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org/joc

Synthesis of 20 ,40 -Propylene-Bridged (Carba-ENA) Thymidine and Its

Analogues: The Engineering of Electrostatic and Steric Effects at the
Bottom of the Minor Groove for Nuclease and Thermodynamic Stabilities
and Elicitation of RNase H
Yi Liu, Jianfeng Xu, Mansoureh Karimiahmadabadi, Chuanzheng Zhou, and
Jyoti Chattopadhyaya*
Bioorganic Chemistry Program, Department of Cell and Molecular Biology, Box 581, Biomedical Center,
Uppsala University, SE-751 23 Uppsala, Sweden

[email protected]
Received June 21, 2010

20 ,40 -Propylene-bridged thymidine (carba-ENA-T) and five 80 -Me/NH2/OH modified carba-ENA-T

analogues have been prepared through intramolecular radical addition to CdN of the tethered oxime-
ether. These carba-ENA nucleosides have been subsequently incorporated into 15mer oligodeoxynucleo-
tides (AON), and their affinity toward cDNA and RNA, nuclease resistance, and RNase H recruitment
capability have been investigated in comparison with those of the native and ENA counterparts. These
carba-ENAs modified AONs are highly RNA-selective since all of them led to slight thermal stabilization
effect for the AON:RNA duplex, but quite large destabilization effect for the AON:DNA duplex. It was
found that different C80 substituents (at the bottom of the minor groove) on carba-ENA-T only led to
rather small variation of thermal stability of the AON:RNA duplexes. We, however, observed that the
parent carba-ENA-T modified AONs exhibited higher nucleolytic stability than those of the ENA-T
modified counterparts. The nucleolytic stability of carba-ENA-T modified AONs can be further
modulated by C80 substituent to variable extents depending on not only the chemical nature but also
the stereochemical orientation of the C80 substituents: Thus, (1) 80 S-Me on carba-ENA increases the
nucleolytic stability but 80 R-Me leads to a decreased effect; (2) 80 R-OH on carba-ENA had little, if any,
effect on nuclease resistance but 80 S-OH resulted in significantly decreased nucleolytic stability; and (3) 80 -
NH2 substituted carba-ENA leads to obvious loss in the nuclease resistance. The RNA strand in all of the
carba-ENA derivatives modified AON:RNA hybrid duplexes can be digested by RNase H1 with high
efficiency, even at twice the rate of those of the native and ENA modified counterpart.

7112 J. Org. Chem. 2010, 75, 7112–7128 Published on Web 10/07/2010 DOI: 10.1021/jo101207d
r 2010 American Chemical Society
Liu et al.
JOC Article
Introduction moiety, such as ENA,11 aza-ENA,15 PrNA,11 BNACOC,16 and
BNANC.17
Oligonucleotides with high affinity and specificity toward
Replacement of the 20 -O- in LNA with 20 -CH2- results in a
complementary DNA or RNA through Watson-Crick hybridiza-
more hydrophobically locked carba-LNA nucleoside, which
tion can be potentially used as diagnostic and therapeutic
has been synthesized recently by us through a radical cycliza-
agents via several different machineries, such as antisense,1
tion strategy.18-20 The advantage of carba-LNA is that
ribozymes, 2 small interfering RNA (siRNA),3 micro-
carba-LNA-modified AONs exhibit similar hybridizing abil-
RNA (miRNA),4 and triple helix-forming oligonucleotides
ity for the complementary RNA as that of LNA counter-
(TFOs).5 In the past two decades, the effort to develop
parts, but the former showed significantly improved
AON with striking target affinity and biological stability
nucleolytic resistance than the latter. Moreover, we found
has resulted in the synthesis of a large number of chemically
that the nucleolytic resistance and hybridizing ability of
modified nucleotides, among which the locked nucleic acid
carba-LNA can be finely modulated by introduction of
(LNA) has attracted extensive attention.6 The 20 ,40 -linkage
hydrophobic methyl, hydrophilic hydroxyl, or zwitterionic
in LNA is able to lock the conformation of the sugar moiety
amino functions at C60 and/or C70 of the carbocyclic moiety.
into a perfect N-type conformation.7 When LNA monomer
This modulation was found to be greatly dependent on not
was incorporated into oligonucleotide, it can spontane-
only the nature but also the steric orientation of the substit-
ously tune its neighboring natural nucleotides from S- to
uents at the chiral C60 and/or C70 position of the carbocyclic
N-conformation, resulting in an A-type or A-type-like duplex
moiety in the carba-LNA.
form consistent with the predominant form of the RNA helix.8
Carba-ENA that contains a 20 ,40 -propyl linkage has also
Hence, LNA-modified AONs exhibit remarkable sequence
been synthesized by us through a radical cyclization re-
selectivity for the complementary RNA over the DNA.6
action21 and also independently by Nielsen’s group through
Though the striking feature of LNA-modified oligos has
a ring-closing metathesis.22,23 Compared with carba-LNA,
given immense opportunity to a broad application in bio-
carba-ENA has a sterically larger and more hydrophobically
technology and therapeutics,9 it is clear that further devel- carbocyclic linkage. This structural feature has rendered
opment is required, due to the low nuclease resistance and carba-ENA incorporated oligos with more improved
hepatotoxicity of LNA.10,11 Thus large numbers of chemi- nucleolytic resistance than those of carba-LNA counterparts.
cally modified analogues with 20 ,40 -linkage have been de- However, none of these works reported any studies on the
signed and synthesized.12 Some of the modifications focus on nuclease stability of parent carba-ENA-modified AONs. In the
introduction of different functions at the C60 in LNA. present study, we report a new synthetic strategy for carba-
For example, Nielsen13 and Swayze14 have independently ENA-T and study the nucleolytic stability as well as other
reported C60 -CH2OH-LNA and C60 -Me-LNA (cEt), C60 - antisense properties of carba-ENA-modified AONs.
CH2OMe-LNA (cMOE), respectively. The cEt and cMOE In addition, C60 - and/or C70 -substitued carba-ENA ana-
modified oligonucleotides showed improved exonucleolytic logues have been synthesized by Nielsen’s group23 and us18
resistance without loss of hybridization behavior compared independently. Our work showed that the C60 substituent on
to the LNA-modified counterpart. Another type of modi- carba-ENA exerted a significant effect on nucleolytic stabi-
fication focuses on expanding ring size or introducing lity and the effect depends on the nature of the substitu-
heteroatoms into different positions on the locked ring ents. Nielsen’s work showed hydrophilic substituents at
C60 and/or C70 led to increased affinity for complementary
(1) (a) Bennett, C. F.; Swayze, E. E. Annu. Rev. Pharmacol. Toxicol. 2010, RNA and DNA. Compared to C60 and C70 substituents on
50, 259–293. (b) Uhlman, E; Peyman, A. Chem. Rev. 1990, 90, 543–584.
(2) Schubert, S.; Kurreck, J. Curr. Drug Targets 2004, 5, 667–681. carba-ENA, the C80 substituent should be more interesting
(3) de Fougerolles, A.; Vornlocher, H. P.; Maraganore, J.; Lieberman, J. because a C80 substituent is located at the bottom of the minor
Nat. Rev. Drug Discovery 2007, 6, 443–453.
(4) Bartel, D. P. Cell 2004, 116, 281–297.
groove, hence it should have a larger effect on thermodynamic
(5) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7, 717–726. stability and other properties of AON:RNA and AON:DNA
(6) (a) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.; duplexes. In this report, we also describe the synthesis of
Kumar, R.; Meldgaard, M. C.; Olsen, E.; Wengel, J. Tetrahedron 1998, 54,
3607–3630. (b) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J. Chem. 80 -NH2/OH/Me-substituted carba-ENA analogues in detail.
Commun. 1998, 455–456. (c) Kaur, H.; Babu, B. R.; Maiti, S. Chem. Rev. Biophysical and biological properties such as target affinity
2007, 107, 4672–4697. (d) Lebreton, J.; Escudier, J. M.; Arzel, L.; Len, C. toward complementary DNA and RNA, nucleolytic stability,
Chem. Rev. 2010, 110, 3371–3418.
(7) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; and the RNase H recruitment capability of AONs with these
Imanishi, T Tetrahedron Lett. 1997, 38, 8735–8738. 80 -substituted carba-ENAs modifications have been tested and
(8) Petersen, M.; Bondensgaard, K.; Wengel, J.; Jacobsen, J. P. J. Am.
Chem. Soc. 2002, 124, 5974–5982.
(9) Veedu, R. N.; Wengel, J. Chem. Biodiversity 2010, 7, 536–542. (16) Hari, Y.; Obika, S.; Ohnishi, R.; Eguchi, K.; Osaki, T.; Ohishi, H.;
(10) Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.; Imanishi, T. Bioorg. Med. Chem. 2006, 14, 1029–1038.
Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F. Nucleic Acids (17) Abdur Rahman, S. M.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita,
Res. 2007, 35, 687–700. K.; Imanishi, T. J. Am. Chem. Soc. 2008, 130, 4886–4896.
(11) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; (18) Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych, O.;
Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003, Chattopadhyaya, J. J. Org. Chem. 2009, 74, 118–134.
11, 2211–2226. (19) Srivastava, P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Wenska,
(12) Zhou, C.; Chattopadhyaya, J. Curr. Opin. Drug Discovery Dev. 2009, M.; Chattopadhyaya, J. J. Am. Chem. Soc. 2007, 129, 8362–8379.
12, 876–898. (20) Xu, J.; Liu, Y.; Dupouy, C.; Chattopadhyaya, J. J. Org. Chem. 2009,
(13) Enderlin, G.; Nielsen, P. J. Org. Chem. 2008, 73, 6891–6894. 74, 6534–6554.
(14) Seth, P. P.; Vasquez, G.; Allerson, C. A.; Berdeja, A.; Gaus, H.; (21) Zhou, C.; Plashkevych, O.; Chattopadhyaya, J. Org. Biomol. Chem.
Kinberger, G. A.; Prakash, T. P.; Migawa, M. T.; Bhat, B.; Swayze, E. E. 2008, 6, 4627–4633.
J. Org. Chem. 2010, 75, 1569–1581. (22) Albæk, N.; Petersen, M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731–
(15) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; 7740.
Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128, (23) Kumar, S.; Hansen, M. H.; Albæk, N.; Steffansen, S. I.; Petersen, M.;
15173–15187. Nielsen, P. J. Org. Chem. 2009, 74, 6756–6769.

J. Org. Chem. Vol. 75, No. 21, 2010 7113

JOC Article Liu et al.

SCHEME 1. Retrosynthetic Analysis to C80 -Modified Carba-ENA phenyl chlorothioformate in pyridine, the key precursor for
Thymidine radical cyclization, 20 -O-phenoxythiocarbonyl (PTC)-40 -
C-(benzyloxyimino)propyl-thymidine 13, was obtained in
88% yield. It has been reported that the configuration of the
oximino-ether group does not affect the chemical yield or
stereoselectivity of radical cyclization,27 thus the Z/E mixture
of 13 was directly subjected to radical cyclization, which was
performed smoothly utilizing tributyltin hydride (Bn3SnH)
with AIBN as radical initiator in degassed toluene at <110 °C,
in order to avoid the decomposition of the starting material.
To avoid the side reactions, starting material was highly
diluted, and Bn3SnH and AIBN were added dropwise slowly
(Experimental Section). As previously reported,28,29 the radi-
cal reaction proceeded in the 6-exo cyclization mode to afford
the sole product 14 in 60% yield. Meanwhile 1D NOE
compared with those of AON with carba-ENA modification. experiments proved that the configuration of C80 was
On the basis of the results provided, we show how the nature R stereochemistry. Subsequently, selective debenzylation of
and stereochemical orientation of C80 substituents on carba- 80 -N-OBn was achieved with 10% Pd/C and ammonium formate
ENA modulate the biophysical and biological properties of as hydrogenolysis reagent at room temperature to produce the
modified AONs, which, in turn, we believe will enable us to highly polar product 15 without any loss of 30 - and 50 -O-
design more promising nucleoside analogues as potential nucleic benzyl groups. The effort to protect 80 -amino with ethyl
acids-based therapeutic or diagnostic reagents. trifluoroacetate20 was unsuccessful because of very low yield;
merely about 20% even when 20 equiv of the reagent was
Results and Discussion added. Instead, the more reactive trifluoroacetic anhydride30
was found to be a superior choice: Treatment of 15 with
1. Synthesis of 80 -Modified-Carba-ENA Thymidine Ana-
trifluoroacetic anhydride in pyridine and CH2Cl2 gave the
logues. As shown in the retrosynthetic analysis in Scheme 1,
expected compound 16a in 60% yield. The structural integrity
the C80 -modified carba-ENA thymidine analogues can readily
of the product was confirmed through 13C NMR experiment,
be accomplished exploiting cyclic ketone 2 that was synthesized
in which two typical quartet peaks around 110 and 150 ppm
from 80 -amino-carba-ENA thymidine 3, which we obtained
for the trifluoroacetyl group (TFA) were observed. Then 16a
through free-radical cyclization of the oxime ether 4. The oxime
was subjected to debenzylation involving 20% Pd(OH)2/C
ether function can be introduced starting from nitrile 5, which in
and cyclohexene as hydrogen donor at reflux and subsequent
turn can be easily obtained from known sugar precursor 619 by a
50 -dimethoxytritylation to give the corresponding product 17a.
simple nucleophilic substituent with cyanide ion.24
Unfortunately, significant loss of TFA during debenzylation has
1.1. Synthesis of 80 -Amino-Carba-ENA Thymidine (15).
been observed, resulting in lower yield (30%) from 16a to 17a.
The synthetic route to 80 -amino-carba-ENA thymidine 15
To overcome this problem, the phenoxyacetyl group (PAC) was
and its phosphoramdite 18a/18b is shown in Scheme 2.
employed as a more stable protective group to afford 16b,31
Treatment of the known sugar precursor 619 with tosyl
which can be debenzylated completely under the same condition
chloride, triethylamine, and DMAP in dichlormethane fur-
without any acylamino cleavage, followed by 50 -dimethoxytri-
nished tosylate 7.24 After workup, the crude tosylate was
tylation to acquire 17b in 72% yield. Phosphitylation of 17a and
directly subjected to substitution with tetrabutylammonium
17b with 2-cyanoethyl N,N-diisopropylphosphoramidochlori-
cyanide (TBACN) in DMF to afford the desired 4-C-cya-
dite was achieved to give the desired amidites 18a and 18b as a
noethyl-D-ribofuranose 8 in an overall yield of 77% from 6.25
diastereomeric mixture in 93% and 70% yield, respectively.
Compound 8 was treated with a mixture of acetic acid, acetic
1.2. Synthesis of Carba-ENA-T 25, 80 -OH-Carba-ENA-T
anhydride, and triflic acid at 10 °C for 1 h to give diacetate 9 as
22a/b, and 80 -Me-Carba-ENA-T 29 Starting from 80 -Amino-
an anomeric mixture. Glycosylation of the crude 9 provided
Carba-ENA-T 15. Transformation of the 80 -amino to ketone
exclusively β-configured thymine nucleoside 10 through a
(15 f 19) function was a key step for the synthesis of carba-
modified Vorbr€ uggen-type reaction in 85% yield in two
ENA-T 25, 80 -OH-carba-ENA-T 22a/b, and 80 -Me-carba-
steps.15 Deacetylation of the 20 -O-acetyl group was achieved
ENA-T 29 starting from 80 -amino-carba-ENA-T 15 (Scheme 3).
by treating compound 10 with 30% methylamine solution in
Oxidation of benzyloxyamine by MCPBA to oxime followed
ethanol at room temperature to furnish the desired product 11
by treatment with Dess-Martin periodinane reagent has been
in 94% yield. Then the nitrile group of 11 was reduced with
shown in the previous study20,32 to be an efficient strategy for
diisobutylaluminum hydride (DIBALH) at -78 °C for 2 h to
the corresponding aldehyde, which was subsequently sub-
(27) Miyabe, H.; Torieda, M.; Inoue, K.; Tajiri, K.; Kiguchi, T.; Naito, T.
jected to oximation with O-benzylhydroxylamine to provide J. Org. Chem. 1998, 63, 4397–4407.
O-benzyl oxime 12 as a mixture of E and Z isomers in 61% (28) Marco-Contelles, J.; Martinez-Grau, A.; Martinez-Ripoll, M.;
yield.26 After esterification of 20 -OH in nucleoside 12 with Cano, H.; Foces-Foces, C. J. Org. Chem. 1992, 57, 403–406.
(29) Marco-Contelles, J.; Pozuelo, C.; Jimeno, M. L.; Martinez, L.;
Martinez-Grau, A. J. Org. Chem. 1992, 57, 2625–2631.
(24) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.; (30) Wenska, M.; Honcharenko, D.; Pathmasiri, W.; Chattopadhyaya, J.
Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003, Heterocycles 2007, 73, 303–324.
11, 2211–2226. (31) Honcharenko, D.; Varghese, O. P.; Plashkevych, O.; Barman, J.;
(25) Åkerfeldt, K. S.; Bartlett, P. A. J. Org. Chem. 1991, 56, 7133–7144. Chattopadhyaya, J. J. Org. Chem. 2006, 71, 299–314.
(26) Kumar, V.; Gauniyal, M. H.; Shaw, K. A. Tetrahedron: Asymmetry (32) Simpkins, N. S.; Stokes, S.; Whittle, A. J. J. Chem. Soc., Perkin
2007, 18, 2069–2078. Trans. I 1992, 2471–2477.

7114 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.
JOC Article
SCHEME 2. Synthesis of 80 -Amino-Carba-ENA Thymidinea

a
Reagents and conditions: (a) tosyl chloride, Et3N, DMAP, CH2Cl2, rt, overnight; (b) TBACN, DMF, rt-35 °C, 77% in two steps; (c) Ac2O, AcOH,
TfOH, 10 °C, 1 h; (d) thymine, BSA, TMSOTf, MeCN, reflux, overnight, 85% in two steps; (e) methylamine solution, methanol, 0 °C, 1 h, 95%; (f) 1 M
DIBAL in toluene, CH2Cl2, -78 °C, 3 h; BnONH2 3 HCl, pyridine, CH2Cl2, reflux, 1 h, 61% in two steps; (g) phenyl chlorothionoformate, pyridine, rt,
2 h, 89%; (h) Bu3SnH, AIBN, toluene, 110 °C, 6 h, 60%; (i) 10% Pd/C, ammonium formate, methanol, rt, 8 h; (j) trifluoroacetic anhydride, pyridine,
CH2Cl2, rt, 2 h, 60% (from 14); (k) phenoxyacetyl chloride, pyridine, rt, overnight, 62% (from 14); (l) 20% Pd(OH)2/C, cyclohexene, ethanol, reflux,
overnight; DMTr-Cl, pyridine, rt, overnight, 30% for 17a, 72% for 17b; (m) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, DIPEA, CH2Cl2,
rt, 3 h, 93% for 18a, 70% for 18b.

this transformation, but in this case, oxidation of benzyloxy- followed by selective 50 -dimethoxytritylation to give 22a (63%)
amine 14 by MCPBA only gave the corresponding oxime in and 22b (83%), respectively, in satisfactory yields. Special care
20% yield. Oxidative deamination33 is another strategy for was taken for the debenzylation of 21a because thermodynami-
transformation of the second amino to ketone directly. Thus, cally favored migration of the 80 -O-toluoyl group to the
different oxidative deamination reagents such as aqueous 30 -hydroxyl group in 21a is possible owing to their cis orientation
hypochlorite,34 trichloroisocyanuric acid,35 and copper- in a spatial proximity. Thus amount of hydrogenolysis reagent
ascorbic acid dyad36 have been tried but neither worked in was increased (20% Pd(OH)2/C, 1500 mg/mmol substrate and
our case. Instead, 3,5-di-tert-butyl-1,2-benzoquinone was ammonium formate, 60 equiv) and reaction time was shortened
found to be a satisfactory oxidative deamination reagent, for debenzylation of 21a,18 under which the benzyl groups were
and it efficaciously oxidized amine 15 to ketone 19 in an completely removed without cleavage and migration of the
acceptable yield.37 Reduction of 19 with NaBH4 furnished toluoyl group. Due to steric hindrance of the 80 -O-toluoyl group,
two diastereomeric products 20a (69%) and 20b (19%). The 30 -O-phosphitylation of 22a with 2-cyanoethyl N,N-diisopropyl-
major product 20a has 80 S stereochemistry, suggesting that phosphoramidochloridite was allowed to proceed overnight to
the hydride attack took place from the back of the carbo- afford the resulting phosphoramidite 30a in 71% yield, whereas
cycle. Clearly, the hydride attack from the back is much more the phosphitylation of 22b under the same condition furnished
favored than the attack from the front of carbocycle, most phosphoramidite 30b in 80% yield in only 3 h.
likely owing to steric hindrance of the bulky 30 -O-benzyl Starting from intermediate 20a, parent carba-ENA-T 25
group in the front face. After protection of free 80 -OH in 20a has been synthesized through radical deoxygenation of the
and 20b with the p-toluoyl group, the obtained compounds 80 -OH in 20a (Scheme 3). Considering the steric proximity
21a (90%) and 21b (89%) were subjected to debenzylation between 30 -O-benzyl and 80 -hydroxyl groups in 20a, the more
reactive and smaller (methylthio)thiocarbonate was chosen
(33) (a) Neumann, R.; Levin, M. J. Org. Chem. 1991, 56, 5707–5710. as the radical-generating group.38 Thus, 20a was converted
(b) Barman, D. C.; Saikia, P.; Prajapati, D.; Sandhu, J. S. Synth. Commun. through the three-component reaction to radical precursor
2002, 32, 3407–3412. (c) Richey, H. G., Jr.; Erickson, W. F. J. Org. Chem.
1983, 48, 4349–4357. 23 (53%), which was subjected to the standard radical
(34) Lee, G. A.; Freedman, H. H. Tetrahedron Lett. 1976, 20, 1641–1644. deoxygenation procedure20 to give the expected parent carba-
(35) Luca, L. D.; Giacomelli, G. Synlett 2004, 12, 2180–2184.
(36) Srogl, J.; Voltrova, S. Org. Lett. 2009, 11, 843–845. ENA thymidine 24 in 74% yield.
(37) (a) Corey, E. J.; Achiwa, K. J. Am. Chem. Soc. 1969, 91, 1429–1432.
(b) Klein, R. F. X.; Bargas, L. M.; Horak, V. J. Org. Chem. 1988, 53, 5994–
5998. (38) Mereyala, H. B.; Pola, P. Synth. Commun. 2002, 32, 2453–2458.

J. Org. Chem. Vol. 75, No. 21, 2010 7115

JOC Article Liu et al.

SCHEME 3. Synthesis of Carba-ENA Thymidine (24) and Its Analougesa

a
Reagents and conditions: (a) (i) 10% Pd/C, ammonium formate, methanol, rt, 8 h; (ii) 3,5-di-tert-butyl-1,2-benzoquinone, methanol, THF, 40 °C,10 h;
oxalic acid, H2O, 40 °C, overnight, 50% in two steps; (b) NaBH4, ethanol, rt, 4 h, 69% for 20a and 19% for 20b; (c) p-toluoyl chloride, pyridine, rt,
overnight, 90% for 21a, 89% for 21b; (d) 20% Pd(OH)2/C, ammonium formate, methanol, reflux; DMTr-Cl, pyridine, rt, overnight, 63% for 22a, 83%
for 22b, 75% for 25, 80% for 29; (e) NaH, CS2, MeI, THF, 40 °C-rt, 53% (from 20a); (f) Bu3SnH, AIBN, toluene, reflux, 1.5 h, 74%; (g) 1.6 M MeLi in
Et2O, THF, -78 °C, 10 h, 58%; (h) methyl oxalyl chloride, pyridine, 40 °C, overnight, 87%; (i) Bu3SnH, AIBN, toluene, reflux, 4 h, 87%; (j) 2-cyanoethyl
N,N-diisopropylphosphoramidochloridite, DIPEA, CH2Cl2, rt, 71% for 30a, 80% for 30b, 84% for 31, 76% for 32.

Finally, carba-ENA-T with a hydrophobic C80 substitu- intermediate 26 reacted with methyl(chlorocarbonyl)-
ent, 80 -Me-carba-ENA-T 29, has been synthesized starting formate in pyridine at 40 °C to give 27 in 87% yield,41 which
from key intermediate ketone 19 by a known strategy.18 was subjected to a standard radical deoxygenation to give two
Instead of methyl magnesium iodide,39 treatment of ketone inseparable diastereomers 28 (80 S:80 R = 5:4) in 87% yield.
19 with methyl lithium in THF for 8 h furnished the sole Relative lack of stereoselectivity during the free-radical reaction is
product 26 (58%),40 in which the 80 S-configuration was likely to be due to higher reaction temperature. To provide appro-
confirmed by 1D NOE experiment. Hence, the methyl anion priately protected building blocks for standard automated solid-
attacked the ketone exclusively from the back of the carbo- phase oligonucleotide synthesis by using the phosphoramidite
cycle, which is consistent with the stereoselective reduction approach, nucleosides 24 and 28 were debenzylated followed by
on ketone 19 by NaBH4. To remove the 80 -hydroxyl group, 50 -O-dimethoxytritylation to give 25 and 29 in 75% and 80%
yield, respectively. Subsequently, the resulting compounds 25 and
29 were converted into the respective 30 -O-phosphoramidites 31
(39) Kakefuda, A.; Yoshimura, Y.; Sasaki, T.; Matsuda, A. Tetrahedron
1993, 49, 8513–8528.
(84%) and 32 (76%) under standard condition.
(40) Matsuda, A.; Itoh, H.; Takenuki, K.; Sasaki, T.; Ueda, T. Chem. 1.3. NMR Characterization of Key Intermediates Involved
Pharm. Bull. 1988, 36, 945–953. in the Synthesis of the 80 -Functionalized Carba-ENA Thymi-
(41) (a) Dolan, S. C.; Macmillian, J. J. Chem. Soc., Chem. Commun. 1985,
1588–1589. (b) Pettit, G. R.; Melody, N.; Herald, D. L.; Knight, J. C.; dines. Structures of all intermediates and final products were
Chapuis, J. J. Nat. Prod. 2007, 70, 417–422. confirmed by 1H, 13C, COSY, 1H-13C HMQC and HMBC
7116 J. Org. Chem. Vol. 75, No. 21, 2010
Liu et al.
JOC Article

FIGURE 1. The NOE contacts of the key intermediates (14, 20a, 21b, 26, and 28). R = Bn, R1 = Tol.

NMR, and mass spectroscopy (see the Supporting Information). be assigned to the R-configuration since compound 21b has
The 1H NMR spectra of all carba-ENA derivatives showed a been found to have the C80 R-configuration: irradiation of
singlet for H10 as found for all 20 ,40 -fused bicyclic nucleosides, H80 in 21b resulted in the enhancement for H20 (3.1%) and
which strongly suggested the North-type sugar pucker.42 H70 (2.7%), but not for H10 . For compound 26, the strong
For compound 14, the correlation between H20 with H80 in NOE enhancement for the 80 -methyl group (5.9%) and
COSY NMR spectra gave evidence to the bicyclic system H70 (4.2%) upon irradiation of H10 clearly indicated the
formed during the free-radical cyclization, which is further S-configuration for C80 . By comparing the observation that
consolidated by the observation of the long-range correlations irradiation on H10 in the major product of 28 caused
between H20 with C70 and between H10 with C80 in HMBC remarkable enhancement for H80 (4.1%) and none for
NMR spectra. In the 1D NOE experiment, irradiation of H10 80 -Me, in contradistinction to the minor product of 28, which
led to NOE enhancement for 80 -NH (0.7%), H20 (1.8%), and led to notable NOE enhancement for 80 -Me (3.6%) but none
H700 (3.9%), but none for H80 (Figure 1), which identified C80 for H80 , it was concluded that the C80 is S in the major
in the 80 R-configuration. product, whereas R in the minor product.
The orientation of C80 substituents in intermediates 20a, On the other hand, the coupling constants also have been
20b, 26, and 28 has been assigned by 1D NOE experiments. used to identify the configuration of chiral center in these
In compound 20a, the strong enhancement for H80 (3.1%) derivatives. The six-membered carbocyclic ring of carba-
and H70 (3.5%) was observed upon irradiation of H10 , ENA adopts a typical chair conformation.18,22 Through
confirming the S-configuration for C80 . Severe overlap of
decoupling experiments, the observation of a small coupling
signals of H80 and H30 in compound 20b made it hard to
constant (∼6 Hz) between H80 and axial H70 and a large one
determine the configuration of C80 by 1D NOE experiments
(∼11 Hz) between H80 and equatorial H700 in 16b and 21b
at this stage. The configuration of C80 in 20b, however, could
implied H80 and equatorial H70 have torsion angles in gauche
(42) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, V. K.; Sing,
but that of H80 and axial H700 in trans, according with the
S. K.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–13253. 80 R-configuration. On the contrary, the 3JH70 0 H80 in 17a is 0 Hz,
J. Org. Chem. Vol. 75, No. 21, 2010 7117
JOC Article Liu et al.

SCHEME 4. Mechanism of Radical Cyclization Reaction tides.44,45 Thus, building block 18a with a trifluoroacetyl-
protecting group was used to synthesize AONs 22-25 following
the standard procedure. Meanwhile, ENA-T phosphoramidite
was synthesized according to a reported procedure11 (see the
Supporting Information), and was incorporated into AONs
30-33 for comparison. The fully deprotected AONs were
purified by 20% denatured PAGE and their structural integrity
was confirmed by MALDI-TOF mass measurement (Table SII
1, Supporting Information).
3. Hybridization Studies of Carba-ENA Analogues-Mod-
ified AONs with Complementary RNA and DNA. The hybri-
dization properties of the modified oligonucleotides toward
complementary DNA and RNA were studied by thermal
denaturation experiments. The melting temperature (Tm)
values of duplex formed by AONs 2-33 with the complemen-
tary DNA or RNA were measured, and their ΔTm values
provided by comparing with the native AON 1 were listed in
Table 1. The ΔTm values of AON 26-29 that have been
reported previously18 are also listed in Table 1 for comparison.
3.1. Comparison of Carba-ENA (Type I) Modification with
which suggested the H80 and axial H700 is nearly 90°, Hence C80 Native Counterpart and ENA (Type VIII) Modification.
has an S stereochemistry (Figures SII 8-13, Supporting Albæk et al. reported that the ΔTm value of duplexes formed
Information). The results obtained from decoupling experiments by parent carba-ENA-U with complementary RNA was
conformed to those from 1D NOE experiments. þ4.5 °C,22 whereas our present result showed that one parent
1.4. Mechanism of the Ring-Closure Reaction. During carba-ENA-T modification in AON:RNA duplex resulted in
radical mediated 6-exo cyclization of compound 13, the in an increase of 1.4 °C in Tm. The notable disparity between the
situ generated C20 carbon radical could attack the oxime exact ΔTm value obtained by us and those by Albæk et al. is
intramolecularly through both faces of CdN, forming two likely due to the sequence difference.
possible transition states TS1 and TS2 (Scheme 4). 1,3- One ENA (type VIII) modification in the AON strand
Diaxial disposition between the forming bulky benzyloxya- resulted in a Tm increase of 3.6 °C for the AON:RNA duplex,
mino substituent and 30 -O-benzyl group in TS 1 is energeti- which is a much higher stabilization effect than the parent
cally disfavored. In contrast, the benzyloxyamino group in carba-ENA modification (þ1.4 °C/modification). We have
TS 2 points equatorially, leading to minimal steric repulsion previously reported20 that in the same AON sequence, LNA
between C80 and C30 substituents. As a result, compound 14 modification (þ4.5 °C/modification) also led to much higher
that has R stereochemistry at chiral C80 has been observed as Tm increase for the AON:RNA duplex than the parent
the exclusive product. carba-LNA modification (þ3.6 °C/modification). Hence, it
2. Synthesis and Purification of Oligonucleotides Contain- seems that the 20 -oxygen on the bicyclic system plays an
ing 80 -Modified Carba-ENA Thymidine Analogues. Through important role in the thermal stability of the AON:RNA
solid-phase DNA synthesis protocol43 on an automated duplex. One of the plausible explanations for the role of
DNA/RNA synthesizer, the phosphoramidites 18b, 30a/b, 20 -oxygen could be that the 20 -oxygen is engaged in the
31, and 32 were incorporated as monosubstitution into a hydrogen bonding to water molecules, whereas it is well-
15mer DNA sequence as used in our former studies.18,20 The known46 the extensive hydration network in the minor groove
coupling time of building blocks 18b, 30b, 31, and 32 was about can reduce the electrostatic repulsion of the internucleotidic
10 min, whereas for 30a the coupling time was prolonged to phosphates, thus stabilizing the AON:RNA duplex. Although
15 min, in view of steric obstruction by the 80 -O-toluoyl group this 20 -oxygen in LNA or ENA incorporated oligos has a Tm
during the coupling. The satisfactory coupling yields (40-75%) increase effect with complementary RNA, it, however, reduces
were obtained for all compounds. The sequences, modification the nucleolytic stability considerably compared to their carbo-
site, and structure of modification are shown in Table 1. For cyclic counterparts (see section 4).
AONs 2-9 the treatment with 33% aqueous ammonia at room 3.2. Comparison of Type IV, Type VI, and Type VII with
temperature overnight was carried out to cleave oligos from the Type I Modifications. One [80 R-OH]-carba-ENA (type IV),
solid support as well as for the removal of the protecting groups. [80 R-Me]-carba-ENA (type VII), and [80 R-NH2]-carba-ENA
On the other hand, considering the stability of PAC and Tol (type VI) modification in AON leads to Tm increase for the
groups, the solid support for AONs 10-17 and 18-21 was AON:RNA duplex around 0.6, 0.5, and 0.8 °C, respectively
incubated in aqueous ammonia at 55 °C for 1 to 3 days. (Table 1), which are slightly lower than the effect imposed by
Unfortunately, the PAC groups in AONs 18-21 are too stable
the parent carba-ENA (type I) modification (þ1.4 °C/
to be removed. A stronger deprotection reagent such as 1 N
modification). This observation indicates the C80 substitu-
NaOH solution, however, led to degradation of oligonucleo-
ents, regardless of hydrophilic hydroxyl and amino group or
hydrophobic methyl group, destabilize the AON:RNA duplexes
(43) Beaucage, S. L.; Caruthers, M. H. Current Protocols in Nucleic Acid slightly when they face away from the vicinal 30 -phosphate.
Chemistry; John Wiley & Sons: New York, 2003.
(44) Ditrich, K.; Ladner, W.; Melder, J. US Patent US6713652 B1, 2000.
(45) Heinz, L. J.; Lunn, W. H. W.; Schoepp, D. D. Patent EP658539, (46) Tereshko, V; Gryaznov, S.; Egli, M. J. Am. Chem. Soc. 1998, 120,
1995. 269–283.

7118 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.
JOC Article
TABLE 1. Thermal Denaturation of Duplexes of Native and Carba-ENA Analogues-Modified AONs with Complementary DNA or RNAa

a
A = native 9-adeninyl, C = 1-cytosinyl, T = 1-thyminyl, “T ” (shown in red) indicates the incorporated modified thymidine monomer with the
specified structure shown in the table. Molecular weights of all antisense sequences are conformed by MALDI-TOF (see Table SII 1, Supporting
Information). Tm values measured as the maximum of the first derivative of the melting curve (A260 nm vs temperature) in medium salt buffer (60 mM
tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2) with temperature range 20 to 70 °C, using 1 μM concentrations of two complementary strands. The
value of Tm given is the average of two or three independent measurements (the error of the three consecutive measurements is within (0.3 °C). bΔTm
values were obtained by comparing the Tm values of modified AONs 2-33 with that of the native AON 1. The ΔTm(aver) value obtained here is the
average value from four AONs incorporated with the same compound at four different modification sites. cΔΔTm = (Tm of AON:RNA) - (Tm of AON:
DNA). dModifications of type VII have been taken from reported work,18 and are used for Tm comparison with our present series of modified AONs.

The less destabilizing effect of the 80 R-amino group than that affinity than when it points away from the neighboring
of the 80 R-methyl or the hydroxyl group is presumably 30 -phosphate.
because in experimental buffer the amino group is partially 3.4. Comparison of Type II with Type VII Modification. It
protonated, and can reduce charge repulsion between phos- seems the orientation of hydrophobic 80 -Me exerts little if
phates on opposition strands through partial charge neutra- any effect on thermal stability of the AON:RNA duplex
lization, which is a favorable contribution to the free energy since we found type II ([80 -56%S/44%R-Me]-carba-ENA)
of duplex formation.47,48 and type VII ([80 R-Me]-carba-ENA) modification lead to very
3.3. Comparison of Type III with Type IV Modification. similar Tm increase, 0.5 and 0.3 °C/modification, respectively.
[80 S-OH]-carba-ENA (type III) modification did not result 3.5. Comparison of Type V with Type VI Modification. To
in any Tm change for the AON:RNA duplex, which is investigate the influence of larger hydrophobic substituent in
contrary to the observation that one [80 R-OH]-carba-ENA the minor groove of the AON:RNA duplex on the thermo-
(type IV) modification led to a 0.6 °C increase in Tm. Hence, dynamic stability, the Tm of duplexes formed by [80 R-NHPAC]-
when the 80 -OH group points toward the vicinal 30 -phos- carba-ENA (type V)-modified AONs 18-21 with comple-
phate, it renders a more negative effect for target RNA mentary RNA has also been measured. It was found that
[80 R-NHPAC]-carba-ENA (type V) modification renders a
(47) Cuenoud, B.; Casset, F.; H€ usken, D.; Natt, F.; Wolf, R. M.;
Altmann, K. H.; Martin, P.; Moser, H. E. Angew. Chem., Int. Ed. 1998, 37, (48) Prakash, T. P.; P€
uschi, A.; Lesnik, E.; Mohan, V.; Tereshko, V.; Egli,
1288–1291. M.; Manoharan, M. Org. Lett. 2004, 6, 1971–1974.

J. Org. Chem. Vol. 75, No. 21, 2010 7119

JOC Article Liu et al.

relatively higher Tm increase than [80 R-NH2]-carba-ENA were taken out at appropriate time intervals and analyzed by
(type VI) modification (Table 1). This is an unusual observa- 20% denaturing PAGE. The gel pictures obtained upon
tion, since the phenyl of PAC in type V modification is autoradiography are shown in Figure SII 14 (Supporting
expected to perturb the hydration network in the minor Information). The [80 R-CH3]-carba-ENA-modified AON 26,
groove, which we expected to result in a destabilization effect. [60 R-CH3,80 R-CH3]-carba-ENA-modified AON 34, and [60 S-
One likely explanation for this unusual observation is that the CH3,80 R-CH3]-carba-ENA-modified AON 35 that have been
phenyl group may intercalate into the hybrid duplex. reported previously18 were also treated with SVPDE in parallel
3.6. The Carba-ENA-Modified AONs Are Highly RNA- for comparison purpose.
Selective. a. Comparison of Carba-ENAs with Native. Unlike The native DNA (AON 1) did not display any 30 -exonuclease
in AON:RNA duplex, carba-ENAs modification in AON: resistance and was completely degraded within ∼4 min, whereas
DNA led to significant Tm decrease compared to the native under the identical condition, all modified AONs exhibit im-
counterpart. Hence, carba-ENAs-modified AONs are more proved exonuclease resistance to variable extents. Due to modi-
RNA-selective than the native. The RNA selectivity of carba- fied nucleotides locating at position T13, the stability of both
ENAs could be due to the fact that the DNA:DNA duplex has a phosphate P14 and P13 (see Figure SII 14 in the Supporting
narrower minor groove (ca. 5-6 Å) than the DNA:RNA hybrid Information for phopshate (P) numbering) has been remark-
(ca. 9-10 Å).49 Hence, the carbocycle of carba-ENA, which is ably improved toward 30 -exonuclease, so two bands corre-
located at the minor groove, may cause more serious perturba- sponding to 14mer and 13mer on PAGE pictures can be
tion in AON:DNA than in the AON:RNA duplex. As a result, observed. However, once the phosphate P13 is cleaved, AON
the Tm value of the carba-ENA-modified AON:DNA homo- could be degraded to the monomer blocks very rapidly.
duplex was pronouncedly decreased compared to that of the 4.1. Relative Nuclease Resistance of Carba-ENA, ENA-
carba-ENA-modified AON:RNA heteroduplex. Modified AONs. Total percentages of integrated 14mer and
b. Comparison of Carba-ENAs with ENA. The magnitude 13mer AONs were plotted against time points to give
of RNA selectivity [denoted by ΔΔTm, ΔΔTm = (Tm of SVPDE digestion curves (Figure 2) for each AON, and
AON:RNA duplex) - (Tm of AON:DNA duplex)] of carba- pseudo-first-order reaction rates were observed by fitting
ENA modifications (types I, II, III, IV, and VII) is around 5- the curves to single-exponential decay functions. The stabi-
6 °C/modification (Table 1), which is significantly higher than lity of these AONs toward SVDPE incubation increases in
that of ENA modification (ΔΔTm = ∼3.5 °C/modification). By the following sequences: AON 1 , ENA-modified AON 30 (k =
analyzing the Tm data, we found the higher RNA selectivity of 1.7704 ( 0.0812 h-1) < [80 R-NHPAC]-carba-ENA-modified
carba-ENA compared to ENA is because the carba-ENA AON 18 (k = 0.6761 ( 0.0386 h-1) ≈ [80 S-OH]-carba-ENA-
modifications result in much more significant Tm decrease for modified AON 10 (k = 0.5131 ( 0.0530 h-1) < [80 R-NH2]-
AON:DNA than the ENA modification (Table 1). Hence, it carba-ENA-modified AON 22 (k = 0.3086 ( 0.0085 h-1) <
seems 20 -oxygen is more important for the thermal stability of the [80 R-Me]-carba-ENA-modified AON 26 (k = 0.1249 ( 0.0042
AON:DNA duplex than for the AON:RNA duplex. h-1) ≈ [80 R-OH]-carba-ENA-modified AON 14 (k = 0.0985 (
c. Comparison of Parent Carba-ENA with Parent Carba- 0.0027 h-1) ≈ [80 R/S-Me]-carba-ENA-modified AON 6 (k =
LNA and LNA. It has been reported previously by us20 that 0.0881 ( 0.0034 h-1) ≈ carba-ENA-modified AON 2 (k =
parent carba-LNA-modified AON (around 5 °C/modification) 0.0827 ( 0.0017 h-1) < [60 S-Me-80 R-Me]-carba-ENA-modified
are also more RNA selective than LNA-modified counter- AON 35 (k = 0.0494 ( 0.0019 h-1) ≈ [60 R-Me-80 R-Me]-carba-
part (3-4 °C/modification). Hence, it seems the relative RNA ENA-modified AON 34 (k = 0.0401 ( 0.0030 h-1).
selectivity of parent carba-ENA, parent carba-LNA, ENA, The above comparison leads us to the following conclusions:
and LNA follows this rank: parent carba-ENA ≈ parent a. Replacement of 20 -Oxygen of ENA and LNA with the
carba-LNA > ENA ≈ LNA. -CH2- Group Significantly Increases the Nuclease Resis-
4. Engineering the 30 -Exonuclease Stability in the Carba- tance. Parent carba-ENA-T (type I)-modified AON 2 was
ENA Analogues-Modified AONs. It is known that 30 -exonu- about 20 times more stable than ENA-T-modified AON 30.
clease SVPDE-mediated phosphate scission involves recog- In our previous study, parent carba-LNA-T-modified AON
nition of the 30 -end nucleotide, followed by the attack by has also been found to be about 50 times nucleolytically more
threonine residue of the enzyme on the phosphate in line with stable than the LNA-T-modified counterpart. These obser-
the 30 -O of the penultimate nucleotide, which subsequently vations highlighted the unique effect of replacement of
departs.50,51 The incorporation of modified nucleotides in 20 -oxygen with the -CH2- group in improving the nuclease
AONs may regulate the enzymatic resistance of the vicinal resistance of vicinal phosphates.
phosphate. Here AONs 2, 6, 10, 14, 18, 22, and 30 with b. 80 R-OH and 80 -Me Subsituents on Carba-ENA Only Have
different modifications at position T13 (position 3 from a Marginal Effect on the Nuclease Resistance. This conclusion
30 -end, see Figure SII 14 in the Supporting Information) were can be confirmed by the fact that AON 6, 14, and 26 showed
adopted to test how these modifications influence the 30 - very similar stability as parent carba-LNA-T-modified AON 2
exonuclease resistance of the modified AONs. These AONs (Figure 2).
(32P-labeled at 50 -end) were incubated with phosphodiesterase c. 80 R-NH2 Substituent on Carba-ENA Significantly
I from Crotalus adamanteus venom (SVPDE) [SVPDE Decreases the Stability. [80 R-NH2]-carba-ENA-modified
6.7 ng/μL, AON 3 μM, 100 mM Tris-HCl (pH 8.0), 15 mM AON 22 is around four times less stable than parent carba-
MgCl2, total reaction volume was 30 μL] at 21 °C. Aliquots LNA-T-modified AON 2. Moreover, [80 R-NHPAC]-carba-
ENA-modified AON 18 was found to be even less stable then
(49) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Biochemistry 1998, 37, AON 22.
73–80.
(50) Culp, J. S.; Butler, L. G. Arch. Biochem. Biophys. 1986, 246, 245–249. d. 80 S Substituent Can Render Totally Different Effect on
(51) Cleland, W. W.; Hengge, A. C. Chem. Rev. 2006, 106, 3252–3278. the Nuclease Resistance Depending upon the Nature of the
7120 J. Org. Chem. Vol. 75, No. 21, 2010
Liu et al.
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FIGURE 2. Amount of remaining initial oligonucleotide (taken 14mer and 13mer together in the calculation of % remaining) during
30 -exonuclease (SVPDE)-promoted digestion. Digestion condition: AON 3 μM (50 -end 32P labeled with specific activity 80 000 cpm), 100 mM
Tris-HCl (pH 8.0), 15 mM MgCl2, SVPDE 6.7 ng/μL, reaction temperature 21 °C, total reaction volume was 30 μL. Molecular structure of T13
modified AON with carba-ENA analogues was also shown in the figure.

Substituent. 80 S-OH substituent obviously destabilizes vicinal chemical effect is probably dictated by the spatial proximity of
phosphate toward 30 -exonuclease given the faster degradation the lipophilic 80 S methyl group to the 30 -phosphate compared
of [80 S-OH]-carba-ENA-modified AON 10, but 80 S-Me sub- to that of the 80 R methyl group, thus sterically preventing the
stituent leads to a stabilization effect because [80 R/S-Me]- binding and processing of the phosphate by SVPDE.
carba-ENA-modified AON 6 is more stable than [80 R-Me]- Compared to 80 -Me, 80 -OH on carba-ENA exhibits a
carba-ENA-modified AON 26. reverse stereochemical effect on the nuclease resistance:
e. Substituent at C60 Is More Efficient than Substituent at 80 S-OH-carba-ENA-modified AON 10 showed a more
C80 To Modulate the Nuclease Resistance of Vicinal Phos- dramatic decrease of exonuclease resistance than 80 R-OH-
phate. From Figure 2, we can see that the modified AONs 34 carba-ENA-modified AON 14. The destabilization effect
and 35 which contain the C60 -Me-carba-ENA modifications caused by 80 S-OH on carba-ENA could be attributed to
showed the best stability among all the studied AONs. Given the closure of 80 S-OH to vicinal 30 -phosphate. Therefore, the
the fact that C60 R-OH-carba-ENA-modified AON showed 80 S-OH could assist enzymatic cleavage of 30 -phosphate
the least nuclease stability among other substituted carba- (P14) by H-bonding interaction.52 An interesting observa-
ENA counterparts,18 it seems that nuclease resistance of carba- tion for SVDPE-mediated digestion of AON 10 is that there
ENA-modified AON is more sensitive to substitution at C60 than is one clear band corresponding to the 13mer formation
at C80 . This is presumably because of the fact that the C60 on the gel (Figure SII 14, Supporting Information), im-
substituent is spatially closer to the phosphate than the C80 plying that the 80 S-OH group can improve the stability of
substituent, and thus bestows greater effect during the phosphate 50 -phosphate (P13), putatively by impairing the binding of
recognition process and hydrolysis by the nuclease. SVPDE to 50 -phosphate.52 It is obvious that the destabiliza-
4.2. The Stereochemical Orientation of the 80 Substituent tion effect on P14 surpasses the stabilization effect on P13.
Influences the Exonuclease Resistance of the Vicinal Scissile Hence, 80 S-OH on carba-ENA makes modified AON less
Phosphate. On the basis of the above comparison, we found the resistant toward 30 -exonuclease.
nuclease recognition and subsequent hydrolysis effect imposed 4.3. The Electrostatic Effect of 80 Substituent Plays an
by 80 -OH and 80 -Me is significantly dependent on their stere- Important Role in Stability Modulation of the Vicinal Phos-
ochemical orientation. AON 6 in which 44% of 80 -Me are in the phate. 80 R-NH2 and 80 R-NHPAC on carba-ENA result in
S configuration was found to be more stable than AON 26 in a significant decrease in the stability of modified AONs.
which all the 80 -Me are in the R configuration, suggesting when This destabilization effect is likely attributed to the electro-
80 -Me points at the 30 -phosphate (80 S), it leads to a more posi- statics effect. The protonated 80 R-amino group, as a zwitterion,
tive effect on the nuclease resistance of vicinal phosphate than
when it points away from the 30 -phosphate (80 R). This stero- (52) Zhou, C.; Chattopadhyaya, J. J. Org. Chem. 2010, 75, 2341–2349.

J. Org. Chem. Vol. 75, No. 21, 2010 7121

JOC Article Liu et al.

FIGURE 3. The Escherichia coli RNase H1 promoted cleavage pattern of AONs 1-33:RNA duplexes. Vertical arrows show the RNase H
cleavage sites, with the relative length of the arrow indicating the extent of the cleavage. The square boxes show the stretch of the modification,
which is resistant to RNase H1 cleavage thereby giving footprints.

could assist binding and interaction between SVPDE and the T nucleotide, toward the 30 -end of the RNA strand. If A8, the
nucleotide, leading to an unfavorable contribution for enzymatic preferred cleavage site for native AON 1:RNA hybrid, is
resistance of the vicinal phosphate. On the other hand, it is located within the suppressed region, the major cleavage site
known that departure of the 30 -oxyanion is the rate-limiting step shifts to the edges of this region (Figure 3). This cleavage
for SVPDE-mediated phosphate cleavage.52 Under the experi- pattern is very similar as that of AON:RNA hybrids contain-
mental condition, the positively charged 80 R-NH2 could exert an ing carba-LNA19 or aza-ENA15 modifications, so one can
induction effect for the stability of the developing 30 -oxyanion, effectively engineer a cleavage at a specific site in the RNA
thus assisting it in its departure. As a result, the potential of the strand by properly choosing the position of modification in
scission of 30 O-P14 bond (Figures 2 and SII 14 in the Support- the complementary antisense strand.53 Interestingly, this site-
ing Information) is increased. Once the amine was converted into specific RNA cleavage property is akin to the hallmark of
the amide in 80 R-NHPAC, which is a better electron-withdraw- RNA cleavage in the RNA catalysis.
ing group than amino, the cleavage of 30 O-P14 should be The RNase H-promoted cleavage rates were determined
further promoted. This expectation is consistent with the result by autoradiography of gels and subsequently plotting the
that [80 R-NHPAC]-carba-ENA-modified AON 18 is even less intact RNA fraction as a function of time (Figure SII 15,
stable then [80 R-NH2]-carba-ENA-modified AON 22. Supporting Information). Fitting the degradation curve to
In summary, the SVPDE resistance can be modulated not single-exponential decay functions gave the digestive rates
only by the stereochemical orientation of the substituents on that are shown as bar plots in Figure 4. It was found that the
carbocyclic moiety but also by their intrinsic electrostatic RNA component in all modified AON:RNA hybrids can be
effect. Careful introduction of an electron-donating substi- degraded by RNase H with similar or even with better
tuent at the appropriate position of the carbocycle of the efficiency as the digestion rate of the native counterpart.
carba-ENA or -LNA with correct stereochemistry seems to Comparison of the cleavage rates of the modified AON:
be an efficient way to design oligonucleotides having high RNA hybrid can be summarized in the following conclu-
30 -exonuclease resistance. sions:
5. RNase H Digestion Study of the Duplexes Formed by (i) ENA (type VIII)-modified hybrids show similar cleav-
Carba-ENA Analogues-Modified AONs with Complementary age efficiency as that of the native counterpart. However, the
RNA. In antisense strategy, RNase H recruitment is a very cleavage rate of parent carba-ENA (type I)-modified hybrids
important property when the modified oligonucleotides are is two times better than that of the ENA-modified counter-
bound to the target RNA in heteroduplex form. So the part, which implies replacement of 20 -O of ENA with 20 -CH2
RNase H-mediated cleavage of the duplexes formed by all leads to a significant improvement on the RNase H recruit-
modified AON 2-33 with complement RNA was studied, ment capability.
applying native DNA (AON 1) as a reference. As the (ii) Just like the parent carba-ENA (type I), type VI
autoradiographs indicated (Figure SII 15, Supporting modification ([80 R-NH2]-carba-ENA) also showed a double
Information), all modified AON:RNA hybrids were excel- RNA hydrolysis rate relative to the native counterpart. It is
lent substrates for RNase H1; however, the cleavage pattern likely that electropositive C80 -NH2 as a zwitterion at the
changes, as the site of incorporation of 80 -modified carba- physiological pH presumably plays a role in promoting
ENA analogues changes, regardless of the modification type RNase H digestion, just like its role in assisting 30 -exonu-
of the substituent. In general, the RNase H-elicited cleavage clease-mediated oligo degradation.
activity (degrading the complementary RNA in the AON:
RNA hybrid duplex) was suppressed within a 4-5 base pairs (53) Pradeepkumar, P. I.; Chattopadhyaya, J. J. Chem. Soc., Perkin
long region that starts from the base opposite to the modified Trans. 2 2001, 2074–2083.

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FIGURE 4. Bar plots of the observed cleavage rates of the RNase H1-promoted degradation of RNA in AON 1-33:RNA hybrid duplexes.
Note that type I modification recruits RNase H at least 2-times better than that of the native.

(iii) For the nonelectropositive group at C80 , type II ([80 - stereochemistry of 80 substituents on carba-ENA: (i) hydro-
CH3]-carba-ENA)-modified AONs and type IV-modified phobic 80 S-Me substituent on carba-ENA increases the
AONs can mediate the recruitment of RNase H to similar nucleolytic stability but 80 R-Me lead to a decrease effect;
degrees as the native counterpart, suggesting that the nature (ii) hydrophilic 80 R-OH on carba-ENA had little if any effect
of the substituent (lipophilic Me versus hydrophilic OH) on nuclease resistance but 80 S-OH resulted in significantly
does not affect the efficiency of RNase H elicitation. decreased nucleolytic stability; and (iii) zwitterionic 80 -NH2
(iv) The RNA cleavage efficiency in the modified AON: and 80 -NH2PAC on carba-ENA led to obvious loss on the
RNA hybrids does not depend on the orientation of the 30 -exonuclease resistance. Hence, it seems that the electrostatic
substituent at C80 . For example, type III ([80 S-OH]-carba- effect of 80 substituents contributes to their modulations on
ENA)-modified AONs and type IV ([80 R-OH]-carba-ENA)- nucleolytic stability.
modified AONs showed the same reaction rate as the native (3) All the carba-ENAs-modified AONs showed better
hybrid. RNase H recruitment capabilities than their native and
(v) Substitution of carba-ENA or its analogues (types I to ENA-modified counterpart. Especially, parent carba-ENA
VI) at position 3 or 10 (from the 30 -end) of the AON sequence (type I) and [80 R-NH2]-carba-ENA (type VI)-modified
gives a much improved RNA cleavage by RNase H1 in the AONs exhibited double RNA hydrolysis rates relative to
corresponding heteroduplexes than at any other sites in the the ENA counterpart.
sequence. Modification in the middle of the AON strand Thus this study shows that carba-ENA and its analogue
produces less effective RNase H1 elicitation. modified AON fulfill at least three most important criteria
for being a winner as potential RNA-directed theraopeutics:
(1) desired RNA selectivity; (2) good nuclease stability, and
Conclusions
(3) much improved RNase H1 elicitation capability com-
In this investigation, five 80 -modified carba-ENA deriva- pared to that of the native and ENA-modified AONs.
tives and parent carba-ENA-T have been synthesized through
an intramolecular radical cyclization. Biological properties of
Implications
AONs containing these modifications have been evaluated and
compared with that of AONs containing other types of The present study shows that C80 substituents of carba-
modifications such as LNA, carba-LNA, and ENA. The ENA impart distinctly different biophysical and biochemical
major conclusions are as follows: effects on the properties of modified nucleic acids compared
(1) All the carba-ENAs-modified AONs showed improved to those of C60 substituents because they have different
RNA affinity over their native counterpart. Interestingly, it was stereochemical locations, hence interact differently, in the
found the nature and the steric orientation of the 80 substituents minor groove. Since the C80 substituent of carba-ENA is
only slightly affect the Tm values of modified AON:RNA located at the bottom and center of the minor groove, and
duplexes. One the contrary, carba-ENAs modifications in the theC60 substituent of carba-ENA is also at the minor groove
AON:DNA homoduplex resulted in significant Tm decrease. (but not at the center) but spatially close to the internucleo-
Hence, parent carba-ENA and 80 -substituted analogues are tidic phosphate, they have different biophysical-chemical
highly RNA selective. effects: it was found that a positively charged amino sub-
(2) The effect of 80 substituents on the nucleolytic stability stituent at the C80 can improve target affinity and RNase H
of AON was found to greatly depend on the nature and recruitment capability, whereas the C60 substituent of carba-ENA
J. Org. Chem. Vol. 75, No. 21, 2010 7123
JOC Article Liu et al.

can render a remarkable effect on nuclease resistance in that acetamide (7.5 mL, 30.35 mmol) was added under N2 followed by
the C60 R-hydrophobic substituent (Me) seems to give the best reflux for 30 min until the suspension became a clear solution.
result. Combining the merits of both C80 and C60 substituents Then the solution was cooled to room temperature and TMSOTf
in one engineering of a carba-ENA nucleotide, such as a C80 R- (2.5 mL, 14.05 mmol) was added dropwise. The obtained mixture
amino-C60 R-Me-carba-ENA moiety, may generate an excel- was refluxed overnight followed by quenching with saturated
aqueous NaHCO3 and extracted with CH2Cl2. The organic layer
lent candidate for AON-based therapeutics, which deserve was dried over MgSO4 and concentrated. The residue was purified
further investigation in the future. On the other hand, the by column chromatography on silica gel (20-33% acetone in
unique location of C80 substituents of carba-ENA (at the petroleum ether, v/v) to obtain 10 (5.07 g, 84.5% in two steps) as a
bottom and center of the minor groove) makes C80 modified white foam. 1H NMR (600 MHz, CDCl3) δ 9.09 (1H, s, H3),
carba-ENA very good chemical models to study the role of 7.38-7.26 (11H, m, aromatic and H6), 6.05 (1H, d, J10 ,20 = 4.8 Hz,
electrostatic and steric effect in the self-assembly as well as to H10 ), 5.45 (1H, dd, J10 ,20 = 4.8 Hz, J20 ,30 = 6.0 Hz, H20 ), 4.62 (1H,
probe various biological functions of nucleic acids. d, Jgem = 11.4 Hz, CH2Bn), 4.52 (1H, d, Jgem = 11.4 Hz, CH2Bn),
4.47 (1H, d, Jgem = 11.4 Hz, CH2Bn), 4.43 (1H, d, Jgem = 11.4 Hz,
CH2Bn), 4.42 (1H, d, J20 ,30 = 6.0 Hz, H30 ), 3.62 (1H, d, Jgem = 9.6
Experimental Section Hz, H50 ), 3.37 (1H, d, Jgem = 9.6 Hz, H500 ), 2.55 (1H, m, H70 ), 2.41
(1H, m, H700 ), 2.21 (1H, m, H60 ), 2.10 (3H, s, acetyl-CH3), 1.85
Note: All individual reaction steps, workup, and product (1H, m, H700 ), 1.60 (3H, s, thymine-CH3). 13C NMR (150 MHz,
characterization for compounds 16b, 17b, 18a/b, 21b, 22a/b, 25, CDCl3) δ 170.3 (CdO, acetyl), 163.9 (C4), 150.7 (C2), 137.3, 137.2
28, 29, 30a/b, 31, 32, and ENA are given in the Supporting (2  Cipso-Bn), 136.3 (C6), 129.0-128.1 (aromatic), 120.3 (CN),
Information. 111.9 (C5), 88.1 (C10 ), 86.3 (C40 ), 78.6 (C30 ), 74.9 (C20 ), 74.8, 73.9
3,5-Di-O-benzyl-4-C-cyanoethyl-1,2-O-isopropylidene-r-D- (CH2Bn), 72.8 (C50 ), 28.6 (C60 ), 21.0 (CH3, acetyl), 12.4 (CH3-
ribofuranose (8). Et3N (15.4 mL, 110.72 mmol) was added drop- thymine), 12.2 (C70 ). MALDI-TOF m/z [C29H31N3O7 þ H]þ
wise to a solution of 6 (8.66 g, 20.89 mmol), p-toluenesulfonyl found 534.224, calcd 534.223.
chloride (7.17 g, 37.61 mmol), and DMAP (255 mg, 2.09 mmol) 1-(3,5-O-Benzyl-4-C-cyanoethyl-2-O-hydroxyl-β-D-ribofuranosyl)-
in anhydrous CH2Cl2 (290 mL) at 0 °C under N2 atmosphere. thymine (11). Compound 10 (2.52 g, 4.72 mmol) was dissolved in
The mixture was allowed to warm to room temperature fol- methanol (13 mL) and methylamine solution (95 mL). The
lowed by stirring overnight at this temperature. The reaction mixture was stirred in an ice bath for 1 h. After evaporation of
was quenched with saturated aqueous NaHCO3 and the solvent, the obtained residue was purified by column chromatog-
obtained mixture was partitioned between CH2Cl2 and water, raphy on silica gel (1% methanol in CH2Cl2, v/v) to obtain 11
then the aqueous layer was extracted again with CH2Cl2. The (2.19 g, 94.7%) as white foam. 1H NMR (500 MHz, CDCl3) δ 9.10
combined organic layer was dried over MgSO4 and evaporated (1H, s, H3), 7.39-7.26 (11H, m, aromatic and H6), 5.84 (1H, d,
to give tosylate 7. Compound 7 was dissolved in anhydrous J10 ,20 = 5.5 Hz, H10 ), 4.78 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.61
DMF (73 mL) and tetrabutylammonium cyanide (14.02 g, 52.23 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.55 (1H, d, Jgem = 11.5 Hz,
mmol) was added. The obtained solution was stirred at room CH2Bn), 4.50 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.41 (1H, dd,
temperature for 2.5 days, then warmed to 35 °C followed by J10 ,20 = 5.5 Hz, J20 ,30 = 6.0 Hz, J20 ,OH = 7.0 Hz, H20 ), 4.22 (1H, d,
stirring at this temperature for 1 day. After evaporation of J20 ,30 = 6.0 Hz, H30 ), 3.60 (1H, d, Jgem = 10.0 Hz, H50 ), 3.53 (1H,
solvent, the residue was dissolved in CH2Cl2 and saturated d, J20 ,OH = 7.0 Hz, 20 -OH), 3.40 (1H, d, Jgem = 10.0 Hz, H500 ),
aqueous NaHCO3. The organic layer was separated and the 2.52 (1H, m, H70 ), 2.39 (1H, m, H700 ), 2.29 (1H, m, H60 ), 1.84
aqueous layer was extracted again with CH2Cl2. The combined (1H, m, H600 ), 1.61 (3H, s, thymine-CH3). 13C NMR (150 MHz,
organic layer was dried over MgSO4 and concentrated. The CDCl3) δ 164.0 (C4), 151.2 (C2), 137.3, 137.2 (2  Cipso-Bn), 136.4
residue was purified by column chromatography on silica gel (C6), 129.1-128.1 (aromatic), 120.4 (CN), 111.5 (C5), 90.3 (C10 ),
(4-6% acetone in petroleum ether, v/v) to obtain 8 (6.85 g, 86.2 (C40 ), 79.8 (C30 ), 75.0 (C20 ), 74.9, 74.1 (CH2Bn), 73.1 (C50 ),
77.4% in two steps) as a light yellow syrup. 1H NMR (500 MHz, 28.8 (C60 ), 12.5 (CH3-thymine), 12.2 (C70 ). MALDI-TOF m/z
CDCl3) δ 7.34-7.23 (10H, m, aromatic), 5.73 (1H, d, J1,2 = 4.0 [C27H29N3O6 þ H]þ found 492.215, calcd 492.213.
Hz, H1), 4.75 (1H, d, Jgem = 12.0 Hz, CH2Bn), 4.60 (1H, dd, 1-(3,5-O-Benzyl-4-C-propionaldehyde-2-O-hydroxyl-β-D-ribofur-
J1,2 = 4.0 Hz, J2,3 = 5.0 Hz, H2), 4.52 (1H, d, Jgem = 12.0 Hz, anosyl)thymine O-Benzyl Oxime (12). DIBALH (11.5 mL, 11.5
CH2Bn), 4.48 (1H, d, Jgem = 12.0 Hz, CH2Bn), 4.43 (1H, d, mmol, 1.0 M solution in toluene) was added dropwise to a solution
Jgem = 12.0 Hz, CH2Bn), 4.00 (1H, d, J2,3 = 5.0 Hz, H3), 3.37 of 11 (1.41 g, 2.87 mmol) in anhydrous CH2Cl2 (29 mL) over 30 min
(1H, d, Jgem = 10.0 Hz, H5), 3.25 (1H, d, Jgem = 10.0 Hz, H50 ), at -78 °C. After being stirred at the same temperature for 2 h under
2.59 (2H, m, H7 and H6), 2.43 (1H, m, H70 ), 1.91 (1H, m, H60 ), nitrogen atmosphere, the mixture was quenched with methanol at
1.58 (3H, s, CH3), 1.31 (3H, s, CH3). 13C NMR (125 MHz, -78 °C, followed by water. The mixture was allowed to warm to
CDCl3) δ 137.6 (2  Cipso-Bn), 128.4-127.7 (aromatic), 120.5 room temperature until a precipitate was formed, which was filtered
(isopropyl), 113.2 (CN), 104.3 (C1), 85.1 (C4), 79.3 (C3), 78.5 though Celite. The filtrate was washed with 10% aqueous acetic
(C2), 73.6 (CH2Bn), 73.0 (C5), 72.4 (CH2Bn), 27.9 (C6), 26.5 acid, saturated aqueous NaHCO3, and brine. The organic layer was
(CH3), 25.8 (CH3), 11.9 (C7). MALDI-TOF m/z [C25H29NO5 þ Na]þ dried over MgSO4 and concentrated to give crude aldehyde as an
found 446.194, calcd 446.200. intermediate. The crude aldehyde was dissolved in anhydrous
1-(2-O-Acetyl-3,5-O-benzyl-4-C-cyanoethyl-β-D-ribofuranosyl)- CH2Cl2 (29 mL) and anhydrous pyridine (1.4 mL, 17.22 mmol),
thymine (10). Compound 8 (4.76 g, 11.24 mmol) was dissolved in to which O-benzyl hydroxylamine hydrochloride (916 mg, 5.74
acetic anhydride (6.3 mL, 67.44 mmol) and acetic acid (63 mL). mmmol) was added. After refluxing for 1 h, the mixture was cooled
This solution was cooled to 10 °C, to which trifilic acid (100 μL, to room temperature and saturated aqueous NaHCO3 was added.
1.12 mmol) was added dropwise, and the obtained mixture was The organic layer was separated, dried over MgSO4, and evapo-
stirred at this temperature for 1 h. The reaction was quenched with rated. The residue was subjected to short column chromatography
saturated aqueous NaHCO3 and extracted with CH2Cl2. The over silica gel (0.9-1.2% methanol in CH2Cl2, v/v) to give 12 (1.04 g,
organic layer was dried over MgSO4 and evaporated to give 60.6% in two steps) as the mixture of Z and E isomers. Isomer I:
crude product 9. The crude product, after coevaporation with 1
H NMR (500 MHz, CDCl3) δ 8.14 (1H, s, H3), 7.44 (1H, t, J70 ,80 =
toluene twice, was dissolved in anhydrous MeCN (146 mL), to 5.4 Hz, J70 0 ,80 = 5.4 Hz, H80 ), 7.37-7.26 (16H, m, aromatic and
which thymine (2.12 g, 16.85 mmol) and N,O-bis(trimethylsilyl)- H6), 5.89 (1H, d, J10 ,20 = 6.0 Hz, H10 ), 5.05 (2H, s, NOCH2Bn),

7124 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.
JOC Article
4.68 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.65 (1H, d, Jgem = 11.5 Hz, 29.1 (C60 ), 20.9 (C70 ), 12.6 (CH3-thymine). MALDI-TOF m/z:
CH2Bn), 4.52 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.49 (1H, d, Jgem = [C41H41N3O8S þ H]þ found 736.270, calcd 736.269.
11.5 Hz, CH2Bn), 4.34 (1H, m, J10 ,20 = 6.0 Hz, J20 ,30 = 6.0 Hz, (1R,3R,4R,5R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-benzy-
J20 ,OH = 8.5 Hz, H20 ), 4.16 (1H, d, J20 ,30 = 6.0 Hz, H30 ), 3.61 (1H, d, loxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (14). Com-
Jgem = 10.0 Hz, H50 ), 3.40 (1H, d, Jgem = 10.0 Hz, H500 ), 2.90 (1H, pound 13 (910 mg, 1.237 mmol) was dissolved in anhydrous toluene
d, J20 ,OH = 8.5 Hz, 20 -OH), 2.38 (1H, m, H70 ), 2.22 (1H, m, H700 ), (130 mL) then the solution was purged with N2 for 40 min. The
2.12 (1H, m, H60 ), 1.70 (1H, m, H600 ), 1.61 (3H, d, J6,CH3 = 0.6 Hz, mixture was refluxed, Bn3SnH (0.8 mL in 20 mL dry toluene) and
thymine-CH3). 13C NMR (150 MHz, CDCl3) δ 163.2 (C4), 150.6 AIBN (40 mg in 10 mL dry toluene) were added dropwise in 5 h, then
(C80 ), 150.5 (C2), 137.7, 137.1, 136.7 (3  Cipso-Bn), 135.8 (C6), the mixture continued to reflux for 1 h. The mixture was cooled to
128.8-127.6 (aromatic), 111.1 (C5), 88.6 (C10 ), 86.7 (C40 ), 80.2 room temperature and solvent was evaporated. The residue was
(C30 ), 75.6 75.1 (2  CH2Bn), 74.7 (C20 ), 73.7 (CH2Bn), 73.5 (C50 ), chromatographed on silica gel (18-30% in ethyl acetate in cyclo-
29.1 (C60 ), 24.0 (C70 ), 12.1 (CH3-thymine). Isomer II: 1H NMR hexane, v/v) to give 14 (433 mg, 60.1%) as a white foam. 1H NMR
(500 MHz, CDCl3) δ 8.20 (1H, s, H3), 7.39-7.26 (16H, m, (500 MHz, CDCl3) δ 8.20 (1H, s, H3), 8.00 (1H, d, J6,CH3 = 1.0 Hz,
aromatic and H6), 6.68 (1H, t, J70 ,80 = 5.5 Hz, J70 0 ,80 = 5.5 Hz, H6), 7.39-7.26 (15H, m, aromatic), 5.99 (1H, s, H10 ), 5.58 (1H, s,
H80 ), 5.87 (1H, d, J10 ,20 = 6.0 Hz, H10 ), 5.05 (2H, s, NOCH2Bn), NH), 4.83 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.80 (1H, d, Jgem = 11.5
4.68 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.63 (1H, d, Jgem = 11.5 Hz, Hz, CH2Bn), 4.61 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.57 (1H, d,
CH2Bn), 4.52 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.48 (1H, d, Jgem = Jgem = 11.5 Hz, CH2Bn), 4.52 (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.44
11.5 Hz, CH2Bn), 4.33 (1H, dd, J10 ,20 = 6.0 Hz, J20 ,30 = 6.0 Hz, (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.25 (1H, d, J20 ,30 = 5.0 Hz, H30 ),
J20 ,OH = 7.5 Hz, H20 ), 4.17 (1H, d, J20 ,30 = 6.0 Hz, H30 ), 3.63 (1H, d, 3.69 (1H, d, Jgem = 11.0 Hz, H50 ), 3.56 (1H, m, J20 ,80 = 3.5 Hz, J70 ,80 =
Jgem = 10.0 Hz, H50 ), 3.41 (1H, d, Jgem = 10.0 Hz, H500 ), 3.93 (1H, 5.0 Hz, J70 0 ,80 = 11.5 Hz, H80 ), 3.54 (1H, d, Jgem = 11.0 Hz, H500 ), 2.82
d, J20 ,OH = 7.5 Hz, 20 -OH), 2.45 (2H, m, H70 and H700 ), 2.08 (1H, (1H, d, J20 ,80 = 3.5 Hz, J20 ,30 = 5.0 Hz, H20 ), 1.86 (1H, m, H60 ), 1.80
m, H60 ), 1.71 (1H, m, H600 ), 1.60 (3H, s, thymine-CH3). 13C NMR (1H, m, H70 ), 1.61 (3H, d, J6,CH3 = 1.0 Hz, thymine-CH3), 1.40
(125 MHz, CDCl3) δ 163.3 (C4), 151.5 (C80 ), 150.6 (C2), 138.0, (1H, m, H60 ), 1.38 (1H, m, H700 ). 13C NMR (125 MHz, CDCl3) δ
137.2, 136.8 (3  Cipso-Bn), 135.9 (C6), 128.8-127.6 (aromatic), 163.8 (C4), 149.7 (C2), 138.1, 137.5, 137.4 (Cipso-Bn), 136.5 (C6),
111.1 (C5), 88.9 (C10 ), 86.8 (C40 ), 80.0 (C30 ), 75.9, 75.0 (2  128.6-127.5 (aromatic), 109.3 (C5), 84.6 (C40 ), 84.4 (C10 ), 76.5,
CH2Bn), 74.8 (C20 ), 73.7 (CH2Bn), 73.3 (C50 ), 28.8 (C60 ), 20.4 73.6 (2  CH2Bn), 73.2 (C30 ), 71.9 (CH2Bn), 70.2 (C50 ), 53.5 (C80 ),
(C70 ), 12.1 (CH3-thymine). MALDI-TOF m/z [C34H37N3O7 þ H]þ 44.7 (C20 ), 26.2 (C60 ), 22.1 (C70 ), 11.9 (CH3-thymine). MALDI-
found 600.272, calcd 600.270. TOF m/z [C34H37N3O6 þ Na]þ found 606.258, calcd 606.257.
1-(3,5-O-Benzyl-2-O-phenoxythiocarbonyl-4-C-propionaldehyde- (1R,3R,4R,5R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-trifluoroa-
β-D-ribofuranosyl)thymine O-Benzyl Oxime (13). To a solution of 12 cetamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (16a). To a
(1.13 g, 1.89 mmol) in anhydrous pyridine (19 mL) was added solution of 14 (110 mg, 0.188 mmol) in anhydrous methanol
dropwise phenyl chlorothionoformate (0.31 mL, 2.27 mmol) in an (4.0 mL) were added 10% Pd/C (94 mg) and ammonium formate
ice bath under N2. The mixture was stirred for 30 min in the ice bath, (475 mg, 7.54 mmol) under nitrogen. The mixture was stirred for
then stirred for a further 2 h at room temperature followed by 8 h at room temperature. The suspension was filtered through a
quenching with methanol. After the mixture was stirred for 30 min, pad of Celite. The filtrate was concentrated and dried over
the solvent was removed. The residue was dissolved in CH2Cl2 and vacuum pump. The obtained crude amine 15 was dissolved in a
washed with saturated aqueous NaHCO3, and then the aqueous mixture of anhydrous CH2Cl2 (3.8 mL) and pyridine (0.12 mL,
layer was extracted with CH2Cl2 twice. The combined organic layer 1.50 mmol) and cooled to 0 °C, to which trifluoroacetic anhy-
was dried over MgSO4 and evaporated. The residue was purified by dride (0.1 mL, 0.752 mmol) was added dropwise over 5 min.
column chromatography on silica gel (11-17% acetone in petro- Then the reaction mixture was allowed to warm to room
leum ether, v/v) to obtain 13 (1.23 g, 88.7%) as the mixture of Z and temperature and stirred at this temperature for 2 h, followed
E isomers. Isomer I: 1H NMR (500 MHz, CDCl3) δ 8.23 (1H, s, H3), by quenching with crushed ice and diluted with CH2Cl2. The
7.44 (1H, d, J6,CH3 = 1.2 Hz, H6), 7.41-7.26 (19H, m, aromatic and aqueous layer was extracted with CH2Cl2 twice. The organic
H80 ), 7.00 (2H, dd, aromatic), 6.35 (1H, d, J10 ,20 = 6.0 Hz, H10 ), 5.95 layers were combined, dried over MgSO4, and concentrated. The
(1H, t, J10 ,20 = 6.0 Hz, J20 ,30 = 6.0 Hz, H20 ), 5.04 (2H, s, NOCH2Bn), residue was purified by column chromatography on silica gel
4.74 (1H, d, Jgem = 11.0 Hz, CH2Bn), 4.54 (1H, d, J20 ,30 = 6.0 Hz, (0.6-0.8% methanol in CH2Cl2, v/v) to obtain 16a (65 mg,
H30 ), 4.53 (1H, d, Jgem = 11.0 Hz, CH2Bn), 4.52 (2H, s, CH2Bn), 60.0% in two steps) as a white foam. 1H NMR (500 MHz, CDCl3)
3.67 (1H, d, Jgem = 10.0 Hz, H50 ), 3.43 (1H, d, Jgem = 10.0 Hz, δ 8.79 (1H, s, H3), 7.89 (1H, d, J6,CH3=1.5 Hz, H6), 7.65 (1H, d,
H500 ), 2.37 (1H, m, H70 ), 2.22 (1H, m, H700 ), 2.05 (1H, m, H60 ), 1.74 J80 ,NH=6.5 Hz, 80 -NH), 7.40-7.27 (10H, m, aromatic), 5.92 (1H,
(1H, m, H600 ), 1.54 (3H, d, J6,CH3 = 1.2 Hz, thymine-CH3). 13C s, H10 ), 4.71 (1H, d, Jgem =11.5 Hz, CH2Bn), 4.59 (1H, d, Jgem =
NMR (125 MHz, CDCl3) δ 194.4 (CdS), 163.3 (C4), 153.4 (Cipso- 11.5 Hz, CH2Bn), 4.54 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.53 (1H,
Bn), 150.6 (C80 ), 150.2 (C2), 137.7, 137.2, 137.0 (3  Cipso-Bn), 135.8 d, Jgem=11.5 Hz, CH2Bn), 4.46 (1H, m, JNH,80=6.5 Hz, J70 ,80 =6.0
(C6), 129.6-126.8, 121.7 (aromatic), 111.6 (C5), 87.3 (C40 ), 85.6 Hz, J70 0 ,80 =11.0 Hz, H80 ), 4.27 (1H, d, J20 ,30 =5.0 Hz, H30 ), 3.74
(C10 ), 82.8 (C20 ), 78.1 (C30 ), 75.6, 75.0, 73.8 (3  CH2Bn), 73.4 (1H, d, Jgem=10.5 Hz, H50 ), 3.59 (1H, d, Jgem=10.5 Hz, H500 ), 2.63
(C50 ), 29.1 (C60 ), 24.1 (C70 ), 12.1 (CH3-thymine). Isomer II: 1H (1H, d, J20 ,30 =5.0 Hz, H20 ), 2.34 (1H, m, J60 ,70 =6.0 Hz, J70 ,80 =6.0
NMR (600 MHz, CDCl3) δ 9.37 (1H, s, H3), 7.43 (1H, s, H6), Hz, J70 ,70 0=13.0 Hz, H70 ), 1.96 (1H, ddd, J60 ,70=6.0 Hz, J60 ,70 0=13.0
7.42-7.30 (18H, m, aromatic), 7.00 (2H, dd, aromatic), 6.68 (1H, t, Hz, J60 ,60 0 =13.0 Hz, H60 ), 1.63 (1H, m, J60 ,70 0 =6.0 Hz, J60 0 ,70 0 =
J70 ,80 = 5.4 Hz, J70 0 ,80 = 5.4 Hz, H80 ), 6.39 (1H, d, J10 ,20 = 6.0 Hz, 6.5 Hz, J70 0 ,80=11.0 Hz, J70 ,70 0=13.0 Hz, H700 ), 1.51 (1H, dd, J60 0 ,70 0=
H10 ), 5.93 (1H, t, J10 ,20 = 6.0 Hz, J20 ,30 = 6.0 Hz, H20 ), 5.10 (2H, s, 6.5 Hz, J60 ,60 0=13.0 Hz, H600 ), 1.46 (3H, d, J6,CH3=1.5 Hz, thymine-
NOCH2Bn), 4.76 (1H, d, Jgem = 11.4 Hz, CH2Bn), 4.60 (1H, d, CH3). 13C NMR (125 MHz, CDCl3) δ 164.0 (C4), 157.2 (CF3CO),
J20 ,30 = 6.0 Hz, H30 ), 4.55 (1H, d, Jgem = 11.4 Hz, CH2Bn), 4.52 150.3 (C2), 137.3, 137.0 (2  Cipso-Bn), 135.4 (C6), 128.7-127.8
(2H, s, CH2Bn), 3.72 (1H, d, Jgem = 10.2 Hz, H50 ), 3.46 (1H, d, (aromatic), 115.8 (CF3CO), 110.2 (C5), 84.9 (C40 ), 84.1 (C10 ), 73.6
Jgem = 10.2 Hz, H500 ), 2.44 (2H, m, H70 and H700 ), 2.04 (1H, m, (CH2Bn), 72.8 (C30 ), 72.4 (CH2Bn), 69.8 (C50 ), 47.4 (C20 ), 44.4
H60 ), 1.75 (1H, m, H600 ), 1.47 (3H, s, thymine-CH3). 13C NMR (125 (C80 ), 26.1 (C60 ), 24.1 (C70 ), 11.9 (CH3-thymine). MALDI-TOF
MHz, CDCl3) δ 195.0 (CdS), 164.5 (C4), 153.7 (Cipso-Bn), 152.0 m/z [C29H30F3N3O6 þ H]þ found 574.212, calcd 574.216.
(C80 ), 151.0 (C2), 138.2, 137.6, 137.4 (3  Cipso-Bn), 136.4 (C6), (1R,3R,4R,5R,8S)-1-(4,40 -Dimethoxytrityloxymethyl)-8-hydroxyl-
130.2-127.4, 122.2 (aromatic), 112.2 (C5), 87.7 (C40 ), 85.8 (C10 ), 5-trifluoroacetamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane
83.3 (C20 ), 78.4 (C30 ), 76.2, 75.4, 74.1 (3  CH2Bn), 73.5 (C50 ), (17a). A mixture of 20% Pd(OH)2/C (100 mg), 16a (95 mg,

J. Org. Chem. Vol. 75, No. 21, 2010 7125

JOC Article Liu et al.

0.166 mmol) in ethanol (6.6 mL), and cyclohexene (13.2 mL) H6), 7.46-7.25 (10H, m, aromatic), 5.57 (1H, s, H10 ), 4.73 (1H, d,
was reflux overnight. Then the suspension was filtered through Jgem=11.5 Hz, CH2Bn), 4.62 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.59
a pad of Celite. The filtrate was concentrated and dried. The (1H, d, Jgem = 11.5 Hz, CH2Bn), 4.55 (1H, d, Jgem = 11.5 Hz,
residue was coevaporated twice with dry pyridine and dissolved CH2Bn), 4.36 (1H, d, J20 ,30 =4.5 Hz, H30 ), 4.22 (1H, m, J20 ,80 =4.0
in the same solvent (1.7 mL), to which 4,40 -dimethoxytrityl Hz, JOH,80 =11.5 Hz, H80 ), 4.03 (1H, d, JOH,80 =11.5 Hz, 80 -OH),
chloride (69 mg, 0.202 mmol) was added, and stirred overnight 3.77 (1H, d, Jgem=11.0 Hz, H50 ), 3.59 (1H, d, Jgem=11.0 Hz, H500 ),
at room temperature. Then solvent was removed and the 2.82 (1H, t, J20 ,80=4.0 Hz, J20 ,30=4.5 Hz, H20 ), 2.03 (2H, m, H70 and
obtained residue was chromatographed on silica gel (0-0.6% H60 ), 1.91 (1H, m, H700 ), 1.49 (3H, d, J6,CH3 = 1.0 Hz, thymine-
methanol in CH2Cl2 containing 1% pyridine, v/v) to obtain 17a CH3), 1.43 (1H, m, H600 ). 13C NMR (125 MHz, CDCl3) δ 163.7
(35 mg, 30.3% in two steps) as a white foam. 1H NMR (600 (C4), 149.9 (C2), 137.1, 136.4 (2  Cipso-Bn), 135.9 (C6),
MHz, CDCl3 þ d4-MeOH) δ 8.24 (1H, d, J80 ,NH =6.0 Hz, 80 - 128.7-127.9 (aromatic), 109.4 (C5), 85.4 (C10 ), 84.5 (C40 ), 75.4
NH), 7.90 (1H, s, H6), 7.43-6.84 (13H, m, aromatic), 5.85 (1H, (C30 ), 74.0, 73.6 (2  CH2Bn), 70.4 (C50 ), 68.7 (C80 ), 46.2 (C20 ),
s, H10 ), 4.63 (1H, d, J20 ,30 =5.4 Hz, H30 ), 4.52 (1H, m, J20 ,80 =2.5 27.3 (C70 ), 23.5 (C60 ), 11.9 (CH3-thymine). MALDI-TOF m/z
Hz, J80 ,NH =6.0 Hz, J70 ,80 =6.0 Hz, J70 0 ,80 =11.5 Hz, H80 ), 3.79 [C27H30N2O6 þ Na]þ found 501.200, calcd 501.200. 20b: 1H
(6H, s, 2  OCH3), 3.35 (1H, d, Jgem=10.8 Hz, H50 ), 3.28 (1H, d, NMR (500 MHz, CDCl3) δ 8.05 (1H, s, H6), 7.35-7.18 (10H,
Jgem=10.8 Hz, H500 ), 2.58 (1H, d, J20 ,80 =2.5 Hz, J20 ,30 =5.4 Hz, m, aromatic), 6.08 (1H, s, H10 ), 4.77 (1H, d, Jgem = 12.0 Hz,
H20 ), 2.16 (2H, m, J70 ,80 =6.0 Hz, J60 ,70 =6.0 Hz, J70 ,70 0 =13.8 Hz, CH2Bn), 4.60 (1H, d, Jgem=12.0 Hz, CH2Bn), 4.55 (2H, d, Jgem=
H70 and 30 -OH), 1.87 (1H, ddd, J60 ,70 =6.0 Hz, J60 ,70 0 =12.6 Hz, 12.0 Hz, 2  CH2Bn), 4.39 (1H, s, H30 ), 4.37 (1H, m, H80 ), 3.69 (1H,
J60 ,60 0 =13.8 Hz, H60 ), 1.61 (1H, m, J70 0 ,80 =11.5 Hz, J60 0 ,70 0 =6.6 d, Jgem=11.0 Hz, H50 ), 3.55 (1H, d, Jgem=11.0 Hz, H500 ), 3.08 (1H,
Hz, J60 ,70 0 =12.6 Hz, J70 ,70 0 =13.8 Hz, H700 ), 1.45 (1H, dd, J60 0 ,70 0 = s, H20 ), 2.04 (1H, m, H70 ), 1.85 (1H, ddd, H60 ), 1.62 (1H, m, H700 ),
6.6 Hz, J60 ,60 0 = 13.8 Hz, H600 ), 1.29 (3H, s, thymine-CH3). 1.41 (1H, dd, H600 ), 1.39 (3H, d, J6,CH3 =1.0 Hz, thymine-CH3).
13
C NMR (125 MHz, CDCl3) δ 164.3 (C4), 158.7, 158.6 (2  13
C NMR (125 MHz, CDCl3) δ 164.5 (C4), 151.0 (C2), 138.1, 137.5
OMe-Cispo), 157.5 (CF3CO), 150.8 (C2), 144.3, 135.5, 135.3 (2  Cipso-Bn), 136.7 (C6), 128.6-127.3 (aromatic), 108.8 (C5),
(DMTr-Cispo), 135.2 (C6), 130.1-124.0 (aromatic), 115.8 84.4 (C40 ), 84.3 (C10 ), 74.3 (C30 ), 73.5, 71.9 (2  CH2Bn), 70.2
(CF3CO), 113.3 (aromatic), 110.4 (C5), 86.6 (DMTr-C), 85.8 (C50 ), 64.3 (C80 ), 49.1 (C20 ), 26.3 (C70 ), 26.1 (C60 ), 12.2 (CH3-
(C40 ), 84.2 (C10 ), 66.5 (C30 ), 63.3 (C50 ), 55.2 (2  OMe), 49.9 thymine). MALDI-TOF m/z [C27H30N2O6 þ H]þ found 479.221,
(C20 ), 44.3 (C80 ), 25.8 (C60 ), 23.8 (C70 ), 11.9 (CH3-thymine). calcd 479.218.
MALDI-TOF m/z [C36H36F3N3O8 þ Na]þ found 718.235, (1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-(4-methyl-
calcd 718.235. benzoate)-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (21a). Com-
(1R,3R,4R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-one-3-(thymin- pound 20a (72 mg, 0.150 mmol) was dissolved in dry pyridine
1-yl)-2-oxa-bicyclo[3.2.1]octane (19). The crude amine 15 (3 mL) and the solution was cooled to 0 °C, then toluoyl chloride
(0.43 mmol) was dissolved in dry methanol (2.6 mL) and dry (0.1 mL, 0.760 mmol) was added. The mixture was allowed to warm
THF (0.4 mL), to which was added 3,5-di-tert-butyl-1,2-benzo- to room temperature followed by stirring at this temperature over-
quinone (114 mg, 0.516 mmol). The mixture was stirred at 40 °C night. Then the reaction was quenched with saturated NaHCO3
for 10 h, then H2O (0.72 mL) and oxalic acid (68 mg, 0.54 mmol) solution. The aqueous layer was exacted with CH2Cl2 three times.
were added followed by stirring at 40 °C overnight. Then addi- The combined organic layer was dried over MgSO4 and concen-
tional H2O (5 mL) was added and extracted with EtOAc three trated. The residue was chromatographed on silica gel (0.8-1.2%
times. The organic layer was dried over MgSO4 and evaporated to methanol in CH2Cl2, v/v) to obtain 21a (81 mg, 90.0%) as a white
dryness. The residue was applied to column chromatography on foam. 1H NMR (500 MHz, CDCl3) δ 8.19 (1H, s, H3), 8.04 (1H, s,
silica gel (0.5-2% methanol in CH2Cl2, v/v) to give compound 19 H6), 7.79-6.98 (14H, m, aromatic), 5.73 (1H, s, H10 ), 5.53 (1H, t,
(102 mg, 50% in two steps). 1H NMR (500 MHz, CDCl3) δ 8.54 J20 ,80 = 4.0 Hz, J70 ,80 = 5.0 Hz, H80 ), 4.72 (1H, d, Jgem = 12.0 Hz,
(1H, s, H3), 8.00 (1H, d, J6,CH3=1.5 Hz, H6), 7.38-7.26 (10H, m, CH2Bn), 4.60 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.55 (1H, d, Jgem=
aromatic), 6.07 (1H, s, H10 ), 4.68 (1H, d, Jgem=11.5 Hz, CH2Bn), 11.5 Hz, CH2Bn), 4.48 (1H, d, Jgem=12.0 Hz, CH2Bn), 4.23 (1H, d,
4.63 (1H, d, J2,3 = 5.5 Hz, H30 ), 4.62 (1H, d, Jgem = 11.5 Hz, J20 ,30 =4.5 Hz, H30 ), 3.78 (1H, d, Jgem=11.0 Hz, H50 ), 3.66 (1H, d,
CH2Bn), 4.56 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.42 (1H, d, Jgem= Jgem=11.0 Hz, H500 ), 3.10 (1H, t, J20 ,80 =4.0 Hz, J20 ,30 =4.5 Hz, H20 ),
11.5 Hz, CH2Bn), 3.87 (1H, d, Jgem =11.0 Hz, H50 ), 3.65 (1H, d, 2.34 (3H, s, Tol-CH3), 2.30 (1H, ddd, J60 ,70 0 = 5.5 Hz, J60 ,70 =
Jgem=11.0 Hz, H500 ), 3.32 (1H, d, J20 ,30=5.5 Hz, H20 ), 2.61 (1H, m, 12.5 Hz, J60 ,60 0 =13.0 Hz, H60 ), 2.15 (1H, m, J70 ,80 =5.0 Hz, J60 0 ,70 =
J60 0 ,70 =9.0 Hz, J60 ,70 =10.5 Hz, J70 ,70 0 =16.5 Hz, H70 ), 2.52 (1H, dd, 6.0 Hz, J60 ,70=12.5 Hz, J70 ,70 0=15.5 Hz, H70 ), 1.42 (1H, dd, J60 ,70 0=5.5
J60 ,70 0 =7.5 Hz, J70 ,70 0 =16.5 Hz, H700 ), 2.15 (1H, m, J60 ,70 0 =7.5 Hz, Hz, J70 ,70 0 =15.5 Hz, H700 ), 1.41 (1H, dd, J60 0 ,70 =6.0 Hz, J60 ,60 0 =13.0
J60 ,70 =10.5 Hz, J60 ,60 0 =13.5 Hz, H60 ), 1.91 (1H, dd, J60 0 ,70 =9.0 Hz, Hz, H600 ), 1.41 (3H, s, thymine-CH3). 13C NMR (125 MHz, CDCl3)
J60 ,60 0 = 13.5 Hz, H600 ), 1.44 (3H, d, J6,CH3 = 1.5 Hz, thymine- δ 166.1 (CdO), 163.6 (C4), 149.8 (C2), 143.5 (Cipso-Tol), 137.6, 137.3
CH3). 13C NMR (125 MHz, CDCl3) δ 204.0 (C80 ), 162.6 (C4), (2  Cipso-Bn), 136.0 (C6), 129.7-127.2 (aromatic), 109.3 (C5), 85.8
148.7 (C2), 136.0, 135.6 (aromatic), 134.3 (C6), 127.7-126.9 (C10 ), 84.8 (C40 ), 73.6 (CH2Bn), 73.1 (C30 ), 72.5 (CH2Bn), 70.3
(aromatic), 109.1 (C5), 84.1 (C40 ), 83.4 (C10 ), 75.1 (C30 ), 72.7 (C50 ), 69.4 (C80 ), 44.7(C20 ), 24.3 (C70 ), 24.0 (C60 ), 21.6 (CH3-Tol),
(CH2Bn), 71.1 (CH2Bn), 68.6 (C50 ), 58.4 (C20 ), 33.5 (C70 ), 27.0 11.8 (CH3-thymine). MALDI-TOF m/z [C35H36N2O7 þ H]þ found
(C60 ), 10.8 (thymine-CH3). MALDI-TOF m/z [C27H28N2O6 þ 597.263, calcd 597.260.
H]þ found 477.203, calcd 477.202. (1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-((methyl-
(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy- thio)thiocarbonyl)oxy-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (23).
3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (20a) and (1R,3R,4R,5R, To a solution of 20a (77 mg, 0.161 mmol) in dry THF (3 mL) was
8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy-3-(thymin-1-yl)-2-oxa- added 60% NaH (32 mg, 1.808 mmol) at 0 °C. After the solution was
bicyclo[3.2.1]octane (20b). NaBH4 (59 mg, 1.55 mmol) was added to stirred at room temperature for 1 h, CS2 (89 μL, 1.62 mmol) was
a solution of 19 (185 mg, 0.338 mmol) in ethanol (10 mL). The added to the suspension at 0 °C, and the resulting mixture was stirred
mixture was stirred at room temperature for 4 h followed by at 40 °C for 4 h. The solution became clear and was cooled by ice to
quenching with acetone. After evaporation of solvent, the residue which methyl iodide (65 μL, 1.05 mmol) was added dropwise at 0 °C
was purified by column chromatography on silica gel (1.1-5% followed by stirring at room temperature overnight. The reaction was
methanol in CH2Cl2, v/v) to obtain two diastereomers 20a (112 mg, quenched by chilled water, then extracted with ethyl acetate three
69.2%) and 20b (30 mg, 18.6%) as a white foam. 20a: 1H NMR times. The combined organic layer was dried over MgSO4 and
(500 MHz, CDCl3) δ 8.33 (1H, s, H3), 7.99 (1H, d, J6,CH3=1.0 Hz, evaporated. The residue was chromatographed over silica gel

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Liu et al.
JOC Article
(0.5-0.7% methanol in CH2Cl2, v/v) to give 23 (48 mg, 52.7%) as a (1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-methoxaly-
yellow foam. 1H NMR (500 MHz, CDCl3) δ 8.04 (1H, d, J6,CH3=1.5 loxy-5-methyl-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (27). 26
Hz, H6), 8.02 (1H, s, H3), 7.37-7.25 (10H, m, aromatic), 6.03 (1H, t, (60 mg, 0.12 mmol) was dissolved in dry pyridine (1.5 mL) to
J20 ,80 =3.5 Hz, H80 ), 5.72 (1H, s, H10 ), 4.80 (1H, d, Jgem =12.0 Hz, which methyl oxalyl chloride (90 μL, 0.974 mmol) was added. The
CH2Bn), 4.60 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.56 (1H, d, Jgem=11.5 mixture was stirred at 40 °C overnight. After evaporation of
Hz, CH2Bn), 4.45 (1H, d, Jgem=12.0 Hz, CH2Bn), 4.24 (1H, d, J20 ,30= solvent, the residue obtained was dissolved in CH2Cl2, washed
4.5 Hz, H30 ), 3.77 (1H, d, Jgem = 11.0 Hz, H50 ), 3.65 (1H, d, with saturated NaHCO3 solution, dried over MgSO4, and eva-
Jgem = 11.0 Hz, H500 ), 3.23 (1H, t, J20 ,80 = 3.5 Hz, J20 ,30 = 4.5 Hz, porated. The residue was purified by column chromatography on
H20 ), 2.50 (3H, s, SCH3), 2.16 (3H, m, H60 , H70 and H700 ), 1.43 (1H, silica gel (0.5-1% methanol in CH2Cl2, v/v) to give 27 (61 mg,
m, H600 ), 1.42 (3H, d, J6,CH3=1.5 Hz, thymine-CH3). 13C NMR (125 87%). 1H NMR (500 MHz, CDCl3): δ 8.68 (1H, s, NH), 8.02 (1H,
MHz, CDCl3) δ 215.1 (CdS), 163.5 (C4), 149.7 (C2), 137.7, 137.3 d, J6,CH3=1.5 Hz, H6), 7.35-7.23 (10H, m, aromatic), 5.70 (1H, s,
(2  Cipso-Bn), 135.9 (C6), 128.7-127.6 (aromatic), 109.4 (C5), 85.5 H10 ), 4.63 (1H, d, Jgem =11.5 Hz, CH2Bn), 4.57 (1H, d, Jgem =
(C10 ), 85.0 (C40 ), 78.1 (C80 ), 73.7 (CH2Bn), 72.6 (C30 ), 72.4 (CH2Bn), 11.5 Hz, CH2Bn), 4.53 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.48 (1H,
70.2 (C50 ), 44.4 (C20 ), 24.0 (C70 ), 23.8 (C60 ), 19.0 (SCH3), 11.8 (CH3- d, Jgem=11.5 Hz, CH2Bn), 4.23 (1H, d, J20 ,30 =4.5 Hz, H30 ), 3.77
thymine). MALDI-TOF m/z [C29H32N2O6S2 þ H]þ found 569.180, (1H, d, Jgem =11.0 Hz, H50 ), 3.59 (1H, d, Jgem =11.0 Hz, H500 ),
calcd 569.177. 3.55 (1H, d, J20 ,30 =4.5 Hz, H20 ), 2.22 (2H, m, H60 and H70 ), 1.83
(1R,3R,4R,8S)-8-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)- (1H, m, H700 ), 1.79 (3H, s, 80 -CH3), 1.46 (3H, d, J6,CH3=1.5 Hz,
2-oxa-bicyclo[3.2.1]octane (24). 23 (64 mg, 0.112 mmol) was dis- thymine-CH3), 1.43 (1H, dd, H600 ). 13C NMR (125 MHz,
solved in dry toluene (5 mL) and the solution was purged with dry CDCl3) δ 162.8 (C4), 157.2, 155.7 (oxalyl CdO), 148.9 (C2),
nitrogen for 30 min. AIBN (9 mg, 0.034 mmol) and Bu3SnH 136.4, 136.0 (2  Cipso-Bn), 134.8 (C6), 127.7-126.4 (aromatic),
(91 μL, 0.338 mmol) were added to the mixture followed by reflux 108.5 (C5), 84.5 (C10 ), 83.2 (C80 ), 82.7 (C40 ), 73.6 (C30 ), 73.0, 72.6
for 1.5 h. The mixture was cooled to room temperature and solvent (2  CH2Bn), 69.1 (C50 ), 51.9 (OCH3), 46.8 (C20 ), 31.0 (C70 ), 23.7
was evaporated. The residue was subject to short column chroma- (C60 ), 22.9 (80 -CH3), 10.9 (CH3-thymine). MALDI-TOF m/z
tography over silica gel (0.5-0.7% methanol in CH2Cl2, v/v) to [C31H34N2O9 þ H]þ found 579.235, calcd 579.234.
obtain 24 (38 mg, 73.5%) as a white foam. 1H NMR (500 MHz, General Procedure for Phosphoramidite Synthesis. DIPEA
CDCl3) δ 8.09 (1H, d, J6,CH3 = 1.0 Hz, H6), 7.98 (1H, s, H3), (5 equiv) and 2-cyanoethyl-N,N-diisopropylphosphoramido-
7.49-7.30 (10H, m, aromatic), 5.84 (1H, s, H10 ), 4.63 (1H, d, chloridite (3 equiv) were added dropwise to a solution of
Jgem=11.5 Hz, CH2Bn), 4.61 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.56 substrate (1 equiv) in dry CH2Cl2 in an ice bath. The reaction
(1H, d, Jgem = 11.5 Hz, CH2Bn), 4.48 (1H, d, Jgem = 11.5 Hz, was allowed to warm to room temperature and stirred at this
CH2Bn), 4.18 (1H, d, J20 ,30 =5.0 Hz, H30 ), 3.71 (1H, d, Jgem=10.5 temperature for 3 h (overnight for 22a). The reaction was
Hz, H50 ), 3.57 (1H, d, Jgem=10.5 Hz, H500 ), 2.60 (1H, m, J20 ,30=5.0 quenched with dry methanol followed by stirring over 15 min.
Hz, H20 ), 1.94 (1H, m, H80 ), 1.87 (1H, m, H60 ), 1.74 (3H, m, H800 , Then the mixture was diluted with ethyl acetate and washed with
H70 and H700 ), 1.46 (3H, d, J6,CH3 =1.0 Hz, thymine-CH3), 1.36 saturated NaHCO3 solution. The organic layer was dried over
(1H, m, H600 ). 13C NMR (125 MHz, CDCl3) δ 163.7 (C4), 149.9 MgSO4 and concentrated. The residue was chromatographed
(C2), 137.8, 137.5 (2  Cipso-Bn), 136.5 (C6), 128.6-127.4 on silica gel to obtain the corresponding phosphoramidite,
(aromatic), 109.1 (C5), 87.5 (C10 ), 85.4 (C40 ), 73.5 (CH2Bn), 73.0 which was first precipitated in n-hexane then dried over P2O5
(C30 ), 71.7 (CH2Bn), 70.7 (C50 ), 42.9 (C20 ), 26.9 (C60 ), 20.7 (C80 ), under vacuum for 3 days before it was used for DNA synthesis.
17.7 (C70 ), 11.8 (CH3-thymine). MALDI-TOF m/z [C27H30N2O5 þ Oligonucleotide Synthesis and Purification. All AONs were
H]þ found 463.226, calcd 463.223. synthesized with an automated DNA/RNA synthesizer based
(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy- on phosphoramidite chemistry. For native A, G, and C building
5-methyl-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (26). 19 (100 mg, blocks, fast deprotecting phosphoramidites (Ac for C, iPr-Pac
0.21 mmol) was dissolved in dry THF (3 mL) and the mixture was for G, Pac for A) were used. Standard DNA synthesis reagents
cooled to -78 °C, then MeLi (393 μL, 1.6 M in Et2O, 0.63 mmol) and cycle were used except that 0.25 M 5-ethylthio-1H-tetrazole
was added dropwise. After the solution was stirred at -78 to was used as the activator and Tac2O as the cap A. For incorpor-
-65 °C for 10 h, the reaction was quenched by addition of ating modified nucleotides, extended coupling time (10 min
saturated NH4Cl solution. Upon warming to room temperature, compared to 25 s for native nucleotides, 15 min for 30a) was
the mixture was diluted with EtOAc and H2O. The aqueous layer used. For AONs 2-9 the protection was performed in 33%
was extracted with EtOAc three times. The combined organic aqueous ammonia at room temperature overnight. For AONs
layers were dried by MgSO4, and then evaporated to dryness. 26 10-13, 14-17, and 18-21 the protection was performed in 33%
(60 mg, 58%) was obtained by chromatography on silica gel aqueous ammonia at 55 °C for 3, 2, and 1 day, respectively. After
(0.5-3% methanol in CH2Cl2, v/v), and starting material (30 mg, deprotection, all crude oligos were purified by denaturing
30%) was recovered. 1H NMR (500 MHz, CDCl3) δ 8.35 (1H, s, PAGE (20% polyacrylamide with 7 M urea), extracted with
H3), 8.06 (1H, d, J6,CH3 = 1.5 Hz, H6), 7.40-7.28 (10H, m, 0.3 M NaOAc, and desalted with a C-18 reverse phase cartridge
aromatic), 5.66 (1H, s, H10 ), 4.90 (1H, s, OH), 4.76 (1H, d, Jgem= to give AONs in >99% purity, and correct masses have been
11.0 Hz, CH2Bn), 4.62 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.58 (1H, d, obtained by MALDI-TOF mass spectroscopy for each of them
Jgem=11.0 Hz, CH2Bn), 4.56 (1H, d, Jgem=11.5 Hz, CH2Bn), 4.35 (Table SII 1,Supporting Information).
(1H, d, J20 ,30 =4.5 Hz, H30 ), 3.77 (1H, d, Jgem=11.0 Hz, H50 ), 3.60 UV Melting Experiments. Determination of the Tm of the
(1H, d, Jgem=11.0 Hz, H500 ), 2.48 (1H, d, J20 ,30 =4.5 Hz, H20 ), 1.98 AON:RNA hybrids or AON:DNA duplex was carried out in the
(1H, ddd, J60 ,70 0 =6.0 Hz, J60 ,70 =13.0 Hz, J60 ,60 0 =13.0 Hz, H60 ), 1.82 following buffer: 60 mM Tris-HCl (pH 7.5), 60 mM KCl, 0.8
(1H, ddd, J70 ,60 0 = 6.0 Hz, J60 ,70 =13.0 Hz, J70 ,70 0 = 14.0 Hz, H70 ), mM MgCl2. Absorbance was monitored at 260 nm in the
1.75 (1H, dd, J60 ,70 0 =6.0 Hz, J70 ,70 0 =14.0 Hz, H700 ), 1.50 (3H, d, temperature range from 20 to 70 °C, using an UV spectro-
J6,CH3=1.5 Hz, thymine-CH3), 1.44 (4H, m, J60 0 ,70 =6.0 Hz, J60 ,60 0 = photometer equipped with a Peltier temperature programmer
13.0 Hz, CH3 and H600 ). 13C NMR (125 MHz, CDCl3) δ 162.7 (C4), with the heating rate of 1 deg/min. Prior to measurements, the
148.8 (C2), 136.1, 135.1 (2  Cipso-Bn), 135.0 (C6), 127.7-127.0 samples (1 μM of AON and 1 μM cDNA or RNA mixture) were
(aromatic), 108.3 (C5), 84.9 (C10 ), 82.6 (C40 ), 74.7 (C30 ), 73.1, 72.6 preannealed by heating to 80 °C for 5 min followed by slow
(2  CH2Bn), 71.2 (C80 ), 69.1 (C50 ), 49.3 (C20 ), 32.2 (C70 ), 26.6 (80 - cooling to 21 °C and 30 min of equilibration at this temperature.
CH3), 23.4 (C60 ), 10.9 (CH3-thymine). MALDI-TOF m/z [C28H32- The value of Tm is the average of two or three independent
N2O6 þ H]þ found 493.235, calcd 493.234. measurements. If error of the first two measurements is >(0.3 °C,

J. Org. Chem. Vol. 75, No. 21, 2010 7127

JOC Article Liu et al.

the third measurement was carried out to check if the error is presence of 0.08 U E. coli RNase H (obtained from USB
indeed within (0.3 °C, otherwise it is repeated again. corporation, Cleveland, Ohio). Prior to the addition of the
32
P Labeling of Oligonucleotides. The oligoribonucleotides enzyme, reaction components were preannealed in the reaction
and oligodeoxyribonucleotides were 50 -end labeled with 32P, buffer by heating at 80 °C for 5 min followed by slow cooling to
using T4 polynucleotide kinase, [γ-32P]ATP, and the standard 21 °C and 30 min equilibration at this temperature. Total
protocol. Labeled AONs and the target RNA were purified by reaction volume was 30 μL. Aliquots of 3 μL were removed
QIAquick Nucleotide Removal Kit, and specific activities were after 5, 10, 15, 30, and 60 min, and the reactions were terminated
measured with a Beckman LS 3801 counter. by mixing with stop solution [containing 0.05 M EDTA, 0.05%
SVPDE Degradation Studies. Stability of the AONs toward (w/v) bromophenol blue, and 0.05% (w/v) xylene cyanole in
30 -exonucleases was tested by using phosphodiesterase I from 80% formamide]. The samples were subjected to 20% 7 M urea
Crotalus adamanteus (obtained from USB corporation, Cleveland, PAGE and visualized by autoradiography. Pseudo-first-order
Ohio). All reactions were performed at 3 μM DNA concentra- reaction rates could be obtained by fitting the digestion curves to
tion (50 -end 32P labeled with specific activity 80 000 cpm) in single-exponential decay functions.
100 mM Tris-HCl (pH 8.0) and 15 mM MgCl2 at 21 °C. An
exonuclease concentration of 6.7 ng/μL was used for digestion of Acknowledgment. Generous financial support from the
oligonucleotides. Total reaction volume was 30 μL. Aliquots Swedish Natural Science Research Council (Vetenskapsradet),
(3 μL) were taken at proper time points and quenched by the Swedish Foundation for Strategic Research (Stiftelsen f€
or
addition of stop solution (4 μL) [containing 0.05 M EDTA,
Strategisk Forskning), and the EU-FP6 funded RIGHT project
0.05% (w/v) bromophenol blue, and 0.05% (w/v) xylene cyanole
in 80% formamide]. Reaction progress was monitored by 20% (Project no. LSHB-CT-2004-005276) is gratefully acknowl-
denaturing (7 M urea) PAGE and autoradiography. edged. Jianfeng Xu and Mansoureh Karimiahmadabadi have
The total percentage of integrated AONs (13- and 14mer) was equally contributed in this work.
plotted against time points to give the digestion curve, and the
1
pseudo-first-order reaction rate could be obtained by fitting Supporting Information Available: H, 13C, 31P, COSY,
curves to single-exponential decay functions. HMQC, and HMBC of all carba-ENA derivatives (SI part I); 1D
Stability Studies in Human Blood Serum. AONs at 2 μM con- NOE spectra of key intermediates 14, 20a, 21b, 26, and 28,
centration (50 -end 32P labeled with specific activity 80 000 cpm) were decoupling experiments of key intermediates 14, 16b, and 21a/b
incubated in 10 μL of human blood serum (male AB, obtained from (SI part II); denaturing PAGE analysis of the SVPDE degradation
Sigma-aldrich) at 21 °C (total reaction volume was 36 μL). Aliquots of AONs with modified carba-LNA analogues; discussion about
(3 μL) were taken at proper time points and quenched with 4 μL of stability of carba-ENA analogues-modified AONs in blood serum;
stop solution [containing 0.05 M EDTA, 0.05% (w/v) bromophenol autoradiograms of 20% denaturing PAGE as well as degradation
blue, and 0.05% (w/v) xylene cyanole in 80% formamide], resolved curves, showing the cleavage kinetics of target RNA in AON:RNA
in 20% polyacrylamide denaturing (7 M urea) gel electrophoresis, hybrid duplexes by E. coli RNase H1; all individual reaction steps,
and visualized by autoradiography. workup, and product characterization for the intermediates
RNase H Digestion Assay. Target 0.1 μM RNA (specific 21b, 22a/b, 25, 28, and 29, ENA and the characterization for
activity 80 000 cpm) and AON (2 μM) were incubated in a final amidites 18a/b, 30a/b, 31, and 32 (SI part II). This
buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10 material is available free of charge via the Internet at http://
mM MgCl2, 0.1 mM EDTA, and 0.1 mM DTT at 21 °C in the pubs.acs.org.

7128 J. Org. Chem. Vol. 75, No. 21, 2010