A Staphylococcus aureus lipoteichoic acid (LTA) derived structural variant with two diacylglycerol residues

Article abstract of DOI:10.1016/j.bmc.2006.05.055

Based on 1,2-O-isopropylidene-sn-glycerol five chiral building blocks containing differently modified glycerol residues were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl β-gentiobioside carrying a pho

Full text of DOI:10.1016/j.bmc.2006.05.055

Bioorganic & Medicinal Chemistry 14 (2006) 6239–6254
A Staphylococcus aureus lipoteichoic acid (LTA) derived
structural variant with two diacylglycerol residues
A. Stadelmaier,a I. Figueroa-Perez,a S. Deininger,b S. von Aulock,b
T. Hartungb and R. R. Schmidta,*
aFachbereich Chemie, Universita¨t Konstanz, Fach M 725, D-78457 Konstanz, Germany
bFachbereich Biologie, Universita¨t Konstanz, Fach M 668, D-78457 Konstanz, Germany
Received 3 February 2006; accepted 30 May 2006
Available online 16 June 2006
Abstract—Based on 1,2-O-isopropylidene-sn-glycerol five chiral building blocks containing differently modified glycerol residues
were required for the synthesis of the target molecule 2. One of these building blocks is diacylglyceryl b-gentiobioside carrying a
phosphite residue at 6b-O position. Ligation of these five building blocks led to the desired glycerol phosphate backbone to which
D-alanyl residues were attached, thus generating after O-deprotection the target molecule 2, a bisamphiphilic structural variant of
Staphylococcus aureus LTA. This compound displayed higher potency in terms of cytokine release by human blood leukocytes than
the monoamphiphilic variant LTA.
Ó 2006 Elsevier Ltd. All rights reserved.
1. Introduction
essentially the same biological activity in terms of initia-
tion of cytokine release by human blood leukocytes as
found for the natural product. Whereas the replacement
of the D-alanine residues by L-alanine residues as in 1b
led to almost complete loss of biological activity.4 Fur-
ther structural modifications of LTA 1a, as for instance
deletion of the gentiobiose moiety or replacement of the
hydrolytically labile ester bond to the D-alanyl residue
by a stable amide bond, led only to minor decrease of
induction of cytokine release by human blood leuko-
cytes.5,13 Thus, the importance of the D-alanyl residues
was displayed. However, none of the investigated struc-
tural modifications so far led to an increased induction
of cytokine release. For this, optimal presentation of
the hydrophilic part of LTA to the receptor should be
of utmost importance; therefore, bisamphiphilic com-
pound 2 (Scheme 2) having two diacylglycerol gentio-
bioside residues at each end of the glycerophosphate
backbone was designed. It was hypothesized that with
two lipid anchors, possibly within the same membrane,
the epitope presentation should be supported due to ste-
rically improved accessibility to the D-alanyl and the a-
O-linked N-acetylglucosamine residues.14
For Gram-negative bacteria, LPS is well established as
the crucial stimulus of the innate immune system, as
injection of LPS into mice causes all known symptoms
of sepsis.1 There is now good evidence that lipoteichoic
acid (LTA) from the cytoplasmic membrane of Gram-
positive bacteria is an immunostimulatory Gram-posi-
tive counterpart to LPS.2–6 The most frequently isolated
Gram-positive pathogen that causes infections is Staph-
ylococcus aureus,7 and the development of antibiotic
resistance in this species is a big problem.8,9 Therefore,
alternatives and adjuvants to antibiotics are required.
To identify these, it is important to understand the path-
ophysiology of this bacterial infection.
The structure of the S. aureus LTA is shown in Scheme
1.3,10,11 For unequivocal bioactivity assignment, besides
an improved isolation procedure,11 the chemical synthe-
sis of the structurally closely related compound 1a
(Scheme 1) was decisive.3–5,12 This compound contains
the hydrolytically labile D-alanine residues in the re-
quired ratio with other substituents at a hexameric glyc-
erophosphate backbone. Compound 1a exhibited
2. Results and discussion
Keywords: Bacteria; Gram-positive; Lipoteichoic acid; Bisamphiphilic;
Synthesis; Cytokine release.
*
The retrosynthesis of compound 2 is shown in Scheme 2.
Disintegrations – lead to building blocks 3 to 8 which
Corresponding author. Tel.: +49 7531 88 2538; fax: +49 7531 88
3135; e-mail: Richard.Schmidt@uni-konstanz.de
0968-0896/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.055

6240
A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
R1 = alkyl
Me
O
P
O
O
OH
R2
=
(~ 70%)
R1
HO
HO
O
O
O
O
R1
O
H
O
O
–
O
HO
O
OR2
HO
+
O
NH3
OH
R2 = H
(~ 15%)
(~ 15%)
n
O
OH
(n = 40-50)
A
O
R2
=
HO
HO
AcHN
O
P
O
O
O
P
O
P
O
O
P
–
P
–
P
O
O
O
O
O
O
O
O
O
O
O
HO
O
O
–
–
HO
HO
O
O
O
O
OH
HO
O
HO
O
O
O
O
O
O
OH
+
+
+
+
O
O
O
O
O
AcHN
NH3
NH3
NH3
NH3
OH
O
O
Me
Me
Me
Me
HO
O
OH
OH
O
HO
HO
1a
1b (L-Ala instead of D-Ala)
Scheme 1. General structure A of lipoteichoic acid (LTA) from Staphylococcus aureus (A) and structure of closely related compound 1a and its
diastereoisomer 1b.
O
OH
4
3
2
H
O
P
O
P
O
P
O
P
3'
1'
O
OH
O
O
b
O
OH
2'
O
1'
O
3'
a
H27C13
H27C13
O
O
2'
HO
HO
O
OH
HO
O
O
O
1
O
O
1
C13H27
O
O
1 O
O
a
O
O
–
HO
OH
O
OH
O
OH
HO
b
HO
C13H27
O
O
O
OH
2
O
+
AcHN
OH
NH3
O
2
O
Me
c
O
4
OH
HO
HO
OBn
P
O
iPr2N
O
OH
O
O
BnO
BnO
3
NZ
H
8
O
O
C13H27
O
C13H27
Me
O
BnO
BnO
BnO
O
BnO
OBn
OBn
P
OBn
P
O
P
NiPr2
OTBDPS
OTBDPS
MMTrO
O
iPr2N
O
iPr2N
O
MMTrO
OH
6
7
OMPM
OBn
5
OMPM
4
O
AcHN
O
OBn
BnO
OH
BnO
BnO
(for 1a, 1b)
OBn
Scheme 2. Structure of target molecule 2 and required building blocks 3–8 for the synthesis.
consider the presence of diacylglyceryl gentiobioside,
glyceryl 2-N-acetylamino-2-deoxy-a-D-glucopyranoside,
D-alanylated glycerol and 2-O-unsubstituted glycerol
residues, respectively, and their sequence specific linkage
via phosphorus diester bonds. Also the most important
aspect, the lability of the D-alanyl residues, which are
readily cleaved at pH 8.5, is taken into account: as tem-
porary protecting groups in building blocks 5 and 6 4-
methoxyphenylmethyl (MPM) groups are chosen; they
can be selectively cleaved by oxidation after completion
of the backbone synthesis. Ensuing attachment of D-al-
anyl residues with Z-protected alanine 8 and then com-
plete O-debenzylation will provide the target molecule 2.
Building blocks 3–5 and, as substitute for 6 and 7, 2,3-
di-O-benzyl-sn-glycerol,4 were successfully employed in
the synthesis of LTAs 1a, b. Because this work has not
been reported in detail,15 the syntheses of compounds
1a, b will also be described.
2.1. Synthesis of building block 3
Benzoylation of gentiobiose with benzoyl chloride in pyr-
idine gave an a-, b-mixture of per-O-benzoylated com-
pound 9 (Scheme 3). Treatment of 9 with hydrazinium
acetate in DMF permitted chemoselective removal of
the anomeric O-benzoyl group furnishing 1-O-unprotect-
ed compound 10. Reaction of 10 with trichloroacetonitri-
le in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6241
2.2. Synthesis of LTA 1a and its diastereomer 1b
Gentiobiose
a
OBz
With building block 3 and the previously synthesized
glycerophosphate oligomer 20,13 possessing the required
protecting group array for regioselective chain extension
and for following D-alanyl residue attachment, the syn-
thesis of 1a, b could be readily completed (Scheme 4).
Ligation of 3 and 20 in the presence of tetrazole and
then oxidation with tert-butylhydroperoxide gave phos-
phate linked intermediate 21, which contains the back-
bone of the target molecule. Treatment of 21 with
ceric(IV) ammonium nitrate (CAN) liberated four of
the glycerol hydroxy groups affording compound 22.
Attachment of the Z-protected D-alanyl residues was
performed with excess 8 in the presence of (ben-
zotriazol-1-yloxy)tripyrrolidinophosphonium hexaflu-
O
BzO
BzO
b
O
O
BzO
a
BzO
BzO
X
BzO
Y
9: X, Y = H, OBz
10: X, Y = H, OH
b
c
11: X, Y = H, OC(NH)CCl3
12
HO
O
d
O
OR6
O
orophosphate18
(PyBOP)/N-methyl-imidazole
as
RO
RO
O
RO
RO
O
O
condensing agent to give the fully protected target mol-
ecule 23a. Hydrogenolysis with Pearlman’s catalyst19 in
a mixture of CH2Cl2/MeOH/H2O (5:5:1) furnished the
desired final product 1a after hydrophobic interaction
(HI) chromatography on octylsepharose in 47% yield.
The structural assignment was based on MS and
NMR data and comparison with naturally occurring
material.3 Compound 22 was similarly transformed with
Z-protected L-alanine via 23b into 1b.
RO
O
e
f
g
O
RO
13: R = R6 = Bz
14: R = R6 = H
15: R = H, R6 = TBDPS
16: R = Bn, R6 = TBDPS
OR6
h
O
BnO
BnO
OR1
O
O
BnO
BnO
OR2
BnO
2.3. Synthesis of building blocks 6 and 7
O
BnO
Previously described 1-O-(tert-butyldiphenylsilyl)-2-O-
(4-methoxybenzyl)-sn-glycerol (24),13 obtained from
1,2-O-isopropylidene-sn-glycerol, was treated with
monomethoxytrityl (MMTf) chloride in CH2Cl2/pyri-
dine to afford fully O-protected glycerol derivative 25
(Scheme 5). Regioselective O-desilylation with TBAF
in THF afforded the 1-O-unprotected building block 6.
17: R1 = R2 = H, R6 = TBDPS
i
j
18: R1 = R2 = COC13H27, R6 = TBDPS
19: R1 = R2 = COC13H27, R6 = H
k
3: R1 = R2 = COC13H27, R6 = P(OBn)NiPr2
Scheme 3. Synthesis of building block 3. Reagents and conditions: (a)
Bz-Cl, pyr, 40 °C (98%); (b) N2H4ÆHOAc, 50 °C, DMF (78%); (c)
CCl3CN, DBU, CH2Cl2 (97%); (d) BF3ÆOEt2, CH2Cl2, ꢀ30 °C (80%);
(e) NaOMe, MeOH (qu); (f) TBDPS-Cl, pyr, ꢀ15 °C (91%); (g) Bn-Br,
NaH, DMF (66%); (h) HOAc, THF/H2O, 80 °C (78%); (i) myristoyl
chloride, NEt3, THF, 50 °C (81%); (j) TBAF, HOAc, THF, 40 °C
(76%); (k) BnOP(NiPr2)2, tetrazole, THF/CH2Cl2 (79%).
Treatment of previously described 3-O-allyl-1-O-(tert-
butyldiphenylsilyl)-sn-glycerol (26)13 with benzyl bro-
mide in DMF afforded per-O-protected glycerol 27. O-
Deallylation with Wilkinson’s catalyst20 in the presence
of DBU as base in ethanol furnished the 3-O-propenyl
derivative which was cleaved with 1 M HCl in acetone
to afford 3-O-unprotected 28. Reaction with MMTr-Cl
in CH2Cl2/pyridine furnished fully protected glycerol
29 which on treatment with TBAF in THF led to 1-O-
unprotected glycerol 30. Phosphitylation with benzyl-
oxy-bis(diisopropylamino)phosphane17 led to the de-
sired phosphite derivative 7.
(DBU) as base afforded trichloroacetimidate 11 as a 1:1
anomeric mixture. Glycosylation of 1,2-O-isopropyl-
idene-sn-glycerol (12)16 with 11 in the presence of
BF3ÆOEt2 as activator gave exclusively b-glycoside 13 in
80% yield. Complete O-debenzoylation with sodium
methoxide in methanol afforded compound 14 which
was regioselectively silylated at the primary hydroxy
group with tert-butyldiphenylsilyl (TBDPS) chloride in
pyridine to give 6b-O-protected compound 15. Following
O-benzylation with benzyl bromide and sodium hydride
as base in DMF as solvent afforded fully O-protected gen-
tiobioside 16. Acid-catalyzed O-deisopropylidenation (!
17) and then treatment with myristoyl chloride in the pres-
ence of triethylamine as base led to introduction of two
myristoyl residues furnishing compound 18. 6b-O-Desily-
lation with tetrabutylammonium fluoride (TBAF) in the
presence of acetic acid in THF as solvent gave 6b-O-un-
protected compound 19 which on reaction with benzyl-
oxy-bis(diisopropylamino)-phosphane17 in the presence
of tetrazole as catalyst furnished building block 3.
2.4. Synthesis of target molecule 2
The assembly of target molecule 2 from building blocks
3–8 was performed stepwise in solution starting with
building block 6 (Scheme 6). Phosphitylation of 6 with
5 in the presence of tetrazole and then oxidation with
tert-butylhydroperoxide gave phosphate intermediate
31 which was O-desilylated with TBAF in THF as sol-
vent to furnish 32 with a free hydroxy group as acceptor
for the next reaction cycles for chain extension. This way,
with 5 intermediates 33–35 were obtained. Following
cleavage of the MMTr group with camphersulfonic acid
(CSA) in methanol afforded 36 which on phosphitylation

6242
A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
O
P
O
P
O
P
O
P
O
P
O
O
OH
O
O
O
O
O
O
BnO
O
O
BnO
OMPM
BnO
OMPM
BnO
OMPM
BnO
OMPM
BnO
O
AcHN
OBn
20
O
3
OBn
BnO
BnO
a
O
O
P
O
P
O
P
O
P
O
P
P
O
O
O
O
O
O
O
O
O
O
O
BnO
BnO
BnO
O
BnO
O
O
BnO
BnO
OMPM
BnO
OMPM
BnO
OMPM
BnO
O
AcHN
OMPM
OBn
O
O
C13H27
O
C13H27
O
BnO
BnO
BnO
O
O
21: R = PMB
22: R = H
BnO
OBn
b
BnO
BnO
O
8, c
O
23a: R =
23b: R = O
NZ
H
NZ
H
Me
Me
d
d
1a
1b
Scheme 4. Synthesis of compounds 1a and 1b. Reagents and conditions: (a) tetrazole, CH2Cl2; t-BuO2H (75%); (b) CAN, MeCN/Tol/H2O, ꢀ10 °C
! rt (67%); (c) PyBOP, Me-Im, CH2Cl2 (23a, 70%; 23b, 62%); (d) Pd(OH)2/C, H2, CH2Cl2/MeOH/H2O (1a, 47%; 1b, 40%).
methanol (! 40) and then with TBAF in THF afforded
RO
OTBDPS
compound 41 which was available to attachment of
building block 3 at each terminus. To this end, 41 was
treated with tetrazole under standard conditions to af-
ford compound 42 in good yield (Scheme 7), which con-
tains the desired glycerophosphate backbone of target
molecule 2. For the attachment of the D-alanyl residues,
the four MPM groups were selectively removed by treat-
ment with CAN in an acetonitrile/toluene/water mixture
as solvent affording compound 43 in 76% yield. D-Alany-
lation was performed with triethylammonium salt of 8 in
the presence of N-methyl-imidazole and PyBOP as con-
densing agent furnishing fully protected target molecule
44 in 49% yield. Hydrogenolytic O-debenzylation (i.e.
cleavage of 27 O-benzyl groups) was performed with
Pearlman’s catalyst19 in a dichloromethane/methanol/
water mixture as solvent. The crude product was purified
by HI chromatography to afford target molecule 2 in
32% yield. Compound 2 was structurally ascertained as
all intermediates by NMR and MS data and most inter-
mediates also by elemental analyses.
OMPM
24: R = H
25: R = MMTr
a
b
6
R1O
OTBDPS
OR2
26: R1 = All, R2 = H
27: R1 = All, R2 = Bn
28: R1 = H, R2 = Bn
29: R1 = MMTr, R2 = Bn
c
d
e
f
MMTrO
OR
OBn
30: R = H
7: R = P(OBn)NiPr2
3. Biological activity
g
The evaluation of the biological activity of compound 2
was performed in comparison with compound 1a
(Fig. 1), which induces a similar pattern of cytokine re-
lease as natural S. aureus LTA.3,4 Measurement of initi-
ation of cytokine release (A, TNFa; B, IL-8) by human
blood leukocytes displayed that bisamphiphilic 2 is more
potent than monoamphiphilic LTA 1a by a factor of
about 10. Hence, the hypothesis that bisamphiphilic
LTA analogues should exhibit stronger biological activ-
ity than monohomophilic LTA 1a was proven to be
correct.
Scheme 5. Synthesis of building blocks 6 and 7. Reagents and
conditions: (a) MMTr-Cl, CH2Cl2/pyr (98%); (b) TBAF, THF
(96%); (c) Bn-Br, NaH, DMF (72%); (d) (Ph3P)3RhCl, DBU, EtOH;
HCl, Me2CO, 70 °C (76%); (e) MMTr-Cl, CH2Cl2/pyr (98%); (f)
TBAF, THF (98%); (g) BnO(iPr2N)2P, tetrazole, THF/CH2Cl2 (qu).
with building block 7, oxidation and then TBDPS group
cleavage led to compounds 37 and 38. Then phosphityla-
tion with building block 4 and oxidation gave pentaphos-
phate intermediate 39. Selective deprotection of the
primary hydroxy groups first by treatment with CSA in

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6243
MMTrO
OH
OMPM
6
5
a
O
P
R
n
O
MMTrO
O
O
OBn
OMPM
OMPM
31: n = 1, R = TBDPS
32: n = 1, R = H
b
b
b
5 a
33: n = 2, R = TBDPS
34: n = 2, R = H
5 a
35: n = 3, R = TBDPS
36: n = 3, R = H
7 a
O
P
O
P
O
O
P
P
OR
OMPM
O
O
O
O
O
O
MMTrO
O
BnO
O
BnO
OMPM
BnO
BnO
OMPM
OMPM
OBn
37: R = TBDPS
38: R = H
b
4 a
O
O
O
P
O
O
P
P
P
P
OR1
R2O
O
BnO
O
O
O
O
O
O
O
O
O
BnO
OMPM
BnO
OMPM
BnO
OMPM
BnO
OMPM
O
AcHN
OBn
O
39: R1 = TBDPS, R2 = MMTr
40: R1 = TBDPS, R2 = H
41: R1 = R2 = H
c
a
OBn
BnO
BnO
Scheme 6. Synthesis of glycerophosphate oligomer 41. Reagents and conditions: (a) tetrazole, CH2Cl2; t-BuO2H (31, 98%; 33, 96%; 35, 94%; 37, 96%;
39, 96%); (b) TBAF, THF (32, 82%; 34, 91%; 36, 92%; 38, 95%; 41, 85%); (c) CSA, MeOH (92%).
4. Conclusion
5. Experimental
5.1. General remarks
In conclusion, retrosynthesis of bisamphiphilic struc-
tural variant 2 of S. aureus LTA led to six building
blocks out of which five required differently modified
chiral glycerol residues. They readily permitted the
assembly of compound 2. The backbone construction
was based on the ligation of different glycerol phosph-
ites possessing, similar to nucleotide synthesis, tempo-
rary O-protecting groups permitting sequence specific
chain extension. Hence, based on this synthesis design
ready access not only to monoamphiphilic S. aureus
LTA 1a, which is also described in detail, but also
to a multitude of structural variants of LTA is avail-
able. As anticipated, bisamphiphilic compound 2 is
more potent in terms of induction of cytokine release
in human leukocytes than natural LTA and its short-
ened version 1a.
Solvents were dried according to standard procedures.
NMR spectroscopic measurements were performed at
22 °C with Bruker DRX600 and Bruker AC250 instru-
ments. TMS or the resonances of the deuterated solvents
were used as internal standard. CDCl3 (d = 7.24 ppm)
was used as external standard; 85% of phosphoric acid
was used as external standard for 31P spectra. MALDI
mass spectra were recorded with a Kratos Kompact
Maldi II spectrometer; 2,5-dihydroxybenzoic acid
(DHB) or p-nitroaniline and NaI were used as matrices
for positive measurements, and trihydroxyacetophenone
(THAP) was used as matrix for negative mode measure-
ments. Optical rotations were measured with a Perkin
Elmer polarimeter 241/MS in a 1-dm cell at 22 °C.

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A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
41
3 a
OBn
O
P
O
P
O
P
O
P
O
BnO
O
OBn
O
O
O
O
O
O
O
O
O
O
O
O
O
BnO
C13H27
O
BnO
OBn
OBn
BnO
BnO
OBn
OBn
O
O
C13H27
O
C13H27
OR
O
AcHN
OBn
C13H27
O
4
O
OBn
BnO
BnO
BnO
O
O
O
BnO
OBn
O
BnO
BnO
42: R = MPM
43: R = H
b
8, c
O
44: R =
NZ
H
Me
d
2
Scheme 7. Synthesis of target molecule 2. Reagents and conditions: (a) tetrazole, CH2Cl2; t-BuO2H (71%); (b) CAN, MeCN/Tol/H2O, ꢀ10 °C ! rt
(76%); (c) PyBOP, Me-Im, CH2Cl2 (49%); (d) Pd(OH)2/C, H2, CH2Cl2/MeOH/H2O (16%).
A
600
450
300
150
0
5.2. General procedure for phosphate formation
TNF
The alcohol and the phosphane (1.2 equiv) were coevap-
orated with dry CH2Cl2 and dried in high vacuum for
1 h. The mixture was dissolved in dry CH2Cl2 and tetra-
zole (2.5 equiv, previously dried for 1 h in high vacuum)
was added. The reaction mixture was stirred for 1.5 h at
room temp (TLC control), and after this time t-BuO2H
(1.3 equiv of 5.5 M solution in decane) was added. The
reaction mixture was stirred for another 30–45 min and
diluted with EtOAc, washed with saturated NaHCO3
solution. The organic phase was dried over MgSO4
and evaporated in vacuo. Purification by flash chroma-
tography on silica gel gave the desired product.
monoamphiphilic LTA
bisamphiphilic LTA
control
3.2 nM
32 nM
320 nM
3.2 µM
40
30
20
10
0
B
IL-8
5.3. General procedure for the removal of tert-butyldi-
phenylsilyl protecting group
monoamphiphilic LTA
bisamphiphilic LTA
The silylated compound was dissolved in THF (p.a.
quality) and treated with TBAF (1.2 equiv of 1 M solu-
tion in THF). The reaction mixture was stirred for 30–
45 min at room temp (monitoring by TLC). After this
time, the reaction mixture was diluted with EtOAc and
washed with saturated NH4Cl solution and water. The
organic phase was dried over MgSO4 and the solvent
evaporated in vacuo. Flash chromatography on silica
gel gave the desired compound.
control
3.2 nM
32 nM
320 nM
3.2 µM
Figure 1. Concentration dependence of TNFa (A) and IL-8 (B) release
in human whole blood in response to 1a and 2 as measured by ELISA.
Data are means ( SEM) of blood from four donors. SEM, standard
error of the mean.
5.4. 6-O-(2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl)-
1,2,3,4-tetra-O-benzoyl-a/b-D-glucopyranose (9)
Thin-layer chromatography (TLC) was performed on
Merck silica gel 60 F254 plastic plates. Compounds
were visualized by treatment with a solution of
(NH4)6Mo7O24 Æ 4H2O (20 g) and Ce(SO4)2 (0.4 g) in
10% sulfuric acid (400 mL). Flash chromatography was
performed on J. T. Baker silica gel 60 (0.040–0.063 mm)
at a pressure of 0.3 bar. Target molecules were purified
by Hydrophobic Interaction Chromatography on
octylsepharose as stationary phase and as elution phase
was used as a gradient of propanol (15–60%) in 0.1 M
ammonium acetate buffer (pH 4.8).
Gentiobiose (14.13 g, 41.28 mmol) was dissolved in pyr-
idine (375 mL), benzoyl chloride was added (50 mL,
0.48 mol) and the reaction mixture was stirred at 40 °C
overnight. The solvent was removed and the crude mate-
rial purified by flash chromatography (petroleum ether/
EtOAc, 2:1) to give 9 in 98% yield (48 g) as a white solid.
TLC (petroleum ether/EtOAc 3:2): Rf = 0.45; a:b ꢁ 1:1.
1
[a]D +15.8 (c 1, CHCl3). H NMR (250 MHz, CDCl3):
d = 3.40–4.17, 4.18–4.30, 4.35–4.65 (m, 6 H, 6a, 6b, 5a,
5b-H), 5.05 (d, 0.5 H, J1,2 = 7.8 Hz, 1b-H), 5.40–5.78,
5.88–6.01 (m, 6 H, 2a, 2b, 3a, 3b, 4a, 4b-H), 6.15 (d,

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6245
0.5 H, J1,2 = 8.2 Hz, 1a), 7.20–7.65, 7.76–8.15 (m, 35 H,
Ph). MALDI-MS (positive Mode, Matrix DHB, diox-
ane): [M+Na]+, m/z = 1198.2; found: m/z = 1198.6,
[M+K]+, m/z = 1214.3; exp.: m/z = 1215.7. C68H54O19
(1175.2): Calcd: C, 69.50; H, 4.63. Found: C, 69.15; H,
4.74.
CDCl3): d = 1.22, 1.26 (2s, 6 H, C(CH3)2), 3.42 (dd, 1
H, Jgem = 10.6 Hz, Jvic = 5.6 Hz, 10-H), 3.62 (dd, 1 H,
Jgem = 8.0 Hz, Jvic = 6.7 Hz, 30-H), 3.69 (dd, 1 H,
Jgem = 10.6 Hz, Jvic = 4.0 Hz, 10-H), 3.81 (dd, 1 H,
Jgem = 8.1 Hz, Jvic = 6.5 Hz, 30-H), 3.85 (dd, 1 H,
Jgem = 11.4 Hz, Jvic = 7.6 Hz, 6a-H), 3.96–4.03 (m, 1
H, 5a-H), 4.05 (dd, 1 H, Jgem = 11.4 Hz, Jvic = 1.5 Hz,
6a-H), 4.09–4.16 (m, 1 H, 5b-H), 4.42 (dd, 1 H,
Jgem = 12.1 Hz, Jvic = 5.1 Hz, 6b-H), 4.59 (dd, 1 H,
Jgem = 12.1 Hz, Jvic = 2.9 Hz, 6b-H), 4.71 (d, 1 H,
J1,2 = 7.9 Hz, 1a-H), 4.97 (d, 1 H, J1,2 = 7.8 Hz, 1b-H),
5.31 (dd, 1 H, J4,5 = J4,3 = 9.7 Hz, 4a-H), 5.37 (dd, 1
H, J2,1 = 8.1 Hz, J2,3 = 9.6 Hz, 2a-H), 5.50 (dd, 1 H,
J2,1 = 8.0 Hz, J2,3 = 9.6 Hz, 2b-H), 5.62 (dd, 1 H,
5.5. 6-O-(2,3,4,6-Tetra-O-benzoyl-b-D-glucopyranosyl)-
2,3,4-tri-O-benzoyl-a/b-D-glucopyranose (10)
A solution of 9 (48 g, 40.85 mmol) in dry DMF
(425 mL) was heated to 50 °C and hydrazinium acetate
was added (5.4 g). After 3.5 h, the solution was diluted
with CH2Cl2 and washed with water. The organic phase
was dried over MgSO4 and the solvent evaporated. The
crude material was purified by flash chromatography
(toluene/EtOAc 7:1) to give 10 in 78% of yield
(34.12 g) as a colourless solid. TLC (toluene/EtOAc
J4,5 = J4,3 = 9.7 Hz,
J3,4 = J3,2 = 9.6 Hz,
4b-H),
3a-H),
5.78
5.89
(dd,
(dd,
1
1
H,
H,
J3,4 = J3,2 = 9.6 Hz, 3b-H), 7.17–7.58, 7.71–8.05 (m, 35
H, Ph). 13C NMR (150.9 MHz, CDCl3): d = 25.38,
26.41 (2 C, C(CH3)2), 62.94 (1 C, C-6b), 66.07 (1 C,
C-30), 68.51 (1 C, C-6a), 68.83 (1 C, C-10), 69.51 (1 C,
C-4b), 69.83 (1 C, C-4a), 71.67 (1 C, C-2a), 71.86 (1
C, C-2b), 72.28 (1 C, C-5b), 72.78 (2 C, C-3a, C-3b),
73.88 (1 C, C-5a), 74.09 (1 C, C-20), 100.93 (1 C, C-
1a), 101.27 (1 C, C-1b), 128.24–133.47 (42 C, Ph),
165.04–166.06 (7 C, CO-Ph). MALDI-MS (positive
Mode, Matrix DHB, THF): [M+Na]+, m/z = 1208.2;
found: m/z = 1208.9, [M+K]+, m/z = 1224.3; found: m/
z = 1225.3. C67H60O20 (1185.2): Calcd: C, 67.90; H,
5.10. Found: C, 67.69; H, 5.01.
1
6:1): Rf = 0.30. [a]D +30.6 (c 0.34, CHCl3). H NMR
(250 MHz, CDCl3): d = 3.14 (br s, 1 H, OH), 3.62–
4.24, 4.38–4.59, 4.63–4.82 (m, 6 H, 6a, 6b, 5a, 5b-H),
4.98 (d, 1 H, J1,2 = 7.6 Hz, 1b-H), 5.09–6.22 (m, 7 H,
1a, 2a, 2b, 3a, 3b, 4a, 4b-H), 7.20–7.65, 7.74–8.17 (m,
30 H, Ph). C61H50O18Æ0.5 H2O (1080.1): Calcd: C,
67.84; H, 4.76. Found: C, 67.80; H, 4.75.
5.6. O-[6-O-(2,3,4,6-tetra-O-benzoyl-b-D-glucopyrano-
syl)-2,3,4-tri-O-benzoyl-a/b-D-glucopyranosyl] trichloro-
acetimidate (11)
Compound 10 (32.11 g, 29.98 mmol) was dissolved in
dry CH2Cl2 (450 mL), and Cl3CCN (30.5 mL, 0.3 mol)
was added; subsequently DBU was added (0.45 mL).
After 2 h, the solvent was removed in vacuo and the rest
was purified by flash chromatography (petroleum ether/
EtOAc 2:1, +1% Et3N) to give 11 in 97% of yield
(35.35 g) as a slightly yellow foam. TLC (petroleum
ether/EtOAc 2:1): Rf = 0.33. [a]D = +26 (c = 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 3.40–4.17, 4.18–4.30,
4.35–4.65 (m, 6 H, 6a, 6b, 5a, 5b-H), 5.05 (d, 0.5 H,
J1,2 = 7.8 Hz, 1b-H), 5.40–5.78, 5.88–6.01 (m, 6 H, 2a,
2b, 3a, 3b, 4a, 4b-H), 6.15 (d, 0.5 H, J1,2 = 8.2 Hz, 1a),
7.20–7.65, 7.76–8.15 (m, 35 H, Ph). C63H50O18NCl3Æ0.5
H2O (1224.5): Calcd: C. 61.80; H: 4.20; N: 1.14; found:
C: 61.68, H: 4.17, N: 0.97.
5.8. 1,2-O-Isopropylidene-3-O-[6-O-(-b-D-glucopyrano-
syl)-b-D-glucopyranosyl]-sn-glycerol (14)
To a solution of 13 (19.14 g, 16.15 mmol) in dry MeOH
(300 mL) and dry CH2Cl2 (20 mL) was added NaH
(0.81 g, 33.75 mmol) and stirred at rt. After 12 h, the
reaction mixture was neutralized with amberlite IR-120
H+, filtered and evaporated in vacuo with a little amount
of Et3N. The solid was dissolved in a mixture of dioxane/
water and lyophilized, 14 was obtained in quantitative
yield (7.37 g) as a yellow solid. TLC (EtOAc/MeOH
5:2): Rf = 0.28. [a]D ꢀ19.1 (c = 1, CHCl3). 1H NMR
(600 MHz, CD3OD): d = 1.31, 1.38 (2s, 6 H, C(CH3)2),
3.14–3.23 (m, 2 H, 2a, 2b-H), 3.26 (m, 1 H, 5b-H), 3.33
(m, 2 H, 3a, 3b-H), 3.42–3.48 (m, 1 H, 5a-H), 3.63 (dd,
1 H, J1 ,2 = 6.0 Hz, Jgem = 10.5 Hz, 10-H), 3.65 (dd, 1
H, Jgem = 11.8 Hz, Jvic = 5.2 Hz, 6b-H), 3.76 (dd, 1 H,
Jgem = 11.6 Hz, Jvic = 5.9 Hz, 6a-H), 3.81 (dd, 1 H,
Jgem = 8.4 Hz, Jvic = 6.1 Hz, 30-H), 3.86 (dd, 1 H,
Jgem = 11.3 Hz, Jvic = 1.6 Hz, 6b-H), 3.89 (dd, 1 H,
Jgem = 10.5 Hz, Jvic = 5.5 Hz, 10-H), 4.07 (dd, 1 H,
Jgem = 8.3 Hz, Jvic = 6.5 Hz, 30-H), 4.14 (dd, 1 H,
Jgem = 11.5 Hz, Jvic = 1.7 Hz, 6a-H), 4.29 (d, 1 H,
J1,2 = 7.8 Hz, 1a-H), 4.31 (m, 1 H, 20-H), 4.36 (d, 1 H,
J1,2 = 7.8 Hz, 1b-H). 13C NMR (150.9 MHz, CD3OD):
d = 25.62 (1 C, C(CH3)2), 27.09 (1 C, C(CH3)2), 62.75
(1 C, C-6b), 67.75 (1 C, C-30), 69.94 (1 C, C-6a), 71.45-
71.59 (3 C, C-10, C-4a/b, C-5b), 74.98, 75.10 (2 C, C-
2a, C-2b), 75.86 (1 C, C-20), 77.06 (1 C, C-5a), 77.85,
78.02 (3 C, C-4a/b, C-3a, C-3b), 104.68 (2 C, C-1a, C-
1b), 104.92 (1 C, C(CH3)2). MALDI-MS (positive Mode,
Matrix DHB, dioxane): [M+Na]+, m/z = 479.4; found:
m/z = 479.0, [M+K]+, m/z = 495.5. Found: m/z = 494.8.
0
0
5.7. 1,2-O-Isopropylidene-3-O-[6-O-(2,3,4,6-tetra-O-ben-
zoyl-b-D-glucopyranosyl)-2, 3, 4-tri-O-benzoyl-b-D-gluco-
pyranosyl]-sn-glycerol (13)
To a solution of 1216 (4.2 mL, 1.2 equiv) and donor 11
(35 g, 28.8 mmol) in dry CH2Cl2 (450 mL) molecular
˚
sieves (3 A) were added and the reaction mixture was
stirred for 15 min at rt under argon atmosphere. Subse-
quently the reaction mixture was cooled to ꢀ30 °C and
BF3ÆEt2O solution (0.2 equiv, 0.75 mL) was added. After
20 min, the reaction mixture was neutralized with NEt3,
evaporated to half volume, rediluted with toluene, and
the solvent was removed in vacuo. The crude material
was purified by flash chromatography (petroleum
ether/EtOAc 2:1, +1% NEt3) to give 13 in 80% of yield
(27.3 g; 23.04 mmol). TLC (toluene/EtOAc 6:1):
1
Rf = 0.3. [a]D +3.6 (c 1, CHCl3). H NMR (600 MHz,

6246
A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
5.9. 1,2-O-Isopropylidene-3-O-[6-O-(6-O-tert-butyldiphe-
nylsilyl-b-D-glucopyranosyl)-b-D-glucopyranosyl]-sn-glyc-
erol (15)
red for 1.5 h at 80 °C. The solvent was removed in
vacuo and the rest was coevaporated twice with
toluene. Purification by flash chromatography (petro-
leum ether/EtOAc 2:1) gave compound 17 (3.02 g) in a
78% yield as a colourless foam. TLC (petroleum ether/
EtOAc 2:1): Rf = 0.25. [a]D +9.4 (c 1, CHCl3). 1H
NMR (600 MHz, CDCl3): d = 1.05 (s, 9 H, t-Bu), 2.0
(br s, 1 H, OH), 2.8 (br s, 1 H, OH), 3.31–3.35 (m, 1
H, 5b-H), 3.38–3.45 (m, 3 H, 10/30-H, 4a-H, 2a-H),
3.50 (dd, 1 H, J2,3 = J2,1 = 8.5 Hz, 2b-H), 3.56 (m, 1
H, 10-H, 30-H), 3.62, 3.65 (m, 2 H, 10/30-H), 3.64 (m, 1
H, 5a-H), 3.65 (m, 1 H, 3b-H), 3.68 (m, 1 H, 3a-H),
3.70 (m, 1 H, 6a-H), 3.73 (m, 1 H, 20-H), 3.74 (m, 1
H, 4b-H), 3.92 (m, 2 H, 6b-H), 4.18–4.23 (m, 1 H,
6a-H), 4.37 (d, 1 H, J1,2 = 7.8 Hz, 1a-H), 4.48 (d, 1 H,
J1,2 = 7.8 Hz, 1b-H), 4.51–4.56, 4.65–4.99 (m, 12 H,
CH2Ph), 7.12–7.46, 7.66–7.77 (m, 40 H, Ph). MALDI-
MS (positive Mode, Matrix DHB, THF): [M+Na]+,
m/z = 1218.5; found: m/z = 1217.2, [M+K]+, m/z = 1234.6;
found: m/z = 1234.1. C73H82O13Si (1195.5): Calcd: C,
73.34; H, 6.91. Found: C, 73.06; H, 6.91.
A solution of 14 (7.35 g, 16.1 mmol) in pyridine (150 mL)
was cooled to ꢀ15 °C and 5 mL TBDPS-Cl (1.2 equiv,
9.2 mmol) was added dropwise. The reaction mixture
was stirred for 72 h, quenched with MeOH, evaporated
in vacuo and coevaporated with toluene. After flash chro-
matography (EtOAc/MeOH 4:1), product 15 (10.2 g, 91
%) was obtained. TLC (EtOAc/MeOH 3:2): Rf = 0.60.
[a]D ꢀ25.5 (c 1, CHCl3). 1H NMR (250 MHz, CD3OD):
d = 1.02 (s, 9 H, t-Bu), 1.31, 1.39 (2s, 6 H, C(CH3)2),
3.18–4.42 (m, 19 H), 7.3–7.46, 7.63–7.80 (m, 10 H, Ph).
MALDI-MS (positive Mode, Matrix DHB, dioxane):
[M+Na]+, m/z = 717.85; found: m/z = 717.1, [M+K]+,
m/z = 733.95; found: m/z = 733.1.
5.10. 1,2-O-Isopropylidene-3-O-[6-O-(2, 3, 4-tri-O-ben-
zyl-6-O-tert-butyldiphenylsilyl-b-D-glucopyranosyl)-
2,3,4-tri-O-benzyl-b-D-glucopyranosyl]-sn-glycerol (16)
To a solution of compound 15 (6.2 g, 8.92 mmol) in
180 mL of dry DMF was added benzyl bromide (10 equiv,
10.6 mL) at 10 °C. Then NaH (15 equiv, 2.1 g) was added
portionwise, and the reaction mixture was stirred for 2 h
at rt. The solvent was evaporated in vacuo; the residue
was redissolved in EtOAc and washed two times with sat-
urated NH4Cl solution. The organic phase was dried over
MgSO4 and the solvent was removed in vacuo. After flash
chromatography (petroleum ether/EtOAc 6:1), com-
pound 16 (7.28 g, 66%) was obtained. TLC (petroleum
5.12. 1,2-Di-O-myristoyl-3-O-[6-O-(2,3,4-tri-O-benzyl-6-
O-tert-butyldiphenylsilyl-b-D-glucopyranosyl)-2,3,4-tri-
O-benzyl-b-D-glucopyranosyl]-sn-glycerol (18)
To a solution of compound 17 (3.5 g, 2.93 mmol) in dry
THF (140 mL), Et3N (4.5 mL, 11 equiv) and myristoyl
chloride (4.76 mL, 6 equiv) were added. The reaction
mixture was stirred at 50 °C; after 4 h it was diluted with
EtOAc and washed with saturated NH4Cl solution. The
organic phase was dried over MgSO4, and the solvent
was evaporated in vacuo. Purification by flash chroma-
tography (petroleum ether/EtOAc 8:1) yielded com-
pound 18 (3.84 g, 81%) as a colourless syrup. TLC
(petroleum ether/EtOAc 5:1): Rf = 0.6. [a]D +5 (c = 1,
1
ether/EtOAc 4:1): Rf = 0.5. [a]D +9.3 (c 1, CHCl3). H
NMR (600 MHz, CDCl3): d = 1.04 (s, 9 H, t-Bu), 1.30,
1.35 (2s, 6 H, C(CH3)2), 3.29 (m, 1 H, 5b-H), 3.42 (m, 1
H, 10/30-H), 3.43 (m, 1 H, 2a-H), 3.46 (m, 1 H, 4a-H),
3.50 (m, 1 H, 2b-H), 3.57 (m, 1 H, 5a-H), 3.63 (m, 1 H,
3b-H), 3.64 (m, 1 H, 10/30-H), 3.65 (m, 1 H, 3a-H), 3.68
(m, 1 H, 6a-H), 3,75 (dd, 1 H, J4,5 = J4,3 = 9.4 Hz, 4b-
H), 3.69–3.95 (m, 4 H, 10/30-H, 6b-H, 10/30-H), 4.16 (m,
1 H, 20-H), 4.22 (m, 1 H, 6a-H), 4.37 (d, 1 H,
J1,2 = 7.8 Hz, 1a-H), 4.46 (d, 1 H, J1,2 = 7.8 Hz, 1b-H),
4.51–4.55, 4.65–4.83, 4.85–4.95, 4.97–5.03 (m, 12 H,
CH2Ph), 7.10–7.43, 7.65–7.79 (m, 40 H, Ph). 13C NMR
(150.9 MHz, CDCl3): d = 19.28 (1 C, C(CH3)3), 25.35 (1
C, C(CH3)2), 26.79 (4 C, C(CH3)2, C(CH3)3), 62.69 (1 C,
C-6b), 66.51 (1 C, C-10/30), 68.25 (1 C, C-6a), 70.16 (1 C,
C-10/30), 74.29 (1 C, C-20), 74.83–76.3 (8 C, CH2Ph, C-
5a, C-5b), 77.59 (1 C, C-4b), 78.09 (1 C, C-4a), 82.13 (1
C, C-2a), 82.41 (1 C, C-2b), 84.65 (1 C, C-3a), 84.87 (1
C, C-3b), 103.69 (1 C, C-1a), 104.03 (1 C, C-1b),
127.58–138.54 (40 C, Ph). MALDI-MS (positive Mode,
Matrix DHB, THF): [M+Na]+, m/z = 1258.6; found: m/
z = 1257.7, [M+K]+, m/z = 1274.7; found: m/z = 1273.9.
C76H86O13Si (1235.6): Calcd: C, 73.88; H, 7.02. Found:
C, 73.95; H, 7.34.
1
CH2Cl2). H NMR (600 MHz, CDCl3): d = 0.86–0.87
(t, 6 H, Me), 1.04 (s, 9 H, t-Bu), 1.09–1.38 (s, 40 H,
CH2-chain), 1.45–1.62 (m, 4 H, COCH2CH2R), 2.12–
2.28 (m, 4 H, COCH2CH2R), 3.30 (m, 1 H, 5b-H),
3.41 (dd, 1 H, J2,3 = J2,1 = 8.5 Hz, 2a-H), 3.47 (m, 2 H,
10/30-H, 4a-H), 3.50 (m, 1 H, 2b-H), 3.55 (m, 1 H, 5a-
H), 3.62 (m, 1 H, 3b-H), 3.65 (m, 1 H, 6a-H), 3.76
(dd, 1 H, J4,5 = J4,3 = 9.4 Hz, 4a-H), 3.92 (m, 1 H, 6b-
H), 3.96 (m, 1 H, 10/30-H), 4.09–4.15 (m, 1 H, 10/30-H),
4.18–4.26 (m, 2 H, 10/30-H, 6a-H), 4.33 (d, 1 H,
J1,2 = 7.8 Hz, 1a-H), 4.42 (d,
1 H, J1,2 = 7.8 Hz,
1b-H), 4.50-4.55, 4.64-4.84, 4.86-4.95, 4.97-5.03 (m, 12
H, CH2Ph), 5.13 (m, 1 H, 20-H), 7.10-7.43, 7.64-7.77
(m, 40 H, Ph). MALDI-MS (positive Mode, Matrix
DHB, THF): [M+Na]+, m/z = 1639.2; found: m/
z = 1638.5, [M+K]+, m/z = 1655.3; found:m/z = 1654.5.
C101H134O15Si (1616.2): Calc.: C, 75.06; H, 8.36. Found:
C, 74.81; H, 8.67.
5.13. 1,2-Di-O-myristoyl-3-O-[6-O-(2,3,4-tri-O-benzyl-b-
D-glucopyranosyl)-2,3,4-tri-O-benzyl-b-D-glucopyrano-
syl]-sn-glycerol (19)
5.11. 3-O-[6-O-(2,3,4-Tri-O-benzyl-6-O-tert-butyldiphe-
nylsilyl-b-D-glucopyranosyl)-2,3,4-tri-O-benzyl-b-D-
glucopyranosyl]-sn-glycerol (17)
Compound 18 (3.84 g, 2.38 mmol) was dissolved in dry
THF (200 mL), CH3COOH (0.6 mL, 4 equiv) and
TBAF (1 M solution, 9.5 mL, 4 equiv) were added and
the reaction mixture was stirred for 72 h at 40 °C. The
To a solution of 16 (4 g, 3.24 mmol) in THF (100 mL),
200 mL of 75% CH3COOH in water was added and stir-

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6247
reaction mixture was diluted with AcOEt (500 mL) and
washed with half saturated NH4Cl solution (300 mL),
the organic phase was dried over MgSO4 and the solvent
was evaporated in vacuo. Purification by flash chroma-
tography (petroleum ether/EtOAc 4:1 ! 3:1) yielded
compound 19 (2.48 g, 76%) as a white solid. TLC (petro-
leum ether/EtOAc 3:1): Rf = 0.45. Mp 89.4 °C. ꢀ[a]D
+11.9 (c 1, CHCl3). 1H NMR (600 MHz, CDCl3):
d = 0.84–0.93 (t, 6 H, Me), 1.11–1.38 (s, 40 H, CH2-
chain), 1.47–1.63 (m, 4 H, COCH2CH2R), 2.02–2.14
(s, 1 H, OH), 2.14–2.29 (m, 4 H, COCH2CH2R), 3.31–
3.36 (m, 1 H, 5b-H), 3.37–3.48 (m, 3 H, 2a-H, 2b-H,
4a-H), 3.48–3.57 (m, 3 H, 5a-H, 4b-H, 10-H), 3.60-3.73
(m, 4 H, 3a-H, 3b-H, 6a-H, 6b-H), 3.81–3.87 (m, 1 H,
6b-H), 3.92 (dd, 1 H, Jgem = 10.9 Hz, Jvic = 4.5 Hz, 10-
H), 4.08 (dd, 1 H, Jgem = 11.1 Hz, Jvic< 1 Hz, 6a-H),
4.11–4.17 (m, 1 H, 30-H), 4.25 (dd, 1 H, Jgem = 11.9 Hz,
Jvic = 3.3 Hz, 30-H), 4.31 (d, 1 H, J1,2 = 7.8 Hz, 1a-H),
4.45 (d, 1 H, J1,2 = 7.8 Hz, 1b-H), 4.51–4.56, 4.61–
4.70, 4.73–4.87, 4.89–4.96 (m, 12 H, CH2Ph), 5.13–
5.19 (m, 1 H, 20-H), 7.13–7.37 (m, 30 H, Ph). 13C
NMR (150.9 MHz, CDCl3): d = 14.10–34.23 (26 C,
CH2-chain), 62.04 (1 C, C-6b), 62.69 (1 C, C-30), 68.05
(1 C, C-10), 68.81 (1 C, C-6a), 69.87 (1 C, C-20), 74.69-
77.0 (8 C, C-5a, C-5b, CH2Ph), 77.58 (1 C, C-4b),
77.79 (1 C, C-4a), 81.88 (1 C, C-2a), 82.08 (1 C, C-
2b), 84.50, 84.55 (2 C, C-3a, C-3b), 103.75 (1 C, C-1a),
103.93 (1 C, C-1b), 127.61–138.43 (36C, Ph), 172.97,
173.29 (2 C, COCH2CH2R). MALDI-MS (positive
Mode, Matrix DHB, THF): [M+Na]+, m/z = 1400.8;
found: m/z = 1399.9, [M+K]+, m/z = 1416.9; found: m/
z = 1417.2. C85H116O15 (1377.8): Calcd: C, 74.10; H,
8.49. Found: C, 74.06; H, 8.61.
J1,2 = 7.8 Hz, 1b-H), 4.45–4.52, 4.62–4.84, 4.84–4.97
(m, 14 H, CH2Ph), 5.08–5.15 (m, 1 H, 20-H), 7.06–7.44
(m, 35 H, Ph). 13C NMR (150.9 MHz, CDCl3):
d = 14.11–34.23 (30 C, 2 (NCH(CH3)2), CH2-chain),
43.1, 43.18 (2 C, 2 (NCH(CH3)2)), 62.46, 62.70 (2 C,
C-10, C-6b), 65.21–65.39 (1 C, POCH2Ph), 67.99 (1 C,
C-30), 68.41 (1 C, C-6a), 69.81 (1 C, C-20), 74.66–77.0
(8 C, C-5a, C-5b, CH2Ph), 77.74/77.85 (1 C, C-4b),
78.05 (1 C, C-4a), 81.91–82.15 (2 C, C-2a, C-2b),
84.51–84.78 (2 C, C-3a, C-3b), 103.65 (1 C, C-1a),
103.92–103.98 (1 C, C-1b), 126.84–138.61 (42 C, Ph),
172.88, 173.24 (2 C, COCH2CH2R). 31P NMR
(242.9 MHz, CDCl3): d = 149.79, 149.89 (2s, 1 P).
C98H136NO16P (1615.1): Calcd: C, 72.88; H, 8.49; N,
0.87. Found: C, 72.61; H, 8.69; N, 0.88.
5.15. Hexaphosphate 21
Compound 2013 (494 mg, 0.202 mmol) and phosphite
amide 3 (424 mg, 1.3 equiv) were coevaporated each
with dry CH2Cl2 (10 mL) and dried for 1 h in high vac-
uum. Compound 3 was dissolved in 15 mL of dry
CH2Cl2 and was added, under argon atmosphere, to
compound 20; tetrazole (29 mg, 2 equiv, dried previous-
ly for 1 h in high vacuum) was also added. The reaction
mixture was stirred at rt under argon atmosphere. After
70 min, t-BuO2H (0.6 mL) was added dropwise and the
reaction mixture was stirred for another 35 min. CH2Cl2
was added and the mixture was washed with saturated
NaHCO3 solution, the organic phase was dried over
MgSO4 and the solvent was removed in vacuo. Purifica-
tion by flash chromatography (toluene/acetone 1:1) gave
compound 21 (602 mg, 75%) as a colourless syrup,
which was stored at ꢀ20 °C. TLC (toluene/acetone
1
5.14. [Benzyloxy]-[diisopropylamino]-[1,2-di-O-myristoyl-
3-O-{6-O-(2,3,4-tri-O-benzyl-b-D-glucopyranosyl)-2,3,4-
tri-O-benzyl-b-D-glucopyranosyl}-sn-glycero]phosphane
(3)
1:1): Rf = 0.35, Rf = 0.42. [a]D +12.1 (c 1, CHCl3). H
NMR (600 MHz, CDCl3): d = 0.81–0.92 (t, 6 H, Me),
1.11–1.35 (m, 40 H, CH2-chain), 1.44–1.61 (m, 4 H,
COCH2CH2R), 1.84–1.98 (m, 3 H, NHAc), 2.09–2.25
(m, 4 H, COCH2CH2R), 3.32 (m, 1 H, 5a/b-H), 3.33
(m, 1 H, 2a-H), 3,35 (m, 1 H, 10-H), 3.37 (m, 1 H, 5a/
b-H), 3.38 (m, 1 H, 2b-H), 3.43, 3.44 (m, 2 H, 4a-H,
4b-H), 3.49 (m, 2 H, 18-H), 3.57 (m, 2 H, 3a-H, 3b-
H), 3.58-3.60 (m, 3 H, 6c-H, 6a-H), 3.63 (m, 4 H, 5-H,
8-H, 11-H, 14-H), 3.64 (m, 1 H, 4c-H), 3.67 (m, 12 H,
OMe), 3.68 (m, 1 H, 3c-H), 3.69 (m, 1 H, 2-H), 3.72
(m, 1 H, 17-H), 3.75 (m, 1 H, 5c-H), 3.83 (m, 1 H, 10-
H), 3.96, 4.04 (m, 18 H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-
H, 12-H, 13-H, 15-H), 4.06, 4.15 (m, 2 H, 30-H), 4.08
(m, 1 H, 6a-H), 4.07, 4.17 (m, 4 H, 16-H, 6b-H), 4.20
(m, 1 H, 1a-H), 4.35 (m, 1 H, 2c-H), 4.41 (m, 1 H, 1b-
H), 4.74 (m, 1 H, 1c-H), 4.29–4.91 (m, 30 H, CH2Ph),
4.93 (m, 12 H, POCH2Ph), 5.06 (m, 1 H, 20-H), 6.69–
6.80 (m, 8 H, PhMPM), 7.03–7.34 (m, 93 H, Ph). 13C
NMR (150.9 MHz, CDCl3): d = 14.11 (2 C, Me), 23.1
(1 C, NHAc), 24.84 (2 C, COCH2CH2R), 22.68/29.14–
31.91 (20 C, CH2-chain), 34.04, 34.22 (2 C, COCH2R),
52.8 (1 C, C-2c), 55.17 (4 C, OMe), 63.2 (1 C, C-30),
65.5–67.2 (12 C, CH2-Glyc, C-6b), 68.1 (1 C, C-10), 68.5
(1 C, C-6c), 68.6 (1 C, C-6a), 69.0 (1 C, C-18), 69.5 (6
C, POCH2Ph), 69.8 (1 C, C-20), 72 (1 C, C-5c), 73.5 (1
C, C-5a/b), 75 (1 C, C-4a/b), 75.4 (4 C, C-5, C-8, C-
11, C-14), 72-77.0 (15 C, CH2Ph), 76.8 (1 C, C-17),
77.5 (1 C, C-4c), 78.2 (1 C, C-2), 81.2 (1 C, C-3c), 81.8
Compound 19 (0.7 g, 0.508 mmol) was dried for 1 h
together with tetrazole (21.4 mg, 0.6 eq) in high vacuum.
Under argon atmosphere benzyloxybis-(diisopropylami-
no)-phosphane (215 mg, 1.3 equiv) dissolved in 10 mL
of dry CH2Cl2 was added. After 30 min of stirring at
rt the tetrazole was dissolved, the reaction mixture was
stirred for 1.5 h; then the mixture was diluted with
CH2Cl2 and poured over a saturated NaHCO3solution.
The organic phase was dried over MgSO4 and removed
in vacuo below 30 °C. Fast purification over flash silica
gel (petroleum ether/EtOAc 5:1, +1% NEt3) yielded
compound 3 (650 mg, 79%) after one co-evaporation
with toluene as a colourless syrup. TLC (petroleum
ether/EtOAc 5:1, +1% NEt3): Rf = 0.9. [a]D +7.5 (c 1,
CHCl3). 1H NMR (600 MHz, CDCl3): d = 0.80–0.92
(t, 6 H, Me), 1.06–1.38 (s, 40 H, CH2-chain), 1.45–1.62
(m,
4
H, COCH2CH2R), 2.10–2.58 (m,
4
H,
COCH2CH2R), 3.38 (m, 1 H, 2a-H), 3.39 (m, 1 H, 5b-
H), 3.41 (m, 1 H, 4a-H), 3.42 (m, 1 H, 2b-H), 3.43 (m,
1 H, 30-H), 3.51 (m, 1 H, 5a-H), 3.54/3.59 (m, 1 H, 4b-
H), 3.61 (m, 2 H, 3a-H, 3b-H), 3.65 (m, 3 H,
NCH(CH3)2, 6a-H), 3.79, 3.89, 4.00 (m, 2 H, 6b-H),
3.92 (m, 1 H, 30-H), 4.07–4.24 (m, 3 H, 6a-H, 10-H),
4.28 (d, 1 H, J1,2 = 7.8 Hz, 1a-H), 4.43 (d, 1 H,

6248
A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
(2 C, C-2a, C-2b), 84.6 (2 C, C-3a, C-3b), 100.05 (1 C, C-
1c), 103.1 (1 C, C-1a), 103.6 (1 C, C-1b). MALDI-MS
(positive Mode, Matrix p-nitroaniline+NaI, THF):
ed. N-Methyl imidazole (180 lL, 40 equiv) was added
dropwise and the reaction mixture was stirred for 2.5–
3 h at rt under argon atmosphere. The reaction mixture
was diluted with CH2Cl2 and washed with saturated
NH4Cl solution. The organic phase was dried over
MgSO4 and the solvent was removed in vacuo. Purifica-
tion by flash chromatography (toluene/acetone 3:1) and
second column (toluene/acetone 3:1) yielded 23a as a
mixture of diastereomers (170 mg, 70%) as a colourless
syrup which was stored at ꢀ20 °C. TLC (toluene/ace-
tone 1:1): Rf = 0.71, Rf = 0.75. [a]D +15 (c 0.15, CHCl3).
1H NMR (600 MHz, CDCl3): d = 0.88 (t, 6 H, Me),
1.12–1.41 (m, 52 H, CH2-chain, Ala-Me), 1.46–1.59
(m, 4 H, COCH2CH2R), 1.86–1.99 (m, 3 H, NHAc),
2.10–2.26 (m, 4 H, COCH2CH2R), 3.33 (m, 1 H, 5a/b-
H), 3.34 (m, 1 H, 2a-H), 3.35 (m, 1 H, 5a/b-H), 3.36
(m, 1 H, 10-H), 3.39 (m, 1 H, 2b-H), 3.44, 3.46 (m, 2
H, 4a-H, 4b-H), 3.52 (m, 2 H, 18-H), 3.57 (m, 2 H,
3a-H, 3b-H), 3.59 (m, 2 H, 6c-H), 3.67 (m, 1 H, 6a-H),
3.69 (m, 2 H, 4c-H, 3c-H), 3.74 (m, 2 H, 2-H, 17-H),
3.76 (m, 1 H, 5c-H), 3.85 (m, 1 H, 10-H), 4.01 (m, 20
H, 1-H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H,
15-H), 4.07 (m, 1 H, 16-H), 4.08 (m, 1 H, 30-H), 4.10
(m, 1 H, 6a-H), 4.16 (m, 1 H, 30-H), 4.18 (m, 3 H, 6b-
H, 16-H), 4.21 (m, 1 H, 1a-H), 4.33 (m, 4 H,
CHNHCbz), 4.34 (m, 1 H, 2c-H), 4.32-4.92 (m, 22 H,
CH2Ph), 4.43 (m, 1 H, 1b-H), 4.79 (m, 1 H, 1c-H),
4.98 (m, 4 H, CH2Cbz), 4.99 (m, 12 H, POCH2Ph),
5.05 (m, 4 H, CH2Cbz), 5.08 (m, 1 H, 20-H), 5.09 (m,
4 H, 5, 8, 11, 14-H), 5.56–6.14 (NH), 7.04–7.46 (m,
105 H, Ph). 13C NMR (150.9 MHz, CDCl3): d = 14.4
(2 C, Me), 18.3 (4 C, Ala-Me), 23.1 (1 C, NHAc), 25.2
(2 C, COCH2CH2R), 29.8 (2 0C, CH2-chain), 34.3,
34.5 (2 C, COCH2R), 49.9 (4 C, CHNHCbz), 53.0 (1
C, C-2c), 63.0 (1 C, C-30), 65.1 (10 C, C-1, C-3, C-4,
C-6, C-7, C-9, C-10, C-12, C-13, C-15), 66.9 (1 C, C-
6b), 67.0 (4 C, CH2Cbz), 67.5 (1 C, C-16), 68.3 (1 C,
C-10), 68.9 (2 C, C-6a, C-6c), 69.1 (1 C, C-18), 70.0 (1
C, C-20), 70.1 (6 C, POCH2Ph), 71.0 (4 C, CH-Ala),
72.0 (1 C, C-5c), 72.2-76.0 (11 C, CH2Ph), 73.8 (1 C,
C-5a/b), 75.3 (1 C, C-4a/b), 76.8 (2 C, C-2, C-17), 77.1
(1 C, C-4a/b), 77.9 (1 C, C-4c), 78.0 (1 C, C-5a/b),
81.1 (1 C, C-3c), 82.1 (2 C, C-2a, C-2b), 84.8 (2 C, C-
3a, C-3b), 100.2 (1 C, C-1c), 103.8 (1 C, C-1a), 104.1
(1 C, C-1b). MALDI-MS (positive Mode, Matrix p-nit-
roaniline+NaI, MeOH): [M+Na]+, m/z = 4340; found:
m/z = 4336. C232H281N5O62P6 (4317.6): Calcd: C,
64.54; H, 6.56; N, 1.62. Found: C, 64.34; H, 6.75; N,
1.51.
[M+Na]+,
m/z = 4002.3;
found:
m/z = 4000.0.
C220H271NO54P6 (3979.3): Calcd: C, 66.40; H, 6.86; N,
0.35. Found: C, 66.40; H, 7.09; N, 0.36.
5.16. Hexaphosphate 22
Compound 21 (417 mg, 0.105 mmol) was dissolved in
acetonitrile/toluene/water (60:3:4, 20 mL) and cooled to
ꢀ10 °C. Ce(NH4)2(NO3)6 (1.15 g, 20 equiv) was added
portionwise and the reaction mixture was stirred for
20 min at ꢀ10 °C, the cooling bath was removed and
the reaction mixture was stirred for another 30–40 min
(TLC-monitoring). After this time, the reaction mixture
was diluted with EtOAc and washed with saturated
NaHCO3 solution, the organic phase was dried over
MgSO4 and evaporated in vacuo. Fast purification on
silica gel (toluene/acetone 1:1 ! 1:3) yielded compound
22 (246 mg, 67%) as a colourless syrup, which was stored
at ꢀ20 °C. TLC (toluene/acetone 1:1): Rf = 0.40,
Rf = 0.45. [a]D +13.6 (c 0.5, CHCl3). 1H NMR
(600 MHz, CDCl3): d = 0.82–0.94 (t, 6 H, Me), 1.09–
1.38 (m, 40 H, CH2-chain), 1.44–1.59 (m, 4 H,
COCH2CH2R), 1.85–1.99 (m, 3 H, NHAc), 2.09–2.26
(m, 4 H, COCH2CH2R), 3.33 (m, 2 H, 2a-H, 5a/b-H),
3.37 (m, 1 H, 2b-H), 3,38 (m, 1 H, 10-H), 3.39 (m, 1 H,
5a/b-H), 3.45 (m, 2 H, 4a-H, 4b-H), 3.52 (m, 2 H, 18-
H), 3.57 (m, 2 H, 3a-H, 3b-H), 3.59 (m, 3 H, 6c-H, 6a-
H), 3.67 (m, 1 H, 4c-H), 3.68 (m, 1 H, 3c-H), 3.73 (m,
2 H, 2-H, 17-H), 3.78 (m, 1 H, 5c-H), 3.85 (m, 1 H, 10-
H), 3.96, 4.07 (m, CH2-Glyc), 4.05 (m, 4 H, 1-H, 3-H),
4.08, 4.16 (m, 2 H, 30-H), 4.00–4.26 (m, 4 H, 6b-H, 16-
H), 4.21 (m, 1 H, 1a-H), 4.31 (m, 1 H, 2c-H), 4.34–4.92
(m, 22 H, CH2Ph), 4.42 (m, 1 H, 1b-H), 4.82 (m, 1 H,
1c-H), 5.01 (m, 12 H, POCH2Ph), 5.07 (m, 1 H, 20-H),
7.04–7.46 (m, 85 H, Ph). 13C NMR (150.9 MHz, CDCl3):
d = 14.1 (2 C, Me), 23.0 (1 C, NHAc), 24.9 (2 C,
COCH2CH2R), 22.7/29.1–29.7/31.9 (20 C, CH2-chain),
34.0, 34.2 (2 C, COCH2R), 52,8 (1 C, C-2c), 62.9 (1 C,
C-30), 65.8 (2 C, C-1, C-3), 66.0-68.2 (10-C, CH2-Glyc
-
OH, C-6b, C-16), 68.5 (1 C, C-6c), 69.0 (1 C, C-18),
69.8 (6 C, POCH2Ph), 69.9 (1 C, C-20), 71.8 (1 C, C-
5c), 72.2–76.0 (11 C, CH2Ph), 73.8 (1 C, C-5a/b), 75.2
(1 C, C-4a/b), 76.6 (2 C, C-2, C-17), 77.1 (1 C, C-4a/b),
78.0 (1 C, C-4c), 78.1 (1 C, C-5a/b), 81.0 (1 C, C-3c),
81.9 (2 C, C-2a, C-2b), 84.5 (2 C, C-3a, C-3b), 99.8 (1
C, C-1c), 103.9 (1 C, C-1a), 104.1 (1 C, C-1b). FAB-
MS (positive Mode): [M+Na]+, m/z = 3519.7; gef.:
m/z = 3522. MALDI-MS (positive Mode, Matrix p-nit-
roaniline+NaI, THF): [M+Na]+, m/z = 3519.7; found:
m/z = 3518. C188H237NO50P6H2O (3514.7): Calcd: C,
64.25; H, 6.85; N, 0.40. Found: C, 64.33; H, 7.31; N,
0.40.
5.18. Target molecule 1a
The diastereomers 23a (83 mg, 0.019 mmol) were dis-
solved in CH2Cl2/MeOH/H2O (7.5:7.5:1.5, 6 mL), treat-
ed with Pearlman’s catalyst (10% in weight) and under
hydrogen atmosphere, with a H2-filled balloon, was stir-
red overnight at rt. The reaction mixture was filtered
through Celite, washed with CH2Cl2/MeOH/H2O
(7.5:7.5:1.5, 2 mL) and the filtrate was diluted with
0.1 M NH4OAc-buffer (pH 4.8). The solvent was lyoph-
ilized and purified using hydrophobic interaction HPLC
on octylsepharose. After lyophilization, compound 1a
(20 mg, 47%) was obtained as white powder. [a]D +6.9
5.17. Hexaphosphate 23a
Compound 22 (197 mg, 0.056 mmol), PyBOP (585 mg,
20 equiv) and Z-D-Ala triethylammonium salt (365 mg,
20 equiv) were dried separately for 3 h in high vacuum.
After this time, 22 was dissolved in dry CH2Cl2 (15 mL),
Z-D-Ala triethylammonium salt and PyBOP were add-

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6249
(c 0.13, H2O). 1H NMR (600 MHz, D2O): d = 0.82–0.94
5.20. Target molecule 1b
(m, 6 H, Me), 1.16–1.43 (m, 40 H, CH2-chain), 1.55–1.69
(m, 16 H, Ala-Me, COCH2CH2R), 2.11 (s, 3 H, NHAc),
2.29–2.47 (m, 4 H, COCH2CH2R), 3.27–4.59 (m, 54 H),
5.10 (br s, 1 H, 1c-H), 5.31–5.43 (m, 5 H, CH-Ala, 20-H).
13C NMR (150.9 MHz, D2O): d = 14.2 (2 C, Me), 15.9
(4 C, Ala-Me), 22.6 (1 C, NHAc), 25.4 (2 C,
COCH2CH2R), 30.5 (20 C, CH2-chain), 34.5 (2 C,
COCH2CH2R), 49.2 (4 C, CHNH3þ), 54.0 (1 C, C-2c),
Compound 1b was obtained using the same procedure
as for 1a. Compound 23b (83 mg, 0.019 mmol) yielded
1b (17 mg, 40%) as white powder. [a]D +5.1 (c 0.17,
1
H2O). H NMR (600 MHz, D2O): d = 0.80–0.96 (br s,
6 H, Me), 1.13–1.46 (m, 40 H, CH2-chain), 1.53–1.77
(m, 16 H, Ala-Me, COCH2CH2R), 2.10 (s, 3 H, NHAc),
2.27–2.49 (m, 4 H, COCH2CH2R), 3.25–4.62 (m, 54 H),
5.09 (br s, 1 H, 1c-H), 5.29–5.46 (m, 5 H, CH-Ala, 20-H).
13C NMR (150.9 MHz, D2O): d = 16.7 (2 C, Me), 18.2
(4 C, Ala-Me), 25.0 (1 C, NHAc), 27.6 (2 C,
COCH2CH2R), 25.5/33.1/34.9 (20 C, CH2-chain), 37.0
(2 C, COCH2CH2R), 51.7 (4 C, CHNH3þ), 56.5 (1 C,
C-2c), 63.7 (1 C, C-6c), 65.1 (1 C, C-18), 66.3 (1 C, C-
30), 66.6 (C–CH2-Glyc), 68.1 (C–CH2-GlycGlcNAc), 69.4 (1
C, C-16), 73.1 (1 C, C-4c), 73.7 (1 C, C-17), 74.0 (1 C,
C-3c), 75.2 (1 C, C-5c), 76.2 (2 C, C-2a, C-2b), 77.0 (4
C, CH-Ala), 78.7 (2 C, C-3a, C-3b), 79.0 (1 C, CHGlcNAc),
99.9 (1 C, C-1c). MALDI-MS (negative Mode, Matrix
THAP, CH3CN/H2O 3:2): [MꢀH]ꢀ, m/z = 2247.9;
found: m/z = 2245.5; [(M-Ala)ꢀH]ꢀ, m/z = 2176.9;
found: m/z = 2174.8. MALDI-MS (positive Mode,
Matrix HCCA, H2O): [M+H]+, m/z = 2249.9; found:
m/z = 2249.9.
60.9 (1 C, C-6c), 62.4 (1 C, C-18), 64.0 (C–CH2-Glyc
,
C-30), 65.5 (C–CH2-GlycGlcNAc), 66.8 (1 C, C-16), 68.2
(1 C, C-10), 70.3 (1 C, C-4c), 71.1 (1 C, C-17), 71.4 (1
C, C-3c), 72.4 (1 C, C-5c), 73.4 (2 C, C-2a, C-2b), 74.4
(4 C, CH-Ala), 75.9 (2 C, C-3a, C-3b), 76.2 (1 C,
C–CHGlcNAc), 97.2 (1 C, C-1c). MALDI-MS
(negative Mode, Matrix THAP, CH3CN/H2O 3:2):
[MꢀH]ꢀ, m/z = 2247.9; found: m/z = 2248.5; [(M-
Ala)ꢀH]ꢀ, m/z = 2176.9; found: m/z = 2177.6.
5.19. Compound 23b
Compound 23b was synthesized following the same
method as for 23a, but using (Z)-L-alanine triethylam-
monium salt. Compound 22 (250 mg, 0.072 mmol) yield-
ed 23b (190 mg, 62%) as a colourless syrup. TLC
(toluene/acetone 1:1): Rf = 0.71, Rf = 0.75. [a]D +8.5 (c
1
0.18, CHCl3). H NMR (600 MHz, CDCl3): d = 0.87 (t,
5.21. 1-O-(tert-Butyldiphenylsilyl)-2-O-(4-methoxyben-
zyl)-3-O-monomethoxytrityl-sn-glycerol (25)
6 H, Me), 1.10–1.40 (m, 52 H, CH2-chain, Ala-Me),
1.46–1.62 (m, 4 H, COCH2CH2R), 1.82–1.99 (m, 3 H,
NHAc), 2.09–2.26 (m, 4 H, COCH2CH2R), 3.32 (m, 1
H, 5a/b-H), 3.33 (m, 1 H, 2a-H), 3,35 (m, 1 H, 10-H),
3.37 (m, 1 H, 5a/b-H), 3.38 (m, 1 H, 2b-H), 3.43, 3.45
(m, 2 H, 4a-H, 4b-H), 3.50 (m, 2 H, 18-H), 3.57 (m, 2
H, 3a-H, 3b-H), 3.59 (m, 2 H, 6c-H), 3.63 (m, 1 H, 6a-
H), 3.67 (m, 1 H, 4c-H), 3.68 (m, 1 H, 3c-H), 3.71 (m,
2 H, 2-H, 17-H), 3.76 (m, 1 H, 5c-H), 3.83 (m, 1 H, 10-
H), 4.01 (m, 20 H, 1-H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-
H, 12-H, 13-H, 15-H), 4.07 (m, 2 H, 30-H, 16-H), 4.09
(m, 1 H, 6a-H), 4.14 (m, 1 H, 6b-H), 4.15 (m, 1 H, 30-
H), 4.17 (m, 1 H, 16-H), 4.21 (m, 2 H, 6b-H, 1a-H),
4.33 (m, 4 H, CHNHCbz), 4.33–4.92 (m, 22 H, CH2Ph),
4.34 (m, 1 H, 2c-H), 4.43 (m, 1 H, 1b-H), 4.79 (m, 1 H, 1c-
H), 4.97 (m, 4 H, CH2Cbz), 4.99 (m, 12 H, POCH2Ph),
5.05 (m, 4 H, CH2Cbz), 5.07 (m, 1 H, 20-H), 5.09 (m, 4
H, 5, 8, 11, 14-H), 5.59–6.17 (NH), 7.04–7.45 (m, 105
H, Ph). 13C NMR (150.9 MHz, CDCl3): d = 14.4 (2 C,
Me), 18.3 (4 C, Ala-Me), 23.1 (1 C, NHAc), 25.2 (2 C,
COCH2CH2R), 22.9, 29.8, 32.1 (20 C, CH2-chain),
34.2, 34.5 (2 C, COCH2R), 50.1 (4 C, CHNHCbz),
53.0 (1 C, C-2c), 63.4 (1 C, C-30), 65.5 (10 C, C-1, C-3,
C-4, C-6, C-7, C-9, C-10, C-12, C-13, C-15), 67.2 (4 C,
CH2Cbz), 67.3 (1 C, C-6b), 67.9 (1 C, C-16), 68.5 (1 C,
C-10), 69.2 (2 C, C-6a, C-6c), 69.3 (1 C, C-18), 70.0 (1
C, C-20), 70.3 (6 C, POCH2Ph), 71.3 (4 C, CH-Ala),
72.2 (1 C, C-5c), 72.6–76.5 (11 C, CH2Ph), 74.0 (1 C,
C-5a/b), 75.5 (1 C, C-4a/b), 77.0 (2 C, C-2, C-17), 77.8
(1 C, C-4a/b), 78.2 (1 C, C-4c), 78.5 (1 C, C-5a/b), 81.6
(1 C, C-3c), 82.7 (2 C, C-2a, C-2b), 85.2 (2 C, C-3a,
C-3b), 100.2 (1 C, c-1c), 104.0 (1 C, C-1a), 104.4 (1 C,
C-1b). MALDI-MS (positive Mode, Matrix p-nitroani-
line+NaI, THF): [M+Na]+, m/z = 4340; found: m/
z = 4337.6. C232H281N5O62P6 (4317.6): Calcd: C, 64.48;
H, 6.51; N, 1.62. Found: C, 64.11; H, 6.68; N, 1.54.

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A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
(484.6): Calcd: C, 76.66; H, 6.61. Found: C, 76.34; H,
6.86.
1.2 equiv) was added dropwise. The reaction mixture
was stirred for 3 h, diluted with EtOAc and washed with
saturated NaHCO3 solution, the organic phase was
dried over MgSO4 and the solvent was removed in vac-
uo. Flash chromatography (petroleum ether/EtOAc 3:1)
yielded 30 (9.86 g, 98%) as a colourless syrup. TLC
(petroleum ether/EtOAc 7:1): Rf = 0.13. [a]D +23.6 (c
5.23. 3-O-Allyl-2-O-benzyl-1-O-tert-butyldiphenylsilyl-
sn-glycerol (27)
To a solution of 2613 (15 g, 40.5 mmol) in DMF were
added benzyl bromide (7.24 mL, 60.7 mmol) and NaH
(2 g, 2 equiv) portionwise. The reaction mixture was stir-
red for 3 h. The solvent was evaporated in vacuo and the
residue was dissolved in EtOAc, washed with saturated
NH4Cl solution and dried over MgSO4. The solvent
was removed in vacuo. Flash chromatography (petro-
leum ether/EtOAc 30:1) yielded 27 (13.4 g, 72%) as a col-
ourless syrup. TLC (petroleum ether/EtOAc 7:1):
Rf = 0.49. [a]D ꢀ1.3 (c 1, acetone). 1H NMR
(250 MHz, CDCl3): d = 1.05 (s, 9 H, t-Bu), 3.52–3.87,
3.96–4.10 (m, 7 H, CH2Glyc–H, CHGlyc–H, CH2CHCH2),
4.64 (s, 2 H, CH2Ph), 5.11–5.35 (m, 2 H, CH2CHCH2),
5.81–6.01 (m, 1 H, CH2CHCH2), 7.21–7.53, 7.61–7.81
(m, 15 H, Ph). C29H36O3Si (460.7): Calcd: C, 75.61; H,
7.88. Found: C, 75.37; H, 7.90.
1
1, CHCl3). H NMR (250 MHz, CDCl3): d = 1.98–2.05
(m,
Jvic = 4.9 Hz, CH2-Glyc–H) 3.30 (dd, 1 H, Jgem = 9.9 Hz,
Jvic = 4.7 Hz, CH2Glyc–H), 3.62–3.79 (m, 3 H, CH2-Glyc
1 H, OH), 3.23 (dd, 1 H, Jgem = 9.9 Hz,
,
CHGlyc), 3.79 (s, 3 H, OMe), 4.54 (d, 1 H, Jgem = 11.7
Hz, CH2Ph), 4.69 (d, 1 H, Jgem = 11.7 Hz, CH2Ph),
6.80–6.88 (m, 2 H, PhMPM), 7.19–7.52 (m, 17 H, Ph).
C30H30O4Æ0.25H2O (459.1): Calcd: C, 78.49; H, 6.70.
Found: C, 78.45; H, 6.82.
5.27. [Benzyloxy]-[diisopropylamino]-[2-O-benzyl-3-O-
monomethoxytrityl-sn-glycero]phosphane (7)
Compound 7 was obtained as described for 3. Tetrazole
(108 mg, 0.6 equiv), 30 (2.03 g, 7.46 mmol), benzyloxy-
bis-(diisopropyl amine)-phosphane (1.37 g, 1.2 equiv).
Flash chromatography (petroleum ether/EtOAc 8:1,
+1% NEt3) yielded 7 (2.4 g, quant.) as a colourless oil.
TLC (petroleum ether/EtOAc 7:1, 1% NEt3):
Rf = 0.54. [a]D ꢀ0.5 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 1.10–1.30 (m, 12 H, CH(CH3)2),
3.19–3.34 (m, 2 H, CH2-Glyc), 3.47–3.91 (m, 8 H, OMe,
CH(CH3)2, CH2-Glyc), 4.51–4.80 (m, 4 H, POCH2Ph,
CH2Ph), 6.73–6.89 (m, 2 H, PhMMTr), 7.13–7.58 (m, 22
H, Ph). C43H50NO5P (691.8): Calcd: C, 74.65; H, 7.28;
N, 2.02. Found: C, 74.64; H, 7.35; N, 2.02.
5.24. 2-O-Benzyl-1-O-tert-butyldiphenylsilyl-sn-glycerol
(28)
Compound 27 (13.3 g, 28.9 mmol) was dissolved in dry
EtOH (300 mL), DBU (648 lL, 0.15 equiv) and
(Ph3P)3RhCl (8 g, 0.3 equiv) were added and the reac-
tion mixture was stirred at 90 °C. After 15 min, the pro-
penyl intermediate was formed (Rf = 0.76, toluene/
acetone 1.5:1); the solvent was removed in vacuo and
the residue was dissolved in a solution of 1 M HCl/ace-
tone (1:9, 200 mL) and stirred at 70 °C for 15 min. The
solution was neutralized with NEt3, diluted with EtOAc
and washed with saturated NaHCO3 solution. After
drying with MgSO4, the solvent was removed in vacuo.
Flash chromatography (petroleum ether/EtOAc 3:1)
yielded 28 (9.2 g, 76 %) as a yellow syrup. The analytical
data are in accordance with those reported in the
literature.21
5.28. Compound 31
Compound 31 was synthesized following the general
coupling procedure. Compound 6 (3.59 g, 7.41 mmol),
tetrazole (780 mg, 11.14 mmol) and phosphite amide
513 (5.6 g, 1.1 equiv) were dissolved in dry CH2Cl2
(150 mL). (TLC, petroleum ether/EtOAc: 7:1, +1%
NEt3; Rf = 0.22). Oxidation with t-BuO2H (4 mL) and
flash chromatography (petroleum ether/EtOAc 3:1)
yielded 31 (7.89 g, 98%) as a colourless syrup. TLC
(petroleum ether/EtOAc 1:1): Rf = 0.69. [a]D ꢀ0.4 (c 1,
CHCl3). 1H NMR (250 MHz, CDCl3): d = 1.01 (s, 9
H, t-Bu), 3.13-3.22 (m, 2 H, 6-H), 3.58–3.88 (m, 13 H,
1, 2-H, 5-H, OMe), 4.03–4.31 (m, 4 H, 3-H, 4-H),
4.41–4.57 (m, 4 H, CH2-Ph), 4.92–5.01 (m, 2 H,
POCH2-Ph), 6.72–6.88 (m, 6 H, PhMPM, MMTr), 7.10–
7.50, 7.60–7.70 (m, 31 H, Ph). MALDI-MS (positive
Mode, Matrix DHB, THF): [M+Na]+, m/z = 1110.3;
found: m/z = 1110.4. C65H71O11PSi Æ0.5 H2O (1096.3):
Calcd: C, 71.21; H, 6.62. Found: C, 71.10; H, 6.68.
5.25. 2-O-Benzyl-3-O-monomethoxytrityl-1-O-(tert-buty-
ldiphenylsilyl)-sn-glycerol (29)
Compound 29 was obtained as described for 25 from
compound 28 (9.15 g, 21.75 mmol) and monomethoxy-
trityl chloride (8.2 g, 26.55 mmol). Flash chromatogra-
phy (PE/EE 20:1) yielded 29 (14.77 g, 98%) as a
colourless syrup. TLC (petroleum ether/EtOAc 7:1):
1
Rf = 0.48. [a]D +1.9 (c 1, CHCl3). H NMR (250 MHz,
CDCl3): d = 0.96 (s, 9 H, t-Bu), 3.21–3.35 (m, 2 H,
CH2Glyc–H) 3.69–3.85 (m, 6 H, CH2-Glyc–H, CHGlyc
–
H, OMe), 4.67 (s, 2 H, CH2PhMPM), 6.74–6.83 (m, 2
H, PhMPM), 7.15–7.51, 7.58–7.69 (m, 27 H, Ph).
C46H48O4Si (693.0): Calcd: C, 79.73; H, 6.98. Found:
C, 79.80; H, 7.09.
5.29. 3-O-[2-O-(4-Methoxybenzyl)-3-O-monomethoxytri-
tyl-sn-glycero-(1)-benzylphospho]-2-O-(4-methoxyben-
zyl)-sn-glycerol (32)
5.26. MPM2-O-Benzyl-3-O-monomethoxytrityl-sn-glyc-
erol (30)
Comoound 32 was synthesized following the general
deprotection procedure. 31 (7.78 g, 7.16 mmol), TBAF-
solution (8.60 mL, 1.2 equiv), THF (150 mL) and flash
chromatography (petroleum ether/EtOAc 1:1 ! 1:3)
Compound 29 (16 g, 23.1 mmol) was dissolved in THF
(300 mL) and TBAF-solution (27 mL, 1 M in THF,

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6251
1
yielded 32 (5 g, 82%) as a colourless syrup. TLC (tolu-
ene/acetone 1:1): Rf = 0.61. [a]D +12.5 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 3.16–3.25 (m, 2 H,
6-H), 3.48–3.89 (m, 13 H, 1-H, 2-H, 5-H, OMe), 4.01–
4.30 (m, 4 H, 3-H, 4-H), 4.39–4.60 (m, 4 H, CH2-Ph),
4.96–5.05 (m, 2 H, POCH2-Ph), 6.76–6.91 (m, 6 H,
PhMPM, MMTr), 7.15–7.49 (m, 21 H, Ph). MALDI-MS
(positive Mode, Matrix DHB, THF): [M+Na]+, m/
z = 871.9; found: m/z = 872.2. C49H53O11PÆ0.5H2O
(857.9): Calcd: C, 68.60; H, 6.35. Found: C, 68.61; H,
6.40.
tone 3:1): Rf = 0.48. [a]D ꢀ0.7 (c 1, CHCl3). H NMR
(250 MHz, CDCl3): d = 1.02 (s, 9 H, t-Bu), 3.13–3.22
(m, 2 H, 12-H), 3.56–3.88 (m, 21 H, 1-H, 2-H, 5-H, 8-
H, 11-H, OMe), 3.90–4.32 (m, 12 H, 3-H, 4-H, 6-H, 7-
H, 9-H, 10-H), 4.39–4.58 (m, 8 H, CH2-Ph), 4.89–5.08
(m, 6 H, POCH2-Ph), 6.69–6.89 (m, 10 H, PhMPM,
MMTr), 7.10–7.50, 7.59–7.71 (m, 45 H, Ph). MALDI-
MS (positive Mode, Matrix DHB, THF): [M+Na]+,
m/z = 1838.97; found: m/z = 1838.8, [M+K]+, m/
z = 1855.1; found: m/z = 1856.3. C101H113O23P3Si
(1816.0): Calcd: C, 66.80; H, 6.27. Found: C, 67.30; H,
6.15.
5.30. Diphosphate 33
5.33. Triphosphate 36
Compound 33 was synthesized following the general
coupling procedure. 32 (5.13 g, 6.04 mmol), tetrazole
(635 mg, 9.07 mmol) and phosphite amide 513 (5.0 g,
1.2 equiv) were dissolved in dry CH2Cl2 (120 mL).
(TLC petroleum ether/EtOAc: 1:1, +1% NEt3,
Rf = 0.68). Oxidation with t-BuO2H (3.5 mL). Flash
chromatography (petroleum ether/EtOAc 2:1 ! 1:1)
yielded 33 (8.4 g, 96%) as a colourless syrup. TLC
(petroleum ether/EtOAc 1:1): Rf = 0.35. [a]D ꢀ1.1 (c 1,
CHCl3). 1H NMR (250 MHz, CDCl3): d = 1.02 (s, 9
H, t-Bu), 3.14–3.22 (m, 2 H, 9-H), 3.59–3.82 (m, 17 H,
1-H, 2-H, 5-H, 8-H, OMe), 3.90–4.32 (m, 8 H, 3-H, 4-
H, 6-H, 7-H), 4.39–4.54 (m, 6 H, CH2-Ph), 4.90–5.02
(m, 4 H, POCH2-Ph), 6.69–6.88 (m, 8 H, PhMPM, MMTr),
7.09–7.48, 7.59–7.70 (m, 38 H, Ph). MALDI-MS (posi-
To a solution of triphosphate 35 (8.66 g, 4.77 mmol) in
CH2Cl2 (50 mL) and MeOH (100 mL), CSA was added
(222 mg, 0.95 mmol, 0.2 equiv); the reaction mixture was
stirred for 2 h at rt. After neutralization with NEt3, the
solvent was removed in vacuo and coevaporated with
toluene. Flash chromatography (toluene/acetone 2.5:1)
yielded 36 (6.8 g, 92%) as a colourless syrup. TLC (tol-
uene/acetone 2.5:1): Rf = 0.30. [a]D ꢀ7.5 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 1.02 (s, 9 H, t-Bu),
2.73–3.0 (br s, 1 H, OH), 3.50–3.86 (m, 20 H, 1-H, 2-
H, 5-H, 8-H, 11-H, 12-H, OMe), 3.91–4.34 (m, 12 H,
3-H, 4-H, 6-H, 7-H, 9-H, 10-H), 4.39–4.60 (m, 8 H,
CH2-Ph), 4.93–5.10 (m, 6 H, POCH2-Ph), 6.71–6.90
(m, 8 H, PhMPM, MMTr), 7.10–7.47, 7.59–7.70 (m, 33
H, Ph). MALDI-MS (positive Mode, Matrix DHB,
THF): [M+Na]+, m/z = 1566.6; found: m/z = 1567.3,
tive Mode, Matrix DHB, THF): [M+Na]
+
,
m/
z = 1474.6; found: m/z = 1475.9. C83H92O17P2Si
(1451.6): Calcd: C, 68.67; H, 6.39. Found: C, 68.34; H,
6.37.
[M+K]+,
m/z = 1582.7;
found:
m/z = 1582.4.
C81H97O22P3Si (1543.6): Calcd: C, 63.02; H, 6.33.
Found: C, 63.02; H, 6.58.
5.31. Diphosphate 34
5.34. Tetraphosphate 37
Compound 34 was synthesized following the general
deprotection procedure. 33 (8.3 g, 5.72 mmol) TBAF-so-
lution (6.9 mL, 1.2 equiv), THF (150 mL) and flash
chromatography (toluene/acetone 2.5:1) yielded 34
(6.34 g, 91%) as a colourless syrup. TLC (toluene/ace-
Compound 37 was synthesized following the general
coupling procedure. Compound 36 (2.5 g, 1.62 mmol),
tetrazole (170 mg, 2.43 mmol) and phosphite amide 7
(1.35 g, 1.2 equiv) were dissolved in dry CH2Cl2
(60 mL). (TLC toluene/acetone 3:1, +1% NEt3,
Rf = 0.62). Oxidation with t-BuO2H (2 mL). Flash chro-
matography (toluene/acetone 2:1) yielded 37 (3.34 g,
96%) as a colourless syrup. TLC (toluene/acetone 2:1):
Rf = 0.42. [a]D ꢀ1.1 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 1.02 (s, 9 H, t-Bu), 3.17–3.25
(m, 2 H, 15-H), 3.59–3.81 (m, 19 H, 1-H, 2-H, 5-H, 8-
H, 11-H, 14-H, OMe), 3.89–4.32 (m, 16 H, 3-H, 4-H,
6-H, 7-H, 9-H, 10-H, 12-H, 13-H), 4.38–4.51 (m, 8 H,
CH2-Ph), 4.51–4.61 (m, 2 H, CH2-Ph), 4.90–5.04 (m, 8
H, POCH2-Ph), 6.69–6.88 (m, 10 H, PhMPM, MMTr),
7.09–7.49, 7.58–7.69 (m, 55 H, Ph). C118H132O28P4Si
(2150.3): Calcd: C, 65.91; H, 6.19. Found: C, 66.09; H,
6.16.
1
tone 1:1): Rf = 0.32. [a]D +5.1 (c 1, CHCl3). H NMR
(250 MHz, CDCl3): d = 2.62–2.71 (br s, 1 H, OH),
3.14–3.23 (m, 2 H, 9-H), 3.50–3.87 (m, 17 H, 1-H, 2-
H, 5-H, 8-H, OMe), 3.90–4.28 (m, 8 H, 3-H, 4-H, 6-H,
7-H), 4.40–4.59 (m, 6 H, CH2-Ph), 4.91–5.07 (m, 4 H,
POCH2-Ph), 6.73–6.89 (m, 8 H, PhMPM, MMTr), 7.11–
7.47 (m, 28 H, Ph). MALDI-MS (positive Mode, Matrix
DHB, THF): [M+Na]+, m/z = 1236.2; found: m/
z = 1236.0, [M+K]+, m/z = 1252.3; found: m/
z = 1252.3. C67H74O17P2 (1213.2): Calcd: C, 66.33; H,
6.15. Found: C, 66.54; H, 6.37.
5.32. Triphosphate 35
Compound 35 was synthesized following the general
coupling procedure. Compound 34 (6.30 g, 5.19 mmol),
tetrazole (563 mg, 8.04 mmol) and phosphite amide 513
(4.42 g, 1.2 equiv) were dissolved in dry CH2Cl2
(120 mL). (TLC toluene/acetone 3:1, +1% NEt3,
Rf = 0.62). Oxidation with t-BuO2H (3.3 mL). Flash
chromatography (toluene/acetone 4.5:1) yielded 35
(8.82 g, 94%) as a colourless syrup. TLC (toluene/ace-
5.35. Tetraphosphate 38
Compound 38 was synthesized following the general
deprotection procedure. 37 (3.3 g, 1.54 mmol), TBAF-so-
lution (1.85 mL, 1.2 equiv), THF (60 mL). Flash chroma-
tography (toluene/acetone 2:1 ! 1:1) yielded 38 (2.78 g,
95%) as a colourless syrup. TLC (toluene/acetone 1:1):

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A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
1
Rf = 0.53. [a]D +2.5 (c 1, CHCl3). H NMR (250 MHz,
CDCl3): d = 2.75–2.99 (br s, 1 H, OH), 3.14–3.26 (m, 2
H, 15-H), 3.50–3.84 (m, 19 H, 1-H, 2-H, 5-H, 8-H, 11-
H, 14-H, OMe), 3.89–4.29 (m, 16 H, 3-H, 4-H, 6-H, 7-
H, 9-H, 10-H, 12-H, 13-H), 4.49–4.63 (m, 10 H, CH2-
Ph), 4.89–5.10 (m, 8 H, POCH2-Ph), 6.70–6.92 (m, 10
H, PhMPM, MMTr), 7.10–7.49 (m, 45 H, Ph). C102H114O28P4Ætol-
uene (2004.0): Calcd: C, 65.33; H, 6.14. Found: C, 65.41; H, 6.12.
tone). 1H NMR (250 MHz, CDCl3): d = 1.86–2.01 (m, 3
H, NHAc), 2.80–3.20 (br s, 2 H, OH), 3.41–3.83 (m, 27
H, 3c-H, 4c-H, 5c-H, 6c-H, 1-H, 2-H, 5-H, 8-H, 11-H,
14-H, 17-H, 18-H, OMe), 3.89–4.84 (m, 38 H, 1c-H,
2c-H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H, 15-
H, 16-H, CH2-Ph), 4.90–5.10 (m, 10 H, POCH2-Ph),
6.71–6.89 (m, 8 H, PhMPM), 7.08–7.45 (m, 53 H, Ph).
5.39. Heptaphosphate 42
5.36. Pentaphosphate 39
Compound 42 was synthesized following the general
coupling
Compound 39 was synthesized following the general
coupling procedure. Compound 38 (2.71 g, 1.42 mmol),
tetrazole (150 mg, 2.14 mmol) and phosphite amide
413 (1.78 g, 1.2 equiv) were dissolved in dry CH2Cl2
(60 mL). (TLC toluene/acetone 3:1, +1% NEt3,
Rf = 0.62). Oxidation with t-BuO2H (2 mL). Flash chro-
matography (toluene/acetone 2:1 ! 1:3) yielded 39
(3.60 g, 96%) as a colourless syrup. TLC (toluene/ace-
tone 1:1): Rf = 0.52, Rf = 0.60. [a]D +12.0 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 1.02 (s, 9 H, t-Bu),
1.90–2.03 (m, 3 H, NHAc), 3.10–3.26 (m, 3 H, 18, 6c-
H), 3.41–3.83 (m, 27 H, 3c-H, 4c-H, 5c-H, 6c-H, 1-H,
2-H, 5-H, 8-H, 11-H, 14-H, 17-H, OMe), 3.88–4.80
(m, 38 H, 1c-H, 2c-H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H,
12-H, 13-H, 15-H, 16-H, CH2-Ph), 4.85–5.05 (m, 10 H,
POCH2-Ph), 6.68–6.88 (m, 10 H, PhMPM, MMTr), 7.02–
7.71 (m, 75 H, Ph). C157H176NO38P5Si (2868.0): Calcd:
C, 65.75; H, 6.19; N, 0.49. Found: C. 66.11; H, 6.44;
N, 0.48.
procedure.
Compound
41
(320 mg,
0.136 mmol), tetrazole (19 mg, 0.272 mmol) and phos-
phite amide 3 (570 mg, 2.5 equiv) were dissolved in
dry CH2Cl2 (10 mL). Oxidation was achieved with t-
BuO2H (1 mL). Flash chromatography (toluene/acetone
3:1 ! 1.5:1) yielded 42 (520 mg,71%) as a colourless syr-
up. TLC (toluene/acetone 2:1): Rf = 0.44, Rf = 0.51,
Rf = 0.59. [a]D +10.34 (c 0.75, acetone). 1H NMR
(600 MHz, CDCl3): d = 0.80–0.91 (t, 12 H, Me), 1.09–
1.35 (m, 80 H, CH2-chain), 1.44–1.52 (m, 8 H,
COCH2CH2R), 1.87–1.98 (m, 3 H, NHAc), 2.09–2.28
(m, 8 H, COCH2CH2R), 3.31, 3.39 (m, 4 H, 2a-H, 2b-
H), 3.33 (m, 2 H, 5a/b-H), 3,36 (m, 2 H, 10-H), 3.38
(m, 2 H, 5a/b-H), 3.45 (m, 4 H, 4a-H, 4b-H), 3.56 (m,
1 H, 6c-H), 3.58 (m, 4 H, 3a-H, 3b-H), 3.60 (m, 2 H,
6a-H), 3.62 (m, 6 H, 2-H, 5-H, 8-H, 11-H, 14-H, 17-
H), 3.64 (m, 1 H, 6c-H), 3.66 (m, 12 H, OMe), 3.68
(m, 1 H, 3c-H), 3.69 (m, 1 H, 4c-H), 3.77 (m, 1 H, 5c-
H), 3.84 (m, 2 H, 10-H), 3.95, 4.03 (m, 24 H, 1-H, 3-H,
4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H, 15-H, 16-H,
18-H), 4.06 (m, 2 H, 30-H), 4.10 (m, 2 H, 6a-H), 4.15
(m, 2 H, 30-H), 4.18 (m, 2 H, 6b-H), 4.21 (m, 2 H, 1a-
H), 4.23 (m, 2 H, 6b-H), 4.33 (m, 1 H, 2c-H), 4.42 (m,
2 H, 1b-H), 4.69/4.73 (m, 1 H, 1c-H), 4.32-4.91 (m, 40
H, CH2Ph), 4.94 (m, 14 H, POCH2Ph), 5.07 (m, 2 H,
20-H), 6.68–6.81 (m, 8 H, PhMPM), 7.05–7.35 (m, 123
H, Ph). 13C NMR (150.9 MHz, CDCl3): d = 53.0 (1 C,
C-2c), 55.7 (4C, OMe), 63.1 (2 C, C-30), 66.0 (12 C,
C–CH2-Glyc), 66.8 (2 C, C-6b), 68.4 (2 C, C-10), 68.9 (1
C, C-6c), 69.0 (2 C, C-6a), 69.7 (7 C, POCH2Ph), 69.9
(2 C, C-20), 72.0–78.0 (20 C, CH2Ph), 72.2 (1 C, C-5c),
74.0 (2 C, C-5a/b), 75.5 (2 C, C-4a/b), 75.9 (6 C, C-2,
C-5, C-8, C-11, C-14, C-17), 77.3 (2 C, C-4a/b), 78.0
(1 C, C-4c), 78.3 (2 C, C-5a/b), 81.4 (1 C, C-3c), 82.2
(4 C, C-2a, C-2b), 84.9 (4 C, C-3a, C-3b), 101.0 (1 C,
C-1c), 104.0 (2 C, C-1a), 104.2 (2 C, C-1b). MALDI-
MS (positive Mode, Matrix p-nitroaniline+NaI, THF):
5.37. Pentaphosphate 40
To a solution of 39 (3.53 g, 1.23 mmol) in CH2Cl2
(60 mL) and MeOH (20 mL), CSA was added (86 mg,
0.37 mmol, 0.3 equiv); the reaction mixture was stirred
for 2 h at rt. After neutralization with NEt3, the solvent
was removed in vacuo and coevaporated with toluene.
Flash chromatography (toluene/acetone 1:1 ! 1:3)
yielded 40 (2.93 g, 92%) as a colourless syrup. TLC (tol-
1
uene/acetone 1:1): Rf = 0.55. [a]D +7.6 (c 1, CHCl3). H
NMR (250 MHz, CDCl3): d = 1.02 (s, 9 H, t-Bu), 1.89–
2.02 (m, 3 H, NHAc), 3.11–3.23 (m, 1 H, 6c-H), 3.40–
3.85 (m, 26 H, 3c-H, 4c-H, 5c-H, 6c-H, 1-H, 2-H, 5-H,
8-H, 11-H, 14-H, 17-H, 18-H, OMe), 3.88–4.80 (m, 38
H, 1c-H, 2c-H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H,
13-H, 15-H, 16-H, CH2-Ph), 4.85–5.11 (m, 10 H,
POCH2-Ph), 6.68–6.89 (m, 8 H, PhMPM), 7.01–7.46,
7.46–7.70 (m, 63 H, Ph). C137H160NO37P5Si (2595.7):
Calcd: C, 63.39; H, 6.21; N, 0.54. Found: C, 63.10; H,
6.46; N, 0.55.
[M+Na]+,
m/z = 5440.1;
found:
m/z = 5445.6.
C305H384NO71P7 (5417.1): Calcd: C, 67.62; H, 7.14; N,
0.26. Found: C, 67.35; H, 7.32; N, 0.20.
5.38. Pentaphosphate 41
5.40. Heptaphosphate 43
Compound 40 (433 mg, 0.17 mmol) was dissolved in
THF (15 mL) and TBAF solution (0.2 mL, 1.2 equiv)
was added. After 45 min, the reaction mixture was dilut-
ed with EtOAc, washed with saturated NaHCO3 solu-
tion and water, and after drying over MgSO4 the
solvent was removed in vacuo. Flash chromatography
(toluene/acetone 1:1.5) yielded 41 (335 mg, 85%) as a
colourless syrup. TLC (toluene/acetone 1:1.5):
Rf = 0.09, Rf = 0.20, Rf = 0.33. [a]D +15.5 (c = 0.75, ace-
Compound 43 was synthesized following the same pro-
cedure used for 22. Compound 42 (476 mg, 0.088 mmol)
was dissolved in CH3CN/toluene/H2O (60:3:4, 15 mL).
Flash chromatography (toluene/acetone 1:1. ! 1:3)
yielded 43 (330 mg, 76%) as a colourless syrup. TLC
(toluene/acetone 1:1.5): Rf = 0.44, Rf = 0.53. [a]D +12.6
(c 0.5, acetone). 1H NMR (600 MHz, CDCl3):
d = 0.80–0.92 (t, 12 H, Me), 1.08–1.36 (m, 80 H, CH2-
chain), 1.43–1.59 (m, 8 H, COCH2CH2R), 1.82–1.97

A. Stadelmaier et al. / Bioorg. Med. Chem. 14 (2006) 6239–6254
6253
(m, 3 H, NHAc), 2.11–2.27 (m, 8 H, COCH2CH2R),
3.34 (m, 2 H, 5a/b-H), 3.35 (m, 4 H, 2a-H, 2b-H), 3.37
(m, 2 H, 5a/b-H), 3.38 (m, 2 H, 10-H), 3.46 (m, 2 H,
4a/b-H), 3.47 (m, 2 H, 4a/b-H), 3.56 (m, 1 H, 6c-H),
3.58 (m, 4 H, 3a-H, 3b-H), 3.59 (m, 2 H, 6a-H), 3.64
(m, 1 H, 6c-H), 3.67 (m, 2 H, 2-H, 17-H), 3.68 (m, 1
H, 3c-H), 3.69 (m, 1 H, 4c-H), 3.80 (m, 1 H, 5c-H),
3.85 (m, 2 H, 10-H), 3.94 (m, 4-H, 6-H, 7-H, 9-H, 10-
H, 12-H, 13-H, 15-H), 3.99 (m, 4 H, 1-H, 3-H, 16-H,
18-H), 4.07 (m, 2 H, 30-H), 4.08 (m, 4 H, 1-H, 3-H,
16-H, 18-H), 4.10 (m, 2 H, 6a-H), 4.15 (m, 2 H, 30-H),
4.16 (m, 2 H, 6b-H), 4.22 (m, 2 H, 1a-H), 4.24 (m, 2
H, 6b-H), 4.31 (m, 1 H, 2c-H), 4.34-4.93 (m, 32 H,
CH2Ph), 4.42 (m, 2 H, 1b-H), 4.83 (m, 1 H, 1c-H),
5.01 (m, 14 H, POCH2Ph), 5.08 (m, 2 H, 20-H), 7.05–
7.47 (m, 115 H, Ph). 13C NMR (150.9 MHz, CDCl3):
d = 53.0 (1 C, C-2c), 63.0 (2 C, C-30), 66.0 (4 C, C-1,
C-3, C-16, C-18), 66.8 (2 C, C-6b), 68.3 (2 C, C-10),
68.4 (CH2-Glyc-OH), 68.9 (1 C, C-6c), 69.0 (2 C, C-6a),
70.0 (7 C, POCH2Ph), 70.1 (2 C, C-20), 72.0-76.4 (16
C, CH2Ph), 72.1 (1 C, C-5c), 73.9 (2 C, C-5a/b), 75.5
(2 C, C-4a/b), 76.0 (2 C, C-2, C-17), 77.3 (2 C, C-4a/
b), 78.1 (1 C, C-4c), 78.3 (2 C, C-5a/b), 81.1 (1 C, C-
3c), 82.2 (4 C, C-2a, C-2b), 84.9 (4 C, C-3a, C-3b),
99.9 (1 C, C-1c), 104.0 (2 C, C-1a), 104.2 (2 C, C-1b).
MALDI-MS (positive Mode, Matrix p-nitroani-
line+NaI, THF): [M+Na]+, m/z = 4959.5; found: m/
z = 4966.1. C273H352NO67P7 (4936.5): Calcd: C, 66.42;
H, 7.19; N, 0.28. Found: C, 66.30; H, 7.38; N, 0.24.
b), 75.3 (2 C, C-4a/b), 76.1 (2 C, C-2, C-17), 77.1 (2 C,
C-4a/b), 77.9 (1 C, C-4c), 78.3 (2 C, C-5a/b), 81.2 (1
C, C-3c), 82.1 (4 C, C-2a, C-2b), 84.7 (4 C, C-3a, C-
3b), 100.6 (1 C, C-1c), 103.8 (2 C, C-1a), 104.2 (2 C,
C-1b). MALDI-MS (positive Mode, Matrix p-nitroani-
line+NaI, THF): [(MꢀH)+2Na]+, m/z = 5802.3; found:
m/z = 5801.3. C317H396N5O79P7 (5757.34): Calcd: C,
66.13; H, 6.93; N, 1.22. Found: C, 65.94; H, 7.08; N,
1.20.
5.42. Target molecule 2
Compound 2 was synthesized following the procedure
used for 1a. Compound 44 (107 mg, 0.019 mmol) yield-
ed 2 (19 mg, 16%) as white powder. 1H NMR
(600 MHz, D2O): d = 0.78–0.96 (m, 12 H, Me), 1.13–
1.46 (m, 80 H, CH2-chain), 1.55–1.74 (m, 20 H, Ala-
Me, COCH2CH2R), 2.11 (s, 3 H, NHAc), 2.27–2.49
(m, 8 H, COCH2CH2R), 3.40–4.60 (m, 72 H), 5.10 (m,
1 H, 1c-H), 5.31–5.45 (m, 6 H, CH-Ala, 20-H). 13C
NMR (150.9 MHz, D2O): d = 14.2 (4 C, Me), 15.9 (4
C, Ala-Me), 22.6 (1 C, NHAc), 25.2 (4 C,
COCH2CH2R), 30.9 (40 C, CH2-chain), 34.7 (4 C,
COCH2R), 49.1 (4 C, CHNH3þ, 60.9 (1 C, C-6c), 64.0
(C–CH2-Glyc), 66.4 (C–CH2-Glyc), 74.1 (4C, CH-Ala).
MALDI-MS (negative Mode, Matrix THAP, CH3CN/
H2O 3:2): [MꢀH]ꢀ, m/z = 3147.0; found: m/z = 3146.0;
[(M-Ala)ꢀH]ꢀ, m/z = 3074.9; found: m/z = 3075.2.
5.43. Measurement of biological activity
5.41. Fully protected target molecule 44
Heparinized whole blood from healthy donors con-
trolled by differential blood cell count was diluted 5-fold
with RPMI 1640 (BioWhittaker Europe) and stimulated
overnight (37 °C, 5% CO2) in polypropylene cups with
equimolar concentrations of 1a and 2. Cytokines were
measured in the supernatants using ELISA antibody
pairs against TNFa and IL-8 (Endogen, Pierce, Perbio
Science, Bonn, Germany).
Compound 44 was synthesized following the procedure
used for 23a. Compound 43 (296 mg, 0.060 mmol) gave
44 (170 mg, 49%, mixture of diastereomers) as a colour-
less syrup. TLC (toluene/acetone 1:1): Rf = 0.60,
Rf = 0.67. [a]D +12.45 (c 0.48, acetone). 1H NMR
(600 MHz, CDCl3): d = 0.80–0.94 (t, 12 H, Me), 1.07–
1.38 (m, 92 H, CH2-chain, Ala-Me), 1.45–1.59 (m, 8
H, COCH2CH2R), 1.85–1.97 (m, 3 H, NHAc), 2.11–
2.28 (m, 8 H, COCH2CH2R), 3.33 (m, 2 H, 5a/b-H),
3.34 (m, 4 H, 2a-H, 2b-H), 3.36 (m, 4 H, 5a/b-H, 10-
H), 3.46 (m, 4 H, 4a-H, 4b-H), 3.58 (m, 4 H, 3a-H,
3b-H), 3.60 (m, 4 H, 6c-H, 6a-H), 3.64 (m, 2 H, 2-H,
17-H), 3.68 (m, 1 H, 3c-H), 3.70 (m, 1 H, 4c-H), 3.76
(m, 1 H, 5c-H), 3.84 (m, 2 H, 10-H), 4,00 (m, 24 H, 1-
H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H, 15-H,
16-H, 18-H), 4.09 (m, 2 H, 30-H), 4.11 (m, 2 H, 6a-H),
4.15 (m, 2 H, 30-H), 4.18 (m, 2 H, 6b-H), 4.22 (m, 2
H, 1a-H), 4.24 (m, 2 H, 6b-H), 4.33 (m, 4 H, CH-
NHCbz), 4.35 (m, 1 H, 2c-H), 4.34-4.93 (m, 32 H,
CH2Ph), 4.42 (m, 2 H, 1b-H), 4.78 (m, 1 H, 1c-H),
4.96 (m, 4 H, CH2-Cbz), 4.98 (m, 14 H, POCH2Ph),
5.03 (m, 4 H, CH2-Cbz), 5.08 (m, 2 H, 20-H), 5.09 (m,
4 H, 5, 8, 11, 14-H), 5.76-6.18 (bs, NH), 7.03–7.46 (m,
135 H, Ph). 13C NMR (150.9 MHz, CDCl3): d = 49.9
(4 C, CH-NHCbz), 53.0 (1 C, C-2c), 63.0 (2 C, C-30),
65.3 (12 C, C-1, C-3, C-4, C-6, C-7, C-9, C-10, C-12,
C-13, C-15, C-16, C-18), 66.8 (2 C, C-6b), 67.1 (4 C,
CH2-Cbz), 68.3 (2 C, C-10), 68.8 (1 C, C-6c), 68.9 (2
C, C-6a), 70.0 (2 C, C-20), 70.3 (7 C, POCH2Ph), 71.0
(4 C, C-5, C-8, C-11, C-14), 72.0 (1 C, C-5c), 73.9 (2
C, C-5a/b), 72.5–75.9 (16 C, CH2Ph), 73.8 (2 C, C-5a/
Acknowledgments
This work was supported by the Deutsche Forschungs-
gemeinschaft and the Fonds der Chemischen Industrie.
We thank L. Cobianchi for help in separating the final
products by HI-chromatography, and Prof. A. Geyer
and A. Friemel for the help in the structural assignments
of the products by NMR experiments.
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