2013 RSC Bio-Organic Group Postgraduate Symposium … · 2013-04-16 · 1 2013 RSC Bio-Organic...

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2013 RSC Bio-Organic Group Postgraduate Symposium

Manchester Institute of Biotechnology, University of Manchester

Thursday 11th April 2013

10:15 Registration, coffee, poster set-up

10:55 Welcome

11:00 – 12:30 Sarah Lovelock (University of Manchester)

Anabaena variabilis Phenylalanine Ammonia Lyase: An investigation into the catalytic

mechanism and applications as a biocatalyst.

Cristina Marculescu (University College London)

The Development of a New Class of Maleimides as Reagents for Protein Modification

Simge Davulcu (University of Bath)

Catalytic conversion of unactivated nitriles into N-substituted amides

Jon Ashley (National University of Singapore)

Hybridised-SELEX: a capillary electrophoresis based method for maximising the number

of aptamers screened

12:30 – 13:40 Lunch, poster session

13:40 – 15:10 Ryan Beattie (University of Bristol)

A Versatile Synthetic Approach to Novel Deoxy Sugar Analogues

Matthew Styles (University of Manchester)

Tailoring Complex Natural Products by Altering the Biosynthetic Machinery that Builds

Them

Oscar Cascón (Cardiff University)

Study of (+)-δ-cadinene synthase mechanism using farnesyl diphosphate analogues.

John M. Wadsworth (University of Edinburgh)

The natural product inhibitor myriocin displays a unique dual mode of action against

serine palmitoyltransferase

15:10 – 15:45 Coffee, poster session

15:45 – 16:30 Plenary lecture: Prof. Nigel Scrutton, Manchester Institute of Biotechnology

Addressing controversies in tunnelling and dynamics in enzyme catalysed H transfer

16:30 – 17:15 Prizes, refreshments

17:30 Close

We are grateful to the following organisations for sponsoring this event:

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Talk 1

Anabaena variabilis Phenylalanine Ammonia Lyase: An investigation into the catalytic mechanism and

applications as a biocatalyst.

Authors: Sarah Lovelock, Rachel Heath, Richard Lloyd and Nicholas Turner.

Presenting author affiliation: University of Manchester

Other affiliations: CoEBio3

OH

O

R

PAL+NH3

-NH3

OH

O

RNH2

Figure 1: The amination of substituted cinnamic acids to yield L-amino acids is catalysed by phenylalanine ammonia lyase

In Nature, the enzyme phenylalanine ammonia lyase (PAL) catalyses the deamination of L-phenylalanine to

yield trans-cinnamic acid and ammonia.1 However, the reaction is reversible under conditions of high

ammonia concentration and synthetically useful conversions in the amination direction can be obtained.

PALs therefore represent potentially attractive biocatalysts for the synthesis of enantiomerically pure L-

amino acids from substituted cinnamic acids and ammonia (figure 1). The broad use of PALs as biocatalysts

for the synthesis of non-natural amino acids is currently limited by their relatively narrow substrate range.

The application of eukaryotic PALs as biocatalysts for the amination of cinnamic acid analogues has been

described previously.2 However, the activity of prokaryotic PALs towards non-natural substrates has not

been investigated. The bacterial PAL from Anabaena variabilis(AvPAL) has recently received attention as a

potential therapeutic enzyme for the metabolic disorder phenylketonuria.3 An available crystal structure of

this enzyme makes it a suitable target for structure guided directed evolution.4 We have examined the

activity and enantioselectivity of AvPAL towards a broad range of non-natural substrates and compared this

activity with the eukaryotic PALs from the yeast Rhodotorula glutinis (RgPAL) and parsley Petroselinum

crispum (PcPAL). AvPAL shows significantly higher activity towards a series of non-natural substrates than

previously described eukaryotic PALs. Interestingly, some non-natural substrates also led to significant

formation of D-amino acids and the catalytic mechanism has been investigated.

Key references:

(1) N. J. Turner, Curr. Opin.Chem. Bio., 2011, 15, 234; J. Ward and R. Wohlgemuth, Curr. Org. Chem, 2010,

14, 1914

(2) A. Gloge, J. Zón, Á. Kövári, L. Poppe, and J. Rétey, Chem. Eur. J., 2000, 6, 3386; C. Paizs, A. Katona and J.

Rétey, Eur. J. Org. Chem, 2006, 5, 1113; A. Gloge, B. Langer, L. Poppe and J. Rétey, Arch. Biochem. Biophys.,

1998, 359, 1; S. Bartsch and U. T. Bornscheuer P.E.D.S., 2010, 23, 929.

(3) M. C. Moffitt, G. V. Louie, M. E. Bowman, J. Pence, J. P. Noel and B. S. Moore, Biochemistry, 2007, 46,

1004

(4) L. Wang, A. Gamez, H. Archer, E. E. Abola, C. N. Sakissian, P. Fitzpatrick, D. Wendt, Y. Zhang, M. Velard, J.

Bliesath, S. M. Bell, J. F. Lemontt, C. R. Scriver and R. C. Stevens, J. Mol. Biol., 2008, 380, 623.

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Talk 2

THE DEVELOPMENT OF A NEW CLASS OF MALEIMIDES AS REAGENTS FOR PROTEIN MODIFICATION

Authors: Cristina Marculescu, Dr. James Baker, Dr. Rachel Morgan, Dr. Lyn Jones.

Presenting author affiliation: University College London, 20 Gordon St., London, WC1H 0AJ.

Other affiliations: Pfizer

In 2009, the Baker group reported on the bromomaleimides as the first of a new class of reagents

that could be efficiently used for the highly selective and reversible modification of cysteine and for the

bridging of disulfide bonds in proteins.1,2,3 Aiming to prove that these transformations are not restricted to

bromomaleimides, the present work presents a library of novel analogues, bearing different leaving groups

on the double bond. By controlling the chemistry of this class of compounds we were able to tune

properties such as selectivity, reactivity, solubility, and cross reactivity with reducing agents.

Fig. 1 Mono- and disubstituted maleimide analogues.

The utility of the novel monosubstitued analogues as protein labelling reagents was shown using a

single cysteine mutant of protein Grb2 (L111C) as a model system. The disubstitued analogues were tested

as cystine bridging reagents therefore the model system used was somatostatin, a 14-aminoacid peptide

containing a disulfide bridge. A novel dual modification of peptides method will be presented.

Fig. 2 Reactivity profiles of mono and di-substituted maleimides with the model systems.

Key references:

(1) Tedaldi, L. M.; Smith, M. E. B.; Nathani, R. I.; Baker, J. R., Chem. Commun. 2009, 6583.

(2) Smith, M. E. B.; Schumacher, F. F.; Ryan, C. P.; Tedaldi, L. M.; Papaioannou, D.; Waksman, G.; Caddick,

S.; Baker, J. R. J. Am. Chem. Soc. 2010, 132, 1960.

(3) Schumacher, F. F.; Nobles, M.; Ryan, C. P.; Smith, M. E. B.; Tinker, A.; Caddick, S.; Baker, J. R. Bioconj.

Chem. 2011, 22, 132.

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Talk 3

Catalytic conversion of unactivated nitriles into N-substituted amides

Authors: Simge Davulcu, Jonathan M J Williams

Presenting author affiliation: University of Bath

The amide bond is essential to sustain life, making up the peptide bonds in proteins such as enzymes. It is

found in numerous natural products and biologically active molecules (Scheme 1). Despite their

importance, no currently used industrial methods for constructing the amide bond are particularly

“practical” or atom–efficient.

Scheme 1. Amide bonds in natural products and biologically active molecules

We have developed a zinc triflate and hydroxylamine hydrochloride catalysed methodolgy for direct

conversion of unactivated nitriles into N-substituted amides (Scheme 2). The reaction proceeds in

environmentally friendly water and provides a straightforward, atom-efficient methodology to synthesise

secondary and tertiary amides from nitriles which is a rarely reported transformation in the literature.

Scheme 2. Amide synthesis from nitriles and amines in water

The zinc triflate in combination with hydroxylamine hydrochloride salt efficiently catalyses the direct

conversion of unactivated nitriles into N-substituted amides with both primary and secondary amines.

Possible mechanisms for this reaction are discussed and evidence for initial amidoxime and amidine

formation pathways are reported. Isolated yields vary from 25-96%.

Key references:

(1) S. Davulcu, C. L. Allen, K. Milne and J. M. J. Williams, ChemCatChem 2013, 5, 435-438.

(2) C. L. Allen and J. M. J. Williams, Chemical Society Reviews 2011, 40, 3405-3415.

(3) C. J. Cobley, M. van den Heuvel, A. Abbadi and J. G. de Vries , Tetrahedron Letters 2000, 41, 2467-2470.

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Talk 4

Hybridised-SELEX: a capillary electrophoresis based method for maximising the number of aptamers

screened

Authors: Jon Ashley, Sam Fong Yau Li

Presenting author affiliation: Department of Chemistry, National University of Singapore, 3 Science Drive 3

Singapore 117543

Aptamers are ssDNA or ssRNA which specifically bind to biomolecules such as proteins, small molecules and

even whole cells. DNA aptamers are usually selected from a library of random sequences using a method

called Systematic evolution of ligands by exponential enrichment (SELEX) (Ellington and Szostak 1990). A

number of post SELEX modifications have appeared in the literature to address the issues associated with

the use of SELEX such as reducing the number of rounds of selection needed. Capillary electrophoresis

based methods such as CE-SELEX and Non-SELEX are attractive alternatives due to the high separation

efficiency, which can allow a selection to be carried out in <5 rounds. Also CE-SELEX and Non-SELEX are

carried out in free solution removing the necessity to immobilize the target and allowing the aptamers to

bind with the whole target (Mendonsa and Bowser 2004; Berezovski, Musheev et al. 2006). Moreover Non-

SELEX removes the need for intermittent amplification reducing the likelihood of contamination. However

due to the small sample sizes associated with CE, the number of sequences screened is limited, reducing

the likely hood of finding aptamers with high binding affinity. In this research, we propose a method which

significantly increases the number of sequences screened during the aptamer selection using capillary

electrophoresis. One round of selection using nitrocellulose filter was combined with subsequent rounds of

selection using Non-SELEX (Fig. 1).

Fig. 1 A general scheme for the hybridized SELEX procedure

The NC filter removes all non-binding sequences in the first round leaving non-specific binding sequences

and the target bound sequences. Non-specific binding sequences are then removed in the capillary

partitioning steps. Aptamers towards Cholesterol esterase were selected in less than 3 rounds of selection.

An Aptamer with a binding affinity KD of 116.6 ± 4.7 nM and good specificity was successfully selected.

Key references:

Ellington, A. D. and J. W. Szostak (1990). "IN VITRO SELECTION OF RNA MOLECULES THAT BIND SPECIFIC LIGANDS."

Nature 346(6287): 818-822.

Mendonsa, S. D. and M. T. Bowser (2004). "In vitro evolution of functional DNA using capillary electrophoresis."

Journal of the American Chemical Society 126(1): 20-21.

Berezovski, M., M. Musheev, et al. (2006). "Non-SELEX selection of aptamers." Journal of the American Chemical

Society 128(5): 1410-1411.

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Talk 5

A Versatile Synthetic Approach to Novel Deoxy Sugar Analogues

Authors: Ryan J. Beattie, Christine L. Willis, M. Carmen Galan

Presenting author affiliation: School Of Chemistry, University of Bristol

Carbohydrates are one of the most abundant and important class of biomolecules, implicated in roles as

energy sources, structural frameworks (e.g. cellulose) and glycobiology as motifs for intracellular

recognition, for example in immune response and metastasis. A subclass within this family are the deoxy

sugars, which are components of a number of important biologically active natural products.1 However

studies into their roles of mediating pharmacological activity are limited by a lack of rapid and versatile

access to defined carbohydrates within this important class of compounds.2

To this end, we have developed methodology based upon Prins cyclisations3 to access stereodefined

tetrahydropyran scaffolds towards deoxy sugars targets and their analogues. A novel cyclisation involving

homoallylic alcohols and silyl acetals has been established, which, with subsequent Tamao-Fleming

oxidation affords lactols that can be converted in the same pot to O-acetates. By varying the reaction

conditions for the cyclisation, different groups (X) may be introduced. The approach is ideally suited for the

divergent syntheses of libraries of analogues for biological evaluation and these studies will be described.

Key references:

(1) Weymouth-Wilson, A. C., Nat. Prod. Rep. 1997, 14, 99.

(2) Kirschning, A.; Jesberger, M.; Schoning, K. U., Synthesis-Stutt. 2001, 507.

(3) Olier, C.; Kaafarani, M.; Gastaldi, S.; Bertrand, M. P., Tetrahedron, 2010, 66, 413.

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Talk 6

Tailoring Complex Natural Products by Altering the Biosynthetic Machinery that Builds Them

Authors: Matthew Styles


Natural products are often referred to as ‘privileged’ structures in that they are predisposed for protein

binding or other biological activity. This high propensity for bioactivity makes them an important source of

drug scaffolds in modern drug design. Nonribosomal peptides represent an important class of secondary

metabolite natural products biosynthesised by huge assembly line enzymes known as nonribosomal

peptide synthetases. These synthetases are capable of producing extremely complex products, containing

not just a huge range of natural and non-proteinogenic amino acids, but a large number of other

modifications such as halogenations and glycosylations which would be practically unattainable from a

purely synthetic standpoint.

The genes responsible for these elaborate systems tend to be clustered together in the organisms that

produce them. This gene organisation, coupled with the modular nature of the nonribosomal peptide

synthetases, has led to the conclusion that it would be possible to gain access to this biochemical space

through manipulation of the genes themselves. It is conceivable to envisage the redesign of such systems in

order to carry out complex biotransformations and produce altered or ‘unnatural’ natural products which

may have optimised or novel activity.

In this work it is shown how we can manipulate two antibiotics, calcium dependent antibiotic (CDA) and

enduracidin. In CDA biosynthesis we modify the adenylation domain responsible for the incorporation of

the polar 3-methyl-glutamate, so that it instead accepts the non-polar 3-methyl-glutamine, an unnatural

non-proteinogenic amino acid not observed in nature.

The biosynthesis of enduracidin is altered by the integration of a putative mannosyl transferase gene,

ram29, taken from the producer of the antibiotic ramoplanin, onto the chromosome of Streptomyces

fungicidicus, the enduracidin producing organism. This results in new variants of enduracidin being

produced with an additional mannosyl group attached to a specific residue.

(1) J. Thirlway, R. Lewis, L. Nunns, M. Al Nakeeb, M. Styles, A.-W. Struck, C. P. Smith and J. Micklefield,

Angewandte Chemie International Edition 2012, 51, 7181-7184.

CDA

CDA3a: R1=H, R

2=OH

CDA4a: R1=CH3, R

2=OH

CDA3a-10Q: R1=H, R

2=NH2

CDA4a-10Q: R1=CH3, R

2=NH2

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Talk 7

Study of (+)-δ-cadinene synthase mechanism using farnesyl diphosphate analogues.

Authors: Oscar Cascón, Juan A. Faraldos, Veronica Gonzalez, David J. Miller, Rudolf K. Allemann*

Presenting author affiliation: School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Park

Place, Cardiff, CF10 3AT, United Kingdom., [email protected]

(+)-δ-Cadinene synthase (DCS) from Gossypium arboreum catalyzes the metal-dependent

cyclisation of (E,E)-farnesyl diphosphate (FDP) to the sesquiterpene δ-cadinene, the parent

hydrocarbon of prominent cotton phyotoalexins such as gossypol. In contrast to other

sesquiterpene cyclases, DCS catalyses the reaction with exquisite fidelity producing > 98% of the

final bicyclic hydrocarbon, but as a consequence, the enzyme leaves no mechanistic traces of its

mode of action.

Here we report a detailed investigation of the catalytic mechanism of DCS using a variety of FDP

analogues as mechanistic probes including isotopically labelled FDPs, fluorinated FDPs and C2,C3

double-bond isomers of FDP and 2F-FDP. Analysis of the reaction products arising from the

incubation of these mechanistic probes with DCS gave results that were not consistent with either

proposed catalytic mechanism; rather they suggest a mechanism for the DCS catalyzed

conversion of FDP to δ-cadinene that involves a 1,6-cyclization of FDP to α-bisabolyl cation.

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Talk 8

The natural product inhibitor myriocin displays a unique dual mode of action against serine

palmitoyltransferase.

Authors: John M. Wadswortha, David J. Clarkea, Stephen A. McMahonb, Ashley E. Beattiea, Pat

Langridge-Smitha, Teresa Dunnc, James H. Naismithb and Dominic J. Campopianoa

Presenting author affiliation: aEaStChem, School of Chemistry, University of Edinburgh, Scotland, EH9 3JJ Other affiliations: aEaStChem, School of Chemistry, University of Edinburgh, Scotland, EH9 3JJ bEaStChem, School of Chemistry, University of St Andrews, Scotland, KY16 9ST cBiochemistry & Molecular Biology, Uniformed Services University of the Health Sciences 4301 Jones Bridge Road, Bethesda, Maryland, 20814

Sphingolipids (SLs) and ceramides are essential components of cellular membranes and important signalling

molecules. Because of a growing appreciation of their diverse biological roles, understanding of the

biosynthesis and regulation of SLs has recently become a key goal in drug discovery. Serine

palmitoyltransferase (SPT) is a pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyses the

condensation between L-serine and a long-chain (C16) acyl thioester. This first step in SL biosynthesis is

conserved in all organisms studied to date, from microbes to man. Myriocin, a natural product from the

fungus Mycelia sterilia, is a potent inhibitor of SPT, however, the molecular details of inhibition are not fully

understood. Myriocin contains a long alkyl chain and a polar head group so displays features of both SPT

substrates. Thus, the prevailing hypothesis is that inhibition of SPT occurs because myriocin acts as a mimic

of a key transition state of the catalytic mechanism. We have used a combination of UV-vis spectroscopy,

mass spectrometry, x-ray crystallography and enzyme inhibition assays to study the interaction between S.

paucimobilis SPT and myriocin. We show that myriocin initially forms an inhibitory PLP:myriocin aldimine

complex in the active site that displays a Ki of 967 nM. Interestingly, this complex is succeptable to

unexpected, slow enzymatic degradation. The mechanism for myriocin breakdown has been elucidated as a

retro-aldol type reaction, which results in cleavage of the C2-C3 bond producing a C18 aldehyde. This

aldehyde is then capable of covalently modifying the active site lysine, forming a second (suicide) inhibitory

complex and rendering the enzyme catalytically inactive. This novel mechanism has implications on the SAR

and design of drugs targeted towards SPT, the role of feedback regulation by long chain aldehydes and

further expands the range of reactions catalysed by this important enzyme.

10

Poster 1

The Development of Biophysical Assays for Protein-Protein Interactions: HIF-1α/p300 and eIF4e/eIF4g

Authors : George Burslem, Hannah Kyle, Thomas Edwards, Adam Nelson, Stuart Warriner, Andrew Wilson

Presenting author affiliation: University of Leeds

Historically medicinal chemistry and drug discovery have targeted enzyme/substrate interactions to meet a

clinical need however protein-protein interactions (PPIs) are implicated in virtually all biological pathways

and are emerging as new targets for medical intervention. Enzyme inhibition assays are often biochemical

in nature and are based on the measurement of either the substrate or product of the enzyme catalysed

process. Often for PPIs no such process exists so only biophysical assays which determine the energy of

interaction (isothermal titration calorimetry) or the proximity of two proteins (fluorescence anisotropy or

surface plasmon resonance) can be used.

Herein we report the development of novel biophysical assays for 2 PPIs of interest to the oncology field,

which could potentially be used to identify inhibitors. We have developed and validated fluorescence

anisotropy assays for the eukaryotic initiation factor (eIF) 4e/eIF4g interaction and the hypoxia inducible

factor (HIF) 1α/p300 interaction as well as developing orthogonal biophysical assays. These assays have also

allowed us to further elucidate the contributions of certain regions of the protein to the interaction affinity,

which could potentially inform the design of inhibitors.

Key references: (1) T.A. Edwards and A.J. Wilson, Amino Acids, 2011, 41, 743 (2) C.J. Brown, C. S. Verma,

M.D. Walkinshaw and D.P. Lane, Cell Cycle, 2009, 8,1905 (3) S.J. Freedman, Z-Y. J. Sun, F. Poy, A.L. Kung,

D.M. Livingston, G. Wagner and M.J. Eck, P. Natl. Sci. USA., 2002, 8, 5367

11

Poster 2

Re-engineering Riboswitches as Novel Gene Expression Tools

Authors: Neil Dixon, Christopher J. Robinson , Phillip Lowe, Ming-Cheng Wu, Helen Vincent, James Leigh

and Jason Micklefield

Presenting author affiliation: The University of Manchester

Other affiliations: BBSRC

The ability to independently control the expression of multiple genes in response to small molecule

inducers or repressors is highly desirable in the study of metabolic pathways; has numerous applications in

the fields of drug discovery and synthetic biology; and has commercial potential in the production of

therapeutic proteins. In light of this, we have developed an entirely novel method of controlling gene

expression by re-engineering naturally occurring RNA regulatory elements known as riboswitches, so that

they no longer respond to intracellular metabolites, but instead can be controlled by synthetic small

molecules which are not naturally present in the cell. Riboswitches are structured regulatory genetic

elements, which specifically bind small molecule metabolites to regulate gene expression through

conformational mechanisms, including transcriptional termination, translational initiation, pre-mRNA

splicing and mRNA self-cleavage.

We chose to study the adenine-responsive riboswitch from the add gene of Vibrio vulnificus, which induces

gene expression upon ligand binding by releasing the ribosome-binding site from a repressor stem. We

selectively mutated residues which contact the adenine ligand, these mutant riboswitches were then

screened for in vivo control of gene expression using a library of over 100 heterocyclic molecules. Mutant

riboswitch-ligand pairs were identified, which were no longer responsive to adenine, but which responded

to specific synthetic small molecules, allowing for highly responsive, dose-dependent and dynamic control

of gene expression in vivo. We have also shown that mutually orthogonal riboswitches can be deployed

within the same bacterial cell to independently control the co-expression of multiple genes in a dose-

dependent fashion, in response to distinct small molecule inducers. Building on these principles, we have

subsequently re-engineered the PreQ1-binding riboswitch from the preC gene of Bacillus subtilis, to

generate a riboswitch which prematurely terminates mRNA transcription upon addition of a novel synthetic

ligand, thereby demonstrating the general applicability of our approach for expanding the repertoire of

novel gene expression tools.

Key references:

1. Dixon N, Duncan J, Geerlings T, Dunstan M, Leys D, Micklefield J. (2010) Re-engineering

orthogonally selective riboswitches. Proceedings of the National Academy of Sciences of the United

States of America. 2010; 107(7): 2830-2835.

2. Dixon N, Robinson C, Geerlings T, Duncan J, Drummond S, Micklefield J. (2012) Orthogonal

riboswitches for tuneable co-expression in bacteria. Angewandte Chemie (International ed. in

English). 2012 April; 51(15): 3620-4.

12

Poster 3

Investigating the Programming of Iterative Polyketide Synthases

Authors: Douglas Roberts, David Ivison, Russell Cox

Presenting author affiliation: University of Bristol

Other affiliations: Leibnez Universität Hannover

Highly-reducing iterative type I polyketide synthases (iPKS) produce a diverse range of chemical structures.

iPKS are able to do this by condensing acetate units together and then further reductive domains are able

to instill additional functionality into the molecule. iPKS differ from other classes of polyketide synthase as

they only have a single copy of each reductive domain. In each round of chain extension these domains

have to be switched on and off, this commonly known as programming. Programming of iPKS is not fully

understood and it remains unclear the role that individual domains have.

Scheme 1: The mechanism of polyketide chain extension and the active domains required for the synthesis of 1

In this project the ER domain of squalestatin tetrakedie synthase (that produces 1) was isolated as a soluble

active protein and was assayed in vitro with synthesized mimics of polyketdide biosyntheis. This

methodology enables a thorough examination of which structural features triggers programming.

Figure 1: Results of in vitro assays of the isolated ER domain of SQTKS with substrate mimics as percentage turnover against time

(mins)

Figure 1 shows results of some of the assays, this shows that the isolated ER domain is unable to turnover

the tetraketide substrate mimic. Assays with further substrate mimics have shown that the programming is

likely to be casused by methylation in the backbone and not by chain-length. It has also been shown by

NMR experiments that the domain is unable to place the correct stereochemistry at the α-position. The

exact reason for this remains unclear but further assays have been designed to test this further.

Key references:

(1) Cox, R.J. Org Biomol Chem, 2007, 5, 2010

13

Poster 4

Approach Towards a Novel Mimetic Heparanase Inhibitor

Authors Garrett T. Pottera, Gavin J. Millera, Gordon C. Jaysonb, John M. Gardinera

Presenting author affiliation: aManchester Institute of Biotechnology - The University of Manchester

Other affiliations: bThe Patterson Institute for Cancer Research

Heparanase (Hpa1) is an enzyme present in nearly all forms of cancer, usually found on the growth front of

tumours.1 It plays a key role in cancerous formation, growth, angiogenesis, metastasis, and resulting

adhesion.2 Hpa1 has a natural affinity for Heparan Sulfate (HS) chains that are ubiquitous to the surface of

cells, where the enzyme docks to and cleaves the chains to release growth factors necessary for tumour

growth. The region of binding requires a specific sulfation and sugar pattern not yet utilised by drug

developers, though other sugar-based heparanase inhibitors are in advanced clinical trials (i.e. PI-88). We

suggest that, by following a more strict interpretation of sulfation and sugar sequencing, by imitating that

which is naturally occuring and favoured by Hpa1, we will increase binding affinity and form a potentially

more effective cancer treatment.

Through the culmination of research done by various groups for over three decades, the trisaccharide core

necessary for recognition and binding by Hpa1 has been elucidated, as has its sulfation pattern.3

Additionally, computer modelling has suggested this core is accurate for enzyme recognition and binding.4

We propose a synthesis of this core with a functional handle attached that could then potentially be

adapted for ADMET purposes and used in chemical biological investigations (Figure 1). This synthesis uses,

in part, selectively protected monomers developed from precursors made within our group.5

Key references:

(1) N. Ilan, M. Elkin, and I. Vlodavsky, Int. J. Biochem. Cell Biol., 2006, 38, 2018–2039.

(2) D. Liu, Z. Shriver, G. Venkataraman, Y. El Shabrawi, and R. Sasisekharan, Proc. Natl. Acad. Sci. U. S. A., 2002, 99, 568–573.

(3) D. Liu and R. Sasisekharan in Chemistry and Biology of Heparin and Heparan Sulfate, ed. H. G. Garg, R. J. Linhardt, and Charles A.

Hales, Elsevier, Amsterdam, 2005, pp. 699–725.

(4) N. Sapay, É. Cabannes, M. Petitou, and A. Imberty, Biopolymers, 2012, 97, 21–34.

(5) S. U. Hansen, G. M. Miller, M. Baráth, K. R. Broberg, E. Avizientye, M. Helliwell, J. Raftery, G. C. Jayson, and J. M. Gardiner, J. Org.

Chem., 2012, 77, 7823–7843.

14

Poster 5

The next generation of CtBP inhibitors and their anti-tumour effects

Authors: Charlotte Mardle, Jeremy Blaydes, Ali Tavassoli

Presenting author affiliation: University of Southampton, School of Chemistry, SO17 1BJ

Other affiliations: University of Southampton, Faculty of Medicine

C-terminal binding proteins (CtBP1 and CtBP2) are transcriptional co-repressors with roles in cell-cycle

regulation and development. They have been identified as a potential target for cancer therapeutics as

their repression targets include pro-apoptotic genes and essential adhesion molecules. The repression of

these genes can promote tumour-cell survival, epithelial-to-mesenchymal transition and the migration of

tumour cells1. The function of CtBPs is dependent on its oligomeric state, as it is CtBP homo-and hetero-

dimer complexes that recruit the transcription factors necessary to mediate transcription. CtBPs dimerise

upon binding to NADH. The increase in NADH in tumour cells, caused by hypoxia and/or increased aerobic

glycolysis, means that they are particularly dependent on CtBP dimerisation compared to normal cells.

Inhibiting CtBP dimerisation will therefore directly target the stress-response pathways in cancer cells.

A small-molecule inhibitor of CtBP dimerisation, which works at low concentrations, could be a useful tool

in the investigation of the roles of CtBPs in cancer and potentially used as a basis for the design of a

therapeutic agent targeting CtBPs.

A cyclic peptide inhibitor of CtBP1 homo-dimerisation has been identified via high-throughput-screening of

a 3.2 million compound library generated by split-inteins (SICLOPPS)2 and a bacterial reverse-two hybrid

system (RTHS). A FRET assay has been developed, based on previously reported work3, to test the ability of

the inhibitor to prevent the binding of CtBP1 to NADH. Currently alanine analogues of the peptide inhibitor

are being synthesised to identify the active motif using the FRET assay and further in vitro techniques.

Furthermore screening to identify specific inhibitors to CtBP2 homo-dimerisation is being undertaken using

SICLOPPs technology.

Key references:

(1) Straza MW, Paliwal S, Kovi RC, Rajeshkumar B, Trenh P, Parker D, et al. Cell Cycle 2010, 9(18): 3740-

3750.

(2) Tavassoli A, Benkovic SJ. Nat Protoc 2007, 2(5): 1126-1133.

(3) Fjeld CC, Birdsong WT, Goodman RH. Proceedings of the National Academy of Sciences 2003,

100(16): 9202-9207.

15

Poster 6

Regioselective N-7 methylation of purine derivatives

Authors: Honorine Lebraud, Benoit Carbain, Bernard T. Golding, Celine Cano, Ian R. Hardcastle, and Roger J. Griffin.

Presenting author affiliation: Northern Institute for Cancer Research at the Newcastle Cancer Centre, School of

Chemistry, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK

As part of a project directed towards the synthesis of purine-based irreversible inhibitors of Nek2,1 a cell

cycle-associated kinase implicated in cancer, control compounds were required to assess the selectivity of a

lead series of 2-aminoaryl-6-ethynylpurines in cellular assays. In particular, derivatives bearing a methyl

substituent at the purine N-7 position were required, as this modification abrogates Nek2-inhibitory activity

without imposing dramatic structural changes elsewhere. Although a number of literature procedures

describe selective N-7 methylation of purines,2 these were found to be of limited value for purines bearing

a diverse range of substituents at the 2- and 6-positions.

New methodology has been developed for the regioselective methylation of substituted purines, inspired

by the N-7 alkylation of guanine residues in DNA following exposure to alkylating agents.3, 4 In the absence

of repair of these lesions by glycosylases, depurination occurs to generate potentially mutagenic abasic

sites.5 We reasoned that analogous N-7 alkylation of purines bearing a suitable N-9 protecting group, would

facilitate cleavage of the N-9 substituent to give the requisite N-7 substituted purines. To this end, the

introduction of a para-methoxybenzyl (PMB) group at the purine N-9 position was readily achieved

employing PMB-chloride in N,N-dimethylformamide. Methylation at the purine N-7 position with

trimethyloxonium tetrafluoroborate, followed by heating in 2,2,2-trifluoroethanol (TFE) under microwave

conditions in a ‘one-pot’ reaction, furnished the target N-7 methylpurines in excellent yields. This new

method was found to have broad applicability, and was also amenable for the introduction of ethyl

substituents at the purine N-7 position utilising triethyloxonium tetrafluoroborate.

This approach enabled the preparation of a defined series of N7-methylpurine derivatives, varied with

respect to substituents at the 2- and 6-positions, for subsequent biological studies. The synthesis and

biological activity of 6-substituted 2-arylamino-N7-methylpurines will be discussed.

References 1. Fry, A. M., The Nek2 protein kinase: a novel regulator of centrosome structure. Oncogene 2002, 21 (40), 6184-94. 2. (a) Dalby, C.; Bleasdale, C.; Clegg, W.; Elsegood, M. R. J.; Golding, B. T.; Griffin, R. J., Regiospecific alkylation of 6-chloropurine and 2,6-dichloropurine at N7 by transient protection of N3/N9 by methylcobaloxime. Angew. Chem. Int. Ed. Engl. 1993, 32 (12), 1696-1697; (b) Kotek, V.; Chudikova, N.; Tobrman, T.; Dvorak, D., Selective Synthesis of 7-Substituted Purines via 7,8-Dihydropurines. Org. Lett. 2010, 12 (24), 5724-5727; (c) Kohda, K.; Baba, K.; Kawazoe, Y., Chemical reactivity of alkylguanines. I. Methylation of O6-methylguanine derivatives. Tetrahedron Lett. 1987, 28 (50), 6285-6288. 3. Barrows, L. R.; Magee, P. N., Nonenzymatic methylation of DNA by S-adenosylmethionine in vitro. Carcinogenesis 1982, 3 (3), 349-351. 4. Boysen, G.; Pachkowski, B. F.; Nakamura, J.; Swenberg, J. A., The formation and biological significance of N7-guanine adducts. Mutat. Res. 2009, 678 (2), 76-94. 5. Gates, K. S.; Nooner, T.; Dutta, S., Biologically Relevant Chemical Reactions of N7-Alkylguanine Residues in DNA. Chem. Res.

Toxicol. 2004, 17 (7), 839-856.

16

Poster 7

SYNTHESIS AND APPLICATION OF GLUCURONIDATED

3'-DEOXY-3'-[19F]FLUOROTHYMIDINE ([19F]FLT)

Suzannah J. Harnor, Tommy Rennison, Roger J. Griffin, David R. Newell,

Céline Cano and Bernard T. Golding

Newcastle Cancer Centre, Northern Institute for Cancer Research, School of Chemistry, Bedson Building,

Newcastle University, Newcastle upon Tyne, NE1 7RU, UK.

Email: [email protected]; Tel: 0191 222 6913

The glucuronidation of endogenous substrates and xenobiotics is a crucial detoxification process during

phase II metabolism, which may give rise to inactive or active metabolites, e.g. morphine-6-glucuronide.[1]

3'-Deoxy-3'-fluorothymidine (FLT, 1) is a nucleoside analogue that bears a fluoro substituent at the 3'-

position of the ribose ring of thymidine. [18F]FLT has been established as a valuable biomarker for imaging

tumour proliferation by positron emission tomography (PET). Conversion of 1 into the 5'-glucuronide is a

major route of metabolism for this nucleoside analogue.

To our knowledge, the chemical synthesis of [19F]FLT-glucuronide 2, has not been reported in the literature.

Therefore, the overall objective was to develop an efficient synthetic route for the preparation of 2, for use

as an HPLC standard in the analysis of plasma samples from patients undergoing [18F]FLT PET scanning. The

de novo synthesis of 2 was successfully achieved in four steps, through glycosylation of FLT 1, with the

trichloroacetimidate of suitably protected glucuronic acid.[2]

References

1. S. De Gregori, M. De Gregori, G. N. Ranzani, M. Allegri, C. Minella and M. Regazzi, Metab. Brain Dis., 2012, 27, 1

2. A. V. Stachulski and G. N. Jenkins, Nat. Prod. Rep., 1998, 15, 173

17

Poster 8

Hit Validation of ERK5 Inhibitors: Expectations and Challenges

Stephanie Myers,1 Lauren Barrett,

1 Ruth Bawn,

1 Betty Cottyn,

1 Bernard T. Golding,

1 Roger J.

Griffin,1 Tim Hammonds,

2 Hing Leung,

3 Nicholas Martin,

1 David Newell,

1 Laurent Rigoreau,

2 Ai-

Ching Wong,2 Ian R. Hardcastle,

1 and Celine Cano.

1

1 Northern Institute for Cancer Research and Newcastle Cancer Centre, Newcastle University, Newcastle Upon Tyne

NE1 7RU, UK 2 CRT Discovery Laboratories, Cambridge, CB1 3LQ

3 The Beatson Institute for Cancer Research, Glasgow University, Glasgow, G61 1BD

[email protected]

Extracellular signal-regulated kinase 5 (ERK5) is a member of the protein kinase superfamily,

which plays an essential role in the transduction of extracellular signals to intracellular effectors.

Activation of the ERK5 signalling pathway is associated with cell survival, proliferation, and

differentiation, and thus ERK5 over-expression may have implications in carcinogenesis.1 High

levels of ERK5 are associated with poor patient prognosis and the presence of bony metastases in

advanced prostate cancer.2 It has recently been discovered that ERK5 is also involved in the

development and progression of hepatocellular carcinoma (HCC).3 Therefore, the discovery and

development of small molecule inhibitors of ERK5 is a desirable therapeutic area.

High throughput screening (HTS) of chemical libraries conducted by Cancer Research Technology

revealed several distinct chemical series as moderate inhibitors of ERK5. Benzo[d]thiazole (1), 4-

aminopyrimidine-5-carbonitrile (2) and 3-cyanopyridine (3) based compounds were selected for hit

validation by resynthesis. The synthesis of these chemically diverse targets will be described.

Various challenges as a result of the high throughput screening approach were encountered, which

will also be discussed.

References:

1. Wang, X.; Tournier, C.; Cell. Signal. 2006, 18, 753

2. McCracken, S.R.C.; Ramsay, A.; Heer, R. et al. Oncogene. 2008, 27, 2978-2988

3. Zen, K.Y.; Yasui, K.; Nakajima, T. et al. Genes, Chrom and Cancer. 2009, 48, 109-120

mailto:[email protected]

18

Poster 9

Design and Synthesis of Stapled α-Helix Mimetics: New Synthetic Tools to Investigate the Role of

HDACs in Cancer

Authors: Naomi S. Allaway*, Peter J. Watson, John W.R. Schwabe, Andrew G. Jamieson

Presenting author affiliation: University of Leicester

In recent years, histone deacetylase (HDAC) enzymes have emerged as important oncology drug targets because they have been linked mechanistically to the pathogenesis of cancer and can be regulated by small molecule inhibitors.1 Indeed, a number of HDAC active site inhibitors are currently in clinical trials and two drugs, vorinostat and romidepsin have been approved for the treatment of cutaneous T-cell lymphomas. However, these inhibitors are not isoform selective and so selective HDAC inhibitors are urgently required to investigate HDAC isoform phenotypes and provide targeted therapeutics.

Class 1 HDACs are generally recruited into large multi-subunit co-repressor complexes for maximal activity. However, little attention has been paid to targeting the disruption of these protein-protein interactions (PPIs) and so represents a novel approach to develop more specific therapeutics and chemical probes. Until Schwabe and co-workers solved the structure of HDAC3 bound to the deacetylase activation domain (DAD) from the human SMRT co-repressor (Figure 1),2 little was known about the structure of HDAC co-repressor complexes. Using this structural knowledge, we initiated a study to determine if a synthetic DAD mimetic could be designed to disrupt the HDAC3-DAD PPI and thus HDAC activity by using a stapled α-helix.

In this poster, we will present our most recent results, including the design, synthesis and biological

evaluation of a novel hydrocarbon stapled α-helix proteomimetic.

(1) B. Venugopal, T. R. J. Evans.; Developing histone deacetylase inhibitors as anti-cancer therapeutics.

Curr. Med. Chem. 2011, 18, 1658-1671.

(2) Watson, P. J.; Fairall, L.; Santos, G. M.; Schwabe J. W. R.; Structure of HDAC3 bound to co-repressor

and inositol tetraphosphate. Nature 2012, 481, 335-340.

(3) Blackwell, H.E. & Grubbs, R.H.; Highly Efficient Synthesis of Covalently Cross-Linked Peptide Helices by

Ring-Closing Metathesis Angew. Chem. Int. Ed. 1998 37, 3281–3284.

(4) Kim, Y. W.; Grossmann, T.N.; Verdine, G.L.; Synthesis of all-hydrocarbon stapled α-helical peptides by

ring-closing olefin metathesis. Nat. Protoc. 2011, 6, 761-771.

Figure 1. HDAC3-DAD complex.

Figure 2. General synthetic scheme of stapled peptide mimetic.3,4

19

Poster 10

Analysis of Exopolysaccharides Produced by Bifidobacteria

Authors: Sohaib Sadiq, Patricia Ruas-Madiedo & Andrew P. Laws

Presenting author affiliation: University of Huddersfield, Queensgate, Huddersfield, West Yorkshire, HD1 3DH

Other affiliations: Spanish Dairy Industry, Instituto de Productos Lacteos de Asturias – Consejo Superior de Investigaciones Cientificas (IPLA – CSIC) Villaviciosa, Asturias, Spain

A number of bifidobacteria are believed to be probiotic organisms which, when consumed, provide a

potential health benefits. It is thought the exopolysaccharides generated by these organisms contribute to

their biological activity. Our work is focused on characterising the exopolysaccharides from Bifidobacteria.

NMR spectroscopy, size exclusion chromatography, HPAEC analysis, monomer and linkage analysis

employing GCMS have been used to analyse the EPS structure of different friendly strains of Bifidobacteria.

NMR and size exclusion chromatography analysis shows the presence of more than one polysaccharide.

Monomer analysis for different batches suggests that each contain variable amounts of rhamnose, glucose

and galactose along with trace levels of uronic acids. The results of the linkage analysis indicate that a

complex mixture of differently linked sugars are present including : terminal rhamnose, 1,2- linked

rhamnose, 1,3-linked rhamnose, terminal hexoses, 1,2,3-linked rhamnose, 1,4-linked hexose, 1,3-linked

hexose, 1,6-linked hexose, N-acetyl sugars and 1,3,4-linked hexoses.

Key references:

(1) Laurence, D.M., Bronwen G.S., Determination of Uronic Acid content of plant cell walls using colorimetric assay, Current Protocols in Food Analytical Chemistry (2001), E3.3.1-F3.3.4, John Wiley and Sons,Inc.

(2) Wilson, D.J., Gabriel, E, Gee, S., Tracing the source of Campy, PLoS Genetics (2008), vol.4, issue 9, e1000203

20

Poster 11

Biosynthesis of [FeFe]-hydrogenase Cyanide Ligands

Authors: Rebecca C. Driesener, Benjamin R. Duffus, Eric M. Shepard, Joan B. Broderick, John W. Peters,

Peter L. Roach

Presenting author affiliation: University of Southampton, Chemistry, Southampton, SO17 1BJ (UK)

Other affiliations: Montana State University, Bozeman, MT 59717 (US)

The radical S-adenosyl-L-methionine (AdoMet) enzyme HydG catalyses L-tyrosine cleavage to yield p-cresol

and the the cyanide and carbon monoxide ligands for assembly of the [FeFe]-hydrogenase cofactor1, 2

(Figure). The HydG primary sequence contains two highly conserved cysteine motifs. These include the

canonical radical AdoMet CX3CX2C motif which coordinates a [4Fe4S] cluster for cosubstrate AdoMet

binding and the C-terminal CX2CX22C motif, thought to coordinate a second iron sulfur cluster. Mutations

in or absence of this C-terminal motif impair cyanide and carbon monoxide synthesis3.

The objective of this project is to investigate the role of the C-terminal cysteine motif in L-tyrosine cleavage

by spectroscopic and kinetic characterisation of wild-type and mutant HydG proteins. Using a Clostridium

acetobutylicum HydG C96/100/103A mutant unable to coordinate the N-terminal AdoMet cluster, we use

UV-Vis and EPR spectroscopy to unambiguously show that the C-terminal C386/389/412 cysteine motif

coordinates a second [4Fe4S] cluster. Spectroscopic comparison to a C-terminal deletion mutant (ΔCTD)

harbouring only the N-terminal cluster shows that both clusters have similar UV-Vis and EPR spectral

properties, but that AdoMet binding can only occur at the N-terminal cluster. A single C386S mutant can

partially coordinate both [4Fe4S] clusters. We further optimised pre-column derivatisation and HPLC

analyses to determine that the C386S and ΔCTD mutant HydG variants bind AdoMet with equal affinity

while the respective affinity for L-tyrosine is 7 and 45 fold reduced compared to wild-type HydG. Under

optimised in vitro conditions, the ΔCTD mutant was found to be completely impaired in cyanide formation,

while L-tyrosine cleavage was not affected. This strongly suggests that HydG harbours two distinct metallo-

active sites; the N-terminal cluster for tyrosine cleavage to p-cresol and the C-terminal cluster for synthesis

of cyanide and carbon monoxide.

(1) Driesener, R. C.; Challand, M. R.; McGlynn, S. E.; Shepard, E. M.; Boyd, E. S.; Broderick, J. B.; Peters, J. W.; Roach, P.

L. Angew. Chem. Int. Ed. 2010, 49, 1687 (2) Shepard, E. M.; Duffus, B. R.; George, S. J.; McGlynn, S. E.; Challand, M. R.;

Swanson, K. D.; Roach, P. L.; Cramer, S. P.; Peters, J. W.; Broderick, J. B. J. Am. Chem. Soc. 2010, 132, 9247 (3) Nicolet,

Y.; Martin, L.; Tron, C.; Fontecilla-Camps, J. C. FEBS Lett. 2010, 584, 4197

21

Poster 12

Aromatic Halogenases for Biocatalysis

Authors: Sarah Shepherd, Chinnan Velmurugan Karthikeyan, Mark Thompson, Matthew Styles, Anna-

Winona Struck and Jason Micklefield.


Halogenated aromatics are key components of pharmaceuticals, polymers, pesticides and many other

products of major industrial importance. Current methods to produce haloaromatics rely on toxic

halogenating agents, deleterious solvents and catalysts. More efficient and sustainable methods of

producing haloaromatics are required that utilise benign inorganic halides, aqueous media and natural

catalysts. These new methods would overcome problems associated with regiocontrol and environmental

issues.

Biohalogenases include heme- and vanadium-dependent haloperoxidases, α-ketoglutarate dependent

halogenases and flavin dependent halogenases. [1] The enzymes that are currently being studied are flavin-

dependent halogenases involved in the formation of Pyrrolnitrin, PrnA [2] as well as PyrH involved in

Pyrroindomycin biosynthesis [3] and a halogenase enzyme found in the Streptomyces toxytricini strain,

STTH.[4]

These enzymes have been used to chlorinate and brominate novel non-indolic substrates and the

regioselectivity of the products has been determined. Mutations have been made to improve activity and

regioselectivity of a less active substrate and work is ongoing to expand the range of substrates accepted

via mutagenesis.

Key references:

1. van Pee, K.H., et al., Biological halogenation has moved far beyond haloperoxidases. Advances in Applied Microbiology, Vol 59, 2006. 59: p. 127-157.

2. Dong, C.J., et al., Tryptophan 7-halogenase (PrnA) structure suggests a mechanism for regioselective chlorination. Science, 2005. 309(5744): p. 2216-2219.

3. Zehner, S., et al., A regioselective tryptophan 5-halogenase is involved in pyrroindomycin biosynthesis in Streptomyces rugosporus LL-42D005. Chemistry & Biology, 2005. 12(4): p. 445-452.

4. Zeng, J. and J.X. Zhan, Characterization of a tryptophan 6-halogenase from Streptomyces toxytricini. Biotechnology Letters, 2011. 33(8): p. 1607-1613.

22

Poster 13

Modulating protein-protein interactions – EB1 and SxIP proteins

Authors Teresa Almeida, Andrew Carnell, Igor Barsukov, Neil Berry

Presenting author affiliation: University of Liverpool

Protein-protein interaction inhibition is currently one of the major challenges in Drug Discovery.1 The

surface of interaction between two proteins is usually large, flat or moderately convex and cannot be

covered with a small molecule with drug like properties. Unlike other drug targets like enzymes, G-protein-

coupled receptors and ion channels there is no convenient natural substrate to act as a starting point for

small molecule design.2 As such discovery of molecules that modulate protein:protein interactions is a

contemporary challenge.

Microtubules are cellular polymers fundamental to many essential processes in eukaryotic cells including

maintenance of cell structure, accurate chromosome segregation, intracellular transport, and cell division.3

EB family members bind to microtubule growing ends and mediate the binding of other microtubule plus

end tracking proteins (+TIPs). Therefore, EB1 family members are considered as a key regulator of dynamic

+TIPs interaction networks at growing microtubule ends. A 4-residue sequence was shown to be

fundamental for this interaction – Ser, X, Ile, Pro – the SxIP motif.4

Working with the crystal structure of the EB1 – MACF complex, a struture-based molecular design approach

has been adopted to identify molecules that can modulate this interaction. Using a combination of

pharmacophore searching, molecular docking, scoring and ranking a virtual screening process has been

performed to identify candidate compounds.

Top scored/ranked compounds, with suitable drug-like properties, will be synthesised and tested. EB1 was

expressed and purified for binding assays using NMR and ITC techniques. The information gathered from

these assays will then be used to identify and further optimise molecular scaffolds for this target.

References

(1) Koes, D. R.; Camacho, C. J. Bioinformatics 2012, 28, 1951. (2) J. Wilson, A. Chemical Society Reviews 2009, 38, 3289. (3) Kumar, P.; Wittmann, T. Trends in Cell Biology 2012, 22, 418. (4) Kumar, P.; Chimenti, M. S.; Pemble, H.; Schönichen, A.; Thompson, O.; Jacobson, M. P.; Wittmann,

T. Journal of Biological Chemistry 2012, 287, 17050.

23

Poster 14

Characterization of Polysaccharides and Sophorolipids produced by the Yeast

Candida Bombicola

Ammar AL-Jasim

The University of Huddersfield, Department of Chemical and Biological Sciences

The yeast Candida bombicola (formerly Torulopsis bombicola) is a non-pathogenic yeast strain which is able

to produce extracellular glycolipidic biosurfactant known as sophorolipids (Fig.1). These types of glycolipids

are produced as a mixture of acidic and lactonic forms when the yeast grown on mixtures of carbohydrate

and fatty acids. It has been shown that SLs are efficient spermicides, anti-HIV drugs, and effective anti-

cancer agents. In addition to their use as deodorants, anti-dandruff agents and in pharmaceuticals field [1-

2]. Sophorolipids are low molecular weight biosurfactants consisted of a hydrophilic portion, the dimeric

sugar known as sophorose and a hydrophobic portion which is usually long chain fatty acid with a hydroxyl

group on either the penultimate or terminal carbon.

Fig.1: Chemical structures of lactonic and acidic sophorolipids.

The building blocks for sophorolipids synthesis are glucose (hydrophilic substrate) and fatty acid

(hydrophobic) and both can be provided in the production medium during biosynthesis. Those building

blocks can either be directly supplemented in the production medium or can be supplied as a triglyceride or

a fatty acid methyl or ethyl ester which will undergo extracellular hydrolysis by a lipase.

Sophorolipids biosynthesis starts with the hydroxylation of fatty acid after conversion of fatty acid to a

hydroxy-fatty acid, then two UDP- activated glucose molecules are added in a serial way and linked

glycosidically to the fatty acid hydroxyl group.

The aims of this study include the purification and identification of sophorolipids structural forms by TLC

and the use of analytical separation techniques (silica gel column chromatography) to separate the possible

sophorolipidic forms produced by fermentation.

Also to characterise and elucidate the chemical structures of possible sophorolipids using various analytical techniques such as, 1H and 13C NMR which will be utilized for the identification of the molecular structures of sophorolipids: as it’s a powerful method which is able to identify functional groups as well as the position of them within the carbohydrate and lipid molecules. Finally to analyse and identify sophorolipids hydroxyl fatty acid moieties by acid methanolysis reaction

using GC-MS as well as the molecular weight distribution of sophorolipids using HPLC method.

24

Poster 15

Continued development of a fluorescence based assay in the search for novel inhibitors of aromatase

(AR)

Authors: Fortuny, N.M., Shah, P.S., Rudra, B.C., Owen, C.P., Ahmed, S.

Presenting author affiliation: Institute of Biomedical and Environmental Health Research (IBEHR), School of

Science, University of the West of Scotland, High Street, Paisley, Renfrewshire, PA1 2BE.

The cytochrome P-450 enzyme aromatase (AR) catalyses the conversion of androgens and androgen precursors (i.e. testosterone and androstenedione) to their respective estrogens (namely, estradiol and estrone) involving the oxidation of the C(19) methyl moiety of the substrates using three mols of NADPH and oxygen; resulting in the release of the estrogen and water. The traditional method of biochemical evaluation of compounds against AR has involved the use of radiolabelled substrate and which therefore leads to the release of tritiated water. A fluorescence-based assay has been reported previously as a high throughput alternative (using AR supersomes), however, the use this technique has (in our hands) proved to be difficult and troublesome, we therefore considered the modification of the assay so as to improve its efficiency so as to be able to compare against radiolabelled assay using cells as the source of AR as opposed to supersomes. Here, we therefore report the results of our initial study to re-develop the fluorescence assay and its use in the biochemical evaluation of both standard inhibitors (namely anastrozole, letrozole and aminoglutethimide) and novel inhibitors of AR. The non-radiolabelled assay involved the use of JEG-3 cells, 7-methoxy-4-trifluoromethyl coumarin as the substrate (resulting in 7-hyroxy-4-trifluoromethyl coumarin as the fluorescent product), media and methanol as the termination solution. The substrate was therefore incubated with the JEG-3 cells followed by the addition of methanol to terminate the reaction. The solution was then read for fluorescence and the inhibitory activity determined from the intensity of the emission. As previously stated, the assay in our hands proved to be difficult and inefficient, we therefore initially considered the basis of the assay and discovered that the reported wavelength stated proved to be the initial problem. That is, emission at 530nm was reported for the detection of the product, however, when we considered the emission spectra of both the substrate and product, we discovered that both possessed similar emission spectra. We therefore considered the excitation wavelength and the emission wavelength in a number of solvents and discovered that the best results (with the substrate and product differing significantly in their emission spectra) were when the excitation was initiated using 340nm. The development of a cell-based assay therefore involved the incubation of the substrate with JEG-3 cells which showed initial product formation but required extensive optimisation so as to produce an operational assay system. Using the assay, standard and novel inhibitors were evaluated and were found to give similar initial screening trends as the radiolabelled assay system, namely that aminoglutethimide is a weak inhibitor of AR in comparison to the triazole-based compounds currently in the clinic. In conclusion, the cell-based fluorescence assay for AR has significant limitations but has proved to be useful in the determination of inhibitory activity of novel inhibitors of AR.

25

Poster 16

Synthesis and biochemical evaluation of sulfamoylated derivatives of thiosemicarbazone-based

compounds as novel inhibitors of estrone sulfatase (ES)

Authors: Vasireddy, V.R., Rudra, B.C., Owen, C.P., Ahmed, S.


Science, University of the West of Scotland, High Street , Paisley, Renfrewshire, PA1 2BE.

The enzyme estrone sulfatase (ES) catalyses the conversion of estrone sulfate to the bio-active estrogen estrone (E1) in adipose and, in particular, breast cancer tissue; E1 is subsequently converted to estradiol (E2) involving the enzyme 17β-hydroxysteroid dehydrogenase type 1. Furthermore, ES has been shown to become the major source of estrogens in postmenopausal women as a result of the decrease in activity of ovarian aromatase (AR) and the expression of ES within estrogen-dependent breast cancer cells. As such, the inhibition of ES in postmenopausal women would be expected to lead to a decrease in E1 biosynthesis and therefore to a decrease in the stimulation of hormone-dependent breast cancer tissue. This would therefore be expected to lead to a decrease in tumour mass. ES has therefore become a major target in the treatment of estrogen-dependent breast cancer and has resulted in the synthesis of a wide range of steroidal and non-steroidal compounds; the majority of these have contained the sulfamate moiety which has been shown to lead to the irreversible inhibition of ES through the formation of an imine moiety. We have, however, recently reported the modelling of novel thiosemicarbazone-based compounds as potential inhibitors of ES and which have been shown to possess alosteric inhibitory activity. Here, we report the synthesis and biochemical evaluation of a series of novel sulfamoylated derivatives of thiosemicarbazone-based compounds as potential inhibitors of ES which would be expected to possess both irreversible inhibition (via the sulfamate moiety) and uncompetitive inhibition (as a result of the thiosemicarbazone moiety). The synthesis of the target compounds involved the initial synthesis of 4-hydroxyphenone-based compounds using Friedal-Crafts acylation. Following the isolation and purification, the 4-hydroxyphenone-based compounds were then heated to reflux in a solution of thiosemicarbazide in absolute ethanol to give the target compounds. The resulting intermediates were then derivatised to the sulfamate containing compound involving a reaction between aminosulfonyl chloride and the thiosemicarbazone-based intermediate. The biochemical evaluation involved the use of radiolabelled estrone sulfate which was incubated with JEG-3 cells as the source of ES at 37oC to give the radiolabelled estrone which was placed in scintillant and counted for 5min. In general, the synthesis of the target compounds were achieved in moderate to excellent yield and without any major problems, however, as previously reported by us, the sulfamoylated derivatives proved to be chemically unstable when dissolved in solvents, including media, as such, the biochemical assays were undertaken with freshly prepared solutions of the target compounds. The evaluation of the compounds showed them to be weak inhibitors of ES in comparison to the standard compounds, estrone-3O-sulfamate (EMATE) which was found to possess 99% inhibiton at 2.5µM. The most potent compound within the current range was found to possess approximately 85% inhibitory activity (IC50=198.01nM). In conclusion, the compounds have proved to be weak inhibtors of ES but have provided us with a new

library of compounds for further study.

26

Poster 17

Synthesis and biochemical evaluation of potential novel inhibitors of carbonic anhydrase type II (CAII)

Authors: Nazeer, A., Bhamare, B., Rudra, B.C., Singh, S., Owen, C.P., Ahmed, S.



Carbonic anhydrase (CA) family of enzymes have been shown to play a major role in malignant neoplasm development, in particular, they have been shown to be expressed in greater amounts within tumour cells. As a result, it has been proposed that the hypoxic microenvironment which has been shown to develop, as well as the associated increase in acidity of the microenvironment of the tumour. As a result, a number of compounds have been evaluated against the CA family of enzymes. In particular, acetazolamide (1) and sulfamate-based compounds, including estrone-3-O-sulfamate (EMATE), 667-COUMATE and COUMATE have all been evaluated and shown to be potent inhibitors of CAII. Here, we report the results of our initial study into the synthesis and evaluation of a series of a novel range of non-sulfamoylated compounds which we propose undergo chelation to the Zn2+ ion found within the active site of CAII, we therefore report the synthesis and biochemical evaluation of a range of thiosemicarbazone-based inhibitors of CA II. In the synthesis of the potential inhibitors of CAII, carbonyl containing starting material was heated to reflux with derivatives of thiosemicarbazide to give the target compounds; the products were found to crystallise out of the reaction solvent and were subsequently recrystallised from aqueous ethanol to give the target compounds. Biochemical evaluation involved a colourimetric assay where 4-nitrophenyl acetate (as the un-natural substrate) was incubated together with the inhibitor and enzyme (human CA II) in Tris-HCl buffer. From the biochemcial evaluation of the synthesised compounds, we observe that the potential inhibitors are weak inhibitors of CA II in comparison to the standard compounds; EMATE and 667-COUMATE were found to possess IC50 values of 9.36nM and 14.86nM respectively. Within the range of compounds synthesised the most potent inhibitors were found to possess IC50 values of 4.40µM and 5.33µM. The inhibitory activity observed within these compounds appear to support our initial hypothesis that the thiosemicarbazone-based compounds are indeed inhibitors of CAII. In conclusion, we have provided a novel series of inhibitors of CA II and therefore offer a new library of compounds in the treatment of a number of diseases including cancer.

27

Poster 18

Sulfamate derivatives of 4-hydroxycoumaric acid-based compounds as inhibitors of carbonic anhydrase II

and estrone sulfatase (ES)

Authors: Bhamare, B., Rudra, B.C., Owen, C.P., Ahmed, S.



Breast cancer cells have been shown to produce estrogens via the action of estrone sulfatase (ES) which

catalyses the conversion of estrone sulfate to estrone, as such, the ES pathway is a source of estrone within

postmenopausal women. Hormone-dependent breast cancer cells continue to be stimulated by the

continued production of estrone and in particular, estradiol (the most potent estrogen). Studies on tumour

cells have also shown an increased expression of carbonic anhydrase (CA) family of enzymes and which has

been proposed to play an important role in tumour cell survival. Both ES and CA family of enzymes would

appear to therefore possess an important role in tumour cell progression. A number of compounds,

including acetazolamide and estrone-3-O-sulfamate (EMATE) have been used to target both the CA and ES

family of enzymes. In our search for potent dual-inhibitors of both CA and ES, we report the use of the

EMATE backbone in the design, synthesis and biochemical evaluation of a series of sulfamoylated

derivatives of a range of 4-hydroxycoumaric acid-based compounds.

Synthesis of the target compound involved the initial synthesis of the 4-hydroxycoumaric acid amide

followed by the conversion of the 4-hydroxy moiety to the sulfamate derivative. Biochemical evaluation of

the target compounds against CA II involved inhibitors dissolved in dimethylsulfoxide (DMSO) (final

[I]=100µM). The assay was carried out (in triplicate) using prepared substrate (4-nitrophenyl acetate),

inhibitor, Tris-HCl; the mixture was incubated at 25°C. The intensity of the colour was then read using

microplate reader at 405nm. The ES assay involved the use of radiolabelled estrone sulfate which was

incubated with JEG-3 cells as the source of ES at 37oC to give the radiolabelled estrone which was placed in

scintillant and counted for 5min.

The results of the compounds considered within the current study show that these compounds are weak

inhibitors of both ES and are good inhibitors of CA II; e.g. the most potent target compound was found to

possess an IC50 value of 6.86nM against CA II but only possessed ~33% inhibitory activity against ES

([I]=2.5µM). Under similar assay conditions, EMATE was found to possess 99% inhibitory activity against ES

and possessed 14.5nM against CAII. As such, the investigation has resulted in the discovery of excellent

lead compounds in the search for more potent and specific inhibitors of CA II.

28

Poster 19

Synthesis and biochemical evaluation of novel potent inhibitors of estrogen synthesis

Authors: Shah, P.S., Nazeer, A., Owen, C.P., Ahmed, S.



The cytochrome P-450 enzyme aromatase (AR) catalyses the conversion of androstenedione to estrone. A number of triazole-based inhibitors have been introduced for use in the clinic, i.e. anastrozole and letrozole, however, there appears to be a lack of structure-activity relationship having been reported for these compounds, as such, the current study considers a series of compounds which use the phenylmethyl imidazole backbone with the phenyl moiety having been substituted with various functional groups so as to provide polar-polar interaction with the active site of AR (in addition to the Fe-azole dative covalent bond). We therefore report the initial results of our efforts to synthesise and evaluate a number of derivatives of phenylmethyl imidazole (1) against AR and their specificity having been determined through against the enzyme 17α-hydroxylase/17,20-lyase (P-45017α). The synthesis of the target compounds involved a reaction between the appropriate phenylmethyl bromide and imidazole using anhydrous potassium carbonate as base in anhydrous tetrahydrofuran (THF). The biochemical evaluation involved the modification of a literature-based AR assay (using 3H-androstenedione) [1], however, JEG3 cells were used as the source of AR; an organic phase (chloroform) was also used to partition out all organic components from the aqueous phase (which therefore contained the 3H2O produced) and counted for 3H. The compounds were also evaluated against P-45017α using 3H-progesterone using rat testicular microsome as previously reported by Owen et al [2]. The reactions proceeded in good yield and without any major problem. The biochemical evaluation of the synthesised compounds shows that a number of the derivatives of 1 proved to be highly potent inhibitors of AR. Anastrozole was found to possess 92% inhibition against AR resulting in an IC50 value of 21.54nM. The most potent compound within the current study was found to be 4-nitrobenzylimidazole which was found to possess 92% inhibitory activity and therefore was found to be equipotent to anastrozole. With regard to the inhibitory activity observed against P-45017α, the compounds were also found to be highly potent inhibitors of the lyase (in comparison to the hydroxylase) component. In conclusion, these compounds have proved to be potent inhibitors of AR, however, they are not suitable for further development as they lack specificity as demonstrated by the inhibitory activity against P-45017α. Key references:

(1) E. A. Thompson and P. K., Siterii, “Utilization of oxygen and reduced nicotinamide adenine dinucleotide

phosphate by human placental microsomes during aromatization of androstenedione” J. Biol. Chem., 1974,

249, 5373-5378.

(2) C.P. Owen, S. Dhanani, C.H. Patel, I. Shahid, S. Ahmed, “Synthesis and biochemical evaluation of a range

of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17α-

hydroxylase/17,20-lyase (P45017α)” Bioorg. Med. Chem. Lett., 2006, 16, 4011-4015.

29

Poster 20

The synthesis and biochemical evaluation of dual inhibitors of the cytochrome P450 enzymes aromatase

and 17α-hydroxylase/17,20-lyase

Authors: Muhammad, R., Lee, W-Y., Shahid, I., Shah, P.S., Pang, S.H-N., Owen, C.P., Ahmed, S.



The conversion of pregnanes and progestins to the corresponding androgen derivative is catalysed by 17α-

hydroxylase/17,20-lyase (P45017α); inhibition of this enzyme has been shown to be of clinical benefit which

has resulted in the use of ketoconazole (KTZ) in the treatment of refractory prostate cancer. The enzyme

aromatase converts androgens to the estrogens; the inhibition of both of these enzymes could result in the

total ablation of estrogen production within breast cancer cells. In the current study, we considered the

use of the backbone of the aromatase inhibitor used in the first line treatment of hormone-dependent

breast cancer so as to design compounds which could possess inhibitory activity against both of aromatase

and P45017α. We therefore report here: the synthesis of a range of n-alkanesulfonate derivatives of 4-

hydroxybenzyl imidazole (1); their biochemical evaluation and; the rationalisation of their inhibitory

activity.

In the synthesis of the potential inhibitors, imidazole was reacted with 4-hydroxybenzyl alcohol to give 1.

Derivatisation of the 4-hydroxy moiety involved reaction between 1 and the appropriate alkanesulfonyl

chloride. Biochemical evaluation of the compounds was undertaken using literature assay procedure using

rat testes homogenate (in the biochemical evaluation of compounds against P45017α) and the use of JEG-3

cells (in the evaluation of compounds against aromatase) and also using radiolabelled substrates to

determine inhibitory activity.

In general, the reactions proceeded in poor to good yield (ranging from 10% to 50%) without any major

problems. Consideration of the inhibitory activity against the two components of P45017α shows that the

compounds, in general, were found to possess greater potency against the lyase component in comparison

to the 17α-OHase component; only butane-1-sulfonic acid 4-imidazol-1-ylmethyl-phenyl ester (5)

(IC50=1.11µM and 1.28µM against 17α-OHase and lyase respectively) was found to possess slightly greater

potency towards17α-OHase. In comparison, KTZ was found to possess IC50 values of 2.66µM and 0.21µM

against 17α-OHase and lyase respectively. In comparison to anastrozole, the compounds were found to be

weak inhibitors of aromatase; the most potent compound evaluated was found to possess 70% inhibitory

activity (at [I]=500nM), under similar conditions, anastrozole was found to possess 99% inhibitory activity.

We undertook a structure-activity relationship determination study and considered factors such as

hydrophobicity and discovered that this appears to be a major factor in determining the overall potency

within the alkyl sulfonate-based compounds.

In conclusion, we have reported a series of compounds in which three of the compounds proved to be

potent inhibitors of the 17α-OHase component in comparison to the standard compound KTZ; however,

the compounds were found to be weak inhibitors of aromatase, nevertheless, the compounds are good

lead compounds in the development of dual inhibitors of aromatase and P45017α.

30

Poster 21

Synthesis and biochemical evaluation of novel dual inhibitors of aromatase (AR) and estrone sulfatase

(ES)

Authors: Rudra, B.C., Bhamare, B., Shah, K., Owen, C.P., Ahmed, S.



The conversion of androgens to the mitogenic estrogens is undertaken by the cytochrome P-450 enzyme

aromatase (AR). Aminogluethimide (AG) was the first generation of AR inhibitor, however, triazole-based

compounds such as anastrozole and letrozole are currently the first line treatment of hormone-dependent

breast cancer. The conversion of estrone sulfate (E1S) to estrone (E1) is catalysed by estrone sulfatase (ES).

After the menopause, ES becomes the major source of estrogen in postmenopausal women and allows the

continued stimulation of estrogen-dependent breast cancer cells. Recently, we reported a novel model for

the inhibition of ES; the model proposes that the C=S moiety within thiosemicarbazone-based compounds

interact with the Ca2+ ion found at the active site. Using the model we designed a number of

thiosemicarbazone-based compounds containing appropriate groups (pyridyl moiety to undergo interaction

with the Fe atom at the centre of the AR active site whilst the C=S would undergo interaction with the Ca2+

present within the active site of ES) so as to stabilise the enzyme-inhibitor complex. Here, we therefore

report the design, synthesis and biochemical evaluation of a number of pyridyl containing derivatives of

madurahydroxylactone (MHL) as potential dual inhibitors of AR and ES.

Synthesis of the target compounds involved a reaction between the appropriate pyridyl carboxyaldehyde-

based starting material with derivatives of thiosemicarbazide to give the crude products which were

recrystallised from aqueous ethanol to give the target compounds. Biochemical evaluation involved the

use of a literature-based a cell-based assay system using 3H-E1S for ES activity whilst the AR assay involved

use of 3H-androstenedione and JEG3 cells.

The biochemical evaluation shows that the compounds synthesised within the current study were found to

be weak inhibitors of both AR and ES in comparison to the standard inhibitors used, namely anastrozole

(IC50 value of 21.54nM) and EMATE (IC50 value of 1.14nM. Under similar conditions, (E)-N-cyclohexyl-2-[1-

(pyridin-3-yl)propylidene]hydrazinecarbothioamide was found to possess IC50 values of 168.45±3.21µM and

73.29nM against ES and AR respectively.

In conclusion, the thiosemicarbazone-based compounds have been found to be weak inhibitors in

comparison to the standard inhibitors but are excellent lead compounds in the further design of novel

potent dual inhibitors of AR and ES.

31

Poster 22

Synthesis and evaluation of a novel range of non-steroidal inhibitors of 17β-hydroxysteroid

dehydrogenase type 3

Authors: Gandhi, J., Pang, S.H-N., Olusanjo, M.S., Owen, C.P., Ahmed, S.



Type 3 17β-hydroxysteroid dehydrogenase (17β-HSD3) enzyme has been shown to be responsible for the conversion of the C(17)=O moiety within androgens (e.g. androstenedione) to the C(17)-βOH (resulting in the biosynthesis of testosterone, the more potent androgen). In the design of novel inhibitors of 17β-HSD3, we argued that mimicking of the steroid structure may allow potential compounds to possess inhibitory activity. We therefore undertook the design, synthesis, biochemical evaluation and rationalisation of the observed inhibitory activity of a series of compounds based upon the backbone of progesterone, in particular, of progesterone. The target compounds and potential inhibitors were synthesised involving the initial oxidative cleavage of the A-ring of progesterone [it should be noted that the C(20) carbonyl moiety was required to be protected through the formation of the ketal]. The resulting progesterone ‘keto-acid’ was subsequently derivatised to the ester functionality involving the reaction (in the absence of an acid catalyst) with a range of alcohols so as to give the appropriate ester. The biochemical evaluation of the synthesised compounds was undertaken using literature procedure using microsomes from rat testes. The assay was quenched using diethyl ether and the substrate and products were separated using thin layer chromatography, each spot cut out and counted for tritium for 4min. The reactions proceeded in good yield and without any major problems. Consideration of the initial screening data shows that the compounds are, in general, weak inhibitors of 17β-HSD3. The most potent was the butyl derivative which was found to possess ~57% inhibitory activity at [I]=100µM, whilst the weakest inhibitory activity was observed with the octyl derivative which was found to possess ~16% inhibitory activity under similar conditions. In comparison, the two standard inhibitors used (namely 4-hydroxynonanophenone and 4-hydroxydecanophenone which have been previously reported by us) were found to possess ~71% and ~62% inhibitory activity under similar conditions. Using the previously derived transition-state for 17β-HSD3, we propose that the weak inhibitory activity observed within the compounds (containing large alkyl chains) is due to steric hindrance between the alkyl chain within the inhibitor and the active site; resulting in a decrease in inhibitory activity. The results of our study show that in the inhibition of 17β-HSD3 and the mimicking of the steroid backbone, in particular, the C(17)=O group within the inhibitors is a factor in the inhibition process. We have therefore provided a novel range of compounds which may be suitable for further development.

32

Poster 23

Anthracene tagged biomolecules for sensing

Authors: G A Bullen, A F A Peacock and J H R Tucker

Presenting author affiliation: University of Birmingham

Anthracene is a well studied aromatic compound with

interesting fluorescence and photodimerisation

properties. Within this study both of these aspects are

explored with respect to potential bio-sensing

applications. Firstly, anthracene has been introduced

into an oligonucleotide sequence to allow for the

identification of the nucleobase located opposite on the

complimentary strand of DNA, by monitoring the

change in fluorescence intensity upon duplex formation. This has been used to identify the nucleobase

present at a single nucleotide polymorphism (SNP) site thought to be associated with Alzheimer’s disease.

Various anthracene probes have been evaluated and initial trials have identified a probe that can allow for

the quantitative determination of the amount of SNP nucleobase and wildtype nucleobase present.

Recently we have begun using this probe to analyse real biological samples from Alzheimer patients. 1-2

A second project involves coupling anthracene to a small biological building block

and exploiting the photodimerisation properties of the anthracene. An anthracene

unit has been attached to the basic region of GCN4, the DNA binding domain of a

well-studied bacterial transcription factor. When the anthracene tagged peptide is

exposed to UV light it is found that no photodimerisation occurs. However, upon

templating to target DNA photodimerisation ought to occur resulting in a loss of

the fluorescence of the anthracene and a decrease in the absorbance of light.

When DNA is present which does not contain the target sequence, dimerisation is

no longer expected to occur allowing for the sensing of target DNA within a

sample. Furthermore the DNA binding domain of GCN4 has a weak affinity for

target DNA as a monomer, however, dimerisation results in significantly enhanced

DNA binding. Therefore the same anthracene-GCN4 adduct can be used as a light triggered DNA binding

agent.

Key references:

(1) J.-L. H. A. Duprey, Z.-y. Zhao, D. M. Bassani, J. Manchester, J. S. Vyle and J. H. R. Tucker, Chem. Commun.,

2011, 47, 6629-6631.

(2) Z.-y. Zhao, M. San, J.-L. H. A. Duprey, J. R. Arrand, J. S. Vyle and J. H. R. Tucker, Bioorganic Medicinal

Chemistry Letters, 2012, 22, 129-132.

A

C

33

Poster 24

Understanding Oligonucleotide Synthesis Authors: James L. Scotson, Andrew P. Laws, Michael I. Page, John H. Atherton and Ben Atherton

Presenting author affiliation: The University of Huddersfield

Oligonucleotides have many uses including DNA sequencing, artificial gene synthesis and as trheraputic

agents. A recent surge in the number of oligonucleotide therapies in stage 2 and 3 clinical trials has lead to

a hugely increased amount of research in the area. Currently, the most common method of oligonucleotide

synthesis is via the ‘phosphoramidite’ method which uses a solid support accross which reagents are

passed sequentially and upon which the oligonucleotide chain grows as detailed in the scheme below.

Synthesis of a phosphorthioate modified DNA oligonucleotide.

Though this method is widely and successfully used to create tailor made oligocnucleotides little is known

about the mechanisms and kinetics of the individual steps in the reactions and what mechanistic studies

there are are in very early stages. Many of the conditions used to generate oligonucleotides have only ever

been implemented on a gram scale in which large excesses of reagents are used to force equilibria and

increase reaction yeilds. This method is not viable if these therapies are to be used on a large scale both in

terms of cost of production and in terms of the economy of the reactions.

It is the focus of this work to take a physical-organic approach to improving oligonucleotide synthesis. The

project will study each reaction individually to elucidate exact mechanisms of reactions to reduce waste

and cost. This work will also aim to offer alternative routes to oligonucleotide synthesis, i.e. novel and more

efficient activators and sulfurising agents.

Key references: 1. M. A. Russell, A. P. Laws, J. H. Atherton and M. I. Page, Org. Biomol. Chem., 2008, 6,

3270-3275

2. J. Hanusek, M. A. Russell, A. P. Laws, P. Jansa, J. H. Atherton, K. Fettes and M. I. Page, Org. Biomol. Chem.,

2007, 5, 478-484

3. M. A. Russell, A. P. Laws, J. H. Atherton and M. I. Page, Org. Biomol. Chem., 2009, 7, 52-57

34

Poster 25

Biotechnological applications for phenylpropanoids derived from biorefining

Authors: Prof Robert Edwards, Keir Bailey

Presenting author affiliation: The University of York

Other affiliations: BBSRC IBTI Club

Biorefining involves "refining" multiple useful

products from biomass. The biorefining of plant

fibres produces by-products, such as

phenylpropanoids. These phenylpropanoid by-products

can be used to produce more useful, high-value

compounds such as curcuminoids.

Curcuminoids are diarylhepatanoids that give turmeric,

the popular curry spice, its distinctive yellow colour. Research has shown these

molecules to have anti-tumour, anti-cancer, anti-oxidant and anti-inflammatory effects as

well as providing neuroprotection.1 Therefore, the ability to sustainably produce these

compounds in high yields is very lucrative.

This project aims to produce a variety of curcuminoids and curcuminoid-like analogues using plant

polyketide synthase (PKS) enzymes and Saccharomyces cerevisiae, with the hope that curcuminoids can

eventually be produced on a large scale by microbial fermentation.2 This involves cloning the two PKS

enzymes, diketide CoA synthase (DCS) and curcumin synthase 1 (CURS1) which produce curcuminoids in

the turmeric plant (Curcuma longa) and expressing them in yeast.3 The engineered yeast will be fed using

various phenylpropanoids and the yield of different curcuminoids will be monitored. The focus will then be

to optimise this metabolic pathway and investigate ways of elaborating the chemical structure of

curcuminoids by adding new enzymes to the system such as glucosyltransferases.

Key references:

(1) B. Aggarwal, C. Sundaram, N. Malani and H. Ichikawa, Adv. Exp. Med. Biol., 2007, 595, 818.

(2) K. Hong and J. Nielsen, Cell Mol. Life Sci., 2012, 69, 2671.

(3) Y. Katsuyama, T. Kita, N. Funa, and S. Horinouchi, J. Biol. Chem, 2009, 284, 11160.

../TAP/Keir%20Bailey_3%20month%20TAP%20Report.docx#_ENREF_2

35

Poster 26

Synthesis of Calpain-1 Inhibitors to Target Rheumatoid Arthritis

Authors: Sarah E. Adams, Maurice B. Hallett, Pierre Rizkallah, David J. Miller and Rudolf K. Allemann

Presenting author affiliation: School of Chemistry, Cardiff University, Cardiff, CF10 3AT, UK

Abstract: The activity of phagocytes such as neutrophils in otherwise healthy tissue is a cause of rheumatoid

arthritis and other inflammatory diseases.1 The calcium activated cysteine protease calpain-1 has been

linked to neutrophil spreading - a process that is a necessary prelude to extravasation from the bloodstream

and transfer to a site of infection or trauma.2

The flattened cell is then able to adhere to the endothelial

membrane of the capilary wall and leave the blood stream. The over stimulation of neutrophils has been

demonstrated to have an effect in enabling the damage inflicted in rhuematoid arthritis and therefore limiting

the rate of cell spreading and extravasation may reduce the symptoms of this and other inflammatory

diseases. As calpain-1 is a known initiator of the cell spreading process it is an attractive target for inhibition.

A series of 24 monohalogenated aromatic mercaptoacrylate compounds was prepared and tested for

inhibition of calpain-1. Variation of both the nature and position of the halogen in these molecules profoundly

affects the affinity for calpain-1; some mercaptoacrylates were found to be highly potent inhibitors of calpain-

1.3 X-ray crystallographic studies of the inhibitor in complex with the calcium binding domain of calpain-1

were also carried out to investigate the mode of inhibition of the enzyme.

Key references:

(1) Edwars, S. W.; Hallett, M. B. Immunol. Today, 1997, 18, 320.

(2) Hallett, M. B.; Dewitt, S.Trends Cell Biol., 2007, 17, 209-214.

(3) Adams, S. E.; Parr, C.; Miller, D. J.; Allemann, R. K.; Hallett, M. B. MedChemComm, 2012, 3, 566-570.

36

Poster 27

Regiospecific isotopic labelling via de novo NADPH biosynthesis

Authors: William M. Dawson, Louis Y.P. Luk, E. Joel Loveridge, Rudolf K. Allemann

Presenting author affiliation: School of Chemistry, Cardiff University

Dihydrofolate reductase (DHFR) is a popular therapeutic target against cancer, malaria and bacterial

infections, as it catalyses the NADPH-dependent reduction of dihydrofolate in nucleic acid and amino acid

biosynthesis. Despite being a well-studied enzyme, knowledge of the transition state is still lacking, and this

has become particulary relevant due to the recent debate surrounding the role of enzyme dynamics on the

reaction coordinate. Measurement of kinetic isotope effects (KIEs) using isotopically labelled substrates

presents an incredibly effective method to characterise the transition state at an atomic-scale resolution.

However, incorporation of isotopic labels into NADPH still remains a challenging task that requires a long

synthetic route with low yields.1 To incorporate isotopic labels into regiospecific positions of the

nicotinamide ring, we have reconstructed the de novo biosynthetic pathway of NADPH in our laboratory.

This work has resulted in the biosynthesis and isolation of key intermediates in the pathway, starting from

simple starting materials.

Key references:

(1) Oberfrank et al.,1984, Eur J Biochem, 140, 157

37

Poster 28

Chemoenzymatic approaches to bio-active monoterpenoids

Authors: Zulfa Yoosuf-Aly, David J. Miller, Juan A. Faraldos and Rudolf K. Allemann

Presenting author affiliation: Cardiff University

Terpenoids are the largest and structurally most diverse family of natural products,1 many of which are a source of commercially valuable fragrances, flavours, drugs, agrochemicals and synthetic building blocks.2

-Pinene (2), a monoterpene found in turpentine, is a precursor to several high-value mono-oxygenated terpenoids such as verbenone (3).3 It is produced naturally through the Mg2+ -dependent transformation of geranyl diphosphate (GDP, 1 -pinene synthase (APS).4 Bark beetle infestations pose a major threat to conifer populations worldwide with millions of acres of pine trees killed by beetles such as Dendroctonus ponderosae.5 The male beetles use (+)--pinene produced by the pines for host selection and then convert it to verbenol, which acts as an attractant for female beetles. (+)-Verbenone (3) is then produced as a dispersal pheromone by the beetles in order to modulate the attack density. In a scheme to limit tree damage from beetle infestation, foresters hang small bags of synthetic verbenone on trees.5

Here a chemoenzymatic synthesis is described that generates (+)-verbenone from GDP in good yield and without the need to isolate the intermediate (+)--pinene.6

OP2O63-

Mg2+

GDP (+)-a-pinene

O

Cr(CO)6

ButOOH

C5H12/

CH3CN2 3(+)-verbenone

1

APS

This approach has been applied to the synthesis of verbenone analogues from synthetic GDP derivatives. These ‘unnatural’ verbenones may display enhanced biological activities.

Key references:

1. J. Degenhardt, T. G. Kollner and J. Gershenzon, Phytochem., 2009, 70, 1621-1637. 2. E. M. Davis, in Comprehensive Natural Products II, Elsevier, 2010, pp. 585-608. 3. S. G. Bell, R. J. Sowden and L. L. Wong, J. Chem. Soc. Chem. Commun., 2001, 635-636. 4. M. A. Phillips, M. R. Wildung, D. C. Williams, D. C. Hyatt and R. Croteau, Arch. Biochem. Biophys., 2003, 411, 267-

276. 5. R. Petkewich, Chem. Eng. News, 2008, 86, 36-37. 6. Z. Yoosuf Aly, J. A. Faraldos, D. J. Miller and R.K. Allemann, Chem. Commun., 2012, 48, 7040-7042.

38

Poster 29

Why isn’t P(III) pyrophosphate an inhibitor or a good substrate for P(V) pyrophosphatase?

Authors: Dharmit Mistry, Professor M.I. Page and Professor D.R. Brown

Presenting author affiliation: Innovative Physical Organic Solutions (IPOS), University of Huddersfield.

The reactions and stabilities of phosphate mono- and di-esters underpin the entire processes of life:- the storage and

manifestation of genetic information, energy transduction, signalling, regulation, differentiation,

compartmentalisation (ionic phosphate esters unable to cross membranes), substrate modification to facilitate

chemical reactions and structural components.

The amazing paradox of the previous paragraph is the remarkable variation from extreme stability – half-lives of

millions of years for some spontaneous hydrolyses, to millisecond turnovers for some enzyme-catalysed reactions.

Phosphate mono- and di-esters show remarkable resistance to spontaneous hydrolysis under normal physiological

conditions, hence their contribution to the stability of genes1. By contrast enzyme-catalysed phosphoryl groups

transfer reactions are highly efficient and show some of the largest enzymatic rate enhancement – up to 1020

. The key

to understanding enzyme catalysis is the charge and geometric complementarity between substrate and enzyme

expressed in the transition state. The processes of phosphorylation and dephosphorylation that occur by associative

type mechanisms usually occur via a transition state with a trigonal bipyramidal geometry, and in the case of

phosphoryl transfer effectively involve a trigonal planar metaphosphate anionic species, PO3-. The important feature

of mono- and diphosphate esters is their invariable negative charge, so a major feature of enzymes catalysing their

reactions is the neutralisation of this charge in the transition state2.

We have been interested in H-phosphonates as phosphorylating agents and have been studying P(III)

pyrophosphonates. Compounds containing ionisable groups will associate/dissociate dependent on the pH of their

surroundings, inevitably causing the compound to change its characteristics. This will in turn affect the rates of any

reaction in which the substrate is involved. P(V) pyrophosphates contain four ionisable protons whereas the P(III)

pyro-di-H-phosphonate only contains two. We have studied the hydrolysis of these two compounds with the use of 31

P

NMR, a pH rate profile has been constructed for the P(III) compound enabling comparison with the P(V)

pyrophosphate. There is a large difference between the two compounds, the hydrolysis rate of the P(V)

pyrophosphate decreases with increasing pH- with increasing ionisation decreasing the rate. However, the P(III) pyro-

di-H-phosphonate shows a characteristic U-shaped pH rate profile with a reaction between the di-anion and

hydroxide.

Most chemical reactions or physical changes are accompanied by a change in heat or enthalpy. Isothermal Titration

Calorimetry (ITC) has been used as a novel method to measure the rates of pyrophosphate hydrolysis with the enzyme

pyrophosphatase (Ppase), which cannot be measured by 31

P NMR. Molar enthalpies of hydrolysis for P(V)

pyrophosphate and P(III) pyro-di-H-phosphonate have been obtained.

The enzyme Ppase uses Mg2+

as a cofactor for hydrolysing pyrophosphate. ITC and NMR have shown conclusively that

pyrophosphate binds in a 1:1 ratio with Mg2+

whereas pyro-di-H-phosphonate exhibits no detectable binding.

Key references:

(1) N. Powles, J. Atherton and M. I. Page, Organic & Biomolecular Chemistry, 2012, 10, 5940-5947.

(2) L. Yang, R.-Z. Liao, J.-G. Yu and R.-Z. Liu, The Journal of Physical Chemistry B, 2009, 113, 6505-6510.

39

Poster 30

Synthesis and Biochemical Evaluation of Molecular Probes for Label-Free Detection of Thrombin

Activity.

Authors: James Murray, Dominika Nowak, Steven Johnson and Robin S Bon.

Presenting author affiliation: School of Chemistry, University of Leeds, Woodhouse Lane, LS2 9JT

Other affiliations: Astbury Centre for Structural and Molecular Biology, University of Leeds, LS2 9JT.

Activity-based protein profiling is a powerful technique for studying enzymatic activities and

annotating their roles in physiological and pathological processes1.. What has yet to be realised, is the

use of activity-based probes to generate protein activity fingerprints of enzymes involved in disease

and incorporation of these probes into point-of-care, biomedical diagnostic devices. We envision that

by assembling activity-based probes, on a gold electrode, label-free detection of enzyme activity and a

route to such diagnostic devices may be achieved.

We shall report the design and synthesis of functionalised self-assembled monolayer (SAM) - forming

alkanethiol-PEG molecules. By changing the terminal group of these molecules, a wide range of

ligation strategies are made available: molecules terminated with amines, azides and alkynes have

been demonstrated for the immobilisation of small molecules and peptides- including, peptidic

thrombin substrates. The kinetic parameters of these peptides have been determined in solution by

the incorporation of a fluorogenic moiety. Surface-phase characterisation of functionalised SAMs has

been realised using surface-plasmon resonance (SPR), cyclic voltammetry (CV) and electrochemical

impedance spectroscopy (EIS). EIS and CV studies showed that our molecules form well-ordered and

stable SAMs. Finally, we shall discuss preliminary results of the use of functionalised SAMs for the

detection of thrombin activity.

(1) Cravatt, B. F.; Wright, A. T.; Kozarich, J. W., Annu. Rev. Biochem., 2008, 77, 383–414.

40

Poster 31

An integrated computational and experimental approach to identify novel ligands that target BACE-1

Authors: Giorgia Magnatti, Colin Fishwick, Nigel Hooper, Adam Nelson

Presenting author affiliation: University of Leeds, School of Chemistry

Other affiliations: Astbury Centre for Structural Molecular Biology, University of Leeds

Introduction: There are numerous complementary approaches to facilitate the identification of novel

ligands for biological targets, including high throughput screening and fragment-based drug discovery.

Computational tools are also employed to predict binding pose and affinity of the new ligands (1). In this

poster an integrated computational approach to identify novel ligands is presented. The approach involves

the design of a virtual library of synthetically accessible lead-like molecules, followed by virtual high

throughput screening (vHTS) against a specific biological target.

Discussion: In our group, diversity-oriented synthesis (DOS) has been employed to explore chemical space

(2) and more recently was focused to explore the chemical space of lead-like molecules. BACE-1 protein is a

well known biological target of pharmaceutical interest involved in Alzheimer’s disease, whose structure is

accessible from the protein data bank. Our in silico approach to identifying novel BACE-1 ligands is

schematically represented as follows.

N

H2N

SO

O

virtual library of synthetically accessible lead-like molecules BACE-1

predictedLE > 0.28

vHTS

prioritised ligandsfor synthesis

N

H2N

ON

N

N

NHN

O

NH2

N

O

N

NH

O

N

F

FF

O

NOH

A virtual library of lead-like molecules was generated based on DOS methods established in our laboratory.

The library was virtually screened against BACE-1 by using eHiTS (3) and two families of potential ligands

were identified with high predicted ligand efficiency (LE). The synthesis of focused libraries based on these

scaffolds is underway.

Future work: An established assay will be employed to evaluate the biological activity of the ligands. The

assay will allow to verify the validity of the integrated approach in identifying new ligands for biological

targets.

Key references: (1) N. Y. Mok, N. M. Hooper, A. P. Johnson, C. W. G. Fishwick et al., J. Med. Chem. 2013, 56, 1843-1852. (2) C. Cordier, A. Nelson et al., Angew. Chemie. Int. Ed. 2009, 48, 104-109, P. MacLellan and A. Nelson, Chem. Commun. 2013, in press. (3) Z. Zsoldos, A. P. Johnson et al., Curr. Protein and Pept. Sc. 2006, 7, 421-435.

http://www.asn.leeds.ac.uk/view_person?id=20

http://www.ingentaconnect.com/content/ben/cpps/2006/00000007/00000005/art00005

41

Poster 32

Could ‘life’ exist in liquid ammonia – conditions found on some planets such as Jupiter and Saturn?

Authors: J. M. Griffin, Prof M. I. Page, Prof J. H. Atherton

Presenting author affiliation: IPOS, School of Applied Sciences, University of Huddersfield, Queensgate,

Huddersfield, West Yorkshire, HD1 3DH

The basic unit of all life is the cell which allows a diverse range of chemical reactions to take place within a

confined environment. Cells are composed of chemical structures which have the unique property in water to

aggregate into spheres, trapping water inside. It is this property that has allowed life to evolve. Liquid

ammonia has many properties similar to water, and having recently explored a variety of chemical reactions

in this non-aqueous solvent, speculation has risen about the possible formation of micelles in liquid

ammonia. The other major contributor to life processes are enzymes. These are nature’s catalysts made up of

proteins which fold up into a unique structure because of their interactions with water. Although there is

wide knowledge of the catalytic activity of enzymes in non-aqueous solvents, the ability for them to function

in liquid ammonia has never been previously studied.

We have initially investigated the catalytic activity of enzymes in liquid ammonia, with on-going work

exploring the formation of micelles to support these enzymes in a cellular type arrangement. This would

satisfy two of the fundamental processes that could allow life to develop in such harsh conditions found on

Jupiter and Saturn.

The background solvolysis rates in liquid ammonia for a variety of simple esters and triglycerides were

initially established. The catalytic effect of various lipases was then investigated, to see if, and to what

extent, any solvolysis rate enhancement due to enzyme activity could occur. Enzymes, more specifically

lipases, have been used extensively for the synthesis of carboxylic amides from simple small chain esters to

more complex triglycerides 1. Candida Antarctica Lipase B has shown activity on the triglyceride tributyrin

in ammonia saturated t-butanol at 40°C, and so this enzyme was to be the initial focus of our studies in liquid

ammonia 2. An immobilized preparation of the enzyme showed rate enhancement on the solvolysis of

tributyrin in liquid ammonia. Rate enhancement was also observed with Candida Rugosa Lipase.

The use of conductivity to monitor the formation of micelles from anionic and cationic surfactants is well

known in aqueous and even anhydrous media 3, 4

. These techniques have been applied to see if these cell-like

structures can form in liquid ammonia.

[1] van Rantwijk, F., Hacking, M.A.P.J., Sheldon, R.A., Monatshefe für Chemie 2000: 131, 549-569

[2] de Zoete, M.C., Kock-van Dalen, A.C., van Rantwijk, F., Sheldon, R.A., Journal of molecular catalysis

B: Enzymatic 1 1996: 109-113

[3] Dubey, N., J.Surface Sci. Technol., Vol 24, No. 3-4 2008: 139-148

[4] Singh, H.N., Saleem, S.M., Singh, R.P., J. Phys Chem 1980: 84, 2191-2194

42

Poster 33

Development of Growth Medium Suitable for Exopolysaccharie production by Bifibacteriaanimalis ssp.

lactis

Authors: Muhannad Alhudhud , Andy Laws and Paul Humphreys

Presenting author affiliation: University of Huddersfield

Complex media are commonly used in studies examining exopolysaccharide production and structure

characterisation by Bifidobacteria animalis ssp. lactis. Quantification and structure analysis of

exopolysaccharide in complex medium can be complicated by interference due to carbohydrate polymers

contained in media components. The use of solid medium have minimized most of their interference,

however, the EPS yield dropped to less than 10 mg per litre of culture. This project was undertaken to

develop a new broth medium for production of exopolysaccharide that provides growth for B. animalis ssp.

lactis strain similar to their levels in BIM media and to demonstrate exopolysaccharide production by B.

animalis ssp. lactis grown in the newly developed medium. The ingredients of BIM broth media accounted

for exopolysaccharide-equivalent where either eliminated when it found not to affect the maximum growth

levels of B. animalis ssp. lactis such as starch, replaced by alternative free of EPS-equivalent interfrences

such as yeast extract which replaced with yeast nitrogen base without amino acids, or treated chemically in

laboratory if there are no suitable alternatives such as beef extract. Growth level of B. animalis ssp. lactis in

newly developed medium was evaluated and found similar to the levels when cultured in BIM media.

Production of exopolysaccharide by B. animalis ssp. lactis was relatively much higher when grown in the

developed media in comparative to their levels when grown on agar media (150 mg/L of culture).

Key references:

1. Kimmel S. A., Roberts R. F., 1998

2. van Neil E.W. J., Hahn-Hagerdal B., 1999

3. Payne J. F., Morris A. E. J., Beers P., 1999

4. Salazar N. et al, 2009

5. Roy D., 2001

43

Poster 34

Developing Regiocomplementary Catechol O-Methyltransferase Enzymes for Biocatalysis

Authors: Matthew R Bennett, Brian Law, Mark Thompson, Colin Levy, David Leys and Jason Micklefield


Other affiliations: CoEBio

Current alkylation methods rely on synthetic agents that are genotoxic as well as lacking stereo, regio and

chemo control. Methyltransferases offer a safer, “greener” and more selective alternative. The catechol O-

methyltransferase (COMT) methylates either meta or para phenolic hydroxyls of catechol based structures.

One example of a substrate of COMT is 3, 4-dihydroxybenzaldehyde (DHBAL), which produces vanillin (meta

methylation), a flavouring agent, and isovanillin (para methylation), a precursor in drug syntheses, in a 3:1

ratio respectively. This lack of regioselectivity limits the enzyme for catalytic purposes.1 Using a directed

mutagenesis strategy, based on a crystal structure of the WT enzyme,2 we produced the

regiocomplementary mutants Y200L and K144A. The Y200L mutant improves methylation in the meta

direction registering a 22:1 (meta: para) ratio and the K144A mutant improves methylation in the para

direction registering a 1:1 ratio (meta: para). Therefore, we have developed COMT mutants, which have

exhibited more desirable regioselectivities for use in the production of both vanillin and isovanillin

regioisomers.

Key references: (1) Vidgren, J.; Svensson, L. A.; Liljas, A., Nature, 1994, 368, 354

(2) Li, K.; Frost, J. Journal of the American Chemical Society 1998, 120, 10545.

44

Poster 35

The design, synthesis and biological evaluation of non-viral gene delivery vectors

Authors: Amanda B. Maginty,1 Dr William P. D. Goldring,1 Dr Michael D. Pungente2

Presenting author affiliation: 1. School of Chemistry and Chemical Engineering Queens University Belfast

Other affiliations: 2. Weill Cornell Medical College in Qatar

Gene therapy involves the transfer of genetic material into a cell, using a delivery vehicle or vector, to

correct either the overexpression, or under-expression of a protein and thus eliminate the genetic

abnormality or damage affecting the healthy function of the cell. The current study involves the synthesis

and application of cationic lipid vectors to achieve the safe and efficient delivery of genetic material into

Chinese hamster ovarian (CHO) cells. These so-called non-viral vectors are characterised by their ease of

preparation and a much lower immune response in comparison to viral vectors. Although viral vectors

possess a natural ability to target and deliver genetic material into cells, their use has been characterised by

a number of serious immune response events.

Our design for a novel, non-viral cationic lipid vector was based on either a macrocyclic or acyclic

hydrophobic domain, linked via ester bonds to a glycerol backbone, together with a cationic head group

based on either a protonated amine or quaternary ammonium salt. The culmination of our research efforts,

which includes the design and optimisation of a synthesis route to a library of cationic lipids, together with

the results of binding, degradation, cytotoxicity and transfection assays, will be presented. The transfection

efficiency and cytotoxicity of our lipids was compared against each other and commercial delivery systems.

In fact, we found a number of our cationic lipids outperformed the gold standard lipofectamine 2000.

Key references:

(1) W. P.D. Goldring, E. Jubeli, R. A. Downs, A. J.S. Johnston, N. Abdul Khalique, L. Raju, D. Wafadari, M.D.

Pungente, Bioorg. Med. Chem. Lett., 2012, 22, 4686–4692

45

Poster 36

Portulaca oleraceae L. as potential candidate inhibitor of HCV NS3

Serine Protease

Sobia Noreen1*, Ishtiaq Hussain

1, Muhammad Ilyas Tariq

1, Qamar-ul-Zaman

2, Tayyab Hussain

3,

Aqeel Khan4 and John M. Gardiner

4

1 Department of Chemistry, University of Sargodha, Sargodha

2 Department of Pharmacy, University of Sargodha, Sargodha

3 Center of Excellence for Molecular Biology (CEMB), University of the Punjab, Lahore

4 Manchester Institute of Biotechnology, The University of Manchester

Hepatitis C virus (HCV) infectivity is a worldwide health problem effecting about 200 million

individuals annually. Viral infection is a causative for chronic liver diseases; liver cirrhosis, hepato-

cellular carcinoma and eventually death. The potential from natural products as anti-HCV drugs is

thus of considerable potential significance. NS3 serine protease (NS3-SP) is a target for screening

of antiviral activity against HCV. The present work was intended to explore plants with anti-HCV

potential, leading to natural chemical entities as lead compounds. Ten plants used for medicinal

purposes against different infections in rural areas of Pakistan for antiviral activities were collected.

The cellular toxicity effects of organic extracts on the viability of Huh-7 were studied through

Trypan blue dye exclusion method. Furthermore, antiviral effects of plant extracts were also

assessed against HCV NS3-SP by transfecting HCV NS3 protease plasmid into liver cells. To

identify the active ingredient, Portulaca oleraceae L. (PO) was fractioned by column

chromatography, purified and characterized by spectral data (UV-VIS, FTIR, EI-MS and NMR).

The results revealed that the PO methanolic extract reduced the HCV NS3 protease expression in a

dose-dependent manner and GAPDH remained constant. The novel antiviral activity of compounds

from PO presents an attractive lead for natural chemical entities for the development of potential

anti-HCV agents.

Conclusion: These results suggest that PO extract and its bioactive constituents contain potential

agents against HCV and combination of PO extract with interferon could offer a future option to

treat chronic HCV.

Key words: Hepatitis C; NS3 Serine Protease; Portulaca oleraceae L; bioactive compounds;

natural products extraction; fractionation; NMR; Structural elucidation

46

Poster 37

A magnetoresponsive drug delivery system using Native Chemical Ligation: towards magnetic HIV

therapy

Authors: Raul Camarillo and Simon J. Webb

Presenting author affiliation: MIB and School of Chemistry, University of Manchester, 131 Princess St.

Manchester M1 7DN. Email:[email protected]

Regardless of existing HIV therapies, complete erradication of this public health concern is still considered

as a challenge. The design and development of new drug delivery systems with particular emphasis on

nanotechnology has been considered a promising strategy to improve the efficacy of existing therapies.

Among the nanoparticulate carriers, liposomes (vesicles) have been found to be the most popular

antiretroviral drug carriers.1 Combining functionalized Fe3O4 with adhesive vesicles has led to interesting

nanoparticle-vesicle assemblies able to be magnetically manipulated and to release their payloads when

subjected to an alternating magnetic field. 2,3

In this context, this project aims to create a covalent bond between iron oxide nanoparticles and lipid

vesicles using native chemical ligation (Fig. 1). Coprecipitation, ultrasonic and thermal decomposition

methods were performed to obtain the required magnetite nanoparticles, and calorimetric measurements

were carried out to determine their efficiency to convert magnetic energy into heat (SAR performance).

Spectrophotometric analysis (TNBS assay and Ellman’s test) were conducted to verify nanoparticle surface

functionalization. Preliminary results suggest that NCL approach might be a promising strategy to create a

strong linkage between cysteine-capped iron oxide nanoparticles (Fig. 1 a) and N-acetyl-S-palmitoyl

cysteine lipid vesicles (Fig. 1 b). Further developments are needed to determine whether or not the

aggregates are the result of a covalent bond (Fig. 1 c) or might be due to non-specific aggregation.

Keyreferences:

[1] Carvalho, F.C.; Mainardes, R.M.; Gremiao, M. P. Exploring the nanotechnology-based drug delivery

systems for AIDS treatment; Kasenga, F., Ed.; In-Tech. 2011.

[2] Mart, R. J.; Liem, K. P.; Webb, S. J., Pharm. Res. 2009, 26, 1701-10.

[3] Mart, R. J.; Liem, K. P.; Webb, S. J., Chem. Commun. 2009, (17), 2287-2289.

47

Poster 38

Aggregation of magnetite nanoparticles and phospholipid vesicles through the use of saccharide coatings

Authors: Thomas Coxon, Dr. Simon Webb, Dr. Julie Gough, Dr. Andrew Almond

Presenting author affiliation: MIB, University of Manchester

A small library of novel compounds has been synthesized using a simple, versatile method. These

compounds are adducts of various hydrazides and saccharides and have been designed with the intention

of coating either magnetite nanoparticles or phospholipid vesicles. For the former, a catechol-containing

hydrazide has been used, and for the latter, a novel lipid hydrazide containing an tetraethylene glycol

spacer. For all adducts, the β-pyranose anomer was found to be the major product, with an anomeric purity

of over 80% in most cases. Further transformation of the resulting adducts has been attempted using trans-

sialidase and galactosyl-transferase enzymes. Finally, these novel adducts have been used to coat both

nanoparticles and vesicles with the aim of lectin-induced aggregation.

Key references:

(1) F. De Cogan, A. Booth, J.E. Gough and S.J. Webb, Angew. Chem. Int. Ed., 2011, 50, 12290-12293

48

Poster 39

Magnetically triggered release of bio-active compounds from magnetic nanoparticle-vesicle aggregates

Authors Andrew Booth, Dr. Simon Webb, Dr. Julie Gough.

Presenting author affiliation: MIB, School of Chemistry and School of Materials Science, University of

Manchester

Controlled release of bioactive compounds is an important goal in the development of selective drug

delivery systems and novel biomaterials for tissue engineering. This project aims to provide temporal and

spatial control over the release of active compounds from phospholipid vesicle carriers by biotin-avidin

mediated aggregation with superparamagnetic iron oxide nanoparticles (SPIONs). The magnetic properties

of the nanoparticles enable release to be triggered by induced local heating in response to an alternating

magnetic field (AMF). Aggregates can also be manipulated in response to a static magnetic field.

Incorporation of these aggregates into hydrogels has created a novel responsive biomaterial. Controlled

release of ascorbic acid-2-phosphate has been used to induce collagen production by chondrocytes,

demonstrating an AMF triggered cellular response in vitro.

An improved version of this system has been produced after extensive assessment of the components and

their performance. Of particular note is the replacement of calcium alginate with hyaluronic acid hydrogels,

which has eliminated gel-induced leakage of vesicle contents, and improved nanoparticle coating that has

improved release.

Key references:

(1) F. de Cogan, A. Booth, J. E. Gough, S. J. Webb, Angew Chem Int Ed Engl 2011, 50, 12290-12293.

(2) F. de Cogan, A. Booth, J. E. Gough, S. J. Webb, Soft Matter 2013, 9, 2245.

49

Poster 40

Synthesis and decomposition of water-soluble sulfonyl hydrazides

Authors: Rebecca K. Booth, Wen Li, Daniele Castagnolo, Luis Martinez Crespo and Simon J. Webb.

Presenting author affiliation: School of Chemistry and Manchester Institute of Biotechnology, University of

Manchester, 131 Princess Street, Manchester, M1 7DN.

Sulfonyl hydrazides are a fascinating class of compound that have been shown to decompose to produce

molecules of increased lipophilicity.1 The decomposition is believed to take place via elimination of a

sulfinate with concomitant formation of a diazene, followed by radical formation or a sigmatropic

rearrangement to eliminate dinitrogen.

Interest in the synthesis and decomposition of sulfonyl hydrazides has grown in recent years due to their

potential applications, including in drug delivery and the ‘caging’ of volatile molecules. Consequently,

further investigations are needed to determine the decomposition pathway for different sulfonyl

hydrazides.

To this end, different synthetic routes towards sulfonyl hydrazides have been investigated, including

towards water-soluble analogues. Studies on their decomposition will be presented, along with structure-

activity relationships for the decomposition of substituted hydrazides. Assays of the stability of the sulfonyl

hydrazides in various solvents, e.g. organic apolar solvents and water, will also be presented.

Subsequently, the synthetic methodology will be applied to the release of drugs and pheromones such as

paracetamol and Lepidoptera sex pheromone, in water under basic conditions

Key references:

(1) Myers, A. G.; Movassaghi, M.; Zheng, B., Journal of the American Chemical Society 1997, 119 (36),

8572-8573; Movassaghi, M.; Ahmad, O. K., The Journal of Organic Chemistry 2007, 72 (5), 1838-1841.

50

Poster 41

Accelerated enzymatic transformation of synthetic glycolipids in artificial lipid rafts

Authors: Faye Craven, Gavin T. Noble, Josef Voglmeir, Robert Šardzik, Sabine L. Flitsch and Simon J. Webb

Presenting author affiliation: Manchester Institute of Biotechnology and School of Chemistry, University of

Manchester, 131 Princess St, Manchester M1 7DN. Email: [email protected]

Several glycolipids have been synthesised and shown to cluster into

artificial “lipid rafts” in solid ordered or liquid ordered phospholipid

bilayers, whilst remaining unclustered in liquid disordered bilayers.1,2

Addition of soluble β-(1,4)-galactosyltransferase (β4GalT1) to a mixture of

UDP-galactose (UDP-Gal) and N-acetylglucosamine (GlcNAc)-capped

glycolipid 1 in phospholipid vesicles was found to catalyse the transfer of

galactose to the headgroup of 1 (Fig. 2). This transformation of the GlcNAc

“glycocalix” into a Gal-β(1,4)-GlcNAc “glycocalyx” made these vesicles

susceptible to agglutination by Erythrina cristagalli lectin.

The rate of enzymatic galactosylation was significantly enhanced when lipid 1 was clustered in lipid rafts

(Fig. 1b); at 1% mol/mol in the membrane, clustered GlcNAc lipid 1 was galactosylated 9-fold faster than

lipid 1 dispersed across the bilayer surface (Fig. 1a). This effect was observed for β4GalT1 but not during the

transformation of an analogous sialic acid-capped lipid 2 by TcTS/fetuin, and it is suggested that the

difference is due to the shape of the active site in β4GalT1.3 Our observations that laterally inhomogeneous

glycolipid distribution can accelerate the rate at which a soluble enzyme transforms a glycolipid may have

wider implications; cellular lipid rafts may act as focal points for the action of soluble cytosolic enzymes.

Figure 2: Enzymatic transformations of synthetic fluorinated glycolipids.

Key references: [1] Liem, K. P.; Noble, G. T.; Flitsch, S. L.; Webb, S. J. Faraday Discuss. 2010, 145, 219-233.

[2] Noble, G. T.; Liem, K. P.; Flitsch, S. L.; Webb, S. J. Org. Biomol. Chem. 2009, 7, 5245-5254 [3]

Ramakrishnan, B.; Balaji, P.V.; Qasba P.K. J. Mol. Biol, 2002, 318, 491.

Figure 1: a. Unclustered glycolipids in a

disordered phospholipid bilayer b.

Clustered glycolipids in an ordered bilayer.

a.

b.

51

Poster 42

The synthesis and rate of hydrolysis of a new beta-sultam;

1,2-thiazetidine-1,1-dioxide-3-carboxylic acid

Authors: Victoria L. Sutcliffe, Michael I. Page and Andrew P. Laws

Presenting author affiliation: School of Applied Sciences, University of Huddersfield, Queensgate,

Huddersfield, United Kingdom, HD1 3DH

Enzyme inhibition forms the basis of much of the medicinal chemistry used in the treatment of

disease. Finding molecules which target specific enzymes is the focus of much research and our

group has spent many years investigating small ring compounds such as β-lactams and β-sultams

as potential inhibitors of β-lactamase enzymes. Structurally β-sultams are similar to β-lactams, the

sulfonyl group substituting for the carbonyl, and they have been shown to have a range of

biological functions including anti-inflammatory behaviour and inhibition of DD-peptidase and

elastase enzymes [1-3]. Hydrolysis occurs by cleavage of the N-S bond with the rate being

dependent on the substituents on the ring. Structural stability is important for potential enzyme

inhibitors and so a detailed understanding of their reactivity is required.

We would like to present the synthesis of a novel β-sultam for use as a potential inhibitor of the β-

lactamase enzyme, BcII. 1,2-thiazetidine-1,1-dioxide-3-carboxylic acid (1) has been prepared by a

four step route from L-Cystine and the final product has been characterised by NMR and MS.

The rate of hydrolysis at a range of pHs has been calculated to produce a pH-rate profile for the

compound. It is relatively stable at physiological pH with the half-life of 1,2-thiazetidine-1,1-dioxide-

3-carboxylic acid being over 2 weeks at pH 7. The rate of hydrolysis increases as pH decreases

and the half-life at pH 2.5 is around 14 minutes. Curve fitting has elucidated the rate constants for

the hydrolysis and shown the pKa of the acid group to be 4.5.

Key references

[1] Ward, R. J., Lallemand, F., de Witte, P., Chrichton, R. R., Piette, J., Tipton, K., Hemming, K., Pitard, A.,

Page, M. I., Corte, L. D., Taylor, D. and Dexter, D., Biochemical Pharmacology 2011, 81(6): 743-751.

[2] Llinás A, Ahmed N, Cordaro M, Laws AP, Frère JM, Delmarcelle M, Silvaggi NR, Kelly JA, Page MI.,

Biochemistry. 2003 44(21):7738-46.

[3] Hinchliffe PS, Wood JM, Davis AM, Austin RP, Beckett RP, Page MI., Org Biomol Chem. 2003 1(1):67-80.

52

List of Delegates

Please email Dr Simon Webb ([email protected]) if you would like to contact one of the

delegates on this list about their work.

Name Affiliation

Sarah Adams Cardiff University

Amar Al-Jasim University of Huddersfield

Muhannad Alhudhud University of Huddersfield

Naomi Allaway University of Leicester

Jon Ashley NUS

Keir Bailey University of York, Department of Biology, CNAP

Adam Baldwin University of Leicester

Lucy Ballamy ConvaTec

Rosie Bamford University of Birmingham

Ashley Beattie The University of Edinburgh

Ryan Beattie University of Bristol

Matthew Bennett University of Manchester

Balkrishna Bhamare University of the West of Scotland

Robin Bon University of Leeds

Andrew Booth University of Manchester

Gemma Bullen University of Birmingham

George Burslem University of Leeds

Raul Camarillo University of Manchester

Dominic Campopiano The University of Edinburgh

Oscar Cascon Cardiff University

David Clarke University of Edinburgh

Thomas Coxon University of Manchester

Faye Craven University of Manchester

Charlotte Dalton University of Manchester

Simge Davulcu University of Bath

William Dawson Cardiff University

Rebecca Driesener University of Southampton

Jolly Gandhi University of the West of Scotland

Joseph Griffin University of Huddersfield

Suzannah Harnor University of Newcastle

Jianjun Hu Institute of Cancer Therapeutics, University of Bradford

Elizabeth Hull UCL

Andrew Jamieson University of Leicester

Andrew Laws Huddersfield University

Honorine Lebraud University of Newcastle

James Leigh University of Manchester

Kate Leigh University of Manchester

Ross Lewin University of Manchester

Sarah Lovelock University of Manchester

Phillip Lowe University of Manchester

Darren Machin University of Leeds

Amanda Maginty Queens University Belfast

Giorgia Magnatti University of Leeds

53

Núria Marí Fortuny University of the West of Scotland

Christopher McLean Edinburgh University

Dharmit Mistry University of Huddersfield

Diana Monteiro School of Chemistry

Phil Morrison School of Chemistry, University of Leeds

Raza Muhammad University of West of the Scotland

James Murray University of Leeds

Stephanie Myers Newcastle University

Riyadh Nahi Cardiff University

Aqsa Nazeer University of the West of Scotland

Sobia Noreen Manchester Institute of Biotechnology, The University of Manchester

James Redman Cardiff University

Douglas Roberts University of Bristol

Glenn Robinson University of Huddersfield

Angelica Jimenez Rosales University of Manchester

Bhanu Rudra University of the West of Scotland

Sohaib Sadiq University of Huddersfield

James Scotson University of Huddersfield

Pallav Shah University of the West of Scotland

Sarah Shepherd University of Manchester

Alison Singer The University of Edinburgh

Rebecca Snell AstraZeneca

Anna-Winona Struck University of Manchester

Matthew Styles University of Manchester

Yunyun Sun Imperial College London

Victoria Sutcliffe University of Huddersfield

Matthew Tozer Peakdale Molecular

Vijayapal Reddy Vasireddy University of the West of Scotland

John Wadsworth Edinburgh University

Daynea Wallock-Richards University of Edinburgh

Menglu Wang University of Edinburgh

Simon Webb MIB, University of Manchester

Lu Shin Wong University of Manchester

Zulfa Yoosuf-Aly University of Cardiff

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