PAPER
Facile Synthesis of a Glycopeptide Building Block of Antifreeze Glycoprotein
1689
peptide 617 in 74% yield. The fully protected glycoside 6
was de-O-acetylated with a catalytic amount of sodium
methoxide in methanol followed by saponification to ob-
tain the desired building block 7 (80%).18 The removal of
Moz-group with trifluoroacetic acid/dichloromethane or
hydrochloric acid/methanol in peptide synthesis is well
documented.9,10 This acidic condition may be hazardous
to the acid-labile glycosidic linkage of the disaccharide.8
The Moz-group of 7 was removed by treatment with for-
mic acid at room temperature for 30 minutes. This condi-
tion was mild enough to leave the glycosidic linkage
intact (as judged by 1H NMR spectrum in the region of the
glycoside bond) and can be applied to a solid phase glyco-
peptide synthesis using disaccharide 7 as a building block.
The mixture was stirred at r.t. After 1 h, a solution of compound 3
(0.70 g, 1.00 mmol) in ClCH2CH2Cl (5 mL) was added at r.t., and
stirred at 60 °C overnight. The resulting solution was diluted with
EtOAc and filtered. The filtrate was washed with aq satd NaHCO3
solution (60 mL), dried (MgSO4), and concentrated in vacuo to give
a brown liquid. The crude product was purified by column chroma-
tography over silica gel (eluent: 8:2 CH2Cl2/EtOAc) to give 5 (0.47
g, 42%) as a foam.
1H NMR (400 MHz, CDCl3): d = 1.02, 1.34, 1.41 (3 d, J = 6.1 Hz,
J = 7.1 Hz, J = 7.2 Hz, 9 H, 2 ¥ Ala-b-CH3, Thr-g-CH3), 1.80, 1.99,
2.00, 2.14 (4 s, 15 H, 5 ¥ COCH3), 3.74 (s, 3 H, CO2CH3), 3.77 (s,
3 H, PhOCH3), 3.88–3.95 (m, 2 H), 4.05–4.38 (m, 9 H), 4.50 (br s,
1 H), 4.61 (m, 1 H), 4.80 (d, 1 H, J = 7.8 Hz, H-1), 4.90–5.10 (m, 3
H, PhCH2, H-3’), 5.17 (dd, 1 H, J = 10.4, 7.8 Hz, H-2’), 5.26 (d, 1
H, J = 3.7 Hz, H-1, overlapped with NH), 5.32 (d, 1 H, J = 3.3, H-
4’), 5.57 (d, 1 H, J = 3.1 Hz, H-4), 6.84 (br d, 1 H, o-MeOC6H4),
7.24 (br d, 1 H, m-MeOC6H4), 7.38 ( br t, 2 Hm, C6H5), 7.50 (br t, 1
Hp, C6H5), 7.97 (br d, 2 Ho, C6H5).
In conclusion, we have described a complete procedure to
obtain Moz-Ala-(Galb1Æ3GalNAca1)Thr-Ala as
a
13C NMR (100 MHz, CDCl3): d = 15.8, 18.4, 18.7, 20.5, 20.6, 20.7,
48.0, 50.7, 52.4, 52.2, 60.5, 60.9, 63.2, 66.7, 66.9, 68.0, 68.8, 69.9,
70.7, 70.9, 74.0, 75.5, 97.8, 101.5, 113.9, 128.1, 128.4, 129.6,
130.0, 133.2, 156.0, 160.6, 166.1,167.5, 169.4, 169.6, 170.2, 170.3,
170.4, 172.1, 173.2.
building block of AFGP. This method can overcome the
previous limitation while using monosaccharide for
AFGP synthesis. Further work on solid phase synthesis is
in progress.
MS: m/z (%) = 1103 ( M++H, 62), 1059 (81), 664 (51), 391 (100),
331 (55).
Column chromatography was performed on silica gel 60 (70–230
and 230–400 mesh, Merck) and preparative HPLC on nucleosil 5
C18. 1,2-Dichloroethane and toluene were dried by CaH2 and dis-
N-(4-Methoxylbenzyloxycarbonyl)-L-alanyl-O-[2,3,4,6-tetra-O-
acetyl-b-D-galacto-pyranosyl-(1Æ3)-2-acetamido-4-O-acetyl-6-
O-benzoyl-2-deoxyl-a-D-galactopyranosyl]-L-threonyl-L-ala-
nine Methyl Ester (6)
To a solution of azide 5 (1.10g, 1.00 mmol) in absolute MeOH (50
mL) under argon were added propane-1,3-dithiol (1.0 mL, 10
mmol) and Et3N (1.4 mL, 10 mmol) at r.t. The mixture was stirred
at r.t. for 24 h. The solvent and excess reagents were removed under
vacumn, and the residue was treated with pyridine (20 mL) and
Ac2O (5 mL). After 1 h, the solution was concentrated in vacuo and
the crude product was purified by column chromatography over sil-
ica gel (eluent:2:8 CH2Cl2/EtOAc ) to yield 6 (0.83 g, 74%) as a col-
orless foam.
1H NMR (400 MHz, CDCl3): d = 1.11, 1.35, 1.40 (3 d, J = 6.2 Hz,
J = 7.0 Hz, J = 7.1 Hz, 9 H, 2 ¥ Ala-b-CH3, Thr-g-CH3), 1.94, 1.97,
1.99, 2.09, 2.12, 2.13 (6 s, 18 H, 5 ¥ COCH3), 3.70 (s, 3 H,
CO2CH3), 3.76 (s, 3 H, PhOCH3), 3.95–4.15 (m, 3 H), 4.15–4.40
(m, 5 H), 4.40–4.58 (m, 3 H), 4.60 (d, 1 H, J = 7.8 Hz, H-1’), 4.88–
5.10 (m, 5 H, PhCH2, H-1, 2’, 3’), 5.27 (d, 1 H, J = 3.0 Hz, H-4’),
5.44 (d, 1 H, J = 2.5 Hz, H-4), 5.62 (br d, 1 H, J = 6.5 Hz, NH), 6.68
(br d, 1 H, J = 8.5 Hz, NH), 6.82 (br d, 2 H, o-MeOC6H4), 7.11 (br
d, 1 H, J = 5.5 Hz, NH), 7.21 (d, 2 H, m-MeOC6H4), 7.36 ( br t, 2
Hm, C6H5), 7.49 (br t, 1 Hp, C6H5), 7.94 (br d, 2 Ho, C6H5).
13C NMR (100 MHz, CDCl3): d = 17.6, 17.7, 17.8, 20.7, 20.7, 23.0,
48.4, 49.1,50.8, 52.8, 55.2, 56.2, 60.6, 63.4, 66.7, 67.0, 67.8, 68.6,
69.3, 70.3, 70.7, 73.4, 75.6, 99.1, 101.2, 113.9, 114.0, 127.7, 128.4,
129.5, 129.6, 129.7, 129.9, 133.2, 156.5, 159.7, 166.4, 169.4, 170.2,
170.3, 170.4, 170.5, 172.8, 173.4.
1
tilled. The H NMR spectra were recorded on a Bruker ASPECT
3000 (400 MHz) spectrometer. The value of d are expressed in ppm
relative to the solvent signal as internal standard (CHCl3, 7.24 ppm;
HOD, 4.7 ppm). Mass spectra were measured with Autospec (mi-
cromass, UK). All reagents are commerically available.
2,3,4,6-Tetra-O-acetyl-b-D-galactopyranosyl-(1Æ3)-4-O-acetyl-
2-azido-6-O-benzoyl-2-deoxyl-a-D-galactopyranosyl Chloride
(3)
Compound 2 (0.68 g, 1.00 mmol) and MeSO2Cl ( 0.16 mL, 2.00
mmol) were dissolved in CH2Cl2 (5 mL) at 0 °C. Et3N (0.42 mL,
3.00 mmol) was added dropwise under N2. After 36 h, the mixture
was diluted with EtOAc (60 mL), the solution was washed with aq
satd NaHCO3 solution (50 mL), dried (MgSO4), and concentrated in
vacuo to give a yellow liquid. The crude product was purified by
column chromatography over silica gel (eluent:7:3 hexane/EtOAc)
to give 3 (0.60 g, 85%) as a colorless foam.
1H NMR (400 MHz, CDCl3): d = 1.96, 2.03, 2.07, 2.13 (4 s, 15 H,
5 COCH3), 3.91 (t, J = 6.6 Hz, 1 H, H-5), 4.04–4.18 (m, 4 H, H-3,
6), 4.33 (d, 2 H, J = 6.1 Hz, H-6’), 4.53 (t, 1 H, J = 6.1 Hz, H-5’),
4.73 (d, 1 H, J = 7.8 Hz, H-1), 4.98 (dd, 1 H, J = 10.4, 3.3 Hz, H-
3’), 5.17 (dd, 1 H, J = 10.4, 7.8 Hz, H-2’), 5.34 (d, 1 H, J = 3.3, H-
4’), 5.62 (d, 1 H, J = 3.1 Hz, H-4), 6.17 (d, 1 H, J = 3.7 Hz, H-1),
7.42 ( br t, 2 Hm, C6H5), 7.55 (br t, 1 Hp, C6H5), 7.99 (br d, 2 Ho,
C6H5).
13C NMR (100 MHz, CDCl3): d = 20.5, 30.6, 20.8, 60.7, 61.0, 62.3,
66.7, 68.7, 70.7, 71.0, 74.8, 93.1, 101.3, 128.4, 129.4, 129.7, 133.3,
166.0, 169.3, 169.5, 170.1, 170.3, 170.4.
MS: m/z (%) = 700 ( M++H, 22), 698 (40), 674 (65), 664 (58), 656
(42), 640 (52), 331 (100).
MS: m/z (%) = 1119 ( M++H, 67), 1141 (M++Na, 88), 1077 (43),
955 (25), 789 (41), 680 (72), 331 (100).
N-(4-Methoxylbenzyloxycarbonyl)-L-alanyl-O-[b-D-galactopy-
ranosyl-(1Æ3)-2-acetamido-2-deoxyl-a-D-galactopyranosyl]-L-
threonyl-L-alanine (7)
Compound 6 (0.56 g, 0.50 mmol) was dissolved in MeOH (15 mL).
A 0.5 M solution of NaOMe in MeOH was added dropwise until
pH = 10.5 (pH paper). The reaction was complete after 6 h. The
mixture was neutralized with AcOH and the solvents were removed
in vacuo. The crude product in MeOH (3 mL) was cooled in an ice-
N-(4-Methoxylbenzyloxycarbonyl)-L-alanyl-O-[2,3,4,6-tetra-O-
acetyl-b-D-galacto-pyranosyl-(1Æ3)-4-O-acetyl-2-azido-6-O-
benzoyl-2-deoxyl-a-D-galactopyranosyl]-L-threonyl-L-alanine
Methyl Ester (5)
Tripeptide 4 (1.32g, 3.00 mmol), tetramethylurea (0.48 mL, 4.00
mmol), AgOTf (0.77 g, 3.00 mmol) were dissolved in anhyd
ClCH2CH2Cl/toluene (1:2, 15 mL) in the presence of Drierite (4 g).
Synthesis 1999, No. 9, 1687–1690 ISSN 0039-7881 © Thieme Stuttgart · New York