Thermodynamics of phenylacetamides synthesis: Linear free energy relationship with the pK of amine

Article abstract of DOI:10.1016/j.molcatb.2011.08.013

The effective equilibrium constants ^K′C expressed through the total concentrations of the reagents for the synthesis of N-phenylacetyl-derivatives in aqueous medium from phenylacetic acid and various primary amino compounds have been determined with penicillin acylase as a catalyst. Broad specificity of penicillin acylase (EC 3.5.1.11) to amino components made possible to investigate the acylation of primary amines with different structures and physicochemical properties. Analysis of different components of the effective standard Gibbs energy change ΔGC ^o′ has revealed favorable thermodynamics for the synthesis of phenylacetamides from unionized substrates forms, however the ionization of reactants carboxy and amino groups in aqueous solutions pushes the equilibrium position to the hydrolysis especially in case of highly basic amines. A linear correlation between the standard Gibbs energy change for amide bond formation from the unionized reagents species and the basicity of amino group was observed: ΔGTo=-3.56pK_amine+7.71(kJ/mol). The established linear free energy relationship (LFER) allows to predict the thermodynamic parameters for direct condensation of phenylacetic acid with any amine of known pK. Condensation of phenylacetic acid and amines with pK value within 1.5-8.5 was shown to be thermodynamically favorable in homogeneous aqueous solution. .

Full text of DOI:10.1016/j.molcatb.2011.08.013

Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Thermodynamics of phenylacetamides synthesis: Linear free energy
relationship with the pK of amine
Dorel T. Gurandaa, Gennadij A. Ushakova,b, Petr G. Yolkin , Vytas K. Svedas
b
a,c,∗
ˇ
a Belozersky Institute of Physicochemical Biology, Lomonosov Moscow State University, Vorob’ev Hills, Moscow 119991, Russia
b Faculty of Chemistry, Lomonosov Moscow State University, Vorob’ev Hills, Moscow 119991, Russia
c Faculty of Bioengineering & Bioinformatics, Lomonosov Moscow State University, Vorob’ev Hills, Moscow 119991, Russia
a r t i c l e i n f o
a b s t r a c t
The effective equilibrium constants KCꢀ expressed through the total concentrations of the reagents for the
synthesis of N-phenylacetyl-derivatives in aqueous medium from phenylacetic acid and various primary
amino compounds have been determined with penicillin acylase as a catalyst. Broad specificity of peni-
cillin acylase (EC 3.5.1.11) to amino components made possible to investigate the acylation of primary
Article history:
Received 28 April 2011
Received in revised form 23 August 2011
Accepted 30 August 2011
Available online 6 September 2011
amines with different structures and physicochemical properties. Analysis of different components of
ꢀ
the effective standard Gibbs energy change ꢀGCo has revealed favorable thermodynamics for the synthe-
Keywords:
sis of phenylacetamides from unionized substrates forms, however the ionization of reactants carboxy
and amino groups in aqueous solutions pushes the equilibrium position to the hydrolysis especially in
case of highly basic amines. A linear correlation between the standard Gibbs energy change for amide
bond formation from the unionized reagents species and the basicity of amino group was observed:
ꢀGTo = −3.56 · pKamine + 7.71 (kJ/mol). The established linear free energy relationship (LFER) allows to
predict the thermodynamic parameters for direct condensation of phenylacetic acid with any amine of
known pK. Condensation of phenylacetic acid and amines with pK value within 1.5–8.5 was shown to be
thermodynamically favorable in homogeneous aqueous solution.
Penicillin acylase/amidase
LFER
Thermodynamics of phenylacetamide
synthesis
Biochemical equilibrium
pH-independent component of standard
Gibbs potential
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
In the early 60s Carpenter had introduced a “nonionized com-
pound convention” by expressing the reactants concentrations
Biochemical equilibrium of enzyme-catalyzed transformations
is one of the major factors which should be considered at ratio-
nal bioprocess design. Nevertheless the state of the art in the area
of thermodynamics of amide and peptide bond formation is rather
limited and controversial [1,2]. The causes of this are mainly related
to experimental problems. Slow attainment of equilibrium, diffi-
culties to determine low equilibrium concentrations of products
at some syntheses as well as side reactions stipulate application
of indirect methods and non-standard experimental conditions.
As a result the experimentally determined equilibrium constants
are effective (apparent), and in many cases reliable only for given
conditions.
in terms of all existing forms including their uncharged species
[3]. This allowed to consider the contribution of the ionization of
reagents and to compare the equilibrium constants of a wide range
of reactions carried out in aqueous solutions at different pH. As
a consequence it was found that the Gibbs energy components
responsible for conversion of the nonionized reactants forms differ
substantially in case of amide and ester hydrolysis, but vary within
a narrow range for peptide and amide hydrolysis [3]. The following
studies showed this convention to be insensitive to the nature of
acyl moiety [4–6], except for formyl amides [7]. Other authors have
revealed that the equilibrium constants referred to the neutral reac-
tants species differ largely, and, moreover, with some exceptions
correlate with the basicity of amino compounds [7–9]. However,
recently most of the earlier published experimental data have been
recalculated in terms of the “non-ionized compound convention”,
and a common, independent on the nature of reagents equilibrium
constant KTo, 103.6 M (ꢀGTo, −20.5 kJ/mol) for hydrolysis of amide
and peptide bonds has been established [10]. This discrepancy is
quite a principal question as there is a need to set up a common
model for quantitative estimation of the equilibrium parameters
for amide bond hydrolysis/synthesis based on the physicochemical
properties of reagents.
The ionization of reagents in aqueous medium affects the equi-
librium of condensation reactions more than other physical factors.
Abbreviations: IS, ionic strength; LFER, linear free energy relationship; PA, peni-
cillin acylase.
∗
Corresponding author at: Belozersky Institute of Physicochemical Biology,
Lomonosov Moscow State University, Vorob’ev Hills, Moscow 119991, Russia.
Tel.: +7 495 9392355; fax: +7 495 9394484.
ˇ
E-mail address: vytas@belozersky.msu.ru (V.K. Svedas).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.08.013

D.T. Guranda et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
49
Scheme 1. Direct condensation of an amine (Nu) with a carboxylic acid (SH) is
characterized by the thermodynamic equilibrium constant KT (I). The reaction (I)
is complicated by ionization of the reagents (I-a and I-b); KSH and KHNu
+
are the
dissociation constants for reacting carboxylic and amino groups.
In this paper the thermodynamics of amide bond formation
between phenylacetic acid and various primary amino compounds
has been studied. The effective equilibrium constants for the syn-
thesis of N-phenylacetyl-derivatives in aqueous medium have been
determined with penicillin acylase EC 3.5.1.11 (PA) as a catalyst.
PA-catalyzed acylations in aqueous medium are widely used for
antibiotic synthesis [11] as well as for chiral resolutions [12–14],
therefore thermodynamic characterization of these biocatalytic
conversions is very important for process optimization.
Fig. 1. Thermodynamics of direct condensation of phenylacetic acid and amines
(Scheme 2, experimental conditions: 298 K, IS 0.1 M. Theoretical curves were calcu-
lated according to Eqs. (4) and (5) using the experimental data from Tables 1 and 2.
2. Equations
The thermodynamic equilibrium constant for direct condensa-
tion of unionized forms of a carboxylic acid and an amine (reaction
I, Scheme 1) is expressed in terms of the activities of reaction com-
ponents:
characterizes the thermodynamic equilibrium of the ꢀreaction for
unionized species; the contribution of ionization ꢀGioon takes into
account the acid–base properties and reflects Gibbs energy change
related to the neutralization of the reagents charged functional
groups at given conditions, i.e. protonation of carboxylic group and
deprotonation of amino group in aqueous solution.
aP · aW
KTo
=
(1)
aSH · aNu
If we take into account the ionization reactions I-a and I-b
(Scheme 1) as well as the water activity (aW) being equal to 1, the
equilibrium constant can be represented as:
KSoH
KHoNu
3. Results and discussion
aP
aS− · aHNu
KTo
=
·
+
+
The effective equilibrium constants KCꢀ for the synthesis of
N-phenylacetyl-derivatives from phenylacetic acid and different
primary amino compounds in aqueous medium have been deter-
mined at 298 K and ionic strength (IS) 0.1 M using PA from
Escherichia coli as a catalyst. Broad specificity of PA to amino com-
ponents made possible to investigate the equilibrium parameters
of acylation of various primary amines (Scheme 2), which differ
largely by structure and basicity of their amino group. The effective
equilibrium constants KCꢀ have been determined by the most rigor-
ous method, when the equilibrium position is attained from both
sides: the side of enzymatic synthesis and the side of enzymatic
hydrolysis (Table 1).
Here we consider the ideal-dilute systems only:
KSoH
KHoNu
1
cP
KTo
=
·
·
(2)
˛
− · ˛HNu
+
cS · cNu
+
S
Hence the experimentally determined and expressed through the
total concentrations of the reagents effective equilibrium constant
makes up
KHoNu
KSoH
+
KCꢀ = KTo
·
· ˛S− · ˛HNu
+
[H+] · [Nu]
[SH]
1
1
KCꢀ = KTo
KCꢀ = KTo
KCꢀ = KTo
·
·
·
·
·
·
[HNu+
1
]
[H+] · [S−
]
1 + ([SH]/[S−]) 1 + ([Nu]/[HNu+])
The values of the thermodynamic equilibrium constant KTo and
the corresponding standard Gibbs energy change ꢀGTo related to
direct condensation of unionized reagents forms for each of the
investigated reactions were calculated on the basis of the experi-
mental data at given pH according to Eqs. (3)–(5) (Table 2). When
considering amines containing additional ionogenic group the cor-
responding microconstants for their zwitterions were taken into
account according to the procedure described earlier (Table 3)
[10,15]. Further, based on the determined intrinsic value ꢀGTo of
each condensationꢀreaction the pH-profile of the effective Gibbs
energy change ꢀGCo has been calculated. According to the obtained
profiles (Fig. 1) the condensation of phenylacetic acid with many of
the amino compounds appears to be thermodynamically unfavor-
able.
1
·
(3)
([S−]/[SH]) + 1 ([HNu+]/[Nu]) + 1
1
1
·
1 + (KSoH/[H+]) 1 + ([H+]/[KHoNu+ ])
1
KCꢀ = KTo
·
(1 + 10(pH−pK )) · (1 + 10(pK
)
−pH)
+
SH
HNu
Assuming designations
ꢀ
ꢁ
ꢀ
ꢀGion = R · T · ln (1 + 10(pH−pK )) · (1 + 10(pK
)
(4)
(5)
−pH)
o
+
SH
HNu
Eq. (3) can be expressed in terms of Gibbs energy change:
ꢀ
ꢀ
ꢀGCo = ꢀGTo + ꢀGioon
This equation combines true thermodynamic and effective equi-
librium parameters of amide bond formation. Evidently, the
pH-independent component of standard Gibbs potential ꢀGTo
It is interesting to consider how much each of the both compo-
ꢀ
nents of ꢀGCo (according to Eq. (5) depends on the nature of amino

50
D.T. Guranda et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
Scheme 2. Penicillin acylase-catalyzed condensation between phenylacetic acid (1) and various amino compounds: p-nitroaniline (2), 4-aminobenzenesulfonic acid (3),
3-aminobenzoic acid (4), O-methylhydroxylamine (5), aniline (6), (S)-phenylalanine amide (7), 2-amino-2-phenylethanol (8), phenylalanine (9), (R)-1-phenylethylamine
(10), (R)-1-(2-naphthyl)ethanamine (11), glycine (12), 3-phenylpropanamine (13).
Table 1
Determination of the equilibrium position of phenylacetamides hydrolysis/synthesis using penicillin acylase as a catalyst (298 K, IS 0.1 M).
Amine
pH
Equilibrium concentration, mM
Method
KCꢀ M−1
Acyl donor
Amine
Amide
(2)
3.6
3.7
98.0
55.0
3.5
5.0
1.43
1.57
Synth.
Hydr.
0.20
0.25
(3)
7.90
17.3
8.10
17.0
0.16
0.81
Synth.
Hydr.
2.5
2.8
(4)
4.7
4.3
5.60
2.90
5.50
2.90
0.062
0.015
Synth.
Hydr.
2.0
1.8
(5)
5.0
35
33.5
35
33.5
7.7
8.25
Synth.
Hydr.
6.3
7.4
(6)
4.3
4.4
3.92
2.20
3.56
2.30
0.052
0.02
Synth.
Hydr.
3.7
3.9
(7)
5.23
5.47
10.9
10.0
9.40
9.20
0.48
0.42
Synth.
Hydr.
4.6
4.6
(8)
6.4
45.6
45.7
42.8
47.1
0.96
1.14
Synth.
Hydr.
0.49
0.53
(9)
7.2
6.67
52.0
43.0
60.0
50.7
1.08
1.15
Synth.
Hydr.
0.35
0.53
(10)
(11)
(12)
(13)
7.0
7.5
59.5
56.2
54.6
55.0
0.62
0.60
Synth.
Hydr.
0.19
0.19
9.58
10.2
9.57
10.9
0.020
0.020
Synth.
Hydr.
0.22
0.18
7.0
7.05
11.4
11.4
11.4
11.4
0.0167
0.0144
Synth.
Hydr.
0.13
0.11
7.1
46.1
4.31
55.6
8.50
0.75
0.010
Synth.
Hydr.
0.29
0.27

D.T. Guranda et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
51
Table 2
Thermodynamics of N-phenylacetamides synthesis by direct condensation at 298 K, IS 0.1 M.
N-phenylacetyl-derivative
Macroscopic pK valuesa
Amineb
pH
Effective (IS 0.1)
Thermodynamic (IS 0)
Contribution
ꢀ
ꢀ
Product
ꢀGCo , kJ/mol
KTo · 10−3 M−1
ꢀGTo , kJ/mol
ꢀGioon, kJ/mol
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
(11)
(12)
(13)
1.0 [23]
–
3.6
3.7
4.2
5.0
4.35
5.35
6.4
7.25
7.0
3.71
−2.43
−2.57
−4.76
−3.31
−3.80
1.76
2.59
4.07
3.99
5.08
0.00027
0.00462
0.00948
0.0574
0.0223
4.10
5.755
34.99
20.87
36.43
3.23
−3.79
−5.57
−10.0
−7.69
−20.6
−21.4
−25.9
−24.4
−26.0
−26.3
−30.9
0.481
1.36
3.00
5.27
4.38
16.8
23.2
28.5
28.7
30.0
31.4
34.2
3.3 [24]; 1.0c
4.4, 3.3 [25]
4.6 [26]
1.0c
3.9c
–
4.6 [27]
–
–
–
7.2 [28]
8.5c
9.3, 2.2 [29]
9.5
9.6
9.8 [30], 2.4 [24]
10.3 [31]
3.5c
–
–
7.6
7.0
7.1
3.7c
–
41.29
265.67
3.27
a
pK values were recalculated for IS 0 M.
First value corresponds to amino group.
b
c
The pK values were calculated using the computer programs developed by ACD Labs (www.acdlabs.com) and SPARC calculator (http://ibmlc2.chem.uga.edu); the pK 4.2
for 1 was from [32].
Table 3
Experimental and estimateda microscopic and macroscopic pK values.
Amine
Microscopic constants
Macroscopic constant
pKoacid
o,m
pK
o,m
base
,m
,m
pK
pKacid
pKbase
pKobase
pKoprod
acid
(3)
(4)
(9)
(12)
1.6
4.0
4.1
4.4 [33]
2.7
3.7
7.4
8.0
1.0
3.1
2.1
2.4
3.3
4.6
9.4
9.8
1.0
3.3 [24]
4.4 [25]
9.3 [29]
9.8 [30]
1.0
3.9
3.5
3.7
3.3 [25]
2.2 [29]
2.4
a
The pK values were calculated using the computer programs developed by ACD Labs (www.acdlabs.com) and SPARC calculator (http://ibmlc2.chem.uga.edu).
compound and what impact it brings to the overall Gibbs energy
change at amide bond formation.
the pKamine, so for highly basic amines its value reaches +25 to
+50 kJ/mol (Fig. 2).
The consideration of the equilibrium position of reaction I for
unionized species (Scheme 1) is reasonable because the kinetically
reactive species in the formation of an amide bond during direct
condensation are the neutral forms of reagents [6,16]. Surprisingly,
the obtained results demonstrated that the thermodynamic param-
eters for the synthesis of N-phenylacetamides (Scheme 2) referred
to unionized species of reaction components linearly depend on
the pK of amino group (Fig. 2):
So, increase of the pK of the amino group of acyl acceptor leads,
on the one hand, to the linear decrease of ꢀGTo favoring synthe-
sis, but, on the other hand, to the exponential growth of ꢀGioon
ꢀGTo = −3.56 · pKamine + 7.71 (kJ/mol)
(6)
This correlation is an example of linear free energy relation-
ship (LFER). Earlier satisfactory linear correlations of the Gibbs
energy of reaction with the basicity of the nucleophile as well
as of the leaving group were disclosed for transacylation reac-
tions [17–19]. A surprising result of the obtained LFER is that the
steric and so-called “␣-effect” of substituents, which are signifi-
cant for kinetic properties [20], apparently are not manifested. So,
O-methoxyamine as well as anilines, amino alcohols, amino acids,
amides and nonfunctionalized amines sufficiently obey this corre-
lation (Fig. 2). The value of thermodynamic Gibbs potential ꢀGTo
for amide bond formation according to Eq. (6) could be within the
range +2 to −35 kJ/mol depending on the acid-base properties of
amines. According to the obtained results, the recently supposed
common value ꢀGTo, −20.5 kJ/mol [10] appear to be rather aver-
aged one and does not reflect the situation in case of amines with
low as well as high pK values.
The ꢀGioon is minimal when pH is equal to half of the sum of
ꢀ
C
pKSH and pKHNu+ , and ꢀGo is equal to ꢀGTo when pKSH > pKHNu+ ,
ꢀ
Fig. 2. Dependence of the effective standard Gibbs energy change ꢀGCo (solid
that is a seldom case in real systems. As a rule pKHNu+ > pKSH
,
line, ꢀ) and of its components: the thermodynamic unionized convention ꢀGTo
(dash-dotted line) and the neutralization contribution ꢀGioon (dash line) for direct
condensation between phenylacetic acid and amino compounds (Scheme 2 on the
pK of their aminogroup at optimal pH, 298 K, IS 0.1 M. The curves were calculated
according to the Eqs. (4)–(6) using experimental data (Tables 1 and 2).
and when the difference between pK values of two functional
groups is increased it leads to higher ꢀGioon and correspondingly to
higher total Gibbs potential. The ꢀGioon for condensation of pheny-
lacetic acid and amines at optimal pH exponentially increases with
ꢀ

52
D.T. Guranda et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
ꢀ
that finally determines the increased total Gibbs potential ꢀGCo
.
column: Chrompack Nucleosil 100 C-18 5 ␮, 150 mm × 4.6 mm,
Phenomenex Luna C-18
␮, 250 mm × 4.6 mm, or Kromasil
5
Evidently, a large difference in the pK values of the carboxylic
and amino groups makes direct condensation thermodynamically
unfavorable. Thus, the equilibrium is shifted to the synthesis for
condensations of carboxylic acid with weakly basic amines [6,9],
and, on the contrary, the condensation of carboxylic acid with
highly basic amines is unfavorable [3,21].
Ethernity C18 5 ␮, 250 mm × 4.6 mm. Mobile phase consisted
of 7 mM phosphate buffer pH 3.0, acetonitrile (26–50%, v/v)
and 0.7 g/l of sodium dodecylsulphate. Retention time was
(in min): (a) for the eluent with 50% CH3CN (Phenomenex
column, flow rate 0.5 ml/min) –phenylacetic acid (9.3), N-
phenylacetyl-O-methylhydroxylamine (7.0); 3-aminobenzoic
acid (7.5), N-phenylacetyl-3-aminobenzoic acid (14.5); pheny-
lalanine (7.8), N-phenylacetyl-phenylalanine (18); aniline
(7.9), N-phenylacetyl-aniline (19); p-nitroaniline (12), N-
phenylacetyl-p-nitroaniline (45); (b) for the eluent with 40%
CH3CN (Kromasil column, flow rate 0.7 ml/min) –phenylalanine
(5.8), phenylacetic acid (7.3); N-phenylacetyl-phenylalanine
(8.7); (c) for the eluent with 20% CH3CN (Phenomenex col-
umn, flow rate 0.5 ml/min) –4-aminosulfonic acid (3.9),
phenylacetic acid (4.7), N-phenylacetyl-4-aminosulfonic acid
(10.4); (d) for the eluent with 26% CH3CN (Chrompack
column, flow rate 1.0 ml/min) –phenylacetic acid (7.8), N-
phenylacetyl-phenylglycinol (13.8), phenylglycinol (18); (e)
for the eluent with 40% CH3CN (Chrompack column, flow rate
1.0 ml/min) –phenylacetamide (2.9), phenylacetic acid (4.4),
3-phenylpropylamine (10), N-phenylacetyl-3-phenylpropylamine
The “real”, practically important value of Gibbs potential change
ꢀ
ꢀGCo can be obtained by summarizing ꢀGTo (pKamine) (Fig. 2, dash-
dotted line) and ꢀGioon (pKamine) (dash line) (see Table 2). The
ꢀ
resulting effective ꢀGCo calculated at optimal pH for amide bond
synthesis, represents a turned bell-shaped dependence upon the
pK of amino group of acyl acceptor with a narrow minimum
equal to −6.3 kJ/mol at pK 4–5 (Fig. 2, solid line). According to
the obtained dependences the condensation of phenylacetic acid
(1) and amines with pK value within range of 1.5–8.5 is ther-
ꢀ
modynamically favorable (ꢀGCo < 0) at 298 K, IS 0.1 M. However
even for the most favorable synthesis a high degree of conver-
sion in a homogeneous reaction mixture could be attained only
by using significant excess of phenylacetic acid. The precipitation
driven synthesis [34,35] can be also an efficient tool to improve
the conversion via direct condensation. Systematic investigation of
the effect of physicochemical characteristics of both carboxylic acid
and amino compound, and the target product on the equilibrium
apparently could allow to estimate thermodynamic parameters of
amide bond synthesis and to find out optimal reaction conditions
on the basis of accessible properties of initial reagents.
(20),
phenylethylamine
1-phenylethylamine
(10.5),
N-phenylacetyl-1-
(16.6),
(19), 1-(2-naphthyl)ethylamine
N-phenylacetyl-1-(2-naphthyl)ethylamine (33.4).
5.3. Solubility measurements
4. Conclusions
The solubility of amides in water was determined by stirring
an excess of amide suspension during 1 h in a thermostatted cell
of a pH-stat (Titrino 718, Metrohm, Switzerland) at correspond-
ing pH, 298 K, 0.1 M KCl. Then suspensions were centrifuged and
supernatants were taken for HPLC-analysis.
Analysis of the different components of standard Gibbs energy
change has revealed thermodynamically favorable synthesis of N-
phenylacetamides from unionized species. However the ionization
of a carboxy group of phenylacetic acid and amino group of amino
compound in aqueous solution seriously influences the overall
Gibbs potential pushing equilibrium in the aqueous reaction sys-
tems to hydrolysis. This effect is especially remarkable in case of
highly basic amines. A linear correlation between the standard
Gibbs energy change for amide bond formation from the unionized
reagents species and the basicity of amino group was observed,
which allows to predict the thermodynamic parameters for direct
condensation of phenylacetic acid with any amine of known pK. In
homogeneous aqueous solution condensation of phenylacetic acid
and amines with pK values within 1.5–8.5 is thermodynamically
favorable and can be used for preparative synthesis using penicillin
acylase as a catalyst.
5.4. Determination of the ionization constants of amino groups
The pK value of amino groups of analyzed amino compounds
was determined by both acidic and alkaline titration of 0.1 M aque-
ous solution of amine (total volume, 10 ml) containing 0.1 M KCl.
The titration was carried out in a thermostatted cell of a pH-stat
(Titrino 718, Metrohm, Switzerland) at 298 K using 2.32 M KOH or
2.61 M HCl solution as a titrant. The pH was changed in the range
of pH 2–12. The titration curves were simulated in MathCad 7Pro.
The ionization constants determined from both acidic and alkaline
titration curves differed from each other no more than by 0.1.
5.5. Determination of the equilibrium position for amines
condensation with phenylacetic acid
5. Materials and methods
5.1. Materials
Determination of the effective equilibrium position at the con-
densation of amines with phenylacetic acid was performed by
monitoring the concentration change of all reaction components in
reaction mixtures of different composition created to achieve equi-
librium from synthesis and hydrolysis side. Each of the reactions
was carried out in a thermostatted cell of a pH-stat (Titrino 718,
Metrohm, Switzerland) at pH-value close to the half of the sum of
the pK values of the carboxylic and amino groups, at 298 K, IS 0.1 M
in aqueous medium (total volume 5 ml) in the presence of 10 ␮M
PA under permanent stirring. The ionic strength (IS) value for each
reaction mixture was adjusted to 0.1 M by 3 M KCl solution. The
enzymatic activity in each reaction mixture was monitored until
the end of conversion in order to be sure that enzyme inactivation
did not take place until equilibrium position was reached. The equi-
librium was assumed to be reached when the concentrations of all
of the reaction components stayed constant during last 30 h of the
Phenylacetic
acid
and
3-phenylpropylamine
were
products of Aldrich; O-methylhydroxylamine-hydrochloride,
(S)-phenylglycinol and (R)-1-(2-naphtyl)ethylamine were prod-
ucts of Fluka; glycine, (S)-phenylalanine and 3-aminobenzoic
acid were products of Acros; (R)-1-phenylethylamine and (S)-
phenylalaninamide were products of Sigma; aniline, p-nitroaniline,
4-aminobenzenesulfonic acid and phenylacetamide were products
of Reakhim. N-phenylacetyl-derivatives of amines were prepared
as described [12]. Wild type penicillin acylase from E. coli was
purified as described earlier [22].
5.2. HPLC analysis
Concentrations of the reactants were determined on a Perkin
Elmer HPLC-system Series 200 at 210 nm using a reversed phase

D.T. Guranda et al. / Journal of Molecular Catalysis B: Enzymatic 74 (2012) 48–53
53
reaction time. In order to exclude errors in determination of equi-
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1313–1320.
librium concentrations two two samples of the liquid phase of each
reaction mixture were prepared by withdrawing the aliquots via
a Chromafil 0.45 ␮m filter (Bester, Amstelveen, The Netherlands)
and were diluted by the eluent in order to obtain samples with the
concentrations of the analyzed components in the range 5 ␮M to
0.5 mM, preferably 0.02–0.1 mM. When the equilibrium concentra-
tion of the amide was more than 10 times lower than that of the
amine and phenylacetic acid the withdrawn samples were ana-
lyzed in the following way: the concentration of the amide was
determined after lower dilution, whereas the concentrations of
amine and acyl donor were determined after higher dilution; in
both cases the analyte concentration was in the range 0.02–0.1 mM.
The obtained samples were subjected to HPLC-analysis to deter-
mine the average concentrations of each reaction component based
on corresponding calibration graphs and to calculate the effec-
tive equilibrium constant values (Table 2). Each of the calibration
graphs was linear through origin in the concentration range 5 ␮M
to 0.5 mM with the correlation coefficient not less than 0.9985.
[7] A.R. Fersht, J. Requena, J. Am. Chem. Soc. 93 (1971) 3499–3504.
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981–984.
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Svedas, Tetrahydron: Asymmetry. 15 (2004) 2901–2906.
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[14] D.T. Guranda, G.A. Ushakov, V.K. Svedas, Acta Naturae. 2 (2010) 94–97.
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Acknowledgements
This work was supported by the Russian Ministry for Sci-
ence and Education (contract # 02.740.11.0866) and the European
Commission (grant agreement n◦ 227279) as a joint EC-Russian
FP7-KBBE-2008-2B project “IRENE”.
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