synthesis directed at the disruption of a protein-protein ... · – hay fever: h1 antagonist...
TRANSCRIPT
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Synthesis directed at the disruption of a protein-protein interaction in asthma
Alan C. Spivey
RSC/BMCS 2nd Symposium on Chemical Biology for Drug
Discovery AstraZeneca, Alderley Park
20th-21st March 2012
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Format of presentation
• Asthma & allergic disorders – Background & biochemical cascade – The hIgE/FceRI protein protein interaction (PPI) – Therapeutic proof-of-concept - Xolair®
• Peptide-based antagonists – Synthesis & activity
• Development of the germyl-Stille Reaction
– A photolabile safety-catch system • The synthesis of aspercyclide A & its C19 methyl ether
– Racemic total synthesis, optical resolution – Asymmetric synthesis – Emerging SAR data
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Asthma & allergic disorders – UK figures
• The prevalence of asthma is increasing worldwide:
– 10.8 million sufferers in the UK (~20%) – 12.5 GP consultations pa (183,000 bed days) – NHS annual asthma bill ~£890 million – Cost of lost work days estimated ~£1.2 billion
Nasser Allergy 2008, 1624 [DOI]
http://dx.doi.org/10.1039/b707517k
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Asthma & allergic disorders - Medication
• The majority of current medications address ‘downstream’ symptoms, e.g. – Asthma: b2-adrenergic agonist bronohidilators e.g. albuterol
– Hay fever: H1 antagonist anti-histamines e.g. diphenhydramine
– Eczema: anti-inflammatories vs. leukotrienes e.g. zafirlukast
– Anaphylactic shock: injection of adrenaline (epinephrine)
Stinson C & EN 1997, Jan 6, 25 [DOI]
http://dx.doi.org/10.1039/b707517k
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The allergic response – Overview of the cascade
Block PPI
Gould Ann.Rev. Immunol. 2003, 21, 579
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Proof-of-principle – Antibody therapy
• XolairTM (Genentech, Novartis) was approved by the European Commission in Oct 2005 for the treatment of chronic asthma in all 25 EU member states
- Humanised monoclonal anti-IgE antibody (~149 kDa) - Prescribed for severe asthma/hey fever - Subcutaneous injections required every 2-4 weeks - Expensive (£10K/patient/year)
http://www.xolair.com/
http://www.xolair.com/
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The IgE-FceRI protein-protein complex (1)
Gould & Sutton Nat. Rev. Immunol. 2008, 8, 205 [DOI]
• The ligand (IgE): – Y-shaped C2 symmetric dimer (L-H)2 – Heavy (e) chain is involved in FceRI
binding – Heavy chain comprises 1 variable (Ve)
and 4 constant (Ce1, Ce2, Ce3, & Ce4) domains
• The receptor (FceR1):
– membrane bound, comprising 4 subunits (abg2)
– extracellular a subunit is involved in IgE binding
– comprises two small (86 residue) globular domains a1 & a2
http://dx.doi.org/10.1038/nri2273
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The IgE-FceRI protein-protein complex (2)
• Characteristics of PPI: – High affinity - Ka 10-10 M-1
– Biphasic interaction where initial ‘sensitisation’ is reversible
– Large surface area - 1830 Å2
– Only a small proportion of cross-linking is required to initiate exocytosis?
• Implications for antagonist design:
– require high binding affinity for competitive inhibition
– require good bio-availability to attain high intra-cellular concentration
PDB 1F6A Jardetzky 2000 [DOI]
Gould & Sutton Nat. Rev. Immunol. 2008, 8, 205 [DOI]
http://www.rcsb.org/pdb/home/home.dohttp://dx.doi.org/10.1038/nri2273
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The IgE-FceRI protein-protein complex (3)
PDB 1F6A Jardetzky 2000 [DOI]
http://www.rcsb.org/pdb/home/home.do
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Peptide-based antagonists of the IgE-FceRI PPI
PDB 1F6A Jardetzky 2000 [DOI]
http://www.rcsb.org/pdb/home/home.do
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• Non-’epitope’ peptides:
• Epitope peptides:
Peptide antagonists of IgE-FceRI PPI
b-hairpin (binds to receptor)
IC50 = 1 μM
Starovasnik,, Biochemistry 2001, 40, 9828 [DOI]
Zeta-loop (binds to receptor)
IC50 = 36 nM
Starovasnik, Structure 2004, 12, 1289 [DOI]
[V6, A12] MCD
IC50 = 0.6 µM
Buku, Chem. Bio. Drug. Des. 2008, 72, 113 [DOI]
a2(C-C’) mimetic (binds to IgE)
IC50 = 30 μM
Sutton, Biochem. Soc. Trans. 1997, 25, 387 [DOI]
Ro 25-7162 a2(C-F) mimetic (binds to IgE)
IC50 = 40 μM
Danho, Proc. Am. Peptide Symp. 1997, June 14-19, 539 [DOI]
http://pubs.acs.org/doi/10.1021/bi0109360http://dx.doi.org/10.1016/j.str.2004.04.015http://www3.interscience.wiley.com/cgi-bin/fulltext/120750790/PDFSTARThttp://pubs.acs.org/doi/pdf/10.1021/ja963884o
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The IgE-FceRI protein-protein complex - Hotspots
PDB 1F6A Jardetzky 2000 [DOI]
http://www.rcsb.org/pdb/home/home.do
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The AB loop - Ce3/Ce4 Hinge region
• Site directed mutant hIgE (Phe349Ala) → just 10% mediator release (degranulation assay) – Presta J. Biol. Chem. 1994, 269, 26368 [DOI]
• Phe349 is situated in the middle of the ‘Omega’ loop at terminus of the Ce3 A-B b-strand:
PDB 1F6A Jardetzky 2000 [DOI]
http://www.jbc.org/content/269/42/26368.full.pdf+htmlhttp://www.rcsb.org/pdb/home/home.do
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A disulfide-constrained A-B loop mimetic
• Simple disulfide constrained A-B loop epitope:
Helm Allergy 1997, 52, 1155 [DOI]
http://dx.doi.org/10.1021/jo026693e
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Tolan amino acids as peptide loop constraints
• ...what if we introduced a more rigid constraint than a disulfide? – b-turn mimetics: e.g. ‘tolan’ amino acid: – Kemp Tetrahedron Lett. 1995, 36, 4175 [DOI]
• ...what about a suite of regioisomeric tolan amino acids & analogues?
With John McKendrick & Ratnasothy Srikaran J. Org. Chem. 2003, 68, 1843 [DOI]
http://dx.doi.org/10.1016/0040-4039(95)00775-8http://dx.doi.org/10.1021/jo026693e
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A tolan-constrained A-B loop mimetic • Synthesis of 2,2’-tolan constrained peptide via ‘on-resin’ Sonogashira macrocyclisation:
– Only rink amide resin allows for cyclisation
With John McKendrick & Ratnasothy Srikaran J. Org. Chem. 2003, 68, 1843 [DOI]
http://dx.doi.org/10.1021/jo026693e
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An array of tolan-constrained A-B loop mimetics
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Synthesis of the tolan amino acids • NB. Installation of Ser353 allows for higher yielding subsequent macrocyclisations :
With Danny Offermann
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Synthesis of hydrogenated 2,2’-tolans • The fully saturated 2,2’-bibenzyl amino acid:
• The partially saturated 2,2’-stilbene amino acid:
With Danny Offermann
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Synthesis of the constrained A-B loop mimetics
With Danny Offermann
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Constrained A-B loop mimetics – ELISA activity • ELISA data for the array of tolan & tolan-derived constrained A-B loop mimetics:
– linker & sequence dependent activity
with Danny Offermann & Jimmy Sejberg J. Org. Chem. 2012, 77, ASAP. IC50 = 660 ±70 mM
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Ongoing work...
• Co-crystallisation with both proteins – with Brian Sutton & Mary Holdom, KCL
• Increase affinity by synthetic mutagenesis? – Use of germyl-Stille coupling...
with Bingli Mo & Jimmy Sejberg
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The germyl-Stille reaction
O
GeR R
Cl
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Where is germanium in the periodic table?
• Group 14: C, Si, Ge, Sn, Pb
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Ge vs Si & Sn
• features: – Susceptibility to ipso-SEAr – intermediate between Si and Sn
– Better stability towards nucleophiles & bases cf. Si & Sn
• e.g. Vasella Helv. Chim. Acta, 1996, 79, 255
– Essentially non-toxic like Si cf. Sn! – Interesting and unique germylene etherate chemistry [Ge(II) cf. carbene) – Expensive: GeCl4 ~£300 /100 g...
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Fluorous-tagged arylgermanes - Synthesis
OOGeCl2·
1)BrMg
OMe
2) 4M HCl in dioxane
(>2eq)
RMgX (Xs.)
R = 2-NapMe [86%]
IC8F17
mW 300 WH2O, c.HCl
20 min
C8F17GeCl3
C8F17Ge
OMe
ClCl
[78%]
[84%]
£
C8F17Ge
OMe
RR1) 4M HCl in dioxane2) Ar-MgBr
R = 2-NapMe, Ar = p-Tol [71%]R = 2-NapMe, Ar = Ph [83%]R = 2-NapMe, Ar = p-ClC6H4 [80%]R = 2-NapMe, Ar = p-CF3C6H4 [84%]R = 2-NapMe, Ar = o-MeOC6H4 [87%]
C8F17Ge
RR
Ar
features • can ‘tune’ steric and electronic
properties of R groups for specific applications
• fluorous-tag allows rapid purification by FC on fluorous SiO2
with David Whitehead & Joseph Hannah
Zhang Chem. Rev. 2004, 104, 2531 [DOI]
http://dx.doi.org/10.1021/cr030600r
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Pd(0) catalysed cross-coupling
• Sn couples readily as SnR3: Stille coupling – review: Stille Angew. Chem. Int. Ed. 1986, 98, 508 [DOI]
• Si requires an eletronegative ligand and nucleophilic activation: Hiyama-Denmark coupling – review: Denmark Aldrichimica Acta 2003, 36, 75 [DOI]
• what is required for Ge coupling? – review: with Chris Gripton & Joseph Hannah Curr. Org. Synth. 2004, 1, 111 [DOI]
http://dx.doi.org/10.1002/anie.198605081http://www.sigmaaldrich.com/aldrich/acta/al_acta_36_3.pdfhttp://www.ch.ic.ac.uk/spivey/publicationpdfs/currorgsynth20041211.pdf
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Requirement for di-halogermanes
• Ge requires 2 × electronegative ligands and nucleophile activation – initial results: (cf. conditions of Hiyama Tetrahedron Lett. 1997, 38, 439 [DOI]
– optimised conditions:
GeR R
MePd(OAc)2 (5 mol%)
PPh3 (10 mol%)NaOH (6 eq)
THF, 70 °C, 24 h
O
Br CF3
CF3(1 eq) Me
CF3
CF3
R2 = Me2 [0%]R2 = Me,Cl [0%]R2 = Cl2 [36%]
OEt
with Chris Gripton
# R Ar Yield/% 1 Me 4-AcC6H4 60 2 Me 3,5-(CF3)2C6H3 63 3 Me 1-Nap 79 4 OMe Ph 36 5 OMe 3-CF3C6H4 51 6 OMe 3,5-(CF3)2C6H3 71 7 OMe 1-Nap 56 8 OMe 3-Py 44 9 OMe 4-NO2C6H4 47
GeCl Cl
R PdCl2(MeCN)2 (10 mol%)dppp (20 mol%)
KF (6 eq)DMF, 120 °C, 8 h
O R
Ar-Br (1eq)ArOEt
http://dx.doi.org/10.1016/S0040-4039(96)02320-9
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A safety-catch germyl-Stille reaction?
• Target:
• Safety-catch R groups must:
• confer stability to a wide range of reaction conditions prior to activation (i.e. retain unique advantages of Ge vs. Si or Sn vis-à-vis stability to strong bases and nucleophiles)
• be selectively activated with in presence of wide range of FGs – ‘magic bullet’ conditions
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Photo-oxidative ‘activation’ of benzylic ‘R’ groups
• 2-naphthylmethyl groups can be selectively 'activated' by photo-oxidation with Cu(II)
• mechanism: – cf. Otsuji Chem. Lett. 1988, 229 [DOI]
with Chris Gripton, Joseph Hannah & Chih-Chung Tseng Appl. Organomet. Chem. 2007, 21, 572 [DOI]
GePh Ph
OMeO
OEt
Ge2-Nap 2-Nap
OMeO
OEt
http://dx.doi.org/10.1246/cl.1988.229http://dx.doi.org/10.1002/aoc.1270
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The photolysis apparatus • ...a tower PC case:
...a Cathodeon HPW 125W lamp
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Convenient monitoring – 19F NMR
• sequential photooxidation of both groups monitored by 19F NMR:
with Chih-Chung Tseng
C8F17Ge
Cu(BF4)2 (4 eq)MeCN:MeOH (3:1)
h, pyrex filter10 min
OMe
2-Nap 2-Nap
OMe ~1 h
OMe
C8F17GeF F
OMe
C8F17Ge
F2-Nap
OMe
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Scope of germyl-Stille aryl-aryl cross-coupling
with Chih-Chung Tseng, Joseph Hannah & Chirs Gripton Chem. Comm. 2007, 2926 [DOI]
Ar
Ge2-Nap2-Nap
R
GeF F
R
R
Ar-Br (2 eq)
C8F17
C8F17
(2 eq)
Cu(BF4)2 (4 eq)MeCN:MeOH (3:1)
h, pyrex filter~2 h
PdCl2(MeCN)2 (10 mol%)P(2-Tol)3 (15 mol%)
TBAF·3H2O (2.7 eq), CuI (1 eq)DMF, 120 °C, 16 h
OMe
# R Ar Yield/% 1 4-OMe 3,5-(CF3)2C6H3 96 2 4-OMe 4-ClC6H4 85 3 4-OMe 4-BnOC6H4 65 4 4-OMe 1-Nap 75 5 4-Me 3,5-(CF3)2C6H3 84 6 4-Me 4-ClC6H4 69 7 4-Me 4-BnOC6H4 48 8 4-Me 1-Nap 71 9 H 3,5-(CF3)2C6H3 74 10 H 4-ClC6H4 63 11 H 4-BnOC6H4 40 12 H 1-Nap 60 13 4-Cl 3,5-(CF3)2C6H3 71 14 4-Cl 4-BnOC6H4 42 15 4-Cl 1-Nap 75 16 2-OMe 3,5-(CF3)2C6H3 65 17 2-OMe 4-ClC6H4 49 18 2-OMe 4-BnOC6H4 11 19 2-OMe 1-Nap 27 20 4-CF3 3,5-(CF3)2C6H3 26 21 4-CF3 4-ClC6H4 11
http://dx.doi.org/10.1039/b707517k
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Total synthesis of aspercyclide A – a natural product antagonist of the
IgE-FceRI PPI
Aspergillus sp.
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Small molecule antagonists of IgE-FceRI PPI
• Fluorescein dyes MW ~700–1100 Da
– e.g. Na2-Rose Bengal – Cheng (Heska Corp.) US patent 5,965,605 12th Oct 1999 – IC50 = 0.5 mM (MW = 1017 Da)
• (+) Aspercyclide A MW 410 Da – Soil fungus metabolite ex. Aspergillus sp. – Singh (Merck) Tetrahedron Lett. 2004, 45, 7605 [DOI] – IC50 of 200 mM (MW = 410 Da)
X-ray
MM2
http://dx.doi.org/10.1016/j.tetlet.2004.08.116
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(±)-Aspercyclide A – Syntetic strategy
• Features & strategy – 11 membered ring – labile paraquinol – trans-alkene – anti-1,2-diol – hindered ester – di-ortho-substituted biaryl ether
• Aryl-alkene Germyl-Stille key step?
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Anti-1,2-diol formation & ring B esterification
• Boeckman modified Takai-Utimoto Condensation – Boeckman J. Org. Chem. 1998, 63, 3524 [DOI]
• Hindered ester formation with di-ortho-substituted benzoate
X-ray with James Carr
via
http://dx.doi.org/10.1021/jo980160h
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Ring A synthesis and vinyl germane formation
• (E)-Vinyl germane synthesis – cf. (E)-selective hydroboration: Srebnik Tetrahedron Lett. 1996, 37, 3283 [DOI]
• Ring A synthesis
– Porco Jr Org. Lett. 2001, 3, 1649 [DOI]
X-ray
with James Carr
http://dx.doi.org/10.1016/0040-4039(96)00576-Xhttp://dx.doi.org/10.1021/ol0159367
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Biaryl ether formation & germyl-Stille macrocyclisation
• Biaryl ether formation – vinyl germane substrate – cf. Kulkarni Tetrahedron 1988, 44, 5145 [DOI]
• germyl-Stille macrocyclisation
with James Carr & Jimmy Sejberg Org. Biomol.Chem. 2011, 6814
http://dx.doi.org/10.1016/S0040-4020(01)86020-8
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Biaryl ether formation & Heck macrocyclisation
• Biaryl ether formation – terminal alkene substrate – Kulkarni Tetrahedron 1988, 44, 5145 [DOI]
• Heck macrocyclisation
– cf. Nolan J. Organomet. Chem. 2003, 687, 269 [DOI]
Entry Additive Ratio A:B:C
Conversion
ArBr - 0 : 1 : 4.2 100%
ArBr AgI (1 eq.) 10 : 5 : 1 30%
ArI AgI (1 eq.) 2.5 : 10 : 1 75% (52% isolated)
Aromatic Finklestein:
Buchwald J. Am. Chem. Soc. 2002, 124, 14884 [DOI] with James Carr
http://dx.doi.org/10.1016/S0040-4020(01)86020-8http://dx.doi.org/10.1016/S0022-328X(03)00375-9http://dx.doi.org/10.1021/ja028865v
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Deprotection & oxidation • Acetal hydrolysis & benzylic oxidation...
• Need a new C19 OH protecting group…
X-ray
with James Carr
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Use of PMB protection – synthesis of (±)-aspercyclide
with James Carr & Daniel Offermann Chem. Commun., 2010, 46, 1777 [DOI]
http://dx.doi.org/10.1039/c003190a
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Aspercyclide A & its C19 Me ether – ELISA activity
• Racemic ‘Nat Prod’ vs. C19 Me ether – ELISA – (±)-C19 Me ether is equipotent wrt parent! – IC50 ~50 mM
• Enantiomers of C19 Me ether – Separation by CSP-HPLC (Chiralpak IA) – ELISA – (+)-C19 Me ether has natural configuration – 10 fold more IC50 ~50 mM
Entry Compound (% ee)
IC50 (µM)
1 (+)-asp (98.4) 40 ± 1
2 (–)-31 (98.8) 483 ± 105
3 (±)-31 56 ± 2
with Jimmy Sejberg, Helena Dennison & Mary Holdom (KCL)
CD curve for (+)-aspercyclide
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Enantioselective synthesis of ()-aspercyclide A
with Jimmy Sejberg
cf. Krische J. Org. Chem. 2011, 76, 2350 [DOI]
http://dx.doi.org/10.1021/jo200068q
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SAR studies - Ongoing work...
• Synthesis of analogues → SAR – with Helena Dennison & Jimmy Sejberg
• Co-crystallisation with protein, SPR , NMR... – with Lucy Smith, IC – with Brian Sutton & Andrew Beavil, KCL
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SAR studies – aspercyclide C19 OMe analogues... • A-ring analogues → SAR
• B-ring analogues → SAR
with Jimmy Sejberg, Helena Dennison & Lucy Smith
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SAR studies – aspercyclide C19 OMe analogues... • Macrocycle ‘strap’ analogues → SAR
• Dibenzofuran A-ring & ‘strap’ analogues → SAR
with Jimmy Sejberg, Helena Dennison & Lucy Smith
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SAR studies – aspercyclide C19 OMe analogues... • Dibenzofuran B-ring & ‘strap’ analogues → SAR
with Jimmy Sejberg, Helena Dennison & Lucy Smith
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Acknowledgements • peptides: John McKendrick & Ratnasothy Srikaran (Birgit Helm @MBB, Sheffield), Daniel Offermann, Bingli Mo,
Jimmy Sejberg & Lucy Smith (Robin Leatherbarrow @IC; Andrew Beavil @KCL; Brian Sutton @KCL; Mary Holdom @KCL)
• Germyl-Stille: Chris Gripton, (Jan Scinski @GSK), Joseph Hannah & Chih-chung (Jimy) Tseng • aspercyclide A: James Carr (Steve Lindell @)Bayer CropScience), Daniel Offermann , Jimmy Sejberg & Helena
Dennison (Keith Spencer & Kevin Foote@Arrow Therapeutics/AstraZeneca; Fabienne Saab @KCL) • X-ray: Chris Frampton @ Pharmorphix & Andrew J.P. White @IC
• EPSRC, Pfizer, Roche, Bayer CropScience, AstraZeneca, Novartis, Wellcome Trust, MRC, EU FP7 Marie Curie
http://portal.bayercropscience/http://www.epsrc.ac.uk/