Synthesis of conformationally locked carba-LNAs through intramolecular free-radical addition to C=N. Electrostatic and steric implication of the carba-LNA substituents in the modified oligos for nuclease and thermodynamic stabilities

pubs.acs.org/joc

Synthesis of Conformationally Locked Carba-LNAs through

Intramolecular Free-Radical Addition to CdN. Electrostatic and Steric

Implication of the Carba-LNA Substituents in the Modified Oligos for

Nuclease and Thermodynamic Stabilities

Jianfeng Xu, Yi Liu,^†Christelle Dupouy,^†and Jyoti Chattopadhyaya*

Department of Bioorganic Chemistry, Box 581, ICM, Biomedical Center, Uppsala University, SE-751 23

Uppsala, Sweden. ^†Drs. Liu and Dupouy contributed equally to the project.

jyoti@boc.uu.se

Received May 14, 2009

The syntheses of the hitherto unavailable parent fully unsubstituted carba-LNA and its C7⁰-amino and/or

C6⁰-hydroxyl substituted derivatives, have been accomplished by the intramolecular 5-exo free-radical

addition to a C4⁰-tethered CdN to give carba-LNAs with variable hydrophilic substituents at C6⁰/C7⁰

(amino and/or hydroxyl). They have been introduced into isosequential antisense oligonucleotides

(AONs) to examine how their relative electrostatic and steric effects in the minor groove of a putative

AON-RNA duplex affect the target affinity, nuclease resistance, and RNase H elicitation. We show that

0

ꢀ

2 -oxygen in LNA is important in stabilizing the DNA/DNA and DNA/RNA duplexes vis-a-vis the

unsubstituted carba-LNA and its other derivatives and that hydrophobic groups at C6⁰/C7⁰in both

carba-LNA and carba-ENA relatively destabilize the AON/DNA duplex more profoundly than those in

the AON/RNA duplexes. Two main factors affect the relative stabilities of AON/DNA versus AON/

ꢀ

RNA duplexes: (i) hydration in the minor groove depending upon hydrophilicity vis-a-vis hydropho-

bicity of the substituents, and (ii) the relative size of the minor groove in the AON/DNA versus AON/

RNA duplexes dictates the steric clash with the substituents depending upon their relative chiralities. We

also show how the chirality and chemical nature of the C6⁰/C7⁰substituents affect the nuclease stability as

well as the thermal stability and the RNase recruitment by AON/RNA duplexes.

Introduction

cancer, viral, or inflammatory diseases as well as for diag-

nostics. Each generation of chemical modification has shown

its own advantages and disadvantages. Thus, DNA phos-

phorothioates (PS-DNA) have been found to exhibit

Chemical modification of oligonucleotides continue to be

potentially important as nucleic acid directed therapeutics,

as antisense (AONs), siRNA or triplexing agents against

6534 J. Org. Chem. 2009, 74, 6534–6554

Published on Web 08/11/2009

DOI: 10.1021/jo901009w

r

2009 American Chemical Society

Xu et al.

JOCArticle

improved nuclease resistance but poor affinity to a comple-

mentary RNA.^1,2The 2⁰-O-substituted nucleic acids in which

the 2⁰-O-substituents drive the ribose to a preferential C3⁰-

endo (North-type) conformation show good affinities to the

RNA target.^3-5The AONs with one or more modifications

at the 3⁰-end with 2⁰-O-substituents, such as 2⁰-O-(3-amino-

propyl)⁶or 2⁰-O-[2-[2-(N,N-dimethylamino)ethoxy]ethyl]⁷),

show improved nuclease resistance.⁸On the other hand, new

conformationally constrained bicyclic ring systems such as

LNA,^9,10ENA,¹¹and their aza-analogues (2⁰-amino-LNA¹²

and aza-ENA¹³) are unique in that they have the continuum

of the dynamic sugar pseudorotamer population locked into

a fixed 3⁰-endo conformation.¹⁴Thus, LNA or ENA inte-

grated oligonucleotides are preorganized to A-type canoni-

cal structure¹⁵and show unusually high affinity toward

complementary RNA strand, but they do not have good

nuclease resistance when compared to that of the PS

AONs.¹⁶

Lately, the synthesis of a carbocyclic uridine analogue for

ENA (i.e., carba-ENA-U) was reported by Nielsen⁰s

group.¹⁷Although the AONs containing carba-ENA-U

showed higher RNA-selective recognition compared to that

of ENA,¹¹no study has so far been conducted on the

nuclease stability. Independently, the syntheses of the con-

formationally locked five-membered 7⁰-methyl-2⁰,4⁰-carbo-

cyclic-LNA thymine analogue (7⁰-methyl-carba-LNA-T)

and six-membered 7⁰-methyl-2⁰,4⁰-carbocyclic-ENA thy-

mine analogue (8⁰-methyl-carba-ENA-T) were reported by

us.¹⁸These unique carbocyclic 2⁰,4⁰-bridged-LNA and -ENA

when incorporated into the modified AONs confer a un-

precedented nuclease resistant property in the blood serum,

without compromising the RNA affinity (compared to those

of the isosequential LNA containing AONs), and yet they

show uncompromising RNase H elicitation compared to

that of the native counterpart. Moreover, the two carbon

atoms in the 2⁰,4⁰-carba-bridge of carba-LNA and carba-

ENA (herein defined as 6⁰-carbon and 7⁰-carbon in case of

carba-LNA, and 6⁰-carbon and 8⁰-carbon in the carba-ENA

derivatives) are only ∼4 A away from the phosphate back-

bone of the AON/RNA heterodupex. This has led us to

functionalize these two carbon atoms in the 2⁰,4⁰-carba-

bridge with a bulky, hydrophobic 7⁰-methyl and/or hydro-

philic 6⁰-hydroxyl group to modulate important electro-

static interactions around the phosphodiester linkage in its

homo or heteroduplex form.¹⁹The thermal denaturation

studies and stability assay of these modified AONs show

that the hydrophobic nature of the 7⁰-methyl group and its

stereochemical configuration play an important role in

both the RNA affinity of modified AONs and their stabi-

lities toward 3⁰-exonuclease. Whereas the stereochemical

orientation of the 6⁰-hydroxyl group in carba-LNA does

not exert a marked effect on the AON/RNA duplex thermal

stability, the corresponding AONs show the chirality-de-

pendent stability of the scissile internucleotidic phosphate

toward 3⁰-exonuclease. A rationalization of these effects

was advanced by arguing that the 7⁰-methyl in carba-LNA

located at the center of the minor grooves could decrease

the magnitude of hydration around the scissile phosphate

and perturb the interaction between nuclease and phos-

phate linkage owing to the steric clash²⁰and hydro-

phobic repulsion, especially when the methyl group is

pointed toward the internucleotidic 3⁰-phosphate in the

steric proximity.

We argued that it would be of considerable interest to

show how the AONs containing modified carba-LNA

behave in the study of RNA affinity and 3⁰-exonuclease

stability when the hydrophobic effect of the 7⁰-methyl

group is reversed to the hydrophilic effect upon substitution

at C7⁰by a positively charged amino group (i.e., 7⁰-amino-

carba-LNA). We furthermore argued that this structure-

activity study could be important if one could utilize the

hitherto new parent unsubstituted carba-LNA as a refer-

ence point. Thus the synthesis of the parent unsubstituted

carba-LNA and 7⁰-amino-carba-LNA analogues appealed

to us for several reasons. First, the 7⁰-amino functionality

could be a well-defined conjugation site in a conformation-

ally restricted LNA-type AON.²¹Second, amine-deriva-

tized AONs have displayed increased thermal affinities

toward complementary AONs and superior nuclease resis-

tance possibly because the introduction of positively

charged moieties partially neutralized the negatively

charged phosphates in the duplex at physiological pH.²²

Third, compared to 2⁰-amino-LNA (aza-LNA)¹²or aza-

ENA,¹³the 7⁰-amino functionality with defined stereo-

chemistry (S or R) at 7⁰-carbon will give more stereochem-

ical information about the minor groove of AON/RNA

heteroduplex, which may give more in-depth understanding

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J. Org. Chem. Vol. 74, No. 17, 2009 6535

Xu et al.

JOCArticle

on the effect of electrostatic modulation by substituents

on the 2⁰,4⁰-bridge of modified carba-LNA at the center

of the minor groove, compared to those of our previous

result.¹⁹

deoxygenation [10 f 9 f 8 f 2 f 5 f 6 f 7 f 1 ]. So in

the synthesis of parent carba-LNA (1), 7⁰-amino derivative

(2) and its oxidized products 5 and 6 may constitute key roles

in our strategy. Similarly, the oxime-ether 11 was expected

to give 6⁰-hydroxy-7⁰-amino derivative 3 [14 f13 f 12a/b f

11 f 4 f 3] by the key free-radical cyclization of the pre-

cursor 12b to CdN of a tethered oximino function to

engineer amino and hydroxyl functionality with both S and

R stereochemistries. Interestingly, the key intermediates 10

and 14 could be made from the common intermediate 15,

which is already known in the literature.^13,19

In the previous study,^18,19an intramolecular free-radical

ring-closure reaction between a radical generated at C2⁰and

a distant double bond at C4⁰was a key step to accomplish

7⁰-methyl-carba-LNA. The intramolecular 5-exo free-radi-

cal addition to a terminal CdC always leads however to an

exocylic diastereomeric 7⁰-methyl group,²³which is often

difficult to manipulate for further transformation and in

the past has forced us to make compromise in our struc-

ture-activity study, since 7⁰-methyl was a constant feature

of our carba-LNA analogues. To circumvent this, we chose

to initiate the 5-exo radical addition reaction to CdN^24-30

of an appropriately protected oximino-ether, with putative

intermediate 8 or 11, as shown in Scheme 1, for the synthesis

of carba-LNAs with or without functionalization at the C6⁰

or C7⁰of the newly formed carbocycle fused to the pentose-

sugar ring to give target compounds 1-3 in Scheme 1.

Interestingly, the benzyl-protected 7⁰-amino-carba-LNAs,

compound 2, can potentially be utilized as key intermediate

to synthesize the parent carba-LNA 1 as outlined in

Scheme 1, which simply could not be afforded by radical

addition/cyclization of a radical center at C2⁰to a distant

double bond appended at C4⁰.^18,19

2.0. Synthesis of Diastereomerically Pure 7⁰-Amino-Carba-

LNAs (2a-7⁰R and 2b-7⁰S). The synthetic route to the 7⁰-

amino-carba-LNAs is shown in Scheme 2. The synthesis

was started from the known precursor 10¹³(Scheme 2).

Deacetylation of 10 with 30% methylamine in methanol at

rt for 1 h gave compound 16 in 95% yield. Reduction of

nitrile group of 16 with DIBALH afforded the aldehyde 9 as

a single product (TLC), which was directly used for the

oximation³¹with O-benzylhydroxylamine after a simple

workup procedure to give oxime 17 as a mixture of Z and

E isomers (60% in two steps). After esterification of 17 with

phenyl chlorothioformate, the key intermediate for radical

cyclization, δ-functionalized O-benzyl oxime 18, was pro-

vided in good yield (40% from compound 10). The radical

was generated by tin hydride reduction of the phenyl

thionocarbonate 18 in toluene at reflux. Herein the oximi-

no-ether of 18 functioned as an effective intramolecular

radical trap to the 5-exo radical cyclization exclusively to

give two enantiomerically pure stereoisomers in which the

major product (60%) is 2a (with 7⁰-amino at the pseudoe-

quatorial position, 7⁰R), along with 2b as the minor product

(4%) (with the 7⁰S stereochemistry). The NMR character-

izations of 2a and 2b have been investigated by ¹H, ¹³C, as

well as COSY, ¹H-¹³C HMQC, long-range ¹H-¹³C corre-

lation (HMBC) experiments and 1D differential NOE

experiment (Supporting Inforamtion). The attempt to

cleave the N-O bond selectively with samarium diiodide³²

without affecting the 3⁰- and 5⁰-benzyl group failed. The use

We herein report the first synthesis of the parent

fully unsubstituted carba-LNA and its C6⁰- and/or C7⁰-

substituted derivatives, which have also led to their intro-

duction into oligonucleotides. The thermodynamic pro-

perties of the various carba-LNA substituted oligonucleo-

tides have been investigated by complexing with the

complementary DNA or RNA, and subsequently their

blood serum and 3⁰-exonuclease stabilities have also been

investigated to assess their potential as nucleic acid direc-

ted therapeutics.

Results and Discussion

of 20% Pd(OH)₂/C and ammonium formate as

a

1.0. Strategy for the Synthesis of Parent Carba-LNA (1)

and Its 7⁰-Amino (2a and 2b) and 6⁰-Hydroxy Analogues (3a

and 3b). We argued that the oxime-ether 8 could give

diastereomerically pure (R or S) 7⁰-amino derivatives (2) by

a key free-radical cyclization step to a tethered CdN, and

once the synthesis of 7⁰-amino-carba-LNA (2) was achieved,

we could easily oxidize the 7⁰-amino group to a 7⁰-oximino

and then to a 7⁰-keto group, which could be reduced to the 7⁰-

hydroxy function (compound 7) under a standard condition,

which could easily give the parent carba-LNA 1 upon

hydrogenolysis reagent at reflux temperature, however,

removed the 3⁰- and 5⁰-benzyl groups easily.¹⁹Therefore,

10% Pd/C was used instead of 20% Pd(OH)₂/C at rt to

reduce 7⁰-N-OBn group in 2a and 2b to 7⁰-amino without

any loss of 3⁰- and 5⁰-benzyl groups; the crude higly

polar putatative 7⁰-amino carba-LNA thus obtained was

not isolated. It was subjected to trifluoroacetylation with

ethyl trifluoroacetate to provide 7⁰-amino trifluoroacetyl

protected compounds 19a/19b (50% in two steps), which

were confirmed by ¹³C and long-range ¹H-¹³C corre-

lation (HMBC) experiments. In the ¹³C spectrum of 19a/

19b, we observed typical quatertet peaks around 110 and

150 ppm for the trifluoroacetyl groups. ³J_HCHMBC corre-

lations between NH and the trifluoroacetyl carbonyl

carbon were further used to prove the presence of

7⁰-trifluoroacetyl (TFA) amino functionality in com-

pounds 19a/19b. Debenzylation of 19a/19b with 20%

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6536 J. Org. Chem. Vol. 74, No. 17, 2009

Xu et al.

JOCArticle

SCHEME 1. Retrosynthetic Analysis of the Target Compounds 1-3^a

^aR, R₁, and R₂have been systematically varied in that R₁= OH (OAc) or H, and R₂= NHR₃, dNOH, dO, OH, or H [ R₃= H, OBn, TFA], and R =

Bn or H or 5⁰-O-DMTr and /or 3⁰-phosphoramidite. When R₁= R₂= H (generalformula, 1), it constitutes the first synthesis of the parent unsubstituted

carba-LNA.

SCHEME 2. Synthesis of Diasteromerically Pure 7⁰R- and 7⁰S-Amino-Carba-LNAs 2a/2b and Their 3⁰-Phosphiteamidites 21a/21b

Pd(OH)₂/C and ammonium formate as hydrogenolysis

reagents at reflux was unsuccessful as a result of the strong

reactivity and poor selectivity for the benzylether group

against the TFA function, which was evident by the fact

J. Org. Chem. Vol. 74, No. 17, 2009 6537

Xu et al.

JOCArticle

that the trifluoroacetyl functionality was also removed at

reflux with these reagent. To circumvent this problem,

cyclohexene³³was used as hydrogen donor instead of

ammonium formate to remove the 3⁰- and 5⁰-benzyl groups

with the 7⁰-TFA group intact. After 5⁰-dimethoxytritylation

and phosphitylation with 2-cyanoethyl N,N-diisopropyl-

phosphoramindochloridite under standard conditions,

phosphoramidites 21a/21b (55% from compounds 19a/b)

were obtained as a diastereomeric mixture.

with 2-cyanoethyl N,N-diisopropylphosphoramindochlori-

dite under standard conditions to give phosphoramidites 24

(84%) as a diastereomeric mixture.

4.0. Synthesis of Diastereomerically Pure 6⁰-OH/7⁰-NH₂-

Carba-LNAs (3a/3b). To introduce the hydroxyl group at

C6⁰, the synthesis was started through Grignard reaction on

the aldehyde generated from the compound 15¹⁹(Scheme 4)

to give 14¹⁹as a pure isomer with 6⁰S-OH stereochemistry.

Acetylation of 14 using a mixture of acetic acid, acetic

anhydride, and triflic acid gave the corresponding triacetate

as an anomeric mixture. The crude triacetate was subjected

to cis-hydroxylation with osmium tetraoxide, followed by

sodium periodate cleavage of the diol to provide crude

aldehyde 25. After a simple workup, the crude aldehyde 25

was oximated with O-benzyl hydroxylamine in DCM and

pyridine at reflux to give the corresponding 6⁰-substituted

oxime 13 (82% from 14). Glycosylation of 13 with thymine

using a modified Vorbruggen reaction gave exclusively β-^D-

ribofuranosyl thymine derivatives 12a (93%).¹³The β-con-

figuration of compound 12a was confirmed by 1D differen-

tial NOE experiment, which shows 4% enhancement of H6

3.0. Synthesis of the Parent Carba-LNA (1). Here we have

successfully utilized the major cyclic product 7⁰R-amino-

carba-LNA 2a as a key intermediate to synthesize the parent

fully unsubstituted carba-LNA (Scheme 3). Oxidation of

benzyloxyamine 2a with MCPBA removed the benzyl group

to give 7⁰-oxime 5 (60%) under a basic condition.^34,35The

structure of oxime 5 was confirmed by ¹H, ¹³C, and HMBC

1

spectrum. In the H spectra, the peak for H7⁰was not

observed whereas the typical peak around 155 ppm for the

oxime-carbon could be found in the ¹³C spectra of com-

pound 5 which shows correlations with H1⁰, H2⁰, H3⁰and

H6⁰in the HMBC experiment. There are several methods

such as hydrolytic, oxidative and reductive methods³⁶to

regenerate ketone from the oxime are available in the

literature. For our nucleosides with complex functionalities,

we have, however, chosen the mild Dess-Martin periodinane

^37-39reagent to transform the 7⁰-oxime 5 to 7⁰-ketone 6. The

reduction of ketone 6 to alcohol with NaBH₄was highly

stereoselective to give compound 7 (40% in two steps) with

the C7⁰(S)-OH stereochemistry as the only product which

was ₀proved by strong NOE enhancement for H7⁰(3.8%)

upon irradiation of H2⁰(d_{H2 -H6}≈ 2.5 A for β anomer,

0

whereas d_{H2 -H6}≈ 5.0 A for R anomer). Deacetylation of 12a

using 30% methylamine in ethanol at rt gave compound 26

(96%), which was selectively esterified with phenyl chlor-

othioformate to give 2⁰-O-phenoxythiocarbonyl (PTC) deri-

vatives 27 (68%). The regioselectivity of this esterification

was probably due to the steric effect of the three hindered

benzyl groups surrounding the 6⁰-hydroxyl function in 26. In

the previous study,¹⁹it has been shown that such an exposed

hydroxyl group does not affect the nature of intramolecular

5-hexenyl radical cyclization leading to the key carbocyclic

compound. In this work, however, it was found that

the unprotected 6⁰-hydroxyl group in 27 indeed affects the

5-hexenyl radical cyclization process to CdN of an oximino-

ether in that we recovered 80% starting material intact and

the rest of the material (20%) was decomposed to an

inseparable mixture. Hence, compound 27 was protected

with an acetyl group with acetic anhydride in dry pyridine to

provide the key radical precursor 12b (81%) for the radical

cyclization. Thus, the 5-exo free-radical cyclization reaction

of 12b went on smoothly in refluxing anhydrous toluene,

using AIBN as the initiator to give two diastereomerically

pure compounds, in which the major product (34%) is 4a

(with 6⁰S and 7⁰S stereochemistries), along with 4b as the

minor product (24%) (with 6⁰S and 7⁰R stereochemistries).

To circumvent the migration of 6⁰-O-acetyl group to 7⁰-

amino, the 6⁰-O acetyl group was first removed with 30%

methylamine before the deprotection of 7⁰-amino with 10%

Pd/C and ammonium formate. After trifluoroacetylation

of the resulting free 7⁰-amino group, the 6⁰-hydroxyl group

was reprotected with acetyl to give 29a/29b (40% from

compound 4a/b), which were debenzylated with 20% Pd-

(OH)₂, using cyclohexene as hydrogen donor. In this

strategy, both 6⁰-O-acetyl and 7⁰-N-trifluoroacetyl could be

kept intact during hydrogenolysis of the benzyl group.

So cyclohexene can serve as a mild hydrogen donor com-

pared to ammonium formate to give much improved selec-

0

(d_{H1 -H7}≈ 2.4 A) when H1⁰was irradiated. The stereose-

lectivity for this reduction was due to the steric hindrance of

bulky benzyl group at the 3⁰-OH which was located above

the carbocycle ring and blocked the hydride attack to C7⁰

from the top. For the same reason, subsequent esterification

of 7 with bulky phenyl chlorothioformate failed due to the

unfavored orientation of hydroxyl group. Hence, sterically

less bulky 7⁰-O-(methylthio)thiocarbonate⁴⁰group in 22 was

introduced, as a radical precursor instead of phenyl thiono-

carbonate, by the three components reaction with more

reactive and less hindered reagents, CS₂and MeI along with

NaH as a proton absorbent. After the standard radical

deoxygenation with tin hydride, the benzyl protected parent

carba-LNA-T (1) was obtained in 40% yield (from compound

7). The chemical nature of the 7⁰-methylene group in carba-

LNA-T (1) wasverifiedbyDEPTandHMQCexperiments. In

the DEPT spectra, C7⁰appeared as a secondary carbon and in

the HMQC spectra, C7⁰has two protons attached. The benzyl

protected parent carba-LNA-T (1) was debenzylated through

catalytic hydrogenolysis with 20% Pd(OH)₂/C and ammo-

nium formate at reflux followed by 5⁰-O-dimethoxytritylation

to give 23 (70% yield in two steps), which was phosphitylated

(33) Hanessian, S.; Liak, T. J.; Vanasse, B. Synthesis 1981, 396–397.

(34) Simpkins, N. S.; Stokes, S. Tetrohedron Lett. 1992, 33, 793–796.

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

Trans. 1 1992, 2471–2477.

ꢀ

(36) Corsaro, A.; Chiacchio, U.; Pistara, V. Synthesis 2001, 13, 1903–

1931.

(37) Bose, D. S.; Narsaiah, A. V. Synth. Commun. 1999, 29, 937–941.

(38) Chaudhari, S. S.; Akamanchi, K. G. Tetrohedron Lett. 1998, 39,

3909–3212.

(39) Chaudhari, S. S.; Akamanchi, K. G. Synthesis 1998, 5, 760–764.

(40) Mereyala, H. B.; Pola, P. Synth. Commun. 2002, 32, 2453–2458.

ꢀ

tivity for O-benzyl group deprotection vis-a-vis a TFA-

amide group. After 5⁰-O-dimethoxytritylations and phosphi-

tylations with 2-cyanoethyl N,N-diisopropylphosphoramin-

dochloridite, the 6⁰-hydroxyl-7⁰-amino-carba-LNA amidite

6538 J. Org. Chem. Vol. 74, No. 17, 2009

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SCHEME 3. Synthesis of Fully Unsubstituted Parent Carba-LNA 1 and 5⁰-O-DMTr-3⁰-Phosphiteamidites 24

0

derivatives 31a/31b (56% from compounds 29a/b) were

afforded.

to strong NOE enhancement for H7⁰(5.3%) (d_{H1 -H7}≈ 2.3

A), suggesting C7⁰is in 7⁰S configuration. For compound 4a/

b, the configuration of C6⁰was determined at the Grignard

reaction stage and was found¹⁹to have 6⁰S configuration.

The configuration of C7⁰was also determined by 1D NOE

experiment as well as by the evaluation of coupling constants

using a Karplus-type equation. For compound 4a, irradiation

0

5.0. NMR Characterization of Key Fused Carbocyclic

Nucleoside Intermediates Involved in the Synthesis. All inter-

mediates and final products were characterized by ¹H, ¹³C,

COSY, ¹H-¹³C HMQC, HMBC and mass spectroscopy [see

Supporting Information].

That the key radical ring-closure reaction successfully

gives the fused carbocyclic products has been proven by

2D proton-proton and proton-carbon correlation experi-

ments. Thus, the ³J_HCHMBC correlations between H1⁰and

C7⁰in compound 2a/b (SI.34 for compound 2a, SI.40 for

compound 2b) (in Scheme 2) and 4a/b (SI.117 for compound

4a, SI.122 for compound 4b) (in Scheme 4) proved that the

oxa-bicyclo [2.2.1] heptane ring systems have indeed been

formed during radical cyclization. This five-membered ring

systems in compound 2a and 4a were further confirmed

of H1⁰leads to 1.9% NOE enhancement for NH (d_{H1 -NH}

≈

2.1 A), but none for H7⁰(d_{H1 -H7}≈3.8 A), whereas irradiation

0

of H7⁰leads to 5.4% NOE enhancement for H6⁰(d_{H6 -H7}

≈

0

2.3 A), suggesting that H6⁰and H7⁰were in cis disposition,

which is consistent with the coupling constants (³J_{6 ,7}= 9.5

Hz, hence dihedral angle for H6⁰-C6⁰-C7⁰-H7⁰≈ 18°). As

for compound 4b, strong NOE enhancement for H7⁰(4.9%)

0

(d_{H1 -H7}≈ 2.3 A) was observed when H1⁰was irradiated,

suggesting C7⁰is in 7⁰R configuration which was further

supported by the observation of the four-bond W-coupling

0

3

w

by observation of J_HHcorrelations between H2⁰and H7⁰

( J) between H3⁰and H7⁰( J_{3 ,7}= 2 Hz). The relatively

0

3

(³J_{2 ,7}= 4.2 Hz for compound 2a and J_{2 ,7}= 4.0 Hz for

compound 4a), whereas the ³J_HHcorrelations of compound

2b and 4b were not observed in the COSY spectrum (SI.38 for

smaller coupling constant between H6⁰and H7⁰( J_{6 ,7}) is 3 Hz,

and hence dihedral angle for H6⁰-C6⁰-C7⁰-H7⁰≈ 113°,

suggesting that H7⁰and H6⁰are in trans disposition. The

coupling constants for all cyclic compounds were obtained

by H-H homodecoupling experiments (SII.11-22) and found

to corroborate very well with the results obtained by the NOE

experiments (SII.3-10).

0

compound 2b, SI.120 for compound 4b) since the ³J_{2 ,7}were

very close to 0 Hz, which suggested that the torsion angle

H2⁰-C2⁰-C7⁰-H7⁰was close to 90°, corroborating the 7⁰S

configuration in compound 2b and the 7⁰R configuration in

compound 4b (both of their amino groups point, however, at

the 3⁰-phosphate). The relative configurations of C7⁰in

compounds 2a/b and 4a/b were also confirmed by 1D

differential NOE experiments (Figure 1). For compound

2a, irradiation of H1⁰leads to NOE enhancement for H2⁰

0

6.0. Stereoselectivity of the Radical Ring-Closure Reaction.

The preference of the 5-hexenyl radical to orchestrate the exo

cyclization over the endo mode can be explained by the

preferred formation of a chairlike transition state, which

suggests that radical ring closures are subject to stereoelectronic

and kinetic control rather than thermodynamic control.⁴¹

0

(1.8%) (d_{H1 -H2}≈ 2.8 A) and NH (0.6%) (d_{H1 -NH}≈ 3.4 A),

but none for H7⁰(d_{H1 -H7}≈ 3.8 A), suggesting C7 is in 7 R

0

configuration. As for compound 2b, irradiation of H1⁰leads

(41) Beckwith, A. L. J. Tetrohedron 1981, 37, 3073–3100.

J. Org. Chem. Vol. 74, No. 17, 2009 6539

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JOCArticle

SCHEME 4. Synthesis of Diasteromerically Pure 6⁰S-Hydroxyl-7⁰R- or -7⁰S-Amino-Carba-LNAs 3a/3b and their 5⁰-O-DMTr-3⁰-

Phosphiteamidites 31a/31b

Beckwith et al. have found that 5-hexenyl intramolecular

radical addition took place via cyclopentylcarbinyl radical

intermediate rather than relatively more stabilized cyclohexyl

radical because of unfavorable nonbonding interaction, desta-

bilizing the transition state for 1,6-ring closure.^30,41The pre-

ference for the exo mode in oximino-ether cyclization is even

more profound, probably because of the additional stabiliza-

tion of the aminyl radical intermediate by the lone pair of the

adjacent oxygen atom. So, no cyclic product was found to have

formed in our studies through the endo mode. The intermediate

aminyl radical preferentially adopts a chairlike conformation

with the substituents in the pseudoequatorial orientations to

avoid the 1,3-diaxial interaction, giving 1,5-cis product as the

major diastereomer.²⁴However, the yield and stereoselectivity

in this process is very structure-dependent.²⁹The substitution

of 5-hexenyl radical and 5-oximinoakyl radical can impact the

stereoselectivity by destabilizing the chairlike conformation by

nonbonding/steric interaction. In our case, the radical cyclic

addition reaction to the oximino-ether were carried out with

two different substrates (18 and 12b) as shown in Scheme 5.

Both of the radical reaction proceeded as exo mode to adopt an

energetically favored chairlike conformation in the transition

states (TS1, TS2, TS3, TS4). As for compound 18, the partici-

pation of TS1 involves a higher energy penalty because the

pseudoaxial orientation of bulky O-benzyl hydroxylamino

leads to 1,3-diaxial interaction with the bulky 3⁰-O-benzyl

group, which gives a transition state with cisoid disposition of

two bulky O-benzyl hydroxylamino and 3⁰-O-benzyl groups.

As a result, the participation of TS1 is energetically disfavored,

which only leads to the formation of the minor product 2b with

7⁰S configuration. In contradistinction, TS2 transition state is

energetically favored because of the pseudoequatorial orienta-

tion of O-benzyl hydroxylamino group leading to a transoid

disposition of two bulky O-benzyl hydroxylamino and 3⁰-O-

benzyl groups, which favors the formation of 2a (7⁰R

configuration) as a major product. These explanations are

neatly corroborated by our experimental observation in that

the ratio of isolated and pure cis product 2a versus trans

product 2b is 15:1. For the cyclization of the radical intermedi-

ate, compound 12b, there are also two transition states (cisoid-

TS3 and transoid-TS4) possible to give two products with two

opposite stereochemistries at C7⁰. However, compared to 18,

the preference of transoid stereoselectivity of radical cyclization

reaction of compound 12b is considerably reduced compared to

cisoid radical cyclization, which is evidenced by the experimen-

tally isolated yields, 4a/4b = 1.4:1, respectively. The reason for

the reduced influence of the transoid transition state of the

radical cyclization is most probably because of the presence of

asymmetric induction effect of the C6⁰chirality, which makes

this reaction overall less stereoselective. In the favored transi-

tion state TS4, due to the presence of 6⁰S-O-acetyl, 1,2-cis

repulsion between acetoxy and 7⁰-O-benzyl hydroxylamino

6540 J. Org. Chem. Vol. 74, No. 17, 2009

Xu et al.

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FIGURE 1. Key NOE contacts of carbocyclic compound (2a/b, 4a/b) and the observation of the four-bond W-coupling (^wJ) between H3⁰and

w

H7⁰( J_{3 ,7}= 2 Hz) in compound 4b. R = Bn.

0

SCHEME 5. Mechanism for the Stereoselectivity of the 5-exo Radical Ring-Closure Reaction to a Tethered -CdN

makes TS4 relatively less favored compared to TS2, although

the bulky O-benzyl hydroxylamino is in pseudoequatorial

orientation for both cases. So, the stereoselectivity for cycliza-

tion of compound 12b is decreased by an additional 1,2-cis

repulsion between 6⁰-acetoxy and 7⁰-O-benzyl hydroxylamino

groups. The foregoing discussion only emphasizes the fact that

the stereoselectivity of this oximino radical cyclic addition is

highly structure-dependent.

been carried out by GAUSSIAN 98 program package⁴²at the

Hartree-Fock level using 6-31G**. Relevant calculated³J_H,H

have been back-calculated from the corresponding theoretical

torsions employing the Haasnoot-de Leeuw-Altona gener-

alized Karplus equation^43-45taking β substituent correction

into account (see Table SII.2 in Supporting Information).

The pseudorotational phase angle (P) is a key parameter

for the conformational analysis of sugar moiety. If the sugar

7.0. Determination of Solution Conformation by ³J

moiety adopts the North-type conformation (C₃-endo), the

0

H,H

Coupling Constants at 600 MHz (¹H). The parent and

hydroxyl/amino-substituted carba-LNA nucleosides have

been investigated in silico using ab initio calculations. The

geometry optimizations of the modified nucleosides have

P value equals -1° to 34°.⁴⁶As is shown in Table SII.2, the

(43) Haasnoot, C. A. G.; de Leeuw, F. A. A. M.; Altona, C. Tetrahedron

1980, 36, 2783–2792.

(44) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1972, 94, 8205–8212.

(45) Huggins, M. L. J. Am. Chem. Soc. 1953, 75, 4123–4126.

(46) De Leeuw, H. P. M.; Haasnoot, C. A. G.; Altona, C. Isr. J. Chem.

1980, 20, 108–126.

(42) Frisch, M. J. et al. Gaussian 98 (Revision A.6); Gaussian, Inc.:

Pittsburgh, PA, 1998.

J. Org. Chem. Vol. 74, No. 17, 2009 6541

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TABLE 1. Thermal Denaturation of Duplexes of the Native, Parent Unsubstituted Carba-LNA, 6⁰- and 7⁰-Substituted Carba-LNAs, and LNA Modified

AONs with Complementary RNA or DNA^a

a

^*Molecular weights of all antisense sequences are conformed by MALDI-TOF (see Table SII.1 in Supporting Information) A = 9-adeninyl, C = 1-

cytosinyl, T = 1-thyminyl, T indicates the carba-LNA modified thymidine monomers with the specified structure shown in the table. “Ref.” column indicates

reference duplex with the native AON 1 for T_mcomparison. 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 20-70 °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). ΔT_mvalues were obtained by comparing the T_mvalues of modified AONs 2-37 with that of the native AON 1. TheΔT_m(aver) value obtained

here is the average value for four AONs incorporated with the same compound at four different modification site. ^bModifications of Type VI-IX have been

taken from reported work,^9,18,19and they are used for T_mcomparison with our present set of modified AONs. (For more detailed comparison with all carba-

LNAs and carba-ENAs, see Table SII.1 in Supporting Information.)

sugar moiety in the parent carba-LNA is indeed conforma-

tionally restricted to the North conformation [pseu-

dorotational phase angle P=16.3°, which is very similar to

the value obtained for LNA (P=19.8°)¹⁸]. Hence, the parent

carba-LNA has similar affinity toward complementary

RNA compared to LNA (see section 9.1). Interestingly, the

chemical nature and the chirality of the substituents at 6⁰and

7⁰has also been found to influence puckering of the furanose

sugar ring, resulting in pseudorotational angles ranging from

16° for compound 1 to 26° for compound 2a (see Table SII.2

8.0. Synthesis and Purification of Carba-LNA Modified

AONs 2-21. The phosphoramidites 21a/b, 24, and 31a/b

were incorporated as mono substitution, but at four different

sites, in a 15-mer DNA sequence on an automated RNA/

DNA synthesizer. Standard DNA synthesis cycle (1 μmol

scale) was applied to synthesize the sequence targeted to the

coding region of SV 40 large T antigen. The sequence,

modification site, and structure of modification are show

in Table 1. The modified building block 21a, 24, and 31a gave

satisfactory coupling yield (60-80%), whereas the phos-

phoramidites 21b and 31b only gave 20% of coupling yield

probably due to the orientation of bulky trifluoroacetyl

0

in Supporting Information), albeit they all adopt C₃-endo

conformation.

6542 J. Org. Chem. Vol. 74, No. 17, 2009

Xu et al.

JOCArticle

amino group in 21b and 31b, pointing toward vicinal

3⁰-phosphate, blocking the coupling to some extent. Clea-

vage from the support and deprotection were carried out by

treating the solid support with 33% aqueous ammonia at rt

for 12 h to obtain the fully deprotected AONs 2-21, which

were purified by 20% denatured PAGE, and their structural

integrity was confirmed by MALDI-TOF mass measure-

ment (see Table SII.1 in Supporting Information). The

AONs 22-37 containing LNA,^9,107⁰(S,R)-methyl-carba-

LNA,¹⁸6⁰S-hydroxyl-7⁰S-methyl-carba-LNA,¹⁹and 6⁰S-hy-

droxyl-7⁰R-methyl-carba-LNA,¹⁹have been reported ear-

lier; they were, however, used in this work for comparison

to clarify the modulation effects of modified carba-LNAs in

general.

9.0. Thermal Denaturation Studies of the Carba-LNA

Modified AONs. The T_mvalues of duplex formed by AONs

2-37 with the complementary RNA or DNA were measured

in the present study and compared with that of the native

AON 1 to provide ΔT_m, which are listed in Table 1. The T_m

value of duplexes formed by AONs 38-76 (see Table SII.1 in

Supporting Information) with the complementary RNA or

DNA are also cited from previous publications^18,19on the

modified carba-LNA containing AONs in order to compare

and shed light on the structure-activities relationship (SAR)

of all 6⁰/7⁰-modified carba-LNAs and carba-ENAs (section

9.6). Comparison of ΔT_m(SAR) of AONs containing dif-

ferently modified carba-LNA derivatives clearly illustrate

that the 2⁰-oxa substituent of LNA is engaged in the hydro-

gen bonding to water molecules, which contributes to the

extensive hydration network in the duplex, reducing the

electrostatic repulsion of the internucleotidic phosphates,

and thereby contributing to an increase in the thermody-

namic stability.⁴⁸In the context of the dsDNA duplex with

carba-LNA and LNA, a net decrease of 1-2 °C in T_m

[ΔT_m(aver)=-1 °C] was observed for the carba-LNA incor-

porated AONs (AON 2-5) in the AON/DNA homodu-

plexes, whereas the LNA incorporated AONs (AON 22-

25) duplexes show 0.5-2 °C increase in T_mcompared to the

native AON (AON 1). This suggests that the 2⁰-oxa sub-

stituent of the bicyclic moiety is much more important for the

formation of stabilized B-type dsDNA than that of stabilized

DNA/RNA hybrid, which was also observed in the six-

membered ring systems.¹⁷The extent of hydration of the

wider minor groove in the DNA/RNA duplex is perhaps

relatively less important compared to that of the relatively

narrow and extensively hydrated minor groove in B-type

DNA/DNA duplex. Hence, substitution of the hydrophilic

2⁰-oxa substituent by the hydrophobic C2⁰-methylene is

relatively less tolerated in the AON/DNA duplexes com-

pared to those of the AON/RNA duplexes. This is why the

carba-LNA incorporated AONs are more complementary

RNA-selective by 1-2 °C/modification compared to that of

the LNA counterpart.

9.2. Comparison of Type I (Parent Carba-LNA) with Type

VII [7⁰(S/R)-Methyl-Carba-LNA]. Type VII modification [7⁰

(S/R)-methyl-carba-LNA)] has one additional methyl group

at C7⁰as a diastereomeric mixture¹⁸(7⁰R vs 7⁰S, 7: 3 by

NMR, inseparable mixture) compared to Type I (parent

unsubstituted carba-LNA). The diastereomeric ratio of 7⁰R

to 7⁰S in Type VII suggested that the major product (70%)

with methyl group at the pseudoequatorial orientation (7⁰R) is

how the hydrophilic positively charged amino group versus

0

ꢀ

the hydrophobic methyl group at C7 vis-a-vis the hydro-

philic hydroxyl group at C6⁰(on the basis of the chiralities of

respective substituents) affects the ele₀ctrostatics, steric, and

0

ꢀ

the spine of hydration around the 3 - vis-a-vis 5 -internu-

cleotidic phosphate, thereby modulating the modified AONs

affinity toward the complementary RNA or DNA, as deter-

mined by the measurement of the relative stabilities of the

resulting hetero and homoduplexes. In this study, native

AON (AON 1), the parent unsubstituted carba-LNAs

(AONs 2-5), and LNA^9,10(AONs 22-25) were used as

reference for the sake of comparison (Table 1). Throughout

the present study, we have used four different single-mod-

ified AONs, each one of them encompassing the minor

groove of the AON/DNA and AON/RNA duplexes,

and therefore ΔT_mof all duplexes have been averaged

[ΔT_m(aver)] in a separate column to reflect the average effect

of all minor groove modifications (see Table 1) in general.

9.1. Comparison of Type I (Parent Carba-LNA) with Type

VI (LNA). The experiments show that a single incorporation

of parent carba-LNA (AON 2-5) increases the stability of

the duplexes formed with the complementatry RNA by 3-4

°C [ΔT_m(aver)=3.6 °C], which means ∼1 °C decrease in ΔT_m

compared to that of the LNA-incorporated isosequential

AONs (compare AONs 2-5 with AONs 22-25). The lack of

pointing away from the vicinal 3⁰-phosphate (d_{(7 S-methyl)-3 P}

0

≈ 4.3 A, d_{(7 R-methyl)-3 P}≈ 5.4 A), whereas the pseudoaxial

methyl group in the 7⁰S minor product (30%) is pointing at the

vicinal 3⁰-phosphate. As a result, the Type I modified AONs

2-5 have slightly higher ΔT_m(aver)=∼ 0.5 °C for AON/RNA

duplex compared to those of Type VII modifications (AONs

26-29). This result is consistent with the previous conclusion

made by Zhou et al.¹⁹in that when the hydrophobic 7⁰-methyl

is pointing at the vicinal 3⁰-phosphate, it can impair the AON/

RNA duplex thermal stability by perturbing the hydration

network in the minor groove.

9.3. Comparison of Type II with Type III and Type IV with

Type V Using Type I (Parent Carba-LNA) as Reference. To

transform the hydrophobic nature of carba-LNA, owing to

the endoxcyclic 2⁰-CH₂group, in Type I to a hydrophilic

substitutent to improve the hydration pattern of duplex, the

amino group was introduced at C7⁰with two different

chiralities (S vs R), and the corresponding diastereomerically

pure 7⁰-amino (S and R) modified carba-LNAs was incor-

porated into Type II and Type III modified AONs. Table 1

shows that the C7⁰-amino functionality in either R (Type II)

or S (Type III) configuration can surprisingly destabilize the

AON/RNA duplex compared to Type I modification. Type

II modification decreases ΔT_m(aver) by 1 °C compared to

Type I (parent carba-LNA), whereas Type III decreases

hydrophilic substituent at C2⁰(as in 2⁰-oxa group in LNA^9,10

)

in the parent unsubstituted carba-LNA leads to a decrease in

the heteroduplex stability with complementary RNA, sug-

gesting that the 2⁰-oxygen of LNA plays an important role in

the thermodynamic stability of the corresponding hetero-

duplex, which is supported by Egli⁰s work on the X-ray

crystal structure of LNA duplex.⁴⁷These workers showed

(47) Egli, M.; Minasov, G.; Teplova, M.; Kumar, R.; Wengel, J. Chem.

Commun. 2001, 651–652.

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

269–283.

J. Org. Chem. Vol. 74, No. 17, 2009 6543

Xu et al.

JOCArticle

ΔT_m(aver) by up to 3 °C compared to Type I (parent carba-

relationships of the thermal affinities of all 13 modi-

fied carba-LNAs^18,19and 5 modified carba-ENAs^18,19

(Figure 2) is made here. The C6⁰and/or C7⁰substituent of

carba-LNA are close to the adjacent phosphodiester linkage

(see above for discussion), which suggests that the hydration

pattern, electrostatics, and steric effects around the adjacent

phosphates could be modulated by introducing a hydropho-

bic sterically bulky methyl, hydrophilic hydroxyl, or posi-

tively charged amino group. In summary, the hydrophilic

amino group and hydrophobic methyl group at 7⁰in mod-

ified carba-LNA have a similar destabilizing effect on T_m,

and the extent of destabilizing effects is dependent on the

orientation of the substituents, suggesting that as for the

7⁰-substituted carba-LNA, the stabilities of DNA/RNA

duplexes is determined by the orientation of the 7⁰-substitu-

ents regardless of their chemical nature. On the contrary,

both the hydrophilic hydroxyl group and hydrophobic

methyl group at C6⁰in modified carba-LNA can stabilize the

duplex regardless of their orientation. As for the modified

carba-ENA, they all show less affinity toward the comple-

mentary RNA compared to the five-membered carba-LNA

anologues. (For more detailed discussion see SII.32-33 in

Supporting Information.)

9.7. Competitive Interplay of Electrostatic versus Steric

Effect Dictates the Overall Stability of the AON/RNA Het-

eroduplexes. To explain how different hydrophilic versus

hydrophobic substitutents in carba-LNAs and carba-ENAs

stabilize or destabilize the T_m, we would like to address the

stereochemical location of the substituents of the 2⁰,4⁰-bridge

in the minor groove of the DNA/RNA duplex. In the

7⁰-CH₃/NH₂substituted carba-LNAs, both the bulky hydro-

phobic methyl group and the hydrophilic amino group,

located in the center of the minor groove of the DNA/

RNA duplex, will either perturb or enhance the hydration

network in the minor groove in competition with the desta-

bilizing steric effect imparted by these groups. The steric

clash resulting from the 7⁰-CH₃/NH₂will lead to slight

extension of the minor groove to accommodate the bulky

substituents, which results in a local or global conforma-

tional change at the cost of the thermal stability. With respect

to the AON/RNA duplex, the steric effect is suggested to be

more prominent than the hydration effect. Hence, the hydro-

philic amino group and hydrophobic methyl group with the

same orientation at C7⁰have a similar destabilizing effect on

the DNA/RNA duplex, which has been emphasized in the

discussion above. Clearly, the hydration effect and the steric

effect are even more pronounced in the DNA/DNA duplexes

because of their relatively narrower minor groove width

compared to that of the DNA/RNA duplex, which explains

why the AON/DNA duplex with 8⁰-CH₃-carba-ENA (Types

XV-XIX) modification give a much larger decrease in

T_mcompared to that in the corresponding AON/RNA

duplex, rendering them a good RNA recognizing agent.

Compared to the 7⁰-substituents at carba-LNA or 8⁰-substit-

uents at carba-ENA that are located at the center of the

minor groove of AON/RNA duplex, substituents at 6⁰of

carba-LNAs and carba-ENAs are located at the border of

grooves, close to the phosphate linkage. Hence, the substi-

tuent at 6⁰(CH₃/OH) has a relatively smaller effect on the

hydration network, which leads to less pronounced effects

conferred by the C6⁰-substituents compared to that of C7⁰-

substituents.

LNA). When the 7⁰-amino group points at the vicinal 3⁰-

phosphate (i.e., 7⁰S) (d_{C7 amino(up)-3 P}≈ 4.2 A for 7⁰S versus

0

d_{C7 amino(down)-3 P}≈ 5.4 A for 7⁰R), the destabilization effect

on the AON/RNA duplex is more pronounced than that for

7⁰R-amino, in which the amino group points away from the

vicinal 3⁰-phosphate, despite the fact that the distance dif-

ference between them is only ∼1.2 A, and hence the direc-

tionality of the amino function in the minor groove is

important. This result can be further corroborated by the

comparison of Type IV (6⁰S-hydroxyl-7⁰S-amino-carba-

LNA) with Type V (6⁰S-hydroxyl-7⁰R-amino-carba-LNA)

in that the 6⁰S-hydroxyl-7⁰R-amino-carba-LNA modified

AONs 18-21 (Type V) also lower ΔT_m(aver) for the AON/

RNA duplex by 2.0 °C compared to 6⁰S-hydroxyl-7⁰S-ami-

no-carba-LNA modified AONs 14-17 (Type IV). The role

conferred by the configuration-dependency of the C7⁰-amino

functionality in the AON/RNA heteroduplex is perhaps owing

to a possible steric effect. As for the AON/DNA duplex with a

narrower minor groove, the role conferred by the configura-

tion is more complicated. It can be seen from Table 1 that with

the 7⁰R-amino carba-LNA (the C7⁰-amino is pointing away

from the 3⁰-phosphate), the hydration in the minor groove of

AON/DNA duplex is promoted (hence increase in ΔT_m(aver)

is found compared to that of Carba-LNA), whereas with the

7⁰S-amino carba-LNA (the C7⁰-amino is pointing toward the

3⁰-phosphate), the AON/DNA duplex hydration is relatively

poorer and therefore destabilizing.

0

9.4. Comparison of Type IV with Type VIII and Type V with

Type IX. It is observed that both the hydrophilic 7⁰-amino

group and the hydrophobic 7⁰-methyl group, when they are

in the pseudoaxial disposition, bringing themselves in close

steric proximity to the vicinal 3⁰-phosphate, can destabilize

the duplex formed with the complementary RNA. We ob-

served similar ΔT_m(aver) when comparing Type IV (6⁰S-

hydroxyl-7⁰S-amino-carba-LNA) with Type VIII (6⁰S-hy-

droxyl-7⁰S-methyl-carba-LNA) and Type V (6⁰S-hydroxyl-

7⁰R-amino-carba-LNA) with Type IX (6⁰S-hydroxyl-7⁰

R-methyl-carba-LNA), suggesting that the hydrophilic ami-

no and hydrophobic methyl group with the same orientation

at C7⁰have a similar destabilizing effect on the AON/RNA

duplex. So the relative stabilities of the corresponding AON/

RNA and AON/DNA duplex depend on the chiralities of the

substituents at C7⁰.

9.5. Comparison of Type II with Type IV and Type III with

Type V Modifications. Both Type IV (6⁰S-hydroxyl-7⁰S-ami-

no-carba-LNA) and Type V (6⁰S-hydroxyl-7⁰R-amino-carba-

LNA) modifications have one additional 6⁰S-hydroxyl group,

which points away from the vicinal 3⁰-phosphate (d_{6 S-hydroxyl-3 P}

0

≈ 6.0 A versus d_{6 S-hydroxyl-5 P}≈ 4.6 A), compared to Type II

(7⁰R-amino-carba-LNA) modification and Type III (7⁰S-ami-

no-carba-LNA) modification, respectively. As a result, the

6⁰S-hydroxyl group of AONs 14-17 (Type IV) and AONs

18-21 (Type V) stabilize the AON/DNA duplex by ∼1 °C in

ΔT_m(aver) compared to that of AONs 6-9(Type II) and AONs

10-13 (Type III), respectively.

9.6. Structure-Activity Relationships of Modified Carba-

LNA and Carba-ENA for Thermal Affinity. To fully illustrate

and understand the functional modulation of the AON

properties rendered by different chiral substituents at the

2⁰,4⁰-fused carbon bridge of carba-LNAs and carba-ENAs,

a brief comparative discussion on the structure-activity

6544 J. Org. Chem. Vol. 74, No. 17, 2009

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FIGURE 2. Increased thermal affinities toward RNA complements relative to that of the native AON (ΔT_m) for all modified carba-LNAs and

carba-ENAs. The ΔT_m(aver) values exhibited here is the average value for four isosequential AONs in which a given modification with the same

compound is incorporated at four different sites covering the minor groove (see Table 1 and Table SII.1 in Supporting Information for more

detailed comparison).

10.0. 3⁰-Exonuclease Stability of Parent Carba-LNA Con-

taining AONs and Modified Carba-LNA Containing AONs.

The metabolism of AONs in vivo involves degradation by

exo- and endonucleases, and the predominate nuclease ac-

tivity in blood serum originates from 3⁰-exonuclease.⁴⁹

Hence the newly synthesized AONs (AON 2, 6, 10, 14, 18)

with a single modification at position T13 (position 3 from

3⁰-end) were utilized to test the 3⁰-exonuclease stability of the

modified carba-LNA AONs. The known AONs containing

LNA (AON 22), 7⁰(S,R)-methyl-carba-LNA (AON 26), 6⁰S-

hydroxyl-7⁰S-methyl-carba-LNA (AON 30), and 6⁰S-hydro-

xyl-7⁰R-methyl-carba-LNA (AON 34) were also used in our

present parallel experiments for comparison to clarify the

stabilizing effects of the modified carba-LNAs. The selected

AONs were labeled at the 5⁰-end with ³²P and then incubated

with phosphodiesterase I from Crotalus adamateas venom

(SVPDE) [SVPDE 6.7 ng/μL, AON 3 μM, 100 mM Tris-HCl

(pH 8.0), 15 mM MgCl₂, total volume 30 μL] at 21 °C.

Aliquots were taken out at appropriate time intervals and

analyzed by 20% denaturing PAGE. The gel pictures were

obtained upon autoradiography and are shown in

Figure 3A. The native DNA (AON 1) and the LNA incor-

porated AON (AON 22) did not have any 3⁰-exonuclease

resistance and were completely degraded in ∼10 min, under

the present condition, whereas the parent carba-LNA AON

(AON 2) and the modified carba-LNA containing AONs

show improved 3⁰-exonuclease resistance to a variable ex-

tent. Because the AONs were modified at position T13, the

phosphate P14 (see the structure in Figure 4 for phosphate

(P) numbering) has considerably improved stability toward

3⁰-exonuclease. T13 modification can also improve the sta-

bility of the phosphate P13 to some extent, which was also

observed in the previous study,¹⁹and hence two bands

corresponding to 14mer and 13mer could be observed on

the PAGE pictures (Figure 3). However, once the P13

cleaved, AON was degraded to the monomer blocks quickly,

and thus no bands corresponding to 12mer to dimer oligos

could be observed.

Total percentages of integrated 14mer and 13mer AONs

were plotted against time points to give SVPDE digestion

curves for each AON in Figure 4, and pseudo-first-order

reaction rates could be obtained by fitting the curves to

single-exponential decay functions. A comparison of diges-

tion rates of AONs with different types of modifications

showed the following results:

(1) Parent unsubstituted carba-LNA incorporated AON

(AON 2) (k = 1.0473 ( 0.0601 h^-1) has greatly improved

resistance to degradation compared to LNA incorporated

AON (AON 22) (k = 52.5135 ( 1.0968 h^-1), which has

stability similar to that of the native AON (AON 1).

(2) All substituted carba-LNA incorporated AONs (AON

6, 10, 14, 18, 26, 30, 34) show higher stability toward

3⁰-exonuclease compared to that of the parent carba-LNA

(49) Shaw, J. P.; Kent, K.; Bird, J.; Fishback, J.; Froehler, B. Nucleic

Acids Res. 1991, 19, 747–750.

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FIGURE 3. Denaturing PAGE analysis of the SVPDE degradation of AONs with functionalized carba-LNA modifications. Digestion

conditions: AON 3 μM (5⁰-end ³²P-labeled with specific activity 80,000 cpm), 100 mM Tris-HCl (pH 8.0), 15 mM MgCl₂, reaction temperature

21 °C, total reaction volume 30 μL. (A) Higher concentration (6.7 ng/μL) of SVPDE was used. (B) Lower concentration (2.2 ng/μL) of SVPDE

was used.

incorporated AON (AON 2), suggesting that the substituents

at 6⁰and 7⁰could perturb the interaction between nuclease and

phosphate linkage and hence stabilize the AONs.

0.0298 h^-1) and 7⁰S-amino-carba-LNA (Type III) modified

AON 10 (k = 0.2768 ( 0.0082 h^-1), respectively, suggesting

that the introduction of the 6⁰S-hydroxyl can improve the

resistance to 3⁰-exonuclease of AONs. The bands corre-

sponding to 13mer in the PAGE pictures of AON 14 and

AON 18 disappeared relatively more slowly than those in the

PAGE pictures of AON 6 and AON 10, respectively

(Figure 3A), suggesting that the introduction of 6⁰S-hydro-

xyl stabilize the AONs mainly by improving the nuclease

(3) By comparing the degradation kinetics of 7⁰R or S-

amino-carba-LNA (Type II and Type III) modified AON 6

and 10 (k = 0.2820 ( 0.0298 and 0.2768 ( 0.0082 h^-1

,

respectively) with 7⁰-methyl-carba-LNA (Type VII) modi-

fied AON 26 (k = 0.1343 ( 0.0052 h^-1), 6⁰S-hydroxyl-7⁰-

amino-carba-LNA (Type IV and Type V) modified AON 14

and 18 (k = 0.1047 ( 0.0046 and 0.1050 ( 0.0087 h^-1

,

resistance of 5⁰-phosphate (P13 in Figure 4) (d_{C6 S-hydroxyl-5 P}

≈ 4.6 A), which was also observed in the previous study.¹⁹

10.1. Role of Configuration of the 7⁰-Amino (S versus R)

Group in Carba-LNAs in the Modulation of Stability toward

3⁰-Exonuclease. There were only slight difference in the

enzymatic stability between 7⁰R-amino-carba-LNA (Type

II) modified AON 6 and 7⁰S-amino-carba-LNA (Type III)

modified AON 10, 6⁰S-hydroxyl-7⁰S-amino-carba-LNA

(Type IV) modified AON 14 and 6⁰S-hydroxyl-7⁰R-amino-

carba-LNA (Type V) modified AON 18 in the digestion rates

of AONs obtained in Figure 4, which seemed to suggest that

the orientation of 7⁰-amino only had a small effect on the

stability toward 3⁰-exonuclease of AONs.

0

respectively) with 6⁰S-hydroxyl-7⁰-methyl-carba-LNA

(Type VIII and Type IX) modified AON 30 and 34 (k =

0.0563 ( 0.0040 and 0.0447 ( 0.0019 h^-1, respectively), it can

be concluded that the hydrophobic methyl substitution at 7⁰

had more positive effects on the enzymatic stability of the

modified carba-LNA containing AONs compared to that of

the hydrophilic amino group, although they both stabilize the

modified carba-LNA containing AONs more compared to the

parent unsubstituted carba-LNA containing AON (AON 2) (k

=1.0473 ( 0.0601 h^-1).

(4) 6⁰S-Hydroxyl-7⁰S-amino-carba-LNA (Type IV) mod-

ified AON 14 (k = 0.1047 ( 0.0046 h^-1) and 6⁰S-hydroxyl-

7⁰R-amino-carba-LNA (Type V) modified AON 18 (k =

0.1050 ( 0.0087 h^-1) were two times more stable than 7⁰R-

amino-carba-LNA (Type II) modified AON 6 (k = 0.2820 (

However, the role of configuration of the 7⁰-amino in the

modulation of nuclease stability could be observed on the

PAGE gel pictures in which the bands corresponding to

6546 J. Org. Chem. Vol. 74, No. 17, 2009

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FIGURE 4. Amount of remaining initial oligonucleotide (taken 14mer and 13mer together in the calculation of percent remaining) during

3⁰-exonuclease (SVPDE)-promoted digestion. Following digestion condition was used: AON 3 μM (5⁰-end ³²P-labeled with specific activity

80000 cpm), 100 mM Tris-HCl (pH 8.0), 15 mM MgCl₂, SVPDE 6.7 ng/μL, reaction temperature 21 °C, total reaction volume 30 μL. Molecular

structure of T13 modified AON with carba-LNA is also shown in the figure.

14mer in the PAGE pictures (Figure 3A) of AONs 10 and 18

(with the 7⁰-amino pointing at the 3⁰-phosphate) disappeared

relatively more slowly than those in the PAGE pictures of

AONs 6 and 14 (with the 7⁰-amino pointing away from the 3⁰-

phosphate), respectively, whereas the bands corresponding

to 13mer in the gel pictures of AONs 10 and 18 disappeared

relatively more rapidly than those found in the PAGE

pictures of AONs 6 and 14, respectively, suggesting that

the amino group can improve nuclease resistance of 3⁰-

phosphate of AONs when it is pointed at the 3⁰-phosphate

These results, taken together, suggested that as the scissile

phosphate came closer to the hydrophilic and positively

charged 7⁰-amino substituent, it became more stable toward

the nuclease degradation. The improved nuclease resistance

conferred by the hydrophilic and positively charged nature

of the amino group could be plausibly explained by the fact

that the amino group at 7⁰could become a zwitterionic form

at the physiological pH, and such zwitterionic AONs may

escape exonuclease degration by displacing a catalytically

important metal ion from the active site,⁸which was also

observed by Griffey⁰s group recently by synthesizing and

analyzing 2⁰-O-aminopropyl ribonucleotides.⁶

10.2. Comparison of Nuclease Resistance of the Parent

Unsubstituted Carba-LNA versus 2⁰-Oxa-LNA (LNA).

Although the parent unsubstituted carba-LNA incorporated

AON (AON 2) was nucleolytically less stable than the other

substituted carba-LNA incorporated AONs (AONs 6, 10,

14, 18, 26, 30, 34), it would however give more accurate

impression on the consequence of substitution of 2⁰-oxa in

LNA with 2⁰-CH₂in the isosteric carba-LNA and rule out

any additional modulation effect conferred by the substitu-

ents at the 2⁰,4⁰-linkage of carba-LNA, when the nucleolytic

stability of the parent carba-LNA incorporated AON is

directly pairwise compared with the isosequential 2⁰-oxa-

LNA (LNA) counterpart.^9,10In the above experiment (with

the high concentration of SVPDE in Figure 3A), the parent

carba-LNA incorporated AON (AON 2) and LNA incor-

porated AON (AON 22) were degraded so fast that only an

approximate degradation rate could be obtained. To give

more accurate degradation kinetics, AON 2 and 22 were

incubated with a lower concentration of SVPDE [SVPDE

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

MgCl₂, 21 °C, total volume 30 μL] compared to the native

(d_{C7 amino(up)-3 P}≈ 4.2 A). In contrast, when 7⁰-amino group

0

is pointed at the 5⁰-phosphate (d_{C7 amino(down)-5 P}≈ 6.8 A) it

can stabilize the 5⁰-phosphate. So, the overall effects of two

opposite orientations of the 7⁰-amino group on the 3⁰-phos-

phate and 5⁰-phosphate were very similar, leading to the

similar degradation kinetic patterns in Figure 4 because

products formed from the cleavage at P14 and P13 were

considered together for rate calculation.

0

To confirm the role of 7⁰-amino stereochemistry in the

modified carba-LNAs, we have estimated the relative rates

of cleavage at P14 only (taking only the percentage of

integrated 14mer AONs formed) for each AON (see Figure

SII.22 in Supporting Information). This shows that when the

7⁰-amino is pointing away from the 3⁰-phosphate [as in 7⁰R-

amino-carba-LNA (Type II) modified AON 6 (k=0.4304 (

0.0308 h^-1) and 6⁰S-hydroxyl-7⁰S-amino-carba-LNA (Type

IV) modified AON 14 (k = 0.2709 ( 0.0097 h^-1)], the

corresponding AONs showed higher digestion rates com-

pared to the AONs with 7⁰-amino pointing at the 3⁰-phos-

phate [as in 7⁰S-amino-carba-LNA (Type III) modified

AON 10 (k=0.2781 ( 0.0084 h^-1) and 6⁰S-hydroxyl-7⁰R-

amino-carba-LNA (Type V) modified AON 18 (k=0.2095 (

0.0153 h^-1)], respectively.

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TABLE 2. Degradation Rates k₁(h^-1) for All Modified Carba-LNAs and Carba-ENAs^a

^aThe degradation rates k (h^-1) value was calibrilated for Types X-XIX, published in our previous study,¹⁹by using the correlation coefficient

obtained from dividing the observed value (k₁) in the present study for Type VII by the observed value (k₂) for Type VII in the earlier study, which has

been used to calibrate other k₂values.

AON (AON 1). The gel pictures obtained by autoradiogra-

phy are shown in Figure 3B and the degradation curves are

shown in Figure SII.21 in Supporting Information. Under

this condition, the parent carba-LNA incorporated AON

(AON 2) (k=0.0015 ( 0.0002 h^-1) was found to be 145 times

more stable than LNA incorporated AON (AON 22) (k=

0.2195 ( 0.0383 h^-1), which on the other hand has degrada-

tion kinetics very similar to that of the native AON (AON 1)

(k=0.7617 ( 0.0368 h^-1). This result simply showed in a

straightforward manner that the lipophilic 2⁰-CH₂- in the

parent carba-LNA (compared to LNA) can indeed improve

the nuclease resistance of the vicinal 3⁰-phosphate probably

by decreasing the magnitude of hydration around it

with the interaction between nuclease and phosphate linkage

by the steric clash effect which was also observed by Imanishi’s

group.²⁰(2) The amino substitution at C7⁰can also improve the

stability of AONs regardless of its orientation (k = 0.2820 (

0.0298 h^-1for Type II versus k = 0.2768 ( 0.0082 h^-1for Type

III) but in a less pronounced way compared to the methyl group

(k = 0.1343 ( 0.0052 h^-1for Type VII), suggesting that the

steric effect conferred by the substituents is more important

than the electrostatic effect on the stability toward nuclease. (3)

The role of hydrophilic modification with a hydroxyl group at

the C6⁰position is more complex. The opposite orientation of

6⁰-hydroxyl group (S versus R) can lead to a completely

opposite effect on the stability of AONs and hence should be

handled very carefully. The C6⁰-hydroxyl pointing at

0

(d_{C2 methylene-3 P}≈ 4.2 A).

5⁰-phosphate (d_{C6 hydroxyl(down)-5 P}≈ 4.6 A) can stabilize the

proximate 5⁰-phosphate (k = 0.0563 ( 0.0040 h^-1for Type

VIII and k = 0.0447 ( 0.0019 h^-1for Type IX), whereas

0

10.3. Structure-Activity Relationships of All Modified

Carba-LNAs and Carba-ENAs for Nuclease Stability. From

the observations on the structure-activity relationships of

all modified carba-LNAs and carba-ENAs for nuclease

stability (Table 2), the following summary may be made:

(1) The methyl substitution can always improve nuclease

resistance of AONs, independent of its location and config-

uration on the 2⁰,4⁰-carba bridge. The improved nuclease

resistance conferred by the lipophilic nature of the methyl group

could be explained by two effects: First, the methyl group

counld decrease the extent of hydration around the vin-

the C6⁰-hydroxyl pointing at 3⁰-phosphate (d_{C6 hydroxyl(up)-3 P}

≈ 4.5 A) makes the phosphate too labile toward the nuclease

(k = 10.4896 ( 1.2408 h^-1for Type X and k = 1.8568 (

0.2288 h^-1for Type XI). How the C6⁰-hydroxyl interfere with

the modulation of the stability of AONs is still very poorly

understood. (See SII.34-35 in Supporting Information for

detailed pairwise comparison.)

0

ꢀ

11.0. Lessons from the Target Affinity vis-a-vis Nuclease

Resistance for the Design of AONs. Combining the target

affinity with nuclease resistance of modified AONs, the

0

cinal phosphate (d_{C7 methyl(up)-3 P}≈ 4.3 A, d_{C6 methyl(down)-5 P}

≈ 4.6 A). Second, the bulky methyl could also interfere

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FIGURE 5. Autoradiograms of 20% denatured PAGE showing the stability of 5⁰-³²P-labeled AONs in human blood serum in the present

study.

following conclusions can be drawn: (1) In the center of the

minor groove, both hydrophobic methyl substitution and

hydrophilic amino substitution are not favored, which

causes loss of target affinity toward RNA. (2) At the border

of minor grooves, the hydrophobic methyl group and the

hydrophilic 6S⁰-hydroxyl (pointing away from 3⁰-phosphate)

can improve the enzymatic stability without loss of target

affinity toward RNA and hence are proposed as suit-

able candidates for future modification in the AONs. (3)

The hydrophilic 6R⁰-hydroxyl (pointing at 3⁰-phosphate)

renders the modified AONs the most susceptible to enzy-

matic degradation, and hence this modification should be

avoided.

12.0. Stability of Parent Carba-LNA and Modified Carba-

LNA AONs in the Human Blood Serum. Blood serum

stabilities of all newly synthesized AONs modified at the

third position (position T13) from the 3⁰-end (AONs 2, 6, 10,

14, 18) and the known AON counterparts (AON 22, 26, 30,

34) with a single modification at position T13 (Table 1) have

also been tested in the present studies. The AONs (³²P-

labeled at 5⁰-end) were incubated with human blood serum

(male, type AB) for up to 48 h at 21 °C, and aliquots were

taken out at regular time intervals and analyzed by 20%

denaturing PAGE. The gel pictures obtained by autoradio-

graphy are shown in Figure 5. [Caution: Although it was

seriously attempted to carry out the experiment under the

same condiction as the previous study,¹⁹the enzymatic

activity of the blood serum may vary from batch to batch

and from donor to donor. So the result obtained in the

present study should/could not be used for direct parallel

comparison with the previous result.¹⁹This study of blood

serum stability should be treated as an independent study

and can only be compared within the present candidates].

Although the quantified data from the gel picture could not

be obtained because the alkaline phosphatase in serum

removed the 5⁰-end ³²P-label gradually, it could be seen by

visualization of the gel that the parent carba-LNA incor-

porated AON (AON 2) (the bands corresponding to 14mer

of parent carba-LNA containing AON 2 in the gel pictures

persisted up to 12 h under the present condition) was more

stable than LNA incorporated AON (AON 22) and

native AON (AON 1), both of which were however degraded

completely within 8 h. All the substituted carba-LNA mod-

ified AONs in present study (AON 6, 10, 14, 18, 26, 30, 34)

were more stable than the parent carba-LNA incor-

porated AON (AON 2) in which the 6⁰S-hydroxyl-7⁰-methyl

carba-LNA AONs (AON 30, 34) were more stable than 6⁰S-

hydroxyl-7⁰-amino substituted carba-LNA containing

AONs (AON 14, 18) with the same configuration at C6⁰

and C7⁰, suggesting that the methyl group has a more

positive effect on stability of AONs than that of the amino

group in human blood serum. However, the extra stabiliza-

tion conferred by the 6⁰S-hydroxyl, which was observed

upon treatment with 3⁰-exonuclease, was not marked in the

stability assay in the blood serum when comparing AON 6

with AON 14 and AON 10 with AON 18 in Figure 5. So, in

summary, the relative stabilities in blood serum for all

modified carba-LNA in present study is as followings:

AON 34 (Type IX) > AON 30 (Type VIII) > AON 18

(Type V) > AON 10 (Type III) > AON 26 (Type VII) ≈

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AON 6 (Type II) > AON 2 (Type I) ≈ AON 14 (Type IV) >

AON 22 (LNA) ≈ AON 1 (native). The effects on the

stability in human blood serum imparted by the substituent

at 2⁰,4⁰-connection follow the similar trend as those observed

upon treatment of 3⁰-exonuclease.

13.0. RNase H Digestion Study of Carba-LNA Modified

AON/RNA Duplexes. The RNase H recruitment capabilities

of a series of four 7⁰-amino-carba-LNA substituted AONs

(Types II-V) and parent carba-LNA substituted AONs

(Type I) were studied, using native AON (AON 1) as a

reference, whereas Type VI containing AONs (LNA) and

Type VII containing AONs (7⁰-methyl-carba-LNA) were

used in a parallel experiment for comparison. The gel

pictures obtained are shown in Figure SII.23, from which

we can see that all modified AONs 2-29 in the correspond-

ing AON/RNA duplexes were good substrates for RNase H,

and the cleavage patterns were all very similar, independent

of the nature of the modification, but were dependent on the

site of modification within the AON. Just as in the previous

study,^18,19the cleavage activity of RNase H was suppressed

within a 5-6 base pairs long region that starts from the base

opposite to the modified T nucleotide in AON/RNA duplex

toward the 3⁰-end of the RNA strand. However, the pre-

ferred cleavage site shifts to the edges of the suppressed

region as is shown in Figure 6. Hence, the overall RNase

recruitment capabilities were not impared significantly com-

pared to native AON 1/ RNA duplex, as is shown in Figure 7.

The cleavage rates of parent carba-LNA modified AONs/

RNA and 7⁰-amino-carba-LNA modified AONs/RNA are

even increased compared to native one, in which the AON

10/RNA (Type III) has three times cleavage rate as that of

native AON 1/RNA. (For detailed discussion see SII.36 in

Supporting Information).

Conclusions

The first syntheses of the parent fully unsubstituted carba-

LNA and its C7⁰-amino substituted and/or C6⁰-hydroxyl

substituted derivatives were accomplished by using an in-

tramolecular 5-exo free-radical addition to a CdN double

bond of the appropriately protected oxime-ether. Various

NMR experiments, including ¹H, ¹³C, DEPT, one bond

¹H-¹³C correlation (HMQC) and long-range ¹H-¹³C

FIGURE 6. Escherichia coli RNase H1 promoted cleavage pattern

of AONs 1-29/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.

FIGURE 7. Bar plots of the observed cleavage rates of the RNase H1 promoted degradation of RNA in AON 1-29/RNA hybrid duplexes.

6550 J. Org. Chem. Vol. 74, No. 17, 2009

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HMBC, have been employed to characterize all synthesized

compounds. The configuration of subsituents in the key

intermediates was determined by NOE and was also corro-

borated by the ³J_{H, H}coupling constants obtained from ¹H

homodecoupling experiments.

(aromatic), 134.8 (C6), 127.7, 127.5, 127.2, 127.0 (aromatic),

116.0 (CN), 110.0 (C5), 89.5 (C1⁰), 83.8 (C4⁰), 75.8 (C3⁰), 73.5

(CH₂Bn), 73.0 (CH₂Bn), 72.8 (C2⁰), 70.8 (C5⁰), 21.6 (C6⁰), 11.1

(T-CH₃). MALDI-TOF m/z: [M þ H]^þ, found 478.200, calcd

478.198.

4⁰-(Benzyloxyimino)ethyl-2⁰-hydroxyl-3⁰,5⁰-di-O-benzyl-thymi-

dine (17). To a solution of 16 (6 g, 12.56 mmol) in dry dichloro-

methane (DCM, 130 mL) was added DIBALH (38 mL, 1.0 M

solution in toluene) dropwise at -78 °C. After stirring at the same

temperature for 4 h, the mixturewas allowed to warm to0 °C, and

then 1 N HCl solution was added to quench the reaction and

stirred for 30 min at 0 °C. The resulting suspension was diluted

with DCM. The organic layer was separated and washed with

brine, dried over MgSO₄, and coevaporated with dry pyridine

twice togive crude compound 9, which was dissolved indry DCM

(310 mL) and dry pyridine (6 mL). To this solution, O-benzyl

hydroxylamine hydrochloride (5 g, 31.4 mmmol) was added.

After heating at reflux for 2 h, the mixture was cooled to rt and

neutralized by the addition of saturated aqueous solution of

NaHCO₃. The organic layer was separated, dried over MgSO₄,

evaporated, and chromatographed over silica gel (0.5-1%

methanol in DCM) to give 17 as the mixture of Z and E isomers

The corresponding phosphoramidites 21a/b, 24, 31a/b

were incorporated as mono substitution in a 15mer DNA

sequence on an automated RNA/DNA synthesizer. The

investigation for the antisense properties of resulting AONs

reveals that the substitution and its orientation at C6⁰and

C7⁰play an important role in the modulation of modified

carba-LNA. The relationship between substitution and tar-

get affinity and nuclease resistance of AONs can be sum-

marized as follows: (i) The hydrophobic methyl group and

the hydrophilic amino group at 7⁰both can impair the

thermal stability of AON/RNA duplex to a similar extent

especially when the substituents point at the vicinal

3⁰-phosphate. (ii) In contrast, substitution at C7⁰with either

CH₃or NH₂can stabilize the carba-LNA modified AONs

compared to the parent carba-LNA modified AON. The

CH₃substitution at C7⁰gives more pronounced positive

effects on stabilities of modified AONs. The orientation of

substituents (CH₃or NH₂) plays a very important role for

the selective stabilization for the 3⁰-phosphate versus

5⁰-phophate in the exonuclease cleavage reaction. (iii) The

introduction of 6⁰S-hydroxyl can not only improve the

thermal affinity toward the complementary RNA of the

corresponding AONs but also stabilize the AONs against

3⁰-exonuclease and hence is recommended as a suitable

modification for carba-LNAs.

For the RNase H mediated RNA cleavage in the AON/

RNA hybrid, the cleavage pattern was not affected by the

nature of different substituents but depended upon the site of

the modification. The RNase H has strong positional pre-

ference for a given active DNA/RNA hybrid. As for the

cleavage rate by RNase H, the parent carba-LNAs and

7⁰-amino carba-LNAs show improved recruitment capabil-

ities compared to LNAs and native AON, in which the

heteroduplex with 7⁰S-amino carba-LNAs (Type III) has

the fastest cleavage rate (AON 10/RNA has three times

cleavage rate as that of native AON 1/RNA).

1

(4.4 g, 7.53 mmol, 60% in two steps from 16). H NMR (500

00

0

MHz, CDCl₃): δ 9.10 (1H, broad, NH), 7.42 (0.5H, dd, J_{7 ,6}=7.5

0

Hz, J_{7 ,6}= 5 Hz, H7 ), 7.35 (1H, s, H6), 7.30-7.16 (15H, m,

0

00

0

aromatic), 6.78 (0.5H, dd, J_{7 ,6}=6 Hz, J_{7 ,6}=5 Hz, H7 ), 5.89 (1H,

0

2xd, J_{1 ,2}=3.5 Hz, H1 ), 5.02, 4.97(2H, 2xs, NOCH₂Bn), 4.69-4.34

(4H, m, CH₂Bn), 4.32 (1H, m, H2⁰), 4.08 (1H, 2xd, J_{2 ,3}= 2 Hz,

0

H3⁰), 3.55-3.31 ₀(3H, m, H5⁰,OH), ₀2₀.81 (0.5H, dd, J_gem=15 Hz,

0

J_{6 ,7}=5 Hz, H6 ), 2.72 (1H, m, H6 ,H6 ), 2.43 (0.5H, dd, J_gem

=

15 Hz, J_{7 ,6}= 7.5 Hz, H6 ), 1.50 (3H, s, T-CH₃). ¹³C NMR

(125 MHz, CDCl₃): δ 162.6 (C4), 149.9 (C2), 146.8 (C7⁰), 146.3

(C7⁰), 136.8, 136.1 (aromatic), 134.8 (C6), 127.7, 127.6, 127.1, 126.6

(aromatic), 110.1 (C5), 88.0 (C1⁰), 87.9 (C1⁰), 85.4 (C4⁰), 85.2 (C4⁰),

78.2 (C3⁰), 74.9 (CH₂Bn), 74.6 (CH₂Bn), 74.0 (C2⁰), 73.9 (C2⁰),

73.6 (CH₂Bn), 72.7 (CH₂Bn), 72.6 (C5⁰), 32.3 (C6⁰), 28.7 (C6⁰), 11.0

(T-CH₃). MALDI-TOF: m/z: [M þ H]^þ, found 586.248, calcd.

586.255.

00

0

00

4⁰-(Benzyloxyimino)ethyl-3⁰,5⁰-Di-O-benzyl-2⁰-O-phenoxythio-

carbonyl-thymidine (18). To a solution of 17 (4.4 g, 7.73 mmol)

in dry pyridine (150 mL) was added phenyl chlorothionoformate

(1.34 mL, 10 mmol) dropwise at 0 °C. After stirring at rt for 2 h,

saturated aqueous solution of NaHCO₃was added, and the

resulting mixture was extracted with DCM three times. The

organic solvent was washed with water, then brine, dried over

MgSO₄and evaporated. The residue was chromatographed over

silica gel (5-20% ethyl acetatein cyclohexane) togive 18 as Z and

E isomers (4.2 g, 5.8 mmol, 75%). Isomer I: ¹H NMR (500 MHz,

CDCl₃): δ 8.70 (1H, broad, NH), 7.52 (1H, app t, J = 6 Hz, H7⁰),

Experimental Section

Note. The individual characterization for all intermediates

are given in Supporting Information (Part I). All individual

reaction steps, workup, and product characterization for the

intermediates in Scheme 3 and the characterization for final

amidites 21a,b, 24, and 31a,b are given in Supporting Informa-

tion (SII.37-46)

1-(2-Hydroxyl-3,5-di-O-benzyl-4-C-cyanomethyl-β-^D-ribofur-

anosyl)-thymine (16). Compound 10 (6.87 g, 13.2 mmol) was

dissolved in methanol (30 mL) and methylamine solution (210

mL). The mixture was stirred at rt for 1 h. After evaporation of

the solvent, the residue was purified by column chromatography

on silica gel (1% methanol in CH₂Cl₂, v/v) to obtain 16 (6 g,

95%) as white foam. ¹H NMR (500 MHz, CDCl₃): δ 10.6 (1H,

7.46 (1H, s, H6), 7.42-7.29 (18H, m, aromatic), 7.03 (2H, d, J=8

0

Hz, aromatic), 6.42 (1H, d, J_{1 ,2}= 6.5 Hz, H1 ), 6.03 (1H, app t,

=

J = 5 Hz, H2⁰), 5.09 (2H, s, NOCH₂Bn), 4.74 (1H, d, J_gem

0

11 Hz, CH₂Bn), 4.58 (1H, d, J_{2 ,3}=5.5 Hz, H3 ), 4.54 (2H, s,

CH₂Bn), 4.50 (1H, d, J_gem= 11 Hz, CH₂Bn), 3.64 (1H, d,

_gem=10 Hz, H5⁰), 3.58 (1H, d, J_gem=10 Hz, H5⁰⁰), 2.75 (1H,

J

dd, J_{6 ,7}= 4.5 Hz, J_gem= 15 Hz, H6⁰), 2.56 (1H, dd, J_{6 ,7}= 7.5

Hz, J_gem=15 Hz, H6⁰⁰), 1.56 (3H, s, T-CH₃). ¹³C NMR (125

MHz, CDCl₃): δ 193.4 (CdS), 162.5 (C4), 152.4 (C2), 149.3

(aromatic), 146.4 (C7⁰), 136.7, 136.0 (aromatic), 134.8 (C6),

128.6, 127.6, 125.8, 120.6 (aromatic), 110.6 (C5), 85.9 (C4⁰),

84.6 (C1⁰), 81.7 (C2⁰), 76.8 (C3⁰), 74.6 (CH₂Bn), 73.9 (CH₂Bn),

72.8 (CH₂Bn), 72.7 (C5⁰), 32.2 (C6⁰), 11.0 (T-CH₃). MALDI-

TOF m/z: [M þ H]^þ, found 722.20, calcd 722.25. Isomer II: ¹H

NMR (500 MHz, CDCl₃): δ 8.70 (1H, broad, NH), 7.42-7.25

0

00

0

broad, NH), 7.55 (1H, s, H6), 7.55-7.29 (10H, m, aromatic),

0

5.93 (1H, d, J_{1 ,2}=4 Hz, H1 ), 4.93 (1H, d, J_gem=11 Hz, CH₂-

Bn), 4.85 (1H, broad, OH), 4.66-4.55 (4H, m, CH₂Bn and H2⁰),

4.38 (1H, d, J_{2 ,3}= 5.5 Hz, H3 ), 3.87 (1H, d, J_gem= 10 Hz, H5⁰),

3.62 (1H, d, J_gem=10 Hz, H5⁰⁰), 2.94 (1H, d, J_gem=17.5 Hz, H6⁰),

2.62 (1H, d, J_gem=17.5 Hz, H6⁰⁰), 1.47 (3H, s, T-CH₃). ¹³C NMR

(125 MHz, CDCl₃): δ 162.6 (C4), 150.4 (C2), 136.5, 136.2

0

(19H, m, aromatic and H6), 7.00 (2H, d, J=8 Hz, aromatic), 6.85

(1H, app t, J = 5 Hz, H7⁰), 6.37 (1H, d, J_{1 ,2}=5.5 Hz, H1 ), 5.96

0

(1H, app t, J=5.5 Hz, H2⁰), 5.11 (2H, s, NOCH₂Bn), 4.75 (1H, d,

J

_gem= 11.5 Hz, CH₂Bn), 4.58 (2H, m, CH₂Bn, H3⁰), 4.46 (2H, s,

J. Org. Chem. Vol. 74, No. 17, 2009 6551

Xu et al.

JOCArticle

CH₂Bn), 3.62 (1H, d, J_gem=10.5 Hz, H5⁰), 3.42(1H, d, J_gem=10.5

(125 MHz, CDCl₃): δ 163.9 (C4), 157.9 (q, J=36 Hz, COCF₃),

150.0 (C2), 137.5, 136.7 (aromatic), 135.5 (C6), 128.6, 128.3,

128.1, 127.8 (aromatic), 116.8, 114.5 (q, J=286 Hz, CF₃), 110.1

(C5), 88.1 (C4⁰), 82.9 (C1⁰), 77.8 (C3⁰), 73.8 (CH₂Bn), 72.4

(CH₂Bn), 66.9 (C5⁰), 47.6 (C7⁰), 46.9 (C2⁰), 36.9 (C6⁰), 12.1 (T-

CH₃). MALDI-TOF m/z: [M þ Na]^þ, found 582.181, calcd

582.183.

(1R,3R,4R,5S,7S)-7-Benzyloxy-1-benzyloxymethyl-5-trifluo-

roacetoxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (19b).

Compound 19b (58 mg, 49% in two steps) was obtained from 2b

after following the same reaction and workup procedure as 19a. ¹H

NMR (500 MHz, CDCl₃): δ 8.41 (1H, s, NH), 7.64 (1H, s, H6),

Hz, H5⁰⁰), 2.86 (1H, dd, J_{6 ,7}=4.5 Hz, J_gem=16 Hz, H6⁰), 2.76

0

(1H, dd, J _{6 ,7}=6 Hz, J_gem=16 Hz, H6⁰⁰), 1.52 (3H, s, T-CH₃).

¹³C NMR (125 MHz, CDCl₃): δ 193.4(CdS), 162.5 (C4), 152.3

(aromatic), 149.9 (C2), 146.0 (C7⁰), 136.7, 136.0 (aromatic), 134.7

(C6), 128.6, 125.8, 120.6 (aromatic), 110.6 (C5), 85.8 (C4⁰), 85.0

(C1⁰), 81.6 (C2⁰), 77.7 (C3⁰), 75.0 (CH₂Bn), 74.0 (CH₂Bn), 72.7-

(CH₂Bn), 71.6 (C5⁰), 28.6 (C6⁰), 11.0 (T-CH₃). MALDI-TOF

m/z: [M þ H]^þ, found 722.28, calcd 722.25.

00

0

(1R,3R,4R,5R,7S)-7-Benzyloxy-1-benzyloxymethyl-5-benzy-

loxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (2a) and

(1R,3R,4R,5S,7S)-7-Benzyloxy-1- benzyloxymethyl-5-benzylo-

xyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (2b). Com-

pound 18 (4 g, 5.5 mmol) was dissolved in dry toluene (545

mL), to which N₂was purged for 40 min. The mixture was

refluxed, and Bn₃SnH (2.7 mL in 40 mL dry toluene) and AIBN

(210 mg in 20 mL dry toluene) were added very slowly in 7 h. After

addition, the reflux was continued for 1 h. The solvent was

evaporated, and the residue was applied to chromatography (5-

20% ethyl acetate in cyclohexane) to give the two diastereomers 2a

7.38-7.33 (8H, m, aromatic), 7.21-7.19 (2H, m, aromatic), 7.06

0

(1H, d, J_NH,H7=9.5 Hz, NH), 5.44 (1H, s, H1 ), 4.67-4.56 (5H, m,

H7⁰, CH₂Bn), 4.16 (1H, s, H3⁰), 3.89 (1H, d, J_gem=11 Hz, H5⁰),

3.79 (1H, d, J_gem=11 Hz, H5⁰⁰), 2.71 (1H, s, H2⁰), 2.34 (1H, dd,

0

_gem=14 Hz, J_{6 ,7}=8.5 Hz, H6 ), 1.72 (1H, dd, J_gem=14 Hz,

0

J

_{6 ,7}=3.5 Hz, H6 ), 1.61 (T-CH₃).¹³C NMR (125 MHz, CDCl₃):δ

00

0

162.5 (C4), 154.6 (q, J = 36 Hz, COCF₃), 148.6 (C2), 136.2, 135.1

(aromatic), 134.2 (C6), 127.8, 127.7, 127.3, 126.9, 126.7 (aromatic),

115.8, 113.5(q, J=286 Hz, CF₃), 108.7 (C5), 87.0 (C4⁰), 84.3 (C1⁰),

78.0 (C3⁰), 72.9 (CH₂Bn), 72.5 (CH₂Bn), 65.9 (C5⁰), 47.9 (C7⁰),

47.7 (C2⁰), 38.9 (C6⁰), 11.2 (T-CH₃). MALDI-TOF m/z: [M þ

Na]^þ, found 582.185, calcd 582.183.

1

(1.87 g, 60%) and 2b (125 mg, 4%). 2a: H NMR (600 MHz,

CDCl₃): δ 9.18 (1H, s, NH), 7.65 (1H, s, H6), 7.65-7.15 (15H, m,

aromatic), 6.11 (1H, s, H1⁰), 5.54 (1H, broad, NH), 4.87 (1H, d,

J_gem=12 Hz, CH₂Bn), 4.78 (1H, d, J_gem=12 Hz, CH₂Bn), 4.51

(1H, d, J_gem=12 Hz, CH₂Bn), 4.47 (2H, d, J_gem= 12 Hz, CH₂Bn),

(1R,3R,4R,5R,7S)-1-(4,4⁰-Dimethoxytrityloxymethyl)-7-hydro-

xyl-5-trifluoroacetoxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]-

heptane (20a). To a solution of 19a (140 mg, 0.25 mmol) in dry

ethanol (5 mL) was added 20% Pd(OH)₂/C (108 mg) and

cyclohexene (2 mL). The mixture was refluxed overnight and then

filtered through a Celite pad, and the organic solvent was

evaporated to dryness. The residue was coevaperated with dry

pyridine twice and dissolved in the same solvent. 4,4⁰-Dimethoxy-

trityl chloride (154 mg, 0.45 mmol) was added and the mixture

was stirred overnight at rt.The solvent was removed . The residue

was diluted with DCM, washed with saturated NaHCO₃. The

oganic layer was separated, dried over MgSO₄, and evaporated.

The residue was applied to column chromatography on silica gel

(0.5-1% methanol in DCM containing 1% pyridine) to give 20a

(111 mg, 65% in two steps). ¹H NMR (500 MHz, CD-

Cl₃þDABCO): δ 7.70 (1H, s, H6), 7.45 (2H, d, J = 7.5 Hz,

aromatic), 7.34-7.19 (8H, m, aromatic andNH), 6.84 (4H, d, J =

7.5Hz, aromatic), 5.81 (1H, s, H1⁰), 4.79 (1H, m, H7⁰), 4.37 (1H, s,

H3⁰), 3.79 (6H, s, 2xOCH₃), 3.54 (1H, d, J_gem=11 Hz, H5₀⁰), 3.36

4.36 (1H, d, J_gem=12 Hz, CH₂Bn), 4.03 (1H, s, H3⁰₀), 3.94 (1H, dt,

0

00

0

J

_{2 ,7}=4.2 Hz, J_{6 ,7}=10.2 Hz, J_{6 ,7}=4.2 Hz, H7 ), 3.71 (1H, d,

J

_gem=10.8 Hz, H5⁰), 3.60 (1H, d, J_gem=10.8 Hz, H5⁰⁰), 2.95 (1H,

0

d, J_{2 ,7}=4.2Hz, H2 ), 1.98 (1H, dd, J_gem=12.6 Hz, J_{6 ,7}=10.2 Hz,

H6⁰), 1.49(3H, s,T-CH₃), 1.12(1H, dd, J_gem= 12.6 Hz, J_{6 ,7}=4.2

Hz, H6⁰⁰). ¹³C NMR (125 MHz, CDCl₃): δ 163.3 (C2), 149.0 (C4),

137.0, 136.7, 136.2 (aromatic), 135.4 (C6), 128.5, 127.7, 127.5,

127.4, 127.3, 127.2, 126.8, 126.5 (aromatic), 108.2 (C5), 86.7 (C4⁰),

82.8 (C1⁰), 77.6 (C3⁰), 75.3 (CH₂Bn), 72.7 (CH₂Bn), 71.0 (CH₂-

Bn), 66.3 (C5⁰), 56.3 (C7⁰), 44.5 (C2⁰), 33.4 (C6⁰), 11.2 (T-CH₃).

MALDI-TOF m/z: [M þ H]^þ, found 570.264, calcd 570.260. 2b:

¹H NMR (600 MHz, CDCl₃):δ 8.45 (1H, broad, NH), 7.66 (1H, s,

H6), 7.37-7.19 (15H, m, aromatic), 5.88 (1H, broad, NH), 5.37

(1H, s, H1⁰), 4.75 (1H, d, J_gem=12 Hz, CH₂Bn), 4.71 (1H, d,

00

0

J

_gem=12 Hz, CH₂Bn), 4.62-4.56 (3H, m, CH₂Bn), 4.46 (1H, d,

_gem=12 Hz, CH₂Bn), 4.04 (1H, s, H3⁰), 3.84 (1H, d, J_gem=10.8

Hz, H5⁰), 3.75 (1H, d, J_gem=10.8 Hz, H5⁰⁰), 3.69(1H, m, H7⁰), 3.16

(1H, s, H2⁰), 2.05 (1H, dd, J_gem=13.2 Hz, J_{6 ,7}=8.4 Hz, H6 ),

00

0

(1H, d, J_gem=11 Hz, H5⁰⁰), 2.93 (1H, d, J_{2 ,7}=4.0 Hz, H2₀), 2.80

0

1.58 (1H, dd, J_gem= 13.2 Hz, J_{6 ,7}= 4.0 Hz, H6 ), 1.56 (3H, s, T-

CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 162.8 (C4), 148.7 (C2),

137.1, 136.6, 135.7 (aromatic), 134.8 (C6), 127.6, 127.5, 127.3,

127.1, 126.8, 126.7 (aromatic), 108.2 (C5), 86.7 (C4⁰), 85.3 (C1⁰),

77.3 (C3⁰), 74.7 (CH₂Bn), 72.7 (CH₂Bn), 71.6 (CH₂Bn), 66.2

(C5⁰), 59.7 (C7⁰), 43.5 (C2⁰), 35.1 (C6⁰), 11.1 (T-CH₃). MALDI-

TOF m/z: [M þ H]^þ, found 570.258, calcd 570.260.

0

(s, DABCO), 2.37(1H, dd, J_gem=14 Hz, J_{6 ,7}=10.0 Hz, H6 ), 1.62

(T-CH₃), 1.52 (1H, dd, J_gem= 14 Hz, J_{6 ,7}=4.5 Hz, H6 ). ¹³C

NMR (125 MHz, CDCl₃þDABCO): δ 163.4 (C4), 157.7

(aromatic), 156.5 (q, J = 36 Hz, COCF₃), 149.3 (C2), 143.5,

134.5 (aromatic), 134.1 (C6), 129.1, 128.0, 127.1, 127.0, 126.1

(aromatic), 113.5(q, J= 286 Hz, CF₃), 112.3 (aromatic), 109.2

(C5), 87.9 (C4⁰), 85.6(DMTr-C), 82.0 (C1⁰), 71.0(C3⁰), 59.7(C5⁰),

54.2 (OCH₃), 48.6 (C2⁰), 46.8 (C7⁰), 45.8 (DABCO), 35.6 (C6⁰),

11.4 (T-CH₃). MALDI-TOF m/z: [M þ Na]^þ, found 704.198,

calcd 704.220.

00

0

(1R,3R,4R,5R,7S)-7-Benzyloxy-1-benzyloxymethyl-5-trifluo-

roacetoxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane

(19a). To a solution of 2a (300 mg, 0.53 mmol) in dry methanol

(15 mL) were added 10% Pd/C (262 mg) and ammonium

formate (661 mg, 10.48 mmol). After stirring at rt for 4 h, the

mixture was filtered through a Celite pad, and the organic

solvent was evaporated to dryness. The residue was dissolved

in anhydrous methanol. Ethyl trifluoroacetate (0.63 mL, 5.3

mmol) and 4-dimethylamino-pyridine (DMAP, 130 mg, 1.06

mmol) were added, and the mixture was stirred at rt overnight.

The solvent was removed and the residue was applied to

chromatography (1-2% methanol in DCM) to give 19a (148

mg, 50% in two steps). ¹H NMR (500 MHz, CDCl₃): δ 8.99 (1H,

s, NH), 7.62 (1H, s, H6), 7.37-7.27 (11H, m, aromatic and NH),

5.92 (1H, s, H1⁰), 4.77 (1H, m, H7⁰), 4.64-4.50 (4H, m, CH₂Bn),

4.13 (1H, s, H3⁰), 3.85 (1H, d, J_gem=11 Hz, H5⁰), 3.73 (1H, d,

(1R,3R,4R,5S,7S)-1-(4,4⁰-Dimethoxytrityloxymethyl)-7-hydro-

xyl-5-trifluoroacetoxyamino-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]-

heptane (20b). Compound 20b (37 mg, 60% in two steps) was

obtained from 19b after following the same reaction and workup

procedure as 20a. ¹H NMR (500 MHz, CDCl₃þDABCO): δ 7.75

0

(1H, s, H6), 7.58 (1H, d, J_NH,H7=9.5 Hz, NH), 7.44 (2H, d, J=7.5

Hz, aromatic), 7.33-7.25 (7H, m, aromatic), 6.85 (4H, m,

aromatic), 5.41 (1H, s, H1⁰), 4.60 (1H, m, H7⁰), 4.37 (1H, s,

H3⁰), 3.80 (6H, s, 2xOCH₃), 3.67 (1H, d, J_gem= 11 Hz, H5⁰), 3.39

(1H, d, J_gem=11 Hz, H5⁰⁰), 2.80 (s, DABCO), 2.68 (1H, s, H2⁰),

2.26 (1H, dd, J_gem=13.5 Hz, J_{6 ,7}=8.5 Hz, H6⁰), 1.65 (1H, dd,

0

_gem=13.5 Hz, J_{6 ,7}=3.2 Hz, H6 ),1.58 (T-CH₃). ¹³C NMR (125

00

0

J

_gem=11 Hz, H5⁰⁰), 3.04 (1H, s, H2⁰), 2.48 (1H, dd, J_gem=13 Hz,

MHz, CDCl₃þDABCO): δ 163.0(C4), 157.8(aromatic), 155.5(q,

J=36 Hz, COCF₃), 149.0, 148.8 (aromatic), 143.4 (C2), 135.0,

_{6 ,7}=11 Hz, H6 ), 1.58 (4H, m, H6 and T-CH₃). ¹³C NMR

0

00

0

6552 J. Org. Chem. Vol. 74, No. 17, 2009

Xu et al.

JOCArticle

134.4 (aromatic), 133.9 (C6), 129.1, 129.0, 126.3, 122.8 (aromatic),

113.5(q, J=286 Hz, CF₃), 112.3(aromatic), 108.9 (C5), 88.0(C4⁰),

85.9 (DMTr-C), 84.8 (C1⁰), 71.6 (C3⁰), 59.4 (C5⁰), 54.3 (OCH₃),

49.2 (C2⁰), 47.8 (C7⁰), 45.8 (DABCO), 38.9 (C6⁰), 11.4 (T-CH₃).

MALDI-TOF m/z: [M þ Na]^þ, found 704.196, calcd 704.220.

(1R,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-

yl)-2-oxa-bicyclo[2.2.1]heptan-5-one Oxime (5). To a solution of

2a (1.4 g, 2.45 mmol) in ethyl acetate (50 mL) were added K₂CO₃

(814 mg, 5.92 mmol) and 77% 3-chloroperbenzoic acid

(mCPBA, 665 mg, 2.98 mmol). After stirring at rt for 1 h, the

white suspension was diluted with ethyl acetate and washed with

water. The organic layer was separated, dried over MgSO₄, and

evaporated. The residue was chromatographed over silica gel

(0.8-2% methanol in DCM) to give compound 5 (702 mg,

NH), 7.66 (1H, s, H6), 7.36-7.30(10H, m, aromatic), 5.78(1H, dd,

0 0

0 0 00 0

_{6 ,7}=6 Hz, J_{6 ,7}=3 Hz, H7 ), 5.48 (1H, s, H1 ), 4.66 (1H, d, J_gem=

J

9.5 Hz, CH₂Bn), 4.60 (2H, 2xd, J_gem=9.5 Hz, CH₂Bn), 4.43 (1H, d,

J_gem= 9.5 Hz, CH₂Bn), 4.10 (1H, s, H3⁰), 3.85 (1H, d, J_gem=9 Hz,

H5⁰), 3.82 (1H, d, J_gem=9 Hz, H5⁰⁰), 3.27 (1H, s, H2⁰), 2.51 (3H, s,

0

SCH₃), 2.44 (1H, dd, J_gem=11.5 Hz,₀J₀_{6 ,7}=6 Hz, H6 ), 2.15 (1H,

dd, J_gem=11.5 Hz, J_{6 ,7}=3 Hz, H6 ), 1.53 (T-CH₃). ¹³C NMR

(125 MHz, CDCl₃): δ 213.7 (CdS), 162.6 (C4), 148.7 (C2), 136.6,

136.1 (aromatic), 134.6 (C6), 127.6, 127.4, 127.0, 126.9, 126.8

(aromatic), 108.6 (C5), 87.2 (C4⁰), 84.0 (C1⁰), 80.4 (C7⁰), 76.5

(C3⁰), 72.8 (CH₂Bn), 71.0 (CH₂Bn), 65.6 (C5⁰), 46.4 (C2⁰), 39.1

(C6⁰), 18.2 (SCH₃), 11.1 (T-CH₃). MALDI-TOF m/z: [M þ H]^þ,

found 555.158, calcd 555.162.

00

0

(1R,3R,4R,7S)-7-Benzyloxy-1-benzyloxymethyl-3-(thymin-1-

yl)-2-oxa-bicyclo[2.2.1]heptane (1). Compound 22 (150 mg, 0.27

mmol) was dissolved in dry toluene (6 mL) and purged with dry

nitrogen for 30 min. AIBN (10 mg, 0.06 mmol) and nBu₃SnH

(216 μL, 0.8 mmol) were added, and the mixture was refluxed for

1.5 h. The mixture was cooled to rt. After evaporation of the

solvent, the residue was chromatographed over silica gel (25-

50% ethyl acetate in cyclohexane) to give compound 1 (72 mg,

1

60%). H NMR (500 MHz, CDCl₃): δ 8.82 (1H, broad, OH),

8.19 (1H, broad, NH), 7.69 (1H, s, H6), 7.37-7.24 (10H, m, aro-

matic), 5.64 (1H, s, H1⁰), 4.64-4.59 (3H, m, CH₂Bn), 4.76 (1H, d,

J_gem=9.5 Hz, CH₂Bn), 4.21 (1H, s, H3⁰), 3.95 (1H, d, J_gem=9.5

Hz, H5⁰), 2.55 (1H, d, J_gem=9.5 Hz, H5⁰⁰), 3.60 (1H, s, H2⁰), 2.72

(1H, d, J_gem=14.5 Hz, H6⁰), 2.55 (1H, d, J_gem=14.5 Hz, H6⁰⁰),

1.54(3H, s, T-CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 162.9 (C4),

156.9 (C7⁰), 148.7 (C2), 136.4, 135.8 (aromatic), 134.6 (C6), 127.6,

127.4, 127.1, 126.8, 126.7 (aromatic), 108.7 (C5), 87.3 (C4⁰), 84.6

(C1⁰), 76.3 (C3⁰), 72.82 (CH₂Bn), 71.1 (CH₂Bn), 65.8 (C5⁰), 49.2

(C2⁰), 33.4 (C6⁰), 11.1 (T-CH₃). MALDI-TOF m/z: [M þ Na]^þ,

found 500.210, calcd 500.180.

1

65%). H NMR (600 MHz, CDCl₃): δ 8.40 (1H, broad, NH),

7.74 (1H, s, H6), 7.36-7.27 (10H, m, aromatic), 5.47 (1H, s,

=

H1⁰), 4.61 (2H, 2xd, J_gem=11.4 Hz, CH₂Bn), 4.55 (1H, d, J_gem

11.4 Hz, CH₂Bn), 4.48 (1H, d, J_gem=11.4 Hz, CH₂Bn), 3.99 (1H,

s, H3⁰), 3.86 (1H, d, J_gem=10.8 Hz, H5⁰), 3.78 (1H, d, J_gem=10.8

0

Hz, H5⁰⁰), 2.75 (1H, d, J_{2 ,7}=4.2 Hz, H2 ), 2.07 (1H, tt, J_{2 ,7}=4.2

0

(1R,3R,4R,5S,7S)-7-Benzyloxy-1-benzyloxymethyl-5-hydroxyl-

3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (7). To a solution of

compound 5 (580 mg, 1.21 mmol) and NaOAc (36 mg) in dry

DCM (55 mL) was added 0.3 M Dess-Martin periodinate solution

in DCM (5.44 mL, 1.63 mmol) dropwise. After stirring at rt for 30

min, the mixture was diluted with DCM and washed with satu-

rated NaHCO₃. The organic solvent was dried over MgSO₄and

evaporated to dryness to give crude compound 6, which

was dissolved in ethanol (15 mL). NaBH₄(187 mg, 4.89 mmol)

was added to the solution. After stirring at rt for 3 h, the solvent

was removed, and the residue was diluted with DCM and washed

with saturated NaHCO₃. The organic layer was separated, dried

over MgSO₄, and then evaporated. The residue was chromato-

graphed over silica gel (0.5-2% methanol in DCM) to give

0

00

Hz, J_{6 ,7}=4 Hz, J_{6 ,7}=12 Hz, J_{7 ,7}= 12 Hz, H7 ), 1.88 (1H, dt,

0

00

0

00

0

00

J_{6 ,7}=4 Hz, J_{6 ,7}=12 Hz, J_{6 ,6}=12 Hz, H6₀)₀, 1.73 (1H, ddd,

0

00

00 00

0

00

J_{6 ,7}=4 Hz, J_{6 ,7}= 8.6 Hz, J_{7 ,7}=12 Hz, H7₀₀), 1.64 (1H, ddd,

00 00

00

0

00

J_{6 ,7}=8.6 Hz, J_{6 ,7}=4 Hz, J_{6 ,6}=12 Hz, H6 ), 1.57 (T-CH₃).

¹³C NMR (125 MHz, CDCl₃): δ 162.8 (C4), 148.9 (C2), 136.8,

136.6 (aromatic), 135.1 (C6), 127.5, 127.4, 127.0, 126.9,

126.7,126.5 (aromatic), 108.1 (C5), 87.3 (C4⁰), 87.2 (C1⁰), 76.5

(C3⁰), 72.7 (CH₂Bn), 70.9 (CH₂Bn), 66.6 (C5⁰), 42.7 (C2⁰), 28.5

(C6⁰), 22.1 (C7⁰), 11.1 (T-CH₃). MALDI-TOF m/z: [M þ H]^þ,

found 449.210, calcd 449.208.

(1R,3R,4R,7S)-1-(4,4⁰-Dimethoxytrityloxymethyl)-7-hydroxyl-

3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane (23). To a solution of

1 (64 mg, 0.14 mmol) in dry methanol (4 mL) were added 20%

Pd(OH)₂/C (72 mg) and ammonium formate (182 mg, 2.8 mmol).

The mixture was refluxed for 2 h and then filtered through a Celite

pad, and the organic solvent was evaporated to dryness. The

residue was coevaperated with dry pyridine twice and dissolved in

the same solvent. 4,4⁰-Dimethoxytrityl chloride (96 mg, 0.28

mmol) was added, and the resulting mixture was stirred overnight

at rt. After the solvent was removed, the residue was diluted with

DCM and washed with saturated NaHCO₃. The organic layer

was dried over MgSO₄and evaperated, and the residue was

applied to column chromatography on silica gel (0.5-1% metha-

nol in DCM containing 1% pyridine) to give 23 (56 mg, 70% in

two steps). ¹H NMR (500 MHz, CDCl₃þ DABCO): δ 7.80 (1H,

s, H6), 7.45 (2H, d, J=7.5 Hz, aromatic), 7.37-7.16 (7H, m,

aromatic), 6.85 (4H, m, aromatic), 5.40 (1H, s, H1⁰), 4.22 (1H, s,

H3⁰), 3.80 (6H, s, 2xOCH₃), 3.64 (1H, d, J_gem=11 Hz, H5⁰), 3.35

1

compound 7 (226 mg, 40% in two steps). H NMR (600 MHz,

CDCl₃): δ 8.21 (1H, broad, NH), 7.64 (1H, s, H6), 7.39-7.25

(10H, m, aromatic), 5.32 (1H, s, H1⁰), 4.64 (2H, d, J_gem=11.4 Hz,

CH₂Bn), 4.59 (2H, d, J_gem=11.4 Hz, CH₂Bn), 4.29 (1H, app t, J=

9.6 Hz, H7⁰), 4.16 (1H, s, H3⁰), 3.88 (1H, d, J_gem=11.4 Hz, H5⁰),

3.84 (1H, d, J_gem=11.4 Hz, H5⁰⁰), 2.82 (1H, s, H2⁰), 2.78 (1H, d,

0

J

_{7 ,OH}=11.4 Hz, OH), 2.32 (1H, dd, J_gem=13.8 Hz, J_{6 ,7}=7.8 Hz,

H6⁰), 1.82 (1H, dd, J_gem=13.8 Hz, J_{6 ,7}=2.4 Hz, H6 ), 1.6 (3H, s,

T-CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 162.5 (C4), 148.6 (C2),

136.5, 135.5 (aromatic), 134.6 (C6), 127.7, 127.6, 127.4, 127.1,

126.8 (aromatic), 108.5 (C5), 87.0 (C4⁰), 83.6 (C1⁰), 78.1 (C3⁰), 72.7

(CH₂Bn), 72.2 (CH₂Bn), 71.3 (C7⁰), 66.2 (C5⁰), 49.0 (C2⁰), 42.0

(C6⁰), 11.2 (T-CH₃). MALDI-TOFm/z:[MþNa]^þ, found 487.180,

calcd 487.185.

00

0

(1R,3R,4R,5S,7S)-7-Benzyloxy-1-benzyloxymethyl-5-((methy-

lthio)thiocarbonyl)oxy-3-(thymin-1-yl)-2-oxa-bicyclo[2.2.1]heptane

(22). To a solution of compound 7(190 mg, 0.41 mmol) in dry THF

(6 mL) was added 60% NaH (50 mg, 1.22 mmol) at 0 °C. After

stirring at rt for 1 h, CS₂(190 μL, 4.1 mmol) was added to the

suspension at 0 °C, and the resulting mixture was stirred at rt for

30 min and then at 40 °C for 2 h. The solution became clear and was

cooled by ice. Methyl iodide (163 μL, 2.61 mmol) was added at

0 °C, and the mixture was stirred at rt for another 3 h. The reaction

was quenched by chilled water and extracted with ethyl acetate

several times. The organic layer was dried over MgSO₄and

evaporated. The residue was chromatographed over silica gel

(20-50% ethyl acetate in cyclohexane) to give compound 22

(1H, d, J_gem=11 Hz, H5⁰⁰), 2.79 (s, DABCO), 2.63 (1H, d, J_{2 ,7}=3

Hz, H2⁰), 2.08 (1H, m, H7⁰), 1.74 (2H, m, H6⁰, H7⁰⁰), 1.60 (T-CH₃),

1.58 (1H, m, H6⁰⁰). ¹³C NMR (125 MHz, CDCl₃þ DABCO): δ

162.9 (C4), 157.7 (aromatic), 149.0 (C2), 143.6 (aromatic), 134.6

(C6), 129.0, 128.0, 127.2, 127.0, 126.1, 124.3, 112.3(aromatic),

108.4 (C5), 87.9 (C4⁰), 87.4 (C1⁰), 85.7 (DMTr-C), 70.9 (C3⁰), 60.0

(C5⁰), 54.2 (OCH₃), 46.0 (DABCO), 45.1 (C2⁰), 28.2 (C6⁰), 22.1

(C7⁰), 11.4 (T-CH₃). MALDI-TOF m/z: [M þ Na]^þ, found

593.198, calcd 593.226.

0

General Procedure for Phosphoramidite Synthesis. To a solu-

tion of substrate (1 equiv) in dry DCM were added DIPEA (5

equiv) and 2-cyanoethyl N,N-diisopropyl phosphoramidochlor-

idite (3 equiv) dropwise in an ice bath. The reaction was allowed

1

(152 mg, 65%). H NMR (500 MHz, CDCl₃): δ 8.40 (1H, s,

J. Org. Chem. Vol. 74, No. 17, 2009 6553

Xu et al.

JOCArticle

to warm to rt and stirred at this temperature for 3 h. After being

quenched with methanol, the mixture was diluted with ethyl

acetate, washed with saturated NaHCO₃solution, dried over

MgSO₄, and concentrated. The residue was applied to short

column chromatography on silica gel to give phosphoramidite,

which was first precipitated in n-hexane and then dried over

P₂O₅on vacuum for three days before it was used for DNA

synthesis.

Oligonucleotide Synthesis and Purification. All AONs were

synthesized using an automated DNA/RNA synthesizer based

on phosphoramidite chemistry. For native A, G, and C building

blcok, 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 incorpora-

ting modified nucleotides, extended coupling time (10 min

comparing to 25 s for native nucleotides) was used. Cleavage

from the support and deprotection were carried out by treating

the solid support with 33% aqueous ammonia at rt for 12 h to

obtain the fully deprotected AONs 2-21, which were purified by

denaturingPAGE(20%polyacrylamidewith7Murea), extracted

with 0.3 M NaOAc, and desalted with a C-18 reverse phase

cartridge to give AONs in >99% purity. Correct masses have

been obtained by MALDI-TOF mass spectroscopy for each of

them (Table SII.1).

exonuclease concentration of 6.7 or 2.2 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 cya-

nole in 80% formamide]. Reaction progress was monitored by

20% denaturing (7 M urea) PAGE and autoradiography.

Stability Studies in Human Blood Serum. AONs at 2 μM

concentration (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 cya-

nole in 80% formamide], resolved in 20% polyacrylamide

denaturing (7 M urea) gel electrophoresis, and visualized by

autoradiography.

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

presence of 0.08 U of E. coli RNase H (obtained from USB

corporation, Cleveland, OH). Prior to the addition of the

enzyme, reaction components were preannealed in the reac-

tion 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 sub-

jected to 20% 7 M urea PAGE and visualized by autoradio-

graphy. Pseudo-first-order reaction rates could be obtained by

fitting the digestion curves to single-exponential decay func-

tions.

The complementary RNA was also synthesized by solid-

supported phosphoramidite approach based on the 2⁰-O-TEM

strategy.^50,51

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 °C/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 equilibration at this temperature.

The value of T_mis the average of two or three independent

measurements. If error of the first two measurements was >

(0.3 °C, the third measurement was carried out to check if the

error is indeed within (0.3 °C; otherwise it was repeated.

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 using a Beckman LS 3801 counter.

Acknowledgment. Generous financial support from the

Swedish Natural Science Research Council (Veten-

skapsradet), 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 acknowledged.

Supporting Information Available: ¹H, ¹³C, ³¹P, DEPT,

COSY, HMQC, HMBC of carba-LNA derivatives (SI part I);

1D NOE spectra, experimental and calculated coupling con-

stants of key intermediates 2a, 2b, 1, 4a, and 4b (SI part II);

autoradiograms of 20% denaturing PAGE as well as degrada-

tion curves, showing the cleavage kinetics of target RNA in

AON/RNA hybrides by E. coli RNase H1, the detailed discus-

sion about the structure-activity relationships of all modified

carba-LNAs and carba-ENAs for thermal affinity, nuclease

stability and the RNase recruitment of DNA/RNA duplexes,

all individual reaction steps, workup, and product characteriza-

tion for the intermediates in Scheme 3 and the characterization

for final amidites 21a,b, 24, 31a,b (SI part II). These materials

are available free of charge via the Internet at http://pubs.acs.

org.

SVPDE Degradation Studies. Stability of the AONs toward

3⁰-exonucleases was tested using phosphodiesterase I from

Crotalus adamanteus (obtained from USB corporation, Cleve-

land, OH). All reactions were performed at 3 μM DNA con-

centration (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

(50) Zhou, C.; Honcharenko, D.; Chattopadhyaya, J. Org. Biomol.

Chem. 2007, 5, 333–343.

(51) Zhou, C.; Pathmasiri, W.; Honcharenko, D.; Chatterjee, S.; Barman,

J.; Chattopadhyaya, J. Can. J. Chem. 2007, 85, 293–301.

6554 J. Org. Chem. Vol. 74, No. 17, 2009

Article Doi

Synthesis of conformationally locked carba-LNAs through intramolecular free-radical addition to C=N. Electrostatic and steric implication of the carba-LNA substituents in the modified oligos for nuclease and thermodynamic stabilities

DOI: 10.1021/jo901009w

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Article abstract of DOI:10.1021/jo901009w

Full text of DOI:10.1021/jo901009w

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