synthesis and crystallization study of the 5-se-thymidine

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Chemistry Theses Department of Chemistry

8-13-2019

Synthesis and Crystallization Study of the 5-Se-Thymidine DNAs and the Complexes with SmallMoleculesShuo [email protected]

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SYNTHESIS AND CRYSTALLIZATION STUDY OF THE 5-SE-THYMIDINE DNAS AND

THE COMPLEXES WITH SMALL MOLECULES

by

SHUO LIU

Under the Direction of Zhen Huang, PhD

ABSTRACT

There are two major challenges in nucleic acid X-ray crystallography: crystallization and

phasing determination. With the introduction of selenium modification, the phasing problem can

be solved rationally, while the crystallization problem can be solved partially. By taking advantage

of the selenium modification, herein we report the synthesis of the Se-DNAs and the

thermostability and crystallization study of the DNA-small molecule complexes. 5-Se-thymidine

was successfully synthesized and incorporated into DNA oligonucleotides via solid-phase

synthesis in order to facilitate the crystallization and phasing. The UV-melting study reveals that

the selenium modification has caused no significate perturbation to the DNA stability. The small

molecules (DB2429 and DB2457) were co-crystalized with Se-DNAs and the Se-DNA crystals

were obtained, which are better than the native ones. In addition, our research suggested that the

DNA oligonucleotides with the 5’-overhang form better crystals than those with the 3’-overhang

or non-overhang. Our X-ray diffraction study is in progress.

INDEX WORDS: Nucleic acid, Selenium derivatization, Small molecule, Crystallization, X-ray

crystallography



by

SHUO LIU

A Thesis Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science

in the College of Arts and Sciences

Georgia State University

2019

Copyright by

Shuo Liu

2019



by

SHUO LIU

Committee Chair: Zhen Huang

Committee: Ming Luo

Markus Germann

Electronic Version Approved:

Office of Graduate Studies

College of Arts and Sciences

Georgia State University

July 2019

iv

DEDICATION

I want to thank my parents here. It is they who support me silently behind me, encourage

me so that I don't have to worry about economic problems. Whenever I encounter a significant

choice, talking to them always makes me determined. In any case, I would like to thank them for

their understanding and support!

I also want to thank my roommates, Wei Liu, Shuliang Zhao, and Xinyu Fu. They have

given me a lot of help in life. Also, let me live in a foreign land, do not feel so lonely.

Thanks to my friend Xu Jin. I have a company when I am bored. We often travel together

and play together.

Thanks to my friends, Weiting Tang, Jia Li, and Ruyu Li. We meet by chance but hit it off.

I miss the days when we traveled like a family.

Thanks to my friend, Mengshi Zhang. She accompanied me when I was most stressed,

encouraged me, and urged me. It was her motivation that allowed me to overcome the difficulties

and eventually complete my graduation thesis.

I am grateful to my family and friends who have accompanied me for two years and brought

me joy. Thank you very much.

v

ACKNOWLEDGMENTS

Unconsciously, my graduate career is nearing completion. As the last answer to my

master's study life, I hope to express my sincere gratitude to the teachers, students, and everybody

who has helped me educated me, and encouraged me!

First of all, I would like to thank my PI, Dr. Zhen Huang. During the past three years, Dr.

Huang has given me a lot of help in study and life. When I was a senior, I participated in the 3+1+2

project of Sichuan University and Georgia State University. He created the opportunity for me to

study abroad. Being in a foreign land is inconvenient. Dr. Huang gave me a lot of support in life,

and I quickly adapted to living abroad. At the same time, He also gave me a lot of help in scientific

research. I am very grateful to the teaching and care of the two teachers!

Then, I want to thank my committee, Dr. Ming Luo and Dr. Markus Germann. They are

busy taking time out to help me complete the graduation reply. I appreciate that.

I would also like to thank my lab mate Lin Qin. As a newcomer to the lab, Lin taught me

how to experiment. He patiently and very seriously informed me how to find problems and solve

problems in the experiment. I have benefited a lot from it. I am very grateful to Qin Lin for helping

me!

Also, my labmate Cen Chen, in the most helpless period of my life, he accompanied me

through. In the experiment, he taught me how to solve problems, and taught me how to deal with

difficulties in life. Thank you very much.

These two years of graduate life are full of bitterness and joy, and many people have helped

me and educated me. A thousand words are combined into four. Thank you very much!

1

TABLE OF CONTENTS

ACKNOWLEDGMENTS ................................................................................... V

TABLE OF CONTENTS ..................................................................................... 1

LIST OF TABLES ................................................................................................ 6

LIST OF FIGURES .............................................................................................. 9

1 INTRODUCTION...................................................................................... 15

1.1 Introduction of Central Dogma and Nucleic Acid Inhibition ............ 15

1.2 Introduction of X-ray Crystallography ................................................ 16

1.2.1 The function and application of X-ray crystallography .................. 16

1.2.2 Challenges and difficulties in nucleic acid X-ray crystallography . 17

1.3 Introduction of Selenium Nucleic Acid ................................................ 19

1.3.1 The chemical and biochemical properties of selenium ................... 19

1.3.2 Introduction of 5-Se-Tymidine modified nucleic acid ..................... 22

1.3.3 Application of selenium nucleic acid in X-ray crystallography ...... 24

2 EXPERIMENT .......................................................................................... 26

2.1 The Synthesis of 5-Se-T phosphorimidite Starting with 5’-DMTr-5-I-

dU 26

2.1.1 Tert-butyldimethylsilyl-(TBDMS-) protection .................................. 27

2.1.2 The introduction of methyl selenium ............................................... 30

2.1.3 Detritylation ....................................................................................... 33

2

2.1.4 The synthesis of phosphoramidite .................................................... 35

2.2 Synthesis and Purification of the Selenium Nucleic Acids ................. 37

2.2.1 Solid phase synthesis of the nucleic acid with selenium modification

37

2.2.2 Deprotection and purification of the nucleic acid with selenium

modified 46

2.3 Crystallization Procedure ...................................................................... 51

2.3.1 Introduction for Hanging Drop Vapor Diffusion Crystallization ... 51

2.3.2 Sample Preparation ........................................................................... 52

2.3.3 Crystallization .................................................................................... 52

2.4 Crystal and Structure Study of DND-small Molecule Complex ........ 53

3 RESULTS ................................................................................................... 55

3.1 The Synthesis of 5-Se-T phosphorimidite Starting with 5-I-dU ......... 55

3.1.1 Tert-butyldimethylsilyl (TBDMS-) protection .................................. 56

3.1.2 The introduction of methyl selenium ............................................... 57

3.1.3 Detert-butyldimethylsilyl protection ................................................. 59

3.1.4 Dimethoxytrityl (DMTr-) protection ................................................. 60

3.2 Solid Phase Synthesis of the Selenium Nucleic Acid ........................... 62

3.2.1 Advantages of solid phase synthesis ................................................. 62

3.2.2 Solid supports .................................................................................... 63

3

3.2.3 The phosphoramidite method ........................................................... 64

3.3 High-Performance Liquid Chromatogram (HPLC) Analysis for

Selenium Modified DNA Sequences .......................................................................... 65

3.3.1 Reverse phase HPLC procedure ....................................................... 65

3.3.2 HPLC chromatogram for W3 (5 '-d(CCAAAGT5- SeTGC)-3') ......... 67

3.3.3 HPLC chromatogram for W4 (5'-d(GCAACTT5 -SeT GG)-3') ......... 68

3.3.4 Summary ............................................................................................ 68

3.4 Mass Spectra of Nucleic Acid ................................................................ 69

3.4.1 MALDI(+)-TOF spectra of W1 (5'-d(CCAAAGTTGC)-3') ............. 69

3.4.2 MALDI(+)-TOF spectra of W2 (5'-d(GCAACTTTGG)-3') ............. 70

3.4.3 MALDI(+)-TOF spectra of 5’-double overhang DNA (5’-

d(CGGCAACTTTGG)-3’) ........................................................................................ 71


d(CGCCAAAGTTGC)-3’) ........................................................................................ 72

3.4.5 MALDI(+)-TOF spectra of single selenium modified 5’-double

overhang DNA (5’-d(CGGCAACTT(5-SeT)GG)-3’) ................................................. 73

3.4.6 MALDI(+)-TOF spectra of double selenium modified 5’-double

overhang DNA (5’-d(CGGCAACT(5-SeT)(5-SeT)GG)-3’) ......................................... 74

3.4.7 MALDI(+)-TOF spectra of triple selenium modified 5’-double

overhang DNA (5’-d(CGGCAAC(5-SeT)(5-SeT)(5-SeT)GG)-3’) .................................. 75

3.4.8 Summary ............................................................................................ 75

4

3.5 Melting Study for Selenium Nucleic Acid ............................................ 75

3.5.1 Melting study for DNA double-strand .............................................. 76

3.5.2 Melting study for DNA and DB2429 complex ................................. 78

3.5.3 Melting study for DNA and DB2457 complex ................................. 80

3.6 Crystallization and Structure Studies .................................................. 82

3.6.1 Crystallization result for 5’-d(CCAAAGTTGC)-3’ and 5’-

d(GCAACTTTGG)-3’ double strand DNA sequence binding with a small molecule.

82

3.6.2 Crystallization study for DNA sequences with 3’-position overhangs

85


87

3.6.4 The effect on the crystallization of different MPD concentration. . 91

3.6.5 Summary ............................................................................................ 95

4 CONCLUSIONS ........................................................................................ 97

REFERENCES .................................................................................................... 99

APPENDICES ................................................................................................... 105

Appendix A Nucleic Acid Mini Screen (NAM) ........................................ 105

Appendix B The Synthesis of the Nucleotide with an Extended

Pyrimidine Ring 107

Appendix B.1 Introduction ......................................................................... 107

5

Appendix B.2 TIPDS Protection ................................................................ 108

Appendix B.3 Adding Ms-Leaving Group ................................................. 109

Appendix B.4 Boc-Protection ..................................................................... 111

Appendix B.5 Selenium Introduction ........................................................ 112

Appendix B.6 Ring Formation Reactions .................................................. 114

Appendix B.7 Ethylene glycol introduction reaction ................................ 118

Appendix B.8 Ms-leaving group introduction reaction............................. 120

Appendix B.9 The synthesis for the nucleotide with an extended pyrimidine

ring .......................................................................................................................... 121

6

LIST OF TABLES

Table 3.1 Mobile phase composition gradient for DMTr-on DNA sequences purification.

........................................................................................................................................... 65

Table 3.2 Mobile phase composition gradient for DMTr-off DNA sequences purification.

........................................................................................................................................... 65

Table 3.3 The Tm and ΔTm data of the four DNA double-strands via melting study ..... 77

Table 3.4 The Tm and ΔTm data of the four DNA double-strands binding with DB2429

via melting study ............................................................................................................... 79

Table 3.5 The Tm and ΔTm data of the four DNA double-strands binding with DB2457

via melting study ............................................................................................................... 81

Table 3.6 DNA sequences we synthesized which can bind with the small molecule DB2429

and DB2457. ..................................................................................................................... 83

Table 3.7 DNA sequences we synthesized which has a GC overhang at 3'-position. ...... 85

Table 3.8 The DNA double brand could be formed by the synthesized DNA sequences. To

be read vividly, the bottom sequence is presented in 3' to 5' direction and the GC overhangs

are highlighted. ................................................................................................................. 85

Table 3.9 Screening result for the DNA double strands with 3'-overhangs. .................... 86

Table 3.10 DNA sequences we synthesized which has a GC overhang at 5'-position. .... 87

Table 3.11 The DNA double brand could be formed by the synthesized DNA sequences.

To be read vividly, the bottom sequence is presented in 3' to 5' direction and the GC

overhangs are highlighted. ................................................................................................ 87

7

Table 3.12 The crystallization result for the single selenium modified DNA double-strand

with 5'-position GC overhangs. The MPD concentration is distributed from 20% to 45%.

The pictures are what the crystals looked like on the third day. ....................................... 91


with 5'-position GC overhangs binding with small molecule DB2429. The MPD

concentration is distributed from 20% to 45%. The pictures are what the crystals looked

like on the third day. ......................................................................................................... 91





Table 3.15 The crystallization result for the double selenium modified DNA double-strand











8

Table 3.18 The crystallization result for the triple selenium modified DNA double-strand











9

LIST OF FIGURES

Figure 1.1 Schematic diagram of the central dogma. The solid arrows indicate the direction

in which genetic information is transmitted under normal conditions, and the dashed arrows

indicate the direction in which information is transmitted under unconventional

circumstances. ................................................................................................................... 15

Figure 2.1 Schematic diagram of the synthetic route of 5'-dimethoxytrityl-5-

methylselenodeoxyuridine phosphoramidite with 5'-dimethoxytrityl-5-iodo-deoxyuridine

as initial reactant. .............................................................................................................. 26

Figure 2.2 The reaction equation for the tert-butyldimethylsilyl protection reaction. ...... 27

Figure 2.3 Reaction mechanism of tert-butyldimethylsilyl protection reaction. .............. 29

Figure 2.4 Reaction equation for introducing methyl selenium reaction. ......................... 30

Figure 2.5 Schematic diagram of the reaction mechanism for methyl selenium introduction

reaction. ............................................................................................................................. 32

Figure 2.6 The reaction equation of detritylation step. ..................................................... 33

Figure 2.7 Schematic diagram of the mechanism of detritylation reaction. ..................... 34

Figure 2.8 Chemical reaction equation for phosphoramidite synthesis. ........................... 35

Figure 2.9 Schematic diagram of the mechanism of the phosphoramidite synthesis reaction.

........................................................................................................................................... 36

Figure 2.10 Schematic diagram of the process of DNA phosphoramidite solid phase

synthesis. ........................................................................................................................... 40

Figure 2.11 Schematic diagram of the reaction mechanism of DMTr protection under acid

catalysis. ............................................................................................................................ 41

10

Figure 2.12 Schematic diagram of the reaction mechanism of the phosphoramidite

activation and coupling step. ............................................................................................. 42

Figure 2.13 Schematic representation of the reaction mechanism of the phosphoramidite

activation and coupling steps. The correct oligonucleotide sequence (top) and the

oligonucleotide sequence lacking one base (below) are deleted due to deletion of the sixth

thymine. ............................................................................................................................ 43

Figure 2.14 Schematic diagram of the reaction mechanism of the acetylation capping step

during the synthesis of oligonucleotide sequences by the phosphoramidite method. ...... 44

Figure 2.15 Schematic diagram of the reaction mechanism of the phosphite triester

oxidation step during the synthesis of the oligonucleotide sequence by the phosphoramidite

method............................................................................................................................... 45

Figure 2.16 Schematic diagram of the protective group structure of adenosine, cytidine and

guanosine commonly used in the synthesis of oligonucleotide sequences by

phosphoramidite solid phase synthesis. ............................................................................ 48

Figure 2.17 A schematic diagram of the reaction mechanism for removing the propionitrile

group protection on the phosphate triester after synthesizing the oligonucleotide sequence

by the phosphoramidite method. ....................................................................................... 49

Figure 2.18 Hanging drop vapor diffusion crystallization ................................................ 51

Figure 2.19 Schematic diagram of the molecular structure of two small molecules DB2429

and DB2457. ..................................................................................................................... 54

Figure 3.1 Schematic diagram of the synthetic route for the synthesis of 5'-dimethoxytrityl-

5-methylseleno-deoxyuridinephosphoramidite using 5-iodo-deoxyuridine as the starting

reactant. ............................................................................................................................. 55

11

Figure 3.2 The reaction equation for the tert-butyldimethylsilyl protection reaction. ...... 56

Figure 3.3 The reaction equation for introducing a methyl selenium reaction. ................ 57

Figure 3.4 The equation for detritylation reaction. ........................................................... 59

Figure 3.5 Chemical reaction equation for dimethoxytrityl protecting reaction. ............. 60

Figure 3.6 Schematic diagram of the mechanism of phosphoramidite synthesis reaction.

........................................................................................................................................... 61

Figure 3.7 HPLC chromatogram of DNA sequence W3 containing only one selenium-

modified thymidine. .......................................................................................................... 67

Figure 3.8 HPLC chromatogram of the DNA sequence W4 of all thymidine-containing

selenium-modified DNA. .................................................................................................. 68

Figure 3.9 MALDI-TOF(+) MS analysis of the DMTr-off DNA W1 (5’-d

(CCAAAGTTGC)-3’). Average mass [M] = 3012.034. Calculated [M + H]+: 3013.041.

Measured [M + H]+: 3006.158. ........................................................................................ 69


(GCAACTTTGG)-3’). Average mass [M] = 3043.046. Calculated [M + H]+: 3044.053.

Measured [M + H]+: 3039.333. ........................................................................................ 70

Figure 3.11 MALDI-TOF(+) MS analysis of the DMTr-off 5’-double overhang DNA (5’-

d (CGGCAACTTTGG)-3’). Average mass [M] = 3630.429. Calculated [M + H]+:

3631.436. Measured [M + H]+: 3628.923. ....................................................................... 71

Figure 3.12 MALDI-TOF(+) MS analysis of the DMTr-off 5’-double overhang DNA

(CGGCAACTTTGG). Average mass [M] = 3661.429. Calculated [M + H]+: 3662.429.

Measured [M + H]+: 3660.152. ........................................................................................ 72

12

Figure 3.13 MALDI-TOF(+) MS analysis of the DMTr-off single selenium modified 5’-

double overhang DNA (CGGCAACTT(5-SeT)GG). Average mass [M] = 3740.400.

Calculated [M + H]+: 3741.400. Measured [M + H]+: 3739.581. ................................... 73

Figure 3.14 MALDI-TOF(+) MS analysis of the DMTr-off double selenium modified 5’-

double overhang DNA (CGGCAACT(5-SeT)(5-SeT)GG). Average mass [M] = 3819.371.

Calculated [M + H]+: 3820.371. Measured [M + H]+: 3818.136. ................................... 74

Figure 3.15 MALDI-TOF(+) MS analysis of the DMTr-off triple selenium modified 5’-

double overhang DNA (CGGCAAC(5-SeT)(5-SeT)(5-SeT)GG). Average mass [M] =

3898.342. Calculated [M + H]+: 3899.342. Measured [M + H]+: 3896.818. .................. 75

Figure 3.16 Normalized thermal-denaturation curves of four DNA double-stranded

molecules containing different numbers of selenium modifications. ............................... 77


molecules containing different numbers of selenium modifications and small molecule

DB2429 complexes. .......................................................................................................... 79


molecules with different numbers of selenium modifications and small molecule DB2457

complexes. ........................................................................................................................ 81

Figure 3.19 Experimental DNA crystals obtained. ........................................................... 84

Figure 3.20 Some good crystals we got by using 5'-position GC overhangs. .................. 90

Figure A.0.1 Nucleic acids mini screen kit conditions. .................................................. 106

Figure B.2 Synthesis route of 5-MeSe-T-phosphorimidite from 5-I-dU. ....................... 107

Figure B.3 TIPDS protection reaction. ........................................................................... 108

Figure B.4 The proposed mechanism of TIPDS protection reaction. ............................. 109

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13

Figure B.5 Ms-leaving group adding reaction. ............................................................... 109

Figure B.6 The proposed mechanism of Ms-leaving group adding reaction. ................. 110

Figure B.7 Boc-protection reaction................................................................................. 111

Figure B.8 The mechanism of the Boc-protection reaction. ........................................... 112

Figure B.9 Selenium introduction reaction. .................................................................... 112

Figure B.10 The proposed mechanism of the selenium introduction reaction. .............. 114

Figure B.11 The resonance structure for acetonitrile carbon anion. The carbon anion should

be relatively stable because of the resonance effect. ...................................................... 115

Figure B.12 The resonance structure for acetonitrile. The cyan carbon contains partial

positive charge, which makes it electrophilic. ................................................................ 115

Figure B.13 Reaction equation of the ring formation reaction for the TiOS with acetonitrile.

......................................................................................................................................... 116

Figure B.14 Reaction equation of the ring formation reaction for the TiOS with cyanamide.

The electrons in the p orbitals could be conjugated to make the expanded ring aromatic.

......................................................................................................................................... 117

Figure B.15 Reaction equation of the ring formation reaction for the TiOS with cyanic acid.

The electrons in the p orbitals could be conjugated to make the expanded ring aromatic.

......................................................................................................................................... 117

Figure B.16 Reaction equation of the ring formation reaction for the TiOS with cyanic acid.

The electrons in the p orbitals might not be conjugated to make the expanded ring aromatic.

......................................................................................................................................... 117

Figure B.17 The overall reaction equations of the ring formation reaction for the TiOS with

ethylene glycol. We shall get a novel nucleoside with nine-member-ring. .................... 118

14

Figure B.18 Ethylene glycol introduction reaction. ........................................................ 119

Figure B.19 The mechanism of the ethylene glycol introduction reaction. .................... 119

Figure B.20 Ms-leaving group introduction reaction. .................................................... 120

Figure B.21The final step for the synthesis is a ring formation reaction. We have both

nucleophile and electrophile in the molecule. And they are not too far away from each other.

Theoretically, we can do the reaction finally. ................................................................. 121

15

1 INTRODUCTION

1.1 Introduction of Central Dogma and Nucleic Acid Inhibition

Nucleic acids are macromolecules that store and transfer genetic information in

cells. They are ubiquitous in living systems. Nucleic acids, in the form of DNA and RNA,

play important roles in central dogma, controlling the synthesis of proteins from the origin1-

3. Inhibition of nucleic acid activities can allow disease intervention. Therefore, nucleic

acid molecules have become important drug targets4, 5. Unlike targeting proteins, inhibiting

nucleic acids offers great advantages: sequence-based targeting, understanding of the

underlying causes of diseases (such as gene expression disorders, viral infections and

cancer)5-7, and easy design of drugs with high specificity.

Figure 1.1 Schematic diagram of the central dogma. The solid arrows indicate the

direction in which genetic information is transmitted under normal conditions, and the

dashed arrows indicate the direction in which information is transmitted under

unconventional circumstances.

Recently, it has been found that functional nucleic acids (such as ribozyme and

DNazyme) can catalyze chemical and biochemical reactions like proteins3, 8-10. The

functional nucleic acids, e.g., ribozymes, DNazymes, antisense nucleic acid molecules,

anti-gene sequences, nucleic acid aptamers, and small interfering RNAs3, 11, can also be

used as drugs to inhibit nucleic acids for treating diseases. Likewise, small molecules can

be designed to inhibit nucleic acids.

DNA RNA Protein

DNA Replication

Transcription

Reverse Transcription

RNA Replication

Translation

16

Small molecules that recognize mixed base pairs nucleic acid sequences can be

used as transcription factor inhibitors by binding to a DNA promoter sequence rather than

by targeting the transcription factor part of the complex. The applications of gene specific

probes of this type are almost infinite.

To further understand the inhibition of functional nucleic acids and small molecules,

the study of the three-dimensional structure of nucleic acids, nucleic acid-small molecules

and nucleic acid-protein complexes by X-ray crystallography is important and has become

a very active research field. Our laboratory has found that selenium modification of nucleic

acids can facilitate crystallization and phasing for nucleic acid structure determination.

Therefore, by introducing selenium modification, we have investigated the Se-

derivatization and crystallization to accelerate the crystal structure determination of DNA-

small molecule complexes.3

1.2 Introduction of X-ray Crystallography

1.2.1 The function and application of X-ray crystallography

Proteins and nucleic acids (especially RNA) can fold into complicated three-

dimensional structures12. The structural information of nucleic acids is essential for gaining

insight into their biological functions. For example, after tRNA discovery, its role has been

a mystery for a long time13. Until 1974, the X-ray crystal structure of L-shaped tRNA was

successfully determined, and the mechanism of tRNA in protein biosynthesis was

gradually revealed13-15. Over the past two decades, due to advances in X-ray diffraction

crystallography, such as the development of crystallization science, the leap of synchrotron

performance, and the use of more advanced purification equipment, nucleic acid structure

detection has also been rapidly developed16. Consequently, more and more nucleic acid

17

molecules with high-resolution structures have been discovered, especially non-coding

RNA molecules. At present, a large number of non-coding regulatory RNAs have been

discovered, such as ribozymes17, nucleic acid switches18, and so on. In addition, there are

many technological advances on potential nucleic acid therapeutics19, including antisense

oligonucleotide chains, small interfering RNAs, microRNAs20, and aptamers. These

studies demand advancements of structural and functional study of nucleic acids. Therefore,

new methods, especially new nucleic acid X-ray crystallographic techniques (such as

selenium modification), are urgently needed5.

1.2.2 Challenges and difficulties in nucleic acid X-ray crystallography

There are two bottlenecks in X-ray crystallography research itself: crystallization

and phase problem. These two bottlenecks much limit the discovery of new structures and

folding states of biological macromolecules (such as proteins and nucleic acids) and the

exploration of biological mechanisms3.

Compared to protein crystallography, nucleic acid X-ray crystallography has its

unique challenges and difficulties. The first is the crystallization of nucleic acids. A crystal

is a combination of a large number of molecules arranged in regular order, and the

molecules are grouped by non-covalent bonds21. The surface of a protein molecule has a

variety of structural and chemical groups that enable it to produce intermolecular

interactions that facilitate crystal growth. The surface of the nucleic acid molecule is

covered with dense, negatively charged phosphate groups, which makes it more difficult

to crystallize into agglomerates. The second difficulty is that the method of rationally

solving the phase problem of nucleic acid crystals has not been well developed so far. In

general, crystallographic phase problems can be solved in several different ways. The

18

phase is determined by multi-wavelength anomalous diffraction (MAD) or single-

wavelength anomalous diffraction (SAD)3. For example, the study of selenomethionine-

derived proteins is the most successful and reasonable method to solve the phase problem

of new protein crystal structure22. Currently, more than two-thirds of novel proteins are

structurally determined by this method23. If a similar structure can be obtained, the phase

problem of the protein structure can also be solved by molecular replacement24. However,

it is somewhat difficult to determine the structure of a nucleic acid molecule having a

highly repetitive unit by molecular replacement25. Heavy metal cation soaking and co-

crystallization have also been successfully applied to protein crystallization, while nucleic

acid molecules with a phosphate backbone are often randomly hydrolyzed under these

conditions. Halogen-derived nucleic acid molecules can cause nucleic acid structure

perturbations and radiation instability. Therefore, due to the lack of convenient and stable

massive atom derivatives, the phase problem of nucleic acid is still a considerable

problem16. In addition to these two major challenges, there are other challenges in nucleic

acid crystallography. For example, it is difficult to obtain consistent and conformational

samples for crystallization and structural studies. In vitro enzymatic synthesis of DNA

sequences over 50 nt (e.g., larger non-coding RNA) produces significant heterogeneity.

Enzymatically synthesized nucleic acid sequences typically carry the 3'-end and/or 5'-end

heterogeneity that is undesired by high-resolution structural analysis. The multi-step

purification process is complicated and inefficient26. To increase the homogeneity of the

transcribed RNA and increase the transcription efficiency, the 2'-methylated DNA of the

last two nucleotides of the 5'-end was reported as a highly efficient template27. Another

difficulty in nucleic acid crystallography is the heterogeneity of the structure. Because a

19

nucleic acid molecule (especially RNA) can be folded into many different three-

dimensional conformations21. In this case, it is necessary to find suitable refolding

conditions in order to obtain conformationally homogenized molecules for crystal

screening and structural studies. In order to solve all the above challenges and difficulties,

our group pioneered and developed a selenium nucleic acid (SeNA) strategy that rationally

solves the phase problem in nucleic acid crystallography5, 28-30.

1.3 Introduction of Selenium Nucleic Acid

1.3.1 The chemical and biochemical properties of selenium

Selenium (Se), like the oxygen and sulfur, is located in the sixth main group of the

periodic table. Therefore, their chemical properties are very similar, and there is a

possibility of mutual substitution. Although as early as 1817, selenium was discovered by

Jons Jakob Berzelius in plants enriched in selenium31. However, due to the toxicity of

selenium itself, there is seldom research work on it in the early years. Between the 1930s

and the 1950s, animal nutritionists and chemists had a great interest and curiosity about the

structure and function of selenium-containing compounds in selenium-containing plants32.

But unfortunately, these studies were not very successful and failed to identify any

selenium-containing compounds. This is most likely because these compounds are unstable

under separation conditions. Since the mid-1950s, the presence of selenium in proteins and

the critical role that selenium plays in a series of physiological processes have been

discovered, and research fields related to selenium have gradually attracted more and more

attention from scientists3, 33, 34.

Although it is toxic in the case of large intakes (it could cause selenium poisoning

if selenium intake exceeds 400 mg per day), selenium is widely recognized as a vital trace

20

element35. Some plants are enriched in specific concentrations of selenium at individual

sites and are somewhat toxic to prevent them from being eaten by animals. Some plants,

such as mad grass, require selenium to grow, and their growth status can indicate the

presence and content of selenium in the soil36. It is also a constituent of the unnatural amino

acids selenocysteine and selenomethionine. In humans, selenium-containing compounds

can also act as cofactors for the reduction of antioxidant enzymes, such as glycoperoxidase

and some forms of thioredoxin reductase found in animals and some plants (these enzymes

are present in all organisms, but only some plants require selenium-containing

compounds)37-39. As a cofactor for three known thyroid deiodinases, selenium-containing

compounds also play an essential role in goiter. The further influence of selenium-

containing compounds on human health, such as cancer, AIDS, tuberculosis, and diabetes,

is still controversial, and research work in this area is continuing40. In general, selenium in

the daily diet is mainly derived from nuts, grains, meat, fish, and eggs.

Like oxygen atoms (atomic radius 0.73 Å) and sulfur atoms (atomic radius 1.02 Å),

because of their similar chemical properties and electron distribution, in addition to

proteins, selenium atoms (atomic radius 1.16 Å) are also used in several transport RNAs.

Find. The predominant forms of selenium in natural transport RNA are 2-selenium-uridine

and 5-methylaminomethyl-2-selenour41. The selenium-modified uridine 2-position

selenium atom is introduced by the substitution of the 2-sulfur-uridine sulfur atom on the

transport RNA under the action of 2-selenium-uridine synthase. The selenium atom donor

is selenium phosphate42, 43. Selenium provides many unique biochemical and biological

functions and properties for RNA and protein. There are approximately 23 known

selenium-containing proteins in mammals44. In most selenium-containing enzymes,

21

selenium is present in the form of a selenocysteine residue. Selenocysteine is considered

to be the 21st natural amino acid present in all organisms45. Also because of its large size

and chemical and electronic arrangement properties, selenium is found in the anti-codon

swing site of several bacterial transfer RNAs in the form of 5-methylamino-2-selenouridine,

including Lysine transfer RNA, glutamate transfer RNA, and glutamine transfer RNA46.

The function of the 2-position selenium atom of these modified nucleic acids is likely to

increase the speed and efficiency of the translation process. In vivo observations indicate

that selenium atoms can provide many useful and unique properties to natural RNA.

Because the mechanism of selenium modification of transfer RNA is still a mystery,

understanding the presence and function of selenium in proteins can be useful information

and inspiration for the functional study of selenium-modified RNA. Selenium atoms in

selenocysteine provide proteins with many unique properties different from oxygen and

sulfur47, 48. The most intuitive difference is that SeH with selenocysteine (pKa value of 5.2)

has a lower pKa value and a higher reduction potential than SH with a thiocysteine (pKa

value of 8.5)49, 50. Thus, at physiological pH, selenocysteine has stronger nucleophilicity

and better reducibility than native cysteine. These differences are the most fundamental

reason to explain the existence of selenocysteine in proteins, especially redox proteins,

because its presence can significantly increase the efficiency of redox reactions51, 52.

Selenium plays a significant role in the redox centers of several important

oxidoreductases53. Compared to a large amount of research work on naturally occurring

selenium-containing modified proteins, the study of selenium-modified naturally occurring

RNA appears to be very limited. Because selenium-modified RNA is difficult to obtain,

and the research is challenging. At the same time, the exciting research and development

22

of selenium-modified proteins has also stimulated the synthesis and research of naturally

occurring and non-natural selenium-modified RNAs.

1.3.2 Introduction of 5-Se-Tymidine modified nucleic acid

Because there are plenty of oxygen atoms can be modified in the nucleoside

molecule, the direct selenium modification method for nucleic acids can be varied

depending on the needs of the chemical system54. My research mainly focuses on the

crystallization and structural studies of DNA containing 5-position selenium-modified

thymidine.

The hydrogen bond is the interaction between the hydrogen bond acceptor and the

donor (X-H). In the hydrogen bond in the conventional sense, X refers to an electron-

positive atom (oxygen atom, nitrogen atom, etc.). However, in recent studies, when X is

an electron-negative atom (such as a carbon atom), the hydrogen bond it generates is more

and more accepted and valued, such as hydrogen bonds in CH···O=C55-58. Interaction of

CH groups with hydrogen bond acceptors (electron donors), such as CH···O=C hydrogen

bonds in proteins56, CH···O=C hydrogen bonds in uracil crystals58, and host-guest systems

CH···Cl hydrogen bonds59 and other non-traditional interactions (such as CH···π

interactions in RNA)60 which plays a vital role in molecular recognition, catalysis, and

DNA double-strand stability in chemical and biological systems61, 62. Because the

negatively charged phosphate group is an excellent electron donor, and phosphorylation

and dephosphorylation are ubiquitous processes in the cell-regulatory mechanism63. Our

group studied the synthesis of a thymidine (5-Se-T) by introducing an atom probe (a

selenium atom) outside the 5-position of a heterocyclic base. The hydrogen bond

interaction between the 5-position methyl group of thymidine and its phosphate backbone

23

(hydrogen bond interaction of CH···O--PO3) exists54. The first synthesis of 5-position

methyl selenium modified thymidine was achieved via a method for introducing into 3',5'-

dibenzoyl-2'-deoxyuridine by electrophilic addition reaction through Mn(OAc)3 assisted

CH3SeSeCH3. The 5-position methyl selenide-modified thymidine dephosphoramidite

monomer was subsequently successfully synthesized and yielded a yield of more than 99%

during the coupling of DNA synthesis. Biophysical and structural studies of thymidine-

modified DNA containing 5-methylselenolenium revealed that Se-modified DNA and

almost unmodified DNA are almost identical. At the same time, the structural analysis of

the crystallography of the thymidine modified by 5-methyl-selenium clearly indicates that

there is a hydrogen bond between the 5-methyl group and the 5'-position phosphate group

(CH3···PO4- interaction)54.

In the double-stranded DNA molecule64, the 5-position methyl group of the

thymidine is usually at a distance of 4 to 5 Å from the nearest oxygen atom on the 5'-

position phosphate group (O--PO3). In order to further extend the methyl group to bring it

closer to the phosphate group and at the same time increase the rotational flexibility of the

methyl group, an atomic bond is introduced between the methyl group of the thymidine

and the 5-position carbon atom65, 66. If the methyl and phosphate groups (O--PO3) are

capable of forming a real hydrogen bond, they can be observed in the crystal structure to

be close to each other, and the density of electron clouds in between them will be high. If

there is no such stable interaction between them, the steric hindrance forces the methyl

group to be more prone to rotation away from the phosphate group and to the direction of

the major groove. Since the position and spatial geometry of atoms and radicals have a

significant influence in this experimental study, it is chosen to insert a selenium atom

24

between the 5-position methyl group and the 5-position carbon atom as an atom probe. And

a brand new methyl-hydrogen phosphate bond (C-H···O--PO3) is successfully discovered

54.

1.3.3 Application of selenium nucleic acid in X-ray crystallography

As mentioned above, in crystal diffraction, selenium atoms can provide an

abnormal dispersion of electron centers, which can solve the phase problem well. The

selenomethionine (Se-Met) modification revolutionized the X-ray crystallography of

proteins. Especially after MAD analysis of selenomethionine-modified RNase H to

successfully obtain its crystal structure, this method is increasingly favored by researchers.

It is estimated that more than two-thirds of newly discovered protein structures are phased

by the Se-Met MAD method3, 5. Similarly, although somewhat time-consuming and labor

intensive, methods for determining the phase of nucleic acids by selenium-modified

proteins bound to nucleic acids have also been developed. Inspired by the revolutionary

advancement of protein X-ray crystallography by the substitution of sulfur atoms with

selenium atoms, our group pioneered the method of determining the phase and crystal

structure by replacing oxygen atoms at different positions in the nucleic acid with selenium

atoms.

This method of selenium modification has excellent potential for the study of the

structure and function of accounting. So far, studies have shown that the crystal samples

obtained by this method are more stable under X-ray irradiation than the conventional

bromine atom modification method67. At the same time, the selenium atom modification

method has more options than some limitations of the bromine modification method,

because many oxygen atoms in the nucleic acid molecule can be substituted. Also, based

25

on the basic X-ray diffraction phase calculation, a modification of a selenium atom can

determine the phase of a 30 or more base DNA or RNA sequence68. Since the selenium

element and the oxygen element are located in the sixth main group of the periodic table,

it is possible to achieve a very significant change in the structure of the nucleic acid

molecule after replacing the oxygen atom with the selenium atom. In fact, this new

modification method has been used in many laboratories for X-ray diffraction

crystallography to analyze the molecular structure of DNA or RNA, such as ribozymes,

human DNA, and riboswitches69, 70. In order to further optimize this method, an enzymatic

process for preparing a selenium-modified nucleic acid molecule by a DNA synthase and

an RNA transcriptase has emerged71.

26

2 EXPERIMENT

2.1 The Synthesis of 5-Se-T phosphorimidite Starting with 5’-DMTr-5-I-dU

Depending on the starting reactant, there are two different synthetic routes to

prepare 5-Se-thymidine-phosphoramidite, via the reaction with 5'-dimethoxytrityl-5-

iododeoxyuridine or 5-iododeoxyuridine as the initial reactant. Both routes have their

advantages and should be selected according to specific experimental needs54, 72.

Figure 2.1 Schematic diagram of the synthetic route of 5'-dimethoxytrityl-5-

methylselenodeoxyuridine phosphoramidite with 5'-dimethoxytrityl-5-iodo-deoxyuridine

as initial reactant.

The synthesis route of 5'-dimethoxytrityl-5-methylselenodeoxyuridine

phosphoramidite with 5'-dimethoxytrityl-5-iodo-deoxyuridine (5'-DMTr-5-I-dU) as initial

reactant is shown in Figure 2.1. There are four steps in the route including tert-

butyldimethylsilyl-(TBDMS-) protection, the introduction of methyl selenium,

detritylation, and the synthesis of phosphoramidite.

27

2.1.1 Tert-butyldimethylsilyl-(TBDMS-) protection

The hydroxyl group at the 3'-position in the starting material is a good nucleophile.

To avoid its influence on the subsequent introduction of methyl selenium, it is necessary

to protect it with tert-butyldimethylsilyl group54, 72.

2.1.1.1 Experimental procedure

Figure 2.2 The reaction equation for the tert-butyldimethylsilyl protection reaction.

The reaction equation of 3’-DMTr-5-Iodo-dU (Compound 1) protection step is

shown in Figure 2.2. 1 g of Compound 1 was mixed with 408.6 mg of imidazole in a 25

mL round bottom flask. 452.1 mg TBDMS-Cl was placed in another 10 mL round bottom

flask. The flasks were dried over high vacuum for 30 min. The flasks were purged with

argon and an argon balloon was placed to maintain constant pressure and to ensure an argon

atmosphere during the reaction. TBDMS-Cl was dissolved in 7.5 mL of anhydrous DMF,

and a DMF solution of TBDMS-Cl was added dropwise to a round bottom flask containing

Compound 1 and imidazole. The reaction was stirred at room temperature overnight. The

progress of the reaction was monitored by TLC. The TLC eluent was a mixed solution of

ethyl acetate and hexane 1:2. After the reaction was completed, the reaction was quenched

by the addition of 0.7 mL of methanol. The reaction solution was diluted with ethyl acetate

and washed with water to remove DMF and other water-soluble impurities. The aqueous

28

phase was extracted three times with ethyl acetate, the aqueous phase was discarded and

the organic phases were combined. The organic phase was washed three times with

saturated brine and the aqueous phase was discarded. The organic phase is dried over

anhydrous magnesium sulfate. After filtration and rotary evaporation, a crude product was

gained. Further purification using a silica gel column yields a product of higher purity. The

eluent of the silica gel column was a mixed solution of ethyl acetate and hexane 1:4. It

should be noted that dimethoxytrityl-(DMTr-)group is unstable under acidic conditions and

easily falls off into carbon cations. Since TBDMS-Cl is acidic, it cannot be mixed with

Compound 1. In addition, when purifying the silica gel column, a base such as N,N-

dimethylethylamine should be used to neutralize the silica gel.

The yield for this step is about 90%.

29

2.1.1.2 Proposed mechanism

Figure 2.3 Reaction mechanism of tert-butyldimethylsilyl protection reaction.

The reaction mechanism of this step is shown in Figure 2.3. First, the imidazole

replaces the chlorine atom of TBDMS-Cl to form a better leaving group, thereby activating

the silicon atom. Then, the hydroxyl group at the 3'-position of the sugar ring attacks the

silicon atom to undergo a substitution reaction. The role of imidazole in the reaction

process is the catalyst and proton acceptor.

30

2.1.2 The introduction of methyl selenium


Figure 2.4 Reaction equation for introducing methyl selenium reaction.

The reaction equation of selenium introduction to 3’-DMTr-5’-TBDMS-5-Iodo-dU

(Compound 2) reaction is shown in Figure 2.4. 500 mg of Compound 2 was weighed into

a 25 mL round bottom flask. Another 23.4 mg of sodium hydride was weighed into another

25 mL round bottom flask. The flasks were dried over high vacuum for 30 min. The flasks

were purged with argon and an argon balloon was placed to maintain constant pressure and

to ensure an argon atmosphere during the reaction. Compound 2 was dissolved in 7.5 mL

of anhydrous tetrahydrofuran treated with sodium metal, and a solution of Compound 2 in

tetrahydrofuran was added dropwise to a round bottom flask containing sodium hydride.

The reaction was stirred at room temperature until no bubbles are produced. The reaction

flask was placed in a cooling bath of dry ice-acetone mixture (-78 °C). After the

temperature was constant, 1.04 mL of a 2.5 M n-butyllithium hexane solution was added

dropwise to the reaction flask using a syringe. The progress of the reaction was monitored

by TLC. The TLC eluent was a mixed solution of ethyl acetate and hexane 1:2. After all

the reactants were converted into intermediate products, 0.215 mL of dimethyl diselenide

liquid was added dropwise to the reaction flask with a syringe. The progress of the reaction

31

was examined by TLC. After the reaction was completed, the reaction was quenched by

the addition of 0.7 mL of saturated brine. The reaction solution was diluted with ethyl

acetate, and washed with water to remove THF and other water-soluble impurities. The

aqueous phase was extracted three times with ethyl acetate, the aqueous phase was

discarded, and the organic phases were combined. The organic phase was washed three

times with saturated brine and the aqueous phase was discarded. The organic phase is dried

over anhydrous magnesium sulfate. After filtration and rotary evaporation, a crude product

was gained. Further purification using a silica gel column yields a product of higher purity.

The eluent of the silica gel column was a mixed solution of ethyl acetate and hexane 1:5.

When purifying on a silica gel column, a base should be used to neutralize the silica gel.

In addition, because dimethyl diselenide has a particularly pungent odor, the contact with

the dimethyl diselenide reaction vessel is oxidized by using a bleaching agent. Selenium-

containing waste liquid should be collected in a specific container to avoid environmental

pollution.

The yield of this step is only 30%-40%.

32


Figure 2.5 Schematic diagram of the reaction mechanism for methyl selenium

introduction reaction.

The mechanism of this step is shown in Figure 2.5. First, sodium hydride removes

active protons in the molecule. Then, n-butyllithium converts iodine at the 5-position of

the base to lithium by a lithium-halogen exchange reaction. Currently, the carbon atom at

the 5-position becomes a suitable nucleophilic reagent, and the methyl selenization reaction

occurs in attacking dimethyl diselenide. Finally, during the quenching process by adding

saturated brine, the molecules acquire protons and restore electrical neutrality.

33

2.1.3 Detritylation


Figure 2.6 The reaction equation of detritylation step.

The reaction equation of the deprotection of 3’-DMTr-5’-TBDMS-5-Se-Thymidine

(Compound 3) is shown in Figure. 2.6. 500 mg of Compound 3 was weighed into a 25 mL

round bottom flask. The flasks were dried over high vacuum for 30 min. The flasks were

purged with argon and an argon balloon was placed to maintain constant pressure and

ensure an argon atmosphere during the reaction. Compound 3 was dissolved in 13 mL of

anhydrous tetrahydrofuran. A round bottom flask containing the compound 3

tetrahydrofuran solution was placed in an ice water bath. After the temperature was

stabilized, 1.36 mL of a 1 M tetrabutylammonium fluoride solution in tetrahydrofuran was

added dropwise to a round bottom flask containing the compound 3 tetrahydrofuran

solution. The flask was removed from the ice water bath and stirred at room temperature

for two hours. The progress of the reaction was monitored by TLC. The TLC eluent was a

mixed solution of ethyl acetate and hexane 2:1. After completion of the reaction, the

reaction was quenched by the addition of 0.7 mL of saturated brine. The reaction solution

was diluted with ethyl acetate, and washed with water to remove THF and other water-

soluble impurities. The aqueous phase was extracted three times with ethyl acetate, the

34

aqueous phase was discarded and the organic phases were combined. The organic phase

was washed three times with saturated brine, and the aqueous phase was discarded. The

organic phase is dried over anhydrous magnesium sulfate. Filtration and rotary evaporation

gave a crude product. Further purification using a silica gel column yields a product of

higher purity. The eluent of the silica gel column was a 4% methanol-dichloromethane

mixed solution. When purifying on a silica gel column, a base should be used to neutralize

the silica gel.

The yield of this step is about 80%.


Figure 2.7 Schematic diagram of the mechanism of detritylation reaction.

The mechanism of this step is shown in Figure 2.7. According to the nature of the

silicon-fluorine bond stronger than the silicon-oxygen bond, the fluoride ion attacks the

silicon atom to undergo a substitution reaction. The process of quenching the reaction by

adding saturated brine to obtain a proton of oxygen anion finally leads to an electrically

neutral hydroxyl group.

35

2.1.4 The synthesis of phosphoramidite


Figure 2.8 Chemical reaction equation for phosphoramidite synthesis.

The reaction equation of the phosphoramidation of 3’-DMTr-5-Se-Thymidine

(Compound 4) is shown in Figure. 2.8. 365 mg of Compound 4 was weighed into a 25 mL

round bottom flask. The flasks were dried over high vacuum overnight. The flasks were

purged with argon and an argon balloon was placed to maintain constant pressure and to

ensure an argon atmosphere during the reaction. 318.7 μL of dimethylethylamine and

180.77 mg of i-Pr2NP(Cl)CH2CH2CN were simultaneously added to the round bottom flask

containing Compound 4 with a syringe. The reaction was stirred at room temperature for

half an hour. The progress of the reaction was monitored by TLC. The TLC eluent was a

mixed solution of ethyl acetate and hexane 2:1. After the reaction was completed, the

solvent was evaporated to dryness using a rotary evaporator. The phosphoramidite solid

crude product was dissolved in 4 mL of dichloromethane. 200 mL of hexane was placed in

a 500 mL beaker, and a solution of the phosphorous amide solid crude product in

dichloromethane was added dropwise with stirring. Stir for 10 minutes until the

precipitated precipitates all adhere to the beaker cup wall. The supernatant was decanted

36

by decantation. The precipitated solid crude product was dissolved in dichloromethane and

dried. Further purification using a silica gel column immediately gave a product of higher

purity. The eluent of the silica gel column was a mixed solution of ethyl acetate and hexane

1:1.5. When purifying on a silica gel column, a base should be used to neutralize the silica

gel.



Figure 2.9 Schematic diagram of the mechanism of the phosphoramidite synthesis

reaction.

The mechanism of this step is shown in Figure 2.9. The chlorine atom is a preferred

leaving group, so the first step in the reaction is that the hydroxyl group attacks the

phosphorus atom to undergo a substitution reaction. The resulting acidic protons are

absorbed by dimethylethylamine, thereby causing the reaction to proceed in the forward

direction.

37

2.2 Synthesis and Purification of the Selenium Nucleic Acids

2.2.1 Solid phase synthesis of the nucleic acid with selenium modification

Millions of oligonucleotides are synthesized and used in the laboratory every year

in the world. For most applications, only a very small amount of DNA is needed. Moreover,

the synthesis of oligonucleotides is mainly carried out at a scale below 40 nmol. This is

enough for most biological and biochemical experiments. But for biophysical studies such

as nuclear magnetic resonance and X-ray diffraction crystallography, more DNA (more

than 10 μmol) needs to be prepared. In extreme cases, it is often required to achieve

quantities of several kilograms for the preparation of drug molecules (e.g., antisense

oligonucleotides). Solid phase synthesis of oligonucleotides has emerged. And almost

exclusively fully automated solid phase synthesis can meet all of the above requirements73,

74.

2.2.1.1 Advantages of solid phase synthesis

Solid phase synthesis is widely used in the synthesis of peptides, oligonucleotides,

oligosaccharides and various combinatorial chemistries. Solid-phase chemical synthesis

was invented by Bruce Merrifield in the 1960s, and he won the 1984 Nobel Prize in

Chemistry.

The starting reagent of the solid phase synthesis method is immobilized on a solid

support in the reaction column and the filter, and other reaction reagents and solvents can

freely pass through the reaction. Relative to solution synthesis, solid phase synthesis can

use a relatively large amount of solution phase reagent to promote the reaction to proceed

more quickly and thoroughly. Moreover, impurities and excess reagents are rinsed off so

38

no additional purification process is required after each step is completed. At the same time,

the solid phase synthesis process can also be fully automated through a computer.

2.2.1.2 Solid supports

The solid support is an insoluble particle and usually has a particle size of 50-200

μm. During the synthesis, oligonucleotides are attached to these particles. There are many

different particles to choose from, but microporous glass (CPG) and polystyrene have

proven to be the most suitable.

2.2.1.2.1 Controlled-pore glass (CPG)

Controlled-pore glass is chemically stable, does not absorb solvent swelling, and

has deep pores that allow for oligonucleotide synthesis reactions. A glass support with a

pore size of 500 Å is suitable for mechanical synthesis of short-chain oligonucleotides.

However, when a 500 Å pore size vector is used to synthesize an oligonucleotide sequence

of more than 40 bases in length, the yield of the synthesis decreases sharply. This is because

the growing oligonucleotide strand blocks the micropores and prevents the reaction reagent

from diffusing into the support. Although larger pore size matrices are more fragile, it is

more appropriate to use controlled-pore glass with a pore size of up to 1000 Å when

synthesizing oligonucleotide sequences of up to 100 bases in length. Moreover, for the

synthesis of longer oligonucleotide sequences, it is even possible to use a vector with a

pore size of 2000 Å.

2.2.1.2.2 Polystyrene (PS)

Highly cross-linked polystyrene beads have good hydrophobicity and thus higher

oligonucleotide synthesis efficiency, especially at very low scales (eg 40 nmol). A solid

phase carrier for the synthesis of a conventional oligodeoxynucleotide is usually prepared

39

by attaching 20-30 μmol of nucleoside per gram of the carrier. The efficiency of synthesis

of higher loading oligonucleotides is significantly reduced. This is because the steric

hindrance effect of the oligonucleotide strands adjacent to each other on the carrier

increases as the load increases. However, polystyrene carriers with a loading of up to 350

μmol / g are used in some applications, especially the synthesis of short-chain

oligonucleotides. This also makes it possible to synthesize a large number of

oligonucleotide sequences.

2.2.1.3 The phosphoramidite method

A variety of solution phase oligonucleotide synthesis methods have been developed

over the years. From the early studies of the hydrogen phosphite and phosphotriester

methods by Michelson and Todd in the 1950s75, 76, and the attempt of Khorana to the

phosphodiester method77, the re-study of the phosphotriester method and the development

of the phosphite triester method in the 1960s and 1970s78-83, all these methods have their

own drawbacks. The phosphoramidite method pioneered by Marvin Caruthers in the early

1980s combined with automated solid phase synthesis became the current universal

choice82.

40

Figure 2.10 Schematic diagram of the process of DNA phosphoramidite solid phase

synthesis.

The phosphoramidite synthesis process is in the direction of synthesis from the 3'-

end to the 5'-end (opposite to the direction of DNA biosynthesis in the DNA replication

process). After each synthesis cycle is completed, one nucleotide is attached. The

phosphoramidite DNA synthesis involves a series of steps, as shown in Figure 2.16. In

41

general, DNA phosphoramidite solid phase synthesis can be divided into four steps:

activation and coupling, capping, oxidation, and DMTr protection. After completing the

four steps, one cycle is completed and the synthetic oligonucleotide strand is increased by

one base. This is repeated until the final completion of the desired oligonucleotide sequence

for synthesis.

2.2.1.3.1 Detritylation of the support-bound 3’-nucleoside

Figure 2.11 Schematic diagram of the reaction mechanism of DMTr protection

under acid catalysis.

At the very beginning of the synthesis of the oligonucleotide strand, a nucleoside

protected by a protecting group has been attached to the vector. A DNA synthesis column

containing one of the four nucleosides A, C, G, and T is selected based on the nucleoside

of the 3'-end of the desired DNA molecule. The nucleoside attached to the carrier is

protected by a DMTr group at the 5'-position to prevent the corresponding polymerization

from occurring during the preparation of the carrier. Prior to synthesis, the DMTr

protecting group must be removed to ensure that the synthesis of the oligonucleotide is

possible. The mechanism of DMTr protection is shown in Figure 2.17. DMTr easily forms

42

stable carbon cations under acidic conditions. The DMTr carbon cation is orange in

solution.

2.2.1.3.2 Activation and coupling (first step)

Figure 2.12 Schematic diagram of the reaction mechanism of the phosphoramidite

activation and coupling step.

After deprotection by DMTr, the nucleoside attached to the carrier is ready to react

with the next base. The reaction form of the base is a nucleoside phosphoramidite monomer.

A large excess of a mixed solution of acetonitrile (an excellent solvent in a nucleic acid

synthesis reaction) corresponding to a nucleoside phosphoramidite monomer and an

activator (tetrazole or a derivative thereof) is injected into a DNA synthesis column to

participate in the reaction. The diisopropylamino group on the nucleoside phosphoramidite

monomer is protonated by the activator to a good leaving group. The phosphorus atom

adjacent to it quickly attacks and undergoes a substitution reaction than the hydroxyl group

on the nucleoside attached to the carrier. A new phosphorus-oxygen bond is formed,

resulting in a phosphite triester attached to the support, as shown in Figure 2.18. The

nucleoside phosphoramidite monomer is quite stable in an inert gas environment. It can be

stored in large quantities, shipped to all parts of the world and can be used for several

43

months in dry conditions. Only when the nucleoside phosphoramidite is protonated will it

become active.

2.2.1.3.3 Capping (second step)

Although the efficiency of the coupling reaction is quite high (up to 99.5%), there

is no guarantee that all of the nucleosides on the support will react with the

phosphoramidite monomer. This means that a small amount of nucleoside remains on the

carrier containing a 5'-terminal hydroxyl group which is not involved in the reaction. If left

untreated, it is possible for these 5'-terminal hydroxyl groups to react with the injected new

phosphoramidite monomer in the next coupling reaction. This results in the deletion of one

base by the synthetic oligonucleotide strand, which corresponds to a deletion mutation in

the target oligonucleotide strand. If these deletion mutations are left unchecked, they will

accumulate as the number of cycles is accumulated, making the final product a complex

mixture of multiple oligonucleotide strands. Most of them are erroneous genetic

information and are very difficult to purify. This will ruin subsequent biochemical

experiments.

Figure 2.13 Schematic representation of the reaction mechanism of the

phosphoramidite activation and coupling steps. The correct oligonucleotide sequence (top)

and the oligonucleotide sequence lacking one base (below) are deleted due to deletion of

the sixth thymine.

The "capping" step is introduced after the end of the coupling reaction to protect

the unreacted 5'-terminal hydroxyl group to avoid deletion mutations. Two capping

44

solutions were used in the synthesizer: acetic anhydride and N-methylimidazole (NMI).

The two reagents (dissolved in tetrahydrofuran to which a small amount of pyridine was

added) were first mixed in a DNA synthesizer and then injected into a synthesis column.

The mixed solution of the electrophile rapidly acetylates the alcohol. The presence of

pyridine ensures that the reaction is carried out under basic conditions to avoid the reaction

of acetic anhydride with N-methylimidazole to produce an acidic material which could

remove the trityl group. The formylation at the 5'-end ensures a smooth follow-up reaction.

The mechanism of the acetylation reaction is shown in Figure 2.20.

Figure 2.14 Schematic diagram of the reaction mechanism of the acetylation

capping step during the synthesis of oligonucleotide sequences by the phosphoramidite

method.

2.2.1.3.4 Oxidation (third step)

The phosphite triester formed in the coupling reaction is unstable under acidic

conditions and must be converted to a more stable pentavalent phosphorus form prior to

the next step of trichloroacetic acid (TCA) deprotection with DMTr. This is achieved by

oxidation of iodine in the presence of water and pyridine. The reaction product

phosphotriester is an effective DNA backbone protected by 2-propionitrile. The

propionitrile group avoids unnecessary reactions that may occur on the phosphate group in

45

subsequent synthesis cycles. The mechanism of the oxidation reaction is shown in Figure

2.21.

Figure 2.15 Schematic diagram of the reaction mechanism of the phosphite triester

oxidation step during the synthesis of the oligonucleotide sequence by the phosphoramidite

method.

In some DNA synthesizers, there is a capping step after the iodine oxidation

reaction. Its purpose is to dry the carrier. Because the moisture contained in the residual

oxidizing solution affects the subsequent coupling reaction. Excess water will react with

the acetylating reagent acetic anhydride to form acetic acid and be washed away by a

tetrahydrofuran-pyridine mixed solution.

2.2.1.3.5 Detritylation (fourth step)

After coupling, capping, and oxidation, the 5'-end DMTr protecting group of the

oligonucleotide strand attached to the carrier needs to be removed to obtain a free hydroxyl

group, so that the new phosphoramidite monomer can be neutralized in the next cycle. An

even reaction occurs. The de-DMTr-protected reaction under trichloroacetic acid in

dichloromethane was very rapid and quantitative. The detached DMTr carbon cation is

orange and can absorb visible light with a wavelength of 495 nm. The intensity of this

absorption is used to test the efficiency of the coupling reaction. Most commercial DNA

synthesizers support the ability to monitor the efficiency of synthesis in real time by

detecting and reporting the yield of each cycle of DMTr.

46

The cycle is repeated, adding one base per cycle to finally synthesize the desired

oligonucleotide sequence.

2.2.1.4 Synthesis of oligonucleotide sequence containing 5-seleno-modified

thymidine using a DNA synthesizer

Our laboratory used a 3400 DNA synthesizer from Applied Biosystems. 25.6 mg

(1 μmol) of dG-CPG powder was weighed in four DNA synthesis columns, respectively.

The four base injection vials of the DNA synthesizer were loaded with enough four

phosphoramidite acetonitrile solutions. A sufficient 0.15 mol/L 5-position selenium-

modified thymidine phosphoramidite acetonitrile solution was placed in the No. 5 solution

bottle. Set the synthesis procedure, the sequence of the synthesis column No. 1 is5'-

d(CCAAAGTTGC)-3'; the sequence of the synthesis column No. 2 is 5'-

d(GCAACTTTGG)-3'; the sequence of the synthesis column No. 3 is 5’-

d(CCAAAGT5TGC)-3’; The sequence of the synthesis column is 5'-5’-

d(GCAACTT5TGG)-3’. To facilitate subsequent purification procedures, DMTr

protection was set up without taking off the last nucleoside. After the DNA was synthesized,

it was blown dry, transferred to a 1.5 mL centrifuge tube, and stored in a -80 ̊C refrigerator.

2.2.2 Deprotection and purification of the nucleic acid with selenium modified

DNA that has just been synthesized from a DNA synthesizer is clearly not directly

available for subsequent biochemical experiments. First, the synthetic oligonucleotide

strand is also attached to the vector and a separate treatment from the vector is required. In

addition, many nucleoside bases are protected by various groups and require deprotection.

Furthermore, some short-chain oligonucleotide strands are inevitably produced during

47

DNA synthesis. Although they are capped, they still need to be removed to avoid

unnecessary trouble for subsequent experiments.

2.2.2.1 Principle of nucleic acid deprotection and purification

2.2.2.1.1 Isolation of the oligonucleotide strand from the vector

After the completion of the synthesis, the obtained 3'-end of the target

oligodeoxynucleotide chain is also linked to a complex on a solid phase carrier. This

complex must be stable during the synthesis of the solid phase oligonucleotide sequence,

but at the end of the synthesis it is necessary to be able to separate under certain conditions.

This complex is usually linked by succinyl. This connection structure can be separated by

reacting at room temperature for 1 h under concentrated ammonia conditions.

In some DNA synthesizers, the separation reaction can be automated. After the

reaction, the ammonia solution containing the oligonucleotide chain was transferred to a

glass bottle. The separation reaction can also be accomplished by manual manipulation.

2.2.2.1.2 Deprotection of oligonucleotide strands

The oligonucleotide strand dissolved in aqueous ammonia requires heat to remove

the heterocyclic base and the protecting group on the phosphate. Water and some volatile

components are then removed by lyophilization. The next step is to purify the

oligonucleotide strand.

2.2.2.1.3 Deprotection of heterocyclic bases

The primary amino group outside the ring of the heterocyclic base (A, C and G) is

a nucleophile and therefore must be protected prior to the DNA synthesis reaction. The

concentrated ammonia water used in the final deprotection treatment was reacted at 55 °C

48

for 5 hours to effectively remove these protecting groups. The most commonly used

protection groups for these heterocyclic bases are shown in Figure 2.25.

Figure 2.16 Schematic diagram of the protective group structure of adenosine,

cytidine and guanosine commonly used in the synthesis of oligonucleotide sequences by

phosphoramidite solid phase synthesis.

The benzoyl protection on A and C is easily removed under aqueous ammonia

conditions. However, the isopropyl formyl protection on G is more stable, so the final step

of the deprotection reaction of the oligonucleotide chain is the reaction in which the

isopropyl formyl group is removed on G. In the case of synthesizing a chemically modified

oligonucleotide sequence, heating in aqueous ammonia may result in decomposition of the

compound. Thus, in this case, a more unstable guanosine protecting group is required. The

most popular active group protected guanosine is dimethylformamide guanosine (DMF

dG). This guanosine makes it possible to deprotect the oligonucleotide chain under

relatively mild conditions (reaction at 55 ºC in concentrated aqueous ammonia for 1 hour).

49

2.2.2.1.4 Deprotection of the phosphate backbone

The phosphate group is always protected by the 2-propionitrile group during the

synthesis, so deprotection is also required after the reaction is completed. The propionitrile

group can be quickly removed under concentrated aqueous ammonia conditions. Since the

cyano group has a strong electron withdrawing property, the hydrogen atom on its adjacent

carbon atom thus exhibits a stronger acidity. The reaction was carried out in accordance

with the β-elimination mechanism.

Figure 2.17 A schematic diagram of the reaction mechanism for removing the

propionitrile group protection on the phosphate triester after synthesizing the

oligonucleotide sequence by the phosphoramidite method.

2.2.2.2 Nucleic acid deprotection and purification steps

To the synthesized DMTr-protected DNA-CPG carrier complex, 1 mL of

concentrated aqueous ammonia was added, and the reaction was heated at 55 °C overnight.

Transfer the supernatant to a 1.5 mL centrifuge tube A. The pellet was washed three

times with 1 mL of 100 mg/mL sodium chloride solution, and the supernatant was

transferred to a 1.5 Ml centrifuge tube B. The solutions of the centrifuge tubes A and B

were repeatedly mixed so that the salt concentration in the final two centrifuge tubes was

adjusted to 50 mg/mL.

Take 0.1 mL of the sample solution and centrifuge to remove the solid support.

Samples were analyzed using analytical HPLC. If the sample is relatively pure (the peak

area ratio of the target DNA is >80%), and the subsequent experiment does not require

50

strict DNA purity, the DNA desalting column can be directly used for desalination

treatment. Otherwise, the sample needs to be purified using preparative HPLC.

The vacuum centrifuge was used for 1 h to remove most of the ammonia and some

volatile impurities from the solution. Separation tests were first performed in preparative

HPLC using a sample solution of about 5%. If the peak condition is normal, all samples

are separated using preparative HPLC. The purified DNA solution needs to be purified by

analytical HPLC. If the purity can meet the requirements of subsequent biochemical

experiments, it should be prepared for DMTr protection after lyophilization. If the purity

cannot meet the requirements of subsequent biochemical experiments, it needs to be re-run.

purification. The DMTr protection was performed by dissolving the sample in 0.5 mL of

ultrapure water and adjusting the pH to 4.5 using a 10% (v/v) aqueous solution of acetic

acid. Heating at 40 ° C for 1 h. Samples were analyzed using analytical HPLC to ensure

that the DMTr protecting groups of all DNA molecules were removed. If multiple peaks

occur, purification by preparative HPLC is required once. Freeze to remove moisture and

volatile impurities. The sample was dissolved in 1 mL of ultrapure water to prepare for salt

removal.

For the desalination treatment, the desalting column was treated with 1 mL of

acetonitrile and 1 mL of 2 mol/L TEA·Ac aqueous solution. The sample is injected into a

desalting column. The desalting column was then rinsed three times with ultrapure water,

1 mL each time. Finally, the DNA was eluted from the desalting column using 1 mL of 30%

aqueous acetonitrile.

Freeze the sample. The DNA sample concentration was adjusted to 1 mmol/L using

an ultraviolet-visible absorber.

51

2.3 Crystallization Procedure

2.3.1 Introduction for Hanging Drop Vapor Diffusion Crystallization

Figure 2.18 Hanging drop vapor diffusion crystallization

Hanging drop vapor diffusion technology is a useful method for macromolecular

crystallization. The principle of vapor diffusion is simple. The droplets consisting of a

mixture of sample and reagent are equilibrated with the liquid reagent container vapor.

Typically, the droplets contain a lower concentration of reagent than the reservoir. In order

to reach equilibrium, the water vapor leaves the water droplets and eventually reaches the

reservoir. As the water leaves the water droplets, the relative supersaturation of the sample

increases. As the water leaves the reservoir, the concentration of both the sample and the

reagent increases. Equilibrium is reached when the concentration of the reagent in the

droplet is approximately the same as the concentration of the reagent in the reservoir.84

There are pretty advantages for hanging drop vapor diffusion method. First, it can

be cost effective. We only need 1 μL of sample for each screening try. Second, the sample

52

and reagents are in contact with a siliconized glass surface. The hydroxy groups in the glass

may accelerate the crystallization process. Third, when we want to access the crystal, it can

be straightforward. We only need to uncover the slide and mount the crystal. Last but not

least, we can perform multiple drops (experiments) with a single reservoir. The glass is

large enough to contain several sample drops simultaneously. It would cost even less, and

the comparison could be very convenient.

2.3.2 Sample Preparation

Macromolecular samples should be homogeneous, as pure as possible (> 95%) and

free of amorphous and particulate materials. The amorphous material is removed by

centrifugation or microfiltration before use. If necessary, we can use HPLC to purify the

sample.

The recommended sample concentration is 5 to 25 mg/mL in deionized water.

Initially, the sample should be free of any unnecessary additives to observe the effects of

the Crystal Screen variable. Ideally, the initial screening should be performed with a sample

that has been dialyzed against water, although the ligand, ion, reducing agent or other

additive required for the sample may be soluble, stable or active.84

2.3.3 Crystallization

Firstly, using a clean pipet tip, pipet 1.0 milliliter of crystallization reagent into

reservoir A1 of the Hampton Research VDX Plate. The recommended reservoir volume is

0.5 to 1.0 milliliters.

Then, clear a Siliconized 22 mm Circle or Square Cover Slide by wiping the cover

slide with lens paper and blowing the cover slide with clean, dry compressed air. Pipet 2

53

microliter of the sample into the center of a Siliconized 22 mm Circle or Square Cover

Slide. The recommended total drop volume is 2 to 20 microliters.

Next, pipet 2 microliters of reagent from reservoir A1 into the drop on the cover

slide containing the sample.

Holding the cover slide with tweezers, the Pen-Vac, or on the edge between our

thumb and forefinger, carefully and without delay invert the cover slide, so the drop is

hanging from the cover slide.

Finally, position the cover slide onto the bead of grease on reservoir A1. Gently

press the slide down onto the grease and twist the slide 45º to ensure a complete seal.

Repeat for reservoirs 2 (A2) through 24 (D6).73

2.4 Crystal and Structure Study of DND-small Molecule Complex

Designing and preparing novel compounds that recognize specific mixed base

sequences in nucleic acid sequences can make DNA a cellular receptor for therapeutic and

biotechnological applications as well as gene-specific probes. For example, if a small

molecule capable of binding to a DNA promoter is developed, regulation of the RNA

transcription process can be achieved. According to the central rule, the synthesis of

proteins is regulated accordingly. Using this idea can cure some diseases. The potential of

gene-specific probes in this area is limitless. The small molecules DB2429 and DB2457

can specifically recognize the presence of A·T base pairs and G·C base pairs in the DNA

minor groove. The 5-position selenium atom is located in the major groove, and the

structure of the DNA-small molecule complex can be obtained by X-ray diffraction

crystallographic analysis to determine whether the small molecule will also interact with

the DNA major groove position and how it acts.

54

Figure 2.19 Schematic diagram of the molecular structure of two small molecules

DB2429 and DB2457.

In this experiment, four DNA sequences were synthesized, in which two natural

DNA sequences were: W1 5'-d(CCAAAGTTGC)-3' and W2 5'-d(GCAACTTTGG)-3'; the

selenium-modified DNA sequences were also two: W3 5 '-d(CCAAAGT5- SeTGC)-3' and

W4 5'-d(GCAACTT5- SeT GG)-3'. W1 and W3 are the same sequences. W2 and W4 are the

same, as well. W1 and W2 are complementary. These four DNA sequences can be

constructed into four double-stranded DNA molecules of W1-W2, W1-W4, W2-W3, and

W3-W4. W1-W2 is natural DNA. W1-W4 and W2-W3 contain a single selenium-modified

thymidine. W3-W4 contains two selenium-modifications.

55

3 RESULTS

3.1 The Synthesis of 5-Se-T phosphorimidite Starting with 5-I-dU

Figure 3.1 Schematic diagram of the synthetic route for the synthesis of 5'-

dimethoxytrityl-5-methylseleno-deoxyuridinephosphoramidite using 5-iodo-deoxyuridine

as the starting reactant.

The synthetic route of synthesizing 5'-dimethoxytrityl-5-methylseleno-

deoxyuridine phosphoramidite with deoxyuridine (dU) as the initial reactant is shown in

Figure 3.1. There are 5 steps totally to synthesis phosphoramidite, including tert-

butyldimethylsilyl (TBDMS-) protection, introduction of methyl selenium, det-

butyldimethylsilyl protection and dimethoxytrityl (DMTr-) protection. An advantage of

this method compared to the former route is that in the step of introducing methyl selenium,

the group at the 5'-position changes from dimethoxytrityl group to tert-butyldimethylsilyl

group. The steric hindrance is significantly smaller, which increases the efficiency of

methyl selenization.

56

3.1.1 Tert-butyldimethylsilyl (TBDMS-) protection


Figure 3.2 The reaction equation for the tert-butyldimethylsilyl protection reaction.

The reaction equation of the protection of 5-Iodo-dU (Compound 6) is shown in

Figure 3.2. 1 g of compound 6 was mixed with 3.08 g of imidazole in a 100 mL round

bottom flask. 3.4 g of TBDMS-Cl was placed in another 10 mL round bottom flask. The

flasks were dried over high vacuum for 30 min. The flasks were purged with argon and an

argon balloon was placed to maintain a constant pressure and to ensure an argon

atmosphere during the reaction. TBDMS-Cl was dissolved in 26 mL of anhydrous DMF,

and a DMF solution of TBDMS-Cl was added dropwise to a round bottom flask containing

compound 6 and imidazole. The reaction was stirred at room temperature overnight. The

progress of the reaction was monitored by TLC. The TLC developing solvent was a mixed

solution of ethyl acetate and hexane 1:2. After the reaction was completed, the reaction

was quenched by the addition of 0.7 mL of methanol. The reaction solution was diluted

with ethyl acetate and washed with water to remove DMF and other water-soluble

impurities. The aqueous phase was extracted three times with ethyl acetate, the aqueous

phase was discarded, and the organic phases were combined. The organic phase was

washed three times with saturated brine and the aqueous phase was discarded. The organic

57

phase is dried over anhydrous magnesium sulfate. After filtration and rotary evaporation,

a crude product was gained. Further purification using a silica gel column yields a product

of higher purity. The eluent of the silica gel column was a mixed solution of ethyl acetate

and hexane 1:5.


3.1.2 The introduction of methyl selenium


Figure 3.3 The reaction equation for introducing a methyl selenium reaction.

The reaction equation of the selenium introduction to 3’,5’-diTBDMS-5-Iodo-dU

(Compound 7) is shown in Figure 3.3. 900 mg of Compound 7 was weighed into a 100 mL

round bottom flask. Another 59 mg of sodium hydride was weighed into another 50 mL

round bottom flask. The flasks were dried over high vacuum for 30 min. The flasks were

purged with argon and an argon balloon was placed in the reaction flask to maintain a

constant pressure and to ensure an argon atmosphere during the reaction. The compound 7

was dissolved in 20 mL of anhydrous tetrahydrofuran treated with sodium metal, and a

solution of the compound 7 in tetrahydrofuran was added dropwise to a round bottom flask

containing sodium hydride. The reaction was stirred at room temperature until no bubbles

are produced. The reaction flask was placed in a cooling bath of dry ice-acetone mixture (-

58

78 ° C). After the temperature was constant, 2.5 mL of a 2.5 mol/L hexane solution of n-

butyllithium was added dropwise to the reaction flask with a syringe. The progress of the

reaction was monitored by TLC. The TLC developing solvent was a mixed solution of

ethyl acetate and hexane 1:5. After all the reactants were converted into intermediate

products, 0.53 mL of dimethyl diselenide liquid was added dropwise to the reaction flask

with a syringe. The progress of the reaction was monitored by TLC. After the reaction was

completed, the reaction was quenched by the addition of 0.7 mL of saturated brine. The

reaction solution was diluted with ethyl acetate, and washed with water to remove THF

and other water-soluble impurities. The aqueous phase was extracted three times with ethyl

acetate, the aqueous phase was discarded and the organic phases were combined. The

organic phase was washed three times with saturated brine and the aqueous phase was

discarded. The organic phase is dried over anhydrous magnesium sulfate. After filtration

and rotary evaporation, a crude product was gained. Further purification using a silica gel

column yields a product of higher purity. The eluent of the silica gel column was a mixed

solution of ethyl acetate and hexane 1:6.5. Because dimethyl diselenide has a certain

pungent odor, it is oxidized by using a bleaching agent in contact with a dimethyl

diselenium reaction container. Selenium-containing waste liquid should be collected in a

specific container to avoid environmental pollution.


59

3.1.3 Detert-butyldimethylsilyl protection


Figure 3.4 The equation for detritylation reaction.

The reaction equation of deprotection of 3’,5’-diTBDMS-5-Se-dU (Compound 8)

is shown in Figure. 3.4. 490 mg of compound 8 was weighed into a 100 mL round bottom

flask. The flasks were dried over high vacuum for 30 min. The flasks were purged with

argon and an argon balloon was placed to maintain a constant pressure and ensure an argon

atmosphere during the reaction. Compound 8 was dissolved in 20 mL of anhydrous

tetrahydrofuran. A round bottom flask containing the compound 8 tetrahydrofuran solution

was placed in an ice water bath. After the temperature was stabilized, 3.56 mL of a 1 mol/L

tetrabutylammonium fluoride in tetrahydrofuran solution was added dropwise to a round

bottom flask containing the Compound 8 tetrahydrofuran solution. The flask was removed

from the ice water bath and stirred at room temperature for two hours. The progress of the

reaction was monitored by TLC. The TLC developer was a 5% methanol in

dichloromethane. After completion of the reaction, the reaction was quenched by the

addition of 0.7 mL of saturated brine. The reaction solution was diluted with ethyl acetate,

and washed with water to remove THF and other water-soluble impurities. The aqueous

phase was extracted three times with ethyl acetate, the aqueous phase was discarded and

60

the organic phases were combined. The organic phase was washed three times with

saturated brine and the aqueous phase was discarded. The organic phase is dried over

anhydrous magnesium sulfate. Filtration and rotary evaporation gave a crude product.

Further purification using a silica gel column yields a product of higher purity. The eluent

of the silica gel column was 6% methanol in dichloromethane.


3.1.4 Dimethoxytrityl (DMTr-) protection


Figure 3.5 Chemical reaction equation for dimethoxytrityl protecting reaction.

The reaction equation of the tritylation of 5-Se-Thymidine (Compound 9) is shown

in Figure. 3.5. 340 mg of compound 9 was mixed with 5 mg of 4-dimethylaminopyridine

(DMAP) in a 25 mL round bottom flask. 538.74 mg DMTr-Cl was placed in another 25

mL round bottom flask. The flasks were dried over high vacuum for 30 min. The flasks

were purged with argon and an argon balloon was placed to maintain a constant pressure

and to ensure an argon atmosphere during the reaction. The DMTr-Cl was dissolved in 6

mL of anhydrous pyridine, and a pyridine solution of DMTr-Cl was added dropwise to a

round bottom flask containing Compound 9 and DMAP using a syringe. The reaction was

stirred at room temperature for 2 h. The progress of the reaction was examined by TLC.

61

The TLC developer was a 5% methanol in dichloromethane. After the reaction was

completed, the reaction was quenched by the addition of 0.7 mL of saturated brine. The

reaction solution was diluted with ethyl acetate and washed with water to remove DMF

and other water-soluble impurities. The aqueous phase was extracted three times with ethyl

acetate, the aqueous phase was discarded and the organic phases were combined. The

organic phase was washed three times with saturated brine and the aqueous phase was

discarded. The organic phase is dried over anhydrous magnesium sulfate. After filtration

and rotary evaporation, a crude product was gained. Further purification using a silica gel

column yields a product of higher purity. The eluent of the silica gel column was 1.5%

methanol in dichloromethane.



Figure 3.6 Schematic diagram of the mechanism of phosphoramidite synthesis

reaction.

62

The reaction mechanism of this step is shown in Figure 3.6. First, DMAP replaces

the chlorine atom of DMTr-Cl to become a better leaving group, thereby activating carbon

atoms. Then, the hydroxyl group at the 3'-position of the sugar ring attacks the carbon atom

to undergo a substitution reaction. The role of DMAP in the reaction process is the catalyst

and proton acceptor.

3.2 Solid Phase Synthesis of the Selenium Nucleic Acid

Millions of oligonucleotides are synthesized and used in the laboratory every year

in the world. For most applications, only a tiny amount of DNA is needed. Moreover, the

synthesis of oligonucleotides is mainly carried out at a scale below 40 nmol. This is enough

for most biological and biochemical experiments. But for biophysical studies such as

nuclear magnetic resonance and X-ray diffraction crystallography, more DNA (more than

10 μmol) needs to be prepared. In extreme cases, it is often required to achieve quantities

of several kilograms for the preparation of drug molecules (e.g., antisense

oligonucleotides). Solid phase synthesis of oligonucleotides has emerged. And almost

exclusively fully automated solid phase synthesis can meet all of the above requirements73,

74.

3.2.1 Advantages of solid phase synthesis

Solid phase synthesis is widely used in the synthesis of peptides, oligonucleotides,

oligosaccharides, and various combinatorial chemistries. Solid-phase chemical synthesis

was invented by Bruce Merrifield in the 1960s, and he won the 1984 Nobel Prize in

Chemistry.

The starting reagent of the solid phase synthesis method is immobilized on a solid

support in the reaction column and the filter, and other reaction reagents and solvents can

63

freely pass through the reaction. Relative to solution synthesis, solid phase synthesis can

use a relatively large amount of solution phase reagent to promote the reaction to proceed

more quickly and thoroughly. Moreover, impurities and excess reagents are rinsed off so

no additional purification process is required after each step is completed. At the same time,

the solid phase synthesis process can also be fully automated through a computer.

3.2.2 Solid supports

The solid support is an insoluble particle and usually has a particle size of 50-200

μm. During the synthesis, oligonucleotides are attached to these particles. There are many

different particles to choose from, but microporous glass (CPG) and polystyrene have

proven to be the most suitable.

3.2.2.1 Controlled-pore glass (CPG)

Controlled-pore glass is chemically stable, does not absorb solvent swelling, and

has deep pores that allow for oligonucleotide synthesis reactions. A glass support with a

pore size of 500 Å is suitable for mechanical synthesis of short-chain oligonucleotides.

However, when a 500 Å pore size vector is used to synthesize an oligonucleotide sequence

of more than 40 bases in length, the yield of the synthesis decreases sharply. This is because

the growing oligonucleotide strand blocks the micropores and prevents the reaction reagent

from diffusing into the support. Although larger pore size matrices are more fragile, it is

more appropriate to use controlled-pore glass with a pore size of up to 1000 Å when

synthesizing oligonucleotide sequences of up to 100 bases in length. Moreover, for the

synthesis of longer oligonucleotide sequences, it is even possible to use a vector with a

pore size of 2000 Å.

64

3.2.2.2 Polystyrene (PS)

Highly cross-linked polystyrene beads have good hydrophobicity and thus higher

oligonucleotide synthesis efficiency, especially at very low scales (eg 40 nmol). A solid

phase carrier for the synthesis of a conventional oligodeoxynucleotide is usually prepared

by attaching 20-30 μmol of nucleoside per gram of the carrier. The efficiency of synthesis

of higher loading oligonucleotides is significantly reduced. This is because the steric

hindrance effect of the oligonucleotide strands adjacent to each other on the carrier

increases as the load increases. However, polystyrene carriers with a loading of up to 350

μmol / g are used in some applications, especially the synthesis of short-chain

oligonucleotides. This also makes it possible to synthesize a large number of

oligonucleotide sequences.

3.2.3 The phosphoramidite method

A variety of solution phase oligonucleotide synthesis methods have been developed

over the years. From the early studies of the hydrogen phosphite and phosphotriester

methods by Michelson and Todd in the 1950s75, 76, and the attempt of Khorana to the

phosphodiester method77, the re-study of the phosphotriester method and the development

of the phosphite triester method in the 1960s and 1970s78-83, all these methods have their

own drawbacks. The phosphoramidite method pioneered by Marvin Caruthers in the early

1980s combined with automated solid phase synthesis became the current universal

choice82.

65

3.3 High-Performance Liquid Chromatogram (HPLC) Analysis for Selenium

Modified DNA Sequences

3.3.1 Reverse phase HPLC procedure

The HPLC equipment used in our lab is a Hewlett Packard Series 1100 HPLC

System. The HPC column is Welch Materials, Inc. UltisilTM XB-C18 chromatographic

column. It’s a reverse phase column.

The mobile phase is a mixture of Buffer A and Buffer B. Buffer A is 20 mM

triethylamine in acetic acid(TEA-Ac) in deionized water solution. Buffer B is 20 mM TEA-

Ac in water/acetonitrile (50%/50%) solution. The mobile phase composition gradient

varies for DMTr-on or DMTR-off DNA analysis because of the polarity difference.

Table 3.1 Mobile phase composition gradient for DMTr-on DNA sequences

purification.

Time B%

0 5

8 60

12 100

20 100

Table 3.2 Mobile phase composition gradient for DMTr-off DNA sequences

purification.

Time B%

0 5

15 40

17 100

66

20 100

The retention time for ideal DNA is about 12 min while that for short oligo usually

is 5 min. If we observe only one single peak at 12 min in the HPLC chromatogram, there

is no short oligo but right DNA.

67

3.3.2 HPLC chromatogram for W3 (5 '-d(CCAAAGT5- SeTGC)-3')

Figure 3.7 HPLC chromatogram of DNA sequence W3 containing only one

selenium-modified thymidine.

68

3.3.3 HPLC chromatogram for W4 (5'-d(GCAACTT5 -SeT GG)-3')

Figure 3.8 HPLC chromatogram of the DNA sequence W4 of all thymidine-

containing selenium-modified DNA.

3.3.4 Summary

The chromatograms are all single peaks, indicating that the DNAs after synthesis

and purification don’t contain too many short oligos. We can do an mass spectra to make

sure that our sample is pure enough for subsequent biochemical experiments. Besides, the

absorption peak at a wavelength of 300 nm is still strong, indicating that the DNA molecule

is indeed selenium modified.

69

3.4 Mass Spectra of Nucleic Acid

3.4.1 MALDI(+)-TOF spectra of W1 (5'-d(CCAAAGTTGC)-3')


(CCAAAGTTGC)-3’). Average mass [M] = 3012.034. Calculated [M + H]+: 3013.041.

Measured [M + H]+: 3006.158.

30

06

.15

80

50

100

150

200

250

300

Inte

ns. [a

.u.]

500 1000 1500 2000 2500 3000 3500 4000 4500m/z

70

3.4.2 MALDI(+)-TOF spectra of W2 (5'-d(GCAACTTTGG)-3')


(GCAACTTTGG)-3’). Average mass [M] = 3043.046. Calculated [M + H]+: 3044.053.

Measured [M + H]+: 3039.333.

30

39

.33

3

29

16

.48

6

15

21

.97

5

28

37

.53

7

24

42

.14

9

0.00

0.25

0.50

0.75

1.00

1.25

4x10

Inte

ns. [a

.u.]

500 1000 1500 2000 2500 3000 3500 4000 4500m/z

71


d(CGGCAACTTTGG)-3’)

Figure 3.11 MALDI-TOF(+) MS analysis of the DMTr-off 5’-double overhang

DNA (5’-d (CGGCAACTTTGG)-3’). Average mass [M] = 3630.429. Calculated [M + H]+:

3631.436. Measured [M + H]+: 3628.923.

3628.923

1815.270

0

500

1000

1500

2000

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500m/z

72


d(CGCCAAAGTTGC)-3’)

Figure 3.12 MALDI-TOF(+) MS analysis of the DMTr-off 5’-double overhang

DNA (CGGCAACTTTGG). Average mass [M] = 3661.429. Calculated [M + H]+:

3662.429. Measured [M + H]+: 3660.152.

3660.152

1831.165

3535.461

2441.0921221.026 3331.065 3848.421

0.0

0.5

1.0

1.5

2.0

2.5

4x10

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500m/z

73

3.4.5 MALDI(+)-TOF spectra of single selenium modified 5’-double overhang

DNA (5’-d(CGGCAACTT(5-SeT)GG)-3’)

Figure 3.13 MALDI-TOF(+) MS analysis of the DMTr-off single selenium modified

5’-double overhang DNA (CGGCAACTT(5-SeT)GG). Average mass [M] = 3740.400.

Calculated [M + H]+: 3741.400. Measured [M + H]+: 3739.581.

3739.581

2080.685

1190.043

1776.626

2275.682

1536.534 2527.5613410.526

0

200

400

600

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500m/z

74

3.4.6 MALDI(+)-TOF spectra of double selenium modified 5’-double overhang

DNA (5’-d(CGGCAACT(5-SeT)(5-SeT)GG)-3’)

Figure 3.14 MALDI-TOF(+) MS analysis of the DMTr-off double selenium

modified 5’-double overhang DNA (CGGCAACT(5-SeT)(5-SeT)GG). Average mass [M] =

3819.371. Calculated [M + H]+: 3820.371. Measured [M + H]+: 3818.136.

3818.136

1190.030

1909.9993693.078

0

1000

2000

3000

4000

5000

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500m/z

75

3.4.7 MALDI(+)-TOF spectra of triple selenium modified 5’-double overhang

DNA (5’-d(CGGCAAC(5-SeT)(5-SeT)(5-SeT)GG)-3’)

Figure 3.15 MALDI-TOF(+) MS analysis of the DMTr-off triple selenium modified

5’-double overhang DNA (CGGCAAC(5-SeT)(5-SeT)(5-SeT)GG). Average mass [M] =

3898.342. Calculated [M + H]+: 3899.342. Measured [M + H]+: 3896.818.

3.4.8 Summary

The measured molecular ion peak is almost the same as the theoretical value

obtained by calculation. According to the HPLC chromatogram and the MS spectra of the

DNA, we can believe that the synthesized DNA molecules are correct, and the selenium

modification is successful. Because the molecular ion peaks are pretty clear, our samples

are pure enough to do the crystal study.

3.5 Melting Study for Selenium Nucleic Acid

Complementary DNA mixtures typically have two different conformations, a

double-stranded structure, and a single-stranded structure. It is not difficult to speculate

from the thermodynamic law that the DNA molecules are mainly in the form of double-

strand in the case of lower temperature, and the DNA molecules are mostly in a single-

3896.818

1947.625

1189.961

0

1000

2000

3000

4000

5000

6000

Inte

ns. [a

.u.]

1000 1500 2000 2500 3000 3500 4000m/z

76

stranded way at a higher temperature and lower concentration. The process of breaking

double-stranded DNA into single strands is known as DNA denaturation, or DNA

denaturing. The melting temperature of DNA refers to the temperature at which 50% of

DNA in a sample has denatured from double-stranded DNA to single-stranded DNA. The

amount of strand separation, or melting, is measured by the absorbance of the DNA

solution at 260nm. Nucleic acids absorb light at this wavelength because of the electronic

structure in their bases, but when two strands of DNA come together, the close proximity

of the bases in the two strands quenches some of this absorbance. When the two strands

separate, this quenching disappears, and the absorbance rises 30%-40%. This is called

Hyperchromicity. The Hypochromic effect is the effect of stacked bases in a double helix

absorbing less ultra-violet light.85, 86

3.5.1 Melting study for DNA double-strand

A mixed buffer solution of pH=7.4, 50 mmol/L Tris-HCl, 100 mmol/L NaCl, and

1 mmol/L EDTA was prepared. The four DNA sequences used in the experiments were all

configured to 1 mmol/L. Add 988 μL of buffer solution to each of the four quartz cuvettes

labeled 1, 2, 3, and 4, and add 6 μL of W1 to the 1 and 2 cuvettes respectively, 6 μL of W2

to the 1 and 3 cuvettes, 6 μL of W3 to the 3 and 4 cuvettes, and 6 μL of W4 to the 2 and 4

cuvettes. The concentration of DNA double strands in the four cuvettes was 6 μmol/L.

Place the four cuvettes in the UV-Vis absorption tester in sequence. Slowly warm up to

90ºC to melt, then cool down to 15ºC to ensure that the DNA is fully integrated into a

double-stranded structure. Set the measured absorption wavelength to 260 nm, the

measurement speed to 0.5ºC/min, the onset temperature to 25ºC, and the termination

temperature to 95ºC.

77


molecules containing different numbers of selenium modifications.

We can obtain not only the trending lines in Figure 3.16 by analyzing the melting

study data but also the thermodynamic data, such as Tm and ΔTm. Tm is the temperature

where the fraction ssDNA rate of change over temperature is the biggest. We regard the

native DNA, W1-W2, as the standard. The Tm difference between the selenium modified

DNA and native DNA is ΔTm.

Table 3.3 The Tm and ΔTm data of the four DNA double-strands via melting study

Sequence ID Tm/ºC ΔTm/ºC

W1-W2 44.8 0

20 30 40 50 60 70 80 90 100

0.0

0.2

0.4

0.6

0.8

1.0R

ela

tive

Ab

so

rba

nce

Temperature(C)

W1-W2

W1-W4

W2-W3

W3-W4

78

W1-W4 41.9 -2.9±0.1

W2-W3 43.7 -1.1±0.1

W3-W4 40.8 -4.0±0.2

The Tm of the DNA double-strand decreases as the number of selenium-containing

modified bases increases. However, the Tm difference isn’t very notable. The selenium

modification affects the thermal stability of DNA double-strand but not too much.

3.5.2 Melting study for DNA and DB2429 complex



configured to 1 mmol/L. Add 976 μL of buffer solution and 12 μL of 0.5 mmol/L DB2429

solution to each of the four quartz cuvettes labeled 1, 2, 3, and 4, respectively, and add 6

μL of W1 to the 1 and 2 cuvettes, 6 μL of W2 to the 1 and 3 cuvettes, 6 μL of W3 to the 3

and 4 cuvettes, and 6 μL of W4 to the 2 and 4 cuvettes. The concentration of DNA double-

strands binding with small molecule in the four cuvettes was 6 μmol/L. Place the four

cuvettes in the UV-Vis absorption tester in sequence. Slowly warm up to 90ºC to melt, then

cool down to 15ºC to ensure that the DNA is fully integrated into a double-stranded

structure. Set the measured absorption wavelength to 260 nm, the measurement speed to

0.5ºC /min, the onset temperature to 25ºC, and the termination temperature to 95ºC.

79


molecules containing different numbers of selenium modifications and small molecule

DB2429 complexes.

The method we calculate Tm and ΔTm of DNA binding with DB2429 is all the

same as what we did for DNA double-strand only.

Table 3.4 The Tm and ΔTm data of the four DNA double-strands binding with

DB2429 via melting study

Sequence ID Tm/ºC ΔTm /ºC

W1-W2-DB2429 56.7 0

W1-W4-DB2429 56.8 0.1±0.1

W2-W3-DB2429 55.9 -0.8±0.1

W3-W4-DB2429 55.8 -0.9±0.1

20 30 40 50 60 70 80 90 100

0.0

0.2

0.4

0.6

0.8

1.0R

ela

tive A

bsorb

ance

Temperature(C)

W1-W2-DB2429

W1-W4-DB2429

W2-W3-DB2429

W3-W4-DB2429

80

The Tm of the DNA double-stranded and small-molecule DB2429 complex are

almost all the same. The thermal stability of the DNA double-stranded isn’t affect by

selenium modification. The ΔTm of the double-stranded DNA binding with the small

molecule is smaller than DNA alone. It can be inferred that DB2429 binding tightly at the

DNA minor groove.

3.5.3 Melting study for DNA and DB2457 complex



configured to 1 mmol/L. Add 976 μL of buffer solution and 12 μL of 0.5 mmol/L DB2457

solution to each of the four quartz cuvettes labeled 1, 2, 3, and 4, respectively, and add 6

μL of W1 to the 1 and 2 cuvettes, 6 μL of W2 to the 1 and 3 cuvettes, 6 μL of W3 to the 3

and 4 cuvettes, and 6 μL of W4 to the 2 and 4 cuvettes. The concentration of DNA double-

strands binding with small molecule in the four cuvettes was 6 μmol/L. Place the four

cuvettes in the UV-Vis absorption tester in sequence. Slowly warm up to 90ºC to melt, then

cool down to 15ºC to ensure that the DNA is fully integrated into a double-stranded

structure. Set the measured absorption wavelength to 260 nm, the measurement speed to

0.5ºC /min, the onset temperature to 25ºC, and the termination temperature to 95ºC.

81


molecules with different numbers of selenium modifications and small molecule DB2457

complexes.

The calculation of Tm and ΔTm of DNA binding with DB2457 is all the same as

DNA binding with DB2429.

Table 3.5 The Tm and ΔTm data of the four DNA double-strands binding with

DB2457 via melting study

Sequence ID Tm/ºC ΔTm /ºC

W1-W2-DB2457 59.8 0

W1-W4-DB2457 59.4 -0.4±0.1

W2-W3-DB2457 59.9 0.1±0.1

W3-W4-DB2457 59.3 -0.5±0.1

20 30 40 50 60 70 80 90 100

0.0

0.2

0.4

0.6

0.8

1.0R

ela

tive

Ab

so

rba

nce

Temperature(C)

W1-W2-DB2457

W1-W4-DB2457

W2-W3-DB2457

W3-W4-DB2457

82

Similar to DB2429, the Tm of the DNA double-stranded and small-molecule

DB2457 complex are also almost the same. The selenium modification doesn’t

significantly change the melting temperature or alter the duplex structure. The ΔTm of the

double-stranded DNA binding with the small molecule is smaller than DNA alone. It can

be inferred that DB2457 binding tightly at the DNA minor groove.

At the temperature range from 40 to 50ºC, there is a shoulder in the trending line.

That might be the effect caused by the small molecule DB2457 released from the DNA

double-strand which means that DB2457 binding with DNA is not as tight as DB2429.

3.6 Crystallization and Structure Studies

For crystal screening, 24 different buffers of Hampton Scientific's Nucleic Acid

Mini Screen Kit reagent were used. The DNA double-strand concentration was 0.2 mmol/L

in deionized water, and the small molecule concentration was 0.3 mmol/L in deionized

water. 500 mL of 15% v/v MPD aqueous solution or 20% v/v MPD aqueous solution was

first added to the crystallization vial during crystal screening. Then 0.5 μL of the DNA

sample solution and 0.5 μL of the crystallization reagent were evenly mixed on the slide

and inverted on the crystallizing cell. Crystal growth was observed every other day.

3.6.1 Crystallization result for 5’-d(CCAAAGTTGC)-3’ and 5’-

d(GCAACTTTGG)-3’ double strand DNA sequence binding with a small

molecule.

As we talked at the very beginning, we synthesized 4 different DNA sequences,

which are listed in the form below. W1 and W2 can form a native DNA double strand. W1

83

with W4 or W2 with W3 can form a DNA double strand with single selenium modified.

W3 and W4 can form a double selenium modified DNA double strand.

Table 3.6 DNA sequences we synthesized which can bind with the small molecule

DB2429 and DB2457.

DNA Sequences Sequence Name

5’-d(CCAAAGTTGC)-3’ W1

5’-d(GCAACTTTGG)-3’ W2

5’-d(CCAAAGT5-SeTGC)-3’ W3

5’-d(GCAACTT5-SeTGG)-3’ W4

We used the synthesized DNA sequences alone or binding with small molecule

trying to create some crystal to do the X-ray diffraction. The figures in the following are

some of the crystals we obtained.

84

Figure 3.19 Experimental DNA crystals obtained.

The crystal quality is scaled by its shape and size. The higher scores the crystal get,

the more possibility to get useful diffraction data. Perfect crystal shall get 5 scores while

poor crystal can only get 1.

W3-W4 Scale: 2

W2-W3-DB2429 Scale: 2 W3-W4-DB2429 Scale: 2

W1-W2-DB2457 Scale: 2



85

From the crystal figure, the crystal is small and numerous, and the shape is irregular.

We sent the crystals to do X-ray diffraction. Unfortunately, we didn't get useful X-ray

diffraction data with those DNA sequences. Subsequent work will continue to optimize

crystallization conditions for better crystals.


Since we cannot get good crystals, we tried to add a GC overhang at 3’-position for

each DNA sequence. As the overhangs introduced, the DNA double-strand shall form a

GC sticky end. The sticky end could connect two DNA double strands easily which may

make it easier to form better crystals. The form below shows the DNA sequences we

synthesized.

Table 3.7 DNA sequences we synthesized which has a GC overhang at 3'-position.


5’-d(CCAAAGTTGCGC)-3’ Top

5’-d(GCAACTTTGGGC)-3’ N

5’-d(GCAAC5-Se TTTGGGC)-3’ S

5’-d(GCAAC5-Se T5-Se TTGGGC)-3’ D

5’-d(GCAAC5-Se T5-Se T5-SeTGGGC)-3’ T

The Top sequence can form a double strand DNA with S, D or T. The newly formed

DNA double-strand should contain single, double or triple selenium modified.

Table 3.8 The DNA double brand could be formed by the synthesized DNA

sequences. To be read vividly, the bottom sequence is presented in 3' to 5' direction and

the GC overhangs are highlighted.

Name DNA Double Brand Sequences

N 5’-d( CCAAAGTTGCGC)-3’

86

3‘-d(CGGGTTTCAACG )-5’

S 5-d( CCAA AGTTGCGC)-3’

3‘-d(CGGGTT(5-SeT)CAACG )-5’

D 5’-d( CCA A AGTTGCGC)-3’

3‘-d(CGGGT(5-SeT)(5-SeT)CAACG )-5’

T 5’-d( CC A A AGTTGCGC)-3’

3‘-d(CGGG(5-SeT)(5-SeT)(5-SeT)CAACG )-5’

However, we couldn’t get any good shape crystals by using these DNA sequences.

Still, we did a screening test. The screening result is shown in the form below.

Table 3.9 Screening result for the DNA double strands with 3'-overhangs.

Although we didn’t have good crystal which can do the X-ray diffraction to finish

the structure study, we can still come to the conclusion that DNAs with selenium modified

could be easier to form crystals. As we can see in the table above, the more selenium atoms

in the DNA double strand, the faster and the more crystal we observed.

87


We cannot get better crystal with 3’-position GC overhangs. So, we had another

trial with 5’-position overhangs. The sequences we synthesized were listed below.

Table 3.10 DNA sequences we synthesized which has a GC overhang at 5'-position.


5’-d(CGCCAAAGTTGC)-3’ Top

5’-d(CGGCAACTTTGG)-3’ N

5’-d(CGGCAAC TT5-Se TGG)-3’ S

5’-d(CGGCAAC T5-Se T5-Se TGG)-3’ D

5’-d(CGGCAAC5-Se T5-Se T5-SeTGG)-3’ T

The Top sequence can form a double strand DNA with S, D or T. The newly formed

DNA double-strand should contain single, double, or triple selenium modified.

Table 3.11 The DNA double brand could be formed by the synthesized DNA

sequences. To be read vividly, the bottom sequence is presented in 3' to 5' direction and

the GC overhangs are highlighted.

Name DNA Double Brand Sequences

N 5’-d(CGCCAAAGTTGC)-3’

3‘-d( GGTTTCAACGGC)-5’

S 5-d(CGCC AAAGTTGC)-3’

3‘-d( GG (5-SeT)TTCAACGGC)-5’

D 5’-d(CGCC A AAGTTGC)-3’

3‘-d( GG(5-SeT)(5-SeT)TCAACGGC)-5’

T 5’-d(CGCC A A AGTTGC)-3’

3‘-d( GG(5-SeT)(5-SeT)(5-SeT)CAACGGC)-5’

88

N Scale: 3 N Scale: 4

N + 2429 Scale: 3 N + 2429 Scale: 3.5

S Scale: 3 S Scale: 3

S + 2429 Scale: 3 S + 2429 Scale: 2.5

89

S + 2457 Scale: 3 S + 2457 Scale: 2.5

D Scale: 3 D Scale: 2.5

D + 2429 Scale: 2.5 D + 2429 Scale: 2.5

D + 2457 Scale: 2.5 D + 2457 Scale: 3

90

We are happy to see that we got some good crystals when we introduced GC

overhangs at 5’-position. We mounted the crystals and tried to do the X-ray diffraction

experiment. However, we still didn’t get good data even if we use the crystals which have

regular shapes.

T Scale: 2.5 T Scale: 3

T + 2429 Scale: 2 T + 2429 Scale: 2

T + 2457 Scale: 2.5 T + 2457 Scale: 2.5

Figure 3.20 Some good crystals we got by using 5'-position GC overhangs.

91

3.6.4 The effect on the crystallization of different MPD concentration.

Table 3.12 The crystallization result for the single selenium modified DNA double-

strand with 5'-position GC overhangs. The MPD concentration is distributed from 20% to

45%. The pictures are what the crystals looked like on the third day.


strand with 5'-position GC overhangs binding with small molecule DB2429. The MPD


like on the third day.



92



Table 3.15 The crystallization result for the double selenium modified DNA double-





93







94

Table 3.18 The crystallization result for the triple selenium modified DNA double-









95



Through the screening result above, we can conclude that higher MPD

concentration can form crystals easier and better. And the sequences with more Se-

modification can create crystal more easily.

3.6.5 Summary

Single overhang and double overhangs DNA can form better crystals than triple

overhangs DNA. The 5’-overhang DNA can form better crystals than 3’-overhang DNA.

Single overhang and double overhangs DNA can form better crystals than triple overhangs

DNA. The 5’-overhang DNA can form better crystals than 3’-overhang DNA. Higher MPD

concentration can form more crystals. The sequences with more Se-modification can form

crystal more easily.

After acquiring the DNA crystal, the structure can be determined by X-ray

diffraction. The crystal structure of each DNA molecule can be obtained by analyzing the

X-ray diffraction data.

96

However, although we sent the DNA crystals to do the X-ray diffraction, we didn’t

get any useful diffraction data.

97

4 CONCLUSIONS

The two major challenges in nucleic acid X-ray crystallography, crystallization and

phasing determination, can be partially solved by introducing selenium modification into

DNA. 5-Se-thymidine was successfully synthesized and incorporated into DNA

oligonucleotides via solid-phase synthesis to facilitate the crystallization and phasing.

Small molecule DB2429 and DB2457 were co-crystalized with Se-DNAs and nice

crystals were obtained, which are better than the native crystals. 5’-d(CCAAAGTTGC)-3’

and 5’-d(GCAACTTTGG)-3’ duplex can crystallize but doesn’t have useful X-ray

diffraction data even that it’s selenium modified. The complex of DNA duplex binding

with small molecule doesn’t have good diffraction data, as well.

The melting study reveals that the selenium modification has no signification

perturbation to the DNA stability.

Since we couldn’t get good structure data from the regular DNA double-strands

(native and Se-modified) after the X-ray diffraction, we tried to introduce overhangs to the

duplex through which we could get a sticky end. The 5’-overhang DNA can form better

crystals than 3’-overhang DNA. Single overhang and double overhangs DNA can form

better crystals than triple overhangs DNA. We also find out that higher MPD concentration

can form more crystals quickly and 35% MPD aqueous solution shall be the best reservoir

solution for crystallization. The sequences with more Se-modification can create crystal

more efficiently which means that selenium atom in the DNA molecule affects

crystallization.

98

After obtaining the DNA crystal, the structure can be determined by X-ray

diffraction. The crystal structure of each DNA molecule can be obtained by analyzing the

X-ray diffraction data.

99

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APPENDICES

Appendix A Nucleic Acid Mini Screen (NAM)

The Nucleic Acid Mini Screen™ is an efficient screen formulated to assist in the

determination of preliminary crystallization conditions of nucleic acid fragments.

Using 1 to 4 mM oligonucleotide stock concentration the screen requires less than

25 microliters of sample.

The screen evaluates sample concentration, temperature, pH, monovalent cations,

divalent cations, and polyamines. To complete the screen, one will need 1) HR2-118

Nucleic Acid Mini Screen, a set of twenty-four, 1 milliliter volumes of reagent and 2) 35%

v/v (+/-)-2-Methyl-2,4-pentanediol, the dehydrant for the reagent well / reservoir (available

separately as HR2-863 35% v/v (+/-)-2-Methyl-2,4-pentanediol).

The composition of the nucleic acid mini screen allows one to apply the formulation

to other nucleic acids such as deoxy- and ribozymes, pseudoknots and tRNAs.

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Figure A.0.1 Nucleic acids mini screen kit conditions.

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Appendix B The Synthesis of the Nucleotide with an Extended Pyrimidine Ring

Appendix B.1 Introduction

By using uridine as the starting reactants, the 3‘,5’-TIPDS-biscyc-selenoate (TiOS)

was synthesized via 4 steps. The 4 steps are TIPDS protection, adding Ms-leaving group,

Boc-protection and selenium introduction. The newly synthesized biscyc-selenoate can be

used to synthesis novel nucleotide with seven or more-member ring as the base group. This

could be a very interesting research. There are many different small molecules we can use

to extend the base group ring. For now, we introduced ethylene glycol to extend the ring.

In the future, we may use many other interesting small molecules such as acetonitrile to

get aromatic extended ring. We can even synthesis sequences containing the new

nucleotide to figure out the interaction between conventional nucleotides.

Figure B.2 Synthesis route of 5-MeSe-T-phosphorimidite from 5-I-dU.

The synthesis route of biscyc-selenoate was shown in figure one. As we can see,

there are 4 steps totally to synthesis the selenoate from uridine. Firstly, we use TIPDS

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group to protect the 3’- and 5’-position hydroxy groups. Secondly, we use Ms-group as the

leaving group at 2’-position. Then, Boc-group is added to protect the nitrogen atom in the

pyrimidine ring. Finally, we introduce selenium into the molecule.

Appendix B.2 TIPDS Protection

Appendix B.2.1 Experimental procedure

Figure B.3 TIPDS protection reaction.

10 g uridine was dissolved in 250 mL pyridine (concentration is about 0.1 M). The

solution was cooled with ice bath. 15 mL TIPDS-Cl was added dropwise. The reaction was

stirred at room temperature overnight. The reaction process was detected by TLC (TLC

condition methanol: methylene chloride=1:20). When there was no uridine spot in the TLC

plate, pyridine was evaporated under reduced pressure. The residue was dissolved in DCM

and washed with 3M HCl. Then washed with sat. NaHCO3 and brine. The solution was

dried by MgSO4. Then concentrated by evaporator after filtration. Finally, purified by

column (with the eluent of methanol: methylene chloride=1:100).

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Appendix B.2.2 Proposed mechanism

Figure B.4 The proposed mechanism of TIPDS protection reaction.

The first step is an SN2 reaction. First, the oxygen atom in the 5’-hydroxy group

attacks the silica atom in the TIPDS-Cl and the chloride atom will be substituted, therefore.

The pyridine here will work as a base, which will take the proton released from the hydroxy

group. Then, 3’-hydroxy will react with the other silica though the same mechanism and

the ring shall be closed.

Appendix B.3 Adding Ms-Leaving Group


Figure B.5 Ms-leaving group adding reaction.

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6 g starting material 3’,5’-TIPDS-uridine was dissolved in 80 mL THF and

cooled down to 0℃. 5.1 mL TEA was added into the solution. Then treated with 1.4 mL

MsCl. Reaction will be finished in about 1 h. The reaction process was detected by TLC

(TLC condition methanol: methylene chloride=1:50). When there was no starting material

spot in the TLC plate, saturated NaCl solution was added to quench the reaction. The

residue was dissolved in DCM and washed with water and brine. The aqueous layer was

extracted by DCM. The organic layer was dried by MgSO4. Then concentrated by

evaporator after filtration. Finally, purified by silica gel column (with the eluent of

methanol: methylene chloride=1:100).


Figure B.6 The proposed mechanism of Ms-leaving group adding reaction.

As shown in Figure B.6, the mechanism of Ms-leaving group adding reation is

addition-elimination. First, the hydrogen atom in the 2’-hydroxy group attacks the sulfur

atom in the Ms-Cl and a hydroxide will be formed during the process. Next, the electron

pair in the hydroxide reforms a double bond which eliminated the chloride.

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Appendix B.4 Boc-Protection


Figure B.7 Boc-protection reaction.

6.7 g starting material 2’-Ms-3’,5’-TIPDS-uridine and 1.4 g DMAP was dissolved

in 60 mL THF. 3.3 mL TEA was injected into the flask. 4.1 mL (Boc)2O was injected. The

reaction was stirred at room temperature for about 1 h. The reaction process was detected

by TLC (TLC condition Methanol: Methylene Chloride=1:100). When there was no

starting material spot in the TLC plate, the solvent was evaporated and redissolved in DCM.

The residue was dissolved in DCM and washed with water and brine. The aqueous layer

was extracted by DCM. The organic layer was dried by MgSO4. Then concentrated by

evaporator after filtration. Finally, purified by silica gel (with the eluent of ethyl acetate:

hexanes=1:7).

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Figure B.8 The mechanism of the Boc-protection reaction.

The mechanism of the Boc-protection reaction is shown in Figure B.8. As we can

see, the DMAP here works as a catalyst. First, DMAP actives the (Boc)2O through an

addition-elimination reaction. The actived Boc-group will react with the nitrogen through

the reaction.

Appendix B.5 Selenium Introduction


Figure B.9 Selenium introduction reaction.

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The reaction was conducted under Ar protection. 0.3 mL Me2Se2 and 3 mL THF

was injected into the round bottom flask. The flask was cooled down to -78℃ by dry using

ice and acetone mixture. 0.7 mL n-BuLi was injected under -78℃, then the solution was

warmed to room temperature. Keep stirring at r.t. for 15 min. 0.56 g starting material 1-N-

Boc-2’-Ms-3’,5’-TIPDS-uridine was dissolved in 3 mL THF and injected into the selenium

solution. The mixture was stirred at r.t. for 15-30 min. The reaction process was detected

by TLC (TLC condition Methanol: Methylene Chloride=1:50). When there was no starting

material spot in the TLC plate, the reaction was quenched with brine. The residue was

dissolved in DCM and washed with water and brine. The aqueous layer was extracted by

DCM. The organic layer was dried by MgSO4. Then concentrated by evaporator after

filtration. Finally, purified by silica gel column (with the eluent of ethyl acetate:

hexanes=1:7).

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Figure B.10 The proposed mechanism of the selenium introduction reaction.

The reaction goes on by two steps. First, the dimethyl diselenide is cleaved by n-

butyl lithium. Methyl selenide could be formed through the reaction. Next, the methyl

selenide attacks the carbonyl carbon at 6’-position. Through the electron transfer, the C-N

bond is open and the 2’-oxygen gets a negative charge. The oxygen becomes a strong

nucleophile, and the MsO-group is a very good leaving group. With an SN2 reaction, the

bicyclic intermediate is created. Then the methyl selenide replace the boc-group. After we

quench the reaction, the intermediate is hydrolyzed into the product.

Appendix B.6 Ring Formation Reactions

The methyl selenide group is a good leaving group, which make the carbonyl

carbon in the selenide ester very electrophilic. In the meantime, the nitrogen atom in imide

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group is supposed to be a nucleophile. If we have a small molecule which has both

electrophilic and nucleophilic group. The nucleophilic group could react with selenide ester

part while the electrophilic group with imide nitrogen. Though such reaction, we could

achieve the ring formation reaction.

Theoretically, all of the small molecule which contains both a good leaving group

and a nucleophilic part should be applicable to the ring formation reaction. Acetonitrile is

a very proper example.

First, the pKa of acetonitrile is only 25, which means that in base condition, the

methyl group might lose a proton and form a carbon cation. The carbon cation is pretty

stable because of the resonance effect, which is shown in the picture below. Carbon cation

can react with the selenide ester and replace the methyl selenide group as the role of a

nucleophile.

Figure B.11 The resonance structure for acetonitrile carbon anion. The carbon

anion should be relatively stable because of the resonance effect.

Second, as we know, though the resonance structure of the acetonitrile shown

below, it’s conspicuous that the carbon in the carbon-nitrogen triple bond shall contain

some positive charge. Then that carbon could be possible to react with the imide nitrogen

in the TiOS molecule as an electrophile.

Figure B.12 The resonance structure for acetonitrile. The cyan carbon contains

partial positive charge, which makes it electrophilic.

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Now we have the eligible small molecule, acetonitrile, which might finish the ring

formation reaction. The probable reaction equation is shown as following.

Figure B.13 Reaction equation of the ring formation reaction for the TiOS with

acetonitrile.

If the reaction goes on well and just reacts as what we assumed, we should get the

ring expanded product finally. The reaction could be divided by two steps. First, in basic

condition, the methyl group attacks the carbonyl carbon. Through addition-elimination

reaction, the methyl selenide group is substituted. Second, the imide nitrogen attacks the

cyan carbon, and the new expanded ring is formatted.

The new product we shall get via TiOS react with acetonitrile isn’t perfect. The

CH2 in the expanded ring blocked the conjugated electrons, which makes the ring not

aromatic. It can be a solution if we transfer the methyl group into amino group or hydroxyl

group. By doing so, the long pair elections in N or O can be conjugated with the other

electrons in the ring. And the ring could be aromatic.

Some of the examples are listed as reaction equations below.

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cyanamide. The electrons in the p orbitals could be conjugated to make the expanded ring

aromatic.


cyanic acid. The electrons in the p orbitals could be conjugated to make the expanded ring

aromatic.


cyanic acid. The electrons in the p orbitals might not be conjugated to make the expanded

ring aromatic.

The small molecules listed above are just some of the possible reagents to expand

the ring. There are still a lot of alternative molecules for us to format the ring. As we talked

a lot before, any molecule that has or could have both leaving group and nucleophilic part

might be a good choice.

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Appendix B.7 Ethylene glycol introduction reaction

We tried with acetonitrile to format the ring before. However, there is no reaction

even if we heat it up overnight. It’s a brand-new molecule we got and we don’t know very

much of the properties. There is one thing we are certain is that the selenide ester can react

with alcohol.

So, we decided to use ethylene glycol to format the ring. The overall reaction is

shown as following.

Figure B.17 The overall reaction equations of the ring formation reaction for the

TiOS with ethylene glycol. We shall get a novel nucleoside with nine-member-ring.

The reaction contains 3 steps totally. First, the ethylene glycol reacts with selenide

ester part. The methyl selenide group is substituted. In Compound 5, both hydroxy group

and imide nitrogen are nucleophiles. We can protect the hydroxy group with mesyl group

to make it a good leaving. Then, it should be possible to finish the ring formation reaction.

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Figure B.18 Ethylene glycol introduction reaction.

0.4 g starting material Ti-OS was dissolved in 3 mL THF and 3 mL ethylene glycol.

0.4 mL TEA was added into the solution. Keep stirring at 65℃ overnight. The reaction

process was detected by TLC (TLC condition methanol: methylene chloride=1:4). When

there was no Ti-OS spot in the TLC plate, the residue was dissolved in DCM and washed

with water and brine. The aqueous layer was extracted by DCM. The organic layer was

dried by MgSO4. Then concentrated by evaporator after filtration. Finally, purified by silica

gel column (with the eluent of ethyl acetate: hexanes=1:1).


Figure B.19 The mechanism of the ethylene glycol introduction reaction.

The ethylene glycol introduction reaction is also an addition elimination reaction.

The oxygen atom in the hydroxy group in ethylene glycol attacks the carbonyl carbon near

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the selenium. Then, the electron pair on the hydroxide reform a carbonyl bond and the

methyl selenide group is substituted.

Appendix B.8 Ms-leaving group introduction reaction


Figure B.20 Ms-leaving group introduction reaction.

0.1 g starting material Ti-OS-EGol was dissolved in 2 mL THF. 0.1 mL TEA was

added into the solution. 0.02 mL MsCl was injected. The reaction was stirred at room

temperature for about 1 h. The reaction process was detected by TLC (TLC condition

methanol: methanol: methylene chloride=1:4). When there was no Ti-OS-EGol spot in the

TLC plate, the residue was dissolved in DCM and washed with water and brine. The

aqueous layer was extracted by DCM. The organic layer was dried by MgSO4. Then

concentrated by evaporator after filtration. Finally, purified by silica gel column (with the

eluent of ethyl acetate: hexanes=1:2).

121

Appendix B.9 The synthesis for the nucleotide with an extended pyrimidine ring

Figure B.21The final step for the synthesis is a ring formation reaction. We have

both nucleophile and electrophile in the molecule. And they are not too far away from each

other. Theoretically, we can do the reaction finally.

Now, we have already got 150 mg Ms-protected Ti-OS-EGol. There is only one

step left to our final product. It’s a ring formation step, as the picture showing above. If

everything is going on well, we shall get the novel eight-member ring nucleotide finally.

It is possible to extend pyrimidine ring via TiOS intermediate. In fact, all of the

small molecules which contain both a good leaving group and a nucleophilic part should

be applicable to the ring formation reaction. Acetonitrile and a series of acetonitrile

derivatives are suitable for the ring formation. However, despite we tried different basic

condition, different solvent, and different temperature, there is still no reaction.

To make the reaction easier, we tried ethylene glycol. After reacting overnight at

65℃, we observed the first step reaction finally. Although we still haven’t done the ring

formation reaction, we believe that we can make it finally and we will keep working on it.