Synthesis of structural variants of Staphylococcus aureus lipoteichoic acid (LTA)

Article abstract of DOI:10.1016/j.tetasy.2004.11.070

Based on 1,2-O-isopropylidene-sn-glycerol, which is readily available from d-mannitol, five chiral building blocks for the construction of structural variants of Staphylococcus aureus LTA designed and synthesized. Ligation of these building blocks led readily to the target molecules 1 and 2. They demonstrated that the d-alanine residues at the glycerophosphate backbone are decisive for the activation of the immune system.

Full text of DOI:10.1016/j.tetasy.2004.11.070

Tetrahedron:
Asymmetry
Tetrahedron: Asymmetry 16 (2005) 493–506
Synthesis of structural variants of Staphylococcus aureus
lipoteichoic acid (LTA)
Ignacio Figueroa-Perez,a Andreas Stadelmaier,a Siegfried Morath,b
Thomas Hartungb and Richard R. Schmidta,*
aFachbereich Chemie, Universita¨t Konstanz, Fach M 725, D-78457 Konstanz, Germany
bFachbereich Biologie, Universita¨t Konstanz, D-78457 Konstanz, Germany
Received 11 November 2004; accepted 25 November 2004
Abstract—Based on 1,2-O-isopropylidene-sn-glycerol, which is readily available from D-mannitol, five chiral building blocks for the
construction of structural variants of Staphylococcus aureus LTA designed and synthesized. Ligation of these building blocks led
readily to the target molecules 1 and 2. They demonstrated that the D-alanine residues at the glycerophosphate backbone are decisive
for the activation of the immune system.
Ó 2004 Elsevier Ltd. All rights reserved.
1. Introduction
idue the a-linked N-acetylglucosamine residue is omitted
and the glycerol moieties are replaced by 2-aminopro-
pane-1,3-diol residues with an enantiomeric configura-
tion to which D-alanine residues are connected via
amide linkages.
The inflammatory response to Gram-negative and
Gram-positive bacteria can hardly be distinguished.
Most of the responses to Gram-negative bacteria could
be attributed to lipopolysaccharides (LPS) and their
lipid A anchor as general principles.1,2 The response to
Gram-positive bacteria could only recently be attributed
clearly to lipoteichoic acid (LTA) for the Staphylococcus
aureus LTA,3 whose structure is shown in Scheme 1.4
For unequivocal bioactivity assignment, besides an im-
proved isolation procedure,5 the chemical synthesis of
the structurally closely related compound A was deci-
sive.6–8 A contains the hydrolytically labile D-alanine
residues in the required ratio with other substituents at
a hexameric glycerophosphate backbone; this com-
pound exhibited the same biological activity in terms
of initiation of cytokine release by human blood leuko-
cytes as found for the natural product.
This way the hydrolytic lability of the D-alanine residues
in S. aureus LTA and also in A and 1, which is already
fast at pH 8.5, can be overcome. Obviously it is of inter-
est to determine the influence of the gentiobiose residue
and of all the other structural entities on the biological
activity. In both molecules the basic backbone and the
D-alanine residues were retained, because their require-
ment for the activation of the immune system has
already been demonstrated.6
2. Results and discussion
2.1. Synthesis of compound 1
For the elucidation of the structural requirements for
bioactivity two structural variants were designed,
namely compounds 1 and 2 (Scheme 2). Compound 1
is closely related to A, however it lacks the phosphoryl-
gentiobiose moiety. In compound 2 more rigorous
changes are proposed: in addition to the gentiobiose res-
The retrosynthesis of 1, taking into consideration the
hydrolytic lability of the D-alanine residues, led to build-
ing blocks 3–6 (Scheme 2) of which 3 and 6 are known
compounds.9,10 Building block 4 was readily obtained
from 1,2-O-isopropylidene-sn-glycerol 811 (Scheme 3).
3-O-Allylation with allyl bromide and sodium hydride
(NaH) as base in DMF as solvent (!9), acid catalyzed
O-deisopropylidenation (!10),12 and then regioselective
1-O-silylation with tert-butyl-diphenylsilyl (TBDPS)
*
Corresponding author. Tel.: +49 7531 88 2538; fax: +49 7531 88
3135; e-mail: richard.schmidt@uni-konstanz.de
0957-4166/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2004.11.070

494
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
O
O
OH
P
R2
HO
HO
H
O
O
O
HO
n
O
O
O
O
O
R2
O
O
R1
HO
O
OH
O
O
R1 =
(D-Ala)
(~ 70%)
(~ 15%)
Me
+
NH3
R1 = H
OH
O
R1 = HO
HO
(α-D-GlcNAc) (~ 15%)
AcHN
R2 = Alkyl and/or branched alkyl chain
n ~ 40-50
A: n = 6; R1 = 1 x H, 4 x D-Ala, 1 x α-D-GlcNAc; R2 = C13H27
Scheme 1. General structure of lipoteichoic acid of Staphylococcus aureus.
O
O
P
O
P
O
P
O
P
O
P
O
P
O
O
O
O
O
O
O
O
O
O
O
O
HO
O
HO
–O
–O
–O
–O
HO
O
O
O
O
O
O
OH
O
O
O
O
AcHN
+
+
+
+
NH3
NH3
NH3
NH3
O
O
1
OH
HO
HO
OBn
O
P
C13H27
C13H27
Pr N
O
O
i
OH
BnO
2
OBn
O
OBn
6
3
P
OBn
OTBDPS
Pr N
O
i
O
2
P
O
OTBDPS
OMPM
iPr2N
O
5
AcHN
O
4
OBn
BnO
BnO
O
O
O
O
P
O
O
P
O
O
P
P
P
P
O
O
O
O
O
O
O
O
O
O
HO
O
HO
O
– O
–O
–O
– O
–O
NH
O
NH
NH
NH
NH
OH
O
O
O
O
O
+
+
+
+
+
NH3
NH3
NH3
NH3
NH3
O
2
6
3
OBn
P
OTBDPS
iPr2N
O
NH
7
O
NZ
H
Scheme 2. Structure of compounds 1 and 2 and their retrosynthesis.

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
495
8: R = H
OAc
O
RO
O
a
9: R = All
AcO
AcO
O
O
NH
CCl3
AllO
11
OTBDPS
Me
Me
N3
OH
14
Ref. 9
b
a
OR
AllO
OH
BnO
OTBDPS
AllO
OH
OBn
O
3
N3
10: R = H
c
15: R = Ac
16: R = Bn
11: R = TBDPS
b
O
RO
RO
OR
d
c
OTBDPS
e
RO
OTBDPS
RO
AcHN
OMPM
OBn
P
O
f
12: R = All
13: R = H
17: R = All
18: R = H
OTBDPS
OMPM
Pr N
O
i
2
d
e
O
POBn
5: R =
4
BnO
BnO
OBn
N
iPr2
Scheme 3. Synthesis of building blocks 3 and 4. Reagents and
conditions: (a) AllBr, NaH, DMF, rt (93%); (b) MeOH, Amberlyte
15 (H+-form), 40 °C (88%); (c) TBDPS-Cl, imidazole, 0 °C, CH2Cl2
(75%); (d) MPM-Cl, NaH, DMF, rt (70%); (e) (Ph3P)3RhCl, DBU,
EtOH, 90 °C; 1 M HCl, Me2CO (71%); (f) tetrazole, CH2Cl2, rt (81%).
Scheme 4. Synthesis of building block 5. Reagents and conditions: (a)
TMSOTf (0.1 equiv), CH2Cl2, rt (84%, a:b = 9:1); (b) NaOMe, MeOH;
BnBr, NaH, DMF, rt (74%); (c) CH3COSH, 40 °C (83%); (d)
(Ph3P)3RhCl, DBU, EtOH, 90 °C; 1 M HCl, Me2CO (77%); (e)
(iPr2N)2P(OBn), tetrazole, CH2Cl2, rt (85%).
chloride in the presence of imidazole as base afforded 2-
O-unprotected glycerol derivative 11, which was also
useful for the synthesis of building block 5. The 4-meth-
oxyphenylmethyl (MPM) group was selected as tempo-
rary protecting group for the 2-hydroxy group of 11,
because it can be selectively removed in the presence
of O-benzyl protecting groups. To this end, 11 was re-
acted with MPM chloride and NaH as base in DMF
to furnish orthogonally protected glycerol derivative
12. 3-O-Deallylation with Wilkinsonꢀs catalyst13 in the
presence of DBU as base in ethanol furnished the 3-O-
propenyl intermediate and then treatment with aqueous
HCl in acetone afforded 3-O-unprotected 13. Phosphit-
ylation with benzyloxy-bis(diisopropylamino)phos-
phane14 in the presence of tetrazole led to the desired
phosphite derivative 4 in good yield.
The construction of target molecule 1 from building
blocks 3–6 was performed stepwise in solution starting
with di-O-benzylglycerol 3 (Scheme 5). Phosphitylation
with 4 in the presence of tetrazole and then oxidation
of the phosphite moiety with tert-butyl hydroperoxide
afforded phosphate intermediate 19, which was desil-
ylated with tetrabutylammonium fluoride (TBAF) in
THF as solvent to furnish O-unprotected 20 as acceptor
for the next reaction cycle for chain extension. Thus, this
phosphitylation/oxidation and deprotection sequence
was repeated three times with 4 (!21–26), then with 5
(!27, 28) and finally the chain extension was terminated
by phosphitylation with 6 and then oxidation affording
29 in very good yield; 29 contains the desired glycero-
phosphate backbone. For the attachment of the D-ala-
nine residues, the four MPM groups were selectively
cleaved with ceric ammonium nitrate (CAN)19 in an ace-
tonitrile–toluene–water mixture as solvent affording
compound 30 in 70% yield. D-Alanylation was per-
formed with the triethylammonium salt of N-benzyl-
oxycarbonyl (Z) protected D-alanine in the presence
of N-methylimidazole and (benzotriazol-1-yloxy)tripyr-
rolidinophosphonium hexafluorophosphate (PyBOP)
as condensing agent furnishing 31 in 70% yield.
Hydrogenolytic O-debenzylation (i.e., cleavage of 15
O-benzyl groups) was performed with Pearlmanꢀs cata-
lyst20 in a dichloromethane–methanol–water mixture
as solvent; the crude product was purified by hydropho-
bic interaction chromatography (HIC) to afford target
molecule 1 in 41% yield. 1 was structurally ascertained
as all intermediates by NMR and MS data and most
intermediates also by elemental analyses.
For the synthesis of building block 5 known glycosyl
donor 1415,16 was reacted with 2-O-unprotected glycerol
derivative 11 as acceptor (Scheme 4); as expected based
on previous experiments,16 with 0.1 equivof TMSOTf
as catalyst the desired a-glycoside 15 was obtained in
high yield (81%, a:b = 9:1). O-Deacetylation with
NaOMe in methanol and then O-benzylation with benz-
yl bromide and NaH as base in DMF furnished tri-O-
benzyl derivative 16. Following known procedures,17,18
treatment of 16 with thioacetic acid transformed the
azido group into the acetylamino group to give 17; the
same result was obtained with 1,3-propane-dithiol as
reducing agent and then N-acetylation. Following the
above described procedures, 3-O-deallylation (!18)
and then phosphitylation afforded the desired building
block 5 again in very good yield.

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I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
3
4, a
O
P
19: R = TBDPS
20: R = H
O
R
BnO
O
O
b
OBn
OMPM
OBn
n
4, a
21: n = 2, R = TBDPS
22: n = 2, R = H
b
b
4, a
23: n = 3, R = TBDPS
24: n = 3, R = H
4, a
25: n = 4, R = TBDPS
26: n = 4, R = H
b
5, a
O
O
1
1'
P
P
O
BnO
O
O
O
OR
OBn
4
OBn
O
OBn
OMPM
1''
AcHN
O
27: R = TBDPS
28: R = H
BnO
BnO
b
OH
6, a
O
P
O
O
P
O
1'
3'
1
P
O
C13H27
O
O
O
O
BnO
O
O
O
C13H27
OBn
OBn
OBn
4
O
OR
OBn
1''
AcHN
O
O
BnO
BnO
OH
29: R = MPM
30: R = H
c
d
Me
CO
31: R =
HNZ
e
1
Scheme 5. Synthesis of target molecule 1. Reagents and conditions: (a) tetrazole, CH2Cl2, rt; t-BuO2H (19: 76%; 21: 95%; 23: 93%; 25: 90%; 27: 89%;
29: 82%); (b) TBAF, THF, rt (20: 87%; 22: 80%; 24: 92%; 26: 93%; 28: 80%); (c) CAN, MeCN/toluene/H2O; rt (70%); (d)
ðdÞ-MeCHðNHZÞCOÀHNEtþ, PyBOP, N-methylimidazole, rt (70%); (e) Pd(OH)2/C, H2, CH2Cl2/MeOH/H2, rt; HI chromatography (41%).
2
3
2.2. Synthesis of compound 2
base (DIPEA) to afford dipeptide 32 (Scheme 6). In
order to distinguish the primary hydroxy groups after
ester group reduction in 32, first O-tritylation with
monomethoxytrityl (MMT) chloride in pyridine (!33)
and then reduction with NaBH4 in ethanol as solvent
was performed leading to 2-aminopropane-1,3-diol
derivative 34. O-Silylation with TBDPS-Cl and imid-
azole (!35) and then O-detritylation with camphor-
sulfonic acid (CSA) as catalyst in dichloromethane and
methanol as nucleophile afforded intermediate 36. Phos-
phitylation as described above led to the desired build-
ing block 7 in very good yield.
The retrosynthesis of target molecule 2 led to building
blocks 3, 6 and 7 (Scheme 2). Hence, because of the sta-
bility of the amide linked D-alanyl residues the protect-
ing group pattern for the synthesis of 2 required only
O-benzyl and N-Z for permanent protection and O-silyl
for temporary protection. The synthesis of building
block 7 was initiated with commercially available D-ser-
ine methyl ester and Z-protected D-alanine, which were
connected with N-hydroxybenzotriazole (HOBt), dic-
€
yclohexylcarbodiimide (DCC) in the presence of Hunigꢀs

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
497
The construction of target molecule 2 from building
blocks 3, 6 and 7 was also performed stepwise starting
from di-O-benzylglycerol 3 (Scheme 7). Phosphitylation
with 7 and oxidation to phosphate and then desilylation
was repeated five times (!37–46); this reaction sequence
for chain extension was terminated by phosphitylation
of 46 with 6 and then oxidation with tert-butyl hydro-
peroxide affording 47, which contained the target mole-
cule in protected form. Hydrogenolytic O-debenzylation
and HIC-purification as described for the isolation of 1
furnished target molecule 2 in 35% yield. Again, struc-
tural assignments were based on NMR and MS data
and on the elemental analyses.
O
OMe
RO
O
32: R = H
a
NH
33: R = MMT
NZ
H
b
OR'
RO
O
34: R = MMT, R' = H
c
d
35: R = MMT, R' = TBDPS
36: R = H, R' = TBDPS
NH
NZ
H
e
The evaluation of the biological activity of 1 and 2 re-
vealed that initiation of cytokine release by human
blood leukocytes is practically the same as observed
for A and for natural LTA.21 Hence, it seems that nei-
ther the gentiobiose or the a-linked N-acetylglucosamine
residues, nor the configuration or the type of functional
group at the glycerol moiety of the glycerophosphate
backbone are required for an immune response. Obvi-
ously, this biological activity is essentially dependent
on the presence of D-alanine residues in a specific align-
ment. Hence, quite a few questions as to the importance
of the other groups and functionalities of LTA remain
to be elucidated.
7
Scheme 6. Synthesis of building block 7. Reagents and conditions: (a)
MMT-Cl, pyr, rt (92%); (b) NaBH4, EtOH (78%); (c) TBDPS-Cl,
imidazole, rt (85%); (d) CSA, CH2Cl2/MeOH, rt (86%); (e) (iPr2N)2-
P(OBn), tetrazole, CH2Cl2; t-BuO2H, CH2Cl2 (82%).
3
7, a
O
1
6
R
n
P
BnO
O
O
O
37: n = 1, R = TBDPS
38: n = 1, R = H
OBn
O
b
OBn
NH
NZ
H
3. Conclusion
7, a
In conclusion, with the help of synthetic structural vari-
ants of S. aureus LTA the prerequisites for the stimul-
ation of the immune system could be further elucidated:
the most important constituents are D-alanine residues.
The synthesis design considers the variable substitution
pattern and the hydrolytic lability of ester bound D-ala-
nyl residues. Hence, based on a small number of versa-
tile building blocks, the synthesis design also opens
ready access to a multitude of structural variants of glyc-
erophosphate backbone containing LTAs.
39: n = 2, R = TBDPS
40: n = 2, R = H
b
b
b
b
7, a
41: n = 3, R = TBDPS
42: n = 3, R = H
7, a
43: n = 4, R = TBDPS
44: n = 4, R = H
4. Experimental
4.1. General
7, a
45: n = 5, R = TBDPS
46: n = 5, R = H
Solvents were dried according to the standard proce-
dures. NMR spectroscopic measurements were per-
formed at 22 °C with Bruker DRX600 and Bruker
AC250 Cryospec instruments. TMS or the resonances
of the deuterated solvents were used as internal stan-
dard. CDCl3 (d = 7.24 ppm) was used as external stan-
dard; 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 mode measure-
ments, and trihydroxyacetophenone (THAP) was used
as matrix for negative mode measurements. Optical
rotations were measured with a Perkin Elmer polarime-
ter 241/MS in a 1-dm cell at 22 °C. Thin layer chroma-
tography (TLC) was performed on Merck silica gel 60
6, a
O
O
P
O
P
1
21
C13H27
O
BnO
O
O
O
O
OBn
O
C13H27
O
OBn
OBn
NH
NZ
H
5
O
47
c
2
Scheme 7. Synthesis of target molecule 2. Reagents and conditions: (a)
t
tetrazole; CH2Cl2; BuO2H, CH2Cl2 (37: 97%; 39: 95%; 41: 77%; 43:
91%; 45: 81%; 47: 82%); (b) TBAF, THF, rt (38: 82%, 40: 78%; 42:
72%; 44: 72%; 46: 78%); (c) Pd(OH)2, H2; CH2Cl2/MeOH/H2O (35%).

498
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
F254 plastic plates. Compounds were visualized by
treatment with a solution of (NH4)6Mo7O24Æ4H2O
(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 octyl-sepharose as sta-
tionary phase and as elution phase was used as a gradi-
ent of propanol (15–60%) in 0.1 M ammonium acetate
buffer (pH = 4.8).
chloride (15 mL, 1.5 equiv) in dry DMF (250 mL),
NaH (2.5 g, 0.1 mol, 1.5 equiv) was added portionwise
and the reaction mixture was stirred for 1 h at room
temp. The DMF was removed in vacuo; the rest was
redissolved in EtOAc and washed with a saturated
NH4Cl solution; the organic phase was dried over
MgSO4 and the solvent was removed in vacuo. After
flash chromatography (petroleum ether/EtOAc 30:1)
compound 12 (24 g, 70%) was obtained as colourless
syrup. TLC (petroleum ether/EtOAc 7:1): Rf = 0.59.
[a]D = À11.8 (c 1, CHCl3). 1H NMR (250 MHz, CDCl3):
d = 1.05 (s, 9H, tBu), 3.50–3.82 (m, 8H, OMe, 1-H, 2-H,
3-H) 3.96–4.02 (m, 2H, CH2CHCH2), 4.58 (s, 2H,
CH2PhPMB), 5.12–5.30 (m, 2H, CH2CHCH2), 5.80–
5.99 (m, 1H, CH2CHCH2), 6.80–6.89 (m, 2H, PhPMB),
7.19–7.28 (m, 2H, PhPMB), 7.30–7.47, 7.61–7.74 (m,
10H, Ph). C30H38O4SiÆ1/4H2O (495.2). Anal. Calcd: C:
72.76 H: 7.84. Found: C: 72.62, H: 7.91.
4.2. General procedures
4.2.1. General procedure for phosphate formation. The
alcohol and the phosphane (1.2 equiv) were coevapo-
rated 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 1 h in high vacuum) was
added. The reaction mixture was stirred for 1.5 h at
room temperature (TLC control), and after this time
t-BuO2H (1.3 equivof 5.5 M solution in decane) was
added. The reaction mixture was stirred for another
30–45 min and was diluted with EtOAc, washed with
saturated NaHCO3 solution. The organic phase was
dried over MgSO4, and evaporated in vacuo. Purifica-
tion by flash chromatography in silica gel gave the de-
sired product.
4.3.3. 2-O-(4-Methoxybenzyl)-1-O-(tert-butyldiphenylsil-
yl)-sn-glycerol 13. Compound 12 (16 g, 32.6 mmol)
was dissolved in dry EtOH (200 mL), DBU (0.48 mL)
and (Ph3P)3RhCl (0.61 g, 0.02 equiv) were added and
the reaction mixture was stirred at 90 °C for 15 min.
The solvent was removed in vacuo and the isomerized
product (Rf = 0.57, petroleum ether/EtOAc 7:1) was
redissolved in 200 mL of 1 M HCl/acetone (1:9) solu-
tion. The reaction mixture was stirred at 70–80 °C for
10 min and then neutralized with Et3N, diluted with
EtOAc and washed with saturated NaHCO3. The
organic phase was dried with MgSO4 and the solvent
removed in vacuo. Flash chromatography (petroleum
ether/EtOAc petroleum ether/EtOAc 4:1) gave 13
(10.5 g, 71%) as colourless syrup. TLC (petroleum
ether/EtOAc 7:1): Rf = 0.16. [a]D = À25.4 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 1.06 (s, 9H, tBu),
2.02 (br s, 1H, OH), 3.57–3.85 (m, 5H, 1-H, 2-H, 3-H)
3.80 (s, 3H, OMe) 4.44, 4.57 (2d, 2H, J = 11.4 Hz,
CH2Ph), 6.81–6.90 (m, 2H, PhPMB), 7.17–7.25 (m, 2H,
PhMPM), 7.33–7.48 (m, 6H, PhTBDPS), 7.63–7.71 (m,
4H, PhTBDPS). C27H35O4Si (451.7). Anal. Calcd: C:
71.80, H: 7.81. Found: C: 71.67, H: 7.65.
4.2.2. General procedure for the removal of tert-butyldi-
phenylsilyl protecting group (B). The silylated com-
pound was dissolved in THF (p.a. quality) and treated
with TBAF (1.2 equivof 1 M solution in THF). The
reaction mixture was stirred for 30–45 min at room tem-
perature (monitoring by TLC). After this time the reac-
tion mixture was diluted with EtOAc and washed with
saturated NH4Cl solution and water. The organic phase
was dried over MgSO4 and the solvent was evaporated
in vacuo. Flash chromatography in silica gel gave the
desired compound.
4.3. Preparation of 4
4.3.1. 3-O-Allyl-1-O-(tert-butyldiphenylsilyl)-sn-glycerol
solution of compound 1012 (22.9 g,
11. To
a
4.3.4. [Benzyloxy]-[diisopropylamino]-[1-O-tert-butyldi-
phenylsilyl-2-O-(4-methoxybenzyl)-sn-glycero] phosphane
4. Tetrazole (517 mg, 0.6 equiv) and compound 13
(5.55 g, 12.32 mmol) were dried separately for 1 h in
high vacuum. After unification of both compounds,
0.17 mol) and imidazole (17.7 g, 0.26 mol) in dry
CH2Cl2 (450 mL), 46 mL of TBDPS-Cl were added at
0 °C and the reaction mixture was stirred for 10 min.
The solution was washed with water; the organic phase
was dried over MgSO4 and then evaporated in vacuo.
Purification by flash silica gel (petroleum ether/EtOAc
10:1) yielded compound 11 (48 g, 75%) as a colourless
syrup. TLC (petroleum ether/EtOAc 5:1): Rf = 0.9.
benzyloxybis(diisopropylamino)phosphane14
(4.8 g,
1.2 equiv) was added (dissolved in 100 mL of dry
CH2Cl2). After 30 min the reaction mixture was diluted
with CH2Cl2 and washed with saturated NaHCO3 solu-
tion. The organic phase was dried over MgSO4 and the
solvent was removed in vacuo below 30 °C. Fast purifi-
cation by flash silica gel (petroleum ether/EtOAc 15:1,
1% Et3N) yielded 4 (6.86 g, 81%) as colourless oil.
TLC (petroleum ether/EtOAc 7:1, 1% NEt3):
Rf = 0.84. [a]D = À4.7 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 1.05 (s, 9H, tBu), 1.10–1.30
(m, 12H, CH(CH3)2), 3.52–3.90 (m, 10H, OMe,
CH(CH3)2, 1-H, 2-H, 3-H), 4.54–4.79 (m, 4H,
POCH2Ph, CH2PhPMB), 6.78–6.88 (m, 2H, PhPMB),
7.19–7.45, 7.62–7.73 (m, 12H, Ph). C40H54NO5PSi
1
[a]D = À3.6 (c 1, CHCl3). H NMR (250 MHz, CDCl3):
d = 1.0 (s, 9H, tBu), 1.49 (d, 1H, J = 5.1 Hz, OH), 3.47–
3.60 (m, 2H, CH2–glycerol), 3.71 (d, 2H, J = 5.5 Hz,
CH2–glycerol), 3.85–3.94 (m, 1H, CH–glycerol), 3.98–
4.02 (m, 2H, CH2CHCH2), 5.15–5.30 (m, 2H,
CH2CHCH2), 5.81–5.97 (m, 1H, CH2CHCH2), 7.35–
7.47, 7.64–7.69 (m, 10H, Ph).
4.3.2. 3-O-Allyl-2-O-(4-methoxybenzyl)-1-O-(tert-butyl-
diphenylsilyl)-sn-glycerol 12. To a solution of com-
pound 11 (26 g, 0.07 mol) and p-methoxybenzyl

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
499
(687.9). Anal. Calcd: C: 69.84, H: 7.91, N: 2.04. Found:
C: 69.69, H: 7.91, N: 2.02.
H, CH2–glycerol), 3.77 (dd, 1H, Jvic = 5.3 Hz,
Jgem = 10.6 Hz, CH2–glycerol), 3.86–3.89 (m, 1H, 5-
H), 3.96–4.04 (m, 4H, CHglycerol, CH2CHCH2–H),
4.36, 4.54 (dd, 2H, J = 12.1 Hz, CH2–Ph), 4.47, 4.77
(dd, 2H, J = 11.0 Hz, CH2–Ph), 4.83 (dd, 2H,
4.4. Preparation of 5
4.4.1. 3-O-Allyl-2-O-(2-azido-2-deoxy-3,4,6-tri-O-acetyl-
a-D-glucopyranosyl)-1-O-tert-butyldiphenylsilyl-sn-glyc-
J = 10.7 Hz,
CH2–Ph),
5.16–5.30
(m,
2H,
CH2CHCH2), 5.25 (d, 1H, J1,2 = 3.7 Hz, 1-H), 5.85–
5.95 (m, 1H, CH2CHCH2), 7.05–7.15, 7.16–7.42,
7.57–7.69 (m, 25H, Ph). 13C NMR (150.9 MHz,
CDCl3): d = 19.21 (1C, C(CH3)3), 26.84 (3C,
C(CH3)3), 63.3 (1C, C-2), 63.87 (1C, C–CH2–glycerol),
67.97 (1C, C-6), 70.67 (1C, C-5), 72.35 (1C,
CH2CHCH2), 73.47–76.79 (3C, CH2–Ph), 77.21 (1C,
C–CH2–glycerol), 78.15 (1C, C-4), 79.89 (1C, C-3),
97.49 (1C, C-1), 116.87 (1C, CH2CHCH2), 127.51–
138.26 (30C, Ph), 134.67 (1C, CH2CHCH2).
C49H57N3O7 (828.1). Anal. Calcd: C: 71.07, H: 6.94,
N: 5.07. Found: C: 70.90, H: 6.96, N: 4.97.
erol 15. To
a solution of acceptor 11 (8.1 g,
21.86 mmol) and donor 1415,16 (9.58 g, 20.18 mmol) in
dry CH2Cl2 (150 mL) was added TMSOTf (0.37 mL,
0.1 equiv) at room temp. After 2 min the reaction mix-
ture was neutralized with Et3N; the solvent was re-
moved in vacuo. Flash chromatography (petroleum
ether/EtOAc 6:1) yielded compound 15 (11.4 g, 76%).
b-Product (Rf = 0.34). TLC (petroleum ether/EtOAc
3:1): Rf = 0.42. [a]D = +107 (c 1, CHCl3). 1H NMR
(600 MHz, CDCl3): d = 1.06 (s, 9H, tBu), 1.95 (s, 3H,
Ac), 2.02 (s, 3H, Ac), 2.09 (s, 3H, Ac), 3.22 (dd, 1H,
J2,1 = 3.6 Hz, J2,3 = 10.7 Hz, 2-H), 3.56–3.66 (m, 2H,
CH2–glycerol), 3.66–3.73 (m, 1H, CH2–glycerol),
3.75–3.86 (m, 2H, 6-H, CH2–glycerol), 3.93–4.12 (m,
4H, 6-H, 20-H, CH2CHCH2–H), 4.19–4.27 (m, 1H, 5-
H), 5.04 (t, 1H, J4,3 = J4,5 = 9.7 Hz, 4-H), 5.13–5.20,
5.21–5.28 (m, 2H, CH2CHCH2), 5.40 (d, 1H,
J1,2 = 3.6 Hz, 1-H), 5.54 (dd, 1H, J = 9.4 Hz,
J = 10.4 Hz, 3-H), 5.83–5.92 (m, 1H, CH2CHCH2).
13C NMR (150.9 MHz, CDCl3): d = 19.15 (1C,
C(CH3)3), 20.53, 20.66, 20.72 (3C, CH3CO), 26.84
(3C, C(CH3)3), 60.79 (1C, C-2), 61.60 (1C, C-6), 64.04
(1C, C–CH2–glycerol), 67.36 (1C, C-5), 68.35 (1C, C-
4), 70.19 (1C, C–CH2–glycerol), 70.23 (1C, C-3),
72.39 (1C, CH2CHCH2), 77.3 (1C, CH), 97.06 (1C,
C-1), 117.1 (1C, CH2CHCH2), 127.86–135.54 (12C,
Ph), 134.39 (1C, CH2CHCH2), 169.58, 169.97, 170.57
(3C, CH3CO). C34H45N3O10Æ1/2H2O (692.8). Anal.
Calcd: C: 58.94, H: 6.69, N: 6.07. Found: C: 58.72,
H: 6.50, N: 5.86.
4.4.3. 2-O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-D-
glucopyranosyl)-3-O-allyl-1-O-tert-butyldiphenylsilyl-sn-
glycerol 17. Compound 16 (7.2 g, 8.7 mmol) was dis-
solved in pyridine/H2O (5:1, 150 mL), 0.7 mL of Et3N
and 1,3-propanedithiol (4.4 mL, 5 equiv) were added.
The reaction mixture was stirred overnight and the sol-
vent was removed in vacuo. The product was coevapo-
rated twice with toluene and redissolved in pyridine/
Ac2O (2:1, 150 mL). The reaction mixture was stirred
for 2 h. The solvent was evaporated in vacuo and the
product was coevaporated with toluene. Purification
by flash chromatography (toluene/acetone 8:1) yielded
compound 17 (5.82 g, 85%) as colourless foam. TLC
(toluene/acetone 2:1): Rf = 0.31. [a]D = +42 (c 1,
1
CHCl3). H NMR (600 MHz, CDCl3): d = 1.04 (s, 9H,
tBu), 1.84 (s, 3H, NHAc), 3.39 (dd, 1H, Jvic < 1 Hz,
Jgem = 10.6 Hz, 6-H), 3.52 (dd, 1H, Jvic = 6.7 Hz,
Jgem = 10.3 Hz, 10-H), 3.61 (dd, 1H, Jvic = 2.9 Hz,
Jgem = 10.8 Hz, 6-H), 3.62–3.69 (m, 3H, 30-H, 10-H, 3-
H), 3.72–3.82 (m, 3H, 30-H, 5-H, 4-H), 3.84 (m, 1H,
20-H), 3.91–4.01 (m, 2H, CH2CHCH2–H), 4.26 (ddd,
1H, J = 3.4 Hz, 2-H), 4.37/4.54 (dd, 1H, J = 12.2 Hz,
CH2–Ph), 4.48/4.78 (dd, 2H, J = 10.9 Hz, CH2–Ph),
4.63/4.79 (dd, 2H, J = 11.5 Hz, CH2–Ph), 4.89 (d, 1H,
J1,2 = 3.5 Hz, 1-H), 5.15–5.28 (m, 2H, CH2CHCH2),
5.79–5.87 (m, 1H, CH2CHCH2), 7.09–7.58, 7.57–7.67
(m, 25H, Ph). 13C NMR (150.9 MHz, CDCl3):
d = 19.20 (1C, C(CH3)3), 23.43 (1C, NHAc), 26.81
(3C, C(CH3)3), 52.77 (1C, C-2), 63.47 (1C, C-30), 68.18
(1C, C-6), 69.65 (1C, C-10), 71.36 (1C, C-5), 72.28 (1C,
CH2CHCH2), 73.32–74.90 (3C, CH2–Ph), 77.97 (1C,
C-4), 78.09 (1C, C-20), 80.91 (1C, C-3), 98.33 (1C, C-
1), 117.39 (1C, CH2CHCH2), 127.56–138.53 (31C, Ph,
CH2CHCH2), 169.81 (1C, COCH3). C51H51NO8SiÆ1/
2H2O (853.1). Anal. Calcd: C: 71.80, H: 7.33, N: 1.64.
Found: C: 71.84, H: 7.27, N: 1.42.
4.4.2. 3-O-Allyl-2-O-(2-azido-3,4,6-tri-O-benzyl-2-deoxy-
a-D-glucopyranosyl)-1-O-tert-butyldiphenylsilyl-sn-glyc-
erol 16. Compound 15 (9.8 g, 14.33 mmol) was dis-
solved in dry MeOH (200 mL) and 4 mL of 0.1 M
NaOMe solution (in MeOH) were added (pH 9). The
reaction mixture was stirred overnight and neutralized
with ion exchange resin IR120 (H+-Form); the solvent
was removed in vacuo. This intermediate (7.4 g,
13.28 mmol Rf = 0.15, toluene/acetone 2:1) was dried
in high vacuum and dissolved in dry DMF (150 mL),
BnBr (8 mL, 66.67 mmol) was added and NaH
(1.28 g, 53.33 mmol) was added portionwise. After
24 h the solvent was removed in vacuo, redissolved
in EtOAc and washed with a saturated solution of
NH4Cl. The organic phase was dried over MgSO4
and the solvent was removed in vacuo. Purification
by flash chromatography (petroleum ether/EtOAc
9:1) yielded 16 (8.17 g, 74%) as colourless syrup.
TLC (petroleum ether/EtOAc 5:1): Rf = 0.53.
4.4.4. 2-O-(2-Acetamido-3,4,6-tri-O-benzyl-2-deoxy-a-
D-glucopyranosyl)-1-O-tert-butyldiphenylsilyl-sn-glycerol
18. Compound 17 (2.7 g, 3.43 mmol) was dissolved in
dry EtOH (40 mL), DBU (77 lL, 0.15 equiv) and
(Ph3P)3RhCl (1 g, 0.3 equiv) were added and the reac-
tion mixture was stirred at 90 °C for 15 min. The solvent
was removed in vacuo, and the reaction intermediate
1
[a]D = +63 (c 1, CHCl3). H NMR (600 MHz, CDCl3):
d = 1.04 (s, 9H, tBu), 3.01 (dd, 1H, J2,1 = 3.6 Hz,
J2,3 = 10.3 Hz, 2-H), 3.36 (dd, 1H, Jvic = 2.0 Hz,
Jgem = 10.9 Hz, 6-H), 3.55 (dd, 1H, Jvic = 3.0 Hz,
Jgem = 10.9 Hz, 6-H), 3.61 (dd, 1H, Jvic = 6.3 Hz,
Jgem = 10.3 Hz, CH2–glycerol), 3.71–3.73 (m, 3H, 4-

500
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
(Rf = 0.76, toluene/acetone 1.5:1) was redissolved in
50 mL of a 1 M HCl/acetone solution (1:9). The reaction
mixture was stirred at 70 °C for 15 min. The reaction
mixture was neutralized with Et3N, diluted with EtOAc
and washed with saturated NaHCO3 solution. The
organic phase was dried over MgSO4, and the solvent
was removed in vacuo. Flash chromatography (tolu-
ene/acetone 2:1) yielded 18 (1.98 g, 77%) as light brown
foam. TLC (toluene/acetone 1.5:1): Rf = 0.55.
[a]D = +62.6 (c 1, CHCl3). 1H NMR (600 MHz, CDCl3):
d = 1.05 (s, 9H, tBu), 1.81 (s, 3H, NHAc), 2.52 (s, 1H,
OH), 3.42 (dd, 1H, Jvic = 1.3 Hz, Jgem = 10.7 Hz, 30-H),
3.60 (dd, 1H, Jvic = 3.7 Hz, Jgem = 10.8 Hz, 30-H),
3.74–3.84 (m, 8H, 10-H, 6-H, 20-H, 3-H, 4-H, 5-H),
4.19 (m, 1H, 2-H), 4.34/4.54 (dd, 2H, J = 12.2 Hz,
CH2–Ph), 4.51/4.80 (dd, 2H, J = 10.9 Hz, CH2–Ph),
4.63/4.81 (dd, 2H, J = 11.6 Hz, CH2–Ph), 5.0 (d, 1H,
J1,2 = 3.6 Hz, 1-H), 5.91 (d, 1H, J = 8.6 Hz, NH),
7.10–7.46, 7.60–7.77 (m, 25H, Ph). 13C NMR
(150.9 MHz, CDCl3): d = 19.12 (1C, C(CH3)3), 23.24
(1C, NHAc), 26.80 (3C, C(CH3)3), 52.93 (1C, C-2),
62.29 (1C, C-10), 64.94 (1C, C-6), 68.26 (1C, C-30),
71.36 (1C, C-20), 73.33–74.83 (3C, CH2–Ph), 78.01–
80.39 (3C, C-3, C-4, C-5), 97.28 (1C, C-1), 127.58–
138.78 (30C, Ph, CH2CHCH2–C), 170.25 (1C, COCH3).
C48H57NO8Si (804.1). Anal. Calcd: C: 71.70, H: 7.15, N:
1.74. Found: C: 71.45, H: 7.20, N: 1.36.
4.5. Preparation of target molecule 1
4.5.1. Benzyl[(2S)-1,2-di-benzyloxy-propan-3-yl]-[(2R)-
1-tert-butyldiphenylsilyloxy-2-(4-methoxybenzyloxy)-pro-
pan-3-yl] phosphate 19. Procedure A. Compound 3
(1.44 g, 5.29 mmol), 4 (3.64 g, 1 equiv). TLC before oxi-
dation (petroleum ether/EtOAc 2:1, 1% Et3N) Rf = 0.74.
Flash chromatography (petroleum ether/EtOAc 3:1)
yielded phosphate 19 (3.5 g, 76%) as colourless syrup.
TLC (petroleum ether/EtOAc 2:1): Rf = 0.41. [a]D = À6
(c 1, CHCl3). 1H NMR (600 MHz, CDCl3): d = 1.03
(s, 9H, tBu), 3.46–3.56 (m, 2H, 6-H), 3.59–3.80 (m,
7H, 1-H, 2-H, 5-H, OMe), 4.07–4.30 (m, 4H, 3-H, 4-
H), 4.41–4.65 (m, 6H, CH2–Ph, CH2–PhOMe), 4.97–
5.04 (m, 2H, POCH2–Ph), 6.75–6.83, 7.12–7.20 (m,
4H, PhPMB), 7.20–7.44, 7.58–7.68 (m, 25H, Ph). 13C
NMR (150.9 MHz, CDCl3): d = 18.68 (1C, C(CH3)3),
26.29 (3C, C(CH3)3), 54.73 (1C, OMe), 62.18 (1C, C-
1), 66.42, 66.51, 66.55 (2C, C-3, C-4), 68.72 (2C,
POCH2–Ph, C-6), 71.34, 71.73, 72.91 (3C, CH2–Ph),
76.1 (1C, C-5), 77.27 (1C, C-2), 113.20 (2C, CPMB),
127.09–137.66 (33C, Ph), 158.63 (1C, C–OMe). 31P
NMR (242.9 MHz, CDCl3): d = 0.39, 0.40 (2s, 1P).
FAB-MS (positive mode, Matrix NBOH+NaI, THF):
[M+Na]+, m/z = 897; found: m/z = 897.
4.5.2. Benzyl[(2S)-1,2-di-benzyloxy-propan-3-yl]-[(2R)-1-
hydroxy-2-(4-methoxybenzyloxy)-propan-3-yl] phosphate
20. Procedure B. Phosphate 19 (2.79 g, 3.19 mmol)
Purification by flash silica gel (petroleum ether/EtOAc
1:1) yielded compound 20 (1.75 g, 87%) as colourless
syrup. TLC (petroleum ether/EtOAc 1:1): Rf = 0.10;
(toluene/acetone 1:1): Rf = 0.63. [a]D = +8.6 (c 1,
CHCl3). 1H NMR (600 MHz, CDCl3): d = 2.15–2.38
(br s, 1H, OH), 3.46–3.73 (m, 5H, 1-H, 2-H, 6-H),
3.73–4.11 (m, 4H, 5-H, OMe), 4.03–4.29 (m, 4H, 3-H,
4-H), 4.41–4.71 (m, 6H, CH2–Ph, CH2–PhOMe), 5.00–
5.12 (m, 2H, POCH2–Ph), 6.79–6.92, 7.12–7.20 (m,
2H, PhPMB), 7.14–7.41 (m, 20H, Ph). 13C NMR
(150.9 MHz, CDCl3,): d = 55.2 (1C, OMe), 61.0 (1C,
C-1), 65.5–67.5 (2C, C-3, C-4), 69.0 (1C, C-6), 69.4
(1C, POCH2–Ph), 71.6–73.8 (3C, CH2–Ph), 76.7 (1C,
C-5), 77.1 (1C, C-2). 31P NMR (242.9 MHz, CDCl3):
d = À1.55, À1.58 (2s, 1P). MALDI-MS (positive mode,
Matrix DHB, THF): [M+Na]+, m/z = 659.7; found:
m/z = 659.5, [M+K]+, m/z = 675.8; found: m/z = 675.8.
C35H41O9P (636.7). Anal. Calcd: C: 66.03, H: 6.49.
Found: C: 65.74, H: 6.50.
4.4.5. [Benzyloxy]-[diisopropylamino]-[2-O-(2-acetamido-
3,4,6-tri-O-benzyl-2-deoxy-a-D-glucopyranosyl)-1-O-tert-
butyldiphenylsilyl-sn-glycero] phosphane 5. Compound
18 (1.92 g, 2.57 mmol) was dried for 1 h in high vac-
uum together with tetrazole (108 mg, 0.6 equiv). Under
argon
atmosphere
benzyloxybis(diisopropyla-
mino)phosphane (1 g, 1.2 equiv) was added (dissolved
in 30 mL of dry CH2Cl2). After 15 min the reaction
mixture was diluted with CH2Cl2 and washed with a
saturated NaHCO3 solution. The organic phase was
dried over MgSO4 and the solvent was removed in va-
cuo below 30 °C. Fast purification by flash silica gel
(toluene/EtOAc 4:1, 1% Et3N) yielded compound 5
(2.27 g, 85%) as colourless oil. TLC (toluene/acetone
1.5:1, 1% NEt3): Rf = 0.71. [a]D = +31.2 (c 1, CHCl3).
1H NMR (600 MHz, DMSO-d6): d = 0.96 (s, 9H,
tBu), 1.0–1.17 (m, 12H, C(CH3)2), 1.80/1.81 (2s, 3H,
NHAc), 3.36–3.41 (m, 1H, 30-H), 3.44–3.49 (m, 1H,
30-H), 3.49–3.59 (m, 3H, CH, 4-H), 3.59–3.65 (m, 1H,
10-H), 3.67–3.82 (m, 4H, 10-H, 3-H, 6-H), 3.87–3.98
(m, 3H, 20-H, 2-H, 5-H), 4.33–4.39, 4.42–4.51, 4.55–
4.74 (m, 8H, CH2–Ph, POCH2–Ph), 4.92 (m, 1H, 1-
H), 7.07–7.43, 7.55–7.65, 7.70–7.82 (m, 30H, Ph). 13C
NMR (150.9 MHz, DMSO-d6): d = 18.72 (1C,
C(CH3)3), 22.53 (1C, NHAc), 24.32 (4C, C(CH3)2),
26.62 (3C, C(CH3)3), 42.40 (2C, C(CH3)2), 52.63 (1C,
C-2), 60.70 (1C, C-10), 61.30/63.78 (1C, C-6), 64.55/
64.67 (1C, POCH2–Ph), 68.33 (1C, C-30), 70.39 (1C,
C-5), 73.85–73.99 (3C, CH2–Ph), 76.43 (1C, C-20),
78.10 (1C, C-4), 79.68 (1C, C-3), 96.17/96.34 (1C, C-
1), 125.30–139.11 (36C, Ph), 169.26 (1C, COCH3).
31P NMR (242.9 MHz, DMSO-d6): d = 148.38 (s),
148.63 (s). C61H77N2O9PSi (1041.3). Anal. Calcd: C:
70.36, H: 7.45, N: 2.69. Found: C: 70.39, H: 7.64, N:
2.54.
4.5.3. Diphosphate 21. Procedure A. Compound 20
(1.67 g, 2.62 mmol) TLC before oxidation (petroleum
ether/EtOAc 1:1, 1% Et3N, Rf = 0.62). Purification by
flash
chromatography
(petroleum
ether/EtOAc
2:1!1:1) yielded compound 21 (3.1 g, 95%) as colour-
less syrup. TLC (petroleum ether/EtOAc 1:1):
Rf = 0.38. [a]D = À4.7 (c 1, CHCl3). 1H NMR
(600 MHz, CDCl3): d = 1.02 (s, 9H, tBu), 3.46–3.55
(m, 2H, 9-H), 3.59–3.77 (m, 11H, 1-H, 2-H, 5-H, 8-H,
OMe), 3.94–4.30 (m, 8H, 3-H, 4-H, 6-H, 7-H), 4.38–
4.62 (m, 8H, CH2–Ph, CH2–PhOMe), 4.92–5.03 (m,
4H, POCH2–Ph), 6.70–6.80 (m, 4H, PhPMB), 7.12–
7.43, 7.58–7.66 (m, 38H, Ph). 13C NMR (150.9 MHz,
CDCl3): d = 19.16 (1C, C(CH3)3), 26.77 (3C, C(CH3)3),

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
501
55.20 (1C, OMe), 62.63 (1C, C-1), 65.81 (2C, C-4, C-6),
67.08 (2C, C-3, C-7), 69.09 (1C, C-9), 69.28 (2C,
POCH2–Ph), 71.81–73.39 (4C, CH2–Ph), 75.42 (1C, C-
5), 76.53 (1C, C-8), 77.71 (1C, C-2), 113.69, 113.74
(4C, C2COMe), 127.58–138.11 (42C, Ph), 159.13,
159.25 (2C, C–OMe). 31P NMR (600 MHz, CDCl3):
d = 0.069–0.041 (m, 2P). MALDI-MS (positive mode,
Matrix DHB, THF): [M+Na]+, m/z = 1262.4; found:
m/z = 1261.8, [M+K]+, m/z = 1278.5; found: m/z =
1277.9. C69H80O15P2Si (1239.4). Anal. Calcd: C: 66.87,
H: 6.51. Found: C: 65.60, H: 6.53.
(1383.3). Anal. Calcd: C: 61.65, H: 6.19. Found: C:
61.68, H: 6.42.
4.5.7. Tetraphosphate 25. Procedure A. Compound 24
(2.37 g, 1.74 mmol). TLC before oxidation (toluene/ace-
tone 1:1, 1% Et3N, Rf = 0.74). After purification in silica
gel (toluene/acetone 3:1) compound 25 (3.1 g, 90%) was
obtained as colourless syrup. TLC (toluene/acetone 1:1):
Rf = 0.70. [a]D = À3.6 (c 1, CHCl3). 1H NMR
(600 MHz, CDCl3): d = 1.02 (s, 9H, tBu), 3.46–3.53
(m, 2H, 15-H), 3.59–3.77 (m, 19H, 1-H, 2-H, 5-H, 8-
H, 11-H, 14-H, OMe), 3.90–4.28 (m, 16H, 3-H, 4-H,
6-H, 7-H, 8-H, 10-H, 12-H, 13-H), 4.38–4.50, 4.52–
4.63 (m, 12H, CH2–Ph, CH2–PhOMe), 4.92–5.03 (m,
8H, POCH2–Ph), 6.71–6.81 (m, 8H, PhPMB), 7.10–7.43
(m, 44H, Ph), 7.59–7.67 (m, 4H, PhTBDPS). 13C NMR
(150.9 MHz, CDCl3): d = 26.77 (3C, C(CH3)3), 55.17
(4C, OMe), 62.6 (1C, C-1), 65.79–67.1 (3C, C-3, C-4,
C-6, C-7, C-8, C-10, C-12, C-13), 69.06 (1C, C-15),
69.41 (4C, POCH2–Ph), 71.86–73.39 (6C, CH2–Ph),
75.36 (3C, C-5, C-8, C-11), 76.79 (1C, C-14), 77.7 (1C,
C-2), 113.76 (8C, C2COMe), 127.59–135.57 (Ph). MAL-
DI-MS (positive mode, Matrix DHB, THF): [M+Na]+,
m/z = 1991.1; found: m/z = 1989.9. C105H122O27P4Si
(1968.1). Anal. Calcd: C: 64.08, H: 6.25. Found: C:
64.18, H: 6.23.
4.5.4. Diphosphate 22. Procedure B. Compound 21
(3.08 g, 2.49 mmol) Purification by flash chromatogra-
phy (toluene/acetone 2:1) yielded product 22 (2.0 g,
80%) as colourless syrup. TLC (toluene/acetone 1:1):
Rf = 0.38. [a]D = +1.5 (c 1, CHCl3). 1H NMR
(600 MHz, CDCl3): d = 3.47–3.80 (m, 13H, 1-H, 2-H,
5-H, 8-H, 9-H, OMe), 3.91–4.22 (m, 8H, 3-H, 4-H, 6-
H, 7-H), 4.41–4.65 (m, 8H, CH2–Ph, CH2–PhOMe),
4.95–5.06 (m, 4H, POCH2–Ph), 6.76–6.88 (m, 4H,
PhPMB), 7.12–7.36 (m, 26H, Ph). 13C NMR
(150.9 MHz, CDCl3): d = 55.24 (2C, OMe), 60.9 (1C,
C-1), 65.30–66.60 (3C, C-3, C-4, C-6), 67.13 (1C, C-7),
69.00 (1C, C-9), 69.42–6953 (2C, POCH2–Ph), 71.70–
73.42 (4C, CH2–Ph), 76.46 (1C, C-5), 76.79 (1C, C-8),
77.30 (1C, C-2), 113.80, 113.86 (4C, C2COMe),
127.61–137.93 (30C, Ph), 159.35 (2C, C–OMe). MAL-
DI-MS (positive mode, Matrix DHB, THF):
[M+Na]+, m/z = 1024.0; found: m/z = 1022.8, [M+K]+,
m/z = 1040.1; found: m/z = 1039.0. C53H62O15P2Æ1/
4H2O (1005.5). Anal. Calcd: C: 63.31, H: 6.27. Found:
C: 63.17, H: 6.14.
4.5.8. Tetraphosphate 26. Procedure B. Compound 25
(3.05 g, 1.55 mmol) Purification by flash chromatogra-
phy (toluene/acetone 2:1!1:1) yielded compound 26
(2.5 g, 93%) as colourless syrup. TLC (toluene/acetone
1:1): Rf = 0.64. [a]D = À0.4 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 3.47–3.82 (m, 21H, 1, 2, 5, 8,
11, 14, 15-H, OMe), 3.91–4.25 (m, 16H, 3, 4, 6, 7, 9,
10, 12, 13-H), 4.40–4.64 (m, 12H, CH2–Ph, CH2–
PhOMe), 4.92–5.09 (m, 8H, POCH2–Ph), 6.71–6.89
(m, 8H, PhPMB), 7.11–7.41 (m, 38H, Ph). MALDI-
MS (positive mode, Matrix DHB, THF): [M+Na]+,
m/z = 1752.7; found: m/z = 1750.2. C89H104O27P4
(1729.66). Anal. Calcd: C: 61.80, H: 6.06. Found: C:
61.71, H: 6.16.
4.5.5. Triphosphate 23. Following procedure A. Com-
pound 22 (2.21 g, 2.21 mmol). TLC before oxidation
(toluene/acetone 2:1, 1% Et3N, Rf = 0.66). Purification
in (toluene/acetone 4:1!3:1) yielded 23 (3.3 g, 93%) as
colourless syrup. TLC (toluene/acetone 3:1): Rf = 0.20.
1
[a]D = À3.5 (c 1, CHCl3). H NMR (250 MHz, CDCl3):
d = 1.02 (s, 9H, tBu), 3.48–3.55 (m, 2H, 12-H), 3.59–3.80
(m, 15H, 1-H, 2-H, 5-H, 8-H, 11-H, OMe), 3.90–4.29
(m, 12H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H), 4.39–4.51,
4.57–4.62 (m, 10H, CH2–Ph, CH2–PhOMe), 4.91–5.03
(m, 6H, POCH2–Ph), 6.72–6.83 (m, 6H, PhPMB), 7.11–
7.44 (m, 37H, Ph), 7.58–7.68 (m, 4H, PhTBDPS). MAL-
DI-MS (positive mode, Matrix DHB, THF):
4.5.9. Pentaphosphate 27. Procedure A. Compound 26
(2.08 g, 1.20 mmol), compound 5 (1.5 g, 1.2 eq). TLC
before oxidation (toluene/acetone 1:1, 1% Et3N,
Rf = 0.64). Purification in silica gel (toluene/acetone
2:1!1:3) gave compound 27 (2.9 g, 89%) as colourless
syrup. TLC (toluene/acetone 1:1): Rf = 0.62; 0.58.
[M+Na]+,
m/z = 1626.7;
found:
m/z = 1627.1.
1
C87H101O21P3SiÆ3/4 H2O (1617.2). Anal. Calcd: C:
64.61 H: 6.39. Found: C: 64.63 H: 6.51.
[a]D = +9.2 (c 1, CHCl3). H NMR (600 MHz, CDCl3):
d = 1.01 (s, 9H, tBu), 1.88–2.02 (m, 3H, NHCOCH3),
3.13–3.21 (m, 1H, 600-H), 3.44–3.55 (m, 4H, 500-H,
600-H, 18-H), 3.55–3.81 (m, 22H, 1-H, 2-H, 5-H, 8-H,
11-H, 14-H, 17-H, 300-H, 400-H, OMe), 3.91–4.14, 4.15–
4.22 (m, 18H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H,
15-H, 16-H), 4.22–4.35, 4.36–4.52, 5.55–4.67, 4.67–4.77
(m, 22H, 100-H, 200-H, 3-H, CH2–Ph, CH2–PhOMe),
4.84–5.02 (m, 10H, POCH2–Ph), 6.68–6.82, 7.01–7.43,
7.52–7.63 (m, 76H, Ph). 13C NMR (150.9 MHz,
CDCl3,): d = 52.7 (200-C), 55.2 (OMe), 61.6 (C-1), 65.9
(C-4, C-6, C-7, C-9, C-10, C-12, C-13, C-15), 67.1 (3,
C-16), 68.0 (C-600), 69.1 (C-18), 69.3 (POCH2–Ph),
71.1–75.3 (CH2–Ph), 75.5–77.0 (C-2, C-5, C-8, C-11,
C-14, C-17), 77.8–81.1 (C-300, C-400), 100.2 (C-100).
4.5.6. Triphosphate 24. Procedure B. Compound 23
(3.32 g, 2.07 mmol). Purification in (toluene/acetone
2:1!1:1) yielded 24 (2.6 g, 92%) as colourless syrup.
TLC (toluene/acetone 1:1): Rf = 0.22. [a]D = +0.6 (c 1,
1
CHCl3). H NMR (250 MHz, CDCl3): d = 2.88 (br s,
1H, OH), 3.47–3.79 (m, 17H, 1-H, 2-H, 5-H, 8-H, 11-
H, 12-H, OMe), 3.91–4.25 (m, 12H, 3-H, 4-H, 6-H,
7-H, 9-H, 10-H), 4.41–4.62 (m, 10H, CH2–Ph, CH2–
PhOMe), 4.93–5.07 (m, 6H, POCH2–Ph), 6.72–6.88
(m, 6H, PhPMB), 7.10–7.38 (m, 31H, Ph). MALDI-MS
(positive mode, Matrix DHB, THF): [M+Na]+,
m/z = 1388.3; found: m/z = 1388.2. C71H83O21P3ÆH2O

502
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
MALDI-MS (positive mode, Matrix DHB, THF):
[M+H3O+]+, m/z = 2704.8; found: m/z = 2704.7.
C144H166NO37P5SiÆH2O (2703.8). Anal. Calcd: C:
63.97, H: 6.26, N: 0.52. Found: C: 64.01, H: 6.40, N:
0.52.
the solvent was evaporated in vacuo. A fast flash chro-
matography (toluene/acetone 1:2!acetone) gave 30
(290 mg, 70%) as colourless syrup, which was stored at
–20 °C. TLC (toluene/acetone 1:2): Rf = 0.47, 0.58.
1
[a]D = +7 (c 0.5, CHCl3). H NMR (600 MHz, CDCl3):
d = 0.80–0.92 (t, 6H, Me), 1.10–1.36 (m, 40H, CH2
chain), 1.47–1.61 (m, 4H, COCH2CH2R), 1.88–1.99
(m, 3H, NHAc), 2.18–2.30 (m, 4H, COCH2CH2R),
3.52 (m, 2H, 18-H), 3.61, 3.66 (m, 2H, 600-H), 3.68 (m,
1H, 400-H), 3.69 (m, 1H, 300-H), 3.73 (m, 2H, 17, 2-H),
3.80 (m, 1H, 500-H), 3.98, 4.08 (m, 4H, 6-H, 7-H, 9-H,
10-H, 12-H, 13-H, 15-H), 4.06 (m, 7H, 10-H, 30-H,
1-H, 3-H), 4.09, 4.19 (m, 2H, 16-H), 4.26 (m, 1H,
30-H), 4.34 (m, 1H, 200-H), 4.38–4.76 (m, 10H, CH2Ph),
4.85/4.89 (m, 1H, 100-H), 5.02 (m, 12H, POCH2Ph),
5.08 (m, 1H, 20-H), 7.05–7.45 (m, 55H, Ph). 13C NMR
(150.9 MHz, CDCl3): d = 14.1 (2C, Me), 22.9 (1C,
NHAc), 24.8 (2C, COCH2CH2R), 22.7–31.9 (20C,
CH2 chain), 34.0, 34.1 (2C, COCH2R), 52.7 (1C,
C-200), 61.6 (1C, C-30), 65.6 (3C, C-1, C-3, C-10), 67.3
(1C, C-16), 68.2 (CH2Glyc.), 68.5 (1C, C-600), 69.0
(1C, C-18), 69.1 (1C, C-20), 69.9 (6C, POCH2Ph), 71.7
(1C, C-500), 72.2–75.5 (5C, CH2Ph), 76.5 (2C, C-2,
C-17), 77.8 (1C, C-400), 80.8 (1C, C-300), 99.5/100.0 (1C,
C-100), 127.6–138.4 (C–Ph), 172.8, 173.2 (2C, COOR).
MALDI-MS (positive mode, Matrix p-nitroani-
line+NaI, THF): [M+Na]+, m/z = 2654.7; found:
m/z = 2656.4. C134H181NO40P6 (2631.7). Anal. Calcd:
C: 61.16, H: 6.93, N: 0.53. Found: C: 60.94, H: 7.38,
N: 0.47.
4.5.10. Pentaphosphate 28. Procedure B. Compound
27 (2.87 g, 1.07 mmol) Purification by flash chromato-
graphy (toluene/acetone 2:1!1:1) yielded compound
28 (2.1 g, 80%) as colourless syrup. TLC (toluene/
acetone 1:1): Rf = 0.4, 0.51, 0.57. [a]D = +15.2 (c 1,
CHCl3). 1H NMR (600 MHz, CDCl3): d = 1.84–2.02
(m, 3H, NHCOCH3), 3.38–3.80, 3.88–4.21 (m, 39H,
300-H, 400-H, 500-H, 600-H, OMe, 1–18-H), 4.21–4.82 (m,
19.5H, 100-H, 200-H, CH2–Ph, CH2–PhOMe), 4.88–5.06
(m, 10.5H, 100-H, POCH2–Ph), 6.69–6.82 (m, 8H, Ph),
7.05–7.36 (m, 58H, Ph). MALDI-MS (positive mode,
Matrix DHB, THF): [M+Na]+, m/z = 2470.4; gef.:
m/z = 2469.9. C128H148NO37P5 (2447.4). Anal. Calcd:
C: 62.82, H: 6.10, N: 0.57. Found: C: 62.82, H: 6.33,
N: 0.58.
4.5.11. Hexaphosphate 29. Procedure A. Compound
28 (530 mg, 0.217 mmol), 6 (211.2 mg, 1.3 equiv). Purifi-
cation by flash chromatography (toluene/acetone 2:1)
gave 29 (550 mg, 82%) as colourless syrup. TLC (tolu-
ene/acetone 1:1): Rf = 0.48, Rf = 0.56. [a]D = À16 (c
1
0.25, CHCl3). H NMR (600 MHz, CDCl3): d = 0.80–
0.90 (t, 6H, Me), 1.15–1.32 (m, 40H, CH2 chain),
1.48–1.63 (m, 4H, COCH2CH2R), 1.90–2.0 (m, 3H,
NHAc), 2.19–2.29 (m, 4H, COCH2CH2R), 3.46–3.55
(m, 2H, 18-H), 3.62 (m, 1H, 600-H), 3.65 (m, 4H, 5-H,
8-H, 11-H, 14-H), 3.68 (m, 1H, 600-H), 3.69 (m, 13H,
OMe, 300-H), 3.71 (m, 1H, 400-H), 3.73 (m, 2H, 17-H,
2-H), 3.78 (m, 1H, 500-H), 3.98–4.07 (m, 22H, 1-H, 3-
H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H, 15-H, 10-
H), 4.07 (m, 1H, 30-H), 4.08 (m, 1H, 16-H), 4.18 (m,
1H, 16-H), 4.26 (m, 1H, 30-H), 4.78 (m, 1H, 200-H),
3.79–4.76 (m, 18H, CH2Ph), 4.78 (m, 1H, 100-H), 4.96
(m, 12H, POCH2Ph), 5.16 (m, 1H, 20-H), 6.73–6.82
(m, 8H, PhMPM), 7.08–7.38 (m, 63H, Ph). 13C NMR
(150.9 MHz, CDCl3,): d = 52.9 (1C, C-200), 55.6 (4C,
OMe), 62.1 (1C, C-30), 66.2 (11C, C-1, C-3, C-4, C-6,
C-7, C-9, C-10, C-12, C-13, C-15, C-10), 66.8 (1C, C-
16), 68.9 (1C, C-600), 69.4 (1C, C-18), 69.8 (1C, C-20),
70.0 (6C, POCH2Ph), 72.0–76.0 (9C, CH2Ph), 72.2
(1C, C-500), 75.9 (4C, C-5, C-8, C-11, C-14), 77.0 (2C,
C-2, C-17), 78.0 (1C, C-400), 81.5 (1C, C-300), 100.7 (1C,
C-100). MALDI-MS (positive mode, Matrix p-nitro-
aniline+NaI, THF): [M+Na]+, m/z = 3136.3; found:
m/z = 3135.2. C166H214NO44P6 (3113.3). Anal. Calcd:
C: 64.04, H: 6.93, N: 0.45. Found: C: 63.97, H: 7.05,
N: 0.41.
4.5.13. Compound 31. Compound 30 (250 mg,
0.095 mmol), PyBOP (992 mg, 20 equiv) and Z-D-ala-
nine triethylammonium salt (619 mg, 20 equiv) were
dried separately in high vacuum for 3 h. The mixture
was dissolved in dry CH2Cl2 (20 mL), N-methylimidaz-
ole (305 lL, 40 equiv) was added dropwise and the reac-
tion mixture was stirred for 2.5–3 h at room temp under
argon atmosphere. The reaction mixture was diluted
with CH2Cl2 and washed with saturated NH4Cl solu-
tion. The organic phase was dried over MgSO4, and
the solvent was removed in vacuo. Flash chromatogra-
phy (toluene/acetone 3:1) and second column (toluene/
acetone 1:1.5) gave 31 (230 mg, 70%) as colourless foam,
which was stored at À20 °C. TLC (toluene/acetone
1:1.5): Rf = 0.62, 0.69. [a]D = +14.95 (c 0.58, acetone).
1H NMR (600 MHz, CDCl3): d = 0.79–0.92 (t, 6H,
Me), 1.07–1.32 (m, 52H, CH2 chain, Ala–Me), 1.45–
1.59 (m, 4H, COCH2CH2R), 1.86–2.01 (m, 3H, NHAc),
2.15–2.29 (m, 4H, COCH2CH2R), 3.49 (m, 2H, 18-H),
3.64 (m, 2H, 600-H), 3.67 (m, 1H, 400-H), 3.69 (m, 3H,
300-H, 17-H, 2-H), 3.80 (m, 1H, 500-H), 4.04 (m, 1H, 30-
H), 4.05 (m, 22H, 10-H, 1-H, 3-H, 4-H, 6-H, 7-H, 9-H,
10-H, 12-H, 13-H, 15-H), 4.06, 4.16 (m, 2H, 16-H),
4.23 (m, 1H, 30-H), 4.30 (m, 1H, 200-H), 4.31 (m, 4H,
CHNHZ), 4.32–4.74 (m, 10H, CH2Ph), 4.87/4.89 (m,
1H, 100-H), 4.96 (m, 4H, CH2-Z), 5.01 (m, 12H,
POCH2Ph), 5.06 (m, 4H, CH2-Z), 5.12 (m, 4H, 5-H, 8-
H, 11-H, 14-H), 5.14 (m, 1H, 20-H), 5.45–6.02 (NH),
7.00–7.46 (m, 75H, Ph). 13C NMR (150.9 MHz, CDCl3):
d = 14.6 (2C, Me), 18.3 (4C, Ala–Me), 23.2 (1C, NHAc),
23.1/29.9–32.3 (20C, CH2 chain), 25.1 (2C,
COCH2CH2R), 34.4 (2C, COCH2R), 49.9 (4C, CH–
4.5.12. Hexaphosphate 30. Compound 29 (490 mg,
0.157 mmol) was dissolved in acetonitrile/toluene/water
(60:3:4, 15 mL), the solution was cooled to 0 °C and
Ce(NH4)2(NO3)6 (1.15 g, 20 equiv) was added portion-
wise. After 10 min the ice bath was removed and the
reaction mixture was stirred for another 50–70 min
(monitoring by TLC). The reaction mixture was diluted
with EtOAc and washed with saturated NaHCO3 solu-
tion. The organic phase was dried over MgSO4 and

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
503
NHZ), 53.0 (1C, C-200), 62.0 (1C, C-30), 65.7 (11C, C-10,
C-1, C-3, C-4, C-6, C-7, C-9, C-10, C-12, C-13, C-15),
67.2 (4C, CH2Z), 67.9 (1C, C-16), 68.5 (1C, C-600), 69.1
(1C, C-18), 69.5 (1C, C-20), 70.1 (6C, POCH2Ph), 70.8
(4C, C-5, C-8, C-11, C-14), 71.9 (1C, C-500), 72.2–76.0
(5C, CH2Ph), 76.8 (2C, C-2, C-17), 78.0 (1C, C-400),
81.1 (1C, C-300), 100.2 (1C, C-100). MALDI-MS (positive
mode, Matrix p-nitroaniline+NaI, THF): [M+Na]+,
m/z = 3475.5; found: m/z = 3478.0. To produce the tri-
ethylammonium salt, the commercial Z-D-alanine was
dissolved in methanol (1.1 equiv NEt3) and coevapo-
rated with toluene. The product was dried in high
vacuum.
Matrix DHB, THF): [M+Na]+, m/z = 347.1; found
m/z = 346.7. C15H20N2O6 (324.3). Anal. Calcd: C: 55.55;
H, 6.22; N, 8.64. Found: C: 55.48; H, 6.17; N, 8.53.
4.6.2. N-Benzyloxycarbonyl-D-alanyl-(O-monomethoxy-
trityl)-D-serine methyl ester 33. To a solution of 32
(2.05 g, 6.3 mmol) in pyridine (20 mL), MMTrCl
(2.33 g, 1.2 equiv) was added and the reaction mixture
was stirred for 2 h at room temperature. The pyridine
was evaporated, the material redissolved in EtOAc
and washed with water. The organic phase was dried
over MgSO4, and the solvent was evaporated in vacuo.
Flash chromatography (petroleum ether/EtOAc 3:1 to
1:1) yielded compound 33 (3.47 g, 92%). TLC (petro-
leum ether/EtOAc 1:1) = 0.48. [a]D = +4.2 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 1.4 (d, 3H, CH3
D-Ala); 3.35 (dd, 1H, CH2 D-Ser); 3.6 (dd, 1H, CH2
D-Ser); 3.75 (s, 6H, COOCH3, CH3O); 4.3 (q, 1H, CH
D-Ala); 4.65 (m, 1H, CH D-Ser); 5.1 (dd, 2H, CH2Ph);
5.35 (d, 1H, NH D-Ala); 6.8 (d, 2H, orthoPhOMe);
7.25 (m, 18H, Ph, NH D-Ser). MALDI-MS (positive
mode, Matrix DHB, THF): [M+Na]+, m/z = 619.7;
found m/z = 619.8. C35H36N2O7 (596.7). Anal. Calcd:
C: 70.45; H, 6.08; N, 4.69. Found: C: 70.52; H, 6.17;
N, 4.56.
4.5.14. Target molecule 1. Compound 31 (105 mg,
0.03 mmol) was dissolved in CH2Cl2/MeOH/H2O
(5:5:1 5 mL) and Pearlmanꢀs catalyst was added
(10.5 mg). The reaction was stirred overnight under H2
atmosphere and after this time was filtered through
Celite with the same solvent mixture. The solution was
diluted with 0.1 M NH4OAc buffer (pH 4.8), frozen
and lyophilized. The crude white solid was purified by
ꢁHydrophobic Interaction Chromatographyꢀ, and after
lyophilization compound
1
(24 mg, 41%) was
1
obtained as white powder. H NMR (600 MHz, D2O):
d = 0.85 (br s, 6H, Me), 1.09–1.42 (m, 40H, CH2 chain),
1.47–1.67 (m, 16H, Ala–Me, COCH2CH2R), 2.03/2.07
(2s, 3H, NHAc), 2.22–2.44 (m, 4H, COCH2CH2R),
3.40–3.50 (m, 1H, 4c-H), 3.58, 3.65 (m, 2H, 18-H),
3.70–4.58 (m, 37H), 4.95–5.08 (m, 1H, 100-H), 5.20–
5.40 (m, 5H, CH-Ala, 20-H). 13C NMR (150.9 MHz,
D2O): d = 16.5 (2C, Me), 18.0 (4C, Ala–Me), 24.8 (1C,
NHAc), 27.5 (2C, COCH2CH2R), 25.3/33.6/34.7
(20C, CH2 chain), 36.7, 36.8 (2C, COCH2CH2R), 51.6
(4C, CHNHþ3 ), 56.3 (1C, C-200), 63.3 (1C, C-600), 64.7
(1C, C-18), 65.8 (1C, C-30), 66.2, 67.9 (C–CH2-Glyc.),
68.9 (1C, C-16), 73.0 (1C, C-400), 73.2 (1C, C-20), 73.4
(1C, C-17), 73.7 (1C, C-300), 74.9 (1C, C-500), 76.6
(4C, CH-Ala), 78.5 (1C, C–CHGlcNAc), 99.7 (1C, C-100).
MALDI-MS (negative mode, Matrix THAP,
CH3CN/H2O 3:2): [MÀH]À, m/z = 1923.7; found: m/z =
1921.5; [(MÀAla)ÀH]À, m/z = 1852.6; found: m/z =
1850.3.
4.6.3.
(2S)-2-N-(N-Benzyloxycarbonyl-D-alanyl)-2-
amino-1-hydroxy-3-(monomethoxytrityloxy)propane 34.
A solution of 33 (3.43 g, 5.75 mmol) in dry EtOH
(130 mL) was cooled to 0 °C and NaBH4 (870 mg,
4 equiv) was added. The reaction mixture was stirred
for 30 min at 0 °C and overnight at room temp. The sol-
vent was evaporated and the residue was redissolved in
EtOAc, washed with water and brine. The organic phase
was dried over MgSO4 and the solvent evaporated in
vacuo. Flash chromatography (petroleum ether/EtOAc
1:1 to 1:3) yielded 34 (2.55 g, 78%) as colourless syrup.
TLC (petroleum ether/EtOAc 1:1) = 0.32. [a]D = +7.8
1
(c 1, CHCl3). H NMR (250 MHz, CDCl3): d = 1.4 (d,
3H, CH3 D-Ala); 3.35 (m, 2H, CH2OH); 3.7 (m, 2H,
CH2OMMTr); 3.75 (s, 3H, CH3O); 4,05 (m, 1H, CH
D-Ala); 4.2 (m, 1H, CH D-Ser); 5.0 (dd, 2H, CH2Ph);
5.45 (d, 1H, NH); 6.7 (d, NH D-Ser); 6.8 (d, 2H, ortho-
PhOMe); 7.25 (m, 17H, Ph). MALDI-MS (positive
mode, Matrix DHB, THF): [M+Na]+, m/z = 591.3;
found m/z = 591.4. C34H54N2O6 (568.7). Anal. Calcd:
C: 71.81; H: 6.38; N: 4.93. Found: C: 71.69; H: 6.14;
N: 4.86.
4.6. Preparation of 7
4.6.1. N-Benzyloxycarbonyl-D-alanyl-D-serine methyl
ester 32. To a solution of D-serine-methyl ester (2 g,
12.8 mmol) and N-benzyloxycarbonyl-D-alanine (3.44 g,
1.2 equiv) in dry DMF, HOBt (2.08 g, 1.2 equiv),
DIPEA (13.1 mL, 6 equiv) and DCC (3.18 g, 1.2 equiv)
were added. After 3 h the reaction mixture was concen-
trated to half its volume and diluted with CH2Cl2,
filtered, washed with water and brine. The organic layer
was dried over MgSO4 and evaporated. Purification by
flash chromatography (toluene/acetone 1:1) yielded 32
(4.15 g, 77%) as white solid. TLC (toluene/acetone
1:1) = 0.29. [a]D = +10.3 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 1.4 (d, 3H, CH3 D-Ala); 3.75
(s, 3H, COOCH3); 3.95 (m, 1H, CH2OH); 4.3 (q, 1H,
CH D-Ala); 4.65 (m, 1H, CH D-Ser); 5.1 (dd, 2H,
CH2Ph); 5.7 (d, 1H, NH D-Ala); 7.25 (d, 1H, NH D-
Ser); 7.35 (m, 5H, Ph). MALDI-MS (positive mode,
4.6.4.
amino-1-tert-butyldimethylsilyloxy-3-(monomethoxytri-
tyloxy)propane 35. To solution of 34 (2.5 g,
(2R)-2-N-(N-Benzyloxycarbonyl-D-alanyl)-2-
a
4.4 mmol) and imidazole (0.43 g, 1.5 equiv) in dry
CH2Cl2 (30 mL) TBDPS-Cl (1.14 mL, 1.05 equiv) was
added. The reaction mixture was stirred for 30 min, then
diluted with CH2Cl2 and washed with water. The organ-
ic phase was dried over MgSO4 and the solvent evapo-
rated in vacuo. Flash chromatography (petroleum
ether/EtOAc 3:1) yielded compound 35 (3.01 g, 85%)
as colourless syrup. TLC (petroleum ether/EtOAc 2:1)
1
= 0.47. [a]D = +0.8 (c 1, CHCl3). H NMR (250 MHz,
CDCl3): d = 0.95 (s, 9H, CCH3); 1.3 (d, 3H, CH3
D-Ala); 3.2 (m, 1H, CH2OTBDPS) 3.45 (m, 1H,

504
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
CH2OTBDPS); 3.75 (m, 4H, CH2OMMTr, OCH3); 3.9
(m, 1H, CH2OMMTr); 4,15 (m, 2H, CH D-Ala, CH);
5.0 (dd, 2H, CH2Ph); 5.2 (d, 1H, NH D-Ala); 6.2 (d,
NH amide); 6.8 (d, 2H, orthoPhOMe); 7.25 (m, 23H,
Ph); 7.6 (d, 4H, orthoPhOSi). MALDI-MS (positive
mode, Matrix DHB, THF): [M+Na]+, m/z = 829.4;
found m/z = 829.4. C50H54N2O6Si (807.1). Anal. Calcd:
C: 74.41; H: 6.74; N: 3.47. Found: C: 74.68; H: 6.64;
N: 4.56.
colourless syrup. TLC (toluene/acetone 3:1) = 0.37.
[a]D = À5.7 (c 1, CHCl3). H NMR (250 MHz, CDCl3):
1
d = 1.05 (s, 9H, CCH3); 1.35 (d, 3H, CH3 D-Ala); 3.5 (d,
2H, 1-H); 3.6–3.8 (m, 3-H, 6-H, 5-H); 4.07–4.30 (m, 5H,
3-H, 4-H, CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (m, 2H,
CH2Ph); 5.0 (m, 4H, POCH2Ph, CH2Z); 5.6 (d, 1H, NH
D-Ala); 6.6 (d, NH amide); 7.3 (m, 26H, Ph); 7.65 (d,
4H, orthoPhOSi). 13C NMR (63 MHz, CDCl3):
d = 18.68 (1C, C (CH3)3), 26.29 (3C, C(CH3)3), 48.96
(1C, CNHR), 50.58 (1C, CNHR), 62.18 (1C, C-1),
66.42, 66.51, 66.55 (2C, C-3, C-4), 68.72 (2C, POCH2–
Ph, C-6), 71.34, 71.73, 72.91 (3C, CH2–Ph), 76.1 (1C,
C-5), 127.09–137.66 (30C, Ph), 173.09 (1C, CO),
178.66 (1C, CO). MALDI-MS (positive mode, Matrix
DHB, THF): [M+Na]+, m/z = 958.4; found m/z =
981.6. C54H63N2O10PSi (959.1). Anal. Calcd: C:
67.62; H: 6.62; N: 2.92. Found: C: 67.48; H: 6.44; N:
2.58.
4.6.5.
(2R)-2-N-[(N-Benzyloxycarbonyl-D-alanyl)-2-
amino]-1-tert-butyldimethylsiyloxy-3-hydroxypropane 36.
To a solution of 35 (2.95 g, 3.66 mmol) in CH2Cl2/
MeOH (30 mL), camphor-10-sulfonic acid (150 mg) was
added. The reaction mixture was stirred for 20 min,
then quenched with saturated solution of NaHCO3
(15 mL). The organic phase was washed with water
and dried over MgSO4; the solvent was evaporated in
vacuo. Flash chromatography (petroleum ether/EtOAc
2:1!1:1) yielded 36 (1.7 g, 87%) as colourless syrup.
TLC (petroleum ether/EtOAc 1:1) = 0.53. [a]D = +9.3
(c 1, CHCl3). 1H NMR (250 MHz, CDCl3): d = 1.05
(s, 9H, CCH3); 1.35 (d, 3H, CH3 D-Ala); 3.7 (m, 4H,
CH2OTBDPS, CH2OH); 4.0 (m, 2H, CH D-Ala, CH);
5.0 (dd, 2H, CH2Ph); 5.2 (d, 1H, NH D-Ala); 6.7 (d,
NH amide); 7.4 (m, 11H, Ph); 7.65 (d, 4H, orthoPhOSi).
MALDI-MS (positive mode, Matrix DHB, THF):
[M+Na]+, m/z = 557.2; found m/z = 557.0. C30H38N2O5-
Si (534.7). Anal. Calcd: C: 67.39; H: 7.16; N: 5.24.
Found: C: 67.38; H: 7.24; N: 5.32.
4.7.2. Compound 38. Procedure B. Phosphate 37
(240 mg, 0.25 mmol). Purification by flash silica gel
(petroleum ether/EtOAc 1:1) yielded compound 38
(130 mg, 82%) as colourless syrup. TLC (toluene/ace-
tone 6:1) = 0.15. [a]D = +8.8 (c 1, CHCl3). 1H NMR
(250 MHz, CDCl3): d = 1.35 (d, 3H, CH3 D-Ala); 1.8–
2.0 (br s, 1H, OH); 3.5 (d, 2H, 1-H); 3.6–3.8 (m, 3H,
6-H, 5-H); 3.8–4.30 (m, 5H, 3-H, 4-H, CH D-Ala);
4.45 (m, 2H, CH2Ph); 4.6 (s, 2H, CH2Ph); 5.0 (m, 4H,
POCH2Ph, CH2Z); 5.7 (d, 1H, NH D-Ala); 7.0 (d, NH
amide); 7.3 (m, 20H, Ph). 13C NMR (63 MHz,
CDCl3): d = 48.96 (1C, CNHR), 50.58 (1C, CNHR),
61.0 (1C, C-1), 65.5, 65.7 (2C, C-3, C-4), 68.7 (1C,
POCH2–Ph), 69.3 (1C, C-6), 71.3–72.9 (3C, CH2–Ph),
76.7 (1C, C-5), 127.09–137.66 (34C, Ph), 173.12 (1C,
CO), 178.87 (1C, CO). MALDI-MS (positive mode,
Matrix DHB, THF): [M+Na]+, m/z = 743.3; found
m/z = 743.4. C38H45N2O10P (720.8). Anal. Calcd: C:
63.32; H: 6.29; N: 3.89. Found: C: 63.44; H: 6.41; N:
3.57.
4.6.6. Benzyloxy-[(2R)-2-N-(N-benzyloxycarbonyl-D-ala-
nyl)-2-amino-1-tert-butyldimethylsilyloxy-propan-3-yloxy]-
diisopropylaminophosphane 7. Compound 36 (1.65 g,
3.09 mmol) and tetrazole (130 mg, 0.6 equiv) were dried
separately in high vacuum for 1 h. To the mixture of the
two dried compounds a solution of benzyloxy(diisopro-
pylamino)phosphane (1.2 g, 1.2 equiv) in dry CH2Cl2
(10 mL) was added and the reaction mixture was stirred
for 45 min at room temp. The reaction was diluted with
CH2Cl2 and washed with saturated NaHCO3 solution.
The organic phase was dried over sodium sulfate and
the solvent was removed in vacuo. Fast flash chroma-
tography (petroleum ether/EtOAc 3:1 1% Et3N) yielded
7 (2.26 g, 95%) as a colourless syrup. TLC (petroleum
ether/EtOAc 1:1 1% Et3N) = 0.82. TLC (petroleum
ether/EtOAc 3:1 1% Et3N) = 0.38. [a]D = +0.9 (c 1,
4.7.3. Diphosphate 39. Procedure A. Compound 38
(130 mg, 0.18 mmol). TLC before oxidation (toluene/
acetone 2:1, 1% Et3N, Rf = 0.73). Purification by flash
chromatography (toluene/acetone 7:2) yielded com-
pound 39 (240 mg, 95%) as colourless syrup. TLC (tolu-
ene/acetone 3:1) = 0.36, 0.28. [a]D = À7.8 (c 1, CHCl3).
1H NMR (250 MHz, CDCl3): d = 1.03 (s, 9H, CCH3);
1.35 (m, 6H, CH3 D-Ala); 3.45 (d, 2H, 1-H); 3.6–3.8
(m, 3H, 9-H, 8-H); 3.8–4.40 (m, 10H, 3-H, 4-H, 6-H,
7-H, CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (m, 2H,
CH2Ph); 5.0 (m, 8H, POCH2Ph, CH2Z); 5.75 (d, 2H,
NH D-Ala); 6.35 (d, NH amide); 7.3 (m, 36H, Ph);
7.65 (d, 4H, orthoPhOSi). 13C NMR (63 MHz, CDCl3):
d = 18.72 (1C, C(CH3)3), 26.3 (3C, C(CH3)3), 48.9 (2C,
CNHR), 50.62 (2C, CNHR), 62.18 (1C, C-1), 66.45,
66.50, 66.55 (4C, C-3, C-4, C-6, C-7), 68.69–69.7 (3C,
POCH2–Ph, C-9), 71.30–72.57 (4C, CH2–Ph), 76.15
(1C, C-8), 127.09–137.66 (40C, Ph), 173.09 (1C, CO),
177.68 (1C, CO). MALDI-MS (positive mode, Matrix
DHB, THF): [M+Na]+, m/z = 1431.5; found: m/z =
1431.6. C75H88N4O17P2Si (1407.6). Anal. Calcd: C:
64.00; H: 6.30; N: 3.98. Found: C: 63.67; H: 6.42; N:
4.16.
1
CHCl3). H NMR (250 MHz, CDCl3): d = 1.05 (s, 9H,
CCH3); 1.10–1.25 (m, 12H, CH(CH3)2); 1.35 (d, 3H,
CH3 D-Ala); 3.55 (m, 4H, H-1, H-2); 4,05 (m, 2H, CH
D-Ala, CH); 5.1 (m, 5H, CH2Ph, NH D-Ala, PhCH2OP);
6.7 (d, NH amide); 7.4 (m, 16H, Ph); 7.65 (d, 4H,
orthoPhOSi). C43H58N3O6PSi (772.0). Anal. Calcd: C:
66.90; H: 7.57; N: 5.44. Found: C: 66.67; H: 7.35; N:
5.32.
4.7. Preparation of target molecule 2
4.7.1. Compound 37. Procedure A. Mixture of 3
(78 mg, 1.1 equiv) and 7 (240 mg, 0.26 mmol). TLC
before oxidation (toluene/acetone 3:1, 1% Et3N,
Rf = 0.74). Flash chromatography (petroleum ether/
EtOAc 2:1) yielded phosphate 36 (240 mg, 97%) as

I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
505
4.7.4. Diphosphate 40. Procedure B. Compound 39
(230 mg, 0.16 mmol). Purification by flash silica gel (tol-
uene/acetone 3:1) yielded 40 (156 mg, 78%) as colourless
syrup. TLC (toluene/acetone 2:1) = 0.36 and 0.40.
12-H, 13-H, CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (s,
2H, CH2Ph); 5.0 (m, 16H, POCH2Ph, CH2Z); 5.75–6.4
(m, 4H, NH); 7.3 (m, 56H, Ph); 7.65 (d, 4H, ortho-
PhOSi). MALDI-MS (positive mode, Matrix DHB,
THF): [M+Na]+, m/z = 2325.8; found: m/z = 2325.7.
C117H138N8O31P4Si (2304.4). Anal. Calcd: C: 60.98; H:
6.04; N: 4.86. Found: C: 60.53; H: 6.37; N: 4.52.
1
[a]D = +3.4 (c 1, CHCl3). H NMR (250 MHz, CDCl3):
d = 1.35 (d, 6H, CH3 D-Ala); 1.9–2.2 (br s, 1H, OH);
3.5–3.8 (m, 5H, 1-H, 9-H, 8-H); 3.8–4.40 (m, 10H, 3-
H, 4-H, 6-H, 7-H, CH D-Ala); 4.45 (m, 2H, CH2Ph);
4.6 (m, 2H, CH2Ph); 5.0 (m, 8H, POCH2Ph, CH2Z);
5.7 (br s, 2H, NH D-Ala); 7.0 (d, NH amide); 7.3 (m,
30H, Ph). 13C NMR (63 MHz, CDCl3): d = 48.78 (2C,
CNHR), 50.58 (2C, CNHR), 61.0 (1C, C-1), 65.51–
66.93 (6C, C-3, C-4, C-6, C-7, CH2Ph(Z)), 68.7 (2C,
POCH2–Ph), 69.3 (1C, C-9); 71.3, 72.9 (2C, CH2–Ph),
77.75 (1C, C-8), 127.09–137.66 (36C, Ph). MALDI-MS
(positive mode, Matrix DHB, THF): [M+Na]+,
m/z = 1191.4; found m/z = 1191.2. C59H70N4O17P2
(1169.2). Anal. Calcd: C: 60.61; H: 6.03; N: 4.79. Found:
C: 60.49; H: 6.24; N: 4.56.
4.7.8. Tetraphosphate 44. Procedure B. Compound 43
(136 mg, 0.065 mmol). Purification by flash silica gel (tol-
uene/acetone 1:3) yielded 44 (86 mg, 72%) as colourless
syrup. TLC (toluene/acetone 1:1) = 0.15. [a]D = +1.2 (c
1
1, CHCl3). H NMR (250 MHz, CDCl3): d = 1.25 (m,
12H, CH3 D-Ala); 2.35 (br s, 1H, OH); 3.5–3.8 (m,
11H, 1-H, 2-H, 5-H, 8-H, 11-H, 14-H, 15-H); 3.8–4.40
(m, 20H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H,
CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (s, 2H, CH2Ph);
5.0 (m, 16H, POCH2Ph, CH2Z); 6.05 (br s, 4H, NH);
7.3 (m, 50H, Ph). MALDI-MS (positive mode, Matrix
DHB, THF): [M+Na]+, m/z = 2087.7; found: m/z =
2087.7. C101H120N8O31P4Si (2066.0). Anal. Calcd: C:
58.72; H: 5.85; N: 5.42. Found: C: 58.59; H: 5.71; N: 5.64.
4.7.5. Triphosphate 41. Procedure A. Compound 40
(156 mg, 0.133 mmol), TLC before oxidation (toluene/
acetone 2:1, 1% Et3N, Rf = 0.75). Purification by flash
chromatography (toluene/acetone 3:1 to 2:1) yielded
compound 41 (184 mg, 77%) as colourless syrup. TLC
(toluene/acetone 2:1) = 0.42, 0.39. [a]D = À6.5 (c 1,
4.7.9. Pentaphosphate 45. Procedure A. Compound 44
(65 mg, 0.031 mmol). TLC before oxidation (toluene/
acetone 2:1, 1% Et3N, Rf = 0.61). Purification by flash
chromatography (toluene/acetone 2:1!1:2) yielded
compound 45 (71 mg, 81%) as colourless syrup.
[a]D = À0.4 (c 1, CHCl3). TLC (toluene/acetone
1:1) = 0.36 and 0.39. 1H NMR (250 MHz, CDCl3):
d = 1.03 (s, 9H, CCH3); 1.25 (m, 15H, CH3 D-Ala);
3.5–3.8 (m, 14H, 1-H, 2-H, 5-H, 8-H, 11-H, 14-H, 17-
H, 18-H); 3.8–4.40 (m, 25H, 3-H, 4-H, 6-H, 7-H, 9-H,
10-H, 12-H, 13-H, 15-H, 16-H, CH D-Ala); 4.45 (s,
2H, CH2Ph); 4.6 (s, 2H, CH2Ph); 5.0 (m, 20H,
POCH2Ph, CH2Z); 5.75–6.4 (m, 5H, NH); 7.3 (m,
66H, Ph); 7.65 (d, 4H, orthoPhOSi). MALDI-MS (posi-
tive mode, Matrix DHB, THF): [M+Na]+, m/z = 2774.0;
found: m/z = 2773.8. C138H163N10O38P5Si (2752.8).
Anal. Calcd: C: 60.21; H: 5.97; N: 5.09. Found: C:
60.48; H: 6.14; N: 5.32.
1
CHCl3). H NMR (250 MHz, CDCl3): d = 1.03 (s, 9H,
CCH3); 1.35 (m, 9H, CH3 D-Ala); 3.5–3.8 (m, 8H,
1-H, 2-H, 5-H, 8-H, 11-H, 12-H); 3.8–4.40 (m, 10H,
3-H, 4-H, 6-H, 7-H, 9-H, 10-H, CH D-Ala); 4.45 (m,
2H, CH2Ph); 4.6 (m, 2H, CH2Ph); 5.0 (m, 12H,
POCH2Ph, CH2Z); 5.75, 6.95 (m, 3H, NH D-Ala); 6.35
(m, NH amide); 7.3 (m, 46H, Ph); 7.65 (d, 4H, ortho-
PhOSi). MALDI-MS (positive mode, Matrix DHB,
THF): [M+Na]+, m/z = 1854.7; found: m/z = 1877.6.
C96H113N6O24P3Si (1856.0). Anal. Calcd: C: 62.13; H:
6.14; N: 4.53. Found: C: 62.05; H: 6.24; N: 4.59.
4.7.6. Triphosphate 42. Procedure B. Compound 41
(184 mg, 0.1 mmol). Purification by flash silica gel (tolu-
ene/acetone 1:1) yielded 42 (115 mg, 72%) as colourless
syrup. TLC (toluene/acetone 1:1) = 0.29. [a]D = +2.8 (c
1
1, CHCl3). H NMR (250 MHz, CDCl3): d = 1.15–1.4
4.7.10. Pentaphosphate 46. Procedure B. Compound
45 (56 mg, 0.02 mmol). Purification by flash silica gel
(toluene/acetone 1:3) yielded 46 (40 mg, 78%) as colour-
less syrup. [a]D = À3.1 (c 1, CHCl3). TLC (toluene/ace-
tone 1:1) = 0.13. 1H NMR (250 MHz, CDCl3):
d = 1.25 (m, 15H, CH3 D-Ala); 3.5–3.8 (m, 14H, 1-H,
2-H, 5-H, 8-H, 11-H, 14-H, 17-H, 18-H); 3.8–4.40 (m,
25H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H, 12-H, 13-H, 15-
H, 16-H, CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (s, 2H,
CH2Ph); 5.0 (m, 20H, POCH2Ph, CH2Z); 5.75–6.4 (m,
5H, NH); 7.3 (m, 66H, Ph). C122H145N10O38P5
(2514.4). Anal. Calcd: C: 58.28; H: 5.81; N: 5.57. Found:
C: 57.99; H: 5.84; N: 5.62.
(m, 9H, CH3 D-Ala); 3.45–3.80 (m, 8H, 1-H, 2-H, 5-H,
8-H, 11-H, 12-H); 3.85–4.40 (m, 15H, 3-H, 4-H, 6-H,
7-H, 9-H, 10-H, CH D-Ala); 4.45 (m, 2H, CH2Ph); 4.6
(m, 2H, CH2Ph); 5.0 (m, 8H, POCH2Ph, CH2Z); 6.05
(br s, 3H, NH D-Ala); 7.3 (m, 40H, Ph). MALDI-MS
(positive mode, Matrix DHB, THF): [M+H]+,
m/z = 1616.6; found: m/z = 1616.6. C80H95N6O24P3
(1617.6). Anal. Calcd: C: 59.40; H: 5.92; N: 5.20. Found:
C: 59.48; H: 5.64; N: 5.12.
4.7.7. Tetraphosphate 43. Procedure A. Compound 42
(115 mg, 0.071 mmol). TLC before oxidation (toluene/
acetone 2:1, 1% Et3N, Rf = 0.71). Purification by flash
chromatography (toluene/acetone 2:1!1:2) yielded
compound 43 (136 mg, 91%) as colourless syrup.
[a]D = À5.2 (c 1, CHCl3). TLC (toluene/acetone
1:1) = 0.36 and 0.39. 1H NMR (250 MHz, CDCl3):
d = 1.03 (s, 9H, CCH3); 1.25 (m, 12H, CH3 D-Ala);
3.5–3.8 (m, 11H, 1-H, 2-H, 5-H, 8-H, 11-H, 14-H, 15-
H); 3.8–4.40 (m, 20H, 3-H, 4-H, 6-H, 7-H, 9-H, 10-H,
4.7.11. Hexaphosphate 47. Procedure A. Compound
46 (40 mg, 0.016 mmol). TLC before oxidation (tolu-
ene/acetone 2:1, 1% Et3N, Rf = 0.61). Purification by
flash chromatography (toluene/acetone 1:1 to 1:2)
yielded compound 47 (36 mg, 82%) as colourless syrup.
[a]D = +13.3 (c 1, CHCl3). TLC (toluene/acetone
1:1) = 0.38 and 0.40. 1H NMR (250 MHz, CDCl3):

506
I. Figueroa-Perez et al. / Tetrahedron: Asymmetry 16 (2005) 493–506
d = 0.90 (t, 6H, Me); 1.25 (m, 55H, CH3 D-Ala, CH2
chain); 1.55 (m, 4H, COCH2CH2R); 2.25 (t, 4H,
COCH2R); 3.5 (m, 2H, H-21); 3.7 (m, 1H, H-20); 3.8–
4.15 (22H, 4-H, 5-H, 6-H, 7-H, 8-H, 9-H, 10-H, 11-H,
12-H, 13-H, 14-H, 15-H, 16-H, 17-H, 18-H, 19-H);
4.2–4.40 (m, 14H, 1-H, 2-H, 3-H, 4-H, 6-H, 16-H, 18-
H, CH D-Ala); 4.45 (s, 2H, CH2Ph); 4.6 (s, 2H, CH2Ph);
5.0 (m, 22H, POCH2Ph, CH2Z); 5.75–6.4 (m, 5H, NH);
7.3 (m, 66H, Ph); 7.65 (d, 4H, orthoPhOSi). MALDI-
MS (positive mode, Matrix DHB, THF): [M+Na]+,
m/z = 3200.3; found: m/z = 3201.1. C160H210N10O45P6
(3179.3). Anal. Calcd: C: 60.45; H: 6.66; N: 4.41. Found:
C: 60.41; H: 6.74; N: 4.62.
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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
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the help in the structural assignments of the products by
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