Intramolecular Free-Radical Cyclization Reactions on ... · The pentose sugar of bicyclic...

Intramolecular Free-Radical Cyclization Reactions on Pentose Sugarsfor the Synthesis of Carba-LNA and Carba-ENA and the Applicationof Their Modified Oligonucleotides as Potential RNA TargetedTherapeuticsChuanzheng Zhou and Jyoti Chattopadhyaya*

Chemical Biology Program, Department of Cell and Molecular Biology, Box 581, Biomedical Center, Uppsala University, SE-751 23Uppsala, Sweden

CONTENTS

1. Introduction 38082. Synthesis of Carba-ENA and Carba-LNA Nucleo-

sides 38102.1. Synthesis of Carba-ENA through Ring

Closure Metathesis 38102.2. Application of Intramolecular Free-Radical

Cyclization on Constructing Bicyclic Nucleo-sides 3810

2.3. Synthesis of Carba-LNA and Carba-ENAthrough Intramolecular Free-Radical Addi-tion to CC 3812

2.4. Synthesis of Carba-LNA and Carba-ENAthrough Intramolecular Free-Radical Addi-tion to CN 3814

2.5. Synthesis of Carba-LNA through Intramolec-ular Free-Radical Addition to CC 3814

2.6. Synthesis of α-L-Carba-LNA Derivativesthrough Intramolecular Free-Radical Addi-tion 3814

2.7. Regio-Selectivity and Stereoselectivity of theFree-Radical Cyclization 3815

3. Conformation of Carba-LNA and Carba-ENANucleosides 3818

4. Orientation of Carbocyclic Moieties of Carba-LNAand α-L-Carba-LNA in Duplex Form 3818

5. Thermal Stability of Modified AON/RNA Duplex 38205.1. Carba-LNA Modification 38205.2. α-L-Carba-LNA Modification 38225.3. Carba-ENA Modification 3822

6. RNA Selectivity of Modified AONs 38227. Nuclease Resistance of Modified Oligonucleo-

tides 38237.1. Carba-LNA Modified Oligonucleotides 38237.2. Carba-ENA Modified Oligonucleotides 38237.3. α-L-Carba-LNA Modified Oligonucleotides 3824

8. RNase H Elicitation of Carba-LNA and Carba-ENAModified AONs 38248.1. Carba-LNA and Carba-ENA Modified AONs 3824

8.2. α-L-Carba-LNA Modified AONs 38259. Biological Evaluation of Antisense Oligonucleo-

tides Containing carba-LNA Derivatives 382510. RNAi Potency of siRNAs Containing Carba-LNA

and Carba-ENA Modifications 382611. Conclusions and Implications 3829Author Information 3830

Corresponding Author 3830Notes 3830Biographies 3830

Acknowledgments 3830References 3830

1. INTRODUCTIONSince 1950s, many different types of conformationally con-strained nucleos(t)ides have been synthesized for structuralstudies or as potential antivirus agents.1−3 In the early 1990s,conformationally constrained nucleosides such as Bc-DNA(Figure 1) were incorporated into antisense oligonucleotides(AONs) with the aim to develop therapeutic AONs thateffectively and specifically recognize target RNA with highaffinity and selectivity.4 Altmann and Marquez subsequentlyshowed that 4′,6′-methanocarba nucleotides5,6 having a North-type (N-type) sugar pucker and 1′,6′-methanocarba nucleotidescharacterized by South-type (S-type) sugar pucker7 (Figure 1)have very different antiviral and antisense activities.8 4′,6′-Methanocarba-T (N-type) showed excellent antiherpeticactivity, but 1′,6′-methanocarba-T (S-type) was devoid of theactivity. Incorporation of 4′,6′-methanocarba-T in DNA/RNAduplexes resulted in Tm increase (∼1.3 °C/modification), whereas1′,6′-methanocarba-T modification induced a small decrease inTm. This observation highlighted the importance of sugar con-formation for RNA targeting, which in turn inspires an upsurgein the synthesis of sugar conformation constrained nucleotidesfor antisense therapeutics.9,10 An important compound thatemerged during this upsurge is locked nucleic acid (LNA). Thesynthesis of LNA was reported first by Imanishi11 in 1997,whereas Wengel’s group12 reported an independent synthesis in1998. In LNA, the 2′-O,4′-C-methylene- across the pentose-sugar forms a fused five-membered ring (Figure 1), con-straining the furanose sugar in a typical North conformation

Received: September 12, 2010Published: April 24, 2012

Review

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© 2012 American Chemical Society 3808 dx.doi.org/10.1021/cr100306q | Chem. Rev. 2012, 112, 3808−3832

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(phase angle P = 17.4°).11 Introduction of LNA to AONsresults in unprecedented increases in the thermal stability ofduplexes toward both DNA and RNA.13 Thermodynamic andstructural studies suggest that LNA stabilizes the duplex byfacilitating preorganization of AONs in RNA-like molecularstructures and/or improving stacking.14,15

Short 2′,4′-linkage (up to eight atoms) always constrains thenucleos(t)ides into a typical North-type sugar conformation.Hence, many types of 2′,4′-locked nucleos(t)ides have beensynthesized in the past decade and their structures are listed inFigure 2. LNA analogues such as 2′-thio-LNA16 (Figure 2B), 2′-amino-LNA17−19 (Figure 2C−E), C3′-branched LNA20 (Figure 2F),C6′-branched LNA21,22 (Figure 2G,H), α-L-LNA,23 and 2′-amino-α-L-LNA24,25 (Figure 2I−K) are 2′,4′-locked nucleosidescontaining a two-atom linkage. These nucleotides are con-strained in a very similar conformation as that of LNA and alsobestow similar high affinity toward complementary RNA. Thepseudorotational phase angles (P) of 2′,4′-locked nucleos(t)idescontaining a three-atom linkage such as 2′-C,4′-C-methyleneoxy-methylene nucleoside26 (Figure 2N), 2′-O,4′-C-ethylene-bridged nucleic acids (ENA)27 (Figure 2O), aza-ENA28−31

(Figure 2P), BNANC and derivatives32 (Figure 2Q), and N-Me-aminooxy-BNA19 (Figure 2R) are also conformationally closeto that of LNA. However, AONs modified with nucleotidecontaining 2′,4′ three-atom linkage generally exhibit slightlylower RNA affinity compared to those of AONs con-

taining 2′,4′ two-atom linkage nucleotide modification but higherselectivity toward target RNA compared to targeting DNA.Incorporation of the 2′,4′-locked nucleotide(s) containing a four-atom linkage such as BNAcoc33 (Figure 2S) and PrNA27 (Figure 2T)into AONs does not lead to an increase in the RNA affinityrendering these modifications unsuitable for modulation of AONor siRNA properties as a potential therapeutic or diagnostic.It is noteworthy that locked nucleos(t)ides described above

have a heteroatom connected to C2′ of the sugar moiety; hencethey are all classified as a group containing 2′,4′-heterocycliclinkages. Their carbocyclic analogues, locked nucleos(t)idescontaining a 2′,4′ carbocyclic ring such as carba-LNA (Figure 2U),carba-ENA (Figure 2V), α-L-carba-LNA (Figure 2W) as wellas their derivatives, have been synthesized in the past fiveyears by our group34−37 and others.38−40 The striking feature ofcarba-LNA and carba-ENA derivatized AONs is that theyrender similar RNA affinity as AONs modified by incorporationof LNA and ENA but show very much improved nucleaseresistance (survival in the blood serum for >48 h). Thecarbocyclic ring of carba-LNA and carba-ENA also allows for arange of substitutions thus providing an effective handle forengineering of new types of substitutions in the minor grooveof AON/RNA or RNA/RNA duplexes to modulate importantantisense or siRNA properties such as target RNA affinity,nuclease resistance, and delivery without impairing their RNaseH or RISC recruitment capabilities. The present review focuses

Figure 1. The structures of early developed conformationally constrained nucleosides.

Figure 2. Structures of 2′,4′-locked nucleosides.

Chemical Reviews Review

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on the synthesis and structure of carba-LNA and carba-ENA aswell as their derivatives that are decorated with various hydro-philic and hydrophobic groups at the C6′/C7′ position of thefused carbocylic ring in carba-LNA and C6′/C7′/C8′ position incarba-ENA. All carba-LNA derivatives and most of the carba-ENAs, except for one case involving ring closure metathesis byGrubb’s catalyst, are synthesized through intramolecular free-radical cyclization reaction. The key synthetic and mechanisticfeatures of the intramolecular free-radical cyclization reactionon the pentose sugar, developed in the Uppsala laboratory, willbe briefly addressed. The recent advances in the therapeuticproperties of AONs and siRNAs with these modifications willalso be discussed. Readers can refer to older reviews2,3,41 aboutthe synthesis and properties of other types of conformationallyconstrained nucleos(t)ides.

2. SYNTHESIS OF CARBA-ENA AND CARBA-LNANUCLEOSIDES

2.1. Synthesis of Carba-ENA through Ring ClosureMetathesis

The 2′,4′ carbocyclic rings in carba-LNA and carba-ENA aregenerally formed by coupling C2′ and C4′ tethered functions.Ring closure metathesis has been found to be very efficient forthis C−C coupling. Carba-ENA series (compounds 3−7, 9−13,

Scheme 1) were first synthesized through the ring-closingmetathesis method by Nielsen et al.38 The key intermediate 2was obtained from uridine in 11 steps, and then it was treatedwith Grubbs' catalyst to furnish the unsaturated three-carbon2′,4′-locked uridine 3 (unsaturated carba-ENA) in 96% yield.Carba-ENA uridine (6) was obtained by hydrogenation of thedouble bond of 3 following a known procedure.38 Treatment ofunsaturated carba-ENA-U (3) with OsO4 gave the 6′,7′-di-OH-carba-ENA-U (4).39 Instead of this approach, a ring-closingenyne metathesis for compound 8 had been employed leadingto the 6′-vinyl-unsaturated carba-ENA 9, which has further beentransformed to C6′-hydroxymethyl-unsaturated carba-ENA-U(10) and C6′R/S-ethyl-carba-ENA-U (11).39

2.2. Application of Intramolecular Free-Radical Cyclizationon Constructing Bicyclic Nucleosides

Ring closure of hex-5-enyl radical was first reported five decadesago by Lamb et al.42 It is an efficient approach for stereo- andregiocontrolled C−C bond formation, and its utility has beenwell recognized in natural product synthesis.43 In the early1990s, this method was introduced by Chattopadhyaya and hisco-workers to synthesize bicyclic nucleosides.44−48 The firstreported bicyclic nucleoside 17 was obtained by treatment ofthe radical precursor 14 with Bu3SnH and AIBN in boilingbenzene (Scheme 2).44 This treatment putatively generated

Scheme 1. Synthesis of carba-ENA Derivatives through Ring-Closing Metathesis



C2′ radical (16) and the addition of the radical to the CC ofC3′-O-allyl occurred in the 5-exo mode, leading to a new 2′,3′-α-fused heterocyclic five-membered ring. The pentose sugar ofbicyclic nucleoside 17 was found in S-conformation. Instead,if starting from radical precursor 19, the obtained bicyclicnucleoside 21 exhibited N-type sugar puckering. Hence, it isnot the 2′,3′-fused ring but the property of the 2′ or 3′ electro-negative substituent that dictates the conformation of thepentose moiety. This is easy to understand given that (1) thefused ring is five-membered and so is flexible, and (2) electro-negative substituent on C2′ or C3′ tends to occupy a pseudo-axial position because of the stabilizing gauche effect.49

If the 2′ (or 3′)-O-allyl lies in the β face in the radicalprecursor (23 and 25, Scheme 2), radical cyclization resultedin a bicyclic nucleoside with a 2′,3′-β-fused heterocyclic five-membered ring (24 and 26).44 The 2′,3′-fused five-memberedring in the bicyclic nucleoside could also be efficiently formedby addition of the C2′ radical to the C3′-O-propynyl (Scheme 3).44

This cyclization took place in the 5-exo mode, resulting in an exo-cyclic methlyene function. The success of this reaction wasrecently exploited for the synthesis of methylene-carba-LNA bySeth et al. (see Section 2.5).40

Another type of bicyclic nucleoside synthesized through free-radical cyclization is 3′,4′-β-fused six-membered bicyclic nucleo-sides 31 (Scheme 4).48 In the radical precursors 29, an allyl isattached to the 5′-O through an ether bond. After treatmentwith Bu3SnH and AIBN, intramolecular cyclization of theobtained hept-6-enyl radical (30) occurred exclusively in the6-exo mode, formatting the 3′,4′-β-fused six-membered hetero-cyclic ring (31). The alkenyl function can also be introducedto the radical precursor through a bond other than an ether.Chattopadhyaya reported that after introduction of the allylfunction to the 5′-O through an ester linkage, the obtainedprecursor (32, Scheme 4) was subjected to radical cyclization,

leading to a 3′,4′-lactone fused bicyclic nucleoside 33,48 which couldbe conveniently hydrolyzed under alkaline conditions to give 3′-C-branched alkyl carboxylic acid 34. However, the stereochemistryof these two cyclizations is different given that C6′ of 31 is inS-configuration but 33 is in R-configuration. This suggests thestereoelectronic modulation on the linkage itself can significantlyinfluence the stereoselectivity during free-radical cyclization. A 2′,3′-lactone fused bicyclic nucleoside 36 has been subsequentlysynthesized from 35 using this method by Camarasa et al.50

Chattopadhayaya and co-workers reported that addition ofC3′ radical to an alkynyl tethered to C2′ through a siloxanelinkage (38, Scheme 5) is also efficient, generating a 2′,3′-fusedfive-membered ring containing siloxane with exocyclic methyl-ene (CH2) function (39) through 5-exo cyclization mode.47

However, if trapping the C3′ radical using silicon-bearing allylgroup (42), a 2′,3′-cis-fused seven-membered ring was formed(43). This indicates the radical cyclization took place in anunusual 7-endo mode in this case.45 Interestingly, by invertingthe configuration of the C2′ tethered silicon-bearing allyl (as in45, Scheme 5), a mixture (1/1) of cis and trans-fused seven-membered rings (46) was obtained. The Si−O and Si−C bondin these siloxane-containing bicyclic nucleosides were cleavedby fluoride ion and by oxidation, respectively, affording C-branched nucleosides 40, 44, 47, and 48. Thus, intramolecularcyclization of siloxane-bearing olefin or alkyne to a prochiralradical center, followed by opening the temporary silicon con-nection, represents an effective and wise strategy for synthesisof C-branched nucleosides. We envision further use of thisintramolecular free-radical cyclization of siloxane-bearing olefinor alkyne for the synthesis of other C-branched carbohydrates.Following Chattopadhyaya’s work, many bicyclic carohydrates

and branched nucleosides have been synthesized through free-radical cyclization strategy.51−53 Impressed by the stereo- andregiospecificity of the above free-radical cyclization reac-tions on the C2′ or C3′ chiral centers of the pentose ring inconstructing these bicyclic nucleosides with predictable stereooutcome, in a preparative scale, vis-a-vis the biochemical successof Imanishi−Wengel’s LNA incorporated oligonucleotides,Chattopadhyaya realized that if the tethered olefin and the radicalcenter are separated by a one-carbon bridge in the pentose ring ofthe nucleoside, free-radical cyclization will lead to a strained carba-bicyclic nucleoside. This idea prompted the synthesis of carba-LNA and carba-ENA by free-radical cyclization, which we arguedmight open a new direction for new types of potential therapeutic

Scheme 2. Synthesis of 2′,3′-Fused Bicyclic Nucleosides through Free-Radical Addition to CC

Scheme 3. Synthesis of 2′,3′-Fused Bicyclic Nucleosidesthrough Free-Radical Addition to CC



oligonucleotides, in which we should be able to create flexibility tosteer required biophysical and biological properties.

2.3. Synthesis of Carba-LNA and Carba-ENA throughIntramolecular Free-Radical Addition to CC

The first carba-LNA and carba-ENA nucleosides synthesized inthe Uppsala laboratory by the free radical approach are 7′R/S-Me-carba-LNA-T (55, Scheme 6) and 8′R-Me-carba-ENA-T(59).34 The synthesis started from compound 49 whose pri-mary alcohol was oxidized to aldehyde by Swern oxidation. Thealdehyde was subjected to successive Wittig reaction, hydro-boration−oxidation, Swern oxidation, and Wittig reaction, givingC4-allylated sugar 52, which was converted to radical precursor53 in four steps. Treatment of compound 53 with AIBN and

Bu3SnH in toluene putatively gave radical intermediate 54. Free-radical cyclization, exclusively through a 5-exo pathway, produced7′R/S-Me-carba-LNA-T (55) as a diastereoisomeric mixture.Through a similar strategy, 7′-Me-carba-LNA-5MeC/A/G havebeen successfully synthesized recently.54

On the other hand, subjecting 52 to another round ofhydroboration−oxidation, Swern oxidation and Wittig reactionfurnished 56 (Scheme 6), starting from which the radical precursor57 was obtained in four steps. Treatment of 57 with AIBN andBu3SnH in toluene led exclusively to 8′R-Me-carba-ENA nucleoside59,34 putatively through 6-exo cyclization of radical intermediate 58.Starting from the aldehyde 50, C6′-modified carba-LNAs

have also been synthesized.36 50 reacted with vinylmagnesiumbromide to introduce 1S-hydroxy-allyl at C4′ as in compound

Scheme 5. Synthesis of Siloxane-Containing Bicyclic Nucleosides through Free-Radical Cyclization Reaction

Scheme 4. Sythesis of Lactone Fused Bicyclic Nucleosides through Free-Radical Cyclization Reaction



60 (Scheme 7A). Generation of C2′ radical (62) throughradical precursor 61 followed by the 5-exo cyclization reactionled to a mixture of two main isomers, 6′S-OH-7′S-Me-carba-LNA-T (63) and 6′S-OH-7′R-Me-carba-LNA-T (64, 63:64 =7:3) as well as about 10% of 6′-OH-carba-ENA-T (65) whichwas formed following the 6-endo cyclization pathway.35 The6′-hydroxy group in 6′-OH-carba-ENA-T (65) was efficientlyremoved by radical deoxygenation resulting in parent carba-ENA-T (66).35 Complete inversion of the configuration of

6′-OH in compound 64 was achieved by successive Dess−Martin oxidation and NaBH4 reduction, giving 6′R-OH-7′R-Me-carba-LNA-T (67, Scheme 7B). Similarly, oxidation the 6′-OHof 63 gave ketone 68. Reduction of the ketone by NaBH4resulted in 6′R-OH-7′S-Me-carba-LNA-T (69). In 69, the6′R-OH and 3′-OH are cis-orientated which allows couplingwith thymidine-5′-phosphorodiamidite resulting in Sp andRp-D2-CNA-T

55−57 (70, Scheme 7C), which in fact are thefirst reported sugar and phosphate double locked nucleotides.58

Scheme 6. Synthesis of 7′-Me-carba-LNA and 8′-Me-carba-ENA through Free-Radical Cyclization to CC

Scheme 7. Synthesis of C6′ and C7′ Modified Carba-LNAs through Free-Radical Cyclization to CC



Other reported carbocyclic nucleosides 6′S-OH-6′-Me-7′S-Me-carba-LNA-T (71) and 6′R-OH-6′-Me-7′S-Me-carba-LNA-T(72)36 have been synthesized using the reaction of ketone 68with methyl magnesium iodide (Scheme 7C). The 6′-OH groupin nucleoside 71 was removed by radical deoxygenation reac-tion, giving 6′R/S-Me-7′S-Me-carba-LNA-T (73) as a diaster-oisomeric mixture together with an unexpected bicyclo[2.2.1]-2′,6′-methylene-bridged hexopyranosyl nucleotide (BHNA, 74),which was found to be formed by a radical rearrangement.59,60

C6′-modified carba-ENAs also have been synthesized byaddition of the C2′ radical to C4′ tethered 1S-hydroxy-butenylfunction as in 77 (Scheme 8).36 The radical cyclization reactiontook place exclusively by the 6-exo pathway, giving 6′S-OH-8′R-Me-carba-ENA-T (78). After oxidation the 6′-OH of 78, theresulting ketone 79 was transformed to 6′R-OH-8′R-Me-carba-ENA-T (80), 6′R-OH-6′-Me-8′R-Me-carba-ENA-T (81), and6′R/S-Me-8′R-Me-carba-ENA-T (82a/b), respectively (Scheme 8).

2.4. Synthesis of Carba-LNA and Carba-ENA throughIntramolecular Free-Radical Addition to CN

Carba-LNA derivatives containing C7′ hydrophilic substitu-tions have been synthesized by Chattopadhyaya et al. throughintramolecular free-radical addition to CN.37 The synthesisstarted from 49 (Scheme 9), which was converted to 83 in sixsteps. Then the C4′-tethered aldehyde reacted with O-benzyl-hydroxylamine, giving oxime 84, which was further transformedto radical precursor 85. After treatment with Bu3SnH andAIBN in toluene, the generated C2′ radical was trapped by theCN (86, Scheme 9). This radical cyclization also took placeexclusively in the 5-exo pathway, giving 7′R-benzyloxyamino-carba-LNA (88) as the major products plus a trace amount of7′S-benzyloxyamino-carba-LNA (87). The 7′-benzyloxyaminofunction of 87 was converted to ketone by mCPBA oxidationto 7′-oxime followed by Dess−Martin oxidation. Reduction ofketone 89 furnished 7′S-OH-carba-LNA (90), which was sub-jected to radical deoxygenation to give parent carba-LNA (91).Subjecting the radical precursor 94 (Scheme 10), which was

obtained by introduction of a hydroxyl group to the C4′tethered oxime as in 84, to radical cyclization afforded 96 and97.37 They are carba-LNA derivatives containing hydrophilicmodifications at both C6′ and C7′.Through intramolecular radical addition to CN, carba-

ENA derivatives containing C8′ hydrophilic modifications have alsobeen synthesized.61 The synthesis started from 51 (Scheme 11A).It was first transformed to 98 in five steps. The C4′ tetherednitrile group of 98 was reduced with DIBALH to aldehydefollowed by oximation with O-benzylhydroxylamine to provideO-benzyl oxime 99. After esterification of the 2′-OH of 99 withphenyl chlorothioformate, the obtained 100 was subjected to

free-radical cyclization. The cyclization proceeded in the 6-exocyclization mode to afford 8′R-benzyloxyamino-carba-ENA (102)as the sole product.Oxidative deamination of the 8′R-benzyloxyamino function

of 102 was achieved by treatment with 3,5-di-tert-butyl-1,2-benzoquinone, furnishing ketone 103. Starting from 103, 8′-OH-carba-ENA 104 and 105, 8′-Me-carba-ENA 107, and parentcarba-ENA 66 have been synthesized (Scheme 11B,C).61

2.5. Synthesis of Carba-LNA through IntramolecularFree-Radical Addition to CC

Another member of the carba-LNA family, methylene-carba-LNA, was synthesized through intramolecular addition of theC2′ radical to a C4′-tethered CC by Seth et al. recently.40

Such an intramolecular addition of the C2′ radical to a C3′-O-tethered alkyne, generating a 2′,3′-fused five-membered hetero-ring with exocyclic olefin function (28, Scheme 3), has beenestablished in 1991 in the Uppsala lab.44 The synthesis ofmethylene-carba-LNA started from 5′-TBDPS-3′-(2-methyl-napthalene)-allo-furanose derivative 108 (Scheme 12). Swernoxidation of the primary alcohol followed by a Wittig reactionprovided an olefin, which was subjected to hydroboration−oxidation using 9-BBN/sodium perborate to give alcohol 109in good yield. A Swern oxidation of 109 followed by a Coery−Fuchs reaction generated dibromo olefin 110. After deprotec-tion of the naphthyl group, treatment with with n-BuLi furnishedalkyne 111, which was further converted to thiobicarbonate 112in four steps. Treatment of 112 with AIBN and Bu3SnH intoluene putatively gave radical intermediates 113 followed byintramolecular free-radical cyclization to give methylene-carba-LNA 114 in satisfactory yield.

2.6. Synthesis of α-L-Carba-LNA Derivatives throughIntramolecular Free-Radical Addition

All of the carba-LNA and carba-ENA nucleos(t)ides discussedabove are in β-D form. It has been reported that α-L-LNA, justlike β-D-LNA, showed striking biochemical features,62−64 whichprompted us to synthesize α-L-carba-LNA nucleos(t)ides65

(Scheme 13). Swern oxidation of the primary alcohol of 115followed by Grignard reaction with vinylmagnesium bromideafforded C4-hydroxylallyl ribofuranose 116 and 117 as dia-stereomers. After transformation to thiocarbonate 118 and 119,respectively, they were subjected to free-radical cyclization bytreating with Bu3SnH and AIBN. Cyclization of 118 occurredwith high stereoselectivity to give 6′R-OH-7′S-Me-α-L-carba-ENA-T (121) as the only product, but cyclization of 119 led to6′S-OH-7′S-Me-α-L-carba-ENA-T (122) as the major productplus tetracyclic minor product 123 (122/123 = 3/1). The 6′-OH in 122 was efficiently removed by radical deoxygenationreaction, giving 7′R-Me-α-L-carba-ENA-T (124) plus a minor

Scheme 8. Synthesis of C6′ and C8′ Modified Carba-ENAs through Free-Radical Cyclization to CC



bicyclo[2.2.1]-2′,6′-methylene-bridged hexopyranosyl nucleoside125 which was supposed to be formed by a radical rearrangementmechanism.65 Similarly, radical deoxygenation of compound 123furnished 6,7′-methylene bridged-α-L-carba-LNA-T (126).Another α-L-carba-LNA nucleoside, α-L-methylene-carba-

LNA, has been synthesized by Seth et al. recently.66 Thesynthesis strategy is the same as synthesis of methylene-carba-LNA 114 except using the sugar 127 as the starting material(Scheme 14). The primary alcohol was first transformed toalkyne (128) in six steps, and then to radical precursor 129 infour steps. Treatment of 129 with Bu3SnH/AIBN in refluxingtoluene provided α-L-methylene-carba-LNA (130).

2.7. Regio-Selectivity and Stereoselectivity of theFree-Radical Cyclization

It is known that intramolecular cyclization in lower alkenyl andalkynyl radicals and related species occurs preferentially in theexomode, giving the thermodynamically less stable exoproductas the major product (Scheme 15).67 The more rapid exocycli-zation is rationalized by better orbital overlap in the exo-transition state.Radical cyclization on nucleoside to synthesize the bicyclic

nucleoside follows the general rule: hex-5-CC(CN orCC) and hept-6-CC(CN) radical cyclization takes placepredominantly in exocyclization mode. Introducing heteroatom

such as O, N, ester to linkage generally has no effect on thecyclization mode, but Si has been found to change the radicalcyclization mode in hept-6-CC radical cyclization as shownin Scheme 5.For five- and six-member ring formation, the ring is always cis-

fused. The face (α or β on the pentose sugar) of the newly formedring is always the same as that of the configuration of the 4′-tethered AB (any double or triple bond). The configuration ofthe radical center in the radical precursor seems to play no role indetermining the stereoselectivity of the carbon−carbon bondformation. However, a larger (e.g., seven-member ring) ring forma-tion on the pentose sugar may lead to both cis- and trans-fusedproducts, as stated above (41 to 43 vs 45 to 46 in Scheme 5).It has been proposed that hex-5-enyl radical cyclization

generally proceeds through a chairlike or boatlike transitionstate.68 Different C5 stereochemistry will be reached throughdifferent transition states (Scheme 16). The chairlike transitionstate is more stable than the boatlike transition state, but thecalculated difference between the two states is very small (lessthan 1 kcal/mol). Hence, many other effectors may compensatefor the formation of the normally unfavorable boatlike transi-tion state. For example, a big substituent X at C4 (Scheme 16)may favor the boatlike transition because it eclipses one of thevinylic hydrogen atoms in the chairlike transition state.

Scheme 9. Synthesis of C7′ Modified Carba-LNAs through Free-Radical Cyclization to CN

Scheme 10. Synthesis of C6′ and C7′ Modified Carba-LNAs through Free-Radical Cyclization to CN



In all the radical precursors (the C2′-O-thiocarbonate) forcarba-LNA and carba-ENA, their JH1′, H2′ and JH2′, H3′ vary from5.5 to 6.5 Hz, suggesting their sugar rings are predominantly inSouth-type conformation. After radical generation, the sugarring must go over a pseudorotational cycle to take up North-type conformation with the 3′-OBn in the axial position, sincethe sugar is constrained in this conformation in the finalproduct. To reduce the total energy of the system, overlappingof the SOMO of C2′ radical with the HOMO of C4′-tetheredAB (any double or triple bond) leads to C−C formation. Forradical cyclization to carba-LNA derivatives, either a chairlike(Scheme 17A) or boatlike (Scheme 17B) transition state couldbe formed. The former leads to the 2′,7′-trans product while thelatter leads to the 2′,7′-cis product. Actually, the 2′,7′-cis productwas identified as the major product (around 70%), so the majorpathway for the cyclization here is through a naturally

unfavorable boatlike transition state. The observation that abulky B (NOBn) led to the significantly increased 2′,7′-cisproduct (to 94%, Scheme 17) suggests it is the steric clashbetween B and the axial 3′-OBn that makes the chairlike stateunfavorable. However, the clash between B and the C6′substitution in the boatlike state is also unfavorable. It wasindeed found in the presence of both bulky B (NOBn) andbulky C6′ substitution (-OAc) that the ratio of cis/trans productdecreased to 1/1. Hence, the interaction of B with surroundingsubstitution plays a very important role in influencing thestereochemical outcome for synthesis of carba-LNA. In turn, wecan modulate the stereochemical outcome of the cis or/andtrans product by introducing the proper substituent to C3′, C6′,and C7′.For radical cyclization to carba-ENA derivatives, a chairlike

transition state is favorable. In this transition state (Scheme 18),

Scheme 11. Synthesis of C8′ Modified Carba-ENA through Free-Radical Cyclization to CN

Scheme 12. Synthesis of Methylene-carba-LNA through Free-Radical Cyclization to CC



the 4′ and 6′ substitutents adopt pseudoequatorial posi-tions and 3′-OBn adopts a pseudoaxial position. Equatorialorientation of the bulky exocyclic B in this transition state(Scheme 18A) is favorable because pseudoaxial orienta-tion of B may increase the system energy by introduc-ing 1,3-pseudodiaxial interaction between B and 3′-OBn(Scheme 18B). This explains why the 2′,8′-cis product(Scheme 18A) is the sole product obtained from hept-6-enyl radical (58, 77) and hept-6-oxime radical (101)cyclization.

As shown in Scheme 4, similar radical precursors 29 and 32lead to similar bicyclic nucleosides 31 and 33 but their C6′configuration is opposite. This cannot be explained using stericclash. Instead, this stereoselectivity could be imposed by theconstraining of the ester bond (Scheme 19). Radical inter-mediate 30 from 29 should adopt a chairlike transition state inwhich the CC adopts an equatorial position. Cyclization of

Scheme 13. Synthesis of α-L-carba-LNA Derivatives through Free-Radical Cyclization to CC

Scheme 14. Synthesis of α-L-Methylene-carba-LNA through Free-Radical Cyclization to CC

Scheme 15. Intramolecular Radical Cyclization OccursPreferentially in the Exo-Modea

aHere, n ≤ 5, AB is any double or triple bond.

Scheme 16. Transition States for the Hex-5-enyl Free-Radical Cyclization



30 leads to 31 with C6′ S-configuration. On the contrary,the rigid ester linkage in 32 enforces only a boatlike transi-tion state. Again, to minimize the system energy, the CCshould adopt an equatorial position. Cyclization leads to33 with C6′ R-configuration. In summary, the stereo-chemical selectivity during synthesis of bicyclic nucleosidethrough radical cyclization was mainly controlled by the stericclash, but some other factors such as stereoelectroniceffects may also play an important role when steric clashis insignificant.

3. CONFORMATION OF CARBA-LNA ANDCARBA-ENA NUCLEOSIDES

Sugar conformation of nucleosides can be characterized by thepseudorotation phase angles (P) and puckering amplitude(Φm).

69 Phase angle P characterizes the sugar puckering mode.P = 0° corresponds to the 2′-exo, 3′-endo puckering mode(North) and P = 180° corresponds to the 2′-endo, 3′-exopuckering mode (South).69 All of the carba-LNA and carba-ENA nucleosides are constrained in the North conformation

(Table 1). The phase angle of parent carba-ENA is 15°, whichis very similar to that of ENA and aza-ENA.70 The presenceof the CC bond in the carbocyclic ring decreases theconstrained force on the sugar since the nucleosides 5, 9, and10 have much higher phase angle values, 27°, 30°, and 30°respectively, than the parent carba-ENA. Nucleoside 4 exhibitsan extremely small phase angle of 7°, suggesting 6′,7′-di-OHsubstitution in carba-ENA remarkably triggers the sugar towardNorth conformation. Other 6′ and/or 7′ modifications on carba-ENA have a medium effect on sugar puckering and their phaseangle P varies from 14° to 22°.Comparatively, substitution on the carbocyclic ring of carba-

LNA nucleosides led to less effect on sugar puckering thansubstitution on the carba-ENA. The phase angles of parent andall modified carba-LNA nucleotides vary in a narrow range from15° to 26°.The puckering amplitudes Φm describes the maximum out-

of-plane pucker.69 The puckering amplitudes of carba-ENA-type nucleosides, just like ENA and aza-ENA, are limited to anarrow range from 45° to 49°,34,36,61,70 whereas the puckeringamplitude of various carba-LNA nucleosides varies from 54° to58°.34,36,37,70 Hence, the pucker degrees of carba-LNAnucleosides are slightly higher than that of carba-ENA nucleo-sides. It seems that different substitutions on the carbocyclicring of carba-LNA and carba-ENA only lead to a marginal effecton the puckering amplitude.

4. ORIENTATION OF CARBOCYCLIC MOIETIES OFCARBA-LNA AND α-L-CARBA-LNA IN DUPLEXFORM

The models of carba-LNA modified DNA/RNA and α-carba-LNA modified DNA/RNA duplex have been built based onthe published structures of LNA modified DNA/RNA71 and

Scheme 17. Transition States of Hex-5-enyl (ynyl) Radical Cyclization To Afford 2′,7′-trans (A) and 2′,7′-cis (B) Products

Scheme 18. Transition States of Hept-6-enyl (ynyl, oxime) Radical Cyclization To Afford 2′,8′-cis (A) and 2′,8′-trans (B)Products

Scheme 19. Stereoelectronic Effects Dictate theStereochemical Selectivity of Free-Radical Cyclization



α-carba-LNA modified DNA/RNA duplex,72 respectively. Asshown in Figure 3A, the carba-LNA modified DNA/RNAduplex adopts an A-like duplex structure which has a wide,shallow minor groove and a narrow, deep major groove. Thecarbocylic moiety of carba-LNA locates on the top of the minorgroove. The α-L-carba-LNA modified DNA/RNA duplex

resembles a native DNA/RNA hybrid, adopting a geometryintermediate between that of A- and B-type duplex forms(Figure 3B). The carbocyclic moiety of α-L-carba-LNA lies atthe bottom of the deep major groove. According to themodels, it is likely that bulky substitution on carba-LNA willbe better tolerated than on α-L-carba-LNA because the bulky

Table 1. The Pseudorotational Phase Angles (P) and Puckering Amplitude (Φm)a of Carba-LNA and Carba-ENA Nucleosides

aAll the phase angle P and puckering amplitude Φm are calculated from structures obtained by ab initio (HF/6-31G**) optimization.



group renders less steric clash in the wider, shallower minorgroove. Hydrophobic substitution will lead to a morenegative effect on carba-LNA than α-carba-LNA since it isknown that the hydration network in the minor groove isimportant for the stability of the DNA/RNA duplex.73

Comparatively, C7′ is closer to the bottom of the groovethan C6′ for both carba-LNA and α-L-carba-ENA. Themodels provide the basis for understanding how differentsubstitutions on the carbocyclic moiety modulate theproperties of modified oligonucleotides.

5. THERMAL STABILITY OF MODIFIED AON/RNADUPLEX

5.1. Carba-LNA Modification

Carba-LNA-type thymidines that have been synthesized inChattoapdhyaya’s laboratory have been incorporated as monosubstitution but at four different sites in a 15mer AONsequence, 5′-d (CTT CAT TTT TTC TTC) (T denotes themodification sites). Relative thermal stability of the modifiedAON/RNA duplexes are compared with the native counterpartto give ΔTm which was calculated by subtraction of the duplexmelting temperatures (Tm) of native AON/DNA from the Tmof the modified AON/DNA. ΔTm listed in Figure 4A for eachmodification is an average ΔTm value of four AONs with themodification at four different sites.In the 15mer AON sequences 5′-d (CTT CAT TTT TTC

TTC), one LNA-T modification leads to Tm increase of about4.5 °C for AON/RNA duplex, whereas one parent carba-LNA-T (91) modification only leads to 3.6 °C increase in Tm.

37

Thus, replacement of 2′-O- with 2′-CH2- function results in a

negative effect on Tm for the AON/RNA duplex, suggesting the2′-oxygen of LNA plays an important role in the enhancedthermodynamic stability of the AON/RNA duplex. The crystalstructure of DNA duplex containing one LNA modificationshowed that the 2′-oxa of LNA is engaged in the hydrogenbonding to several water molecules.74 It is conceivable that thehydration network is likely to reduce the electrostatic repulsionof the internucleosidic phosphates, and thereby contribute toan increase in the thermodynamic stability for AON/RNAduplex (which in turn can have negative implications on thenuclease stability; see discussion below).Just like the parent carba-LNA itself, all C6′ and/or C7′

substituted carba-LNA modified AON/RNAs exhibit remarkablyhigher Tm than native duplex. However, it was found thatsubstitution at the C6′ and/or C7′ of carba-LNA rendersdifferent effects on the thermodynamic stability of the AON/RNA duplex depending both on the nature (hydrophobic versushydrophilic) and stereochemical orientation of the substitu-ent.36,37 Compared with parent carba-LNA, both the hydro-phobic 7′-Me and hydrophilic 7′-NH2 substitution on carba-LNAresult in Tm decrease and the extent of the decrease dependson the configuration. When the 7′-Me and 7′-NH2 substituentspoint to the vicinal 3′-phosphate (compounds 64, 67 and 87,97), they cause a decrease of the Tm by about 2−3 °C comparedto the parent carba-LNA modified AON/RNA, but when the7′ substituent points away from the vicinal 3′-phosphate(compounds 63, 69 and 88, 96), a much less pronounceddestabilization effect (around −1 °C) is observed.36,37 The Tm

decrease caused by C7′ substitution could be explained by theinfluence of (A) steric hindrance, and/or (B) hydration effects.

Figure 3. Models of carba-LNA/RNA (A) and α-carba-LNA/RNA (B) duplex. Sequence of the DNA strand: 5′-CTGATATGC. All three T aremodified by carba-LNA-T or α-carba-LNA-T but only one is highlighted on the model.



On the one hand, the C7′ substituent could render a steric clashin the minor groove of the AON/RNA duplex, leading to slightinduced-expansion of the minor groove to accommodate bulkysubstituents, which results in perturbation of local or globalconformation with an energy penalty at the cost of reduction ofthe thermal stability. On the other hand, the C7′ substituentcould potentially influence the hydration network in the minor

groove, thereby contributing to the energetic stabilization ordestabilization by promoting or perturbing the electrostaticinteractions. Since hydrophilic C7′-NH2 substitution results ina more significant destabilization effect on Tm than hydro-phobic C7′-Me substitution when they have the same stericorientation,37 the relative contribution of steric clash versusthe perturbed hydration is apparently difficult to dissect.

Figure 4. Comparison of (A) thermal stability: [ΔTm (ΔTm = Tm of modified AON/RNA hybrid − Tm of native AON/RNA hybrid)], (B) RNAselectivity: [ΔΔTm (ΔΔTm = ΔTm of AON/RNA hybrid − ΔTm of AON/DNA duplex)], and (C) nuclease stability [t1/2 of carba-LNA and carba-ENA modified AONs upon SVPDE digestion] of AONs containing different modifications. AON sequence is 5′-d (CTT CAT TTT TTC TTC); Tdenotes the carba-LNA and carba-ENA modification sites within the modified AONs.



The RNA affinity of methylene-carba-LNA modified AONhas been studied by Seth et al. using a 12mer DNA sequenced(GCGTTUTTTGCT) (U denotes the methylene-carba-LNA-Umodification).40 Methylene-carba-LNA modified AON/RNAshowed similar thermal stability as the LNA modifiedcounterpart. Hence, methylene modification at C7′ on carba-LNA seems better than both hydrophobic Me group and hydro-philic amino group. One obvious reason is methylene substi-tution renders less steric clash. Another explanation comesfrom the crystal structure of the duplex:40 it was found that themethylene group participates in the formation of a CH···O typehydrogen bond with the O4′ of the 5-terminal nucleotides of aneighboring duplex. Hence, the methylene-carba-LNA moietyretains a negative polarization in the minor groove, and thedisruption of hydration network by methylene substitution isminimal compared to other modifications. Using the same sequence,they also found the RNA affinity of both 7′R-Me-carba-LNAand 7′S-carba-LNA is less than LNA. Comparatively, 7′R-Me-carba-LNA is higher than 7′S-carba-LNA.40 This result isconsistent with the above conclusion obtained by Chattopad-hyaya from the 15mer AON sequence.36

On the contrary, both the hydrophilic hydroxyl group andhydrophobic methyl group substitution at C6′ of carba-LNAcan stabilize the duplex regardless of their orientation.36 Forexample, both 6′S-Me-7′S-Me-carba-LNA (63) and 6′R-Me-7′S-Me-carba-LNA (69) modified AONs showed slightly higherRNA affinity than the parent carba-LNA modified counterpart.Though 6′S-OH, Me-7′S-Me-carba-LNA (71) and 6′R-OH, Me-7′S-Me-carba-LNA (72) have opposite C6′ configuration, theyhave the same affinity toward complementary RNA, which iseven as good as LNA. Methylation of C6′ on LNA (cEt) hasalso been found to result in no negative effect on RNA affinityno matter what the configuration of the C6′-Me is.22 Obviously,a C6′ substituent of carba-LNA is located at the edge of theminor groove, thereby resulting in much less steric clashcompared with C7′ substituents, but how the C6′ substituentsrender stabilizing effect on AON/RNA duplex needs furtherelucidation. A practical conclusion that can be drawn fromthese observations36 is that introduction of modification at C6′can be used as an efficient strategy to fine-tune the electrostaticsof the backbone in order to further develop carba-LNAnucleotides with high affinity toward target RNA.

5.2. α-L-Carba-LNA Modification

Unlike C7′ and/or C6′ substituted carba-LNA modified AON/RNAs which exhibit higher Tm than native duplex, C7′ and/orC6′ substituted α-L-carba-LNA modification in the 15mer AONsleads to Tm decrease for the AON/RNA duplex (Figure 4A).65

Though 6′R-OH-7′S-Me-α-L-carba-LNA (121) and 6′S-OH-7′S-Me-α-L-carba-LNA (122) have an additional 6′-OH com-pared with 7′-Me-α-L-carba-LNA (124), AONs containingone 121 or 122 modification exhibit the same RNA affinity asthe 124 modified counterpart, around −3 °C/modification.This observation is consistent with the results obtained by Sethet al. recently that 6′-Me-α-L-LNA exhibited the same RNAaffinity as α-L-LNA and LNA.75 These results reveal that theC6′ substituent on α-L-carba-LNA and α-L-LNA which liesalong the edge of the major groove in the AON/RNA hybriddoes not impair RNA affinity.One 7′-methylene-α-L-carba-LNA modification in the 12mer

DNA sequence d(GCGTTUTTTGCT) leads to a 10 °Cdecrease in Tm for the AON/RNA duplex compared to thenative duplex.66 However, α-L-LNA modification in the same

sequence leads to Tm increase by 4.6 °C/modification. Hence,the destabilization effect caused by substituted α-L-carba-LNAshould be attributed to C7′ substitution. Comparatively, C7′hydrophobic substitution on α-L-carba-LNA seems much moreharmful to the thermodynamic stability of the AON/RNAduplex than C7′ substitution on carba-LNA. It is conceivablethat the C7′ substitution on α-L-carba-LNA, which locates inthe bottom of a narrow, deep major groove, creates a moresignificant steric clash and hydration perturbation than C7′substitution on carba-LNA that lies in the wide, shallow minorgroove of the AON/RNA duplex.

5.3. Carba-ENA Modification

Carba-ENA nucleosides that have been synthesized inChattopadhyaya’s laboratory34,36,61 have also been incorporatedas mono substitutions at four different sites of the 15mer AON[5′-d (CTT CAT TTT TTC TTC) where T denotes the sitesof modifications]. Generally speaking, all AONs incorporatedwith the carba-ENA derivatives show slightly higher RNAaffinity (Figure 4A) than the native AON but much lower DNAaffinity compared to carba-LNA modified counterparts.One parent carba-ENA-T modification (66) increases the

thermodynamic stability of the AON/RNA duplex on averageby 1.4 °C/modification.61 Further substitution on the C6′ and/or C8′ of the carbocyclic linkage of carba-ENA only slightlydestabilizes (down to −1 °C/modification) or stabilizes (upto +1 °C/modification) the AON/RNA duplex depending mostlyon the substitution site (C6′ or C8′) and orientation of thesubstituents but not so much on its nature (hydrophobic Meversus hydrophilic OH or NH2). For example, when C8′-OH orC8′-Me points at the 3′-phosphate (105 and 107, respectively),they lead to a more significant destabilization effect than whenthey point away from the 3′-phosphate (104 and 59).61 On theother hand, C6′-OH or C6′-Me substation leads to a slightstabilization effect when they point toward the 3′-phosphate,whereas when they point away from the 3′-phosphate, theyexert an obvious destabilization effect (78 vs 80, 82a vs 82b).36

Carba-ENA nucleosides 3, 4, 6, 9, 10, 1138,39 have beenincorporated as mono or triple substitution in 9mer AONsequences: 5′-d (GTG ATA TGC) (T denotes the modificationsites). One parent carba-ENA-U (6) modification in this 9merAON resulted in Tm increase by 4.0 °C/modification for theAON/RNA duplex.38 In view of the fact that one parent carba-ENA-T (66) modification in the 15mer AON sequence onlyled to a 1.4 °C increase in Tm, we can clearly see how significantthe Tm is sequence-dependent. C6′,C7′-di-OH substitution oncarba-ENA-U (4) can further slightly increase the Tm (+0.5to +1 °C/modification).39 On the contrary, introduction of theCC double bond on the carbocyclic ring of carba-ENA-U (3)leads to a slight decrease of the Tm (−0.5 to −1 °C/modification) compared to parent carba-ENA.38

6. RNA SELECTIVITY OF MODIFIED AONS

For all of the carba-LNA, α-L-carba-LNA, and carba-ENAmodified AON/DNA duplexes, the Tm is lower than that of themodified AON/RNA hybrid,34,36,37,61,65 so all of them are RNAselective. The magnitude of RNA selectivity (denoted byΔΔTm, ΔΔTm = ΔTm of AON/RNA − ΔTm of AON/DNAduplex) are compared in Figure 4B.The 7′-NH2 substitution in carba-LNA (87 and 88) and 8′-

NH2 substitution in carba-ENA (102) significantly reduce themagnitude of RNA selectivity (Figure 4B).37,61 Presumably, the7′-NH2 and 8′-NH2 group can reduce the repulsion of two



strands by neutralizing the negative charge on the phosphatebackbone to a higher extent in the AON/DNA duplex than inthe AON/RNA duplex because the minor groove of the formeris much narrower than the latter. For Me and OH modifiedcarba-LNA and carba-ENA, the RNA selectivity was found notto be significantly dependent on the chemical nature andorientation of substituent on the carbocyclic moiety.Generally, carba-ENA derivatives significantly decrease the

Tm of AON/DNA (∼ −4 to −5 °C/modification). As a result,the RNA selectivity (ΔΔTm) of carba-ENA derivativesmodified AONs is around 5 °C/modification.36,61 On the con-trary, carba-LNA derivatives only slightly increase or decreasethe Tm of AON/DNA, leading to medium RNA selectivity,around 3−4 °C/modification.36,37 Hence, carba-ENA-typemodified AONs show lower RNA binding affinity but betterRNA selectivity than carba-LNA-type modified AONs.34,36 It iswell-known that a typical B-type DNA/DNA duplex has anarrower minor groove (ca. 5−6 Å) than its DNA/RNA hybrid(ca. 9−10 Å).76 Thus, the larger six-membered carbocyclic ringin carba-ENA may produce larger steric perturbations in thenarrow minor groove of DNA/DNA than the relatively smallerfive-membered carbocyclic ring of carba-LNA. This may and infact has been shown to result in an overall larger destabilizationeffect for carba-ENA nucleosides modified AON/DNAduplexes compared to that of carba-LNA nucleosides modifiedAON/DNA duplexes. On the other hand, the AON/RNAhybrid has a relatively wider minor groove, and the steric clashbestowed by carba-ENA and carba-LNA lead to much smalldifferences in the thermal stability of AON/RNA than AON/DNA. The overall steric effect results in better RNA selectivityof carba-ENA over carba-LNA.α-L-Carba-LNA nucleosides modified AONs are also RNA

selective,65 but the magnitude of their RNA selectivity (2−3 °C/modification) is much lower than carba-LNA-type and carba-ENA-type modified AONs (Figure 4B).

7. NUCLEASE RESISTANCE OF MODIFIEDOLIGONUCLEOTIDES

Therapeutic oligonucleotides should be stable enough duringdelivery and after internalization of cells to fulfill their role.Unmodified AONs (i.e., single-stranded native DNA) are verylabile toward intracellular and extracellular nucleases.77 It hasbeen demonstrated that the nuclease resistance of AONcorrelates well with the magnitude and duration of the genesilencing effect.77 Hence, satisfactory nuclease resistance is arequirement for good pharmacokinetics of modified AON inantisense technology.78 It was found that the predominatenuclease activity in human blood serum is 3′ exonuclease.79

Chattopadhyaya et al. have compared the nuclease stabilities ofdifferent carba-LNA nucleosides and carba-ENA nucleosidesmodified AON sequences, 5′-d (CTT CAT TTT TTC TTC)(T denotes the modification), by treating them with 3′exonuclease, SVPDE under identical conditions.36,37,61 Theirstabilities (t1/2) toward SVPDE treatment are compared inFigure 4C.

7.1. Carba-LNA Modified Oligonucleotides

It has been reported that LNA modification only slightlyincreases80 the nuclease resistance for AON. All of the carba-LNA modified AONs are significantly more stable than theLNA-modified counterpart. This is a striking merit of carba-LNA compared to standard LNA. Moreover, AONs containingdifferent carba-LNA derivatives show completely different

nuclease resistance, which highlights that the substituent onthe carbocyclic ring of carba-LNA can significantly modulate itsnuclease resistance.Replacing the 2′-O in LNA with 2′-CH2, the obtained parent

carba-LNA was found to be 60 times more stable towardSVPDE treatment than LNA.37 All C6′ and/or C7′ substitutedcarba-LNA except C6′R-OH-carba-LNA (compounds 67 and69) modified AONs are more stable than the parent carba-LNAmodified counterpart, suggesting the steric clash imposed bythe substitution in the carbocyclic ring contributes greatly tothe improved nuclease resistance. Substituent C6′R-OHdecreases the stability,36,81 but C6′S-OH substitution, whichpoints to the 5′-phosphate, can significantly improve thenuclease resistance.36 It is conceivable that C6′R-OHsubstitution can assist the departure of the 3′-O− anion duringSVPDE digestion through intramolecular hydrogen bondformation, whereas C6′S-OH substitution retards the bindingof nuclease to the phosphate. Hydrophobic C6′-methylsubstitution renders the best effect on nuclease resistance forcarba-LNA and carba-ENA modified AON.36

Both hydrophobic (methyl) and hydrophilic (amino)substitution at C7′ of carba-LNA imparts a positive effect onnuclease stability with the C7′-Me-carba-LNA (55) being in thisrespect slightly better than C7′-NH2-carba-LNA (87 and 88).37

Generally for carba-LNA, C7′ substitution leads to a lesssignificant effect on nuclease stability than substitution at C6′when the substituents are the same. Unlike the substitution atC6′, the orientation of substituent at C7′ also seems to be notso important in view that C7′S-NH2 (87) and C7′S-Me (69)exert similar effect as C7′R-NH2 (88) and C7′R-Me (67)substitutions, respectively.37

To address why carba-LNA modified AONs exhibitconsiderably improved 3′-exonuclease stability than the LNAmodified counterpart, a Michaelis−Menten kinetic analysis ofSVPDE digestion of model dinucleotides 131−135 has beencarried out by Chattopadhyaya and Zhou.81 The results of theseexperiments had shown that 7′S-Me-carba-LNA modifieddimers 133 and 135 were 620- and 330-fold more stablerespectively than native dimer 131, whereas LNA modifieddimers 132 and 134 were only 6- and 50-fold more stable,respectively, than the native dimer 131 (Figure 5). Hence, 7′S-Me-carba-LNA modified dimers are much more nucleaseresistant than the LNA modified counterpart by ∼100 fold. ApKa measurement showed that the pKa of 3′-OH (13.53) of 7′S-Me-carba-LNA is 1.4 unit higher than that of 3′-OH of LNA(12.10).81 Since the scission of the 3′O−P bond has beenproven to be the rate limiting step upon SVPDE digestion,81 itseems it is the relatively lower acidity of the of 3′-OH of 7′S-Me-carba-LNA that makes the internucleotide phosphate of133 more nuclease resistant (decreased Kcat) than LNAmodified counterpart 132. On the contrary, the improvedstability of internucleotide phosphate for 3′-end modified dimer135 compared with that of dimer 134 comes not from thedecreased Kcat but increased KM, suggesting the 7′S-Me-carba-LNA modification retards binding of 3′-exonuclease to the5′-OH linked phosphate more remarkably than LNA.81 Insummary, both an electrostatic effect and steric clash contributeto the improved nuclease resistance for carba-LNA modifiedantisense oligonucleotides.

7.2. Carba-ENA Modified Oligonucleotides

As shown in Figure 4C, parent carba-ENA (66) modified AONwas found to be around 15 times more stable than parent



carba-LNA (91) modified AON toward SVPDE treatment.61

This can be easily understood in terms of their relativehydrophobic character: Propylene linkage between C2′ and C4′positions in carba-ENA is one carbon longer compared to theethylene linkage in carba-LNA. Thus, the propylene linkage incarba-ENA is more hydrophobic than carba-LNA and also mayrender a more pronounced steric hindrance to prevent nucleasebinding and attacking on the vicinal phosphodiester linkage.But unlike carba-LNA, neither hydrophobic nor hydrophilic

substitution at the C6′ and/or C8′ of carba-ENA renders asignificant effect on the nuclease resistance: C6′-Me sub-stitution on carba-ENA (82) only results in a 2-fold increase inthe stability;36 C8′-Me substitution (59 and 107) renders noobvious effect; hydrophilic substitution (OH, NH2) at C6′ andC8′ even leads to slightly decreased nuclease resistance.36,61

7.3. α-L-Carba-LNA Modified Oligonucleotides

Upon SVPDE treatment, α-L-LNA modified AON is morestable than the LNA modified counterpart and the former isless stable than α-L-carba-LNA modified AONs.65 However, thenuclease resistance of modified α-L-carba-LNA is not as good ascarba-LNA and carba-ENA derivatives, indicating the C6′ and/or C7′ substitutions on α-L-carba-LNA lead to a less positiveeffect on the nuclease resistance than C6′ and/or C7′ substitu-tions on carba-LNA nucleotides.

8. RNASE H ELICITATION OF CARBA-LNA ANDCARBA-ENA MODIFIED AONS

8.1. Carba-LNA and Carba-ENA Modified AONs

In antisense strategy, RNase H recruitment is a very importantapproach leading to target RNA degradation. Digestion ofcarba-LNA and carba-ENA modified AON/RNA duplex byEscherichia coli RNase H1 has been evaluated.36,37,61,65 It shouldbe noted that the activity obtained by E. coli RNase H1 may notreveal the real activity of RNase H elicitation in human thoughE. coli RNase H1 have similar nuclease properties as humanRNase H.82 It was found the carba-LNA-type and carba-ENA-type modified AONs are (1) good substrates for E. coli RNase

H1, and (2) the cleavage patterns are all very similar,independent of the nature of the modification type. Generally,as a result of modification in the AON strand, the cleavage ofAON/RNA hybrid duplex by RNase H is suppressed within a5−6 base pairs long region toward the 3′-end of the RNAstrand starting from the base opposite to the modification sitein the AON strand of the duplex. This is presumably because ofthe fact that the 5−6 basepairs from the site of modificationwith North-sugar constrained nucleotides take up a local RNA/RNA type conformation.71 This local alteration of theconformation in the modified AON/RNA hybrid cannot berecognized and cleaved by RNase H.For carba-LNA and carba-ENA derivatives modified AON/

RNA, the RNase H digestion rates are however dependent onthe site of modification within the AON strand.36 Generally,single modification at the 3′-end or 5′-end of AON strandsshow relatively higher RNase H recruitment ability comparedto those containing modification in the middle.36

The RNase H recruitment of AON (5′-d (CTT CAT TTTTTC TTC, T denotes modification site) containing differentcarba-LNA and carba-ENA nucleosides modifications arecompared in Figure 6. It seems that different types of carba-LNA and carba-ENA modifications in the AON strand canslightly influence the digestion rate. LNA modified AON/RNAwas digested by RNase H with a slower digestion rate thandigestion of native AON/RNA, but both parent carba-LNA(91) and parent carba-ENA (66) modified AON/RNAduplexes have been found to undergo digestion faster thanthe native counterpart.37,61 7′S-NH2-carba-LNA (87) and 8′R-NH2-carba-ENA (102) modification in AON strands have alsobeen found to accelerate the digestion of the complementaryRNA strand in the AON/RNA hybrid. However, when the C7′-NH2 substituent points away from the 3′ phosphate like in 7′R-NH2-carba-LNA (88) and 6′S-OH-7′S-NH2-carba-LNA (96),AONs with these modifications show the lowest RNase Hrecruitment ability.61 Other types of substituted carba-LNA andcarba-ENA generally render an insignificant effect on RNase Hrecruitment efficiency.36,37,61

Figure 5. Comparison of Michaelis−Menten parameters of digestion of native and 7′S-Me-carba-LNA and LNA modified dinucleotidesmonophosphates 131−135 by 3′-exonuclease, SVPDE.



8.2. α-L-Carba-LNA Modified AONs

Wengel et al. have reported that an AON sequenced(CACACTCAATA) fully modified by α-L-LNA can induceRNase H-mediated cleavage of complementary RNA, albeitwith an efficiency that is substantially reduced compared withnative DNA/RNA hybrid.23 A structural study based on theNMR showed that α-L-LNA modification does not alter theminor groove of AON/RNA too much, which provides areasonable explanation for the observation given the fact thatRNase H binds the AON/RNA duplex in the minor groove.83

However, Chattopadhyaya et al. found a α-L-LNA or α-L-carba-LNA modification in the 15mer AON sequence d(CTT CATTTT TTC TTC) leads to RNase H activity suppressed within a5−6 base pairs long region,65 just in the same way as for LNAand carba-LNA derivatives. It is conceivable that full modi-fication of this AON with α-L-LNA will completely abolishRNase H recruitment. Given that the concentration of RNaseH used in Wengel’s study is more than 100-fold higher thanthat Chattopadhyaya et al. used, the above contradiction couldbe attributed to the different enzyme concentrations used in thetwo experiments. If this is true, α-L-LNA modification in AON/RNA does not abolish the RNase H elicitation but needs moreenzyme for efficient RNA degradation. However, since theRNase H concentration in the actual biological system is sup-posed to be extremely low, heavy or full α-L-LNA modificationshould be avoided while designing therapeutic AON.The RNase H recruitment of AON sequences (5′-d (CTT

CAT TTT TTC TTC, T denotes modification site) containingdifferent α-L-carba-LNA derivatives modifications have beenstudied by Chattopadhyaya et al.65 Here, their RNase Hrecruitment ability is compared with that of carba-LNA andcarba-ENA (Figure 6). The reaction rates of RNase H-mediated

hydrolysis of α-L-LNA and 7′R-Me-α-L-carba-LNA (124)modified AON/RNA65 are much higher than digestion of thenative AON/RNA hybrid and ∼2 times higher than that ofLNA and 7′-Me-carba-LNA (55) respectively, suggesting thatthe locked ring of LNA and carba-LNA that lies in the minorgroove of AON/RNA hybrid may impart RNase H elicitation,but it is not the case for α-L-LNA and α-L-carba-LNA-typewhose locked ring is located in the major groove. The reactionrates of RNase H-mediated hydrolysis of 6′S-OH-7′S-Me-α-L-carba-LNA (122) and 6′R-OH-7′S-Me-α-L-carba-LNA (121)modified AON/RNA have been found to be slightly lower thanthat of the native counterpart.

9. BIOLOGICAL EVALUATION OF ANTISENSEOLIGONUCLEOTIDES CONTAINING CARBA-LNADERIVATIVES

Though more than 30 carba-LNA and carba-ENA deriva-tives have been synthesized, only a few including methylene-carba-LNA, 7′-Me-carba-LNA, and 8′-Me-carba-ENA have beensubjected to biological evaluation for antisense and RNAipotency.The biological property of AONs containing methylene-

carba-LNA and 7′R-Me-carba-LNA has been studied by Sethet al. recently.40 They designed two AONs (14mer and 18mer;see Figure 7) to target mouse PTEN mRNA, and two modifiedresidues were introduced to both the 5′ end and 3′ end of theseAONs (Figure 7). Down regulation of PTEN mRNA was firststudied in brain endothelial cells. For the 14mer AONs, it wasfound that the activity follows this rank: LNA (IC50 = 2.8 μM) >methylene-carba-LNA (3.9 μM) > 7′R-Me-carba-LNA (7.9 μM).These activities just parallel the Tm's of corresponding AON/RNA hybrids. The 18mer AONs were less active than the

Figure 6. Comparison of RNase H digestion rates of AON/RNA heteroduplexes containing different types of modifications. AON sequence is5′-d (CTT CAT TTT TTC TTC) and T denotes the modification site. Dotted line indicates the rate of RNase H digestion of native AON/RNAhybrids.



14 mer AONs. Comparatively, the methylene-carba-LNA modified18mer (D1) was more potent than LNA (D3) and 7′R-Me-carba-LNA (D2) modified ones (Figure 7).The potency of the same AONs has also been tested in

animal experiments. Dose-dependent down regulation ofPTEN mRNA in liver tissue was observed for all the modifiedAONs.40 The methylene-carba-LNA modified 14mer and18mer showed ED50 of 4.9 mg/kg and 4 mg/kg, respectively,which is very similar to that of LNA modified counterparts(Figure 7). 7′R-Me-carba-LNA was less potent. A very inte-resting observation is that at a high dose (e.g., 15 mg/kg), LNAmodified AONs were found to be highly toxic, but 7′R-Me-carba-LNA and methylene-carba-LNA modified ones onlyshowed modest toxicity. It is noteworthy that all of the AONsused in this work contain phosphorothioate (PS) internucleosidiclinkages. Hence, the results cannot reveal the effect of betternuclease resistance of carba-LNA analogues compared to LNA.The expansion of a CAG repeat in exon 1 of the Huntingtin

(HTT) gene leads to Huntington’s disease. However,expression of wild-type (WT) HTT is necessary to supportnormal development of function. Thus, potential therapeuticstrategies include reducing expression of mutant HTT allele.LNA modified antisense oligonucleotides have been shown tosuccessfully inhibit expression of the mutant HTT allele.84

Recently, AONs containing 7′-Me-carba-LNA have beensubjected to evaluation for the same purpose.85 7′-Me-carba-LNA, LNA, and several other types of modification have beenintroduced into a 19mer AON consisting of repeating GCTsequence. The cells employed for this evaluation are patient-derived fibroblast cells which contain 69 CAG repeats in themutant allele and 17 in the WT allele. After transfection ofthese AONs into the cell followed by 4 days of incubation, IC50values were calculated from Western blot quantification. Asshown in Figure 8, native AON I, ENA modified AON V, and2′F-RNA modified AON VI does not show any allele selectivity.However, the IC50 for 7′-Me-carba-LNA modified AON IV was15 nM and revealed >6.6-fold selectivity, which is even betterthan standard LNA and cET modified counterparts. The AONshaving the same sequence and modification but PS internucleosidic

linkages have been also prepared. It was found that PS-substituted AON VII, VIII, IX still achieved potent and selectiveinhibition of HTT, but the difference between 7′-Me-carba-LNAmodification and LNA, cEt became smaller. Just as expected, allof the PS-AONs displayed some toxicity.The expansion of a CAG repeat is also involved in other

diseases such as Spinocerebellar ataxi-3. The CAG repeat inATX3 gene is typically less than 31. Individuals with more than 52repeats show full penetrance. Inhibition of WT and mutant ATX3by 7′-Me-carba-LNA modified AON IV and cEt modified III(Figure 8) has also been studied by Corey et al.86 In contrast toallele-selective inhibition of HTT, both AON IV and III showedpotent but nonselective inhibition of WT and mutant ATX3.

10. RNAI POTENCY OF SIRNAS CONTAININGCARBA-LNA AND CARBA-ENA MODIFICATIONS

siRNA has been proven to have the potency to knockdownvirtually any specific genes in mammalian cells and hencerepresents an important new therapeutic strategy.87 However,native siRNA have many problems as potential therapeuticstools including nuclease degradation, off-targeting, delivery, andso on. An efficient solution to the challenges is chemical modi-fication.88 Several of carba-LNA and carba-ENA nucleosideshave been incorporated to siRNAs to target different mRNAs incell culture. It was found that carba-LNA and carba-ENAnucleoside modification can render striking features for siRNAsuch as improved potency and nuclease reisitance and de-creased off-target effect.89−92

Using the 2′-O-TEM based RNA synthesis strategy that hasbeen developed in our lab,93,94 7′-Me-carba-LNA and 8′R-Me-carba-ENA modified siRNAs have been synthesized to targetthe eGFP gene in HeLa cells.89 These HeLa cells stablyexpressing eGFP were transfected with 10 nM siRNAscomplexed with INTERFERin. After 72 h post-transfection,eGFP levels were evaluated and compared with that obtained inexperiments employing siRNAs incorporated at differentmodification sites with 19 other types of modified nucleotidessuch as 2′-F, 2′-OMe,95 4′-modified RNA (HM),96 LNA;

Figure 7. Targeting mouse PTEN mRNA by methylene-carba-LNA, 7′R-Me-carba-LNA, and LNA modified 14mer and 18mer AONs. Underlinedletters indicate modified nucleosides. All internucleosidic linkages are phosphorothioate; IC50 values were determined in brain endothelial cells usingelectroporation.



α-L-LNA, aza-ENA,28 UNA,97 HNA,98 ANA,99 etc. Thisexhaustive study provides valuable guidance to chemicallymodify siRNAs as potential therapeutics.89 It was found thatregardless of the modification type, the modifications in thepassenger strand are well tolerated, and even siRNAs withheavily modified passenger strands support target geneknockout to below 20%. Modification at the 3′-end of passengerstrand has been found to be a very effective strategy to improvesiRNA performance especially when the modification canthermally destabilize siRNA duplex. It is likely that the 3′-endmodification increases siRNA performance by favoring guidestrand incorporation to RISC.89 However, not only modificationtype but also modification position as well as number ofmodifications in the guide strand have been found to have astrong impact on siRNA silencing activity:89 (1) Extensivemodification of the guide strand is generally accompanied byreduced activity. (2) Multimodification in the seed region(positions 2−8 counting from 5′-end) result in reduced activity,but single modifications in the seed region are generally toleratedfor HNA, ANA, 2′-OMe, 7′-Me-carba-LNA, and 8′R-Me-carba-ENA, whereas single LNA, aza-ENA modification in theseed region have a negative effect on silencing activity. (3)Modification in the central position of guide strand is verysensitive to modification type. Modifications that arecapable of perfect base-pairing to the target gene aregenerally well tolerated; otherwise, the modification in thecentral position of guide strand impairs silencing activity.(4) Most modification types are well tolerated in the3′-region of the guide strand. Hence extensive modificationin the 3′-region of the guide strand could be preferred toimprove the 3′-exonuclease resistance.The potency of downregulation of eGFP by siRNAs con-

taining different chemical modifications in the guide strandhave been compared, and it was found that siRNAs containing7′-Me-carba-LNA or 8′R-Me-carba-ENA modification are ob-viously higher than other types of modifications (Figure 9).89

siRNAs containing one 7′-Me-carba-LNA (JC-F1, Figure 9) or8′R-Me-carba-ENA (JC-S1) modification at the position 3(from 5′ end) of guide strand showed the best gene silencing

efficiency, which is much higher than that of siRNAscontaining UNA, aza-ENA, or LNA modification in thesame region. They are compatible with the siRNAs contain-ing a HNA modification in the seed region as well as withunmodified siRNA (Figure 9). Interestingly, some siRNAssuch as JC-F1/W131 and JC-S1/DO003 that formed byannealing JC-F1 and JC-S1 respectively with modified passengerstrands showed even better silencing potency than the nativecounterpart.89

In this study, LNA modified siRNA was found to havesignificantly improved serum stability than native siRNA. Thestability of 7′-Me-carba-LNA modified siRNA should be at leastequal or better than the LNA modified one. However bothLNA and 7′-Me-carba-LNA modified siRNA did not showbetter eGFP knockdown activity than native siRNA, suggestingthe activity of siRNA in cell culture is not related to the serumstability of siRNA. Hence, we cannot attribute the improvedactivity of 7′-Me-carba-LNA modified siRNA compared to LNAmodified counterpart to the better nuclease resistance of theformer.It is known100 that interaction between the seed region of the

guide strand and the complementary sites in the target mRNAis very important for the specificity of gene deregulation.Chemical modification in the seed region has been proven tobe an efficient approach to reduce the off-target effects.101

Recently, 7′-Me-carba-LNA and 8′R-Me-carba-ENA togetherwith other eight types of modifications have been incorporatedinto the seed region of the guide strand of siRNA to evaluatetheir off-target effects.90 Generally, siRNA potency was foundto be positively correlated to off-target effects for mostinvestigated siRNA. However, reduced off-targeting can beachieved by properly incorporating some types of modificationsto specific positions in the seed region. For example, a single 2′-OMe modification at position 2, a single 7′-Me-carba-LNA,8′R-carba-ENA, aza-ENA, or HNA modification at position 3can obviously reduce off-target effects. A single UNA modi-fication, however, at position 7 resulted in the most potentactivity. Since a single nucleotide modification of carba-LNAor carba-ENA in the seed region is well tolerated,89 it seems

Figure 8. AONs containing LNA, 7′-Me-carba-LNA modifications support allele-selective HTT inhibition. Underlined letters indicate modifiednucleosides. For AONs VII, VIII, and IX, all internucleosidic linkages are phosphorothioate.



that incorporating a single carba-LNA or carba-ENAmodification at position 3 of the guide strand can producesiRNAs with high knockdown activity but reduced off-targeteffects.Silencing HIV-1 by carba-LNA modified siRNA in HEK293T

cells culture has been studied by us recently.91 One 21mersiRNA, 5′-U1A2G3C4C5A6G7A8G9A10G11C12U13C14C15C16A17-G18G19U20U21 (guide strand), was selected as the target againstthe HIV-1 TAR1 region. 7′-Me-carba-LNA and LNA wereintroduced into position U1, U13, U20 or U1 + U20 in the guidestrand. After 48 h post-transfection using Lipofectamine, theIC50 values were evaluated by p24 ELISA (Figure 10). 7′-Me-carba-LNA modified siRNA showed very similar silencing po-tency as the LNA modified counterpart when their modificationpositions are the same. However their silencing potency varieda lot with the change of the modification positions. Modifica-tion at position 1 and position 13 resulted in a minimum effectand the silencing efficiency was found similar as native siRNA,whereas modification at position 20 and especially 20 plus 1 led

to increased silencing potency. Serum stability study revealedthat 7′-Me-carba-LNA modification confers exceptionalstability in a position dependent manner:91 Doublemodification at position 20 and 1 is the best, followed bysingle modification at position 20, and single modification atposition 1 showed the least stability though it is still 2 timesmore stable than the unmodified one. Comparatively, LNAmodified siRNAs were found similar or only slightly lessstable than 7′-Me-carba-LNA modified counterparts inserum, which is contradictory to the fact that 7′-Me-carba-LNA modified antisense oligonucleotides is significantlymore stable LNA modified ones. This is because degradationof double-stranded siRNA in serum follows differentmechanisms than degradation of single-stranded antisenseolionucleotides.102−104

In this study,91 the toxicity of these modified siRNAs wasstudied by MTT assay. It was found all the 7′-Me-carba-LNAand LNA modified siRNAs were just the same as native siRNA,so none of them confer cellular toxicity.

Figure 9. (A) Comparison of silencing eGFP activity of modified siRNAs in HeLa cells. The eGFP levels shown here are the values after72 h post-transfection of 10 nM siRNAs complexed with INTERFERin. The sequences of siRNA and structures of modifications areshown in (B).



A pair of members of the carba-LNA family, 6′R-O-Tol-7′S-Me-carba-LNA and 6′S-O-Tol-7′S-Me-carba-LNA, have alsobeen incorporated to siRNA to targeting the HIV-1 TAR1region.92 Compared to 7′-Me-carba-LNA, both 6′R-O-Tol-7′S-Me-carba-LNA and 6′S-O-Tol-7′S-Me-carba-LNA have a addi-tional bulky and hydrophobic C6′-O-Tol group. When modifiedat the end of the guide strand, for example, position 1 andposition 20, the silencing potency of 6′R-O-Tol-7′S-Me-carba-LNA and 6′S-O-Tol-7′S-Me-carba-LNA is more orless than 7′S-Me-carba-LNA. However, modification inthe middle, position 13, 6′R-O-Tol-7′S-Me-carba-LNA wasfound to be 2.4-fold more efficient than 7′-Me-carba-LNA,but 6′S-O-Tol-7′S-Me-carba-LNA was found to be 10-foldless efficient than 7′-Me-carba-LNA, so the differentconfiguration of C6′-O-Tol substitution leads to a 24-folddifference in the silencing potency. A model study showedthe 6′R-O-Tol is exposed toward the edge of the RNAduplex, while 6′S-O-Tol is located in the minor groove.6′S-O-Tol-7′S-Me-carba-LNA modification at the position13 is in the vicinity of the RISC cleavage site, and itmay inhibit RISC mediated hydrolysis. This study high-lights the possibility of using carba-LNA derivatives asmolecular probes to address the mechanism of someimportant biology processes.

11. CONCLUSIONS AND IMPLICATIONSSynthesis of carba-LNA- and carba-ENA-type modifiednucleosides has been achieved so far by free-radicalcyclization strategy and ring-closing metathesis. Free-radicalcyclization approach also allowed us to synthesize α-L-carba-LNA nucleos(t)ides. In the duplex form, the carbocyclicmoiety of carba-LNA and carba-ENA modifications is foundlocated in the minor groove, whereas the carbocyclic moietyof α-L-carba-LNA lies in the major groove. The C7′ of carba-LNA and α-L-carba-LNA, C8′ of carba-ENA are in the centerof the grooves, which provide very good scaffolds for furtherfunctionalization to study how different chemical environ-ments in the grooves modulate target RNA recognition aswell as how they affect interaction with different enzymes.

This is one of the advantages of carba-LNA and carba-ENAover LNA.Another advantage of carba-LNA and carba-ENA over LNA

is the carba-LNA and carba-ENA modified antisenseoligonucleotides are significantly more stable in serum thanthe LNA modified counterpart. It is known the antisenseactivity is correlated directly with nuclease resistance for anti-sense oligonucleotides.77 So far, the limited biological evalua-tion data showed when containing phosphorothioate (PS)internucleosidic linkages, carba-LNA and LNA modified AONshave similar silencing efficiency,40,85 but when containingnormal phosphate internucleosidic linkages, carba-LNA modi-fied AONs showed slightly better silencing potency than LNAmodified ones.85 However, it is not clear how much of theincreased silencing potency should be attributed to its improvednuclease stability. Hence, the effect of nuclease stability of carba-LNA modified AON on the in vitro and in vivo kinetics of genesilencing needs to be addressed and compared with the LNAmodified counterpart. Since PS-modified oligonucleotidesoften show some level of toxicity, development of nucleasestable oligonucleotides with high affinity to target RNA butwithout PS modification continues to be a perspective trend.From this point, carba-LNA modifications deserve moreattention in the field of antisense oligonucleotides basedtherapeutics.Though single-strand RNA is very susceptible to intracellular

nucleases, double-strand siRNA is fairly stable once enteringinto the cells.105 The major degradation of siRNA occurs duringdelivery.106 The mostly used delivery agents such DOTAP andLipofectamine 2000 do not protect the siRNA from degrada-tion,107 so improvement of nuclease stability for siRNA is alsoimportant for successful therapeutics. Not like in antisenseoligonucleotides, one or two carba-LNA modifications in the 3′end and/or 5′ end of siRNA does not significantly improve thenuclease stability compared with standard LNA. This is becausesiRNA is digested in serum by RNase A like endonuclease butnot by exonuclease. Comparison of nuclease stability of carba-LNA heavily modified siRNA with the LNA modifiedcounterpart and studying the effect of siRNA nuclease stabilityon in vitro and in vivo gene silencing remain to be done. Onthe other hand, the interesting observation that significantdiastereomeric bias with C6′-O-Tol-carba-LNA modifiedsiRNA92 suggests modification on the carbocyclic moiety ofcarba-LNA is an efficient strategy to modulate the RNAipotency. A detailed structure−activity relationship basedon the available carba-LNA derivatives may providevaluable principles on how to modify siRNA to get highsilencing potency. In this aspect, practical conclusion whichcan be drawn from above observations is that introduc-tion of various types of modifications at C6′ and C7′ incarba-LNA provides unique possibilities, compared tothe LNA type molecules, to mine differently active molec-ules for “designer” thermodynamic and nuclease stabilityand delivery, which are essential for synthetic oligonucleo-tides to be successful as potential therapeutics. This isbecause there is a choice to fine-tune the electrostaticsof the backbone in order to further develop carba-LNAnucleotides with high affinity toward target RNA and to preparefavorable conjugates built on the carba-bridge for one of thecarba-LNA derivatives to be successful in the ADME-Toxstudies.

Figure 10. Targeting HIV-1 TAR1 region by LNA and carba-LNAmodified siRNA. IC50 values were determined from p24 ELISA ofHEK 293T cells culture supernatant. The sequence of guidesequence is 5′-U1A2G3C4C5A6G7A8G9A10G11C12U13C14C15C16-A17G18G19U20U21 and modifications locate in position 1, 13, 20 orboth 1 and 20.



AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected].

Notes

The authors declare no competing financial interest.

Biographies

Chuanzheng Zhou was born in Hubei, China, in 1978. He earned hisbachelor degree in chemistry from Nankai University in2001 followed by a master's degree in organic chemistry in 2004 underthe supervision of Prof. Zhen Xi. From October 2004 to September 2010Chuanzheng Zhou worked under the supervision of Prof. JyotiChattopadhyaya at the Chemical Biology Program, Biomedical Center,Uppsala University on nucleic acids chemistry and structure leading toa Ph.D. degree in bioorganic chemistry in 2010. At present he is apostdoctoral fellow with Prof. Marc Greenberg at the Johns HopkinsUniversity.

Jyoti Chattopadhyaya obtained his Ph.D. from National Chemicallaboratory and Pune University in 1974 followed by a posdoctoraltraining with Prof. Collin B. Reese, FRS, in London University atKing’s College (1974−1979). He has been holding a full-Chair ofBioorganic Chemistry since 1982 at Uppsala University. His researchinterests include chemical synthesis of DNA and RNA and theirderivatives in order to modulate the biophysical, structural, andbiological properties, stereoelectronic effects in nucleotides and howthey assist in the preorganization of DNA and RNA, synthesis ofnucleos(t)ide and nucleotide analogues as potential therapeutics,carbohydrate and heterocyclic chemistry as well as solution structuresof nucleic acids by NMR spectroscopy. He has supervised 30 Ph.D.students and is the author of more than 400 scientific publications

(http://www.boc.uu.se). He has received many awards includingHumboldt Research Award and Sorm Award.

ACKNOWLEDGMENTS

Generous financial support from the Swedish Natural ScienceResearch Council (Vetenskapsradet), the Swedish Foundationfor Strategic Research (Stiftelsen for Strategisk Forskning), andthe EU-FP6 funded RIGHT project (Project No. LSHB-CT-2004-005276) and Uppsala University is gratefully acknowl-edged. Authors thank very warmly their co-workers who havebeen involved in this carba-LNA/ENA project. Authors alsothank Oleksandr Plashkevych for his proofreading of themanuscript.

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