Overcoming equilibrium issues with carbonyl reductase enzymes

Article abstract of DOI:10.1021/op200241u

We report herein the screening, optimisation and scale up to 100 g of a bioreduction process that employs an in situ product removal (ISPR) technique to overcome the inherent equilibrium problem associated with the coupled-substrate approach to biocatalyt

Full text of DOI:10.1021/op200241u

ARTICLE
pubs.acs.org/OPRD
Overcoming Equilibrium Issues with Carbonyl Reductase Enzymes
Susan J. Calvin,† David Mangan,*,‡ Iain Miskelly,‡ Thomas S. Moody,‡ and Paul J. Stevenson†
†School of Chemistry and Chemical Engineering, Queen’s University Belfast, David Keir Building, Stranmillis Road, Belfast,
Northern Ireland BT95AG.
‡Almac, Biocatalysis Group, David Keir Building, Stranmillis Road, Belfast, Northern Ireland BT95AG.
ABSTRACT: We report herein the screening, optimisation and scale up to 100 g of a bioreduction process that employs an in situ
product removal (ISPR) technique to overcome the inherent equilibrium problem associated with the coupled-substrate approach
to biocatalytic carbonyl reduction. This technique allowed the valuable chiral alcohol, (S)-2-bromo-2-cyclohexen-1-ol, to be isolated
in 88% yield and 99.8% ee without the need for further purification, validating the general applicability of this experimental setup.
’ INTRODUCTION
α-Halogenated, α,β-unsaturated compounds represent a useful
class of intermediates in organic synthesis. Their versatility extends
from their ability to serve as α-ketovinyl anion equivalents as well as
enabling access to α-substituted enones. α-Bromocyclohex-2-
enone (1) is frequently encountered as the starting point in a
number of natural product syntheses includingthe total synthesis of
the sesquiterpenes echinopines A and B1 and (+)-trans-195A,2 the
name assigned to a decahydroquinoline alkaloid isolated from the
skin of dendrobatid frogs, as shown in Figure 1. Nicolaou and co-
workers generated chiral alcohol (S)-3 in 90% yield and g95% ee,2
while Blechert and co-workers used a modified CBS reagent to
afford (S)-3 in 95% yield and 99% ee.3 Unfortunately, both
procedures necessitated subsequent purification by chromato-
echinopines A and B, which require (S)-3 or (S)-4 as intermediates
graphic means.
Figure 1. Recent natural product synthesis targets, (+)-trans-195A and
derived from ketones 1 or 2, respectively.
Almac recently had a requirement to synthesise kilogram quanti-
ties of α-halo, α,β-unsaturated chiral alcohols (S)-3 or (S)-4 shown
in Figure 1. These were required as a key intermediate for the syn-
thesis of a novel therapeutic agent currently under development.
The interest of the synthetic organic chemistry community in
biocatalysis has exploded over the past decade.3 This is true, in
particular, for carbonyl reductase (CRED) enzymes, which have
now become the method of choice for the asymmetric reduction
of prochiral ketones.4 These enzymes have received much atten-
tion by both academic and industrial groups and many cloned re-
ductases have been reported.5 In order for this approach to be
considered economically viable at scale, CRED enzymes require
cofactor recycling, and several methods have been employed to
date, mostnotably glucose dehydrogenase(GDH)6 and isopropyl
alcohol (IPA), (Scheme 1).7 The use of IPA as the cofactor re-
cycle system is generally preferred not only for economic reasons
(using one enzyme as opposed to two), but also because it has the
added advantage that pH control is not required; a most desirable
attribute during the optimisation phase of process development.
Most crucially, in this case bioreduction would offer access to the
required chiral alcohols (S)-3 and/or (S)-4 without the need for
chromatographic purification ensuring ease of scalability.
commercially available, 2-cyclohexen-1-one (5) as shown in
Scheme 2. Compounds 1 and 2 were obtained in 36% and 74%
yield respectively. These reactions were not optimised in any way.
Almac’s CESK-5000 carbonyl reductase enzyme kit8 was screened
for the reduction of ketones 1 and 2. All of the reactions were per-
formed utilising the coupled-enzyme approach, employing a
glucose/GDH system, to regenerate the required cofactor. How-
ever, for enzymes that exhibit tolerance towards IPA, a second
reaction was performed in tandem utilising the coupled-substrate
approach. Racemic standards were prepared by carrying out
NaBH4-mediated reductions of ketones 1 and 2. Absolute stere-
ochemistry was assigned by comparison of [α]D measurements
with known literature values. Selections of the screening results for
ketones 1 and 2 are shown in Tables 1 and 2, respectively.
As can be seen from Table 1, entry 6 shows that CRED A-601
exhibited high conversion and high enantioselectivity generating
the required (S)-isomer quantitatively in 98.8% ee, albeit in the
glucose/GDH-coupled system.
As can be seen from Table 2 entry 4, (S)-4 was also formed in
high ee, albeit in lower yield than (S)-3 under screening conditions.
For this reason, the decision was made to move forward with
α-bromoketone 1 as the preferred CRED substrate.
’ RESULTS AND DISCUSSION
Screening. The desired ketone substrates were synthesised
Received: August 31, 2011
according to known literature precedents from the cheap,
Published: November 04, 2011
r
2011 American Chemical Society
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Scheme 1. Cofactor recycle systems used in CRED asym-
metric reductions of prochiral ketones5
Table 1. Selection of results from the CRED library screen of
ketone 1
entry enzyme cofactor cofactor recycle % conversiona
% eeb
1
A101
A201
A301
A401
A501
A601
A131
A161
A171
A231
A411
A441
A451
A461
A481
A511
N701
NADP+
NADP+
NADP+
NADP+
NADP+
NADP+
NAD+
NADP+
NAD+
NADP+
NAD+
NAD+
NAD+
NAD+
NAD+
NAD+
NADP+
GDH
IPA
85.3
6.0
98.0 (S)
94.7 (S)
63.2 (S)
40.7 (R)
92.1 (S)
98.8 (S)
18.8 (S)
55.4 (R)
90.6 (S)
34.7 (R)
91.7 (S)
97.0 (R)
72.6 (S)
97.2 (R)
68.2 (S)
87.8 (R)
86.4 (S)
2
3
GDH
GDH
GDH
GDH
IPA
1.4
4
83.4
4.1
5
6
99.9
97.8
82.8
77.9
100.0
5.6
7
8
GDH
GDH
GDH
GDH
GDH
IPA
9
10
11
12
13
14
15
16
17
59.9
30.8
28.1
1.5
GDH
GDH
GDH
GDH
Scheme 2. Synthesis of ketones 1 and 2, precursors to chiral
alcohols 3 and 4, respectively
19.4
8.9
a Conversion determined by GC analysis on a Supelco Beta Dex 225
(30 m  0.25 mm  0.25 μm). b Ee determined by GC analysis on a
Supelco Beta Dex 225 (30 m  0.25 mm  0.25 μm).
Table 2. Selection of results from the CRED library screen of
ketone 2
Process Development. Ascanbeseenfrom Tables1and2, the
lead enzyme/substrate combination during the screening phase of
this process was found to be A-601 acting on ketone 1 using the
glucose/GDH recycle system (99.9% conversion, 98.8% ee). The
use of IPA for cofactor regeneration resulted in a marginally poorer
result (95.7% conversion, 96.8% ee). The main advantage of IPA
over glucose/GDH for cofactor recycling is cost. In addition, the
absence of a requirement for pH control, the potential to use IPA as
both cofactor and also organic cosolvent, and the added simplicity
of optimising a one-enzyme versus a two-enzyme system encour-
aged us to proceed with the IPA dependent system.
Brief optimisation studies were carried out to assess the effect
of a number of variables, namely, temperature, pH, % IPA loading
(v/v), % substrate loading (w/v), and cosolvent. The thermo-
stability of the enzyme was first assessed, as once this has been
set, all further optimisation could be carried out using an iso-
thermal parallel reactor. These reactions were carried out under
the screening conditions employed during the enzyme selection
phase of development.
entry enzyme cofactor cofactor recycle conversion (%)a ee (%)b
1
A101
A201
A401
A501
A601
A131
A151
A161
A171
A231
A441
A451
NADP+
NADP+
NADP+
NADP+
NADP+
NAD+
NAD+
NADP+
NAD+
GDH
GDH
GDH
GDH
GDH
GDH
GDH
GDH
GDH
GDH
GDH
GDH
45.2
33.4
2.7
96.5 (S)
67.5 (S)
64.7 (R)
97.3 (S)
95.2 (S)
22.6 (S)
84.6 (S)
23.0 (R)
92.0 (S)
90.2 (R)
98.1 (R)
73.8 (S)
2
3
4
3.7
5
53.7
93.4
4.6
6
7
8
80.2
8.3
9
10
11
12
NADP+
NAD+
NAD+
94.8
47.0
8.6
a Conversion determined by GC analysis on a Supelco Beta Dex 225
(30 m  0.25 mm  0.25 μm). b Ee determined by GC analysis on a
Supelco Beta Dex 225 (30 m  0.25 mm  0.25 μm).
As can be seen from Figure 2, this process benefits from an in-
crease in temperature up to 30 °C. Beyond this point, the rate of
conversion decreases rapidly with increasing temperature. This is
undoubtedly due to the denaturation of the carbonyl reductase
enzyme. All subsequent reactions were carried out at 30 °C.
The effect of pH on reaction conversion was also assessed by
carrying out five parallel reactions covering the pH range of 6À8.
The results are depicted in Figure 3.
Figure 3 shows that the rate of conversion of 1 to (S)-3 de-
creases rapidly once the buffer pH becomes even slightly alkaline.
It is worth noting also that although the rate of reaction remained
high in slightly acidic media (pH = 6.5/6), the presence of small
quantities (<10% total GC area) of unidentified side products
was observed in these cases, suggesting that the vinyl bromide
moiety is inherently unstable under acidic conditions. In any
case, the optimum level was found to be pH 7, and all subsequent
reactions were carried out under this condition.
The effect of IPA loading on the process was explored with
another parallel set of experiments, the results of which are outlined
in Figure 4.
Figure 4 shows that the optimum IPA loading (v/v with re-
spect to buffer) was 20%. The unusual trend observed here at
lower IPA loadings can be explained by the fact that a 1% IPA
loading is approximately equal to 0.75 mol equivalents with
respect to the substrate and is therefore depleted before reaction
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Figure 4. Effect of IPA loading on the conversion of ketone 1 to alcohol
(S)-3.
Figure 2. Thermostability assessment for A-601 in the conversion of
ketone 1 to alcohol (S)-3.
Figure 5. Effect of concentration on the A-601 reduction of ketone 1 to
afford alcohol (S)-3.
Figure 3. Effect of buffer pH in the A-601-mediated conversion of
ketone 1 to alcohol (S)-3.
Gratifyingly, the reaction could be driven to completion by re-
ducing the pressure to 350 mbar. Unfortunately, it was found to
be necessary to maintain the level of IPA in the reaction as this
was concomitantly removed with the acetone. This required
completion. It is also evident that higher IPA loadings (30% and
50%) produce a detrimental effect on reaction rate.
The final parameter that was assessed in this fashion was sub-
strate loading (v/v with respect to buffer). In an effort to move
towards a more practically useful, economical process, the cell-
free extract (CFE) form of the enzyme that was used up until this
point was replaced with crude cell pellet. The results are shown in
Figure 5.
As is clearly evident from this data, more attractive volume
efficiency results in poor conversion, even after 48 h. The in-
crease in substrate concentration markedly amplifies an under-
lying problem associated with the coupled-substrate approach to
cofactor recycling. The obvious drawback of employing the IPA
cofactor regeneration approach is the competition that occurs be-
tween all four components of the system; substrate, product, co-
substrate, and coproduct. The thermodynamic equilibrium that
is established determines the maximum conversion to product
that can occur and is independent of the kinetics or catalyst used.
It has been demonstrated that removal of acetone from the equi-
librium can drive the desired reaction to completion.9À11 This is a
form of ISPR, a concept first pioneered in the area of biotech-
nology in the 1960s.12
1
regular in situ sampling of the reaction by H NMR spec-
troscopy and addition of the appropriate quantity of IPA to main-
tain the optimum 20% v/v level. An alternative solution to the
problem is illustrated in Figure 7.
In the experimental setup shown in Figure 7, the saturation
flask contains the desired IPA/H2O ratio (20% v/v). This allows
the level of IPA in the chemical reactor to remain relatively con-
stant throughout the course of the reaction.
Up until this point, all optimization experiments were conducted
with DMSO as the organic cosolvent. It was deemed prudent to
investigate while another non-volatile solvent might confer a
positive effect on the reaction rate, thereby allowing further reduc-
tion in key cost contributors such as enzyme loading and/or
NADP+ loading prior to scale-up.13 Of the cosolvents tested, only
DMF achieved comparable results. It was decided to proceed
with DMSO as cosolvent.
Scale-Up. Following a trial reaction on a 10-g scale, the re-
action was performed on 100 g of 1. The reaction profile is shown
in Figure 8. There is no negative impact on reaction rate observed
when operating at this scale. It is envisaged that when required,
this reaction will scale to kilogram quantities without the need for
further optimisation.
A series of experiments were conducted in an effort to monitor
the effect of reducing the atmospheric pressure in the reaction
vessel. Temperature was maintained constant throughout. A
representative sample of the data obtained is shown in Figure 6.
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mass input of substrate. For example, 100 g of substrate in 1 L
equates to 10 volumes.
Chemicals and Enzymes. Chemicals were purchased from
Sigma Aldrich. CESK-5000 enzyme screening kit and subsequent
gram quantities of CRED enzyme A601 were supplied by Almac.
Analytical Methods. Conversion and enantiomeric excess
measurements were determined by GC analysis using a Perkin
Elmer AutoSystem XLGC equipped with Supelco β-DEX 225
column (30 m  0.25 mm  0.25 μm). Hydrogen was used as a
carrier gas. Method: 80 °C hold for 3 min, 5 °C minÀ1 until
180 °C, hold for 2 min; inlet temperature 250 °C; detector tem-
perature 280 °C; flow rate: 3.4 mL minÀ1. Retention times for the
reduction of (S)-3 and (R)-3 from 2-bromo-cyclohexen-1-one
(1) were 19.6, 21.3, and 31.4 min, respectively. Retention times
for the reduction of (S)-4 and (R)-4 from 2-iodo-cyclohexen-1-
one (2) were 18.6, 19.1, and 23.6 min, respectively. Assignment of
absolute stereochemistry for each enantiomer was achieved by
carrying out the required selective enzymatic reductions at
500 mg scale and comparison of [α]D measurement with known
literature values.3,14
Figure 6. Effect of reduced pressure at a range of substrate loadings on
the reduction of ketone 1 to afford alcohol (S)-3.
2-Bromocyclohex-2-enone (1). A solution of Br2 (108 mL,
2.11 mol) in CH2Cl2 (1.5 L) was added dropwise to a solution of
α-cyclohexen-1-one (5) (200 mL, 2.07 mol) in CH2Cl2 (1.5 L)
at 0 °C over 45 min. The solution was stirred for 1.5 h before
dropwise addition of triethylamine (480 mL, 3.44 mol). The re-
action mixture was allowed to warm to room temperature and
stirred for a further 1.5 h before quenching with 2 M HCl
(250 mL). The layers were separated, and the aqueous phase was
extracted with CH2Cl2 (3 Â 250 mL). The combined organic
phases were washed with aqueous NaOH (pH 12.5) to remove
phenolic impurities and subsequently washed with brine(300 mL),
dried over anhydrous sodium sulfate, filtered, and concentrated in
vacuo. Recrystallisation using EtOAc/hexane (1:4 v/v) afforded
the product as an off-white solid (130.5 g, 36%). Mp 40À42 °C, lit.
Figure 7. IPA/H2O-saturated air sparge.
1
38À40 °C15; H NMR (300 MHz, CDCl3): δ = 2.09 (2H, m,
CH2), 2.42À2.52 (2H, m, CH2), 2.65 (2H, t, J = 6.4, CH2), 7.44
(1H, t, J = 4.4, CH); 13C (100 MHz, CDCl3): δ = 22.7, 28.4, 38.4,
123.9, 151.2, 191.3, lit.16
Racemic 2-Bromo-2-cyclohexen-1-ol (3). Cerium(III)
chloride heptahydrate (2.13 g, 5.71 mmol) was added to a solu-
tion of 2-bromocyclohex-2-enone (1) (1.00 g, 5.78 mmol) in
methanol (15 mL) at 0 °C. The reaction mixture was stirred for
30 min before slow addition of NaBH4 (0.26 g, 6.88 mmol). The
reaction mixture was allowed to warm to room temperature and
stirred for 2 h before quenching with ice water (10 mL). The reac-
tion mixture was extracted with diethyl ether (3 Â 40 mL), and the
combined organic phases were washed with brine (30 mL) and
dried over anhydrous Na2SO4, filtered, and concentrated in vacuo
to afford the product as a pale-yellow crystalline solid (0.96 g, 95%).
1H NMR (300 MHz, CDCl3): δ = 1.62À1.77 (2H, m), 1.83À1.98
(2H, m), 2.03À2.13 (2H, m), 2.40 (1H, d, J = 4.2), 4.22 (1H, t, J =
4.3), 6.21 (1H, t, J = 4.2). 13C NMR (100 MHz, CDCl3): δ = 17.6,
27.8, 32.0, 69.9, 125.8, 132.6, lit.;2 HRMS (EI): calcd for C6H9BrO
[M]+ 175.9837, found 175.9845.
Figure 8. Reaction profile of the bioreduction of ketone 1 to alcohol
(S)-3 at 100-g scale.
’ CONCLUSION
The synthesis, screening, and process development work de-
scribed herein has resulted in an efficient, scalable, environmen-
tally friendly route to the required enantioenriched alcohol, (S)-3.
The use of IPA/H2O-saturated air sparging has effectively
overcome the inherent unfavorable equilibrium of the system
of interest.
2-Iodo-2-cyclohexen-1-one (2). To a solution of 2-cyclohex-
en-1-one (5) (1.58 g, 16.4 mmol) in THF/H2O (80 mL, 1:1 v/v)
was added K2CO3 (2.73 g, 19.7 mmol), I2 (6.26 g, 24.7 mmol)
and 4-(dimethylamino)pyridine (DMAP) (0.4 g, 3.3 mmol).
The reaction mixture was stirred for 3 h at room temperature and
then diluted with EtOAc (80 mL). The organic phase was washed
with saturated Na2S2O3 (60 mL), 0.1 M HCl (60 mL) and brine
(60 mL) before it was dried over anhydrous Na2SO4, filtered, and
’ EXPERIMENTAL SECTION
1
General. H NMR spectra were recorded at 300 MHz on a
Bruker AV-300 spectrometer. 13C NMR spectra were recorded at
100 MHz. All chemical shifts are relative to internal TMS. The
term ‘volumes’ is used throughout this paper to describe the
quantity of solvent used in a given experiment with respect to
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concentrated in vacuo to afford the product as a low-melting solid,
which was subsequently recrystallised from hexane/EtOAc
(4:1 v/v) (2.70 g, 74%). H NMR (300 MHz, CDCl3): δ =
1.98À2.07 (2H, m), 2.37À2.42 (2H, m), 2.60 (2H, t, J = 6.7),
7.72 (1H, t, J = 4.4); 13C (100 MHz, CDCl3): δ = 23.3, 30.3, 37.7,
104.3, 159.9, 192.6, lit.17
(4) (a) Brown, G.; Mangan, D.; Miskelly, I.; Moody, T. S. Org. Process
Res. Dev. 2011, 5, 1036. (b) Matsuda, T.; Yamanaka, R; Nakamura, K.
Tetrahedron: Asymmetry 2009, 20, 513. (c) Borges, K. B.; de Souza
Borges, W.; Durꢀan-Patrꢀon, R.; Pupo, M. T.; Bonato, P. S.; Collado, I. G.
Tetrahedron: Asymmetry 2009, 20, 385. (d) Kaluzna, I. A.; Rozzell, J. D.;
Kambourakis, S. Tetrahedron: Asymmetry 2005, 16, 3682. (e) Nakamura,
K.; Yamanaka, R.; Matsuda, T.; Harada, T. Tetrahedron: Asymmetry 2003,
14, 2659.
(5) (a) Yang, Y.; Zhu, D.; Piegat, T. J.;Hua, L. Tetrahedron: Asymmetry
2007, 18, 1799. (b) Padha, S. K.; Kaluzna, I. A.; Buisson, D.; Azerad,
R.; Stewart, J. D. Tetrahedron: Asymmetry 2007, 18, 2133. (c) Panizza,
P.; Onetto, S.; Rodríguez, S. Biocatal. Biotransform. 2007, 25, 414.
(d) Weckbecker, A.; Hummel, W. Biocatal. Biotransform. 2006, 24, 380.
(e) Hanson, R. L.; Goldberg, S.; Goswami, A.; Tully, T. P.; Patel, R. N.
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Weuster-Botz, D. Tetrahedron: Asymmetry 2004, 15, 3591.
(6) (a) Kosjek, B.; Nti-Gyabaah, J.; Telari, K.; Dunne, L.; Moore,
J. C. Org. Process Res. Dev. 2008, 12, 584. (b) Zhu, D.; Yang, Y.; Hua, L.
J. Org. Chem. 2006, 71, 4202.
(7) (a) Lavandera, I.; H€oller, B.; Kern, A.; Ellmer, U.; Glieder, A.; de
Wildeman, S.; Kroutil, W. Tetrahedron: Asymmetry 2008, 19, 1954.
(b) Matsuda, T.; Yamagishi, Y.; Koguchi, S.; Iwai, N.; Kitazume, T.
Tetrahedron Lett. 2006, 47, 4619. (c) Amidjojo, M.; Weuster-Botz, D.
Tetrahedron: Asymmetry 2005, 16, 899.
(8) See: www.almacgroup.com/biocatalysis.
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2002, 74, 1035.
1
Racemic 2-Iodo-2-cyclohexen-1-ol (4). 2-Iodo-2-cyclohex-
en-1-one (2) was reduced as for 3 to yield the title compound as a
1
colourless crystalline solid (1 g, 90%). H NMR (300 MHz,
CDCl3): δ = 1.59À2.15 (6H, m), 2.28 (1H, s), 4.20 (1H, s), 7.72
(1H, t, J = 4.4); 13C (100 MHz, CDCl3): δ = 17.7, 29.4, 32.0,
72.1, 103.6, 141.0, lit.18
Screening Conditions for the Enzymatic Reduction of
2-Bromocyclohex-2-enone (1). A solution of 2-bromocyclohex-
2-enone (1) (20 mg) in DMSO (50 μL) was added to a solution of
NADP+ or NAD+ (2 mg), depending on cofactor preference, in
pH 7 KH2PO4 buffer (0.1 M, 1.5 mL). Lyophilized CRED
biocatalyst (2 mg), glucose (50 mg), and GDH (3 mg) were added.
The vial was sealed and shaken overnight at 30 °C. MtBE (1.5 mL)
was added to the vial, and the organic layer was separated, filtered
through a cotton wool plug containing anhydrous MgSO4, and
analysed by GC.
(S)-2-Bromo-2-cyclohexen-1-ol ((S)-3). CRED A601 cell
paste (20 g) was resuspended in a solution of pH 7 KH2PO4
buffer (3.9 L, 0.1 M) containing NADP+ (1.0 g, 1.3 mmol) and
shaken at 20 °C for 30 min. The suspension was transferred to a
5-L reactor to which was added a solution of 2-bromocyclohex-2-
enone (1) (100 g, 0.57 mol) dissolved in IPA (800 mL) and
DMSO (200 mL). In addition, polypropylene glycol (1.5 mL) was
added to prevent excessive foaming. The reaction mixture was
stirred mechanically using an overhead stirrer and incubated at
35 °C for 24 h. The depletion of IPA and water was counter-
balanced by bubbling a pressurised airflow saturated with 2-
propanol and water (1:4 v/v) through the reaction mixture. Upon
completion, the resulting mixture was extracted with MtBE (3 Â 5L)
and washed with water (2 Â 2.5 L) and brine (2 Â 2.5 L) and dried
over MgSO4. Solvent was removed under reduced pressure to afford
the title compound as a colourless oil (89 g, 88%) with an
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1
enantiomeric excess of 99.8% as determined by GC analysis. H
NMR (300 MHz, CDCl3):δ=1.62À1.77(2H, m), 1.83À1.98 (2H,
m), 2.03À2.13 (2H, m), 2.40 (1H, d, J = 4.2), 4.22 (1H, t, J = 4.3),
6.21 (1H, t, J = 4.2). 13C NMR (100 MHz, CDCl3): δ = 17.6, 27.8,
32.0, 69.9, 125.8, 132.6, lit.;1 [α]D20 = À80.3, (c 1.77, CHCl3); lit.2
[α]2D0 = À77 (c 1.74, CHCl3).
’ AUTHOR INFORMATION
Corresponding Author
david.mangan@almacgroup.com
’ ACKNOWLEDGMENT
This study was partially financed by the European Regional
Development Fund under the European Sustainable Competi-
tiveness Programme for Northern Ireland.
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