Monomers for preparation of amide-linked RNA: Asymmetric synthesis of all four nucleoside 5′-azido 3′-carboxylic acids

Article abstract of DOI:10.1021/jo0515879

Recent discovery of RNA interference as an efficient and naturally occurring mechanism of gene regulation has reinvigorated the interest in chemically modified RNA. For potential in-vivo applications small interfering RNAs require chemical modifications t

Full text of DOI:10.1021/jo0515879

Monomers for Preparation of Amide-Linked RNA: Asymmetric
Synthesis of All Four Nucleoside 5′-Azido 3′-Carboxylic Acids
Eriks Rozners* and Yang Liu
Department of Chemistry and Chemical Biology, Northeastern University, Boston, Massachusetts 02115
e.rozners@neu.edu.
Received July 29, 2005
Recent discovery of RNA interference as an efficient and naturally occurring mechanism of gene
regulation has reinvigorated the interest in chemically modified RNA. For potential in-vivo
applications small interfering RNAs require chemical modifications to fine-tune the thermal stability
and increase the cellular delivery and potency and in vivo half-life of the RNA duplexes. From this
perspective, amides as neutral and hydrophobic internucleoside linkages in RNA are highly
interesting modifications for RNA interference. Amides are remarkably good mimics of the
phosphodiester backbone of RNA and can be prepared using a relatively straightforward peptide
coupling chemistry. However, the progress in the field has been hampered by the shortage of efficient
methods to synthesize the monomeric building blocks for such couplings, the nucleoside amino
acid equivalents. Herein, we report enantioselective synthesis of 5′-azido 3′-carboxylic acid
derivatives of all four natural ribonucleosides. The key transformations in our synthesis are a double
asymmetric ene reaction and a stereoselective iodolactonization that form the basic carbon skeleton
of the modified ribose. Standard nucleoside synthesis is followed by a short and highly efficient
protecting group manipulation to give the enantiomerically pure (>98%) title compounds in 9-10
steps and 15-19% overall yields starting from small achiral molecules. The present results are a
significant improvement over our first-generation racemic synthesis and compare favorably with
the previously reported synthesis from nucleoside and carbohydrate precursors.
Introduction
linkages.2,3 Of many nonionic DNA analogues tested,
amide3-5 (Figure 1, 3′-CH2-CO-NH-5′ (1a) and 3′-CH2-
NH-CO-5′ (2a)), methylene(methylimino)6 (3′-CH2-
N(CH3)-O-5′), formacetal7,8 (3′-O-CH2-O-5′), and thio-
Nucleic acid analogues where the phosphodiesters are
replaced with nonionic non-phosphorus linkages began
to attract wide interest about a decade ago as potential
nuclease-resistant antisense compounds.1-3 Short hybrid
DNA-RNA A-form duplexes, where the DNA strands
have a few selected phosphodiesters replaced by neutral
synthetic linkages, have been the most extensively
studied model systems for testing the properties of such
(4) (a) Idziak, I.; Just, G.; Damha, M.; Giannaris, P. A. Tetrahedron
Lett. 1993, 34, 5417-5420. (b) Lebreton, J.; Waldner, A.; Lesueur, C.;
De Mesmaeker, A. Synlett 1994, 137-140. (c) De Mesmaeker, A.;
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S. M. Angew. Chem., Int. Ed. Engl. 1994, 33, 226-229. (d) De
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Wolf, R. M. Angew. Chem., Int. Ed. Engl. 1996, 35, 2790-2794. (e) De
Mesmaeker, A.; Lebreton, J.; Jouanno, C.; Fritsch, V.; Wolf, R. M.;
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10.1021/jo0515879 CCC: $30.25 © 2005 American Chemical Society
Published on Web 10/27/2005
J. Org. Chem. 2005, 70, 9841-9848
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Rozners and Liu
sulfone (3′-CH2-SO2-CH2-5′) linked DNA and RNA.
Substitution of a single phosphodiester with the sulfone
linkage strongly destabilized DNA-DNA and DNA-RNA
duplexes.16a Although the crystal structure of dinucleo-
side analogue r(GSO2C) revealed a slightly distorted
Watson-Crick miniduplex,16b all sulfone-linked octamers
did not base pair with complementary oligonucleotides
but self-associated and formed a very stable self-folded
structure instead.16c,d Recently, we showed that form-
acetal17 and both isomeric amide18 (Figure 1, 1b and 2b)
linkages were well-accommodated in all RNA-RNA
duplexes. Whereas the 1b-modified duplexes have ther-
mal stability similar to the nonmodified controls, the 2b
modification remarkably stabilizes the RNA-RNA du-
plexes (∆tm up to over 2 °C per modification).18 This is in
contrast to the results by De Mesmaeker and co-workers
in the DNA series where both 1a- and 2a-modified
duplexes show thermal stability similar to the nonmodi-
fied DNA.4,5
FIGURE 1. Amides as internucleoside linkages.
formacetal8 (3′-S-CH2-O-5′) analogues form thermally
stable double helices.
Emerging RNA-based gene control technologies,9 es-
pecially, the discovery of RNA interference,10 have in-
vigorated the interest in chemically modified RNA.11,12
Modifications of the sugar-phosphate backbone such as
phosphorothioates,12a 2′-O-alkyl,12a-c 2′-F,12a-c and 4′-
thio12d RNA, and even locked nucleic acid (LNA)12a,e are
generally well-tolerated in RNA interference. Moreover,
small interfering RNAs (siRNAs) having boranophos-
phate-modified internucleoside linkages are at least as
active as the native siRNAs.12f Despite the potentially
interesting applications in RNA interference, there are
relatively few reports on synthesis and properties of RNA
analogues having dephospho linkages. Ribonucleoside
dimers having thioformacetal13 and sulfide14 (3′-CH2-
CH2-S-5′) linkages have been prepared, incorporated in
short DNA fragments, and shown to destabilize short
DNA-RNA duplexes. The analysis of these data, how-
ever, is complicated because the ribonucleoside dimers
were incorporated in DNA, which may cause complex
interplay of alternating (DNA and RNA) sugar composi-
tions.15 Benner and co-workers16 studied dimethylene
Except for the extensively studied peptide nucleic
acids, which form stable Watson-Crick base paired
helices with complementary DNA, RNA, and itself,19,20
only a few studies report on nucleic acid analogues having
uniformly modified amide backbones. All amide-linked
DNA (Figure 1, analogue of 1a) forms stable duplexes
with the complementary unmodified RNA and DNA.21
Robins and co-workers22 prepared all amide-linked uri-
dine pentamer (Figure 1, analogue of 1b) but did not
disclose the biochemical properties of this analogue.
A practical advantage of oligoamide analogues of
nucleic acids is that they could be prepared using a
relatively straightforward peptide coupling chemistry.
The synthetic challenge, that so far has hindered the
progress in the field, is the synthesis of the highly
modified monomers, C-branched nucleoside amino acids
(such as 3, Figure 2) required for the peptide type
couplings.
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pounds: nucleosides or carbohydrates. Several research
groups have synthesized compounds related to 3. Von
Matt and co-workers synthesized 2′-deoxy21 and 2′-OMe23
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9842 J. Org. Chem., Vol. 70, No. 24, 2005

Toward Total Synthesis of Amide-Linked RNA
SCHEME 1a
FIGURE 2. Retrosynthetic analysis of amide-linked RNA.
workers24 prepared a series of ribonucleosides 3 (base )
adenine and thymine) starting from nucleosides24a,b or
protected xylose.24c Both groups used the Wittig reaction
followed by a stereoselective hydrogenation to form the
new carbon-carbon bond at C3′. The advantage of
traditional carbohydrate-based routes is that the stereo-
chemical relationships are already set or can be easily
adjusted in the chiral pool starting materials. However,
such routes are frequently lengthy and laborious because
the multifunctional and sensitive starting materials limit
the synthetic methodology that can be used and require
extensive protecting group manipulations.
In a preliminary paper, we recently reported de novo
synthesis of the racemic uridine derivative of 3 (Figure
2, base ) Ura, R ) tert-butyldimethylsilyl (TBS)).25
Although conceptually similar approaches have been
frequently used to prepare carbocyclic nucleosides,26
applications of total synthesis principles to modify natu-
ral nucleosides are relatively rare.27,28 The most relevant
precedent is of Lavallee and Just27 who synthesized 3′-
carbomethoxymethyl thymidine using a radical cycliza-
tion as the key step. In the present paper we report a
full account on further developments of our total syn-
thesis route that culminated in asymmetric synthesis of
all four protected nucleoside azido acids (Figure 2, 3, base
) Ura, Cyt, Ade, Gua, R ) Ac). The synthesis was
accomplished in 9-10 steps and 15-19% overall yield,
which constitutes a significant improvement over the
previously reported syntheses.24,25
a Conditions: (a) for 7b:9, CH2Cl2, rt, 3 days, 89% (dr 98:2); (b)
Me2NH-HCl, AlMe3, toluene, 70-90 °C, 2 days, 79%; (c) I2,
NaHCO3, THF/H2O (2:1), 0 °C, 26 h, 65%; (d) NaN3, DMF, 50 °C,
23 h, 78%.
Such an approach allows all four nucleoside derivatives
to be made from the same modified glycosyl donor 4.
Adjustment of functional groups in 4 reveals the iodo-
lactone 5 as the key intermediate. Opening of the five-
membered tetrahydofuran ring (iodolactonization) identi-
fies the unsaturated carboxylic acid 6 (or a derivative
thereof) as the next intermediate. These disconnections
reduce the complexity from two heterocycles and four
stereogenic centers in 3 to only two stereogenic centers
in the acyclic 6, which can be further disconnected to
simple organic compounds 7 and 8 to be joined in a
stereoselective ene reaction.
Double Asymmetric Ene Reaction. In our prelimi-
nary paper,25 reaction of ethyl glyoxylate 7a with alkene
8 in the presence of the bis(oxazolinyl)copper(II) catalyst
9, developed by Evans and co-workers,29 gave the chiral
hydroxy ester 6a in 70% yield after 4 days (Scheme 1).
The use of anhydrous 9 was essential; the reaction
catalyzed by the aqua complex of 9 was unacceptably slow
(30% after 7 days). Enantioselective HPLC (Chiralcel
OD-H) established that 6a was formed along with its
enantiomer in a ratio of 95:5.
Results and Discussion
In the present study, this result was substantially
improved by performing the reaction in a double asym-
metric mode. Whitesell and co-workers30 have described
a series of menthol-derived auxiliaries for carbonyl ene
reactions. We chose the modestly selective (1′S,2′R,5′S)-
menthyl glyoxalate 7b because the low cost and easy
large-scale preparation of 7b were considered more
important than a higher asymmetric induction achievable
by more expensive alternatives (such as 8-phenylmenthyl
Retrosynthetic Analysis. Our retrosynthetic analy-
sis of 3 (Figure 2) disconnects the heterocyclic base first.
(24) (a) Robins, M. J.; Sarker, S.; Xie, M.; Zhang, W.; Peterson, M.
A. Tetrahedron Lett. 1996, 37, 3921-3924. (b) Peterson, M. A.; Nilsson,
B. L.; Sarker, S.; Doboszewski, B.; Zhang, W.; Robins, M. J. J. Org.
Chem. 1999, 64, 8183-8192. (c) Robins, M. J.; Doboszewski, B.;
Timoshchuk, V. A.; Peterson, M. A. J. Org. Chem. 2000, 65, 2939-
2945.
(25) Rozners, E.; Liu, Y. Org. Lett. 2003, 5, 181-184.
(26) (a) Shin, K. J.; Moon, H. R.; George, C.; Marquez, V. E. J. Org.
Chem. 2000, 65, 2172-2178. (b) Trost, B. M.; Madsen, R.; Guile, S.
D.; Brown, B. J. Am. Chem. Soc. 2000, 122, 5947-5956. (c) Crimmins,
M. T.; King, B. W.; Zuercher, W. J.; Choy, A. L. J. Org. Chem. 2000,
65, 8499-8509.
(29) (a) Evans, D. A.; Burgey, C. S.; Paras, N. A.; Vojkovsky, T.;
Tregay, S. W. J. Am. Chem. Soc. 1998, 120, 5824-5825. (b) Evans, D.
A.; Tregay, S. W.; Burgey, C. S.; Paras, N. A.; Vojkovsky, T. J. Am.
Chem. Soc. 2000, 122, 7936-7943. (c) Johnson, J. S.; Evans, D. A. Acc.
Chem. Res. 2000, 33, 325-335.
(30) (a) Whitesell, J. K.; Bhattacharya, A.; Aguilar, D. A.; Henke,
K. J. Chem. Soc., Chem. Commun. 1982, 989-990. (b) Whitesell, J.
K.; Lawrence, R. M.; Chen, H.-H. J. Org. Chem. 1986, 51, 4779-4784.
(27) Lavallee, J. F., Just, G. Tetrahedron Lett. 1991, 32, 3469-3472.
(28) (a) Trost, B. M.; Shi, Z. J. Am. Chem. Soc. 1996, 118, 3037-
3038. (b) Hager, M. W.; Liotta, D. C. J. Am Chem. Soc. 1991, 113,
5117-5119. (c) Svansson, L.; Kvarnstroem, I.; Classon, B.; Samuelsson,
B. J. Org. Chem. 1991, 56, 2993-2997.
J. Org. Chem, Vol. 70, No. 24, 2005 9843

Rozners and Liu
SCHEME 2a
glyoxylate). Interestingly, the reaction of 7b with alkene
8 in the presence of catalyst 9 gave 6b in a higher yield
(89%) over a shorter period of time (3 days) than in the
case of ethyl glyoxylate (Scheme 1). In the 1H NMR
spectrum, the peaks due to the (2S,3S) diastereomer of
6b were barely visible and could not be reliably inte-
grated (see Supporting Information). Assuming a 2%
cutoff for the sensitivity of NMR integration, the NMR
suggested that the diastereomer ratio (dr) was better
than 98:2. This result was later confirmed by enantiose-
lective HPLC. The work of Mikami et al. has established
good precedent for the regioselectivity and the anti
diastereoselectivity in the carbonyl ene reactions of this
a Conditions: (a) DIBAL-H, toluene, -78 °C, 110 min; (b) Ac2O/
pyridine (1:1), rt, 23 h, 86% (two steps); (c) for 12a, O,O′-
bis(trimethylsilyl)uracil, TMSOTf, CH2Cl2, rt, 3 h, > 99%; for 12b′,
O,N-bis(trimethylsilyl)cytosine, TMSOTf, ClCH2CH2Cl, reflux, 50
min, >99%; for 12c, 6-N-benzoyladenine, SnCl4, CH3CN, rt, 30
min, 60%; for 12d, BSA, 2-N-acetyl-6-O-diphenylcarbamoylgua-
nine, ClCH2CH2Cl, reflux, 10 min, add to 4 in ClCH2CH2Cl, add
TMSOTf, reflux 1 h, 56%; (d) TMSCl, 1 h, propionic anhydride,
16 h, H2O, 20 min, pyridine, rt, 61%.
1
type.31 The H NMR spectrum of 6b also indicated the
presence of <10% of a minor compounds, possibly the syn
diastereomers, which were separated by flash chroma-
tography at later stages.
Stereoselective Iodolactonization and Synthesis
of Modified Ribose. Next we planned to construct the
tetrahydofuran ring by stereoselective iodolactonization.
From the literature precedents32 we anticipated that
under thermodynamic control the C4-C5 trans isomer
would be the major product. Iodolactonization of ethyl
(6a), menthyl (6b), or trimethylsilyl esters gave com-
plex mixtures that did not contain the desired lactone.
Iodolactonization (under conditions given in Scheme 1)
of carboxylic acid 6d gave a mixture of C4-C5 trans and
cis lactones 5a and 5b in a ratio of 1.5:1 (56% yield).
Eventually, the best results were achieved with amides
as the cyclization precursors.33 Thus, treatment of ester
6b with the aluminum amide reagent prepared from
AlMe3 and dimethylamine hydrochloride gave the di-
methylamide 6c in 79% yield. Iodolactonization of 6c
gave the desired iodolactone as a nonseparable mixture
of C4-C5 trans (5a) and cis (5b) diastereomers in a ratio
of 4:1. Providing that the hydrolysis of the intermediate
iminium ion 10 is slow enough to allow for thermody-
namic control in the iodolactonization of amide 6c, it is
conceivable that the observed 4:1 stereoselectivity reflects
the sterically most favorable trans arrangement of the
two alkyl substituents on the relatively flat five-mem-
bered ring in 10.32,33 A brief study of reaction conditions
revealed that a tetrahydrofuran (THF)/water mixture
was the best solvent; MeCN, CH2Cl2, MeOH, dimethyl-
formamide (DMF), dimethoxyethane, and diethyl either
mixture of 5a and 5b in 58% yield and 2:1 ratio. Because
we could not efficiently separate the trans and cis isomers
of 5 (or later intermediates) by flash column chromatog-
raphy, the following synthetic steps were carried out on
mixtures (for clarity only the major diastereomer is
shown in Scheme 2). Separation of isomers was achieved
at later steps after the synthesis of nucleosides. Treat-
ment of iodolactones 5 with sodium azide gave the
mixture of trans and cis azidolactones 11a,b. Selective
reduction of the lactone group34 was immediately followed
by acetylation of the hydroxyl groups to give the glycosyl
acetate 4 as a mixture of four diastereomers (Scheme 2).
Synthesis of Modified Nucleosides. Following the
standard Vorbru¨ggen methodology,35 coupling of bis-
(trimethylsilyl) heterocycles with 4 in the presence of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) gave
the modified pyrimidine nucleosides 12a and 12b′. For
the synthesis of adenosine derivative 12c, we used the
tin tetrachloride mediated reaction of 4 with 6-N-ben-
zoyladenine.36 To achieve a regioselective synthesis of
guanosine derivative 12d, we used the methodology
developed by Zou and Robins.37 Thus, the coupling of 4
with persilylated 2-N-acetyl-6-O-diphenylcarbamoylgua-
nine gave 12d as a single 9-N-isomer. At this stage the
3′,4′-cis and -trans isomers of purine nucleosides were
separated by silica gel chromatography, giving 12c and
12d as single diastereomers. The somewhat lower yield
of the purine nucleosides reflected the removal of the
undesired 3′,4′-cis diastereomers. The pyrimidine nucleo-
sides 12a and 12b′ were still inseparable mixtures of
isomers.
alone or with water present reduced the yield. Sodium
-
bicarbonate was the best base; the use of NHEt3+HCO3
,
NEt3, K2CO3, and NBu4+OH- reduced both yield and
stereoselectivity. Whereas dimethyl and pyrrolidinyl
amides performed equally well, other amide derivatives
gave inferior results: EtNH-, low yield and selectivity;
PhNH-, no product; MeO(Me)N-, yield 50%. The best
results were achieved at 0 to -10 °C; lower or higher
temperatures reduced the yield and selectivity. Finally,
the use of N-iodosuccinimide instead of iodine gave the
Protecting Group Manipulation and Final Steps.
The most important issue after the synthesis of nucleo-
sides was the design of a protecting group scheme for the
final compounds, azido acids 3a-d. The need for exten-
sive protection-deprotection of hydroxyl and amino
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1990, 46, 265-276.
(35) (a) Vorbru¨ggen, H. Acc. Chem. Res. 1995, 28, 509-520. (b)
Vorbru¨ggen, H.; Ruh-Pohlenz, C. Handbook of Nucleoside Synthesis;
Wiley-Interscience: New York, 2001; p 631.
(36) Saneyoshi, M.; Satoh, E. Chem. Pharm. Bull. 1979, 27, 2518-
2521.
(37) Zou, R.; Robins, M. J. Can. J. Chem. 1987, 65, 1436-1437.
9844 J. Org. Chem., Vol. 70, No. 24, 2005

Toward Total Synthesis of Amide-Linked RNA
SCHEME 3a
SCHEME 4a
a Conditions: (a) TBAF/AcOH (1:1), THF, rt, (13a) 2.75 h, 79%,
(13b) 5 h, 99%, (13c) 4 h, 93%, (13d) 5 h, 98%; (b) TEMPO (cat.),
(diacetoxyiodo)benzene, CH3CN/H2O (1:1), (3a) rt, 41 h, 80%, (3d)
0 °C, 4 days, 87% or (i) Dess-Martin periodinane, CH2Cl2, 1.5 h;
(ii) NaClO2, 2-methyl-2-butene, NaH2PO4, tert-butyl alcohol/H2O
(1:1), rt, 25 min, (3b) 86%, (3c) 86%.
functions is one of the most severe problems in carbohy-
drate chemistry. In our initial design, published in the
preliminary paper,25 we removed both the TBDPS ether
and the 2′-O-Ac groups from 12a and used a two-step
silylation-selective deprotection sequence to install the
2′-O-TBS protection. The overall yield of this sequence
was 37%, reflecting the difficulty of protecting group
manipulations on advanced carbohydrate intermediates.
Herein we describe a second-generation design that
retains the base labile 2′-O- and N-acyl protections and
minimizes the protection-deprotection steps. Because
the 2′-O-Ac group was required for the stereoselective
synthesis of nucleosides and the 2-N-acetyl and 6-O-
diphenylcarbamoyl (DPC) groups were required for the
regioselective synthesis of the guanosine derivative 12d,
we envisioned that the most efficient design would keep
these base labile protections in the final products. We
were also encouraged by our previous successful experi-
ence with 2′-O- and N-acyl groups in synthesis of native38
and modified17,18 RNA. Thus, the cytidine 12b′ was
further converted into 4-N-propionyl derivative 12b,
which was isolated as a single diastereomer after silica
gel chromatography.
Next we investigated selective removal of the TBDPS
ether in the presence of the 2′-O-Ac group in 12a, which
was a problem during our preliminary work.25 Treatment
of 12a with tetrabutylammonium fluoride (TBAF) in THF
resulted in a mixture of compounds. Along with the
expected 13a and its 3′,4′-cis isomer, the mixture con-
tained the fully deprotected diol (ca 5%) and another
byproduct (25%). The NMR spectrum of the latter was
consistent with structure 14a, where the acetyl group had
migrated to the primary alcohol (Scheme 3). It is conceiv-
able that the spatial 1,7 relationship of the acetyl and
the TBDPS ether cause this unexpected side reaction.
Similar results were obtained using the less basic NH4F
in methanol39 and the weakly acidic HF in pyridine and
HSiF6 in acetonitrile.40 The strongly acidic 1% HCl in
methanol caused complete deprotection. After consider-
a Conditions: (a) NH3/MeOH, rt, 24 h, >90%; (b) 3a, HCTU,
DIEA, DMF, rt, 50 min, 83%; (c) NH3/MeOH, rt, 20 min, >99%
able experimentation we found that a mixture of TBAF
and acetic acid (1:1) in THF cleanly removed the TBDPS
group in 12a without any side reactions at the 2′-O-Ac.41
At this stage the 3′,4′-isomers of 13a were separated by
silica gel chromatography. The buffered TBAF reagent
was also successful with the base-protected nucleosides
12b-d, yielding all four primary alcohols 13a-d as pure
diastereomers.
The last step, oxidation of alcohol to carboxylic acid,
was achieved for 13b and 13c in a two-step process using
Dess-Martin periodinane followed by NaClO2. For 13a
and 13d, a combination of TEMPO (catalytic) and (diace-
toxyiodo)benzene gave somewhat better results.
Stability of Protecting Groups and Internucleo-
side Amide Linkage. The conditions for removal of the
base labile groups were established using the guanosine
derivative 12d (Scheme 4). Thus treatment of 12d with
an anhydrous solution of NH3 in methanol (saturated at
0 °C) resulted in a clean and complete removal of all three
protecting groups (2′-O-Ac, 2-N-Ac, and 6-O-DPC) in less
than 24 h at room temperature. The estimated half-life
(from thin-layer chromatography (TLC)) of formation of
the fully deprotected guanosine derivative 15d was less
than 5 h. Under the same conditions cytidine 12b and
adenosine 12c were rapidly and fully deprotected within
3 and 6 h, respectively.
To check the stability of the 2′-O-Ac group and the
internucleoside amide linkage under the deprotection
conditions, we prepared dimer 17 (Scheme 4). 2-(6-
Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluroni-
um hexafluorophosphate (HCTU) mediated coupling of
uridine azido acid 3a with amine 16 gave the dimer 17
in good yield. Exposure of 17 to a saturated solution of
NH3 in methanol at room temperature resulted in a rapid
(<20 min) removal of the 2′-O-Ac group to form 18.
Further exposure of 18 to the same deprotection solution
(38) (a) Rozners, E.; Westman, E.; Stro¨mberg, R. Nucleic Acids Res.
1994, 22, 94-99. (b) Rozners, E.; Renhofa, R.; Petrova, M.; Popelis,
Y.; Kumpins, V.; Bizdena, E. Nucleosides Nucleotides 1992, 11, 1579-
1593.
(39) Zhang, W.; Robins, M. J. Tetrahedron Lett. 1992, 33, 1117-
1180.
(40) (a) Pilcher, A. S.; Hill, D. K.; Shimshock, S. J.; Waltermire, R.
E.; DeShong, P. J. Org. Chem. 1992, 57, 2492-2495. (b) Pilcher, A. S.;
DeShong, P. J. Org. Chem. 1993, 58, 5130-5134.
(41) (a) Hayward, C. M.; Yohannes, D.; Danishefsky, S. J. J. Am.
Chem. Soc. 1993, 115, 9345-9346. (b) Smith, A. B.; Chen, S. S.-Y.;
Nelson, F. C.; Reichert, J. M.; Salvatore, B. A. J. Am. Chem. Soc. 1995,
117, 12013-12014. (c) Panek, J. S.; Liu, P. J. Am. Chem. Soc. 2000,
122, 11090-11097.
J. Org. Chem, Vol. 70, No. 24, 2005 9845

Rozners and Liu
CH2Cl2 (10 mL) were stirred for 4 h. The green solution was
filtered through a 25 mm hydrophobic poly(tetrafluoroethyl-
ene) (PTFE) membrane filter (0.45 µm, Waters). AgSbF6 (0.93
g, 2.7 mmol) in CH2Cl2 (40 mL) was added to the clear filtrate,
the mixture was stirred for 3 h, carefully decanted from the
majority of precipitate, and filtered through a 25 mm hydro-
phobic PTFE membrane filter (0.45 µm, Waters). To the clear
green solution were added 1-(tert-butyldiphenylsilanoxy)-3-
pentene25 (8; 4.32 g, 13.32 mmol) and freshly distilled
(1′S,2′R,5′S)-menthyl glyoxalate30b (7b; 28 g, 132 mmol), and
the mixture was stirred for 3 days at room temperature. The
solvent was evaporated, and the residue was purified by silica
gel column chromatography (5-20% of ethyl ether in hexanes,
stepwise gradient by 5%) to afford 6b as oil. Yield: 6.36 g,
89%. TLC Rf ) 0.33 hexanes/ethyl ether (7:3). 1H NMR (CDCl3,
300 MHz): δ 7.69-7.66 (m, 4H), 7.45-7.34 (m, 6H), 5.72-
5.60 (m, 1H), 5.18-5.00 (m, 2H), 4.83-4.74 (m, 1H), 4.24-
4.21 (m, 1H), 3.80-3.65 (m, 2H), 2.89-2.81 (m, 2H), 1.98-
1.66 (m, 6H), 1.54-1.35 (m, 2H), 1.06 (s, 9H),1.10-0.75
(overlapping m, 2H), 0.90 (t, J ) 6.7 Hz, 6H), 0.76 (d, J ) 7.2
Hz, 3H). TLC Rf ) 0.33 hexanes/ether (7:3). HRMS (ESI).
Calcd for C33H48O4Si [M + H]+: 537.3400. Found: 537.3348.
resulted in no observable cleavage (TLC and NMR) of
the internucleoside amide even after 6 days at room
temperature. However, treatment of 18 with a mixture
of aqueous NH3 and methanol (3:1) at 65 °C resulted in
about 80% degradation of the amide after 16 h. Thus,
the internucleoside amide linkages 1b (as in dimer 18)
are notably more reactive than regular alkyl amides. This
must be a result of neighboring group assistance due to
a spatial 1,5 relationship of amide and 2′-OH. Neverthe-
less, anhydrous NH3 in methanol at room temperature
provides a safe reactivity window for removal of all
protecting groups without detectable cleavage of the
internucleoside amides.
Conclusions
We have developed the asymmetric synthesis of all four
enantiomerically pure (>98%) nucleoside 5′-azido 3′-
carboxylic acids 3a-d in nine steps (10 for cytidine)
starting from 7b and 8. The number of steps and the
overall yields (3a, 19%; 3b, 16%; 3c, 15%; 3d, 15%)
compare favorably with the previously reported synthesis
from D-xylose24 and our first-generation racemic synthe-
sis.25 It is conceivable that our synthesis may be further
improved by employing an alternative and more efficient
electrophilic cyclization. The asymmetric synthesis has
several important advantages over the traditional routes
that start from sugars or nucleosides. Carbohydrate
routes are constrained by the choice of the chiral pool
starting material and, once developed, are difficult to
change and improve. In contrast, the asymmetric syn-
thesis is extremely flexible for optimization. Because the
upstream starting materials are usually small simple
molecules, it is much easier to find a new reaction that
provides a better synthesis of a key intermediate. We
have recently demonstrated such an improvement in the
asymmetric synthesis of modified nucleosides related to
3a-d, the isomeric 3′-azido 5′-carboxylic acids.42 Because
the key 3′-C alkyl substituent does not directly partici-
pate in the chemical transformations, it is conceivable
that the synthesis of 3a-d reported herein could be
modified to provide other nucleoside analogues with
different 3′-C alkyl substitution patterns. Finally, the
asymmetric synthesis provides access to both enanti-
omers of the modified nucleosides by choosing the ap-
propriate chiral catalyst and auxiliary, which is not
possible starting from sugars or nucleosides. Such an
option may be highly useful for potential applications of
amide-modified RNA in biotechnology, biomedicine, and
nanotechnology. Current work in our laboratory is fo-
cused on solid-phase synthesis, properties, and applica-
tions of oligoamides derived from the nucleoside 5′-azido
3′-carboxylic acids 3a-d reported herein.
1
The H NMR spectrum contained minor impurities: <2% of
the (2S,3S) diastereomer (well-separated doublet at 0.72 ppm)
and <10% total of not identified compounds, presumably the
syn diastereomers (see Supporting Information). The following
two experiments (for NMR spectra, see Supporting Informa-
tion) were used to clarify the sense of asymmetric induction
by menthyl auxiliaries and catalyst 9 (matched vs mismatched
combinations) and to assign the 1H NMR spectra of the
diastereomers. Reaction of (1′R,2′S,5′R)-menthyl glyoxylate
(the enantiomer of 7b) catalyzed by SnCl4 gave the products
with dr of 3:1 (the major product was the enantiomer of 6b).
The mismatched double asymmetric reaction of (1′R,2′S,5′R)-
menthyl glyoxylate (the enantiomer of 7b) catalyzed by 9 gave
the same products with dr of 1:5. In all cases, the observed
stereoselectivity was in good agreement with our previous
study25 and literature precedents.29,30 13C NMR (CDCl3, 75.4
MHz): δ 174.2, 135.7, 133.9, 129.7, 127.8, 118.2, 75.9, 72.7,
61.4, 47.1, 43.9, 41.1, 34.2, 33.9, 31.5, 27.0, 26.3, 23.4, 22.1,
20.9, 19.4, 16.4.
For experimental procedure for synthesis of 6c, see Sup-
porting Information.
(3R,4S,5S)-4-[2-(tert-Butyldiphenylsilanoxy)ethyl]-3-
hydroxy-5-iodomethyl-2-dihydrofuranone (Mixture of 5a
and 5b). Iodine (0.47 g, 1.85 mmol) was added to the solution
of (2R,3R)-3-[2-(tert-butyldiphenylsilanoxy)ethyl]-2-hydroxy-
4-pentenoic acid dimethylamide (6c; 0.20 g, 0.47 mmol) in THF
(2 mL) and saturated aqueous NaHCO3 (1 mL) at 0 °C. The
mixture was stirred for 26 h at 0 °C. Saturated aqueous
Na2S2O3 (3 mL) and ether (3 mL) were added to quench the
reaction. The aqueous layer was extracted with ether (5 × 3
mL). The combined organic layers were dried (Na2SO4),
concentrated, and purified by silica gel column chromatogra-
phy (5-20% of ethyl acetate in hexanes, stepwise gradient by
5%) to afford a nonseparable mixture of C4-C5 trans (5a) and
cis (5b) iodolactones in a ratio of 4:1. Yield: 0.16 g, 65%. TLC
Rf ) 0.27 CH2Cl2/2-propanol (100:1). IR: 3430, 1778 cm-1. 1H
NMR (CDCl3, 300 MHz; major diastereomer (C3-C4 trans)):
δ 7.68-7.65 (m, 4H), 7.48-7.38 (m, 6H), 4.54-4.48 (m, 1H),
4.32-4.26 (m, 1H), 3.78-3.65 (m, 3H), 3.44-3.28 (m, 2H),
2.63-2.54 (m, 1H), 2.07-1.96 (m, 1H), 1.72-1.61 (m, 1H), 1.07
(s, 9H). 13C NMR (CDCl3, 75.4 MHz): δ 175.6, 135.7, 132.8,
132.8, 130.1, 128.0, 81.7, 69.1, 62.1, 43.7, 28.6, 27.0, 19.2, 6.2.
Experimental Section
(2R,3R)-3-[2-(tert-Butyldiphenylsilanoxy)ethyl]-2-hy-
droxy-4-pentenoic Acid (1′S, 2′R, 5′S)-Menthyl Ester (6b).
The reaction was done in a glovebox under an atmosphere of
dry nitrogen. 2,2′-Isopropylidenebis[(R)-4-phenyl-2-oxazoline]
(9; 0.45 g, 1.35 mmol) and CuCl2 (0.18 g, 1.35 mmol) in dry
For experimental procedures for synthesis of 4, 11a, and
11b, see Supporting Information.
2′-O-Acetyl-5′-azido-3′-[2-(tert-butyldiphenylsilanoxy)-
ethyl]-3′,5′-dideoxyuridine (12a). A solution of 1,2-di-O-
acetyl-5-azido-3-[2-(tert-butyldiphenylsilanoxy)ethyl]-3,5-dide-
oxyribofuranose (4; 0.31 g, 0.58 mmol), O,O′-bis(trimethyl-
silyl)uracil35a (0.30 g, 1.17 mmol), and trimethylsilyl trifluo-
romethanesulfonate (0.26 g, 1.17 mmol) in CH2Cl2 (20 mL) was
(42) Compare the routes in: (a) Rozners, E.; Xu, Q. Org. Lett. 2003,
5, 3999-4001. (b) Xu, Q.; Rozners, E. Org. Lett. 2005, 7, 2821-2824.
9846 J. Org. Chem., Vol. 70, No. 24, 2005

Toward Total Synthesis of Amide-Linked RNA
stirred for 3 h. CH2Cl2 (80 mL) and saturated aqueous
NaHCO3 (80 mL) were added. The aqueous layer was extracted
with CH2Cl2 (3 × 80 mL). The combined organic layers were
dried (Na2SO4), concentrated, and purified by silica gel column
chromatography (1-3% of 2-propanol in CH2Cl2 stepwise
gradient by 1%) to afford 12a. Yield: 0.34 g, 99%. TLC Rf )
0.37 CH2Cl2/2-propanol (94:6). IR: 2104, 1745 cm-1. 1H NMR
(CDCl3, 300 MHz; major diastereomer): δ 10.02 (s, H), 7.65-
7.61 (m, 4H), 7.50-7.35 (m, 7H), 5.82-5.72 (m, 2H), 5.26 (d,
J ) 6.3 Hz, 1H), 4.05-4.00 (m, 1H), 3.80-3.62 (m, 3H), 3.47
(dd, J ) 4.2 Hz, 13.5 Hz, H), 2.68-2.58 (m, 1H), 2.02 (s, 3H),
1.72-1.48 (m, 2H), 1.06 (s, 9H). MS (ESI). Calcd for C29H35N5O6-
Si: 577.2. Found [M + H]+: 578.3.
2′-O-Acetyl-5′-azido-3′-[2-(tert-butyldiphenylsilanoxy)-
ethyl]-3′,5′-dideoxycytidine (12b′). Trimethylsilyl trifluo-
romethanesulfonate (0.34 g, 1.52 mmol) was added to a
solution of 4 (0.40 g, 0.76 mmol) and O,N-bis(trimethylsilyl)-
cytosine43 (0.39 g, 1.52 mmol) in 1,2-dichloroethane (10 mL)
at 0 °C. The solution was refluxed for 50 min, cooled to room
temperature, and diluted with cold CH2Cl2 (100 mL). Satu-
rated aqueous NaHCO3 (100 mL) was added. The aqueous
layer was extracted with CH2Cl2 (4 × 100 mL). The combined
organic layers were dried (Na2SO4), concentrated and purified
by silica gel column chromatography (4-8% of methanol in
CH2Cl2 stepwise gradient by 4%) to afford 12b′. Yield: 0.44 g,
99%. TLC Rf ) 0.14 CH2Cl2/2-propanol (94:6). IR: 2102, 1643
cm-1. 1H NMR (CDCl3, 300 MHz; major diastereomer): δ 7.66-
7.57 (m, 5H), 7.43-7.32 (m, 6H), 5.87 (d, J ) 7.5 Hz, H), 5.80
(s, H), 5.34 (d, J ) 5.7 Hz, H), 4.05-4.00 (m, 1H), 3.78-3.59
(m, 3H), 3.49 (dd, J ) 4.7 Hz, 13.7 Hz, H), 2.50-2.40 (m, 1H),
2.02 (s, 3H), 1.61-1.45 (m, 2H), 1.03 (s, 9H). MS (ESI). Calcd
for C29H36N6O5Si: 576.3. Found [M + H]+: 577.4.
as a single 3′,4′-trans diastereomer. Yield: 362 mg, 56%. TLC
1
Rf ) 0.42 CH2Cl2/2-propanol (94:6). IR: 2102, 1745 cm-1. H
NMR (CDCl3, 300 MHz): δ 8.18 (s, H), 8.01(s, H), 7.62-7.57
(m, 4H), 7.45-7.22 (m, 16H), 5.95 (s, H), 5.43 (d, J ) 5.7 Hz,
H), 4.19-4.13 (m, 1H), 3.79-3.56 (m, 4H), 3.08 (s, H), 2.42(s,
3H), 2.06 (s, 3H), 1.84-1.57 (m, 2H), 1.01 (s, 9H). 13C NMR
(CDCl3, 75.4 MHz): δ 169.8, 156.4, 154.2, 152.2, 150.4, 142.4,
141.9, 135.6, 135.6, 133.5, 133.4, 129.9, 129.3, 127.9, 127.9,
127.1, 121.4, 89.7, 83.8, 78.4, 61.8, 55.9, 52.3, 39.1, 27.7, 26.9,
25.2, 20.7, 19.2. MS (ESI). Calcd for C45H47N9O7Si: 853.3.
Found [M + H]+: 854.3.
2′-O-Acetyl-5′-azido-3′-(2-hydroxyethyl)-3′,5′-dideoxy-
uridine (13a). Acetic acid (33 µL, 0.58 mmol) was added to 1
M TBAF solution in THF (0.58 mL). To this solution of 1 M
TBAF/HOAc (1:1 mol/mol) was added 12a (67 mg, 0.12 mmol)
dissolved in THF (34 mL). The reaction mixture was stirred
for 2.75 h, diluted with ethyl acetate (100 mL), and applied
directly to silica gel chromatography (1-3% of methanol in
ethyl acetate, stepwise gradient by 2%) to give 41 mg of crude
product, which was purified by silica gel column (6-15% of
2-propanol in CH2Cl2 stepwise gradient by 3%) to give the
desired trans-alcohol product. This product was further puri-
fied by preparative HPLC (SUPELCOSIL PLC-SI, silica gel,
12 µm, 25 cm × 21.1 cm, elution with 4% methanol in CH2-
Cl2) to afford 13a as a single 3′,4′-trans diastereomer. Yield:
29 mg, 75%. TLC Rf ) 0.37 CH2Cl2/2-propanol (90:10). IR:
1
2108, 1680 cm-1, H NMR (CDCl3, 300 MHz): δ 9.23 (s, 1H),
7.54 (d, J ) 8.1 Hz, 1H), 5.75 (d, J ) 8.1 Hz, 1H), 5.69 (s, 1H),
5.53 (d, J ) 5.7 Hz, 1H), 4.10-4.06 (m, 1H), 3.86-3.55 (m,
4H), 2.68-2.58 (m, 1H), 2.15 (s, 3H), 1.76-1.52 (m, 2H). 13C
NMR (CDCl3, 75.4 MHz): δ 170.2, 163.6, 150.2, 140.7, 102.6,
91.9, 83.0, 77.9, 60.3, 51.7, 39.0, 27.2, 20.9. MS (ESI). Calcd
for C13H17N5O6 + Na: 362.1. Found [M + Na]+: 362.1.
2′-O-Acetyl-5′-azido-3′-[2-(tert-butyldiphenylsilanoxy)-
ethyl]-3′,5′-dideoxy-6 -N-benzoyladenosine (12c). SnCl4
(86.3 mg, 0.33 mmol) was added to a solution of 4 (87 mg, 0.17
mmol) and 6-N-benzoyladenine (39.6 mg, 0.17 mmol) in CH3-
CN (5 mL). After stirring for 30 min, CH2Cl2 (25 mL) and
saturated aqueous NaHCO3 (25 mL) were added. The aqueous
layer was extracted with CH2Cl2 (4 × 25 mL). The combined
organic layers were dried (Na2SO4), concentrated and purified
by silica gel column chromatography (2-10% of 2-propanol in
2:1 hexanes/ethyl acetate, stepwise gradient by 2%) to afford
12c as a single 3′,4′-trans diastereomer. Yield: 70 mg, 60%.
For experimental procedures for synthesis of 13b-d, see
Supporting Information.
2′-O-Acetyl-5′-azido-3′-carboxymethyl-3′,5′-dideoxyuri-
dine (3a). (Diacetoxyiodo)benzene (96.7 mg, 0.3 mmol) and
TEMPO (6.2 mg, 0.04 mmol) were added to the solution of 13a
(34 mg, 0.1 mmol) in CH3CN/H2O (1.2 mL/1.2 mL). After
stirring for 41 h, the mixture was freeze-dried and purified
by silica gel column chromatography using a gradient (3-9%,
stepwise by 2%) of methanol (containing 8% H2O) in CH2Cl2
to afford 3a. Yield: 28 mg, 80%; ee was not determined because
no baseline separation was achieved (see Supporting Informa-
tion). HPLC: Rt) 76.7 min (major enantiomer) and 81.1 min
(minor enantiomer) on Chiralcel OD-H 4.6 × 150 mm column
equipped with Chiralcel OD 4.6 × 50 mm precolumn; eluent,
0.5% of acetic acid and 10% of ethanol in hexanes; flow rate,
0.75 mL/min. TLC Rf ) 0.31 MeOH/CH2Cl2 (10:90). IR: 2108,
1711 cm-1. Anal. Calcd for (C13H15N5O7 + H2O): C, 42.05; H,
TLC Rf ) 0.39 CH2Cl2/2-propanol (94:6). IR: 2102, 1736 cm-1
.
1H NMR (CDCl3, 300 MHz): δ 8.79 (s, H), 8.26 (s, H), 8.06-
8.03 (m, 2H), 7.65-7.20 (m, 13H), 6.09 (s, 1H), 5.59 (d, J )
5.7 Hz, 1H), 4.23-4.17 (m, 1H), 3.79-3.55 (m, 4H), 3.28-3.18
(m, 1H), 2.10 (s, 1H), 1.85-1.57 (m, 2H), 1.04 (s, 9H). 13C NMR
(CDCl3, 75.4 MHz): δ 170.0, 164.7, 152.7, 151.3, 149.7, 141.9,
135.6, 135.6, 133.8, 133.5, 133.3, 132.9, 130.0, 129.0, 128.0,
127. 9, 123.4, 89.9, 83.9, 78.4, 61.5, 52.3, 39.1, 27.7, 26.9, 20.8,
19.2. MS (ESI). Calcd for C37H40N8O5Si + Na: 727.3. Found
[M + Na]+: 727.2.
1
4.61; N, 18.86. Found: C, 42.05; H, 4.28; N, 18.46. H NMR
(CDCl3/CD3OD, 5:1, 300 MHz): δ 7.56 (d, J ) 8.1 Hz, 1H),
5.78 (d, J ) 8.1 Hz, 1H), 5.78 (d, J ) 2.4 Hz, 1H), 5.48 (dd, J
) 1.8 Hz, J ) 6.9 Hz, 1H), 4.11-4.06 (m, 1H), 3.79 (dd, J )
2.7 Hz, J ) 13.2 Hz, 1H), 3.56 (dd, J ) 4.2 Hz, J ) 13.5 Hz,
1H), 2.96-2.86 (m, 1H), 2.59-2.35 (m, 2H), 2.13 (s, 3H). 13C
NMR (CDCl3/CD3OD, 75.4 MHz): δ 173.1, 170.0, 164.0, 150.6,
140.4, 102.8, 90.5, 82.0, 77.6, 51.8, 38.4, 30.2, 20.3. HRMS
(ESI). Calcd for C13H15N5O7 [M + H]+: 354.1049. Found:
354.1028.
2′-O-Acetyl-5′-azido-3′-[2-(tert-butyldiphenylsilanoxy)-
ethyl]-3′,5′-dideoxy-2-N-acetyl-6-O-diphenylcarbamoylgua-
nosine (12d). Bis(trimethylsilyl)acetamide (BSA; 0.62 g, 3.06
mmol) was added to a solution of 2-N-acetyl-6-O-diphenylcar-
bamoylguanine37 (0.59 g, 1.53 mmol) in 1,2-dichloroethane (22
mL). The mixture was refluxed for 10 min, cooled to room
temperature, and added to a solution of 4 (0.40 g, 0.76 mmol)
in 1,2-dichloroethane (8 mL). Trimethylsilyl trifluoromethane-
sulfonate (0.34 g, 1.53 mmol) was added dropwise; the brown
solution was refluxed for 1 h, cooled to room temperature, and
diluted with CH2Cl2 (150 mL). Saturated aqueous NaHCO3
(150 mL) was added, and the aqueous layer was extracted with
CH2Cl2 (3 × 150 mL). The combined organic layers were dried
(Na2SO4) and concentrated. The white residue was dissolved
in CH2Cl2 (100 mL) and filtered. The filtrate was concentrated
and purified by silica gel column chromatography (7-21% of
2-propanol in hexanes, stepwise gradient by 7%) to afford 12d
2′-O-Acetyl-5′-azido-3′-carboxymethyl-3′,5′-dideoxy-4-
N-propionylcytidine (3b). Dess-Martin periodinane (15 wt
% solution in CH2Cl2, 0.38 mL, 0.18 mmol) was added to the
solution of 2′-O-acetyl-5′-azido-3′-(2-hydroxyethyl)-3′,5′-dideoxy-
4-N-propionylcytidine (13b; 36 mg, 0.091 mmol) in CH2Cl2 (1.9
mL). The solution was stirred for 1.5 h. Saturated aqueous
Na2S2O3 (0.6 mL) was added to quench the reaction. The
aqueous layer was extracted with CH2Cl2 (4 × 2 mL). Com-
bined organic layers were washed by aqueous phosphate buffer
(pH ) 9.2; 4 × 1 mL). The organic layer was concentrated to
give the intermediate aldehyde as a white residue that was
used in the next step without further purification.
(43) Nishimura, T.; Iwai, I. Chem. Pharm. Bull. 1964, 12, 352-356.
J. Org. Chem, Vol. 70, No. 24, 2005 9847

Rozners and Liu
To the solution of aldehyde in tert-butyl alcohol/H2O (1:1,
5.5 mL) were added a mixture of 2-methyl-2-butene (2 M in
THF, 0.23 mL, 0.46 mmol), NaH2PO4 (27.4 mg, 0.23 mmol),
and NaClO2 (technical 80%, 36.1 mg, 0.32 mmol). After stirring
for 25 min, saturated aqueous Na2S2O3 (1 mL) was added. The
solution was freeze-dried and purified by silica gel column
chromatography starting with 3% of methanol in CH2Cl2
followed by a gradient (3-9%, stepwise by 2%) of methanol
(containing 8% H2O) in CH2Cl2 to afford 3b. Yield: 32 mg, 86%,
ee 97.8% (see Supporting Information). HPLC: Rt ) 45.2 min
(major enantiomer) and 64.2 min (minor enantiomer) on
Chiralcel OD-H 4.6 × 150 mm column equipped with Chiralcel
OD 4.6 × 50 mm precolumn; eluent, 0.5% of acetic acid and
10% of ethanol in hexanes; flow rate, 0.75 mL/min. TLC Rf )
0.38 MeOH/CH2Cl2 (10:90). IR: 2106, 1728 cm-1. Anal. Calcd
for (C16H20N6O7 + 0.5H2O): C, 46.04; H, 5.07; N, 20.14.
TLC Rf ) 0.43 MeOH/CH2Cl2 (10:90). IR: 2106, 1705 cm-1
.
Anal. Calcd for (C21H22N8O7 + H2O): C, 50.60; H, 4.45; N,
1
22.48. Found: C, 50.97; H, 4.14; N, 22.14. H NMR (CDCl3/
CD3OD, 5:1, 300 MHz): δ 8.04 (s, 1H), 8.38 (s, 1H), 8.10-8.07
(m, 2H), 7.65-7.51(m, 3H), 6.17 (s, 1H), 5.82 (d, t, J ) 6 Hz,
1H), 4.29-4.23 (m, 1H), 3.82-3.60 (m, 2H), 3.43-3.33 (m, 1H),
2.69-2.46 (m, 2H), 2.18 (s, 3H). 13C NMR (CDCl3, 75.4 MHz):
δ 174.8, 170.0, 165.5, 152.3, 151.8, 149.9, 142.3, 133.4, 133.0,
128.8, 128.4, 123.6, 89.3, 83.2, 78.4, 52.1, 38.9, 30.2, 20.7.
HRMS (ESI). Calcd for C21H20N8O6 [M + H]+: 481.1584.
Found: 481.1573
2′-O-Acetyl-5′-azido-3′-carboxymethyl-3′,5′-dideoxy-2-
N-acetyl-6-O-diphenylcarbamoylguanosine (3d). (Diace-
toxyiodo)benzene (73.8 mg, 0.23 mmol), TEMPO (4.76 mg, 0.03
mmol), and NaOAc (12.5 mg, 0.15 mmol) were added to the
solution of 2′-O-acetyl-5′-azido-3′-(2-hydroxyethyl)-3′,5′-dideoxy-
2-N-acetyl-6-O-diphenylca rbamoylguanosine (13d; 47 mg,
0.076 mmol) in CH3CN/H2O (1.2 mL/1.2 mL). After stirring
for 4 days at 0 °C, the mixture was freeze-dried and purified
by silica gel column chromatography starting with 3% metha-
nol in CH2Cl2 followed by a gradient (3-9%, stepwise by 2%)
of methanol (containing 8% H2O) in CH2Cl2 to afford 3d.
Yield: 42 mg, 87%; ee 95% (see Supporting Information).
HPLC: Rt ) 51.0 min (minor enantiomer) and 66.8 min (major
enantiomer) on Chiralcel OD-H 4.6 × 150 mm column equipped
with Chiralcel OD 4.6 × 50 mm precolumn; eluent, 0.5% of
acetic acid and 10% of ethanol in hexanes; flow rate, 0.75 mL/
1
Found: C, 45.83; H, 4.88; N, 19.93. H NMR (CDCl3/CD3OD,
5:1, 300 MHz): δ 8.08 (d, J ) 7.5 Hz, H), 7.55 (b, 1H), 5.84 (s,
1H), 5.56(d, J ) 6.0 Hz, H), 4.19-4.13 (m, 1H), 3.90-3.84 (m,
1H), 3.67-3.62 (m, 1H), 2.89-2.79 (m, 1H), 2.57-2.32 (m, 1H),
2.15 (s, 3H), 1.21 (t, J ) 7.5 Hz, 3H). 13C NMR (CDCl3/CD3-
OD, 75.4 MHz): δ 174.9, 169.8, 163.0, 155.8, 150.6, 144.4, 97.3,
91.6, 82.7, 77.8, 51.7, 38.3, 30.6, 20.4, 8.7. HRMS (ESI). Calcd
for C16H20N6O7 [M + H]+: 409.1471. Found: 409.1442.
2′-O-Acetyl-5′-azido-3′-carboxymethyl-3′,5′-dideoxy-6-
N-benzoyladenosine (3c). Dess-Martin periodinane (15 wt
% solution in CH2Cl2, 0.25 mL, 0.12 mmol) was added to the
solution of 2′-O-acetyl-5′-azido-3′-(2-hydroxyethyl)-3′,5′-dideoxy-
6-N-benzoyladenosine (13c; 28 mg, 0.06 mmol) in CH2Cl2 (1.3
mL). The reaction mixture was stirred for 1 h and diluted by
CH2Cl2 (5 mL). Saturated aqueous NaHCO3 containing an
excess of Na2S2O3 (4 mL) was added to quench the reaction.
This mixture was stirred for 20 min. The organic layer was
washed by saturated aqueous NaHCO3 containing an excess
of Na2S2O3 (4 mL) and aqueous phosphate buffer (pH ) 9.2; 4
mL), dried (Na2SO4), concentrated to give the intermediate
aldehyde as a white residue that was used in the next step
without further purification.
min. TLC Rf ) 0.5 MeOH/CH2Cl2 (10:90). IR: 2104, 1741 cm-1
.
Anal. Calcd for (C29H27N9O8 + H2O): C, 53.79; H, 4.51; N,
1
19.47. Found: C, 53.95; H, 4.32; N, 19.36. H NMR (CDCl3/
CD3OD, 5:1, 300 MHz): δ 8.12 (s, H), 7.35-7.14 (m, 10H), 5.23
(s, H), 5.70 (d, J ) 5.7 Hz, 1H), 4.14-4.08 (m, 1H), 3.66-3.50
(m, 2H), 3.41(b, 1H), 2.57-2.36 (m, 2H), 2.31(s, 3H), 2.05(s,
3H). 13C NMR (CDCl3/CD3OD, 75.4 MHz): δ 174.4, 170.3,
156.2, 154.1, 151.8, 150.3, 143.8, 141.8, 129.4, 127.3, 121.5,
90.3, 83.6, 78.7, 52.6, 39.2, 30.4, 25.2, 20.7. HRMS (ESI). Calcd
for C29H27N9O8 [M + H]+: 630.2061. Found: 630.2003.
To the solution of aldehyde in tert-butyl alcohol/H2O (1:1,
3.8 mL) were added a mixture of 2-methyl-2-butene (2 M in
THF, 0.15 mL, 0.3 mmol), NaH2PO4 (18 mg, 0.15 mmol), and
NaClO2 (technical 80%, 23.7 mg, 0.21 mmol). After stirring
for 1 h, saturated aqueous Na2S2O3 (2 mL) was added. The
solution was freeze-dried and purified by silica gel column
chromatography starting with 3% of methanol in CH2Cl2
followed by a gradient (3-9%, stepwise by 2%) of methanol
(containing 8% H2O) in CH2Cl2 to afford 3c. Yield: 25 mg, 86%;
ee was not determined because no separation was achieved
(see Supporting Information). HPLC: Rt ) 83.2 min (single
peak) on Chiralcel OD-H 4.6 × 150 mm column equipped with
Chiralcel OD 4.6 × 50 mm precolumn; eluent, 0.5% of acetic
acid and 10% of ethanol in hexanes; flow rate, 0.75 mL/min.
Acknowledgment. We thank Northeastern Univer-
sity (startup funds and RSDF grant to E.R.) and the
donors of the Petroleum Research Fund, administered
by the ACS (37599-AC1), for support of this research.
We thank Prof. Paul Vouros and James Glick for mass
spectroscopy experiments.
Supporting Information Available: Experimental pro-
cedures, spectral data, and copies of 1H and 13C aeNMR data.
This material is available free of charge via the Internet at
http://pubs.acs.org.
JO0515879
9848 J. Org. Chem., Vol. 70, No. 24, 2005