1	Synthesis and Screening in Micro Reactors Paul Watts University of Hull, UK LRIG Oxford, 7 November 2002
2	Spot the Difference!!
3	Micro Reactors Defined as a series of interconnecting channels formed in a planar surface Channel dimensions of 10-300 mm Fabricated from polymers, metals, quartz, silicon or glass Various pumping techniques available
4	Electroosmotic Flow (EOF) Above pH 2 the negative glass surface is neutralised by a diffuse layer of positive ions Application of an electric field causes this layer to move towards the negative electrode Within the channel this causes the bulk liquid to move
5	Advantages of EOF Direction and magnitude of flow readily changed by altering the applied voltage Reproducible flow rates (ml min^-1) The pumping technique has no mechanical or moving parts Not limited by back pressure effects Automated power supply with multiple electrodes Plugs of fluid are transported without significant hydrodynamic dispersion Chemical reaction in an electric field
6	Automated Power Supply Computer control enables continuous flow, stopped flow and injection techniques to be used
7	Operational Advantages of Micro Reactors Spacial and temporal control over reagents and products Reduced exposure to hazardous chemicals Ability to scale out Use of EOF to move solvents and reagents Computer control
8	Chemical Advantages of Micro Reactors Rapid optimisation of reactions Ability to generate reagents in situ Unique thermal and concentration gradients Product formed in higher purity Enhanced yield of product Electrophoretic separation can enable isolation of pure products Electrochemical effects
9	Micro Reactor Chemistry
10	Wittig Reaction Formation of C-C double bonds
11	Wittig Reaction Manifold
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13	Second Generation Reactor Design
14	Carbanion Chemistry Michael Addition Investigated the use of Hunigs base Bulk reaction: 98 % yield >99 % trans isomer observed
15	Michael Addition in a Micro Reactor 5.0 M solutions in EtOH were used and the reaction conducted for 20 min Continuous flow: 56 % Stopped flow 2.5 sec ON/ 5 sec OFF: 95 % conversion
16	Michael Addition in a Micro Reactor Extended the technique to substituted diketones: Bulk: 91 % Micro reactor: Continuous flow: 40 % Stopped flow: 100 % (2.5/5) Bulk: 78 % Micro reactor: Continuous flow: 15 % Stopped flow: 34 % (2.5/5) 100 % (2.5/10)
17	Solid Phase Peptide Synthesis Advantages Ease of product purification Reduced product loss during purification Automation Combinatorial synthesis Disadvantages Expensive polymer support required Must link amino acid with solid support Need to remove support from product By-products can accumulate on the resin Observed discrepancies in product functionality
18	Carbodiimide Coupling Methodology Most common method of peptide bond formation Activation of the carboxylic acid using DCC or EDCI DMAP may be used as a catalyst Some deprotection observed
19	Micro Reactor Manifold Reaction conducted for 20 minutes at room temperature Analysed by HPLC 5 equivalents of DCC using stopped flow technique All reagents maintained at 389 V/cm, 93 % conversion
20	Pentafluorophenyl Esters Excellent leaving groups Easily prepared from protected amino acids Continuous flow of reagents Pentafluorophenyl ester 389 V/cm Amine 333 V/cm Quantitative conversion in 20 min
21	Dmab Deprotection 2 % hydrazine in DMF used in SPPS Within a micro reactor: One equivalent of hydrazine Continuous flow of both reagents at 389 V/cm Quantitative deprotection
22	Fmoc Deprotection 2 % DBU in DMF used in SPPS Continuous flow of reagents 96 % Conversion Dmab ester 313 V/cm DBU 363 V/cm Pentafluorophenyl ester 333 V/cm
23	Tripeptide Synthesis Continuous flow PFP ester 417 V/cm Amine 417 V/cm DBU 182 V/cm PFP ester 333 V/cm 30 % conversion
24	Combinatorial Synthesis Reactor design
25	Peptides Derived from a-Amino Acids Is the degree of racemisation observed comparable to that of bulk reactions? Diastereomers separable by GC-MS Continuous flow of reagents PFP ester 313 V/cm, Amine 556 V/cm Quantitative conversion At 0.1 M concentration of reagents - 4.2 % racemisation At 0.5 M concentration of reagents - 7.8 % racemisation
26	Kinetic Studies 100 % Peptide bond formation in a micro reactor Batch reactions gave 40-50 % yield in 24 hour Typically conducted at higher concentrations 0.1 M solutions reacted in bulk reaction Conversion monitored with time Only ca. 5 % conversion to dipeptide in 20 min
27	Kinetics of Pentafluorophenyl Esters 0.1 M solutions reacted in bulk reaction
28	Immobilisation of Cells
29	Immunoassay Screening
30	The Future - Drug Discovery
31	Conclusions Rapid optimisation of reactions High throughput synthesis Combinatorial synthesis Ability to generate reagents in situ Product formed in higher purity Enhanced conversion of product Electrochemical effects Atom efficient reactions Green chemistry Biological Applications Drug Discovery
32	Research Workers and Collaborators Prof. Stephen Haswell Dr. Gillian Greenway Dr. Tom McCreedy Prof. Paul Fletcher Dr. Xunli Zhang Dr. Xizeng Feng Dr. Ping He Dr. Colin Ellis Dr. Vinod George Mairead Kelly Gary Hayes Charlotte Wiles Sarah Painter Nicola Horton Bashar Al-Gailani Novartis GSK Astra Zeneca Avecia Thomas Swan EPSRC