Novel and convergent synthesis of modified glycosphingolipids, galactosyl-5-aza-sphinganines, by a diversity-oriented method

Article abstract of DOI:10.1016/j.tet.2011.05.054

The stereocontrolled synthesis of β-galactosyl-5-aza-sphinganines was accomplished with high efficiency by a novel and convergent strategy. The truncated β-galactosphinganine 5 was easily acylated with different long chains. Moreover, the simple conversion of thiazole to formyl enabled 5-aza-alkylchains to be introduced via an amino-reduction reaction. The overall yield for the synthesis of β-galacto-5-aza-sphinganines 9 and 13 was about 30-35% starting from 5. Preliminary results concerning the β- transgalactosylation of a truncated sphinganine catalyzed by galactosidases are also presented and discussed.

Full text of DOI:10.1016/j.tet.2011.05.054

Tetrahedron 67 (2011) 5176e5183
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Novel and convergent synthesis of modified glycosphingolipids,
galactosyl-5-aza-sphinganines, by a diversity-oriented method
*
Bimalendu Roy, Salim Ferdjani, Charles Tellier, Claude Rabiller
ꢀ
ꢀ
ꢁ
Biotechnologie, Biocatalyse et Bioregulation, UMR 6204 CNRS, Universite de Nantes, 2 rue de la Houssiniere, F-44322 Nantes Cedex 03, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 April 2011
Received in revised form 5 May 2011
Accepted 11 May 2011
Available online 23 May 2011
The stereocontrolled synthesis of
ciency by a novel and convergent strategy. The truncated
b
-galactosyl-5-aza-sphinganines was accomplished with high effi-
-galactosphinganine 5 was easily acylated
with different long chains. Moreover, the simple conversion of thiazole to formyl enabled 5-aza-al-
kylchains to be introduced via an amino-reduction reaction. The overall yield for the synthesis of
galacto-5-aza-sphinganines 9 and 13 was about 30e35% starting from 5. Preliminary results concerning
the -transgalactosylation of a truncated sphinganine catalyzed by galactosidases are also presented and
b
b
-
b
Keywords:
Glycolipids
Glycosphinganines
Reductive amination
discussed.
Ó 2011 Published by Elsevier Ltd.
1. Introduction
potentimmunostimulatory properties, being able toactivatenatural
killer T (NKT) cells to produce cytokines.8
b
-Galactosyl ceramides (
b
-GalCer), an important class of glyco-
Due to the biological significance of b-GalCer derivatives, many
lipids are abundant in the myelin sheath where they are constituted
by a large variety of galactosphingolipids differing in chain length,
with or without alkenestructures and having hydroxylgroups either
in the main chain or in the acyl chain.1 Since the building of myelin
requires an interaction between cerebroside and cerebroside sul-
fate,2 the lack of synthesis of these compounds is responsible for
many neurodegenerative pathologies.3 Alterations in the metabo-
lism of sphingolipids or glycosphingolipids are mainly disorders of
the degradation of these compounds caused by defects in the system
of lysosomal sphingolipid degradation, with subsequent accumu-
strategies have been proposed for their synthesis9e12 and the need
for a large library of derivatives has arisen. However, many reported
syntheses suffer from a lack of flexibility to access various chain
lengths or functionalized chains.13 Thus, recent methods have in-
volved in their first steps, the coupling of an activated glycoside
moiety with truncated precursors of sphingoids acting as acceptors.
Then, the truncated galactosylceramide precursor thus obtained is
(i) N-acetylated and (ii) alkylated, or vice versa. Among the acceptors
designed for this purpose, olefinic ones have been extensively used
(this is the case, for example, of the truncated D-ribo-sphingosine,
lation of non-degradable storage material in organs.4
b
-GalCer is
(2S,3S,4R)-2-aminohex-5-ene-1,3,4-triol) because they enable long
alkene chains to be introduced via olefinic cross-metathesis with
a high E-stereoselectivity.14e22 Numerous methods are available to
synthesize truncated sphingoids, generally starting from natural
chiral sources (mainly carbohydrates and amino-acids)5,23e31 or
from asymmetric CeC bond formation.10,32e34 Using this later ap-
proach, a very efficient diastereoselective strategy described by
Dondoni et al. afforded optically pure sphingoid precursors bearing
a thiazole ring.35,36 Since the thiazole is a ‘masked’ aldehyde
group,37,38 this strategy allows the introduction of an olefinic long
chain via Wittig reactions. Unfortunately, the relatively low yields
and stereoselectivity of the Wittig reaction have limited the appli-
cation of this method. However, this approach was used for the
also known to act as an alternative ligand for the HIV-1 glycoprotein
gp120 responsible for the entry of the virus into epithelial cells.5,6 In
addition, b-GalCer derivatives could act as possible ligands for the
adhesion of Helicobacter pylori to cells in the gastric system.7 While
glycosphingolipids are present in animals, glycophytosphingolipids
are mainly found in plants and sometimes in bacteria and marine
organisms. Unlike sphingolipids, phytosphingolipids have a satu-
rated lipidic chain (generally C18). These kinds of GalCer, which are
constituents of cell membranes, also play important biological roles.
For example, the
(known as KRN7000), extracted from a marine sponge, exhibits
a-galactosyl form of D-ribo-phytosphingosine
synthesis of D-erythro-C20-sphingosine and of D-ribo-C18-phytos-
phingosine.39 Curiously, this powerful strategy used for the prepa-
ration of ceramides has not been applied to the synthesis of
* Corresponding author. Tel.: þ33 251 12 57 32; fax: þ33 251 12 57 22; e-mail
address: claude.rabiller@univ-nantes.fr (C. Rabiller).
0040-4020/$ e see front matter Ó 2011 Published by Elsevier Ltd.
doi:10.1016/j.tet.2011.05.054

B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
5177
glycoceramides. With the aim of extending the library of available
-GalCer derivatives, we report here the synthesis of diversely
substituted -galactosyl-5-aza-sphinganines using the approach
developed by Dondoni. The choice of -galactosyl-5-aza-sphinga-
nines as target compounds was motivated by the following reasons:
6-tetra-O-pivaloyl-
b
-
D
-galactopyranosyl)-3-(thiazol-2-yl)-1,3-propan-
b
ediol 5 was obtained in good yield (82%) with high stereoselectivity
since the presence of the
-anomer was not observed on the 1H and
13C NMR spectra. Starting from 5, we developed two methods to
obtain -galactosyl-5-aza-sphinganines. The first one, shown in
b
a
b
b
Scheme 2 enabled the heptadecanoyl chain to be introduced after
Boc deprotection and the reaction of heptadecanoïc acid with the
amino group in the presence of EDC.
(i) their greater water solubility compared to the parent
lactosyl sphinganines.
b-ga-
(ii) an easier synthesis than that of
b
-galactosyl sphingosides since
The
(2S,3S)-3-O-benzyl-2-heptadenanoylamino-1-O-(2,3,4,6-
-galactopyranosyl)-3-(thiazol-2-yl)-1,3-prop-
these compounds are devoid of a 4-hydroxy group.
tetra-O-pivaloyl-
b-D
anediol 6 thus obtained in good yields was then submitted to
a thiazolyl to formyl transformation.43 The amination reduction re-
action carried out on the formyl group of (2S,3S)-2-O-benzyl-3-
(iii) recent work has shown that the presence of the 4-hydroxy
group is not absolutely necessary to obtain galactoceramides
able to stimulate iNKT cells.40
heptadecanoylamino-4-O-(2,3,4,6-tetra-O-pivaloyl-b-D-galactopyr-
anosyl)-butanal 7 in thepresence ofdiverse primaryaliphatic amines
(hexanamine to pentadecanamine) in reductive medium afforded
the corresponding (2S,3R)-5-aza-3-O-benzyl-2-heptadecanoy-
2. Results and discussion
lamino-1-O-(2,3,4,6-tetra-O-pivaloyl-b-D-galactopyranosyl)-1,3-alc-
anediols 8 in very good conditions. Then, the deprotection of 8 was
achieved by the removal of the pivaloyl groups (NaOMe/MeOH) fol-
lowed by the Pd/C catalyzed hydrogenation to cleave the benzyl
group. Previous attempts to reverse the order of the two operations
The synthetic pathways and the
nines prepared are described in Schemes 1e3.
2-(Trimethylsilyl)thiazole (2-TST) was prepared according to
a well known procedure starting from commercially available
b-galactosyl-5-aza-sphinga-
i : BnCl
ii : PTSA, MeOH
Boc
S
Boc
Boc
Ref 41
N
HN
N
S
S
O
HO
O
+
CHO
SiMe3
N
N
N
2-TST
1
OBn
OH
2
3
TMSOTf
Dry CH2Cl2
OPiv
O
PivO
PivO
Boc
OPiv
O
PivO
PivO
HN
S
82 %
+
O
3
N
PivO
CCl3
NH
O
PivO
OBn
4
5
Scheme 1. Synthesis of truncated
b
-D-galactosylsphinganine 5.
O
O
i. CH3I, Acetonitrile
OPiv
O
C
16H33
i. 33% TFA
PivO
ii.NaBH4
iii. HgCl2
OPiv
O
C
S
16H33
PivO
PivO
HN
HN
ii. C16H33COOH, EDC
O
PivO
O
CHO
OBn
5
N
67%
73%
PivO
PivO
OBn
7
6
O
O
i : NaOMe, MeOH
ii : Pd/C, H2
OPiv
C16H33
PivO
PivO
OH
C16H33
HO
HO
R-NH2
HN
HN
O
7
O
O
O
R
R
NaBH3CN
N
N
H
PivO
H
HO
8
86%
9a-e
BnO
HO
8a R = C15H31
8b R = C14H29
8c R = C12H25
8d R = C8H17
8e R = C5H11
Scheme 2. Synthesis of b-D-galactosyl-5-aza-ceramides 9 via the introduction of (i) the acyl chain and (ii) the 5-aza-alkyl chain.
2-bromothiazole via nBuLi lithiation and trimethylsilylation.41
Then, the highly diastereoselective condensation of 2-TST with
Garner’s aldehyde 1 afforded the O-,N-protected (2S,3S)-2-amino-
3-thiazolylpropan-1,3-diol 2 (de¼0.92), the later being easily
purified by crystallization.35 Next, the 2-hydroxyl group was
benzylated and further cleavage of the O-,N-acetonide in the
presence of catalytic amounts of PTSA in methanol afforded 3.
This compound was used as an acceptor in the coupling reaction
were unsuccessful, probably due to the bulkiness of compounds 8.
Even when carried out in the correct order, the reduction still
remained a slow rate reaction (see Experimental section). Never-
theless, the deprotection of
heptadecanoylamino-1-O-( -galactopyranosyl)-1,3-alcanediols 9
in nearly quantitative yields.
8
afforded (2S,3R)-5-aza-2-
b-D
Starting from 5, we also developed a second strategy, which con-
sisted of i: transforming the thiazole into formyl followed by the
subsequent aminationereduction introduction of the fatty chain and
ii: acylation of the amino group with different chain lengths (see
Scheme 3). Whichever method was used, the overall yields (around
with 2,3,4,6-tetra-O-pivaloyl-a-D-galactopyranosyl trichloroace-
timidate 4.42 Using TMSOTf as a catalyst in dry CH2Cl2, the
target (2S,3S)-3-O-benzyl-2-tert-butyloxycarbonylamino-1-O-(2,3,4,

5178
B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
i. CH3I, Acetonitrile
OPiv
O
ii. NaBH4
iii. HgCl2
OPiv
O
PivO
PivO
Boc
PivO
PivO
Boc
HN
HN
S
O
O
N
O
69%
PivO
PivO
OBn
10
OBn
5
O
i: 33% TFA
ii: R'COOH, EDC
PivO
OPiv
O
OPiv
O
PivO
PivO
Boc
PivO
R'
C14H29NH2
HN
HN
10
O
O
C
14H29
C14H29
NaBH3CN
N
H
N
PivO
PivO
H
81%
OBn
O
OBn
12
11
12a R' = C21H43
12b R' = C24H49
OH
HO
HO
R'
HN
13a-b
Pd/C, H2
O
O
C14H29
NaOMe, MeOH
N
H
HO
OH
Scheme 3. Synthesis of b-D-galactosyl-5-aza-ceramides 9 via the introduction of (i) the 5-aza-alkyl chain and (ii) the acyl chain.
32e37% starting from 3) for compounds 9 and 12 remained almost
identical.
Our results clearly indicate the power of Dondoni’s approach to
prepare truncated sphingoids. Obviously, one of the limitations of the
methodstillremainsinthestereoselectivityoftheglycosyl/truncated
sphingoid coupling. Although the NMR spectra of compounds 5
acceptors in transglycosylation reactions catalyzed by
dases (see Scheme 4). We have tested this idea by using the mutant
E382G glycosynthase from Tt Gly Thermus thermophilus -galacto-
sidase prepared in our laboratory,47 in the presence of
-galactosyl
fluoride as donor. The (2S,3S)-2-amino-1-O-( -galactopyr-
b-galactosi-
b
b
a
a
b-D
anosyl)-3-(thiazol-2-yl)-1,3-propanediol 15 was thus obtained as
revealed the absence of the
a-anomer, the presence of small amounts
asingleregioisomer witha disappointing15%yield, despitetheuse of
(<0.1%) of the latter cannot be excluded. This is an important point to
a 10-fold amount of a-galactosyl fluoride (see Scheme 4).
consider for therapeutic applications of galactosylceramide since in
Moreover, prohibitive quantities of the enzyme (see
Experimental section) had to be used in order to compete with the
some cases
sponding
a-anomers were 1000-fold more active than the corre-
b
-ones. Thus, traces of
a-anomers and not the major
spontaneous hydrolysis of the
a
-galactosyl fluoride. In our efforts to
OH
O
OH
HO
OH
OH
O
Boc
HN
OH
HO
F
H2N
S
H2N
S
S
HO
10 equivalents
HO
O
N
N
N
OH
Yield 15 %
E352G glycosynthase
from
TtbGly Thermus thermophilus
OH
OBn
OH
3
14
15
Scheme 4. Attempts to synthesize unprotected truncated
b-D-galactosylsphinganine 15 via glycosynthase catalysis.
b
-anomers could be responsible for the activity. From this point of
view, only enzymatic reactions can provide absolute stereo-
selectivity. Clearly, hypothetical -anomers present in compounds 9
for instance could be removed by hydrolysis catalyzed by an -gal-
actoceramidase. We shall perform this experiment in the near future
after testing the possible therapeutic activity of our -galactosyl-5-
aza-sphinganines. A most promising strategy would be to use the
transglycosylation power of -galactoceramidases to build the
provide glycosidases possessing high transglycosylation power, we
have recently developed new directed evolution strategies leading
to mutant glycosidases, called ‘transglycosidases’ since they are
devoid of their original hydrolytic activity.48,49 This method will be
applied to generate an enzyme capable of catalyzing this target
reaction more efficiently.
In summary, we have shown that the use of truncated thiazo-
lylsphinganines, prepared according to Dondoni’s procedure pro-
vides a general and flexible approach to the synthesis of diversely
a
a
b
b
b-
glycosidic bond with complete stereoselectivity. Moreover, this ‘in
water’ strategy offers the possibility of reducing the number of steps
in organic solvents. While retaining glycoceramidases were able to
accept some simple alcohols (like ethanol, for example), these gly-
cosylhydrolasescouldnottransferceramidesduetotheirinsolubility
in water. Meanwhile, Withers et al. recently showed that glyco-
synthases created from Rhodococcus sp. endo-glycoceramidase
substituted b-galactosyl-5-aza-sphinganines. The acyl chain can be
extended to the desired length as well as the 5-aza-alkyl chain via
an amino-reduction reaction. The amino group present in the alkyl
chain of these new
b
-D-GalCer should improve their solubility in
water. Some promising developments of the method using
b
-ga-
lactosidases as catalysts in the glycosylation step have also been
presented.
(EGCII) were able to transfer
saccharyl
D-erythro-sphingosine to GM3 oligo-
a
-fluoride with high yields.44 Unfortunately, due to the
high lipid chain specificity of this enzyme, these glycosynthases did
notenablealibraryofglycoceramidestobesynthesized. Forexample,
3. Experimental
3.1. General
the replacement of
D-erythro-sphingosine by phytosphingosine
resulted in a 10,000-fold decrease of the kcat/Km value.45 However,
a directed evolution approach applied to the E351S glycosynthase led
to variants that were able to utilize phytosphingosine at improved
rates.46 A more general enzymatic approach could involve using
truncated sphingoids, like 2-amino-3-thiazolylpropan-1,3-diol 14 as
All reactions were monitored by thin-layer chromatography (TLC)
on silica gel 60 F254 with detection by fluorescence and/or by charring
followed by immersion in a 10% ethanolic solution of sulfuric acid. An
orcinol dip, prepared by the careful addition of sulfuric acid(20 mL) to

B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
5179
an ice-cold solution of 3,5-dihydroxytoluene (360 mg) in ethanol
(250 mL) and water (10 mL), was used to detect deprotected com-
pounds by charring. All reagents and solvents were dried prior to use
according to standard methods. Commercial reagents were used
without further purification unless otherwise stated. Flash chroma-
tographywasperformedwithsilicagel230e400.Opticalrotationwas
measured at the sodium D-line at room temperature.1H and 13C NMR
spectra were recorded on a spectrometer (Bruker) at 400 MHz and
500 MHz. Assignment of 1H and 13C spectrawas achieved by means of
conventional2Dexperiments(COSY, HMQC,HOHAHA, andTOCSY). In
4.22 (m, 2H, H-6a, H-5), 3.98 (m, 2H, H-6b, H0-1ab), 3.51 (dd, 1H,
J¼3.7, 8.53 Hz, H-6b), 1.33 (s, 9H, (CO)OC(CH3)3), 1.24, 1.20, 1.18, 1.06
(4s, 36H, 4ꢁ COC(CH3)3). 13C NMR (400 MHz, CDCl3):
d 177.9 (2),
177.8, 177.3 (4ꢁ COC(CH3)3),176.0, 163.7 ((CO)OC(CH3)3), 137.2,
129.3, 128.4 (2), 128.3, 128.0, 120.2, 119.7, 101.1 (C-1), 80.13, 78.05,
77.2 (C0-2), 72.9 (CH2C6H5), 71.4 (C-5), 71.0 (C-3), 70.8 (C-2), 69.0
(C0-1), 66.6 (C-4), 61.0 (C-6), 59.6 (C0-2), 54.4, 41.1, 39.0, 38.9, 38.8,
38.7, 28.1, 27.1, 27.0 (3), 24.8. Elemental analysis: calcd for
C44H66N2O13S (862.42): C, 61.23; H, 7.71; N, 3.25; S, 3.72; found C,
61.31; H, 7.68; N, 3.30; S, 3.65.
thecaseof b
-glycolipid,1HNMRparametersweredenoted asHforthe
galactosyl moiety and H0 for sphinganine. For 13C NMR assignment,
similar primes were used.
3.4. (2S,3S)-3-O-Benzyl-2-heptadecanoylamino-1-O-(2,3,4,6-
tetra-O-pivaloyl-b-D-galactopyranosyl)-3-(thiazol-2-yl)-1,3-
The thiazolyl compound 2 was prepared according to Dondoni’s
propanediol 6
procedure.35,36
Compound 5 (350 mg, 0.405 mmol) and 50% TFA in CH2Cl2
(10 mL) were stirred for 3 h. When TLC showed complete conver-
sion of the starting material, the solution was diluted with
dichloromethane (25 mL) and washed successively with ice-cold aq
NaHCO3 (2ꢁ25 mL) and brine (25 mL). The organic layer was sep-
arated, dried over MgSO4, and evaporated under vacuum. The crude
amine resulting from the Boc deprotection was used in the next
step without any further purification.
3.2. (2S,3S)-3-O-Benzyl-2-tert-butyloxycarbonylamino-3-
(thiazol-2-yl)-1,3-propanediol 3
A
catalytic amount of p-toluenesulfonic acid (150 mg,
0.87 mmol) was added to a solution of acetonide 2 (1.75 g,
4.32 mmol) in methanol (20 mL) and the reaction mixture was
stirred at room temperature for 3 h. Then, methanol was removed
at low temperature under reduced pressure. The residue was dis-
solved in ether, and the organic layer was washed with aq NaHCO3
(5%), brine, dried over Na2SO4, and concentrated under vacuum.
The residue was purified on silica gel column with ethyl acetate in
hexane (1:2) as an eluent. Pure compound 3 (1.42 g, 90%,
To a solution of the crude amine and heptadecanoic acid (220 mg,
0.810 mmol) in dry DMF (10 mL), was added EDC (107
mL,
0.607 mmol) at 0 ꢀC under a dry nitrogen atmosphere. The reaction
mixture was kept at 0 ꢀC for 30 min and stirred overnight at room
temperature. After removal of the solvent, the residue was dissolved
in dichloromethane (30 mL) and washed with brine (2ꢁ15 mL). The
organic layer was separated, dried over MgSO4, and concentrated
under reduced pressure. The crude product was purified by flash
3.89 mmol) was thus obtained as a white solid. Mp 122 ꢀC.
20
[
a]
þ36ꢀ (c 1.5, CHCl3). 1H NMR (400 MHz, CDCl3):
d 7.82 (d, 1H,
D
J¼3.24 Hz, thiazole-H), 7.39 (m, 6H), 5.19 (br s, 1H, NH), 5.01 (d, 1H,
J¼5.32 Hz, H-3), 4.78 (d, 1H, J¼11.6 Hz, C6H5CH2), 4.62 (d, 1H,
J¼11.6 Hz, C6H5CH2), 4.16 (m, 1H, H-2), 3.96 (dd, 1H, J¼3.4 Hz, 1-Ha),
3.69 (dd, 1H, J¼4.5 Hz, 1-Hb), 1.41 (s, 9H, 3ꢁ CH3). 13C NMR
chromatography using n-hexane/EtOAc (2:1) to afford pure 6 (315 g,
20
D
75%) as a white solid. Mp 81 ꢀC. [
a
]
þ5.6 (c 1.0, CHCl3). 1H NMR
(CDCl3, 400 MHz):
d
7.73, 7.38 (2d, 2H, J¼3.12 Hz, Thiazole-H), 7.34
(400 MHz, CDCl3):
d
170.8 (CO of Boc), 155.7 (C-3), 142.9, 137.1,
(m, 5H, C6H5), 5.42 (d,1H, J¼2.9 Hz, H-4), 5.22 (dd,1H, J¼7.7,10.4 Hz,
H-2), 5.13 (dd, 1H, J¼3.2, 10.4 Hz, H-3), 4.99 (d, 1H, J¼8.4 Hz, H0-3),
4.63, 4.59 (2d, 2H, J¼11.4 Hz, CH2C6H5), 4.56 (d, 1H, J¼7.7 Hz, H-1),
4.51 (m, 1H, H0-2), 4.33 (dd, 1H, J¼3.1, 9.4 Hz, H0-1a), 4.1 (dd, 1H,
J¼6.5,10,8 Hz, H-6a), 4.05 (dd,1H, J¼7.3,10.8 Hz, H-6b), 3.97 (m,1H,
H-5), 3.67 (dd,1H, J¼3.5, 9.5 Hz, H0-1b), 2.02 (m, 2H, COCH2),1.43 (m,
2H, COCH2CH2),1.27 (m, 35H),1.18,1.17,1.13 (3s, 27H, 3ꢁ COC(CH3)3),
128.8, 128.3, 119.9, 79.7, 72.9, 62.3, 55.2 (C(CH3)3), 28.5 (C(CH3)3).
Elemental analysis: calcd for C18H24N2O4S (364.14): C 59.32, H 6.64,
N 7.69, S 8.80; found C 59.13, H 6.57, N 7.58, S 8.91.
3.3. (2S,3S)-3-O-Benzyl-2-tert-butyloxycarbonylamino-1-O-
(2,3,4,6-tetra-O-pivaloyl-b-D-galactopyranosyl)-3-(thiazol-2-
yl)-1,3-propanediol 5
0.89 (t, 3H, CH3). 13C NMR (400 MHz, CDCl3):
d 177.9, 177.4, 176.9 (2)
(4ꢁ COC(CH3)3),172.74 (COC16H33),171.2 (C0-3),141.7,137.7,128.6 (2),
128.1 (3), 120.2, 101.3 (C-1), 77.3 (C0-1), 73.0 (CH2C6H5), 71.2 (C-5),
71.0 (C-3), 69.2 (C-2), 67.8 (C0-3), 66.7 (C-4), 61.1 (C-6), 53.0 (C0-2),
36.7 (COCH2), 25.5 (COCH2CH2), 29.4, 27.1, 14.2 (CH3). Elemental
analysis: calcd for C56H90N2O12S (1015.38): C 66.24, H 8.93, N 2.76, S
3.16; found C 66.31, H 9.08, N 2.74, S 3.16.
To a solution of 2,3,4,6-tetra-O-pivaloyl-
(2.8 g, 5.42 mmol) in dry CH2Cl2 (30 mL), CCl3CN (2.7 mL,
27.0 mmol), and DBU (405 L, 2.71 mmol) were added. The mixture
b-D-galactopyranose
m
was stirred at room temperature for 25 min and the solvents were
evaporated. The residue was loaded on a flash silica gel column and
eluted with n-hexane/EtOAc (15:1) to afford pure tri-
chloroacetamidate 4 (2.26 g, 63%). Then, a mixture of acceptor 3
3.5. Thiazolyl to formyl conversion: synthesis of (2S,3S)-2-O-
benzyl-3-heptadecanoylamino-4-O-(2,3,4,6-tetra-O-pivaloyl-
ꢂ
(500 mg, 1.37 mmol), donor 4 (2.26 g, 3.42 mmol) and activated 4 A
molecular sieves (1 g) in dry CH2Cl2 (50 mL) was stirred under
b-D-galactopyranosyl)-butan-1-al 7
nitrogen atmosphere for 1 h. TMSOTf (61
mL, 0.342 mmol) was
added and the mixture was stirred for 3 h at ꢂ15 ꢀC. When TLC (n-
hexane/EtOAc, 4:1) showed complete consumption of the acceptor,
the mixture was filtered through a pad of CeliteÒ and the filtrate
was diluted with CH2Cl2 (20 mL) and washed successively with aq
NaHCO3 (2ꢁ30 mL) and brine (30 mL). The organic layer was sep-
arated, dried (Na2SO4), and evaporated to give a syrup. The crude
product was purified by flash chromatography using n-hexane/
To a solution of compound 6 (300 mg, 0.296 mmol) in dry
CH3CN (5 mL), MeI (184 L, 2.963 mmol) was added and the solu-
m
tion was refluxed for 12 h. When TLC showed complete conversion
of the starting material, the solvents were evaporated and dried
over vacuum. Next, the methylated thiazolyl ammonium iodide
was redissolved in dry methanol (5 mL) and NaBH4 (22 mg,
0.592 mmol) was added at 0 ꢀC. The mixture was kept for 5 min at
this temperature, warmed to room temperature and stirred for
30 min. Then, methanol was evaporated and the crude product was
dissolved in 20 mL of CH3CN/H2O (8:2). After the introduction of
HgCl2 (66 mg, 0.246 mmol), the mixture was stirred at room tem-
perature for 1 h. Then, the solvents were evaporated and the resi-
due was portioned between EtOAC (30 mL) and water (30 mL). The
EtOAc (6:1) to afford pure 5 (350 mg, 0.405, 82%) as a colorless
25
foam. [
a]
þ66ꢀ (c 1.0, CHCl3). 1H NMR (CDCl3, 300 MHz):
d 7.75,
D
7.38 (2d, 2H, J¼3.12 Hz, Thiazole-H), 7.31 (m, 5H, C6H5), 5.79 (d, 1H,
J¼2.9 Hz, H-4), 5.09 (dd, 1H, J¼7.4, 10.4 Hz, H-2), 5.07 (dd, 1H, J¼2.9,
10.4 Hz, H-3), 4.86 (d, 1H, J¼8.0 Hz, H0-3), 4.62, 4.52 (2d, 2H,
J¼11.4 Hz, CH2C6H5), 4.49 (d, 1H, J¼8.0 Hz, H-1), 4.38 (m, 1H, H0-2),

5180
B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
organic layer was separated, washed with brine (2ꢁ30 mL), dried
(t, 6H, 2ꢁ CH3). 13C NMR (500 MHz, CDCl3):
d 177.8, 177.2, 176.8,
over MgSO4, and concentrated under reduced pressure. The residue
176.7 (4ꢁ COC(CH3)3), 173.3 (COCH2CH2), 138.2, 128.4 (2), 127.9 (2),
127.8 (C6H5), 101.5 (C-1), 77.2, 75.7, 72.0, 71.1, 70.8, 69.1, 66.6, 61.0,
50.7, 50.1 39.0, 38.8, 38.7, 38.6, 36.9, 31.9, 29.7, 29.6, 29.3, 28.3, 27.1,
25.7, 22.6, 14.0. Elemental analysis: calcd for C68H120N2O12
(1157.68): C 70.55, H 10.45, N 2.42; found C 70.42, H 10.29, N 2.35.
was purified on a silica gel column using n-hexane/EtOAc (3:1) to
20
D
afford pure aldehyde 7 (207 mg, 73%) as a colorless oil. [
a
]
þ27ꢀ
(c 1.5, CH2Cl2). 1H NMR (CDCl3, 400 MHz):
d
9.56 (1H, CHO), 7.35 (m,
5H, C6H5), 5.79 (d, 1H, J¼8.4 Hz, NH), 5.42 (d, 1H, J¼3.12 Hz, H-4),
5.19 (dd, 1H, J¼7.55, 10.47 Hz, H-2), 5.13 (dd, 1H, J¼3.14, 10.47 Hz, H-
3), 4.65, 4.59 (2d, 2H, J¼11.43 Hz, CH2C6H5), 4.54 (d, 1H, J¼7.52 Hz,
H-1), 4.46 (m, 1H, H0-3), 4.14 (m, 2H, H-5, H0-2), 4.05, 3.96 (dd, 2H,
J¼7.14, 10.89 Hz, H-6ab), 3.85 (dd, 1H, J¼3.09, 7.9 Hz, H0-4a), 3.64
(dd, 1H, J¼4.38, 9.59 Hz, H0-4b), 2.09 (t, 2H, COCH2), 1.58 (m, 2H,
COCH2CH2), 1.28 (m, 36H),1.19,1.14 (3s, 27H, 3ꢁ COC(CH3)3), 0.90 (t,
20
D
3.6.3. Compound 8c. [
a
]
þ6.1 (c 1.8, CHCl3). 1H NMR (CDCl3,
500 MHz):
d
7.38e7.34 (m, 6H, C6H5, NH), 5.41 (d, 1H, J¼3.3, Hz, H-
4), 5.19 (dd, 1H, J¼7.6, 10.5 Hz, H-2), 5.12 (dd, 1H, J¼3.3, 10.5 Hz, H-
3), 4.70 (d, 1H, J¼11.5 Hz, CH2C6H5), 4.57 (d, 1H, J¼7.6 Hz, H-1), 4.56
(dd, 1H, J¼11.5 Hz, CH2C6H5), 4.38 (m, 1H, H0-2), 4.05 (m, 4H), 3.71
(m, 1H), 3.58 (dd, 1H, J¼5.8, 9.9 Hz, H0-1), 2.90 and 2.80 (2dd, 2H,
J¼4.0, 12.8 Hz), 2.59 (m, 2H), 2.13 (t, 3H, COCH2), 1.6 (m, 4H), 1.46
(m, 2H), 1.26 (m, 54H), 1.17 (2), 1.12 (3s, 27H, 3ꢁ COC(CH3)3), 0.89 (t,
3H, CH3). 13C NMR (400 MHz, CDCl3):
d 200.5 (CHO), 177.7, 177.1,
176.8, 176.7 (4ꢁ COC(CH3)3), 172.8 (COCH2CH2), 136.9, 128.6 (2),
128.3, 128.1 (2) (C6H5), 101.0 (C-1), 82.4 (C0-2), 77.2, 73.5 (CH2C6H5),
71.2 (C-3), 70.6 (C-2), 69.0, 66.8, 66.5, 60.9, 48.6, 39.0, 38.8, 38.7,
38.6, 36.5, 31.8, 29.6, 29.4, 29.3, 27.1, 27.0, 25.3, 22.6, 14.0 (CH3).
Elemental analysis: calcd for C54H89NO13 (960.28): C 67.54, H 9.34,
N 1.46; found C 66.99, H 9.08, N 2.74.
6H, 2ꢁ CH3). 13C NMR (500 MHz, CDCl3):
d 177.7, 177.1, 176.8, 176.7
(4ꢁ COC(CH3)3), 173.4 (COCH2CH2), 138.1, 128.4 (2), 127.9 (2), 127.8
(C6H5), 101.5 (C-1), 77.2, 75.7, 72.1, 71.1, 70.8, 69.1, 68.6, 66.6, 60.9,
50.6, 50.1, 39.0, 38.8, 38.7, 38.6, 36.9, 31.9, 29.7, 29.5 (2), 29.4, 29.3,
27.1(2), 27.0 (2), 25.6, 22.6, 14.0. Elemental analysis: calcd for
C66H116N2O12 (1129.63): C 70.17, H 10.35, N 2.48; found C 69.83, H
10.40, N 2.73.
3.6. General procedure for reductive amination. Synthesis of
(2S,3R)-5-aza-3-O-benzyl-2-heptadecanoylamino-1-O-
(2,3,4,6-tetra-O-pivaloyl-
alcanediols 8aee
b-D-galactopyrano-syl)-1,3-
20
D
3.6.4. Compound 8d. [
a
]
þ4.1 (c 1.0, CHCl3). 1H NMR (CDCl3,
400 MHz):
d
7.44 (br s, 1H, NH), 7.31 (m, 5H, C6H5), 5.37 (d, 1H,
A solution of aldehyde 7 (120 mg, 0.125 mmol) and alcanamine
[for example, pentadecanamine (56 mg, 0.250 mmol) in MeOH
(5 mL)] was stirred at room temperature for 2 h. When TLC showed
complete formation of the intermediate imine, NaBH3CN (11 mg,
0.1873 mmol) was added at the same temperature and the reaction
mixture was stirred for an additional period of 2 h at room tem-
perature. Then, MeOH was evaporated under vacuum and 10% HCl
(10 mL) was added to the residue. The aqueous solution was stirred
for 0.5 h at room temperature and aq Na2CO3 (ca. 2.5 g in 10 mL
H2O) was added under cooling to make the solution alkaline. The
aqueous solution was extracted with CH2Cl2 (30 mLꢁ2), and the
combined organic layers were washed with brine (15 mL), dried
over Na2SO4, and concentrated. The crude product was purified on
a silica gel column with n-hexane/AcOEt (1:1) to afford pure 8a
(127 mg, 87%). The same procedure was used for the synthesis of
8bee (yields, 8b 81%, 8c 79%, 8d 85%, 8e 88%).
J¼3.2 Hz, H-4), 5.16 (dd, 1H, J¼7.8, 10.5 Hz, H-2), 5.08 (dd, 1H, J¼3.2,
10.5 Hz, H-3), 4.66 (d, 1H, J¼11.5 Hz, CH2C6H5), 4.54 (d, 1H, J¼7.8 Hz,
H-1), 4.50 (m, 2H, CH2C6H5, H0-2), 4.36 (m, 1H, H-5), 4.01 (m, 4H),
3.68 (m, 1H), 3.54 (dd, 1H, J¼5.9, 9.6 Hz, H0-1), 2.87, 2.77 (2dd, 2H,
J¼3.8, 12.7 Hz), 2.55 (m, 2H), 2.08 (t, 2H, COCH2), 1.56 (m, 2H), 1.43
(m, 2H), 1.22 (m, 42H), 1.14, 1.13, 1.09 (3s, 27H, 3ꢁ COC(CH3)3), 0.85
(t, 6H, 2ꢁ CH3). 13C NMR (400 MHz, CDCl3):
d 177.9, 177.3, 176.9 (2)
(4ꢁ COC(CH3)3), 173.4 (COCH2), 138.4, 128.6 (2), 128.1 (2), 127.9
(C6H5) 101.6 (C-1), 75.9, 75.7, 72.2, 71.3, 71.0, 69.3, 68.8, 66.8, 61.2,
50.8, 50.3, 39.2, 38.9 (2), 37.1, 32.0 (2), 29.8, 29.7, 29.5, 29.4, 27.4,
27.3, 27.2, 25.8, 22.8, 14.2. Elemental analysis: calcd for
C62H108N2O12 (1073.52): C 69.37, H 10.14, N 2.61; found C 69.39, H
10.09, N 2.55.
20
3.6.5. Compound 8e. [
400 MHz):
a]
þ3.8 (c 0.8, CHCl3). 1H NMR (CDCl3,
D
d
7.36 (m, 6H, C6H5, NH), 5.41 (d, 1H, J¼3.2 Hz, H-4), 5.19
(dd, 1H, J¼7.5, 10.4 Hz, H-2), 5.13 (dd, 1H, J¼3.2, 10.4 Hz, H-3), 4.69
(d, 1H, J¼11.5 Hz, CH2C6H5), 4.60 (d, 1H, J¼11.5 Hz, CH2C6H5), 4.57
(d, 1H, J¼7.4 Hz, H-1), 4.3 (m, 1H, H0-2), 4.09 (m, 4H), 3.73 (m, 1H),
3.60 (dd, 1H, J¼5.4, 9.8 Hz, H0-1), 2.94 and 2.79 (2dd, 2H, J¼3.9,
12.7 Hz), 2.7e2.59 (m, 2H), 2.14 (t, 2H, COCH2),1.56 (m, 4H),1.26 (m,
38H), 1.17, 1.13 (2s, 18H, 2ꢁ COC(CH3)3), 0.89 (t, 6H, 2ꢁ CH3). 13C
20
D
3.6.1. Compound 8a. [
a
]
þ3.7 (c 1.2, CHCl3). 1H NMR (CDCl3,
500 MHz):
d
7.48e7.36 (m, 6H, C6H5, NH), 5.41 (d, 1H, J¼3.3 Hz, H-
4), 5.20 (dd, 1H, J¼7.6, 10.5 Hz, H-2), 5.12 (dd, 1H, J¼3.3, 10.5 Hz, H-
3), 4.70 (d, 1H, J¼11.5 Hz, CH2C6H5), 4.57 (d, 1H, J¼7.6 Hz, H-1), 4.56
(d, 1H, J¼11.5 Hz, CH2C6H5), 4.39 (m, 1H, H0-2), 4.07 (m, 4H), 3.70
(m, 1H), 3.58 (dd, 1H, J¼5.9, 9.9 Hz, H0-1), 2.90 and 2.81 (2dd, 2H,
J¼4.09, 12.8 Hz), 2.58 (m, 2H), 2.13 (t, 3H, COCH2), 1.60 (m, 4H), 1.47
(m, 2H), 1.27 (m, 61H), 1.17 (2), 1.13 (3s, 27H, 3ꢁ COC(CH3)3), 0.89 (t,
NMR (400 MHz, CDCl3):
d
177.7, 177.1, 176.9, 176.6 (4ꢁ COC(CH3)3),
173.9 (COCH2), 137.9, 128.5 (2), 128.1 (2), 127.9 (C6H5) 101.3 (C-1),
77.2, 75.2, 72.3, 71.1, 70.7, 69.2, 68.2, 66.6, 60.9, 50.5, 49.9, 39.0, 38.8,
38.7, 38.6, 36.7, 31.9, 29.6 (3), 29.5, 29.3 (3), 27.1 (2), 27.0, 25.6, 22.6,
22.4, 14.0, 13.9. Elemental analysis: calcd for C59H102N2O12
(1031.44): C 68.70, H 9.97, N 2.72; found C 67.34, H 9.93, N 3.30.
6H, 2ꢁ CH3). 13C NMR (500 MHz, CDCl3):
177.7, 177.1, 176.7 (2) (4ꢁ
d
COC(CH3)3), 173.3 (COCH2CH2), 138.2, 128.4 (2), 127.9 (2), 127.8
(C6H5) 101.4 (C-1), 77.2, 75.6, 72.0, 71.1, 70.8, 69.1, 68.6, 66.6, 60.9,
50.6, 50.1, 39.0, 38.8, 38.7, 38.6, 36.9, 31.9, 29.6, 29.5, 29.4, 29.3,
29.3, 27.1, 27.0, 25.7, 22.6, 14.0. Elemental analysis: calcd for
C69H122N2O12 (1171.71): C 70.73, H 10.49, N 2.39; found C 69.34, H
10.44, N 2.59.
3.7. Synthesis of (2S,3R)-5-aza-2-heptadecanoylamino-1-O-
(b-D
-galactopyranosyl)-1,3-alcanediols 9aee
20
3.6.2. Compound 8b. [
500 MHz):
a]
þ2.1ꢀ (c 1.0, CHCl3). 1H NMR (CDCl3,
To a solution of 8a (112 mg, 0.095 mmol) in MeOH (10 mL),
D
d
7.46 (br s, 1H, NH), 7.34 (m, 5H, C6H5), 5.39 (d, 1H,
NaOMe (50 mg) was added and the suspension was stirred at room
temperature for 24 h. At this time, TLC (1:2 n-hexane/EtOAc)
showed complete conversion of the starting material. Solvents
were evaporated in vacuo and co-evaporated with toluene. The
resulting syrup (55 mg, 0.4 mmol) was dissolved in CH3OH and 20%
Pd(OH)2/C (50 mg) was added. The reaction mixture was stirred at
room temperature under a positive pressure of hydrogen for 12 h.
J¼3.2 Hz, H-4), 5.18 (dd, 1H, J¼7.8, 10.5 Hz, H-2), 5.10 (dd, 1H, J¼3.2,
10.5 Hz, H-3), 4.68 (d, 1H, J¼11.6 Hz, CH2C6H5), 4.55 (d, 1H, J¼7.8 Hz,
H-1), 4.53 (m, 2H, CH2C6H5, H0-2), 4.39 (m, 1H, H-5), 4.04 (m, 4H),
3.71 (m, 1H), 3.56 (dd, 1H, J¼5.9, 9.6 Hz, H0-1), 2.89, 2.79 (2dd, 2H,
J¼3.94, 12.7 Hz), 2.57 (m, 2H), 2.10 (t, 3H, COCH2), 1.58 (m, 4H), 1.45
(m, 2H), 1.24 (m, 64H), 1.15, 1.15, 1.11 (3s, 27H, 3ꢁ COC(CH3)3), 0.87

B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
5181
The reaction mixture filtered through a CeliteÒ bed and evaporation
to dryness afforded the pure 9a (65 mg, 65%) as a white foam.
analysis: calcd for C42H65NO14 (807.96): C 62.43, H 8.11, N 1.72;
found C 62.39, H 7.54, N 1.59.
25
D
3.7.1. Compound 9a. [
a
]
þ21.3 (c 1.4, CHCl3). 1H NMR (CDCl3,
3.9. Synthesis of (2S,3S)-5-aza-3-O-benzyl-2-tert-
butyloxycarbonylamino-1-O-(2,3,4,6-tetra-O-pivaloyl-b-D-
galactopyranosyl)-1,3-nonadecananediol 11
400 MHz):
d
4.41 (m), 3.94 (m, 2H), 3.59e3.37 (5H), 2.75 (m, 2H),
2.19 (m, 2H), 1.48 (m, 2H), 1.18 (m), 0.80 (t, 6H). 13C NMR (400 MHz,
CDCl3): 172.0, 101.0, 78.0, 76.0, 74.5, 72.0, 71.5, 69.5, 61.5, 48.5,
d
39.0, 32.0, 30.0, 27.0, 26.5, 23.5, 14.0. HRMS calcd for C42H88N3O8
(MþNH4)þ: 763.1250; found m/z 763.1241. Elemental analysis:
calcd for C42H84N2O8 (744): C 67.74, H 11.29, N 3.76; found C 67.58,
H 11, 10 N 3.87.
A solution of aldehyde 10 (535 mg, 0.662 mmol) and tetrade-
canamine (282 mg, 1.32 mmol) in MeOH (10 mL) was stirred at
room temperature for 2 h. When TLC showed complete formation
of the intermediate imine, NaBH3CN (62 mg, 0.993 mmol) was
added at the same temperature and the reaction mixture was
stirred for an additional 2 h at room temperature. Then, MeOH was
evaporated under vacuum and 10% HCl (20 mL) was added to the
residue. The aqueous solution was stirred for 0.5 h at room tem-
perature and aq Na2CO3 (ca. 2.5 g in 50 mL H2O) was added under
cooling to make the solution alkaline. The aqueous layer was
extracted with CH2Cl2 (500 mLꢁ2), and the combined organic
layers were washed with brine (35 mL), dried over Na2SO4, and
concentrated. The crude product was purified on a silica gel column
with n-hexane/AcOEt (1:1) as an eluent to afford pure 11 (565 mg,
20
3.7.2. Compound 9c. [
400 MHz):
a
]
D
þ19.4 (c 1.2, CHCl3). 1H NMR (CDCl3,
d
4.2 (m), 3.9 (m), 3.63e3.5 (m), 2.52e2.21 (m), 1.90 (m),
1.2, 1.02e0.89 (m), 0.77 (t). 13C NMR (400 MHz, CDCl3):
d 169.5,
104.0, 75.0, 74.5, 73.0, 71.0 (2), 70.0 (2), 61.0, 49.0, 40.0, 35.0, 32.0,
30.0, 28.0, 27.5, 22.0, 14.0. HRMS calcd for C39H82N3O8 (MþNH4)þ:
720.5758; found m/z 720.5749. Elemental analysis: calcd for
C39H78N2O8 (702): C 66.67, H 11.11, N 3.99; found C 67.01, H 10.85, N
3.81.
Compounds 9b, 9d, and 9e gave 1H and 13C spectra similar to
those of 9a and 9c.
85%). [a] d 7.35 (m,
20 e3.9 (c 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz):
D
5H, C6H5), 5.47 (d,1H, J¼8.1 Hz, NH), 5.41 (d,1H, J¼3.2 Hz, H-4), 5.24
(dd, 1H, J¼7.6, 10.5 Hz, H-2), 5.13 (dd, 1H, J¼3.2, 10.5 Hz, H-3), 4.7,
4.6 (2d, 2H, J¼11.2 Hz, CH2C6H5), 4.56 (d, 1H, J¼7.9 Hz, H-1), 4.2 (m,
1H, H0-2), 4.0 (m, 4H, H-6ab, H01a), 3.67 (m, H-5), 3.35 (dd, 1H,
J¼4.2, 9.4 Hz, H0-1b), 2.8 (m, 2H), 2.6 (m, 2H), 1.4 (s, 9H, (CO)
OC(CH3)3), 1.27 (m, 27H), 1.23, 1.20, 1.18, 1.13 (4s, 27H, 4ꢁ
25
3.7.3. Compound 9b. [
a
]
þ24.3 (c 1.2, CHCl3), elemental analysis:
D
calcd for C41H82N2O8 (730): C 67.40, H 11.23, N 3.84; found C 67.11, H
10.95, N 3.91.
25
D
3.7.4. Compound 9d. [
a
]
þ20.9 (c 1.1, CHCl3), elemental analysis:
calcd for C35H70N2O8 (646): C 65.02, H 10.84, N 4.33; found C 64.85,
H 10.95, N 4.17.
COC(CH3)3). 13C NMR (400 MHz, CDCl3):
d 177.7 (2), 177.2, 176.8,
176.7, 138.2, 101.1 (C-1), 77.2, 72.8, 71.0, 70.8, 69.0, 68.5, 61.0, 50.0,
39.0, 38.8, 38.7 (2), 31.9, 29.6 (5), 29.5, 29.3, 28.3 (3), 27.1 (12),
22.66, 14.0. Elemental analysis: calcd for C56H96N2O13 (1005.36): C
66.90, H 9.62, N 2.79; found C 66.83, H 9.61, N 2.79.
25
3.7.5. Compound 9e. [
a
]
þ18.6 (c 0.9, CHCl3), elemental analysis:
D
calcd for C32H64N2O8 (604): C 63.58, H 10.60, N 4.64; found C 63.41,
H 10.95, N 4.58.
3.10. Synthesis of (2S,3R)-5-aza-3-O-benzyl-2-alcanoylamino-
3.8. Thiazolyl to formyl conversion: synthesis of (2S,3S)-2-O-
benzyl-3-tert-butyloxycarbonylamino-4-O-(2,3,4,6-tetra-O-
1-O-(2,3,4,6-tetra-O-pivaloyl-b-D-galactopyranosyl)-1,3-
nonadecananediol 12
pivaloyl-b-D-galactopyranosyl)-2,4-butan-1-al 10
3.10.1. Compound 12a (alcanoylamino group¼eCOeC21H43). A so-
lution of compound 11 (250 mg, 0.248 mmol) and 50% TFA in
CH2Cl2 (10 mL) was stirred for 3 h. When TLC showed complete
conversion of the starting material, the solution was diluted with
dichloromethane (25 mL) and washed successively with ice-cold
NaHCO3 (2ꢁ50 mL) and brine (50 mL). Then, the organic layer
was separated, dried over MgSO4, and evaporated under vacuum.
The crude amine produced was used for the next step without any
further purification.
To a solution of compound 5 (700 mg, 0.81 mmol) in dry CH3CN
(10 mL), MeI (504 L, 8.11 mmol) was added and the solution was
m
refluxed for 12 h. When TLC showed complete conversion of the
starting material, the solvents were evaporated under vacuum. The
residue was dissolved in dry methanol (10 mL) and NaBH4 (61 mg,
1.62 mmol) was added at 0 ꢀC. After 5 min reaction at 0 ꢀC, the
mixture was warmed to room temperature and stirred for 30 min.
Then, MeOH was evaporated and the crude product was dissolved
in 20 mL of CH3CN/H2O (8:2). Next, HgCl2 (263 mg, 0.97 mmol) was
added and the mixture was stirred at room temperature for an
additional 1 h period. The solvents were evaporated and the residue
was portioned between EtOAC (50 mL) and water (50 mL). The
organic layer was separated, washed with brine (2ꢁ35 mL), dried
over MgSO4, and concentrated under reduced pressure. The residue
To a solution of the crude amine and docosanoic acid (170 mg,
0.248 mmol) in dry DMF (10 mL) was added EDC (55
mL,
0.298 mmol) at 0 ꢀC under a nitrogen atmosphere. After 30 min, the
reaction mixture was warmed to 15 ꢀC and stirred overnight. The
solvents were evaporated and the residue was dissolved in
dichloromethane (30 mL) and washed with brine (25 mL). The or-
ganic layer was dried over MgSO4 and concentrated under reduced
pressure. The crude product was purified by flash chromatography
was purified on a silica gel column using n-hexane/EtOAc (5:1) to
20
afford pure 10 (537 mg, 82%) as a colorless oil. [
a]
þ16.4 (c 1.5,
D
CHCl3). 1H NMR (CDCl3, 400 MHz):
d
9.61 (d, 1H, J¼2.94 Hz, CHO),
using n-hexane/EtOAc (2:1) to afford pure 12a (250 mg, 82%) as
20
D
7.35 (m, 5H, C6H5), 5.42 (d, 1H, J¼3.42 Hz, H-4), 5.22 (dd, 1H, J¼7.8,
10.5 Hz, H-2), 5.13 (dd, 1H, J¼3.3, 10.4 Hz), 4.89 (d, 1H, J¼8.6 Hz,
H02), 4.65 and 4.60 (2d, 2H, J¼11.2 Hz, CH2C6H5), 4.55 (d, 1H,
J¼7.81 Hz, H-1), 4.21 (m, 1H, H-5), 4.13 (m, 1H, H0-3), 4.06 (dd, 1H,
J¼7.1, 11.0 Hz, H-6b), 3.98 (m, 1H, H-6a), 3.83 (dd, 1H, J¼2.9, 8.2 Hz,
H0-4a), 3.6 (dd, 1H, J¼3.9, 9.4 Hz, H0-4b), 1.4 (s, 9H, OC(CH3)3), 1.26,
1.19, 1.18, 1.13 (4s, 36H, COC(CH3)3). 13C NMR (400 MHz, CDCl3):
a colorless oil. [
d
a
]
þ5.8ꢀ (c 1.3, CHCl3). 1H NMR (CDCl3, 400 MHz):
7.5 (d, 1H, J¼8.5 Hz, NH), 7.35 (m, 5H, C6H5), 5.40 (d, 1H, J¼3.2 Hz,
H-4), 5.20 (dd, 1H, J¼7.7, 10.5 Hz, H-2), 5.12 (dd, 1H, J¼3.2, 10.5 Hz,
H-3), 4.7 (d, 1H, J¼11.6 Hz, CH2C6H5), 4.57 (d, 1H, J¼7.5 Hz, H-1), 4.5
(m, 2H, CH2C6H5, H0-2), 4.4 (m, 1H, H-5), 4.06 (m, 4H), 3.7 (m, 1H),
3.58 (dd,1H, J¼6.1, 9.4 Hz), 2.81, 2.79 (2dd, 2H, J¼3.91,12.5 Hz), 2.56
(m, 2H), 2.12 (t, 3H, COCH2), 1.59 (m, 2H), 1.45 (m, 4H), 1.26 (m,
75H),1.17, 1.16,1.12 (3s, 27H, 3ꢁ (CO)OC(CH3)3), 0.89 (t, 6H, 2ꢁ CH3).
d
200.6 (CHO), 177.8, 177.2, 176.9, 176.8 (4ꢁ COC(CH3)3), 155.0, 136.9,
128.5 (2C), 128.2 (2C), 101.0 (C-1), 82.4, 77.2, 73.6, 71.1, 70.7, 68.8,
67.3, 66.6, 61.0, 41.0, 28.2, 27.14, 27.10, 27.06, 27.05. Elemental
13C NMR (400 MHz, CDCl3):
COC(CH3)3), 173.3 (COCH2CH2), 138.2, 128.4 (2), 127.9 (2), 127.7
d
177.7 (2), 177.1, 176.7 (2), (4ꢁ

5182
B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
(C6H5) 101.4 (C-1), 77.2, 75.7, 72.0, 71.1, 70.8, 69.1, 68.6, 66.6, 61.0,
50.7, 39.0, 38.8, 38.7 (2), 31.9, 29.7 (5), 29.6, 29.3, 28.3 (3), 27.1 (12),
22.6, 14.0. Elemental analysis: calcd for C73H130N2O12 (1227.81): C
71.41, H 10.67, N 2.28; found C 71.21, H 10.60, N 2.20.
2 and 1-Hb). 13C NMR (400 MHz, D2O):
d 177.2, 140.3, 125.5 (thia-
zolyl), 56.4 (C-3), 52.8 (C-1), 40.3 (C-2). Elemental analysis: calcd
for C6H10N2O2S (174.04): C 41.36, H 5.79, N 16.08, S 18.40; found C
41.12, H 5.23, N 15.4, S 18.21.
3.10.2. Compound 12b (alcanoylamino group¼eCOeC24H49). Same
3.13. Enzymatic synthesis of (2S,3S)-2-amino-1-O-(
galactopyranosyl)-3-(thiazol-2-yl)-1,3-propanediol 15
b-D-
20
experimental procedure as for 12a (yield 81%). [
a
]
D
þ7.1ꢀ (c 1.0,
CHCl3). 1H NMR (CDCl3, 400 MHz):
d
7.5 (d, 1H, J¼8.5 Hz, NH), 7.35
(m, 5H, C6H5), 5.40 (d, 1H, J¼3.2 Hz, H-4), 5.20 (dd, 1H, J¼7.7,
10.5 Hz, H-2), 5.12 (dd, 1H, J¼3.2, 10.5 Hz, H-3), 4.7 (d, 1H, J¼11.6 Hz,
CH2C6H5), 4.57 (d, 1H, J¼7.5 Hz, H-1), 4.5 (m, 2H, CH2C6H5, H0-2), 4.4
(m, 1H, H-5), 4.06 (m, 4H), 3.7 (m, 1H), 3.58 (dd, 1H, J¼6.1, 9.4 Hz),
2.81, 2.79 (2dd, 2H, J¼3.91, 12.5 Hz), 2.56 (m, 2H), 2.12 (t, 3H,
COCH2), 1.59 (m, 2H), 1.45 (m, 4H), 1.26 (m, 75H), 1.17, 1.16, 1.12 [3s,
27H, 3ꢁ (CO)OC(CH3)3], 0.89 (t, 6H, 2ꢁ CH3). 13C NMR (400 MHz,
a-D-Galactosyl fluoride (83 mg, 0.46 mmol) and (1S,2S)-2-
amino-3-(thiazol-2-yl)-1,3-propanediol 14 (91 mg, 0.27 mmol)
were dissolved in phosphate buffer (50 mmol/L, pH 7.0, 8 mL). Then,
E338G glycosynthase solution47 (3 mL), was added. The reaction
was allowed to proceed at 55 ꢀC for 12 h. At this time, the Gal-F had
completely disappeared. After elimination of the solvent under
diminished pressure, the crude mixture was dissolved in D2O and
the yield (15%) was determined by 1H NMR spectroscopy. At this
stage, we did not perform any purification for compound 15. Nev-
ertheless, NMR parameters were extracted from the mixture using
TOCSY NMR experiments.
CDCl3):
d
177.7 (2), 177.1, 176.7 (2), (4ꢁ COC(CH3)3), 173.3
(COCH2CH2),138.2,128.4 (2),127.9 (2),127.7 (C6H5) 101.4 (C-1), 77.2,
75.7, 72.0, 71.1, 70.8, 69.1, 68.6, 66.6, 61.0, 50.7, 39.0, 38.8, 38.7 (2),
31.9, 29.7 (5), 29.6, 29.3, 28.3 (3), 27.1 (12), 22.6, 14.0. Elemental
analysis: calcd for C76H136N2O12 (1269.89): C 71.88, H 10.79, N 2.21;
found C 71.90, H 10.65, N 2.23.
1H NMR (D2O, 400 MHz):
d
8.26, 8.21 (2d, 2H, J¼3.9 Hz, thia-
zolyl-H),4.60 (d, 1H, J¼7.8 Hz, H-1), 4.22e4.17 (m, 2H, H0-3 and H0-
1a), 4.01 (dd, 1H, H0-1b), 3.95 (m, 1H, H-4), 3.60e3.90 (6H, H-2, H-3,
H-5, H-6ab, H0-2).
3.11. Synthesis of (2S,3R)-2-alcanoylamino-5-aza-1-O-
galactopyranosyl-1,3-nonadecanediols 13aeb
b-D-
Acknowledgements
To a solution of 12a (95 mg, 0.077 mmol) in MeOH (10 mL),
NaOMe (50 mg) was added and the suspension was stirred at room
temperature for 24 h. At this time, TLC (1:2 n-hexane/EtOAc)
showed complete conversion of the starting material. Solvent was
evaporated in vacuo and co-evaporated with toluene. The resulting
syrup (55 mg, 0.4 mmol) was dissolved in CH3OH and Pd(OH)2/C
(20%, 50 mg) was added. The reaction mixture was stirred at room
temperature under a positive pressure of hydrogen for 12 h. It was
then filtered through a CeliteÒ bed and evaporated to dryness to
We are grateful to ANR (Glycenli project), the French Ministry of
Education and the University of Nantes for financial support.
Supplementary data
1H and 13C spectra of compounds 3e14 (only some examples of
galactosyl ceramides 8, 9,12, and 13 are given). Supplementary data
associated with this article can be found, in the online version, at
doi:10.1016/j.tet.2011.05.054.
afford the pure target (2S,3S)-5-aza-2-docosanoylamino-1-O-(b-D-
galactopyranosyl)-1,3-nonadecanediol (13a) (45 mg, 72%) as
a white foam.
25
D
[a
]
þ27.7 (c 1.0, CHCl3). 1H NMR (CDCl3, 400 MHz):
d 4.67 (d,
References and notes
1H, H-1), 4.40 (m), 4.09e4.07 (m), 3.95 (m, 1H, H-4), 3.68e3.35 (m),
2.75 (m), 2.19 (m), 1.49, 1.35e11.18 (m), 0.80 (t). 13C NMR (400 MHz,
1. Stoffel, W.; Bosio, A. Curr. Opin. Neurobiol. 1997, 7, 654e661.
2. Boggs, J. M.; Wang, H. M.; Gao, W.; Arvanitis, D. N.; Gong, Y. P.; Min, W. X.
Glycoconjugate J. 2004, 21, 97e110.
3. Kolter, T.; Sandhoff, K. Angew. Chem., Int. Ed. 1999, 38, 1532e1568.
4. Kolter, T.; Sandhoff, K. BBA-Biomembranes 2006, 1758, 2057e2079.
5. Villard, R.; Hammache, D.; Delapierre, G.; Fotiadu, F.; Buono, G.; Fantini, J.
ChemBioChem 2002, 3, 517e525.
CDCl3):
d 174.0 (COeNH), 101.5 (C-1), 75.0 (C-5), 74.5, 73.5 (C-3),
71.0 (C-2), 70.0 (C-4 and C0-1), 61.0 (C-6), 49.0, 40.0, 35.0, 32.0, 31.0,
27.0, 27.5, 22.5, 14.0. HRMS calcd for C46H96N3O8 (MþNH4)þ:
819.2313; found m/z 819.2307.
6. Nolting, B.; Yu, J. J.; Liu, G. Y.; Cho, S. J.; Kauzlarich, S.; Gervay-Hague, J. Langmuir
2003, 19, 6465e6473.
25
3.11.1. Compound 13b. [
400 MHz):
a
]
D
þ25.1 (c 1.0, CHCl3). 1H NMR (CDCl3,
7. Santacroce, P. V.; Basu, A. Angew. Chem., Int. Ed. 2003, 42, 95e98.
8. Van Kaer, L. Nat. Rev. Immunol. 2005, 5, 31e42.
9. Lu, X. Q.; Bittman, R. Tetrahedron Lett. 2005, 46, 3165e3168.
10. Cai, Y.; Ling, C. C.; Bundle, D. R. Org. Biomol. Chem. 2006, 4, 1140e1146.
11. Jeon, J.; Shin, M.; Yoo, J. W.; Oh, J. S.; Bae, J. G.; Jung, S. H.; Kim, Y. G. Tetrahedron
Lett. 2007, 48, 1105e1108.
12. Kim, S.; Lee, N.; Lee, S.; Lee, T.; Lee, Y. M. J. Org. Chem. 2008, 73, 1379e1385.
13. Vankar, Y. D.; Schmidt, R. R. Chem. Soc. Rev. 2000, 29, 201e216.
14. Rich, J. R.; Bundle, D. R. Org. Lett. 2004, 6, 897e900.
15. Jacques, S.; Rich, J. R.; Ling, C. C.; Bundle, D. R. Org. Biomol. Chem. 2006, 4,
142e154.
16. Rich, J. R.; Wakarchuk, W. W.; Bundle, D. R. Chem.dEur. J. 2006, 12, 845e858.
17. Bundle, D. R.; Rich, J. R.; Jacques, S.; Yu, H. N.; Nitz, M.; Ling, C. C. Angew. Chem.,
Int. Ed. 2005, 44, 7725e7729.
d
4.66 (d, 1H, H-1), 4.40 (m), 4.09e4.06 (m), 3.96 (m, 1H,
H-4), 3.68e3.35 (m), 2.75 (m), 2.19 (m), 1.49, 1.35e11.18 (m), 0.80
(t). 13C NMR (400 MHz, CDCl3):
d 174.1 (COeNH), 101.7 (C-1), 75.2
(C-5), 74.5, 73.6 (C-3), 71.0 (C-2), 70.0 (C-4 and C0-1), 61.1 (C-6),
49.0, 40.0, 35.0, 32.0, 31.0, 27.0, 27.5, 22.5, 14.0. HRMS calcd. For
C49H102N3O8 (MþNH4)þ: 861.2673; found m/z 861.2662.
3.12. Synthesis of (2S,3S)-2-amino-3-(thiazol-2-yl)-1,3-
propanediol 14
Compound 2 (1 g, 3.18 mmol) was dissolved in a mixture of
dioxan (10 mL) and 1 N HCl (5 mL), refluxed for 1 h, and cooled to
25 ꢀC. A solution of 2 N NaOH was added until pH 10 was reached.
The mixture was extracted with CH2Cl2 (3ꢁ250 mL). The organic
layers were washed with brine (250 mL), dried over MgSO4, and
concentrated in vacuo. After silica-gel column chromatography
18. Rai, A. N.; Basu, A. Org. Lett. 2004, 6, 2861e2863.
19. Chaudhari, V. D.; Kumar, K. S. A.; Dhavale, D. D. Org. Lett. 2005, 7, 5805e5807.
20. Yamamoto, T.; Hasegawa, H.; Hakogi, T.; Katsumura, S. Org. Lett. 2006, 8,
5569e5572.
21. Nussbaumer, P.; Ettmayer, P.; Peters, C.; Rosenbeiger, D.; Hogenauer, K. J. Chem.
Soc., Chem. Commun. 2005, 5086e5087.
22. Peters, C.; Billich, A.; Ghobrial, M.; Hogenauer, K.; Ullrich, T.; Nussbaumer, P.
J. Org. Chem. 2007, 72, 1842e1845.
(CH2Cl2/MeOH 4:1), compound 14 was obtained as a pale yellow
20
liquid (512 mg, 91%). [
a
]
þ51ꢀ (c 1.3, MeOH). 1H NMR (400 MHz,
23. Wild, R.; Schmidt, R. R. Tetrahedron: Asymmetry 1994, 5, 2195e2208.
24. Wild, R.; Schmidt, R. R. Liebigs Ann. Chem. 1995, 755e764.
25. Schmidt, R. R.; Bar, T.; Wild, R. Synthesis 1995, 868e876.
26. Lin, C. C.; Fan, G. T.; Fang, J. M. Tetrahedron Lett. 2003, 44, 5281e5283.
D
D2O):
d
8.27, 8.23 (2d, 2H, J¼3.88 Hz, thiazolyl-H), 5.89 (d, 1H,
J¼3.24 Hz, OH), 4.07 (m,1H, H-3), 3.95 (m,1H,1-Ha), 3.82 (m, 2H, H-

B. Roy et al. / Tetrahedron 67 (2011) 5176e5183
5183
27. Murata, K.; Toba, T.; Nakanishi, K.; Takahashi, B.; Yamamura, T.; Miyake, S.;
Annoura, H. J. Org. Chem. 2005, 70, 2398e2401.
28. Rai, A. N.; Basu, A. J. Org. Chem 2005, 70, 8228e8230.
29. Chang, C. W.; Chen, Y. N.; Adak, A. K.; Lin, K. H.; Tzou, D. L. M.; Lin, C. C. Tet-
rahedron 2007, 63, 4310e4318.
40. Lacone, V.; Hunault, J.; Pipelier, M.; Blot, V.; Lecourt, T.; Rocher, J.; Turcot-Du-
bois, A. L.; Marionneau, S.; Douillard, J. Y.; Clement, M.; Le Pendu, J.; Bonneville,
M.; Micouin, L.; Dubreuil, D. J. Med. Chem. 2009, 52, 4960e4963.
41. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P. J. Org. Chem. 1988,
53, 1748e1761.
30. Cai, Y.; Ling, C. C.; Bundle, D. R. Carbohydr. Res. 2009, 344, 2120e2126.
31. Ghosal, P.; Kumar, V.; Shaw, A. K. Tetrahedron 2010, 66, 7504e7509.
32. Yen, V. H.; Micouin, L.; Ronet, C.; Gachelin, G.; Bonin, M. ChemBioChem 2003, 4,
27e33.
33. Enders, D.; Terteryan, V.; Palecek, J. Synthesis 2008, 2278e2282.
34. Grondal, C.; Enders, D. Adv. Synth. Catal. 2007, 349, 694e702.
35. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Medici, A. J. Chem. Soc., Chem. Commun.
1988, 10e12.
36. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Pedrini, P. J. Org. Chem. 1990, 55,
1439e1446.
37. Dondoni, A.; Marra, A. Chem. Rev. 2004, 104, 2557e2599.
38. Dondoni, A. Org. Biomol. Chem. 2010, 8, 3366e3385.
39. Dondoni, A.; Perrone, D.; Turturici, E. J. Chem. Soc., Perkin Trans. 1 1997,
2389e2393.
42. Schmidt, R. R.; Michel, J. Angew. Chem., Int. Ed. Engl. 1980, 19, 731e732.
43. Dondoni, A.; Fantin, G.; Fogagnolo, M.; Medici, A.; Pedrini, P. Synthesis 1988,
685e689.
44. Vaughan, M. D.; Johnson, K.; DeFrees, S.; Tang, X. P.; Warren, R. A. J.; Withers, S.
G. J. Am. Chem. Soc. 2006, 128, 6300e6301.
45. Caines, M. E. C.; Vaughan, M. D.; Tarling, C. A.; Hancock, S. M.; Warren, R. A. J.;
Withers, S. G.; Strynadka, N. C. J. J. Biol. Chem. 2007, 282, 14300e14308.
46. Hancock, S. M.; Rich, J. R.; Caines, M. E. C.; Strynadka, N. C. J.; Withers, S. G. Nat.
Chem. Biol. 2009, 5, 508e514.
47. Dion, M.; Fourage, L.; Hallet, J.-N.; Colas, B. Glycoconjugate J. 1999, 16, 27e37.
48. Feng, H. Y.; Drone, J.; Hoffmann, L.; Tran, V.; Tellier, C.; Rabiller, C.; Dion, M.
J. Biol. Chem. 2005, 280, 37088e37097.
49. Osanjo, G.; Dion, M.; Drone, J.; Solleux, C.; Tran, V.; Rabiller, C.; Tellier, C. Bio-
chemistry 2007, 46, 1022e1033.