Preparation of zwitterionic ribonucleoside phosphoramidites for solid-phase siRNA synthesis

Article abstract of DOI:10.1021/jo800451m

(Chemical Equation Presented) RNA oligomers, carrying 2′-O-modified nucleosides, proved to be extremely useful in different antisense strategies, including RNAi. The 2′-O-alkyl modification, carrying an amino functionality, deserves special attention due

Full text of DOI:10.1021/jo800451m

Preparation of Zwitterionic Ribonucleoside Phosphoramidites for
Solid-Phase siRNA Synthesis
Romualdas Smicius and Joachim W. Engels*
Institut fu¨r Organische Chemie und Chemische Biologie, Johann Wolfgang Goethe-UniVersita¨t,
Max-Von-Laue-Strasse 7, 60438 Frankfurt am Main, Germany
Joachim.Engels@chemie.uni-frankfurt.de
ReceiVed March 4, 2008
RNA oligomers, carrying 2′-O-modified nucleosides, proved to be extremely useful in different antisense
strategies, including RNAi. The 2′-O-alkyl modification, carrying an amino functionality, deserves special
attention due to its ability to neutralize the negatively charged phosphate backbone, leading to improved
physicochemical and pharmaceutical properties of antisense agents. Here, we report a very short,
convenient, and straightforward synthesis of phosphoramidites for all four 2′-aminoethyl-modified natural
ribonucleosides, where the aminoethyl group is introduced in a single alkylation step.
Introduction
the thermal stability of RNA duplexes,9,10 presumably due to
neutralization of the negatively charged phosphate backbone.
On the basis of these features, we decided to develop an easy
access to the 2′-aminoethyl modification of all four natural
ribonucleosides and their appropriate phosphoramidite building
blocks for the solid-phase synthesis of oligonucleotides.
Generally, 2′-O-aminoethyl nucleosides are synthesized by
alkylation of the 2′-OH group with methyl bromoacetate9–12 or
allylvinyl carbonate12 followed by transformation of the cor-
responding functional groups to the 2-aminoethyl functionality.
In the case of the 2′-O-methoxycarbonylmethyl group these steps
involve reduction of the ester group to an alcohol, conversion
of the latter to tosyl or mesyl derivatives, their replacement with
azide, and, finally, reduction of the azide to an amino group,
followed by acylation for protection. Conversion of the 2′-O-allyl
group to aminoethyl includes two additional stepssdihydroxylation
of the bond and oxidative cleavage of the 1,2-diol to aldehydesand
further steps are the same as for the ester.
Since its discovery RNAi has already proved to be one of
the most powerful tools for investigating gene function and has
shown great promise as being therapeutic for diseases, such as
cancer and virus infections.1,2 Despite great hopes of using
siRNA for therapeutic purposes, some serious problems exist,
such as activation of immune response, low serum stability, and
poor in vivo delivery.3–5 One way to try to overcome these
limitations is to design chemically modified siRNAs. Among
many possibilities the 2′-O-position of the sugar moiety in
nucleosides has already proved to be optimal for oligonucleotide
modifications in antisense technology.1,3,6 Alkyl substituents
with basic groups, such as amino, in this position enhance the
resistance to nucleases,6–9 and additionally they contribute to
(1) Bumcrot, D.; Manoharan, M.; Koteliansky, V.; Sah, D. W. Y. Nat. Chem.
Biol. 2006, 2, 711–719.
(2) Dorsett, Y.; Tuschl, T. Nat. ReV. Drug Disc. 2004, 3, 318–329.
(3) De Paula, D.; Bentley, M. V. L. B.; Mahato, R. I. RNA 2007, 13, 431–
456.
(8) Teplova, M.; Wallace, S. T.; Tereschko, V.; Minasov, G.; Symons, A. M.;
Cook, P. D.; Manoharan, M.; Egli, M. PNAS 1999, 96, 14240–14245.
(9) Odadzic, D.; Bramsen, J. B.; Smicius, R.; Bus, C.; Kjems, J.; Engels,
J. W. Bioorg. Med. Chem. 2008, 16, 518–529.
(10) Klo¨pffer, A. E.; Engels, J. W. ChemBioChem 2004, 5, 707–716.
(11) Cuenoud, B.; Casset, F.; Hu¨sken, D.; Natt, F.; Wolf, R. M.; Altmann,
K. H.; Martin, P.; Moser, H. E. Angew. Chem., Int. Ed. 1998, 37, 1288–1291.
(12) Jin, S.; Miduturu, C. V.; McKinney, D. C.; Silverman, S. K. J. Org.
Chem. 2005, 70, 4284–4299.
(4) Kurreck, J. Eur. J. Biochem. 2003, 270, 1628–1644.
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928–935.
(6) Manoharan, M. Biochim. Biophys. Acta 1999, 1489, 117–130.
(7) Griffey, R. H.; Monia, B. P.; Cummins, L. L.; Freier, S.; Greig, M. J.;
Guinosso, C. J.; Lesnik, E.; Manalili, S. M.; Mohan, V.; Owens, S.; Ross, B. R.;
Sasmor, H.; Wancewicz, E.; Weiler, K.; Wheeler, P. D.; Cook, P. D. J. Med.
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4994 J. Org. Chem. 2008, 73, 4994–5002
10.1021/jo800451m CCC: $40.75 2008 American Chemical Society
Published on Web 06/04/2008

Zwitterionic Ribonucleoside Phosphoramidites
For pyrimidine nucleosides in the alkylation step only uridine
or thymidine with protected 3′- and 5′-hydroxyl groups of the
sugar moiety (TIPDS (1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)
protection), as well as the N(3)-position of the nucleobase, were
utilized.11 Correspondingly cytidine or 5-methylcytidine deriva-
tives were synthesized from 2′-O-trifluoroacetylaminoethylu-
ridine or -thymidine derivatives, transforming the 4-oxo group
to triazol-1-yl12 or amidine-protected amino group.11 As an
alternative to alkylation, the 2′-O-aminoethylthymidine deriva-
tive was synthesized from 2,2′-anhydrothymidine with the borate
of hydroxyethylphthalimide13 or hydroxyethylphthalimide in the
presence of Ti(O-i-Pr)4.14
In contrast to uridine and thymidine, adenosine was alkylated
with methyl bromoacetate without any protection.9,12 2′-O-
Methoxycarbonylmethyladenosine was isolated and purified
from the resulting mixture of 2′-, 3′-, and 5′-O-isomers by
column chromatography.
In the synthesis of 2′-O-aminoethylguanosine unprotected
2-aminoadenosine was alkylated with benzyl 2-bromoethyl ether
and, after isolation of the 2′-O-isomer from the resulting mixture,
was enzymatically (with adenosine deaminase) converted to the
guanosine derivative. Furthermore, after protection of ribose and
nucleobase moieties, the benzyl protecting group was cleaved
by hydrogenolysis, yielding the 2′-O-hydroxyethyl derivative,
which then was converted to the aminoethyl derivative using
the steps described above.12
The last and, probably, the most versatile reported approach
to 2′-O-aminoethyl nucleosides includes the preparation of 2′-
trifluoroacetylamino(or N-phthalimidino)ethyl-1′,3′,5′-triacetyl-
ribose and introduction of the corresponding nucleobase under
Vorbru¨ggen conditions.15–17 But again, synthesis of the ribose
moiety includes the same steps as for uridine or thymidine
(TIPDS protection, alkylation with methyl bromoacetate, etc.).
It is worth mentioning that in some cases the number of synthetic
steps was reduced by replacement of the hydroxy group with a
phthalimido functionality under Mitsunobu conditions.9,10,14–16
The advantage here is that the amino group already is protected
(with phthaloyl residue) and could be used further without
additional deprotection/protection steps.
with derivatives containing a longer alkyl chain.7,18 In that way
the desired 2-aminoethyl group could be introduced in a single
step. Surprisingly, only in one report did we find a brief note,
that attempts to alkylate the 2′-OH group of adenosine with
BrCH2CH2NPth gave no results.12 Due to these insufficient data,
we decided to study this type of reaction. We chose 3′,5′-TIPDS
protected uridine and, as an alkylating agent, the already
mentioned BrCH2CH2NPth. For initial testing of the appropriate
conditions we used NaH as a base and carried out the reaction
in solvents such as CH2Cl2, THF, and DMF. Unfortunately,
these attempts were all unsuccessful. Using THF or CH2Cl2 at
temperatures below 20-25 °C the reaction did not take place,
and at higher temperatures (above 25-30 °C) decomposition
of the uridine derivative as well as elimination of BrCH2-
CH2NPth to N-vinylphthalimide occurred, yielding complex
mixtures. Carrying out the reaction in DMF, we observed
decomposition of starting materials already at temperatures close
to 0 °C. Thus, for further testing of this reaction with other
reagents we used only CH2Cl2 and THF as solvents and the
same base, NaH. Because our attempts to alkylate the 2′-OH
group with BrCH2CH2NPth failed, we decided to test in this
reaction more reactive alkylating agents. For this purpose we
synthesized a series of XCH2CH2NPth type compounds with
increasing reactivity, where X is iodo, mesyl, tosyl, tresyl, and
finally the trifluoromethanesulfonyl group.19,20 Surprisingly to
us, only with the last, most active species, the trifluoromethane-
sulfonyl derivative, did alkylation of 2′-OH took place and the
2′-O-phthalimidoethyluridine derivative 2a was successfully
isolated in moderate yield (Scheme 1). The aforementioned
phthalimidoethyl triflate21 is easy to synthesize and purify by
column chromatography (see the Experimental Section), and it
stays stable at least over 1 year when stored at -20 °C under
argon.
Interestingly, carrying out the reaction in THF product 2a
was isolated in a much higher yield (65%), than in CH2Cl2
(30%).
Along with the desired compound 2a, the double-alkylated
side product 2a-1 was formed (Scheme 2). In contrast to the
yields of the main product 2a, the yield of compound 2a-1 was
higher in CH2Cl2 (12%), than in THF (5%).
The same reaction conditions were applied for cytidine, but
in that case we used not only 3′,5′-protection on the sugar moiety
but also protection on the nucleobase (compound 1b; Scheme
1). This avoids additional synthetic steps to protect the amino
group of cytosine afterward and also improves solubility in THF
or CH2Cl2. Thus, we successfully synthesized the 2′-O-phthal-
imidoethyl cytidine derivative 2b (Scheme 1) in the same way
as for uridine. The yield of the compound 2b, as for 2a, was
also higher in THF (60%) than in CH2Cl2 (35%). Along with
the main product we isolated two side products, 2b-1 and 2b-2
(Scheme 2), but whereas the first species was obtained in THF
(5% yield) as well as in CH2Cl2 (12% yield), the formation of
the second species was observed only in CH2Cl2 (11% yield).
In summarizing the syntheses described above, it is clear that
for different nucleosides different approaches have to be used,
which is a great disadvantage when several 2′-O-aminoethyl-
modified nucleosides are required to be synthesized. Further-
more, all reported methods are multistep procedures, making
these syntheses prolonged and material consuming. Here we
report a short and very convenient synthesis of all four building
blocks of 2′-O-aminoethyl nucleosides useful in the solid-phase
synthesis of oligonucleotides.
Results and Discussion
For us, the shortest and most straightforward way to 2′-O-
aminoethyl nucleosides seemed to be the direct alkylation of
the 2′-OH group of nucleosides with XCH2CH2NPth (X )
leaving group, NPth ) N-phthalimide) type reagents, in analogy
Inspired by the successful syntheses of the 2′-O-phthalimi-
douridine and -cytidine derivatives 2a,b, we also attempted to
(13) Manoharan, M.; Prakash, T. P.; Barber-Peoc’h, I.; Bhat, B.; Vasquez,
G.; Ross, B. S.; Cook, P. D. J. Org. Chem. 1999, 64, 6468–6472.
(14) Blommers, M. J. J.; Natt, F.; Jahnke, W.; Cuenoud, B. Biochemistry
1998, 37, 17714–17725.
(18) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron Lett. 1991,
32, 7171–7174.
(19) March, J. AdVanced Organic Chemistry; Wiley-Interscience: Weinheim,
Germany, 1985.
(15) Sollogoub, M.; Dominguez, B.; Fox, K. R.; Brown, T. Chem. Commun.
2000, 2315–2316.
(20) Carey, F. A.; Sundberg, R. J. Organische Chemie; VCH: Weinheim,
Germany, 1995.
(21) Yasaka, Y.; Tanaka, M.; Matsumoto, T.; Katakawa, J.; Tetsumi, T.;
Shono, T. Anal. Chem. 1990, 6, 49–52.
(16) Osborne, S. D.; Powers, V. E. C.; Rusling, D. A.; Lack, O.; Fox, K. R.;
Brown, T. Nucleic Acid Res. 2004, 32, 4439–4447.
(17) Buchini, S.; Leumann, C. J. Eur. J. Org. Chem. 2006, 3152, 3168.
J. Org. Chem. Vol. 73, No. 13, 2008 4995

Smicius and Engels
SCHEME 1
SCHEME 2
synthesize the purinic nucleosides adenosine and guanosine.
Here again we used THF as a solvent and NaH as a base, and
as in the case for U and C, we used disilyl protection on the
sugar moiety and nucleobases were also protected (adenine with
benzoyl, guanine with isobutyryl groups). Unfortunately, syn-
theses of 2′-phthalimidoethyl derivatives of those compounds
were unsuccessful. Complex mixtures of products (predomi-
nantly alkylated on the base moiety) formed in the reaction of
3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N6-benzoylad-
enosine with phthalimidoethyl triflate. When dibenzoyl adenos-
ine 1e was used, the desired product 2e was isolated only in
18% yield. Also, in this reaction we observed the formation of
3′,5′-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)-N6-benzoylad-
enosine, as well as a complex mixture of compounds similar to
that mentioned above. In contrast, 3′,5′-(1,1,3,3-tetraisopropyl-
disiloxane-1,3-diyl)-N2-isobutyrylguanosine did not react at all.
Surprisingly, when these alkylation reactions were per-
formed with A and G having only a protected sugar moiety
(compounds 1c,d), the desired 2′-O-alkylated products 2c,d
were formed and isolated in 65% and 50% yields, respec-
tively, and no double-alkylated products were observed
(Scheme 1). Thus, using phthalimidoethyl triflate we suc-
cessfully synthesized 2′-O-phthalimidoethyl derivatives of all
four main natural ribonucleosides. Furthermore, we protected
nucleobase moieties of compounds 2c,d to the corresponding
acyl derivatives 3c,d (Scheme 3). Notably, for adenine along
with the monobenzoylated derivative 3c, the dibenzoylated
compound 2e formed (16%).
4a, 82% for 4b, and 97% for 4c). For deprotection of the
guanosine derivative 3d, due to its low solubility, triethy-
lamine trihydrofluoride in THF at 60 °C was used (82% yield
for 4d). Protection of the 5′-OH group of nucleosides 4a-d
with 4,4′-dimethoxytrityl chloride in pyridine yielded the
corresponding 5′-DMTr derivatives 5a-d (82-90% yields).
Finally, in the last step all four compounds were phosphi-
tylated with 2-cyanoethyl N,N,N′,N′-tetraisopropylphospho-
rodiamidite using 4,5-dicyanoimidazole (DCI) as the activator
to obtain the corresponding phosphoramidites 6a-d (80%
yields for 6a-c, 72% for 6d).
The overall yields were 35% for the final uridine and cytidine
derivatives 6a,b, 27% for adenosine 6c, and 23% for guanosine
6d, and the synthesis of each phosphoramidite included only
five (for U and C) or six steps (for A and G). For comparison,
the overall yields in previously reported syntheses of phos-
phoramidites of 2′-O-aminoethyl-modified natural ribonucleo-
sides12 were at least 2 times lower (17% for U, 9% for C, 12%
for A, and 6% for G), while the number of synthetic steps nearly
doubled (11-12).
All of the phosphoramidites 6a-d are being used for the
syntheses of modified 21-22-mers of siRNAs for testing in
RNAi assays, and some of these results have already been
published.9 One should consider that for the preparation of
oligonucleotides, containing 2′-aminoethyl-modified nucleosides,
full removal of the phthaloyl protection from amino groups
requires, after the solid-phase synthesis, prolonged (48 h)
treatment with ammonium hydroxide at high temperature (40
°C).
Further steps to the 2′-phthalimidoethyl phosphoramidites
6a-d are shown in Scheme 4. TIPDS protection was removed
from the derivatives of uridine (2a), cytidine (2b), and
adenosine (3c) using TBAF in THF at 0 °C (yields 78% for
4996 J. Org. Chem. Vol. 73, No. 13, 2008

Zwitterionic Ribonucleoside Phosphoramidites
SCHEME 3
SCHEME 4
8.55 g, 0.03 mol) in 30 mL of CH2Cl2 was cooled to 0 °C and a
solution of N-(2-hydroxyethyl)phthalimide (5 g, 0.0262 mol) and
N,N-diisopropylethylamine (4.48 mL, 3.38 g, 0.0262 mol) in 100
mL of CH2Cl2 was added dropwise. The reaction mixture was
warmed to room temperature, stirred for 1 h, washed with saturated
NaHCO3 (2 × 200 mL), dried over MgSO4, and evaporated at room
or lower temperature (heating must be avoided) to dryness. The
residue was purified by column chromatography using CH2Cl2 as
the eluant to yield 7.8 g (92%) of phthalimidoethyl triflate as a
grayish solid (yield 60%, mp 79 °C (hexane)19). Rf ) 0.6 (CH2Cl2).
1H NMR (250 MHz, DMSO-d6): δ 3.86 (2H, t, J ) 4.9 Hz,
CH2Pth), 4.40 (2H, t, J ) 4.9 Hz, OCH2), 7.82 (4H, m, Pth H4);
ESI-MS: m/z calcd 323.3 [M]+, found 323.5. Anal. Calcd for
C11H8F3NO5S: C, 40.87; H, 2.49; N, 4.33. Found: C, 41.04; H,
2.67; N, 4.37.
Conclusion
We have described here a very short, convenient, and
straightforward approach to phosphoramidites of all four 2′-
aminoethyl-modified natural ribonucleosides, where the 2′-O-
phthalimidoethyl group was introduced in a single alkylation
step. Also, we show that it is possible to synthesize 2′-modified
analogues of U, C, A, and G using the same alkylation procedure
for all of them, and there is no greatly observable difference in
reactivity.
Experimental Section
Phthalimidoethyl Triflate. This compound was synthesized
according to the procedure described in ref 21 with some modifica-
tions. A solution of trifluoromethanesulfonic anhydride (5 mL,
J. Org. Chem. Vol. 73, No. 13, 2008 4997

Smicius and Engels
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)uridine (2a). A suspension of NaH (0.72 g, 18 mmol, 60%
dispersion in oil) in 15 mL of THF was cooled to -40 °C, and a
solution of 1a (3.65 g, 7.5 mmol) in 25 mL of THF was added
dropwise. The mixture was slowly warmed to 0 °C, stirred for 1 h,
stirred additionally for 0.5 h at room temperature, and cooled to 0
°C. Then a one-third portion of a solution of phthalimidoethyl triflate
(3.39 g, 10.5 mmol) in 10 mL of THF was added dropwise and the
reaction mixture was stirred at 0 °C for 1 h. After addition of 20
mL of CH2Cl2, the reaction mixture was stirred at 0 °C for 1 h, the
rest of the phthalimidoethyl triflate solution in THF was added
dropwise over 1 h, and the mixture was further stirred at 0 °C for
3 h. The reaction mixture was neutralized with glacial acetic acid
and evaporated to dryness. The residue was dissolved in 100 mL
of CH2Cl2, and this solution was washed with saturated NaHCO3
(2 × 150 mL) and then with saturated NaCl (1 × 150 mL), dried
over MgSO4, and evaporated to dryness. The residue was purified
by column chromatography using 5-30% AcOEt in CH2Cl2 as the
eluant and collecting fractions with Rf ) 0.7 (1/1 AcOEt/CH2Cl2)
to produce 3.22 g (65%) of 2a as a white foam (30% yield, when
the reaction was carried out in CH2Cl2). 1H NMR (400 MHz,
DMSO-d6): δ 0.67 (1H, q, J ) 7.3 Hz, SiCHCH3), 0.78 (3H, d, J
) 7.3 Hz, SiCHCH3), 0.83-1.01 (24H, m, TIPDS H24), 3.74-3.98
(6H, m, 4′,5′-H2, R,ꢀ-(CH2)2), 4.05-4.10 (2H, m, 2′,5′′-H2), 4.19
(1H, dd, J ) 9.1, 4.8 Hz, 3′-H), 5.51 (1H, d, J ) 8.1 Hz, 5-H),
5.54 (1H, s, 1′-H), 7.61 (1H, d, J ) 8.1 Hz, 6-H), 7.85 (4H, m, Pth
H4), 11.36 (1H, s, NH). 13C NMR (63 MHz, DMSO-d6): δ 11.6,
12.13, 12.18, 12.5 (SiCHCH3), 16.52, 16.67, 16.73, 16.77, 17.01,
17.06, 17.13, 17.24 (SiCHCH3), 37.6 (C-ꢀ), 59.5 (C-5′), 67.4 (C-
R), 68.3 (C-3′), 80.7 (C-4′), 81.0 (C-2′), 88.8 (C-1′), 100.9 (C-5),
122.9, 131.5, 134.3 (Pth), 139.6 (C-6), 149.9 (C-2), 163.2 (C-4),
167.7 (CO); ESI-MS: m/z calcd 660.3 [M + H]+, found 660.3.
Anal. Calcd for C31H45N3O9Si2: C, 56.42; H, 6.87; N, 6.37. Found:
C, 56.26; H, 6.68; N, 6.11.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O,N3-bis(2-phthal-
imidoethyl)uridine (2a-1). This compound was isolated from the
reaction described above (synthesis of 2a) collecting fractions with
Rf ) 0.9 (1/2 AcOEt/CH2Cl2) to produce 0.31 g (5%) of 2a-1 as a
white foam (yield 12%, when reaction was carried out in CH2Cl2).
1H NMR (400 MHz, DMSO-d6): δ 0.63 (1H, q, J ) 7.6 Hz,
SiCHCH3), 0.78 (3H, d, J ) 7.6 Hz, SiCHCH3), 0.87 (3H, d, J )
7.6 Hz, SiCHCH3), 0.95-1.03 (21H, m, TIPDS H21), 3.69-3.96
(10H, m, 2′,3′,4′,5′-H4, R,ꢀ-(CH2)2, ꢀ′-CH2, R′-CH), 4.08-4.14 (3H,
m, 3′,5′′-H2, R′-CH), 5.50 (1H, s, 1′-H), 5.53 (1H, d, J ) 8.1 Hz,
5-H), 7.62 (1H, d, J ) 8.1 Hz, 6-H), 7.80 (4H, m, Pth H4), 7.84
(4H, m, Pth H4); 13C NMR (63 MHz, DMSO-d6): δ 11.6, 12.2,
12.7 (SiCHCH3), 16.48, 16.67, 16.79, 17.03, 17.07, 17.14, 17.26
(SiCHCH3), 35.2 (C-ꢀ′), 37.7 (C-ꢀ), 39.1 (C-R′), 59.3 (C-5′), 67.7
(C-R), 67.8 (C-3′), 81.0 (C-2′), 81.7 (C-4′), 88.9 (C-1′), 99.9 (C-
5), 122.7, 122.9, 131.4, 131.6, 134.2, 134.4 (Pth), 137.4 (C-6), 149.9
(C-2), 162.0 (C-4), 167.68, 167.75 (CO); ESI-MS: m/z calcd 832.3
[M]+, found 832.8. Anal. Calcd for C41H52N4O11Si2: C, 59.11; H,
6.29; N, 6.73. Found: C, 59.45; H, 6.35; N, 6.34.
was washed with saturated NaHCO3 (2 × 200 mL) and then with
saturated NaCl (1 × 200 mL), dried over MgSO4, and evaporated
to dryness. The residue was purified by column chromatography
using 5-70% AcOEt in CH2Cl2 as eluant and collecting fractions
with Rf ) 0.4 (1/1 AcOEt/CH2Cl2) to produce 3.43 g (60%) of 2b
as a white foam (yield 35%, when reaction was carried out in
1
CH2Cl2). H NMR (400 MHz, DMSO-d6): δ 0.63 (1H, q, J ) 7.3
Hz, SiCHCH3), 0.77 (3H, d, J ) 7.3 Hz, SiCHCH3), 0.83-0.88
(10H, m, TIPDS H10), 1.02-1.06 (14H, m, TIPDS H14), 3.78-4.09
(7H, m, 2′,4′,5′-H3, R,ꢀ-(CH2)2), 4.13 (1H, dd, J ) 9.6, 4.3 Hz,
3′-H), 4.20 (1H, m(d), 5′′-H), 5.68 (1H, s, 1′-H), 7.37 (1H, d, J )
7.6 Hz, 5-H), 7.52 (2H, dd, J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz
H2), 7.64 (1H, m, Bz H1), 7.85 (4H, m, Pth H4), 8.02 (2H, dd,
J2-3,6-5 ) 7.3 Hz, J2,6-4 ) 1.3 Hz, Bz H2), 8.12 (1H, d, J ) 7.6
Hz, 6-H), 11.31 (1H, s, NH). 13C NMR (63 MHz, DMSO-d6): δ
11.6, 12.3, 12.4, 12.5 (SiCHCH3), 16.51, 16.67, 16.72, 17.02, 17.08,
17.16, 17.27 (SiCHCH3), 37.7 (C-ꢀ), 59.3 (C-5′), 67.57 (C-R), 67.59
(C-3′), 81.0 (C-4′), 81.4 (C-2′), 89.1 (C-1′), 95.7 (C-5), 122.9 (Pth),
128.38, 128.42 (Bz), 131.6 (Pth), 132.7, 133.0 (Bzl), 134.2 (Pth),
143.4 (C-6), 154.0 (C-2), 163.2 (C-4), 167.3, 167.7 (CO). ESI-
MS: m/z calcd 762.3 [M]+, found 762.6. Anal. Calcd for
C38H50N4O9Si2: C, 59.82; H, 6.61; N, 7.31. Found: C, 60.08; H,
6.81; N, 7.13.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O,N4-bis(2-phthal-
imidoethyl)-N4-benzoylcytidine (2b-1). This compound was isolated
from the reaction described above (synthesis of 2b), collecting
fractions with Rf ) 0.9 (1/1 AcOEt/CH2Cl2) to produce 0.35 g (5%)
of 2b-1 as a white foam (yield 12%, when reaction was carried
out in CH2Cl2). 1H NMR (400 MHz, DMSO-d6): δ 0.62 (1H, q, J
) 7.3 Hz, SiCHCH3), 0.77 (3H, d, J ) 7.3 Hz, SiCHCH3), 0.86
(3H, d, J ) 7.3 Hz, SiCHCH3), 0.94-1.01 (21H, m, TIPDS H21),
3.68-3.90 (7H, m, 2′,4′,5′-H3, R,ꢀ-(CH2)2), 4.00-4.15 (4H, m,
3′,5′′-H2, ꢀ′-CH2), 4.31 (1H, m, R′-CH), 4.45 (1H, m, R′-CH), 5.52
(1H, s, 1′-H), 6.38 (1H, d, J ) 8.1 Hz, 5-H), 7.40 (2H, dd, J3,5-4
) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz H2), 7.53-7.58 (2H, m, Bz H1,
6-H), 7.74 (4H, m, Pth H4), 7.86 (4H, m, Pth H4), 7.92 (2H, dd,
J2-3,6-5 ) 7.3 Hz, J2,6-4 ) 1.3 Hz, Bz H2). 13C NMR (63 MHz,
DMSO-d6): δ 11.5, 12.1, 12.2, 12.6 (SiCHCH3), 16.41, 16.59, 16.70,
16.90, 16.99, 17.17 (SiCHCH3), 34.8 (C-ꢀ′), 37.6 (C-ꢀ), 41.0 (C-
R′), 59.2 (C-5′), 67.6 (C-R), 67.8 (C-3′), 81.0 (C-2′), 81.5 (C-4′),
88.9 (C-1′), 96.7 (C-5), 122.7, 122.9 (Pth), 128.1, 128.9 (Bz), 131.4,
131.5 (Pth), 132.1 (Bz), 134.1, 134.2 (Pth), 135.8 (Bz), 136.1 (C-
6), 149.1 (C-2), 156.7 (C-4), 167.6, 167.8, 175.4 (CO). ESI-MS:
m/z calcd 936.4 [M + H]+, found 936.3. Anal. Calcd for
C48H57N5O11Si2: C, 61.58; H, 6.14; N, 7.48. Found: C, 61.70; H,
6.22; N, 7.40.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O,N3-bis(2-phthal-
imidoethyl)-N4-benzoylcytidine (2b-2). This compound was iso-
lated from the reaction described above (synthesis of 2b), when
it was carried out in CH2Cl2, collecting fractions with Rf ) 0.7
(1/1 AcOEt/CH2Cl2) to produce 11% of 2b-2 as a white foam.
1H NMR (400 MHz, DMSO-d6): δ 0.60 (2H, m (q), 2 ×
SiCHCH3), 0.72 and 0.74 (total 6H, each d, J ) 7.1 Hz, 2 ×
SiCHCH3), 0.81-0.86 (13H, m, TIPDS H13 (1 × SiCHCH3, 4 ×
SiCHCH3)), 0.94-1.01 (7H, m, TIPDS H21 (1 × SiCHCH3, 2 ×
SiCHCH3)), 3.74-4.00 (10H, m, 2′,3′,4′,5′-H4, R,ꢀ-(CH2)2, ꢀ′-CH2),
4.07 (1H, m(d), 5′′-H), 4.22 (1H, m, R′-CH), 4.43 (1H, m, R′-CH),
5.47 (1H, s, 1′-H), 5.51 (1H, d, J ) 7.3 Hz, 5-H), 7.35 (2H, dd,
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)-N4-benzoylcytidine (2b). A suspension of NaH (0.72 g, 18
mmol, 60% dispersion in oil) in 25 mL of THF was cooled to -40
°C, and a solution of 1b (4.42 g, 7.5 mmol) in 25 mL of THF was
added dropwise. The mixture was slowly warmed to 0 °C and stirred
for 2 h. Then a solution of phthalimidoethyl triflate (3.39 g, 10.5
mmol) in 12 mL of THF was added dropwise in three parts every
1 h to the reaction mixture with stirring at 0 °C. After addition of
the last part of the solution of phthalimidoethyl triflate the reaction
mixture was stirred for 1 h at 0 °C and cooled to -40 °C and NaH
(0.17 g, 4.3 mmol, 60% dispersion in oil) was added. The reaction
mixture was slowly warmed to 0 °C, and a solution of phthalimi-
doethyl triflate (1.37 g, 4.3 mmol) in 5 mL of THF was added
dropwise. The reaction mixture was additionally stirred for 2 h at
0 °C, neutralized with glacial acetic acid, and evaporated to dryness.
The residue was dissolved in 150 mL of CH2Cl2, and this solution
J3,5-4 ) 7.8 Hz, J3-2, 5-6 ) 7.1 Hz, Bz H2), 7.41 (2H, d, J2-3, 6-5
)
7.1 Hz, Bz H2), 7.50 (1H, m, Bzl-H1), 7.65 (1H, d, J ) 7.3 Hz,
6-H), 7.81 (4H, m, Pth H4), 7.86 (4H, m, Pth H4), 13C NMR (63
MHz, DMSO-d6): δ 11.49, 11.51, 12.1, 12.4 (SiCHCH3), 16.38,
16.54, 16.63, 16.70, 16.79, 16.90, 17.10, 17.28 (SiCHCH3), 36.6
(C-ꢀ′), 37.6 (C-ꢀ), 45.4 (C-R′), 59.1 (C-5′), 67.42 (C-R), 67.48
(C-3′), 80.7 (C-2′), 81.2 (C-4′), 88.7 (C-1′), 100.4 (C-5), 122.9,
123.0 (Pth), 128.5 (Bz), 131.49, 131.56 (Pth), 131.74 (Bz), 134.25,
134.29 (Pth), 134.8 (Bzl), 140.6 (C-6), 153.3 (C-2), 166.1 (C-4),
167.7, 167.8, 168.0, 171.2 (CO); ESI-MS: m/z calcd 936.4 [M +
4998 J. Org. Chem. Vol. 73, No. 13, 2008

Zwitterionic Ribonucleoside Phosphoramidites
H]+, found 936.3. Anal. Calcd for C48H57N5O11Si2: C, 61.58; H,
6.14; N, 7.48. Found: C, 61.83; H, 6.21; N, 7.51.
NMR (63 MHz, DMSO-d6): δ 11.7, 12.2, 12.3, 12.6 (SiCHCH3),
16.54, 16.68, 16.81, 17.04, 17.07, 17.10, 17.23 (SiCHCH3), 37.7
(C-ꢀ), 59.8 (C-5′), 67.6 (C-R), 69.4 (C-3′), 80.6 (C-4′), 81.0 (C-
2′), 86.3 (C-1′), 116.7 (C-5), 122.9, 131.5 (Pth), 133.9 (C-8), 134.3
(Pth), 150.4 (C-2), 153.6 (C-4), 156.6 (C-6), 167.7 (CO); ESI-MS:
m/z calcd 699.3 [M + H]+, found 699.5. Anal. Calcd for
C32H46N6O8Si2: C, 54.99; H, 6.63; N, 12.02. Found: C, 54.73; H,
6.60; N, 11.95.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)adenosine (2c). A suspension of NaH (0.26 g, 6.5 mmol, 60%
dispersion in oil) in 10 mL of THF was cooled to -40 °C, and a
solution of 1c (2.55 g, 5 mmol) in 25 mL of THF was added
dropwise. The mixture was slowly warmed to 0 °C and stirred for
1 h, and a solution of phthalimidoethyl triflate (2.1 g, 6.5 mmol) in
12 mL of THF was added dropwise in two parts every 1 h to the
reaction mixture with stirring at 0 °C. After addition of the last
part of the solution of phthalimidoethyl triflate the reaction mixture
was stirred for 2 h at 0 °C and for 0.5 h at room temperature. Then
the mixture was cooled to -40 °C, NaH (0.12 g, 3 mmol, 60%
dispersion in oil) was added to it, and the mixture was slowly
warmed to 0 °C and stirred for 0.5 h. After dropwise addition of a
solution of phthalimidoethyl triflate (0.97 g, 3 mmol) in 3 mL of
THF the reaction mixture was additionally stirred for 1 h at 0 °C,
neutralized with glacial acetic acid, and evaporated to dryness. The
residue was dissolved in 100 mL of CH2Cl2, and this solution was
washed with saturated NaHCO3 (2 × 150 mL) and then with
saturated NaCl (1 × 150 mL), dried over MgSO4, and evaporated
to dryness. The residue was purified by column chromatography
using 20-100% AcOEt in CH2Cl2 and then 0-3% CH3OH in
AcOEt as eluants and collecting fractions with Rf ) 0.8 (3%
CH3OH/AcOEt) to produce 2.22 g (65%) of 2c as a white foam.
1H NMR (400 MHz, DMSO-d6): δ 0.77 (1H, q, J ) 7.1 Hz,
SiCHCH3), 0.85 (3H, d, J ) 7.1 Hz, SiCHCH3), 0.93 (3H, d, J )
7.3 Hz, SiCHCH3), 0.96-1.01 (21H, m, TIPDS H21), 3.76-3.89
(4H, m, 4′,5′-H2, ꢀ-CH2), 3.94 (1H, m, R-CH), 4.00-4.06 (2H, m,
5′′-H, R-CH), 4.47 (1H, d, J ) 4.8 Hz, 2′-H), 4.90 (1H, dd, J )
8.8, 4.8 Hz, 3′-H), 5.88 (1H, s, 1′-H), 7.30 (2H, s, NH2), 7.81 (4H,
m, Pth H4), 7.96 (1H, s, 2-H), 8.15 (1H, s, 8-H). 13C NMR (63
MHz, DMSO-d6): δ 11.8, 12.1, 12.3, 12.6 (SiCHCH3), 16.60, 16.73,
16.83, 16.86, 17.04, 17.09, 17.24 (SiCHCH3), 37.9 (C-ꢀ), 60.0 (C-
5′), 67.7 (C-R), 69.8 (C-3′), 80.4 (C-4′), 81.0 (C-2′), 87.6 (C-1′),
119.2 (C-5), 122.9, 131.4, 134.2 (Pth), 139.2 (C-8), 148.4 (C-4),
152.3 (C-2), 156.1 (C-6), 167.7 (CO). ESI-MS: m/z calcd 683.3
[M + H]+, found 683.6. Anal. Calcd for C32H46N6O7Si2: C, 56.28;
H, 6.79; N, 12.31. Found: C, 56.18; H, 6.76; N, 12.44.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)-N6,N6-dibenzoyladenosine (2e). A suspension of NaH (0.086
g, 2.15 mmol, 60% dispersion in oil) in 3 mL of THF was cooled
to -60 °C, and a solution of 1e (1.03 g, 1.433 mmol) in 12 mL of
THF was added dropwise. The mixture was warmed to 0 °C, stirred
for 10 min, and cooled to -30 °C. Then a solution of phthalimi-
doethyl triflate (0.6 g, 1.86 mmol) in 5 mL of THF was added
dropwise. The reaction mixture was stirred as it was slowly warmed
to 0 °C (in 2 h) and then cooled to -20 °C. Glacial acetic acid
(0.02 mL, 0.35 mmol) was added to the reaction mixture, and this
mixture was then warmed to room temperature and evaporated to
dryness. The residue was dissolved in 50 mL of CH2Cl2, and this
solution was washed with saturated NaHCO3 (2 × 150 mL) and
then with saturated NaCl (1 × 100 mL), dried over MgSO4, and
evaporated to dryness. The residue was purified by column
chromatography using 5-20% AcOEt in CH2Cl2 as eluant and
collecting fractions with Rf ) 0.75 (1/9 AcOEt/CH2Cl2) to produce
0.23 g (18%) of 2e as a white foam. 1H NMR (400 MHz, DMSO-
d6): δ 0.75-0.87 (8H, m, TIPDS H8 (2 × SiCHCH3, 2 ×
SiCHCH3)), 0.92 and 0.94 (6H, m, 2 × SiCHCH3), 0.99-1.02 (7H,
m, TIPDS H14 (2 × SiCHCH3, 4 × SiCHCH3)), 3.75-3.92 (4H,
m, 4′,5′-H2, ꢀ-CH2), 3.93-4.08 (3H, m, 5′′-H, R-CH2), 4.71 (1H,
d, J ) 4.8 Hz, 2′-H), 4.91 (1H, dd, J ) 8.9, 4.8 Hz, 3′-H), 6.02
(1H, s, 1′-H), 7.48 (4H, dd, J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.5 Hz, 2
× Bz H2), 7.61 (2H, m, 2 × Bz H1), 7.80 (4H, dd, J2-3,6-5 ) 7.5
Hz, J2,6-4 ) 1.3 Hz, 2 × Bz H2), 7.82 (4H, m, Pth H4), 8.51 (1H,
s, 2-H), 8.64 (1H, s, 8-H). 13C NMR (63 MHz, DMSO-d6): δ 11.8,
12.2, 12.3, 12.6 (SiCHCH3), 16.59, 16.72, 16.84, 16.88, 16.93,
16.98, 17.08, 17.21 (SiCHCH3), 37.7 (C-ꢀ), 59.6 (C-5′), 67.1 (C-
R), 70.5 (C-5′), 80.0 (C-2′), 80.6 (C-4′), 88.1 (C-1′), 123.0 (Pth),
127.4 (C-5), 128.97, 129.02 (Bz), 131.5 (Pth), 133.37, 133.43 (Bz),
134.3 (Pth), 145.1 (C-8), 150.9 (C-4), 151.4 (C-6), 151.8 (C-2),
167.7, 172.0 (CO). ESI-MS: m/z calcd 891.3 [M + H]+, found
891.6. Anal. Calcd for C46H54N6O9Si2: C, 62.00; H, 6.11; N, 9.43.
Found: C, 62.27; H, 6.21; N, 9.33.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)guanosine (2d). A suspension of NaH (0.48 g, 12 mmol, 60%
dispersion in oil) in 45 mL of THF was cooled to -20 °C, and 1d
(2.63 g, 5 mmol) was added. The mixture was slowly warmed to
0 °C, stirred at this temperature for 1 h and then additionally stirred
for 0.5 h at room temperature, and cooled to 0 °C. A solution of
phthalimidoethyl triflate (2.26 g, 7 mmol) in 5 mL of THF was
added dropwise in two parts every 1 h to the reaction mixture with
stirring at 0 °C. After addition of the last part of the solution of
phthalimidoethyl triflate the reaction mixture was stirred for 2 h at
0 °C and 0.5 h at room temperature. Then the mixture was cooled
to -20 °C, NaH (0.12 g, 3 mmol, 60% dispersion in oil) was added
to it, and this mixture was slowly warmed to 0 °C and stirred for
0.5 h. After dropwise addition of a solution of phthalimidoethyl
triflate (0.97 g, 3 mmol) in 3 mL of THF the reaction mixture was
additionally stirred for 2 h at 0 °C, neutralized with glacial acetic
acid, and evaporated to dryness. The residue was purified by column
chromatography as eluant using 1-15% CH3OH in AcOEt and
collecting fractions with Rf ) 0.7 (1/3 CH3OH/AcOEt). The
fractions containing the mixture of the starting material and the
product were collected and purified again by column chromatog-
raphy. The product was crystallized from methanol to produce
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)-N6-benzoyladenosine (3c). To a solution of 2c (2.4 g, 3.51
mmol) in 40 mL of pyridine cooled to 0 °C was added benzoyl
chloride (0.41 mL, 0.49 g, 3.51 mmol) dropwise, and the mixture
was stirred at 0 °C for 1 h. Then benzoyl chloride (0.41 mL, 0.49 g,
3.51 mmol) was again added dropwise; after this mixture was stirred
at 0 °C for 1 h, it was evaporated to dryness. The residue was
dissolved in 150 mL of CH2Cl2, and this solution was washed with
saturated NaHCO3 (2 × 200 mL) and then with saturated NaCl (1
× 200 mL), dried over Na2SO4, and evaporated to dryness. The
residue was purified by column chromatography using 5-100%
AcOEt in CH2Cl2 and then 1-5% CH3OH in AcOEt as eluants
and collecting fractions with Rf ) 0.6 (1/1 AcOEt/CH2Cl2) to
produce 1.81 g (66%) of 3c as a white foam (also, during
1
purification 2e (0.5 g (16%)) was isolated). H NMR (400 MHz,
DMSO-d6): δ 0.80 (1H, m (q), 2 × SiCHCH3), 0.88 (3H, d, J )
7.1 Hz, SiCHCH3), 0.94-1.04 (23H, m, TIPDS H23), 3.78-3.90
(4H, m, 4′,5′-H2, ꢀ-CH2), 3.95-4.09 (3H, m, 5′′-H, R-CH2), 4.62
(1H, d, J ) 5.1 Hz, 2′-H), 4.94 (1H, dd, J ) 9.1, 5.1 Hz, 3′-H),
6.01 (1H, s, 1′-H), 7.57 (2H, dd, J3,5-4 ) J3-2,5-6 ) 7.6 Hz, Bz
H2), 7.67 (1H, m, Bz H1), 7.81 (4H, m, Pth H4), 8.07 (2H, d, J2-3,6-5
) 7.6 Hz, Bz H2), 8.49 (1H, s, 8-H), 8.54 (1H, s, 2-H), 11.21 (1H,
s, NH). 13C NMR (63 MHz, DMSO-d6): δ 11.8, 12.1, 12.3, 12.6
(SiCHCH3), 16.59, 16.72, 16.83, 16.87, 17.02, 17.06, 17.08, 17.20
(SiCHCH3), 37.8 (C-ꢀ), 59.8 (C-5′), 67.7 (C-R), 80.49 (C-4′), 80.53
(C-2′), 87.9 (C-1′), 122.9 (Pth), 125.9 (C-5), 128.5 (Bz), 131.4 (Pth),
1
1.75 g (50%) of 2d as a white crystals. Mp: >260 °C. H NMR
(400 MHz, DMSO-d6): δ 0.71-0.81 (4H, m, TIPDS H4 (SiCHCH3,
SiCHCH3)), 0.89-0.96 (10H, m, TIPDS H10 (SiCHCH3, 3 ×
SiCHCH3)), 1.00-1.06 (14H, m, TIPDS H14 (2 × SiCHCH3, 4 ×
SiCHCH3)), 3.76-3.95 (5H, m, 4′,5′-H2, R-CH, ꢀ-CH2), 4.00-4.06
(2H, m, 5′′-H, R-CH), 4.24 (1H, d, J ) 4.8 Hz, 2′-H), 4.38 (1H,
dd, J ) 8.6, 4.8 Hz, 3′-H), 5.72 (1H, s, 1′-H), 6.45 (2H, s, NH2),
7.68 (1H, s, 8-H), 7.83 (4H, m, Pth H4), 10.67 (1H, s, NH); 13C
J. Org. Chem. Vol. 73, No. 13, 2008 4999

Smicius and Engels
132.8 (Bz), 134.3 (Pth), 143.3 (C-8), 150.3 (C-4), 151.20 (C-6),
151.26 (C-2), 165.5, 167.7 (CO). ESI-MS: m/z calcd 787.3 [M +
H]+, found 787.5. Anal. Calcd for C39H50N6O8Si2: C, 59.52; H,
6.40; N, 10.68. Found: C, 59.53; H, 6.52; N, 10.56.
66.9 (C-R), 67.3 (C-3′), 82.0 (C-2′), 84.0 (C-4′), 88.4 (C-1′), 95.8
(C-5), 122.9 (Pth), 128.4 (Bz), 131.6 (Pth), 132.7, 133.1 (Bz), 134.2
(Pth), 145.0 (C-6), 154.3 (C-2), 163.1 (C-4), 167.3, 167.8 (CO).
ESI-MS: m/z calcd 519.2 [M - H]-, found 519.2. Anal. Calcd. for
C26H24N4O8: C, 60.00; H, 4.65; N, 10.76. Found: C, 59.73; H, 4.77;
N, 10.52.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-phthalimido-
ethyl)-N2-isobutyrylguanosine (3d). To a solution of 2d (1.75 g,
2.5 mmol) in 15 mL of pyridine cooled to 0 °C was added isobutyryl
chloride (0.66 mL, 0.67 g, 6.25 mmol) dropwise. The mixture was
stirred at 0 °C for 1 h and then at room temperature for 2 h and
evaporated to dryness. The residue was dissolved in 150 mL
CH2Cl2, and this solution was washed with saturated NaHCO3 (2
× 250 mL) and then with saturated NaCl (1 × 250 mL), dried
over Na2SO4, and evaporated to dryness. The residue was purified
by column chromatography using 10-80% AcOEt in CH2Cl2 as
eluant and collecting fractions with Rf ) 0.75 (AcOEt) to produce
1.63 g (85%) of 3d as a white foam. 1H NMR (400 MHz, DMSO-
d6): δ 0.75-0.83 (4H, m, TIPDS H4 (SiCHCH3, SiCHCH3)),
0.91-1.04 (24H, m, TIPDS H24), 1.14 and 1.16 (total 6H, each d,
J ) 6.8 Hz, COCH(CH3)2), 2.79 (1H, septet, J ) 6.8 Hz, CHCO),
3.75-3.95 (5H, m, 4′,5′-H2, R-CH, ꢀ-CH2), 4.03-4.08 (2H, m,
5′′-H, R-CH), 4.35 (1H, dd, J ) 4.8, 1 Hz, 2′-H), 4.43 (1H, dd, J
) 8.6, 4.8 Hz, 3′-H), 5.80 (1H, d, J ) 1 Hz, 1′-H), 7.81 (4H, m,
Pth H4), 7.99 (1H, s, 8-H), 11.60 (1H, s, NH), 12.09 (1H, s, NH).
13C NMR (63 MHz, DMSO-d6): δ 11.8, 12.2, 12.3, 12.6
(SiCHCH3), 16.58, 16.72, 16.84, 16.86, 17.07, 17.12, 17.23
(SiCHCH3), 18.83, 18.86 (COCH(CH3)2), 34.7 (COCH(CH3)2), 37.6
(C-ꢀ), 60.0 (C-5′), 67.6 (C-R), 69.6 (C-3′), 80.8 (C-2′), 81.1 (C-
4′), 86.3 (C-1′), 120.2 (C-5), 122.9, 131.4, 134.2 (Pth), 136.4 (C-
8), 148.0 (C-2), 148.2 (C-4), 154.7 (C-6), 167.7, 180.1 (CO). ESI-
MS: m/z calcd 769.3 [M + H]+, found 769.2. Anal. Calcd for
C36H52N6O9Si2: C, 56.23; H, 6.82; N, 10.93. Found: C, 56.51; H,
7.10; N, 10.65.
2′-O-(2-Phthalimidoethyl)-N6-benzoyladenosine (4c). To a solu-
tion of 3c (1.81 g, 2.3 mmol) in 20 mL of THF cooled to 0 °C was
added 5.1 mL of 1 M TBAF/THF dropwise. The mixture was stirred
at 0 °C for 0.5 h, neutralized with glacial acetic acid, evaporated
to dryness, and dried. The residue was purified by column
chromatography using 1-7% MeOH in AcOEt as eluant and
collecting fractions with Rf ) 0.3 (3% MeOH/AcOEt) to produce
1.21 g (97%) of 4c as a white foam. 1H NMR (400 MHz, DMSO-
d6): δ 3.56 (1H, m, 5′-H), 3.68 (1H, m, 5′′-H), 3.73-3.77 (3H, m,
R-CH, ꢀ-CH2), 3.93-3.98 (2H, m, 4′-H, R-CH), 4.35 (1H, m (dd),
3′-H), 4.60 (1H, dd, J ) 5.6, 5.1 Hz, 2′-H), 5.13 (1H, t, J ) 5.6
Hz, 5′-OH), 5.25 (1H, d, J ) 5.6 Hz, 3′-OH), 6.07 (1H, d, J ) 5.6
Hz, 1′-H), 7.59 (2H, dd, J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz
H2), 7.68 (1H, m, Bz H1), 7.75 (4H, m, Pth H4), 8.09 (2H, dd,
J2-3,6-5 ) 7.3 Hz, J2,6-4 ) 1.4 Hz, Bz H2), 8.61 (1H, s, 2-H), 8.65
(1H, s, 8-H), 11.17 (1H, s, NH). 13C NMR (63 MHz, DMSO-d6):
δ 37.2 (C-ꢀ), 61.0 (C-5′), 66.6 (C-R), 68.8 (C-3′), 81.2 (C-2′), 85.8
(C-1′), 85.9 (C-4′), 122.7 (Pth), 125.5 (C-5), 128.4 (Bz), 131.2 (Pth),
132.4, 133.3 (Bz), 134.1 (Pth), 142.8 (C-8), 150.1 (C-4), 151.4 (C-
2), 151.8 (C-6), 165.5, 167.7 (CO). ESI-MS: m/z calcd 543.2 [M
- H]-, found 543.2. Anal. Calcd for C27H24N6O7: C, 59.56; H,
4.44; N, 15.43. Found: C, 59.48; H, 4.70; N, 15.20.
2′-O-(2-Phthalimidoethyl)-N2-isobutyrylguanosine (4d). To a
solution of 3d (1.63 g, 2.12 mmol) in 30 mL of THF was added
triethylamine trihydrofluoride (0.28 mL, 0.273 g, 1.7 mmol). The
reaction mixture was stirred at 60 °C for 2 h and evaporated to
dryness. The residue was purified by column chromatography using
1-20% CH3OH in AcOEt as eluant and collecting fractions with
Rf ) 0.15 (10% CH3OH/AcOEt). The product was crystallized from
2′-O-(2-Phthalimidoethyl)uridine (4a). To a solution of 2a (3.22
g, 4.88 mmol) in 30 mL of THF cooled to 0 °C was added 10.7
mL of 1 M TBAF/THF dropwise. The mixture was stirred at 0 °C
for 0.5 h, neutralized with glacial acetic acid, evaporated to dryness,
and dried. The residue was purified by column chromatography
using 1-5% MeOH in AcOEt as eluant and collecting fractions
with Rf ) 0.4 (3% MeOH/AcOEt). The product was crystallized
from ethyl acetate to produce 1.59 g (78%) of 4a as white crystals.
1
water to produce 0.92 g (82%) of 4d as a white solid. H NMR
(250 MHz, DMSO-d6): δ 1.13 (3H, d, J ) 6.8 Hz, COCHCH3),
1.16 (3H, d, J ) 6.8 Hz, COCHCH3), 2.79 (1H, septet, J ) 6.8
Hz, CHCO), 3.53 (1H, m, 5′-H), 3.60 (1H, m, 5′′-H), 3.66-3.73
(3H, m, R-CH, ꢀ-CH2), 3.90-3.93 (2H, m, 4′-H, R-CH), 4.26 (1H,
m, 3′-H), 4.39 (1H, dd, J ) 6.6, 4.8 Hz, 2′-H), 5.06 (1H, t, J ) 5.3
Hz, 5′-OH), 5.19 (1H, d, J ) 4.5 Hz, 3′-OH), 5.81 (1H, d, J ) 6.6
Hz, 1′-H), 7.71 (4H, m, Pth H4), 8.19 (1H, s, 8-H), 11.55 (1H, s,
NH), 11.92 (1H, s, NH). 13C NMR (63 MHz, DMSO-d6): δ 18.76,
18.79 (COCH(CH3)2), 34.6 (COCH(CH3)2), 37.2 (C-ꢀ), 61.1 (C-
5′), 66.3 (C-R), 68.8 (C-3′), 81.7 (C-2′), 86.1 (C-4′), 84.4 (C-1′),
119.9 (C-5), 122.5, 131.1, 134.0 (Pth), 137.3 (C-8), 147.8 (C-2),
148.5 (C-4), 154.5 (C-6), 167.5, 179.9 (CO). ESI-MS: m/z calcd
527.2 [M + H]+, found 527.2. Anal. Calcd for C24H26N6O8: C,
54.75; H, 4.98; N, 15.96. Found: C, 54.52; H, 5.12; N, 15.84.
1
Mp: 151 °C. H NMR (400 MHz, DMSO-d6): δ 3.53 (1H, m, 5′-
H), 3.62 (1H, m, 5′′-H), 3.71-3.83 (4H, m, 4′-H, ꢀ-CH2, R-CH),
3.87 (1H, m, R-CH), 3.96 (1H, dd, J ) 5.1, 4.8 Hz, 2′-H), 4.08
(1H, m (dd), 3′-H), 5.06 (1H, d, J ) 5.8 Hz, 3′-OH), 5.10 (1H, t,
J ) 5.1 Hz, 5′-OH), 5.55 (1H, d, J ) 8.1 Hz, 5-H), 5.77 (1H, d, J
) 5.1 Hz, 1′-H), 7.83 (4H, br s, Pth H4), 7.84 (1H, d, J ) 8.1 Hz,
6-H), 11.22 (1H, s, NH). 13C NMR (63 MHz, DMSO-d6): δ 37.2
(C-ꢀ), 60.4 (C-5′), 66.4 (C-R), 68.2 (C-3′), 81.0 (C-2′), 84.9 (C-
4′), 86.1 (C-1′), 101.7 (C-5), 122.9, 131.5, 134.3 (Pth), 140.2 (C-
6), 150.4 (C-2), 163.0 (C-4), 167.7 (CO). ESI-MS: m/z calcd 416.1
[M - H]-, found 415.9. Anal. Calcd for C19H19N3O8: C, 54.68; H,
4.59; N, 10.07. Found: C, 54.85; H, 4.66; N, 10.24.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)uridine (5a).
To a solution of 4a (1.47 g, 3.52 mmol) in 15 mL of pyridine was
added 4,4′-dimethoxytrityl chloride (1.43 g, 4.23 mmol). The
mixture was stirred at room temperature for 3 h and evaporated to
dryness. The residue was dissolved in 75 mL of CH2Cl2, and this
solution was washed with saturated NaHCO3 (2 × 150 mL), dried
over Na2SO4, and evaporated to dryness. The residue was purified
by column chromatography using 20-100% AcOEt in CH2Cl2 as
eluant and collecting fractions with Rf ) 0.35 (1/1 AcOEt/CH2Cl2)
2′-O-(2-Phthalimidoethyl)-N4-benzoylcytidine (4b). To a solution
of 2b (3.12 g, 4.1 mmol) in 30 mL of THF cooled to 0 °C was
added 9 mL of 1 M TBAF/THF dropwise. The mixture was stirred
at 0 °C for 1 h, neutralized with glacial acetic acid, and evaporated
to dryness. The residue was purified by column chromatography
using 1–12% MeOH in CH2Cl2 as eluant and collecting fractions
with Rf ) 0.35 (3% MeOH/AcOEt). The product was crystallized
from a methanol/water (6/1) mixture to produce 1.74 g (82%) of
4b as white crystals. 1H NMR (400 MHz, DMSO-d6): δ 3.60 (1H,
m, 5′-H), 3.77 (1H, m, 5′′-H), 3.82-3.87 (3H, m, 4′-H, ꢀ-CH2),
3.91-3.95 (3H, m, 2′-H, R-CH2), 4.06 (1H, m (dd), 3′-H), 4.97
(1H, d, J ) 6.3 Hz, 3′-OH), 5.20 (1H, t, J ) 5.1 Hz, 5′-OH), 5.80
(1H, d, J ) 2.3 Hz, 1′-H), 7.31 (1H, d, J ) 7.3 Hz, 5-H), 7.53
(2H, dd, J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz H2), 7.64 (1H, m,
Bz H1), 7.84 (4H, m, Pth H4), 8.03 (2H, d, J2-3,6-5 ) 7.3 Hz, J2,6-4
) 1.3 Hz, Bz H2), 8.50 (1H, d, J ) 7.3 Hz, 6-H), 11.25 (1H, s,
NH). 13C NMR (63 MHz, DMSO-d6): δ 37.4 (C-ꢀ), 59.3 (C-5′),
1
to produce 2.19 g (86%) of 5a as a white foam. H NMR (400
MHz, DMSO-d6): δ 3.20 (1H, dd, J ) 10.6, 2.5 Hz, 5′-H), 3.27
(1H, dd, J ) 10.6, 4.3 Hz, 5′′-H), 3.76 (6H, s, 2 × OCH3),
3.80-3.85 (3H, m, R-CH, ꢀ-CH2), 3.88-3.94 (2H, m, 4′-CH,
R-CH), 4.03 (1H, dd, J ) 4.8, 3.8 Hz, 2′-H), 4.18 (1H, m (dd),
3′-H), 5.14 (1H, d, J ) 6.6 Hz, 3′-OH), 5.24 (1H, d, J ) 8.1 Hz,
5-H), 5.75 (1H, d, J ) 3.8 Hz, 1′-H), 6.91 and 6.92 (total 4H, each
d, J ) 8.8 Hz, DMTr H4), 7.24-7.27 (5H, m, DMTr H5), 7.31-7.35
(2H, m, DMTr H2), 7.37-7.40 (2H, m, DMTr H2), 7.64 (1H, d, J
) 8.1 Hz, 6-H), 7.84 (4H, m, Pth H4), 11.30 (1H, s, NH). 13C NMR
5000 J. Org. Chem. Vol. 73, No. 13, 2008

Zwitterionic Ribonucleoside Phosphoramidites
(63 MHz, DMSO-d6): δ 37.2 (C-ꢀ), 54.9, 55.0 (OCH3), 62.7 (C-
5′), 66.7 (C-R), 68.5 (C-3′), 80.8 (C-2′), 82.6 (C-4′), 85.8 (DMTr),
87.1 (C-1′), 101.4 (C-5), 113.2 (DMTr), 122.9 (Pth), 126.8, 127.7,
127.9, 129.7 (DMTr), 131.5, 134.3 (Pth), 135.1, 135.3 (DMTr),
140.2 (C-6), 144.6 (DMTr), 150.2 (C-2), 158.1 (DMTr), 163.0 (C-
4), 167.8 (CO). ESI-MS: m/z calcd 720.2 [M + H]+, found 720.4.
Anal. Calcd for C40H37N3O10: C, 66.75; H, 5.18; N, 5.84. Found:
C, 66.36; H, 5.39; N, 5.46.
2.1 mmol), and the mixture was stirred at room temperature for
4 h. Then 4,4′-dimethoxytrityl chloride (0.071 g, 0.21 mmol) was
added once more; the mixture was stirred for an additional 2 h and
evaporated to dryness. The residue was dissolved in 75 mL of
CH2Cl2, and this solution was washed with saturated NaHCO3 (2
× 150 mL), dried over Na2SO4, and evaporated to dryness. The
residue was purified by column chromatography using 10-100%
AcOEt in CH2Cl2 and then 1-7% CH3OH in AcOEt as eluants
and collecting fractions with Rf ) 0.25 (AcOEt) to produce 1.3 g
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N4-ben-
zoylcytidine (5b). To a solution of 4b (1.89 g, 3.63 mmol) in 20
mL of pyridine was added 4,4′-dimethoxytrityl chloride (1.48 g,
4.36 mmol). The mixture was stirred at room temperature for 3 h
and evaporated to dryness. The residue was dissolved in 100 mL
of CH2Cl2, and this solution was washed with saturated NaHCO3
(2 × 150 mL), dried over Na2SO4, and evaporated to dryness. The
residue was purified by column chromatography using 20-100%
AcOEt in CH2Cl2 and then 1-5% CH3OH in AcOEt as eluants
and collecting fractions with Rf ) 0.6 (AcOEt) to produce 2.69 g
1
(90%) of 5d as a white foam. H NMR (400 MHz, DMSO-d6): δ
1.14 and 1.15 (total 6H, each d, J ) 6.8 Hz, COCH(CH3)2), 2.72
(1H, septet, J ) 6.8 Hz, CHCO), 3.15 (1H, dd, J ) 10.6, 3.3 Hz,
5′-H), 3.26 (1H, dd, J ) 10.6, 6.1 Hz, 5′′-H), 3.74-3.77 (9H, m,
R-CH, ꢀ-CH2, 2 × OCH3,), 3.94 (1H, m, R-CH), 4.01 (1H, m,
4′-H), 4.28 (1H, m (dd), 3′-H), 4.51 (1H, dd, J ) 5.6, 4.8 Hz, 2′-
H), 5.22 (1H, d, J ) 5.6 Hz, 3′-OH), 5.86 (1H, d, J ) 5.6 Hz,
1′-H), 6.83 and 6.85 (total 4H, each d, J ) 8.9 Hz, DMTr H4),
7.19-7.29 (7H, m, DMTr H7), 7.35 (2H, m, DMTr H2), 7.74 (4H,
m, Pth H4), 8.04 (1H, s, 8-H), 11.52 (1H, s, NH), 11.97 (1H, s,
NH). 13C NMR (63 MHz, DMSO-d6): δ 18.78, 18.86 (CO-
CH(CH3)2), 34.7 (COCH(CH3)2), 37.3 (C-ꢀ), 54.95, 54.97 (DMTr),
63.9 (C-5′), 66.7 (C-R), 69.2 (C-3′), 81.0 (C-2′), 84.0 (C-4′), 85.1
(C-1′), 85.5 (DMTr), 113.1 (DMTr), 120.3 (C-5), 122.6 (Pth), 126.6,
127.66, 127.74, 129.7 (DMTr), 131.3, 134.1 (Pth), 135.35, 135.43
(DMTr), 137.4 (C-8), 144.7 (DMTr), 147.9 (C-2), 148.5 (C-4),
154.6 (C-6), 158.02, 158.03 (DMTr), 167.7, 180.0 (CO). ESI-MS:
m/z calcd 829.3 [M + H]+, found 829.4. Anal. Calcd for
C45H44N6O10: C, 65.21; H, 5.35; N, 10.14. Found: C, 65.48; H,
5.63; N, 9.88.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)uridine-3′-
O-(cyanoethyl-N,N-diisopropyl)phosphoramidite (6a). To a solution
of 5a (0.36 g, 0.5 mmol) in 6 mL of CH2Cl2 were added
2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.24 mL,
0.226 g, 0.75 mmol) and 4,5-dicyanoimidazole (0.068 g, 0.575
mmol). The reaction mixture was stirred for 3 h at room temper-
ature. Then the mixture was diluted with 60 mL of CH2Cl2, washed
with saturated NaHCO3 (2 × 150 mL), and then with saturated
NaCl (1 × 150 mL), dried over Na2SO4, and evaporated to dryness.
The mixture of diastereomers was purified by column chromatog-
raphy using 20-100% AcOEt in CH2Cl2 (with 0.1% Et3N) as eluant
and collecting fractions with Rf ) 0.6 and 0.7 (1/1 AcOEt/CH2Cl2)
to produce 0.372 g (81%) of 6a as a white foam. 31P NMR (400
MHz, CDCl3): δ 149.3 and 149.8. ESI-MS: m/z calcd for
C49H54N5O11P 920.4 [M + H]+, found 920.5.
1
(90%) of 5b as a yellowish foam. H NMR (400 MHz, DMSO-
d6): δ 3.30-3.37 (2H, m, 5′-H2), 3.77 (6H, s, 2 × OCH3), 3.87
(2H, t, J ) 5.6 Hz, ꢀ-CH2), 3.94-4.05 (4H, m, 2′,4′-H2, R-CH2),
4.27 (1H, m, 3′-H), 5.07 (1H, d, J ) 7.1 Hz, 3′-OH), 5.82 (1H, d,
J ) 0.8 Hz, 1′-H), 6.93 (4H, d, J ) 9.1 Hz, DMTr H4), 7.15 (1H,
d, J ) 7.3 Hz, 5-H), 7.26-7.30 (5H, m, DMTr H5), 7.34-7.38
(2H, m, DMTr H2), 7.41-7.43 (2H, m, DMTr H2), 7.54 (2H, dd,
J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz H2), 7.65 (1H, m, Bz H1),
7.84 (4H, m, Pth H4), 8.03 (2H, dd, J2-3,6-5 ) 7.3 Hz, J2,6-4 ) 1.3
Hz, Bz H2), 8.34 (1H, d, J ) 7.3 Hz, 6-H), 11.29 (1H, s, NH). 13
C
NMR (63 MHz, DMSO-d6): δ 37.4 (C-ꢀ), 54.97, 54.98 (DMTr),
61.5 (C-5′), 67.1 (C-R), 67.7 (C-3′), 81.77 (C-2′), 81.87 (C-4′),
86.0 (DMTr), 88.8 (C-1′), 96.0 (C-5), 113.3 (DMTr), 122.9 (Pth),
126.8, 127.8, 127.9 (DMTr), 128.4 (Bz), 129.6, 129.8 (DMTr),
131.7 (Pth), 132.7, 133.1 (Bz), 134.2 (Pth), 135.2, 135.5 (DMTr),
144.3 (C-6, DMTr), 154.2 (C-2), 158.14, 158.16 (DMTr), 163.2
(C-4), 167.2, 167.9 (CO). ESI-MS: m/z calcd 823.3 [M + H]+,
found 823.3. Anal. Calcd for C47H42N4O10: C, 68.60; H, 5.14; N,
6.81. Found: C, 68.37; H, 5.36; N, 6.71.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N6-benzoyl-
adenosine (5c). To a solution of 4c (1.21 g, 2.22 mmol) in 15 mL
of pyridine was added 4,4′-dimethoxytrityl chloride (0.92 g, 2.7
mmol). The mixture was stirred at room temperature for 4 h and
evaporated to dryness. The residue was dissolved in 50 mL CH2Cl2,
and this solution was washed with saturated NaHCO3 (2 × 100
mL), dried over Na2SO4, and evaporated to dryness. The residue
was purified by column chromatography using 10-100% AcOEt
in CH2Cl2 and then 0.5-2% CH3OH in AcOEt as eluants and
collecting fractions with Rf ) 0.7 (AcOEt) to produce 1.54 g (82%)
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N4-ben-
zoylcytidine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite (6b).
To a solution of 5b (0.411 g, 0.5 mmol) in 6 mL of CH2Cl2 were
added 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.24
mL, 0.226 g, 0.75 mmol) and 4,5-dicyanoimidazole (0.068 g, 0.575
mmol). The reaction mixture was stirred for 3 h. Then the mixture
was diluted with 60 mL of CH2Cl2, washed with saturated NaHCO3
(2 × 150 mL) and then with saturated NaCl (1 × 150 mL), dried
over Na2SO4, and evaporated to dryness. The mixture of diaster-
eomers was purified by column chromatography using 10-70%
AcOEt in CH2Cl2 (with 0.1% Et3N) as eluant and collecting
fractions with Rf ) 0.4 and 0.5 (1/1 AcOEt/CH2Cl2) to produce
0.41 g (80%) of 6b as a white foam. 31P NMR (400 MHz, CDCl3):
δ 148.7 and 149.8. ESI-MS: m/z calcd for C56H59N6O11P 1023.4
[M + H]+, found 1023.5.
1
of 5c as a white foam. H NMR (400 MHz, DMSO-d6): δ 3.23
(2H, d, J ) 4.6 Hz, 5′-H2), 3.73 (6H, s, 2 × OCH3), 3.76-3.83
(4H, m, R-CH, ꢀ-CH2), 3.97 (1H, m, R-CH), 4.07 (1H, dd, J )
9.3, 4.6 Hz, 4′-H), 4.46 (1H, m (dd), 3′-H), 4.77 (1H, dd, J ) 5.1,
4.8 Hz, 2′-H), 5.30 (1H, d, J ) 6.1 Hz, 3′-OH), 6.08 (1H, d, J )
4.8 Hz, 1′-H), 6.84 and 6.85 (total 4H, each d, J ) 8.9 Hz, DMTr-
H4), 7.19-7.28 (7H, m, DMTr H7), 7.36 (2H, m, DMTr H2), 7.58
(2H, dd, J3,5-4 ) 7.8 Hz, J3-2,5-6 ) 7.3 Hz, Bz H2), 7.67 (1H, m,
Bz H1), 7.75 (4H, m, Pth H4), 8.09 (2H, dd, J2-3,6-5 ) 7.3 Hz,
J2,6-4 ) 1.3 Hz, Bz H2), 8.53 (1H, s, 8-H), 8.55 (1H, s, 2-H), 11.18
(1H, s, NH). 13C NMR (63 MHz, DMSO-d6): δ 37.4 (C-ꢀ), 54.97,
54.98 (DMTr), 63.4 (C-5′), 67.0 (C-R), 69.2 (C-3′), 80.4 (C-2′),
83.5 (C-4′), 85.5 (DMTr), 86.4 (C-1′), 113.1 (DMTr), 122.8 (Pth),
125.7 (C-5), 126.6, 127.6, 127.7 (DMTr), 128.44, 128.49 (Bz),
129.7 (DMTr), 131.3 (Pth), 132.4, 133.4 (Bz), 134.4 (Pth), 135.4,
135.5 (DMTr), 143.2 (C-8), 144.7 (DMTr), 150.3 (C-4), 151.4 (C-
2), 151.8 (C-6), 158.0 (DMTr), 165.5, 167.7 (CO). ESI-MS: m/z
calcd 847.3 [M + H]+, found 847.4. Anal. Calcd for C48H42N6O9:
C, 68.07; H, 5.00; N, 9.92. Found: C, 67.80; H, 5.24; N, 9.67.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N2-isobu-
tyrylguanosine (5d). To a solution of 4d (0.92 g, 1.75 mmol) in 12
mL of pyridine was added 4,4′-dimethoxytrityl chloride (0.71 g,
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N6-benzoyl-
adenosine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite (6c). To
a solution of 5c (0.423 g, 0.5 mmol) in 5 mL of CH2Cl2 were added
2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (0.24 mL,
0.27 g, 0.75 mmol) and 4,5-dicyanoimidazole (0.068 g, 0.575
mmol), and the reaction mixture was stirred for 4 h at room
temperature. Then the mixture was diluted with 50 mL of CH2Cl2,
washed with saturated NaHCO3 (2 × 100 mL), and then with
saturated NaCl (1 × 100 mL), dried over Na2SO4, and evaporated
to dryness. The mixture of diastereomers was purified by column
J. Org. Chem. Vol. 73, No. 13, 2008 5001

Smicius and Engels
chromatography using 5-50% AcOEt in CH2Cl2 (with 0.1% Et3N)
as eluant and collecting fractions with Rf ) 0.55 and 0.7 (1/1
AcOEt/CH2Cl2) to produce 0.42 g (80%) of 6c as a white foam.
31P NMR (400 MHz, acetone-d6): δ 149.7 and 149.8. ESI-MS: m/z
calcd for C57H59N8O10P 1047.4 [M + H]+, found 1047.5.
31P NMR (400 MHz, acetone-d6): δ 149.7 and 150.2. ESI-MS: m/z
calcd for C54H61N8O11P 1029.4 [M + H]+, found 1029.6.
Acknowledgment. We thank the RIGHT EU-Project No.
LSHB-CT-2004-005276 for financial support and Wacker
Chemie AG (Germany) for providing 1,3-dichloro-1,1,3,3-
tetraisopropyldisiloxane.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-phthalimidoethyl)-N2-isobu-
tyrylguanosine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramid-
ite (6d). To a solution of 5d (0.41 g, 0.5 mmol) in 5 mL of CH2Cl2
were added 2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodia-
midite (0.24 mL, 0.23 g, 0.75 mmol) and 4,5-dicyanoimidazole
(0.068 g, 0.575 mmol), and the reaction mixture was stirred for
3 h at room temperature. Then the mixture was diluted with 30
mL of CH2Cl2, washed with saturated NaHCO3 (2 × 100 mL),
and then with saturated NaCl (1 × 100 mL), dried over Na2SO4,
and evaporated to dryness. The mixture of diastereomers was
purified by column chromatography using 5-100% AcOEt in
CH2Cl2 (with 0.1% Et3N) as eluant and collecting fractions with
Rf ) 0.5 (AcOEt) to produce 0.37 g (72%) of 6d as a white foam.
Supporting Information Available: Text giving details of
the synthesis, purification, and characterization of N4-benzoyl-
cytidine, N6,N6-dibenzoyladenosine, and compounds 1a-e and
1
figures giving H NMR, 13C NMR, and HSQC NMR spectra
of the new compounds 2-5 and 31P NMR spectra for phos-
phoramidites 6a-d. This material is available free of charge
via the Internet at http://pubs.acs.org.
JO800451M
5002 J. Org. Chem. Vol. 73, No. 13, 2008