Synthesis of 2′,4′-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

pubs.acs.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

jyoti@boc.uu.se

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 C8⁰substituents (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 C8⁰substituent to variable extents depending on not only the chemical nature but also

the stereochemical orientation of the C8⁰substituents: Thus, (1) 8⁰S-Me on carba-ENA increases the

nucleolytic stability but 8⁰R-Me leads to a decreased effect; (2) 8⁰R-OH on carba-ENA had little, if any,

effect on nuclease resistance but 8⁰S-OH resulted in significantly decreased nucleolytic stability; and (3) 8⁰-

NH₂substituted 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

2010 American Chemical Society

r

Liu et al.

JOCArticle

Introduction

moiety, such as ENA,¹¹aza-ENA,¹⁵PrNA,¹¹BNA^{COC 16}

BNA^{NC 17}

, and

.

Oligonucleotides with high affinity and specificity toward

complementary DNA or RNA through Watson-Crick hybridiza-

tion can be potentially used as diagnostic and therapeutic

agents via several different machineries, such as antisense,¹

ribozymes,²small interfering RNA (siRNA),³micro-

RNA (miRNA),⁴and triple helix-forming oligonucleotides

(TFOs).⁵In the past two decades, the effort to develop

AON with striking target affinity and biological stability

has resulted in the synthesis of a large number of chemically

modified nucleotides, among which the locked nucleic acid

(LNA) has attracted extensive attention.⁶The 2⁰,4⁰-linkage

in LNA is able to lock the conformation of the sugar moiety

into a perfect N-type conformation.⁷When LNA monomer

was incorporated into oligonucleotide, it can spontane-

ously tune its neighboring natural nucleotides from S- to

N-conformation, resulting in an A-type or A-type-like duplex

form consistent with the predominant form of the RNA helix.⁸

Hence, LNA-modified AONs exhibit remarkable sequence

selectivity for the complementary RNA over the DNA.⁶

Though the striking feature of LNA-modified oligos has

given immense opportunity to a broad application in bio-

technology and therapeutics,⁹it is clear that further devel-

opment is required, due to the low nuclease resistance and

hepatotoxicity of LNA.^10,11Thus large numbers of chemi-

cally modified analogues with 2⁰,4⁰-linkage have been de-

signed and synthesized.¹²Some of the modifications focus on

introduction of different functions at the C6⁰in LNA.

For example, Nielsen¹³and Swayze¹⁴have independently

reported C6⁰-CH₂OH-LNA and C6⁰-Me-LNA (cEt), C6⁰-

CH₂OMe-LNA (cMOE), respectively. The cEt and cMOE

modified oligonucleotides showed improved exonucleolytic

resistance without loss of hybridization behavior compared

to the LNA-modified counterpart. Another type of modi-

fication focuses on expanding ring size or introducing

heteroatoms into different positions on the locked ring

Replacement of the 2⁰-O- in LNA with 2⁰-CH₂- results in a

more hydrophobically locked carba-LNA nucleoside, which

has been synthesized recently by us through a radical cycliza-

tion strategy.^18-20The advantage of carba-LNA is that

carba-LNA-modified AONs exhibit similar hybridizing abil-

ity for the complementary RNA as that of LNA counter-

parts, but the former showed significantly improved

nucleolytic resistance than the latter. Moreover, we found

that the nucleolytic resistance and hybridizing ability of

carba-LNA can be finely modulated by introduction of

hydrophobic methyl, hydrophilic hydroxyl, or zwitterionic

amino functions at C6⁰and/or C7⁰of the carbocyclic moiety.

This modulation was found to be greatly dependent on not

only the nature but also the steric orientation of the substit-

uents at the chiral C6⁰and/or C7⁰position of the carbocyclic

moiety in the carba-LNA.

Carba-ENA that contains a 2⁰,4⁰-propyl linkage has also

been synthesized by us through a radical cyclization re-

action²¹and also independently by Nielsen’s group through

a ring-closing metathesis.^22,23Compared with carba-LNA,

carba-ENA has a sterically larger and more hydrophobically

carbocyclic linkage. This structural feature has rendered

carba-ENA incorporated oligos with more improved

nucleolytic resistance than those of carba-LNA counterparts.

However, none of these works reported any studies on the

nuclease stability of parent carba-ENA-modified AONs. In the

present study, we report a new synthetic strategy for carba-

ENA-T and study the nucleolytic stability as well as other

antisense properties of carba-ENA-modified AONs.

In addition, C6⁰- and/or C7⁰-substitued carba-ENA ana-

logues have been synthesized by Nielsen’s group²³and us¹⁸

independently. Our work showed that the C6⁰substituent on

carba-ENA exerted a significant effect on nucleolytic stabi-

lity and the effect depends on the nature of the substitu-

ents. Nielsen’s work showed hydrophilic substituents at

C6⁰and/or C7⁰led to increased affinity for complementary

RNA and DNA. Compared to C6⁰and C7⁰substituents on

carba-ENA, the C8⁰substituent should be more interesting

because a C8⁰substituent is located at the bottom of the minor

groove, hence it should have a larger effect on thermodynamic

stability and other properties of AON:RNA and AON:DNA

duplexes. In this report, we also describe the synthesis of

8⁰-NH₂/OH/Me-substituted carba-ENA analogues in detail.

Biophysical and biological properties such as target affinity

toward complementary DNA and RNA, nucleolytic stability,

and the RNase H recruitment capability of AONs with these

8⁰-substituted carba-ENAs modifications have been tested and

(1) (a) Bennett, C. F.; Swayze, E. E. Annu. Rev. Pharmacol. Toxicol. 2010,

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.

(3) de Fougerolles, A.; Vornlocher, H. P.; Maraganore, J.; Lieberman, J.

Nat. Rev. Drug Discovery 2007, 6, 443–453.

(4) Bartel, D. P. Cell 2004, 116, 281–297.

(5) Buchini, S.; Leumann, C. J. Curr. Opin. Chem. Biol. 2003, 7, 717–726.

(6) (a) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;

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.

Commun. 1998, 455–456. (c) Kaur, H.; Babu, B. R.; Maiti, S. Chem. Rev.

2007, 107, 4672–4697. (d) Lebreton, J.; Escudier, J. M.; Arzel, L.; Len, C.

Chem. Rev. 2010, 110, 3371–3418.

(7) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.;

Imanishi, T Tetrahedron Lett. 1997, 38, 8735–8738.

(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.

(10) Swayze, E. E.; Siwkowski, A. M.; Wancewicz, E. V.; Migawa, M. T.;

Wyrzykiewicz, T. K.; Hung, G.; Monia, B. P.; Bennett, C. F. Nucleic Acids

Res. 2007, 35, 687–700.

(11) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.;

Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003,

11, 2211–2226.

(12) Zhou, C.; Chattopadhyaya, J. Curr. Opin. Drug Discovery Dev. 2009,

12, 876–898.

(13) Enderlin, G.; Nielsen, P. J. Org. Chem. 2008, 73, 6891–6894.

(14) Seth, P. P.; Vasquez, G.; Allerson, C. A.; Berdeja, A.; Gaus, H.;

Kinberger, G. A.; Prakash, T. P.; Migawa, M. T.; Bhat, B.; Swayze, E. E.

J. Org. Chem. 2010, 75, 1569–1581.

(16) Hari, Y.; Obika, S.; Ohnishi, R.; Eguchi, K.; Osaki, T.; Ohishi, H.;

Imanishi, T. Bioorg. Med. Chem. 2006, 14, 1029–1038.

(17) Abdur Rahman, S. M.; Seki, S.; Obika, S.; Yoshikawa, H.; Miyashita,

K.; Imanishi, T. J. Am. Chem. Soc. 2008, 130, 4886–4896.

(18) Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych, O.;

Chattopadhyaya, J. J. Org. Chem. 2009, 74, 118–134.

(19) Srivastava, P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Wenska,

M.; Chattopadhyaya, J. J. Am. Chem. Soc. 2007, 129, 8362–8379.

(20) Xu, J.; Liu, Y.; Dupouy, C.; Chattopadhyaya, J. J. Org. Chem. 2009,

74, 6534–6554.

(21) Zhou, C.; Plashkevych, O.; Chattopadhyaya, J. Org. Biomol. Chem.

2008, 6, 4627–4633.

(22) Albæk, N.; Petersen, M.; Nielsen, P. J. Org. Chem. 2006, 71, 7731–

7740.

(23) Kumar, S.; Hansen, M. H.; Albæk, N.; Steffansen, S. I.; Petersen, M.;

Nielsen, P. J. Org. Chem. 2009, 74, 6756–6769.

(15) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.;

Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128,

15173–15187.

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

Liu et al.

JOCArticle

SCHEME 1. Retrosynthetic Analysis to C8⁰-Modified Carba-ENA

Thymidine

phenyl chlorothioformate in pyridine, the key precursor for

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,²⁷thus 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,^28,29the radi-

cal reaction proceeded in the 6-exo cyclization mode to afford

the sole product 14 in 60% yield. Meanwhile 1D NOE

experiments proved that the configuration of C8⁰was

R stereochemistry. Subsequently, selective debenzylation of

8⁰-N-OBn was achieved with 10% Pd/C and ammonium formate

as hydrogenolysis reagent atroom temperatureto produce the

highly polar product 15 without any loss of 3⁰- and 5⁰-O-

benzyl groups. The effort to protect 8⁰-amino with ethyl

trifluoroacetate²⁰was unsuccessful because of very low yield;

merely about 20% even when 20 equiv of the reagent was

added. Instead, the more reactive trifluoroacetic anhydride³⁰

was found to be a superior choice: Treatment of 15 with

trifluoroacetic anhydride in pyridine and CH₂Cl₂gave the

expected compound 16a in 60% yield. The structural integrity

of the product was confirmed through ¹³C NMR experiment,

in which two typical quartet peaks around 110 and 150 ppm

for the trifluoroacetyl group (TFA) were observed. Then 16a

was subjected to debenzylation involving 20% Pd(OH)₂/C

and cyclohexene as hydrogen donor at reflux and subsequent

5⁰-dimethoxytritylation to give the corresponding product 17a.

Unfortunately, significant loss of TFA during debenzylation has

been observed, resulting in lower yield (30%) from 16a to 17a.

To overcome this problem, the phenoxyacetyl group (PAC) was

employed as a more stable protective group to afford 16b,³¹

which can be debenzylated completely under the same condition

without any acylamino cleavage, followed by 5⁰-dimethoxytri-

tylation to acquire 17b in 72% yield. Phosphitylation of 17a and

17b with 2-cyanoethyl N,N-diisopropylphosphoramidochlori-

dite was achieved to give the desired amidites 18a and 18b as a

diastereomeric mixture in 93% and 70% yield, respectively.

1.2. Synthesis of Carba-ENA-T 25, 8⁰-OH-Carba-ENA-T

22a/b, and 8⁰-Me-Carba-ENA-T 29 Starting from 8⁰-Amino-

Carba-ENA-T 15. Transformation of the 8⁰-amino to ketone

(15 f 19) function was a key step for the synthesis of carba-

ENA-T 25, 8⁰-OH-carba-ENA-T 22a/b, and 8⁰-Me-carba-

ENA-T 29 starting from 8⁰-amino-carba-ENA-T 15 (Scheme 3).

Oxidation of benzyloxyamine by MCPBA to oxime followed

by treatment with Dess-Martin periodinane reagent has been

shown in the previous study^20,32to be an efficient strategy for

compared with those of AON with carba-ENA modification.

On the basis of the results provided, we show how the nature

and stereochemical orientation of C8⁰substituents on carba-

ENA modulate the biophysical and biological properties of

modified AONs, which, in turn, we believe will enable us to

design more promising nucleoside analogues as potential nucleic

acids-based therapeutic or diagnostic reagents.

Results and Discussion

1. Synthesis of 8⁰-Modified-Carba-ENA Thymidine Ana-

logues. As shown in the retrosynthetic analysis in Scheme 1,

the C8⁰-modified carba-ENA thymidine analogues can readily

be accomplished exploiting cyclic ketone 2 that was synthesized

from 8⁰-amino-carba-ENA thymidine 3, which we obtained

through free-radical cyclization of the oxime ether 4. The oxime

ether function can be introduced starting from nitrile 5, which in

turn can be easily obtained from known sugar precursor 6¹⁹by a

simple nucleophilic substituent with cyanide ion.²⁴

1.1. Synthesis of 8⁰-Amino-Carba-ENA Thymidine (15).

The synthetic route to 8⁰-amino-carba-ENA thymidine 15

and its phosphoramdite 18a/18b is shown in Scheme 2.

Treatment of the known sugar precursor 6¹⁹with tosyl

chloride, triethylamine, and DMAP in dichlormethane fur-

nished tosylate 7.²⁴After workup, the crude tosylate was

directly subjected to substitution with tetrabutylammonium

cyanide (TBACN) in DMF to afford the desired 4-C-cya-

noethyl-^D-ribofuranose 8 in an overall yield of 77% from 6.²⁵

Compound 8 was treated with a mixture of acetic acid, acetic

anhydride, and triflic acid at 10 °C for 1 h to give diacetate 9 as

an anomeric mixture. Glycosylation of the crude 9 provided

exclusively β-configured thymine nucleoside 10 through a

€

modified Vorbruggen-type reaction in 85% yield in two

steps.¹⁵Deacetylation of the 2⁰-O-acetyl group was achieved

by treating compound 10 with 30% methylamine solution in

ethanol at room temperature to furnish the desired product 11

in 94% yield. Then the nitrile group of 11 was reduced with

diisobutylaluminum hydride (DIBALH) at -78 °C for 2 h to

the corresponding aldehyde, which was subsequently sub-

jected to oximation with O-benzylhydroxylamine to provide

O-benzyl oxime 12 as a mixture of E and Z isomers in 61%

yield.²⁶After esterification of 2⁰-OH in nucleoside 12 with

(27) Miyabe, H.; Torieda, M.; Inoue, K.; Tajiri, K.; Kiguchi, T.; Naito, T.

J. Org. Chem. 1998, 63, 4397–4407.

(28) Marco-Contelles, J.; Martinez-Grau, A.; Martinez-Ripoll, M.;

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.

(30) Wenska, M.; Honcharenko, D.; Pathmasiri, W.; Chattopadhyaya, J.

Heterocycles 2007, 73, 303–324.

(31) Honcharenko, D.; Varghese, O. P.; Plashkevych, O.; Barman, J.;

Chattopadhyaya, J. J. Org. Chem. 2006, 71, 299–314.

(32) Simpkins, N. S.; Stokes, S.; Whittle, A. J. J. Chem. Soc., Perkin

Trans. I 1992, 2471–2477.

(24) Morita, K.; Takagi, M.; Hasegawa, C.; Kaneko, M.; Tsutsumi, S.;

Sone, J.; Ishikawa, T.; Imanishi, T.; Koizumi, M. Bioorg. Med. Chem. 2003,

11, 2211–2226.

˚

(25) Akerfeldt, K. S.; Bartlett, P. A. J. Org. Chem. 1991, 56, 7133–7144.

(26) Kumar, V.; Gauniyal, M. H.; Shaw, K. A. Tetrahedron: Asymmetry

2007, 18, 2069–2078.

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

Liu et al.

JOCArticle

SCHEME 2. Synthesis of 8⁰-Amino-Carba-ENA Thymidine^a

^aReagents 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; BnONH₂HCl, pyridine, CH₂Cl₂, reflux, 1 h, 61% in two steps; (g) phenyl chlorothionoformate, pyridine, rt,

3

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-

amine 14 by MCPBA only gave the corresponding oxime in

20% yield. Oxidative deamination³³is another strategy for

transformation of the second amino to ketone directly. Thus,

different oxidative deamination reagents such as aqueous

hypochlorite,³⁴trichloroisocyanuric acid,³⁵and copper-

ascorbic acid dyad³⁶have been tried but neither worked in

our case. Instead, 3,5-di-tert-butyl-1,2-benzoquinone was

found to be a satisfactory oxidative deamination reagent,

and it efficaciously oxidized amine 15 to ketone 19 in an

acceptable yield.³⁷Reduction of 19 with NaBH₄furnished

two diastereomeric products 20a (69%) and 20b (19%). The

major product 20a has 8⁰S stereochemistry, suggesting that

the hydride attack took place from the back of the carbo-

cycle. Clearly, the hydride attack from the back is much more

favored than the attack from the front of carbocycle, most

likely owing to steric hindrance of the bulky 3⁰-O-benzyl

group in the front face. After protection of free 8⁰-OH in 20a

and 20b with the p-toluoyl group, the obtained compounds

21a (90%) and 21b (89%) were subjected to debenzylation

followed by selective 5⁰-dimethoxytritylation to give 22a (63%)

and 22b (83%), respectively, in satisfactory yields. Special care

was taken for the debenzylation of 21a because thermodynami-

cally favored migration of the 8⁰-O-toluoyl group to the

3⁰-hydroxyl group in 21a is possible owing to their cis orientation

in a spatial proximity. Thus amount of hydrogenolysis reagent

was increased (20% Pd(OH)₂/C, 1500 mg/mmol substrate and

ammonium formate, 60 equiv) and reaction time was shortened

for debenzylation of 21a,¹⁸under which the benzyl groups were

completely removed without cleavage and migration of the

toluoyl group. Due to steric hindrance of the 8⁰-O-toluoyl group,

3⁰-O-phosphitylation of 22a with 2-cyanoethyl N,N-diisopropyl-

phosphoramidochloridite was allowed to proceed overnight to

afford the resulting phosphoramidite 30a in 71% yield, whereas

the phosphitylation of 22b under the same condition furnished

phosphoramidite 30b in 80% yield in only 3 h.

Starting from intermediate 20a, parent carba-ENA-T 25

has been synthesized through radical deoxygenation of the

8⁰-OH in 20a (Scheme 3). Considering the steric proximity

between 3⁰-O-benzyl and 8⁰-hydroxyl groups in 20a, the more

reactive and smaller (methylthio)thiocarbonate was chosen

as the radical-generating group.³⁸Thus, 20a was converted

through the three-component reaction to radical precursor

23 (53%), which was subjected to the standard radical

deoxygenation procedure²⁰to give the expected parent carba-

ENA thymidine 24 in 74% yield.

(33) (a) Neumann, R.; Levin, M. J. Org. Chem. 1991, 56, 5707–5710.

(b) Barman, D. C.; Saikia, P.; Prajapati, D.; Sandhu, J. S. Synth. Commun.

2002, 32, 3407–3412. (c) Richey, H. G., Jr.; Erickson, W. F. J. Org. Chem.

1983, 48, 4349–4357.

(34) Lee, G. A.; Freedman, H. H. Tetrahedron Lett. 1976, 20, 1641–1644.

(35) Luca, L. D.; Giacomelli, G. Synlett 2004, 12, 2180–2184.

(36) Srogl, J.; Voltrova, S. Org. Lett. 2009, 11, 843–845.

(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

Liu et al.

JOCArticle

SCHEME 3. Synthesis of Carba-ENA Thymidine (24) and Its Analouges^a

^aReagents 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 C8⁰substitu-

ent, 8⁰-Me-carba-ENA-T 29, has been synthesized starting

from key intermediate ketone 19 by a known strategy.¹⁸

Instead of methyl magnesium iodide,³⁹treatment of ketone

19 with methyl lithium in THF for 8 h furnished the sole

product 26 (58%),⁴⁰in which the 8⁰S-configuration was

confirmed by 1D NOE experiment. Hence, the methyl anion

attacked the ketone exclusively from the back of the carbo-

cycle, which is consistent with the stereoselective reduction

on ketone 19 by NaBH₄. To remove the 8⁰-hydroxyl group,

intermediate 26 reacted with methyl(chlorocarbonyl)-

formate in pyridine at 40 °C to give 27 in 87% yield,⁴¹which

was subjected to a standard radical deoxygenation to give two

inseparable diastereomers 28 (8⁰S:8⁰R = 5:4) in 87% yield.

Relative lack of stereoselectivity during the free-radical reaction is

likely to be due to higher reaction temperature. To provide appro-

priately protected building blocks for standard automated solid-

phase oligonucleotide synthesis by using the phosphoramidite

approach, nucleosides 24 and 28 were debenzylated followed by

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

(84%) and 32 (76%) under standard condition.

(39) Kakefuda, A.; Yoshimura, Y.; Sasaki, T.; Matsuda, A. Tetrahedron

1993, 49, 8513–8528.

1.3. NMR Characterization of Key Intermediates Involved

in the Synthesis of the 8⁰-Functionalized Carba-ENA Thymi-

dines. Structures of all intermediates and final products were

(40) Matsuda, A.; Itoh, H.; Takenuki, K.; Sasaki, T.; Ueda, T. Chem.

Pharm. Bull. 1988, 36, 945–953.

(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.;

Chapuis, J. J. Nat. Prod. 2007, 70, 417–422.

1

confirmed by H, ¹³C, COSY, H-¹³C HMQC and HMBC

7116 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.

JOCArticle

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).

The ¹H NMR spectra of all carba-ENA derivatives showed a

singlet for H1⁰as found for all 2⁰,4⁰-fused bicyclic nucleosides,

which strongly suggested the North-type sugar pucker.⁴²

For compound 14, the correlation between H2⁰with H8⁰in

COSY NMR spectra gave evidence to the bicyclic system

formed during the free-radical cyclization, which is further

consolidated by theobservation of the long-rangecorrelations

between H2⁰with C7⁰and between H1⁰with C8⁰in HMBC

NMR spectra. In the 1D NOE experiment, irradiation of H1⁰

led to NOE enhancement for 8⁰-NH (0.7%), H2⁰(1.8%), and

H7⁰⁰(3.9%), but none for H8⁰(Figure 1), which identified C8⁰

in the 8⁰R-configuration.

The orientation of C8⁰substituents in intermediates 20a,

20b, 26, and 28 has been assigned by 1D NOE experiments.

In compound 20a, the strong enhancement for H8⁰(3.1%)

and H7⁰(3.5%) was observed upon irradiation of H1⁰,

confirming the S-configuration for C8⁰. Severe overlap of

signals of H8⁰and H3⁰in compound 20b made it hard to

determine the configuration of C8⁰by 1D NOE experiments

at this stage. The configuration of C8⁰in 20b, however, could

be assigned to the R-configuration since compound 21b has

been found to have the C8⁰R-configuration: irradiation of

H8⁰in 21b resulted in the enhancement for H2⁰(3.1%) and

H7⁰(2.7%), but not for H1⁰. For compound 26, the strong

NOE enhancement for the 8⁰-methyl group (5.9%) and

H7⁰(4.2%) upon irradiation of H1⁰clearly indicated the

S-configuration for C8⁰. By comparing the observation that

irradiation on H1⁰in the major product of 28 caused

remarkable enhancement for H8⁰(4.1%) and none for

8⁰-Me, in contradistinction to the minor product of 28, which

led to notable NOE enhancement for 8⁰-Me (3.6%) but none

for H8⁰, it was concluded that the C8⁰is S in the major

product, whereas R in the minor product.

On the other hand, the coupling constants also have been

used to identify the configuration of chiral center in these

derivatives. The six-membered carbocyclic ring of carba-

ENA adopts a typical chair conformation.^18,22Through

decoupling experiments, the observation of a small coupling

constant (∼6 Hz) between H8⁰and axial H7⁰and a large one

(∼11 Hz) between H8⁰and equatorial H7⁰⁰in 16b and 21b

implied H8⁰and equatorial H7⁰have torsion angles in gauche

but that of H8⁰and axial H7⁰⁰in trans, according with the

(42) Obika, S.; Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, V. K.; Sing,

S. K.; Wengel, J. J. Am. Chem. Soc. 1998, 120, 13252–13253.

3

8⁰R-configuration. On the contrary, the J_{H7 H8}in 17a is 0 Hz,

00

0

J. Org. Chem. Vol. 75, No. 21, 2010 7117

Liu et al.

JOCArticle

SCHEME 4. Mechanism of Radical Cyclization Reaction

tides.^44,45Thus, 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 (T_m)

values of duplex formed by AONs 2-33 with the complemen-

tary DNA or RNA were measured, and their ΔT_mvalues

provided by comparing with the native AON 1 were listed in

Table 1. The ΔT_mvalues of AON 26-29 that have been

reported previously¹⁸are also listed in Table 1 for comparison.

3.1. Comparison of Carba-ENA (Type I) Modification with

Native Counterpart and ENA (Type VIII) Modification.

Albæk et al. reported that the ΔT_mvalue of duplexes formed

by parent carba-ENA-U with complementary RNA was

þ4.5 °C,²²whereas our present result showed that one parent

carba-ENA-T modification in AON:RNA duplex resulted in

an increase of 1.4 °C in T_m. The notable disparity between the

exact ΔT_mvalue obtained by us and those by Albæk et al. is

likely due to the sequence difference.

One ENA (type VIII) modification in the AON strand

resulted in a T_mincrease of 3.6 °C for the AON:RNA duplex,

which is a much higher stabilization effect than the parent

carba-ENA modification (þ1.4 °C/modification). We have

previously reported²⁰that in the same AON sequence, LNA

modification (þ4.5 °C/modification) also led to much higher

T_mincrease for the AON:RNA duplex than the parent

carba-LNA modification (þ3.6 °C/modification). Hence, it

seems that the 2⁰-oxygen on the bicyclic system plays an

important role in the thermal stability of the AON:RNA

duplex. One of the plausible explanations for the role of

2⁰-oxygen could be that the 2⁰-oxygen is engaged in the

hydrogen bonding to water molecules, whereas it is well-

known⁴⁶the extensive hydration network in the minor groove

can reduce the electrostatic repulsion of the internucleotidic

phosphates, thus stabilizing the AON:RNA duplex. Although

this 2⁰-oxygen in LNA or ENA incorporated oligos has a T_m

increase effect with complementary RNA, it, however, reduces

the nucleolytic stability considerably compared to their carbo-

cyclic counterparts (see section 4).

which suggested the H8⁰and axial H7⁰⁰is nearly 90°, Hence C8⁰

has an S stereochemistry (Figures SII 8-13, Supporting

Information). The results obtained from decoupling experiments

conformed to those from 1D NOE experiments.

1.4. Mechanism of the Ring-Closure Reaction. During

radical mediated 6-exo cyclization of compound 13, the in

situ generated C2⁰carbon radical could attack the oxime

intramolecularly through both faces of CdN, forming two

possible transition states TS1 and TS2 (Scheme 4). 1,3-

Diaxial disposition between the forming bulky benzyloxya-

mino substituent and 3⁰-O-benzyl group in TS 1 is energeti-

cally disfavored. In contrast, the benzyloxyamino group in

TS 2 points equatorially, leading to minimal steric repulsion

between C8⁰and C3⁰substituents. As a result, compound 14

that has R stereochemistry at chiral C8⁰has been observed as

the exclusive product.

2. Synthesis and Purification of Oligonucleotides Contain-

ing 8⁰-Modified Carba-ENA Thymidine Analogues. Through

solid-phase DNA synthesis protocol⁴³on an automated

DNA/RNA synthesizer, the phosphoramidites 18b, 30a/b,

31, and 32 were incorporated as monosubstitution into a

15mer DNA sequence as used in our former studies.^18,20The

coupling time of building blocks 18b, 30b, 31, and 32 was about

10 min, whereas for 30a the coupling time was prolonged to

15 min, in view of steric obstruction by the 8⁰-O-toluoyl group

during the coupling. The satisfactory coupling yields (40-75%)

were obtained for all compounds. The sequences, modification

site, and structure of modification are shown in Table 1. For

AONs 2-9 the treatment with 33% aqueous ammonia at room

temperature overnight was carried out to cleave oligos from the

solid support as well as for the removal of the protecting groups.

On the other hand, considering the stability of PAC and Tol

groups, the solid support for AONs 10-17 and 18-21 was

incubated in aqueous ammonia at 55 °C for 1 to 3 days.

Unfortunately, the PAC groups in AONs 18-21 are too stable

to be removed. A stronger deprotection reagent such as 1 N

NaOH solution, however, led to degradation of oligonucleo-

3.2. Comparison of Type IV, Type VI, and Type VII with

Type I Modifications. One [8⁰R-OH]-carba-ENA (type IV),

[8⁰R-Me]-carba-ENA (type VII), and [8⁰R-NH₂]-carba-ENA

(type VI) modification in AON leads to T_mincrease for the

AON:RNA duplex around 0.6, 0.5, and 0.8 °C, respectively

(Table 1), which are slightly lower than the effect imposed by

the parent carba-ENA (type I) modification (þ1.4 °C/

modification). This observation indicates the C8⁰substitu-

ents, regardless of hydrophilic hydroxyl and amino group or

hydrophobic methyl group, destabilize the AON:RNA duplexes

slightly when they face away from the vicinal 3⁰-phosphate.

(43) Beaucage, S. L.; Caruthers, M. H. Current Protocols in Nucleic Acid

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,

1995.

(46) Tereshko, V; Gryaznov, S.; Egli, M. J. Am. Chem. Soc. 1998, 120,

269–283.

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

Liu et al.

JOCArticle

TABLE 1. Thermal Denaturation of Duplexes of Native and Carba-ENA Analogues-Modified AONs with Complementary DNA or RNA^a

^aA = 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). T_mvalues measured as the maximum of the first derivative of the melting curve (A₂₆₀nm 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 T_mgiven is the average of two or three independent measurements (the error of the three consecutive measurements is within (0.3 °C). ^bΔT_m

values were obtained by comparing the T_mvalues of modified AONs 2-33 with that of the native AON 1. The ΔT_m(aver) value obtained here is the

average value from four AONs incorporated with the same compound at four different modification sites. ^cΔΔT_m= (T_mof AON:RNA) - (T_mof AON:

DNA). ^dModifications of type VII have been taken from reported work,¹⁸and are used for T_mcomparison with our present series of modified AONs.

The less destabilizing effect of the 8⁰R-amino group than that

of the 8⁰R-methyl or the hydroxyl group is presumably

because in experimental buffer the amino group is partially

protonated, and can reduce charge repulsion between phos-

phates on opposition strands through partial charge neutra-

lization, which is a favorable contribution to the free energy

of duplex formation.^47,48

3.3. Comparison of Type III with Type IV Modification.

[8⁰S-OH]-carba-ENA (type III) modification did not result

in any T_mchange for the AON:RNA duplex, which is

contrary to the observation that one [8⁰R-OH]-carba-ENA

(type IV) modification led to a 0.6 °C increase in T_m. Hence,

when the 8⁰-OH group points toward the vicinal 3⁰-phos-

phate, it renders a more negative effect for target RNA

affinity than when it points away from the neighboring

3⁰-phosphate.

3.4. Comparison of Type II with Type VII Modification. It

seems the orientation of hydrophobic 8⁰-Me exerts little if

any effect on thermal stability of the AON:RNA duplex

since we found type II ([8⁰-56%S/44%R-Me]-carba-ENA)

and type VII ([8⁰R-Me]-carba-ENA) modification lead to very

similar T_mincrease, 0.5 and 0.3 °C/modification, respectively.

3.5. Comparison of Type V with Type VI Modification. To

investigate the influence of larger hydrophobic substituent in

the minor groove of the AON:RNA duplex on the thermo-

dynamic stability, the T_mof duplexes formed by [8⁰R-NHPAC]-

carba-ENA (type V)-modified AONs 18-21 with comple-

mentary RNA has also been measured. It was found that

[8⁰R-NHPAC]-carba-ENA (type V) modification renders a

€

(47) Cuenoud, B.; Casset, F.; Husken, D.; Natt, F.; Wolf, R. M.;

Altmann, K. H.; Martin, P.; Moser, H. E. Angew. Chem., Int. Ed. 1998, 37,

1288–1291.

€

(48) Prakash, T. P.; Puschi, A.; Lesnik, E.; Mohan, V.; Tereshko, V.; Egli,

M.; Manoharan, M. Org. Lett. 2004, 6, 1971–1974.

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

Liu et al.

JOCArticle

relatively higher T_mincrease than [8⁰R-NH₂]-carba-ENA

(type VI) modification (Table 1). This is an unusual observa-

tion, since the phenyl of PAC in type V modification is

expected to perturb the hydration network in the minor

groove, which we expected to result in a destabilization effect.

One likely explanation for this unusual observation is that the

phenyl group may intercalate into the hybrid duplex.

were taken out at appropriate time intervals and analyzed by

20% denaturing PAGE. The gel pictures obtained upon

autoradiography are shown in Figure SII 14 (Supporting

Information). The [8⁰R-CH₃]-carba-ENA-modified AON 26,

[6⁰R-CH₃,8⁰R-CH₃]-carba-ENA-modified AON 34, and [6⁰S-

CH₃,8⁰R-CH₃]-carba-ENA-modified AON 35 that have been

reported previously¹⁸were also treated with SVPDE in parallel

for comparison purpose.

3.6. The Carba-ENA-Modified AONs Are Highly RNA-

Selective. a. Comparison of Carba-ENAs with Native. Unlike

in AON:RNA duplex, carba-ENAs modification in AON:

DNA led to significant T_mdecrease compared to the native

counterpart. Hence, carba-ENAs-modified AONs are more

RNA-selective than the native. The RNA selectivity of carba-

ENAs could be due to the fact that the DNA:DNA duplex has a

The native DNA (AON 1) did not display any 3⁰-exonuclease

resistance and was completely degraded within ∼4min, whereas

under the identical condition, all modified AONs exhibit im-

proved exonuclease resistance to variable extents. Due to modi-

fied nucleotides locating at position T13, the stability of both

phosphate P14 and P13 (see Figure SII 14 in the Supporting

Information for phopshate (P) numbering) has been remark-

ably improved toward 3⁰-exonuclease, so two bands corre-

sponding to 14mer and 13mer on PAGE pictures can be

observed. However, once the phosphate P13 is cleaved, AON

could be degraded to the monomer blocks very rapidly.

4.1. Relative Nuclease Resistance of Carba-ENA, ENA-

Modified AONs. Total percentages of integrated 14mer and

13mer AONs were plotted against time points to give

SVPDE digestion curves (Figure 2) for each AON, and

pseudo-first-order reaction rates were observed by fitting

the curves to single-exponential decay functions. The stabi-

lity of these AONs toward SVDPE incubation increases in

the following sequences: AON 1,ENA-modified AON 30 (k=

1.7704 ( 0.0812 h^-1) < [8⁰R-NHPAC]-carba-ENA-modified

AON 18 (k = 0.6761 ( 0.0386 h^-1) ≈ [8⁰S-OH]-carba-ENA-

modified AON 10 (k = 0.5131 ( 0.0530 h^-1) < [8⁰R-NH₂]-

carba-ENA-modified AON 22 (k = 0.3086 ( 0.0085 h^-1) <

[8⁰R-Me]-carba-ENA-modified AON 26 (k = 0.1249 ( 0.0042

h^-1) ≈ [8⁰R-OH]-carba-ENA-modified AON 14 (k = 0.0985 (

0.0027 h^-1) ≈ [8⁰R/S-Me]-carba-ENA-modified AON 6 (k=

0.0881 ( 0.0034 h^-1) ≈ carba-ENA-modified AON 2 (k =

0.0827 ( 0.0017 h^-1) < [6⁰S-Me-8⁰R-Me]-carba-ENA-modified

AON 35 (k = 0.0494 ( 0.0019 h^-1) ≈ [6⁰R-Me-8⁰R-Me]-carba-

ENA-modified AON 34 (k = 0.0401 ( 0.0030 h^-1).

The above comparison leads us to the following conclusions:

a. Replacement of 2⁰-Oxygen of ENA and LNA with the

-CH₂- Group Significantly Increases the Nuclease Resis-

tance. Parent carba-ENA-T (type I)-modified AON 2 was

about 20 times more stable than ENA-T-modified AON 30.

In our previous study, parent carba-LNA-T-modified AON

has also been found to be about 50 times nucleolytically more

stable than the LNA-T-modified counterpart. These obser-

vations highlighted the unique effect of replacement of

2⁰-oxygen with the -CH₂- group in improving the nuclease

resistance of vicinal phosphates.

b. 8⁰R-OH and 8⁰-Me Subsituents on Carba-ENA Only Have

a Marginal Effect on the Nuclease Resistance. This conclusion

can be confirmed by the fact that AON 6, 14, and 26 showed

very similar stability as parent carba-LNA-T-modified AON 2

(Figure 2).

˚

narrower minor groove (ca. 5-6A) than the DNA:RNA hybrid

(ca. 9-10 A).⁴⁹Hence, the carbocycle of carba-ENA, which is

˚

located at the minor groove, may cause more serious perturba-

tion in AON:DNA than in the AON:RNA duplex. As a result,

the T_mvalue of the carba-ENA-modified AON:DNA homo-

duplex was pronouncedly decreased compared to that of the

carba-ENA-modified AON:RNA heteroduplex.

b. Comparison of Carba-ENAs with ENA. The magnitude

of RNA selectivity [denoted by ΔΔT_m, ΔΔT_m= (T_mof

AON:RNA duplex) - (T_mof AON:DNA duplex)] of carba-

ENA modifications (types I, II, III, IV, and VII) is around 5-

6 °C/modification (Table 1), which is significantly higher than

that of ENA modification (ΔΔT_m= ∼3.5 °C/modification). By

analyzing the T_mdata, we found the higher RNA selectivity of

carba-ENA compared to ENA is because the carba-ENA

modifications result in much more significant T_mdecrease for

AON:DNA than the ENA modification (Table 1). Hence, it

seems 2⁰-oxygen is more important for the thermal stability of the

AON:DNA duplex than for the AON:RNA duplex.

c. Comparison of Parent Carba-ENA with Parent Carba-

LNA and LNA. It has been reported previously by us²⁰that

parent carba-LNA-modified AON (around 5 °C/modification)

are also more RNA selective than LNA-modified counter-

part (3-4 °C/modification). Hence, it seems the relative RNA

selectivity of parent carba-ENA, parent carba-LNA, ENA,

and LNA follows this rank: parent carba-ENA ≈ parent

carba-LNA > ENA ≈ LNA.

4. Engineering the 3⁰-Exonuclease Stability in the Carba-

ENA Analogues-Modified AONs. It is known that 3⁰-exonu-

clease SVPDE-mediated phosphate scission involves recog-

nition of the 3⁰-end nucleotide, followed by the attack by

threonine residue of the enzyme on the phosphate in line with

the 3⁰-O of the penultimate nucleotide, which subsequently

departs.^50,51The incorporation of modified nucleotides in

AONs may regulate the enzymatic resistance of the vicinal

phosphate. Here AONs 2, 6, 10, 14, 18, 22, and 30 with

different modifications at position T13 (position 3 from

3⁰-end, see Figure SII 14 in the Supporting Information) were

adopted to test how these modifications influence the 3⁰-

exonuclease resistance of the modified AONs. These AONs

(³²P-labeled at 5⁰-end) were incubated with phosphodiesterase

I from Crotalus adamanteus venom (SVPDE) [SVPDE

6.7 ng/μL, AON 3 μM, 100 mM Tris-HCl (pH 8.0), 15 mM

MgCl₂, total reaction volume was 30 μL] at 21 °C. Aliquots

c. 8⁰R-NH₂Substituent on Carba-ENA Significantly

Decreases the Stability. [8⁰R-NH₂]-carba-ENA-modified

AON 22 is around four times less stable than parent carba-

LNA-T-modified AON 2. Moreover, [8⁰R-NHPAC]-carba-

ENA-modified AON 18 was found to be even less stable then

AON 22.

(49) Gyi, J. I.; Lane, A. N.; Conn, G. L.; Brown, T. Biochemistry 1998, 37,

73–80.

(50) Culp, J. S.; Butler, L. G. Arch. Biochem. Biophys. 1986, 246, 245–249.

(51) Cleland, W. W.; Hengge, A. C. Chem. Rev. 2006, 106, 3252–3278.

d. 8⁰S Substituent Can Render Totally Different Effect on

the Nuclease Resistance Depending upon the Nature of the

7120 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.

JOCArticle

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. 8⁰S-OH substituent obviously destabilizes vicinal

phosphate toward 3⁰-exonuclease given the faster degradation

of [8⁰S-OH]-carba-ENA-modified AON 10, but 8⁰S-Me sub-

stituent leads to a stabilization effect because [8⁰R/S-Me]-

carba-ENA-modified AON 6 is more stable than [8⁰R-Me]-

carba-ENA-modified AON 26.

e. Substituent at C6⁰Is More Efficient than Substituent at

C8⁰To Modulate the Nuclease Resistance of Vicinal Phos-

phate. From Figure 2, we can see that the modified AONs 34

and 35 which contain the C6⁰-Me-carba-ENA modifications

showed the best stability among all the studied AONs. Given

the fact that C6⁰R-OH-carba-ENA-modified AON showed

the least nuclease stability among other substituted carba-

ENA counterparts,¹⁸it seems that nuclease resistance of carba-

ENA-modified AON is more sensitive to substitution at C6⁰than

at C8⁰. This is presumably because of the fact that the C6⁰

substituent is spatially closer to the phosphate than the C8⁰

substituent, and thus bestows greater effect during the phosphate

recognition process and hydrolysis by the nuclease.

4.2. The Stereochemical Orientation of the 8⁰Substituent

Influences the Exonuclease Resistance of the Vicinal Scissile

Phosphate. On the basis of the above comparison, we found the

nuclease recognition and subsequent hydrolysis effect imposed

by 8⁰-OH and 8⁰-Me is significantly dependent on their stere-

ochemical orientation. AON 6 in which 44% of 8⁰-Me are in the

S configuration was found to be more stable than AON 26 in

which all the 8⁰-Me are in the R configuration, suggesting when

8⁰-Me points at the 3⁰-phosphate (8⁰S), it leads to a more posi-

tive effect on the nuclease resistance of vicinal phosphate than

when it points away from the 3⁰-phosphate (8⁰R). This stero-

chemical effect is probably dictated by the spatial proximity of

the lipophilic 8⁰S methyl group to the 3⁰-phosphate compared

to that of the 8⁰R methyl group, thus sterically preventing the

binding and processing of the phosphate by SVPDE.

Compared to 8⁰-Me, 8⁰-OH on carba-ENA exhibits a

reverse stereochemical effect on the nuclease resistance:

8⁰S-OH-carba-ENA-modified AON 10 showed a more

dramatic decrease of exonuclease resistance than 8⁰R-OH-

carba-ENA-modified AON 14. The destabilization effect

caused by 8⁰S-OH on carba-ENA could be attributed to

the closure of 8⁰S-OH to vicinal 3⁰-phosphate. Therefore, the

8⁰S-OH could assist enzymatic cleavage of 3⁰-phosphate

(P14) by H-bonding interaction.⁵²An interesting observa-

tion for SVDPE-mediated digestion of AON 10 is that there

is one clear band corresponding to the 13mer formation

on the gel (Figure SII 14, Supporting Information), im-

plying that the 8⁰S-OH group can improve the stability of

5⁰-phosphate (P13), putatively by impairing the binding of

SVPDE to 5⁰-phosphate.⁵²It is obvious that the destabiliza-

tion effect on P14 surpasses the stabilization effect on P13.

Hence, 8⁰S-OH on carba-ENA makes modified AON less

resistant toward 3⁰-exonuclease.

4.3. The Electrostatic Effect of 8⁰Substituent Plays an

Important Role in Stability Modulation of the Vicinal Phos-

phate. 8⁰R-NH₂and 8⁰R-NHPAC on carba-ENA result in

a significant decrease in the stability of modified AONs.

This destabilization effect is likely attributed to the electro-

statics effect. The protonated 8⁰R-amino group, as a zwitterion,

(52) Zhou, C.; Chattopadhyaya, J. J. Org. Chem. 2010, 75, 2341–2349.

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

Liu et al.

JOCArticle

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

nucleotide, leading to an unfavorable contribution for enzymatic

resistance of the vicinal phosphate. On the other hand, it is

known that departure of the 3⁰-oxyanion is the rate-limiting step

for SVPDE-mediated phosphate cleavage.⁵²Under the experi-

mental condition, the positively charged 8⁰R-NH₂could exert an

induction effect for the stability of the developing 3⁰-oxyanion,

thus assisting it in its departure. As a result, the potential of the

scission of 3⁰O-P14 bond (Figures 2 and SII 14 in the Support-

ing Information) is increased. Once the amine was converted into

the amide in 8⁰R-NHPAC, which is a better electron-withdraw-

ing group than amino, the cleavage of 3⁰O-P14 should be

further promoted. This expectation is consistent with the result

that [8⁰R-NHPAC]-carba-ENA-modified AON 18 is even less

stable then [8⁰R-NH₂]-carba-ENA-modified AON 22.

In summary, the SVPDE resistance can be modulated not

only by the stereochemical orientation of the substituents on

carbocyclic moiety but also by their intrinsic electrostatic

effect. Careful introduction of an electron-donating substi-

tuent at the appropriate position of the carbocycle of the

carba-ENA or -LNA with correct stereochemistry seems to

be an efficient way to design oligonucleotides having high

3⁰-exonuclease resistance.

T nucleotide, toward the 3⁰-end of the RNA strand. If A8, the

preferred cleavage site for native AON 1:RNA hybrid, is

located within the suppressed region, the major cleavage site

shifts to the edges of this region (Figure 3). This cleavage

pattern is very similar as that of AON:RNA hybrids contain-

ing carba-LNA¹⁹or aza-ENA¹⁵modifications, so one can

effectively engineer a cleavage at a specific site in the RNA

strand by properly choosing the position of modification in

the complementary antisense strand.⁵³Interestingly, this site-

specific RNA cleavage property is akin to the hallmark of

RNA cleavage in the RNA catalysis.

The RNase H-promoted cleavage rates were determined

by autoradiography of gels and subsequently plotting the

intact RNA fraction as a function of time (Figure SII 15,

Supporting Information). Fitting the degradation curve to

single-exponential decay functions gave the digestive rates

that are shown as bar plots in Figure 4. It was found that the

RNA component in all modified AON:RNA hybrids can be

degraded by RNase H with similar or even with better

efficiency as the digestion rate of the native counterpart.

Comparison of the cleavage rates of the modified AON:

RNA hybrid can be summarized in the following conclu-

sions:

5. RNase H Digestion Study of the Duplexes Formed by

Carba-ENA Analogues-Modified AONs with Complementary

RNA. In antisense strategy, RNase H recruitment is a very

important property when the modified oligonucleotides are

bound to the target RNA in heteroduplex form. So the

RNase H-mediated cleavage of the duplexes formed by all

modified AON 2-33 with complement RNA was studied,

applying native DNA (AON 1) as a reference. As the

autoradiographs indicated (Figure SII 15, Supporting

Information), all modified AON:RNA hybrids were excel-

lent substrates for RNase H1; however, the cleavage pattern

changes, as the site of incorporation of 8⁰-modified carba-

ENA analogues changes, regardless of the modification type

of the substituent. In general, the RNase H-elicited cleavage

activity (degrading the complementary RNA in the AON:

RNA hybrid duplex) was suppressed within a 4-5 base pairs

long region that starts from the base opposite to the modified

(i) ENA (type VIII)-modified hybrids show similar cleav-

age efficiency as that of the native counterpart. However, the

cleavage rate of parent carba-ENA (type I)-modified hybrids

is two times better than that of the ENA-modified counter-

part, which implies replacement of 2⁰-O of ENA with 2⁰-CH₂

leads to a significant improvement on the RNase H recruit-

ment capability.

(ii) Just like the parent carba-ENA (type I), type VI

modification ([8⁰R-NH₂]-carba-ENA) also showed a double

RNA hydrolysis rate relative to the native counterpart. It is

likely that electropositive C8⁰-NH₂as a zwitterion at the

physiological pH presumably plays a role in promoting

RNase H digestion, just like its role in assisting 3⁰-exonu-

clease-mediated oligo degradation.

(53) Pradeepkumar, P. I.; Chattopadhyaya, J. J. Chem. Soc., Perkin

Trans. 2 2001, 2074–2083.

7122 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.

JOCArticle

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-

CH₃]-carba-ENA)-modified AONs and type IV-modified

AONs can mediate the recruitment of RNase H to similar

degrees as the native counterpart, suggesting that the nature

of the substituent (lipophilic Me versus hydrophilic OH)

does not affect the efficiency of RNase H elicitation.

(iv) The RNA cleavage efficiency in the modified AON:

RNA hybrids does not depend on the orientation of the

substituent at C8⁰. For example, type III ([8⁰S-OH]-carba-

ENA)-modified AONs and type IV ([8⁰R-OH]-carba-ENA)-

modified AONs showed the same reaction rate as the native

hybrid.

(v) Substitution of carba-ENA or its analogues (types I to

VI) at position 3 or 10 (from the 3⁰-end) of the AON sequence

gives a much improved RNA cleavage by RNase H1 in the

corresponding heteroduplexes than at any other sites in the

sequence. Modification in the middle of the AON strand

produces less effective RNase H1 elicitation.

stereochemistry of 8⁰substituents on carba-ENA: (i) hydro-

phobic 8⁰S-Me substituent on carba-ENA increases the

nucleolytic stability but 8⁰R-Me lead to a decrease effect;

(ii) hydrophilic 8⁰R-OH on carba-ENA had little if any effect

on nuclease resistance but 8⁰S-OH resulted in significantly

decreased nucleolytic stability; and (iii) zwitterionic 8⁰-NH₂

and 8⁰-NH₂PAC on carba-ENA led to obvious loss on the

3⁰-exonuclease resistance. Hence, it seems that the electrostatic

effect of 8⁰substituents contributes to their modulations on

nucleolytic stability.

(3) All the carba-ENAs-modified AONs showed better

RNase H recruitment capabilities than their native and

ENA-modified counterpart. Especially, parent carba-ENA

(type I) and [8⁰R-NH₂]-carba-ENA (type VI)-modified

AONs exhibited double RNA hydrolysis rates relative to

the ENA counterpart.

Thus this study shows that carba-ENA and its analogue

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

(3) much improved RNase H1 elicitation capability com-

pared to that of the native and ENA-modified AONs.

Conclusions

In this investigation, five 8⁰-modified carba-ENA deriva-

tives and parent carba-ENA-T have been synthesized through

an intramolecular radical cyclization. Biological properties of

AONs containing these modifications have been evaluated and

compared with that of AONs containing other types of

modifications such as LNA, carba-LNA, and ENA. The

major conclusions are as follows:

Implications

The present study shows that C8⁰substituents of carba-

ENA impart distinctly different biophysical and biochemical

effects on the properties of modified nucleic acids compared

to those of C6⁰substituents because they have different

stereochemical locations, hence interact differently, in the

minor groove. Since the C8⁰substituent of carba-ENA is

located at the bottom and center of the minor groove, and

theC6⁰substituent of carba-ENA is also at the minor groove

(but not at the center) but spatially close to the internucleo-

tidic phosphate, they have different biophysical-chemical

effects: it was found that a positively charged amino sub-

stituent at the C8⁰can improve target affinity and RNase H

recruitment capability, whereas the C6⁰substituentofcarba-ENA

(1) All the carba-ENAs-modified AONs showed improved

RNA affinity over their native counterpart. Interestingly, it was

found the nature and the steric orientation of the 8⁰substituents

only slightly affect the T_mvalues of modified AON:RNA

duplexes. One the contrary, carba-ENAs modifications in the

AON:DNA homoduplex resulted in significant T_mdecrease.

Hence, parent carba-ENA and 8⁰-substituted analogues are

highly RNA selective.

(2) The effect of 8⁰substituents on the nucleolytic stability

of AON was found to greatly depend on the nature and

J. Org. Chem. Vol. 75, No. 21, 2010 7123

Liu et al.

JOCArticle

can render a remarkable effect on nuclease resistance in that

the C6⁰R-hydrophobic substituent (Me) seems to give the best

result. Combining the merits of both C8⁰and C6⁰substituents

in one engineering of a carba-ENA nucleotide, such as a C8⁰R-

amino-C6⁰R-Me-carba-ENA moiety, may generate an excel-

lent candidate for AON-based therapeutics, which deserve

further investigation in the future. On the other hand, the

unique location of C8⁰substituents of carba-ENA (at the

bottom and center of the minor groove) makes C8⁰modified

carba-ENA very good chemical models to study the role of

electrostatic and steric effect in the self-assembly as well as to

probe various biological functions of nucleic acids.

acetamide (7.5 mL, 30.35 mmol) was added under N₂followed by

reflux for 30 min until the suspension became a clear solution.

Then the solution was cooled to room temperature and TMSOTf

(2.5 mL, 14.05 mmol) was added dropwise. The obtained mixture

was refluxed overnight followed by quenching with saturated

aqueous NaHCO₃and extracted with CH₂Cl₂. The organic layer

was dried over MgSO₄and concentrated. The residue was purified

by column chromatography on silica gel (20-33% acetone in

petroleum ether, v/v) to obtain 10 (5.07 g, 84.5% in two steps) as a

1

white foam. H NMR (600 MHz, CDCl₃) δ 9.09 (1H, s, H3),

0

7.38-7.26 (11H, m, aromatic and H6), 6.05 (1H, d, J_{1 ,2}= 4.8 Hz,

0

H1⁰), 5.45 (1H, dd, J_{1 ,2}= 4.8 Hz, J_{2 ,3}= 6.0 Hz, H2 ), 4.62 (1H,

d, J_gem= 11.4 Hz, CH₂Bn), 4.52 (1H, d, J_gem= 11.4 Hz, CH₂Bn),

4.47 (1H, d, J_gem= 11.4 Hz, CH₂Bn), 4.43 (1H, d, J_gem= 11.4 Hz,

0

CH₂Bn), 4.42 (1H, d, J_{2 ,3}= 6.0 Hz, H3 ), 3.62 (1H, d, J_gem= 9.6

Hz, H5⁰), 3.37 (1H, d, J_gem= 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

(1H, m, H7⁰⁰), 1.60 (3H, s, thymine-CH₃). ¹³C NMR (150 MHz,

CDCl₃) δ 170.3 (CdO, acetyl), 163.9 (C4), 150.7 (C2), 137.3, 137.2

(2 ꢀ C_ipso-Bn), 136.3 (C6), 129.0-128.1 (aromatic), 120.3 (CN),

111.9 (C5), 88.1 (C1⁰), 86.3 (C4⁰), 78.6 (C3⁰), 74.9 (C2⁰), 74.8, 73.9

(CH₂Bn), 72.8 (C5⁰), 28.6 (C6⁰), 21.0 (CH₃, acetyl), 12.4 (CH₃-

thymine), 12.2 (C7⁰). MALDI-TOF m/z [C₂₉H₃₁N₃O₇þ H]^þ

found 534.224, calcd 534.223.

Experimental Section

Note: All individual reaction steps, workup, and product

characterization for compounds 16b, 17b, 18a/b, 21b, 22a/b, 25,

28, 29, 30a/b, 31, 32, and ENA are given in the Supporting

Information.

3,5-Di-O-benzyl-4-C-cyanoethyl-1,2-O-isopropylidene-r-^D-

ribofuranose (8). Et₃N (15.4 mL, 110.72 mmol) was added drop-

wise to a solution of 6 (8.66 g, 20.89 mmol), p-toluenesulfonyl

chloride (7.17 g, 37.61 mmol), and DMAP (255 mg, 2.09 mmol)

in anhydrous CH₂Cl₂(290 mL) at 0 °C under N₂atmosphere.

The mixture was allowed to warm to room temperature fol-

lowed by stirring overnight at this temperature. The reaction

was quenched with saturated aqueous NaHCO₃and the

obtained mixture was partitioned between CH₂Cl₂and water,

then the aqueous layer was extracted again with CH₂Cl₂. The

combined organic layer was dried over MgSO₄and evaporated

to give tosylate 7. Compound 7 was dissolved in anhydrous

DMF (73 mL) and tetrabutylammonium cyanide (14.02 g, 52.23

mmol) was added. The obtained solution was stirred at room

temperature for 2.5 days, then warmed to 35 °C followed by

stirring at this temperature for 1 day. After evaporation of

solvent, the residue was dissolved in CH₂Cl₂and saturated

aqueous NaHCO₃. The organic layer was separated and the

aqueous layer was extracted again with CH₂Cl₂. The combined

organic layer was dried over MgSO₄and concentrated. The

residue was purified by column chromatography on silica gel

(4-6% acetone in petroleum ether, v/v) to obtain 8 (6.85 g,

77.4% in two steps) as a light yellow syrup. ¹H NMR (500 MHz,

CDCl₃) δ 7.34-7.23 (10H, m, aromatic), 5.73 (1H, d, J_1,2= 4.0

Hz, H1), 4.75 (1H, d, J_gem= 12.0 Hz, CH₂Bn), 4.60 (1H, dd,

J_1,2= 4.0 Hz, J_2,3= 5.0 Hz, H2), 4.52 (1H, d, J_gem= 12.0 Hz,

CH₂Bn), 4.48 (1H, d, J_gem= 12.0 Hz, CH₂Bn), 4.43 (1H, d,

J_gem= 12.0 Hz, CH₂Bn), 4.00 (1H, d, J_2,3= 5.0 Hz, H3), 3.37

(1H, d, J_gem= 10.0 Hz, H5), 3.25 (1H, d, J_gem= 10.0 Hz, H5⁰),

2.59 (2H, m, H7 and H6), 2.43 (1H, m, H7⁰), 1.91 (1H, m, H6⁰),

1.58 (3H, s, CH₃), 1.31 (3H, s, CH₃). ¹³C NMR (125 MHz,

CDCl₃) δ 137.6 (2 ꢀ C_ipso-Bn), 128.4-127.7 (aromatic), 120.5

(isopropyl), 113.2 (CN), 104.3 (C1), 85.1 (C4), 79.3 (C3), 78.5

(C2), 73.6 (CH₂Bn), 73.0 (C5), 72.4 (CH₂Bn), 27.9 (C6), 26.5

(CH₃), 25.8 (CH₃),11.9(C7).MALDI-TOFm/z[C₂₅H₂₉NO₅þ Na]^þ

found 446.194, calcd 446.200.

1-(3,5-O-Benzyl-4-C-cyanoethyl-2-O-hydroxyl-β-^D-ribofuranosyl)-

thymine (11). Compound 10 (2.52 g, 4.72 mmol) was dissolved in

methanol (13 mL) and methylamine solution (95 mL). The

mixture was stirred in an ice bath for 1 h. After evaporation of

solvent, the obtained residue was purified by column chromatog-

raphy on silica gel (1% methanol in CH₂Cl₂, v/v) to obtain 11

(2.19 g, 94.7%) aswhite foam. ¹H NMR (500MHz, CDCl₃) δ9.10

(1H, s, H3), 7.39-7.26 (11H, m, aromatic and H6), 5.84 (1H, d,

J_{1 ,2}= 5.5 Hz, H1⁰), 4.78 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.61

(1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.55 (1H, d, J_gem= 11.5 Hz,

CH₂Bn), 4.50 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.41 (1H, dd,

0

J_{1 ,2}= 5.5 Hz, J_{2 ,3}= 6.0 Hz, J_{2 ,OH}= 7.0 Hz, H2⁰), 4.22 (1H, d,

0

J_{2 ,3}= 6.0 Hz, H3 ), 3.60 (1H, d, J_gem= 10.0 Hz, H5⁰), 3.53 (1H,

0

d, J_{2 ,OH}= 7.0 Hz, 2⁰-OH), 3.40 (1H, d, J_gem= 10.0 Hz, H5⁰⁰),

0

2.52 (1H, m, H7⁰), 2.39 (1H, m, H7⁰⁰), 2.29 (1H, m, H6⁰), 1.84

(1H, m, H6⁰⁰), 1.61 (3H, s, thymine-CH₃). ¹³C NMR (150 MHz,

CDCl₃) δ164.0 (C4), 151.2 (C2), 137.3, 137.2 (2 ꢀ C_ipso-Bn), 136.4

(C6), 129.1-128.1 (aromatic), 120.4 (CN), 111.5 (C5), 90.3 (C1⁰),

86.2 (C4⁰), 79.8 (C3⁰), 75.0 (C2⁰), 74.9, 74.1 (CH₂Bn), 73.1 (C5⁰),

28.8 (C6⁰), 12.5 (CH₃-thymine), 12.2 (C7⁰). MALDI-TOF m/z

[C₂₇H₂₉N₃O₆þ H]^þfound 492.215, calcd 492.213.

1-(3,5-O-Benzyl-4-C-propionaldehyde-2-O-hydroxyl-β-^D-ribofur-

anosyl)thymine O-Benzyl Oxime (12). DIBALH (11.5 mL, 11.5

mmol, 1.0 M solution in toluene) was added dropwise to a solution

of 11 (1.41 g, 2.87 mmol) in anhydrous CH₂Cl₂(29mL) over30min

at -78 °C. After being stirred at the same temperature for 2 h under

nitrogen atmosphere, the mixture was quenched with methanol at

-78 °C, followed by water. The mixture was allowed to warm to

room temperature until a precipitate was formed, which was filtered

though Celite. The filtrate was washed with 10% aqueous acetic

acid, saturated aqueous NaHCO₃, and brine. The organic layer was

dried over MgSO₄and concentrated to give crude aldehyde as an

intermediate. The crude aldehyde was dissolved in anhydrous

CH₂Cl₂(29 mL) and anhydrous pyridine (1.4 mL, 17.22 mmol),

to which O-benzyl hydroxylamine hydrochloride (916 mg, 5.74

mmmol) was added. After refluxing for 1 h, the mixture was cooled

to room temperature and saturated aqueous NaHCO₃was added.

The organic layer was separated, dried over MgSO₄, and evapo-

rated. The residue was subjected to short column chromatography

over silica gel (0.9-1.2% methanol in CH₂Cl₂, v/v) togive12 (1.04g,

60.6% in two steps) as the mixture of Z and E isomers. Isomer I:

1-(2-O-Acetyl-3,5-O-benzyl-4-C-cyanoethyl-β-^D-ribofuranosyl)-

thymine (10). Compound 8 (4.76 g, 11.24 mmol) was dissolved in

acetic anhydride (6.3 mL, 67.44 mmol) and acetic acid (63 mL).

This solution was cooled to 10 °C, to which trifilic acid (100 μL,

1.12 mmol) was added dropwise, and the obtained mixture was

stirred at this temperature for 1 h. The reaction was quenched with

saturated aqueous NaHCO₃and extracted with CH₂Cl₂. The

organic layer was dried over MgSO₄and evaporated to give

crude product 9. The crude product, after coevaporation with

toluene twice, was dissolved in anhydrous MeCN (146 mL), to

which thymine (2.12 g, 16.85 mmol) and N,O-bis(trimethylsilyl)-

¹H NMR (500 MHz, CDCl₃) δ8.14 (1H, s, H3), 7.44 (1H, t, J_{7 ,8}

0

=

5.4 Hz, J_{7 ,8}= 5.4 Hz, H8⁰), 7.37-7.26 (16H, m, aromatic and

00

0

H6), 5.89 (1H, d, J_{1 ,2}= 6.0 Hz, H1⁰), 5.05 (2H, s, NOCH₂Bn),

0

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

Liu et al.

JOCArticle

4.68 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.65 (1H, d, J_gem= 11.5 Hz,

CH₂Bn), 4.52 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.49 (1H, d, J_gem=

29.1 (C6⁰), 20.9 (C7⁰), 12.6 (CH₃-thymine). MALDI-TOF m/z:

[C₄₁H₄₁N₃O₈S þ H]^þfound 736.270, calcd 736.269.

0

11.5 Hz, CH₂Bn), 4.34 (1H, m, J_{1 ,2}= 6.0 Hz, J_{2 ,3}= 6.0 Hz,

(1R,3R,4R,5R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-benzy-

loxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (14). Com-

pound 13 (910 mg, 1.237 mmol) was dissolved in anhydrous toluene

(130 mL) then the solution was purged with N₂for 40 min. The

mixture was refluxed, Bn₃SnH (0.8 mL in 20 mL dry toluene) and

AIBN (40 mg in 10 mL dry toluene) were added dropwise in 5 h, then

the mixture continued to reflux for 1 h. The mixture was cooled to

room temperature and solvent was evaporated. The residue was

chromatographed on silica gel (18-30% in ethyl acetate in cyclo-

hexane, v/v) to give 14 (433 mg, 60.1%) as a white foam. ¹H NMR

_{2 ,OH}=8.5Hz, H2⁰), 4.16(1H, d,J_{2 ,3}=6.0Hz, H3 ),3.61(1H, d,

0

J

_gem= 10.0 Hz, H5⁰), 3.40 (1H, d, J_gem= 10.0 Hz, H5⁰⁰), 2.90 (1H,

d, J_{2 ,OH}= 8.5 Hz, 2⁰-OH), 2.38 (1H, m, H7⁰), 2.22 (1H, m, H7⁰⁰),

0

2.12 (1H, m, H6⁰), 1.70 (1H, m, H6⁰⁰), 1.61 (3H, d, J_6,CH= 0.6 Hz,

3

thymine-CH₃). ¹³C NMR (150 MHz, CDCl₃) δ 163.2 (C4), 150.6

(C8⁰), 150.5 (C2), 137.7, 137.1, 136.7 (3 ꢀ C_ipso-Bn), 135.8 (C6),

128.8-127.6 (aromatic), 111.1 (C5), 88.6 (C1⁰), 86.7 (C4⁰), 80.2

(C3⁰), 75.6 75.1 (2 ꢀ CH₂Bn), 74.7 (C2⁰), 73.7 (CH₂Bn), 73.5 (C5⁰),

1

29.1 (C6⁰), 24.0 (C7⁰), 12.1 (CH₃-thymine). Isomer II: H NMR

(500 MHz, CDCl₃) δ 8.20 (1H, s, H3), 8.00 (1H, d, J_6,CH= 1.0 Hz,

(500 MHz, CDCl₃) δ 8.20 (1H, s, H3), 7.39-7.26 (16H, m,

3

H6), 7.39-7.26 (15H, m, aromatic), 5.99 (1H, s, H1⁰), 5.58 (1H, s,

NH), 4.83 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.80 (1H, d, J_gem= 11.5

Hz, CH₂Bn), 4.61 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.57 (1H, d,

0

00

0

aromatic and H6), 6.68 (1H, t, J_{7 ,8}= 5.5 Hz, J_{7 ,8}= 5.5 Hz,

0

H8⁰), 5.87 (1H, d, J_{1 ,2}= 6.0 Hz, H1⁰), 5.05 (2H, s, NOCH₂Bn),

4.68 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.63 (1H, d, J_gem= 11.5 Hz,

0

J

_gem= 11.5 Hz, CH₂Bn), 4.52 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.44

0

CH₂Bn), 4.52 (1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.48 (1H, d, J_gem

0

=

11.5 Hz, CH₂Bn), 4.33 (1H, dd, J_{1 ,2}= 6.0 Hz, J_{2 ,3}= 6.0 Hz,

(1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.25 (1H, d, J_{2 ,3}= 5.0 Hz, H3⁰),

0

J

_{2 ,OH}=7.5Hz, H2⁰), 4.17(1H, d,J_{2 ,3}=6.0Hz, H3 ),3.63(1H, d,

0

3.69 (1H, d, J_gem=11.0Hz,H5 ),3.56(1H,m,J_{2 ,8}=3.5Hz,J_{7 ,8}

0

=

5.0 Hz, J_{7 ,8}= 11.5 Hz, H8 ), 3.54 (1H, d, J_gem=11.0Hz,H5⁰⁰), 2.82

0

_gem= 10.0 Hz, H5⁰), 3.41 (1H, d, J_gem= 10.0 Hz, H5⁰⁰), 3.93 (1H,

00

0

d, J_{2 ,OH}= 7.5 Hz, 2⁰-OH), 2.45 (2H, m, H7⁰and H7⁰⁰), 2.08 (1H,

0

(1H, d, J_{2 ,8}= 3.5 Hz, J_{2 ,3}= 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

(125 MHz, CDCl₃) δ 163.3 (C4), 151.5 (C8⁰), 150.6 (C2), 138.0,

137.2, 136.8 (3 ꢀ C_ipso-Bn), 135.9 (C6), 128.8-127.6 (aromatic),

111.1 (C5), 88.9 (C1⁰), 86.8 (C4⁰), 80.0 (C3⁰), 75.9, 75.0 (2 ꢀ

CH₂Bn), 74.8 (C2⁰), 73.7 (CH₂Bn), 73.3 (C5⁰), 28.8 (C6⁰), 20.4

(C7⁰),12.1(CH₃-thymine). MALDI-TOF m/z[C₃₄H₃₇N₃O₇þ H]^þ

found 600.272, calcd 600.270.

(1H, m, H7⁰), 1.61 (3H, d, J_6,CH= 1.0 Hz, thymine-CH₃), 1.40

3

(1H, m, H6⁰), 1.38 (1H, m, H7⁰⁰). ¹³C NMR (125 MHz, CDCl₃) δ

163.8 (C4), 149.7 (C2), 138.1, 137.5, 137.4 (C_ipso-Bn), 136.5 (C6),

128.6-127.5 (aromatic), 109.3 (C5), 84.6 (C4⁰), 84.4 (C1⁰), 76.5,

73.6 (2 ꢀ CH₂Bn), 73.2 (C3⁰), 71.9(CH₂Bn), 70.2 (C5⁰), 53.5 (C8⁰),

44.7 (C2⁰), 26.2 (C6⁰), 22.1 (C7⁰), 11.9 (CH₃-thymine). MALDI-

TOF m/z [C₃₄H₃₇N₃O₆þ Na]^þfound 606.258, calcd 606.257.

(1R,3R,4R,5R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-trifluoroa-

cetamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (16a). To a

solution of 14 (110 mg, 0.188 mmol) in anhydrous methanol

(4.0 mL) were added 10% Pd/C (94 mg) and ammonium formate

(475 mg, 7.54 mmol) under nitrogen. The mixture was stirred for

8 h at room temperature. The suspension was filtered through a

pad of Celite. The filtrate was concentrated and dried over

vacuum pump. The obtained crude amine 15 was dissolved in a

mixture of anhydrous CH₂Cl₂(3.8 mL) and pyridine (0.12 mL,

1.50 mmol) and cooled to 0 °C, to which trifluoroacetic anhy-

dride (0.1 mL, 0.752 mmol) was added dropwise over 5 min.

Then the reaction mixture was allowed to warm to room

temperature and stirred at this temperature for 2 h, followed

by quenching with crushed ice and diluted with CH₂Cl₂. The

aqueous layer was extracted with CH₂Cl₂twice. The organic

layers were combined, dried over MgSO₄, and concentrated. The

residue was purified by column chromatography on silica gel

(0.6-0.8% methanol in CH₂Cl₂, v/v) to obtain 16a (65 mg,

60.0% in two steps) as a white foam. ¹H NMR (500 MHz, CDCl₃)

1-(3,5-O-Benzyl-2-O-phenoxythiocarbonyl-4-C-propionaldehyde-

β-^D-ribofuranosyl)thymine O-Benzyl Oxime (13).To a solution of 12

(1.13 g, 1.89 mmol) in anhydrous pyridine (19 mL) was added

dropwise phenyl chlorothionoformate (0.31 mL, 2.27 mmol) in an

ice bath under N₂. The mixture was stirred for 30 min in the ice bath,

then stirred for a further 2 h at room temperature followed by

quenching with methanol. After the mixture was stirred for 30 min,

the solvent was removed. The residue was dissolved in CH₂Cl₂and

washed with saturated aqueous NaHCO₃, and then the aqueous

layer was extracted with CH₂Cl₂twice. The combined organic layer

was dried over MgSO₄and evaporated. The residue was purified by

column chromatography on silica gel (11-17% acetone in petro-

leum ether, v/v) to obtain 13 (1.23 g, 88.7%) as the mixture of Z and

Eisomers. Isomer I: ¹H NMR (500 MHz, CDCl₃) δ8.23(1H,s,H3),

7.44 (1H, d, J_6,CH= 1.2 Hz, H6), 7.41-7.26 (19H, m, aromatic and

3

H8⁰), 7.00 (2H, dd, aromatic), 6.35(1H, d, J_{1 ,2}=6.0 Hz, H1 ), 5.95

0

(1H,t, J_{1 ,2}=6.0Hz, J_{2 ,3}=6.0Hz, H2 ),5.04(2H, s, NOCH₂Bn),

0

4.74 (1H, d, J_gem= 11.0 Hz, CH₂Bn), 4.54 (1H, d, J_{2 ,3}= 6.0 Hz,

H3⁰), 4.53 (1H, d, J_gem= 11.0 Hz, CH₂Bn), 4.52 (2H, s, CH₂Bn),

3.67 (1H, d, J_gem= 10.0 Hz, H5⁰), 3.43 (1H, d, J_gem= 10.0 Hz,

H5⁰⁰), 2.37 (1H, m, H7⁰), 2.22 (1H, m, H7⁰⁰), 2.05 (1H, m, H6⁰), 1.74

δ 8.79 (1H, s, H3), 7.89 (1H, d, J_6,CH=1.5 Hz, H6), 7.65 (1H, d,

3

_{8 ,NH}=6.5 Hz, 8⁰-NH), 7.40-7.27 (10H, m, aromatic), 5.92 (1H,

=

0

J

(1H, m, H6⁰⁰), 1.54 (3H, d, J_6,CH= 1.2 Hz, thymine-CH₃). ¹³C

s, H1⁰), 4.71 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.59 (1H, d, J_gem

11.5 Hz, CH₂Bn), 4.54 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.53 (1H,

3

NMR (125 MHz, CDCl₃) δ 194.4 (CdS), 163.3 (C4), 153.4 (C_ipso

-

Bn), 150.6 (C8⁰), 150.2 (C2), 137.7, 137.2, 137.0 (3 ꢀ C_ipso-Bn), 135.8

(C6), 129.6-126.8, 121.7 (aromatic), 111.6 (C5), 87.3 (C4⁰), 85.6

(C1⁰), 82.8 (C2⁰), 78.1 (C3⁰), 75.6, 75.0, 73.8 (3 ꢀ CH₂Bn), 73.4

0

d, J_gem=11.5 Hz, CH₂Bn), 4.46 (1H, m, J_NH,8=6.5 Hz, J_{7 ,8}=6.0

Hz, J_{7 ,8}=11.0 Hz, H8 ), 4.27 (1H, d, J_{2 ,3}=5.0 Hz, H3 ), 3.74

(1H, d, J_gem=10.5 Hz, H5⁰), 3.59 (1H, d, J_gem=10.5 Hz, H5⁰⁰), 2.63

0

(1H, d, J_{2 ,3}=5.0 Hz, H2 ), 2.34 (1H, m, J_{6 ,7}=6.0 Hz, J_{7 ,8}=6.0

00

Hz, J_{7 ,7}=13.0 Hz, H7 ), 1.96 (1H, ddd, J_{6 ,7}=6.0 Hz, J_{6 ,7}=13.0

00

Hz, J_{6 ,6}=13.0 Hz, H6 ), 1.63 (1H, m, J_{6 ,7}=6.0 Hz, J_{6 ,7}

6.5 Hz, J_{7 ,8}=11.0 Hz, J_{7 ,7}=13.0 Hz, H7 ), 1.51 (1H, dd, J_{6 ,7}

0

00

0

1

0

(C5⁰), 29.1 (C6⁰), 24.1 (C7⁰), 12.1 (CH₃-thymine). Isomer II: H

0

00

0

NMR (600 MHz, CDCl₃) δ 9.37 (1H, s, H3), 7.43 (1H, s, H6),

7.42-7.30 (18H, m, aromatic), 7.00 (2H, dd, aromatic), 6.68 (1H, t,

0

00

0

00 00

=

00

J_{7 ,8}= 5.4 Hz, J_{7 ,8}= 5.4 Hz, H8⁰), 6.39 (1H, d, J_{1 ,2}= 6.0 Hz,

0

00

0

00

0

00

00 00

H1⁰), 5.93 (1H, t, J_{1 ,2}= 6.0 Hz, J_{2 ,3}= 6.0 Hz, H2 ), 5.10 (2H, s,

NOCH₂Bn), 4.76 (1H, d, J_gem= 11.4 Hz, CH₂Bn), 4.60 (1H, d,

6.5 Hz, J_{6 ,6}=13.0 Hz, H6 ), 1.46(3H, d, J_6,CH=1.5 Hz, thymine-

0

00

3

CH₃). ¹³C NMR (125 MHz, CDCl₃) δ 164.0 (C4), 157.2 (CF₃CO),

150.3 (C2), 137.3, 137.0 (2 ꢀ C_ipso-Bn), 135.4 (C6), 128.7-127.8

(aromatic), 115.8 (CF₃CO), 110.2 (C5), 84.9 (C4⁰), 84.1 (C1⁰), 73.6

(CH₂Bn), 72.8 (C3⁰), 72.4 (CH₂Bn), 69.8 (C5⁰), 47.4 (C2⁰), 44.4

(C8⁰), 26.1 (C6⁰), 24.1 (C7⁰), 11.9 (CH₃-thymine). MALDI-TOF

m/z [C₂₉H₃₀F₃N₃O₆þ H]^þfound 574.212, calcd 574.216.

J_{2 ,3}= 6.0 Hz, H3⁰), 4.55 (1H, d, J_gem= 11.4 Hz, CH₂Bn), 4.52

(2H, s, CH₂Bn), 3.72 (1H, d, J_gem= 10.2 Hz, H5⁰), 3.46 (1H, d,

0

J

_gem= 10.2 Hz, H5⁰⁰), 2.44 (2H, m, H7⁰and H7⁰⁰), 2.04 (1H, m,

H6⁰), 1.75 (1H, m, H6⁰⁰), 1.47 (3H, s, thymine-CH₃). ¹³C NMR (125

MHz, CDCl₃) δ 195.0 (CdS), 164.5 (C4), 153.7 (C_ipso-Bn), 152.0

(C8⁰), 151.0 (C2), 138.2, 137.6, 137.4 (3 ꢀ C_ipso-Bn), 136.4 (C6),

130.2-127.4, 122.2 (aromatic), 112.2 (C5), 87.7 (C4⁰), 85.8 (C1⁰),

83.3 (C2⁰), 78.4 (C3⁰), 76.2, 75.4, 74.1 (3 ꢀ CH₂Bn), 73.5 (C5⁰),

(1R,3R,4R,5R,8S)-1-(4,4⁰-Dimethoxytrityloxymethyl)-8-hydroxyl-

5-trifluoroacetamino-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane

(17a). A mixture of 20% Pd(OH)₂/C (100 mg), 16a (95 mg,

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

Liu et al.

JOCArticle

0.166 mmol) in ethanol (6.6 mL), and cyclohexene (13.2 mL)

was reflux overnight. Then the suspension was filtered through

a pad of Celite. The filtrate was concentrated and dried. The

residue was coevaporated twice with dry pyridine and dissolved

in the same solvent (1.7 mL), to which 4,4⁰-dimethoxytrityl

chloride (69 mg, 0.202 mmol) was added, and stirred overnight

at room temperature. Then solvent was removed and the

obtained residue was chromatographed on silica gel (0-0.6%

methanol in CH₂Cl₂containing 1% pyridine, v/v) to obtain 17a

H6), 7.46-7.25 (10H, m, aromatic), 5.57 (1H, s, H1⁰), 4.73 (1H, d,

_gem=11.5 Hz, CH₂Bn), 4.62 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.59

J

(1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.55₀(1H, d, J_gem= 11.5 Hz,

0

CH₂Bn), 4.36 (1H, d, J_{2 ,3}=4.5 Hz, H3 ), 4.22 (1H, m, J_{2 ,8}=4.0

0

Hz, J_OH,8=11.5 Hz, H8 ), 4.03 (1H, d, J_OH,8=11.5 Hz, 8 -OH),

3.77 (1H, d, J_gem=11.0 Hz, H5⁰), 3.59 (1H, d, J_gem=11.0 Hz, H5⁰⁰),

0

2.82 (1H, t, J_{2 ,8}=4.0 Hz, J_{2 ,3}=4.5 Hz, H2 ), 2.03 (2H, m, H7 and

H6⁰), 1.91 (1H, m, H7⁰⁰), 1.49 (3H, d, J_6,CH=1.0 Hz, thymine-

3

CH₃), 1.43 (1H, m, H6⁰⁰). ¹³C NMR (125 MHz, CDCl₃) δ 163.7

(C4), 149.9 (C2), 137.1, 136.4 (2 ꢀ C_ipso-Bn), 135.9 (C6),

128.7-127.9 (aromatic), 109.4 (C5), 85.4 (C1⁰), 84.5 (C4⁰), 75.4

(C3⁰), 74.0, 73.6 (2 ꢀ CH₂Bn), 70.4 (C5⁰), 68.7 (C8⁰), 46.2 (C2⁰),

27.3 (C7⁰), 23.5 (C6⁰), 11.9 (CH₃-thymine). MALDI-TOF m/z

[C₂₇H₃₀N₂O₆þ Na]^þfound 501.200, calcd 501.200. 20b: ¹H

NMR (500 MHz, CDCl₃) δ 8.05 (1H, s, H6), 7.35-7.18 (10H,

m, aromatic), 6.08 (1H, s, H1⁰), 4.77 (1H, d, J_gem= 12.0 Hz,

1

(35 mg, 30.3% in two steps) as a white foam. H NMR (600

MHz, CDCl₃þ d⁴-MeOH) δ 8.24 (1H, d, J_{8 ,NH}=6.0 Hz, 8⁰-

0

NH), 7.90 (1H, s, H6), 7.43-6.84 (13H, m, aromatic), 5.85 (1H,

s, H1⁰), 4.63 (1H, d, J_{2 ,3}=5.4 Hz, H3 ), 4.52 (1H, m, J_{2 ,8}=2.5

0

00

0

Hz, J_{8 ,NH}=6.0 Hz, J_{7 ,8}=6.0 Hz, J_{7 ,8}=11.5 Hz, H8 ), 3.79

(6H, s, 2 ꢀ OCH₃), 3.35 (1H, d, J_gem=10.8 Hz, H5⁰), 3.28 (1H, d,

J_gem=10.8 Hz, H5⁰⁰), 2.58 (1H, d, J_{2 ,8}=2.5 Hz, J_{2 ,3}=5.4 Hz,

0

H2⁰), 2.16 (2H, m, J_{7 ,8}=6.0 Hz, J_{6 ,7}=6.0 Hz, J_{7 ,7}=13.8 Hz,

0

00

CH₂Bn), 4.60 (1H, d, J_gem=12.0 Hz, CH₂Bn), 4.55 (2H, d, J_gem

=

12.0 Hz, 2 ꢀ CH₂Bn), 4.39 (1H, s, H3⁰), 4.37 (1H, m, H8⁰), 3.69 (1H,

d, J_gem=11.0 Hz, H5⁰), 3.55 (1H, d, J_gem=11.0 Hz, H5⁰⁰), 3.08 (1H,

s, H2⁰), 2.04 (1H, m, H7⁰), 1.85 (1H, ddd, H6⁰), 1.62 (1H, m, H7⁰⁰),

H7⁰and 3⁰-OH), 1.87 (1H, ddd, J_{6 ,7}=6.0 Hz, J_{6 ,7}=12.6 Hz,

0

00

0

00

0

00

00 00

J_{6 ,6}=13.8 Hz, H6 ), 1.61 (1H, m, J_{7 ,8}=11.5 Hz, J_{6 ,7}=6.6

0

00

0

00

00 00

Hz, J_{6 ,7}=12.6 Hz, J_{7 ,7}=13.8 Hz, H7 ), 1.45 (1H, dd, J_{6 ,7}

=

1.41 (1H, dd, H6⁰⁰), 1.39 (3H, d, J_6,CH=1.0 Hz, thymine-CH₃).

6.6 Hz, J_{6 ,6}= 13.8 Hz, H6⁰⁰), 1.29 (3H, s, thymine-CH₃).

¹³C NMR (125 MHz, CDCl₃) δ 164.3 (C4), 158.7, 158.6 (2 ꢀ

OMe-C_ispo), 157.5 (CF₃CO), 150.8 (C2), 144.3, 135.5, 135.3

(DMTr-C_ispo), 135.2 (C6), 130.1-124.0 (aromatic), 115.8

(CF₃CO), 113.3 (aromatic), 110.4 (C5), 86.6 (DMTr-C), 85.8

(C4⁰), 84.2 (C1⁰), 66.5 (C3⁰), 63.3 (C5⁰), 55.2 (2 ꢀ OMe), 49.9

(C2⁰), 44.3 (C8⁰), 25.8 (C6⁰), 23.8 (C7⁰), 11.9 (CH₃-thymine).

MALDI-TOF m/z [C₃₆H₃₆F₃N₃O₈þ Na]^þfound 718.235,

calcd 718.235.

0

00

3

¹³C NMR (125 MHz, CDCl₃) δ164.5 (C4), 151.0 (C2), 138.1, 137.5

(2 ꢀ C_ipso-Bn), 136.7 (C6), 128.6-127.3 (aromatic), 108.8 (C5),

84.4 (C4⁰), 84.3 (C1⁰), 74.3 (C3⁰), 73.5, 71.9 (2 ꢀ CH₂Bn), 70.2

(C5⁰), 64.3 (C8⁰), 49.1 (C2⁰), 26.3 (C7⁰), 26.1 (C6⁰), 12.2 (CH₃-

thymine). MALDI-TOF m/z [C₂₇H₃₀N₂O₆þ H]^þfound 479.221,

calcd 479.218.

(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-(4-methyl-

benzoate)-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (21a). Com-

pound 20a (72 mg, 0.150 mmol) was dissolved in dry pyridine

(3 mL) and the solution was cooled to 0 °C, then toluoyl chloride

(0.1 mL, 0.760 mmol) was added. The mixture was allowed to warm

to room temperature followed by stirring at this temperature over-

night. Then the reaction was quenched with saturated NaHCO₃

solution. The aqueous layer was exacted with CH₂Cl₂three times.

The combined organic layer was dried over MgSO₄and concen-

trated. The residue was chromatographed on silica gel (0.8-1.2%

methanol in CH₂Cl₂, v/v) to obtain 21a (81 mg, 90.0%) as a white

foam. ¹H NMR (500 MHz, CDCl₃) δ 8.19 (1H, s, H3), 8.04 (1H, s,

(1R,3R,4R,8S)-8-Benzyloxy-1-benzyloxymethyl-5-one-3-(thymin-

1-yl)-2-oxa-bicyclo[3.2.1]octane (19). The crude amine 15

(0.43 mmol) was dissolved in dry methanol (2.6 mL) and dry

THF (0.4 mL), to which was added 3,5-di-tert-butyl-1,2-benzo-

quinone (114 mg, 0.516 mmol). The mixture was stirred at 40 °C

for 10 h, then H₂O (0.72 mL) and oxalic acid (68 mg, 0.54 mmol)

were added followed by stirring at 40 °C overnight. Then addi-

tional H₂O (5 mL) was added and extracted with EtOAc three

times. The organic layer was dried over MgSO₄and evaporated to

dryness. The residue was applied to column chromatography on

silica gel (0.5-2% methanol in CH₂Cl₂, v/v) to give compound 19

(102 mg, 50% in two steps). ¹H NMR (500 MHz, CDCl₃) δ 8.54

H6), 7.79-6.98 (14H, m, aromatic), 5.73 (1H, s, H1⁰), 5.53 (1H, t,

0

J_{2 ,8}=4.0 Hz, J_{7 ,8}=5.0 Hz, H8 ), 4.72 (1H, d, J_gem=12.0 Hz,

0

CH₂Bn), 4.60 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.55 (1H, d, J_gem=

11.5 Hz, CH₂Bn),₀4.48 (1H, d, J_gem=12.0 Hz, CH₂Bn), 4.23 (1H, d,

(1H, s, H3), 8.00 (1H, d, J_6,CH=1.5 Hz, H6), 7.38-7.26 (10H, m,

3

aromatic), 6.07 (1H, s, H1⁰), 4.68 (1H, d, J_gem=11.5 Hz, CH₂Bn),

J

_{2 ,3}=4.5 Hz, H3 ), 3.78 (1H, d, J_gem=11.0 Hz, H5⁰), 3.66 (1H, ₀d,

4.63 (1H, d, J_2,3= 5.5 Hz, H3⁰), 4.62 (1H, d, J_gem= 11.5 Hz,

CH₂Bn), 4.56 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.42 (1H, d, J_gem=

0

J_gem=11.0 Hz, H5⁰⁰), 3.10 (1H, t, J_{2 ,8}=4.0 Hz, J_{2 ,3}=4.5 Hz, H2 ),

0

11.5 Hz, CH₂Bn), 3.87 (1H, d, J_gem=11.0 Hz, H5⁰₀), 3.65 (1H, d,

0

2.34 (3H, s, Tol-CH₃), 2.30 (1H, ddd, J_{6 ,7}= 5.5 Hz, J_{6 ,7}

00

0

=

0

J

J_{6 ,7}=9.0 Hz, J_{6 ,7}=10.5 Hz, J_{7 ,7}=16.5 Hz, H7 ), 2.52 (1H, dd,

_gem=11.0 Hz, H5⁰⁰), 3.32 (1H, d, J_{2 ,3}=5.5 Hz, H2 ), 2.61 (1H, m,

0

00

0

00

0

12.5 Hz, J_{6 ,6}=13.0 Hz, H6 ), 2.15 (1H,₀m, J_{7 ,8}=5.0 Hz, J_{6 ,7}

0

00

0

00

6.0 Hz, J_{6 ,7}=12.5 Hz, J_{7 ,7}=15.5 Hz, H7 ), 1.42(1H, dd, J_{6 ,7}=5.5

00

0

00

0

00

0

00

0

00

0

00

0

00

Hz, J_{7 ,7}=15.5 Hz, H7 ), 1.41 (1H, dd, J_{6 ,7}=6.0 Hz, J_{6 ,6}=13.0

Hz, H6⁰⁰), 1.41 (3H, s, thymine-CH₃). ¹³C NMR (125 MHz, CDCl₃)

δ166.1 (CdO), 163.6 (C4), 149.8 (C2), 143.5 (C_ipso-Tol), 137.6, 137.3

(2 ꢀ C_ipso-Bn), 136.0 (C6), 129.7-127.2 (aromatic), 109.3 (C5), 85.8

(C1⁰), 84.8 (C4⁰), 73.6 (CH₂Bn), 73.1 (C3⁰), 72.5 (CH₂Bn), 70.3

(C5⁰), 69.4 (C8⁰), 44.7(C2⁰), 24.3 (C7⁰), 24.0 (C6⁰), 21.6 (CH₃-Tol),

11.8 (CH₃-thymine). MALDI-TOF m/z [C₃₅H₃₆N₂O₇þ H]^þfound

597.263, calcd 597.260.

J_{6 ,7}=7.5 Hz, J_{7 ,7}=16.5 Hz, H7₀), 2.15 (1H, m, J_{6 ,7}=7.5 Hz,

0

00

_{6 ,7}=10.5 Hz, J_{6 ,6}=13.5 Hz, H6 ), 1.91 (1H, dd, J_{6 ,7}=9.0 Hz,

0

J

00

0

00

J_{6 ,6}= 13.5 Hz, H6 ), 1.44 (3H, d, J_6,CH= 1.5 Hz, thymine-

3

13

CH₃). C NMR (125 MHz, CDCl₃) δ 204.0 (C8⁰), 162.6 (C4),

148.7 (C2), 136.0, 135.6 (aromatic), 134.3 (C6), 127.7-126.9

(aromatic), 109.1 (C5), 84.1 (C4⁰), 83.4 (C1⁰), 75.1 (C3⁰), 72.7

(CH₂Bn), 71.1 (CH₂Bn), 68.6 (C5⁰), 58.4 (C2⁰), 33.5 (C7⁰), 27.0

(C6⁰), 10.8 (thymine-CH₃). MALDI-TOF m/z [C₂₇H₂₈N₂O₆þ

H]^þfound 477.203, calcd 477.202.

(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-((methyl-

thio)thiocarbonyl)oxy-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (23).

To a solution of 20a (77 mg, 0.161 mmol) in dry THF (3 mL) was

added 60% NaH (32 mg, 1.808 mmol) at 0 °C. After the solution was

stirred at room temperature for 1 h, CS₂(89 μL, 1.62 mmol) was

added to the suspension at 0 °C, and the resulting mixture was stirred

at 40 °C for 4 h. The solution became clear and was cooled by ice to

which methyl iodide (65 μL, 1.05 mmol) was added dropwise at 0 °C

followed by stirring at room temperature overnight. The reaction was

quenched by chilled water, then extracted with ethyl acetate three

times. The combined organic layer was dried over MgSO₄and

evaporated. The residue was chromatographed over silica gel

(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy-

3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (20a) and (1R,3R,4R,5R,

8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy-3-(thymin-1-yl)-2-oxa-

bicyclo[3.2.1]octane (20b). NaBH₄(59 mg, 1.55 mmol) was added to

a solution of 19 (185 mg, 0.338 mmol) in ethanol (10 mL). The

mixture was stirred at room temperature for 4 h followed by

quenching with acetone. After evaporation of solvent, the residue

was purified by column chromatography on silica gel (1.1-5%

methanol in CH₂Cl₂, v/v) to obtain two diastereomers 20a (112 mg,

69.2%) and 20b (30 mg, 18.6%) as a white foam. 20a: ¹H NMR

(500 MHz, CDCl₃) δ 8.33 (1H, s, H3), 7.99 (1H, d, J_6,CH=1.0 Hz,

3

7126 J. Org. Chem. Vol. 75, No. 21, 2010

Liu et al.

JOCArticle

(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-

loxy-5-methyl-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (27). 26

(60 mg, 0.12 mmol) was dissolved in dry pyridine (1.5 mL) to

which methyl oxalyl chloride (90μL, 0.974 mmol) was added. The

mixture was stirred at 40 °C overnight. After evaporation of

solvent, the residue obtained was dissolved in CH₂Cl₂, washed

with saturated NaHCO₃solution, dried over MgSO₄, and eva-

porated. The residue was purified by column chromatography on

silica gel (0.5-1% methanol in CH₂Cl₂, v/v) to give 27 (61 mg,

87%). ¹H NMR (500 MHz, CDCl₃): δ 8.68 (1H, s, NH), 8.02 (1H,

yellow foam. ¹H NMR (500 MHz, CDCl₃) δ 8.04 (1H, d, J_6,CH=1.5

3

Hz, H6), 8.02 (1H, s, H3), 7.37-7.25 (10H, m, aromatic), 6.03 (1H, t,

0

J_{2 ,8}=3.5 Hz, H8 ), 5.72 (1H, s, H1 ), 4.80 (1H, d, J_gem=12.0 Hz,

CH₂Bn), 4.60 (1H, d, J_gem=11.5 Hz, CH₂Bn),4.56(1H,d,J_gem=11.5

0

=

Hz, CH₂Bn), 4.45 (1H, d, J_gem=12.0 Hz, CH₂Bn), 4.24 (1H, d, J_{2 ,3}

4.5 Hz, H3⁰), 3.77 ₀₀(1H, d, J_gem= 11.0 Hz, H5⁰), 3.65 (1H, d,

0

J_gem=11.0 Hz, H5 ), 3.23 (1H, t, J_{2 ,8}=3.5 Hz, J_{2 ,3}=4.5 Hz,

H2⁰), 2.50 (3H, s, SCH₃), 2.16 (3H, m, H6⁰, H7⁰and H7⁰⁰), 1.43 (1H,

m, H6⁰⁰), 1.42 (3H, d, J_6,CH=1.5 Hz, thymine-CH₃). ¹³C NMR (125

3

d, J_6,CH=1.5 Hz, H6), 7.35-7.23 (10H, m, aromatic), 5.70(1H, s,

=

MHz, CDCl₃) δ 215.1 (CdS), 163.5 (C4), 149.7 (C2), 137.7, 137.3

(2 ꢀ C_ipso-Bn), 135.9 (C6), 128.7-127.6 (aromatic), 109.4 (C5), 85.5

(C1⁰), 85.0 (C4⁰), 78.1 (C8⁰),73.7(CH₂Bn), 72.6 (C3⁰), 72.4 (CH₂Bn),

70.2 (C5⁰), 44.4 (C2⁰), 24.0 (C7⁰), 23.8 (C6⁰), 19.0 (SCH₃), 11.8 (CH₃-

thymine). MALDI-TOF m/z [C₂₉H₃₂N₂O₆S₂þ H]^þfound 569.180,

calcd 569.177.

(1R,3R,4R,8S)-8-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-yl)-

2-oxa-bicyclo[3.2.1]octane (24). 23 (64 mg, 0.112 mmol) was dis-

solved in dry toluene (5 mL) and the solution was purged with dry

nitrogen for 30 min. AIBN (9 mg, 0.034 mmol) and Bu₃SnH

(91 μL, 0.338 mmol) were added to the mixture followed by reflux

for 1.5 h. The mixture was cooled to room temperature and solvent

was evaporated. The residue was subject to short column chroma-

tography over silica gel (0.5-0.7% methanol in CH₂Cl₂, v/v) to

obtain 24 (38 mg, 73.5%) as a white foam. ¹H NMR (500 MHz,

3

H1⁰), 4.63 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.57 (1H, d, J_gem

11.5 Hz, CH₂Bn), 4.53 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.48 (1H,

0

d, J_gem=11.5 Hz, CH₂Bn), 4.23 (1H, d, J_{2 ,3}=4.5 Hz, H3 ), 3.77

(1H, d, J_gem=11.0 Hz, H5⁰), 3.59 (1H, d, J_gem=11.0 Hz, H5⁰⁰),

0

3.55 (1H, d, J_{2 ,3}=4.5 Hz, H2 ), 2.22 (2H, m, H6 and H7 ), 1.83

(1H, m, H7⁰⁰), 1.79 (3H, s, 8⁰-CH₃), 1.46 (3H, d, J_6,CH=1.5 Hz,

3

thymine-CH₃), 1.43 (1H, dd, H6⁰⁰). ¹³C NMR (125 MHz,

CDCl₃) δ 162.8 (C4), 157.2, 155.7 (oxalyl CdO), 148.9 (C2),

136.4, 136.0 (2 ꢀ C_ipso-Bn), 134.8 (C6), 127.7-126.4 (aromatic),

108.5 (C5), 84.5 (C1⁰), 83.2 (C8⁰), 82.7 (C4⁰), 73.6 (C3⁰), 73.0, 72.6

(2 ꢀ CH₂Bn), 69.1 (C5⁰), 51.9 (OCH₃), 46.8 (C2⁰), 31.0 (C7⁰), 23.7

(C6⁰), 22.9 (8⁰-CH₃), 10.9 (CH₃-thymine). MALDI-TOF m/z

[C₃₁H₃₄N₂O₉þ H]^þfound 579.235, calcd 579.234.

General Procedure for Phosphoramidite Synthesis. DIPEA

(5 equiv) and 2-cyanoethyl-N,N-diisopropylphosphoramido-

chloridite (3 equiv) were added dropwise to a solution of

substrate (1 equiv) in dry CH₂Cl₂in an ice bath. The reaction

was allowed to warm to room temperature and stirred at this

temperature for 3 h (overnight for 22a). The reaction was

quenched with dry methanol followed by stirring over 15 min.

Then the mixture was diluted with ethyl acetate and washed with

saturated NaHCO₃solution. The organic layer was dried over

MgSO₄and concentrated. The residue was chromatographed

on silica gel to obtain the corresponding phosphoramidite,

which was first precipitated in n-hexane then dried over P₂O₅

under vacuum for 3 days before it was used for DNA synthesis.

Oligonucleotide Synthesis and Purification. All AONs were

synthesized with an automated DNA/RNA synthesizer based

on phosphoramidite chemistry. For native A, G, and C building

blocks, fast deprotecting phosphoramidites (Ac for C, iPr-Pac

for G, Pac for A) were used. Standard DNA synthesis reagents

and cycle were used except that 0.25 M 5-ethylthio-1H-tetrazole

was used as the activator and Tac₂O as the cap A. For incorpor-

ating modified nucleotides, extended coupling time (10 min

compared to 25 s for native nucleotides, 15 min for 30a) was

used. For AONs 2-9 the protection was performed in 33%

aqueous ammonia at room temperature overnight. For AONs

10-13, 14-17, and 18-21 the protection was performed in 33%

aqueous ammonia at 55 °C for 3, 2, and 1 day, respectively. After

deprotection, all crude oligos were purified by denaturing

PAGE (20% polyacrylamide with 7 M urea), extracted with

0.3 M NaOAc, and desalted with a C-18 reverse phase cartridge

to give AONs in >99% purity, and correct masses have been

obtained by MALDI-TOF mass spectroscopy for each of them

(Table SII 1,Supporting Information).

CDCl₃) δ 8.09 (1H, d, J_6,CH= 1.0 Hz, H6), 7.98 (1H, s, H3),

3

7.49-7.30 (10H, m, aromatic), 5.84 (1H, s, H1⁰), 4.63 (1H, d,

J_gem=11.5 Hz, CH₂Bn), 4.61 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.56

(1H, d, J_gem= 11.5 Hz, CH₂Bn), 4.48 (1H, d, J_gem= 11.5 Hz,

0

CH₂Bn), 4.18 (1H, d, J_{2 ,3}=5.0 Hz, H3 ), 3.71 (1H, d, J_gem=10.5

0

Hz, H5⁰), 3.57 (1H, d, J_gem=10.5 Hz, H5⁰⁰), 2.60 (1H, m, J_{2 ,3}=5.0

0

Hz, H2⁰), 1.94 (1H, m, H8⁰), 1.87 (1H, m, H6⁰), 1.74 (3H, m, H8⁰⁰,

H7⁰and H7⁰⁰), 1.46 (3H, d, J_6,CH=1.0 Hz, thymine-CH₃), 1.36

3

(1H, m, H6⁰⁰). ¹³C NMR (125 MHz, CDCl₃) δ 163.7 (C4), 149.9

(C2), 137.8, 137.5 (2 ꢀ C_ipso-Bn), 136.5 (C6), 128.6-127.4

(aromatic), 109.1 (C5), 87.5 (C1⁰), 85.4 (C4⁰), 73.5 (CH₂Bn), 73.0

(C3⁰), 71.7 (CH₂Bn), 70.7 (C5⁰), 42.9 (C2⁰), 26.9 (C6⁰), 20.7 (C8⁰),

17.7 (C7⁰), 11.8 (CH₃-thymine). MALDI-TOF m/z [C₂₇H₃₀N₂O₅þ

H]^þfound 463.226, calcd 463.223.

(1R,3R,4R,5S,8S)-8-Benzyloxy-1-benzyloxymethyl-5-hydroxy-

5-methyl-3-(thymin-1-yl)-2-oxa-bicyclo[3.2.1]octane (26). 19 (100 mg,

0.21 mmol) was dissolved in dry THF (3 mL) and the mixture was

cooled to -78 °C, then MeLi (393 μL, 1.6 M in Et₂O, 0.63 mmol)

was added dropwise. After the solution was stirred at -78 to

-65 °C for 10 h, the reaction was quenched by addition of

saturated NH₄Cl solution. Upon warming to room temperature,

the mixture was diluted with EtOAc and H₂O. The aqueous layer

was extracted with EtOAc three times. The combined organic

layers were dried by MgSO₄, and then evaporated to dryness. 26

(60 mg, 58%) was obtained by chromatography on silica gel

(0.5-3% methanol in CH₂Cl₂, v/v), and starting material (30 mg,

30%) was recovered. ¹H NMR (500 MHz, CDCl₃) δ 8.35 (1H, s,

H3), 8.06 (1H, d, J_6,CH= 1.5 Hz, H6), 7.40-7.28 (10H, m,

=

3

aromatic), 5.66 (1H, s, H1⁰), 4.90 (1H, s, OH), 4.76 (1H, d, J_gem

11.0 Hz, CH₂Bn), 4.62 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.58 (1H, d,

J

_gem=11.0 Hz, CH₂Bn), 4.56 (1H, d, J_gem=11.5 Hz, CH₂Bn), 4.35

0

(1H, d, J_{2 ,3}=4.5 Hz, H3 ), 3.77 (1H, d, J_gem=11.0 Hz, H5⁰₀), 3.60

UV Melting Experiments. Determination of the T_mof the

AON:RNA hybrids or AON:DNA duplex was carried out in the

following buffer: 60 mM Tris-HCl (pH 7.5), 60 mM KCl, 0.8

mM MgCl₂. Absorbance was monitored at 260 nm in the

temperature range from 20 to 70 °C, using an UV spectro-

photometer equipped with a Peltier temperature programmer

with the heating rate of 1 deg/min. Prior to measurements, the

samples (1 μM of AON and 1 μM cDNA or RNA mixture) were

preannealed by heating to 80 °C for 5 min followed by slow

cooling to 21 °C and 30 min of equilibration at this temperature.

The value of T_mis the average of two or three independent

measurements. If error of the first two measurements is >(0.3 °C,

0

(1H, d, J_gem=11.0 Hz, H5⁰⁰), 2.48 (1H, d, J_{2 ,3}=4.5 Hz, H2₀), 1.98

0

00

0

00

(1H, ddd, J_{6 ,7}=6.0 Hz, J_{6 ,7}=13.0 Hz, J_{6 ,6}=13.0 Hz, H6 ), 1.82

0

(1H, ddd, J_{7 ,6}=6.0 Hz, J_{6 ,7}=13.0 Hz, J_{7 ,7}=14.0 Hz, H7 ),

0

1.75 (1H, dd, J_{6 ,7}=6.0 Hz, J_{7 ,7}=14.0 Hz, H7 ), 1.50 (3H, d,

00

0

00

0

00

0

00

J

_6,CH=1.5 Hz, thymine-CH₃), 1.44 (4H, m, J_{6 ,7}=6.0 Hz, J_{6 ,6}=

3

13.0 Hz, CH₃and H6⁰⁰).¹³C NMR (125 MHz, CDCl₃) δ162.7 (C4),

148.8 (C2), 136.1, 135.1 (2 ꢀ C_ipso-Bn), 135.0 (C6), 127.7-127.0

(aromatic), 108.3 (C5), 84.9 (C1⁰), 82.6 (C4⁰), 74.7 (C3⁰), 73.1, 72.6

(2 ꢀ CH₂Bn), 71.2 (C8⁰), 69.1 (C5⁰), 49.3 (C2⁰), 32.2 (C7⁰), 26.6 (8⁰-

CH₃), 23.4 (C6⁰), 10.9 (CH₃-thymine). MALDI-TOF m/z [C₂₈H₃₂-

N₂O₆þ H]^þfound 493.235, calcd 493.234.

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

Liu et al.

JOCArticle

the third measurement was carried out to check if the error is

indeed within (0.3 °C, otherwise it is repeated again.

presence of 0.08 U E. coli RNase H (obtained from USB

corporation, Cleveland, Ohio). Prior to the addition of the

enzyme, reaction components were preannealed in the reaction

buffer by heating at 80 °C for 5 min followed by slow cooling to

21 °C and 30 min equilibration at this temperature. Total

reaction volume was 30 μL. Aliquots of 3 μL were removed

after 5, 10, 15, 30, and 60 min, and the reactions were terminated

by mixing with stop solution [containing 0.05 M EDTA, 0.05%

(w/v) bromophenol blue, and 0.05% (w/v) xylene cyanole in

80% formamide]. The samples were subjected to 20% 7 M urea

PAGE and visualized by autoradiography. Pseudo-first-order

reaction rates could be obtained by fitting the digestion curves to

single-exponential decay functions.

³²P Labeling of Oligonucleotides. The oligoribonucleotides

and oligodeoxyribonucleotides were 5⁰-end labeled with ³²P,

using T4 polynucleotide kinase, [γ-³²P]ATP, and the standard

protocol. Labeled AONs and the target RNA were purified by

QIAquick Nucleotide Removal Kit, and specific activities were

measured with a Beckman LS 3801 counter.

SVPDE Degradation Studies. Stability of the AONs toward

3⁰-exonucleases was tested by using phosphodiesterase I from

Crotalus adamanteus (obtained from USB corporation, Cleveland,

Ohio). All reactions were performed at 3 μM DNA concentra-

tion (5⁰-end ³²P labeled with specific activity 80 000 cpm) in

100 mM Tris-HCl (pH 8.0) and 15 mM MgCl₂at 21 °C. An

exonuclease concentration of 6.7 ng/μL was used for digestion of

oligonucleotides. Total reaction volume was 30 μL. Aliquots

(3 μL) were taken at proper time points and quenched by

addition of stop solution (4 μL) [containing 0.05 M EDTA,

0.05% (w/v) bromophenol blue, and 0.05% (w/v) xylene cyanole

in 80% formamide]. Reaction progress was monitored by 20%

denaturing (7 M urea) PAGE and autoradiography.

The total percentage of integrated AONs (13- and 14mer) was

plotted against time points to give the digestion curve, and the

pseudo-first-order reaction rate could be obtained by fitting

curves to single-exponential decay functions.

Stability Studies in Human Blood Serum. AONs at 2 μM con-

centration (5⁰-end ³²P labeled with specific activity 80 000 cpm) were

incubated in 10 μL of human blood serum (male AB, obtained from

Sigma-aldrich) at 21 °C (total reaction volume was 36 μL). Aliquots

(3 μL) were taken at proper time points and quenched with 4 μL of

stop solution [containing 0.05 M EDTA, 0.05% (w/v) bromophenol

blue, and 0.05% (w/v) xylene cyanole in 80% formamide], resolved

in 20% polyacrylamide denaturing (7 M urea) gel electrophoresis,

and visualized by autoradiography.

Acknowledgment. Generous financial support from the

Swedish Natural Science Research Council (Vetenskapsradet),

€

the Swedish Foundation for Strategic Research (Stiftelsen for

Strategisk Forskning), and the EU-FP6 funded RIGHT project

(Project no. LSHB-CT-2004-005276) is gratefully acknowl-

edged. Jianfeng Xu and Mansoureh Karimiahmadabadi have

equally contributed in this work.

Supporting Information Available: ¹H, ¹³C, ³¹P, COSY,

HMQC, and HMBC of all carba-ENA derivatives (SI part I); 1D

NOE spectra of key intermediates 14, 20a, 21b, 26, and 28,

decoupling experiments of key intermediates 14, 16b, and 21a/b

(SI part II); denaturing PAGE analysis of the SVPDE degradation

of AONs with modified carba-LNA analogues; discussion about

stability of carba-ENA analogues-modified AONs in blood serum;

autoradiograms of 20% denaturing PAGE as well as degradation

curves, showing the cleavage kinetics of target RNA in AON:RNA

hybrid duplexes by E. coli RNase H1; all individual reaction steps,

workup, and product characterization for the intermediates

21b, 22a/b, 25, 28, and 29, ENA and the characterization for

final amidites 18a/b, 30a/b, 31, and 32 (SI part II). This

material is available free of charge via the Internet at http://

pubs.acs.org.

RNase H Digestion Assay. Target 0.1 μM RNA (specific

activity 80 000 cpm) and AON (2 μM) were incubated in a

buffer containing 20 mM Tris-HCl (pH 7.5), 20 mM KCl, 10

mM MgCl₂, 0.1 mM EDTA, and 0.1 mM DTT at 21 °C in the

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

Article Doi

Synthesis of 2′,4′-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

DOI: 10.1021/jo101207d

Source and publish data:

Authors:

Article abstract of DOI:10.1021/jo101207d

Full text of DOI:10.1021/jo101207d

Products guided by the article

R&D Labs maybe for 1254203-74-1

Relevant to this article

Hot Product