Synthesis of Proteophosphoglycans
pyranosyl}phosphate (4). To a mixture of compounds 2 (100
mg, 0.17 mmol) and 3 (62.4 mg, 0.17 mmol) in acetonitrile (2
mL) was added 1H-tetrazole (0.72 mL, 3 wt % solution in
acetonitrile). The mixture was stirred for 4 h at room temper-
ature under an atmosphere of argon. TLC analysis showed the
complete disappearance of the starting material. The reaction
mixture was cooled (-40 °C), a solution of t-BuOOH in decane
(77 µL, 5.5 M) was added dropwise, and stirring was continued
at -40 °C for 3 h. The solvent was evaporated under reduced
pressure, and the residue was purified by a size-exclusion
column chromatograph over Sephadex LH-20 (eluent, CH2Cl2/
MeOH, v/v, 1/1) to give compound 4 (102 mg, 73%) as a
colorless syrup. Rf 0.22 (hexane/EtOAc, 2/3). [R]D +22.3 (c 5.53,
CHCl3). 1H NMR (300 MHz; CD3OD) δH: 7.75 (d, 2H, Ar-H, J
) 7.3 Hz), 7.61 (d, 2H, Ar-H, J ) 6.8 Hz), 7.40-7.20 (m, 9H,
Ar-H), 6.00-5.80 (m, 1H, OCH2CHdCH2), 5.62 (dd, 1H, H-1,
J1,2 ) 1.5 Hz, J1,P ) 7.3 Hz), 5.36-5.04 (m, 7H, H-3, H-2, H-4,
OCH2-CHdCH2, OCH2Ph), 4.66-4.55 (m, 3H, OCH2CHdCH2,
R-CH), 4.48-3.91 (m, 8H, CH2 of Fmoc, CH of Fmoc, OCH2 of
Ser, H-5, H-6, H-6′, diastereomers), 2.10-1.94 (s, 12H, CH3-
CO). 13C NMR (75 MHz; DMSO-d6) δC: 170.6, 170.2, 170.0,
169.6, 132.8, 118.7, 118.5, 95.6, 79.5, 70.6, 68.8, 68.7, 68.4, 67.4,
66.0, 65.3, 63.6, 62.1, 28.7, 21.2, 21.1, 21.0, 19.7. 31P NMR (121
MHz; CDCl3) δP: -3.19, -3.29 (diastereoisomers). Anal. Calcd
for C42H46NO17P: C, 58.13; H, 5.34; N, 1.61. Found: C, 57.96;
H, 5.26; N, 1.61. HR MALDI-TOF MS (m/z): calcd for C42H46-
NO15P, 890.2485 (M + Na); found, 890.2479.
MHz, CDCl3) δC: 171.5, 170.5, 169.6, 169.2, 92.4, 92.3, 71.2,
68.3, 67.9, 61.9, 61.4, 52.9, 52.9, 45.2, 45.1, 44.0, 44.9, 44.3,
44.2, 24.3, 24.2, 24.2, 24.1, 24.0, 23.5, 23.4, 23.0, 22.4, 22.4,
20.8, 20.5, 20.4, 20.4. 31P NMR (121 MHz; CD3OD) δP: 8.60.
HR MALDI-TOF MS (m/z): calcd for C26H48N3O9P, 600.3128
(M + Na); found, 600.3112. (3,4,6-Tri-O-acetyl-2-acetamido-
R-D-glucopyranosyl)bis-N,N-diisopropyl phosphoramidite (389
mg, 0.67 mmol) and benzyl alcohol (66 µL, 0.64 mmol) were
stirred in DCM (4 mL) at room temperature under argon for
5 min. 1H-Tetrazole (1.8 mL, 0.64 mmol) was added dropwise,
and stirring was continued for 2 days. The reaction mixture
was refluxed for 2 h, and then the solvent was evaporated
under reduced pressure. The residue was purified by silica gel
column chromatography (eluent, hexane/EtOAc/Et3N, 75/20/
5, v/v/v) to give 7 as a colorless oil (157 mg, 42%). Rf 0.33
1
(hexane/EtOAc, 3/2). H NMR (300 MHz; CDCl3) δH: 5.25 (d,
1H, NH, J ) 5.4 Hz), 5.40-4.85 (m, 3H, H-3, H-1, H-4), 4.80-
4.45 (m, 2H, CH2Ph), 4.40-4.00 (m, 2H, H-2, H-5, H-6), 3.87
(d, 1H, H-6′, J ) 11.7 Hz), 3.80-3.60 (m, 2H, CH of isopropyl),
2.10-1.84 (s, 12H, CH3CO), 1.30-1.15 (s, 12H, CH3 of isopro-
pyl). HR MALDI-TOF MS (m/z): calcd. for C27H41N2O10P,
607.2499 (M + Na); found, 607.2478. To a mixture of com-
pounds 7 (88 mg, 0.15 mmol) and 3 (55 mg, 0.15 mmol) in DCM
(4 mL) under an atmosphere of nitrogen was added 1H-
tetrazole solution (1.32 mL, 3 wt % solution in acetonitrile).
The reaction mixture was stirred for 12 h, cooled to -40 °C,
t-BuOOH (90 µL, 6 M solution in decane) was added, and
stirring was continued for 2 h. The solvent was removed under
reduced pressure, and the residue was purified by silica gel
column chromatography (eluent, EtOAc/Hexane, 4/1, v/v). The
resulting product was further purified by size-exclusion column
chromatography over Sephadex LH-20 (eluent, MeOH/DCM,
1/1, v/v) to give compound 8 (53 mg, 41%) as an oil. Rf 0.11
(hexane/EtOAc, 1/4). [R]D 28.1 (c 0.15, CH3OH). 1H NMR (500
MHz; CD3OD) δH: 7.73 (d, 2H, Ar-H, J ) 9 Hz), 7.60 (d, 2H,
Ar-H, J ) 9 Hz), 7.40-7.18 (m, 9H, Ar-H), 5.85-5.75 (m, 1H,
OCH2CH ) CH2), 5.70-5.50 (dd, 1H, H-1, J ) 3.4 Hz,
diastereomers), 5.35-4.80 (m, 6H, H-3, H-4, POCH2Ph, OCH2-
CHdCH2), 4.70-3.60 (m, 12H, H-2, OCH2CHdCH2, R-CH,
H-5, H-6, H-6′, OCH2 of Ser, CH2 of Fmoc, CH of Fmoc), 2.02-
1.80 (s, 15H, CH3CO). 13C NMR (75 MHz; CD3OD) δC: 173.4,
172.3, 171.8, 171.2, 170.4, 145.2, 145.2, 142.6, 133.1, 129.6,
129.4, 128.8, 128.8, 128.2, 126.3, 126.2, 120.9, 118.8, 102.9,
74.3, 72.9, 70.2, 69.9, 68.3, 67.2, 63.2, 57.2, 56.0, 56.0, 55.4,
22.7, 20.6, 20.6, 20.5. 31P NMR (121 MHz; CD3OD) δP: -3.05,
-3.07 (diastereomers). HR MALDI-TOF MS (m/z): calcd for
C42H47N2O16P, 889.2663 (M + 2Na); found, 889.2597. Anal.
Calcd for C42H47N2O16P: C, 58.2; H, 5.47; N, 3.23; P, 3.57.
Found: C, 58.19; H, 5.43; N, 3.21; P, 3.54.
NH2AlaPheAlaCONH2 (9). Peptide 9 was synthesized
using Rink Amide Resin under standard automated Fmoc
protocol. Removal of Fmoc was accomplished with a solution
of NMP/Piperidine (80/20, v/v). After completion of the auto-
mated synthesis on a 0.25 mmol scale, the peptide resin was
washed into a peptide synthesis vessel with methanol and
dried under vacuum. This dry peptide resin was kept in DCM
for 2 h and then treated with the cleavage cocktail of CF3-
COOH/H2O/triisopropylsilane (10 mL, 95/2.5/2.5, v/v/v) for 1
h. The resin was removed by filtration, the filtrate was
concentrated under reduced pressure and cooled to 0 °C, and
ice-cold tert-butyl methyl ether (15 mL) was added to give a
thick, white suspension, which was transferred to a polypro-
pylene conical tube. The tube was centrifuged and the ether
decanted. This procedure was repeated twice to give compound
9 as a white precipitate (72 mg). 1H NMR (300 MHz; CD3OD)
δH: 7.23-7.01 (m, 5H, Ar-H), 4.56-4.48 (m, 1H, R-CH), 4.23-
4.01 (m, 1H, R-CH), 3.80-3.69 (m, 1H, R-CH), 3.04 (dd, 1H,
CHHPh of Phe, J ) 14.9, 7.5 Hz), 2.82 (dd, 1H, CHHPh of
Phe, J ) 14.9, 9.4 Hz), 1.33 (d, 3H, CH3 of Ala, J ) 7.6 Hz),
1.20 (d, 3H, CH3 of Ala, J ) 7.5 Hz). 13C NMR (75 MHz; CD3-
OD) δC: 177.1, 172.9, 171.3, 138.2, 130.3, 129.6, 127.9, 56.3,
38.6, 18.3, 17.7.
Benzyl-{{[(S)-2-carboxy-2-(9-fluorenylmethoxycarbo-
nylamino)]ethyl}-2,3,4,6-tetra-O-acetyl-R-D-manno-
pyranosyl}phosphate (5). To a stirred solution of 4 (40 mg,
0.04 mmol) in DCM (0.5 mL) was added a mixture of Pd(PPh3)4
(2 mg, 0.001 mmol), Bu3SnH (24.7 µL, 0.09 mmol), and AcOH
(6 µL, 0.11 mmol) in THF (0.5 mL). The reaction mixture was
stirred at room temperature for 48 h, after which the solvent
was removed by coevaporation with toluene under reduced
pressure. The crude product was purified by silica gel column
chromatography (eluent, MeOH/DCM, 5/95, v/v) to give 5 as a
1
colorless syrup (23 mg, 82%). Rf 0.19 (MeOH/DCM, 5/95). H
NMR (300 MHz, CD3OD) δ: 7.71 (d, 2H, Ar-H, J ) 7.5 Hz),
7.60 (d, 2H, Ar-H, J ) 7.2 Hz), 7.40-7.18 (m, 9H, Ar-H), 5.60,
5.56 (2xbd, 1H, H-1, J ) 6.3, 5.7 Hz, diastereomers), 5.32-
5.14 (m, 3H, H-2, H-3, H-4), 5.12-5.02 (m, 2H, OCH2Ph),
4.50-3.88 (m, 9H, R-CH, CH of Fmoc, CH2 of Fmoc, H-5, H-6,
H-6′, OCH2 of Ser), 2.04, 1.93, 1.92, 1.89 (s, 12 H, CH3CO).
13C NMR (75 MHz, DMSO-d6) δC: 170.7, 170.6, 170.2, 170.2,
170.0, 169.9, 144.6, 144.5, 141.4, 136.5, 129.1, 128.9, 128.7,
128.5, 127.7, 125.8, 120.7, 95.2, 70.1, 69.8, 69.7, 69.6, 69.0, 68.8,
68.6, 68.5, 66.4, 65.3, 65.1, 62.0, 56.7, 47.3, 41.1, 40.8, 40.5,
40.2, 39.9, 39.7, 39.4, 21.2, 21.1, 21.0. 31P NMR (121 MHz, CD3-
OD) δP: -2.39, -2.43 (diastereomers). HR MALDI-TOF MS
(m/z): calcd for C39H42NO17P, 873.6712 (M + 2Na); found,
873.6701. Anal. Calcd for C39H42NO17P: C, 56.59; H, 5.11; N,
1.69; P, 3.74. Found: C, 56.53; H, 5.09; N, 1.67; P, 3.75.
Benzyl-{{[(S)-2-allyloxycarbonyl-2-(9-fluorenylmethoxy-
carbonylamino)]ethyl}-3,4,6-tri-O-acetyl-2-acetamido-r-
D-glucopyranosyl}phosphate (8). A solution of compound
6 (581 mg, 1.67 mmol) and bis-N,N-diisopropyl chlorophos-
phoramidite (424 mg, 1.59 mmol) in DCM (15 mL) was stirred
at room temperature under argon for 5 min. Diisopropylethyl-
amine (2.9 mL) was added dropwise, and stirring was contin-
ued for 2 days. Then, the reaction mixture was heated under
reflux for 2 h after which the solvent was evaporated under
reduced pressure. The residue was purified by silica gel column
chromatography (eluent, hexane/EtOAc/Et3N, 75/20/5, v/v/v)
to give (3,4,6-tri-O-acetyl-2-acetamido-R-D-glucopyranosyl)bis-
N,N-diisopropyl phosphoramidite as a colorless oil (432 mg,
48%). Rf 0.36 (hexane/EtOAc, 3/2). [R]D +30.0 (c 3.9, CHCl3).
1H NMR (300 MHz; CDCl3) δH: 5.57 (d, 1H, NH, J ) 5.4 Hz),
5.24 (t, 1H, H-3, J ) 6.0 Hz), 5.16-5.12 (m, 2H, H-1, H-4),
4.32-4.21 (m, 2H, H-2, H-5), 4.08-4.04 (m, 2H, H-6, H-6′),
3.56-3.48 (m, 4H, CH of isopropyl), 2.06, 2.01, 2.00, 1.91 (4s,
12H, CH3CO), 1.90 (m, 24H, CH3 of isopropyl). 13C NMR (75
J. Org. Chem, Vol. 70, No. 5, 2005 1695