Synthesis of structures corresponding to the capsular polysaccharide of Neisseria meningitidis group A

Article abstract of DOI:10.1039/b507898a

Four differently substituted trimers of the CPS repeating unit have been synthesised in order to investigate the dependence on oligosaccharide size, acetylation and mode of phosphorylation of glycoconjugate vaccines against Neisseria meningitidis group A.

Full text of DOI:10.1039/b507898a

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A R T I C L E
Synthesis of structures corresponding to the capsular polysaccharide
of Neisseria meningitidis group A
Rikard Sla¨ttega˚rd,a Peter Teodorovic,a Henok Hadgu Kinfe,b Neil Ravenscroft,b
David W. Gammonb and Stefan Oscarson*a
a Department of Organic Chemistry, Arrhenius Laboratory, Stockholm University, S-106 91,
Stockholm, Sweden. E-mail: s.oscarson@organ.su.se; Fax: +46 8154 908; Tel: +46 8162 480
b Department of Chemistry, University of Cape Town, Rondebosch, 7701, South Africa.
E-mail: raveneil@science.uct.ac.za; Fax: +27 2168 974 99; Tel: +27 2165 043 54
Received 6th June 2005, Accepted 23rd August 2005
First published as an Advance Article on the web 13th September 2005
Four differently substituted trimers of the CPS repeating unit have been synthesised in order to investigate the
dependence on oligosaccharide size, acetylation and mode of phosphorylation of glycoconjugate vaccines against
Neisseria meningitidis group A. A spacer-containing starting monomer, a H-phosphonate elongating monomer and a
6-O-phosphorylated H-phosphonate cap monomer have been synthesised and coupled together to afford, after
deprotection, the target trimer structures differing in their acetylation and phosphorylation substitution
pattern.
Introduction
Results and discussion
In order to circumvent the problems associated with the acid
lability of the native anomeric phosphodiester linkage during
the synthesis, an approach based on 2-azido-2-deoxy-glucose
derivatives has been developed and used.10,11 The properties
of the azido group (electron-withdrawing, non-participating)
strongly stabilize the anomeric linkage as further proven by Pozs-
gay and coworkers in their synthesis of trimer 14.12 A common
precursor for both the starting monomer and the elongating
monomer was synthesized by transformation of the known
tetraacetate 1 into the corresponding ethyl thioglycoside (→2,
68%),13,14 followed by deacetylation, regioselective silylation and
acetylation to give 3 (99% over three steps) (Scheme 1).
Meningitis is caused both by viruses and bacteria. The two
major types of bacteria causing meningitis are Haemophilus
influenzae and Neisseria meningitidis. In the case of H. influenzae
only one serotype, type b, is important, whereas with N.
meningitidis, five groups, i.e., A, B, C, W and Y-135, are
associated with meningococcal meningitis. The various groups
have different geographical prevalence, e.g., group B (MenB)
and C (MenC) in Europe and North America and group
A (MenA) in Africa and South America.1,2 The serotyping
is based on the structure and antigenicity of the capsular
polysaccharide (CPS) surrounding the bacteria, and the purified
polysaccharide itself can be used as a vaccine. Especially
efficient vaccines (glycoconjugate vaccines) can be made by
attaching the saccharide to a carrier protein, which induces a
T-cell dependent immune response with memory and is also
effective in small children.3 Such vaccines against H. influenzae
type b (Hib) have been commercially available for more than
ten years and have more or less eradicated disease caused by this
bacterium in countries that have implemented mass vaccination.
Recently a Hib glycoconjugate vaccine based on synthetic
oligosaccharides was licensed.4 For MenC, three monovalent
glycoconjugate vaccines are commercially available,5 and devel-
opment of vaccines against the other groups are under way,6
MenB being a severe problem since its CPS structure is identical
to a human carbohydrate structure. Another issue is the poor
stability of MenA vaccines due to the inherent instability of
the anomeric phosphate diester linkages of the CPS.7 The
repeating unit of MenA is a monosaccharide, 2-acetamido-2-
deoxy-a-D-mannopyranose linked 1 → 6 via a phosphodiester
bridge.8 In the native polysaccharide the 3-OH is acetylated to
an extent of about 90%.9 The immunological importance of this
acetylation has not been completely investigated, but there are
indications that it is not of major significance. To investigate
the various characteristics of an optimal glycoconjugate vaccine
and to develop routes to more stable analogues of the native
structure we now report on the synthesis of a number of
trimers, 13, 14, 21 and 23, of the repeating unit of the MenA
CPS. These will be conjugated to carrier proteins and used
in immunological experiments to evaluate the importance of
oligosaccharide chain length, acetates, and terminal phosphates
on the immune response.
Scheme 1 Synthesis of monosaccharide precursor. (i) EtSH, BF3OEt,
CH2Cl2; (ii) NaOMe, MeOH; (iii) TBDMSCl, pyridine; (iv) Ac2O.
NIS-promoted coupling (NIS = N-iodosuccinimide) of donor
3 with a spacer, Z-protected ethanolamine (Z = benzyloxycar-
bonyl), gave the a-linked glycoside 4 in 86% yield (Scheme 2).
Interestingly, if the corresponding donor with 3,4-di-O-benzyl
protection is used in the coupling instead, a high amount
of b-linked glycoside is also formed.12 Removal of the silyl
protecting group with TREAT–HF (triethylamine tris(hydrogen
fluoride))15 then afforded the starting acceptor 5 (97%) without
any indication of acetyl migration. Compound 3 was also
processed to give the elongating monomer by hydrolysis of
the thioglycoside (→6, 82%) and then phosphitylation using
PCl3–imidazole–Et3N16 to give the a-anomeric H-phosphonate
7 (97%). Earlier, with 3-O-acetyl-4-O-benzyl-protected deriva-
tives, the exclusive formation of the a-H-phosphonate was an
issue,10 problems also encountered in the first synthesis of MenA
structures.17 However, here with 3,4-di-O-acetyl-protection, this
was not a problem. When the hydrolysis reaction was performed
at −20 ◦C exclusively, the a-anomer was formed. Formation of
the first phosphate diester bridge then proceeded smoothly by
activation of the H-phosphonate 7 with pivaloyl chloride in the
3 7 8 2
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

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Scheme 2 Synthesis of a repeating unit dimer. (i) HO(CH2)2NHZ,
NIS–AgOTf, CH2Cl2; (ii) TREAT–HF, THF; (iii) NIS–AgOTf, wet
CH2Cl2; (iv) imidazole, PCl3, Et3N, MeCN; (v) PivCl, pyridine; (vi)
I2, pyridine–H2O.
presence of acceptor 5 followed by oxidation using iodine in
pyridine–water to yield the dimer 8 in 96% yield (Scheme 2).16
This procedure could then be iterated to give oligomers.
Even with these stabilized anomeric phosphodiester linkages,
there is still a large difference in lability as compared to
non-anomeric phosphates. Thus, the yields decrease as the
number of phosphates present in the molecules to be coupled
increases,11,12,18 and cannot compete with the ones obtained in
nucleotide chemistry. Even so, an acceptable yield was obtained
in the next coupling. Removal of the silyl group from compound
8 gave derivative 9 (91%). Compound 9 can be processed to
yield an analogue of the dimer present in the native MenA CPS,
but is also a new acceptor and was elongated once more with
H-phosphonate 7 to afford the trimer 10 (62%, Scheme 3). The
stabilizing azido functions were now transformed into the native
acetamido group through reduction followed by acetylation
(→11, 89%). Catalytical hydrogenolysis of the Z-group (→12,
83%) and subsequent removal of the silyl group (TREAT–HF)
then gave the first target structure 13 (85%). Treatment of 13 with
sodium methoxide afforded the deacetylated target structure
14 (94%), having NMR-data identical to those published by
Pozsgay and co-workers.12
In the first attempt at synthesis of the target structures
with terminal phosphates at the non-reducing end, derivative
10 was desilylated and tried as an acceptor. However, the
yield after phosphorylation using dibenzyl N,N-diisopropyl
phosphoramidite19 was quite low and an alternative pathway
involving an already 6-O-phosphorylated “cap” H-phosphonate
donor was investigated. Precursor 3 was thus desilylated (→15)
and the obtained 6-OH derivative phosphorylated to yield
compound 16 (Scheme 4), which was then transformed as
discussed for derivative 3 into the H-phosphonate 18 (37%
overall yield from 3). Once more only the a-anomer was
obtained.
Scheme 3 Synthesis of target trimers 13 and 14. (i) PivCl, pyridine; (ii)
I2, pyridine–H2O; (iii) NaBH4, NiCl2, MeOH; (iv) Ac2O; (v) H2, Pd/C,
MeOH; (vi) TREAT–HF, THF; (vii) NaOMe, MeOH.
Scheme 4 Synthesis of phosphorylated elongating monomer. (i)
TREAT–HF, THF; (ii) (BnO)2PN(CH(CH3)2)2, tetrazole, CH2Cl2; (iii)
mCPBA; (iv) NIS–AgOTf, wet CH2Cl2; (v) imidazole, PCl3, Et3N,
MeCN.
(→22, 80%) and then hydrogenolysed to give the deacetylated
target structure 23 (64%). Attempts to deacetylate compound
21 resulted in low yields of 23.
In conclusion, a straightforward synthesis of oligomers of
the MenA CPS has been developed. The pathway allows
for synthesis of both acetylated and non-acetylated structures
and also introduction of a terminal phosphate. Furthermore,
inherent in the design is the possibility of synthesising analogues
differing in the nature of the 2-amino group.
Experimental
Elongation of acceptor 9 with 18 then proceeded in good
yield (59%) to give compound 19 (Scheme 5), which could be
converted into target structures as discussed above. Reduction
of the azido groups followed by acetylation yielded derivative
20 (64%), which was either directly hydrogenolysed to give
the acetylated target structure 21 (85%), or first deacetylated
General methods
TLC was carried out on Merck precoated 60 F254 plates using
AMC (ammonium molybdate–cerium(IV) sulfate–10% sulfuric
acid; 100 g : 2 g : 2 L) or 8% H2SO4 for visualization. Column
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7
3 7 8 3

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9H), 1.30 (t, 3H), 2.05 (s, 3H), 2.10 (s, 3H), 2.55–2.75 (m, 2H),
3.60–3.75 (m, 2H), 4.10 (m, 1H), 4.15–4.20 (m, 1H), 5.20–5.25
(m, 1H), 5.30–5.35 (m, 1H); HRMS calcd for C18H33N3NaO6SSi
[M + Na]+ 470.1757, found 470.1750.
2-(Benzyloxycarbonyl)aminoethyl 3,4-di-O-acetyl-2-azido-2-
deoxy-a-D-mannopyranoside (5). Compound
3
(527 g,
1.18 mmol) was dissolved in CH2Cl2 (10 mL) containing
˚
MS (4 A). Benzyl N-(2-hydroxyethyl) carbamate (300 mg,
1.54 mmol) was added and the mixture was stirred under
argon at −20 ◦C for 30 min before NIS (345 mg, 1.53 mmol)
and AgOTf (catalytic amount) were added. After 30 min,
the reaction mixture was neutralized with Et3N, diluted with
CH2Cl2, washed with Na2S2O3 (10%), filtered (silica) and
concentrated. Chromatography (1 : 0 → 1 : 1 toluene–EtOAc)
gave 2-(benzyloxycarbonyl) aminoethyl 3,4-di-O-acetyl-2-azido-
6-O-(tertbutyldimethylsilyl)-2-deoxy-a-D-mannopyranoside (4,
595 mg, 1.02 mmol, 86%); [a]D +46◦ (c 1.0, CHCl3); NMR: 13C,
d −5.38, −5.34 (CH3Si), 18.4 ((CH3)3CSi), 20.6, 20.8 (CH3CO),
25.9 ((CH3)3CSi), 40.7 (HOCH2CH2NH), 61.6, 62.6, 66.6, 66.9,
67.6, 71.3, 71.8 (C-2–6, PhCH2O, OCH2CH2N), 98.0 (C-1),
128.2, 128.3, 128.6, 136.5 (aromatic C), 156.4 (NHCOOCH2),
1
169.6, 170.1 (CH3CO); H, d 0.03 (s, 3H), 0.04 (s, 3H), 0.89
(s, 9H), 2.03 (s, 3H), 2.09 (s, 3H), 2.35 (s, 1H), 3.32–3.40 (m,
1H), 3.44–3.52 (m, 1H), 3.58–3.66 (m, 3H), 3.72–3.80 (m, 2H),
3.98–4.00 (m, 1H), 5.11 (s, 2H), 5.18–5.23 (m, 1H), 5.32–5.36
(m, 1H), 7.16–7.36 (m, 5H). To a solution of compound 4
(575 mg, 0.99 mmol) in THF (5 mL), TREAT–HF (0.81 mL,
4.97 mmol) was added. The mixture was stirred under argon at
rt overnight. Concentration and silica gel chromatography (2 :
1 → 0 : 1 toluene–EtOAc) gave 5 (448 mg, 0.96 mmol, 97%);
[a]D +60◦ (c 1.0, CHCl3); NMR: 13C, d 20.7, 20.8 (CH3CO), 40.7
(HOCH2CH2NH), 61.4, 61.6, 66.4, 67.0, 67.5, 70.9, 71.0 (C-2–6,
PhCH2O, OCH2CH2N), 98.2 (C-1), 128.3, 128.3, 128.7, 136.5
(aromatic C), 156.5 (NHCOOBn), 170.1, 170.5 (CH3CO); 1H, d
2.04 (s, 3H), 2.1 (s, 3H), 3.32–3.46 (m, 2H), 3.54–3.60 (m, 2H),
3.62–3.78 (m, 3H), 4.02–4.04 (m, 1H), 5.06–5.18 (m, 3H), 5.22–
5.28 (m, 1H), 5.36–5.40 (m, 1H), 7.28–7.38 (m, 5H); HRMS
calcd for C20H26N4NaO9 [M + Na]+ 489.1597, found 489.1574.
Scheme 5 Synthesis of target trimers 21 and 23. (i) PivCl, pyridine; (ii)
I2, pyridine–H2O; (iii) NaBH4, NiCl2, MeOH; (iv) Ac2O; (v) H2, Pd/C,
MeOH; (vi) NaOMe, MeOH.
chromatography was performed on silica gel (0.040–0.063 mm,
Amicon) or reversed phase gel (C18 60A 40–63 lm). NMR
spectra were recorded in CDCl3 (Me4Si, d = 0.00) or D2O
(acetone 13C d = 30.89, 1H = 2.22) at 25 ◦C on a Varian 300 MHz
or 400 MHz instrument. For 31P NMR spectra, H3PO4 (d = 0.00)
was used as a reference. Organic solutions were concentrated at
30 ◦C under reduced pressure.
3,4-Di-O-acetyl-2-azido-6-O-(tertbutyldimethylsilyl)-2-deoxy-
a-D-mannopyranosyl hydrogen-phosphonate, triethylammonium
salt (7). NIS (394 mg, 1.75 mmol) and AgOTf (catalytic
amount) were added to a solution of 3 (653 mg, 1.46 mmol) in
wet CH2Cl2 (10 mL). The reaction mixture was stirred at −20 ◦C
for 30 min. CH2Cl2 was added and the mixture was washed with
Na2S2O3 (10%), filtered (silica) and concentrated. The residue
was purified by chromatography (1 : 0 → 1 : 1 toluene–EtOAc)
to give 3,4-di-O-acetyl-2-azido-6-O-(tertbutyldimethylsilyl)-2-
deoxy-a-D-mannopyranoside (6, 487 mg, 1.21 mmol, 82%);
NMR: 13C, d −5.29, −5.22 (CH3Si), 18.6 ((CH3)3CSi), 20.7,
20.9 (CH3CO), 26.0 ((CH3)3CSi), 62.1, 63.2, 66.9 71.0, 71.6
Ethyl 3,4-di-O-acetyl-2-azido-6-O-(tertbutyldimethylsilyl)-2-
deoxy-1-thio-a-D-mannopyranoside (3). To a solution of 1
(5.04 g, 13.5 mmol) in CH2Cl2 (60 mL) was added EtSH
˚
(1.6 mL, 21.6 mmol) and MS (4 A). The mixture was stirred
under argon at rt for 30 min. BF3-etherate (4.8 mL, 38.1 mmol)
dissolved in CH2Cl2 (10 mL) was then added during 1 h. After
another 7 h the mixture was diluted with CH2Cl2, washed with
saturated NaHCO3, filtered through silica and concentrated.
Silica gel chromatography (1 : 0 → 1 : 1 toluene–EtOAc) of
the residue gave ethyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-thio-
a-D-mannopyranoside14 (2, 3.46 g, 9.22 mmol, 68%); NMR: 13C,
d 14.8 (SCH2CH3), 20.6, 20.7, 20.8 (CH3CO), 25.6 (SCH2CH3),
62.3, 62.9, 66.2, 69.0, 71.4 (C-2–6), 82.5 (C-1), 169.6, 170.0,
170.7 (CH3CO). To a solution of 2 (3.71 g, 9.88 mmol) in MeOH
(30 mL), NaOMe (1 M) was added. After 1 h the mixture was
neutralized with AcOH and concentrated. The dry residue was
dissolved in pyridine (15 mL) and tertbutyldimethylsilyl chloride
(1.94 g, 12.87 mmol) was added. The reaction mixture was stirred
at rt overnight. Acetylation with acetic anhydride followed by
dilution with toluene, filtration through silica, concentration and
purification by silica gel chromatography (1 : 0 → 1 : 1 toluene–
EtOAc) gave 3 (4.357 g, 9.73 mmol, 99%); [a]D +109◦ (c 1.0,
CHCl3); NMR: 13C, d −5.33, −5.28 (CH3Si), 14.7 (SCH2CH3),
18.4 ((CH3)3CSi), 20.7, 20.9 (CH3CO), 25.0 (SCH2CH3), 25.9
((CH3)3CSi), 62.6, 62.9, 67.0, 71.7, 71.9 (C-2–6), 81.5 (C-1),
1
(C-2–6), 92.7 (C-1), 169.8, 170.3 (CH3CO); H, d 0.05 (s, 3H),
0.06 (s, 3H), 0.89 (s, 9H), 2.03 (s, 3H), 2.08 (s, 3H), 3.65–3.70
(m, 2H), 4.00–4.02 (m, 2H), 5.19–5.23 (m, 2H), 5.42–5.47
(m, 1H). A mixture of imidazole (915 mg, 13.45 mmol), PCl3
(335 lL, 3.84 mmol) and Et3N (2.0 mL, 14.35 mmol) in MeCN
(25 mL) was stirred at 0 ◦C for 30 min. A solution of compound
6 (388 mg, 0.96 mmol) in MeCN (25 mL) was added during
◦
30 min at 0 C. The reaction mixture was then stirred at rt for
10 min, quenched with TEAB (triethylammonium bicarbonate)
(0.5 M) and concentrated. The residue was diluted (CHCl3),
washed with TEAB (0.5 M), filtered (cotton) and concentrated.
Chromatography (1 : 0 → 10 : 1 CHCl3–MeOH + 1.0% Et3N)
of the residue gave 7 (499 mg, 0.93 mmol, 97%); [a]D +67◦ (c 1.0,
CHCl3); NMR: 13C, d −5.43, (CH3Si), 8.98, 18.3 ((CH3)3CSi),
20.6, 20.8 (CH3CO), 25.8 ((CH3)3CSi), 45.7, 62.4, 62.5, 66.5,
1
71.1, 72.1 (C-2–6), 93.1 (C-1), 169.4, 170.0 (CH3CO); H, d
−0.06 (s, 3H), −0.05 (s, 3H), 0.79 (s, 9H), 1.94 (s, 3H), 1.99 (s,
1
169.7, 170.1 (CH3CO); H, d 0.05 (s, 3H), 0.05 (s, 3H), 0.90 (s,
3H), 3.59–3.63 (m, 2H), 3.93–4.00 (m, 2H), 5.21–5.28 (m, 1H),
3 7 8 4
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7

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5.37–5.41 (m, 1H), 5.51–5.50 (m, 1H); 31P, d 0.28; HRMS calcd
for C16H29N3O9PSi [M + H]− 466.1416, found 466.1400.
2-(Benzyloxycarbonyl)aminoethyl (2-acetamido-3,4-di-O-
acetyl-6-O-(tertbutyldimethylsilyl)-2-deoxy-a-D-mannopyranosyl
phosphate)-(1
D-mannopyranosyl phosphate)-(1 → 6)-(2-acetamido-3,4-di-O-
acetyl-2-deoxy-a-D-mannopyranoside) bis-triethylammonium
→
6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-a-
2-(Benzyloxycarbonyl)aminoethyl (3,4-di-O-acetyl-2-azido-2-
deoxy-a-D-mannopyranosyl phosphate)-(1 → 6)-(3,4-di-O-acetyl-
2-azido-2-deoxy-a-D-mannopyranoside) triethylammonium salt
(9). A mixture of 5 (217 mg, 0.47 mmol) and 7 (325 mg,
0.60 mmol) was dissolved in pyridine (3 mL). Pivaloyl chloride
(144 lL, 1.18 mmol) was added and the mixture was stirred
unde◦r argon at rt for 1 h. The reaction mixture was cooled to
−40 C and a solution of I2 (143 mg, 0.56 mmol) in pyridine–
H2O◦(3 mL 49 : 1) was added. The oxidation was completed
at 0 C and the mixture was diluted with CHCl3, washed with
Na2S2O3 (10%) and cold TEAB (0.5 M). Filtration (cotton),
concentration and chromatography (1 : 0 → 10 : 1 CHCl3–
MeOH + 0.5% Et3N) gave 2-(benzyloxycarbonyl)aminoethyl
(3,4-di-O-acetyl-2-azido-6-O-(tertbutyldimethylsilyl)-2-deoxy-
a-D-mannopyranosyl phosphate)-(1 → 6)-(3,4-di-O-acetyl-2-
azido-2-deoxy-a-D-mannopyranoside) triethylammonium salt
(8, 469 mg, 0.45 mmol, 96%); [a]D +52◦ (c 1.0, CHCl3); NMR:
13C, d −5.48, −5.38 (CH3Si), 9.27, 18.4 ((CH3)3CSi), 20.6, 20.7,
20.8, 20.8 (CH3CO), 25.9 ((CH3)3CSi), 40.7 (HOCH2CH2NH),
45.8, 61.7, 62.2, 64.5, 66.3, 66.6, 66.7, 67.3, 69.8, 69.9, 71.2, 71.3,
71.8 (C-2–6, 2ꢀ–6ꢀ, PhCH2O, OCH2CH2N), 94.1, 97.9 (C-1, 1ꢀ),
128.1, 128.2, 128.6, 136.8 (aromatic C), 156.7 (NHCOOBn),
salt (11). Compound 10 (83 mg, 0.056 mmol) was dissolved
in MeOH (3 mL) and NiCl2(H2O)6 was added (catalytic
amount). Reduction was performed by adding NaBH4 in small
amounts over a period of 1 h at 0 ◦C. The mixture was then
subjected to acetic anhydride followed by dilution (MeOH) and
concentration. The residue was purified on silica gel (1 : 0 → 5
: 1 CHCl3–MeOH + 0.5% Et3N) and on LH-20 gel (MeOH +
1.5% Et3N), to give compound 11 (76 mg, 0.050 mmol, 89%);
[a]D +63◦ (c 1.0, CHCl3); NMR: 13C, d −5.44, −5.38 (CH3Si),
8.46, 18.4 ((CH3)3CSi), 20.9, 21.0, 21.0, 21.1, 23.0, 23.0, 23.0,
23.3, 23.9 (CH3CO, CH3CONH), 26.0 ((CH3)3CSi), 40.8
(HOCH2CH2NH), 46.1, 50.1, 50.4, 50.7 (C-2, 2ꢀ, 2ꢀꢀ), 59.1,
61.6, 64.5, 65.0, 65.7, 65.9, 66.7, 67.1, 70.0, 70.2, 70.3, 71.3,
77.4 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, PhCH2O, OCH2CH2N), 94.9, 95.1,
99.1 (C-1, 1ꢀ, 1ꢀꢀ), 128.0, 128.1, 128.6, 136.8 (aromatic C), 156.6
(NHCOOBn), 169.5, 169.6, 170.0, 170.5, 170.6, 170.8, 171.3
(CH3CO, CH3CONH); 31P, d −3.75, −3.39; HRMS calcd for
C52H79N4O30P2Si [M + H]− 1329.3956, found 1329.3984.
2-Aminoethyl (2-acetamido-3,4-di-O-acetyl-6-O-(tertbutyl-
dimethylsilyl)-2-deoxy-a-D-mannopyranosyl phosphate)-(1 →
6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-a-D-mannopyranosyl
1
169.3, 169.7, 170.0, 170.1 (CH3CO); H, d 0.00 (s, 3H), 0.02
(s, 3H), 0.88 (s, 9H), 1.98 (s, 3H), 2.00 (s, 3H), 2.06 (s, 3H),
2.08 (s, 3H), 3.30–3.38 (m, 1H), 3.42–3.52 (m, 1H), 3.56–3.64
(m, 1H), 3.66–3.68 (m, 2H), 3.74–3.80 (m, 1H), 3.90–4.04 (m,
5H), 4.12–4.14 (m, 1H), 5.1 (s, 2H), 2.16–2.22 (m, 1H), 5.29
(m, 1H), 5.32–5.47 (m, 3H), 5.51–5.54 (m, 1H), 5.63–5.68 (m,
1H), 7.28–7.38 (m, 5H); 31P, d −3.61. Compound 8 (472 mg,
0.46 mmol) was dissolved in THF (10 mL) and TREAT–HF
(372 lL, 2.28 mmol) was added. The mixture was stirred at rt
for 24 h followed by concentration and purification on silica gel
(1 : 0 → 5 : 1 CHCl3–MeOH + 0.5% Et3N) to give 9 (390 mg,
0.42 mmol, 91%); [a]D +34◦ (c 1.0, CHCl3); NMR: 13C, d
10.6, 20.6, 20.8, 20.8, (CH3CO), 40.7 (HOCH2CH2NH), 46.1,
61.5, 61.7, 62.2, 62.3, 64.6, 66.6, 66.6, 66.8, 67.4, 69.9, 70.0,
70.9, 71.2, 71.9, (C-2–6, 2ꢀ–6ꢀ, PhCH2O, OCH2CH2N), 94.0,
98.0 (C-1, 1ꢀ), 128.1, 128.2, 128.6, 136.6 (aromatic C), 156.6
(NHCOOBn), 169.9, 170.3 (CH3CO); 31P, d −3.51; HRMS
calcd for C30H39N7O18P [M]− 816.2089, found 816.2078.
phosphate)-(1
→
6)-(2-acetamido-3,4-di-O-acetyl-2-deoxy-a-
D-mannopyranoside) bis-triethylammonium salt (12). To a
solution of compound 11 (37 mg, 0.024 mmol) in MeOH
(1.5 mL) was added amberlite IR-45(OH−) resin (40 mg) and
palladium on activated carbon. The mixture was hydrogenolysed
at 100 psi overnight, diluted (MeOH), centrifuged and
concentrated. Purification on reversed phase gel (1 : 0 → 0 :
1 H2O–MeOH) gave 12 (28 mg, 0.020 mmol, 83%); [a]D +60◦
(c 1.0, MeOH); NMR (D2O): 13C, d −5.9, −5.4 (CH3Si), 8.89,
18.7 ((CH3)3CSi), 20.9, 21.0, 22.4, 22.5 (CH3CO, CH3CONH),
26.0 ((CH3)3CSi), 39.7 (HOCH2CH2NH), 47.3, 50.6, 51.4 (C-2,
2ꢀ, 2ꢀꢀ), 62.4, 64.4, 64.7, 64.9, 65.9, 66.2, 66.6, 70.1, 70.8, 71.0,
71.2, 71.5 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, OCH2CH2N), 95.4, 95.5, 99.1
(C-1, 1ꢀ, 1ꢀꢀ), 173.1, 173.3, 173.5, 173.6, 173.8, 174.9, 175.1,
175.2 (CH3CO, CH3CONH); 31P, d −3.04, −2.87; HRMS calcd
for C44H72N4O28P2Si [M + H]− 1195.3589, found 1195.3567.
2-(Benzyloxycarbonyl)aminoethyl (3,4-di-O-acetyl-2-azido-6-O-
(tertbutyldimethylsilyl)-2-deoxy-a-D-mannopyranosyl phosphate)-
(1 → 6)-(3,4-di-O-acetyl-2-azido-2-deoxy-a-D-mannopyranosyl
phosphate)-(1 → 6)-(3,4-di-O-acetyl-2-azido-2-deoxy-a-D-man-
nopyranoside) bis-triethylammonium salt (10). To a mixture of
5 (273 mg, 0.51 mmol) and 9 (357 mg, 0.39 mmol) in pyridine
(3 mL) was added pivaloyl chloride (119 lL, 0.97 mmol).
After 2 h the mixture was cooled to −40 ◦C and a solution
of I2 (119 mg, 0.47 mmol) in pyridine–H2O (3 mL 49 : 1)
was added. The mixture was diluted with CHCl3 when the
2-Aminoethyl (2-acetamido-3,4-di-O-acetyl-2-deoxy-a-D-
mannopyranosyl phosphate)-(1
acetyl-2-deoxy-a-D-mannopyranosyl phosphate)-(1
→
6)-(2-acetamido-3,4-di-O-
6)-(2-
→
acetamido-3,4-di-O-acetyl-2-deoxy-a-D-mannopyranoside) bis-
triethylammonium salt (13). A solution of TREAT–HF (17 lL,
0.10 mmol) in THF (1.5 mL) was treated with Et3N (17 lL,
0.12 mmol). This solution was added to compound 12 (28 mg,
0.020 mmol). After 30 minutes of stirring at rt the mixture
was concentrated and purified on reversed phase gel (1 : 0 →
0 : 1 H2O–MeOH) which gave 13 (22 mg, 0.017 mmol, 85%);
[a]D +45◦ (c 1.0, MeOH); NMR (D2O): 13C, d 8.87, 20.9, 20.9,
22.3, 22.4 (CH3CO, CH3CONH), 39.6 (HOCH2CH2NH), 47.3,
50.6, 51.3, 51.4 (C-2, 2ꢀ, 2ꢀꢀ), 60.2, 64.3, 64.5, 64.9, 66.2, 66.4,
69.9, 70.0, 70.5, 70.5, 70.7, 70.8, 71.0, 71.6 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ,
OCH2CH2N), 95.3, 95.3, 99.5 (C-1, 1ꢀ, 1ꢀꢀ), 173.3, 173.3, 173.6,
◦
temperature reached −10 C. Extraction with Na2S2O3 (10%),
cold TEAB (0.5 M), filtration (cotton) and chromatography
(1 : 0 → 5 : 1 CHCl3–MeOH + 0.5% Et3N) gave 10 (351 mg,
0.24 mmol, 62%); [a]D +68◦ (c 1.0, CHCl3); NMR: 13C, d −5.53,
−5.42 (CH3Si), 10.1, 18.3 ((CH3)3CSi), 20.5, 20.6, 20.6, 20.8
(CH3CO), 25.9 ((CH3)3CSi), 40.6 (HOCH2CH2NH), 45.9, 61.7,
62.1, 62.3, 64.2, 66.2, 66.4, 66.5, 67.1, 69.7, 70.4, 71.0, 71.2,
71.4, 71.6, 77.4 (C-2–6, 2ꢀ–6ꢀ, 2ꢀꢀ–6ꢀꢀ, PhCH2O, OCH2CH2N),
93.8, 94.0, 97.8 (C-1, 1ꢀ, 1ꢀꢀ), 128.0, 128.2, 128.5, 136.9 (aromatic
C), 156.8 (NHCOOBn), 169.3, 169.6, 169.7, 169.8, 169.9, 170.0
1
173.7, 173.8, 175.0, 175.1, 175.3 (CH3CO, CH3CONH); H, d
2.03 (s, 3H), 2.08 (s,3H), 2.09 (s, 3H), 2.14 (s, 3H), 2.14 (s, 3H),
2.18 (s, 3H), 2.24 (s, 3H), 2.24 (s, 3H), 2.24 (s, 3H), 3.28–3.36
(m, 2H), 3.68–3.84 (m, 3H), 4.00–4.18 (m, 5H), 4.27–4.33
(m, 1H), 4.57–4.64 (m, 2H), 5.22–5.30 (m, 1H), 5.31–5.37 (m,
2H), 5.44–5.49 (m, 1H); 31P, d −3.02, −2.95; HRMS calcd for
C38H59N4O28P2 [M + H]− 1081.2729, found 1081.2687.
2-Aminoethyl (2-acetamido-2-deoxy-a-D-mannopyranosyl
phosphate)-(1 → 6)-(2-acetamido-2-deoxy-a-D-mannopyranosyl
phosphate)-(1 → 6)-(2-acetamido-2-deoxy-a-D-mannopyranoside)
bis-triethylammonium salt (14). Compound 13 (22 mg,
1
(CH3CO); H, d −0.05 (s, 3H), −0.03 (s, 3H), 0.80 (s, 9H),
1.90–2.00 (m, 18H), 3.23–3.31 (m, 1H), 3.36–3.45 (m, 1H),
3.50–3.64 (m, 4H), 3.69–3.76 (m, 1H), 3.79–3.97 (m, 7H), 4.02–
4.15 (m, 3H), 5.00–5.06 (m, 2H), 5.12–5.21 (m, 2H), 5.24–5.31
(m, 1H), 5.33–5.37 (m, 2H), 5.38–5.45 (m, 3H), 6.04–6.10 (m,
1H), 7.18–7.30 (m, 5H); 31P, d −3.89, −3.56; HRMS calcd for
C46H66N10NaO27P2Si [M + Na]− 1303.3241, found 1303.3197.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7
3 7 8 5

View Article Online
0.017 mmol) was dissolved in MeOH (1 mL) and NaOMe (1 M)
was added. The mixture was concentrated after 30 min and the
residue was purified on reversed phase gel (H2O → MeOH).
The fractions containing the product were freeze dried to give
14 (14 mg, 0.016 mmol, 94%); [a]D +12.4◦ (c 0.5, MeOH);
NMR (D2O): 13C, d 22.6 (CH3CONH), 40.1 (HOCH2CH2NH),
53.1, 53.8, 53.9 (C-2, 2ꢀ, 2ꢀꢀ), 60.8, 65.3, 66.6, 67.0, 69.1, 69.3,
69.5, 72.2, 73.0, 74.1 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, OCH2CH2N), 95.8,
95.8, 99.6 (C-1, 1ꢀ, 1ꢀꢀ), 175.5, 175, 5, 175.6 (CH3CONH); 31P,
d −2.36, −2.22; HRMS calcd for C26H47N4O22P2 [M + H]−
829.2090, found 829.2064.
ranosyl phosphate)-(1 → 6)-(3,4-di-O-acetyl-2-azido-2-deoxy-a-
D-mannopyranoside) bis-triethylammonium salt (19). A mix-
ture of 18 (96 mg, 0.13 mmol) and 9 (103 mg, 0.11 mmol)
was dissolved in pyridine (3 mL). Pivaloyl chloride (34 lL,
0.28 mmol) was added and the mixture was stirred under
argon at rt for 2 h. The mixture was cooled to −40 ◦C and a
solution of I2 (34 mg, 0.13 mmol) in pyridine–H2O (3 mL 49 :
1) was added. The oxidation was completed at −10 ◦C and the
mixture was diluted with CHCl3, washed with Na2S2O3 (10%)
and cold TEAB (0.5 M). Filtration (cotton), concentration
and chromatography (1 : 0 → 5 : 1 CHCl3–MeOH + 0.5%
Et3N) gave 19 (106 mg, 0.065 mmol, 59%); [a]D +80◦ (c 1.0,
CHCl3); NMR: 13C, d 10.2, 20.6, 20.7, 20.8 (CH3CO), 40.6
(HOCH2CH2NH), 45.9, 57.9, 61.7, 62.3, 64.4, 65.5, 65.7, 66.1,
66.5, 66.6, 67.2, 69.4, 69.5, 70.3, 71.0, 71.4 (C-2–6, 2ꢀ–6ꢀ, 2ꢀꢀ–6ꢀꢀ,
PhCH2O, OCH2CH2N), 93.9, 94.0, 97.8 (C-1, 1ꢀ, 1ꢀꢀ), 128.0,
128.1, 128.2, 128.5, 128.6, 136.0, 136.9 (aromatic C), 156.8
(NHCOOBn), 169.6, 169.7, 169.8, 169.9 (CH3CO); 31P, d −4.13,
−3.58, −1.52.
Ethyl 3,4-di-O-acetyl-2-azido-2-deoxy-6-O-dibenzyloxyphos-
phoryl-1-thio-a-D-mannopyranoside (16). To compound
3
(305 mg, 0.68 mmol) dissolved in THF (6 mL), TREAT–HF
(0.55 ml, 3.38 mmol) was added. The mixture was stirred
at rt overnight. Concentration and chromatography (10 : 1
→ 0 : 1 toluene–EtOAc) gave ethyl 3,4-di-O-acetyl-2-azido-2-
deoxy-1-thio-a-D-mannopyranoside (15, 171 mg, 0.51 mmol,
75%); NMR: 13C, d 14.7 (SCH2CH3), 20.6, 20.8 (CH3CO), 25.4
(SCH2CH3), 61.3, 63.0, 66.6, 71.1, 71.3 (C-2–6), 82.2 (C-1),
2-Aminoethyl (2-acetamido-3,4-di-O-acetyl-2-azido-2-deoxy-
1
a-D-mannopyranosyl 1,6-bisphosphate)-(1 → 6)-(2-acetamido-
170.0, 170.6 (CH3CO); H, d 1.26–1.30 (t, 3H), 2.05 (s, 3H),
3,4-di-O-acetyl-2-azido-2-deoxy-a-D-mannopyranosyl
phos-
2.08 (s, 3H), 2.32 (s, 1H), 2.56–2.68 (m, 2H), 3.58–3.67 (m, 2H),
4.09–4.14 (m, 2H), 5.24–5.35 (m, 3H). To compound 15 (171 mg,
0.51 mmol) dissolved in CH2Cl2 (10 mL), tetrazole (125 mg,
1.78 mmol) and dibenzyl N,N-diisopropyl phosphoramidite19
(264 lL, 0.76 mmol) were added. The reaction mixture was
stirred for 30 min at rt. mCPBA (176 mg, 1.02 mmol) was added
at 0 ◦C and stirring was continued for 30 min. The mixture was
diluted (CH2Cl2), washed with Na2S2O3 (10%) and NaHCO3.
Filtration (Na2SO4), concentration and chromatography (10 : 1
→ 2 : 1 toluene–EtOAc) gave 16 (200 mg, 0.34 mmol, 67%);
[a]D +92◦ (c 1.0, CHCl3); NMR: 13C, d 14.6 (SCH2CH3), 20.5,
20.6 (CH3CO), 25.3 (SCH2CH3), 62.9, 65.7, 66.1, 69.4, 69.4,
69.5, 71.4, (C-2–6, PhCH2O), 82.0 (C-1), 127.9, 128.0, 128.5,
128.5, 128.6, 128.6, 128.7, 135.8, 135.9 (aromatic C), 169.5, 169.9
(CH3CO); 31P, d −1.36; HRMS calcd for C26H32N3NaO9PS [M +
Na]+ 616.1495, found 616.1487.
phate)-(1 → 6)-(2-acetamido-3,4-di-O-acetyl-2-azido-2-deoxy-
a-D-mannopyranoside) tris-triethylammonium salt (21). Com-
pound 19 (76 mg, 0.047 mmol) was dissolved in MeOH (3 mL)
and NiCl2(H2O)6 was added (catalytic amount). Reduction was
performed by adding NaBH4 in small amounts over a period
of 30 min at 0 ◦C. The mixture was then subjected to acetic
anhydride followed by dilution (MeOH) and concentration. The
residue was purified on silica gel (1 : 0 → 5 : 1 CHCl3–MeOH +
0.5% Et3N) and on LH-20 gel (MeOH + 1.5% Et3N), to give
compound 2-(benzyloxycarbonyl) aminoethyl (2-acetamido-
3,4-di-O-acetyl-2-azido-2-deoxy-6-O-dibenzyloxyphosphoryl-
a-D-mannopyranosyl phosphate)-(1 → 6)-(2-acetamido-3,4-di-
O-acetyl-2-azido-2-deoxy-a-D-mannopyranosyl
phosphate)-
(1 6)-(2-acetamido-3,4-di-O-acetyl-2-azido-2-deoxy-a-D-
→
mannopyranoside) bis-triethylammonium salt (20, 50 mg,
0.030 mmol, 64%); NMR (D2O): 13C, d 8.89, 20.7, 20.8, 22.4
(CH3CO, CH3CONH), 40.7 (HOCH2CH2NH), 47.3, 50.7,
51.4 (C-2, 2ꢀ, 2ꢀꢀ), 64.5, 65.9, 66.1, 66.5, 66.6, 67.2, 67.3, 69.7,
69.8, 70.5, 70.8, 70.9, 71.1 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, PhCH2O,
OCH2CH2N), 95.2, 95.3, 98.8 (C-1, 1ꢀ, 1ꢀꢀ), 128.2, 128.9, 129.0,
129.3, 129.5, 129.7, 135.7, 135.8, 137.1 (aromatic C), 158.7
(NHCOOBn), 172.8, 172.9, 173.2, 173.3, 173.4, 173.4, 174.8,
174.9, 175.0 (CH3CO, CH3CONH); 31P, d −3.05, −2.75,
−1.20. To a solution of compound 20 (46 mg, 0.027 mmol)
in MeOH (2 mL) was added Amberlite IR-45(OH−) resin
(46 mg) and palladium on activated carbon. The mixture
was hydrogenolysed at 100 psi overnight, diluted (MeOH),
centrifuged and concentrated. Purification on a reversed phase
silica gel column (1 : 0 → 0 : 1 H2O–MeOH) gave 21 (34 mg,
0.023 mmol, 85%); [a]D +43◦ (c 1.0, MeOH); NMR (D2O):
13C, d 8.89, 20.9, 20.9, 22.4, 22.4 (CH3CO, CH3CONH), 39.6,
(HOCH2CH2NH), 47.3, 50.6, 51.3, 51.4 (C-2, 2ꢀ, 2ꢀꢀ), 63.6,
64.4, 64.8, 66.1, 66.2, 66.4, 69.9, 70.0, 70.6, 70.7, 71.0 (C-3–6,
3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, OCH2CH2N), 95.3, 95.4, 99.1 (C-1, 1ꢀ, 1ꢀꢀ), 173.2,
173.3, 173.4, 173.6, 173.7, 173.7, 175.0, 175.1, 175.2 (CH3CO,
CH3CONH); 31P, d −3.08, −2.95, 0.05; HRMS calcd for
C38H59N4O31P3 [M + H]2− 580.1157, found 580.1142.
2-Aminoethyl (2-acetamido-2-azido-2-deoxy-6-O-phosphoryl-
a-D-mannopyranosyl 1,6-bisphosphate)-(1 → 6)-(2-acetamido-
2-azido-2-deoxy-a-D-mannopyranosyl phosphate)-(1 → 6)-(2-
acetamido-2-azido-2-deoxy-a-D-mannopyranoside) tris-sodium
salt (23). Compound 20 (50 mg, 0.030 mmol) was dissolved
in MeOH (3 mL) and NaOMe (1 M) was added. The reaction
mixture was stirred for 1 h at rt. Concentration and puri-
fication on reversed phase gel (1 : 0 → 0 : 1 H2O–MeOH)
gave 2-(benzyloxycarbonyl)aminoethyl (2-acetamido-2-azido-
2-deoxy-6-O-dibenzyloxyphosphoryl-a-D -mannopyranosyl
3,4-Di-O-acetyl-2-azido-2-deoxy-6-O-dibenzyloxyphosphoryl-
a-D-mannopyranosyl hydrogenphosphonate, triethylammonium
salt (18). Compound 16 (281 mg, 0.47 mmol) was dissolved
in wet CH2Cl2 (10 mL) and cooled to −20 ◦C. NIS (137 mg,
0.61 mmol) and AgOTf (catalytic amount) were added and
the mixture was stirred for 30 min at −20 ◦C. The mixture
was diluted (CH2Cl2), washed with Na2S2O3 (10%), filtered
(Na2SO4) and concentrated. Chromatography (2 : 1 → 0 :
1 toluene–EtOAc) gave 3,4-di-O-acetyl-2-azido-2-deoxy-6-O-
dibenzyloxyphosphoryl-a-D-mannopyranoside (17, 201 mg,
0.37 mmol, 79%); NMR: 13C, d 20.7, 20.7, 62.5, 66.2, 66.3,
66.4, 68.6, 68.7, 69.6, 69.7, 69.8, 69.8, 71.0 (C-2–6, PhCH2O),
92.5 (C-1), 128.0, 128.1, 128.3, 128.6, 128.7, 128.7, 135.6, 135.7
(aromatic C), 169.8, 170.1 (CH3CO); 31P, d −1.87. A mixture of
imidazole (349 mg, 5.13 mmol), PCl3 (128 lL, 1.47 mmol) and
Et3N (765 lL, 5.49 mmol) in MeCN (10 mL) was stirred at 0 ◦C
for 30 min. A solution of compound 17 (201 mg, 0.37 mmol) in
MeCN (10 mL) was added during 30 min at 0 ◦C. The reaction
mixture was then stirred at rt for 10 min, quenched with TEAB
(0.5 M) and concentrated. The residue was diluted (CHCl3),
washed with TEAB (0.5 M), filtered (cotton) and concentrated.
Chromatography (1 : 0 → 10 : 1 CHCl3–MeOH + 1.0% Et3N)
of the residue gave 18 (243 mg, 0.34 mmol, 92%); [a]D +55◦ (c
1.0, CHCl3); NMR: 13C, d 9.34, 20.6, 20.7 (CH3CO), 45.9, 62.4,
62.5, 65.6, 65.7, 69.4, 69.5, 69.9, 70.0, 70.9 (C-2–6, PhCH2O),
93.0 (C-1), 128.0, 128.0, 128.1, 128.5, 128.5, 128.6, 135.8
(aromatic C), 169.5, 169.9 (CH3CO); 31P, d −1.32, 0.29; HRMS
calcd for C24H28N3O12P2 [M]− 612.1148, found 612.1129.
2-(Benzyloxycarbonyl)aminoethyl (3,4-di-O-acetyl-2-azido-2-
deoxy-6-O-dibenzyloxyphosphoryl-a-D-mannopyranosyl phos-
phate)-(1 → 6)-(3,4-di-O-acetyl-2-azido-2-deoxy-a-D-mannopy-
3 7 8 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7

View Article Online
phosphate)-(1
→
6)-(2-acetamido-2-azido-2-deoxy-a-D-
References
mannopyranosyl phosphate)-(1 → 6)-(2-acetamido-2-azido-
2-deoxy-a-D-mannopyranoside) bis-sodium salt (22, 30 mg,
0.024 mmol, 80%); NMR (D2O): 13C, d 22.6 (CH3CONH),
40.7 (HOCH2CH2NH), 53.1, 53.8, 54.0 (C-2, 2ꢀ, 2ꢀꢀ), 65.1, 66.4,
66.5, 66.8, 67.5, 68.9, 69.1, 69.6, 71.0, 71.1, 71.9, 72.0, 72.3
73.1 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, PhCH2O, OCH2CH2N), 95.6, 95.8,
99.4 (C-1, 1ꢀ, 1ꢀꢀ), 128.1, 128.4, 129.0, 129.0, 129.4, 129.5, 129.8,
135.7, 135.8, 137.1 (aromatic C), 158.8 (NHCOOBn), 175.3
(CH3CONH); 31P, d −2.35, −2.23, −0.96. Compound 22 (30 mg,
0.024 mmol) was dissolved in MeOH (2 mL). Amberlite IR-
45(OH−) resin (30 mg) and palladium on activated carbon were
added. The mixture was hydrogenolysed at 100 psi overnight,
diluted (MeOH), centrifuged and concentrated. Purification on
reversed phase gel (1 : 0 → 0 : 1 H2O–MeOH) gave 23 (15 mg,
0.015 mmol, 64%); [a]D +13.6◦ (c 0.5, MeOH); NMR (D2O):
13C, d 22.6, 22.6, 22.6 (CH3CONH), 39.7 (HOCH2CH2NH),
49.5, 53.0, 53.7, 53.8 (C-2, 2ꢀ, 2ꢀꢀ), 63.8, 64.2, 65.1, 65.2, 65.4,
66.5, 66.7, 67.0, 68.9, 69.2, 69.5, 72.2, 72.3, 72.9, 73.1, 73.2,
73.4, 73.4 (C-3–6, 3ꢀ–6ꢀ, 3ꢀꢀ–6ꢀꢀ, OCH2CH2N), 95.9, 99.6 (C-1,
1ꢀ, 1ꢀꢀ), 175.4, 175.5, 175.5 (CH3CONH); 31P, d −2.35, −2.30,
2.2; HRMS calcd for C26H46N4Na2O25P3 [M + 2Na]− 953.1459,
found 953.1440.
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Acknowledgements
We are thankful to the Swedish Research Council and the Na-
tional Research Foundation South Africa for financial support.
Dr Petter Tollba¨ck is acknowledged for recording the HRMS
spectra.
19 K.-L. Yu and B. Fraser-Reid, Tetrahedron Lett., 1988, 29, 979.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 3 7 8 2 – 3 7 8 7
3 7 8 7