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
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
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
Biocatalysis and Solid Phase Synthesis
Concept C versus Concept D
O
Target Molecule
X
Biocatalitically-Triggered
Safety-Catch Linker
MeO
H
H
N
O
N
O
= Merrifield resin or Wang resins
O
O
X = O, NH, NR
HO
Immobilized
PGA
O
Only when
is a soluble linear polymer (PEG
= polyethyleneglycol) high yields of the product
could be achieved (Concept D)
-
MeO
O
X
Target Molecule
N
O
MeO
O
O
O
O
NH
O
H
N
O
H
+
NH2
HX
U. Grether, H. Waldmann Chem. Eur. J. 2001, 7, 959-971
Target Molecule
Soluble Polymeric Supports
Advantages
1- Easy monitoring of the support functional groups by
common analytical techniques (e.g. 1H NMR)
2-Reactivity similar to the solution, homogeneous phase
3 – 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
x
y
+
O
i) Enzymatic hydrolysis (PGA)
n
O
O
ii) Filtration of the Enzyme
R
Soluble PS Copolymer Substrate
O
n
OH
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
n
+
HO
OH
NaOH/H2O
70°C
Cl
O
OH
n
83-89%
DICD/DPTS
24 h
70-90%
n=1-3
x
x
Toluene
O
O
O
60-80%
n
O
n
O
n=1-3
n=1-3
O
n
O
y
AIBN/70°C
+ y
O
O
n=1-3
x = 0.6-0.93
Introduction of
phenylacetic ester
monomers and
copolymerization
with styrene at
several loadings
Characterization by 1H NMR Spectroscopy
Hb
Hm
Hc
Ha
Hd
Hi
Hd
O
He
Hg
Ha Hb
O
Hm
He
Hi
Hf
80
Hf,g
Hc
O
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
Mn
Mw
(number average) (weight average)
0
PDI
Polydispersity Index
4
Mw / Mn
8
12
t (min)
Values between
1.5 and 2.4
Properties as Supports
High Comonomer Loading (60:40)
60
Bad precipitation in MeOH:
centrifugation needed
40
O
O
O
Low Comonomer Loading (93:7)
93
7
Excellent precipitation in MeOH
Sample Molecular Weight Distribution
Mn = 11080 ; Mw = 18950 ; PD = 1.7
O
O
O
Medium Comonomer Loading (80:20)
80
Good precipitation in MeOH
20
Sample Molecular Weight Distribution
Mn = 9080 ; Mw = 17630 ; PD = 1.9
O
O
O
Copolymer Substrate Hydrolysis by PGA
80
20
30
Conversion(%)
Immobilized on Eupergit (brown)
Immobilized on Agarose (yellow)
20
10
0
0
400
800
0
400
800
1200
t (s)
O
O
0
ln((P0-P)/P0)
O
Hydrolysis Conditions
-Temperature: 37°C
-Mechanical stirring
-Mixed solvent system
(aqueous buffer 80/
DMF 20)
1200
-0,1
-0,2
-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
Chemical Refunctionalization
Soluble Copolymer
(S,R)* (S,R)*
i)Enantioselective Enzymatic Cleavage
ii) Immobilized Enzyme Recovery
iii) Optically-Active Substrate and Soluble
Copolymer Recovery
Soluble Copolymer
(S)*
(R)*
(R)*
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
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
Alternative Synthesis of Copolymer/Substrate
1 - Copolymerization
[
m
+
]n
[
]m
AIBN
toluene 70 °C
48 h
n
OH
OH
O
O
2 - Functionalization
[
]n
[
]m
1-DMAP / Pyridine / Me3SiCl
2- DMF / (COCl)2 a 0 °C
3- Et3N / CH2Cl2
HOOC
OH
O
OH
O
Good yields
O
Good purity
Efficient Polymer Functionalization: 1H NMR and IR
]85 [
[
A
]15
B+C
B
A
O
C
OH
Primary OH
1H
NMR: CDCl3, solution
[
]85 [
IR: KBr, diffuse reflectance,
polymer powder
]15
D
O
OH
D
8
6
A
A+C B
4
B
C
O
O
2
 (ppm)
Ester carbonyl
Efficient Control of Polydispersity
[
RAFT reagent
]85
[
AIBN
toluene 70 °C
48 h
+
OH
OH
O
O
S
]15
Functionalization
“as usual”
S
Reversible Addition-Fragmentation
Chain Transfer (RAFT) Polymerization
[
]85
[
]15
Achieved control of Polydispersity:<1.2
Achieved control of Degree of polymerization (50 to 500)
O
OH
O
C. Barner-Kowollik, S. Perrier, J. Polym. Sci. A 2008, 46, 5715-5723
O
Copolymer/Substrate Solubility Tests
Phenylacetate Copolymer
(R,S)-Mandelate Copolymer
Solvent
Ratio (%)
Solubility
Solvent
Ratio (%)
Solubility
MeCN
100
+
MeCN
100
-
MeCN/H2O
50/50
-/+
DMF
100
++
DMF
100
+++
DMA
100
+++
DMF/H2O
70/30
++
DMA/H20
80/20
++
DME
100
+++
DMA/H20
20/80
+
DME/H2O
70/30
++
DMSO
100
-/+
DMSO
100
+
THF
100
+++
DMSO/H2O
50/50
-/+
THF/H20
60/40
+
DMF / Water
Best Solvent
DMA / Water
Best Solvent
Stability of Immobilized PGA in DMA/Water
Residual activity %
120
20 % DMA
100
30 % DMA
80
60 % DMA
60
80 % DMA
40
20
0
0
500
1000
Time (min)
1500
2000
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
Enantioselectivity 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 = 25°C
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
(R,S)-Mandelate - Copolymer
Hydrolysis Rate (mol/min) 0.73
Hydrolysis Rate (mol/min) 0.04
Conversion (5h)
43%
Conversion (30h)
41%
ee%
21%
ee%
18%
E
1.77
E
1.61
Immobilized PGA = 100U
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
1-DMAP / Pyridine / Me3SiCl
2- DMF / (COCl)2 a 0 °C
OH
O
m
CH2Cl2
HOOC
OH
DL-Mandelic Acid
ClOC
O
Et3N / CH2Cl2
OSiMe3
OH
O
O
+
[
]n
[
]m
AIBN
toluene 70 °C
n
O
48 h
80-85 %
2 -Copolymerization
OH
O
O
- Difficult to precipitate
- Low yield
- Impurities