Reaction of poly-L-lysine with aryl acetates and aryl methyl carbonates. A mechanistic study

Article abstract of DOI:10.1002/poc.1285

The pH profile (log k vs. pH) of the reactions of poly-L-lysine (PL) with a series of aryl acetates and aryl methyl carbonates in aqueous solution show the same conformational changes as those determined by potentiometric titrations. When PL is a random coil, the most probable mechanism for the reactions studied is through the formation of a tetrahedral intermediate and its breakdown to products as the rate-determining step. The tetrahedral intermediate is stabilized by a hydrogen bond interaction between the nitro groups in the substrate and the NH group of the principal chain or some NH₂ groups of the lateral chains. Copyright

Full text of DOI:10.1002/poc.1285

Research Article
Received: 10 September 2007,
Revised: 5 October 2007,
Accepted: 9 October 2007,
Published online in Wiley InterScience: 4 December 2007
(www.interscience.wiley.com) DOI 10.1002/poc.1285
Reaction of poly-L-lysine with aryl
acetates and aryl methyl carbonates.
A mechanistic study
Enrique A. Castroa, Gerardo R. Echevarriab, Alejandra Opazoa,
a
a
*
´
Paz S. Robert and Jose G. Santos
The pH profile (log k vs. pH) of the reactions of poly-L-lysine (PL) with a series of aryl acetates and aryl methyl
carbonates in aqueous solution show the same conformational changes as those determined by potentiometric
titrations. When PL is a random coil, the most probable mechanism for the reactions studied is through the formation
of a tetrahedral intermediate and its breakdown to products as the rate-determining step. The tetrahedral
intermediate is stabilized by a hydrogen bond interaction between the nitro groups in the substrate and the NH
group of the principal chain or some NH2 groups of the lateral chains. Copyright ß 2007 John Wiley & Sons, Ltd.
Supplementary electronic material for this paper is available in Wiley InterScience at http://www.mrw.interscience.wiley.com/
suppmat/0894-3230/suppmat/
Keywords: kinetics; mechanism; aminolysis; poly-L-lysine; Brønsted plots
A few works about the kinetics of the aminolysis of esters with
INTRODUCTION
polymeric amines have been described. At this respect, it is
noteworthy the systematic study of Arcelli[9] on the aminolysis
reactions of aryl acetates with PEI, both in the absence[9a] and
presence[9b] of electrolytes, where different mechanisms were
observed in both cases. A work on the reaction of PAA with a
series of aryl acetates and aryl methyl carbonates shows a
concerted aminolysis mechanism.[10]
Taking into account that polyamine basicity changes with the
dissociation degree it is possible to obtain a basicity range of the
nucleophilic groups without a change in the polymer structure.
This allows the use of Brønsted-type plots for the mechanistic
studies of reactions with polyelectrolytes.[9,10]
It is known that poly-L-lysine (PL) can exist in three different
conformations: coil, a-helix and b-fold. In aqueous solution, at
room temperature and acid or neutral pH, this polymer adopts
a disordered statistic coil, whereas in basic media, when the
lateral amino groups are not charged, it adopts an ordered
a-helix conformation, stabilized by intramolecular hydrogen
bonds.[11–17]
The aminolysis mechanisms of aryl acetates,[18] alkyl aryl
carbonates[19–21] and diaryl carbonates[20b,22,23] with monomeric
amines have been well established. Some of these reactions are
The reactions of some substrates with macromolecules can be
important models to understand the behaviour of complex
enzymatic systems. In general, a polymer can concentrate or
disregard a reactive molecule in its environment and if some
catalytically active function is incorporated they can be able to
react with substrates, showing a kinetic and mechanistic effect on
the reaction.[1–3]
The hydrolysis of esters in the presence of polyamines is
a model of enzymatic reactions, such as in those described
for quimiotripsine.[4] In this model, the macromolecule that
represents the enzyme with the active site is a polyelectrolyte,
carrying out the hydrolysis according to a Michaelis–Menten
mechanism.[4b]
In general, the kinetic and mechanistic studies of these
reactions suggest pre-equilibrium, acylation and deacylation
steps;[4b] nevertheless, the studies have been restricted mainly to
the deacylation or hydrolysis step because the acylation or
aminolysis steps are fast.[4a]
Some works with amino-functionalized polyelectrolytes[5,6]
(with imidazole, benzylimidazole and pyridines among others)
show an increase in the catalytic activity relative to the mono-
meric counterpart. In electrolytes such as poly(ethylenimine)[7]
(PEI) and poly(allylamine)[5] (PAA), modified with hydrophobic
groups (alkyl or benzyl), the results show that a hydrophobic
environment increases the esters hydrolysis. In all these cases
the bond formation between the substrate and the macromol-
ecule is influenced by ionic, hydrogen bond and hydrophobic
interactions.[5b]
´
Facultad de Qu´ımica, Pontificia Universidad Catolica de Chile, Casilla 360,
*
Santiago 6094411, Chile.
E-mail: jgsantos@uc.cl
a
E. A. Castro, A. Opazo, P. S. Robert, J. G. Santos
Facultad de Qu´ımica, Pontificia Universidad Catolica de Chile, Casilla 360,
Santiago 6094411, Chile
´
A first approach toward the understanding of the reactions
involving acetyl groups transfer was obtained by the kinetic and
mechanistic studies of the aminolysis of esters using monomeric
amines.[8]
b
G. R. Echevarria
Departamento de Qu´ımica F´ısica, Universidad de Alcala, 28871 Alcala de
Henares, Spain
´
´
J. Phys. Org. Chem. 2008, 21 62–67
Copyright ß 2007 John Wiley & Sons, Ltd.

A MECHANISTIC STUDY OF THE REACTION OF POLY-L-LYSINE
known to proceed through a zwitterionic tetrahedral intermedi-
ate (Tꢀ) and others by a concerted pathway (a single step).
Structure–reactivity correlations, such as the Brønsted-type
relationship, have helped to clarify these mechanisms.[18–23]
In this work, we report a kinetic study of the reactions
in aqueous solution of 4-nitrophenyl acetate (NPA), 2,4-
dinitrophenyl acetate (DNPA), 2,4,6-trinitrophenyl acetate (TNPA),
4-nitrophenyl methyl carbonate (NPC), 2,4-dinitrophenyl methyl
carbonate (DNPC) and 2,4,6-trinitrophenyl methyl carbonate
(TNPC) with PL of two different polymerization degrees
(Scheme 1). This, together with the investigation on the
dissociation behaviour of PL, will allow the determination of
the mechanism and the assessment of the kinetic effects of (i) the
neighbourhood (micro-environment surrounding) of the reaction
site (ii) the PL polymerization degree and (iii) the leaving and
non-leaving groups of the substrate.
Since the a values for PL for the two polymerization degrees
were closely similar, in the concentration range stated at each pH,
these values were averaged as am. Table 1 (in Supplementary
Material) shows the pKapp and am values of PL at different pH and
at various polymer concentration ranges.
Kinetics
PL solutions were prepared freshly in aqueous solutions of the
corresponding external buffer 0.01 M, ionic strength 0.1 M (KCl)
and the desired pH. The reactions were initiated by addition of
30 mL of a stock solution of the corresponding substrate into
2.5 mL of the polymer solutions thermostated at 25.0 ꢀ 0.18C.
The initial concentration of the substrates was 5 ꢁ 10ꢂ5 M. The
kinetic measurements were carried out spectrophotometrically
by following the production of 4-nitrophenoxide (400 nm),
2,4-dinitrophenoxide (355 nm) and 2,4,6-trinitrophenoxide
(355 nm) anions, by means of a Hewlett-Packard 8453 diode
array spectrophotometer. Under amine excess, pseudo-first-order
rate coefficients (kobs) were found for all reactions. The plots of
kobs versus total amine concentration were linear, with kNobs as
slope. Phosphate, borate and carbonate buffers (0.01 M) were
used at appropriate pH ranges.
EXPERIMENTAL
Materials
Poly-L-lysine hydrobromide, from Sigma, of polymerization
degrees 402 (PL-402) and 277 (PL-277), was used without further
purification. NPA, DNPA and TNPA were synthesized as described
previously.[24] NPC, DNPC and TNPC were synthesized by a
standard procedure.[25] All other reagents were of analytical
grade.
The experimental conditions and the values of kobs and kNobs
for the reactions of PL with TNPA, DNPA, NPA, TNPC, DNPC and
NPC are summarized in Tables 2–5 (in Supplementary Material).
Product studies
pKapp determination
The presence of 4-nitrophenoxide, 2,4-dinitrophenoxide and
2,4,6-trinitrophenoxide anions as products of the reactions was
determined spectrophotometrically by comparison of the UV–Vis
spectra at the end of the reactions with those of authentic
samples under the same experimental conditions.
The potentiometric titration of PL was carried out in aqueous
solution, at 25.0 ꢀ 0.18C, under nitrogen, by means of a Radio-
meter autotitrator equipped with a PHM-62 pH-meter, an ABU-11
autoburette, a TTT-60 titrator, an REA-160 recorder, a TTA-60
thermostatic support and a G-2040 glass and a K-4040 calomel
electrodes. In each experiment 10 mL of the polymer solution at
different concentrations (5.1 ꢁ 10ꢂ4–4.24 ꢁ 10ꢂ3 M, expressed as
amine groups), and an ionic strength of 0.1 M, maintained with
KCl, was titrated with NaOH (0.01–0.1 M). The polymer concen-
tration range in the potentiometric titration was similar to that in
the kinetic measurements. The apparent dissociation constant
(pKapp) was calculated according to Eqn (1),[26] where a is the
dissociation degree. The latter is the quotient between the free
amine groups and the total PL concentration (expressed as
monomeric units); the concentration of free amino groups was
determined from the added NaOH volume at each pH.[26,27]
RESULTS AND DISCUSSION
Potentiometric titrations
Due to the complexity of macromolecular systems and to the
great number of ionizing groups the potentiometric behaviour of
polyamines is quite different than those for monomeric amines.
The ionization of a group is affected by the proximity of other
ionizing groups and by the presence of hydrophobic moieties
and hydrogen bonds in others.[27]
A difference with monomeric amines is that, in poly-
amines[27–29] and polyaminoacids[11,12] the dissociation equi-
librium of the ionizing groups in a polyacid (including the
conjugate acid of a polybase) is cooperative and complex due to
the strong electrostatic interactions between the charged groups
in the chain.
pKapp ¼ pH þ logð1 ꢂ aÞ=a
(1)
Figures 1 and 2 show the titration curves of PL-402 and PL-277,
as pKapp versus a plots. It can be observed that pKapp is a function
of both concentration and dissociation degree. The dependence
of pKapp on the polymer concentration has been described for
PEI,[28] PAA[10] and polyacids.[27] On the other hand, the
comparison between both figures shows an independence of
the polymerization degree.
For low dissociation degrees (a < 0.2) Figs. 1 and 2 show
that pKapp increases as a increases; for a values in the 0.2–0.6
range a relatively constant pKapp is observed and for a > 0.6 a
decrease in pKapp is observed as a increases. This can be
Scheme 1. Structures of the polymer (PL) and the substrates used in this
work
J. Phys. Org. Chem. 2008, 21 62–67
Copyright ß 2007 John Wiley & Sons, Ltd.
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E. A. CASTRO ET AL.
Eqn (2), where k0 and kNobs are the rate coefficients for
spontaneous hydrolysis and aminolysis of the substrates,
respectively. These results suggest that there is no substrate–
polyelectrolyte association because the presence of this
association shows limiting plots, such as those described for
PEI in the absence of KCl.[9a]
kobs ¼ k0 þ kNobs½Nꢃtot
ð2Þ
No differences in kNobs values were observed when reactions
were carried out with PL of different polymerization degrees
(refer to Tables 2–5 in Supplementary Material).
For most of the reactions studied k0 is negligible in Eqn (2),
except for those at higher pH media, where the two terms are
important.
The nucleophilic rate constant (kN) is obtained by the quotient
between the kNobs values (Tables 2–5 in Supplementary Material)
and the corresponding am values (of Table 1 in Supplementary
Material). Tables 1 and 2 show the values of pKapp (of Table 1 in
Supplementary Material) and kN for the reactions of PL with NPA,
DNPA, TNPA, NPC, DNPC and TNPC at the different pH values.
The nucleophilic rate constants for the reaction of PL with
carbonates are smaller than those for the corresponding acetates
at the same pH values. This result can be explained through the
larger electron-donating effect exerted by MeO in the carbonates
relative to Me in the acetates, rendering the former carbonyl
carbon atom less positively charged and, therefore, less prone to
amine attack. This is in accordance with what has been found for
the reactions with PAA[10] and monomeric amines.[18f,g,19a]
Figure 1. Titration curves of PL-402 in aqueous solution at 258C and
ionic strength 0.1 M (KCl). 0.51 ꢁ 10ꢂ3 M (*), 1.69 ꢁ 10ꢂ3 M (~),
3.05 ꢁ 10ꢂ3 M (5), 4.24 ꢁ 10ꢂ3 M (&). The solid line is from Reference [12]
explained by the presence of a transition or conformational
change. The regions described correspond to random coil, the
random coil to a-helix transition and a-helix, respectively, in
accordance with literature.[11,12] The transition zone has been
reported in the a 0.2–0.5 range for PL (MW 110 000) in KBr 1 M[11]
and a 0.4–0.75 range for PL (PD 450) 1.91–2.87 ꢁ 10ꢂ2 M in KCl
0.1 M.[12] The differences in the assignment of zones are related to
the experimental conditions, such as pH, solvent, temperature,
polymer concentration and type and concentration of added
salt.[29]
pH profile
The results are also in accordance with those reported by
circular dichroism,[13,16] optical rotatory dispersion
[15]
´
and
The behaviour of log kN versus pH for the PL reactions (both
fractions) with the series of acetates and methyl carbonates are
shown in Figs. 3 and 4, respectively.
ultrasonic studies,[17] which describe random coil and transition
in the 8–10 pH range.
It can be observed in Figs. 3 and 4 that for pH values lower than
ꢄ9 the log kN values increase with pH, probably due to the
increase in free amine fraction. In this region, the presence of the
protonated e-amine lateral groups leads to the formation of a
statistical coil. The pH region ranged 9–10 is characterized by a
Kinetic study
A linear dependence of the pseudo-first-order rate constant (kobs
on PL concentration at each pH was observed, according to
)
Table 1. Nucleophilic rate constants (kN) for the reactions of
NPA, DNPA, TNPA, NPC, DNPC and TNPC with PL-402 at
different pH values, in aqueous solution, 25.08C, ionic strength
0.1 M
kN (sꢂ1 Mꢂ1
)
pH
pKapp NPA DNPA TNPA NPC DNPC
TNPC
7.0
7.5
8.0
8.5
9.0
9.5
8.39 0.179
2.05 6.67 0.069
3.73 12.4 0.110
7.95 25.5 0.675
0.309
0.610
2.29
3.67
6.77
8.01
5.34
10.9
21.5
30.0
1.39
2.37
6.14
12.6
18.9
14.1
18.0
48.7
8.71
9.05
9.46
9.63
9.63
—
—
1.72 18.0
1.38 24.4
1.50
54.7 0.859
54.5 1.11
77.7 0.85
49.1 0.87
57.9 1.48
145 1.86
—
10.0 9.60
1.64 14.3
2.15 19.3
4.60 40.0
7.60 72.0
10.5
11.0
11.5
—
—
—
Figure 2. Titration curves of PL-277 in aqueous solution at 258C and
ionic strength 0.1 M (KCl). 0.51 ꢁ 10ꢂ3 M (*), 1.69 ꢁ 10ꢂ3 M (~),
3.05 ꢁ 10ꢂ3 M (5), 4.24 ꢁ 10ꢂ3 M(&)
152
230
—
3.50
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Copyright ß 2007 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2008, 21 62–67

A MECHANISTIC STUDY OF THE REACTION OF POLY-L-LYSINE
Table 2. Nucleophilic rate constants (kN) for the reactions of
NPA, DNPA, TNPA, NPC, DNPC and TNPC with PL-277 at
different pH values, in aqueous solution, 25.08C, ionic strength
0.1 M
kN (sꢂ1 Mꢂ1
)
pH
pKapp NPA DNPA TNPA NPC DNPC
TNPC
7.0
7.5
8.0
8.5
9.0
9.5
8.39 0.205
8.71 0.424
9.05 0.651
9.46 2.19
9.63 1.66
9.63 1.85
2.05
—
—
18.7
18.9
16.5
13.8
13.3
—
7.17 0.077
0.132
31.3 0.675
55.4 0.891 3.20
48.8 1.01
—
—
—
1.28
1.73
7.71
9.38
15.0
—
25.3
56.8
76.0
—
6.36
7.48
5.20
8.53
—
52.9
35.1
51.8
—
—
10.0 9.60 1.46
1.19
1.12
2.80
3.80
Figure 4. Plots of log kN versus pH for the reactions of TNPC (~), DNPC
(*) and NPC (*) with PL, in aqueous solution, at 258C and ionic strength
0.1 M (KCl)
10.5
11.0
11.5
—
—
—
1.53
—
6.70
63.0
369
—
210
small decrease in log kN, due to the presence of a less reactive
specie.[11,12,14–17] Above pH ꢄ 10 the log kN values increase with
pH, probably due to the fact that the polymer adopts its a-helix
conformation.[11,12,14–17]
Mechanism
The Brønsted-type plots for the reactions studied are linear in the
7.0–9.0 pH range (Figs. 5 and 6). In this pH range, the PL is a
statistical coil, in accordance with the titration study (refer to
above). The slope (bnuc) values found are 1.1, 1.0 and 0.9 for NPC,
DNPC and TNPC, respectively, and 0.9, 0.9 and 0.8 for NPA, DNPA
and TNPA, respectively; these values are subject to an error of
ꢀ0.1.
These pH profiles are in agreement with those described in our
studies for the Schiff base formation between 50 pyridoxal
phosphate and PLs of several polymerization degrees.[30]
The leaving group effect on kN for the series of acetates and
methyl carbonates studied is in accordance with those discussed
for the reactions of the same substrates with PAA[10] and
monomeric amines,[18c,e,f,g,19b,20b] which show a kN increase with
the decrease in basicity of the leaving group. On the other hand,
the change of CH3 by OCH3 as the non-leaving group reduces the
kN value due to the electron donation of the latter that renders a
less reactive carbonyl group in the same way as observed for the
reactions with PAA,[10] pyridines[18c,d,f,19a,b] and secondary
alicyclic amines.[18e,g,20a]
Considering the bnuc values found (0.8–1.1), in the same range
of those obtained for the reactions of the same and similar
substrates with monomeric amines[18–23] and with that found for
the reaction of NPA with PEI[9b]; the most probable mechanism for
the reactions studied in this work is that shown in Scheme 2
(shown for a lysine group), where a tetrahedral intermediate (Tꢀ)
is formed and its breakdown to products is the rate-determining
step, when PL is a random coil.
Figure 3. Plots of log kN versus pH for the reactions of TNPA (~), DNPA
(*) and NPA (*) with PL, in aqueous solution, at 258C and ionic strength
0.1 M (KCl)
Figure 5. Brønsted-type plots (log kN vs. pKapp) for the reactions of TNPC
(~), DNPC (*) and NPC (*) with PL, in aqueous solution, at 258C and
ionic strength 0.1 M (KCl)
J. Phys. Org. Chem. 2008, 21 62–67
Copyright ß 2007 John Wiley & Sons, Ltd.
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E. A. CASTRO ET AL.
formation as rate limiting), with an expected b value range of
0.1–0.3.[18–23]
The absence of a biphasic plot for the dinitro derivatives
and the b values found for the trinitro and dinitro deriva-
tives suggest that for all these reactions breakdown of Tꢀ to
products is the rate-limiting step. This means that for these
reactions kꢂ1 > k2 in Scheme 1. In order to explain these results,
we propose that the intermediate Tꢀ could be stabilized by a
hydrogen bond interaction between the nitro groups in the
substrate and the NH group of the main chain or some NH2
groups of the lateral chains. Due to this interaction, the rate
constants k and k2 would be smaller than those expected for
ꢂ1
the reactions of these substrates with a monomeric amine.
Nevertheless, this interaction should diminish the rate of
expulsion from Tꢀ of the leaving group (k2) more than that of
the amine (kꢂ1), so that for the reactions of these substrates with
Figure 6. Brønsted-type plots (log kN vs. pKapp) for the reactions of TNPA
(~), DNPA (*) and NPA (*) with PL, in aqueous solution, at 258C and
ionic strength 0.1 M (KCl)
PL, k is still larger than k2.
ꢂ1
CONCLUSIONS
Stepwise aminolyses of aryl acetates (with monomeric amines)
show values of pK0a (pKa at the curvature centre of the
Brønsted-type plot) about 4–5 pKa units greater than the pKa
of the conjugate acid of the leaving group.[18] However, when the
amine group is in the backbone of the polymer this difference is
about 3–3.5 pKa units.[9b] Considering the corresponding pKa of
the leaving groups (around 7, 4 and 0.3 for 4-nitro-, 2,4-dinitro-
and 2,4,6-trinitro-phenols, respectively),[31] we should expect pK0a
values around 4.3–5.3 for TNPA, 8.0–9.0 for DNPA and 11–12 for
NPA; similar ranges can be expected for methyl carbonates.
The expected pK0a value for the nitro derivatives is larger than
the largest pKa of the measured range and, therefore, the b values
correspond to b2 (Tꢀ breakdown to products as rate limiting).
Nevertheless, if the mechanism is that of Scheme 1, for the pKapp
studied, the Brønsted plot for the dinitro derivatives should be
biphasic and the b for the trinitro derivatives should be b1 (Tꢀ
(i) The mechanism of the aminolysis (PL) of the series of aryl
acetates and aryl methyl carbonates studied is stepwise, with the
formation of a tetrahedral intermediate and its breakdown to
products as the rate-determining step, when PL is a random coil.
(ii) The presence of the nitro groups in the substrates stabilize the
tetrahedral intermediate by a hydrogen bond interaction
between the nitro groups in the substrate with the NH group
of the principal chain or some NH2 groups of the lateral chains.
Acknowledgements
We thank MECESUP of Chile (projects PUC-0004 and RED QUI-
MICA UCH-01), FONDECYT of Chile (project 1020538) and DGI of
Spain (project CTQ2006-07643) for financial support.
Scheme 2. Mechanism for the reactions of PL with aryl acetates and aryl methyl carbonates
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Copyright ß 2007 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2008, 21 62–67

A MECHANISTIC STUDY OF THE REACTION OF POLY-L-LYSINE
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