Synthesis and enzymatic deprotection of fully protected 2′-5′ oligoadenylates (2-5A): Towards a prodrug strategy for short 2-5A

Article abstract of DOI:10.1002/cbdv.201100144

Fully protected pA2′p5′A2′p5′A trimers 1a and 1b have been prepared as prodrug candidates for a short 2′-5′ oligoadenylate, 2-5A, and its 3′-O-Me analog, respectively. The kinetics of hog liver carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH 7.5) at 37° has been studied. The deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial removal of the 3′-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage resulted in 2′, 5′→3′,5′ phosphate migration and release of adenosine as side reactions.

Full text of DOI:10.1002/cbdv.201100144

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669
Synthesis and Enzymatic Deprotection of Fully Protected 2’-5’
Oligoadenylates (2-5A): Towards a Prodrug Strategy for Short 2-5A
by Emilia Kiuru*a), Mikko Oraa), Leonid Beigelmanb), Lawrence Blattb), and Harri Lçnnberga)
a) Department of Chemistry, University of Turku, FIN-20014 Turku
(phone: þ358-2-333 6780; fax: þ358-2-333 6776; e-mail: emilia.kiuru@utu.fi)
b) AliosBiopharma, 260 E. Grand Ave, 2nd Floor, South San Francisco, CA 94080, USA
(phone: þ1-650-635 5500; fax: þ1-650-872 0584; e-mail: lbeigelman@aliosbiopharma.com)
Fully protected pA2’p5’A2’p5’A trimers 1a and 1b have been prepared as prodrug candidates for a
short 2’-5’ oligoadenylate, 2-5A, and its 3’-O-Me analog, respectively. The kinetics of hog liver
carboxyesterase (HLE)-triggered deprotection in HEPES buffer (pH 7.5) at 378 has been studied. The
deprotection of 1a turned out to be very slow, and 2-5A never appeared in a fully deprotected form. By
contrast, a considerable proportion of 1b was converted to the desired 2-5A trimer, although partial
removal of the 3’-O-[(acetyloxy)methyl] group prior to exposure of the adjacent phosphodiester linkage
resulted in 2’,5’!3’,5’ phosphate migration and release of adenosine as side reactions.
Introduction. – Interferons produced and secreted by cells in response to the
presence of infectious agents activate synthetases which produce 2’-5’ oligoadenylates
(2-5A) [1]. Intracellular endoribonuclease RNase L activated by 2-5A, in turn,
catalyzes the cleavage of viral RNA resulting in apoptosis [2–4]. Recently, several
structurally modified 2’-5’-adenylate trimers have been prepared to increase the
stability of 2-5A in serum and cytoplasm without loss of activity [5–12]. The results
obtained show that the 3’- and 5’-terminal adenosines in a 2-5A trimer may be
substituted at 3’-O, whereas the 3’-OH of the intervening adenosine is essential for
RNase L activation.
Protection of 2-5A with biodegradable groups that facilitate the uptake to
cytoplasm and release the parent 2’-5’-nucleotide drug in an intact form offer an
alternative approach to therapeutic use of 2-5A. We have previously reported on
removal of esterase-labile protecting groups from dimeric adenylyl-2’,5’-adenosines 2a
and 2b [13]. The present work is aimed at evaluating the feasibility of an enzyme-
triggered prodrug strategy for the trimeric 2-5A. The crucial factor is the timing of the
exposure of the 3’-OH and the adjacent phosphodiester linkage. The negatively
charged phosphate group must be protected to enhance the internalization and the 3’-
OH to prevent its attack on the neighboring protected phosphate linkage. The
phosphate protecting group must be removed before the 3’-OH protection, since
otherwise the extremely fast attack of the free 3’-OH group on the adjacent
phosphotriester moiety would lead to isomerization of the 2’,5’ linkage to a 3’,5’
linkage and cleavage of the PꢀO(5’) bond [14]. In the present work, (acetyloxy)methyl
(AcOCH2) and (pivaloyloxy)methyl (PivOCH2) groups have been used for the 3’-O-
protection. The bulky t-Bu group is in all likelihood removed by esterases less readily
ꢀ 2012 Verlag Helvetica Chimica Acta AG, Zꢁrich

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than the Ac group. To find out which one of these is more appropriate for the purpose,
trimers bearing 3’-O-[(acetyloxy)methyl] and 3’-O-[(pivaloyloxy)methyl] protection
have been prepared. On using the AcOCH2 protection, the 3’-OH of the 5’-terminal
nucleoside has, however, been protected as a methyl ether, since 2-5A has been shown
to tolerate this modification without loosing its ability to activate RNase L [12]. The
internucleosidic phosphodiester linkages have been protected with 3-acetyloxy-2,2-
bis(ethoxycarbonyl)propyl groups and the 5’-terminal monophosphate group with 3-
[(acetyloxy)methoxy]-2,2-bis(ethoxycarbonyl)propyl group. Our previous studies [15]
indicate that the removal of the second 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl
group, i.e., conversion of phosphodiester to monoester, is exceedingly slow, but
replacing the Ac group with an AcOCH2 group markedly accelerates the reaction. The
hog liver esterase (HLE) triggered multistep release of 2-5A from the protected
trimers 1a and 1b was followed by HPLC/MS.

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Results and Discussion. – Preparation of Protected 2-5A Trimers 1a and 1b. The
preparations of diethyl 2-[(acetyloxy)methyl]-2-(hydroxymethyl)malonate (3) [15],
diethyl 2-{[(acetyloxy)methoxy]methyl}-2-(hydroxymethyl)malonate (4) [15], N6-(4-
methoxytrityl)-3’-O-[(pivaloyloxy)methyl]adenosin-2’-yl 2’,3’-di-O-levulinoyl-N6-(4-
methoxytrityl)adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate
(5a) [13], and 3’-O-[(acetyloxy)methyl]-N6-(4-methoxytrityl)adenosin-2’-yl 2’,3’-di-O-
levulinoyl-N6-(4-methoxytrityl)adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)-
propyl phosphate (5b) [13] have been described earlier. 2’-O-Levulinoyl-N6-(4-
methoxytrityl)-3’-O-methyladenosine (6b) was prepared as depicted in Scheme 1. 2’-
O-Levulinoyl-N6-(4-methoxytrityl)-3’-O-[(pivaloyloxy)methyl]adenosine (6a) was ob-
tained by temporary trimethylsilylation of the 5’-OH function of 2’-O-levulinoyl-3’-O-
methyladenosine [13] and subsequent 4-methoxytritylation of the adenine moiety.
Scheme 1
i) TBDMSCl ((t-Bu)Me2SiCl), Py. ii) (4-Methoxyphenyl)(diphenyl)methyl chloride (MMTrCl), Py. iii)
Lev2O (Lev¼levulinoyl¼4-oxopentanoic acid), 4-(dimethylamino)pyridine (DMAP), dioxane, Py. iv)
Bu4NF, THF, AcOH.
Compounds 6a and 6b were phosphitylated with chlorobis(diethylamino)phosphine
((Et2N)2PCl). The Et2N ligands were then replaced with 3 in the case of 6a and with 4 in
the case of 6b, using 1H-tetrazole as an activator (Scheme 2). The resulting phosphite
triesters were oxidized to phosphate esters 10a and 10b with I2 in aqueous THF

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Scheme 2
i) (Et2N)2PCl, Et3N, CH2Cl2. ii) 3 with 6a, 4 with 6b, 1H-tetrazole (TetH), MeCN. iii) I2, THF, H2O,
lutidine. iv) NH2NH3OAc, Py with 10a, NH2NH3OAc, CH2Cl2, THF with 10b. Piv¼pivaloyl¼2,2-
dimethylpropanoyl.
containing 2,6-lutidine. The levulinoyl (Lev) group was removed by hydrazinium
acetate (NH2NH3OAc) treatment to afford building blocks 11a and 11b.
Trimers 1a and 1b were assembled as described in [13] for the synthesis of 2a and 2b.
Accordingly, the 2’-OH function of N6-(4-methoxytrityl)-3’-O-[(pivaloyloxy)methyl]-
adenosine 5’-{bis[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] phosphate} (11a) and
N6-(4-methoxytrityl)-3’-O-methyladenosine 5’-(bis{[3-(acetyloxy)methoxy]-2,2-bis(eth-
oxycarbonyl)propyl} phosphate) (11b) were phosphitylated with (Et2N)2PCl in the
presence of Et3N and the remaining Et2N ligands were replaced sequentially with 2a
and 3 in the case of 11a, and with 2b and 3 in the case of 11b (Scheme 3). In all these
replacements, 1H-tetrazole was used as an activator. Oxidation of the phosphite
triesters to phosphate triesters 12a and 12b, and removal of the Lev and (4-
methoxyphenyl)(diphenyl)methyl (MMTr) groups completed the synthesis to give 1a
and 1b, respectively, as a mixture of (RP)- and (SP)-diastereoisomers. For kinetic
studies, one of the diastereoisomers was separated by reversed-phase (RP) HPLC.
Enzymatic Deprotection of Trimer 1a. Hydrolysis of the protected 2-5A trimer 1a in
a HEPES buffer containing hog liver carboxyesterase (HLE; 2.6 units mlꢀ1) was
followed at pH 7.5 and 378 by analyzing the composition of the aliquots withdrawn from

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Scheme 3
i) (Et2N)2PCl, Et3N, CH2Cl2. ii) 2a (1 equiv.) with 11a and 2b (1 equiv.) with 11b, TetH, MeCN. iii) 3,
TetH, MeCN. iv) I2, THF, H2O, lutidine. v) NH2NH2, AcOH, Py with 12a, NH2NH3OAc, CH2Cl2, THF
with 12b. vi) 80% AcOH.
the reaction mixture at appropriate time intervals by HPLC. The products were
characterized by MS analysis (HPLC/ESI-MS). Fig. 1 shows the HPLC traces of the
reaction mixture at 2 and 8 d. During the first 2 d, only one intermediate accumulated
(cf. Reaction A in Scheme 4). The compound exhibited a [MþH]þ peak at m/z 1967.3,
referring to 1a that had lost one of the phosphate protecting group.
Evidently, enzymatic deacetylation ([MþH]þ transient occurrence at m/z 2169.4,
referring to a monodeacetylated product, was detected) triggered loss of HCHO and
concomitant elimination of the remnant of the protecting group as diethyl 2-
methylidenemalonate, as discussed in more detail in [16]. The mere HPLC/ESI-MS
data do not allow us to decide which one of the three possible diesters, 13a, 13b, or 13c,
was obtained. We are biased to assume that 13a was first formed, but the other
possibilities cannot be strictly excluded.
Loss of the next phosphate protecting group (Reaction B, Scheme 4) resulted in
appearance of three chromatographic signals (tR 42.0, 47.6, and 34.5 min), which all
exhibited the [MþH]þ peak at m/z 1723.3 (see the lower HPLC traces in Fig. 1). Our
previous studies with thymidine 5’-(bis{3-[(acetyloxy)methoxy]-2,2-bis(ethoxycarbo-
nyl)propyl} phosphate) [15] have shown that the enzymatic deacetylation of the
negatively charged phosphodiester is 103 times slower than the deacetylation of the
neutral phosphotriester. Accordingly, the two major chromatographic signals (tR 42.0

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Fig. 1. RP-HPLC Traces for the HLE-catalyzed hydrolysis of 2-5A trimer 1a at pH 7.5 and 37.08 (I ¼ 0.1m
with NaCl). The upper and lower traces refer to aliquots withdrawn at 2 and 8 d, respectively.
Scheme 4. HLE-Triggered Deprotection of 2-5A Trimer 1a
and 47.6 min) most likely refer to 14a and 14b, having one of the internucleosidic
phosphodiester linkages deprotected in addition to the 5’-phosphate. The minor signal

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675
(tR 34.5 min) then refers to a trimer bearing either deprotected internucleosidic
phosphates (i.e., 14c) or fully deprotected terminal phosphate (i.e., 14d).
The next phosphate protecting group was removed only very slowly (Reaction C,
Scheme 4). Two HPLC signals referring to the expected quasi-molecular-ion ([M þ
H]þ ) peak at m/z 1479.0 appeared. In other words, two of the three possible trianions
15a–15c seemed to be formed. Even a prolonged treatment (2 weeks in HLE; 2.6 units
mlꢀ1) resulted in only partial cleavage of the third phosphate protection. The fully
phosphate deprotected 2-5A never appeared, but, in parallel to formation of 14 and 15,
compounds 13 and 14 lost a (pivaloyloxy)methyl group, evidently from the 5’-terminal
Scheme 5. Side Reactions Taking Place during the HLE-Triggered Deprotection of Trimer 1a

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nucleotide (Scheme 5). The latter assumption is based on the fact that, in addition to
the [MþH]þ and [Mþ2 H]2þ peaks at m/z 1609.1 and 927.7, referring to 16 and 17,
respectively, m/z values corresponding to dimers 18 ([MþH]þ at m/z 955.7) and 19
([MþH]þ at m/z 711.5), monomer 20 ([MþH]þ at m/z 916.6), and trimers 21 ([Mþ
2 H]2þ at m/z 683.5) were detected. All these products were obtained by an attack of
the exposed 3’-OH of trimer 16 or 17 on the adjacent protected phosphate group to give
a pentacoordinated phosphorane intermediate [14]. Breakdown of this intermediate
leads to i) isomerization of the 2’,5’ bond to a 3’,5’ bond, ii) cleavage of dimer 18 or 19
(Reaction E, Scheme 5) with concomitant formation of a cyclic phosphotriester that
rapidly gives phosphodiesters 20 (Reaction F), and iii) cleavage of the phosphate
protecting group (Reaction G), resulting in a cyclic phosphotriester, which is
immediately hydrolyzed to isomeric phosphodiesters 21. Unfortunately, the desired
fully deprotedted 2-5A is never detected.
Enzymatic Deprotection of Trimer 1b. As indicated above, 3’-O-[(pivaloyloxy)-
methyl] protection did not afford a viable prodrug strategy for 2-5A. This group was
removed from the 5’-terminal nucleotide too fast, compared to the removal of the
adjacent phosphate protecting group. In addition, the 5’-monophosphate group was not
exposed, but the deprotection stopped at the diester level. For these reasons, another
prodrug candidate, 1b, was prepared: i) the 3’-OH of the 5’-teminal nucleotide was
protected permanently as a methyl ether, ii) the 3’-OH of the intevening nucleotide was
protected with a more labile 3’-O-[(acetyloxy)methyl] group, and iii) the 3-(acetyloxy)-
2,2-bis(ethoxycarbonyl)propyl groups at the 5’-terminal phosphate were replaced with
more labile 3-[(acetyloxy)methoxy]-2,2-bis(ethoxycarbonyl)propyl groups. Previous
studies have shown that replacement of the 3-AcO group with a 3-[(acetyloxy)me-
thoxy] group accelerated the first enzymatic deacetylation of bis-substituted thymidine
5’-monophosphate by a factor of 25, and the deacetylation of the resulting negatively
charged diester even more [15].
Fig. 2 shows the HPLC traces of the reaction mixture of trimer 1b treated with HLE
under the same conditions as for 1a. As seen, 1b rapidly gave a monodeacetylated
triester 22 ([MþH]þ at m/z 2057.6; Reaction H in Scheme 6), which then underwent
two parallel reactions: i) the HLE-triggered deacetylation and concomitant half-acetal
hydrolysis to dideacetylated triester 23 ([M þ H]þ at m/z 1985.1; Reaction I) and ii) the
retro-aldol condensation/elimination to the monoacetylated diester 24 ([MþH]þ at
m/z 1855.2; Reaction J). Both products, 23 and 24, were subsequently converted to the
deacetylated 5’-diester 25 ([MþH]þ at m/z 1783.0; Reactions K and L, resp.) and
further to monoester 26 ([MþH]þ at m/z 1581.0; Reaction M), bearing a fully
deprotected 5’-terminal phosphate group.
The final steps on the way to the fully deprotected 2-5A analog differed markedly
depending on which one of the remaining internucleosidic phosphodiester protecting
group was removed next. In case the 3’-terminal phosphodiester linkage was first
exposed (Reaction N, Scheme 7), to give 27 ([MꢀH]ꢀ at m/z 1334.9; tR 36.5 min), the
desired fully deprotected 2-5A analog was eventually obtained without any side
reactions. The last phosphate protection was removed (Reaction O), and the fully
phosphate deprotected trimer 28 obtained ([MꢀH]ꢀ at m/z 1090.7; tR 27.0 min) was
finally converted to the desired fully deprotected trimer 29 ([MꢀH]ꢀ at m/z 1018.6; tR
23.5 min) by removal of the 3’-O-[(acetyloxy)methyl] group (Reaction P).

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Fig. 2. HPLC Traces for the HLE-triggered deprotection of 2-5A trimer 1b at pH 7.5 and 37.08 (I ¼ 0.1m
with NaCl). The traces from top to bottom refer to aliquots withdrawn at 15 s, 3 h, 8d, and 13 d,
respectively. Signals marked with x refer to dephosphorylated products.

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Scheme 6. First Stages of HLE-Triggered Deprotection of 2-5A Trimer 1b
Unfortunately, 1b was not quantitatively converted to 29, but several side reactions
depicted in Schemes 8–10 took place. First, deprotection of one of the internucleosidic
phosphodiester linkages of 24 (Reaction Q) competed with the formation of 26. Doubly
deprotected compounds 30/31 ([MþH]þ at m/z 1610.9; tR 41 min) were accumulated
(Scheme 8), and they then lost the remaining 5’-terminal phosphate protecting group.
Compound 30, with a deprotected 3’-terminal diester bond, was converted to 27 via the
deacetylated 5’-diesters 32 ([MþH]þ at m/z 1538.7) and it, hence, gave the desired
fully deprotected trimer 29 (Reactions R and S), as indicated in Scheme 7. Compound
31, with a still protected 3’-terminal phosphodiester linkage, was, in turn, converted via
33 to 34 (Reactions R and T), which subsequently yielded several products as indicated
in Scheme 9.
Second, compound 34, which, in principle, may also be obtained by deprotection of
the 5’-terminal internucleosidic phosphodiester bond of 26 (Reaction U, Scheme 9),

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Scheme 7. Late Stages of HLE-Triggered Deprotection of 2-5A Trimer 1b
reacts by two alternative routes (Scheme 9). When the remaining 3’-terminal
phosphodiester protecting group is removed before the 3’-O-[(acetyloxy)methyl]
group (Reaction V), the desired trimer 29 is obtained, as depicted in Scheme 7. By
contrast, if the 3’-OH is first exposed (in the case of 35; Reaction W), isomerization (!
36) and cleavage of the 2’-terminal phosphoester linkage expectedly take place as a
consequence of the facile attack of the 3’-OH on the neighboring phosphotriester.
Evidently, the latter reactions really took place, since the m/z value referring to 35
([MꢀH]ꢀ at m/z 1262.8) was detected among the HPLC signals.
Finally, compound 26 also appeared to loose the 3’-O-[(acetyloxy)methyl] group
before either of the internucleosidic phosphate protecting groups (! 37; Reaction X),
since HPLC signals referring to trimers 38/39 ([MꢀH]ꢀ at m/z 1262.8) and dimers 40/
41 ([MꢀH]ꢀ at m/z 1257.9), i.e., the products expected to be obtained by an attack of

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Scheme 8. Side Reactions R, S, and T of HLE-Triggered Deprotection of 1b
the exposed 3’-OH on the adjacent phosphotriester (Reactions Y and Z), were
observed (Scheme 10).
Conclusions. – The results of the present study indicate that an esterase activity-
dependent prodrug strategy appears feasible for 2-5A, although the present protecting
group scheme, including bis{3-[(acetyloxy)methoxy]-2,2-bis(ethoxycarbonyl)propyl}
protection of the 5’-terminal monophosphate group, 3-(acetyloxy)-2,2-bis(ethoxycar-
bonyl)propyl protection of the internucleosidic phosphodiester linkages, 3’-O-methyl
protection of the 5’-terminal nucleotide and 3’-O-[(acetyloxy)methyl] protection of the
intervening nucleoside, still suffers from formation of several by-products. The main
problem is a too slow removal of the protecting group from the 3’-terminal
phosphodiester bond compared to the removal of the neighboring 3’-O-[(acetyloxy)-
methyl] group. Evidently, protection of this phosphodiester linkage with the more
labile 3-[(acetyloxy)methoxy]-2,2-bis(ethoxycarbonyl)propyl markedly improves the
situation. The advantage of the phosphate protecting groups used in the present study is
that the rate of the enzymatic step that triggers the removal and the subsequent
chemical step, which eventually results in exposure of the negatively charged
phosphate, may be tuned separately. The enzymatic reaction is susceptible to steric
properties of the acyl group, while the departure of the remnant of the protecting group
may be affected by the polar properties of the 2-substituents.

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681
Scheme 9. Side Reactions U, V, and W of HLE-Triggered Deprotection of 1b
Experimental Part
General. MeCN, CH2Cl2, and pyridine were dried over 4-ꢂ and 1,4-dioxane over 3-ꢂ molecular
sieves. Et3N was dried by refluxing over CaH2 and distilled before use. HPLC: Merck Hitachi LaChrom
D7000 with L-7455 UV detector and L-7100 pump. 1H-, 13C-, 31P-, and 2D-NMR spectra: Bruker Avance
500 NMR spectrometer; d in ppm, J in Hz. The assignments of the NMR signals are based on 2D-COSY
and HSQC spectra. LC/ESI-MS: Perkin-Elmer Sciex-API-365 triple-quadrupole. HR-ESI-MS: Bruker
Daltonics micrOTOF-Q.
2’-O-Levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-[(pivaloyloxy)methyl]adenosine
(6a). 2’-O-Levulinoyl-3’-O-[(pivaloyloxy)methyl]adenosine [13] (1.9 mmol, 0.9 g), dried over P2O5
overnight, was dissolved in dry pyridine (12 ml). The soln. was cooled on an ice-bath, and Me3SiCl
(9.4. mmol, 1.19 ml) was added. The mixture was stirred for 2.5 h at r.t. (4-Methoxyphenyl)(diphenyl)-

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Scheme 10. Side Reactions X, Y, and Z of HLE-Triggered Deprotection of 1b
methyl chloride (MMTrCl; 2.3 mmol, 0.70 g) was added, and the mixture was stirred over three nights at
378. The solvent was removed by evaporation under reduced pressure, and the residual oil was
partitioned between H2O and AcOEt. The org. layer was washed with sat. aq. NaHCO3, dried (Na2SO4),
and evaporated to dryness. The Me3Si group was removed by treatment with Bu4NF in THF under acidic
conditions. Accordingly, Bu4NF (2.8 mmol, 0.74 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 2 h. Sat. aq. NaHCO3 soln.
was added, and the mixture was extracted with CH2Cl2. The org. phase was dried (Na2SO4) and
evaporated to dryness. The product was purified by column chromatography (CC; silica gel; CH2Cl2
containing 3% MeOH) to give 6a (1.17 g, 83%). Yellowish foam. 1H-NMR (500 MHz, CDCl3): 8.02 (s,
HꢀC(2)); 7.80 (s, HꢀC(8)); 7.24–7.36 (m, 12 H of MMTr); 7.05 (s, HꢀN6); 6.81–6.84 (m, 2 H of MMTr);
6.62 (dd, J ¼ 12, 2, HOꢀC(5’)); 6.03 (d, J¼7.5, HꢀC(1’)); 5.68 (dd, J¼7.5, 5.3, HꢀC(2’)); 5.55 (d, J¼6.5,
1 H of OCH2O); 5.12 (d, J¼6.5, 1 H of OCH2O); 4.82 (dd, J¼5.3, 1, HꢀC(3’)); 4.35 (m, HꢀC(4’)); 3.93–
3.97 (m, HꢀC(5’)); 3.81 (s, MeO of MMTr); 3.72–3.78 (m, HꢀC(5’’)); 2.53–2.78 (m, 2 CH2 of Lev); 2.20
(s, Me of Lev); 1.25 (s, Me3C of Piv). 13C-NMR (126 MHz, CDCl3): 206.1 (C¼O of Lev); 177.7 (C¼O of
Piv); 171.5 (C¼O of Lev); 158.4 (MMTr); 154.6 (C(6)); 151.9 (C(2)); 147.3 (C(4)); 144.9 (MMTr); 139.8

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
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(C(8)); 136.9, 130.2, 128.9, 128.0, 127.0 (MMTr); 122.5 (C(5)); 113.2 (MMTr); 88.9 (OCH2O); 88.9
(C(1’)); 87.3 (C(4’)); 78.2 (C(3’)); 74.6 (C(2’)); 71.1 (MMTr); 62.7 (C(5’)); 55.2 (MeO of MMTr); 38.8
(Me3C of Piv); 37.7 (CH2C¼O of Lev); 29.8 (Me of Lev); 27.5 (CH2COO of Lev); 27.0 (Me of Piv). HR-
ESI-MS: 752.3312 (Mþ, C41H46N5O9þ ; calc. 752.3290).
2’-O-Levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-[(pivaloyloxy)methyl]adenosine 5’-
{Bis[3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl] Phosphate} (10a). Compound 6a (1.5 mmol, 1.10 g,
dried over P2O5 overnight) was dissolved in dry CH2Cl2 (7 ml) under N2. Anh. Et3N (7.3 mmol, 1.02 ml)
and chlorobis(diethylamino)phosphine ((Et2N)2PCl; 2.1 mmol, 0.43 ml) were added, and the mixture
was stirred for 2 h. The product was isolated by passing the mixture through a short silica gel column with
a 7:3 mixture of AcOEt and hexane containing 0.5% Et3N. The solvent was removed under reduced
pressure, and the residue was co-evaporated from dry MeCN to remove traces of Et3N. The formation of
the phosphitylated product was verified by 31P-NMR spectroscopy. 31P-NMR (202 MHz, CD3CN): 133.3.
The phosphitylated nucleoside was dissolved in dry MeCN (1 ml) under N2. Diethyl 2-[(acetyloxy)-
methyl]-2-(hydroxymethyl)propanedioate (3; 4.2 mmol, 1.10 g, co-evaporated twice with dry MeCN and
dried over P2O5 overnight), dissolved in dry MeCN (2 ml), and 1H-tetrazole (4.4 mmol, 9.76 ml of 0.45m
soln. in MeCN) were added. The course of the reaction was followed by 31P-NMR spectroscopy. The
spectrum was recorded after 0.5 h. 31P-NMR (202 MHz, CD3CN): 138.8. The phosphite ester formed was
oxidized with I2 (0.1m) in a mixture of THF, H2O, and 2,6-lutidine (4 :2 :1 (v/v/v), 10 ml) by stirring
overnight at r.t. Aq. 5% NaHCO3 soln. 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 CC (silica gel; 5%
MeOH in CH2Cl2). The purification was repeated with CH2Cl2/AcOEt 1:1 and then changing to 5%
MeOH in CH2Cl2 to give 10a (0.44 g, 22%). Clear oil. 1H-NMR (500 MHz, CDCl3): 8.03 (s, HꢀC(2)); 7.95
(s, HꢀC(8)); 7.23–7.37 (m, 12 H of MMTr); 6.94 (s, HꢀN6); 6.80–6.83 (m, 2 H of MMTr); 6.09 (d, J¼3.5,
HꢀC(1’)); 5.76 (dd, J¼5.5, 3.5, HꢀC(2’)); 5.34 (d, J¼6.5, 1 H of OCH2O); 5.20 (d, J¼6.5, 1 H of
OCH2O); 4.97 (m, HꢀC(3’)); 4.51–4.62 (m, 2 CH2OAc, 2 POCH2C); 4.17–4.33 (m, HꢀC(4’), HꢀC(5’),
HꢀC(5’’), and 4 MeCH2O); 3.81 (s, MeO of MMTr); 2.77–2.80 (m, 2 H of CH2CH2 of Lev); 2.66–2.70
(m, 2 H of CH2CH2 of Lev); 2.20 (s, Me of Lev); 2.05 (s, AcO); 2.01 (s, AcO); 1.20–1.32 (m, 4 MeCH2,
3 Me of Piv). 31P-NMR (202 MHz, CD3CN): ꢀ2.59. HR-ESI-MS: 1320.4812 (Mþ, C63H79N5O24Pþ ; calc.
1320.4847).
N6-[(4-Methoxyphenyl)(diphenyl)methyl]-3’-O-[(pivaloyloxy)methyl]adenosine 5’-(Bis{3-[(acetyl-
oxy)methyl]-2,2-bis(ethoxycarbonyl)propyl} Phosphate) (11a). Compound 10a (0.3 mmol, 0.44 g) was
dissolved in a soln. of NH2NH2 ·H2O (3.9 mmol, 0.12 ml) in pyridine (4 ml) and AcOH (1 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 2 h. The reaction was quenched with 0.1m NaH2PO3 soln., and the mixture was
extracted with CH2Cl2. The org. phase was washed with H2O, dried (Na2SO4), and evaporated to dryness.
The product was purified by CC (silica gel; CH2Cl2 containing 3–5% MeOH) to give 11a (0.35 g, 88%).
Clear oil. 1H-NMR (500 MHz, CDCl3): 8.02 (s, HꢀC(2)); 7.98 (s, HꢀC(8)); 7.23–7.37 (m, 12 H of MMTr);
6.96 (s, HꢀN6); 6.80–6.83 (m, 2 H of MMTr); 5.93 (d, J¼5.0, HꢀC(1’)); 5.51 (d, J¼6.3, 1 H of OCH2O);
5.42 (d, J¼6.3, 1 H of OCH2O); 4.76 (dd, J¼5.5, 5.0, HꢀC(2’)); 4.64 (m, HꢀC(3’)); 4.50–4.63 (m,
2 CH2OAc, 2 POCH2C); 4.37 (m, HꢀC(4’)); 4.19–4.31 (m, 4 MeCH2O, HꢀC(5’) and HꢀC(5’’)); 3.88 (d,
J ¼ 5, HOꢀC(2’)) 3.81 (s, MeO of MMTr); 2.05 (s, AcO); 2.03 (s, AcO); 1.22–1.32 (m, 4 MeCH2O, 3 Me
of Piv). 13C-NMR (126 MHz, CDCl3): 178.0 (C¼O of Piv); 170.1 (C¼O of Ac); 166.4 (C(¼O)OEt) 158.3
(MMTr); 154.3 (C(6)); 152.1 (C(2)); 148.3 (C(4)); 145.2 (MMTr); 138.8 (C(8)); 135.9, 130.2, 128.9, 127.9,
126.9 (MMTr); 123.7 (C(5)); 113.2 (MMTr); 89.4 (C(1’)); 89.0 (OCH2O); 81.3 (C(4’)); 78.7 (C(3’)); 74.0
(C(2’)); 71.0 (MMTr); 67.2 (C(5’)); 65.4 (POCH2C); 62.3 (MeCH2); 61.2 (CH2OAc); 58.0 (C); 55.2
(MeO of MMTr); 38.8 (Me3C of Piv); 27.0 (Me of Piv); 20.6 (Ac); 13.9 (MeCH2). HR-ESI-MS:
1222.4485 (Mþ, C58H73N5O22Pþ ; calc. 1222.4479).
5’-O-[(tert-Butyl)(dimethyl)silyl]-3’-O-methyladenosine (7). Commercially available 3’-O-methyl-
adenosine (3.6 mmol, 1.01 g) was co-evaporated twice from anh. pyridine, and the residue was dissolved
in the same solvent (7 ml). t-Bu(Me2)SiCl (TBDMSCl; 1.1 equiv.; 4.0 mmol, 0.60 g) was added, and the
mixture was stirred overnight at r.t. The reaction was quenched with MeOH, and the mixture evaporated
to dryness. The residue was purified by CC (silica gel; CH2Cl2 containing 10% MeOH). 1H-NMR
(500 MHz, MeOD): 8.41 (s, HꢀC(2)); 8.23 (s, HꢀC(8)); 6.06 (d, J¼4.2, HꢀC(1’)); 4.77 (dd, J¼4.2, 4.6,

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
HꢀC(2’)); 4.22 (m, HꢀC(4’)); 4.06 (dd, J¼4.6, 5.0, HꢀC(3’)); 4.03 (dd, J¼11.5, 3.4, HꢀC(5’)); 3.87 (dd,
t
J¼11.5, 3.0, HꢀC(5’’)); 3.50 (s, 3’-MeO); 0.96 (s, Bu); 0.14 (s, Me2Si). 13C-NMR (126 MHz, MeOD):
155.9 (C(6)); 152.5 (C(2)); 149.1 (C(4)); 139.3 (C(8)); 119.0 (C(5)); 88.8 (C(1’)); 82.7 (C(4’)); 78.9
(C(3’)); 73.3 (C(2’)); 62.3 (C(5’)); 57.0 (MeO); 25.0 (Me3C); 17.9 (Me3C); ꢀ6.7 (Me2Si).
5’-O-[(tert-Butyl)(dimethyl)silyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-methyladenosine
(8). Compound 7 was co-evaporated twice from anh. pyridine, and the residue was dissolved in dry
pyridine (6 ml). MMTrCl was added, and the mixture was stirred over three nights 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 CC (silica gel; CH2Cl2 containing 2–3% MeOH) to afford 8
1
(180 g, 75% yield from 3’-O-methyladenosine). White foam. H-NMR (500 MHz, CDCl3): 8.06 (br. s,
HꢀC(2), HꢀC(8)); 7.34–7.37 (m, 4 H of MMTr); 7.23–7.30 (m, 8 H of MMTr); 6.96 (br. s, HꢀN); 6.81 (d,
J ¼ 8.8, 2 H of MMTr); 5.98 (d, J¼5.6, HꢀC(1’)); 4.73 (m, HꢀC(2’)); 4.27 (m, HꢀC(4’)); 4.16 (d, J¼6.5,
HOꢀC(2’)); 4.05 (m, HꢀC(3’)); 3.91 (dd, J¼11.2, 4.2, HꢀC(5’)); 3.78–3.81 (m, MeO, HꢀC(5’’)); 3.52 (s,
3’-MeO); 0.90 (s, tBu); 0.09, 0.10 (2s, Me2Si). 13C-NMR (126 MHz, CDCl3): 158.3 (MMTr); 154.1 (C(6));
152.1 (C(2)); 148.5 (C(4)); 145.2 (MMTr); 138.3 (C(8)); 137.2 (MMTr); 130.2 (MMTr); 128.9 (MMTr);
127.9 (MMTr); 126.9 (MMTr); 121.3 (C(5)); 113.1 (MMTr); 89.3 (C(1’)); 83.1 (C(4’)); 80.0 (C(3’)); 74.3
(C(2’)); 71.0 (MMTr); 63.0 (C(5’)); 58.1 (3’-MeO); 55.2 (MMTr); 25.9 (Me3C); 18.3 (Me3C); ꢀ5.5
(Me2Si).
5’-O-[(tert-Butyl)(dimethyl)silyl]-2’-O-levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-
methyladenosine (9). Levulinic anhydride was prepared by dissolving levulinic acid (6.7 mmol, 0.73 g) in
dry 1,4-dioxane (10 ml) on an ice bath and adding N,N’-dicyclohexylcarbodiimide (DCC; 3.4 mmol,
0.70 g) in small portions within 1 h. The soln. was stirred 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 8 (2.7 mmol, 1.80 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, 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 CC (silica gel; CH2Cl2 containing 1–
2% MeOH to give 9 (184 g, 89%). White foam. 1H-NMR (500 MHz, CDCl3): 8.10 (s, HꢀC(2)); 8.06 (s,
HꢀC(8)); 7.23–7.38 (m, 12 H of MMTr); 6.92 (s, 2 HꢀN6); 6.82 (m, 2 H of MMTr); 6.19 (d, J¼4.0,
HꢀC(1’)); 5.74 (dd, J ¼ 4.5, 4.0, HꢀC(2’)); 4.30 (m, HꢀC(3’)); 4.18 (m, HꢀC(4’)); 4.01 (dd, J¼11.5, 3.0,
HꢀC(5’)); 3.83 (dd, J¼11.5, 3.0, HꢀC(5’’)); 3.80 (s, 3 H of MMTr); 3.42 (s, MeO); 2.62–2.81 (m, 4 H of
Lev); 2.18 (s, 3 H of Lev); 0.94 (s, tBu); 0.12 (s, MeSi); 0.11 (s, MeSi). 13C-NMR (126 MHz, CDCl3): 206.1
(C¼O of Lev); 171.7 (C¼O of Lev); 158.3 (MMTr); 154.1 (C(6)); 152.4 (C(2)); 148.5 (C(4)); 145.2
(MMTr); 138.4 (C(8)); 137.2 (MMTr); 130.2 (MMTr); 128.9 (MMTr); 127.9 (MMTr); 126.8 (MMTr);
121.2 (C(5)); 113.1 (MMTr); 86.5 (C(1’)); 82.9 (C(4’)); 77.7 (C(3’)); 74.5 (C(2’)); 71.0 (MMTr); 62.2
(C(5’)); 58.8 (MeO); 55.2 (MMTr); 37.8 (Lev); 29.8 (Lev); 27.8 (Lev); 26.0 (TBDMS); 18.4 (TBDMS);
ꢀ5.3 (TBDMS); ꢀ5.5 (TBDMS).
2’-O-Levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-methyladenosine (6b). Compound 9
was desilylated with Bu4NF in THF under acidic conditions. Accordingly, to a soln. of Bu4NF (3.6 mmol,
0.94 g) in dry THF (30 ml), AcOH (6 ml) and 9 were added, and the mixture was stirred over two nights
at r.t. Sat. aq. NaHCO3 soln. was added, and the mixture was extracted with CH2Cl2. The org. phase was
washed with sat. aq. NaHCO3 and sat. aq. NaCl, and dried (Na2SO4) and evaporated to dryness. The
product was purified by CC (silica gel; CH2Cl2 containing 1–3% MeOH) to give 6b (1.25 g, 80%). White
1
foam. H-NMR (500 MHz, CDCl3): 7.99 (s, HꢀC(2)); 7.77 (s, HꢀC(8)); 7.21–7.34 (m, 12 H of MMTr);
7.00 (s, HꢀN6); 6.80 (m, 2 H of MMTr); 6.59 (dd, J¼12.0, 2.0, HOꢀC(5’)); 6.00 (d, J¼7.5, HꢀC(1’)); 5.71
(dd, J ¼ 7.5, 5.0, HꢀC(2’)); 4.33–4.36 (m, HꢀC(3’), HꢀC(4’)); 3.98 (m, HꢀC(5’)); 3.78 (s, 3 H of MMTr);
3.69 (m, HꢀC(5’’)); 3.44 (s, MeO); 2.53–2.75 (m, 4 H of Lev); 2.17 (s, 3 H of Lev). 13C-NMR (126 MHz,
CDCl3): 206.1 (C¼O of Lev); 171.6 (C¼O of Lev); 158.4 (MMTr); 154.6 (C(6)); 151.9 (C(2)); 147.3
(C(4)); 145.0 (MMTr); 139.8 (C(8)); 136.9 (MMTr); 130.2 (MMTr); 128.9 (MMTr); 128.0 (MMTr); 127.0
(MMTr); 122.5 (C(5)); 113.2 (MMTr); 89.1 (C(1’)); 86.1 (C(4’)); 79.4 (C(3’)); 75.0 (C(2’)); 71.1 (MMTr);
63.2 (C(5’)); 58.5 (MeO); 55.2 (MMTr); 37.7 (Lev); 29.8 (Lev); 27.6 (Lev). HR-ESI-MS: 652.2718 (Mþ,
C31H38N7Oþ9 ; calc. 652.2726).

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
685
2’-O-Levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-3’-O-methyladenosine 5’-(Bis{3-[(acetyl-
oxy)methoxy]-2,2-bis(ethoxycarbonyl)propyl} Phosphate) (10b). Compound 6b (1.5 mmol, 0.99 g, dried
over P2O5 over three nights) was dissolved in dry CH2Cl2 (3 ml) under N2. Anh. Et3N (7.60 mmol,
1.056 ml) and (Et2N)2PCl (2.13 mmol, 447 ml) were added, and the mixture was stirred for 2 h. The
product was isolated by passing the mixture through a short silica-gel column eluting with dry AcOEt
containing 1% Et3N. The solvent was removed under reduced pressure, and the residue was co-
evaporated from dry MeCN to remove traces of Et3N. The identity of the product was checked by 31P-
NMR spectroscopy. 31P-NMR (202 MHz, CD3CN): 133.7. The phosphitylated nucleoside was dissolved in
dry MeCN (1 ml) under N2. Diethyl 2-{[(acetyloxy)methoxy]methyl}-2-(hydroxymethyl)propanedioate
(4; 3.8 mmol, 0.11 g, co-evaporated twice with dry MeCN and dried over P2O5 over three nights) and 1H-
tetrazole (3.80 mmol, 8.440 ml of 0.45m soln. in MeCN) were added. The course of the reaction was
followed by 31P-NMR spectroscopy. The spectrum was recorded after 1 h. 31P-NMR (202 MHz, CD3CN):
139.0. The phosphite ester formed was oxidized with I2 (0.1m) in a mixture of THF, H2O, and 2,6-lutidine
(4 :2 :1 (v/v/v); 10 ml) by stirring overnight at r.t. Aq. 5% NaHCO3 soln. 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 CC (silica gel; CH2Cl2/AcOEt 1:1) to give 10b (0.85 g, 43%). Clear oil. 1H-NMR
(400 MHz, CDCl3): 8.01 (s, HꢀC(2)); 7.92 (s, HꢀC(8)); 7.19–7.35 (m, 12 H of MMTr); 6.92 (s, HꢀN6);
6.77–6.81 (m, 2 H of MMTr); 6.06 (d, J¼3.2, HꢀC(1’)); 5.76 (dd, J¼5.2, 3.2, HꢀC(2’)); 5.20–5.25 (m,
2 OCH2O); 4.50–4.53 (m, 2 POCH2C); 4.39 (m, HꢀC(3’)); 4.32 (m, HꢀC(5’)); 4.09–4.27 (m, 14 H,
HꢀC(4’), HꢀC(5’’), MeCH2O, CH2O); 3.78 (s, MeO of MMTr); 3.41 (s, 3’-MeO); 2.63–2.79 (m, CH2CH2
of Lev); 2.17 (s, Me of Lev); 2.06 (s, Ac); 2.04 (s, Ac); 1.18–1.28 (m, 4 MeCH2). 13C-NMR (101 MHz,
CDCl3): 206.0 (C¼O Lev); 171.7 (C¼O of Lev); 170.2 (MeC¼O); 166.6 (C(¼O)OEt) 158.3 (MMTr);
154.2 (C(6)); 152.4 (C(2)); 148.3 (C(4)); 145.2 (MMTr); 139.0 (C(8)); 137.2, 130.2, 128.9, 127.9, 126.8
(MMTr); 121.5 (C(5)); 113.2 (MMTr); 89.1 (OCH2O); 88.8 (OCH2O); 87.5 (C(1’)); 80.6 (C(4’)); 78.1
(C(3’)); 73.7 (C(2’)); 71.0 (MMTr); 67.1 (C(5’)); 65.2 (POCH2C); 62.1 (MeCH2); 61.8 (CH2OAc); 59.1
(3’-MeO); 55.2 (MeO of MMTr); 53.4 (C); 37.8 (CH2 of Lev); 29.7 (Me of Lev); 27.8 (CH2 of Lev); 20.9
(Ac); 13.9 (MeCH2). 31P-NMR (162 MHz, CDCl3): ꢀ2.15. HR-ESI-MS: 1280.4538 (Mþ, C60H75N5O24Pþ
; calc. 1280.4534).
N6-[(4-Methoxyphenyl)(diphenyl)methyl]-3’-O-methyladenosine 5’-(Bis{3-[(acetyloxy)methoxy]-
2,2-bis(ethoxycarbonyl)propyl} Phosphate) (11b). Compound 10b (0.66 mmol, 0.85 g, dried over P2O5
over three nights) was dissolved in dry CH2Cl2 (16 ml). NH2NH3OAc (1.19 mmol, 0.11 g) in dry MeOH
(2 ml) was added. After stirring the mixture for 3 h, another portion of NH2NH3OAc (0.60 mmol,
0.055 g) in dry MeOH (1 ml) was added, and the reaction was allowed to proceed for 1 h. The reaction
was quenched with acetone, and the mixture was evaporated to dryness. The product was purified by CC
(silica gel; CH2Cl2/AcOEt 1:4) to give 11b (0.70 g, 89%). Clear oil. 1H-NMR (500 MHz, CDCl3): 8.01 (s,
HꢀC(2)); 7.96 (s, HꢀC(8)); 7.20–7.35 (m, 12 H of MMTr); 6.94 (s, HꢀN6)); 6.77–6.81 (m, 2 H of MMTr);
5.91 (d, J¼5.5, HꢀC(1’)); 5.20–5.25 (m, 2 OCH2O); 4.80 (m, HꢀC(2’)); 4.49–4.56 (m, 2 POCH2C); 4.33
(m, HꢀC(4’)); 4.10–4.30 (m, HꢀC(5’), HꢀC(5’’), HꢀC(3’), 4 MeCH2O, 2 CH2O); 3.84 (br. s, HOꢀC(2’));
3.78 (s, MeO of MMTr); 3.54 (s, 3’-MeO); 2.06 (s, Ac); 2.05 (s, Ac); 1.20–1.29 (m, 4 MeCH2O). 13C-NMR
(126 MHz, CDCl3): 170.3 (MeC¼O); 166.6 (C(¼O)OEt); 158.3 (MMTr); 154.2 (C(6)); 152.2 (C(2));
148.5 (C(4)); 145.2 (MMTr); 138.8 (C(8)); 137.2, 130.2, 128.9, 127.9, 126.9 (MMTr); 121.5 (C(5)); 113.2
(MMTr); 89.5 (C(1’)); 88.7 (OCH2O); 80.6 (C(4’)); 79.7 (C(3’)); 73.3 (C(2’)); 71.0 (MMTr); 67.1 (C(5’));
65.3 (POCH2C); 62.2 (CH2Me); 61.8 (CH2OAc); 58.9 (3’-MeO); 55.2 (MeO of MMTr); 53.5 (C); 20.9
(Ac); 13.9 (MeCH2). HR-ESI-MS: 1182.4123 (Mþ, C55H69N5O22Pþ ; calc. 1182.4166).
Assembly of Trimer 1a. Compound 11a (0.29 mmol, 0.340 g, dried over P2O5 overnight) was dissolved
in dry CH2Cl2 (2 ml) under N2. Anh. Et3N (1.42 mmol, 0.198 ml) and (Et2N)2PCl (0.34 mmol, 0.072 ml)
were added. The mixture was stirred for 2.5 h. The product was isolated by passing the mixture through a
short silica-gel column with AcOEt/hexane 7:3, containing 0.5% Et3N. The solvent was removed under
reduced pressure. The product was co-evaporated from dry MeCN to remove traces of Et3N. The
formation of the phosphitylated product was verified by 31P-NMR spectroscopy. 31P-NMR (202 MHz,
CD3CN): 137.4, ꢀ2.43.
The phosphitylated nucleoside was dissolved in dry MeCN (400 ml) under N2. 3’-O-[(Pivaloyloxy)-
methyl]-N6-[(4-methoxyphenyl)(diphenyl)methyl]adenosin-2’-yl 2’,3’-di-O-levulinoyl-N6-[(4-methoxy-

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CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
phenyl)(diphenyl)methyl]adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl)propyl phosphate (2a;
0.16 mmol, 0.270 g, dried over P2O5 overnight) in dry MeCN (600 ml) and 1H-tetrazole (0.19 mmol,
0.422 ml of 0.45m soln. in MeCN) were added. The reaction was allowed to proceed for 10 min. Then, 3
(0.29 mmol, 0.080 g; co-evaporated twice with dry MeCN and dried over P2O5 overnight) in dry MeCN
(200 ml) and 1H-tetrazole (0.22 mmol, 0.487 ml of 0.45m soln. in MeCN) were added, and the mixture was
stirred for 10 min before the spectrum was recorded. 31P-NMR (202 MHz, CD3CN): 151.6, 151.3, 150.8,
150.1, 140.5, 140.4, ꢀ2.1– ꢀ2.6 (m).
The phosphite ester formed was oxidized with I2 (0.1m) in a mixture of THF, H2O, and 2,6-lutidine
(4 :2 :1 (v/v/v); 7 ml) by stirring overnight at r.t. Aq. 5% NaHCO3 soln. 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 CC (silica gel; CH2Cl2/AcOEt 1:1!AcOEt!CH2Cl2 containing 10% MeOH) to give
12a (diastereoisomer mixture; 0.30, 33%). Yellowish oil. The completion of the oxidation was checked by
31P-NMR spectroscopy. 31P-NMR (202 MHz, CD3CN): ꢀ1.5 – ꢀ3.0 (m).
The levulinoyl groups were removed by treatment with NH2NH3OAc. Accordingly, 12a (0.100 g) was
dissolved in a soln. of NH2NH2 ·H2O (2.50 mmol, 0.078 ml) in 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 mixture was
extracted with CH2Cl2. The org. phase was washed with H2O, dried (Na2SO4), and evaporated to dryness.
The product was purified by CC (silica gel; CH2Cl2 containing 5% MeOH). The compound was then
subjected to detritylation. The product was dissolved in 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 silica-gel choromatography with CH2Cl2 containing 10–20% MeOH,
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 from H2O to MeCN in 30 min. Overall yield of 1a starting from 12a was
39% (27 mg). To study the removal of the enzyme labile protecting groups, 2 mg of the slowest migrating
diastereoisomer was separated on a Sun FireTM Prep C18 column (250ꢁ10 mm, 5 mm, flow rate 3.0 ml
1
minꢀ1) eluting with 40–80% MeCN in 30 min. H-NMR (500 MHz, CD3CN): 7.98–8.25 (m, 3 HꢀC(2)
and 3 HꢀC(8)); 6.19–6.53 (m, 3 NH2); 5.90–6.18 (d, 3 HꢀC(1’)); 5.17–5.59 (m, HꢀC(2’), HꢀC(3’),
2 OCH2O); 4.85–5.20 (m, HꢀC(3’)); 4.61 (m, HꢀC(2’)); 4.09–4.58 (m, HꢀC(2’), HꢀC(3’), HOꢀC(2’),
HOꢀC(3’), 4 OCH2OAc, 4 POCH2C, HꢀC(4’), 3 HꢀC(5’), 3 HꢀC(5’’), 8 MeCH2); 1.94–2.04 (m,
4 AcO); 1.13–1.27 (m, 42 H of Me3C of Piv, MeCH2). 31P-NMR (202 MHz, CDCl3): ꢀ2.7– ꢀ2.0 (m).
(Multiplicity of some signals is due to the presence of (RP)- and (SP)-diastereoisomers.) HR-ESI-MS:
2210.6789 (Mþ, C86H123N15O47Pþ3 ; calc. 2210.6903).
Assembly of Trimer 1b. Compound 11b (0.42 mmol, 0.500 g, dried over P2O5 overnight) was
dissolved in dry CH2Cl2 (3 ml) under N2. Anh. Et3N (2.11 mmol, 294 ml) and (Et2N)2PCl (0.59 mmol,
125 ml) were added. The mixture was stirred for 2.5 h. The product was isolated by passing the mixture
through a short silica-gel column with AcOEt/hexane 9 :1, containing 1% Et3N. The solvent was removed
under reduced pressure. The formation of the phosphitylated product was verified by 31P-NMR
spectroscopy. 31P-NMR (202 MHz, CD3CN): 139.2, ꢀ2.3. The compound was co-evaporated from dry
MeCN to remove traces of Et3N and dissolved in dry MeCN (0.5 ml) under N2. Dry CH2Cl2 (0.5 ml) was
added, because the compound was not completely dissolved. 3’-O-[(Acetyloxy)methyl]-N6-[(4-methoxy-
phenyl)(diphenyl)methyl]adenosine-2’-yl 2’,3’-di-O-levulinoyl-N6-[(4-methoxyphenyl)(diphenyl)methyl]-
adenosin-5’-yl 3-(acetyloxy)-2,2-bis(ethoxycarbonyl) phosphate (2b; 0.25 mmol, 0.42 g, dried over P2O5
overnight) was dissolved in a mixture of dry MeCN (2 ml) and dry CH2Cl2 (0.5 ml), and 1H-tetrazole
(0.51 mmol, 1.128 ml of 0.45m soln. in MeCN) was added. The course of the reaction was followed by 31P-
NMR spectroscopy. 1H-Tetrazole (0.25 mmol, 0.564 ml of 0.45m soln. in MeCN) was added after 50 min,
and the reaction was allowed to proceed for 15 min. Compound 3 (0.42 mmol, 0,110 g; co-evaporated
twice with dry MeCN and dried over P2O5 overnight) and 1H-tetrazole (0.76 mmol; 1.692 ml of 0.45m
soln. in MeCN) were added, and the mixture was stirred for 30 min before the spectrum was recorded.
31P-NMR (202 MHz, CD3CN): 140.2–140.6 (m), ꢀ2.2– ꢀ2.6 (m). The phosphite ester formed was
oxidized with I2 (0.1 mol lꢀ1) in a mixture of THF, H2O, and 2,6-lutidine (4 :2 :1, (v/v/v); 7 ml) by stirring
overnight at r.t. Aq. 5% NaHCO3 soln. 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 CC (silica gel;

CHEMISTRY & BIODIVERSITY – Vol. 9 (2012)
687
CH2Cl2/AcOEt 3 :7!CH2Cl2 containing 5% MeOH) to give 12b (diastereoisomer mixture; 0.58 g,
43%). Yellowish oil. The completion of the oxidation was checked by 31P-NMR spectroscopy. 31P-NMR
(202 MHz, CD3CN): ꢀ1.9 – ꢀ2.9 (m).
To remove the levulinoyl groups, 12b was evaporated once from dry MeCN and dissolved in dry
CH2Cl2. NH2NH3OAc (0.74 mmol, 0.070 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, the mixture was stirred for 20 min and
evaporated to dryness. The product was purified by silica-gel cromatography with CH2Cl2 containing 5%
MeOH. The crude product was obtained in 0.510 g yield. A part of the compound (0.280 g) 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 by
HPLC on a Sun FireTM Prep C18 column (250ꢁ10 mm, 5 mm, flow rate 3.0 ml minꢀ1), with a linear
gradient from 17 to 100% MeOH within 20 min, and by isocratic elution with MeOH for 6 min. Overall
yield of 1b starting from 12b was 38% (81 mg). To study the removal of the enzyme-labile protecting
groups, the diastereoisomers were separated on a Sun FireTM Prep C18 column (250ꢁ10 mm, 5 mm, flow
rate 3.0 ml minꢀ1) with 55% MeCN for 30 min. 1H-NMR (500 MHz, CD3OD): 8.07–8.28 (m, 3 HꢀC(2),
3 HꢀC(8)); 6.19–6.26 (m, HꢀC(1’)); 6.08–6.12 (m, HꢀC(1’)); 5.90–5.95 (m, HꢀC(1’)); 5.19–5.69 (m,
HꢀC(2’), 3 OCH2O); 4.80–5.00 (m, HꢀC(3’)); 4.71–4.75 (m, HꢀC(2’)); 4.09–4.63 (m, HꢀC(2’),
2 HꢀC(3’), 3 HꢀC(4’), 3 HꢀC(5’), 3 HꢀC(5’’), 4 CH2O, 4 POCH2C, 8 MeCH2); 3.49–3.58 (m, 3’-MeO);
1.95–2.17 (m, 5 AcO); 1.15–1.28 (m, 8 MeCH2). 31P-NMR (202 MHz, CDCl3): ꢀ2.6– ꢀ1.4 (m).
(Multiplicity of some signals is due to the presence of (RP)- and (SP)-diastereoisomers.) HR-ESI-MS:
2129.6054 (Mþ, C80H114N15O47Pþ3 ; calc. 2129.6205).
Removal of the Enzyme-Labile Protecting Groups. The removal of the protecting groups was
followed by HPLC and HPLC/MS methods. The reactions were carried out in sealed tubes immersed in a
thermostated water-bath (37.0ꢂ0.18). The hydronium ion concentration of the reaction solns. (3.0 ml)
was adjusted with N-[2-hydroxyethyl]piperazine-N-[2-ethanesulfonic acid] (HEPES) buffer (0.040/
0.024m; pH 7.5). The ionic strength of the solns. was adjusted to 0.1m with NaCl. The hydronium ion
concentrations of the buffer solns. were calculated with the aid of the known pKa values of the buffer
acids under the exper. conditions. The initial substrate concentration was 0.15 mm. The Ac 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 1m aq. HCl to inactivate enzyme and to quench the
hydrolysis, cooled in an ice-bath and filtered with minisart RC 4 filters (0.45 mm). The composition of the
samples was analyzed on an ODS Hypersil C18 column (4ꢁ250 mm 5 mm, flow rate 1 ml minꢀ1), with a
AcOH/AcONa buffer (0.045/0.015m) and MeCN, containing NH4Cl (0.1m). A good separation of the
product mixtures of 1 was obtained by a 5-min isocratic elution with the buffer containing 2% MeCN,
followed by a linear gradient (23 min) up to 40.0% MeCN. The reaction products were identified by LC/
MS using a mixture of H2O and MeCN, containing HCOOH (0.1%) as eluent (Gemini C18 column (2ꢁ
150 mm, 5 mm; flow rate 200 ml minꢀ1). Signals were recorded on a UV detector at a wavelength of
260 nm. The reaction products were identified by means of LC/MS. A mixture of H2O and MeCN
containing HCOOH (0.1%) was used as eluent. The enzymatic deacetylations obeyed first-order kinetics
at the HLE concentrations employed.
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Received April 27, 2011