Five- and six-membered conformationally locked 2′,4′- carbocyclic ribo-thymidines: Synthesis, structure, and biochemical studies

Article abstract of DOI:10.1021/ja071106y

Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant double bond at C4′ and the radical center at C2′ of the ribofuranose ring of thymidine have been used as key steps to synthesize North-type conformationally c

Full text of DOI:10.1021/ja071106y

Published on Web 06/07/2007
Five- and Six-Membered Conformationally Locked
2′,4′-Carbocyclic ribo-Thymidines: Synthesis, Structure, and
Biochemical Studies
Puneet Srivastava, Jharna Barman, Wimal Pathmasiri, Oleksandr Plashkevych,
Małgorzata Wenska, and Jyoti Chattopadhyaya*
Contribution from the Department of Bioorganic Chemistry, Box 581, Biomedical Center,
Uppsala UniVersity, SE-75123 Uppsala, Sweden
Received February 15, 2007; E-mail: jyoti@boc.uu.se
Abstract: Two unusual reactions involving the 5-hexenyl or the 6-heptenyl radical cyclization of a distant
double bond at C4′ and the radical center at C2′ of the ribofuranose ring of thymidine have been used as
key steps to synthesize North-type conformationally constrained cis-fused bicyclic five-membered and six-
membered carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-ENA-T) in high yields.
Their structures have been confirmed unambiguously by long range 1H-13C NMR correlation (HMBC),
TOCSY, COSY, and NOE experiments. The carbocyclic-LNA-T and carbocyclic-ENA-T were subsequently
incorporated into the antisense oligonucleotides (AONs) to show that they enhance the Tm of the modified
AON/RNA heteroduplexes by 3.5-5 °C and 1.5 °C/modification for carbocyclic-LNA-T and carbocyclic-
ENA-T, respectively. Whereas the relative RNase H cleavage rates with carbocyclic-LNA-T, carbocyclic-
ENA-T, aza-ENA-T, and LNA-T modified AON/RNA duplexes were found to be very similar to that of the
native counterpart, irrespective of the type and the site modification in the AON strand, a single incorporation
of carbocyclic-LNA and carbocyclic-ENA into AONs leads to very much more enhanced nuclease stability
in the blood serum (stable >48 h) as compared to that of the native (fully degraded <3 h) and the LNA-
modified AONs (fully degraded <9 h) and aza-ENA (≈85% stable in 48 h). Clearly, remarkably enhanced
lifetimes of these carbocyclic-modified AONs in the blood serum may produce the highly desired
pharmacokinetic properties because of their unique stability and consequently a net reduction of the required
dosage. This unique quality as well as their efficient use as the AON in the RNase H-promoted cleavage
of the target RNA makes our carbocyclic-LNA and carbocyclic-ENA modifications excellent candidates as
potential antisense therapeutic agents.
the phosphorothioate7 backbone-modified oligonucleotides have
found some use in therapeutics.8 Recent years have seen
Introduction
Antisense1 oligonucleotides (AON) can potentially inhibit
protein synthesis by translation arrest/steric blocking or by
RNase H-mediated degradation of the AON/RNA hybrid.2 Other
methods of gene silencing include formation of triplexes by
base-pairing with double-helical DNA (antigene effects),3 or
RNA interference (RNAi), by a short double-stranded RNA
(siRNA).4 The in ViVo application of the gene silencing
technology warrants chemical modification of the antisense/
antigene or siRNA strand to enhance the target affinity,
specificity, stability in the blood serum, and tissue specific
delivery in order to improve overall pharmacokinetic properties.5
Various modifications of oligonucleotide involving sugar,
phosphodiester linkage, and nucleobase are known.6 Of these
development of conformationally constrained bicyclic (Figure
1) and tricyclic nucleotides,9-11 in which the sugar is locked in
a definite puckered conformation. Such oligonucleotides show
promising properties with respect to target RNA binding and
nuclease resistance.12,13 Among several molecules reported, short
nucleotides containing LNA12 (tBNA14) modifications have
shown unprecedented thermal stability (+3 to +8 °C per modifi-
cation depending upon the sequence context). The enhanced
target binding property of the North-conformationally con-
strained bicyclic sugar units in these nucleotides has been
(7) Eckstein, F. Annu. ReV. Biochem. 1985, 54, 367-402.
(8) Holmlund, J. T. Ann. N.Y. Acad. Sci. 2003, 1002 (Therapeutic Oligonucle-
otides), 244-251.
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Meldgaard, M.; Olsen, C. E.; Wengel, J. Tetrahedron 1998, 54 (14), 3607-
3630.
(13) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; Sone,
J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003, 11
(10), 2211-2226.
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T. Tetrahedron Lett. 1997, 38 (50), 8735-8738.
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9
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J. AM. CHEM. SOC. 2007, 129, 8362-8379
10.1021/ja071106y CCC: $37.00 © 2007 American Chemical Society

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
Figure 1. Structures of various bicyclic North-type conformationally constrained R/â-D/L-pentofuranosyl nucleosides: (A) LNA;12,14 (B) amino-LNA;16 (C)
xylo-LNA;21 (D) R-L-LNA;22 (E) â-bicyclonucleoside;23 (F) 1′,2′-oxetane-bridged;17 (G) 1′,2′-azetidine-bridged;18 (H) ENA;13,24-26 (I) aza-ENA;20 (J) PrNA;13
(K) unsaturated carbocyclic analogue of LNA;27 (L) saturated carbocyclic analogue of LNA.27
attributed to the improved stacking between the nearest neigh-
bors and quenching of concerted local backbone motions by
LNA nucleotides in ssLNA so as to reduce the entropic penalty
in the free energy of stabilization for the duplex formation with
RNA.15 These bicyclic constrained analogues have thus been
extensively used to facilitate the down-regulation of genes.5 The
features of LNA/BNA have led to the synthesis of a number of
closely related analogues, in which the 2′,4′-bridge has been
altered16 or a new type of 1′,2′-bridged constraint has been
introduced, such as in the 1′,2′-oxetane17 or 1′,2′-azetidine18
analogue. Such modifications show similar or moderately
depressed Tm properties when compared to LNA, but the
nuclease resistance or RNase H recruitment properties (for
example, ENA,19 PrNA,13 and aza-ENA20) have turned out to
be relatively more favorable than those exhibited by the LNA-
containing AONs.
generally using the electrostatic interactions29 to neutralize, for
example, the phosphates charge, as well as to influence
interaction of modified oligonucleotides with other nucleotides
and/or enzymes present in the system.30 We and others31,32 have
argued that replacement of the hydrophilic 2′-oxygen of LNA12
or ENA19 or 2′-nitrogen from their amino analogues16 by the
hydrophobic carbocyclic analogues would steer both target
affinity as well as the nuclease stability in the blood serum
because of change in the immediate shell of hydration.
Recently,27 the ring-closing metathesis approach had been
employed to synthesize two carbocyclic analogues of ENA with
three carbons locking the C2′ and C4′ (compounds K/L in Figure
1). These carbocyclic analogues had been incorporated into
AONs with a natural phosphodiester backbone which leads to
increased thermal stability (Tm) by 2.5-4.5 °C/modification with
the complementary RNA. However, no blood serum or 3′-
exonuclease stability or RNase H recruitment capability of these
carbocyclic analogues has so far been reported.
Here we report a novel synthetic strategy for the carbocyclic
analogues of LNA12 and ENA13 thymidines (carbocyclic-LNA-T
and carbocyclic-ENA-T), which have been accomplished using
an intramolecular free-radical ring-closure reaction between a
radical generated at C2′ and a strategically placed double bond
in the modified pentofuranose moiety of the nucleoside. We
have subsequently incorporated carbocyclic-LNA-T [T(5-carbo)]and
carbocyclic-ENA-T [with T(6-carbo)] into the AONs and studied
their blood serum stabilities, as well as their RNase H recruit-
ment capabilities, and compared these properties with iso-
sequential LNA12,14- and aza-ENA20-containing counterparts.
Studies28 with modified nucleotides show that substituents
play an important role in conformational steering, controlling
hydration, inducing hydrophobic/hydrophilic interactions, and
(15) Petersen, M.; Nielsen, C. B.; Nielsen, K. E.; Jensen, G. A.; Bondensgaard,
K.; Singh, S. K.; Rajwanshi, V. K.; Koshkin, A. A.; Dahl, B. M.; Wengel,
J.; Jacobsen, J. P. J. Mol. Recognit. 2000, 13 (1), 44-53.
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10039.
(17) Pradeepkumar, P. I.; Cheruku, P.; Plashkevych, O.; Acharya, P.; Gohil,
S.; Chattopadhyaya, J. J. Am. Chem. Soc. 2004, 126, 11484-11499.
(18) Honcharenko, D.; Varghese, O. P.; Plashkevych, O.; Barman, J.; Chatto-
padhyaya, J. J. Org. Chem. 2006, 71, 299-314.
(19) Koizumi, M. Curr. Opin. Mol. Ther. 2006, 8, 144-149.
(20) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Honcharenko,
D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128 (47), 15173-15187.
(21) Babu, B. R.; Raunak; Poopeiko, N. E.; Juhl, M.; Bond, A. D.; Parmar, V.
S.; Wengel, J. Eur. J. Org. Chem. 2005, 11, 2297-2321.
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(9), 1656-1659.
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T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. Lett. 2001, 12 (1), 73-
76.
Results and Discussion
The intramolecular radical addition reactions to the tethered
double bond involving both 5-hexenyl and 6-heptenyl radicals33
(25) Morita, K.; Yamate, K.; Kurakata, S.; Abe, K.; Imanishi, T.; Koizumi, M.
Nucleic Acids Res. Sup. 2002, 2, 99-100.
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Koizumi, M. Nucleosides, Nucleotides Nucleic Acids 2003, 22, 1619-1621.
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Manoharan, M.; Egli, M. Nat. Struct. Mol. Biol. 1999, 6 (6), 535-539.
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M. Org. Lett. 2003, 5 (12), 2017-2020.
9
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A R T I C L E S
Srivastava et al.
have been well studied by Beckwith,34,35 Baldwin,36-38 Giese,39
Curran,40,41 Stork,42 and more recently by Rajanbabu.43-46 The
efficiency and regiochemistry47 of such intramolecular cycliza-
tion addition reactions have been shown to be controlled by (i)
the initial radical structure, (ii) the steric effects resulting from
the olefin substitution pattern,34 and (iii) the geometric con-
straints on the chain linking the radical center, and the tethered
double bond.47
In the kinetically controlled rearrangement of variously
substituted 5-hexenyl radicals, the preference for the exo versus
endo mode, i.e., the formation of the five-membered over the
six-membered ring, is well understood in terms of a relatively
strain-free chairlike transition state47 accommodating the ste-
reoelectronic requirements of radical addition to the double
bond. The model also provides satisfactory explanation for the
observed stereoselectivities in the cyclization of 2-, 3-, and
4-substituted hexenyl radicals (5-hexenyl nomenclature48) on
account of the preferred adoption of pseudo-equatorial positions
by the substituents in the respective transition states.45
It is known43 that the ring-closure reaction involving cyclic
5-hexenyl radicals (i.e., radical as a part of the cyclic ring) is
similar to that of the open-chain systems except that the ring
imposes steric constraints on the stereochemical outcome of the
reaction. The initial radical forms the most stable structure with
bulky substituents in the equatorial/pseudo-equatorial position,45
when the radical center is a part of the sugar ring. Ring-closure
reaction occurs via attack of a radical center oriented to the
pseudo-equatorial position on the axially substituted alkenyl
chain resulting in the cis-fused rings.35 It is also known that
the alkenyl chain preferentially occupies an axial position, since
an equatorial alkenyl chain results in poor overlap of the
semioccupied molecular orbitals and π* orbitals.35
The substituents play their role on account of their steric bulk
or stereoelectronic nature as they interact with the ring atoms/
substituents at different positions of alkenyl chain during
intramolecular 5-hexenyl cyclization.49 For example, it has been
shown that substituents on the C1 and C4 atoms (5-hexenyl
nomenclature48) of the initial radical are the major factors
dictating stereochemistry of the newly formed 1,5-bond. Thus
when both C1 and C4 substituents are present, predominantly
a 1,5-cis isomer is formed.44 With no substitution at C4 (i.e.,
C4-deoxy), a mixture of 1,5-cis and 1,5-trans-fused products is
formed. In the presence of vinylic oxygen the boatlike transition
state is stabilized resulting in formation of 1,5-trans isomer.46
In contradistinction, the formation of six-membered rings by
free radical reactions involving 6-exo cyclizations of heptenyl
system presents at least two problems: first, the rate constant
of 6-hepetenyl cyclization is ≈40 times slower than the
corresponding 5-hexenyl cyclization; thus, the competing radical
quenching by reduction with nBu3SnH becomes a serious
problem. Second, the endo mode of cyclization is only ≈7 times
less rapid than the exo mode of cyclization; thus, formation of
the endo products also competes with the six-membered
products.48 Thus far, most of the fused bicyclic ring formation
reactions studied have been of 1,2-type,51-58 i.e., the radical
center is located at the neighboring carbon to the tethered double
bond.
Here we present two unusual reactions involving the 5-hex-
enyl or the 6-heptenyl radical cyclization by forming a C-C
bond from a distant double bond at C4′ toward the C2′ radical
center of the ribofuranose ring of thymidine (Scheme 1). The
2′,4′-free-radical cyclization is a key step in our synthetic
strategy to efficiently yield North-type conformationally con-
strained cis-fused bicyclic five-membered and six-membered
carbocyclic analogues of LNA (carbocyclic-LNA-T) and ENA
(carbocyclic-ENA-T), as it has been originally used in the
construction of conformationally constrained nucleosides by
Wengel’s12 and Imanishi’s14 group in the ionic ring-closure
reaction. To the best of our knowledge the only other similar
case of radical cyclization reported had involved a tethered
double bond (butenyl side chain) and a cyclic radical (Figure
2) in the constrained norbornyl system.50 The double-bond chain
and the free radical in the norbornyl system were positioned at
C1 and C3 to each other with the stereochemistry being dictated
by topological and steric constraints with four asymmetric
centers, giving the 1,6-cis-fused (37%) and 1,6-trans-fused
(25%) cyclohexyl ring through the participation of presumably
chairlike 6-heptenyl cyclization. Clearly, in our case (Figure 3)
the site of the propenyl or the butenyl substituent at C4′ with
respect to the radical center at C2′ in the flexible five-membered
pentofuranose ring59 led us to assume that our free-radical
cyclization reaction may have poorer steric and stereoelectronic
control on the formation of a chairlike or/and boatlike transition
state to give the 1,3-cis-fused ring-closure product(s).
1.0. Synthesis of the Carbocyclic-LNA-T Phosphoramidite
(14). The synthesis starts from a known sugar precursor 112
which was selectively benzylated using a reported procedure20
to give the corresponding benzylated product 2. The primary
alcohol in sugar 2 was oxidized to the corresponding aldehyde
(33) Hanessian, S.; Dhanoa, D. S.; Beaulieu, P. L. Can. J. Chem. 1987, 65 (8),
1859-1866.
(34) Beckwith, A. L. J. Tetrahedron 1981, 37 (18), 3073-3100.
(35) Beckwith, A. L. J.; Phillipou, G.; Serelis, A. K. Tetrahedron Lett. 1981,
29, 2811-2814.
(50) Bakuzis, P.; Campos, O. O. S.; Bakuzis, M. L. F. J. Org. Chem. 1976, 41
(20), 3261-3264.
(51) Lopez, J. C.; Fraser-Reid, B. J. Am. Chem. Soc. 1989, 111 (9), 3450-
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(36) Baldwin, J. E. J. Chem. Soc. Chem. Commun. 1976, 18, 738-741.
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R. C. J. Chem. Soc. Chem. Commun. 1976, 18, 736-738.
(39) Giese, B. Angew. Chem. 1983, 95 (10), 771-782.
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(52) Lopez, J. C.; Gomez, A. M.; Fraser-Reid, B. J. Chem. Soc. Chem. Commun.
1993, 9, 762-764.
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3871-3878.
(42) Stork, G.; Baine, N. H. J. Am. Chem. Soc. 1982, 104 (8), 2321-2323.
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300.
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Tetrahedron 1992, 48 (2), 349-370.
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47 (46), 9675-9690.
(46) RajanBabu, T. V.; Fukunaga, T.; Reddy, G. S. J. Am. Chem. Soc. 1989,
111 (5), 1759-1769.
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3941.
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5272.
(48) Motherwell, W. B.; Crich, D. Free radical chain reactions in organic
synthesis; Academic Press: New York, 1992, 0-12-508760-8.
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(59) Thibaudeau, C.; Acharya, P.; Chattopadhyaya, J. Stereoelectronic Effects
in Nucleosides & Nucleotides and their Structural Implications; Uppsala
University Press: Uppsala, 1999, 91-506-1351-0.
9
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2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
Scheme 1 a
a Reagents and conditions: (i) NaH, BnBr, -5 °C to rt overnight; (ii) oxalyl chloride, DMSO, -78 °C, DIPEA, DCM; (iii) PPh3+CH3Br-, 1.6 M butyllithium
in hexane, THF, -78 °C to rt overnight; (iv) 9-BBN, THF, 3 N NaOH, 33% H2O2; (v) oxalyl chloride, DMSO, -78 °C, DIPEA, DCM, PPh3+CH3Br-, 1.6
M butyllithium in hexane, THF, -78 °C to rt overnight; (vi) acetic acid, acetic anhydride, triflic acid; (vii) persilylated thymine, TMSOTf, CH3CN, 0 °C
to rt overnight; (viii) 27% methanolic NH3, overnight; (ix) DMAP, phenyl chlorothionoformate, pyridine; (x) Bu3SnH, toluene, AIBN, 4 h; (xi) 20% Pd(OH)2/
C, Ammonium Formate, Methanol, Reflux, 8 h; (xii) DMTr-Cl, Pyridine, Overnight; (xiii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, DIPEA,
dry THF, overnight. The IUPAC numbering according to von Bayer nomenclature for bicyclic systems is used in the Experimental Section. Abbreviations:
Bn (benzyl); Ac (acetyl); 9-BBN (9-borobicyclo(3.3.1)nonane); THF (tetrahydrofuran); AIBN, [2,2′-azobis(2-methyl-propionitrile)]; DMTr (4,4′-dimethoxytrityl;
DIPEA, diisopropylethylamine).
modified Vorbruggen reaction62 involving in situ silylation of
thymine and subsequent trimethylsilyl triflate-mediated coupling
to give thymine nucleoside 8 in 80% yield in two steps from 6.
The â-configuration of the product 6 was confirmed by a 1D
differential NOE experiment, which showed 3% enhancement
of H2′ and a 1% enhancement of H3′ upon irradiation of H6
(dH6-2′ ≈ 2.3 Å for â anomer). Deacetylation of compound 8
Figure 2. Heptenyl cyclization of 7-norborneyl radical.50
using 27% methanolic ammonia overnight and subsequent
esterification using phenyl chlorothioformate yielded the desired
precursor 10 for radical cyclization. The key free-radical
cyclization reaction was carried out using Bu3SnH with radical
initiator AIBN at 115 °C in degassed (N2) toluene. To ensure
that the radical generated has an adequate lifetime to capture
the double bond before it is quenched by a hydrogen radical,
the concentrations of Bu3SnH and AIBN were maintained
through high dilution and slow dropwise addition. The 5-hexenyl
type exo mode cyclization of the radical to the C4′-propenyl
double bond yielded exclusively the expected34 five-membered
2′,4′-cis-fused carbocyclic product with a bicyclo[2.2.1]heptane
skeleton as an inseparable diastereomeric mixture of compounds
11a (major compound 70%, 7′R) and 11b (minor compound
30%, 7′S).
3 employing Swern oxidation.60 The vinyl chain at C4 was then
introduced by the Wittig reaction61 on the crude aldehyde 3 to
give the vinyl sugar 4 (87% in two steps from 2). The olefin 4
was converted to the C4-hydroxyethyl derivative via successive
hydroboration-oxidation using 9-BBN/NaOH-H2O2 to give 5
in 95% yield, which was again subjected to Swern oxidation60/
Wittig61 reaction to give the required C4-allylated sugar 6 (70%
in two steps from 5) with a strategically placed propenyl side
chain at C4 for 5-hexenyl type free radical cyclization.
Compound 6 was subjected to acetolysis using a mixture of
acetic anhydride, acetic acid, and triflic acid to give the
corresponding diacetate 7 quantitatively as an R/â anomeric
mixture (single spot on TLC and proven by 1H NMR). The crude
diacetate 7, after bicarbonate workup, was subjected to a
The formation of the bicyclic nucleosides 11a and 11b, with
a North-fused conformationally constrained pentofuranosyl
(60) Mancuso, A. J.; Brownfain, D. S.; Swern, D. J. Org. Chem. 1979, 44 (23),
4148-4150.
(61) Wittig, G.; Schlosser, M. Angew. Chem. 1960, 72, 324.
(62) Vorbru¨ggen, H.; Ho¨fle, G. Chem. Ber 1981, 114, 1256-1258.
9
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A R T I C L E S
Scheme 2 a
Srivastava et al.
a Reagents and conditions: (i) 9-BBN, THF, overnight, 3 M NaOH, 33% H2O2; (ii) oxalyl chloride, DMSO, -78 °C, DIPEA, CH2Cl2; (iii) PPh3+CH3Br-,
THF, 1.6 M butyllithium in hexane, -78 °C to rt overnight; (iv) acetic acid, acetic anhydride, triflic acid; (v) persilylated thymine, TMSOTf, CH3CN, 0 °C
to rt overnight; (vi) 27% methanolic NH3, overnight; (vii) DMAP, phenyl chlorothionoformate, pyridine, rt overnight; (viii) Bu3SnH, toluene, AIBN, 4 h,
reflux; (ix) 20% Pd(OH), ammonium formate, methanol, reflux,12 h; (x) DMTr-Cl, pyridine, rt overnight; (xi) 2-cynoethyl N,N-diisopropylphosphorami-
dochloridite, DIPEA, dry THF. The IUPAC numbering according to von Bayer nomenclature for bicyclic systems is used in the Experimental Section.
Abbreviations: Bn (benzyl); Ac (acetyl); 9-BBN (9 borobicyclo(3.3.1)nonane); THF (tetrahydrofuran); AIBN, [2,2′-azobis(2-methyl-propionitrile)]; DMTr
(4,4′-dimethoxytrityl); DIPEA (diisopropylethylamine).
moiety, was confirmed by long range 1H-13C NMR correlation
esterification using phenyl chlorothioformate yielded the ester
21 (60% yield). Purified ester was then subjected to free-radical
cyclization utilizing tributyltin hydride in the presence of radical
initiator AIBN. Though for the heptenyl type of cyclization the
endo mode is only 7 times less rapid than the exo mode
cyclization in addition to the competing 1,5-hydrogen abstrac-
tion,48 the reaction gave, however, exclusively exo-product 22
in modest 76% yield. The bicyclic nucleoside 22 was depro-
tected using Pd(OH)2/C and ammonium formate in methanol
to give the corresponding dihydroxy compound 23 with 6-oxa-
bicyclo[3.2.1]octane in 82% yield. Dimethoxytritylation (80%)
followed by phosphitylation using standard conditions20 gave
the fully protected phosphoramidite 25 in 84% yield.
1
(HMBC)63 and H-1H (TOCSY)64 for both the isomers (see
Section 4). A 1D differential NOE experiment (Figure 5)
established that the exocyclic methyl at C7′ is in close proximity
to H1′ in the major isomer (d7′-Me/H1′ ) 2.8 Å for R and 4.4 Å
for S-configuration at C7′) of the bicyclic structure. The benzyl
groups in the bicyclic nucleosides 11a/11b were deprotected
using Pd(OH)2/C and ammonium formate in methanol to give
the corresponding dihydroxy compounds 12a and 12b, respec-
tively (see Section 4). Several attempts to separate this diaster-
eomeric mixture of 12a/12b failed in our hands. We therefore
subjected it directly to 5′-dimethoxytritylation (74%) to give
13 followed by 3′-phosphitylation (80%) to give the phosphor-
amidite 14 using standard conditions.18
3.0. Mechanism of the Ring-Closure Reaction. Two pos-
sible transition states TS 1 and TS 2 could be involved in the
radical cyclization of the C4′-propenyl system through the 5-exo-
hexenyl intermediate (structure A in Figure 3) in which case
these transition states should represent the low-energy chair
forms with a newly developing C7′ methyl substituent in the
pseudo-equatorial (TS 1) or pseudoaxial (TS 2) orientation.
Although both transition states are probable, the experimental
evidence (7:3 proportion of major to minor products) suggests
that the absence of 1,3-diaxial interactions of the methyl group
at C7′ (C7′ in R-configuration) with the bulky protective Bn
group at the pseudoaxial O3′ (11a) is energetically more
favorable and the product with methyl group at C7′ in
S-configuration (11b) is the minor product. We anticipate that
the energy of stabilization of the transition state TS 1 is slightly
2.0. Synthesis of Carbocyclic-ENA-T Phosphoramidite
(25). For the synthesis of carbocyclic-ENA-T analogue, a
heptenyl type of free-radical intermediate was warranted, and
thus the C4-allylated sugar 6 obtained in the previous scheme
was subjected to another round of hydroboration-oxidation
followed by Swern oxidation60 and Wittig61 reaction, as for 4,
5, and 6 in Scheme 1, to yield 15 (90%), 16 and 17 (72% in
two steps), respectively (Scheme 2). The sugar 17 was subjected
to acetolysis followed by a modified Vorbruggen-type62 cou-
pling, as for compounds 7 and 8 in Scheme 1, to give 18 and
â-configured thymine nucleoside 19 in 70% yield in two steps.
Deacetylation with 27% methanolic ammonia followed by
(63) Bax, A.; Summers, M. J. Am. Chem. Soc. 1986, 108, 2093-2094.
(64) Bax, A.; Davis, D. G. J. Magn. Reson. 1985, 65, 355-360.
9
8366 J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
Figure 3. (A) 5-Exo-cyclization through two transition states TS 1 and TS 2 leading to favored five-membered carbocyclic 2′,4′-cis-fused bicyclic system
with R-configuration of C7′ chiral center as well as to the counterpart with the disfavored S-configuration of C7′. (B) 6-exo-Heptenyl cyclization through two
transition states TS 3 and TS 4 leading to favored carbocyclic 2′,4′-cis-fused bicyclic system with R-configuration of C8′ and its counterpart with C8′ chiral
center in the disfavored S-configuration.
more because of the absence of any nonbonding/steric interac-
tion compared to that in the TS 2.
Carbocyclic-LNA-T (12a and 12b) and the Six-Membered
Carbocyclic-ENA-T (23). The 1H spectrum at 600 MHz of the
ring-closure reaction of the parent olefin 10 revealed that the
product formed is an intractable diastereomeric mixture of the
sugar-fused five-membered bicyclic 3′,5′-di-O-benzyl protected
nucleosides 11a/11b (Scheme 1). However, because of the
overlap of the H7′ and H2′ peaks in 11a/11b, firm NMR
evidence could not be obtained for the carbon-carbon ring
closure between C2′ and C7′. It is noteworthy that the high-
resolution mass spectrometry would not be able to discriminate
between the mass of the 2′-deoxy counterpart of 10 (formed as
a result of 2′-deoxygenation and subsequent quenching by a
hydrogen radical)65 and the cyclized product 11a/11b. The H7′
and H2′ peaks in the deprotected compounds 12a/12b were
however fully resolved and hence could be successfully used
for full NMR characterization.
Similar considerations can be used to understand the mech-
anism of the 6-exo-heptenyl cyclization of substituted nucleoside
radical B (Figure 3). For cyclization to occur, two favorable
(out of four possible) configurations obtained by rotation around
C6′-C7′ in the side chain, C4′-C6′-C7′-C8′, and around
C7′-C8′ in the C6′-C7′-C8′-C9′, represent probable transi-
tion states TS 3 and TS 4. The position of C7′-C8′ double
bound is apparently in a close proximity of the forming radical
at C2′ which makes the cyclic product arising from the TS 3
transition state (Figure 3) a preferable reaction path. The newly
developing C8′ methyl substituent thus takes up the equatorial
position, in which the 1,3-diaxial interaction with the C3′ axial
substituent (OBn) in the newly formed fused cyclohexyl ring
is absent, which makes it more favored then the product with
chiral C8′ in S-configuration.
The orientation of the transition state, TS 4 in Figure 3, on
the other hand, is also in a chair conformation, with site of attack
in the double bond (C8′) and the radical center at C3′ in the
steric proximity in order to ensure 6-exo-heptenyl cyclization
with minimal entropic penalty. This orientation in TS 4,
however, exhibits two 1,3-diaxial interactions, one between the
3′-O-benzyl substituent and the newly developing axial methyl
substituent at C8′ and, second, between the newly forming C8′
methyl substituent and the axial proton at C6′. The presence of
two 1,3-diaxial interactions would result in a energetically
disfavored transition state, as shown in TS 4, compared to that
in TS 3, which explains why we observe the exclusive formation
of the cyclic product with equatorial methyl at C8′ (Figure 3).
4.0. Assignment of 1H and 13C Chemical Shifts and
Evidence for the Ring Closure in the Five-Membered Fused
The 1H spectrum (Figure S22 in Supporting Information (SI))
showed the presence of two diastereomers, a major (12a) as
well as a minor isomer (12b) in ca. 7:3 ratio. The upfield H2′
at δ 2.43 along with H7′ at δ 2.65 and their proton-proton
couplings, proven by detailed double decoupling (Figures S24
and S26 in SI) and by COSY experiments (Figures S30-S32
in SI), shows that the C2′ substituent is the C7′ methine carbon.
The NOE enhancement (∼12%, corresponds to ca. 2.6 Å)
between H6 (thymine) and H3′ (Figures S35 and S36 in SI), of
3
12a/12b, in addition to JH1′,H2′ ) 0 Hz, further confirms that
the sugar is indeed locked by the fused carbocycle in the North
conformation as observed for other North-locked nucleosides
such as ENA,13 LNA,12 and aza-ENA.20 This further shows that
the 1-thyminyl moiety is in â-configuration and anti-conforma-
(65) Barton D, H. R.; Mccombie, S. W. J. Chem Soc., Perkin Trans. 1 1975,
1574.
9
J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007 8367

A R T I C L E S
Srivastava et al.
tion across the glycoside bond. The fact that the NOE enhance-
ment of 6.5% for H1′ upon irradiation on CH3 at C7′ of 12a
(Figure S35 in SI) is found, shows that the methyl group on
C7′ is in close proximity of H1′ (ca. 2.8 Å), thereby confirming
the R-configuration for C7′. The NOE enhancement of 4.5%
for H7′ in 12b (Figure S36 in SI) upon irradiation on H1′, on
the other hand, confirms that the H7′ is in close proximity of
H1′ (ca. 2.2 Å) and hence the S-configuration is assigned for
(for details of theoretical simulations see Experimental Section).
Our combined theoretical and experimental analyses have shown
that the sugar pucker conformation in 12a, 12b, and 23 is indeed
restricted to North-type and both the ENA- and LNA-type
carbocyclic analogues have the sugar moiety locked in exactly
the same North conformation as in the ENA-T,13 aza-ENA-
T,20 LNA-T,12 and 2′-amino-LNA-T16 counterparts. Further
details on the investigation of the nature of the major and minor
isomers of carbocyclic-LNA-T (compounds 12a and 12b) and
conformations of the aglycon and six-membered ring in car-
bocyclic-ENA-T (23) are provided in SI (Discussions S1 and
S2 as well as in Table S3 and S4 and Figures S73-S75 in SI).
6.0. Synthesis and Thermal Denaturation Studies of AONs
1-17. The phosphoramidites 14 and 23 were incorporated as
mono substitution in a 15 mer DNA sequence through automated
synthesis on Applied Biosystems 392 RNA/DNA synthesizer
for further studies. The stepwise coupling yields of the modified
phosphoramidite were ≈96% and 98%, respectively. Dicy-
anoimidazole was used as the activating agent for 14, whereas
tetrazole was used to activate 23 with 10 min coupling time for
modified phosphoramidites, followed by deprotection of all
base-labile protecting groups with 33% aqueous ammonia at
55 °C to give AONs 1-17 (Table 1). The sequence is targeted
to the coding region of the SV40 large T antigen (TAg)68,69
and has been used in the study of antisense activity of (N)-
methanocarba-T-substituted oligonucleotide70 and as well as in
the study of antisense and nuclease stability assays of oxetane71-
modified, azetidine18-modified, and aza-ENA20-modified oli-
gonucleotides.
Thermal denaturation studies of AONs (Table 1) containing
carbocyclic-LNA-T or carbocyclic-ENA-T have showed an
increase of 3.5 to 5 °C/modification (AONs 6-9 in Table 1)
and 1.5 °C/modification (AONs 10-13 in Table 1), respectively,
for the AONs duplexes with complementary RNA. However, a
net decrease of 1 to 5 °C/modification has been observed for
the AONs duplexes with complementary DNA. This may be
due to increase in steric clash in the shallow minor groove of
AON/DNA duplex. A comparative study of carbocyclic-LNA-T
with LNA-T in our lab showed only ∼ 1 °C decrease in Tm
with complementary RNA (Table 1) which is indeed surprising
meaning that the lack of hydrophilic substituent at 2′ (as in LNA)
does not impart any significant decrease in duplex stability with
complementary RNA. A comparison of six-membered carbocy-
clic-ENA-T with aza-ENA-T20 counterpart has not shown any
sequence dependent change in Tm (+1.5 °C/modification
independent of the sequence) while in the case of aza-ENA-
T20 containing counterpart the Tm significantly varied (2.5-
4.0 °C/modification) depending on the sequence.
2
C7′. JHC HMBC correlations between H7′ and C2′ for
compounds 12a/12b (Figures S38 and S39 in SI) unequivocally
proves that the oxa-bicyclo[2.2.1]heptane ring system has been
formed in the ring-closure reaction (Scheme 1).
For compound 23, the upfield H2′ shift at δ 2.26 along with
H8′ at δ 2.20 and their vicinal proton-proton couplings, proven
by the double decoupling (Figure S58 in SI) as well as COSY
experiments (Figures S62 and S63 in SI), shows that the C2′
substituent is the C8′ methine carbon. Strong NOE enhancement
(8.6%, corresponding to ca. 2.6 Å) between H6 (thymine) and
H3′ (Figure S66 in SI) in compound 23, in addition to 3JH1′,H2′
) 0 Hz, further confirms that the sugar is indeed locked in the
North-type conformation and that the 1-thyminyl moiety is in
â-configuration and anti conformation across the glycoside bond.
The NOE enhancement of 3.0% for H1′ upon irradiation at CH3-
(C8′) (Figure S66 in SI) proved that the CH3(C8′) group is in
close proximity of H1′; hence, the C8′ chiral center is in
R-configuration. Vicinal coupling of H2′ with H8′ as evidenced
by double decoupling experiments (Figure S58 in SI) and COSY
spectra (Figures S62 and S63 in SI) also unequivocally showed
that the bicyclo[3.2.1]octane ring system has indeed been formed
in the ring-closure reaction (Scheme 2). This evidence was
further corroborated by the observation of the long range 1H-
13C connectivity of H8′ with C2′, C3′, and C1′, that of H7′ with
C2′, and that of H2′ with CH3(C8′), C7′, and C8′ in the HMBC
experiment. (Figures S68 and S69 in SI).
Detailed NMR characterization by 1D and 2D NMR spectra
to show the fused carbocyclic nature of compounds 12a, 12b,
and 23 are available in SI including chemical shifts in Tables
S1 and S2 in SI, spin-spin simulations (Figures S25, S27, and
S59 in SI), COSY (Figures S30-32, S62, and S63 in SI) and
TOCSY (Figures S33, S34, S64, and S65 in SI) to show the
proton-proton connectivity, HMQC (Figure S37, S67 in SI)
to show proton-carbon connectivity, and finally HMBC (Figure
S38, S39, S68, and S69 in SI) to establish long-range proton-
carbon correlation on the basis long-range coupling constant to
unequivocally prove the formation of the bicyclic system in 12a/
12b as well as in 23. For the complete NMR characterization
of compounds 12a/12b and 23, see Discussion S1 in SI.
5.0. Molecular Structures of Carbocyclic-LNA-T and
Carbocyclic-ENA-T Based on NMR, ab Initio, and MD
7.0. Stability of the Carbocyclic-Modified AONs in Human
Blood Serum. The stability of AONs toward various exo- and
endonucleases is necessary in order to develop therapeutic
oligonucleotides (antisense,2 RNAi,72 microRNA73,74 or triplex-
3
Calculations. Initial dihedral angles from the observed JH,H
couplings (Table S2 in SI) had been derived (Step I) using the
Haasnoot-de Leeuw-Altona generalized Karplus equation66,67
(Table S3 in SI) and utilized as constraints in the NMR-
constrained simulated annealing (SA) molecular dynamics (MD)
simulation (0.5 ns, 10 steps) followed by 0.5 ns NMR
constrained simulations using torsional constraints to yield
NMR-defined molecular structures of the respective compounds
(68) Graessmann, M.; Michaels, G.; Berg, B.; Graessmann, A. Nucleic Acids
Res. 1991, 19 (1), 53-9.
(69) Wagner, R. W.; Matteucci, M. D.; Lewis, J. G.; Gutierrez, A. J.; Moulds,
C.; Froehler, B. C. Science 1993, 260 (5113), 1510-13.
(70) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J.; Wagner,
R. W.; Matteucci, M. D. J. Med. Chem. 1996, 39 (19), 3739-3747.
(71) Pradeepkumar, P. I.; Chattopadhyaya, J. J. Chem. Soc., Perkin Trans. 2
2001, 2074-2083.
(66) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C. Tetrahedron 1980,
(72) Grunweller, A.; Wyszko, E.; Bieber, B.; Jahnel, R.; Erdmann Volker, A.;
Kurreck, J. Nucleic Acids Res. 2003, 31 (12), 3185-3193.
(73) Ambros, V. Cell (Cambridge, MA, U.S.) 2001, 107 (7), 823-826.
(74) Ruvkun, G. Science (Washington, DC, U.S.) 2001, 294 (5543), 797-799.
36, 2783-2792.
(67) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94 (23), 8205-
1822.
9
8368 J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
Table 1. Thermal Denaturation of Native and Modified AONs in the Duplexes with Complementary RNA or DNA Targetsa
MALDI-MS found/calcd
AON
sequence
Tm/
°
C with RNA§
∆Tm
Tm
/
°
C with DNA§
∆Tm
*
[M +
H]+/(m/z)+
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
3-d(CTTCTTTTTTACTTC)-5
44
48
49
49
49
47.5
49
45
47
46.5
45.0
46
45
44
44
43.0
43.5
39.5
40.0
39.5
44.5
42.5
42
4449.7/4448.7
3-d(CTT(LNA)CTTTTTTACTTC)-5
3-d(CTTCTT(LNA)TTTTACTT C)-5
3-d(CTTCTTTT(LNA)TTACTTC)-5
3-d(CTTCTTTTTT(LNA)ACTTC)-5
3-d(CTT(5-carbo)CTTTTTTACTTC)-5
3-d(CTTCTT(5-carbo)TTTTACTTC)-5
3-d(CTTCTTTT(5-carbo)TTACTTC)-5
3-d(CTTCTTTTTT(5-carbo)ACTTC)-5
3-d(CTT(6-carbo)CTTTTTTACTTC)-5
3-d(CTTCTT(6-carbo)TTTTACTTC)-5
3-d(CTTCTTTT(6-carbo)TTACTTC)-5
3-d(CTTCTTTTTT(6-carbo)ACTTC)-5
3-d(CTT(aza-ENA)CTTTTTTACTTC)-5
3-d(CTTCTT(aza-ENA)TTTTACTTC)-5
3-d(CTTCTTTT(aza-ENA)TTACTTC)-5
3-d(CTTCTTTTTT(aza-ENA)ACTTC)-5
+4
+2
4475.48/4474.43
4475.48/4474.43
4475.48/4474.43
4475.48/4474.43
4489.40/4488.75
4489.26/4488.75
4489.21/4488.75
4489.45/4488.75
4503.42/4502.75
4503.25/4502.75
4503.37/4502.75
4503.26/4502.75
4489.7/4491.1
+5
+1.5
0.0
+5
+5
+1
0.00
+3.5
+5
-1.00
-1.00
-2.00
-1.5
-5.5
-5.0
-5.5
-0.5
-2.5
-3
48
+4
47.5
45.5
45.5
45.5
45.5
48
46.5
47.5
48
+3.5
+1.5
+1.5
+1.5
+1.5
+4
+2.5
+3.5
+4
4489.7/4490.7
4489.7/4490.7
4489.7/4490.8
42
-3
a Tm values measured as the maximum of the first derivative of the melting curve (A260 vs temperature) and are average of at least three runs recorded
in medium salt buffer (60 mM Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2, and 2 mM DTT) with temperature range 20 to 70 °C using 1 µM
concentrations of the two complementary strands; ∆Tm ) Tm relative to RNA compliment; ∆Tm* ) Tm relative to DNA compliment. Tm performed with
complementary DNA or RNA strand. Abbreviations: T(LNA): LNA-T12(compound A in Figure 1); T(5-carbo): carbocyclic-LNA-T (compound 12, Scheme 1);
T
(6-carbo): carbocyclic-ENA-T (compound 23, Scheme 2); T(aza-ENA): aza-ENA-T.20 (compound I in Figure 1).
ing agents3). The first generation nuclease resistant antisense
phosphorothioates7 were followed by 2′-O-alkylated modifica-
tions.31 Recent conformationally constrained molecules (LNA,75-77
ENA,13 bicyclic,70 and tricyclic,78 aza-ENA,20 oxetane,71 aze-
tidine,18 etc.) have also shown enhanced nuclease stability as
compared to the natural deoxy counterpart. Egli et al.79 have
demonstrated that charge effects and hydration properties are
important factors in influencing the nuclease stability of AONs
with a normal phosphodiester backbone. Here, we report a
comparative study involving our carbocyclic-LNA (12a/12b)
and carbocyclic-ENA (23)-modified AONs (Table 1) with those
of LNA and aza-ENA-modified AONs in human blood serum
which is mainly composed of 3′-exonucleases.
Modified sequences (Table 1), 32P-labeled at 5′-ends, were
digested in human blood serum at 21 °C as shown in the PAGE
autoradiograms (Insets A, B, C, and D in Figure 4). In order to
understand the extent of transmission of the stereoelectronic
effects of the modified locked sugar to the neighboring
nucleotides toward the 3′- or at the 5′-end, we introduced
modifications further away from the 3′-end of the AONs. The
assessment of the modified AON stability in blood serum, owing
to various modifications, T(LNA) versus T(aza-ENA) versus T(5-carbo)
versus T(6-carbo), Vis-a`-Vis the native counterpart (AON 1),
becomes very clear as we compare the digestions with (i) AONs
2 [with T(LNA)], (ii) AON 6 [with T(5-carbo)], and (iii) AON 10
[with T(5-carbo)] and AON 14 [with T(aza-ENA)], each having a
specific single modification at the position 3 from the 3′-end
(Table 1), showing that the site of the 3′-exonuclease-promoted
hydrolysis in blood serum was dictated by the site of the
incorporation of the modification in the AON (Table 1).
The observations are as follows: (i) the modified AON 2
(full length AON ) ‘n’) with T(LNA) at position 3 from the 3′-
end gave the n - 1 fragment in 35 min, and n - 2 fragment in
5 h as the major product, which was then completely degraded
in 9 h to give various fragments ranging from n - 2 to n - 8
(Inset A in Figure 4). Thus, a comparison of the blood serum
cleavage pattern of the native AON 1 with that of the LNA-
modified AON 2 showed that the latter is only slightly more
stable than the native counterpart. (ii) On the other hand, the
20
modified AON 14 with T(aza-ENA) at position 3 from the 3′-
end showed full hydrolysis of the 3′-terminal nucleotide within
5 h to give the AON fragments with an n - 1 (ca. 85%) and n
- 2 sequence (ca. 15%), which were further hydrolyzed to ca.
65% and 35% respectively after 12 h. No further cleavage had
been observed until 48 h (Inset D in Figure 4 and plots of percent
AONs remaining as a function of time in Figure S78 in SI).
This means that AON 14 with an n - 1 nucleotide sequence
was being hydrolyzed steadily to give the AON with an n - 2
nucleotide sequence. (iii) In contradistinction, the isosequential
AONs with the five- and six-membered carbocyclic modifica-
tions [AON 6 with T(5-carbo) and AON 10 with T(6-carbo)] showed
only one cleavage site to give a single AON product with n -
1 nucleotide after 2 h, which remained completely stable until
48 h (PAGE autoradiograms in Insets B and C in Figure 4 for
up to 48 h, and plots of percentage AONs remaining versus
time are shown in Inset A in Figure S76 and Inset A in Figure
S77 in SI). Such blood-serum stabilities were also observed for
other AONs with five- and six-membered carbocyclic modifica-
tions at position 6 (AONs 7/11), position 8 (AONs 8/12), and
position 10 (AONs 9/13) (PAGE autoradiograms in Insets B
and C in Figure 4, and plots in Insets B-D in Figures S76 and
S77 in SI) as well as for aza-ENA-T containing AONs (15/16/
17) (PAGE autoradiograms in Inset D, Figure 4, and plots in
Insets B-D in Figure S78). The plots (Figures S76 and S77 in
SI) obtained as a result of quantification of the PAGE autora-
diograms (Figure 4) in carbocyclic-modified AONs 6 (Inset B
in Figure 4) and 10 (Inset B in Figure 4) clearly showed that
the resistance toward nucleases was transmitted toward the
(75) Braasch, D. A.; Jensen, S.; Liu, Y.; Kaur, K.; Arar, K.; White, M. A.;
Corey, D. R. Biochemistry 2003, 42 (26), 7967-7975.
(76) Elmen, J.; Thonberg, H.; Ljungberg, K.; Frieden, M.; Westergaard, M.;
Xu, Y.; Wahren, B.; Liang, Z.; Orum, H.; Koch, T.; Wahlestedt, C. Nucleic
Acids Res. 2005, 33 (1), 439-447.
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2002, 30 (9), 1911-1918.
(78) Steffens, R.; Leumann, C. J. J. Am. Chem. Soc. 1999, 121 (14), 3249-
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M. Biochemistry 2005, 44 (25), 9045-9057.
9
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A R T I C L E S
Srivastava et al.
Figure 4. Autoradiograms of 20% denaturing PAGE showing degradation patterns of 5′-32P-labeled AONs in human blood serum (Table 1 for all AON
sequences). Inset A: AON 1 and LNA-modified AONs 2-5. Inset B: Carbocyclic-LNA-modified AONs 6-9. Inset C: Carbocyclic-ENA-modified AONs
10-13. Inset D: aza-ENA-modified AONs 14-17. Time points are taken after 0, 1/2, 1 h, 2 h, 5 h, 7 h, 9 h, 12 h, for AONs 1-5 and 0 h, 6 h, 8 h, 12 h,
24, 36, and 48 h of incubation for AONs 6-17 at 21 °C (see Experimental Section for details).
neighboring 3′ nucleotide of the carbocyclic modification site
(i.e., n - 1 fragment, when n ) full length 15mer AON and
the carbocyclic modification site is at position 3 from the 3′-
end). In contrast, with the aza-ENA-modified AON 14 (Inset
D in Figure 4, Figure S78 in SI) hydrolysis proceeded both at
the 3′-phosphate of the modification site and at that of the 3′-
neighbor, giving first an n - 1 fragment as the predominant
product, which was further hydrolyzed with time giving the n
- 2 fragment (when n ) full length 15mer AON and the aza-
ENA modification site is at position 3 from the 3′-end). The
residual fragments so formed were stable against further
hydrolysis for up to 48 h. Similar results were obtained for
AONs 7-9 (five-membered carbocyclic modifications at posi-
tion 6, 8, and 10 from the 3′-end giving n - 4, n - 6, and n -
8 fragments, respectively, Figure S76 in SI), AONs 11-13 (six-
membered carbocyclic modifications at position 6, 8, and 10
from the 3′-end giving n - 4, n - 6, and n - 8 fragments,
respectively, Figure S77 in SI) and AONs 15-17 (six-membered
aza-ENA modifications at position 6, 8, and 10 from 3′-end
giving n - 4/n - 5, n - 6/n - 7, and n - 8/n - 9 fragments,
respectively. Figure S78 in SI). The fact that in the case of five-
and six-membered carbocyclic modifications, the 3′-phosphate
of the modified nucleotide was fully stable, whereas the 3′-
phosphate of the neighboring residue, which is next to the
modified nucleotide at the 3′-end, was unstable and fully
hydrolyzed, compared to that of aza-ENA modification in which
both 3′-phosphates of the modified nucleotide as well as that
of the neighbor residue were unstable, shows that the stereo-
electronic effect of the locked sugar into the N-type conforma-
tion (carbocyclic versus aza-ENA) at the modification site is
transmitted differently to alter the phosphate backbone torsions
toward the 3′-end thus modulating its susceptibility differently
toward hydrolytic cleavage by 3′-nucleases. It is noteworthy
that the transmission of the stereoelectronic effect in aza-ENA-T
is very comparable to those for oxetane-T-modified and
azetidine-T-modified isosequential AONs.
7.1. Mechanism of the Modified AON Stability in Blood
Serum. 7.1.1. Effect of 2′-Heteroatom versus 2′-Carbo
Substitution in the Modified Nucleotide in the AON. The
3′-terminal nucleotide is hydrolyzed by a 3′-exonuclease in AON
substituted by a five- or six-membered carbocyclic residue at
position 3 (AONs 6 and 10) to give only the n - 1 fragment.
AONs with single substitution at either position 6 (AONs 7 and
11), 8 (AONs 8 and 12), or 10 (AONs 9 and 13) similarly give
only n - 4, n - 6, or n - 8 fragments, respectively. The residual
AON sequences that remained at the 5′-end of the modification
site were found to be intact for more than 48 h in human blood
serum (see PAGE autoradiograms in Insets B and C in Figure
4, and for plots of percentage of oligonucleotide left as a
function of time in Insets A-D in Figures S76 and S77 in SI).
However AONs containing a single six-membered aza-ENA-T
substitution (i.e., AONs 14-17 with single modification incor-
porated at positions 3, 6, 8, and 10, respectively) showed
progressive cleavage with time until the site of modification
(Figure 4, Inset D and for plot of percentage of oligonucleotide
left as a function time in Insets: A-D in Figure S78 in SI).
9
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2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
This was an interesting observation since the isosequential
at the role of ammonium ions in providing the stability.30
Subsequently, it was shown that the native ribonucleoside with
a 2′-O-alkyl substitutent either by its bulk (pentoxy > propoxy
> methoxy > deoxy)31 or by its stereoelectronic modulation
(2′-O-GE)29 of the hydration can bring about nucleolytic
stability.
North-constrained 1′,2′-oxetane17 or 1′,2′-azetidine18 with identi-
cal modification sites exhibited the 3′-exonuclease-promoted 3′-
O-P bond hydrolysis right at the 3′-end of the modification
site itself (i.e., at the n - 2 site). In contradistinction,
isosequential AON with aza-ENA-T modification at position 3
from the 3′-end (i.e., AON 14) gives predominantly AON with
an n - 1 residue (Figure 4, Inset D), which is steadily
hydrolyzed to give the n - 2 product, but the corresponding
carbocyclic AONs (AONs 6 and 10) give only the fully stable
n - 1 fragment. This suggests that the enzymes of human serum
recognize and confer different grades of stability against
hydrolysis for relatively more flexible 1′,2′-conformational
constraint as in 1′,2′-oxetane17 or 1′,2′-azetidine18 compared to
the stronger conformational constraints imposed by 2′,4′-
modifications as in aza-ENA or in five- and six-membered
carbocyclic-ENA/LNA.
Since our carbocyclic AONs were completely devoid of polar
effect at C2′,28 the above explanations invoking charge or steric
effects is not applicable to explain the unprecedented nuclease
stability of these carbocyclic AONs in the blood serum. The
enhanced stabilities of the carbocyclic-AONs with respect to
their bicyclic 2′-O-(LNA and ENA) and aza-ENA analogues
suggests that the accessibility of water to the 2′-O- in LNA or
to the 2′-N- in the aza-ENA substituent in a modified nucleotide
is most probably more important in order to cleave the vicinal
3′-phosphodiester bond by the exonucleases of blood serum.
7.1.2. Solvation Free-Energy Calculation To Elucidate
Relative Hydrophobicity of the 2′-Substituent. In order to
understand how the nature of 2′-substitution in the modified
AON affects the relative access of water to the scissile
phosphate, we have analyzed the solvation free-energies of
different modified nucleosides (Table S5 in SI) utilizing Baron
and Cossi’s implementation of the polarizable conductor CPCM
model85 of solvation on the ab initio optimized (HF, 6-31G**
basis set) molecular geometries. This allowed us to understand
the lipophilic Versus hydrophilic nature of different 2′,4′-
constrained modifications in LNA/ENA/2′-amino-LNA/aza-
ENA/carbocyclic-LNA/carbocyclic-ENA in a comparative man-
ner. Table S5 in SI thus shows that the energy of solvation of
the five-membered (12a and 12b) and six-membered (23)
carbocyclic nucleosides compared to their oxygen and nitrogen
containing counterparts decreases in the following order: six-
membered carbocyclic-ENA-T (-12.2 kcal mol-1) > five-
membered carbocyclic-LNA-T (-12.9 kcal mol-1) > aza-
ENA-T (-15.2 kcal mol-1) > ENA-T (-15.6 kcal mol-1) >
LNA-T (-16.8 kcal mol-1). This suggests that our five-
membered and six-membered carbocyclic nucleosides, on ac-
count of their hydrophobic nature, are not as well solvated as
compared to their LNA, ENA, and aza-ENA counterparts,
thereby showing that hydration around a scissile phosphate is
most probably important for the nuclease-promoted hydrolysis.
This leads us to speculate that this hydration around the C2′
substituent with heteroatoms such as 2′-O- in LNA or to the
2′-N- in aza-ENA in a modified AON is utilized by the
exonuclease to capture a water molecule to hydrolyze the 3′O-
PO3-R bond of the vicinal phosphate, which is not possible
with our carbocyclic-AONs.
Interestingly, a single modification of AON with our car-
bocyclic-LNA-T or with carbocyclic-ENA-T nucleotides at
position 3 from the 3′-end in to the AON 6 or AON 10 has
provided the highest degree of exonuclease stability which was
earlier achieved by employing four 2′-O-[2-(guanidinium)ethyl]
(2′-O-GE)29 modifications (including the 3′-terminal modifica-
tion). Other carbocyclic modifications have also shown nuclease
resistance, but less efficiently.80-83 The 2′- or 6′-alkoxy-
substituted carbocyclic nucleotide units (three units at the 3′-
end) in the modified AON enhanced stability of AON on fetal
calf serum from 2.5 times for [6′R-carbocyclic-2′-deoxy]-T
substitution to 24 times for [6′R-carbocyclic-2′-O-(CH2)4-NH2
or 2′-O-(CH2)3-Ph]-T substitution), compared to that of the
native form.83 It was suggested that replacement of substituents
involved in the natural enzyme-substrate complex results in
poor recognition and processing by the nucleolytic enzymes,
thereby resulting in the nuclease stability. Thus, the nuclease
stability was enhanced when a 2′-bulky substituent was intro-
duced in the carbocyclic nucleosides.
The AONs containing five-membered carbocyclic-LNA Ver-
sus AONs with the six-membered carbocyclic-ENA show
enhanced, but identical, blood serum stability, thereby showing
that the steric bulk is relatively unimportant.31 This is in sharp
contrast to the conclusion drawn by comparison of the LNA
versus ENA24 modified AONs, in that the latter is 2.5-3 times
more stable than the former,13 apparently, according to the
authors, owing to an extra methylene linker. The 3′-exonuclease
(SVPDE) stability had also been observed to be higher for the
2′-O-GE29 and 2′-O-aminopropyl modifications,84 as well as for
the 4′-R-C-aminoalkylthymidine AONs,30 which showed com-
plete nuclease resistance upon incorporation of five modified
nucleotides as mixmers, compared to the native counterpart.
Notably, nuclease digestion studies involving 4′-R-C-ami-
noalkylthymidine AONs showed that longer chain alkyls were
less potent in providing stability against nuclease, and hinted
8.0. 3′-Exonuclease Stability Assay (with snake venom
phosphodiesterase). The stability of AONs of carbocyclic
analogues of LNA and ENA (Table 1) toward 3′-exonuclease
was investigated using snake venom phosphodiesterase over a
period of 72 h at 21 °C (PAGE autoradiogram in Figure S79 in
SI) and compared with isosequential LNA and aza-ENA
modified AONs under identical conditions. The results obtained
were similar to that obtained in human blood serum assay
(PAGE autoradiogram in Figure 4) showing identical cleavage
sites and identical resistance pattern, i.e., cleavage of the
phosphodiester bond next to the modification site toward the
(80) Renneberg, D.; Bouliong, E.; Reber, U.; Schumperli, D.; Leumann Christian,
J. Nucleic Acids Res. 2002, 30 (13), 2751-2757.
(81) Bolli, M.; Trafelet, H. U.; Leumann, C. Nucleic Acids Res. 1996, 24 (23),
4660-4667.
(82) Perbost, M.; Lucas, M.; Chavis, C.; Pompon, A.; Baumgartner, H.; Rayner,
B.; Griengl, H.; Imbach, J. L. Biochem. Biophys. Res. Commun. 1989, 165
(2), 742-747.
(83) Altmann, K.-H.; Bevierre, M.-O.; De Mesmaeker, A.; Moser, H. E. Bioorg.
Med. Chem. Lett. 1995, 5 (5), 431-436.
(84) Teplova, M.; Wallace, S. T.; Tereshko, V.; Minasov, G.; Symons, A. M.;
Cook, P. D.; Manoharan, M.; Egli, M. Proc. Natl. Acad. Sci. U.S.A. 1999,
96 (25), 14240-14245.
(85) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102 (11), 1995-2001.
9
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A R T I C L E S
Srivastava et al.
Figure 5. The RNase H1 cleavage pattern of AONs 1-17/RNA heteroduplexes. Vertical arrows show the RNase H cleavage sites, with the relative length
of the arrow showing the extent of the cleavage. The square boxes around a specific sequence shows the stretch of the RNA, which is resistant to RNase
H cleavage thereby giving footprints (see PAGE autoradiograms in Figure 6).
3′ end and subsequent resistance to degradation for up to 72 h
(PAGE autoradiogram in Figures S79 in SI and plots of
percentage of oligonucleotide left as function of time in Figures
S80-S82 in SI). These experiments clearly show that the effect
of a hydrophobic environment imparted by the carbocyclic
analogues of LNA and ENA is important in dictating the
nuclease stability of the modified AON.
9.0. RNase H Digestion Studies. In the antisense strategy,
the RNase H recruitment by the modified AON, with long
nuclease stability as exhibited by our carbocyclic modifications,
and the target RNA heteroduplexes is an important step for the
design of potential therapeutics for engineering a specific gene
silencing effect.2
Hence, RNase H-mediated cleavage of the RNA strand
complementary to the modified AON strands 2-17 (with
carbocyclic-LNA, carbocyclic-ENA, aza-ENA, and LNA modi-
fications) has been studied here, using the native AON/RNA
duplex as a reference, in order to address the issue if the
recruitment of RNase H by the heteroduplexes is in anyway
compromised as a result of carbocyclic modifications incorpo-
rated in the AON strand. We have used E. coli RNase H in our
studies on account of its availability, and the fact that its
properties are known to be not very different from the
mammalian enzyme.77 The, RNA complementary to AONs
(AONs 1-17 in Table 1) formed duplexes and were found to
be good substrates for RNase H but with varying cleavage sites
(Figure 5) depending on the site of modification in the AON
strand as shown in PAGE autoradiograms (Insets A-D in Figure
6). The main observations are as follows:
uniquely identical cleavage footprint patterns having 5-6 nt
gaps (Insets A-D in Figure 6). It is remarkably surprising that
the RNase H enzyme could not distinguish the functional
differences exhibited by the North-conformationally constrained
LNA, carbocyclic-LNA, carbocyclic-ENA, and aza-ENA modi-
fications, compared to nucleases.
This suggests that the RNase H does not recognize the
hydrophobic (as in carbocyclic-LNA, carbocyclic-ENA) or
hydrophilic (LNA and aza-ENA) character of the substituent at
the 2′-position of the modified nucleosides, but interrogates only
at the subtle difference in the flexibility of the North-type sugar
puckering (2′,4′ versus 1′,2′) in that we observed different
cleavage sites and footprint pattern (4-5 nt gaps) for iso-
sequential 1′,2′-constrained North-East sugar puckered oxetane71
and azetidine18 modified AONs compared to those of the more
rigid 2′,4′-constrained systems (LNA, carbocyclic-LNA, car-
bocyclic-ENA, and aza-ENA modified AONs).
The cleavage rates of RNase H digestion were determined
by densitometric quantification of gels and subsequently by
plotting the uncleaved RNA fraction as a function of time (Insets
E-H in Figure 6). The reaction rates were obtained by fitting
the degradation curves to single-exponential decay functions.77
The relative cleavage rates with carbocyclic-LNA, carbocyclic-
ENA, aza-ENA, and LNA modified AON/RNA duplexes were
found to be very similar to that of the native counterpart
irrespective of the type and the site of modification in the AON
strand (compare the relative rates in Figure 7).
The main conclusion which can be drawn from these studies
is that even though all AONs used (Table 1) recruited RNase
H almost as efficiently as that of the native counterpart (Figure
7), it is only the carbocyclic-LNA and the carbocyclic-ENA
modified AONs (AON 6-13) which have shown (Figure 4)
much enhanced nuclease stability in the blood serum (ca. 48 h)
as compared to that of the native and the LNA-modified AONs
(fully degraded <12 h) and aza-ENA (≈85% stable in 48 h)
(Figure 8, Figures S76-S78 in SI). Clearly, the enhanced
(i) The RNA complement of the unmodified native AON
1/RNA duplex was cleaved quite randomly with a slight
preference after A-8 position.
(ii) For the isosequential AONs with single modification at
different sites (Table 1) containing either LNA (AONs 2-5),
carbocyclic-LNA-T (AONs 6-9), carbocyclic-ENA-T (AONs
10-13), or aza-ENA-T AONs 14-17) modifications showed
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8372 J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
Figure 6. Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5′-32P-labeled target RNA by E. coli RNase H1 in the AONs 1-17
after 2, 5, 10, 15, 30, and 60 min of incubation. Conditions of cleavage reactions: RNA (0.8 µΜ) and AONs (4 µΜ) in buffer containing 20 nM Tris-HCl
(pH 8.0), 20 mM KCl, 10 mM MgCl2, and 0.1 mM DTT at 21 °C; 0.04 U of RNase H1. Inset A: LNA-T modified AONs 2-5 with native AON 1. Inset
B: carbocyclic-LNA-T modified AONs 6-9. Inset C: carbocyclic-ENA-T modified AONs 10-13 with native AON 1. Inset D: aza-ENA-T modified
AONs 14-17 with native AON 1. The graphs in Insets E, F, G, and H show the kinetics of RNase H1-mediated cleavage of the target RNA, the remaining
fraction of target RNA is measured densitometrically and plotted as a function of time fitted to a single-exponential decay function. Inset E: AON 1 with
AONs 2-5. Inset F: for AON 1 with AONs 6-9. Inset G: AON 1 and AONs 10-13. Inset H: AON 1 and AONs 14-17.
lifetime of these carbocyclic-modified AONs in the blood serum
(Figure 8, Figures S76-S78 in SI) may produce the highly
desired pharmacokinetic properties because of their stability and
consequently a net reduction of the required dosage. This makes
9
J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007 8373

A R T I C L E S
Srivastava et al.
Figure 7. Bar plots of the observed cleavage rates of the RNase H promoted degradation of AONs 2-17/RNA heteroduplexes with various modifications
(LNA-T, carbocyclic-LNA-T, carbocyclic-ENA-T, and aza-ENA-T) in the AON strand at position 3, 6, 8, and 10 from the 3′-end, in comparison to that of
the native counterpart AON 1. The observed initial cleavage rates (s-1) of AONs 1-17/RNA heteroduplexes by E. coli RNase H are found to be very
similar, while in the human blood serum (Figures 8-11) the degree of stability varied widely for the carbocyclic Versus heterocyclic modified AONs
reflecting their respective hydrophobic/hydrophilic properties.
Figure 8. The percent of AONs 6-17 left after 48 h of incubation in the human blood serum at 21 °C. Note that, under similar condition, the native (AON
1) and LNA-containing AONs (AONs 2-5) were fully degraded after 12 h, and shown in blank (red colored). Note that the human blood serum stable
product (i) is (n - 1) for the AONs 6/10/14 (AONs substituted at position 3 from the 3′-end) after the hydrolysis of the 3′-terminal nucleotide, (ii) (n - 4)
product for the AON 7/11/15 (AONs substituted at position 6 from the 3′-end), (iii) the (n - 6) product for the AONs 8/12/16 (AONs substituted at position
8 from the 3′-end), whereas (iv) the (n - 8) product was formed from AONs 9/13/17 (AONs substituted at position 10 from the 3′-end). Thus the concentration
of these final hydrolysis products formed as a result of introduction of a specific modification at a particular site was taken as 100%. Also note: cleavage
of one nucleotide from the 3′-end is (n - 1), and the cleavage of two nucleotides from the 3′-end is (n - 2), and so on, where ‘n’ is the full length AON.
Conclusions
our carbocyclic-LNA, carbocyclic-ENA, and aza-ENA modified
AONs the best candidates for antisense therapeutics because of
their efficient exploitation in the enzymatic turnover. In this
context, it may be noted that the modified AONs with all
thymidines replaced with the conformationally constrained
(North- or South-locked) 2′-deoxy-methanocarba-nucleoside70
or LNAs77 or tricyclo-DNA80 did not recruit RNase H.
1. In order to develop gene silencing agents with natural
phosphodiester linkages, three factors are perhaps most impor-
tant: stability, delivery, and RNase H recruitment. In this regard,
we have designed and synthesized AONs containing five-
membered (12a/12b) and six-membered (23) carbocyclic ana-
logues of LNA (carbocyclic-LNA-T) and ENA (carbocyclic-
9
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2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
ENA-T), which are both nuclease resistant and capable of
eliciting an RNase H response.
LNA and carbocyclic-ENA modifications excellent candidates
as potential antisense or RNAi therapeutic agents.
2. The synthesis of these novel conformationally constrained
carbocyclic analogues have been achieved using free-radical
C-C bond formation as a key step. Various NMR experiments
Experimental Section
3,5-Di-O-benzyl-4-C-vinyl-1,2-O-isopropylidene-r-D-ribofura-
nose (4). Oxalyl chloride (10.7 mL, 125 mmol) was added to
dichloromethane (350 mL) cooled at -78 °C. DMSO (15 mL, 200
mmol was added dropwise to this solution over 30 min. After stirring
for 20 more min a solution of 2 (20 g, 50 mmol) in dichloromethane
(100 mL) was added dropwise to this mixture in about 20 min and
stirred at -78 °C for another 30 min. DIPEA (60 mL, 350 mmol) was
added to this cooled mixture and allowed to warm to room temperature.
Water was added to the reaction and twice extracted with dichlo-
romethane (100 mL). The organic layer was washed with water and
brine, dried over MgSO4, and concentrated under reduced pressure. In
another reaction BuLi (1.6 M solution in hexane, 95 mL, 150 mmol)
was added to a precooled suspension of methyl-triphenylphosphonium
bromide (54 g 150 mmol) at 0 °C in dry THF and stirred at room
temperature for 1 h. The yellow solution so obtained was cooled to
-78 °C and a solution of crude aldehyde in dry THF was then added
dropwise in about 20 min and stirred at -78 °C overnight. The reaction
was quenched with saturated aqueous NH4Cl, stirred for about 1 h at
room temperature, extracted with ether (3 × 200 mL), dried over
MgSO4 and, concentrated under reduced pressure. The crude material
was purified by column chromatography on silica gel (0-10% ethyl
acetate in cyclohexane, v/v) to give 2 as a colorless oil (17 g, 43 mmol,
1
including H homodecoupling experiments, 1D nuclear Over-
hauser effect spectroscopy (1D NOESY), 2D total correlation
spectroscopy (TOCSY), 2D COSY, and 13C NMR experiments,
including distortionless enhancement by polarization transfer
(DEPT), as well as long-range 1H-13C HMBC correlation (2JH,C
and 3JH,C), and one-bond heteronuclear multiple-quantum coher-
ence (HMQC) have been employed to show unambiguously that
indeed a carbon-carbon bond formation has taken place
between C2′ and the olefinic side chain tethered at C4′ to give
the North-type conformationally constrained bicyclic nucleo-
sides.
3. The molecular structures of the five-membered (12a/12b)
and six-membered (23) carbocyclic analogues have also been
studied using ab initio and MD simulations to understand the
structural reasons behind the regio- and stereochemistry of the
cyclization products.
4. Sixteen single modified AONs (15mer) containing the
carbocyclic analogues (carbocyclic-LNA-T and carbocyclic-
ENA-T) and aza-ENA20 modification have been tested and have
shown good target affinity as shown by the increase (compared
to the native counterpart) in thermal stability of duplexes with
their complementary RNAs (Tm increase by 3.5-5 °C and
1.5 °C/modification) for carbocyclic-LNA-T and carbocyclic-
ENA-T, respectively.
1
87%). Rf ) 0.60 (20% ethyl acetate in cyclohexane, v/v). H NMR
(600 MHz, CDCl3) δ: 7.3 (10 H, m), 6.20 (1H, dd, JH6,H7 ) 11 Hz, 18
Hz, H6), 5.76 (1H, d, JH1,H2 ) 3.9 Hz, H1), 5.22 (1H, dd, 1.8 Hz, 17.5
Hz, H7), 5.25 (1H, dd, J ) 1.8 Hz, 11 Hz, H7), 4.76 (1H, d, Jgem) 12
Hz, CH2Bn), 4.59 (1H, d, Jgem) 12 Hz, CH2Bn), 4.57 (1H, app t, J )
4.6 Hz, H2), 4.5 (1H, d, Jgem) 12 Hz, CH2Bn), 4.40 (1H, d, Jgem ) 12
Hz, CH2Bn), 4.25 (1H, d, J ) 4.9 Hz, H3), 3.32 (2H, s, H5), 1.52 (3H,
s, CH3), 1.28 (3H, s, CH3); 13C NMR (125.7 MHz) δ: 138.0, 137.8,
(Bn), 135.4 (C6), 128.3, 127.9, 127.8, 127.5 (aromatic), 116.3(C7),
113.3, 103.8 (C1), 86.4 (C4), 78.3 (C2), 77.2 (C3), 73.4 (CH2Bn), 72.7
(C5), 72.4 (CH2Bn), 26.0 (CH3), 25.6 (CH3).
5. The AONs containing the carbocyclic analogues of ENA
and LNA have shown unprecedented nuclease stability, starting
from the 3′-neighboring nucleotide of the modification site till
the 5′-end; thus, the nuclease stable part of the AON depends
upon where the site of the modification is introduced in the
modified AON.
3,5-Di-O-benzyl-4-C-hydroxyethyl-1,2-O-isopropylidene-r-D-ri-
bofuranose (5). To a solution of 2 in dry THF (200 mL) was added
0.5 M solution of 9-BBN (250 mL, 128 mmol), and the mixture was
stirred overnight. Water was added till gas evolution stopped, 3 N NaOH
solution (50 mL) was added, and then slowly 33% aqueous H2O2 was
added while the temperature was maintained at about 50 °C. The
mixture was stirred for about 30 min at room temperature and then
partitioned between ethyl acetate and water and extracted twice with
ethyl acetate (100 mL). The organic layer was dried, evaporated, and
purified over silica gel (20-30% ethyl acetate in cyclohexane, v/v) to
give 5 as colorless oil (16 g, 39 mmol, 95%). Rf ) 0.40 (5% methanol
6. The modified AONs have shown recruitment of RNase H
almost as efficiently as by native deoxy/RNA duplexes and
brought about degradation of the RNA target.
7. A comparison of the nuclease stability of carbocyclic
analogues with LNA and aza-ENA and its correlation with the
energy of solvation indicates that it is the relative hydrophobicity
of the carbocyclic modification leading to a change of hydration
pattern around the modified nucleotide in the AONs which is
probably responsible for the high nuclease stability of the
modified AONs.
1
in dichloromethane, v/v). H NMR (270 MHz, CDCl3) δ: 7.3 (10H,
m), 5.75 (1H, d, JH1,H2 ) 4 Hz, H1), 4.77 (1H, d, Jgem ) 12 Hz, CH2-
Bn), 4.67 (1H, app t, J ) 4.8 Hz, H2), 4.55 (1H, d, Jgem ) 12 Hz,
CH2Bn), 4.52 (1H, d, Jgem ) 12 Hz, CH2Bn), 4.41 (1H, d, Jgem ) 12
Hz, CH2Bn), 4.12 (1H, d, JH2,H3 ) 5.57 Hz, H3), 3.8 (2H, m, H7),
3.51 (1H, d, J ) 10.3 Hz, H5), 3.3 (1H, d, J ) 10.4 Hz, H5), 3.0 (1H,
br s, 7-OH), 2.51 (1H, ddd, J ) 3.4 Hz, 6.4 Hz, 15 Hz, H6), 1.80 (1H,
ddd, J ) 4 Hz, 8.5 Hz, 15 Hz, H6), 1.63 (3H, s, CH3), 1.31 (3H, s,
CH3); 13C NMR (67.9 MHz, CDCl3) δ: 137.9 (Bn), 127.7, 127.8, 127.9,
128.5 (aromatic), 113.6 (isopropyl), 104.4 (C1), 87.3 (C4), 79.3 (C2),
78.6 (C3), 73.6 (CH2Bn), 73.3 (C5), 72.5 (CH2Bn), 58.8 (C7), 34.0
(C6), 26.5 (CH3), 26.4 (CH3); MALDI TOF m/z [M + Na]+ found
437.14, calcd 437.19.
4-C-Allyl-3,5-di-O-benzyl-1,2-O-isopropylidene-r-D-ribofura-
nose (6). Oxalyl chloride (6.2 mL, 72.46 mmol) was added to
dichloromethane (200 mL) cooled at -78 °C. DMSO (11 mL, 145
mmol) was added dropwise to this solution over 30 min After stirring
for an additional 20 min, a solution of 5 (15 g, 36.23 mmol) in
Implication. Remarkably, a single incorporation of carbocy-
clic-LNA/-ENA-T into AONs leads to very much more en-
hanced nuclease stability in the blood serum (stable >48 h)
[compared to those of the native (fully degraded <3 h) and the
LNA-modified AONs (fully degraded <9 h) and aza-ENA
(≈85% stable in 48 h)]. This enhanced stability of the
carbocyclic-LNA/-ENA-T-containing AONs however do not
compromise the recruitment of the RNase H to cleave the
complementary RNA in the modified AON/RNA heteroduplex,
compared to that of the native. This enhanced lifetime of these
carbocyclic-modified AONs in the blood serum may produce
the highly desired pharmacokinetic properties and consequently
a reduction of the required dosage and the toxicity while down-
regulating a message in ViVo, which may make the carbocyclic-
9
J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007 8375

A R T I C L E S
Srivastava et al.
dichloromethane (100 mL) was added dropwise to this mixture in about
20 min and stirred at -78 °C for another 45 min. DIPEA (35 mL, 200
mmol) was added to this cooled mixture and allowed to warm to room
temperature. Water was next added to the reaction and twice extracted
with dichloromethane (100 mL). The organic layer was washed with
water and brine, dried over MgSO4, and concentrated under reduced
pressure. In another reaction BuLi (1.6 M solution in hexane, 78 mL,
126 mmol) was added to a precooled suspension of methyl-triph-
enylphosphonium bromide (45 g,126 mmol) at 0 °C in dry THF and
stirred at room temperature for 1 h. The yellow solution so obtained
was cooled to -78 °C, and a solution of crude aldehyde in dry THF
was then added dropwise in about 20 min and stirred at -78 °C
overnight. The reaction was quenched with saturated aqueous NH4Cl,
stirred for about 1 h at room temperature, extracted with ether (3 ×
200 mL), dried over MgSO4, and concentrated under reduced pressure.
The crude material was purified by column chromatography on silica
gel (0-10% ethyl acetate in cyclohexane, v/v) to give 6 as a yellowish
oil (12 g, 29 mmol, 70%). Rf ) 0.60 (20% ethyl acetate in cyclohexane,
v/v). 1H NMR (270 MHz, CDCl3) δ: 7.35 (10H, m, Bn), 5.95 (1H, m,
H7), 5.77 (1H, d, JH1,H2 ) 4 Hz, H1), 5.09 (2H, m, H8), 4.78 (1H, Jgem
) 12.1 Hz, CH2Bn), 4.63 (1H, app t, J ) 4.3 Hz, H2), 4.58 (1H, d,
Jgem ) 12.1 Hz, CH2Bn), 4.54 (1H, d, Jgem ) 12 Hz, CH2Bn), 4.41
(1H, d, Jgem ) 12 Hz, CH2Bn), 4.18 (1H, d, JH2,H3 ) 5.2 Hz, H3), 3.45
(1H, d, JH5,H5 ) 10.4 Hz, H5), 3.32 (1 H, d, JH5,H5 ) 10.4 Hz, H5),
2.96 (1H, dd, JH6,H6 ) 14.7 Hz, JH6,H7 ) 7.4 Hz, H6), 2.39 (1H, dd,
JH6,H6 ) 14.7 Hz, JH6,H7 ) 8.5 Hz, H6), 1.60 (3H, s, CH3), 1.33 (3 H,
s, CH3); 13C NMR (67.9 MHz) δ: 138.3 (Bn), 134.0 (C7), 127.6, 127.8,
128.4, 128.6 (aromatic), 117.6 (C8), 113.3 (isopropyl), 104.2 (C1), 86.4
(C4), 79.6 (C2), 78.3 (C3), 73.5 (CH2Bn), 72.5 (C5), 72.3 (CH2Bn),
37.0 (C6), 26.8 (CH3), 26.3 (CH3); MALDI-TOF m/z [M]+ Found 410.2,
calcd 410.1.
(1.17 g, 9.6 mmol). Then dropwise was added phenyl chlorothiono-
formate (1.6 mL, 11.53 mmol), and reaction was stirred overnight.
Reaction was quenched with saturated solution of NaHCO3 and
extracted with dichloromethane. The organic layer was dried over
MgSO4, concentrated, and chromatographed over silica gel (10-30%
ethyl acetate in cyclohexane, v/v) to give 10 as yellowish foam (4.1 g,
6.72 mmol, 70%). Rf ) 0.60 (30% ethyl acetate in cyclohexane, v/v).
1H NMR (600 MHz) δ: 8.58 (1H, s, N-H), 7.48 (1H, s, H6), 7.19
(15H, m), 6.44 (1H, d, JH1′,H2′ ) 6.1 Hz, H1′), 5.97 (1H, app t, J ) 5.8
Hz, H2′), 5.83 (1H, m, H7′), 5.09 (2H, m, H8′-H8′’), 4.77 (1H, d, Jgem
) 11.2 Hz, CH2Bn), 4.55 (3H, m, CH2Bn, H3′), 3.71 (1H, d, JH5′,H5′′
)
10.3 Hz, H5′′), 3.48 (1H, d, JH5′,H5′′ ) 10.3 Hz, H5′), 2.66 (1H, dd,
JH6,H6 )14.6 Hz, JH6,H7 ) 8.1 Hz, H6′′), 2.35 (1 H, dd, JH6,H6 ) 14.6
Hz, JH6,H7 ) 8.1 Hz, H6′), 1.51 (3H, CH3, thymine); 13C NMR (125.7
MHz) δ: 194.4 (>CdS), 163.4 (C4), 150.3 (C2), 137.1, 137.2
(aromatic), 135.6 (C6), 132.4 (C7′), 121.6, 126.7, 127.3, 127.6, 127.8,
128.0, 128.1, 128.4, 128.6, 129.3, 129.5(aromatic), 118.8 (C8′), 111.5
(C5′), 87.1 (C4′), 85.1 (C2′), 82.9 (C1′), 77.8 (C3′), 74.9 (C-CH2Bn),
73.7 (C5′), 73.7 (C-CH2Bn), 37.3 (C6′), 11.9 (CH3, thymine); MALDI-
TOF m/z [M + H]+ found 615.16 calcd 615.21.
(1R,3R,4R,5R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-
yl)-2-oxa-bicyclo[2.2.1]heptane (11a/11b). Compound 10 (3.0 g, 4.8
mmol) was dissolved in 150 mL of dry toluene and purged with dry
nitrogen for 30 min. Bu3SnH (1.3 mL, 4.8 mmol) was dissolved in 20
mL of toluene, and half of this solution was added dropwise to refluxing
solution in over 30 min. AIBN (0.920 g, 4.8 mmol) was dissolved in
20 mL of dry toluene and added to the above solution dropwise, and
simultaneously was added the remaining solution of Bu3SnH over 60-
70 min. After 60 min of reflux, the solution was cooled, CCl4 (10 mL)
was added, and the mixture stirred for 20 min. A solution of iodine in
dichloromethane was added to the above solution until a faint coloration
persisted, and then solvent was evaporated. The solid so obtained was
taken up in ethyl acetate and repeatedly washed with saturated aqueous
solution of potassium fluoride till white flocculent precipitate was seen.
The organic layer was dried evaporated and chromatographed over silica
gel (10-60% ethyl acetate in cyclohexane, v/v) to give a diastereomeric
mixture 11a/11b in 73% yield (1.6 g, 3.5 mmol). Rf ) 0.40 (30% ethyl
acetate in cyclohexane, v/v). 13C NMR (125.7 MHz) δ: 163.8, 149.7,
137.7, 137.4, 136.3, 136.2, 128.5, 128.4, 128.3, 127.8, 127.8, 127.7,
127.7, 127.6, 127.4, 109.0, 88.8, 88.6, 84.1, 78.6, 78.1, 77.1, 73.6, 71.9,
71.7, 67.5, 67.4, 47.9, 47.8, 38.6, 37.6, 33.5, 28.7, 20.0, 15.4, 12.0,
11.9; MALDI-TOF m/z [M + H]+ found 463.11 calcd 463.22.
1-[4-C-Allyl-3,5-di-O-benzyl-2-O-acetyl-â-D-ribofuranosyl]-thym-
ine (8). Acetic anhydride (17 mL, 175 mmol) and acetic acid (87 mL)
were added to 4 (6.0 g, 14 mmol) and cooled, and triflic acid (0.1 mL,
0.7 mmol) was added to it and stirred. After 30 min the reaction was
quenched with cold saturated NaHCO3 solution and extracted with
dichloromethane. The organic layer was dried and evaporated. The crude
was coevaporated with dry CH3CN thrice and dissolved in the same.
Thymine (2.4 g, 19 mmol) and N,O-bis(trimethylsilyl)acetamide (9.6
mL, 38 mmol) were added to this solution and refluxed for 45 min till
the suspension becomes a clear solution. This solution was cooled to
0 °C, and TMSOTf (3.5 mL, 17.5 mmol) was added dropwise and
stirred overnight. The reaction was quenched with saturated NH4Cl
solution and extracted with dichloromethane The organic layer dried,
evaporated, and chromatographed over silica gel (2-6% methanol in
dichloromethane, v/v) to give 8 as a white foam (6 g, 11 mmol, 80%).
Rf ) 0.60 (5% methanol in dichloromethane, v/v). 1H NMR (500 MHz)
δ: 7.48 (1H, s, H6), 7.3 (10H, m), 6.25 (1H, d, JH1′,H2′ ) 5.5 Hz, H1′),
5.83 (1H, m, H7′), 5.41 (1H, app t, 5.5 Hz, H2′), 5.09 (2H, m, H8′,
8′′), 4.63 (1H, d, Jgem ) 11.5 Hz, CH2Bn), 4.50 (1H, d, Jgem ) 11.5
Hz, CH2Bn), 4.47 (1H, d, Jgem ) 11.5 Hz, CH2Bn), 4.45 (1H, d, Jgem
) 11.5 Hz, CH2Bn), 4.37 (1H, d, J2H′,3H′ ) 5.94 Hz, H3′), 3.67 (1H, d,
JH5′,H5′′ ) 10.5 Hz, H5′′), 3.38 (1H, d, JH5′,H5′′ ) 10.5 Hz, H5′), 2.65
(1H, dd, JH6,H7 ) 6 Hz, JH6′,H6′′ )15 Hz, H6′′), 2.29 (1H, dd, 8 Hz, 14
Hz, H6′), 2.08 (3H, acetyl), 1.49 (3H, CH3-thymine); 13C NMR (125.7
MHz) δ: 170.1(>CdO acetyl), 163.9 (C4), 150.5 (C2), 135.6, 137 2,
137.5 (aromatic), 132.5 (C6), 127.6, 127.8, 128.0, 128.0, 128.4, 128.6
(aromatic), 118.6 (C8′), 111.3 (C5), 86.9 (C4′), 85.9 (C1′), 77.6 (C3′),
75.1 (C2′), 74.3 (CH2Bn), 73.5 (CH2Bn), 72.9 (C5′), 37.2 (C6′), 20.7
(CH3, acetyl), 11.9 (CH3, thymine): MALDI-TOF m/z [M + H]+ found
521.14 calcd 521.22.
(1R,3R,4R,5R,7S)-7-Hydroxy-1-hydroxymethyl-3-(thymin-1-yl)-
2-oxa-bicyclo[2.2.1]heptane (12). To a solution of 11a/11b (1.5 g,
3.24mmol) in dry methanol were added 20% Pd(OH)2/C (1.6 g) and
ammonium formate (4.0 g, 65 mmol) and refluxed. The same amount
of ammonium formate and 20% Pd(OH)2/C was added twice after 3 h
and the reaction refluxed for 8 h. After the reaction was finished as
seen by TLC, the suspension was filtered over celite and organic phase
evaporated and chromatographed over silica gel (2-7% methanol in
dichloromethane, v/v) to obtain 12a/12b (0.70 g, 2.4mmol, 76%) as a
1
white powder. Rf ) 0.50 (7% methanol in dichloromethane, v/v). H
NMR (600 MHz, D2O) of 12a δ: 7.81 (1H, s, H6, thymine), 5.78 (1H,
s, H1′), 4.18 (1H, d, 4.4 Hz, H3′), 3.85 (1H, d, JH5′,5′′ ) 12.6 Hz, H5′),
3.83 (1 H, d, JH5′,5′′ ) 12.6 Hz, H5′′), 2.65 (1H, m, H7′), 2.43 (1H, d,
JH2′ H7′ ) 4.4 Hz, H2′), 2.04 (1H, dd, JH6′,H6′′ ) 12.3 Hz, JH7′,H6′′ ) 10.6
,
Hz, H6′′), 1.89 (3H, d, J ) 1.25 Hz, thymine CH3), 1.23 (3H, d, JH7′,CH3
) 7.3 Hz, C7′-CH3), 1.17 (1H, dd, JH6′,H6′′ ) 12.3 Hz, JH7′,H6′′ ) 4.9
Hz, H6′); 13C NMR (125.7 MHz, D2O) δ: 166.64 (C4), 151.05 (C2),
137.33 (C6), 109.7 (C5), 90.6 (C4′), 83.7 (C1′), 71.7 (C3′), 58.7 (C5′),
49.7 (C7′), 36.0, (C6′), 28.1 (C7′), 14.4 (C7′-methyl) 11.5 (thymine
CH3); MALDI-TOF m/z [M + H]+ found 283.94, calcd 283.14.
1-[4-C-Allyl-3,5-di-O-benzyl-2-O-phenoxythiocarbonyl-â-D-ribo-
furanosyl]-thymine (10). Compound 8 (5.0 g, 9.6 mmol) was treated
with 27% methanolic ammonia solution overnight. After evaporation
of the solvent, the crude was coevaporated thrice with dry pyridine
and dissolved in the same. To this precooled solution was added DMAP
9
8376 J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
(1R,3R,4R,5R,7S)-7-Hydroxy-1-(4,4′-dimethoxytrityloxymethyl)-
dichloromethane (50 mL) was added dropwise to this mixture over 20
min and stirred at -78 °C for another 45 min. DIPEA (13 mL, 100
mmol) was added to this cooled mixture and allowed to warm to room
temperature. Water was added to the reaction and twice extracted with
dichloromethane (100 mL). The organic layer was washed with water
and brine, dried over MgSO4, and concentrated under reduced pressure.
In another reaction BuLi (1.6 M solution in hexane, 22 mL, 35 mmol)
was added to a precooled suspension of methyl-triphenylphosphonium
bromide (12.5 g, 35 mmol) at 0 °C in dry THF and stirred at room
temperature for 1 h. The yellow solution so obtained was cooled to
-78 °C, and a solution of crude aldehyde in dry THF was then added
dropwise over 20 min and stirred at -78 °C overnight. The reaction
was quenched with saturated aqueous NH4Cl, stirred for about 1 h at
room temperature, extracted with ether (3 × 100 mL), dried over
MgSO4, and concentrated under reduced pressure. The crude material
was purified by column chromatography on silica gel (0-10% ethyl
acetate in cyclohexane, v/v) to give 17 as a yellowish oil (3.5 g, 8.4
3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (13). Compound 12 (0.70
g, 2.4 mmol) was evaporated twice with dry pyridine and suspended
in the same. 4,4′-Dimethoxytrityl chloride (1.26 g, 3.7 mmol) was added
and stirred overnight. Reaction was quenched with saturated aqueous
NaHCO3 and extracted thrice with dichloromethane (50 mL). The
organic phase was dried, evaporated, and chromatographed over silica
gel (1% pyridine/ethyl acetate in cyclohexane, v/v) to give 13 (1.0 g,
1.8 mmol, 74%) as yellow foam. Rf ) 0.60 (5% methanol in
1
dichloromethane, v/v). H NMR (600 MHz) δ: 8.52 (NH thymine),
7.81 (1H, s, H6 thymine), 6.88-7.34 (13H, m), 5.78 (1H, s, H1′), 4.31
(1H, s, H3′), 3.66 (1H, d, JH5′,H5′′ ) 12.6 Hz, H5′′), 3.55 (1H, d, JH5′,H5′′
) 12.6 Hz, H8′), 2.65 (1H, m, H7′), 2.4 (1H, d, JH2′,H3′′ ) 4.4 Hz,
H2′), 2.04 (1H, dd, JH6′,H6′′ ) 12.3 Hz, JH7′,H6′′ ) 10.6 Hz, H6′′), 1.89
(3H, d, J ) 1.3 Hz, thymine CH3), 1.23 (3H, d, 7.3 Hz, CH3-C7′),
1.17 (1H, dd, JH6′,H6′′ ) 12.3 Hz, JH6′,H7′ ) 4.9 Hz, H6′); 13C NMR
(600 MHz, D2O) δ: 164 (C4), 158.7, 149. 8 (C2), 135.6, 135.7, 135.9
(aromatic), 130 (C6), 113.3, 127.1, 128, 128.1 (aromatic), 109.4 (C5),
89.4 (C4′), 84.1 (C1′), 73.1 (C3′), 61 (C5′), 55.2 (OCH3), 50.4 (C2′),
37.4 (C6′), 28.5 (C7′), 15.3 (C7′-methyl), 12.3 (CH3, thymine); MALDI-
TOF m/z [M + H]+ found 585.22, calcd 585.25.
(1R,3R,4R,5R,7S)-7-(2-(Cyanoethoxy-(diisopropylamino)-phos-
phinoxy)-1-(4,4′-dimethoxytrityloxymethyl)-3-(thymin-1-yl)-2-oxa-
bicyclo[2.2.1]heptane (14). Compound 13 (0.5 g, 0.85 mmol) was
dissolved in 6 mL of dry THF, DIPEA (0.57 mL, 0.2 mmol) was added
at 0 °C followed by 2-cyanoethyl N,N-diisopropyl phosphoramidoch-
loridite (0.4 mL, 1.7 mmol), and reaction was stirred overnight at room
temperature. Methanol (0.5 mL) was added followed by aqueous
saturated NaHCO3 and extracted twice (25 mL) with dichloromethane.
The organic phase was dried, evaporated, and chromatographed on silica
gel (1% Et3N, ethyl acetate in cyclohexane, v/v) to give 14 (0.5 g,
80%) as a mixture of four isomers; 31P NMR (109.4 MHz, CDCl3):
148.82, 149.19, 149.36, 150.08. The four signals in 31P NMR have
been integrated and are found to be in the ratio of 7:3 as found in the
1H NMR spectrum of 12a/12b. The integration is included in the SI
(Inset in Figure S42). MALDI-TOF m/z [M + H]+ found 785.67, calcd
785.36.
1
mmol, 72%). Rf ) 0.40 (30% ethyl acetate in cyclohexane, v/v). H
NMR (270 MHz, CDCl3) δ: 7.35 (10H, m), 5.84 (1H, m, H8), 5.76
(1H, d, JH1,H2 ) 4 Hz, H1), 4.95 (2H, m, H9, 9′), 4.80 (1H, d, Jgem
)
12.1 Hz, CH2Bn), 4.62 (1H, app t, J ) 4.5 Hz, H2), 4.60 (1H, d, Jgem
) 12.1 Hz, CH2Bn), 4.53 (1H, d, Jgem) 11.9 Hz, CH2Bn), 4.4 (1H, d,
Jgem ) 12 Hz, CH2Bn), 4.15 (1H, d, JH2,H3 ) 5.2 Hz, H3), 3.51 (1H, d,
JH5,H5′ ) 10.3 Hz, H5), 3.31 (1H, d, JH5,H5′ ) 10.3 Hz, H5′), 2.35 (2H,
m, H6 and H6′), 2.1 (1H, m, H7′), 1.70 (1H, m, H7′), 1.62 (3H, s,
CH3), 1.33 (3H, s, CH3); 13C NMR (67.9 MHz) δ: 139.1 (C8), 138.2,
128.4, 127.8, 127.7 (aromatic), 114.2 (C9), 113.1, 104.1 (C1), 86.7
(C4), 79.3 (C2), 78.7(C3), 73.5 (CH2Bn), 72.8 (C5), 72.4 (CH2Bn) 30.9
(C7), 27.8 (C6), 26.70 (CH3), 26.3 (CH3).
1-[3,5-Di-O-benzyl-4-C-penten-yl-2-hydroxy-â-D-ribofuranosyl]-
thymine (20). Acetic anhydride (9.5 mL, 50 mmol) and acetic acid
(50 mL) were added to 4 (3.5 g, 8.2 mmol) and cooled on ice bath,
and triflic acid (0.03 mL, 0.4 mmol) was added to it and stirred. After
30 min, the reaction was quenched with cold saturated NaHCO3 solution
and extracted with dichloromethane. The organic layer was dried and
evaporated. The crude product was coevaporated with dry CH3CN thrice
and dissolved in the same. Thymine (1.5 g, 12.37 mmol) and N,O-bis-
(trimethylsilyl)acetamide (5.0 mL, 24.75 mmol) were added to this
solution and refluxed for 45 min till the suspension became a clear
solution. This solution was cooled to 0 °C, and TMSOTf (1.71 mL,
9.9 mmol) was added dropwise and stirred overnight. The reaction was
quenched with saturated NH4Cl solution and extracted with dichlo-
romethane. The organic layer was dried, evaporated, and treated with
27% methanolic ammonia overnight. After completion of the reaction,
the solvent was evaporated and the residue purified over silica gel (0-
3% methanol in dichloromethane, v/v) to give 21 as a white foam (2.8
g, 5.77 mmol, 70% in three steps). Rf ) 0.50 (5% methanol in
3,5-Di-O-benzyl-4-C-hydroxypropyl-1,2-O-isopropylidene-r-D-ri-
bofuranose (15). A 0.5 M solution of 9-BBN (88 mL, 44 mmol) was
added dropwise to a solution of 6 (6 g, 14 mmol) in dry THF (100
mL) and stirred overnight. Water was added till gas evolution stopped,
3 N NaOH solution (25 mL) was then added, and slowly 33% aqueous
H2O2 was added while the temperature was maintained at not more
than 50 °C. The mixture was stirred for about 30 min at room
temperature, partitioned between ethyl acetate and water, and extracted
twice with ethyl acetate (100 mL). The organic layer was dried,
evaporated, and purified over silica gel (30-35% ethyl acetate in
cyclohexane, v/v) to give 15 as a colorless oil (5.4 g, 13 mmol, 90%);
Rf ) 0.40 (50% ethyl acetate in cyclohexane, v/v). 1H NMR (270 MHz
CDCl3) δ: 7.32 (10H, m, benzyl) 5.75 (1H, JH1,H2 ) 4 Hz, H1) 4.76
(1H, d, Jgem ) 12.1 Hz, CH2Bn), 4.62 (1H, app t, 4.6 Hz, H2), 4.6
(1H, d, Jgem ) 12.3 Hz, CH2Bn), 4.52 (1H, d, Jgem ) 12.3 Hz, CH2-
Bn), 4.52 (1H, d, Jgem ) 12 Hz, CH2Bn) 4.41 (1H, d, Jgem ) 12 Hz,
CH2Bn), 4.15 (1H, d, J ) 5.3 Hz, H3), 3.83 (2H, m, H8,8′) 3.49 (1H,
d, JH5′,H5′′ ) 10.3 Hz, H5′), 3.3 (1H, d, JH5′,H5′′ ) 10.3 Hz, H5′′), 2.95
(2H, m, H6′, H7′) 1.72 (2H, m, H6′′,7′′), 1.60 (3H, CH3), 1.31 (3H,
CH3); 13C NMR (67.9 MHz) δ: 138.1, 128.4, 127.9, 127.7, 127.6, 113.1
(aromatic), 104.1 (C1), 87.1 (C4), 79.1 (C2), 78.6 (C3), 73.5 (CH2-
Bn), 72.5 (CH2Bn), 72.4 (C5), 62.7 (C8), 27.8 (C7), 26.9 (C6), 26.5
(CH3), 26.0 (CH3); MALDI TOF m/z [M + Na]+ found 451.12, calcd
451.2.
1
dichloromethane, v/v). H NMR (270 MHz, CDCl3) δ: 7.51 (1H, s,
H-6), 7.3 (10H, m, benzyl), 6.00 (1H, d, JH1′,H2′ ) 5.44 Hz, H1′), 5.76
(1H, m, H8′), 4.94 (2H, m, H9′ and H9′′), 4.84 (1H, d, Jgem ) 11.8 Hz,
CH2Bn), 4.58 (1H, d, Jgem ) 11.8 Hz, CH2Bn), 4.52 (1H, d, Jgem ) 12
Hz, CH2Bn), 4.48 (1H, d, Jgem ) 12 Hz, CH2Bn), 4.41 (1H, app t, J )
5.6 Hz, H2′), 4.15 (1H, d, JH2′,H3′ ) 5.8 Hz, H3′), 3.69 (1H, d, JH5′,H5′′
) 9.9 Hz, H5′′), 3.41 (1H, d, JH5′,H5′′ ) 9.9 Hz, H5′), 2.22 (1H, m,
H6′), 2.04 (2H, m, H7′ and H7′′), 1.63 (1H, m, H6′), 1.49 (3H, thymine
CH3); 13C NMR (67.9 MHz, CDCl3) δ: 164.2 (C2), 151.4 (C4), 138.5
(C8′), 136 (C6), 137.4, 130.0, 128.7, 128.5, 128.1, 127.7 (aromatic),
114.6 (C9′), 111.0 (C5), 88.8 (C1′), 87.3 (C4′), 79.1 (C3′), 75.1 (C2′),
73.3 (C5′), 73.6 (CH2Bn), 73.4 (CH2Bn), 31.9 (C6′), 27.7 (C7′), 12.1
(CH3, thymine); MALDI-TOF m/z [M + H]+ found 493.11, calcd
493.17.
3,5-Di-O-benzyl-4-C-penten-yl-1,2-O-isopropylidene-r-D-ribo-
furanose (17). Oxalyl chloride (2.48 mL, 29.20 mmol) was added to
dichloromethane (100 mL) and cooled at -78 °C. DMSO (3.3 mL,
46.72 mmol) was added dropwise to this solution over 30 min. After
stirring for an additional 20 min, a solution of 2 (5 g, 11.68 mmol) in
1-[3,5-Di-O-benzyl-4-C-penten-yl-2-O-phenoxythiocarbonyl-â-D-
ribofuranosyl]-thymine (21). The nucleoside 20 (2.8 g, 5.7mmol) was
evaporated thrice with dry pyridine and dissolved in the same. To this
precooled solution was added DMAP (0.69 g, 5.7 mmol), dropwise
was added phenyl chlorothionoformate (1.15 mL, 8.55 mmol), and the
9
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A R T I C L E S
Srivastava et al.
reaction was stirred overnight. The reaction was quenched with saturated
solution of NaHCO3 and extracted with dichloromethane. Organic layer
was dried over MgSO4, concentrated, and chromatographed over silica
gel (10-30% ethyl acetate in cyclohexane, v/v) to give 21 as a
yellowish foam (2.1 g, 3.42 mmol, 60%). Rf ) 0.60 (30% ethyl acetate
(1R,2R,5R,7R,8S)-5-(4,4′-Dimethoxytrityloxymethyl)-8-hydroxy-
7-(thymin-1-yl)-6-oxa-bicyclo[3.2.1]octane (24). Compound 23 (0.5
g, 1.68 mmol) was evaporated twice with dry pyridine and then
suspended in the same. 4,4′-dimethoxytrityl chloride was added and
stirred at room temperature overnight. Reaction was quenched with
aqueous NaHCO3 and extracted with dichloromethane. The organic
layer was dried, evaporated, and chromatographed on silica gel (1%
pyridine, ethyl acetate in cyclohexane, v/v) to give 26 (0.8 g, 1.34 mmol,
80%) as a yellowish foam. Rf ) 0.60 (30% ethyl acetate in cyclohexane,
v/v). 1H NMR (600 MHz, CDCl3 + DABCO) δ: 7.8 (1H, s, H6), 6.83-
7.4 (13 H, m), 5.77 (1H, s, H1′), 4.44 (1H, d, JH2′,H3′ ) 5.2 Hz, H3′),
3.78 (6H, s, 2× OCH3), 3.36 (1H, d, JH5′,H5′′ ) 10.6 Hz, H5′), 3.26
(1H, d, JH5′,H5′′ ) 10.6 Hz, H5′′), 2.30 (1H, m, H8′), 2.27 (1H, d, JH2′,H3′
) 5.2 Hz, H2′), 1.77 (1H, m, H6′′), 1.64 (1H, m, H7′), 1.36 (1H, m,
H7′′), 1.25 (2H, m, H6′ and H7′), 1.1 (3H, d, JH8′,CH3 ) 7 Hz, C8′-
CH3); 13C NMR (125.7 MHz) δ: 164.1 (C4), 158.6 (C2), 158.6, 149.9,
144.3, 135.9 (C6), 135.4, 135.3, 130.0, 128.1, 128.0, 127.1, 113.2,
109.4, 84.8 (C1′), 84.8 (C4′), 67.9 (C3′), 54.5 (C5′), 55.2 (OCH3), 50.8
(C2′), 46.9 (DABCO), 26.9 (C7′), 26.4 (C6′), 25.0 (C8′), 18.9 (C8′-
CH3), 11.9 (CH3, thymine); MALDI-TOF m/z [M + Na]+ found 621.59,
calcd 621.29.
(1R,2R,5R,7R,8S)-8-(2-(Cyanoethoxy(diisopropylamino)-phosphi-
noxy)-5-(4,4′-dimethoxytrityloxymethyl)-7-(thymin-1-yl)-6-oxa-bicyclo-
[3.2.1]octane (25). Compound 24 (0.5 g, 0.83 mmol) was dissolved in
6 mL of dry THF, diisopropylethylamine (0.75 mL, 4.18 mmol) was
added, and the reaction was cooled in an ice bath. To this was added
2-cynoethyl N,N-diisopropylphosphoramidochloridite (0.36 mL, 1.66
mmol), and the mixture was stirred overnight at room temperature.
Reaction was quenched by addition of cold aqueous NaHCO3 and
extracted with dichloromethane. The organic layer was dried, evapo-
rated, and chromatographed over silica gel (1% Et3N, ethyl acetate in
cyclohexane, v/v) to give 27 (0.55 g, 0.7 mmol 84%); 31P NMR (109.4
MHz, CDCl3) δ 149.25, 150.26; MALDI-TOF m/z [M + H]+ found
799.64, calcd 799.9.
UV Melting Experiments. All 17 modified AONs have been
purified as a single component on PAGE (20% polyacrylamide with 7
M urea), extracted with 0.3 M NaOAc, and desalted with a C-18 reverse
phase cartridge to give AONs in >99% purity, and correct masses have
been obtained by MALDI-TOF mass spectroscopy for each one of them
(Table 1). Determination of the Tm of the AON/RNA hybrids was
carried out in the following buffer: 57 mM Tris-HCl (pH 7.5), 57 mM
KCl, 1 mM MgCl2. Absorbance was monitored at 260 nm in the
temperature range from 20 °C to 90 °C using an UV spectrophotometer
equipped with a Peltier temperature programmer with the heating rate
of 1 °C per minute. Prior to measurements, the samples (1 µM of AON
and 1 µM RNA mixture) were preannealed by heating to 90 °C for 5
min followed by slow cooling to 4 °C and 30 min equilibration at this
temperature. and are the average of at least three independent runs.
32P Labeling of Oligonucleotides. The oligoribonucleotides and
oligodeoxyribonucleotides were 5′-end labeled with 32P using T4
polynucleotide kinase, [γ-32P]ATP, and the standard protocol.71 Labeled
AONs and the target RNA were purified by 20% denaturing PAGE,
and specific activities were measured using a Beckman LS 3801
counter.
Exonuclease Degradation Studies. Stability of the AONs toward
3′-exonucleases was tested using snake venom phosphodiesterase
(SVPDE) from Crotalus adamanteus. All reactions were performed at
3 µM DNA concentration (5′-end 32P labeled with specific activity
80 000 cpm) in 56 mM Tris-HCl (pH 7.9) and 4.4 mM MgCl2 at 21 °C.
An exonuclease concentration of 17.5 ng/µL was used for digestion of
oligonucleotides. Total reaction volume was 14 µL. Aliquots were taken
at 1, 2, 24, 48, and 72 h and quenched by addition of the same volume
of 50 mM EDTA in 95% formamide. Reaction progress was monitored
by 20% denaturing (7 M urea) PAGE and autoradiography.
1
in cyclohexane, v/v). H NMR (600 MHz, CDCl3) δ: 8.32 (1H, br s,
NH), 7.48 (1H, s, H6), 7.48-6.99 (15H, m), 6.4 (1H, d, JH1′,H2′ ) 6.3
Hz, H1′), 5.94 (1H, app t, J ) 6.0 Hz, H2′), 5.77 (1H, m, H8′), 4.97
(2H, m, H9′ and H9′′), 4.77(1H, d, Jgem) 12 Hz, CH2Bn), 4.55 (3H, d,
Jgem ) 12 Hz, CH2Bn), 4.53 (1H, d, JH2′,H3′ ) 5.8 Hz, H3′), 3.74 (1H,
d, JH5′,H5′′ ) 9.9 Hz, H5′′), 3.47 (1H, d, JH5′,H5′′ ) 9.9 Hz, H5′), 2.22
(1H, m, H7′), 2.06 (1H, m, H7′′), 1.94 (1H, m, H6′), 1.66 (1H, m,
H6′′), 1.53 (3H, thymine CH3); 13C NMR (125.7 MHz, CDCl3) δ: 194.4
(CdS), 163.3 (C4), 153.3, 150.2 (C2), 138.1 (C8′), 137.3, 137.0, 135.6
(C6), 129.5-121.9 (aromatic), 114.8 (C9′), 111.5 (C5), 87.6 (C4′), 85.1
(C1′), 82.9 (C2′), 78.1 (C3′), 74.9 (CH2Bn), 73.7 (C5′), 73.6 (CH2Bn),
31.6 (C6′), 27.6 (C7′), 12.0 (CH3 thymine); MALDI-TOF m/z [M +
H]+ found 629.09, calcd 629.73.
(1R,2R,5R,7R,8S)-8-Benzyloxy-5-benzyloxymethyl-7-(thymin-1-
yl)-6-oxa-bicyclo[3.2.1]octane (22). 21 (2.1 g, 3.3 mmol) was dissolved
in 200 mL of dry toluene, purged for 30 min, and then refluxed. Bu3-
SnH (0.8 mL, 3.0 mmol) dissolved in 25 mL of dry toluene was added
dropwise to this solution and refluxed for 15 min. AIBN (0.768 g,
4mmol) was dissolved in dry toluene and added dropwise to the
refluxing solution over 60 min. Simultaneously was added Bu3SnH (0.8
mL, 3.3 mmol) dissolved in 25 mL of dry toluene, and the reaction
was refluxed till starting material was exhausted in about an additional
60 min. The reaction was cooled, and 25 mL of CCL4 was added
followed by addition of a solution of iodine dissolved in ether till the
color persisted. The solvent was evaporated over vacuum and the
residue taken up in diethyl ether and repeatedly washed with a saturated
solution of potassium fluoride. The ether layer was dried, evaporated,
and chromatographed over silica gel (0-20% ethyl acetate in cyclo-
hexane, v/v) to give 22 as a white powder (1.19 g, 2.5 mmol, 76%). Rf
1
) 0.40 (30% ethyl acetate in cyclohexane, v/v). H NMR (600 MHz,
CDCl3) δ: 8.71 (1H, br s, NH), 8.06 (1H, s, H6), 7.3 (10H, m), 5.82
(1H, s, H1′), 4.56 (2H, dd, Jgem ) 10.8 Hz, CH2Bn), 4.54 (1H, d, Jgem
) 10.8 Hz, CH2Bn), 4.47(1H, d, Jgem ) 10.8 Hz, CH2Bn), 4.17 (1H, d,
JH2′,H3′ ) 5.4 Hz, H3′), 3.69 (1H, d, JH5′,H5′′ ) 10.8 Hz, H5′), 3.55 (1H,
d, JH5′,H5′′ ) 10.8 Hz, H5′′), 2.31 (1H, d, JH2′,H3′ ) 4.8 Hz, H2′), 2.22
(1H, m, H8′), 1.83 (1H, m, H7′), 1.66 (1H, m, H6′′), 1.43 (3H, s, CH3
thymine), 1.33 (2H, m, H6′, H7′′), 1.11 (3H, JCH3,H8′ ) 7.2 Hz, CH3-
C8′); 13C NMR (125.7 MHz, CDCl3) δ: 164.1 (C4), 149.9 (C2) 137.4,
136.6 (C6), 128.5, 128.3, 128.0, 127.8, 127.3, 109.1(C5), 84.7 (C1′),
84.6 (C4′), 73.5(C3′), 73.51 (CH2Bn), 71.7 (CH2Bn), 70.6 (C5′), 48.4
(C2′), 27.0 (C6′), 26.5 (C7′), 25.6 (C8′), 18.9(C8′, methyl), 11.8 (CH3,
thymine); MALDI-TOF m/z [M + H]+ found 477.56, calcd 477.39.
(1R,2R,5R,7R,8S)-8-Hydroxy-5-hydroxymethyl-7-(thymin-1-yl)-
6-oxa-bicyclo[3.2.1]octane (23). Compound 22 (1.2 g, 2.5 mmol) was
dissolved in dry methanol. 20% Pd(OH)2/C (1.3 g) and ammonium
formate (3.4 g, 50 mmol) were added and refluxed. The same amount
of ammonium formate and 20% Pd(OH)2/C was added thrice after every
3 h, and the reaction was refluxed overnight. After the reaction was
finished as seen by TLC, the suspension was filtered over celite and
the organic phase evaporated and chromatographed over silica gel (2-
7% methanol in dichloromethane, v/v) to give 25 as a white powder
(0.60 g, 2.0 mmol, 82%). Rf ) 0.50 (10% methanol in dichloromethane,
v/v). 1H NMR (600 MHz, D2O) δ: 8.19 (1H, s, H6), 5.77 (1H, s, H1′),
4.25 (1H, d, JH2′,H3′ ) 5.2 Hz, H3′), 3.71 (2H, s, H5′ and H5′′), 2.26
(1H, d, JH2′,H3′ ) 5.2 Hz, H2′), 2.20 (1H, m, H8′), 1.86 (3H, s, CH3-
thymine), 1.68 (1H, m, H6′), 1.36 (1H, m, H7′), 1.25 (2H, m, H7′′,
H6′′), 1.05 (3H, d, JCH3,H8′ ) 7 Hz, C8′-CH3); 13C NMR (125.7 MHz,
CDCl3) δ: 165.0 (C4), 150.8 (C2), 137.4 (C6), 108.0 (C5), 86.0 (C4′),
84.2 (C1′), 65.5 (C3′), 62.3 (C5′), 51.6 (C2′), 27.3 (C6′), 26.6 (C7′),
25.5 (C8′), 19.9 (C8′-CH3), 13.3 (CH3, thymine); MALDI-TOF m/z
[M]+ found 296.97, calcd 296.14.
Stability Studies in Human Serum. AONs (6 µL) at 2 µM
concentration (5′-end 32P labeled with specific activity 80 000 cpm)
9
8378 J. AM. CHEM. SOC. VOL. 129, NO. 26, 2007

2′
,4′
-Carbocyclic ribo-Thymidines
A R T I C L E S
3J ) P1 cos2(φ) + P2 cos(φ) + P3 +
were incubated in 26 µL of human blood serum (male AB) at 21 °C
(total reaction volume was 36 µL), and the experiments were repeated
twice up to 48 h. Aliquots (3 µL) were taken at 0, 30 min, 1, 2, 5, 7,
8, 9, 12, 24, 36, 48 h, and quenched with 7 µL of solution containing
8 M urea and 50 mM EDTA, resolved in 20% polyacrylamide
denaturing (7 M urea) gel electrophoresis and visualized by autorad-
iography. The 32P label at the 5′-terminus was cleaved gradually by
the phosphatases present in the blood serum, which resulted in a lower
radioactive count as the digestion progressed with time. Since no new
prominent cleavage products were found toward the later time points,
we considered those prominent stable initial fragments as 100% during
the last time points (12-48 h).
(∆øigroup (P4 + P5 cos2(êi φ + P6 |∆øigroup|))
∑
where P1 ) 13.70, P2 ) -0.73, P3 ) 0.00, P4 ) 0.56, P5 ) -2.47, P6
) 16.90, P7 ) 0.14 (parameters from ref 66), and øigroup ) ∆øiR-substituent
- P7 ∆ø â-substituent where ∆øi are taken as Huggins electronegativi-
∑
i
ties.90
Acknowledgment. Generous financial support from the
Swedish Natural Science Research Council (Vetenskapsrådet),
the Swedish Foundation for Strategic Research (Stiftelsen fo¨r
Strategisk Forskning), and the EU-FP6 funded RIGHT project
(Project no. LSHB-CT-2004-005276) is gratefully acknowl-
edged. We would like to thank Mr. O.P. Varghese for providing
aza-ENA-T modified AONs for this study. Puneet Srivstava
(P.S.) has planned and carried through total synthesis of
carbocyclic nucleosides, their incorporations into oligos, enzy-
mology, physicochemical studies, and NMR characterization.
Małgorzata Wenska was responsible for the scale-up of com-
pound 23 using P.S.’s procedure. Jharna Barman has performed
enzymological experiments with P.S. Wimal Pathmasiri has
performed detailed NMR characterization by 500/600 MHz
NMR and simulation. Oleksandr Plashkevych has performed
molecular structure analysis based on the NMR experiments
and ab initio and MD simulations.
Theoretical Calculations. Carbocyclic analogues of the ENA and
LNA nucleosides have been investigated in silico using ab initio and
molecular dynamics techniques. Their molecular structures have been
refined using Amber force field and available structural information
from NMR experiments according to the following protocol: (i) Derive
3
initial dihedral angles from the observed JH,H using Haasnoot-de
Leeuw-Altona generalized Karplus equation;66,67 (ii) perform NMR-
constrained molecular dynamics (MD) simulation (0.5 ns, 10 steps)
and simulated annealing (SA) followed by 0.5 ns NMR-constrained
simulations at 298 K using the NMR-derived torsional constraints from
Step i to yield NMR-defined molecular structures; (iii) acquire 6-31G**
Hartree-Fock optimized ab initio gas-phase geometries in order to
compare the experimentally derived torsions with the ab initio geometry;
(iv) analyze the full conformational hyperspace using 2 ns NMR/ab
initio-constrained MD simulations of compounds 12a, 12b, and 23
followed by full relaxation of the constraints.
Supporting Information Available: 1H and 13C NMR spectra
of compounds 2-17 and 20-24; 31P NMR of compounds 14
and 25; 1D NOE of 12a/12b and 23; DEPT spectra of
compounds 12a/12b and 23; COSY spectra of 12a/12b and 23;
TOCSY spectra of 12a/12b and 23; HMQC spectra of 10, 11a/
11b, 12a/12b, 21, 22, and 23; HMBC spectra of 10, 11a/11b,
The geometry optimizations of the modified nucleosides have been
carried out by GAUSSIAN 98 program package86 at the Hartree-Fock
level using 6-31G** basis set. The atomic charges and optimized
geometries of compounds 12a, 12b, and 23 were then used as AMBER87
force field parameters employed in the MD simulations. The protocol
of the MD simulations is based on Cheathan-Kollman’s88 procedure
employing a modified version of Amber 1994 force field as it is
implemented in AMBER 7 program package.87 Periodic boxes contain-
ing 718 (12a, 12b) and 753 (23) TIP3P89 water molecules, to model
explicit solvent around the compounds, were generated using xleap
extending 12.0 Å from these molecules in three dimensions in both
the NMR-constrained and -unconstrained MD simulations. The SA
protocol included ten repeats of 25 ps heating steps from 298 to 400 K
followed by fast 25 ps cooling steps from 400 to 298 K. During these
SA and NMR-constrained MD simulations, torsional constraints of 50
kcal mol-1 rad-2 were applied. The constraints were derived from the
experimental 3JH2′,H3′ and available 3JH2′,H7′/H8 coupling constants using
Haasnoot-de Leeuw-Altona generalized Karplus equation.66,67 Ten
SA repeats were followed by 0.5 ns MD run at a constant 298 K
temperature applying the same NMR constraints.
3
12a/12b, 21, 22, and 23; JHH simulations of compounds 12a/
12b and 23; plots of percentage remaining of AONs 6-17 in
human blood serum with time; SVPDE digestion profile of
AONs 1-17 (denaturing PAGE autoradiograms); plots of
percentage remaining of AONs 6-17 in SVPDE; correlation
of experimental 3JH1′,H2′ and 3JH2′,H3′ vicinal coupling constants
in carbocyclic-ENA-T (12a,12b) and carbocyclic LNA-T (23)
as well as their 2′-O- and 2′-N-analogues (ENA-T, aza-ENA-
T, LNA-T, and 2′-amino-LNA-T); overlay of 2500 molecular
structures of the carbocyclic-ENA-T nucleoside collected every
0.2 ps of the last 500 ps (1.5-2.0 ns) of its MD simulation and
1
3
their analysis; tables of H chemical shifts and JHH coupling
constants for compounds 11, 12a/12b, 22, and 23; table of
experimental and theoretical 3JHH vicinal coupling constants and
tables of sugar torsions, pseudorotational phase angle, sugar
puckering amplitude, and solvation energy (Esolv) calculated
using Baron and Cossi’s implementation of the polarizable
conductor CPCM model;85 and a complete discussion of
structure assignment of bicyclic system for compounds 12a/
12b and 23 as well as their molecular structures based on NMR,
ab initio, and MD calculations. This material is available free
of charge via the Internet at http://pubs.acs.org.
Generalized Karplus Parametrization. Relevant vicinal proton
3JH,H coupling constants have been back-calculated from the corre-
sponding theoretical torsions employing Haasnoot-de Leeuw-Altona
generalized Karplus equation66,67 taking into account â substituent
correction in form:
(86) Frisch, M. J., et al. Gaussian 98 (ReVision A.6). Gaussian, Inc.: Pittsburgh,
PA, 1998.
(87) Case, D. A., et al. AMBER 7. University of California: San Francisco,
2002.
(88) Cheatham, T. E., III; Kollman, P. A. J. Am. Chem. Soc. 1997, 119, 4805-
JA071106Y
4825.
(89) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein,
M. L. J. Chem. Phys. 1983, 79, 926-935.
(90) Huggins, M. L. J. Am. Chem. Soc. 1953, 75, 4123-4126.
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