8774 J. Am. Chem. Soc., Vol. 119, No. 38, 1997
Seitz and Wong
24H), 3.49 (m, 1H, H-3′), 3.38-3.36 (m, 1H, H-4′, H-5′), 2.96 (t, 2H,
Kꢀ, Jꢀ,δ ) 7.5), 2.82 (dd, 1H, Nâa, Jâa,âb ) 15.5, Jâa,R ) 6.5), 2.73 (dd,
U/mL, 1 µL) were added. The mixture was shaken at 37 °C for 16 h
and directly subjected to GPC (Biogel P4, 0.05M NH4OAc). After
lyophilization 4.4 mg of a white fluffy powder were obtained. Yield:
99%. tR: 19.3 min [Vydac C18, 250 × 4 mm, 0 min (0% B) and 1
min (0% B) and 30 min (20% B), A ) 2% MeCN, 0.1% TFA in H2O,
B ) 2% H2O, 0.1% TFA in MeCN)]. ESI-MS (pos): 1789 (M (1 ×
13C) + H+), calcd 1789.0.
1H, Nâb, Jâb,âa ) 15.5, Jâb,R ) 7.0), 2.42 (t, 2H, HYCRONH-2, J2,3
)
6.7), 2.34-2.21 (m, 2H, 2 × Pâa), 2.01-1.91 (m, 12H, 2 × Ac, 2 ×
Pγ, 2 × Pâb), 1.78-1.71 (m, 1H, Kâa), 1.67-1.62 (m, 3H, Kδ, Kâb),
1.43-1.37 (m, 5H, Kγ, Aâ, Jâ,R ) 7.2), 1.19 (d, 3H, Tγ, Jγ,â ) 6.3),
1.12 (d, 3H, Tγ, Jγ,â ) 6.3). ESI-MS (pos): 1357 (M + Na+) calcd
1356.8, 1335 (M + H+) calcd 1334.8.
Ac-Lys-Pro-Pro-Asn-Thr(Sialr2f3Galâ1f4(Fucr1f3)GlcNAcâ)-
Thr-Ser-Ala-HYCRON (26). To a solution of 24 (2.5 mg, 1.3 µmol)
and GDP-Fuc (1.2 mg, 1.9 µmol) in 200 µL of buffer (0.05 M MES,
15 mM MnCl2, pH 6.0), R1,3-Fucosyltransferase (Cytel, 2.16 U/mL,
13 µL) and alkaline phosphatase (Boehringer Mannheim, 1 U/mL, 0.5
µL) were added. The mixture was shaken at 37 °C for 1 d before
GDP-Fuc (1.0 mg, 1.6 µmol) and fucosyltransferase (10 µL, 22 mU)
were added a second time. The mixture was shaken for 3 more days
at 37 °C. Purification was achieved by GPC using a Biogel P2 column
(0.05 M NH4OAc) followed by a second GPC with a Biogel P4 column
(0.05 M NH4OAc). The product fractions were lyophilized to yield
1.4 mg of a white fluffy powder. Yield: 55%. tR: 30.5 min [Microsorb,
C18, 5µ, 250 × 4 mm, 0 min (0% B) and 2 min (0% B) and 30 min
(10% B) and 40 min (40% B), A ) 2% MeCN, 0.1% TFA in H2O, B
) 2% H2O, 0.1% TFA in MeCN)]. 1H NMR (500 MHz, D2O,
characteristic signals): 5.86 (m, 2H, HYCRONH-15, HYCRONH-16),
5.05 (mc, 1H, FucH-1), 2.96 (t, 2H, Kꢀ, Jꢀ,δ ) 7.5), 2.87-2.81 (m, 1H,
Nâa), 2.77-2.70 (m, 2H, Nâb, SialH-3a), 2.42 (t, 2H, HYCRONH-2, J2,3
) 6.7), 2.34-2.21 (m, 2H, 2 × Pâa), 2.00, 1.98, 1.97 (3 × s, 3 × Ac),
1.39, 1.28, 1.21, 1.13 (4 × d, 2 × Tγ, Aâ, FucH-6). ESI-MS (neg):
1954 (M (1 × 13C) + Na+ - 2H+), calcd 1954.1), 1933 (M (1 × 13C)
- H+), calcd 1933.1, 1641 (M - Sial - H+), calcd 1641.0.
Ac-Lys-Pro-Pro-Asn-Thr(Galâ1f4GlcNAcâ)-Thr-Ser-Ala-OH
(23). A solution of 18 (6.8 mg, 6.4 µmol), UDP-Gal (5.1 mg, 8.2 µmol)
and 1 mg (1 U) of â1,4-galactosyltransferase (Sigma) in 550 µL of
buffer (50 mM HEPES, 5 mM MnCl2, pH 7.0) was shaken for 21 h at
37 °C. After lyophilization the residue was purified by GPC (Biogel
P2, 0.1M NH4HCO3). Lyophilization of the product fractions gave
7.8 mg of a white fluffy powder. Yield: 100%. tR: 14.4 min [Vydac
C18, 250 × 4 mm, 0 min (0% B) and 2 min (0% B) and 30 min (10%
B), A ) 2% MeCN, 0.1% TFA in H2O, B ) 2% H2O, 0.1% TFA in
MeCN)]. ESI-MS (pos): 1245 (M + Na+), calcd 1245.7, 1223 (M +
H+), calcd 1223.7.
Ac-Lys-Pro-Pro-Asn-Thr(âGlcNAc)-Thr-Ser-Ala-OH (18).
A
solution of 15 (18.0 mg, 10.99 µmol) in dry methanol (1 mL) was
adjusted to pH 8.5 by addition of a 0.1 M solution of sodium methylate
in methanol. After 2 h of stirring, the solution was acidified to pH 6
by addition of acetic acid. The solution was concentrated in Vacuo to
give 17.6 mg of material which was dissolved in TFA/ethanedithiol/
anisole (40:1:1, 6.3 mL) and stirred for 1.5 h. The mixture was
concentrated in Vacuo, and the residue was dissolved in MeOH (3 mL).
The product precipitates upon addition of ether. The precipitate was
collected and purified by GPC (Biogel P2, 0.1M NH4HCO3). Lyo-
philization of the product fraction gave 10.2 mg of a white fluffy
powder. Yield: 88%. tR: 16.0 min (Vydac C18, 250 × 4 mm, 0 min
(0% B) and 2 min (0% B) and 30 min (10% B), A ) 2% MeCN, 0.1%
TFA in H2O, B ) 2% H2O, 0.1% TFA in MeCN)]. 1H NMR (500
MHz, 1H,1H-COSY, D2O): 4.71-4.68 (m, 2H, NR, PR), 4.56-4.54 (m,
3H, H-1′, TR, KR), 4.45-4.37 (m, 3H, SR (4.44), TR (4.38), PR), 4.30
(mc, 1H, Tâ), 4.24 (mc, 1H, Tâ), 4.11 (q, 1H, AR, JR,â ) 7.2), 3.88-
3.77 (m, 5H, H-6′a, Sâ, Pꢀa), 3.72-3.60 (m, 4H, H-6′b, H-2′, Pꢀb), 3.51
(mc, 1H, H-3′), 3.39 (mc, 2H, H-4′, H-5′), 2.97 (t, 2H, Kꢀ, Jꢀ,δ ) 7.5),
2.83 (dd, 1H, Nâa, Jâa,âb ) 15.6, Jâa,R ) 6.6), 2.74 (dd, 1H, Nâb, Jâb,âa
) 15.7, Jâb,R ) 7.2), 2.34 (mc, 1H, Pâa), 2.26 (mc, 1H, Pâa), 2.03-1.93
(m, 10H, 2 × Ac, 2 × Pγ), 1.90-1.87 (m, 2H, 2 × Pâb), 1.75 (mc, 1H,
Kâa), 1.66 (mc, 3H, Kâb, Kδ), 1.45 (mc, 2H, Kγ), 1.30 (d, 3H, Aâ, Jâ,R
) 7.2), 1.21 (d, 3H, Tγ, Jγ,â ) 6.2), 1.14 (d, 3H, Tγ, Jγ,â ) 6.2). ESI-
MS (neg): 1058 (M - H+) calcd 1058.6.
Lipase WG-Catalyzed Removal of the HYCRON Linker. Ac-
Lys-Pro-Pro-Asn-Thr(âAc3GlcNAc)-Thr-Ser-Ala-OH (21). The gly-
copeptide 16 (12.6 mg, 8.0 µmol) was dissolved in a solution of lipase
WG (Sigma, EC 3.1.1.3, 10 U/mL) in phosphate buffer (0.2 M NaH2-
PO4, 10 U/mL, pH 7.0, 3.1 mL) and shaken at 37 °C for 2 d. After
lyophilization the crude was purified by GPC (50 mM NH4OAc). The
product fractions were lyophilized to yield 7.3 mg of 20. Yield: 77%.
tR: 18.3 min [Microsorb C18, 250 × 4 mm, 0 min (0% B) and 1 min
(0% B) and 30 min (20% B), A ) 2% MeCN, 0.1% TFA in H2O, B
) 2% H2O, 0.1% TFA in MeCN)]. 1H NMR (500 MHz, D2O): 5.21
(t, 1H, H-3′, J3′,2′ ) J3′,4′ ) 9.6), 4.97 (t, 1H, H-4′, J4′,3′ ) J4′,5′ ) 9.7),
4.71-4.67 (m, 2H, PR, NR), 4.57 (d, 1H, TR, J ) 3.7), 4.54 (dd, 1H,
KR, J1 ) 5.3, J2 ) 8.6), 4.45 (t, 1H,SR, JR,â ) 5.3), 4.41-4.36 (m, 3H,
H-1′, TR, PR), 4.31-4.24 (m, 2H, 2 × Tâ), 4.13-4.19 (m, 2H, AR,
Ac-Lys-Pro-Pro-Asn-Thr(Sialr2f3Galâ1f4GlcNAcâ)-Thr-Ser-
Ala-OH (25). R 2,3-Sialyltransferase (Cytel, 3 U/mL, 15 µL) and
CMP-NeuNAc (4.9 mg, 7.4 µmol) were added to a solution of 23 (7.1
mg, 5.8 µmol) in 820 µL of buffer (0.1 M HEPES, 5 mM MnCl2, 0.2%
Triton X-100, pH 7.0). The mixture was shaken at 37 °C for 29 h
before alkaline phosphatase (Boehringer Mannheim, 1 U/mL, 2.5 µL)
was added. A second addition of CMP-NeuNAc (3.6 mg, 5.4 µmol)
and sialyltransferase (15 µL, 45 mU) was added after 46 h. The mixture
was lyophilized after two more days. Twofold GPC (Biogel P4, 0.1M
NH4HCO3) and lyophilization of the product fractions gave 6.7 mg of
product and 1.4 mg (20%) of starting material as white fluffy powders.
Yield: 77%. tR: 13.1 min [Vydac C18, 250 × 4 mm, 0 min (0% B)
and 2 min (0% B) and 30 min (10% B), A ) 2% MeCN, 0.1% TFA
in H2O, B ) 2% H2O, 0.1% TFA in MeCN)]. 1H NMR (500 MHz,
H-6′a), 3.94-3.76 (m, 7H), 3.65-3.58 (m, 2H), 2.97 (t, 2H, Kꢀ, Jꢀ,δ
)
7.5), 2.82 (dd, 1H, Nâa, Jâa,âb ) 15.7, Jâa,R ) 6.7), 2.73 (dd, 1H, Nâb,
Jâb,âa ) 15.7, Jâb,R ) 7.2), 2.36-2.22 (m, 2H, 2 × Pâa), 2.07-1.95 (m,
19H, 5 × Ac, 2 × Pγ), 1.92-1.86 (m, 2H, 2 × Pâb), 1.80-1.72 (m,
1H, Kâa), 1.69-1.59 (m, 3H, Kδ, Kâb), 1.49-1.39 (m, 5H, Kγ), 1.30
(d, 3H, Aâ, Jâ,R ) 7.1), 1.21 (d, 3H, Tγ, Jγ,â ) 6.4), 1.15 (d, 3H, Tγ,
Jγ,â ) 6.4). MALDI-MS (R-cyanocinnamic acid): 1186 (M + H+),
calcd 1186.7.
D2O): 4.72-4.68 (m, 2H, NR, PR), 4.58-4.50 (m, 4H, GlcNAcH1, GalH1
,
Enzymatic Solution-Phase Synthesis of O-SLex Octapeptides.
Ac-Lys-Pro-Pro-Asn-Thr(Galâ1f4GlcNAcâ)-Thr-Ser-Ala-
HYCRON (22). A solution of 17 (9.5 mg, 7.1 µmol), UDP-Gal (5.3
mg, 9.0 µmol), and â1,4-galactosyltransferase (Sigma, 1 mg, 1 U) in
600 µL of buffer (50 mM HEPES, 5 mM MnCl2, pH 7.0) was shaken
for 16 h at 37 °C. After lyophilization the residue was purified by
GPC (Biogel P4, 0.05M NH4OAc). Lyophilization of the product
fractions furnishes 9.3 mg of a white fluffy powder. Yield: 87%. tR:
20.2 min [Vydac C18, 250 × 4 mm, 0 min (0% B) and 1 min (0% B)
and 30 min (20% B), A ) 2% MeCN, 0.1% TFA in H2O, B ) 2%
H2O, 0.1% TFA in MeCN)]. ESI-MS (neg): 1495 (M - H-), calcd
1494.9.
TR, KR), 4.45 (t, 1H, SR, JR,â ) 5.4), 4.41 (dd, 1H, PR, JR,â1 ) 8.4, JR,â2
) 5.3), 4.38 (d, 1H, TR, JR,â ) 4.5), 4.29 (dq, 1H, Tâ, Jâ,γ ) 6.3, Jâ,R
) 4.0), 4.24 (dq, 1H, Tâ, Jâ,γ ) 6.3, Jâ,R ) 4.6), 4.11 (q, 1H, AR, JR,â
) 7.2), 4.08 (dd, 1H, J1 ) 9.9, J2 ) 3.0), 3.96-3.76 (m, 10H), 3.72-
3.52 (m, 14H), 2.98 (t, 2H, Kꢀ, Jꢀ,δ ) 7.5), 2.84 (dd, 1H, Nâa, Jâa,δb
)
15.6, Jâa,R ) 6.6), 2.78-2.69 (m, 2H, Nâb, SialH3a), 2.35 (mc, 1H, Pâa),
2.27 (mc, 1H, Pâa), 2.03-1.96 (m, 13H, 3 × Ac, 2 × Pγ), 1.94-1.86
(m, 2H, 2 × Pâb), 1.79-1.74 (m, 2H, Kâa, SialH3b), 1.68-1.65 (m, 3H,
Kâb, Kδ), 1.47-1.43 (mc, 2H, Kγ), 1.32 (d, 3H, Aâ, Jâ,R ) 7.2), 1.22
(d, 3H, Tγ, Jγ,â ) 6.4), 1.14 (d, 3H, Tγ, Jγ,â ) 6.4). MALDI-MS (R-
cyanocinnamic acid): 1536 (M + Na+), calcd 1536.8, 1514 (M + H+),
calcd 1513.8.
Ac-Lys-Pro-Pro-Asn-Thr(Sialr2f3Galâ1f4GlcNAcâ)-Thr-Ser-
Ala-HYCRON (24). To a solution of 22 (3.7 mg, 2.5 µmol) and CMP-
NeuNAc (2.1 mg, 3.2 µmol) in 350 µL of buffer (0.1 M HEPES, 5
mM MnCl2, 0.2% Triton X-100, pH 7.0) R2,3-sialyltransferase (Cytel,
3 U/mL, 6.5 µL) and alkaline phosphatase (Boehringer Mannheim, 1
Ac-Lys-Pro-Pro-Asn-Thr(Sialr2f3Galâ1f4(Fucr1f3)GlcNAcâ)-
Thr-Ser-Ala-OH (1). GDP-Fuc (3.2 mg, 6.0 µmol) was dissolved in
a solution of glycopeptide 25 (6.5 mg, 4.3 µmol) in buffer (660 µL, 50
mM MES, 15 mM MnCl2, pH 6.0). R1,3-Fucosyltransferase (Cytel,
2.16 U/mL, 43 µL) and alkaline phosphatase (Boehringer Mannheim,