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“ ”

« » ( 340 - 278 ) ‘The way ahead is long; I see no ending, yet high and low I’ll search with my will unbending’

Li Sao, Qu Yuan (340 BC - 278 BC)

To My Family

List of Original Publications

This thesis is based on the following original publications referred by the Roman numerals. I Zhou, C.; Liu, Y.; Andaloussi, M.; Badgujar, N.; Plashkevych,

O.; Chattopadhyaya, J. Fine Tuning of Electrostatics around the Internucleotidic Phosphate through Incorporation of Modified 2�, 4�-Carbocyclic-LNAs and -ENAs Leads to Significant Modulation of Antisense Properties. J. Org. Chem. 2009, 74, 118-134.

II Zhou, C.; Plashkevych, O.; Chattopadhyaya, J.

Unusual Radical 6-endo Cyclization to Carbocyclic-ENA and Elucidation of Its Solution Conformation by 600 MHz NMR and ab initio Calculations. Org. Biomol. Chem. 2008, 6, 4627-4633.

III Zhou, C.; Plashkevych, O.; Liu, Y.; Badgujar, N.; Chat-topadhyaya, J. Synthesis and Structure of New Methylene-Bridged Hexopyranosyl Nucleoside (BHNA). Heterocycles 2009, 78, 1715-1728.

IV Zhou, C. and Chattopadhyaya, J.

New Methylene-Bridged Hexopyranosyl Nucleoside Modified Oligonucleotides (BHNA): Synthesis and Biochemical Studies. Arkivoc 2009(3), 171-186.

V Zhou, C.; Plashkevych, O.; Chattopadhyaya, J.

Double Sugar and Phosphate Backbone-Constrained Nucleotides: Synthesis, Structure, Stability, and Their Incorporation into Oli-godeoxynucleotides. J. Org. Chem. 2009, 74, 3248-3265. [JOC Featured Article]

VI Zhou, C.; Honcharenko, D. and Chattopadhyaya, J. 2-(4-Tolylsulfonyl)ethoxymethyl (TEM) - a New 2�-OH Protect-ing Group for Solid-Supported RNA Synthesis. Org. Biomol. Chem. 2007, 5, 333-343.

VII Zhou, C.; Pathmasiri, W.; Honcharenko, D.; Chatterjee, S.; Bar-

man, J. and Chattopadhyaya, J. High-quality Oligo-RNA Synthesis Using the New 2�-O-TEM Protecting Group by Selectively Quenching the Addition of p-Tolyl Vinyl Sulphone to Exocyclic Amino Functions. Can. J. Chem. 2007, 85, 293-301.

VIII Bramsen, J. B.; Laursen, M. B.; Nielsen, A. F.; Hansen, T. B.;

Bus, C.; Langkjær, N.; Babu, B. R.; Højland, T.; Abramov, M.; Van Aerschot, A.; Odadzic, D.; Smicius, R.; Haas, J.; Andree, C.; Barman, J.; Wenska, M.; Srivastava, P.; Zhou, C.; Honcharenko, D.; Hess, S.; Muller, E.; Bobkov, G. V.; Mikhailov, S. N.; Fava, E.; Meyer, T. F. Chattopadhyaya, J.; Zerial, M.; Engels, J. W.; Herdewijn, P.; Wengel, J. and Kjems, J. A Large-scale Chemical Modification Screen Identifies De-sign Rules to Generate siRNAs with High Activity, High Stability and Low Toxicity. Nucleic Acids Res. 2009, 37, 2867-2881.

Reprints were made with permission from the publishers.

List of publications, not included in this thesis. IX Zhou, C. and Chattopadhyaya, J.

The Synthesis of Therapeutic Locked Nucleos(t)ides. Curr. Opin. Drug Discov. Dev. 2009, 12, 876-898. [Invited

Review] X Zhou, C. and Chattopadhyaya, J.

Why carba-LNA Modified Oligonucleotides Show Considerably Improved 3� Exonuclease Stability Compared to that of the LNA Modified or the Native Counterparts? A Michaelis-Menten Ki-netic Analysis. J. Org. Chem., submitted.

XI Honcharenko, D.; Zhou, C. and Chattopadhyaya, J.

Modulation of Pyrene Fluorescence in DNA Probes Depends upon the Nature of the Conformationally Restricted Nucleotide. J. Org. Chem. 2008, 73, 2829-2842.

XII Barman, J.; Acharya, S.; Zhou, C.; Chatterjee, S.; Engström, Å.

and Chattopadhyaya, J. Non-identical Chemical Characters of the Internucleotidic Phos-phates can Modulate the Non-enzymatic Reactivity of the Phos-phodiester Bonds in RNA. Org. Biomol. Chem. 2006, 4, 928-941.

XIII Mook, O.R.F.; Vreijling, J.; Wengel, S.L.; Wengel, J.; Zhou, C.;

Chattopadhyaya, J.; Baas, F.; Fluiter, K. In vivo Efficacy and Off-target Effects of Locked Nucleic Acid (LNA) and Unlocked Nucleic Acid (UNA) Modified siRNA and Small Internally Segmented Interfering RNA (sisiRNA) in Mice Bearing Human Tumor Xenografts. Manuscript.

Contibution Report

The author wishes to clarify his contributions to the research presented in the present thesis: Paper I: Designed research with M.A., Y.L. and J.C. Synthesized carba-LNA nucleosides and their incorporations into oligos, thermal denaturation studies as well as all of the enzymology stuides in this paper. Analyzed data and wrote the first draft of manuscript.

Paper II: Designed the research with J.C. Performed all the exprimental work except the theoretical calculation part that was done by O.P. Analyzed data and wrote the first draft of manuscript.

Paper III: Designed the research with J.C. and O.P. Synthesized and charac-terized BHNA nucleoside. Analyzed data and wrote the first draft of manu-script with O.P.

Paper IV: Designed and performed all the research. Analyzed data and wrote the first draft of manuscript.

Paper V: Initiated the research project with J.C. Designed and performed all the research except the theoretical calculation part that was done by O.P. Analyzed data and wrote the first draft of manuscript.

Paper VI: Initiated the research project with J.C. Designed and performed all the research except the RNase H digestion part that was done by D.H. Ana-lyzed data and wrote the first draft of manuscript.

Paper VII: Designed the research. Performed all the research except charac-terization of new compounds by NMR and Maldi-Tof mass spectoscopy that was done by the coworkers. Analyzed data and wrote the first draft of manu-script.

Paper VIII: Synthesized and purified siRNAs with carba-LNA, carba-ENA and aza-ENA modifications.

Contents

1. Introduction to nucleic acids .....................................................................13 1.1 Composition of DNA and RNA .........................................................13 1.2 Structural properties of nucleic acids .................................................14

1.2.1 Base pairs ..................................................................................14 1.2.2 Sugar conformations .................................................................15 1.2.3 Phosphate backbone conformations..........................................15 1.2.4 Helical structures of nucleic acids.............................................16

1.3 Chemical synthesis of oligonucleotide for therapeutic application....17 1.3.1 Oligonucleotide-based therapeutics ..........................................17

1.3.1.1 Antisense oligonucleotide.............................................17 1.3.1.2 Triple-helix forming oligoncucleotide (antigene

strategy) .......................................................................18 1.3.1.3 Ribozyme and DNA enzyme........................................18 1.3.1.4 siRNA and miRNA.......................................................18

1.3.2 Chemical synthesis of unmodified oligonucleotide ..................19 1.3.3 Modified nucleoside, nucleotide and oligonucleotide...............20

1.3.3.1 Base modification .........................................................21 1.3.3.2 Sugar modification .......................................................21 1.3.3.3 Internucleotidic linkage modification...........................22

1.4 Overview of the thesis........................................................................22

2. Conformationally constrained oligonucleotides as antisense therapeutics...............................................................................................23 2.1 A brief introduction to conformationally constrained nucleos(t)ides.23

2.1.1 LNA and its application in therapeutics and biotechnology .....25 2.2 The synthesis and structure of carba-LNA, carba-ENA, BHNA and

D2-CNA nucleos(t)ides (Papers I-III & V) ........................................27 2.2.1 Synthesis of carba-LNA, carba-ENA and BHNA nucleosides .27 2.2.2 Synthesis of D2-CNA nucleotide ..............................................31 2.2.3 Structure of carba-LNA, carba-ENA, BHNA and D2-CNA

nucleos(t)ides............................................................................32 2.3 The antisense properties of carba-LNA, BHNA and D2-CNA

modified oligonucleotides (Papers I, IV & V) ...................................34 2.3.1 Synthesis of modified AONs and their chemical stabilities......34

2.3.2 Affinity and specificity of modified AONs toward complementary RNA................................................................37

2.3.3 Nucleolytical stabilities of modified AONs..............................39 2.3.4 RNase H-mediated RNA degradation in AON/RNA duplex....40

3. Synthesis of conformationally constrained siRNAs as potential RNAi therapeutics...............................................................................................42 3.1 2�-OH protecting groups for RNA synthesis ......................................42 3.2 2�-O-TEM based RNA synthesis strategy (Papers VI & VII) ............44

3.2.1 Synthesis of 2�-O-TEM protected building blocks....................45 3.2.2 Solid-supported RNA synthesis and post-synthesis treatment..46 3.2.3 Deprotecting problems and solutions........................................48

3.3 RNAi performance of carba-LNA, carba-ENA and aza-ENA modified siRNAs (Paper VIII) ...........................................................48 3.3.1 Potency of modified siRNAs ....................................................49 3.3.2 Cellular toxicity of modified siRNAs .......................................50

4. Sammanfattning ........................................................................................51

5. Acknowldegements...................................................................................53

6. Reference ..................................................................................................55

Abbreviations

1D One dimensional 2D Two dimensional A Adenosine Ac Acetyl Ade Adenine Aza-ENA 2�-N,4�-C-Ethylene bridged nucleic acid AON Antisense oligonucleotide Bc-DNA Bicyclo-deoxyribonucleic acid BHNA bicyclo[2.2.1]-2�,5�-methylene-bridged hexopyrano-

syl nucleic acid Bn Benzyl BNA Bridged nucleic acid C Cytidine Carba-LNA Carbocyclic locked nucleic acid Carba-ENA Carbocyclic 2�-O,4�-C-ethylene bridged nucleic acid CEM 2-Cyanoethoxymethyl Cyt Cytosine CD Circular dichroism D2-CNA Double sugar and phsophate backbone-constrained

nucleic acid DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCA Dichloroacetic acid DNA Deoxyribonucleic acid Dmf Dimethylaminomethylene DMSO Dimethyl sulfoxide DMTr 4�,4�-Dimethoxytrityl ENA 2�-O,4�-C-Ethylene bridged nucleic acid ETT 5-Ethylthio-1H-Tetrazole G Guanosine Gua Guanine HIV Human immunodeficiency virus HNA Hexitol nucleic acid HPLC High performance liquid chromatography IC50 Half maximal inhibitory concentration LCAA-CPG Long chain amino alkyl-controlled pore glass LNA Locked nucleic acid

mRNA Messenger RNA ncRNA Non-coding RNA NMR Nuclear magnetic resonance nOe Nuclear Overhauser effect P Pseudorotation phase angle Pac Phenoxyacetyl PAGE Polyacrylamide gel electrophoresis PNA Peptide nucleic acid RNA Ribonucleic acid RNAi RNA inteference RP Reverse phase siRNA Small inteferencing RNA SVPDE Snake venom phosphodiesterase T Thymidine TBAF Tert-n-butylammonium fluoride tBDMS tert-butyldimethylsilyl TEM 2-(4-tolylsulfonyl)ethoxymethyl TFO Triple-helix forming oligonucletide Thy Thymine Tol Toluoyl TOM [(triisopropylsilyl)oxy]methyl THF Tetrahydrofurane TVS p-Tolyl vinyl sulphone Τm Melting temperature U Uridine Ura Uracil φm Puckering amplitude

13

1. Introduction to nucleic acids

The nucleic acids are biological polymers. They naturally exist as Deoxyri-bonucleic acid (DNA) and Ribonucleic acid (RNA). In living organism, DNA exists as double helix inside cell nucleus and its main role is the long-term storage of genetic information, whereas different types of RNAs have been found and each has different functions. For example, messenger RNA (mRNA) is transcribed from DNA, and carries the genetic information that will be translated in ribosome to proteins; transfer RNA (tRNA) is a small RNA (about 74-95 nucleotides) and serves as transporters of amino acids in the translation process; ribosomal RNA (rRNA) is a component of the ri-bosomes, and has been claimed to be a catalyst for peptide bond formation in the ribosome.1,2 tRNA, rRNA as well as the intron in pre-mRNA are not translated into proteins thus called non-coding RNA (ncRNA).3,4 Many new types of ncRNAs such as siRNA,5,6 miRNA,7-13 piRNA,14-16 small nucleolar RNA (snoRNA),17 riboswitch18-20 have been discovered in the past decades. Most ncRNAs act as molecular switches that regulate gene expression,21 however, the functions of many ncRNAs are still unknown.3 In viruses, RNA also serves as genetic carrier. The retrovirus family uses the reverse tran-scriptase to transcribe single-stranded RNA into double-stranded DNA.

1.1 Composition of DNA and RNA DNA and RNA are made from repeating units of nucleotides, which has three components: a heterocyclic nitrogenous base, a β−D-pentafuranose sugar moiety, either in ribo or 2�-deoxy form, linked at C1� to the nucleobase and a phosphate-diester linkage (Figure 1). The base is normally one of 9-adeninyl (Ade), 1-cytosinyl (Cyt), 9-guaninyl (Gua) or 1-thyminyl (Thy) in DNA, while the 1-Thy is replaced by 1-uracilyl (Ura) in RNA. There are also many modified and hypermodified nucleobases which occur in various tRNAs.22 The presence or absence of 2�-OH in ribo sugar makes DNA and RNA structurally and functionally different. For example, DNA is double stranded while RNA, in most case, is a single-stranded molecule containg many types of secondary structures consisting of haipin loops of different sizes, bulge, internal loop, junction and so on23; the structure of RNA is collections of short helices packed together into structures akin to proteins and as the result, RNAs can achieve chemical catalysis,24-26 like enzymes;

14

the additional 2�-OH makes RNA less stable than DNA because of neighbouring group participation.27

Figure 1. (A) General structure of nucleic acids. (B) Atomic numbering and defini-tion of torsion angles for one nucleotide.

1.2 Structural properties of nucleic acids In nature, nucleic acids exist in high ordered structure through self-assembly. Hydrogen bonding and base stacking are the major driving force for this self-assembly. The structural properties of nucleotide can affect this self-assembly process dictating the final tertiary structure of nucleic acids. The molecular geometry and conformational properties of the nucleobase, the furanose sugar and the phosphate linkage are summarized28 below.

1.2.1 Base pairs Purine and pyrimidine can form base pair through hydrogen bonds. There are many different types of base pairs and amongst them, Watson-Crick,29 Hoogsteen and wobble base pairing30-32 are the most common ones (Figure 2). A complete graphalic list of different base pairs can be found in page 120 of reference28.

Figure 2. Some representative types of base pairs.

BaseO

RO

OPOO

BaseO

RO

O

BaseO

RO

OPOO

BaseO

RO

OPOO

POO

5'

N

NN

N

NH2

NH

NN

N

O

NH2

N

N

NH2

O

NH

N

O

O

NH

N

O

O

Adenine (Ade) Guanine (Gua)Cytosine (Cyt)

Thymine (Thy) Uracil (Ura)

nucleotide

(A) (B)

NN

O

OC1'

N

N

N

NH

H

C1'

N H

H

ThyAde

NN

N

OC1'

H

H

N

N

N

NC1'

N

Gua

O

H

H

HCyt

Watson-Crick G-C

NN

N

N O

NH2

H

N

N

O

Ura

O

H

GuaC1'

C1'

G-U Wobble

N

NN

C1'

H

H Cyt

O

H

Hoogsteen

NN

N

OC1'

HH

N

N

N

N

C1'N

Gua

O

HH

HCyt

C-G-C triplet

NN

O

OC1'

N

N

N

NH

H

C1'

NH

H

ThyAde

N

NO

C1'

Thy

OH

T-A-T triplet

Hoogsteen

N

N

N

NC1'N

Gua

O

H

H

H

NN

NN'1C

N

Gua O

H

H H

N

N

N

N

'1CN

Gua

O

H

H

H

NN

N NC1'

N

GuaO

H

HHM+

Hoogsteen

Watson-Crick A-T

G-quadruplexes

NN

N

N O

HInoC1'

I-C WobbleN

N

N

C1'

H

H

Cyt

O

15

1.2.2 Sugar conformations A planar conformation is energetically unfavorable for the pentafuranose sugar moiety of nucleotide because in this arrangement, all the substituents attached to carbon atoms are fully eclipsed. This system reduces its energy by puckering. The concept of pseudorotation has been introduced to describe the conformation of ribose and deoxyribose rings in nucleotide with two variable parameters, the pseudorotation phase angle (P = (v4+v1-v3-v0)/2v2 (sin36+sin72)) and the puckering amplitude (φm = v2/cos P).33 The pseudoro-tation cycle of sugar part of nucleotide is shown in Figure 3. The deoxyribo-furanosyl sugars in DNA adopt preferentially 3�-endo, 2�-exo twist form (of-ten referred to as North or N-type conformation), whereas ribofuranosyl sugars in RNA are in 3�-exo, 2�-endo twist form (often referred to as South or S-type conformation). The conformation of the pentofuranose sugars are not static and in solution are involved in a two-state North South equilib-rium. The dynamic nature of this equilibrium was evidenced first by Chat-topadhyaya’s lab34-36 by temperature-dependent NMR studies, which showed how different stereoelectronic effects (Gauche and Anomeric Effects)36 actu-ally control this two-state dynamic North South equilibrium. They also determined the energy penalty for each types of stereoelectronic effects.37,36 However, the placement of different substituents in the sugar ring38-41 as well as the aromatic nature of the nucleobase can drive the sugar ring to adopt predominanly one conformation.42-46

1.2.3 Phosphate backbone conformations There are six sugar phosphate backbone torsions [α, β, γ, δ, ε, ξ] present in nucleic acids (Figure 1B). These torsion angles are often roughly described

O

H

R

R'H

N

H

OH

H

T23

3'-endo

2'-exo

O

R

R'

H

N

H

OH

H

2'-endo

3'-exoH

OH

R

H

R'H

HO

N

H

Oo18o

36o

54o

72o

90o

108o

126o

144o

162o180o

198o216o

234o

252o

270o

288o

306o

324o

342oE3

T43

E4

T40

E0

T10

E1

T12

E2T23

E3T43

E4

T40

E0

T10

E1

T21

E2

O

H

R

H

R'

OHN

H

H

West (W)East (E)

North (N)

Sourth (S)

O4'-endo

O4'-exo

Figure 3. Pseudoro-tation cycle of the furanose ring in nu-cleosides. E = enve-lope; T = twist. The North type (-1º � P � 34º) and South type (137º � P � 194º) pseudorotamers commonly populated in β-D-oligonucleotides are shaded.

16

as being in a conformational hyperspace47 such as cis (c) = 0 ± 30º,+gauche (g+) = 60 ± 30º, +anticlinal (a+) = 120 ± 30º, trans (t) = 180 ± 30º, -anticlinal (a-) = 240 ± 30º, -gauche (g-) = 300 ± 30º. The typical range of phsophate backbone torisions for A and B families of DNA and RNA can be found on page 230, reference28.

The base is attached to sugar by a glycosidic bond. The trosion angle about this bond is defined χ [O4�-C1�-N9-C4] for purines and [O4'-C1'-N1-C2] for pyrimidines. Torsion χ can adopt two main conformation called syn (-90º � χ � 90º) and anti (90º � χ � 180º). Anti orientation is more favorable compared to syn orientation because in the anti conformation there is no particular steric hindrance between sugar and nucleobase. However, the an-gle of χ can be tuned by the sugar pucker as well as the base types (purine or pyrimidine)48,49,50,51 and substituent on the base (compare adenosine in anti but 8-bromoadenosine is in syn).52

Figure 4. Structure of double helix of B type DNA-DNA (A), DNA-RNA (B), A type RNA-RNA (C). The strands in red are DNAs and strands in cyan are RNAs. These are solution structures53 from Protein Data Bank (1axp, 1rrr, 1rrd for DNA-DNA, DNA-RNA and RNA-RNA respectively).

1.2.4 Helical structures of nucleic acids DNA is found in single, double helix and multiple stranded (triplexes54,55 and quadruplexes56,57) structures. There are three types of double helical struc-tures for DNA: A-, B- and Z-DNA. B-DNA is the dominant form which has wide major groove (ca 22Å) and narrow minor groove (ca 12Å).58 Whereas A-DNA has shallow, wide minor groove (ca 10Å) and a narrower, deeper major groove (ca 4Å).28 RNA helix exists predominantly in A- form. NMR59,53 and crystallography studies60-62 show the DNA-RNA duplex adopt a intermediate conformation between A and B but closer globally to A than the B form. Represent structures of B-DNA, A-RNA and DNA-RNA hy-dride are shown in Figure 4.

(A) DNA-DNA (B) DNA-RNA (C) RNA-RNA

Major Groove�

Minor Groove�

Major Groove�

� Minor Groove

Major Groove�

� Minor Groove

17

1.3 Chemical synthesis of oligonucleotide for therapeutic application

1.3.1 Oligonucleotide-based therapeutics An oligonucleotide is a short segment of RNA or DNA. Oligonucleotide has extensive applications. It can be used as primer in polymerase chain reaction and in automated DNA sequencing; as probe for detecting DNA or RNA (e.g. Southern blot); as integrated part of artificial gene; it is also used in DNA chips for high-throughput detection of genetic diseases. The most im-portant application of oligonucleotide has been recognized in the field of drug design and development in that it can be designed to down-regulate any gene of interest via antisense,63-65 antigene66,67, ribozyme,68 DNAzyme,69 RNA interference (RNAi)70 or miRNA approaches13,12,11 (Figure 5).

Figure 5. Different oligonucleotide-based therapeutic strategies.

1.3.1.1 Antisense oligonucleotide Antisense technology exploits oligonucleotide, typically 15-20 nucleotides, to bind to target RNA sequences through Watson-Crick hybridization, result-ing in degradation of target RNA by RNase H or disablement of the target RNA.65,63 The potential of antisense oligonucleotides (AON) was first dem-onstrated in 1977 by Paterson, Roberts and Kuff who reported that single stranded DNA can be used to inhibit mRNA translation in a cell-free sys-tem.71 Subsequently, Zamecnik and Stephenson in 1978 showed that a 13-mer DNA could inhibit viral replication in cell culture.72 This work, for the

18

first time, suggested that oligonucleotide could be used as putative drug. Presently, two antisense oligonucleotide-based drugs have been approved: First is Vitravene (Fomivirsen), which was used to treat cytomegalovirus retinitis.73 Second is Macugen®, which was used for the treatment of ne-ovascular age-related macular degeneration.74 There are many other an-tisense oligonucleotides under clinical trials for the treatment of cancer, viral infection, asthma and psoriasis disease.75 Very recently, antisense oligonu-cleotides have been designed and used for inhibiting miRNA function effi-ciently.76

1.3.1.2 Triple-helix forming oligoncucleotide (antigene strategy) In the antigene approach,66 a synthetic triple-helix forming oligonucleotide (TFO) was designed to bind to complementary sequence selectively to ge-nomic double-stranded DNA (generally uninterrupted ho-mopurine•homopyrimidine sequence), which have been shown to perturb the transcription. Using this strategy, not only transcriptional activation and deactivation77,78 but also target DNA cleavage79 as well as targeted mutagenesis80 can be achieved. But the therapeutic application of antigen approach is limited by significant obstacles including target sequence limita-tion, target DNA accessibility, triple-helix stability81 and delivery problem. Consequently, there is still no antigene olignonucleotide in clinical trials.

1.3.1.3 Ribozyme and DNA enzyme In the early 1980s, Cech24 and Altman25 discovered independently that RNA molecules can catalyze RNA degradation even in the absence of protein component. The RNA molecule with enzyme-like activity was named ri-bozyme. Since then, different types of natural ribozymes have been discov-ered and several of them such as hammerhead ribozyme82 and hairpin ri-bozyme83 have been found to bind target RNA through Watson-Crick hy-bridization and cleavage the target RNA site specifically. Ribozyme was soon thought to be promising therapeutic molecule. Several ribozymes have been subjected to clinical trials.68 In addition, in vitro selection techniques have extended the range of catalytically active nucleic acids even to DNA oligonucleotides with enzymatic properties, named DNA enzymes, or DNAzyme.84 DNAzymes are much more easier to be synthesized and han-dled, also less susceptible to degradation by endogenous nucleases, making them considerably more prospective in therapeutic application than ri-bozymes.69

1.3.1.4 siRNA and miRNA In 1998, Fire and Mello found that double strand RNA could silence gene expression in Caenorhabditis elegans.5 This phenomenon was named RNA inteference by them. Soon after, Tuschl and Zamore et al. demonstrated that RNA interference is mediated by 21- and 22-nucleotide85-87 and short syn-

19

thetic siRNA could induce RNAi in mammalian cells.88 This discovery has provided powerful new tools for biological research and drug discovery. siRNA is much more potent than antisense oligonucleotide or ribozyme89, which means siRNA molecules may function at much lower concentrations than other oligonucleotide based strategies. The speed with which RNAi therapeutics is progressing towards the clinic is impressive. Now there are many RNAi based drugs under clinical tries.70,90

MiRNAs are a family of 21-25-nucleotide samll RNAs. They bind to the 3�-untranslated region of mRNAs by base-pairing interactions. In cases of perfect or near-perfect complementarity to the miRNA, target mRNA can be cleavaed; otherwise, their tanslation is repressed.13,11,12 Since the discovery of the first miRNA in 1993,8,91 hundreds of miRNAs have been identified in plants and animals.13 Studies have shown that many miRNAs are associ-ated with human diseases.92 Hence, the development of miRNA pro-vides another oligonucleotide based therapeutic approach. The first miRNA targeting drug has been entered human studies.93

1.3.2 Chemical synthesis of unmodified oligonucleotide The first oligonucleotide was synthesized by Todd and Michelson in 195594 through phosphotriester approach. Very soon, the field of oligonucleotide synthesis became dominated by a different approach introduced by Khorana.95-97 Based on this approach, which now is known as the phosphodiester approach, Khorana and his co-workers have synthesized several biologically relevant oligodeoxyribonucleotides which have been used to solve many fundamental problems in biology such as elucidation of the genetic code.98 In 1981, Beaucage and Caruthers reported the phosphoramidite approach for the oligoncleotide synthesis.99 Formation of phosphate linkage by phosphoramidite approach is fast, high yielding and it very soon became the predominant method in the field of oligonucleotide synthesis.100

During the chemical synthesis of oligonucleotides, the large number of couplings needed to assemble useful sequences was daunting. This was be-cause each step required workup, purification, and the labor and cumulative loss of material became significant problems. This problem was first ad-dressed by Merrifield with the introduction of solid-phase peptide synthe-sis.101 The method of solid supported synthesis was introduced to the field of oligonucleotide synthesis by Letsinger and Mahadevan in 1965.102 Combining the solid-supported synthesis and phosphoramidite approach, gene machine was developed in 1980's.103 Using the gene machine, which now is generally called DNA/RNA synthesizer, one 20mer oligodeoxyribonucleotides can be assembled in the lab in less than 2h with yield in any average step more than 98%! The typical oligonucleotide synthesis cycle on DNA/RNA synthesizer is shown in Figure 6. After

20

completion of the assemably, the solid supports are treated with ammonia to cleave the oligos from the solid support and at the same time to deprotect the protecting group on the base and phosphate linkage. The crude product thus obtained is purified by HPLC or PAGE giving pure oligonucleotide.104

For RNA synthesis, the only difference is that the 2�-OH should be protected during the sequence assembly and the protection group should be removed under mild conditions under which the deprotected oligoribonucleotide is stable.105

1.3.3 Modified nucleoside, nucleotide and oligonucleotide Modified nucleos(t)ide, oligonucleotide have a variety of uses. They can be used as chemical probes in diagnostics106-108; Some modified nucleosides have broad therapeutic applications such as cytosine arabinoside (Ara (C))109; modified oligonucleotides could be potential antigene,66 an-tisense64,65,110 or RNAi111-114 based drugs; they can be used as tools for struc-ture determination115 and study of protein-nucleic acid interactions; they can also be used as tools for understanding mechanisms of Ribozyme,116,117 tRNA processing (RNAse P RNA)118 and cell signaling processes.119

For synthesis of modified oligonucleotides, the nucleoside is first modi-fied. Then the modified nucleoside is converted to building block, phos-phoramidite. In the end, the modified phosphoramidite is incorporated into oligonucleotides through solid-supported synthesis. The modification in nucleoside can be on the nucleobase, sugar moiety or phosphate linkage120 and they are briefly discussed below.

O

RO

HO Bp

O

RO

DMTrO Bp

PCeON

step 2Coupling

O

RO

O Bp

O

RO

DMTrO Bp

PCeO

step 3Cappling

O

RO

O BpH3C

O

O

RO

O Bp

O

RO

DMTrO Bp

PCeO O

O

RO

DMTrO Bpstep 4Oxidation

step 1Deblocking

I2, H2O

TCA, CH2Cl2

tetrazoleAc2O, NMI

building block

solid support

Figure 6. Synthetic cycle of solid-supported oligonucleotide synthesis on DNA/RNA synthesizer based on phosphoramidite. For DNA, R = H; For RNA, R = OP (2�-OH is protected by proper group).

21

1.3.3.1 Base modification The reported base modifications121 can be generally classified into two cate-gories: (1) modifications that enhance base stacking by expanding the π–electron cloud such as 5-propyne-pyrimidine122 modification, 7-iodo-7-deaza-purine modifications123 and size-expanded base124-127; (2) modifica-tions with additional or deleted amino group such as 2-amino-adenine128,129 and 2-aminopurine130,131 (Figure 7A).

Figure 7. Graphically showing representative modifications on base, sugar or phos-phate linkage moiety of nucleotide.

1.3.3.2 Sugar modification Modification on the sugar moiety (Figure 7B) has attracted great interest in the past two decades because AONs with this type of modifications gener-ally have potential antisense properties. The 2�-O-modifictations are consid-ered the second generation of AON132,133. The 2�-O-modifications with ap-propriate electronegative substituents can confer an RNA-like 3�-endo sugar puckering, which results in a considerable improvement of binding affinity to the target RNA.134 The size, electronegativity as well as the conformation of the 2�-substituents are all very important factors for the target affinity and nuclease resistance.135,136 Relatively smaller 2�-substitutions with higher elec-tronegativity such as 2�-F or 2�-O-methyl with ribo configuration stabilize the duplex with complementary RNA but have poorer nucleases resis-tance.134 The bulkier 2�-substitutions improve nucleases resistance, but at the expense of a loss of the target affinity.135

Recently developed LNA137 (also called BNA138) is another type of sugar-modified nucleoside. By linking the 2�-oxygen and 4�-carbon with a methyl-ene group, a rigid bicyclic ring is formed, which results in typical 3�-endo locked conformation of the sugar puckering.139 Thus, oligonucleotides con-taining LNA are preorganized in the A-type canonical structure,140 and thus have unusually high affinity toward complementary RNA strand (3-8 �C per modification).141

The pentafuranose sugar can be replaced by 1,5-anhydrohexitol and the obtained pyranose nucleoside (HNA) can stabilize the DNA or RNA du-

NH

N

O

O

R

N

NN

NH2RN

NN

N

NH2

NH2

NH

NH

O

O

N

NN

N

NH2

NH2

(A) Base modifications - Some examples

O

R

Base

(B) Sugar modifications - Some examples

R = F, OMe, OMOE

OBaseO

O

Base

LNA HNA

(C) Internucleotidic linkage modifications - Some examples

OPO

O S

OPO CH3

OPO

O BH3O

NH

N

NH2

O

RNH

N O

NHX

NHPO OO

HN

O

OPO

O NHRCH2PO OO

N

NN

N

NH2

22

plexes.142-144 It is because,145 firstly, the six-membered ring system adopts a rigid chair conformation, requiring a less negative entropy change during the duplex formation; and secondly, the interstrand phosphate distance in the six-membered pyranosyl-modified system is larger than in the natural nu-cleic acid duplexes giving relatively less interstrand charge repulsion com-pared to the native counterpart. Both LNA and HNA are important members of the family of conformationally constrained nucleoside.

1.3.3.3 Internucleotidic linkage modification Linkage modifications (Figure 7C) involve changes to the nonbridging oxy-gen atoms, such as phosphorothioate (P=S),146,147 methylphosphonate (Me-P),148,149 borane phosphonate (P-BH3),150,151 cationic phosporamidite152,153 as well as changes to the bridging oxygen atoms such as N3'�P5' phosphormi-date.154,155 An alternative approach involves replacing the oligonucleotide backbone entirely, such as in the case of peptide nucleic acid (PNA).156,157

The phosphate backbone modifications render oligonucleotides impressed therapeutic properties such as enhanced binding affinities, improved nucle-ase resistance, and membrane permeability, and thus this type of modifica-tion has attracted great interest in the field of oligonucleotide-based thera-peutics.158

1.4 Overview of the thesis In this thesis we present the utilities of conformationally constrained nu-cleos(t)ides modified DNA and RNA as potential antisense or RNA interfer-ence agents. This thesis consists of two parts. The first part describes the synthesis and structure of the conformationally constrained bicyclic nucleo-sides such as carba-LNA or ENA and their derivatives (Papers I & II), me-thylene-bridged hexopyranosyl nucleoside (BHNA, Papers III & IV), and tricyclic nucleotide such as double-constrained nucleotide (D2-CNA, Paper V). These nucleos(t)ides have been also incorporated into oligodeoxyribonu-cleotides whose antisense properties such as affinity toward complementary DNA or RNA, nuclease resistance and RNase H elicitation have been stud-ied. In the second part we present the solid-supported RNA synthesis using the 2-(4-tolylsulfonyl)ethoxymethyl (TEM) as the protecting group for 2�-OH (Papers VI & VII). Using this strategy, siRNAs modified with carba-LNA, carba-ENA and aza-ENA nucleosides have been synthesized and their RNA siliencing activities as well as cellular toxicity have been studied (Pa-per VIII).

23

2. Conformationally constrained oligonucleotides as antisense therapeutics

2.1 A brief introduction to conformationally constrained nucleos(t)ides Since exogenetic unmodified oligonucleotides can be degraded very fast by cellular nuclease,159 they are required to be modified for efficient therapeutic applications. Among different types of modified therapeutic oligonucleo-tides, more and more attention has been focused on conformationally con-strained nucleotides in the past two decades.160-163 Conformationally con-strained nucleotides were obtained by covalently linking two atoms (gener-ally, between two carbons or between a carbon and a oxygen) of the nu-cleos(t)ide by an alkyl chain to lock a specific conformation amongst all of the flexible pseudorotamers that exist in the conformational equilibrium of the pentofuranose moiety or across the sugar-phosphate backbone.

The first locked nucleoside is nucleobase to sugar locked nucleoside, 3, 5�-cycloadenosine (Figure 8A), which was synthesized by Todd in 1951.164 Since then, many nucleobase to sugar locked nucleosides have been synthe-sized for structural studies or just as synthetic intermediates.165-171 Shortly after some 2�, 3�-dideoxynucleosides were discovered to inhibit the infectiv-ity and cytopathicity of HIV in the middle of 1980s,172 the first sugar locked nucleoside, 2�, 3�-exo-methylene nucleoside (Figure 8E), was synthesized by Okabe173 in 1989. With the development of antisense technologies and gene machine, some sugar pucker locked nucleosides such as Bc-DNA (Figure 8I)174,175 have been synthesized and incorporated into oligonucleotides to complex with the target RNA and DNA to query into the proof of concept for the down-regulation of genes in the antisense and triplexing strategies in the beginning of 1990s. In 1994, Altmann and Marquez showed that 4�, 6�-methanocarba nucleoside (in N-type pucker, Figure 8D) and 1�, 6�-methanocarba nucleoside (in S-type pucker) had totally different antiviral and antisense activities.176-178 These works highlighted the importance of sugar pucker in therapeutic nucleos(t)ide and inspired an upsurge in synthe-sis of sugar moiety locked nucleos(t)ides. This work also showed the advan-tages of N-locked over the S-locked nucleoside containing AONs in making the former more RNA-selective over the latter. Soon after,

24

Figure 8. Chemical structures of conformationally constrained nucleos(t)ides.

OO

O

NH

N

O

O

OMe

OHO

OH

NH

N

O

OO

χ :anti, P = 27o(N-type)

(A) Base to sugar locked

O

N

NN

N

OH OH

NH2

N

NN

N

NH2

O

OH

HO O/S

OHO

OH

NH

N

O

O

ORO

OR

N

N

O

O

OHO

OH

NH

N

O

O

N

NN

N

NH2

O

OH

OH

N

NN

N

NH2

O

OH

HO

ON

NHN

N

O

O

OH OHThe first natural

locked nucleoside

OH

OHO

OH

N

HN

O

OH

Oχ

χ: Syn

P = 272o (W-type)

χ

χ :Anti, P = 210o(43T)

OHχ :Anti

χ

χ :Anti, N-type

χ

R

O

OPO

O

OH

ONH

N

O

O

OH

(B) Base to phosphate locked

N

NN

N

NH2

O

OH

OPO

O

OH

χ

χ :Anti, N-type

O

OPO

O

OH

ONH

N

O

O

5'dT

(C) Phosphate to sugar locked

O

OP

O

O

T

OO

O

U

POO

HO

O

OH

O

OP

O

O

T

α(g+)

β(t)

O

OH

OOP

O

O

Tαβ

OOH

γ

δ(cis), ε(t), ζ(g+/g-)

OC

PO

OPhO

O

O

S

BP

O

OO

O

O

O

U

POO

HO

OO

δ(a+)ε(g−)ζ(g−/a+)

OHO

OP

O

OO

T

εζ

O

O

T

PO

O

HO

O α(g+/g−), β(g−/c), γ(g−)

S-type

OT

PO O

HO

OO

N-type

S-type

δ

OO T

O O

OO

T

O

O

(E) 2', 3' -Locked

OHO U

OHO U

X X

OHO T

OO

N

OHO T

OHO B

OHO B

S-typeN-typeχ :Anti,P = 228o(4E)

Planar sugar E-type χ :Anti,P = 17o(N-type)

OHO U

O

N-type

OHO B

N-type

α,β-CNA

( )n

OB

O

OB

O

(D) Methanocarba Nucleosides

R = H or OHN-type S-type

HO B

N-type

HO

OH

T

R R

R = H or OH

χ

O

X

O B

O

(F) 2', 4'-Locked

X = O, NH, S, CH2

O

O

O B

OR

OO T

O

R1R2 R3

R4

R1, R2, R3, R4 = H/ Me/OH/NH2

OO B

O O

OO B

O OO

OO T

OR3

OO T

O X

R1R2

X = O, NH, CH2

OO B

O N OR

R1, R2, R3 = H/ OH /Me,

O B

O

O

O

O BX

OO

O

O

O B

O

N-type

N-type

P = 17o(N-type)P = 17o(N-type)

N-type

N-type

S-typeS-type

X = O, NH N-type

N-type N-type

OO T

O

OHO

TO

OOH

O

OOH T

OHOHOHHO

OH

(H) 3', 4' -Locked

OO B

O O

OHO B

X

OHO T

O

OHO T

O

OHO B

OO T

O

OO TO

O

O

W-type

OHO T

O

E-type S-type

OO T

O O

ORS-type

P = 174o(S-type) P = 207o(S-type) N-type

O T

O

OO

(G) 2', 5'-Locked

O U

O

NOO T

O

OO

S -typeS -typeS -type

O

HO A

OHN-type

O

O

O T

OR

N-type

O

OB

O

O

OB

O

OT

N

O

OT

O

N

Bc-DNA Tc-DNA

(I) 3', 5'-Locked

OT

O

OO

OR

O

OHB

H OHO

OO U

R

P = 128oP = 18-28o(N-type)

2E or 2E

N-type P = 115o(E-type)

N -type

OT

OH

OH

OU

OH

O

H

AcHNAcO

P = 174o (S-type)

OO U

OO

OO U

N

OO B

O X

(J) 1', 2'/3'/4' -Locked

OO

O

OO

B

E-type χ: anti, S-typeS-type (North-East)

χ

O

OH OH

OHB

(K) 4', 5'-Locked

OXU

OH

OHX

OH

OHB

OU

OH

OH

SO

OH

BF F

OH

O

O O

NH

N

O

O

N

MeH

H

χ

(N) Double Locked

ON

N

O

O

H/Me

χ

χ: -116 (anti)S-type

O

OH

O

O

T O

O

O TO

HOHO

(L) tricyclic locked on sugar

χ: -151 (anti)P =182o (S-type)P =90

o (E-type)

O U

O

O

(M) Locked Hexose nucleoside

O TO

OH

HOχ O

O

O

OU

Ref. 164 Ref. 165 Ref. 166 Ref. 171

Ref. 167, 251 Ref. 168 Ref. 169

Ref. 170 Ref. 252 Ref. 253 Ref. 254

Ref. 258 Ref. 259Ref. 260 Ref. 261 Ref. 262

Ref. 263

Ref. 264

Ref. 264Ref. 265 Ref. 266

Ref. 247

Ref. 175, 245 Ref. 176, 246

Ref. 249

Ref. 255Ref. 256

Ref. 257

Ref. 173, 186 Ref. 185 Ref. 187 Ref. 188Ref. 189

Ref. 190 Ref. 191, 192 Ref. 192 Ref. 193 Ref. 194

Ref. 137-139, 195, 196, 210

Ref. 197, 198 Ref. 197 Ref. 197

Ref. 199 Ref. 200Ref. 201 Ref. 202-204

Ref. 205

Ref. 206 Ref. 207, 208

Ref. 209, 210Ref. 209

Ref. 212 Ref. 213 Ref. 214

Ref. 215 Ref. 216

Ref. 217, 218 Ref. 219Ref. 220 Ref. 221 Ref. 222

Ref. 223 Ref. 224 Ref. 225 Ref. 226 Ref. 227

Ref. 228 Ref. 229Ref. 174 Ref. 230 Ref. 212

Ref. 231 Ref. 232Ref. 233

Ref. 234, 235

Ref. 179 Ref. 180-182 Ref. 184 Ref. 183

Ref. 236, 237 Ref. 238-240 Ref. 241 Ref. 242 Ref. 243

Ref. 245 Ref. 244

Ref. 217

Ref. 267

Ref. 243

Ref. 269Ref. 268

25

LNA was synthesized by by Imanishi in 1997139 and Wengel in 1998137 in-dependently, and showed excellent antisense properties. The development of LNA and discovery of RNAi in 19985 advanced studies in this field to a new stage. In the past twenty years, many sugar locked (including 1�, 2�-locked,179-182 1�, 3�-locked,183 1�, 4�-locked,184 2�, 3�-locked,173,185-194 2�, 4�-locked,139,137,195-211 2�, 5�-locked,212-214 3�, 4�-locked,215-227 3�, 5�-locked,174,228-230,212,231-235 4�, 5�-locked,236-243 locked hexose nucleoside,244,245 methanocarba nucleosides176,177,246-250,178), nucleobase to sugar locked,251-254 nucleobase to phosphate locked,255-257 phosphate to sugar locked258-266 and several dou-ble217,267 and tricyclic268,269,243 locked nucleos(t)ides have been synthesized and some of them have been found to have good antisense and/or RNAi properties. The chemical structures of representative locked nucleosides and nucleotides are shown in Figure 8.

2.1.1 LNA and its application in therapeutics and biotechnology The most important member of conformationally constrained nucleoside family is LNA. In LNA, the 2�, 4�-locked ring results in typical 3�-endo sugar puckering.139 AONs conataining LNA modifications are preorganized in the A-type canonical structure and exhibit unprecedented affinity toward com-plementary DNA and RNA compared with all of the previously reported molecules.141 LNA has a higher affinity for RNA than for DNA; therefore it is RNA selective and has attracted broad applications in RNA-directed therapeutics and biotechnology.270,271

LNA modified AONs have been reported as a reliable and potent ap-proach for inhibition of gene expression.272 in vivo studies showed that LNA modified AONs were more potent but less toxic than phosphorothioate modified counterpart.273 Gapmer design is required for RNase H activa-tion.274 In a non-cellular experiment, LNA-DNA-LNA gapmers has been proved to be more potent activator of RNase H-mediated RNA degradation than the corresponding 2�-O-methyl-RNA-contaning gapmers.275 LNA modi-fied AONs have been shown to target a broad range of mRNAs in different cells276-279 as well as viral RNAs.280

LNA modified AONs have also been used for targeting non-coding RNA. Telomerase is a cancer-specific ribonucleoprotein that is critical for tumor cell prolification. Elayadi et al. reported that a short full LNA modified oli-gonucleotide can inhibit human telomerase activity with very low IC50 value.281 Very recently, applications of LNA modified AONs in miRNA inhibition have been reported.282 Lund and colleagues demonstrated that LNA-modified oligonucleotide mediates the specific inhibition of both ex-ogenous and endogenous miRNA in a dose-dependent manner in Drosophila melanogaster cells.283 In addition, LNA-modified oligonucleotides effi-ciently inhibited miRNA-122 in African green monkeys, resulting in a long-lasting decrease in total plasma cholesterol with no evidence of LNA-

26

associated toxicities.284 LNAs have been reported as a powerful tool for miRNA detection; for example, an LNA-modified oligonucleotide probe has been demonstrated to be > 10-fold more sensitive than its natural counterpart in Northern blot analyses of miRNA, without compromising probe specific-ity.285 The in situ detection of miRNA in animal embryos using an LNA-modified oligonucleotide probe has also been reported.286

LNA can also be used in the DNAzyme-mediated gene-silencing ap-proach. Incoporation of LNA into the two arms of 10-23 DNAzyme pro-duced an LNAzyme which confered marked resistance to nucleolytic degra-dation and at the same time, increased cleavage activity.287 Kjems et al. de-signed one LNAzyme for inhibition of HIV-1 and it was found to exhibit a modest effect compared to DNAzyme which didn’t show any effect.280 An-tagomirzymes are miRNA-silencing DNAzymes. LNA modifications in the variable flanking regions of antagomirzymes resulted in considerably en-hanced silencing efficiency compared with their native counterparts.288

LNA-modified siRNA has been demonstrated to have promising thera-peutic value. Wahlestedt and colleagues have designed an LNA-modified siRNA that targets Severe Acute Respiratory Syndrome (SARS).289 The LNA-modified siRNAs exhibited improved target knockdown efficiency compared with the unmodified counterpart. Additionally, LNAs could re-duce off-target effects if the modification site was chosen appropriately, and this conclusion has been verified by an in vivo study.290 Segmented interfer-ing siRNA (sisiRNA) is a novel siRNA design that completely eliminates off-target effects caused by the sense strand.291 However unmodified sisiRNA has been found to be less efficient as a result of serum instability.291 LNA modification could increase the stability of sisiRNA in serum; no cyto-toxic side effects or growth inhibitions have been observed in cells treated with LNA-modified sisiRNA compared with the unmodified counterpart, which suggested that LNA modifications were essential for sisiRNA de-sign.291 Another significant feature of sisiRNA was that this form of RNA allowed heavier LNA modification in the antisense strand, and hence strengthen stabilization of the duplex.291

Though LNA modified AONs and siRNAs exhibit higher nucleolytical stability than native counterpart, they do not have nuclease resistance as good as that of the phosphorothioate modified counterpart.292 Further im-provement on its nucleolytical stability but without any compromise on its affinity and selectivity toward complementary DNA and RNA will undoubt-edly endow LNA much more therapeutic applications in the future. Re-cently, our group have synthesized carbocyclic-LNA (carba-LNA) through free radical cyclization reaction and found carba-LNA modified AONs had much better nuclease resistance than LNA modified conunterparts.209 On the other hand, carba-LNA modified AONs have been found to exhibit very similar affinity toward target RNA as LNA and can initiate RNase H-mediated RNA degradation. The striking properties of carba-LNA inspired

27

us to further introduction of functional groups to the C6� and C7� of carba-LNA, with the aim to develop perspective candidates for oligonucleotide-based therapeutic applications.

2.2 The synthesis and structure of carba-LNA, carba-ENA, BHNA and D2-CNA nucleos(t)ides (Papers I-III & V)

2.2.1 Synthesis of carba-LNA, carba-ENA and BHNA nucleosides The synthesis carba-LNA nucleosides (Scheme 1) has been started from D-glucose, which was first converted to known precursor 1 in 8 steps.293,202

Scheme 1. Synthesis of C6�-OH carba-LNA derivatives through radical cyclization strategy.

D-GlucoseOBnO

OBnHO

OO

OBnO

OO

HO

1 3

4 5 6 7

OBn

OBnO

OAc

AcO

OBn

OAc OBnO

OAc

AcO

OBn

OBnO

OH

HO

OBn

OBnO

O

HO

OBn

OBnO

OBnO

OO

2

6'S

8 steps oxidation MgBr

Grignard reaction

T T TPTC-Cl

OPh

S

Vorbruggenreaction

radical cyclization O

BnO

OBnCH3

HO

TOBnO

OBn

HO

CH3

TOBnO

OBn

HO

OBnO

OBn CH3O

TOBnO

OBnO

7' S7' R 6'S 7' 8'6'S 6'S

OBnO

HO

OBn

77%

2'6' 7'

8'

T

84% in two step

91% in two step

100% 92%

T

8 9 10 11

oxidation

T

14 15

Acetylation

OBnO

OBnCH37' S

T

oxidation

O

12

OBnO

OBnCH3HO 7' S

6'R

T

13 (54% puls 40% substrate 9)

reduction

OBnO

OBn CH3HO

T

6'R7' R

reduction

100%

90%

16

7' R

62%

9: 10: 11 = 80: 11:9

28

Compound 1 was oxidized, through Swern oxidation to the corresponding aldehyde 2. The crude aldehyde was subjected to Grignard reaction with vinylmagnesium bromide to give the C6-hydroxyl olefin 3 in high yield (84% in two steps). Acetylation of 3 gave the corresponding triacetate 4 as an anomeric mixture, which was subjected to a modified Vorbruggen reac-tion to give exclusively �-D-ribofuranosyl thymine derivative 5. After deace-tylation of 5, the obtained compound 6 was esterified with phenyl chlorothioformate with high selectivity and high yield to give the key pre-cursor 7 for the free-radical cyclization. The free radical cyclization was carried out in toluene in presence of n-Bu3SnH and a catalytic amount of AIBN by reflux, putatively through a radical intermediate 8, to give three products 9, 10 and 11. Product 9 was separated to pure form by short column chromatography, but products 10 and 11 were obtained as an inseparable mixture. The relative ratio of products 9:10:11 was 80:11:9 (Paper I & II).

Figure 9. Transition states for 5-exo and 6-endo radical cyclization.

Product 9 and 10 were obtained through 5-exo cyclization pathway, whereas products 11 resulted from 6-endo cyclization pathway. This phe-nomenon was contrary to radical cyclization of precursor 17, which yielded 100% 5-exo cyclization product (Figure 9A).209 The only difference between radical intermediates 8 and 18 is that in 8, one additional OH at C6� is at-tached. The C6�-OH group affects the outcome of the regioselectivity of free radical cyclization. It suggested the C6�-OH can stabilize inductively the developing negative charge on the olefine moiety in the 6-endo cyclization transition state or intermediate (Figure 9B), and thus lowering the activation energy of 6-endo cyclization. As the result, 6-endo cyclization product 11 is formed (Paper II).

In carba-LNA nucleoside derivative 9, the C6�-OH has the S configuration. Inversion of the C6�-OH configuration was achieved by oxidation to ketone 12 followed by reduction. As the result, C6�R-OH nucleoside 13 was ob-

Oδ

δ

TBnO

BnO

HOO

δδ

TBnO

BnO

HO

6-endo cyclizationtransition state

5-exo cyclizationtransition state

2' 2'Products 9 and 10

Product 11

(B)

91% 9%

(A)

Oδ

δ

TBnO

BnO

2'

TOBnO

OBn

18

21 22

TOBnO

OBn Me

20

100%

TOBnO

OBn

17

O OPh

S 195-exo cyclization

6'

29

tained in 54% yield accompanied by recovery of 40% of substrate 9. Simi-larly, oxidation of mixture of 10 and 11 gave ketones 14 and 15, which for-tunately could be separated in pure form by short column chromatography. Reduction of ketone 14 resulted in exclusive C6�R-OH nucleoside 16 (Paper I).

Scheme 2. Synthesis of carba-ENA nucleoside.

The 6-endo cyclization pathway provided an alternative strategy for syn-thesis of carba-ENA.204 Thus, compound 15 was reduced with NaBH4, re-sulting in exclusive C6�R-OH nucleoside 23. Radical deoxygenation of the C6�R-OH gave compound 25, which was debenzylated, giving carba-ENA nucleoside 26 (Scheme 2, Paper II).

Scheme 3. Synthesis of C6�-Me carba-LNA derivatives and BHNA.

Synthesis of C6�-Me carba-LNA derivatives have been achieved accord-ing to the strategy shown in Scheme 3. The ketone 12 was treated with methyl magnesium iodide in anhydrous THF to give 6�-Me-6�-OH bifunc-tional carba-LNA nucleosides 27a (18%) and 27b (43%). To obtain the C6�-Me carba-LNA nucleoside, the major product 27b was further subjected to radical deoxygenation reaction. Thus, compound 27b was first transformed to radical precursor 28, which was subsequently subjected to standard radical deoxygenation condition, giving C6�-Me carba-LNA nucleosides 29a (18%), 29b (15%) together with a new bicyclo[2.2.1]-2�,5�-methylene-bridged hexopyranosyl nucleoside (BHNA) 30 (27%) (Paper I & III).

27a (18%)

27b (43%)

OBnO

OBn

CH3HO

H3C

OBnO

OBn

CH3O

H3C

OBnO

OBn

CH3H3C

MeO

O

O

6'R

6'

94%

7'S

7'S

T

MeMgI

OBnO

OBnCH3HO

H3C6'S

T

12

T

T

radical deoxygenation

28

29a 6'S (18%) 29b 6'R (15%)

OH3C

CH3H

RO

RO

30 R = Bn (27%)31 R = H BHNA

6'R

T

debenzylation

TOBnO

OBnHO

TOBnO

OBnOPhO

S

TORO

OR

reduction

23 24 25 R = Bn26 R = H (carba-ENA)

82% 63% 72%6'R15

PTC-Clradical deoxygenation

30

The unusual product BHNA 30 was supposed to be obtained through a radical rearrangement process (Figure 10A). Treatment of radical precursor 28 with Bu3SnH in presence of a catalytic amount of AIBN in anhydrous toluene resulted in the putative radical intermediate 32, which could either be reduced directly by H• to give C6� reduced product (Path “A” in Figure 10A), or/and it could induce scission of C4�-O4� bond to give C4� radical, which upon quenching by H• could give BHNA (Path “B” in Figure 10A). On the contrary, radical intermediate 34 only lead to C6� radical reduced product 35 (Figure 10B), which suggested that a stable tertiary radical is a prerequisite to ensure the rearrangement to take place. Intermediate 36 also has one stable tertiary C6� radical, but it did not produce any trace of rear-rangement product (Figure 10C). Ab initio calculations showed that in inter-mediate 32, the single electron occupied p-orbital of C6� is nearly coplanar (φ = 5º) with the �*O4�-C4� orbital. This coplanarity favors the rupture of C4�-O4� bond and formation of new C6�-O4�. Whereas in intermediate 36, there is a dihedral angle of 51º between the single electron occupied p-orbital of C6� with the �*O4�-C4� orbital, thus radical rearrangement was precluded (Pa-per III).

Figure 10. The putative radical intermediates and plausible radical rearrangement to BHNA.

OBnO

OBn

CH3

Me

OMeOO

O

OBnO

OBnCH3

Me

OMe

CH3H

T

BnO

BnO

28

29a 6'S (18%)29b 6'R (15%)

BHNA 30 (27%)

OBnO

OBn

CH3MeH

O

OBn

CH3Me

H

32

33

6'

4'

4'

6'

Path"A"

T

T

T

T

OBn

Path"B"

6'

OBnO

OBn

CH36'

OBnO

OBn CH36'

34 35

Path"A"

H

T T OBnO

OBnH3CCH3

6'

37a: 6'R (75%)37b: 6'S (16%)

OBnO

OBnCH3

6'

36

H3C

Path "A"

H

T T

(A)

(B) (C)

31

2.2.2 Synthesis of D2-CNA nucleotide Although so many sugar puckering constrained nucleos(t)ides and phsophate backbone constrained nucleotides have been reported, a hyper-constrained system constructed by fusing a locked sugar moiety with a cyclic phosphate modification in the same nucleotide has not been studied before our investi-gation (paper V). Synthesis of such a nucleotide and studies of its properties will shed light on how a hyper-constrained modification can modulate the therapeutic performance of nucleic acids.

Scheme 4. Synthesis of D2-CNA nucleotides.

The carba-LNA nucleoside 38 is very good substrate for constructing a double sugar and phosphate backbone-constrained nucleotide because firstly, the sugar is constrained in N-type by the 2�, 4� bridge; secondly, the cis fused 3�, 6� vicinal diol system serves as a very good skeleton for constructing a six-membered cyclic 1,3,2-dioxaphosphorinane ring. Thus, compound 38 was coupled with phosphorodiamidite 39, followed by oxidation with I2/THF-H2O-Pyridine to give two pure P-diastereomers, protected Sp-D2-CNA dimer 40 (39%) and Rp-D2-CNA dimer 41 (16%). Then pure 40 was treated with 1M TBAF/THF to deprotect the 3�-O-tBDMS group, but unex-pectedly, two P-diastereomers 42 and 43 were obtained. Similarly, treatment of pure 41 with 1M TBAF/THF also furnished two products 42 and 43. This unexpected observation suggests that fluoride anion can mediated isomeriza-tion between Sp-D2-CNA 42 and Rp-D2-CNA 43. This isomerization process has been studied in NMR tube and we found in THF-d8 solution containing fluoride anion at 25 �C, equilibrium between Sp-D2-CNA 42 and Rp-D2-CNA 43 can be reached in 2 hours, giving equilibrium constant K298k = 1.92, which

ODMTrO

OHHO

ODMTrO

OCH3O

POO

PNO

O

OtBDMS

T

N

O

OtBDMS

T

T

CH3

ODMTrO

OCH3O

POO

O

OtBDMS

T

T

40 (39%)

41 (16%)

6'R

Sp

Rp

ORO

OCH3O

POO

O

OH

T

T

T

38

39

TBAF/THF

RpTBAF/THF

42 R = DMTr44 R = H Sp-D2-CNA

ORO

OCH3O

POO

O

OtBDMS

T

T

Sp

43 R = DMTr45 R = H Rp-D2-CNA

32

corresponds to G298k = −0.39 Kcal/mol. Thus, Sp-D2-CNA 42 is more ther-mally stable than Rp-D2-CNA 43. Finally, the 5�-O-(4�,4�-Dimethoxytrityl, DMTr) groups in 42 and 43 were removed by treatment with 2% TFA in dichlormethane to give unprotected Sp-D2-CNA 44 and Rp-D2-CNA 45 quan-titatively (Paper V).

2.2.3 Structure of carba-LNA, carba-ENA, BHNA and D2-CNA nucleos(t)ides All the carba-LNA nucleosides 9, 10, 13, 16, 27a/b, 29a/b, carba-ENA nu-cleoside 26, BHNA 31 and D2-CNA 44 and 45 have been characterized by 1H, 13C, DEPT, COSY, 1H-13C HMQC, HMBC NMR experiments using 500 and 600 MHz NMR as well as mass spectroscopy. D2-CNA 44 and 45 have also been characterized by 31P and 1H[31P] experiments. The stereochemistry of the chiral centers was assigned by 1D-nOe, 2D-NOESY experiments.

All the vicinal coupling constants 3JH,H, 3JH,P, 3JC,P in these nucleos(t)ides have been determined by homodecoupling or heterodecoupling experiments. On the other hand, ab initio optimized molecular structures of these nu-cleos(t)ides were obtained utilizing HF/6-31G* and/or B3LYP/6-31++G* geometry optimization by GAUSSIAN 98.294 Subsequently, theoretical vici-nal coupling constants 3JH,H have been back-calculated using Haasnoot-de Leeuw-Altona generalized Karplus equation33,295 from the corresponding torsional angles of the optimized molecular structures. Theoretical vicinal coupling constants 3JH,P, 3JC,P were calculated based on modified Karplus equation.296 The experimental vicinal coupling constants were found to be generally well reproduced by this theoretical approach. The convergence between experimentral and theoretical values was a good indicator of the rigidity of these conformationally constrained nucleos(t)ides.

For carba-LNA nucleosides 9, 10, 13, 16, 27a/b, 29a/b, their sugars are, just like LNA, constrained in N-type. But the chemical nature and the orien-tation of the substituents has also been found to influence puckering of the furanose sugar ring, resulting in pseudorotational angles ranging from 16° (compound 10) to 22° (compound 27b, Table 1, Paper I).

Table 1. Phase angle (P) and the puckering amplitude (φm) of carba-LNA nucleoside derivatives.

Nucleosides 9 10 13 16 27a 27b 29a 29b P 18.48 16.57 18.45 16.87 20.35 21.97 18.79 19.51 φm 55.48 55.72 56.30 56.46 56.88 56.77 55.95 56.62

The optimized molecular structure of carba-ENA is shown in Figure 11A.

The six-membered carbocyclic moiety adopts a perfect chair conformation. The furanose ring of carba-ENA is locked in the N-type conformation char-acterized by pseudorotational phase angle P = 19.6� and the puckering am-

33

plitude φm = 45.9�. The sugar pucker parameters are very similar to that of ENA and aza-ENA (Paper I).297

Figure 11. ab initio optimized molecular structures of constrained nucleos(t)ides carba-ENA (A), BHNA (B), Sp-D2-CNA (C) and Rp-D2-CNA (D).

The optimized molecular structure of BHNA is shown in Figure 11B. The additional methylene bridge between C2� and C6� in BHNA makes the six-membered ring completely rigid and it adopts a twisted conformation. The base moiety occupies the axial position while 3�-hydroxymethyl and 4�-hydroxyl take up the equatorial positions. These structural features make BHNA very distinct from all the other hexopyranosyl nucleosides (Paper III).

The furanose rings in both Sp and Rp-D2-CNA are locked in tightly con-strained North conformation with phase angle P = 11º and puckering ampli-tude φm = 54º. The six-membered 1,3,2-dioxaphosphorinane in D2-CNA adopts half chair conformation (Figure 11C&D). The hyper-constrained D2-CNA system does induce a change in the sugar conformation of the neighboring 3�-end nucleoside: N-type puckered nucleosides at the 3�-end are roughly 61% in Sp-D2-CNA and 56% in Rp-D2-CNA, which are quite differ-ent from the equilibrium observed in case of the native deoxynucleotides (30- 40% North)34 (Paper V).

34

2.3 The antisense properties of carba-LNA, BHNA and D2-CNA modified oligonucleotides (Papers I, IV & V)

2.3.1 Synthesis of modified AONs and their chemical stabilities Carba-LNA nucleosides 9, 10, 13, 16, 27a/b, 29a/b, BHNA 31 and Sp-D2-CNA 44 have been converted to their phosphoramidites 46–55 (Figure 12). 52 and 53 were inseparable and correspondingly used as a mixture (Paper I, IV & V).

Figure 12. Phosphoramidites of carba-LNA, BHNA, Sp-D2-CNA nucleosides.

Using solid-supported DNA synthesis strategy, these phosphoramidites have been incorporated into AONs. The sequences of AONs are shown in Table 2. The carba-LNA nucleosides as well as BHNA nucleoside contain carbocylic rings and thus are supposed to be chemically stable. Indeed, AONs containing these modifications can be synthesized and deprotected using standard procedures without observation of any decomposition. In AONs 18–21 and AONs 45–48, the vicinal C6�-OH or C3�-OH were sup-posed to intramolecularly attack the vicinal phosphate, leading to strand scission under alkali conditions. However we have found these AONs com-pletely stable in saturated aqueous ammonia. Hence AONs 1–40 were depro-tected with saturated ammonia followed by purification with PAGE to give the pure products (Paper I & IV).

The cyclic phosphate ring in D2-CNA has been found to be very unstable under alkali conditions. Incubation of Sp-D2-CNA 44 and Rp-D2-CNA 45 with aqueous 1mM NaOH solution at 21 �C resulte in very fast degradation, k = 0.42 h-1 and k = 0.49 h-1 for 44 and 45 respectively (Paper V). Under the

ODMTrO

OCH3O

POO

O

O

T

T

Sp

PCeON

ODMTrO

O

O

Me

CH3H

T

O

DMTrO

54

T

R3R4

R2

R1

PCeON

PCeO

N

46 R1=Me, R2=H, R3=OTol, R4=H47 R1=H, R2=Me, R3=OTol, R4=H48 R1=Me, R2=H, R3=H, R4=OTol49 R1=H, R2=Me, R3=H, R4=OTol50 R1=Me, R2=H, R3=OH, R4=Me51 R1=Me, R2=H, R3=Me, R4=OH52 R1=Me, R2=H, R3=Me, R4=H53 R1=Me, R2=H, R3=H, R4=Me

carba-LNA derivativesBHNA

Sp-D2-CNA55

35

Table 2. Sequences of modified AONs and Tm values of their duplexes with com-plementary RNA and DNA targets.a

aTm values measured as the maximum of the first derivative of the melting curve (A260nm vs temperature) in medium salt buffer (60 mM Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2) with temperature range 20 to 70 °C using 1M concentrations of the two complementary strands. The value of Tm given is the average of two or three independent measurements.

With RNA With DNA Entry

Modified Carbocyclic structures

Sequence Tm Tmc Tm Tmc

AON 1 Native AON 3’-d(CTT CTT TTT TAC TTC) 44.5 45.5 AON 2 3’-d(CTT CTT TTT TAC TTC) 48.5 +4.0 47.5 +2.0 AON 3 3’-d(CTT CTT TTT TAC TTC) 49.5 +5.0 46.0 +1.5 AON 4 3’-d(CTT CTT TTT TAC TTC) 49.3 +4.8 45.8 +0.3 AON 5

O

O

O T

OLNA

3’-d(CTT CTT TTT TAC TTC) 48.6 +4.1 46.6 +1.1 AON 6 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.5 46.0 0.7 AON 7 3’-d(CTT CTT TTT TAC TTC) 48.5 +3.6 44.5 -0.8 AON 8 3’-d(CTT CTT TTT TAC TTC) 48.5 +3.8 44.0 -1.6 AON 9

O

O T

OCH3

I

3’-d(CTT CTT TTT TAC TTC) 48.0 +3.3 43.0 -2.3 AON 10 3’-d(CTT CTT TTT TAC TTC) 47.5 +3.0 47.5 +2.1 AON 11 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.1 48.0 +2.3 AON 12 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.2 47.0 +1.4 AON 13

O

OCH3

TO

HO

7'S6'S

II 3’-d(CTT CTT TTT TAC TTC) 48.5 +3.9 44.5 -0.9 AON 14 3’-d(CTT CTT TTT TAC TTC) 46.5 +1.7 45.0 -0.3 AON 15 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.1 44.0 -1.7 AON 16 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.4 43.0 -2.5 AON 17

O

O

TO

CH3

HO6'S 7'RIII

3’-d(CTT CTT TTT TAC TTC) 48.0 +3.1 42.5 -2.9 AON 18 3’-d(CTT CTT TTT TAC TTC) 47.5 +2.9 47.0 +1.6 AON 19 3’-d(CTT CTT TTT TAC TTC) 49.0 +0.5 46.0 +0.5 AON 20 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.2 45.5 -0.1 AON 21

O

OCH3

TO

HO 7'S6'R

IV

3’-d(CTT CTT TTT TAC TTC) 48.5 +3.6 44.0 -0.7 AON 22 3’-d(CTT CTT TTT TAC TTC) 46.0 +1.5 44.5 -0.8 AON 23 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.3 43.0 -2.4 AON 24 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.3 42.5 -2.8 AON 25

O

O

TO

CH3HO 7'R

6'R

V

3’-d(CTT CTT TTT TAC TTC) 47.0 +2.3 42.0 -3.4 AON 26 3’-d(CTT CTT TTT TAC TTC) 47.0 +2.3 46.0 0.5 AON 27 3’-d(CTT CTT TTT TAC TTC) 48.0 +3.2 44.0 -1.4 AON 28 3’-d(CTT CTT TTT TAC TTC) 48.0 +3.3 43.5 -1.9 AON 29

O

OCH3

TO

H3C7'S80% S6'

VI

3’-d(CTT CTT TTT TAC TTC) 48.0 +3.1 42.5 -3.1 AON 30 3’-d(CTT CTT TTT TAC TTC) 48.5 +3.6 47.5 +2.0 AON 31 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.5 46.5 +0.8 AON 32 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.5 45.5 0 AON 33

O

OCH3

TO

HO

H3C 7'S6'S

VII

3’-d(CTT CTT TTT TAC TTC) 49.0 +4.4 45.0 +0.7 AON 34 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.3 45.5 +0.2 AON 35 3’-d(CTT CTT TTT TAC TTC) 49.0 +4.5 44.5 -0.8 AON 36

O

OCH3

TO

H3C

HO 7'S6'R

VIII

3’-d(CTT CTT TTT TAC TTC) 49.0 +4.2 44.0 -1.7 AON 37 3’-d(CTT CTT TTT TAC TTC) 39.2 -5.5 39.7 -5.8 AON 38 3’-d(CTT CTT TTT TAC TTC) 40.6 -4.1 35.2 -10.3AON 39 3’-d(CTT CTT TTT TAC TTC) 40.4 -4.3 34.0 -11.5AON 40

O

Me

CH3H

T

O

O

BHNA 3’-d(CTT CTT TTT TAC TTC) 40.4 -4.3 35.0 -10.5

AON 41 3’-d(CTT CTT TTT TAC TTC) 39.9 -4.5 42.7 -2.1 AON 42 3’-d(CTT CTT TTT TAC TTC) 37.7 -6.7 37.7 -7.1 AON 43 3’-d(CTT CTT TTT TAC TTC) 37.0 -7.4 35.8 -9.7 AON 44

O

O

O

PO

O

O

T

MeSP

5'

Sp-D2-CNA 3’-d(CTT CTT TTT TAC TTC) 37.6 -6.8 37.6 -7.2

AON 45 3’-d(CTT CTT TTT TAC TTC) 38.6 -5.8 39.0 -5.8 AON 46 3’-d(CTT CTT TTT TAC TTC) 33.9 -10.5 27.7 -17.1AON 47 3’-d(CTT CTT TTT TAC TTC) 33.6 -10.8 25.3 -19.5AON 48

6'O

O

OHP

O

O

O

T

MeO

carba-LNA-6', 5'-

5'

3’-d(CTT CTT TTT TAC TTC) 32.4 -12.0 26.5 -18.3

36

same condition, methyl-triester dimer Tp(OMe)T (56, Figure 13) has been found to be nearly 200 times more stable, while DNA dimer TpT is com-pletely stable. This result suggested that the conformationally constrained phosphotriesters in 44 and 45 were much more unstable toward nucleophile than conformationally unconstrained phosphotiresters. Digestion of com-pound 44 under this condition gives products 57:58:59 (Figure 13) in the ratio of 74:8:18 while digestion of compound 45 gives products 57:58:59 = 86:10:4. Hence, upon attack by hydroxide on phosphorus in 44 and 45, scis-sion of P-3�O bond is always the major reaction pathway to give the major product, carba-LNA-T-6�p5�-T (57). Product 58 which was formed by scis-sion of bond P-6�O bond has also been observed but it was only in about 10% yield. In concentrated aqueous ammonia at room temperature, hydroly-sis of D2-CNAs has been found very fast; the t1/2 were 12 min and 6 min for Sp-D2-CNA 44 and Rp-D2-CNA 45 respectively (Paper V).

Figure 13. Chemical structure of compounds 56-59.

But Sp-D2-CNA 44 and Rp-D2-CNA 45 have been found to be very stable toward weak nucleophile under a neutral condition such as 0.5M hydrazine hydrate in pyridine/AcOH (4/1, v/v, pH = 5.6). Under this condition at room temperature, Sp-D2-CNA 44 (t1/2 = 178 h) was more stable than Rp-D2-CNA 45 (t1/2 = 99 h). D2-CNA has also been found to be stable toward relatively strong non-nucleophilic organic base such as DBU. In 0.5M DBU in dry MeCN at 21 �C, degradation of Sp-D2-CNA 44 (t1/2 = 154 h) and Rp-D2-CNA 45 (t1/2 = 330 h) were very slow. The satisfing stability of D2-CNA under these conditions allowed us to deprotect Sp-D2-CNA modified AONs 41–44 using these conditions: (1) the solid supports of AONs 41–44 were first treated with Et3N/MeCN (3/2, v/v) for 2h to remove the cyanoethyl group on the phosphate linkage; (2) then treated with 0.5M DBU in dry MeCN for 24 h to cleave the oligos from the solid support; and (3) finally treated with 0.5M hydrazine hydrate in Pyridine/AcOH (4/1, v/v) for 24 h at r.t to remove the protecting groups on the nucleobases to give the crude oligonucleotides, which were purified by RP-HPLC to give pure products. Alternatively, treatment of solid supports of AONs 41–44 with aqueous ammonia at room

OHO

OHCH3O

PO

OO

OH

T

T

O

OHO

OCH3HO

PO

OO

OH

T

T

O

OHO

OHCH3O

PO

T

O

OHO

O

T

OHO

O

PO

OO

OH

T

T

OMe

Tp(OMe)T 56

carba-LNA-Tp T58

carba-LNA-T6' p5'T 57

carba-LNA-Tp6'p 3'T59

37

temperature for 4h resusted in crude products of carba-LNA-6�p5�-modified AONs 45–48, which have been found to be contaminated with roughly 20% of carba-LNA-modified AONs 18–21. AON 46 can be separated from AON 19 by RP-HPLC but separation of AON 45 from AON 18, AON 47 from AON 20, AON 48 from AON 21 failed and so the mixtures were used for thermal denaturing studies (Paper V). The integrity of structures of all the AONs has been verified by the MALDI-TOF mass measurements.

2.3.2 Affinity and specificity of modified AONs toward complementary RNA The Tm values of duplexes formed by LNA and carba-LNA derivatives modified AONs with complementary RNA and DNA targets are shown in Table 2. All the carba-LNA derivatives modified AONs have been found to exhibit less affinity toward RNA target than LNA modified counterparts (Paper I). Comparison of Tms of AONs containing different carba-LNA de-rivatives clearly suggested that the nature of the modification (-OH versus -CH3) and their respective stereochemical orientations at C6� and C7� in the carbocylic moiety of carba-LNA do modulate the AONs affinity toward target RNA to some extent: (1) The [6�S-OH, 7�R-Me]-carba-LNA modified (Type III) AONs 14–17 have significantly decreased Tms (-1.5�C to -2�C) compared to [6�S-OH, 7�S-Me]-carba-LNA modified (Type II) AONs 10-13, which suggests that when the 7�-methyl group is pointed toward the vicinal 3�-phosphate, it destabilizes the AON/RNA duplex greatly. This result can be further corroborated by the finding that [6�R-OH, 7�R-Me]-carba-LNA modified (Type V) AONs 22–25 also have -1.5�C to -2�C lower Tms com-pared to [6�R-OH, 7'S-Me]-carba-LNA modified (Type IV) AONs 18–21; (2) [6�R-OH, 7�S-Me]-carba-LNA modified (Type IV) AONs 18–21 have simi-lar Tms as [6�S-OH, 7�S-Me]-carba-LNA modified (Type II) AONs 10–13. [6�R-OH, 7�R-Me]-carba-LNA modified (Type V) AONs 22–25 have also similar Tms as [6�S-OH, 7�R-Me]-carba-LNA modified (Type III) AONs 14–17, thereby suggesting that the stereochemical orientation of C6'-OH in carba-LNA does not exert marked effect on the AON/RNA duplex thermal stability; (3) Type VII ([6�S-OH/Me, 7�S-Me]-carba-LNA) modification has one additional C6�-methyl group which points at the vicinal 3�-phosphate compared to Type II ([6�S-OH, 7�S-Me]-carba-LNA) modification, and as a result, the Type VII modified AONs 30–33 have a slightly higher 0.5�C) Tms, at least not less than those in Type II modified AONs 10–13. Similarly, [6�R-OH/Me, 7�S-Me]-carba-LNA (Type VIII) modified AONs 34–36 have similar or a slightly higher Tms than [6�R-OH, 7�S-Me]-carba-LNA (Type IV) modified AONs 18–21. This means that the orientation of the C6�-Me in either S or R configuration in carba-LNA does not impair the AON/RNA

38

duplex thermal stability, and in some cases, it can even have the stabilization effect (Paper I).

BHNA modified AONs 37–40 destabilized the AON/RNA duplex by -4.3 to –5.5ºC per modification. The specificity of BHNA modified AON toward RNA has also been studied. Thus AON 40 was used to target RNAs 2–4 (Table 3) which have one mismatch just opposite the BHNA modification. The Tm values of duplexes of AON 40/RNA 2, AON 40/RNA 3, AON 40/RNA 4 are –6.5, -10.6, -8.9ºC lower respectively than fully-matched du-plex AON 40/RNA 1, which is very similar to the native counterpart AON 1, in that AON 1/RNA 2, AON 1/RNA 3, AON 1/RNA 4 showed Tm decrease –2.9, -11.5, -9.2ºC respectively than fully-matched duplex AON 1/RNA 1. Hence, BHNA mdified AON has the same high specificity toward target RNA as the native AON. This observation also suggested that 1-thyminyl moiety of BHNA can base-pair well with the opposite complementary nu-cleobase moiety in the AON/RNA hybrid, and hence BHNA can only cause local conformational heterogeneity in the AON/RNA duplex. This conclu-sion has been further confirmed by CD measurement in that both AON 5/RNA 1 exhibited CD spectra mimicking the natural AON 1/RNA 1 hybrid duplex (Paper IV).

Table 3. Tm values of duplexes formed by BHNA modified AON with complemen-tary RNA or RNAs with one base mismatch.a

a Medium salt buffer is shown in footnote of Table 2; b The value in parentheses is Tm which is the difference between mismatched and matched duplexes.

The Sp-D2-CNA has been found to affect the thermal stability of AON/RNA duplex in sequence-dependent manner (Table 2). When located at the end of AON sequence as in AON 41, generally, it destabilizes the AON/RNA duplex by -4.5�C /modification compared to the native counter-part AON 1. Whereas when it is located in the middle of AON sequences, as in AONs 42–44, it exerts much more negative effect (ca. −7�C /modification) for the thermal stability of AON/RNA duplex. The carba-LNA-6�p5�-phosphodiester modified pure AON 46–48 even exert a higher thermal destabilization effect (-10 to -12 �C /modification) on the AON/RNA duplex (Table 2). Comparison of Tm values of AONs 46–48 with AONs 43–44 and AONs 19–21 clearly demonstrates how the topology of phosphate linkage modulates the thermal stability of AON/RNA duplex. CD spectra showed that base stacking in dimers carba-LNA-TpT, Sp-D2-CNA-TpT, carba-LNA-T-6'p 5'-T follow this rank: carba-LNA-TpT >> Sp-D2-CNA-

Complementary RNA : 5’-r(GAA GAA AAA XUG AAG)

X = A(RNA 1,paired) G(RNA 2) C(RNA 3) U(RNA 4)

AON 1 44.7 41.8 (-2.9)b 33.2 (-11.5) b 35.5 (-9.2) b

AON 40 40.4 33.9 (-6.5) b 29.8 (-10.6) b 31.5 (-8.9) b

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TpT > carba-LNA-T-6'p5'-T. Therefore, Sp-D2-CNA in AONs 41–44 and carba-LNA-6'p5'-phosphodiester modification in AONs 45–48 inflict a stacking geometry distortion in AON/RNA duplexes, making base pairing with the complementary nucleobases in the opposite strand impossible (Pa-per V).

2.3.3 Nucleolytical stabilities of modified AONs The metabolism of AONs in vivo involves degradation by exo- and endocu-cleases and the predominant nuclease activity is of 3�-exonuclease.159 Hence the AONs with a single modification at position T13 (position 3 from 3�-end) were 5�-end 32P-labeled and incubated with phosphodiesterase I from Crota-lus adamanteus venom (SVPDE) [SVPDE 6.7 ng/ l, AON 3M, 100 mM Tris-HCl (pH 8.0), 15 mM MgCl2, total volume 30 l) at 21�C.

For carba-LNA modified AONs, their stabilities (k, pseudo first order di-gestion rate by SVPDE) follow this rank (Paper I): [6�R/S-Me, 7�S-Me]-carba-LNA modified (Type VI) AON 26 (k = 0.0218 ± 0.0013 h-1) > [6�S-OH, 7�S-Me]-carba-LNA (Type II) modified AON 10 (k = 0.0532 ± 0.0044 h-1) >[6�S-OH, 7�R-Me]-carba-LNA (Type III) modified AON 14 (k = 0.0605 ± 0.0032 h-1) > AON 30 (k = 0.091 ± 0.005 h-1) > [7�-Me]-carba-LNA (Type I) modified AON 6 (k = 0.1525 ± 0.0097 h-1) >> [6�R-OH, 7�R-Me]-carba-LNA modified (Type V) AON 22 (k = 2.11 ± 0.26 h-1) > [6�R-OH, 7�S-Me]-carba-LNA modified (Type IV) AON 18 (k = 11.92 ± 1.41 h-1) > native AON 1. The relationship between substitutions and stabilities are summer-ized below: (i) C6�-Methyl substitution exert a unprecedented nucleotytical stabilization effect for vicinal phosphate linkage no matter its orientation; (ii) When C7�-methyl points at the vicinal 3�-phosphate (C7�R), had more posi-tive effect on the nucleotytical stability of the vicinal 3�-phosphate than when it points away (C7�S) from the phosphate; (iii) When C6�-OH pointed at the vicinal 3�-phosphate, it destabilizes the 3�-phosphate significantly; (iv) When the C6�-OH pointed away from the vicinal 3�-phosphate, it stabilizes both 5�-phosphate and 3�-phosphate. Hence the exonuclease resistance of carba-LNA derivatives is dependent not only on the lipophilic versus hydrophilic nature of the substituent, but also significantly on the stereochemical orientation vis-à-vis scissile phosphate (Paper I).

The stabilities of AONs with different carba-LNA modifications in hu-man blood serum have also been studied. Because of the presence of alkaline phosphatase in serum that removed the 5�-end 32P-label gradually, it is im-possible to get the quantified data from the gel picture to compare their rela-tive blood serum stability. But by visualization of the gel (Figure 2 in Paper I), it looks likely that in the human blood serum, the relative order of stabil-ity of modified AONs was nearly the same as that found upon treatment with SVPDE.

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The BHNA modified AON 37 are much more stable than native AON 1 upon SVPDE treatment or in human blood serum (Paper IV). By comparison of AON 37 with carba-LNA modified AONs, we found AON 37 is nucleo-tytically less stable than AON 26, AON 10, AON 14, AON 30, AON 6 but more stable than AON 22 and AON 18.

2.3.4 RNase H-mediated RNA degradation in AON/RNA duplex In the antisense strategy, RNase H-mediated target RNA degradation is a very precious property when the modified AONs are bound to the target RNA in heteroduplex form.63 All duplexes formed by AONs 1-40 with com-plementary RNA have been found to be excellent substrates for E. coli RNase H1 (Paper I & IV). The cleavage pattern was not affected by the na-ture of different substituents on carba-LNA, but depended upon the site of the modification. The E. coli RNase H1 promoted cleavage has a preference at the middle site A8 of the RNA strand for native AON 1/ RNA duplex. If AON strand was modified with carba-LNA or BHNA, the cleavage activity of RNase H was suppressed within a 4-5 base pairs long region that starts from the base opposite the modified nucleotide, towards the 3�-end of the RNA strand. If A8 is included within the suppressed region, the major cleav-age site shifts to the edges of the suppressed region (Figure 14).

Figure 14. The Escherichia coli RNase H1 promoted cleavage pattern of AONs 1-40/RNA duplexes. Vertical arrows show the RNase H cleavage sites, with the rela-tive length of the arrow showing the extent of the cleavage. The square boxes show the stretch of the modification, which is resistant to RNase H1 cleavage thereby giving footprints.

The modification site but not the modification type can significantly modulate the cleavage rates. Exclusively, the AON/RNA having the modifi-cation at position 6 (AONs 3/7/11/15/19/23/27/31/34/38)