comprehensive natural products chemistry || dna with altered backbones in antisense applications

7.08DNA with Altered Backbones inAntisense ApplicationsYOGESH S. SANGHVIIsis Pharmaceuticals, Carlsbad, CA, USA

6[97[0 INTRODUCTION 174

6[97[0[0 Principles 1756[97[0[1 Scope of the Chapter 176

6[97[1 WHY ALTER THE DNA BACKBONE< 176

6[97[1[0 Increased Stability Towards Nucleolytic De`radation 1766[97[1[1 Enhanced Af_nity For Tarèt RNA 1776[97[1[2 Improved Cellular Uptake 1786[97[1[3 Reduced Cost and Synthetic Feasibility 178

6[97[2 CLASSIFICATION 178

6[97[2[0 Phosphate!modi_ed Linkaès 1896[97[2[0[0 Modi_cation of a nonbridìn` phosphateÐoxyèn atom 1806[97[2[0[1 Modi_cations of the bridìn` 2?!oxyèn atom 1846[97[2[0[2 Modi_cation of the bridìn` 4?!oxyèn atom 1866[97[2[0[3 Modi_cations of the 4?!carbon 187

6[97[2[1 Nonphosphate or Dephosphono Linkaès 1876[97[2[1[0 Nitroèn!containin` linkaès 1886[97[2[1[1 Silicon!containin` linkaès 2926[97[2[1[2 Sulfur!containin` linkaès 2936[97[2[1[3 Oxyèn!containin` linkaès 2936[97[2[1[4 All!carbon linkaès 2946[97[2[1[5 Nonphosphate suàr linkaè "PNA# 295

6[97[3 DESIGN CONSIDERATIONS 295

6[97[4 CONCLUSIONS 296

6[97[5 REFERENCES 297

6[97[0 INTRODUCTION

Antisense!based oligonucleotide technology is a _eld of research that has intensi_ed the interestin chemical and biological sciences since the mid!0879s[ Evidence of this is the tremendous growthin the number of research publications and review0Ð4 articles[ In order to obtain a complete pictureof the scope and dimension of this technology\ readers should consult specialized books5Ð09 on thistopic[ Antisense technology represents a major paradigm shift for pharmaceutical! and bio!technology!based drug discovery\ with enormous potential well into the twenty!_rst century[Advances in the automated synthesis of oligonucleotides "oligos# and commercialization of naturaland modi_ed nucleic acid building blocks\ allow the generation and screening of unprecedentednumbers of novel synthetic oligos[ The main emphasis of this chapter will be on advances made

174

175 DNA with Altered Backbones in Antisense Applications

towards development of new synthetic methodologies to construct novel oligos containing alteredor modi_ed backbone linkages[ In addition\ this chapter will focus on the success and failure ofthese altered backbones to serve the intended biological functions[ What follows is a discussionand summary of mainly published results together with some unpublished work at the author|sinstitution[

6[97[0[0 Principles

Antisense oligos are short "½19 mer# synthetic single!stranded molecules\ analogues of naturalnucleic acids\ which inhibit or modulate gene expression[ The inhibition of gene expression refersto the binding of an antisense oligo in a sequence!speci_c manner to preselected RNA targets andtherefore blocking the translation to the corresponding protein "Figure 0#[ The underlying principlefor this blockage is governed by simple WatsonÐCrick hydrogen bonding base!pair rules betweenthe antisense oligo and the cellular mRNA[ As the short synthetic oligomer binds to the {{sense||mRNA and prevents its use\ it is called an {{antisense|| oligo[ These senseÐantisense relationshipsare depicted in Figure 0 for the inhibition of gene expression[

Figure 0 Schematic presentation of the antisense concept "top#\ WatsonÐCrick base!pairing "bottom left] AÐT\ GÐC#\ and structure of the phosphorothioate linkage "bottom right# representing the _rst generation of

antisense oligonucleotides[

In principle\ this {{oligo drugÐmRNA|| binding recognition motif provides many orders of mag!nitude higher a.nity and speci_city than can be achieved using traditional drug!design approaches[Interestingly\ both antisense oligos and mRNA targets share common nucleic acid chemistries\ suchas sugars\ bases\ and backbones\ and since key nucleic acid interactions are well understood\ therational design of antisense oligomers is possible[ An antisense oligo can be designed to target anymRNA\ potentially creating highly speci_c drugs for any disease in which genomic sequence

176DNA with Altered Backbones in Antisense Applications

information is known[ As a result\ antisense oligomers have been reported as potential antiviral\antitumor\ and anti!in~ammatory agents with signi_cant in vitro and in vivo pharmacologicalactivities[0

6[97[0[1 Scope of the Chapter

The content of this chapter is restricted to the modi_cations of the sugarÐphosphate moiety thatconnects the bases in natural nucleic acids[ A variety of linkage changes replacing one or both ofthe nonbridging oxygen atoms\ or one or more of the four bridging atoms which connect "2?: 4?#the two sugar moieties are described in this chapter[ This account does not include developments inmodi_cations of unusual or special linkages\ such as 1?: 4? or 2?: 2? linked oligos\ short "2!atoms#or long "4!atoms or more# linkages\ a!oligos\ and L!oligos[8 In addition\ modi_cations of sugar andheterocyclic bases are not covered[ Chapter 6[98 in this volume describes such base modi_cations[

Clearly\ most of the modi_cations described herein can be directly or indirectly utilized for otherapplications\ such as construction of synthetic ribozymes\ triplexes\ circular oligos\ and otherstructured oligos[ In order to keep this review short and focused\ only antisense!related applicationsare discussed[

6[97[1 WHY ALTER THE DNA BACKBONE<

If natural oligos are considered prototypic\ then the available chemically modi_ed antisenseoligomers can be regarded as _rst!\ second!\ or third!generation antisense molecules\ dependingupon the complexity of modi_cations introduced[ Early experimentation with unmodi_ed oligoscon_rmed that use of natural DNA as an antisense molecule had signi_cant limitations[ Forexample\ natural DNA or RNA oligos are rapidly degraded under physiological conditions by avariety of cellular nucleases that primarily hydrolyze the phosphodiester internucleosidic linkage[As such\ unmodi_ed oligos have only limited utility in advancing antisense research past rudimentarylevels[ As a consequence\ a signi_cant number of modi_cations of the phosphate backbone havebeen made[00 A detailed review of the synthesis of such modi_ed oligomers is beyond the scope ofthis chapter[ However\ it is worth highlighting some of the more promising modi_cations andproviding references for some of the well!known modi_cations[ It is believed that for a successfuloutcome of any backbone modi_cation\ the modi_ed oligomer must meet the following criteria\discussed individually[

6[97[1[0 Increased Stability Towards Nucleolytic Degradation

A major problem in using oligo!containing natural phosphodiester "PO# linkages in antisensetherapeutics\ is that these molecules are nuclease sensitive\ due to serum and intracellular nucleasesthat will hydrolyze the PO linkages\ resulting in small fragments of nucleotide molecules[ Therefore\synthesis of nuclease!resistant antisense oligomers that retain the a.nity and speci_city for thecomplementary RNA has been a major focus of antisense research[

As a result\ two important _rst!generation modi_cations were made to a PO linkage and gave riseto the phosphorothioates01 "PS# and methylphosphonates02 "MP#[ Both modi_cations successfullyincreased the nuclease stability of oligomers relative to PO linked oligomers[ A detailed account ofPS and MP oligomers can be found under individual sections[

It was recognized early on that the modi_cations of the 2?!hydroxy group of oligo\ as well asmodi_cations of the last few 2?!terminal bases or internucleosidic linkages\ enhanced their stabilityagainst 2?!exonuclease activity[03 However\ use of a {{2?!end cap|| strategy in antisense oligos maynot be adequate for their use in vivo\ because cellular nucleases also have signi_cant levels ofendonuclease activity[ On the other hand\ e}orts have been made to generate a {{chimeric antisensemolecule|| "Figure 1# that displays desirable stability in vitro and in vivo[04 Commonly\ these oligoscontain at least two or more types of modi_cations[ First\ a short segment of PS modi_cation "4Ð09 base pairs# is present for RNase H activity in the middle of the oligo\ which is ~anked at the 2?!and 4?!ends by segments of another type of modi_cation that provides not only nuclease resistance\but high a.nity too[ In a chimeric oligo\ MP modi_cation was introduced at the 2?! and 4?!ends


with the central core of PS modi_cation[ Such chimeric oligos have shown favorable half!lives inanimals\ with potent biological activity as well as reduced binding to plasma proteins[05

Figure 1 Chimeric oligonucleotide strategy[

In the 0869s\ the _rst e}orts to replace completely the PO bridge with nonphosphorus "dephos!phono# linkages were made\ with an aim towards gaining nuclease resistance[ With the advent ofantisense research\ a tremendous growth in the synthesis of novel dephosphono linkages hasoccurred[ Over 59 types of linkages have been studied since 0883[06 Comprehensive reviews on thistopic have been published[07Ð14 Nuclease stability remains a high priority towards the design of novelantisense agents\ which becomes clear during the rest of the discussions in this section[

6[97[1[1 Enhanced Af_nity For Target RNA

Target sites for antisense are discovered using gene walks along the mRNA[15 This requiressynthesis of a large number of oligos[ The a.nity of an antisense oligo for its RNA complement istraditionally assessed by Tm or melting temperature analysis[16 The Tm values are highly reproducibleunder _xed salt and duplex concentration[ Therefore\ in order to predict the binding a.nity of anymodi_ed antisense oligo to its RNA target\ it is necessary to evaluate hybridization e.ciency ofsuch hybrids[

Typically RNAÐRNA duplexes show higher Tm compared to DNAÐRNA and DNAÐDNAduplexes\ because of their constrained structure[ Based on this fact\ chemists working on antisensedrug development have pursued several approaches to producing high!a.nity molecules that containthe traditional modi_cation around the phosphate atom "via automated procedures#\ 1?!modi!_cations of the sugar\ and complete replacement of the backbone linkage[ A more detailed accountof how to tune in high a.nity "Tm# into oligos is discussed under each subheading[ Most of theresults are described in terms of Tm values increasing or decreasing upon hybridization to thecomplement RNA[ For example\ in a closely related group of oligos an increase in Tm of 2Ð4 >C"DTm# corresponds roughly to a 09!fold enhancement in association constant[


6[97[1[2 Improved Cellular Uptake

Unlike traditional drugs\ which are of low molecular weight and are uncharged\ certain antisenseagents are polyanionic and of high molecular weight\ which are regarded as presenting a majorimpediment to traverse through the cellular membrane[ Nevertheless\ phosphorothioate antisenseoligos are taken up reasonably well by most cell types[ On the other hand\ the transport anddistribution of antisense oligos into cellular machinery can be altered by suitable chemical modi!_cations of sugarÐbase or Ðbackbone[ Adsorptive endocytosis and receptor!mediated transport arethe two uptake mechanisms by which natural and modi_ed oligos are internalized[17 Surprisingly\the concentration of free oligos in cytoplasm is low due to their compartmentalization in endocyticvesicles[ Nevertheless\ oligos do escape the endosomal compartment and produce a biological e}ect[

Various oligo modi_cation strategies have been utilized to improve cellular uptake and cyto!plasmic transport of antisense molecules[ These include covalent conjugation of various chemicalfunctionality to an antisense agent to alter its hydrophobicity\ charge and amphipathicity[18 Attach!ment of speci_c ligands "such as folic acid and vitamins# to an antisense oligo that binds to certaincellular receptors\ may enhance the internalization of such polyanionic molecules[ Attempts havebeen made to increase the membrane permeability of oligos by neutralization of the charge on thePO backbone\ such as MP and phosphotriesters[ A report indicated that MPs are internalized viaan active uptake process and passive di}usion does not play a signi_cant role in their uptake[29

Generally\ cellular uptake is time! and temperature!dependent[ It is also in~uenced by celltype\ cell!culture conditions\ media\ and sequence "e[g[\ length#[20 Again\ signi_cant di}erences insubcellular distribution between various types of cells have been noted^ therefore\ uptake of antisenseoligos in vitro may not predict their activity in vivo[

6[97[1[3 Reduced Cost and Synthetic Feasibility

Development of a cost!e}ective antisense oligo depends upon improving the synthesis and chem!istry of its production\ increasing the potency and thus reducing the e}ective dose\ and modulatingthe pharmacokinetic parameters to increase the half!life and concentration at the target site[ DNAsynthesis and puri_cation technologies can provide oligonucleotides in multigram quantities\su.cient for preclinical and clinical studies[21 Furthermore\ instrumentation for large!scale synthesisis commercially available allowing 099 mmol of 19!mer PS oligos to be prepared in a single run[Pharmacia Biotech AB of Sweden has developed22 such an instrument\ the OligoProcess\ shown inFigure 2[ One of these mega!synthesizers is currently in use at the Isis GMP manufacturing facilitiesin Carlsbad\ CA\ producing ½09 kg of PS oligos to support human clinical trials of _ve antisenseoligos in 0885[ Through improved and optimized automated synthesis on OligoProcess and puri!_cation conditions\ the cost of a 19!mer PS oligo manufactured on a 0 mol "6[4 kg crude:run# scaleis estimated to be about US,299:g\ making it very attractive for therapeutic use[23

Use of certain nonphosphate linkages may have a potential advantage to be more economicaland easier to synthesize on a large scale in solution phase rather than the traditional solid!supportsynthesis of _rst generation PS oligos[24 Additionally\ use of a nonionic\ achiral backbone\ whichexhibits high a.nity for target RNA could result in shortening the length of the oligos withoutcompromising the activity\ thus decreasing the production costs[25 In selecting a completely neutraloligo\ water solubility must also be considered[ Therefore\ an appropriate combination of neutral"nonphosphate# and charged "anionic or even cationic# backbones could be expected to haveimproved potency and pharmacokinetic properties[

6[97[2 CLASSIFICATION

The _rst use of a synthetic antisense oligonucleotide was reported by Zamecnik and Stephensonin 0867[26 The apparently simple principle and potential power of the approach led to a rapidproliferation of research[ Particularly in the area of backbone modi_cations\ research has been ona fast track due to its usefulness\ as discussed in Section 6[97[1[ Another obvious reason for thegrowth of backbone modi_cations is because it does not directly interfere with the key element ofrecognition i[e[\ WatsonÐCrick base pairing[ Broadly stated\ all backbone modi_cations used forantisense research can be classi_ed under two headings] _rst\ phosphorus!containing linkages


Figure 2 The OligoProcess instrument installed at Isis Pharmaceuticals[

and modi_cations thereof\ and second\ nonphosphate or dephosphono linkages containing otherheteroatoms "Figure 3#[

Figure 3 The various sites of backbone modi_cations[

6[97[2[0 Phosphate!modi_ed Linkages

Modi_cation of the phosphate backbone is viewed as one of the most fundamental and best!studied modi_cations in antisense research[ The impetus for the PO modi_cation derives fromdevelopment in two areas[ Overall improvements in automated oligo synthesis represents the _rst\and advanced understanding of their pharmacological and toxicological pro_le in vitro and in vivorepresents the second[ These two aspects taken together allowed the launching of several human


clinical trials with _rst!generation antisense oligos "Table 0#[27 This section is subdivided into fourtypes of modi_cation\ depending upon where the modi_cation is placed in relation to the centralphosphorus atom[

Table 0 List of human clinical trials with _rst!generation antisense oligonucleotides] phosphorothioates"Figure 0#[

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐMolecular Route"s# of

Oliònucleotide tarèt Disease indications administration Status Sponsor*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐGem 80 gag HIV intravenous\ II Hybridon

subcutaneous

ISIS 1811 HCMV IE CMV retenitis III Isisgene product

ISIS 1291 Intercellular renal allograft\ intravenous\ II Isis:Boehringeradhesion rheumatoid arthritis\ subcutaneous Ingelheimmolecule!0 Crohn|s disease\

ulcerative colitis\psoriasis

LR!2990 C!myb cancer "CML# intravenous I:II Lynx

LR!2179 C!myc restenosis intracoronary I Lynx

ISIS 2410:CPG protein kinase cancer "various# intravenous I Isis:Ciba53017A C!a Geigy

ISIS 4021:CPG Craf kinase cancer "various# intravenous I Isis:Ciba58735A Geigy

GPs 9082 HIV intravenous I Chugai

< Ha!ras bladder cancer bladder I Rhoneinstillation Poulenc Rorer

G2028 BCL!1 cancer intravenous I:IIa Genta*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

6[97[2[0[0 Modi_cation of a nonbridging phosphateÐoxygen atom

The replacement of a nonbridging phosphateÐoxygen atom with a heteroatom or a functionalgroup is one of the earliest synthetic manipulations during automated synthesis of oligos\ thereforemaking them easily accessible[ It must be realized that any modi_cation of the negatively chargedoxygen atom could change the overall charge on the backbone[ As a consequence\ replacement ofthis atom with an alkyl group will create a neutral backbone\ and replacement with an alkylaminewill create a cationic linkage[ In principle\ three types of molecules can be envisioned by replacementof a nonbridging oxygen atom] "i# negatively charged oligos\ "ii# positively charged oligos\ and "iii#neutral oligos[ The synthesis of these three and related modi_cations are discussed below\ and Table1 summarizes their properties[

"i# Anionic linkaès

These oligos are the closest analogues to the natural DNA in terms of structure and chargedensity[ The chemical synthesis of polyanionic oligos is straightforward and has been reviewedextensively[42 This class of oligos can be further divided into six groups\ dependent upon the natureof the substituent\ and are discussed below[

"a# Phosphorothioates "PS#[ One of the most versatile and bene_cial modi_cations has beenthe replacement of one of the nonbridging oxygen atoms with a sulfur atom\ thus creating aphosphorothioate group[ Although _rst introduced into DNA enzymatically by Eckstein and co!workers\43 the phosphoramidite method has greatly facilitated the synthetic aspects of the chemistry[


Table 1 Properties of the oligos containing modi_cations of nonbridging phosphateÐoxygen atom "chargeand stereochemistry are omitted for clarity#[

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐName Xa Ya Tm

b RNase Hc NSd Chirality Char`ee Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐPhosphorothioate O S − yes ¦ yes !ve 01Phosphorodithioate S S −− yes ¦ no !ve 28\ 39\ 30Boranophosphate O BH2 − < ¦ yes !ve 31Phosphoro~uoridate O F < < < yes !ve 32\ 33Phosphoroselenoate O Se < < < yes !ve 34Phosphoramidate O NHR ¦f < ¦ yes ¦ve 35Aminoalkylphosphonate O CH1NR ¦f < ¦ yes ¦ve 36Methylphosphonate O Me ¦f no ¦ yes no 02Phosphoramidate "PN# O NH1 − no ¦ yes no 37\ 38Phosphoramidimidate NR NHR < < < no no 49Phosphotriester O OR − < ¦ yes no 40\ 41*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaSee structure for atom label[ bMelting temperature\ − acceptable\ −− bad\ ¦ good\ ¦¦ very good\ < not reported[ cActivity ofRNase H mediated cleavage[ dNuclease stability[ eCharge on the backbone[ fTm of chirally pure compound[

The chemistry of PS oligos has been reviewed\ with particular attention to use of improved reagents\reduction of cost in scale!up\ and superior puri_cation methodologies[44 The current automatedsynthesis is nonstereospeci_c\ resulting in the creation of a pair of diastereomers so that for n PSlinkages\ 1n diastereomers will be formed[ Stereospeci_c synthesis of PS oligos has been reported45

and also reviewed[46

Oligos containing PS linkages show considerable reduction in the rate of hydrolysis by a varietyof nucleases[ Furthermore\ nuclease stability of diastereomerically pure PS oligos has beenreported[46 This stability has made them particularly attractive for antisense applications[ Generally\Tm of PS oligos are slightly lower than the PO!linked oligos[ However\ lower a.nity of PS oligoscan be compensated by increasing the length of the oligo or increasing the GC content[ Chirallypure all!Sp!PS oligos are slightly more stable than those with all!Rp chirality\ which can be utilizedwhen a practical synthesis becomes available[ The modest gain in the a.nity of chirally pure PSoligos has resulted in development of newer methodologies to prepare them\ and has generatedsome controversy about their clinical applications[47

The synthesis of PS oligos via a phosphotriester approach in solution may prove to be a superiormethod for the synthesis of large quantities of modi_ed oligos and their analogues[ Advancesreported by scientists from this author|s institution and by Reese and co!workers further strengthenthis notion of solution phase synthesis48\59 and proves its utility towards manufacturing of modi_edantisense agents[ High e.ciency liquid phase methodology using soluble polyethylene glycol supporthas been developed by Bonora and Zaramella for the large!scale production of oligos[50 This methodis certainly attractive for the preparation of short oligos and\ if combined with other processes\ itmay become very valuable in making antisense molecules[

Kool and co!workers have reported an exciting method to produce short\ sequence!de_ned oligosusing synthetically prepared circular DNA[51 This novel approach to DNA synthesis may beapplicable to the synthesis of antisense molecules\ such as PS oligos[ A more detailed account ofthis possibility is discussed in Chapter 6[00 of this volume[

One of the mechanisms by which a PS oligo exerts its antisense e}ect is due to activation ofRNase H\ resulting in cleavage of mRNA on formation of hybrid duplexes[ Synthesis and study ofchimeric oligos is yet another active area of antisense research[ Therefore\ increasing the hybrid!ization potential of the antisense e}ector via incorporation of helix!stabilizing modi_cations "e[g[\1?!O!allyl\ neutral dephosphono linkages#\ in the ~anking region have successfully produced mol!ecules that are able to direct RNase H to cleave targeted mRNA[ Examples of this approachincorporating alkoxy substitutions with PO linkages in the ~anking regions of an oligo have beenelegantly demonstrated by Monia et al[\ by enhanced antitumor activity in vivo[52 Additionally\ suchmolecules have limited binding to proteins resulting in a favorable pro_le for sulfur!related toxicity[In addition\ methylene "methylimino# linkage and methylphosphonates have been used in the


~anking regions as nonionic modi_cations25 to lower charge density by incorporating nonioniclinkages[ It is believed that this will result in reduced binding to proteins[

Studies of PS oligos in animals and humans have shown that they have a very acceptabletherapeutic index[ Toxicity has only been observed at a very high dose[53 Moreover\ progress inscale!up and cost reduction has been substantial enough to provide con_dence that cost of therapywith these _rst!generation antisense drugs will not be prohibitive[ As a result\ several PS oligos arein full scale clinical development "see Table 0# and moving along rapidly towards NDA "New DrugApplication# _ling[

"b# Phosphorodithioates "PS1#[ In an e}ort to circumvent the problem of chirality with PS oligos\Caruthers and co!workers28Ð30 synthesized oligos in which two of the nonbridging oxygens ofinternucleotidic phosphate linkage were replaced by sulfur\ resulting in a phosphorodithioate\ whichis now devoid of chirality[ These isoelectronic analogues of DNA are nuclease resistant and are ofinterest as inhibitors of viral gene expression[ PS1 oligos containing pyrimidines are readily syn!thesized via a triester solid!phase method\ providing PS1 oligo free "³9[4)# of PS contaminations[54

While PS1 oligos containing mixed bases are not straightforward to make\ enough progress hasbeen made by Dahl|s and Caruthers| groups independently to obtain such analogues in smallamounts\ enough for biological screening[

Convincing antisense activity has not been reported with a PS1 oligo\ perhaps for the followingreasons[ First\ hybridization Tm of PS1 oligos is lower relative to the PS and PO oligos[ It is about0Ð1 >C:base destabilizing compared to its PO analogue[ Second\ because PS1 oligos have a highersulfur content\ they appear to bind proteins tighter than the PS oligos\ and to activate Rnase Hmediated cleavage with reduced e.ciency compared to the PS analogue[28Ð30 Finally\ synthesis offully mixed bases in 19!mer sequence has not been accomplished\ where all linkages are PS1 and notcontaminated with PS linkages[ The stability of PS1 oligos is under investigation55 and improvedprocedures are needed to guarantee its integrity[ These properties taken together argue against PS1

becoming a signi_cant antisense agent["c# Boranophosphates "BP#[ The use of boron neutron capture therapy for the treatment of

malignant cancer has encouraged Shaw and co!workers31 to investigate boron incorporation intoDNA[ They have shown that BP dimers are very stable to nucleases[ The BP oligos were synthesizedfrom 1!boranotriphosphates by template!directed primer extension using DNA polymerase in vitro[However\ chemical synthesis of BP oligos "T09# has been reported via automated H!phosphonatechemistry[56 Like PS oligos\ BP oligos are chiral and up until 0886 a stereospeci_c chemical synthesishad not been reported\ except one short communication describing chromatographic separation ofdiastereomers at a dimer level[57 The enzymatic preparation of BP oligos provided an Sp con!_guration at the chiral center\ and exerts no change in Tm compared to DNA[ In summary\ due totheir chirality issue and lack of a good synthetic procedure to provide enough materials for antisense!based gene walk\ the therapeutic use of BP oligos may not progress as rapidly as PS oligos[

"d# Phosphoro~uoridates "PF#[ Nucleoside phosphoro~uoridates were synthesized for the _rsttime by Wittmann58 in 0852\ but it is only in the 0889s that their potential in oligos began to beexplored[ The attractive features of PF linkages are that they bear a high resemblance to naturalPO linkages\ that they may provide a new means for a covalent attachment of molecular probes toproteins\ or that the ~uorine may serve as a reporter group for studying conformational propertiesof nucleic acids[ As indicated\ synthesis of PF oligos is still in its infancy and a full!length "½19!mer# oligo has yet to be made[ However\ Dabkowski and Tworowska32 and Stawinski and co!workers33 independently reported a novel entry into PF!linked dimers[ The key reaction was anoxidation of H!phosphonate with iodine in the presence of triethylamine trishydro~uoride furnishinga PF dimer in quantitative yield[ Similarly\ phosphoro~uorodithioate was also prepared[ Insummary\ the PF modi_cation is certainly an interesting mimic of natural PO linkage and may _ndapplications in molecular biology\ but their application in antisense research is remote at thistime[ Interestingly\ Stec and co!workers pointed out that the PF!linked dimer was unstable undersolvolytic conditions and undergoes rapid base!catalyzed hydrolysis[69

"e# Phosphoroselenoates "PSe#[ Despite the biological importance of selenium and an indicationthat metabolism of selenium!containing compounds may involve PSe intermediates\ PSe oligos havereceived relatively little attention[ Synthesis of a PSe analogue was accomplished on an automatedDNA synthesizer by replacing the sulfurization reagent with potassium selenocyanate\ but the yieldwas low\ possibly due to poor solubility of selenone in organic solvent[ This problem was solvedwhen Stawinski and Thelin60 reported the use of 2H!0\1!benzothiaselenol!2!one as selenizing reagent[It has been demonstrated that PSe linkage is not nearly as stable as PS linkage\ and liberates freeselenium upon degradation\ which happens to be toxic[ Stein and co!workers have described theuse of PSe antisense oligos and concluded against their use for the following reasons[34 First\ Tm


results were lower than both PS and PO oligos[ Second\ PSe analogues were found to be non!sequence!speci_c inhibitors with less activity and much more toxic compared to PS analogues[Therefore\ PSe analogues are not suitable for antisense constructs[

"ii# Cationic linka`es

Introduction of cationic groups at various positions throughout the oligo provides a method forproducing molecules with widely di}ering charge densities[ Cationic molecules have previously beenused to enhance the delivery of a variety of conjugates into cells\ such as liposome!mediatedtransfection protocols for PS oligos[61 Certain cationic pendant groups have been conjugated tooligos in attempts to improve their cellular uptake or activity in vivo[62 Other methods which utilizebase modi_cations for introduction of a positive charge have been reviewed elsewhere[63

"a# Phosphoramidates and aminoalkylphosphonates[ An interesting way to alter natural anionicPO backbone is to replace it with cationic groups[ Letsinger et al[ _rst reported64 the synthesis of acationic oligo containing alternating PO and basic aminoalkylphosphoramidate linkages\ whichappeared to be electrically neutral at pH 6[ The Tm of these oligos were found to be higherthan those of the unmodi_ed oligos at low ionic strength[ These studies were carried out withphosphoramidate analogues prepared without stereocontrol at the P0N bonds[ In view of this\Letsinger and co!workers reported35 their study with an oligo containing anionic and stereo!uniformcationic linkages\ showing high binding a.nity "DTm¦1[2 >C:modi_cation# for RNA target[ Theisomeric oligo with opposite con_guration binds much more poorly[ Similar observations weremade by Fathi et al[ incorporating an aminoethylphosphonate[36 Their results with alternatingcationic:anionic linked oligos indicated that the Rp isomers were capable of forming more stableduplexes with RNA than their natural counterparts[ The Sp isomers did not form stable hybridsunder similar conditions[ Additionally\ they indicated that these oligos were stable to S0 nucleaseand were taken up by JY cells somewhat better than the control PO oligos[ This novel approachcertainly has distinct advantages over _rst!generation PS oligos and may o}er a di}erent perspectivein terms of its interaction with proteins[ However\ the vastly di}erent a.nities of Rp and Sp isomersemphasizes that a stereospeci_c synthesis must be developed before their true potential for antisensetherapeutics is realized[

A polycationic oligo consisting of PO!linked 01!mer with a cationic tail at the 2?!end and acholesteryl group at the 4?!end was synthesized and shown to inhibit HSV!0 in Vero cells[65 Therefore\an appropriate mix of positive and negative charge densities on an antisense oligo may result insome unusual properties\ such as higher a.nity and improved uptake[

"iii# Neutral linka`es

As indicated earlier\ the polyanionic nature of DNA backbone was assumed to be a drawbackfor its e.cient uptake into cells[ Therefore\ neutral oligos were made in the hope that they wouldenter the cells via passive di}usion[ Neutralization of the charge in natural DNA can be accomplishedin several ways[ Various approaches towards generating neutral oligos are discussed below\ includingtheir antisense!related properties "see Table 1#[

"a# Methylphosphonates "MP#[ These nonionic derivatives were _rst introduced by Miller andTs|o using modi_ed triester chemistry[66 However\ later methods utilize phosphorus"V# derivativesin which active esters of 2?!methylphosphonates are coupled to 4?!alcohols[ It must be realized thatmost of the older syntheses of MPs resulted in diastereomeric mixtures of oligos[ Nevertheless\stereospeci_c solid!phase syntheses of both Rp and Sp MP oligos are now available[67

MP oligos appear to be completely resistant to hydrolysis by endo! and exonucleases in cell!basedexperiments[ The hybrids formed between MP oligo and RNA are not substrate for Rnase Hmediated cleavage[ However\ {{chimeric oligos|| which contain 3Ð5 contiguous PO linkages ~ankedby MP linkages\ when bound to RNA\ do cleave via RNase H activity "see Figure 1#[ It has beenreported by Lebedev and Wickstrom46 that all!Rp oligos hybridize much more strongly to acomplementary RNA target than do the racemic oligos or all!Sp oligos[ As expected\ the nonionicnature of MP oligos allows duplex formation in the presence of low or no salt[ This salt independenceis believed to be due to reduced charge repulsion between the two oligos[

A number of studies68 have shown that MPs are e}ective inhibitors of viral protein synthesis incell culture systems "e[g[\ VSV\ HSV\ HIV!0#[ Although MP oligos have been studied extensively in


cell culture systems\ their in vivo activity has not been followed up in depth[ A paper published in0883 describes inhibition of HSV!0 replication in a mouse model\79 but animal studies have beenfew[

The reasons for the lack of intense progress in this area could be summarized as follows[ First\ itwas believed that MPs were taken up by passive di}usion\ but later studies indicated nonreceptorendocytosis\ possibly absorptive or ~uid phase endocytosis[29\70\71 Release of MP oligos from theendosomal pathway presumably limits the e.cacy of these compounds in vivo[ Second\ MPs arenot substrate for RNase H\ therefore\ antisense activity is due to steric block only[ Again\ lowera.nity of racemic MP limits their use in vivo[ Finally\ special procedures for synthesis and depro!tection may be required for their production[70\71 In summary\ chirally pure Rp MP will _nd itsutility in the antisense area as more material becomes available[ Scientists from Genta\ Inc[ havepresented interesting results using chirally pure chimeric 1?!O!methyl MP:PO oligos\ inhibitingBCL1 protein expression in cell culture and in SCID mice[72

"b# Phosphoramidates "PN#[ Substitution of an amino or aminoalkyl group for one of the twononbridging oxygen atoms of the PO linkage creates a nonionic PN internucleosidic linkage[ In thisregard\ Vasseur and co!workers37 and Iyer et al[38 independently reported a novel nonionic P0NH1

linkage[ It was suggested that the P0NH1 group could hydrogen bond with water and thus increasetheir water solubility[ The small size of the NH1 group is believed to create the least steric hindrancearound the PN backbone\ while hybridizing to complementary RNA[ In brief\ syntheses of P0NH1

oligos were accomplished on an automated solid!support DNA synthesizer using both H!phos!phonate and phosphoramidite chemistries\ in combination with highly base!labile protection strat!egy[ Oligos containing P0NH1 links formed less stable duplexes with RNA targets "DTm

−1[9 >C:modi_cation# than the corresponding PO oligos[ However\ these oligos were resistant tocleavage by various nucleases[ It was also concluded that this modi_cation does not allow RNaseH mediated cleavage of the target RNA[ Vasseur and co!workers73 have described a dramaticchange in the a.nity "DTm ¦0[1 >C:modi_cation# by changing the anomeric con_guration to ain P0NH1 oligos[ They did not provide any explanation for these unexpected results in thiscommunication[ Syntheses of chimeric PN building blocks have also been reported74 containingP0NH0OMe and P0NH"CH1#4NH acridine functionalities[ However\ these analogues form lessstable duplexes with complementary RNA targets[ Given the nature of synthesis and properties ofPN oligos\ it is unlikely that they will make a useful contribution towards the antisense approach[

"c# Phosphoramidimates "PN1#[ Fischer and Caruthers have reported the _rst synthesis of a PN1

dinucleotide\ derived from an aliphatic amine[49 Their rationale for synthesizing such linkages is inanticipated nuclease resistance\ potential for creating a neutral or electropositive backbone bychanging substituents\ and providing a ~exible site for further conjugations[ This dimeric PN1 linkedmolecule was found to be unstable under acidic and basic conditions\ making it very challenging toincorporate into an antisense oligo[

"d# Phosphotriesters "POR#[ These oligos were made as nonionic analogues of DNA even beforeMP analogues were introduced[ Speci_cally\ POR analogues\ containing O!methyl phosphate or O!methyl phosphorothioate\ have not been rigorously evaluated for their antisense properties becauseof their di.cult syntheses[ The main problem is their base lability during deprotection conditionsor cleavage from support[ Noyori and co!workers40 and Iyer et al[41 independently reported the useof alkyloxycarbonyl and N!pent!3!enoyl protected nucleosides\ respectively[ In addition\ Tang andco!workers have reported75 the use of thionotriester containing oligos for antisense utility[ Theseoligos show improved nuclease stability\ cellular association\ and binding a.nity[ There was alsoan implication of these S!triester PS oligos undergoing slow hydrolysis to all!PS oligos\ and possiblyfunctioning as prodrugs for corresponding antisense PS oligos[ In summary\ many synthetic hurdlesmust be overcome before one can make POR oligos routinely\ in su.cient quantity and of goodenough quality\ for antisense applications[

6[97[2[0[1 Modi_cations of the bridging 2?!oxygen atom

The desire for substituting the bridging 2?!oxygen atom of the PO linkage was born out of chiralityproblems associated with replacement of nonbridging oxygen atoms[ Replacement of 2?!oxygen ina PO linkage should result in nuclease stability of the modi_ed backbone and may have potentialto alter the sugar conformation in a favorable manner for higher a.nity[ As a consequence\ threemajor types of modi_cations have been investigated "Table 2# for their application in antisenseresearch[


Table 2 Properties of the oligos containing modi_cations of bridging!2?!oxygen atom[

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐName Xa Tm

b RNase Hc NSd Chirality Char`ee Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐPhosphoramidate NH ¦¦ no ¦¦ no !ve 76Phosphorothioate S < < < no !ve 77Phosphinate CH1 − no ¦ no !ve 78\ 89*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaSee structure for atom label[ bMelting temperature\ − acceptable\ −− bad\ ¦ good\ ¦¦ very good\ < not reported[ cActivity ofRNase H mediated cleavage[ dNuclease stability[ eCharge on the backbone[

"i# Phosphoramidates "NP#

Synthesis of NP oligos has been discovered and perfected by Gryaznov and co!workers\76 utilizingsolid!phase methodology and 4?!dimethoxytrityl!protected!2?!amino!1?\2?!deoxyribonucleosides askey monomer units[ Oligo chain assembly was based upon a carbon tetrachloride driven oxidativecoupling of the appropriately protected 2?!amino!nucleosides with the 4?!H!phosphonate diestergroup\ resulting in the formation of an internucleosidic NP link "0#[ Uniformly modi_ed NP oligospossess some very attractive features for antisense applications\80 such as negatively charged andachiral phosphorus\ good solubility in water\ and resistance to nuclease digestion\ buttressed by ahigh a.nity for RNA target[ Studies have shown that the sugar ring conformation changes frompredominantly C!1?!endo to C!2?!endo when the 2?!O is replaced with 2?!N functionality\ therebyforming a very stable A!type of duplex with complement RNA[81 Additionally\ placement of a 1?!electronegative "F or OMe\ X or Y in "0## substituent in combination with the NP backboneenhanced the Tm up to 3 >C per modi_cation[82

These oligos were used in various in vitro and in vivo antitumor systems as antisense drugsaddressed to di}erent targeted regions of c!myb\ c!myc\ and bcr!bl mRNAs[80 In certain examples\up to 89) protein reduction was observed[ However\ NP oligos do not activate RNase H andobserved antisense activity was entirely due to high a.nity for the mRNA target[ In summary\ well!established synthesis protocol and attractive antisense properties\ including preorganization\ of NPoligos make them very promising candidates for antisense applications[

"ii# Phosphorothioates "SP#

Analogues where one of the two bridging oxygen atoms in the phosphodiester linkage is replacedby a sulfur atom are particularly attractive for several reasons] "i# they are achiral at phosphorus


and thus\ no diastereomers are created during their synthesis^ "ii# they are electronically and stericallysimilar to the natural congener^ "iii# they provide nuclease resistance to the oligo^ and "iv# they aresusceptible to speci_c cleavage under mild conditions[ These compounds are synthetically chal!lenging to prepare and only two practical syntheses have been reported[ First\ Cosstick and co!workers disclosed a procedure to prepare SP dimers from the corresponding thioesters\ utilizing aregioselective MichaelisÐArbuzov reaction[77 The method appears to be compatible with purineand pyrimidine nucleosides\ and does not require protection of the hydroxy groups[ Second\ acomplementary procedure has been reported by Reese and co!workers which prepares SP linkagefrom thioethers using sulfenyl chlorides under acidic conditions[83 Since a good protocol to prepareSP oligos is available\ synthesis of mixed base 19!mer sequences for antisense research should bepossible[

"iii# Phosphinates "CP#

The altered DNA of this class contains a tetravalent carbon moiety in place of the divalent oxygenmoiety at the 2?!position of an intermediate linkage[ Collingwood and Baxter have reported thesynthesis and incorporation of CP!linked dimers into oligos[78 The synthesis requires base!mediatedcoupling of a 4?!phosphonous ethyl ester with a 2?!C!aldehydo nucleoside to provide a good yieldof dimer[ Oligos containing CP!linked dimers were evaluated for their antisense properties[ Therewas an overall destabilizing e}ect "DTm −1[9 >C:modi_cation# when hybridized to RNA target[However\ they were found to be stable to the cleavage by 2?!exonucleases[ Abundant patentliterature78\89 is available for the CP!linked oligos claiming the use of these molecules for antisensetherapy[

6[97[2[0[2 Modi_cation of the bridging 4?!oxygen atom

The rationale for studying 4?!O!modi_ed linkage is quite similar to the rationale of the 2?!O!modi_cations discussed earlier[ General properties of these oligos are listed in Table 3[

Table 3 Properties of the oligos containing modi_cations of bridging 4?!oxygen atom[


b RNase Hc NSd Chirality Char`ee Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐPhosphoramidate NH < < < no !ve 84Phosphorothioate S < < < no !ve 85Phosphinate CH1 − < ¦ no !ve 78*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaSee structure for atom label[ bMelting temperature\ − acceptable\ −− bad\ ¦ good\ ¦¦ very good\ < not reported[ cActivity ofRNase H mediated cleavage[ dNuclease stability[ eCharge on the backbone[

"i# Phosphoramidates

It has previously been shown that oligomers bearing nucleoside units linked by 2?!O0P0N!4?bonds could be synthesized and are stable under neutral and alkaline conditions[ Mag and Engelshave reported84 the synthesis of several dimeric phosphoramidates\ using the Staudinger reactionfollowed by MichaelisÐArbuzov type transformation[ However\ this chemistry has not been appliedto the synthesis of uniformly modi_ed oligos^ thus\ their true potential for antisense research remainsuninvestigated[


"ii# Phosphorothioates

Synthesis of 2?!O0P0S!4? linked oligos has not been reported "0886#[ However\ Liu and Reesereported85 synthesis of uridylyl!"2?: 4?#!"4?!thiourine#\ which shows exceptional lability to basetreatment\ and under neutral conditions as well[ Due to the lability of this linkage and lack of viableoligo synthesis\ it may not be worthy of consideration towards antisense utility[

"iii# Phosphinates

Like many other modi_cations\ replacement of the 4?!oxygen atom with a 4?!methylene grouphas been super_cially studied[ Incorporation of a 2?!O0P0CH1!4? linked dimer had a destablizinge}ect on the duplex formation[78 In a ribozyme!related study\ Matulic!Adamic et al[ reported86

synthesis of a 4?!deoxy!4?!di~uoromethylphosphonate nucleotide analogue\ using a versatile ~uorosugar synthon[ This synthetic pathway may be utilized for making an antisense construct[

6[97[2[0[3 Modi_cations of the 4?!carbon

Saha et al[ reported87 a provocative modi_cation of the DNA backbone\ in which a methyl groupwas introduced at the 4?!carbon of the phosphodiester linkage[ They believed that the presence of a4?!Me group would reduce recognition by nucleases and yet be small enough to conserve hybridstability[ Cellular transport may be facilitated by the lipophilic Me substituent\ while adequatewater solubility can be retained via the negative charge of the PO linkage[ It is worth noting thatthis linkage contains a chiral carbon atom[ This communication reveals that 4?!Me substitutionprovides extra stability to 2?!exonuclease cleavage and does not adversely a}ect the Tm "DTm

−9[1 >C:modi_cation#[ In view of these bene_cial results\ it may be worthy of consideration toprepare enantiomerically pure backbone and study its antisense potential[

Wang et al[ reported88\099 incorporation of chirally pure 4?!"R#!C!branched "allyl group# and 4?!"S#!C!branched "methoxy\ amino\ cyano\ and allyl groups# building blocks into oligos[ Preliminaryresults indicate that such modi_cations have a positive e}ect toward nuclease stability and com!parable a.nity for RNA target[ The results of incorporation of these modi_cations in antisenseconstructs are eagerly awaited[ Properties of the oligos containing modi_cations of the 4?!carbonare shown in Table 4[

Table 4 Properties of the oligos containing modi_cations of 4?!carbon[


b RNase Hc NSd Chirality Char`ee Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ4?!Methyl "R:S# Me − < ¦ yes !ve 874?!Methoxymethyl "S# CH1OMe − < ¦ yes !ve 88\ 0994?!Aminomethyl "S# CH1NH1 − < ¦ yes !ve 88\ 0994?!Cyanomethyl "S# CH1CN − < ¦ yes !ve 88\ 0994?!Allyl "R or S# CH1CH�CH1 − < ¦ yes !ve 88\ 099*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaSee structure for atom label[ bMelting temperature\ − acceptable\ −− bad\ ¦ good\ ¦¦ very good\ < not reported[ cActivity ofRNase H mediated cleavage[ dNuclease stability[ eCharge on the backbone[

6[97[2[1 Nonphosphate or Dephosphono Linkages

Since the early 0889s\ there has been a steady rise in the number of publications and specializedreviews relating to the synthetic methodologies and application of nonionic:achiral linkages thatreplace the natural PO backbone[00\06 The data concerning these surrogates of PO linkage\ as isdiscussed in this section\ crystallize an understanding of the minimal structural features required


not only to mimic a natural PO bond\ but also to preorganize these surrogates for the best antisenseproperties[

It is now well documented that replacement of the PO backbone with a nonphosphate backbonehas several distinct advantages in terms of their antisense properties] it automatically confersresistance to cellular nucleases because they are known to cleave only PO linkages^ it may provideincreased cellular uptake due to reduced charge density compared to polyanionic _rst!generationPS oligos^ it avoids the chirality imposed by the use of PS oligos^ and major advantages may berealized in the economics of large!scale solution!phase synthesis[

Chemistries required to produce these linkages are often quite challenging^ however\ with advancesin synthetic:enzymatic methodologies it has become more routine[ The most frequently usedapproach is to prepare a nucleosidic dimer containing the backbone of choice and then incorporateit into an oligo using standard phosphoramidite chemistry[ Such procedures allow the synthesis ofa polymer linked with alternating PO and novel linkages[ Synthesis of fully substituted polymerswas not always possible[ Laboratories at Isis Pharmaceuticals25 and several others090Ð093 have sincetaken a major step forward by preparing fully modi_ed polymers devoid of the phosphorus!containing linkage\ called {oligosides[||

This section is limited to developments describing the use of four!atom linkers connecting thetwo sugar moieties of a nucleosidic dimer in natural con_guration[ The subsections are createdaccording to the type of heteroatom present in these novel linkers[ A summary of the key propertiesof the nonphosphate backbone!linked oligos is presented in Table 5[

Table 5 Properties of the oligos containing nonphosphate backbones "Figure 3#[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐName 2?:4? Linka`e Tm

a RNase Hb NSc Chirality Char`ed Ref[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐAmide 0 NRCOCH1CH1 − < ¦ no no 08Amide 1 CH1CH1NHCO − < ¦ no no 08Amide 2 CH1CONHCH1 ¦ yes ¦¦ no no 094Amide 3 CH1NHCOCH1 ¦ < ¦ no no 094Amide 4 CONHCH1CH1 − < ¦ no no 094Guanidine 0 NHC"!NR#NHCH1 − < ¦ no no 095Guanidine 1 NHC"!NH1#NHCH1 ¦ < ¦ no ¦ve 090Urea NHCONHCH1 − < ¦ no no 096Carbamate OCONRCH1 − < ¦ no no 08Amino 0 NHCH1CH1CH1 − < ¦ no no 093Amino 1 CH1CH1NHCH1 − < ¦ no no 093Amino 2 CH1NHCH1CH1 − < ¦ no no 093Amino 3 CH1N"Me#CH1CH1 − < ¦ no ¦ve 097Amino 4 CH1CH1N"Me#CH1 − < ¦ no ¦ve 097MMI CH1N"Me#OCH1 ¦¦e yes ¦¦ no no 098Silyl OSiR1OCH1 − < ¦ no no 009Sul_de CH1CH1SCH1 − < ¦ no no 000\ 001Sulfone CH1SO1CH1CH1 − < ¦ no no 002Sulfonate OSO1CH1CH1 − < ¦ no no 003Sulfonamide NHSO1CH1CH1 − < ¦ no no 003Formacetal OCH1OCH1 − no ¦ no no 07Thioformacetal SCH1OCH1 ¦ no ¦ no no 07Ether 0 OCH1CH1O − < ¦ no no 004Ether 1 OCH1CH1CH1 − < ¦ no no 005Carbon 0 CH1CH1CH1CH1 − < ¦ no no 006Carbon 1 CH1COCH1CH1 − < ¦ no no 08Carbon 2 CH1CH�CHCH1 ¦ < ¦ no no 007Carbon 3 CH1COCH�CH < < < no no 008*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaMelting temperature\ − acceptable\ Ð bad\ ¦ good\ ¦¦ very good\ < not reported[ bActivity of RNase H mediatedcleavage[ cNuclease stability[ dCharge on the backbone[ eTm of chirally pure compound[

6[97[2[1[0 Nitrogen!containing linkages

This is one of the most active areas of study for research groups in search of novel surrogates ofphosphate backbone[ This section is further divided into _ve major types of nitrogen!containinglinkages\ depending upon the nature of the functionality produced[


"i# Amides

De Mesmaeker et al[ designed and synthesized all possible permutations and combinations of anamide functionality within the four!atom space between the two sugar units[08 Indeed\ they havesuccessfully synthesized _ve ~avors of these amide!linked dimers since the early 0889s[ An excellentreview of the syntheses and their antisense properties has been published[094 Readers are encouragedto read on this subject to understand the breadth and scope of this large undertaking by Cibascientists\ which clearly lays the foundation for the future of backbone modi_cations[ This hardwork has paid o} through the emergence of an ideal amide linkage "2?!CH1CONH!4?# that appearsto be very attractive for antisense constructs "1#[ The usefulness of this linkage stems from itspreorganization of sugar into a high N!pucker resulting in an A!type of duplex formation "DTm

¦9[0 >C:modi_cation#[019

Generally\ all amide linkages are resistant to endo! and exonucleases\ and are not a substrate forRNase H in a fully alternating motif[ RNase H activity can be restored when these modi_cationsare placed in the ~anking region of a chimeric oligo\ away from the cleavage site on RNA comp!lement[ The chimeric oligos containing amide linkages have shown good antisense activity incell!based experiments[010 In view of these results\ reports of the in vivo pharmacokinetic andpharmacodynamic behavior are anxiously awaited[

With the utility of amide backbones established\ a novel approach to their synthesis was the focusof attention for Robins et al[\ described in an elegant manner\ via coupling of a 4?!amino nucleosidewith 1?\2?!fused!n!butyrolactone nucleoside[011 This procedure describes the synthesis of a riboanalogue of amide!linked dimer\ which may be somewhat better than previously described amidemodi_cations\ because a 1?!electronegative substituent has been reported8 to enhance the a.nityfor RNA target\ an asset for antisense applications[

Along similar lines\ Wengel and co!workers also made amide!linked dimers containing a pip!erazine skeleton[012 Not surprisingly\ all modi_cations were very destabilizing when hybridized tocomplement RNA[ Therefore\ special attention should be paid to the fact that too much rigiditymay be bad for duplex formation[ One needs to achieve a _ne balance between rigidity and ~exibility[

"ii# Guanidines

In 0882 Herdewijn and co!workers _rst synthesized thymidine dimers containing a variety of N!substituted guanidine linkages as a neutral analogue of PO backbone[095 However\ most of thesemodi_cations were quite destabilizing on duplex formation with RNA target\ and therefore researchwas not pursued[

Following the unsuccessful attempts of incorporating N!substituted guanidines\ a year laterBruice and co!workers looked at the incorporation of a free guanidine moiety into an oligo as asource of positive charge and generated renewed interest in this area of research[090 They havepublished a route for the synthesis of a dimeric unit containing a guanidinium ð2?!NHC"�NH¦

1 #NH!4?Ł linker\ and suggested that the methodology is applicable to the preparation of uniformly modi_edpolymer[ A pentameric DNA linked via 3!guanidine residues was shown to form a base pairspeci_cally with poly rA with an unprecedented a.nity at ×099 >C[ However\ this molecule did


not bind to poly G\ C\ or U tracts[ They also proposed a model for this unusual high!a.nityinteraction\ which takes the general conformation of the nucleic acid[ This study was unique\ andwarrants the synthesis of a biologically relevant mixed!base sequence for its evaluation as anantisense construct[

"iii# Ureas

The logic for synthesizing urea "2?!NRCONR!4?# linked dimers was born out of adding morerigidity to the amide "2?!CH1CONH!4?# linked dimer and studying the e}ects on hybridization[Therefore\ various N!substituted urea analogues were synthesized as dimeric nucleosides and incor!porated into oligos[096\013 As expected\ most of the urea!linked oligos were destabilizing whenhybridized to RNA complements\ compared to amide or PO linkages[ Clearly\ introduction ofextensive conformational restrictions for torsional angles in the backbone has a negative e}ect onduplex formation[ Again\ substitution on the 2?!N of the urea linkage was severely destabilizing\whereas substitution on the 4?!N of the urea linkage was well!tolerated in terms of Tm[ The stabilityof urea!linked oligos in fetal calf serum was increased by a factor of 04 compared to the unmodi_edPO!linked oligos[ These results taken together indicate that urea!linked oligos may not be a goodchoice for antisense applications[

"iv# Carbamates

Various syntheses of 2?: 4? carbamate!bridged "2?!OCONH!4? and 2?!NHCO!O!4?# dimers andpolymers "up to 5!mer# have been reported since the late 0869s[ Interestingly\ some work waspublished even before the antisense concept was realized[ The chemistry of the carbamate!linkedoligo is straightforward and can be performed either in solution or on solid!support[ A detailedaccount of their synthesis is reviewed elsewhere by Sanghvi and Cook[06 The use of carbamate!linkingchemistry has been extended towards the solid!support synthesis of novel oligosides containingmorpholino subunits linked by carbamate bridges[ Further details and applications of the mor!pholino type oligosides have been published by Summerton and co!workers[091

Carbamate!linked "2?!O!CONH!4?# polymers with alternating PO linkages were studied for theirantisense properties[ The overall Tm were destabilized "DTm −1[9 to −3[9 >C:modi_cation# whenhybridized to complement RNA[ Isomeric carbamates "2?!NRCOO!4?# proved to be worse[08 Noduplex formation was observed with 4?!modi_cations[ The thiocarbamates 2?!SCONH!4?# also hada negative e}ect on the Tm due to longer bond lengths between the 2?C0S atoms compared to2?C0O and reduced rotational barrier around the 2?!SCO bond[08 Once again\ increasing the overalllength of the backbone generally had a negative impact on the a.nity for RNA target[ Althoughcarbamates provided enhanced nuclease resistance\092 their poor a.nity for RNA targets makesthem useless for antisense applications[

"v# Amines

Researchers have long been interested in utilizing amino!linked oligos in order to control the netcharge distribution[ It was believed that appropriate substitution of nitrogen atoms could result inconsiderable change in pKa values\ thus creating a wide range of backbones from neutral toprotonated types[ All of this eventually would assist in an improved uptake of these molecules[ Inaddition\ electrostatic and hydration factors between the neutral or protonated nitrogen atom35\090

of the amino linkers and the polyanionic backbone of the RNA strand are expected to enhancefurther the thermal stability of these duplexes[

Several reports have come out of the Sterling group of scientists093 describing the solution!phasesynthesis of amine!bridged dimers and polymers "up to trimers# and studies of their antisenseproperties[ These linkages are all secondary amines] "i# 2?!CH1NHCH1!4?\ "ii# 2?!CH1CH1NH!4?\and "iii# 2?!NHCH1CH1!4?\ and therefore should be neutral at physiological pH[ All of the abovemodi_cations\ when incorporated into oligos as dimeric nucleosides\ destabilized the duplex by−1[9 to −4[9 >C per modi_cation[ Further substitution of these nitrogen atoms by a methyl groupwould make them tertiary amines\ thus a cationic linkage instead of neutral[ De Mesmaeker et al[prepared097 two such linkages "i# 2?!CH1N"Me#CH1!4? and "ii# 2?!CH1CH1N"Me#!4?[ Both of these


modi_cations were equally destabilizing on duplex formation[ Unpublished modeling results indi!cated that these protonated sites were too far away from the negatively charged PO linkage of thecomplement RNA\ therefore charge neutralization could not possibly occur to improve a.nity[ Inaddition\ presence of a C0C bond within the backbone allows free rotation around the torsionalangles\ which could lead to a more ~exible backbone than necessary[ Further restriction of ~exibilitywas achieved with substitution of a methylene group with a carbonyl group "2?!CH1CH1N"Me#!4?to 2?!CH1CON"Me#!4?#\ which resulted in immediate correction of Tm in a positive manner[097

The author|s experience with hydroxylamine!linked oligos also follows a similar trail[ In the0889s\ substantial progress has been made in utilizing nitrogen!containing backbones\ resulting inan extensive structureÐactivity relationship "SAR# with a broad antisense database[ Some of the keylinkages studied at the author|s institute are] "all 2?: 4?# !CH1N!O\ !CH1NHO!\ !CH1N"Me#O!"MMI#\ !CH1ON"Me#!\ !ON"Me#CH1!\ and !CH1N"Me#N"Me#![ As most of the syntheses arepublished\06 only unpublished results will be discussed herein[

A number of scientists believe that methylene methylimino "MMI# is one of the most promisingbackbone modi_cations for incorporation into antisense oligos "Figure 4"a##[098 The reasons are asfollows[ MMI linkages provide a very high degree of nuclease resistance\ as hydroxyamino linkagesare not a substrate for cellular nucleases[ Furthermore\ it was found that an MMI linkage confersnuclease stability to the adjacent phosphodiester\ which allows the use of dimeric strategy\ creatingan antisense molecule with alternating MMI and PO linkages[ The resulting alternating antisenseoligos demonstrated excellent water solubility\ good a.nity\ and speci_city for complementaryRNA\ while reducing the net negative charge of the oligo by 49)[ In vitro studies indicated thatMMI oligos of this type maintain\ or in some cases increase\ biological activity "e[g[\ inhibition ofPKC!a and H!ras# relative to parent PS oligos[ Additionally\ signi_cant bene_ts may be realized inthe economics of large!scale synthesis in solution or even on solid!support[

Figure 4 Structure and properties of MMI "a# and PNA "b# linkages[

The conformational ~exibility vs[ rigidity of MMI linkage\ as well as the northern sugar puckeris believed to play a crucial role in the modulation of its binding a.nity for RNA target[25 ExtensiveNMR and modeling studies have indicated that the 2?!methylene group of the MMI linkage shiftedthe sugar conformation to a desired 2?!endo pucker\ via reduced ring `auche e}ects\ thus helpingthe molecule to preorganize into a preferred A!geometry for duplex formation[014 MMI backbonestudies have now been extended to include modi_cations of the sugar moieties via the introductionof an electronegative 1?!substituent\ with an aim to enhance further the N!type pucker[015 It is well!known that thermal stability of hybrids of 1?!OMe or 1?!F RNA with complementary RNA areconsiderably higher "DTm ¦0[9Ð1[9 >C:modi_cation# than that of the corresponding DNAÐRNAduplexes[016 In brief\ six dimers with various combinations of 1?!sugar modi_cations "see Figure


4"a## were synthesized\ their e}ects on hybridization "Table 6# to RNA were studied\ and their )N!pucker was estimated by NMR "Table 7#[ The novel MMI!sugar modi_ed oligos were found tohybridize to their complement RNA with highly improved a.nity and speci_city compared tounmodi_ed DNA[ The best modi_cation exhibited a DTm of ½4 >C:modi_cation compared to anantisense PS oligo[ This increased stability has been attributed to hydrophobic interactions betweensubstituents in the minor groove\ and especially to the higher percentage of the 2?!endo sugarconformation\ which resulted in a decreased entropic motion of the sugar while maintaining anoptimal preorganized A!type duplex[ In essence\ an appropriate combination of backbone andsugar modi_cations allows one to dial in desirable a.nity in any antisense construct[017

Table 6 E}ect of 1? sugar modi_cation on hybridization of modi_ed backbones "see Figure 4"a# for thestructure of MMI dimer#[

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐModi_cations DTm:modi_cation "vs[ RNA#

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ1? of top 1? of bottom Oligo seq[ Oligo seq[ Oligo seq[

Backbone suàr "x# suàr "y# A B C Averaè*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐDNA!"PO# : : 9[9 9[9 9[9DNA!"PS# : : −0[01MMI H H ¦9[02 −9[12 ¦0[40 ¦9[1

MMI H F ¦0[72 ¦0[99 −9[04 ¦0[3MMI H Me ¦1[16 ¦0[56 ¦9[76 ¦0[8

MMI F F ¦2[16 ¦1[19 ¦0[36 ¦1[7MMI F Me ¦2[63 ¦2[90 ¦0[84 ¦2[2

MMI Me F ¦2[02 ¦1[49 ¦0[45 ¦1[7MMI Me Me ¦2[60 ¦1[67 ¦0[74 ¦2[1

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐOligo sequence A containing 4 dimeres] GCG T�T T�T T�T T�T T�T GCG[ Oligo sequence B containing 0 dimer] CTC GTA CCT�T TC CGG TCC[ Oligo sequence C containing 1 dimers] CTC GTA C T�T T�T C CGG TCC[

Table 7 Sugar conformation in MMI dimers "estimated from proton NMR coupling constants#[*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

Backbone modi_cation*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ

Suàr modi_cationa DNA MMI*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐ1? of top unit "x# none none none none F F OMe1? of bottom unit "y# none none OMe F F OMe OMe) of N!conformationb

Top unit 24 57 55 69 85 87 84Bottom unit 17 20 54 81 85 60 65

*ÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐÐaSee Figure 4"a# for the structure of MMI dimer[ bFractional popoulation ")# of C!2?!endo form calculated by PSEUROT 5[1[

These results encouraged the author and his co!workers to take the MMI linked oligos intoanimal experiments[ The preliminary results indicate that full alternating MMI!PO linked oligos"19!mer# have a distinct biodistribution pattern compared to PS oligos[ At 13 h\ MMI!PO oligoconcentration in kidneys is sixfold less than the PS oligos and also has lower accumulation in livercompared to all!PS oligos[

6[97[2[1[1 Silicon!containing linkages

The dialkylsilyl internucleoside linkage\ _rst described in 0874 by Ogilvie and Cormier\018 appearedto be attractive due to its neutral\ achiral\ size similarity to phosphorus\ lipophilic properties\ andstraightforward synthesis[ Saha et al[009 and Maddry et al[029 independently developed convenientsynthetic procedures\ which allow solution!phase and solid!support synthesis of silicon!linked poly!mers "up to 09!mer#[ Although these oligos demonstrated good stability\ their binding a.nity waspoor[ In addition\ mediocre yields\ polarity concerns\ and suboptimal binding a.nity were thecauses of discontinued interest in their use towards antisense research[ Nevertheless\ use of 0H!18Silong!range HMQC NMR spectroscopy may be useful for structural determination of silicon!containing molecules[020


6[97[2[1[2 Sulfur!containing linkages

There has been an equal amount of excitement in the sulfur!linked backbones "see Table 5#\compared to the nitrogen!containing linkages[ The key reason is that sulfur is the closest isostericand isoelectronic replacement for the natural phosphorus atom\ o}ering a variety of options to alterisosteric linkages chemically\ thus extending the library of neutral linkers available for developmentof antisense molecules[

"i# Sul_des and sulfones

Some of the early work in this area comes from Just and co!workers describing synthesis and useof 2?!CH1CH1S!4? linked dimers[ A review article has been published by Just and Kawai entailingtheir synthetic approaches to obtaining such dimers\ and they extended their studies to include the1?!sugar modi_cations\ which\ as expected\ enhanced the binding a.nity towards RNA targets[000\001

Benner and co!workers also reported a full account of the synthesis of dimers containing 2?!CH1SCH1!4? linkages at the same time[ Their synthetic e}orts have been published in detail descri!bing sequences leading to the fully modi_ed octamers in ½0mg quantities[002 These two groupshave further oxidized their sul_de!linked molecules to sulfone!linked oligosides[ The 2?!CH1CH1SO1!4? linked sulfone described by Just et al[000\001 was found to be base!labile and degraded uponexposure to ammonia at room temperature[ However\ cleavage problems were circumvented bychanging the reaction sequences\ i[e[\ deblock 2?!CH1CH1S!4? linked oligo _rst and then oxidize thelast step[ The Tm data with these oligos indicated that they bind to RNA poorly "DTm −04 >C#\possibly due to steric interaction of SO1 group and base residues[

It is important to note that Egli and co!workers021 published the _rst X!ray crystal structure of anonionic backbone\ which happens to be a 2?!CH1SO1CH1!4? linked ribo dimer placed in the middleof an oligo[ These results allay concerns that the atomic substitutions in the sulfone backbone mayprevent the formation of WatsonÐCrick type double helix with a complementary strand[ The sulfoneoctamer prepared by Benner and co!workers displayed a thermal transition in the UV spectrumabove 54 >C with a large hyperchromicity\ suggesting some secondary structure\ possibly a tighthairpin[002 Interestingly\ the Tm of this octamer did not change either in the presence of bothcomplementary RNA and DNA or in their absence[ In summary\ oligos linked via sul_des andsulfones are di.cult to prepare and show very unusual self!association properties which discouragestheir use in antisense\ as clearly stated by Egli and co!workers[021

"ii# Sulfonates and sulfonamides

Like sulfones\ sulfonates "2?!OSO1CH1!4?# and sulfonamides "2?!NHSO1CH1!4?# linkages areisoelectronic to the natural 2?!O!PO1!O!4? linkage[ Unlike silicon!linked oligos\ sulfonyl!linkedoligos are expected to be very polar nonionic congeners[ In addition\ they possess extra hydrogenbonding capacity which may help towards solubility in water[ These oligomers should also sharethe nuclease stability demonstrated by other nonionic linkages[

Widlanski and co!workers003 described the synthesis of such linkages "e[g[\ 2?!XSO1CH1!4?^ X�Oor NH# in 0883\ via standard dimer approach[ However\ in the case of sulfonate!linked DNA\ itwas demonstrated that such oligosides can be prepared via solid!support synthesis[022 Maddry et al[also prepared identical sulfonyl!dimers\ following a slightly di}erent synthetic strategy[018 Asexpected\ these oligos were quite resistant to nucleases but hybridized with lower a.nity with targetDNA[ On a side note\ Widlanski and co!workers tested oligos containing sulfonamide linkages inplace of the sulfonate linkage\ expecting to alter the proteinÐDNA interactions\ based on the factthat sulfonamide was a better H!bonding acceptor[023 Indeed\ they were able to modulate proteinÐDNA binding\ which in turn can provide important information about the location\ nature\ andrelative strength of proteins and the speci_c PO linkage of the DNA to which they bind[ Anothersulfur!based linkage "2?!OSO1NH!4?# was reported by Trainor and co!workers in 0881[024 Notsurprisingly\ this modi_cation too had a destabilizing e}ect on duplex formation[

6[97[2[1[3 Oxygen!containing linkages

A simple yet provocative replacement of a phosphorus atom with a methylene group created anovel oxygen!containing linkage\ commonly known as formacetal "2?!OCH1O!4?#[ Conceptually\


other ether!linked dimers "2?!OCH1CH1O!3?\ ethylene glycol^ 2?!OCH1CH1CH1!3?\ 2?!CH1CH1

CH1UC!3?\ propoxy type# were visualized and _nally synthesized[ The chemistry and antisenseproperties "see Table 5# of these analogues are summarized in this section[

"i# Formacetals

This modi_cation was synthesized and studied by two groups "Matteucci et al[07 and Van Boomet al[025# simultaneously and independently in 0889[ Several publications have appeared on thissubject and have been reviewed by Matteucci^07 therefore\ a short summary should su.ce herein[The formacetal!linked oligos bind in a sequence!speci_c manner\ but their a.nity appears to besomewhat inferior relative to the unmodi_ed oligo[ They proposed that this helical instability is dueto a strong stereoselective e}ect\ which results in an energy penalty for the `auche conformationsrequired for helix formation\ pointing out the complexity of phosphate isostere design[026

This work took an interesting turn when 2?! and 4?!oxygen atoms of the formacetal linkage werereplaced by sulfur\ one at a time\ thus creating 2?!SCH1O!4? and 2?!OCH1S!4? linked thioformacetals[Again\ synthesis of these linkages has been optimized by more than one group "Matteucci07 andDucharme and Harrison027#[ The binding a.nity of 2?!OCH1S!4? linked oligos was found to be poorand not worth pursuing[ On the contrary\ the 2?!SCH1O!4? linked oligos had a positive e}ect onbinding to the complement RNA "DTm ¦9[7 >C:modi_cation#[07

It is noteworthy that a subtle change like this one in the backbone could have a dramatic e}ecton its a.nity for RNA target[ The di}erences can be highlighted in terms of longer C0S bondlength\ change of sugar pucker\ and the torsion angles around the anomeric XCH1O center[ A 4?!thio derivative produces a bad steric clash with adjacent ribose oxygen\ destabilizing the optimal`auche conformation of these two heteroatoms[ In conclusion\ the 2?!thioformacetal linkage appearsto be the best among this series of modi_cations for antisense applications\ perhaps even better incombination with 1?!sugar modi_cations "e[g[\ OMe\ F#[

"ii# Ethers

Teng and Cook reported the synthesis of some unusual ether!linked dimers utilizing Vorbru�ggen!type glycosylation reactions[004 To be speci_c\ they prepared a 2?!OCH1CH1O!3? and 2?!CH1CH1

CH1O!3? linked analogue of thymidine dimers[ Incorporation of these dimers into oligos had adestabilizing e}ect when hybridized to RNA "DTm −2[9 >C:modi_cation#[ Detailed NMR andmodeling studies of these dimers indicated that the newly formed 3?!oxofuran ring lies in a high S!pucker causing its destabilization during binding to RNA[

E}orts from other researchers005 include reversal of the 2?!CH1CH1CH1O!3? to 2?!OCH1CH1CH1!3? and its thio analogue "2?!SCH1CH1CH1!3?#\ which improved Tm compared to its isomeric struc!tural analogue\ but Tm remained low compared to unmodi_ed PO DNA[ Once again\ extra ~exibilityvia freedom of rotation around the a:b torsional angles could be the reason for compromised a.nityfor RNA target[ This can be further justi_ed by relatively better Tm with formacetal linkage\ whichmay have reduced freedom of rotation due to the presence of the 4?!O!atom\ compared to 2?!OCH1CH1CH1!3? linked molecules[

6[97[2[1[4 All!carbon linkages

Replacement of the four!atom PO linkage with an all!carbon backbone is synthetically challengingand may turn out to be futile because of too much freedom of rotation and hydrophobic interactions[Nevertheless\ _rst Butter_eld and Thomas\006 and later Lebreton et al[028 have reported the synthesisof all!carbon backbone "2?!CH1CH1CH1!4?#[ The latter group investigated its a.nity for RNAtarget and found it to be very destabilizing "DTm −2[1 to −4[8 >C:modi_cation depending uponsequence#[ Subsequently\ the Ciba group reported08 the Tm studies with 2?!CH1COCH1!4? linkeddimers placed in an oligo[ This modi_cation had slightly improved a.nity "DTm −1[4 >C:mod!i_cation# compared to all!carbon linkage\ due to reduced rotational freedom around the linkage[Based on this observation\ Ciba scientists postulated that an all!carbon linked backbone maybe tuned for higher a.nity if they introduced a double bond in this linkage\ such as 2?!CH1

CH1CHCH1!3?\ creating cis and trans isomers[ Indeed\ they synthesized two thymidine dimers


containing cis and trans linked all!carbon backbones and studied hybridization[007 As predicted\ thetrans!isomer was able to bind to the complementary RNA "DTm −9[5 >C:modi_cation# with animproved a.nity compared to 2?!CH1CH1CH1!4? linked oligos[ On the other hand\ the cis isomerhad a larger destabilizing e}ect "DTm −0[2 >C:modi_cation#[ Lee and Wiemer also reported008 asynthesis of carbon!bridged ribonucleoside dimer "2?!CH1COCH1CH!3?#\ which may be worthyof incorporating into oligos[ In summary\ straight alkane!type linkages are unattractive for antisenseutility[ Appropriate conformational restriction "e[g[\ cis!isomer# of all!carbon backbone may help\but overall increased hydrophobicity should be a concern in designing such linkages\ whichinvariably results in poor water solubility[

6[97[2[1[5 Nonphosphate sugar linkage "PNA#

PNA "peptide nucleic acid# is a DNA analogue in which the phosphate sugar backbone is replacedby a structurally homomorphous pseudopeptide chain\ consisting of N!"1!aminoethyl#glycine units"Figure 4"b##[ Unlike DNA\ the PNA backbone carries no charges and has no chiral centers[ Allfour natural DNA bases are retained\ and PNA recognizes sequence!complementary nucleic acidsspeci_cally\ forming WatsonÐCrick base pairs[ The synthesis of PNA has been reviewed039 andreaders are referred to these reviews for a full account[

PNA exhibited exceptional hybridization properties\ with Tm well above those of the cor!responding natural unmodi_ed duplexes\ and stability towards nucleases or proteases[ Despite thesefeatures\ initial e}orts to use PNA as an antisense construct have been hampered by poor watersolubility\ self!aggregation properties\ and inability to activate RNase H[ Tam and co!workers030

and van Boom and co!workers031 have independently reported their syntheses of a PNAÐDNAhybrid molecule[ They reasoned that natural DNA or PS oligos\ covalently linked at both ends "2?and 4?# to short segments "3!units# of PNA\ would give access to a chimeric type of oligo "PNAÐDNAÐPNA# discussed earlier in Section 6[97[1[0\ a strategy that maintains high a.nity and nucleasestability to PNA in ~anking regions\ and RNase H mediated cleavage via the PO or PS linkedcentral portion of the chimera "see Figure 1#[ Interestingly\ half PNAÐhalf DNA hybrid was ableto bind to RNA target with similar a.nity to an unmodi_ed DNA[ Uhlmann et al[032 have reportedthe synthesis and properties of DNAÐPNA chimeras that are taken up by cells to a similar extentas PS oligos\ are largely nuclease resistant\ and have excellent binding a.nity to complementaryRNA[ However\ the evidence from cell!based experiments indicated that the uptake of pure PNAis very poor^ therefore\ microinjection techniques have been utilized to demonstrate the antisensee.cacy of PNA[

6[97[3 DESIGN CONSIDERATIONS

This section summarizes the key elements of novel backbone design considerations for antisenseapplications\ with an emphasis towards pinpointing pitfalls for novices in this area of research[Synthetic backbone linkages have become one of the most attractive areas of antisense!basedoligonucleotide research[ These isosteres of natural phosphate backbone as discussed in Section6[97[2 should allow us to begin to understand the key structural elements required for an {{ideal||backbone linkage[

Generally\ any modi_cation of the phosphate backbone guarantees nuclease resistance to someextent\ and complete replacement of the phosphate backbone with a nonphosphate linker assurescomplete nuclease stability[ Therefore\ it is almost given that any modi_cation of the backbonewould result in stability towards nucleolytic degradation\ and should not be a concern anymoretowards their design[ However\ one should avoid ester and peptide linkages because they may besubstrate for cellular esterases and peptidases[

The chirality of _rst!generation PS oligos appears to be of great concern to some researchers andhas been the subject of many reviews[ Clearly\ with a dozen or more PS oligos undergoing clinicaltrials successfully as mixtures of diastereoisomers\ the concern may be only academic[ On the otherhand\ not all of the properties of chirally pure PS oligos are yet known[ Thus\ the quest for novelroutes towards the synthesis of chirally pure PS oligos should continue[45\46 There has been someexcitement in the use of all!Rp!methylphosphonate due to improved antisense e.cacy[ Since MPoligos are neutral and may avoid protein binding completely\ an e}ort towards synthesis of chirallypure Rp dimers or oligomers should be bene_cial[ Once again\ chirality on C!4?!carbon was intro!


duced with some of the interesting backbone modi_cations[ In summary\ it is the author|s personalbias that chirality in backbone linkers should be avoided[

Solubility of antisense oligo in water is another important feature which should be considered inall design aspects[ As has been seen with silyl\ sulfone and all!carbon containing backbones\ someanalogues have poor water solubility[ In addition\ completely neutral or unchanged antisensemolecules have had solubility problems "e[g[\ methylphosphonates#[ The general recommendationshould be the presence of some charge "positive or negative# on the antisense molecule\ enough todissolve the oligo in water[ The experience at the author|s institution with the use of neutral oligos"e[g[\ MMI and PNA#\ usually 19!mer in length\ has shown that at least four charges on the moleculeare required for e.cient solubility[ Particularly for MMI\ an alternating PO:MMI motif works verywell in all oligos made thus far[ Therefore\ the message is to incorporate charged species in antisenseoligos[

The correct balance of conformational ~exibility vs[ rigidity of the backbone linkage is one of themost important issues in design considerations[ The concept of conformational restriction is aproven method for enhancing the binding a.nity of a ligand for its receptor[ Therefore\ anappropriate preorganization of the backbone linkage can result in enhanced binding a.nity andspeci_city for the target RNA[ The basic structure of RNA is well understood^ however\ those ofits secondary and tertiary structural components are not\ which plays a major role in recognitionduring the binding process[ From what is known about RNA modi_ed oligo interactions\ anRNA mimic "e[g[\ 1?!F\ OMe\ O!alkyl\ O"CH#1OMe\ and others# would serve as a better _t forhybridization\ mainly due to its preorganized structure in N!type sugar conformation\ resulting inA!type duplex formation[ Additionally\ modi_cations of the sugar residue "e[g[\ MMI\ amide!2"1#\ phosphoramidate NP "0#\ 0\4!anhydrohexitol "HNA^ "2##\ and others# can further help thispreorganization of the antisense molecule[ Van Aerschot et al[033 have reported an elegant exampleof the preorganization with a 0\4!anhydrohexitol "5?: 3?# "2# linked oligo[ These oligos form verystable A!type duplexes with RNA "DTm:HNA unit 0[9Ð4[9 >C depending on the chain length andthe sequence# with good base pair speci_city[ The enhanced a.nity of the HNA for the RNAtarget can be further explained by increased hydrophobicity in the minor groove of the duplex[Interestingly\ in HNA\ the base moiety is at the C!1? axial position instead of the C!0? positionof the pyranose ring[ This unusual baseÐsugar connection causes a distortion of the backboneconformation and gives rise to a favorable helicity for the duplex formation[13 Similarly\ the 1?!~uoro modi_ed phosphoramidate NP oligos "0# were found to enhance the Tm "DTm:modi_cation3[9Ð4[9 >C# due to their preferred C!2?!endo conformation[ The phosphoramidate NP oligosÐRNAduplex may be further stabilized due to increased hydrogen bonding in the minor groove due to the2?!amino group[034 It seems that no single modi_cation can confer all the desirable properties on anoligo^ therefore\ one must utilize a combination of sugarÐbackbone modi_cations[ None of theheterocyclic base modi_cations is discussed in this chapter\ but they do have a signi_cant con!tribution towards design constraints[035 Ultimately\ all second! and third!generation antisense agentsare going to be hyper!modi_ed molecules armed with exact amounts of charge\ hydration\ andpreorganization for high a.nity and sequence speci_city for its complement target RNA[

6[97[4 CONCLUSIONS

Chemical modi_cations of phosphate backbone at nearly every position have been considered\and numerous potentially interesting nonphosphate backbone linkages have been prepared[


Ironically\ there have been more failures than successes reported in this chapter[ Now that theconcepts and principles of preorganization are better understood\ the design of superior linkagesshould be more amenable and reduce the number of failures[ Some of these nonphosphate modi!_cations provide enhanced stability to nucleases\ improved a.nity "Tm#\ and in vitro activity[However\ the most rewarding part of this monumental synthetic e}ort to produce nonphosphatelinkages is yet to come\ which is their in vivo activity based on antisense principles[ On the otherhand\ it is important to note that phosphorothioates have performed exceptionally well as the _rst!generation antisense agents[ As a result\ over a dozen PS oligos as potential antisense drugs areundergoing human clinical trials for various diseases[ Again\ much remains to be learned about PSoligos\ but there is no question that novel antisense agents with improved properties have beenidenti_ed[ Although it appears that some of the second! and third!generation modi_cations haveoutperformed _rst!generation PS oligos\ it is quite unlikely that any single modi_cation will solveall issues[ Therefore\ an appropriate combination of baseÐsugar heterocycle modi_cations have tobe utilized to create a future generation of antisense oligos with improved therapeutic properties[There is every reason to be optimistic that antisense inhibition with altered oligos will be availableto the medicinal chemists of the future[

ACKNOWLEDGMENTS

I would like to thank members of the MMI team "Eric Swayze\ Bal Bhat\ Didier Peoc|h\ and StuDimock# for their excellent e}orts in making this project successful and allowing me to includesome of their unpublished results[ Additionally\ terri_c support from the scientists in the Chemistryand Biology Departments at Isis Pharmaceuticals is much appreciated[ The author also wishes toacknowledge Dan Cook and Herb Boswell for their comments and careful reading of the manuscript[Special thanks are also due to Mrs Anna Alessi for typing this manuscript immaculately[

6[97[5 REFERENCES

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Chim[ Pays!Bas\ 0884\ 003\ 184[


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