Solution- and Solid-Phase Glycosylations
J . Org. Chem., Vol. 64, No. 16, 1999 5929
none ketyl; ethyl acetate, toluene, and CH2Cl2 were freshly
distilled from CaH2. Amine-free DMF and 20% piperidine in
DMF were purchased from PerSeptive Systems. Rink Amide
polystyrene resin (FmocNH capped, 0.46 mmol/g) was pur-
chased from NovaBiochem. On-bead product quality control
was performed by Kaiser tests, % N determination, and IR
analyses. Products were cleaved off resin by treatment with
20% TFA-CH2Cl2 for 30 min and analyzed by solution-phase
1H and 13C NMR spectroscopy and liquid chromatography (LC)
with light-scattering (LSD) and mass spectrometry (MS)
detectors. Conversion yields and purities were estimated by
integrating the LSD traces. 1H NMR spectra were obtained
at 300 MHz, and 13C NMR spectra were obtained at 75.4 MHz.
Im m obiliza tion of Meth yl 3-Azid o-3-d eoxy-4-O-m eth yl-
â-D-glu cop yr a n osid u r on ic Acid on Rin k Am id e Resin (6).
Rink Amide polystyrene resin (500 mg, 0.23 mmol) was treated
with 4 mL of a 20% solution of piperidine in DMF for 30 min.
The resin was washed with DMF (3×), THF (3×), and CH2Cl2
(3×). Removal of the Fmoc group was confirmed by a positive
Kaiser test of the resin. To the resin were sequentially added
amine-free DMF (4 mL), methyl 3-azido-3-deoxy-4-O-methyl-
â-D-glucopyranosiduronic acid (114 mg, 0.46 mmol), HATU
(174 mg, 0.46 mmol), and DIPEA (distilled from CaH2, 80 µL,
0.46 mmol). The reaction mixture was stirred overnight. The
resin was washed with DMF (3×), THF (3×), and CH2Cl2 (3×)
and dried under high vacuum. Complete derivatization of the
resin amino groups was confirmed by a negative Kaiser test.
IR (bead, cm-1): 2191, 2104. Anal. Calcd for C8H14N4O5 (on
bead): N, 2.58. Found: N, 2.54 (98% loading). A sample of
the resin was cleaved with 20% TFA-CH2Cl2 for analytical
analysis. ES for C8H14N4O5: [MH] calcd m/z 247, found 247.
1H NMR (CDCl3): 6.64 (s, br, 1H), 6.32 (s, br, 1H), 5.14 (d, J
) 5.1 Hz, 1H), 3.98 (d, J ) 7.5 Hz, 1H), 3.50 (d, J ) 9.0 Hz,
1H), 3.28 (s, br, 6H), 3.19 (t, J ) 9.6 Hz, 1H), 3.06-2.94 (m,
2H). 13C NMR (CDCl3): 169.9, 103.3, 80.2, 74.5, 71.2, 67.8,
59.7, 56.6.
Typ ica l Glycosyla tion P r oced u r e. Rink Amide resin (20
mg; 9 µmol) containing the glycosyl acceptor was dried under
high vacuum and kept under argon. To the resin was added a
solution of the glycosyl donor 1-5 (4 equiv; concentration of
0.14 M) and 2,6-di-tert-butyl-4-methylpyridine (4.5 mg; 2 equiv)
in a 5:1 mixture of methylene chloride and ethyl acetate (total
volume of 300 µL). The mixture was stirred at room temper-
ature for 5 min and cooled to -78 °C. Trifluoromethanesulfonic
anhydride (7 µL; 4 equiv) was slowly added and the system
was kept at -70 °C for 1 h. The temperature was slowly raised
to -45 °C and kept constant for 5.5 h. The reaction was
quenched with 100 µL of a 2:1 mixture of methanol and DIPEA
at -45 °C. The reaction mixture was allowed to warm to room
temperature and the resin was washed with DMF (3×), THF
(2×), CH3OH (2×), and CH2Cl2 (2×). Data for 7: IR (bead,
cm-1) 2193, 2105. Anal. Calcd for C22H29F3N5O13 (on bead): N,
2.71. Found: N, 2.69 (99% loading). For analytical purposes,
a sample of 7 was cleaved with 20% TFA-CH2Cl2. LC-MS for
the cleaved material is reproduced in Figure 4 (Supporting
Information). ES for C22H30F3N5O13: [MH] calcd m/z 630, found
630. 1H NMR (CDCl3): 9.06 (d, J ) 9 Hz, 0.8H), 6.75 (s, br,
1H), 6.28 (s, br, 1H), 5.26 (t, J ) 10.2 Hz, 1H), 5.03 (t, J ) 9.9
Hz, 1H), 4.96 (d, J ) 8.4 Hz, 1H), 4.34 (d, J ) 7.2 Hz, 1H),
4.20 (dd, J ) 12.6 Hz, 5.1 Hz, 1H), 4.11 (dd, J ) 12.6 Hz, 2.4
Hz, 1H), 4.04 (m, 1H), 3.80-3.6 (m, 3H), 3.51 (s, 3H), 3.49 (s,
3H), 3.45-3.40 (m, 1H), 3.30 (q, J ) 7.8 Hz, 1H), 2.05 (s, 3H),
2.00 (s, 3H), 1.97 (s, 3H). 13C NMR (CDCl3): 170.6, 170.4,
170.2, 169.3, 155.0 (J ) 38 Hz), 113.0 (J ) 288 Hz), 102.6,
100.4, 81.2, 78.9, 74.6, 72.0, 71.7, 68.4, 66.8, 62.0, 60.3, 57.3,
54.4, 20.7, 20.5, 20.4.
frequency tags. The MicroKans were then sealed, drained,
dried under vacuum, and scanned into the Irori combinatorial
chemistry software through an Irori AccuTag 100 scanning
station.
F or m a tion of Am id e Gr ou p s (14). The cans containing
13 were sorted into 8 different containers, each corresponding
to a different carboxylic acid. To each container were added
in sequence 9 mL of DMF, 0.225 mmol of R1COOH, 0.225 mmol
of HATU (85 mg), and 0.225 mmol of DIPEA (40 µL). The
containers were shaken overnight at room temperature. The
supernatants were drained, and the cans were washed with
DMF (2×), MeOH (2×), THF (2×), and CH2Cl2 (2×).
P er -O-Acetyla tion (15). The MicroKans were combined
and treated with 30 mL of CH2Cl2, 30 mL of toluene, 9 mL of
Ac2O, 9 mL of pyridine, and 30 mg of DMAP for 3 h at room
temperature. The supernatant was drained, and the cans were
washed with CH2Cl2 (4×) and THF (2×).
Data for R1 ) 4-phenyl-benzoyl: IR (bead, cm-1) 2189, 2105.
Anal. Calcd for C33H39N5O13 (on bead): N, 2.60. Found: N, 2.50
(96% loading). For analytical purposes, a sample of the resin
was cleaved with 20% TFA-CH2Cl2. ES for C33H39N5O13: [MH]
1
calcd m/z 714, found 714. H NMR (CDCl3-DMSO-d6): 8.56
(d, J ) 9.3 Hz, 1H), 7.92 (d, J ) 8.1 Hz, 2H), 7.73 (two sets of
t, J app ) 8.4 Hz, 4H), 7.59 (s, br, 0.8H), 7.50 (t, J ) 7.2 Hz,
2H), 7.41 (t, J ) 7.2 Hz, 1H), 7.34 (s, br, 0.8H), 5.34 (t, J ) 9.6
Hz, 1H), 5.10 (d, J ) 8.7 Hz, 1H), 5.00 (t, J ) 9.9 Hz, 1H),
4.45 (d, J ) 7.5 Hz, 1H), 4.26 (dd, J ) 12 Hz, 4.5 Hz, 1H),
4.14-4.10 (m, 2H), 3.85-3.74 (m, 3H), 3.59 (t, J ) 9.6 Hz, 1H),
3.51 (s, 3H), 3.49 (s, 3H), 3.29 (m, 1H), 2.08 (s, 3H), 2.06 (s,
3H), 2.04 (s, 3H). 13C NMR (CDCl3-DMSO-d6): 169.7, 169.4,
169.2, 168.9, 166.2, 142.7, 139.2, 133.3, 128.7, 127.7, 127.5,
126.7, 126.2, 102.0, 100.1, 79.9, 78.8, 74.2, 72.7, 71.0, 68.4, 66.2,
61.9, 59.2, 56.4, 54.1, 20.4, 20.3, 20.2.
Red u ction of Azid e Gr ou p s a n d Ur ea F or m a tion (17).
The MicroKans were combined and treated with 7.5 mL of
THF, 30 mL of EtOH, 7 mL of water, and 22.5 mL of a 1.0 M
solution of PMe3 in THF. The MicroKans were shaken for 4 h
at room temperature. The supernatant was drained, and the
cans were washed with THF (2×), DMF (2×), and CH2Cl2 (3×)
and dried under high vacuum overnight. The resin yielded a
strong Kaiser test. Data for R1 ) 4-phenylbenzoyl: IR (bead,
cm-1) no absorption in the 2200-2000 cm-1. Anal. Calcd for
C
33H41N3O13 (on bead): N, 1.60. Found: N, 1.57 (98% loading).
For analytical purposes, a sample of the resin was cleaved with
20% TFA-CH2Cl2. ES for C33H41N3O13: [MH] calcd m/z 688,
found 688.
The MicroKans were sorted into 6 different containers. To
each container were added in sequence 12 mL of toluene, 6
mmol of R2NCO, and 0.2 mL of DIPEA. The reaction vessels
were shaken for 2 h. The supernatant was drained, and the
cans were washed with DMF (3×), MeOH (2×), and CH2Cl2
(4×). The cans were dried under vacuum and kept under
argon. Data for R1 ) 4-phenylbenzoyl and R2 ) 3-(trifluoro-
methyl)phenyl: Anal. Calcd for C41H45F3N4O14 (on bead): N,
1.99. Found: N, 1.90 (95% loading). For analytical purposes,
a sample of the resin was cleaved with 20% TFA-CH2Cl2. ES
1
for C41H45F3N4O14: [MH] calcd m/z 875, found 875. H NMR
(DMSO-d6): 8.47 (d, J ) 9.6 Hz, 1H), 7.9-7.1 (m, 15H), 5.28
(t, J ) 9.9 Hz, 1H), 5.14 (d, J ) 8.4 Hz, 1H), 4.98 (t, J ) 9.9
Hz, 1H), 4.45 (d, J ) 6.3 Hz, 1H), 4.27 (dd, J ) 12.6 Hz, 4.5
Hz, 1H), 4.12 (d, br, J app ) 11.1 Hz, 1H), 4.04 (q, J ) 9.9 Hz,
1H), 3.9-3.6 (m, 5H), 3.5-3.3 (m, 2H), 3.44 (s, 3H), 3.25 (s,
3H), 2.06 (s, 3H), 2.02 (s, 3H), 1.85 (s, 3H). 13C NMR (DMSO-
d6): 170.5, 170.2, 170.0, 169.7, 166.5, 154.6, 142.7, 141.2, 139.3,
133.2, 129.7, 129.2, 128.2, 128.0, 127.0, 126.6, 126.3, 121.2,
117.4, 113.7, 103.0, 99.7, 77.9, 78.3, 75.2, 73.4, 71.1, 68.7, 62.1,
59.0, 56.5, 54.7, 54.2, 20.7, 20.6, 20.5.
Rem ova l of Ba se-Sen sitive Gr ou p s (13). The glycosy-
lated resin 7 (1.0 g) was treated with 16 mL of 0.5 M LiOH in
1:1 THF-MeOH overnight. The resin was drained and washed
with 1:1 THF-MeOH (3×), THF (3×), and CH2Cl2 (3×). As
expected, the resin gave a strongly positive Kaiser test. Anal.
Calcd for C14H25N5O9 (on bead): N, 3.22. Found: N, 3.20 (99%
loading).
Su p p or tin g In for m a tion Ava ila ble: Text and figures
giving experimental procedures and characterization data for
glycosylation studies and solid-phase derivatization studies (10
pages). This material is available free of charge via the
Internet at http://pubs.acs.org.
Design of Libr a r y. Derivatized resin 13 (20 mg; 9 µmol)
was added to each of 48 Irori MicroKans containing radio
J O9903499