Synthesis and Biological Evaluation of Pyrrole–Imidazole Polyamide Probes for Visualization of Telomeres

130 Pages·2019·7.55 MB·English

by  Yusuke Kawamoto

Springer Theses
Recognizing Outstanding Ph.D. Research
Yusuke Kawamoto
Synthesis and
Biological Evaluation
of Pyrrole–Imidazole
Polyamide Probes
for Visualization of
Telomeres
Springer Theses
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Yusuke Kawamoto
Synthesis and Biological
–
Evaluation of Pyrrole
Imidazole Polyamide Probes
for Visualization
of Telomeres
Doctoral Thesis accepted by
Kyoto University, Kyoto, Japan
123
Author Supervisor
Dr. Yusuke Kawamoto Prof. Hiroshi Sugiyama
Department ofChemistry andBiochemistry Department ofChemistry
University of California Graduate Schoolof Science
SanDiego, La Jolla, CA,USA KyotoUniversity
Kyoto, Japan
ISSN 2190-5053 ISSN 2190-5061 (electronic)
SpringerTheses
ISBN978-981-13-6911-7 ISBN978-981-13-6912-4 (eBook)
https://doi.org/10.1007/978-981-13-6912-4
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’
Supervisor s Foreword
Over the several decades, varied molecules to target specific sequences on nucleic
acids have been developed. They have been used for therapeutics of cancer and
other diseases, visualization of specific regions, regulation of genes, and so on. In
this area, we have worked on synthetic molecules recognizing DNA-specific
sequences, named pyrrole (Py)-imidazole (Im) polyamides.
Inthisthesis,Dr.YusukeKawamotoreportsPy-Impolyamidestargetinghuman
telomeres.Theauthordemonstratespolyamidestargetinglongerbasepairsthanthe
previously reported ones and their cellular application. In Chap. 1, the author
discussesthehistoryandrecentstudiesofPy-Impolyamidestohelpreaderstolearn
the background. In Chap. 2, the author reports the development of the facile syn-
thetic method of tandem hairpin Py-Im polyamides targeting 12 bp in human
telomere repetitive sequence and the method to visualize telomeres under mild
condition without the harsh denaturation. In Chap. 3, the author reports longer
tandem trimer Py-Im polyamides targeting 18 bp in telomere sequence and shows
that extending the target base pairs decreases the background signals of fluores-
centlylabeledpolyamidesandincreasesthespecificity.InChap.4,tandemtetramer
Py-Im polyamides targeting 24 bp are reported. 24 bp is the longest reported
binding site for synthetic, non-nucleic-acid-based, sequence-specific DNA-binding
molecules. Visualization of telomeres in cells and high-throughput sequencing
technique demonstrate that tandem tetramer polyamides have higher specificity to
telomeres than shorter polyamides as discussed in Chaps. 2 and 3.
This thesis clearly demonstrates the development and optimization of tandem
hairpin Py-Im polyamides and visualization of telomeres in cells using the poly-
amides. I believe this can be applied to future studies on DNA-binding molecules
and their cellular applications.
Kyoto, Japan Prof. Hiroshi Sugiyama
November 2018
v
Parts of this thesis have been published in the following journal articles:
1. “Sequence-specific DNA Binding Pyrrole–Imidazole Polyamides and Their
Applications” Kawamoto, Y.; Bando, T.; Sugiyama, H. Bioorg. Med. Chem.
2018, 26, 1393–1411.
2. “Development of a New Method for Synthesis of Tandem Hairpin
Pyrrole-ImidazolePolyamideProbes TargetingHuman Telomeres”Kawamoto,
Y.; Bando, T.; Kamada, F.; Li, Y.; Hashiya, K.; Maeshima, K.; Sugiyama,
H. J. Am. Chem. Soc. 2013, 135, 16468–16477.
3. “Tandem Trimer Pyrrole-Imidazole Polyamide Probes Targeting 18 Base Pairs
in Human Telomere Sequences” Kawamoto, Y.; Sasaki, A.; Hashiya, K.; Ide,
S.; Bando, T.; Maeshima, K.; Sugiyama, H. Chem. Sci. 2015, 6, 2307–2312.
4. “Targeting 24 bp within Telomere Repeat Sequences with Tandem Tetramer
Pyrrole–ImidazolePolyamideProbes”Kawamoto,Y.;Sasaki,A.;Chandran,A.;
Hashiya,K.;Ide,S.;Bando,T.;Maeshima,K.;Sugiyama,H.J.Am.Chem.Soc.
2016, 138, 14100–14107.
vii
Acknowledgements
First, I would like to deeply appreciate my research supervisor, Professor Hiroshi
Sugiyama, for teaching me about science and valuable suggestions about my
research.Hisguidanceanddiscussionshelpedmetosolvevariousproblems.Ialso
deeply appreciate Associate Professor Toshikazu Bando for teaching me about
chemical synthesis and his carefully considered feedback and valuable comments
andsuggestions,especiallyaboutthedesignofcompoundsandexperimentalplans.
I would also like to appreciate other Sugiyama group staffs, Associate Professors
MasayukiEndoandAsakoYamayoshi,andAssistantProfessorsSoyoungParkand
Namasivayam Ganesh Pandian for their helpful advice and discussions.
I would like to thank senior laboratory members, Dr. Gengo Kashiwazaki,
Dr. Yuki Suzuki, and Dr. Hironobu Morinaga, for their coaching of me.
IamgratefultoMs.YasukoNiimi,Ms.TakakoFutamata,Ms.ChikakoBamba,
Ms. Kumi Hidaka, and Ms. Tomoko Emura for their kind support of my study as
secretaries. I am also grateful to laboratory technicians, Ms. Kaori Hashiya for
performing solid-phase synthesis, and Mr. Shinsuke Sato for helping me in cell
culturing.
I would like to express my special thanks to laboratory mates,
Dr. Anandhakumar Chandran for performing high-throughput sequencing and its
analysis, Dr. Yue Li for supporting the synthesis of our compounds, Dr. Junichi
Taniguchi for helpful discussions and suggestions about our compounds and their
cellular evaluations, and Mr. Sefan Asamitsu for supporting SPR experiments and
helpful discussions about G-quadruplexes. I am also grateful to all the members
of the Sugiyama’s laboratory who supported and gave comments about my work.
I am very much indebted to our collaborators Professor Kazuhiro Maeshima,
Assistant Professor Satoru Ide, Ms. Asuka Sasaki, and Ms. Fukumi Kamada
(National Institute of Genetics) for cellular experiments, capturing excellent cell
images, and their helpful discussions. Thanks to their data, we successfully pub-
lishedmanyexcellentpapers,someofwhichareincludedinmythesis.Iwouldlike
to thank Professor Peter B. Dervan (California Institute of Technology) for wel-
comingmetovisithislaboratoryandvaluablediscussionandsuggestionaboutthe
ix
x Acknowledgements
design of pyrrole-imidazole polyamides. I also appreciate Assistant Professor
Shigeo S. Sugano (Ritsumeikan University) for evaluating our compounds using
plants and helpful suggestion about analysis of our high-throughput sequencing
data.
I would like to thank Professor Hanbin Mao, Mr. Shankar Mandal, and
Mr. Mohammed Enamul Hoque (Kent State University) for their teaching and
helpfuldiscussionsabouttheresearchofG-quadruplexusingtheiropticaltweezers
and our compounds and helping a lot all about my staying in the USA.
I also thank Japan Society for the Promotion of Science for financial support
(Research Fellowship for Young Scientists), our Department of Chemistry for
financial support of overseas collaborative research, and WPI-iCeMS for financial
support of visiting laboratories in the USA.
Finally, I will give a deep appreciation to my family for their constant support
and encouragement of my motivation.
Contents
1 Introduction: Sequence-Specific DNA Binding Pyrrole–Imidazole
Polyamides and Their Applications. . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1.2 Hairpin Structure and Pairing Rule of Py–Im Polyamides . . . . . . 3
1.3 Varied Structures of Py–Im Polyamides. . . . . . . . . . . . . . . . . . . 8
1.4 Regulation of Gene Expression by Py–Im Polyamides . . . . . . . . 11
1.5 Repression of Gene Expression by Sequence-Specific DNA
Alkylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
1.6 Transcriptional Activation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
1.7 Py–Im Polyamides for Imaging . . . . . . . . . . . . . . . . . . . . . . . . . 20
1.8 Other Functions and Applications of Py–Im Polyamides. . . . . . . 25
1.9 Conclusion and Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
1.10 Scope of the Work . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2 Development of a New Method for Synthesis of Tandem Hairpin
Pyrrole–Imidazole Polyamide Probes Targeting Human
Telomeres . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
2.2 Results and Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
2.2.1 Synthesis of Conjugates 1–4. . . . . . . . . . . . . . . . . . . . . . 45
2.2.2 UV-Visible Absorption Spectra and Fluorescence
Spectra . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
2.2.3 Binding Affinities and Specificities of Tandem
Hairpin PI Polyamides . . . . . . . . . . . . . . . . . . . . . . . . . . 49
2.3 Conclusion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.4 Supporting Information. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
2.4.1 Materials for the Synthesis . . . . . . . . . . . . . . . . . . . . . . . 54
2.4.2 Materials for the Analysis of Spectra and Thermal
Denaturation Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . 55
xi