Synthesis of the lipoteichoic acid of the Streptococcus species DSM 8747

Article abstract of DOI:10.1016/j.bmc.2010.04.006

The lipoteichoic acid (LTA) of the Streptococcus species DSM 8747 consists of a β-d-galactofuranosyl diacylglycerol moiety (with different acyl groups) that is linked via 6-O to a poly(glycerophosphate) backbone; about 30% of the glycerophosphate moieties carry at 2-O hydrolytically labile d-alanyl residues. As typical LTA for this array of compounds LTA 1a was synthesized. To this end, from d-galactose the required galactofuranosyl building block 5 was obtained. The anomeric stereocontrol in the glycosylation step with 1,2-O-cyclohexylidene-sn-glycerol (4) was based on anchimeric assistance, thus finally leading to the unprotected core glycolipid 16. Regioselective protection and deprotection procedures permitted the defined attachment of the pentameric glycerophosphate 3 to the 6-hydroxy group of the galactose residue. Introduction of four d-alanyl residues led after global deprotection and purification to target molecule 1a possessing on average about two d-alanyl residues at 2-O of the pentameric glycerophosphate backbone, thus being in close accordance with the structure of the natural material.

Full text of DOI:10.1016/j.bmc.2010.04.006

Bioorganic & Medicinal Chemistry 18 (2010) 3696–3702
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of the lipoteichoic acid of the Streptococcus species DSM 8747
Yan Qiao a, Buko Lindner b, Ulrich Zähringer b, Peter Truog c, Richard R. Schmidt a,
*
a Fachbereich Chemie, Universität Konstanz, Fach 725, D-78457-Konstanz, Germany
b Laborgruppe Immunchemie, Forschungszentrum Borstel, Leibniz-Zentrum für Medizin und Biowissenschaften, Parkallee 1-40, D-23845-Borstel, Germany
c LUNAMed AG, CH-7000 Chur, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
The lipoteichoic acid (LTA) of the Streptococcus species DSM 8747 consists of a b-D-galactofuranosyl
Received 20 January 2010
Revised 29 March 2010
Accepted 1 April 2010
Available online 8 April 2010
diacylglycerol moiety (with different acyl groups) that is linked via 6-O to a poly(glycerophosphate)
backbone; about 30% of the glycerophosphate moieties carry at 2-O hydrolytically labile -alanyl resi-
dues. As typical LTA for this array of compounds LTA 1a was synthesized. To this end, from -galactose
the required galactofuranosyl building block 5 was obtained. The anomeric stereocontrol in the glycosyl-
ation step with 1,2-O-cyclohexylidene-sn-glycerol (4) was based on anchimeric assistance, thus finally
leading to the unprotected core glycolipid 16. Regioselective protection and deprotection procedures
permitted the defined attachment of the pentameric glycerophosphate 3 to the 6-hydroxy group of the
D
D
Keywords:
Lipoteichoic acid (LTA)
Synthesis
Glycosidation
Glycophospholipids
Gram-positive bacteria
galactose residue. Introduction of four
D-alanyl residues led after global deprotection and purification
to target molecule 1a possessing on average about two
D
-alanyl residues at 2-O of the pentameric
glycerophosphate backbone, thus being in close accordance with the structure of the natural material.
Ó 2010 Published by Elsevier Ltd.
1. Introduction
previous studies of Staphylococcus aureus revealed that a backbone
length of about five to six residues is sufficient for biological activ-
A unique lipoteichoic acid (LTA) was isolated from the cell wall
of the Streptococcus species DSM 8747 and structurally assigned
by Roethlisberger et al.1,2 (Fig. 1, 1). The LTA comprises a linear
(1–3)-linked poly(glycerophosphate) chain which is partly substi-
ity.7 Hence, we decided to synthesize LTA 1a having five glycero-
phosphate moieties with up to four
before final deprotection. Thus, the hydrolytic lability of the
D
-alanyl residues attached
-ala-
D
nyl residues and their importance for biological activity has been
taken into account and after workup of the target compound at
tuted with 2-O-D-alanyl residues; this part is phosphodiester
linked to the 6-hydroxy group of 3-O-(b-
D
-galactofuranosyl)-1,2-
least the average number of
D-alanyl residues should be available
di-O-acyl-sn-glycerol; thus, 1 belongs to the type I LTA structures.3
(1a ꢀ0.3 n = 1.5
D
-Ala residues). For the acyl chain, myristoyl
Noteworthy for the structure of 1 is the average length of only
(C14) residues were chosen.
about ten glycerophosphate residues with about three D-alanyl res-
idues attached and the core of the lipid anchor is proposed to pos-
sess the rare mono-hexosyl-1,2-diacyl-sn-glycerol structure. This
Gram-positive bacterium is genetically closely related to Strepto-
coccus pneumoniae that encomprises in its cell wall a structurally
totally different LTA, belonging to type IV LTA.3b A total synthesis
of this LTA has been recently accomplished,4 thus the previous
structural assignment was confirmed, yet the biological studies
with this synthetic material led to unexpected results.4,5 Hence,
the synthesis of the Streptococcus species DSM 8747 was of great
interest as well.
2. Results and discussion
The synthesis design of LTA 1a is outlined in the retro-synthetic
scheme (Scheme 1). For a convergent synthesis of the molecule, a
disconnection between the glycolipid core and the oligo(glycero-
phosphate) moiety is proposed leading to intermediates 2 and 3;
their further disintegration furnishes building blocks 4–7. Thus,
for the b-linkage in intermediate 2 neighbouring group participa-
tion for the galactofuranosyl donor 5 is employed. For convenience,
the totally benzoylated donor is used in the reaction with known
1,2-cyclohexylidene-sn-glycerol (4)8 as acceptor, thus requiring
in the following steps for the galactosyl residue an exchange of
protecting groups, that is, temporary protection at 6-O and benzyl
protection at 2-, 3-, and 5-O. Known intermediate 37,9 with tempo-
rary 2-O-MPM (4-methoxyphenylmethyl) protection permits
selective cleavage in the presence of O-benzyl protecting groups,
As indicated in Figure 1, LTA 1 varies in the acyl residues in
the chain length of the glycerophosphate backbone, as well as in
the number and location of the attached D-alanyl residues that
are hydrolytically labile and cleaved rapidly even at pH 8.5.6 Our
* Corresponding author. Tel.: +49 7531 882538; fax: +49 7531 883135.
E-mail address: richard.schmidt@uni-konstanz.de (R.R. Schmidt).
thus allowing introduction of the D-alanyl residues as the second
0968-0896/$ - see front matter Ó 2010 Published by Elsevier Ltd.
doi:10.1016/j.bmc.2010.04.006

Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
3697
O
R1
O
O
R2
O
OH
O
n ~ 7-17, average ~ 10
O
O
P
O-
O
P
OH
OH
R: ~ 30% D-Ala, ~ 70% H
R1, R2: C11 (~ 6%), C13 (~ 23%), C15 (~ 47%), C17
(~ 23%) including unsaturation
HO
O
O
O
O
O-
OR
OR
1
n-1
Figure 1. Structure of LTA found in Streptococcus species DSM 8747.
O
O
C13H27
C13H27
O
O
OH
O
O
O
P
O-
O
O
P
O-
O
P
O-
O
P
O-
OH
OH
P
O-
O
O
O
HO
O
O
O
O
O
O
O
OR
OR
OH
OR
OR
1a: R = H,
D-Ala (minimum 1.5
D-Ala)
O
O
O
C13H27
C13H27
O
OBn
O
O
O
P
OBn
O
P
OBn
O
P
OBn
O
P
OBn
OBn
O
OBn
BnO
O
OH
OMPM
O
O
OMPM
O
O
OMPM
O
O
OMPM
O
P
N
OBn
2
BnO
3
OBn
O
OBz
NH
CCl3
HO
O
O
O
P
OH
OTBDPS
OMPM
BnO
N
O
OBn
4
OBz
OBz
7
OBz
5
6
Scheme 1. Retro-synthetic scheme for the synthesis of target molecule 1a.
last step and thereafter global hydrogenolytic O-debenzylation un-
der mild conditions. This way, as previously found by us in the syn-
thesis of S. aureus LTA,7 another type I LTA, no or little loss of the
group in the presence of ethylene glycol as nucleophile and p-tol-
uenesulfonic acid (p-TsOH) as catalyst afforded glycerol derivative
14. Treatment of this compound with myristic acid in the presence
of dicyclohexylcarbodiimide (DCC) as condensing agent and
4-dimethylaminopyridine (DMAP) as catalyst provided diacylglyc-
erol derivative 15. Hydrogenlytic O-debenzylation of 15 with Pd/C
as catalyst in the presence of trifluoroacetic acid (TFA) gave the
glycolipid 16 that was of interest for comparisons in the biological
studies.
For the regioselective attachment of the glycerophosphate
backbone to the glycolipid anchor 16, 6-O-silylation with tert-
butyldimethylsilyl chloride (TBDMSCl) with NEt3 as base and
DMAP as catalyst was carried out affording selectively protected
compound 17 (Scheme 3). Following O-benzylation with benzyl
bromide and NaH as base gave fully protected derivative 18 that
was desilylated by treatment with tetra-n-butylammonium fluo-
ride (TBAF), thus affording 6a-O-unprotected intermediate 19.
Reaction with bis(diisopropylamino) benzyloxyphosphine18 in
the presence of bis(diisopropyl) ammonium tetrazolide as catalyst
hydrolytically labile D-alanyl residues is expected. Therefore, the
previously prepared building blocks 67,9,10 and 77,8,11 will be em-
ployed for the synthesis of 3.
For the synthesis of the phosphitylated glycolipid core structure
2, D-galactose was transformed into octyl b-D-galactofuranoside (8)
and its per-O-benzoyl derivative 9 following a reported procedure
(Scheme 2).12 Acetolysis of 9 with acetic anhydride/sulfuric acid
furnished, contrary to the literature reports claiming the require-
ment of trifluoroacetic anhydride for the acidolysis,13 the desired
1-O-acetyl protected intermediate 1014 that was transformed by
treatment with HBr and acetic anhydride and then with water into
1-O-unprotected intermediate 11.15 Reaction with trichloroaceto-
nitrile in the presence of DBU as base afforded quantitatively
O-galactofuranosyl trichloroacetimidate 516 as anomeric mixture.
Glycosylation of 48 with TMSOTf as catalyst in dichloromethane
as solvent afforded the desired b-D-galactofuranoside 12 in high
yield. The NMR data of 12 [1H NMR: dH-1a = 5.29 (s); 13C NMR:
dC-1 = 105.96, JC,H = 176.7 Hz] are in accordance with those
gave the desired 6-O-phosphitylated b-
glycerol 2.
D-galactofuranosyl-diacyl-
reported for b-
D
-galactofuranosides.15,17 In order to avoid rear-
Reaction of previously synthesized 37,9 with 2 in the presence of
tetrazole as catalyst afforded the phosphite triester intermediate
that was oxidized with tert-butyl hydroperoxide to the corre-
sponding phosphate 20 and obtained as mixture of diastereomers.
rangement reaction in this molecule, the benzoyl groups were
removed under Zemplén conditions and then replaced by benzyl
groups to give intermediate 13. Cleavage of the cyclohexylidene

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Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
All attempts to record well resolved 1H NMR spectra in deuter-
D-Galactose
ated THF, DMSO, water, methanol and mixtures of these solvents
remained unsuccessful due to micelle and aggregate formation of
the glycolipid part. The solvent for HIC (propanol/water, 1:1) was
found to be the best for the NMR experiments at concentrations
that were sufficient for heteronuclear correlated 1H, 13C, 31P NMR
spectroscopy. This way, a peak assignment by homo- and hetero-
nuclear correlated NMR spectroscopy was possible (Table 1). The
NMR data are in good accordance with those reported for the nat-
ural material,1,2 thus confirming the previous structure assign-
ment. In the 1H MNR spectra of 1a three different signals for 2-H
of the glycerol (Gro) residue were obtained. One signal at d 5.38
(C-2Gro = 74.9) is assigned to O-acylated 2-H of the diacylglycerol
(DAG) residue. The signal at d 5.30 (C-2Gro = 70.1) originates from
the glycerophosphate repeating units having 2-O-alanyl residues
and the signal at d 4.33 (C-2Gro = 75.0) is due to O-unsubstituted
glycerophosphate residues. In agreement with this also for 2-H of
the alanyl residues two different signals were observed: One signal
at d 4.26 (C-2Ala = 49.5) is due to ester bound 2-O-alanyl residues
and the other signal at d 3.73 (C-2Ala = 51.2) originates from non-
bound alanine, that is known to be co-eluting with LTA form the
HIC column.6 From the signals at d 5.38 and 5.30 an intensity ratio
of about 1:2 was observed, indicating that 1a contains about two
O-alanyl residues. This is in agreement with the ESI-MS data.
Hence, about two O-alanyl residues were lost in the deprotection
and/or purification procedure of 1a that seems to be more sensitive
to O-de-alanylation than the LTA of S. aureus.7,9
Ref. [12]
O
C8H17
O
OR
8: R = H
a
OR
OR
OR
9: R = Bz
b
X
O
OBz
10: X, Y = H, OAc
11: X, Y = H, OH
c
Y
OBz
OBz
OBz
d
O
5: X, Y = H,
NH
Cl3C
4, e
O
O
OR
O
12: R = Bz
13: R = Bn
O
f
OR
OR
OR
g
O
O
OBn
OH
OH
OBn
OBn
14
OBn
3. Summary
C13H27CO2H, h
In conclusion, following the retro-synthetic design the LTA 1a of
the Streptococcus species DSM 8747 was successfully synthesized.
The expected partial hydrolytic cleavage of D-alanyl residues dur-
ing final deprotection and/or purification could be compensated
by introducing beforehand a higher degree of alanylation. Thus,
the ratio of the constituents of 1a with on average two D-alanyl res-
O
O
O
OR
O
C13H27
C13H27
O
15: R = Bn
i
O
OR
OR
OR
16: R = H
idues is in good agreement with the natural compound.
Scheme 2. Synthesis of the core glycolipid 16. Reagents and conditions: (a) BzCl, Py
(qu); (b) Ac2O, H2SO4, 0 °C (95%); (c) HBr, Ac2O, DCM, H2O (90%); (d) CCl3CN, DBU,
DCM (qu); (e) TMSOTf (0.1 equiv), DCM (89%); (f) NaOMe, MeOH; then BnBr, NaH,
DMF (96%); (g) p-TsOH, DCM–CH3CN, ethylene glycol (76%); (h) DCM, DMAP, DCC
(qu); (i) Pd/C, H2, TFA (cat.), EtOAc–MeOH (90%).
4. Experimental details
4.1. General
Solvents were dried according to standard procedures. NMR
spectroscopic measurements were performed at 22 °C with Bruker
DRX 600, Bruker Avance 600 cryo, Bruker 400 Avance, Varian Mer-
cury 300 and Bruker AC250 spectrometers. Tetramethylsilane
(TMS) or the resonances of the deuterated solvents were used as
internal standards: CDCl3, (d = 7.24 ppm) was used as an external
standard, 85% of phosphoric acid was used as an external standard
for 31P spectra. MALDI mass spectra were recorded with a Kratos
Kompact MALDI II spectrometer; 2,5-dihydroxybenzoic acid
(DHB) or p-nitroaniline and NaI were used as matrices for positive
mode measurements, and trihydroxyacetophenone (THAP) was
used as the matrix for negative mode measurements. HRMS spec-
tra were recorded with a Bruker ESI-MS mass spectrometer. High
resolution Electrospray Ionization Fourier Transform Ion Cyclotron
Mass Spectrometry (ESI FT-ICR MS) for 1a was performed in the
negative ion mode on a 7 Tesla APEX Qe (Bruker Daltonics). Sam-
ples preparation and instrumental settings were the same as de-
scribed previously.4 Optical rotations were measured at 22 °C
with a Büchi Polar-Monitor using the sodium D line. Thin-layer
chromatography (TLC) was performed on Merck Silica Gel (60
F254) plastic plates; compounds were visualized by treatment with
a solution of (NH4)6Mo7O24ꢁ4H2O (20 g) and Ce(SO4)2 (0.4 g) in
sulfuric acid (10%, 400 mL) and then by heating to 120 °C. Flash
Oxidative cleavage of the MPM groups with ceric ammonium ni-
trate (CAN) in an acetonitrile/toluene/water mixture liberated four
hydroxy groups of the glycerophosphate backbone furnishing com-
pound 21. For the attachment of the
acted with the triethylammonium salt of N-benzyloxycabonyl
(Cbz) protected -alanine and benzotriazol-1-yl-oxytripyrrolidino-
D-alanyl residues, 21 was re-
D
phosphonium hexafluorophosphate (PyBOP) as coupling reagent in
the presence of N-methylimidazole, thus affording compound 22
as fully protected derivative of the target molecule. Hydrogenolytic
cleavage of the six O-benzyl and four Z groups was performed with
Pearlman’s catalyst in a mixture of dichloromethane/methanol/
water, thus providing after hydrophobic interaction chromatogra-
phy (HIC) with an ammonium acetate/n-propanol gradient (from
85:15 to 40:60) pure target compound 1a with about two D-alanyl
residues attached. This material will be used for biological studies.
The charge deconvoluted ESI FT-ICR mass spectrum obtained in
the negative ion mode revealed molecular mass peaks being in
agreement for 1a with zero to four alanyl residues with the highest
intensity for two alanyl residues. Hence, as expected, alanyl resi-
dues have been lost in the deprotection and/or purification proce-
dure. The MS data confirm that on average at least two alanyl
residues are attached.

Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
3699
16
a
17: R = H, R6 = TBDMS
O
b
c
d
O
O
OR
O
C13H27
18: R = Bn, R6 = TBDMS
19: R = Bn, R6 = H
O
C13H27
O
OR
OR
R6O
2: R = Bn, R6 = P-OBn
NiPr2
3, e
O
O
O
C13H27
C13H27
O
OBn
O
O
O
O
P
OBn
OBn
OBn
20: R = MPM
P
O
OBn
BnO
O
O
O
f
OBn
OR
4
21: R = H
g
22: R = D-Ala
h
O
O
C13H27
C13H27
O
O
OH
O
O
O
P
O-
O
P
O-
O
P
O-
O
P
O-
O
P
O-
OH
OH
O
O
O
HO
O
O
O
O
O
O
O
OR
OR
OH
OR
OR
O
1a: R = H (~ 50%); R =
(~ 50%)
+
NH3
Me
Scheme 3. Synthesis of the target molecule 1a. Reagents and conditions: (a) TBDMSCl, DMAP, Et3N, DCM (63%); (b) BnBr, NaH, DMF (90%); (c) TBAF, DCM (76%); (d)
BnOP(NiPr2)2, diisopropanylammonium tetrazolide (81%); (e) tetrazole, DCM, then t-BuOOH (90%); (f) CAN, CH3CN, toluene, H2O (85%); (g) N-methylimidazole, Cbz-D-alanine
triethylammonium salt, PyBOP, DCM (74%); (h) Pd(OH)2, H2, DCM/MeOH/H2O (7:3:2) (28%).
chromatography was performed on MN Silica Gel 60 (230–400
mesh) at a pressure of 0.2 bar. Target molecules were purified by
hydrophobic interaction chromatography on octyl-sepharose as
the stationary phase and the elution phase was used as a gradient
of propanol (15–60%) in 0.1 mol/L ammonium acetate buffer (pH
4.8).
4.1.4. 3-O-(2,3,5,6-Tetra-O-benzoyl-
O-cyclohexylidene-sn-glycerol (12)
To a solution of 5 (2.6 g, 3.5 mmol) and 1,2-O-cyclohexylidene-
sn-glycerol (4) (750 mg, 4.3 mmol; commercial material) in CH2Cl2
(60 mL) was added TMSOTf (70 lL, 0.35 mmol) under argon. The
a,b-D-galactofuranosyl)-1,2-
reaction was stirred for 3 h and then quenched with NEt3
(0.2 mL). The solvent was evaporated in vacuo. Flash silica gel col-
umn chromatography (petroleum ether/ethyl acetate = 5:1) gave
12 (2.2 g, 89%) as oil. 1H NMR (400 MHz, CDCl3): d 8.01–7.81 (m,
8H), 7.51–7.19 (m, 12H), 6.0–5.97 (m, 1H), 5.55 (d, 1H,
J = 5.0 Hz), 5.41 (d, 1H, J = 1.0 Hz), 5.29 (s, 1H, anomeric proton),
4.71–4.63 (m, 2H), 4.58 (dd, 1H, J = 3.8, 5.0 Hz), 4.25 (m, 1H),
3.97 (dd, 1H, J = 6.4, 8.3 Hz), 3.78–3.71 (m, 2H), 3.52 (dd, 1H,
J = 5.7, 10.4 Hz), 1.54 (m, 8H), 1.33 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 166.12, 165.75, 165.65, 165.40, 133.52, 133.40, 133.26,
133.12, 129.97, 129.85, 129.73, 129.56, 129.48, 129.03, 128.88,
128.46, 128.43, 128.41, 128.38, 110.08, 105.96 (JC,H = 176.7 Hz,
anomeric carbon), 81.86, 81.63, 77.51, 77.35, 77.23, 77.03, 76.71,
74.02, 70.37, 67.97, 66.27, 63.53, 36.40, 35.01, 25.13, 24.01,
23.84. ESI HRMS Calcd for C43H42O12: [M+Na]+ m/z 773.2568.
Found: [M+Na]+ m/z 773.2730.
4.1.1. 2,3,5,6-Tetra-O-benzoyl-a,b-D-galactofuranosyl acetate
(10)
To a solution of 9 (2.1 g, 3 mmol) in CH2Cl2 (21 mL) at 0 °C, ace-
tic anhydride (1.13 mL) and concd H2SO4 (0.08 mL) were added
and the mixture stirred for 30 min. The temperature was then
allowed to rise to 25 °C and after 4 h the reaction mixture was
quenched by adding excess satd NaHCO3. The mixture
was extracted with ethyl acetate and the organic layer was washed
successively with ice water, aqueous NaHCO3 and water and then
dried over MgSO4 and concentrated in vacuo to give crude 10. After
purification as described14 the NMR data are identical with those
reported in the literature14 for 10.
4.1.2. 2,3,5,6-Tetra-O-benzoyl-a,b-D-galactofuranose (11)
Compound 11 was obtained from 10 following the reported
procedure.15
4.1.5. 3-O-(2,3,5,6-Tetra-O-benzyl-b-
cyclohexylidene-sn-glycerol (13)
D-galactofuranosyl)-1,2-O-
4.1.3. O-(2,3,5,6-Tetra-O-benzoyl-
trichloroacetimidate (5)
a
,b-
D
-galactofuranosyl)
Compound 12 (2.2 g, 3 mmol) in MeOH (20 mL) was treated
with NaOMe (0.5 mL, l M in MeOH), then the solvent was removed
after 2 h. The crude debenzoylation compound was dissolved in
DMF (20 mL), BnBr (1.8 mL, 15 mmol) and NaH (800 mg, 60% in
Reaction of 11 with CCl3CN and DBU in CH2Cl2 at room temper-
ature afforded known 516 in practically quantitative yield.

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Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
Table 1
138.18, 137.58, 137.50, 128.49, 128.44, 128.41, 128.33, 128.30,
NMR data of 1aa,b,c
128.06, 127.94, 127.93, 127.89, 127.72, 127.66, 106.20 (anomeric
carbon), 87.65, 82.40, 81.99, 77.41, 77.29, 77.09, 76.77, 76.44,
73.47, 73.31, 72.17, 71.91, 70.49, 70.43, 70.03, 63.85. ESI HRMS
Calcd for C37H42O8: [M+Na]+ m/z 637.7138. Found: [M+Na]+ m/z
637.7444.
1H NMR data
Chemical shift
13C NMR data
Chemical shift
Proton
b- -Galf
d (ppm)
Carbon
d (ppm)
D
1-H
2-H
3-H
4-H
5-H
6a-H
6b-H
4.97
4.04
4.10
3.97
3.89
3.9c
3.9c
C-1
C-2
C-3
C-4
C-5
C-6
108.3
81.8
76.8
84.3
70.8
67.0
4.1.7. 3-O-(2, 3, 5, 6-Tetra-O-benzyl-b-
di-O-myristoyl-sn-glycerol (15)
D-galactofuranosyl)-1,2-
Compound 14 (1.5 g, 2.4 mmol) was dissolved in DCM (30 mL)
and DCC (1.5 g, 7 mmol), DMAP (30 mg, 0.24 mmol) and myristic
acid (1.6 g, 7.2 mmol) were added; after stirring overnight the
reaction was quenched by adding 0.2 mL MeOH and 0.2 mL HOAc,
then concentrated under reduced pressure and filtered through
Celite (washing with petroleum ether/EtOAc = 8:1). Flash silica
gel column chromatography (petroleum ether/ethyl acetate =
10:1) gave 15 (2.4 g, 98%) as oil. 1H NMR (400 MHz, CDCl3): d
7.27-7.18 (m, 20H), 5.16 (m, 1H), 4.99 (s, 1H, anomeric proton),
4.65–4.34 (m, 7H), 4.24–4.28 (m, 2H), 4.10 (dd, 1H, J = 6.4,
12.0 Hz), 4.03–4.01 (m, 1H), 3.95–3.89 (m, 2H), 3.70–3.66 (m,
2H), 3.61–3.58 (m, 2H), 3.49 (dd, 1H, J = 5.0, 11.0 Hz), 2.23–2.18
(m, 4H), 1.55–1.50 (m, 4H), 1.18 (m, 40H), 0.81 (m, 6H). 13C NMR
Ala (due to heterogeneity, two-different types of Ala and three of Gro were
identified) (see MS)
C-1
170.5
51.2
49.5
16.0
16.8
2-H(free)
2’-H(bound)
3-H(free)
30-H(bound)
Gro
3.76
4.26
1.59
1.47
C-2(free)
(bound)
C-20
C-3(free)
(bound)
C-30
1,10-H
3.4–3.8c
5.38
5.30
C-1
64c
2-HO-Acyl
2-HO-Ala
2-HOH
C-2O-Acyl
C-2O-Ala
C-2OH
C-3
74.9
71.4
70.1
65c
4.33
3-H
3.4–3.8c
(101 MHz, CDCl3)
d 173.40, 173.01, 138.39, 138.29, 137.82,
Fatty acids (14:0)
137.58, 128.43, 128.37, 128.34, 128.28, 128.27, 127.95, 127.86,
127.74, 127.63, 127.59, 106.02 (anomeric carbon), 88.20, 82.67,
81.18, 77.38, 77.06, 76.74, 76.25, 73.45, 73.33, 72.06, 71.87,
70.85, 69.72, 65.30, 62.62, 34.34, 34.14, 31.95, 29.72, 29.68,
29.53, 29.39, 29.33, 29.17, 29.14, 24.97, 24.92, 22.72, 14.15. ESI
C-1
C-2
C-3
170.7
34.6
25.3
2-H
3-H
4-H
4-H–13-H
14-H
31P
2.28
1.59
1.2
1.02–1.08
0.811
C-4ꢁ ꢁ ꢁC-13
22.8ꢁ ꢁ ꢁ24.50c
C-14
14.3
HRMS Calcd for C65H94O10
:
[M+Na]+ m/z 1057.6739. Found:
+0.82 ppm (br s)
[M+Na]+ m/z 1057.6745.
1H, 13C, 31P NMR [600 MHz/150.9 MHz/243 MHz, n-propanol-d6/D2O approx.
a
1:1 (V/V), 300 K]; 13C assignments based on HMQC and HSQC-DEPT.
b
4.1.8. 3-O-(b-D-Galactofuranosyl)-1,2-di-O-myristoyl-sn-glycerol
Other signals: n-propanol (d, ppm), d1-H 4.10, dC-1 64.04, d2-H 1.20, dC-2 30.3, d3-H
(16)
0.78, dC-3 23.5.
c
Non-resolved signals overlapping with signals of the n-propanol solvent.
Pd/C (100 mg) was added to 15 (2 g, 1.9 mmol), TFA (0.1 mL) in
EtOAc–MeOH (84 mL, 20:1 = V:V), and the reaction mixture was
stirred overnight under hydrogen atmosphere. The reaction was
filtrated through Celite and washed with MeOH. After removal of
the solvent under reduced pressure, 16 was obtained as solid
oil, 20 mmol) were added and the reaction mixture stirred
overnight, then poured into ice water (100 mL) and extracted with
EtOAc. The obtained extract was washed with water and brine,
dried over MgSO4, and evaporated in vacuo. The residue was
purified by silica gel column chromatography (petroleum ether/
ethyl acetate = 8:1) to obtain 13 (2.5 g, 96%) as oil. 1H NMR
(400 MHz, CDCl3): d 7.38–7.29 (m, 20H), 5.12 (s, 1H, anomeric
proton), 4.77–4.47 (m, 7H), 4.34–4.28 (m, 2H), 4.16–4.14 (m, 1H),
4.07–4.03 (m, 3H), 3.87–3.71 (m, 5H), 3.48 (dd, 1H, J = 6.4,
10.5 Hz), 1.65–1.58 (m, 8H), 1.43 (m, 2H). 13C NMR (101 MHz,
CDCl3) d 138.41, 138.27, 137.86, 137.63, 128.43, 128.39, 128.35,
128.31, 128.29, 127.97, 127.93, 127.85, 127.76, 127.64, 127.59,
109.85, 106.34 (anomeric carbon), 88.39, 82.74, 80.97, 77.40,
77.09, 76.77, 76.24, 73.99, 73.46, 73.34, 72.10, 71.92, 70.86,
67.87, 66.49, 36.44, 35.01, 25.19, 24.05, 23.87. ESI HRMS Calcd
for C43H50O8: [M+Na]+ m/z 717.3398. Found: [M+Na]+ m/z
717.3415.
(1.1 g, 90%). ½a D
ꢂ
ꢃ19.3 (c 0.9, CHCl3); 1H NMR (400 MHz, CDCl3):
d 5.24–5.22 (m, 1H), 5.0 (s, 1H, anomeric proton), 4.37 (dd, 1H,
J = 3.7, 12.0 Hz), 4.16–4.05 (m, 4H), 3.94 (m, 1H), 3.82–3.75 (m,
3H), 3.65 (dd, 1H, J = 5.3, 11.0 Hz), 3.41 (br s, 4H), 2.35–2.30 (m,
4H), 1.62–1.60 (m, 4H), 1.27 (m, 40H), 0.89 (m, 6H). 13C NMR
(101 MHz, CDCl3) d 173.70, 173.40, 108.57 (anomeric carbon),
86.82, 79.68, 78.39, 77.34, 77.03, 76.71, 70.99, 69.80, 66.07,
64.05, 62.52, 34.30, 34.12, 31.93, 29.71, 29.69, 29.67, 29.52,
29.37, 29.31, 29.16, 29.12, 24.92, 24.88, 22.70, 14.12. ESI HRMS
Calcd for C37H70O10: [M+Na]+ m/z 697.4861. Found: [M+Na]+ m/z
697.4900.
4.1.9. 3-O-(6-O-tert-Butyldimethylsilyl-b-
1,2-di-O-myristoyl-sn-glycerol (17)
D-galactofuranosyl)-
Triethylamine (0.2 mL, 1.2 mmol), 4-dimethylaminopyridine
(95 mg, 0.8 mmol) and tert-butyldimethylsilyl chloride (142 mg,
0.9 mmol) were added to a solution of 16 (530 mg, 0.78 mmol) in
dry dichloromethane (4 mL). After stirring for 6 h at room temper-
ature, the solution was diluted with EtOAc and washed with sat.
aqueous NaHCO3 and water and then dried over MgSO4. After
concentration in vacuo, the residue was separated by flash silica
gel column chromatography (petroleum ether/ethyl acetate =
4.1.6. 3-O-(2,3,5,6-Tetra-O-benzyl-b-D-galactofuranosyl)-sn-
glycerol (14)
Galactofuranoside 13 (2.4 g, 3.4 mmol) was dissolved in DCM–
CH3CN (60 mL, 1:1) and reacted with ethanediol (2.4 mL) under
acidic condition (pH 1, 800 mg of p-TsOH); after 5 h at room tem-
perature, the reaction was quenched by adding sat. NaHCO3 and
worked up as usual. Flash silica gel column chromatography
(petroleum ether/ethyl acetate = 2:1) gave 14 (1.5 g, 76%) as oil.
1H NMR (400 MHz, CDCl3): d 7.37–7.32 (m, 20H), 5.10 (s, 1H, ano-
meric proton), 4.75–4.34 (m, 8H), 4.22 (dd, 1H, J = 3.8, 6.2 Hz), 4.05
(dd, 1H, J = 2.6, 6.2 Hz), 4.01 (m, 1H), 3.85–3.78 (m, 2H), 3.75–3.57
(m, 6H), 2.56 (br s, 3H). 13C NMR (101 MHz, CDCl3) d 138.29,
4:1–1:1) to give 17 (390 mg, 63%) as a waxy material. ½aꢂD ꢃ17.3
(c 1.3, CHCl3); 1H NMR (400 MHz, CDCl3): d 5.09 (m, 1H), 4.90
(s, 1H, anomeric proton), 4.25 (dd, 1H, J = 4.3, 11.8 Hz), 4.01–3.99
(m, 2H), 3.94 (m, 1H), 3.89 (s, 1H), 3.82 (m, 1H), 3.72 (dd, 1H,
J = 5.9, 11.0 Hz), 3.61 (d, 1H, J = 2.3 Hz), 3.60 (s, 1H), 3.50 (dd, 1H,
J = 4.6, 11.0 Hz), 2.22–2.19 (m, 4H), 1.52–1.49 (m, 4H), 1.18–1.15

Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
3701
(m, 40H), 0.81–0.76 (m, 15H), ꢃ0.02 (s, 6H). 13C NMR (100 MHz,
CDCl3): d 173.40, 173.13, 108.75 (anomeric carbon), 86.72, 78.86,
78.64, 77.52, 77.48, 77.36, 77.25, 77.21, 77.05, 76.73, 71.74,
69.77, 65.86, 63.93, 62.25, 34.29, 34.12, 31.96, 29.72, 29.69,
29.67, 29.52, 29.39, 29.32, 29.17, 29.13, 25.86, 24.91, 22.72,
18.27, 14.15, ꢃ5.33. HRMS Calcd for C43H84O10Si: [M+Na]+ m/z
811.5834. Found: [M+Na]+ m/z 811.6856.
yielded 2 as oil (230 mg, 81%). This mixture of diastereomers was
immediately used in the reaction with 3 to afford 20.
4.1.13. Compound 20
To a solution of glycerol moiety 3 (48 mg, 0.03 mmol) and phos-
phite 2 (50 mg, 0.04 mmol) in DCM (2 mL), tetrazole (0.2 mL,
0.45 M in CH3CN) was added; the reaction mixture was stirred
t
for 6 h under Ar and then BuOOH (20
lL, 5.4 M in decane) was
added. After 2 h, the mixture was diluted with DCM and washed
with saturated aqueous NaHCO3 and water. The organic phase
was dried over MgSO4 and the solvent was evaporated in vacuo
at less than 30 °C. Purification by flash silica gel column chroma-
tography (toluene/acetone = 3:1) yielded the phosphate 20 as oil
(70 mg, 90%, mixture of diastereomers). 1H NMR (400 MHz, CDCl3):
d 7.37–7.21 (m, 70H), 6.82 (m, 8H), 5.27 (m, 1H), 5.04–5.01 (m,
10H, include anomeric proton), 4.71–4.48 (m, 17H), 4.33–3.99
(m, 24H), 3.76-3.56 (m, 21H, include CH3-O-Ph), 2.34–2.31 (m,
4H), 1.63 (m, 4H), 1.30 (m, 40H), 0.94–0.92 (m, 6H). 13C NMR
4.1.10. 3-O-(6-O-tert-Butyldimethylsilyl-2,3,5-tri-O-benzyl-b-
glactofuranosyl)-1,2-di-O-myristoyl-sn-glycerol (18)
D-
To a solution of 17 (560 mg, 0.7 mmol) and benzyl bromide
(0.5 mL, 4 mmol) in DMF (3 mL) was added 60% NaH (120 mg,
3 mmol). The resulting suspension was stirred for 2 h at room tem-
perature, poured into ice water (10 mL), and extracted with EtOAc.
The obtained extract was washed with water and brine, dried over
MgSO4, and evaporated in vacuo. The residue was purified by silica
gel column chromatography (petroleum ether/ethyl acetate =
15:1) to give 18 (700 mg, 90%) as a colourless syrup. ½aꢂD ꢃ11.6
(c 1.3, CHCl3); 1H NMR (400 MHz, CDCl3): d 7.30–7.21 (m, 15H),
5.20 (m, 1H), 5.02 (s, 1H, anomeric proton), 4.65–4.37 (m, 6H),
4.24–4.20 (m, 2H), 4.16–4.12 (m, 1H), 4.05 (dd, 1H, J = 2.6,
7.2 Hz), 3.98–3.93 (m, 2H), 3.75–3.71 (m, 3H), 3.54–3.50 (m, 2H),
2.24–2.19 (m, 4H), 1.57–1.54 (m, 4H), 1.20 (m, 40H), 0.87–0.74
(m, 15H), ꢃ0.02 (s, 6H). 13C NMR (100 MHz, CDCl3): d 173.40,
172.98, 138.51, 137.93, 137.68, 128.45, 128.43, 128.37, 128.34,
128.30, 127.98, 127.96, 127.92, 127.88, 127.85, 127.82, 127.80,
127.76, 127.69, 127.66, 127.63, 127.60, 106.17 (anomeric carbon),
88.34, 82.58, 80.50, 77.42, 77.10, 76.78, 73.67, 72.10, 71.89, 69.75,
63.28, 62.68, 34.38, 34.18, 31.99, 29.75, 29.72, 29.56, 29.42, 29.36,
29.21, 29.17, 25.98, 25.01, 24.96, 22.75, 18.31, 14.18, ꢃ5.33, ꢃ5.39.
HRMS Calcd for C64H102O10Si: [M+Na]+ m/z 1081.6242. Found:
[M+Na]+ m/z 1081.5713.
(101 MHz, CDCl3)
d 173.38, 172.98, 159.36, 138.16, 138.03,
137.91, 137.64, 137.45, 135.72, 129.65, 129.54, 128.62, 128.48,
128.42, 128.39, 128.35, 128.21, 127.98, 127.94, 127.79, 127.70,
127.64, 113.83, 106.17 (anomeric carbon), 87.77, 77.40, 77.29,
77.08, 76.77, 75.45, 73.45, 72.24, 72.08, 71.93, 69.65, 69.47,
69.13, 65.89, 62.61, 55.22, 34.34, 34.14, 31.96, 29.73, 29.69,
29.55, 29.40, 29.35, 29.19, 29.16, 24.98, 24.93, 22.73, 14.16. ESI
HRMS (neg. ion mode) Calcd for C154H197O39P5: [MꢃBn]ꢃ m/z
2734.1578. Found: m/z 2734.1578.
4.1.14. Compound 21
At ꢃ10 °C, Ce(NH4)2(NO3)6 (124 mg, 0.23 mmol) was added por-
tion wise to a solution of 20 (80 mg, 0.028 mmol) in CH3CN/tolu-
ene/H2O (60:3:4, 10 mL), the reaction was stirred for 30 min
(TLC-monitoring) and then diluted with EtOAc and washed with
saturated NaHCO3 solution. The organic phase was dried over
MgSO4 and the solvent was evaporated in vacuo at less than
30 °C. Purification by flash silica gel column chromatography (tol-
uene/acetone = 1:1) yielded 21 as oil (60 mg, 85%, mixture of dia-
stereomers). 1H NMR (400 MHz, CDCl3): d 7.37–7.21 (m, 50H),
5.26 (m, 1H), 5.08 (m, 12H), 4.64–4.61 (m, 12H), 4.33–3.99 (m,
30H), 3.78–3.58 (m, 12H), 2.34–2.30 (m, 4H), 1.62 (m, 4H), 1.30
(m, 40H), 0.94–0.92 (m, 6H). ESI HRMS (neg. ion mode) Calcd for
C122H165O35P5: [M–Bn]ꢃ m/z 2253.9177. Found: m/z 2253.9277.
4.1.11. 3-O-(2,3,5-Tri-O-benzyl-b-
myristoyl-sn-glycerol (19)
D-glactofuranosyl)-1,2-di-O-
The solution of 18 (900 mg, 0.85 mmol) in DCM (5 mL) was
treated with TBAF (1 M solution in THF, 4 mL). The reaction mix-
ture was stirred overnight at room temperature, then diluted with
EtOAc and washed with saturated aqueous NH4Cl and water, the
organic phase was dried over MgSO4 and the solvent was evapo-
rated in vacuo. Purification by silica gel column chromatography
(petroleum ether/ethyl acetate = 10:1) yielded 19 (600 mg, 76%)
as
a
colourless syrup.
½
a D
ꢂ
ꢃ15.1 (c 1.0, CHCl3); 1H NMR
4.1.15. Compound 22
(400 MHz, CDCl3): d 7.31–7.22 (m, 15H), 5.20 (m, 1H), 5.02 (s,
1H, anomeric proton), 4.57–4.42 (m, 6H), 4.34–4.27 (m, 2H),
4.16–4.12 (m, 2H), 3.98–3.96 (m, 2H), 3.71–3.56 (m, 5H), 2.28–
2.23 (m, 4H), 1.55 (m, 4H), 1.20 (m, 40H), 0.84–0.82 (m, 6H). 13C
NMR (101 MHz, CDCl3) d 173.55, 173.12, 138.22, 137.46, 137.30,
128.53, 128.45, 128.12, 128.08, 127.93, 127.84, 106.10 (anomeric
carbon), 87.92, 81.77, 77.37, 77.25, 77.05, 76.73, 72.12, 69.78,
65.41, 62.66, 61.83, 34.16, 31.96, 29.73, 29.69, 29.54, 29.40,
29.34, 29.16, 24.98, 24.93, 22.73, 14.16. HRMS Calcd for
C58H88O10: [M+Na]+ m/z 967.6270. Found: [M+Na]+ m/z 967.6251.
N-Cbz-D-Alanine triethylammonium salt (82 mg, 0.26 mmol)
and PyBOP (131 mg, 0.26 mmol) were added to a solution of 21
(40 mg, 0.013 mmol) in dry DCM (4 mL) under Ar, and then N-
methylimidazole (40 lL, 0.52 mmol) was added drop wise. After
5 h, the reaction mixture was diluted with DCM and washed with
saturated NH4Cl solution. The organic phase was dried over MgSO4
and the solvent was evaporated in vacuo at less than 30 °C. Purifi-
cation by flash silica gel column chromatography (toluene/ace-
tone = 3:1–1:1) yielded a diastereomeric mixture of 22 as oil
(40 mg, 74%, mixture of diastereomers). 1H NMR (400 MHz, CDCl3):
d 7.34–7.21 (m, 70H), 5.26 (m, 1H), 5.07–5.03 (m, 20H), 4.63–4.61
(m, 4H), 4.58–3.98 (m, 35H), 3.75–3.56 (m, 10H), 2.32–2.30 (m,
4H), 1.62 (m, 4H), 1.36–1.28 (m, 52H), 0.94–0.90 (m, 6H). ESI HRMS
(neg. ion mode) Calcd for C166H209N4O47P5: [M–Bn]ꢃ m/z
3074.2233. Found: m/z 3074.1780.
4.1.12. 3-O-[6-O-(Benzyloxy-diisopropylaminophoshinyl)-3,5,6-
tri-O-benzyl-b-D-galactofuranosyl]-1,2-di-O-myrisotyl-sn-
glycerol (2)
Bis(diisopropylamino)benzyloxyphosphine (165 mg, 0.5 mmol)
and diispropylammonium tetrazolide (83 mg, 0.5 mmol) were
added to the solution of 19 (230 mg, 0.24 mmol) in DCM (5 mL);
the reaction was kept at room temperature under Ar for 1 h and
then diluted with DCM, washed with saturated aqueous NaHCO3
and water. The organic phase was dried over MgSO4 and the sol-
vent was evaporated in vacuo below 30 °C. Purification by flash sil-
ica gel column chromatography (petroleum ether/NEt3 = 8:1)
4.1.16. Compound 1a
The diasteromers of 22 (40 mg, 0.013 mmol) were dissolved in
DCM/MeOH/H2O (7:3:2, 24 mL) and then treated with Pearlman’s
catalyst (5 mg) under a hydrogen atmosphere. After stirring over-
night at room temperature, the reaction mixture was filtrated
through Celite, washed with DCM/MeOH/H2O (7:3:2), and the

3702
Y. Qiao et al. / Bioorg. Med. Chem. 18 (2010) 3696–3702
filtrate was concentrated under reduced pressure below 30 °C. The
crude residue (1a) was purified with hydrophobic interaction chro-
matography (HIC) based on the protocol of W. Fischer19 with the
following modifications. Briefly, the compound was dispersed in
0.5 mL 50 mM NH4OAc pH 4.7/n-propanol 85:15 (v/v) (solvent A
for HIC) under ultrasonication (5 min). The procedure was repeated
twice and the resulting 1.5 mL suspension was centrifuged (Biofuge
Heraeus, 13,000 rpm, 5 min, room temperature). The sediment was
dissolved three times in 0.5 mL solvent A and again centrifuged. The
resulting clear 2.5 mL supernatant was divided into two aliquots
(1.25 mL each) and injected for two hydrophobic interaction chro-
matography (HIC) runs. The HIC system (Gilson) was equipped with
a HiPrep 16/10 octyl agarose column (16 ꢄ 100 mm, Amersham
Pharmacia Biotech). The HIC operated at a flow of 0.5 mL/min (4–
7 bar) solvent A isocratic (0% B) for 75 min, then raised in
250 min linear to 100% solvent B [50 mM NH4OAc pH 4.7/n-propa-
nol 40:60 (v/v)] staying isocratic for additional 75 min at 100% B.
The synthetic LTA was monitored by its UV absorbance (254 nm)
H. Moll, H. Käbner and Dr. N. Gisch for excellent LTA purification
and help in MS and NMR analysis.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.006.
References and notes
1. Roethlisberger, P.; Iida-Tanaka, N.; Hollemeyer, K.; Heinzle, E.; Ishizuka, I.;
Fischer, W. Eur. J. Biochem. 2000, 267, 5520.
2. Roethlisberger, P. Ph. D Thesis, ETH Zurich, Switzerland, 1995.
3. (a) Fischer, W.; Behr, T.; Hartmann, R.; Peter-Katalinic´, J.; Egge, H. Eur. J.
Biochem. 1993, 215, 851; (b) Greenberg, J. W.; Fischer, W.; Joiner, K. A. Infect.
Immun. 1996, 64, 3318; (c) Fischer, W. Microb. Drug. Resist. 1997, 3, 309.
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Schmidt, R. R. Angew. Chem., Int. Ed. 2010, 49, 2583.
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Leukocyte Biol. 2004, 75, 671.
6. Morath, S.; Geyer, A.; Hartung, T. J. Exp. Med. 2001, 193, 393.
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945. Angew. Chem., Int. Ed. 2003, 42, 916.
8. This compound is commercially available.
9. Figueroa-Perez, I.; Stadelmaier, A.; Morath, S.; Hartung, T.; Schmidt, R. R.
Tetrahedron: Asymmetry 2005, 16, 493.
10. Baeschlin, D. K.; Chaperon, A. R.; Charbonneau, V.; Green, L. G.; Ley, S. V.;
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and fractions (3 mL) were collected. Aliquots of 30 lL were tested
for organic phosphate by a photometric test.20 The phosphate posi-
tive fractions were combined that gave 6.3 mg (28%) of pure 1a. For
the NMR data see Table 1.—ESI HRMS (neg. ion mode) Calcd for
C64H124N4O39P5 [MꢃH]ꢃ m/z 1727.5607. Found: m/z 1727.6639.
Besides this compound in the ESI-FT-ICR mass spectrum (see
Supplementary data) further species of LTA with different
degree in alanylation are present: completely de-alanylated m/z
1443.4973 (rel. intensity 10%), (ii), mono-alanylated m/z
1514.5413 (13%), di-alanylated m/z 1585.5783 (40%), tri-alanylated
m/z 1656.6066 (30%), These data reflect the degree in de-alanyla-
tion since they are also in agreement with the NMR analysis, in
11. Wickberg, B. Acta Chem. Scand. 1958, 12, 1187.
12. Ferrières, V.; Bertho, J.-N.; Plusquellec, D. Carbohydr. Res. 1998, 311, 25.
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16. Wang, H.; Ning, J. Carbohydr. Res. 2003, 338, 1033.
which also this kind and degree of heterogeneity in the
tion was observed.
D-alanyla-
´
17. (a) Gandolfi-Donadlo, L.; Gola, G.; de Lederkremer, R. M.; Gallo-Rodriguez, C.
Carbohydr. Res. 2006, 341, 2487; (b) Gallo-Rodriguez, C.; Gil-Libarona, M. A.;
Mendoza, V. M.; de Lederkremer, R. M. Tetrahedron 2002, 38, 9373; (c)
Completo, G. C.; Lowary, T. L. J. Org. Chem. 2008, 73, 4513; (d) Lee, Y. J.; Lee, B.-
Y.; Jeon, H. B.; Kim, K. S. Org. Lett. 2006, 8, 3971.
Acknowledgements
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This work was supported by the Fonds der Chemischen Indust-
rie and by LUNAMed AG, Switzerland. We wish to thank B. Wegner,