Total synthesis of α-linked rha-rha-gal undecaprenyl diphosphate Found in Geobacillus stearothermophilus

Article abstract of DOI:10.1002/ejoc.201101021

A (2+1) glycosylation approach was employed to construct the core structure of a trisaccharide found in the soil bacterium Geobacillus stearothermophilus. The phospholipid was assembled by the reaction of a peracetylated trisaccharide bearing an anomeric

Full text of DOI:10.1002/ejoc.201101021

FULL PAPER
DOI: 10.1002/ejoc.201101021
Total Synthesis of α-Linked Rha–Rha–Gal Undecaprenyl Diphosphate Found
in Geobacillus stearothermophilus
Annika Holkenbrink,[a] Dennis C. Koester,[a] Johannes Kaschel,[a] and Daniel B. Werz*[a]
Keywords: Total synthesis / Protecting groups / Carbohydrates / Glycolipids / Phospholipids / Geobacillus stearothermophilus
A (2+1) glycosylation approach was employed to construct
the core structure of a trisaccharide found in the soil bacte-
rium Geobacillus stearothermophilus. The phospholipid was
assembled by the reaction of a peracetylated trisaccharide
bearing an anomeric hydroxy group with phosphoramidite
and subsequent oxidation to the phosphate. The final cou-
pling step with undecaprenyl monophosphate followed by
global acetate deprotection completed the total synthesis of
the highly amphiphilic target molecule.
our studies on the chemical synthesis of the trisaccharidic
undecaprenyl diphosphate 1 consisting of one d-galactose
(Gal) and two l-rhamnose (Rha) units. Compound 1 is as-
sumed to be a key intermediate in the construction of the
S-layer of Geobacillus stearothermophilus.[3]
Introduction
Carbohydrates have attracted much attention especially
because of their appearance on the surface of membranes
and bacterial cell walls. In the recent past many glycoconju-
gates have been used for vaccination and bacterial detec-
tion. Glycoconjugates – especially glycolipids and glycopro-
teins – play a major role in cell–cell communication in eu-
karyotes and prokaryotes. Glycosylation is the most com-
mon posttranslational modification of proteins although it
was considered to be restricted to the eukaryotic kingdom
for decades.[1] Today it is known that O- and N-linked gly-
coproteins are also widely found in prokaryotes. Prominent
examples are surface-layer (S-layer) glycoproteins of Bacil-
laceae, which are able to self-assemble into 2D arrays. The
assembly of the glycan donors is assumed to take place on
the polyisoprenyl carrier undecaprenyl diphosphate and is
mediated by a variety of glycosyltransferases.[2] Several en-
zymes for the S-layer glycoprotein glycan biosynthesis of
the Gram-positive soil bacterium Geobacillus stearothermo-
philus have been identified.[3] However, for an in depth
mode of action study of these glycosyltransferases, undeca-
prenyl-linked glycan substrates are required. A more de-
tailed understanding of these processes is the basis for engi-
neering tailormade glycan motifs on S-layer proteins, which
are applied in microbiology, nanobiotechnology, and vacci-
nology.[4,5] Due to the tiny amounts isolated from natural
sources, isolation and subsequent purification is hardly pos-
sible. To access usable quantities of the desired compound,
organic synthesis is the method of choice. Herein, we report
Results and Discussion
Our retrosynthetic analysis of 1 dissects the target mole-
cule into a disaccharide 4, a galactose building block 5, a
phosphoramidite 6, and an undecaprenyl monophosphate 3
(Scheme 1). Only acetate and benzyl groups are present as
permanent protecting groups in these carbohydrate building
blocks.
As the global final deprotection step, only a deacetyl-
ation under basic conditions was considered because a final
hydrogenation would destroy the π systems of the polyiso-
prenyl moiety. Therefore, all benzyl groups have to be re-
moved and replaced by acetate groups before the undeca-
prenyl unit is introduced.
For the construction of the diphosphate unit, we envi-
sioned the use of phosphoramidite chemistry. Such a PIII
species is known for the construction of phosphate linkages.
For this purpose, 6 is a well-known reagent[6] and it has
shown to be superior to the use of PV reagents such as tet-
rabenzyl diphosphate,[7] which led to only traces of the de-
sired monophosphate. The last building block in our retro-
synthetic analysis is undecaprenyl monophosphate 3.
A commonly applied synthesis of 3 starts with undeca-
prenol,[8] but as 3 is commercially available, we decided to
use it readily for the phosphate coupling step.
[a] Institut für Organische und Biomolekulare Chemie, Georg-
August-Universität Göttingen,
The assembly of the trisaccharide moiety commenced
with the creation of 4 (Scheme 2). The glycosylation of per-
acetylated rhamnosyl trichloroacetimidate donor 7[9] with
rhamnose acceptor 8,[10] bearing a p-methoxyphenyl (MP)
Tammannstr. 2, 37077 Göttingen, Germany
Fax: +49-551-399476
E-mail: dwerz@gwdg.de
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201101021.
Eur. J. Org. Chem. 2011, 6233–6239
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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A. Holkenbrink, D. C. Koester, J. Kaschel, D. B. Werz
FULL PAPER
Scheme 2. Synthesis of 4.
anomeric methoxyphenyl group and subsequently treated
with trichloroacetonitrile to obtain trichloroacetimidate do-
nor 4 in 77% yield over two steps.
To access 5 we made use of an epoxide opening sequence
of galactal 11, which was synthesized by fluorenylmethy-
loxycarbonyl (Fmoc) protection of galactal 10.[11] We ap-
plied dimethyldioxirane (DMDO) as the epoxidizing agent,
which is known to epoxidize glycal double bonds stereose-
Scheme 1. Retrosynthetic analysis of 1.
moiety as anomeric protecting group, afforded disaccharide lectively.[12] As we envisioned, the enolether double bond in
9 in 77% yield with perfect α selectivity. Disaccharide 9 was 11 was epoxidized diastereoselectively; a subsequent epox-
subjected to CAN in acetonitrile for deprotection of the ide opening afforded benzylic alcohol 12 in a total yield of
Scheme 3. Synthesis of 16.
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Synthesis of α-Linked Rha–Rha–Gal Undecaprenyl Diphosphate
84% in favor of the β diastereomer (3:1). Both α and β cleaved under slightly basic conditions. Therefore, we per-
diastereomers were valuable in the next steps and were eas- formed a subsequent peracetylation giving rise to the acet-
ily separated by column chromatography (Scheme 3). The ate-protected trisaccharide 15 as an anomeric mixture (1:1).
anomeric benzyl protecting group of the reducing monosac- The removal of the anomeric acetate group, which led to
charide unit was introduced as a late stage protecting group, trisaccharide 16 (α/β = 1:1), was achieved with piperidine
which could be easily removed by hydrogenolysis. Acetate in tetrahydrofuran (THF, Scheme 3).[15]
protection of the 2-hydroxy group leading to 13, and the
Compound 16 was finally subjected to coupling with
removal of the Fmoc group[13] with 20% piperidine in N,N- phosphoramidite iPr2NP(OBn)2 with tetrazole as the acti-
dimethylformamide (DMF) yielded 5, which bears a free vating agent. The resulting PIII species was oxidized by
3-hydroxy group, in 92% yield. Due to the basic conditions tBuOOH to the corresponding monophosphate 17 in 22%
used for the removal of Fmoc, traces of an acetate-migrated yield (over two steps) with the α-anomer as the major
product were observed; however, this undesired product stereoisomer (Scheme 4).[6] We observed large amounts of
was easily removed by column chromatography.
byproducts with other oxidizing agents. The use of mCPBA
Compounds 4 and 5 were coupled in a trimethylsilyl tri- resulted in a complex mixture of products and the forma-
fluoromethanesulfonate (TMSOTf)-catalyzed (2+1) glycos- tion of several sideproducts. However, with tBuOOH the
ylation, which proceeded smoothly to give 14 in 74% 31P NMR spectrum showed complete conversion of PIII to
yield.[14]
Hydrogenolysis with Pearlman’s catalyst removed all
PV.
The undecaprenyl chain was introduced after global
benzyl protecting groups. Acetate was chosen as the final benzyl deprotection to afford 2 by hydrogenolysis, which
stage protection group because it is known to be easily used Pd/C in methanol. The final coupling of undecaprenyl
Scheme 4. Final steps in the synthesis of 1.
Eur. J. Org. Chem. 2011, 6233–6239
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A. Holkenbrink, D. C. Koester, J. Kaschel, D. B. Werz
FULL PAPER
purified by column chromatography on silica gel (pentane/EtOAc
= 5:1) to afford 9 (829 mg, 77%). H NMR (300 MHz, CDCl3): δ
monophosphate 3 with the trisaccharidic monophosphate
17 was effected by the activation of 1,1Ј-carbonyldiimid-
azole (CDI) and tin(II) chloride,[16] which furnished the per-
acetylated diphosphate 18 in 28% yield. Tin(II) chloride
was necessary as a coordinating agent to promote the phos-
phate–phosphate coupling, otherwise it was impossible to
isolate any product. Furthermore, it was more effective to
activate 3 than 2. The activation of 2 with CDI gave much
lower yields. It is widely known that the yields in these cou-
1
= 1.20 (d, J = 6.3 Hz, 3 H, 6Ј-H3), 1.29 (d, J = 6.2 Hz, 3 H, 6-H3),
1.97 [s, 3 H, O(CO)CH3], 2.05 [s, 3 H, O(CO)CH3], 2.07 [s, 3 H,
O(CO)CH3], 2.22 [s, 3 H, O(CO)CH3], 3.55 (dd, J = 9.6, 9.6 Hz,
1 H, 4-H), 3.74 (s, 3 H, OCH3), 3.82–3.99 (m, 2 H, 5-H, 5Ј-H), 4.29
(dd, J = 9.6, 3.0 Hz, 1 H, 3-H), 4.66 (d, J = 10.8 Hz, 1 H, CH2Ph),
4.81 (d, J = 10.8 Hz, 1 H, CH2Ph), 5.06 (dd, J = 10.0, 5.1 Hz, 1 H,
4Ј-H), 5.08 (d, J = 1.8 Hz, 1 H, 1Ј-H), 5.26 (dd, J = 10.0, 3.4 Hz,
1 H, 3Ј-H), 5.29–5.32 (m, 2 H, 1-H, 2-H), 5.33 (dd, J = 3.4 H,
pling reactions – even in the case of monosaccharides – do 1.8 Hz, 1 H, 2Ј-H), 6.79 (d, J = 9.0 Hz, 2 H, PhH), 6.93 (d, J =
9.0 Hz, 2 H, PhH), 7.23–7.33 (m, 5 H, PhH) ppm. 13C NMR
(125 MHz, CDCl3): δ = 17.4 (C-6Ј), 18.0 (C-6), 20.8, 20.9, 20.9,
21.1 [O(CO)CH3], 55.7 (OCH3), 67.3, 68.5 (C-5, C-5Ј)*, 69.1 (C-
3Ј), 69.8 (C-2Ј), 70.7 (C-4Ј), 71.9 (C-2), 75.6 (CH2Ph), 76.6 (C-3),
80.5 (C-4), 96.2 (C-1), 99.4 (C-1Ј), 114.6, 117.6, 127.7, 127.8, 128.3
(CtertPh), 137.7, 149.8, 155.0 (CquatPh), 169.7, 169.9, 170.0, 170.4
often not exceed 40–50%.[6,7] Finally, global acetate depro-
tection under mild conditions using MeOH/H2O/Et3N
(7:3:1)[17] gave access to the desired highly amphiphilic tar-
get 1 in 64% yield. Common saponification conditions
using sodium methoxide in methanol resulted in the com-
plete destruction of 1.
[O(CO)CH3] ppm. [α]2D0 = –67.7 (c = 0.73, CHCl ). IR (film): ν =
˜
3
3063, 2638, 1972, 1609, 910 cm–1. HRMS (ESI): calcd. for
C34H42O14Na [M + Na]+ 697.24668; found 697.24692.
Conclusions
2,3,4-Tri-O-acetyl-α-
L-rhamnopyranosyl-(1Ǟ3)-2-O-acetyl-4-O-
benzyl-α- -rhamnopyranosyl Trichloroacetimidate (4): Disaccharide
L
We have reported a strategy for the construction of an α-
linked undecaprenyl diphosphate trisaccharide consisting of
l-Rha(α1Ǟ3)-l-Rha(α1Ǟ3)-d-Gal from the soil bacterium
Geobacillus stearothermophilus. For the crucial phosphoryl-
ation step, we made use of phosphoramidite chemistry fol-
lowed by subsequent oxidation of PIII to PV. Tin(II) chlo-
ride was a crucial additive as a coordinating agent for the
phosphates in the final coupling reaction. The target com-
pound 1 is an important chemical tool to study the bacte-
9 (828 mg, 1.22 mmol, 1.0 equiv.) was dissolved in acetonitrile/
water (72 mL, 1:1), and cerium ammonium nitrate (2.10 g,
3.38 mmol, 3.2 equiv.) was added. The mixture was stirred at r.t.
for 2 h, poured into brine, and extracted twice into ethyl acetate.
The combined organic layers were washed twice with water, dried
with Na2SO4, and concentrated by rotary evaporation. After purifi-
cation by column chromatography on silica gel (pentane/EtOAc =
2:1) the hemiacetal was obtained (605 mg, 87%). 1H NMR
(300 MHz, CDCl3): δ = 1.19 (d, J = 6.3 Hz, 3 H, 6Ј-H3), 1.31 (d,
rial S-layer glycoprotein glycan biosynthesis in Geobacillus J = 6.2 Hz, 3 H, 6-H3), 1.97 [s, 3 H, O(CO)CH3], 2.06 [s, 3 H,
O(CO)CH3], 2.07 [s, 3 H, O(CO)CH3], 2.21 [s, 3 H, O(CO)CH3],
2.75 (d, J = 3.7 Hz, 1 H, OH), 3.50 (dd, J = 9.7, 9.5 Hz, 1 H, 4-
H), 3.85–4.05 (m, 2 H, 5-H, 5Ј-H), 4.18 (dd, J = 9.7, 3.3 Hz, 1 H,
3-H), 4.65 (d, J = 11.0 Hz, 1 H, CH2Ph), 4.79 (d, J = 11.0 Hz, 1 H,
CH2Ph), 5.03 (d, J = 1.7 Hz, 1 H, 1Ј-H), 5.05 (dd, J = 10.2, 9.7 Hz,
1 H, 4Ј-H), 5.12 (d, J = 2.2 Hz, 1 H, 1-H), 5.18 (dd, J = 3.3, 2.2 Hz,
1 H, 2-H), 5.23 (dd, J = 10.2, 3.4 Hz, 1 H, 3Ј-H), 5.30–5.33 (dd, J
= 3.4, 1.7 Hz, 1 H, 2Ј-H), 7.25–7.33 (m, 5 H, PhH) ppm. 13C NMR
(125 MHz, CDCl3): δ = 17.3, 17.9 (C-6, C-6Ј), 20.7, 20.8, 20.8, 21.0
[O(CO)CH3], 67.2, 67.8 (C-5, C-5Ј)*, 69.1, 69.7, 70.7, 72.3, 75.5,
76.1 (C-2, C-2Ј, C-3, C-3Ј, C-4Ј)*, 80.7 (C-4), 91.9 (C-1), 99.3 (C-
1Ј), 127.7, 127.9, 128.4 (CtertPh), 137.8 (CquatPh), 169.9, 170.1,
stearothermophilus. Biological studies are underway and will
be reported in due course.
Experimental Section
General: All reactions were performed in flame-dried glassware un-
der an argon atmosphere. The solvents were dried by standard pro-
cedures and distilled before use. 1H, 13C, and 31P NMR spectra
were obtained with Varian Mercury 300 MHz, Varian Unity
300 MHz, Varian Inova 500 MHz, and Varian Inova 600 MHz
spectrometers using the solvent as an internal standard. Assign-
ments of the respective signals were made by the combination of
H,H-COSY, heteronuclear single quantum coherence, and HMBC
experiments. Unsecured assignments are labelled with an asterisk
*. HRMS (ESI) was carried out with a FTICR instrument
(Bruker). IR spectra were measured with a Bruker Vector 22 spec-
trometer and a JASCO FT/IR-4100 spectrometer with a Pike
GladiATR unit. Optical rotations were obtained with a polarimeter
model 241 from Perkin–Elmer. Concentrations are given in g/
100 mL.
170.1, 170.6 [O(CO)CH ] ppm. IR (KBr): ν = 3467, 3065, 1658,
˜
3
1534, 1498, 1182, 912, 811 cm–1. HRMS (ESI): calcd. for
C27H36O13Na [M + Na]+ 591.20481; found 591.20503.
The hemiacetal (322 mg, 0.57 mmol, 1.0 equiv.) was dissolved in
anhydrous dichloromethane (15 mL) and cooled to 0 °C. DBU
(13 μL, 0.09 mmol, 0.15 equiv.) and Cl3CCN (1.10 mL, 11.4 mmol,
20 equiv.) were added. The mixture was stirred at 0 °C for 30 min
before the solvent was evaporated under reduced pressure. Purifica-
tion by column chromatography on silica gel (pentane/EtOAc = 5:1
Ǟ 2:1) gave 4 (360 mg, 89%). 1H NMR (300 MHz, CDCl3): δ =
1.15 (d, J = 6.0 Hz, 3 H, 6Ј-H3), 1.35 (d, J = 6.0 Hz, 3 H, 6-H3),
1.97 [s, 3 H, O(CO)CH3], 2.05 [s, 3 H, O(CO)CH3], 2.06 [s, 3 H,
p-Methoxyphenyl 2,3,4-Tri-O-acetyl-α-
2-O-acetyl-4-O-benzyl-α- -rhamnopyranoside (9): Rhamnosyl tri-
chloroacetimidate 7 (956 mg, 2.20 mmol, 1.4 equiv.) and rhamno-
L-rhamnopyranosyl-(1Ǟ3)-
L
side 8 (642 mg, 1.60 mmol, 1.0 equiv.) were coevaporated three O(CO)CH3], 2.23 [s, 3 H, O(CO)CH3], 3.58 (dd, J = 9.9, 9.3 Hz,
times with toluene (3ϫ1 mL) and dried under high vacuum for
one hour. Compounds 7 and 8 were dissolved in anhydrous CH2Cl2
(25 mL), cooled to 0 °C, and TMSOTf (113 mg, 92 μL, 0.51 mmol,
0.32 equiv.) was added. The reaction mixture was stirred at 0 °C
for 3 h before pyridine (1 mL) was added to stop the reaction. The
solvents were removed by rotary evaporation, and the residue was
1 H, 4-H), 3.87-3.98 (m, 2 H, 5-H, 5Ј-H), 4.21 (dd, J = 9.3, 3.6 Hz,
1 H, 3-H), 4.65 (d, J = 10.5 Hz, 1 H, CH2Ph), 4.78 (d, J = 10.5 Hz,
1 H, CH2Ph), 5.04 (dd, J = 9.9, 9.4 Hz, 1 H, 4Ј-H), 5.05 (d, J =
2.0 Hz, 1 H, 1Ј-H), 5.20 (dd, J = 9.9, 3.6 Hz, 1 H, 3Ј-H), 5.31 (dd,
J = 3.6, 1.8 Hz, 1 H, 2-H), 5.33 (dd, J = 3.6, 2.0 Hz, 1 H, 2Ј-H),
6.15 (d, J = 1.8 Hz, 1 H, 1-H), 7.23-7.34 (m, 5 H, PhH), 8.66 (s,
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Synthesis of α-Linked Rha–Rha–Gal Undecaprenyl Diphosphate
1 H, NH) ppm. 13C NMR (125 MHz, CDCl3): δ = 17.3 (C-6Ј), 17.9 solvents were removed under reduced pressure, and the residue was
(C-6), 20.7, 20.7, 20.8, 20.9 [O(CO)CH3], 67.4 (C-5Ј), 69.0 (C-3Ј),
purified by column chromatography on silica gel (pentane/EtOAc =
69.6 (C-2Ј), 70.3 (C-2), 70.5 (C-4), 70.9 (C-5), 75.2 (C-2), 75.8 6:1 Ǟ 5:1 Ǟ 3:1) to afford 13 (541 mg, 95%). 1H NMR (300 MHz,
(CH2Ph), 80.1 (C-4), 90.8 (CCl3), 94.6 (C-1), 99.2 (C-1Ј), 128.0, CDCl3): δ = 2.00 [s, 3 H, O(CO)CH3], 3.62–3.72 (m, 3 H, 5-H, 6-
128.1, 128.5 (CtertPh), 137.5 (CquatH), 160.0 [O(NH)CCCl3], 169.8, H2), 4.02 (d, J = 3.2 Hz, 1 H, 4-H), 4.22 (dd, J = 7.5, 7.3 Hz, 1 H,
170.0, 170.1, 170.2 [O(CO)CH3] ppm. [α]2D0 = –40.0 (c = 0.5,
CH–Fmoc), 4.34 (d, J = 10.2 Hz, 1 H, CH2Ph), 4.40 (d, J =
10.2 Hz, 1 H, CH2Ph), 4.42 (d, J = 10.9 Hz, 1 H, CH2Ph), 4.45–
4.50 (m, 3 H, 1-H, CH2–Fmoc), 4.59 (d, J = 12.4 Hz, 1 H, CH2Ph),
4.73 (d, J = 10.9 Hz, 1 H, CH2Ph), 4.79 (dd, J = 10.3, 3.2 Hz, 1 H,
3-H), 4.88 (d, J = 12.4 Hz, 1 H, CH2Ph), 5.50 (dd, J = 10.3, 7.7 Hz,
1 H, 2-H), 7.19–7.40, 7.54–7.60, 7.69–7.75 (m, 23 H, PhH) ppm.
13C NMR (125 MHz, CDCl3): δ = 20.9 [O(CO)CH3], 46.6 (CH–
Fmoc), 68.1 (C-6), 69.5 (C-2), 70.1, 70.2 (CH2Ph), 73.3 (C-5), 73.5
(CH2–Fmoc), 73.7 (C-4), 75.1 (CH2Ph), 77.9 (C-3), 99.8 (C-1),
120.0, 125.0, 125.0, 127.1, 127.1, 127.5, 127.6, 127.6, 127.7,127.7,
127.8, 128.0, 128.1, 128.2, 128.2, 128.3, 128.3 (CtertPh), 137.1,
137.7, 141.1, 141.2, 142.9, 143.1 (CquatPh), 154.4 (CO–Fmoc),
169.2 [O(CO)CH3] ppm. [α]2D0 = –10.0 (c = 0.17, CHCl3). IR (KBr):
CHCl ). IR (KBr): ν = 3091, 2939, 2122, 1696, 1608, 1498, 1160,
˜
3
940 cm–1. HRMS (ESI): calcd. for C29H36O13NCl3Na [M + Na]+
734.11444; found 734.11427.
4,6-Di-O-benzyl-3-O-fluorenymethoxycarbonyl-D-galactal (11): Gal-
actal 10 (1.23 g, 3.77 mmol, 1.0 equiv.) was dissolved in pyridine
(50 mL), and FmocCl (1.95 g, 7.52 mmol, 2.0 equiv.) was added at
r.t. in one portion. The reaction mixture was stirred for 2 h before
the solvent was removed under reduced pressure. Purification by
column chromatography on silica gel (pentane/EtOAc = 15:1 Ǟ
1
8:1 Ǟ 5:1) gave 11 (1.75 g, 85%). H NMR (300 MHz, CDCl3): δ
= 3.71 (dd, J = 10.4, 4.5 Hz 1 H), 3.84 (dd, J = 10.4, 7.6 Hz, 1 H),
4.05 (dd, J = 3.6, 3.6 Hz, 1 H), 4.19–4.35 (m, 2 H), 4.35–4.43 (m,
2 H), 4.43–4.60 (m, 3 H), 4.74 (d, J = 11.7 Hz, 1 H), 4.83 (dd, J =
6.2, 3.5 Hz, 1 H), 5.38 (dd, J = 3.8, 3.8 Hz, 1 H), 6.43–6.47 (m,
1 H), 7.23–7.43 (m, 14 H), 7.60 (dd, J = 7.4, 3.4 Hz, 2 H), 7.72–
7.79 (m, 2 H) ppm. 13C NMR (125 MHz, CDCl3): δ = 46.5, 50.3,
64.9, 67.6, 69.6, 73.3, 75.2, 97.7, 119.7, 119.8, 123.5, 124.7, 126.8,
126.9, 126.9, 127.2, 127.5, 127.6, 127.6, 127.7, 127.8, 128.1, 128.2,
135.7, 137.5, 137.7, 141.1, 141.2, 143.1, 143.1, 144.4, 145.8, 149.5,
ν = 2870, 1753, 1453, 1370, 1268, 1072 cm–1. HRMS (ESI): calcd.
˜
for C44H42O9Na [M + Na]+ 737.2721; found 737.2722.
Benzyl 2-O-Acetyl-4,6-di-O-benzyl-β-D-galactopyranoside (5): Mono-
saccharide 13 (551 mg, 0.77 mmol, 1.0 equiv.) was dissolved in
DMF (24 mL), and piperidine (4.8 mL, 48.5 mmol, 63 equiv.) was
added. The mixture was stirred at r.t. for 20 min. The solvents were
evaporated and purification by column chromatography on silica
gel (pentane/EtOAc = 3:1 Ǟ 1:1) gave 5 (348 mg, 92%) together
with a minor amount of an isomer of 5 with a migrated acetyl
group. 1H NMR (300 MHz, CDCl3): δ = 2.05 [s, 3 H, O(CO)CH3],
2.26 (d, J = 10.3 Hz, OH), 3.57 (ddd, J = 10.3, 10.0, 3.6 Hz, 1 H,
3-H), 3.62–3.71 (m, 3 H, 5-H, 6-H2), 3.88 (d, J = 3.6 Hz, 1 H, 4-
H), 4.40 (d, J = 7.9 Hz, 1 H, 1-H), 4.48 (d, J = 11.6 Hz, 1 H,
CH2Ph), 4.53 (d, J = 11.6 Hz, 1 H, CH2Ph), 4.59 (d, J = 12.4 Hz,
1 H, CH2Ph), 4.66 (d, J = 11.9 Hz, 1 H, CH2Ph), 4.72 (d, J =
11.9 Hz, 1 H, CH2Ph), 4.87 (d, J = 12.4 Hz, 1 H, CH2Ph), 5.04 (dd,
J = 10.0, 7.9 Hz, 1 H, 2-H), 7.25–7.38 (m, 15 H, PhH) ppm. 13C
NMR (125 MHz, CDCl3): δ = 21.0 [O(CO)CH3], 68.3 (C-6), 70.2
(CH2Ph), 73.0, 73.5, 73.5, 73.6 (C-2, C-3, C-5, CH2Ph), 75.5
(CH2Ph), 76.6 (C-4), 99.5 (C-1), 127.5, 127.6, 127.7, 127.7, 127.7,
127.8, 127.9, 127.9, 128.1, 128.1, 128.2, 128.4, 128.4, 128.5
(CtertPh), 137.2, 137.6, 137.9 (CquatPh), 170.9 [O(CO)CH3] ppm.
154.6 ppm. [α]2D0 = –33 (c = 0.23, CHCl ). IR (ATR): ν = 3063,
˜
3
2854, 1808, 1644, 1607, 1497, 1384, 1350 cm–1. HRMS (ESI): calcd.
for C35H32O6Na [M + Na]+ 571.2091; found 571.2094.
Benzyl 3-O-Fluorenylmethoxycarbonyl-4,6-di-O-benzyl-β-D-galacto-
pyranoside (12): Galactal 11 (1.06 g, 1.93 mmol, 1.0 equiv.) was co-
evaporated with toluene (3ϫ1 mL) and dried under high vacuum
for 1 h. Anhydrous dichloromethane (50 mL) was added and the
solution was cooled to 0 °C. Dimethyldioxirane in acetone (0.07 m,
41 mL, 2.90 mmol, 1.5 equiv.) was added. After stirring for 20 min
at 0 °C, the solvents were removed under reduced pressure at 0 °C.
The residue was redissolved in anhydrous dichloromethane
(50 mL), benzyl alcohol (10 mL) was added and the mixture was
stirred at r.t. overnight. The solvents were removed under reduced
pressure. The two epimers [260 mg (20%, α) and 837 mg (64%, β)]
were obtained after purification by column chromatography on sil-
ica gel (pentane/EtOAc = 6:1 Ǟ 4:1). Analytical data for the β-
anomer: 1H NMR (300 MHz, CDCl3): δ = 2.33 (d, J = 2.9 Hz,
1 H, OH), 3.62–3.72 (m, 3 H, 5-H, 6-H2), 3.98 (d, J = 3.1 Hz, 1 H,
4-H), 4.05 (ddd, J = 10.5, 8.1, 2.9 Hz, 1 H, 2-H), 4.25 (dd, J = 7.1,
6.8 Hz, 1 H, CH–Fmoc), 4.38 (d, J = 8.1 Hz, 1 H, 1-H), 4.39–4.56
(m, 5 H, CH2–Fmoc, CH2Ph), 4.61 (d, J = 11.8 Hz, 1 H, CH2Ph),
4.67 (d, J = 10.7 Hz, 1 H, CH2Ph), 4.72 (dd, J = 10.5, 3.1 Hz, 1 H,
3-H), 4.92 (d, J = 11.8 Hz, 1 H, CH2Ph), 7.17–7.40, 7.55–7.75 (m,
23 H, PhH, Ar–Fmoc) ppm. 13C NMR (125 MHz, CDCl3): δ =
46.8 (CH–Fmoc), 68.3 (C-6), 69.7 (C-2), 69.9 (CH2Ph), 71.0
(CH2Ph), 73.4 (C-5), 73.5 (CH2–Fmoc), 73.8 (C-4), 75.2 (CH2Ph),
79.5 (C-3), 101.9 (C-1), 120.0, 125.0, 125.1, 127.1, 127.6, 127.7,
127.7, 127.8, 127.8, 127.9, 128.1, 128.1, 128.2, 128.3, 128.3, 128.3,
128.4, 128.4, 128.4 (CtertPh), 136.8, 137.7, 137.8, 141.1, 141.2,
142.8, 143.4 (CquatPh), 154.6 (CO–Fmoc) ppm. [α]2D0 = –5.8 (c =
[α]2D0 = –35.0 (c = 0.3, CHCl ). IR (KBr): ν = 3429, 2888, 1719,
˜
3
1369, 1271, 1085 cm–1. HRMS (ESI): calcd. for C29H32O7Na [M +
Na]+ 515.2040; found 515.2042.
Benzyl 2,3,4-Tri-O-acetyl-α-
4-O-benzyl-α- -rhamnopyranosyl-(1Ǟ3)-2-O-acetyl-4,6-di-O-
benzyl-β- -galactopyranoside (14): Disaccharide trichloroacetimid-
L-rhamnopyranosyl-(1Ǟ3)-2-O-acetyl-
L
D
ate 4 (320 mg, 0.45 mmol, 1.3 equiv.) and galactoside 5 (170 mg,
0.35 mmol, 1.0 equiv.) were coevaporated with toluene (3ϫ1 mL)
and dried under high vacuum for 1 h. Compounds 4 and 5 were
dissolved in anhydrous dichloromethane (5 mL), cooled to 0 °C,
and TMSOTf (10 mg, 8 μL, 0.05 mmol, 0.13 equiv.) was added.
The reaction mixture was stirred at 0 °C for 3 h before pyridine
(1 mL) was added. The solvents were removed under reduced pres-
sure, and the residue was purified by column chromatography on
silica gel (pentane/EtOAc = 3:1 Ǟ 1:1) to afford 14 (350 mg, 74%).
1H NMR (600 MHz, CDCl3): δ = 1.16 (d, J = 6.1 Hz, 3 H, 6ЈЈ-
H3), 1.28 (d, J = 6.4 Hz, 3 H, 6Ј-H3), 1.95 [s, 3 H, O(CO)CH3], 2.04
[s, 3 H, O(CO)CH3], 2.06 [s, 3 H, O(CO)CH3], 2.07 [s, 3 H, O(CO)
CH3], 2.16 [s, 3 H, O(CO)CH3], 3.47 (dd, J = 9.7, 9.2 Hz, 1 H, 4Ј-
H), 3.59-3.64 (m, 3 H, 5-H, 6-H2), 3.74 (dd, J = 10.2, 2.6 Hz, 1 H,
3-H), 3.76-3.84 (m, 3 H, 4-H, 5Ј-H, 5ЈЈ-H), 3.98 (dd, J = 9.7,
3.4 Hz, 1 H, 3Ј-H), 4.36 (d, J = 8.1 Hz, 1 H, 1-H), 4.44 (d, J =
11.7 Hz, 1 H, CH2Ph), 4.47 (d, J = 11.7 Hz, 1 H, CH2Ph), 4.56 (d,
0.5, CHCl ). IR (KBr): ν = 3443, 3030, 2867, 1746, 1452, 1264,
˜
3
1073 cm–1. HRMS (ESI): calcd. for C42H40O8Na [M + Na]+
695.2615; found 695.2616.
Benzyl 2-O-Acetyl-3-O-fluorenylmethoxycarbonyl-4,6-di-O-benzyl-
β-D-galactopyranoside (13): Monosaccharide 12 (535 mg,
0.80 mmol, 1.0 equiv.) was dissolved in a mixture of acetic anhy-
dride (10 mL) and pyridine (5 mL) and stirred at r.t. for 3 h. The
Eur. J. Org. Chem. 2011, 6233–6239
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6237

A. Holkenbrink, D. C. Koester, J. Kaschel, D. B. Werz
FULL PAPER
J = 12.2 Hz, 1 H, CH2Ph), 4.57 (d, J = 10.8 Hz, 1 H, CH2Ph), 4.60 2,3,4-Tri-O-acetyl-α-
(d, J = 10.8 Hz, 1 H, CH2Ph), 4.78 (d, J = 11.5 Hz, 1 H, CH2Ph), -rhamnopyranosyl-(1Ǟ3)-2,4,6-tri-O-acetyl-
4.84 (d, J = 12.2 Hz, 1 H, CH2Ph), 4.85 (d, J = 11.5 Hz, 1 H, (16): Compound 15 (85 mg, 0.24 mmol) was dissolved in anhydrous
CH2Ph), 4.88 (m, 2 H, 1Ј-H, 1ЈЈ-H), 5.00 (m, 1 H, 2Ј-H), 5.03 (dd, tetrahydrofuran (2 mL). Piperidine (100 μL, 1.0 mmol, 1 equiv.)
J = 10.3, 10.0 Hz, 1 H, 4ЈЈ-H); 5.20 (dd, J = 10.3, 3.7 Hz, 1 H, 3ЈЈ- was added dropwise at r.t. The mixture was stirred for 2.5 h before
L
-rhamnopyranosyl-(1Ǟ3)-2,4-di-O-acetyl-α-
L
D
-galactopyranose
H), 5.26 (m, 1 H, 2ЈЈ-H), 5.46 (dd, J = 10.2, 8.1 Hz, 1 H, 2-H),
7.17-7.35 (m, 20 H, PhH) ppm. 13C NMR (125 MHz, CDCl3): δ =
17.2 (C-6ЈЈ), 18.1 (C-6Ј), 20.8, 20.8, 20.9, 21.0 [O(CO)CH3], 67.3
the solvents were evaporated. Purification by column chromatog-
raphy on silica gel (pentane/EtOAc = 1:1 Ǟ 1:2) gave 16 (43 mg,
1
53%) as a 1:1 mixture of anomers. H NMR (600 MHz, CDCl3):
(C-5ЈЈ), 68.4 (C-6), 68.8 (C-5Ј), 69.1 (C-3ЈЈ), 69.6 (C-2ЈЈ), 70.0 δ = 1.13 (d, J = 6.2 Hz, 3 H), 1.19 (d, J = 6.2 Hz, 3 H), 1.95 (s,
(CH2Ph), 70.7 (C-4ЈЈ), 71.4 (C-2), 72.0 (C-2Ј), 73.5 (CH2Ph), 73.8 3 H), 2.04 (s, 3 H), 2.05 (s, 3 H), 2.11 (s, 3 H), 2.12 (s, 3 H), 2.13
(C-5), 74.9 (CH2Ph), 75.4 (CH2Ph), 76.0 (C-4), 76.3 (C-3Ј), 78.7
(s, 3 H), 2.15 (s, 3 H), 2.15 (s, 3 H), 3.72–3.92 (m, 4 H), 4.00 (d, J
(C-3), 80.1 (C-4Ј), 98.5 (C-1Ј), 99.1 (C-1ЈЈ), 99.8 (C-1), 127.4, 127.5, = 7.1 Hz, 1 H), 4.02 (d, J = 7.1 Hz, 1 H), 4.27 (dd, J = 10.3, 3.5 Hz,
127.5, 127.6, 127.7, 127.7, 127.8, 128.2, 128.2, 128.3, (CtertPh),
137.2, 137.6, 137.8, 138.0 (CquatPh), 169.5, 169.6, 169.8, 169.9
1 H), 4.39 (dd, J = 6.5, 6.2 Hz, 1 H), 4.81 (d, J = 1.4 Hz, 1 H), 4.96
(d, J = 1.4 Hz, 1 H), 4.98–5.13 (m, 5 H), 5.37 (d, J = 3.8 Hz, 1 H),
[O(CO)CH ] ppm. IR (KBr): ν = 2935, 1747, 1696, 1497, 1454, 5.48 (dd, J = 3.3, 3.0 Hz, 1 H) ppm. 13C NMR (125 MHz, CDCl3):
˜
3
1370, 1226, 1073 cm–1. [α]2D0 = –51.0 (c = 0.35, CHCl3). HRMS
(ESI): calcd. for C56H66O19Na [M + Na]+ 1065.40905; found
1065.40901.
δ = 17.3, 17.5, 20.7, 20.8, 20.8, 20.9, 20.9, 20.9, 21.0, 21.1, 62.3,
66.9, 67.2, 67.5, 68.5, 70.0, 70.8, 71.0, 71.3, 71.9, 74.5, 90.6, 92.0.
98.5, 98.5, 169.5, 169.9, 169.9, 170.2, 170.2, 170.3, 170.5 ppm. IR
(film): ν = 3278, 2857, 2123, 2032, 1966, 1327 cm–1. HRMS (ESI):
˜
2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranosyl-(1Ǟ3)-2,4-di-O-acetyl-α-
-galactopyranosyl
calcd. for C33H48O22Na [M + Na]+ 831.2529; found 831.2513.
L
-rhamnopyranosyl-(1Ǟ3)-2,4,6-tri-O-acetyl-
D
Acetate (15): Trisaccharide 14 (72 mg, 0.069 mmol) was dissolved
in methanol (3 mL) and dichloromethane (1 mL). Pd(OH)2/C (20%
Pd, 10 mol-%) was added under an argon atmosphere. The argon
atmosphere was exchanged with H2 and the mixture was stirred at
r.t. overnight. The slurry was filtered through Celite, and the sol-
vents were evaporated to afford the debenzylated product (47 mg,
quant.). 1H NMR (300 MHz, CD3CN): δ = 1.10–1.15, 1.19–1.23
(m, 12 H, 6-H3, 6Ј-H3), 1.91–2.09 [m, 30 H, O(CO)CH3], 3.41–3.51
(m, 5 H), 3.57–3.66 (m, 6 H), 3.74–3.84 (m, 6 H), 3.88–4.00 (m,
6 H), 4.54 (d, J = 8.4 Hz, 1 H), 4.79 (d, J = 1.6 Hz, 1 H), 4.86 (d,
2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranosyl-(1Ǟ3)-2,4-di-O-acetyl-α-
-galactopyranosyl
L
-rhamnopyranosyl-(1Ǟ3)-2,4,6-tri-O-acetyl-α-
D
Dibenzyl Phosphate (17): Trisaccharide 16 (42 mg, 52 μmol,
1.0 equiv.) was dissolved in anhydrous acetonitrile (3 mL). Tetraz-
ole (333 μL, 150 μmol, 2.8 equiv.) and 6 (74 μL, 220 μmol, 4.3
equiv.) were added dropwise. The reaction mixture was stirred at
r.t. for 5 h, cooled to –10 °C, and tBuOOH (75 μL, 410 μmol,
7.9 equiv.) was added. After stirring for 90 min at –10 °C, ethyl
ether was added, the phases were separated, and the organic layer
was washed with Na2S2O4 solution, NaHCO3 solution, and brine.
J = 1.8 Hz, 1 H), 4.90–5.08 (m, 10 H), 5.17 (d, J = 3.2 Hz, 1 H), The organic layer was dried with Na2SO4 and concentrated. Purifi-
5.26–5.31 (m, 3 H) ppm. 13C NMR (125 MHz, CD3CN): δ = 17.6,
17.6, 17.9, 17.9, 20.7, 20.7, 20.7, 20.8, 20.9, 21.1, 62.1, 62.2, 68.2,
68.2, 70.0, 70.3, 70.4, 70.7, 70.7, 70.7, 70.9, 70.9, 71.6, 72.0, 72.1,
cation by column chromatography on silica gel (pentane/EtOAc =
2:1 Ǟ 1:1) gave 17 (12 mg, 22%) as an anomeric mixture (α/β =
6:1). Analytical data for the α-anomer: 1H NMR (300 MHz,
73.2, 73.3, 73.3, 73.9, 76.5, 76.6, 77.2, 77.5, 81.6, 91.5, 96.6, 100.6, CDCl3): δ = 1.11 (d, J = 6.3 Hz, 3 H), 1.20 (d, J = 6.30 Hz, 3 H),
100.7, 100.7, 100.9, 171.4, 171.6, 171.6, 171.8, 171.9, 172.2 ppm.
1.89 (s, 3 H), 1.91 (s, 3 H), 1.94 (s, 3 H), 2.01 (s, 3 H), 2.11 (s, 3 H),
2.12 (s, 3 H), 2.14 (s, 3 H), 2.15 (s, 3 H), 3.72–3.97 (m, 4 H), 4.04
(d, J = 5.4 Hz, 1 H), 4.14 (dd, J = 10.5, 3.4 Hz, 1 H), 4.25 (dd, J
= 6.4, 6.2 Hz, 1 H), 4.79 (d, J = 1.8 Hz, 1 H), 4.90 (d, J = 1.6 Hz,
1 H), 4.93–5.14 (m, 10 H), 5.30–5.36 (m, 1 H), 5.89 (dd, J = 6.6,
3.2 Hz, 1 H, 1-H), 7.28–7.34 (m, 10 H) ppm. 13C NMR (125 MHz,
CDCl3): δ = 17.3, 17.5, 20.4, 20.6, 20.7, 20.8, 20.9, 20.9, 21.0, 21.0,
61.8, 67.2, 67.6, 68.5, 69.0, 69.2, 69.6, 69.6, 69.6, 69.7, 70.0, 70.6,
70.7, 71.2, 71.8, 74.4, 77.2, 94.6 (d, JPC = 5.7 Hz), 98.4, 98.7, 127.7,
127.8, 127.9, 128.5, 128.5, 128.6, 128.6, 128.7, 128.7, 135.2, 135.3,
169.5, 169.9, 169.9, 170.0, 170.1, 170.2, 170.2, 170.2 ppm. 31P
NMR (121 MHz, CDCl3): δ = –1.4 (α), –2.1 (β) ppm. IR (ATR):
IR (film): ν = 3382, 2982, 2259, 2129, 1970, 1645, 1433, 1137 cm–1.
˜
HRMS (ESI): calcd. for C28H42O19Na [M + Na]+ 705.2213; found
705.2213.
The debenzylated trisaccharide (162 mg, 0.24 mmol) was dissolved
in acetic anhydride (5 mL) and pyridine (6 mL). A spatula tip of
4-(dimethylamino)pyridine (DMAP) was added at r.t., and the mix-
ture was stirred overnight. The solvents were removed under re-
duced pressure. Purification by column chromatography on silica
gel (pentane/EtOAc = 1:1) and size-exclusion HPLC (recycling 3
times, chloroform) gave 15 (177 mg, 87%). 1H NMR (300 MHz,
CDCl3): δ = 1.87 (s, 6 H), 1.94 (s, 6 H), 1.96 (s, 6 H), 1.99 (s, 3 H),
2 01 (s, 3 H), 2.02 (s, 3 H), 2.04–2.05 (m, 15 H), 2.08 (s, 6 H), 2.09
(s, 3 H), 2.11 (s, 3 H), 3.65–4.12 (m, 15 H), 4.17–4.24 (m, 1 H), 4.74
(sbr, 1 H), 4.79 (d, J = 1.7 Hz, 1 H), 4.85 (dd, J = 3.5, 1.8 Hz, 1 H),
4.87–5.04 (m, 7 H), 5.06 (dd, J = 3.5, 1.1 Hz, 1 H), 5.15 (d, J =
3.9 Hz, 1 H), 5.18 (d, J = 1.1 Hz, 1 H), 5.22 (dd, J = 10.2, 8.5 Hz,
1 H), 5.27 (d, J = 3.2 Hz, 1 H), 5.33 (d, J = 3.3 Hz, 1 H), 5.53 (d,
J = 8.4 Hz, 1 H), 6.25 (d, J = 3.5 Hz, 1 H) ppm. 13C NMR
(125 MHz, CDCl3): δ = 17.3, 17.4, 20.4, 20.6, 20.7, 20.7, 20.7, 20.7,
20.7, 20.7, 20.8, 20.8, 20.8, 20.9, 20.9, 20.9, 20.9, 21.0, 21.0, 60.3,
61.5, 61.7, 67.0, 67.2, 67.2, 67.6, 67.7, 68.5, 68.6, 69.0, 69.1, 69.3,
69.7, 69.9, 70.6, 70.6, 70.7, 71.1, 71.2, 71.2, 71.6, 71.7, 72.1, 74.2,
76.2, 77.3, 89.7, 91.9, 98.5, 98.6, 98.7, 99.0, 168.6, 168.8, 169.3,
169.4, 169.5, 169.7, 169.8, 169.8, 169.9, 170.0, 170.0, 170.0, 170.1,
ν = 2924, 2341, 2160, 1456 cm–1. HRMS (ESI): calcd. for
˜
C48H61O25PNa [M + Na]+ 1091.3132; found 1091.3157.
2,3,4-Tri-O-acetyl-α-
L
-rhamnopyranosyl-(1Ǟ3)-2,4-di-O-acetyl-α-
-galactopyranosyl
L
-rhamnopyranosyl-(1Ǟ3)-2,4,6-tri-O-acetyl-α-
D
Phosphate (2): Dibenzylated phosphate 17 (13 mg, 12 μmol,
1.0 equiv.) was dissolved in methanol (3 mL) and dichloromethane
(1 mL). Pd/C (10% Pd, 10 mol-%) was added under an argon atmo-
sphere. The argon atmosphere was exchanged with hydrogen and
the reaction mixture was stirred at r.t. overnight. The slurry was
filtered through Celite. Evaporation of the solvents afforded 2
(10.7 mg, quant.). Analytical data for the α-anomer: 1H NMR
(300 MHz, CD3OD): δ = 1.13 (d, J = 6.3 Hz, 3 H), 1.17 (d, J =
6.6 Hz, 3 H), 1.96 (s, 3 H), 2.02 (s, 3 H), 2.05 (s, 3 H), 2.12 (s, 3 H),
2.12 (s, 3 H), 2.13 (s, 3 H), 2.16 (s, 3 H), 2.17 (s, 3 H), 3.83–3.89
(m, 1 H), 3.95 (dd, J = 10.0, 3.3 Hz, 1 H), 3.98–4.06 (m, 2 H), 4.15–
4.21 (m, 1 H), 4.40 (dd, J = 10.5, 3.5 Hz, 1 H), 4.54 (dd, J = 6.7,
170.1, 170.1, 170.2, 170.2, 170.2 ppm. IR (ATR): ν = 2923, 2341,
˜
2148, 1973, 1665, 1434 cm–1. HRMS (ESI): calcd. for C36H50O23Na
[M + Na]+ 873.2635; found 873.2633.
6238
www.eurjoc.org
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2011, 6233–6239

Synthesis of α-Linked Rha–Rha–Gal Undecaprenyl Diphosphate
2005, 3, 225–237; c) J. Eichler, M. W. W. Adams, Microbiol.
Mol. Biol. Rev. 2005, 69, 393–425.
[2] a) C. Schäffer, P. Messner, Glycobiology 2004, 14, 31R–42R; b)
P. Messner, K. Steiner, K. Zarschler, C. Schäffer, Carbohydr.
Res. 2008, 343, 1934–1951.
[3] K. Steiner, R. Novotny, D. B. Werz, K. Zarschler, P. H. See-
berger, A. Hofinger, P. Kosma, C. Schäffer, P. Messner, J. Biol.
Chem. 2008, 283, 21120–21133.
6.4 Hz, 1 H), 4.96–5.10 (m, 10 H), 5.47 (d, J = 2.5 Hz, 1 H), 5.68
(dd, J = 7.2, 3.0 Hz, 1 H, 1-H) ppm. 13C NMR (125 MHz,
CD3OD): δ = 17.6, 17.7, 20.6, 20.6, 20.7, 20.7, 20.8, 20.8, 20.9,
21.0, 49.3, 49.5, 54.8, 68.4, 68.5, 68.6, 70.1, 71.1, 71.4, 71.9, 72.7,
73.6, 75.7, 93.5 (d, JPC = 5.0 Hz), 99.8, 100.3, 171.3, 171.5, 171.6,
171.7, 172.0, 172.1, 172.2, 172.3 ppm. 31P NMR (121 MHz,
CD OD): δ = –0.5 (α) ppm. IR (ATR): ν = 3726, 3599, 2360, 2341,
˜
3
2077, 1748, 1541, 1225 cm–1. HRMS (ESI): calcd. for C34H49O25P
[M – H]+ 887.2228; found 887.2227.
[4] a) J. Stevens, O. Blixt, T. M. Tumpey, J. K. Taubenberger, J. C.
Paulson, I. A. Wilson, Science 2006, 312, 404–410; b) S. Elliott,
T. Lorenzini, S. Asher, K. Aoki, D. Brankow, L. Buck, L.
Busse, D. Chang, J. Fuller, J. Grant, N. Hernday, M. Hokum,
S. Hu, A. Knudten, N. Levin, R. Komorowski, F. Martin, R.
Navarro, T. Osslund, G. Rogers, N. Rogers, G. Trail, J. Egrie,
Nat. Biotechnol. 2003, 21, 414–421; c) D. B. Werz, P. H. See-
berger, Chem. Eur. J. 2005, 11, 3194–3206.
[5] a) P. H. Seeberger, D. B. Werz, Nature 2007, 446, 1046–1051;
b) C. Schäffer, R. Novotny, S. Küpcü, S. Zayni, A. Scheberl, J.
Friedmann, U. B. Sleytr, P. Messner, Small 2007, 3, 1549–1559;
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4, 751–763.
[6] a) M. S. VanNieuwenhze, S. C. Mauldin, M. Zia-Ebrahimi,
J. A. Aikins, L. C. Blaszczak, J. Am. Chem. Soc. 2001, 123,
6983–6988; b) I. Brockhausen, E. A. Larsson, O. Hindsgaul,
Bioorg. Med. Chem. Lett. 2008, 18, 804–807; c) R. Woodward,
W. Yi, L. Li, G. Zhao, H. Eguchi, P. R. Sridhar, H. Guo, J. K.
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418–423.
[7] Imperiali et al. used tetrabenzyl pyrophosphate for the phos-
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Soc. 2005, 127, 13766–13767.
Diammonium Salt of 2,3,4-Tri-O-acetyl-α-
(1Ǟ3)-2,4-di-O-acetyl-α- -rhamnopyranosyl-(1Ǟ3)-2,4,6-tri-O-acet-
yl-α- -galactopyranosyl Undecaprenyl Diphosphate (18): The am-
L-rhamnopyranosyl-
L
D
monium salt of 3 (5.0 mg, 5.7 μmol, 1.1 equiv.) and 2 (5.0 mg,
5.4 μmol, 1.0 equiv.) were each coevaporated three times with tolu-
ene and dried under high vacuum overnight. Undecaprenyl phos-
phate 3 was dissolved in THF (1.2 mL), and CDI (4.6 mg, 28 μmol,
5.5 equiv.) was added. After stirring for 24 h at ambient tempera-
ture the MS did not show complete conversion. Therefore, CDI
(5.8 mg, 36 μmol, 6.6 equiv.) was added again. The activation was
finished overnight. The reaction was quenched with methanol
(3.5 μL, 16 equiv.) and stirred for 15 min. The solvents were evapo-
rated, and the residue was dried under high vacuum for 1 hour. The
residue was dissolved in DMF (620 μL) and added to 2 followed by
anhydrous tin(II) chloride (2 mg, 11 μmol, 2 equiv.). The reaction
mixture was stirred at ambient temperature for one week, quenched
with ethylenediaminetetraacetic acid (EDTA) solution (10 mm,
2.2 mL) and purified by column chromatography on silica gel
(CHCl3/MeOH/2 m NH4OH, 90:10:1 Ǟ 50:10:1 Ǟ 50:50:1) to ob-
tain 18 (2.8 mg, 28%). Analytical data for the α-anomer: 1H NMR
(600 MHz, CD3OD): δ = 1.09–1.17 (m), 1.55–1.75 (m), 1.91–2.17
(m), 3.81–3.86 (m), 3.94 (dd, J = 10.8, 4.1 Hz), 3.96–4.05 (m), 4.19
(dd, J = 10.0, 6.7 Hz), 4.35 (dd, J = 9.8, 3.7 Hz), 4.48 (dd, J = 6.3,
6.0 Hz), 4.87–5.16 (m), 5.36–5.48 (m), 5.74–5.78 (m) ppm. 31P
NMR (162 MHz, CDCl3): δ = –10.8, –13.2 ppm. HRMS (ESI):
calcd. for C89H138O28P2 [M – H]+ 1715.8777; found 1715.8784.
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Diammonium Salt of α-
L-Rhamnopyranosyl-(1Ǟ3)-α-L-rhamnopyr-
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anosyl-(1Ǟ3)-α- -galactopyranosyl Undecaprenyl Diphosphate (1):
D
Peracetylated diphosphate 18 (2.0 mg, 1.1 μmol, 1 equiv.) was dis-
solved in MeOH/H2O/Et3N (7:3:1, 1.1 mL) and stirred at ambient
temperature for two weeks. After dialysis (molecular weight cut-off
500) and lyophilisation, 1 was obtained (1.0 mg, 64%). HRMS
(ESI): calcd. for C73H122O20P2 [M
1379.7925.
–
H]+ 1379.7932; found
Supporting Information (see footnote on the first page of this arti-
cle): NMR spectra of the new compounds.
Acknowledgments
We are grateful to the Deutsche Forschungsgemeinschaft (DFG)
(SFB 803 and Emmy Noether Fellowship to D. B. W.), the Fonds
der Chemischen Industrie (Liebig Fellowship to D. B. W. and Ph.D.
Fellowship to D. C. K.), the Konrad-Adenauer-Stiftung (Ph.D. Fel-
lowship to A. H.), and BASF AG for the donation of chemicals.
Furthermore, we thank Jun.-Prof. Dr. Christian Ducho for helpful
advice and Prof. Dr. Lutz F. Tietze for generous support.
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Received: July 14, 2011
Published Online: September 15, 2011
Eur. J. Org. Chem. 2011, 6233–6239
© 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
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