Scheme 2a
a Reagents and conditions: (a) deblocking 3% TCA in CH2Cl2; (b) coupling (1) 1, HgCl2, DIEA, DMF, (2) 20% thiophenol in DMF; (c)
capping CF3CO2Et, DIEA, DMF; (d) coupling of 5′-end monomer (1) 2, HgCl2, DIEA, DMF, (2) 20% thiphenol in DMF; (e) deprotection
and cleavage (1) 3% TCA in CH2Cl2, (2) NH4OH/EtOH (3/1), (3) 1 M TBAF in THF.
acetyl protecting group was removed, and the resulting 3′-
amino group was allowed to react with fluorenylmethyloxy-
carbonyl isothiocyanate (Fmoc-NCS)15 in DCM to give the
desired building block 1.16 To place a hydroxyl group at the
5′-end of the RNG, a building block with a 5′-hydroxyl group
was required. Needed compound 2 was obtained from
intermediate 5 by protection of the 5′-hydroxyl moiety with
an MMTr group followed by conversion of the 3′-position
to N-Fmoc-protected thiourea.17
Long chain alkylamine controlled pore glass (LCAA-CPG)
was chosen for the solid support. The attachment of the 3′-
end nucleoside onto LCAA-CPG followed the standard
procedure for DNA/RNA synthesis.7b Selective silylation of
5′-monomethoxytritylaminouridine was accomplished using
TBSCl, AgNO3, and pyridine in THF to give the 2′-OTBS
derivative. This compound was condensed with LCAA-CPG
via 3′-succinate to provide solid-support-attached 3′-end
monomer 9 (23.5 µmol/g).
A typical solid-phase synthesis is outlined in Scheme 2.
Both trimeric (I) and pentameric (II) uridyl RNG were
synthesized on a 2.0 µmol scale: 85 mg of LCAA-CPG
support containing the 3′-end nucleoside (9) was placed in
a solid-phase synthesis vessel and treated with 3% trichlo-
roacetic acid in DCM (2.0 mL, 10 s, 3 times) to remove the
MMTr group. Next, there was added 1 (27.3 mg, 0.030
mmol) in DMF (0.5 mL) followed by a solution of mercury-
(II) chloride (60 mM in DMF, 0.5 mL) and N,N-diisopro-
pylethylamine (120 mM in DMF, 0.5 mL). The mixture was
agitated at room temperature for 4 h. Preliminary study
indicated that this single coupling reaction gave an average
coupling yield of 77% as determined from the released
MMTr cation. This relatively low coupling yield is probably
due to the bulkiness of the adjacent 2′-OTBS group of
incoming nucleoside monomer, hindering the attack of the
terminal 5′-amino group to carbodiimide. The coupling
reaction was repeated and resulted in an average of 90% in
(15) Kearney, P. C.; Fernandez, M.; Flygare, J. A. J. Org. Chem. 1998,
63, 196.
(16) Physical data for 1: 1H NMR (400 MHz DMSO-d6) δ (ppm) 11.67
(s, 1 H), 11.45 (d, 1 H, J ) 1.9 Hz), 10.12 (d, 1 H, J ) 8.0 Hz), 8.02 (d,
1 H, J ) 8.0 Hz), 7.89 (m, 2 H), 7.80 (m, 2 H), 7.45-7.14 (m, 16 H), 6.83
(m, 2 H), 5.69 (d, 1 H, J ) 2.6 Hz), 5.60 (dd, 1 H, J ) 8.0, 1.9 Hz), 5.06
(ddd, 1 H, J ) 6.1, 8.6, 8.0 Hz), 4.67 (dd, 1 H, J ) 2.6, 6.1 Hz), 4.42-
4.34 (m, 2 H), 4.27 (t, 1 H, J ) 7.3 Hz), 4.08 (ddd, 1 H, J ) 8.6, 4.2, 4.1
Hz), 3.70 (s, 3 H), 3.05 (t, 1 H, J ) 8.1 Hz), 2.53 (ddd, 1 H, J ) 4.2, 12.8,
8.1 Hz), 2.36 (ddd, 1 H, J ) 4.1, 12.8, 8.1 Hz), 0.82 (s, 9 H), 0.07 (s, 3 H),
0.02 (s, 3 H); 13C NMR (100 MHz DMSO-d6) δ (ppm) 179.86, 163.00,
157.41, 153.49, 150.23, 146.06, 143.17, 143.13, 141.42, 140.72, 140.68,
137.59, 129.59, 128.26, 127.85, 127.69, 127.12, 126.07, 125.43, 125.39,
120.17, 113.03, 101.84, 91.23, 81.03, 73.70, 69.81, 67.37, 56.72, 54.91,
45.98, 44.65, 25.51, 17.59, -5.14, -5.24; FAB-MS (HR) m/z 910.3698,
calcd for C51H56N5O7SiS (M + H)+ 910.3670.
(17) Physical data for 2: 1H NMR (400 MHz DMSO-d6) δ (ppm) 11.73
(s, 1 H), 11.48 (d, 1 H, J ) 2.0 Hz), 10.09 (d, 1 H, J ) 7.3 Hz), 7.90 (m,
2 H), 7.85 (d, 1 H, J ) 8.3 Hz), 7.81 (m, 2 H), 7.46-7.20 (m, 16 H), 6.89
(m, 2 H), 5.76 (d, 1 H, J ) 2.9 Hz), 5.35 (dd, 1 H, J ) 8.3, 2.0 Hz), 5.14
(dt, 1 H, J ) 6.0, 7.3 Hz), 4.58 (dd, 1 H, J ) 2.9, 6.0 Hz), 4.39 (dd, 1 H,
J ) 8.0, 10.2 Hz), 4.35 (dd, 1 H, J ) 7.4, 10.2 Hz), 4.27 (dd, 1 H, J ) 7.4,
8.0 Hz), 4.18, (ddd, 1 H, J ) 7.3, 3.6, 2.2 Hz), 3.72 (s, 3 H), 3.44, (dd, 1
H, J ) 3.6, 11.0 Hz), 3.37 (dd, 1 H, J ) 2.2, 11.0 Hz), 0.80 (s, 9 H), 0.06
(s, 3 H), 0.00 (s, 3 H); 13C NMR (100 MHz DMSO-d6) δ (ppm) 180.24,
162.91, 158.30, 153.57, 150.27, 144.10, 143.70, 143.18, 140.75, 140.72,
140.18, 134.38, 130.18, 128.02, 127.97, 127.93, 127.19, 128.15, 127.02,
125.49, 120.22, 113.27, 101.83, 90.04, 86.50, 81.03, 73.79, 67.41, 62.63,
55.56, 55.05, 45.97, 44.86, 25.43, 17.60, -5.23, -5.33; FAB-MS (HR)
m/z 911.3535, calcd for C51H55N4O8SiS (M + H)+ 911.3510.
Org. Lett., Vol. 2, No. 1, 2000
83