Synthesis and evaluation of sphingolipid analogues modification of the hydroxy group at C(1) of 7-oxasphingosine, and of the hydroxy group at C(1) and the amide group of 7-oxaceramides

Article abstract of DOI:10.1002/cbdv.200900013

The analogues 7-9 of 7-oxaceramide and 7-oxasphingosine were synthesized from the known azidosphingosine 21. The 1,4-disubstituted 1,2,3-triazole analogues 10-16 of ceramides were synthesized by the click reaction of the known azide 24. None of the analogues 7-15 was active as inhibitor of SPHK type 1 and of acid sphingomyelinase, whereas 16 is a weak inhibitor of SPHK1. Triazoles 10, 11, and 15 did not inhibit ceramide phosphorylation by CerK, and none of 7, 8, and 10-15 activated invariant natural killer T (iNKT) cell clones when presented by human CD1d-transfected antigen-presenting cells (APC) or by plate-bound human CD1d [55]. Triazoles 14 and 15 prevent binding of α-galactosylceramide (α-GalCer) to plate-bound human CD1d and subsequent T-cell response to α-GalCer. Only 15 reduced activation by α-GalCer significantly and independently of the cytokine measured.

Full text of DOI:10.1002/cbdv.200900013

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
705
Synthesis and Evaluation of Sphingolipid Analogues
Modification of the Hydroxy Group at C(1) of 7-Oxasphingosine, and of the Hydroxy
Group at C(1) and the Amide Group of 7-Oxaceramides
by Thresen Mathewa), Andreas Billichb), Marco Cavallaric), Frederic Bornancinb),
Peter Nussbaumerb), Gennaro De Liberoc), and Andrea Vasella*a)
a) Laboratorium fꢀr Organische Chemie, Departement Chemie und Angewandte Biowissenschaften,
ETH-Zꢀrich, Wolfgang-Pauli-Strasse 10, CH-8093 Zꢀrich
(e-mail: vasella@org.chem.ethz.ch)
b) Novartis Institutes for BioMedical Research, Brunner Strasse 59, A-1235 Vienna
c) Department of Biomedicine, University Hospital Basel, CH-4031 Basel
The analogues 7–9 of 7-oxaceramide and 7-oxasphingosine were synthesized from the known
azidosphingosine 21. The 1,4-disubstituted 1,2,3-triazole analogues 10–16 of ceramides were synthesized
by the click reaction of the known azide 24. None of the analogues 7–15 was active as inhibitor of SPHK
type 1 and of acid sphingomyelinase, whereas 16 is a weak inhibitor of SPHK1. Triazoles 10, 11, and 15 did
not inhibit ceramide phosphorylation by CerK, and none of 7, 8, and 10–15 activated invariant natural
killer T (iNKT) cell clones when presented by human CD1d-transfected antigen-presenting cells (APC)
or by plate-bound human CD1d [55]. Triazoles 14 and 15 prevent binding of a-galactosylceramide (a-
GalCer) to plate-bound human CD1d and subsequent T-cell response to a-GalCer. Only 15 reduced
activation by a-GalCer significantly and independently of the cytokine measured.
Introduction. – The ability of sphingolipids and glycosphingolipids to modulate
apoptosis [1–7] and immune responses [8–13] is likely to depend on specific receptor
interactions with both the polar head group and the lipid tails of the sphingosine and
fatty acid moieties. The exploration of these interactions is based on crystal-structure
analyses of receptors in complex with the lipids [14][15], and on the synthesis and
biological evaluation of analogues [16]. We disclosed the synthesis of a few analogues
of sphingosine (Sph; 1) and ceramide (Cer; 2; Fig. 1) viz., 7-oxasphingosine (3), the
corresponding ceramide 4, the thioamide 5, and the N-methyl Cer 6 [17]. 7-
Oxasphingosine (3) was synthesized to facilitate the incorporation of modified lipid
moieties and the exploration of their interaction with receptors. The analogues 5 and 6
were prepared to investigate the H-bond donor and acceptor roles of the amide
function in Cer, as thioamide 5 was expected to be a weaker H-bond acceptor than Cer
[18], while N-methylation lowers the energy difference between the (E)- and (Z)-
isomers [19] and offsets the H-bond-donating properties of the amide moiety.
The apoptosis-inducing properties of the analogues 3–6 were compared to those of
Sph 1 and Cer 2 using a human neuroblastoma (SK-N-BE) and a murine-
promyelocyte-derived (32d) cell line. In both SK-N-BE and 32d cells, 7-oxasphingosine
(3) exhibited toxicity equal to that of sphingosine, even in the presence of N,N-
dimethylsphingosine. Compounds 4–6 do not differ in their effect on the viability of
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. Structures of sphingosine 1, ceramide 2, 7-oxasphingosine 3, 7-oxaceramide 4, the thioamide 5, and
the N-methylceramide 6
cells and in their ability to trigger cell proliferation. However, in the presence of N,N-
dimethylsphingosine (DMS), an inhibitor of sphingosine kinase (SPHK), Cer 2
showed stronger effects than the thio-ceramide 5 in 32d cells, while 5 was efficacious in
SK-N-BE cells, where it showed an IC50 value of 3 nm, as compared to 100 nm for Cer.
The N-methylamide 6 behaved similarly to Cer 2 [17]. The data suggest that there is
neither a major difference in the degradation of Cer and its 7-oxa- and N-methyl
analogues to the respective Sph derivatives, nor in the recognition of the resulting Sph
and oxasphingosine by SPHK. The greater potency shown by the thio-Cer 5 in SK-N-
BE cells devoid of SPHK activity (as the result of DMS treatment) suggests that 5 may
have a higher affinity than Cer to death effectors, such as ion channels, protein kinase,
and phosphatase, or a decreased capacity to be phosphorylated by either ceramide
kinase or SPHK.
We wished to further modify the H-bond-accepting and -donating properties of Cer
and Sph, and considered the urea 7, the sulfonamide 8, the Sph analogue 9, and the
triazoles 10–16 to be of interest (Fig. 2).
The urea derivative 7 was expected to be a better H-bond acceptor [20] and a
poorer H-bond donor than the corresponding amide, considering that ureas have
higher pKa values (ca. 26–27) than amides (ca. 20–23) [20][21], while the biological
effects of replacing a CH2 by an NH group remained to be explored. The lower pKa
values of sulfonamides (ca. 12) [22] should make 8 a better H-bond donor than the
corresponding amide. The sulfonyl group is a poor H-bond acceptor as compared to the
C¼O group [23], but the change of geometry may be more significant, as the
tetrahedral S- and the pyramidal N-atoms of sulfonamides [24] differ significantly from
the trigonal geometry of the amide moiety [25].
In the 1-(dimethylamino)-deoxy-7-oxasphingosine (9), the C(1)ꢀOH group of Sph
is replaced with a basic functionality; the corresponding ammonium salt may act as

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
707
Fig. 2. Analogues of 7-oxasphingosine and 7-oxaceramide with a modified C(1)ꢀOH, or amide moiety
a good H-bond donor, but the analogue can no longer be phosphorylated at
OꢀC(1).
1,2,3-Triazoles are non-hydrolysable bioisosters of amides [26][27], readily
obtained by the click reaction [26–32]. They mimic geometric and electronic features
of a configurationally biased amide bond, and participate in dipole–dipole interactions
[31][33] and H-bonding. They may act as H-bond acceptors and as (weak) donors,
depending on their substitution. In 1,4-disubstituted 1,2,3-triazoles, N(2) and N(3)
exhibit H-bond accepting properties, whereas the strong dipole moment of triazole
causes the polarization of HꢀC(5) that may act as a week H-bond donor [26–36].
The triazole ring of the envisaged analogues 10–16 (Fig. 2) orients the acyl chain in
a way that prevents a parallel orientation with the alkyl chain of the Sph moiety, and its
polar substituents may serve as H-bond acceptors in a position differing from the C¼O
group of the C(2)-acylamino chain. These features are of interest, as the crystal
structure of a-GalCer bound to the human and mouse CD1d receptor shows that the
two alkyl chains diverge from each other [14], suggesting that the configuration of the
amide moiety, besides its H-bond-acceptor and -donor properties, plays an important
role in interacting with receptors. The triazoles should be readily available from the
known azide intermediate in the synthesis of 7-oxaceramide [17]. After we had
completed the synthesis of the triazoles 10–16, Kim et al. published a synthesis of
related ceramides [37] and O-glycosides [38] that stimulated cytokine production,
comparable to the one of a-GalCer, and exhibited a stronger Th2 cytokine response.
Biological properties for which we considered testing these analogues comprise T-
cell suppression [40], and the interaction with sphingosine and ceramide kinases, and
with acid sphingomyelinase [41].
Results and Discussion. – Synthesis. The amine 18 (Scheme 1) was obtained in high
yield by reduction of the protected azido-7-oxasphingosine 17 that had been prepared
in 94% yield by isopropylidenation of the derivative 21 [17]. Coupling of 18 with

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 1
a) 2,2-Dimethoxypropane, TsOH·H2O, acetone, 258; 94%. b) LiAlH4, Et2O, 08; 97% of 18; 90% of 9. c)
C16H33NCO, Et3N, CH2Cl2, 10–258; 77%. d) 60% CF3CO2H, THF; 89% for 7; 94% for 8. e) C16H33SO2Cl,
Et3N, CH2Cl2, 0–258; 89%. f) TsCl, Et3N, 4-(Dimethylamino)pyridine (DMAP), CH2Cl2, 108; 79%.
g) Me2NH, THF, 258; 60%.
hexadecyl isocyanate [42] yielded 77% of the protected urea 19 that was smoothly
deprotected by 60% CF3COOH in THF to give the desired analogue 7 (89%).
Condensation of 18 with hexadecyl sulfonyl chloride [43] in the presence of Et3N yielded
89% of the protected sulfonamide 20 that was de-isopropylidenated to the sulfonamide 8
(94%). The chemical shifts and coupling constants of the analogues 7 and 8 of 7-
oxaceramide, and the known Cer 2 and 7-oxaceramide 4 are summarized in Table 1.
To obtain the 1-(dimethylamino) deoxy-7-oxasphingosine (9), we monotosylated
the azido-7-oxasphingosine 21 [17] (Scheme 1) in the presence of Et3N and 4-
(dimethylamino)pyridine (DMAP) at 108 to obtain 79% of 22. Replacing Et3N by
pyridine or by EtN(i-Pr)2 resulted in a slower reaction, and increasing the temperature
above 358 led to tosylation of both OH groups, while addition of a catalytic amount of
DMAP increased the rate of tosylation without impairing the selectivity. The expected
selective tosylation of the primary OH group is evidenced by the HOꢀC(3) doublet at
2.26 ppm (J(3,OH)¼4.5 Hz) and the downfield shift for C(1) from 62.47 (21) to
68.58 ppm (22). Treating 22 with Me2NH in THF provided the protected azido-

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
709
1
Table 1. Selected H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] for the Ceramide 2, 7-
Oxaceramide 4, Urea 7, and Sulfonamide 8 (in CDCl3)
2a)
4b)
7c)
8
HꢀC(1)
H’ꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
NH
HOꢀC(3)
HOꢀC(1)
J(1,1’)
J(1,2)
J(1’,2)
J(2,3)
J(3,4)
J(4,5)
J(5,6)
J(NH,2)
J(OH,3)
J(OH,1)
J(OH,1’)
3.93
3.96–3.88
3.83
3.90
3.71
3.49–3.40
4.44
5.78
5.93
3.98
5.32
3.07
3.68
3.88
4.31
5.51
5.76
2.03
6.21
2.70
2.70
11.2
3.4
3.4
3.6
6.5
3.74–3.65
3.96–3.88
4.38
5.79
5.91
3.98
6.33
3.26
2.97
3.69
3.77
4.36
5.78
5.89
3.97
1.81
1.81
1.81
2.81
11.7
4.5
d
)
)
)
10.8
d
d
4.2
3.0
3.8
5.2
15.6
5.4
–
–
–
–
3.3
d
4.0
5.3
15.4
5.1
)
5.7
15.9
5.1
8.7
–
15.4
6.8
7.3
7.2
d
d
)
)
)
)
d
3.6
7.5
–
–
d
a) From [16a,b]. b) From [17]. c) Three drops of CD3OD were added. d) Not assigned.
dimethylamino derivative 23, and its reduction with LiAlH4 provided the dimethyl-
amino compound 9 in a yield of 42–43% from 21.
The 1,4-disubstituted 1,2,3-triazoles 30–34 (cf. Scheme 2) were obtained in yields of
81–91% as single regioisomers by cycloaddition of the azido derivatives 24 [17] to the
alkynes 25–29 [26–32] in the presence of CuSO4 and ascorbic acid [32], either in H2O
(for 30–32) or in H2O/i-PrOH 1:1 (for 33–34) at 55–608.
The alkynone 25 was obtained in 60% yield by acylation of stearoyl chloride with
(trimethylsilyl)acetylene [44][45] in the presence of AlCl3 in CH2Cl2. Nonadec-1-yne
(26) was prepared in 71% yield from octadecanal by a Corey–Fuchs reaction [46],
whereas the ester 27 was obtained by Mitsunobu reaction of hexadecan-1-ol with
propargylic acid (90%) [47]. Amide 28 was synthesized by coupling [48] between
hexadecanamine and propargylic acid (84%).
The triazoles 30–34 were debenzylated by treatment with AlCl3 in the presence of
anisole [49] to afford the desired triazole derivatives 10, 12–14, and 16 (Scheme 2 and
Table 2). The structure of these 1,4-disubstituted 1,2,3-triazoles is evidenced by the
chemical shift for C(4) and C(5) of the triazolyl unit, resonating at 139.5–151.1 and
126.9–129.7 ppm, respectively1).
1
)
Typical chemical shifts for C(4) and C(5) of 1,4-disubstituted 1,2,3-triazoles are 143–148 and 117–
126 ppm, respectively, differing from those of 1,5-disubstituted 1,2,3-triazoles (131–139 and 130–
147 ppm, resp.) [50].

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Scheme 2
a) CuSO4, Ascorbic acid, H2O (H2O/iPrOH 1:1 for 33–34), 608; 91% of 30; 81% of 31; 84% of 32; 90%
of 33; 84% of 34. b) AlCl3, Anisole, 1,2-dichloroethane, 258; 78% of 10; 71% of 11; 86% of 12; 91% of 13;
89% of 14; 80% of 15; 70% of 16. c) NaBH4, THF/H2O (3 :2), 08–258, 24 h; 88%. d) Lawessonꢂs
reagent, toluene, 758; 84%. e) Bu4NF·3 H2O, THF, AcOH, 258; 97%.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
711
1
Table 2. Selected H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of the Ceramide 2, 7-
Oxaceramide 4, and the 1,2,3-Triazole Analogues 10–16 (in CDCl3)
2a)
4b)
10
4.37–4.30
11c)
12
4.29
13
4.36
14
4.35
15
4.27
16
4.31
HꢀC(1)
H’ꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
HOꢀC(3)
HOꢀC(1)
J(1,1’)
J(1,2)
J(1’,2)
J(2,3)
J(3,4)
J(4,5)
J(5,6)
J(NH,2)
J(OH,3)
J(OH,1)
J(OH,1’)
3.93
3.96–3.88
4.30
3.68
3.88
4.31
5.51
5.76
2.03
2.70
2.70
11.2
3.4
3.4
3.6
6.5
3.74–3.65
3.96–3.88
4.38
5.79
5.91
3.98
3.26
2.97
4.15–4.08
4.67–4.63
4.75
5.73
5.88
3.92
3.18
2.91
4.15
4.75
4.87
5.71
5.84
3.91
4.14–4.06
4.46
4.74
5.67
5.83
3.90
3.88
3.59
11.1
4.13
4.64
4.79
5.77
5.92
3.93
3.38
3.19
4.03
4.08
4.60
4.69
5.69
5.84
3.90
3.35
3.11
4.12
4.57
4.74
5.68
5.83
3.90
3.70
3.44
4.71–4.65
4.71–4.65
5.71
5.86
3.91
4.41
4.17
11.4
d
)
)
)
)
)
)
)
d
d
d
d
d
d
d
d
)
)
)
)
)
)
)
)
11.8
12.0
12.0
d
d
d
d
d
d
5.7
3.6
5.2
2.7
4.8
2.4
5.7
3.6
5.4
3.6
d
d
d
d
d
4.0
5.3
15.4
5.1
)
)
)
)
)
5.7
15.6
4.8
–
5.7
15.6
5.1
–
6.9
3.0
3.0
5.7
15.3
5.4
–
5.1
15.3
5.4
–
4.8
4.8
6.3
5.6
15.6
5.4
–
15.4
6.8
15.6
5.1
–
15.6
4.8
–
7.3
7.2
d
d
d
d
d
d
)
)
)
)
)
)
)
)
)
)
)
)
)
4.2
–
6.2
d
d
d
d
3.6
7.5
–
–
d
d
d
–
a) From [16a,b]. b) From [17]. c) Not assigned. d) NMR Spectrum was recorded at 458.
The protected 4-acyltriazole 30 was reduced with NaBH4 to the corresponding
alcohols 35 (88%) that were debenzylated to the triols 11. The thioamide 36 was
obtained in 84% yield from amide 33 by treatment with Lawessonꢂs reagent, and
deprotected to 15 (Scheme 2).
Biological Results. – Phosporylation by Ceramide Kinase (CerK). Compounds 7–
15 were tested for activity as inhibitors of SPHK type 1 and of acid sphingomyelinase
according to established assay methods [51][52]; none of them was found to be active.
The monosubstituted triazolyl derivative 16 was found to be a weak inhibitor of
SPHK1, inhibiting the enzyme by 62% at 100 mm and by 25% at 10 mm. For compounds
10, 11, and 15, we checked whether they would be accepted as substrate by ceramide
kinase [53]; however, no phosphorylation of the compounds was detectable.
T-Cell Suppression. Compounds 7, 8, and 10–15 did neither show any activatory
capacity on iNKT-cell clones when presented by human CD1d-transfected antigen-
presenting cells (APC) nor by plate-bound human CD1d (data not shown). None of the
compounds proved cytotoxic for APC or iNKT cells at tested doses (up to 20 mg/ml), as
assessed by flow cytometry (data not shown).
Lack of stimulatory activity may not exclude the capacity of these compounds to
bind to CD1d. This was investigated using a competition assay to evaluate the capacity
of the compounds to prevent binding of a-GalCer to CD1d and, thus, to prevent T-cell

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
activation. Only compound 15 was able to compete, although this competition was only
achieved at 15-fold molar excess over a-GalCer (Fig. 3,a and b; data not shown). In
additional experiments, we investigated whether 15 was capable of competing with a-
GalCer in living cells. In contrast to the strong inhibition seen in the plate-bound assay,
15 was incapable of reducing T-cell activation at all tested doses of a-GalCer.
Fig. 3. Triazoles 14 and 15: competition with a-GalCer. Ceramide analogues 14 and 15 were tested for
their capacity to prevent binding of a-GalCer to plate-bound human CD1d and subsequent T-cell
response to a-GalCer. Supernatants were taken after 24 h, and released human IL-4 (a) and human GM-
CSF (b) were measured by ELISA and expressed as pg/mlꢁSD of duplicates. Only 15 was significantly
reducing activation by a-GalCer independently of the cytokine measured (**: P<0.01; *: P<0.05).
This unexpected discrepancy between plate-bound and living cell competition
assays might be explained with the instability of the thioamide moiety of 15 inside living
cells. To test the stability of the thioamide 15, we performed plate-bound assays at pH 4
and 7. At pH 4, an increase in cytokine release as compared to pH 7 was seen, and a
partial degradation of 15 was thus assumed (Fig. 4,a and b). In living cells, 15 requires
trafficking to lysosomes, where lipid antigens are loaded onto CD1d molecules [54].
Therefore, it is tempting to speculate that the acidic late-endosomal microenvironment
affects the stability of 15.
We thank Novartis AG, Basel, for a fellowship, the ETH-Zꢀrich for financial support, and Dr. Bruno
Bernet for checking the analytical data.
Experimental Part
1. Synthesis. General. THF was distilled from Na and benzophenone, CH2Cl2 from P2O5, and MeOH
and MeCN from CaH2. Reactions were carried out under Ar, unless stated otherwise. Qual. TLC:
precoated silica-gel plates (Merck silica gel 60 F254); detection by heating with ꢃmostainꢂ (400 ml of 10%
H2SO4 soln., 20 g of (NH4)6Mo7O24 ·6 H2O, 0.4 g of Ce(SO4)2). Flash chromatography (FC): silica gel
Fluka 60 (0.04–0.063 mm). Optical rotations: 1-dm cell at 258, 589 nm. FT-IR Spectra: KBr or ca. 2%
soln. in CHCl3, ˜n in cmꢀ1. 1H- and 13C-NMR Spectra: chemical shifts d in ppm rel. to Me4Si as external
standard, and coupling constants J in Hz. HR-MALDI-MS: in gentisic acid (¼2,5-dihydroxybenzoic
acid, DHB) matrix.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
713
Fig. 4. Triazole 15 is sensitive at low pH. A competition experiment was performed at pH 4 or 7.
Supernatants were taken after 24 h, and released human IL-4 (a) and human GM-CSF (b) were
measured by ELISA, and expressed as pg/mlꢁSD of duplicates. At pH 4, triazole 15 shows less
competition than at pH 7, independently of the cytokine measured (*: P<0.05).
(E)-2-Azido-2,4,5-trideoxy-1,3-O-isopropylidene-6-O-undecyl-d-erythro-hex-4-enitol (17). An ice-
cold soln. of 21 (253 mg, 0.77 mmol) in acetone (4 ml) was treated with 2,2-dimethoxypropane (402.3 mg,
3.86 mmol) and TsOH·H2O (14.7 mg, 0.077 mmol), stirred at r.t. for 2.5 h, and evaporated. A soln. of the
residue in Et2O (100 ml) was washed with H2O (2ꢂ50 ml), and the aq. phase was extracted with Et2O
(2ꢂ50 ml). The combined org. layers were washed with brine, dried (Na2SO4), and evaporated. FC
(AcOEt/hexane 1:9) gave 17 (266 mg, 94%). Colourless oil. Rf (AcOEt/hexane 1:9) 0.33. [a]2D5 ¼ ꢀ15.1
(c¼2.0, CHCl3). IR (CHCl3): 3000w, 2925s, 2856m, 2110s, 1602w, 1465w, 1382w, 1371w, 1304w, 1264m,
1
1160w, 1101m, 1018m, 979w, 939w. H-NMR (CDCl3, 300 MHz): 5.98 (dtd, J¼15.5, 5.3, 0.9, HꢀC(5));
5.75 (ddt, J¼15.6, 6.6, 1.5, HꢀC(4)); 4.12 (dd, J¼9.6, 6.3, HꢀC(3)); 4.00 (br. dd, J¼5.4, 1.2, 2 HꢀC(6));
3.94 (dd, Jꢃ11.6, 5.6, HꢀC(1)); 3.66 (dd, J¼11.4, 10.2, H’ꢀC(1)); 3.42 (t, J¼6.8, 2 HꢀC(1’)); 3.30 (td,
J¼9.8, 5.5, HꢀC(2)); 1.63–1.52 (m, 2 HꢀC(2’)); 1.47, 1.41 (2s, Me2C); 1.36–1.21 (m, 16 H); 0.88 (t, J¼
6.6, Me). 13C-NMR (CDCl3, 75 MHz): 132.05 (d, C(4)); 128.40 (d, C(5)); 98.84 (s, Me2C); 73.17 (d,
C(3)); 70.76 (t, C(6)); 70.46 (t, C(1’)); 62.30 (t, C(1)); 58.40 (d, C(2)); 31.98, 29.80, 29.68, 29.57, 29.41,
26.24, 22.77 (several t); 28.83, 19.16 (2q, Me2C); 14.22 (q, Me). HR-ESI-MS: 390.2725 (100, [MþNa]þ
,
C20H37N3NaOþ3 ; calc. 390.2727). Anal. calc. for C20H37N3O3 (367.53): C 65.36, H 10.15, N 11.43; found: C
65.39, H 10.05, N 11.60.
(E)-2-Amino-2,4,5-trideoxy-1,3-O-isopropylidene-6-O-undecyl-d-erythro-hex-4-enitol (18). A stir-
red suspension of LiAlH4 (54.7 mg, 1.44 mmol) in Et2O (10 ml) was cooled to 08, treated dropwise with a
soln. of 17 (265 mg, 0.72 mmol) in Et2O (5 ml), stirred at 08 for 1 h, and treated dropwise with H2O
(0.4 ml), 1n NaOH (0.8 ml), and H2O (1.2 ml). The suspension was then filtered through Celite, and the
filtrate was extracted with AcOEt (2ꢂ100 ml). The combined org. layers were washed with brine, dried
(Na2SO4), and evaporated to afford crude 18 (245 mg, 99.5%) that was used without further purification.
A pure sample of 18 was obtained by FC (MeOH/CH2Cl2 1:19). Colourless oil. Rf (MeOH/CH2Cl2 1:19)
0.24. [a]2D5 ¼ ꢀ2.3 (c¼1.0, CHCl3). IR (CHCl3): 3479w (br.), 3377w, 3000m, 2928s, 2856s, 1666w, 1655w,
1602w, 1463w, 1458w, 1382m, 1373m, 1264m, 1213m, 1158m, 1103m, 1078m, 1016m, 975w, 941w. 1H-NMR
(CDCl3, 300 MHz): 5.91 (br. dt, Jꢃ15.6, 5.3, HꢀC(5)); 5.68 (ddt, Jꢃ15.6, 7.5, 1.4, HꢀC(4)); 3.98 (br. d,
J¼6.0, 2 HꢀC(6)); 3.91 (dd, J¼9.3, 7.5, HꢀC(3)); 3.86 (dd, Jꢃ11.3, 5.6, HꢀC(1)); 3.54 (dd, J¼11.3,
10.4, H’ꢀC(1)); 3.42 (t, Jꢃ6.6, 2 HꢀC(1’)); 2.71 (td, Jꢃ9.9, 5.2, HꢀC(2)); 1.63–1.51 (m, 2 HꢀC(2’))
1.48, 1.41 (2s, Me2C); 1.42 (br. s, NH2); 1.36–1.20 (m, 16 H); 0.87 (t, Jꢃ6.7, Me). 13C-NMR (CDCl3,
75 MHz): 131.88 (d, C(4)); 129.60 (d, C(5)); 98.45 (s, Me2C); 77.38 (d, C(3)); 70.89 (t, C(6)); 70.58 (t,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
C(1’)); 65.57 (t, C(1)); 49.12 (d, C(2)); 32.01, 29.84, 29.74, 29.62, 29.45, 26.29, 22.81 (several t); 29.41, 19.23
(2q, Me2C); 14.26 (q, Me). HR-MALDI-MS: 342.3000 (100, [MþH]þ , C20H40NO3þ ; calc. 342.3003).
(E)-2,4,5-Trideoxy-2-[3-(hexadecyl)ureido]-1,3-O-isopropylidene-6-O-undecyl-d-erythro-hex-4-eni-
tol (19). An ice-cold soln. of 18 (66 mg, 0.193 mmol) in CH2Cl2 (2 ml) was treated with ice-cold soln. of
hexadecyl isocyanate (56.9 mg, 0.21 mmol), followed by Et3N (54 ml, 0.39 mmol), stirred for 18 h, and
evaporated. A soln. of the residue in CH2Cl2 (50 ml) was washed with H2O (20 ml). The aq. phase was
extracted with CH2Cl2 (3ꢂ20 ml). The combined org. phases were dried (Na2SO4) and evaporated. FC
(AcOEt/hexane 1:4!3 :7!1:1) gave pure 19 (90 mg, 77%). White fluffy powder. Rf (CH2Cl2/MeOH
95 :5) 0.80. [a]2D5 ¼ þ15.7 (c¼0.3, CHCl3). IR (ATR): 3320w, 2957w, 2918s, 2850s, 1622s, 1575s, 1467m,
1378w, 1370w, 1290w, 1261w, 1243m, 1199m, 1158w, 1108m, 1077m, 1022m, 967w, 940w, 869w, 720w, 657w.
1H-NMR (CDCl3, 300 MHz): 5.87 (dt, J¼15.6, 5.5, HꢀC(5)); 5.72 (dd, J¼15.6, 6.5, HꢀC(4)); 4.52 (t, J¼
5.2, HNꢀC(1’’)); 4.36 (d, J¼7.2, HNꢀC(2)); 4.12 (dd, J¼9.1, 6.5, HꢀC(3)); 4.00 (dd, J¼11.0, 4.9,
1 HꢀC(1)); 3.97–3.89 (m, 2 HꢀC(6)); 3.66 (dd, J¼10.8, 9.0, 1 HꢀC(1)); 3.60–3.52 (m, HꢀC(2)); 3.39
(t, J¼6.7, 2 HꢀC(1’)); 3.10 (q, Jꢃ6.7, 2 HꢀC(1’’)); 1.59–1.50 (m, 2 HꢀC(2’)); 1.47–1.38 (m,
2 HꢀC(2’’)); 1.47, 1.41 (2s, Me2C); 1.36–1.16 (m, 42 H); 0.87 (t, J¼6.7, 2 Me). 13C-NMR (CDCl3,
75 MHz): 157.26 (s, C¼O); 131.09 (d, C(4)); 129.78 (d, C(5)); 98.80 (s, Me2C); 73.45 (d, C(3)); 70.77 (t,
C(6)); 70.71 (t, C(1’)); 63.56 (t, C(1)); 49.23 (d, C(2)); 40.71 (t, C(1’’)); 32.03 (2t); 30.28, 29.89 (2t); 29.82,
29.75, 29.68, 29.48 (several t); 28.56, 19.78 (2q, Me2C); 27.07, 26.32 (2t); 14.26 (q, Me). HR-MALDI-MS:
631.6369 (100, [MþNa]þ , C37H72N2NaO4þ ; calc. 631.5390). Anal. calc. for C37H72N2O4 (608.9786): C
72.97, H 11.92, N 4.60; found: C 73.01, H 12.06, N 4.55.
(E)-2,4,5-Trideoxy-2-[3-(hexadecyl)ureido]-6-undecyl-d-erythro-hex-4-enitol (7). A soln. of 19
(40 mg, 0.066 mmol) in THF (1 ml) was treated with 60% aq. CF3COOH (2 ml), stirred at 258 for
30 min, and evaporated. A soln. of the residue in CH2Cl2/MeOH 95 :5 (0.5 ml) was filtered through a
short pad of silica gel (Lichoprep-NH2) (CH2Cl2/MeOH 97:3) to yield pure 7 (33 mg, 89%). White shiny
crystals. M.p. 102.68. Rf (CH2Cl2/MeOH 95 :5) 0.14. [a]2D5 ¼ þ1.6 (c ¼ 0.25, THF). IR (ATR): 3369m,
2925w, 2916s, 2849s, 2795w, 1589s, 1520w, 1464w, 1360w, 1321w, 1261m, 1223w, 1141w, 1100m, 1083m,
1051m, 986w, 956w, 884w, 812w, 721m, 673w. 1H-NMR (CDCl3/CD3OD (2 drops), 300 MHz): 5.89 (dt, J ¼
15.9, 5.4, HꢀC(5)); 5.78 (br. dd, J¼15.6, 5.4, irrad. at 4.36!d, J¼15.9, HꢀC(4)); 4.36 (t, Jꢃ4.2, irrad. at
3.77!d, J¼5.2, HꢀC(3)); 3.97 (d, J¼5.1, irrad. at 5.89!s, 2 HꢀC(6)); 3.83 (dd, J ¼ 10.8, 4.2,
1 HꢀC(1)); 3.77 (q, J¼3.8, irrad. at 4.36!t, J¼3.6, HꢀC(2)); 3.69 (dd, J ¼ 10.8, 3.0, 1 HꢀC(1)); 3.42 (t,
J¼6.8, 2 HꢀC(1’)); 3.13 (t, J¼7.1, 2 H ꢀC(1’’)); 1.62–1.52 (m, 2 HꢀC(2’)); 1.52–1.42 (m, 2 HꢀC(2’’));
1.46–1.24 (m, 42 H); 0.87 (t, Jꢃ6.8, 2 Me). 13C-NMR (CDCl3, 75 MHz): 158.84 (s, C¼O); 131.59 (d,
C(4)); 128.94 (d, C(5)); 73.87 (d, C(3)); 70.99 (t, C(6)); 70.62 (t, C(1’)); 62.80 (t, C(1)); 55.50 (d, C(2));
40.74 (t, C(1’’)); 31.98 (2t); 30.03–29.41 (several t); 26.96, 26.20 (2t), 22.76 (2t); 14.21 (q, 2 He). HR-ESI-
MS: 591.5071 (100, [M þ Na]þ , C34H68N2NaO4þ ; calc. 591.5077). Anal. calc. for C34H68N2O4 (568.9147): C
71.78, H 12.05, N 4.92; found: C 71.88, H 12.04, N 4.86.
(E)-2,4,5-Trideoxy-2-(hexadecanesulfonamido)-1,3-O-isopropylidene-6-O-undecyl-d-erythro-hex-4-
enitol (20). An ice-cold soln. of 18 (50 mg, 0.15 mmol) in CH2Cl2 (5 ml) was treated successively with
Et3N (102 ml, 0.732 mmol) and hexadecylsulfonyl chloride (118.9 mg, 0.37 mmol). The mixture was
stirred at 258 for 72 h, diluted with CH2Cl2 (50 ml), and washed with H2O (20 ml). The aq. phase was
extracted with CH2Cl2 (2ꢂ50 ml). The combined org. phases were washed with brine, dried (Na2SO4),
and evaporated. FC (AcOEt/hexane 1:9!1:4) gave pure 20 (82 mg, 89%). White fluffy powder. Rf
(CH2Cl2/MeOH 95 :5) 0.79. IR (ATR): 2954w, 2919s, 2850s, 2795w, 1467w, 1440w, 1351w, 1301w, 1272w,
1
1219w, 1121s, 1089m, 1062m, 1034m, 1020w, 987w, 959w, 889w, 860w, 773m, 724m. H-NMR (CDCl3,
300 MHz): 5.94 (dt, J¼15.3, 4.8, HꢀC(5)); 5.74 (ddt, J¼15.3, 7.2, 1.2, HꢀC(4)); 4.37 (d, J¼8.4, NH);
4.08 (dd, J¼12.0, 5.8, 1 HꢀC(1)); 4.03 (dd, J¼9.9, 7.5, HꢀC(3)); 3.96 (dd, J¼5.1, 1.2, 2 HꢀC(6)); 3.67
(dd, J¼11.4, 9.6, 1 HꢀC(1)); 3.41 (t, J¼6.9, 2 HꢀC(1’)); 3.26 (qd, Jꢃ9.6, 5.1, HꢀC(2)); 3.04–2.88 (m,
2 HꢀC(1’’)); 1.80–1.72 (m, 2 HꢀC(2’’)); 1.60–1.51 (m, 2 HꢀC(2’)); 1.47, 1.41 (2s, Me2C); 1.37–1.19 (m,
42 H); 0.87 (t, J¼6.6, Me). 13C-NMR (CDCl3, 75 MHz): 132.69 (d, C(4)); 128.19 (d, C(5)); 98.89 (s,
Me2C); 73.45 (d, C(3)); 71.09 (t, C(6)); 70.26 (t, C(1’)); 64.39 (t, C(1)); 53.83 (t, C(1’’)); 51.90 (d, C(2));
32.03 (2t); 29.88, 29.70, 29.53, 29.48, 29.32 (several t); 28.69, 19.48 (2q, Me2C); 28.43, 26.31, 23.80 (3t);
22.82 (2t); 14.26 (q, Me). HR-ESI-MS: 634.4838 (100, [MþNa]þ , C36H69NNaO4Sþ ; calc. 634.4840).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
715
Anal. calc. for C36H69NO4S (612.01): C 70.65, H 11.36, N 2.29, S 5.24; found: C 70.61, H 11.34, N 2.35, S
5.13.
(E)-2,4,5-Trideoxy-2-(hexadecanesulfonamido)-6-O-undecyl-d-erythro-hex-4-enitol (8). A soln. of
20 (31.7 mg, 0.05 mmol) in THF (1 ml) was treated with 60% aq. CF3COOH (2 ml), stirred at 258 for
30 min, and evaporated. A soln. of the residue in AcOEt (20 ml) was washed with brine. The aq. phase
was extracted with AcOEt (2ꢂ10 ml), and the combined org. phases were dried (Na2SO4), and
evaporated. The crude was filtered through a short pad of silica gel (AcOEt/hexane 1:1) to yield pure 8
(28 mg, 94%). Colourless crystals. M.p. 69.78. Rf (CH2Cl2/MeOH 95 :5) 0.14. Rf (CH2Cl2/MeOH 9 :1)
0.50. [a]2D5 ¼ þ2.3 (c ¼ 0.15, CHCl3). IR (ATR): 3465w, 3276w, 2953w, 2918s, 2848s, 2795w, 1465w, 1441w,
1350w, 1303w, 1271w, 1219w, 1121s, 1089m, 1062m, 1034m, 1020w, 987w, 959w, 889w, 865w, 773m, 723m.
1H-NMR (CDCl3, 300 MHz): 5.93 (dt, J ¼ 15.9, 5.4, HꢀC(5)); 5.78 (br. dd, J¼15.9, 5.7, HꢀC(4)); 5.32
(d, J¼8.7, exchange with D2O, NH); 4.44 (br. s, HꢀC(3)); 3.98 (d, J¼5.1, 2 HꢀC(6)); 3.90 (dd, J ¼ 11.7,
4.5, 1 HꢀC(1)); 3.71 (dd, J ¼ 11.1, 3.3, 1 HꢀC(1)); 3.49–3.40 (m, HꢀC(2)); 3.43 (t, J¼6.6, 2 HꢀC(1’));
3.10–3.04 (m, 2 HꢀC(1’’)); 3.07 (br. s, exchange with D2O, OH); 2.81 (br. s, exchange with D2O, OH) ;
1.87–1.77 (m, 2 HꢀC(2’’)); 1.62–1.53 (m, 2 HꢀC(2’)); 1.44–1.25 (m, 42 H); 0.87 (t, Jꢃ6.8, 2 Me).
13C-NMR (CDCl3, 75 MHz): 130.59 (d, C(4)); 130.10 (d, C(5)); 74.28 (d, C(3)); 71.21 (t, C(6)); 70.54 (t,
C(1’)); 62.64 (t, C(1)); 58.34 (d, C(2)); 54.02 (t, C(1’’)); 32.10 (2t); 29.88–29.74 (several t); 29.55 (2t);
29.35, 28.51 (2t); 26.34, 23.85 (2t), 22.87 (2t); 14.30 (q, 2 Me). HR-MALDI-MS: 612.4632 (100, [M þ
Na]þ , C33H67NNaO5Sþ ; calc. 612.4638). Anal. calc. for C33H67NO5S (589.9538): C 67.18, H 11.45, N 2.37;
found: C 67.16, H 11.55, N 2.42.
(E)-2-Azido-2,4,5-trideoxy-1-O-[(4-methylphenyl)sulfonyl]-6-O-undecyl-d-erythro-hex-4-enitol
(22). A soln. of 21 (200.5 mg, 0.61 mmol) in CH2Cl2 (5 ml) was treated with TsCl (128.4 mg, 0.67 mmol),
followed by Et3N (0.17 ml, 1.23 mmol) and DMAP (7.5 mg, 0.061 mmol), stirred at 108 for 30 min,
diluted with Et2O (50 ml), and washed with H2O (50 ml). The aq. phase was extracted with Et2O (3ꢂ
50 ml). The combined org. phases were washed with brine, dried (Na2SO4), and evaporated. FC (AcOEt/
hexane 1:9!1:4) gave pure 22 (233 mg, 79%). Colourless oil. IR (CHCl3): 3329w (br.), 2923s, 2853s,
2098s, 1465w, 1375m, 1266m, 1180w, 1168m, 1110m, 1096w, 1070m, 1064m, 1012m, 972m, 852w. 1H-NMR
(CDCl3, 300 MHz): 7.80 (d, J ¼ 8.4, 2 arom. H); 7.36 (d, J ¼ 8.5, 2 arom. H); 5.87 (dtd, J¼15.6, 5.1, 0.7,
HꢀC(5)); 5.71 (ddt, J¼15.6, 6.5, 1.3, HꢀC(4)); 4.22 (dd, J¼10.6, 3.8, 1 HꢀC(1), HꢀC(3)); 4.09 (dd, J¼
10.6, 7.4, 1 HꢀC(1)); 3.95 (d, J¼5.1, 2 HꢀC(6)); 3.64 (ddd, J¼7.3, 5.6, 3.7, HꢀC(2)); 3.40 (t, J¼6.7,
2 HꢀC(1’)); 2.45 (s, Me); 2.27 (d, J¼4.3, OH); 1.57 (quint., J¼6.9, 2 HꢀC(2’)); 1.34–1.23 (m, 16 H);
0.87 (t, J¼6.7, Me). 13C-NMR (CDCl3, 75 MHz): 145.13, 132.33 (2s); 131.70 (d, C(4)); 129.86 (2d); 128.89
(d, C(5)); 127.88 (2d); 71.49 (d, C(3)); 70.81 (t, C(6)); 70.08 (t, C(1’)); 68.58 (t, C(1)); 63.93 (d, C(2));
31.78 (t); 29.59–29.23 (several t); 26.04, 22.58 (2t), 21.58 (q, Me); 14.01 (q, Me). HR-MALDI-MS:
504.2502 (100, [MþNa]þ , C24H39N3NaO5Sþ ; calc. 504.2508).
(E)-2-Azido-1,2,4,5-tetradeoxy-1-(dimethylamino)-6-O-undecyl-d-erythro-hex-4-enitol (23). A mix-
ture of 22 (190 mg, 0.39 mmol) and Me2NH (40% in H2O, 2 ml) was stirred at 808 for 15 h, cooled to 258,
diluted with H2O (5 ml), and extracted with AcOEt (3ꢂ50 ml). The combined org. phases were washed
with brine, dried (Na2SO4), and evaporated. The residue was filtered through a short pad of silica gel
(AcOEt) to afford pure 23 (83 mg, 60%). Colourless oil. IR (CHCl3): 3329w (br.), 3140w (br.), 2923s,
1
2852s, 2803w, 2099s, 1465w, 1365w, 1266m, 1108s, 1041m, 1012m, 972m, 832w, 721w. H-NMR (CDCl3,
300 MHz): 5.95 (dt, J¼15.9, 5.3, HꢀC(5)); 5.78 (dd, J¼15.5, 6.2, HꢀC(4)); 4.19 (t, Jꢃ6.8, HꢀC(3));
4.01 (d, J¼5.4, 2 HꢀC(6)); 3.43 (t, J¼6.7, 2 HꢀC(1’)); 3.37 (td, J¼7.6, 5.8, HꢀC(2)); 2.68–2.55 (m,
2 HꢀC(1)); 2.32 (s, Me2N); 1.58 (quint., J¼6.9, 2 HꢀC(2’)); 1.35–1.23 (m, 16 H); 0.88 (t, J¼6.6, Me).
13C-NMR (CDCl3, 75 MHz): 131.70 (d, C(4)); 130.29 (d, C(5)); 76.25 (d, C(3)); 70.65, 70.60 (2t, C(6),
C(1’)); 62.32 (t, C(1)); 61.13 (d, C(2)); 46.02 (q, Me2N); 32.02 (t); 29.88–29.46 (several t); 26.30, 22.82
(2t), 14.27 (q, Me). HR-MALDI-MS: 355.3062 (100, [MþH]þ , C19H39N4O2þ ; calc. 355.3073). Anal. calc.
for C19H38N4O2 (354.53): C 64.37, H 10.80, N 15.80; found: C 64.62, H 10.85, 15.96.
(E)-2-Amino-1,2,4,5-tetradeoxy-1-(dimethylamino)-6-O-undecyl-d-erythro-hex-4-enitol (9). A sus-
pension of LiAlH4 (14.4 mg, 0.38 mmol) in Et2O (5 ml) was treated with a soln. of 23 (66 mg, 0.19 mmol)
in Et2O (5 ml) and stirred at 08 for 1 h. The mixture was treated dropwise with H2O (0.1 ml), 1m NaOH
(0.2 ml), and H2O (0.3 ml). The suspension was filtered through Celite, washing with AcOEt. After
evaporation of the combined filtrate and washings, a soln. of the residue in AcOEt (20 ml) was washed

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
with H2O (5 ml). The aq. phase was extracted with AcOEt (3ꢂ30 ml). The combined org. phases were
washed with brine, dried (Na2SO4), and evaporated. FC (CH2Cl2/MeOH/Et3N 95 :5 :1) afforded pure 9
(56 mg, 90%). Colourless oil. IR (CHCl3): 3330w (br.), 3148w (br.), 2923s, 2852s, 2802w, 1465w, 1365w,
1266m, 1108s, 1041m, 1012m, 972m, 832w, 721w. 1H-NMR (CDCl3, 300 MHz): 5.86 (dt, J¼15.3, 5.4,
HꢀC(5)); 5.67 (br. dd, J¼15.3, 6.3, HꢀC(4)); 4.13 (t, J¼6.4, HꢀC(3)); 4.13 (br. s, exchange with D2O,
NH2, OH); 3.96 (d, J¼5.7, 2 HꢀC(6)); 3.38 (t, J¼6.6, 2 HꢀC(1’)); 2.96 (q, Jꢃ6.9, HꢀC(2)); 2.53 (dd,
J¼12.6, 6.6, 1 HꢀC(1)); 2.43 (dd, J¼12.9, 7.5, 1 HꢀC(1)); 2.28 (s, Me2N); 1.54 (quint., J¼6.9,
2 HꢀC(2’)); 1.31–1.23 (m, 16 H); 0.84 (t, J¼6.6, Me). 13C-NMR (CDCl3, 75 MHz): 131.41 (d, C(4));
130.00 (d, C(5)); 76.37 (d, C(3)); 70.62 (t, C(6), C(1’)); 62.80 (t, C(1)); 51.53 (d, C(2)); 45.72 (q, Me2N);
31.95 (t); 29.80–29.38 (several t); 26.23, 22.74 (2t), 14.20 (q, Me). HR-MALDI-MS: 329.3162 (100, [Mþ
H]þ , C19H41N2O2þ ; calc. 329.3168). Anal. calc. for C19H40N2O2 ·0.75 H2O (342.04): C 66.72, H 12.23, N
8.19; found: C 66.56, H 12.04, N 8.31.
Icos-1-yn-3-one (25). A 50-ml two-necked flask was charged with AlCl3 (490 mg, 3.675 mmol) in
CH2Cl2 (5 ml), cooled to ꢀ158 and treated with (trimethylsilyl)acetylene (0.59 ml, 4.2 mmol). The
resulting reddish yellow mixture was treated with a (slow addition) ice-cooled soln. of stearoyl chloride
(1.06 g, 3.5 mmol) in CH2Cl2 (5 ml), stirred at ꢀ158 for 2 h (TLC (Et2O/hexane 1:25, visualized with
KMnO4) showed complete conversion of stearoyl chloride). The reaction was quenched with slow
addition of ice-cold 10% aq. HCl (3 ml), and the mixture was diluted with H2O (20 ml) and extracted
with Et2O (3ꢂ150 ml). The combined org. phase was washed with sat. aq. NaHCO4 and brine, dried
(Na2SO4), and evaporated. FC (Et2O/hexane 1:25) yielded pure 25 (60%). Colourless powder. M.p. 38–
398. Rf (Et2O/hexane 1:25) 0.17. IR (CHCl3): 3299m, 3028w, 2927s, 2855s, 2097m, 1681s, 1466m. 1H-NMR
(CDCl3, 300 MHz): 3.20 (s, HꢀC(1)); 2.58 (t, J¼7.5, 2 HꢀC(1’)); 1.67 (quint., J¼7.5, 2 HꢀC(2’)); 1.24–
1.17 (m, 28 H); 0.88 (t, J¼6.3, Me). 13C-NMR (CDCl3, 300 MHz): 187.81 (s, C¼O); 81.65 (s, C(2)); 78.40
(d, C(1); 45.65 (d, C(1’)); 32.11 (t, C(2’)); 29.87–29.07 (several t); 23.95, 22.88 (2t); 14.30 (q, Me). HR-EI-
MS: 292.2746 (Mþ ), 291.2686 ([MꢀH]þ ), 263.2375 ([MꢀC2H5]þ ), 249.2215 ([MꢀC3H7]þ ), 235.2056
([MꢀC4H9]þ ), 221.1894 ([MꢀC5H11]þ ), 207.1751 ([MꢀC6H13]þ ), 179.1444 ([MꢀC7H15]þ ), 165.1295
([MꢀC8H17]þ ), 151.1147 ([MꢀC9H19]þ ).
Nonadec-1-yne (26). Compound 26 was prepared according to the Corey–Fuchs method from
octadecanal (stearaldehyde), which, in turn, was prepared by pyridinium chlorochromate-mediated
oxidation of the commercially available octadecan-1-ol according to the procedure reported in [37][48].
A mixture of PPh3 (391 mg, 1.49 mmol) and CBr4 (247 mg, 0.745 mmol) in CH2Cl2 (2 ml) was cooled to
08, treated with octadecanal (100 mg, 0.373 mmol) in one lot, and stirred at 08 for 0.5 h and at 258 for
0.5 h. The mixture was diluted with hexane, and the supernatant was filtered through Celite. The solid was
dissolved in CH2Cl2, reprecipitated by the addition of hexane, and filtered. The process was repeated
until the 1,1-dibromononadec-1-ene was completely washed out. The combined filtrates were evaporated
to afford the crude 1,1-dibromoundec-1-ene, which was dissolved in dry THF (3 ml) under N2 and cooled
to ꢀ788. The soln. was treated dropwise with 1.6m BuLi in hexane (0.93 ml, 1.49 mmol), stirred for 1 h,
warmed to 258, stirred for 4 h, treated dropwise with H2O, and extracted with hexane. Evaporation and
FC (hexane) gave pure 26 (75 mg, 76%) as a white solid, whose spectral and physical data were identical
to those reported in [37]. M.p. 36–378. Rf (hexane) 0.47. IR (ATR): 3287w, 2953w, 2915s, 2848s, 2113w,
1472m, 1462m, 1278w, 729w, 719m, 684m, 666m, 652m, 628s. 1H-NMR (CDCl3, 300 MHz): 2.18 (td, J ¼
6.9, 3.0, 2 HꢀC(3)); 1.94 (t, J¼2.4, HꢀC(1)); 1.55–1.26 (m, 30 H); 0.88 (t, J¼6.6, Me). 13C-NMR
(CDCl3, 300 MHz): 84.83 (s, C(2)); 68.05 (s, C(1)); 32.04 (t); 29.80 (several t); 29.63 (t); 29.48 (t); 29.24
(t); 28.89 (t); 28.61 (t); 22.83 (t); 18.54 (t); 14.27 (q, Me).
Hexadecyl Prop-2-ynoate (27). A mixture of diethyl azodicarboxylate (DEAD) (736.5 mg,
4.229 mmol) and prop-2-ynoic acid (0.26 ml, 4.229 mmol) in THF (10 ml) was treated dropwise at 08
with a mixture of hexadecan-1-ol (512.6 mg, 2.114 mmol) and PPh3 (1.109 g, 4.229 mmol) in THF
(10 ml), stirred for 1.5 h, and evaporated under reduced pressure to give a colourless residue, which was
filtered through a short pad of silica gel using Et2O/hexane 1:9 as eluent to afford pure 27 (560 mg, 90%).
1
Colourless gum. Rf (Et2O/hexane 1:9) 0.54. H-NMR (CDCl3, 300 MHz): 4.19 (t, J ¼ 6.7, 2 HꢀC(1’));
2.87 (s, HꢀC(1)); 1.67 (quint., J¼7.2, 2 HꢀC(2’)); 1.38–1.21 (m, 26 H); 0.88 (t, J¼6.7, Me). 13C-NMR
(CDCl3, 300 MHz): 152.70 (s, C¼O); 74.82 (s, C(2)); 74.39 (d, C(1); 66.55 (d, C(1’)); 32.04 (t, C(2’));
29.86–29.28 (several t); 28.42 (t); 25.86 (t); 22.82 (t); 14.27 (q, Me).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
717
N-Hexadecylprop-2-ynamide (28). A mixture of prop-2-ynoic acid (0.1 ml, 1.62 mmol) and N,N’-
dicyclohexylcarbodiimide (DCC; 401.17 mg, 1.944 mmol) in CH2Cl2 (10 ml) was treated dropwise over
10 min, at 08, with a soln. of hexadecylamine (391.2 mg, 1.62 mmol) in CH2Cl2 (5 ml), and stirred for 1 h.
The mixture was allowed to warm to 258 and stirred for 1.5 h. The mixture was filtered through a sintered
funnel (No. 3) to remove the precipitated dicyclohexylurea, and the filtrate was evaporated. FC (silica
gel; AcOEt/hexane 1:9!1:4) afforded pure 28 (400 mg, 84%). Colourless solid. Rf (AcOEt/hexane
1:1) 0.42. 1H-NMR (CDCl3, 300 MHz): 5.92 (s, NH); 3.29 (td, J ¼ 7.1, 6.1, 2 HꢀC(1’)); 2.76 (s, HꢀC(1));
1.52 (quint., J¼7.1, 2 H ꢀC(2’)); 1.31–1.25 (m, 26 H); 0.87 (t, J¼6.7, Me). 13C-NMR (CDCl3, 300 MHz):
151.97 (s, C¼O); 77.34 (s, C(2)); 72.77 (d, C(1); 39.84 (d, C(1’)); 31.84 (t); 30.46 (t); 29.58–29.10 (several
t); 26.71, 26.37 (2t), 14.02 (q, Me). HR-EI: 293.2701 (Mþ ), 292.2635 ([MꢀH]þ ), 264.2323 ([MꢀC2H5]þ ),
250.2167 ([MꢀC3H7]þ ), 236.2012 ([MꢀC4H9]þ ), 222.1856 ([MꢀC5H11]þ ), 96.0453 ([MꢀC14H29
H]þ ), 83.0365 ([MꢀC15H31 þH]þ ).
þ
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-(4-octadecanoyl-1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-
hex-4-enitol (30). A mixture of 24 (151 mg, 0.297 mmol) and 25 (130.5 mg, 0.446 mmol) was treated with
a soln. of CuSO4 (0.7 mg, 0.0045 mmol) in H2O (0.5 ml) and ascorbic acid (10.5 mg, 0.06 mmol) in H2O
(0.5 ml). The mixture was heated to 608, stirred for 20 h, allowed to cool to r.t., diluted with H2O (5 ml),
and extracted with Et2O (2ꢂ30 ml). The org. phases were washed with brine, dried (Na2SO4), and
evaporated. FC (silica gel; Et2O/hexane 1:4) yielded pure 30 (216 mg, 91%). White solid. M.p. 53.78. Rf
(Et2O/hexane 3 :7) 0.20. [a]2D5 ¼ ꢀ58.0 (c¼1.0, CHCl3). IR (CHCl3): 3160w, 3030w, 3008w, 2927s, 2855s,
1684m, 1602w, 1529w, 1496w, 1467w, 1455w, 1365w, 1107m, 1027w, 974w, 911w, 836w, 781s. 1H-NMR
(CDCl3, 300 MHz): 8.17 (s, C¼CHN); 7.35–7.17 (m, 10 arom. H); 5.71 (dt, J¼15.6, 5.1, HꢀC(5)); 5.49
(br. dd, J¼15.6, 8.1, HꢀC(4)); 4.87 (dt, J ¼ 15.3, 8.7, 5.7, HꢀC(2)); 4.59 (d, J¼11.7, PhCH); 4.49 (d, J¼
11.7, PhCH); 4.44 (d, J¼11.7, PhCH); 4.29 (d, J¼12.0, PhCH); 4.29 (t, Jꢃ7.4, HꢀC(3)); 4.09 (br. dd, J¼
10.5, 6.9, 1 HꢀC(1)); 3.91 (br. dd, J¼10.2, 3.6, 1 HꢀC(1)); 3.86 (br. d, J¼5.4, 2 HꢀC(6)); 3.35–3.21
(AB, 2 HꢀC(1’)); 3.09 (br. t, J¼7.5, 2 HꢀC(2’’)); 1.80–1.69 (m, 2 HꢀC(2’)); 1.43–1.58 (m, 2 HꢀC(3’’));
1.40–1.20 (m, 44 H); 0.88 (t, Jꢃ6.8, 2 Me). 13C-NMR (CDCl3, 75 MHz): 195.7 (s, C¼O); 147.79 (s,
C¼CHN); 137.42, 137.34 (2s); 134.57 (d, C(4)); 128.66 (4d); 128.16 (2d); 128.07 (2d); 127.91 (2d); 127.06
(d, C¼CHN); 126.17 (d, C(5)); 78.28 (d, C(3)); 73.62, 71.0 (2t, 2 PhCH2); 70.75, 70.16 (2t, C(6), C(1’));
67.58 (t, C(1)); 64.74 (d, C(2)); 38.80 (t); 32.11 (2t); 29.89–29.54 (several t); 26.38, 24.17 (2t); 22.88 (2t);
14.31 (q, 2 Me). HR-MALDI-MS: 800.6286 (100, [M þ H]þ , C51H82N3O4þ ; calc. 800.6305). Anal. calc. for
C51H81N3O4 (800.2065): C 76.55, H 10.20, N 5.25; found: C 76.31, H 10.43, N 5.14.
(E)-2,4,5-Trideoxy-2-(4-octadecanoyl-1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-hex-4-enitol
(10). A soln. of 30 (50 mg, 0.062 mmol) in ClCH2CH2Cl (1 ml) was treated with anisole (0.08 ml,
0.75 mmol) and AlCl3 (75 mg, 0.56 mmol), stirred at 258 for 24 h, treated with 1m HCl (0.5 ml), diluted
with H2O, and extracted with AcOEt (3ꢂ25 ml). The combined org. layers were washed with sat. aq.
NaHCO3 soln. and brine, dried (Na2SO4), and evaporated. FC (silica gel; AcOEt/hexane 1:1) gave pure
10 (30 mg, 78%). White solid. M.p. 808. Rf (AcOEt/hexane 1:1) 0.28. [a]2D5 ¼ þ3.0 (c ¼ 0.25, CHCl3). IR
(CHCl3): 3284w (br.), 2954m, 2920s, 2849s, 1688m, 1530w, 1468m, 1403w, 1366w, 1333w, 1251w, 1183w,
1118m, 1104m, 1081m, 1045m, 1015m, 973m, 778w, 720m. 1H-NMR (CDCl3, 300 MHz): 8.38 (s,
C¼CHN); 5.88 (dtd, J¼15.6, 5.1, 0.9, HꢀC(5)); 5.73 (br. ddt, J¼15.5, 5.8, 1.25, HꢀC(4)); 4.75 (br. q, Jꢃ
4.6, HꢀC(3)); 4.67–4.63 (m, HꢀC(2)); 4.37–4.30 (m, 1 HꢀC(1)); 4.15–4.08 (m, 1 HꢀC(1)); 3.92 (d,
J¼5.1, 2 HꢀC(6)); 3.36 (t, J¼6.3, 2 HꢀC(1’)); 3.18 (d, J¼4.5, HOꢀC(3)); 3.08 (t, J¼7.5, 2 HꢀC(2’’));
2.91 (t, J¼5.7, HOꢀC(1)); 1.77–1.67 (m, 2 HꢀC(2’)); 1.58–1.50 (m, 2 HꢀC(3’’)); 1.38–1.25 (m, 44 H);
0.86 (t, Jꢃ6.8, 2 Me). 13C-NMR (CDCl3, 75 MHz): 195.49 (s, C¼O); 147.20 (s, C¼CHN); 131.10 (d,
C(4)); 129.12 (d, C¼CHN); 126.52 (d, C(5)); 72.45 (d, C(3)); 70.95, 70.08 (2t, C(6), C(1’)); 65.99 (d,
C(2)); 61.24 (t, C(1)); 39.69 (t); 31.99 (2t); 29.78–29.43 (several t); 26.21, 24.01 (2t); 22.77 (2t); 14.22 (q,
2 Me). HR-MALDI-MS: 620.5350 (100, [M þ H]þ , C37H70N3O4þ ; calc. 620.5366). Anal. calc. for
C37H69N3O4 (619.9615): C 71.68, H 11.22, N 6.78; found: C 71.79, H 11.26 , N 6.69.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-[4-(1-hydroxyoctadecyl)-1H-1,2,3-triazol-1-yl]-6-O-undecyl-
d-erythro-hex-4-enitol (35). A soln. of 30 (100 mg, 0.125 mmol) in THF/H2O 3 :2 (1.5 ml) was cooled to
08, treated with NaBH4 (4.8 mg, 0.125 mmol), stirred for 30 min, then warmed to r.t., and stirred for 24 h.
The mixture was neutralized with 1m HCl, diluted with H2O, and extracted with AcOEt (2ꢂ50 ml). The
combined org. layers were washed with sat. aq. NaHCO3 soln. and brine, dried (Na2SO4), and

718
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
evaporated. FC (silica gel; Et2O/hexane 8 :2) gave pure 35 (88 mg, 88%). White solid. M.p. 64.28. Rf
(Et2O/hexane 4 :1) 0.46. [a]2D5 ¼ ꢀ10.1 (c¼1.1, CHCl3) IR (CHCl3): 3609w, 3432w (br.), 2927s, 2855s,
1
1496w, 1465m, 1455m, 1365w, 1099m, 1048m, 1028m, 972w, 783m. H-NMR (CDCl3, 300 MHz): 7.57 (s,
C¼CHN); 7.36–7.19 (m, 10 arom. H); 5.66 (dt, J¼15.6, 5.1, HꢀC(5)); 5.48 (br. dd, J¼15.6, 8.1,
HꢀC(4)); 4.87–4.79 (m, HꢀC(2), HꢀC(1’’)); 4.60 (d, J¼11.7, PhCH); 4.50 (d, J¼11.7, PhCH); 4.45 (d,
J¼11.7, PhCH); 4.30 (d, J¼11.7, PhCH); 4.28 (t, Jꢃ8.0, HꢀC(3)); 4.09 (br. dd, J¼10.5, 6.9, 1 HꢀC(1));
3.91 (br. dd, J¼10.2, 3.6, 1 HꢀC(1)); 3.86 (br. d, J¼5.4, 2 HꢀC(6)); 3.35–3.24 (AB, 2 HꢀC(1’)); 2.26
(br. s, OH); 1.80–1.69 (m, 2 HꢀC(2’)); 1.43–1.58 (m, 2 HꢀC(2’’)); 1.40–1.20 (m, 44 H); 0.88 (t, Jꢃ6.8,
2 Me). 13C-NMR (CDCl3, 75 MHz): 151.13 (s, C¼CHN); 137.43 (d, C(4)); 137.36 (2s); 133.65, 128.35
(4d); 127.73 (4d); 127.58 (2d); 127.34 (d, C¼CHN); 120.86 (d, C(5)); 78.37 (d, C(3)); 73.24, 70.72 (2t,
2 PhCH2); 70.59, 70.04 (2t, C(6), C(1’)); 67.71 (t, C(1)); 67.05 (d, C(2’’)); 64.08 (d, C(2)); 37.23 (t); 31.82
(2t); 29.61–29.27 (several t); 26.08, 25.40 (2t); 22.59 (2t); 14.03 (q, 2 Me). HR-MALDI-MS: 824.6262
(100, [M þ Na]þ , C51H83N3NaO4þ ; calc. 824.6281). Anal. calc. for C51H83N3O4 (802.2224): C 76.36, H
10.43, N 5.24; found: C 76.39, H 10.49, N 5.28.
(E)-2,4,5-Trideoxy-2-[5-(1-hydroxyoctadecyl)-1H-1,2,3-triazol-1-yl]-6-O-undecyl-d-erythro-hex-4-
enitol (11). A soln. of 35 (47 mg, 0.059 mmol) in ClCH2CH2Cl (1 ml) was treated with anisole (76 ml,
0.703 mmol) and AlCl3 (70.3 mg, 0.53 mmol), stirred at 258 for 5 h, treated with 1m HCl (0.5 ml), diluted
with H2O, and extracted with AcOEt (3ꢂ25 ml). The combined org. layers were washed with 2% aq.
NaOH soln., H2O, and brine, dried (Na2SO4), and evaporated. FC (silica gel; AcOEt/hexane 1:1) gave
pure 11 (26 mg, 71%). White powder which, at r.t., was insoluble in solvents other than THF. Compound
11 dissolves partially in CHCl3 when heated to 458. 1H-NMR Measurements in (D8)THF were not useful,
as many signals were overlapping with the THF signals. M.p. 95–99.58. Rf (AcOEt) 0.40. IR (ATR): 3275
(br.), 3135w, 3119w, 2955w, 2916s, 2849s, 2795w, 1470m, 1364w, 1328w, 1261w, 1220w, 1117m, 1104m,
1087m, 1030m, 998w, 974w, 962w, 842w, 808w, 718w, 703w. 1H-NMR (CDCl3, 300 MHz, 458): 7.69 (s,
C¼CHN); 5.84 (dt, J¼15.6, 5.1, HꢀC(5)); 5.71 (dd, J¼15.6, 8.1, HꢀC(4)); 4.87 (m, HꢀC(3)); 4.75 (m,
HꢀC(2)); 4.52–4.48 (m, HꢀC(1’’)); 4.30 (m, 1 HꢀC(1)); 4.15 (m, 1 HꢀC(1)); 3.91 (dd, J¼4.8, 0.9,
2 HꢀC(6)); 3.65 (d, J¼0.9, 1 H); 3.47–3.43 (m, 2 HꢀC(1’)); 3.39 (t, J¼6.3); 1.85–1.48 (m, 2 HꢀC(2’),
2 HꢀC(2’’)); 1.30–1.26 (m, 44 H); 0.89 (t, Jꢃ ꢃ6.6, 2 Me). HR-MALDI-MS: 644.5325 (100, [M þ Na]þ
,
C37H71N3NaOþ4 ; calc. 644.5342). Anal. calc. for C37H71N3O4 ·0.5 H2O: C 70.43, H 11.50, N 6.66; found: C
70.26, H 11.13, N 6.37.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-(4-heptadecyl-1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-
hex-4-enitol (31). A mixture of 24 (53 mg, 0.104 mmol) and 26 (41.5 mg, 0.157 mmol) was treated with a
soln. of CuSO4 (0.25 mg, 0.0016 mmol) in H2O (0.3 ml) and ascorbic acid (4.0 mg, 0.021 mmol) in H2O
(0.5 ml), heated to 558, stirred for 48 h, allowed to cool to r.t., diluted with H2O (2 ml), and extracted
with Et2O (2ꢂ15 ml). The org. phases were washed with brine, dried (Na2SO4), and evaporated. FC
(silica gel; Et2O/hexane 1:1) yielded pure 31 (65 mg, 81%). White amorphous solid. Rf (Et2O/hexane
1:1) 0.37. [a]2D5 ¼ ꢀ11.3 (c¼1.0, CHCl3). IR (CHCl3): 3026w, 2927s, 2855s, 1496w, 1467w, 1455m, 1365w,
1100m, 1049w, 1028w, 973w. 1H-NMR (CDCl3, 300 MHz): 7.38 (s, C¼CHN); 7.35–7.27 (m, 6 arom. H);
7.25–7.18 (m, 4 arom. H); 5.67 (dt, J ¼ 15.6, 5.4, HꢀC(5)); 5.48 (br. dd, J¼15.9, 8.1, HꢀC(4)); 4.79 (td,
Jꢃ6.6, 3.9, HꢀC(2)); 4.56 (d, J¼11.7, PhCH); 4.49 (d, J¼12.3, PhCH); 4.44 (d, J¼12.3, PhCH); 4.31 (d,
J¼11.4, PhCH); 4.31 (t, J¼8.1, HꢀC(3)); 4.09 (br. dd, J¼10.2, 6.3, 1 HꢀC(1)); 3.93 (br. dd, J¼9.9, 3.6,
1 HꢀC(1)); 3.85 (br. d, J¼5.4, 2 HꢀC(6)); 3.34–3.22 (AB, 2 HꢀC(1’)); 2.68 (br. t, J¼15.6, 2 HꢀC(1’’));
1.68–1.58 (m, 2 HꢀC(2’)); 1.55–1.48 (m, 2 HꢀC(2’’)); 1.26 (m, 44 H); 0.88 (t, Jꢃ6.7, 2 Me). 13C-NMR
(CDCl3, 75 MHz): 147.91 (s, C¼CHN); 137.55, 137.47 (2s); 133.58 (d, C(4)); 128.34 (4d); 127.70 (4d);
127.58 (2d); 127.48 (d, C¼CHN); 121.05 (d, C(5)); 78.56 (d, C(3)); 73.33, 70.83 (2t, 2 PhCH2); 70.51, 70.18
(2t, C(6), C(1’)); 68.07 (t, C(1)); 64.05 (d, C(2)); 32.04 (2t); 29.84–29.42 (several t); 26.33, 25.85 (2t),
22.83 (2t); 14.28 (q, 2 Me). HR-MALDI-MS: 772.6336 (100, [MþH]þ , C50H82N3O3þ ; calc. 772.6356).
Anal. calc. for C50H81N3O3 (772.1964): C 77.77, H 10.57, N 5.44; found: C 77.51, H 10.45, N 5.41.
(E)-2,4,5-Trideoxy-2-(4-heptadecyl-1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-hex-4-enitol (12).
A soln. of 31 (53 mg, 0.063 mmol) in CH2Cl2 (1 ml) was treated with anisole (89.7 ml, 0.826 mmol) and
AlCl3 (82.4 mg, 0.618 mmol), stirred at 258 for 24 h, and treated with 1m HCl (0.5 ml), diluted with H2O
(3 ml), and extracted with AcOEt (2ꢂ20 ml). The combined org. layers were washed with brine, dried
(Na2SO4), and evaporated. FC (silica gel; AcOEt/hexane 1:9!1:1) afforded pure 12 (33 mg, 86%).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
719
White solid. M.p. 69.88. Rf (AcOEt/hexane 1:1) 0.17. [a]2D5 ¼ þ8.0 (c¼0.3, CHCl3). IR (CHCl3): 3397w
(br.), 3017m, 2927s, 2855s, 1466m, 1366w, 1110m, 974w, 767s. 1H-NMR (CDCl3, 300 MHz): 7.51 (s,
C¼CHN); 5.83 ((dtd, J¼15.6, 5.1, 0.9, HꢀC(5)); 5.67 (br. dd, J¼15.6, 5.7, HꢀC(4)); 4.74 (br. dd, J¼9.9,
5.1, HꢀC(3)); 4.46 (dtd, J ¼ 11.1, 5.7, 3.6, HꢀC(2)); 4.29 (td, J¼12.0, 5.7, 1 HꢀC(1)); 4.14–4.06 (m,
1 HꢀC(1)); 3.90 (d, J¼4.8, 2 HꢀC(6)); 3.88 (d, Jꢃ6.9, HOꢀC(3)); 3.59 (t, J¼3.0, HOꢀC(1)); 3.35 (t,
J¼6.6, 2 HꢀC(1’)); 2.64 (t, J¼7.8, 2 HꢀC(1’’)); 1.67–1.49 (m, 2 HꢀC(2’), 2 HꢀC(2’’)); 1.39–1.22 (m,
44 H); 0.87 (t, Jꢃ6.8, 2 Me). 13C-NMR (CDCl3, 75 MHz): 147.72 (s, C¼CHN); 130.43 (d, C(4)); 129.72
(d, C¼CHN); 121.75 (d, C(5)); 72.44 (d, C(3)); 70.86, 70.29 (2t, C(6), C(1’)); 65.85 (d, C(2)); 61.63 (t,
C(1)); 32.03 (t); 29.83–29.48 (several t); 26.28 (t); 25.71 (t); 22.82 (2t); 14.26 (q, 2 Me). HR-MALDI-MS:
592.5403 (100, [MþH]þ , C36H70N3Oþ3 ; calc. 592.5417). Anal. calc. for C36H69N3O3 (591.9514): C 73.04, H
11.75, N 7.10; found: C 73.00, H 11.64, N 6.97.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-[4-(hexadecyloxycarbonyl)-1H-1,2,3-triazol-1-yl]-6-O-undec-
yl-d-erythro-hex-4-enitol (32). A mixture of 24 (50 mg, 0.098 mmol) and 27 (145 mg, 0.49 mmol) was
treated with a soln. of CuSO4 (0.3 mg, 0.0019 mmol) in H2O (0.5 ml) and ascorbic acid (3.5 mg,
0.02 mmol) in H2O (0.5 ml). The mixture was stirred at 608 for 36 h, allowed to cool to 258, diluted with
H2O (2 ml), and extracted with AcOEt (2ꢂ25 ml). The combined org. layers were washed with NaHCO3
soln. and brine, dried (Na2SO4), and evaporated. FC (silica gel; Et2O/hexane 1:9!3 :7) to yield pure 32
(66 mg, 84%). White solid. M.p. 46.28. Rf (Et2O/hexane) 0.34. [a]2D5 ¼ ꢀ7.0 (c¼0.235, CHCl3). IR (ATR):
2922w, 2852s, 1741w, 1720w, 1542m, 1496w, 1454w, 1390w, 1366w, 1219m, 1196w, 1105w, 1036w, 974w, 772s,
735w, 697w. 1H-NMR (CDCl3, 300 MHz): 8.18 (s, C¼CHN); 7.35–7.28 (m, 6 arom. H); 7.22–7.17 (m, 4
arom. H); 5.71 (dt, J ¼ 15.6, 5.1, HꢀC(5)); 5.49 (br. dd, J¼15.6, 8.1, HꢀC(4)); 4.88 (td, Jꢃ6.6, 3.3,
HꢀC(2)); 4.69 (d, J¼11.7, PhCH); 4.50 (d, J¼12.0, PhCH); 4.44 (d, J¼12.0, PhCH); 4.37–4.27 (m,
PhCH, HꢀC(3), 2 HꢀC(1’’)); 4.11 (dd, J¼10.2, 6.3, 1 HꢀC(1)); 3.92 (dd, J¼10.2, 3.3, 1 HꢀC(1)); 3.86
(br. d, J¼4.8, 2 HꢀC(6)); 3.35–3.22 (AB, 2 HꢀC(1’)); 1.83–1.73 (m, 2 HꢀC(2’’)); 1.58–1.48 (m,
2 HꢀC(2’)); 1.39–1.21 (m, 42 H); 0.88 (t, Jꢃ6.5, 2 Me). 13C-NMR (CDCl3, 75 MHz): 160.86 (s, C¼O);
139.78 (s, C¼CHN); 137.17 (d, C(4)); 137.08 (2s); 134.35, 128.40 (5d); 127.87 (2d); 127.79 (d, C¼CHN);
127.63 (2d); 126.72 (d, C(5)); 78.05 (d, C(3)); 73.33 (t, PhCH2); 70.75 (t, C(6)); 70.49 (t, PhCH2); 69.87 (t,
C(1’)); 67.32 (t, C(1)); 65.25 (t, C(1’’)); 64.49 (d, C(2)); 31.82 (2t); 29.60–29.26 (several t); 28.59 (t); 26.08,
25.81 (2t), 22.56 (2t); 14.03 (2q, 2 Me). HR-MALDI-MS: 802.6078 (100, [MþH]þ , C50H80N3O5þ ; calc.
802.6098). Anal. calc. for C50H97N3O5 (802.1794): C 74.86, H 9.93, N 5.24; found: C 74.86, H 10.00, N 5.04.
(E)-2,4,5-trideoxy-2-[4-(hexadecyloxycarbonyl)-1H-1,2,3-triazol-1-yl]-6-O-undecyl-d-erythro-hex-
4-enitol (13). A soln. of 32 (46 mg, 0.057 mmol) in CH2Cl2 (2 ml) was treated with anisole (75 ml,
0.694 mmol) and AlCl3 (76.5 mg, 0.573 mmol), stirred at r.t. for 24 h, treated dropwise with 1m HCl
(0.1 ml), diluted with H2O (3 ml), and extracted with AcOEt (2ꢂ20 ml). The combined org. layers were
washed with NaHCO3 soln. and brine, dried (Na2SO4), and evaporated. FC (silica gel; AcOEt/hexane
3 :7!1:1) afforded pure 13 (32.3 mg, 91%). White solid. M.p. 66.58. Rf (AcOEt/hexane 1:1) 0.18.
[a]2D5 ¼ þ6.2 (c ¼ 0.59, CHCl3). IR (CHCl3): 3600w, 3396 (br.), 2928s, 2855m, 1721w, 1543w, 1466w,
1398w, 1363w, 1255w, 1162w, 1106w, 1042w, 973w. 1H-NMR (CDCl3, 300 MHz): 8.40 (s, C¼CHN); 5.92
(dtd, J¼15.6, 5.4, 0.9, HꢀC(5)); 5.77 (br. dd, J¼15.3, 5.7, HꢀC(4)); 4.79 (t, Jꢃ5.1, HꢀC(3)); 4.64 (td,
J¼5.1, 3.3, HꢀC(2)); 4.36 (dd, J ¼ 11.8, 5.2, 1 HꢀC(1)); 4.32 (t, J¼6.9, 2 HꢀC(1’’)); 4.13 (dd, J ¼ 12.0,
2.7, 1 HꢀC(1)); 3.93 (d, J¼5.1, 2 HꢀC(6)); 3.51–3.43 (br. s, HOꢀC(3)); 3.38 (t, J¼6.6, 2 HꢀC(1’));
3.19 (br. s, HOꢀC(1)); 1.81–1.71 (m, 2 HꢀC(2’’)); 1.60–1.50 (m, 2 HꢀC(2’)); 1.42–1.25 (m, 42 H); 0.87
(t, Jꢃ6.6, 2 Me). 13C-NMR (CDCl3, 75 MHz): 160.69 (s, C¼O); 139.50 (s, C¼CHN); 131.16 (d, C(4));
129.00 (d, C¼CHN); 128.39 (d, C(5)); 72.55 (d, C(3)); 70.85, 70.00 (2t, C(6), C(1’)); 65.83 (d, C(2));
65.45 (t, C(1’’)); 61.19 (t, C(1)); 31.81 (t, C(2’)); 29.59–29.52 (several t); 29.44 (2t); 29.25, 29.20 (2t); 28.54
(t); 26.04 (t); 25.78 (t); 22.59 (2t); 14.01 (q, 2 Me). HR-MALDI-MS: 622.5143 (100, [M þ H]þ
,
C36H68N3Oþ5 ; calc. 622.5159). Anal. calc. for C36H67N3O5 (621.36143): C 69.52, H 10.86, N 6.76; found: C
69.69, H 10.87, N 6.64.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-{4-[(hexadecylamino)carbonyl]-1H-1,2,3-triazol-1-yl}-6-O-
undecyl-d-erythro-hex-4-enitol (33). A mixture of 24 (85 mg, 0.167 mmol) and 28 (49.1 mg, 0.167 mmol)
in i-PrOH (2 ml) was treated with a soln. of CuSO4 (0.4 mg, 0.0025 mmol) in H2O (0.5 ml) and ascorbic
acid (6.0 mg, 0.33 mmol) in H2O (0.5 ml), and stirred at 608 for 24 h. The mixture was allowed to cool to
r.t. and extracted with AcOEt (2ꢂ50 ml). The combined org. layers were washed with brine, dried

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
(Na2SO4), and evaporated. FC (silica gel; Et2O/hexane 3 :7) yielded pure 33 (134 mg, 100%). White
solid. M.p. 93.58. Rf (Et2O/hexane 1:1) 0.23. [a]2D5 ¼ ꢀ8.3 (c ¼ 2.3, CHCl3). IR (CHCl3): 3621m, 3419w,
3013m, 2974s, 2928s, 2856s, 1663m, 1576m, 1508w, 1454m, 1391w, 1368w, 1240m, 1046s, 974w, 877m, 668w,
570w. 1H-NMR (CDCl3, 300 MHz): 8.19 (s, C¼CHN); 7.35–7.18 (m, 10 arom. H); 5.70 (dt, J ¼ 15.6, 5.4,
HꢀC(5)); 5.48 (ddt, J¼15.9, 8.1, 1.2, HꢀC(4)); 4.85 (td, Jꢃ6.9, 3.6, HꢀC(2)); 4.59 (d, J¼11.7, PhCH);
4.50 (d, J¼12.0, PhCH); 4.44 (d, J¼12.0, PhCH); 4.30 (d, J¼11.7, PhCH); 4.29 (t, J¼7.5, HꢀC(3)); 4.08
(dd, J¼10.2, 6.6, 1 HꢀC(1)); 3.90 (dd, J¼10.2, 3.6, 1 HꢀC(1)); 3.85 (dt, J¼5.4, 1.5, 2 HꢀC(6)); 3.47–
3.40 (m, 2 HꢀC(1’’)); 3.32–3.20 (AB, 2 HꢀC(1’)); 1.66–1.48 (m, 2 HꢀC(2’), 2 HꢀC(2’’)); 1.40–1.20 (m,
42 H); 0.88 (t, Jꢃ6.3, 2 Me). 13C-NMR (CDCl3, 75 MHz): 159.92 (s, C¼O); 143.05 (s, C¼CHN); 137.17,
137.09 (2s); 134.28 (d, C(4)); 128.40 (4d); 127.81 (4d); 127.64 (2d); 126.90 (d, C¼CHN); 125.56 (d, C(5));
78.08 (d, C(3)); 73.49 (t, PhCH2); 70.84 (t, C(6)); 70.53 (t, PhCH2); 70.03 (t, C(1’)); 67.57 (t, C(1)); 64.68
(d, C(2)); 31.26 (t, C(1’’)); 32.04 (2t); 29.82–29.47 (several t); 27.12, 26.31 (2t); 22.83 (2t), 14.03 (2q,
2 Me). HR-MALDI-MS: 801.6235 (100, [MþH]þ , C50H81N4O4þ ; calc. 801.6258). Anal. calc. for
C50H80N4O4 (801.1946): C 74.96, H 10.06, N 6.99; found: C 74.89, H 10.08, N 6.98.
(E)-2,4,5-Trideoxy-2-{4-[(hexadecylamino)carbonyl]-1H-1,2,3-triazol-1-yl}-6-O-undecyl-d-erythro-
hex-4-enitol (14). A soln. of 33 (65 mg, 0.081 mmol) in CH2Cl2 (3 ml) was treated with anisole (133 ml,
1.217 mmol) and AlCl3 (129.8 mg, 0.974 mmol), and stirred at r.t. for 24 h. The mixture was cooled to 08,
treated dropwise with 10% HCl (0.5 ml), diluted with H2O (3 ml), and extracted with AcOEt (5ꢂ
20 ml). The combined org. layers were dried (Na2SO4) and evaporated. FC (silica gel; AcOEt/hexane
1:1!AcOEt) afforded pure 14 (45 mg, 89%). White solid. M.p. 958. Rf (AcOEt) 0.43. [a]2D5 ¼ þ10.1 (c ¼
0.65, CHCl3). IR (CHCl3): 3691w, 3415w, 2928s, 2855m, 1661w, 1577w, 1509w, 1466w, 1368w, 1213w,
1
1107w, 1048w, 970w, 757s, 670w, 438m. H-NMR (CDCl3, 300 MHz): 8.55 (s, C¼CHN); 7.28 (t, J ¼ 5.7,
NH); 5.86 (dt, J¼15.3, 5.1, HꢀC(5)); 5.71 (br. dd, J¼15.6, 5.1, HꢀC(4)); 4.71–4.65 (m, HꢀC(2),
HꢀC(3)); 4.41 (d, J¼4.8, HOꢀC(3)); 4.35 (dt, J ¼ 12.0, 4.8, 1 HꢀC(1)); 4.17 (dd, J¼6.3, 4.8,
HOꢀC(1)); 4.03 (ddd, J ¼ 11.4, 6.0, 2.4, 1 HꢀC(1)); 3.91 (d, J¼5.4, 2 HꢀC(6)); 3.42–3.32 (m,
2 HꢀC(1’), 2 HꢀC(1’’)); 1.64–1.49 (m, 2 HꢀC(2’), 2 HꢀC(2’’)); 1.38–1.21 (m, 42 H); 0.87 (t, Jꢃ6.8,
2 Me). 13C-NMR (CDCl3, 75 MHz): 160.70 (s, C¼O); 142.31 (s, C¼CHN); 130.60 (d, C(4)); 129.57 (d,
C¼CHN); 126.24 (d, C(5)); 72.19 (d, C(3)); 70.68, 70.13 (2t, C(6), C(1’)); 66.02 (d, C(2)); 60.93 (t, C(1));
39.28 (t, C(1’’)); 31.82 (2t); 29.60–29.25 (several t); 26.86, 26.04 (2t); 22.59 (2t); 14.01 (q, 2 Me). HR-
MALDI-MS: 643.5122 (100, [M þ Na]þ , C36H68N4NaOþ4 ; calc. 643.5138). Anal. calc. for C36H68N4O4
(620.9495): C 69.63, H 11.04, N 9.02; found: C 69.53, H 11.04 , N 9.02.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-{4-[(hexadecylamino)thiocarbonyl]-1H-1,2,3-triazol-1-yl}-6-
O-undecyl-d-erythro-hex-4-enitol (36). A mixture of 33 (83.7 mg, 0.1045 mmol) and Lawessonꢂs reagent
(29.6 mg, 0.073 mmol) in toluene (2 ml) was stirred for 2 h at 758 and then evaporated. The residue after
FC (silica gel; Et2O/hexane 1:4) gave pure 36 (79 mg, 93%). Pale yellow crystals. M.p. 39.98. Rf (Et2O/
hexane 1:1) 0.50. [a]2D5 ¼ ꢀ3.1 (c ¼ 0.45, CHCl3). IR (CHCl3): 3610w, 3448 (br.), 3361w, 3164w, 3016m,
2928s, 2856m, 2401w, 1562w, 1512w, 1455w, 1405w, 1366w, 1221w, 1101w, 1045w, 972w, 731w, 668w.
1H-NMR (CDCl3, 300 MHz): 8.89 (t, Jꢃ5.6, NH); 8.37 (s, C¼CHN); 7.36–7.19 (m, 10 arom. H); 5.73 (dt,
J ¼ 15.6, 5.4, HꢀC(5)); 5.50 (ddt, J¼15.3, 8.1, 1.2, HꢀC(4)); 4.82 (td, J¼6.9, 3.9, HꢀC(2)); 4.59 (d, J¼
11.4, PhCH); 4.50 (d, J¼12.3, PhCH); 4.45 (d, J¼12.0, PhCH); 4.30 (d, J¼11.4, PhCH); 4.28 (t, J¼8.1,
HꢀC(3)); 4.07 (dd, J¼10.2, 6.6, 1 HꢀC(1)); 3.90 (dd, J¼10.5, 3.9, 1 HꢀC(1)); 3.88 (d, J¼3.0,
2 HꢀC(6)); 3.87–3.79 (m, 2 HꢀC(1’’)); 3.34–3.22 (AB, 2 HꢀC(1’)); 1.81–1.71 (m, 2 HꢀC(2’’)); 1.57–
1.48 (m, 2 HꢀC(2’)); 1.46–1.20 (m, 42 H); 0.88 (t, Jꢃ6.3, 2 Me). 13C-NMR (CDCl3, 75 MHz): 184.28 (s,
C¼S); 147.87 (s, C¼CHN); 137.09 (2s); 134.27 (d, C(4)); 128.41 (5d); 127.91 (2d); 127.84 (2d); 127.68 (d,
C¼CHN); 126.84 (d, C(5)); 77.95 (d, C(3)); 73.32 (t, PhCH2); 70.75 (t, C(6)); 70.42 (t, PhCH2); 69.91 (t,
C(1’)); 67.33 (t, C(1)); 64.68 (d, C(2)); 44.88 (t, C(1’’)); 31.82 (2t); 29.60–29.46 (several t); 29.26, 28.13
(2t); 26.99, 26.01 (2t); 22.59 (2t), 14.03 (q, 2 Me). HR-MALDI-MS: 817.6009 (100, [MþH]þ
,
C50H81N4O3Sþ ; calc. 817.6029). Anal. calc. for C50H80N4O3S (817.2602): C 73.48, H 9.87, N 6.86; found:
C 73.49, H 9.61, N 6.82.
(E)-2,4,5-Trideoxy-2-{4-[(hexadecylamino)thiocarbonyl]-1H-1,2,3-triazol-1-yl}-6-O-undecyl-d-er-
ythro-hex-4-enitol (15). A soln. of 36 (70 mg, 0.0857 mmol) in CH2Cl2 (6 ml) was treated with anisole
(75 ml, 0.685 mmol) and AlCl3 (114.2 mg, 0.857 mmol), and stirred at r.t. for 11 h. The mixture was cooled
to 08, treated dropwise with 10% HCl (1.1 ml), and extracted with AcOEt (5ꢂ30 ml). The combined org.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
721
layers were dried (Na2SO4) and evaporated. FC (silica gel; AcOEt/hexane 1:4!1:3) gave pure 15
(43.7 mg, 80%) as pale yellow amorphous solid, which solidified after keeping at ꢀ208 for 2 d. M.p. 45.48.
Rf (AcOEt/hexane 1:4) 0.35. [a]2D5 ¼ þ8.9 (c ¼ 0.65, CHCl3). IR (CHCl3): 3609w, 3362w, 3164w, 3015w,
2928s, 2856m, 1562w, 1511w, 1456w, 1406w, 1368w, 1306w, 1217w, 1111w, 1077w, 1045w, 973w, 748s, 668.
1H-NMR (CDCl3, 300 MHz): 8.93 (t, J ¼ 5.7, NH); 8.48 (s, C¼CHN); 5.84 (dtd, J¼15.6, 5.4, 0.9,
HꢀC(5)); 5.69 (br. dd, J¼15.6, 6.0, HꢀC(4)); 4.69 (t, Jꢃ5.6, HꢀC(3)); 4.60 (td, J¼5.7, 3.6, HꢀC(2));
4.27 (dd, J ¼ 12.3, 6.0, 1 HꢀC(1)); 4.08 (dd, J ¼ 12.0, 3.9, 1 HꢀC(1)); 3.90 (d, J¼5.4, 2 HꢀC(6)); 3.80
(dd, J¼13.2, 7.2, 2 HꢀC(1’’)); 3.35 (t, J ¼ 6.6, 2 HꢀC(1’), HOꢀC(3)); 3.11 (br. s, HOꢀC(1)); 1.79–1.69
(m, 2 HꢀC(2’’)); 1.58–1.48 (m, 2 HꢀC(2’)); 1.42–1.25 (m, 42 H); 0.87 (t, Jꢃ6.6, 2 Me). 13C-NMR
(CDCl3, 75 MHz): 183.88 (s, C¼S); 147.72 (s, C¼CHN); 131.21 (d, C(4)); 129.28 (d, C¼CHN); 128.27 (d,
C(5)); 72.32 (d, C(3)); 70.99, 70.14 (2t, C(6), C(1’)); 66.30 (d, C(2)); 61.26 (t, C(1)); 45.21 (t, C(1’’)); 32.03
(2t); 29.81–29.77 (several t); 29.65 (2t); 29.47 (2t); 28.28, 27.17(2t); 26.24 (t); 22.82 (2t); 14.27 (q, 2 Me).
HR-MALDI-MS: 659.4892 (100, [M þ Na]þ , C36H68N4NaO3Sþ ; calc. 659.4910). Anal. calc. for
C36H68N4O3S (637.0151): C 67.88, H 10.76, N 8.80; found: C 67.66, H 10.63, N 8.60.
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-[4-(trimethylsilyl)-1H-1,2,3-triazol-1-yl]-6-O-undecyl-d-eryth-
ro-hex-4-enitol (34). A mixture of 24 (86 mg, 0.17 mmol) and 29 (1.2 ml, 8.47 mmol) in H2O/i-PrOH 1:2
(0.5 ml) was treated with a soln. of CuSO4 (0.5 mg, 0.0032 mmol) in H2O (0.25 ml) and ascorbic acid
(9 mg, 0.051 mmol) in H2O (0.25 ml), and stirred at 558 for 2 d. The mixture was allowed to cool to 258,
diluted with H2O (5 ml), and extracted with AcOEt (2ꢂ50 ml). The org. phases were dried (Na2SO4)
and evaporated. The residue was filtered through a short pad of silica gel (first using AcOEt/hexane 1:9
as eluent to separate the unreacted 24 (30 mg) and then using AcOEt/hexane 1:4). The fractions that
contain 34 were concentrated to yield pure 34 (50 mg, 75%, based on recovery of 24), which was taken to
the next step without further purification. Colourless oil. Rf (AcOEt/hexane 1:4) 0.22. IR (CHCl3):
3692w, 3068w, 3031w, 3010w, 2958m, 2928s, 2856s, 1603w, 1495w, 1484w, 1455w, 1364w, 1258w, 1171w,
1102s, 1028w, 1002w, 974w, 845s, 701w, 432w. 1H-NMR (CDCl3, 300 MHz): 7.63 (s, C¼CHN); 7.35–7.17
(m, 10 arom. H); 5.69 (dt, J ¼ 15.3, 5.1, HꢀC(5)); 5.50 (ddt, J¼15.6, 8.1, 1.2, HꢀC(4)); 4.90 (td, Jꢃ6.9,
4.2, HꢀC(2)); 4.60 (d, J¼11.7, PhCH); 4.49 (d, J¼12.0, PhCH); 4.44 (d, J¼12.0, PhCH); 4.32 (t, J¼8.7,
HꢀC(3)); 4.30 (d, J¼11.7, PhCH); 4.10 (dd, J¼10.2, 6.9, 1 HꢀC(1)); 3.96 (dd, J¼10.2, 4.2, 1 HꢀC(1));
3.85 (dd, J¼5.7, 1.2, 2 HꢀC(6)); 3.32–3.24 (AB, 2 HꢀC(1’)); 1.58–1.49 (m, 2 HꢀC(2’)); 1.34–1.21 (m,
16 H); 0.88 (t, Jꢃ6.8, Me); 0.31 (s, Me3Si). 13C-NMR (CDCl3, 75 MHz): 145.75 (s, C¼CHN); 137.57,
137.48 (2s); 133.51 (d, C(4)); 129.46 (d, C¼CHN); 128.31 (4d); 127.67 (2d); 127.57 (4d); 127.36 (d, C(5));
78.63 (d, C(3)); 73.15 (t, PhCH2); 70.70 (t, C(6)); 70.40 (t, PhCH2); 70.04 (t, C(1’)); 67.91 (t, C(1)); 63.52
(d, C(2)); 31.81 (t); 29.62–29.52 (several t); 29.44, 26.33 (2t), 26.11, 22.59 (2t); 14.03 (q, Me); ꢀ1.16 (q, 3
Me). HR-MALDI-MS: 606.4075 (100, [MþNa]þ , C36H56N3O3Siþ ; calc. 606.4091).
(E)-1,3-Di-O-benzyl-2,4,5-trideoxy-2-(1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-hex-4-enitol
(37). A soln. of 34 (46.5 mg, 0.077 mmol) in THF (2 ml) was treated with Bu4NF·3 H2O (48.3 mg,
0.153 mmol) and AcOH (10 ml), and stirred at 258 for 36 h. The mixture was diluted with AcOEt (50 ml)
and washed with H2O (5 ml). The aq. layer was separated and extracted with AcOEt (2ꢂ25 ml). The
combined org. layers were washed with sat. aq. NaHCO3 soln. and brine, dried (Na2SO4), and
evaporated. The residue was filtered through a short pad of silica gel using AcOEt/hexane 1:1 as eluent
to get crude 37 (39.5 mg, 97%), which was used directly without further purification. Colourless oil. Rf
(AcOEt/hexane 3 :7) 0.29. 1H-NMR (CDCl3, 300 MHz): 7.69 (d, J¼0.9, HC¼CHNC(2)); 7.65 (d, J¼0.9,
HC¼CHNC(2)); 7.36–7.27 (m, 6 arom. H); 7.25–7.18 (m, 4 arom. H); 5.68 (dt, J ¼ 15.6, 5.1, HꢀC(5));
5.48 (ddt, J¼15.6, 7.8, 1.2, HꢀC(4)); 4.88 (td, Jꢃ6.6, 3.9, HꢀC(2)); 4.61 (d, J¼12.0, PhCH); 4.50 (d, J¼
12.0, PhCH); 4.45 (d, J¼12.0, PhCH); 4.34 (t, Jꢃ7.7, H ꢀC(3)); 4.32 (d, J¼11.4, PhCH); 4.13 (dd, J¼
10.2, 6.3, 1 HꢀC(1)); 3.95 (dd, J¼10.2, 3.6, 1 HꢀC(1)); 3.85 (dt, J¼5.1, 1.8, 2 HꢀC(6)); 3.33–3.21 (AB,
2 HꢀC(1’)); 1.58–1.48 (m, 2 HꢀC(2’)); 1.33–1.26 (m, 16 H); 0.88 (t, Jꢃ6.8, Me). 13C-NMR (CDCl3,
75 MHz): 137.48, 137.37 (2s); 133.88 (d, HC¼CHNC); 133.37 (d, HC¼CHNC); 128.37 (4d); 127.76 (2d);
127.70 (2d); 127.58 (2d); 127.24 (d, C(4)); 123.94 (d, C(5)); 78.46 (d, C(3)); 73.39 (t, PhCH2); 70.82 (t,
C(6)); 70.51 (t, PhCH2); 70.12 (t, C(1’)); 68.01 (t, C(1)); 64.11 (d, C(2)); 32.03 (t, C(2’)); 29.82 (2t); 29.75
(2t); 29.65, 29.46 (2t); 26.31, 22.83 (2t); 14.29 (q, Me). HR-MALDI-MS: 534.3681 (100, [MþH]þ
,
C33H48N3Oþ3 ; calc. 534.3696).

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
(E)-2,4,5-Trideoxy-2-(1H-1,2,3-triazol-1-yl)-6-O-undecyl-d-erythro-hex-4-enitol (16). A soln. of 37
(39 mg, 0.073 mmol) in CH2Cl2 (3 ml) was treated with anisole (63.7 mg, 0.59 mmol) and AlCl3 (97.4 mg,
0.731 mmol), and stirred at 258 for 24 h. The mixture was cooled to 08 and treated dropwise with 1m HCl
(0.4 ml). The mixture was diluted with AcOEt (50 ml). The aq. layer was separated and extracted with
AcOEt (2ꢂ25 ml). The combined org. layers were washed with brine, dried (Na2SO4), and evaporated.
FC (silica gel; AcOEt/hexane 1:1!AcOEt) gave pure 16 (18 mg, 70%). Colourless oil that solidified
upon standing at 58. M.p. 47.68. Rf (AcOEt) 0.25. [a]2D5 ¼ þ12.0 (c¼0.45, CHCl3). IR (CHCl3): 3609w,
3409 (br.), 3014w, 2929s, 2856m, 2397w, 1456w, 1413w, 1366w, 1278w, 1113w, 1073m, 973w, 750m, 669w.
1H-NMR (CDCl3, 300 MHz): 7.82 (d, J¼0.9, HC¼CHNC(2)); 7.64 (d, J¼0.9, HC¼CHNC(1)); 5.83
((dtd, J¼15.6, 5.1, 0.9, HꢀC(5)); 5.68 (ddt, J¼15.6, 5.7, 1.2, HꢀC(4)); 4.74 (br. dd, J¼10.2, 5.1,
HꢀC(3)); 4.57 (td, J ¼ 5.4, 3.6, HꢀC(2)); 4.31 (td, J¼12.0, 5.7, 1 HꢀC(1)); 4.12 (ddd, J ¼ 12.3, 6.0, 3.6,
1 HꢀC(1)); 3.90 (d, J¼4.8, 2 HꢀC(6)); 3.70 (d, J¼4.2, HOꢀC(3)); 3.44 (t, Jꢃ6.2, HOꢀC(1)); 3.35 (t,
J¼6.9, 2 HꢀC(1’)); 1.58–1.49 (m, 2 HꢀC(2’)); 1.32–1.26 (m, 16 H); 0.87 (t, Jꢃ6.8, 2 Me). 13C-NMR
(CDCl3, 75 MHz): 133.16 (d, HC¼CHNC(2)); 130.74 (d, HC¼CHNC(2)); 129.50 (d, C(4)); 124.59 (d,
C(5)); 72.47 (d, C(3)); 70.90, 70.21 (2t, C(6), C(1’)); 65.92 (d, C(2)); 61.60 (t, C(1)); 32.02 (t, C(2’)); 29.74
(4t); 26.62, 29.46 (2t); 26.25, 22.81 (2t); 14.27 (q, Me). HR-MALDI-MS: 354.2746 (100, [MþH]þ
,
C19H36N3Oþ3 ; calc. 354.2757). Anal. calc. for C36H69N3O3 (353.4995): C 64.56, H 9.98, N 11.89; found: C
64.31, H 10.27, N 11.64.
2. Biological Tests. Materials and Methods. T-Cell Clones and APC. Human CD1d-restricted iNKT-
cell clones were derived and cultivated as described in [55]. Human CD1d-transfected C1R or THP1
cells were used as APC in the experiments.
Abbreviations. a-GalCer, a-galactosylceramide; APC, antigen-presenting cells; BSA, bovine serum
albumin; FACS, fluorescence-activated cell sorting; FCS, fetal calf serum; IEF, isoelectric focusing;
iNKT cells, invariant natural killer T cells; PBMC, peripheral blood mononuclear cells; PBS, phosphate-
buffered saline; TCR, T-cell receptor.
Cytotoxicity Assay. Sonicated compounds were incubated overnight with T cells or APC at 378. Cells
were labelled with 5 mg/ml propidium iodide (Sigma-Aldrich, St. Louis, Missouri, USA) or 7-
aminoactinomycin D (Invitrogen, Carlsbad, California, USA), and cell death was assessed by flow
cytometry on a CYANTM ADP (Beckman Coulter, Fullerton, California, USA) cytometer.
Antigen Presentation Assay. Human CD1d transfectants (2.5·104/well) in RPMI-1640 medium
containing 10% FCS were incubated during the assay at 378 with sonicated compounds at the indicated
concentrations. T Cells (7.5·104/well) were added after indicated time periods. Supernatants were
harvested after 24 h, and released cytokines were measured by ELISA.
Antigen Competition Assay. Human CD1d transfectants (2.5·104/well) in RPMI-1640 medium
containing 10% FCS were incubated during the assay at 378 with sonicated antigen at the indicated
concentrations. Compounds were given 4.5 h before a-GalCer. T Cells (7.5·104/well) were added 0.5 h
after a-GalCer. Supernatants were harvested after 24 h, and released cytokines were measured by
ELISA.
Plate-Bound Human CD1d Activation Assay. MaxiSorpTM ELISA plates (Nunc, Roskilde, Denmark)
were coated overnight at r.t. with 10 mg/ml BIR1.4 (anti-BirA, generated in our laboratory) monoclonal
antibody (mAb). IEF-Purified soluble recombinant human CD1d (established in our laboratory with a
BirA tag) was incubated overnight at r.t. on washed BIR1.4-coated plates at pre-titrated batch-
dependent concentrations (twofold molar excess). Sonicated compounds were added after washing to
plate-bound human CD1d overnight at r.t. T Cells (1.5·105/well) were plated in RPMI-1640 medium
containing 5% HS and 100 U/ml human IL-2 after extensive washing. Supernatants were harvested after
24 h incubation at 378, and released cytokines were measured by ELISA.
Plate-Bound Human CD1d Competition Assay. MaxiSorpTM ELISA plates were coated overnight at
r.t. with 10 mg/ml BIR1.4 mAb. IEF-Purified soluble recombinant human CD1d was incubated overnight
at r.t. on washed BIR1.4-coated plates at pre-titrated batch-dependent concentrations (twofold molar
excess). Sonicated compounds were added after washing and given 4.5 h in advance of a-GalCer to plate-
bound human CD1d before overnight competition at r.t. T Cells (1.5·105/well) were plated in RPMI-1640
medium containing 5% HS and 100 U/ml human IL-2 after extensive washing. Supernatants were
harvested after 24 h incubation at 378, and released cytokines were measured by ELISA.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
723
ELISA. MaxiSorpTM ELISA plates were coated overnight at 48 with 1 mg/ml 8D4-8 (anti-human IL-4;
BD, Franklin Lakes, New Jersey, USA) mAb, with 1 mg/ml MAb1 (anti-human TNF-a; BD) mAb, with
2 mg/ml 6804 (anti-human GM-CSF, R&D, Minneapolis, Minneapolis, USA) mAb, or with 3 mg/ml HB-
8700 (anti-human IFN-g, ATCC) mAb diluted in PBS.
Coated wells were blocked with 0.05% Tween-20 and 10 mg/ml BSA in PBS (PBST/BSA) and then
incubated with the supernatants of the antigen presentation assays.
1 mg/ml MP4-25D2 (anti-human IL-4 biotin labelled; BD) mAb, 0.5 mg/ml MAb11 (anti-human
TNF-a biotin labelled, BD) mAb, 1 mg/ml 3209 (anti-human GM-CSF biotin labelled, R&D) mAb, or
0.72 mg/ml g69 [56] (anti-human IFN-g biotin labeled, provided by G. Garotta) mAb with 0.3 mg/ml
streptavidin-HRP (Zymed, Invitrogen), and o-Phenylenediamine dihydrochloride (Sigma, according to
the manufacturerꢂs instructions) as substrate were used to detect human IL-4, human TNF-a, or human
IFN-g, resp.
The O.D.s of the developed ELISA were read in a SpectraMaxꢄ 190 spectrophotometer at 490 nm
(Molecular Devices, Sunnyvale, California, USA), then converted to concentrations and expressed as
mean pg/mlꢁSD of duplicates or triplicates using the SoftMax Pro 5 program by comparison to
standards of human IL-4 (produced in our laboratory), recombinant human TNF-a (Immunokontact,
AMS Biotechnology, Switzerland), human GM-CSF (produced in our laboratory), and recombinant
human IFN-g (BenderMedSystems, Vienna, Austria).
Data and Statistical Analysis. Data are at least of two independent experiments and expressed as
meanꢁstandard deviation (SD). Single data points were compared using two-tailed Studentꢂs test.
Results in figures are flagged with a single asterisk (*) when the P value is less than 0.05, with two
asterisks (**) when the P value is less than 0.01.
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Received December 31, 2008