564 J . Org. Chem., Vol. 61, No. 2, 1996
Sjo¨lin et al.
[CDCl3 (δC 77.0) as internal standard], MeOH-d4 [CD3OD (δC
49.15)], or acetone-d6 [CD3COCD3 (δC 29.92)]. Ions for positive
fast atom bombardment mass spectra were produced by a
beam of Xe atoms (6 keV) from a matrix of glycerol and
thioglycerol.
Analytical and preparative HPLC separations were per-
formed on Kromasil C-8 columns (100 Å, 50 µm, 4.6 or 20 ×
250 mm, respectively) with flow rates of 1.5 or 11 mL/min,
detection at 214 nm, and the solvent systems A, aqueous 0.1%
CF3CO2H; and B, 0.1% CF3CO2H in MeCN. The homogeneity
of the glycopeptides was determined by analytical HPLC using
a linear gradient of 0-100% B in A during 60 min.
mixture was filtered and concentrated. Flash column chro-
matography (heptane-ethyl acetate, 2:1) of the residue gave
1
5 (108 mg, 82%): [R]25 +23° (c 0.26, CDCl3); H NMR (500
D
MHz, CDCl3) δ 6.25 (d, J ) 8.8 Hz, 1H, NH), 5.27 (d, J ) 2.8
Hz, 1H, H-4), 5.11 (dd, J ) 8.0, 10.4 Hz, 1H, H-2), 4.96 (dd, J
) 3.3, 10.4 Hz, 1H, H-3), 4.74 (dd, J ) 5.7, 10.9 Hz, 1H,
OCOCH2CH), 4.64 (dd, J ) 1.8, 8.7 Hz, 1H, H-R), 4.55 (dd, J
) 5.4, 10.9 Hz, 1H, OCOCH2CH), 4.23 (t, J ) 5.5 Hz, 1H,
OCOCH2CH), 4.19 (d, J ) 7.9 Hz, 1H, H-1), 4.00 (m, 1H, H-â),
3.91 (m, 2H, H-6,6′), 3.36 (bt, J ) 6.4 Hz, 1H, H-5), 2.13, 2.04,
1.99, and 1.77 (4s, each 3H, Ac), 1.43 (d, J ) 6.6 Hz, 3H, H-γ);
13C NMR (75 MHz, CDCl3) δ 170.2, 170.2, 169.0, 164.8, 155.8,
143.8, 143.7, 141.6, 141.3, 127.9, 127.2, 127.1, 124.9, 124.6,
120.0, 101.0, 78.5, 70.7, 70.7, 68.6, 66.9, 66.2, 61.7, 58.4, 47.3,
20.6, 20.1, 17.9; MS (FAB): calcd for C39H37NO14F5 838 (M +
H+), found 838. Anal. Calcd for C39H36NO14F5: C, 55.9; H,
4.3; N, 1.7. Found: C, 56.2; H, 4.1; N, 1.6.
N-(9-Fluorenylmethoxycarbonyl)-D-allo-threonine23 (Fmoc-
D-allo-Thr), 2,3,4,6-tetra-O-acetyl-R-D-galactopyranosyl bro-
mide11 (1), and N-(9-fluorenylmethoxycarbonyl)-L-threonine
pentafluorophenyl ester10 (2) were prepared according to the
indicated literature methods.
Nr-Acetyl-L-p h en yla la n yl-3-O-(2,3,4,6-tetr a -O-a cetyl-â-
D-ga la ctop yr a n osyl)-L-th r eon yl-L-a la n in e Am id e (6), Nr-
Acetyl-D-p h en yla la n yl-3-O-(2,3,4,6-tetr a -O-a cetyl-â-D-ga -
lactopyr an osyl)-L-th r eon yl-L-alan in e Am ide (7), Nr-Acetyl-
L -p h e n yla la n yl-3-O-(2,3,4,6-t e t r a -O-a ce t yl-â-D -ga la c-
top yr a n osyl)-D-a llo-th r eon yl-L-a la n in e Am id e (8), a n d
Nr-Acetyl-L-p h en yla la n yl-3-O-(2,3,4,6-tetr a -O-a cetyl-â-D-
ga la ctop yr a n osyl)-L-th r eon yl-D-a la n in e Am id e (9). The
acetylated glycopeptides 6-9 were prepared, as described
previously,13 by manual Fmoc solid phase synthesis on an
aminomethylated polystyrene resin functionalized with the
Rink linker. Cleavage from the resin was effected with TFA-
H2O, 9:1, and glycopeptides 6-9 were obtained in 45-60%
yields after purification with reversed-phase HPLC. HRMS
(FAB): calcd for C32H45N4O14 709.2932 (M + H+), found for 6:
709.2941, 7: 709.2916, 8: 709.2930, and 9: 709.2940. 1H
NMR data for compounds 6-9 are given in Table 1.
Nr-Acetyl-L-p h en yla la n yl-3-O-(â-D-ga la ctop yr a n osyl)-
L-th r eon yl-L-a la n in e Am id e (10), Nr-Acetyl-D-p h en yla la -
n yl-3-O-(â-D-galactopyr an osyl)-L-th r eon yl-L-alan in e Am ide
(11), Nr-Acetyl-L-p h en yla la n yl-3-O-(â-D-ga la ctop yr a n o-
syl)-D-a llo-th r eon yl-L-a la n in e Am id e (12), a n d Nr-Acetyl-
L-p h en yla la n yl-3-O-(â-D-ga la ctop yr a n osyl)-L-th r eon yl-D-
a la n in e Am id e (13). Deacetylation of 6-9 with 6 mM
methanolic sodium methoxide as described below and purifica-
tion by reversed-phase HPLC gave the glycopeptides 10-13
in quantitative yields. HRMS (FAB): calcd for C24H37N4O10
541.2510 (M + H+), found for 10: 541.2513, 11: 541.2507,
12: 541.2501, and 13: 541.2507. 1H NMR data for compounds
10-13 are given in Table 2.
N R-Acetyl-L-phenylalanyl-(2-amino-Z-but-2-enoyl)-L-ala-
nine Amide (14). Methanolic sodium methoxide (2 M, 1500
µL) was added to glycopeptide 6 (31 mg, 44 µmol) in MeOH
(31 mL). The time-course of the reaction was monitored by
reversed-phase analytical HPLC, and after 16 h the solution
was neutralized with Duolite (H+) resin. Purification by
reversed-phase HPLC gave 10 (4.1 mg, 7.6 µmol) and 14 (3.0
mg, 8.3 µmol). Compound 14: 1H NMR (500 MHz, acetone-
d6) δ Phe 7.80 (bs, 1H, NH), 4.49 (q, J ) 6.8 Hz, 1H, H-R),
3.18 (dd, AB-type, J ) 6.9, 13.8 Hz, 1H, H-â), 3.07 (dd, AB-
type, J ) 8.1, 13.8 Hz, 1H, H-â′), 1.93 (s, 3H, Ac); ∆Aba 8.67
(bs, 1H, NH), 6.51 (q, J ) 7.0 Hz, 1H, H-â), 1.49 (d, J ) 7.1
Hz, 1H, H-γ); Ala 7.52 (bd, J ) 6.7 Hz, 1H, NH), 6.99 and
6.20 (2 bs, 2H, CONH2), 4.33 (qv, J ) 7.3 Hz, 1H, H-R), 1.34
(d, J ) 7.3 Hz, 1H, H-â).
Meth od s Used for Dea cetyla tion of 6. (A) Hydrazine
hydrate:14 A solution of 6 (5 mg) in N2H4‚H2O-MeOH (1:5, 5
mL) was stirred at 20 °C for 3 h. Acetone was then added,
and the solution was concentrated. (B) Ammonia:8 A solution
of 6 (5.5 mg) in methanolic ammonia (8 mL, saturated at 0
°C) was stirred in a stoppered flask at 20 °C for 6 h and then
concentrated. (C) Sodium methoxide:8 Methanolic sodium
methoxide (2 M, 15 µL) was added to a solution of 6 (5 mg) in
MeOH (5 mL) so that the pH reached 10.5-11 (wet pH paper).
After 30 min at 20 °C the solution was neutralized with Duolite
(H+) resin, filtered, and concentrated. (D) Sodium hydroxide:
A solution of 6 (5 mg) in aqueous 0.1 M sodium hydroxide (5
N-(9-Flu or en ylm eth oxycar bon yl)-D-a llo-th r eon in e P en -
ta flu or op h en yl Ester (3). 1,3-Diisopropylcarbodiimide (570
µL, 1.62 mmol) was added to Fmoc-D-allo-Thr (429 mg, 1.26
mmol) and pentafluorophenol (680 mg, 1.62 mmol) in ethyl
acetate (15 mL) at 0 °C. After 1 h the mixture was allowed to
attain room temperature during 2 h before removal of pre-
cipitated diisopropylurea by filtration. The solution was
diluted with CH2Cl2 (85 mL) and washed with saturated
aqueous NaHCO3 (100 mL). The aqueous phase was extracted
with CH2Cl2 (70 mL), and the combined organic phases were
dried, filtered, and concentrated. Flash column chromatog-
raphy (heptane-ethyl acetate, 1:1) of the residue gave 3 (0.48
g, 75%): [R]25 +20° (c 0.61, CDCl3); 1H NMR (300 MHz,
D
CDCl3) δ 5.67 (bs, 1H, NH), 4.76 (bs, 1H, H-R), 4.33 (bs, 1H,
H-â), 4.51 (d, J ) 6.7 Hz, 2H, OCOCH2CH), 4.26 (t, J ) 6.7
Hz, 1H, OCOCH2CH), 1.39 (bd, J ) 5.4 Hz, 3H, H-γ); 13C NMR
(75 MHz, CDCl3) δ 166.6, 156.2, 143.6, 143.5, 141.4, 127.8,
127.1, 125.0, 120.3, 68.6, 67.7, 59.4, 47.1, 19.2. Anal. Calcd
for C25H18NO5F5: C, 59.2; H, 3.6; N, 2.8. Found: C, 59.4; H,
3.6; N, 2.6.
N-(9-F lu or en ylm eth oxyca r bon yl)-3-O-(2,3,4,6-tetr a -O-
acetyl-â-D-galactopyr an osyl)-L-th r eon in e P en taflu or oph e-
n yl Ester (4). Silver triflate (0.64 g, 2.5 mmol) was added to
111 (1.10 g, 2.68 mmol), 210 (0.833 g, 1.64 mmol), and molecular
sieves (4 Å, 1.1 g) in dry CH2Cl2 (15 mL) at 0 °C. After 40
min the mixture was allowed to attain room temperature
during 70 min, and it was then diluted with CH2Cl2 (50 mL)
and filtered. The solution was washed with saturated aqueous
NaHCO3 (80 mL), the aqueous phase was extracted with CH2-
Cl2 (30 mL), and the combined organic phases were dried,
filtered, and concentrated. Flash column chromatography
(heptane-ethyl acetate, 3:1) of the residue gave 4 (0.78 g,
57%): [R]25 -24° (c 0.30, CDCl3); 1H NMR (300 MHz, CDCl3)
D
δ 5.73 (d, J ) 9.0 Hz, 1H, NH), 5.38 (d, J ) 2.9 Hz, 1H, H-4),
5.18 (dd, J ) 7.8, 10.3 Hz, 1H, H-2), 5.04 (dd, J ) 3.4, 10.5
Hz, 1H, H-3), 4.72 (dd, J ) 2.5, 9.1 Hz, 1H, H-R), 4.58 (dd, J
) 2.7, 6.1 Hz, 1H, H-â), 4.52 (d, J ) 7.8 Hz, 1H, H-1), 4.50
(dd, AB-type, J ) 7.2, 10.8 Hz, 1H, OCOCH2CH), 4.43 (dd,
AB-type, J ) 7.4, 10.5 Hz, 1H, OCOCH2CH), 4.27 (t, J ) 7.1
Hz, 1H, OCOCH2CH), 4.03 (m, 2H, H-6,6′), 3.89 (t, J ) 6.4
Hz, 1H, H-5), 2.17, 2.07, 2.01, and 2.00 (4s, each 3H, Ac), 1.31
(d, J ) 6.2 Hz, 3H, H-γ); 13C NMR (75 MHz, CDCl3) δ 170.3,
170.3, 170.1, 169.4, 166.3, 156.5, 143.8, 143.6, 141.3, 127.8,
127.1, 125.1, 120.0, 98.6, 73.7, 70.7, 70.6, 69.0, 67.4, 66.7, 60.8,
58.6, 47.2, 20.8, 20.6, 20.5, 20.4, 16.7; MS (FAB): calcd for
C39H37NO14F5 838 (M + H+), found 838. Anal. Calcd for
C39H36NO14F5: C, 55.9; H, 4.3; N, 1.7. Found: C, 55.9; H, 4.4;
N, 1.4.
N-(9-F lu or en ylm eth oxyca r bon yl)-3-O-(2,3,4,6-tetr a -O-
a cetyl-â-D-ga la ctop yr a n osyl)-D-a llo-th r eon in e P en ta flu o-
r op h en yl Ester (5). A solution of 111 (110 mg, 0.268 mmol)
and 3 (80 mg, 0.158 mmol) in CH2Cl2 (5 mL) was stirred with
molecular sieves (3 Å, 200 mg) for 1 h at 20 °C under argon.
Silver silicate12 (250 mg, activated under vacuum at 100 °C
for 48 h) was then added and after a further 4 h at 20 °C the
(23) Chang, C.-D.; Waki, M.; Ahmad, M.; Meienhofer, J .; Lundell,
E. O.; Haug, J . D. Int. J . Pept. Protein Res. 1980, 15, 59-66.