M. Ziemniak et al. / Bioorg. Med. Chem. 23 (2015) 5369–5381
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was extracted three times with 5 cm3 of diethyl ether. The pro-
duct was separated by IC on Sephadex resin using a linear TEAB
gradient of 0–1.4 M. 44.2 mg of product (31%, 0.053 mmol, tri-
4.2.16. 1P,4P-(7-methylguanosin-50-yl) (1,4-boranotetraphos-
phate), m7GpBH3pppBH3m7G (8)
Method 1: Prepared from m7GpBH3 (19, 75 mg, 0.16 mmol, tri-
ethylammonium salt) was isolated as
a
white powder.
ethylamine
salt)
and
P1,P2-di(1-imidazolyl)pyrophosphate
Diastereoisomers D1 and D2 were then separated on semi-prepar-
ative RP HPLC and, after repeatedly freeze-drying, isolated as
ammonium salts (D1: 12.2 mg, 0.02 mmol; D2: 16.5 mg,
0.027 mmol). MS ESI: [M]ꢁ found 532.05751, calcd 532.05763;
1H NMR: (400 MHz, D2O) (D1) d 9.12 (s, 1H), 6.08 (d, J = 3.0 Hz,
1H), 4.69 (dd, J = 4.4 Hz, J = 3.0 Hz, 1H), 4.58 (m, 1H), 4.42 (m,
1H), 4.35–4.25 (m, 2H), 4.16 (s, 3H), 2.32 (t, J = 20.3 Hz, 2H)
(D2) d 9.21 (s, 1H), 6.08 (d, J = 3.3 Hz, 1H), 4.68 (dd, J = 4.5 Hz,
J = 3.3 Hz, 1H), 4.49 (m, 1H), 4.44 (m, 1H), 4.37–4.22 (m, 2H),
4.16 (s, 3H), 2.34 (t, J = 20.3 Hz, 2H); 31P NMR (160 MHz, D2O)
(D1) d 82.49 (br s, 1P), 15.15 (dt. J = 20.3 Hz, J = 8.8 Hz, 1P), 8.57
(ddt, J = 30.0 Hz, J = 20.3 Hz, J = 8.8 Hz, 1P) (D2) d 82.51 (br s,
1P), 15.17 (dt. J = 19.8 Hz, J = 8.6 Hz, 1P), 8.63 (ddt, J = 30.1 Hz,
J = 20.3 Hz, J = 8.8 Hz, 1P).
(48 mg, 0.15 mmol, disodium salt) after 70 h. 90 mg (0.033 mmol,
41%, triethylammonium salt) of the product (white solid) was iso-
lated. The diastereisomer ratio was 8:3:4 (D1/D2/D3). Method 2:
105 mg (0.083 mmol, TEA salt) of the GpBH3pppBH3G (24) was dis-
solved in 1 cm3 anhydrous DMSO and 0.19 cm3 (0.167 mmol;
20 equiv) of methyl iodide was added. The reaction was performed
for 2 h, quenched by the addition of deionised water (15 cm3) and
the pH was adjusted to 7.0 using 1 M KOH water solution. The
reaction mixture was extracted three times with 5 cm3 of diethyl
ether. After separation, 760 optical units of a white solid (TEA salt,
purity 63%, 24% yield) were isolated. Purification of diastereomers:
760 optical units of the product (purity 63%) were purified on
semi-preparative HPLC (0%–30% of acetonitrile in 180 min). After
repeatedly freeze-drying, it was isolated as ammonium salts (D1:
RT = 64.0 min, 4.6 mg; D2: RT = 65.6 min, 1.6 mg; D3:
RT = 68.9 min, 4.2 mg). 1H (400 MHz, D2O): (D1) d 9.15 (s, 1H),
9.10 (s, 1H), 6.04 (d, J = 3.0 Hz, 1H), 6.00 (d, J = 3.5 Hz, 1H), 4.66
(m, 1H), 4.63 (m, 1H), 4.59 (m, 1H), 4.46 (m, 1H), 4.41 (br s, 2H),
4.37–4.25 (m, 4H), 4.14 (s, 6H), 0.37 (m, 6H) (D2): d 9.08 (s, 2H),
6.04 (d, J = 3.2 Hz, 2H), 4.68 (dd, J = 4.8 Hz, J = 3.2 Hz, 2H), 4.61
(m, 2H), 4.40 (m, 2H), 4.36–4.23 (m, 4H), 4.13 (s, 6H), 0.28 (m,
6H). 31P (160 MHz, D2O): d 80.69 (m, 2P), ꢁ26.20 (d, J = 25.9 Hz,
4.2.14. General procedure for synthesis of m7Gppppm7G-analog
ues 6–9
0.5–0.75 equiv of an appropriate P1,P2-diimidazolyl derivative
and 8 equiv of anhydrous MgCl2 (or ZnCl2 in the case of phospho-
rothioates) were added to anhydrous DMF and vigorously stirred
until both substrates dissolved. In the second step, 1 equiv of
nucleoside monophosphate was added to the mixture and the
reaction was performed until HPLC measurements indicated the
formation of the desired products. Then, the reaction was
quenched via the addition of either MQ water or a solution of
EDTA and NaHCO3. The products were purified by ion-exchange
chromatography on Sephadex resin using a linear gradient of
TEAB 0–1.2 M and subsequently all diastereoisomers were sepa-
rated via semi-preparative RP-HPLC.
2P). (D3)
d 9.19 (s, 1H), 5.99 (d, J = 3.2 Hz, 1H), 4.68 (dd,
J = 4.8 Hz, J = 3.2 Hz, 2H), 4.63 (br s, 2H), 4.51–4.25 (m, 8H), 4.13
(s, 6H), 0.28 (m, 6H). 31P (160 MHz, D2O): (D1) d 80.64 (m, 2P),
ꢁ26.04 (d, J = 28.8 Hz, 2P) (D2) d 80.64 (m, 2P), ꢁ26.04 (d,
J = 28.8 Hz, 2P) (D3) d 80.69 (m, 2P), ꢁ26.20 (d, J = 25.9 Hz, 2P).
4.2.17. 1P,4P-(7-methylguanosin-50-yl) (1,4-thiotetraphosphate),
m7GpSpppSm7G (6)
Prepared from m7GpS (165 mg, 0.33 mmol, triethylammonium
salt) and P1,P2-di(1-imidazolyl)pyrophosphate (48 mg, 0.15 mmol,
disodium salt) after 45 h. 211 mg (0.16 mmol, 48%, triethylammo-
nium salt) of the product (yellowish solid) was isolated. The
diastereomer ratio was 3:8:4 (D1/D2/D3). 10 mg of the product
was purified on semi-preparative HPLC to obtain pure diastere-
omers. After repeatedly freeze-drying, it was isolated as ammo-
nium salts (D1+D2: RT = 58.2 min, 3.15 mg; D3: RT = 49.4 min,
1.4 mg). 1H (400 MHz, D2O): (D1/D2) d 9.32 (s, 1H), 9.29 (s, 1H),
6.03 (m, 2H), 4.69 (m, 2H), 4.56 (m, 2H), 4.45–4.27 (m, 6H), 4.15
(s, 3H), 4.14 (s, 3H) (D3) d 9.28 (s, 1H), 6.04 (d, J = 3.5, 2H), 4.69
(dd, J = 5.0, 3.5 Hz, 2H), 4.55 (m, 2H), 4.45–4.40 (m, 4H), 4.37–
4.29 (m, 2H), 4.15 (s, 6H). 31P (160 MHz, D2O): (D1/D2) d 44.11
(m, 2P), ꢁ23.40 (m, 2P) (D3) d 40.27 (d, J = 14.7 Hz, 2P), ꢁ23.40
(m, 2P)
4.2.15. 1P,4P-(7-methylguanosin-50-yl) (2,3-methylene-,1,4-
boranotetraphosphate), m7GpBH3pCH2ppBH3m7G (9)
Method 1: Prepared from m7GpBH3 (19, 75 mg, 0.16 mmol, tri-
ethylammonium salt) and P1,P2-di(1-imidazolyl)methylenebispho-
sphonate (48 mg, 0.15 mmol, disodium salt) after 70 h. The
diastereoisomer ratio was 8:3:4 (D1/D2/D3). HPLC yield was 91%.
57 mg (26%, 0.04 mmol, triethylammonium salt) of the product
(white solid) was isolated. Method 2: 100 mg (0.079 mmol, TEA
salt) of GpBH3pCH2ppBH3G (25) was dissolved in 1 cm3 anhydrous
DMSO and 0.18 cm3 (1.58 mmol; 20 equiv) of methyl iodide was
added. The reaction was performed for 2 h, quenched by the addi-
tion of deionised water (15 cm3) and the pH was adjusted to 7.0
using 1 M KOH water solution. The reaction mixture was extracted
three times with 5 cm3 of diethyl ether. After separation, 590 opti-
cal units of white solid (purity 45%, 14%, triethylammonium salt)
were isolated. Purification of diastereoisomers: 590 optical units
of the product (purity 45%) were purified on semi-preparative
HPLC. After repeatedly freeze-drying, they were isolated as ammo-
nium salts (D1: RT = 67.3 min, 4 mg; D2: RT = 68.7 min, mg; D3
RT = 71.2 min, 3.1 mg). 1H (400 MHz, D2O): (D1) d 9.17 (s, 1H),
9.10 (s, 1H), 6.05 (d, J = 2.5 Hz, 1H), 6.04 (d, J = 3.2 Hz, 1H), 4.64
(m, 2H), 4.58 (m, 1H), 4.46 (m, 1H), 4.40 (m, 2H), 4.34–4.23 (m,
4H), 4.14 (s, 6H), 2.67 (t, J = 20.9 Hz, 2H), 0.37 (m, 6H) (D2) d
9.08 (s, 2H), 6.05 (d, J = 2.5 Hz, 2H), 4.64 (dd, J = 4.3 Hz, J = 3.0 Hz,
2H), 4.56 (m, 2H), 4.40 (m, 2H), 4.30–4.24 (m, 4H), 4.12 (s, 6H),
2.53 (t, J = 20.8 Hz, 2H), 0.27 (m, 6H) (D3): 1H (400 MHz, D2O): d
9.16 (s, 2H), 6.05 (d, J = 2.5 Hz, 2H), 6.01 (d, J = 2.5 Hz, 2H), 4.63
(m, 2H), 4.50–4.40 (m, 4H), 4.35–4.27 (m, 4H), 4.15 (s, 6H), 2.52
(t, J = 20.8 Hz, 2H), 0.36 (m, 6H). 31P (160 MHz, D2O): (D1) d
79.23 (m, 2P), 3.68 (m, 2P) (D2) d 79.23 (m, 2P), 3.68 (m, 2P)
(D3) d 82.59 (m, 2P), 7.74 (m, 2P).
4.2.18. 1P,4P-(7-methylguanosin-50-yl) (2,3-methylene-1,4-thio-
tetraphosphate), m7GpSpCH2ppSm7G (7)
Prepared from m7GpS (23, 165 mg, 0.33 mmol, triethylammo-
nium salt) and P1,P2-di(1-imidazolyl)methylenebisphosphonate
(54 mg, 0.165 mmol, disodium salt) after 18 h. 162 mg (0.06 mmol,
37%, triethylammonium salt) of the product (yellowish solid) was
isolated. 10 mg of the product was purified on semi-preparative
HPLC to obtain pure diastereomers. After repeatedly freeze-drying,
it was isolated as ammonium salts (D1: RT = 64.1 min. 1.0 mg; D2
RT = 68.7 min, 2.1 mg; D3: RT = 76.8 min, 1.3 mg). 1H (400 MHz,
D2O): (D1) d 9.33 (s, 2H), 6.07 (d, J = 3.3 Hz, 2H), 4.71 (m, 2H), 4.58
(m, 2H), 4.44–4.26 (m, 6H), 4.16 (s, 6H), 2.69 (t, J = 21.0 Hz, 2H).
(D2) d 9.34 (s, 1H), 9.32 (s, 1H), 6.07 (d, J = 3.3 Hz, 1H), 6.06 (d,
J = 3.3 Hz, 1H), 4.70 (m, 2H), 4.59 (m, 1H), 4.59 (m, 1H), 4.54 (m,
1H), 4.45–4.25 (m, 6H), 4.17 (s, 3H), 4.15 (s, 3H), 2.69 (m, 2H) (D3)
d 9.29 (s, 2H), 6.05 (d, J = 3.0 Hz, 2H), 4.68 (m, 2H), 4.54 (m, 2H),