Application of polymer-supported enzymes and reagents in the synthesis of γ-aminobutyric acid (GABA) analogues

Article abstract of DOI:10.1055/s-2002-34249

Polymer-supported pig liver esterase was used for the resolution of meso-diesters. The enzyme can be recovered quantitatively from the reaction mixture by filtration and reused without significant loss of activity. Further transformation of the resulting enantiomerically enriched carboxylic acids through the application of polymer-supported reagents and scavengers provides a number of GABA-analogues.

Full text of DOI:10.1055/s-2002-34249

LETTER
1641
Application of Polymer-Supported Enzymes and Reagents in the Synthesis of
-Aminobutyric Acid (GABA) Analogues
S
ynthesi
a
s
of -Amin
n
obutyric
A
cid
(
GA
R
BA
)
A
nalogues. Baxendale, Martin Ernst, Wolf-Rüdiger Krahnert, Steven V. Ley*
Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, UK
Fax +44(1223)336442; E-mail: svl1000@cam.ac.uk
Received 29 July 2002
mationally locked compounds containing a rigid back-
Abstract: Polymer-supported pig liver esterase was used for the
resolution of meso-diesters. The enzyme can be recovered quantita-
tively from the reaction mixture by filtration and reused without sig-
nificant loss of activity. Further transformation of the resulting
enantiomerically enriched carboxylic acids through the application
of polymer-supported reagents and scavengers provides a number
of GABA-analogues.
bone.6 Here we wish to report the first combined
application of polymer-supported enzymes and reagents
in the synthesis of structurally defined GABA-analogues.
The initial step of our synthetic route involved the desym-
metrisation of commercially available meso-diester 1 with
polymer-supported pig liver esterase on Eupergit®
(Scheme 1).7 The reaction was carried out in an aqueous
phosphate buffer system, which was kept at pH 7 by con-
tinuous addition of 0.5 N NaOH by a pH stat. After com-
pletion of the reaction, the enzyme was removed by
filtration and reused in subsequent runs. We noticed an in-
crease in the rate of hydrolysis with each successive cycle
of the enzyme. The reaction required 26 hours to reach
completion when the enzyme was used for the first time,
only 12 hours in the second cycle and 9 hours in the third
cycle. This was probably due to a preadjustment of con-
formation of the enzyme by incorporation of water mole-
cules after reaction. In every cycle the hydrolysis
proceeded without any decrease of the enantiomeric ex-
cess of the product. The desired carboxylic acid 2 was iso-
lated in 90% ee from the acidified reaction mixture
(saturated with sodium chloride).8 The enantiomeric ex-
cess could be increased to >95% ee by twofold recrystal-
Key words: polymer-supported enzymes, pig liver esterase, poly-
mer-supported reagents, GABA-analogues, desymmetrisation
Enzymes have become valuable tools in asymmetric or-
ganic synthesis, notably in the desymmetrisation of meso-
compounds.1 In this way, achiral starting materials can be
converted into enantiomerically enriched compounds in
up to quantitative yield. However, the high cost of enzyme
preparations have made recycling of the biocatalyst an im-
portant issue. Since filtration is a simple way of recover-
ing a catalyst from a reaction mixture, enzymes have been
covalently linked to a multitude of insoluble supports and
adsorbed onto various materials. However, immobilising
enzymes can modify their properties and care has to be
taken not to alter the selectivity and the activity of the na-
tive catalyst.2
With the need for automated parallel synthesis of large lisation from benzene.
numbers of pharmaceutically interesting compounds,
Reduction of the carboxylic acid 2 to 3 and 4 was achieved
polymer-supported reagents have been developed that
display high degree of versatility and applicability in re-
cent years.3 Compared to traditional solution phase and
solid phase chemistry, supported reagents offer many ad-
vantages for example the reactions can be monitored by
conventional techniques (TLC, LC-MS, GC, NMR) and
no additional steps are required in order to attach and sub-
sequently remove the substrate from the support. As yet,
polymer-supported enzymes have not been effectively
integrated into the toolbox of reagents for multistep syn-
thesis.
using BH3 SMe2 in THF.9 This reaction was quenched
with a small amount of water and the resulting boric acid
scavenged using the boron specific resin Amberlite
IRA-743.10 The ratio of 3 and 4 depended on the excess of
BH3 SMe2 used. Any attempts to apply polymer-support-
ed reducing agents in this reaction failed. Both alcohol 3
and lactone 4 were transformed into bromide 5 using an
excess of thionyl bromide in methanol. Neutralisation was
achieved by addition of poly(4-vinylpyridine)11 to the re-
action mixture. The conversion of bromide 5 into azide 6
proceeded smoothly in 84% yield by application of poly-
Analogues of the neurotransmitter -aminobutyric acid mer-supported azide12 and iodide13 in acetonitrile. Reduc-
(GABA) have been used to explore the binding mode and tion of azide 6 using either Staudinger conditions
structure-activity relationship of the parent compound.4 involving polymer-supported triphenylphosphine14 or
They have also been investigated as therapeutic agents for Pd/C under a hydrogen atmosphere furnished a mixture of
a range of central nervous system disorders.5 Specific the desired amino acid ester and the corresponding lac-
conformations of GABA can be mimicked using confor- tam. To suppress lactamisation, 6 was reacted with Pd/C
and Boc-anhydride under hydrogen atmosphere to con-
vert the amine in situ into the Boc-protected derivative 7.
Excess Boc2O was scavenged using an amino resin.15
Synlett 2002, No. 10, Print: 01 10 2002.
Art Id.1437-2096,E;2002,0,10,1641,1644,ftx,en;D11602ST.pdf.
Finally, application of a catch and release technique for
© Georg Thieme Verlag Stuttgart · New York
ISSN 0936-5214

1642
S. V. Ley et al.
LETTER
removal of the protecting group and saponification of the
methyl ester using Amberlyst A-15 and HCl gave the
GABA-analogue 8 as its hydrochloride in 67% yield.16
PLE
0.1 M phosphate buffer,
0.5 M NaOH, pH 7, r. t.
H3CO2C
CO2CH3
HO2C
CO2CH3
98%
The presented reaction sequence affords the desired prod-
uct without the requirement of any column chromatogra-
phy and therefore demonstrates clearly the advantages of
polymer-supported enzymes and reagents. We next ex-
panded this reaction sequence to analogues with a four,
five and six membered ring. The synthetic strategy was al-
tered since these analogues are more conveniently pre-
pared using a combination of polymer-supported and
traditional solution phase chemistry.
1
2
90% ee
>95% ee after recryst.
1.
2.
BMS, THF,
-10 °C → r. t.
NBn3+OH-
+
HO
CO2CH3
67%
O
O
3
4
-
NMe3+N3
1. SOBr2, MeOH, r.t.
NMe3+I-
Starting from meso-diesters 9a–d the enzymatic hydroly-
sis of these compounds proceeded smoothly and the en-
zyme could be reused after its isolation by filtration
(Scheme 2). The enantiomeric ratios are similar to those
observed with the corresponding non-immobilized en-
zyme.17 Meso-cyclopentane dicarboxylic acid dimethyl
ester 9c and meso-cyclohexane dicarboxylic acid dimeth-
yl ester 9d proved to be poor substrates for the polymer-
supported pig liver esterase providing enantiomeric ex-
cesses of 10% and 68% respectively. The enatiomeric ra-
tios were determined by 1H NMR analysis of the
corresponding salt formed with (+)-(R)- -methylbenzyl-
amine. We were able to confirm the absolute stereochem-
istry of 10a by a X-ray structure of the corresponding
diastereomeric amide formed from 12a and (+)-(R)- -me-
thylbenzylamine.18 In case of 10b the absolute stereo-
chemistry was assigned by comparison of the optical
rotations of 10b and 13b with literature values.19 The
N
2.
CH3CN, 50 °C
85%
Br
CO2CH3
84%
5
1. H2, Pd/C, Boc2O, MeOH
NH2
2.
N
H
N3
CO2CH3
95%
6
1.
SO3H
CH2Cl2, r. t.
2. 1 N HCl, reflux
BocHN
CO2CH3
HCl·H2N
CO2H
67%
7
8
[α]D25 = -35°
(c = 0.25, 1 N HCl)
Scheme 1 Synthesis of GABA-analogue 8 with polymer-supported
reagents and enzymes.
1. BMS, THF,
-10 °C → r. t.
PLE
0.1 M phosphate buffer,
0.5 M NaOH, pH 7, r. t.
2.
n
n
NBn3+OH-
n
98%
3.
4.
SO3H or ∆
H3CO2C
CO2CH3
HO2C
CO2CH3
H3CO2C
CO2H
O
O
O
O
N
n=1,2,3,4
9a-d
10a: 98%, 90% ee
10b: n=1, 99%, 86% ee
10c: n=2, 94%, 10% ee
10d: n=3, 97%, 68% ee
11a: 68%
11b: n=1, 62%
11c: n=2, 88%
11d: n=3, 57%
N
O
N-K+
NH2
n
n
EtOH, reflux
O
DMF, reflux
PhthN
CO2H
12a: 80%
HO2C
NPhth
H2N
CO2H
HO2C
NH2
12b: n=1, 70%
13a: 89%
13b: n=1, 90%
12c: n=2, 24%
12d: n=3, 40%
13c: n=2, 73%
13d: n=3, 81%
13a·HCl: [α]D25 = -36.1° (c = 0.7, 1 N HCl)
13b·HCl: [α]D25 = 20.4° (c = 0.61, MeOH)
13d·HCl: [α]D25 = 3.3° (c = 1.1, MeOH)
F
F
SO3H
97%
NH2
NH2
MeOH, reflux
CO2CH3
N
N
H
MeOH, reflux
PhthN
O
N
95%
14
H
15
[α]D25 = 51.4°
(c = 0.5, CHCl3)
Scheme 2 Synthesis of GABA-analogues 13a–d with polymer-supported reagents and enzymes.
Synlett 2002, No. 10, 1641–1644 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of -Aminobutyric Acid (GABA) Analogues
1643
measured rotations for 10b and 13b indicate a reversal of
the selectivity of the immobilized enzyme in contrast to
the non-immobilized pig liver esterase.
1. NEt3, ClCO2CH3, THF
2. NaBH4, THF/MeOH
n
3. 100°C neat or
SO3H
HO2C
10a
CO2CH3
H3CO2C
CO2H
10b: n=1
The reduction of the carboxylic acid function was
achieved using the same procedure as for compound 2.
The resulting mixture of lactone and corresponding al-
cohol was transformed into the pure lactones 11a–d by
heating the mixture neat or by reaction with Amberlyst
A-15 in CH2Cl2. Remaining non-reduced carboxylic acids
10a–d could be scavenged with polymer-supported
DMAP. A small amount of impurity in this reaction was
responsible for the formation of side products in the fol-
lowing lactone ring-opening step and therefore made
chromatographic purification of the resulting carboxylic
acids 12a–d necessary (for an alternative approach see be-
low). The opening of the lactone ring in 11a–d was
achieved by reaction with potassium phthalimide in re-
fluxing DMF.6a This reaction proceeded smoothly in the
case of the GABA-analogues with a three and a four mem-
bered ring 12a and 12b due to the strained ring system. In
the case of the five membered ring 12c the yield is poor
and epimerisation at the α-position of the carboxylic acid
occurs due to the long reaction time (24 h).20 All carbox-
ylic acids were isolated by an aqueous basic-acidic work-
up sequence. The phthalimide protecting group could be
removed by refluxing 12a–d with aminomethylated poly-
styrene resin21 in EtOH. GABA-analogues 13a–d were
obtained after hot filtration, washing and evaporation in
73–90% yield.22 Other polymer-supported amines and hy-
drazines did not react or resulted in scavenging of the
starting material. In contrast, deprotection of the amino
group of methyl ester 14 (obtained from 12a by acid catal-
ysed esterification) with trisamine resin23 furnished lac-
tam 15 selectively.24
10c: n=2
10d: n=3
n
O
O
11a: 71%
O
O
11b: n=1, 80%
11c: n=2, 85%
11d: n=3, 88%
Scheme 3 Improved synthesis of lactones 11a–d.
Acknowledgement
We would like to thank the German Academic Exchange Service
(DAAD) for the support of this work by a fellowship within the
postdoc-program (to WRK). We also gratefully acknowledge the fi-
nancial support of Pfizer Global Research and Development for a
Postdoctoral Fellowship (to IRB), the BP Endowment and the No-
vartis Research Fellowship (to SVL).
References
(1) (a) Mohr, P.; Waespe-Sarcevic, N.; Tamm, C.; Gawronska,
K.; Gawronski, J. K. Helv. Chim. Acta 1983, 66, 2501.
(b) Walser, P.; Renold, P.; N’Goka, V.; Hosseinzadeh, F.;
Tamm, C. Helv. Chim. Acta 1991, 74, 1941. (c) Schneider,
M.; Engel, N.; Hönicke, P.; Heinemann, G.; Görisch, H.
Angew. Chem., Int. Ed. Engl. 1984, 23, 67.
(2) (a) Mosbach, K. FEBS Lett. 1976, 62, E80. (b) Worsfold, P.
Pure Appl. Chem. 1995, 67, 597.
(3) For recent reviews see: (a) Ley, S. V.; Baxendale, I. R.;
Bream, R. N.; Jackson, P. S.; Leach, A. G.; Longbottom, D.
A.; Nesi, M.; Scott, J. S.; Storer, R. I.; Taylor, S. J. J. Chem.
Soc., Perkin Trans. 1 2000, 3815. (b) Kirschning, A.;
Monenschein, H.; Wittenberg, R. Angew. Chem. Int. Ed.
2001, 40, 650. (c) Guino, M.; Brule, E.; De Miguel, Y. R.
Chimica Oggi–Chem. Today 2002, 20, 23. (d) Ley, S. V.;
Baxendale, I. R.; Brusotti, G.; Caldarelli, M.; Massi, A.;
Nesi, M. Farmaco 2002, 57, 321.
(4) (a) Allan, R. D.; Curtis, D. R.; Headley, P. M.; Johnston, G.
A. R.; Lodge, D.; Twitchin, B. J. J. Neurochem. 1980, 34,
652. (b) Kusama, T.; Spivak, C. E.; Whiting, P.; Dawson, V.
L.; Schaeffer, J. C.; Uhl, G. R. Br. J. Pharmacol. 1993, 109,
200. (c) Kusama, T.; Wang, T.-L.; Guggino, W. B.; Cutting,
G. R.; Uhl, G. R. Eur. J. Pharmacol. 1993, 245, 83.
(5) Allan, R. D.; Johnston, G. A. R. Med. Res. Rev. 1983, 3, 91.
(6) (a) Kennewell, P. D.; Matharu, S. S.; Tazlor, J. B.; West-
wood, R.; Sammes, P. G. J. Chem. Soc., Perkin Trans. 1
1982, 2563. (b) Paulini, K.; Reissig, H.-U. Liebigs Ann.
Chem. 1991, 455. (c) Morikawa, T.; Sasaki, H.; Hanai, R.;
Shibuya, A.; Taguchi, T. J. Org. Chem. 1994, 59, 97.
(d) Galeazzi, R.; Mobili, G.; Orena, M. Tetrahedron:
Asymmetry 1997, 8, 133. (e) Duke, R. K.; Allan, R. D.;
Chebib, M.; Greenwood, J. R.; Johnston, G. A. R.
As mentioned above, the reduction of the carboxylic acids
10a–d was usually accompanied by the formation of an
impurity, which caused purification problems in the fol-
lowing ring-opening step. To circumvent this problem, we
developed a reaction sequence, which furnishes the lac-
tones 11a–d in good yields and purity without costly pu-
rification (Scheme 3). Acids 10a–d were transformed into
their corresponding ethyl carbonates, which were reduced
using NaBH4 in THF by slow addition of a small amount
of MeOH.25 After evaporation, the crude products were
redissolved again in a petrol ether/ethyl acetate mixture
(1:1) and filtered through a short plug of silica. The alco-
hols obtained were transformed into the lactones 11a–d
by acid catalysis or by heating them neat to 100 °C.
The short syntheses of GABA-analogues shown above
represent an useful application of polymer-supported re-
agents. The advantages of this strategy are not only the
circumvention of classical purification methods like col-
umn chromatography and distillation, but also the intro-
duction of chirality by use of polymer-supported
enzymes. This type of polymer-supported reagent has to
date not been employed in a polymer-supported reaction
sequence and represents an extension to the toolbox of
available reagents for potential automated syntheses.
Tetrahedron: Asymmetry 1998, 9, 2533. (f) Forti, L.;
Ghelfi, F.; Levizzani, S.; Pagnoni, U. M. Tetrahedron Lett.
1999, 40, 3233. (g) Bonnaud, B.; Cousse, H.; Mouzin, G.;
Briley, M.; Stenger, A.; Fauran, F.; Couzinier, J.-P. J. Med.
Chem. 1987, 30, 318. (h) Galeazzi, R.; Mobili, G.; Orena,
M. Tetrahedron 1999, 8, 261.
Synlett 2002, No. 10, 1641–1644 ISSN 0936-5214 © Thieme Stuttgart · New York

1644
S. V. Ley et al.
LETTER
(7) Purchased from Fluka Cat. No. 46064; solid support:
methacrylamide/allyl glycidylether/methylene-bis-
acrylamide; loading: ~500 U/g.
(+)-(R)- -methylbenzylamine have been deposited with the
Cambridge Crystallographic Data Centre as supplementary
publication No. CCDC-189257. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK (Fax: +44(1223)336033;
deposit@ccdc.cam.ac.uk).
(8) Enantiomeric ratio of 2 using non-immobilized pig liver
esterase: 100% ee.1a
(9) Born, M.; Tamm, C. Synthesis 1991, 435.
(10) Purchased from Aldrich Cat. No. 21,644-5.
(11) Purchased from Fluka Cat. No. 81393; solid support: poly(4-
vinylpyridine)/25% DVB.
(12) Purchased from Aldrich Cat. No. 36,834-2; solid support:
styrene/DVB; loading: 3.8 mmol/g.
(19) For compound 10b we measured [ ]D25 = –3.6 (c 0.95,
CHCl3), which is the opposite of the rotations published in
references 1a,c.
(20) According to the 13C NMR spectrum the ratio of the
diastereomers is 1:1.7.
(13) Purchased from Fluka Cat. No. 57895; solid support:
styrene/>20% DVB; loading: 2.9 mmol/g.
(14) Purchased from Fluka Cat. No. 93093; solid support:
styrene/2% DVB; loading: 3 mmol/g.
(21) Purchased from Novabiochem Cat. No. 01-64-0177; solid
support: styrene/2% DVB; loading: 2.0–3.0 mmol/g.
(22) Lit. values: 13a·HCl: [ ]D25 = –38.1 (c 1, 1 N HCl);6d
13b·HCl: [ ]D25 = 24.1 (c 0.4, MeOH);6h analytical data for
13d: 1H NMR (400 MHz, D2O): = 1.25–1.71 (m, 8 H),
2.08–2.19 (m, 1 H), 2.43–2.50 (m, 1 H), 2.97 (dd, J = 12.8,
5.9 Hz, 1 H), 3.05 (dd, J = 12.8, 7.7 Hz, 1 H); 13C NMR (100
MHz, D2O): = 21.9, 24.5, 26.5, 27.3, 35.6, 40.6, 47.6,
183.6; 13d HCl: [ ]D25 = 3.3 (c 1.1, MeOH).
(15) Purchased from Novabiochem Cat. No. 01-64-0178; solid
support: styrene/1% DVB; loading 2.0–3.5 mmol/g.
(16) Lit. value 8: [ ]D25 = –38.1 (c 1, 1 N HCl).6d
(17) Enantiomeric ratios of 10a–d using non-immobilized pig
liver esterase: 10a: 100% ee, 10b: 90% ee, 10b: 9% ee, 10c:
78% ee.1a
(18) For the observed inconsistencies compare reported optical
rotation in references 1a and 1c. We wish to report the
following rotation 10a: [ ]D25 = –17.4 (c 0.8, CHCl3).
Crystallographic data of the amide formed from 12a and
(23) Purchased from Novabiochem Cat. No. 01-64-0170; solid
support: styrene/1% DVB; loading: 2.2 mmol/g.
(24) Lit. value 15: [ ]D25 = 49.2 (c 1, CHCl3).6h
(25) Soai, K.; Yokoyama, S.; Mochida, K. Synthesis 1987, 647.
Synlett 2002, No. 10, 1641–1644 ISSN 0936-5214 © Thieme Stuttgart · New York