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Novel Methods for Synthesis of High Quality Oligonucleotides

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Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 179 Novel Methods for Synthesis of High Quality Oligonucleotides ANDREY SEMENYUK ACTA UNIVERSITATIS UPSALIENSIS ISSN 1651-6206 UPPSALA ISBN 91-554-6672-9 2006 urn:nbn:se:uu:diva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o my mother and my beloved wife Natalia THE ORIGINAL PUBLICATIONS This thesis is based on the following original publications, which are re- ferred to by the Roman numerals: I Andrey Semenyuk, Matilda Ahnfelt, Camilla Estmer-Nilsson, Xiao-Yong Hao, Andras Földesi, Yu-Shu Kao, Hong-Huei Chen, Wei-Chen Kao, Konan Peck, Marek Kwiatkowski. Cartridge- based high-throughput purification of oligonucleotides for reli- able oligonucleotide arrays. Anal. Biochem. 2006, 356, 132–141. II Andrey Semenyuk, Andras Földesi, Tommy Johansson, Camilla Estmer-Nilsson, Peter Blomgren, Mathias Brännvall, Leif A. Kirsebom, Marek Kwiatkowski. Synthesis of RNA using 2´-O- DTM protection. J. Am. Chem. Soc 2006; 128(38), 12356– 12357. III Andrey Semenyuk, Marek Kwiatkowski. A base-stable di- thiomethyl linker for solid-phase synthesis of oligonucleotides. Manuscript submitted to Tetrahedron Lett. IV Andrey Semenyuk, Jörg Shlingemann, Aida Zuberovic, Marek Kwiatkowski. Two Hydrophobic Group Strategy for Efficient Purification of Oligonucleotides. Manuscript. Contents Introduction...................................................................................................11 Solid-phase oligonucleotide synthesis......................................................12 5´ Hydroxyl protection ........................................................................15 Aglycone protection.............................................................................16 Phosphate protection............................................................................17 2´ Hydroxyl protection ........................................................................17 Oligosynthesis cycle and deprotection conditions...............................26 Purification of oligonucleotides ...............................................................29 Functionalized solid support as a purification aid ...............................31 Oligonucleotide purification using RPC..............................................31 Present work .................................................................................................33 Paper I. A method for RPC oligonucleotide purification.........................33 Paper II. Solid-phase RNA synthesis using 2´-O-tert-butyldithiomethyl (2´-O-DTM) protecting group..................................................................37 Paper III and IV. Synthesis of 3´-O-alkyldithiomethyl analogues and their application in oligonucleotide synthesis...................................................43 Summary in Swedish ....................................................................................52 Acknowledgements.......................................................................................54 References.....................................................................................................56 Abbreviations 2´-OH 2´ hydroxyl 2c1peoc (2-cyano-1-phenylethoxy)carbonyl 5´-OH 5´ hydroxyl ACE bis(2-acetoxyethoxy)methyl aq. aqueous BF3·OEt2 trifluoroborate diethyl etherate CEE 1-(2-chloroethoxy)ethyl CEM 2-cyanoethoxymethyl CMES carbomethoxyethylsulfonyl CNEE 1-(2-cyanoethoxy)ethyl CPES 4-cyanophenylethylsulfonyl CPG controlled pore glass CPSEC 2-(4-chlorophenyl)sulfonylethoxycarbonyl Ctmp 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl DBU 1,8-Diazabicyclo[5.4.0]undec-7-ene DCA dichloroacetic acid DCM dichloromethane DMAP N,N-Dimethyl-4-aminopyridine dmf N,N-dimethylformamidine DMF N,N-dimethylformamide DMTr bis(4-O-methoxyphenyl)phenylmethyl (4,4´-dimethoxytrityl) DNA deoxyribonucleic acid Dnseoc 2-([5-(dimethylamino)naphthalen-1-yl]sulfonyl)ethoxycarbonyl DOD bis(trimethylsiloxy)cyclododecyloxysilyl DTM tert-butyldithiomethyl DTT DL-1,4-dimercapto-3,4-dihydroxybutane (1,4-dithio-DL-threitol) Fmoc 9-fluorenylmethoxycarbonyl Fpmp 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl HCl hydrochloric acid HIFA 2,6-dicarbomethoxyphenoxymethyl (hydroxyisophthalate formalde- HPLC high performance liquid chromatography IE ion exchange iPr-Pac 4-iso-propylphenoxyacetyl MDMP 3-methoxy-1,5-dicarbomethoxypentan-3-yl MEM Methoxyethoxymethyl MMTr (4-O-methoxyphenyl)diphenylmethyl (4-methoxytrityl) Mthp 4-methoxytetrahydropyran-4-yl MTM methylthiomethyl nbm 4-nitrobenzyloxymethyl NMI N-methylimidazole NMP N-methyl-2-pyrrolidone NPE 4-nitrophenylethyl NPEOC 4-nitrophenylethyloxycarbonyl NPES 4-nitrophenylethylsulfonyl Pac phenoxyacetyl PAGE polyacrylamide gel electrophoresis Px 9-phenylxanthen-9-yl (pixyl) RNA ribonucleic acid RP reversed-phase RPC reversed-phase cartridge SEM trimethylsilylethoxymethyl Tac 4-tert-butylphenoxyacetyl TBAF tetrabutylammonium fluoride TBDMS tert-butyldimethylsilyl TCA trichloroacetic acid TCEP tris(2-carboxyethyl)phosphine TEA triethylamine TEA·3HF triethylamine trihydroflouride TEAA triethylammonium acetate TFA trifluoroacetic acid Thf tetrahydrofuranyl THF Tetrahydrofurane Thp tetrahydropyran-4-yl TMG N,N,N´,N´-tetramethylguanidine TMTr tris(4-O-methoxyphenyl)methyl (4,4´,4´´-trimethoxytrityl) TOM tri-iso-propylsilyloxymethyl trityl triarylmethyl tRNA transfer RNA Introduction Synthetic oligonucleotides are widely used in biochemical and medical re- search as tools for DNA detection and amplification. The application of syn- thetic oligonucleotides, both ribo- and deoxyribo-, has been further extended with findings that oligonucleotides can interfere with gene expression [1, 2]. The potential therapeutic uses antisense oligonucleotides, aptamers, ri- bozymes, and small interfering RNAs (reviewed in [3-8]). Intensive development of diagnostic and therapeutic applications places serious purity demands on synthetic oligonucleotides. Oligonucleotides of high purity provide higher sensitivity of DNA detection techniques [9] and efficacy of antisense therapeutics. The amount and nature of impurities in synthetic oligonucleotides are directly correlated with the efficiency of their synthesis and purification. State-of-the-art DNA synthesis employs the effi- cient, commercially available solid-phase phosphoramidite method that can produce DNA with over 99% stepwise yields according to calculations based on trityl release. However, the synthesized oligonucleotide can be damaged or modified during its assembly. This process proceeds regardless of the coupling efficiency, as unwanted reactions may affect the already completed part of the sequence. The major problem is depurination [10, 11] that takes place during detritylation of the growing oligonucleotide chain. Frequently employed trityl-based purification methods are unable to remove tritylated failure fragments arising from depurination. To date, cartridge-based oli- gonucleotide purification is commonly used for fast product enrichment. It can be performed automatically and is able to purify many mixtures simulta- neously. Although, a number of cartridge systems are commercially avail- able, there is no suitable method for an automated and parallel purification of 5´-amino- or 5´-thiol-modified oligonucleotides that are commonly used for making oligonucleotide arrays. The present study was dedicated to the development of methods improv- ing quality of synthetic oligonucleotides. The research focused both on oli- gonucleotide synthesis and purification methods. The developed procedure for cartridge-based purification allowed automated purification of many tritylated oligonucleotides at a time regardless of their 5´-terminal modifica- tion. This procedure combined with the disiloxyl linker led to oligonucleo- tides of higher purity compared to that of oligonucleotides purified by RP HPLC. Additionally, the dithiomethyl linker was developed as an alternative to the disiloxyl linker. The former does not require anhydrous cleavage con- 11 ditions and is stable towards concentrated aqueous ammonia used to hydro- lyze abasic sites. Compared to DNA the synthesis of RNA oligonucleotides is more prob- lematic due to the presence of an extra hydroxyl group that has to be pro- tected. As the 2´-OH protecting group
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