E. Łodyga-Chruścińska et al. / Journal of Inorganic Biochemistry 105 (2011) 1212–1219
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Scheme 1. Synthesis of the histidine modified 2′-deoxyadenosine. Reagents and conditions: (i) Ac2O, pyridine; (ii) 1-phenoxycarbonyltetrazole, dioxane, 35 °C or 1-methyl-3-
phenoxycarbonyl-imidazolium chloride, CH2Cl2, room temp.; (iii) dihydrochloride histidine methyl ester, pyridine, 35 °C, 72 h; (iv) 0.2 M KOH/pyridine (1:1 v/v), room temp.
2.1.1. Synthesis of N-[(9-β-D-2′-deoxyribofuranosylpurin-6-yl)-carbamoyl]
histidine (his6dA) 3′,5′-di-O-acetyl-2′-deoxyadenosine (2)
2.1.3. Synthesis of methyl ester of N-[(9-β-3′,5′-di-O-acetyl-D-2′-
deoxyribofuranosylpurin-6-yl)-carbamoyl]-histidine (4)
To the solution of 2′-deoxyadenosine (2.01 g, 8 mmol) in 25 mL of
anhydrous pyridine 7.5 mL of acetic anhydride (80 mmol) was slowly
dropped and the reaction mixture was stirred for 2 h at r.t. (room
temperature). Then, after cooling in the ice bath, 100 mL of CH2Cl2 was
added and the solution was washed with 10% aq. NaHCO3 (25 mL) and
with water (20 mL). The organic layer was evaporated to dryness, co-
evaporated with toluene (3×10 mL) and the residue was crystallized
from ethanol to give 2.25 g of acetylated 2′-deoxyadenosine 2 (yield 85%)
(Scheme 1).
3′,5′-Di-O-acetyl-N6-phenoxycarbonyl-2′-deoxyadenosine 3
(650 mg, 1.4 mmol) was treated with 2 M excess of dihydrochloride
histidine methyl ester (678 mg, 2.8 mmol) in anhydrous pyridine
(10 mL) at 35 °C. After 72 h, TLC analysis (CHCl3/MeOH, 95:5) revealed
no starting nucleoside in the reaction mixture. The solvent was
evaporated and the residue, after coevaporation with toluene
(2×15 mL), was chromatographed over silica gel column using 0–2%
methanol in chloroform. Pure product was obtained as white foam
(470 mg, yield 63.2%). TLC Rf: 0.17 (CHCl3/MeOH 90:10 v/v), 0.09
(AcOEt/MeOH 90:10 v/v).
TLC (thin layer chromatography) Rf (retention factor): 0.43
(CHCl3/MeOH 80:20 v/v); mp. (melting point)=153–154 °C, (mp.lit.
152–153 °C) [27].
MS FAB (Fast Atom Bombardment Mass Spectrometry) m/z: 531.2
([M+H]+; MW (molecular weight) for C22H26N8O8 530; 1H NMR
(250 MHz, CDCl3) δ 2.05 (s, 3H, CH3COO), 2.15 (s, 3H, CH3COO), 2.65
(ddd, 1H, JH2′,H3′=2.7 Hz, JH2′,H1′=6.3 Hz, Jgem=14.1 Hz, H2′), 3.03
(ddd, 1H, JH2″,H3′ =6.6 Hz, JH2″,H1′ =7.5 Hz, Jgem =14.1 Hz, H2″),
3.16–3.38 (m, 2H, CH2-β), 3.75 (s, 3H, OCH3), 4.26–4.48 (m, 3H, H4′,
H5′, H5″), 4.94 (dt, 1H, JCHα,CHβ=5.9 Hz, JCHα,NH =7.5 Hz, CH-α), 5.46
(m, 1H, H3′), 6.47 (dd, 1H, JH1′,H2′=6.3 Hz, JH1′,H2″=7.5 Hz, H1′), 6.88
(s, 1H, Im-CH-5), 7.59 (s, 1H, Im-CH-2), 8.46 (s, 1H, H2), 8.49 (s, 1H, H8),
9.39 (bs, 1H, NHCO), 10.24 (d, 1H, JNHα,CHα=7.5 Hz, NH-α); 13C NMR
(63 MHz, CDCl3) δ 20.97 (CH3COO), 21.14 (CH3COO), 30.06 (Cβ), 37.50
(C2′), 52.73 (OCH3), 53.67 (Cα), 63.96 (C5′), 74.65 (C3′), 82.85 (C4′),
84.90 (C1′), 119.26 (Im-CH-5), 121.04 (C5), 131.95 (Im-CH-4), 135.41
(Im-CH-2), 141.88 (C8), 150.40 (C4), 150.46 (NHCONH), 151.34 (C2),
154.25 (C6), 170.56 (CH3COO), 170.84 (CH3COO), 172.52 (COOCH3).
1H NMR (250 MHz, CDCl3, s = singlet, dd = doublet of doublets,
ddd = doublet of doublets of doublets, m = multiplet, dt = doublet of
triplets, and bs = broad singlet) δ 2.09 (s, 3H, CH3COO), 2.14 (s, 3H,
CH3COO), 2.63 (ddd, 1H, JH2′,H3′ =2.5 Hz, JH2′,H1′ =6.0 Hz, Jgem
13.8 Hz, H2′), 2.96 (ddd, 1H, JH2″,H3′=6.3 Hz, JH2″,H1′=8.0 Hz, Jgem
14.0 Hz, H2″), 4.32–4.46 (m, 3H, H4′, H5′, H5″), 5.46 (dt, 1H, JH3′,H2′
=
=
=
JH3′,H4′=2.3 Hz, JH3′,H2″=6.3 Hz, H3′), 5.99 (bs, 2H, NH-6), 6.46 (dd, 1H,
JH1′,H2′=6.0 Hz, JH1′,H2″=8.0 Hz, H1′), 7.99 (s, 1H, H2), 8.35 (s, 1H, H8).
2.1.2. Synthesis of 3′,5′-di-O-acetyl-N6-phenoxycarbonyl-2′-
deoxyadenosine (3)
a) with 1-phenoxycarbonyltetrazole [28].
3′,5′-Di-O-acetyl-2′-deoxyadenosine (1.16 g, 3.5 mmol) was dissolved
in anhydrous dioxane (35 mL) and treated with 1-phenoxycarbonyl-
tetrazole (1.9 g, 10 mmol) at 35 °C. The reaction was monitored with
TLC (CHCl3/MeOH 90:10 v/v) and after 24 h, when no nucleoside
substrate was detected, the reaction mixture was evaporated to
dryness and the residue was chromatographed on silica gel column
using 0–2% methanol in chloroform. Nucleoside 3 was obtained as
white foam (1.20 g, yield 75%).
b) with 1-metyl-3-phenoxycarbonylimidazolium chloride [29].
3′,5′-Di-O-acetyl-2′-deoxyadenosine (1.68 g, 5 mmol) was dissolved
in dry CH2Cl2 (50 mL) and then 1-metyl-3-phenoxycarbonylimida-
zolium chloride (3.6 g, 15 mmol) was added. After the reaction
mixture was stirred for 2 h at r.t., the solution was evaporated in
vacuum and the residue dissolved in small amount of EtOAc.
Purification on a silica gel column using the same solvent as the
eluent gave the nucleoside 3 in 78% yield (1.77 g). TLC Rf: 0.63
(CHCl3/MeOH 90:10), 0.42 (AcOEt); 1H NMR δH (250 MHz, CDCl3;
Me4Si) 2.07 (3H, s, CH3COO), 2.14 (3H, s, CH3COO), 2.67 (1H, ddd,
JH2′,H3′=2.5 Hz, JH2′,H1′=6.0 Hz, Jgem=14.2 Hz, H2′), 2.98 (1H, ddd,
JH2″,H3′=6.5 Hz, JH2″,H1′=7.7 Hz, Jgem=14.2 Hz, H2″), 4.27–4.49
2.1.4. Synthesis of N-[(9-β-D-2′-deoxyribofuranosylpurin-6-yl)-carbamoyl]
histidine (his6dA, 5)
Protected nucleoside 4 (159 mg, 0.3 mmol) was dissolved in pyridine
(6 mL) and 6 mL of 0.2 M KOH aqueous solution was added. After stirring
for 24 h at r.t., TLC analysis (CHCl3/MeOH, 80:20) revealed no starting
protected nucleoside in the reaction mixture. The reaction mixture was
evaporated to give an oil residue which was purified by silica gel
chromatography (eluent 0–20% MeOH in CHCl3). Deprotected nucleoside
5 was obtained as white foam (85 mg, yield 66%). TLC Rf: 0.02 (CHCl3/
MeOH 80:20 v/v), m/z (MS, CI) 433 [M+H]+; MW for C17H20N8O6 432;
1H NMR (250 MHz, D2O, pt = pseudotriplet) δ 2.50–2.55 (m, 1H, H2″),
2.81–2.86 (m, 1H, H2′), 3.22–3.31 (m, 2H, CH2-β), 3.71–3.75 (m, 2H, H5′,
H5″), 4.10–4.14 (m, 1H, H4′), 4.55 (pt, 1H, JHα,Hβ=6.3 Hz, CH-α), 4.60–
4.62 (m, 1H, H3′), 6.49 (pt, 1H, JH1′H2″=7.0 Hz, H1′), 7.08 (s, 1H, Im-CH-5),
8.30 (s, 1H, H2); 8.35 (s, 1H, H8), 8.50 (s, 1H, Im-CH-2). 13C NMR (63 MHz,
D2O) δ 27.79 (Cβ), 39.04 (C2′), 55.08 (Cα), 61.57 (C5′), 71.12 (C3′), 84.82
(C1′), 87.46 (C4′), 116.87 (Im-CH-5), 120.26 (C5), 129.39 (Im-CH-4),
133.10 (Im-CH-2), 142.28 (C8), 149.42 (C4), 149.76 (C2), 150.95 (C6),
154.63 (NHCONH), 171.28 (COOH).
(3H, m, H4′, H5′, H5″), 5.44 (1H, m, H3′), 6.49 (1H, dd, JH1′,H2′
=
6.1 Hz, JH1′,H2″ =7.7 Hz, H1′), 6.80–7.47 (5H, m, PhO), 8.26 (1H, s,
H2), 8.81 (1H, s, H8), 9.34 (1H, bs, CONH).
Concentration of the ligand stock solution was determined by
potentiometric titrations. A stock solution of copper(II) was prepared