1. i
The Application of Organocatalysis in
Preparing Important Antimicrobial
Biomolecules
A Thesis Presented for the Award of Master of Science
David Malone, BSc.(Hons.)
Department of Science
Institute of Technology Tallaght, Dublin
For Research Carried Out Under the Guidance of
Dr. Fintan Kelleher
July 2015
2. ii
DECLARATION
I hereby certify that the material, which I now submit for the assessment on the progress
of study leading to the award of Master of Science (Research), is entirely my own work
and has not been taken from the work of others save to the extent that such work has
been cited and acknowledged within the text of my own work. No portion of the work
contained in this thesis has been submitted in support of an application for another
degree or qualification to this or any other institution or university.
Signed: _______________________ Date: ____________
David Malone
I hereby certify that the unreferenced work described in this thesis and being submitted
for the award of a Master of Science (Research), is entirely the work of David Malone.
No portion of the work contained in this thesis has been submitted in support of an
application for another degree or qualification to this or any other institution or
university.
Signed: _______________________ Date: ____________
Dr. Fintan Kelleher
3. iii
ACKNOWLEDGEMENTS
Firstly, I would like to thank my supervisor Dr. Fintan Kelleher, who over the past two
years has provided not only expert knowledge and supervision but has also been a vital
source of motivation throughout my studies.
I would also like to express my thanks to Dr. Brian Murray for all the time he has given
up over the past years to impart his endless NMR knowledge. Thank you also to the
technical staff at ITTD, Ross Fitzgerald, John Jones, David Saville, Hugh Gallagher,
and Aine McParland, who have shown an enormous amount of patience to deal with my
million-and-one requests.
A big thanks has to go to all my fellow postgraduates, especially the old organic crowd,
Kim Manzor, Darren Crowe and John Moran, who have been ever present since I began
my Masters studies and were always a source of knowledge and entertainment. Thanks
also to Tony, Rachel, Aoife, Louise and the newbies Jessica, Gemma, Aga, and Jordan,
who have provided the endlessly strange, comical and entertaining lunchtime
conversations. A special mention has to go to Arundhuti, my desk buddy, who has been
forced to listen to me ramble on about everything and anything from over the partition
for the past two years.
I would like to thank all the lads: Barry, Mark, Daragh, Dee, Liam, Gavin, Morris,
Aido, Gearóid, Fintan, Diarmuid and Paul, who have never failed to provide much-
needed distraction on the weekends.
Finally, I would like to express my gratitude to those closest to me, my parents Declan
and Marie, brothers Emmet and Aidan, sister Susan and girlfriend Alison, who have
always provided encouragement, support and limitless patience throughout both my
undergraduate and postgraduate studies.
4. iv
ABSTRACT
The Application of Organocatalysis in Preparing Important
Antimicrobial Biomolecules
David Malone, BSc.(Hons)
Due to the emergence of a number of antibiotic resistant bacteria such as MRSA
(methicillin-resistant Staphylococcus Aureus), it is important that new antibiotics are
developed, and those that are already in use, such as the antibacterial peptide nisin, are
modified to become more stable and effective. An important residue involved in the
biological mode of action of nisin, which consists of a 34-amino-acid chain, is
lanthionine (two alanine residues cross-linked by a thioether). This thesis will outline
the details of the synthetic efforts used to stereoselectively synthesise (S,R)-lanthionine.
The methodologies detailed in Chapter One demonstrate the difficulties faced in the
stereoselective synthesis of SPPS-compatible lanthionine residues. It was found that the
most common methodologies used, which involve the sulfa-Michael addition of
protected cysteine residues to protected β-haloalanines, result in the synthesis of an
undesirable mixture of lanthionine diastereoisomers due to the dehydrohalogenation of
the β-haloalanines to form didehydroalanines (Dha).
The synthesis of various protected Dhas, a protected cysteine and the subsequent
optimisation of the sulfa-Michael addition are detailed in Chapters Two and Three. This
optimisation resulted in a 50:50 diastereoisomeric mixture of protected (S,R):(R,R)-
lanthionine in yields of up to 77%, which is a substantial increase in the poor yields of
17% obtained while following literature methods.
Chapter Four outlines the synthetic methods used to synthesise and utilise a cinchona
alkaloid based organocatalyst to control the stereochemistry of the lanthionine product
to yield the desired (S,R) diastereoisomer exclusively. While Chapter Five outlines
potential future options that could be utilised to optimise lanthionine synthesis.
Chapter Six details the full synthetic methodologies and characterisation data of all of
the compounds prepared.
5. v
ABBREVIATIONS
AA
A
Amino acid
AcOH Acetic acid
Ala Alanine
AMR Antimicrobial resistance
Ar Aromatic
AviCys Aminovinyl cysteine
Bu3P Tributylphosphine
o
C Degrees Celsius/ Centigrade
CBr4 Carbon tetrabromide
CDCl3 Deuterated chloroform
CHCl3 Chloroform
CH3CN Acetonitrile
cm Centimetre(s)
Cs2CO3 Cesium carbonate
DCM Dichloromethane
Dha Dehydroalanine/Didehydroalanine
Dhb Dehydrobutyrine/Didehyrdobutyrine
DIAD Di-iso-propyl azodicarboxylate
DMAc Dimethylacetamide
DMF N,N-Dimethylformamide
DPPA Diphenylphosphoryl azide
Et3N Triethylamine
6. vi
EtOAc Ethyl acetate
g Gram(s)
HCl Hydrochloric acid
H2O Water
HPLC High performance liquid chromatography
hr Hour(s)
MeAviCys Aminovinyl methyl-cysteine
MeOH Methanol
min Minute(s)
mmol Millimole(s)
mol Mole(s)
M.pt. Melting point
MRSA Methicillin-resistant Staphylococcus aureus
Na2CO3 Sodium carbonate
NaHCO3 Sodium hydrogen carbonate
NaOH Sodium hydroxide
nm Nanometer(s)
NMM N-Methylmorpholine
NOE Nuclear overhauser effect
PPh3 Triphenylphosphine
ppm Parts per million
r.t. Room temperature
SPPS Solid-phase peptide synthesis
7. vii
TFA Trifluoroacetic acid
THF Tetrahydrofuran
TLC Thin layer chromatography
Protecting Groups
Alloc Allyloxycarbonyl
Bn Benzyl
Boc t-Butoxycarbonyl
DNs Dinitrobenzenesulfonyl
Fmoc 9-Fluorenylmethyloxycarbonyl
MeO Methyl ester
Ms Methanesulfonyl
Trt Triphenylmethyl (trityl)
8. viii
Table of Contents
Cover Page i
Declaration ii
Acknowledgements iii
Abstract iv
Abbreviations v
Table of Contents viii
Chapter 1 Introduction
1.1 Introduction 2
1.2 Bacterial cells 3
1.3 Bacterial cell wall 3
1.3.1 Gram-positive bacterial cell walls 4
1.3.2 Gram-negative bacterial cell walls 4
1.3.3 The plasma membrane 5
1.3.4 Peptidoglycan 5
1.3.5 The lipid bilayer 6
1.3.5.1 Lipid II 8
1.4 Antibiotics 9
1.4.1 Modes of action of antibiotics 9
9. ix
1.4.2 Antibiotic resistance 12
1.5 Antibiotic classes 12
1.5.1 β-lactam 12
1.5.2 Aminogylcosides 13
1.5.3 Peptide antibiotics 13
1.6 Lantibiotics 14
1.6.1 Type A lantibiotics 15
1.6.2 Type B lantibiotics 16
1.7 Nisin 17
1.7.1 Nisin’s mechanism of action 18
1.7.1.1 Inhibition of cell wall synthesis 18
1.7.1.2 Pore formation 19
1.7.2 Chemical synthesis of nisin 19
1.8 Solid-phase peptide synthesis 20
1.9 Orthogonally protected lanthionines 22
1.10 Synthesis of protected lanthionines via the alkylation of
cysteines with β-haloalanines 24
1.10.1 Protected β-chloroalanine as the electrophile 24
1.10.2 Protected β-iodoalanine as the electrophile 26
1.10.3 Protected β-bromoalanine as the electrophile 27
1.10.4 Aziridine ring-opening 28
10. x
1.11 Synthesis of lanthionines via sulfa-Michael addition of
protected cysteines to protected dehydroalanines 29
1.11.1 Intramolecular Michael addition studies 31
1.12 Intermolecular Michael addition of gluthathione to Dha 32
1.13 Organocatalysis 33
1.14 Cinchona alkaloids 34
1.14.1 Cinchona alkaloids as Michael addition reaction catalysts 35
1.15 Project Aims 36
Chapter 2 Lanthionine Synthesis
2.0 Introduction 39
2.1 Synthesis of protected Dha residues 39
2.1.1 Synthesis of protected Dha 48 40
2.1.2 Synthesis of protected Dha 50 42
2.2 Synthesis of starting materials for lanthionine 57 43
2.2.1 Synthesis of protected Dha 55 44
2.2.2 Synthesis of protected cysteine 56 48
2.3 Synthesis of protected lanthionine 57 52
2.3.1 Optimisation of lanthionine 57 synthesis 53
2.3.2 Optimised lanthionine 57 synthesis 57
2.4 Chapter conclusions 64
11. xi
Chapter 3 Catalyst Synthesis
3.0 Introduction 67
3.1 Synthesis of cinchona alkaloid catalyst 46 67
3.2 Chapter conclusions 73
Chapter 4 Organocatalytic Michael Additions
4.1 Introduction 75
4.2 Attempted synthesis of lanthionine 57 using quinine as the catalyst 75
4.3 Attempted synthesis of lanthionine 57 using 46 as the catalyst 79
4.4 Chapter conclusions 81
Chapter 5 Future Work
5.0 Introduction 86
5.1 Optimisation of substrate 23 86
5.2 Optimisation of catalyst 46 87
Chapter 6 Experimental Procedures
6.0 General procedures 90
(R,R’)-Cystine bis-t-butyl ester (64). 91
14. 2
1.1 Introduction
An increase in antimicrobial resistance (AMR) has developed due to the over
prescription of broad-spectrum antibiotics, with over 50% of antibacterial prescriptions
in the US being deemed unnecessary.1
This has led to approximately 60% of
staphylococcal infections in US hospitals being caused by methicillin-resistant
Staphylococcus aureus (MRSA) alone, with 23,000 deaths deemed to be AMR
related.1,2
Therefore, it is important that new antibiotics are developed to combat the
emergence of the resistant bacterial strains, while those already in use are modified to
become more compatible as antibiotics for use in humans or animals.
With healthcare provision specifically to treat AMR in the EU totalling over €1.5
billion, coupled with the annual death rate of 25,000, an “Action Plan” has been
proposed to control the situation.3
While some of the “key actions” that have been laid
out include promoting awareness of AMR and improving hygiene standards across
healthcare facilities, the area of greatest interest to this report is the development of
novel antibiotics through research and innovation.
The identification and development of novel antibiotics is imperative for the treatment
of these resistant bacterial strains. Identifying antibiotics with a new and unique mode
of action has the greatest potential for the successful treatment of microbes possessing
AMR.4
One of the main areas being researched is the development of antibacterial
peptides that have proven toxicity towards pathogens. One such family of peptides of
interest are the lantibiotics.5
The exact lantibiotic in question, where this report will
focus, is nisin. Nisin, also known as additive E234, has been used for decades as a
tinned food preservative. It is of interest due to its activity at nanomolar concentrations,
its dual mechanism of action, as well as its apparent non-toxicity to humans. A key
feature in the 34-amino-acid chain of nisin is the bis-amino acid lanthionine, which is
involved in the formation of the ring structures that are vital for its biological
mechanism of action (see section 1.7.1), and also where lantibiotics get their name.6
15. 3
1.2 Bacterial cells
A bacterial cell is a unicellular prokaryotic microorganism. Each bacterial cell is
encased within a cell wall with varying degrees of protection from outer membranes or
capsules. The cell itself is comprised of a number of integral parts, namely; plasma
membrane, nucleoid, cytoplasm, ribosomes, flagella, pili, gas vacuole and periplasmic
space (Figure 1).
Figure 1: Bacterial cell (diagram by Campbell).7
1.3 Bacterial cell wall
The cell wall of a bacterium serves many and varying purposes, but it mainly gives
protection to the cell contents. The wall itself differs depending on the whether the
bacterium is Gram-positive or Gram-negative. In order for the bacterium to be
destroyed the cell wall must first be breached, requiring disparate approaches due to the
differences in cell wall structure between Gram-positive and Gram-negative bacteria.
16. 4
1.3.1 Gram-positive bacterial cell walls
The cell wall of a Gram-positive bacterium (Figure 2) is relatively thick at
approximately 30-100 nm. It is composed primarily of thick layers of peptidoglycans
which are cross-linked by the DD-transpeptidase enzyme which adds rigidity to the cell
wall. Large amounts of teichoic acids and lipoteichoic acids are also dispersed across
the cell wall adding further rigidity.
Figure 2: The Gram-positive bacterial cell wall (diagram by Esko et al.).8
1.3.2 Gram-negative bacterial cell walls
The cell wall of a Gram-negative bacterium (Figure 3) is comparatively more complex
than that of a Gram-positive cell wall, while also being comparatively thinner at
approximately 20-30 nm. The principle differences in the cell wall structure are that
while the Gram-negative cell walls have a far thinner layer of peptidoglycan, they
possess an additional layer known as the outer plasma membrane. This outer plasma
membrane is essentially a second lipid bilayer coupled with proteins and lipid
polysaccharides (LPS).
17. 5
Figure 3: The Gram-negative bacterial cell wall (diagram by Esko et al.).8
1.3.3 Plasma membrane
The plasma membrane is a ubiquitous structure in all prokaryotic and eukaryote cells.
The membrane is mainly composed of a phospholipid bilayer interspersed with various
proteins. The main function of this plasma membrane is to act as a semi-permeable
barrier, separating the inner cytoplasm of the cell from the outer cell and deciding what
is allowed to enter and exit the cell.9
It is this membrane barrier that is the innermost
defence against attack from antibiotics and other foreign substances.
1.3.4 Peptidoglycan
Peptidoglycan (Figure 4) is found in high abundance in Gram-positive bacteria, as
stated in section 1.3.1, and accounts for up to 50% of the entire mass of the bacterium. It
is also present in Gram-negative bacteria, as stated in section 1.3.2, but in much smaller
18. 6
quantities. The peptidoglycan polymer consists of β-(1-4)-linked N-acetylglucosamine
and N-acetylmuramic acid units. Its main functions for the cell include maintaining the
shape of the cell, counteracting the osmotic pressures from within the cell and also
serving as an anchor for the teichoic acid, proteins and other polysaccharides that
maintain the cells cohesion.10
Figure 4: N-acetylglucosamine (orange) and N-acetylmuramic acid (blue) residues
cross-linked via peptide chains (diagram by Esko et al.).8
1.3.5 Lipid bilayer
A highly porous second lipid bilayer is present in Gram-negative bacteria, again
composed of a double layer of phospholipids. Phospholipids (Figure 5) are long chains
of fatty acids attached to a glycerol backbone. They possess hydrophobic chains (tails)
that are attached to the glycerol backbone (hydrophilic head).
19. 7
Figure 5: Phospholipid structure.11
The phospholipids align tail to tail in two layers to form a lipid bilayer (Figure 6).
Figure 6: The phospholipid bilayer (diagram by Campbell).7
The hydrophilic phospholipid heads are orientated outwards towards the aqueous
environment, both inside and outside the cell, while the highly hydrophobic tails are
orientated inwards to form a passage that impedes water-soluble substances from
entering the cell.9
20. 8
1.3.5.1 Lipid II
Lipid II is a cell-wall precursor that is essential for the biosynthesis of bacterial cell
walls. It is found in very low abundances (less than 1mol% of membrane
phospholipids12
) as a membrane-anchored lipid. Lipid II is the target for multiple
classes of antibiotic, most notably the glycopeptide antibiotic vancomycin and the
lantibiotic nisin. The lipid II moiety consists of one N-acetylglucosamine (GluNAc),
one N-acetylmuramic acid (MurNAc), two pyrophosphate molecules, a pentapeptide
side-chain and an anchor consisting of a chain of 11 isoprene monomers (polyisoprene)
(Figure 7).13
Figure 7: Structure of Lipid II (diagram by Royet and Dziarski).13
21. 9
1.4 Antibiotics
Antibiotics can be defined as any synthetic, semi-synthetic or naturally occurring
substance whose purpose is to kill or prevent the growth of microorganisms through
varying modes of action.14
1.4.1 Modes of action of antibiotics
Antibiotics available today differ greatly from each other in terms of both their chemical
structure and their mode of action in destroying or inhibiting the growth of
microorganisms (Table 1).15
22. 10
Mechanism of Action Antibiotic
Inhibition of synthesis or damage to cell
wall
Penicillins
Cephalosporins
Monobactams
Carbapenems
Vancomycin
Inhibition of synthesis of, or damage to,
cytoplasmic membrane
Polymyxins
Polyene
Inhibition of synthesis of, or metabolism
of, nucleic acids
Quinolones
Rifampin
Nitrofurantoins
Nitroimidazoles
Inhibition of protein biosynthesis
Aminoglycosides
Tetracyclines
Chloramphenicol
Erythromycin
Modification of energy metabolism
Sulfonamides
Trimethoprim
Dapsone
Table 1: Modes of action of antibiotics and the antibiotic agents.16
23. 11
1.4.2 Antibiotic resistance
Antibiotic resistance is a mechanism developed by bacteria against a hostile antibiotic
entity to prevent the mechanism of action of the antibiotic from destroying or inhibiting
the growth of the bacterium. Resistance can occur due to a multitude of factors but is
most commonly acquired through four distinctive pathways (Figure 8):17,18,19
Enzymatic inactivation – the bacteria produces an enzyme that either chemically alters
the antibiotic’s binding site (e.g. streptomycin) or fully degrades the antibiotic itself
(e.g. penicillin via β-lactamases).
Efflux pumps – the bacteria has a reverse transport system that can selectively pump
the antibiotic out of the cell across the cell membrane (e.g. tetracycline).
Target site modification – the bacteria modifies the binding site of the antibiotic
through gene mutation or enzymatic interactions (e.g. vancomycin).
Modified cell permeability – the bacteria modifies its cell membranes making it less
likely to allow the passage of the antibiotic into the cell (e.g. tetracycline).
Figure 8: Pathways for bacterial antibiotic resistance (diagram by Abreu et al.).20
24. 12
1 2
3
1.5 Antibiotic classes
Due to the presence of different species of bacteria (Gram-positive and Gram-negative)
and the emergence of antibiotic resistance, there has been a need for the development of
different classes of antibiotic to counteract them.
1.5.1 The β-Lactams
β-Lactams are the largest and most widely used family of antibiotics currently on the
market. Some of the main antibiotics in this family are penicillins 1, cephalosporins 2
and carbapenems 3. They are used as broad-spectrum antibiotics with their mode of
action derived from the β-lactam ring present in their structures (Figure 9). Their mode
of action involves the inhibition of peptidoglycan synthesis causing the failure of the
bacterial cell wall. This class is highly susceptible to resistance via β-lactamase
production by the bacteria.21
Figure 9: General structure of penicillins 1, cephalosporins 2 and carbapenems 3,
with the β-lactam substructure highlighted in red.
25. 13
4
5
1.5.2 Aminoglycosides
Aminoglycosides are extremely potent broad-spectrum antibiotics. They act as protein
synthesis inhibitors, eventually causing cell death. This class is highly susceptible to
resistance via aminoglycoside-modifying enzymes.22
Some of the main antibiotics in
this family are streptomycin 4 and gentamicin 5 (Figure 10).
Figure 10: Structure of the aminoglycosides streptomycin 4 and gentamicin 5.
1.5.3 Peptide antibiotics
Antimicrobial peptides (AMPs) are naturally synthesised by a variety of strains of
bacteria. The main mode of action of peptide-based antibiotics usually involves pore
formation in the outer cell membrane or the inhibition cell wall synthesis.23
Bacteriocins, as they are known, synthesised by a bacterium are shown to be highly
potent weapons against competing bacterial species, being active in pico- and nano-
26. 14
HOOC
S
COOH
NH2 NH2
HOOC
S
COOH
NH2 NH2
HO
O
NH2
HO
O
NH2
6 7
8 9
molar concentrations.24
One such sub-category of peptide antibiotics that are at the
forefront of research into new therapies are lantibiotics.
1.6 Lantibiotics
Lantibiotics are polypeptide bacteriocins that are synthesised by Gram-positive bacteria
such as Lactococcus, Staphylococcus, Bacillus and Streptomyces species. They are
considered small peptides consisting of between 19-38 amino acids which undergo
considerable post-translational modifications, including the formation of thioether
bridges that form ring structures, vital for their biological mode of action.25
The ring
structures are formed via the unusual amino acids lanthionine 6 and β-methyl-
lanthionine 7 (Figure 11), from which lantibiotics get their name: lanthionine-
containing antibiotics. Lantibiotics are also known to contain other unusual amino acids
including didehydroalanine (Dha) 8 and didehydrobutyrine (Dhb) 9 (Figure 11).
Lantibiotics are categorised into two groups based on their structural features and mode
of action; Type A and Type B.
Figure 11: Structure of lanthionine 6, β-methyllanthionine 7, didehydroalanine
8 and didehydrobutyrine 9.
27. 15
1.6.1 Type A lantibiotics
Type A lantibiotics are flexible, amphipathic peptides synthesised by Gram-positive
bacteria. The mode of action of type A lantibiotics involves the formation of pores in
the bacterial membranes allowing the diffusion of small ions such as H+
, K+
and PO4
3+
through the membrane, resulting in cell death.26
Nisin is the most relevant and important
lantibiotic and will therefore be discussed in detail in section 1.7; however, discussed
below are several other important type A lantibiotics.
Subtilin
Subtilin is a 32-amino-acid lantibiotic produced by Bacillus subtilis that consists of one
lanthionine, four β-methyl lanthionines, two Dha and one Dhb residues (Figure 12).27
Subtilin is active towards a broad range of Gram-positive bacteria including
Streptococci and Staphylococci species.
Figure 12: Amino acid sequence of subtilin with the unusual amino acids
highlighted in colour.27
Epidermin
Epidermin is a 22-amino-acid lantibiotic produced by Staphylococcus epidermis that
consists of two lanthionines, one β-methyl lanthionine and one Dhb residue (Figure
13).27
Also present in the structure of epidermin is the highly unusual AviCys residue at
28. 16
the C-terminus. Epidermin is highly active towards other Gram-positive bacteria such as
Streptococci and Staphylococci species, most notably showing therapeutic potential
against MRSA.28
Figure 13: Amino acid sequence of epidermin with the unusual amino acids
highlighted in colour.27
1.6.2 Type B lantibiotics
Type B lantibiotics are rigid globular peptides synthesised by Gram-positive bacteria.
The mode of action involves the inhibition of peptidoglycan synthesis by forming
complexes with membrane-bound substrates, leading to cell death via lysis.29
Discussed
below are several important Type B lantibiotics.
Mersacidin
Mersacidin is a 20-amino-acid lantibiotic produced by a Bacillus sp. strain whose
structure consists of one Dha, three β-methyl lanthionine and one Dha residue (Figure
14).27
Also present in the structure of mersacidin is the highly unusual MeAviCys
residue at the C-terminus. Mersacidin is active towards a broad range of Gram-positive
bacteria, most notably the Staphylococcus and Strepococcus species.
29. 17
Figure 14: Amino acid sequence of mersacidin with the unusual amino acids
highlighted in colour.27
1.7 Nisin
Nisin is a 34-amino-acid lantibiotic produced by Lactococcus lactis that consists of one
lanthionine, four β-methyl lanthionine, two Dha and one Dhb residue (Figure 15).27
Nisin was discovered in 192730
but its structure was not determined until 1971 by Gross
and Morell.31
It is now known to contain five ring structures (A-E) formed by thioether
bridges via lanthionine and β-methyl lanthionine residues. Nisin has been used
extensively for several decades as a food preservative and can still be widely seen today
as the food additive E234. One advantage of nisin is its antimicrobial activity at
nanomolar concentrations.32
Interestingly, nisin does not contain any aromatic amino
acid residues and is cationic due to the three lysine residues present.
Figure 15: Amino acid sequence of Nisin with the unusual amino acids highlighted
in colour.27
30. 18
1.7.1 Nisin’s mechanism of action
Nisin possesses a dual mechanism of action against Gram-positive bacteria, cell wall
inhibition and pore formation, and it is thought that its nanomolar activity is due to this
dual mechanism.
1.7.1.1 Inhibition of cell wall synthesis
The inhibition of the bacterial cell wall comes from the binding of rings A and B of
nisin to the pyrophosphate moiety of lipid II through five hydrogen bonds as observed
by 1
H NMR spectroscopy.13
These five hydrogen bonds are provided by residues 2
(Dhb), 4 (Ile), 5 (Dha), 6 (Leu) and 8 (Abu) (Figure 16).
Figure 16: Hydrogen bonding (five yellow dashed lines) between nisin and the
pyrophosphate moiety of lipid II (diagram by Breukink and Kruijff).13
The intermolecular interaction between nisin and lipid II results in the inhibition of
peptidoglycan, which is vital for the stability of bacterial cell walls, causing cell death.
31. 19
1.7.1.2 Pore formation
The second mode of action of nisin takes place once eight nisin molecules have been
bound to the pyrophosphate layer. The nisin molecules bend at the “hinge region”
(residues 20 – 22) which forces rings D and E through the bilayer, forming a stable pore
from which the cytoplasmic material and ions flow through, resulting in cell death
(Figure 17). Studies have found that nisin in the presence of lipid II, that is available for
targeted pore formation, has a threefold increase in activity compared to nisin in the
absence of lipid II. They have also shown that the presence of high levels of lipid II
have resulted in an increase in the lifespan of the pores from milliseconds to
seconds.33,34
Figure 17: Pore formation by Nisin (diagram by Wiedemann et al.).33
1.7.2 Chemical synthesis of nisin
The total synthesis of nisin was first published in 1988 by Shiba et al.35
They utilised
methodology developed by Harpp and Gleason36
to synthesise the difficult lanthionine
and β-methyl lanthionine residues using a desulfurisation technique. This oxidation
reaction (Scheme 1) involves the addition of tris(diethylamino)phosphine to a disulfide
11 that reacts with one of the two bridging sulfurs, breaking the S-S bond. This reaction
produces a new S-P bond and also a thiolate anion 12. This thiolate anion acts as a
nucleophile, attacking intramolecularly at the CH2 adjacent to the S-P bond, producing
the lanthionine or β-methyl lanthionine product 13 (Scheme 1).37
32. 20
Gly Ala Leu Met Gly CysCysBoc OBn
Gly Ala Leu Met Gly CysCysBoc OBn
Gly Ala Leu Met Gly CysCysBoc OBn
Gly Ala Leu Met Gly AlaAlaBoc OBn
(i) I 2, MeOH; (ii) P(Et 2NH) 3
(i)
(ii)
STrt AcmS
S S
S P(Et2N)3 S
S
Acm = -CH2-NH-CO-CH3
10
11
12
13
Scheme 1: Desulfurisation of cystine 11 to form a lanthionine 13.38
1.8 Solid-phase peptide synthesis
Solid-phase peptide synthesis (SPPS) was developed by Merrifield39
in 1963 as a
method to chemically synthesise peptides. The methodology behind SPPS requires the
33. 21
attachment of a peptide to a solid support known as a resin, via a linker, at the C-
terminus. Once attached the sequential addition of amino acids can be performed to
synthesise a linear polypeptide chain.40
The most common form of SPPS used today is
Fmoc-SPPS (Figure 18), which uses the 9-fluorenylmethyloxycarbonyl (Fmoc) moiety
as the N-terminal protecting group. To carry out the additions the Fmoc group is
removed using a piperidine solution (Step 1) and the next N-terminal-protected amino
acid is added (Step 2), extending the chain. The advantage of using a solid resin as an
anchor is that it allows the washing and filtration of the peptide chain after each addition
without the requirement for the peptide chain to be in solution, which ultimately results
in the loss of product. Upon completion, the desired chain of the polypeptide can be
cleaved from the resin using appropriate conditions (Step 3), depending on the linker,
which in the majority of Fmoc-SPPS reactions is treatment with trifluoroacetic acid
(TFA).41
34. 22
Figure 18: Fmoc-SPPS scheme.42
1. 9 Orthogonally protected lanthionines
The synthesis of the rings of nisin has also been achieved through the application of
solid-phase peptide synthesis (SPPS) using orthogonally protected lanthionines (Figure
19) or β-methyl lanthionine.
Step 1
Deprotection
Activation
Step 2
Coupling
Deprotection
Step 3
Deprotection + Cleavage
35. 23
Figure 19: Orthogonally protected lanthionine and removal conditions of selected
protecting groups.
An orthogonally protected lanthionine is one where each of the two amino and carboxyl
termini are protected using different protecting groups, each with independent removal
conditions. This enables the selective removal of any of the protecting groups while
maintaining the stability of the others (Figure 20).43
Factors such as pH, temperature
and the reagents used must also be carefully considered to ensure a fully selective
removal of any one of the protecting groups.
36. 24
Figure 20: The selective removal of protecting groups A, B and C from functional
groups X, Y and Z using different reaction conditions a-c (diagram by
Schelhaas and Waldmann).43
1.10 Synthesis of protected lanthionines via the alkylation of cysteines with β-
haloalanines
To date, a number of methods have been utilised in the synthesis of orthogonally
protected lanthionines, however, the main method investigated is the alkylation reaction
of protected cysteines with protected β-haloalanines. The use of β-chloroalanine, β-
iodoalanine, and β-bromoalanine as an electrophile has been thoroughly investigated
with varying degrees of success.
1.10.1 Protected β-chloroalanine as the electrophile
The use of a β-chloroalanine as the electrophilic partner was first investigated by du
Vigneaud and Brown in 1940 (Scheme 2).44,45
37. 25
MeOOC
Cl
NHTrt
MeOOC
NHTrt
HS
COOMe
NHFmoc
MeOOC
S
COOMe
NHTrt NHFmoc
KOH
Scheme 2: Synthesis of lanthionine 17 from β-chloroalanine 14 and cysteine 16.
Their lanthionine synthesis involved the reaction of, for example, the protected cysteine
16 with protected β-chloroalanine 14. The reaction was undertaken in the presence of
aqueous KOH producing highly basic conditions. This basic environment caused the
rapid dehydrohalogenation of 14, resulting in the formation of the Dha intermediate 15.
The subsequent 1,4-Michael addition of the thiolate anion of 16 to Dha 15 resulted in a
diastereoisomeric mixture of the protected lanthionine 17. In the mechanism of the
sulfa-Michael addition reaction, the stereoselective reprotonation step showed no facial
preference on enolate 18 due to a lack of chiral control, producing a 50:50 mixture of
diastereoisomers (Scheme 3).
1415
16 17
38. 26
S
COOMe
NHTrt NHFmoc
O
O
S
COOMe
NHTrt NHFmoc
MeOOC
S
COOMe
NHTrt NHFmoc
MeOOC
H
H
and
Scheme 3: Reprotonation of lanthionine enolate 18 resulting in
diastereoisomers of 17.
1.10.2 Protected β-iodoalanine as the electrophile
The use of protected β-iodoalanines in lanthionine synthesis has also been extensively
investigated. Previous research by Dugave and Menez46
, and also by Goodman et al.47
,
showed that the reaction of N-trityl-protected β-iodoalanines with protected cysteines in
the presence of Cs2CO3 resulted in the synthesis of a protected lanthionine in high-yield
and high stereoselectivity. A doubling of the peaks reported in the 1
H NMR spectrum
was attributed to the presence of rotamers and not diastereoisomers. However, more
recent investigations by Tabor48
have shown that a rearrangement reaction occurs
during the formation of β-iodoalanine 23 from the mesylate 20 (Scheme 4). The
rearrangement occurs via an aziridine intermediate 21 due to the competitive attack at
the α-position by the iodide anion, facilitated by the adjacent electron-withdrawing ester
group. This rearrangement results in the formation of unwanted α-iodo-β-alanine 22b as
the major product and the desired β-iodoalanine 23 as the minor product via attack at
the less hindered β-position of aziridine 21. 22b, in the presence the iodide anion, can
also undergo racemisation through an SN2 reaction at the chiral centre to form the
enantiomeric α-iodo-β-alanine 22a. When this mixture of 22a, 22b and 23 was carried
forward the resulting lanthionine consists of an inseparable mixture of the
diastereoisomeric norlanthionine 25 and the desired lanthionine 26 (Scheme 4).
18
17
17
40. 28
BnOOC
OH
NHBoc
BnOOC
Br
NHBoc
HS
COOAllyl
NHFmoc
S
COOAllyl
NHFmoc
BnOOC
NHBoc
(i)
(ii)
(i) CBr4, PPh3, DCM; (ii) TBAHS, NaHCO3, EtOAc, H2O
subsequent reaction with the protected cysteine 29 under mildly basic conditions
(NaHCO3). Under these mildly basic conditions β-bromoalanine 28 did not undergo
dehydrohalogenation to the same extent as the β-chloroalanine and β-iodoalanine
methodologies. It also did not result in the formation of any aziridine intermediates
which could result in the formation of regioisomers, allowing the reaction to produce
the desired lanthionine diastereomer 30 as the major product. This methodology was
further enhanced by the introduction of the phase transfer catalyst tetrabutylammonium
hydrogensulfate (TBAHS) in addition to the sodium hydrogen carbonate (Scheme 5).
Scheme 5: Synthesis of orthogonally protected lanthionine 30 from protected β-
bromoalanine 28 and protected cysteine 29.38
1.10.4 Aziridine ring-opening
The synthesis of protected lanthionines is also possible through the ring opening of
protected aziridine 35 with N-protected cysteine 36 (Scheme 6). Following
methodology reported by Vederas et al.50
the protected aziridine is prepared by the
cyclisation of N-trityl protected serine 32 via mesylate intermediate 33, under basic
conditions to form protected aziridine 34. The trityl protecting group on the nitrogen of
34 was substituted by the 2,4-dinitrobenzenesulfonyl (DNs) group under basic
conditions, to form aziridine 35. 35 was then reacted with Fmoc-protected cysteine 36
27
28 29 30
41. 29
HO OH
O
NHTrt
AllylO OH
O
NHTrt
AllylO OMs
O
NHTrt
AllylO
O
N
Trt
AllylO
O
N
DNs
HS
COOH
NHFmoc
AllylO S
COOH
O
NHDNs NHFmoc
(i) (ii), (iii)
(iv)
(v)
(i) Allyl chloride, NaHCO3; (ii) methanesulfonyl chloride, Et3N, 0 o
C; (iii) 3:3:1 TFA:DCM:MeOH, 0 o
C;
(iv) DNs-Cl, Na2CO3; (v) BF3.OEt2, 0 o
C.
in the presence of the Lewis acid BF3.OEt2. This method yields the diastereomerically
pure protected lanthionine 37, though in a poor yield of only 40%.
Scheme 6: Synthesis of lanthionine 37 by reaction of protected aziridine 35 with
protected cysteine 36.50
1.11 Synthesis of lanthionines via sulfa-Michael addition of protected cysteines to
protected dehydroalanines
The synthesis and use of chiral Dha molecules in the formation of lanthionines has
been investigated by Avenoza et al.51
and Zurbano et al.52
They have concluded that
the stereoselective synthesis of lanthionines is possible using a Dha protected with a
chiral substituted oxazolidinone, without the use of a chiral catalyst (Scheme 7).
31 32 33
343536
37
42. 30
HS
NHBoc
CO2Me
O
N
S
NHBoc
O
MeO
OH
CO2Me CO2Me
O
N
S
NHBoc
O
MeO
OH
CO2Me CO2Me
HS
NHBoc
CO2Me
O
N
O
MeO
OH
CO2Me
H2N
S
NH2
HO2C HO2C
(i)
(ii)
(ii)
(i)
(i) DBU, THF, -78 o
C; (ii) 6 M HCl, reflux temp.
Scheme 7: Zurbano’s lanthionine synthesis using chiral Dha 38.52
The synthesis was carried out through one of two reaction pathways which yielded the
same lanthionine product 43. The first reaction proceeded through the reaction of
protected L-cysteine 39 with Dha 38, which was protected with a substituted
oxazolidinone, to yield protected lanthionine 40 as a single diastereoisomer. The second
reaction proceeded in a similar fashion but this time Dha 38 was reacted with protected
D-cysteine 41, the enantiomer of 39, to yield protected lanthionine 42. The final step of
both reactions was the removal of the protecting groups by treating 40 or 42 with 6 M
HCl, which hydrolysed them to a single unprotected lanthionine diastereomer 43.
38
39
40
41
42
43
43. 31
H2N
N
H
N
O
O
H
N
N
H
H
N
O
O
O
N
H
O
O
HS
NH2
H2N
N
H
N
O
O
H
N
N
H
H
N
O
O
O
N
H
O
O
NH2
S
(i)
(i) H2O, pH 8.6
Although excellent yields and diastereomeric ratios were achieved through this
methodology, the resulting lanthionine was not suitable for peptide synthesis. This is
due to the lack of SPPS-compatible protecting groups on the amino and carboxylic acid
termini and also because the resulting (S,S) lanthionine 43 produced has the opposite
stereochemistry to that which is required.
1.11.1 Intramolecular Michael addition studies
The synthesis of lanthionines via the intramolecular Michael addition of cysteine
residues to a Dha in a polypeptide chain has also been studied. A report by Bradley et
al.53
details the synthetic method used (Scheme 8). The polypeptide 44, under mildly
basic conditions, was found to rapidly cyclise to form a single diastereomer of the
meso-lanthionine resulting in the formation of the thioether-bridged polypeptide 45 in
65% yield. It is probable that the incorporation of the L-proline group has a strong effect
on the conformational selectivity of the reaction.
Scheme 8: Intramolecular Michael addition of Dha and cysteine residues in a
polypeptide.53
44
45
44. 32
GSH
Step 1
Dha analog of GSH
Step 2
GSG
HOOC
NH2
N
H
O
SH
H
N
O
COOH
HOOC
NH2
N
H
O
H
N
O
COOH
HOOC
NH2
N
H
O
H
N
O
COOH
HOOC
NH2
H
N
O
N
H
O
COOH
S
H
1.12 Intermolecular Michael addition of glutathione to a Dha
A study by Younis et al.54
has investigated the formation of a thioether-bridged
molecule (GSG) synthesised via a sulfa-Michael addition of glutathione (GSH) with a
Dha analog of GSH (Figure 21).
Figure 21: Synthesis of GSG from the sulfa-Michael addition of two glutathione
moieties.54
45. 33
The methodology put forward follows a two-step reaction. Step 1 involves the
elimination of hydrogen sulfide from GSH to form a Dha analog of GSH. One method
used to accomplish this involves the use of an extremely strong base capable of the
deprotonation of the α-proton of the cysteine sub-unit in GSH, which is predicted to
have a pKa of above 21.55
A second method to produce the Dha analog of GSH,
investigated by Asquith and Carthew56
, was the use of an excellent leaving group in the
β-position of the cysteine sub-unit. The leaving group chosen to replace the natural thiol
of GSH was the dinitrophenyl group. Step two of the methodology involved the sulfa-
Michael addition of a molecule of GSH, acting as the Michael donor, with the Dha
analog of GSH, acting as the Michael acceptor. The resulting thioether-bridged
molecule GSG was synthesised in an unreported ratio of diastereoisomers.
1.13 Organocatalysis
Organocatalysis describes the use of typically substoichiometric quantities of organic
molecules to increase the rate of a chemical reaction.57
Organocatalysts are generally
categorised into five main subsections depending on their catalytic mode of action
(Table 2).58
Mode of catalysis Activation method
(i) Enamine catalysis HOMO activation
(ii) Iminium catalysis
LUMO activation(iii) Hydrogen bonding catalysis
(iv) Counter-ion catalysis
(v) Singly occupied molecular orbital catalysis SOMO activation
Table 2: Main organocatalytic modes and their activation method.
However, it is common for organocatalysts to contain more than one activating group
that can contribute to the overall catalyst effect of the molecule; these are known as
bifunctional catalysts. For example, a urea catalyst that also contains an amine can be
considered to be both a hydrogen-bonding catalyst through the urea but also a Brønsted
base catalyst through the amine.59
46. 34
N
S
N
H H
R2
R1
S
O
R4
N
O
H
R3
O O
N N
H
R6
H
R5
thiourea sulfonamide squaramide
N
N
R'
R
X
X = OH, NH2, thiourea, sulfonamide, squaramide.
R = OH, OMe, thiourea.
R' = C2H2, C2H3.
C6'
C9
1.14 Cinchona Alkaloids
Cinchona alkaloids (Figure 22) are a well-known and well-studied group of
organocatalysts that have been used extensively in promoting stereoselective conjugate
additions and intramolecular Michael addition reactions.60,61
Figure 22: Cinchona alkaloid structures.62
Up until the 1990s cinchona alkaloids had been mainly used as phase-transfer catalysts.
However, since then, numerous synthetic cinchona alkaloids have been developed by
functionalising the C9 or C6’ positions. The functionalisation using a sulfonamide,
thiourea, or squaramide moiety (Figure 23) allows the cinchona alkaloid to act as a
bifunctional organocatalyst.
Figure 23: Thiourea, sulfonamide and squaramide functional groups.63
47. 35
R
PG
S
N
NN
S
CF3
F3C
N
OMe
PG
PG
O
H H
H
S R
N
NN
S
CF3
F3C
N
OMe
PG
O
H H
H
S
R
PG
PG
O
1.14.1 Cinchona alkaloids as Michael addition reaction catalysts
When used to catalyse a Michael addition reaction the bifunctional catalyst activates the
Michael acceptor via hydrogen-bonding of the functional group on C9 or C6’ with the
carbonyl group of the Michael acceptor. Concurrently the Michael donor is activated via
interactions with the quinuclidine tertiary amine of the cinchona alkaloid (Step 1).63,64
Once the deprotonated Michael donor has undergone the addition to the activated
acceptor the hydrogen on the newly formed quaternary ammonium cation of the
cinchona alkaloid is available for the facially controlling the reprotonation step (Step 2)
(Figure 24).
Figure 24: Proposed mechanism of the stereoselective conjugate addition reaction
catalysed by bifunctional cinchona alkaloid catalysts.63
A number of factors can influence the interactions between the hydrogen-bond donor
functional group and the electrophile. Two of the most significant, as outlined by
Ingemann and Hiemstra,63
are the acidity of the hydrogen on the nitrogen adjacent to the
R group and the distance between the two hydrogens of the thiourea. The study shows,
Step 1
Step 2
48. 36
N
NN
R
S
N
OMe
H H
R = 4-Me-C6H4
R = Ph
R = 4-Cl-C6H4
R = 3,5-(CF3)2-C6H3
R = t
Bu
pKa = 16.2
pKa = 15.8
pKa = 15.2
pKa = 13.2
pKa = 19.5
that the presence of highly electron-withdrawing groups at the R-position, such as 3,5-
(CF3)2-C6H3, result in lower pKa values (higher acidities) compared to the presence of
highly electron-donating groups, such as t
Bu, which result in higher pKa values (lower
acidities) (Figure 25). Investigations by Cheng et al.65
and Deng et al.66
have also
shown a correlation between the strength of the hydrogen-bonding (i.e. the acidity of the
hydrogens) and the enantioselectivity of the reaction, with the catalysts capable of
stronger hydrogen bonding resulting in better selectivity.
Figure 25: pKa values of the cinchona alkaloid thiourea protons depending on the
R group.63
1.15 Project aims
The main objective of this project was to develop novel methodology for the
stereoselective synthesis of protected lanthionines and β-methyl lanthionines that could
subsequently be used in SPPS for the preparation of lantibiotics. As stated previously in
section 1.10 the current methodology used in lanthionine and β-methyl lanthionine
synthesis has significant problems due to the dehydrohalogenation of the β-haloalanines
to Dhas which form undesired diastereomeric products. It is the aim of this project to
eliminate the need to use the unstable β-haloalanines by using an organocatalyst to
control the stereoselectivity of the Michael addition reaction used in the formation of
lanthionines. The use of a bifunctional cinchona-alkaloid-based thiourea organocatalyst
(Figure 26) would allow the far more stable protected Dhas to be used in place of the β-
haloalanines as the starting material (Scheme 9).
49. 37
N
N
H
N
H
S
CF3
F3C
N
OMe
C6'
C9
O
PG
NH
PG
O
O
PG
NH
PG
O
HS O
PG
HN
PG
O
S O
PG
HN
PG
O
(i)
(i) cinchona alkaloid catalyst, solvent.
Figure 26: Structure of initial cinchona-alkaloid-based thiourea catalyst 46.
Scheme 9: Proposed stereoselective synthesis of lanthionines using protected Dhas
and protected cysteines using a bifunctional catalyst to control the
stereochemistry.
To investigate the effect of the chosen catalyst on the lanthionine-forming Michael
addition reaction, it was intended to first carry out a reaction without a catalyst with
identical starting materials to determine the uncatalysed diastereomeric ratio of the
products. This would give a baseline level from which to compare all catalysed
reactions and determine the success or failure of any catalyst tested.
46
51. 39
HO
HN
O
O
O
O
HN
O
O
O
O
HO
HN
O
O
O
O
HN
O
O
O
O
dehydration
dehydration
2.0 Introduction
This chapter outlines the synthetic methods used to synthesise a number of protected
Dha residues in order to investigate their inherent stability once isolated, which will
ultimately determine their potential use in lanthionine synthesis. It also covers, in detail,
the methodology used in the synthesis of an orthogonally protected lanthionine without
control of the stereochemistry. As discussed in section 1.15 an orthogonally protected
lanthionine must be synthesised without stereocontrol to determine the baseline ratio of
the (S,R) to (R,R) diastereoisomers produced.
2.1 Synthesis of protected Dha residues.
Before the synthesis of a protected Dha suitable for use in SPPS was carried out, a
selection of other protected serine residues 47 and 49 were first synthesised and
subsequently reacted to form their corresponding protected Dha residues 48 and 50
(Figure 27). Once isolated, they were analysed for their stability and ultimately
examined for the potential application of the Dhas in the synthesis of protected
lanthionines.
Figure 27: Target molecules: protected serines 47 and 49 and their respective
protected Dha residues 48 and 50.
47 48
49 50
52. 40
HO
NH2
O
O
HO
HN
O
O
O
O
HN
O
O
O
O
(i)
(ii)
MsO
HN
O
O
O
O
(iii)
(i) Boc2O, Et3N, DCM; (ii) methanesulfonyl chloride, Et3N, DCM, 0 o
C; (iii) Et3N, DCM.
2.1.1 Synthesis of protected Dha 48
The synthesis of protected serine 47 was carried out via methodology reported by
Sacramento et al.67
and was subsequently converted to Dha 48. (Scheme 10)
Scheme 10: Synthesis of protected Dha 48.
The first step in the synthesis of protected Dha 48 involved the protection of the N-
terminus of the salt of serine 51 with the Boc protecting group. Serine 51 was purchased
with the C-terminus protected by a methyl ester group. This reaction was carried out in
DCM using two mole equivalents of triethylamine at 0 o
C to yield the protected serine
47 in an excellent yield of 82%. Confirmation of the formation of protected serine 47
was found through the comparison of the 1
H and 13
C NMR spectra of 47 with those
from the literature67
(Tables 3 and 4).
51
47
5248
α
α
αα
β
β
ββ
53. 41
α-proton
(ppm)
β-protons
(ppm)
Boc-protons
(ppm)
Serine 47 4.35 4.01 – 3.80 1.45
Literature67
4.39 3.98 – 3.88 1.46
Table 3: Comparison of 1
H NMR chemical shifts of protected serine 47 with
literature values.
α-carbon
(ppm)
β-carbon
(ppm)
Boc-CH3
(ppm)
Boc-Cq
(ppm)
Serine 47 55.6 62.7 28.1 80.0
Literature67
55.7 63.4 28.2 80.3
Table 4: Comparison of 13
C NMR chemical shifts of protected serine 47 with
literature values.
The protected serine 47 then proceeded through a two-step reaction to yield protected
Dha 48. The first step involved the conversion of the hydroxyl group of 47 to a mesylate
to form 52. This was achieved using methanesulfonyl chloride to add the mesylate
group at 0 o
C, and triethylamine as the base. The mesylate intermediate 51 was carried
onto the next step without isolation or purification to form Dha 48 in high yield (76 %).
The formation of 48 could be clearly seen through the comparison of its 1
H NMR
spectrum with that of 47. A downfield shift can be seen in the two β-hydrogens as well
as the disappearance of the α-hydrogen signal (Table 5), which is characteristic of the
formation of Dhas from serines.
β-hydrogens
(ppm)
α-hydrogen
(ppm)
47 4.01 – 3.80 4.35
48 6.16 and 5.71 -
Table 5: 1
H NMR signal comparisons between 47 and 48.
54. 42
HO O
O
NH2
HO O
O
HN
O
O
MsO O
O
HN
O
O
O
O
HN
O
O
(i)
(ii)
(iii)
(i) Alloc-Cl, Na2CO3, CH3CN:H2O (1:2); (ii) methanesulfonyl chloride, Et3N, DCM, 0 o
C;
(iii) Et3N, DCM.
It was found that, once isolated, if Dha 48 was left as a concentrated oil that it appears
to polymerise to form an insoluble, uncharacterisable gel which was unusable in any
further reactions. It was therefore always stored as a solution in approximately 1.0 g/10
ml DCM, which prevented any degradation from occurring.
2.1.2 Synthesis of protected Dha 50
The synthesis of Dha 50 was undertaken due to the alloc protecting group on the N-
terminus allowing a closer comparison with an SPPS-compatible Dha, which will also
contain an alloc group on the N-terminus. The synthesis of Dha 50 was carried out via a
one-pot synthesis from L-serine 53 (Scheme 11).
Scheme 11: Synthesis of protected Dha 50.
50
49
53
54
β
β
β β
αα
α
α
55. 43
The first step in the synthesis of 50 involved the protection of the N-terminus of the
protected serine 53 with an alloc protecting group using triethylamine as the base in
DCM to form protected serine 49. To confirm that the reaction had gone to completion
a minute sample was analysed via LC-MS. A mass of 204.0871 m/z was detected for
the [M+H]+
species, compared to a calculated mass of 204.0872 m/z, confirming the
formation of protected serine 49. Serine 49 was converted to its corresponding protected
Dha 50 through a mesylate intermediate 54. This was carried out using methanesulfonyl
chloride to form 54, which was subsequently eliminated using triethylamine to yield
Dha 50 in 70% yield. The successful formation of Dha 50 was determined through
analysis by LC-MS with a mass of 186.0764 m/z detected for the [M+H]+
species,
compared to a calculated mass of 186.0766 m/z.
As with Dha 48, it is found that Dha 50 appears to polymerise to form an insoluble,
uncharacterisable gel which was unusable in any further reactions. It was therefore also
always stored as a solution in approximately 1.0 g/10 ml DCM which prevented any
degradation from occurring.
The synthesis of both Dha 48 and 50 highlighted that once careful control of the storage
conditions is maintained that Dhas can be both synthesised in large quantities and stored
for an extended period of time without risk of degradation, making them ideal
candidates as lanthionine starting materials.
2.2 Synthesis of starting materials for lanthionine 57
The synthesis of orthogonally protected lanthionine 57 could be achieved by the
Michael addition of protected Dha 55 as the electrophile and protected cysteine 56 as
the nucleophile, as shown in the retrosynthetic scheme (Figure 28). Dha 55 is used as
the protecting groups alloc and allyl provide a reaction site with no facial preference at
the Michael addition reaction site and the subsequent stereocontrolling reprotonation
step as shown previously (Scheme 3). Also the use of cysteine 56 is important as the
Fmoc protecting group results in lanthionine 57 being suitable for use in SPPS. The
alloc and allyl protecting groups are also very commonly used as SPPS-compatible
protecting groups in the synthesis of protected lanthionines.48
56. 44
S
COOt
BuAllylOOC
NHAlloc NHFmoc
NHAlloc
AllylOOC
HS
COOt
Bu
NHFmoc
Figure 28: Retrosynthesis of lanthionine 57 from Dha 55 and cysteine 56.
2.2.1 Synthesis of protected Dha 55
The synthesis of 55 began with the protection of L-serine with the alloc protecting group
on the N-terminus to form 59, followed by the placement of the allyl ester protecting
group on the C-terminus to form 60 (Scheme 12).68,69
The methodology that was
followed outlines the synthesis of a protected serine that retained the (R)-
stereochemistry at the α-position, so therefore called for the use of D-serine. However,
due to the destruction of the stereochemistry in the formation of 55 from 60 the use of
L-serine was decided upon due to its far lower cost. The successful synthesis of 60 was
confirmed through analysis of the 1
H NMR spectrum with the presence of multiplets at
5.83 ppm and 5.21 ppm (allyl and alloc vinyl protons), a doublet at 4.59 ppm (allyl CH2,
J = 5.5 Hz), and a doublet at 4.50 ppm (alloc CH2, J = 5.5 Hz), all indicating successful
addition of the allyl and alloc groups. Definitive confirmation was obtained through LC-
MS where a mass of 252.0841 m/z was detected for the [M+Na]+
species, compared to a
calculated value of 252.0848 m/z.
55 56
57
57. 45
HO
COOH
NH2
HO
COOH
NHAlloc
HO
COOAllyl
NHAlloc
MsO
COOAllyl
NHAlloc
COOAllyl
NHAlloc
(i) (ii)
(iii)
(iv)
(i) Alloc-Cl, Na2CO3, CH3CN:H2O (1:2); (ii) Allyl-Br, NaHCO3, DMF; (iii) methanesulfonyl chloride,
Et3N, DCM, 0 o
C; (iv) Et3N, DCM.
Scheme 12: Synthesis of protected Dha 55.
As with the synthesis of the previous Dha residues, the protected serine 60 then
proceeded through a two-step reaction to yield protected Dha 55 in 88% yield. The first
step involved the conversion of the hydroxyl group of 60 to a mesylate to form 61. The
mesylate intermediate 61 was carried onto the next step without isolation or purification
to form 55 in high yield (88%). The resulting brown oil was immediately purified via
flash column chromatography to yield a clear colourless oil. The oil was subsequently
stored in 10 ml of DCM per gram of oil to prevent degradation as discussed in section
2.1. The formation of 55 from 60 could be clearly seen through the comparison of their
1
H NMR spectra (Figure 29). A large downfield shift can be seen in the two β-
hydrogens as well as the disappearance of both the α-hydrogen signal and the hydroxyl
proton signal (Table 6), all of which would be expected upon the formation of Dha 55.
55
58 59 60
61
β β β
ββ
α α α
α α
58. 46
β-hydrogens
(ppm)
α-hydrogen
(ppm)
hydroxyl hydrogen
(ppm)
60 3.92 and 3.81 4.36 3.47
55 6.25 and 5.83 - -
Table 6: 1
H NMR signal comparisons between serine 60 and Dha 55.
The synthesis of 55 could also be confirmed through a comparison of the IR spectra.
The OH/NH stretching frequency appearing at 3431 cm-1
in 60 appears in significantly
reduced intensity in the IR spectrum of 55, indicating the hydroxyl group has indeed
been removed. Again the conclusive evidence of the formation of 55 was through LC-
MS, with a mass of 234.0735 m/z detected for the [M+Na]+
species, compared to a
calculated mass of 234.0742 m/z.
60. 48
NHAlloc
AllylOOC
NHAlloc
AllylOOC
OH
NHAlloc
AllylOOC
Br
(i)
(i) CBr4, PPh3, 0o
C, DCM; (ii) Et3N, DCM.
(ii)
X
The formation of 55 from 60 was also attempted through a one-pot reaction using β-
bromoalanine intermediate 62 in place of the mesylate intermediate (Scheme 13). It was
carried out using carbon tetrabromide in the presence of triphenylphosphine at 0 o
C to
form β-bromoalanine intermediate 62. An equivalent of triethylamine was added after 4
hours to eliminate HBr and form Dha 55. However, upon work-up of the mixture it was
found by 1
H NMR spectroscopy that 55 was not formed. Due to the high yielding and
simple methodology of using the mesylate intermediate, the β-bromoalanine
intermediate pathway was not further investigated.
Scheme 13: Attempted synthesis of Dha 55 via β-bromoalanine intermediate 62.
2.2.2 Synthesis of protected cysteine 56
The synthesis of 56 was carried out by protection of the C- and N-termini of L-cysteine
63 with the t-butyl ester and Fmoc protecting groups respectively, producing 65.
Subsequent reduction of 65 yielded protected cysteine 56 (Scheme 14).70,72
60 5562
61. 49
HOOC
S S
COOH
NH2 NH2
But
OOC
S S
COOt
Bu
NH2 NH2
But
OOC
S S
COOt
Bu
NHFmoc NHFmoc
HS
COOt
Bu
NHFmoc
(i)
(ii)
(iii)
(i) Perchloric acid, t-butyl acetate; (ii) Fmoc-Cl, Et3N, NMM, THF; (iii) Tributylphosphine/H2O in THF.
Scheme 14: Synthesis of protected cysteine 56.
The initial step involved the protection of both C-termini of 63 with t-butyl esters using
t-butyl acetate in the presence of 70% perchloric acid. The di-t-butyl-ester-protected
cystine 64 was isolated as a white solid, in 21% yield (Lit. 90%70
). The successful
addition of the t-butyl group was seen from the 1
H NMR spectrum as a singlet at 1.48
ppm. The singlet produced by the Me3 of t-butyl acetate that is unattached to the cystine
appears at 1.27 ppm. The formation of 64 was also confirmed due to the presence of a
C=O stretching band at 1737 cm-1
in the IR spectrum, a band characteristic of an ester.
LC-MS was also used to confirm successful synthesis with a mass value of 353.1567
m/z detected for the [M+H]+
species, compared to the calculated value of 353.1569 m/z.
Attempts were made to improve the poor yields obtained from the synthesis of cystine
64 through the use of fresh bottles of both t-butyl acetate and 70% perchloric acid but
without success. The reaction was also allowed stir for two and three times the duration
reported in the literature method, again resulting in no improvement in yield. Due to
low yield of this reaction the HCl salt of cystine 64 was reluctantly purchased from a
commercial source in small quantities, due to its high cost.
Step two in the synthesis of protected cysteine 56 involved the protection of both N-
termini using the Fmoc group. This was carried out using Fmoc-Cl in the presence of
triethylamine (to remove the HCl salt from the cystine 64) and N-methylmorpholine
63 64
6556
α
α αα
αα α
β β β β
β β β
62. 50
producing 65 in a yield of 78%. The addition of the Fmoc group was confirmed by 1
H
NMR spectroscopy with the aromatic peaks found between 7.74 – 7.27 ppm, the CH2 as
a multiplet at 4.36 and the CH peak as a triplet at 4.19 ppm (J = 7.18 Hz). The effect on
the chemical shifts after the Fmoc addition could be seen on the α-hydrogens of the
cystine which moved downfield from 3.71 ppm to 4.58 ppm and on the β-hydrogens of
the cystine which shifted from 3.11-2.98 ppm to 3.27-3.14 ppm (Table 7). Again the
conclusive evidence for the formation of 31 was achieved through LC-MS where a mass
of 797.2936 m/z was detected for the [M+H]+
species, compared to the calculated mass
of 797.2930.
β-hydrogens
(ppm)
α-hydrogens
(ppm)
64 3.11 – 2.98 3.71
65 3.27 – 3.14 4.58
Table 7: 1
H NMR signal comparison between 64 and 65.
The third and final step in the synthesis of 56 involved the reduction of the disulfide
bond in 65 to form two molecules of 56 using tributylphosphine and H2O, using THF as
the solvent, in 81% yield (Scheme 15).
63. 51
P
BuBu
Bu
S
But
OOC
NHFmoc
COOt
Bu
NHFmoc
S
S
But
OOC
NHFmoc
COOt
Bu
NHFmoc
P
Bu
Bu
Bu
S
SH
But
OOC
NHFmoc
P
O Bu
Bu
Bu
(i)
(ii)
(i) THF; (ii) H2O, THF.
2 X
S
But
OOC
NHFmoc
H
O
H
P
Bu
Bu
Bu
O
H
H
S
But
OOC
NHFmoc
P
Bu
Bu
Bu
O
H
Scheme 15: Reduction of the disulfide bond of 65 to form cysteine 56.71
Once the reaction was complete 56 was used immediately without purification or
analysis due to its susceptibility to oxidation, reforming cystine 65. After many
repetitions of the reaction, wide variations in yield were seen using this methodology. It
was found that the success of the reaction was highly dependent on the purity of the
tributylphosphine, which had a strong tendency to degrade upon exposure to air. This
66 65
5
67
56 68
64. 52
But
OOC
S S
COOt
Bu
NHFmoc NHFmoc
HS
COOt
Bu
NHFmoc
Zinc dust, AcOH
O
O
NHAlloc
S
COOt
Bu
NHFmoc
O S
COOt
Bu
NHFmoc
O
NHAlloc
H
H
AllylOOC
S
COOt
Bu
NHFmocNHAlloc
(i)
(i) Cs2CO3 (0.12 mole eq.), DMF.
methodology however was still preferred to the alternative of using zinc dust and glacial
acetic acid as the cleavage cocktail as this produced a lower yield and a considerable
number of impurities (Scheme 16).72
Scheme 16: Reduction of cystine 65 to cysteine 56 using zinc dust.
2.3 Synthesis of protected lanthionine 57
The synthesis of lanthionine 57 proceeded through the Michael addition reaction of
protected Dha 55 and protected cysteine 56 under basic conditions (Scheme 17).48,69
Scheme 17: Synthesis of diastereoisomeric lanthionines 57 via Michael addition
of cysteine 56 to Dha 55.
65 56
5655
69
57
65. 53
The initial synthesis of 57 was carried out via methodology developed by Tabor.69
The
reaction was carried out at ambient temperature in DMF using 0.12 mole equivalents of
Cs2CO3 as a catalytic base. The bicarbonate formed via the deprotonation of the thiol by
Cs2CO3 is expected to act as the proton source for the stereocontrolling reprotonation
step of 69. Upon completion of an acidic work-up a yield of 17% was achieved,
compared to a 100% yield reported.
2.3.1 Optimisation of lanthionine 57 synthesis
Due to the non-reproducibility of the reported yields, new methodology was developed
by optimisation of the reaction conditions and the reagents in order to consistently
produce acceptable yields. The main four factors investigated were:
(i) Choice of solvent;
(ii) Choice of base;
(iii) Reaction temperature;
(iv) Number of equivalents of base used.
(i) Choice of solvent
A selection of solvents were investigated as alternatives to using DMF. It was hoped
that a lower boiling solvent would show equally good or better results. Due to the
boiling point of DMF being 153 o
C it required multiple water washes in the work-up of
57, which could have accounted for the significant drop in the isolated yield. Upon
performing the Michael addition of 55 and 56 using different solvents, but continuing to
use 0.12 mole eq. of Cs2CO3 and maintaining the temperature at ambient temperature, it
was shown that the use of CH3CN as the solvent resulted in the highest yield of 35%
(Table 8). This result proved very useful moving forward as its lower boiling point of
82 o
C allows CH3CN to be removed in vacuo, which negates the use of copious
amounts of water that was necessary to remove the DMF. Due to this increase in
66. 54
percentage yield, CH3CN was used as the solvent for all further lanthionine syntheses
and optimisations.
Table 8: Effect of solvents on % yield of lanthionine 57.
(ii) Choice of base
The choice of base was investigated in a similar fashion to that of the solvent. Identical
reactions involving the Michael addition of 55 and 56 were carried out except using a
different carbonate base in each case (Table 9).
Table 9: Effect of carbonate bases on % yield of lanthionine 57.
Cs2CO3 was discovered to have a far greater effect in the formation of lanthionine 57
than either the Na2CO3 or K2CO3. This result can be explained through what is known
as the “Cesium Effect”.73,74
This effect is due to cesium salts being far more soluble, and
therefore reactive, in aprotic solvents than the rest of the alkali metal salts (Table 10).75
Solvent Cs2CO3
(Mole eq.)
Temperature
(o
C)
Yield
55 + 56 DMF 0.12 r.t. 17%
55 + 56 THF 0.12 r.t. 14%
55 + 56 CH3CN 0.12 r.t. 35%
55 + 56 Toluene 0.12 r.t. 6%
Solvent Base
(0.12 mole eq.)
Temperature
(o
C)
Yield
55 + 56 CH3CN Na2CO3 r.t. 10%
55 + 56 CH3CN K2CO3 r.t. 18%
55 + 56 CH3CN Cs2CO3 r.t. 35%
55 + 56 CH3CN None r.t. 10%
67. 55
Solvent Cs2CO3
(g/10 ml)
K2CO3
(g/10 ml)
DMF 1.195 0.075
DMSO 3.625 0.470
DMAc 0.490 0.046
Sulfolane 3.950 0.160
NMP 7.224 0.237
Table 10: Comparison of the dissolution of alkali salts in aprotic solvents.75
As CH3CN is also an aprotic solvent it would be expected that this trend would also be
true for it. Due to this result it was decided that Cs2CO3 would be used for all further
lanthionine syntheses and optimisations.
(iii) Reaction temperature
The ideal temperature to conduct the lanthionine reaction was the next parameter to be
investigated. Again identical reactions involving the Michael addition of 55 and 56 were
carried out except with careful control of temperature (Table 11).
Table 11: Effect of temperature on % yield.
This investigation indicated that the formation of lanthionine 57 using CH3CN as the
solvent and 0.12 mole eq. of Cs2CO3 as the base proceeded most successfully when
conducted at 0 o
C. It has also shown that moderate yields are achieved at 25 o
C but as
the temperature is increased there is a dramatic decrease in yield, with no lanthionine
recovered from the reaction at 45 o
C. Analysis via LC-MS and NMR show that neither
the lanthionine product nor either starting material were present. Due to the marked
Solvent Cs2CO3
(Mole eq.)
Temperature
(o
C)
Yield
55 + 56 CH3CN 0.12 0 60%
55 + 56 CH3CN 0.12 r.t. 35%
55 + 56 CH3CN 0.12 35 11%
55 + 56 CH3CN 0.12 45 0%
68. 56
increase in yield from the reduction in temperature to 0 o
C, this reaction condition was
used in the further lanthionine syntheses and optimisation.
(iv) Number of mole equivalents of base used
With the appropriate solvent, base and temperature selected the next step was to
investigate the optimal number of mole equivalents of Cs2CO3 needed to ensure the
maximum yield of lanthionine 57. The same template as the previous three
investigations was used to carry out this study. Identical reactions involving the Michael
addition of 55 and 56 were carried out except using a different number of mole
equivalents of Cs2CO3 in each case (Table 12).
Table 12: Effect of the quantity of Cs2CO3 used on % yield.
It was shown from the results that, excluding the highest value, as the equivalents of
Cs2CO3 increased so did the % yield. This was an unexpected outcome as the Cs2CO3 in
this reaction should be acting catalytically and therefore should not ultimately increase
the final yield, unless other factors were influencing the reaction. One such possibility
was that the highly unstable protected cysteine 56 was becoming oxidised back to
cystine 65 before the Michael addition could take place. This would account for the
lower yielding reaction at 0.12 mole eq., but also account for the increase in relative
yield as the equivalents of Cs2CO3 were increased. This could be due to the larger
quantity of Cs2CO3 being available to react at the beginning of the reaction, before a
significant amount of 56 has been converted to 65. An explanation for the reaction using
2 mole eq. of Cs2CO3 producing 0% yield could again be due to the “Cesium effect”.
Solvent Cs2CO3
(Mole eq.)
Temperature
(o
C)
Yield
55 + 56 CH3CN 0.12 0 60%
55 + 56 CH3CN 0.50 0 65%
55 + 56 CH3CN 1.00 0 77%
55 + 56 CH3CN 2.00 0 0%
69. 57
Studies carried out in industry76
have shown that a 50 mM solution of Cs2CO3 in
CH3CN has a pH of approximately 13 (Figure 30).
Figure 30: pH value of 50 mM solutions of various bases in CH3CN.76
The lanthionine reaction using 2 mole eq. of Cs2CO3 in CH3CN equates to
approximately a 100 mM solution, resulting in a higher pH, possibly resulting in the
degradation of the starting materials and/or product. The most likely source of
degradation could be from the removal of the Fmoc protecting group from both the
cysteine starting material and the lanthionine product that has been formed. This is a
possibility as the standard removal conditions of the Fmoc group is piperidine in an
aprotic solvent i.e. 20% piperidine in DMF.
2.3.2 Optimised synthesis of lanthionine 57
Once the optimisation of the reaction conditions and reagents was finalised it was found
that the synthesis of protected lanthionine 57 from protected Dha 55 and protected
70. 58
cysteine 56 was most successful when performed in CH3CN, at 0 o
C and using 1 eq. of
Cs2CO3 as the base (Scheme 18).
Scheme 18: Optimised synthesis of diastereoisomeric lanthionines 57.
The synthesis proceeded through protected cysteine 56 being fully dissolved in CH3CN
with 1 mole eq. of Cs2CO3 and cooled to 0 o
C. The reaction vessel was purged with N2
to ensure an oxygen-free environment to prevent the oxidation of cystine 56 to cystine
65. Once fully purged, Dha 55 was dissolved in a small amount of CH3CN and was
added dropwise into the reaction mixture via a syringe to maintain the N2 environment.
Upon completion of the work-up and product purification by flash column
chromatography, lanthionine 57 was reproducibly recovered in yields of up to 77%.
The successful formation of lanthionine 57 was confirmed through comparison of 1
H
and 13
C NMR spectra with literature values (Tables 13 + 14).
55 56
57
(i) Cs2CO3 (1 mole eq.), CH3CN, 0 o
C
71. 59
Table 13: Comparison of 1
H NMR chemical shifts of lanthionine 57 with
literature values.
α-carbons
(ppm)
β-carbons
(ppm)
Dha-side cysteine-side Dha-side cysteine-side
Lanthionine 57 53.9 and 53.8 54.4 and 54.3 35.8 and 35.5 35.7 and 35.3
Literature69
53.8 and 53.7 54.4 and 54.3 35.8 and 35.4 35.7 and 35.3
Table 14: Comparison of 13
C NMR chemical shifts of lanthionine 57 with
literature values.
Each of the protecting groups Fmoc, t-butyl, alloc and allyl can also be clearly identified
in the full 1
H NMR spectra of lanthionine 57 (Figure 31) with major functional groups
also visible in the IR spectrum (Figure 32).
α-hydrogens
(ppm)
β-hydrogens
(ppm)
Dha-side cysteine-side
Lanthionine 57 4.54 – 4.67 4.49 2.94 – 3.10
Literature69
4.61 4.47 2.91 – 3.09
73. 61
Figure 32: IR spectrum of lanthionine 57. (A) N-H stretch (carbamate), (B) C-H
stretch (Fmoc), (C) C=O stretch (ester and carbamate), (D) C=C stretch (Fmoc),
(E) C-O stretch (ester and carbamate).
74. 62
For definitive proof of the formation of lanthionine 57, LC-MS was again used. A mass
of 633.2243 m/z was detected for the [M+Na]+
species, compared to the calculated mass
of 633.2247 m/z.
Purity of the lanthionine 57 product was confirmed using UHPLC analysis: the
diastereomeric mixture resulted in a single sharp peak that could not be separated
(Figure 33). Multiple mobile phases were attempted using combinations of CH3CN,
H2O, DCM, MeOH and EtOAc without the successful separation of the peak. As well as
interchanging mobile phases, a broad range of flow rates and injection volumes were
also attempted, again to no avail.
Mobile Phase: 70:30 (CH3CN : H2O)
Flow rate: 1 mL/min
Detector: 254 nm
Figure 33: UHPLC chromatograph of diastereoisomeric lanthionines 57.
Datafile Name:2-05-00
Sample Name:2-05
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
0
250
500
750
1000
1250
1500
1750
2000
2250
mV
Detector A 254nm
75. 63
13
C NMR was used to detect the diastereoisomers present in the lanthionine 57 mixture.
A doubling of peaks can be seen in a large number of the carbon peaks, most notably
and significantly in the α-carbons and β-carbons, which is very characteristic of
diastereoisomers (Figure 34).77
Figure 34: 13
C NMR chemical shifts and doubling of the (i) α-carbons and (ii) β-
carbons of diastereoisomeric lanthionines 57.
According to a report by Tabor69
the ratio of diastereoisomers can be estimated via the
shape of β-hydrogen multiplet peak in the 1
H NMR spectrum (Figure 35). The literature
peak shown (ii) is an excerpt from the 1
H NMR spectrum of a 50:50 mixture of
diastereoisomers of lanthionine 55. As can be seen in the comparison it would appear
that there is a roughly 50:50 ratio of the (S,R)-lanthionine and (R,R)-lanthionine present
in lanthionine 57 due to the high degree of similarity in shape of the β-hydrogen peaks.
The report shows that a mixture of 85:15 (S,R)-lanthionine to (R,R)-lanthionine results
in a broader, less defined multiplet than that of a 50:50 mixture.
(R,R)(S,R) (S,R) (R,R)
76. 64
Figure 35: Comparison of the β-hydrogen signals in 1
H NMR spectrum of (i)
lanthionine 57 and (ii) literature.69
2.4 Chapter conclusion
This chapter has detailed the synthetic methods used to synthesise protected Dha 48
and Dha 50. This investigation determined that not only was it possible to
synthesise Dhas in excellent yields but that once they are stored correctly that Dhas
will remain un-degraded for periods of over six months, making them ideal starting
materials.
This chapter has also detailed the methodology that was first utilised in the
synthesis of protected Dha 55 and protected cysteine 56, followed by their
subsequent use in the synthesis of the desired diastereomeric mixture of lanthionine
57. The methodology investigated for the synthesis of lanthionine 57 was the sulfa-
Michael addition reaction using the following specific conditions: DMF as solvent,
0.12 mole eq. of Cs2CO3 as a catalytic base and the reaction temperature at ambient
temperature (25 o
C). The use of this method resulted in the successful synthesis of
the desired lanthionine 57 but very inconsistently and in extremely poor yields of
17%.
An investigation into the optimisation of the reaction conditions of the sulfa-
Michael addition of 55 and 56 was carried out to provide methodology that would
(ii)(i)
77. 65
result in consistently high yields of lanthionine 57. The parameters that were
investigated were:
the reaction solvent (CH3CN, DMF, THF, Toluene);
the base (Cs2CO3, K2CO3, Na2CO3, none);
mole equivalents of base (none, 0.12, 0.5, 1.0, 2.0);
the reaction temperature (0 o
C, 25 o
C, 35 o
C, 45 o
C).
It was determined that the optimum conditions required the use of CH3CN as the
solvent, 1 mole eq. of Cs2CO3 as the base and a reaction temperature of 0 o
C. The
use of this methodology resulted in the synthesis of lanthionine 57 in consistently
high yields of up to 77%, compared to yields of approx. 17% achieved using
literature methods, which is a 4.5-fold improvement. The lanthionine produced was
also analysed using polarimetery, with an [α]25
D of -10o
recorded. This value will be
used in future chapters to analyse the diastereomeric ratio of lanthionines produced
when organocatalysts are used to attempt to influence the stereochemistry.
Due to the susceptibility of lanthionine to undergo oxidation it was always
refrigerated and stored under an N2 environment to ensure its stability.78
79. 67
(S)
S
(R) COOt
BuAllylOOC
NHAlloc NHFmoc
3.0 Introduction
This chapter outlines the synthetic methodology used in the synthesis of the bifunctional
cinchona alkaloid catalysts that were used to control the stereochemistry of the Michael
addition reaction of Dha 55 and cysteine 56 to form the single (S,R) lanthionine
diastereomer 70 (Figure 36).
Figure 36: Structure of (S, R)-lanthionine.
3.1 Synthesis of cinchona alkaloid catalyst 46
The utilisation of catalyst 46 to control the stereochemistry of Michael addition
reactions has been widely investigated to date.60,61
Its proven application as a
stereocontrolling bifunctional catalyst, utilising both the hydrogen bonding of the
thiourea and the Lewis-base properties of the quinuclidine, makes it an ideal starting
point in this investigation. The route used for the synthesis of cinchona alkaloid 46
(Figure 37) was developed by Soós et al.79
The synthesis proceeded through the
conversion of quinine 71 to a 9-azidoepiquinine 74 via a Mitsunobu reaction. The
resulting azide 74 could then be converted to amine 76 via a Staudinger reduction
reaction (Scheme 19).
70
80. 68
N
N
H
N
H
S
CF3
F3C
N
OMe
C9
C6'
Figure 37: Structure of cinchona alkaloid based thiourea catalyst 46.
The first step in the reaction involved using Mitsunobu reaction conditions of di-iso-
propyl azodicarboxylate (DIAD), triphenylphosphine (PPh3) and diphenyl phosphoryl
azide to yield the 9-azidoepiquinine 74 intermediate (Scheme 19). Without purification
or recovery, 74 was carried on to the next step in the reaction, a Staudinger reduction,
using an extra equivalent of triphenylphosphine in THF at 50 o
C. The result of the
Staudinger reduction is an inversion of stereochemistry at the C9 position to yield 9-
aminoepiquinine 76 in poor yields of up to 8% compared to literature values of 71%.79
46
81. 69
N
H2N
N
OMe
N
N
OMe
HO
N
N
OMe
O
P
PhO
O
PhO N NH
Ph3P
Pri
O2C
CO2
i
Pr
N3
N
N3
N
OMe
N
N
N
OMe
P
Ph
Ph
Ph
N2
(i)
(ii)
(iii)
(i) DPPA, DIAD, PPh3, THF; (ii) PPh3, THF, 50 o
C; (iii) H2O, THF.
P
O
Ph Ph
Ph
P
O
Ph Ph
Ph
Scheme 19: Synthesis of 9-aminoepiquinine 76 via Mitsunobu and Staudinger
reactions.
Through analysis by LC-MS it was seen that a large quantity of quinine 71 were still
present at the end of the reaction, indicating the reaction has not gone to completion. In
an effort to try and ensure a maximum amount of 76 was produced, the reaction was
71 72 73
7475
76
77
77
82. 70
repeated on numerous occasions utilising a fresh bottle of DIAD and commercially
dried THF. Very strict control of both the N2 atmosphere and temperature was
undertaken but this resulted in no measurable difference in the final yield.
After purification via flash column chromatography a very small quantity of 76 was
recovered. The conclusive identification of 76 proved difficult due to little observable
difference in Rf values, IR spectra, 1
H NMR spectra and 13
C NMR spectra between 71
and 76. However, through the comparison of the 1
H and 13
C NMR spectra of 76 with
that of literature values79
(Table 15) it was concluded that the formation of 76 was
successful.
N
H2N
N
OMe
9
3'
8
4a'
4'
6'
Proton and/or
Carbon #
76
(ppm)
Lit. values
(ppm)
9
1
H
13
C
4.75
49.9
4.72
51.9
8
1
H
13
C
3.28 - 3.18
63.2
3.16
62.2
3’
1
H
13
C
7.61
121.1
7.61
120.2
4’ 13
C 149.0 148.3
4a’ 13
C 130.3 129.4
Table 15: 1
H and 13
C NMR spectra comparison of 76 with literature values.
Definitive proof of the formation of 76 was found through analysis by LC-MS with a
mass of 324.2098 m/z detected for the [M+H]+
species, compared to the calculated mass
of 324.2076 m/z.
Due to poor yields and the irreproducibility of the reaction it was decided to purchase 9-
aminoepiquinine 76 in extremely small quantities due to its excessive cost. This was to
ensure the second step of the synthesis, the addition of the thiourea-containing
83. 71
N
N
H
N
H
S
CF3
F3C
N
OMe
N
H2N
N
OMe
F3C
F3C
N C S
THF
functional group using 3,5-bis(trifluoromethyl)phenyl isothiocyanate 44, can be
successfully achieved to produce the bifunctional catalyst 46 in sufficient yields
(Scheme 20).
Scheme 20: Synthesis of bifunctional cinchona-alkaloid-based thiourea
catalyst 46.
The sample of 9-aminoepiquinine that was purchased came as the HCl salt and therefore
it was required to first desalt using 1 mole equivalent of triethylamine. The unsalted
catalyst was then reacted with the isothiocyanate 78 in THF for approximately 15 hr at
ambient temperature. Upon analysis via TLC it was found that the reaction produced
numerous side products that were removed through flash column chromatography using
a mobile phase of EtOAc:MeOH:concentrated aqueous NH4OH (300:5:1) to yield
catalyst 46 in yields of up to 62%.
The successful synthesis of catalyst 46 was confirmed through comparison of the 1
H
and 13
C NMR spectra of 46 with that of literature values79
(Table 16).
76 46
78
84. 72
4'
N
N
H
N
H
S
CF3
F3C
N
OMe4a'
9
8
3'2''
3''
4''
6'
Proton and/or
Carbon #
46
(ppm)
Lit. values
(ppm)
9
1
H
13
C
6.45
56.4
6.32
55.4
C=S 13
C 182.5 181.6
8
1
H
13
C
3.61 - 3.53
61.5
3.39
60.7
3’
1
H
13
C
7.61 - 7.59
120.6
7.55
120.2
4’ 13
C 147.0 146.6
4a’ 13
C 130.1 129.2
2’’
1
H
13
C
8.14
123.6
8.11
122.6
3’’ 13
C
132.6
(quartet, 2
JCF = 35 Hz)
131.8
(quartet, J = 33 Hz)
4’’
1
H
13
C
7.59 - 7.61
117.8
(septet, 3
JCF = 3.8 Hz)
7.59
116.9
(septet, 3
JCF = 3.7 Hz)
Table 16: Comparison of the 1
H and 13
C NMR spectra of 46 with literature
values.
85. 73
The successful synthesis of 46 was definitively confirmed through analysis by LC-MS
with a mass of 595.1955 m/z detected for the [M+H]+
species, compared to a calculated
mass of 595.1966 m/z.
3.2 Chapter conclusion
This chapter has detailed the initial methodology that has been used in the synthesis of
the bifunctional cinchona-alkaloid-based thiourea catalyst 46. It has shown that, to date,
the synthesis of 9-aminoepiquinine 76 has proved problematic with very poor yields
being achieved. Purification and characterisation also proved very difficult due to the
high degree of similarity between 76 and the quinine starting material. However through
comparison with literature results and analysis by LC-MS it was confirmed that small
quantities of 76 were being produced in the reaction.
The methodology used to synthesise 46 from a commercially obtained sample of the 76
salt was also examined. Unlike the previous step, the addition of isothiocyanate 78
resulted in 46 being produced in acceptable yields of up to 62%. The reaction however
did produce multiple side products that required further purification via flash column
chromatography. Confirmation was again obtained through comparison with literature
results and analysis by LC-MS.
87. 75
N
N
OMe
HO
AllylOOC
HS
NHFmoc
COOt
Bu
NHAlloc
AllylOOC
NHFmoc
COOt
Bu
NHAlloc
S
4.1 Introduction
This chapter outlines the methodologies that were used in the attempted synthesis of
protected lanthionine 57 from protected cysteine 56 and protected Dha 55 using either
quinine or cinchona alkaloid derivative 46 as an organocatalyst. The use of these
catalysts was to attempt to control the stereochemistry of the sulfa-Michael addition
reaction to form the desired (S,R)-lanthionine product exclusively. The successful
stereoselective synthesis of an orthogonally protected SPPS-compatible (S,R)-
lanthionine would negate the need for the tedious, time-consuming synthesis of the
highly unstable orthogonally protected β-bromoalanine starting material which is
currently the most efficient procedure for lanthionine synthesis (see section 1.10).
4.2 Attempted synthesis of lanthionine 57 using quinine as the catalyst
As a starting point for the eventual use of catalyst 46 in the attempt to control the
stereochemical outcome of the sulfa-Michael addition of cysteine 56 and Dha 55 to
form orthogonally protected lanthionine 57, it was decided that quinine would first be
investigated (Scheme 21).
Scheme 21: Synthesis of lanthionine 57 using quinine as a catalyst.
55 56
71
57
88. 76
N
N
H
N
H
S
CF3
F3C
N
OMe
N
N
OMe
HO
Quinine was deemed an ideal catalyst to begin the investigation as the aromatic
quinoline rings and bicyclic quinuclidine rings of quinine also form the chiral backbone
of catalyst 46 (Figure 38).
Figure 38: Quinoline and quinuclidine backbone, highlighted in red, of quinine
71 and catalyst 46.
According to numerous reports80,81,82
it is the tertiary amine of the quinuclidine that is
responsible for both the activation of the thiol of cysteine 56 via deprotonation and also
the subsequent facial-selective reprotonation of the lanthionine once the sulfa-Michael
addition has occurred, thus controlling the stereochemistry. However, due to quinine 71
possessing only the quinuclidine rings and not the hydrogen-bond donor group on the
C9 position it was expected that the reaction would proceed with little or no
stereoselectivity.
It was found however that the reaction involving cysteine 56 and Dha 55 using 0.12
mole equivalents of quinine as the catalyst produced an extremely poor yield of 12%,
with no stereoselectivity compared to that of the reaction carried out using Cs2CO3. A
specific rotation value of -10o
confirms an identical ratio of diastereoisomers present in
the lanthionine mixture as that obtained from the non-stereocontrolled reaction using
Cs2CO3. Analysis of the 13
C NMR spectra of the lanthionine product also shows that
signals from both the α-carbons and β-carbons show no discernible difference from the
Cs2CO3-catalysed reaction (Figure 39).
4671
89. 77
Figure 39: Comparison of 13
C NMR spectra of (i) α-carbons and (ii) and β-
carbons of 57 synthesised using quinine (red) or Cs2CO3 (blue)
as the catalyst.
It was found upon analysis of the reaction mixture by TLC and LC-MS that large
quantities of both cleaved cysteine 56 and Dha 55 were still present. Increasing the
quantity of quinine (to 1 mole equivalent) resulted in no increase in the yield of
lanthionine, with cleaved cysteine 56 and Dha 55 still present in large quantities. From
this it was concluded that the quinine was not catalysing the reaction and that all
lanthionine formation was due to the spontaneous uncatalysed Michael addition of
cysteine 56 and Dha 55. This theory was confirmed through the addition of 0.12, 0.50
and 1.0 mole equivalents of Cs2CO3 resulting in yields and specific rotations
corresponding to those obtained without the use of quinine (Table 17).
(ii)(i)
(S,R)
(S,R)
(R,R)
(R,R)
90. 78
Quinine
(Mole eq.)
Cs2CO3
(Mole eq.)
Yield [α]25
D
55 + 56 0.12 0 12% -10o
55 + 56 1.0 0 12% -10o
55 + 56 1.0 0.12 60% -10o
55 + 56 1.0 0.5 65% -10o
55 + 56 1.0 1.0 77% -10o
Table 17: Catalytic effect of quinine in CH3CN at 0 o
C.
To investigate if an increase in reaction temperature was necessary for the reaction to
proceed using quinine as a catalyst, each reaction was increased to 40 o
C once
lanthionine synthesis ceased through observation by TLC. It was observed that no
additional lanthionine was produced through this increase in temperature with cleaved
cysteine 56 and Dha 55 still present in large quantities.
A series of reactions between protected Dha 55 and protected cysteine 56 using quinine
as the catalyst were also carried out in toluene (Table 18) in accordance with several
literature reports81,83,84
that demonstrate successful and high-yielding Michael addition
reactions involving thiols and α,β-unsaturated species using quinine and other cinchona
alkaloid derivatives in this solvent. Despite the success of these reports the use of
toluene proved to be even less successful, as protected Dha 55 was highly insoluble in
toluene and resulted in yields of ≈ 6% when using both 0.12 and 1.0 mole equivalents of
quinine. It was also shown that even after the addition of 1.0 mole equivalent of Cs2CO3
that no observable difference in yield was detected. This was thought to be largely due
to insolubility of Cs2CO3 in toluene.
Quinine
(Mole eq.)
Cs2CO3
(Mole eq.)
Yield [α]25
D
55 + 56 0.12 0 ≈ 6% -10o
55 + 56 1.0 0 ≈ 6% -10o
55 + 56 1.0 1.0 ≈ 6% -10o
55 + 56 0 1.0 ≈ 6% -10o
Table 18: Catalytic effect of quinine in toluene at 0 o
C.
91. 79
N
NN
S
CF3
F3C
N
OMe
FmocHN
COOt
Bu
NHAlloc
AllylOOC
NHFmoc
COOt
Bu
NHAlloc
S
O
O
H H H
S
N
NN
S
CF3
F3C
N
OMe
FmocHN
COOt
Bu
NHAlloc
O
O
H H H
S
N
NN
S
CF3
F3C
N
OMe
O
O
H H H
S
COOt
Bu
NHFmocNHAlloc
AllylOOC
NHAlloc
HS
NHFmoc
COOt
Bu
(i)
(i) Cinchona alkaloid catalyst, CH3CN, 0 o
C.
4.3 Attempted synthesis of lanthionine 57 using 46 as the catalyst
Although quinine failed to catalyse the Michael addition of cysteine 56 with Dha 55 it
was decided to still attempt the use of catalyst 46 as several publications66,79
have
observed that cinchona alkaloid derivatives may still catalyse a reaction in which
quinine either fails to do so, or produces very poor yields and diastereomeric ratios. The
reaction using catalyst 46 was expected to proceed through the sulfa-Michael addition
of cysteine 56 to protected Dha 55 (Scheme 22).
Scheme 22: Proposed mechanism and transition state models for the formation
of protected lanthionine 57 using catalyst 46.
92. 80
(i) (ii)
Unfortunately, as with quinine, the use of catalyst 46 resulted in extremely poor yields
of 12% with no stereoselectivity observed through analysis of the 13
C NMR spectra
(Figure 40) and through analysis of the specific rotation of the product, which was again
-10o
.
Figure 40: Comparison of 13
C NMR spectra of (i) α-carbons and (ii) β-carbons
of 57 synthesised using 46 (red) or Cs2CO3 (blue) as the catalyst.
As with the reactions using quinine, it was observed through LC-MS and TLC that large
quantities of unreacted cleaved cystine and Dha were still present in the reaction
mixture. It was again the case that upon the addition of 1 mole equivalent of Cs2CO3
that the reaction went to completion with yields corresponding to the yields obtained
from the reaction carried out without the presence of catalyst 46 (Table 19).
46
(Mole eq.)
Cs2CO3
(Mole eq.)
Yield [α]25
D
55 + 56 0.12 0 12% -10o
55 + 56 0.12 1 77% -10o
Table 19: Catalytic effect of catalyst 46 in CH3CN at 0 o
C.
(S,R) (R,R) (S,R) (R,R)
93. 81
This has resulted in the conclusion that, as with the use of quinine, catalyst 46 is
exerting no catalytic effect on the reaction and all lanthionine formed prior to the
addition of Cs2CO3 was formed through the non-catalysed reaction of cysteine 56 with
Dha 55.
4.4 Chapter conclusion
This chapter has detailed the synthetic methodology used in the attempted
stereoselective synthesis of protected lanthionine 57 from protected cysteine 56 and
protected Dha 55 using an organocatalyst.
It began by investigating the application of the cinchona alkaloid quinine as the chosen
catalyst. It was expected that the amine of the quinuclidine functional group would
catalyse the reaction via the deprotonation of the thiol functional group on cysteine 56,
which would then undergo a Michael addition to Dha 55. The resulting lanthionine
enolate 69 would then be reprotonated by the newly formed quaternary ammonium
cation of the quinuclidine. As quinine is only a monofunctional catalyst, (i.e. it has no
secondary function to interact with the Dha to control the facial-selective reprotonation),
it was not expected that a dramatic change in the resulting stereochemistry of the
lanthionine would occur. Results have shown that quinine failed to carry out the
deprotonation of cysteine 56 and therefore only spontaneous uncatalysed Michael
addition reactions occurred, resulting in poor yields of 12% with no observable
stereocontrol, consistent with results achieved through the completely uncatalysed
reaction.
This chapter also investigated the application of the bifunctional cinchona alkaloid 46 as
the chosen catalyst. As with the use of quinine, it was expected that the amine of the
quinuclidine functional group would catalyse the reaction via the deprotonation of the
thiol functional group on cysteine 56, which would then undergo a Michael addition to
Dha 55. Due to catalyst 46 being a bifunctional catalyst, (i.e. containing a secondary
thiourea functional group that interacts with Dha 55 through hydrogen-bonding to
control the stereoselective reprotonation step of enolate 69 by the quaternary
ammonium cation), it was expected that there would be a substantial influence on the
94. 82
N
N
MeO OHH
N
N
OMe
OHH
N
N
MeO
HO
H
stereochemistry of the lanthionine product. However, as with quinine, catalyst 46 also
failed to carry out the deprotonation of cysteine 56 and only spontaneous uncatalysed
Michael addition reactions occurred, resulting in poor yields of 12% with no observable
stereocontrol, consistent with results achieved through the completely uncatalysed
reaction using Cs2CO3.
An investigation by Bürgi and Baiker85
into the conformation of cinchona alkaloids in
polar solvents could shed light on the potential reason for the failure of 46 to catalyse
the reaction. Through NMR analysis and sophisticated ab initio calculations they found
that the most stable form of the cinchona alkaloid quinidine in a polar solvent, such as
CH3CN, is either the anti-closed or syn-closed confirmation (Figure 41). It was also
shown that the most stable form of quinidine in a non-polar solvent such as toluene,
which is the solvent used in the majority of cinchona-alkaloid-catalysed reactions, is the
anti-open conformation (Figure 41).
Figure 41: Structural conformations of the cinchona alkaloid quinidine.
It can be seen in the structures of anti-closed and syn-closed that the forward-facing
quinoline group could be sterically hindering the access of 56 to the tertiary amine of
the quinuclidine, which is required in order for it to be deprotonated and ultimately
catalyse the sulfa-Michael addition. The anti-open conformation has only the forward-
facing proton at the chiral centre, which will not contribute to any steric hindrance. This
anti-closed syn-closed anti-open
95. 83
trend of polar solvents conferring a negative effect on the catalytic function of cinchona
alkaloids can be seen in the investigation by Unhale et al.83 They have observed that
even when using relatively small side-groups the yields in polar solvents such as
CH3CN and CH2Cl2 are up to 30% less than the same reaction carried out in non-polar
solvents such as toluene and m-xylene. These steric issues would be further exacerbated
by the extremely bulky Fmoc and t-butyl protecting groups present on 56, potentially
stopping the catalytic effect completely.
A study by Tárkányi et al.86
has also shown that catalyst 46 participates in both
intermolecular interactions with other molecules of 46 and intramolecular interactions
that form dimers (Figure 42).
Figure 42: Intermolecular and intramolecular interactions of catalyst 46.
It was observed through NOE analysis that intermolecular interactions occur via
hydrogen-bonding between the thiourea and quinoline functional groups as well as π-π
interactions between the two quinoline groups. It is also observed that intramolecular
interactions occur via hydrogen-bonding of the thiourea functional group with the
tertiary amine of the quinuclidine. Although no studies have been carried out on the
effect this dimerisation has on the catalytic function of cinchona alkaloids it could again
be causing steric issues in the ability of the tertiary amine of the quinuclidine to
96. 84
deprotonate the thiol of 56, and also could prevent the hydrogen-bond donors on 46
from interacting with Dha 55, therefore reducing the catalytic effect even further.
In H2O the amine of a quinuclidine molecule has a pKa of approximately 11.3.
However, several publications87,88
report that the quinuclidine amine in quinine has a
pKa of between 4.1 – 5.07. A report by Aggarwal et al.89
has shown that the addition of
functional groups, especially electron-withdrawing groups, causes the pKa of the
quinuclidine to drop significantly. A drop from 11.3 to 9.9 is seen with the addition of a
hydroxyl group, and a larger drop to 6.9 with the addition of a ketone. This trend could
explain the very low pKa’s recorded for the quinuclidine of quinine due to multiple
substituents on the ring. This low reported pKa, compared to a pKa of 8.0 for the thiol of
56, would prevent the deprotonation of the thiol by the quinuclidine.
An alternative explanation for catalyst 46 being unsuccessful in catalysing the reaction
could be down to non-equivalent changes in pKa of the quinuclidine tertiary amine and
the thiol of cysteine when dissolved in CH3CN as opposed to H2O. Although reliable
pKa values could not be extrapolated for the quinuclidine amine and cysteine thiol in
CH3CN, it is possible that the pKa of the thiol of 56 could be higher than that of the
amine, therefore preventing deprotonation. Again, due to the need to produce an
extremely high pH of approx. 13 (5 mmol Cs2CO3 in 100 ml CH3CN) to result in
optimal yields (as discussed in section 2.3.1), it is possible that the basicity of the
quinuclidine is not sufficient for deprotonation to take place.
The pKa of the thiol could also be dramatically changed due to solvent interactions and
the electronic effects of the protecting groups on 56 in CH3CN.
It is plausible that one, or a combination, of these factors could be the reasoning behind
the poor performance of both 46 and 71 in successfully catalysing the sulfa-Michael
addition.
98. 86
5.0 Introduction
The future work on this project would consist of two discrete sections; first would be
the optimisation of the current substrates and the protecting groups that are used on the
N-termini and C-termini to facilitate better binding to the catalyst. The second section is
that the catalyst itself will undergo optimisation, namely in the position and type of
functional group that is added to impart the bifunctionality of the catalyst and facilitate
stronger binding to the substrates.
5.1 Optimisation of substrate 55
The optimisation of the protecting groups that are used on both the C-terminus and N-
terminus of the Dha substrate would be an ideal way to potentially increase the strength
of substrate-catalyst interaction, thereby enhancing the ultimate stereoselectivity of the
sulfa-Michael addition reaction between protected Dha 55 and protected cysteine 56.
Possible alternative protecting groups that could be utilised to increase the substrate-
catalyst interactions are Weinreb amides and oxazolidinones due to their additional
hydrogen-bond acceptor (Figure 43).
Two hydrogen bond acceptors One hydrogen bond acceptor
NHAlloc
N
O
O
O
O
NHAlloc
NHAlloc
O
O N
O
Figure 43: Increased the number of hydrogen-bond acceptors using Weinreb
amide 79 and oxazolidinone 80, as compared to the allyl protected 55.
5579
80
