Synthesis and evaluation of sphinganine analogues of KRN7000 and OCH

Article abstract of DOI:10.1021/jo051147h

The phytosphingosine-containing α-galactosylceramides (α-GalCers), KRN7000 and OCH, have been shown to activate NKT cells via interaction with CD1d, a member of the CD1 family of antigen presenting proteins. Evidence from KRN7000 stimulation of NKT cells

Full text of DOI:10.1021/jo051147h

Synthesis and Evaluation of Sphinganine Analogues of KRN7000
and OCH
Rachel M. Ndonye,† Douglas P. Izmirian,† Matthew F. Dunn,† Karl O. A. Yu,‡
Steven A. Porcelli,‡ Archana Khurana,§ Mitchell Kronenberg,§ Stewart K. Richardson,† and
Amy R. Howell*,†
Department of Chemistry, University of Connecticut, Storrs, Connecticut 06269-3060, Department of
Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx,
New York 10461, and Division of Developmental Immunology, La Jolla Institute for Allergy and
Immunology, 10355 Science Center Drive, San Diego, California 92121
amy.howell@uconn.edu
Received June 7, 2005
The phytosphingosine-containing R-galactosylceramides (R-GalCers), KRN7000 and OCH, have been
shown to activate NKT cells via interaction with CD1d, a member of the CD1 family of antigen
presenting proteins. Evidence from KRN7000 stimulation of NKT cells suggests that R-GalCers
may have applications in the treatment or prevention of a range of viral, bacterial, and autoimmune
conditions. Moreover, OCH, a truncated analogue of KRN7000, appears to induce a TH2 bias, which
could have implications for the treatment of autoimmune and inflammatory conditions. We have
prepared the direct sphinganine-containing analogues of KRN7000 and OCH, 1 and 2, and found
them to be comparable in activity to the parent compounds in inducing the release of IL-2, IL-4,
and IFNγ. In addition, compound 2 leads to a cytokine bias similar to that seen with OCH. This is
significant because sphinganines are more easily accessed than phytosphingosines, which should
facilitate SAR studies.
Introduction
standing of the role of CD1d antigen presentation and
NKT cell function. Activation of NKT cells in vivo by
administering KRN7000 to mice leads to the production
of several cytokines, including IFNγ and IL-4. KRN7000
stimulation has provided evidence suggesting that NKT
cell-mediated pathways may be used to inhibit hepatitis
B virus replication5,6 or protect against diabetes,7-9
malaria,10-12 and tuberculosis.12 A truncated analogue of
Studies have identified the CD1 family of proteins as
novel, antigen-presenting molecules encoded by genes
located outside of those coding for major histocompat-
ibility complexes.1 CD1d is a member of the CD1 family
that has been shown to be an antigen presenting protein
for the T cell receptors (TCR) of natural killer (NK) T
cells.2-4 CD1d activated NKT cells have been demon-
strated to play a role in a variety of immune responses.
The R-galactosylceramide (R-GalCer), KRN7000, has
proved to be an invaluable tool for enhancing under-
(5) Baron, J. L.; Gardiner, L.; Nishimura, S.; Shinkai, K.; Locksley,
R.; Ganem, D. Immunity 2002, 16, 583-594.
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Med. 2000, 192, 921-930.
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holm, M.; Holmberg, D.; Cardell, S. L. J. Immunol. 2004, 173, 3112-
3118.
† University of Connecticut.
‡ Albert Einstein College of Medicine.
§ La Jolla Institute for Allergy and Immunology.
(1) Martin, L. H.; Calabi, F.; Milstein, C. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 9154-9158.
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O. V.; Miura, T.; Haba, T.; Scherer, D. C.; Wei, J.; Kronenberg, M.;
Koezuka, Y.; Van Kaer, L. Nature Med. (N.Y., NY, U.S.A.) 2001, 7,
1052-1056.
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J. R.; Brutkiewicz, R. R. Science 1995, 268, 863-865.
10.1021/jo051147h CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/05/2005
10260
J. Org. Chem. 2005, 70, 10260-10270

Sphinganine Analogues of KRN7000 and OCH
SCHEME 1
KRN7000, OCH, was found to selectively induce IL-4, as
opposed to IFNγ, and to offer protection in mice against
experimental autoimmune encephalomyelitis (EAE), an
animal model of multiple sclerosis.13 More recently, OCH
has been shown to offer protection against diabetes in
NOD mice14 and against collagen induced arthritis.15
These results illustrate the importance of the CD1d
pathway of immunomodulation, and they also demon-
strate the potential for using structural analogues of
KRN7000 to modulate the responses of NKT cells in
potentially useful ways.
To further elucidate the role of CD1d antigen presen-
tation and NKT cell responses, access to glycosyl cera-
mide tools is critical. In addition, the development of
more extensive libraries of R-GalCer structural variants
would be of great value in searching for compounds with
optimal bioactivities for a range of applications. Both
KRN7000 and OCH have a phytosphingosine (aminotriol)
sphingoid base moiety. The preparation of phytosphin-
gosines is not trivial.16 In contrast, sphinganines (ami-
nodiols) are generally more readily prepared.17 If sphin-
ganine-containing R-GalCers were shown to mirror the
potency and types of activation seen with phytosphin-
gosine-containing compounds, access to biological tools
and targeted structure-activity studies would be more
straightforward. In this paper we report the preparation
and initial biological evaluation of the direct sphinganine
analogues 1 and 2 of KRN7000 and OCH, respectively.
derived Weinreb amide 3 as a template.18 Thus, the
sphingoid bases 8a and 8b required for the synthesis of
R-GalCers 1 and 2 were prepared as shown in Scheme
1. Double deprotonation of 3 with the sacrificial base sec-
butylmagnesium chloride under conditions previously
described,18,19 followed by addition of n-pentadecylmag-
nesium bromide or n-hexylmagnesium bromide (prepared
from the corresponding alkylbromides), provided amino-
hydroxyketone 4a or 4b, respectively. Silyl protection of
4 gave ketones 5. Diastereoselective reduction of 5 with
lithium tri-tert-butoxyaluminum hydride under condi-
tions described by Hoffman20 produced anti-alcohols 6 in
excellent yields. Benzyl protection of the secondary
alcohol, followed by cleavage of both the Boc and silyl
protecting groups with trifluoroacetic acid (TFA), gave
the required protected sphinganines 8a and 8b.
The modest yield in the benzylation of 6 was found to
be a result of migration of the TBDMS moiety (∼30%
migration) to the secondary alcohol (giving 7′) under the
reaction conditions. Although 7a could be separated with
difficulty from 7a′ (and 8a could be readily separated
from 8a′), 7b and 7b′ were essentially inseparable, as
were 8b and 8b′. However, following acylation (Scheme
3), pure 11b was easily isolated. The yield quoted for 7b
1
is based on the H NMR ratios of 7b/7b′ (2:1) and the
combined isolated yield (86%). We have found that the
unwanted migration of the silyl group can be circum-
(14) Mizuno, M.; Masumura, M.; Tomi, C.; Chiba, A.; Oki, S.;
Yamamura, T.; Miyake, S. J. Autoimmun. 2004, 23, 293-300.
(15) Chiba, A.; Oki, S.; Miyamoto, K.; Hashimoto, H.; Yamamura,
T.; Miyake, S. Arthritis Rheum. 2004, 50, 305-313.
(16) Howell, A. R.; Ndakala, A. J. Curr. Org. Chem. 2002, 6, 365-
391.
Results and Discussion
We have recently reported straightforward access to
sphinganines and C3 protected sphinganines using serine-
(17) Howell, A. R.; So, R. C.; Richardson, S. K. Tetrahedron 2004,
60, 11327-11347.
(11) Gonzalez-Aseguinolaza, G.; Van Kaer, L.; Bergmann, C. C.;
Wilson, J. M.; Schmeig, J.; Kronenberg, M.; Nakayama, T.; Taniguchi,
M.; Koezuka, Y.; Tsuji, M. J. Exp. Med. 2002, 195, 617-624.
(12) Chackerian, A.; Alt, J.; Perera, V.; Behar, S. M. Inf. Immunol.
2002, 70, 6302-6309.
(13) Miyamoto, K.; Miyake, S.; Yamamura, T. Nature 2001, 413,
531-534.
(18) So, R. C.; Ndonye, R.; Izmirian, D. P.; Richardson, S. K.;
Guerrera, R. L.; Howell, A. R. J. Org. Chem. 2004, 69, 3233-3235.
(19) Liu, J.; Ikemoto, N.; Petrillo, D.; Armstrong, J. D., III Tetra-
hedron Lett. 2002, 43, 8223-8226.
(20) Hoffman, R. V.; Maslouh, N.; Cervantes-Lee, F. J. Org. Chem.
2002, 67, 1045-1056.
J. Org. Chem, Vol. 70, No. 25, 2005 10261

Ndonye et al.
SCHEME 2
SCHEME 3
vented by employing TBDPSCl, rather than TBDMSCl,
as illustrated for the conversion of 4a to 8a in Scheme 2.
Thus, after protection of 4 with a TBDPS group, reduc-
tion and benzylation of 9 proceeded in a straightforward
manner. No significant silyl migration was observed in
the benzylation step. One-step cleavage of the silyl- and
Boc-protecting groups of 10 was achieved by using in situ
generation of HCl.
Sphinganines 8 were converted to R-GalCer analogues
1 and 2 as shown in Scheme 3. Compound 8a was
acylated with the p-nitrophenyl (PNP) ester of hexa-
cosanoic acid, and 8b was similarly reacted with the
corresponding ester of tetracosanoic acid. The resulting
ceramides 11a and 11b, respectively, were glycosylated
with use of â-tetrabenzylgalactosyl fluoride in the pres-
ence of silver perchlorate and tin(II) chloride. Although
the R-selectivity was high, the yields were low. Global
deprotection of 12a and 12b provided analogues 1 and
2, respectively.
In identifying KRN7000 as a lead clinical candidate
several related sphinganine analogues were prepared and
assayed.21,22 Although the sphinganine-containing com-
pounds showed tumor inhibitory properties and compa-
rable abilities to stimulate mouse spleen cells, they were
not as potent as the aminotriols. More recently, two of
the Kirin aminodiols (C24 acyl/C18 sphinganine and C14
acyl/C16 sphinganine) were assayed for their ability to
induce the release of IL-2 in mouse VR14+ NKT cell
hybridomas,23 a response more directly related to CD1d-
mediated effects. The longer chain sphinganine-contain-
ing R-GalCer elicited a greater level of response than the
shorter analogue, but not as great a response as KRN7000,
consistent with the trend seen in evaluation of T cell
proliferation. However, the direct analogue 1 of KRN7000
was not evaluated.24 Moreover, in efforts to gain an
understanding of the role of CD1d in immune responses,
it is also appropriate to evaluate other markers related
to NKT-mediated responses. Since KRN7000 is the
R-GalCer used most widely in studies related to CD1d,
and since OCH has been shown to display both similari-
ties and differences compared to KRN7000, we decided
to compare the sphinganine analogues 1 and 2 of
KRN7000 and OCH for their ability to induce the release
of IL-2, IL-4, and IFNγ. These cytokines are well-known
products of activated NKT cells and are frequently used
in evaluating R-GalCers.
KRN7000 was available to us, and OCH25 was pre-
pared by using a combination of literature procedures,
as shown in Scheme 4. Garner’s aldehyde 13, prepared
in three steps as described by Taylor and co-workers,26
was converted by minor modifications of the procedure
of Imashiro et al.27 to ribo-phytosphingosine 14. Acylation
gave OCH ceramide 15. Following an approach similar
to that used by Savage and co-workers in preparing a
biotinylated KRN7000 from its ceramide,28 global protec-
tion of 15, followed by selective deprotection and glyco-
(24) A preparation of compound 1 and some cytokine data are
described in Biomera patent WO 2004028475.
(25) Murata, K.; Toba, T.; Nakanishi, K.; Takahashi, B.; Yamamura,
T.; Miyake, S.; Annoura, H. J. Org. Chem. 2005, 70, 2398-2401.
(26) Campbell, A. D.; Raynham, T. M.; Taylor, R. J. K. Synthesis
1998, 1707-1709.
(21) Morita, M.; Motoki, K.; Akimoto, K.; Natori, T.; Sakai, T.; Sawa,
E.; Yamaji, K.; Koezuka, Y.; Kobayashi, E.; Fukushima, H. J. Med.
Chem. 1995, 38, 2176-2187.
(22) Motoki, K.; Kobayashi, E.; Uchida, T.; Fukushima, H.; Koezuka,
Y. Bioorg. Med. Chem. Lett. 1995, 5, 705-710.
(27) Imashiro, R.; Sakurai, O.; Yamashita, T.; Horikawa, H. Tetra-
hedron 1998, 54, 10657-10670.
(23) Brossay, L.; Naidenko, O.; Burdin, N.; Matsuda, J.; Sakai, T.;
Kronenberg, M. J. Immunol. 1998, 161, 5124-5128.
(28) Zhou, X.-T.; Forestier, C.; Goff, R. D.; Li, C.; Teyton, L.;
Bendelac, A.; Savage, P. B. Org. Lett. 2002, 4, 1267-1270.
10262 J. Org. Chem., Vol. 70, No. 25, 2005

Sphinganine Analogues of KRN7000 and OCH
SCHEME 4
sylation gave protected OCH 16. Conversion to OCH was
achieved by a two-stage deprotection.
IL-2 production is typically measured to assess the
relative abilities of preparations of R-GalCers to stimulate
immortalized NKT cells (NKT cell hybridomas). The
relative potencies of KRN7000, OCH, 1, and 2 were
measured with use of the VR14+ iNKT hybridoma DN3A4-
1.2 and three different CD1d-expressing antigen present-
ing cells: A20.mCD1d (a B cell lymphoma), RMA-
S.mCD1d (a lymphoma), and JAWS II (a myeloid/
dendritic cell line). IL-2 production was measured by
ELISA from co-cultured supernatants of the hybridoma
and one of the three antigen presenting cells previously
pulsed with varying amounts of lipid. The relative
potencies of the lipids (based on reciprocal effective
concentrations for half-maximal response, 1/EC50) in the
presence of the RMA-S and JAWS II antigen presenting
cells (APC’s) showed the sphinganine analogues 1 and 2
to be essentially as potent as the parent phytosphin-
gosine-containing R-GalCers, KRN7000 and OCH (Figure
1). Moreover, the shorter chain analogue 2, like the
corresponding OCH, was slightly less effective at eliciting
IL-2 release than KRN7000. Similar results were ob-
tained by using A20.CD1d APCs, with all of the com-
pounds showing potent stimulation of NKT cells, even
in the 100 nM range (see the Supporting Information).
Thus, with respect to this assay for NKT cell activation,
the more easily accessible sphinganine-containing R-Gal-
Cers mirrored the activities of the corresponding phyto-
sphingosine-containing compounds.
FIGURE 1. Relative potencies of KRN7000, OCH, 1, and 2
in two CD1d-restricted antigen presentation systems. IL-2
production was measured from co-cultured supernatants of the
VR14+ iNKT hybridoma DN3A4-1.2 and one of two CD1d
antigen-presenting cells (RMA-S.mCD1d or JAWS II) previ-
ously pulsed with varying amounts of lipid. IL-2 levels were
fitted to 4-parameter dose/response curves. Relative potencies
of each lipid (reciprocal effective concentrations in nM for half-
maximal response, 1/EC50) are displayed for RMA-S.mCD1d
and JAWS II.
and residue Asp80 showed H-bonding with both the 3′-
and 4′-OH’s of the phytosphingosine. However, given the
similar responses of NKT cell hybridoma DN3A4-1.2 to
sphinganine analogues 1 and 2 compared to their
phytosphingosine counterparts, it is evident that the
interaction with the 4′-OH is not crucial to activity. It is
noteworthy that an analogue lacking both the 3′- and 4′-
OH’s on the sphingoid base has been shown to be
inactive.23
Immortalized T cell hybridomas tend to produce IL-2,
regardless of the cytokines that were produced by the
parent cell they were derived from. Therefore, assay of
the activation of T cell hybridomas, including those
derived from NKT cells, is generally restricted to mea-
surement of IL-2 secretion. By contrast, primary NKT
cells from mice and humans tend to produce IL-4 and
IFNγ when stimulated, plus a variety of other cytokines
and chemokines that have been less well studied. T cell
cytokine responses have been categorized into two broad
groups. TH1 responses, typified by the release of IFNγ
and several other cytokines, are important for fighting
These results are interesting in light of recently
published structural studies.29,30 Zajonc et al. recently
reported a crystal structure of mouse CD1d in complex
with a variant of KRN7000 shortened in the acyl chain.
Crucial H-bonding interactions were seen between CD1d
residue Arg79 and the 3′-OH of the phytosphingosine,
(29) Zajonc, D. M.; Cantu, C., III; Mattner, J.; Zhou, D.; Savage, P.
B.; Bendelac, A.; Wilson, I. A.; Teyton, L. Nat. Immunol. 2005, 6, 810-
818.
(30) Koch, M.; Stronge, V. S.; Shepherd, D.; Gadola, S. D.; Matthew,
B.; Ritter, G.; Fersht, A. R.; Besra, G. S.; Schmidt, R. R.; Jones, E. Y.;
Cerundolo, V. Nat. Immunol. 2005, 6, 819-826.
J. Org. Chem, Vol. 70, No. 25, 2005 10263

Ndonye et al.
FIGURE 2. C57BL/6 splenocytes were incubated with varying doses of lipid for 48 h. Supernatant levels of IL-4 and IFNγ were
measured by ELISA. Means of duplicate wells are shown; SEM were typically <10% of the mean.
intracellular infections by bacteria and viruses, and over-
exuberant TH1 responses are typical of several autoim-
mune diseases. By contrast, the secretion of IL-4 char-
acterizes TH2 responses. Although excessive TH2 responses
can cause allergy, they are important for stimulating
humoral immune responses and for the elimination of
several protozoan parasites. OCH, truncated in both the
acyl chain and sphingoid base in comparison to KRN7000,
generated intense interest because, although it was not
as potent as KRN7000 in inducing NKT cell responses,
it consistently induced a TH2 bias of NKT cell cytokine
responses, as reflected in the proportionately larger
production of IL-4 in comparison to IFNγ.13 In the
phytosphingosine series, recent results appear to dem-
onstrate that shortening of either the acyl or the sphin-
goid base chains induces a TH2 bias.31,32 This suggests
that OCH or related compounds (i.e., those that elicit an
increased ratio of IL-4 to IFNγ secretion) might be
potential chemotherapeutic agents for autoimmune or
inflammatory conditions mediated by TH1 cytokines, as
TH2 cytokines can inhibit production of TH1 cytokines and
in some cases reverse their effects. Moreover, ready
access to compounds that further probe this hypothesis
would be timely. To examine the effects of the sphinga-
nine compounds on the production of cytokines by NKT
cells, we measured the levels of IL-4 and IFNγ in
supernatants of mouse splenocytes cultured in the pres-
ence of the various R-GalCer analogues. Sphinganine
analogues 1 and 2, although not as potent, paralleled the
parent phytosphingosines in their production of IL-4 and
IFNγ (Figure 2). There was an indication of a TH2 bias
of both OCH and 2 in comparison to the longer chain
variants. For example, at the 100 nm dose, the ratio of
IL-4 to IFNγ production with OCH or 2 was approxi-
mately twice that observed for KRN7000 or 1 (Figure 2).
This was consistent with the similar degree of TH2
cytokine bias that has been seen in vitro in other
studies.13,31,32 It should be noted that previous studies of
the TH2 skewing effect of some R-GalCer analogues have
shown that this effect tends to be more pronounced in
vivo when analyzed by injection of compounds into mice
and measurement of serum cytokine levels.32,33
In summary, the results reported here support the
utility of sphinganine-containing R-GalCers for examin-
ing the role of R-GalCer analogues in modulating immune
response. The sphinganine-containing compounds are
more readily prepared. Although not quite as potent as
the corresponding phytosphingosine-containing R-Gal-
Cers in some assays, such as the in vitro splenocyted
stimulation assay, 1 and 2 mirror these compounds in
the cytokine responses elicited.
Experimental Section
[2-Hydroxy-1-(methoxymethylcarbamoyl)ethyl]-
carbamic Acid tert-Butyl Ester (3). Boc-L-serine (8.00 g,
39.0 mmol) was dissolved in dry CH2Cl2 (153 mL) and the
solution cooled to -15 °C under N2. N,O-Dimethylhydroxyl-
amine hydrochloride (3.92 g, 40.0 mmol) was added, followed
by N-methylmorpholine (4.42 mL, 40.2 mmol). After 5 min
1-(3-methylaminopropyl-3-ethylcarbodiiimide hyrdrochloride
(7.70 g, 40.2 mmol) was added in five portions over 30 min.
After being stirred for 1 h at -15 °C, the reaction was
quenched with HCl (1 M, 25 mL), and the layers were
separated. The aqueous layer was extracted with CH2Cl2 (3 ×
50 mL). The organic extracts were combined, washed with
saturated NaHCO3 (23 mL) and H2O (23 mL), dried (MgSO4),
and concentrated to provide 3 as a white solid (8.33 g, 86%):34
1H NMR (400 MHz, CDCl3) δ 5.71 (br s, 1H), 4.95 (br s, 1H),
3.82-3.78 (m, 5H), 3.23 (s, 3H), 2.90 (br s, 1H), 1.43 (s, 9H).
(2S)-2-(N-tert-Butoxycarbonyl)amino-1-hydroxyocta-
decan-3-one (4a). [2-Hydroxy-1-(methoxymethylcarbamoyl)-
ethyl]carbamic acid tert-butyl ester (3) (1.6 g, 6.5 mmol) was
dissolved in dry THF (13 mL) under N2. The resulting solution
was cooled to -15 °C and s-BuMgCl (2.0 M in Et2O, 6.5 mL,
13 mmol) was added dropwise, affording a clear solution. After
5 min, pentadecylmagnesium bromide (0.42 M in THF, 20 mL,
8.4 mmol) was added at -15 °C. The resulting solution was
allowed to warm to room temperature and stirred overnight.
The mixture was cooled to -15 °C, and HCl (1 M, 15 mL) was
added, followed by EtOAc (15 mL). The two layers were
separated and the aqueous layer extracted with CH2Cl2 (3 ×
30 mL). The organic extracts were combined, washed with H2O
(30 mL), dried (MgSO4), and concentrated. Purification by flash
chromatography on silica gel (petroleum ether/EtOAc 80:20)
afforded 4a as a white solid (1.8 g, 70%): mp 48-50 °C; [R]25
D
(31) Goff, R. D.; Gao, Y.; Mattner, J.; Zhou, D.; Yin, N.; Cantu, C.,
III; Teyton, L.; Bendelac, A.; Savage, P. A. J. Am. Chem. Soc. 2004,
126, 13602-13603.
+28.0 (c 1.0, CHCl3); IR (KBr) 3420, 2923, 2853, 1710, 1169
1
cm-1; H NMR (400 MHz, CDCl3) δ 5.48 (br s, 1H), 4.26 (m,
(32) Yu, K. O. A.; Im, J. S.; Molano, A.; Dutronc, Y.; Illarionov, P.
A.; Forestier, C.; Fujiwara, N.; Arias, I.; Miyake, S.; Yamamura, T.;
Chang, Y.-T.; Besra, G. S.; Porcelli, S. A. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 3383-3388.
(33) Yu, K. O. A.; Porcelli, S. A. Unpublished observations
(34) Bold, G. V.; Allmendinger, T.; Herold, P.; Moesch, L.; Schar, P.
H.; Duthaler, O. R. Helv. Chim. Acta 1992, 75, 865-882.
10264 J. Org. Chem., Vol. 70, No. 25, 2005

Sphinganine Analogues of KRN7000 and OCH
1H), 3.94 (m, 2H), 2.63 (br s, 1H), 2.55 (m, 2H), 1.57 (m, 2H),
2.51 (m, 2H), 1.58 (m, 2H), 1.43 (s, 9H), 1.30-1.25 (m, 6H),
0.87-0.84 (m, 12H), 0.01 (s, 6H); 13C NMR (100 MHz, CDCl3)
δ 208.1, 155.5, 79.8, 63.6, 61.4, 40.3, 31.7, 29.0, 28.5, 25.9, 23.5,
22.6, 18.3, 14.2, -5.4; MS (EI) m/z 314 (M+ - C5H10), 274, 218,
174, 73, 57 (100). Anal. Calcd for C20H41NO4Si: C, 61.97; H,
10.66; N, 3.61. Found: C, 62.24; H, 10.27; N, 3.77.
1.45 (s, 9H), 1.27-1.22 (m, 24H), 0.88 (t, J ) 6.8 Hz, 3H); 13
C
NMR (100 MHz, CDCl3) δ 208.1, 164.0, 80.3, 70.1, 68.9, 63.3,
61.6, 52.3, 49.8, 39.9, 31.9, 29.7, 29.6, 29.4, 29.3, 29.2, 28.3,
23.5, 22.7, 14.1; MS (EI) m/z 327 (M+ - t-Bu-CH3), 281, 239,
86, 57 (100). Anal. Calcd for C23H45NO4: C, 69.13; H, 11.35; N,
3.51. Found: C, 69.12; H, 11.06; N, 3.60.
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-bu-
tyldimethylsilanyloxyoctadecan-3-ol (6a). LiAl(Ot-Bu)3H
(19.0 g, 74.8 mmol) was added to dry EtOH (105 mL) at -78
°C under N2. Then, a solution of (2S)-2-(N-tert-butoxycarbonyl)-
amino-1-tert-butyldimethylsilanyloxyoctadecane-3-one (5a) (6.40
g, 12.5 mmol) in dry EtOH (105 mL) was added dropwise. After
being stirred for 6 h at -78 °C the reaction mixture was diluted
with CH2Cl2 (30 mL), and 10% citric acid (310 mL) was added.
This mixture was stirred for 1.5 h at room temperature then
extracted with CH2Cl2 (3 × 100 mL). The combined organic
layers were washed with H2O (100 mL) and brine (100 mL),
dried (MgSO4), and concentrated. Purification by flash chro-
matography on silica gel (petroleum ether/EtOAc 98:2 to 95:
(2S)-2-(N-tert-Butoxycarbonyl)amino-1-hydroxynonan-
3-one (4b). [2-Hydroxy-1-(methoxymethylcarbamoyl)ethyl]-
carbamic acid tert-butyl ester (3) (4.3 g, 17.5 mmol) was
dissolved in dry THF (60 mL) under N2. The resulting solution
was cooled to -15 °C and s-BuMgCl (2.0 M in Et2O, 17.5 mL,
35.0 mmol) was added dropwise. After 10 min hexylmagnesium
bromide (0.65 M in THF, 35.0 mL, 22.8 mmol) was added
dropwise at -15 °C. The resulting solution was allowed to
warm to room temperature and was stirred for 4 h. The gray
solution was cooled to -15 °C and saturated aqueous NH4Cl
(40 mL) was added slowly, in one portion, followed by EtOAc
(75 mL) and H2O (75 mL). The aqueous layer was extracted
with EtOAc (3 × 75 mL). The combined organic layers were
washed with H2O (2 × 75 mL), dried (MgSO4), and concen-
trated. Purification by flash chromatography on silica gel
(petroleum ether/EtOAc 90:10) provided 4b as a pale yellow
5) afforded 6a as a colorless oil (5.52 g, 86%): [R]25 +21.3 (c
D
1.0, CHCl3); IR (neat) 3448, 2925, 2854, 1698, 1499, 1468 cm-1
;
1H NMR (400 MHz, CDCl3) δ 5.32 (d, J ) 6.6 Hz, 1H), 3.97
(dd, J ) 10.6, 2.8 Hz, 1H), 3.83 (d, J ) 9.3 Hz, 1H), 3.65 (m,
1H), 3.51 (br s, 1H), 3.01 (br s, 1H), 1.53 (br s, 4H), 1.46 (s,
9H), 1.26 (m, 24H), 0.92 (s, 9H), 0.89 (t, J ) 7.7 Hz, 3H), 0.09
(s, 6H); 13C NMR (100 MHz, CDCl3) δ 155.9, 79.6, 74.5, 63.7,
54.1, 35.1, 32.1, 29.9, 29.8, 29.8, 29.6, 28.6, 26.2, 26.0, 22.9,
18.3, 14.3, -5.4, -5.4. Anal. Calcd for C29H61NO4: C, 67.52;
H, 11.92; N, 2.72. Found: C, 67.89; H, 12.17; N, 3.07.
oil (3.6 g, 75%): [R]25 +36.3 (c 0.5, CHCl3); IR (neat) 3422,
D
1
2931, 1709, 1500 cm-1; H NMR (400 MHz, CDCl3) δ 5.67 (d,
J ) 5.6 Hz, 1H), 4.32 (br s, 1H), 3.94 (dd, J ) 11.2, 3.2 Hz,
1H), 3.88 (dd, J ) 11.2, 3.6 Hz, 1H), 2.81 (br s, 1H), 2.55 (m,
2H), 1.58 (m, 2H), 1.44 (s, 9H), 1.34-1.21 (m, 6H), 0.87 (t, J )
6.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 208.3, 156.2, 80.4,
63.3, 61.8, 40.1, 31.7, 29.0, 28.5, 23.6, 22.6, 14.2; MS (EI) m/z
200 (M+ - C5H10), 160, 113, 104, 60, 57 (100). Anal. Calcd for
C14H27NO4: C, 61.51; H, 9.96; N, 5.12. Found: C, 61.70; H,
9.67; N, 5.01.
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-bu-
tyldimethylsilanyloxynonan-3-ol (6b). LiAl(Ot-Bu)3H (23.2
g, 91.3 mmol) was added in six portions over 15 min to a
solution of (2S)-2-(N-tert-butoxycarbonyl)amino-1-tert-butyldi-
methylsilanyloxynonan-3-one (5b) (7.10 g, 18.3 mmol) in dry
EtOH (300 mL) at -78 °C under N2. After being stirred for 5
h, the mixture was quenched slowly with aqueous citric acid
(10%, 100 mL). The mixture was warmed to room temperature
and allowed to stir for 1.5 h. The mixture was then diluted
with H2O (1 L) and CH2Cl2 (400 mL). The aqueous layer was
extracted with CH2Cl2 (3 × 200 mL). The combined organic
layers were washed with H2O (3 × 200 mL) and brine (200
mL), dried (MgSO4), and concentrated. Purification by flash
chromatography on silica gel (petroleum ether/EtOAc 95:5)
(2S)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldi-
methylsilanyloxyoctadecan-3-one (5a). A catalytic amount
of DMAP was added to a solution of (2S)-2-(N-tert-butoxycar-
bonyl)amino-1-hydroxyoctadecan-3-one (4a) (6.00 g, 15.0 mmol)
and imidazole (3.07 g, 45.1 mmol) in dry DMF (17 mL) under
N2. After 20 min TBDMSCl (2.71 g, 18.0 mmol) was added,
and the reaction was stirred overnight. The reaction mixture
was diluted with saturated aqueous NH4Cl (39 mL) and the
aqueous layer extracted with CH2Cl2 (3 × 30 mL). The organic
extracts were combined, washed with H2O (30 mL) and brine
(30 mL), dried (MgSO4), and concentrated. Purification by flash
chromatography on silica gel (petroleum ether/EtOAc 98:2 to
95:5) provided 5a as a colorless oil (6.42 g, 83%): [R]25D +36.6
(c 1.0, CHCl3); IR (neat) 3432, 2926, 2855, 1712, 1491, 1470
gave 6b as a colorless oil (5.7 g, 80%): [R]25 +34.5 (c 0.5,
D
CHCl3); IR (CDCl3) 3448, 2930, 1697, 1501 cm-1; 1H NMR (400
MHz, CDCl3) δ 5.32 (d, J ) 7.2 Hz, 1H), 3.97 (dd, J ) 10.4,
2.4 Hz, 1H), 3.83 (d, J ) 9.6 Hz, 1H), 3.66 (m, 1H), 3.51 (br s,
1H), 3.02 (br s, 1H), 1.54 (br s, 2H), 1.46 (s, 9H), 1.32-1.30
(m, 8H), 0.94-0.87 (m, 12H), 0.09 (s, 6H); 13C NMR (100 MHz,
CDCl3) δ 155.9, 79.5, 74.4, 63.7, 54.1, 35.1, 32.0, 29.5, 28.6,
26.1, 26.0, 22.8, 18.3, 14.3, -5.4, -5.4; MS (EI) m/z 316, 276
(M+ - C7H14O), 218, 174, 144, 116, 75, 57 (100). Anal. Calcd
for C20H43NO4Si: C, 61.65; H, 11.12; N, 3.59. Found: C, 61.67;
H, 10.79; N, 3.78.
1
cm-1; H NMR (400 MHz, CDCl3) δ 5.49 (d, J ) 7.0 Hz, 1H),
4.26 (t, J ) 3.3 Hz, 1H), 4.05 (d, J ) 10.3 Hz, 1H), 3.82 (dd, J
) 10.2, 3.6 Hz, 1H), 2.62-2.43 (m, 2H), 1.58 (m, 2H), 1.45 (s,
9H), 1.26 (br s, 24H), 0.88 (t, J ) 5.7 Hz, 3H), 0.86 (s, 9H),
0.03 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 208.0, 155.5, 79.8,
63.6, 61.3, 40.3, 32.1, 29.8, 29.8, 29.8, 29.6, 29.6, 29.5, 29.4,
28.5, 25.9, 23.5, 22.9, 18.3, 14.3, -5.4. Anal. Calcd for C29H59-
NO4Si: C, 67.78; H, 11.57; N, 2.73. Found: C, 67.39; H, 11.18;
N, 3.08.
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-bu-
tyldimethylsilanyloxy-3-benzyloxyoctadecane (7a). (2S,-
3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldimethyl-
silanyloxyoctadecan-3-ol (6a) (5.40 g, 10.5 mmol) was dissolved
in dry DMF (35 mL), and the solution was cooled to 0 °C and
stirred under N2. Tetrabutylammonium iodide (6.17 g, 16.7
mmol) was added, followed by NaH (60% in mineral oil, 0.50
g, 20.9 mmol). After 10 min benzylbromide (2.80 g, 16.7 mmol)
was added dropwise via syringe. After the addition the cooling
bath was removed, and the reaction mixture was stirred at
room temperature for 2 h. The reaction was quenched with
saturated aqueous NH4Cl (90 mL), and the mixture was
extracted with Et2O (4 × 100 mL). The combined organic
extracts were washed with H2O (100 mL) and brine (100 mL),
dried (MgSO4), and concentrated. Purification by flash chro-
matography on silica gel (petroleum ether/EtOAc 99:1 to 97:
(2S)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldi-
methylsilanyloxynonan-3-one (5b). A catalytic amount of
DMAP was added to a solution of (2S)-2-(N-tert-butoxycarbo-
nyl)amino-1-hydroxynonan-3-one (4b) (3.2 g, 11.5 mmol) and
imidazole (2.4 g, 34.6 mmol) in dry DMF (12 mL) under N2.
After 10 min TBDMSCl (2.1 g, 13.9 mmol) was added in one
portion and the mixture was stirred for 24 h. The mixture was
slowly diluted with saturated NH4Cl (30 mL), and the solution
was extracted with CH2Cl2 (3 × 75 mL). The combined organic
layers were washed with H2O (75 mL) and brine (75 mL), dried
(MgSO4), and concentrated. Purification by flash chromatog-
raphy on silica gel (petroleum ether/EtOAc 100:0 to 98:2)
provided 5b as a colorless oil (4.2 g, 93%): [R]25D +33.0 (c 0.5,
CHCl3); IR (CDCl3) 3433, 2930, 1712, 1492 cm-1; 1H NMR (400
MHz, CDCl3) δ 5.47 (d, J ) 6.8 Hz, 1H), 4.25 (m, 1H), 4.03
(dd, J ) 10.4, 2.8 Hz, 1H), 3.80 (dd, J ) 10.4, 4.0 Hz, 1H),
3) afforded 7a as a clear oil (3.3 g, 53%): [R]25 +5.66 (c 1.0,
D
CHCl3); IR (neat) 3450, 3031, 2925, 2854, 1720, 1497 cm-1
;
J. Org. Chem, Vol. 70, No. 25, 2005 10265

Ndonye et al.
1H NMR (400 MHz, CDCl3) δ 7.32 (m, 5H), 4.65 (br s, 1H),
4.49 (s, 2H), 3.79 (m, 2H), 3.58 (m, 1H), 3.47 (br s, 1H), 1.49
(m, 2H), 1.39 (s, 9H), 1.21 (br s, 26H), 0.85 (s, 9H), 0.82 (t, J
) 4.5 Hz, 3H), 0.00 (s, 6H); 13C NMR (100 MHz, CDCl3) δ
155.8, 138.9, 128.8, 128.3, 127.8, 79.2, 78.6, 72.6, 62.2, 53.8,
32.1, 30.7, 30.1, 29.9, 29.9, 29.8, 29.8, 29.6, 28.6, 25.5, 25.1,
22.9, 18.4, 18.3, 14.3, -4.3, -5.2. Anal. Calcd for C36H67NO4-
Si: C, 71.35; H, 11.14; N, 2.31. Found: C, 71.21; H, 11.22; N,
2.44.
matography on silica gel (petroleum ether/EtOAc 90:10) af-
forded 9 as a clear oil (5.1 g, 88%): [R]25D +32.4 (c 2.28, CHCl3);
IR (neat) 3433, 3072, 3050, 2925, 2855, 1712, 1499, 1172, 1113
cm-1; 1H NMR (400 MHz, CDCl3) δ 7.60 (m, 4H), 7.39 (m, 6H),
5.53 (d, J ) 7.7 Hz, 1H), 4.33 (m, 1H), 4.04 (dd, J ) 10.6, 3.1
Hz, 1H), 3.90 (dd, J ) 10.9, 3.8 Hz, 1H), 2.51 (m, 2H), 1.58
(m, 2H), 1.44 (s, 9H), 1.26 (m, 24H), 1.03 (s, 9H), 0.89 (t, J )
7.0 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 207.4, 155.3, 135.5,
132.8, 129.9, 129.9, 127.8, 79.5, 64.2, 61.1, 60.3, 41.3, 40.0, 31.9,
29.7, 29.6, 29.6, 29.4, 29.4, 29.3, 29.2, 29.0, 28.3, 27.6, 26.7,
26.2, 23.3, 22.7, 22.6, 20.9, 20.4, 19.4, 19.2, 18.7, 14.3; HRMS
(FAB) calcd for C39H64NO4Si (M+ + H) 638.4605, found
638.4600. Anal. Calcd for C39H63NO4Si: C, 73.42; H, 9.95; N,
2.20. Found: C, 73.16; H, 9.58; N, 2.31.
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-bu-
tyldimethylsilanyloxy-3-benzyloxynonane (7b). (2S,3R)-
2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldimethylsilanyl-
oxynonan-3-ol (6b) (0.65 g, 1.66 mmol) was dissolved in dry
DMF (7 mL), and the solution was cooled to 0 °C under N2.
Tetrabutylammonium iodide (0.92 g, 2.49 mmol) was added
at once, followed by the dropwise addition of benzylbromide
(0.29 mL, 2.59 mmol). NaH (60% in mineral oil, 0.09 g, 2.32
mmol) was then added. The cloudy mixture was stirred at 0
°C for 20 min and then allowed to warm to room temperature
and stirred for 2 h. The pale yellow solution was diluted with
saturated aqueous NH4Cl (90 mL), and the mixture was
extracted with Et2O (4 × 100 mL). The combined organic
extracts were washed with H2O (100 mL) and brine (100 mL),
dried (MgSO4), and concentrated. Purification by flash chro-
matography on silica gel (petroleum ether/EtOAc 100:0 to 98:
2) provided a pale yellow oil that was an inseparable mixture
of regioisomers 7b and 7b′ (2:1 based on 1H NMR) (0.68 g,
86%). 7b: 1H NMR (500 MHz, CDCl3) δ 7.33-7.28 (m, 5H),
4.71 (br s, 1H), 4.55 (s, 2H), 3.84 (br s, 2H), 3.64 (br s, 1H),
3.53 (br s, 1H), 1.44 (s, 9H), 1.27 (br s, 10H), 1.04-0.67 (m,
12H), 0.06 (s, 6H); 13C NMR (125 MHz, CDCl3) δ 155.8, 138.9,
128.5, 128.0, 127.7, 79.2, 78.6, 72.3, 62.2, 53.8, 32.0, 30.7, 29.7,
28.6, 26.1, 25.4, 22.8, 18.4, 14.3, -5.20, -5.31.
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyl-
diphenylsilanyloxyoctadecan-3-ol. Dry EtOH (17 mL) was
cooled to -78 °C and stirred under N2 for 20 min. LiAl(O-t-
Bu)3H (3.1 g, 12.4 mmol) was added at -78 °C and stirred for
20 min. (2S)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldiphe-
nylsilanyloxyoctadecan-3-one (9) (1.32 g, 2.1 mmol) in dry
EtOH (17 mL) was added dropwise to the stirred solution. The
temperature was maintained at -78 °C, and the mixture was
stirred for 6 h. Aqueous citric acid (10%, 50 mL) was added,
and the reaction mixture was allowed to warm to room
temperature over 1.5 h. The cloudy suspension was extracted
with CH2Cl2 (3 × 100 mL). The organic extracts were com-
bined, washed with H2O (100 mL) and brine (100 mL), dried
(MgSO4), and concentrated. Purification by flash chromatog-
raphy on silica gel (petroleum ether/EtOAc 95:5) afforded
(2S,3R)-2-(N-tert-butoxycarbonyl)amino-1-tert-butyldiphenyl-
silanyloxyoctadecan-3-ol as a clear oil (1.1 g, 82%): [R]25D +17.4
(c 1.35, CHCl3); IR (neat) 3447, 3071, 3050, 2925, 1698, 1499,
1
1172, 1113 cm-1; H NMR (300 MHz, CDCl3) δ 7.62 (m, 4H),
7.41 (m, 6H), 5.30 (d, J ) 8.1 Hz, 1H), 3.93 (m, 1H), 3.90 (m,
1H), 3.67 (m, 1H), 3.57 (br s, 1H), 2.88 (d, J ) 8.1 Hz, 2H),
1.44-1.06 (m, 45H), 0.87 (t, J ) 7.0 Hz, 3H); 13C NMR (75
MHz, CDCl3) δ 155.9, 135.8, 132.8, 132.7, 130.2, 130.1, 128.1,
128.0, 79.6, 74.0, 64.4, 54.7, 34.7, 32.1, 31.1, 29.8, 29.6, 28.6,
27.1, 26.1, 22.9, 19.4, 14.3; HRMS (FAB) calcd for C39H66NO4-
Si (M+ + H) m/z 640.4761, found 640.4738. Anal. Calcd for
C39H65NO4Si: C, 73.19; H, 10.24; N, 2.19. Found: C, 73.30;
H, 10.17; N, 2.36.
(2S,3R)-2-Amino-3-benzyloxyoctadecan-1-ol (8a). Tri-
fluoroacetic acid and H2O (19:1, 20 mL) were added to (2S,3R)-
2-(N-tert-butoxycarbonyl)amino-1-tert-butyldimethylsilanyloxy-
3-benzyloxyoctadecane (7a) (4.50 g, 7.60 mmol), and the
solution was stirred under N2 for 2 h. Saturated NaHCO3 was
added dropwise to pH ∼8.0; then, the solution was extracted
with CH2Cl2 (4 × 100 mL). The combined organic extracts were
washed with H2O (100 mL), dried (MgSO4), and concentrated
to give 8a as a pale yellow oil (2.74 g, 93%): [R]25 -8.90 (c
D
1.0, CHCl3); IR (neat) 3351, 3285, 2916, 1647, 1602, 1469 cm-1
;
(2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyl-
diphenylsilanyloxy-3-benzyloxyoctadecane (10). (2S,3R)-
2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldiphenylsilanyl-
oxyoctadecan-3-ol (1.33 g, 2.1 mmol) was dissolved in dry DMF
under N2 (10 mL), and tetrabutylammonium iodide (1.15 g,
3.1 mmol) and benzyl bromide (0.53 g, 3.1 mmol) were added.
The reaction mixture was then cooled to 0 °C, and NaH (60%
in mineral oil, 62 mg, 2.6 mmol) was added in two portions.
The reaction mixture was stirred for 45 min at 0 °C and then
at room temperature for 45 min. EtOAc (250 mL) was added,
and the solution was washed with saturated aqueous NH4Cl
(50 mL), H2O (4 × 50 mL), and brine (50 mL), dried (MgSO4),
and concentrated. Purification by flash chromatography on
silica gel (petroleum ether/EtOAc 99:1) afforded 10 as a clear
1H NMR (400 MHz, CDCl3) δ 7.31 (m, 5H), 4.50 (m, 2H), 3.69
(m, 3H), 3.01 (br s, 1H), 2.47 (br s, 3H), 1.35 (m, 2H), 1.20 (m,
26H), 0.82 (t, J ) 5.9 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ
138.6, 128.7, 128.0, 128.0, 82.5, 72.5, 63.7, 54.5, 32.1, 30.7, 30.1,
29.9, 29.9, 29.8, 29.8, 29.6, 25.6, 22.9, 14.3; MS (EI) m/z 250,
221, 168, 112, 91(100), 84; HRMS (FAB) calcd for C25H46NO2
(M+ + H) 392.3529, found 392.3510.
(2S,3R)-2-Amino-3-benzyloxynonan-1-ol (8b). TFA (11.4
mL) and H2O (0.6 mL) were added to a mixture of (2S,3R)-2-
(N-tert-butoxycarbonyl)amino-1-tert-butyldimethylsilanyloxy-
3-benzyloxynonane (7b) and its regioisomer 7b′. The yellow
solution was stirred for 2 h. Most of the TFA was evaporated
at 30 °C under reduced pressure. Residual TFA was neutral-
ized with aqueous NaOH (7.5% w/v, 75 mL). The mixture was
extracted with EtOAc (4 × 60 mL). The combined organic
extracts were washed with NaOH (7.5%, 2 × 50 mL), dried
(MgSO4), and concentrated. Purification on silica gel (CH2Cl2/
MeOH 95:5) provided an inseparable mixture of regioisomers
8b and 8b′ as a light yellow oil (0.42 g, 65%). The mixture
was used directly in the synthesis of 11b.
(2S)-2-(N-tert-Butoxycarbonyl)amino-1-tert-butyldiphen-
ylsilanyloxyoctadecan-3-one (9). DMAP (0.23 g, 0.18 mmol)
was added to a stirred solution under N2 of (2S)-2-(N-tert-
butoxycarbonylamino)-1-hydroxyoctadecan-3-one (4a) (3.7 g,
9.2 mmol) and imidazole (1.9 g, 28 mmol) in dry DMF (25 mL).
TBDPSCl (3.1 g, 11 mmol) was then added dropwise, and the
reaction mixture was stirred overnight, then diluted with
EtOAc (400 mL). The solution was washed with H2O (6 × 75
mL), dried (MgSO4), and concentrated. Purification by chro-
oil (1.6 g, 80%): [R]25 +8.47 (c 3.05, CHCl3); IR (neat) 3450,
D
3070, 3048, 3030, 2925, 2854, 1719, 1497, 1365, 1172, 1112
1
cm-1; H NMR (400 MHz, CDCl3) δ 7.65 (m, 4H), 7.63-7.27
(m, 11H), 4.71 (d, J ) 8.0 Hz, 1H), 4.54 (d, J ) 11.3 Hz, 1H),
4.49 (d, J ) 11.3 Hz, 1H), 3.86 (m, 2H), 3.72 (dd, J ) 9.0, 4.1
Hz, 1H), 3.58 (m, 1H), 1.42 (s, 9H), 1.42 (s, 2H), 1.26 (br s,
26H), 1.05 (s, 9H), 0.88 (t, J ) 7.0 Hz, 3H); 13C NMR (100 MHz,
CDCl3) δ 155.8, 138.8, 135.9, 133.5, 130.0, 128.5, 128.0, 127.7,
79.3, 79.1, 72.3, 63.1, 53.7, 32.2, 30.7, 30.1, 29.9, 29.9, 29.8,
29.6, 28.6, 27.1, 25.6, 22.9, 19.5, 14.4; HRMS (FAB) calcd for
C46H72NO4Si (M+ + H) 730.5231, found 730.5246.
(2S,3R)-2-Amino-3-benzyloxyoctadecan-1-ol (8a). Acetyl
chloride (6.35 mL, 73.3 mmol) was added to MeOH (63 mL),
and the solution was allowed to cool to room temperature
under N2. (2S,3R)-2-(N-tert-Butoxycarbonyl)amino-1-tert-bu-
tyldiphenylsilanyloxy-3-benzyloxyoctadecane (10) (1.6 g, 2.12
10266 J. Org. Chem., Vol. 70, No. 25, 2005

Sphinganine Analogues of KRN7000 and OCH
mmol) dissolved in dry Et2O (63 mL) was added, and the
solution was stirred for 2 d at room temperature. The reaction
mixture was concentrated then taken up in CH2Cl2 (10 mL),
and saturated NaHCO3 (20 mL) was carefully added, followed
by NaOH (1 M, 10 mL). The solution was stirred for 1 h. The
solution was then diluted with CH2Cl2 (100 mL), and the two
layers were separated. The aqueous layer was further ex-
tracted with CH2Cl2 (3 × 50 mL). The combined organic
extracts were dried (MgSO4) and concentrated. Purification
by flash chromatography on silica gel (CH2Cl2/MeOH 90:10)
afforded 8a as a pale yellow oil (0.59 g, 71%) giving the same
spectral characteristics as described above.
2H), 1.52 (br s, 4H), 1.26 (br s, 70H), 0.89 (t, J ) 6.4 Hz, 6H);
13C NMR (100 MHz, CDCl3) δ 173.0, 138.9, 138.7, 128.6, 128.6,
128.6, 128.5, 128.4, 128.0, 128.0, 127.9, 127.9, 127.7, 127.7,
99.7, 79.0, 78.4, 75.1, 74.9, 73.7, 73.6, 73.2, 72.2, 70.2, 37.0,
32.1, 30.2, 29.9, 29.9, 29.9, 29.8, 29.7, 29.6, 25.9, 25.2, 22.9,
14.3. Anal. Calcd for C85H129NO8: C, 78.96; H, 10.06; N, 1.08.
Found: C, 78.78; H, 9.71; N, 1.36.
(2S,3R)-3-Benzyloxy-2-(N-tetracosanoylamino)-1-(2,3,4,6-
tetra-O-benzyl-R-D-galactopyranosyl)nonane (12b). (2S,-
3R)-3-Benzyloxy-2-(N-tetracosanoylamino)-1-nonanol (11b) (0.27
g, 0.44 mmol) was dissolved in dry THF (6.5 mL) and stirred
under N2. Freshly ground molecular sieves (4Å, 1.64 g), silver
perchlorate (0.27 g, 1.31 mmol), and tin(II) chloride (0.25 g,
1.31 mmol) were added successively to give a mixture that was
stirred for 30 min. The mixture was cooled to -15 °C, and a
solution of â-tetrabenzylgalactosyl fluoride (0.35 g, 0.66 mmol),
in dry THF (6.5 mL), was added dropwise. After 10 min, the
mixture was allowed to warm to room temperature and was
stirred for 2 h. The mixture was filtered through Celite and
the filter cake washed with Et2O. The filtrate was washed with
brine (2 × 50 mL), dried (MgSO4), and concentrated. Purifica-
tion by flash chromatography on silica gel (petroleum ether/
EtOAc 95:5 to 90:10) provided 12b as a white solid (0.11 g,
15%): [R]25D +21.1 (c 0.5, CHCl3); IR (NaCl) 3321, 2919, 2850,
1638 cm-1; 1H NMR (400 MHz, CDCl3) δ 7.38-7.25 (m, 25H),
6.07 (d, J ) 8 Hz, 1H), 4.92 (d, J ) 12 Hz, 1H), 4.82-4.35 (m,
10H), 4.16-3.88 (m, 7H), 3.68-3.34 (m, 4H), 2.00 (m, 2H),
1.51-1.22 (m, 52H), 0.88-0.83 (m, 6H); 13C NMR (125 MHz,
CDCl3) δ 173.0, 138.9, 138.9, 138.7, 138.7, 137.9, 128.6, 128.6,
128.6, 128.5, 128.4, 128.1, 128.0, 127.9, 127.9, 127.8, 127.7,
99.8, 79.0, 78.4, 75.1, 74.9, 73.7, 73.6, 73.2, 72.2, 70.2, 69.6,
69.3, 51.7, 37.0, 32.1, 32.0, 30.9, 29.9, 29.9, 29.8, 29.7, 29.6,
29.6, 25.9, 25.1, 22.9, 22.8, 14.3, 14.3; HRMS (FAB) calcd for
C74H108NO8 (M+ + H) 1138.8075, found 1138.8070.
(2S,3R)-1-(R-D-Galactopyranosyl)-2-hexacosanoylami-
nooctadecan-3-ol (1). Pd(OH)2 (20% on carbon, 123 mg)
was added to a stirred solution of (2S,3R)-3-benzyloxy-2-
(N-hexacosanoylamino)-1-(2,3,4,6-tetra-O-benzyl-R-d-galacto-
pyranosyl)octadecane (12a) (30 mg, 0.025 mmol) in ethanol
(1.6 mL) and CHCl3 (0.4 mL). The mixture was placed under
a H2 atmosphere, and stirring was continued vigorously for
24 h. The mixture was filtered through Celite, and the filter
cake was washed with CHCl3 and MeOH. The filtrate was
evaporated, and purification by flash chromatography on silica
gel (CH2Cl2/MeOH 95:5 to 90:10) provided 1 as a white solid
(17.5 mg, 93%): mp 168-170 °C; [R]24D +39.8 (c 0.5, pyridine);
1H NMR (400 MHz, pyr-d5) δ 8.54 (d, J ) 8.7 Hz, 1H), 5.48 (br
s, 5H), 5.08 (br s, 2H), 4.68 (br s, 1H), 4.57 (dd, J ) 6.7, 2.4
Hz, 2H), 4.52 (m, 4H), 4.48 (m, 1H), 4.44 (br s, 1H), 2.51 (m,
2H), 1.87 (m, 4H), 1.47 (br s, 70H), 0.90 (t, J ) 4.9 Hz, 6H);
13C NMR (100 MHz, pyr-d5) δ 173.8, 102.5, 73.5, 72.3, 72.0,
71.4, 70.9, 70.1, 63.1, 55.3, 37.2, 35.5, 32.5, 32.5, 30.6, 30.5,
30.4, 30.3, 30.3, 30.2, 30.1, 30.0, 30.0, 27.0, 26.8, 23.3, 14.7;
HRMS (FAB) calcd for C50H100NO8 (M+ + H) 842.7449, found
842.7464.
(2S,3R)-3-Benzyloxy-2-(N-hexacosanoylamino)octade-
can-1-ol (11a). p-Nitrophenyl hexacosanoate (1.26 g, 2.43
mmol) was added to a solution under N2 of (2S,3R)-2-amino-3-
benzyloxyoctadecan-1-ol (8a) (0.80 g, 2.03 mmol) in dry pyri-
dine (40 mL). The reaction was left to stir for 18 h. The solution
was concentrated, and the residue was purified by flash
chromatography on silica gel (petroleum ether/EtOAc 90:10
to 70:30) to provide 11a as a white solid (1.11 g, 70%): mp
85-87 °C; [R]25 -24.6 (c 1.0, CHCl3); IR (KBr) 3317, 3033,
D
2919, 2849, 1638, 1549 cm-1
;
1H NMR (400 MHz, CDCl3) δ
7.33 (m, 5H), 6.09 (d, J ) 7.2 Hz, 1H), 4.62 (d, J ) 11.6 Hz,
1H), 4.36 (d, J ) 11.6 Hz, 1H), 3.94 (m, 2H), 3.63 (br s, 1H),
3.55 (d, J ) 9.1 Hz, 1H), 2.04 (m, 2H), 1.68 (m, 1H), 1.53 (m,
3H), 1.22 (br s, 70H), 0.85 (t, J ) 5.6 Hz, 6H); 13C NMR (100
MHz, CDCl3) δ 173.6, 138.2, 128.9, 128.3, 128.1, 82.2, 73.0,
62.4, 52.3, 36.9, 32.1, 31.6, 30.0, 29.9, 29.8, 29.7, 29.6, 29.5,
25.9, 25.9, 22.9, 14.3. Anal. Calcd for C51H95NO3: C, 79.52; H,
12.43; N, 1.82. Found: C, 79.54; H, 12.31; N, 1.87.
(2S,3R)-3-Benzyloxy-2-(N-tetracosanoylamino)-1-
nonanol (11b). (2S,3R)-2-Amino-3-benzyloxynonan-1-ol (8b)
(0.22 g, 0.82 mmol) was dissolved in dry pyridine (5 mL) under
N2. p-Nitrophenyl tetracosanoate (0.48 g, 0.98 mmol) was
added in one portion. An additional portion of dry pyridine (2
mL) was added to aid in dissolution. After being stirred for 24
h, the mixture was diluted with EtOAc (50 mL) and washed
with HCl (1 M, 3 × 50 mL), NaOH (7.5% w/v, 3 × 75 mL), and
H2O (2 × 50 mL). The organic layer was dried (MgSO4) and
concentrated. Purification on silica gel (petroleum ether/EtOAc
70:30 to 60:40) provided 11b as a white solid (0.27 g, 53%):
mp 87.0-88.5 °C; [R]25 -28.4 (c 0.5, CHCl3); IR (KBr) 3439,
D
1
2928, 2842, 1633 cm-1; H NMR (400 MHz, CDCl3) δ 7.43-
7.28 (m, 5H), 6.13 (d, J ) 4 Hz, 1H), 4.65 (d, J ) 12 Hz, 1H),
4.40 (d, J ) 12 Hz, 1H), 3.98 (br s, 2H), 3.74-3.51 (m, 2H),
3.18 (br s, 1H), 2.18-1.98 (m, 2H), 1.77-0.78 (m, 58H); 13C
NMR (100 MHz, CDCl3) δ 173.6, 138.2, 128.9, 128.3, 128.1,
82.2, 73.0, 62.4, 52.3, 37.0, 32.1, 32.0, 31.6, 30.0, 29.9, 29.7,
29.6, 29.6, 29.5, 25.9, 22.9, 22.8, 14.3, 14.3; HRMS (FAB) calcd
for C40H74NO3 (M+ + H) 616.5669, found 616.5647.
(2S,3R)-3-Benzyloxy-2-(N-hexacosanoylamino)-1-(2,3,4,6-
tetra-O-benzyl-R-D-galactopyranosyl)octadecane (12a).
Tin(II) chloride (0.2 g, 1.05 mmol), silver perchlorate (0.22 g,
1.06 mmol), and freshly ground 4Å molecular sieves (1.62 g)
were added to a solution under N2 of (2S,3R)-3-benzyloxy-2-
(N-hexacosanoylamino)-1-octadecanol (11a) (0.27 g, 0.35 mmol)
in dry THF (6.5 mL), and the mixture was stirred for 30 min
and then cooled to -10 °C. A solution of â-benzylgalactosyl
fluoride (0.29 g, 0.54 mmol) in dry THF (6.5 mL) was added.
The reaction mixture was then warmed gradually to room
temperature and stirred for 2 h. The reaction mixture was
filtered through Celite and the filter cake washed with CH2-
Cl2. The filtrate was washed with brine, dried (MgSO4), and
concentrated. The residue was purified by flash chromatog-
raphy on silica gel (petroleum ether/EtOAc 95:5 to 85:15) to
(2S,3R)-1-(R-D-Galactopyranosyl)-2-tetracosanoylami-
nononan-3-ol (2). (2S,3R)-3-Benzyloxy-2-(N-tetracosanoyl-
amino)-1-(2,3,4,6-tetra-O-benzyl-R-d-galactopyranosyl)nonane
(12b) (0.08 g, 0.07 mmol) was dissolved in EtOH (4 mL) and
CHCl3 (1 mL). Pd(OH)2 (20% on carbon, 0.32 g) was added in
one portion, and the mixture was stirred under H2. After 24
h, the mixture was filtered through Celite and the filter cake
washed with a combination of EtOH and CHCl3. The filtrate
was concentrated, and the residue was purified on silica gel
(CH2Cl2/MeOH 95:5 to 91.5:8.5) to provide a white solid (0.04
g, 89%): IR (KBr) 3347, 2919, 2850, 1678 cm-1; 1H NMR (400
MHz, pyr-d5) δ 8.52 (d, J ) 8 Hz, 1H), 6.54 (br s, 2H), 6.45 (br
s, 1H), 6.35 (br s, 1H), 6.14 (d, J ) 4 Hz, 1H), 5.48 (d, J ) 4
Hz, 1H), 4.76-4.25 (m, 9H), 3.65 (s, 1H), 2.50 (t, J ) 4 Hz,
2H), 1.87-0.81 (m, 58H); 13C NMR (100 MHz, pyr-d5) δ 173.8,
102.5, 73.5, 72.3, 72.0, 71.4, 70.9, 70.0, 63.1, 55.3, 37.2, 35.5,
provide 12a as a white solid (0.11 g, 26%): mp 71-73 °C; [R]25
D
+18.5 (c 1.0, CHCl3); IR (KBr) 3310, 3030, 2925, 2850, 1640,
1
1542 cm-1; H NMR (400 MHz, CDCl3) δ 7.30 (m, 25H), 6.05
(d, J ) 8.8 Hz, 1H), 4.94-4.36 (m, 10H), 4.17 (br s, 1H), 4.03
(dd, J ) 10.1, 2.8 Hz, 1H), 3.98-3.87 (m, 4H), 3.68 (dd, J )
11.0, 3.7 Hz, 1H), 3.59 (dd, J ) 11.0, 6.0 Hz, 1H), 3.52 (dd, J
) 9.3, 6.8 Hz, 2H), 3.38 (dd, J ) 9.3, 5.9 Hz, 1H), 1.99 (m,
J. Org. Chem, Vol. 70, No. 25, 2005 10267

Ndonye et al.
32.5, 30.4, 30.3, 30.2, 30.1, 30.0, 26.9, 26.8, 23.3, 23.3, 14.6;
HRMS (FAB) calcd for C39H77NO8 (M+ + H) 688.5727, found
688.5717.
(2S,3S,4R)-2-(tert-butoxycarbonyl)amino]-1,3,4-nonanetriol (0.63
g, 2.15 mmol) in CH2Cl2 (37 mL). The solution was stirred for
30 min then neutralized with saturated aqueous NaHCO3 (270
mL) to pH ∼8.0. The resulting solution was concentrated under
high vacuum to provide a white solid. Purification by flash
chromatography on silica gel (CH2Cl2/MeOH 90:10 to 85:15)
provided 14 as a pale yellow oil (0.22 g, 53%): IR (neat) 3115,
(4R,1′Z)-3-(tert-Butoxycarbonyl)-2,2-dimethyl-4-(1′-hep-
tenyl)oxazolidine. n-BuLi (2.5 M in hexane, 14.4 mL, 36.0
mmol) was added to freshly prepared hexylphosphonium
bromide (16.4 g, 38.4 mmol) in dry THF (250 mL) at -78 °C
under N2. The resulting orange solution was allowed to warm
to 0 °C and stirred for 30 min. The solution was then cooled
to -78 °C, and Garner’s aldehyde (13) (5.5 g, 24 mmol) in dry
THF (24 mL) was added dropwise. After being stirred for 2 h
at room temperature, the reaction was diluted with saturated
aqueous NH4Cl (30 mL), and the layers were separated. The
aqueous layer was extracted with CH2Cl2 (3 × 30 mL). The
combined organic extracts were washed with brine (50 mL),
dried (MgSO4), and concentrated. Purification by flash chro-
matography on silica gel (petroleum ether/EtOAc 99:1 to 98:
2) provided (4R,1′Z)-3-(tert-butoxycarbonyl)-2,2-dimethyl-4-(1′-
1683, 1443, 1206, 1139 cm-1 1H NMR (500 MHz, pyr-d5) δ
;
4.66 (dd, J ) 11.5, 3.5 Hz, 1H), 4.60-4.56 (m, 1H), 4.50-4.44
(m, 2H), 4.10 (t, J ) 6.5 Hz, 1H), 3.64 (br s, 4H), 2.20 (t, J )
3.0 Hz, 1H), 1.76 (m, 2H), 1.58 (m, 1H), 1.32 (m, 5H), 0.89 (t,
J ) 6.5 Hz, 3H); 13C NMR (125 MHz, pyr-d5) δ 73.8, 73.0, 59.3,
56.5, 35.3, 32.3, 25.7, 23.0, 14.3; HRMS (FAB) calcd for C9H22-
NO3 (M+ + H) 192.1600, found 192.1601.
(2S,3S,4R)-2-Tetracosanoylamino-1,3,4-nonanetriol (15).
p-Nitrophenyl tetracosanoate (0.63 g, 1.2 mmol) was added to
a solution of (2S,3S,4R)-2-amino-1,3,4-nonanetriol (14) (0.20
g, 1.0 mmol) in dry pyridine (20 mL). The reaction was stirred
for 18 h, concentrated, and purified by flash chromatography
on silica gel (CH2Cl2/MeOH 98:2 to 95:5) to provide 15 as a
white solid (0.40 g, 67%): mp 117-119 °C; [R]25D -6.51 (c 1.0,
heptenyl)oxazolidine as a pale yellow oil (5.0 g, 70%): [R]25
D
+75.5 (c 1.0, CHCl3); IR (neat) 2979, 2930, 2862, 1700, 1457,
1
1385 cm-1; H NMR (500 MHz, CDCl3) δ 5.46-5.39 (m, 2H),
pyr); IR (KBr) 3350, 2908, 1648, 1543, 1453 cm-1 1H NMR
;
4.70-4.65 (m, 1H), 4.06 (dd, J ) 8.5, 6.5 Hz, 1H), 3.65 (dd, J
) 8.5, 3.0 Hz, 1H), 2.09 (br s, 2H), 1.60-1.26 (m, 21H), 0.88
(t, J ) 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 152.2, 132.0,
131.0, 79.9, 69.3, 54.8, 31.7, 29.9, 29.6, 28.7, 27.7, 22.8, 14.3;
MS (EI) m/z 226 (M+ - C5H11), 67, 57 (100). Anal. Calcd for
C17H31NO3: C, 68.65; H, 10.51; N, 4.71. Found C, 68.55; H,
10.48; N, 4.64.
(500 MHz, pyr-d5) δ 7.93 (d, J ) 8.5 Hz, 1H), 5.15 (br s, 3H),
4.80 (m, 1H), 4.34 (dd, J ) 11.0, 4.5 Hz, 1H), 4.27 (dd, J )
10.5, 5.0 Hz, 1H), 4.15 (m, 1H), 4.10 (m, 1H), 2.41 (t, J ) 7.5
Hz, 2H), 2.12 (m, 1H), 1.82 (m, 4H), 1.64 (br s, 1H), 1.40 (m,
44H), 1.02 (t, J ) 6.0 Hz, 3H), 0.99 (t, J ) 6.0 Hz, 3H); 13C
NMR (125 MHz, pyr-d5) δ 173.8, 77.2, 73.5, 62.6, 54.2, 37.3,
34.4, 32.9, 32.6, 30.5, 30.4, 30.3, 30.3, 30.2, 30.1, 26.9, 26.7,
23.5, 23.4, 14.7, 14.7; HRMS (FAB) calcd for C33H68NO4 (M+
+ H) 542.5148, found 542.5139.
(2R,3Z)-2-(tert-Butoxycarbonyl)amino-3-nonen-1-ol. p-
TsOH (0.44 g, 2.31 mmol) was added to a stirred solution under
N2 of (4R,1′Z)-3-(tert-butoxycarbonyl)-2,2-dimethyl-4-(1′-hep-
tenyl)oxazolidine (4.40 g, 14.8 mmol) in MeOH:H2O (73 mL,
9:1) and the reaction was stirred for 48 h. The reaction mixture
was concentrated under high vacuum to provide a white solid,
which was then dissolved in CH2Cl2 (100 mL). The resulting
solution was washed with brine (30 mL), dried (MgSO4), and
concentrated. Purification by flash chromatography on silica
gel (petroleum ether/EtOAc 85:15 to 70:30) afforded (2R,3Z)-
2-(tert-butoxycarbonyl)amino-3-nonen-1-ol as a white solid (3.1
g, 82%): mp 98-100 °C; [R]25D +27.9 (c 1.0, CHCl3); IR (KBr)
(2S,3S,4R)-1,3,4-Tris(tert-butyldimethylsilanyloxy)-2-
tetracosanoylaminononane. A solution of (2S,3S,4R)-2-
tetracosanoylamino-1,3,4-nonanetriol (15) (0.19 g, 0.32 mmol)
and 2,6-lutidine (0.34 mL, 2.90 mmol) in dry CH2Cl2:DMF (4.5
mL, 1:2) was cooled to 0 °C and TBDMSOTf (0.6 mL, 2.6 mmol)
was added dropwise under N2. After 10 min, the cooling bath
was removed, and the reaction was stirred at room tempera-
ture for 5 h. The reaction mixture was quenched with MeOH,
poured into H2O (10 mL), and extracted with Et2O (3 × 30
mL). The organic extracts were washed with H2O (30 mL),
saturated aqueous NaHCO3 (30 mL), and brine (30 mL), dried
(MgSO4), and concentrated. Purification by flash chromatog-
raphy on silica gel (petroleum ether/EtOAc 99:1 to 97:3)
provided (2S,3S,4R)-1,3,4-tris(tert-butyldimethylsilanyloxy)-2-
3376, 2948, 1686, 1530 cm-1 1H NMR (500 MHz, CDCl3) δ
;
5.59 (dd, J ) 17.5, 7.5 Hz, 1H), 5.27 (t, J ) 10.0 Hz, 1H), 4.80
(br s, 1H), 4.49 (br s, 1H), 3.59 (m, 2H), 2.90 (br s, 1H), 2.13
(m, 2H), 1.46 (s, 9H), 1.35 (m, 6H), 0.89 (t, J ) 6.0 Hz, 3H);
13C NMR (125 MHz, CDCl3) δ 156.5, 135.0, 126.3, 80.1, 66.8,
50.9, 31.7, 29.4, 28.6, 28.2, 22.8, 14.3; MS (EI) m/z 226 (M+
CH2OH), 170 (100), 126, 57. Anal. Calcd for C14H27NO3: C,
65.33; H, 10.57; N, 5.55. Found: C, 65.24; H, 10.20; N, 5.67.
-
tetracosanoylaminononane as a clear oil (0.32 g, 74%): [R]25
D
+1.50 (c 1.0, CHCl3); IR (neat) 3440, 3341, 2925, 2854, 1687,
1
1495, 1469 cm-1; H NMR (500 MHz, CDCl3) δ 5.82 (d, J )
8.5 Hz, 1H), 3.95 (m, 1H), 3.88 (dd, J ) 10.0, 4.0 Hz, 1H), 3.83
(d, J ) 7.0 Hz, 1H), 3.69 (dd, J ) 7.5, 4.0 Hz, 1H), 3.64 (dd, J
) 10.0, 4.0 Hz, 1H), 2.15 (t, J ) 7.5 Hz, 2H), 1.60 (m, 2H),
1.56-1.39 (m, 3H), 1.30-1.26 (m, 45H), 0.94-0.89 (m, 33H),
0.14-0.02 (m, 18H); 13C NMR (125 MHz, CDCl3) δ 172.5, 75.6,
61.6, 52.8, 37.4, 32.4, 32.4, 32.2, 29.9, 29.9, 29.9, 29.7, 29.7,
29.6, 29.6, 26.3, 26.3, 26.3, 26.1, 26.0, 22.9, 22.8, 18.6, 18.5,
18.4, 14.3, 14.3, -3.28, -3.59, -4.40, -4.96, -4.99, -5.34.
Anal. Calcd for C51H109NO4Si3: C, 69.24; H, 12.42; N, 1.58.
Found: C, 68.89; H, 12.08; N, 1.57.
(2S,3S,4R)-3,4-Bis(tert-butyldimethylsilanyloxy)-2-tet-
racosanoylamino-1-nonanol. A solution of (2S,3S,4R)-1,3,4-
tris(tert-butyldimethylsilanyloxy)-2-tetracosanoylamino-
nonane (0.30 g, 0.29 mmol) in dry THF (3.5 mL) was cooled to
-10 °C, and a solution of TFA:H2O (1.3 mL, 9:1) was added
dropwise under N2. The reaction mixture was gradually
warmed to 10 °C and stirred for 2.5 h, then quenched with
NaOH (1 M, 1 mL). The reaction mixture was diluted with
Et2O (30 mL), washed with H2O (10 mL), saturated aqueous
NaHCO3 (10 mL), and brine (10 mL), dried (MgSO4), and
concentrated. Purification by flash chromatography on silica
gel (petroleum ether/EtOAc 95:5 to 85:15) provided (2S,3S,4R)-
3,4-bis(tert-butyldimethylsilanyloxy)-2-tetracosanoylamino-1-
nonanol as a clear oil (0.19 g, 70%): [R]25D -10.4 (c 1.0, CHCl3);
(2S,3S,4R)-2-(tert-Butoxycarbonyl)amino-1,3,4-nonane-
triol. (2R,3Z)-2-(tert-Butoxycarbonyl)amino-3-nonen-1-ol (1.50
g, 5.84 mmol) was dissolved in t-BuOH:H2O (58 mL, 1:1) under
N2 and methanesulfonamide (0.56 g, 5.84 mmol) added. The
reaction mixture was cooled to 0 °C, and AD-mix-â (8.17 g)
was added. The resulting mixture was stirred at 0 °C for 36 h
and at room temperature for 8 h, then quenched with solid
Na2SO3 (8.8 g) and extracted with EtOAc (3 × 50 mL). The
organic extracts were washed with NaOH (1 M, 30 mL), H2O
(30 mL), and brine (30 mL), dried (MgSO4), and concentrated.
Purification by flash chromatography on silica gel (petroleum
ether/EtOAc 50:50 to 30:70) provided (2S,3S,4R)-2-(tert-bu-
toxycarbonyl)amino-1,3,4-nonanetriol as a white solid (1.2 g,
70%): mp 103-105 °C; [R]25 +8.32 (c 1.0, CHCl3); IR (KBr)
D
3363, 2922, 1674, 1544 cm-1
;
1H NMR (500 MHz, CDCl3) δ
5.55 (d, J ) 8.0 Hz, 1H), 4.20 (br s, 1H), 3.94 (d, J ) 5.0 Hz,
1H), 3.83 (m, 3H), 3.71-3.64 (m, 4H), 1.65 (br s, 1H), 1.43 (s,
9H), 1.31 (br s, 6H), 0.87 (t, J ) 6.5 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 156.6, 80.3, 76.1, 73.2, 62.0, 52.9, 33.0, 32.0,
28.6, 25.8, 22.8, 14.2; MS (EI) m/z 207, 187, 117, 86 (100), 57.
Anal. Calcd for C14H29NO5: C, 57.71; H, 10.03; N, 4.81.
Found: C, 57.57; H, 9.74; N, 4.83.
(2S,3S,4R)-2-Amino-1,3,4-nonanetriol (14). A solution of
TFA:CH2Cl2 (15 mL, 1:1) was added to a solution under N2 of
10268 J. Org. Chem., Vol. 70, No. 25, 2005

Sphinganine Analogues of KRN7000 and OCH
IR (neat) 3298, 2925, 2854, 1649, 1511, 1465, 1254 cm-1; H
NMR (500 MHz, CDCl3) δ 6.25 (d, J ) 7.5 Hz, 1H), 4.22 (dd,
J ) 11.5, 2.5 Hz, 1H), 4.07 (m, 1H), 3.92 (t, J ) 2.5 Hz, 1H),
3.77 (ddd, J ) 15.0, 6.0, 2.5 Hz, 1H), 3.60 (br s, 1H), 3.15 (br
s, 1H), 2.19 (t, J ) 7.0 Hz, 2H), 1.30-1.26 (m, 50H), 0.89-
0.83 (m, 24 H), 0.12-0.09 (m, 12H); 13C NMR (125 MHz,
CDCl3) δ 172.9, 76.7, 63.9, 51.5, 37.2, 34.6, 32.2, 32.2, 30.3,
30.0, 29.9, 29.9, 29.7, 29.6, 29.6, 26.3, 26.3, 26.2, 25.9, 25.8,
22.9, 22.8, 18.4, 18.4, 14.4, 14.3, -3.51, -3.81, -4.28, -4.65.
Anal. Calcd for C45H95NO4Si2: C, 70.15; H, 12.43; N, 1.82.
Found: C, 69.82; H, 12.04; N, 1.74.
32.2, 32.1, 29.9, 29.9, 29.8, 29.6, 29.6, 29.5, 25.9, 25.8, 22.9,
22.9, 14.3, 14.3; HRMS (FAB) calcd for C67H102NO9 (M+ + H)
1065.7555, found 1065.7513.
1
(2S,3S,4R)-1-(R-D-Galactopyranosyl)-2-tetracosanoyl-
aminononan-3,4-diol (OCH). Pd(OH)2 (20% on carbon, 224
mg) was added to a vigorously stirred solution of (2S,3S,4R)-
2-tetracosanoylamino-1-(2,3,4,6-tetra-O-benzyl-R-D-galactopy-
ranosyl)nonan-3,4-diol (47 mg, 0.045 mmol) in EtOH (3.0 mL)
and CHCl3 (0.75 mL). The mixture was placed under H2, and
stirring was continued for 24 h. The mixture was then filtered
through Celite, and the filter cake was rinsed with CHCl3 and
MeOH. The filtrate was concentrated, and the residue was
purified by flash chromatography on silica gel (CH2Cl2/MeOH
95:5 to 85:15) to provide OCH31 as a white solid (25.0 mg,
81%): mp 125-127 °C; [R]25D +37.1 (c 1.0, pyr); IR (KBr) 3428,
(2S,3S,4R)-3,4-Bis(tert-butyldimethylsilanyloxy)-2-tet-
racosanoylamino-1-(2,3,4,6-tetra-O-benzyl-R-D-galactopy-
ranosyl)nonane (16). Tin(II) chloride (0.11 g, 0.58 mmol),
silver perchlorate (0.12 g, 0.59 mmol), and freshly ground 4Å
molecular sieves (0.90 g) were added successively to a solution
under N2 of (2S,3S,4R)-3,4-bis(tert-butyldimethylsilanyloxy)-
2-tetracosanoylamino-1-nonanol (0.18 g, 0.20 mmol) in dry
THF (3.6 mL). The reaction mixture was stirred for 30 min,
then cooled to -10 °C, and a solution of â-tetrabenzylgalactosyl
fluoride (0.16 g, 0.30 mmol) in dry THF (3.6 mL) was added.
The reaction mixture was warmed gradually to room temper-
ature and stirred for 2 h. The reaction mixture was then
filtered through Celite, and the filter cake was washed with
CH2Cl2. The filtrate was washed with brine (30 mL), dried
(MgSO4), and concentrated under reduced pressure. Purifica-
tion by flash chromatography on silica gel (petroleum ether/
EtOAc 95:5 to 85:15) provided 16 as a clear oil (0.10 g, 37%):
1
2934, 2856, 1648, 1544, 1466 cm-1; H NMR (500 MHz, pyr-
d5) δ 8.44 (d, J ) 9.0 Hz, 1H), 5.51 (d, J ) 4.5 Hz, 2H), 5.26
(m, 2H), 4.68 (m, 2H), 4.55 (d, J ) 3.5 Hz, 1H), 4.51 (t, J ) 6.0
Hz, 1H), 4.40 (m, 4H), 4.30 (m, 2H), 3.64 (d, J ) 3.0 Hz, 1H),
3.44 (br s, 1H), 2.47 (t, J ) 7.0 Hz, 2H), 2.26 (m, 1H), 1.91-
1.77 (m, 4H), 1.74 (br s, 1H), 1.64 (m, 1H), 1.32-1.23 (m, 44H),
0.88 (t, J ) 7.0 Hz, 3H), 0.82 (t, J ) 7.0 Hz, 3H); 13C NMR
(125 MHz, pyr-d5) δ 173.7, 102.0, 77.3, 73.5, 73.0, 72.1, 71.5,
71.3, 71.3, 70.8, 69.1, 63.2, 51.9, 37.3, 34.8, 32.9, 32.6, 32.4,
30.5, 30.5, 30.4, 30.3, 30.3, 30.2, 30.1, 27.6, 26.9, 26.6, 23.5,
23.4, 14.8, 14.7; HRMS (FAB) calcd for C39H78NO9 (M+ + H)
704.5677, found 704.5662.
Antigen Presentation Assays. Bulk splenocytes were
isolated from normal, 8-12 week old female C57BL/6 mice
(Jackson Labs). Cells were plated at 300 000 per well in 96-
well flat-bottom tissue culture plates in 200 µL complete
medium: RPMI-1640 supplemented with 10 mM HEPES, 2
mM L-glutamine, 0.1 mM nonessential amino acids, 55 µM
2-mercaptoethanol, 100 U/mL of penicillin and 100 µg/mL of
streptomycin (Gibco), and 10% heat-inactivated fetal calf
serum (Gemini). Cultures contained varying doses of glycolip-
ids, diluted from 100 µM stocks in 100% DMSO. After 48 h in
a 37 °C, 5% CO2 humidified incubator, cell-free supernatants
were collected and assayed for mouse IL-4 and IFNγ content,
using standard sandwich ELISA. Capture and biotinylated
antibodies were obtained from BD Pharmingen, cytokine
standards from Peprotech, streptavidin-horseradish peroxidase
from Zymed, and TMB-turbo substrate from Pierce. Absor-
bance at 450 nm was monitored with a microplate reader
(Titertek).
[R]25 +5.80 (c 1.0, CHCl3); IR (neat) 3031, 2924, 2853, 1681,
D
1496, 1462 cm-1; 1H NMR (500 MHz, CDCl3) δ 7.30-7.23 (m,
20H), 6.00 (d, J ) 7.5 Hz, 1H), 4.93 (d, J ) 11.5 Hz, 1H), 4.84
(d, J ) 3.5 Hz, 1H), 4.79 (m, 2H), 4.73-4.65 (m, 2H), 4.56 (d,
J ) 11.5 Hz, 1H), 4.49 (d, J ) 12.0 Hz, 1H), 4.40 (d, J ) 12.0
Hz, 1H), 4.08 (m, 1H), 4.04 (dd, J ) 10.0, 3.5 Hz, 1H), 3.97-
3.86 (m, 6H), 3.66 (m, 1H), 3.50 (m, 2H), 2.01 (t, J ) 7.5 Hz,
2H), 1.30-1.25 (m, 50H), 0.91-0.86 (m, 24H), 0.08-0.03 (m,
12H); 13C NMR (125 MHz, CDCl3) δ 173.4, 139.0, 138.9, 138.9,
138.1, 128.7, 128.6, 128.6, 128.5, 128.4, 128.2, 128.0, 128.0,
127.9, 127.8, 127.8, 127.7, 100.6, 79.3, 77.9, 76.8, 76.0, 75.2,
75.0, 73.8, 73.7, 73.2, 70.1, 69.9, 69.0, 52.0, 37.0, 33.4, 32.4,
32.2, 30.0, 30.0, 29.9, 29.8, 29.7, 29.6, 26.4, 26.4, 26.0, 25.9,
23.0, 22.9, 18.6, 18.4, 14.4, 14.4, -3.39, -3.57, -4.38, -4.68.
Anal. Calcd for C79H129NO9Si2: C, 73.38; H, 10.06; N, 1.08.
Found: C, 72.99; H, 9.68; N, 1.08.
Alternatively, lipid presentation by CD1d was tested
by using a hybridoma-based assay. The CD1d-transfected
RMA-S.mCD1d clone was provided by Dr. S. Behar.35 The VR-
14+ iNKT hybridoma DN3A4-1.2 has been described previ-
ously.36 Both clones were maintained in complete RPMI
medium as above. The CD1d+ mouse dendritic cell line JAWS
II was obtained from the American Type Culture Collection
and maintained in MEMR medium with ribonucleosides,
deoxyribonucleosides, 4 mM L-glutamine, 1 mM sodium pyru-
vate (Gibco), 20% heat-inactivated fetal calf serum, and 5 ng/
mL mouse GM-CSF (Peprotech). Antigen presenting cells were
plated at 50 000 per well on 96-well flat bottom plates in
complete RPMI medium with varying amounts of glycolipids
for 6 h at 37 °C. The cells were then washed three times with
PBS (430g, 3 min). These were then fixed with 0.05% glut-
araldehyde in PBS (grade I, Sigma) to prevent further antigen
processing or loading, followed by quenching with 0.2 M
L-lysine (pH 7.4) for 2 min and washes in complete medium.
Fifty thousand iNKT hybridoma cells were then added in 100
µL complete medium for 12 h. Cell-free supernatants were
collected and tested for the presence of IL-2 by ELISA.
Antibodies were obtained from BD Pharmingen, mouse IL-2
(2S,3S,4R)-2-Tetracosanoylamino-1-(2,3,4,6-tetra-O-ben-
zyl-R-D-galactopyranosyl)nonan-3,4-diol. (2S,3S,4R)-3,4-
Bis(tert-butyldimethylsilanyloxy)-2-tetracosanoylamino-
1-(2,3,4,6-tetra-O-benzyl-R-D-galactopyranosyl)nonane (16) (0.10
g, 0.07 mmol) was dissolved under N2 in THF (4 mL), and the
solution was cooled to 0 °C. TBAF (1.0 M in THF, 0.28 mL,
0.28 mmol) was added, and the cooling bath was removed. The
reaction mixture was stirred for 2 h at room temperature then
diluted with H2O (10 mL) and extracted with CH2Cl2 (3 × 20
mL). The combined extracts were washed with H2O (20 mL)
and brine (20 mL), dried (MgSO4), and concentrated. Purifica-
tion by flash chromatography on silica gel (petroleum ether/
EtOAc 80:20 to 70:30) provided (2S,3S,4R)-2-tetracosanoyl-
amino-1-(2,3,4,6-tetra-O-benzyl-R-D-galactopyranosyl)nonan-
3,4-diol as a clear oil (0.070 g, 82%): [R]25D +32.7 (c 1.0, CHCl3);
1
IR (neat) 3319, 2918, 2850, 1637, 1543, 1468 cm-1; H NMR
(500 MHz, CDCl3) δ 7.55-7.26 (m, 20H), 6.37 (d, J ) 8.5 Hz,
1H), 4.94-4.85 (m, 3H), 4.77 (m, 2H), 4.68 (d, J ) 11.5 Hz,
1H), 4.57 (d, J ) 11.5 Hz, 1H), 4.48 (d, J ) 11.5 Hz, 1H), 4.40
(d, J ) 11.5 Hz, 1H), 4.22 (m, 1H), 4.06 (dd, J ) 10.0, 4.0 Hz,
1H), 3.97 (br s, 1H), 3.93-3.86 (m, 4H), 3.80 (d, J ) 8.5 Hz,
1H), 3.53-3.45 (m, 4H), 2.14-2.10 (m, 4H), 1.27 (br s, 50H),
0.91-0.84 (m, 6H); 13C NMR (125 MHz, CDCl3) δ 173.4, 138.7,
138.6, 138.1, 137.8, 128.7, 128.7, 128.7, 128.5, 128.4, 128.3,
128.2, 128.1, 127.9, 127.9, 127.7, 99.5, 79.6, 76.5, 76.3, 75.0,
74.7, 74.5, 73.9, 73.5, 73.0, 70.3, 70.2, 69.2, 49.8, 37.0, 33.4,
(35) Behar, S. M.; Prodrebarac, T. A.; Roy, C. J.; Wang, C. R.;
Brenner, M. B. J. Immunol. 1999, 162, 161-167.
(36) Brossay, L.; Tangri, S.; Bix, M.; Cardell, S.; Locksley, R.;
Kronenberg, M. J. Immunol. 1998, 160, 3681-3688.
J. Org. Chem, Vol. 70, No. 25, 2005 10269

Ndonye et al.
cytokine standard from R&D Systems, streptavidin-alkaline
phosphatase from Zymed, and 4-nitrophenyl phosphate sub-
strate from Sigma. Absorbance at 405 nm was monitored with
a microplate reader (Titertek). IL-2 versus logarithm of lipid
concentration plots and classical 4-parameter dose/response
curves were drawn with Prism 4.02 (GraphPad). Relative
potencies of the individual lipids were estimated by the
reciprocals of the effective concentrations at 50% maximal
response (1/EC50).
A.R.H.; AI45889: S.A.P.; AI45053: M.K.) and for a
predoctoral fellowship for D.P.I. (NIH AI054346).
Supporting Information Available: General experimen-
tal information and copies of high-resolution 13C NMR spectra
for those new compounds for which elemental analyses are not
1
reported; copies of high-resolution H NMR spectra for OCH
and analogues 1 and 2; and results for hydbridoma assay with
A20.CD1d APC’s. This material is available free of charge via
the Internet at http://pubs.acs.org.
Acknowledgment. The authors are grateful for
financial support provided by NIH NIAID (AI057519:
JO051147H
10270 J. Org. Chem., Vol. 70, No. 25, 2005