Synthesis of amine- and thiol-modified nucleoside phosphoramidites for site-specific introduction of biophysical probes into RNA

Article abstract of DOI:10.1021/jo050061l

For studies of RNA structure, folding, and catalysis, site-specific modifications are typically introduced by solid-phase synthesis of RNA oligonucleotides using nucleoside phosphoramidites. Here, we report the preparation of two complete series of RNA nu

Full text of DOI:10.1021/jo050061l

Synthesis of Amine- and Thiol-Modified Nucleoside
Phosphoramidites for Site-Specific Introduction of Biophysical
Probes into RNA
Shengxi Jin,† Chandrasekhar V. Miduturu,† David C. McKinney, and Scott K. Silverman*
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, Illinois 61801
scott@scs.uiuc.edu
Received January 11, 2005
For studies of RNA structure, folding, and catalysis, site-specific modifications are typically
introduced by solid-phase synthesis of RNA oligonucleotides using nucleoside phosphoramidites.
Here, we report the preparation of two complete series of RNA nucleoside phosphoramidites; each
has an appropriately protected amine or thiol functional group. The first series includes each of
the four common RNA nucleotides, U, C, A, and G, with a 2′-(2-aminoethoxy)-2′-deoxy substitution
(i.e., a primary amino group tethered to the 2′-oxygen by a two-carbon linker). The second series
encompasses the four common RNA nucleotides, each with the analogous 2′-(2-mercaptoethoxy)-
2′-deoxy substitution (i.e., a tethered 2′-thiol). The amines are useful for acylation and reductive
amination reactions, and the thiols participate in displacement and oxidative cross-linking reactions,
among other likely applications. The new phosphoramidites will be particularly valuable for enabling
site-specific introduction of biophysical probes and constraints into RNA.
CHART 1. RNAs with Site-Specific 2′-Tethered
Amine or 2′-Tethered Thiol Groups
Introduction
Appending chromophores and other biophysical probes
and constraints onto biological macromolecules enables
us to develop a detailed understanding of their structure,
folding, and catalysis.1 For such purposes, an important
prerequisite is the development of the chemical methods
necessary to synthesize the modified biomolecules. Here,
we describe the preparation of eight modified RNA nu-
cleoside phosphoramidites. These reagents collectively
allow the solid-phase synthesis of RNA oligonucleotides
that incorporate a primary amine or thiol group tethered
to the ribose 2′-oxygen of any specific nucleotide via a
two-carbon tether (Chart 1). The ribose 2′-position is
particularly useful for chemical modification because of
its location in the minor groove of A-form duplex nucleic
acid, where it points outward into solution.2 The ribose
2′-position is also readily modified synthetically, as
described in this work. Both established3 and emerging4
* To whom correspondence should be addressed. Phone: (217) 244-
4489. Fax: (217) 244-8024.
† S.J. and C.V.M. are co-first authors of this paper.
(1) (a) Cohen, S. B.; Cech, T. R. J. Am. Chem. Soc. 1997, 119, 6259-
6268. (b) Glick, G. D. Biopolymers 1998, 48, 83-96. (c) Sigurdsson, S.
T. Methods 1999, 18, 71-77. (d) Silverman, S. K.; Cech, T. R.
Biochemistry 1999, 38, 14224-14237. (e) Silverman, S. K.; Deras, M.
L.; Woodson, S. A.; Scaringe, S. A.; Cech, T. R. Biochemistry 2000, 39,
12465-12475. (f) Silverman, S. K.; Cech, T. R. RNA 2001, 7, 161-
166. (g) Young, B. T.; Silverman, S. K. Biochemistry 2002, 41, 12271-
12276. (h) Smalley, M. K.; Silverman, S. K. 2005, submitted for
publication.
(2) Saenger, W. Principles of Nucleic Acid Structure; Springer-
Verlag: New York, 1984.
10.1021/jo050061l CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/21/2005
4284
J. Org. Chem. 2005, 70, 4284-4299

Amine- and Thiol-Modified RNA Phosphoramidites
ligation techniques are used to construct RNAs larger
than those that can be prepared by direct solid-phase
synthesis (<100 nt),5 as required for the study of many
catalytically active RNAs.6 In conjunction with such
techniques, the modified nucleoside phosphoramidites
reported here should be immediately useful in ongoing
biochemical and biophysical studies of nucleic acids.
We chose to prepare phosphoramidites with relatively
short two-carbon tethers rather than longer tethers for
two specific reasons. First, for some purposes, such as
certain fluorescence applications, a long tether can
provide too little interaction between the RNA and a
chromophore appended onto the tether. Because a spacer
of variable length can typically be incorporated during
the reaction with a chromophore reagent,1h the most
generally useful tethers for such applications are rela-
tively short. Second, for applications involving structural
constraints, a long tether may introduce too much flex-
ibility into the RNA, thereby precluding the goal of a
structural constraint. In contrast, a short tether permits
more structural control over the RNA conformation. For
such applications, the two-carbon tethers in Chart 1 are
the shortest chemically stable tethers that retain the 2′-
oxygen atom (thereby avoiding certain concerns over
destabilizing RNA structure with unnatural functional
groups7) and also provide reactive amine or thiol func-
tional groups for subsequent derivatization. Our syn-
thetic routes to the nucleoside phosphoramidites that are
required to incorporate these tethers into RNA are
designed to be maximally convergent. Experimental
protocols for preparing sufficient quantities of each
phosphoramidite (at least 100 mg) for conventional solid-
phase RNA oligonucleotide synthesis are described.
approach has been the reaction of a pyrimidine nucleoside
2′,3′-stannylene derivative8 with an alkyl halide. The
reaction proceeds with a modest chemical yield and little,
if any, selectivity between 2′- and 3′-alkylation,9 in those
cases for which the yield and selectivity were reported.10
We chose to avoid such alkylation routes to prevent
separating mixtures of 2′- and 3′-isomers. For the two-
carbon tethers, others have reported nucleophilic attack
on a 2,2′-anhydrothymidine derivative, either by a boron
derivative of 2-hydroxyethylphthalimide11 or by the latter
reagent itself in the presence of Ti(OiPr)4.12 The former
procedure requires a sealed stainless steel bomb heated
to 150 °C and provides only a 21% yield of 2′-modified
product after silica gel column chromatography; the yield
is 18% for a three-carbon tether and anhydrouridine.13
The latter procedure with Ti(OiPr)4 is reported to produce
a 32% yield, but no experimental protocol was provided.
An alternative approach to the 2′-modified pyrimidine
nucleosides first protects the 3′- and 5′-hydroxyl groups
as a TIPDS silyl derivative (see below); this step is
followed by the alkylation of the 2′-hydroxyl group with
an R-bromo ester.14,15 Then, reduction of the ester pro-
duces the 2′-OCH2CH2OH substitutent for further ma-
nipulation. In one case, the tethered hydroxyl group was
activated with TsCl and displaced with NaN3 to introduce
the nitrogen, but the communication provided no experi-
mental protocols.14 In another case, phthalimide was used
in a Mitsunobu reaction to introduce the nitrogen, and
again no experimental protocol was provided.15
On the basis of these limited precedents, we decided
to protect the pyrimidine 3′- and 5′-hydroxyl groups at
the outset and to employ a different 2′-alkylation strat-
egy. Our route began with the simultaneous TIPDS
(tetraisopropyldisiloxane-1,3-diyl) protection16 of the 3′-
and 5′-hydroxyl groups of uridine U1 (Scheme 1).17 The
TIPDSCl2 silylating reagent is commercially available,
albeit expensive. As an alternative, TIPDSCl2 may
conveniently be generated from the corresponding bis-
silane and CCl4 using substoichiometric PdCl2.18 The
crude TIPDS-uridine was immediately protected as its
Results and Discussion
Pyrimidine Nucleoside Phosphoramidites with
2′-Tethered Amines. A small number of pyrimidine
nucleosides with a primary amino group tethered via the
ribose 2′-oxygen have been reported previously. For
tethers longer than two carbon atoms, the only reported
(8) Wagner, D.; Verheyden, J. P. H.; Moffatt, J. G. J. Org. Chem.
1974, 39, 24-30.
(9) Manoharan, M.; Tivel, K. L.; Andrade, L. K.; Cook, P. D.
Tetrahedron Lett. 1995, 36, 3647-3650.
(10) 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. Chem. 1996, 39, 5100-5109.
(3) (a) Moore, M. J.; Sharp, P. A. Science 1992, 256, 992-997. (b)
Moore, M. J.; Query, C. C. In RNA-Protein Interactions: A Practical
Approach; Smith, C. W. J., Ed.; Oxford University Press: Oxford, U.K.,
1998; pp 75-108. (c) Moore, M. J. Methods Mol. Biol. 1999, 118, 11-
19. (d) Moore, M. J.; Query, C. C. Methods Enzymol. 2000, 317, 109-
123.
(4) (a) Silverman, S. K. Org. Biomol. Chem. 2004, 2, 2701-2706.
(b) Flynn-Charlebois, A.; Wang, Y.; Prior, T. K.; Rashid, I.; Hoadley,
K. A.; Coppins, R. L.; Wolf, A. C.; Silverman, S. K. J. Am. Chem. Soc.
2003, 125, 2444-2454. (c) Flynn-Charlebois, A.; Prior, T. K.; Hoadley,
K. A.; Silverman, S. K. J. Am. Chem. Soc. 2003, 125, 5346-5350. (d)
Wang, Y.; Silverman, S. K. J. Am. Chem. Soc. 2003, 125, 6880-6881.
(e) Wang, Y.; Silverman, S. K. Biochemistry 2003, 42, 15252-15263.
(f) Ricca, B. L.; Wolf, A. C.; Silverman, S. K. J. Mol. Biol. 2003, 330,
1015-1025. (g) Prior, T. K.; Semlow, D. R.; Flynn-Charlebois, A.;
Rashid, I.; Silverman, S. K. Nucleic Acids Res. 2004, 32, 1075-1082.
(h) Coppins, R. L.; Silverman, S. K. Nat. Struct. Mol. Biol. 2004, 11,
270-274. (i) Coppins, R. L.; Silverman, S. K. J. Am. Chem. Soc. 2004,
126, 16426-16432. (j) Coppins, R. L.; Silverman, S. K. J. Am. Chem.
Soc. 2005, 127, 2900-2907. (k) Wang, Y.; Silverman, S. K. Biochemistry
2005, 44, 3017-3023. (l) Semlow, D. R.; Silverman, S. K. J. Mol. Evol.
2005, in press.
(11) (a) 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. (b) Prakash, T. P.; Puschl, A.; Lesnik, E.; Mohan, V.; Tereshko,
V.; Egli, M.; Manoharan, M. Org. Lett. 2004, 6, 1971-1974.
(12) Blommers, M. J.; Natt, F.; Jahnke, W.; Cuenoud, B. Biochem-
istry 1998, 37, 17714-17725.
(13) Ross, B. S.; Springer, R. H.; Tortorici, Z.; Dimock, S. Nucleosides
Nucleotides 1997, 16, 1641-1643.
(14) 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.
(15) (a) Sollogoub, M.; Dominguez, B.; Fox, K. R.; Brown, T. Chem.
Commun. 2002, 2315-2316. (b) Osborne, S. D.; Powers, V. E.; Rusling,
D. A.; Lack, O.; Fox, K. R.; Brown, T. Nucleic Acids Res. 2004, 32,
4439-4447.
(16) (a) Markiewicz, W. T.; Markiewicz, M. Nucleic Acids Res., Spec.
Publ. 1978, 4, 185. (b) Markiewicz, W. T. J. Chem. Res., Synop. 1979,
24-25. (c) Markiewicz, W. T. J. Chem. Res., Miniprint 1979, 181-
197. (d) Markiewicz, W. T.; Biala, E.; Adamiak, R. W.; Grzeskowiak,
K.; Kierzek, R.; Kraszewski, A.; Stawinski, J.; Wiewiorowski, M.
Nucleic Acids Symp. Ser. 1980, 115-127.
(5) Marshall, W. S.; Kaiser, R. J. Curr. Opin. Chem. Biol. 2004, 8,
222-229.
(6) (a) Cech, T. R. Annu. Rev. Biochem. 1990, 59, 543-568. (b)
Michel, F.; Ferat, J. L. Annu. Rev. Biochem. 1995, 64, 435-461. (c)
Frank, D. N.; Pace, N. R. Annu. Rev. Biochem. 1998, 67, 153-180.
(7) Pham, J. W.; Radhakrishnan, I.; Sontheimer, E. J. Nucleic Acids
Res. 2004, 32, 3446-3455.
(17) Robins, M. J.; Wilson, J. S.; Hansske, F. J. Am. Chem. Soc.
1983, 105, 4059-4065.
J. Org. Chem, Vol. 70, No. 11, 2005 4285

Jin et al.
SCHEME 1
N3-methoxycarbonylvinyl (mocvinyl) derivative,19 U2;20
this protection was necessary to avoid undesired deriva-
tization of N3 in the subsequent alkylation step. Pal-
ladium-catalyzed allylation21 of the free 2′-hydroxyl group
of U2 readily produced U3 in high yield. This 2′-O-allyl
nucleoside was ozonolyzed and treated with NaBH4 to
provide 2′-(2-hydroxyethyl) nucleoside U4. The ozonolysis
conditions also removed the mocvinyl group, presumably
by the oxidative cleavage of the CdC double bond and
the subsequent breakdown of the hemiaminal. TIPDS-
protected nucleoside U4 has been accessed before by
different routes,22-24 but at most partial experimental
protocols were provided; in all cases, our route is one to
five steps shorter.
group, which was protected as the trifluoromethyl amide,
and the TIPDS 3′,5′-protecting group was removed with
TBAF, producing U7.14 The Staudinger reaction was used
instead of H2 reduction over Pd/C because, in our hands,
the latter method led to ∼15% hydrogenation of the uracil
C5dC6 double bond.25 Trifluoromethyl amide protection
was chosen because the trifluoromethyl group is readily
removed by transamidation during the standard NH3
deprotection of RNA oligonucleotides following solid-
phase synthesis.26
Selective reprotection of the 5′-hydroxyl group of U7
as its 4,4′-dimethoxytrityl (DMT) derivative led to U8,
which was phosphitylated with 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite using 4,5-dicyanoim-
idazole (DCI) as the activator27 to provide uridine-
tethered amine nucleoside phosphoramidite U9. Starting
with uridine U1, the overall route provides phosphor-
amidite U9 in a 20% overall yield in 9 steps with 7
chromatographic purifications; 440 mg of U9 was ob-
tained. This route to the 2′-(2-aminoethoxy)-substituted
pyrimidine phosphoramidite represents a substantial
improvement in yield and convenience for moderate-scale
laboratory synthesis when compared to previous reports
for similar compounds, as cited above.
The tethered hydroxyl group of U4 was exchanged for
an azido moiety by mesylation to U523 and displacement
with sodium azide in the presence of 18-crown-6, forming
U6. In a one-pot procedure, the azide of U6 was reduced
via the Staudinger reaction (PPh3, H2O) to the amino
(18) Ferreri, C.; Costantino, C.; Romeo, R.; Chatgilialoglu, C.
Tetrahedron Lett. 1999, 40, 1197-1200.
(19) Faja, M.; Ariza, X.; Ga´lvez, C.; Vilarrasa, J. Tetrahedron Lett.
1995, 36, 3261-3264.
(20) Costa, A. M.; Faja, M.; Farra`s, J.; Vilarrasa, J. Tetrahedron
Lett. 1998, 39, 1835-1838.
(21) (a) Lakhmiri, R.; Lhoste, P.; Sinou, D. Tetrahedron Lett. 1989,
30, 4669-4672. (b) Sproat, B. S.; Iribarren, A.; Beijer, B.; Pieles, U.;
Lamond, A. I. Nucleosides Nucleotides 1991, 10, 25-36. (c) Kachalova,
A. V.; Zatsepin, T. S.; Romanova, E. A.; Stetsenko, D. A.; Gait, M. J.;
Oretskaya, T. S. Nucleosides, Nucleotides Nucleic Acids 2000, 19,
1693-1707.
(25) (a) Watkins, B. E.; Kiely, J. S.; Rapoport, H. J. Am. Chem. Soc.
1982, 104, 5702-5708. (b) Johnson, D. C., II; Widlanski, T. S. Org.
Lett. 2004, 6, 4643-4646.
(26) (a) Imazawa, M.; Eckstein, F. J. Org. Chem. 1979, 44, 2039-
2041. (b) Pieken, W. A.; Olsen, D. B.; Benseler, F.; Aurup, H.; Eckstein,
F. Science 1991, 253, 314-317. (c) Benseler, F.; Williams, D. M.;
Eckstein, F. Nucleosides Nucleotides 1992, 11, 1333-1351.
(27) Vargeese, C.; Carter, J.; Yegge, J.; Krivjansky, S.; Settle, A.;
Kropp, E.; Peterson, K.; Pieken, W. Nucleic Acids Res. 1998, 26, 1046-
1050.
(22) Douglas, M. E.; Beijer, B.; Sproat, B. S. Bioorg. Med. Chem.
Lett. 1994, 4, 995-1000.
(23) Maglott, E. J.; Glick, G. D. Nucleic Acids Res. 1998, 26, 1301-
1308.
(24) Dobson, N.; McDowell, D. G.; French, D. J.; Brown, L. J.; Mellor,
J. M.; Brown, T. Chem. Commun. 2003, 1234-1235.
4286 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
SCHEME 2
U9 was converted to its triazolide derivative U10 to
obtain the cytidine phosphoramidite analogue (Scheme
1). This reagent can be incorporated as usual during
solid-phase RNA synthesis and upon standard oligo-
nucleotide deprotection with NH3 leads to cytidine at the
desired nucleotide position.28 This highly convergent
route is greatly preferred over the independent synthesis
of the cytidine nucleoside phosphoramidite starting from
cytidine.29 Such an independent route would entail nearly
twice the number of reaction steps as synthesis of the
uridine nucleoside phosphoramidite U9 plus its one-step
conversion to the triazolide U10. Starting from uridine
U1, phosphoramidite U10 was obtained in a 10% overall
yield in 10 steps with 8 chromatographic purifications;
100 mg of U10 was prepared.
Pyrimidine Nucleoside Phosphoramidites with
2′-Tethered Thiols. The route to the pyrimidine nucleo-
sides with 2′-tethered thiols started with intermediate
U4 from the pyrimidine-tethered amines strategy (Scheme
2). Displacement of the hydroxyl group with trityl mer-
captan under Mitsunobu conditions produced trityl-
protected thiol U11.22 The conversion of U4 to U11
appears to be the first reported use of trityl mercaptan
as the nucleophile in a Mitsunobu reaction. Intermediate
U11 was treated with TBAF to remove the 3′,5′-protect-
ing group, providing diol U12. As was done for the amine
route, the 5′-hydroxyl was reprotected as its DMT deriva-
tive to produce U13, which was phosphitylated to provide
uridine-tethered thiol nucleoside phosphoramidite U14.
The overall route from uridine U1 to U14 is 8 steps with
7 chromatographic purifications and proceeds in a 13%
overall yield; 340 mg of U14 was produced. Compounds
U11-U14 were previously accessed by a different route
with little experimental information provided;22 our route
to U11 is three steps shorter and has a higher yield
starting from uridine (30% vs 16-20%). The cytidine
phosphoramidite analogue was obtained from U14 as the
triazolide derivative U15, in a manner analogous to the
tethered amine route. A cytidine-tethered thiol was
prepared previously by a completely different route that
did not involve the uridine triazolide.29 The route from
U1 to U15 is 9 steps with 8 chromatographic purifications
in 7% overall yield; 112 mg of U15 was prepared.
The trityl group has been used previously for thiol
protection of nucleosides.30,31 For the 2′-tethered thiol, we
chose trityl rather than tert-butyl disulfide protection23
because the S-trityl deprotection procedure (treatment
with AgNO331) avoids the use of reducing agents such as
DTT. Application of such reducing agents would compli-
cate the subsequent use of oligonucleotides with tert-butyl
disulfide protecting groups in RNA ligation reactions
catalyzed by protein enzymes, which are normally used
in DTT-containing buffers.3
Adenosine Nucleoside Phosphoramidite with 2′-
Tethered Amine. Our preparation of the adenosine
nucleoside phosphoramidite with a 2′-tethered amine
followed a route related to that used for the pyrimidines
(Scheme 3). Adenosine A1 was alkylated preferentially
at the 2′-position10,32 with NaH and methyl bromoacetate
to provide A2 in a 47% yield.33 Although ethyl bromo-
acetate had a slightly higher yield of 58%, greater than
50% of the ethyl ester analogue of A2 underwent trans-
esterification during silica gel chromatography with
MeOH/CH2Cl2 as the eluent (for separation purposes,
methanol proved to be superior to ethanol as the chro-
matography cosolvent). Therefore, although the yield was
(30) (a) Connolly, B. A.; Rider, P. Nucleic Acids Res. 1985, 13, 4485-
4502. (b) Manoharan, M.; Tivel, K. L.; Ross, B.; Cook, P. D. Gene 1994,
149, 147-156.
(31) Hamm, M. L.; Piccirilli, J. A. J. Org. Chem. 1997, 62, 3415-
3420.
(32) (a) Manoharan, M.; Guinosso, C. J.; Cook, P. D. Tetrahedron
Lett. 1991, 32, 7171-7174. (b) Manoharan, M.; Johnson, L. K.; Tivel,
K. L.; Springer, R. H.; Cook, P. D. Bioorg. Med. Chem. Lett. 1993, 3,
2765-2770.
(33) Kawasaki, A. M.; Casper, M. D.; Prakash, T. P.; Manalili, S.;
Sasmor, H.; Manoharan, M.; Cook, P. D. Tetrahedron Lett. 1999, 40,
661-664.
(28) (a) Reese, C. B.; Skone, P. A. J. Chem. Soc., Perkin Trans. 1
1984, 1263-1271. (b) Xu, Y.-Z.; Zheng, Q.; Swann, P. F. J. Org. Chem.
1992, 57, 3839-3845. (c) Shah, K.; Wu, H.; Rana, T. M. Bioconjugate
Chem. 1994, 5, 508-512. (d) Obika, S.; Uneda, T.; Sugimoto, T.; Nanbu,
D.; Minami, T.; Doi, T.; Imanishi, T. Bioorg. Med. Chem. Lett. 2001, 9,
1001-1011. (e) Chirakul, P.; Sigurdsson, S. T. Org. Lett. 2003, 5, 917-
919.
(29) Goodwin, J. T.; Osborne, S. E.; Scholle, E. J.; Glick, G. D. J.
Am. Chem. Soc. 1996, 118, 5207-5215.
J. Org. Chem, Vol. 70, No. 11, 2005 4287

Jin et al.
SCHEME 3
slightly lower, we chose to use methyl bromoacetate in
the alkylation reaction so that the product, A2, was
homogeneous and fully characterizable by 2D NMR
spectroscopy. In the 2D HMBC spectrum of A2, both of
the long-range (four-bond) H-C-O-C couplings between
H2′ and 2′-OCH2 and between 2′-OCH2 and C2′ were
clearly observed, thereby confirming the identity of A2
as the 2′-alkylated isomer.
Nucleoside A2 was doubly protected with tert-butyl-
dimethylsilyl (TBS) at its 3′- and 5′-positions to produce
A3. We used double TBS protection rather than 3′,5′-
TIPDS protection because the site-selectivity of TIPDSCl2
for the 3′- and 5′-positions was not required (the
2′-hydroxyl was already substituted) and TBSCl is less
expensive than TIPDSCl2. Nucleoside A3 was reduced
with LiAlH4 to provide the 2′-(2-hydroxyethyl) nucleoside
A4. In our hands, NaBH4 was ineffective for reductions
of this type.33
Our overall strategy to the 2′-(2-hydroxyethyl)adeno-
sine nucleoside is an alternative to the previously used
2′-O-allylation of adenosine followed by ozonolysis and
reduction of the tethered aldehyde group, which was
performed with a different combination of hydroxyl and
nucleobase protecting groups.34 Here, we favored adeno-
sine 2′-alkylation with the R-bromo ester because we
found that the 2′-isomer, A2, and its 3′-isomer could
easily be separated by silica gel chromatography, which
was not the case for the 2′-O-allyl derivatives.34 We also
found that direct 2′-alkylation of adenosine with func-
tionalized alkyl halides such as N-(2-bromoethyl)phthal-
imide, which was at least moderately successful with
longer-chain substituted alkyl halides,9,10,32 was not
preparatively useful for the two-carbon tether (R. L.
Coppins and S. K. Silverman, data not shown).
Protection of nucleobase N6 of A4 as its isobutyryl
amide was achieved with transient TMS protection35 of
the 2′-tethered hydroxyl group, leading to A5. The 2′-
tethered hydroxyl group was exchanged for an azido
group by mesylation to A6, which was followed by azide
displacement yielding A7. The amine was obtained by
reduction with H2 over Pd/C and then protected as
trifluoromethyl amide A8. Removal of the 3′- and 5′-silyl
groups with TBAF, yielding A9, was followed by 5′-DMT
protection to produce A10 and phosphitylation to yield
adenosine tethered amine nucleoside phosphoramidite
A11. The route from adenosine A1 to A11 takes 10 steps
with 8 chromatographic purifications in a 12% overall
yield; 198 mg of phosphoramidite A11 was prepared.
Adenosine Nucleoside Phosphoramidite with 2′-
Tethered Thiol. Adenosine nucleoside derivatives with
2′-OCH2CH2SR substituents appear to be unreported.
The adenosine-tethered thiol phosphoramidite was pre-
pared from intermediate A5 by a route analogous to that
used for the pyrimidine thiol phosphoramidites (Scheme
4). Mitsunobu displacement with trityl mercaptan pro-
duced A12 from A5; A12 was 3′,5′-deprotected to form
(34) Prakash, T. P.; Kawasaki, A. M.; Fraser, A. S.; Vasquez, G.;
Manoharan, M. J. Org. Chem. 2002, 67, 357-369.
(35) Ti, G. S.; Gaffney, B. L.; Jones, R. A. J. Am. Chem. Soc. 1982,
104, 1316-1319.
4288 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
SCHEME 4
A13, and then A13 was 5′-DMT-protected to produce
A14. Finally, phosphitylation yielded the adenosine-
tethered thiol nucleoside phosphoramidite A15. Phos-
phoramidite A15 was obtained in 8 steps with 7 chro-
matographic purifications from adenosine A1 in a 12%
overall yield; 102 mg of A15 was synthesized.
provide G6, which was converted via the mesylate G7 to
the azide G8. Reduction with H2 over Pd/C to the amine,
protection as the trifluoromethyl amide, and removal of
the 3′- and 5′-silyl groups with TBAF produced G9. The
5′-hydroxyl was protected as its DMT derivative, yielding
G10. To complete the route, the nucleoside was phosphi-
tylated to produce the guanosine-tethered amine nucleo-
side phosphoramidite G11. The 10-step route from 2,6-
diaminopurine ribonucleoside G1 to G11 was achieved
with 8 chromatographic purifications in a 6% overall
yield. This includes the modest 33% yield for the first-
step conversion of G1 into the 2′-alkylated G2. A total of
160 mg of G11 was synthesized.
Guanosine Nucleoside Phosphoramidite with 2′-
Tethered Thiol. The last of the eight phosphoramidites,
the guanosine-tethered thiol, was prepared starting from
intermediate G6 (Scheme 6). Exchange of the 2′-tethered
hydroxyl group for the trityl-protected thiol group was
achieved under Mitsunobu conditions, and the 3′- and 5′-
silyl groups were removed with TBAF to produce G12.
5′-DMT protection led to G13, and phosphitylation
provided the guanosine-tethered thiol nucleoside phos-
phoramidite G14. The overall 8-step route from G1 to
G14 proceeded with 7 chromatographic purifications in
a 7% overall yield; 109 mg of G14 was prepared.
Solid-Phase RNA Synthesis Using the Modified
Phosphoramidites. The new nucleoside phosphoramid-
ites were successfully incorporated into RNA oligonucleo-
tides by solid-phase synthesis. For the tethered amine
series, each of the four nucleoside phosphoramidites,
U14, U15, A11, and G11, was incorporated into a
separate oligonucleotide, as detailed in the Experimental
Section. This demonstrates that all nucleobase deprotec-
tions and the U-triazolide-to-C conversion were successful
in the context of oligonucleotides. For the tethered thiol
series, adenosine phosphoramidite A15 was used for
solid-phase synthesis, thereby establishing that the thiol
modification is successfully incorporated. Subsequently,
in a larger RNA to which the tethered thiol oligonucleo-
tide derived from A15 was ligated,40 a standard AgNO3
deprotection procedure31 was employed, which demon-
Guanosine Nucleoside Phosphoramidite with 2′-
Tethered Amine. Direct 2′-alkylation of unprotected
guanosine is impractical because of the unwanted reac-
tivity of the nucleobase.36,37 Therefore, our route to the
guanosine derivatives began with commercially available
2,6-diaminopurine ribonucleoside G1 (Scheme 5). Nucleo-
side G1 was preferentially 2′-alkylated with NaH and
benzyl 2-bromoethyl ether to provide G2, which was
obtained free of the 3′-isomer after silica gel chromatog-
raphy.38 Although the yield of G2 was modest (33%), the
reaction and separation were readily performed on a
large scale, and for expediency we judged this route
preferable to multistep options involving protecting
groups (e.g., initial 3′,5′-TIPDS protection of G1, then 2′-
alkylation and 3′,5′-deprotection). The enzymatic conver-
sion of the 2,6-diaminopurine derivative G2 to the
guanosine analogue with adenosine deaminase (ADA)
provided 2′-functionalized guanosine nucleoside G3.38,39
Protection of the 3′- and 5′-hydroxyl groups using 5 equiv
of TBSCl was accompanied by guanine O6-silylation to
form G4. This compound appears to be stable to silica
gel chromatography, which is somewhat unexpected on
the basis of literature reports for similar compounds.37
Isobutyryl protection of the guanosine N2 was achieved
along with removal of the O6-TBS group, producing G5.
The 2′-tethered benzyl group was hydrogenolyzed to
(36) Wagner, E.; Oberhauser, B.; Holzner, A.; Brunar, H.; Issakides,
G.; Schaffner, G.; Cotten, M.; Knollmuller, M.; Noe, C. R. Nucleic Acids
Res. 1991, 19, 5965-5971.
(37) (a) Grøtli, M.; Douglas, M. E.; Beijer, B.; Sproat, B. Bioorg. Med.
Chem. Lett. 1997, 7, 425-428. (b) Grøtli, M.; Douglas, M.; Beijer, B.;
Garc´ıa, R. G.; Eritja, R.; Sproat, B. J. Chem. Soc., Perkin Trans. 1 1997,
2779-2788.
(38) Gundlach, C. W., IV; Ryder, T. R.; Glick, G. D. Tetrahedron
Lett. 1997, 38, 4039-4042.
(39) Robins, M. J.; Zou, R.; Hansske, F.; Wnuk, S. F. Can. J. Chem.
1997, 75, 762-767.
J. Org. Chem, Vol. 70, No. 11, 2005 4289

Jin et al.
SCHEME 5
SCHEME 6
strates that the free thiol moiety can be readily un-
masked after solid-phase synthesis.
upon storage for months at -20 °C or below when the
compounds are suitably protected from moisture.
For practical purposes, we generally store large amounts
of all nucleosides as the immediate precursors to the
phosphoramidites (i.e., as U8, U13, A10, A14, G10, and
G13), which obviates the issue of long-term phosphor-
amidite stability. Nevertheless, we have not noticed
substantial decomposition of any of the phosphoramidites
Conclusions
We have described a comprehensive and streamlined
set of experimental procedures for synthesizing all four
standard RNA nucleoside phosphoramidites, each with
either a 2′-tethered amine or a 2′-tethered thiol functional
group. The synthetic routes are maximally convergent
and provide sufficient amounts of phosphoramidite for
(40) Silverman, S. K.; Cech, T. R. Biochemistry 1999, 38, 8691-
8702.
4290 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-hy-
droxyethyl)uridine (U4). A solution of U3 (2.80 g, 4.58
mmol) in CH2Cl2 (50 mL) was cooled to -78 °C. Ozone was
bubbled through the solution for 20 min until a faint blue color
persisted. The ozonide was reduced by the addition of NaBH4
(0.976 g, 25.8 mmol) dissolved in EtOH (40 mL). After the
mixture was stirred for 3.5 h, the solution was quenched with
saturated aqueous NH4Cl (100 mL), and the cloudy white
mixture was diluted with EtOAc (200 mL). The organic layer
was separated, and the aqueous layer was extracted with
EtOAc (2 × 50 mL). The combined organic extracts were
washed with saturated aqueous NaCl (75 mL), dried over Na2-
SO4, and concentrated under vacuum. The residual colorless
viscous oil was purified via chromatography with 0-7% MeOH
in CH2Cl2 as the eluant to yield 1.60 g (62%) of U4 as a white
foam: Rf 0.42 (1:19 MeOH/CH2Cl2); 1H NMR (500 MHz, CDCl3)
δ 9.08 (br s, 1H), 7.89 (d, J ) 8.0 Hz, 1H), 5.73 (br s, 1H), 5.69
(dd, J ) 8.0, 1.6 Hz, 1H), 4.26 (ABq, J ) 13.6 Hz, 1H), 4.19
(ABqd, J ) 10.0, 4.0 Hz, 1H), 4.15 (ABqd, J ) 10.0, 2.0 Hz,
1H), 4.03-3.88 (m, 4H), 3.74-3.70 (m, 2H), 2.92 (br s, 1H),
1.10-1.02 (m, 28H); 13C NMR (125 MHz, CDCl3) δ 163.2, 150.1,
139.2, 101.7, 89.4, 82.9, 81.8, 73.0, 68.1, 61.6, 59.2, 17.4, 17.3,
17.2, 17.1, 17.0, 16.92, 16.90, 16.7, 13.4, 13.0, 12.8, 12.5. FAB-
HRMS [M + H]+ calcd for C23H43N2O8Si2 531.2558, found
531.2557.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-meth-
anesulfonylethyl)uridine (U5). A portion of U4 (2.20 g, 4.14
mmol, combined from two preparations of U4 as described
above) was coevaporated with pyridine (10 mL) and dissolved
in CH2Cl2 (35 mL). The solution was cooled to 0 °C, and Et3N
(0.69 mL, 4.96 mmol) and methanesulfonyl chloride (0.38 mL,
4.96 mmol) were added. After 45 min, the solution was
quenched with MeOH (5 mL) and concentrated under vacuum.
The residual colorless viscous oil was purified via chromatog-
raphy with 0-50% EtOAc in CH2Cl2 as the eluant to produce
2.10 g (82%) of U5 as a white foam: Rf 0.36 (1:4 EtOAc/CH2-
Cl2); 1H NMR (400 MHz, CDCl3) δ 9.60 (br s, 1H), 7.89 (d, J )
8.0 Hz, 1H), 5.72 (s, 1H), 5.68 (d, J ) 8.0 Hz, 1H), 4.45-4.25
(dt, J ) 4.2, 2.0 Hz, 2H), 4.24 (ABq, J ) 13.6 Hz, 1H), 4.18
(ABqd, J ) 9.6, 4.5 Hz, 1H), 4.13-4.09 (m, 3H), 3.95 (ABqd,
J ) 13.6, 2.0 Hz, 1H), 3.87 (d, J ) 4.0 Hz, 1H), 3.09 (br s, 3H),
1.01 (m, 28H); 13C NMR (125 MHz, CDCl3) δ 163.8, 150.2,
139.2, 101.7, 88.5, 82.9, 81.6, 69.4, 68.9, 68.1, 59.2, 37.6, 17.4,
17.3, 17.2, 17.1, 17.0, 16.9, 16.8, 16.7, 13.3, 13.0, 12.8, 12.3.
EI-HRMS [M + H]+ calcd for C24H45N2O10Si2S 609.2336, found
609.2333.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-azi-
doethyl)uridine (U6). NaN3 (0.88 g, 13.5 mmol) and 18-
crown-6 (5 mg, 0.019 mmol) were added to a solution of U5
(1.65 g, 2.71 mmol) in DMF (25 mL). The solution was stirred
at 60 °C for 6 h. The solvents were removed under vacuum,
and the residual colorless viscous oil was partitioned between
EtOAc (75 mL) and water (75 mL). The organic layer was
separated, and the aqueous layer was extracted with EtOAc
(2 × 75 mL). The combined organic extracts were washed with
saturated aqueous NaCl (75 mL), dried over Na2SO4, and
concentrated under vacuum to yield 1.57 g (104% of the
theoretical amount) of U6 as a pale yellow foam. Most of this
crude sample was used in the next reaction step without
further purification: Rf 0.50 (3:1 CH2Cl2/EtOAc); 1H NMR (400
MHz, CDCl3) δ 8.84 (br s, 1H), 7.91 (d, J ) 8.0 Hz, 1H), 5.74
(s, 1H), 5.68 (d, J ) 8.0 Hz, 1H), 4.25 (ABq, J ) 14.0 Hz, 1H),
4.193 (d, J ) 4.0 Hz, 1H), 4.190 (m, 1H), 4.03 (ddd, J ) 6.4,
4.0, 2.8 Hz, 2H), 3.97 (ABqd, J ) 13.6, 1.6 Hz, 1H), 3.86 (d,
J ) 3.2 Hz, 1H), 3.41 (ABqdd, J ) 13.2, 6.0, 4.0 Hz, 2H), 1.10-
0.93 (m, 28H); 13C NMR (100 MHz, CDCl3) δ 163.3, 149.9,
139.5, 101.6, 88.9, 82.8, 81.6, 70.5, 68.1, 59.3, 50.8, 17.5, 17.4,
17.3, 17.2, 17.1, 17.0, 16.8, 16.7, 13.4, 13.0, 12.8, 12.4. EI-
HRMS [M + H]+ calcd for C23H42N5O7Si2 556.2623, found
556.2622.
several solid-phase synthesis coupling reactions. The
resulting oligonucleotides are immediately useful in
ongoing studies of RNA structure, folding, and catalysis.
Experimental Section
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-N3-(meth-
oxycarbonylvinyl)uridine (U2). TIPDSCl2 (2.88 mL, 9.01
mmol) was added dropwise over 5 min to a solution of uridine
U1 (2.00 g, 8.20 mmol) in pyridine (50 mL) at 0 °C. (Alterna-
tively, the TIPDSCl2 can be generated in situ as described in
the text; we have successfully used the procedure of ref 18 on
the 10-25 mmol scale.) The colorless solution was warmed to
room temperature, stirred for 10 h, and then quenched with
water (5 mL). Pyridine was removed under vacuum, and the
residual white solid was partitioned between EtOAc (100 mL)
and water (100 mL). The organic layer was washed with cold
1 N HCl (2 × 50 mL), saturated aqueous NaHCO3 (2 × 50
mL), and saturated aqueous NaCl (50 mL). The organic layer
was dried over Na2SO4 and concentrated under vacuum to
produce 4.20 g of a crude white solid that was used without
further purification. This solid was dissolved in 42 mL of 1:5
CH2Cl2/CH3CN and cooled to ∼13-15 °C (chilled water bath;
at higher temperature, methyl propiolate reacts considerably
with the 2′-hydroxyl group, whereas at lower temperature, the
overall reaction rate is very low). DMAP (0.30 g, 2.40 mmol)
was added to the solution; this was followed by the addition
of methyl propiolate (0.25 mL, 2.40 mmol) dropwise over a
period of 2 min. An additional aliquot of methyl propiolate
(2.40 mmol) was added every 30 min until TLC indicated
complete consumption of 3′,5′-O-(tetraisopropyldisiloxane-1,3-
diyl)uridine; three additional aliquots were required. The
solution was quenched with MeOH (8 mL) and concentrated
under vacuum. The residual red viscous oil was purified via
chromatography using 0-30% EtOAc in hexanes as the eluant
to yield 3.20 g (68%) of U2 as a white foam: Rf 0.28 (3:1
hexanes/EtOAc); 1H NMR (500 MHz, CDCl3) δ 8.27 (d, J )
15.0 Hz, 1H), 7.71 (d, J ) 8.0 Hz, 1H), 7.08 (d, J ) 15.0 Hz,
1H), 5.76 (d, J ) 8.0 Hz, 1H), 5.74 (br s, 1H), 4.37 (dd, J )
9.0, 4.7 Hz, 1H), 4.22 (ABqd, J ) 13.3, 1.5 Hz, 1H), 4.17 (d,
J ) 5.0 Hz, 1H), 4.14-4.09 (m, 1H), 4.00 (ABqd, J ) 13.5, 3.0
Hz, 1H), 3.78 (s, 3H), 2.84 (s, 1H), 1.10-1.00 (m, 28H); 13C
NMR (125 MHz, CDCl3) δ 167.6, 161.1, 149.2, 138.2, 134.0,
113.6, 101.0, 91.4, 82.0, 75.3, 68.8, 60.0, 51.8, 17.4, 17.3, 17.21,
17.20, 16.98, 16.91, 16.8, 16.7, 13.3, 12.8, 12.7, 12.4. FAB-
HRMS M+ calcd for C25H42N2O9Si2 570.7801, found 570.7804.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-allyl-
N3-(methoxycarbonylvinyl)uridine (U3). A solution of U2
(3.20 g, 5.60 mmol) and allyl methyl carbonate (1.30 g, 11.20
mmol) in THF (20 mL) was added dropwise via cannula over
15 min, rinsing the flask containing U2 with THF (3 × 2 mL),
to a suspension of 1,4-bisphosphinobutane (92 mg, 0.20 mmol)
and Pd2(dba)3 (52 mg, 0.06 mmol) in THF (6 mL). After the
mixture was stirred at room temperature for 12 h, the solution
was quenched with MeOH (4 mL) and concentrated under
vacuum. The residual green viscous oil was purified via
chromatography with 20% EtOAc in hexanes as the eluant to
produce 2.90 g (85%) of U3 as a white foam: Rf 0.50 (4:1
hexanes/EtOAc); 1H NMR (400 MHz, CDCl3) δ 8.26 (d, J ) 15
Hz, 1H), 7.93 (d, J ) 8.3 Hz, 1H), 7.08 (d, J ) 15 Hz, 1H),
5.92 (ddt, J ) 17.3, 10.5, 5.2 Hz, 1H), 5.74 (s, 1H), 5.73 (d,
J ) 8.3 Hz, 1H), 5.39 (dd, J ) 17.3, 1.7 Hz, 1H), 5.21 (dd, J )
10.5, 1.2 Hz, 1H), 4.39 (m, 2H), 4.25 (ABqd, J ) 14 Hz, 1H),
4.19 (ABqd, J ) 9.6, 1.6 Hz, 1H), 4.14 (ABqd, J ) 9.6, 4.0 Hz,
1H), 3.96 (ABqd, J ) 14.0, 2.0 Hz, 1H), 3.86 (d, J ) 4 Hz, 1H),
3.77 (s, 3H), 0.99-1.10 (m, 28H); 13C NMR (125 MHz, CDCl3)
δ 167.5, 161.1, 149.1, 137.8, 134.13, 134.10, 113.5, 117.6, 100.7,
89.6, 81.9, 80.9, 71.2, 67.9, 59.2, 51.7, 17.44, 17.40, 17.25, 17.20,
17.0, 16.9, 16.7, 13.4, 13.0, 12.8, 12.3. FAB-HRMS M+ calcd
for C28H47N2O9Si2 611.2821, found 611.2820.
2′-O-(2-(Trifluoroacetamido)ethyl)uridine (U7). Tri-
phenylphosphine (1.38 g, 5.26 mmol) and water (0.24 mL, 13.2
J. Org. Chem, Vol. 70, No. 11, 2005 4291

Jin et al.
mmol) were added to a solution of crude U6 (1.46 g, assumed
2.63 mmol) dissolved in THF (25 mL). The solution was stirred
at 45 °C for 4.5 h. After the solution was cooled to room
temperature, ethyl trifluoroacetate (1.56 mL, 13.2 mmol) and
Et3N (1.83 mL, 13.2 mmol) were added, and the mixture
continued to be stirred at room temperature for 10 h. The
solution was cooled to 0 °C, and 1 M TBAF in THF (5.25 mL,
5.25 mmol) was added. The solution was warmed to room
temperature over 5 h, and the solvents were removed under
vacuum. The residual colorless viscous oil was purified via
chromatography with 5% MeOH in EtOAc as the eluant to
produce 0.95 g (94%) of U7 as a white foam: Rf 0.35 (1:19
and 4.21 (each td, J ) 7.5, 3.0 Hz, total 1H), 3.98-3.80 (m,
3H), 3.79 and 3.78 (each s, total 6H), 3.75-3.45 (m, 7H), 2.80
and 2.63 (t with J ) 6.0 Hz and m, total 2H), 1.20, 1.19, 1.18,
1.17, and 1.08 (each d, J ) 7.0 Hz, total 12H); 13C NMR (100
MHz, acetone-d6) δ 163.5, 159.8, 159.7, 151.3, 145.7, 140.5,
136.3, 136.1, 131.13, 131.10. 129.10, 129.07, 128.7, 127.8,
114.0, 102.4, 89.1, 87.5, 83.07, 83.00, 81.95, 81.90, 70.8, 69.0,
62.2, 59.3, 59.1, 55.5, 43.9, 43.8, 40.3, 25.00, 24.96, 24.90, 24.80,
20.72, 20.70; 19F NMR (376 MHz, acetone-d6) δ -76.71 and
-76.68; 31P NMR (202 MHz, acetone-d6) δ 151.2 and 150.1.
FAB-HRMS [M + Na]+ calcd for C43H51N5O10F3NaP 908.3224,
found 908.3223.
1
MeOH/EtOAc); H NMR (400 MHz, acetone-d6) δ 10.06 (br s,
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)-4-(1,2,4-triazolyl)uridine-3′-O-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (U10). A portion of 1,2,4-
triazole (0.80 g, 11.6 mmol) was dissolved in acetonitrile (15
mL) and cooled to 0 °C. POCl3 (200 µL, 2.0 mmol) was added
dropwise over 10 min to the cloudy white mixture, and the
mixture was stirred at 0 °C for 20 min. A portion of Et3N (8.0
mL, 57.4 mmol) was added, and the mixture was stirred at 0
°C for 3 h. A solution of U9 (0.200 g, 0.22 mmol) in CH3CN
(0.5 mL) was transferred into the reaction flask via cannula,
rinsing the flask containing U9 with CH3CN (2 × 0.3 mL).
The mixture was stirred at 0 °C for 1.5 h and diluted with
10% Et3N in EtOAc (25 mL). Saturated aqueous NaHCO3 (25
mL) was added to the mixture resulting in two clear layers.
The organic layer was separated, washed with saturated
aqueous NaHCO3 (25 mL), dried over Na2SO4, and concen-
trated under vacuum. The residual pale yellow foam was
purified via chromatography with the column packed as
described for U9 (the sample was loaded in 1:4 hexanes/CH2-
Cl2). The column was eluted with 1:4 hexanes/CH2Cl2 contain-
ing 3% Et3N to yield 0.100 g (45%) of U10 as a white foam: Rf
0.42 (1:5:4 Et3N/hexanes/CH2Cl2). NMR spectroscopy revealed
the presence of both diastereomers: 1H NMR (500 MHz,
acetone-d6) δ 9.241 and 9.240 (each s, total 1H), 8.90 and 8.86
(each d, J ) 7.0 Hz, total 1H), 8.23 and 8.22 (each s, total 1H),
7.56 and 7.52 (each d, J ) 7.5 Hz, total 2H), 7.45-7.31 (m,
9H), 6.94 (m, 4H), 6.56 and 6.50 (each d, J ) 7.5 Hz, total
1H), 5.92 (s, 1H), 4.81 and 4.72 (td, J ) 9.5, 4.5 Hz, total 1H),
4.38-4.28 (m, 2H), 4.15 (m, 1H), 4.00 (m, 1H), 3.81 and 3.78
(each s, total 6H), 3.75-3.57 (m, 8H), 2.65 (m, 1H), 1.22 (d,
J ) 6.5 Hz, 6H), 1.17 (d, J ) 6.5 Hz, 6H); 13C NMR (125 MHz,
acetone-d6) δ 159.5, 159.2, 154.2, 154.0, 147.6, 144.8, 143.3,
135.9, 135.5, 130.56, 130.46, 128.6, 128.2, 127.3, 118.5, 113.4,
106.8, 94.2, 91.4, 90.9, 87.1, 87.2, 82.2, 81.9, 81.1, 69.2, 69.1,
68.7, 60.5, 60.3, 58.6, 58.4, 54.9, 46.3, 43.3, 43.2, 43.1, 39.7,
39.6, 39.5, 26.1, 24.3, 20.2; 19F NMR (470 MHz, acetone-d6) δ
-76.75 and -76.72; 31P NMR (202 MHz, acetone-d6) δ 151.7
and 149.5. ESI-HRMS [M + H]+ calcd for C45H53N8O9F3P
937.3625, found 937.3594.
3′,5′-O-(Tetraisopropyldisiloxane-1,3-diyl)-2′-O-(2-(tri-
phenylmethylthio)ethyl)uridine (U11). Diisopropyl azodi-
carboxylate (1.1 mL, 5.6 mmol) was added to a solution of
triphenylphosphine (1.48 g, 5.6 mmol) in THF (30 mL) cooled
to 0 °C. After 0.5 h, a solution of U4 (1.50 g, 2.82 mmol) and
trityl mercaptan (1.55 g, 5.6 mmol) in THF (20 mL) was added
via cannula, and the flask containing the latter reagents was
rinsed with THF (2 × 5 mL). After 40 min, the solution was
quenched with MeOH (2 mL) and concentrated under vacuum.
The resulting colorless viscous oil was purified via chroma-
tography with 10-30% EtOAc in hexanes as the eluant to
produce 1.90 g (85%) of U11 as a white foam: Rf 0.35 (3:1
hexanes/EtOAc); 1H NMR (500 MHz, CDCl3) δ 9.43 (s, 1H),
7.88 (d, J ) 8.0 Hz, 1H), 7.42-7.18 (m, 15H), 5.67 (dd, J )
6.0, 2.0 Hz, 1H), 5.66 (s, 1H), 4.22 (ABq, J ) 13.5 Hz, 1H),
4.12 (s, 1H), 3.95 (dd, J ) 13.5, 2.0 Hz, 1H), 3.76-3.68 (m,
4H), 2.47-2.44 (m, 2H), 1.10-0.97 (m, 28H); 13C NMR (125
MHz, CDCl3) δ 164.1, 150.0, 145.1, 139.9, 129.9, 128.1, 126.8,
101.6, 89.2, 82.6, 81.8, 70.0, 68.4, 66.7, 59.6, 32.2, 17.7, 17.6,
1H), 8.59 (br s, 1H), 8.07 (d, J ) 8.0 Hz, 1H), 5.92 (d, J ) 4.0
Hz, 1H), 5.57 (d, J ) 8.0 Hz, 1H), 4.39 (br s, 1H), 4.34 (br t,
J ) 5.2 Hz, 1H), 4.23 (br s, 1H), 4.13 (dd, J ) 4.8, 3.2 Hz, 1H),
3.99-3.93 (m, 2H), 3.89 (br s, 1H), 3.84-3.78 (m, 2H), 3.65-
3.49 (m, 2H); 13C NMR (125 MHz, acetone-d6) δ 163.8, 157.9,
151.4, 141.1, 117.2, 102.4, 88.5, 85.7, 83.3, 69.5, 69.3, 61.2, 40.5.
19F NMR (470 MHz, acetone-d6) δ -76.9. EI-HRMS [M + H]+
calcd for C13H17N3O7F3 384.1022, found 384.1018.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)uridine (U8). 4,4′-Dimethoxytrityl chloride (0.53 g, 1.56
mmol) and DMAP (2 mg, 0.016 mmol) were added to a solution
of U7 (0.50 g, 1.3 mmol) in pyridine (10 mL). The solution was
stirred at room temperature for 20 h, and the solvents were
removed under vacuum. The orange viscous oil was purified
by column chromatography. The silica gel was swirled with
10% Et3N in hexanes (∼1 mL of solvent per 1 g of silica gel)
for several minutes, and the solvents were removed under
vacuum until a freely flowing powder was obtained. The
column was packed with this prewashed silica gel using CH2-
Cl2. After the sample had been loaded, the column was eluted
with a gradient of 0-5% MeOH in CH2Cl2 to produce 0.67 g
(75%) of U8 as a white foam: Rf 0.32 (1:19 MeOH/CH2Cl2); 1H
NMR (400 MHz, CDCl3) δ 8.05 (d, J ) 8.0 Hz, 1H), 7.63 (br s,
1H), 7.39-7.37 (m, 2H), 7.32-7.27 (m, 7H), 6.86-6.82 (m, 4H),
5.85 (d, J ) 1.2 Hz, 1H), 5.31 (d, J ) 8.5 Hz, 1H), 4.42 (dd,
J ) 8.4, 5.2 Hz, 1H), 4.05-4.01 (m, 2H), 3.96 (dd, J ) 6.4, 4.0
Hz, 1H), 3.93 (d, J ) 6.4 Hz, 1H), 3.79 (s, 6H), 3.64 (br s, 1H),
3.62-3.59 (m, 2H), 3.53 (ABqd, J ) 11.2, 2.5 Hz, 1H); 13C NMR
(125 MHz, CDCl3) δ 163.1, 158.73, 158.69, 150.8, 144.2, 139.8,
135.2, 135.0, 130.1, 130.0, 128.1, 128.0, 127.2, 113.3, 102.4,
88.2, 87.1, 83.3, 83.1, 69.2, 68.4, 60.8, 55.2, 39.6; 19F NMR (470
MHz, CDCl3) δ -76.1. FAB-HRMS [M + H]+ calcd for
C34H34N3O9F3 685.2248, found 685.2247.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)phos-
phoramidite (U9). A portion of U8 (0.42 g, 0.60 mmol) was
coevaporated with pyridine (2 × 10 mL) and dissolved in CH2-
Cl2 (10 mL). The solution was cooled to 0 °C, and 2-cyanoethyl
N,N,N′,N′-tetraisopropylphosphorodiamidite (0.27 mL, 0.82
mmol) and 4,5-dicyanoimidazole27 (DCI, 90 mg, 0.76 mmol)
were added. The cloudy mixture was warmed to room tem-
perature, stirred for 10 h, and diluted with CH2Cl2 (75 mL)
and saturated aqueous NaHCO3 (100 mL). The layers were
separated, and the aqueous layer was extracted with CH2Cl2
(3 × 50 mL). The combined organic extracts were washed with
saturated aqueous NaCl (100 mL), dried over Na2SO4, and
concentrated under vacuum. The residual colorless viscous oil
was purified via chromatography; the column was packed with
silica gel using 5% Et3N in hexanes. After the sample had been
loaded in CH2Cl2, the column was eluted with 0-3% Et3N in
CH2Cl2 to produce 0.44 g (81%) of U9 as a colorless oil: Rf
0.80 (1:1 EtOAc/CH2Cl2). NMR spectroscopy revealed the
presence of both diastereomers: 1H NMR (500 MHz, acetone-
d6) δ 8.67 and 8.64 (each br s, total 1H), 7.98 and 7.94 (each
d, J ) 8.0 Hz, total 1H), 7.52-7.47 (m, 2H), 7.41-7.31 (m,
7H), 7.28-7.24 (m, 1H), 6.93-6.89 (m, 4H), 5.94 and 5.93 (each
d, J ) 3.0 Hz, total 1H), 5.22 and 5.21 (each d, J ) 8.0 Hz,
total 1H), 4.67 and 4.59 (each ddd, J ) 9.5, 7.0, 5.0 Hz, total
1H), 4.31 and 4.27 (each dd, J ) 5.0, 2.5 Hz, total 1H), 4.26
4292 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
17.5, 17.4, 17.3, 17.2. 17.1. 17.0, 13.6, 13.3, 13.1, 12.7. ESI-
HRMS [M + Na]+ calcd for C42H56N2O7SSi2Na 811.3245, found
811.3296.
(m, 1H), 2.45 (m, 2H), 1.18 (m, 12H); 13C NMR (125 MHz,
acetone-d6) δ 162.5, 159.0, 150.5, 145.2, 140.2, 130.5, 129.8,
128.4, 128.3, 127.1, 126.9, 113.3, 101.7, 88.0, 87.0, 82.2, 82.3,
82.1, 70.3, 69.3, 69.1, 66.2, 62.2, 43.2, 32.1, 24.2, 20.1; 31P NMR
(202 MHz, acetone-d6) δ 150.8 and 150.4. ESI-HRMS [M +
H]+ calcd for C60H66N4O9SP 1049.4288, found 1049.4281.
2′-O-(2-(Triphenylmethylthio)ethyl)uridine (U12). TBAF
(1 M) in THF (5.6 mL, 5.6 mmol) was added dropwise over 5
min to a solution of U11 (1.90 g, 2.41 mmol) in THF (30 mL)
at 0 °C. The solution was stirred at room temperature for 6 h,
diluted with EtOAc (20 mL), washed with saturated aqueous
NaHCO3 (2 × 25 mL), dried over Na2SO4, and concentrated
under vacuum. The resulting colorless viscous oil was purified
via chromatography with 0-5% MeOH in CH2Cl2 as the eluant
to produce 0.96 g (72%) of U12 as a white foam: Rf 0.33 (1:19
MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 9.81 (br s, 1 H),
7.75 (d, J ) 8.0 Hz, 1H), 7.41-7.19 (m, 15H), 5.69 (d, J ) 8.0
Hz, 1H), 5.65 (d, J ) 4.0 Hz, 1H), 4.20 (t, J ) 5.2 Hz, 1H),
4.01 (br dt, J ) 5.6, 2.4 Hz, 1H), 3.94 (dd, J ) 5.2, 3.5 Hz,
1H), 3.93 (ABqd, J ) 12.2, 1.5 Hz, 1H), 3.77 (ABqd, J ) 11.2,
2.0 Hz, 1H), 3.60 (dt, J ) 10.0 Hz, 5.6 Hz, 1H), 3.38 (br s, 1H),
3.23 (ddd, J ) 10.5, 8.0, 5.0 Hz, 1H), 3.12 (br s, 1H), 2.54 (ddd,
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)-4-(1,2,4-triazolyl)uridine-3′-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (U15). A portion of
1,2,4-triazole (0.65 g, 9.4 mmol) was dissolved in acetonitrile
(13 mL) and cooled to 0 °C. POCl3 (165 µL, 1.8 mmol) was
added dropwise over 10 min to the cloudy white mixture, and
the mixture was stirred at 0 °C for 20 min. A portion of Et3N
(5.1 mL, 36.6 mmol) was added, and the mixture was stirred
at 0 °C for 3 h. A solution of U14 (0.197 g, 0.188 mmol) in
CH3CN (1.5 mL) was transferred into the reaction flask via
cannula; the flask containing U14 was rinsed with CH3CN
(2 × 0.2 mL). The mixture was stirred at 0 °C for 1.5 h and
diluted with 10% Et3N in EtOAc (25 mL). Saturated aqueous
NaHCO3 (25 mL) was added to the mixture resulting in two
clear layers. The organic layer was separated, washed with
saturated aqueous NaHCO3 (25 mL), dried over Na2SO4, and
concentrated under vacuum. The residual pale yellow foam
was purified via chromatography with the column packed as
described for U9 (the sample was loaded in 3:2 hexanes/CH2-
Cl2). The column was eluted with 1:11:8 to 1:8:11 Et3N/
hexanes/CH2Cl2 to yield 0.112 g (54%) of U15 as a white
foam: Rf 0.55 (1:11:8 Et3N/hexanes/CH2Cl2). NMR spectros-
copy revealed the presence of both diastereomers: 1H NMR
(500 MHz, acetone-d6) δ 9.24 and 9.23 (each s, total 1H), 8.89
and 8.83 (each d, J ) 7.0 Hz, total 1H), 8.22 and 8.21 (each s,
total 1H), 7.57-7.23 (m, 24H), 6.93 (m, 4H), 6.53 and 6.46
(each d, J ) 7.0 Hz, total 1H), 5.91 and 5.90 (each s, total 1H),
4.77 and 4.64 (each td, J ) 9.0, 5.0 Hz, total 1H), 4.30 (m,
1H), 4.15 (ABqd, J ) 10.5, 4.5 Hz, 1H), 3.96-3.82 (m, 2H),
3.81 and 3.80 (each s, total 6H), 3.78-3.56 (m, 6H), 2.85 and
2.82 (each s, total 1H), 2.68 (m, 1H), 2.62 (m, 1H), 2.53 (m,
1H), 1.19 (d, J ) 7.5 Hz, 3H), 1.14 (d, J ) 7.0 Hz, 3H), 1.10 (d,
J ) 7.0 Hz, 3H). 1.05 (d, J ) 6.5 Hz, 3H); 13C NMR (125 MHz,
acetone-d6) δ 159.4, 159.1, 154.2, 147.7, 145.3, 144.8, 143.2,
135.5, 135.4, 130.6, 130.5, 130.4, 130.3, 129.8, 128.6, 128.2,
127.3, 126.9, 113.3, 94.0, 90.7, 87.1, 82.2, 69.1, 68.9, 60.5, 60.3,
58.6, 58.4, 54.9, 43.3, 43.2, 32.1, 24.5, 20.0, 18.6; 31P NMR (202
MHz, acetone-d6) δ 151.2 and 149.6. ESI-HRMS [M + Na]+
calcd for C62H66N7O8PSNa 1122.4329, found 1122.4363.
J ) 13.0, 7.4, 5.4 Hz, 1H), 2.40 (dt, J ) 13.0, 6.0 Hz, 1H); 13
C
NMR (100 MHz, CDCl3) δ 163.6, 150.4, 144.7, 142.2, 129.8,
128.3, 127.1, 102.6, 90.7, 85.3, 81.2, 69.6, 68.9, 61.6, 32.3. ESI-
HRMS [M + Na]+ calcd for C30H30N2O6SNa 569.1722, found
569.1714.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)uridine (U13). A portion of U12 (0.90 g, 1.65
mmol) was coevaporated from pyridine (3 mL) and dissolved
in pyridine (3 mL). The solution was cooled to 0 °C, and 4,4′-
dimethoxytrityl chloride (1.00 g, 2.95 mmol) was added. After
the mixture was stirred at room temperature for 12 h, the
solvents were removed under vacuum. The residual orange
foam was purified via chromatography with the column packed
as described for U9. The column was eluted with a gradient
of 0-5% MeOH in CH2Cl2 to produce 0.95 g (68%) of U13 as
a pale yellow foam: Rf 0.45 (1:19 MeOH/CH2Cl2); 1H NMR (500
MHz, acetone-d6) δ 10.07 (br s, 1H), 7.733 (s, 1H), 7.85 (d, J )
8.5 Hz, 1H), 7.48-7.21 (m, 24H), 6.92-6.90 (m, 4H), 5.89 (d,
J ) 3.0 Hz, 1H), 5.26 (d, J ) 8.0 Hz, 1H), 4.41 (q, J ) 5.5 Hz,
1H), 4.06 (dt, J ) 6.5, 3.0 Hz, 1H), 3.95 (dd, J ) 4.5, 3.0 Hz,
1H), 3.71 (br q, J ) 7.5 Hz, 1H), 3.65 (ABqd, J ) 10.5, 7.0 Hz,
1H), 3.54 (ABqt, J ) 10.5, 6.5 Hz, 1H), 3.79 (s, 6H), 3.47 (ABqd,
J ) 11.0, 3.0 Hz, 1H), 3.34 (ABqd, J ) 11.0, 3.0 Hz, 1H), 2.86
(br s, 1H); 13C NMR (125 MHz, acetone-d6) δ 163.0, 159.1,
150.6, 145.2, 135.9, 135.7, 130.4, 129.8, 128.4, 128.2, 128.1,
127.1, 126.9, 113.4, 101.8, 87.8, 86.8, 83.3, 82.4, 69.1, 67.1, 62.6,
54.9, 32.0, 18.5. ESI-HRMS [M + Na]+ calcd for C51H48N2O8-
SNa 871.3029, found 871.3060.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)uridine-3′-O-(2-cyanoethyl-N,N-diisopropyl)-
phosphoramidite (U14). A portion of U13 (0.30 g, 0.35 mmol)
was coevaporated with pyridine (2 × 5 mL) and dissolved in
CH2Cl2 (4 mL). The solution was cooled to 0 °C, and 2-cyano-
ethyl N,N,N′,N′-tetraisopropylphosphorodiamidite (0.15 mL,
0.45 mmol) and 4,5-dicyanoimidazole (50 mg, 0.42 mmol) were
added. The cloudy mixture was warmed to room temperature
and stirred for 6 h. The mixture was diluted with 5% Et3N in
EtOAc (25 mL) and washed with saturated aqueous NaHCO3
(2 × 25 mL). The organic layer was dried over Na2SO4 and
concentrated under vacuum. The residual pale yellow foam
was purified via chromatography with the column packed as
described for U9 (the sample was loaded in 1:15:4 Et3N/
hexanes/CH2Cl2). The column was eluted with a gradient from
1:15:4 to 1:7:12 Et3N/hexanes/CH2Cl2 to produce 0.31 g (84%)
of U14 as a white foam: Rf 0.51 (1:7:12 Et3N/hexanes/CH2-
Cl2). NMR spectroscopy revealed the presence of both diaster-
eomers: 1H NMR (500 MHz, acetone-d6) δ 10.10 (br s, 1H),
7.93 and 7.86 (each d, J ) 8.0 Hz, total 1H), 7.53-7.22 (m,
24H), 6.87 (m, 4H), 5.93 and 5.91 (each d, J ) 3.0 Hz, total
1H), 5.23 and 5.21 (each d, J ) 2.5 Hz, total 1H), 4.57 and
4.49 (each m, total 1H), 4.21 and 4.15 (each m, total 1H), 4.08
(m, 1H), 3.88 (m, 1H), 3.79 and 3.78 (each s, total 6H), 3.68
(m, 1H), 3.63 (m, 4H), 3.55-3.43 (m, 2H), 2.71 (m, 1H), 2.62
2′-O-(Methoxycarbonylmethyl)adenosine (A2). A 60%
dispersion of sodium hydride in mineral oil (1.26 g, 32 mmol)
was added to a suspension of adenosine (6.15 g, 23.0 mmol) in
DMF (150 mL) at 0 °C. The suspension was stirred at 0 °C for
1 h, and methyl bromoacetate (3.3 mL, 34.5 mmol) was added.
The mixture was warmed to room temperature, stirred for 8
h, and quenched with acetic acid (3 mL) and methanol (10 mL).
The solvents were removed under vacuum, and the resulting
paste was purified via chromatography (dry-load) with 1-10%
MeOH in CH2Cl2 as the eluant to produce 3.65 g (47%) of A2
1
as a white foam: Rf 0.35 (1:9 MeOH/CH2Cl2); H NMR (400
MHz, CDCl3) δ 8.32 (s, 1H), 7.87 (s, 1H), 6.58 (br s, 1H), 5.96
(br s, 2H), 5.94 (d, J ) 7.5 Hz, 1H), 4.73 (dd, J ) 7.6, 4.3 Hz,
1H), 4.43 (d, J ) 5.5 Hz, 1H), 4.40-4.31 (m, 4H), 3.97-3.91
(m, 2H), 3.74 (s, 3H), 3.72 (br s, 1H); 13C NMR (125 MHz,
CDCl3) δ 172.2, 156.1, 152.3, 148.3, 141.0, 121.1, 89.0, 87.4,
84.6, 70.8, 69.1, 63.2, 52.5. ESI-HRMS [M + H]+ calcd for
C13H18N5O6 340.1257, found 340.1266. The HMBC spectrum
was also recorded (see text).
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(methoxycar-
bonylmethyl)adenosine (A3). tert-Butyldimethylsilyl chlo-
ride (5.08 g, 33.7 mmol) and imidazole (2.27 g, 16.2 mmol) were
added to a stirred solution of A2 (3.65 g, 10.8 mmol) in DMF
(50 mL). After 16 h, the solvents were removed under vacuum,
and the resulting paste was partitioned between water (100
mL) and CH2Cl2 (100 mL). The aqueous layer was extracted
with CH2Cl2 (3 × 70 mL). The combined organic extracts were
J. Org. Chem, Vol. 70, No. 11, 2005 4293

Jin et al.
washed with saturated aqueous NaCl (100 mL), dried over
MgSO4, and concentrated under vacuum. The resulting oil was
purified via chromatography with 0-3% MeOH in CH2Cl2 as
the eluant to produce 5.67 g (93%) of A3 as a white solid: Rf
dried over MgSO4, and concentrated under vacuum. The
residual yellow viscous oil was purified via chromatography
with 1:1 EtOAc/CH2Cl2 as the eluant to produce 0.57 g (82%)
1
of A6 as a colorless oil: Rf 0.52 (1:1 EtOAc/CH2Cl2); H NMR
1
0.54 (1:9 MeOH/CH2Cl2); H NMR (400 MHz, CDCl3) δ 8.34
(500 MHz, CDCl3) δ 8.84 (br s, 1H), 8.67 (s, 1H), 8.36 (s, 1H),
6.16 (d, J ) 3.5 Hz, 1H), 4.52 (dd, J ) 5.3, 4.8 Hz, 1H), 4.34
(dd, J ) 4.5, 3.8 Hz, 1H), 4.33 (t, J ) 4.0 Hz, 2H), 4.09 (ddd,
J ) 6.2, 3.0, 2.4 Hz, 1H), 3.98 (ABqd, J ) 11.6, 3.3 Hz, 1H),
3.89 (m, 2H), 3.76 (ABqd, J ) 11.6, 2.6 Hz, 1H), 3.16 (m, 1H),
2.98 (s, 3H), 1.27 (d, J ) 6.8 Hz, 3H), 1.27 (d, J ) 6.8 Hz, 3H),
0.90 (s, 9H), 0.89 (s, 9H), 0.09 (s, 3H), 0.08 (s, 3H), 0.07 (s,
3H), 0.07 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 176.2, 152.5,
150.8, 149.3, 141.4, 122.5, 86.7, 84.8, 82.7, 69.6, 68.5, 68.5, 61.5,
37.5, 35.9, 25.9, 25.6, 19.1, 18.3, 17.95, -4.7, -5.0, -5.5. EI-
HRMS M+ calcd for C29H53N5O8SSi2 687.3153, found. 687.3143.
(s, 1H), 8.20 (s, 1H), 6.22 (d, 1H, J ) 4.1 Hz), 5.78 (br s, 2H),
4.58 (dd, J ) 4.6, 3.5 Hz, 1H), 4.57 (dd, J ) 4.7, 3.3 Hz, 1H),
4.28 (ABq, J ) 16.7 Hz, 1H), 4.20 (ABq, J ) 16.8 Hz, 1H),
4.14 (q, J ) 3.4 Hz, 1H), 3.96 (ABqd, J ) 11.4, 3.6 Hz, 1H),
3.77 (ABqd, J ) 11.5, 3.1 Hz, 1H), 3.64 (s, 3H), 0.93 (s, 9H),
0.91 (s, 9H), 0.14 (s, 3H), 0.13 (s, 3H), 0.09 (s, 3H), 0.08 (s,
3H); 13C NMR (125 MHz, CDCl3) δ 170.1, 155.3, 152.9, 149.7,
139.7, 120.1, 86.7, 85.1, 82.1, 70.2, 67.6, 61.9, 51.2, 25.9, 25.7,
18.4, 18.1, -4.7, -4.9, -5.4, -5.5. FAB-HRMS [M + H]+ calcd
for C25H46N5O6Si2 568.2984, found 568.2986.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-hydroxy-
ethyl)adenosine (A4). Lithium aluminum hydride (0.99 g,
25 mmol) was added to a solution of A3 (5.67 g, 9.98 mmol) in
ether (45 mL) cooled to 0 °C. After 2 h, the solution was
quenched at 0 °C by the slow addition of saturated aqueous
NH4Cl (100 mL). After the solution was warmed to room
temperature, it was diluted with ether (100 mL). The aqueous
layer was extracted with ether (6 × 100 mL). The combined
organic extracts were washed with saturated aqueous NaCl
(200 mL), dried over MgSO4, and concentrated under vacuum
to produce 5.07 g (94%) of A4 as a colorless oil: Rf 0.61 (1:9
MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 8.29 (s, 1H), 8.19
(s, 1H), 6.18 (d, J ) 3.4 Hz, 1H), 6.04 (br s, 2H), 4.49 (dd, J )
5.4, 5.0 Hz, 1H), 4.32 (dd, J ) 4.6, 3.6 Hz, 1H), 4.12 (dd, J )
5.80, 2.8 Hz, 1H), 3.99 (ABqd, J ) 11.6, 3.2 Hz, 1H), 3.80-
3.70 (m, 5H), 3.65 (br s, 1H), 0.92 (s, 9H), 0.91 (s, 9H), 0.10 (s,
9H), 0.06 (s, 3H). 13C NMR (100 MHz, CDCl3) δ 155.5, 152.9,
149.1, 138.9, 119.9, 87.5, 84.6, 82.9, 72.6, 69.6, 61.7, 61.5, 26.0,
25.7, 18.4, 18.0, -4.6, -4.8, -5.4, -5.5. EI-HRMS M+ calcd
for C24H45N5O5Si2 539.2959, found 539.2953.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-azidoethyl)-
N6-isobutyryladenosine (A7). NaN3 (0.26 g, 4.0 mmol) was
added to a solution of A6 (0.55 g, 0.80 mmol) in DMF (8 mL),
and the mixture was heated at 50 °C for 3 h. The solvents
were removed under vacuum, and the resulting pale orange
residue was partitioned between CH2Cl2 (100 mL) and water
(100 mL). The aqueous layer was extracted with CH2Cl2 (2 ×
50 mL). The combined organic extracts were washed with
saturated aqueous NaCl (50 mL), dried over MgSO4, and
concentrated under vacuum to produce 0.49 g (97%) of A7 as
a colorless oil: Rf 0.78 (1:1 EtOAc/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 8.71 (s, 1H), 8.45 (br s, 1H) 8.38 (s, 1H), 6.20 (d, J )
4.0 Hz, 1H), 4.49 (dd, J ) 5.3, 4.9 Hz, 1H), 4.37 (dd, J ) 4.4,
3.8 Hz, 1H), 4.16, (ddd, J ) 5.9, 3.3, 2.9 Hz, 1H), 4.03 (ABqd,
J ) 11.5, 3.5 Hz, 1H), 3.84-3.78 (m, 3H), 3.45 (ABqt, J ) 13.6,
5.1 Hz, 1H), 3.37 (ABQt, J ) 13.4, 4.8 Hz, 1H), 3.21 (br septet,
J ) 6.7 Hz, 1H), 1.31 (d, J ) 6.8 Hz, 6H), 0.94 (s, 9H), 0.93 (s,
9H), 0.11 (s, 6H,), 0.12 (s, 3H), 0.11 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 176.2, 152.4, 150.7, 149.3, 141.6, 122.6, 87.1,
84.6, 82.2, 69.6, 69.6, 61.4, 50.6, 35.8, 25.8, 25.5, 19.0, 18.2,
17.9, -4.8, -5.1, -5.6. FAB-HRMS [M + H]+ calcd C28H51N8O5-
Si2 635.3520, found 635.3523.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-hydroxy-
ethyl)-N6-isobutyryladenosine (A5). Trimethylsilyl chloride
(3.0 mL, 23.6 mmol) was added to a solution of A4 (2.41 g,
4.46 mmol) in pyridine (20 mL) resulting in a cloudy mixture.
After 1 h, isobutyric anhydride (3.0 mL, 18.1 mmol) was added.
After an additional 5 h, the mixture was cooled to 0 °C, and
water (4 mL) was added. After an additional 30 min, concen-
trated NH4OH (4 mL) was added, and the sample was
concentrated under vacuum. The resulting paste was parti-
tioned between CH2Cl2 (100 mL) and water (100 mL). The
aqueous layer was extracted with CH2Cl2 (3 × 100 mL). The
combined organic extracts were washed with saturated aque-
ous NaHCO3 (50 mL) and saturated aqueous NaCl (50 mL),
dried over MgSO4, and concentrated under vacuum. The
residual colorless viscous oil was purified via chromatography
with 40-80% EtOAc in CH2Cl2 as the eluant to yield 2.44 g
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-(trifluoro-
acetamido)ethyl)-N6-isobutyryladenosine (A8). Pd/C (10%,
0.20 g, 0.19 mmol) was added to a solution of A7 (0.48 g, 0.76
mmol) in MeOH (9 mL). The flask was evacuated and placed
under an atmosphere of hydrogen. The consumption of A7 was
monitored by TLC. After 1.5 h, ethyl trifluoroacetate (1.0 mL,
8.4 mmol) and Et3N (0.7 mL, 5.0 mmol) were added. After 4
h, silica gel (3 g) was added for dry loading, and the solvents
were removed under vacuum. The sample was purified via
chromatography with 1:4 EtOAc/CH2Cl2 as the eluant to
produce 0.44 g (81%) of A8 as a pale yellow oily solid: Rf 0.75
1
(1:1 EtOAc/CH2Cl2); H NMR (500 MHz, CDCl3) δ 9.17 (br s,
1H), 8.62 (s, 1H), 8.35 (s, 1H), 7.38 (br t, J ) 4.8 Hz, 1H), 6.12
(d, J ) 3.6 Hz, 1H), 4.48 (dd, J ) 5.3, 4.9 Hz, 1H), 4.28 (dd,
J ) 4.4, 3.9 Hz, 1H), 4.08 (ddd, J ) 5.3, 2.7, 2.6 Hz, 1H), 3.94
(ABqd, J ) 11.6, 3.1 Hz, 1H), 3.80-3.75 (m, 1H), 3.73 (ABqd,
J ) 11.7, 2.5 Hz, 1H), 3.72-3.68 (m, 1H), 3.60-3.50 (m, 1H),
3.50-3.40 (m, 1H), 3.20 (m, 1H), 1.23 (d, J ) 6.9 Hz, 3H), 1.22
(d, J ) 6.8 Hz, 3H), 0.86 (s, 9H), 0.84 (s, 9H), 0.06 (s, 3H),
0.04 (s, 3H), 0.04 (s, 3H), 0.03 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 176.4, 157.2, 152.3, 150.6, 149.4, 141.3, 122.5, 115.6,
87.3, 84.9, 82.4, 69.9, 68.1, 61.4, 39.6, 35.8, 25.8, 25.4, 19.0,
18.2, 17.8, -4.9, -5.1, -5.6, -5.7; 19F NMR (470 MHz, CDCl3)
δ -76.2. FAB-HRMS [M + H]+ calcd for C30H52N6O6F3Si2
705.3439, found 704.3437.
1
(90%) of A5 as a white foam: Rf 0.39 (4:1 EtOAc/CH2Cl2); H
NMR (500 MHz, CDCl3) δ 8.70 (s, 1H), 8.43 (br s, 1H), 8.37 (s,
1H), 6.22 (d, J ) 3.5 Hz, 1H), 4.51 (dd, J ) 5.6, 5 Hz, 1H),
4.34 (dd, J ) 4.3, 3.3 Hz, 1H), 4.15 (dt, J ) 4.5, 2.5 Hz, 1H),
4.02 (ABqd, J ) 11.6, 2.8 Hz, 1H), 3.81-3.74 (m, 5H), 3.24
(septet, J ) 6.7 Hz, 1H), 2.55 (br s, 1H), 1.32 (d, J ) 6.7 Hz,
3H), 1.30 (d, J ) 6.7 Hz, 3H), 0.93 (s, 9H), 0.92 (s, 9H), 0.12
(s, 6H), 0.11 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 176.3, 152.7,
150.4, 149.7, 141.0, 121.8, 87.6, 84.7, 83.0, 72.4, 69.3, 61.7, 61.3,
36.0, 26.0, 25.6, 19.2, 18.8, 18.4, 18.0, -4.7, -4.9, -5.4, -5.6.
EI-HRMS M+ calcd for C28H51N5O6Si2 609.3377, found.
609.3376.
2′-O-(2-(Trifluoroacetamido)ethyl)-N6-isobutyrylade-
nosine (A9). TBAF (1 M) in THF (1.4 mL, 1.36 mmol) was
added dropwise over 5 min to a solution of A8 (0.440 g, 0.62
mmol) in THF (7 mL) cooled to 0 °C. The solution was warmed
to room temperature. After 3 h, silica gel (2 g) was added for
dry loading, and the solvents were removed under vacuum.
The sample was purified via chromatography with 0-6%
MeOH in CH2Cl2 as the eluant to produce 0.253 g (86%) of A9
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-methane-
sulfonylethyl)-N6-isobutyryladenosine (A6). Et3N (0.7 mL,
5 mmol) was added to a solution of A5 (0.61 g, 1.0 mmol) in
CH2Cl2 (10 mL) cooled to 0 °C and followed by the addition of
methanesulfonyl chloride (0.23 mL, 3.0 mmol). After 1 h, the
solution was diluted with CH2Cl2 (50 mL), and washed with
saturated aqueous NaHCO3 (50 mL). The aqueous layer was
extracted with CH2Cl2 (3 × 25 mL), and the combined organic
extracts were washed with saturated aqueous NaCl (50 mL),
1
as a clear colorless oil: Rf 0.17 (1:1 EtOAc/CH2Cl2); H NMR
(500 MHz, CDCl3) δ 8.69 (s, 1H), 8.65 (br s, 1H), 8.12 (s, 1H),
4294 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
6.00 (d, J ) 6.9 Hz, 1H), 5.87 (br s, 1H), 4.80 (dd, J ) 6.9, 4.7
Hz, 1H), 4.64 (br d, J ) 3.8 Hz, 1H), 4.36 (d, J ) 1.6 Hz, 1H),
3.99 (d, J ) 12.9 Hz, 1H), 3.80 (d, J ) 12.5 Hz, 1H), 3.70-
3.63 (m, 2H), 3.60-3.54 (m, 1H), 3.47-3.40 (m, 1H), 3.26
(septet, J ) 6.1 Hz, 1H), 1.32 (d, J ) 6.8 Hz, 3H), 1.31 (d, J )
6.8 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 177.3, 157.7, 151.9,
150.3, 149.8, 143.1, 123.3, 115.8, 89.1, 87.7, 81.7, 70.1, 68.5,
62.4, 39.9, 35.9, 19.0; 19F NMR (470 MHz, CDCl3) δ -76.1.
FAB-HRMS [M + H]+ calcd for C18H24N6O6F3 477.1709, found
477.1707.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)-N6-isobutyryladenosine (A10). 4,4′-Dimethoxytrityl
chloride (0.19 g, 0.57 mmol) was added to a solution of A9
(0.249 g, 0.52 mmol) in pyridine (5 mL) cooled to 0 °C. The
resulting solution was warmed to room temperature and
stirred for 24 h, and the solvents were removed under vacuum.
The residual yellow gum was purified via chromatography with
the column packed as described for U8. The column was eluted
with a gradient of 0-6% MeOH in CH2Cl2 to yield 0.292 g
(71%) of A10 as a yellow foam: Rf 0.35 (1:9 MeOH/CH2Cl2);
1H NMR (500 MHz, CDCl3) δ 8.88 (br s, 1H), 8.60 (s, 1H), 8.22
(s, 1H), 7.37-7.15 (m, 9H), 6.75 (d, J ) 8.9 Hz, 4H), 6.13 (d,
J ) 2.6 Hz, 1H), 4.58-4.55 (m, 2H), 4.25 (br q, J ) 4.0 Hz,
1H), 3.92 (ABqdd, J ) 10.0, 5.0, 3.5 Hz, 1H), 3.81 (ABqdd,
J ) 11.0, 7.5, 3.5 Hz, 1H), 3.72 (s, 6H), 3.54 (m, 1H), 3.51 (dd,
J ) 11.0, 3.5 Hz, 1H), 3.48 (m, 1H), 3.37 (dd, J ) 10.5, 4.0 Hz,
1H), 3.18 (septet, J ) 7.5 Hz, 1H), 1.24 (d, J ) 6.8 Hz, 6H);
13C NMR (125 MHz, CDCl3) δ 176.7, 158.8, 157.8, 152.6, 151.1,
149.6, 144.7, 141.9, 135.9, 135.8, 130.3, 130.2, 128.4, 128.1,
127.2, 123.0, 116.2, 113.4, 88.2, 86.7, 84.0, 82.2, 69.9, 69.0, 63.1,
55.4, 40.2, 36.3, 19.4; 19F NMR (470 MHz, CDCl3) δ -76.1.
ESI-HRMS [M + H]+ calcd for C39H42N6O8F3 779.3016, found
779.3013.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)-N6-isobutyryladenosine-3′-O-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (A11). A portion of A10
(0.214 g, 0.30 mmol) was coevaporated with pyridine (5 mL)
and dissolved in CH2Cl2 (6 mL). The solution was cooled to 0
°C, and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodi-
amidite (220 µL, 0.69 mmol) and 4,5-dicyanoimidazole (40 mg,
0.34 mmol) were added. The cloudy mixture was warmed to
room temperature, stirred for 4 h, and diluted with CH2Cl2
(50 mL) and saturated aqueous NaHCO3 (50 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 25 mL). The combined
organic extracts were washed with saturated aqueous NaCl
(25 mL), dried over Na2SO4, and concentrated under vacuum.
The residual colorless viscous oil was purified via chromatog-
raphy on Et3N-washed silica gel with 1:6:3 to 1:3:6 Et3N/
hexanes/CH2Cl2 as the eluant to yield 0.198 g (73%) of A11 as
a yellow gum. TLC revealed the presence of both diastereo-
mers: Rf 0.48 and 0.38 (1:3:6 Et3N/hexanes/CH2Cl2). NMR
spectroscopy also revealed the presence of both diastereo-
mers: 1H NMR (500 MHz, acetone-d6) δ 9.31 (br s, 1H), 8.54
and 8.53 (each s, total 1H), 8.46 and 8.45 (each s, total 1H),
7.47-7.21 (m, 9H), 6.87-6.81 (m, 4H), 6.25 and 6.24 (each d,
J ) 4.5 Hz, total 1H), 5.08-5.05 (m, total 1H), 4.94 and 4.88
(each dt, J ) 10.5, 5.0 Hz, total 1H), 4.41 and 4.36 (each td,
J ) 4.5, 3.5 Hz, total 1H), 3.93 (m, total 1H), 3.84 (m, total
1H), 3.78 and 3.77 (each s, total 3H), 3.75-3.73 (m, total 1H),
3.72-3.67 (m, total 2H), 3.55-3.53 (m, total 3H), 3.44-3.40
(m, total 2H), 2.89 and 2.86 (br s, total 1H), 2.82-2.79 (m,
total 1H), 2.62 (t, J ) 6.0 Hz, total 2H), 1.24-1.12 (m, 18H);
13C NMR (125 MHz, acetone-d6) δ 175.8, 158.97, 158.95, 152.1,
151.6, 150.1, 145.3, 143.1, 142.9, 136.08, 136.02, 135.98,
130.39, 130.32, 130.29, 128.40, 128.33, 127.9, 126.9, 123.5,
118.2, 113.2, 88.1, 87.8, 86.4, 83.5, 80.47, 80.44, 71.63, 71.53,
68.63, 68.49, 63.3, 63.1, 58.82, 58.66, 54.8, 43.33, 43.24, 39.73,
39.61, 35.2, 24.41, 24.35, 24.22, 24.16, 20.04, 19.98, 18.9; 19F
NMR (470 MHz, acetone-d6) δ -76.77 and -76.80; 31P NMR
(202 MHz, acetone-d6) δ 150.7 (just one peak observed). FAB-
HRMS [M + Na]+ calcd for C48H58N8O9PNaF3 1001.3914, found
1001.3914.
3′,5′-Bis-O-(tert-butyldimethylsilyl)-2′-O-(2-(triphenyl-
methylthio)ethyl)-N6-isobutyryladenosine (A12). Diethyl
azodicarboxylate (DEAD, 1.13 mL, 7.1 mmol) was added to a
solution of triphenylphosphine (1.86 g, 7.1 mmol) in THF (30
mL) cooled to 0 °C. The DEAD was prepared as described;41
the use of commercially available diisopropyl azodicarboxylate
(see procedures for U11 and G12) was not tested. After 1 h, a
solution of A5 (2.88 g, 4.72 mmol) and trityl mercaptan (1.95
g, 7.1 mmol) in THF (20 mL) was added through a cannula;
the flask containing the latter reagents was rinsed with THF
(6 × 5 mL). After 1 h, the solution was quenched with MeOH
(10 mL) and concentrated under vacuum. The resulting oil was
purified via chromatography with 10-40% EtOAc in hexanes
as the eluant to produce 3.29 g (80%) of A12 as an off-white
foam: Rf 0.38 (1:2 EtOAc/hexanes); 1H NMR (500 MHz, CDCl3)
δ 8.68 (s, 1H), 8.63 (br s, 1H), 8.31 (s, 1H), 7.37-7.19 (m, 15H),
6.07 (d, J ) 3.8 Hz, 1H), 4.44 (dd, J ) 5.1, 4.9 Hz, 1H), 4.18
(dd, J ) 4.4, 4.1 Hz, 1H), 4.10 (ddd, J ) 5.1, 3.4, 2.7 Hz, 1H),
3.99 (ABqd, J ) 11.5, 3.9 Hz, 1H), 3.77 (ABqd, J ) 11.5, 2.7
Hz, 1H), 3.47 (dt, J ) 9.8, 6.8 Hz, 1H), 3.34 (dt, J ) 9.9, 6.8
Hz, 1H), 3.23 (m, 1H), 2.42 (t, J ) 6.9 Hz, 2H), 1.33 (d, J )
6.8 Hz, 3H), 1.32 (d, J ) 6.8 Hz, 3H), 0.93 (s, 9H), 0.91 (s,
9H), 0.11 (s, 6H), 0.10 (s, 3H), 0.06 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 176.0, 152.5, 150.8, 149.2, 144.6, 141.6, 129.5, 127.8,
126.6, 122.4, 87.0, 85.0, 82.0, 69.6, 69.4, 66.6, 61.7, 36.1, 31.6,
26.0, 25.7, 19.2, 19.2, 18.4, 18.0, -4.6, -4.9, -5.5. EI-HRMS
M+ calcd for C47H65N5O5SSi2 867.4244, found 867.4242.
2′-O-(2-(Triphenylmethylthio)ethyl)-N6-isobutyryl ad-
enosine (A13). TBAF (1 M) in THF (8.4 mL, 8.4 mmol)
dropwise over 10 min was added to a solution of A12 (3.29 g,
3.8 mmol) in THF (40 mL) cooled to 0 °C. After 2 h, the solvents
were removed under vacuum. The resulting oil was purified
via chromatography with 0-5% MeOH in CH2Cl2 as the eluant
to yield 2.08 g (85%) of A13 as a white foam: Rf 0.23 (1:19
MeOH/CH2Cl2); 1H NMR (500 MHz, CDCl3) δ 8.68 (s, 1H), 8.45
(br s, 1H), 7.99 (s, 1H), 7.40-7.21 (m, 15H), 6.11 (dd, J ) 12,
2 Hz, 1H), 5.84 (d, J ) 7.5 Hz, 1H), 4.59 (dd, J ) 7.5, 4.4 Hz,
1H), 4.35 (d, J ) 3.5 Hz, 1H), 4.34 (br s, 1H), 3.96 (td, J )
12.5, 2.5 Hz, 1H), 3.76 (dt, J ) 12.5, 1.5 Hz, 1H), 3.26 (septet,
J ) 6.5 Hz, 1H), 3.19 (dt, J ) 10.0, 5.0 Hz, 1H), 3.10 (br s,
1H), 2.86 (ddd, J ) 9.7, 8.6, 4.6 Hz, 1H), 2.54 (ddd, J ) 13.6,
8.4, 4.9 Hz, 1H), 2.32 (dt, J ) 13.8, 4.4 Hz, 1H), 1.33 (d, J )
6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ 176.3, 151.9, 150.2,
149.9, 144.2, 143.0, 129.3, 128.0, 126.8, 123.4, 89.4, 88.0, 81.0,
70.4, 69.2, 67.0, 63.1, 36.0, 31.8, 19.2, 19.1. FAB-HRMS M+
calcd for C35H38N5O5S 640.2593, found 640.2596.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)-N6-isobutyryladenosine (A14). Et3N (1 mL) and
4,4′-dimethoxytrityl chloride (0.58 g, 1.68 mmol) were added
to a solution of A13 (0.98 g, 1.53 mmol) in CH2Cl2 (15 mL)
cooled to 0 °C. The solution was warmed to room temperature,
stirred for 24 h, and quenched with MeOH (2 mL). The sample
was purified via chromatography using the dry loading
technique (4 g of silica gel) with the column packed as
described for U8 (the sample was loaded in 10:9 hexanes/CH2-
Cl2). The column was eluted with a gradient of Et3N/hexanes/
CH2Cl2 from 1:10:9 to 1:1:18 to produce 0.905 g (63%) of A14
as a yellow foam: Rf 0.17 (1:10:9 Et3N/hexanes/CH2Cl2); 1H
NMR (500 MHz, CDCl3) δ 8.58 (s, 1H), 8.35 (br s, 1H), 8.10 (s,
1H), 7.42-7.16 (m, 24H), 6.81-6.80 (m, 4H), 6.04 (d, J ) 4.3
Hz, 1H), 4.43 (t, J ) 4.5 Hz, 1H), 4.36 (q, J ) 5.0 Hz, 1H),
4.20 (q, J ) 4.2 Hz, 1H), 3.78 (s, 6H), 3.55 (ABqt, J ) 10.5, 5.0
Hz, 1H), 3.49 (ABqd, J ) 10.6, 3.5 Hz, 1H), 3.38 (ABqd, J )
10.7, 4.4 Hz, 1H), 3.18 (br septet, J ) 6.0 Hz, 1H), 3.12 (ABqdd,
J ) 10.1, 7.9, 5.3 Hz, 1H), 2.87 (br d, J ) 5.5 Hz, 1H), 2.57 (m,
1H), 2.42 (ABqt, J ) 13.5, 5.5 Hz, 1H), 1.31 (d, J ) 6.9 Hz,
3H), 1.30 (d, J ) 6.9 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
175.9, 158.6, 152.5, 151.0, 149.2, 144.5, 144.4, 141.5, 135.6,
135.5, 130.0, 129.5, 128.1, 128.0, 127.9, 127.0, 126.8, 122.6,
(41) Simionescu, C. I.; Comanita, E.; Pastravanu, M.; Comanita, B.
Acta Chim. Hung. 1990, 127, 41-44.
J. Org. Chem, Vol. 70, No. 11, 2005 4295

Jin et al.
113.2, 86.8, 86.6, 84.0, 81.4, 69.6, 69.5, 67.0, 62.9, 55.2, 36.1,
31.9, 19.1. FAB-HRMS [M + Na]+ calcd for C56H55N5O7NaS
964.3720, found 964.3718.
6.45 (br s, 2H), 5.80 (d, J ) 6.4 Hz, 1H), 5.08-5.04 (m, 2H),
4.37 (s, 1H), 4.35 (m, 1H), 4.23 (dd, J ) 4.5, 3.0 Hz, 1H), 3.88
(q, J ) 3.2 Hz, 1H), 3.72 (m, 1H), 3.62-3.55 (m, 2H), 3.55-
3.44 (m, 4H); 13C NMR (100 MHz, DMSO-d6) δ 156.9, 154.2,
151.5, 138.2, 135.6, 128.3, 127.6, 127.5, 116.7, 86.0, 84.6, 81.5,
72.1, 69.2, 69.1, 69.0, 61.5. ESI-HRMS [M + H]+ calcd for
C19H24N5O6 418.1727, found 418.1735.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)-N6-isobutyryladenosine-3′-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (A15). A portion of A14
(0.116 g, 0.12 mmol) was coevaporated with pyridine (3 mL)
and dissolved in CH2Cl2 (3.4 mL). The solution was cooled to
0 °C, and 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphorodi-
amidite (50 µL, 0.16 mmol) and 4,5-dicyanoimidazole (19 mg,
0.16 mmol) were added. The cloudy mixture was warmed to
room temperature, stirred for 4 h, and diluted with CH2Cl2
(50 mL) and saturated aqueous NaHCO3 (50 mL). The aqueous
layer was extracted with CH2Cl2 (2 × 25 mL). The combined
organic extracts were washed with saturated aqueous NaCl
(25 mL), dried over Na2SO4, and concentrated under vacuum.
The crude product was purified via chromatography with the
column packed as described for U8 (the sample was loaded in
8:1 hexanes/CH2Cl2). The column was eluted with 1:8:1 to 1:5:4
Et3N/hexanes/CH2Cl2 to produce 0.102 g (73%) of A15 as a
white foam: Rf 0.46 (1:5:4 Et3N/hexanes/CH2Cl2). NMR spec-
troscopy revealed the presence of both diastereomers: 1H NMR
(400 MHz, CDCl3) δ 8.59 and 8.56 (each s, total 1H), 8.29 and
8.28 (each br s, total 1H), 8.14 and 8.09 (each s, total 1H),
7.42-7.15 (m, 24H), 6.81-6.77 (m, 4H), 6.04 and 6.01 (each
d, J ) 5.6 and 4.8 Hz, total 1H), 4.67 and 4.56 (each t, J ) 4.2
and 4.8 Hz, total 1H), 4.53-4.43 (m, 1H), 4.36 and 4.30 (each
q, J ) 4.0 Hz, total 1H), 3.77 and 3.76 (each s, total 6H), 3.70-
3.48 (m, 4H), 3.34-3.21 (m, 3H), 2.57-2.33 (m, 6H), 1.31 (d,
J ) 6.8 Hz, 6H), 1.15 (d, J ) 6.8 Hz, 3H), 1.16 (d, J ) 6.8 Hz,
3H), 1.11 (d, J ) 6.8 Hz, 3H), 1.02 (d, J ) 6.8 Hz, 3H); 13C
NMR (125 MHz, CDCl3) δ 175.9, 158.5, 158.5, 152.4, 151.1,
151.0, 149.2, 149.2, 144.6, 144.5, 144.5, 144.4, 144.4, 142.0,
141.8, 135.6, 135.5, 130.1, 130.0, 129.5, 129.5, 128.3, 128.1,
127.9, 127.8, 126.7, 126.6, 122.6, 117.7, 117.3, 113.1, 87.2, 87.0,
86.6, 86.5, 83.6, 80.7, 80.3, 71.1, 71.0, 70.6, 69.6, 69.3, 66.6,
62.9, 62.4, 58.8, 58.7, 57.9, 57.8, 55.2, 55.1, 52.6, 46.8, 43.3,
43.2, 43.1, 43.0, 36.0, 31.7, 31.6, 24.7, 24.6, 24.5, 24.5, 20.8,
20.3, 20.2, 20.1, 20.0, 19.0; 31P NMR (162 MHz, CDCl3) δ 151.5
and 150.9. FAB-HRMS [M + H]+ calcd for C65H73N7O8PS
1142.4979, found 1142.4982.
3′,5′,O6-Tris(tert-butyldimethylsilyl)-2′-O-(2-benzyloxy-
ethyl)guanosine (G4). tert-Butyldimethylsilyl chloride (0.72
g, 4.8 mmol) and imidazole (0.55 g, 7.9 mmol) were added to
a solution of G3 (0.53 g, 1.3 mmol) in DMF (15 mL). The
solution was stirred at room temperature for 12 h, diluted with
CH2Cl2 (15 mL), and quenched with MeOH (1 mL) and water
(20 mL). The aqueous layer was extracted with CH2Cl2 (3 ×
20 mL). The combined organic extracts were washed with
saturated aqueous NH4Cl (20 mL) and saturated aqueous
NaCl (20 mL) and dried over MgSO4. The solvents were
removed under vacuum to produce 0.78 g (73%) of G4 as a
white powder: Rf 0.55 (1:9 MeOH/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 7.83 (s, 1H), 7.30-7.24 (m, 5H), 6.36 (br s, 2H), 5.97
(d, J ) 4.0 Hz, 1H), 4.52 (s, 2H), 4.48 (t, J ) 4.8 Hz, 1H), 4.24
(t, J ) 4.0 Hz, 1H), 4.08 (dt, J ) 5.6, 2.9 Hz, 1H), 3.93 (ABqd,
J ) 11.4, 3.3 Hz, 1H), 3.81-3.74 (m, 3H), 3.61 (t, J ) 4.0 Hz,
2H), 0.92 (s, 18H), 0.91 (s, 9H), 0.11 (s, 3H), 0.10 (s, 6H), 0.09
(s, 3H), 0.08 (s, 6H); 13C NMR (100 MHz, CDCl3) δ 159.4, 153.7,
151.5, 138.3, 136.2, 128.6, 127.9, 127.8, 117.7, 86.7, 84.8, 82.8,
73.4, 70.4, 70.2, 69.6, 62.1, 26.3, 26.0, 25.9, 18.7, 18.4, -4.3,
-4.6, -5.1, -5.2. ESI-HRMS [M + H]+ calcd for C37H66N5O6-
Si3 760.4321, found 760.4298.
3′,5′-Bis(tert-butyldimethylsilyl)-2′-O-(2-benzyloxyethyl)-
N2-isobutyrylguanosine (G5). Isobutyryl chloride (0.50 mL,
5.0 mmol) was added to a solution of G4 (0.78 g, 1.03 mmol)
in pyridine (5 mL) cooled to 0 °C. After the mixture was stirred
for 45 min, the colorless solution was quenched with saturated
aqueous NaHCO3 (25 mL) and extracted with EtOAc (2 × 25
mL). The combined organic extracts were dried over Na2SO4
and concentrated under vacuum. The residual yellow foam was
purified via chromatography with 1-5% MeOH in CH2Cl2 as
the eluant to yield 0.73 g (98%) of G5 as a white foam: Rf
1
0.75 (1:19 MeOH/CH2Cl2); H NMR (500 MHz, CDCl3) δ 8.92
(br s, 1H), 8.05 (s, 1H), 7.29 (s, 1H), 7.28-7.24 (m, 5H), 5.98
(d, J ) 4.0 Hz, 1H), 4.52 (ABq, J ) 12.4 Hz, 1H), 4.43 (ABq,
J ) 12.0 Hz, 1H), 4.42 (d, J ) 3.0 Hz, 1H), 4.10 (t, J ) 4.0 Hz,
1H), 4.07 (dt, J ) 5.5, 3.0 Hz, 1H), 3.91 (ABqd, J ) 11.6, 2.8
Hz, 1H), 3.76 (ABqd, J ) 11.6, 3.0 Hz, 1H), 3.74 (br q, J ) 3.0
Hz, 2H), 3.55 (t, J ) 5.0 Hz, 2H), 2.52 (septet, J ) 7.0 Hz,
1H), 1.16 (d, J ) 7.0 Hz, 3H), 1.14 (d, J ) 6.5 Hz, 3H), 0.91 (s,
9H), 0.90 (s, 9H), 0.10-0.09 (m, 12H); 13C NMR (125 MHz,
CDCl3) δ 178.9, 155.9, 148.2, 147.8, 138.2, 137.2, 128.6, 127.9,
127.8, 121.6, 86.5, 85.3, 83.6, 73.5, 70.1, 69.7, 62.1, 36.5, 25.9,
25.6, 18.8, 18.8, 18.3, 18.0, -4.7, -4.9, -5.4, -5.5. ESI-HRMS
[M + H]+ calcd for C35H58N5O7Si2 716.3875, found 716.3860.
2′-O-(2-Benzyloxyethyl)-2-aminoadenosine (G2). A 60%
dispersion of sodium hydride in mineral oil (0.27 g, 6.6 mmol)
was added to a suspension of 2,6-diaminopurine riboside (1.56
g, 6.0 mmol) in DMF (20 mL). After the mixture was stirred
for 1 h, benzyl 2-bromoethyl ether (0.94 mL, 6.0 mmol) was
added dropwise over 5 min, and the pale yellow mixture was
stirred for 18 h. The solvents were removed under vacuum,
and the residual yellow solid was purified via chromatography
with 5-30% MeOH in CH2Cl2 as the eluant to produce 0.81 g
1
(33%) of G2 as a white solid: Rf 0.65 (1:9 MeOH/CH2Cl2); H
NMR (500 MHz, DMSO-d6) δ 7.94 (s, 1H), 7.29-7.17 (m, 5H),
6.79 (br s, 2H), 5.84 (d, J ) 6.5 Hz, 1H), 5.74 (br s, 2H), 5.45
(dd, J ) 6.0, 5.0 Hz, 1H), 5.07 (dd, J ) 4.5, 1.5 Hz, 1H), 4.47
(dd, J ) 6.5, 5.5 Hz, 1H), 4.36 (s, 2H), 4.26 (m, 1H), 3.92 (m,
1H), 3.72 (ABqd, J ) 11.1, 14.1 Hz, 1H), 3.60-3.52 (m, 3H),
3.49 (t, 4.5 Hz, 2H); 13C NMR (125 MHz, DMSO-d6) δ 160.9,
156.9, 152.2, 138.9, 136.7, 128.9, 128.2, 128.0, 114.1, 86.8, 85.7,
81.6, 72.0, 69.8, 69.7, 69.5, 62.3. FAB-HRMS [M + H]+ calcd
for C19H25N6O5 417.1886, found 417.1888.
3′,5′-Bis(tert-butyldimethylsilyl)-2′-O-(2-hydroxyethyl)-
N2-isobutyrylguanosine (G6). Pd/C (10%, 1.0 g, 0.94 mmol)
was added to a solution of G5 (0.73 g, 1.02 mmol) in EtOH
(10 mL). The flask was evacuated and placed under an
atmosphere of hydrogen. The consumption of G5 was moni-
tored by TLC. After 12 h, 10 g of Celite was added, and the
mixture was filtered and concentrated under vacuum. The
residue was purified via chromatography with 0-5% MeOH
in CH2Cl2 as the eluant to produce 0.56 g (88%) of G6 as white
foam: Rf 0.45 (1:19 MeOH/CH2Cl2); 1H NMR (500 MHz, CDCl3)
δ 10.37 (br s, 1H), 8.04 (s, 1H), 6.01 (s, 1H), 4.38 (t, J ) 5.5
Hz, 1H), 4.07 (d, J ) 7.0 Hz, 1H), 4.05 (d, J ) 5.0 Hz, 1H),
3.91 (q, J ) 5.07 Hz, 1H), 3.75 (ABq, J ) 5.0 Hz, 1H), 3.79-
3.71 (br m, 6H), 2.75 (septet, J ) 6.5 Hz, 1H), 1.18 (d, J ) 7.0
Hz, 6H), 0.87 (s, 9H), 0.86 (s, 9H), 0.06 (s, 6H), 0.05 (s, 3H),
0.04 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 180.1, 156.3, 148.4,
147.9, 137.1, 121.3, 88.2, 84.4, 84.2, 72.7, 69.3, 61.7, 61.2, 36.3,
26.3, 25.6, 19.3, 19.2, 19.1, 19.0, 18.6, 18.2, -4.4, -4.8, -5.2,
-5.3. ESI-HRMS [M + H]+ calcd for C28H52N5O7Si2 626.3405,
found 626.3394.
2′-O-(2-Benzyloxyethyl)guanosine (G3). A solution of
adenosine deaminase type II (20 mg, 30 units) in 0.1 M Tris,
pH 7.5 (4 mL), 0.1 M sodium phosphate, pH 7.4 (0.2 mL), and
DMSO (1.0 mL) was added to a solution of G2 (0.65 g, 1.6
mmol) in 0.1 M Tris, pH 7.5 (6 mL), 0.1 M sodium phosphate,
pH 7.4 (0.4 mL), and DMSO (1.5 mL). The cloudy white
mixture was stirred at room temperature for 36 h, and the
solvents were removed under vacuum. The pale yellow viscous
oil was purified via chromatography with 5-15% MeOH in
CH2Cl2 as the eluant to yield 0.53 g (82%) of G3 as white
powder: Rf 0.35 (1:9 MeOH/CH2Cl2); 1H NMR (400 MHz,
DMSO-d6) δ 10.62 (br s, 1H), 7.95 (s, 1H), 7.27-7.17 (m, 5 H),
4296 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
3′,5′-Bis(tert-butyldimethylsilyl)-2′-O-(2-methanesul-
fonylethyl)-N2-isobutyrylguanosine (G7). A solution of G6
(0.50 g, 0.80 mmol) in CH2Cl2 (10 mL) was cooled to 0 °C.
Portions of Et3N (0.15 mL, 1.07 mmol) and methanesulfonyl
chloride (84 µL, 1.09 mmol) were added. After 1 h, the colorless
solution was diluted with CH2Cl2 (25 mL), washed with
saturated aqueous NaHCO3 (2 × 25 mL), dried over Na2SO4,
and concentrated under vacuum to produce 0.55 g (98%) of
G7 as a pale yellow foam: Rf 0.45 (1:24 MeOH/CH2Cl2); 1H
NMR (400 MHz, CDCl3) δ 12.18 (br s, 1H), 9.90 (br s, 1H),
8.06 (s, 1H), 5.90 (s, 1H), 4.38-4.31 (m, 4H), 3.99 (t, J ) 8.8
Hz, 1H), 3.94 (m, 1H), 3.90 (d, J ) 4.8 Hz, 1H), 3.73 (d, J )
11.2 Hz, 1H), 3.07 (s, 3H), 2.67 (septet, J ) 6.8 Hz, 1H), 1.14
(d, J ) 6.8 Hz, 3H), 1.12 (d, J ) 6.8 Hz, 3H), 0.87 (s, 9H), 0.82
was added. After the solution was stirred at room temperature
for 18 h, the solvents were removed under vacuum. The
residual yellow foam was purified via chromatography with
the column packed as described for U9. The column was eluted
with a gradient of 0-5% MeOH in CH2Cl2 to produce 0.240 g
(83%) of G10 as a white foam: Rf 0.45 (1:19 MeOH/CH2Cl2);
1H NMR (500 MHz, acetone-d6) δ 10.68 (br s, 1H), 8.91 (br s,
1H), 8.01 (s, 1H), 7.45-7.17 (m, 9H), 6.81 (m, 4H), 6.03 (d,
J ) 3.0 Hz, 1H), 4.70 (t, J ) 11.0 Hz, 2H), 4.61 (t, J ) 7.5 Hz,
1H), 4.21 (m, 1H), 3.98 (m, 2H), 3.74(s, 6H), 3.60 (m, 2H), 3.46-
3.37 (m, 2H), 3.20 (q, J ) 7.0 Hz, 1H), 2.88 (septet, J ) 7.0
Hz, 1H), 1.22 (d, J ) 4.0 Hz, 3H), 1.20 (d, J ) 3.5 Hz, 3H); 13
C
NMR (125 MHz, acetone-d6) δ 180.3, 159.0, 158.9, 155.4, 148.7,
148.5, 145.4, 137.7, 136.1, 136.0, 130.3, 128.4, 128.0, 127.0,
121.3, 113.2, 87.3, 86.3, 83.8, 82.3, 70.1, 68.8, 63.9, 54.8, 46.4,
40.0, 35.9, 29.7, 18.7, 18.6; 19F NMR (470 MHz, acetone-d6) δ
-76.7. ESI-HRMS [M + H]+ calcd for C39H42N6O9F3 795.2965,
found 795.2978.
(s, 9H), 0.08 (s, 3H), 0.06 (s, 3H), 0.02 (s, 3H), 0.01 (s, 3H); 13
C
NMR (100 MHz, CDCl3) δ 180.2, 156.0, 148.4, 147.8, 136.8,
121.6, 88.1, 84.4, 83.7, 69.7, 69.5, 68.8, 60.9, 51.0, 37.7, 36.3,
26.3, 25.8, 19.2, 19.1, 18.7, 18.2, -4.4, -4.7-5.1, -5.2. ESI-
HRMS [M + H]+ calcd for C29H54N5O9Si2S 704.3181, found
704.3171.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)-N2-isobutyrylguanosine-3′-O-(2-cyanoethyl-N,N-
diisopropyl)phosphoramidite (G11). A portion of G10
(0.160 g, 0.20 mmol) was coevaporated with pyridine (2 × 5
mL) and dissolved in CH2Cl2 (2 mL). The solution was cooled
to 0 °C, and 2-cyanoethyl N,N,N′,N′-tetraisopropylphospho-
rodiamidite (87 µL, 0.27 mmol) and 4,5-dicyanoimidazole (27
mg, 0.24 mmol) were added. The colorless solution was warmed
to room temperature, stirred for 6 h, diluted with 5% Et3N in
EtOAc (25 mL), and washed with saturated aqueous NaHCO3
(2 × 25 mL). The organic layer was dried over Na2SO4 and
concentrated under vacuum. The residual pale yellow solid was
purified by chromatography with the column packed as
described for U9 (the sample was loaded in 4:15 hexanes/CH2-
Cl2). The column was eluted with a gradient from 1:4:15 Et3N/
hexanes/CH2Cl2 to 1:19 Et3N/CH2Cl2 to produce 0.160 g (80%)
of G11 as a white foam: Rf 0.46 (1:19 Et3N/CH2Cl2). NMR
spectroscopy revealed the presence of both diastereomers: 1H
NMR (500 MHz, acetone-d6) δ 8.87 (br s, 1H), 7.99 and 7.94
(each s, total 1H), 7.50-7.23 (m, 9H), 6.87 (m, 4H), 6.01 and
6.00 (each d, J ) 4.5 Hz, total 1H), 4.73 (dt, J ) 8.5 Hz, 5.0
Hz, 1H), 4.66 and 4.59 (each dt, J ) 9.5 Hz, 5.5 Hz, total 1H),
4.38 and 4.37 (each m, total 1H), 4.04 (m, 1H), 3.98 (m, 2H),
3.90 (m, 1H), 3.78 and 3.77 (each s, total 6H), 3.74-3.40 (m,
8H), 2.86 (septet, J ) 7.0 Hz, 1H), 2.79 (t, J ) 10.5 Hz, 1H),
2.59 (m, 1H), 1.18 (m, 16H), 1.08 (s, 1H), 1.07 (s, 1H); 13C NMR
(125 MHz, acetone-d6) δ 180.2, 159.0, 155.2, 148.7, 148.5, 145.3,
145.2, 136.9, 136.8, 136.0, 135.9, 130.3, 128.4, 128.3, 127.0,
121.6, 118.6, 118.3, 113.3, 87.5, 86.5, 83.5, 83.2, 81.5, 81.2, 71.4,
71.3, 68.6, 68.4, 63.5, 63.2, 59.1, 59.0, 58.5, 58.4, 54.9, 52.8,
43.3, 43.2, 39.8, 35.8, 24.5, 20.0, 18.6; 19F NMR (470 MHz,
acetone-d6) δ -76.7 (just one peak observed); 31P NMR (202
MHz, acetone-d6) δ 151.2 and 150.6. ESI-HRMS [M + H]+ calcd
for C48H59N8O10F3P 995.4044, found 995.4040.
2′-O-(2-(Triphenylmethylthio)ethyl)-N2-isobutyrylgua-
nosine (G12). Diisopropyl azodicarboxylate (123 µL, 0.64
mmol) was added to a solution of triphenylphosphine (0.124
g, 0.50 mmol) in THF (4 mL) cooled to 0 °C. After 30 min, a
solution of G6 (0.153 g, 0.24 mmol) and trityl mercaptan (0.178
g, 0.62 mmol) in THF (2 mL) was added via cannula; the flask
containing the latter reagents was rinsed with THF (2 × 2
mL). After 30 min, 1 M TBAF in THF (0.48 mL, 0.48 mmol)
was added to the solution. After 3 h, the solvents were removed
under vacuum, and the residue was purified via chromatog-
raphy with 0-2% MeOH in CH2Cl2 as the eluant to yield 0.156
g (97%) of G12 as a white foam: Rf 0.33 (1:19 MeOH/CH2Cl2);
1H NMR (500 MHz, CDCl3) δ 9.69 (br s, 1H), 8.12 (s, 1H), 7.31-
7.13 (m, 15H), 5.86 (d, J ) 6.0 Hz, 1H), 5.23 (br s, 1H), 4.43
(br s, 1H), 4.39 (q, J ) 5.0 Hz, 1H), 4.17 (br s, 1H), 3.95 (br s,
1H), 3.88 (ABq, J ) 12.5 Hz, 1H), 3.74 (br ABqd, J ) 12.0, 1.5
Hz, 1H), 3.19 (m, 3H), 2.77 (septet, 1H, J ) 7.0 Hz), 2.41 (ABqt,
J ) 13.5, 6.0 Hz, 1H), 2.32 (ABqt, J ) 13.5, 5.5 Hz, 1H), 1.21
(d, J ) 6.5 Hz, 3H), 1.20 (d, J ) 6.9 Hz, 3H); 13C NMR (125
MHz, CDCl3) δ 178.9, 155.1, 147.6, 147.2, 144.2, 139.1, 129.3,
3′,5′-Bis(tert-butyldimethylsilyl)-2′-O-(2-azidoethyl)-
N2-isobutyrylguanosine (G8). NaN3 (0.25 g, 4.0 mmol) and
18-crown-6 (50 mg, 0.19 mmol) were added to a solution of
G7 (0.55 g, 0.78 mmol) in DMF (6 mL). The cloudy pale yellow
mixture was stirred at 60 °C for 5 h, diluted with EtOAc (25
mL), washed with saturated aqueous NaHCO3 (2 × 25 mL),
dried over Na2SO4, and concentrated under vacuum. The pale
yellow foam was purified via chromatography using a gradient
of 20-40% EtOAc in CH2Cl2 as the eluant to produce 0.35 g
1
(70%) of G8 as a white foam: Rf 0.75 (1:2 EtOAc/CH2Cl2); H
NMR (400 MHz, CDCl3) δ 12.37 (s, 1H), 10.35 (s, 1H), 8.14 (s,
1H), 5.98 (d, J ) 2.4 Hz, 1H), 4.39 (dd, J ) 7.4, 5.2 Hz, 1H),
4.05 (td, J ) 9.2, 2.4 Hz, 1H), 3.98 (dd, J ) 12.0, 2.4 Hz, 1H),
3.89 (dt, J ) 9.2, 2.4 Hz, 1H), 3.99 (ABqd, J ) 11.6, 2.4 Hz,
1H), 3.85 (dd, J ) 4.4, 2.0 Hz, 1H), 3.85 (m, 1H), 3.75 (ABqd,
J ) 11.2, 2.4 Hz, 1H), 3.71 (m, 1H), 2.76 (septet, J ) 6.8 Hz,
1H), 1.15 (d, J ) 6.8 Hz, 3H), 1.12 (d, J ) 6.8 Hz, 3H), 0.87 (s,
9H), 0.85 (s, 9H), 0.08 (s, 3H), 0.07 (s, 3H), 0.031 (s, 3H), 0.028
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 180.2, 156.2, 148.4,
148.3, 137.3, 121.4, 87.0, 84.1, 83.7, 69.7, 69.0, 68.8, 61.3, 51.0,
36.2, 26.3, 25.8, 19.2, 18.7, 18.2, -4.4, -4.7, -5.2. ESI-HRMS
[M + H]+ calcd for C28H51N8O6Si2 651.3470, found 651.3467.
2′-O-(2-(Trifluoroacetamido)ethyl)-N2-isobutyrylgua-
nosine (G9). Pd/C (10%, 0.37 g, 0.35 mmol) was added to a
solution of G8 (0.350 g, 0.53 mmol) in EtOH (13 mL). The flask
was evacuated and placed under an atmosphere of hydrogen.
The consumption of G8 was monitored by TLC. After 12 h, 10
g of Celite was added, and the mixture was filtered and
concentrated under vacuum. The pale yellow foam was dis-
solved in THF (5 mL), and ethyl trifluoroacetate (0.28 mL, 2.4
mmol) and Et3N (0.34 mL, 2.4 mmol) were added. After 12 h,
the solution was cooled to 0 °C, and 1 M TBAF in THF (1.24
mL, 1.24 mmol) was added dropwise over 5 min. The solution
was warmed to room temperature and stirred for 6 h. The
solvents were removed under vacuum, and the residual yellow
foam was purified via chromatography using a gradient of
5-15% MeOH in CH2Cl2 as the eluant to yield 0.190 g (73%)
of G9 as a white foam: Rf 0.23 (1:19 MeOH/CH2Cl2); 1H NMR
(400 MHz, DMSO-d6) δ 12.06 (br s, 1H), 11.62 (br s, 1H), 9.25
(s, 1H), 5.88 (d, J ) 6.0 Hz, 1H), 5.26 (d, J ) 4.8 Hz, 1H), 5.07
(t, J ) 5.2 Hz, 1H), 4.38 (t, J ) 4.8 Hz, 1H), 4.30 (ABq, J )
4.8 Hz, 1H), 3.92 (ABq, J ) 4.8 Hz, 1H), 3.67 (m, 1H), 3.55
(m, 4H), 2.74 (septet, J ) 6.8 Hz, 1H), 1.11 (s, 3H), 1.09 (s,
3H); 13C NMR (100 MHz, DMSO-d6) δ 180.8, 155.5, 149.5,
148.9, 138.1, 120.8, 86.7, 85.2, 82.2, 69.5, 67.9, 61.8, 35.4, 19.49,
19.48l; 19F NMR (376 MHz, DMSO-d6) δ -74.9. ESI-HRMS
[M + H]+ calcd for C18H24N6O7F3 493.1659, found 493.1664.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(trifluoroacetamido)-
ethyl)-N2-isobutyrylguanosine (G10). A portion of G9
(0.180 g, 0.37 mmol) was coevaporated from pyridine (2 × 5
mL) and dissolved in pyridine (3 mL). The solution was cooled
to 0 °C, and 4,4′-dimethoxytrityl chloride (0.15 g, 0.44 mmol)
J. Org. Chem, Vol. 70, No. 11, 2005 4297

Jin et al.
127.9, 126.8, 122.0, 88.1, 86.8, 81.7, 70.2, 69.2, 66.5, 62.5, 36.2,
31.9, 18.9. ESI-HRMS [M + H]+ calcd for C35H38N5O6S
656.2543, found 656.2543.
supernatant was transferred by pipet into two 1.5 mL eppen-
dorf tubes; the solid support and vial were rinsed with water
(2 × 0.25 mL). The combined solutions were evaporated to
dryness using an evaporating centrifuge. The residue was
dissolved in a total volume of 1.0 mL of 1 M TBAF in THF
and stirred at 37 °C for 12 h. One milliliter of 1 M Tris, pH
7.5, was added to the solution, and the solution was evaporated
to half the original volume. Additional 1 M Tris, pH 7.5, was
added to a total volume of 2.0 mL, and the oligonucleotide was
desalted on a NAP-25 column using water as the eluant. The
fractions containing the desired RNA oligonucleotide (as
judged by UV absorbance at 260 nm) were combined and
evaporated to dryness. The oligonucleotide was then purified
by 20% denaturing PAGE using 1× TBE (89 mM each Tris
and boric acid, pH 8.3, containing 2 mM EDTA) as the running
buffer. The gel electrophoresis and oligonucleotide extraction
and precipitation procedures have been described previously.4e
The solid-phase coupling efficiency for each of the modified
phosphoramidites was >90% as judged by the appearance of
abort bands on the polyacrylamide gels. The yield of the
purified oligoribonucleotide was typically 150-200 nmol, which
is similar to the yields for unmodified oligoribonucleotides that
are synthesized in a similar fashion.
Oligonucleotide Sequences and Mass Spectrometry
Data. Five RNA oligonucleotides were prepared using the
above procedure incorporating phosphoramidites U9, U10,
A11, G11, and A15. The first four oligonucleotides have one
of the four 2′-tethered amine nucleotides (U, C, A, or G), and
the fifth oligonucleotide has the adenosine 2′-tethered thiol
nucleotide with the thiol group protected as the S-trityl
derivative. The oligonucleotide sequences and mass spectrom-
etry data are given below. 5′-32P radiolabeling of each oligo-
nucleotide after PAGE purification revealed that each oligo-
nucleotide is >99% pure (see Supporting Information). The
oligonucleotide with the 2′-tethered thiol was ligated to a larger
strand of RNA with T4 DNA ligase and a DNA splint,40 and
the S-trityl group was subsequently deprotected with AgNO3
essentially as described31 (data not shown).
Oligonucleotide with 2′-Tethered Amine U. The se-
quence is 5′-UUCUGUUGAXAUGGAUGCAGUUCA-3′, where
X denotes the modified nucleotide from incorporation of
phosphoramidite U9. MALDI-MS calcd 7675.6, found 7677.2.
Oligonucleotide with 2′-Tethered Amine C. The se-
quence is 5′-UUCUGUUGAUAUGGAUGXAGUUCA-3′, where
X denotes the modified nucleotide from incorporation of
phosphoramidite U10. MALDI-MS calcd 7675.6, found 7672.9.
Oligonucleotide with 2′-Tethered Amine A. The se-
quence is 5′-GGAAUUGCGGGAXAG-3′, where X denotes the
modified nucleotide from incorporation of phosphoramidite
A11. MALDI-MS calcd 4961.1, found 4962.1.
Oligonucleotide with 2′-Tethered Amine G. The se-
quence is 5′-GGAAUUXCGGGAAAG-3′, where X denotes the
modified nucleotide from incorporation of phosphoramidite
G11. MALDI-MS calcd 4961.1, found 4961.9.
Oligonucleotide with 2′-Tethered Thiol A. The sequence
is 5′-GGAAUZGCGGGAXAG-3′, where X denotes the modified
nucleotide (S-trityl protecting group remaining) from incor-
poration of phosphoramidite A15 and Z denotes a 2′-amino-
2′-deoxyuridine nucleotide (C. V. Miduturu and S. K. Silver-
man, manuscript in preparation). MALDI-MS calcd 5219.3,
found 5218.7.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)-N2-isobutyrylguanosine (G13). A portion of
G12 (0.150 g, 0.23 mmol) was coevaporated from pyridine (3
mL) and dissolved in pyridine (3 mL). The solution was cooled
to 0 °C, and 4,4′-dimethoxytrityl chloride (0.089 g, 0.26 mmol)
was added. After the mixture was stirred at room temperature
for 12 h, the solvents were removed under vacuum. The
residual pale yellow foam was purified via chromatography
with the column packed as described for U9. The column was
eluted with a gradient of 0-5% MeOH in CH2Cl2 to yield 0.150
g (68%) of G13 as a white foam: Rf 0.55 (1:19 MeOH/CH2Cl2);
1H NMR (500 MHz, CDCl3) δ 7.80 (s, 1H), 7.46 (dd, J ) 7.0,
1.5 Hz, 2H), 7.36-7.11 (m, 24H), 6.82-6.75 (m, 4H), 5.83 (d,
J ) 6.5 Hz, 1H), 4.54 (m, 1H), 4.34 (m, 1H), 4.15 (d, J ) 2.0
Hz, 1H), 3.75 (s, 3H), 3.74 (s, 3H), 3.40 (ABqd, J ) 10.5, 2.0
Hz, 1H), 3.39 (ABqt, J ) 10.5, 5.0 Hz, 1H), 3.15 (ABqd, J )
10.5, 3.5 Hz, 1H), 3.07-3.02 (m, 1H), 2.61 (m, 2H), 2.37 (ABqt,
J ) 13.0, 5.5 Hz, 1H), 1.91 (septet, J ) 7.0 Hz, 1H), 0.91 (d,
J ) 7.0 Hz, 3H), 0.81 (d, J ) 7.0 Hz, 3H); 13C NMR (125 MHz,
CDCl3) δ 179.1, 158.9, 156.7, 148.5, 147.5, 145.1, 144.6, 138.7,
136.1, 135.8, 130.3, 130.2, 129.7, 128.2, 127.3, 127.1, 122.2,
113.5, 86.5, 84.5, 69.7, 67.5, 55.5, 36.4, 32.1, 18.9. ESI-HRMS
[M + H]+ calcd for C56H56N5O8S 958.3850, found 958.3817.
5′-O-(4,4′-Dimethoxytrityl)-2′-O-(2-(triphenylmethyl-
thio)ethyl)-N2-isobutyrylguanosine-3′-O-(2-cyanoethyl-
N,N-diisopropyl)phosphoramidite (G14). A portion of G13
(0.150 g, 0.16 mmol) was coevaporated with pyridine (2 × 5
mL) and dissolved in CH2Cl2 (1 mL). The solution was cooled
to 0 °C, and 2-cyanoethyl N,N,N′,N′-tetraisopropylphospho-
rodiamidite (65 µL, 0.21 mmol) and 4,5-dicyanoimidazole (24
mg, 0.22 mmol) were added. The colorless solution was warmed
to room temperature, stirred for 6 h, diluted with 5% Et3N in
EtOAc (25 mL), and washed with saturated aqueous NaHCO3
(2 × 25 mL). The organic layer was dried over Na2SO4 and
concentrated under vacuum. The residual pale yellow solid was
purified via chromatography with the column packed as
described for U9 (the sample was loaded in 14:6 hexanes/CH2-
Cl2). The column was eluted with a gradient from 1:14:6 to
1:3:16 Et3N/hexanes/CH2Cl2 to produce 0.109 g (61%) of G14
as a white foam: Rf 0.44 (1:3:16 Et3N/hexanes/CH2Cl2). NMR
spectroscopy revealed the presence of both diastereomers: 1H
NMR (500 MHz, acetone-d6) δ 7.98 and 7.96 (each s, total 1H),
7.50 and 7.49 (each s, 2H), 7.36-7.23 (m, 24H), 6.87 (m, 4H),
5.96 and 5.92 (each d, J ) 6.0 Hz, total 1H), 4.63 and 4.57
(each t, J ) 5.5 Hz, total 1H), 4.53 (m, 1H), 4.35 and 4.28 (each
ABqd, J ) 7.5, 4.0 Hz, total 1H), 3.78 (s, 6H), 3.72-3.59 (m,
5H), 3.45-3.32 (m, 3H), 2.77 (septet, J ) 7.0 Hz, 1H), 2.72
(m, 1H), 2.60 (t, J ) 6.0 Hz, 1H), 2.36 (m, 2H), 1.16 (m, 16H),
1.07 (d, J ) 7.0 Hz, 2H); 13C NMR (125 MHz, acetone-d6) δ
180.2, 159.0, 155.2, 148.7, 148.5, 145.3, 145.2, 136.9, 136.8,
136.0, 135.9, 130.3, 128.4, 128.3, 127.0, 121.6, 118.6, 118.3,
113.3, 87.5, 86.5, 83.5, 83.2, 81.5, 81.2, 71.4, 71.3, 68.6, 68.4,
63.5, 63.2, 59.1, 59.0, 58.5, 58.4, 54.9, 52.8, 46.1, 43.3, 43.2,
39.8, 35.8, 24.5, 20.0, 18.6; 31P NMR (202 MHz, acetone-d6) δ
151.13 and 151.06. FAB-HRMS [M + H]+ calcd for C65H73N7O9-
PS 1158.4928, found 1158.4929.
Solid-Phase RNA Oligonucleotide Synthesis. Acetoni-
trile (synthesis grade), the standard 2′-TOM-phosphoramidetes
for A, C, G, and U, 5-ethylthio-1H-tetrazole, and other
standard solutions were from a commercial supplier. RNA
oligonucleotides were synthesized on the 1 µmol scale. The
standard coupling time of 6 min was used for the four standard
phosphoramidites, and an increased coupling time of 10 min
was used for the modified phosphoramidites. After the solid-
phase synthesis, the solid support was transferred to a screw-
cap glass vial and incubated at room temperature for 16 h with
1.5 mL of methylamine solution, prepared by mixing equal
volumes of 40% aqueous methylamine and 33% methylamine
in ethanol. After the vial was cooled briefly on ice, the
Acknowledgment. This research was supported by
the Burroughs Wellcome Fund (New Investigator Award
in the Basic Pharmacological Sciences), the March of
Dimes Birth Defects Foundation (Research Grant
5-FY02-271), the National Institutes of Health (GM-
65966), the American Chemical Society Petroleum
Research Fund (38803-G4), and the UIUC Department
of Chemistry (all to S.K.S.). S.K.S. is the recipient of a
fellowship from The David and Lucile Packard Founda-
4298 J. Org. Chem., Vol. 70, No. 11, 2005

Amine- and Thiol-Modified RNA Phosphoramidites
Supporting Information Available: General experimen-
tion. We are grateful to Rebecca Coppins for the initial
investigations into 2′-alkylations of uridine nucleosides
and to Mary Smalley for the PAGE purification of the
RNA oligonucleotides prepared using phosphoramidites
U10 and A11. We thank members of the Silverman lab
for discussions.
1
tal procedures, H and 31P NMR spectra, and 5′-32P radiola-
beling to demonstrate oligoribonucleotide purity. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO050061L
J. Org. Chem, Vol. 70, No. 11, 2005 4299