Synthesis and enzymatic deprotection of biodegradably protected dinucleoside-2′,5′-monophosphates: 3-(Acetyloxy)-2,2- bis(ethoxycarbonyl)propyl phosphoesters of 3′-O-(acyloxymethyl)adenylyl- 2′,5′-adenosines

Article abstract of DOI:10.1002/cbdv.201000288

As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2-5A and its congeners, adenylyl-2′, 5′-adenosines bearing a 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group at the phosphate moiety, and an (acetyloxy)methyl- or a (pivaloyloxy)methyl- protected 3′-OH group of the 2′-linked nucleoside have been prepared. The enzyme-triggered removal of these protecting groups by hog liver carboxyesterase at pH 7.5 and 37° has been studied. The (acetyloxy)methyl group turned out to be too labile for the 3′-O-protection, being removed faster than the phosphate-protecting group, which results in 2′,5′- to 3′,5′-isomerization of the internucleosidic phosphoester linkage. In addition, the starting material was unexpectedly converted to the 5′-O-acetylated derivative. (Pivaloyloxy)methyl group appears more appropriate for the purpose. The fully deprotected 2′,5′-ApA was accumulated as a main product, although, even in this case, the isomerization of the starting material takes place.

Full text of DOI:10.1002/cbdv.201000288

266
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Synthesis and Enzymatic Deprotection of Biodegradably Protected
Dinucleoside-2’,5’-monophosphates: 3-(Acetyloxy)-2,2-
bis(ethoxycarbonyl)propyl Phosphoesters of 3’-O-(Acyloxymethyl)adenylyl-
2’,5’-adenosines
by Emilia Kiuru*a), Mikko Oraa), Leonid Beigelmanb), Lawrence Blattb), and Harri Lçnnberga)
a) Department of Chemistry, University of Turku, FI-20014 Turku
(fax: þ35823336700; e-mail: emilia.kiuru@utu.fi)
b) AliosBiopharma, 260 E. Grand Ave, 2nd Floor, South San Francisco, CA 94080, USA
As a first step towards a viable prodrug strategy for short oligoribonucleotides, such as 2–5A and its
congeners, adenylyl-2’,5’-adenosines bearing a 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group at the
phosphate moiety, and an (acetyloxy)methyl- or a (pivaloyloxy)methyl-protected 3’-OH group of the 2’-
linked nucleoside have been prepared. The enzyme-triggered removal of these protecting groups by hog
liver carboxyesterase at pH 7.5 and 378 has been studied. The (acetyloxy)methyl group turned out to be
too labile for the 3’-O-protection, being removed faster than the phosphate-protecting group, which
results in 2’,5’- to 3’,5’-isomerization of the internucleosidic phosphoester linkage. In addition, the starting
material was unexpectedly converted to the 5’-O-acetylated derivative. (Pivaloyloxy)methyl group
appears more appropriate for the purpose. The fully deprotected 2’,5’-ApA was accumulated as a main
product, although, even in this case, the isomerization of the starting material takes place.
Introduction. – The 5’-triphosphates of 2’,5’-oligoadenylates, collectively known as
2–5A, are activators of an intracellular endoribonuclease RNase L, which cleaves both
messenger and ribosomal RNA, resulting in apoptosis [1][2]. The formation of 2–5A is
triggered by interferon in response to various stimuli, including viral infection.
Interferon induces 2–5A synthetase that, upon activation by double-stranded RNA,
converts ATP into 2’,5-linked oligoribonucleotides, and 2–5A phosphodiesterase then
produces 2–5A from longer oligomers [3]. Accordingly, activation of RNase L by
synthetic 2–5A might offer a way to combat against viral diseases. In addition, 2–5A
activation has been reported to result in apoptosis in metastatic prostate cell lines [4].
The major limitations of 2–5A for therapeutic applications include short biological
half-life and ionic structure that leads to insufficient internalization to cytoplasm [3].
Numerous structurally modified analogues of 2–5A have been prepared to increase the
stability in serum and cytoplasm [5–12], but no prodrug strategy for the enhancement
of cellular uptake has been introduced. In principle, masking of the negative charges of
the sugar-phosphate backbone with biodegradable lipophilic protecting groups should
improve the internalization, but the presence of the 3’-OH groups complicates the
situation.
The attack of the neighboring OH group on the protected phosphodiester linkage is
extremely fast, resulting in both 2’!3’ phosphate migration and cleavage of the P-O5’
bond [13]. Accordingly, the 3’-OH functions must also be protected, and these
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
267
protecting groups should be removed more slowly than the protecting groups of the
phosphodiester linkages. As a first step towards a viable prodrug strategy for 2–5A, we
now report on our studies on a dimeric model compound, viz. adenylyl-2’,5’-adenosine,
which has both the phosphodiester linkage and the 3’-OH group of the 2’-linked
nucleoside protected with an esterase-labile protecting group. For the phosphate
protection, 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl group [14] has been used. This
group undergoes esterase-catalyzed deacetylation, and the remnants of the group are
removed by retro-aldol condensation and concomitant phosphate elimination. Two
different esterase-labile groups, viz. a (pivaloyloxy)methyl and an (acetyloxy)methyl
(AcOCH2) group, have been used for the protection of the neighboring OH function to
find out whether a successful deprotection strategy could be based on one of these
groups. As mentioned above, the phosphate-protecting group should be removed
before exposure of the 3’-OH group, and the enzymatic removal of the (pivaloyloxy)-
methyl group expectedly is slower than the removal of the AcOCH2 group. For this
purpose, protected dinucleoside-2’,5’-monophosphates 1a and 1b have been prepared,
and their deprotection by hog liver carboxyesterase (HLE) has been studied.
Results and Discussion. – Syntheses. 3-(Acetyloxy)-2,2-bis(ethoxycarbonyl)propyl
protected 3’-O-(acyloxymethyl)adenylyl-2’,5’-adenosines, 1a and 1b, were assembled
from the appropriately protected nucleosides and 2-(acetyloxymethyl)-2-(hydroxyme-
thyl)malonate as outlined in Scheme 1. Accordingly, the 2’-OH function of 3’-O-
(acetyloxymethyl)-N6-(4-methoxytrityl)adenosine (2a) or its 3’-O-(pivaloyloxymethyl)
counterpart (2b) was first phosphitylated with bis(diethylamino)phosphorochloridite
((Et2N)2PCl) in the presence of Et3N, and the remaining Et2N ligands were then
sequentially replaced with 2’,3’-di-O-levulinoyl-N6-(4-methoxytrityl)adenosine (4) and
diethyl 2-(acetyloxymethyl)-2-(hydroxymethyl)malonate (5) using 1H-tetrazole as an
activator. The resulting phosphite triesters 6a and 6b were oxidized to phosphate esters
7a and 7b, respectively, with I2 in aqueous THF containing 2,6-lutidine. The course of
the phosphitylation, coupling, and oxidation steps described above were monitored by
31P-NMR spectroscopy. The formation of N,N,N’,N’-tetraethylphosphorodiamidites, 3a

268
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Scheme 1
i) (Et2N)2PCl, Et3N, CH2Cl2. ii) 1H-Tetrazole (TetH), MeCN. iii) TetH, MeCN. iv) I2, THF, H2O, 2,6-
lutidine. v) Bu4NF, AcOH, THF. vi) NH2NH3OAc, MeOH, CH2Cl2 for 8a; NH2NH2, AcOH, pyridine for
8b. vii) 80% AcOH.
and 3b, was accompanied by appearance of a 31P-NMR resonance at 137.6 and
137.2 ppm, respectively. The phosphite triester 6a resonated at 140.7 and 140.5 ppm
((RP)- and (SP)-diastereoisomers, resp.) and 6b at 140.6 and 140.5 ppm, and the
phosphate triesters, 7a and 7b, at ꢀ2.4 and ꢀ2.5, and ꢀ2.4 and ꢀ2.6 ppm,
respectively. The silyl group of the fully protected dimer 7b was removed with Bu4NF

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
269
in the presence of AcOH to yield 8b. Compound 7a was desilylated to 8a with Et3N·3
HF, to keep the AcOCH2 group intact [15]. The levulinoyl groups were finally removed
with NH2NH3OAc and the 4-methoxytrityl groups with aqueous AcOH, to give 1a and
1b as a mixture of (RP)- and (SP)-diastereoisomers. Fig. 1 shows the RP-HPLC traces of
the purified products 1a and 1b.
Fig. 1. Analytical RP-HPLC traces of a) the two diastereoisomers of 3-(acetyloxy)-2,2-bis(ethoxycarbo-
nyl)propyl-protected 3’-O-(acyloxymethyl)adenylyl-2’,5’-adenosines 1a (tR 32.77 and 35.08 min) and b) 1b
(tR 41.52 and 44.35 min) at 260 nm. Thermo ODS Hypersil C18 column (4ꢁ250 mm, 5 mm; flow rate
0.95 ml minꢀ1); buffer: AcOH/AcONa 0.045 :0.015 mol lꢀ1), containing NH4Cl (0.1 mol lꢀ1); buffer A:
buffer in 2% MeCN, buffer B: buffer in 60% MeCN. Isocratic elution with A for 5 min and gradient
elution from 0 to 71% B in 45 min.

270
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
5’-O-[(tert-Butyl)(dimethyl)silyl]-N6-(4-methoxytrityl)-3’-O-(pivaloyloxymethyl)-
adenosine (2b) used for the assembly of the corresponding phosphate-protected
dinucleoside monophosphate, 1b, was obtained as depicted in Scheme 2. Adenosine
was converted to its 5’-O,N6-bis(4-methoxytrityl) derivative 9 [16] and subjected to
alkylation with (pivaloyloxy)methyl chloride to obtain a mixture of 2’-O- and 3’-O-
(pivaloyloxymethyl) derivatives. The latter reaction turned out to be somewhat
difficult. The best yield was obtained by deprotonating the OH group with 1 equiv. of
NaH in THF and then treating the nucleoside with (pivaloyloxy)methyl chloride in the
presence of a catalytic amount of NaI. Even then the 3’-O-alkylated product 10 was
obtained only in a 21% total yield. The yield of the undesired 2’-O-alkylated product
was 7%. Unexpectedly, a significant amount of 3’-O- and 2’-O-pivalates was formed,
which explains the low yield of the pivaloyloxymethylated products. The 2’-OH
function of 10 was then protected with a levulinoyl group, and the 4-methoxytrityl
groups were removed with acid to obtain 11. The exposed 5’-OH function was protected
t
with a BuMe2Si group and the 6-amino group with a 4-methoxytrityl group to give
compound 12. Removal of the levulinoyl group afforded the desired product 2b.
Scheme 2
i) Monomethoxytrityl chloride (¼(4-methoxyphenyl)(diphenyl)methyl chloride) (MMTrCl), pyridine.
ii) PivOCH2Cl (¼(pivaloyloxy)methyl chloride¼(2,2-dimethylpropanoyl)methyl chloride), NaH, NaI,
THF. iii) 1. Lev2O (Lev¼levunoyl¼4-oxopentanoyl), dioxane, pyridine; 2. 80% AcOH. iv) 1. tBuMe2-
SiCl (TBDMSCl), pyridine; 2. MMTrCl, pyridine. v) NH2NH2, AcOH, pyridine.
Attempts to prepare the corresponding 3’-O-(acetyloxymethyl) derivative 2a in a
similar manner failed. This nucleoside was obtained as depicted in Scheme 3.
Adenosine was converted to 2’-O,5’-O,N6-tris(4-methoxytrityl)adenosine (13) by
treating with 3 equiv. of 4-methoxytrityl chloride in pyridine. Compound 13 was
deprotonated with 1 equiv. of NaH and alkylated with MeSCH2Cl to obtain the 3’-O-
[(methylthio)methyl] derivative 14. After acid-catalyzed detritylation to 15, the 5’-O
was protected with a tBuMe2Si group, and the N6-atom with a 4-methoxytrityl group to

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
271
yield 16. 2’-O Position was protected with a levulinoyl group to obtain 17. The MeSCH2
group was transformed to a ClCH2 group with SO2Cl2 and then to an AcOCH2 group
(!18) with AcOK in the presence of dibenzo-18-crown-6. Removal of the levulinoyl
group with NH2NH3OAc in MeOH/CH2Cl2 gave 2a, leaving the AcOCH2 group intact.
Scheme 3
i) MMTrCl, pyridine. ii) 1. NaH, DMF, 2. MeSCH2Cl, DMF. iii) 80% AcOH. iv) 1. TBDMSCl, pyridine,
2. MMTrCl, pyridine. v) Lev2O, dioxane, pyridine. vi) 1. SO2Cl2, CH2Cl2, 2. AcOK, dibenzo-18-crown-6,
CH2Cl2. vii) H2NNH3OAc, CH2Cl2, MeOH.
The 5’-linked nucleoside 4 was obtained by protecting the 2’- and 3’-OH groups of
5’-O,N6-bis(4-methoxytrityl)adenosine (9) with levulinoyl groups, detritylating the
product, and re-introducing the 4-methoxytrityl group at N6 by using Me3Si group for a
transient protection [17] at 5’-O (Scheme 4). The preparation of diethyl 2-(acetyloxy-
methyl)-2-(hydroxymethyl)malonate (5) has been described previously [14e].
Scheme 4
i) 1. Lev2O, dioxane, pyridine, 2. 80% AcOH. ii) 1. Me3SiCl, pyridine, 2. MMTrCl, pyridine, 3. Bu4NF,
AcOH, THF.

272
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Enzymatic Deprotection. The deprotection of protected adenylyl-2’,5’-adenosines
1a and 1b was followed in HEPES buffer at pH 7.5 and 378 in the presence of hog liver
carboxyesterase (HLE; 2.6 units mlꢀ1). The composition of the aliquots withdrawn at
appropriate intervals from the mixture was determined by RP-HPLC, and the products
were characterized by mass spectrometric analysis (HPLC/ESI-MS) and/or by spiking
with authentic samples (in the case of 2’,5’-ApA, 3’,5’-ApA, adenosine, and inosine).
Fig. 2 shows the HPLC traces for the mixture of the enzymatic deprotection of 3’-O-
(acetyloxymethyl)adenosin-2’-yl adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)
phosphate (1a) at various times after the addition of the enzyme. These data show that
three initial products are formed in parallel: deacetylated phosphotriester 20a,
phosphate-protected adenylyl-2’,5’-adenosine 21, and a monoacetylated derivative of
21, most likely the 5’-O-Ac derivative 21-Ac (Scheme 5). Triester 20a then gives diester
22a by a HOꢀ ion-catalyzed loss of HCHO and concomitant elimination of the remnant
Fig. 2. RP-HPLC Traces for the HLE-catalyzed hydrolysis of 3’-O-(acetyloxymethyl)adenosin-2’-yl
adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate (1a) at pH 7.5 and 37.08 (I¼
0.1 mol lꢀ1 with NaCl). For the chromatographic conditions, see the Exper. Part.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
273
Scheme 5. HLE-Triggered Deprotection

274
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
of the phosphate-protecting group as diethyl 2-methylidenemalonate [14a]. Finally,
enzymatic deacetylation of the 3’-O-(acetyloxymethyl) group, followed by rapid
hydrolysis of the resulting half-acetal gives the desired product, 2’,5’-ApA (23).
The competing route via the 3’-O-deprotected triester 21 also yields 2’,5’-ApA (23),
besides several additional products, viz. 3’,5’-ApA (24), and adenosine 2’-O- and 3’-O-
[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] phosphates 25 and 26, respectively. All
these compounds are formed via a phosphorane intermediate obtained by an attack of
the 3’-OH function at the P-atom. This intermediate is decomposed, in addition to
acyclic 2’,5’- and 3’,5’-phosphotriesters, to two different 2’,3’-cyclic phosphotriesters
obtained by removal of either the phosphate protecting group or the 5’-linked
nucleoside. Hydrolysis of these cyclic esters then gives 2’,5’- and 3’,5’-ApA (23 and 24,
resp.), and diesters 25 and 26. The mechanisms of the hydrolysis of ribonucleoside 2’-
and 3’-phosphotriesters have been discussed previously in more detail [13][18].
Consistent with earlier findings [14e], the enzymatic deacetylation of 25 and 26 is much
slower than the deacetylation of triester 1a, and they are, hence, accumulated as
virtually stable products.
In spite of the fact that 2’,5’-ApA (23) is formed via both of the routes indicated in
Scheme 5, its proportion in the product mixture remains very low. The main reason is
that, in addition to the products mentioned above, monoacetylated derivatives of
products 23–26 were accumulated in a considerable amount. A likely explanation for
this unexpected finding is that the Ac group of the 3’-O-(acetyloxymethyl) moiety
migrates to the 5’-OH group. Accordingly, parallel to the formation of 22a and 21, the
starting material is converted to the 5’-O-acetylated derivative of 21, which is then
hydrolyzed along the pathway depicted for the breakdown of 21. As seen from Fig. 2,
upon 95% disappearance of the diastereoisomeric starting material, the main products
were adenosine (25%) and acetylated derivatives of 2’,5’-ApA (8%) and 3’,5’-ApA
(15%), and acetylated diesters 25 and 26 (23%). In addition, small amounts of 3’,5’-
ApA (24; 3%), 2’,5’-ApA (23; 6%), diesters 25 and 26 (8%), and intermediate 22a
(3%) were accumulated. Even though this kind of Ac migration is not possible with 2–
5A; owing to the absence of free 5’-OH function, the removal of the protecting group at
3’-O still appears too facile compared to the phosphate deprotection to afford a viable
pro-drug strategy for 2’,5’-ApA (23).
To decelerate the removal of the 3’-O-protecting group, the AcOCH2 group was
replaced with a more bulky (pivaloyloxy)methyl group. Fig. 3 shows the HPLC traces
for the mixture of the enzymatic deprotection of 3’-O-(pivaloyloxymethyl)adenosin-2’-
yl adenosine-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl) phosphate (1b). These data
reveal that even the 3’-O-(pivaloyloxy)methyl group is removed somewhat too fast
compared to the removal of the phosphate-protecting group to allow exposure of 2’,5’-
ApA (23) as the sole product. In fact, deprotection of the 3’-OH function is twice as
rapid as the phosphate deprotection, but, since also the former route yields 23 in
addition to its 3’,5’-isomer, 24 and diesters 25 and 26, 23 is the main product of the
enzymatic deprotection. As seen from Fig. 3, virtually all the starting material, 1b, had
been disappeared in 24 h. At this stage, the product mixture consisted of diesters 22b
(24%), 25 (12%), and 26 (12%) in addition to 2’,5’-ApA (23; 20%), 3’,5’-ApA (24;
8%), adenosine (16%), and inosine (7%). Owing to slow enzymatic conversion of 22b
to 23, the proportion of the latter compounds was, however, increased with time, being

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
275
Fig. 3. RP-HPLC Traces for the PLE-catalyzed hydrolysis of 3’-O-(pivaloyloxymethyl)adenosin-2’-yl
adenosine-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate (1b) at pH 7.5 and 37.08 (I¼
0.1 mol lꢀ1 with NaCl). For the chromatographic conditions, see the Exper. Part.
finally 37%. Prolonged treatment with HLE also resulted in partial deamination of
adenosine to inosine.
Conclusions. – Protected adenylyl-2’,5’-adenosines bearing a 3-(acetyloxy)-2,2-
bis(ethoxycarbonyl)propyl group at the phosphate moiety and an (acetyloxy)methyl
(i.e., 1a) or a (pivaloyloxy)methyl group (i.e., 1b) at the neighboring 3’-O have been
prepared. During the HLE-catalyzed deprotection of 1a, the AcOCH2-protected
compound was unexpectedly converted to an acetylated one, most likely by migration
of the Ac group to 5’-O of the 2’-linked nucleoside and hydrolytic removal of the
remaining 3’-O-(hydroxymethyl) group. The exposed 3’-OH function readily attacks on
the still protected phosphate linkage, resulting in cleavage and isomerization of the
2’,5’-phosphoester linkage to a 3’,5’-linkage. Accordingly, 2’,5’-ApA (23) is released
only as a minor product. With the 3’-O-(pivaloyloxymethyl)-protected compound, 1b,
the desired 2’,5’-ApA (23) is the main product, although, even in this case, the
departure of the 3’-O-(pivaloyloxymethyl) group competes with the deprotection of
the phosphate group, leading to partial isomerization and cleavage of the internucleo-
sidic phosphoester linkage.
The authors thank Ms. Anna Rossi, B. Sc., for her skillful assistance on HPLC analyses.

276
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Experimental Part
General. Chemicals were purchased from SigmaꢀAldrich, Fluka, and Merck. MeCN, CH2Cl2, and
pyridine were dried over 4-ꢂ molecular sieves. Dioxane was dried over 3-ꢂ molecular sieves. Et3N was
dried by refluxing over CaH2 and distilled before use. Column chromatography (CC): Fluka silica gel 60
(SiO2; 230–400 mesh). 1H-, 13C-, 31P-, and 2D-NMR spectra: Bruker Avance 500 NMR spectrometer; the
chemical shifts are given in ppm with reference to internal TMS; the coupling constants Jare given in Hz.
LC/ESI-MS: Perkin-Elmer Sciex-API-365 triple-quadrupole. HPLC: Merck Hitachi LaChrom D7000
with L-7455 UV-detector and L-7100 pump. HR-ESI-MS: Bruker Daltonics micrOTOF-Q.
5’-O,N6-Bis[(4-methoxyphenyl)(diphenyl)methyl]adenosine; 9). Adenosine (26.2 mmol, 7.00 g) was
co-evaporated twice from dry pyridine and dissolved in the same solvent (40 ml). A soln. of 4-
methoxytrityl chloride (MMTrCl; 57.7 mmol, 17.8 g) in pyridine (60 ml) was added, and the mixture was
stirred overnight at 358. MeOH (25 ml) was added, and the stirring was continued for 0.5 h. The solvent
was removed by evaporation under reduced pressure, and the residual oil was partitioned between H2O
and CHCl3. The org. layer was washed with sat. aq. NaHCO3 and sat. aq. NaCl, dried (Na2SO4) and
evaporated to dryness. The residue was co-evaporated with toluene and purified by SiO2 chromatography
1
using CH2Cl2 containing 1–2% MeOH as eluent to afford 9 (15.2 g, 71%). Yellowish foam. H-NMR
(500 MHz, CDCl3): 8.07 (s, HꢀC(8)); 8.02 (s, HꢀC(2)); 7.17–7.39 (m, 24 H of MMTr); 7.09 (br. s,
HNꢀC(6)); 6.79–6.84 (m, 4 H of MMTr); 6.64 (br. s, HOꢀC(2’)); 5.95 (d, J¼6.0, HꢀC(1’)); 4.79 (m,
HꢀC(2’)); 4.42–4.44 (m, HꢀC(4’)); 4.37 (dd, J¼5.5, 2.0, HꢀC(3’)); 3.80 (s, 2 MeO of MMTr); 3.70 (br. s,
HOꢀC(3’)); 3.49 (dd, J¼10.5, 3.5, 1 H of CH2(5’)); 3.25 (dd, J¼10.5, 3.5, 1 H of CH2(5’)). 13C-NMR
(126 MHz, CDCl3): 158.7 (MMTr); 158.4 (MMTr); 154.3 (C(6)); 151.6 (C(2)); 147.9 (C(4)); 145.0, 143.9
(MMTr); 138.1 (C(8)); 137.1, 134.8, 130.4, 130.2, 128.9, 128.2, 127.9, 127.0 (MMTr); 121.2 (C(5)); 113.2
(MMTr); 91.0 (C(1’)); 86.8 (C(4’)); 86.5 (MMTr); 76.1 (C(2’)); 72.9 (C(3’)); 71.1 (MMTr); 63.7 (C(5’));
55.2 (MeO of MMTr). HR-ESI-MS: 812.3420 (C50H46N5Oþ6 ; calc. 812.3443).
5’-O,N6-Bis(4-methoxytrityl)-3’-O-(pivaloyloxymethyl)adenosine (¼ 3’-O-{[(2,2-Dimethylpropanoyl)-
oxy]methyl}-N-[(4-methoxyphenyl)(diphenyl)methyl]-5’-O-[(4-methoxyphenyl)(diphenyl)methyl]ade-
nosine; 10). Compound 9 (0.60 mmol, 0.49 g), dried over P2O5 overnight, was dissolved in dry THF
(5 ml), and 1 equiv. of NaH (24 mg of 60% dispersion, 0.60 mmol) was added. After stirring for 1 h at r.t.,
the mixture was added into a mixture of (pivaloyloxy)methyl chloride (0.66 mmol, 94 ml) and NaI
(9 mg). The reaction was allowed to proceed for 5 h and quenched by adding H2O. The mixture was
extracted three times with Et2O, and the Et2O layer was dried (Na2SO4) and evaporated to dryness. The
products were separated by SiO2 chromatography AcOEt/petroleum ether (PE) 1:1 (v/v). Three
products were obtained. According to 1H-NMR data, in addition to the desired product 10 (0.12 g, 21%
yield), its 2’-O-isomer (0.04 g) and, unexpectedly, 5’-O,N6-bis(4-methoxytrityl)-3’-O-pivaloyladenosine
1
(0.20 g) were formed. H-NMR (500 MHz, CDCl3): 8.02 (s, HꢀC(2)); 8.01 (s, HꢀC(8)); 7.22–7.38 (m,
24 H of MMTr); 7.02 (s, NH); 6.80–6.84 (m, 4 H of MMTr); 5.93 (d, J¼6.5, HꢀC(1’)); 5.43 (d, J¼6.5, 1 H
of OCH2O); 5.39 (d, J¼6.5, 1 H of OCH2O); 4.93 (dd, J¼6.5, 5.5, HꢀC(2’)); 4.74 (d, J¼5.5, HOꢀC(2’));
4.51 (dd, J¼5.5, 3.0, HꢀC(3’)); 4.37–4.38 (m, HꢀC(4’)); 3.80 (s, 2 MeO of MMTr); 3.48 (dd, J¼10.5, 4.0,
1 H of CH2(5’)); 3.28 (dd, J¼10.5, 4.0, 1 H of CH2(5’)); 1.17 (s, Me3C of Piv). 13C-NMR (126 MHz,
CDCl3): 177.8 (CO of Piv); 158.7 (C(6)); 158.3 (MMTr); 152.0 (C(2)); 148.3 (C(4)); 145.13 (MMTr);
138.6 (C(8)); 126.9, 127.9, 128.2, 128.9, 130.2, 130.3, 134.9 (MMTr); 121.3 (C(5)); 113.2 (MMTr); 89.7
(C(1’)); 88.7 (OCH2O); 86.8 (MMTr); 83.6 (C(4’)); 79.3 (C(3’)); 74.7 (C(2’)); 71.1 (MMTr); 63.2 (C(5’));
55.2 (MeO of MMTr); 38.7 (Me3C of Piv); 27.0 (Me of Piv). HR-ESI-MS: 926.4115 (C56H56N5Oþ8 ; calc.
926.4123).
2’-O-Levulinoyl-3’-O-(pivaloyloxymethyl)adenosine (¼ 3’-O-{[(2,2-Dimethylpropanoyl)oxy]meth-
yl}-2’-O-(4-oxopentanoyl)adenosine; 11). Lev2O was prepared by dissolving levulinic acid (5.6 mmol,
0.65 g) in dry 1,4-dioxane (10 ml) on an ice bath and adding dicyclohexylcarbodiimide (DCC; 2.8 mmol,
0.58 g) into the soln. in small portions within 1 h. The ice bath was removed, and the reaction was allowed
to proceed at r.t. for 2 h. Precipitated dicyclohexylurea was filtered off and washed with 5 ml of dry
dioxane. The filtrate was added to a soln. of 10 (2.3 mmol, 2.1 g, dried over P2O5 overnight) in dry
pyridine (9 ml), and a cat. amount of 4-(dimethylamino)pyridine (DMAP) was added. After stirring
overnight at r.t., the mixture was evaporated to dryness. The residue was dissolved in CH2Cl2, washed

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
277
with sat. aq. NaHCO3 and sat. aq. NaCl, and dried (Na2SO4). The org. phase was evaporated to dryness
and co-evaporated with toluene. The compound was subjected to detritylation without purification.
Accordingly, the crude product was dissolved in 80% (v/v) aq. AcOH (50 ml), and after stirring for 5 h at
408, the mixture was evaporated to dryness. The residue was dissolved in CH2Cl2 and washed three times
with H2O. The org. phase was dried (Na2SO4) and evaporated to dryness. The product was purified by
SiO2 chromatography, eluting with CH2Cl2 containing 5% MeOH, to yield 11 (0.91 g, 84%). White foam.
1H-NMR (500 MHz, CDCl3): 8.33 (s, HꢀC(2)); 7.83 (s, HꢀC(8)); 6.54 (m, HOꢀC(5’)); 6.01 (d, J¼7.5,
HꢀC(1’)); 5.74 (dd, J ¼ 7.5, 5.5, HꢀC(2’)); 5.64 (m, NH); 5.54 (d, J¼6.5, 1 H of OCH2O); 5.12 (d, J¼6.5,
1 H of OCH2O); 4.80 (m, HꢀC(3’)); 4.35 (m, HꢀC(4’)); 3.98 (m, 1 H of CH2(5’)); 3.77 (m, 1 H of
CH2(5’)); 2.49–2.75 (m, CH2CH2 of Lev); 2.16 (s, Me of Lev); 1.23 (s, Me3C of Piv). 13C-NMR (126 MHz,
CDCl3): 206.1 (CO of Lev); 177.8 (CO of Piv); 171.5 (CO of Lev); 155.9 (C(6)); 152.7 (C(2)); 148.7
(C(4)); 140.6 (C(8)); 121.3 (C(5)); 88.9 (OCH2O); 88.9 (C(1’)); 87.3 (C(4’)); 78.3 (C(3’)); 74.3 (C(2’));
62.7 (C(5’)); 38.9 (Me3C of Piv); 37.7 (CH2 of Lev); 29.8 (Me of Lev); 27.5 (CH2 of Lev); 27.0 (Me of
Piv). HR-ESI-MS: 480.2072 (C21H30N5Oþ8 ; calc. 480.2089).
5’-O-[(tert-Butyl)dimethylsilyl]-2’-O-levulinoyl-N6-(4-methoxytrityl)-3’-O-(pivaloyloxymethyl)ade-
nosine (¼ 5’-O-[(tert-Butyl)(dimethyl)silyl]-3’-O-{[(2,2-dimethylpropanoyl)oxy]methyl}-N6-[(4-me-
thoxyphenyl)(diphenyl)methyl]-2’-O-(4-oxopentanoyl)adenosine; 12). Compound 11 (1.7 mmol, 0.82 g)
t
was co-evaporated twice from dry pyridine and dissolved in the same solvent (5 ml). BuMe2SiCl
(2.1 mmol; 0.31 g) was added, and the mixture was stirred overnight at r.t. The reaction was quenched
with MeOH, and the mixture was evaporated to dryness. The residue was dissolved in CH2Cl2 and washed
with sat. aq. NaHCO3 and sat. aq. NaCl. The org. phase was dried (Na2SO4) and evaporated to dryness.
The product was purified by SiO2 chromatography, eluting with CH2Cl2 containing 10% MeOH, and
subjected directly to tritylation. Accordingly, the product (1.6 mmol, 0.94 g) was co-evaporated twice
from dry pyridine and dissolved in dry pyridine (6 ml). MMTrCl (1.9 mmol, 0.59 g) was added, and the
mixture was stirred over two nights at 408. The reaction was quenched with MeOH, and the mixture was
evaporated to dryness. The residue was dissolved in CH2Cl2 and washed with H2O and sat. aq. NaCl. The
org. phase was dried (Na2SO4) and evaporated to dryness. The product was purified by SiO2
chromatography, eluting with CH2Cl2 containing 1–3% MeOH, to yield 12 (1.27 g, 85%). Yellowish
1
foam. H-NMR (500 MHz, CDCl3): 8.06 (s, HꢀC(2)); 8.03 (s, HꢀC(8)); 7.20–7.35 (m, 12 H of MMTr);
6.89 (s, NH); 6.79 (m, 2 H of MMTr); 6.18 (d, J¼5.0, HꢀC(1’)); 5.68 (m, HꢀC(2’)); 5.37 (d, J¼6.5, 1 H of
OCH2O); 5.18 (d, J¼6.5, 1 H of OCH2O); 4.78 (m, HꢀC(3’)); 4.20 (dd, J¼7.5, 3.0, HꢀC(4’)); 3.92 (dd,
J¼11.3, 3.3, 1 H of CH2(5’)); 3.80 (dd, J¼11.3, 3.3, 1 H of CH2(5’)); 3.78 (s, MeO of MMTr); 2.56–2.75
(m, CH2CH2 of Lev); 2.14 (s, Me of Lev); 1.21 (s, Me3C of Piv); 0.89 (s, Me3CSi); 0.07 (s, MeSi); 0.05 (s,
MeSi). 13C-NMR (126 MHz, CDCl3): 206.1 (CO of Lev); 177.8 (CO of Piv); 171.7 (CO of Lev); 158.3
(MMTr); 154.1 (C(6)); 152.5 (C(2)); 148.8 (C(4)); 145.2 (MMTr); 138.5 (C(8)); 137.2, 130.2, 128.9, 127.9,
126.9 (MMTr); 121.2 (C(5)); 113.2 (MMTr); 88.3 (OCH2O); 85.9 (C(1’)); 83.9 (C(4’)); 76.2 (C(3’)); 74.9
(C(2’)); 71.0 (MMTr); 62.6 (C(5’)); 55.2 (MeO of MMTr); 38.8 (Me3C of Piv); 37.8 (CH2 of Lev); 29.7
(Me of Lev); 27.7 (CH2 of Lev); 27.0 (Me of Piv); 25.9 (Me3CSi); 18.4 (Me3CSi); ꢀ5.4, ꢀ5.5 (MeSi).
HR-ESI-MS: 866.4145 (C47H60N5O9Siþ ; calc. 866.4155).
5’-O-[(tert-Butyl)dimethylsilyl]-N6-(4-methoxytrityl)-3’-O-(pivaloyloxymethyl)adenosine (¼ 5’-O-
[(tert-Butyl)(dimethyl)silyl]-3’-O-{[(2,2-dimethylpropanoyl)oxy]methyl}-N6-[(4-methoxyphenyl)(diphe-
nyl)methyl]adenosine; 2b). Compound 12 was dissolved in a soln. of NH2NH2 ·H2O (6.0 mmol, 0.29 ml)
in pyridine (10 ml) and AcOH (2 ml) on an ice bath, and the mixture was stirred for 1.5 h. The ice bath
was removed, and the reaction was allowed to proceed at r.t. for 5 h. The reaction was quenched with sat.
aq. NaHCO3, and the mixture was extracted with CH2Cl2. The org. phase was washed with sat. aq. NaCl
and dried (Na2SO4) and evaporated to dryness. The product was purified by SiO2 chromatography,
eluting with CH2Cl2 containing 3% MeOH, to give 2b (1.04 g, 92%). Yellowish foam. 1H-NMR
(500 MHz, CDCl3): 8.03 (s, HꢀC(2)); 8.01 (s, HꢀC(8)); 7.23–7.37 (m, 12 H of MMTr); 6.97 (s, NH); 6.81
(d, J ¼ 9, 2 H of MMTr); 5.95 (d, J¼5.5, HꢀC(1’)); 5.44–5.49 (m, OCH2O); 4.75 (m, HꢀC(2’)); 4.51 (dd,
J¼5.3, 2.8, HꢀC(3’)); 4.41 (d, J¼5.0, OH); 4.30 (m, HꢀC(4’)); 3.88 (dd, J¼11.3, 3.8, 1 H of CH2(5’));
3.80–3.83 (m, HꢀC(5’’), MeO of MMTr); 1.25 (s, Me3C of Piv); 0.86 (s, Me3CSi); 0.08 (s, MeSi); 0.04 (s,
MeSi). 13C-NMR (126 MHz, CDCl3): 177.9 (CO of Piv); 158.3 (MMTr); 154.2 (C(6)); 152.1 (C(2)); 148.3
(C(4)); 145.2 (MMTr); 138.4 (C(8)); 137.2, 130.2, 128.9, 127.9, 126.9 (MMTr); 121.2 (C(5)); 113.2

278
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
(MMTr); 89.3 (C(1’)); 88.5 (OCH2O); 84.4 (C(4’)); 79.0 (C(3’)); 74.9 (C(2’)); 71.0 (MMTr); 62.8 (C(5’));
55.2 (MeO of MMTr); 38.7 (Me3C of Piv); 27.0 (Me of Piv); 25.8 (Me3CSi); 18.3 (Me3CSi); ꢀ5.5 (MeSi).
2’-O,5’-O,N6-Tris(4-methoxytrityl)adenosine (¼ N6-[(4-Methoxyphenyl)(diphenyl)methyl]-2’,5’-bis-
O-[(4-methoxyphenyl)(diphenyl)methyl]adenosine; 13). Adenosine (18.7 mmol, 5.00 g) was dried on
P2O5 overnight. The nucleoside was co-evaporated from dry pyridine and dissolved in the same solvent
(50 ml). MMTrCl (59.9 mmol, 18.5 g) was added, and the mixture was stirred at 608 overnight. The
reaction was quenched by adding MeOH (50 ml), and the volatiles were removed under reduced
pressure. The residue was dissolved in CH2Cl2, washed with H2O and brine, and the org. layer was dried
(Na2SO4) and evaporated to dryness. The residue was purified in three portions by SiO2 chromatography,
using CH2Cl2 containing 0–2% MeOH as eluent, to give 13 was (10.3 g, 50%). 1H-NMR (500 MHz,
CDCl3): 7.98 (s, HꢀC(2)); 7.97 (s, HꢀC(8)); 7.00–7.43 (m, 36 H of MMTr); 6.84, 6.73, 6.66 (3d, J¼8.5, 2 H
of MMTr); 6.38 (d, J¼7.5, HꢀC(1’)); 5.09 (dd, J¼7.5, 4.5, HꢀC(2’)); 4.08 (dd, J¼3.0, 3.5, HꢀC(4’)); 3.77,
3.78, 3.79 (3s, 3 H of MMTr); 3.31 (dd, J¼11.0, 3.5, 1 H of CH2(5’)); 3.00 (dd, J¼11.0, 3.0, 1 H of
CH2(5’)); 2.92 (d, J¼4.5, HꢀC(3’)); 2.32 (s, OH). 13C-NMR (126 MHz, CDCl3): 159.2, 158.6, 158.3
(MMTr); 154.1 (C(6)); 152.4 (C(2)); 149.4 (C(4)); 145.3, 144.4, 143.9, 143.8, 143.0 (MMTr); 139.1 (C(8));
137.3, 135.2, 134.5, 130.4, 130.2, 130.2, 128.9, 128.4, 128.3, 128.1, 128.0, 127.9, 127.8, 127.5, 127.4, 127.2, 127.0,
126.8 (MMTr); 121.2 (C(5)); 113.5, 113.1, 113.1 (MMTr); 87.5 (C(1’)); 86.8, 86.1 (MMTr); 84.3 (C(4’));
77.0 (C(2’), under CDCl3); 71.0 (MMTr); 70.6 (C(3’)); 63.8 (C(5’)); 55.2 (MeO of MMTr). HR-ESI-MS:
1084.4649 (C70H62N5Oþ7 ; calc. 1084.4644).
2’-O,5’-O,N6-Tris(4-methoxytrityl)-3’-O-[(methylsulfanyl)methyl]adenosine (¼ N6-[(4-Methoxyphe-
nyl)(diphenyl)methyl]-2’,5’-bis-O-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-[(methylsulfanyl)methyl]-
adenosine; 14). Compound 13 (1.72 mmol, 1.86 g) was dried by co-evaporation from dry pyridine and
twice from dry MeCN. The residue was dissolved in dry DMF (4.0 ml), and the soln. was cooled to 08 on
an ice-bath. NaH (3.4 mmol, 0.137 g of 60% dispersion in oil) and NaI (1.5 mmol, 0.230 g) were added.
The mixture was stirred for 0.5 h in an ice-bath, after which MeSCH2Cl (2.1 mmol, 170 ml) was added, and
the stirring was continued for 4 h at r.t. Another portion of MeSCH2Cl (1.0 mmol, 85 ml) was added, and
the stirring was continued for 0.5 h. The reaction was quenched by H2O. The mixture was extracted three
times with Et2O. The combined org. phase was washed with brine, dried (Na2SO4), and evaporated to
dryness. SiO2 chromatography with 30–40% AcOEt in PE gave 14 (0.86 g, 43%) yield. 1H-NMR
(500 MHz, CDCl3): 7.77 (s, HꢀC(2)); 7.70 (s, HꢀC(8)); 6.95–7.43 (m, 36 H of MMTr); 6.85 (s, NH); 6.83,
6.77, 6.64 (3d, J¼9.0, 2 H of MMTr); 6.05 (d, J¼6.5, HꢀC(1’)); 5.33 (dd, J¼6.5, 5.0, HꢀC(2’)); 4.58 (d,
J¼11.5, 1 H of OCH2S); 4.20 (m, HꢀC(4’)); 4.18 (d, J¼11.5, 1 H of OCH2S); 3.71, 3.78, 3.81 (3s, 3 H of
MMTr); 3.57 (dd, J¼4.5, 2.0, HꢀC(3’)); 3.37 (dd, J¼10.5, 5.5, 1 H of CH2(5’)); 3.17 (dd, J¼10.5, 4.5, 1 H
of CH2(5’)); 2.08 (s, MeS). HR-ESI-MS: 1144.4692 (C72H66N5O7Sþ ; calc. 1144.4677).
3’-O-[(Methylsulfanyl)methyl]adenosine (15). Nucleoside 14 (2.15 mmol, 2.46 g) was dissolved in
80% AcOH, the mixture was stirred overnight at r.t., evaporated to dryness, and co-evaporated twice
from H2O. The product was purified by SiO2 chromatography, eluting with 10–20% MeOH in CH2Cl2, to
yield 15 (0.49 g, 70%). 1H-NMR (500 MHz, CD3OD): 8.33 (s, HꢀC(2)); 8.21 (s, HꢀC(8)); 5.98 (d, J¼6.5,
HꢀC(1’)); 4.92 (d, J¼12.0, 1 H of OCH2S); 4.87–4.89 (m, 1 H of OCH2S, HꢀC(2’)); 4.52 (dd, J¼5.5, 2.5,
HꢀC(3’)); 4.29 (m, HꢀC(4’)); 3.91 (dd, J¼12.5, 2.5, 1 H of CH2(5’)); 3.78 (dd, J¼12.5, 2.5, 1 H of
CH2(5’)); 2.22 (s, MeS). 13C-NMR (126 MHz, CD3OD): 156.1 (C(6)); 152.2 (C(2)); 149.0 (C(4)); 140.5
(C(8)); 121.2 (C(5)); 89.8 (C(1’)); 84.9 (C(4’)); 75.6 (C(3’)); 74.6 (OCH2S); 73.6 (C(2’)); 61.9 (C(5’)); 12.4
(MeS). HR-ESI-MS: 328.1067 (C12H18N5O4Sþ ; calc. 328.1074).
5’-O-[(tert-Butyl)dimethylsilyl]-3’-O-[(methylsulfanyl)methyl]-N6-(4-methoxytrityl)adenosine (¼ 5’-
O-[(tert-Butyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-[(methylsulfanyl)methyl]-
adenosine; 16). Nucleoside 15 (2.5 mmol, 0.81 g) dried on P2O5 overnight was dissolved in dry pyridine
t
(9.0 ml). BuMe2SiCl (3.0 mmol, 0.45 g) was added, and the mixture was stirred overnight at r.t. The
reaction was quenched with MeOH, and the mixture was evaporated to dryness. The product was
purified by eluting through a thin layer of SiO2 with 10% MeOH in CH2Cl2. The volatiles were removed
under reduced pressure, and the residue was dried by coevaporating twice with anh. pyridine and
dissolved in the same solvent (10 ml). MMTrCl (2.7 mmol, 0.84 g) was added, and the mixture was stirred
overnight at 408. The reaction was quenched with MeOH, and the mixture was evaporated to dryness.
The residue was dissolved in CH2Cl2 and washed with H2O, sat. aq. NaHCO3, and sat. aq. NaCl. The org.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
279
layer was dried (Na2SO4) and evaporated to dryness. The product was purified by SiO2 chromatography,
1
eluting with 1–2% MeOH in CH2Cl2, to give 16 (1.56 g, 88%). Yellowish foam. H-NMR (500 MHz,
CDCl3): 8.05 (s, HꢀC(2)); 8.04 (s, HꢀC(8)); 7.23–7.38 (m, 12 H of MMTr); 6.97 (s, NH); 6.81 (d, J¼7.0,
2 H of MMTr); 5.99 (d, J¼5.5, HꢀC(1’)); 4.88 (d, J¼11.5, 1 H of OCH2S); 4.83 (d, J¼11.5, 1 H of
OCH2S); 4.71 (m, HꢀC(2’)); 4.52 (dd, J¼5.0, 3.0, HꢀC(3’)); 4.37 (d, J¼5.5, OH); 4.31 (m, HꢀC(4’));
3.89 (dd, J¼11.5, 4.0, 1 H of CH2(5’)); 3.83 (dd, J¼11.5, 3.0, 1 H of CH2(5’)); 3.80 (s, 3 H of MMTr); 2.23
(s, MeS), 0.87 (s, Me3CSi); 0.10 (s, MeSi); 0.06 (s, MeSi). 13C-NMR (126 MHz, CDCl3): 158.3 (MMTr);
154.2 (C(6)); 152.0 (C(2)); 148.4 (C(4)); 145.2 (MMTr); 138.2 (C(8)); 137.2, 130.2, 128.9, 127.9, 126.9
(MMTr); 121.2 (C(5)); 113.2 (MMTr); 89.5 (C(1’)); 84.3 (C(4’)); 76.2 (C(3’)); 75.3 (OCH2S); 75.0
(C(2’)); 71.0 (MMTr); 62.8 (C(5’)); 55.2 (MeO of MMTr); 25.9 (Me3CSi); 18.3 (Me3CSi); 14.2 (MeS),
ꢀ5.5, ꢀ5.6 (MeSi). HR-ESI-MS: 714.3140 (C38H48N5O5SSiþ ; calc. 714.3140).
5’-O-[(tert-Butyl)dimethylsilyl]-2’-O-levulinoyl-N6-(4-methoxytrityl)-3’-O-[(methylsulfanyl)methyl]-
adenosine (¼ 5’-O-[(tert-Butyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-[(meth-
ylsulfanyl)methyl]-2’-O-(4-oxopentanoyl)adenosine; 17). Levulinic acid (5.5 mmol, 0.63 g) was dissolved
in dry dioxane (10.0 ml). The mixture was stirred on an ice-bath, DCC (2.7 mmol, 0.56 g) was added
portionwise within 1 h, and stirring was continued at r.t. for 2 h. Dicyclohexylurea was filtered off and
washed with dioxane (5.0 ml). The washings were combined to the filtrate, and 16 (2.2 mmol, 1.56 g;
dried on P2O5) in dry pyridine (9.0 ml) was added. A cat. amount of DMAP was added, and the mixture
was stirred overnight at r.t. The mixture was evaporated to dryness, the residue was dissolved in CH2Cl2,
washed with sat. aq. NaHCO3 and sat. aq. NaCl, and the org. phase was dried (Na2SO4). The product was
purified by SiO2 chromatography, eluting with 1% MeOH in CH2Cl2, to yield 17 (1.57 g, 88%). 1H-NMR
(500 MHz, CDCl3): 8.11 (s, HꢀC(2)); 8.06 (s, HꢀC(8)); 7.23–7.37 (m, 12 H of MMTr); 6.94 (s, NH); 6.81
(d, J¼9.0, 2 H of MMTr); 6.23 (d, J¼4.5, HꢀC(1’)); 5.69 (dd, J ¼ 4.5, 5.0, HꢀC(2’)); 4.79 (dd, J¼5.0, 5.5,
HꢀC(3’)); 4.75 (d, J¼11.5, 1 H of OCH2S); 4.60 (d, J¼11.5, 1 H of OCH2S); 4.22 (m, HꢀC(4’)); 4.01 (dd,
J¼11.5, 2.5, 1 H of CH2(5’)); 3.86 (dd, J¼11.5, 2.5, 1 H of CH2(5’)); 3.80 (s, 3 H of MMTr); 2.61–2.80 (m,
4 H of Lev); 2.18 (s, MeS); 2.17 (s, 3 H of Lev); 0.94 (s, Me3CSi); 0.13 (s, MeSi); 0.11 (s, MeSi). HR-ESI-
MS: 812.3474 (C43H54N5O7SSiþ ; calc. 812.3508).
3’-O-(Acetyloxymethyl)-5’-O-[(tert-butyl)dimethylsilyl]-2’-O-levulinoyl-N6-(4-methoxytrityl)adeno-
sine (¼ 3’-O-[(Acetyloxy)methyl]-5’-O-[(tert-butyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)-
methyl]-2’-O-(4-oxopentanoyl)adenosine; 18). Nucleoside 17 (1.04 mmol, 0.84 g) was dried on P2O5
overnight and dissolved in dry CH2Cl2 (7.0 ml) under N2. SO2Cl2 in CH2Cl2 (1.24 mmol, 1.14 ml of 1 mol
lꢀ1 soln.) was added dropwise, and the mixture was stirred for 1 h at r.t. The volatiles were removed under
reduced pressure. The residue was dissolved in CH2Cl2 (3.0 ml) and added dropwise to a mixture of
AcOK (1.78 mmol, 0.175 g) and dibenzo-18-crown-6 (0.77 mol, 0.28 g) in dry CH2Cl2. After 2 h stirring at
r.t., the mixture was diluted with AcOEt and washed with H2O and brine. The org. phase was dried
(Na2SO4). The mixture was concentrated under reduced pressure, and the crown ether precipitate was
removed by filtration. The product was purified on a SiO2 column, eluting with CH2Cl2 containing 1%
MeOH, to give 18 (0.78 g, 91%). 1H-NMR (500 MHz, CDCl3): 8.06 (s, HꢀC(2)); 8.04 (s, HꢀC(8)); 7.23–
7.37 (m, 12 H of MMTr); 6.93 (s, NH); 6.81 (d, J¼9.0, 2 H of MMTr); 6.19 (d, J¼4.5, HꢀC(1’)); 5.71 (dd,
J ¼ 4.5, 5.0, HꢀC(2’)); 5.30 (d, J¼6.5, 1 H of OCH2O); 5.27 (d, J¼6.5, 1 H of OCH2O); 4.80 (dd, J¼5.0,
5.5, HꢀC(3’)); 4.21 (m, HꢀC(4’)); 3.97 (dd, J¼11.5, 3.0, 1 H of CH2(5’)); 3.83 (dd, J¼11.5, 3.0, 1 H of
CH2(5’)); 3.81 (s, 3 H of MMTr); 2.61–2.78 (m, 4 H of Lev); 2.17 (s, 3 H of Lev); 2.11 (s, Me of Ac); 0.92
(s, Me3CSi); 0.11 (s, MeSi); 0.09 (s, MeSi). 13C-NMR (126 MHz, CDCl3): 206.1 (CO of Lev); 171.7 (CO of
Lev); 170.4 (CO of Ac); 158.3 (MMTr); 154.1 (C(6)); 152.5 (C(2)); 148.6 (C(4)); 145.2 (MMTr); 138.4
(C(8)); 137.2 (MMTr); 130.2 (MMTr); 128.9 (MMTr); 127.9 (MMTr); 126.9 (MMTr); 121.2 (C(5)); 113.2
(MMTr); 88.3 (OCH2O); 86.2 (C(1’)); 83.5 (C(4’)); 76.2 (C(3’)); 74.8 (C(2’)); 71.0 (MMTr); 62.1 (C(5’));
55.2 (MMTr); 37.8 (Lev); 29.8 (Lev); 27.7 (Lev); 25.9 (TBDMS); 21.0 (MeCOOCH2); 18.4 (TBDMS);
ꢀ5.5 (TBDMS); ꢀ5.4 (TBDMS). HR-ESI-MS: 824.3641 (C44H54N5O9Siþ ; calc. 824.3685).
3’-O-[(Acetyloxy)methyl]-5’-O-[(tert-butyl)dimethylsilyl]-N6-(4-methoxytrityl)adenosine (¼ 3’-O-
[(Acetyloxy)methyl]-5’-O-[(tert-butyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]adeno-
sine; 2a). Nucleoside 17 (0.78 mmol, 0.64 g) was dissolved in dry CH2Cl2 (18 ml). NH2NH3OAc
(1.17 mmol; 0.107 g) in dry MeOH (2.0 ml) was added, and the mixture was stirred at r.t. for 1 h. The
reaction was quenched with 1.5 equiv. of acetone, and the mixture was stirred for 15 min and evaporated

280
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
to dryness. The product was purified on a SiO2 column, eluting with CH2Cl2 containing 1–2% MeOH, to
afford 2a (0.44 g, 78%). 1H-NMR (500 MHz, CDCl3): 8.03 (s, HꢀC(2)); 8.01 (s, HꢀC(8)); 7.23–7.37 (m,
12 H of MMTr); 6.96 (s, NH); 6.81 (d, J¼9.0, 2 H of MMTr); 5.94 (d, J¼6.0, HꢀC(1’)); 5.46 (d, J¼6.5,
1 H of OCH2O); 5.44 (d, J¼6.5, 1 H of OCH2O); 4.71 (m, HꢀC(2’)); 4.51 (m, HꢀC(3’)); 4.46 (d, J¼5.5,
OH); 4.32 (m, HꢀC(4’)); 3.87 (dd, J¼11.5, 4.0, 1 H of CH2(5’)); 3.81–3.83 (m, 3 H of MMTr, HꢀC(5’’));
3.81 (s, 3 H of MMTr); 2.14 (s, Me of Ac); 0.85 (s, Me3CSi); 0.08 (s, MeSi); 0.03 (s, MeSi). 13C-NMR
(126 MHz, CDCl3): 170.5 (CO); 158.3 (MMTr); 154.2 (C(6)); 152.0 (C(2)); 148.0 (C(4)); 145.1 (MMTr);
138.3 (C(8)); 137.2, 130.2, 128.9, 127.9, 126.9 (MMTr); 121.2 (C(5)); 113.2 (MMTr); 89.5 (C(1’)); 88.5
(OCH2O); 84.6 (C(4’)); 79.3 (C(3’)); 75.2 (C(2’)); 71.0 (MMTr); 62.8 (C(5’)); 55.2 (MeO of MMTr); 25.8
(Me3CSi); 21.1 (Ac); 18.2 (Me3CSi); ꢀ5.5, ꢀ5.6 (MeSi). HR-ESI-MS: 726.3294 (C39H48N5O7Siþ ; calc.
726.3318).
2’,3’-Di-O-levulinoyladenosine (¼2’,3’-Bis-O-(4-oxopentanoyl)adenosine; 19). Lev2O was prepared
by dissolving levulinic acid (29.6 mmol, 3.43 g) in dry 1,4-dioxane (40 ml) on an ice-bath and adding DCC
(14.8 mmol, 3.05 g) into the soln. in small portions within 1 h. The ice-bath was removed, and the reaction
was allowed to proceed at r.t. for 2 h. Precipitated dicyclohexylurea was filtered off, and the precipitate
was washed with 10 ml of dry dioxane. The filtrate was added to a soln. of 9 (7.4 mmol, 6.00 g) in dry
pyridine (30 ml), and a cat. amount of DMAP was added. After 2 h at r.t., the mixture was evaporated to
dryness. The residue was dissolved in CH2Cl2, and washed with sat. aq. NaHCO3 and sat. aq. NaCl. The
org. phase was dried (Na2SO4) and evaporated to dryness. The compound was subjected to detritylation
without purification. The crude product was dissolved in 80% (v/v) aq. AcOH (80 ml). After stirring
overnight at r.t., the mixture was evaporated to dryness. The product was purified by SiO2
chromatography, eluting with CH2Cl2 containing 5–10% MeOH, to give 19 (2.79 g, 81%). White foam.
81% yield (2.79 g). 1H-NMR (500 MHz, CDCl3): 8.34 (s, HꢀC(2)); 7.88 (s, HꢀC(8)); 6.71 (br. d, J¼10.0,
OH); 6.06 (d, J¼7.5, HꢀC(1’)); 6.01 (dd, J¼7.5, 5.0, HꢀC(2’)); 5.73 (dd, J¼5.0, 1.0, HꢀC(3’)); 4.38–4.39
(m, HꢀC(4’)); 4.00 (dd, J¼13.5, 1.5, 1 H of CH2(5’)); 3.85 (m, 1 H of CH2(5’)); 2.67–2.84 (m, 3 CH2 of
Lev); 2.54–2.56 (m, CH2 of Lev); 2.25 (s, Me of Lev); 2.17 (s, Me of Lev). 13C-NMR (126 MHz, CDCl3):
206.4 (OC¼O); 206.3 (OC¼O); 171.8 (CC¼O); 171.2 (CC¼O); 156.0 (C(6)); 152.6 (C(2)); 148.6 (C(4));
140.5 (C(8)); 121.1 (C(5)); 88.8 (C(1’)); 86.6 (C(4’)); 73.0 (C(2’)); 72.9 (C(3’)); 62.7 (C(5’)); 37.8
(CH2C¼O of Lev); 37.7 (CH2C¼O of Lev); 29.9 (Me of Lev); 29.8 (Me of Lev); 27.7 (CH2C¼OO of Lev);
27.4 (CH2C¼OO of Lev). HR-ESI-MS: 464.1797 (C20H26N5Oþ8 ; calc. 464.1776).
2’,3’-Di-O-levulinoyl-N6-(4-methoxytrityl)adenosine (¼ N-[(4-Methoxyphenyl)(diphenyl)methyl]-
2’,3’-bis-O-(4-oxopentanoyl)adenosine; 4). Compound 19 (3.2 mmol, 1.5 g) was evaporated twice from
dry pyridine and dissolved in the same solvent (25 ml). Me3SiCl (8.1 mmol, 1.03 ml) was added, and the
mixture was stirred for 1.5 h. Another portion of Me3SiCl (8.1 mmol, 1.03 ml) was added, and stirring was
continued for 1 h. MMTrCl (3.6 mmol, 1.1 g) was added, and the mixture was stirred overnight at 358.
Since the reaction was not completed according to TLC analysis, the mixture was stirred at 458 for 7 h.
Then, MMTrCl (0.7 mmol, 0.2 g) was added, and the mixture was stirred at 408 overnight. Sat. aq.
NaHCO3 was added, and the mixture was stirred for 10 min and extracted with AcOEt. The org. phase
was washed with sat. aq. NaHCO3, dried (Na2SO4) and evaporated to dryness. The product was purified
by SiO2 chromatography, eluting with CH2Cl2 containing 3% MeOH. The products were not separated,
and the purification was repeated, eluting with AcOEt containing 5% MeOH. Two products were
obtained: 4 (0.67 g) and its 5’-O-trimethylsilyl derivative (0.96 g). The Me3Si group was removed from
the latter by treatment with Bu4NF in THF under acidic conditions. Accordingly, Bu4NF (2.5 mmol,
0.65 g) was dissolved in dry THF (16 ml), and AcOH (3 ml) was added. The nucleoside was added, and
the mixture was stirred at r.t. for 15 min. Sat. aq. NaHCO3 soln. was added, and the mixture was extracted
with CH2Cl2. The org. phase was washed with sat. aq. NaCl soln., dried (Na2SO4), and evaporated to
1
dryness to yield 4. Yellowish foam. H-NMR (500 MHz, CDCl3): 8.03 (s, HꢀC(2)); 7.83 (s, HꢀC(8));
7.23–7.36 (m, 12 H of MMTr); 7.04 (s, NH); 6.83–6.84 (m, 2 H of MMTr, OH); 6.06 (d, J¼8.0, HꢀC(1’));
5.94 (dd, J¼8.0, 5.5, HꢀC(2’)); 5.71 (dd, J¼5.5, 0.5, HꢀC(3’)); 4.36–4.37 (m, HꢀC(4’)); 3.95 (dd, J¼13.0,
1.0, 1 H of CH2(5’)); 3.79–3.83 (m, MeO of MMTr, HꢀC(5’’)); 2.53–2.80 (m, 4 CH2 of Lev); 2.24 (s, Me
of Lev); 2.19 (s, Me of Lev). 13C-NMR (126 MHz, CDCl3): 206.4 (OC¼O); 206.2 (OC¼O); 171.7
(CC¼O); 171.2 (CC¼O); 158.3 (MMTr); 154.6 (C(6)); 151.9 (C(2)); 147.2 (C(4)); 144.9 (MMTr); 139.8
(C(8)); 136.8 (MMTr); 130.1, 128.8, 127.9, 126.9 (MMTr); 122.4 (C(5)); 113.2 (MMTr); 88.7 (C(1’)); 86.6

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
281
(C(4’)); 73.1 (C(2’)); 72.9 (C(3’)); 71.1 (MMTr); 62.6 (C(5’)); 55.2 (MeO of MMTr); 37.7 (CH2C¼O of
Lev); 37.6 (CH2C¼O of Lev); 29.8 (Me of Lev); 29.8 (Me of Lev); 27.6 (CH2C¼OO of Lev); 27.4
(CH2C¼OO of Lev). HR-ESI-MS: 736.2944 (C40H42N5Oþ9 ; calc. 736.2977).
3’-O-(Acetyloxymethyl)-5’-O-[(tert-butyl)dimethylsilyl]-N6-(4-methoxytrityl)adenosin-2’-yl 2’,3’-Di-
O-levylinoyl-N6-(4-methoxytrityl)adenosin-5’-yl 3-Acetyloxy-2,2-bis(ethoxycarbonyl) Phosphate (¼ Di-
ethyl ({[{[(2R,3R,4R,5R)-4-[(Acetyloxy)methoxy]-5-({[(tert-butyl)(dimethyl)silyl]oxy}methyl)-2-(6-
{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,
5R)-5-(6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]te-
trahydrofuran-2-yl}methoxy)phosphoryl]oxy}methyl)[(acetyloxy)methyl]propanedioate; 7a). Nucleo-
side 2a (0.69 mmol, 0.500 g) was dried on P2O5 overnight and dissolved in dry CH2Cl2 (3 ml) under
N2. Et3N (3.45 mmol; 0.479 ml) and (Et2N)2PCl (0.90 mmol, 0.188 ml) were added, and the mixture was
stirred under N2 for 2 h. The product was isolated by passing the mixture through a short SiO2 column
with AcOEt/hexane containing 0.5% Et3N 7:3. The solvent was removed under reduced pressure, and
the residue was co-evaporated from dry MeCN and dry CH2Cl2 to remove the traces of Et3N. The
identitiy of the product (3’-O-[(acetyloxy)methyl]-2’-O-[bis(diethylamino)phosphanyl]-5’-O-[(tert-bu-
tyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]adenosine; 3a) was checked by 31P- and
1H-NMR spectroscopy. 1H-NMR (500 MHz, CD3CN): 8.14 (s, HꢀC(2)); 7.88 (s, HꢀC(8)); 7.25–7.41 (m,
12 H of MMTr); 6.93 (s, NH); 6.86 (d, J¼9.0, 2 H of MMTr); 6.05 (d, J¼6.5, HꢀC(1’)); 5.41 (d, J¼6.5,
1 H of OCH2O); 5.35 (d, J¼6.5, 1 H of OCH2O); 4.96 (ddd, J¼11.0, 6.0, 4.5, HꢀC(2’)); 4.51 (dd, J¼4.5,
2.5, HꢀC(3’)); 4.12 (m, HꢀC(4’)); 3.97 (dd, J¼11.5, 4.5, 1 H of CH2(5’)); 3.86 (dd, J¼11.5, 3.5, 1 H of
CH2(5’)); 3.78 (s, 3 H of MMTr); 2.85–2.97 (m, 2 CH2 of EtN); 2.65–2.72 (m, 2 CH2 of NEt); 2.17 (s, Me
of Ac); 1.00 (t, J¼7.0, 2 Me of EtN); 0.95 (s, 3 Me of TBDMS); 0.77 (t, J¼7.5, 2 Me of NEt); 0.12 (s, Me
of TBDMS); 0.11 (s, Me of TBDMS). 31P-NMR (202 MHz, CD3CN): 137.6.
Compound 3a was dissolved in dry MeCN (1.0 ml) under N2. 1H-Tetrazole (0.68 mmol, 1.51 ml of
0.45 mol lꢀ1 soln. in MeCN) and 4 (0.48 mmol, 0.355 g) in MeCN (1.0 ml) were added. The reaction was
allowed to proceed for 25 min. After this period, 1H-tetrazole (0.78 mmol, 1.74 ml of 0.45 mol lꢀ1 soln. in
MeCN) and diethyl 2-(acetyloxymethyl)-2-(hydroxymethyl)malonate (5; 0.69 mmol, 0.180 g) were
added. The course of the reaction was monitored by 31P-NMR spectroscopy. After 1 h, 31P-NMR signals
(202 MHz, CD3CN) at 140.7 and 140.5 ppm were observed. The phosphite ester (diethyl
({[{[(2R,3R,4R,5R)-4-[(acetyloxy)methoxy]-5-({[(tert-butyl)(dimethyl)silyl]oxy}methyl)-2-(6-{[(4-me-
thoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,5R)-5-(6-
{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]tetrahydro-
furan-2-yl}methoxy)phosphanyl]oxy}methyl)[(acetyloxy)methyl]propanedioate; 6a) formed was oxi-
dized with I2 (0.2 g) in a mixture of THF (4.0 ml), H2O (2.0 ml), and 2,6-lutidine (1.0 ml). The oxidation
was allowed to proceed overnight. The excess of I2 was destroyed with 5% NaHSO3. The mixture was
extracted twice with CH2Cl2. The org. phase was washed with brine, dried on Na2SO4 and evaporated to
dryness. The crude product was purified on a SiO2 column eluting with a 4 :1 mixture of AcOEt and
1
CH2Cl2. The overall yield of 7a starting from 2a was 40% (0.49 g). H-NMR (500 MHz, CDCl3): 8.12,
8.10, 8.08, 8.06, 8.04, 8.01, 8.00, 7.98 (8s, 2 HꢀC(2), 2 HꢀC(8)); 7.22–7.38 (m, 24 H of MMTr); 6.95, 6.94,
6.92, 6.91 (4s, 2 NH); 6.79–6.83 (m, 4 H of MMTr); 6.21 (d, J¼1.5, 0.5 H of HꢀC(1’)); 6.19 (d, J¼5.5,
0.5 H of HꢀC(1’)); 6.19 (d, J¼2.0, 0.5 H of HꢀC(1’)); 6.12 (d, J¼5.5, 0.5 H of HꢀC(1’)); 5.83 (dd, J¼5.5,
5.5, 0.5 H of HꢀC(2’)); 5.80 (dd, J¼5.5, 5.5, 0.5 H of HꢀC(2’)); 5.70 (m, HꢀC(3’)); 5.50 (m, HꢀC(2’));
5.44 (d, J¼6.5, 0.5 H of OCH2OAc); 5.43 (d, J¼6.5, 0.5 H of OCH2OAc); 5.39 (m, HꢀC(3’)); 5.36 (d, J¼
6.5, 0.5 H of OCH2OAc); 5.28 (d, J¼6.5, 0.5 H of OCH2OAc); 4.57–4.69 (m, POCH2C, CH2OAc); 4.33–
4.43 (m, HꢀC(4’), HꢀC(5’), HꢀC(5’’)); 4.12–4.25 (m, HꢀC(4’), 2 MeCH2O); 4.00 (m, 1 H of CH2(5’));
3.83 (m, 1 H of CH2(5’)); 3.80, 3.80, 3.80, 3.79 (4s, 2 MeO of MMTr); 2.56–2.83 (m, 4 CH2 of Lev); 2.21,
2.17, 2.15, 2.13 (4s, 2 Me of Lev); 2.10, 2.08, 2.00, 1.92 (4s, 2 Me of Ac); 1.15–1.25 (m, 2 MeCH2); 0.88, 0.87
(2s, Me3CSi); 0.08, 0.06, 0.03, 0.01 (4s, 2 MeSi). 31P-NMR (202 MHz, CD3CN): ꢀ2.4 and ꢀ2.5.
(Multiplicity of some signals is due to the presence of (RP)- and (SP)-diastereoisomers.) HR-ESI-MS:
1768.6720 (C90H105N10O24PSiþ ; calc. 1768.6805).
3-Acetyloxy-2,2-bis(ethoxycarbonyl) 3’-O-(Acetyloxymethyl)-N6-(4-methoxytrityl)adenosin-2’-yl
2’,3’-Di-O-levylinoyl-N6-(4-methoxytrityl)adenosin-5’-yl Phosphate (¼ Diethyl ({[{[(2R,3R,4R,5R)-4-
[(Acetyloxy)methoxy]-5-(hydroxymethyl)-2-(6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-pu-

282
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
rin-9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,5R)-5-(6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}-
9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]tetrahydrofuran-2-yl}methoxy)phosphoryl]oxy}methyl)[(acet-
yloxy)methyl]propanedioate; 8a). Compound 7a (0.27 mmol, 0.47 g), dried over P2O5 overnight, was
dissolved in dry THF (5 ml). Et3N·3 HF (1.06 mmol, 173 ml) was added, and the mixture was stirred for
3 d at r.t. The mixture was neutralized by adding aq. Et3NHOAc (2.0 mol lꢀ1) in small portions. The
mixture was evaporated to dryness, and the residue was then dissolved in CH2Cl2 and washed with H2O.
The org. phase was evaporated to dryness. The product was purified by SiO2 chromatography, eluting
with 3% MeOH in CH2Cl2, to give 8a (0.41 g, 93%). 1H-NMR (500 MHz, CDCl3): 8.07, 8.04, 8.03, 8.02,
8.00, 7.98, 7.96, 7.95 (8s, 2 HꢀC(2), 2 HꢀC(8)); 7.19–7.35 (m, 24 H of MMTr); 7.04 (br. d, J ¼ 3.0, NH);
6.94 (br. s, NH); 6.76–6.81 (m, 4 H of MMTr); 6.45 (m, HOꢀC(5’)); 6.17 (d, J¼5.5, 0.5 H of HꢀC(1’));
6.14 (d, J¼5.5, 0.5 H of HꢀC(1’)); 6.06 (d, J¼7.0, 0.5 H of HꢀC(1’)); 5.82 (dd, J¼5.5, 5.5, 0.5 H of
HꢀC(2’)); 5.77 (dd, J¼5.5, 5.5, 0.5 H of HꢀC(2’)); 5.71 (m, 0.5 H of HꢀC(2’)); 5.66 (m, 0.5 H of
HꢀC(2’)); 5.54 (d, J¼6.8, 0.5 H of OCH2OAc); 5.53 (m, 0.5 H of HꢀC(3’)); 5.43 (m, 0.5 H of HꢀC(3’));
5.36 (s, 1 H of OCH2OAc); 5.24 (d, J¼6.5, 0.5 H of OCH2OAc); 4.77 (dd, J¼5.0, 1.5, 0.5 H of HꢀC(3’));
4.68 (dd, J¼5.0, 1.5, 0.5 H of HꢀC(3’)); 4.61 (s, 0.5 H of CH2OAc); 4.02–4.53 (m, 1.5 H of CH2OAc,
POCH2C, 2 HꢀC(4’), 1.5 CH2(5’), 1.5 CH2(5’’)); 3.86–3.95 (m, 1 H of CH2(5’)); 3.75–3.79 (m, 2 MeO of
MMTr); 3.65–3.72 (m, 1 H of CH2(5’’)); 2.51–2.82 (m, 4 CH2 of Lev); 2.19, 2.18, 2.13, 2.12 (4s, Me of
Lev); 2.08, 2.06, 1.98, 1.95 (4s, 2 Me of Ac); 1.13–1.21 (m, 2 MeCH2). 13C-NMR (126 MHz, CDCl3):
206.3, 206.3, 206.1, 206.1 (CO of Lev); 171.8, 171.7, 171.6, 171.5 (CO of Lev); 170.6, 170.4, 170.1, 170.0
(CO of Ac); 166.3, 166.2 (C¼OOEt), 158.4, 158.3 (MMTr); 154.6, 154.2 (C(6)); 152.6, 151.7 (C(2)); 148.7,
147.3 (C(4)); 145.1, 145.1, 144.9 (MMTr); 140.5, 138.8 (C(8)); 137.2, 137.0, 130.2, 128.9, 128.8, 128.0, 127.9,
127.9, 127.0, 126.9, 126.9 (MMTr); 122.5, 121.3 (C(5)); 113.2, 113.2, 113.2 (MMTr); 89.3 (C(1’)); 88.9, 88.2
(OCH2O); 86.5, 86.2 (C(4’)); 85.9 (C(1’)); 80.7, 80.1 (C(4’)); 78.8, 78.3, 77.1–77.3 (C3’, under CDCl3),
73.3, 73.0 (C(2’)); 71.1, 71.1, 71.0 (MMTr); 70.5, 70.4 (C(2’)); 67.5, 67.2 (C(5’)); 65.7 (POCH2C), 62.4
(C(5’)); 62.3 (MeCH2); 62.0, 61.1 (CH2OAc); 58.0 (ꢀCꢀ); 55.2 (MeO of MMTr); 37.7, 37.6 (CH2C¼O of
Lev); 29.8, 29.7 (Me of Lev); 27.6, 27.5, 27.5 (CH2C¼OO Lev, 21.0, 21.0, 20.6, 20.6 (Ac); 13.9 (MeCH2).
31P-NMR (202 MHz, CDCl3): ꢀ2.3, ꢀ2.7. (Multiplicity of some signals is due to the presence of (RP)-
and (SP)-diastereoisomers.) HR-ESI-MS: 1675.5600 (C84H89N10NaO24Pþ ; calc. 1675.5687).
3-Acetyloxy-2,2-bis(ethoxycarbonyl) 3’-O-(Acetyloxymethyl)-N6-(4-methoxytrityl)adenosin-2’-yl
2’,3’-Di-O-levylinoyl-N6-(4-methoxytrityl)adenosin-5’-yl Phosphate (¼ Diethyl {[({[(2R,3R,4R,5R)-4-
[(Acetyloxy)methoxy]-2-(6-amino-9H-purin-9-yl)-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxy}{[(2R,3S,
4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxytetrahydrofuran-2-yl]methoxy}phosphoryl)oxy]methyl}-
[(acetyloxy)methyl]propanedioate; 1a). Compound 8a (0.35 mmol, 0.58 g) was evaporated from dry
MeCN, and the residue was dissolved in dry CH2Cl2 (8 ml). NH2NH3OAc (1.17 mmol, 0.107 g) in dry
MeOH (0.9 ml) was added, and the mixture was stirred at r.t. for 2.5 h. The reaction was quenched with
acetone, and the mixture was stirred for 20 min and evaporated to dryness. The product was purified on a
SiO2 column eluting with CH2Cl2 containing 5% MeOH. The compound was then subjected to
detritylation with 80% (v/v) aq. AcOH (10 ml). After stirring overnight at r.t., the mixture was
evaporated to dryness, and the residue was co-evaporated twice with H2O. The product was purified first
by SiO2 chromatography, eluting with CH2Cl2 containing 10–20% MeOH, and then by HPLC on a Sun
FireTM Prep C18 column (250ꢁ10 mm, 5 mm; flow rate 3.0 ml minꢀ1; 150ꢁ4.6 mm, 5 mm; flow rate 1.0 ml
minꢀ1), using a linear gradient elution from 33 to 100% MeOH in 20 min, to afford 1a (153 mg, 39%).
1H-NMR (500 MHz, CD3CN): 7.99–8.25 (m, 2 HꢀC(2), 2 HꢀC(8)); 6.43, 6.34, 6.27, 6.18 (4 br. s, 2 NH,
HOꢀC(5’)); 6.09 (m, HꢀC(1’)); 5.96 (d, J¼4.5, 0.5 H of HꢀC(1’)); 5.91 (br. d, J¼4.0, 0.5 H of HꢀC(1’));
5.54 (m, HꢀC(2’)); 5.47 (d, J ¼ 6.5, 0.5 H of OCH2O); 5.40 (d, J ¼ 7.0, 0.5 H of OCH2O); 5.30 (m, 1 H of
OCH2O); 4.66 (dd, J¼5.0, 2.5, 0.5 H of HꢀC(3’)); 4.61–4.65 (m, 0.5 H of HꢀC(3’), 0.5 H of HꢀC(2’));
4.54 (m, 0.5 H of HꢀC(2’)); 4.46–4.51 (m, 1 H of CH2(5’), 1 H of CCH2OAc); 4.38–4.44 (m, 1 H of
CH2(5’’), 0.5 H of CCH2OAc); 4.28–4.35 (m, 0.5 H of HꢀC(3’), 0.5 H of CCH2OAc, 1 H of POCH2C,
HꢀC(4’)); 4.10–4.25 (m, 0.5 H of HꢀC(3’), 2 MeCH2, 1 H of POCH2C); 4.08 (m, 0.5 H of HꢀC(4’)); 4.03
(m, 0.5 H of HꢀC(4’)); 3.85 (m, 1 H of CH2(5’)); 3.70 (m, 1 H of CH2(5’)); 2.10 (s, 1.5 H of AcO); 2.07 (s,
1.5 H of AcO); 2.01, 1.97 (2s, Me of OCH2OAc), 1.13–1.23 (m, 2 MeCH2). 13C-NMR (126 MHz,
CD3CN): 170.4, 170.1 (CO of Ac); 166.4, 166.3 (C¼OOEt), 156.5, 156.0 (C(6)); 152.9, 152.5 (C(2)); 148.6
(C(4)); 140.8, 140.6, 139.6, 139.3 (C(8)); 119.7, 120.6 (C(5)); 88.7 (C(1’)); 88.3 (OCH2O); 87.99 (C(1’));

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
283
85.8, 85.7, 82.4, 82.0 (C(4’)); 78.4, 78.1 (C(3’)); 76.7, 76.5 (C(2’)); 74.0, 73.9 (C(2’)); 70.2, 70.0 (C(3’)); 67.8,
67.6 (POCH2C); 65.5, 65.4 (C(5’), POCH2C); 62.4, 62.3 (MeCH2); 61.8, 61.7 (C(5’)); 60.9, 60.8
(CH2OAc); 57.9 (ꢀCꢀ); 20.3 (Ac); 19.8 (OCH2OAc); 13.2 (MeCH2). 31P-NMR (202 MHz, CD3CN):
ꢀ2.5, ꢀ2.4. (Multiplicity of some signals is due to the presence of (RP)- and (SP)-diastereoisomers.) HR-
ESI-MS: 913.2707 (C34H46N10O18Pþ ; calc. 913.2724).
3-(Acetyloxy)-2,2-bis(ethoxycarbonyl) 5’-O-[(tert-Butyl)dimethylsilyl]-N6-(4-methoxytrityl)-3’-O-
(pivaloyloxymethyl)adenosin-2’-yl 2’,3’-Di-O-levylinoyl-N6-(4-methoxytrityl)adenosin-5’-yl Phosphate
(¼ Diethyl [(Acetyloxy)methyl]({[{[(2R,3R,4R,5R)-5-({[(tert-butyl)(dimethyl)silyl]oxy}methyl)-4-
{[(2,2-dimethylpropanoyl)oxy]methoxy}-2-(6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-purin-
9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,5R)-5-(6-{[(4-methoxyphenyl)(diphenyl)methyl]amino}-
9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]tetrahydrofuran-2-yl}methoxy)phosphoryl]oxy}methyl)pro-
panedioate; 7b). Nucleoside 2b (0.73 mmol, 0.600 g) was dried over P2O5 overnight and dissolved in dry
CH2Cl2 (4 ml) under N2. Et3N (3.91 mmol, 0.543 ml) and (Et2N)2PCl (0.94 mmol, 0.197 ml) were added,
and the mixture was stirred under N2 for 2 h. The product was isolated by passing the mixture through a
short SiO2 column with AcOEt/hexane containing 0.5% Et3N 7:3. The solvent was removed under
reduced pressure, and the residue was co-evaporated from dry MeCN and dry CH2Cl2 to remove the
traces of Et3N. The identitiy of the product (2’-O-[bis(diethylamino)phosphanyl]-5’-O-[(tert-butyl)(di-
methyl)silyl]-3’-O-{[(2,2-dimethylpropanoyl)oxy]methyl}-N6-[(4-methoxyphenyl)(diphenyl)methyl]ade-
nosine; 3b) was checked by 31P- and 1H-NMR spectroscopy. 1H-NMR (500 MHz, CD3CN): 8.13 (s,
HꢀC(2)); 7.88 (s, HꢀC(8)); 7.24–7.41 (m, 12 H of MMTr); 6.92 (s, HNꢀC(6)); 6.83–6.87 (m, 2 H of
MMTr); 6.05 (d, J¼6.5, HꢀC(1’)); 5.47 (d, J¼6.5, 1 H of OCH2O); 5.34 (d, J¼6.5, 1 H of OCH2O); 4.97
(m, HꢀC(2’)); 4.50 (dd, J¼4.8, 2.8, HꢀC(3’)); 4.23 (m, HꢀC(4’)); 3.98 (dd, J¼11.5, 4.5, 1 H of CH2(5’));
3.84 (dd, J¼11.5, 3.5, 1 H of CH2(5’)); 3.78 (s, 3 H of MMTr); 2.84–2.98 (m, 4 H of EtN); 2.62–2.70 (m,
4 H of EtN); 1.22 (s, Me3C); 1.00 (t, J¼7.5, 6 H of EtN); 0.94 (s, Me3CSi); 0.75 (t, J¼7.5, 6 H of EtN); 0.11
(s, 2 MeSi). 31P-NMR (202 MHz, CD3CN): 137.2.
Compound 3b was dissolved in dry MeCN (1.0 ml) under N2. 1H-Tetrazole (0.73 mmol, 1.630 ml of
0.45 m soln. in MeCN) and 4 (0.55 mmol; 0.400 g) in dry MeCN (1.0 ml) were added. The reaction was
allowed to proceed for 15 min. After this period, 1H-tetrazole (0.73 mmol, 1.630 ml of 0.45 m soln. in
MeCN) and diethyl 2-(acetyloxymetyl)-2-(hydroxymethyl)malonate (0.86 mmol, 0.230 g) in dry MeCN
were added. The course of the reaction was monitored by 31P-NMR spectroscopy. After 40 min, 31P-NMR
signals (202 MHz, CD3CN) at 140.6 and 140.5 ppm were observed.
The phosphite ester (diethyl [(acetyloxy)methyl]({[{[(2R,3R,4R,5R)-5-({[(tert-butyl)(dimethyl)si-
lyl]oxy}methyl)-4-{[(2,2-dimethylpropanoyl)oxy]methoxy}-2-(6-{[(4-methoxyphenyl)(diphenyl)methyl]-
amino}-9H-purin-9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,5R)-5-(6-{[(4-methoxyphenyl)(diphenyl)-
methyl]amino}-9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]tetrahydrofuran-2-yl}methoxy)phosphanyl]-
oxy}methyl)propanedioate; 6b) formed was oxidized with I2 (0.2 g) in a mixture of THF (4.0 ml), H2O
(2.0 ml), and 2,6-lutidine (1.0 ml). The oxidation was allowed to proceed overnight. The oxidized
product, 7b, exhibited 31P-NMR signals (202 MHz, CD3CN) at ꢀ2.4 and ꢀ2.6 ppm. The excess of I2 was
destroyed with 5% NaHSO3. The mixture was extracted three times with CH2Cl2. The org. phase was
dried (Na2SO4) and evaporated to dryness. The crude product was purified by SiO2 chromatography,
eluting first with a 1:1 mixture of AcOEt and CH2Cl2, then with AcOEt, and finally with AcOEt
containing 5% MeOH, to yield 7b (0.50 g, 35%). 1H-NMR (500 MHz, CDCl3): 7.98–8.07 (m, 2 HꢀC(2),
2 HꢀC(8)); 7.21–7.38 (m, 24 H of MMTr); 6.94 (br. s, NH); 6.90 (br. s, NH); 6.78–6.82 (m, 4 H of
MMTr); 6.19 (d, J¼2.0, HꢀC(1’)); 6.13 (d, J¼5.5, HꢀC(1’)); 5.78 (dd, J¼5.3, 5.3, HꢀC(2’)); 5.68 (dd, J¼
5.3, 5.3, HꢀC(2’)); 5.58 (m, HꢀC(3’)); 5.52 (d, J¼6.3, 1 H of OCH2O); 5.32 (d, J¼6.3, 1 H of OCH2O);
5.03 (m, HꢀC(3’)); 4.54–4.65 (m, CCH2OAc, POCH2C); 4.17–4.38 (m, 2 HꢀC(4’), 1 H of CH2(5’), 1 H
of CH2(5’’), 2 MeCH2); 3.94 (m, 1 H of CH2(5’)); 3.78–3.83 (m, 1 H of CH2(5’’), 2 MeO of MMTr); 2.55–
2.78 (m, 2 CH2CH2 of Lev); 2.18, 2.14 (2s, 2 Me of Lev); 2.01 (s, Me of Ac); 1.23 (2s, Me3C of Piv); 1.15–
1.25 (m, 2 MeCH2); 0.83 (s, Me3CSi); 0.03 (s, 2 MeSi). 31P-NMR (202 MHz, CDCl3): ꢀ2.4, ꢀ2.6.
(Multiplicity of some signals is due to the presence of (RP)- and (SP)-diastereoisomers.)
3-(Acetyloxy)-2,2-bis(ethoxycarbonyl) 2’,3’-Di-O-levylinoyl-N6-(4-methoxytrityl)adenosin-5’-yl N6-
(4-Methoxytrityl)-3’-O-(pivaloyloxymethyl)adenosin-2’-yl Phosphate (¼ Diethyl [(Acetyloxy)me-
thyl]({[{[(2R,3R,4R,5R)-4-{[(2,2-dimethylpropanoyl)oxy]methoxy}-5-(hydroxymethyl)-2-(6-{[(4-me-

284
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
thoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)tetrahydrofuran-3-yl]oxy}({(2R,3R,4R,5R)-5-(6-
{[(4-methoxyphenyl)(diphenyl)methyl]amino}-9H-purin-9-yl)-3,4-bis[(4-oxopentanoyl)oxy]tetrahydro-
t
furan-2-yl}methoxy)phosphoryl]oxy}methyl)propanedioate; 8b). The BuMe2Si group was removed by
treatment with Bu4NF in THF under acidic conditions. Accordingly, Bu4NF (0.8 mmol, 0.22 g) was
dissolved in dry THF (6.8 ml), and AcOH (1.2 ml) was added. Compound 7b (0.3 mmol, 0.50 g) was
added, and the mixture was stirred at r.t. for 2 d. NaHCO3 soln. (1%) was added, and the mixture was
extracted twice with CH2Cl2. The org. phase was dried (Na2SO4) and evaporated to dryness. The product
was purified by SiO2 chromatography, eluting with CH2Cl2 containing 3–5% MeOH, to afford a
diastereoisomeric mixture (1:1) of 8b (0.33 g, 70%). Clear oil. 1H-NMR (500 MHz, CDCl3): 8.04, 8.03,
8.03, 8.01, 8.00, 7.98, 7.95, 7.95 (8s, 2 HꢀC(2), 2 HꢀC(8)); 7.19–7.35 (m, 24 H of MMTr); 7.04 (m, NH);
6.92 (br. s, NH); 6.77–6.81 (m, 4 H of MMTr); 6.48 (dd, J ¼ 2.0, 11.5, 0.5 H of HOꢀC(5’)); 6.40 (dd, J¼
2.5, 11.5, 0.5 H of HOꢀC(5’)); 6.16 (d, J¼5.5, 0.5 H of HꢀC(1’)); 6.14 (d, J¼5.0, 0.5 H of HꢀC(1’)); 6.04
(d, J¼6.5, 0.5 H of HꢀC(1’)); 6.03 (d, J¼7.0, 0.5 H of HꢀC(1’)); 5.80 (t, J¼5.5, 0.5 H of HꢀC(2’)); 5.77 (t,
J¼5.5, 0.5 H of HꢀC(2’)); 5.71 (m, 0.5 H of HꢀC(3’)); 5.64–5.67 (m, 0.5 H of HꢀC(3’), 0.5 H of
OCH2O); 5.50–5.56 (m, 0.5 H of HꢀC(2’), 0.5 H of OCH2O); 5.46 (m, 0.5 H of HꢀC(2’)); 5.29 (d, J¼6.5,
0.5 H of OCH2O); 5.19 (d, J¼6.5, 0.5 H of OCH2O); 4.75 (dd, J¼1.5, 5.5, 0.5 H of HꢀC(3’)); 4.70 (dd,
J¼1.5, 5.5, 0.5 H of HꢀC(3’)); 4.50–4.55 (m, 1 H of CCH2OAc, 1 H of POCH2C); 4.08–4.46 (m, 1 H of
OCH2OAc, POCH2C, 2 HꢀC(4’), HꢀC(5’), HꢀC(5’’), 2 MeCH2); 3.88 (m, 0.5 H of HꢀC(5’), 0.5 H of
HꢀC(5’’)); 3.75–3.78 (m, 2 MeO of MMTr); 3.65–3.72 (m, 0.5 H of HꢀC(5’), 0.5 H of HꢀC(5’’)); 2.55–
2.80 (m, 2 CH2CH2 of Lev); 2.18, 2.17, 2.13, 2.12 (4s, 2 Me of Lev); 1.99, 1.95 (2s, Me of AcO); 1.20, 1.19
(2s, Me3C of Piv); 1.13–1.22 (m, 2 MeCH2). 13C-NMR (126 MHz, CDCl3): 206.3, 206.3, 206.1, 206.1 (CO
of Lev); 177.7, 177.9 (CO of Piv); 171.8, 171.7, 171.6, 171.5 (CO of Lev); 170.1, 170.0 (CO of Ac); 166.3,
166.2, 166.1, 166.0 (C¼OOEt); 158.4, 158.3 (MMTr); 154.6, 154.2 (C(6)); 152.6, 151.7 (C(2)); 148.7, 147.3
(C(4)); 145.2, 145.1, 145.1, 145.0 (MMTr); 140.5, 140.3, 138.6 (C(8)); 137.2, 137.0, 130.2, 128.9, 127.9, 127.9,
126.9, 126.9 (MMTr); 122.5, 121.3 (C(5)); 113.2, 113.2 (MMTr); 89.3 (C(1’)); 89.1, 88.9 (OCH2O); 86.8,
86.6 (C(4’)); 86.0, 85.9 (C(1’)); 80.7 (C(4’)); 78.8, 78.6 (C(3’)); 73.3 (C(2’)); 71.0 (MMTr); 67.2 (C(5’));
65.4 (POCH2C), 62.3 (CH2CH3), 61.2 (CH2OAc), 58.0 (ꢀCꢀ); 55.2 (MeO of MMTr); 38.8 (Me3C of
Piv); 37.8, 37.7, 37.7 (CH2C¼O of Lev); 29.8, 29.8 (Me of Lev); 27.6, 27.5, 27.5 (CH2C¼OO of Lev); 27.0
(Me of Piv); 20.6, 20.6 (Ac); 13.9 (MeCH2). 31P-NMR (202 MHz, CDCl3): ꢀ2.2, ꢀ2.7. (Multiplicity of
some signals is due to the presence of (RP)- and (SP)-diastereoisomers.) HR-ESI-MS: 1717.6164
(C87H95N10NaO24Pþ ; calc. 1717.6151).
3-(Acetyloxy)-2,2-bis(ethoxycarbonyl) adenosin-5’-yl 3’-O-(pivaloyloxymethyl)adenosin-2’-yl Phos-
phate (¼ Diethyl [(Acetyloxy)methyl]{[({[(2R,3S,4R,5R)-5-(6-amino-9H-purin-9-yl)-3,4-dihydroxyte-
trahydrofuran-2-yl]methoxy}{[(2R,3R,4R,5R)-2-(6-amino-9H-purin-9-yl)-4-{[(2,2-dimethylpropanoyl)-
oxy]methoxy}-5-(hydroxymethyl)tetrahydrofuran-3-yl]oxy}phosphoryl)oxy]methyl}propanedioate; 1b).
To remove the levulinoyl groups, 8b (0.087 mmol, 0.100 g) was dissolved in a soln. of NH2NH2 ·H2O
(2.50 mmol, 0.078 ml) in a mixture of pyridine (4 ml) and AcOH (1 ml) on an ice-bath, and the mixture
was stirred for 1 h. The ice-bath was removed, and the reaction was allowed to proceed at r.t. for 3 h. The
reaction was quenched with 0.1m NaH2PO3 soln. (25 ml), and the product was extracted into CH2Cl2. The
org. phase was washed with H2O, dried (Na2SO4), and evaporated to dryness. The product was purified
by SiO2 chromatography, eluting with CH2Cl2 containing 5% MeOH. The compound was then subjected
to detritylation with 80% (v/v) aq. AcOH (10 ml). After stirring overnight at r.t., the mixture was
evaporated to dryness. The residue was co-evaporated twice with H2O. The product was purified first by
SiO2 chromatography, eluting with CH2Cl2 containing 10–20% MeOH, and then by HPLC on a Thermo
Hypersil HypurityTM Elite C18 column (150ꢁ4.6 mm, 5 mm, flow rate 1.0 ml minꢀ1), using a linear
gradient elution from H2O to MeCN in 30 min, to give 1b (24 mg, 29%). 1H-NMR (500 MHz, CD3CN):
8.23, 8.20, 8.20, 8.11 (4s, 2 HꢀC(2)); 8.11, 8.06, 8.06, 8.00 (4s, 2 HꢀC(8)); 6.49, 6.38, 6.33, 6.22 (4 br. s,
2 NH, HOꢀC(5’)); 6.10 (m, HꢀC(1’)); 5.96 (d, J¼4.5, 0.5 H of HꢀC(1’)); 5.91 (br. d, J¼4.0, 0.5 H of
HꢀC(1’)); 5.52–5.60 (m, HꢀC(2’), 0.5 H of OCH2O); 5.50 (d, J ¼ 7.0, 0.5 H of OCH2O); 5.27 (m, 1 H of
OCH2O); 4.70 (dd, J¼5.0, 2.5, 0.5 H of HꢀC(3’)); 4.67 (dd, J¼5.0, 2.0, 0.5 H of HꢀC(3’)); 4.63 (m, 0.5 H
of HꢀC(2’)); 4.53 (m, 0.5 H of HꢀC(2’)); 4.42–4.52 (m, HꢀC(5’), 1 H of CCH2OAc); 4.30–4.42 (m, 1 H
of OCH2OAc, 0.5 H of HꢀC(3’), POCH2C); 4.29 (m, HꢀC(4’)); 4.07–4.25 (m, 0.5 H of HꢀC(3’),
HꢀC(5’’), 2 MeCH2, 0.5 H of HꢀC(4’)); 4.03 (m, 0.5 H of HꢀC(4’)); 3.85 (m, 1 H of CH2(5’)); 3.69 (m,

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
285
1 H of CH2(5’)); 1.96 (s, Me of AcO); 1.23, 1.21 (2s, Me3C of Piv); 1.13–1.23 (m, 2 MeCH2). 13C-NMR
(126 MHz, CD3CN): 177.6, 177.6 (CO of Piv); 170.1, 170.0 (CO of Ac); 166.4, 166.3 (C¼OOEt); 156.5,
156.0 (C(6)); 152.9, 152.5 (C(2)); 149.7, 148.6 (C(4)); 140.8, 140.5, 139.8, 139.1 (C(8)); 119.7, 120.6 (C(5));
88.8 (OCH2O); 88.8, 88.0, 88.0 (C(1’)); 85.9, 85.7, 82.4, 82.3 (C(4’)); 78.3, 78.0 (C(3’)); 76.7, 76.5, 74.0, 73.9
(C(2’)); 70.2, 70.0 (C(3’)); 67.8, 65.5 (C(5’)); 65.4, 65.3 (POCH2C), 62.4, 62.3 (MeCH2); 61.8, 61.7 (C(5’));
60.9, 60.8 (CH2OAc), 57.9, 57.8 (ꢀCꢀ); 38.5, 38.5 (Me3C of Piv); 26.2, 26.2 (Me of Piv); 19.8, 19.8 (Ac);
13.2, 13.2 (MeCH2). 31P-NMR (202 MHz, CD3CN): ꢀ2.4, ꢀ2.4. (Multiplicity of some signals is due to
the presence of (RP)- and (SP)-diastereoisomers.) HR-ESI-MS: 955.3215 (C37H52N10O18Pþ ; calc.
955.3193).
Kinetic Measurements. The reactions were carried out in sealed tubes immersed in a thermostated
H2O bath (37.0ꢂ0.18). The hydroxonium ion concentration of the reaction solns. (3.0 ml) was adjusted
with 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) buffer (0.036/0.024 mol lꢀ1
;
pH 7.5). The ionic strength of the solns. was adjusted to 0.1m with NaCl. The hydroxonium ion
concentration of the buffer solns. was calculated with the aid of the known pKa values of the buffer acids
under the experimental conditions. The initial substrate concentration was ca. 0.15 mmol lꢀ1. The AcO
group was removed with hog liver carboxyesterase (HLE; 2.6 U mlꢀ1). The samples (200 ml) withdrawn
at appropriate intervals were made acidic (pH 2) with 1 m aq. HCl to inactivate the enzyme and to quench
the hydrolysis, cooled in an ice-bath and filtered with minisart RC 4 filters (0.20 mm). The reaction
products were identified by the mass spectra (LC/MS) using a mixture of H2O, MeCN, and HCOOH
(0.1%) as an eluent (Gemini C18 column (2ꢁ150 mm 5 mm; flow rate 200 ml minꢀ1). The composition of
the samples was analyzed on a Thermo ODS Hypersil C18 column (4ꢁ250 mm, 5 mm; flow rate 0.95 ml
minꢀ1), using AcOH/AcONa buffer (0.045/0.015 mol lꢀ1) and MeCN, containing ammonium chloride
(0.1m). A good separation of the product mixtures was obtained on using a 5 min isocratic elution with
the buffer containing 2% MeCN, followed by a linear gradient (45 min) up to 43% MeCN. Signals were
recorded on a UV detector at a wavelength of 260 nm. The enzymatic deacetylations obeyed first-order
kinetics at the HLE concentrations employed.
REFERENCES
[1] A. Zhou, B. A. Hassel, R. H. Silverman, Cell 1993, 72, 753.
[2] R. H. Silverman, Cytokine Growth Factor Rev. 2007, 18, 381.
[3] K. Kubota, K. Nakahara, T. Ohtsuka, S. Yoshida, J. Kawaguchi, Y. Fujita, Y. Ozeki, A. Hara, C.
Yoshimura, H. Furukawa, H. Haruyama, K. Ichikawa, M. Yamashita, T. Matsuoka, Y. Ijima, J. Biol.
Chem. 2004, 279, 37832.
[4] Y. Xiang, Z. Wang, J. Murakami, S. Plummer, E. A. Klein, J. D. Carpten, J. M. Trent, W. B. Isaacs, G.
Casey, R. H. Silverman, Cancer Res. 2003, 63, 6795.
[5] J. Imai, M. I. Johnston, P. F. Torrence, J. Biol.Chem. 1982, 257, 12739; J. Imai, P. F. Torrence,
Biochemistry 1984, 23, 766; W. Xiao, G. Li, K. Lesiak, B. Dong, R. H. Silverman, P. F. Torrence,
Bioorg. Med. Chem. Lett. 1994, 4, 2609; M. R. Player, P. F. Torrence, Pharmacol. Ther. 1998, 78, 55.
[6] B. Bayard, C. Bisbal, M. Silhol, J. Cnockaert, G. Huez, B. Lebleu, Eur. J. Biochem. 1984, 142, 291; C.
Bisbal, M. Silhol, M. Lemaitre, B. Bayard, T. Salehzada, B. Lebleu, T. D. Perree, M. G. Blackburn,
Biochemistry 1987, 26, 5172.
´
[7] K. Kariko, J. Ludwig, Biochem. Biophys. Res. Commun. 1985, 128, 695.
[8] H. Sawai, H. Taira, K. Ishibashi, M. Itoh, J. Biochem. 1987, 101, 339.
[9] E. N. Kalinichenko, T. L. Podkopaeva, M. Kelve, M. Saarma, I. A. Mikhailopulo, Biochem. Biophys.
Res. Commun. 1990, 167, 20.
[10] R. W. Sobol, E. E. Henderson, N. Kon, J. Shao, P. Hitzges, E. Mordechai, N. L. Reichenbach, R.
Charubala, H. Schirmeister, W. Pfleiderer, R. J. Suhadolnik, J. Biol. Chem. 1995, 270, 5963; R.
Charubala, W. Pfleiderer, Helv. Chim. Acta 1992, 75, 471.
[11] H. Cramer, J. R. Okicki, T. Rho, X. Wang, R. H. Silverman, W. D. W. Heston, Nucleosides,
Nucleotides Nucleic Acids 2007, 26, 1471.
[12] K. Morita, M. Kaneko, S. Obika, T. Imanishi, Y. Kitade, M. Koizumi, ChemMedChem 2007, 2, 1703.

286
CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
[13] M. Kosonen, M. Oivanen, H. Lçnnberg, J. Org. Chem. 1994, 59, 3704; M. Kosonen, H. Lçnnberg, J.
Chem. Soc., Perkin Trans. 2 1995, 1203.
[14] a) M. Ora, E. Mꢃki, P. Poijꢃrvi, K. Neuvonen, M. Oivanen, H. Lçnnberg, J. Chem. Soc., Perkin
Trans. 2 2001, 881; b) P. Poijꢃrvi, E. Mꢃki, J. Tomperi, M. Ora, M. Oivanen, H. Lçnnberg, Helv.
Chim. Acta 2002, 85, 1869; c) P. Poijꢃrvi, M. Oivanen, H. Lçnnberg, Lett. Org. Chem. 2004, 1, 183;
d) P. Poijꢃrvi, P. Heinonen, P. Virta, H. Lçnnberg, Bioconjugate Chem. 2005, 16, 1564; e) M. Ora, S.
Taherpour, R. Linna, A. Leisvuori, E. Hietamꢃki, P. Poijꢃrvi-Virta, L. Beigelman, H. Lçnnberg, J.
Org. Chem. 2009, 74, 4992; f) A. Leisvuori, Y. Aiba, T. Lçnnberg, P. Poijꢃrvi-Virta, L. Blatt, L.
Beigelman, H. Lçnnberg, Org. Biomol. Chem. 2010, 8, 2131.
[15] N. Parey, C. Baraguey, J.-J. Vasseur, F. Debart, Org. Lett. 2006, 8, 3869.
[16] P. Herdewinjn, R. Pauwels, M. Baba, J. Balzarini, E. De Clercq, J. Med. Chem. 1987, 30, 2131.
[17] G. S. Ti, B. L. Gaffney, R. A. Jones, J. Am. Chem. Soc. 1982, 104, 1316.
[18] M. Kosonen, K. Hakala, H. Lçnnberg, J. Chem. Soc., Perkin Trans. 2 1998, 663; M. Kosonen, R.
Seppꢃnen, O. Wichmann, H. Lçnnberg, J. Chem. Soc., Perkin Trans. 2 1999, 2433; T. Lçnnberg, J.
Kiiski, S. Mikkola, Org. Biomol. Chem. 2005, 3, 1089; T. Lçnnberg, J. Korhonen, J. Am. Chem. Soc.
2005, 127, 7752.
Received October 7, 2010