Synthesis of stable C-phosphonate analogues of Neisseria meningitidis group A capsular polysaccharide structures using modified Mitsunobu reaction conditions

Article abstract of DOI:10.1039/b614038f

Examples of synthetic C-phosphonate analogues of microbial polysaccharide structures containing inter-residue phosphodiester linkages are most rare. The successful construction of such analogues of the Neisseria meningitidis Group A capsular polysaccharid

Full text of DOI:10.1039/b614038f

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www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of stable C-phosphonate analogues of Neisseria meningitidis group
A capsular polysaccharide structures using modified Mitsunobu reaction
conditions
Peter Teodorovic´, Rikard Sla¨ttega˚rd and Stefan Oscarson*
Received 27th September 2006, Accepted 26th October 2006
First published as an Advance Article on the web 9th November 2006
DOI: 10.1039/b614038f
Examples of synthetic C-phosphonate analogues of microbial polysaccharide structures containing
inter-residue phosphodiester linkages are most rare. The successful construction of such analogues of
the Neisseria meningitidis Group A capsular polysaccharide is described. Using a modified Mitsunobu
reaction (tris(4-chlorophenyl)phosphine, DIAD, excess of Et3N) between an anomeric C-phosphonate
monoester and a 6-OH ManNAc acceptor a high yield (88%) of a dimer was obtained. Transformation
of the dimer into a new 6-OH acceptor through deacetylation and further reaction with the elongating
C-phosphonate monomer employing the same conditions afforded the trimer in 92% yield. Iteration of
the procedure then afforded the tetramer with a coupling yield of 85%. The di-, tri- and tetramer were
deprotected to give target structures ready for conjugation to a carrier protein and subsequent
immunological evaluation.
repeating unit of the Neisseria meningitidis Group A capsular
Introduction
polysaccharide, needed for vaccine development. The crucial
Bacterial meningitis is caused mainly by three types of bacteria,
coupling step is performed using a C-phosphonate monoester and
Streptococcus pneumoniae, Haemophilus influenzae and Neisseria
modified Mitsunobu conditions. The synthetic strategy also allows
for continued synthesis of larger oligomers and polycondensation.
meningitidis. With N. meningitidis five serogroups, i.e., A, B, C,
Y and W-135, are associated with meningococcal meningitis.1,2
The target structures will be conjugated to a carrier protein and
The serotyping is based on the structure and antigenicity of the
the resulting glycoconjugates evaluated as vaccine candidates in
capsular polysaccharide (CPS) surrounding the bacteria. Efficient
mice.
bacterial glycoconjugate vaccines can be produced by attaching
the polysaccharide to a carrier protein.3 Against N. meningitidis
Results and discussion
Group C (MenC), there are now three monovalent glycoconjugate
vaccines commercially available,4 and development of vaccines
against the other serogroups are under way.5 However, Group B
represents a major issue, since its CPS show molecular mimicry
to human carbohydrate structure. Another problem encountered
when trying to make multivalent glycoconjugate vaccines against
N. meningitidis is the poor stability of Group A CPS. The repeat-
ing unit of the Group A CPS is a monosaccharide, 2-acetamido-2-
deoxy-a-D-mannopyranose, linked 1 → 6 via a phosphodiester
bridge,6 and there is an inherent instability of the anomeric
phosphate diester linkages of the CPS.7 Hence, there is an interest
in stable analogues of these structures. In a programme directed
towards development of stable glycoconjugate vaccine candidates
against MenA, we have earlier synthesised a variety of MenA
trisaccharide structures8 and were now interested in exploring
C-phosphonate analogues. However, in spite of the obvious
applicability of and interest in such derivatives very few have
been synthesised, probably because of their difficult preparation.
To our knowledge there are only two publications describing
synthesis of structures with inter-residue C-phosphonate linked
oligosaccharides.9,10 Herein we describe the efficient synthesis
of C-phosphonate analogues of di-, tri- and tetramers of the
For the construction of the elongating C-phosphonate monomer
a modified version of a published approach was applied
(Scheme 1).11 After desilylation of precursor 1 to afford the 2-OH
compound 2, azide displacements using Mitsunobu conditions
gave the 2-azido-2-deoxymannopyranoside 3 (86%). An orthogo-
nal protecting group, to allow later 6-O elongation, was introduced
by acetolysis to afford the 6-O-acetate 4 (84%). Azide reduction
followed by acetylation gave 5 (76%), from which the ethyl esters
were removed to yield the elongating monomer 6.
The starting spacer-containing monomer acceptor 9 was al-
ready known.12 In the esterification of anomeric C-phosphonates
various examples involving spacer and fatty alcohols have been
published,13 but, as mentioned in the introduction, esterifications
using sugar alcohols are most rare. In the publication by Nikolaev
and co-workers both DCC and Mitsunobu conditions were
employed with yields in the range of 50–70%. However, when
esterifications with acceptors already containing phosphonates
were performed, the yields dropped to 10–30%.9 Our first attempt
to esterify compound 6 with acceptor alcohol 9 using DCC as
condensation reagent gave no product at all. Consequently, the
methyl monoester 8 was synthesised (Scheme 1) and tried in
the same reaction, which afforded dimer 10 but in a very low
yield (around 20%), the pyrophosphonate dimer being the major
product according to MALDI-TOF MS. Other condensation
Department of Organic Chemistry, Arrhenius Laboratory, Stockholm Uni-
versity, S-106 91, Stockholm, Sweden. E-mail: s.oscarson@organ.su.se;
Fax: +46 8162 480; Tel: +46 8154 908
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Scheme 1 Synthesis of monosaccharide elongating precursor: (i) TBAF;
(ii) Ph3P, DIAD, DPPA, THF; (iii) HOAc, Ac2O, H2SO4; (iv) (a) NaBH4,
NiCl2·6H2O, (b) Ac2O; (v) Me3SiBr; (vi) MeC(OMe)3, HOAc; (vii) PhSH,
Et3N.
reagents (TIPSCl, MSNT) as well as displacements reactions using
various leaving groups (triflate, tosylate) at the 6-position of 9 gave
even less product. Hence, Mitsunobu conditions were tested using
Ph3P and DIAD, which gave a good yield (47%) of 10.14 Under
these conditions the pyrophosphate was not a competing side-
product since it is the acceptor alcohol that is transformed into a
leaving group.
During the course of this work a synthesis of a similar MenA
C-phosphonate dimer analogue was published using the same
Mitsunobu conditions in the esterification step in an excellent yield
of 97%.10 The structure of both the acceptor (b-linked spacer) and
donor (6-O-benzylated) differs slightly, but the large difference in
yield is still difficult to explain.
In the beginning of the 1990s, Campbell described the
use of tris(4-chlorophenyl)phosphine together with a large ex-
cess of triethylamine to improve Mitsunobu esterification of
phosphonates.15 The application of these conditions to our
reaction proved to be an excellent combination. The desired
dimer 10 was obtained in an 89% yield (Scheme 2). Efforts
have to be made to exclude external nucleophiles in the reaction
mixture to obtain an optimal yield. We found 6-chlorination
when acidic chloroform had been used as solvent in a preceding
chromatography purification as well as re-acetylation when acetate
from the prior deacetylation was still present.
One major problem in the synthesis of anomeric phosphate
diester structures is the formation of oligomers since already
formed diesters are easily cleaved during the reaction conditions.16
As mentioned above this might be a problem also for continued
C-phosphonate formation.9 Most pleasingly, using the optimized
Mitsunobu conditions, the yield in the trimer formation was found
to be comparable and even higher than in the dimer formation.
The dimer 10 was transformed into a new acceptor (→11) by
Scheme
2
Synthesis of
a
repeating unit di-, tri- and tetramers:
(i) (p-ClC6H4)3P, DIAD, Et3N, THF; (ii) KOH, MeOH; (iii) PhSH, DBU,
CH3CN; (iv) H2, Pd/C, HCl.
deacetylation (Scheme 2). Using the modified Mitsunobu reaction
between 8 and 11 then afforded the trimer 14 in 92% yield.
The procedure could be iterated. Deacetylation of 14, created a
new acceptor 15 and a Mitsunobu reaction between 8 and 15
formed the tetramer 18 in 85% yield. The identity of the products
was proven mainly by MS since the NMR spectra were quite
complex due to phosphonate diester diastereomers. However, after
subsequent removal of the methyl esters using thiophenol and
DBU, the NMR data of compounds 12, 16 and 20 could be
interpreted. Final deprotection of these compounds by catalytic
hydrogenolysis afforded the unprotected dimer 13, trimer 17 and
tetramer 21 in 79, 62 and 58% overall yield from compounds 11,
15 and 19, respectively, all target products ready for conjugation
to a carrier protein and immunological studies.
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To conclude, an efficient synthesis of C-phosphonate analogues
of the Neisseria meningitidis Group A capsular polysaccharide
has been developed using modified Mitsunobu conditions in the
formation of the inter-saccharide phosphonate ester linkages.
The building blocks can be prepared in gram quantities and
the yields are excellent also for the formation of tri- and
tetramers and the approach is suitable also for formation of longer
oligomers.
4.18 (m, 4H), 4.21–4.37 (m, 3H), 4.54–4.64 (m, 2H), 4.72–4.78
(m, 2H), 7.25–7.37 (m, 10H); 13C NMR (CDCl3) d 16.2, 16.3
(CH3CH2O), 20.6 (CH3CO), 27.6 (d, J = 141 Hz, C-7), 60.4,
60.5, 61.7, 61.8, 62.5, 69.3, 71.9, 72.2, 73.4, 74.0, 77.6, 77.8 (C-
1–6, PhCH2O, CH3CH2O), 127.8–128.3, 137.0, 137.4 (aromatic
C), 170.4 (CH3CO); 31P NMR (CDCl3) d 27.5; HRMS: calc. for
C27H36N3NaO8P [M + Na]+: 584.2137, found: 584.2116.
Diethyl C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-
D-mannopyranosyl)methanephosphonate (5). To a stirred solu-
tion of 4 (2.50 g, 4.45 mmol) in MeOH (125 mL) a catalytic amount
of NiCl2·6H2O was added, followed by portions of NaBH4 (in total
0.34 g, 8.99 mmol) every 10 min until no starting material was
observed on TLC (2 : 1 toluene–EtOAc). Ac2O (3 mL, 31.8 mmol)
was added and after 50 min the reaction mixture was diluted with
toluene, filtered through a plug of silica gel and concentrated. The
residue was purified by silica gel chromatography (1 : 0 → 0 : 1
toluene–EtOAc) to give 5 (1.95 g, 3.38 mmol, 76%); [a]D +37◦ (c
1.0, CHCl3); 13C NMR (CDCl3) d 16.1, 16.2, 16.3 (CH3CH2O),
20.6 (CH3CO), 23.0 (CH3CONH), 28.5 (d, J = 142 Hz, C-7),
48.7 (d, C-2), 61.2, 61.3, 61.8, 61.8, 61.9, 67.1, 72.0, 72.1, 72.4,
72.6, 75.3 (C-1, C-3–6, PhCH2O, CH3CH2O), 127.4–129.9, 137.1,
137,4 (aromatic C), 170.0, 170.6 (CH3CO, CH3CONH; 31P NMR
(CDCl3) d 29.5; HRMS: calc. for C29H40NNaO9P [M + Na]+:
600.2339, found: 600.2346.
Experimental
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
chromatography was performed on silica gel (0.040–0.063 mm,
Amicon), reversed phase gel (C18 60A 40–63 lm) or liphophilic
Sephadex gel (LH20, bead size 20–100 lm). NMR spectra were
recorded in CDCl3 (Me4Si, d = 0.00) or D2O (acetone 13C d =
30.89, H = 2.22) at 25 ◦C on a Varian 300 MHz or 400 MHz
1
instrument. For 31P NMR spectra, H3PO4 (d = 0.00) was used
as reference. Organic solutions were concentrated at 30 ◦C under
reduced pressure.
Diethyl C-(3,4,6-tri-O-benzyl-a-D-glucopyranosyl)methane-
phosphonate (2). TBAF (2.6 g, 8.24 mmol) was added to a
solution of compound 1 (5.36 g, 7.68 mmol) in THF (150 mL).
After 20 min the solvent was removed under reduced pressure and
the product was purified by silica gel column chromatography (1 :
0 → 0 : 1 toluene–EtOAc) to give 2 (4.44 g, 7.60 mmol, 99%).
NMR data were in agreement with those reported earlier.11
Dimethyl C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-
a-D-mannopyranosyl)methanephosphonate (7). To a solution of 5
(250 mg, 0.43 mmol) in dry CH2Cl2 (5 mL), bromotrimethylsilane
(0.28 mL, 2.16 mmol) was added. After the addition was complete,
the mixture was stirred under argon at rt for 1 h. The solution
was cooled to 0 ◦C and Et3N (1 mL) was added followed
by addition of water (1 mL). After 10 min, the solvents were
removed under reduced pressure and the residue was desalted by
reversed phase chromatography (1 : 0 → 1 : 1 H2O–MeOH). The
product C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-
D-mannopyranosyl)methanephosphonic acid (6) was methylated
without further purification. To the product from the reaction
described above were added AcOH (5.5 mL) and trimethy-
lorthoacetate (11.5 mL). The mixture was heated at reflux for
30 min and then concentrated. (The reaction had to be monitored
carefully and stopped exactly when the reaction was completed.
Otherwise the result is a mixture of the desired product and
the deacetylated product) The residue was purified by silica gel
chromatography (1 : 0 → 0 : 1 toluene–EtOAc) to give 7 (200 mg,
Diethyl C-(2-azido-3,4,6-tri-O-benzyl-2-deoxy-a-D-manno-
pyranosyl)methanephosphonate (3). DIAD (2.4 mL, 12.19 mmol)
was added dropwise to a cooled (−5 ◦C) solution of Ph3P (3.1 g,
11.82 mmol) in THF (50 mL). After 30 min, a solution of 2 (5.57 g,
9.53 mmol) in THF (20 mL) was added. After an additional
10 min, diphenyl phosphorazidate (DPPA; 2.4 mL, 11.12 mmol)
was added and the reaction mixture was allowed to attain rt. After
stirring overnight under argon at rt, the solvent was evaporated
and the residue was purified by silica gel chromatography (1 :
0 → 0 : 1 toluene–EtOAc) to give 3 (5.0 g, 8.21 mmol, 86%) as a
colourless oil. [a]D +20◦ (c 1.0, CHCl3); 13C NMR (CDCl3) d 16.4,
16.5 (CH3CH2O), 27.9 (d, J = 141 Hz, C-7), 60.9, 61.0, 61.9, 62.0,
62.1, 62.2, 68.8, 69.5, 72.4, 73.5, 73.7, 73.9, 74.3, 78.2 (C-1–6,
PhCH2O, CH3CH2O), 127.7–128.6, 137.4, 137.9, 138.2 (aromatic
C); 31P NMR (CDCl3) d 27.9; HRMS: calc. for C32H40N3NaO7P
[M + Na]+: 632.2502, found: 632.2513.
0.36 mmol, 84% over two steps); [a]D +36◦ (c 1.0, CHCl3); H
1
NMR (CDCl3) d 1.83 (s, 3H), 2.03 (s, 3H), 2.05–2.09 (m, 2H), 3.51
(t, J = 3.6 Hz, 1H), 3.67–3.75 (dd, J = 23.2 Hz, J = 11.2 Hz,
6H), 4.05–4.19 (m, 4H), 4.28–4.35 (m, 2H), 4.49–4.62 (m, 5H),
5.82 (d, J = 9.2 Hz, 1H), 7.22–7.34 (m, 10H); 13C NMR (CDCl3) d
20.9 (CH3CO), 23.3 (CH3CONH), 28.0 (d, J = 141 Hz, C-7), 48.8
(d, J = 14.8 Hz, C-2), 52.1 (d, J = 6.5 Hz, CH3O), 52.9 (d, J =
6.2 Hz, CH3O), 61.9, 66.8, 66.9, 72.0, 72.5, 72.6, 73.0, 75.4 (C-1,
C-3–6, PhCH2O), 128.0–128.9 137.1, 137.5 (aromatic C), 170.0,
170.7 (CH3CO, CH3CONH); 31P NMR (CDCl3) d 31.6; HRMS:
calc. for C27H36NNaO9P [M + Na]+: 572.2026, found: 572.2000.
Diethyl C-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-D-
mannopyranosyl)methanephosphonate (4). Compound 3 (3.2 g,
5.25 mmol) was dissolved in HOAc–Ac2O (1:1, 32 mL). 10 drops
of 1% H2SO4 in Ac2O was added. After stirring overnight the
mixture was poured into a separation funnel containing ice and
CH2Cl2. The organic phase was separated, filtered through a plug
of silica gel and concentrated. The residue was purified by column
chromatography (1 : 0 → 0 : 1 toluene–EtOAc) to give 4 (2.48 g,
◦
1
4.42 mmol, 84%). [a]D +70 (c 1.0, CHCl3); H NMR (CDCl3) d
1.32 (t, J = 7.2 Hz, 6H), 2.04 (s, 3H), 2.05–2.16 (m, 2H), 3.72–
3.75 (m, 2H), 3.91–3.94 (m, 1H), 3.98 (t, J = 3.6 Hz, 1H), 4.06–
Methyl C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-
D-mannopyranosyl)methanephosphonate triethylammonium salt
(8). To a solution of 7 (200 mg, 0.36 mmol) in THF (0.5 mL)
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were added thiophenol (149 lL, 1.45 mmol) and Et3N (304 lL,
2.18 mmol). After 48 h the reaction mixture was directly put on
a silica gel column and eluted (1 : 0 → 5 : 1 CHCl3–MeOH +
1.5% Et3N) to give 8 (208 mg, 0.33 mmol, 92%); [a]D +15◦ (c 1.0,
(NHCOOBn), 172.6, 173.6 (CH3CONH); 31P NMR (CDCl3)
d 21.6. To a solution of compound 12 (12 mg, 0.011 mmol) in
MeOH (4.5 ml), HCl in water (40 ll, 1 M) was added followed by
a catalytic amount of palladium on activated carbon. The mixture
was hydrogenolysed at 100 psi overnight, diluted (MeOH) and
centrifuged to remove the activated carbon. Concentration and
purification on a reversed phase silica gel column (H2O) gave
13 (6 mg, 0.009 mmol, 82%) after freeze-drying; [a]D +17◦ (c
1
CHCl3); H NMR (CDCl3) d 1.94 (s, 3H), 1.96 (s, 3H), 3.41 (t,
J = 6.4 Hz, 1H), 3.53 (d, J = 10.4 Hz, 3H), 3.84–3.86 (m, 1H),
4.05 (m, 1H), 4.16–4.20 (m, 2H), 4.34 (dd, J = 11.6 Hz, J = 6 Hz,
1H), 4.42 (t, J = 11.2 Hz, 2H), 4.63–4.72 (m, 3H), 7.21–7.26 (m,
10H); 13C NMR (CDCl3) d 8.88 [(CH3CH2)3N], 20.8 (CH3CO),
23.5 (CH3CONH), 28.5 (d, J = 129 Hz, C-7), 45.3 [(CH3CH2)3N],
50.3 (C-2), 51.6 (d, J = 6.1 Hz, CH3O), 63.1, 72.2, 73.4, 73.5,
76.3, (C-1, C-3–6, PhCH2O), 127.7–128.6, 137.9, 138.0 (aromatic
C), 169.9, 170.7 (CH3CO, CH3CONH); 31P NMR (CDCl3) d
20.4; HRMS: calc. for C26H33N1O9P1 [M]−: 534.1898, found:
534.1902.
1
0.5, H2O); H NMR (D2O) d 2.08 (s, 3H), 2.09 (s, 3H), 2.16 (d,
J = 7.2 Hz, 1H), 2.21 (d, J = 7.2 Hz, 1H), 3.28–3.32 (m, 2H),
3.64–3.66 (m, 2H), 3.71–3.76 (m, 3H), 3.79 (m, 1H), 3.82 (m,
1H), 3.85 (m, 1H), 3.88 (m, 1H), 3.99–4.05 (m, 3H), 4.07–4.11
(m, 1H), 4.14–4.17 (m, 2H), 4.23–4.25 (m, 1H), 4.42 (m, 2H),
4.86 (m, 1H); 13C NMR (D2O) d 8.8 [(CH3CH2)3N], 22.5, 22.6
(CH3CONH), 27.7 (d, J = 134 Hz, C-7ꢀ), 39.5 (OCH2CH2NH),
47.2 [(CH3CH2)3N], 52.8 (C-2), 53.2 (d, J = 10 Hz, C-2ꢀ), 60.0 (C-
6ꢀ), 63.6, 63.9, 66.8, 67.8, 69.1, 69.6, 72.1, 73.1, 74.5 (C-3–6, C-1ꢀ,
C-3ꢀ–5ꢀ, OCH2CH2NH), 99.3 (C-1), 174.8, 175.4 (CH3CONH);
31P NMR (D2O) d 23.1; HRMS: calc. for C19H35N3O13P [M]−:
544.1907, found: 544.1943.
2-(Benzyloxycarbonyl)aminoethyl 6-O-[methyl C-(6-O-acetyl-2-
acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methane-
phosphonate] - 2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-manno-
pyranoside (10). To a solution of compound 8 (100 mg,
0.157 mmol) and compound 9 (70 mg, 0.121 mmol) in THF
(0.5 mL), tris(4-chlorophenyl)phosphine (62 mg, 0.17 mmol),
Et3N (84 lL, 0.60 mmol) and DIAD (33 lL, 0.168 mmol) were
added. After stirring under argon at rt for 30 min, the residue
was directly purified by silica gel chromatography (1 : 0 →
20 : 1 CHCl3–MeOH) followed by further purification on a
LH-20 gel column (MeOH) to give the product 10 (118 mg,
0.108 mmol, 89%) as a diastereomeric mixture; HRMS: calc. for
C58H70N3NaO16P [M + Na]+: 1118.4391, found: 1118.4399.
2-(Benzyloxycarbonyl)aminoethyl 6-O-[methyl C-(6-O-[methyl
C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-manno-
pyranosyl)methanephosphonate]-2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-D-mannopyranosyl)methanephosphonate]-2-acetamido-3,4-
di-O-benzyl-2-deoxy-a-D-mannopyranoside (14). To a solution
of 11 (38 mg, 0.036 mmol) and 8 (27 mg, 0.042 mmol) in THF
(0.5 mL), tris(4-chlorophenyl)phosphine (19 mg, 0.052 mmol),
Et3N ( 25 lL, 0.18 mmol) and DIAD (10 lL, 0.051 mmol) were
added. The mixture was stirred under argon at rt for 40 min
followed by purification on silica gel column (1 : 0 → 20 : 1
CHCl3–MeOH). The products were further purified on a LH-20
gel column (MeOH) to give 14 (52 mg, 0.033 mmol, 92%); [a]D
+38◦ (c 1.0, CHCl3). HRMS: calc. for C82H99N4NaO23P2 [M +
Na]+: 1593.6157, found: 1593.6151.
2-(Benzyloxycarbonyl)aminoethyl 6-O-[methyl C-(2-acetamido-
3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methanephospho-
nate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranoside
(11). To a solution of 10 (113 mg, 0.103 mmol) in MeOH (3 mL),
KOH (206 lL) from a stock solution of KOH dissolved in MeOH
(1 M) was added. After 30 min the reaction mixture was directly
purified by silica gel chromatography (1 : 0 → 10 : 1 CHCl3–
MeOH) followed by further purification on a LH-20 gel column
(MeOH) to give 11 (93 mg, 0.088 mmol, 86%); [a]D +48◦ (c 1.0,
CHCl3); HRMS: calc. for C56H69N3O15P1 [M + H]+: 1054.4461,
found: 1054.4430.
2-(Benzyloxycarbonyl)aminoethyl 6-O-[C-(6-O-[C-(2-acetamido-
3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methanephospho-
nate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)-
methanephosphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-
mannopyranoside bis(triethylammonium) salt (16). To a solution
of 14 (59 mg, 0.038 mmol) in MeOH (3 ml), KOH (76 lL)
from a stock solution of KOH dissolved in MeOH (1 M) was
added. After 30 min the reaction mixture was purified by silica gel
chromatography (1 : 0 → 10 : 1 CHCl3–MeOH) followed by further
purification on a LH-20 gel column (MeOH) to give 2-(benzyloxy-
carbonyl)aminoethyl 6-O-[methyl C-(6-O-[methyl C-2-acetamido-
3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methanephospho-
nate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)-
methanephosphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-
mannopyranoside 15 (49 mg, 0.032 mmol, 84%); HRMS: calc.
for C80H99N4O22P2 [M + H]+: 1529.6221, found: 1529.6244. To a
solution of 15 (36 mg, 0.024 mmol) in CH3CN (0.5 ml), thiophenol
(96 lL, 0.94 mmol) and DBU (70 lL, 0.47 mmol) were added.
The reaction mixture was stirred under argon at rt for 2 h and the
product was then purified by silica gel chromatography (1 : 0 →
5 : 1 CHCl3–MeOH + 1.5% Et3N) and further purified on a LH-20
gel column (MeOH) to give 16 (30 mg, 0.018 mmol, 75%); [a]D
+60◦ (c 1.0, MeOH); 13C NMR (CDCl3) d 9.06 [(CH3CH2)3N],
23.2, 23.3, 23.5 (CH3CONH), 29.9 (m, C-7ꢀ, C-7ꢀꢀ), 40.8
2-Aminoethyl 6-O-[C-(2-acetamido-2-deoxy-a-D-mannopyra-
nosyl)methanephosphonate]-2-acetamido-2-deoxy-a-D-mannopyra-
noside triethylammonium salt (13). To a solution of 11 (44 mg,
0.042 mmol) in CH3CN (0.5 ml), thiophenol (86 lL, 0.84 mmol)
and DBU (125 lL, 0.84 mmol) were added. The reaction mixture
was stirred under argon at rt for 2 h and then the product was
directly purified by silica gel chromatography (1 : 0 → 5 : 1
CHCl3–MeOH + 1.5% Et3N). Further purification on a LH-20
gel column (MeOH) gave 2-(benzyloxycarbonyl)aminoethyl
6-O-[C-(2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyra-
nosyl)methanephosphonate]-2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-D-mannopyranoside (12, 41 mg, 0.36 mmol, 86%); [a]D
+32◦ (c 1.0, H2O); 13C NMR (CD3OD) d 8.88 [(CH3CH2)3N],
22.4 22.6 (CH3CONH), 29.9 (d, J = 135 Hz, C-7ꢀ), 41.4
(OCH2CH2NH), 45.3 [(CH3CH2)3N], 50.6, 51.2, 51.3, 54.7, 61.1,
64.6, 67.3, 67.4, 72.0, 72.2, 72.3, 72.9, 74.2, 74.6, 75.2, 75.9, 76.0,
77.8, 78.9 (C-2–6, C-1–6, PhCH2O, OCH2CH2NH), 100.3 (C-1),
125.1–130.4, 138.2, 139.4, 139.6, 139.7, 139.9 (aromatic C), 158.7
4488 | Org. Biomol. Chem., 2006, 4, 4485–4490
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(OCH2CH2NH), 48.5, 49.6 (C-2, C-2, C-2ꢀꢀ), 59.0, 63.5, 63.7,
66.8, 71.1, 71.5, 71.8, 72.2, 72.6, 72.9, 73.9, 74.2, 74.7, 75.2, 76.1
(C-3–6, C-1ꢀ, C-3ꢀ–6ꢀ, C-1ꢀꢀ, C-3ꢀꢀ–6ꢀꢀ, PhCH2O, OCH2CH2NH),
100.0 (C-1), 127.1–128.8, 136.7, 137.6, 138.1, 138.5, 138.7,
138.9 (aromatic C), 156.6 (NHCOOBn), 169.8, 170.8, 171.2
(CH3CONH); 31P NMR (CDCl3) d 21.2, 22.4; HRMS: calc. for
C78H92N4O22P2 [M]2−: 749.2845, found: 749.2836.
C-(6-O-[methyl
C-(2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-
mannopyranosyl)methanephosphonate]-2-acetamido-3,4-di-O-
benzyl-2-deoxy-a-D-mannopyranosyl)methanephosphonate]-2-
acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)metha-
nephosphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-manno-
pyranoside (19, 39 mg, 0.019 mmol, 73%). To compound 19
(39 mg, 0.019 mmol) dissolved in CH3CN (0.5 mL), thiophenol
(80 lL, 0.78 mmol) and DBU (116 lL, 0.78 mmol) were added.
The mixture was stirred under argon at rt for 2 h and then
put on top of a silica gel column and eluted (1 : 0 → 5 : 1
CHCl3–MeOH + 1.5% Et3N). Additional purification of the
product on a LH-20 gel (MeOH) afforded compound 20 (30 mg,
0.013 mmol, 68%); 13C NMR (CDCl3) d 9.00 [(CH3CH2)3N],
23.2, 23.3, 23.5 (CH3CONH), 30.2 (m, C-7, C-7ꢀꢀ, C-7ꢀꢀꢀ), 40.8
(OCH2CH2NH), 47.0 [(CH3CH2)3N], 48.8, 49.6 (C-2, C-2ꢀ, C-2ꢀꢀ,
C-2ꢀꢀꢀ), 59.1, 63.9, 66.8, 71.1, 71.5, 72.3, 72.6, 72.9, 73.4, 73.9,
75.2, 76.1 (C-3–6, C-1ꢀ , 3ꢀ–6ꢀ , C-1ꢀꢀ, C-3ꢀꢀ–6ꢀꢀ, C-1ꢀꢀꢀ, C-3ꢀꢀꢀ–6ꢀꢀꢀ,
PhCH2O, OCH2CH2NH), 100.0 (C-1), 127.3–128.8, 136.7, 137.6,
138.1, 138.5, 138.9 (aromatic C), 156.5 (NHCOOBn), 170.6,
170.7, 171.2 (CH3CONH); 31P NMR (CDCl3) d 18.7 (2P), 21.4;
HRMS: calc. for C101H121N5O29P3 [M + 2H]−: 1960.7366, found:
1960.7297.
2-Aminoethyl 6-O-[C-(6-O-[C-(2-acetamido-2-deoxy-a-D-
mannopyranosyl)methanephosphonate]-2-acetamido-2-deoxy-a-D-
mannopyranosyl)methanephosphonate]-2-acetamido-2-deoxy-a-D-
mannopyranoside bis(triethylammonium) salt (17). To a solution
of 16 (30 mg, 0.018 mmol) in MeOH (4.5 ml), HCl in water (50 lL,
1M) was added followed by a catalytic amount of palladium on
activated carbon. The mixture was hydrogenolysed at 100 psi
overnight, diluted (MeOH) and centrifuged to remove the
activated carbon. Concentration and purification on a reversed
phase silica gel column (H2O) gave 17 (15 mg, 0.015 mmol,
83%) after freeze-drying; [a]D +70◦ (c 0.5, H2O); ); 1H NMR
(D2O) d 2.05 (s, 6H), 2.06 (s, 3H), 2.14–2.18 (m, 4H), 2.71 (s,
2H), 3.26–3.27 (m, 2H), 3.61–3.86 (m, 10H), 3.97–4.23 (m, 10H),
4.38–4.40 (m, 2H), 4.44 (m, 1H), 4.84 (m, 1H); 13C NMR (D2O)
d 8.8 [(CH3CH2)3N], 22.5, 22.7, 23.9 (CH3CONH), 27.8 (d, J =
134 Hz, C-7ꢀ, C-7ꢀꢀ), 39.5 (OCH2CH2NH), 47.1 [(CH3CH2)3N],
52.8 (C-2), 53.2 (d, J = 9.7 Hz, C-2ꢀ, C-2ꢀꢀ), 60.9 (C-6ꢀꢀ), 63.6, 64.0,
66.9, 67.3, 67.8, 69.2, 69.5, 69.7, 72.1, 72.2, 73.2, 74.2, 74.5 (C-3–6,
C-1ꢀ, C-3ꢀ–6ꢀ, C-1ꢀꢀ, C-3ꢀꢀ–5ꢀꢀ, OCH2CH2NH2), 99.4 (C-1), 174.8,
175.4 (CH3CONH); 31P NMR (D2O) d 22.72, 22.75; HRMS: calc.
for C28H51N4O20P2 [M 2+ H]+: 825.2572, found: 825.2608.
2-Aminoethyl 6-O-[C-(6-O-[C-(6-O-[C-(2-acetamido-2-deoxy-
a-D-mannopyranosyl)methanephosphonate]-2-acetamido-2-deoxy-
a-D-mannopyranosyl)methanephosphonate]-2-acetamido-2-deoxy-
a-D-mannopyranosyl)methanephosphonate]-2-acetamido-2-deoxy-
a-D-mannopyranoside tris(triethylammonium) salt (21). A cata-
lytic amount of palladium on activated carbon and HCl (50 lL,
1 M) was added to a solution of compound 20 (29 mg, 0.013 mmol)
in MeOH (4.5 mL). The mixture was hydrogenolysed at 100 psi
overnight, diluted (MeOH) and centrifuged to remove the acti-
vated carbon. Concentration of the supernatant and purification
on a reversed phase silica gel column (H2O) gave 21 (15 mg,
2-(Benzyloxycarbonyl)aminoethyl 6-O-[methyl C-(6-O-[methyl
C-(6-O-[methyl C-(6-O-acetyl-2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-D-mannopyranosyl)methanephosphonate]-2-acetamido-3,4-
di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methanephosphonate]-
2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methane-
phosphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopy-
ranoside (18). Compound 15 (35 mg, 0.023 mmol) and
compound 8 (29 mg, 0.046 mmol) were dissolved in THF
(0.5 mL). To this solution, tris(4-chlorophenyl)phosphine (17 mg,
0.046 mmol), DIAD (9 lL, 0.046 mmol) and Et3N (16 lL,
0.115 mmol) were added. The reaction mixture was stirred under
argon at rt for 30 min. The mixture was then directly purified
by silica gel chromatography (1 : 0 → 20 : 1 CHCl3–MeOH).
Concentration of the fractions containing the product and further
purification on a LH-20 gel column (MeOH) gave 18 (40 mg,
0.020 mmol, 87%); HRMS: calc. for C106H131N5O30P3 [M + H]+:
2046.8086, found: 2046.8000.
0.011 mmol, 85%) after freeze-drying; [a]D +13◦ (c 1.0, H2O); 13
C
NMR (D2O) d 8.6 [(CH3CH2)3N], 22.6, 22.7 (CH3CONH), 27.4,
28.7 (C-7ꢀ, C-7ꢀꢀ, C-7ꢀꢀꢀ), 39.7 (OCH2CH2NH), 47.3 [(CH3CH2)3N],
53.0 (C-2), 53.6 (m, C-2ꢀ, C-2ꢀꢀ, C-2ꢀꢀꢀ), 61.1, 63.8, 64.2, 67.2, 67.4,
67.4, 68.0, 69.4, 69.8, 69.8, 69.9, 73.5, 74.4, 74.5, 74.7 (C-3–6, C-
1ꢀ, C-3ꢀ–6ꢀ, C-1ꢀꢀ, C-3ꢀꢀ–6ꢀꢀ , C-1ꢀꢀꢀ, C-3ꢀꢀꢀ–6ꢀꢀꢀ, OCH2CH2NH), 99.6
(C-1), 175.1, 175.8, 175.9 (CH3CONH); 31P NMR (D2O) d 22.2
(2P), 22.7; HRMS: calc. for C37H65N5O27P3 [M + 2H]−: 1106.3242,
found: 1106.3206.
Acknowledgements
We are thankful to the Swedish Research Council and the National
Research Foundation South Africa for financial support.
2-(Benzyloxycarbonyl)aminoethyl 6-O-[C-(6-O-[C-(6-O-[C-(2-
acetamido-3,4-di-O-benzyl-2-deoxy-a-D-mannopyranosyl)methane-
phosphonate]-2-acetamido-3,4-di-O-benzyl-2-deoxy-a-D-manno-
pyranosyl)methanephosphonate]-2-acetamido-3,4-di-O-benzyl-2-
deoxy-a-D-mannopyranosyl)methanephosphonate]-2-acetamido-3,4-
di-O-benzyl-2-deoxy-a-D-mannopyranoside tris(triethylammonium)
salt (20). To a solution of compound 18 (54 mg, 0.026 mmol) in
MeOH (1.5 mL), KOH (32 lL) from a stock solution of KOH, in
MeOH (1M) was added. After 20 min the mixture was purified
by silica gel chromatography (1 : 0 → 10 : 1 CHCl3–MeOH). The
product was then further purified on a LH-20 gel (MeOH) to give
2-(benzyloxycarbonyl)aminoethyl 6-O-[methyl C-(6-O-[methyl
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The Royal Society of Chemistry 2006
©