Synthesis of the core structure of the lipoteichoic acid of streptococcus pneumoniae

Article abstract of DOI:10.1002/chem.201001204

Streptococcus pneumoniae LTA is a highly complex glycophospholipid that consists of nine carbohydrate residues: three glucose, two galactosamine and two 2-acetamino-4-amino-2,4,6-trideoxygalactose (AATDgal) residues that are each differently linked, one r

Full text of DOI:10.1002/chem.201001204

FULL PAPER
DOI: 10.1002/chem.201001204
Synthesis of the Core Structure of the Lipoteichoic Acid of Streptococcus
pneumoniae
Christian Marcus Pedersen,[a] Ignacio Figueroa-Perez,[a] Joshodeep Boruwa,[a]
Buko Lindner,[b] Artur J. Ulmer,[b] Ulrich Zꢀhringer,[b] and Richard R. Schmidt*[a]
Dedicated to Professor Richard Neidlein on the occasion of his 80th birthday
Abstract: Streptococcus pneumoniae
LTA is a highly complex glycophospho-
lipid that consists of nine carbohydrate
residues: three glucose, two galactosa-
mine and two 2-acetamino-4-amino-
2,4,6-trideoxygalactose (AATDgal) res-
idues that are each differently linked,
one ribitol and one diacylated glycerol
(DAG) residue. Suitable building
blocks for the glucose and the AATDg-
al residues were designed and their
synthesis is described in this paper.
These building blocks permitted the
successful synthesis of the core struc-
O)DAG in a suitably protected form
for further chain extension (1b, 1c)
and as unprotected glycolipid (1a) that
was employed in biological studies.
These studies revealed that 1a as well
as 1 lead to interleukin-8 release, how-
ever not via TLR2 or TLR4 as recep-
tor.
ture GlcbCAHTUNGTRNE(NNUG 1-3)AATDgalbAHCTUNRTGENN(GUN 1-3)GlcaACHTNGUTREN(NGUN 1-
Keywords: carbohydrates · glycoli-
pids · glycosidation · receptor rec-
ognition · total synthesis
Introduction
colipidic core structure comprising a trisaccharide linked to
diacylglycerol (Scheme 1, 1).[4] This structural analysis was
confirmed by our recent total synthesis of compound 1 with
Streptococcus pneumoniae, one of the most common Gram-
positive pathogens, colonizes the upper respiratory tract
where it causes severe infections; life-threatening diseases
like pneumonia, bacteremia, and meningitis. When it gains
access to the lower respiratory tract or the bloodstream,[1]
high mortality rates are often observed.[2,3]
+
R=H, X=NH3 and n=1.[5] The synthesis of the glycolipid
core structure 1a and particularly of the required building
blocks as well as further biological studies are reported in
the present paper.
Ideal precursors for the construction of pneumococcal
LTA 1 are the O-benzyl and N-benzyloxycarbonyl (Z) pro-
tected derivatives of 1a, namely 1b and 1c (Scheme 1). Ret-
rosynthesis of these compounds leads to commercially avail-
able 1,2-O-cyclohexylidene-sn-glycerol, to two glucosyl
donors for constituents A and C, as for instance 2 and 4
having O-allyl as temporary protecting groups, and to a 2-
acetamino-4-amino-2,4,6-trideoxygalactose (AATDgal) de-
rivative for constituent B, as for instance 3 having temporary
orthogonal protecting groups at 1-O (4-methoxyphenyl=
MP) and at 3-O (allyloxycarbonyl=Alloc) to permit its use
as donor and acceptor. In addition, for 3 Z-protection at the
4-amino group and trichloroethoxycarbonyl (Troc) protec-
tion at the 2-amino group are proposed; thus through anchi-
meric assistance b-linkage in the glycosylation step and fi-
nally chemoselective Troc group removal and N-acetylation
without affecting the Z-protected amino group at C-4 are
ensured. Other orthogonal temporary protecting group pat-
terns for the AATDgal residue B were probed as well in
As for all Gram-positive bacteria, the cell wall of S. pneu-
moniae consists of several layers of peptidoglycan, covalent-
ly linked to teichoic acid and of lipoteichoic acid (LTA) that
is anchored in the cell membrane. Structural analysis of
pneumococcal LTA of the R6 strain revealed that it contains
phosphodiester interlinked pseudopentasaccharide repeating
units carrying each two phosphocholine residues and a gly-
[a] Dr. C. M. Pedersen, Dr. I. Figueroa-Perez, Dr. J. Boruwa,
Prof. Dr. R. R. Schmidt
Fachbereich Chemie, Universitꢀt Konstanz, Fach 725
78457 Konstanz (Germany)
Fax : (+49)7531-883135
E-mail: richard.schmidt@uni-konstanz.de
[b] Dr. B. Lindner, Prof. Dr. A. J. Ulmer, Prof. Dr. U. Zꢀhringer
Leibniz-Zentrum fꢁr Medizin und Biowissenschaften
Forschungszentrum Borstel, Parkallee 1-40, 23845 Borstel (Germany)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201001204.
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12627

Scheme 1. Structure of the LTA of Streptococcus pneumoniae (1), the derived core structure (1a) and protected forms (1b, 1c) for repeating unit attach-
ment. A retrosynthetic Scheme for the synthesis of 1a–c.
this demanding synthetic endeavour.[6] A further aim of this
work was to provide building blocks for constituents G and
H of the DEFGH repeating unit,[5] that take into account
the linkage differences, particularly between B and G.
chloride afforded compounds 15 and 15a,[17] respectively.
Cleavage of the MP group by treatment first with N-bromo-
succinimide (NBS) in acetone and then with aqueous
NaHCO3 furnished the 1-O-unprotected intermediates that
were immediately transformed with CCl3-CN and DBU as
base into trichloroacetimidates 4[18] and 4a;[18–20] the 1-O-un-
protected 6-O-allyl-2,3,4-tri-O-benzyl-d-glucopyranose inter-
mediate could also be obtained form the corresponding
methyl glucoside.[21] Compounds 4 and 4a are useful build-
ing blocks for constituents A in the core structure and H in
the repeating unit. Intermediate 11 was also transformed via
per-O-benzylation (! 16),[22] MP-cleavage (! 17)[23] into
known trichloroacetimidate 4b[24] that is useful as building
block for constituent H in repeating unit termination.
2-Acetamino-4-amino-2,4,6-trideoxy-d-galactopyranose
(AATDgal), that is a constituent of LTA 1, is present on the
cell surface of a number of bacterial saccharides.[4,25,26]
Hence, several reports on the synthesis of derivatives of this
compound appeared.[27–33] However, due to a specific pro-
tecting group pattern required for the building blocks of
constituents B and G of 1 a different route was designed. As
discussed above, for constituent B glycosyl donor 3 might
serve all the demands; however, for constituent G due to a-
linkage an azido group in 2-position and a phthalimido
group in 4-position will be ideal. Hence, in this synthetic en-
deavour a versatile intermediate was designed that serves
Results and Discussion
Synthesis of 1a–c: The synthesis of glucose derived building
blocks 2 and 4 followed essentially published procedures
(Scheme 2). Hence, glucose was transformed into 3-O-allyl
protected furanoside 5 and then into pyranoside 6.[7] Modifi-
cations of the procedures for the introduction of the 4-me-
thoxyphenyl (MP) group (! 7),[8] O-deacetylation and per-
O-benzylation (! 8),[9] then MP cleavage (! 9)[10] and fi-
nally reaction with trichloroacetonitrile in the presence of
DBU as base led to trichloroacetimidate 2[11] in excellent
yield. For the synthesis of 4, per-O-acetylated glucopyranose
10 was transformed into MP glucoside 11;[12] O-deacetyla-
tion and 4,6-O-benzylidenation afforded 2,3-O-unprotected
glucoside 12[13] that was O-benzylated to give fully protected
derivative 13.[14] Reductive opening of the benzylidene ring
with H3B·THF complex in the presence of dibutylborane tri-
fluoromethanesulfonate (triflate) as activator[15] furnished 6-
O-unprotected compound 14.[16] 6-O-Allylation with allyl
bromide or silylation with tert-butyldiphenylsilyl (TBDPS)
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Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
could be regioselectively tosylated at 6-O, thus providing
after reacetylation derivative 28 that gave on treatment with
TBAI in refluxing acetonitrile 6-iodo derivative 29. Follow-
ing reductive deiodination as described above and then O-
deacetylation afforded compound 30. Regioselective 3-O-
benzoylation with benzoyl chloride in pyridine at ꢀ308C led
to 4-O-unprotected 31 that was transformed with trifluoro-
methanesulfonyl (Tf) anhydride in pyridine into triflate in-
termediate 32; following reaction with potassium phthal-
imide in DMF at room temperature led to 27b in good over-
all yield. Thus, versatile intermediates were available for the
construction of the designed building blocks for constituents
B and G of 1.
For the synthesis of the desired building block for constit-
uent G cleavage of the MP group in 27a,b was required
(Scheme 4). To this end, two procedures were probed. The
standard procedure, that is, treatment of 27a with ceric(IV)
ammonium nitrate (CAN) in acetonitrile at ꢀ158C led to
33a in good yield. Alternatively, treatment of 27b with bis-
(trifluoracetoxy)iodobenzene[35] and BF3·OEt2 in CH2Cl2
was investigated, thus affording after hydrolysis 33b in even
better yield. Both compounds were readily transformed into
the desired glycosyl donors 34a,b, that were successfully
employed in the total synthesis of 1.[5]
For the synthesis of building block 3 the phthaloyl group
was cleaved from 27a or 27b by treatment with ethylenedia-
mine in butanol at 958C,[36] thus liberating the amino group;
then treatment with benzyloxycarbonyl chloride in the pres-
ence of NaHCO3 in THF/H2O afforded Z-protected 35. Hy-
drogenation with Raney-nickel as catalyst liberated from
the azido group the latent amino group; following reaction
with 2,2,2-trichloroethoxycarbonyl (Troc) chloride with
NaHCO3 as acid scavenger gave N-Troc protected 38. 3-O-
Allyloxycarbonyl (Alloc) protection was performed with
Alloc-Cl in pyridine affording compound 39. Treatment of
39 with CAN in MeCN/H2O led to 1-O-unprotected deriva-
tive 40 that gave with trichloroacetonitrile in the presence
of DBU as base the desired trichloroacetimidate 3 as glyco-
syl donor. 3-O-Acetylation of 35 and then MP cleavage
under standard conditions provided 1-O-unprotected deriva-
tive 36 that was transformed into trichloroacetimidate 37.
This glycosyl donor was also probed in the construction of
CBA-DAG intermediate 1b.
With these building blocks in hand the synthesis of the
target molecule 1b could be undertaken (Scheme 5). Glyco-
sylation of 1,2-O-cyclohexylidene-sn-glycerol with glucosyl
donor 2 in the presence of TMSOTf as catalyst at 08C af-
forded a 3.2:1 anomeric mixture from which the a-anomer
41 was separated (1H NMR: J1,2 =3.6 Hz; 13C NMR: d C-1=
97.45 ppm). Cleavage of the cyclohexylidene group with
aqueous acetic acid at 808C furnished 42 that gave with myr-
istic acid and dicyclohexylcarbodiimide (DCC) as condens-
ing agent diacyl glycerol (DAG) derivative 43. 3-O-Deallyla-
tion with PdCl2, NaOAc and aqueous acetic acid[37] afforded
acceptor 44 that was available for further chain extension.
Preliminary experiments on a convergent approach for the
synthesis of 1b via the disaccharide X obtained from 4 and
Scheme 2. Synthesis of glucosyl donors 2 and 4, 4a, 4b. a) 4-Methoxyphe-
nol, TfOH, CH2Cl2, 08C (89%); b) NaOMe, MeOH; NaH, BnBr, DMF
(71%); c) CAN, MeCN/H2O (69%); d) CCl3CN, DBU, CH2Cl2 (87%);
e) BH3·THF, Bu2BOTf (75%); f) NaH, All-Br, DMF (85%); g) TBDPS-
Cl, imidazole, DMF (92%); h) NBS, Me2CO, ꢀ158C, aq. NaHCO3;
CCl3CN, DBU, CH2Cl2 (4: 86%; 4a: 98%); i) CAN, MeCN/H2O (79%);
j) CCl3CN, DBU, CH2Cl2 (89%).
both demands. To this end, as shown in Scheme 3, inexpen-
sive glucosamine was selected as precursor. Transformation
of glucosamine into per-O-acetylated azido derivative 18
followed known procedures.[34] Fischer-type glycosylation of
MP-OH afforded 4-methoxyphenyl glucoside 19 as anomeric
mixture (a/b ~4.5:1), that could be separated; in the follow-
ing reactions mainly the a-anomer was used. Cleavage of
the O-acetyl groups under Zemplꢃn conditions and then
treatment with 4-methoxybenzylidene dimethyl acetal in the
presence of p-toluenesulfonic acid (p-TsOH) as catalyst af-
forded 4,6-O-methoxybenzylidene derivative 20. 3-O-Acety-
lation with acetic anhydride in pyridine (! 21) and then re-
ductive opening of the arylidene group with H3B·THF as re-
ducing agent and Bu2BOTf as activator of the a-anomer of
21 afforded 6-O-unprotected 4-O-(4-methoxyphenylmethyl)
(MPM)-protected derivative 22. 6-O-Tosylation with Ts-Cl
in pyridine (! 23), tosylate/iodide exchange with tetrabuty-
lammonium iodide (TBAI) (! 24), and then treatment with
sodium cyanoborohydride in DMPU as solvent at 958C led
to 6-deoxy derivative 25 in very good overall yield. Selective
cleavage of the MPM group with 2,3-dichloro-5,6-dicyano-
quinone (DDQ) in CH2Cl2/H2O furnished 4-O-unprotected
derivative 26 that was transformed under Mitsunobu condi-
tions with phthalimide into the versatile 2,4-diamino-2,4,6-
trideoxy-d-galactose intermediate 27a. Alternatively and
more straightforward O-deacetylated 2-azidoglucoside 19
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R. R. Schmidt et al.
cleophile to afford acceptor
46b. Glycosylation with gluco-
syl donor 4 by taking again ad-
vantage of the nitrile effect[38]
at ꢀ408C furnished mainly the
desired b-anomer 47b (a/b
~1:4.5) in high yield (13C NMR:
d C-1a = 96.7; C-1b = 101.9;
C-1c = 103.9 ppm). Treatment
of 47b with zinc in acetic anhy-
dride[39] led to replacement of
the Troc group by the acetyl
group affording the desired in-
termediate 48b. 6c-O-Deallyla-
tion under standard conditions
provided target molecule 1b
that is a decisive intermediate
in the total synthesis of 1.[5]
Hence, 1b was transformed by
treatment with bis-diisopropyla-
minocyanoethoxyphosphine in
the presence of diisopropyl am-
monium tetrazolide[40] into the
phosphite derivative 1c that
was required for the chain ex-
tension in LTA total synthesis.[5]
1b is also available for global
hydrogenolytic
deprotection
with Pearlmanꢄs catalyst afford-
ing CBA-DAG 1a that is of in-
terest for biological studies.
The positive ion ESI FT-ICR
mass spectrum of the purified
glycolipid 1a revealed abundant
molecular and adduct ion peaks
being in perfect agreement with
the masses calculated of the
Scheme 3. Synthesis of 2,4-diamino-2,4,6-trideoxy-d-galactose intermediates 27a,b. a) TfN3; Ac2O, Pyr (73%);
b) 4-methoxyphenol, TfOH, CH2Cl2, 08C (84%); c) NaOMe, MeOH; MeOC6H4-CH(OMe)2, p-TsOH, DMF,
AHCTUNGTRENNUNG
408C (76%); d) Ac2O, Pyr (95%); e) BH3·THF, Bu2BOTf, CH2Cl2, 08C (76%); f) Ts-Cl, Pyr (79%); g)
TBAI, MeCN, reflux (85%); h) NaBH3CN, DMPU, 958C (86%); i) DDQ, CH2Cl2/H2O (88%); j) PPh3,
PhthNH, DIAD, Tol, 508C (74%); k) NaOMe, MeOH; Ts-Cl, Pyr, Ac2O, DMAP (95%); l) TBAI, MeCN,
reflux (98%); m) NaBH3CN, DMPU, 95%; MaOMe, MeOH (86%); n) BzCl, Pyr, ꢀ308C (61%); o) Tf2O,
Pyr (crude prod.); p) PhthNK, DMF, room temperature (84%, two steps).
the a-anomer of 35 and the derived donor Y failed due to
problems with the solubility of the starting materials and
due to insufficient anomeric selectivity. Therefore, a linear
synthesis of 1b was envisaged. To this end, 44 was glycosy-
lated first with donor 37; this azide route furnished the de-
sired b-linked disaccharide 45a (13C NMR: d C-1a = 96.6;
C-1b = 101.1 ppm) in acceptable yield. 3b-O-Deacetylation
(! 46a) and then glycosylation with glycosyl donor 4a in
MeCN at ꢀ408C furnished due to the nitrile effect[38] b-link-
age affording trisaccharide 47a (13C NMR: d C-1a = 96.4;
C-1b = 101.1; C-1c = 104.2 ppm). Treatment of 47a with
pure thioacetic acid at 408C led to azide reduction and liber-
ation of the amino group and concomitant N-acetylation,
thus affording the decisive intermediate 48a. Alternatively,
the Troc route was investigated, that is, glycosylation of ac-
ceptor 44 with glucosyl donor 3 that furnished under
TMSOTf catalysis the desired b-linkage in disaccharide 45b
in almost quantitative yield (13C NMR: d = C-1a 96.8; C-1b
target compound (C51H94O18N2), measured [M+H]+ =
1023.654 Da (calcd 1023.658 Da), measured [M+Na]+ =
1045.635 Da (calcd 1045.640 Da). For NMR spectroscopy
methanol as solvent gave the best spectral resolution of 1a.
The assignment of the individual resonances was in good
agreement with that obtained for 1.[5] As 1, 1a showed high
coupling constants (3J1,2 8.5 and 7.7 Hz) of the anomeric 1b-
H and 1c-H resonances of sugar residues B and C (b-config-
3
uration), whereas J1,2 = 3.7 Hz for 1a-H of sugar residue A
indicated a-anomeric linkage to DAG (Table 1). In the
HMQC experiment all protons could be correlated to the
carbon signals allowing the determination of the substitution
pattern. It is noteworthy that some, especially the anomeric
1
signals of the H and 13C resonances showed slightly differ-
ent chemical shifts when compared with 1.[5] This can be ex-
plained by the use of two different solvent systems (metha-
nol for 1a and methanol/water for 1). However, this solvent
change was required in order to improve spectral resolution
and to assign all signals unambiguously.
=
101. ppm). The Alloc group in 45b was selectively
cleaved with Pd
ACHTUNGTRENNUNG(PPh3)4 and sodium toluenesulfinate as nu-
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Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
Table 1. 1H, 13C NMR chemical shift assignment for 1a.[a]
Assignment
C
B
A
b-Glc
b-AATGalNAc
d [ppm] J [Hz]
a-Glc
d [ppm]
d [ppm]
J [Hz]
J [Hz]
1-H
2-H
3-H
4-H
5-H
6-H
4.820
J1,2 8.5
J2,3 10.3 3.219
4.387
J1,2 7.7
J2,3 9.3
4.816
3.529
3.390
J1,2 3.7
J2,3 9.6
3.241
3.28[b]
3.28[b]
3.678
3.83[b]
3.378
4.00
1.32
J4,5 ~1.2 3.352
J5,6 6.8
J4,5 9.7
3.61
3.71–3.69
3.67–3.63
C-1
C-2
C-3
C-4
C-5
C-6
101.3
75.6
78.8
72.1
72.3
63.5
106.6
55.8
63.3
50.5
70.3
22.5
102.2
73.3
70.6
78.3
74.2
63.2
Gro
Fatty acids (14:0)
1-H
1’-H
2-H
3-H
3’-H
4.241
4.497
5.290
3.68[b]
3.903
J1,2 3.2, J1,1’ 11.7 2-H
2.37
1.64
1.46
1.37 … 1.31
0.93
J1’,2 6.5
3-H
4-H
5- … 13-H
J2,3’ 5.4, J3,3’ 10.4 14-H
C-1
C-2
C-3
64.5
73.6
67.9
C-1
C-2
C-3
C-4
C-5 … -12
C-13
C-14
n. d.
35.7
26.6
33.6
32 … 30.5
24.5
15.2
Scheme 4. Synthesis of 2,4-diamino-2,4,6-trideoxygalactose derived
donors 34a,b, 37 and 3. a) CAN, MeCN, H2O ꢀ158C (75%); b) PhI-
ACHTUNGTRENNUNG(O2C-CF3)2, BF3·OEt2, CH2Cl2, H2O (87%); c) CCl3-CN, DBU, CH2Cl2
(34a: 90%; 34b: 72%); d) H2N-CH2-CH2-NH2, BuOH, 958C (68%); Z-
Cl, NaHCO3, THF/H2O (88%); e) Ac2O, Pyr; CAN, MeCN/H2O, ꢀ158C
(84%); f) CCl3-CN, DBU, CH2Cl2 (87%); g) Raney-Ni, H2, EtOH;
NaHCO3, Troc-Cl (91%); h) Alloc-Cl, Pyr, (85%); i) CAN, MeCN/H2O
(crude prod.); j) CCl3-CN, DBU, CH2Cl2 (89%, two steps).
NAc (C=O)
NAc (CH3)
13C
n.d.[c]
1H: 2.014 13C: 24.92
[a] 700.75 MHz. Homo- (1H) and 1H,13C-heteromuclear NMR spectra
(HMQC) were recorded at 300 K in [D4]MeOH (dH =3.34 ppm, dC =
49.86 ppm). For further details see Experimental Section. [b] Non-re-
solved multiplets. [c] n.d.=not determined.
Biological studies: The induction of innate immune respons-
es by 1a was tested in human peripheral blood cells using a
whole blood assay (data not given)) and stimulation of iso-
lated human mononuclear cells (MNCs) (Figure 2). Both
tests revealed that 1a stimulates interleukin-8 (IL-8) release.
Hence, it is assumed that monocytes are the source of IL-8
producing cells because these cells are the prominent innate
immune cells within the MNC preparation that are able to
produce IL-8. The lipophilic part of 1a is suggested to be re-
sponsible for the biological activity. However, the nature of
the cells as well as the receptors remain to be investigated.
In comparison to lipopolysaccharide (LPS) and lipopeptide
(Pam3C-SK4) the concentration required for cell stimulation
was rather high (10 mgmLꢀ1). Therefore, the presence of
these bacterial cell wall products was excluded, as previously
reported.[5] It was found that 1a did not sense toll-like re-
ceptor2 (TLR2) as well as TLR4/MD2/CD14 indicating that
the preparations were free of contaminating bacterial LPS
and lipopeptide, respectively (Figure 1). In addition, these
results clearly prove that neither TLR2 nor TLR4 are the
signalling receptors of the lipophilic part of 1a.
gated with transfected HEK 293-TLR4/MD-2/CD14 cells.
Therefore, it was postulated that the immune-stimulatory
activity of 1 is mediated by other, so far unidentified recep-
tor(s). In the present work it is shown that also the glycoli-
pid core structure 1a consisting of a trisaccharide bound to
the lipid anchor exhibits qualitatively and quantitatively the
same biological profile as 1 and activates the release of IL-8
in MNCs (Figures 1 and 2). These results indicate that the
lipid anchor and part of the attached oligosaccharide medi-
ate the biological activities of LTA observed in cells. For 1
as well as for 1a it was found that this activity is neither cor-
related to TLR2 nor to TLR4 indicating that other receptors
of the innate immune system, such as the lectin pathway of
the complement system, might be the most likely PRR for 1
and 1a. Based on the biological activities observed for these
two compounds it is speculated that this biological profile is
identical also for bacterial ꢅhighly purifiedꢄ natural LTA
preparations, free of lipopeptide contaminations, as has
been isolated from lgt-mutant bacteria.[43–46] The TLR2-relat-
ed activation, originally assigned to natural LTA, is rather
due to minor contaminations of highly active lipoproteins of
Staphylococcus aureus strains.[47] Since these observations
Although it has often been postulated that so called
“highly purified” natural LTA activates pattern recognition
receptors (PRRs) of the innate immune system via
TLR2,[41,42] we could not obtain such TLR2-mediated activi-
ty with synthetic Streptococcus pneumoniae LTA 1.[5] In ad-
dition, 1 did not show any TLR4-related activity, as investi-
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12631

R. R. Schmidt et al.
linkage, and on the anomeric
effect for the GlcaACHTNUTGRNEUNG(1-O)DAG
linkage. Via 1b the 6c-O-phos-
phitylated intermediate 1c re-
quired for the total synthesis of
1[5] and the totally unprotected
core trisaccharide a-linked to
DAG, respectively, are readily
available. The biological studies
with 1a and comparison of the
results with those obtained for
1
exhibited that both com-
pounds stimulate IL-8 release,
however not via TLR2. Hence,
the results strongly support the
view that previous reports on
natural LTA-based signalling
via TLR2 are due to lipopep-
tide contaminations.[43]
Experimental Section
General: Solvents were dried accord-
ing to standard procedures. NMR
spectroscopic measurements were per-
formed at 228C with Bruker DRX600,
Bruker Avance 600 cryo, Bruker 400
Avance, Varian Mercury 300 and
Bruker AC250 instruments. TMS or
the resonances of the deuterated sol-
vents were used as an internal stan-
dard. CDCl3 (d=7.24 ppm) was used
as an external standard; 85% of phos-
phoric 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-ni-
troaniline and NaI were used as matri-
ces for positive measurements, and tri-
hydroxyacetophenone (THAP) was
used as the matrix for negative mode
measurements. HRMS spectra were
recorded with a Bruker ES-MS spec-
trometer. Optical rotations were mea-
sured with a Perkin–Elmer polarime-
Scheme 5. Synthesis of target compounds 1a, 1b, 1c. a) TMSOTf (0.05 equiv), 08C, CH2Cl2 (84%, a/b 3.2:1);
b) HOAc/H2O, 80 8C (78%); c) C13H27CO2H, DCC, CH2Cl2/DMF (75%); d) PdCl2, HOAc, NaOAc, AcOEt
(68%); e) TMSOTf (0.1 equiv), CH2Cl2, ꢀ408C (45a: 66%; 45b: 93%); f) NaOMe, MeOH (98%); g) [Pd-
(PPh3)4], p-TolSO2Na, THF/MeOH, 08C (59%); h) TMSOTf (0.1 equiv), MeCN or EtCN, ꢀ408C (47a: 79%;
47b: 83%, a/b 1:4.5); i) AcSH, 408C (72%); j) Zn, Ac2O, NEt3 (86%); k) TBAF, HOAc, THF (79%); l)
PdCl2, MeOH/CH2Cl2, 08C (79%); m) H2, Pd(OH)2, CH2Cl2/MeOH/H2O!THF/H2O; n)
(iPr2N)2POCH2CH2CN, tetrazole, iPr2NH, CH2Cl2/MeCN (86%).
could also be corroborated by other investigators[44] we con-
ter 241/MS in a 1-dm cell at 228C. Thin-layer chromatography (TLC)
was performed on E. Merck Silica Gel 60 F254 plastic plates. The com-
clude that not only synthetic but also pure natural LTA, as
can be isolated from lgt-mutant bacteria, does not activate
the immune system via TLR2.
pounds were visualized by
a
treatment with
a
solution of
(NH4)6Mo7O24·4H2O (20 g) and CeAHCTUNGTRE(NNNUG SO4)2 (0.4 g) in 10% H2SO4
(400 mL). Flash silica gel column chromatography was performed on J. T.
Baker Silica Gel 60 (0.040–0.063 mm) at a pressure of 0.3 bar. The depro-
tected synthetic LTA trisaccharide 1a (~10 mg) was purified in two steps
first by hydrophobic interaction chromatography (HIC) as described.[4,5]
Since TLC and NMR analysis revealed impurities still to be present after
HIC, 1a was further purified by preparative liquid chromatography
(PLC) on TLC plates (silica gel 60 F254, 0.25 mm, MERCK) developed
with chloroform/methanol/water (100:100:30, v/v/v). The target com-
pound 1a (Rf =0.55) was scrapped off and eluted from the plate to give
0.46 mg of pure 1a suitable for NMR, MS and biological experiments.
Conclusion
The core trisaccharide a-glycosidically linked to DAG 1a
was successfully synthesized. Starting from glucose the re-
quired glucose building blocks and from glucosamine versa-
tile AATDgal building blocks were obtained. The anomeric
stereocontrol in the construction of the target molecule was
ESI-MS analysis and NMR spectra of compound 1a: High-resolution
electrospray ionization Fourier transform ion cyclotron mass spectrome-
try (ESI FT-ICR MS) was performed in the positive ion mode on a 7 T
based on the nitrile effect for the Glcb
age, on anchimeric assistance for the AATDgalbACHTUNGTRENNUNG
ACHTUNGTRENNUNG
12632
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Chem. Eur. J. 2010, 16, 12627 – 12641

Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
Figure 2. Induction of IL-8 release in human MNC by synthetic 1a. After
incubation for 16 h the release of IL-8 into the culture supernatant was
determined by ELISA. Each result represents the mean ꢁSD of dupli-
cate cultures.
NaOMe solution was added until pH 9 was reached; the reaction mixture
was stirred for 2 h and then amberlite IR120 acid resin was added until
neutralization. The mixture was filtered and the solvent evaporated in
vacuo. The product was dried in vacuo for 2 h and the product redis-
solved in DMF, then BnBr (8.9 mL, 3.9 equiv) was added and the mixture
cooled to 08C. Then NaH 60% (3.5 g, 4.5 equiv) was added portionwise
and the reaction mixture stirred overnight. MeOH was added to quench
the reaction, the mixture was poured into water and extracted with
EtOAc; the organic phase was washed with water and brine, dried over
sodium sulfate and the solvent was removed in vacuo. Flash chromatog-
raphy (petroleum ether/EtOAc 8:1) yielded 8 (10.97 g, 95%). The physi-
cal data are in accordance with those reported.[9]
3-O-Allyl-2,4,6-tri-O-benzyl-a,b-d-glucopyranose (9): To a ꢀ108C cooled
solution of 8 (10.95 g, 10.35 mmol) in acetonitrile (270 mL), CAN (20.1 g,
2.00 equiv, dissolved in 90 mL of water) was added dropwise. The reac-
tion mixture was stirred at that temperature for 2 h and the reaction was
neutralized with saturated NaHCO3 solution: The mixture was extracted
with EtOAc three times, the organic phase dried over sodium sulfate and
the solvent removed in vacuo. Flash chromatography (petroleum ether/
EtOAc 5:1) yielded 9 (6.65 g, 74%). The physical data are in accordance
with those reported.[10]
Figure 1. Induction of IL-8 release in TLR2 or TLR4/MD2/CD14 transi-
ent transfected HEK 293 cells by synthetic 1a. The negative response in
both cases shows that the 1a preparation was free of contaminating lipo-
peptide and LPS, respectively.
APEX Qe (Bruker Daltonics, Billerica, USA). Samples preparation and
instrumental settings were the same as described previously.[5] One- and
two-dimensional homo- and heteronuclear NMR spectra were obtained
with an Avance III 700 MHz spectrometer (Bruker, Rheinstetten, Ger-
many) equipped with quadrupol-resonance cryogenic probe QXI. Puri-
fied 1a (0.42 mg) was dissolved in 250 mL methanol ([D4]MeOD, 99.96%,
Eurisotope) and transferred to a 3 mm NMR Tube (Deutero GmbH,
Kastellaun). 1H and 13C Chemical shifts were referenced to internal
methanol (dH 3.34, dC 49.86 ppm). For homo- and heteronuclear correlat-
ed 2D experiments (COSY, TOCSY, HMQC) Bruker standard software
TOPSPIN (Version 2.1.1) was used to acquire and process all data.
O-(3-O-Allyl-2,4,6-tri-O-benzyl-a,b-d-glucopyranosyl) trichloroacetimi-
date (2): To a solution of 9 (6.08 g, 12.4 mmol) in CH2Cl2 (220 mL),
Cl3CCN (24.85 mL, 20 equiv) and DBU (0.18 mL, 0.1 equiv) were added.
The reaction mixture was stirred for 2 h and the solvent evaporated in
vacuo. Flash chromatography (petroleum ether/EtOAc 6:1) yielded 2
(6.8 g, 88%). The physical data are in accordance with those reported.[11]
4-Methoxyphenyl 2,3,4-tri-O-benzyl-b-d-glucopyranoside (14): A solution
of 13[14] (2.5 g, 5.08 mmol) in CH2Cl2 (20 mL) was cooled to 08C and
BH3·THF complex (50.75 mL, 10 equiv, 1 m solution) was added drop-
wise and the reaction mixture stirred at 08C for 2 h. The reaction was
neutralized with Et3N, then MeOH was added to quench the excess of
borane. The solvent was evaporated in vacuo and the residue coevaporat-
ed several times with MeOH. Flash chromatography (petroleum ether/
EtOAc 5:1) yielded 14 (1.89 g, 75%). The physical data are in accord-
ance with those reported.[16]
4-Methoxyphenyl 2,4,6-tri-O-acetyl-3-O-allyl-a,b-d-glucopyranoside (7):
To a solution of 6[7] (17.73 g, 45.65 mmol) and 4-methoxyphenol (11.33 g,
2 equiv) triflic acid (0.95 mL, 0.17 equiv) was added dropwise at 08C. The
reaction mixture was stirred at this temperature for 3 h. Et3N was added
to neutralize the reaction and the solvent was removed in vacuo. Flash
chromatography (petroleum ether/EtOAc 2:1) yielded 7 (15.00 g, 73%).
TLC (petroleum ether/EtOAc 2:1): Rf =0.35. The physical data are in ac-
cordance with those reported.[8]
4-Methoxyphenyl 2,3,4-tri-O-benzyl-6-O-tert-butyldiphenylsilyl-b-d-glu-
copyranoside (15a): To a solution of 14 (1.89 g, 3.82 mmol) in CH2Cl2
(15 mL), imidazole (0.39 g, 1.5 equiv) and TBDPSCl (1.09 mL, 1.1 equiv)
were added and the reaction stirred for 1 h. After this time the mixture
was diluted with CH2Cl2 and washed with water and brine; the organic
phase was dried over sodium sulfate and the solvent evaporated in vacuo.
4-Methoxyphenyl 3-O-allyl-2,4,6-tri-O-benzyl-a,b-d-glucopyranoside (8):
7 (15.19 g, 33.57 mmol) was dissolved in MeOH and a freshly prepared
Chem. Eur. J. 2010, 16, 12627 – 12641
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12633

R. R. Schmidt et al.
Flash chromatography (petroleum ether/EtOAc 10:1) yielded 15a
(2.57 g, 92%). The physical data are in accordance with those report-
ed.[17]
1.5 equiv) and p-TsOH (0.63 g, 0.1 equiv) were added. The reaction mix-
ture was stirred overnight at 408C and the mixture was poured into
water and extracted twice with EtOAc. The organic phase was washed
with water and brine, dried over sodium sulfate and the solvent was
evaporated in vacuo. Flash chromatography (petroleum ether/EtOAc
3:1) yielded 20 (12.05 g, 76%).TLC (toluene/EtOAc 5:1); Rf =0.25;
[a]D = +15.3 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3) b-isomer: d
O-(6-O-Allyl-2,3,4-tri-O-benzyl-a,b-d-glucopyranosyl) trichloroacetimi-
date (4): To a cooled solution of 6-O-allyl-2,3,4-tri-O-benzyl-d-glucopyra-
nose[17] (370 mg, 0.75 mmol) in CH2Cl2 (5 mL) and trichloroacetonitrile
(0.4 mL, 5 equiv) was added DBU (23 mg, 0.2 equiv) and the reaction
was stirred at room temperature over night. The reaction was concentrat-
ed on celite and purified by flash chromatography (EtOAc/petroleum
ether, 1:10 to 1:5 + 1% Et3N) to give 4 as colourless syrup (460 mg,
98%). [a]D = +86.5 (c = 1, CHCl3); 1H NMR (300 MHz, CDCl3): d =
8.38 (s, 1H, NH), 7.50–7.28 (m, 15H, Ar), 6.62 (d, J=3.5 Hz, 1H, 1-H),
5.95 (m, 1H, All), 5.33 (m, 1H, All), 5.23 (m, 1H, All), 5.05 (d, J=
11.0 Hz, 1H, Bn), 4.99 (d, J=10.7 Hz, 1H, Bn), 4.92 (d, J=11.0 Hz, 1H,
Bn), 4.80 (d, J=11.7 Hz, 1H, Bn), 4.74 (d, J=11.7 Hz, 1H, Bn), 4.73 (d,
J=10.7 Hz, 1H, Bn), 4.18–3.78 (m, 7H, 2-H, 3-H, 4-H, 5-H, 6-H,
-CH2CHCH2), 3.71 ppm (dd, J=1.8, 10.8 Hz, 1H, 6’-H); 13C NMR
(75 MHz, CDCl3): d = 161.3 (C=N), 138.7, 138.3, 138.0 (Ar), 134.5 (All),
128.5–127.6 (15 C, Ar), 117.4 (All), 94.4 (C-1), 91.4 (-CCl3), 81.4, 79.4,
76.6 (C-2, C-3, C-4), 75.6 (Bn), 75.4 (Bn), 73.1 (C-5), 72.9 (Bn), 72.4
(CH2CHCH2), 68.1 ppm (C-6). elemental analysis calcd (%) for
C32H34Cl3NO6 (700.2 gmolꢀ1): C 60.53, H 5.40, N 2.21; found: C 60.85, H
5.36, N 2.01.
= 7.40–6.90 (m, 8H, Ar), 5.50 (m, 2H, 3-H, CHAr), 5.00 (d, 1H, J1,2
=
8.0 Hz, 1-H), 4.40 (dd, 1H, J6a,6b =10.5, J6a,5 =4.9 Hz, 6a-H), 3.80 ppm (m,
11H, 2-H, 5-H, 4-H, 6a-H, 2ꢆCH3O); MALDI-MS (positive mode,
matrix DHB, THF): m/z: 452.1; found: 452.1 [M+Na]+; elemental analy-
sis calcd (%) for C21H23N3O7 (429.42 gmolꢀ1): C 58.74, H 5.40, N 9.79;
found: C 58.73, H 5.48, N 9.77.
4-Methoxyphenyl 3-O-acetyl-2-azido-2-deoxy-4,6-O-(4-methoxybenzyli-
dene)-a,b-d-glucopyranoside (21): Compound 20 (17.2 g, 40.1 mmol was
dissolved in pyridine (180 mL) and Ac2O (120 mL) was added. The reac-
tion mixture was stirred for 5 h, the solvent was removed in vacuo and
the residue coevaporated several times with toluene. Flash chromatogra-
phy (petroleum ether/EtOAc 3:1) yielded 21 (20.7 g, 97%). TLC (tolu-
ene/acetone 6:1): Rf =0.56; [a]D = +5.3 (c
=
1, CHCl3); 1H NMR
(250 MHz, CDCl3) b-isomer: d = 7.40–6.90 (m, 8H, Ar), 5.50 (m, 2H, 3-
H, CHPh), 5.00 (d, 1H, J1,2 =8.0 Hz, 1-H), 4.40 (dd, 1H, J6a,6b =10.5,
J
6a,5 =4.9 Hz, 6a-H), 3.80 ppm (m, 11H, 2-H, 5-H, 4-H, 6a-H, 2ꢆCH3O);
MALDI-MS (positive mode, matrix DHB, THF): m/z: 494.2; found:
494.3 [M+Na]+; elemental analysis calcd (%) for C23H25N3O8
(471.46 gmolꢀ1): C 58.59, H 5.34, N 8.91; found: C 58.83, H 5.49, N 8.77.
O-(2,3,4-Tri-O-benzyl-6-O-tert-butyldiphenylsilyl-a,b-d-glucopyranosyl)
trichloroacetimidate (4a): To a solution of 15a (2.5 g, 3.41 mmol) in ace-
tone (60 mL) cooled to ꢀ158C NBS (0.85 g, 1.4 equiv) was added and
the reaction stirred in the darkness for 2 h. The reaction mixture was
neutralized with saturated NaHCO3 solution and extracted twice with
CH2Cl2; the organic phase was dried over sodium sulfate and the solvent
evaporated in vacuo. Flash chromatography (petroleum ether/EtOAc
5:1) yielded the pyranose (2.01 g, 86%). The physical data are in accord-
ance with those reported.[19] This compound was transformed into 4a as
previously described.[20]
4-Methoxyphenyl
3-O-acetyl-2-azido-2-deoxy-4-O-(4-methoxybenzyl)-
a,b-d-glucopyranoside (22): To a solution of 21 (19.6 g, 36.7 mmol) in
CH2Cl2 (150 mL) borane tetrahydrofuran (184 mL, 5 equiv, 1 m solution
in THF) was added. The reaction mixture was cooled with an ice bath
and Bu2BOTf (36.7 mL, 1 equiv, 1 m solution in THF) was added drop-
wise. The reaction mixture was stirred at 08C for 1 h and then neutralized
with Et3N. MeOH was added to quench excess borane and the solvent
was evaporated in vacuo; the residue was coevaporated several times
with MeOH. Flash chromatography on silica gel (petroleum ether/
EtOAc 3:1) yielded 22 (14.95 g, 76%). TLC (toluene/EtOAc 2:1): Rf =
0.41; [a]D = +9.5 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3): d =
7.20–6.60 (m, 8H, Ar), 6.00 (dd, 1H, J3,2 =10.5, J3,4 =9.1 Hz, 3-H), 5.60
(d, 1H, J1,2 =3.5 Hz, 1-H), 4.60 (s, 2H, CH2Ar), 3.95 (m, 2H, 5-H, 6b-H),
3.85 (m, 2H, 6a-H, 4-H), 3.80 (s, 3H, CH3O), 3.70 (s, 3H, CH3O), 3.35
O-(2,3,4,6-Tetra-O-benzyl-a,b-d-glucopyranosyl)
trichloroacetimidate
(4b): To a ꢀ158C cooled solution of 16[22] (10.0 g, 15.4 mmol) in acetoni-
trile (270 mL), CAN (20.34 g, 2.4 equiv dissolved in 70 mL H2O) was
added dropwise. The reaction mixture was stirred at ꢀ158C for 3 h and
the reaction quenched with saturated NaHCO3 solution. The mixture was
extracted three times with CH2Cl2, the organic phase dried over sodium
sulfate and the solvent evaporated in vacuo. Flash chromatography (pe-
troleum ether/EtOAc 2:1) yielded 17 (6.2 g, 74%). The physical data are
in accordance with those reported.[23] This compound was transformed
into 4b as previously described.[22]
(dd, 1H,
J2,3 =10.5, J1,2 =3.5 Hz, 2-H), 2.00 ppm (s, 3H, CH3CO);
MALDI-MS (positive mode, matrix DHB, THF): m/z: 496.2; found:
496.3 [M+Na]+, 512.2 [M+K]+; elemental analysis calcd (%) for
C23H27N3O8 (473.48 gmolꢀ1): C 58.34, H 5.75, N 8.87; found: C 58.41, H
5.92, N 8.77.
4-Methoxyphenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-a,b-d-glucopyrano-
side (19): Compound 18[34] (16.50 g, 44.20 mmol) was dissolved in CH2Cl2
(160 mL) and p-methoxyphenol (11.0 g, 2 equiv) was added; the mixture
was cooled to 08C and TfOH (0.92 mL, 0.17 equiv) was added dropwise;
the reaction mixture was stirred for 3 h at 08C and another 3 h at room
temperature. After this time a saturated solution of NaHCO3 was added
and the phases separated. The organic phase was washed with water and
dried with sodium sulfate, the solvent evaporated in vacuo and the crude
product purified by flash chromatography (petroleum ether/EtOAc 3:1)
to yield 19 (16.2 g, 84%). TLC (toluene/EtOAc 5:1): Rf =0.25; [a]D =
+15.3 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3) a-isomer: d 7.40–
6.90 (m, 4H, Ar), 5.70 (dd, 1H, J3,2 =10.5, J3,4 =10 Hz, 3-H), 5.50 (d, 1H,
4-Methoxyphenyl 3-O-acetyl-2-azido-2-deoxy-4-O-(4-methoxybenzyl)-6-
O-(4-methylbenolsulfonyl)-a,b-d-glucopyranoside (23): To a solution of
22 (13.6 g, 25.4 mmol) in pyridine (100 mL) cooled to 08C, TsCl (9.68 g,
2 equiv dissolved in 50 mL of pyridine) was added dropwise. The reaction
mixture was stirred overnight and the solvent was evaporated in vacuo.
The crude material was dissolved in EtOAc and washed with 1 n HCl so-
lution and water. The product was purified by flash chromatography (pe-
troleum ether/EtOAc 3:1) to yield 23 (12.8 g, 79%). TLC (toluene/
EtOAc 3:1): Rf =0.34; [a]D = +7.5 (c = 1, CHCl3); 1H NMR (250 MHz,
CDCl3): d
9.1 Hz, 3-H), 5.50 (d, 1H, J1,2 =3.5 Hz, 1-H), 4.50 (s, 2H, CH2Ar), 4.35
(m, 1H, 6b-H), 4.20 (m, 1H, 6a-H), 4.10 (m, 1H, 5-H), 3.90 (t, 1H, J3,4
10.5 Hz, 4-H), 3.80 (s, 3H, CH3O), 3.70 (s, 3H, CH3O), 3.35 (dd, 1H,
3,2 =10.5, J1,2 =3.5 Hz, 2-H), 2.50 (s, 3H, CH3(Ts)), 2.00 ppm (s, 3H,
= 7.20–6.60 (m, 12H, Ar), 6.00 (dd, 1H, J3,2 =10.5, J3,4 =
J
J
1,2 =3.5 Hz, 1-H), 5.15 (t, 1H, J4,3 =J4,5 =10 Hz, 4-H), 4.30 (dd, 1H,
=
6a,6b =12.2, J6b,5 =4.5 Hz, 6a-H), 4.20 (m, 1H, 5-H), 4.10 (dd, 1H, J6a,6b
=
12.2, J6a,5 =2 Hz, 6a-H’), 3.80 (s, 3H, CH3O), 3.50 (dd, 1H, J2,1 =3.5, J2,3
=
J
10.6 Hz, 2-H), 2.00 ppm (m, 9H, 3ꢆCH3-Ac); MALDI-MS (positive
mode, matrix DHB, THF): m/z: 460.1; found: 460.1 [M+Na]+; elemental
analysis calcd (%) for C19H23N3O9 (437.4 gmolꢀ1): C 52.17, H 5.30, N
9.61; found: C 52.03, H 5.45, N 9.76.
CH3CO); MALDI-MS (positive mode, matrix DHB, THF): m/z: 650.2;
found: 650.2 [M+Na]+; elemental analysis calcd (%) for C30H33N3O10S
(627.66 gmolꢀ1): C 57.41, H 5.30, N 6.69; found: C 57.28, H 5.12, N 6.89.
4-Methoxyphenyl 2-azido-2-deoxy-4,6-(4-methoxybenzylidene)-a,b-d-glu-
copyranoside (20): To a solution of 19 (16.1 g, 36.81 mmol) in MeOH
(100 mL) NaOMe was added until pH 9. The mixture was stirred for 2 h
and the amberlite IR 120 (H+ form) was added until neutralization. The
mixture was filtered and concentrated in vacuo. The crude residue was
dissolved in DMF (200 mL) and anisaldehyde dimethylacetal (9.4 mL,
4-Methoxyphenyl 3-O-acetyl-2-azido-2,6-dideoxy-6-iodo-4-O-(4-methoxy-
benzyl-a,b-d-glucopyranoside (24): To
a
solution of 23 (12.6 g,
20.1 mmol) in acetonitrile (300 mL) TBAI (9.64 g, 1.3 equiv) was added
and the reaction mixture was refluxed overnight. The solvent was evapo-
rated in vacuo and the residue dissolved in EtOAc and washed with
water twice. The organic phase was dried over sodium sulfate and the sol-
12634
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ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12627 – 12641

Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
vent evaporated in vacuo. Flash chromatography (petroleum ether/
EtOAc 7:1) yielded 24 (9.93 g, 85%). TLC (toluene/EtOAc 5:1): Rf =
0.41; [a]D = +11.5 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3): d =
7.20–6.90 (m, 8H, Ar), 6.10 (dd, 1H, J3,2 =10.5, J3,4 =9.1 Hz, 3-H), 5.60
(d, 1H, J1,2 =3.5 Hz, 1-H), 4.60 (s, 2H, CH2Ar), 3.75 (m, 8H, 2 ꢆ CH3O,
5-H, 6b-H), 3.40 (m, 3H, 2-H, 4-H, 6a-H), 2.00 ppm (s, 3H, CH3CO);
MALDI-MS (positive mode, matrix DHB, THF): m/z: 606.1; found:
606.3 [M+Na]+; elemental analysis calcd (%) for C23H26IN3O7
(583.37 gmolꢀ1): C 47.35, H 4.49, N 7.20; found: C 47.58, H 4.63, N 7.32.
The reaction was diluted with water and extracted with EtOAc. The or-
ganic phase was washed successively with water to remove DMF, HCl
(1 m) and brine, dried (MgSO4) and concentrated in vacuo. Flash chro-
matography (petroleum ether/EtOAc 4:1 to 2:1) yielded 27b (3.521 g,
overall 84%) as a solid. M.p. 92–938C; [a]D = +137.7 (c = 1, CHCl3);
1H NMR (600 MHz, CDCl3): d = 7.83 (d, 2H, J=7.5 Hz, Bz), 7.83 (m,
2H, Phth), 7.74 (brs, 1H, Phth), 7.46 (t, 1H, J=7.5 Hz, Bz), 7.28 (dd,
2H, J=7.8 Hz, Phth), 7.13 (d, 2H, J=9.2 Hz, MP), 6.87 (d, 2H, J=
9.2 Hz, MP), 6.06 (dd, 1H, J=6.4 Hz, 11.0 Hz, 3-H), 5.78 (d, 1H, J=
4.0 Hz, 1-H), 5.16 (dd, 1H, J=3.3 Hz, 6.4 Hz, 4-H), 4.79 (dd, 1H, J=
3.9 Hz, 11.0 Hz, 2-H), 4.62 (m, 1H, 5-H), 3.79 (s, 3H, OMe), 1.13 ppm (d,
3H, J=6.7 Hz, 6-H); 13C NMR (150.9 MHz, CDCl3): d = 168.8 (2 C, C=
O), 165.2 (C=O), 155.4 (MP), 150.8 (MP), 134.6 (Ar), 133.5 (Ar), 129.8
(2 C, Ar), 129.0 (Ar), 128.5 (2 C, Ar), 123.7 (2 C, Ar), 117.8 (2 C, Ar),
114.9 (2 C, Ar, 98.2 (C-1), 68.5 (C-3), 64.7 (C-5), 58.6 (C-2), 55.8 (OMe),
52.4 (C-4), 16.6 ppm (C-6); HRMS: m/z: 551.1537; found: 551.1620
[M+Na]+; elemental analysis calcd (%) for C28H24N4O7 (528.5128): C
63.63, H 4.58, N 10.60; found: C 63.20, H 4.73, N 10.46.
4-Methoxyphenyl
3-O-acetyl-2-azido-2,6-dideoxy-4-O-(4-methoxyben-
zyl)-a,b-d-glucopyranoside (25): To a solution of 24 (9.90 g, 15.3 mmol)
in DMPU (100 mL) sodium cyanoboronhydride (6.75 g, 7 equiv) was
added and the reaction mixture stirred overnight at 958C. The mixture
was poured into water and extracted twice with EtOAc. The organic
phase was washed with water, dried over sodium sulfate and the solvent
removed in vacuo. Flash chromatography (petroleum ether/EtOAc 6:1)
yielded 25 (6.85 g, 86%). TLC (toluene/EtOAc 5:1): Rf =0.38; [a]D =
+11.1 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3): d = 7.20–6.90 (m,
4-Methoxyphenyl 3,4-di-O-acetyl-2-azido-2-deoxy-6-O-(4-methylbenzyl-
8H, Ar), 6.00 (dd, 1H, J3,2 =10.5, J3,4 =9.1 Hz, 3-H), 5.50 (d, 1H, J1,2
=
sulfonyl)-a,b-d-glucopyranoside (28): To
a solution of 19 (8.57 g,
3.5 Hz, 1-H), 4.60 (s, 2H, CH2Ar), 4.10 (m, 1H, 5-H), 3.80 (s, 3H,
CH3O), 3.70 (s, 3H, CH3O), 3.40 (m, 2H, 2-H, 4-H), 2.00 (s, 3H,
CH3CO), 1.30 ppm (d, 3H, J6,5 =6.6 Hz, 6-H); MALDI-MS (positive
mode, matrix DHB, THF): m/z: 650.2; found: 650.2 [M+Na]+; elemental
analysis calcd (%) for C23H27N3O7 (457.48 gmolꢀ1): C 60.38, H 5.95, N
9.19; found: C 60.56, H 6.12, N 8.96.
19.6 mmol) in MeOH (100 mL) NaOMe was added until pH 9. The mix-
ture was stirred for 2 h and then Amberlite IR120 (H+ form) was added
until neutralization. The mixture was filtered and concentrated in vacuo.
The crude residue was dissolved in pyridine (100 mL) and cooled to 08C,
where TsCl (1.05 equiv, 20.6 mmol, 3.92 g) was added. The reaction mix-
ture was allowed to reach room temperature and after reaction overnight
Ac2O (50 mL) was added together with DMAP (ca. 200 mg). After disap-
pearing of intermediates from TLC the reaction was concentrated and
purified by column chromatography (petroleum ether/EtOAc 5:1 to 3:1)
to yield 28 (10.23 g, 95%, a/b 2:1). 1H NMR (600 MHz, CDCl3) a-
isomer: d = 7.67 (d, 2H, J=8.4 Hz, Ar), 7.25 (d, 2H, J=8.4 Hz, Ar),
6.91 (m, 2H, Ar), 6.75 (m, 2H, Ar), 5.55 (dd, 1H, J=9.4 Hz, 10.4 Hz, 3-
H), 5.34 (d, 1H, J=3.5 Hz, 1-H), 4.99 (dd, 1H, J=9.7 Hz, 4-H), 4.98–4.12
(m, 3H, 5-H, 6-H), 3.71 (s, 3H, Me), 3.31 (dd, 1H, J=3.3 Hz, 10.6 Hz, 2-
H), 2.38 (s, 3H, OMe), 2.04 (s, 3H, Ac), 1.91 ppm (s, 3H, Ac); b-isomer:
7.67 (d, 2H, J=8.4 Hz, Ts), 7.21 (d, 2H, J=8.4 Hz, Ts), 6.91 (m, 2H,
MP), 6.75 (m, 2H, MP), 4.94 (dd, 1H, J=9.6 Hz, 9.6 Hz, 3-H), 4.86 (dd,
1H, J=9.7 Hz, 4-H), 4.69 (d, 1H, J=8.1 Hz, 1-H), 4.12–3.98 (m, 2H, 6-
H), 3.75 (m, 1H, 5-H), 3.72 (s, 3H, Me), 3.61 (dd, 1H, J=8.1 Hz,
10.0 Hz, 2-H), 3.34 (s, 3H, OMe), 2.02 (s, 3H, Ac), 1.93 ppm (s, 3H, Ac);
13C NMR (150.9 MHz, CDCl3) a-isomer: d 170.1, 169.4, 155.8, 150.1,
145.1, 132.5, 129.9, 128.1, 118.0, 114.80, 97.5, 70.2, 68.3, 67.9, 67.0, 60.6,
55.7, 21.7, 20.7, 20.5 ppm; b-isomer: d = 169.9, 169.6, 156.0,.150.6, 145.2,
132.5, 129.9, 128.1, 118.8, 114.7, 72.1, 71.7, 68.4, 67.4, 63.4, 55.7, 21.7, 20.7,
20.5 ppm; HRMS: m/z: 572.1309 [M+Na]+; found: 572.1305; elemental
analysis calcd (%) for C24H27N3O10S (549.55 gmolꢀ1): C 52.45, H 5.03, N
7.65; found: C 52.46, H 5.03, N 7.43.
4-Methoxyphenyl 3-O-acetyl-2-azido-2,6-dideoxy-a,b-d-glucopyranoside
(26): To a solution of 25 (6.80 g, 14.9 mmol in CH2Cl2/water 20:1 (84 mL)
DDQ (6.75 g, 2 equiv) was added. The reaction mixture was stirred for
2 h and saturated NaHCO3 was added to quench the reaction. The organ-
ic phase was washed with water and dried over sodium sulfate. The sol-
vent was removed in vacuo. Flash chromatography (petroleum ether/
EtOAc 3:1) yielded 26 (4.4 g, 88%). TLC (toluene/EtOAc 3:1): Rf =0.29;
[a]D = +19.2 (c= 1, CHCl3); 1H NMR (250 MHz, CDCl3): d = 7.20–
6.90 (m, 4H, Ar), 5.70 (dd, 1H, J3,2 =10.5, J3,4 =9.1 Hz, 3-H), 5.50 (d, 1H,
J
1,2 =3.5 Hz,1-H), 4.10 (m, 1H, 5-H), 3.60 (s, 3H, CH3O), 3.50 (m, 2H, 2-
H, 4-H), 2.00 (s, 3H, CH3CO), 1.20 ppm (d, 3H, J6,5 =6.6 Hz, 6-H);
MALDI-MS (positive mode, matrix DHB, THF): m/z: 360.1; found:
360.2 [M+Na]+; elemental analysis calcd (%) for C15H19N3O6
(337.33 gmolꢀ1): C 53.41, H 5.68, N 12.46; found: C 53.48, H 5.79, N
12.71.
4-Methoxyphenyl
3-O-acetyl-2-azido-2,4,6-trideoxy-4-phthalimido-a-d-
galactopyranoside (27a): To a solution of 26 (4.3 g, 12.7 mmol), phthali-
mide (3.75 g, 25.4 mmol), triphenylphosphine (5.01 g, 19.1 mmol) in dry
toluene (90 mL) DIAD was added and the reaction mixture stirred for
1.5 h at 608C. The mixture was diluted with CH2Cl2 and washed with
water. The organic phase was dried over sodium sulfate and the solvent
removed in vacuo. Flash chromatography (petroleum ether/EtOAc 4:1)
yielded 27a (4.41 g, 74%). TLC (toluene/EtOAc 3:1): Rf =0.59; [a]D =
+8.2 (c= 1, CHCl3); 1H NMR (250 MHz, CDCl3): d = 8.00–6.90 (m,
8H, Ar), 5.85 (dd, 1H, J3,2 =10.4, J3,4 =6.5 Hz, 3-H), 5.70 (d, 1H, J1,2
3.7 Hz, 1-H), 5.00 (dd, 1H, J4,3 =6.4, J4,5 =3.4 Hz, 4-H), 4.50 (m, 2H, 2-H,
5-H), 3.80 (s, 3H, CH3O), 2.00 (s, 3H, CH3CO), 1.10 ppm (d, 3H, J6,5
6.6 Hz, 6-H); MALDI-MS (positive mode, matrix DHB, THF): m/z:
489.1; found: 489.3 [M+Na]+; elemental analysis calcd (%) for
C23H22N4O7 (466.44 gmolꢀ1): C 59.22, H 4.75, N 12.01; found: C 59.46, H
5.02, N 11.83.
4-Methoxyphenyl 3,4,di-O-acetyl-2-azido-2-deoxy-6-iodo-a,b-d-glucopyr-
anoside (29): The tosylate 28 (12.24 g, 22.3 mmol) was dissolved in
MeCN (50 mL) and TBAI (2.2 equiv, 52 mmol, 19.2 g) added. The reac-
tion mixture was refluxed overnight, concentrated and purified by flash
chromatography (EtOAc/petroleum ether 6:1 to 2:1) to give 29 (11.04 g,
=
=
98%). 1H NMR (600 MHz, CDCl3) a-isomer: d
= 7.12 (d, 2H, J=
9.1 Hz, C6H4), 6.86 (d, 2H, J=9.1 Hz, C6H4), 5.68 (dd, 1H, J=10.7 Hz, 3-
H), 5.50 (d, 1H, J=3.4 Hz, 1-H), 4.97 (dd, 1H, J=9.2, 9.7 Hz, 4-H), 3.93
(m, 1H, m, 5-H), 3.78 (s, 2H, OMe), 3.43 (dd, 1H, J=3.4, 10.7 Hz, 2-H),
3.29 (dd, 1H, J=2.9, 11.1 Hz, 6-H), 3.14 (dd, 1H, J=7.0, 11.1 Hz, 6-H),
2.11 (s, 3H, Ac), 2.07 ppm (s, 3H, Ac); 13C NMR (150.9 MHz, CDCl3): d
= 169.6 (Ac), 169.1 (Ac), 155.7 (Ar), 150.1 (Ar), 118.0 (2 C, Ar), 114.7
(2 C, Ar), 97.7 (C-1), 72.7 (C-4), 69.8 (C-3), 69.1 (C-5), 60.9 (C-2), 55.7
(OMe), 20.7 (Ac), 20.6 (Ac), 3.68 ppm (C-6); b-isomer: 1H NMR
(600 MHz, CDCl3) b: d = 7.17 (d, 2H, J=9.0 Hz, C6H4), 6.86 (d, 2H, J=
9.0 Hz, C6H4), 504 (dd, 1H, J=9.2, 10.1 Hz, 3-H), 4.87 (dd, 1H, J=9.2,
9.7 Hz, 4-H), 4.84 (d, 1H, J=8.1 Hz, 1-H), 3.78 (dd, 1H, J=8.1, 10.1 Hz,
2-H), 3.78 (s, 3H, OMe), 3.63 (m, 1H, 5-H), 3.33 (dd, 1H, J=2.5,
11.1 Hz, 6-H), 3.16 (dd, 1H, J=8.8, 11.0 Hz, 6-H), 2.17 (s, 3H, Ac),
2.04 ppm (s, 3H, Ac); 13C NMR (150.9 MHz, CDCl3): d = 169.6 (2 C,
Ac), 156.0 (Ar), 150.7 (Ar), 118.8 (2 C, Ar), 114.6 (2 C, Ar), 101.5 (C-1),
74.0 (C-5), 72.2 (C-4), 63.8 (C-2), 55.8 (OMe), 20.6 (2 C, Ac), 2.33 ppm
4-Methoxyphenyl 2-azido-3-O-benzoyl-2,4,6-trideoxy-4-phthalimido-a-d-
galactopyranoside (27b): The mono-benzoylated sugar 31 (3.188 g,
7.98 mmol) was dissolved in CH2Cl2 (50 mL) containing pyridine (1 mL,
2 equiv) and cooled by and ice bath followed by addition of Tf2O
(2.0 mL, 1.5 equiv) by syringe. After 1 h the reaction was finished (TLC:
petroleum ether/EtOAc 3:1) and the reaction was quenched with water
and worked up by washing with HCl (1 m), water and brine followed by
drying and concentration in vacuo to give the crude triflate 32 as a foam
which was used directly in the next reaction without further purification.
The triflate 32 was dissolved in DMF (20 mL), cooled by an ice bath fol-
lowed by addition of potassium phthalimide (3.0 g, 2 equiv). The reaction
mixture was allowed to reach room temperature and stirred overnight.
Chem. Eur. J. 2010, 16, 12627 – 12641
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12635

R. R. Schmidt et al.
(C-6); elemental analysis calcd (%) for C17H20IN3O7: C 40.41, H 3.99, N
8.32; found: C 42.38, H 4.55, N 8.05.
2-Azido-3-O-benzoyl-2,4,6-trideoxy-4-phthalimido-a,b-d-galactopyranose
(33b): The OMP-protected sugar 27b (1.54 g, 2.9 mmol) and bis(trifluor-
oacetoxy)iodobenzene (2 equiv, 2.51 g) were dissolved in CH2Cl2 (50 mL)
and water (10 mL) was added. The mixture was stirred vigorously and
BF3·OEt2 (1.8 equiv, 0.7 mL) was added slowly by syringe. The reaction
was quenched with Et3N after 1 h, worked up by washing with water,
brine and drying over MgSO4. After concentration in vacuo the red
crude product was purified by flash chromatography (EtOAc/petroleum
ether 1:2) to give the desired reducing sugar 33b containing some red im-
purities which could partly be removed by dissolving it in CH2Cl2 and
stirring it with activated carbon overnight. After filtration and concentra-
tion a pink foam of 33b (1.06 g, 87%) was obtained consisting of an a/b
4-Methoxyphenyl 2-azido-2,6-dideoxy-a-d-glucopyranoside (30): To a so-
lution of 29 (23.34 g, 46.4 mmol) in DMPU (100 mL) sodium cyanobor-
onhydride (14 g, 4.8 equiv) was added and the reaction stirred overnight
at 958C. The mixture was poured into water and extracted twice with
EtOAc, the organic phase was washed with water 4 times, brine, dried
over MgSO4 and concentrated in vacuo to give the crude product which
was deacetylated under standard Zemplꢃn conditions. Flash chromatog-
raphy (petroleum ether/EtOAc 1:1) yielded 30 (11.81 g, 86%) as a solid.
M.p. 104–1058C); [a]D = +113 (c= 1, CHCl3); 1H NMR (600 MHz,
CDCl3): d = 7.03 (d, 2H, J=9 Hz, MP), 6.84 (d, 2H, J=9 Hz, MP), 5.40
(d, 1H, J=3.7 Hz, 1-H), 4.15 (m, 1H, 3-H), 3.92 (m, 1H, 5-H), 3.78 (s,
3H, OMe), 3.30 (m, 2H, 2-H, 4-H), 1.30 ppm (d, 3H, J=6.3 Hz, 6-H);
13C NMR (151 MHz, CDCl3): d = 155.4 (MP), 150.6 (MP), 118.1 (2 C,
MP), 114.7 (2 C, MP), 97.7 (C-1), 76.5 (C-4), 71.7 (C-3), 68.0 (C-5), 63.1
(C-2), 55.7 (OMe), 17.5 ppm (C-6); HRMS: m/z: 318.1060; found:
318.1057 [M+Na]+.
1
mixture (1:1.7). H NMR (250 MHz, CDCl3): d =8.00–7.25 (m, 9H, Phth,
Bz), 5.84 (dd, 1H, J=6.5 Hz, 11.4 Hz, 3a-H), 5.63 (d, 1H, J=3.4 Hz, 1a-
H), 5.32 (dd, 1H, J=6.9 Hz, 10.0 Hz, 3b-H), 5.09 (dd, 1H, J=3.7 Hz,
6.8 Hz, 4a-H), 4.97 (dd, 1H, J=3.0 Hz, 7.0 Hz, 4b-H), 4.86–4.66 (m, 1H,
1b-H, 5a-H, 5b-H), 4.14–4.02 (m, 1H, 2a-H, 2b-H), 1.17 (d, 3H, J=
6.6 Hz, 6b-H), 1.10 ppm (d, 3H, J=6.6 Hz, 6a-H).
O-(3-O-Acetyl-2-azido-2,4,6-trideoxy-4-phthalimido-a,b-d-galactopyrano-
syl) trichloroacetimidate (34a): To a solution of 33a (0.90 g, 2.50 mmol)
and trichloroacetonitrile (6.57 mL, 20 equiv) in CH2Cl2 (20 mL), DBU
(49 mL, 0.1 equiv) was added. The reaction mixture was stirred for 1 h
and the solvent evaporated in vacuo. A fast purification by flash chroma-
tography (petroleum ether/EtOAc 5:1) yielded 34a (1.13 g, 90%). TLC
(toluene/EtOAc 3:1): Rf =0.42; [a]D = +3.8 (c = 1, CHCl3); a-isomer:
1H NMR (250 MHz, CDCl3): d =8.80 (s, 1H, NH), 8.00–7.40 (m, 4H,
Ph), 6.60 (d, 1H, J1,2 =3.7 Hz, 1-H), 5.90 (dd, 1H, J3,2 =11.4, J3,4 =6.5 Hz,
3-H), 5.00 (dd, 1H, J4,3 =6.4, J4,5 =3.4 Hz, 4-H), 4.50 (m, 1H, 5-H); 4.25
(dd, 1H, J2,1 =3.6, J2,3 =10.7 Hz, 2-H), 2.00 (s, 3H, CH3CO), 1.10 ppm (d,
3H, J6,5 =6.6 Hz, 6-H); MALDI-MS (positive mode, matrix DHB, THF):
m/z: 526.0; found: 526.1 [M+Na]+ and 383.1 (loss of imidate); elemental
analysis calcd (%) for C18H16Cl3N5O6 (504.71 gmolꢀ1): C 42.84, H 3.20, N
13.88; found: C 42.59, H 3.42, N 13.83.
Alternatively the acetylated product could be isolated by flash chroma-
tography (EtOAc/petroleum ether 7:1 to 4:1). M.p. 94–958C; [a]D
=
1
+190 (c = 1, CHCl3); H NMR (600 MHz, CDCl3): d = 7.05 (d, 2H, J=
9 Hz, MP), 6.84 (d, 2H, J=9 Hz, MP), 5.64 (dd, 1H, J=9.4 Hz, 10.4 Hz,
3-H), 5.44 (d, 1H, J=3.4 Hz, 1-H), 4.86 (dd, 1H, J=9.6 Hz, 9.6 Hz, 4-H),
4.08 (m, 1H, 5-H), 3.77 (s, 3H, OMe), 3.39 (dd, 1H, J=3.5 Hz, 10.4 Hz,
2-H), 2.11 (Ac), 2.05 (Ac), 1.18 ppm (d, 3H, J=6.4 Hz, 6-H); 13C NMR
(150.9 MHz, CDCl3): d
= 170.2 (Ac), 170.0 (Ac), 155.6 (MP), 150.4
(MP), 117.9 (MP), 114.9 (MP), 97.5 (C-1), 73.9 (C-4), 70.3 (C-3), 66.3 (C-
5), 61.2 (C-2), 55.8 (OMe), 20.9 (Ac), 20.8 (Ac), 17.4 ppm (C-6); elemen-
tal analysis calcd (%) for C24H27N3O10S (549.55 gmolꢀ1): C 53.82, H 5.58,
N 11.08; found: C 53.75, H 5.47, N 10.84.
4-Methoxyphenyl 2-azido-3-O-benzoyl-2,6-dideoxy-a-d-glucopyranoside
(31): The diol 30 (4.709 g, 15.9 mmol) was dissolved in dry pyridine
(100 mL) containing 4 ꢇ molecular sieves (2 g). The mixture was stirred
for 2 h at room temperature and cooled to ꢀ308C where BzCl (1.1 equiv,
15.5 mmol, 2.2 mL) was added slowly by syringe. The reaction was
quenched after 1 h with water and concentrated in vacuo, dissolved in
EtOAc, washed with water, HCl (1 m), NaHCO3 (sat) and brine. The or-
ganic phase was dried over MgSO4, concentrated in vacuo and purified
by flash chromatography (EtOAc/petroleum ether 1:5 to 1:3) to give 31
(3.88 g, 61%). M.p. 164–1658C. The side products and starting material
O-(2-Azido-3-O-benzoyl-2,4,6-trideoxy-4-phthalimido-a,b-d-galactopyra-
nosyl) trichloroacetimidate (34b): Crude 33b (1.06 g) was dissolved in
dry CH2Cl2 (15 mL) and Cl3CCN (2.7 mL). DBU (0.1 equiv, 35 mL) was
added and the red solution turned dark. After completion, estimated
from TLC, the reaction was concentrated on silica gel and purified by
flash chromatography (EtOAc/petroleum ether 1:4 + 1% Et3N) to give
34b (1.017 g, 72%) as a foam; a/b mixture (1:7). 1H NMR (400 MHz,
CDCl3): d =8.78 (s, 1H, =NH), 7.93–7.23 (m, 9H, Ar), 6.73 (d, 1H, J=
3.8 Hz, 1a-H), 5.90 (dd, J=6.5, 11.4 Hz, 3a-H), 5.84 (d, 1H, J=8.3 Hz,
1b-H), 5.42 (dd, 1H, J=7.3, 10.8 Hz, 5.25–5.02 (m, 3H, 4a-H, 4b-H, 2b-
H), 4.67 (m, 1H, 5a-H), 4.19 (m, 1H, 5b-H); 1.22 (d, 3H, J=6.4 Hz, 6b-
H), 1.15 ppm (d, 3H, J=6.4 Hz, 6a-H).
were collected, debenzoylated and recycled. [a]D
= +230 (c = 1,
CHCl3); 1H NMR (600 MHz, CDCl3): d =8.11 (d, 2H, J=8.0 Hz, Bz),
7.62 (t, 1H, J=7.5 Hz, Bz), 7.48 (dd, 2H, J=7.5 Hz, 8.0 Hz, Bz), 7.08 (d,
2H, J=8.8 Hz, MP), 6.86 (d, 2H, J=8.8 Hz, MP), 5.70 (dd, 1H, J=
9.9 Hz, 9.9 Hz, 3-H), 5.48 (d, 1H, J=3.3 Hz, 1-H), 4.03 (m, 1H, 5-H),
3.79 (s, 3H, OMe), 3.53 (m, 2H, 2-H, 4-H), 1.36 ppm (d, 3H, J=6.1 Hz,
6-H); 13C NMR (150.9 MHz, CDCl3): d = 170.0 (C=O), 167.4 (Ar), 150.4
(Ar), 133.6 (Ar), 130.1 (Ar), 129.2 (Ar), 128.5 (Ar), 118.0 (Ar), 114.7
(Ar), 97.7 (C-1), 75.2 (C-4), 74.3 (C-3), 68.7 (C-5), 61.3 (C-2), 55.5
(OMe), 17.5 ppm (Me); HRMS: m/z: 422.1313; found: 422.1313
[M+Na]+.
4-Methoxyphenyl 2-azido-4-benyloxycarbonylamino-2,4,6-trideoxy-a-d-
galactopyranoside (35): To a solution of 27a (2.59 g, 5.55 mmol) in n-bu-
tanol (300 mL) ethylendiamine (64 mL) was added and the reaction mix-
ture stirred for 12 h at 958C. The solvent was evaporated and the residue
filtered through a pad of silica gel (toluene/acetone 2:1) to yield the
amino intermediate (1.11 g, 68%) which without any further purification
was directly used in the next step. 27b was transformed into this inter-
mediate by the same procedure. The amine (1.18 g, 4.0 mmol) was dis-
solved in THF/H2O (4:1, 50 mL) containing NaHCO3 (1.0 g, 11.9 mmol)
and cooled by an ice bath followed by addition of Z-Cl (0.90 mL,
6.3 mmol). After 20 min at 08C no starting material was observed by
TLC (EtOAc) and the reaction mixture was diluted with EtOAc, washed
with water, NH4Cl (aq, sat) and brine followed by drying (MgSO4) and
concentration in vacuo. The crude compound was purified by flash chro-
matography (petroleum ether/EtOAc 5:1 to 2:1) to give 35 (1.455 g,
3-O-Acetyl-2-azido-2,4,6-trideoxy-4-phthalimido-a,b-d-galactopyranose
(33a): To a solution of 27a (1.6 g, 3.43 mmol) in acetonitrile (50 mL)
cooled to ꢀ158C, an aqueous solution of CAN (4.51 g, 2.4 equiv, in
10 mL H2O) was added. The reaction mixture was stirred for 1.5 h and a
saturated NaHCO3 solution was added to quench the reaction. The mix-
ture was extracted 3 times with CH2Cl2, the organic phase was dried over
sodium sulfate and the solvent was removed in vacuo. Flash chromatog-
raphy (toluene/acetone 5:1) yielded 33a (0.93 g, 75%). TLC (toluene/
1
= +77.0 (c =
1, CHCl3); 1H NMR
acetone 5:1): Rf =0.29; [a]D = +8.8 (c =1, CHCl3); H NMR (250 MHz,
85%). M.p. 46–478C; [a]D
CDCl3): d =8.00–7.40 (m, 4H, Ph), 5.85 (dd, 1H, J3,2 =11.4, J3,4 =6.5 Hz,
3-H), 5.50 (d, 1H, J1,2 =3.7 Hz, 1-H), 5.00 (dd, 1H, J4,3 =6.4, J4,5 =3.4 Hz,
4-H), 4.50 (m 1H, 5-H), 4.20 (dd, J2,1 =3.6, J2,3 =10.7 Hz, 2-H), 2.00 (s,
3H, CH3CO), 1.10 ppm (d, 3H, J6,5 =6.6 Hz, 6-H). MALDI-MS (positive
mode, matrix DHB, THF): m/z: 383.1; found: 383.1 [M+Na]+; elemental
analysis calcd (%) for C16H16N4O6 (360.32 gmolꢀ1): C 53.33, H 4.48, N
15.55; found: C 53.49, H 4.22, N 15.83.
(400 MHz, CDCl3): d =7.35 (m, 5H, Ph), 7.02 (d, 2H, J=8.9 Hz, MP),
6.84 (d, 2H, J=8.9 Hz, MP), 5.46 (brd, 1H, J=9.4 Hz, NH), 5.31 (d, 1H,
J=3.5 Hz, 1-H), 5.16 (d, 1H, J=12.0 Hz, Bn), 5.12 (d, 1H, J=12.0 Hz,
Bn), 4.41 (m, 1H, 3-H), 4.29 (m, 1H, 5-H), 4.13 (brd, 1H, J=3.2, 9.0 Hz,
4-H), 3.82 (brd, 1H, J=4.7 Hz, OH), 3.77 (s, 3H, OMe), 3.34 (dd, 1H,
J=3.7, 10.8 Hz, 2-H), 1.17 ppm (d, 3H, J=6.5 Hz, 6-H); 13C NMR
(100 MHz, CDCl3): d = 157.1, 155.4, 150.7, 136.04, 128.6, 128.4, 128.2,
12636
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12627 – 12641

Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
118.2, 114.8, 98.1, 68.4, 67.6, 65.7, 60.2, 56.0, 16.6 ppm; HRMS (amine in-
Bn), 4.78 (d 1H, J=12.0 Hz, Troc), 4.62 (d, 1H, J=12.0 Hz, Troc), 4.27
(brm, 1H, 3-H), 4.19–4.08 (brm, 1H, 4-H, 5-H), 3.97 (brm, 1H, 2-H),
3.77 (s, 3H, OMe), 3.36 (d, 1H, J=6.4 Hz, OH), 1.16 ppm (d, 1H, J=
6.4 Hz, 6-H); 13C NMR (101 MHz, CDCl3): d = 158.2, 155.5, 155.2, 150.5,
136.1, 128.7, 128.4, 128.3, 118.2, 114.8, 97.5, 95.4, 74.9, 69.1, 67.6, 66.0,
55.8, 55.7, 53.0, 16.7 ppm.
termediate): m/z: 452.1588; found: 452.1676 [M+Na]+.
3-O-Acetyl-2-azido-4-benzoyloxycarbonylamino-2,4,6-trideoxy-a,b-d-gal-
actopyranose (36): a) 3-O-Acetylation of 35: Compound 35 (1.46 g,
3.40 mmol) was dissolved in pyridine (10 mL) and Ac2O (7 mL) was
added. The reaction mixture was stirred for 2 h at room temperature and
the solvent evaporated in vacuo. Flash chromatography (petroleum
ether/EtOAc 3:1) yielded 36 (1.40 g, 88%). TLC (petroleum ether/
4-Methoxyphenyl 3-O-allyloxycarbonyl-4-benzyloxycarbonylamino-2,4,6-
trideoxy-2-(2,2,2-trichloroethoxycarbonylamino-a-d-galactopyranoside
(39): Compound 38 (1.00 g, 1.72 mmol) was dissolved in CH2Cl2 and
cooled with an ice bath followed by addition of AllocCl (0.56 mL,
3 equiv) and pyridine (0.68 mL, 5 equiv). The reaction was allowed to
reach room temperature over 1 h and stirred further for 1 h before it was
diluted with EtOAc, washed with water, HCl (aq 1m), water and brine,
followed by drying (MgSO4), concentration in vacuo on silica gel and pu-
rification by flash chromatography (petroleum ether/EtOAc 5:1 to 1:1) to
1
EtOAc 3:1): Rf =0.19; [a]D = +8.8 (c = 1, CHCl3); H NMR (250 MHz,
CDCl3): d =7.50–6.80 (m, 9H, Ph), 5.45 (dd, 1H, J3,2 =11.3, J3,4 =3.9 Hz,
3-H), 5.40 (d, 1H, J1,2 =3.7 Hz, 1-H), 5.15 (m, 3H, CH2Ar, NH), 4.40 (m,
1H, 5-H), 4.30 (m, 1H, 4-H), 3.75 (s, 3H, CH3O), 3.50 (dd, 1H, J1,2 =3.7,
J
2,3 =11.3 Hz, 2-H), 2.00 (s, 3H, CH3CO), 1.20 ppm (d, 3H, J6,5 =6.6 Hz,
6-H); MALDI-MS (positive mode, matrix DHB, THF): m/z: 493.2;
found: 493.1 [M+Na]+; elemental analysis calcd (%) for C23H26N4O7
(470.48 gmolꢀ1): C 58.72, H 5.57, N 11.91; found: C 58.97, H 5.68, N
11.99.
give 39 (977 mg, 85%). [a]D
= +90.0 (c =
1, CHCl3); 1H NMR
(400 MHz, CDCl3): d =7.40–7.28 (m, 5H, Ph), 6.96 (d, 2H, J=9.1 Hz,
MP), 6.82 (d, 2H, J=9.1 Hz, MP), 5.91 (m, 1H, All), 5.48 (d, 1H, J=
9.5 Hz, NH), 5.41 (d, 1H, J=3.7 Hz, 1-H), 5.36 (dd, 1H, J=1.3, 17.3 Hz,
All), 5.29 (d, 1H, J=9.3 Hz, NH), 5.26 (brd, 1H, J=10 Hz, All), 5.15
(dd, 1H, J=4.0, 11.4 Hz, 3-H), 5.14 (d, 1H, J=12.2 Hz, Bn), 5.10 (d, 1H,
J=12.2 Hz, Bn), 4.84 (d, 1H, J=12.0 Hz, Troc), 4.64 (brd, 2H, J=
5.6 Hz, All), 4.36 (m, 2H, 4-H, 5-H), 4.22 (ddd, 1H, J=3.7, 9.8 Hz, 2-H),
3.77 (s, 3H, OMe), 1.16 ppm (d, 3H, J=6.4 Hz, 6-H); 13C NMR
(101 MHz, CDCl3): d = 156.9 (C=O), 155.6 (C=O), 154.8 (C=O), 154.5
(Ar), 150.3 (Ar), 136.3 (Ar), 131.5 (All), 128.7 (Ar), 128.3 (Ar), 128.2
(Ar), 119 L (All), 118.1 (Ar), 114.9 (Ar), 97.3 (C-1), 95.5 (CCl3), 74.8
(CH2-CCl3), 73.7 (C-3), 69.1 (CH2CH=CH2), 67.3 (CH2-Ph), 65.8 (C-5),
55.8 (OMe), 52.8 (C-4), 50.4 (C-2), 16.5 ppm (C-6); HRMS: m/z:
685.0909; found: 685.0910 [M+Na]+.
b) Cleavage of the MP group: To a solution of the 3-O-acetyl derivative
(1.4 g, 2.98 mmol) in acetonitrile (42 mL, cooled to ꢀ158C) a solution of
CAN (3.92 g, 2.4 equiv in 14 mL of water) was added dropwise and the
reaction mixture was stirred for 2 h. NaHCO3 was added then to quench
the reaction and the mixture was extracted with CH2Cl2; the organic
phase was dried over sodium sulfate and the solvent evaporated in vacuo.
Flash chromatography (petroleum ether/EtOAc 2:1) yielded 36 (0.91 g,
84%). TLC (toluene/EtOAc 2:1): Rf =0.21; [a]D
CHCl3); 1H NMR (250 MHz, CDCl3): d =7.45 (m, 5H, Ph), 5.40 (d, 1H,
1,2 =10 Hz, NH), 5.30–5.00 (m, CH2Ph-Cbz, 1a-H, 3a-H), 4.80–4.60 (m,
= +4.5 (c = 1,
J
5b-H, 3b-H, 1b-H), 4.50 (m, 1H, 5a-H), 4.22 (m, 1H, 4a-H), 4.20 (m,
1H, 4b-H), 3.75 (m, 2b-H), 3.50 (m, 1H, 2a-H), 2.00 (s, 3H, CH3CO a
and b), 1.20 ppm (d, J6,5 =6.6 Hz, 6a-H, 6b-H); MALDI-MS (positive
mode, matrix DHB, THF): m/z: 387.1; found: 387.1 [M+Na]+; elemental
analysis calcd (%) for C163H20N4O6 (364.35 gmolꢀ1): C 52.74, H 5.53, N
15.38; found: C 52.79, H 5.58, N 15.59.
O-(3-O-Allyloxycarbonyl)-4-benzyloxycarbonylamino-2,4,6-trideoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-a-d-galactopyranosyl) trichloroace-
timidate (3): Compound 39 (977 mg, 1.48 mmol) was dissolved in MeCN
(20 mL) containing water (8 mL) cooled with an ice bath and CAN
(2.0 g, 2.5 equiv) dissolved in water (10 mL) was added slowly by syringe.
After the addition no starting material was observed by TLC (EtOAc/pe-
troleum ether 1:3) and the reaction was quenched by addition of
NaHCO3 (sat). The reaction mixture was diluted with EtOAc, washed
with water and brine followed by drying (MgSO4) and concentration in
vacuo on silica gel. After a short path column the reddish product 40 was
concentrated and dissolved in CH2Cl2 (10 mL) followed by addition of
Cl3CCN (1.5 mL, 10 equiv) and DBU (25 mL). After 1 h the reaction was
concentrated on silica gel and purified by a short path column (petro-
leum ether/EtOAc 5:1 to 3:1 containing 1% Et3N) to give 3 (917 mg,
O-(3-O-Acetyl-2-azido-4-benzyloxycarbonylamino-2,4,6-trideoxy-a,b-d-
galactopyranosyl) trichloroacetimidate (37): To a solution of 36 (0.85 g,
2.33 mmol) in CH2Cl2 (30 mL) Cl3CCN (2.34 mL, 10 equiv) and DBU
(35 mL, 0.1 equiv) were added and the reaction mixture stirred for 1 h at
room temperature. The solvent was evaporated in vacuo. Flash chroma-
tography (petroleum ether/EtOAc 4:1) yielded 37 (1.03 g, 88%). [a]D
=
+12.3 (c = 1, CHCl3); 1H NMR (250 MHz, CDCl3) a-isomer: d = 8.70
(s, 1H, NH-imidate), 7.45 (m, 5H, Ph), 6.30 (d, 1H, J1,2 =3.9 Hz, 1-H),
5.15 (m, 4H, CH2Ph-Cbz, 3-H, NH), 4.35 (m, 2H, 4-H, 5-H), 3.80 (dd,
J
2,1 =3.9, J2,3 =11.2 Hz, 3-H), 2.00 (s, 3H, CH3CO), 1.20 (d, J6,5 =6.6 Hz,
6-H); b-isomer: d 8.70 (s, 1H, NH-imidate), 7.45 (m, 5H, Ph), 5.60 (d,
1H, J1,2 =8.5 Hz, 1-H), 5.20 (m, 3H, CH2Ph-Cbz, NH), 4.80 (dd, J2,1 =3.9,
89%) which was used directly in the following glycosylation. [a]D
=
+78.1 (c= 1, CHCl3); 1H NMR (300 MHz, CDCl3): d =8.74 (m, 1H,
NH), 7.40–7.28 (m, 5H, Ar), 6.38 (d, 1H, J=3.6 Hz, 1-H), 5.92 (m, 1H,
All), 5.40–5.19 (m, 4H, All, 2ꢆNH), 5.12 (s, 2H, CH2Ar), 5.04 (dd, 1H,
J=4.0, 11.7 Hz, 3-H), 4.71 (s, 2H, CH2CCl3), 4.65 (brd, 2H, J=5.7 Hz,
All), 4.49–4.29 (m, 3H, 2-H, 4-H, 5-H), 1.24 ppm (d, 3H, J=6.4 Hz, 6-
H); 13C NMR (75 MHz, CDCl3): d = 160.7, 156.9, 154.7, 154.4 (4ꢆC=O),
136.2 (Ar), 131.4 (all), 128.7, 128.3, 128.2 (Ar), 119.2 (All), 95.4
(CNCCl3), 95.2 (C-1), 90.9 (CH2CCl3), 74.8 (-CH2CHCH2), 73.3 (C-3),
69.2 (CH2CCl3), 67.9 (C-5), 67.4 (CH2Ph), 52.4 (C-2), 49.9 (C-4),
16.6 ppm (C-6); elemental analysis calcd (%) for C23H25Cl6N3O9
(700.2 gmolꢀ1): C 39.74, H 3.60, N 6.00; found: C 39.45, H 3.63, N 5.71.
J
J
2,3 =10.7 Hz, 3-H), 4.20 (m, 1H, 4-H), 3.90 (m, 1H, 5-H), 3.75 (dd, 1H,
2,1 =8.6, J2,3 =10.7 Hz, 2-H), 2.00 (s, 3H, CH3CO), 1.20 ppm (d, J6,5
=
6.6 Hz, 6-H); MALDI-MS (positive mode, matrix DHB, THF): m/z:
530.1; found: 530.1 [M+Na]+; elemental analysis calcd (%) for
C18H20Cl3N3O6 (508.74 gmolꢀ1): C 42.50, H 3.96, N 13.77; found: C 42.71,
H 4.12, N 13.59.
4-Methoxyphenyl 4-benzyloxycarbonylamino-2-(2,2,2-trichloroethoxycar-
bonylamino)-2,4,6-trideoxy-a-d-galactopyranoside (38): The azide 35
(3.00 g, 7.00 mmol) was dissolved in EtOH (50 mL) and Raney nickel
(approx. 2 g) was added. The reaction mixture was stirred under H2 pres-
sure until all starting material was consumed, followed by filtration
through celite and concentration. The crude product was dissolved in
CH2Cl2 and stirred vigorously with water containing NaHCO3 (1.76 g,
1.5 equiv). TrocCl (1.4 mL, 1.5 equiv) was added slowly by syringe and
the reaction was followed by TLC (petroleum ether/EtOAc 1:3). After
complete reaction the phases were separated and the organic phase
washed with water and brine, dried (MgSO4) and concentrated in vacuo
to give a crude product that was purified by flash chromatography (pe-
troleum ether/EtOAc 3:1 to 2:1) to give 38 (3.675 g, 91%). M.p. 648C;
3-O-(3-O-Allyl-2,4,6-tri-O-benzyl-a-d-glucopyranosyl)-1,2-O-cyclohexyli-
dene-sn-glycerol (41): To a solution of 1,2-O-cyclohexylidene-sn-glycerol
(1.72 g, 10 mmol) and donor 2 (7.6 g, 1.2 equiv) in CH2Cl2 (160 mL),
TMSOTf (0.092 mL, 0.05 equiv) was added dropwise at 08C and the cool-
ing was removed. After 15 min the reaction mixture was neutralized with
Et3N and the solvent evaporated in vacuo. Flash chromatography (petro-
leum ether/EtOAc 4:1) yielded 41 (3.76 g, 58%). Overall yield 84%, a/b
3.2:1. TLC (petroleum ether/EtOAc 3:1): Rf =0.42; 1H NMR (600 MHz,
CDCl3): d =7.40–7.15 (m, 15H, Ph), 6.00 (m, 1H, CH-All), 5.25–5.15 (m,
2H, CH2-All), 4.88 (d, J1,2 =3.6 Hz, 1-H), 4.85, 4.75, 4.65, 4.50 (6d, 6H,
J=12 Hz, OCH2Ph), 4.45 (m, 1H, OCH2-All), 4.34 (m, 1H, 2-H), 4.25
(m, 1H, OCH2-All), 4.00 (dd, 1H, Jgem =8.4, Jgem =6.6 Hz, 1Gro-H), 3.80
1
[a]D = +82.8 (c= 1, CHCl3); H NMR (400 MHz, CDCl3): d =7.40–7.30
(m, 5H, Ph), 6.96 (d, 2H, J=8.9 Hz, MP), 6.82 (d, 2H, J=8.9 Hz, MP),
5.59 (d, 1H, J=9.0 Hz, NH), 5.49 (d, 1H, J=9.3 Hz, NH), 5.40 (d, 1H,
J=3.0 Hz, 1-H), 5.20 (d, 1H, J=12.2 Hz, Bn), 5.09 (d, 1H, J=12.2 Hz,
Chem. Eur. J. 2010, 16, 12627 – 12641
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12637

R. R. Schmidt et al.
(t, 1H, J3,2 =J3,4 =9.0 Hz, 3-H), 3.76–3.70 (m, 3H, 6-H, 1Gro-H, 5-H), 3.63–
3.56 (m, 4H, 4-H, 6’-H, 3Gro-H), 3.49 (dd, 1H, J2,1 =3.6, J2,3 =9.6 Hz, 2-H),
1.55 (m, 8H, CH2), 1.35 ppm (m, 2H, CH2); 13C NMR (150.9 MHz,
CDCl3): d = 116.6 (1 C, CH2-All), 110.6 (1 C, C-Cy), 97.5 (1 C, C-1),
81.7 (1 C, C-3), 79.7 (1 C, C-2), 77.6 (1 C, C-4), 74.5 (1 C, CH2-All), 74.3
(1 C, C-2), 75.1, 73.5, 73.0 (3 C, CH2Ph), 70.3 (1 C, C-5), 69.0 (1 C, C-
3-O-[(3-O-Acetyl-2-azido-4-benzyloxycarbonylamino-2,4,6-trideoxy-b-d-
galactopyranosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl]-1,2-di-O-
myrisoyl-sn-glycerol (45a): To
a solution of acceptor 44 (0.45 g,
0.48 mmol) and donor 37 (0.34 g, 1.4 equiv) in acetonitrile cooled to
ꢀ408C TMSOTf (8.6 mL, 0.1 equiv) was added and the reaction stirred
for 30 min at this temperature. Et3N was added to quench the reaction
and the solvent was evaporated in vacuo. Flash chromatography (petro-
leum ether/EtOAc 7:1) yielded 45a (0.405 g, 66%). TLC (petroleum
ether/EtOAc 7:1): Rf =0.21; [a]D = +21.3 (c = 1, CHCl3); 1H NMR
(250 MHz, CDCl3): d =7.40 (m, 20H, Ph), 5.05 (m, 5H, 3b-H, 1b-H, 4b-
NH, CHPh-Cbz, 2Gro-H), 4.90 (m, 2H, CHPh-Cbz, 1a-H), 4.78 (d, 1H,
J=10.2 Hz, CHPh), 4.66–4.48 (m, 5H, 2ꢆCH2Ph, CHPh), 4.26 (m, 2H,
4b-H, 3Gro-H), 4.15 (dd, 1H, Jgem =9.0, J3,2 =7.0 Hz, 1Gro-H), 3.80–3.58 (m,
6H, 6a-H, 4a-H, 2a-H, 5a-H, 1’Gro-H), 3.55 (m, 2H, 3Gro-H), 3.43 (m, 1H,
3
Gro), 68.5 (1 C, C-6), 66.7 (1 C, C-1Gro), 36.5, 35.0, 25.2, 24.1, 23.9 ppm (5
C, CH2); MALDI-MS (positive mode, matrix DHB, THF): m/z: 667.3;
found: 667.0 [M+Na]+; elemental analysis calcd (%) for C39H48O8
(644.79 gmolꢀ1): C 63.75, H 7.50; found: C 63.81, H 7.68.
3-O-(3-O-Allyl-2,4,6-tri-O-benzyl-a-d-glucopyranosyl)-sn-glycerol (42): A
solution of 41 (0.8 g, 1.24 mmol) in 80% aqueous acetic acid (15 mL) was
stirred at 808C for 3 h. After this time the solution was concentrated in
vacuo and then diluted with toluene and concentrated to dryness in
vacuo. Flash chromatography (petroleum ether/EtOAc 1.5:1) yielded 42
(0.55 g, 78%) as a colourless syrup. TLC (petroleum ether/EtOAc 3:1):
Rfa =0.36; Rfb = 0.42; 1H NMR (600 MHz, CDCl3): d =7.40–7.15 (m,
15H, Ph), 6.00 (m, 1H, CH-All), 5.25–5.15 (m, 2H, CH2-All), 4.88 (d,
5b-H), 3.40 (dd, 1H,
J2,1 =9.0, J2,3 =10.8 Hz, 2b-H), 2.25 (t, 4H,
COCH2R), 2.00 (s, 3H, CH3-Ac), 1.55 (m, 4H, COCH2CH2R), 1.25 (m,
40H, CH2-chain), 0.90 ppm (m, 9H, 2ꢆCH3, 6c); 13C NMR (150.3 MHz,
CDCl3):
d = 172.2 (2ꢆRCH2CH2CO), 165.0 (BnOC(O)N), 139.66–
128.44 (Ph), 101.1 (C-1b), 96.6 (C-1a), 80.5 (C-2a), 80.0–66.3 (CH2Ph-
Cbz, C-4a, C-1Gro, C-2Gro, C-3Gro, 3ꢆCH2Ph, C-3a, C-5a, C-6a, C-5b,C-3b),
60.6 (C-2b), 56.3 (C-4b), 37.3 (CH3-Ac), 32.9–15.1 ppm (C-6b, CH2-chain,
2ꢆCH3); MALDI-MS (positive mode, matrix DHB, THF): m/z: 1329.8;
found: 1330.0 [M+Na]+; elemental analysis calcd (%) for C76H110N2O16
(1307.69 gmolꢀ1): C 69.80, H 8.48, N 2.14; found: C 70.05, H 8.63, N 2.35.
J
1,2 =3.6 Hz, 1-H), 4.85, 4.75, 4.65, 4.60, 4.50 (6d, 6H, J=12 Hz,
OCH2Ph), 4.45 (m, 1H, OCH2-All), 4.25 (m, 1H, OCH2-All), 4.00 (dd,
1H, Jgem =8.4, Jgem =6.6 Hz, 1Gro-H), 3.76–3.70 (m, 5H, 6’-H, 1Gro-H, 5-H,
3-H, 2Gro-H), 3.63–3.56 (m, 4H, 4-H, 6-H, 3Gro-H), 3.49 ppm (dd, 1H,
J
2,1 =3.6, J2,3 =9.6 Hz, 2-H); 13C NMR (150.9 MHz, CDCl3): d = 116.6 (1
C, CH2-All), 97.5 (1 C, C-1), 81.7 (1 C, C-3), 79.7 (1 C, C-2), 77.6 (1 C, C-
4), 74.5 (1 C, CH2-All), 74.3 (1 C, C-2), 75.1, 73.5, 73.0 (3 C, CH2Ph), 70.3
(1 C, C-5), 68.5 (1 C, C-6), 62.0 (1 C, C-1Gro), 61.7 ppm (1 C, C-3Gro);
MALDI-MS (positive mode, matrix DHB, THF): m/z: 587.3; found:
587.3 [M+Na]+; elemental analysis calcd (%) for C33H40O8
(564.67 gmolꢀ1): C 70.19, H 7.14; found: C 70.38, H 7.38.
3-O-[(3-O-Allyloxycarbonyl-4-benzyloxycarbonylamino-2,4,6-trideoxy-2-
(2,2,2-trichloroethoxycarbonylamino)-b-d-galactopyranosyl)-(1–3)-2,4,6-
tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-sn-glycerol (45b):
The acceptor 44 (707 mg, 0.75 mmol) and the donor 3 (576 mg, 1.1 equiv)
were coevaporated with toluene and dried under vacuum followed by ad-
dition of dry CH2Cl2 (9 mL) cooling to 08C and addition of TMSOTf
(7 mL in 1 mL CH2Cl2). After 10 min TLC (petroleum ether/EtOAc 5:1)
showed disappearance of starting material and formation of one spot.
The reaction was quenched with Et3N, concentrated on silica gel and pu-
rified by flash chromatography to give 45b (1.035 g, 93%) with a minor
amount of trichloroacetamide. [a]D = +30.4 (c = 1, CHCl3); 1H NMR
(400 MHz, CDCl3): d =7.50–7.10 (m, 20H, Ar), 5.89 (m, 1H, All), 5.32
(dd, 1H, J=17.3, 1 Hz, All), 5.24 (d, 1H, J=10.5 Hz, All), 5.19 (m, 1H,
2Gro-H), 5.11 (d, 1H, J=12.0 Hz, Z-CH2), 5.04 (d, 1H, J=12.0 Hz, Z-
CH2), 5.00–4.30 (m, 10H, 3ꢆPhCH2, Cl3CCH2, C=C-CH2), 4.74 (d, 1H,
J=3.4 Hz, 1a-H), 4.72 (d, 1H, J=8.6 Hz, 1b-H), 4.48 (dd, 1H, J=3.5,
11 Hz, 3b-H), 4.35 (dd, 1H, J=3.5, 12 Hz, 1Gro-H), 4.18 (brdd, J=3.5,
10 Hz), 4.15–4.07 (m, 2H, 1’Gro-H, 4a-H), 3.74–3.63 (m, 5H, 3Gro-H, 2b-H,
5b-H, 5a-H, 6’aH), 3.55 (m, 3H, 2a-H, 4a-H, 6’’a-H), 3.39 (dd, 1H, J=
5.5, 10.8 Hz, 3’Gro-H), 2.25 (m, 4H, COCH2R), 1.56 (m, 4H,
COCH2CH2R), 1.24 (m, 40H, CH2), 1.13 (d, 3H, J=6.3 Hz, 6b-H),
0.87 ppm (t, 6H, J ꢂ 6.8 Hz, CH3); 13C NMR (101 MHz, CDCl3): d =
173.5 (C=O acyl), 173.2 (C=O acyl), 156.9, 154.8, 154.4 (3ꢆC=O, Z,
Alloc, Troc), 138.5 (Ar), 137.9 (Ar), 136.4 (Ar), 131.6 (Alloc), 129–127
(21 C, Ar), 118.9 (Alloc), 101.9 (C-1b), 96.8 (C-1a), 95.7 (Cl3C-), 81.2 (C-
2a), 79.5 (C-3a), 76.3 (C-3b), 75.6 (C-4a), 75.0 (CH2), 74.8 (CH2), 73.7
(CH2), 72.6 (CH2), 70.3, 69.9, 69.3, 69.0 (4ꢆC, C-2Gro, C-5a, C-5b, CH2),
68.4 (C-6a), 67.2 (CH2), 66.5 C-3Gro), 62.6 (C-1Gro), 53.4 (C-2b), 52.4 (C-
4b), 34.4, 34.2, 32.0, 29.8, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2, 25.0, 22.8 (CH2-
chain), 16.6 (C-6b), 14.3 ppm (2ꢆCH3); HRMS: m/z: 1505.6767; found:
1505.6790 [M+Na]+.
3-O-(3-O-Allyl-2,4,6-tri-O-benzyl-a-d-glucopyranosyl)-1,2-di-O-myristo-
yl-sn-glycerol (43): To a solution of 42 (0.55 g, 0.97 mmol) in CH2Cl2/
DMF (15 mL, 6:1) DCC (0.52 g, 2.6 equiv), myristoic acid (0.56 g,
2.5 equiv dissolved in DMF) and DMAP (0.06 g, 0.5 equiv) were added.
The reaction mixture was stirred overnight, filtered and washed with
water and brine. The organic phase was dried over sodium sulfate and
the solvent evaporated in vacuo. Flash chromatography (petroleum
ether/EtOAc 8:1) yielded 43 (0.72 g, 75%) as a milky solid. TLC (petro-
1
leum ether/EtOAc 8:1): Rf =0.36; H NMR (600 MHz, CDCl3): d =7.40–
7.15 (m, 15H, Ph), 6.00 (m, 1H, CH-All), 5.25–5.15 (m, 2H, CH2-All),
5.05 (m, 1H, 2Gro-H), 4.88 (d, J1,2 =3.6 Hz, 1-H), 4.85, 4.75, 4.65, 4.60, 4.50
(6d, 6H, J=12 Hz, OCH2Ph), 4.45 (m, 1H, OCH2-All), 4.25 (m, 1H,
OCH2-All), 4.00 (dd, 1H, J=8.4, J=6.6 Hz, 1Gro-H), 3.80 (t, 1H, J3,2
=
J
3,4 =9.0 Hz, 3-H), 3.76–3.70 (m, 3H, 6b-H, 1’Gro-H, 5-H), 3.63–3.56 (m,
4H, 4-H, 6a-H, 3-H), 3.49 (dd, 1H, J2,1 =3.6, J2,3 =9.6 Hz, 2-H), 2.25 (t,
4H, COCH2R), 1.55 (m, 4H, COCH2CH2R), 1.25 (m, 40H, CH2-chain),
0.90 ppm (t, 6H, Me). MALDI-MS (positive mode, matrix DHB, THF):,
m/z: 1007.7; found: 1007.8 [M+Na]+; elemental analysis calcd (%) for
C61H92O10 (985.38 gmolꢀ1): C 74.35, H 9.41; found: C 74.38, H 9.63.
1,2-Di-O-myristoyl-3-O-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)-sn-glyc-
erol (44): To a solution of 43 (0.70 g, 0.71 mmol) in EtOAc (15 mL),
NaOAc (0.35 g, 6 equiv), 90% HOAc (14 mL) and PdCl2 (0.25 g,
1.5 equiv) were added. The reaction mixture was stirred overnight and
filtered over celite. The filtrate was neutralized with solid NaHCO3 and
extracted several times with EtOAc, the organic phase was then washed
with water and brine, dried over sodium sulfate and the solvent was
evaporated in vacuo. Flash chromatography (petroleum ether/EtOAc
8:1) yielded 44 (2.10 g, 68%). TLC (petroleum ether/EtOAc 8:1): Rf =
3-O-[(2-Azido-4-benzyloxycarbonylamino-2,4,6-trideoxy-b-d-galactopyra-
nosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-
sn-glycerol (46a): To an ice bath cooled solution of 45a (0.4 g,
0.31 mmol) in MeOH (10 mL) sodium methanolate (0.1 mL, 0.05 m) was
added until neutralization. The mixture was filtered and the solvent
evaporated in vacuo to give 46a (0.38 g, 98%). TLC (petroleum ether/
0.27; [a]D
= +11.8 (c =
1, CHCl3); 1H NMR (250 MHz, CDCl3): d
=7.40–7.15 (m, 15H, Ph), 5.05 (m, 1H, 2Gro-H), 4.88 (d, J1,2 =3.6 Hz, 1-
H), 4.85, 4.75, 4.65, 4.60, 4.50 (6d, 6H, J=12 Hz, OCH2Ph), 4.00 (dd, 1H,
EtOAc 5:1): Rf =0.26; [a]D
= +22.5 (c =
1, CHCl3); 1H NMR
J
gem =8.4, J3,2 =6.6 Hz, 1Gro -H), 3.80 (t, 1H, J3,2 =J3,4 =9.0 Hz, 3-H), 3.76–
(250 MHz, CDCl3): d =7.40 (m, 20H, Ph), 5.05 (m, 5H, 3b-H, 1b-H, 4b-
NH, CHPh-Cbz, 2Gro-H), 4.90 (m, 2H, CHPh-Cbz, 1a-H), 4.78 (d, 1H,
J=10.2 Hz, CHPh), 4.66–4.48 (m, 5H, 2ꢆCH2Ph, CHPh), 4.26 (m, 2H,
4b-H, 3a-H), 4.15 (dd, 1H, J=9, 7 Hz, 1Gro-H), 3.8–3.58 (m, 7H, 3b-H,
6a-H, 4a-H, 2a-H, 5a-H, 1’Gro-H, 3’Gro-H), 3.55 (m, 2H, 3Gro-H), 3.43 (m,
1H, 5b-H), 3.4 (dd, 1H, J2,1 =9, J2,3 =10.8 Hz, 2b-H), 2.25 (m, 4H,
COCH2R), 1.55 (m, 4H, COCH2CH2R), 1.25 (m, 40H, CH2-chain),
3.70 (m, 3H, 6-H, 1’Gro-H, 5-H), 3.63–3.56 (m, 4H, 4-H, 6’-H, 3Gro-H),
3.49 (dd, 1H, J2,1 =3.6, J2,3 =9.6 Hz, 2-H), 2.25 (t, 4H, COCH2R), 1.55
(m, 4H, COCH2CH2R), 1.25 (m, 40H, CH2-chain), 0.90 ppm (t, 6H, Me).
MALDI-MS (positive mode, matrix DHB, THF): m/z: 967.6; found:
967.7 [M+Na]+; elemental analysis calcd (%) for C58H88O10
(945.31 gmolꢀ1): C 73.69, H 9.38; found: C 73.42, H 9.48.
12638
www.chemeurj.org
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 12627 – 12641

Synthesis of the Core Structure of the Lipoteichoic Acid
FULL PAPER
0.90 ppm (m, 9H, 2ꢆCH3, 6b); 13C NMR (150.9 MHz, CDCl3): d = 172.2
(2ꢆRCH2CH2CO), 165.0 (BnOC(O)N), 139.7–128.4 (Ph), 104.2 (C-1c),
101.1 (C-1b), 96.4 (C-1a), 84.6 (C-4c), 81.9 (C-2c), 80.5 (C-2a), 80.0–65.3
(CH2Ph, Cbz, C-4a, C-1Gro, C-2Gro, C-3Gro, 6ꢆCH2Ph, C-3a, C-5a, C-6a, C-
5b, C-3b, C-4c, C-5c), 61.9 (C-6c), 60.6 (C-2b), 56.3 (C-4b), 32.9–15.1 ppm
(C-6b, CH2-chain); MALDI-MS (positive mode, matrix DHB, THF):
m/z: 1287.8; found: 1287.8 [M+Na]+, 1303.9 [M+K]+; elemental analysis
calcd (%) for C74H108N2O15 (1265.65 gmolꢀ1): C 70.22, H 8.60, N 2.21;
found: C 70.28, H 8.65, N 2.32.
(3 mL) and cooled to ꢀ408C where TMSOTf (5 mL in propionitrile) was
added slowly. TLC (petroleum ether/EtOAc 2:1) showed full conversion
of the acceptor after 5 min and after additional 15 min the reaction was
quenched by addition of Et3N. The reaction mixture was concentrated in
vacuo on silica gel and purified by column chromatography (petroleum
ether/EtOAc 7:1 to 5:1) to give 47b (295 mg, 83%, a/b 1:4.5). [a]D
=
+39.7 (c =1, CHCl3); 1H NMR (400 MHz, CDCl3): d =7.36–7.05 (m,
35H), 5.83 (m, 1H, All), 5.22 (dd, 1H, J=1.5, 17.2 Hz, All), 5.15–4.94
(m, 4H, 2Gro-H, CH2, All), 4.90–4.35 (m, 15H, CH2, 2ꢆNH), 4.74 (d, J=
5 Hz, 1a-H), 4.60 (d, 1H, J
ꢂ
8.1 Hz,1b-H), 4.30 (dd, 1H, J=3.7,
3-O-[(4-Benzyloxycarbonylamino-2,4,6-trideoxy-2-(2,2,2-trichloro-ethoxy-
carbonylamino)-b-d-galactopyranosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-glu-
copyranosyl)]-1,2-di-O-myristoyl-sn-glycerol (46b): Disaccharide 45a was
dissolved in TMF/MeOH (2:1, 60 mL) and degassed with argon (g) for
11.9 Hz, 1Gro-H), 4.24 (d, 1H, J=10.4 Hz, CH2), 4.18 (d, 1H, J=7.5 Hz,
1c-H), 4.10–3.90 (m, 5H, 4b-H, 1Gro-H, CH2), 3.70–3.20 (m, 16H, 2a-H,
4a-H, 5a-H, 6’a-H, 6’’a-H, 2b-H, 3b-H, 5b-H, 2c-H, 3c-H, 4c-H, 5c-H, 6’c-
H, 6’’c-H, 2’Gro-H, 2’’Gro-H), 2.17 (m, 4H, COCH2R), 1.48 (m, 4H,
COCH2CH2R), 1.16 (m, 40H, CH2-chain), 1.12 (d, 3H, J=6.3 Hz, 6b-H),
0.81 ppm (t, 6H, 2ꢆCH3); 13C NMR (101 MHz, CDCl3): d = 173.3 (C=O
acyl), 173.0 (C=O acyl), 156.6 (C=O Z), 154.5 (C=O Troc), 139.0 (Ar),
138.7 (Ar), 138.4 (Ar), 138.2 (Ar), 138.1 (Ar), 137.0 (Ar), 136.6 (Ar),
135.0 (All), 128.9–127.3 (35 C, Ar), 116.6 (All), 103.9 (JCꢀH =163 Hz, C-
1c), 101.9 (JCꢀH =165 Hz, C-1b), 96.7 (JCꢀH =172 Hz, C-1a), 95.6 (Cl3C),
84.5 (C-4c), 82.4 (C-2a), 79.4 (C-3a), 78.1, 77.3, 75.5, 75.2, 75.1 (2 C),
74.4, 74.2, 73.6, 72.4, 71.9, 70.2, 69.8, 68.3, 66.7 (CH2 Z, CH2-Troc, C-4a,
6ꢆBn, C-5a, C-6a, C-5b, C-3b, C-5c, C-6c), 69.5 (C-2Gro), 66.4 (C-3Gro),
62.5 (C-1Gro), 55.4 (C-4b), 55.0 (C-2b), 34.3–22.7 (CH2-chain), 16.6 (C-
6b), 14.2 ppm (2ꢆCH3); HRMS: m/z: 1893.8807; found: 1893.8959
[M+Na]+.
15 min. [PdACHTUNGTRENNUNG(Ph3)4] (74 mg, 0.1 equiv) and sodium p-toluenesulfinate
(114 mg, 1 equiv) were added at 08C. TLC (petroleum ether/EtOAc 3:1)
showed full conversion after 15 min and the reaction was concentrated in
vacuo on silica gel followed by flash chromatography (petroleum ether/
EtOAc 1:3 to 1:1) to give 46b (530 mg, 59%). [a]D = +34.1 (c = 1,
CHCl3); 1H NMR (400 MHz, CDCl3): d =7.45–7.10 (m, 20H, Ar), 5.16
(m, 2H, CH Z, 2Gro-H), 5.08 (m, 2H, CH Z, NH), 4.91 (m, 3H, NH, 2ꢆ
CH2), 4.83 (d, 1H, J=3.4 Hz, 1a-H), 4.72–4.56 (m, 4H, 2ꢆCH2, CH2),
4.59 (d, 1H, J=8.4 Hz, 1b-H), 4.46 (d, 1H, J=12 Hz, CH2), 4.37 (d, 1H,
J=10.7 Hz, CH2), 4.35 (dd, 1H, J=3.5, 12 Hz, 1Gro-H), 4.13 (dd, 1H, J=
6, 12 Hz, 1Gro-H), 4.08 (t, 1H, J=9.2 Hz, 3a-H), 3.95 (brd, 1H, J=9 Hz,
4b-H), 3.72–3.62 (m, 4H, 5a-H, 5b-H, 6’a-H, 3Gro-H), 3.62–3.54 (m, 3H,
2a-H, 4a-H, 6’’a-H), 3.50–3.35 (m, 3H, 2b-H, 3b-H, 3Gro-H), 3.29 (brs,
1H, OH), 2.23 (m, 4H, COCH2R), 1.55 (m, 4H, COCH2CH2R), 1.24 (m,
40H, CH2-chain), 1.13 (d, 3H, J=6.3 Hz, 6b-H), 0.88 ppm (t, 6H, 2ꢆ
CH3); 13C NMR (101 MHz, CDCl3): d = 173.5 (C=O acyl), 173.1 (C=O
acyl), 157.9 (C=O, Z), 155.9 (C=O, Troc), 138.6 (Ar), 137.9 (Ar), 137.7
(Ar), 136.2 (Ar), 129.0–127.7 (20 C, Ar), 102.2 (C-1b), 96.6 (C-1a), 95.5
(Cl3C-), 81.1 (C-2a), 80.3 (C-3a), 75.6 (C-4a), 75.1 (CH2), 75.0 (CH2), 73.7
(CH2), 73.6 (C-4a), 72.1 (CH2), 70.5, 69.9, 69.7 (C-2Gro), C-5a, C-5b), 68.3
(C-6a), 67.5 (CH2, 66.5 (C-1Gro), 62.6 (C-3Gro), 56.7 (C-2b), 55.2 (C-4b),
34.4, 34.2, 32.1, 29.8, 29.8, 29.6, 29.5, 29.4, 29.3, 29.2, 25.0, 22.8 (CH2-
chain), 16.7 (C-6b), 14.3 ppm (2ꢆCH3); HRMS: m/z: 1421.6555; found:
1421.6593 [M+Na]+.
3-O-[(6-O-tert-Butyldiphenylsilyl-2,3,4-tri-O-benzyl-b-d-glucopyranosyl)-
(1–3)-(ꢀ2-acetamino-4-benzyloxycarboylamino-2,4–6-trideoxy-b-d-galac-
topyranosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myr-
istoyl-sn-glycerol (48a): Compound 47a (0.4 g, 0.208 mmol) was dissolved
in AcSH (3 mL, distilled until colourless) and the reaction mixture was
stirred for 16 h at 408C. The solvent was evaporated in vacuo and puri-
fied by flash chromatography (toluene/EtOAc 3:1) to give 48a (0.29 g,
72%). TLC (toluene/EtOAc 3:1): Rf =0.19; [a]D =ꢀ9.3 (c = 1, CHCl3);
1H NMR (600 MHz, CDCl3): d =7.40 (m, 45H, Ph), 5.05 (m, 5H, 1b-H,
4b-NH, 2b-NH, CHPh-Cbz, 2Gro-H), 5.00–4.48 (m, 14H, 6ꢆCH2Ph, 1c-H,
1a-H), 4.15–4.40 (m, 5H, 1Gro-H, 4b-H, 3a-H, 3b-H, 2b-H), 4.00–3.43 (m,
13H, 3Gro-H, 5b-H, 6a-H, 4a-H, 2a-H, 5a-H, 1’Gro-H, 2c-H, 3c-H, 4c-H,
6c-H), 3.4 (m, 1H, 2c-H), 2.25 (m, 4H, COCH2R), 1.55 (m, 7H,
3-O-[(6-O-tert-Butyldiphenylsilyl-2,3,4-tri-O-benzyl-b-d-glucopyranosyl)-
(1–3)-(2-azido-4-benzyloxycarboylamino-2,4,6-trideoxy-b-d-galactopyra-
nosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-
sn-glycerol (47a): To a solution of acceptor 46a (0.38 g, 0.304 mmol) and
donor 4a (0.38 g, 1.5 equiv) in acetonitrile (15 mL) cooled to ꢀ408C.
TMSOTf (8.2 mL, 0.1 equiv) was added and the reaction mixture stirred
for 30 min, then Et3N was added to neutralize the reaction. The solvent
was evaporated in vacuo. Flash chromatography (petroleum ether/EtOAc
10:1) yielded 47a (0.46 g, 79%). TLC (petroleum ether/EtOAc 10:1):
Rf =0.19; [a]D = +12.3 (c = 1, CHCl3); 1H NMR (600 MHz, CDCl3): d
=7.40 (m, 45H, Ph), 5.05 (m, 4H, 1b-H, 4b-NH, CHPh-Cbz, 2Gro-H),
5.00–4.48 (m, 14H, 6ꢆCH2Ph, 1c-H, 1a-H), 4.15 (dd, 1H, J=9.0, 7.0 Hz,
COCH2CH2R, CH3C(O)N), 1.25 (m, 49H, CH2-chain,
CACHTUNGTRENNUNG(CH3)3),
0.90 ppm (m, 9H, 2ꢆCH3, 6b-H); 13C NMR (150.3 MHz, CDCl3): d =
172.2 (2ꢆRCH2CH2CO), 165 (BnOC(O)N), 157.0 (CH3CO), 139.7–128.4
(Ph), 103.9 (C-1c), 102.8 (C-1b), 96.4 (C-1a), 84.6 (C-4c), 81.9 (C-2c),
80.5 (C-2a), 80.0–65.3 (CH2Ph-Cbz, C-4a, C-1Gro, C-2Gro, C-3Gro, 6ꢆ
CH2Ph, C-3a, C-5a, C-6a, C-5b, C-3b, C-3c, C-5c), 61.9 (C-6c), 58.3 (C-
2b), 55.4 (C-4b), 32.9–15.1 ppm (C-6b, CH3C(O)N, CH2-chain, 2ꢆCH3, C-
AHCTUNGTRENNG(NU CH3)3); MALDI-MS (positive mode, matrix DHB, THF): m/z: 1958.1;
found: 1958.2 [M+Na]+, 1974.1 [M+K]+; elemental analysis calcd (%)
for C117H154N2O20Si (1936.56 gmolꢀ1): C 72.56, H 8.02, N 1.45; found: C
72.53, H 8.05, N 1.62.
1
Gro-H), 4.00–3.58 (m, 11H, 6a-H, 4a-H, 2a-H, 5a-H, 1’Gro-H, 2c-H, 3c-H,
3-O-[(6-O-Allyl-2,3,4-tri-O-benzyl-b-d-glucopyranosyl)-(1–3)-(2-acetyl-
amino-4-benzyloxycarbonylamino-2,4,6-trideoxy-b-d-galactopyranosyl)-
(1–3)-(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-sn-glyc-
erol (48b): Trisaccharide 47b (108 mg, 57 mmol) was dissolved in Ac2O
(2 mL) containing Et3N (10 mL, 2 equiv). Freshly activated Zn (100 mg)
was added and the reaction mixture stirred for 14 h at room temperature
followed by filtration through celite, concentration in vacuo on silica gel
and purification by flash chromatography (petroleum ether/EtOAc 3:1 to
1:1) to give 48b (86 mg, 86%). The reaction could be carried out in
45 min by using ultra sonic bath conditions. This however causes a slight
4c-H, 6c-H), 3.55–3.43 (m, 3H, 5b-H, 3Gro-H), 3.4 (m, 2H, 2c-H, 2b-H),
2.25 (m, 4H, COCH2R), 1.55 (m, 4H, COCH2CH2R), 1.25 (m, 49H,
CH2-chain,
CACHTUNGTRENNUNG
(CH3)3), 0.90 ppm (m, 9H, 2ꢆCH3, 6b-H); 13C NMR
(150.9 MHz, CDCl3): d = 172.2 (2ꢆRCH2CH2CO), 165 (BnOC(O)N),
139.66–128.44 (Ph), 104.2 (C-1c), 101.1 (C-1b), 96.4 (C-1a), 84.6 (C-4c),
81.9 (C-2c), 80.5 (C-2a), 80.0–65.3 (CH2Ph-Cbz, C-4a, C-1Gro, C-2Gro, C-
3
Gro, 6ꢆCH2Ph, C-3a, C-5a, C-6a, C-5b, C-3b, C-3c, C-5c), 61.9 (C-6c),
60.6 (C-2b), 50.4 (C-4b), 32.9–15.1 ppm (C-6b, CH2-chain, 2ꢆCH3, C-
ACHTUNGTRENNUNG(CH3)3); MALDI-MS (positive mode, matrix DHB, THF): m/z: 1942.1;
found: 1942.3 [M+Na]+, 1958.1 [M+K]+; elemental analysis calcd (%)
for C115H150N4O19Si (1920.52 gmolꢀ1): C 71.92, H 7.87, N 2.92; found: C
72.13, H 8.05, N 3.02.
decrease in yield. [a]D
= +38 (c =
1, CHCl3); 1H NMR (400 MHz,
CDCl3): d =7.36–7.05 (m, 35H), 5.83 (m, 1H, All), 5.22 (d, 1H, J=
17.2 Hz, All), 5.15–4.94 (m, 4H, 2Gro-H, CH2, All), 4.90–4.35 (m, 13H,
CH2, 2ꢆNH), 4.82 (d, 1H, J=9.1 Hz, 1b-H), 4.69 (d, J ꢂ 5 Hz, 1a-H),
4.28 (m, 1H, 1Gro-H), 4.24 (d, 1H, J=10.4 Hz, CH2), 4.24 (d, 1H, J ꢂ
8.7 Hz, 1c-H), 4.10–3.90 (m, 5H, 3a-H, 4b-H, 1Gro-H, 3b-H, CH2), 3.70–
3.20 (m, 15H, 2a-H, 4a-H, 5a-H, 6’a-H, 6’’a-H, 2b-H, 5b-H, 2c-H, 3c-H,
4c-H, 5c-H, 6’c-H, 6’’c-H, 3’Gro-H, 3’’Gro-H), 2.18 (m, 4H, COCH2R), 1.53
(s, 3H, NAc), 1.50 (m, 4H, COCH2CH2R), 1.17 (m, 40H, CH2-chain),
3-O-[(6-O-Allyl-2,3,4-tri-O-benzyl-b-d-glucopyranosyl)-(1–3)-(4-benzyl-
oxycarbonylamino-2,4,6-trideoxy-2-(2,2,2-trichloroethoxycarbonyl-
amino)-b-d-galactopyranosyl)-(1–3)-2,4,6-tri-O-benzyl-a-d-glucopyrano-
syl)-1,2-di-O-myristoyl-sn-glycerol (47b): Donor 4 (300 mg, 0.473 mmol)
and acceptor 46b (261 mg, 0.186 mmol) were coevaporated with toluene
and dried under vacuum. The mixture was dissolved in propionitrile
Chem. Eur. J. 2010, 16, 12627 – 12641
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12639

R. R. Schmidt et al.
1.04 (brd, 3H, J=6 Hz, 6b-H), 0.80 ppm (t, 6H, 2ꢆCH3); 13C NMR
(101 MHz, CDCl3): d = 173.4 (C=O, aryl), 170.6 (NAc), 156.7 (C=O, Z),
138.9 (Ar), 138.9 (Ar), 138.6 (Ar), 138.3 (Ar), 138.1 (Ar), 137.9 (Ar),
3-O-{[2,3,4-Tri-O-benzyl-6-O-(2-cyanoethoxydiisopropylaminophosphin-
yl)-b-d-glucopyranosyl]-(1–3)-(2-acetamino-4-benzyloxycarbonylamino-
2,4,6-trideoxy-b-d-galactopyranosyl)-(1–3)-(2,4,6-tri-O-benzyl-a-d-gluco-
pyranosyl)}-1,2-di-O-myristoyl-sn-glycerol (1c): Trisaccharide 1b (65 mg,
38 mmol) was dissolved in CH2Cl2/MeCN (1:1, 4 mL) together with 2-cya-
noetyl-N,N,N’,N’-tetraisopropylphosphordiamidite (50 mL) and diisopro-
pylammonium tetrazolide (10 mg, 1.7 equiv). After 3.5 h at room temper-
ature the reaction was finished (TLC: EtOAc/toluene 1:2) and the mix-
ture concentrated in vacuo on silica gel and purified by flash chromatog-
raphy (toluene/EtOAc 1:0 to 3:1 + 1% Et3N) to give the phosphorami-
dite 1c (72 mg, 86%) as a mixture of two diastereomers. 1H NMR
(600 MHz, CDCl3): d = 7.35–7.05 (m, 35H, Ar), 5.64 (d, 0.25H, J=
8.8 Hz, NH), 5.43 (d, 0.25H, J=8.8 Hz, NH), 5.11 (m, 1H, 2Gro-H), 5.10–
4.98 (m, 1.8H, CH2 Z, NH, Bn), 4.98–4.88 (m, 1.7H, 1b-H, Bn), 4.86–4.75
(m, 2.6H, Bn), 4.74–4.60 (m, 2.6H, 1a-H, Bn), 4.60–4.49 (m, 3H, Bn),
4.48–4.37 (m, 3.25H, 1c-H, Bn), 4.34–4.28 (m, 2.4H), 4.13–3.90 (m, 4H,
4b-H, 1Gro-H, 3a-H, CH2CH2CN), 3.79 (m, 1H, 3Gro-H), 3.72–3.24 (m,
16H, 2a-H, 4a-H, 5a-H, 6’a-H, 6a-H, 2b-H, 5b-H, 2c-H, 3c-H, 4c-H, 5c-H,
136.7 (Ar), 135.1 (All), 129.0–127.5 (35 C, Ar), 116.7 (All), 104.1 (JCꢀH
=
165 Hz, C-1c), 101.6 (JCꢀH =166 Hz, C-1b), 97.0 (JCꢀH =170 Hz, C-1a),
84.7 (C-4c), 82.3 (C-2c), 80.8 (C-2a), 79.1 (C-3a), 78.2, 77.4, 76.3, 76.0,
75.5, 75.2, 75.1, 74.9, 74.5, 73.7, 72.6, 70.4, 69.9, 68.3, 66.7 (CH2 Z, C-4a,
6ꢆBn, C-5a, C-6a, C-5b, C-3b, C-3c, C-5c, C-6c), 69.6 (C-2Gro), 66.4 (C-
3Gro), 62.5 (C-1Gro), 55.3 (C-4b), 54.6 (C-2b), 34.33–23.5 (CH2-chain), 22.8
(NAc), 16.8 (C-6b), 14.2 ppm (2ꢆCH3); HRMS: m/z: 1776.9665; found:
1777.0540 [M+Na]+.
3-O-[(2,3,4-Tri-O-benzyl-b-d-glucopyranosyl)-(1–3)-(2-acetylamino-4-
benzyloxycarbonylamino-2,4,6-trideoxy-b-d-galactopyranosyl)-(1–3)-
(2,4,6-tri-O-benzyl-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-sn-glycerol
(1b): a) From 48a: To a solution of 48a (0.27 g, 0.139 mmol) in THF
(5 mL) AcOH (9.5 mL, 1.2 equiv) and TBAF (0.167 mL, 1.2 equiv) were
added and the reaction mixture stirred for 2 d. The mixture was diluted
with CH2Cl2 and washed with water, the aqueous phase was then extract-
ed twice with CH2Cl2, the unified organic phase was dried over sodium
sulfate and the solvent evaporated in vacuo. Flash chromatography (tolu-
ene/EtOAc 1:1) yielded 1b (0.186 g, 79%).
6’c-H, 6c-H, 3’Gro-H, 2ꢆCH
4H, COCH2R), 1.70, 1.65–1.46 (m, 7H, COCH2CH2R, NAc), 1.19 (m,
40H, CH2 chain), 1.17 (4ꢆs, 12H, CH(CH3)2), 1.06 (m, 3H, 6b-H),
149.7,
ACTHUNGTREN(NUNG CH3)2), 2.50 (m, 2H, CH2CH2CN), 2.19 (m,
AHCTUNGTRENNUNG
0.82 ppm (t, 6H, 2ꢆCH3); 31P NMR (162 MHz, CDCl3): d
=
146.8 ppm; HRMS: m/z: 1932.0; found: 1932.6 [M+Cl]ꢀ; HRMS: m/z:
1999.4; found: 1999.2 [M+(Me2CH)2NH2]+.
b) From 48b: Trisaccharide 48b (120 mg, 69 mmol) was dissolved in
MeOH/CH2Cl2 (1:1, 3 mL) and PdCl2 (3 mg, 0.3 equiv) was added at 08C.
After 2 h additional catalyst (10 mg) was added and the reaction mixture
was left for further 2 h. Then it was concentrated in vacuo on silica gel
and purified by flash chromatography (petroleum ether/EtOAc 2:1) to
Transient transfected HEK293 cells with human TLR2: Transfection and
culture of HEK293 cells were performed as described.[48] In short
HEK293 cells were transiently transfected with either TLR2 or TLR4/
MD2/CD14 using Polyfect (Quiagen, Hilden, Germany). After 6 h of
transfection, cells were washed and stimulated for further 24 h with the
ligands. IL-8 content in the culture supernatants were quantified using a
commercial ELISA (Invitrogen GmbH, Karlsruhe, Germany).
give 1b (93 mg, 79%). [a]D
= +31.5 (c =
1, CHCl3); 1H NMR
(400 MHz, CDCl3): d =7.32–7.08 (m, 35H, Ar), 5.18 (d, 1H, J=8.1 Hz,
NH), 5.11 (m, 1H, 2Gro-H), 5.10 (d, 1H, J ꢂ 12 Hz, Bn Z), 5.02 (d, 1H, J
ꢂ 12 Hz, Bn Z), 5.00 (d, J=10.8 Hz, NH), 4.88 (d, 1H, J=10.7 Hz, Bn),
4.83 (d, 1H, J=8.2 Hz, 1b-H), 4.79–4.61 (m, 5H, Bn), 4.73 (d, 1H, J=
3.6 Hz, 1a-H), 4.56–4.46 (m, 4H, Bn), 4.43 (d, J=11.7 Hz, Bn), 4.38 (d,
1H, J=12 Hz, Bn), 4.37 (d, 1H, J=7.6 Hz, 1c-H), 4.30 (d, 1H, J=
10.2 Hz, Bn), 4.28 (dd, 1H, J=3.7, 11.9 Hz, 1Gro-H), 4.22 (bdd, 1H, J=
4.3, 10.8 Hz (JNꢀH), 4b-H), 4.16 (dd, 1H, J=5.7, 8.9 Hz, OH), 4.10–3.93
(m, 1H, 1Gro-H, 3a-H, 3b-H), 3.75 (m, 1H, 3Gro-H), 3.67–3.16 (m, 14H,
2a-H, 4a-H, 5a-H, 6’aH, 6’’a-H, 2b-H, 5b-H, 2c-H, 3c-H, 4c-H, 5c-H, 6’c-
H, 6’’c-H, 3’Gro-H), 2.17 (m, 4H, COCH2R), 1.56 (s, 3H, NAc), 1.49 (m,
4H, COCH2CH2R), 1.17 (m, 40H, CH2-chain), 0.99 (d, 3H, J=6.1 Hz,
Human mononuclear cells: Human mononuclear cells (MNC) were iso-
lated from peripheral blood of healthy donors and stimulated as de-
scribed.[49] Briefly, 100 mL of RPMI-1640 medium containing antibiotic,
10% heat-inactivated human serum, and the stimulus were added to 96-
well U-form cell-culture microplates and then 1.5ꢆ104 MNC in 50 mL
RPMI-1640 culture medium was added. After incubation for 16 h the
cell-free supernatants were harvested and the IL-8 concentration was
quantified using a commercial ELISA (Invitrogen GmbH, Karlsruhe,
Germany).
6b-H), 0.80 ppm (t, 6H, 2ꢆCH3); 13C NMR (101 MHz, CDCl3): d
=
173.4 (C=O, acyl), 171.0 (C=O, NAc), 157.5 (C=O, Z), 139.0 (Ar), 138.8
(Ar), 138.5 (Ar), 138.2 (Ar), 137.9 (Ar), 137.9 (Ar), 136.8 (Ar), 129.0–
127.3 (35 C, Ar), 105.0 (JCꢀH =162 Hz, C-1c), 101.5 (JCꢀH =161 Hz, C-1b),
97.0 (JCꢀH =171 Hz, C-1a), 84.4 (C-4c), 82.2 (C-2c), 80.6 (C-2a), 79.2 (C-
3a), 78.1, 77.4, 76.6, 75.7, 75.4, 75.3, 74.8, 74.4, 73.7, 72.8, 70.5, 70.2, 68.3,
66.1, 66.5 (CH2 Z, C-4a, 6ꢆBn, C-5a, C-6a, C-5b, C-3b, C-3c, C-5c, C-6c),
69.9 (C-1Gro), 62.4 (C-1Gro), 61.4 (C-3Gro), 55.6 (C-4b), 55.0 (C-2b), 34.4,
32.1, 29.8, 29.8, 29.7, 29.5, 29.4, 29.3, 29.2, 25.0, 22.8 ppm (CH2-chain),
23.4 (NAc), 16.6 (C-6b), 14.3 (2ꢆCH3); HRMS: m/z: 1720.9612; found:
1720.9566 [M+Na]+.
Acknowledgements
The work was supported by the Deutsche Forschungsgemeinschaft and
the Fonds der Chemischen Industrie. C.M.P. is particularly grateful for a
fellowship from the Danish Agency for Science, Technology and Innova-
tion. We would like to thank B. Wegner and N. Gisch for preparing and
analyzing HIC purified 1a, as well as B. Kunz and H. Kꢀßner for techni-
cal assistance running the MS and NMR experiments, respectively.
3-O-[(b-d-Glucopyranosyl)-(1–3)-(2-acetylamino-4-amino-2,4,6-trideoxy-
b-d-galactopyranosyl)-(1–3)-a-d-glucopyranosyl)]-1,2-di-O-myristoyl-sn-
glycerol (1a): Protected trisaccharide 1b (15 mg, 8.8 mmol) was dissolved
in a mixture of CH2Cl2/MeOH/H2O (5:5:1) and Pearlmanꢄs catalyst
[Pd(OH)2] was added together with H2 (1 atm). The reaction was fol-
lowed by MS (MALDI-TOF) and due to incomplete reaction after 15 h
the Pd(OH)2 was replaced by fresh catalyst and the solvent changed to
THF/H2O (2:1). After additional 20 h under H2 pressure (40 atm) only
the product and some monobenzylated products were observed in the
MS. The catalyst was again removed by filtration and the procedure re-
peated. When no further reaction took place the reaction mixture was fil-
tered and concentrated to give the crude product which was purified by
HIC chromatography (gradient from 15 to 60% n-propanol) to give
1.9 mg of deprotected trisaccharide 1a containing very minor monoben-
zylated side products. Further purification was done by preparative TLC
(CHCl3/MeOH 95:5). For NMR data see Table 1. HRMS (C51H94N2O18,
positive mode): m/z: 1023.6579; found: 1023.654 [M+H]+, 1045.635
[M+Na]+.
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Published online: September 28, 2010
Chem. Eur. J. 2010, 16, 12627 – 12641
ꢂ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
12641