Harkiss, Alexander Hugh (2017) Synthesis of novel fluorescent heterocyclic-derived α-amino acids ...

213 Pages·2017·9.18 MB·English

by  Alex Harkiss

Harkiss, Alexander Hugh (2017) Synthesis of novel fluorescent
heterocyclic-derived α-amino acids and the total syntheses of piperidine
natural products. PhD thesis.

http://theses.gla.ac.uk/8629/

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Synthesis of Novel Fluorescent
Heterocyclic-Derived α-Amino Acids and
the Total Syntheses of Piperidine
Natural Products

Alexander Hugh Harkiss, MSci

A thesis submitted in part fulfilment of the
requirements of the degree of Doctor of Philosophy

School of Chemistry
College of Science and Engineering
University of Glasgow
September 2017
1

Abstract
During the course of this PhD, methodology for the synthesis of a series of novel, highly fluorescent
pyridine-derived α-amino acids was developed. Enone-derived α-amino acids were subjected to an
inverse electron demand hetero-Diels-Alder cycloaddition and aromatisation reaction, which led to
a twelve-membered library of pyridine analogues. The optical properties of these compounds were
analysed, with several exhibiting interesting fluorescent characteristics. One of the analogues was
incorporated into a cell penetrating pentapeptide via solid phase peptide synthesis. The resulting
hexapeptide was incubated with human fibroblast cells and fluorescence microscopy was used to
show accumulation of the peptide in the cells.

The total syntheses of piperidine containing natural products, spruce alkaloid and (+)-241D were
also examined during this PhD. A short nine-step linear sequence was developed giving spruce
alkaloid and (+)-241D in 21% and 19% overall yield, respectively. A base mediated 6-endo-trig
cyclisation was employed as the key-step followed by stereoselective ketone reduction to complete
the total syntheses. The scope of the cyclisation and reduction was examined with a range of enone
side-chains resulting in a small library of novel 4-hydroxy-2,6-disubstituted piperidines.
2

Table of Contents
Abstract ............................................................................................................................................... 1
Table of Contents ................................................................................................................................ 2
Acknowledgements ............................................................................................................................. 5
Author’s Declaration ........................................................................................................................... 6
Abbreviations ...................................................................................................................................... 7
1.0 Introduction ................................................................................................................................ 11
1.1 α-Amino Acids ......................................................................................................................... 11
1.2 Fluorescent α-Amino Acids ..................................................................................................... 12
1.3 Natural α-Amino Acids and Fluorescent Analogues ............................................................... 12
1.4 Coumarin-Derived Fluorescent α-Amino Acids....................................................................... 17
1.5 Xanthone and Acridone-Derived Fluorescent α-Amino Acids ................................................ 21
1.6 Flavone-Derived Fluorescent α-Amino Acids .......................................................................... 23
1.7 BODIPY-Derived Fluorescent α-Amino Acids .......................................................................... 25
1.8 Aminophthalimide- and Aminonaphthyl-Derived Fluorescent α-Amino Acids ...................... 29
1.9 Polyaromatic-Derived Fluorescent α-Amino Acids ................................................................. 35
1.10 Summary ............................................................................................................................... 41
2.0 Synthesis of Novel Fluorescent Heterocyclic-Derived α-Amino Acids ........................................ 43
2.1 Previous Work in the Sutherland Group ................................................................................. 43
2.2 Proposed Research ................................................................................................................. 44
2.3 Synthesis of Pyridine-Derived α-Amino Acids ......................................................................... 45
2.3.1 Synthesis of N-Trityl Protected Enone-Derived α-Amino Acids ....................................... 45
2.3.2 Synthesis of N-Cbz Protected Enone-Derived α-Amino Acids ......................................... 47
2.3.3 Synthesis of N-Boc Protected Enone-Derived α-Amino Acids ......................................... 48
2.3.4 Inverse Electron Demand Hetero-Diels-Alder (HDA ) Cycloaddition ............................ 50
INV
2.3.5 Synthesis of N-Cbz Protected Pyridine-Derived α-Amino Acids ...................................... 53
2.3.6 Deprotection to Form the Parent Pyridine-Derived α-Amino Acids ................................ 55
2.4 Optical Properties of Pyridine-Derived α-Amino Acids ........................................................... 56
2.4.1 Absorption and Emission Data for Neutral and Electron-Rich Pyridine Derivatives ........ 56
2.4.2 Absorption and Emission Data for Electron-Poor Pyridine Derivatives ........................... 58
2.4.3 Methoxyphenyl 151 and Naphthyl 152 Dilution Study .................................................... 60
2.4.4 Methoxyphenyl 151 pH Study .......................................................................................... 63
2.4.5 Other Optical Properties .................................................................................................. 65
2.4.6 Summary .......................................................................................................................... 66
2.5 Synthesis of Second Generation Pyridine-Derived α-Amino Acids ......................................... 67
3

2.5.1 Synthesis of N-Cbz Protected Enone-Derived α-Amino Acids ......................................... 67
2.5.2 Inverse Electron Demand Hetero-Diels-Alder (HDA ) Cycloaddition ............................ 68
INV
2.5.3 Synthesis of N-Cbz Protected Pyridine-Derived α-Amino Acids ...................................... 68
2.5.4 Aryl Nitro Reduction with Tin(II) Chloride Dihydrate ....................................................... 69
2.5.5 Deprotection to Form the Parent Pyridine-Derived α-Amino Acids ................................ 69
2.6 Optical Properties of Second Generation Pyridine-Derived α-Amino Acids ........................... 70
2.6.1 Absorption and Emission Data for Second Generation Pyridine Derivatives .................. 70
2.6.2 Other Optical Properties .................................................................................................. 73
2.6.3 Summary .......................................................................................................................... 74
2.7 Synthesis of Fmoc Protected Pyridine-Derived α-Amino Acids .............................................. 75
2.7.1 Solid Phase Peptide Synthesis .......................................................................................... 75
2.7.2 SPPS - Boc Protection Method ......................................................................................... 75
2.7.3 SPPS - Fmoc Protection Method ...................................................................................... 75
2.7.4 Synthesis of N-Fmoc Protected Pyridine-Derived α-Amino Acids ................................... 76
2.8 Synthesis of Novel Fluorescent Cell Penetrating Hexapeptide ............................................... 76
2.8.1 SPPS of Cell Penetrating Pentapeptide VPTLK ................................................................. 76
2.8.2 Synthesis of Hexapeptide 181 .......................................................................................... 79
2.9 Cell Study with Hexapeptide 181 ............................................................................................ 80
2.10 Synthesis of Pyrimidine-Derived α-Amino Acids................................................................... 85
2.10.1 Initial Synthesis of Pyrimidine-Derived α-Amino Acids.................................................. 85
2.10.2 Alternative Synthesis Towards Pyrimidine-Derived α-Amino Acids .............................. 87
2.11 Future Work .......................................................................................................................... 89
2.11.1 Proposed Synthesis of Pyrimidine-Derived α-Amino Acids ........................................... 89
2.11.2 Proposed Synthesis of β-Carboline-Derived α-Amino Acids .......................................... 90
2.11.3 Proposed Synthesis of Benzotriazole-Derived α-Amino Acids ....................................... 91
2.13 Conclusions ........................................................................................................................... 93
3.0 Total Syntheses of 4-Hydroxy-2,6-Disubstituted Piperidine Natural Products ........................... 94
3.1 Previous Work in the Sutherland Group ................................................................................. 94
3.2 Proposed Research ................................................................................................................. 95
3.3 Previous Synthesis of Spruce Alkaloid .................................................................................... 96
3.4 Previous Syntheses of (+)-241D .............................................................................................. 98
3.5 Towards the Total Synthesis of Spruce Alkaloid ................................................................... 100
3.5.1 First Approach Towards the Total Synthesis of Spruce Alkaloid .................................... 100
3.5.2 Revised Approach Towards the Total Synthesis of Spruce Alkaloid .............................. 104
3.6 Total Synthesis of Spruce Alkaloid and (+)-241D .................................................................. 106
3.6.1 Synthesis of Enones ....................................................................................................... 106
4

3.6.2 6-Endo-Trig Cyclisation of Enones 258 and 259 ............................................................. 108
3.6.3 Stereoselective Reduction ............................................................................................. 109
3.7 Expanding the Scope of 4-Hydroxy-2,6-Disubstituted Piperidine Synthesis ........................ 110
3.7.1 Synthesis of Enones ....................................................................................................... 110
3.7.2 Scope of 6-Endo-Trig Cyclisation .................................................................................... 111
3.7.3 Selective NOE Assignments ............................................................................................ 111
3.7.4 Stereoselective Reduction ............................................................................................. 112
3.7.5 Selective NOE Assignments ............................................................................................ 113
3.8 Stereoselective Acid Mediated 6-Endo-Trig Cyclisation ....................................................... 114
3.9 Future Work .......................................................................................................................... 117
3.10 Conclusions ......................................................................................................................... 118
4.0 Experimental ............................................................................................................................. 119
4.1 General Experimental ........................................................................................................... 119
4.2 Experimental Procedures ...................................................................................................... 119
4.3 Experimental for the Synthesis of Hexapeptide 181 ............................................................ 199
4.4 Cell-Hexapeptide 181 Study .................................................................................................. 201
5.0 References ................................................................................................................................ 202
6.0 Appendices ................................................................................................................................ 209
Appendix 1 - Genetically Encoded Proteinogenic α-Amino Acids .............................................. 209
Appendix 2 - Pentapeptide Test Cleavage Data .......................................................................... 210
Appendix 3 - Fluorescence Lamp Settings .................................................................................. 211
5

Acknowledgements
First off, I would like to thank my supervisor Dr Andy Sutherland for giving me the opportunity to
work in his group. Throughout my PhD, he has provided me with a limitless amount of knowledge
and expertise allowing me to get the most out of this experience. Whether it was practical work,
report writing or presentation preparation he always had constructive opinions on how to improve.
I would also like to thank my second supervisor Professor Richard Hartley for providing me with
invaluable advice regarding annual report feedback. Thanks to the EPSRC and Cancer Research UK
for funding my research.

A big thank you to all the staff working in stores (Ted, Bruce and Paul), the mass spectroscopy
service (Jim, Andy and Harry), the NMR service (Dr Adams) and the IT service (Stuart and Arlene)
for their support. Massive thanks to Dr Astrid Knuhtsen, Dr Andrew Jamieson, Carol-Anne Smith
and Dr Mathis Riehle. Without their help and expertise in the final months of my PhD I would not
have had the positive ending I had been striving for.

Of course, I must thank all of the Sutherland group members past and present for all the laughs and
making the experience so much more enjoyable. To Mark, Lynne, Ewen, Filip, Salaheddin, Nikki,
Tomas, Mohamed, Kerry, Tim, Réka, Marytn, Maria, and last but not least Team Big Boys (Lewis
AJ
and Jonny), cheers! Same goes for the other half of the Loudon lab, thanks to all the past and
present members of the Hartley group. A big thanks to the two ERASMUS students Ariyada
Souvanlasy and Laetitia Baud, and MSci student Christina Bell, all of whom assisted my projects.
Thanks to everyone who was part of the 14/15 Alchemist Committee, making it an enjoyable and
rewarding experience.

A massive thank you to my parents, Sally and Jim, for supporting me throughout my PhD, even
although they still have no idea what I have been up to. Thanks to my other family members who
have helped in one way or another during my PhD, my brother Andrew, Aunt Christine, cousin Peter
and my late gran. I also appreciated the time spent out of the lab playing for Westerlands AFC or
with my good friends Stevie and Richard as well as the other lads from back home when we had
the chance to get together.

Finally, thanks to my lovely girlfriend Kim for putting up with me over the years, coming home late
and constantly whining about my research not working. She has always been there for me and has
been massively supportive and understanding since I started my PhD, very much appreciated.

“Boys can do anything, when they put their mind to it.” - Sally Harkiss
6

Author’s Declaration
I declare that, except where explicit reference is made to the contribution of others, this thesis
represents the original work of Alexander H. Harkiss and has not been submitted for any other
degree at the University of Glasgow or any other institution. The work upon which is based was
carried out at the University of Glasgow in the Loudon laboratory under the supervision of Dr
Andrew Sutherland between October 2013 and May 2017. Aspects of the work described herein
have previously been published elsewhere as stated below.

L. Gilfillan, R. Artschwager, A. H. Harkiss, R. M. L. Liskamp and A. Sutherland, Synthesis of Pyrazole
Containing α-Amino Acids via a Highly Regioselective Condensation/Aza-Michael Reaction of β-Aryl
α,β-Unsaturated Ketones, Org. Biomol. Chem., 2015, 13, 4514–4523.

Signature

Printed name ALEXANDER HUGH HARKISS
7

Abbreviations
°C Degrees centigrade
9-BBN 9-Borabicyclo[3.3.1]nonane
Ac Acetyl
Anap 3-[(6-Acetyl-2-naphthalenyl)amino] alanine
Ar Aromatic
BADAN 6-Bromoacetyl-2-dimethylaminonaphthalene
BINAP 2,2′-Bis(diphenylphosphino)-1,1′-binaphthalene
Boc tert-Butyloxycarbonyl
BODIPY Boron-dipyrromethene
br Broad
c Concentration
CaM Calmodulin
Cbz Carboxybenzyl
CI Chemical ionisation
COSY Correlated spectroscopy
CPME Cyclopentyl methyl ether
CPP Cell penetrating peptide
d Doublet
DAPA Dimethylaminophthalimide
DAST Diethylaminosulfur trifluoride
DCC N,N'-Dicyclohexylcarbodiimide
DDQ 2,3-Dichloro-5,6-dicyano-1,4-benzoquinone
DEPT Distortionless enhancement polarisation transfer
DHFR Dihydrofolate reductase
DIAD Diisopropyl azodicarboxylate
DIBAL-H Diisobutylaluminium hydride
DIC N,N′-Diisopropylcarbodiimide
DIPEA Diisopropylethylamine
DMA Dimethylacetamide
DMAP 4-Dimethylaminopyridine
DMEM Dulbecco’s Modified Eagle’s Medium
DMF Dimethylformamide
DMSO Dimethyl sulfoxide
DNA Deoxyribonucleic acid
EI Electron impact
8

EPL Expressed protein ligation
ESI Electrospray ionisation
ESIPT Excited state intramolecular proton transfer
Et Ethyl
FAB Fast atom bombardment
FBS Fetal bovine serum
Fmoc Fluorenylmethyloxycarbonyl
FRET Förster resonance energy transfer
g Grams
GFP Green fluorescent protein
GMO Genetically modified organism
h Hour
HBTU (2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate)
HCTU O-(1H-6-Chlorobenzotriazole-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate
HDA Hetero-Diels-Alder
HDA Inverse electron demand hetero-Diels-Alder
INV
HeLa Human epithelial cells
HIV Human immunodeficiency virus
HOMO Highest occupied molecular orbital
HPLC High-performance liquid chromatography
HSQC Heteronuclear single quantum correlation spectroscopy
hTERT Human telomerase reverse transcriptase
HWE Horner-Wadsworth-Emmons
Hz Hertz
IC Half maximal inhibitory concentration
50
IR Infrared
J NMR spectra coupling constant
K Dissociation constant
d
KHMDS Potassium hexamethyldisilazane
KR Lysine and arginine rich peptide
LC-MS Liquid chromatography-mass spectroscopy
LiHMDS Lithium hexamethyldisilazane
LUMO Lowest unoccupied molecular orbital
m Multiplet
M Molar