J. Steger, R. Micura / Bioorg. Med. Chem. 19 (2011) 5167–5174
5171
4.1.4. 6-N-[(Di-n-butylamino)methylene]-30-{[N-(9-fluorenyl)
methoxycarbonyl-
-isoleucyl]amino}-30-deoxy-50-O-(4,40-di
methoxytrityl)-b- -adenosine (4)
N-(9-Fluorenyl)methoxycarbonyl- (179 mg,
0.506 mmol) was three times co-evaporated with dry tetrahydro-
furan and then dissolved in 8.2 mL dry tetrahydrofuran. After cool-
ing the solution to 0 °C, 1-hydroxybenzotriazole hydrate (103 mg,
0.673 mmol) was added. The solution was stirred for 10 min and
Rf = 0.31. 1H NMR (600 MHz, CDCl3):
d
0.97 (dt, J1 = 7.5 Hz,
L
J2 = 15.7 Hz, 6H, N(CH2CH2CH2CH3)2); 1.35–1.41 (m, 4H,
N(CH2CH2CH2CH3)2); 1.65–1.68 (m, 4H, N(CH2CH2CH2CH3)2); 1.73
(m, 4H, OOCCH2CH2CH2CH2COO); 2.42–2.44 (m, 2H, OOCCH2CH2
CH2CH2COO); 2.65–2.67 (m, 2H, OOCCH2CH2CH2CH2COO); 3.41 (t,
J = 7.3 Hz, 2H, N(CH2CH2CH2CH3)); 3.47–3.49 (m, 2H, H(a)-C(50),
H(b)-C(50)); 3.68–3.73 (m, 2H, N(CH2CH2CH2CH3)); 3.78 (s, 6H,
D
L-isoleucine
2ꢁ O–CH3(DMT)); 3.81 (t, J = 6.7 Hz, 2H, H2C(
a, Gly)); 4.14 (dt,
N,N-diisopropylcarbodiimide (104
lL, 0.673 mmol) was added.
J1 = 3.8 Hz, J2 = 7.7 Hz, 1H, H–C(40)); 4.23 (t, J = 6.7 Hz, 1H, H–C(9,
Fmoc)); 4.47 (d, J = 6.8 Hz, 2H, O–CH2(Fmoc)); 5.23 (q, J = 7.2 Hz,
1H, H–C(30)); 5.41 (s, br, 1H, HN(Gly)); 5.84 (d, J = 2.7 Hz, 1H, H–
C(20)); 6.16 (d, J = 2.8 Hz, 1H, H–C(10)); 6.40 (d, J = 6.5 Hz, 1H,
HN–C(30)); 6.80 (dd, J1 = 2.7 Hz, J2 = 8.9 Hz, 4H, H–C(ar)); 7.19 (t,
J = 7.2 Hz, 1H, H–C(ar)); 7.25–7.32 (m, 8H, H–C(ar)); 7.39–7.42
(m, 4H, H–C(ar)); 7.58 (d, J = 6.8 Hz, 2H, H–C(ar)); 7.77 (d,
J = 6.1 Hz, 2H, H–C(ar)); 8.07 (s, 1H, H–C(8)); 8.52 (s, 1H, H–
C(2)); 9.00 (s, 1H, HC@N–C(6)) ppm. 13C NMR (150 MHz, CDCl3):
d 13.76, 13.96 (N(CH2CH2CH2CH3)2); 19.86, 20.25 (N(CH2CH2CH2
CH3)2); 23.77, 23.95 (OOCCH2CH2CH2CH2COO); 29.33, 31.08
(N(CH2CH2CH2CH3)2); 32.86 (OOCCH2CH2CH2CH2COO); 33.26
After 10 more minutes compound 1 (286 mg, 0.389 mmol), which
was dissolved in 11.1 mL dry tetrahydrofuran, was slowly dropped
into the solution. Then, 15 min later trimethylphosphine in tetra-
hydrofuran (848 lL, 0.848 mmol, 1.0 M in tetrahydrofuran) was
added. The solution was allowed to warm to room temperature
and stirred for 16 h. The solvent and the volatile compounds were
evaporated under reduced pressure and the residue was dissolved
in dichloromethane. The solution was extracted with water and
with half saturated sodium bicarbonate solution. The organic lay-
ers were dried over Na2SO4 and evaporated. Compound 4 was puri-
fied by column chromatography on SiO2 with dichloromethane/
methanol, 99.5:0.5–97:3. Yield: 304 mg of 4 as white foam (75%).
TLC (8% methanol in dichloromethane): Rf = 0.50. 1H NMR
(600 MHz, CDCl3): d 0.78 (d, J = 6.7 Hz, 3H, H3C(c0, Ile)); 0.87 (t,
J = 7.2 Hz, 3H, H3C(d, Ile)); 0.97 (q, J = 6.5 Hz, 6H, N(CH2CH2CH2
(OOCCH2CH2CH2CH2COO); 44.88 (C(a, Gly)); 45.31 (N(CH2CH2CH2
CH3)); 47.15 (C(9, Fmoc)); 50.75 (C(30)); 51.97 (N(CH2CH2CH2
CH3)); 55.28 (2ꢁ O–CH3(DMT)); 63.22 (C(50)); 67.32 (O–
CH2(Fmoc)); 75.29 (C(20)); 82.26 (C(40)); 86.87; 87.57 (C(10));
100.07; 113.24, 120.14, 124.93, 126.13, 126.96, 127.19, 127.93,
128.25, 129.22, 130.21 (C(ar)); 135.58; 135.66; 137.01; 138.79;
139.91 (C(8)); 140.38; 141.39; 142.00; 143.62; 143.65; 144.47;
151.17; 153.00 (C(2)); 156.88; 158.43; 158.60; 160.17; 169.01;
169.42; 171.50 ppm. ESI-MS (m/z): [M+H]+ calcd for
CH3)2); 1.07–1.12 (m, 1H, H(a)-C(
N(CH2CH2CH2CH3)2); 1.49 (m, 1H, H(b)-C(
c
,
Ile)); 1.36–1.44 (m, 4H,
, Ile)); 1.67–1.72 (m,
c
4H, N(CH2CH2CH2CH3)2); 1.75–1.76 (m, 1H, H–C(b, Ile)); 3.33–
3.36 (m, 1H, H(a)-C(50)); 3.41–3.46 (m, 3H, N(CH2CH2CH2CH3),
H(b)-C(50)); 3.69 (dt, 1H, J1 = 7.1 Hz, J2 = 13.7 Hz, N(H(a)CH(b)CH2
CH2CH3)); 3.75–3.80 (m, 7H, N(H(a)CH(b)CH2CH2CH3), 2ꢁ O–
C69H69F5N8O11, 1282.33. Found 1281.39.
CH3(DMT)); 4.04 (t, J = 8.0 Hz, 1H, H–C(a, Ile)); 4.18 (t, J = 6.7 Hz,
1H, H–C(9, Fmoc)); 4.33–4.34 (m, 3H, H–C(40), O–CH2(Fmoc));
4.69–4.72 (m, 1H, H–C(30)); 4.77 (m, 1H, H–C(20)); 5.47 (d,
J = 8.9 Hz, 1H, HN(Ile)); 6.05 (s, 1H, H–C(10)); 6.14 (s, 1H, HO–
C(20)); 6.78 (d, J = 7.8 Hz, 4H, H–C(ar)); 6.84 (d, J = 5.6 Hz, 1H,
HN–C(30)); 7.18–7.33 (m, 10H, H–C(ar)); 7.38 (t, J = 7.1 Hz, 3H,
H–C(ar)); 7.55 (dd, J1 = 7.7 Hz, J2 = 16.8 Hz, 2H, H–C(ar)); 7.73
(dd, J1 = 7.6 Hz, J2 = 15.6 Hz, 2H, H–C(ar)); 8.18 (s, 1H, H–C(8));
8.54 (s, 1H, H–C(2)); 9.07 (s, 1H, HC@N–C(6)) ppm. 13C NMR
(150 MHz, CDCl3): d 11.34 (C(d, Ile)); 13.74, 13.98 (N(CH2CH2CH2
CH3)2); 15.37 (C(c0, Ile)); 19.83, 20.27 (N(CH2CH2CH2CH3)2); 25.07
4.1.6. 6-N-[(Di-n-butylamino)methylene]-30-{[N-(9-fluorenyl)
methoxycarbonyl-L
-alanyl]amino}-30-deoxy-50-O-(4,40-dimeth-
oxytrityl)-20-O-[1,6-dioxo-6-(pentafluorophenyloxy) hexyl]-b-
D-
adenosine (6)
Compound 3 (227 mg, 0.227 mmol) was dissolved in 2.9 mL dry
N,N-dimethylformamide and 2.9 mL dry pyridine. After addition of
4-(N,N-dimethylamino)pyridine (28 mg, 0.229 mmol) and adipic
acid bis(pentafluorophenyl)ester (533 mg, 1.11 mmol) the solution
was stirred for 2 h at room temperature. The reaction mixture was
evaporated under reduced pressure and afterwards the residue
was co-evaporated three times with toluene and once with dichlo-
romethane. Compound 6 was purified by column chromatography
on SiO2 with dichloromethane/acetone, 95:5–80:20. Yield: 195 mg
of 6 as white foam (66%). TLC (15% acetone in dichloromethane):
(C(
c, Ile)); 29.34, 31.05 (N(CH2CH2CH2CH3)2); 37.51 (C(b, Ile));
45.31 (N(CH2CH2CH2CH3)); 47.17 (C(9, Fmoc)); 51.93 (C(30));
52.00 (N(CH2CH2CH2CH3)); 55.22 (2ꢁ O–CH3(DMT)); 59.66 (C(
a,
Ile)); 63.11 (C(50)); 67.16 (O–CH2(Fmoc)); 74.64 (C(20)); 80.06;
83.12 (C(40)); 86.53; 91.42 (C(10)); 113.20, 119.99, 120.05, 125.07,
126.43, 126.87, 127.10, 127.16, 127.77, 127.79, 127.89, 128.26,
130.16 (C(ar)); 135.64; 139.51 (C(8)); 141.27; 141.34; 143.66;
143.82; 144.45; 150.58; 152.47 (C(2)); 156.47; 158.55; 158.89;
160.45; 171.92 ppm. ESI-MS (m/z): [M+H]+ calcd for C61H70N8O8,
1044.26. Found 1043.55.
Rf = 0.40. 1H NMR (600 MHz, CDCl3):
d 0.94–0.99 (m, 6H,
N(CH2CH2CH2CH3)2); 1.28–1.29 (m, 3H, H3C(b, Ala)); 1.36–1.40
(m, 4H, N(CH2CH2CH2CH3)2); 1.65–1.68 (m, 8H, N(CH2CH2CH2
CH3)2, OOCCH2CH2CH2CH2COO); 2.42 (t, J = 4.5 Hz, 2H, OOCCH2CH2
CH2CH2COO); 2.60 (t, J = 4.6 Hz, 2H, OOCCH2CH2CH2CH2COO); 3.41
(t, J = 6.9 Hz, 2H, N(CH2CH2CH2CH3)); 3.46 (m, 2H, H(a)-C(50), H(b)-
C(50)); 3.65–3.72 (m, 2H, N(CH2CH2CH2CH3)); 3.78 (s, 6H, 2ꢁ O–
4.1.5. 6-N-[(Di-n-butylamino)methylene]-30-{[N-(9-fluorenyl)
CH3(DMT)); 4.15–4.22 (m, 3H, H–C(a
, Ala), H–C(40), H–C(9, Fmoc));
methoxycarbonyl-
L
-glycyl]amino}-30-deoxy-50-O-(4,40-dimeth-
4.37 (m, 1H, O–H(a)CH(b)(Fmoc)); 4.45–4.48 (m, 1H, O–H(a)CH(b)
(Fmoc)); 5.28–5.25 (m, 1H, H–C(30)); 5.27 (s, br, 1H, HN(Ala));
5.84–5.85 (m, 1H, H–C(20)); 6.16 (m, 1H, H–C(10)); 6.52 (s, br, 1H,
HN–C(30)); 6.80 (d, J = 8.4 Hz, 4H, H–C(ar)); 7.19 (t, J = 6.8 Hz, 1H,
H–C(ar)); 7.25–7.32 (m, 8H, H–C(ar)); 7.39–7.42 (m, 4H, H–
C(ar)); 7.57 (d, J = 6.8 Hz, 2H, H–C(ar)); 7.76 (d, J = 6.8 Hz, 2H, H–
C(ar)); 8.08 (s, 1H, H–C(8)); 8.52 (s, 1H, H–C(2)); 8.99 (s, 1H,
HC@N–C(6)) ppm. 13C NMR (150 MHz, CDCl3): d 13.75, 13.93
(N(CH2CH2CH2CH3)2); 17.71 (C(b, Ala)); 19.86, 20.24 (N(CH2CH2
CH2CH3)2); 23.74, 23.97 (OOCCH2CH2CH2CH2COO); 29.33, 31.08
(N(CH2CH2CH2CH3)2); 32.84 (OOCCH2CH2CH2CH2COO); 33.28
(OOCCH2CH2CH2CH2COO); 45.33 (N(CH2CH2CH2CH3)); 47.15 (C(9,
oxytrityl)-20-O-[1,6-dioxo-6-(pentafluorophenyloxy) hexyl]-b-
D-
adenosine (5)
Compound 2 (221 mg, 0.224 mmol) was dissolved in 2.9 mL dry
N,N-dimethylformamide and 2.9 mL dry pyridine. After addition of
4-(N,N-dimethylamino)pyridine (27 mg, 0.221 mmol) and adipic
acid bis(pentafluorophenyl)ester (524 mg, 1.10 mmol) the solution
was stirred for 2 h at room temperature. The reaction mixture was
evaporated under reduced pressure and afterwards the residue
was co-evaporated three times with toluene and once with dichlo-
romethane. Compound 5 was purified by column chromatography
on SiO2 with dichloromethane/acetone, 95:5–80:20. Yield: 192 mg
of 5 as white foam (67%). TLC (15% acetone in dichloromethane):
Fmoc)); 50.47 (C(a
, Ala)); 50.66 (C(30)); 51.98 (N(CH2CH2CH2CH3));