Synthesis of 7-aza- and 7-thiasphingosines, and evaluation of their interaction with sphingosine kinases and with T-cells

Article abstract of DOI:10.1002/cbdv.200900039

The synthesis of 7-oxasphingosine (3) and 7-oxaceramide (4) was improved by starting from the 4-methoxybenzyl-protected d-galactal 9. The sphingosine analogues 5-7 and 24 were synthesized via the azido alcohol 13. The 7-thiasphingosine 5 is a poorer subst

Full text of DOI:10.1002/cbdv.200900039

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
725
Synthesis of 7-Aza- and 7-Thiasphingosines, and Evaluation of Their
Interaction with Sphingosine Kinases and with T-Cells
by Thresen Mathewa), Ce´lia Billauda), Andreas Billichb), Marco Cavallaric), 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 synthesis of 7-oxasphingosine (3) and 7-oxaceramide (4) was improved by starting from the 4-
methoxybenzyl-protected d-galactal 9. The sphingosine analogues 5–7 and 24 were synthesized via the
azido alcohol 13. The 7-thiasphingosine 5 is a poorer substrate for both isoforms of sphingosine kinase
(SPHK) than sphingosine, but showed a slight preference for SPHK2. The sulfone 6 and the 7-aza
compounds 7 and 24 were not phosphorylated by either SPHK1 or SPHK2, and none of 5–7 and 24
activated invariant natural killer T (iNKT) cell clones when presented by human CD1d-transfected
antigen-presenting cells (APC) or by plate-bound human CD1d. Only 7 and 24 associated with plate-
bound recombinant CD1d prevented stimulation of iNKT cells by a-galactosylceramide (a-GalCer).
Introduction. – Sphingolipids are structural components of the cell and exert
multiple functions in cell signalling, including the role of d-erythro-sphingosine (1) and
ceramide (2) in the regulation of apoptosis [1–7], and the role of sphingosine-1-
phosphate and ceramide in immune responses [8–13]. These functions are thought to
depend on specific interactions between receptors, and both the lipid tail (lipid tails for
ceramide) and the polar head group of sphingosine and ceramide [14]. We began
exploring these interactions by synthesizing and evaluating analogues, and so far
reported on modifications of the polar head group and on the replacement of the
C(7)H2 group by an O-atom, as in 3 and 4, a replacement that did not appear to affect
the tested biological properties [15]. This may be different for analogues where the
C(7)H2 group is replaced by bulkier, more polar, or charged groups, and we considered
the 7-thia and 7-aza analogues 5–7 of interest.
During the synthesis of 7-oxasphingosines we protected the OH groups by
benzylation, deprotecting them by cleaving the benzyl ether moieties with AlCl3 in the
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢀrich

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
presence of anisole [16]. For some analogues, these conditions led to cleavage of the
allylic CꢀO bond, substantially lowering the overall yield, and suggesting to replace the
benzyl (Bn) by 4-methoxybenzyl (PMB) groups that are cleaved under milder
conditions, and to otherwise follow the established synthetic route [15]; the expected
improvement should facilitate the synthesis of the envisaged analogues 5–7. We now
describe the modified synthesis of 7-oxasphingosine and 7-oxaceramide, the synthesis
of 7-thia- and 7-azasphingosines, and the evaluation of these analogues as substrates of
sphingosine kinases (SPHK), as CD1d ligands, and as activators of iNKT cells.
Synthesis. – To improve the synthesis of 7-oxasphingosine and 7-oxaceramide, we
started from PMB-protected d-galactal 9 [17–20] rather than from the benzylated
analogue that we used in the past. Galactal 9 was obtained in 90% yield from d-galactal
81), itself available in an overall yield of 75% from galactose [21][22] (Scheme 1).
Scheme 1
a) NaH, 4-methoxybenzyl (PMB) chloride, Bu4NI, DMF; 90%. b) HgSO4, 0.02n H2SO4, 1,4-dioxane;
92%. c) MsCl, pyridine, CH2Cl2; 96%. d) NaBH4, MeOH/CH2Cl2 26 :4; 93%. e) NaN3, DMF, 1408; 89%.
f) NaH, C11H23Br, DMF; 78%. g) LiAlH4, Et2O; 99%. h) CF3COOH (TFA)/CH2Cl2 1:9; 74% of 3; 75%
of 4. i) C17H35COCl, pyridine, CH2Cl2; 95%.
1
)
Prepared similarly as the d-glucal analogue (see [17][18]).

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
727
Treatment of 9 with HgSO4 and dilute H2SO4 [23][24] provided the a,b-unsaturated
aldehyde 10 (Scheme 1). Mesylation of 10 to 11, followed by reduction with NaBH4,
yielded the allylic alcohol 12 in 93% yield. Substitution of the MsO group of 12 with
NaN3 yielded 89% of the azido alcohol 13 that was O-alkylated with 1-bromoundecane
to the azido ether 14 (78%). Reduction of 14 with LiAlH4 yielded the PMB-protected
amino alcohol 15 (99%) that was debenzylated by treatment with 10% CF3COOH
(TFA) in CH2Cl2 to afford the 7-oxasphingosine 3 (74%), while N-acylation of 15
followed by deprotection provided the 7-oxaceramide 4 in 71% yield. As expected,
exchanging the Bn for PMB groups improved the overall yield of 4 from 8, from 22 to
36%.
To synthesize the 7-thia analogue 5 and the corresponding sulfone 6, we
transformed the allylic alcohol 13 into the methanesulfonate 17 (Scheme 2). The
methanesulfonate was obtained in a high yield, and reacted smoothly with the in situ
generated thiolate of 1-sulfanylundecane to yield 87% of the protected 7-thio ether 18.
Scheme 2
i
a) Ms2O, Pr2NEt, CH2Cl2; 99%. b) C11H23SH, NaH, DMF; 87%. c) TFA/CH2Cl2 1:9. d) PMe3, THF/
H2O 4 :1; 83.5% of 5; 84% of 6. e) Oxone, MeOH/CH2Cl2 1:2; 78%.
Cleavage of the PMB group of 18 by treatment with CF3COOH/CH2Cl2 and
Staudinger reduction [25] of the azide 19 gave the 7-thiasphingosine 5 (84% yield from
18). Oxidation of the thio ether 18 with oxone led to the protected sulfone 20 (78%),
and debenzylation of 20 (CF3COOH/CH2Cl2), followed by a Staudinger reduction,
provided the crystalline sulfone 6 in 84% yield. The structure of 6 was established by X-
ray crystal structure analysis (Fig. 1)2).
The 7-azasphingosine 7 was similarly obtained from the methanesulfonate 17 via
the corresponding in situ generated bromide that was treated with N-Boc-protected
undecylamine to yield the protected 7-azasphingosine 22 (Scheme 3). Staudinger
2
)
The crystallographic data have been deposited with the Cambridge Crystallographic Data Centre
(CCDC) as deposition No. CCDC-716357. Copies of the data can be obtained free of charge via
http://www.ccdc.cam.ac.uk/data_request/cif (or from the Cambridge Crystallographic Data Centre,
12 Union Road, Cambridge CB21EZ; fax: þ44(1223)336033; e-mail: deposit@ccdc.cam.ac.uk).

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 1. X-Ray structure of the sulfone 6
reduction of 22 afforded the partially protected amine 23. To remove the PMB and the
Boc groups, we treated 23 with 10% TFA in CH2Cl2 to obtain the 7-trifluoroacetamide
24 (60% from 22), as evidenced by a 19F-NMR signal at ꢀ75.5 ppm, two 13C qs at 157.58
(2J(C,F)¼36.7 Hz) and 116.3 ppm (1J(C,F)¼286.0 Hz), and the HR-ESI-MS peak at
m/z 397.2650. The trifluoroacetamide 24 was hydrolyzed by treatment with Amberlyst
A-26 (OHꢀ form) [26] to provide the 7-azasphingosine 7 in 95% yield.
Scheme 3
a) LiBr, THF; BocNHC11H23, NaH, DMF; 70%. b) PMe3, THF/H2O 4 :1. c) TFA/CH2Cl2 1:9; 60% from
22. d) Amberlyst A-26 (OHꢀ form), MeOH; 95%.
1
The H-NMR chemical shifts and coupling constants for sphingosine (1) and the
analogues 3, 5, 7, and 24 in CDCl3, and for 6 in CD3OD are listed in Table 1. The d-
erythro-configuration of 5–7 and 24 is evidenced by J(2,3)¼3.6–6.0 Hz, in agreement
with J(2,3) for 1 and 3. HOꢀC(1) and HOꢀC(3) of 1, 3, 5, and 7 resonate at similar
fields, but those of 24 are shifted downfield, in agreement with the acceptor effect of the
trifluoroacetamido group.
Biological Results. – Phosporylation by Sphingosine Kinase (SPHK). The relative
rates for phosphorylation of the sphingosine derivatives by SPHK1 and SPHK2, as
determined in comparison to sphingosine (1) [27] are shown in Table 2. While
sphingosine is phosphorylated with similar efficiency by both SPHK isoforms [28], the
7-oxasphingosine was phosphorylated faster by SPHK1 than by SPHK2. 7-Oxasphin-
gosine (3) was phosphorylated by SPHK1 with a similar rate as sphingosine. The 7-
thiasphingosine (5) proved a poorer substrate than sphingosine for both isoforms, but
showed a slight preference for SPHK2. Both the sulfone 6 analogue, and the 7-aza

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
729
Table 1. Selected 1H-NMR Chemical Shifts [ppm] and Coupling Constants [Hz] of d-erythro-
Sphingosine (1) and Its Analogues 3, 5–7, and 24 (in CDCl3)
1 [27]
3 [15]
5
6a)
7
24
4.04
HaꢀC(1)
HbꢀC(1)
HꢀC(2)
HꢀC(3)
HꢀC(4)
HꢀC(5)
CH2(6)
HOꢀC(3)
HOꢀC(1)
J(1a,2)
3.67
3.60
2.87
4.04
5.46
5.75
2.04
1.95
1.95
5.9
3.71–3.61
3.71–3.61
2.90
4.14
5.76
3.64
3.64
2.83
4.11
5.58
5.77
3.14
2.52
2.52
4.8
3.66
3.70–3.61
3.70–3.61
2.89
4.11
5.68
5.87
3.27
1.88
1.88
3.52
2.81
4.13
5.98
5.81
3.92–3.78
–
–
4.8
6.3
3.67
3.92
4.38
5.71
5.93
3.25
3.25
3.25
2.3
5.89
3.98
2.25–1.82
2.25–1.82
5.1
5.3
5.3
b
J(1b,2)
4.5
)
4.8
3.3
J(2,3)
J(3,4)
5.1
6.9
5.1
6.3
5.7
6.6
5.1
6.3
6.0
6.7
3.6
5.2
J(4,5)
J(5,6)
15.5
7.0
15.6
5.1
15.3
7.2
15.6
7.2
15.6
6.0
15.5
5.8
a) Recorded in CD3OD. b) Not assigned.
Table 2. Rate of Phosphorylation of d-erythro-Sphingosine (1) and Its Analogues 3, 5–7, and 24 by
SPHK1 and SPHK2 (values relative to the rate for 1)
Compound
SPHK1
SPHK2
1
3
1
0.86
1
0.081
5
0.12
0.18
6
7
24
not detectable
not detectable
not detectable
not detectable
not detectable
not detectable
compounds 7 and 24 were not phosphorylated by either SPHK1 or SPHK2. The results
clearly show that the electronic environment and/or nature of the groups near C(7) of
sphingosine do have an effect on the biological activity.
CD1d Binding. Compounds 5–7 and 24 did neither activate iNKT-cell clones when
presented by human CD1d-transfected antigen-presenting cells (APC) nor by plate-
bound human CD1d (data not shown). Compounds 5, 7, and 24 were cytotoxic for APC
and iNKT cells above 5 mg/ml as assessed by flow cytometry (data not shown), and,
therefore, cytotoxicity could mask their stimulatory capacity. Next, binding to CD1d
was evaluated in cell-free competition assays. When T cells were stimulated with plate-
bound recombinant CD1d and a-GalCer, only the 7-azasphingosines 7 and 24 were
partially inhibitory at a 20-fold molar excess (Fig. 2).
The large molar excess needed to compete with a-GalCer could be attributed to the
fact that these lipids have only one tail, and thus bind weakly to CD1d, whereas a-
GalCer binds with high affinity because it has two lipid tails. The compounds were also
tested for inhibition of the response to a-GalCer using living APC. This is a more

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
Fig. 2. The 7-azasphingosines 7 and 24 are able to compete with a-GalCer for plate-bound human CD1d.
Weak reduction of both a) human IL-4 and b) GM-CSF release is observed.
sensitive assay than the one using plate-bound CD1d, but could be affected by other
types of lipid influence on APC. All compounds were carefully applied at nontoxic
doses. As observed with plate-bound assays, also with living APC, compounds 7 and 24
slightly inhibited T-cell response (Fig. 3). This was observed with all three tested
human cytokines (IL-4, IFN-g, and TNF-a).
Fig. 3. The 7-azasphingosines 7 and 24 are able to compete with a-GalCer for human CD1d on living
antigen-presenting cells (APC). Reduction of iNKT activation is seen by release of a) human IL-4, b)
IFN-g, and c) TNF-a. Compounds 7 and 24 affect potency and/or efficacy of a-GalCer differently
*
^
^
regarding the cytokine measured ( ¼no competitor, ¼24, ¼7).
In conclusion, both 7-azasphingosines 7 and 24 do not stimulate iNKT cells but bind
to human CD1d, and might be used to influence iNKT-cell responses.
We thank Novartis AG, Basel, for a fellowship, the ETH-Zꢀrich for financial support, Dr. Bruno
Bernet for checking the analytical data, and Dr. W. Bernd Schweizer for the X-ray analysis.

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
731
Experimental Part
1. Synthesis. – General. THF was distilled from Na and benzophenone, CH2Cl2 from P2O5, and
MeOH from CaH2. Reactions were carried out under N2, unless stated otherwise. Qual. TLC: precoated
silica-gel plates (Merck silica gel 60 F254); detection by heating with ꢂmostainꢃ (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). M.p.: uncorrected. Optical rotations: 1-dm cell at 258, 589 nm. FT-IR Spectra: ATR or
1
ca. 2% soln. in CHCl3, ˜n in cmꢀ1. H- and 13C-NMR spectra: chemical shifts d in ppm rel. to TMS as
external standard and coupling constants J in Hz. HR-MALDI-MS: in gentisic acid (¼2,5-
dihydroxybenzoic acid, DHB) matrix.
3,4,6-Tris-O-(4-methoxybenzyl)-d-galactal (9) [18]. A soln. of 8 (1.526 g, 10.44 mmol) in DMF
(20 ml) was cooled to 08 and treated with NaH (60% in oil; 2.08 g, 52.21 mmol) in small portions. After
the evolution of gas had ceased (90 min), the mixture was treated with 4-methoxybenzyl (PMB) chloride
(7.08 ml, 52.21 mmol) and Bu4NI (200 mg, 5.22 mmol), and stirred at r.t. for 14 h. After dilution with
AcOEt and H2O, the layers were separated. The org. phase was washed with brine (2ꢁ30 ml), and the
aq. phase was extracted with AcOEt (2ꢁ30 ml). The combined org. phases were dried (Na2SO4) and
evaporated. FC (hexane/AcOEt 7:3) gave 9 (4.88 g, 92%). Pale yellow syrup. Rf (hexane/AcOEt 7:3)
0.42. [a]2D5 ¼ ꢀ43.2 (c ¼ 2.0, CHCl3). IR (ATR): 2921w, 2853w, 1643w, 1611m, 1585w, 1511s, 1463m,
1442w, 1301m, 1243s, 1171m, 1084s, 1056m, 1030s, 909w, 816s, 730m. 1H-NMR (300 MHz, CDCl3): 7.27–
7.20 (m, 6 arom. H); 6.89–6.83 (m, 6 arom. H); 6.34 (dd, J¼6.0, 1.5, HꢀC(1)); 4.83–4.81 (m, HꢀC(2));
4.79 (d, J¼11.1), 4.57 (d, J¼11.7) (ArCH2); 4.56 (s, ArCH2); 4.43, 4,33 (2d, J¼11.4, ArCH2); 4.16–4.12
(m, HꢀC(4), HꢀC(5)); 3.90–3.87 (m, HꢀC(3)); 3.81, 3.80, 3.79 (3s, 3 MeO); 3.71 (dd, J¼9.9, 2.7,
HaꢀC(6)); 3.56 (dd, J¼10.2, 5.1, HbꢀC(6)). 13C-NMR (75 MHz, CDCl3): 159.05 (2s); 158.94 (s); 143.96
(d, C(1)); 130.51, 130.44, 129.95 (3s); 129.70, 129.43, 128.92 (6d); 113.66 (4d); 113.58 (2d); 100.10 (d,
C(2)); 75.73 (d, C(4)); 73.03 (d, C(3)); 72.88 (t, 3 ArCH2); 70.51 (d, C(5)); 68.17 (t, C(6)); 55.25 (q, 3
MeO). HR-MALDI-MS: 529.2188 (100, [MþNa]þ , C30H34NaO7þ ; calc. 529.2197). Anal. calc. for
C30H34O7 (506.59): C 71.13, H 6.76; found: C 70.95, H 6.79.
(E)-2,3-Dideoxy-4,6-bis-O-(4-methoxybenzyl)-d-threo-hex-2-enose (10).
A soln. of 9 (2 g,
3.947 mmol) in 1,4-dioxane (40 ml) was treated with 0.02n H2SO4 (8 ml) and HgSO4 (12 mg,
0.039 mmol), and stirred at 258 for 23 h. The mixture was neutralised by the addition of sat. aq.
NaHCO3 soln. (20 ml). After extraction with AcOEt (2ꢁ20 ml), the org. phase was dried (Na2SO4) and
evaporated. FC (pentane/AcOEt 7:3) gave 10 (> 92%). Colourless oil. Rf (hexane/AcOEt 7:3) 0.14. IR
(ATR): 3452 (br.), 2910w, 2863w, 2836w, 1686m, 1611m, 1585w, 1511s, 1463w, 1442w, 1421w, 1364w,
1301m, 1244s, 1173m, 1087s, 1029s, 979m, 816s, 757w. 1H-NMR (300 MHz, CDCl3): 9.55 (d, J¼7.8,
HꢀC(1)); 7.21 (d, J ¼ 8.7, 4 arom. H); 6.87 (d, J¼7.8, 4 arom. H); 6.75 (dd, J¼15.6, 5.7, HꢀC(3)); 6.32
(ddd, J¼15.9, 7.8, 1.2, HꢀC(2)); 4.56, 4.36 (2d, J¼11.1, ArCH2); 4.46, 4.40 (2d, J¼11.4, ArCH2); 4.25 (td,
J ¼ 6.3, 1.2, HꢀC(4)); 3.83–3.80 (m, HꢀC(5)); 3.81 (s, 2 MeO); 3.54 (dd, J¼9.9, 4.8, HaꢀC(6)); 3.45
(dd, J¼9.6, 5.4, HbꢀC(6)); 2.58 (d, J¼5.1, OH). 13C-NMR (75 MHz, CDCl3): 192.93 (d, C(1)); 159.39,
159.22 (2s); 153.22 (d, C(3)); 133.55 (d, C(2)); 129.62 (2d); 129.54 (2d); 129.46, 129.07 (2s); 113.87 (2d);
113.77 (2d); 78.09 (d, C(4)); 73.15 (t, ArCH2); 72.16 (d, C(5)); 71,85 (t, ArCH2); 69.62 (t, C(6)); 55.33,
55.28 (2q, 2 MeO). HR-MALDI-MS: 409.1616 (100, [MþNa]þ , C22H26NaO6þ ; calc. 409.1622).
(E)-2,3-Dideoxy-4,6-bis-O-(4-methoxybenzyl)-5-O-(methylsulfonyl)-d-threo-hex-2-enose (11). An
ice-cold soln. of 10 (100 mg, 0.258 mmol) in CH2Cl2 (1 ml) was treated successively with pyridine (55 ml,
0.68 mmol) and MsCl (34 ml, 0.43 mmol), stirred at 08, and allowed to warm to 258 over 3.5 h. The
mixture was diluted with H2O. After extraction with CH2Cl2 (3ꢁ20 ml), the combined org. phases were
washed with brine (2ꢁ20 ml), dried (Na2SO4), evaporated, and dried under high vacuum overnight.
Crude 11 (115 mg, 96%) was used for the next step without further purification. Rf (hexane/AcOEt 7:3)
0.24. IR (ATR): 3002w, 2921m, 2852w, 1737w, 1689m, 1611m, 1585w, 1512m, 1463w, 1360m, 1302w, 1245s,
1
1170s, 1096m, 1029s, 969m, 918s, 813s, 748m. H-NMR (300 MHz, CDCl3): 9.55 (d, J¼7.8, HꢀC(1));
7.22–7.19 (m, 4 arom. H); 6.87 (d, J¼8.7, 4 arom. H); 6.73 (dd, J¼15.6, 5.1, HꢀC(3)); 6.36 (ddd, J¼15.9,
7.8, 1.2, HꢀC(2)); 4.81–4.76 (m, HꢀC(5)); 4.57–4.36 (m, HꢀC(4), 2 ArCH2); 3.79 (s, 2 MeO); 3.67 (dd,
J¼11.1, 3.0, HaꢀC(6)); 3.57 (dd, J¼11.1, 6.6, HbꢀC(6)); 2.97 (s, MsO). 13C-NMR (75 MHz, CDCl3):
192.58 (d, C(1)); 159.46, 159.30 (2s); 150.39 (d, C(3)); 134.19 (d, C(2)); 129.65 (2d); 129.48 (2d); 128.99,

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
128.61 (2s); 113.87 (2d); 113.82 (2d); 81.14 (d, C(5)); 76.50 (d, C(4)); 73.18, 72.09 (2t, 2 ArCH2); 68.23 (t,
C(6)); 55.31, 55.25 (2q, 2 MeO); 38.53 (q, MsO). HR-MALDI-MS: 487.1389 (100, [MþNa]þ
,
C23H28NaO8Sþ ; calc. 487.1397).
(E)-2,3-Dideoxy-4,6-bis-O-(4-methoxybenzyl)-5-O-(methylsulfonyl)-d-threo-hex-2-enitol (12). An
ice-cold soln. of 11 (115 mg, 0.247 mmol) in MeOH/CH2Cl2 26 :4 (3 ml) was treated with NaBH4 (9.4 mg,
0.247 mmol), stirred at 08 for 50 min, diluted with H2O, and evaporated. A soln. of the residue in CHCl3
was washed with H2O. The combined org. phases were dried (Na2SO4) and evaporated. Crude 12
(108 mg, 93%) was used for the next step without further purification. Rf (hexane/AcOEt 6 :4) 0.24. IR
(ATR): 3453 (br.), 3006w, 2936w, 2867w, 1665w, 1611m, 1585w, 1512s, 1463w, 1420w, 1346m, 1302m,
1244s, 1170s, 1086m, 1029s, 968m, 915s, 816s, 756w, 663w, 636w. 1H-NMR (300 MHz, CDCl3): 7.24–7.19
(m, 4 arom. H); 6.89–6.30 (m, 4 arom. H); 5.96 (br. dt, J¼15.6, 5.8, HꢀC(2)); 5.61 (ddt, J¼15.6, 7.5, 1.5,
HꢀC(3)); 4.71 (td, J¼5.7, 3.9, HꢀC(5)); 4.55, 4.27 (2d, J¼11.4, ArCH2); 4.46, 4.37 (2d, J¼11.1,
ArCH2); 4.15 (br. s, 2 HꢀC(1)); 4.11 (br. t, Jꢂ6.6, HꢀC(4)); 3.79 (s, 2 MeO); 3.66–3.64 (m, 2 HꢀC(6));
2.95 (s, MsO); 2.44 (br. s, OH). 13C-NMR (75 MHz, CDCl3): 159.16, 159.10 (2s); 135.37 (d, C(3)); 129.57
(2s); 129.49 (2d); 129.41 (2d); 125.40 (d, C(2)); 113.73 (2d); 113.71 (2d); 83.22 (d, C(5)); 77.61 (d, C(4));
73.03, 70.44 (2t, 2 ArCH2); 68.91 (t, C(6)); 62.37 (t, C(1)); 51.27 (q, 2 MeO); 38.64 (q, MsO). HR-
MALDI-MS: 489.1549 (100, [MþNa]þ , C23H30NaO8Sþ ; calc. 489.1554).
(E)-2-Azido-2,4,5-trideoxy-1,3-bis-O-(4-methoxybenzyl)-d-erythro-hex-4-enitol (13). A soln. of
NaN3 (464 mg, 7.13 mmol) and 12 (555 mg, 1.13 mmol) in dry DMF (10 ml) was heated to 1408 for
4.5 h, cooled to 258, and diluted with H2O. The aq. phase was extracted with Et2O. The combined org.
phases were dried (Na2SO4) and evaporated to give crude 13 (525 mg, 89%), which was used for the next
step without further purification. Rf (hexane/AcOEt 3 :2) 0.30. [a]2D5 ¼ ꢀ27.7 (c ¼ 1.9, CHCl3). IR
(ATR): 3402 (br.), 2922w, 2857w, 2097m, 1665m, 1611m, 1585w, 1512s, 1463w, 1386w, 1301m, 1244s,
1
1173m, 1088m, 1030s, 974m, 816s, 756w, 660w. H-NMR (300 MHz, CDCl3): 7.31–7.17 (m, 4 arom. H);
6.92–6.83 (m, 4 arom. H); 5.90 (dt, J¼15.9, 5.1, HꢀC(5)); 5.66 (ddt, J¼15.6, 7.8, 1.5, HꢀC(4)); 4.64, 4.52
(2dd, J¼11.7, ArCH2); 4.47, 4.42 (2dd, J¼11.4, ArCH2); 4.19 (dd, J¼4.8, 1.5, 2 HꢀC(6)); 3.95 (dd, J¼7.8,
5.7, HꢀC(3)); 3.80, 3.79 (2s, 2 MeO); 3.65–3.51 (m, 2 HꢀC(1), HꢀC(2)); 1.90 (br. s, OH). 13C-NMR
(75 MHz, CDCl3): 159.18, 159.09 (2s); 135.14 (d, C(4)); 129.84, 129.75 (2s); 129.24 (2d); 129.20 (2d);
127.07 (d, C(5)); 113.70 (2d); 113.67 (2d); 78.30 (d, C(3)); 72.91, 70.08 (2t, 2 ArCH2); 68.80 (t, C(1));
64.09 (d, C(2)); 62.55 (t, C(6)); 55.16 (q, 2 MeO). HR-MALDI-MS: 436.1850 (100, [MþNa]þ
,
C22H27N3NaOþ5 ; calc. 436.1843). Anal. calc. for C22H27N3O5 (413.20): C 63.91, H 6.58, N 10.16; found: C
63.84, H 6.67, N 10.04.
(E)-2-Azido-2,4,5-trideoxy-1,3-bis-O-(4-methoxybenzyl)-6-O-undecyl-d-erythro-hex-4-enitol (14).
A soln. of 13 (500 mg, 1.21 mmol) in DMF (15 ml) was treated with NaH (60% in oil; 145 mg,
3.63 mmol), stirred at r.t. for 15 min, treated with 1-bromoundecane (542 ml, 2.42 mmol), stirred at 258
for 20 h, and treated with MeOH (10 ml). The mixture was diluted with H2O and extracted with AcOEt.
The combined org. phases were washed with H2O and brine, dried (Na2SO4), and evaporated. FC
(pentane/AcOEt 95 :5) gave 14 (536 mg, 78%). Colourless oil. Rf (hexane/AcOEt 95 :5) 0.17. [a]D25
¼
ꢀ25.9 (c ¼ 0.75, CHCl3). IR (ATR): 2923m, 2853m, 2097m, 1739w, 1612m, 1586w, 1512s, 1484w, 1362w,
1301m, 1245s, 1172m, 1095m, 1035s, 973w, 819m, 757w, 721w. 1H-NMR (300 MHz, CDCl3): 7.26–7.16 (m,
4 arom. H); 6.91–6.83 (m, 4 arom. H); 5.81 (dt, J ¼ 15.6, 5.1, HꢀC(5)); 5.67 (ddt, J¼15.6, 7.8, 1.2,
HꢀC(4)); 4.54, 4.28 (2d, J¼11.4, ArCH2); 4.45 (s, ArCH2); 4.01 (dd, J¼5.4, 1.2, 2 HꢀC(6)); 3.95 (dd, J¼
7.8, 5.7, HꢀC(3)); 3.80, 3.79 (2s, 2 MeO); 3.65–3.51 (m, 2 HꢀC(1), HꢀC(2)); 3.42 (t, J¼6.6,
2 HꢀC(1’)); 1.64–1.54 (m, 2 HꢀC(2’)); 1.38–1.20 (m, 16 H); 0.88 (t, J ¼ 6.8, Me). 13C-NMR (75 MHz,
CDCl3): 159.09, 158.99 (2s); 133.00 (d, C(4)); 129.83 (d, C(5)); 129.74, 128.27 (2s); 129.17 (4d); 113.70
(2d); 113.67 (2d); 78.46 (d, C(3)); 73.01, 70.60 (2t, 2 ArCH2); 70.44 (t, C(1)); 70.14 (t, C(6)); 68.98 (d,
C(2)); 64.23 (t, C(1’)); 55.26 (q, 2 MeO); 31.99 (t); 29.83–29.42 (several t); 26.31, 22.77 (2t); 14.23 (q,
Me). HR-MALDI-MS: 590.3564 (100, [MþNa]þ , C33H49N3NaO5þ ; calc. 590.3564). Anal. calc. for
C33H49N3O6 (567.76): C 69.81, H 8.70, N 7.40; found: C 69.99, H 8.71, N 7.26.
(E)-2-Amino-2,4,5-trideoxy-1,3-bis-O-(4-methoxybenzyl)-6-O-undecyl-d-erythro-hex-4-enitol (15).
A suspension of LiAlH4 (133 mg, 3.49 mmol) in dry Et2O (5 ml) was cooled to 08, treated dropwise
with a soln. of 14 (500 mg, 0.88 mmol) in dry Et2O (5 ml), allowed to warm to 258, and stirred for 14 h.
The mixture was cooled to 08, and treated dropwise with H2O (750 ml), 1n NaOH (1.5 ml), and H2O (900

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
733
ml). The suspension was filtered through Celite, and the filtrate was extracted with AcOEt (3ꢁ50 ml).
The combined org. phases were dried (MgSO4) and evaporated to afford crude 15 (475 mg, >99%)
which was used for the next step without further purification. Rf (hexane/AcOEt 2 :3) 0.15. IR (ATR):
3290w (br.), 2922s, 2852m, 1612w, 1586w, 1513s, 1464m, 1376w, 1301w, 1246s, 1172m, 1083m, 1036s, 975w,
819m, 756w, 721w, 637w. 1H-NMR (300 MHz, CDCl3): 7.32–7.17 (m, 4 arom. H); 6.92–6.82 (m, 4 arom.
H); 5.82 (dt, J¼15.3, 5.4, HꢀC(5)); 5.66 (ddt, J¼15.6, 8.1, 1.5, HꢀC(4)); 4.51, 4.25 (2d, J¼11.4, ArCH2);
4.41 (s, ArCH2); 4.01 (dd, J¼5.4, 1.2, 2 HꢀC(6)); 3.80, 3.79 (2s, 2 MeO); 3.63 (t, J¼6.6, 2 HꢀC(1’)); 3.56
(dd, J¼9.3, 4.2, HaꢀC(1)); 3.47–3.40 (m, HbꢀC(1), HꢀC(3)); 3.10–3.04 (m, HꢀC(2)); 1.64–1.54 (m,
2 HꢀC(2’)); 1.38–1.20 (m, 16 H); 0.88 (t, J ¼ 6.8, Me). 13C-NMR (75 MHz, CDCl3): 159.02 (2s); 132.46
(d, C(4)); 130.42 (2s); 129.80 (d, C(5)); 129.25 (4d); 113.69 (4d); 80.76 (d, C(3)); 72.97 (t, ArCH2); 71.62
(t, C(1)); 70.63 (t, ArCH2); 70.13 (t, C(6)); 63.11 (t, C(1’)); 55.32 (q, 2 MeO); 54.39 (d, C(2)); 32.03 (t);
29.81–29.46 (several t); 26.36, 22.82 (2t); 14.27 (q, Me). HR-MALDI-MS: 542.3831 (100, [MþNa]þ
,
C33H51NNaOþ5 ; calc. 542.3840).
(E)-2-Amino-2,4,5-trideoxy-6-O-undecyl-d-erythro-hex-4-enitol (3).
A soln. of 15 (60 mg,
0.11 mmol) in CH2Cl2 (4.5 ml) was treated dropwise with TFA (0.5 ml, 6.5 mmol), stirred at 258 for
15 min., and poured into sat. aq. NaHCO3 soln. (30 ml). The aq. layer was extracted with AcOEt (2ꢁ
25 ml). The combined org. layers were dried (Na2SO4) and evaporated. FC (AcOEt/pentane 1:1) gave 3
(24.7 mg, 74%) whose spectral and physical data are in accordance with those reported in [15].
(E)-1,3-Bis-O-(4-methoxybenzyl)-2,4,5-trideoxy-2-(octadecanoylamino)-6-O-undecyl-d-erythro-
hex-4-enitol (16). A soln. of crude 15 (250 mg, 0.46 mmol) in dry CH2Cl2 (10 ml) was treated with
pyridine (186 ml, 2.31 mmol) and stearoyl chloride (209 mg, 0.69 mmol), stirred at 258 for 6 h, diluted
with H2O, and extracted with CH2Cl2 (3ꢁ100 ml). The combined org. layers were dried (Na2SO4) and
evaporated. Residual pyridine was removed by azeotropic distillation with toluene. FC (AcOEt/hexane
1:3) gave 16 (353 mg, 95%). White fluffy powder. M.p. 83.98. Rf (AcOEt/hexane 1:3) 0.21. [a]2D5 ¼ ꢀ25.4
(c¼0.6, CHCl3). IR (ATR): 3297w, 2953w, 2915s, 2848s, 1703w, 1644s, 1614w, 1586w, 1543m, 1515m,
1469m, 1364w, 1302w, 1253m, 1172w, 1119m, 1093m, 1033m, 888w, 811m, 718w. 1H-NMR (CDCl3,
400 MHz): 7.20–7.16 (m, 4 arom. H); 6.87–6.83 (m, 4 arom. H); 5.78 (dt, J¼15.6, 6.0, HꢀC(5)); 5.72 (d,
J¼9.3, NH); 5.64 (ddt, J¼15.6, 7.9, 1.2, HꢀC(4)); 4.52, 4.41, 4.35 (3d, J¼11.4, 3 ArCH); 4.24–4.18 (m,
HꢀC(2)): 4.22 (d, J¼11.6, ArCH); 3.97 (dd, J¼5.6, 0.9, 2 HꢀC(6)); 3.93 (t, J¼7.6, HꢀC(3)); 3.80, 3.79
(2s, 2 MeO); 3.75 (dd, J¼9.6, 4.0, HaꢀC(1)); 3.48 (dd, J¼9.6, 4.0, HbꢀC(1)); 3.44–3.35 (AB,
2 HꢀC(1’)); 2.32 (t, J¼7.5, 2 HꢀC(1’’)); 2.13–2.01 (m, 2 HꢀC(2’)); 1.61–1.53 (m, 4 H); 1.35–1.30 (m,
42 H); 0.89 (t, J¼6.8, Me); 0.88 (t, J¼6.8, Me). 13C-NMR (CDCl3, 75 MHz): 172.57 (s, C¼O); 159.28,
159.19 (2s); 132.17 (d, C(4)); 130.36 (s); 130.23 (d, C(5)); 130.20 (s); 129.41 (2d); 129.36 (2d); 113.78
(2d); 113.76 (2d); 78.54 (d, C(3)); 72.75 (t, ArCH2); 70.66 (t, C(6)); 70.51 (t, C(1’)); 70.24 (t, ArCH2);
68.07 (t, C(1)); 55.24 (q, 2 MeO); 51.48 (d, C(2)); 36.95 (t, C(2’’)); 33.86 (2t); 31.94 (t); 29.81–29.16
(several t); 26.26, 25.75 (2t); 22.70 (2t); 14.11 (q, 2 Me). HR-MALDI-MS: 830.6270 (100, [MþNa]þ
,
C51H85NNaOþ6 ; calc. 830.6275). Anal. calc. for C51H85NO6 (808.22): C 75.79, H 10.60, N 1.73; found: C
75.98, H 10.69, N 1.62.
(E)-2,4,5-Trideoxy-2-[(octadecanoyl)amino]-6-O-undecyl-d-erythro-hex-4-enitol (4). An ice-cold
soln. of 16 (60 mg, 0.074 mmol) in CH2Cl2 (4.5 ml) was treated dropwise with TFA (0.5 ml, 6.5 mmol),
stirred at 08 for 30 min, and then at 258 for 30 min, and poured into sat. aq. NaHCO3 soln (30 ml). The aq.
layer was extracted with AcOEt (2ꢁ25 ml). The combined org. layers were dried (Na2SO4) and
evaporated. FC (CH2Cl2/MeOH 4 :1) gave 4 (31.5 mg, 75%) whose spectral and physical data are in
accordance with those reported in [15].
(E)-2-Azido-2,4,5-trideoxy-1,3-bis-O-(4-methoxybenzyl)-6-O-(methylsulfonyl)-d-erythro-hex-4-eni-
tol (17). A soln. of 13 (1.64 g, 3.97 mmol) in CH2Cl2 was cooled to 08 and treated successively with Ms2O
(1.036 g, 5.95 mmol) and EtN(i-Pr)2 (0.98 ml, 5.95 mmol). The mixture was stirred for 2.5 h, diluted with
CH2Cl2, and washed with H2O. After separation of the layers, the aq. phase was extracted with CH2Cl2
(3ꢁ100 ml). The combined org. phases were washed with brine, dried (Na2SO4), and evaporated to
afford crude 17, which was filtered through a short pad of silica gel (AcOEt/hexane 2 :3) to yield anal.
pure 17 (1.95 g, >99%). Rf (AcOEt/hexane 3 :7) 0.19. IR (ATR): 3010–2835w, 2097w, 1745w, 1715w,
1611m, 1585w, 1512s, 1463w, 1353m, 1301m, 1274m, 1244s, 1171s, 1087m, 1064m, 1030s, 972m, 927s, 816s,
750s. 1H-NMR (400 MHz, CDCl3): 7.25–7.19 (m, 4 arom. H); 6.89–6.86 (m, 4 arom. H); 5.92–5.81 (m,

734
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
HꢀC(4), HꢀC(5)); 4.74 (d, J¼4.5, 2 HꢀC(6)); 4.52, 4.32 (2d, J¼11.4, ArCH2); 4.46, 4.44 (2d, J¼11.6,
ArCH2); 4.00 (td, J¼5.5, 1.0, HꢀC(3)); 3.81, 3.80 (2s, 2 MeO); 3.63 (td, J¼6.0, 4.6, HꢀC(2)); 3.56–3.54
(m, 2 HꢀC(1)); 3.02 (s, MsO). 13C-NMR (100 MHz, CDCl3): 159.39 (2s); 133.37 (d, C(4)); 129.74, 129.70
(2s); 129.39 (4d); 127.44 (d, C(5)); 113.88 (4d); 77.71 (d, C(3)); 73.08, 70.74 (2t, 2 ArCH2); 69.00 (t, C(6));
68.68 (t, C(1)); 63.95 (d, C(2)); 55.29 (q, 2 MeO); 38.06 (q, MsO). HR-MALDI-MS: 514.1620 (100, [Mþ
Na]þ , C23H29N3NaO7Sþ ; calc. 514.1624).
(E)-2-Azido-2,4,5,6-tetradeoxy-1,3-bis-O-(methoxybenzyl)-6-(undecylsulfanyl)-d-erythro-hex-4-
enitol (18). A suspension of NaH, (60% in oil, 112 mg, 2.8 mmol) in DMF (3 ml) was cooled to 08, treated
with a soln. of C11H23SH (263.8 mg, 0.54 mmol) in DMF (2 ml), stirred for 5 min, treated dropwise with a
soln. of 17 (459 mg, 0.934 mmol), stirred for 75 min, treated dropwise with ice-cold H2O (3 ml), diluted
with H2O (20 ml) and brine (5 ml), and extracted with AcOEt (3ꢁ100 ml). The combined org. phases
were washed with brine, dried (Na2SO4), and evaporated. FC (hexane/Et2O 1:0!4 :1) gave 18 (472 mg,
87%). Pale yellow oil. Rf (Et2O/hexane 15 :85) 0.26. [a]2D5 ¼ ꢀ29.3 (c ¼ 0.53, CHCl3). IR (CHCl3): 3002w,
2923s, 2853s, 2095m, 1612w, 1586w, 1512s, 1464w, 1441w, 1419w, 1362w, 1301w, 1245s, 1172m, 1081m,
1034s, 970m, 819m, 757w, 720w, 637w. 1H-NMR (CDCl3, 400 MHz): 7.28–7.19 (m, 4 arom. H); 6.91–6.85
(m, 4 arom. H); 5.73 (dtd, J ¼ 15.2, 7.2, 0.4, HꢀC(5)); 5.51 (ddt, J¼15.4, 8.2, 1.1, HꢀC(4)); 4.55, 4.29 (2d,
J¼11.4, ArCH2); 4.48, 4.44 (2d, J¼11.7, 2 ArCH2); 3.92 (ddd, J¼8.2, 5.3, 0.4, HꢀC(3)); 3.81, 3.80 (2s,
2 MeO); 3.66–3.61 (m, HꢀC(2)); 3.59 (dd, J¼9.6, 4.4, HaꢀC(1)); 3.54 (dd, J¼9.8, 6.9, HbꢀC(1)); 3.23–
3.11 (m, 2 HꢀC(6)); 2.48 (t, J¼7.4, 2 HꢀC(1’)); 1.59 (q, J¼7.3, 2 HꢀC(2’)); 1.35–1.22 (m, 16 H); 0.89 (t,
J¼6.7, Me). 13C-NMR (CDCl3, 100 MHz): 159.34, 159.27 (2s); 132.84 (d, C(4)); 129.94, 129.90 (2s);
129.30 (4d); 128.72 (d, C(5)); 113.86 (2d); 113.84 (2d); 78.39 (d, C(3)); 73.07, 70.05 (2t, 2 ArCH2); 69.10
(t, C(1)); 64.32 (d, C(2)); 55.26 (q, 2 MeO); 33.41 (t, C(6)); 31.94, 30.10 (2t); 29.65–29.33 (several t);
28.99, 24.17, 22.71 (3t); 14.14 (q, Me). HR-MALDI-MS: 622.3075 (100, [MþK]þ , C33H49KN3O4Sþ ; calc.
622.3081). Anal. calc. for C33H49N3O4S (583.3444): C 67.89, H 8.46, N 7.20; found: C 68.14, H 8.52, N 6.94.
(E)-2-Azido-2,4,5,6-tetradeoxy-6-(undecylsulfanyl)-d-erythro-hex-4-enitol (19). A soln. of 18
(58.5 mg, 0.1 mmol) in CH2Cl2 (4.5 ml) was cooled to 08 and treated with TFA (0.5 ml, 6.5 mmol).
The mixture was stirred for 45 min, and diluted with CH2Cl2 (20 ml) and H2O. After separation of the
layers, the aq. phase was extracted with CH2Cl2 (3ꢁ30 ml). The combined org. phases were washed with
brine, dried (Na2SO4), and evaporated to afford crude 19 (34.3 mg, >99%), which was pure enough to
be used directly in the next step. IR (ATR): 3371w (br.), 2922s, 2852s, 2099s, 1464m, 1419w, 1265m,
1063m, 1008m, 969m, 721w. 1H-NMR (CDCl3, 300 MHz): 5.84 (dtd, J¼15.3, 7.2, 1.2, HꢀC(5)); 5.65 (ddt,
J¼15.0, 6.6, 0.9, HꢀC(4)); 4.32 (t, J¼6.0, HꢀC(3)); 3.80 (br. d, J¼4.9, 2 HꢀC(1)); 3.53 (q, J¼5.2,
HꢀC(2)); 3.16 (d, J¼7.1, 2 H ꢀC(6)); 2.46 (t, J¼7.5, 2 HꢀC(1’)); 2.21 (br. s, HOꢀC(3)); 2.09 (br. s,
HOꢀC(1)); 1.61–1.52 (m, 2 HꢀC(2’)); 1.39–1.20 (m, 16 H); 0.88 (t, J¼6.7, Me). 13C-NMR (CDCl3,
75 MHz): 130.73 (d, C(4)); 130.45 (d, C(5)); 72.73 (d, C(3)); 66.47 (d, C(2)); 62.36 (t, C(1)); 32.20 (t,
C(6)); 31.79, 31.08 (2t); 29.50 (2t); 29.22 (2t); 29.43, 29.18 (2t); 28.80, 22.58 (2t); 14.01 (q, Me). HR-
MALDI-MS: 366.2198 (100, [M þ Na]þ , C17H33N3NaO2Sþ ; calc. 366.2191).
(E)-2-Amino-2,4,5,6-tetradeoxy-6-(undecylsulfanyl)-d-erythro-hex-4-enitol (5). A soln. of crude 19
(34.3 mg, 0.1 mmol) in THF (4 ml) was cooled to 08 and treated dropwise with 1m PMe3 in THF (0.15 ml,
0.15 mmol). The mixture was warmed to 258 and stirred for 1 h, when the TLC showed complete
conversion of 19, treated with H2O (1 ml), and stirred for 15 h. The mixture was diluted with AcOEt
(30 ml) and H2O. After separation of the layers, the aq. phase was extracted with AcOEt (3ꢁ30 ml). The
combined org. phases were washed with H2O and brine, dried (Na2SO4), and evaporated. FC (CH2Cl2/
MeOH 100 :0!9 :1) yielded 5 (26.5 mg, 83.5%). Colourless solid. M.p. 628. Rf (CH2Cl2/MeOH 4 :1)
0.12. [a]2D5 ¼ þ3.3 (c ¼ 0.25, CHCl3). IR (ATR): 3358 (br.), 3326 (br.), 3286 (br.), 3261 (br.), 3176w,
2955w, 2917s, 2850s, 1615w, 1579w, 1467w, 1431w, 1337w, 1101w, 1052m, 1038m, 1021m, 994m, 967m,
1
957m, 889w, 812w, 720w, 646w. H-NMR (CDCl3, 300 MHz): 5.77 (dtd, J¼15.3, 7.2, 0.9, HꢀC(5)); 5.58
(br. dd, J¼15.3, 6.6, HꢀC(4)); 4.11 (t, Jꢂ5.7, HꢀC(3)); 3.64 (d, J¼4.8, 2 HꢀC(1)); 3.14 (d, J ¼ 7.2,
2 HꢀC(6)); 2.83 (q, Jꢂ5.1, HꢀC(2)); 2.52 (br. s, exchanged with D2O, NH2, 2 OH); 2.45 (t, J ¼ 7.3,
2 HꢀC(1’)); 1.60–1.50 (m, 2 HꢀC(2’)); 1.38–1.25 (m, 16 H); 0.87 (t, J¼6.6, Me). 13C-NMR (CDCl3,
75 MHz): 132.04 (d, C(4)); 129.30 (d, C(5)); 74.60 (d, C(3)); 63.85 (t, C(1)); 56.05 (d, C(2)); 33.56 (t,
C(6)); 32.00 (t, C(1’)); 31.43 (t); 29.73–29.41 (several t); 29.03, 22.81 (2t); 14.26 (q, Me). HR-MALDI-

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
735
MS: 318.2465 (100, [M þ H]þ , C17H36NO2Sþ calc. 318.2467). Anal. calc. for C17H35NO2S (317.53): C
64.30, H 11.11, N 4.41; found: C 64.21, H 11.11, N 4.41.
(E)-2-Azido-2,4,5,6-tetradeoxy-1,3-bis-O-(methoxybenzyl)-6-(undecylsulfonyl)-d-erythro-hex-4-
enitol (20). A soln. of 18 (82 mg, 0.14 mmol) in CH2Cl2/MeOH 2 :1 (6 ml) was treated with oxone
(432 mg, 0.7 mmol), stirred at 258 for 24 h, and filtered through a short pad of silica gel (filter cake was
washed with AcOEt/CH2Cl2 1:2). The combined filtrate and washings were evaporated. FC (AcOEt/
hexane 1:4) gave 20 (67 mg, 78%). Colourless syrup. Rf (AcOEt/hexane 1:4) 0.15. [a]2D5 ¼ ꢀ16.7 (c ¼
0.55, CHCl3). IR (ATR): 2923m, 2854w, 2096m, 1612w, 1586w, 1512m, 1464w, 1403w, 1361w, 1301m,
1245s, 1173m, 1127m, 1033s, 977m, 892w, 819s, 757w, 721w. 1H-NMR (CDCl3, 400 MHz): 7.26–7.17 (m, 4
arom. H); 6.90–6.83 (m, 4 arom. H); 5.89–5.78 (m, HꢀC(4), HꢀC(5)); 4.53, 4.32 (2d, J¼11.4, ArCH2);
4.45 (s, ArCH2); 3.99 (dt, J¼6.0, 2.0, HꢀC(3)); 3.80, 3.79 (2s, 2 MeO); 3.71 (d, J¼5.8, 2 HꢀC(6)); 3.65
(td, J¼6.1, 4.5, HꢀC(2)); 3.58 (dd, J ¼ 10.0, 4.8, HaꢀC(1)); 3.54 (dd, J ¼ 10.0, 6.4, HbꢀC(1)); 2.97–2.91
(m, 2 HꢀC(1’)); 1.86–1.78 (m, 2 HꢀC(2’)); 1.45–1.38 (m, 2 HꢀC(3’)); 1.32–1.25 (m, 14 H); 0.88 (t, J¼
6.9, Me). 13C-NMR (CDCl3, 100 MHz): 159.38 (2s); 136.84 (d, C(4)); 129.69, 129.46 (2s); 129.40 (2d);
129.37 (2d); 122.05 (d, C(5)); 113.90 (2d); 113.88 (2d); 77.67 (d, C(3)); 73.10, 70.61 (2t, 2 ArCH2); 68.79
(t, C(1)); 63.98 (d, C(2)); 56.26 (t, C(6)); 55.27 (q, 2 MeO); 51.74 (t, C(1’)); 31.88 (t); 29.54–29.08 (several
t); 28.51, 22.66, 21.87 (3t); 14.09 (q, Me). HR-MALDI-MS: 638.3242 (100, [M þ Na]þ , C33H49N3NaO6Sþ
;
calc. 638.3240). Anal. calc. for C33H49N3O6S (615.82): C 64.36, H 8.02, N 6.82; found: C 64.49, H 8.07, N
6.76.
(E)-2-Azido-2,4,5,6-tetradeoxy-6-(undecylsulfonyl)-d-erythro-hex-4-enitol (21). An ice-cold soln. of
20 (65 mg, 0.1 mmol) in CH2Cl2 (4.5 ml) was treated dropwise with TFA (0.5 ml), stirred for 1 h, diluted
with CH2Cl2 (20 ml), and washed with H2O (20 ml). The aq. phase was extracted with CH2Cl2 (3ꢁ
50 ml). The combined org. phases were washed with brine, dried (Na2SO4), and evaporated to afford
crude 21 (37 mg, >99%), which was anal. pure to be used directly in the next step. IR (ATR): 3433 (br.),
2922s, 2853m, 2098s, 1465w, 1405w, 1281s, 1162w, 1125s, 1064m, 1009m, 975m, 891w, 763w, 721w, 654w.
1H-NMR (CDCl3, 300 MHz): 6.00 (dd, J¼15.6, 5.4, HꢀC(4)); 5.91 (dt, J¼15.6, 6.9, HꢀC(5)); 4.38 (q,
Jꢂ4.7, addn. of CD3OD!t, J¼5.8, HꢀC(3)); 3.82 (t, Jꢂ4.7, addn. of CD3OD!d, J¼5.2, 2 HꢀC(1));
3.73 (d, Jꢂ6.3, 2 HꢀC(6)); 3.54 (q, J¼4.8, HꢀC(2)); 3.37 (d, Jꢂ4.5, exchange with CD3OD,
HOꢀC(3)); 3.01–2.95 (m, 2 HꢀC(1’)); 2.77 (t, Jꢂ5.2, exchange with CD3OD, HOꢀC(1)); 1.87–1.77
(m, 2 HꢀC(2’)); 1.48–1.38 (m, 2 HꢀC(3’)); 1.34–1.26 (m, 14 H); 0.87 (t, J¼6.5, Me). 13C-NMR (CDCl3,
75 MHz): 139.02 (d, C(4)); 118.87 (d, C(5)); 72.00 (d, C(3)); 66.23 (d, C(2)); 62.20 (t, C(1)); 55.98 (t,
C(6)); 52.28 (t, C(1’)); 31.99 (t); 29.66, 29.62 (2t); 29.41 (2t); 29.20, 28.56, 22.79, 21.99 (4t); 14.26 (q, Me).
HR-MALDI-MS: 398.2086 (100, [M þ Na]þ , C17H33N3NaO4Sþ ; calc. 398.2089).
(E)-2-Amino-2,4,5,6-tetradeoxy-6-(undecylsulfonyl)-d-erythro-hex-4-enitol (6). An ice-cold soln. of
crude 21 in THF (4 ml) was treated dropwise with 1m PMe3 in THF (0.16 ml, 0.16 mmol) and stirred at 258
for 1 h. When the TLC showed complete conversion of 21, the mixture was treated with H2O (1 ml),
stirred for 15 h, and diluted with AcOEt (20 ml). After separation of the layers, the aq. phase was
extracted with AcOEt (3ꢁ20 ml). The combined org. phases were washed with H2O and brine, dried
(Na2SO4), and evaporated. FC (CH2Cl2/MeOH 1:0!9 :1) yielded 6 (31 mg, 84%). M.p. 98.68. Rf
(CH2Cl2/MeOH 4 :1) 0.12. [a]2D5 ¼ þ9.1 (c ¼ 0.15, CHCl3). IR (ATR): 3460 (br.), 3407 (br.), 3371 (br.),
3348 (br.), 3314w, 3290w, 3066m (sh), 2920s, 2848s, 2718m (sh), 1585w, 1467w, 1320m, 1287m, 1275m,
1255w, 1119s, 1048m, 992m, 980m, 947w, 764w, 725w. 1H-NMR (CD3OD, 300 MHz): 5.98 (br. dd, J¼15.6,
6.3, HꢀC(4)); 5.81 (dtd, J¼15.3, 7.2, 0.9, HꢀC(5)); 4.13 (br. t, Jꢂ5.1, HꢀC(3)); 3.92–3.78 (AB,
2 HꢀC(6)); 3.66 (dd, J¼11.1, 4.8, HaꢀC(1)); 3.52 (dd, J¼10.8, 6.3, HbꢀC(1)); 3.10–3.05 (m,
2 HꢀC(1’)); 2.81 (td, J¼6.6, 5.1, HꢀC(2)); 1.84–1.74 (m, 2 HꢀC(2’)); 1.52–1.41 (m, 2 HꢀC(3’));
1.41–1.29 (m, 14 H); 0.89 (t, J¼6.7, Me). 13C-NMR (CD3OD, 75 MHz): 141.14 (d, C(4)); 119.21 (d,
C(5)); 73.66 (d, C(3)); 63.78 (t, C(1)); 57.64 (t, C(6)); 56.67 (d, C(2)); 52.26 (t, C(1’)); 32.79 (t); 30.45–
29.25 (several t); 23.48, 22.42 (2t); 14.23 (q, Me). HR-MALDI-MS: 351.2360 (100, [M þ Na]þ
,
C17H37NO4Sþ ; calc. 351.2443).
X-Ray Analysis of 6. Single crystal was obtained by isothermal crystallization from MeOH and H2O.
A soln. of 6 in MeOH (ca. 5 mg/300 ml), in a microtube (diameter ca. 5 mm), was immersed in a chamber
containing H2O and kept for 7 d undisturbed. The resulting plates of 6 were good enough for X-ray
crystallographic analysis. Dimensions: 0.4ꢁ0.4ꢁ0.005 mm. Colourless crystals: C17H35NO4S (349.534),

736
CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
triclinic P1, a¼4.7826(5), b¼6.2512(8), c ¼ 16.624(2) ꢄ, a¼90.343(9), b¼96.817(10), g¼98.494(4)8,
V¼487.94(10) ꢄ3, Z¼1, Dcalc ¼1.190 Mg/m3. All reflections were measured using a Bruker Nonius-
Kappa CCD diffractometer (MoKa radiation, l¼0.71073) at 203 K. 2764 measured reflections, 2764
independent reflections, 2499 observed reflections. Refinement of F2: full-matrix least-squares refine-
ment, R(all)¼0.0596, R(gt)¼0.0522, wR(ref)¼0.1433, wR(gt)¼0.1353. All diagrams and calculations
were performed using maXus (Bruker Nonius, Delft & MacScience, Japan). Programme used to solve
structure: SIR97; programme used to refine structure: SHELXL-97.
(E)-2-Azido-6-{[(tert-butoxy)carbonyl](undecyl)amino}-2,4,5,6-tetradeoxy-1,3-bis-O-(methoxyben-
zyl)-d-erythro-hex-4-enitol (22). A soln. of 17 (100 mg, 0.2 mmol) in THF (2 ml) was treated with LiBr
(dried in a Kugelrohr at 2508 for 24 h and cooled in a vacuum desiccator over KOH; 177 mg, 2 mmol),
and stirred at 258 for 2.5 h when TLC (AcOEt/pentane 1:4) showed complete conversion of 17. The
mixture was diluted with H2O and extracted with Et2O (3ꢁ50 ml). The combined org. phases were dried
(Na2SO4) and evaporated to yield the crude bromide, which was used for the next step without
purification. An ice-cold suspension of NaH (60% in oil, 24.4 mg, 0.609 mmol) in DMF (1 ml) was
treated with C11H23NHBoc (83 mg, 0.3 mmol) and stirred for 15 min. The mixture was treated with a soln.
of the above crude bromide in DMF (1 ml) and stirred at 08 for 3 h. After the portionwise addition of
crushed ice and sat. aq. NH4Cl soln. and extraction with Et2O (3ꢁ25 ml), the combined org. phases were
dried (Na2SO4) and evaporated. FC (Et2O/pentane 3 :17) gave crude 22 (93 mg, 70%). Colourless syrup
solidifying upon standing. Rf (AcOEt/hexane 1:4) 0.23. IR (ATR): 2924m, 2853m, 2097m, 1612w, 1586w,
1513s, 1464m, 1412w, 1365w, 1301m, 1246s, 1172m, 1090m, 1035m, 976w, 819m, 764w. 1H-NMR (CDCl3,
400 MHz): 7.32–7.16 (m, 4 arom. H); 6.90–6.83 (m, 4 arom. H); 5.70 (dt, J¼15.7, 5.6, HꢀC(5)); 5.52 (br.
dd, J¼15.6, 8.2, HꢀC(4)); 4.52, 4.27 (2d, J¼11.5, ArCH2); 4.45 (s, ArCH2); 3.92 (dd, J ¼ 8.0, 5.4,
HꢀC(3)); 3.89–3.82 (m, 2 HꢀC(6)); 3.80, 3.79 (2s, 2 MeO); 3.62 (m, 2 HꢀC(1), HꢀC(2)); 3.20–3.11
(m, 2 HꢀC(1’)); 1.52–1.44 (m, 2 HꢀC(2’)); 1.46 (s, Me3C); 1.33–1.19 (m, 16 H); 0.88 (t, J¼6.6, Me).
HR-MALDI-MS: 689.4249 (100, [M þ Na]þ , C38H58N4NaO6Sþ ; calc. 689.4254).
(E)-2-Amino-6-{[(tert-butoxy)carbonyl](undecyl)amino}2,4,5,6-tetradeoxy-1,3-bis-O-(methoxyben-
zyl)-d-erythro-hex-4-enitol (23). An ice-cold soln. of crude 22 (150 mg, 0.225 mmol) in THF (4 ml) was
treated dropwise with 1m PMe3 in THF (0.45 ml, 0.45 mmol) and stirred at 258 for 1 h when the TLC
showed complete conversion of 22. The mixture was treated with H2O (1 ml), stirred for 10 h, and diluted
with AcOEt. After the separation of the layers, the aq. phase was extracted with AcOEt (3ꢁ100 ml).
The combined org. phases were washed with H2O, and brine, dried (Na2SO4), and evaporated to afford
crude 23 (114 mg, >99%), which was used for the next step without further purification. Rf (CH2Cl2/
MeOH 95 :5) 0.25. IR (ATR): 3387w, 2924m, 2853m, 1690s, 1612m, 1586w, 1512m, 1463m, 1411m, 1389w,
1364w, 1301m, 1245s, 1170s, 1078m, 1035s, 975w, 875w, 844m, 819s, 772w, 721w. 1H-NMR (CDCl3,
400 MHz): 7.24–7.16 (m, 4 arom. H); 6.88–6.83 (m, 4 arom. H); 5.69 (dt, J¼15.4, 5.7, HꢀC(5)); 5.51 (br.
dd, J¼15.5, 8.1, HꢀC(4)); 4.49, 4.40 (2d, J¼11.4, ArCH2); 4.43, 4.24 (2d, J¼11.4, ArCH2); 3.88 (br. s,
2 HꢀC(6)); 3.80, 3.79 (2s, 2 MeO); 3.81–3.75 (m, HꢀC(3)); 3.55 (dd, J¼9.1, 4.0, HꢀC(1)); 3.43 (dd, J¼
9.1, 6.7, H’ꢀC(1)); 3.16 (br. s, exchange with D2O, NH2); 3.05 (td, J¼6.5, 4.0, HꢀC(2)); 1.52–1.48 (m,
2 HꢀC(1’)); 1.46 (s, Me3C); 1.33–1.19 (m, 18 H); 0.88 (t, J¼6.9, Me). 13C-NMR (CDCl3, 100 MHz):
159.21, 159.15 (2s); 155.41 (s, C¼O); 131.94 (d, C(4)); 130.48 (2s); 129.31 (d, 4 arom. C and C(5)); 113.79
(2d); 113.78 (2d); 80.66 (d, C(3)); 79.39 (s, Me3C); 72.97 (t, ArCH2); 71.58 (t, C(1)); 70.03 (t, ArCH2);
55.26 (q, 2 MeO); 54.37 (d, C(2); 46.89 (t, C(6)); 31.91 (t); 29.66–29.34 (several t); 28.50 (q, Me3C); 26.91,
22.69 (2t); 14.11 (q, Me). HR-MALDI-MS: 641.4531 (100, [M þ H]þ , C38H61N2O6þ ; calc. 641.4530).
Anal. calc. for C38H60N2O6 (640.8928): C 71.21, H 9.44, N 4.37; found: C 71.00, H 9.49, N 4.27.
(E)-2-Amino-2,4,5,6-tetradeoxy-6-{[(trifluoromethyl)carbonyl](undecyl)amino}-d-erythro-hex-4-
enitol (24). A soln. of 23 (70 mg, 0.11 mmol) in CH2Cl2 (4.5 ml) was cooled to 08, treated with CF3COOH
(0.5 ml), stirred for 45 min, diluted with CH2Cl2 (20 ml), and washed with H2O (3ꢁ20 ml). The
combined aq. phases were extracted with CH2Cl2 (3ꢁ100 ml). The combined org. phases were washed
with brine, dried (Na2SO4), and evaporated. FC (silica gel (NH2), CH2Cl2/MeOH 9 :1) yielded 24 (21 mg,
65%). Colourless gum solidifying into a glassy substance upon cooling. Rf (CH2Cl2/MeOH 4 :1) 0.12.
[a]2D5 ¼ ꢀ4.5 (c ¼ 0.25, CHCl3). IR (ATR): 3505w, 3272m, 3107w, 2922s, 2853m, 2681w, 1696s, 1561m,
1468w, 1459w, 1366w, 1342w, 1295w, 1206m, 1183s, 1168s, 1123w, 1079w, 1055w, 1032w, 974w, 910w, 875w,
747w, 728w. 1H-NMR (CDCl3, 300 MHz): 5.93 (dt, J¼15.6, 6.6, HꢀC(5)); 5.71 (br. dd, J¼15.6, 5.4,

CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
737
HꢀC(4)); 4.38 (t, Jꢂ4.5, HꢀC(3)); 4.04 (dd, J¼11.4, 4.8, HaꢀC(1)); 3.92 (q, Jꢂ3.5, HꢀC(2)); 3.67 (dd,
J¼11.7, 3.6, HbꢀC(1)); 3.25 (d, J ¼ 6.3, 2 HꢀC(6)); 3.25 (br. s, exchange with D2O, NH2, 2 OH); 2.57 (t,
J ¼ 7.3, 2 HꢀC(1’)); 1.50–1.42 (m, 2 HꢀC(2’)); 1.32–1.22 (m, 16 H); 0.88 (t, J¼6.5, Me). 19F-NMR
(CDCl3, 300 MHz): ꢀ75.5 (s, CF3). 13C-NMR (CDCl3, 100 MHz): 157.58 (q, 2J(C,F) ¼ 36.7, C¼O);
131.81 (d, C(4)); 130.73 (d, C(5)); 116.30 (q, 1J(C,F) ¼ 286.0, CF3); 73.31 (d, C(3)); 61.18 (t, C(1)); 54.22
(d, C(2)); 51.12 (t, C(6)); 49.98 (t, C(1’)); 32.29 (t); 29.99–29.72 (several t); 27.64, 23.07 (2t); 14.49 (q,
Me). HR-ESI-MS: 397.2650 (100, [M þ H]þ , C19H36F3N2O3þ ; calc. 397.2678).
(E)-2-Amino-2,4,5,6-tetradeoxy-6-(undecylamino)-d-erythro-hex-4-enitol (7). A soln. of 24 (5 mg,
0.0126 mmol) in MeOH (1 ml) was treated with Amberlyst A-26 (OHꢀ form) (300 mg) and stirred at 258
for 7 h when MALDI-MS showed the complete conversion of 24. The mixture was filtered and the filtrate
evaporated to dryness. FC (NH2 silica gel, CH2Cl2/MeOH 1:0!85 :15) gave 7 (3.0 mg, 79%). [a]D25
¼
þ3.6 (c ¼ 0.135, CHCl3). IR (ATR): 3350w, 3279w, 3172w, 2953m, 2921s, 2852s, 1574w, 1464m, 1409w,
1377w, 1304w, 1260w, 1095w, 1027m, 974m, 867w, 802m, 720w. 1H-NMR (CDCl3, 400 MHz): 5.87 (dtd, J¼
15.6, 6.0, 1.0, HꢀC(5)); 5.68 (ddt, J¼15.5, 6.7, 1.3, HꢀC(4)); 4.11 (t, J¼6.0, HꢀC(3)); 3.70–3.61 (m,
2 HꢀC(1)); 3.27 (dd, J¼6.0, 0.4, 2 HꢀC(6)); 2.89 (q, J¼5.3, HꢀC(2)); 2.60 (t, J ¼ 7.3, 2 HꢀC(1’)); 1.88
(br. s, NH2, HOꢀC(1), HOꢀC(3), NH); 1.49 (quint., J ¼ 7.1, 2 H ꢀC(2’)); 1.34–1.26 (m, 18 H); 0.88 (t,
J¼6.9, Me). 13C-NMR (CDCl3, 100 MHz): 131.61 (d, C(4)); 131.20 (d, C(5)); 75.02 (d, C(3)); 64.20 (t,
C(1)); 56.08 (d, C(2)); 51.07 (t, C(6)); 49.65 (t, C(1’)); 31.90 (t); 29.98–29.33 (several t); 27.37, 22.67 (2t);
14.09 (q, Me). HR-MALDI-MS: 301.2850 (100, [M þ H]þ , C17H37N2O2þ ; calc. 301.2855).
2. Biological Tests. – 2.1. Phosphorylation by Sphingosine Kinase (SPHK). The phosphorylation
reactions were performed essentially as described in [28]. Briefly, the cytoplasmic fraction of
recombinant HEK-293 cells overexpressing human SPHK1 or SPHK2 was incubated at 308 in total
volumes of 100 ml with sphingosine derivatives (20 mm; added from stock solns. in DMSO), 1 mm of ATP,
and 2 mCi [g-32P]ATP in 50 mm Hepes buffer (pH 7.4) containing 15 mm MgCl2, 0.005% Triton X-100,
10 mm KCl, 10 mm NaF, and 1.5 mm semicarbazide. Following incubations for different time points up to
2 h, lipids were extracted and separated by TLC plates (Merck). Radiolabeled sphingosine phosphate
derivatives were visualized and quantified using a Molecular Dynamics Storm PhosphorImager
(Sunnyvale, CA). The rate of phosphorylation was calculated and is reported for the derivatives as
value relative to the rate for sphingosine (for which the rate was 41 and 25 nmol/(min·mg) with SPHK1
and SPHK2, resp.).
2.2. T-Cell Suppression: T-Cell Clones and Antigen-Presenting Cells (APC). Human CD1d-restricted
iNKT-cell clones were established and cultured as described in [29]. Human CD1d-transfected THP1
(THP1-CD1d) cells or monocyte-derived dendritic cells were used as antigen-presenting cells (APCs) in
the experiments.
Cytotoxicity Assays. To test cell toxicity sonicated compounds were incubated overnight with
different numbers of cells. Cells were labelled with 5 mg/ml propidium iodide (Sigma-Aldrich) or 7-
aminoactinomycin D (Invitrogen, Carlsbad CA), and cell death was assessed by flow cytometry on a
CYANTM ADP cytometer (Beckman Coulter, Fullerton CA).
Antigen Presentation and Competition Assays with Living APC. THP1-CD1d Cells (2.5·104/well) in
RPMI-1640 medium containing 10% fetal calf serum (FCS) were incubated during the assays at 378 with
sonicated compounds at the indicated concentrations in the presence of T cells (7.5·104/well). For
competition assays, a fixed dose of the compounds was given 4.5 h before addition of different doses of a-
GalCer. Supernatants were harvested after 24 h, and released cytokines were measured by ELISA.
Plate-Bound Human CD1d Activation and Competition Assays. All incubations were performed
overnight at r.t. with extensive washing between each step. Plates were coated with 10 mg/ml
BIR1.4 monoclonal antibody (mAb). Soluble recombinant human CD1d purified by isoelectric focusing
was incubated on BIR1.4-coated plates at twofold molar excess. Sonicated compounds were added and
given 4.5 h in advance of a-GalCer to plate-bound human CD1d before competition. T Cells (1.5·105/
well) were plated in RPMI-1640 medium containing 5% HS and 100 U/ml human IL-2. After 24 h at 378,
supernatants were harvested, and released cytokines were measured by ELISA.
ELISA. Plates coated with 8D4-8 (anti-human IL-4 mAb, BD), MAb1 (anti-human TNF-a mAb,
BD), 6804 (anti-human GM-CSF mAb, R&D), or HB-8700 (anti-human IFN-g mAb, ATCC) were
blocked, and then incubated with the freshly collected T-cell supernatants. Detection of cytokines was

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CHEMISTRY & BIODIVERSITY – Vol. 6 (2009)
performed using MP4-25D2 (anti-human IL-4 biotin labelled mAb, BD), MAb11 (anti-human TNF-a
biotin-labelled mAb, BD), 3209 (anti-human GM-CSF biotin labelled mAb, R&D), or g69 [30] (anti-
human IFN-g-biotin labelled mAb, provided by G. Garotta) with streptavidin-HRP (Zymed, Invitrogen)
and o-phenylenediamine dihydrochloride (Sigma, according to the manufacturerꢃs instructions) as
substrate.
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Received January 19, 2009