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Styrene-Based Copolymers as Soluble Platforms for the Biocatalytic Transformation of Organic Substrates with Immobilized Enzymes

Dario Pasini

Dipartimento di Chimica Organica Università degli Studi di Pavia APIB-2009 Pavia, 3 rd June 2009

Overview

1) Biocatalysis, Solid Phase Synthesis and Soluble Polymers 2) Soluble Polymer-Achiral Substrate / Immobilized Enzyme 3) Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer 4) Conclusions and Outlook

General concepts involved in the use of supported organic targets and their biocatalytic transformations

Crosslinked Polymers-Substrate / Free Enzyme Concept B Tentagel and Argogel resins (polyethylene glycol chains grafted onto classical polystyrene/divinylbenzene cores) •High swelling characteristics in aqueous solvents •Low loading capacity •Limited success in combination with biocatalysis PEGA 1900 (Copolymer Acrylamide/PEG) used in Enzymatic Solid Phase synthesis of peptides and resolution of racemates.

A. Basso, P. Braiuca, C. Ebert, L. Gardossi, P. Linda J. Chem. Technol. Biotechnol. 2006 ,

, 1626-40

Biocatalysis and Solid Phase Synthesis Concept C versus Concept D

N H O O MeO

Immobilized PGA

N H O O MeO O O O X Target Molecule

Biocatalitically-Triggered Safety-Catch Linker

O HO O H N NH 2 O

= Merrifield resin or Wang resins

X = O, NH, NR

Only when is a soluble linear polymer (PEG = polyethyleneglycol) high yields of the product could be achieved (Concept D)

X Target Molecule N H O O HX MeO NH O

Target Molecule O

U. Grether, H. Waldmann Chem. Eur. J. 2001 ,

, 959-971

Soluble Polymeric Supports

Advantages 1

- Easy monitoring of the support functional groups by common analytical techniques (e.g. 1 H NMR)

-Reactivity similar to the solution, homogeneous phase

– Facile product/reagent separation by precipitation of the polymer in a non -solvent

Soluble Polymeric Supports

POLYETHYLENE GLYCOLS POLYSTYRENES n

- Soluble in water and most organic solvents - Insoluble in diethyl ether Low loading capacity - Soluble in non polar organic solvents - Insoluble in MeOH - Good loading capacity

D. E. Bergbreiter Chem. Rev. 2002 ,

102

, 3345-3384

Soluble PS Copolymer-Substrate / Immobilized Enzyme Concept D

Immobilized Enzyme

x y OH R O O n O

i) Enzymatic hydrolysis (PGA) ii) Filtration of the Enzyme

Soluble PS Copolymer Substrate

x y O OH n

iii) Recovery of the Copolymer by precipitation

iv) Isolation of substrate

from the solution

D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato Adv. Synth. Catal. 2007 ,

349

, 971-978

Monomer and Polymer Synthesis

O OH Cl + HO n OH NaOH/H 2 O 70°C

83-89%

O n=1-3 n OH DICD/DPTS 24 h

70-90%

n=1-3 O n O O x + y O n O O n=1-3 AIBN/70

C Toluene

60-80%

x y O n O O n=1-3 x = 0.6-0.93

Introduction of phenylacetic ester monomers and copolymerization with styrene at several loadings

Ha Hb Hc

Characterization by 1 H NMR Spectroscopy

Hm Hd Hi He Hd He O Hg Hf Hi O O Hm Hc Ha Hb Hf,g 80 20 O O O

Excellent agreement between feed and observed ratios of monomers

8.0

7.5

7.0

6.5

6.0

5.5

5.0

4.5

4.0

3.5

3.0

2.5

2.0

1.5

Gel permeation chromatography Solvent Polymer Average Molecular Mass

n (number average)

w (weight average)

PDI Polydispersity Index

4 8 12 t (min)

w /

n Values between 1.5

and 2.4

Properties as Supports

High Comonomer Loading (60:40) 60 40 Bad precipitation in MeOH: centrifugation needed O O O Low Comonomer Loading (93:7) 93 7 O O O Medium Comonomer Loading (80:20) 80 20 O O O Excellent precipitation in MeOH

Sample Molecular Weight Distribution M n = 11080 ; M w = 18950 ; PD = 1.7

Good precipitation in MeOH

Sample Molecular Weight Distribution M n = 9080 ; M w = 17630 ; PD = 1.9

Copolymer Substrate Hydrolysis by PGA

Immobilized on Eupergit (brown) Immobilized on Agarose (yellow)

20 20 10 O O O

Hydrolysis Conditions -Temperature: 37 °C -Mechanical stirring -Mixed solvent system (aqueous buffer 80/ DMF 20)

0 0 0 0 -0,1 -0,2 400 400 800 800 t (s) 1200 1200 -0,3 -0,4

Quantitative release First order kinetics

D. Pasini, M. Filippini, I. Pianetti, M. Pregnolato,

Adv. Synth. Catal.

2007

349

, 971 – 978.

Enantiomeric Resolution Strategy

Immobilized enzyme Soluble Copolymer (S,R)* (S,R)* i)Enantioselective Enzymatic Cleavage ii) Immobilized Enzyme Recovery iii) Optically-Active Substrate and Soluble Copolymer Recovery Soluble Copolymer (R)* (R)* (S)* i) Chemical Cleavage ii) Soluble Copolymer Recovery Soluble Copolymer (R)* Possible application to enantioselective resolution of racemic carboxylic acids?

Enzymatic Hydrolysis of (R,S)-Methyl mandelate Concept A

From E. Coli on activated agarose gel

= OMe, OEt, OPr,

, Opr,

iso

, OBut,

, NH 2 , NHPr,

, NHPr,

iso

S. Rocchietti et al. Enzyme Microb. Technol. 2002 ,

, 88-93

m Alternative Synthesis of Copolymer/Substrate

1 - Copolymerization

[ ] n [ ] m + n AIBN toluene 70 °C 48 h OH OH O O

2 - Functionalization

1-DMAP / Pyridine / Me 3 SiCl 2- DMF / (COCl) 2 a 0 °C 3- Et 3 N / CH 2 Cl 2 [ HOOC OH ] n [ ] m O O O OH Good yields Good purity

Efficient Polymer Functionalization: 1 H NMR and IR

[ ] 85 [ ] 15 A B+C

1 H NMR: CDCl 3 , solution

[ D A+C B ] 85 [ A ] 15 O B C OH Primary OH

IR: KBr, diffuse reflectance, polymer powder

A O O B C O D OH

6 4 2



(ppm)

Ester carbonyl

Efficient Control of Polydispersity

[ ] 85 [ ] 15 + RAFT reagent AIBN toluene 70 °C 48 h OH OH O O S S

Reversible Addition-Fragmentation Chain Transfer

(

RAFT

)

Polymerization

Achieved control of Polydispersity:<1.2

Achieved control of Degree of polymerization (50 to 500) [ ] 85 [ Functionalization “as usual” ] 15 C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A 2008 ,

, 5715-5723 O O O OH

Copolymer/Substrate Solubility Tests

Phenylacetate Copolymer Solvent

MeCN MeCN/H 2 O DMF DMF/H 2 O DME DME/H 2 O DMSO DMSO/H 2 O

Ratio (%)

100 50/50 100 70/30 100 70/30 100 50/50

Solubility

+ -/+ +++ ++ +++ ++ + -/+

(R,S)-Mandelate Copolymer Solvent

MeCN DMF DMA DMA/H 2 0 DMA/H 2 0 DMSO THF THF/H 2 0

Ratio (%)

100 100 100 80/20 20/80 100 100 60/40

Solubility

++ +++ ++ + -/+ +++ +

DMF / Water Best Solvent DMA / Water Best Solvent

Stability of Immobilized PGA in DMA/Water 120 100 80 60 40 20 0 0 500 1000

Time (min)

1500 2000 20 % DMA 30 % DMA 60 % DMA 80 % DMA

Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer Hydrolysis Conditions -Temperature: 25 °C -Mechanical stirring -Mixed solvent system (aqueous buffer 80/ DMA 20)

Analytical Control

Conversion monitoring

HPLC: Merck Hitachi LaChrom L-7000 Column: AGILENT ZORBAX C18; 4,6 x 250mm  = 220 nm Flow: 1 ml/min Method (Gradient elution): A: 98% phosphate buffer 10 mM pH 3,2 B: 2% CH 3 CN T = 25 °C

Enantioselectivity monitoring

HPLC: Merck Hitachi LaChrom L-7000 Column: REGIS (S,S) Whelko-O1; 4,6 x 250mm  = 220 nm Flow: 2 ml/min Method: 90% Hexane10 mM-10%Ammonium acetate 100 mM in Ethanol T = 25 °C R S Esters Acids

Preliminary Data Results

(R,S)-Methyl mandelate Free

Hydrolysis Rate (  mol/min) 0.73 Conversion (5h) 43% ee% 21% E 1.77

Immobilized PGA = 100U

(R,S)-Mandelate - Copolymer

Hydrolysis Rate (  mol/min) 0.04 Conversion (30h) 41% ee% 18% E 1.61

Immobilized PGA = 200U

Same Hydrolysis Conditions in Aqueous Buffer 80 / DMA 20

Conclusions and Perspectives 1 – The use of Polystyrene Soluble Polymers as Tags for Substrates in combination with Immobilized Enzymes is feasible 2- In a biocatalytic reaction on a racemate, Enantioselectivity seems to be retained (more experiments needed to confirm preliminary data) 3- Work-up, recovery and refunctionalization of the Soluble Polymer need to be optimized

Acknowledgments

Dep. Organic Chemistry Prof. Dario Pasini Dr. Carmine Coluccini Dr. Claudio Cornaggia Michele Petenzi Dep. Pharmaceutical Chemistry Prof. Massimo Pregnolato Prof. Daniela Ubiali Dr. Teodora Bavaro Dr. Davide A. Cecchini Dr. Chiara Savarino Visit:

www.unipv.it/labt

Classical Synthesis of Copolymer/Substrate

1 –Functionalization of monomer

HOOC OH

-Mandelic Acid 1-DMAP / Pyridine / Me 3 SiCl 2- DMF / (COCl) 2 a 0 °C CH 2 Cl 2 ClOC + OSiMe 3 [ ] n [ OH O Et 3 N / CH 2 Cl 2 m ] m n 80-85 % AIBN toluene 70 °C 48 h

2 -Copolymerization

O O O OH O O O - Difficult to precipitate - Low yield - Impurities OH