Synthesis of 3',5'-cyclic phosphate and thiophosphate esters of 2'-C-methyl ribonucleosides

Article abstract of DOI:10.1002/hlca.201200099

2'-C-Methylnucleosides are known to exhibit antiviral activity against Hepatitis C virus. Since the inhibitory activity depends on their intracellular conversion to 5'-triphosphates, dosing as appropriately protected 5'-phosphates or 5'-phosphorothioates appears attractive. For this purpose, four potential pro-drugs of 2'-C-methylguanosine, i.e., 3',5'-cyclic phosphorothioate of 2'-C-methylguanosine and 2'-C,O⁶-dimethylguanosine, 1 and 2, respectively, the S-[(pivaloyloxy)methyl] ester of 2'-C,O⁶- dimethylguanosine 3',5'-cyclic phosphorothioate and the O-methyl ester of 2'-C,O⁶-dimethylguanosine 3',5'-cyclic phosphate, 3 and 4, respectively, have been prepared.

Full text of DOI:10.1002/hlca.201200099

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Helvetica Chimica Acta – Vol. 95 (2012)
Synthesis of 3’,5’-Cyclic Phosphate and Thiophosphate Esters of 2’-C-Methyl
Ribonucleosides
by Anna Leisvuoria), Zafar Ahmeda), Mikko Ora*a), Leonid Beigelmanb), Lawrence Blattb), and
Harri Lçnnberga)
a) University of Turku, Department of Chemistry, FI-20014 Turku
(phone: þ 358-2-3336706; fax: þ 358-2-3336700; e-mail: mikora@utu.fi)
b) AliosBiopharma, 260 E. Grand Ave, 2nd Floor, South San Francisco, CA 94080, USA
2’-C-Methylnucleosides are known to exhibit antiviral activity against Hepatitis C virus. Since the
inhibitory activity depends on their intracellular conversion to 5’-triphosphates, dosing as appropriately
protected 5’-phosphates or 5’-phosphorothioates appears attractive. For this purpose, four potential pro-
drugs of 2’-C-methylguanosine, i.e., 3’,5’-cyclic phosphorothioate of 2’-C-methylguanosine and 2’-C,O6-
dimethylguanosine, 1 and 2, respectively, the S-[(pivaloyloxy)methyl] ester of 2’-C,O6-dimethylguano-
sine 3’,5’-cyclic phosphorothioate and the O-methyl ester of 2’-C,O6-dimethylguanosine 3’,5’-cyclic
phosphate, 3 and 4, respectively, have been prepared.
Introduction. – Inhibitors of RNA-dependent RNA polymerase of Hepatitis C virus
(HCV) constitute a class of potential drugs to combat against this worldwide viral
infection [1 – 3]. Several of the known inhibitors are derivatives of 2’-C-methyl
ribonucleosides, including 2’-C-methyl-3’-O-(l-valinyl)cytidine (Vabilopicitabine) [4],
4’-azido-3’,5’-bis-O-(2-methylpropanoyl)cytidine [5], and 7-deaza-2’-C-methyladeno-
sine in addition to unsubstituted 2’-C-methyl ribonucleosides [6]. All these nucleosides
need, however, to be converted to 5’-triphosphates to exhibit the desired inhibition.
Since the first phosphorylation giving a nucleoside 5’-monophosphate usually limits the
rate of this conversion, appropriately protected nucleoside 5’-monophosphates expect-
edly exhibit enhanced antiviral activity [1]. For example, the potency of 2’-C-
methylguanosine in cell lines is dramatically increased by conversion to 5’-(O-
arylphosphoramidates) derived from l-alanine alkyl esters [7]. Similarly, various alkyl
[8], aryl [8], acyloxymethyl [9] and 2-(acylthio)ethyl esters of 3’,5’-cylic phosphates of
base-modified 2’-C-methyl ribonucleosides appear to be more potent inhibitors of
HCV RNA replication than the parent cyclic phosphodiester. Encouraged by these
results, we now report on syntheses of the 3’,5’-cyclic phosphorothioates of 2’-C-
methylguanosine, 2’-C,O6-dimethylguanosine, 1 and 2, respectively, the S-(pivaloyloxy-
methyl), and the O-methyl ester of 2’-C,O6-dimethylguanosine 3’,5’-cyclic phosphate, 3
and 4, respectively (Fig.). While all these nucleotides may exhibit antiviral activity,
compound 3 expectedly is the most potent prodrug candidate. The (pivaloyloxy)methyl
group is removed by esterase-dependent deacylation and the remaining S-(hydroxy-
methyl) group by subsequent chemical hydrolysis. The exposed 3’,5’-cyclic phosphoro-
thioate diester may then undergo diesterase-catalyzed ring-opening to a 5’-phosphoro-
thioate.
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

Helvetica Chimica Acta – Vol. 95 (2012)
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Figure. Structures of potential prodrugs of HCV antiviral 2’-C-methylguanosine nucleotides
Results and Discussion. – 2’-C-Methylguanosine (5) was prepared by glycosylation
of persilylated N2-acetylguanine, i.e., 9-(trimethylsilyl)-6-(trimethylsilyloxy)-2-[(N-
(trimethylsilyl)acetamido]purine, with 1,2,3,5-tetra-O-benzoyl-2-C-methylribofura-
nose, as described by Li and Piccirilli [10]. 2’-C,O6-Dimethylguanosine (6) was, in
turn, obtained by glycosylation of 6-chloroguanine with the same glycosyl donor,
followed by treatment with MeONa in MeOH, as described previously by McGuigan et
al. [7]. Conversion of 2’-C-methylguanosine (5) and 2’-C,O6-dimethylguanosine (6) to
N2-(4-methoxytritylated) form, aimed at allowing their 5’-phosphitylation and sub-
sequent intramolecular cyclization, is depicted in Scheme 1. Accordingly, 2’-, 3’-, and 5’-
OH groups of 5 and 6 were protected with trimethylsilyl (TMS) groups, and the amino
function was then alkylated with 4-methoxytrityl (¼(4-methoxyphenyl)diphenyl,
MMTr) chloride.
Finally, the TMS protecting groups were removed. With N2-(4-methoxytrityl)-2’-
C,O6-dimethyl-2’,3’,5’-tris-O-(trimethylsilyl)guanosine, the removal of the 2’-O-SiMe3
group turned out to be surprisingly difficult. Treatment with Bu4NF (TBAF) in THF
was required instead of the NH3 treatment, which usually is sufficient to remove the
TMS protecting groups from sugar OH functions.
Conversions of the tritylated 2’-C-methyl nucleosides 7 and 8 to 3’,5’-cyclic
phosphorothioates 1 and 2, respectively, and 3’,5’-cyclic (S-[(pivaloyloxy)methyl]
phosphorothioate) and 3’,5’-cyclic O-methyl phosphate, 3 and 4, respectively, are
outlined in Schemes 2 – 5. Phosphitylation of N2-(4-methoxytrityl)-2’-C-methylguano-
sine (7) with 2-cyanoethyl N,N-diisopropyl(chloro)phosphoramidite (Scheme 2) and
subsequent 1H-tetrazole (TetH)-promoted intramolecular replacement of the 3’-OH
group with the (iPr)2N ligand gave a 3’,5’-cyclic phosphite triester, which was sulfurized

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Helvetica Chimica Acta – Vol. 95 (2012)
Scheme 1. Preparation of 2’-C-Methyl- and 2’-C,O6-Dimethylguanosine
with 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent) in MeCN to N2-(4-
methoxytrityl)-2’-C-methylguanosine cyclic 3’,5’-(O-(2-cyanoethyl) phosphoro-
thioate). The 2-cyanoethyl group was cleaved during chromatographic purification
(SiO2; CH2Cl2 containing 1% Et3N). The MMTr group was then removed by treatment
with aqueous 80% AcOH to give 2’-C-methylguanosine 3’,5’-cyclic phosphorothioate
(1).
The same method was applied for preparation of N2-(4-methoxytrityl)-2’-C,O6-
dimethylguanosine 3’,5’-cyclic phosphorothioate (2). Accordingly, N2-(4-methoxytri-
tyl)-2’-C,O6-dimethylguanosine (8) was phosphitylated with 2-cyanoethyl N,N-diiso-
propylchlorophosphoramidite and the 3’-OH group was replaced with (iPr)2N ligand
using tetrazole as an activator (Scheme 3). The phosphite triester obtained was
sulfurized to thiophosphate ester 9 with elemental sulfur. The desired fully deprotected
3’,5’-cyclic phosphorothioate 2 was obtained by removal of the 2-cyanoethyl group with
Et3N in CH2Cl2 and of the MMTr group with aq. AcOH.

Helvetica Chimica Acta – Vol. 95 (2012)
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Scheme 2. Preparation of 2’-C-Methylguanosine 3’,5’-Cyclic Phosphorothioate
i) Et3N, CH2Cl2. ii) TetH, MeCN. iii) 3H-1,2-Benzodithiol-3-one 1,1-dioxide. iv) SiO2 Chromatography
with CH2Cl2/Et3N. v) 80% AcOH.
Scheme 3. Preparation of 2’-C,O6-Dimethylguanosine 3’,5’-Cyclic Phosphorothioate
The intranucleosidic 3’,5’-phosphorothioate linkage of N2-(4-methoxytrityl)-2’-
C,O6-dimethylguanosine 3’,5’-cyclic phosphorothioate (2) was protected with an
esterase-labile (pivaloyloxy)methyl group by treating 2 with iodomethyl pivalate in
MeCN (Scheme 4). The MMTr group was then removed by treatment with aq. 80%

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Scheme 4. Preparation of the of 2’-C,O6-Dimethylguanosine 3’,5’-Cyclic (S-(Pivaloyloxy)methyl Phos-
phorothioate)
AcOH to afford 2’-C,O6-dimethylguanosine cyclic 3’,5’-(O-[(pivaloyloxy)methyl]
phosphorothioate) (3).
To prepare 2’-C,O6-dimethylguanosine 3’,5’-cyclic (O-methyl phosphate) (4), N2-(4-
methoxytrityl)-2’-C,O6-dimethylguanosine (8) was phosphitylated with methyl-N,N-
diisopropyl(chloro)phosphoramidite, and the (iPr)2N ligand then resplaced the 3’-OH
group, as described above. The resulting cyclic phosphite triester was oxidized to
phosphate ester with I2 in aqueous THF containing 2,6-lutidine, and the MMTr group
was removed by AcOH treatment to afford 2’-C,O6-dimethylguanosine cyclic 3’,5’-(O-
methyl phosphate) (4; Scheme 5).
Scheme 5. Preparation of the of 2’-C,O6-Dimethylguanosine 3’,5’-Cyclic (Methyl Phosphate)
Experimental Part
General. Chemicals were purchased from SigmaꢀAldrich, Fluka, and Glen Research. MeCN,
CH2Cl2, THF, and pyridine were dried over 4-ꢂ molecular sieves. Et3N was dried by refluxing over CaH2
and distilled before use. Column chromatography (CC): silica gel 60 (SiO2; 230 – 400 mesh; Fluka).
HPLC: Perkin-Elmer LC 250 with LC-290 UV detector. 1H-, 13C-, and 31P-NMR spectra: Bruker Avance
500 or 400 NMR spectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. HR-ESI-MS: Bruker
Daltonics micrOTOF-Q; in m/z. LC-ESI-MS: Perkin-Elmer Sciex-API-365 triple-quadrupole spectrom-
eter; in m/z.

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N-[(4-Methoxyphenyl)(diphenyl)methyl]-2’-C-methylguanosine (7). 2’-C-Methylguanosine (5;
0.520 g, 1.74 mmol) was co-evaporated 3 times with dry pyridine and dissolved in the same solvent
(10.0 ml). The mixture was cooled on an ice bath, and TMSCl (1.99 ml, 15.74 mmol) was added. The ice
bath was removed, and the mixture was stirred at r.t. for 2 h. The mixture was cooled on an ice bath and
(4-methoxyphenyl)(diphenyl)methyl chloride (MMTrCl; 2.70 g, 8.74 mmol) in pyridine was added. After
stirring overnight (20 h) at r.t., the mixture was cooled on an ice bath, and cold H2O (3 ml) was added.
After 5 min stirring, concentrated aq. NH3 (33%; 5 ml) was added, and stirring was continued for 30 min.
The solvent was removed by evaporation under reduced pressure. The residue was dissolved in CH2Cl2,
and the org. phase was washed with H2O, dried (Na2SO4) and evaporated to dryness. The product was
purified by CC (SiO2; MeOH/CH2Cl2 1:9) to yield 7 (0.37 g, 38%). Solid. 1H-NMR (400 MHz, CD3OD):
8.16 (s, HꢀC(8)); 7.37 – 7.19 (12 H, MMTr); 6.85 (d, J ¼ 8.91, MMTr); 5.30 (s, HꢀC(1’)); 3.97 (d, J ¼ 9.1,
HꢀC(3’)); 3.92 (dd, J ¼ 12.5, 2.1, HꢀC(5’)); 3.87 (m, HꢀC(4’)); 3.75 – 3.72 (m, Me of MMTr, HꢀC(5’’));
0.55 (s, 2’-Me). 13C-NMR (101 MHz, CD3OD): 158.48 (C(6)); 157.97 (MMTr); 151.07 (C(4)); 150.11
(C(2)); 144.94 and 144.85 (MMTr); 136.72 (MMTr); 136.28 (C(8)); 130.04; 128.64, 127.45, 126.45
(MMTr); 116.10 (C(5)); 112.76 (MMTr); 90.17 (C(1’)); 82.36 (C(4’)); 78.89 (C(2’)); 71.56 (C(3’)); 70.38
(Spiro Cq of MMTr); 59.23 (C(5’)); 54.32 (MeO of MMTr); 18.48 (2’-Me). HR-ESI-MS: 570.2381 ([M þ
H]þ, C31H32N5Oþ6 ; calc. 570.2353).
N2-(4-Methoxytrityl)-2’-C,O6-dimethylguanosine (¼6-Methoxy-N-[(4-methoxyphenyl)(diphenyl)-
methyl]-9-(2-C-methyl-b-d-ribofuranosyl)-9H-purin-2-amine; 8). To a precooled soln. of 2’-C,O6-
dimethylguanosine (6; 1.30 g, 4.17 mmol) in dry pyridine, TMSCl (2.65 ml, 20.88 mmol) was added,
and the mixture was stirred at r.t. for 2 h. MMTrCl (1.28 g, 4.17 mmol) was added and the mixture was
kept at 508 for 20 h. The solvent was removed by evaporation under reduced pressure. The residue was
dissolved in CH2Cl2 and washed twice with sat. aq. NaHCO3 and brine, and evaporated to dryness. To
remove the TMS groups, the residue, N2-(4-methoxytrityl)-2’,O6-dimethyl-2’,3’,5’-tris-O-(trimethylsilyl)-
guanosine (ESI-MS (pos.): 800.9 ([M þ H]þ)) was dissolved in dry THF, and Bu4NF hydrate was added.
The soln. was stirred for 90 min at r.t. The mixture was evaporated to dryness, and the residue was
dissolved in CH2Cl2 and treated with sat. aq. NaHCO3. The org. phase was evaporated to dryness. The
residue was purified by CC (SiO2; CH2Cl2 containing 5% MeOH) to give 8 (1.27 g, 53%). White solid.
1H-NMR (500 MHz, CD3OD): 8.27 (s, HꢀC(8)); 7.41 – 7.16 (MMTr); 6.80 (d, J ¼ 5.0, MMTr); 5.77 (br. s,
HꢀC(1’)); 4.11 (d, J ¼ 10.0, HꢀC(3’)); 3.98 – 3.96 (m, HꢀC(4’), HꢀC(5’’)); 3.79 (d, J ¼ 15.0, HꢀC(5’));
3.74 (s, MeO); 3.31 (s, MeO of MMTr); 0.79 (br. s, 2’-Me). 13C-NMR (500 MHz, CD3OD): 160.0 (C(6));
158.30 (MMTr); 152.59 (C(2)); 145.90 and 145.85 (C(4) and MMTr); 137.93 and 139.91 (MMTr and
C(8)); 130.04, 128.64, 127.45 and 126.15 (MMTr); 113.79 (C(5)); 112.49 (MMTr); 90.17 (C(1’)); 82.36
(C(4’)); 78.91 (C(2’)); 71.87 (C(3’)); 70.35 (Cq of MMTr); 59.45 (C(5’)); 54.28 (MeO of MMTr); 53.02
(MeO); 18.79 (2’-Me). HR-ESI-MS: 584.2363 ([M þ Na]þ, C32H34N5O6þ ; calc. 584.2509).
2’-C-Methylguanosine 3’,5’-Cyclic Phosphorothioate (¼(4aR,6R,7R,7aR)-6-(2-Amino-1,6-dihydro-
6-oxo-9H-purin-9-yl)-tetrahydro-7-hydroxy-7-methyl-4H-furo[3,2-d][1,3,2]dioxaphosphinine-2-thiolate
2-Oxide; 1). Compound 7 (250 mg, 0.438 mmol), dried (P2O5) overnight, was dissolved in dry CH2Cl2
(3 ml) under Ar. Dry Et3N (0.288 ml, 2.06 mmol) and 2-cyanoethyl N,N-diisopropyl(chloro)phosphor-
amidite (0.103 ml, 0.46 mmol) were added, and the mixture was stirred at r.t. for 40 min. The product was
isolated by passing the mixture through a short SiO2 column with dry AcOEt containing 0.5% Et3N. The
solvent was removed under reduced pressure. The product was co-evaporated with dry MeCN (2 ꢁ
20 ml) to remove the traces of Et3N. The phosphitylated nucleoside was dissolved in dry MeCN
(60 ml) under Ar, and 1H-tetrazole (1.03 mmol, 2.25 ml of 0.45 mol/dm3 soln. in MeCN) was added. The
mixture was stirred for 3 h, and then 3H-1,2-benzodithiol-3-one 1,1-dioxide (185 mg, 0.877 mmol) was
added, and stirring was continued overnight. The mixture was evaporated to dryness. The crude was
purified by CC (SiO2; CH2Cl2 containing 15% MeOH and 0.5% Et3N). The 2-cyanoethyl group was
cleaved during CC and, hence, N2-(4-methoxytrityl)-2’-C-methylguanosine 3’,5’-cyclic phosphorothioate
was obtained as white solid in 32% yield (90 mg).
To remove the MMTr group, N2-(4-methoxytrityl)-2’-C-methylguanosine 3’,5’-cyclic phosphoro-
thioate (71 mg, 0.08 mmol) was treated with aq. 80% AcOH (2.0 ml) at r.t., and the mixture was stirred
overnight. The mixture was evaporated to dryness, and the residue was co-evaporated twice with H2O.
The crude was purified CC(SiO2;CH2Cl2 containing 10% MeOH). The triethylammonium salt of 1 was

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Helvetica Chimica Acta – Vol. 95 (2012)
obtained as solid form in 73% (38 mg). The 13C-NMR spectra of 1 could not be obtained due to a poor
solubility. For the same reason, the triethylammonium cation could not be exchanged by cation exchange
chromatography.1H-NMR (400 MHz, D2O): 7.90 and 7.84 (br. s, HꢀC(8)); 5.90 (br. s, HꢀC(1’)); 4.97
(HꢀC(3’)); 4.50 – 4.35 (HꢀC(4’), HꢀC(5’’)); 4.21 (HꢀC(5’)); 3.37 (br. s, 2’-OH) and 1.00 (br. s, 2’-Me).
31P-NMR (162 MHz, DMSO): 56.28 and 55.06. HR-ESI-MS: 398.0299 ([M þ Na]þ, C11H14N5NaO6PSþ;
calc. 398.0300).
N2-(4-Methoxytrityl)-2’,O6-dimethylguanosine 3’,5’-Cyclic O-(2-Cyanoethyl) Phosphorothioate (¼ 3-
{[(4aR,6R,7R,7aR)-Tetrahydro-7-hydroxy-6-(6-methoxy-2-{[(4-methoxyphenyl)(diphenyl)methyl]ami-
no}-9H-purin-9-yl)-7-methyl-2-sulfido-4H-furo[3,2-d][1,3,2]dioxaphosphinin-2-yl]oxy}propanenitrile;
9). Compound 8 (0.625 g, 1.07 mmol) was dried (P2O5) overnight and dissolved in dry CH2Cl2 (5 ml).
Anh. Et3N (0.75 ml, 5.39 mmol) and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (0.26 ml,
1.17 mmol) were added, and the mixture was stirred for 60 min under Ar at r.t. The phosphitylated
product was isolated by passing the mixture through a short SiO2 column with dry AcOEt containing
0.5% Et3N. The solvent was removed under reduced pressure, and the crude was co-evaporated twice
with dry MeCN. The residue was dissolved in dry MeCN (60 ml) and 0.45 mol/dm3 soln. of 1H-tetrazole
in MeCN (5.95 ml, 5.80 mmol) was added. After 2.5 h stirring at r.t., dry pyridine (80 ml) and elemental
sulfur (245 mg, 7.65 mmol) were added, and stirring was continued overnight. After filtration, the mixture
was evaporated to dryness. The residue was dissolved in CH2Cl2, and the org. layer was washed with aq.
NaHCO3 and brine, dried (Na2SO4), and evaporated to dryness. The crude was purified by CC (SiO2;
CH2Cl2 containing 3% MeOH and 0.5% pyridine). Compound 9 was obtained as a mixture of (RP)- and
1
(SP)-diastereoisomers in 21% yield (156 mg by repeating the synthesis). H-NMR (500 MHz, CDCl3):
7.58 (s, HꢀC(8)); 7.38 – 7.20 (m, MMTr); 6.85 – 6.78 (m, MMTr); 6.53 (s, HꢀC(1’)); 5.76 (br. s, NH); 4.55 –
4.37 (m, HꢀC(3’), HꢀC(5’), HꢀC(5’’), CH2O); 3.90 (dd, J ¼ 6.3 and 6.3, HꢀC(4’)); 3.83 (s, MMTr, 6-
MeO); 2.82 – 2.75 (m, CH2CN); 1.09 (s, 2’-Me). 13C-NMR (126 MHz, CDCl3): 160.50 (C(6)); 158.20 and
158.20 – 158.13 (MMTr); 158.10 (C(4)); 152.17 (C(2)); 145.80 (C(8)); 144.46 (MMTr); 135.69 (MMTr);
130.5 – 126.5 (MMTr); 118.32 (CN); 116.62 (C(5)); 113.5 – 113.0 (MMTr); 94.70 (C(1’)); 86.76 (Cq of
MMTr); 80.90 (C(3’)); 70.61 and 70.05 (C(4’), C(2’)); 57.67 (C(5’)); 55.29 (MeO of MMTr); 54.05
(MeO); 51.99 (CH2O); 19.73 (2’-Me); 19.58 (CH2CN). 31P-NMR (202 MHz, CD3OD): 83.57 and 67.84.
HR-ESI-MS: 715.2087 ([M þ H]þ, C35H36N6O7PSþ; calc. 715.2104).
2’-C,O6-Dimethylguanosine 3’,5’-Cyclic Phosphorothioate (¼(4aR,6R,7R,7aR)-6-(2-Amino-6-meth-
oxy-9H-purin-9-yl)-tetrahydro-7-hydroxy-7-methyl-4H-furo[3,2-d][1,3,2]dioxaphosphinine-2-thiolate 2-
Oxide; 2). To remove the 2-cyanoethyl group, 9 (230 mg, 0.32 mmol) was dissolved in a mixture of dry
CH2Cl2 (1.5 ml) and dry Et3N (2.0 ml). The mixture was stirred at r.t. for 18 h, evaporated to dryness, and
co-evaporated with dry MeCN. N2-(4-Methoxytrityl)-2’-C,O6-dimethylguanosine 3’,5’-cyclic phosphoro-
thioate was obtained as white solid foam and used without further purification. The residue was dissolved
in 80% aq. AcOH (10 ml), and the mixture was stirred at r.t. for 5 h and evaporated to dryness. The crude
product was purified by HPLC eluting with 15% MeOH in H2O. The diastereoisomers were not
separated. The product was dissolved in H2O and passed through Dowex Naþ-form (50 W, 100 – 200
1
mesh). The product was obtained as white solid in 32% yield (42 mg). H-NMR (500 MHz, CD3OD):
7.95 (s, HꢀC(8)); 5.94 (s, HꢀC(1’)); 4.61 – 4.22 (HꢀC(3’), HꢀC(4’), HꢀC(5’’), HꢀC(5’)); 4.07 (s, 6-MeO);
1.06 (s, 2’-Me). 13C-NMR (126 MHz, CD3OD): 161.34 (C(6)); 160.34 (C(2)); 153.00 (C(4)); 138.12
(C(8)); 114.25 (C(5)); 94.33 (C(1’)); 80.40 (C(3’)); 77.06, 77.29 (C(2’)); 72.50 (C(4’)); 66.90, 69.85 (C(5’));
52.80 (MeO); 18.51 (2’-Me). 31P-NMR (202 MHz, DMSO): 55.97. HR-ESI-MS: 388.0482 ([M ꢀ H]ꢀ,
C12H15N5O6PSꢀ; calc. 388.0486).
2’-C,O6-Dimethylguanosine Cyclic S-3’,5’-([(Pivaloyloxy)methyl] Phosphorothioate) (¼{[(4aR,
6R,7R,7aR)-6-(2-Amino-6-methoxy-9H-purin-9-yl)-tetrahydro-7-hydroxy-7-methyl-2-oxido-4H-fu-
ro[3,2-d][1,3,2]dioxaphosphinin-2-yl]sulfanyl}methyl 2,2-Dimethylpropanoate; 3). Chloromethyl piva-
late (1.0 ml, 6.90 mmol) was added to a mixture of NaI (2.08 g, 13.80 mmol) and dry MeCN (10 ml). The
mixture was stirred at r.t. overnight in dark. The mixture was evaporated to dryness, dissolved in CH2Cl2,
and washed with 5% aq. NaHSO3 and brine. The org. layer was dried (Na2SO4) and evaporated to
dryness. The iodomethyl pivalate obtained was used as such for the next step. N2-(4-Methoxytrityl)-2’-
C,O6-dimethylguanosine 3’,5’-cyclic phosphorothioate was dissolved in dry MeCN (3 ml), and the
iodomethyl pivalate (56 mg, 0.23 mmol) was added. The mixture was stirred for 2.5 h at r.t. Sat. aq.

Helvetica Chimica Acta – Vol. 95 (2012)
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NaHCO3 soln. was added, and the crude product was extracted with CH2Cl2. The org. layer was dried
(Na2SO4) and evaporated to dryness. The residue was dissolved in 80% aq. AcOH (2.0 ml), and the
mixture was stirred at r.t. for 20 h. The mixture was evaporated to dryness and, the residue was co-
evaporated twice with H2O. The crude product 3 was purified by CC (SiO2;CH2Cl2 containing 10%
1
MeOH) to give 3 (15 mg, 14%). White solid. H-NMR (500 MHz, CD3OD): 7.95 (s, HꢀC(8)); 5.93 (s,
HꢀC(1’)); 5.58 – 5.54 (m, SCH2); 4.80 – 4.69 (m, HꢀC(3’), HꢀC(4’), HꢀC(5’’)); 4.45 (m, HꢀC(5’)); 4.06 (s,
MeO); 1.20 (s, C(Me)3); 1.10 (s, 2’-Me). 13C-NMR (126 MHz, CD3OD): 177.48 (C¼O); 161.50 (C(6));
160.22 (C(2)); 152.66 (C(4)); 139.14 (C(8)); 129.34 (C(5)); 95.15 (C(1’)); 81.87 (C(3’)); 76.76 and 76.70
(C(2’)); 71.00, 70.93, 70.80 and 70.81 (C(4’), C(5’)); 60.23, 60.20 (SCH2); 52.87 (MeO); 38.52 (Me3C);
25.85 (Me3C); 18.18 (2’-Me). 31P-NMR (202 MHz, CD3OD): 23.13. HR-ESI-MS: 504.1323 ([M þ H]þ,
C18H27N5O8PSþ; calc. 504.1318).
2’-C,O6-Dimethylguanosine Cyclic 3’,5’-(O-Methyl Phosphate) (¼(4aR,6R,7R,7aR)-6-(2-Amino-6-
methoxy-9H-purin-9-yl)-tetrahydro-2-methoxy-7-methyl-4H-furo[3,2-d][1,3,2]dioxaphosphinin-7-ol 2-
Oxide; 4). Compound 8 (1.80 mmol, 1.05 g) dried (P2O5) overnight was dissolved in dry CH2Cl2 (8 ml)
under N2 . Dry Et3N (9.00 mmol, 1.25 ml) and methyl N,N-diisopropylchlorophosphoramidite
(1.99 mmol, 385 ml) were added, and the mixture was stirred at r.t. for 30 min. The product was isolated
by passing the mixture through a short SiO2 with dry hexane containing 30% dry AcOEt and 0.5% dry
Et3N. The solvent was removed under reduced pressure, and the residue was co-evaporated three times
with dry MeCN to remove the traces of Et3N. The phosphitylated nucleoside was dissolved in dry MeCN
(70 ml) under N2, and 1H-tetrazole (4.50 mmol, 10.00 ml of 0.45 mol/dm3 soln. in MeCN) was added.
The mixture was stirred for 3 h. After 2 h stirring at r.t., I2 (0.1m) in a mixture of THF, H2O, and 2,6-
lutidine (4 :2 :1 (v/v/v); 7 ml) was added, and stirring was continued overnight (16 h). The 3’,5’-cyclic O-
methyl phosphate of N2-(4-methoxytrityl)-2’-C,O6-dimethylguanosine was isolated by CH2Cl2/5% aq.
NaHSO3 workup and subsequent CC(SiO2; CH2Cl2 containing 5% MeOH). The identity of the product
was verified by HR-ESI-MS (660.2207 ([M þ H]þ; calc. 660.2218)). To remove the MMTr group, the
product (0.53 mmol, 0.35 g) was dissolved in aq. 80% AcOH (5.0 ml), and the mixture was stirred
overnight at r.t. (24 h). The mixture was evaporated to dryness, and the residue was co-evaporated twice
with H2O. The diastereoisomers were separated by HPLC eluting with 39% aq. MeOH. The product was
obtained in 60% yield (60 mg þ 63 mg). 1H-NMR (500 MHz, CD3OD): 8.00 (s, HꢀC(8)); 6.02 (s,
HꢀC(1’)); 4.71 – 4.66 (m, HꢀC(3’), HꢀC(4’), HꢀC(5’’)); 4.40 (m, HꢀC(5’)); 4.07 (s, COMe); 3.94, 3.91
(2s, POMe); 1.09 (s, 2’-Me). 13C-NMR (126 MHz, CD3OD): 161.40 (C(6)); 160.52 (C(2)); 153.00 (C(4));
138.14 (C(8)); 114.32 (C(5)); 94.53 (C(1’)); 82.56, 82.49 (C(3’)); 76.64, 76.59 (C(2’)); 70.75, 70.71, 69.99,
69.89 (C(4’), C(5’)); 53.65, 53.60 (POMe); 52.90 (COMe); 18.29 (2’-Me). 31P-NMR (202 MHz, CD3OD):
ꢀ 3.56. HR-ESI-MS: 388.0992 ([M þ H]þ, C13H19N5O7Pþ; calc. 388.1022).
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Received February 29, 2012