Novel Methods for Synthesis of High Quality Oligonucleotides

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine 179

ANDREY SEMENYUK

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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 dithiomethyl 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 research as tools for DNA detection and amplification. The application of synthetic 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 efficient, 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 oligonucleotide 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 available, 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 oligonucleotide 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 modification. This procedure combined with the disiloxyl linker led to oligonucleotides 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 hydrolyze abasic sites. Compared to DNA the synthesis of RNA oligonucleotides is more problematic due to the presence of an extra hydroxyl group that has to be protected. As the 2´-OH protecting group remains until the completed oligonucleotide sequence is cleaved from the solid phase, and aglycones are deprotected; it should meet very demanding stability requirements. On the other hand the 2´-OH protecting group should be readily cleavable under mild conditions which do not threaten RNA integrity. It is a challenging task to find a group that fits the aforementioned somewhat contradictory demands. To date, there are several commercial methods for RNA solid-phase phosphoramidite synthesis available [12-15]. However, RNA synthesis still lacks suitable method matching DNA synthesis efficiency, and the search for an appropriate 2´ hydroxyl protecting group continues. Herein, a method of RNA synthesis was developed using 2´-O-tert- butyldithiomethyl (2´-O-DTM) protecting group which is orthogonal to the protecting groups used in DNA oligosynthesis and is removed under mild aqueous conditions. The method provides efficient synthesis of RNA with quality comparable to that of synthetic DNA and allows application of the automated trityl-based purification. Therefore, both ribo- and deoxyribo- oligonucleotides can be obtained in higher quality at about the same cost and time as crude oligonucleotides.

Solid-phase oligonucleotide synthesis The development of oligonucleotide synthesis methods started from the synthesis of thymidilyl-(3ó5´)-thymidine [12] after the structure of DNA was revealed by Watson and Crick [13]. Initially, three different approaches has been used to create phosphate linkages: phosphodiester [14, 15], H- phosphonate [16-18] and phosphotriester [19-21]. The former two approaches did not require internucleotide linkage protection (Scheme1) and were thus favored over the phosphotriester method (Scheme 2). It became obvious that rapid and nearly quantitative coupling reactions are needed to build long oligonucleotides with satisfactory overall yields. As a logical result phosphite triester approach [22] emerged later. More reactive 3´-O-phosphorochloridites reacted rapidly with the corresponding 3´- protected nucleoside yielding elongated oligonucleotides after subsequent oxidation of the resulting phosphite (Scheme 3). Unfortunately, due to their high reactivity, phosphorochloridites could neither be isolated nor stored, so they were obtained in situ from 5´-O-protected nucleosides by phosphityla- tion with protected phosphorodichloridate. As a result symmetrical by- products with 3ó3´ and 5ó5´ internucleotide linkages were also formed.

12 PG O B O PG O B PG O B O O OH O O + R P O O P O O B O B O O O O P O B O R O O PG PG O PG

Scheme 1. Phosphodiester (R = OH) and H-phosphonate (R = H) oligosynthesis methods

PG O B O

PG O B OH O

HO B + O O Cl PG O P O PG O P O Cl O Cl O P O + O B O

HO B O OH

O PG

Scheme 2. Phosphotriester oligonucleotide synthesis

PG O B O PG O B OH O PG O B + O HO B O O PG O P PG O Cl O P R O PG O P R O P O + O B O O B O HO B O O OH PG O PG

i Scheme 3. Phosphite triester (R = Cl) and phosphoramidite (R = N Pr2) oligosynthesis methods

13 OMe

MeO O B O

O O P CN N

O O

HN HN O O N N N N NH O NH B = N N N N O N N N O H

ABz CBz GiBu T

Figure 1. Amidites used in the traditional solid-phase DNA synthesis

In order to meet the requirements for automation, the phosphoramidite method of nucleotide coupling was developed [23] as an elegant modification of the phosphite triester approach (Scheme 3). This new class of intermediates gave rise to the traditional solid-phase oligosynthesis method (Fig.1), as phosphoramidites are suitable for both storage and handling under normal conditions and react rapidly with the corresponding nucleophile (e.g. the 5´ hydroxyl group of the oligonucleotide linked to the solid-phase support) upon activation with 1H-tetrazole [24]. Usually, phosphoramidites are obtained in the reaction of a protected nucleoside with either chloro- phosphine [25] or diamidophosphite [26] and can be isolated and stored as solids at –20qC. The growing oligonucleotide chain has to be isolated from the synthesis reagents after each reaction. The concept of using an organic polymer as a support for oligonucleotide synthesis was adapted from Merrifield’s approach of peptide synthesis [27], which gave the basic principle employed in the modern solid-phase oligosynthesis. Currently CPG [28, 29] and highly cross-linked polystyrene [30], containing the 3´-terminal nucleoside attached via an ester linkage, are used as solid supports for oligosynthesis. A great amount of studies were conducted to find optimal protecting groups of phosphoramidite building blocks and conditions for their removal. Some strategies will be mentioned here, and more comprehensive informa- tion can be found in extensive reviews on oligonucleotide and solid-phase synthesis [31-37].

14 PG O B O

O O PG P N O PG

PG PG NH NH O O N N N N NH NH B = N N N N O N N PG N O H

APG CPG GPG U

Figure 2. Amidites used for solid-phase RNA synthesis

A successful synthesis of oligonucleotides, besides amidite reactivity, depends on the integrity of other groups of the molecule. Protecting groups have to be introduced at several places of the nucleoside phosphoramidite molecule: 5´ hydroxyl; exocyclic amino groups of cytidine, adenosine and guanosine; phosphoramidite moiety; and 2´ hydroxyl of ribonucleosides (Fig.2). Some studies reported the necessity of guanosine lactam group protection [38-40], but replacing DMAP by NMI in the capping mixture [38], and introduction of a capping step before oxidation in the oligosynthesis procedure allowed this group to be left unprotected [41].

5´ Hydroxyl protection The trityl group has found a broad application as it can be selectively introduced at the 5´ hydroxyl of a nucleoside and its sensitivity to acids is adjusted with substituents at the aromatic rings [42]. Among the variety of studied structures DMTr (Fig.3a, R = R´ = OCH3, R´´ = H), MMTr (Fig.3a, R = OCH3, R´ = R´´ = H) and to some extent Px (Fig.3b) [43] groups are commonly used in the synthesis of oligonucleotides, as they offer a good balance between nucleoside stability and ease of trityl group removal. Fast trityl cleavage within seconds under mild acidic conditions (2% TCA in DCM) is used to liberate the 5´ hydroxyl of the growing oligonucleotide chain after every cycle, so the chain can be elongated further. Moreover, an + orange and yellow color formation upon the release of DMTr (Omax = 498 + nm) and MMTr (Omax = 478 nm) respectively, provides a possibility to evaluate oligonucleotide synthesis in real time. This is particularly useful for solid-phase synthesis, and the yield of oligonucleotide can be calculated spectrophotometrically [44] or by measuring conductivity [45].

15 R'

Ph R O

R'' a b

Figure 3. Widely used trityl-based groups

The 5´-O-trityl group is extensively used for purification of oligonucleotides [46], as its hydrophobicity allows discrimination between capped sequences that failed in elongation and full-length oligonucleotides. Furthermore, trityl groups with long alkyl chain lipophilic handles were applied to ensure purification of long oligonucleotides due to the further increased hydrophobicity difference of oligonucleotides bearing such trityl groups compared to non- tritylated fragments [47-52].

Aglycone protection DNA and RNA molecules are composed of four types of nucleotides, which differ in their heterocyclic bases. To avoid undesirable side reactions, exocyclic amino-groups of cytosine, adenine and guanine must be temporarily blocked during oligonucleotide assembly steps. Benzoyl is commonly used to protect exocyclic amines of adenosine and cytidine, and isobutyryl – of guanosine. These groups are removed from an oligonucleotide after its assembly during 8–12 hour incubation in concentrated aq. ammonia at 55qC. O O N

R dmf Pac (R = H) Tac (R = t-Bu) iPr-Pac (R = i-Pr)

Figure 4. Base-labile protecting groups

More labile groups, such as acetyl, Pac, Tac and dmf (Fig.4) [53-55], can be introduced to facilitate the deprotection of oligonucleotides under milder conditions and in shorter time [56], which is particularly important for oligonucleotides that contain functions unstable in ammonia [57-60]. Acetyla- tion of the exocyclic amine of guanine was reported when iPr-Pac- (Fig.4) and Tac- protected nucleoside phosphoramidites were used [55]. Thus, cap-

16 ping should be performed with the corresponding phenoxyacetic anhydride to avoid formation of acetylated oligonucleotides. Protected purine nucleotides, and especially N6-benzoyl adenine derivatives, are prone to depurination [10, 61, 62]. The influence of aglycone protecting group on the depurination rate was studied, and it was reported that phenoxyacetyl derivatives and dmf protecting group increase stability of purines towards acids [54, 56, 63].

Phosphate protection Among a variety of proposed protecting groups for oligosynthesis the 2- cyanoethyl group [64, 65] has found the widest application. The 2- cyanoethyl group is easily cleaved from phosphates with ammonia, which is used for hydrolysis of ester bonds releasing oligonucleotides from the solid support and for the subsequent deprotection of aglycones. The methyl group was also used for the protection of phosphates and was applied in the synthesis of oligonucleotides [66-68]. However, removal of the methyl group from internucleotide phosphates required thiolates [66, 69], which was considered less practical. The 2-cyanoethyl phosphate protection is cleaved by E- elimination retaining other phosphate substituents, whereas a deprotection mechanism based on nucleophilic substitution can result in the internucleotide linkage cleavage [70, 71]. Other phosphate protection alternatives, such as 2,2,2-trichloroethyl [72, 73], 2,2,2-tribromoethyl [73, 74], benzyl [12, 66, 73], p-chlorophenyl [71, 73, 75] and p-nitrophenylethyl [73, 76] were studied, but their application was not followed in conventional oligosynthesis.

2´ Hydroxyl protection The chemical synthesis of RNA is challenged by the presence of the 2´ hydroxyl group (Fig.2) that has to be protected. The protecting group should be stable during all oligonucleotide assembly stages and alkaline conditions used for removal of aglycone protecting groups. At the same time, the protection must be readily cleavable under mild conditions which do not alter oligonucleotide integrity. It is practical to employ protecting groups which were already established for the DNA synthesis. This would avoid a development of new synthesis procedures and suitable conditions for deprotection of the novel protecting groups. Moreover, finding new protecting groups with utility comparable to that of the 5´-O-DMTr or 2-cyanoethyl could be very difficult. In addition to the mentioned requirements on stability, the 2´-OH protecting group should not introduce steric hindrance to the phosphoramidite moiety in order to produce oligonucleotides with more than 99% stepwise yields.

17 Acyl and sulfonyl protecting groups An attempt to use benzoyl for 2´-OH protection was presented [77], but the strategy of using acyl protecting groups was not followed. It was reported that these groups underwent facile 2´l3´ isomerization [77-79] (Scheme 4) and their removal conditions resulted in internucleotide chain cleavage [77] (Scheme 5).

O O O

OH O O O O OH

R O R OH O R

Scheme 4. Acyl group isomerization

The 2´-O-NPES group (Fig.5) was tested, and it did not undergo 2´o3´ migration [80-83]. The removal of NPES was performed by anhydrous DBU via E-elimination [83] which also led to the formation of anhydrouridine derivatives (Scheme 6). This problem was circumvented with O4-protection of uridine. The latter, in turn, can be removed only at elevated temperature (50qC) upon the treatment with DBU [83], conditions which may damage deprotected RNA (Scheme 5).

B B O O B O O HO B O OH Base O O O P O O P + O O O O B O B O OH O O P O O O OH O OH

B B O O + O OH OH O O P O O P O OH OH

Scheme 5. Hydrolysis of internucleotide chain in basic media

Various sulfonyl groups were studied and CPES and CMES protecting groups (Fig.5) were proposed later to facilitate oligosynthesis with reduced internucleotide cleavage and to avoid the formation of anhydronucleosides [84]. To minimize side reactions, such as formation of anhydronucleosides and internucleotide bond cleavage, the deprotection sequence strictly fol-

18 lowed the order of 1) phosphate, 2) 2´-OH and 3) aglycone deblocking. The CPES group was more stable towards bases than NPES, and thus required longer deprotection times. In contrast, the 2´-O-CMES group was too base- labile to allow its application in oligosynthesis [84]. These studies were conducted using dimers and no evidence was presented that longer oligonucleotides would not have substantial internucleotide bond cleavage under the proposed deprotection pattern. Also the time and conditions for complete deprotection of 2´-O-NPES, and especially 2´-O-CPES, from moderate size and long oligonucleotides are not expected to favor this strategy.

O O

HN N TBAF O N N MMTr O MMTr O O O or O DBU OH O NPES OH

Scheme 6. Formation of anhydrouridine from the corresponding 2´-O-NPES protected nucleoside

19 O S O O S O OMe O O O O O Thf Thp Mthp

R CMES

NPES (R = NO ) Cl Si 2 O O CPES (R = CN) CEE SEM

OR1 MeO2C Si N O R2 MeO2C TBDMS HIFA R3

Fpmp (R1 = Me, R2 = F, R3 = H) OMe Ctmp (R1 = Me, R2 = Cl, R3 = Me) Cpep (R1 = Et, R2 = H, R3 = Cl) MeO2C CO2Me

O MDMP O O O

O O Si CN CN O O O

ACE TOM CEM CNEE

NO 2 O O O O O O O NO R 2

MEM nbm NEBE (R = H) FNEBE (R = F)

Figure 5. Groups proposed for 2´ hydroxyl protection

Acid-labile protecting groups Acetal- and ketal- type protecting groups have received considerable atten- tion. Regardless of the risk that internucleotide linkages undergo acid cata- lyzed 3´o2´ migration, the development of these groups and their removal conditions was very extensive.

20 The proposed Thp [85, 86] and Mthp [87] groups (Fig. 5) for 2´ hydroxyl protection that could be cleaved under relatively mild acidic conditions (0.01 N HCl, 20qC, pH 2), were found incompatible with DMTr and Px protecting groups used to block 5´ hydroxyl [88]. To minimize unwanted cleavage of the 2´-OH protecting group milder conditions using zinc bromide in various solvents [89-93] were applied for Px and DMTr removal. These conditions were investigated for solid-phase oligosynthesis using the 2´-O-Thf nucleoside phosphoramidites [94-96]. Unfortunately the zinc bromide method required long time for the removal of DMTr [94, 95, 97] leading again to the partial loss of the 2´ hydroxyl protecting group [93, 96, 97]. An attempt to use more acid-labile TMTr group (Fig.3a, R = R´ = R´´ = OCH3) for the 5´ hydroxyl protection [98] was not successful, as this group was found too unstable [92]. The search for an acid-labile group that is stable under conditions required for the 5´-O-DMTr (or 5´-O-Px) removal led to the development of the Ctmp [99] and Fpmp [100] groups (Fig.5). The latter had the same acidic hydrolysis properties as the Ctmp but was more readily accessible [100]. These groups were designed so they were unaffected under the relatively strong acidic conditions applied for removal of the acid-labile 5´ hydroxyl protecting group. The stability of the Ctmp and Fpmp groups in strong acid resulted from the inductive effect of the protonated nitrogen atom which retards the hydrolysis of the acetal. In turn, the nitrogen atom becomes largely unprotonated at pH 2.0 – 2.5 allowing the Ctmp and Fpmp groups to be hydrolyzed at the rate similar to the Mthp group [99]. The CEE group (Fig.5) was also tested as a 2´-OH protecting group [101]. The stability of this group is about threefold higher than that of the Thp towards the treatment with DCA in DCM used for removal of the 5´-O-DMTr group [101]. An approach, where an acid-labile 5´-OH protecting group is replaced by the protection which is orthogonal to an acid-labile 2´-OH protecting group, was also explored. The combination of 5´-O-levulinyl (Fig.6) and 2´-O-Mthp [102], 5´-O-levulinyl and 2´-O-DMTr [103], 5´-O-levulinyl and 2´-O-Thf [104, 105] was tested for oligonucleotide synthesis. Removal of the levulinyl group was performed by hydrazine hydrate in pyridine – acetic acid solution. This treatment, however, resulted in partial cleavage of N6-benzoyl group of adenine [105]. Application of the levulinyl group is not attractive, as this group cannot be selectively introduced onto the 5´ hydroxyl and often requires prolonged time for its removal [104]. Compared to DMTr or Px, the levulinyl group also lacks intrinsic absorbance in the UV or visible region to provide an aid in monitoring of the oligosynthesis efficiency. Thus, it was proposed to use the base-labile 5´-O-Fmoc or 5´-O-CPSEC group (Fig.6), whereas the 2´ hydroxyl was protected with the Px group or its derivatives [106, 107]. However, the 2´-O-Px protection is not practical, as Px introduces a great steric hindrance to the nearby phosphoramidite and therefore

21 affects its coupling efficiency, and the resulting hydrophobicity of the 2´-O- Px oligonucleotide hampers its handling.

O O O O S O O Cl O O Levulinyl CPSEC Fmoc

N SiMe3 O O Si O O CN O O S O SiMe O 3 O 2c1peoc DOD Dnseoc

O NO NO 2 O 2

NPE NPEOC

Figure 6. Alternative protecting groups used for RNA synthesis

A protecting group pair, 5´-O-Fmoc and 2´-O-Mthp, was investigated for solid-phase oligonucleotide synthesis [108, 109]. Nucleoside phosphoramidite couplings could reach 96% stepwise yields within 10 minutes of coupling time but the use of the 5´-O-Fmoc protection was not orthogonal to the phosphate protecting groups employed in the study. Thus, 2-cyanoethyl groups were cleaved during the oligonucleotide assembly during the Fmoc removal step upon the treatment with DBU [108]. An attempt to utilize a methyl group for the protection of internucleotide linkages resulted in me- thylation of the N3-position of thymidine residues [108]. Similarly, 5´-O- Dnseoc and 5´-O-2c1peoc (Fig.6) were used together with 2´-O-Mthp in the solid-phase synthesis [110-113]. The stepwise deprotection of the terminal Dnseoc and 2c1peoc groups was achieved with 0.1 M DBU in acetonitrile within 140 seconds. In those strategies NPE and NPEOC protecting groups (Fig.6) were employed to block phosphate and aglycone functions accordingly. Recently, a silyl-based DOD group (Fig.6) was introduced as a 5´-OH protecting group [114, 115]. The protection was readily cleavable with fluo-

22 ride reagent within 35 seconds. In this strategy the acid-labile ACE orthoester (Fig.5) was introduced onto 2´ hydroxyl. The presence of the effi- ciently cleavable DOD function on 5´ hydroxyl allowed usage of 2´-OH acid-labile protecting group without the risk of its cleavage during oligonucleotide assembly stages. On the other hand 2-cyanoethyl groups are cleavable with fluoride reagents therefore methyl groups were employed to protect phosphates. The deprotection of the 2´-O-ACE group is performed in two stages, where firstly esters are hydrolyzed during the deprotection of aglycones which results in tenfold more acid-labile 2´-O-bis(2- hydroxyethoxy)methyl and allows subsequent hydrolysis of the 2´-O- orthoester in buffered media (pH 3.8) at 60qC. The increased lability of the resulting 2´-O-orthoester is based on its facilitated protonation after removal of electron withdrawing acetyl groups. The strategy of rendering the 2´ hydroxyl protecting group more labile prior to its removal, was initially presented with introduction of the 2´-O- MDMP group (Fig.5) [116]. The stability of the MDMP group in acid was similar to the Mthp group. However, after ammonolysis the MDMP was converted to the corresponding diamide, according to the authors, which was cleaved at a much faster rate (ca. 17-fold). Later, the principle was also used in the design of 2´-O-HIFA (Fig.5) as an alternative acetal protecting group [117]. The HIFA group in its bis ester form was very stable towards acid (the half-life was 86 h at pH 1) and after conversion to bis acid form had a half- life of its cleavage 124 minutes under the same conditions. The markedly increased rate of the acid hydrolysis of the HIFA group could be ascribed to either intramolecular general acid catalysis [118] or electrostatic facilitation of the acetal oxygen protonation [119]. The search for a suitable 2´-OH acid-labile protecting group which is suf- ficiently stable during DMTr removal is still continued. A study of different substituted acetal groups was conducted [120]. The selected NEBE and FNEBE protecting groups (Fig.5) were employed for RNA synthesis [120, 121]. These acetals were stabilized against acids by the NPEOC protecting group, which was also employed for protection of aglycones. The removal of NPEOC during the deprotection of aglycones rendered the 2´-OH protecting groups more acid-labile. However, the proposed 2´-OH protecting groups were not completely stable under DMTr removal conditions. In order to minimize the loss of the 2´-OH protecting groups 1.3% DCA was used, and the use of anhydrous reagents was required. This method, as many other methods involving acid-labile 2´-OH protecting groups, has found limited application due to the poor stability of the proposed protecting groups towards acids. Incompatibility with the trityl group, and as a result with trityl- based oligonucleotide purification, hampers these methods to be widely applied.

23 Protecting groups cleavable by oxidation, reduction or photolytic hydrolysis Groups that are stable under both acidic and basic conditions were also employed to protect 2´-OH. Examples include 4-methoxybenzyl [122, 123] and MEM (Fig.5) [124]. These groups were removed with triphenylmethyl tetra- fluoroborate in aq. acetonitrile by a hydride transfer mechanism [125]. These studies were not continued and it is questionable if complete deprotection of oligonucleotides longer than nonamer is possible. Benzyl and 2-nitrobenzyl were also suggested as groups stable in acidic and basic media for the protection of 2´ hydroxyl [126, 127]. Removal of the benzyl group by catalytic hydrogenation was already problematic for moderate size oligonucleotides, and it also resulted in partial reduction of 5,6- double bonds of cytosine and uracil residues [128]. The 2-nitrobenzyl group was cleaved photolytically with irradiation of long wave UV light in buffered media (pH 3.5) [129, 130]. The subsequently proposed 2´-O-nbm (Fig.5) protecting group [131], where 2-nitrobenzyl protects formacetal rather than 2´ hydroxyl, solved the problem of steric hindrance of the amidite moiety influenced by the neighboring 2´-O-(2-nitrobenzyl) group. However, the photolabile types of protecting groups could not be cleaved quantitatively in reasonable time, and the removal difficulty is increased further with the size of the oligonucleotides. Moreover, in order to increase the efficiency of photolysis acidic conditions are required, which results in incompatibility of these methods with trityl-based oligonucleotide purification.

Fluoride-labile protecting groups The 2´-O-TBDMS group (Fig.5) introduced for the protection of hydroxyl functions of ribonucleosides in the mid-seventies [132] was applied for the synthesis of oligoribonucleotides [60, 133-138], and despite its limitations and drawbacks, the TBDMS group is still in frequent use. Thus, the TBDMS group is prone to 2´l3´ isomerization [139-141] and is partially cleaved by ammonia during the deprotection step of aglycones [142]. To avoid the loss of the 2´-O-TBDMS groups from oligoribonucleotides, milder conditions were applied for deprotection of aglycones [58, 143, 144] and later, labile aglycone protecting groups were employed [57]. It is noteworthy that the coupling efficiency of the 2´-O-TBDMS phosphoramidites was least efficient in a comparative study with deoxynucleoside, 2´-O-methyl and 2´-O- Thp derivatives [145]. To circumvent the limitations of the TBDMS approach, the alternative silyl-based 2´-O-TOM group (Fig.5) was introduced [146-148]. The strategy of using protected formacetal rather than direct protection of 2´ hydroxyl was employed by analogy with the method of using the 2´-O-nbm group [131] (Fig.5). The obtained phosphoramidites allowed for better coupling

24 yields due to the less steric hindrance compared to the 2´-O-TBDMS protected derivatives, and the problem of silyl migration was also solved. A silyl-based group (SEM; Fig.5) was proposed [149, 150], which can be removed by BF3·OEt2. The SEM group was stable under basic and all but the harshest acidic conditions. It was employed for the synthesis of a U10 ho- mopolymer which was deprotected with only 74% yield. There was no study dealing with longer oligonucleotides of various sequences reported to date. Recently, fluoride-labile CNEE [120, 151] and CEM [152] groups (Fig.5) were proposed for the protection of 2´-OH. The CNEE protecting group was stable towards ammonia and had a half-life of 240 minutes in 0.5 M DBU in acetonitrile. The stability of the CNEE group in TEA·3HF and in 25% aq. ammonia suggests that its complete removal with TBAF from oligonucleotides longer than decamers might be problematic, as the 2´-O-CNEE group appeared to be more stable towards fluoride reagents than the 2´-O-TBDMS group and thus will require even more prolonged time for its deprotection. On the other hand, the CNEE stability in acidic media was found limited [120]. Whereas the stabilizing electron withdrawing effect of cyano function is more pronounced compared to chloride, the destabilizing influence of alkyl in the 2´-O-formacetal moiety should not be disregarded. For example, the observed half-lives for 2´-O-CEE and 2´-O-CNEE in 0.05N HCl in methanol were 10 and 56 minutes respectively. Half of the 2´-O-CEE groups were cleaved in 80% aq. acetic acid within 6 min, which indicates that the 2´-O-CNEE group will be prone to partial acidic hydrolysis during DMTr removal [120]. The 2´-O-CEM group was found more base-labile than CNEE and was not stable during treatment with methylamine in aq. ethanol. Therefore, to avoid the loss of the 2´-O-protecting group, the deprotection of aglycones was performed with ammonia – ethanol (3:1) rather than aq. ammonia. Al- though it was stated that the loss of CEM groups under these conditions was less than 5% and no cleavage of internucleotide linkages was observed [152], for oligonucleotides bearing twenty such protecting groups it will result in the situation when almost every molecule will have inside the sequence at least one unprotected 2´-OH exposed to basic conditions. So, it is expected that the amount of failure fragments under such circumstances will be increased and the synthesis of long oligonucleotides will be impossible. The removal of silyl-based 2´ hydroxyl protecting groups was initially performed with TBAF in dry THF. However, tetraalkylammonium fluorides are hygroscopic, and the presence of moisture decreases base strength of the fluoride dramatically which results in a great decrease of silyl cleavage efficiency. Thus, the removal of silyl-based protecting groups with TBAF was sometimes inefficient. For example, the application of TBAF for the deprotection of tRNA resulted in incomplete cleavage of TBDMS groups [60]. The removal efficiency of the silyl-based protecting groups was improved by using TEA·3HF [60, 153]. The proposed TEA·3HF was not as sensitive to

25 water contamination as TBAF and it deprotected long RNA oligonucleotides more rapidly [60, 153]. Later, it was reported that neat TEA·3HF can result in RNA degradation and reduction of the total yield, therefore an alternative mixture (TEA·3HF/NMP) was proposed [154].

A strategy of using protected protecting groups and its perspectives Direct protection of 2´-OH was sometimes accompanied with undesired effects such as 2´l3´ isomerization or an increase of steric hindrance resulting in lower phosphoramidite coupling yields. The difficulty of designing a protecting group which is completely stable both in acidic and basic media but is rapidly cleavable under neutral and mild conditions is an additional problem. To avoid a premature cleavage of the 2´-O-protecting group, additional protecting group that protects the 2´-OH protecting group have frequently been used. For example, due to the field effect ester groups increased stability of MDMP, ACE and HIFA towards acids, whereas hydrolysis of those esters enabled rapid removal of the 2´ hydroxyl protecting groups under mild acidic conditions. The use of protected formacetal as in the 2´-O-TOM protection assured higher coupling efficiency compared to the TBDMS chemistry and solved the silyl migration problem. The versatil- ity provided in the design of protected protecting group can be helpful to adjust the protecting group properties. Thus far, it seems likely that protected formacetal is the most appropriate choice for the 2´-OH protecting group. Firstly, it introduces a non-migrating C–O bond and has minimal steric effect on the neighboring phosphoramidite group. Secondly, the formacetal group does not lead to the formation of op- tical isomers which might hinder isolation and characterization of phosphoramidites and their intermediates. Finally, the hemiformacetal, which is formed upon the deprotection, is unstable and degrades rapidly releasing 2´ hydroxyl. Thus, the deprotection rate of formacetal derived protecting group could be high and will largely depend on the group that protects the formacetal group.

Oligosynthesis cycle and deprotection conditions Oligonucleotide of the desired sequence is built on an insoluble polymer derivatized with the 3´-terminal nucleoside of the sequence attached via an ester bond, and the synthesis proceeds from 3´- to 5´-end via consecutive incorporations of nucleotides introduced in the amidite form. In modern solid-phase phosphoramidite oligosynthesis four reactions are performed in order to elongate an oligonucleotide by one nucleotide (Scheme 7, R is hydrogen or protected hydroxyl). The cycle starts from detritylation of the material bound to the support, liberating 5´ hydroxyls for the attachment of the next nucleotide of the sequence. Free hydroxyls then react with the phosphoramidite added during

26 the coupling step, and the reaction is usually promoted with tetrazole or its derivatives (reviewed in [155]). All molecules that failed to react are capped with acetyl to prevent their further elongation. The capping step is essential for oligonucleotide purification as it largely prevents formation of sequences containing deletions. The cycle is completed with an oxidation step forming stable phosphotriester. The synthesized oligonucleotide is cleaved from the solid support with concentrated ammonia and is further incubated in ammonia solution at 55qC for 8–12 hours for deprotection of exocyclic amines of aglycones. For oligoribonucleotides (Scheme 7, R is protected hydroxyl), an additional step of 2´-O-protecting groups’ removal is performed after the deprotection of aglycones.

27 1) Detritylation

O O H O DMTr H OH N N O O O O O O R R B B

2) Coupling

DMTr O B O

O R P CE O N(iPr) 2 O CE O OH O DMTr H O O P N H O N O O O O O R O O R B R B B

3) Capping

O O H OH O O N H O N O O O R O O R B B

4) Oxidation

O CE O CE O O DMTr O O DMTr H O P H O P N O N O O O O O O O O R O O R R B R B B B

Scheme 7. The solid-phase phosphoramidite oligosynthesis cycle

28 Purification of oligonucleotides Oligonucleotide synthesis proceeds with stepwise elongation of the molecule by one nucleotide during each cycle. Therefore, the efficiency of the coupling, capping and detritylation reactions altogether has a substantial impact on the resulting oligonucleotide purity and yield, as the synthesis impurities accumulate with every cycle. Oligonucleotide impurities originate from: x incomplete addition of nucleotides forming so-called truncated fragments, whose synthesis has been prematurely terminated by a subsequent capping step; x inefficient capping of sequences that have failed to incorporate a nucleotide forming oligonucleotides with internal deletions known as (n – x) fragments [156, 157]; x inefficient detritylation, also resulting in (n – x) fragments [157, 158]; x premature detritylation during coupling resulting in so-called (n + x) fragments which have duplicated nucleotides in the sequence [157, 159]; x depurination during the detritylation step, forming oligonucleotides with abasic sites [10, 11, 160, 161], which are cleaved later by ammonia during the deprotection stage (Scheme 8); x incomplete deprotection; x oligonucleotide derivatization with reagents used or formed in the synthesis and deprotection steps [162, 163]. Generally, impurities resulting from failed couplings and depurination are present in the greatest amounts in synthetic oligonucleotides.

CE CE O O CE CE + O P O 5' O P O 5' H O O 3' O P O O 3' O P O O O O O O B OH

aq. NH3

CE O O O P O 5' 3' O P O + HO O O O

Scheme 8. Depurination and abasic site cleavage

29 Commercial oligonucleotides are usually purified with RP HPLC or using RPC [164-167]. The 5´-O-protecting group is used to discriminate between the full-length product and failure sequences [168-170]. For this purpose the detritylation of the last incorporated nucleotide is omitted, leaving DMTr on the 5´-end of the synthesized oligonucleotide. The difference in chromatographic mobility between oligonucleotide strands that possess DMTr and those that do not, is great enough (Fig.7) to allow fast oligonucleotide purification using RPC without the need of the purification process monitoring. The only drawback of systems using distinction between tritylated and non- tritylated oligonucleotides is that they are unable to remove tritylated failure fragments which arise, for example, from depurination, as these fragments have chromatographic mobility very similar to the full-length product due to the presence of DMTr.

tritylated fragments

non-tritylated fragments

0 5 10 15 20 25 30 35

Figure 7. HPLC chromatogram of oligonucleotide mixture after the synthesis with “trityl on” option and deprotection with ammonia

There are “trityl off” oligonucleotide purification methods, such as PAGE and IE HPLC, which do not employ trityl-based distinction. These can yield purer oligonucleotides but are difficult to automate. Moreover, PAGE and HPLC methods are laborious and susceptible to human error, and that can easily outbalance gained extra purity. Precipitation of oligonucleotides with butanol at low temperature [171], cannot be regarded as an alternative purification method. Since the precipitation is unable to give comparable product purity, it therefore only represents fast and cheap enrichment of oligonucleotides and is generally employed to desalt oligonucleotide mixtures.

30 Functionalized solid support as a purification aid Depurination occurs during the detritylation steps and results in oligonucleotide strand breakage forming 5´-tritylated and 5´-phosphorylated fragments (Scheme 8). The truncated fragments that bear trityl groups pose a problem for trityl-based oligonucleotide purification, as these impurities are difficult to remove. Abasic sites, which arise from depurination, can be used to introduce a hydrophobic tag into oligonucleotides [172] for distinction between depuri- nated and undamaged sequence. Alternatively, it was proposed to treat the protected oligonucleotide bound to the support with 1 M lysine (pH 9) at 60qC for 90 minutes in order to hydrolyze abasic sites and remove tritylated truncated fragments [173]. It was stated that the truncated fragments can be removed under these conditions without significant loss of the product. To avoid the loss of full-length oligonucleotides during the cleavage of abasic sites it is desirable to use a linker with increased stability towards nucleophiles and basic media. The use of urethane-linked support was proposed for oligosynthesis [174], as it allowed treatment of the synthesized oligonucleotide with pyridine-2-carbaldoxime and TMG in dioxane – water for 16 hours at ambient temperature to cleave protecting groups, whereas succinate was not stable under those conditions. However, the urethane linked oligonucleotide requires long time for its release from the support (70% yield after 24 hour incubation in concentrated aq. ammonia at 56qC). Alternatively, the silyl-based linker was introduced for the synthesis of oligonucleotides [175]. Abasic sites were cleaved with TEA – ethanol mixture, and the pre-purified oligonucleotide was released from the support within 30 minutes using TBAF in dry DMF.

Oligonucleotide purification using RPC The RPC used for oligonucleotide purification are small disposable RP col- umns or membranes [164-167]. During the purification process a sequence of washes with ion-pair buffers containing different amounts of organic solvent is applied to the cartridge. It is possible to remove non-tritylated fragments with a certain percentage of organic solvent (acetonitrile is commonly used for this purpose), keeping tritylated oligonucleotide adsorbed to the cartridge support. Oligonucleotides can be detritylated on the RPC support without being eluted. Thus, minimizing oligonucleotide exposure to acidic conditions reduces the risk of glycosylic bond cleavage (Scheme 8). Purified and detritylated oligonucleotides are eluted, if necessary, even in salt-free media. Oligonucleotide purification using RPC can be combined with the method of abasic site cleavage [176], permitting the desired sequence to be isolated from all types of impurities. Speed and simplicity together with abil-

31 ity for automation, good product yield and purity favor RPC methods over other purification methods.

32 Present work

Paper I. A method for RPC oligonucleotide purification Several cartridge systems are commercially available and all of them utilize the same principle for detritylation of oligonucleotides. It became a standard to use aq. TFA for removal of the trityl group from oligonucleotides adsorbed on the cartridge during RPC purification. Our studies revealed that 5% aq. TFA applied on the cartridge required prolonged time for complete cleavage of DMTr from non-modified oligonucleotides and failed to detritylate 5´-amino-modified oligonucleotides bearing the MMTr group. Thus, oligonucleotide probes intended for array immobilization lack an option for fast and parallel purification, as they have the 5´-terminal amino group. These probes are widely used in biomedical research and applications, but the absence of efficient purification system reflects on their quality and cost. Rapid detritylation of both non-modified and amino-modified oligonucleotides can be achieved during solid-phase oligosynthesis. The low efficiency of detritylation on the cartridge could be due to reversibility of the reaction. Usage of aq. acid leads to the condition in which both products, trityl and the deprotected oligonucleotide, stay adsorbed to the cartridge support and therefore may undergo re-association. It is not surprising that amino-modified oligonucleotides have more pronounced effect of the counter reaction, as the amino group is a stronger nucleophile than hydroxyl. The role of the organic solvent during detritylation, which is performed on oligonucleotide synthesizer, is essential. The organic solvent removes the liberated trityl, whereas the growing oligonucleotide chain stays attached to the solid support, and therefore makes the detritylation process irreversible. To obtain a similar effect for detritylation using RPC, where an oligonucleotide is not covalently bound to the solid phase, additional steps were incorporated into purification procedure to prevent premature elution of the product. Similar to the procedure of oligonucleotide precipitation with alco- hol from salt solution, oligonucleotides are precipitated on the separating support by addition of pure acetonitrile. Indeed, it was possible to successfully detritylate the oligonucleotides precipitated on the RPC support with 2% TCA in DCM within 2 – 5 minutes. The released DMTr cation was removed from the cartridge, and the process could be monitored either with the naked eye or by spectrophotometry. The results supported our idea that a reverse reaction plays a crucial role in detritylation process. Accordingly, it

33 was found that MMTr-amino oligonucleotides were also effectively detritylated under these conditions, which permitted RPC purification of oligonucleotide probes used for immobilization on arrays. Drying of the adsorbed oligonucleotide was a critical step for the successful separation, as incompletely dried cartridge could suffer up to 50% of oligonucleotide losses. Unwanted elution of the product was undoubtedly caused by the admixture of the applied organic solvent with the aq. ion-pair buffer remained on the cartridge, which worked as an oligonucleotide eluting gradient. Our purification procedure was combined with the previously described- method [175] that allowed removal of tritylated impurities prior to the cartridge-based purification. The original method was modified to give better efficiency of cleavage of abasic sites. A study revealed that concentrated aq. ammonia performed best compared to ethanol solutions of a variety of organic bases (TEA, DBU, TMG), therefore concentrated aq. ammonia was used instead of the TEA – ethanol mixture proposed in the original method. The ammonia incubation of the support was performed at ambient temperature for 1 hour followed by treatment of the washed and dried support with TBAF. The released oligonucleotide mixture was incubated in ammonia for deprotection of aglycones and the resulting solution was purified using RPC. Initially, a silica-based RP material was tested as a cartridge separating support. The support performed well but did not tolerate ammonia and fluoride reagent present in the oligonucleotide solution. To avoid a cumbersome step of removal of the aforementioned contaminations a polystyrene-based RP support was employed. Working with several commercial cartridges it was found that many of them suffered from severe product losses (up to 90%) so, that in some cases purified oligonucleotide was of substantially lower quality than the crude mixture. The possible explanation of such results was ascribed earlier that these cartridges cannot tolerate ammonia. It was found that ammonia, as well as high pH buffer (pH 11.3) used in the study, behaved as a high-elution- strength mobile phase and did not require organic solvent presence for oligonucleotide elution [167]. In contrast, oligonucleotide losses were pre- vented in our study, when purification followed the described conditions. Among all cartridges in the study only Nensorb cartridge and a cartridge developed in the present study had high recovery yields and resulted in oligonucleotides with purities comparable to HPLC purified mixtures. In turn, HPLC purified 61-mers of the same purine-rich sequence obtained from commercial sources had substantially lower quality. The study presented in Paper I aimed to develop an efficient cartridge purification system that can be arranged in an array format and operated automatically in parallel. A practical solution was found for high-throughput purification of oligonucleotides including 5´-amino- and 5´-thiol-modified molecules using small disposable cartridges packed with 150 mg of polysty-

34 rene RP material. The procedure was performed for purification of oligonucleotides made at 0.2 Pmol scale within 30 minutes with low consumption of solvents. The described new detritylation procedure enabled fast cartridge purification of oligonucleotides with good recovery and purity, comparable to oligonucleotides purified using HPLC. Preliminary data of oligonucleotide arrays, made of 5´-amino-modified oligonucleotides that were purified using the present method, demonstrated better linearity of signal versus probe concentration. Undoubtedly, at equal probe concentrations signals were higher for the aforementioned probes compared to the probes that were just desalted.

35 36 Paper II. Solid-phase RNA synthesis using 2´-O-tert- butyldithiomethyl (2´-O-DTM) protecting group There are several methods to produce synthetic RNA but they all have certain limitations and drawbacks. For example, methods employing silyl-based 2´ hydroxyl protecting groups cannot involve relatively strong bases, such as concentrated aq. ammonia, since these groups are sensitive to basic media. Therefore, these methods cannot use standard aglycone protecting groups [58]. Instead, more base-labile protecting groups are employed for the protection of aglycones, and aq. ammonia is replaced with different mixtures performing cleavage under milder conditions [154]. In addition, RNA oligonucleotides protected with silyl-based 2´-OH protecting groups might be hard to deprotect completely [60]. The method utilizing 2´-O-ACE protecting groups cannot employ the 5´- O-DMTr group and the phosphate protecting cyanoethyl group due to their incompatibility with the chosen strategy. The aforementioned standard groups are replaced with the DOD and the methyl group respectively. As a result, besides a set of new reagents and conditions that are less convenient, a hardware modification is required, as the fluoride reagent used for the stepwise removal of the 5´-O-DOD group is incompatible with parts made of glass. All commercially available methods for the synthesis of RNA lack the ability to preserve the 5´-O-DMTr group for the subsequent trityl-based oligonucleotide purification. For instance, the method employing the 2´-O- ACE protecting group does not have the acid-labile DMTr. The 5´-O-DOD group, as a 5´-terminal hydrophobic alternative to the trityl group, cannot be preserved due to its lability towards bases. On the other hand, silyl-based 2´ hydroxyl protecting groups allow retention of 5´-O-DMTr, but significant loss (more than 50%) of the DMTr group during the deprotection of 2´ hydroxyls was observed in our studies. Performing cartridge-based separation of such mixtures, where the major part of the product is detritylated, would be characterized by low recovery yields. Thus, the option of high-throughput cartridge-based purification of RNA oligonucleotides is lost, if existing commercial methods are applied for the synthesis of RNA.

37 DMTr O B O

O O S S P N O CE

O O N(Ph)2

HN Ph O HN Ph O O O N N N N NH N NH O N O B = N N N O N O N N N N N N H H

1a 1b 1c 1d 1e The lack of an RNA synthesis method which preserves the 5´-O-DMTr group for subsequent trityl-based purification and produces deprotected oligoribonucleotides within short time in aqueous and homogeneous media, stimulated the development of the alternative 2´ hydroxyl protecting group used in this study. A disulfide-based t-butyldithiomethyl (DTM) group was constructed and employed in the present method to block 2´ hydroxyl of the phosphoramidites 1a-e. The protecting group was designed in the way that it represents a protected protecting group derived from formacetal type protecting groups. In contrast to the protecting groups which utilize protection of the 2´ hydroxyl via formacetal, a thioformacetal group was employed. The obtained O,S-acetal is stable towards acids used in oligosynthesis, and the presence of tert-butyl stabilized the disulfide bond towards bases and nucleophiles. The stability of the 2´-O-DTM group allowed keeping the standard protecting groups used in the DNA oligosynthesis. The 2´-O-DTM group is cleaved in two consecutive reactions. The disulfide bond cleavage yields unstable thiohemiacetal which releases the 2´ hydroxyl upon degradation (Scheme 9). Under appropriate conditions these reactions proceed rapidly making 2´-O-DTM a convenient group for oligonucleotide protection.

B B O B O O

O S S O SH OH

Scheme 9. Deprotection of the 2´-O-DTM nucleotides

The 2´-O-DTM group is introduced using 2´-O-methylthiomethyl (2´-O- MTM) nucleoside derivatives (Scheme 10). The synthesis route gives possibility to incorporate any alkyldithiomethyl group in order to adjust the protecting group properties by selecting different thiols. The synthesis started

38 with activation of 2´-O-MTM nucleosides into the reactive 2´-O-halomethyl intermediates, which can be performed using various halogenating reagents, such as sulfuryl chloride, halogens, N-halogensuccinimides or even cupric bromide [177-179]. The obtained intermediate reacted with potassium thiotosylate with subsequent formation of the desired alkyldithiomethyl group upon reaction with thiol.

O B O B O B O O O Si Si Si O O O O Si O O S Si O O Cl Si O O S S O

DMTr O B O O B O Si O O S S O P Si O O S N O CE S

Scheme 10. Synthesis of the 2´-O-DTM amidites

Stability studies of different alkyldithiomethyl groups revealed that primary thiols failed in production of stable derivatives. Analogues 2a-b were degraded by ammonia and iodine, and the corresponding amidites were unstable in acetonitrile. According to the literature [180] the presence of electron withdrawing substituents decrease the disulfide bond stability in basic media. Thus, the presence of the electronegative oxygen atom of the O,S-acetal function should also destabilize the disulfide bond against bases and nucleophiles. To increase the stability of the disulfide bond, tert-butyl mercaptan was used as it has a pronounced electron donating effect and also creates steric hindrance around the disulfide bond. The choice of tert-butyl mercaptan as tertiary thiol was due to its simplest structure and commercial avail- ability. However, special precautions had to be taken working with this compound. To avoid contamination of the working atmosphere with the strong and unpleasant odor that can be mistakenly regarded as a gas leakage, all reactions involving its presence have to be made under the fume hood with minimal exposure to the air. Evaporation of the excess of the thiol required permanganate or other oxidizing agent trap. It is also suggested to use sodium tert-butyl thiolate instead of the free thiol during preparation of the phosphoramidites.

39 DMTr O B O

OH O S S OH

2a, B= T 2b, B= Cbz The obtained 2´-O-DTM protected phosphoramidites were stable as solids during storage at –20qC but gradually degraded in acetonitrile at ambient temperature. Whereas the stability of all nucleoside phosphoramidites is limited under these conditions, the degradation of guanosine amidite 1d did not fit the common pattern (Fig.8). As the 2´-O-DTM phosphoramidites were used immediately after they were dissolved, the instability of the guanosine amidite 1d did not significantly influence oligosynthesis.

80 6a in MeCN 6b in MeCN 70 6b in THF 60 6b in DMF 6c in MeCN 50 6d in MeCN 40 11 in MeCN 30

20 Decomposition (%) Decomposition 10

0 0 20 40 60 80 100 120 140 160 180 Time (h)

Figure 8. Decomposition rates of the 2´-O-DTM phosphoramidites.

The lowered stability of the amidites 1d and somewhat 1b led to a hypothesis that it was influenced by the unprotected lactam function. The hypothesis was supported experimentally, as the guanosine amidite with O6-protection (1e) was more stable. Noteworthy, the stability of 1d was much greater in THF and DMF used as alternative solvents (Fig.8). The DTM group can be rapidly cleaved with thiols or other reducing agents (Fig.9) commonly used to break the disulfide bond. The cleavage leads to the unstable thiohemiacetal which regenerates 2´ hydroxyl upon degradation (Scheme 9). The kinetics of the DTM group cleavage was stud-

40 ied under various conditions. The two most popular reagents, DTT and TCEP (Fig.9), were used to establish times for oligoribonucleotide deprotection at different values of pH and temperature. The data obtained from the deprotection of dimers revealed that the 2´-O-DTM group cleavage time depends on the nucleotide composition.

HO B O

O O S S O P O O C O

OH OH

3a, B= T 3b, B= U 3c, B= C 3d, B= G Thus, 2´-O-DTM-adenilyl-(3´o5´)-cytidine (3a) was deprotected fastest and its deprotection rate was about twofold faster than that of the slowest cytid- inyl-(3´o5´)-cytidine derivative (3c). Increasing pH or temperature acceler- ated the rate of DTM cleavage. Obviously, the nucleophilicity of DTT is higher in basic media, but the stability of the thiohemiacetal intermediate was also lowered at more basic pH. Unlike hemiacetals, this intermediate formed upon cleavage of the disulfide bond was quite stable in acidic media. Whereas it is possible to break the disulfide bond with TCEP even at low pH, most probably, protonation of the sulfur atom of the resulting intermediate prevents regeneration of the 2´ hydroxyl.

COOH HS SH SH HO HO OH P HOOC COOH 2-mercaptoethanol DTT TCEP

Figure 9. Reagents for disulfide bond cleavage

Deprotection rates were also dependent on oligonucleotide lengths. Com- pared to deprotection of dimers, which required less than 10 minutes, a 45- mer was deprotected within 90 minutes under similar conditions. This difference was due to the number of DTM groups to be cleaved. As the cleaving reagent should be used in excess for rapid deprotection, its concentration has to be adjusted according to the concentration of DTM groups. For example, 50 mM DTT was enough to deprotect 0.2 mM dimer (250-fold excess); but that concentration was low to deprotect 0.2 mM 41-mer which has 40 times more DTM groups (about 6-fold excess). The degradation of the thiohe-

41 miacetals from long oligonucleotides might also be slowed down. As a result, deprotection times of synthesized RNA oligonucleotides were about 1 – 1.5 hour at pH ~7.5, 55qC, which can be shortened by either raising the temperature or adjusting to more basic pH. These aqueous and slightly basic conditions yielded RNA with the DMTr group retained on the 5´-end of the oligonucleotides. The obtained deprotected RNA oligonucleotides with retained 5´-O-DMTr were subjected to the RPC purification, which can be performed in parallel. In particular, RNA molecules synthesized with the present method can make use of all advantages of the previously described cartridge-based oligopurification method.

42 Paper III and IV. Synthesis of 3´-O-alkyldithiomethyl analogues and their application in oligonucleotide synthesis The successful development of the 2´-O-DTM protecting group, which with- stands oligosynthesis conditions and treatment with ammonia, stimulated application of the dithiomethyl group as a linker for reversible conjugation of oligonucleotides to a functionality of choice. The ability of the dithiomethyl function to regenerate unmodified oligonucleotide upon its cleavage is hard to overestimate, in respect that such orthogonal linkers is problematic to find. In Paper III the synthesis of the dithiomethyl linkers is described which was performed according to Scheme 11 starting from MTM derivative 4. An attempt to utilize the compound 8 in solid-phase synthesis of oligonucleotides revealed that the amidite degraded rapidly in acetonitrile. Moreover, the disulfide bond in the precursor 5 was completely cleaved during incubation in ammonia – ethanol (1:1) at 55qC overnight. Such remarkable stability difference compared to the 2´-O-DTM function was ascribed to the presence of the hydroxyl group in 5 and the phosphoramidite moiety in 8 in close proximity to the disulfide bond. It was assumed that intramolecular cleavage of the S–S bond in the compound 8 is achieved by nucleophilic attack of phosphorus. On the other hand, the mechanism of the disulfide stability influenced by the hydroxyl group present nearby in 5 is not clear. Reports of the nucleophilic cleavage of such substituted disulfides are controversial. The increased cleavage rates of disulfides with neighboring amino or hydroxyl groups have been ascribed by some authors to the direct intramolecular hydrogen catalysis [181], whereas others reported no evidence for such direct participation and explained observed reactivity solely by polar and steric effects [180]. However, elongation of the alkyl chain of 5 resulted in stable analogues 6 and 9, in which the disulfide bond was not destabilized by the presence of the formed carbamide moiety. Thus, derivatives 6 and 9, together with separately synthesized 7 and 10, were stable and allowed application of amidites 9 and 10 in solid-phase oligosynthesis.

43 MMTr O T i, thiol = MMTr O T O HS OH O

O S O S S OH 4 5

i, thiol = OH HS ii

MMTr O T MMTr O T O O O O S OH O S S S O N OH 7 H 6

MMTr O T O iii 5 O S N S O P O CE 8

MMTr O T O iii O 6 O S N S O N O P H O CE 9

MMTr O T O iii N 7 O S O P S O CE 10

Scheme 11. Synthesis of amidites used in the present studies: (i) a) SO2Cl2, TEA in 1,2-dichloroethane; b) p-MePhSK in DMF; d) thiol, DIEA; (ii) a) CDI in dioxane; b) 5-aminopentanol in Py; (iii) (iPr)2NP(Cl)OCH2CH2CN, TEA in CH2Cl2.

Reversible conjugation was modeled by the synthesis of the oligonucleotide 11, in which decathymidilic acid was attached to pentathymidilic acid via the dithiomethyl linker (Scheme 12).

44 5' O 3' HO TTTTTTTTTT O S S O N O TTTTT OH H 11

O 5' 3' 5' 3' + HO TTTTTTTTTT O SH HS O N O TTTTT OH H 12 13

5' 3' HO TTTTTTTTTT OH

Scheme 12. Cleavage of the dithiomethyl linker in conjugate 11

Cleavage of the linker proceeded in the same manner as the 2´-O-DTM group deprotection. The intermediate thiohemiformacetal 12 was formed, yielding unmodified oligonucleotide upon degradation (Fig.10). Compared to the succinyl and disiloxyl linkages which were used to attach oligonucleotide to the solid phase, the present alternative is stable both in acid and in concentrated aq. ammonia but can be rapidly cleaved with DTT or TCEP in aq. medium. Due to such orthogonal properties the dithiomethyl linker allows for its combination with acid- and base-labile groups and can be used in applications demanding stable covalent attachment of oligonucleotides to either solid phase or other molecules of choice with the option of subsequent release of oligonucleotide bearing unmodified 3´ hydroxyl. The disulfide bond in dithiomethyl linker was cleaved as fast as in the case of the 2´-O-DTM protecting group, but the 3´-terminal thiohemiformacetal required longer time for degradation compared to that in 3a–d. Similar to the 2´-O-DTM group the rate of dithiomethyl linker cleavage can be increased by raising the temperature or/and by adjusting to more alkaline conditions.

45 Figure 10. Deprotection of 11 studied by RP HPLC. (A) crude oligonucleotide 11, (B) 0.2 mM 11 incubated with 50 mM DTT, 5 min, 20qC, pH 10; (C) 0.2 mM 11 incubated with 50 mM DTT, 120 min, 20qC, pH 10; (D) 0.2 mM 11 incubated with 2 mM DTT, 30 min, 20qC, pH 8.5. HPLC: Chromolith Performance RP-18e (100 x 4.6 mm) with linear gradient from 5% MeCN in 0.1 M TEAA (pH 7.0) to 40% MeCN in 0.1 M TEAA (pH 7.0) in 30 min, 1.0 mL/min. Ordinate axes correspond to absorbance at 260 nm.

The synthesized dithiomethyl linker was employed for solid support derivatization in order to remove tritylated truncated sequences and base-labile protecting groups prior oligonucleotide release from the solid phase. A polystyrene-based support [182] functionalized with hydroxyl groups according to the described procedure [175] was used to synthesize purine rich oligonucleotide, 5´-(AG)30T, utilizing the amidite 10 at the first coupling. The same sequence was synthesized as a reference starting from the succinyl amidite 14 (Scheme 13).

1. synthesis of 5'-(AG)30T with 14 as a linker DNA-1

2. cleavage with 32% aq. NH3 O followed by incubation H N OH at 55oC overnight (16 h) PS N H O

1. synthesis of 5'-(AG)30T with 10 as a linker DMTr O T O DNA-2 O 2. incubation with 32% aq. NH3 o O O O CE at 55 C overnight (16 h) O P O N(iPr) 3. cleavage with 50 mM DTT 2 (TCEP) in aq. NH at 55oC 14 3 within 10 min

Scheme 13. Synthesis of (AG)30T on polystyrene support using succinyl and dithiomethyl linker.

46 Figure 11. Analysis of (AG)30T synthesized on the polystyrene support. (A) DNA-1; (B) DNA-2; (C) supernatant taken after incubation of the support-linked DNA-2 in 32% aq. NH3 for 4 h at 20qC; (D) supernatant taken after consecutive incubation of the support-linked DNA-2 in fresh portion of 32% aq. NH3 for 1 h at 55qC; (E) supernatant taken after consecutive incubation of the support-linked DNA-2 in fresh portion of 32% aq. NH3 overnight (16 h) at 55qC. Absorbance scale given for chromatogram B corresponds with that of chromatograms C, D and E. HPLC: Phenome- nex Luna 5P C18 (250 x 2 mm) with linear gradient from 5% MeCN in 0.1 M TEAA (pH 7.0) to 40% MeCN in 0.1 M TEAA (pH 7.0) in 30 min, 0.6 mL/min.

Unfortunately, for the chosen polystyrene support incomplete detritylation was observed during oligosynthesis using standard DNA cycle and 2% TCA in DCM for detritylation. To minimize formation of (n – x) fragments the detritylation step was modified with prolonged waiting and extended wash- ing of the support column with 2% TCA solution to ensure complete removal of liberated trityl. However, extended detritylation led to enhanced depurination that is very favorable on chosen sequence. Despite resulting substantial contamination of the product with failure sequences generated during prolonged detritylation, these impurities were successfully removed during incubation of the oligonucleotide in ammonia prior its cleavage from the solid phase (Fig.11). Ammonia incubation was made in a way to reveal the condition needed to cleave abasic sites completely and remove all failure fragments unattached to the solid phase. In a separate experiment, the same purine-rich oligonucleotides were synthesized on the polystyrene support but without a cleavable linker. The obtained data indicated that ammonia incubation at ambient temperature as well as treatment with TEA or piperidine in ethanol at 55qC was insufficient to wash away all such impurities (Fig.12), and the efficiency of solid supported oligonucleotide purifications reported previously [173-175] was not perfect. Thus, besides the advantage of rapid

47 cleavage in aqueous media, the dithiomethyl linker allows complete removal of apurinic failure fragments with ammonia. Complete cleavage of the dithiomethyl linker with either TCEP or DTT, yielding unmodified oligonucleotide, takes less than 10 minutes at 55qC; and the deprotected and partially purified oligonucleotides can be obtained within the same time as it is needed to obtained crude deprotected product only.

Figure 12. Removal of failure fragments from oligonucleotide (AG)30 attached to the polystyrene support via phosphate linkage. The solid phase was divided and incubated with corresponding reagents. Supernatant fractions were renewed after every incubation under given conditions. Axes Y and X correspond to absorbance at 260 nm and retention time (min), respectively. HPLC: Phenomenex Luna 5P C18 (250 x 2 mm) with linear gradient from 5% MeCN in 0.1 M TEAA (pH 7.0) to 40% MeCN in 0.1 M TEAA (pH 7.0) in 30 min, 0.6 mL/min.

In Paper IV a strategy of using 3´-terminal hydrophobic group along with the 5´-O-DMTr group for cartridge-based purification of oligonucleotides is presented. The amidite containing a hydrocarbon side chain (15) was used to introduce hydrophobic handle onto 3´-end of the oligonucleotide 16. Incor-

48 poration of two molecules of 15 adjusted hydrophobic properties of the oligonucleotide in such a way that all failure fragments lacking 3´-terminal hydrophobic moiety, including tritylated oligonucleotides, were easily washed away from the cartridge while the product bearing the 3´-terminal function was unaffected. Thereby, the cartridge-based purification is able to remove both tritylated and non-tritylated failure fragments. Oligonucleotides can be synthesized on any commercial support containing succinyl linker regardless of the first incorporated nucleoside and no ammonia stable support is needed. For the synthesis of the oligonucleotide 16 standard ABI CPG synthesis column was used and T10 linker was constructed to allow monitoring of the released 3´-terminal hydrophobic thiol.

N O DMTr P O O NH

O C H CN 15 31

O C15H31 O C15H31 5' 3' O HN O HN DMTr O (AG) T O S O 30 S O N (CH ) O P O O H 2 5 O P O O O P O T O 10 O 16 Purification of the obtained 16 proceeded according to the modified version of the procedure described in Paper I. The novel procedure includes additional steps of cleavage of the dithiomethyl linker and removal of sequences lacking 3´-terminal hydrophobic group. Thus, ammonia solution of the crude oligonucleotide mixture after deprotection was applied on the cartridge, where all possible oligonucleotide impurities were adsorbed along with the desired product (Scheme 14). Presence of the 3´-terminal function, much more hydrophobic than trityl, allowed successful removal of the fragments (IV) along with the fragments (III) otherwise inseparable from the desired product in trityl-based purification methods. Cleavage of the dithiomethyl linker in the step b resulted in formation of 3´-unmodified oligonucleotides where only the full-length product is tritylated and can be easily separated from the remained failure fragments. Note, that the released 3´-terminal hydrophobic group was not eluted together with the product in the step e, as it is even more hydrophobic than the starting oligonucleotide 16 (Fig.12). The presented purification method using a 3´-terminal hydrophobic group attached via the dithiomethyl linker further enhanced applicability of this

49 chemistry, as the unmodified oligonucleotide was produced directly on the cartridge in the step b within 30 minutes. Thus, the dithiomethyl linker represents a group that is orthogonal to all other protecting groups used in solid-phase oligonucleotide synthesis but at the same time is cleavable under mild aqueous conditions, which can be used for oligonucleotide purification, reversible labeling and conjugation.

A B Abs at 260 nm 260 at Abs Abs at 260 nm Abs at 260

0 5 10 15 20 25 30 0 5 10 15 20 25 30 Time (min) Time (min)

Figure 12. HPLC analysis of oligonucleotide 16. (A) crude 16; (B) elution of all material from the cartridge with 80% MeCN in 0.1 M TEAA after treatment with DTT. HPLC: Hamilton PRP-1 with linear gradient from 5% MeCN in 0.1 M TEAA (pH 7.0) to 80% MeCN in 0.1 M TEAA (pH 7.0) in 30 min, 0.5 mL/min.

50 3' 5' (I) R-SS-CH2-O- -O-DMTr 3' 5' (II) R-SS-CH2-O- -OH 3' 5' (III) HO- -OH 3' 5' (IV) HO- -O-DMTr

a) 30% MeCN in 0.1 M TEAA

3' 5' (I) R-SS-CH2-O- -O-DMTr 3' 5' (II) R-SS-CH2-O- -OH

b) 0.15M DTT (or TCEP), pH 10

3' 5' HO- -O-DMTr (I) 3' 5' HO- -OH (II)

c) 15% MeCN in 0.1 M TEAA

3' 5' HO- -O-DMTr (I)

d) 2% TCA in DCM

3' 5' HO- -OH (I)

e) 15% MeCN in 0.1 M TEAA

Scheme 14. Cartridge-based oligonucleotide purification using two hydrophobic group strategy. For the sake of clarity the released 3´-terminal hydrophobic group after step b is not presented on the scheme, though it stays on the cartridge and can be eluted with 80% MeCN in 0.1 M TEAA. R = 3´-terminal part of 16.

51 Summary in Swedish

Nya metoder för syntes av oligonukleotider med högre kvalitet beskrivs i denna studie. Den första delen av arbetet beskriver en metod för att rena oligonukleotider med omvänd fas-cartridge. Den framtagna metoden innefat- tar en mycket effektiv och mild detritylering av oligonukleotider på en cartridge support, vilket möjliggör en snabb rening av oligonukleotider obero- ende av vilken 5´-modifiering de har. Tiol- och aminmodifierade oligonukleotider detritylerades och renades med samma höga effektivitet som omodi- fierade oligonukleotider. Den beskrivna metoden möjliggör alltså en snabb och parallel automatisk rening av oligonukleotider som tidigare inte var möj- lig. I kombination med metoden för att avlägsna depurinerade fragment kun- de denna metod framställa oligonukleotider med en renhet som var överläg- sen renheten hos oligonukleotider renade med omvänd fas-HPLC. I den andra delen av denna studie diskuteras en metod för att syntetisera RNA där tert-butylditiometyl (DTM) användes som skyddande grupp för 2´- OH. DTM-gruppens stabilitet under oligonukleotidsyntes och under avlägs- nande av skyddande grupper med ammoniak, samt dess förmåga att snabbt kunna klyvas av under milda betingelser gjorde att kvaliteten på det synteti- serade RNAt var jämförbar med kvaliteten hos syntetiserat DNA. Fördelen med 2´-O-DTM-gruppen är att den är helt ortogonal mot de skyddande grupper som används inom den traditionella DNA-syntesen. Syntesen kan därför göras med de standardmetoder som finns för att syntetisera DNA – ingen förändring behövs av procedurerna för sammansättning och klyvning från fast fas, eller efterföljande borttaganing av skyddande grupper såsom aglykoner och fosfater. Eftersom RNA-oligonukleotider syntetiseras med en kvarlämnad 5´ tritylgrupp kan de renas med den ovan beskrivna ”cartridge”- baserade reningen. Fosforamiditsyntesen är optimerad för storskalig produk- tion och gör det möjligt att introducera andra alkylditiometylgrupper med olika egenskaper. Den tredje delen av avhandlingen beskriver syntesen av en ditiometyllänk och dess användning för reversibla konjugeringar av oligonukleotider. En ditiometylgrupp, klyvbar under milda betingelser, kopplades till 3´- hydroxiden på tritylerade nukleosider via 3´-O-methyltiometylderivat. Olika alkylsubstituenters påverkan av disulfidbindningens stabilitet studerades, och stabila analoger användes för att utföra oligonukleotidsyntesen. Två tillämp- ningar där den beskrivna ditiometyllänken användes utvecklades: 1) rening av oligonukleotider fastlänkade till en fast fas, och 2) en cartridge-baserad

52 rening av tritylerade oligonukleotider med ytterligare en hydrofob grupp i 3´-änden.

53 Acknowledgements

This work has been performed at the Department of Genetics and Pathology of Uppsala University and was supported by Quiatech AB. First of all, I would like to thank my supervisor, Dr. Marek Kwiatkowski, who gave me the opportunity to work in such a beautiful town as Uppsala. I will remember that you did not hesitate to come to Moscow and share your thoughts; to give me inspiration and guide towards oligonucleotide chemistry. Of course, my PhD would not be complete without your challenging ideas and our scientific debates. I would like to express my gratefulness to my second supervisor, Prof. Ulf Landegren, for letting me join his enormous group and always giving valuable suggestions. I will not forget people from Quiatech for their help in projects and social events. My best wishes to the chemistry guys Tommy and Johan, with special thanks to Andras for his valuable help with NMR. I also wish Matilda, Camilla and Peter all the best! I am very grateful to Jenny for her outgoing- ness. Thank you Jenny that you always had time to fix things and keep ma- chines alive! I will definitely miss Karen’s encouraging humor. I hope, Karen, that you are doing well! I’m also thankful to acquaintance with Torbjörn, Anders, Yong, Per Johan and Karina. I am grateful to Mathias for correcting my language in this thesis and to Kalle for his valuable help with the Swedish summary. I am also indebted to Tim for our fruitful discussions and his honesty. I thank you, Tim and Lei, for your endless energy and enthusiasm! You brought quite spectrum of fun and warmth.

I thank my Russian friends in Uppsala: Boriss, Leonid, Yura, Ruslan and Oksana, Damir, Oleg, Kristina and Slava, Nadya and Mitya... for being friends and not allowing me to become stale. Thank you all and good luck with your research! And I would not miss to thank ...... for being kind to me and sharing beautiful moments of life. Thanks a lot! I’d like to thank Alexander Papchikhin who contacted Krayevski’s lab and then all things have started. I’m also in debt to Yura Sharkin for encouraging me to go to Sweden.

54 Very many thanks go to my teacher Svetlana Petrovna who fostered my cu- riosity in chemistry and led me to the Chemistry Department at Lomonosov Moscow State University where I also met my wife, Natalia. I am deeply thankful to my beloved Natalia for her endless patience and love. This thesis would never be made without her emotional support and kindness. Thank you, my love, for your unquenchable energy which is enough for us both! I would like to express my special gratitude to my mother who gave me birth and led me to the scientific world.

55 References

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