BBSRC DTP Studentships Cell-friendly surfaces: phosphonic acid ...

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Dec 5, 2012 (4 years and 8 months ago)

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BBSRC DTP Studentships


Cell
-
friendly surfaces: phosphonic acid polymers for tissue scaffolds

Prof Peter Budd

(School of Chemistry
)
Prof Sandra Downes (School of Materials), Prof David Watts, Dentistry


Metallation
-
Enhanced Biotransformations: Expanding
the Molecular Scope of Biocatalysis for the
Production of Amine Building Blocks

Supervisors:
Prof Jonathan Clayden (School of Chemistry), Prof Nicholas Turner (School of Chemistry)


METAL COMPLEX LIGHT
-
DRIVEN BIOCATALYSTS

Supervisors:
Dr Benjamin J Coe
(School of Chemistry), Prof Nigel S Scrutton (Faculty of Life Sciences)


Conformational Dynamics of a Multidomain Phosphatase

Supervisors:
Dr Alistair Fielding (School of Chemsitry), Dr

Lydia Tabernero (Faculty of Life Sciences) Prof Philip
Woodman (Facult
y of Life Sciences)


Elucidating the roles of protein regulation by phosphorylation of histidine residues in mammalian cells

Supervisors:
Dr Claire Eyers (School of Chemistry), Professor Simon Hubbard (Faculty of Life Sciences)


The synthesis of novel
graphene bioconjugates for applications in biotechnology

Supervisors:
Prof. Sabine L. Flitsch (School of Chemistry), Dr Christopher Blanford (School of Materials), Dr
Sarah Haigh (School of Materials)


Raman spectroscopy and surface enhanced Raman scatteri
ng for direct monitoring of microbial
biotransformations

Supervisors:
Prof Roy Goodacre (School of Chemistry), Prof Nicholas Turner (School of Chemistry)


Integrated bio
-

and chemo
-
catalysis for accelerated synthesis

Supervisors: Prof Michael Greaney
(School of Chemistry), Prof Nicholas Turner (School of Chemistry)


Metabolic profiling of mammalian cells
-

towards single cell characterisation

Supervisors:
Dr Nick Lockyer (School of Chemistry), Prof Roy Goodacre (School of Chemistry)


Luminescent Enzyme

Biosensors Based on Upconverting Lanthanide Nanoparticles

Supervisors:
Dr

Louise Natrajan (School of Chemistry), Dr Sam Hay (Faculty of Life Sciences)


Structural, spectroscopic and computational studies of biological and bio
-
inspired solar water
splitting
catalysts

Supervisors: Dr

Patrick O’Malley (School of Chemsitry), Dr Robin Pritchard (School of Chemistry)


Evolved Cytochromes P450 in a chemo
-
enzymatic approach for the generation of novel antibacterials for
industry

Supervisors: Prof David Pro
cter (School of Chemistry, Prof Sabine Flitsch (School of Chemistry)


Enzyme
-
coupled Tandem Processing of Organosilicon Novel Compounds

Supervisors: Dr Peter Quayle (School of Chemistry), Dr Lu Shin Wong (School of Chemistry)


Novel synthetic biology appro
aches for generating multifunctional catalysts

Supervisors: Prof Nicholas Turner (School of Chemistry), Prof Jon Lloyd (School of Earth, Atmospheric and
Environmental Sciences)


Development of novel biocatalysts for ‘difficult’ strategic bond
functionalization reactions

Supevisors: Dr Roger Whitehead (School of Chemistry), Professor Nicholas Turner (School of Chemistry)






Cell
-
friendly surfaces: phosphonic acid polymers for tissue scaffolds


Prof Peter Budd

(School of Chemistry
)
Prof Sandra
Downes (School of Materials), Prof David Watts, Dentistry




Background:
Tissue engineering concerns the repair of bone or other body tissues. A tissue scaffold is a
structure which supports the three dimensional growth of tissue cells. A good tissue sca
ffold has (1) high
porosity and appropriate pore size for implanting (“seeding”) cells and for nutrients to diffuse through the
structure, (2) surface characteristics that encourage the development of cells, (3) sufficient mechanical strength
to maintain i
ntegrity whilst the new tissue grows and (4) biodegradability, so that the scaffold breaks down once
the newly formed tissue is able to take over the mechanical load. In previous work in the School of Materials,
polymer scaffolds have been prepared compris
ing poly(

-
caprolactone) (PCL) together with a copolymer of vinyl
phosphonic acid and acrylic acid (PVPA
-
AA).
1

The polymer complex has been shown to have osteoconductive
and osteoinductive properties. Two patents covering the invention have been filed. Ho
wever, only one PVPA
-
AA
composition has been available for study, and the underlying science has not been fully elucidated.



Aim:

The proposed project is focussed on gaining a fundamental understanding of the influence of phosphonic
acid and carboxylic a
cid functionality on the activity of osteoblasts (bone
-
forming cells) and osteoclasts (bone
-
resorbing cells), advancing the basic science in support of a research programme aimed at developing synthetic
bone graft substitutes and bone void fillers for orth
opaedic and dental applications.



Objectives:

(1) To prepare a range of well
-
defined copolymers varying in vinyl phosphonic acid (VPA) content
and molar mass. This will involve (1a)
Optimisation of procedures for copolymer synthesis and purification,
esta
blishing preferred monomers (
e.g.
, direct polymerization of acid monomers, or hydrolysis of precursor
polymer prepared from dimethyl ester or dichloride monomers), initiator (
e.g.
, azo or other radical initiator) and
reaction conditions; (1b)
Optimisation of methodologies for characterization of
copolymers with respect to
composition (quantitative
31
P NMR), microstructure (
1
H and
13
C NMR) and molar mass distribution (aqueous gel
permeation chromatography). (2) To prepare and characterize scaffo
lds, and substrates for control experiments,
from PCL and PVPA
-
AA copolymers. This will include electrospinning of PCL fibres and functionalisation with
PVPA
-
AA by a variety of methods. (3) To undertake
in vitro

tests of biocompatibility, seeking to establ
ish the
roles of hydrophilicity, chelation of calcium ions, and other factors. This will include the culture of human primary
osteoblast cells on various substrates and assessment of cell number and mineralisation. (4) To undertake
biodegradation studies,
seeking to illuminate the fate of phosphonic acid and carboxylic acid residues.



Multidisciplinary training:
The proposed PhD project begins with basic polymer science, requiring the synthesis
and characterization of a range of novel copolymers. This aspe
ct of the work will be carried out within the Organic
Materials Innovation Centre, in the School of Chemistry, and led by PMB (Professor of Polymer Chemistry).
Specialised techniques are available for polymer characterization, such as gel permeation chroma
tography. The
student will be integrated into a research group focussed on developing novel polymers for a variety of
applications. DW (Professor of Biomaterials Science in the School of Dentistry) has considerable experience in
the development of biomate
rials for dental and orthopaedic applications and will provide advice and support in
relation to polymer preparation and to materials science aspects of scaffold formation and biodegradation. The
production of scaffolds by electrospinning, and biocompatibi
lity studies, will be carried out in the School of
Materials under the supervision of SD (Professor of Biomaterials), building on work by previous PhD students
and postdoctoral researchers. The PhD project, whilst being focussed on basic science, will run
in parallel with,
and interact strongly with, a programme of work aimed at specific applications, including the development of a
bone void filler, which is the subject of a current BBSRC super follow
-
on
-
fund application, with support from
Smith & Nephew. T
he student will develop a broad range of skills in synthesis, measurement and analysis,
across the chemical, materials and biosciences.



Impact:
The development of an entirely new biomaterial for clinical use is very rare, largely because of the
regulator
y routes it must undergo. However, there is a clinical need for synthetic bone graft substitutes with the
osteoconductive, osteoinductive and mechanical properties required for the treatment of challenging defects, and
bone void filler materials for use wi
th surgical implants. Preliminary results with PVPA
-
AA are extremely
promising and, if borne out by this and other further work, will lead to improved products for a wide range of
surgical procedures.



1.

A.K. Bassi, J.E. Gough and S. Downes,
J. Tiss.
Eng. Regen. Med.
, 2011 (DOI: 10.1002/term.491)



P

For further details please contact

Peter.Budd@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

eter.Budd@manchester.ac.uk



Metallation
-
Enhanced Biotransformations: Expanding the Molecular Scope of

Biocatalysis for the
Production of Amine Building Blocks

Supervisors:
Prof Jonathan Clayden (School of Chemistry), Prof Nicholas Turner (School of Chemistry)


Overview
. Typically over 75% of late stage drug candidates incorporate amine functionality,[1]
and of the top 200
drugs by sales over 40 contain a pyrrolidine, piperidine or piperazine ring.[2] Recent advances in biocatalysis
(many of them developed in the Turner group[3]) have provided important new approaches to the efficient and
economical synthe
sis of chiral amines, especially using methods for deracemization and desymmetrisation.
However, because these methods rely on enzymatic redox processes they invariably generate molecules
carrying C

H bonds adjacent to the nitrogen atom. Nonetheless, it i
s becoming increasingly clear that amines
carrying quaternary centres (i.e. without adjacent C

H bonds) are of value in the construction of drug candidates
(known examples include CETP and ras FTPase inhibitors) and in the manufacture of pharmaceutical pro
ducts.
These compounds are unavailable by biocatalysis alone, but by enhancing biocatalytic methods with new
organolithium chemistry, industrially
-
relevant biotechnological routes to them become possible. Recent
developments in organolithium chemistry, so
me from the Clayden group,[4] show that it is possible to insert a
wide range of carbon
-
based substituents stereospecifically into the CH bond adjacent to nitrogen, oxygen, or
sulfur functionality. By coupling the biological and chemical transformations i
n sequence, it becomes possible to
generate by biocatalysis important new ranges of target compounds, both with proven and proposed
pharmaceutical relevance, including acyclic amines carrying quaternary centres, and nitrogen heterocycles with
quaternary ce
ntres within the ring.

Project plan

1. Suitable precursors for organolithium transformations that are also plausible products of biocatalytic
desymmetrisation or deracemisation methods will be identified. Priority will be given to substituted piperidines
or
pyrrolidines. These will be functionalised chemically and subjected to conditions for C

C bond formation by
rearrangement or alkylation, confirming the viability of the stereospecific C

H to C

C interconversion as a means
to generate quaternary centres.

2. Once an initial collection of viable substrates have been identified, the student will proceed to develop
enzymatic methods for their enantioselective synthesis, establishing scope for variation of the substituted amines
and optimising methods for thei
r generation. Known enzymatic systems (e.g. transaminases, imine reductases,
amine oxidases) will be employed, and new enzymes will be evolved, with tailored properties, using random
mutagenesis coupled with high
-
throughput screening. With enantiomericall
y pure materials in hand the
asymmetric C
-
C bond formation steps will be demonstrated, generating a family of tertiary carbinamines with
drug
-
like substitution patterns. As the scope and limitations of the organolithium processes becomes clear, new
enzymat
ic methods will be developed to address deficiencies in the availability of suitable compounds.

3. As a demonstration of the applicability of the method, target amines, either known drug components or close
analogues / compounds with known activity, will
be identified and synthesised by the tandem biocatalytic
-
organometallic methodology. These include ras
-
farnesyl transferase inhibitors, CETP inhibitors, and pyrrolidines
or piperidines containing quaternary centres. Practical conditions will be establishe
d for the production of these
compounds on a commercially relevant scale. Publication of this work will bring the methods to visibility among
industrial medicinal and synthetic chemists.

4. Not only are industrially
-
relevant drug targets plausible products

from this work, but so are chiral ligands for
metal
-
catalysed processes, in particular those related to QUINAP. During the course of the project, we will
investigate possibility of using organolithium methods
followed by

biocatalytic deracemisation to mak
e such
atropisomeric compounds. Preliminary work in this area is ongoing at present in the hands of a visiting Spanish
Erasmus student.

Multidisciplinary training
.
In the MIB, the student will receive training in a wide range of core biological
techniques
including microbiology, protein expression and purification, enzyme kinetics, high
-
throughput
screening (solid
-
phase assays and use of plate readers), protein modelling, preparative biotransformations, chiral
HPLC/GC and LC
-
MS. The synthetic aspects of th
e project and the mathematical skills used in the kinetic
analysis of the racemisation rates etc. will contribute to a broad multidisciplinary training. We envisage recruiting
a student with a first degree in chemistry, and the student will be able to rapi
dly acquire additional biological skills
by working alongside PDRAs and PhDs in the Turner group.
The students will also gain experience in the
techniques of organic synthesis within the Clayden group in the chemistry building. Both aspects of the projec
t
will also involve extensive use of analytical techniques such as NMR and MS. In order to gain an insight into the
commercial applications of the work, we are currently in discussion with Peakdale Molecular (an SME
specializing in the synthesis of pharmac
eutical intermediates) about the possibility of providing a three
-
month
placement in their laboratories in Derbyshire, during which time he/she will be able to explore industrial
opportunities for application of combined biocatalytic/chemical approaches to

pharmaceutical synthesis.

References:
1. MDL Drug Data Report. MDL Information System Inc. San Leandro CA;

2.
http://cbc.arizona.edu/njardarson/group/top
-
pharmaceuticals
-
po
ster
;

3. Turner, N. J.; Truppo, M. D. in
Chiral Amine Synthesis
, ed Nugent; Wiley
2010
.

4. Clayden, J. et al.
J. Am. Chem. Soc.
2012
,
134
, 7286 and references therein.



For further details please contact

j.p.clayden@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

METAL COMPLEX LIGHT
-
DRIVEN BIOCATALYSTS

Supervisors:
Dr Benjamin J Coe (School of Chemistry), Prof Nigel S Scrutton (Faculty of Life Sciences)


Overview.
Redox enzymes can be photoactivated by dyes which inject electrons into the protein cofactors in the
presence of a sacrificial electron donor
.

Such an approach to biocatalysis is relatively unexplored, but very
promising for producing organic compounds of industrial importance. The premise of this proposal is that
enzymes can be activated efficiently, and even selectively, by transition metal
-
ba
sed chromophores.

Challenges.

This work is based on sound precedents, but is ambitious nonetheless. Experiments will involve
photoactivators free in solution, as well as covalently linked systems prepared by appropriate mutation and dye
labelling of an en
zyme near to its active site. Identifying an effective photoactivator for a particular enzyme
requires careful consideration of redox potentials and optical absorption properties. Extensive directed synthetic
organic and inorganic chemistry, the preparatio
n of mutant enzymes and a very broad range of physical
measurements will be pursued.

Background and state
-
of
-
the
-
art.

Complexes of metals such as ruthenium often show strong, low energy
absorptions with intramolecular charge
-
transfer character. Such chrom
ophores are structurally versatile, and
proven sensitizers in solar cells
1

and related applications like visible
-
light driven H
2

production.
2

Scrutton and
colleagues, as well as others, have used purely organic photoactivators with enzymes such as cytochromes or
neuronal nitric oxide synthase (NOS), with near
-
UV laser excitation
.
3

A number of reports have concerned the
photoreduction (and occas
ionally also oxidation) of cytochromes by non
-
covalently bound Ru
II

2,2

-
bipyridyl
complexes.
4

In such cases, the binding between the complex dye and enzyme is purely electrostatic. More
precise positioning of the chromophore with respect to the active sit
e has been achieved by labeling via
brominated or carboxylic acid derivatives.
5

Other relevant, noteworthy studies have involved “molecular wires”
terminated with either
Ru
II

or Re
I
-
based chromophores and their interactions with cytochromes P450 and NOS
enzymes via both covalent and non
-
covalent binding.
6

Research programme.

Very recently, we have used Ru
II

or Ir
III

polypyridyl

photosensitizers to activate the flavin
dependent oxidoreductase enzymes pentaerythritol tetranitrate reductase (PETNR) and the t
hermophilic old
yellow enzyme (TOYE).
7

These preliminary studies provide important proof
-
of
-
principle, while extensive further
work is required in order to progress towards systems of potential industrial relevance. In this new project, we will
target a ra
nge of enzymes including PETNR, a cytochrome P450 that is involved in terpenoid biosynthesis, and
alcohol dehydrogenase. PETNR is a flavin
-
containing enzyme that catalyzes the reduction of activated alkenes;
this requires the simultaneous delivery of two e
lectrons, and one ambitious goal of this project is to achieve this
by using a bimetallic complex containing two linked chromophoric units. With metal complexes, extensive tuning
of redox and photoexcitation behaviour is
possible via changes in ligand stru
cture,
and there is a vast background literature on
which we will draw to design and
synthesise a range of potential photo
-
activators (
e.g.

1

and
2
). Other aspects
such as the shape, size and charge of a
chromophore can be expected to have
pronounced effec
ts on its potential to
efficiently photoactivate a particular
enzyme. The nature of the sacrificial electron donor and any added electron
-
transfer shuttle will be further
important variables that can be used to optimise performance. One of the established
key requirements for
effective electron
-
transfer is that the distance between the donor and acceptor units must not exceed about 14 Å,
so this consideration will be used to guide the selection of sites for enzyme mutation and covalent linkage
formation.

Mu
ltidisciplinary training.
Synthetic organic and inorganic chemistry will be carried out under the supervision of
Dr Coe in Chemistry. This work will also involve a broad range of characterisation techniques like NMR
spectroscopy and electrochemistry, compl
emented by time
-
dependent density functional theory and other
computations. Biological studies will be supervised by Prof Scrutton with assistance from Dr Derren Heyes in
MIB. The combination of experiments with enzymes and sensitizers either free in solut
ion or covalently linked,
and laser spectroscopic measurements will provide a very extensive training. This project will therefore appeal to
an ambitious, versatile and suitably talented student.





1.

e.g.

A. Hagfeldt
et al.
,
Chem. Rev.

2010
,
110
, 6595.

2.

e.g.

E Reisner
et al.
,
Chem. Commun.

2009
, 550.

3.

e.g.

A B Kotlyar
et al
.,
Eur. J. Biochem
.
2000
, 267, 5805; I Szundi
et al
.,
Biochemistry
2001
,

40
, 2186; H M
Girvan
et al.
,
J. Am. Chem. Soc.
2007
,
129
, 6647;
A J Dunford,
et al.
,
J. Biol. Chem.

2007
,
282
, 24816; D J
Heyes
et al.
,
Chem. Commun
.
2009
, 1124.

4.

e.g.

D Zaslavsky
et al.
,
Biochemistry
1998
,
37,
14910; R C Sadoski,
et al.
,
Biochemistry
2000
,
39,
4231;

R C
Sadoski
et al.
,

J. Biol. Chem.

2001
,
276
, 33616;
F Millett, B Durham,

Biochemistry

2002
,
41
, 11315;
G
Engstrom
et al.
,

J. Biol. Chem.

2002
,
277
, 310272; S E Brand
et al.
,
Biochemistry
2007,
46,
14610.

5.

e.g.

R
-
Q Liu
et al.
,

Biochimie
1995
,

77
, 549; K
-
F Wang
et al.
,

Biochemistry
1996,
35,
15107; K
-
F Wang
et al.
,
J. Biol. Chem.

1999
,
274
,
38042.

6.

J J Wilker
et al.
,
Angew. Chem. Int. Ed.

1999
,
38
, 90; W Belliston
-
Bittner
et al.
,

J. Am.
Chem. Soc.
2005
,
127
,
15907; S M Contakes
et al.
,
Struct. Bonding

2007
,
123
, 177.

7.

M K Peers
et al.
, results to be published.


For further details please
contact

b.coe@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

N
N
R
u
N
N
N
N
N
N
N
N
R
u
N
N
N
N
N
N
1
I
r
N
N
N
N
N
N
+
+
3
+
N
N
N
N
4
+
2


Elucidating the roles of protein regulation by phosphorylation of histidine residues in mammalian cells

Supervisors:
Dr Claire Eyers (School of Chemistry), Professor Simon Hubbard (Faculty of Life Sciences)


Background:

Unlike mammalian cells where phos
phorylation
-
dependent signalling events are primarily
regulated by receptor tyrosine kinases and serine/threonine kinases, bacterial systems have only a limited
repertoire of these enzymes. Rather, they rely on ‘two
-
component’ signalling systems that gene
rally comprise an
auto
-
phosphorylating receptor histidine kinase and a response regulator containing a conserved aspartate
residue to which the phosphate from the catalytic histidine is transferred. Phosphate transfer in this manner
permits the transductio
n of signals from the cell surface to the appropriate functional molecule within the cell.
Transmembrane histidine kinases of this sort are not thought to exist in mammalian cells, where their role of
extracellular sensing is performed by receptor tyrosin
e kinases. However, accumulating evidence suggests that
phosphorylation of histidine on intracellular proteins in mammalian cells may be responsible for certain signalling
events, including neuronal cell and T
-
cell receptor signalling, and differential reg
ulation of cell proliferation. While
preliminary studies suggest that phosphohistidine accounts for ~6% of the phosphoamino acid content in certain
eukaryotes, identification of specific sites of histidine phosphorylation has remained elusive due to the ac
id
-
labile
nature of the modification.

Workplan:

We have previously demonstrated that the protein histidine
kinase PilS will use ATPγS as a substrate in place of ATP, generating a
more stable histidine thiophosphorylated substrate. Subsequent alkylation
with para
-
nitrobenzyl mesylate (PNBM) creates a th
iophosphote ester
epitope against which a commercial generic anti
-
thiophosphate ester
antibody is immunoreactive (Fig. 1). By adapting this strategy, we will use
mass spectrometry to identify novel substrates of exogenous recombinant
bacterial and/or yeast

histidine kinases from human cells. Cell extracts will
be depleted of endogenous ATP and heat treated to inhibit catalytic
activity of endogenous kinases before performing
in vitro

phosphorylation
using ATPγS. The thiophosphorylated substrates will be alk
ylated and the
anti
-
thiophosphate ester antibody used for immunoprecipitation prior to
proteolysis, reverse phase chromatography and tandem mass spectrometric
analysis. We will also assess the prevalence of native mammalian histidine
kinase activity using
non
-
heat treated extracts in the absence of exogenous protein kinases.

Bioinformatics analysis and structural modelling of the identified sites and the proteins on which they reside will
be used to determine the putative physiological role of histidine ph
osphorylation (e.g. cell signalling, trafficking
etc.) and the mechanisms of protein regulation (e.g. enzymatic regulation, protein binding etc.) Should precise
functions be implicated, mutational analysis may be used to confirm function. The motifs surrou
nding the
identified sites will also be investigated with a view to defining one or more putative consensus motifs.
Identification of such motifs and attendant computational approaches would enable the prediction of additional
putative sites of modificatio
n. Equally, we can also place this data in to the context of existing phospho
-
driven
signalling networks looking for synergies and/or novel pathways.

It is only be undertaking such a systems, broad brush approach to identifying sites of histidine phosphorylation in
human cells that we may be able to define physiological roles for this poorly characterised post
-
translational
modification, opening the fie
ld significantly for further study.


Multi
-
disciplinarity:
This is a “wet” and “dry” multidisciplinary project, involving analytical biochemistry,
molecular biology and bioinformatics to elucidate the extent, roles and regulation of histidine phosphorylat
ion in
human cell systems. The protein kinase biochemistry and proteomics training and support will come from Eyers,
with Hubbard leading the bioinformatics. In addition to the direct training within our groups, we aim to target the
student to MSc courses
on which we already teach from the Bioinformatics and Systems Biology MSc
programme to boost the student in areas where there are knowledge gaps, since we are unlikely to recruit a
student with experience in the range of subjects covered in this proposal.

This project fits the BBSRC strategic area to support world class Bioscience Underpinning Bioscience.
Additionally, it will also provide training in handling large proteomics datasets as outlined in BBSRC’s strategic
plan.


For further details please cont
act

Claire.Eyers@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/


Figure 1.
α
J
瑨iophosphaíe esíer
an瑩body tes瑥rn bloí
of in瑡cí and
瑲ínca瑥d

⡋䄩E
domain o映偩l匠景llowing
in vitro
phosphorylation with

ATPγS.


Conformational Dynamics of a Multidomain Phosphatase


Supervisors:
Dr Alistair Fielding (School of Chemsitry), Dr

Lydia Tabernero (Faculty of Life Sciences) Prof Philip
Woodman (Faculty of Life Sciences)

HD
-
PT
P has four domains Bro1 and V domains, a Pro
-
rich region (PRR), and a protein tyrosine phosphatase
domain (PTP). The Bro1 and V domains are essential and sufficient for driving EGFR sorting, and act as a
docking platform for all the relevant ESCRT complexe
s. Preliminary biophysical and structural analyses of the
HD
-
PTP V domain indicate that it exists in a open L
-
shape conformation. However, there is certain degree of
flexibility in solution between the two arms of the V domain and probably between the Bro1

and the V domain.
We believe that this flexibility may be key in controlling binding specificity to the different biological partners and
that it may offer a mechanism of regulation of exchange between ESCRTs. The Woodman group has now
identified various

ECRT components that interact in the V or Bro
-
V domains and they have mapped the minimal
binding regions to 12
-
20 residues. Next, we would like to quantify the degree of flexibility of the V domain both
alone and in the presence of interacting partners. I
n addition the coupling between the Bro1 and V domains is
critical for selective binding to some ESCRTs but not others, therefore we will study the flexibility between these
two domains and the effect on binding.


For this, we propose to exploit the advant
ages of the electron paramagnetic resonance (EPR) spectroscopy to
measure the distributions of distances in solution between the two arms of the V
-

domain under different
conditions and to quantify the variations induced by the binding of interacting partn
ers using synthetic peptides of
the minimal binding regions. The V domains contains three Cys residues, one on each arm and one at the hinge
region, which offer ideal locations for specific labelling with MTSL (1
-
oxyl
-
2,2,5,5
-
tetramethylpyrroline
-
3
-
methyl
-
methanethiosulfonate) and use them in double electron electron resonance (DEER) measurements. Stable MTSL
conformations and distance distributions will be calculated using a rotamer library.


Initially we will establish distance constraints on the native p
rotein and then through a series of competition
experiments aimed at elucidating binding modes. The orientation of spin labels of the protein environment will be
established using a multifrequency approach with measurements at 9
-
, 34
-

and 95

GHz. Also, li
ne width
analysis of singly labelled mutants at multiple frequencies will be used to gain dynamic information about motion
of the nitroxide on the nanosecond time scale and side chain internal motions. This will allow a fuller picture of
the internal motio
ns of the protein.


Potential applications will be the design of mutants that have impaired flexibility (protein engineering) based on
the EPR results. These can then be tested by other biochemical and biophysical techniques (MALS, AUC) and
the molecular s
tructure evaluated by X
-
ray crystallography. Functional assays with the mutants will evaluate
disruption of EGFR trafficking and sublocalisation to different endosomal compartments to test the loss of
functional interactions with the various ESCRTs.


Supervisors:

Alistair Fielding will supervise the advanced EPR experiments, data analysis and simulation. The student will also
attend a workshop provided by EPSRC national service and co
-
taught by the PI.

Lydia Tabernero will supervise the expression and
purification of recombinant protein and various mutants and
biophysical and biochemical characterisation on binding interactions (MALS, AUC, SPR).

Phil Woodman will supervise the functional studies in cells and EM imaging localisation experiments.


Consuma
bles costs:

We request an addition 3 K p.a. to make a contribution to the liquid helium costs, required for the extensive use
of cryogenic EPR experiments.

References

Doyotte
et al
., 2008, PNAS, 105:6308.

Stefani F
.
et al
.,
Curr Biol.

2011, 21:1245.

Ichioka
et al
., 2007, Arch Biochem Biophys 457:142.


For further details please contact

alistair.fielding@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/





The synthesis of novel graphene bioco
njugates for applications in biotechnology


Supervisors:
Prof. Sabine L. Flitsch (School of Chemistry), Dr Christopher Blanford (School of Materials), Dr
Sarah Haigh (School of Materials)


Graphene is a material of superlatives. This single layer of carbon atoms forms the thinnest, strongest, stiffest
and most stretchable material known. These physical and chemical properties make graphene an interesting
component of smart biomaterials such
as biocatalysts, nanoscale diagnostic tools and sensors, vector for
delivery of pharmaceuticals or genes and as foundation for solid
-
phase and combinatorial synthesis of
biomolecules. The full potential of graphene in biotechnology has yet to be realised a
nd the goal of the project will
be to generate graphene
-
bioconjugates through a combination of chemical and biochemical techniques for
application in biotechnology, particularly biocatalysis. This project builds on the international leadership the
Universi
ty of Manchester has both in graphene research and in biocatalysis through CoEBio3.The Flitsch and
Blanford group have recently developed chemical methods for the selective covalent functionalization of
graphene (Fig1) which now allows us to make libraries

of graphene derivatives (with different X
-
groups as in Fig
1).


Figure 1

The project will begin by establishing chemical coupling routes to generate bioconjugates incorporating a range
of biomolecules available in the group starting from small
biomolecules such as peptides and sugars to proteins
as shown in Figure 1. In the second part, these novel conjugates will be characterised using a range of state
-
of
-
the
-
art spectroscopic and imaging techniques such as NMR, mass spectrometry and atomic for
ce microscopy. In
the third part we will investigate applications in biotechnology with a focus on three areas: (i) hosting multistage
chemical and biocatalytic reactions on a single particle that then could be used in applications such as novel thin,
toug
h, flexible wound dressing with built
-
in complex biofunctionality; (ii) as biosensors incorporating
electrochemically driven transformations; and (iii) as platforms for synthetic biology inside and outside cellular
environments. This project will fashion t
he tools to integrate graphene with the biological world.

Project roles

Prof. Sabine Flitsch

will provide training in biological chemistry and is an expert in bioconjugation to surfaces, in
particular of peptides, carbohydrates and proteins. The project wi
ll be able to benefit from interaction with the
Centre of Excellence in Biotransformations (CoEBio3;

http://www.coebio3.org/
) which will guide the choice of
enzymes to match the requirements of end users such as the
chemical and pharmaceutical Industries and to
predict where future commercial applications may exist.

Dr Christopher Blanford

is an expert in the specific and directed attachment of metal
-
containing enzymes to
conductive materials including carbon. This pr
oject will be strengthened by the methods and findings from his
current EPSRC project on energy production through enzyme catalysis. He will provide training in graphene
modification and enzyme attachment.

Dr Sarah Haigh

is a member of the University of Ma
nchester’s graphene working group, led by Nobel laureate
Prof. Andre Geim. She is an expert in analytical electron microscopy and will lead the training on the imaging of
graphene and the graphene

protein composite products and the development of new imagi
ng methods. Dr Haigh
will provide guidance on graphene production and ensure the project work is informed by the latest developments
in the graphene research. Drs Haigh and Blanford will lead the development of the tools to analyse graphene and
its bioconj
ugates.

References



“Efficient electrocatalytic oxygen reduction by the ‘blue’ copper oxidase, laccase, directly attached to
chemically modified carbons.” C.F. Blanford, C.E. Foster, R.S. Heath and F.A. Armstrong.
Faraday
Discussions

140, 319

335 (2009).




Enzymatic catalysis on conducting graphite particles.” K.A. Vincent, X. Li, C.F. Blanford, N.A. Belsey, J.H.
Weiner and F.A. Armstrong.
Nature Chemical Biology

3(12), 760

761 (2007).



“Glycoprotein labeling using engineered variants of galactose oxidase obt
ained by directed evolution.” J.B.
Rannes, A. Ioannou, S.C. Willies, G. Grogan, C. Behrens, S.L. Flitsch and N.J. Turner.
Journal of the
American Chemical Society

133, 8436

8439 (2011).



“Accelerated Enzymatic Galactosylation of

N
-
Acetylglucosaminolipids i
n Lipid Microdomains

,
G.T.
Noble

,

F.L. Craven

,

J. Voglmeir

,
R.Šardzík

,
S.L. Flitsch

, and

S.J.

Webb


Journal of the American
Chemical Society

134

(31), 13010

13017 (2012)

“Chemoenzymatic Synthesis of O
-
Mannosylpeptides in Solution and on Solid Phase” R. Sardzik, S.L.Flitsch et al,
Journal of the American Chemical Society

134(10), 4521
-
4524 (2012).



For further details please contact

Sabine.Flitsch@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

Raman spectroscopy and
surface enhanced Raman scattering for direct monitoring of microbial
biotransformations

Supervisors:
Prof Roy Goodacre (School of Chemistry), Prof Nicholas Turner (School of Chemistry)

Background

The use of biocatalysts, both as enzymes and also engineered

whole cells, in the manufacture of chemicals
offers major advantages in terms of enhanced reaction selectivity, cost of raw materials, lower energy costs,
safety and importantly sustainability. However, a major limitation at present is the time taken to d
evelop
biocatalytic processes which currently restricts their application mainly to 2
nd

generation manufacturing
processes. Within direct evolution experiments sets of potential candidate enzymes are generated and assessed
with the aim of selecting the enz
yme which has enhanced fidelity for the substrate and high product yield. Part of
this process involves the assessment of the enzyme activity and this necessitates the production of an easy to
use assay. For many biotransformations these are bespoke and c
oupled to a colorimetric assay. Of course the
structure of the substrate and product is different and these could be measured using biophysical methods such
as Raman spectroscopy. Raman spectroscopy provides vibrational structural information on molecules

is
quantitative and presents itself as an ideal approach for direct ‘label
-
free’ analysis.

Approach

Whilst Raman spectroscopy has been used to monitor chemical processes direct measurement from bacterial
colonies has not been generated due to the complex

nature of the system. This PhD is to develop Raman
spectroscopy for the direct analysis from colonies and in particular to focus on surface enhanced Raman
scattering (SERS). Laser excitation will be directed on top of colonies and back scattered Raman l
ight collected;
we have lasers in the deep UV (244 nm) to near IR (532, 785, 830 nm) available.

SERS is a powerful approach for boosting the usually rather weak Raman cross section and in our hands 10
4
-
10
6

enhancements are routinely observed for gold and s
ilver nanoparticles (e.g., for colloidal and thin films, see
Anal.
Chem
. 2012,
84
,
7899
-
7905;
Analyst

2012,
137
,
2791
-
2798). As proof
-
of
-
concept we have
generated preliminary data showing that the
-
lactam ring
in ampicillin resulting in new carboxylic acid and
tertiary amide vibrations can be readily monitored
using SERS (see figure for SERS spectra before
and after (60 min) enzyme hydrolysis
as well as
the specific vibrational changes with respect to
time).

In order to deconvolve the SERS spectra so that
the signal from substrate consumption and
product generation is seen chemometrics will be
used and in particular linear and non
-
linear
regres
sion methods such as PLS, KPLS, ANNs,
SVR as detailed in (
J. Phy. Chem. C

2010,
114
,
7285
-
7290). Known levels of substrate and
product from reactions will be generated via LC
-
MS from colony extracts. Additional
benchmarking will include artificial mixture
s of the
substrates/products as well as measuring colony
extracts and the colonies directly.

Supervisory team

Goodacre

has extensive and internationally leading experience in all types of Raman spectroscopies and
especially SERS (>50 papers to date; www.b
iospec.net/pubs.htm), as well as chemometrics (>> 100 papers).
His research group has excellent spectroscopic and laboratory facilities. Facilities include 2 x Renishaw Raman
microscopes (one with a 785 nm laser and the other with 633, 785 and 830 nm lase
r lines) and UV Raman
microscope (244 nm), as well as 6 portable Raman probes covering different excitation wavelengths (532, 633,
785 nm). In addition, for SERS all facilities required for the preparation of samples, including colloid preparation,
are ava
ilable as are the UV/Vis spectrometers and scanning electron microscope (SEM) that we will use for
characterizing the morphologies of the colloid samples.

Turner

has an impressive track record in directed evolution and industrially
-
relevant biotransformati
ons. He is PI
of a very recently awarded BBSRC LoLa with Industrial Partnership from GSK which aims to develop analytical
approaches for rapid evolution of enzymes for specific fine chemical manufacture in microbial systems. Model
biotransformation system
s that will be studied include (but are not limited to) carboxylic acid reductase and
ammonia lyases where colorimetric assays will be used to benchmark the Raman/SERS spectra.


For further details please contact

roy.goodacre@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

Integrated bio
-

and chemo
-
catalysis

for accelerated synthesis


Supervisors: Prof Michael Greaney (School of Chemistry), Prof Nicholas Turner (School of Chemistry)


Project Outline
:

We aim to integrate contemporary TM
-
catalysed C
-
C bond forming reactions with biocatalytic
redox reactions, for the rapid assembly of enantiomerically pure biologically active molecules. The transformation
shown in Scheme 1 is illustrative: The generation
of
-
amino radicals through photoreductive C
-
H cleavage has
great potential as a route into functionalised chiral amines


building blocks demanded by all sectors of the
chemical industry. The power of this transformation, however, has never been adequatel
y captured in synthesis
due to low yields and operational restrictions due to dedicated photochemical equipment. Recent work from the
groups of Macmillan and Stephenson
1

has shown that photoredox catalysis (PRC) can dramatically improve the
efficiency and
usability of this
reaction. We will investigate PRC by
taking simple secondary amines,
cheap and readily available
-
arylation
under photoredox conditions to
afford the racemic functionalised
amines (Scheme 1). This ambitious
transfor
mation represents a formal
oxidative C
-
H cross
-
coupling, and
will be supported by research into other Pd
-
catalysed oxidative C
-
H activation systems.
2

Whilst PRC is an
emerging area for synthesis, existing systems already demonstrate mild conditions of temp
erature and reagents,
offering excellent prospects for developing integration with biocatalysis. Integration with biocatalyis would follow
two pathways: (i) enzyme catalysed deacylation followed by deracemisation with MAO
-
N/ammonia
-
borane
affords valuable
arylated products as single enantiomers for further N
-
functionalisation. We shall also explore the
use of Rhf for P450 catalysed C
-
H oxidation at selected sp
3

C
-
H bonds around the cyclic amine structure. The
hydroxylation reaction would be expected to be b
oth enantioselective (kinetic resolution) as well as
diastereoselective and hence will generate chiral secondary alcohols. These low
-
molecular weight, polar, 3D
building blocks are central to current lead
-
oriented synthesis thinking in medicinal chemistry,
3

which has identified
a tendency in many successful synthetic methods (e.g. biaryl coupling) to produce molecules that have poor
drug
-
like qualities. New catalytic chemistry that can rapidly assemble small, chiral, polar molecules will be integral
to deve
lopments in medicinal chemistry in the next decade.


Multidisciplinary Training:
The successful project will require innovations in biocatalysis, chemo
-
catalysis and
their integration. Training in these areas will take place in the following research group
s:

The
Greaney laboratory

will provide comprehensive training in both theory and practice of synthetic organic
chemistry. Research in TM catalysed heterocycle synthesis is a core discipline in the Greaney group, and will
provide a supporting framework for
the student to pursue their PhD in integrated catalysis. The Greaney group
has established expertise in the form of co
-
workers (8 x PhD and 6 x PDRA), chemicals and equipment, which
are expected to create synergies for the rapid application of the novel ca
talytic methods discovered by the PhD
student in the course of their degree. MFG was recently awarded an
EPSRC Leadership Fellowship

for his
synthetic chemistry research that frees him from all teaching and administrative commitments, enabling him to
concentrate full time on research and the training of his postgraduate research team. Experienced PDRA
supervision in the area of TM
-
cata
lysis is available from Dr Thomas Storr and Dr Christopher Smith.

The
Turner laboratory

is leading research into integrated biocatalysis and will provide a world
-
class research
environment for biocatalyst discovery, optimisation and application. Training i
n enzyme over
-
expression, isolation
and purification will be fundamental to the project, along with molecular biology skills in directed evolution of wild
-
type enzymes to improve enantioselectivity. The project will require extensive analytical chemistry,
with both
laboratories providing training in mass spectrometry, chiral HPLC and NMR.


1. a) McNally
et al. Science
,
2011
,
334
, 1114

; b) Dai
et al.

J. Org. Chem.

2012
,
77
, 4425.

2. Pintori & Greaney
J. Am. Chem. Soc.

2011
,
133
, 1209.

3. Nadin
et al.

Angew.

Chem. Int. Ed.

2012
,
51
, 1114.


For further details please contact

Michael.greaney@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

Scheme
7.
PRC / enzymatic oxidation

Metabolic profiling of mammalian cells
-

towards single cell characterisation


Supervisors:
Dr Nick Lockyer (School of Chemistry), Prof Roy Goodacre (School of Chemistry)


OUTLINE:
Protein pharmaceuticals are increasing in importance and the production of high fidelity
biopharmaceuticals is essential for therapy. Thus i
n the context of industrial bioprocessing (a key component of
the BBRSC Industrial Biotechnology Priority),

there is an urgent need for new phenotyping data to speed up the
cell selection process and improve manufacturing productivity. Productivity depends on the cellular phenotype,
media design, feeding strategy and environmental regulators. Cell line enginee
ring and improved manufacturing
design requires fundamental understanding of the underpinning biology. There is a clear need for more robust,
sensitive, predictive indicators of cell line performance in subsequent manufacturing scale bioprocessing.
Phen
otypic heterogeneity within populations limits the efficiency of bioprocessing activities and prevents detailed
observations of cell response to growth conditions. The industrial production of biopharmaceuticals urgently
needs solutions to the challenge o
f biological heterogeneity and increasing manufacturing costs, which include
the use of new cell lines and optimised growth conditions.



Single cell analysis is an emerging discipline, offering the potential to understand the pathways and interactions
between intracellular components or compartments and their environment at the most fundamental level. The
aim of this proposal is to develop

a new approach for metabolite profiling down to the single cell level allowing us
to address a basic scientific question relating to the molecular response of cells to external challenges. The
approach is based on a new generation of secondary ion imagin
g mass spectrometry (SIMS) instrument
developed in our laboratory. This novel instrumentation is capable of producing both surface
-
specific mass
spectral data;
e.g.
of membrane lipids which are known to depend growth temperature, and also 3D mass
spectral

images of whole, unlabelled cells. This information is not currently available through other analytical
techniques and Manchester is at the forefront of such analysis. We have very recently demonstrated the
application of this new technology in metaboli
c profiling of multicellular spheroids, differentiating
e.g.

different
oxygen potential and drug response. This has been benchmarked against established methods including GC
-
MS
and LC
-
MS, which also provide complementary metabolomics data at the population level. We (RG & AJD) have
established protocols for metab
olite quenching, extraction and quantitative analysis from the CHO system. In this
project we will extend this MS methodology towards single cell metabolic profiling, focusing of CHO cells (using
CHO cell lines expressing commercially
-
relevant recombinant
proteins with parallel analyses of non
-
transfected
parental cells) exposed to different growth temperatures, oxygen potential, media
etc.
The focus will be on
perturbations to the central carbon metabolism and on membrane lipid composition in relation to c
ell phenotype
and growth conditions. This project marks a convergence of new technology, fundamental science and industrial
relevance that demonstrates the timeliness of the proposed work.


Milestones:
[1] Benchmark SIMS performance against GC
-
MS and LC
-
MS for CHO cell extracts; [2] Compare
extract and whole CHO cell analysis using SIMS; [3] Obtain metabolic profiles of cells grown under different
conditions; [4] Determine the level of phenotypic heterogeneity, or otherwise, that can be achieved on limi
ted cell
numbers, down to the single cell.


TRAINING:
The applicants have a track record of successful co
-
supervision (NPL & RG, AJD & RG) and project
management. The project environment is highly multidisciplinary, encompassing the development of analy
tical
instrumentation and methodology (NPL & RG), metabolomics and data analysis (RG) and biotechnology with a
strong industrial activity (AJD). The student will be based in the MIB, benefiting from the multidisciplinary
environment. Training in laborat
ory skills will be overseen by the relevant applicants, with generic skill training
co
-
ordinated through the main supervisor (NPL).


JUSTIFICATION OF RESOURCES:
The project relies on a number of high technology analytical and
bioprocessing facilities. Ce
ll
-
culture costs will be kept to a minimum using 96
-
providing 100 cell/well. On this basis
to provide cell culture support of suspension CHO cells in shake flasks and
bioreactors (medium, flasks, sterile disposables)

will cost £
1000/yr. GC
-
MS and LC
-
MS are vital to provide
benchmark data and aid identification of specific metabolites. Access charges will be £2000/yr. Access to the
SIMS instrument is not charged as a facility (the most comparably commercial service is ~£1500/da
y), but rather
through a direct contribution towards running costs and consumables (~£20k/yr in total). Based on a 20% usage
of this instrument the cost to this project is £4000/yr. This covers cryogens and high purity compressed gas
(£800/yr) and replace
ment ion sources (£1000/yr), electron and ion detectors (£1200/yr), vacuum hardware
(valves, pump servicing, gauge heads
etc
) (£1000/yr). Included in these costs are associated labware/chemicals
and PPE as appropriate for a project of this type.


For furt
her details please contact

nick.lockyer@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

Luminescent Enzyme Biosensors Based on Upconverting Lanthanide Nanoparticles


Supervisors:
Dr

Louise Natrajan (School of Chemistry), Dr Sam Hay (Faculty of Life Sciences)


Outline

Rare
-
earth upconversion
nanophosphors (UCNPs) are rapidly emerging as an important class of nanoparticles
with potential applications in biological imaging, medicine, security inks, dyes for solar cells, lasers, lighting,
displays and near infra
-
red (NIR) quantum counters.
[1]

Whe
n UCNPs are excited with NIR light they exhibit
efficient photoluminescence in the visible spectrum due to photon upconversion (UC). This UC process, which
can be tuned by doping the UCNPs with various lanthanides, is a non
-
linear process involving the abs
orption of
two or more low energy photons. It is based on the sequential absorption and energy transfer of NIR photons
leading to population of a real (long
-
lived,

s) metastable excited state rather than to a virtual excited state in
two
-
photon absorption
.
[2]

As a consequence of their UC, UCNPs are particularly promising as bioimaging probes
as they exhibit no auto
-
fluorescence (
i.e
. noise) and the NIR photons they are excited with can penetrate far
deeper into biological tissues or highly
-
absorbing/turbid

solutions and materials than visible photons. It is now
possible to synthesise water
-
soluble nanoparticles by functionalising then with
e.g
. carboxylate
-
terminated
groups and there has been some success in covalently coupling them to peptides and antibodi
es.
[3]



In this project, we propose to explore strategies to couple active enzymes
to rare
-
earth (Yb, Er, Tm, Tb) UCNPs. As enzymes often have unparalleled
substrate specificity/selectivity and sub
-
nM substrate affinity, they offer a sensitive
and sele
ctive method of sensing biomolecules. By using enzymes using or
containing chromophoric substrates/substrate analogues or intrinsic cofactors such
as haem (Fe
-
porphyrin), flavin (isoalloxazine) or vitamin B12 (cobalamin), it will be
possible to affect FRET

(Förster resonance energy transfer)
[4]

between the UNCP
(donor) and enzyme/substrate chromophore (acceptor). As the spectral absorption
properties of enzyme chromophores typically change (peak shift for haem and
vitamin B12 and bleach for flavin) during e
nzyme turnover,
the FRET efficiency of
the UNCP
-
enzyme conjugate will be sensitive to the presence of enzyme
substrates.

This is the basis for our proposed new class of luminescence enzyme
-
based biosensors. If the enzyme/substrate chromophore is fluorescen
t (
e.g
. flavin, Zn
-
porphyrin), emission from
the enzyme will also be monitored allowing a ratiometric assay to be developed.

There are three main challenges in this project: (
i
) to successful develop method(s) to make the rare
-
earth
UNCPs water soluble
without drastically reducing their UC efficiency


this is not
unprecedented; (
ii
) to design linkers that can covalently bind to enzymes without
deleteriously effecting the enzyme activity


solvent
-
exposed cysteine residues can be
engineered into the reco
mbinant enzymes using site
-
directed mutagenesis and these
bound
e.g
. through Michael addition to maleimide
-
functionalised linkers; and (
iii
) to
characterise and fine tune (through iterations of (
i
) and (
ii
)) the photophysics of the
UNCP
-
linker
-
enzyme conju
gates.

The choice of UCNPs as donors in this system will allow us to fine tune the
emission wavelength of the FRET donor (
viz
. blue, Tm
3+
, green Tb
3+
, Er
3+
, red Er
3+

and
variations thereof). To develop this technology, Dr Natrajan has received pump
-
prime

funds from the Royal Society and the UoM Dean’s strategic fund pool to purchase suitable picosecond pulsed
diode laser sources (405, 640, 980 nm) and a NIR detector that are compatible with current instrumentation and
detectors in the applicants laborator
ies and the Photon Science Institute (PSI); we also have access to a ultra
-
fast Ti:Sapphire laser in the PSI. The PI also has considerable expertise in lanthanide compounds and their
photophysics and the initial synthetic aspects of the project will be pe
rformed in the School of Chemistry. A large
catalogue of recombinant enzymes with various chromophoric cofactors and/or substrates is available to the Co
-
I
in the molecular enzymology group in the MIB. Suitable enzymes will be screened for linker
-
binding a
nd activity
and any protein engineering will be performed in the MIB.

Specific aims of the project are
:



To functionalise rare
-
earth UNCPs to make them water soluble AND able to covalently bind active enzymes



To characterise the photophysics of these
systems using both continuous wave and time
-
dependent
experiments



To tune the UNCPs (
e.g
. by lanthanide doping) and linker groups (
e.g
. in terms of length and conjugation) to
optimise efficient FRET from the UNCPs to a number of common enzyme cofactors.



To

characterise the enzyme activity (
e.g
. in terms of Michaelis Menten
k
cat

and
K
m

parameters) of enzymes
coupled to these nanoparticles



To test the potential of the synthesised UNCPs for biosensing applications


Further possibilities to expand this work
exist with collaborators at The University of Strasbourg (Dr Loic
Charbonniere), in the form of a three month secondment for the student to develop FRET based assays with
these systems. Funding for this will either be available from the PI’s grants as list
ed above and/or requested from
the EU f
-
element COST network (CM1006); the PI sits on the management committee of this new COST action.


[1] C. Li, J. Lin,
J. Mater. Chem.,

2010,
20
, 6831; [2] L.S. Natrajan
et al
.,
Dalton Trans.,
2010,
39
, 10837; [3] Y.
Su
n, T. Yang, W. Feng, C. Li, F. Li,
J. Am. Chem. Soc.,

2011,
133
, 17122; [4] H.N. Barnhill, S. Claudel
-
Gillet, R.
Zeissel, L.J. Charbonniere, Q. Wang,
J. Am.
Chem. Soc.
, 2007,
129
, 7799


For further details please contact

Louise.natrajan@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manches
ter.ac.uk/

N
O
O
C
y
s
N
O
O
S
H
S
E
n
z
N
P
+
Structural, spectroscopic and computational studies of biological and bio
-
inspired solar water splitting
catalysts


Supervisors: Dr

Patrick O’Malley (School of Chemsitry), Dr Robin Pritchard (School of Chemistry)


Efficient harnessing of solar
energy is widely believed to have the greatest potential to meet current and future
global energy demands. Enough energy strikes the earth in one hour to power the planet for one year at current
consumption rates. The key to implementation lies in our abil
ity to store the energy once captured and as such
the search for an efficient means to transform captured solar energy to fuel production remains one of mankind’s
greatest scientific challenges.

A particularly attractive proposal would be the use of abund
ant solar energy to generate molecular hydrogen by
the splitting of equally abundant water molecules into molecular hydrogen and oxygen in an affordable manner.
This necessitates the development of an efficient and cheap catalyst for this process. Here we
can learn from
green plants and algae which have been splitting water affordably for millions of years using abundant materials.
This water splitting reaction is performed by a MnO
5
Ca complex called the oxygen evolving complex (OEC) of
Photosystem II in green plants, algae and cyanobacteria. While there have been many attempts to build synthetic
systems, “artificial leafs” , based on this photosynthetic reaction centre only very rece
ntly have some viable
systems appeared. Of particular interest has been the use of a Cobalt OEC used in an artificial leaf
manufactured by Nocera et al. (1) This Cobalt
-
based catalyst was shown to be able to split water efficiently at
room temperature und
er very mild conditions and has many characteristics in common with the natural enzyme.
Further improvements in catalyst design will depend on an in depth understanding of the mechanism of water
splitting used by both the natural and synthetic OEC.

The current project aims to further this aim by using density functional theory (DFT) based electronic structure
calculations to investigate the electronic structure of the natural and synthetic catalysts and to explore
mechanisms of water splitting. We pl
an to synthesise the Cobalt OEC complex and determine its structure using
X
-
ray crystallography. The project is linked to parallel Electron Paramagnetic Resonance (EPR) spectroscopy
investigations on these catalysts by the groups of Professors Wraight and
Dikanov at the University of Illinois,
Urbana. Dr O’Malley has a long standing collaborative relationship with these groups in the area of biological
electron transfer (see references 2 and 3 for recent joint papers in this area). EPR spectroscopic invest
igations
are a particularly fruitful experimental avenue of investigation as both
55
Mn and
59
Co have magnetic nuclei This
makes possible direct interrogation of the unpaired spin density in these catalysts which can be directly
compared with DFT calculated

values. Such a joint experimental/theoretical approach is a very powerful means
of characterising the reactivity and mechanistic details of biological transition metal complexes.

The project will combine synthesis and construction of appropriate mode
ls with EPR spectroscopic
characterisation and computational DFT calculations. The DFT calculations will be performed under Dr
O’Malley’s supervision, the Co complex will be synthesised in Dr Pritchard’s laboratory and its structure
determined using X
-
ray
crystallography. The biological PS II preparations will be prepared in Prof Wraight’s group
and the EPR studies will be performed by Prof Dikanov. The student’s role will be the determination of the crystal
structure of the Co complex, the construction of
appropriate models based on available X
-
ray crystal structures of
the photosystem II OEC and the performance of DFT calculations using these models. Analysis will concentrate
on spin density distributions and calculated EPR parameters such as hyperfine co
uplings, g
-
values and zero
-
field
splittings. These will then be analysed and compared with experimental values obtained in the Wraight and
Dikanov groups at the University of Illinois, Urbana. The synergism between theory and experiment will generate
ideas

for the next
-
stage calculations and experiments. The student will receive a wide multidisciplinary exposure
to the disciplines of solar energy, photosynthesis, model generation, DFT calculations and EPR spectroscopy. It
is expected that the student will
visit the experimental groups at Illinois in the later years of the project to further
enhance training in biological preparation and EPR spectroscopy.


(1)
Reece, S.Y;Hamel J.A;Sung, K;Jarvi T.D; Esswein, A.J;Pijpers, J.J.H; Nocera,D.G.,

Science 334 (2
011) 645.


(2)

Lin, Myra T; Samoilova, Rimma I.; Lin, Tsu
-
J; Narasimhulu, K..; Gennis, Robert B.; Dikanov, Sergei A.;
O’Malley, Patrick J.,
Biochemistry,

(2012), 51(18), 3827
-
3838.



(3)Martin, Erik; Samoilova, Rimma I.; Narasimhulu, Kupala V.; Lin, Tzu
-
J
en; O'Malley, Patrick J.; Wraight, Colin
A.; Dikanov, Sergei A., Journal of the American Chemical Society (2011), 133(14), 5525
-
5537.



For further details please contact

patrick.omalley@manches
ter.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/


Evolved Cytochromes P450 in a chemo
-
enzymatic approach for the
generation of novel antibacterials for
industry


Supervisors: Prof David Procter (School of Chemistry, Prof Sabine Flitsch (School of Chemistry)


Project outline


The increasing emergence of multi
-
drug
-
resistant microorganisms has led the World Health
Org
anisation to plead for action against antimicrobial resistance. In industry, focus has turned to previously
discovered classes of antibacterial agents that have novel modes of action; one such class is the pleuromutilins.
The natural product pleuromutilin
has an inhibitory effect against the bacteria
Staphylococcus aureous

(including
Methicillin
-
resistant
S. aureous
, MRSA) and is known to prevent bacterial protein synthesis by binding to the 50S
ribosomal subunit of bacteria. The stoichiometric binding of one pleuromutilin derivative to one peptidyl
transferase site per ribosome sterically hinders the binding of bact
erial transfer RNA, thus inhibiting peptide
synthesis. The 3.5 Å structure of a pleuromutilin analogue bound to a 50S ribosomal subunit has recently been
determined.
Crucially, their novel mode of action make pleuromutilins less susceptible to cross
-
resist
ance than
other antibacterials.

Although pleuromutilin is a promising candidate for development as a new antibacterial


in fact most
pharmaceutical companies have a pleuromutilin programme (
e.g.

GSK, Pfizer, Novartis, Nabriva
etc
)


poor
pharmacokinetic p
roperties plague the pleuromutilin class. In particular,
rapid metabolism by P450

mediated
oxidation at several sites (e.g. C2 and C8) on the mutilin skeleton results in high excretion rates and limits oral
availability
. Although by making minor changes to

the periphery of the mutilin core, industrial chemists

have
developed some commercial pleuromutilins for veterinary medicine and a topical cream for human use
(retapamulin, GSK
-

the first new topical antibiotic to be approved in almost 20 years; $19 M sa
les in 2009),
orally

available pleuromutilins for human use remain elusive.



In this project, we will turn the susceptibility of the
pleuromutilin skeleton to P
450

mediated oxidation to
our advantage

in an innovative, combined chemo

enzymatic approach
:
oxidation of both
natural

and
unnatural

skeletons at
unnatural

oxidation sites
using
mutant
P450 enzymes
will allow unprecedented
access to modified mutilins that will be used to
discover novel antibacterial pleuromutilin analogues
with improved
pharmacokinetic properties
for industrial
application. The innovative use of biotechnology in our
combined chemo

enzymatic approach to
pleuromutilins of industrial value is outlined in
Figure
1
.


Despite the enormous potential of P450s, the enzymes have y
et to find extensive use in academia or in industry.
The Flitsch and Turner groups lead the world in overcoming the factors that have so far limited the use of
cytochromes P450 as biocatalysts.
[1]

For example, the team have recently identified
Cytochrome P
450 RhF

as
an
excellent candidate for further development by directed evolution.
[2]

Our studies on the oxidation of natural and
unnatural mutilin skeletons with mutant (and wild type) P450s will help the enzyme class gain widespread
acceptance by industry.

The Procter group are international leaders in the synthetic chemistry of pleuromutilin
and recently reported a ‘cascade’ approach that assembles the complex structure of pleuromutilin in a single
reaction using a single reagent.
[3]

The team therefore hav
e unique access to unnatural mutilin substrates with
unprecedented skeletons for biocatalytic oxidation. Crucially, the team also have the synthetic expertise to
convert the products of P450 oxidation at unnatural sites to active pleuromutilin antibacteria
ls, possessing novel
structures, for industrial exploitation.


Multidisciplinary training


The team features world leaders in the development and exploitation of new
synthetic technology (Procter) and the harnessing of wild type and mutant P450s (Flitsch
and Turner). The
student will receive training in a number of state

of

the

art techniques including directed evolution, development
of high
-
through
-
put assays, advanced asymmetric and target synthesis.
The project will suit students with a
chemistry backgr
ound looking to address biological problems, or biologists with a good background in chemistry.



[1] O’Reilly
et al
. “Cytochromes P450 as useful biocatalysts: addressing the limitations”
Chem. Commun.

2011
,
47
, 2490.

[2] O’Reilly
et al
. “
Catalytic Promiscuity of Cytochrome P450 RhF”
Chem. Commun.

2012
,
in press

[
3] Helm
et al.

“A dialdehyde cyclization cascade approach to pleuromutilin”
Angew. Chem. Int. Ed.

2009
,
48
,
9315.


For further details please contact

david.j.procter@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manches
ter.ac.uk/

biotechnology
fermentation
natural

mutilin core
chemotechnology
synthesis
unnatural

mutilin cores
mutant P450
enzymes
(by directed
evolution)

unnatural
oxidation
of
natural
cores

unnatural
oxidation
of
unnatural
cores
biotechnology
HO
HO
OH
HO
unnatural
cores –
after oxidation at
unnatural
sites
industrial
application
chemotechnology
synthesis
Figure 1
Enzyme
-
coupled Tandem Processing of Organosilicon Novel Compounds


Supervisors: Dr Peter Quayle (School of Chemistry), Dr Lu Shin Wong (School of Chemistry)


Introduction.
In synthetic chemistry, there has been increasing prominence in the use
of engineeredenzymes to
execute chemical transformations.
1,2

These biocatalysts are attractive since they offer highly efficient synthesis in
terms of yields and selectivity; together with an inherent environmental sustainability. However, one area where
biocatalysis has been relatively unexplored is in silicon
-
containing (organosilicon) chemistry. Such organosilicon
compounds are present in a huge variety of consumer products and play a major role in organic synthesis.
Furthermore, such compounds exhibit
desirable physical properties that are difficult to achieve by other materials.
Marine sponges use silicon (in the form of silica) as part of their inorganic skeleton utilise an enzymes termed
“silicateins” to polymerise silicic acid (H4SiO4) into silica.
This family of enzymes are uniquely interesting as they
catalyse a reaction that is rare in living systems, the formation of Si
-
O bonds.
3

Crucially, they also accept organic
silanes such as Si(OEt)4, SiPh(OEt)3 and SiMe3OH, alluding to the possibility of u
tilising these proteins for a
wider range of organic chemistry. Harnessing the ability to form (and hydrolyse) these bonds starting from their
corresponding silanol and alcohol with a high level of control would open new avenues in synthetic methodology.



This research therefore proposes to exploit the silicateins through rational modification of their
structure and apply them to the development of new synthetic reactions. The goals of this project will be
two
-
fold: (1) to develop new biocatalysts fo
r organosilicon chemistry with wide scope yet high
selectivity; (2) apply these biocatalysts to novel processes for the production of useful synthetic
intermediates which may be of use in the synthesis of molecules of biological relevance. In particular,
t
his project will aim to employ these enzymes for the bio
-
activation of unreactive organosilanes towardfs
Pd
-
mediated reactios.


Experimental Plan.
We have recently succeeded in the heterologous production of soluble and bioactive
silicatein
-
α, the
prototypical member of this family, which serves as the ideal starting point for this project. From
here, this research will be divided into two interlinked, but potentially independent, work packages (WPs).


1) The development of organosilicon biocatalyst
s.
As noted above, there is a need for new biocatalysts to
enable the attachment of silyl groups on to organic molecules with high enantio
-
, regio
-

and diastereoselectivity.
Additionally, there is a need to manipulate these groups under mild conditions whe
re other conditions may
degrade a sensitive molecule. Thus,
this WP proposes to generate and screen

recombinant silicateins to
progress our knowledge of organosilicon biocatalysis, which will lead on novel and economically
valuable biocatalysts
. To furnish

such biocatalysts, recombinant directed evolution methods such as iterative
saturation mutagenesis (ISM) will be employed. Screening experiments will then be performed firstly with
relatively simple silanes such as the commonly used silyl protecting group
s (e.g. TES
-
OEt), then progressing on
to more complex substrates. An emphasis will be placed on using high
-
throughput screening methodologies for
these assays (e.g. spectrometric assays on 96
-
well plates). Attempts will be made to characterise enzyme
toler
ance towards a variety of solvents,5 as a means to alter the reaction equilibrium in favour of either silylation
or hydrolysis; and allow the use of these enzymes on poorly water soluble.


2) Coupling biological and chemical catalysis.

This WP will apply
the silicatein enzyme (and any relevant
mutants) to explore a range of avenues in synthetic chemistry, thus developing new tandem biological
-
chemical
synthetic methodologies. Firstly, this WP will investigate whether it will be possible to
generate functio
nalised
enol
-
silyl ethers directly from carbohydrates
using these enzyme systems. Such a development will be a
major advance, as it will enable the synthesis of valuable synthetic intermediates from readily available
precursors in an environmentally friend
ly manner. Additionally,
the biocatalytic synthesis of dixosilanes,
potential precursors to useful reactive intermediates such as

ortho
-
quinomethides, will also be
established
. Given that the mode of action of the
silicateins

most likely proceeds via the i
ntermediacy of a
pentavalent silicon species, we also wish to
establish whether the enzyme system will also promote
palladium
-
mediated cross coupling of

hypervalent silicon species
. This will be a novel departure in the use
of enzyme systems in catalysis a
s it combines bio
-
catalysis with more traditional transition metal
-
mediated
processes


such a combination could have major implications in synthetic design and execution.


Management and Training.
The overall project will be led and managed jointly by PQ
and LSW. LSW will lead
the research on protein mutagenesis, screening and enzyme substrate synthesis. PQ will direct both the
synthetic work and select suitable synthetic targets in order to exemplify the utility of the methodology developed
above. PQ wil
l also lead the use of the enzyme system for the bio
-
activation of the products produced above in
Pd
-
mediated cross coupling reactions. T
his is a highly multidisciplinary project crossing the boundaries of
physical and life sciences. The student will thus
be trained in a wide variety of areas in core bioscience
skills including structural biology, protein engineering, high
-
throughput screening and organic
chemistry/catalysis


skills which will be critical for the underpinning of the industrial and pharmace
utical
sectors.


References.
(1)
Chem. Soc. Rev.
,
2009
,
38
, 3117; (2)
Curr. Opin. Chem. Biol.
,
2009
,
13
, 43; (3)
Chem.

Rev.
,
2008
,
108
, 4915; (4)
Nat. Prod. Rep.
,
2008
,
25
, 455; (5)
J. Inorg.
Biochem.
,
2003
,
96
, 401


For further details please contact

Peter.quayle@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac
.uk/

Novel synthetic biology approaches for generating multifunctional catalysts


Supervisors: Prof Nicholas Turner (School of Chemistry), Prof Jon Lloyd (School of Earth, Atmospheric and
Environmental Sciences)


The biocatalysis group led by Professor Ni
ck Turner at the Manchester Institute of Biotechnology, in combination
with the Geomicrobiology Research Group in the School of Earth, Atmospheric and Environmental Sciences,
have pioneered the exciting combination of molecular biology and bionanomineralo
gy, to engineer organisms
uniquely equipped for multistep catalysis.


Initial proof of concept experiments focused on the

deracemization of
racemic amines, using a catalyst

engineered to express

monoamine oxidase

alongside enzymatically precipitated
nano
-
p
alladium

(
Foulkes et al., 2011
).


Through this studentship, we will build on the success of our earlier work
to extend this concept into a platform technology based on a

catalytically self
-
sufficient P450 enzyme from
a

Rhodococcus

sp.

(
Roberts et al., 2002
), useful for a range of targeted biotransformations required by the
chemical and pharmaceutical industries, combined with the biosynthesis of functional bionanominerals, produced
by subsurface metal
-
reducing bacteria.


The recombinant protein will be expr
essed in one of several strains
capable to reducing either precious metals or metalloids (Lloyd et al., 2011), using broad
-
host range plasmids
and expressed, alongside the precipitation of catalytically active bioreduced precious metal catalysts or quantum

dots.


The former have the potential to supply electrons directly to the P450 enzyme, negating the longstanding
problem of cofactor delivery for biocatalysis using this widely used enzyme system. We know from recent work
that the optical properties of the

biological quantum dots can be tuned through bioprocess control, and will
therefore be used as a light activated electron donor, both with and without added electron mediators.

The
efficiency of electron transfer via these novel approaches will be compare
d with the current state
-
of
-
the
-
art, which
involves use of an

in vitro

cofactor recycling system for NADPH. Test conversions (e.g. enantioselective
hydroxylation of alpha
-

and beta
-

ionone) will be used to benchmark the process.

The most successful
biomine
ral/biocatalyst combination will have the potential to be scaled up with industrial partners to determine the
process economics.


The student will therefore benefit from the unique infrastructure present in two recognized centres of research
excellence (Ma
nchester Institute of Biotechnology and the Williamson Research Centre for Molecular
Environmental Science), and work at a unique interface between applied catalysis and geobiology/nanomaterial
science. Training will be given in molecular biology, analyti
cal chemistry, microbial physiology, biomaterials
synthesis and characterization (including synchrotron techniques) and nanotechnology. With such a broad
training base, the student will be well qualified to work in many areas of the biosciences at the end
of the
studentship, especially in the rapidly developing field of industrial biotechnology.


References

Roberts, G.A., Grogan, G., Greter, A., Flitsch, S.L., Turner, N.J. (2002) Identification of a new class of
cytochrome P450 from a
Rhodococcus

sp.
J
Bacteriol.

184 3898
-
908.

Foulkes, J.M., KJ Malone, M Harfouche, NJ Turner and JR Lloyd

(2011) Engineering a novel biometallic whole
cell catalyst for enantioselective deracemisation reactions. ACS Catalysis 1 11 1589
-
1594

Lloyd, J.R., Byrne, J.M. and
Coker, V.S. (2011) Biotechnological synthesis of functional nanomaterials. Current
Opinion in Biotechnology 22 509

515 DOI 10.1016/j.copbio.2011.06.008

For further details please contact

Nicholas.Tur
ner@manchester.ac.uk
;

Jon.Lloyd@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manchester.ac.uk/

Development of novel biocatalysts for ‘difficult’ strategic bond functionalization reactions


Supevisors: Dr Roger Whitehead (School of Chemistry), Professor Nicholas Turner (School of Chemistry)


An underpinni
ng theme of research in the synthetic, medicinal and biological chemistry fields is the efficient
generation of what are sometimes referred to as ‘privileged structures’: these are usually poly
-
functionalised,
heteroatom containing compounds which can be e
mployed for the preparation of specific drug targets or,
alternatively, compound libraries suitable for biological screening. Over billions of years, the natural biosphere
has employed a ‘privileged structure based approach’ to the synthesis of its own, h
ighly diverse library of
metabolites
-

some members of which have provided the foundations for the discovery of a number of vital life
-
saving medicines.

The monoamine oxidases (MAO’s) are a class of (historically)
catabolic enzymes

which catalyse the oxidation of
monoamines (classic examples being the neurotransmitters adrenaline and serotonin), thereby facilitating their
deactivation and ultimate extrusion from a cell or organism. Although catabolic, in an evolutionary sense, thes
e
enzymes are quite promiscuous regarding their substrate tolerance and have the capability of generating
versatile, high
-
energy species which can be exploited in useful anabolic/synthetic processes. An example of a
natural monoamine oxidation
which leads
, ultimately, to the
generation of an array of
biologically important natural
products is the conversion of
hygrine (
1
) to tropinone (
2
), a
precursor for several tropane
alkaloids.

The goal of this cross
-
disciplinary project is to
generate a “toolbox” of

MAO enzymes which can
be used, on a preparative scale, for the generation
of aminogenic “privileged structural motifs” as
exemplified by the 4
-
piperidones
4

and
5
,
tetrahydro
-
1,3
-
oxazine
6

and amino
-
aldehyde
7
.
These “motifs” are recurring features in a n
umber of
common therapeutic agents as well as synthetic
intermediates of interest to the pharmaceutical and
agrochemical industries. They are also present in
many bioactive secondary metabolites where they
share the common biosynthetic feature of being
de
rived from a pivotal monoamine oxidation
followed by either C
-
C / C
-
O bond formation or
hydrolytic cleavage.


This project, which is carefully structured in order to accomplish a series of strategic goals, will benefit from the
symbiotic research expertise

of Professor Nick Turner (biocatalysis) and Dr Roger Whitehead (organic
synthesis). The
first stage

of the programme will concern the synthesis of a “bespoke” array of tertiary amines
with generic structure
3
. The individual components of this group have

been chosen in order to allow rapid
access to important “privileged structures” (e.g.
4
-
7
)
via

MAO
-
catalysed activation of specific C
-
N bonds: key
features of the oxidative activation steps are: i) that they have no effective synthetic counterpart; ii) th
at they lead,
ultimately, to the generation of enantiomerically pure (or highly enriched) compounds. Working closely with the
group of Professor Nicholas Turner, the
second stage

of the programme will involve screening existing MAO
libraries for the desire
d activity as well as the generation of novel variants capable of selective C
-
N oxidation.
During this phase of the project we will not only exploit the ability of the enzyme to generate optically pure
“privileged structures”, but we will also employ MAO i
n a high
-
throughput screen. Variants capable of selectively
oxidizing the starting tertiary amines
as well as

the target compounds will allow for the detection of activity and
the rapid selection of effective biocatalysts. Finally, during the third stage o
f the project, active variants will be
optimised using directed evolution leading ultimately to the generation of a “toolbox” of highly efficient MAO
biocatalysts. The high substrate concentrations tolerated by the enzyme will allow for efficient scale
-
up
and
isolation of desired compounds.

This is a cross
-
disciplinary project which will provide a PhD student with excellent ‘state
-
of
-
the
-
art’ training in the
combined fields of advanced organic synthesis and biocatalyst development. In a broader context, a

successful
outcome to the project will have widespread impact on two fronts:



it will demonstrate the extensive and, so far, mostly untapped potential of MAO’s as versatile catalysts
for useful preparative scale chemical reactions;



it will provide rese
archers in the synthetic, medicinal and biological chemistry fields with an efficient
route to privileged structural motifs which would otherwise be difficult, or impossible, to obtain.



For further details please contact

roger.whitehead@manchester.ac.uk


Please see the following website for details of the application process.

http://www.dtpstudentships.manches
ter.ac.uk/




N
X
N
R
2
R
1
R
2
R
1
N
X
R
1
R
2
O
R
3
R
4
N
R
2
O
R
3
R
1
R
3
O
NH
H
X
O
R
1
/R
2

l
i
n
ke
d
3
4
5
6
X
X
7