Novel approach to the synthesis of aliphatic and aromatic keto acids

Daniele Balducci and Francesca Paradisi*

UCD School of Chemistry and Chemical Biology, University College Dublin, Belfield, Dublin 4 Ireland.

Fax: 00353 1 716 2501; Tel: 00353 1 716 2967; E-mail: Francesca.paradisi@ucd.ie

Thematic area: Chemical/Structural Biology and Catalysis

Keto acids are very useful substrates in the synthesis of chiral amino acids as

suitable dehydrogenases enzyme can be utilised as biocatalysts (Scheme 1). [1-2]

OH

O

O

R OH

NH 2

O

R

NADH NAD(P)+

NH 3 H 2O

aminoacid dehydrogenase

Scheme 1

In the literature different methods have been reported for the synthesis of either

aromatic or aliphatic keto acids, with yields that vary from very good to very poor.[3]

Here, an efficient synthesis of keto-acids is achieved employing a N-acetyl

diketopiperazin scaffold. The synthesis encompasses both aromatic and aliphatic

substrates proving to be versatile and innovative with excellent carbon economy and

recycling of the glycine by-product (Scheme 2).

HN

NH

O

O

Ac2O

reflux AcN

NAc

O

O

CH2Cl2/RCH O

t-BuOK/tBuOH

AcN

NH

O

O

R

HCl 6M

reflux 2h

R

OH

COOH

NH3+

COOH

+

CH3COOH

Scheme 2

______________________________________________1 P. Busca, F. Paradisi, E. Moynihan, A. R. Maguire, P. C. Engel “Enantioselective Synthesis of Non-natural Amino Acids using Phenylalanine Dehydrogenases Modified by Site-directed Mutagenesis”Organic and Biomolecular Chemistry 2004, 2, 2684 - 26912 F. Paradisi, S. Collins, A. R. Maguire, P. C. Engel “The use of phenylalanine dehydrogenase mutants asefficient biocatalysts for the synthesis of non-natural phenylalanine derivatives” J. Biotechnol.,2007,128, 408-411.3A. J.L. Cooper, J.Z. Ginos, A. Meister “Synthesis and Properties of the -Keto Acids” Chem. Rev. 1983,83, 321-358

PEAT_SA: A program for high-throughput screening of the effect of singlepoint mutations and its application to drug resistant mutations in HIV

Protease.

Michael Johnston, Chresten Søndergaard, Tommy Carstensen, Jens Nielsen

Principal Investigator: Jens Nielsen

Affiliated School: School of Biomolecular and Biomedical Sciences

Thematic Area: Structural Biology and Catalysis

Abstract:

The ability to quickly determine the change in the biophysical properties of a protein

due to a single-point mutation (SPM) is invaluable in protein engineering and the

drug-discovery process.

Here we introduce a new application, called PEAT_SA (Protein Engineering and

Analysis Tool -Structural Analysis), which can calculate the effect of multiple SPM’s

on a proteins stability and dynamics, the pKa’s of its titratable groups and its

interaction with ligands.

We illustrate the use of PEAT_SA by applying it to the problem of drug resistant

mutations in HIV Protease, an enzyme which plays a key role in the HIV life-cycle.

Potent drugs, termed protease inhibitors (PIs), have been developed which target

this enzyme and consequently cause a drastic decrease in HIV replication. However

HIV rapidly accumulates mutations and under the selective pressure of drug

treatment strains often appear which substantially decrease the effectiveness of PI’s.

Here we use PEAT_SA to examine the effect of a large set of mutations on the

intrinsic

properties of the protease and its interaction with a number of the currently

approved PI’s. The set includes both generic mutations (side-chain deletion) and

those known to result in drug resistance.

We correlate our results with the large amount of experimental data available to

investigate the programs ability to predict the effect of SPM’s.

Stereoselective Synthesis and Biological Evaluation of Novel Aromatic

Lipoxin A4 Analogues

Colm Duffy, Syed Tasadaque A. Shah, Surendra Singh, Timothy P. O'Sullivan, Paola

Maderna, Michael Scannell, Catherine Godson, and Patrick J. Guiry

Centre for Synthesis and Chemical Biology, Conway Institute of Biomolecular and Biomedical Research,

School of Chemistry and Chemical Biology, University College Dublin, Belfield D 4, Ireland,

email:colm.duffy@ucd.ie

Lipoxins (LX) are bioactive eicosanoids that activate human monocytes, inhibit

neutrophils and serve as regulators of inflammation. The native lipoxins LXA4 and

LXB4 demonstrate potent anti-inflammatory and pro-resolution bioactions. Moreover,

LXA4 is rapidly regulated by conversion to inactive LX metabolites via local

metabolism that involves dehydrogenation as the predominant route.

Very recently, we have demonstrated the bioactivity of chemically stable aromatic

LXA4 and LXB4 analogues. These aromatic analogues were found to result in a

significant increase of phagocytosis of apoptotic polymorphonuclear leukocytes

(PMN), comparable to the effect of native LXA4. A goal of this study was to

synthesise substituted aromatic ring analogues of LXA4 in order to carry out the

structure activity relationships. Here we report on the stereoselective synthesis of

new aromatic LXA4 analogues. The Sharpless asymmetric epoxidation, along with

necessary protection/deprotection, asymmetric reduction of an alkyl aryl ketone, and

a Pd-catalysed Heck reaction will be discussed.

OH

OH

HO OH O

LXA4

References:

[1]. O’Sullivan, T. P.; Vallin, K. S. A.; Shah, S. T. A.; Fakhry, j.; Maderna, P.; Scannell, M.; Sampaio,

A. L. F.; Perretti, M.; Godson, C.; Guiry, P. J. J. Med. Chem. 2007, 50, 5894.

[2]. Kieran, N. E.; Maderna, P.; Godson, C. Kidney Int. 2004, 65, 1145.

[3]. Takano, T.; Fiore, S.; Maddox, J. F.; Brady, H. R.; Petasis, N. A.; Serhan, C. N. J. Exp. Med.

1997, 185, 1693.

Dimethylamino-Functionalised Titanocenes as Anticancer Drug Candidates

Megan Hogan, James Claffey, Brendan Gleeson, Denise Wallis, Matthias Tacke*Centre for Synthesis and Chemical Biology (CSCB)

Conway Institute for Biomedical and Biomolecular ResearchSchool of Chemistry and Chemical Biology, UCD, Belfield, Dublin 4, Ireland

e-mail: megan.hogan@ucd.ie

The anti-tumor activity of metallocene dihalides has well been established.Titanocene dichloride reached clinical trials, but failed Phase II clinical trials inpatients with metastatic renal cell carcinoma or metastatic breast cancer due to lowanti-proliferative activity and adverse side effects. The work presented hereillustrates that suitable modification of titanocene dichloride may enhance thecytotoxic properties of this compound and overcome the toxicity effects bycontrolling the physiological uptake.

The titanocenes presented here were obtained from the carbolithiation of 6-substituted fulvenes and different lithiated heterocycles, followed by atransmetallation reaction with TiCl4, resulting in various dimethylamino-functionalisedtitanocenes[1-2]. Cytotoxicity of the compounds is expressed as the IC50 value asdetermined from the dose-response curves of the MTT-based assays when thecompounds were tested against epithelial pig kidney carcinoma (LLC-PK) cells. Todate, the most cytotoxic titanocene of this study (depicted below) was found to bealmost as cytotoxic as cisplatin when tested on the LLC-PK cell line[3].

The synthesis of dimethylamino-functionalised titanocenes via carbolithiation is quickand reproducible, thereby enabling the synthesis of a number of titanocenes withdifferent substitution patterns. The most cytotoxic of these substitutions may thenbe used to obtain novel achiral titanocenes via hydridolithiation of the appropriatelysubstituted fulvene with Superhydride, enabling chemotherapy against renal cellcancer (RCC) in the nearby future.

Titanocene M: A dimethylamino-functionalised titanocene with an IC50 value of 5.4μM.

References:[1] M. Hogan, J. Claffey, E. Fitzpatrick, T. Hickey, C. Pampillón, M. Tacke. Metal-Based Drugs, 2008,

Article ID 754358, 7 pages. http://dx.doi.org/10.1155/2008/754358.[2] M. Hogan, J. Claffey, C. Pampillón, M. Tacke, Med. Chem., 2008, 4, 91.[3] M. Hogan, J. Claffey, C. Pampillón, R. W. G. Watson, M. Tacke Organometallics, 2007, 26, 2501 –

2506.

Inhibition of subtilisin Carlsberg by Z-Ala-Ala-Phe-glyoxal

Nicole Howe, Louis Rogers and J. Paul. G. Malthouse

UCD Conway Institute of Biomolecular and Biomedical Research, Centre for Synthesis andChemical Biology, School of Biomolecular and Biomedical science. University College

Dublin, Belfield, Dublin 4, Ireland.

Substrate derived glyoxal inhibitors are potent inhibitors of the serine proteinase

chymotrypsin. With chymotrypsin the serine hydroxyl reacts with the keto carbon of

the glyoxal inhibitor (RCOCHO) [1] and a range of inhibitor adducts are formed [2].

It is therefore not suprising that the glyoxal inhibitor, Z-Ala-Pro-Phe-glyoxal, is an

extremely efficient inhibitor of chymotrypsin with a Ki value of 25 8 nM 3.

However, with subtilisin, inhibition with Z-Ala-Pro-Phe-glyoxal was much weaker with

a Ki value of 2.3 0.2 M 4. The aim of this study is to determine whether other

glyoxal inhibitors can be more effective inhibitors of subtilisin and to determine the

structure of these glyoxal-subtilisin complexes by NMR. It is also intended to

undertake similar studies with metalloproteinases such as stromelysin-1.

References:

1. Spink, E., Hewage, C., Malthouse, J.P.G., Biochemistry (2007); 46(44): 12868-74.

2. Spink, E., Hewage, C., Malthouse, J.P.G., J Biol Chem. (2007) 16; 282(11): 7852-61

3. Djurdjevic-Pahl, A., Hewage, C., Malthouse, J.P.G., Biochem. J. (2002) 362, 339-347.

4. Djurdjevic-Pahl, A., Hewage, C., Malthouse, J.P.G., Biochim Biophys Acta (2005) .1749 (1):

33-41

(A) Chemical/structural biology and Catalysis

The four glutamate dehydrogenases (GDHs) of halophilic organismHalobacterium salinarum

Izyanti Ibrahim, Paul C. EngelConway Institute of Biomolecular and Biomedical Research, University College of Dublin, Belfield, Dublin

4, Republic of IrelandP.I.: Paul C. Engel

Chemical/Structural Biology and Catalysis

Two strains of the halophilic archaeon, Halobacterium salinarum (NRC-36014 andNRC-1), were reported to have more than two glutamate dehydrogenase (gdh)genes in their genome. Strain NRC-36014 contains four gdh genes (gdhA1, gdhA2,gdhB and gdhX) whereas strain NRC-1 contains only three (gdhA1, gdhA2 andgdhB).

The coenzyme specificity has been assigned to two of the GDH proteins, GDHA1 andGDHX, which are NADP+-specific and NAD+-specific respectively. The objectives ofthis research are to study the expression of these four gdh genes by culturing bothstrains in different conditions (i.e. different pH, temperature, salinity and nutrientsupplement), and to determine the biochemical properties of the other two GDHproteins, GDHA2 and GDHB. As no over-expression system was used, cell cultureswere harvested at the growth periods which showed the highest expression levels foreach of these four GDH(s). GDHA1 and GDHX showed the highest expression after72 hours, whereas GDHB was highest after 144 hours. Afterwards, the GDHs wereseparated by a hydrophobic Sepharose-4B column and further purified using a dye-ligand column (DLC) and heat treatment.

DLC and heat treatment are new purification procedures, using betaine to substitutefor salt and exploiting the thermophilic character of these GDH(s). Many of thebiochemical properties of GDHA1 and GDHX differ, i.e. pH optima, heat stability andthe effect of salt concentrations. However, both GDH(s) have the temperatureoptimum of 70C for activity. Biochemical characteristics of the other two GDH(s)(GDHA2 and GDHX) are yet to be analysed.

Oxidative Coupling of 2-Aminophenol and its Silver(I) Complex

Komala Pandurangan, Grace G. Morgan and Helge Müller-Bunz.School of Chemistry and Chemical Biology and Centre for Synthesis and Chemical Biology, University

College Dublin, Belfield, Dublin 4, Ireland.Email: komala.pandurangan@ucd.ie

Actinomycin D (2) is a member of an interesting class of natural products in whichthe yellow–red 2-aminophenoxazine-3-one chromophore is linked to two cyclicpeptides.1 The final step of the biosynthesis of Actinomycin D involves the oxidativecoupling of two molecules of a substituted 2-aminophenol and is catalysed byphenoxazinone synthase, Scheme 1. Actinomycin D inhibits DNA-directed RNAsynthesis and is used in the clinical treatment of a variety of cancers including Wilm’stumour.2-4

Scheme 1. Final step of the biosynthesis of Actinomycin D.

Six different forms of the dimer, 1-6, have been previously reported which arestructural analogues to the actinomycin chromophore but with a variety of redox andtautomeric arrangements.

1 2 3

4 5 6

We have synthesized a dimeric form of 2-aminophenol, 1, by reaction of 2-aminophenol with chloranil in an attempt to make a disubstituted ligand

from chloranil. We isolated the silver (I) complex of the dimeric 2-aminophenol and our structural results on the new complex will be

reported here.

References:

1) C. E. Barry, P. G. Nayer and T. P. Begley, Biochemistry, 1989, 28, 6323.2) U. Hollstein, Chem. Rev., 1974, 74, 625.3) E. Katz and H. Weissbach, J. Biol. Chem., 1962, 237, 882.4) T. Horvath, J. Kaizer and G. Speier, J. Molec. Cat. A: Chem, 2004, 215, 9.

CONHR

NH 2

OH O 2 O

N

O

NH 2

CONHR CONHR

O

2 P h e n o x a z in o n e s y n th a s e

R = -T h r -D V a l-P ro -S a r -M e V a l

Glutamate dehydrogenases from Plasmodium falciparum: targets for

antimalarial drug design?

Aparicio, I.M.1*; McMillan, P.J.2; Müller, S.2 & Engel, P.C.1♦

1. Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland

2. Wellcome Centre for Molecular Parasitology, University of Glasgow, Scotland, United Kingdom

Every year 300-500 million people suffer from malaria, resulting in over one

million deaths (WHO, annual reports). The development of new and more effective

plasmodicidal drugs is, therefore, crucial for the control of the disease.

In the mammalian host, the parasite spends most of its cycle inside

erythrocytes, where it is exposed to intense oxidative stress. The anti-oxidant

defences include redox systems using NADPH as reductant, which in turn requires a

recycling system for NADP+/NADPH. Apparently in Plasmodium the glutamate

dehydrogenase (GDH) reaction is the main source of NADPH. This reaction is absent

in erythrocytes, pointing to Plasmodium GDH as a putative target for antimalarial

drugs. Our previous data suggest that compounds with a lower affinity for the

mammalian enzyme selectively inhibit GDH in the parasite, a crucial feature for a

valid antimalarial drug target.

In P. falciparum, three GDH genes were identified and annotated (PlasmoDB

data). One of these enzymes, a NADP(H)-dependent GDH, was expressed and

characterised (Wagner et al., 1998 ; Werner et al., 2005). Recently, we obtained the

expression of the second enzyme. Preliminary data indicate that this is a dual-

specificity GDH, using both NAD(H) and NADP(H) as co-factors. The cloning of the

third gene is currently on the way.

Stable lineages of parasites bearing a knock-out construction for each enzyme

were recently obtained. Their genetic and phenotypic profile are currently being

determined, and should provide key information for the rationale of drug design,

clarifying whether only one GDH or maybe all three must be targeted.

* Presenting author (poster) - e-mail: Isabela.AparicioTodorov@ucd.ie

contact #: 087 988 2378

♦ PI - Paul C. Engel

Category – Chemical/Structural Biology and Catalysis

Directed evolution of an existing phenylalanine dehydrogenase, L307V, for

enantiopure synthesis of phenylalanine analogues substituted at the

2- and 3-position of the aromatic ring.

Awadelkarim, M.A., Engel, P.C.

Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Ireland

Recent advances in molecular biology techniques coupled to high-throughput

screening methods have paved the way for developing novel biocatalysts with

desired specificity and stability under different reaction conditions.

As described in the literature, biocatalysis has now become a powerful industrial tool

which can be applied in the synthesis of fine chemicals, pharmaceuticals and

agrochemicals, owing to the unique features of biocatalysts which make them

superior to traditional non-biological catalysts. Seah (1997) has engineered

phenylalanine dehydrogenases (PheDHs) from Bacillus sphaericus on the basis of

homology modelling with the resolved 3-D structure of glutamate dehydrogenase

(GDH) from Clostridium symbiosum. So far, these mutants proved to be useful as

biocatalysts for the synthesis of enantiopure non-natural amino acids.

In the present work, an existing mutant, L307V PheDH, was predicted to have a

deep binding pocket for access of substrates with large aromatic side-chains, and

might prove useful for the synthesis of phenylalanine analogues substituted at the 2-

and 3-position of the aromatic ring. Kinetic characterisation of the enzyme should

distinguish the effects of the mutation at position 307 on the affinity and on the

maximum catalytic rate of the enzyme for substrates substituted at the 2- or 3-

positions of the aromatic ring. Furthermore, attempts to enhance the activity of the

existing mutant by random mutagenesis and screening assisted by an automated

colony picker will be reported.

UCD Conway PI: Professor Paul C. Engel

Category: Chemical/ Structural Biology and Catalysis

E-mail: Mohamed.Awadelkarim@ucd.ie

Contact number: 087-61-83803

THE SYNTHESIS AND APPLICATION OF TRIDENTATE BIS(OXAZOLINE)

LIGANDS IN ASYMMETRIC CATALYSIS

Miriam C. Aylward and Patrick J. Guiry*

Centre for Synthesis and Chemical Biology, University College Dublin, Belfield, Dublin 4, Ireland,

Miriam.Aylward@ucd.ie

Asymmetric catalysis has proved to be a very successful approach for the

preparation of enantiomerically pure compounds. It is an attractive tool in both

academic and industrial laboratories as a small amount of enantiomerically pure

material can produce large quantities of enantiopure product. [1] Due to their ready

accessibility, modular nature and applicability in a wide range of metal-catalyzed

transformations, chiral oxazoline ring containing compounds are a versatile class of

ligands for asymmetric catalysis. [2]

One of the most successful applications of the oxazoline containing tridentate ligand

1, [3] the proline-oxazoline derivative 2 and the thiophene-oxazoline ligand 3 is in

the chromium catalyzed asymmetric Nozaki-Hiyama-Kishi allylation of various

aldehydes. [4]

NH

O

N

N

O

R2

R1 O N

NH

R

O

NR'

NH

O

N

N

O

R2

R1

1 2 3

S

This poster will describe a new facile synthesis of the tridentate ligand 1 and its

application into metal catalysed asymmetric transformations.

[1] Catalytic Asymmetric Synthesis, 2nd Edition, Ed. Oijima, I. Wiley-VH, New

York, 2000.

[2] McManus, H.A.; Guiry, P. J. Recent Developments in the Application of

Oxazoline-

Containing Ligands in Asymmetric Catalysis, Chem. Rev. 2004, 104, 4151.

[3] McManus, H.A.; Cozzi, P.G.. Guiry, P.J.. Application of Tridentate Bis(oxazoline) in Catalytic

Asymmetric Nozaki-Hiyama Allylation and Crotylation: An Example of High Enantioselection

with a Non-Symmetric Bis(oxazoline) Ligand, Adv. Synth. Catal., 2006, 348, 551

[4] Hargaden, G, C.; Guiry, P. Eur. J. Org. Chem., 2007, 25, 4235

13C NMR STUDIES OF THERMOLYSIN-GLYOXAL INHIBITOR COMPLEXES IN

PRESENCE OF HIGH SALT CONCENTRATIONS

Mariangela Ceruso, Chandralal Hewage and J. Paul G. Malthouse

University College Dublin, UCD School of Biomolecular and Biomedical science,UCD Centre for Synthesis

and Chemical Biology,

UCD Conway Institute, Belfield, Dublin 4, Ireland.

Specific peptide glyoxals have been found to be potent inhibitors of the thiol- [1] and

serine-proteinases [2]. We are currently synthesising a range of glyoxal inhibitors

and we are using thermolysin to determine if they are effective metalloproteinase

inhibitors.However, the low solubility of thermolysin would prevent us from using

NMR to determine how glyoxal inhibitors interact with the metalloproteinases. It has

been reported that high concentration of salts can improve the solubility of

thermolysin [3]. If this is true, then it could let us use thermolysin concentrations

high enough for NMR studies. But the increased S/N obtained using a high

concentration of thermolysin and inhibitor could be offset by the decreased S/N due

to the high salt concentration.

Therefore in this study we are trying to determine how salt concentrations affect the

signal to noise in 13C NMR. We hope that these studies will allow us to find suitable

conditions to study our glyoxal-thermolysin inhibitor complexes by NMR.

References

5. Lowther, J., Djurdjevic-Pahl, A., Hewage, C., Malthouse, J.P.G., Biochem. J. (2002). 366 (1):

983-987.

6. Spink, E., Cosgrove, S., Rogers, L., Hewage, C., Malthouse, J.P.G., J. Biol. Chem. (2007). 282

(11): 7852-7861.

7. Inouye, K., Kuzuya, K., Tonomura, B., J. Biochem (1998). 123: 847-852

Aknowledgements

We would like to thank Science Foundation Ireland for generous funding.

Uptake Studies of Azurin into MCF-7 Cells: Towards a system for In-

cell NMR

Elysian Chow, Peter B. Crowley.

UCD School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin,

Belfield, Dublin 4.

Azurin is a copper-containing redox protein released by the pathogenic bacterium

Pseudomonas aeruginosa. Azurin is known to enter the cytosol of human breast

cancer MCF-7 cells via endocytosis, travel to the nucleus, enhance the intracellular

levels of p53 and thereby initiate the apoptotic process. The purpose of this project

is to take advantage of Azurin uptake into human cells and to study its interactions

using in-cell NMR. By elucidating the interactions of Azurin in vivo, the regions that

are crucial for activity can be identified. (1)

We have expressed and purified 15N labelled and unlabelled Azurin. An in vitro [15N,1H]-HSQC spectrum of Azurin was obtained. The uptake of Azurin into MCF-7 cells

was examined using confocal microscope imaging and flow cytometry analysis. In-

cell NMR of Azurin in Escherichia coli cells was also examined. In contrast to the in

vitro experiment, the resonances became undetectable in vivo. We hypothesize that

the lack of signal was due to Azurin’s interaction with other macromolecules in the

cell. To test this hypothesis, mutational studies will be carried out to disrupt these

interactions.

Reference:

1. T. Yamada, M. Goto, V. Punj, O Zaborina, M. L. Chen, K. Kimbara, D.Majumdar, E. Cunningham, T.

K. D. Gupta & A. M. Chakrabarty. (2002) PNAS. 99; 14098-14103

UCD Conway PI: Dr. Peter CrowleyCategory: Chemical/Structural Biology and Catalysise-mail: elysian.chow@ucd.ie

Absolute Protein Quantification in the Context of Non-Clinical Drug SafetyEvaluation

Authors: Ben C. Collins1, Peter J. Stone2, Ning Tang3, Thomas Y. Lau1, Stephen R.Pennington4, William M. Gallagher1.

Conway Institute PI: William M. Gallagher - UCD School of Biomolecular and Biomedical Science,UCD Conway Institute, University College Dublin, Ireland1; Agilent Techologies UK Ltd., Cheadle, UnitedKingdom2; Agilent Technologies Inc., Santa Clara, USA3; UCD School of Medicine and Medical Science,UCD Conway Institute, University College Dublin, Ireland4.Thematic Area: (A) Chemical/Structural

Biology and Catalysis

Toxicity issues remain a significant problem for drug development efforts.Specifically, current early biomarkers of toxicity are insufficient and this isdemonstrated by the high failure rate of candidate therapeutics due to safety/toxicityissues. Predtox is a collaborative project partly funded by the EU involving aconsortium of industrial and academic partners throughout Europe.

The aim of this consortium is to assess the value of combining data generated from‘omics technologies (proteomics, transcriptomics, metabonomics) with conventionaltoxicology to facilitate more informed decision making in preclinical safety evaluation.We present a proof of principle experiment whereby candidate protein markers in arat liver lysate generated from ‘omics approaches can be validated by absoluteprotein quantification using peptide multiple reaction monitoring (MRM). Catalase(Swiss-Prot Acc. P04762) was selected as a candidate protein relevant to preclinicaldrug safety based on previous proteomics data indicating up-regulation on treatmentwith troglitazone. Liver lysates prepared from rats treated for 14 days with high dosetroglitazone or vehicle control were subjected to tryptic digest. Appropriate peptidesand MRM transitions were determined using the Peptide Selector tool of theSpectrum Mill Proteomics Workbench (Agilent Technologies) and isotopically labelledsynthetic analogues were purchased for use as internal standards. The digests wereanalysed using a 6410 triple-quadrupole mass spectrometer connected online to a1200 Series nanoflow HPLC via a Chipcube interface (Agilent Technologies).

Endogenous peptides were quantified with respect to a standard curve from serialdilutions of the synthetic peptides. We have demonstrated absolute quantification ofa toxicologically relevant protein with low attomolar sensitivity and linearity over 6orders of dynamic range.Funding is acknowledged from the FP6 Integrated Project, InnoMed, as well as aResearch Demonstratorship from University College Dublin.

Title: Titration_DB - Storage and Analysis of Database of Protein TitrationCurves

Damien Farrell, Emanuel Sa-Miranda, Jens Erik Nielsen

School: School of Biomolecular & Biomedical Science

Research Group: Jens Nielsen Group, Conway

Research Area: Chemical/Structural Biology and Catalysis

Abstract

NMR pH titration experiments are routinely used to measure protein pKa values – a

measurement of the protonation state of each amino acid residue inside the protein.

Accurate pKa values are essential in benchmarking pKa prediction routines and

ultimately in building complete electrostatic models of proteins.

The importance of retaining the raw data has been overlooked and it is often

discarded. pKa values are largely dependent on the titration curve fit and are open to

re-interpretation. The titration curves themselves along with the protein structure

can also give information on, for example, electrostatic interaction energies and on

the nature of the electric field in the protein. We have developed a new data

repository of primary NMR titration data accessible via web interface. Titration curves

have been measured for many proteins and reported in published figures, allowing

us to extract the original data.

This data will be available for download and users will be able to add their data and

do fitting/analyses using our own software, PEAT. A description of the data format

and the fitting procedure is described here. Some novel methods for analysis of the

datasets are also summarized.

The Synthesis and Resolution of Novel P,N Ligands and Their Application inCopper-Catalysed Asymmetric Boration Reactions

William J. Fleming*, Elena Fernandez+, Patrick J. Guiry*

*Centre for Synthesis and Chemical BiologyUniversity College Dublin, Dublin 4, Ireland.

+Dept. de Quimica Fisica i Inorganica, Facultat de Quimica,Universitat Rovira i Virgili, C/Marcel.li Domingo s/n, 43007,

Tarragona, Spain.

The asymmetric boration of olefins is established as a very important transformation

due to the wide utility of the borated compounds en route to a variety of organic

molecules.1 This transformation is somewhat limited due to a heavy reliance on

expensive rhodium metal as part of the catalytic system.

This challenge has been efficently challenged by Yun and Lee using inexpensive

copper as the metal.2 As part of our investigation into the applications of novel

Quinazolinap ligands of type 1, these and other commercially available ligands were

applied to a copper-catalysed variant of the asymmetric boration of α-β unsaturated

olifins (Scheme 1). Quinazolinap ligands have previously demonstrated utility in

asymmetric hydorboration2 and allylic alkylation reactions with impressive levels of

enantioselectivities being obtained.3

N

N R

PPh2

R'

1R = Alkyl, Ar.

R' = H, Cl

R' R

OB2Pin2, MeOH,

CuCl, Ligand, Base

THF, 6 h, RT

R' R

OBpin

or

R' R

OBpin

Scheme 1

This poster will outline the synthetic methodology towards ligands of type 1, their

resolution and our results to date in applying them in the the asymmetric boration of

olefins. (Scheme 1).

1. Marder, T.B. Organomet Chem., 2008, 34, 46-57.

2. Lee, J.; Yun, J.; Angew. Chem. Int. Ed., 2008, 47, 145-147

3. Connolly, D. J.; Lacey, P. M.; McCarthy, M.; Saunders, C. P.; Carroll, A.-M.; Goddard, R.; Guiry,

P. J. J. Org. Chem.; 2004; 69(20); 6572-6589.

4. Fekner, T.; Muller-Bunz, H.; Guiry, P. J. Org. Lett.; 2006; 8(22); 5109-5112

A radiation bystander effect can promote chromosomal instability throughbridge breakage fusion events.

Sheeona Gorman, Miriam Tosetto, Adam Dunlop, Fiona Lyng, Orla Howe,KieranSheehan, Diarmuid O’Donoghue, John Hyland,Hugh Mulcahy, Jacintha O’Sullivan,

Centre for Colorectal Disease, St. Vincents University Hospital, Elm Park, Dublin 4, Ireland,Radiation & Environmental Science Centre, Dublin Institute of Technology and St Lukes Hospital, Dublin,

National Centre for Medical Genetics, Our Lady's Children's Hospital, CrumlinAbstract: There is now clear evidence that ionizing radiation can induce biological

effects in unirradiated cells. This so called bystander response can induce

chromosomal instability in vivo but the mechanisms behind these genomic changes

are not understood. Our hypothesis was that untargeted effects of ionizing radiation

may cause chromosomal instability via the initiation of bridge breakage fusion events

in unirradiated cells and this may have important consequences in vivo.

Design: An ex vivo colorectal cancer model was used to investigate the bystander

response. Resected tumour and matching adjacent normal explant tissue were

treated with different doses of radiation or not irradiated as a control. Conditioned

media was removed from treated and untreated cultured tissue and incubated with a

control colorectal cell line population (sw480). The levels of telomere lengths,

anaphase bridging and micronuclei formation were then assessed in the recipient

cells. Multicoloured fluorescence in situ hybridisation (mFISH) was used to assess

chromosomal rearrangements in bystander cells.

Results: Shortened telomeres, increased anaphase bridging and increased

micronucleus formation were observed in sw480 cells exposed to conditioned media

from treated explant tumour and normal tissue compared to control.(p <0.05)

Karyotype analysis using mFISH showed that chromosome 5 and 8 were most

frequently involved in aberration in bystander cells. Genes located on these

chromosomes may provide novel targets to be investigated in mediation of

untargeted effects of ionizing radiation.

Conclusion: This study provides the first evidence that a bridge breakage fusion

(BBF) cycle is induced by a radiation bystander effect. These early genomic instability

events can lead to chromosomal rearrangements and we believe that acceleration of

instability in bystander cells might have important consequences in vivo such as

increased risk of second cancers and may govern response to neoadjuvant

radiotherapy.

adh2 from Halobacterium sp NRC-1: cloning and over-expression inHaloferax volcanii

Gucciardo Gabriele

Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, UniversityCollege Dublin, Dublin, Ireland

Dr. Francesca ParadisiChemical/structural Biology and Catalysis

One of the major achievement in the development of green chemistry is finding

cleaner and cheaper catalysts which will minimise waste and reaction times. The

main goal of our group is to implement the use of enzymes as biocatalysts. To

overcome some of the enzymes drawbacks such as stability in organic solvents and

versatility, we have focussed our effort on Halophilic archaea organisms which as

source of enzymes with unique biophysical characteristiscs. Halophilic proteins are

more resistant to organic solvents with respect to their mesophilic counterpart.

Alcohol dehydrogenase (ADH) from Halobacterium sp NRC-1 is the class of enzymes

investigate in this project. The over-expression of halophilic proteins is generally

achieved by using E. coli [1]. The expression system is well known and optimised to

deliver high levels of protein. Two enzymes were over-expressed using a pET

system, aryl alcohol dehydrogenase (aad) and alcohol dehydrogenase 4 (adh4), from

Halobacterium sp. NRC-1.

However, this method was inadequate to over-express a third ADH, alcohol

dehydrogenase 2 (adh2). To express adh2 we used an halophilic shuttle vector, pRV-

1, develop by A. Large et all [2]. The vector was introduced into Haloferax volcanii

DS70, the over-expression of the gene is under a tryptophan promoter. Here we

present our most recent results.

Acknowledgment

EPA for funding the project, Dr Peter A Lund for the kind donation of pRV-1 vector.

[1] Kim, H.J., et al., Halophile aldehyde dehydrogenase from Halobacterium salinarum. J ProteomeRes, 2006. 5(1): p. 192-5.

[2]. Large, A., et al., Characterization of a tightly controlled promoter of the halophilic archaeonHaloferax volcanii and its use in the analysis of the essential cct1 gene. Mol Microbiol, 2007.66(5): p. 1092-106.

Apparent Diffusion Coefficient demonstrates greater sensitivity to MultipleSclerosis (MS) lesions than Fractional Anisotropy and conventional

Magnetic Resonance ImagingLonergan, Roisin

Background

Up to one-third of MS patients develop internuclear ophthalmoplegia (INO).Lesions

causing INO are located within the medial longitudinal fasciculus (MLF). The relative

efficacy of conventional and more sophisticated MR techniques to demonstrate these

lesions is unknown.

Objectives

To investigate the relative specificity of DTI (diffusion tensor imaging) in the

detection and quantification of MLF disruption in INO subjects.

Methods

We compared the ability of DTI to display MLF lesions with that of T2 weighted,

proton density and FLAIR MR sequences. Twelve patients with INO and twelve

control subjects had all procedures. Apparent diffusion coefficient (ADC) and

fractional anisotropy (FA) values were recorded in regions of interest extending from

medulla to midbrain. Reconstructed fiber tracts were validated by a neuroanatomist.

A blinded neuroradiologist identified MLF lesions on conventional MR images.

Results

DTI identified areas of abnormality in the MLF region for all INO subjects, while the

MLF in 50% of INO subjects was reported as normal following conventional imaging.

Subjects demonstrated significantly reduced ADC values compared to healthy

controls on both sides [(Right INO = 1.0166; Right Control = 0.9318; p = 0.019) (Left INO

= 0.9771; Left Control = 0.9286; p = 0.029)]. Lower FA values between subjects

compared to healthy controls were not found to be significant.

Conclusions

Compared to conventional MRI, ADC values identify areas of abnormality with

greater sensitivity, allowing confirmation of lesions for a range of clinical signs. We

propose that DTI could enable more accurate monitoring of disease progression and

response to therapy, and guide treatment choices.

New Oxazoline-containing Ligands for Asymmetric Catalysis

Mc Keon, Sean, Guiry, Patrick*.

Centre for Synthesis and Chemical Biology, School of Chemistry and Chemical Biology, UniversityCollege Dublin, Belfield, Dublin 4, Ireland.

Email: sean.mckeon@ucdconnect.ie

Asymmetric metal catalysis is an effective and efficient method of preparing

enantiopure compounds.1 The asymmetry of a metal-catalysed process is controlled

by the organic groups bound to the metal, as they control the binding of the

reactants and their reaction paths through steric and electronic interactions. Chiral

tridentate ligands are believed to form a deeper concave pocket around the metal

centre.

The oxazoline unit has been used successfully in a wide range of metal-catalysed

asymmetric transformations and the tridentate example 1 has been prepared within

the Guiry research group.2 During that study, non-C2-symmetric examples (R1 ≠ R2)

were found to induce optimal enantioselectivities in the Cr-catalysed Nozaki-Hiyama

Kishi (NHK) reaction.3 This poster describes the synthesis of non-C2-symmetric

ligand classes 1 and 2. These ligands offer a further electronic desymmetrisation,

which is hoped to increase the enantiometric excess achieved in metal catalysed

asymmetric transformations.

(1) Catalytic Asymmetric Synthesis, 2nd Edition, Ed. Oijiama, I. Wiley-VH, New York, 2000.(2) Mc Manus, H. A.; Guiry, P. J. Chem. Rev. 2004, 104, 4151-4202.

(3) Hargaden, G.C.; Guiry, P.J; Adv. Synth. Cat. 2007, 349, 2407-2424.

N

NH

N N OO

R1 R2

NH

S N N O

R1 R2

NH

N N OO

R1 R2

1 2 3

In-vitro reactivation of inclusion bodies and homologous expression ofrecombinant glutamate dehydrogenases from Halobacterium salinarum

Munawar N., Engel P.C.School of Biomolecular & Biomedical Sciences, University College Dublin, Dublin Ireland.

Ingoldsby et al (2005) reported four glutamate dehydrogenase genes (GDH A, A2, B

and X) after amplifying the genomic DNA of colourless strain 36014 of Halobacterium

salinarum. Most typical enzymes from halophilic archaea require high concentrations

of salt for activity and stability. Expressed in E.coli, these enzymes are inactive unless

refolded in the presence of salt in-vitro. The over-expression of four recombinant

GDH(s) in E.coli produced inclusion bodies, and in-vitro refolding protocols explored

so far have yielded only insoluble aggregates or soluble inactive protein.

Owing to this problem we need a homologous expression system to keep these

proteins in their active native state. Haloferax volcanii is an obligate halophile and is

particularly suited for genetic analysis in archaea as tools for its genetic manipulation

are well developed. Transformation is straight forward, there are several plasmid

vectors and a number of antibiotic selective markers available. NAD+-specific GDHX

from Halobacterium salinarum has been cloned successfully in pRV1-tna plasmid

having a tryptophane-inducible promoter. Transformation was performed by the

polyethylene glycol (PEG) method and transformants were selected on 18% modified

growth medium (MGM) having novobiocin as a selective marker. Haloferax volcanii

having pRV1-tna GDHX plasmid and induced by 4 mM tryptophane has 50 times

more NAD+-specific GDH activity than cells without the plasmid. We are currently

trying to improve expression levels using different concentrations of tryptophane.

The most recent results will be reported.

UCD Conway PI: Professor Paul C. EngelCategory: Chemical/Structural Biology and CatalysisEmail Address: nayla.munawar@ucd.ie

Title: Kinetic Studies of Phenylalanine Dehydrogenase from Bacillussphaericus with a Variety of Substrates.

Rajesh Narayanan and Paul C. Engel( School of Biomolecular and Biomedical Science,Conway Institute )

Detailed kinetic studies have been carried out on wild type phenylalanine

dehydrogenase from Bacillus sphaericus. Full initial rate equations have been

extracted from the wild type enzyme by systematically varying the concentration of

both the coenzyme, NAD and the substrate, phenylalanine and doing the same for

analogues.

The kinetic constants have been used to apply mechanistic tests to find out the order

of substrate binding. From the initial experiments, we were able to rule out some of

the candidate mechanisms to explain its order of substrate binding. The coenzyme

analogue Deamino NAD was prepared chemically from NAD with nitrous acid and

purified chromatographically to use for the experiments.

Initial results were suggested a random order mechanism by the enzyme for this two

substrate reaction. On the basis of this mechanism, the dissociation constants of all

the binary and ternary enzyme substrate complexes can be calculated from the

parameters obtained through the initial experiments.

Principal Investigator- Prof: Paul C. Engel.Area of Research - Chemical /structural Biology and CatalysisRajesh.Narayanan@ucdconnect.ie

Alanine Scan of Hen Egg White Lysozyme (HEWL)O’Meara F, Bradley J, Kukic P, Nielsen JE.

School of Biomolecular and Biomedical Science, Conway Institute, University College Dublin.PI: JensNielsen

Category: Chemical/Structural Biology and Catalysis

Enzymes catalyze chemical reactions by preferentially lowering the energy of the

transition state (TS) of the reaction compared to the energy of the enzyme-substrate

(ES) and enzyme-product (EP) ground states. The rate of enzymatic catalysis is

related to the energy difference between the ES state and the TS, and it is of

significant interest to construct enzymes that minimize this energy difference and

thus speed up catalysis.

It is likely that enzymes achieve preferential stabilisation of the TS using electrostatic

pre-organisation (1), but exactly how the differential stabilisation is achieved is

poorly understood. In the present project we attempt to understand the preferential

stabilisation of the TS in Hen Egg White Lysozyme (HEWL) by mutating every single

residue in the enzyme to alanine.

Each mutant is compared to the wild type (wt) for changes in secondary structure

using CD spectropolarimetry and changes in the rate of catalysis (kcat parameter).

Using this information along, with X-ray crystal and NMR structures of the WT, a

map of residues that are important for catalysis, but do not significantly affect the

structural stability of the enzyme, can be created. This helps us understand what

type of chemical interactions between active site residues and residues further away

are important for catalysis.

We can also use the data to test the pKD (2), FOLD-X (3), and Goodvibes (4)

software tools, which were designed to estimate changes in pKa values, stability and

dynamics respectively in mutated proteins.

Almost 50 mutants have already been expressed and purified. Data from these

mutants are presented here.

1. Warshel A., Electrostatic origin of the catalytic power of enzymes and the role ofpreorganized active sites. J Biol Chem, 1998. 273(42): p. 27035-8.2. Tynan-Connolly BM, Nielsen JE, pKD: re-designing protein pKa values. Nucleic Acids Res. 2006Jul 1;343. Guerois R, Nielsen JE & Serrano L. Predicting changes in the stability of proteins and proteincomplexes: A study of more than 1000 mutations. J Mol Biol 2002 Jul 5;320(2):369-874. Carstensen T, Nielsen JE, http://polymerase.ucd.ie/goodvibes/

A Novel One-pot Protection/ Deprotection Synthetic MethodologyCatalysed by ZrCl4 for the Synthesis of Key intermediate of Lipoxin

Analogues and Mosquito Attractant Pheromones

Surendra Singh and Patrick J. Guiry*

Centre for Synthesis and Chemical Biology, University College Dublin,Belfield, Dublin4, IrelandEmail: patrick.guiry@ucd.ie, surendra.singh@ucd.ie

Name of PI /Research Group(s): Prof. Pat Guiry

Thematic areas for your research: Chemical/Structural Biology and Catalysis

The protection or deprotection of a functional group is one of the most important

and widely carried out synthetic transformations in preparative organic chemistry.1,2

We have recently reported the synthesis and biological evaluation of aromatic

analogues of lipoxin A4.3 We have found that a catalytic quantity of ZrCl4 (20 mol %)

was an efficient catalyst for the one-pot esterification and deprotection of (5S,6R)-

5,6-diacetoxyoct-7-enoic acid in a good yields for the key intermediate of Lipoxin A4.

The catalyst (10–20 mol %) was sufficient to deprotect 1,3-dioxalane, bis-TBDMS

ethers and diacetate functional groups in excellent yields.4 We have also explored

this methodology for the synthesis (–)-erythro-6-acetoxy-5-hexadecanolide as a

mosquito attractant pheromones in a seven steps.

OMe

OHO OH

OH

Lipoxin A4

O OC10H21

OH

Mosquito attractant pheromones

HO OH

OMe

OCOOH

AcO OAc ZrCl4 (20 mol%)+

O OOH

CH3OH, 25 °C, 48 h

Yield 62% Yield 13%

HO OH

OMe

O

COORR1O OR1

R = Me, nPr, iPr, Et, PhenylethylR1 = TBDMS, Ac, C(Me)2

ZrCl4 (20 mol%)

CH3OH, 3560 °C, 48 hYield 8093%

References:

[1] Greene, T. W.; Wuts, P. G. M. Protective Groups in Organic Synthesis; 3rd ed.; John Wiley andSons: New York, 1999.

[2] Kocienski, P. J. Protecting Groups; 1st ed.; Georg Thieme Verlag: Stuttgart, Germany, 1994.[3] T. P. O’Sullivan; K. S. A. Vallin; S. T. A. Shah; J. Fakhry; P. Maderna; M. Scannell; A. L. F. Sampaio;

M. Perretti; C. Godson and P. J. Guiry, J. Med. Chem, 2007, 50, 5894[4] S. Singh; C. D. Duffy; S. T. A. Shah and P. J. Guiry, J. Org. Chem, 2008, ASAP

Structural studies of Human Obestatin using NMR Spectroscopy andMolecular Modelling

Subasinghage AP1, Flatt PR2, Green BD3, and Hewage CM1*

1. UCD School of Biomolecular and Biomedical Science, UCD Centre for Synthesis and ChemicalBiology, UCD Conway institute, University College Dublin, Belfield, Dublin 4, Ireland

2. School of Biomedical Sciences, University of Ulster, Cromore Road, BT52 1SA, NorthernIreland, UK

3. Queens University Belfast, David Keir Building, Stranmillis Road, Belfast, BT9 5AG, NorthernIreland, UK

Obestatin, 23 residue peptide hormone, was first discovered in rat stomach. Human

form of this peptide sequence has three mutations with the primary sequence of

FNAPFDVGIKLSG VQYQQHSQAL. This peptide is derived from the preproghrelin, and

was initially proven to be involved in the regulation of energy balance, reduction of

food intake and body weight and therefore considered that it has opposite actions of

ghrelin. Recent studies have shown that it promotes pancreatic β-cell and human

islet cell survival and stimulate the expression of main regulatory β-cell genes in the

pancreas. However the biological activities of the obestatin still remain unknown.

Therefore, to understand the

basic structural requirements for the

biological activity of human obestatin,

the solution structure was investigated

by proton NMR spectroscopy and

molecular modelling using various

solution conditions. The calculated

structure of human Obestatin is

characterized by a continuous well

defined alpha helix between residues

Pro4 and Gln15 in 33% TFE solution.

NMR based conformational

features obtained from this project

could help to reveal the structural

motif(s) which would be useful in

understanding the biological role of

Obestatin and its important actions.

Cloning and overexpression of alcohol dehydrogenase (ADH4) from Halobacteriumsp. NRC-1 for use in chemical synthesis

L. M. Timpson and F. Paradisi

Centre for Synthesis and Chemical Biology, UCD School of Chemistry and Chemical Biology, University College Dublin,Ireland.

Prompted by the current requirement for environmentally favourable processes, organic

chemistry is embracing enzymatic asymmetric synthesis [1]. Of industrial importance is the

biocatalytic reduction of ketones to valuable chiral alcohols [2]. This work focuses on the

investigation of alcohol dehydrogenase 4 (ADH4) from the extreme halophile, Halobacterium sp.

NRC-1 as a potential candidate for this transformation.

The gene encoding adh4 has been cloned from the wild type organism into the expression

vector pET21a and overexpressed in E. coli BL21(DE3). The protein was predominantly

overexpressed as insoluble inclusion bodies. Reduction of the growth temperature allowed for a

slight increase in solubility. Activity testing has been performed on the protein soluble fraction

and results have been compared with those obtained when the wild type organism was

employed. Refolding and reactivation from inclusion bodies has also been investigated.

This study is financed by Science Foundation Ireland (SFI).

[1] A. Schmid, J. S. Dordick, B. Hauer, A. Kiener, M. Wubbolts, B. Witholt, Nature, 2001, 409, 258-268.

[2] W. Hummel, M. Kula, Eur. J. Biochem., 1989, 184, 1-13.

NMR based docking studies of glucose-dependent insulinotropic polypeptide (GIP)with the N-terminal domain of its receptor

Venneti KC1, Malthouse JPG1, O’Harte FPM2, Hewage CM1

1UCD School of Biomolecular and Biomedical Science, UCD Centre for Synthesis and Chemical Biology, UCD ConwayInstitute of Biomolecular and Biomedical Research, Belfield, Dublin-4, Ireland

2School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, BT52 1SA, Northern Ireland, UK

Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone synthesised in gut K-

cells and simulates insulin secretion and releases it after meal ingestion. GIP due to its

insulinotropic properties has glucose lowering ability and hence, considered as a potential target

for type 2 diabetes. It has found links to fat metabolism and obesity. GIP enhances glucose

stimulated insulin secretion by interacting with hetero trimetric G-protein coupled receptor

located on the pancreatic -cell. However the exact mechanism of interaction is not understood.

Therefore, it is important to determine the mechanism of action of GIP with its receptor and to

identify the important structural features of GIP responsible for receptor recognition. The

docking interactions between the GIP ligand and N-terminal domain of GIP receptor were

determined in full rotation mode by using HEX software and two-step search was used for

obtaining good resolution of docking correlations. The results of this study indicate that the

docking conformation of GIP is stabilised by the presence of hydrophobic and intermolecular

hydrogen bonding interactions. The ligand-receptor complex model highlights the possible

interactions between the residues Glu19, Gln20, Lys30 and Lys33 of the GIP-ligand with the

residues Gln30, Asn120, His115 and Leu111 of the N-terminal domain of the GIP receptor

respectively. The results obtained in this study could be useful and may facilitate in the

development of therapeutic drugs in the treatment of type 2 diabetes and other related

disorders.