0.4 mmol) was added
slowly and the reaction was stirred for 30 min. A solution of the bromide 18 (179 mg, 0.15 mmol) in
DMF was then added dropwise and the mixture allowed to attain room temp and stirred overnight.
EtOAc and water were added and the aqueous layer was washed with EtOAc. The combined organic
layers were washed with water, brine, dried over MgSO4 and the solvent was removed. Flash
chromatography (petroleum ether-EtOAc 4:1) gave 44a (42 mg, 39%) as a colourless oil; [α]D +60 (c,
CHCl3); IR (film) cm−1: 2923, 2853, 1752, 1697, 1457, 1375, 1177, 1040; 1H-NMR (500 MHz, CDCl3)

Molecules 2013, 18
11214
δ 5.60–5.80 (1H, overlapping signals, H-1), 5.41–5.31 (1H, overlapping signals, H-3), 5.24–5.14 (1H,
overlapping signals, H-4), 5.03 (1H, dd, J = 9.8, 5.5, H-2), 4.72 (1H, overlapping signals, H-5), 3.90–4.10
(2H, overlapping signals, CHO, CHN), 3.74 (3H, s, OMe), 2.08–2.00 (9H, overlapping signals, OAc),
1.05–1.80 (40H), 0.88 (3H, t, J = 6.7, CH3); 13C-NMR (125MHz, CDCl3) δ 169.8, 169.5, 169.4, 167.9
(C=O), 92.5, 83.3 (C-1), 80.1, 76.6 (C-3'), 70.0 (C-2), 69.5 (C-3), 69.1 (C-4), 68.9 (C-5), 58.6 (C-2'),
52.9 (OMe), 32.1, 29.9, 29.7, 29.5, 28.6 (each CH2), 22.8, 20.8, 20.7 (2s), 14.3 (each CH3);
ESI-HRMS calcd for C41H71NO12SNa 824.4595, found m/z 824.4586 [M+Na]+.
Methyl
((2S,3R)-2-(N-nonadecanoylamino)-3-hydroxyicosan-1-yl)
2,3,4-tri-O-acetyl-thio-D-
glucopyranosiduronate (46a). N-Hydroxysuccinimide (0.76 g, 6. 6 mmol) and EDC (1.25 g, 6.6 mmol)
were added to a solution of nonadecanoic acid (1.87 g, 6.6 mmol) in CH2Cl2 (50 mL) and the mixture
was stirred overnight at room temp. The solvent was concentrated under reduced pressure and the
resulting residue was dissolved in CH2Cl2. The solution was washed with water, dried over MgSO4
and concentrated under reduced pressure to yield 45 as a white solid (2.45 g, 98%). This intermediate
was used without further purification; 1H-NMR (500 MHz, CDCl3) δ 2.83 (4H, s, O=CCH2CH2C=O),
2.60 (2H, t, J = 7.5, CH2C=O), 1.79–1.69 (2H, m, CH2), 1.39 (2H, dd, J = 14.9, 7.0, CH2), 1.27 (29H,
13
s, each alkyl CH2), 0.88 (3H, t, J = 6.9, CH3); C-NMR (125 MHz, CDCl3) δ 169.1, 168.6 (each
C=O), 31.9, 30.9, 29.6, 29.6, 29.6, 29.5, 29.3, 29.0, 28.8, 25.6, 24.6, 22.7 (each CH2), 14.1 (CH3).
Compound 44a (30 mg, 0.04 mmol) was taken up in formic acid (3 mL) and stirred vigorously for 30
min. Toluene (5 mL) was added and the solvents were removed under reduced pressure. The resulting
residue was taken up in water and basified with solid NaHCO3. The mixture was then extracted into
CH2Cl2, dried over MgSO4 and the solvent was removed under reduced pressure. The resulting residue
was taken up in CH2Cl2 (2 mL) and DIPEA (22 µL, 0.13 mmol) was added. To this was added a
solution of 45 (37 mg, 0.09 mmol) in CH2Cl2 (1.5 mL) and the mixture was stirred at room temp for 16
h. The reaction mixture was partitioned between EtOAc and satd NaHCO3. Phases were separated and
the aqueous phase extracted into EtOAc. The combined organic phases were washed with brine, dried
over MgSO4 and the solvent was removed under reduced pressure. Flash chromatography (petroleum
ether-EtOAc 3:1) gave 46a (19 mg, 57% over two steps) as a white solid; [α]D +48 (c 1.0 in CHCl3);
1
IR (film) cm−1: 3294 (br), 2917, 2850, 1751, 1650, 1467, 1219, 1043; H-NMR (500 MHz, CDCl3) δ
6.12 (1H, d, J = 8.5, NH), 5.69 (1H, d, J = 5.2, H-1), 5.29 (1H, t, J = 9.0, H-3), 5.21 (1H, t, J = 9.0, H-4),
4.99 (1H, dd, J = 9.0, 5.2, H-2), 4.74 (1H, d, J = 9.0, H-5), 4.01 (1H, br s, CHO), 3.79–3.73 (3H, m,
OMe), 3.68 (1H, br s, CHN), 3.03 (1H, dd, J = 14.0, 8.0, SCH2), 2.84 (1H, dd, J = 14.0, 3.5, SCH2),
2.19 (2H, d, J = 4.3, CH2), 2.08, 2.04, 2.03 (each 3H, each s, each CH3), 1.62 (4H, s, 2 × CH2), 1.45
(2H, m, CH2), 1.32–1.22 (60H, overlapping signals, each CH2), 0.87 (6H, t, J = 6.9, 2 × CH3);
13C-NMR (125 MHz, CDCl3) δ 173.7, 169.8, 169.5, 169.4, 167.9 (each C=O), 83.5 (C-1), 73.6 (CHO),
70.0 (C-2), 69.2 (2s) (C-3 & C-5), 68.8 (C-4), 54.0 (CHN), 53.0 (OMe), 36.7, 34.0, 31.9 (each CH2),
29.7 (3s), 29.6, 29.4 (2s), 26.0, 25.7, 22.7 (each CH2), 20.7, 20.6 (2s), 14.1 (each CH3); ESI-HRMS
calcd. for C52H94NO11S 940.6548, found m/z 940.6578 [M-H]−.
Methyl
((2S,3R)-2-(N-nonadecanoylamino)-3-hydroxyicosan-1-yl)
2,3,4-tri-O-acetyl-thio-D-
glucopyranosiduronate (8). Protected lipid derivative 46a (3.0 mg, 0.3 µmol) was dissolved in
anhydrous EtOAc (200 µL) and LiI (15 mg, 0.11 mmol) was added. The reaction mixture was heated

Molecules 2013, 18
11215
at 70 °C for 6 h. Upon cooling the reaction mixture was washed with H2O, brine, dried over MgSO4
and concentrated under reduced pressure. The resulting residue was then taken up in a guanidine-
guanidinium nitrate solution (2 mL, 1M in CH2Cl2-MeOH 1:9) the reaction was stirred at room temp
for 1 h. The reaction was neutralised by the addition of Amberlite® resin IR-20, filtered and the
solvent was removed under reduced pressure. The crude product was purified using lipophilic
1
Sephadex® LH-20 to give the title compound (1.4 mg) as a white powder; H-NMR (500 MHz,
CDCl3-MeOD 2:1) δ 5.21 (1H, d, J = 4.5), 4.31 (1H, br s), 3.78 (1H, br s), 3.58 (2H, br s), 3.45 (1H, s),
3.31–3.50 (2H, overlapping signals), 2.55–2.80 (2H, m, SCH2), 0.85-2.15 (66H), 0.65 (6H, br s,
2 CH3); 13C-NMR (125 MHz, CDCl3-MeOD 2:1) δ 175.5 (C=O), 87.6 (C-1), 73.5 (CHOH), 71.4 (C-2),
70.4 (C-3), 69.0 (C-5), 67.2 (C-4), 53.6 (CHN), 35.3, 31.3 (CH2S), 34.0, 29.4, 26.1, 26.0, 22.9, 22.7,
19.3 (each CH2), 14.1 (CH3); ESI-HRMS calcd for C45H86NO8S 800.6074, found m/z 800.6077 [M-H]−.
4. Conclusions
Glycolipids with O-, S- and SO2-linkages, analogous to antigen components of Spingomonadacaece
have been prepared via the anomerisation of β-O-glycosides, β-S-glycosides or β-S-glycosyl thiols.
This chemistry was more effective for the preparation of glucuronic acid or galacturonic acid derivatives
than for glucose or galactose derivatives, consistent with a chelation induced anomerisation [37]. Although
not always required, there can be an advantage to using benzoylated substrates in these reactions, as
opposed to acetylated substrates [38]. The reasons for this are not fully understood. The synthesis of
the uronic acid based glycosyl thiols from the β-precursor using TiCl4 is interesting as there are
relatively few syntheses of α-glycosyl thiols reported to date [39] and such building blocks have wider
potential, including S-disaccharide synthesis, for example. Triazole containing mimetics of the natural
glycolipids were also prepared by CuAAC. The glycolipid antigens are being evaluated currently for
their effects on iNKT cells.
Acknowledgments
The research described herein has been supported by the Programme for Research in Third-Level
Institutions Cycle 3 (PRTLI), administered by the Higher Education Authority and Science Foundation
Ireland (06/CHO032 & 12/IA/1398).
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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