dna recognition and antimicrobial compounds

310 Pages·2017·25.48 MB·English

UNIVERSITY OF
LINCOLN

D R A
NA ECOGNITION ND
A C
NTIMICROBIAL OMPOUNDS:
E T V
XPLORING HE ERSATILITY
O P
F EPTIDES

A I
BHISHEK YER

Promoters: Prof. Dr. Annemieke Madder
Dr. Ishwar Singh

Dissertation submitted in fulfilment of the requirements for the degree of
Doctor of Philosophy (PhD)

D O S C P
OCTOR F CIENCE: HEMISTRY / HARMACY
2016

STATEMENT OF ORIGINALITY

“I, Abhishek Iyer, hereby declare that this submission is my own work and to the best of my
knowledge it contains no materials previously published or written by another person, or
substantial proportions of material which have been published or accepted for the award of any
other degree or diploma at University of Lincoln or any other educational institution, except
where references have been made in the thesis. Any contribution made to the research by
others, with whom I have worked at the University of Lincoln or elsewhere, is explicitly
acknowledged in the thesis. I also declare that the intellectual content of this thesis is the
product of my own work, except to the extent that assistance from others in the project's design
and conception or in style, presentation and linguistic expression is acknowledged.”

Signed ……………………………………………..............

Date ……………………………………………..............

I

APPRECIATION AND ACKNOWLEDGEMENTS

After having written nearly three hundred pages as part of this PhD thesis, I am at a loss for words when
it comes to writing this section. I am extremely grateful and would like to thank every single person I
have met along the way for making this, by far, the best four years of my life. It is not possible for me
to mention everyone by name, but I would like to mention the names of those people without whom
this PhD would not have been possible.

First and foremost, I would like to thank my promoters Prof. Dr. Annemieke Madder and Dr. Ishwar
Singh. I consider myself very fortunate for having the opportunity to work with two remarkable, very
knowledgeable and unique individuals. I would like to thank Annemieke for her unwavering support
and for always having faith in me even during those times when I lost confidence in myself. She has
taught me, by example, that being a good human being is as important as being a good chemist and it
is something I will remember for the rest of my life. I would like to thank Ishwar for his out-of-the-box
thinking and ideas that he has shared with me. It has been wonderful discussing chemistry with Ishwar
and I am grateful to him for always taking time out and answering patiently all the queries I have had
in the last two years.

Among my colleagues and friends in Ghent I would first like to thank Yara Ruiz for the countless
discussions we have had in the last four years – related and unrelated to chemistry. She has taught me
a lot in Ghent as well as in Lincoln but also shown me that there is more to a PhD than just academics.
I am very fortunate to have worked with Yara and am even more happy to have her as a very good
friend. I am proud of my former master students – Lars, Dorien and Cecilia (for a short while) all of
whom are doing PhDs. I would like to wish them all the best in their endeavours. Dorien, I would like
to thank in particular for all the work that you have done for me for the articles as well as preparing and
submitting countless mass samples on my behalf in the last two years. I would like to thank everyone
in Ghent – Matthias, Lidia, Duchan, Vicky, Bram, Nathalie, Eri, Eva, Margarida, Smita, An, Kurt,
Lieselot, Ellen, Willem, Maxime and Emily for the atmosphere in the lab. Last, but not the least, I would
like to thank Jos for taking care of all the equipment and consumables and without whom the lab is
simply incomplete. I would like to thank Prof. Bruno De Geest for his valuable advice and help for
testing the peptides for cell uptake. I would also like to thank Maji for his help from time to time and
Tim for the NMR measurements. A big thanks to Jan for running countless mass samples for four long
years and for advice whenever I have needed it. Thanks to Tom and Veerle for all the administrative
tasks.

I am fortunate to have been part of the PhosChemRec funded by a Marie Curie Initial Training Network.
I would like thank the people from the network – Marta, Plamena, Alice, Zeyed, Martina, Charlotte,
Emmanuel, Chris, Sylvain, Christina, Ute, Daniela, Luigi and Pierre-Yves for the time we spent in the
network meetings – sharing chemistry as well as personal experiences.

From my friends in Lincoln, I would first like to thank Anish for the wonderful time we have shared in
the past year and a half and all the light moments amidst tons of work and late night hours – which I
will forever cherish. I would also like to thank Shreesha for helping me out initially, Paolo, Ranga and
Louis for his witty remarks and everyone for maintaining a great atmosphere in the lab. Special thanks
to Dr. Stephen Prior for some fantastic NMR work and Charlie for testing more than two hundred
samples for biological activity. Without your contributions, this PhD would be incomplete. I would also
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like to thank Dr. Timea Palmai-Pallag for testing the compounds for toxicity and Dr. Edward Taylor
for advice regarding my project in Lincoln.

I would like to thank the members of the jury – Dr. Enrico Ferrari, Dr. Tobias Gruber, Dr. M. Frances
Heaney and Prof. Dr. José Martins for taking out valuable time to read, correct and be a part of my jury.

I would like to thank my non-lab friends in Lincoln – Rashmi firstly for always being there for me and
taking care of me. Mihir, Nivedita, Toral, Fawad, Khyati and Ramola we have spent some good time
together and it has made my stay in Lincoln truly wonderful.

Last, but not the least, I would like to thank my family, particularly my mother for always wanting me
to study hard and believing that I was capable of things even I did not think I could do at the time. My
father for his patience, advice and values and my sister for being the joy of the house even when all of
us were going through a tough time.

My PhD has been a long and tiring but amazing journey. I’ve learnt not just a lot of Chemistry but have
grown as a person. It has broadened my perspective of life and given me a great platform and
opportunity to contribute to science and help others less fortunate than me – something I will always
keep in mind.

Abhishek Iyer

Lincoln, 15th August 2016

IV
LIST OF ABBREVIATIONS

AA amino acid
Aba 4-acetamidobenzoic acid
A. baumannii Acinetobacter baumannii
Ac O acetic anhydride
2
AcOH acetic acid
Ahx 6-aminohexanoic acid
Alloc allyloxycarbonyl (protecting group)
Boc tert-butoxycarbonyl (protecting group)
b-HLH-ZIP basic helix-loop-helix zipper
bZIP basic region leucine zipper
CD cyclodextrin
CuAAC copper azide-alkyne cycloaddition
Dab diaminobutyric acid
DCC dicyclohexylcarbodiimide
DCM dichloromethane
DIC diisopropylcarbodiimide
DIPEA/DIEA diisopropylethylamine
DMAP 4-dimethylaminopyridine
DMF N,N-dimethylformamide
DMSO dimethylsulfoxide
DNA deoxyribonucleic acid
DPC DNA-peptide/protein/polyamide cross-link
dsDNA double stranded DNA
E. coli Escherichia coli
EDC 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (hydrochloride salt)
EDT ethanedithiol
EMSA electrophoretic mobility shift assay
ESI Electrospray ionization
EtOAc ethyl acetate
V
EM exact mass
Et O Diethyl ether
2
Fmoc 9-fluorenylmethoxycarbonyl
Gaba γ-aminobutylbenzoic acid
HATU N-[(dimethylamino-1H-1,2,3-triazolo[4,5-b]pyridin-1-ylmethylene]-
N-methylmethanaminium hexafluorophosphate N-oxide
HBTU N-[(1H-benzotrioazole-1-yl)(dimethylamino) methylene]-N-
methylmethanaminium hexafluorophosphate N-oxide
HCA α-cyano-4-hydroxycinnaminic acid
hν UV-light
HOAt 1-hydroxy-7-azabenztriazole
HOBt 1-hydroxybenzotriazole
HPLC High-performance liquid chromatography
HRMS high resolution MS
ICL interstrand cross-link
K. pneumoniae Klebsiella pneumoniae
LC Liquid Chromatography
MALDI matrix assisted laser desorption ionisation
MALDI-TOF Matrix-assisted laser desorption/ionization time of flight
MBC Minimum Bactericidal Concentration
MeCN acetonitrile
MeOH methanol
MIC Minimum Inhibitory Concentration
MoeA Moenomycin A
mRNA messenger RNA
MRSA Methicillin resistant Staphylococcus aureus
MS Mass Spectrometry
MW molecular weight or microwave
mQ/ milliQ, deionized H O
2
m/z mass to charge ratio
NBS N-Bromosuccinimide
NMP N-Methyl-2-pyrrolidine
VI
NMR nuclear magnetic resonance
Oligo oligonucleotide
Oxyma Ethyl (hydroxyimino)cyanoacetate
PAGE polyacrylamide gel electrophoresis
P. aeruginosa Pseudomonas aeruginosa
Pbf 2,2,4,6,7-Pentamethyl-dihydrobenzofurane-5-sulfonyl
PBS Phosphate-buffered saline
PDB protein data bank
PEG polyethyleneglycol
Pet ether Petroleum ether
ppm parts per million
PS polystyrene
PyBOP (Benzotriazol-1-yloxy)tripyrrolidinophosphonium
hexafluorophosphate
Rf ratio to front
rt room temperature
r.t. retention time
RNA Ribonucleic acid
ROS Reactive oxygen species
RP reversed phase
S. aureus Staphylococcus aureus
SPPS Solid Phase Peptide Synthesis
ssDNA single stranded DNA
TBE TrisBoratEDTA (buffer) or tris(hydroxymethyl aminomethane,
B(OH) , ethylenediamine tetraacetic acid
3
TCEP tris(2-carboxyethyl)phosphine
TEA triethylamine
TEAA tetraethylammonium acetate
tBu tert-Butyl
TF transcription factor
TFA trifluoroacetic acid
TIS triisopropylsilane
VII
Tris Trizma hydrochloride buffer solution
tRNA transfer RNA
Tris tris(hydroxymethyl)aminomethane)
Trt trityl or triphenylmethyl
UV Ultraviolet
XL cross-link

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