or metallacarborane modification

Accepted Manuscript Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane and/or metallacarborane modification Agnieszka B. Olejniczak, Ryszard Kierzek, Eric Wickstrom, Zbigniew J. Lesnikowski PII:

S0022-328X(13)00401-4

DOI:

10.1016/j.jorganchem.2013.05.022

Reference:

JOM 18044

To appear in:

Journal of Organometallic Chemistry

Received Date: 25 March 2013 Revised Date:

16 May 2013

Accepted Date: 18 May 2013

Please cite this article as: A.B. Olejniczak, R. Kierzek, E. Wickstrom, Z.J. Lesnikowski, Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane and/or metallacarborane modification, Journal of Organometallic Chemistry (2013), doi: 10.1016/ j.jorganchem.2013.05.022. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Graphical abstract synopsis

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Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane and/or metallacarborane modification

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Agnieszka B. Olejniczak, Ryszard Kierzek, Erick Wickstrom, Zbigniew J. Lesnikowski*

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DNA-oligomers modified simultaneously with both, carborane and metallacarborane boron clusters have been synthesized for the first time. The antisense activity of these bioorganicinorganic constructs and ability to silence the insulin receptor substrate 1 (IRS-1) gene over

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expressed in several types of tumors was tested in preliminary experiments.

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Graphical abstract

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Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane and/or metallacarborane modification

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Agnieszka B. Olejniczak, Ryszard Kierzek, Erick Wickstrom, Zbigniew J. Lesnikowski*

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DNA-oligomers modified simultaneously with both, carborane and metallacarborane boron clusters have been synthesized for the first time. The physicochemical and biochemical properties of these bioorganic-inorganic constructs have been studied. The antisense

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activity and ability to silence the insulin receptor substrate 1 (IRS1) gene over expressed in several types of tumors was tested in

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preliminary experiments.

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Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane

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and/or metallacarborane modification

1

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Agnieszka B. Olejniczak1, Ryszard Kierzek3, Eric Wickstrom4, Zbigniew J. Lesnikowski2* Screening Laboratory and 2Laboratory of Molecular Virology and Biological Chemistry, Institute of

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Medical Biology, Polish Academy of Sciences, 106 Lodowa St., Lodz 93-232, Poland; Institute of Bioorganic Chemistry, Polish Academy of Sciences, Laboratory of RNA Chemistry, 12/14 Noskowskiego, Poznan 61-704, Poland; 4

Thomas Jefferson University, Department of Biochemistry and Molecular Biology, Laboratory of

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Nucleic Acid Therapeutics, 233 S. 10th Street, Philadelphia PA 19107-5541, USA

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Dedicated to Prof. Dr. V. I. Bregadze on the occasion of his 75th birthday

*To whom correspondence should be addressed: phone: 48 42 272 36 29, fax: 48 42 272 36 30, e-mail: [email protected]

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ABSTRACT

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Modified oligonucleotides are potential candidates for therapeutic nucleic acids technology and applications as tools in molecular biology. Despite broad use and extensive studies of the modified DNA/RNA-oligomers there is a continuous need to improve their properties. These properties, related

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mainly to migration through the cell membranes, resistance to nucleolytic digestion and specific interaction with target sequence, can be improved by chemical modifications of the oligonucleotide

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backbone. In this study, we synthesized for the first time DNA-oligomers modified simultaneously with both, carborane and metallacarborane boron clusters and examined the physicochemical and biological properties of these bioorganic-inorganic constructs. The ability to silence the insulin receptor substrate 1 (IRS-1) gene overexpressed in several types of tumors by selected oligomers was tested in preliminary

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experiments. The lipophilicity and resistance to enzymatic degradation of the modified oligomers was higher than the unmodified counterparts. Effective formation of duplexes with complementary

KEYWORDS

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sequences was demonstrated, thought antisense activity was not observed.

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Boron cluster, metallacarborane, DNA-oligonucleotide, antisense, insulin receptor substrate (IRS-1)

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ACCEPTED MANUSCRIPT ABBREVIATIONS A: adenine, AC: ammonium citrate, AON: antisense oligonucleotide, AS: antisense, ATT: 6-Aza-2thiothymine, B: purine or pyrimidine nucleobase, BEMC: 5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxapentoxy-, CBM: (o-carboran-1-yl)methyl-, C: cytosine, FAB: fast atom bombardment, G: guanine, 3-

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HPA: 3-hydroxypicolinic acid, HRP: hors radish peroxidase, IRS-1: insulin receptor substrate 1, MALDI: matrix assisted laser desorption ionization, ODN: oligodeoxyribonucleotide, ODU: optical density unit, PEAGE: polyacrylamide gel electrophoresis, RP-HPLC: reversed phase high performance

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liquid chromatography, Rt: retention time on HPLC column, siRNA: small interfering ribonucleic acids, SVPDE: snake venom phosphodiesterase, T: tymine, Tm: melting temperature, TEAB:

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triethylammonium bicarbonate, THA: 2,4,6- trihydroxyacetophenone, TLC: thin layer chromatography, U: unit.

INTRODUCTION

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Synthetic nucleic acids, in addition to their application as indispensable tools in molecular biology [1] and medical diagnostics [2,3], and as platform for new materials [4,5], constitute a class of potential therapeutics with a broad spectrum of applications ranging from treatment of infectious diseases and

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cancers to genetic disorders [6-8]. The major classes of “therapeutic nucleic acids” (therapeutics based on nucleic acid structure) include antisense oligonucleotides (AON) [9], triple helix forming oligomers

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[10], small interfering ribonucleic acids (siRNA) [11], ribozymes [12], and emerging new types of potential therapeutic nucleic acids such as microRNA [13,14]. The biological activity of therapeutic nucleic acids depends on silencing of the genes participating in pathological process [15]. Independently of the mechanism of action, a key step crucial for therapeutic nucleic acids activity includes recognition of a target RNA/DNA sequence by the oligonucleotide drug utilizing specificity of the nucleobase interactions [16,17]. To improve and optimize pharmacological properties of nucleic acids as therapeutic agents an array of modifications has been designed and

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ACCEPTED MANUSCRIPT synthesized, involving modification of sugar, phosphodiester linkage and nucleobases [6-9, 17-20]. However, in spite of these efforts only few nucleic acid based drugs such as fomivirsen (Vitravene®), mipomersen (Kynamro®) or Macugen®, have found some use as therapeutics or had clinical trial experience so far [18, 21-25].

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It has been shown that modification of oligonucleotides’ lipophilicity through masking the negative charge of the internucleotide phosphodiester groups or by an attachment of lipophilic substituents can improve their properties including increased duplex stability, enhanced nuclease resistance, improved

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cellular uptake, and often increased overall biological activity against specific targets [26]. Several lipophilic groups [27,28] have been used with some successes as lipophilic modification. Another

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approach is based on a pro-drug strategy involving conversion of the parent oligonucleotide to a lipophilic precursor [29].

In our research we have focused on development of boron clusters as modifying units for DNA/RNAoligomers (5,30-32). They consist a vast family of boron containing caged compounds [33,34]. Of this

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the icosahedral dicarba-closo- dodecacarborane (C2B10H12) is used most frequently for modification of biological molecules [35-39]. One of the most important features of ortho-carborane system (1,2C2B10H12) is high lipophilicity and susceptibility to transformation of closed cage cluster (closo-) into

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ionic, open cage form nido-7,8-C2B9H12 (–1), followed by the ability to form a “sandwich” type metal complexes (metallacarboranes) [34].

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Methods for the attachment of boron clusters and boron cluster metal complexes to nucleosides and nucleotides have been developed and used for modification of DNA-oligomers [30, 40-42]. Several advantageous properties of these modifications were discovered, these include: increased resistance to enzymatic digestion, increased lipophilicity and ability to form duplexes with complementary templates. In this work we present a further progress on the synthesis of DNA-heterooligonucleotides modified with both carborane and metallacarborane clusters, and report on some pertinent physicochemical and substrate properties of these modifications.

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MATERIALS AND METHODS Materials 5'-O-Dimethoxytrityldeoxynucleoside 3'-O-(N,N-diisopropyl-β-cyanoethyl)phosphoramidite (B = T, C,

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A, G) was obtained from Beckman Instruments Inc. (2500 Harbor Boulevard, Fullerton CA 926343100, lot No S906649, S905558, S906593 and S907546, respectively). 5'-O-Dimethoxytrityl thymidineloaded 0.2 µmol CPG (500 Å pore size) columns were purchased from Beckman Instruments Inc.

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(2500 Harbor Boulevard, Fullerton CA 92634-3100). Uridine was purchased from Avocado Research Chemicals Ltd. (Heysham, England). Column chromatography was performed on silica gel 70-230 or

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230-400 mesh obtained from Sigma-Aldrich (Steinheim, Germany). TLC was performed on silica gel F254 plates purchased from Sigma-Aldrich (Steinheim, Germany). Solvents were purchased in the highest available quality. C18 reverse phase column Econosil 5 µm, 4.7 x 250 mm was obtained from Altech Associates Applied Science Ltd. (Carnforth, England). Phosphodiesterase I (EC 3.1.4.1) type IV

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(crude dried venom from Crotalus atrox) was purchased from Sigma Co. (St. Louis, MO). Acrylamide was purchased from Sigma Co. (Deisenhofen, Germany), and urea was bought from POCh (Lublin, Poland). Polyacrylamide gel electrophoresis was performed using a C.B.S. Scientific Co. apparatus

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(Del Mar, CA). Ethidium bromide for staining the slab gels was purchased from Sigma Chemical Co. (St. Louis, MO). UV measurements were performed on GBC Cintra10 UV-VIS spectrometer

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(Dandenong, Australia). Tm measurements were performed on Beckman DU 640 spectrometer with a thermoprogrammer. 1H, 11B, and 13C NMR spectra were recorded with a Bruker Avance DPX 250 MHz spectrometer. The spectra were recorded at 250.13, 80.25, and 60.90 MHz, respectively. Tetramethylsilane and BF3/(C2H5)2O were used as standards for 1H/13C and 11B, respectively. 31P NMR analyses were performed using Bruker AC 200, operating at 81.01 MHz. 85% H3PO4 was used as external standard. Fast atom bombardment (FAB) mass spectra were recorded on a Finnigan MAT spectrometer (Bremen, Germany), matrix assisted laser desorption ionization (MALDI) spectra were 5

ACCEPTED MANUSCRIPT recorded on Voyager Elite (Perseptive Biosystems, Framingham, MA). Calculation of the theoretical molecular mass for oligonucleotides 9-16 was performed using the option “Analyze Structure” in the ChemDraw program, the masses correspond to molecular weights based on the average mass of the elements consisting of natural isotopes. Human MCF-7 cells (breast adenocarcinoma, pleural effusion,

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ER+ PR+ Her2-) were obtained from American Type Culture Collection (HTB-22, Manassas, VA) ™). DMEM/F12 culture medium, calf serum, glutamine, penicillin, and streptomycin were obtained from Sigma (St. Louis, MO). Oligofectamine and 4-12% polyacrylamide Tris-glycine gels obtained from

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Invitrogen (Carlsbad, CA). Complete Mini protease inhibitor cocktail tablets were obtained from Roche Applied Science (Indianapolis, IN). Anti-IRS-1 monoclonal antibody was obtained from Santa Cruz

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Biotechnology (Santa Cruz, CA). Anti-GAPDH monoclonal antibody was obtained from Ambion (Austin, TX). HRP-conjugated secondary antibody and SuperSignal® West Femto substrate for HRP were obtained from Pierce Chemicals (Rockford, IL).

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Methods

2’-O-(o-carboran-1-yl)methyluridine (1). The title compound was prepared according to the literature procedure [41,43].

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5’-O-Dimethoxytrityl-2’-O-(o-carboran-1-yl)methyluridine (2). The title compound was prepared as described previously [41].

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5’-O-Dimethoxytrityl-2’-O-(o-carboran-1-yl)methyluridine 3'-O-(N,N-diisopropyl-β-cyanoethyl)phosphoramidite (3). The title compound was prepared as described previously [41]. 2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-O-deoxyguanosine (4). The title compound was prepared as described previously [44]. 5’-O-Dimethoxytrityl-2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-Odeoxyguanosine (5). 2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-O-deoxyguanosine (4) (0.387 g, 0.57 mmol) was evaporated with anhydrous pyridine (3 × 5 mL), then dissolved in the

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ACCEPTED MANUSCRIPT same solvent (20 mL). To the resultant solution, triethylamine (0.33 mL), and 4,4’-dimethoxytrityl chloride (0.579 g, 1.71 mmol) were added. After stirring for 12 h at ambient temperature the reaction was quenched with methanol (5 mL), then the solvents were evaporated. The oily residue was dissolved in dichloromethane (30 mL) containing 1% triethylamine. The resultant solution was washed with a

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saturated solution of sodium bicarbonate (3 × 20 mL). The organic fraction was separated, dried over magnesium sulfate, then filtered. Magnesium sulfate was washed with dichloromethane containing 1% triethylamine. Filtrate and washings were combined, then the solvents were evaporated under reduced

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pressure, next the oily residue was co-evaporated with toluene (3 × 5 mL) to remove traces of pyridine. The crude product 5 was purified by silica gel column chromatography (15 g, 230-400 mesh). As an

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eluting solvent system the gradient of methanol in dichloromethane (0-6%) was used. Fractions containing 5 were collected then solvent was evaporated under reduced pressure. Yield 0.22 g (39%). TLC (acetonitrile/dichloromethane): Rf=0.5; 1H NMR (250.13 MHz, CDCl3, TMS): δ 7.62 (s, 1H, H-8); 7.45-7.19 (m, 9H, H-arom. of DMT group), 6.83-6.79 (m, 4H, H-arom. in α of CH3O), 6.26 (t, 1H, H-

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1’), 4.69 (bs, 1H, H-3’), 4.23-4.17 (m, 5H, 1H-4’, 1H-CH-metallacarborane, 1H-CH-metallacarborane, 2H-CH2-linker), 3.82 (bs, 2H, CH2-linker), 3.76 (s, 6H, 2 x CH3O), 3.71-3.69 (m, 2H, H-5’,5’’), 3.703.56 (m, 2H, CH2-linker), 3.39-3.35 (m, 2H, CH2-linker), 2.70-2.64 (m, 2H, H-2’,2’’), 3.00-1.00 (m,

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17H, BH-metallacarborane), 13C NMR (60.90 MHz, CDCl3, TMS): δ 158.41 (C4 of 4-CH3OPh), 157.37 (C-6), 155.29 (C-4), 148.77 (C-2), 144.58 (C-8), 135.73, 130.00, 129.06, 128.09, 127.78, 126.76,

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113.11 (C of Ph), 86.29 (C-4’), 86.07 (C-methylidene of DMT group), 83.58 (C-1), 72.75 (CH2-linker), 72.42 (C-3’), 70.90 (CH2-linker), 68.03 (C-5’), 64.03 (CH2-linker), 55.21 (CH3O), 54.21 (CHmetallacarborane), 46.99 (CH-metallacarborane), 43.83 (CH2-linker), 40.29 (C-2’); 11B-NMR (80.25, CDCl3, BF3/(C2H5)2O): δ 23.41 (B-8), 10.00-(–25.00) (B-8’, 10, 4, 7, 9, 12, 10’, 4’, 7’, 9’, 12’, 5, 11, 6, 5’, 11’, 6’); UV (95% C2H5OH): λmin=225, 294 nm, λmax=236, 272, 313 nm; FAB-MS (-VE, Gly): 978.3 [M-1]–, 676.1 [M-DMT]–.

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ACCEPTED MANUSCRIPT 5’-O-Dimethoxytrityl-2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-Odeoxyguanosine 3'-O-(N,N-diisopropyl-β-cyanoethyl)-phosphoramidite (6). 5’-O-Dimethoxytrityl-2-N{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-O-deoxyguanosine (5) (0.08 g, 0.082 mmol) was dissolved in anhydrous dichloromethane freshly distilled over CaH2 (4 mL). To the resultant

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solution, N,N-diisopropylethylamine (0.071 mL, 0.41 mmol) was added under argon followed by the addition of the phosphitylating agent, (β-cyanoethyl) (N,N-diisopropylamino)chlorophosphine (0.055 mL, 0.25 mmol). The reaction progress was monitored by TLC using dichloromethane/methanol (9:1)

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as eluting solvent system. After stirring for 4 h under argon at ambient temperature, the reaction was quenched with anhydrous methanol (0.5 mL). The resultant solution was washed with 5% sodium

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bicarbonate (3 × 5 mL). The organic layer was dried over magnesium sulfate then drying agent was filtered off and washed with dichloromethane containing 1% triethylamine. Filtrate and washings were combined and evaporated to dryness under reduced pressure. The crude product was immediately purified by silica gel column chromatography (10 g, 230-400 mesh). As an eluting solvent system a

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gradient of methanol in dichloromethane (0-8%) containing 1% triethylamine was used. Fraction containing product were collected then solvent was evaporated under reduced pressure yielding 6 as a dry yellow foam. The product dried under high vacuum and stored at –20 °C. Yield 0.049 g (51%). TLC

148.47.

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(dichloromethane/methanol, 9:1): Rf=0.63; 31P NMR (81.01 MHz, CD3OD, 85% H3PO4): δ 148.30,

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Unmodified antisense octadecanucleotide 5’-d(gcactagatcgccgtttt)-3’ (9), 5'-d(gcactacttcgccgtttt)-3’ with 2 mismatches (10) and target sequence 5’-r(cccacggcgaucuagugc)-3’ (17). The title DNAolignucleotides were from DNA Sequencing and Oligonucleotide Synthesis Lab, Institute of Biochemistry and Biophysics PAS (Warsaw, Poland). The target RNA oligonucleotide was the kind gift of Dr Marek Kwiatkowski of Uppsala University, Department of Genetics and Pathology, Rudbeck Laboratory (Uppsala, Sweden), present address: Uppsala Biomedicinska Centrum (BMC), Department of Cell and Molecular Biology (Uppsala, Sweden). 8

ACCEPTED MANUSCRIPT Antisense 5’-BEMC-containing octadecanucleotides 5’-d[(BEMCg)cactagatcgccgtttt]-3’ (11) and sequence with 2 mismatches 5’-d[(BEMCg)cactacttcgccgtttt]-3’ (12) (37). The 5’-BEMCmodified oligonucleotides 11 and 12 were synthesized using a Beckman Oligo 1000 DNA

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synthesizer. Columns loaded with controlled pore glass functionalized with 5'-Odimethoxytrityl thymidine (0.2 µmol) were used as a solid support (8). Suitable 5'-O-

dimethoxytrityl-2’-O-deoxynucleoside 3'-(N,N-diisopropyl-β-cyanoethyl)phosphoramidites (7,

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B = ABz, GPr, CBz, T) were prepared as a 0.5 g/10 mL solution in anhydrous acetonitrile. Elongation of the oligomers with natural nucleotides was performed using a standard β-

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cyanoethyl 0.2 µmol DNA synthesis cycle (45) without changes in condensation time. Coupling of the 5’-terminal modified monomer 5’-O-dimethoxytrityl-2-N-{5-[3-cobalt bis(1,2dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-O-deoxyguanosine 3'-O-(N,N-diisopropyl-βcyanoethyl)phosphoramidite (6) as well as oxidation step were performed manually (capping

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step was omitted). After sixteen coupling cycles, detritylation and washing with acetonitrile the column was detached from the DNA synthesizer and dried under high vacuum (5 min). Modified monomer 6 (10 mg, 0.0085 mmol) was dissolved in anhydrous acetonitrile (100 µL)

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followed by addition of tetrazole (0.042 mL, 0.5 M, 1.48 mg). A solution of activated 6 was applied to the column and the coupling reaction was performed for 30 min. The column was

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washed with anhydrous acetonitrile (2 × 5 mL) followed by drying under high vacuum. The oxidation step was performed using tert-butyl hydroperoxide solution (1 mL, 0.5 M) for 1 min. followed by washing with acetonitrile (1 × 5 mL) and drying under high vacuum. Oligonucleotides were then cleaved from the support by 1 h incubation with concentrated aqueous ammonia solution (30%, 1 mL) at room temperature then the base deprotection was achieved by incubation of the resultant solution at 50 °C for 2 h. The solution of crude 5’-Odimethoxytrityl protected oligonucleotide 11 or 12 was degassed with a stream of argon, and 9

ACCEPTED MANUSCRIPT evaporated to dryness under vacuum, then re-dissolved in water. Resultant solution of crude 5’O-protected oligonucleotides 11 and 12 having 69.50 and 26.43 A260 optical density units, respectively, were purified using HPLC C18 reverse phase column (RP-HPLC) using conditions described in “HPLC analysis” section below. Fractions containing the desired

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product were collected, and the buffer was evaporated under vacuum. The residue was coevaporated with 95% ethyl alcohol to remove triethylammonium bicarbonate (TEAB), then

detritylation was performed using a 80% acetic acid (1.0 mL) at room temperature for 20 min.

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Next the acetic acid solution was evaporated to dryness under vacuum and the totally

deprotected oligonucleotides were purified by RP-HPLC using the same conditions as above.

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Fractions containing the desired product were collected, and the buffer was evaporated under vacuum. The residue was co-evaporated with 96% ethyl alcohol to remove TEAB then was dissolved in deionized water and lyophilized. Both oligonucleotides were stored as dry solid at 20 °C. When needed, they were re-dissolved in water, stored as frozen solution, and re-

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lyophilized as soon as possible. The yield of purified modified oligonucleotide 11 was 13.42 ODUA260, and oligonucleotide 12 5.13 ODUA260, respectively. 11: UV (H2O): λmin =230 nm, λmax=261 nm; RP-HPLC (condition as above) Rt =16.53 min., MALDI-MS 5876 [M]. 12: UV

5826 [M].

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(H2O): λmin =240 nm, λmax=262 ; RP-HPLC (condition as above) Rt =15.97 min; MALDI-MS

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Antisense 2’-CBM-containing octadecanucleotides 5’-d[gcactagatcgccgt(u2’CBMu2’CBM)t]-3’ (13) and sequence with 2 mismatches 5’-d[gcactacttcgccgt(u2’CBMu2’CBM)t]-3’ (14). The first two coupling steps with modified monomer 5’-O-dimethoxytrityl-2’-O-(o-carboran-1-yl)methyluridine 3'-O-(N,Ndiisopropyl-β-cyanoethyl)phosphoramidite (3) performed to incorporate two 2’-CBM modifications at the 3’-end of the oligomer, as well as oxidation and capping step were done manually. Columns loaded with controlled pore glass functionalized with 5'-O-dimethoxytrityl thymidine (0.2 µmol) were used as a solid support (8). Direct detritylation of the 5'-O-dimethoxytrityl thymidine attached to the solid support 10

ACCEPTED MANUSCRIPT with 3 % dichloroacetic acid (DCA) in dichloromethane (1 × 5 mL) was performed, before manual coupling of the modified monomer 3. The column was then washed with acetonitrile (1 × 5 mL), and dried with argon stream (3 min). Monomer 3 (24 mg, 0.0027 mmol) was dissolved in anhydrous acetonitrile (70 µL) followed by addition of S-ethylthiotetrazole (0.13 mL, 0.5 M, 8.61 mg). A solution

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of activated 3 was applied to the column, and the coupling reaction was performed for 15 min. The column was washed with anhydrous acetonitrile (2 × 5 mL) followed by drying with a stream of argon. The oxidation step was performed using a standard oxidizing solution containing 0.1 M iodine in

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tetrahydrofuran/pyridine/water (13:6:1) (1 mL) for 30 sec followed by washing with acetonitrile (2 × 5 mL), and drying with a stream of argon. Capping step was done with a standard capping mixture of 1 M

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acetic anhydride in tetrahydrofuran/pyridine (1:8) (0.5 mL), and 0.5 M 4-(dimethylamino)pyridine (DMAP) in tetrahydrofuran (0.5 mL) for 30 sec, followed by wash of the column with acetonitrile (1 × 5 mL), and drying under high vacuum (5 min). The manual detritylation, coupling with monomer 3, oxidation, and capping steps were repeated, then the column was attached to the Beckman Oligo 1000

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DNA synthesizer, and elongation of the oligonucleotide chain continued with unmodified monomers. Suitable 5'-O-dimethoxytrityl-2’-O-deoxynucleoside 3'-(N,N-diisopropyl-β cyanoethyl)phosphoramidites (7, B = T, CAc, ABz, GiBu) were prepared as a 0.5 g/10 mL solution in

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anhydrous acetonitrile. Elongation of the oligomers with natural nucleotides was performed using a standard β-cyanoethyl 0.2 µmol DNA synthesis cycle (41, 45) without changes in condensation time.

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Oligonucleotides were cleaved from the support by 1 h incubation with concentrated aqueous ammonia solution (30%, 1 mL) at room temperature, then base deprotection was achieved by incubation of resultant solution at 55°C for 18 h. The solution of crude 5’-O-dimethoxytrityl protected oligonucleotides 13 and 14 was degassed with a stream of argon and evaporated to dryness under vacuum, then re-dissolved in water. The resultant solution of crude 5’-O-dimethoxytrityl protected oligonucleotide 13 and 14 bearing two modifications: at 2nd and 3th location from 3’-end of the 18-mer having 49.25 and 80.94 ODUA260, respectively, were purified using HPLC C18 reverse phase column 11

ACCEPTED MANUSCRIPT (RP-HPLC) using conditions described in “HPLC analysis” section below. Fractions containing the desired product were collected, and the buffer was evaporated under vacuum. The residue was coevaporated with 95% ethyl alcohol to remove TEAB, then detritylation was performed using a 80% acetic acid (1.0 mL) at room temperature for 20 min. Next, the acetic acid solution was evaporated to

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dryness under vacuum and the totally deprotected oligonucleotides were purified by RP-HPLC using the same conditions as above. Fractions containing the desired product were collected, and the buffer was evaporated under vacuum. The residue was co-evaporated with 95% ethyl alcohol to remove TEAB.

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The oligonucleotides were then re-dissolved in water and lyophilized. All oligonucleotides were stored as lyophilized powder at –20 °C. When needed, they were re-dissolved in water, stored as frozen

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solution, and re-lyophilized as soon as possible. The final yield of purified modified oligonucleotides 13 and 14 bearing modification at 2nd and 3th location was 4.82, and 9.79 ODUA260, respectively. The characteristics of oligonucleotides 13 and 14 are presented in Table 1.

Antisense 5’-BEMC- and 2’-CBM-containing octadecanucleotides 5’-

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d[(BEMCg)cactagatcgccgt(u2’CBMu2’CBM)t]-3’ (15) and sequence with 2 mismatches 5’d[(BEMCg)cactacttcgccgt(u2’CBMu2’CBM)t]-3’ (16). The synthesis of oligomers 15 and 16 containing two 2’-CBM modifications at 3’-end, and one 5’-BEMC modification at 5’-end was performed merging

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protocols for the synthesis of 5’-BEMC oligomers 11 and 12, and for 2’-CBM oligomers 13 and 14. Thus first, trinucleotide containing two 2’-CBM modification was obtained manually, then the

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unmodified part of the oligomer was synthesized using DNA synthesizer analogously as described for 13 and 14. Then finally, after detritylation of the 5’-terminal 2’-deoxycytidine at location 17th and washing with acetonitrile the column was detached from the DNA synthesizer and dried under high vacuum (25 min), and the 5’-terminal BEMC group was attached manually as described for 11 and 12. Oligonucleotides 15 and 16 were then cleaved from the support by 1 h incubation with concentrated aqueous ammonia solution (1 mL) at room temperature, then base deprotection was achieved by incubation of resultant solution at 55 ºC for 18 h. The solution of crude 5’-O-dimethoxytrityl protected

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ACCEPTED MANUSCRIPT oligonucleotides 15 and 16 was degassed with a stream of argon and evaporated to dryness under vacuum, then re-dissolved in water. The resultant solution of crude 5’-O-dimethoxytrityl protected oligonucleotide 15 and 16 bearing two 2’-CBM modifications: at 2nd and 3th location from 3’-end and one 5’-BEMC modification at 18th location of the 18-mer having 91.31 and 46.12 ODUA260,

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respectively, were purified using HPLC C18 reverse phase column (RP-HPLC) using conditions described in “HPLC analysis” section below. Fractions containing the desired product were collected, and the buffer was evaporated under vacuum. The residue was co-evaporated with 95% ethyl alcohol to

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remove TEAB, then detritylation was performed using a 80% acetic acid (1 mL) at room temperature for 20 min. Next, the acetic acid solution was evaporated to dryness under vacuum and the totally

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deprotected oligonucleotides were purified by RP-HPLC using the same conditions as above. Fractions containing the desired product were collected, and the buffer was evaporated under vacuum. The residue was co-evaporated with 95% ethyl alcohol to remove TEAB. The oligonucleotides were then redissolved in water and lyophilized. All oligonucleotides were stored as lyophilized powder at -20 °C.

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When needed, they were re-dissolved in water, stored as frozen solution, and re-lyophilized as soon as possible. The final yield of purified modified oligonucleotides 15 and 16 bearing modification at 2nd and 3th location was 11.43, and 2.72 ODUA260, respectively. The characteristics of oligonucleotides 15 and 16

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are presented in Table 1.

HPLC analysis and purification of oligonucleotides 11-16. HPLC analysis, and purification were

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performed on the Hewlett-Packard 1050 system, using Altech Econosil C18 5 µm, 4.7 x 250 mm column. All analyses were run at room temperature using conditions as follows: 20 min. from 0% D to 100% D, 5 min. 100% D, 5 min. from 100% D to 0% D. Flow rate 1mL/min. UV detection at λ=268 nm, buffer A: CH3CN/H2O (2:98) containing 0.1 M TEAB (pH 7.0), buffer D: CH3CN/H2O (60:40) containing 0.1 M TEAB (pH 7.0). The same conditions were used for both, “trityl on” and “trityl off” oligomers and for the analytical and semi-preparative runs. For semi-preparative purifications of 5’-Odimethoxytrityl protected or deprotected oligonucleotides the obtained amount (30-90 ODU260 of the

13

ACCEPTED MANUSCRIPT crude preparation) was divided into 10 ODU260 fractions, then was loaded into the HPLC column. MALDI-MS oligonucleotide analyses. Mass spectrometry analysis of oligonucleotides 9-16

were

performed on a Voyager Elite (Perseptive Biosystems, Framingham, MA) instrument. Typically, oligonucleotide stock solutions (1 µL) in water (0.01 ODUA260/µL H2O) were mixed with 2,4,6-

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trihydroxyacetophenone monohydrate solution (1 µL) in water/acetonitrile (1:1, 10 mg/mL) containing ammonium citrate (THA/AC, oligomers: 11 and 13), 3-hydroxypicolinic acid/ammonium citrate (3HPA/AC, oligomers 12, 15 and 16) or 6-aza-2-thiotymine/ammonium citrate (ATT/AC, oligomers 9,

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10, 14) to yield a final concentration of corresponding oligomers 5 ODUA260/mL. The resulting solutions

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were applied into the MALDI probe, then the ionization was performed at 337 nm. Melting temperature (Tm) measurements. The olignucleotides were melted in a buffer containing 0.1 M of sodium chloride, 0.9 mM of potassium chloride, 0.4 mM of phosphate buffer, and 0.5 mM of Na2EDTA, pH 7.0. The oligonucleotide single strand concentrations were calculated from hightemperature (>80oC) absorbancies and single strand extinction coefficients approximated by a nearest-

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neighbor model. The absorbance vs. temperature melting curves were measured at 260 nm with a heating rate of 1oC/min from 0 to 90 oC on Beckman DU 640 spectrometer with a thermoprogrammer.

6

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There were nine melts for the duplexes in a standard buffer at the concentrations ranging from 10-3-10M RNA. The melts curves were analyzed and thermodynamic parameters were calculated using

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MeltWin 3.5 program.

Resistance of unmodified octadecanucleotides 5’-d(gcactagatcgccgtttt)-3’ (9), 5’-BEMC-containing 5’d[(BEMCg)cactagatcgccgtttt]-3’ (11), two 2’-CBMs-containing 5’-d[gcactagatcgccgt(u2’CBMu2’CBM)t]-3’ (13) and double, 3’,5’- modified octadecanucleotides 5’-d[(BEMCg)cactagatcgccgt(u2’CBMu2’CBM)t]-3’ (15) 12 toward phosphodiesterase I (SVPDE, EC 3.1.4.1). To 100 mM Tris-HCl buffer (225 µL, pH 8.9) containing 20 mM MgCl2, water solution of oligonucleotide 9, 11, 13 and 15 (0.75 ODUA260, 16-22 µL) and water solution of uracil (0.2 ODUA260, 2,1 µL) used as internal standard, was added. The

14

ACCEPTED MANUSCRIPT resultant mixtures were treated with 2 x 10-4 unit (20 µL) of phosphodiesterase (crude dried venom from Crotalus atrox dissolved in water) and incubated at 38 °C. After 30, 60, 120, 240 and 360 min 44-50 µL aliquots were withdrawn, heated up at 80 °C and than stored at -70 °C prior to analysis by HPLC. HPLC conditions are described in “HPLC analysis” section below. A blank reactions without enzyme and a

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control reaction with unmodified oligomer (9) and enzyme were assayed concurrently.

PAGE analysis. Samples of anti-IRS unmodified oligonucleotides 9, 10 and modified oligomers 11-16 (0.1-0.3 ODU in 5-8 µL formamide containing 0.03% bromophenol blue and 0.03% xylene cyanol),

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were analyzed by electrophoresis using a 20% polyacrylamide denaturing gel containing urea (7 M) for

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16 h at 5 mA. The samples were visualized by staining with ethidium bromide.

Antisense activity. Effects of antisense treatment were measured essentially as described [46]. MCF-7 human breast epithelial cells were grown in DMEM/F12 medium supplemented with 5% calf serum, 50 U/mL penicillin, 5 µg/mL streptomycin, and 2 mM glutamine under 5 % CO2 in a humidified incubator at 37oC. For transfection, 70% confluent cultures were used. Oligonucleotides were complexed with

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Lipofectamine transfection agent according to manufacturer’s instructions, then added to cells to a final oligonucleotide concentration of 1.0 µM in DMEM medium. Cells were treated for 96 hr at 37 oC, then washed and incubated in growth medium for 96 hr at 37 oC. Treated cells were then lysed in 50 mM

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HEPES-KOH, pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 10 mM EGTA, pH 7.5, 10% glycerin, 1% Triton

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X-100 with a Complete Mini protease inhibitor cocktail tablet. Protein concentrations were quantitated with Bio-Rad Protein Assay (Richmond, CA). 100 µg aliquots of lysates were electrophoresed on 412% polyacrylamide Tris-glycine gels. Proteins were electroeluted onto nitrocellulose membranes, then incubated with anti-IRS-1, or anti-GAPDH after stripping, followed by HRP-conjugated secondary antibody, as described (n). SuperSignal® West Femto band intensities were quantitated with an Image Station 2000R (Kodak, Rochester, NY).

15

ACCEPTED MANUSCRIPT RESULTS The modified nucleoside monomers, 2’-O-(o-carboran-1-yl)methyluridine (1) [40,42] and 2-N-{5-[3cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}-2’-O-deoxyguanosine (4) [44] were obtained according to the literature procedures. 5’-O-Dimethoxytrityl-2’-O-(o-carboran-1-yl)methyluridine 3'-O-

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(N,N-diisopropyl-β-cyanoethyl)phosphoramidite (3) was obtained from 1 in a two-step procedure as described previously (41). 5’-O-Dimethoxytrityl-2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxapentoxy}-2’-O-deoxyguanosine 3'-O-(N,N-diisopropyl-β-cyanoethyl)-phosphoramidite (6) was prepared

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analogously as described earlier for thymidine derivative: 5’-O-dimethoxytrityl-4-O-{5-[3-cobalt

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bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}thymidine 3'-O-(N,N-diisopropyl-β-cyanoethyl)-phosphoramidite [42] (Scheme 1). Thus, first, the 5’-hydroxyl function of 4 was protected with the dimethoxytrityl group and next the corresponding partially protected nucleoside 5 was reacted with (βcyanoethyl) (N,N-diisopropylamino)-chlorophosphine according to the standard procedure [47,48] yielding the phosphoramidite monomer (6)

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The modified material bearing two 2’-O-(o-carboran-1-yl)methyl- (2'-CBM) groups at the 2th and 3th location (3'-end), the fully complementary to the target sequence 17, oligomer 13 and forming 2

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mismatches with the target, oligomer 14, one 5-[3-cobalt bis(1,2-dicarbollide)-8-yl]-3-oxa-pentoxy}(BEMC) group at the 18th location (5’-end), oligomers 11 and 12, respectively, and possessing both 2’-

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CBM and BEMC modifications oligomers 15 and 16 (Table 1), were obtained by solid phase automatic synthesis using a standard β-cyanoethyl cycle [45]. The oligonucleotides were purified by two-step trityl-on/trityl-off HPLC procedure. The homogeneity of all oligonucleotides was checked by polyacrylamide gel electrophoresis (PAGE, Figure 1), and their integrity by MALDI-MS (Table 1). Mass spectrometry analysis proved that carborane cage of 2’-CBM modification is in nido-status (Scheme 2) for all 2’-CBM bearing oligomers 13-16. The yield for the coupling reaction involving modified monomer 3 and 6 was lower than for unmodified ones, though the molar excess of 3 and 6 in

16

ACCEPTED MANUSCRIPT the reaction mixture was higher and coupling time was longer (30 min) than for unmodified monomers. Melting temperature (Tm) measurements of the duplexes between modified oligomers 11-16 were compared to those formed between corresponding unmodified octadecanucleotides 9 and 10 (Table 1) and the complementary template ribo-oligonucleotide 17 (Table 2). The measurements of

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thermodynamic stability of the duplexes were performed in standard condition, in phosphate buffer containing 100 mM of sodium chloride. The thermodynamic parameters (entropy, enthalpy and free energy) were calculated by two methods. The first approach was based on fitting the experimental and

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theoretical melting curves (average of curve fits) and the second method of collecting thermodynamic parameters is based on correlation of melting temperature and concentration of duplex (for non-self

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complementary duplexes TM-1 vs. log CT/4). Meaningful effects of 2’-CBM and 5’-BEMC group on Tm were noted depending on location of the modification within the oligonucleotide chain. The Tm value for modified oligonucleotide 11 bearing only BEMC modification at 5’end, and fully complementary to the target sequence 17, was lower than for unmodified oligomer 19 (∆Tm = -3.3 °C) Table 2).

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Interestingly for oligonucleotide bearing two 2’-CBM groups at 3’-end an effect of the modification on Tm was much less pronounced and in general stability of duplexes formed by modified oligonucleotide 13 was comparable to this formed by unmodified partner 9 (∆Tm = -0.5 °C). As expected, highest

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decrease of Tm was observed for the oligonucleotide 15 modified at both 3’-and 5’-ends (∆Tm = -5.1

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°C). However, an effect of oligonucleotide chemical modifications with boron clusters was relatively small in comparison with the effect of imperfect complementarity, thus the presence of 2 mismatches in the oligonucleotides 10, 12, 14 and 16 used as controls cases the decrease of Tm by 15.4-18.5 °C. The decrease of stability of duplexes formed by oligonucleotides with 2 mismatches caused by boron cluster modification is similar, though slightly higher, as for oligonucleotides complementary to the target oligomer 17 and changes in the same order: two 2’-CBM modifications at 3’-end < one BEMC modification at 5’-end < two 2’-CBM modifications at 3’ and one BEMC modification at 5’-end.

17

ACCEPTED MANUSCRIPT To test resistance of 2’-CBM/BEMC modified oligonucleotides 11, 13 and 15 towards 3'-exonuclease, phosphodiesterase I from snake venom (SVPDE), was used. Modified oligonucleotides 11 or 13 bearing modification at 3’- or 5’-end only were digested almost with the same rate as unmodified oligomer 9 (Figure 2). Though, a noticeable effect on increase of oligonucleotide stability was observed for

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oligomer 15 bearing modifications at both, 3- and 5’-ends, in this case the two-phase digestion process was observed contrary to one-phase digestion for 9, 11 and 13. Due to the biphasic character of the process establishing of the exact half-life time (t1/2) for the whole process is difficult. The

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oligonucleotides’ t1/2 increased in order: 13 (15 min) ~ 9 (18.8 min) ~ 11 (20.6 min) < < 15 (ca. 134 min). It is worthy to notice that in spite of the lowest calculated t1/2 for oligonucleotide 13

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approximately 8% of the intact oligomer still persisted in the reaction mixture after 60 min of incubation while unmodified oligomer 9 and modified at 5’-end oligomer 11 were completely digested (Figure 2).

Antisense activity of modified oligonucleotides was assayed in human MCF-7 breast cancer cells

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(Figure 3), as reported previously for siRNA-IGF1 tetrapeptide conjugates [46]. No apparent effect was

lysis.

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DISCUSSION

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detected, either by unmodified oligodeoxynucleotides or by carborane-modified sequences prior to cell

Modified oligomers’ synthesis. The 2’-CBM and BEMC modified oligonucleotides 11-16 were prepared by automated solid phase synthesis (Scheme 2 and Table 1). Manual step was used for the insertion of modified monomer 3 or 6 bearing 2’- CBM or BEMC group, respectively. Important consideration is the ribonucleoside structure of 3, and metallacarborane modification in 6 since the presence of bulky substituent at the 2’-position or N2 may affect the rate of coupling reaction occurring at the 3’-position. The problem of insufficient yields in condensation step during chemical synthesis of oligonucleotides in ribo-series is well recognized [41, 49, 50]. To address the lower coupling efficiency problem higher 18

ACCEPTED MANUSCRIPT concentration of the modified monomers (3, 3.4 g/10 mL and 6 1.0 g/10 mL vs. 0.5 g/10 mL for unmodified counterparts) and prolonged coupling time (30 min) was employed for incorporation of 3 or 6. Under this condition the final yield of HPLC purified 11-16 was in the range of 3-13 ODUA260, for comparison the expected yield of unmodified counterparts is in the range 11-33 ODUA260. It should be

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remarked that albeit o-carborane modification in the monomer 3 exists in closo-form, though due to the treatment of the oligonucleotide product with concentrated solution of ammonia in water during the cleavage from the solid support and deprotection, it is transformed into a nido-counterpart. The change

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is due to the properties of the o-carborane cage. o-Carborane cage can exist in two distinctive forms, neutral closo- and ionic nido-. The nido-form results from removal, often under alkaline conditions, of

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boron 3 or 6 from the neutral closo-cluster, which leads to the nido-o-carborane cage. An effect of boron cluster modification on duplex lipophilicity. The lipophilicity of 2’-CBM/BEMC oligonucleotides 11-16 measured as affinity to a RP-HPLC C18 column (retention time, Rt) was, as observed previously [29,30], substantially higher than unmodified sequence. Lipophilicity increased in

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the order: 9,10 < 13,14 < 11,12 ~ 15, 16. The HPLC analysis indicated not only high affinity of derivatives containing 2’-CBM/BEMC modification to C18 resin but that the affinity increases in spite the presence of negative charge on both types of modifications (Scheme 2). This phenomenon may be

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attributed to the unique character of boron clusters allowing for distribution of the electric charge within the cage leading to low density of surface charge.

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Duplex stability. Several factors exerting an effect on the transition of polynucleotides between ordered (double helix) and disordered (coiled) forms have been defined [51]. Among the most frequently discussed are: hydrogen bonding, both between complementary bases and those due to sugar hydroxyl groups, electrostatic interactions, hydration and hydrophobic interactions. Due to the unique structure of 2’-CBM modifications it may contribute to all of these factors and affect the duplex stability: 1) The ocarboran-1-yl cluster is linked to the 2’ position of uridine - the change of thymine nucleic base for uracil, and bulky substituent at position 2’ may affect hydrogen bonding. 2) There is an additional

19

ACCEPTED MANUSCRIPT negative charge at nido- o-carborane cluster of 2’-CBM group - the electrostatic interactions, in the form of electrostatic repulsion of the negatively charged phosphate groups may decrease stability of the double stranded structure. 3) The nido- o-carborane cluster has both lipophilic and hydrophilic properties - it may affect hydration and hydrophobic interactions [30]. The differences in the

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thermodynamic parameters smaller than 15% mean that a melting process was performed according to a two-state model. It is interesting that two-state melting duplexes were observed for complementary DNA/RNA duplexes as well as for duplexes containing internal tandem 5’d(CT)/3’r(CU) mismatch and

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TTT trinucleotide dangling end on 3’-site of DNA fragment and CCC on 5’-end of RNA fragment (except 15/17 and 16/17 duplex).

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The analysis of Table 2 demonstrated that the presences of one or more boron-modified nucleotides destabilize thermodynamic stability of the studied duplexes. Destabilization mostly depends on a position of modified nucleotides within duplexes [52, 53]. The presence of boron-modified nucleotides within Watson-Crick base pair destabilizes complementary duplexes as well as those duplexes

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containing internal tandem 5’d(CT)/3’r(CU) mismatches. The duplexes 11/17 and 12/17 were by 2.31 and 2.42 kcal/mol less stabile (∆∆Go37) than the referential unmodified duplexes 9/17 and 10/17, relatively. Destabilization of 11/17 and 12/17 duplexes is similar and high, and it can be a consequence

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of the presence of boron-modified nucleotides within a complementary fragment of duplexes. The presence of modification within G-C base pair presumably makes hydrogen bonds weaker or more

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likely prevents the formation of some hydrogen bonds. Such high change of the thermodynamic stability of 11/17 and 12/17 duplexes could be a consequence of breaking of at least 2 hydrogen bonds within GC base pair [54,55]. Placing two modified nucleotides in duplexes 13/17 and 14/17 within terminal trinucleotide mismatches d(TTT)/r(CCC) results in much lower destabilization. The thermodynamic stability of the modified duplexes 13/17 and 14/17 change (∆∆Go37) by 0.12 and 0.56 kcal/mol in comparison to unmodified duplexes 9/17 and 10/17, respectively. When the modified nucleotides were present at both sides of duplexes, the stabilization effects were to

20

ACCEPTED MANUSCRIPT some degree additive. However, in the case of the 15/17 duplex, double modification lowered duplex stability by 1.03 kcal/mol compared with a calculation on the basis of the effects measured in duplexes 11/17 and 13/17 separately. In case of duplex 16/17, the total destabilization effect (∆∆Go37) was different by 0.17 kcal/mol, relatively to the combined destabilization effects measured in 12/17 and

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14/17 duplexes separately. The comparison of thermodynamic stabilities of 9/17 and 10/17 duplexes, as well as those containing boron-modified nucleotides at the same position, duplexes 11/17 and 12/17, 13/17 and 14/17, 15/17 and

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16/17, demonstrates that the presence of internal tandem mismatch 5’d(CT)/3’rCU results in similar duplexes destabilization. The destabilization (∆∆Go37) is in the range of 6.20 and 7.29 kcal/mol. As it

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was reported, the influence of tandem mismatches on thermodynamic stability of RNA duplexes is strongly affected by the nature of the base pair adjacent to tandem mismatches. For the system 5’GUCG/3’CUCC (underlined indicated tandem mismatch), we observed destabilization (∆∆Go37) of 3.20 kcal/mol [56,57]. In the measured duplexes, destabilization is much larger (range between 6.20 and

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7.29 kcal/mol) and it could be a result of different sequence of adjacent base pairs and/or one strand being RNA, whereas the second one being DNA.

It is difficult to compare the results presented in this paper with those reported in literature, since the

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experiments concern DNA/RNA duplexes that are not commonly used as subject of the thermodynamic studies [58]. Additionally, in duplexes there are present one or/and two structural motives tandem

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mismatch 5’d(CT)/3’r(CU) and terminal mismatch d(TTT)/r(CCC)], effecting thermodynamic stability of the investigated duplexes. The information about the influence of boron-modified nucleotides on duplex stability is very limited and does not allow formation of clear conclusions. The studies of various RNA duplexes indicated that the presence of such structural motives in a duplex, i.e. as modified nucleotides, single or multiple mismatches destabilize RNA duplexes [52, 53, 59]. Similar observation is reported herein, however, the nature of modification and duplex is different. The presented above data suggest that, from a thermodynamic point of view, it is optimal to place a 21

ACCEPTED MANUSCRIPT boron cluster of modified nucleotides outside of the complementary region formed by antisense oligonucleotides, such as in duplexes 13/17 and 14/17, which resulted in the smallest destabilization of the formed duplexes. Resistance of 2’-CBM/BEMC oligonucleotides 9, 11, 13 and 15 to digestion by SVPD.

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Phosphodiesterase I from snake venom (SVPDE), often used to test susceptibility to degradation by 3’exonucelases, was applied to evaluate the resistance of 2’-CBM/BEMC oligonucleotides to enzymatic digestion. SVPDE is an exonuclease that successively digests oligonucleotides in the 3'->5' direction

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and is nonprocessive enzyme [60]. Besides, it is known that SVPDE has endonucleolytic activity in addition to exonuclease activity. This activity is probably responsible for the initial degradation of the

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2’-end modified oligonucleotides by endonucleolytic removing of the 2’-end protection leading to truncated product susceptible to further exonucleolytic digestion [61]. As expected unmodified oligonucleotide 9 and oligomer modified at 5’-end 11 were digested fast and with similar rate. Surprisingly only moderate increase in stability in the presence of SVPDE was observed for oligomer

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13 modified at 3’-end contrary to earlier observations [41]. Perhaps the resistance towards SVPDE activity is affected not only by 3’-end modification but also sequence dependent oligonucleotide conformation and ability to form higher ordered structures. This hypothesis is supported by exceptional

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nucleolytic stability of the oligomer 15 modified with BEMC groups at 5’-end and two 2’-CBM groups at 3’-end. The analogous effect of possible oligonucleotide folding caused by boron cluster modification,

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on oligonucleotide resistance towards SVPDE digestion was observed earlier for 2’-CBM modified oligiomers [41].

Antisense activity. Lack of antisense activity in MCF-7 cells, no significant reduction of IRS-1 protein was detected. The inactivity of the unmodified oligodeoxynucleotide could reflect vulnerability to nucleases in the calf serum and in the cells. In the earlier protected siRNA-peptide study in MCF-7 cells, IRS-1 knockdown was observed. Similarly, a separate study of antisense knockdown of IRS-1 in human endothelial cells by a phosphorothioate 25-mer against a different site in the IRS-1 mRNA

22

ACCEPTED MANUSCRIPT reported efficacy [62]. In the present study, nuclease resistance imparted by the carborane modifications might have been insufficient for knockdown efficacy in the MCF-7 cells. Therefore it is of interest that siRNA duplexes targeted against BACE1 gene modified at various positions with a highly lipophilic, electrically neutral, para-carborane system (1,12-C2B10H12, CB), with boron cluster in

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closo-form, used instead of hydrophilic, ionic and negatively charged orto-carborane (1,2-C2B9H12, CBM) with boron cluster in nido-form applied in the present work, expressed specific and high

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silencing activity in model HeLa cells system [63].

CONCLUSIONS

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DNA-oligomers modified simultaneously with both, carborane and metallacarborane boron clusters have been synthesized for the first time. The lipophilicity and resistance to enzymatic degradation by SVPDE of the modified oligomers was higher than the unmodified counterparts, especially in the case of simultaneous modification at 3’- and 5’-ends with 2’-CBM and BEMC groups, respectively. An

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effective formation of the duplexes with complementary sequences was proved. Antisense activity against IRS-1 gene tested with the oligomers modified with two 2’-CBM modifications at 3’-end was not detected. These studies indicate that the boron cluster bearing oligomers have properties that might

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be exploited further with the optimized sequences and judicially chosen boron cluster modification in

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the design of novel therapeutic nucleic acids.

23

ACCEPTED MANUSCRIPT ACKNOWLEDGMENT This work was supported by the National Science Center (NCN), Grant No N N204 531739 to ABO and ZJL and Grant No UMO-2011/03/B/ST5/01098 to RK. Partial contribution of the Statutory Fund of

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IMB PAS is also gratefully acknowledged.

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[42] A.B. Olejniczak, J. Plesek, O. Kriz, Z.J. Lesnikowski, Angew. Chem. Int. Ed. Engl., 42 (2003) 5740-5743. [43] W. Tjarks, A.K.M. Anisuzzaman, L. Liu, A.H. Soloway, R.F. Barth, D.J. Perkins, D.A. Adams, J. Med. Chem., 35 (1992) 1628-1633. [44] A.B. Olejniczak, J. Plesek, Z.J. Lesnikowski, Chem. Eur. J., 13 (2007) 311-318.

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ACCEPTED MANUSCRIPT [45] Oligo 1000 DNA Synthesizer Operating Instructions, Beckman Instruments Inc., Fullerton, CA, 1992. [46] G. Cesarone, O.M. Edugupanti, C-P. Chen, E. Wickstrom, Bioconjugate Chem., 18 (2007) 18311840. [47] T. Atkinson, M. Smith, Solid phase synthesis of oligodeoxyrubonucleotides by the phosphite-

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triester method. In: Oligonucleotide Synthesis: A Practical Approach., M. Gait, Ed., IRL, Oxford, pp 35-81,1984.

[48] S.L. Beaucage, Oligodeoxyribonucleotide synthesis. In: Methods in Molecular Biology, Vol. 20, Protocols for Oligonucleotides and Analogs. Synthesis and Properties. S. Agrawal, Ed., Humana Press Inc., Totowa, NJ, pp 33-61, 1993.

(1994).

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[50] A. Somoza, Chem. Soc. Rev., 37 (2008) 2668-2675.

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[49] Z. Shabarova, A. Bogdanov, A. Advanced Organic Chemistry of Nucleic Acids., VCH, Weinheim,

[51] V.A. Bloomfield, D.M. Crothers, I. Tinoco, Eds., Nucleic Acids - Structures, Properties and Functions, University Science Books, Sausalito, California, pp 259-334, 2000. [52] E. Kierzek, R. Kierzek, Nucleic Acids Res. 31 (2003) 4472-4480.

[53] Z. Ziomek, E. Kierzek, E. Biala, R. Kierzek, Biophys. Chem. 97 (2002) 233-241. [54] E. Kierzek, A. Pasternak, K. Pasternak, Z. Gdaniec, I. Yildirim, D.H. Turner, R. Kierzek,

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Biochemistry, 48 (2009) 4377-4387.

[55] D.H. Turner, N. Sugimoto, R. Kierzek, S.D. Dreiker, J. Am. Chem. Soc. 109 (1987) 3783-3785. [56] M. Wu, J.A. McDowell, D.H. Turner, Biochemistry 34 (1995) 3204-3211. [57] T. Xia, J.A. McDowell, D.H. Turner, Biochemistry 36 (1997) 12486-12497

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[58] N. Sugimoto, S. Nakano, M. Katoh, A. Matsumura, H. Nakamuta, T. Ohmichi, M. Yoneyama, M. Sasaki, Biochemistry 34 (1995) 11211-11216.

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[59] R. Kierzek, M.E. Burkard, D.H. Turner, Biochemistry 38 (1999) 14214-14223. [60] R.S. Brody, K.G. Doherty, P.D. Zimmerman, J. Biol. Chem., 261 (1986) 7136-714. [61] B.D. McLennan, B.G. Lane, Can. J. Biochem. 46 (1968) 93-107. [62] S. Al-Mahmood, S. Colin, N. Farhat, E. Thorin, C. Steverlynck, S. Chemtob, J Pharmacol Exp Ther., 329 (2009) 496-504.

[63] A. Kwiatkowska, M. Sobczak, B. Mikołajczyk, S. Janczak, A.B. Olejniczak, M. Sochacki, Z.J. Leśnikowski, B. Nawrot, B. Bioconjugate Chem., - submitted.

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TABLES.

Table 1. HPLC, Tm, and MS characteristics of anti-IRS unmodified oligonucleotides 9, 10 and carborane/metallcarborane modified oligonucleotides 11-16.

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____________________________________________________________________________________ Oligomer Base composition Rta Tmb Molecular formula MSc [min] [°C] [M] ____________________________________________________________________________________ 9 5’-d(gcactagatcgccgtttt)-3’ 9.37 63.6 C175H223N62O109P17 5465.00 9.17 48.2

C174H224N57O111P17

5417.00d

16.53 60.3

C183H251B18CoN62O111P17

5876.00

15.97 41.8

C182H252B18CoN57O113P17

5826.00e

12.09 63.1

C179H241B18N62O111P17

5761.00f

C178H242B18N57O113P17

5711.00d

15 5’-d[(BEMCg)cactagatcgccgt(u2’CBMu2’CBM)t]-3’ 19.95 58.5

C187H269B36CoN62O113P17

6168.00f

16 5’-d[(BEMCg)cactacttcgccgt(u2’CBMu2’CBM)t]-3’ 16.32 40.6

C186H270B36CoN57O115P17

6126.00g

10 5'-d(gcactacttcgccgtttt)-3’ 11 5’-d[(BEMCg)cactagatcgccgtttt]-3’ 12 5’-d[(BEMCg)cactacttcgccgtttt]-3’

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13 5’-d[gcactagatcgccgt(u2’CBMu2’CBM)t]-3’

12.51 46.9

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14 5’-d[gcactacttcgccgt(u2’CBMu2’CBM)t]-3’

AC C

____________________________________________________________________________ a 20 min. from 0% D to 100% D, 5 min. 100% D, 5 min. from 100% D to 0% D. Flow rate 1mL/min. UV detection at λ=268 nm, buffer A: CH3CN/H2O (2:98, v/v) containing 0.1 M triethylammonium bicarbonate (TEAB) (pH 7.0), buffer D: CH3CN/H2O (60:40, v/v) containing 0.1 M TEAB (pH 7.0); b Tm in phosphate buffer pH 7.0 at 0.9 mM KCl, 100 mM NaCl and 0.5 mM EDTA, ribooligonucleotide complementary strand 5’-r(cccacggcgatctagtgc)-3 (17) in 1:1 molar ratio was used; cMatrix Assisted Laser Desorption Ionization (MALDI)-MS; d[M+1], e[M-1], f[M+2], e[M-2], g[M+5]; d = deoxyribooligonucleotide.

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DuplexesA

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Table 2. Thermodynamic parameters of helix formation.

Average of curve fits (TM-1 vs log CT plots)B

5’-3’ 3’-5’

-∆ ∆G037 (kcal/mol)

TMC (0C)

∆∆G037D (kcal/mol)

∆TMC,D (0C)

369.8±38.9 (372.7±25.4)

16.90±0.96 (16.99±0.60)

63.5 (63.6)

0

0

114.5±11.6 (116.6±6.0)

322.0±35.3 (328.7±18.2)

14.62±0.63 (14.68±0.36)

60.6 (60.3)

2.31

-3.3

d(GCACTAGATCGCCGTU2’CBMU2’CBMT) (13) r(CGUGAUCUAGCGGCACCC) (17)

129.3±9.4 (133.4±6.4)

363.4±28.0 (375.6±19.2)

16.59±0.68 (16.87±0.46)

63.2 (63.1)

0.12

-0.5

d(BEMCGCACTAGATCGCCGTU2’CBMU2’CBMT) (15) r(CGUGAUCUAGCGGCACCC) (17)

91.6±16.4 (107.8±3.7)

254.3±50. (304.0±11.4)

12.74±0.66 (13.53±0.21)

59.5 (58.5)

3.46

-5.1

98.0±12.3 (103.9±3.7)

283.8±38.4 (302.2±11.6)

9.98±0.42 (10.14±0.10)

48.3 (48.2)

0

0

77.3±5.8 (79.0±3.4)

224.3±18.9 (229.8±11.1)

7.72±0.08 (7.72±0.04)

41.9 (41.8)

2.42

-6.4

91.1±7.7 (98.2±2.7)

263.2±24.0 (285.8±8.7)

9.46±0.25 (9.58±0.05)

47.3 (46.9)

0.56

-1.3

69.8±5.9 (69.9±4.9)

201.1±19.1 (201.9±15.9)

7.37±0.14 (7.33±0.08)

40.8 (40.6)

2.81

-7.6

-∆ ∆S0 (eu)

d(GCACTAGATCGCCGTTTT) (9) r(CGUGAUCUAGCGGCACCC) (17)

131.6±13.0 (132.6±8.5)B

d(BEMCGCACTAGATCGCCGTTTT) (11) r(CGUGAUCUAGCGGCACCC) (17)

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d(GCACTACTTCGCCGTTTT) (10) r(CGUGAUCUAGCGGCACCC) (17) d(BEMCGCACTACTTCGCCGTTTT) (12) r(CGUGAUCUAGCGGCACCC) (17)

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d(GCACTACTTCGCCGTU2’CBMU2’CBMT) (14) r(CGUGAUCUAGCGGCACCC) (17)

AC C

d(BEMCGCACTACTTCGCCGTU2’CBMU2’CBMT) (16) r(CGUGAUCUAGCGGCACCC) (17)

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-∆ ∆H0 (kcal/mol)

A - solutions is 4 mM Na2HPO4/NaH2PO4, 100 mM NaCl, 0.9 mM KCl and 0.5 mM Na2EDTA, pH 7; B - thermodynamic parameters in parenthesis were calculated from TM-1 vs log CT plots; C – calculated for 10-4 M oligonucleotide concentration; D – calculated based on TM-1 vs log CT plots values; d = deoxyribooligonucleotide, r = ribooligonucleotide.

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SCHEMES

O

O

NH N O

NH N O

NH N O

i

O

DMTO

H

OH O

ii

O

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HO

O

DMTO

O

O O

OH O

NCCH2CH2O P 1

N(iPr)2 3

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2

O

O N HO

N

NH N

NH

O

N

O Co

O 4

OH

i

DMTO

N

NH NH

= C or CH

O Co

5 ii

O

N N DMTO

O

NH N

NH

O

O Co

6

O

NCCH2CH2O P

N(iPr)2

AC C

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i. DMTCl/Py ii. (iPr)2NP(Cl)OCH2CH2CN in CH2Cl2/diisopropylethylamine

O

O

OH

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= BH,

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N

Scheme 1. Synthesis of 5’-protected 2’-O-deoxyuridine and 2’-O-deoxyguanosine phosphoramidites 3 and 6, bearing 2’-O-(o-carboran-1-yl)- (2’-CBM) or 2-N-{5-[3-cobalt bis(1,2-dicarbollide)-8-yl](BEMC) groups, respectively, monomers for modified oligonucleotide synthesis.

ACCEPTED MANUSCRIPT O N N

T O O

HO

iv

i, ii, iii

NH O

N

O Co

O O

the cycle i, ii, iii repeated 17 times

O

O P OH

NH N O

O d(CACTAGATCGCCGT) O

8

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DMTO

NH

O

O O

15

O

O P OH

= BH,

= C or CH

O

O O O P OH

i. Detritylation ii. Coupling iii. Oxidation and capping iv. Cleavage and deprotection

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O T

O

O

N

NH N O DMTO

O

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b

NH N O

N

DMTO

O

NH

N

NH O

O

6

O

NCCH2CH2O P

NCCH2CH2O P

AC C 2'-CBM =

Co

DMTO

O O

NCCH2CH2O P

N(iPr)2

BEMC=

N(iPr)2

7a, B=T 7b, B=CBz 7c, B=ABz 7d, B=GiPr

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N(iPr)2 3

TE D

O O

B

O

O

O Co

Scheme 2. Solid phase synthesis of example CBM and BMC modified octadecanucleotide 15.

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ACCEPTED MANUSCRIPT

10

11

12

13

14

15

16

TE D

9

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FIGURES

Figure 1. PAGE analysis of anti-IRS unmodified oligonucleotides 9, 10 and modified

AC C

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oligomers 11-16 (0.1-0.3 ODUA260), ethidium bromide staining.

31

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ACCEPTED MANUSCRIPT

100

9 11 13 15

SC

60

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% of intact oligonucleotide

80

40

0

100

200

300

Time [min]

AC C

EP

0

TE D

20

Figure 2. Resistance of unmodified (9), BEMC- (11), CBM- (13) and double BEMC/CBM- (15) modified oligonucleotides to digestion by phosphodiesterase I (SVPDE, EC 3.1.4.1).

32

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ACCEPTED MANUSCRIPT

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Figure 3. Western blot of proteins from human MCF-7 breast cancer cells treated with unmodified or carborane-modified antisense oligonucleotides targeted against IRS-1 mRNA. Cells were treated

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with unmodified complementary oligonucleotide (ODN) 9, doublemismatch ODN 10, carborane-modified complementary ODN 13, or

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carborane-modified double-mismatch ODN 14, targeted against insulin

receptor

substrate

1

r(cccacggcgaucuagugc)-3’ (17)

(IRS-1)

mRNA

fragment

5’-

for 6 hr, then cultured in fresh

medium for 96 h prior to cell lysis.

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Synthesis, physicochemical and biochemical studies of anti-IRS-1 oligonucleotides containing carborane and/or metallacarborane modification

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HIGHLIGHTS

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Agnieszka B. Olejniczak, Ryszard Kierzek, Erick Wickstrom, Zbigniew J. Lesnikowski*

The most important contributions of the submitted work are the following: • •

AC C

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•

DNA modified simultaneously with carborane and metallacarborane complex are described Physicochemical and biochemical properties of these modifications have been studied Antisense activity against IRS-1 gene was tested