Dario Pasini

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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, 3rd June 2009
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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
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Crosslinked Polymers-Substrate / Free
Enzyme Concept B

• Tentagel and Argogel resins (polyethylene glycol
  chains grafted onto classical polystyrene/divinylb
  enzene cores)
• High swelling characteristics in aqueous solvents
• Low loading capacity
• Limited success in combination with biocatalysis
• PEGA1900 (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, 81,
1626-40
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Biocatalysis and Solid Phase Synthesis Concept C
versus Concept D
Biocatalitically-Triggered Safety-Catch Linker
X O, NH, NR
Only when is a soluble linear polymer
(PEG polyethyleneglycol) high yields of the
product could be achieved (Concept D)
ImmobilizedPGA
-

U. Grether, H. Waldmann Chem. Eur. J. 2001, 7,
959-971
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Soluble PS Copolymer-Substrate / Immobilized
Enzyme Concept D
Immobilized Enzyme

i) Enzymatic hydrolysis (PGA)
ii) Filtration of the Enzyme
Soluble PS Copolymer -Substrate
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
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Monomer and Polymer Synthesis
83-89
70-90
Introduction of phenylacetic ester monomers and
copolymerization with styrene at several loadings
y
x
60-80
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Gel permeation chromatography
Solvent
Polymer
Average Molecular Mass
Mn (number average)
Mw (weight average)
PDI Polydispersity Index
Values between 1.5 and 2.4
Mw / Mn
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Properties as Supports
High Comonomer Loading (6040)
Bad precipitation in MeOH centrifugation needed
Low Comonomer Loading (937)
Excellent precipitation in MeOH
Sample Molecular Weight Distribution Mn 11080
Mw 18950 PD 1.7
Good precipitation in MeOH
Medium Comonomer Loading (8020)
Sample Molecular Weight Distribution Mn 9080
Mw 17630 PD 1.9
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Copolymer Substrate Hydrolysis by PGA
Immobilized on Eupergit (brown) Immobilized on
Agarose (yellow)
Hydrolysis Conditions -Temperature
37C -Mechanical stirring -Mixed solvent system
(aqueous buffer 80/ DMF 20)
Quantitative release First order kinetics
D. Pasini, M. Filippini, I. Pianetti, M.
Pregnolato, Adv. Synth. Catal., 2007, 349, 971
978.
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Enantiomeric Resolution Strategy
Immobilized enzyme
Soluble Copolymer
i)Enantioselective Enzymatic Cleavage ii)
Immobilized Enzyme Recovery iii) Optically-Active
Substrate and Soluble Copolymer Recovery
Chemical Refunctionalization
(S)
(R)
i) Chemical Cleavage ii) Soluble Copolymer
Recovery
(R)
Possible application to enantioselective
resolution of racemic carboxylic acids?
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Enzymatic Hydrolysis of (R,S)-Methyl
mandelate Concept A
From E. Coli on activated agarose gel
R OMe, OEt, OPr,n, Opr,iso, OBut,n, NH2,
NHPr,n, NHPr,iso
S. Rocchietti et al. Enzyme Microb. Technol.
2002, 31, 88-93
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Alternative Synthesis of Copolymer/Substrate
1 - Copolymerization
n
m
2 - Functionalization
Good yields
Good purity
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Efficient Polymer Functionalization 1H NMR and IR
A
BC
B
A
C
Primary OH
1H NMR CDCl3, solution
IR KBr, diffuse reflectance, polymer powder
D
B
C
A
AC
B
D
Ester carbonyl
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Efficient Control of Polydispersity
RAFT reagent

Functionalization as usual
Reversible Addition-Fragmentation Chain Transfer
(RAFT) Polymerization
Achieved control of Polydispersitylt1.2 Achieved
control of Degree of polymerization (50 to 500)
C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A
2008, 46, 5715-5723
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Copolymer/Substrate Solubility Tests
Phenylacetate Copolymer
(R,S)-Mandelate Copolymer
Solvent Ratio () Solubility
MeCN 100
MeCN/H2O 50/50 -/
DMF 100
DMF/H2O 70/30
DME 100
DME/H2O 70/30
DMSO 100
DMSO/H2O 50/50 -/
Solvent Ratio () Solubility
MeCN 100 -
DMF 100
DMA 100
DMA/H20 80/20
DMA/H20 20/80
DMSO 100 -/
THF 100
THF/H20 60/40
DMF / Water Best Solvent
DMA / Water Best Solvent
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Stability of Immobilized PGA in DMA/Water
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Enzymatic Hydrolysis of (R,S)-Mandelate Copolymer
Hydrolysis Conditions -Temperature
25C -Mechanical stirring -Mixed solvent system
(aqueous buffer 80/ DMA 20)
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Analytical Control
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-10Ammonium acetate 100 mM in
Ethanol T 25C
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 CH3CN T 25C
R
S
Acids
Esters
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Preliminary Data Results
(R,S)-Mandelate - Copolymer
Hydrolysis Rate (?mol/min) 0.04
Conversion (30h) 41
ee 18
E 1.61
(R,S)-Methyl mandelate Free
Hydrolysis Rate (?mol/min) 0.73
Conversion (5h) 43
ee 21
E 1.77

Immobilized PGA 100U
Immobilized PGA 200U
Same Hydrolysis Conditions in Aqueous Buffer 80 /
DMA 20
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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
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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
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Classical Synthesis of Copolymer/Substrate
1 Functionalization of monomer
m
n
2 -Copolymerization
- Difficult to precipitate
- Low yield
- Impurities