Synthesis and characterization of oligonucleotides containing 2'-0-substitued adenosines

Tremendous interest has been shown in small oligonucleotides as antisense inhibitors of gene expression in the last few years. Considerable efforts have been made to stabilize the duplex formed between the antisense oligonucleotide and its target. The work presented in this dissertation is concerned with the same problem. However, a somewhat different approach was considered to address this issue. The primary goal of the research was to incorporate a 2'-O-aralkyl nucleoside into DNA as well as a DNA:RNA hybrid and to study in detail the manner in which 2'-O-aralkyl substituent would influence the structure of the ribonucleotide to which it is attached and the nucleic acid duplexes with which it interacts. This could, in principle, lead to duplex stabilization or destabilization. The information gained from this study will enable evaluation of the criteria required for helix stabilization by intercalation. These studies will also be useful in elucidating the nature of non-intercalative modes of interaction between duplexes and an aromatic ring. 2'-O-Phenethyladenosine was synthesized and incorporated in DNA:DNA and DNA:RNA duplexes. The ultraviolet (UV) melting temperature studies (Tm) indicated that the modification destabilized both duplexes. The detailed conformation of the modified duplexes was studied using high resolution nuclear magnetic resonance (NMR) and molecular modeling. These studies revealed that the benzene ring preferred the major groove of the duplex over the minor groove or the intercalation inside the duplex. The hydrophobicity of the benzene was the driving force for this location of benzene ring. These studies indicated that the benzene ring was too small for stable intercalation. Consequently, an anthraquinone was conjugated to the 2'-hydroxyl of adenosine via a methylene bridge. This compound, which incorporated into the oligonucleotides, showed significant stabilization of the duplex. Detailed NMR studies and molecular modeling studies revealed the anthraquinone was intercalated inside the helix despite a short methylene linker. The observation made in the dissertation research are unique and will have significant impact on the design of the next generation of nucleic acid binding molecules like intercalator and groove binders.

THE SYNTHESIS AND CHARACTERIZATION OF OLIGONUCLEOTIDES CONTAINING 2'-O-SUBSTITUTED ADENOSINES by Hemant M. Deshmukh A dissertation submitted to the faculty of The University of Utah in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Medicinal Chemistry The University of Utah August 1994 Copyright © Hemant M. Deshmukh 1994 All rights reserved THE UNIVERSITY OF UTAH GRADUATE SCHOOL SUPERVISORY COMMITTEE APPROVAL of a dissertation submitted by Hemant M. Deshmukh This dissertation has been read by each member of the following supervisory committee and by majority vote has been found to be satisfactory. L)" .)) /) " , ~/j, I ,/. / "1,-,,,., // I 'u U "x" , ",' ( I L /Y ) I" ( / // .) (, l ~ U14fj/J:ri£ c'A<J l /; $1 ,;[31 rliy //:. //' r ./ .,' /' ///;~d~~ 6::>Darrell R. Davis QJameS A. McCloskey 2.3 !v)~ I (991 Frederick G. West I THE UNIVERSITY OF UTAH GRADUATE SCHOOL FINAL READING APPROVAL To the Graduate Council of the University of Utah: I have read the dissertation of Hemant M. Deshmukh in its final form and have found that (1) its [onnat, citations and bibliographic style are consistent and acceptable; (2) its illustrative materials including figures, tables, and charts are in place; and (3) the final manuscript is satisfactory to the supervisory committee and is ready for submission to The Graduate School. / \ Arthur D. Br~om =. Chair, Supervisory Committee Approved for the Major Department > ) '/ '&f~/;ir, /~<);/( ./://L .. Arthur D. Broom Chair/Dean Approved for the Graduate Council Ann W. Hart Dean of The Graduale School ABSTRACf Tremendous interest has been shown in small oligonucleotides as antisense inhibitors of gene expression in the last few years. Considerable efforts have been made to stabilize the duplex formed between an antisense oligonucleotide and its target. The work presented in this dissertation is concerned with the same problem. However, a somewhat different approach was considered to address this issue. The primary goal of the research was to incorporate a 2'-O-aralkyl nucleoside into DNA as well as a DNA:RNA hybrid and to study in detail the manner in which the 2'-O-aralkyl substituent would influence the structure of the ribonucleotide to which it is attached and the nucleic acid duplexes with which it interacts. This could, in principle, lead to duplex stabilization or destabilization. The information gained from this study will enable evaluation of the criteria required for helix stabilization by intercalation. These studies will also be useful in elucidating the nature of non-intercalative modes of interaction between duplexes and an aromatic ring. 2'-O-Phenethyladenosine was synthesized and incorporated into DNA:DNA and DNA:RNA duplexes. The ultraviolet (UV) melting temperature studies (T m) indicated that the modification destabilized both duplexes. The detailed conformation of the modified duplexes was studied using high resolution nuclear magnetic resonance (NMR) and molecular modeling. These studies revealed that the benzene ring preferred the major groove of the duplex over the minor groove or the intercalation inside the duplex. The hydrophobicity of the benzene was the driving force for this location of benzene ring. These studies indicated that the benzene ring was too small for stable intercalation. Consequently, an anthraquinone was conjugated to the 2'-hydroxyl of adenosine via a methylene bridge. This compound, when incorporated into the oligonucleotides, showed significant stabilization of the duplex. Detailed NMR studies and molecular modeling studies revealed the anthraquinone was intercalated inside the helix despite a short methylene linker. The observations made in the dissertation research are unique and will have significant impact on the design of the next generation of nucleic acid binding molecules like intercalator and groove binders. v TABLE OF CONTENTS ABSTRACT ........................................................................................ iv LIST OF TABLES ................................................................................ viii LIST OF FIGURES ................................................................................ ix ACKNOWLEDGMENTS ...................................................................... Xlll Chapter 1. INTRODUCTION ......................................................................... 1 Introduction to antisense oligonucleotides..................................... 1 Modifications on phosphodiester backbone................................... 6 2'-O-alkyl oligonucleotides: A historical perspective ......................... 9 Earlier studies on 2'-0-methyl ribonucleosides ............................... 12 Synthesis of 2'-O-alkyl ribonucleosides ...................................... 14 Applications of oligonucleotides containing 2'-O-alkyl ribonucleotides .................................................................. 21 Covalent modification of oligonucleotides with intercalating agents .............................................................. 24 Detection of intercalation by physicochemical techniques.............. .... 31 NMR spectroscopic studies on DNA ......................................... 35 Assignment of exchangeable protons ......................................... 36 Assignment of nonexchangeable protons.... ................ ................ 37 NMR spectroscopic studies on RNA ......................................... 42 Assignment of exchangeable protons..... ................................... 44 Assignment of nonexchangeable protons....... ........... .................. 44 Conformational studies on DNA:RNA hybrids ............................. 47 2. STATEMENT OF THE PROBLEM .................................................. 52 Goals and objectives.............................. ............................. 52 Rationale for the thesis compounds.......................................... 54 3. SYNTHESIS OF 2'-O-ALKYL ADENOSINES AND THEIR INCORPORATION INTO OLIGONUCLEOTIDES ............................... 65 Synthesis of 2'-0-phenethyladenosine and 2' -0 -(p-azidophenethyl)adenosine ............................................ 65 Protection of 2'-0-phenethyladenosine............ ..... ............. ........ 69 Protection of 2'-0-(p-azidophenethyl)adenosine ............................ 71 4. CHARACTERIZATION OF OLIGONUCLEOTIDES CONTAINING 2'-O-PHENETHYLADENOSINE BY UV AND CD SPECTROSCOPy ................................................. 74 Synthesis and purification of oligonucleotides .............................. 74 The melting temperatures (Tm) measurement ................................ 76 Circular dichroism spectroscopy............................................. 82 5. CHARACfERIZA TION OF OLIGONUCLEOTIDES BY HIGH RESOLUTION NMR SPECTROSCOPy ............................................ 87 Purification of oligonucleotides... ............................................ 87 One-dimensional NMR spectroscopy ......................................... 90 Phosphorus NMR spectroscopy .............................................. 95 Characterization of oligonucleotides using two-dimensional NMR spectroscopy............................................................. 95 Model for destabilization of the phenethyl 12-mer ......................... 108 Conclusion ...................................................................... 111 6. CHARACTERIZATION OF DNA:RNA HyBRIDS .............................. 113 Synthesis and purification of DNA:RNA hybrids ......................... 113 UV temperature melting (T m) studies.......... .......... ...... ............. 117 High resolution NMR studies ................................................ 119 Model for stability of the phenethyl hybrid ................................. 132 Conclusions .................................................................... 134 7. SYNTHESIS AND CHARACfERIZA TION OF OLIGONUCLEOTIDE CONTAINING 2'-O-(ANTHRAQUINONYLMETHYL)ADENOSINE ........ 135 Synthesis of 2'-O-(anthraquinonylmethyl)adenosine..................... 135 Synthesis and purification of anthra 12-mer............................... 136 The UV melting studies.... ........................... ............... ........ 138 The circular dichroism studies.. ..... ....................... ......... ........ 142 High resolution NMR studies ................................................ 142 Two-dimensional NMR spectroscopy ....................................... 146 Concl usion..................................................................... 155 8. EXPERIMENTAL ...................................................................... 157 General experimental. .......................................................... 157 T m determination ............................................................... 158 HPLC purification and analysis of oligomers ............................... 158 Nuclease digestion and analysis of modified and unmodified oligomers .......................................................... 161 NMR experiments on oligonucleotides ...................................... 161 Molecular modeling ............................................................ 162 Synthetic procedures ........................................................... 163 REFERENCES ................................................................................... 171 VII LIST OF TABLES 1.1 Various 2'-O-alkyl nucleosides synthesized using strongly alkaline conditions ........................................................................ 19 1.2 T mS of various octathymidylates conjugated to intercalating agents ................ 32 2.1 Sequences of various oligonucleotides ................................................. 59 4.1 Sequences of various oligonucleotides and their T ms ................................ 78 4.2 Various 2'-O-alkyl oligonucleotides and their T ms ................................... 83 5.1 Nonexchangeable IH chemical shifts (ppm) of d(CGCACATGTGCG) ................................................................ 109 5.2 Nonexchangeable IH chemical shifts (ppm) of d(CGCA*CATGTGCG) ............................................................... 110 6.1 Sequences of various oligonucleotides and their Tms in oC ....................... 114 6.2 Nonexchangeable IH chemical shifts (ppm) of d {(CGCACA)r(UG UGCG)} 2 in D20 ............................................... 129 6.3 Nonexchangeable IH chemical shifts (ppm) of d{ (CGCA *CA)r(UGUGCG) 12 in D20 .............................................. 130 8.1 Gradient used in the purification of oligonucleotides ............................... 159 8.2 Gradient used in the analysis of oligonucleotides ................................... 160 LIST OF FIGURES 1.1 Watson-Crick base pairing ............................................................... 2 1.2 Principle of the antisense oligonucleotides. Adapted from reference 1.. ........... 3 1. 3 Modifications on the phosphodiester backbone. Adapted from reference 6 ........ 7 1.4 Structures of various 2'-O-methyl nucleosides synthesized using diazomethane and SnCh. Adapted from reference 80.............................. 15 1.5 Structure of key intermediate (I) and conjugation reaction of the 2'-O-alkylated sugar with base. These compounds were synthesized by Haner and Keller... ............. ........ ..... ........... ............... 17 1.6 Structures of various 2'-O-alkyl nucleosides synthesized using conjugation of 2'-O-alkylated sugar with base. These compounds were synthesized by Haner and Keller ......................................................................... 18 1.7 Synthesis of 2'-O-alkyl nucleosides using 2',3'-dibutylstannylene derivative .................................................................................. 20 1.8 Principle of affinity chromatography using 2'-O-alkyl oligonucleotides Adapted from reference 119............................................................ 25 1. 9 Structures of the intercalators used to conjugate oligonucleotides .................. 27 1.10 NOE observable distances in DNA. Different arrows represent pathways available for sequential assignments. Adapted from reference 156 ................ 40 2.1 Structures of 2'-O-phenethyladenosine and 2'-O-(p-azidophenethyl)adenosine ..................................................... 53 2.2 Energy minimized conformation for 2'-O-phenethyladenosine. Starting sugar conformation was C2'-endo ........................................... 56 2.3 Energy minimized conformation for 2'-O-phenethyladenosine. Starting sugar conformation was C3'-endo ........................................... 57 2.4 Illustration of the phenethyl ring (<1» acting as an extra base ....................... 60 2.5 Structure of 2'-O-(anthraquinonylmethyl)adenosine ................................ 62 2.6 Energy minimized conformation for 2'-O-(anthraquinonylmethyl) adenosine. Starting sugar conformation was C2'-endo ............................ 63 2.7 Energy minimized confonnation for 2'-O-(anthraquinonylmethyl)adenosine. Starting sugar conformation was C3'-endo ........................................... 64 3.1 Fonnation of styrene in the synthesis of 2'-O-phenethyladenosine ................ 67 3.2 Various alkylating agents used in the synthesis of 2'-O-phenethyladenosine and 2'-O-(p-azidophenethyl)adenosine ................................................ 68 3.3 Protection strategy for 2'-O-phenethyladenosine ..................................... 70 3.4 Reaction ofphosphitylating agent with 2'-O-(p-azidophenethyl) adenosine ............................................................... 72 4.1 Sugars puckers observed in RNA and DNA .......................................... 79 4.2 Structures of different ribonuc1eotides that were incorporated into DNA .................................................................................. 81 4.3 The UV melting curves (Tm) for various 2'-O-alkyl nuc1eosides containing oligonucleotides ............................................................. 84 4.4 The Circular Dichroism spectra for the standard 12-mer (A) and the phenethyl 12-mer (B) at various temperatures ........................................ 86 5.1 Size exclusion HPLC of the fraction. A = Before heating and cooling. B = After heating and cooling.......................................................... 89 5.2 Imino proton spectra for the standard 12-mer at various temperatures ............ 91 5.3 Imino proton spectra for the phenethyl 12-mer at various temperatures ........... 93 5.4 NOE difference spectra for the phenethyI12-mer. A = Irradiation at 13.4 ppm. B = Irradiation at 13.1 ppm ................................................ 94 5.5 Expanded contour plot (aromatic-HI' region) of the NOESY spectrum for the standard 12-mer ...................................................................... 97 5.6 Expanded contour plot (aromatic-HI' region) of the NOESY spectrum for the phenethyl 12-mer ................................................................ 98 5.7 Expanded contour plot (HI'-H2' region) of the DQF-COSY spectrum for the phenethyl 12-mer. Arrow indicates H1'-H2' cross peak for A4 ....................................................................... 100 5.8 Expanded contour plot (aromatic-H2' region) of the NOESY spectrum for the phenethyl 12-mer. Arrow indicates connectivities of the benzylic proton................................................................. 102 5.9 Expanded contour plot (HS-H6 region) of the DQF-COSY spectrum for the standard 12-mer. ................................................................... 103 5.10 Expanded contour plot (H5-H6 region) of the DQF-COSY spectrum for the phenethyl 12-mer. .................................................................. 104 x 5.11 Stacked plot (H5-H6 region) of the DQF-COSY spectrum for the phenethyl 12-mer. Arrow indicates H5 of C5 ...................................... 106 5.12 Stereo view ofthe model for the phenethyI12-mer. Arrow indicates proximity of the benzene ring to C5 .................................................. 107 6.1 Reverse phase HPLC of the standard hybrid (A) and the phenethyl hybrid (B) at 550 C ..................................................................... 116 6.2 The UV T m curves for various oligonucleotides. The T mS were taken in 0.1 M NaCl containing 10 mM phosphate buffer (pH 7) ....................... 118 6.3 Expanded contour plot (aromatic-HI' region) of the NOESY spectrum for the standard hybrid... ........ ....... ......................................... .......... 120 6.4 Expanded contour plot (aromatic-H2' region) of the NOESY spectrum for the standard hybrid.......... ................ ........... ............... ................. 122 6.5 Expanded contour plot (H}'-H2' region) of the DQF-COSY spectrum for the standard hybrid..... ..... ................ ......... ............... ................... 124 6.6 Expanded contour plot (aromatic-HI' region) of the NOESY spectrum for the phenethyl hybrid .................................................................... 125 6.7 Expanded contour plot (HI'-H2' region) of the DQF-COSY spectrum for the phenethyl hybrid .................................................................... 127 6.8 Plot of the pseudorotation angle against coupling constant. Adapted from reference 194 ........................................................... 128 6.9 Expanded contour plot (aromatic-H2' region) of the NOESY spectrum for the phenethyl hybrid. Arrow indicates connectivities of the benzylic proton .................................................................................... 131 6.10 Expanded contour plot (H5-H6 region) of the DQF-COSY spectrum for the standard hybrid (A) and the phenethyl hybrid (B) .............................. 133 7.1 Synthesis of 2'-O-(anthraquinonylmethyl)adenosine from 2',3'- dibutylstannylene adenosine ........................................................... 137 7.2 Enzymatic hydrolysis of the anthra 12-mer. The peak corresponding to 9.68 minutes indicates 2'-O-(anthraquinony1methyl)adenosine ................ 139 7.3 The electro spray mass spectrum for the anthra 12-mer............................ 140 7.4 The UV T m curves for the anthra 12-mer and the standard 12-mer. The Tms were taken in 0.1 M NaCI containing 10 mM phosphate buffer ............ 141 7.5 The circular dichroism (CD) spectra for the anthra 12-mer at various temperatures ............................................................................. 143 7.6 Imino proton spectra for the anthra 12-mer at various temperatures .............. 144 xi 7.7 Imino proton spectra for the anthra 12-mer at various temperatures. These spectra were taken in the presence of magnesium ........................... 147 7.8 Aromatic portion of the one-dimensional spectra for the anthra 12-mer at various temperatures ................................................................. 148 7.9 Expanded contour plot (aromatic-HI I region) of the NOESY spectrum for the anthra 12-mer. ...................................................................... 149 7.10 Molecular model for intercalation of the anthraquinone inside the helix Only six nucleosides were used for molecular modeling. Arrow indicates the anthraquinone ring... .................................. ... .......... .... 151 7.11 Molecular model for intercalation of the anthraquinone inside the helix. Expansion of the region near intercalation. ..... .... ..... ............. ........ ... .... 152 7.12 Expanded contour plot (aromatic-H2' region) of the NOESY spectrum for the anthra 12-mer ....................................................................... 153 7.13 Expanded contour plot (HI'-Hi region) of the DQF-COSY spectrum for the anthra 12-mer ....................................................................... 154 Xll ACKNOWLEDGMENTS My sincere appreciation is extended to my advisor, Dr. A. D. Broom. His guidance and understanding have made a lasting impression on me. I hope one day I become as good a mentor as he was to me. I would like to thank all the students and post-docs that I have shared the past five years with. The assistance of Dr. Davis and Jay Olsen in the understanding of NMR is greatly appreciated. I am also appreciative of the faculty and staff that have contributed so much to my education. I would like to thank all the members of my family in India for their encouragement and support. They have always backed my decisions and I am sure they will continue to do so in future. My most enjoyed moments in this country came when I was with my best friends, the BBB group. Their friendship and support are greatly appreciated. I would also like to thank Aptekaka and Kaku for not letting me feel homesick throughout my stay in Salt Lake City. Were it not for my friends and family this dissertation would not have been possible. CHAPTER 1 INTRODUCTION Introduction to antisense oli~onucleotides Discovering a new drug by rational design has always been the dream of the medicinal chemist. How one understands the biological system at molecular level often determines the success of this drug design process. Oligonucleotides are studied in variety of different fields in various aspects. These studies have created a large body of literature, so it is not surprising that the medicinal chemists turned their attention towards oligonucleotides for rational drug design. The new technology that has emerged in attempting therapeutic intervention at the nucleic acid level is termed 'antisense' technology. The complementary nature of nucleic acids makes antisense technology possible. The DNA double helix is held together by Watson-Crick base pairing in which adenosine and thymidine or guanosine and cytosine interact through hydrogen bonding (Figure 1.1). During protein synthesis, two strands of the DNA unwind and one of the strands, termed as coding or sense strand, acts as template for the synthesis of a complementary mRNA (messenger RNA) molecule in a process called transcription. The mRNA is then translated into protein on ribosomes. The process of producing a protein from a DNA genetic code is called gene expression. It is possible to suppress the gene expression by using a synthetic oligonucleotide that hybridizes to a specific mRNA target by Watson­Crick base pairing. This oligonucleotide, which is predominantly DNA, is called as antisense oligonucleotide (Figure 1.2).1 2 CH3 f N\\\ ),--N N I 0 '-0 N=< "V H ~ OH OH Figure 1.1 Watson-Crick base pairing. DNA DNA NORMAL GENE EXPRESSION Nucleus I I I I I I I I I I I > .------:----~ mRNA I I I I I I I I I I Cytoplasm Protein INHHIBmON OF GENE EXPRESSION > I I I I I I I I • - - - - - - -I- - - ~ mRNA No Protein 3 ) Antisense '"') oligonucleotide Figure 1.2 Principle of antisense oligonucleotides. Adapted from reference 1. 4 Two mechanisms have been proposed for the action of antisense drugs. Formation of the RNA:DNA duplex may exert a direct steric effect, blocking the factors required for translation or impeding translocation of the ribosome along the mRNA resulting in chain termination. Alternatively, the mRNA may be cleaved by RNAse H at the site of the RNA:DNA heteroduplex.2 RNAse H is the enzyme required for DNA replication. It is thought to digest the short RNA primers used to initiate DNA syntheses. This enzyme is ubiquitous in nature and also appears in the cytoplasm. Comparison of the inhibition data with and without RNAse H indicates that the RNAse H mechanism is more important than the steric blocking mechanism.2 Antisense technology offers the following advantages: Simplicity. Simple base pairing rules govern the interaction between antisense oligonucleotides and their target. Consequently, at least in principle, only the sequence of the target needs to be known. Specificity. Statistically, the base sequence of a 17-mer oligonucleotide occurs just once in the entire sequence of human genome.3 As a result extremely selective intervention is theoretically possible by a 17-mer antisense oligonucleotide. Effectiveness. One mRNA gives rise to multiple copies of protein. Hence, inhibiting mRNA is a much more effective way of intervention than inhibiting multiple copies of protein. These advantages make the use of antisense oligonucleotides as therapeutic agents extremely attractive. However, several conditions need to fulfilled for antisense drug therapy of be successful. One must be able to synthesize the oligonucleotides in large quantity and then the oligonucleotides must be formulated appropriately. If one assumes these hurdles will be overcome, then an antisense drug must satisfy following requirements. Stability. An antisense oligonucleotide must be stable both in vivo and in vitro, in the extra- and intracellular milieu. The information necessary for an oligonucleotide to 5 bind its target is carried by the sequence of that oligonucleotide. Consequently, it becomes vitally important that breakdown of oligonucleotide does not occur. Unfortunately, nucleases readily degrade normal oligonucleotides in vitro and in vivo.3 3'-Exonuclease activity predominates in serum whereas cells and other bodily fluids show both 3'- and 5'-exonuclease activities. Typically, half lives of phosphodiester oligodeoxynucleotides range from 15 to 60 min in most sera. Oligoribonucleotides are significantly less stable than oligodeoxynucleotides. The rate of degradation varies as a function of the sequence, length of the oligonucleotide and the type of the sera used in the study.I,4,5 Oligonucleotides with altered backbone are compounds of choice for improving the stability of antisense 0ligonucleotides.6 Modifications at the 2'--position of the sugar also have been shown to enhance nuclease resistance.7 Besides these internal modifications, attachments of a number of pendant groups to 3' and/or 5' ends have been shown to improve nuclease stability.8-13 Cellular uptake. Antisense oligonucleotides must traverse cellular membranes to reach specific intracellular targets at a reasonable therapeutic concentrations. The permeation of oligonucleotides through membranes was thought to be difficult due to their poly anionic nature. However, oligonucleotides rapidly accumulate inside the cell. It was shown that the oligonucleotides are actively transported inside the cell and an 80 KD protein is responsible for their binding and intemalization.14 Fluorescently labeled oligonucleotides are found to rapidly concentrate into the nucleus following microinjection into cytoplasm. 15-17 However, when fluorescently labeled oligonucleotides are placed in tissue culture media they accumulate in endosomes and lysosomes.18-21 3'-Derivatization of oligonucleotides seems to improve both the permeability and nuclease stability. I Other means employed to enhance penetration include incorporation in liposomesI 8,22 and covalent binding to carriers like poly-L-Iysine.23,24 Affinity. Antisense oligonucleotides must hybridize or bind with significant affinity with target sequences so expression of the gene is inhibited. Affinity results from 6 the hydrogen bonding between complementary base pairs. Consequently, affinity increases with the length and GC content of the oligonucleotide. Affinity also depends upon salt concentration and pH. The hybridization requirements limit the extent to which oligonucleotides can be modified as it is mandatory to retain hydrogen bonding capabilities of bases. RNAs possess a complex tertiary structure and the hybridization requirements dictate that one must choose an accessible region of the target. Introduction of a phosphate residue at the 5'-end markedly stabilizes the duplex.25 Covalent attachment of an intercalator is another common way of improving affinity.9-11,26-33 Oligonucleotides with 2'-O-alkyl substituents34 and a-anomeric nucleosides also have shown improved affinity towards their RNA targets. 35,36 Specificity. Antisense oligonucleotides must hybridize selectively to specific target sequences. Reduction in non-specific binding to other cellular sites (proteins, lipids and nucleic acids) will enhance potency and minimize toxicity resulting from disruption of normal cellular metabolism. Specificity of an antisense oligonucleotide increases with its length, but only until a certain length.37 Within certain limits, specificity can be enhanced by controlling base sequence. Sequences of A and T are thought to increase the specificity by spreading recognition and binding over a larger number of bases and by decreasing affinity with the targets containing mismatches.37 Diaminopurine, which provides an extra hydrogen bond when substituted for adenosine, increases the selectivity.38 Inosine, however, reduces the selectivity due to its ability to hydrogen bond with almost all the bases.39 Modifications on the phos.phodiester backbone Modifications on the phosphodiester backbone offer two distinct advantages. First, it improves nuclease resistance and second, it retains the hydrogen bonding capabilities of the bases. Figure 1.3 shows the variety of modifications performed on the phosphodiester backbone. B1 B2 B1 X II O-p-O­I 0- B2 A I X-Z-y- I B X= 0 = S = CH3 = NH-R = BH3 = OR z C C C C S S S S S S S S NH NCH3 0 C C Phospodiester Phsphorothioate Methylphosphonate Phospho rami date Boranophosphate Phosphotriester X Y A 0 0 H S 0 H 0 S H CH2 0 H 0 0 0 0 CH2 0 0 NH 0 NH CH2 0 CH2 CH2 0 0 0 0 CH2 CH2 0 CH2 CH2 CH2 0 CH2 0 CH2 0 0 0 0 NH B H FOImacetal H 3'-Thiofonnacetal H 5' -Thiofonnacetal H 5' -Thioether 0 Sulfate 0 Sulfonate 0 Sulfanate 0 Sulfonamide 0 Sulfone Sulfite Sulfoxide Sulfide Hydroxylamine MMI MOM! Carbonate 5'-N-carbamate Figure 1.3 Modifications on the phosphodiester backbone. Adapted from reference 6. 7 8 Of all these modifications, phosphorothioate and methylphosphonates are the most studied oligonucleotides because of their availability through automated DNA synthesis. Both oligonucleotides show nuclease resistance.6 Substitution of one phosphate oxygen atom by sulfur or methyl introduces chirality at the phosphorus. Thus, an oligo with n phosphodiester linkages contains a mixture of 2n distereomers; and only one of them is the most active molecule. In the case of methylphosphonate oligonucleotides, the all-Rp oligonucleotides have significantly higher Tm than the all Sp isomer.25,40 Conversely, with a single phosphorothioate linkage, the Sp isomer has higher T m than the Rp isomer.41 These results complement each other because the Rp isomer in the case of methylphosphonates has the same absolute configuration as the Sp isomer in case the of phosphorothioate. The stability of the duplexes formed by methylphosphonate oligomers is not affected significantly by changes in salt concentration. Consequently, the T mS of methylphosphonate duplexes are similar to those of unmodified duplexes at high salt concentration, but are significantly higher than unmodified duplexes at low salt concentration.42 Phosphorothioate duplexes, in general, show less affinity towards their target than unmodified duplexes.43 Phosphorothioate-RNA duplexes activate RNAse H and exhibit their biological activity mainly by this mechanism.44AS Methylphosphonates do not activate RNAse H and depend upon sterlc impedance mechanism for biological activity.46 The phosphorothioate oligomers are taken up by the cell by an active transport mechanism similar to phosphodiester 0ligomers. 14 However, they do not penetrate as efficiently as phosphodiester oligomersY The methylphosphonate oligomers penetrate inside the cell by passive diffusion.47 This may be due to their non-ionic nature. The uptake does not show any dependence upon chain length in the range of two to nine bases.48 Methylphosphonate oligonucleotides show lower solubility in water than phosphodiester oligonucleotides; consequently, one or more negative charges are introduced in the form of phosphates or phosphodiester linkages. These 9 methylphosphonate oligonucleotides are tested in variety of cell-free and cell culture systems for their antisense activity. They are effective over the range of 30-150 j.l.M.42 The lowest concentration (30 j.l.M) was used in inhibition of the initiation codon region of chloramphenicol acetyltransferase mRNA.49 The highest concentration (l50j.l.M) inhibited VSV mRNA. 50 However, even the lowest effective concentration (30j.l.M) is too high for therapeutic activity. The higher concentration may be needed to compensate for the lack of RNAse H activation. Also, higher concentration may be needed to drive the binding equilibrium towards the bound state. The effective concentration is significantly reduced when methylphosphonate oligonucleotides are covalently linked to reactive groups like psoralen.51,52 On incubation of Vero cells with 150-300 IlM, cell growth was not reduced after 24 h and only slight reduction was observed after 48 hours.53 Phosphorothioate oligonucleotides bind to a variety of proteins. They have been shown to induce nonspecific inhibition of protein synthesis in cell free protein translation experiments.44 The nonspecific effects are length dependent; as the length increases the toxicity also increases. Phosphorothioate oligonucleotides have also been shown to inhibit DNA polymerases, reverse transcriptases and nucleases.54,55 Despite the potential nonspecific interactions, a wide variety of compounds exhibit excellent therapeutic indices.3 Currently phosphorothioates are the most active antisense oligonucleotides against HIV. In general, phosphorothioate oligonucleotides are effective in the low rnicromolar range in cells infected de novo with mv. The effective concentration depends upon chain length. Increasing the chain length decreases the effective concentration until 21 nucleotides. All these oligonucleotides also show high dose dependence.44 2'-O-alkyl olifWnucleotides; A historical perspective Smith and Dunn in 1959 observed unusually stable dinucleotides in alkaline hydrolysis of wheat germ and rat liver RNA. 56 They characterized these dinucleotides and suggested that the dinucleotides contained 2'-O-methyl ribonucleosides. This small 10 report marked the beginning of a very important but still only partially understood area of 2'-O-alkyl biochemistry. This area has expanded considerably now; however, this growth was not continuous and there were two distinct phases of 2'-O-methyl development. The fIrst phase of growth occurred from 1959 to 1974 and the second, from 1988 until today. The first phase of growth started in 1959 with the discovery of 2'-O-methyl ribonucleosides by Smith and Dunn. The initial report of Smith and Dunn was followed by a number of articles describing identifIcation of 2'-O-methyl ribonucleosides in RNA isolated from variety of sources.57-61 With the increasing number of articles it was realized that occurrence of 2'-O-methyl ribonucleosides was not a rare phenomenon and these compounds must have a defInite role in RNA biochemistry. However, biologists could only hypothesize about the possible role of 2'-O-methyl ribonucleosides without any experimental proof. The experimentation was ruled out due to the unavailability of 2'­O- methyl ribonucleosides form synthetic sources. Fortunately this changed in 1966 when Broom and Robins synthesized 2'-O-methyl adenosine from adenosine using diazomethane in a homogeneous mixture of water and 1,2 dimethoxyethane.62 The first synthesis aroused a tremendous activity in the 2'-O-methyl ribonucleotide area. Two communities of scientists became very active; one, the synthetic chemists and two, the biochemists. Synthetic chemists started developing alternate routes for the synthesis of 2'-O-methyl ribonucleosides to achieve superior yields and biochemists started looking into the biochemical role of these compounds. By 1974, effects of these nucleosides on different biological systems were studied and some conclusions were reached. This slowed down the flurry of activity in 2'-O-alkyl ribonucleosides area. The second period of 2'-O-alkyl ribonucleoside growth started in 1988. This period marks the use of 2'-O-alkyl ribonucleosides for regulation as well as characterization of other biological systems. Some key developments in molecular biology over 14 years were responsible for this new role of 2'-O-alkyl ribonucleosides. 11 Paterson and co-workers observed that mRNA in its hybrid fonn with its complementary DNA was not translated in eukaryotic cell-free systems, while heat dissociation of the hybrid reinstated the activity.63 Exploiting this observation, they arrested translation of B­globin mRNA by hybridizing it with cDNA. They tenned this technique as hybrid arrested translation (HART). HART was also useful in precise mapping of specific protein-coding regions in restriction fragments of adenovirus 2 DNA. Hastie and Held used the same technique to relate a specific DNA sequence to the corresponding mRNA and polypeptide product in situations where neither the DNA or the mRNA was available in pure forn1. 64 These reports initiated the use of oligonucleotides to study biochemistry of RNA. At around same time Zamecnik and Stephenson used a synthetic 13-mer to inhibit Rous Sarcoma virus replication and cell transfonnation in Chick embryo fibroblast tissue culture.65 This article suggested the possibility of using synthetic oligonucleotides in therapeutics. A few years later natural regulation of gene expression by RNA was also discovered.66 All these observations laid the foundation of what later emerged as 'antisense technology'. Soon it was realized that the nonnal oligonucleotides with a phosphodiester backbone were not very efficient because of their rapid digestion by nucleases. The instability of nonnal oligonucleotides made scientists look for an alternative. An ideal alternative would 1) be stable, both in vivo and in vitro, in the extra- and intracellular milieu. 2) traverse cellular membranes to reach specific intracellular targets at reasonable therapeutic concentration. 3) hybridize or bind with significant affinity to target sequences so gene expression is inhibited. 4) hybridize selectively to specific target sequences. 5) be easy to synthesize. 2'-O-alkyl oligonucleotides met these criteria and consequently were chosen to replace the nonnal oligonucleotides. The use of 2'-O-alkyl ribonucleosides as antisense 12 probes led to the development of better synthetic routes and by now 2'-O-alkyl ribose­containing oligonucleotides have been established as efficient antisense probes. Earlier studies on 2'-Q-methyl ribonucleosides Within a few years after the first synthesis of 2'-0-methyl ribonucleosides, the following observations were made which helped in understanding the biochemical functi<?ns of 2'-0-methyl ribonucleosides. 1) 2'-0-Methyl ribonucleoside-5'-diphosphates were substrates for polynucleotide phosphorylase, but their incorporation was lower than the corresponding ri bonucleosides. 67 ,68 2) Homopolymers of 2'-Q-methyl ribonucleosides were stable to alkaline hydrolysis conditions and resistant to nucleases.68 3) They formed more stable duplexes with complementary RNA than the corresponding DNA analogs.67 4) In the presence of normal ribonucleosides and polynucleotide phosphorylase the 2'-0-methyl ribonucleosides could form either a homopolymer or heteropolymer. They could also inhibit the incorporation of normal nucleosides.69 5) Polymers containing 2'-0-methyl ribonucleosides could act as a template for DNA dependent RNA polymerases with the exception of poly (2'-0-methyladenylic acid) or poly (2'-0-methylinosinic acid).70 6) Low levels of 2'-0-methyl ribonucleosides in an RNA polymer did not eliminate its template activity for protein synthesis and under certain conditions could be stimulatory.71 7) Poly (2'-0-methyladenosine) exhibited more secondary structure than poly A. 72 From these observations it was concluded that 2'-0-methyl ribonucleosides resemble more closely ribonucleotides than deoxyribonucleotides.68 The roles of 2'-0- 13 methyl ribonucleosides are to improve stability of the RNA without affecting its activity and to impart a characteristic secondary structure to RNA. There were conflicting opinions regarding the role of 2'-O-alkyl substituents in stabilizing the duplex. Shugar et al. observed by CD spectroscopy that the stacking of uracil rings in 2'-0-meUpU is more than that in UpU.73,74 Hence they concluded that the possible source of enhanced stability of 2'-O-alkyl polynucleotides is a change in conformation of the sugar, leading to more extensive base stacking. Drake et al. observed a similar improved stacking effect by CD spectroscopy when 3'-side nucleotide was methylated.75 They observed that methylated nucleotides were more abundant in the middle of the dihydrouridine loop and at the turning points from double-stranded regions to the looped single-stranded regions in tRNA. They hypothesized that the role of methylated nucleosides would be to compensate for the reduced stacking in the dihydrouridine region and to provide stability to the turning points from double to single stranded regions. The NMR and UV experiments by Alderfer et al. on polyA and poly(2'-0-methyl A) contradict the above results.16 They proposed that the stacking decreases in the order poly d A > poly rA > poly r(2'-0-methyl A) > poly r(2'-0-ethyl A). They explained the increased stability of poly 2'-0- methyl adenosine on the basis of thermodynamic considerations. An interesting explanation was offered by Bobst et aI. for stabilization of duplexes by poly(2'-0-methyl A).72 According to their hypothesis, electrostatic forces are responsible to a large extent for the stabilization of poly A duplexes. Attractive forces between protonated adenine residues on one strand and negatively charged phosphate residue on the other strand, and repulsive forces between the phosphates groups of both strands are of major importance. The number of adenine residues which have to be positively charged to keep the two strands together is smaller for poly Am duplexes which makes that duplex more stable. 14 Synthesis of 2'-O-alkyl ribonucleosides Using diazomethane The very fIrst 2'-O-alkyl ribonucleoside was synthesized using diazomethane in a homogeneous mixture of water and 1,2 dimethoxyethane.62 2'-O-Methyl derivatives of other naturally occurring bases were also synthesized.77-79 Diazomethane is not very selective. Along with the desired 2'-0- methyl product 3'-O-methyl, 5'-O-methyl and 2',3'-dimethyl compounds are also obtained. Alkylation of the base is a problem in guanosine. This necessitates complicated purification procedures. The main disadvantage of diazomethane, however, is its hazardous nature and potential for explosion. The selectivity problem of diazomethane was partially solved by Robins and co­workers. 80 They used SnCh along with diazomethane to give exclusively 2'- and 3'-0- alkyl compounds. Various compounds synthesized by this method are shown in Figure 1.4. This procedure was also used to synthesize 2'-0-benzyl, 2'-0-(p-nitrobenzyl) and 2'-0-(p-methoxybenzyl) ribonucleosides. 81 -83 Recently, Leonard et al. used nonhazardous trimethylsilyl diazomethane instead of diazomethane to methylate guanosine.84 Conjugation of 2'-O-alkylated sugar with base In this approach, the 2'-0-alkylated sugar is reacted with base to form a glycoside bond. Generally the coupling is performed in presence of a catalyst like SnCh or titanium chloride. Tong et al. used an appropriately protected sugar to synthesize 3'-O-methyl guanosine.85 Haines used this approach to synthesized 2'-O-methyluridine and its isomer 3 (2-0-methyl-B-D-ribofuranosyl)cytidine. 86 HO OCH3 NH2 NH2 H N~ I~ N~ ~I N, N N N I B= N5' OCH3 N~ O.?--N I O~N I I R NH2 Cl SH SCH2C6H4N02-p 0 HNJN,) H NhN N 2 I 0 0 HN~ HNA.NH OJ..N I YO Figure 1.4 Structures of various 2'-O-methyl nuc1eosides synthesized using diazomethane and SnC12" Adapted from reference 80. 15 16 Recently, Keller and Haner took similar approach to synthesize various 2'-0- substituted uridines and adenosines.87,88 The structure of their key intermediate I, which bears an acetate at 2'-hydroxyl, is shown in Figure 1.5. Acetate at 2' hydroxyl served two purposes. First, it acted as a linker for tethering various groups and second, participation by the ester carbonyl facilitates the attack of the base from the B face. Figure 1.6 shows a variety of compounds synthesized by this method. A major disadvantage of this method is synthesis of the key intermediate I which requires multiple synthetic steps. Also, the coupling reaction produces a and B anomers which require chromatographic separation. Usinf: alkylatinf: af:ents in stronf:ly alkaline medium An observation that N3 of cytosine is quite inert to alkylating agents under strongly alkaline conditions led to the development of this route.89 Table 1.1 shows a variety of compounds synthesized using this method. This method avoids hazardous chemicals like diazomethane. However, similar to diazomethane alkylation, this reaction also gives rise to multiple product resulting from 3' and 5' alkylation. The yield of the 2'-alkyl compound can be improved by using low temperature. Sproat et al. circumvented the multiple alkylation problem by simultaneously protecting 3' and 5' hydroxyls with tetraisopropyldisiloxyl (TIPS) group.90 This protecting group is unstable to strongly alkaline conditions, hence a sterically hindered base 2-tert. -butylimino-2-dieth y lamino-l ,3-dimethy lperh ydro 1, 3, 2-diazaphosp horin (BDDDP) was used. Usinf: organotin compounds with alkylatinf: agent This route of synthesis uses 2', 3'-stannylene derivatives of nucleosides as the starting material (Figure 1.7). The 2', 3'-stannylene function acts as the activator. o HNy O~N) BZOy0'l NaOMe, (Me2N)C2H4N BiS(trimethYISilY!)lhyminy IW 0 Me2SiOTf,50°C BzO O~OMe BzodMeo BzO O~ OMe 1 ACOH/~ H2S04 O°C BZOo a OA°e BzO O~OMe .. N6-Benzoyl­bis( trimethylsilyl) adenine, Sn04' RT ... o HN~ O~N HOD ° HO O~OMe NHBz N~N ~ Jl .. ~ N N BzOD ° BzO O~OMe Figure 1.5 Structure of key intermediate (I) and conjugation reaction of the 2'-alkylated sugar with base. These compounds were synthesized by Haner and Keller. 87 ....... -l H N }-N~ N~ ~ Jl .. J N N H0-C; 0 HO O~NHR <1 ~N N~ oANJ HOD 0 HO o~ NHR H 0 ~~N o n-Octyl Figure 1.6 Structures of various 2'-O-alkyl nucleosides synthesized using conjuation of 2'-O-alkylated sugar with base.These compounds were synthesized by Haner and Keller. 88 18 19 Table 1.1 Various 2'-O-alkyl nucleosides synthesized using strongly alkaline conditions Starting Material Base Alkylating agent Major product Ref. Cytidine 1 NNaOH Dimethyl sulfate 2'-0-Methy1cytidine 91 Uridine 1NKOH 2'-0-Methyluridine J\rabinofuranosyl 1NKOH Dimethyl sulfate 2'-0-Methyl- 92 cytosine arabinofuranosyl ~osine Adenosine 2NKOH Dimethyl sulfate 2'-0-Methyladenosine 93 NaH Methyl iodide Adenosine-3', 5'_ 4NNaOH Methyl iodide 2'-0-Methyladenosine- 94 cyclic phosphate 3', 5'-cyclic phosphate 2-Amino 6-chloro- BDDDP Methyl iodide 2'_0_ Methy 19uanosine 90 3', 5'-TIPS purine ribonucleoside 6-Chloropurine-3', BDDDP Methyl iodide 2'-0-Methy ladenosine 90 5'-TIPS 2'-0-Methylinosine ribonucleoside Adenosine NaH Halide 2'-0-Methyladenosine 95 2'-0-Ethyladenosine 2'-0-Propyladenosine 2'-0-Butyladenosine 2'-0-Pentyladenosine 2' -0-N ony ladenosine 2'-0-Allyladenosine 2'_0_ Benzy ladenosine HO HO HO OH HO OR Catalyst or Heat + HO HO o 0 '\./ Sn BU/ 'Bu R-X RO OH Figure 1.7 Synthesis of 2'-O-alkyl nucleosides using 2',3'-dibutylstannylene derivative. 20 21 Often a high temperature96-98 or catalyst99 is needed for this reaction to proceed at a reasonable rate. 2'-O-(p-Nitrobenzyl)97, 2'-O-(4-substituted 2-picolyl-l-oxide)98 and 2'-O-(anthraquinonylmethyl)99 derivatives of uridine and adenosine are synthesized using this method. Only monoalkylation is observed in this reaction. This approach affords alkylation of the 2'-hydroxyl under neutral conditions and thus offers an alternate route for substituting 2'-hydroxyl with alkali sensitive compounds. Applications of olilionucleotides containinli 2'-O-alkyl ribonucleosides Therapeutic applications Zamecnik and Stephenson in 1974 showed that the synthetic oligonucleotides can be used for therapeutic purposes.65 For antiviral activity, oligonucleotides can be directed towards mRNA which directs the synthesis of proteins essential for viral replication or growth. For anticancer activity, oligonucleotides can be directed against mRNA which gives rise to faulty proteins. Morvan et al. compared 12-mer analogs representative of seven different classes of structurally modified nucleosides and complementary to the same target. 100 These seven oligonucleotides were tested for their binding affinity, resistance to nucleases in a cell-free medium (RPMI 1640 + 10% inactivated fetal calf serum) and inhibition of HIV-l replication in de novo infected MT4lymphocytes. The viral target was the splice acceptor site of the pre-mRNA coding for the regulatory protein tat. Their study included 2'-0- methyl and 2'-0-allyl oligoribonucleotides. These 2'-O-alkyl RNAs were found to form the most stable duplexes with complementary RNA strands. Half-lives of these oligonucleotides in RPMI 1640 medium were approximately double as compared to standard DNA. Their activity against HIV infection was superior than all the other classes except phosphorothioates. 2'-O-Alkyl oligonucleotides do not activate RNAse H, which may explain their lower activity despite higher affinity towards the target. In this 22 study scrambled sequences of same base compositions were used as the controls. These controls showed roughly the same activity as the antisense oligonucleotides, indicating that all the oligonucleotides exhibit non sequence-specific activity. However, direct correlation was observed between activity and their stability. Monia et al. designed chimeric oligonucleotides containing 2'-O-alkyl ribonucleosides to activate RNAse H without compromising their high affinity and nuclease resistance. lOl They tested these chimeric 2'-O-alkyl -phosphorothioate oligonucleotides for their affinity towards the target, ability to activate RNAse Hand activity against Ha-ras oncogene containing a GGC to GTC point mutation at codon 12. In vitro assay indicated that a stretch of at least 5 deoxy linkages at the center of the chimera was essential for RNAse H activation. A chimera with 7 deoxy linkages showed optimal activity. However, this activity was much lower than corresponding all-deoxy analogs. All the 2'-modified oligonucleotides bound to RNA with greater affinity. The order of affinity was 2'-O-methyl > 2'-O-propyl > 2'-O-pentyl > 2'-deoxy. Chimeric 2'­O- methyl oligonucleotides with 1 to 3 deoxy linkages were ineffective against Ha-ras gene expression. Chimeras containing 5 or more deoxy linkages showed marked inhibition of Ha-ras and this inhibition was dose-dependent. Activity against Ha-ras was affected by the nature of modification. An inverse relationship was observed between the length of the alkyl chain at the 2'-position and antisense activity. This inverse relationship was explained on the basis of binding affinities. Oligonucleotides with smaller alkyl chains showed greater affinity towards the complementary strand and consequently, were more effective. These results indicate that 2'-O-alkyl oligonucleotides can be successfully used for therapeutic applications. Applications in molecular biolo~y The credit for establishing 2'-O-alkylribonucleotides as antisense probes in molecular biology goes to Angus Lamond and Brian Sproat. Sproat, a nucleoside 23 chemist, synthesized various 2'-O-alkyl nucleosides which were utilized by Lamond, a molecular biologist, to study the biochemistry and cell biology of RNA processing. Splicing is the most important RNA processing reaction in eukaryotic cells. Splicing takes place in the nucleus within a large RNA-protein complex termed a spliceosome. The major subunits of spliceosomes are VI, V2, V4/U6 and U5 small nuclear ribonucleoprotein particles (snRNPs). Each snRNP consist of an RNA -protein complex containing one or more snRNAs and a set of proteins ('sm' proteins). 102 Each of the snRNPs is essential for splicing and their function and structure show a high level of evolutionary conservation. 103 Antisense 2'-O-alkyl RNAs complementary to each of the splicing snRNAs were developed. 104-107 These probes inhibited the splicing reaction in many cases. The binding of antisense probes to different regions of the snRNA in a snRNP inhibited different stages of splicing, e.g., in case of V2 snRNA, antisense complementary to the pre-mRNA branch site blocked stable binding of U2 to the intron whereas antisense probes targeted to the 5'-terminal sequence arrested spliceosome formation. 106 The probe targeted against the internal domain of V2 snRNA inhibited none of the actions mentioned above but selectively inhibited the second catalytic step of splicing. Oligo (2'-O-methyl)RNA complementary to the splice sites in B-goblin pre-mRNA inhibited the splicing reaction in vitro .108 Cotten et al. utilized oligo 2'-O-methyl or 2'-0- ethyl RNAs complementary to V7 snRNA to study the splicing reaction. 109 They showed that both forms of 2'-O-alkyl RNAs were more potent inhibitors of V7 RNA than either RNA or phosphorothioate DNA of identical sequences. Their results suggest that V7 snRNA is not involved in splicing but is an essential factor in the 3'-processing reaction that forms mature 3'-termini of histone mRNAs. The stability and nuclease resistance of 2'-O-alkyl ribonucleotides have also been exploited to localize RNA in mammalian cells by in situ hybridization. 1 10 Antisense probes bound to RNA in situ can be visualized by fluorescence microscopy. The 24 fluorochrome can be attached either directly to RNA or indirectly through avidin-biotin conjugation. Nucleoplasmic and nucleolar snRNA, 5S and 28S RNA have been localized in this way.lIl,Il2 The RNAs not exposed to the fixation procedure were localized by microinjecting antisense 2'-O-allyl RNA coupled to fluorochrome into living cells. 1 13 Applications in affinity chromato~aphy The stable binding of antisense 2'-O-alkylribonucleotides to snRNA has been used for affinity chromatography. 104,106,110, 114 Figure 1.8 describes the principle behind using 2'-O-alkyl oligonucleotides for affinity chromatography. 2'-O-alkyl RNA containing reporter groups such biotin were used to either purify or deplete snRNPs from in vitro extracts. 1 10,114 2'-O-Methyl or 2'-O-allyl RNAs coupled to biotin were used to purify nucleoplasmic snRNAs involved in splicing and nucleolar snRNPs involved in pre­mRNA processing. 104,106 Ryder et al. used the same technique to purify assembled spliceosomes and for selective as well as efficient depletion of each of the splicing snRNPs from HeLa nuclear extracts.1l4-116 These depletion studies demonstrated that each snRNP is essential for splicing and acts at different stages during splicing. Antisense affinity chromatography has also been used to enrich a rare species prior to further analysis. Trans-splicing is a very rare phenomenon in mammalian cells. Bruzik et al. used 2'-O-methyl RNA to enrich RNAs that have undergone trans-splicing. 1 17 These purified RNAs were then amplified by peR and characterized in detail. Wasserman and SteizI18 used 2'-O-methyl RNA coupled to biotin to enrich specific snRNA complexes that had been cross-linked to psoralen. The cross-linked complexes were then analyzed by two-dimensional PAGE. Covalent modification of oli&onucleotides with intercalating agents Short oligonucleotides are preferred for antisense inhibition of gene expression since their use offers various advantages. As described before, short oligonucleotides, I ,• RNA-Protein complex and impurities Pure RNA-protein complex Column containing 2'-O-alkyl RNA I l Pass the impure RNA-protein complex through 2'-O-alkyl RNA column RNA-protein complex binds to the column while impurities pass on I­• • 11- Impurities Figure 1.8 Principle of affinity chromatography using 2'-O-alkyl oligonucleotides. Adapted from reference 119. 25 26 approximately 17 nucleotides in length, can cause extremely selective inhibition of the human genome. The synthesis and purification become easier with smaller lengths. The probability of forming hairpin structures is relatively small and if the target sequence occurs just once, the specificity improves with short oligonucleotides. This is true because the free energy cost due to a mismatch does not change appreciably when an oligonucleotide is elongated.120 However, short oligonucleotides suffer from one disadvantage. They exhibit lower binding affinities towards their target. To overcome this disadvantage, often an intercalator is covalently attached to short oligonucleotides Use of the intercalator offers various advantages: 1) The duplex between the oligonucleotide and its target is stabilized without affecting the specificity. 120 2) The nonpolar nature of the intercalator enhances the cellular uptake of the oligonucleotides9 3) The intercalator protects the oligonucleotide from 3'- or 5'-exonucleases depending upon its position. 121 Design of the oligonucleotide-intercalator conjugate involves careful consideration of several variables. These variables include the structure of intercalator, the length of spacer between intercalator and oligonucleotide and the position of intercalator on the oligonucleotide. The structures of various intercalators that have been introduced into oligonucleotide are shown in Figure 1.9. An intercalator exerts its effect by inserting itself between adjacent base-pairs of the duplex. The driving force for this insertion is hydrophobicity. An intercalator should be adequately hydrophobic so that the energy gained by favorable interactions with bases is more than that lost due to the disruption of helix. The planar shape of the intercalator, therefore, becomes important as it maximizes the favorable interaction by positioning the intercalator exactly parallel to bases. It is critical that the intercalator and the bonds connecting it to the oligonucleotide remain intact o ~ ~ o Anthraquinone ® OCH3 ~ I ~ ~ CI ~ N'-': .& Acridine >=N o t:~¢ o I I o 0 ~ 0 0- Psoralen Phenazine ~ HN~N~R I Proflavine CH3 ~N ~~~ CNKN) ~ H CH 3 Orthophenanthroline Oxazolopyridocarbozole Ellipticine Phenanthridine Porphyrin Figure 1.9 Structures of the intercalators used to conjugate oligonucleotides. 27 28 during deprotection conditions, if the intercalator is attached to the oligonucleotide before deprotection. For example, the bond connecting 9-arnino with the spacer in 2-methoxy-6- chloro-9-amino acridine is susceptible to aqueous ammonia treatment. As a result, deprotection of bases is carried out with NaOH.121 Conversely, some of the intercalators are stable to all the conditions employed in the automated DNA synthesizer, e.g., anthraquinone. Such intercalators can be conjugated 0 a mononucleotide and the mononucleotide can then be incorporated into the oligonucleotide by automated DNA synthesizer. 87 ,99 An intercalator bearing an aryl azide can be conjugated to a mononucleotide. However, its incorporation into DNA by automated DNA synthesizer is not possible because aryl azide reacts with phosphorodiamidite.122,123 Intercalators are also used for irreversible modification of the complementary strand. Although an aryl azide can not be incorporated into oligonucleotides during automated synthesis, one can incorporate it after the oligonucleotide is fully synthesized. For example, proflavine substituted with an azido group was conjugated to an oligonucleotide after the oligonucleotide was completely synthesized. This group on irradiation cross-linked two strands of the duplex. 124 Rabbit globin mRNA was inhibited by cross-linking with an antisense oligonucleotide. Psoralen was used as the cross­linking agent in this case.125 Oligonucleotides containing porphyrins and phenanthroline are used for site-directed cleavage of the target oligonucleotide. Oligothymidylates covalently linked to iron-porphyrins were targeted for their nuclease activity against complementary sequences. 126,127 Cleavage of poly(dA) and poly(rA) was observed in presence of oxygen and a reducing agent. No cleavage was observed when oligothymidylate was targeted against poly(dT). 1,1O-Phenanthroline was covalently attached to the 3'-termini of two oligothymidylates. In the presence of Cu2+ and 3- mercaptopropionic acid, hybridization dependent cleavage of poly(dA) and a 27 nucleotide long DNA containing an octaadenylate was observed. 128 An oligothymidylate­phenanthroline complex was also used for the scission of lac operon. 129 29 Length of the spacer plays a key role in determining the success of the intercalator-oligonucleotide design. It provides the flexibility needed for an intercalator to get inserted inside the helix. A short linker may not have conformational flexibility and therefore may introduce a major distortion into the helix, especially if it is introduced in the middle. At the same time, too long a linker may give an intercalator too much conformational flexibility. For example, in case of cross-linking two strands using psoralen, length of the linker determined rate and extent of the reaction. 52 Psoralen was conjugated to synthetic oligonucleotides with aminoethylene and aminobutylene linkers. One oligonucleotide without a linker was also synthesized. The order of cross-linking was aminoethylene-linked oligomer> aminobutylene-linked oligomer> linkerless oligomer. The decreased cross-linking of aminobutylene-linked oligomer probably reflects the greater degrees of freedom of the psoralen ring in the aminobutylene-linked oligomer. Asseline et al. observed that length of the linker between the acridine ring and the oligonucleotide greatly affects the stability of the complex. 130 The T m value for an oligothymidylate-propylene-acridine conjugate was about 13 °C lower than that for oligothymidylate-pentamethylene-acridine. The short linker (propylene) reduces the overlap between intercalated acridine and consecutive base pairs resulting into reduced stability of the conjugate. The length of the linker can affect the mode of interaction between intercalator and oligonucleotide without affecting the stability of the conjugate. Gautier et al. did not observe any change in the Tm values of the complex formed between oligothymidylate and oxazolopyridocarbazole (OPC) even if the length of the polymethylene linkers between them was changed from 5 to 7.26 However, the absorption and fluorescence spectra of the OPC changed with the change in the linker length. The difference in the spectra resulted from the difference in the interaction. Oligonucleotide-pentylene-OPC conjugate intercalated between the duplex whereas oligonucleotide-heptylene-OPC conjugate interacted only with single stranded 30 oligonucleotide. Oligonucleotide conjugated to OPC via ten methylene linker did not exhibit any of the above interactions. Exhaustive survey of literature indicates that polymethylenes, especially pentamethylenes, are very common linkers. Their popularity may be because they afford maximum conformational flexibility. For example, an oligonucleotide attached to porphyrins via a linker containing an amide bond showed less reactivity than the one containing just polymethylene. 127 Site of attachment of an intercalator on the oligonucleotide can vary considerably as oligonucleotides offer multiple sites for conjugation. Intercalator site can affect the chemistry of conjugation as well as the mode of interaction between intercalator and oligonucleotide. However, 3' and 5' ends have been the most frequently chosen sites of attachment. There are various reasons for this preference. Simple and high-yielding chemical reactions such as esterification and phosphoramidate formation are involved in conjugation at 3' or 5' ends,t21 One avoids all the chemical reactions involved in the automated synthesis. However, preserving the hydrogen bonding pattern and the normal geometry of the duplex are the important reasons for the preference of 3' or 5' end. Attachment of the intercalator to the 5'-end is much easier than 3'-attachment because one can utilize solid-phase chemistry by synthesizing a phosphoramidite of the intercalator­linker complex.131 Intercalators have also been attached to 2'-hydroxyl, I'-carbon or internucleotidic phosphates. Conjugation of the intercalator at I' or 2' of the sugar requires that the intercalator should be stable to the conditions employed in the automated DNA synthesizer. Anthraquinone is stable to those conditions and therefore has been introduced at both I' and 2' position. However, I' and 2' conjugation is not very easy. Dan et al. synthesized deoxyuridine bearing an anthraquinone at the I'-position by a multi-step procedure. 132 The overall yield of the fully protected compound was 26%. Synthesis of the uridine-anthraquinone conjugate from the 2'-position with a methylene linker is not very difficult. 133 However, yields are relatively low due to the formation of 31 the 3'-isomer. Also, chromatography is required to separate the two isomers. Keller and Haner introduced an acetyl linker between anthraquinone and 2'-oxygen by a very tedious synthetic route. 87,88 (This procedure is described in the 2'-O-alkyl oligonucleotide section.) Asseline et al. have studied the effect of the attachment site on the interaction between intercalator and 0Iigonucleotide.134 They synthesized octathymidylates attached to acridine at 3'-phosphate, 5'-phosphate and internucleotidic fourth phosphate by pentamethylene linkers. These modified octathymidylates were then hybridized with poly(rA). The stabilization afforded by 3'- and 5'- conjugated acridines was more than that afforded by internucleotidic acridine. Fluorescence and absorption spectroscopic studies showed that the acridine ring was experiencing at least two different environments when it was linked to 3'-phosphate and internucleotidic fourth phosphate. Molecular modeling studies implied that acridine entered through the minor groove when linked to 3'-phosphate. 5'-Linked acridine, however, entered though the major groove. The situation became complicated when acridine was linked to internucleotidic phosphate because such linkage made the phosphorus chiral. Both distereomers showed intercalation; however, they entered through different grooves. Detection of intercalation by physicochemical techniques Absorption spectroscopy Intercalation leads to a strong perturbation in the absorption spectrum of the intercalator. Strong hypochromism is observed along with a red-shift of the absorption spectrum of the intercalator.121 The melting temperature (Tm) value obtained by measuring the absorption at the increasing temperatures is used in estimating degree of stabilization afforded by the intercalator. Table 1.2 gives the melting temperatures observed for a series of octathymidylates linked to various intercalators. 32 Table 1.2 Tms of various octathymidylates conjuated to intercalating agents SEQUENCE poly poly As dAg Ref. dA rA B-(Tp)?T 18.1 16.1 10 11.2 28 B-Acr.ms.(pT)8 28.5 28.5 14.8 20 B-(Tp )g.ms.Acr 34.4 36.4 21.6 21.9 a-(Tp)7T 14 22.5 23 13.5 a-Acr.ms·(pT)s 25 38 29.6 18.5 a-(Tp )g.ms.Acr 25 37.5 35.6 27 N3<1>(sp )B-(Tp )g.ms.Acr 27 135 a-(Tp)?T 13.5 a-(Tp )g.ms.Acr 17.1 a-Acr.ms.(pT)S 27 a-Acr.ms. (pT)S(sp )<1>N3 28.8 (TpTp h.ms.Acr 33 33 32 (TpMeTph.mS.Acr (pseudoaxial) 18 24 (TpMeTp h.mS.Acr (pseudoequitorial) 41 43 (TpMeTp h.ms.Acr(pseudoaxial) 23 26 (TpOEtTp h.ms.Acr (pseudoequitorial) 39 41 (TpONeopTp h.msAcr (pseudoaxial) 17 21 (TpONeopTp h.ms.Acr (pseudoequitorial) 37 38 Tg.ms.Ellepticine 17 16 136 m = number of methylenes. Acr = Acridine 33 Fluorescence spectroscopy Fluorescence spectroscopy can be used to study the binding of an oligonucleotide­intercalator conjugate to its complementary sequences. The nature of the first two base pairs on the intercalator side is critical to determine the fluorescence quantum yield. 121 Ifonly one GC base pair is present at the terminal or penultimate position, fluorescence quenching is observed. If only an AT pair is involved, then fluorescence is usually increased. These properties are used to detect the intercalation. Fluorescence quenching of the acridine ring was observed when a complementary parallel B-d-AGA TTTGAG was added to the oligo-a-deoxynucleotide linked to acridine at the 5'-end.130 No effect was observed when an anti-parallel strand was added, indicating that (a)DNA:(B)DNA hybridize in a parallel orientation. Also, quenching of the fluorescence indicated that intercalation occurred between the terminal AT and GC base pairs. However, the quenching was not complete, implying that the oligonucleotide-acridine conjugate existed in at least two equilibrium conformations. Fluorescence polarization and fluorescence anisotropy decays can also be used to study the intercalation. Fluorescence polarization of the intercalating agent is increased upon complex formation. fluorescence anisotropy decay measurements reveal that this increase can be accounted for by both an increase in the rotational correlation time of the aromatic ring and a partial immobilization on the time scale of the fluorescence decay.130 Circular dichroism spectroscopy Intercalation of a planar aromatic ring between the base pairs of DNA induces a circular dichroism in its absorption bands as a result of an asymmetric perturbation of the aromatic ring by neighboring base pairs. 121 Covalent attachment of an acridine derivative to an oligonucleotide induces a circular dichroism in the visible absorption band. This circular dichroism of the conjugate was strongly perturbed upon binding to a complementary sequence. The observed changes depended greatly upon local 34 conformation of the duplex. 137 For example, the circular dichroism spectra of an oligothymidylate-acridine conjugate bound to poly(rA) and poly(dA) had opposite signs in the 400-450 nm range. Circular dichroism was also used to analyze the binding properties of oligonucleotides with a modified backbone covalently linked to an acridine derivative. 32 Alternating phosphodiester-phosphotriester backbones were synthesized with pure diastereoisomers of dinucleotide units. 121 The diastereoisomers of the oligonucleotides were shown to bind with quite different affinities. Circular dichroism was also used to demonstrate the triple helix formation when acridine substituted oligothymidylates bind to poly(dA) but not to poly(rA). The double and triple helices are characterized by different induced circular dichroism in the acridine absorption bands.32,138 Nuclear Ma~etic Resonance spectroscopy NMR studies of the oligonucleotide-intercalator conjugate unequivocally demonstrate intercalation. An intercalator inserts itself between two adjacent base pairs. As a result, the distance between these two base pairs is increased. This should result in the loss of NOE cross-peaks between aromatic protons and sugar protons of the involved bases. At the same time, additional cross-peaks should be observed between the intercalator protons and the protons of the bases. Stacking should also result into the change of chemical shift values of the aromatic protons. Tetrathymidylate-acridine conjugate hybridized to complementary tetradenylate was studied by NMR. Upfield shifts of the proton resonances of both the bases and the acridine ring was observed. 31 P NMR was used to determine the site of intercalation. 139 31p_1H and 1H-1H correlation spectroscopies were used to demonstrate that several intercalated complexes coexisted and that the exchange rate between these different conformers was fast on the NMR time scale. Using two-dimensional NMR techniques, Lancelot et al. 140 showed that the duplex between a hexamer-acridine conjugate and its complementary sequence adopted a B form geometry. All the deoxyribose sugars 35 preferred C2'-endo geometry. NOE connectivities between the nucleoside and the acridine protons revealed an intercalation of the acridine ring between the terminal A T and GC base pairs. NMR spectroscopic studies of DNA Until the late seventies DNA molecules were considered to adopt an averaged right handed structure, either A form or B form, irrespective of their sequence. This popular notion was dismissed completely by several studies performed in the early eighties. Dickerson and Drew observed local variations in DNA conformation depending upon their sequences. 141 Conformational transitions in DNA sensitive to base modification, environment142 and topological state143-145 of the DNA were also observed. Wang et al. accidentally discovered Z-DNA, which adopted a left handed conformation.145 Most of these studies were carried out using single-crystal X-ray crystallography. These studies gave a new insight into DNA structure, however, they raised questions about the structural and dynamic properties of DNA in solution. Conventional spectroscopic techniques were inadequate to answer these questions and consequently, development of new spectroscopic techniques was essential. At around the same time, NMR spectroscopy was experiencing a revolution in terms of technology and instrumentation. Two significant developments in the NMR spectroscopy were development of spectrometers operating at 500 MHz for protons and application of two-dimensional NMR techniques to macromolecules like proteins. These developments made NMR as an ideal candidate for the DNA structure determination. The use of NMR for oligonucleotides did not become routine, however, until the advent of the automated DNA synthesizer. The automated DNA synthesizer made available milligram quantities of DNA essential for NMR spectroscopy. Prior to that, most IH NMR spectroscopy of nucleic acids was performed on tRNA and synthetic RNA polymers.146 Much of the early work focused on observation of exchangeable imino protons, and 36 assignments were based upon chemical shift and ring current shift arguments. Assignments of non-exchangeable protons were also based upon chemical shift arguments. In 1982, Feigon et al. I47 reported the fIrst two-dimensional spectra for a DNA duplex. Even though assignments were not made, the quality of the spectra was suffIcient to reveal COSY Hl'-H2' and Hl'-H2" coupling as well as NOEs from base protons to HI '. These two-dimensional techniques were subsequently applied by many scientists to biologically relevant DNA sequences. Assignment of these spectra was vastly important even though chemical shifts per se are poor indicators of structure. However, chemical shifts were essential to interpret vast number of NOEs between specifIc protons that are close in space. These proximity constraints were very valuable in building the three-dimensional structure of the DNA. Assi~nment of exchan~eable protons The complementary strands of the duplex DNA are held together by the hydrogen­bonded imino protons. These protons (T-N3H in the dA-T base pair and G-NIH in the dG-dC base pair) are extremely de shielded as a result of hydrogen bonding and in-plane ring-current shifts from both bases in the base pair. These imino protons resonate in the 11 to 15 ppm region of the spectrum. These resonances are not observed even in freshly prepared D20 solutions indicating transient helix opening and rapid solvent exchange. As a result, the imino proton spectrum is recorded in H20 solutions. The presence of narrow resolved imino protons in H20 suggests that base-pair lifetimes are long on the msec time scale. 148 The earlier assignments of the imino protons were based upon the ring-current calculations. It was predicted that the G-NIH proton of the dG-dC base pair would resonate at higher fIelds than the T_N3H.I49 Subsequent work has shown that the T-N3H resonates between 13-14 ppm whereas G-NIH resonates between 12-13 ppm. The most effective method of assigning imino protons uses the Nuclear Overhauser Effect (NOE). 37 T-N3H is close to H2 of A and G-NIH is close to C-NH2. The strong NOE between these proton pairs are used in assigning imino protons unambiguously. 150 Assignment of imino proton signals is vitally important for various reasons. The distance between imino protons of adjacent base-pairs varies between 3.5 to 4.8 or more. 148 Since several1H-lH distances within the base pair are much shorter than this distance, pronounced spin diffusion within the base pair is observed. Thus, selective saturation of each imino resonance in turn produces a unique set of sequential connectivities that invariably leads to specific assignments if the sequence is known. This process becomes rather time-consuming for larger DNA duplexes. After few unsuccessful attempts, all the connectivities are now observed in a single NOESY experiment. I51 This route of sequential assignments of DNA provides an alternative to the traditional nonexchangeable proton route. The appearance of imino proton signal and the strength of the given base pair were shown to be related. The linewidth of the imino signal is inversely proportional to the base pair strength. 152 Strength of the base pair can also be evaluated on the basis of the chemical shift. It has been shown that upfield shifts of >0.5 ppm are associated with a decrease in the strength of a base pair. 153 The line width of the imino proton resonances increases with the temperature due to the increase in exchange rate with water. The line width when plotted against temperature gives a curve which is used to calculate the melting temperature (T m) of the duplex. The imino proton spectrum in H20 plays a very important role in the base pairing scheme. A Watson-Crick base pairing is identified by strong NOE between T-N3H and H2 of A whereas a Hoogsteen base pair is identified by strong NOE between N3H and H8 of A. I46 Structural variations in the DNA are usually manifested in the exchangeable resonances. For example, intercalation will result in large up field shifts of imino resonances adjacent to the intercalation siteI54 and protonation of bases will usually result in downfield ShI'tfS 0 f teh am'm o resonances an d appearances 0 fnew' Im I.n O resonances. 155 38 Assiwment of nonexchan&eable protons DNA consists of four nucleosides. Each nucleoside has two subunits, base and sugar. Adenosine and cytosine contain two aromatic protons whereas guanosine and thymidine contain only one. Thymidine also has a methyl group. Of all these protons, only cytidine has two protons on vicinal carbons and thus forms an independent spin system. The deoxyribose sugar unit is common to all the nucleosides. It consists of seven protons which form a coupled spin system. All these protons, except 5' and 5", have very characteristic chemical shifts which divide the one-dimensional proton spectrum into the following six different regions. 156 1) 1.0 to 1.7 ppm - methyl of T 2) 1.7 to 3.0 ppm - deoxyribose 2' H and 2" H 3) 3.6 to 4.6 ppm - deoxyribose 4' H, 5' Hand 5" H 4) 4.6 to 5.1 ppm - deoxyribose 3' H 5) 5.2 to 6.2 ppm - deoxyribose 1'H and H5 of C 6) 7.0 to S.O ppm - Aromatic protons of the bases ( H8 and H2 of A, HS of G, H60fT and C) Although all these protons can be identified by their chemical shifts, as the DNA gets longer these regions become crowded and significant overlap is observed. This calls for additional techniques to assign the spectra. The Nuclear Overhauser Effect (NOE) was the first technique to be applied to DNA for sequential assignment of DNA spectra. IS? The coupled spin within a spin system can be determined by selectively decoupling individual resonances, and NOE connectivities between spatially proximal protons can be determined by selective saturation. This technique, if applied in one-dimensional spectra, becomes rather time consuming and is applicable to short DNAs in the four to six base-pair range. Two­dimensional NMR is an essential requirement for assigning longer DNAs. Complete and 39 unambiguous assignments of all the protons in the DNA are possible by the combination of NOESY and COSY spectra at high resolution. NOESY spectra contain information about the protons relatively close in space. If the distance between two protons is less than 4 angstrom then they give a cross-peak. This rationale can be used in assigning DNA because if the DNA is right handed and close to the B-conformations, then the following protons are close to each other in space. 158 (Figure 1.10) 1) 5-methyl of Tor H5 of C and the H8/H6 of the preceding (5' side) base. 2) H8/H6 of a given base and its own HI'. 3) H8/ H6 of a given base and HI' of the preceding base. 4) H8/H6 of a given base and its own H2', H2". 5) H8/ H6 of a given base and H2', H2" of the preceding base. All these protons are expected give NOESY cross-peaks. These cross-peaks play an important role in sequential assignments of the DNA because they allow one to connect the different nucleosides within the DNA. Such a connection is not possible in COSY spectra as the phosphodiester bond isolates the spin system of one nucleoside from another. The NOESY spectrum provides two independent routes for assigning DNA. One can either walk through the NOESY spectrum using H6/H8 and HI' connectivities or obtain the same information using H6/H8 and H2'/H2" connectivities (Figure 1.10). Although the shortest intra- and intemucleotide base-sugar connectivities are H6/H8-H2' and H6/H8-H2" respectively, the H8/H6-Hl' connectivities are preferred for sequential assignments.146 The H8/H6-H2'/H2" region is complicated, but is useful to confirm the assignments based on the aromatic-HI' region and to resolve ambiguities. Some of the intensity in the H6/H8-Hl' region arises from spin-diffusion, but this does not cause any ambiguities in assignment since the indirect pathways also give the correct connectivities. It is not always possible to find either the 3' or 5' end of the DNA to initiate the walking process. This is not a problem as one can start at any nucleoside in the DNA and proceed o HN I HN~N 2 o 1,.,0 p /'0 o Figure 1.10 NOE observable distances in DNA. Different arrows represent pathways available for assignment. Adapted from reference 156. 40 41 in either direction. The connectivities between 5-methyl of T with its own H6 and H8/H6 of preceding base form a good starting points as these connectivities are normally very well resolved. Although the COSY spectrum fails to connect two neighboring bases, it is very useful in confIrming the assignments made from the NOESY spectrum. The variety of COSY experiments which can be performed on DNA include phase-sensitive COSY, double quantum-filtered COSY and relay COSY.156 Phase-sensitive COSY divides the spectrum into positive (absorptive) and negative (dispersive) peaks. It improves the resolution of the peaks as a result of both the narrower absorption lines and the antiphase intensity distribution in the absorption mode multiplets. Phase-sensitive COSy spectra can be considerably improved with the use of two-quantum filters. The phase-sensitive COSy spectrum contains pronounced tails of the intense diagonal dispersion lines which masks the cross-peaks close to diagonal. In double-quantum filtered COSY the diagonal is suppressed and cross-peaks near the diagonal are clearly visible. Relay COSy establishes connectivities between protons which are not coupled to each other but share a common coupling partner. Thus, it allows one to connect HI' directly to H3' (or H2' to H4') of the same sugar resulting into an easier identifIcation of all the protons in an isolated spin-system. The COSy spectra are valuable for assigning H5 and H6 of cytidines and for obtaining coupling constants (J value) between various sugar protons. The identification of cytidines in COSY acts as a check point and removes the ambiguities in NOESY assignments. The coupling constants between sugar protons provide information regarding the sugar conformation. This is very important because the conformation of sugar pucker determines the overall geometry of the DNA. Although one can derive coupling constants from COSY experiments, they are seldom accurate. The presence of large linewidths precludes the accurate measurement of the coupling constant.159 The 42 intensity of the cross-peaks also depends upon the coupling constants and is used for detennining conformation of the sugar. l60 NMR spectroscopic studies of RNA Chemically, RNA differs from DNA only at the 2' position where it has a hydroxyl group. This substitution, seemingly very minor at first glance, has a major impact on both of the structure of RNA and its elucidation by NMR. Although structural elucidation of DNA by NMR has become routine, the reports on RNA are very few. RNA performs a variety of functions, from storage and translation of the genetic material (mRNA, tRNA) to enzymatic activity (ribozymes). Despite such versatility very little is known about the three-dimensional structure of RNA. This comes as a surprise since our ability to understand biological roles of RNA will be greatly improved with the knowledge of RNA structure. However, various factors are responsible for the slow progress in the field of RNA structure elucidation by NMR. These factors are described in next few paragraphs. Automated DNA synthesizers require aqueous ammonia for extended periods to remove oligomers from the column and some of the amino protecting groups at the end of the synthesis.161 These conditions cause severe degradation of the RNA resulting a lower yield of the full-length material. Presence of a bulky 2'-protecting groups requires longer coupling period. Longer coupling time leads to unwanted side reactions and consequent decrease in the yield of full-length material. In short, whereas automated DNA synthesis has become a relatively trivial process, RNA synthesis still is a complicated operation. All the sugar protons in RNA, with the exception of HI " resonate between approximately 4 and 5 ppm.162 Since all these sugar protons are coupled to each other and are close in space, this region of the spectrum is extremely crowded even in two­dimensional NOESY and homonuclear COSY spectra. A different chemical shift of HI' from the rest suggests that the HI '-H2' cross-peaks have the best potential for resolution. 43 Unfortunately, RNA normally exhibits A type geometry in which HI' and H2' are very weakly coupled.162 Such weak coupling precludes the appearance of HI '-H2' cross­peaks in COSY. Thus, identification of the proton type and individual spin system becomes impossible by methods that are applicable to DNA, and considerable efforts are needed to distinguish between different sugar protons and individual coupling system. The replacement of uracil for thymidine in RNA adds to these problems. The 5-methyl group of thymidine, which acts as a good starting point for sequential assignments in DNA, is now absent and the presence of hydrogen at the 5-position in uridine makes its aromatic spin system equivalent to that of cytidine. Consequently, straightforward assignment of cytidine aromatic protons from COSY is seldom possible. Thus, unambiguous assignment of an RNA spectrum is a much more complicated process than that of DNA. RNA exhibits considerably more structural diversity than DNA. There is a greater tendency to adopt secondary or tertiary structure e.g. pseudoknots, hairpins, bulges, internal loops etc. 163 Whereas a given DNA molecule usually adopts either a duplex form or a random coil form it is not at all necessary that a given RNA molecule will always form a duplex structure. Each RNA may be different structurally and types of connectivities observed in one RNA may not necessarily be observed in another RNA made up of different sequence. Thus, although structural diversity makes RNA molecules more interesting, it also makes them much more difficult to assign spectroscopically. RNA is inherently less stable than DNA. The phosphodiester bond in RNA is much more susceptible to hydrolysis due to the presence of the 2'-hydroxyl. Also, RNA is susceptible to various RNAses which are ubiquitous in nature. Handling of RNA, therefore, is much more difficult than DNA. The presence of the 2'-hydroxyl requires an additional protecting group. Synthesis of a 2'-protected nucleoside is a non-trivial task due to the presence of the 44 chemically almost equivalent 3'-hydroxyl. The procedure either requires a few additional steps or chromatographic separation of 2'- and 3'-isomers. Despite all these difficulties, scientists were successful in elucidating structures of some RNA molecules using high resolution NMR spectroscopy. Various techniques applied in assigning RNA protons are described briefly in next few paragraphs. Assignment of exchangeable protons The presence of secondary and tertiary structures in RNA makes assignment of exchangeable imino protons very important. These assignments playa very important role in identifying unusual base pairs, such as mismatches or base triples. 160,164 Imino protons in RNA are assigned in exactly same manner as in DNA. Assi~ment of nonexchangeable protons Three steps are involved in sequence-specific assignment of RNA. 165 The first step involves assigning each sugar proton to a particular type, e.g., H2', H3' etc., distinguishing adenine H2 from H8 and distinguishing uracil from cytidine. Step two involves identification of individual sugar spin system and the final step is to determine sequence specific assignment. Identification of base and sugar protons Assignment of base and sugar protons in a helical region is a straightforward process. The experiments used in DNA proton assignment are applicable here as well. The adenine H2 is identified by its NOE connectivity with U N3H imino protons. Other strategies used to distinguish H2 form H8 and cytosine from uridine involve using deuteriation of base protons and 13C NMR spectroscopy. The H8 proton of adenine is more susceptible to exchange than H2.165 Complete deuteration of the H8 proton in A TP is achieved by bubbling deuterium gas in presence of a catalyst (palladium) at room 45 temperature for 24 h. The H2 position, under these conditions, is only 70% deuterated. The same conditions are used to deuterate H5 of cytosine and uracil. Comparison between spectra of labeled and nonlabeled oligonucleotides leads to unambiguous assignment of all these base protons. 13C spectroscopy is very valuable in unambiguous assignments in RNA due to crowding in the proton NMR spectrum. In proton the spectrum, all the base protons, except H5 of C and U, resonate between 7 to 8 ppm. However, in 13C NMR spectroscopy, C2 carbons resonate approximately 15 ppm downfield from C8 and C6 resonances.166 This fact is used in two-dimensional 1H-13C heteronuclear correlation spectroscopy. Adenine H2-C2 cross-peaks are well separated from the rest and can be identified unambiguously. The H5 of C resonate in a separate spectral region from the H5 ofU. 13C-IH correlation is even more useful in assigning sugar protons. Unlike proton resonances, carbons resonances of the sugar are well separated from each other. 165 Consequently, in two-dimensional 13C-IH correlation spectroscopy, resolution of sugar protons is much better. Similar to sugar protons in DNA, sugar carbons in RNA exhibit characteristic chemical shifts. The C4' exhibits the farthest downfield shift followed by C2' and C3'. C5' displays the most up field carbon shift. This leads to immediate identification of various sugar protons, except H2' and H3'. Distinction between H2'­C2' and H3'-C3' cross-peaks can be made by careful analysis of peak shape. Identification of the su~ar spin system Sugar spin systems can be identified unambiguously by using scalar connectivities between the protons on each sugar. Double helical RNA exhibits A type of geometry in which the sugar pucker is C3' endo. The HI '-H2' coupling in C3' endo sugar pucker is less than 2 Hz. 167 As a result these cross-peaks are absent in homonuclear COSY spectra except for sugars at the end which undergo fast exchange between C2' endo and 46 C3' endo conformations. These weak couplings can be best observed by two­dimensional double quantum spectroscopy.168 The delay in the excitation sequence can be tuned for optimal excitation of 2-quantum states corresponding to protons with only small couplings. Relaxation degrades the signal for tuning delays longer than 30-50 ms, but delays of 20-40 ms provide good sensitivity for cross-peaks corresponding to 3-5 Hz. The C3' endo sugar pucker allows relatively large couplings between H2'-H3' (5 Hz) and H3'-H4' (8-10 Hz).167 These connectivities can be observed in 31P-decoupled double quantum filtered COSY spectra.169 Phosphorus decoupling is necessary to reduce multiplet complexity. These COSY techniques are not applicable to RNA above 6000 dalton molecular weight, and identification of sugar spin systems for these RNA relies on NOE connectivities. Although these though-space NOESY connectivities are inherently less reliable, sugar spin system identification from NOESY spectrum represent an important step towards establishing complete spectral assignment. 165 Sequence-specific resonance assignments Sequence-specific resonance assignments can be obtained either from through­space NOESY connectivities or from through-bond connectivities between sugar protons and the phosphorus. In the case of NOESY, the strategy used in DNA for the assignment of aromatic and HI' protons from double-stranded region is also applicable in RNA. Each aromatic proton is close in proximity to two HI' protons, its own and the preceding (5' side). However, this HI '-aromatic proton distance in RNA is greater than that in DNA. As a result longer mixing periods are needed to observe this connectivity and the peaks obtained are basically via spin diffusion through H2' protons.162 The A type of geometry in RNA is responsible for some additional cross-peaks in the NOESY spectra. For example, pyrimidine H5 gives strong cross-peaks with H2' of its own and H2' of the preceding base. These peaks are often very weak in DNA. Also strong interstrand and 47 intrastrand cross-peaks are observed for adenine H2 proton and HI'. These adenine cross-peaks can be confusing sometimes and may lead to erroneous assignments. The sequential assignments made using NOESY rely on conformation assumptions. This can lead to misassignments in the presence of unusual conformations. The second procedure for sequence-specific assignment of proton resonances in oligonucleotides is based on the observation of through-bond connectivities between the phosphorus and sugar protons.165 The phosphorus nuclei are coupled to the preceding H3' protons and the following HS', HS" and often H4' protons. As a result, one can walk: from H3' of a base to internucleotide phosphorus and to HS', HS" of the next base. This walking process provides the sequence-specific assignments. This procedure does not rely on conformational assumptions and is applicable to both single and double stranded regions. The problem with this procedure is obtaining a high-quality 31p_1H correlation spectra at molecular weights above 5000 daltons due to the unfavorable 31 P relaxation. This procedure also requires complete identification of all sugar spin systems beforehand. Conformational studies on DNA:RNA hybrids DNA:RNA hybrids playa central role in the transfer of biological information. They are formed during transcription of DNA into RNA and during reverse transcription of viral RNA into DNA. These hybrids have one pure DNA strand and one pure RNA strand. A different type of hybrid is observed during DNA replication. DNA polymerase requires a short RNA primer for initiation of DNA synthesis. The nascent DNA, therefore, contains an RNA fragment covalently attached to it. This type of hybrid is known as 'Okazaki fragment,.170 Knowledge of the three-dimensional structure of DNA:RNA hybrids is of primary importance in understanding the mechanisms of transcription and polymerization processes. Also, structural information regarding DNA:RNA hybrids is important in antisense inhibition of gene expression. The inhibitory 48 activity of antisense oligonucleotides depends greatly upon their ability to form a DNA:RNA hybrid that could be cleaved by RNAse H.2 As a result, rational design of new modified oligonucleotides will be facilitated by knowledge of the structural features of the DNA:RNA hybrids. Until the application of high resolution NMR and molecular modeling in elucidation of macromolecule structure, DNA:RNA hybrids were studied by a variety of different methods. These studies often gave conflicting results. The first X-ray crystallographic studies on DNA:RNA hybrids were performed by Milman and co­workers. l71 From their data they concluded that the hybrid did not adopt a B form geometry. Tunis and Hearst studied a calf thymus DNA:RNA hybrid through optical rotatory dispersion and concluded that the hybrid assumed a conformation similar to double stranded RNA.I72 The X-ray diffraction pattern of poly(rI).poly(dC) was found to be more similar to A form than B form. The first circular dichroism study of hybrids was carried out by Gray and Ratliff on poly[d(AC).r(GU)] and poly[r(AC).d(GT)].173 They concluded that the spectrum of poly[r(AC).d(GT)] resembled more closely the spectrum of the double-stranded RNA duplex poly[r(AC)r(GU)] than the reverse poly[d(AC).r(GU)]. Pardi et al. used NMR for elucidation of hybrid structure. 174 They measured coupling constants between various sugar protons to deduce the sugar pucker. Their data indicated that the sugar puckers of both strands are in an A form RNA-like conformation. All these studies indicated that both strands of the hybrid assumed a single conformation. This was contradicted first by Zimmerman and Pheiffer.175 They showed that the RNA:DNA hybrid poly(rA).poly(dT) could adopt two conformations, depending upon its degree of hydration. Fibers yielded a uniform A RNA-like pattern at 79% relative humidity. However, under high humidity DNA adopted a B form and RNA assumed an A form in the same duplex. This observation was later supported by 31p NMR176, CDl77 and Raman spectroscopy studies178 on poly(rA).poly(dT). Arnot et al. 179 carried out X- 49 ray diffraction studies on poly(dA).poly(rU) and poly(dI).poly(rC). They concluded that in both duplexes the DNA strand assumed a B like C2'-endo conformation, whereas the RNA strand assumed an A like C3'-endo conformation. One-dimensional NMR and CD studies of Imbach et al. contradicted all the results listed above. I80 They concluded that both the RNA and DNA sugars in a synthetic hexamer hybrid assumed a B form conformation in solution. Gupta et al. performed one­dimensional NMR studies on poly(rA)poly(dT).181 They also concluded that both the strands assumed a B form geometry due to the absence of adenine H8 to H3' NOEs in RNA strand. These were the only two reports suggesting that both the strands assume B form conformation. Recent studies performed on DNA:RNA hybrids using high resolution NMR seemed to have ended this conflict. Reid's group have determined the solution structure of the DNA:RNA hybrid d(GTCACATG):r(CAUGUGAC) by means of two-dimensional nuclear overhauser effect spectra, restrained molecular dynamics and full-relaxation matrix simulation of the two-dimensional NOE spectra. 182 They concluded that the DNA:RNA hybrid assumed neither B form nor A form in solution, but an intermediate heteromerous duplex structure. The sugars of the RNA strand exhibited a normal N type C3'-endo geometry, but the sugars of the DNA strand exhibited neither C2'-endo nor C3'-endo conformations. They showed an unexpected intermediate 04'-endo conformation. Detailed structural information was obtained from NOESY experiments. The intensities of the cross-peaks were converted into distance information. These distances were used in conjunction with restrained molecular dynamics algorithms to generate coordinates that are consistent with the initial distance estimates. The solution structure of the DNA:RNA hybrid showed overall geometry that was close to A form. However, the minor groove width was intermediate between that of A form and B form. Both the DNA and RNA strands showed marked sequence-dependent variations in their helical parameters. 50 Reid and co-workers have also described a model for the mechanism of RNAse H. RNAse H an enzyme which selectively cleaves the RNA strand of the DNA:RNA hybrid at the end of the DNA polymerization. Prior to this report, few attempts had been made to model the catalytic mechanism and the specific interactions of the enzyme with the sugar­phosphate backbone in the DNA:RNA hybrid. All these models failed to explain how RNAse H discriminates between DNA:RNA hybrid and pure RNA duplex. However, a three-dimensional model of the hybrid generated by this study adequately explains the discrimination. According to the authors, the size of the minor groove, which is intermediate between A form and B form, is the important structural factor in complex formation and acts as a discriminator between DNA:RNA hybrids and pure RNA:RNA duplexes. Lane and co-workers determined the solution conformation of the DNA:RNA hybrid d(GTGAACIT).r(AAGUUCAC) at approximately same time. Their results are identical to those of Reid et al. and support the conclusions that size of the minor groove is the discriminating factor. 183 Reid's group have also performed structural studies on the chimeric DNA:RNA hybrids. They studied two self-complementary DNA-RNA-DNA duplex chimeras and a non self-complementary chimera. 184 Their data indicate normal Watson-Crick hydrogen bonding and base stacking at the junction. Preliminary qualitative two-dimensional NMR data suggest that the internal RNA segments contain C3'-endo sugar conformation except for the first RNA residues following the 3' end of the DNA block, which, unlike the other ribonucleotides, exhibit detectable HI '-H2' coupling. The nucleosides of the two flanking DNA segments appear to adopt a fairly normal C2'-endo conformation except at the junction, where the last DNA residue adopts an intermediate sugar conformation. The junction residues exhibit quite different NMR behavior, but these effects do not appear to propagate into the DNA or RNA segments. The circular dichroism spectra of these d-r-d chimeras display a mixture of characteristic A-form and B-form absorption bands. The 51 data indicate that A-form and B-form conformations can coexist in a single short continuous nucleic acid duplex. CHAPTER 2 STATEMENT OF TIIE PROBLEM Goals and objectives As described in the preceding chapter, scientists have shown tremendous interest in small oligonucleotides for antisense inhibition of gene expression. Considerable efforts have been made to stabilize the duplex formed between an antisense oligonucleotide and its target. The work presented in this dissertation is concerned with the same problem. However, a somewhat different approach was considered to address this issue. The primary goal of the research was to incorporate a 2'-O-aralkyl nucleoside into DNA as well as a DNA:RNA hybrid and to study in detail the manner in which the 2'­O- aralkyl substituent would influence the structure of ribonucleotide to which it is attached and the nucleic acid duplexes with which it interacts. This could, in principle, lead to duplex stabilization or destabilization. The information gained from this study will enable evaluation of the criteria required for helix stabilization by intercalation. These studies will also be useful in elucidating the nature of non-intercalative modes of interactions between duplexes and an aromatic ring The original objectives of the dissertation research were to: a) Synthesize 2'-O-phenethyladenosine and 2'-O-(p-azidophenethyl)adenosine (Figure 2.1); b) Incorporate these compounds into oligonucleotides; c) Study the effect of modification on stability of the duplex by UV and CD spectroscopy; N ) N HO OH 0 N ) N HO OH 0 Figure 2.1 Structures of 2'-O-phenethyladenosine and 2'-O-(p-azidophenethyl) adenosine. 53 d) Study the effect of modification on the conformation of the duplex by high resolution NMR; and e) Develop principles from the above studies to apply to the design next generation of intercalators and groove binders. Rationale for the thesis compounds 54 2'-O-Phenethyladenosine and 2'-O-(p-azidophenethyl)adenosine contain a benzene ring as the aromatic portion of the 2'-0-substituent. The 2'-hydroxyl was chosen as the site of attachment since it offers several advantages. Oligonucleotides containing 2'-0- alkyl ribonucleosides exhibit enhanced stability against degradation by nucleases.7 Such oligonucleotides also form stable duplexes with complementary nucleic acids.34 Improvement in stability and binding affinity are the most desirable features of an antisense oligonucleotide construct. A large body of literature is available on the synthesis of 2'-0-alkyl nucleosides. As a result, one has a choice of several synthetic procedures. Once the 2'-0-alkylated nucleoside is synthesized, it can be incorporated into oligonucleotides using automated synthesis. The 2'-O-phenethyladenosine served as a good starting model. Partial stacking of the benzene ring in 2'-O-benzyladenosine and 2'-O-(p-nitrobenzyl)uracil is already known.81 ,82 The benzene ring, thus, possesses the required hydrophobicity and planarity to stack under the bases. The ethylene bridge provided additional flexibility to the model. Thus, the minimum requirements for the intercalation were fulfilled. The 2'-O-(p­azidophenethyl) adenosine was synthesized to cross-link two strands of the duplex by irradiation should intercalation take place. Once introduced into the duplex, various options should be open to the benzene ring. It is possible that the hydrophobicity of the benzene ring is not sufficient for intercalation. In that case it can extend into solvent surrounding the duplex. It can also lie in one of the grooves in contact with one or more bases of the oligonucleotides. 55 Molecular modeling studies on B-DNA containing 2'-O-phenethyladenosine indicated that it can choose either major or minor groove, depending upon the conformation of the sugar. The C3'-endo sugar conformation puts the benzene ring in the minor groove. The benzene ring in this case interacts with the nucleosides on the 5'-side of the modified base. The C2'-endo sugar conformation puts the benzene ring in the major groove and the benzene ring in this case interacts with the nucleosides on the 3'-side. However if the benzene ring intercalates, the only available site for intercalation and that site is between adenosine and the base on the 3' side. Detailed study of the oligonucleotides containing 2'-O-phenethyladenosine should provide an insight regarding the nature of interaction between benzene and the duplex. The results obtained should be very valuable in designing different nucleic acid binding molecules such as intercalators and groove binders. Molecular modeling studies were performed to obtain the minimum energy conformation of 2'-O-phenethyladenosine. Both the C2'-endo and C3'-endo sugar conformations were used as the starting conformations. A molecular dynamics simulation was carried out for 50 pecoseconds at 300 OK. The family of low energy conformations obtained in the dynamics simulation were minimized again. One of the minimum energy conformations obtained in each case is shown in Figures 2.2 and 2.3. Almost all these minimum energy conformations show stacking between the phenethyl benzene ring and the purine ring of the adenosine. The molecular modeling studies, therefore, substantiated the notion that the benzene ring prefers a stacked position. Close examination of minimum energy conformers uexpectedly revealed that all of the conformers exhibit C2'­endo sugar pucker in contrast to the usual 3'-endo pucker typical of a ribonucleotide. 164 Presence of a C2'-endo sugar pucker suggests that if the benzene ring fails to intercalate it should lie in the major groove. In fact, later during the analysis of the conformation by NMR, it was learned that the actual conformation of the phenethyl sugar was indeed close to C2'-endo and the benzene ring lay inside the major grove. This unique observation will 56 Figure 2.2 Energy minimized conformation for 2'-O-phenethyladenosine. Starting sugar conformation was C2'-endo. 57 Figure 2.3 Energy minimized conformation for 2'-O-phenethyladenosine. Starting sugar conformation was C3'-endo. 58 significantly affect the design of the next generation of nucleic acid binding molecules. The sequences of the oligonucleotides used in the study are shown in Table 2.1. Sequence II is the modified oligonucleotide and sequence I is the control. Sequences I and II are self-complementary and have alternate purine-pyrimidine bases. Such sequences are easier to interpret using high resolution NMR.185 The self-complementary nature of the oligonucleotides introduces two modifications in the duplex. Interaction between these two modifications may increase the complexity of the model. Therefore, it becomes important that two modifications do not interact with one another. This was assured by introducing the modified nucleosides at the fourth position. Selection of the fourth position also assured that the modified nucleosides were situated in the double­strand region of the helix .. Intercalation of the phenethyl benzene ring, were it to occur, would be spatially similar to introduction of an additional base in the modified strand (Figure 2.4). Such introduction should cause considerable perturbation in the normal geometry of the duplex. This perturbation, in principle, can be confined largely to the duplex sugar backbone by adding an extra base on the complementary strand. Oligonucleotide N was synthesized to test this hypothesis. Oligonucleotide ill served as the control for this experiment. Oligonucleotide V and VI were synthesized to study the effect of the modification on the DNA:RNA hybrids. A different type of geometry exhibited by DNA:RNA hybrids was the reason for studying these hybrids. The DNA:RNA hybrids are known to assume an overall conformation which is close to A-form. 182 As a result, these studies will provide the information regarding the effect of the geometry on the mode of interaction between the benzene and the helix. These studies were important from the antisense perspective also since most of the antisense construct are designed to target mRNA. The oligonucleotides used in the study are chimeric in nature. The first six nucleotides contain deoxyribose whereas the next six nucleotides contain ribose. The self­complementary nature of the oligonucleotide ensures that the DNA portion hybridizes with 59 Table 2.1 Sequences of various oligonucleotides SEQUENCE NAME I 5' d C G C A CAT G T G C G 3' Standard 12-mer IT 5'd CGC A*CA TGT GCG 3' Modified 12-mer A *= 2'-O-Alkyladenosine ITl 5'd CGC ACA TGe TGC G 3' Standard 13-mer N 5'd CGC A*CA TGe TGC G 3' Modified 13-mer A *= 2'-O-AJ!cyladenosine V 5' d(C G C A C A) r(U G U G C G) 3' Standard hybrid VII 5' d(C G C A * C A) r(U G U G C G) 3' Modified hybrid A *= 2'-O-Alkyladenosine 60 C GCACATGTGCG 1 2 3 4 5 6 7 8 9 10 11 12 C G C A <I> C A T G T G C G ~ 1 2 3 4 4' 5 6 7 8 9 10 11 12 ~ G C G T C G T A C <I> A C G C C G C A <I> C A T G C T G C G I I I ~ I I I I I I I 1 2 3 4 4' 5 6 7 8 9 10 11 12 13 Figure 2.4 illustration of the phenethyl ring (<I» acting as an extra base. 61 the RNA portion such that the DNA:RNA structure would result in the duplex form. The sequences of these hybrids were identical to those of standard 12-mer and phenethyl 12- mer. This allowed a direct comparison of the results obtained in the DNA:RNA hybrid with those obtained in the DNA:DNA duplex. Soon after the thesis research was started, Yamana et al. published an article describing intercalation of anthraquinone tethered at 2'-hydroxyl of uridine.99 The oligonucleotide was stabilized by approximately 18°C relative to the unsubstituted oligonucleotides. Keller and Haner introduced the anthraquinone at the 2'-position of various nucleoside via an acetyl linker. 88 They also observed stabilization of the duplex compared to the control oligonucleotide. These authors suggested that the intercalation was responsible for the stabilization, but provided no direct evidence in support of their hypothesis. 2'-0-(Anthraquinonylmethyl)adenosine (Figure 2.5) was synthesized and incorporated into oligonucleotides of the same sequence as used in the phenethyl derivative in order to determine whether the observation of Yamana et al. was general and, if so, whether the stabilization did, in fact, resulted from intercalation. Molecular modeling studies were also performed on 2'-0- (anthraquinonylmethyl)adenosine. The minimum energy conformations are shown in Figures 2.6 and 2.7. Both the C2'-endo and C3 '-endo conformations of the sugar were used as the starting conformation. All the minimum energy conformations show complete stacking of anthraquinone ring under the purine ring. The sugar pucker observed in the minimum energy conformers was C2'-endo. The complete set of oligonucleotides was subjected to detailed structural analysis by high resolution NMR and UV spectroscopy. The oligonucleotides were studied by CD spectroscopy also to gain an insight regarding the overall conformation of the molecule. 62 N ) N HO o OH 0 o Figure 2.5 Structure of 2'-O-(anthraquinonylmethyl)adenosine. Figure 2.6 Energy minimized confonnation for 2'-O-(anthraquinonylmethyl) adenosine. Starting sugar confonnation was C2'-endo. 63 Figure 2.7 Energy minimized conformation for 2'-O-(antbraquinonyl­methyl) adenosine. Starting sugar conformation was C3'-endo. 64 CHAPTER 3 SYNTHESIS OF 2'-O-ALKYL ADENOSINES AND THEIR INCORPORA nON INTO OLIGONUCLEOTIDES Synthesis of 2'-O-phenethyladenosine and 2' -O-(p-azidophenethynadenosine As described in the fIrst chapter, several methods are available for the synthesis of 2'-O-alkyl nuc1eosides. Each method is associated with some distinct advantages but suffers from drawbacks as welL The choice of the method, therefore, depends on what one has to accomplish and how quickly it has to be accomplished. The choice of the method also depends upon the stability of the reagents. If one wants to avoid formation of the 3'-isomer completely, then the method developed by Sproat et al. is the best.90 However, this method involves numerous steps. For example, the first step in this method is simultaneous protection of 3' and 5' hydroxyl. The protecting group is removed later in another reaction, thus increasing number of steps. Increase in the number of steps causes signifIcant consumption of time. Also, some of the reagents used in above synthesis, e.g., BDDDP, are very expensive. If the 2'-substituent is sensitive to alkaline conditions, then the 2',3'-dibutylstannylene derivative of the nucleoside can be used as the starting material. However, the fastest route of synthesis for 2'-O-alkyl nucleosides is the one which utilizes strongly basic conditions obtained by using NaH as a base. For example, synthesis of 2'-O-(p-methoxybenzyl)adenosine was complete in an hour in presence of NaH at -5 oC83, whereas it took four hours at 110 °C to synthesize 2'-O-(0-nitrobenzyl)uridine using the 2',3'-dibutylstannylene derivative.97 Also, the low temperatures used in the NaH method keep formation of 3 '-isomer to a minimum level. 66 However, the NaH method requires that the alkylating agent is stable to strongly basic conditions. Both phenethyl and p-azidophenethyl groups are stable to strongly alkaline conditions. As a result, these conditions were employed in the synthesis of title compounds. Substitution of a phenethyl group on the 2'-oxygen of the ribonucleotide presents some unique problems. Alkylation with phenethyl proceeds much slower than that with methyl or benzyl groups. The rate of alkylation can be increased by using a reactive derivative of the phenethyl group; e.g., phenethyl triflate or phenethyl mesylate. However, under the strongly basic conditions used in alkylation, elimination competes with substitution. The elimination reaction involves abstraction of a benzylic proton and subsequent formation of styrene (Figure 3.1). The rate of elimination can be kept to a minimum by using a less reactive phenethyl derivative; e.g., phenethyl iodide or phenethyl bromide. Thus, the intrinsic low reactivity of the phenethyl group and competing elimination reaction impose exactly opposite conditions on the kind ofphenethyl derivative to be used in the alkylation reaction. To obtain the best possible yield in the alkylation reaction, different derivatives of phenethyl and p-azidophenethyl were tried. Figure 3.2 shows the reaction conditions, various alkylating agents used and the yields obtained in each case. p-Azidophenethyl triflate as well as p-azidophenethyl mesylate underwent complete elimination and failed to give any substitution product. On the other hand, p-azidophenethyl iodide and phenethyl bromide reacted very slowly and gave very minor amounts of the required 2'-O-alkylated product. Phenethyl tosylate and p-azidophenethyl tosylate, however, showed a reasonable balance between elimination and substitution. In case of p-azidophenethyl tosylate 8% yield of the desired 2'-isomer was obtained. This yield, although very low, was not prohibitive due to the low costs of starting materials. Even in cases of p-azidophenethyl tosylate and phenethyl tosylate, elimination proceeded at a greater rate than substitution at all temperatures. As a result, three 67 ~Base j H H Styrene Figure 3.1 Fonnation of styrene in the synthesis of 2'-O-Phenethyladenosine. uo NU2 NU2 NaH ~ N R-o-CH2CH2X uo 80 OU 80 R x yield N3 OTf - N3 OMs - N3 OTs 8% N3 I <1% H OTs 16% H Be 1% Figure 3.2 Various alkylating agents used in the synthesis of 2'-O-phenethyladenosine and 2'-O-(p-azidophenethyl)adenosine. R 0\ 00 69 equivalents of phenethyl tosylate and two equivalents of NaH were used in the reaction. Adenosine was dissolved in dry dimethyl formamide (DMF) by heating the suspension of adenosine in DMF. The reaction rate was significantly reduced if a suspension of adenosine was used instead of a solution. The reaction was started at 0 oC, but the temperature was raised slowly to about 60 °C in 4 h. Two equivalents of phenethyl tosylate or p-azidophenethyl tosylate were added in several portions to the reaction during this period. A second equivalent of NaH was added after cooling the reaction at 25 °C and heating was continued for half an hour. The third equivalent of the alkylating agent was added at this time and the reaction was continued overnight. Protection of 21-0-phenethyladenosine The exocyclic amino group and the 51-hydroxyl of the adenosine, if left unprotected, react with the reagents used in the automated DNA synthesis. As a result, these groups require protection prior to their incorporation via automated DNA synthesis. The scheme for protection of 21-O-phenethyladenosine is shown in Figure 3.3. The exocyclic amino group on the adenosine was protected by a benzoyl group in 65 % yield following the procedure developed by Ti et al.186 The 51-hydroxyl was protected by dimethoxytrityl group (dmtr) in 60% yield. The procedure for dimethoxytritylation was reported by Wu et al.I87 The final reaction performed on the protected nucleoside before it could be utilized in the automated DNA synthesis is phosphitylation of the 31-hydroxyl. The phosphitylation reaction involves attachment of an activated phosphorus on the 31- hydroxyl. N, N, N', N'-tetraisopropyl B-cyanoethoxy phosphorodiamidite was used as the phosphitylating reagent. The 31-phosphitylated compound was synthesized according to procedure reported by Kierzek et aI. in 67 % yield.I88 NH-CO-Ph NA...~ ~NJlN 1) TMS-CI 2) PhCOC,. I iPr iPr -N P-O"""-CN iPr -N iPr ~ DmtrO~ ? O..r-Q .P, _( ~CN NH-CO-Ph t~~ HotJ HO O~ ! Dmtr-CI NH-CO-Ph t~~ N N DmtrO~ HOO~ Figure 3.3 Protection strategy for 2'-O-phenethyladenosine. 70 71 Protection of 2'-O-Cp-azidophenethyl)adenosine The scheme used for the protection of 2'-O-phenethyladenosine was also used for the protection of 2'-O-(p-azidophenethyl)adenosine. However, the phosphitylation reaction failed to give the expected 3'-phosphoramidite. On addition of phosphitylating reagent to the protected 2'-O-(p-azidophenethyl)adenosine, a compound with greater polarity than the parent protected compound was apparent in the reaction mixture within an hour. This was very surprising because 3'-phosphoramidites of the nucleosides are significantly less polar than the parent compounds and exhibit high Rf value on thin-layer chromatography. Also, the 3'-phosphitylation reaction proceeds very slowly and the product starts appearing after 6 or 7 h. The phosphorus NMR study of this compound indicated that it contained a shielded phosphorus with chemical shift of approximately 40 ppm downfield with respect to H3P04. Upon addition of one more equivalent of the phosphitylating reagent, this high polarity compound disappeared and two low polarity compounds, similar to the 3'-phosphoramidite isomers, were obtained. The phosphorus NMR study on these low polarity compounds indicated that they contained shielded phosphorus (chemical shift of approximately 40 ppm) along with expected deshielded phosphorus characteristic of P-O bonds (chemical shifts of approximately 150 ppm). Comparative study of two phosphitylating reactions, one with protected 2'-O-(p­azidophenethyl) adenosine and other with protected 2'-O-benzyladenosine, showed no high-polarity product formation in case of 2'-O-benzyladenosine. This implied involvement of azido group in the reaction. A plausible structure for the high-polarity compound in shown in Figure 3.4. This structure is based upon all the observation listed above. Further evidence for this structure comes from the reaction reported by Gil yarov et al. 123 They reported synthesis of various compounds containing a double bond between phosphorus and nitrogen. This P-N double bond was formed by reacting the aryl azide with various trivalent phosphorus compounds. (iPr ) 2 I N NH-CO-Ph ~~J P-O ............... CN N I (iPr )2 mml'W HOO~N3 ... NH-CO-Ph N~N,) ~NJlr{ mml'-y°~ (r' )2 ''1-( ~ . ~CN HO O~N=~-O • (iPr )2 Figure 3.4 Reaction of phosphitylating agent with 2'-O-(p-azidophenethyl)adenosine. -.] tv 73 The reaction of aryl azides with phosphorodiamidite imply that aryl azides can not be used in any automated DNA synthesis scheme which utilizes phosphoramidite chemistry. One can introduce an aryl azide into an oligonucleotide only after the oligonucleotide is completely synthesized. Thus, only 2'-O-phenethyladenosine could be incorporated into oligonucleotides. Four oligonucleotides, whose sequences are shown in Table 2.1, were synthesized. All the four oligonucleotides were characterized by UV and high-resolution NMR spectroscopy. CHAPTER 4 CHARACfERIZATION OF OLIGONUCLEOTIDES CONTAINING 2'-0-PHENETHYLADENOSINE BY UV AND CD SPECTROSCOPY Synthesis and purification of oli~nucleotideS The oligonucleotides, standard as well as 2'-0-phenethyl-containing, were synthesized on an automated DNA synthesizer. Preliminary syntheses were carried out on IJlM scale because the amount obtained in 1 JlM synthesis is sufficient to carry out UV and CD spectroscopic studies. The sequences of the oligonucleotides are shown in the Table 2.1. One can easily observe from these sequences that all the nucleosides except 2'­O- phenethyladenosine are deoxynucleotides. The phosphoramidite of the 2'-0-phenethyl­adenosine was synthesized in the laboratory. For the standard deoxynucleotides, commercially available phosphoramidites were used. A standard 1 min period was used to couple two deoxynucleotides. However, the phosphoramidite of the 2'-0- phenethyladenosine was thought to be hindered and consequently, the coupling reaction was allowed to proceed for 10 minutes. The analysis of coupling efficiency indicated that the 2'-0-phenethyladenosine phosphoramidite coupled less efficiently than the deoxynucleoside phosphoramidite as judged by the trityl release assay. Such less efficient coupling is a very common phenomenon in the automated synthesis of RNA. The yield of the coupling reaction was improved by purifying the phosphoramidite by column chromatography. The oligonucleotides were treated with aqueous ammonia at 55°C for 18 h to remove acyl protecting groups and to remove the oligonucleotide from the column. 75 The oligonucleotide synthesized from the automated DNA synthesizer often contains failure sequences. The full-length material is separated from the failure sequences either by high performance liquid chromatography (HPLC) or by gel electrophoresis. The purification in this case was carried out by on a preparative reversed phase (CIS) HPLC column. The ammonium bicarbonate buffer (50 rnM, pH 7) along with a gradient of acetonitrile was used to purify the oligonucleotides. Two ammonium bicarbonate buffers were made; one contained 5% acetonitrile, whereas other contained 20%. Both the buffers were filtered through a membrane filter which also degassed the buffers. These two buffers were used to increase the acetonitrile concentration. Such premixing of acetonitrile with buffer ensures that precipitation of the buffer sa