Cooperativity in benzotriazole-amine complexes: Allosteric tuning of molecular recognition interfaces

Article abstract of DOI:10.1002/poc.1715

Benzotriazole forms with amines stable complexes that in some cases separate spontaneously as crystals from solutions of polar organic solvents. The stoichiometries and the binding constants of these complexes have been determined by ¹H NMR analysis in deuterochloroform. The quantitative analysis of the equilibria involved in the complexation process sheds light on the behaviour of benzotriazoles in solutions and gives an estimate of the influence of allosteric effects when multiple interactions are present. Benzotriazole forms with amines and phenols stable complexes. The binding constants of the complexes have been determined in solution giving an estimate of how allosteric effects can tune hydrogen bond strength. Copyright

Full text of DOI:10.1002/poc.1715

Research Article
Received: 7 January 2010,
Revised: 15 February 2010,
Accepted: 22 February 2010,
Published online in Wiley Online Library: 21 June 2010
(wileyonlinelibrary.com) DOI 10.1002/poc.1715
Cooperativity in benzotriazole–amine
complexes: allosteric tuning of molecular
recognition interfaces
b
c
Cristiano Zontaa,b , Ottorino De Lucchi , Riccardo Motterle
*
and Siro Serafinic
Benzotriazole forms with amines stable complexes that in some cases separate spontaneously as crystals from
solutions of polar organic solvents. The stoichiometries and the binding constants of these complexes have been
determined by 1H NMR analysis in deuterochloroform. The quantitative analysis of the equilibria involved in the
complexation process sheds light on the behaviour of benzotriazoles in solutions and gives an estimate of the
influence of allosteric effects when multiple interactions are present. Copyright ß 2010 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this paper.
Keywords: benzotriazole; cooperative effects; non-covalent interactions; supramolecular chemistry
for biological systems. Studies of this phenomenon often refer
INTRODUCTION
to simple systems investigated with the use of theoretical
calculations or evaluating hydrogen bond geometries in the solid
state.[30–43] A more limited number of studies on the subject have
been carried out in solution. This is due to the technical
difficulties arising for both the multiple binding constants to
measure and the small energies involved. However, by means of
biological and supramolecular systems, it was possible to
experimentally measure the relatively low energetic contribution
of this effect in the overall energy.[17–28,44–52]
Recently, we have been involved in a study that disclosed
new complexes of benzotriazole (BTZ) with amines in organic
solvents.[53] It was observed that mixtures of BTZ, dicyclohex-
ylamine (DCH) and phenol (PHE) in isopropylacetate (IPAC) led to
the formation of an unexpected precipitate which could not be
the DCH ꢀ PHE salt known to be highly soluble. 1H NMR analysis
of the redissolved crystal showed the formation of the
DCH ꢀ BTZ ꢀ PHE complex. In the absence of PHE, mixtures of
BTZ and DCH in IPAC led to the formation of another unexpected
The experimental quantification of the role of intermolecular
interactions has been a subject of intense research.[1–3] The study
of interactions such as hydrogen bonds,[4,5] halogen bonds,[6–8]
cation–p[9–12] and p–p[13–15] interactions, just to cite the most
studied, led to the development of accurate models which have
been implemented in force-fields used in molecular
mechanics.[16] However, in systems that feature multiple inter-
actions, the affinities that one would expect from simply
summing all the single association energies sometimes do not
match the overall observed energy.[17–23] If the sum exceeds the
expected mathematical result, the phenomenon is referred to as
positive cooperativity. Two types of cooperative effects are
described depending upon the implication of conformational or
electronic distribution changes (induction) in the implicated
structure(s).[17–28] While conformation-mediated cooperativity
occurs when the interactions among the different subunits are
modulated by restrictions in translation or rotational mobility,
induction-mediated cooperativity is related to changes in the
electronic structure of the molecule(s) generating the complex.
The latter type is thought to be most important in peptides where
hydrogen bonding is ubiquitously involved.[20–29] In the above-
mentioned systems, the firstly established hydrogen bond
between two amide subunits induces polarization through the
amide bond, favouring the formation of a second hydrogen bond.
In general terms, the first intermolecular interaction is able to
modify the electronic charge distribution of the involved
structures facilitating a second binding event to occur. Overall,
this phenomenon introduces an additional contribution to the
total free energy, which could not be predicted considering the
interactions of the single entities. However, experimental
evidence and rationalization of this effect is difficult to obtain
`
Correspondence to: C. Zonta, Dipartimento di Scienze Chimiche, Universita di
*
Padova, Via Marzolo 1, I-30139, Padova, Italy
E-mail: cristiano.zonta@unipd.it
a
b
c
C. Zonta
Dipartimento di Scienze Chimiche, Universita di Padova, Via Marzolo 1,
I-30139, Padova, Italy
`
C. Zonta, O. De Lucchi
Dipartimento di Chimica, Universita Ca’ Foscari di Venezia, Dorsoduro 2137,
I-30123 Venezia, Italy
`
R. Motterle, S. Serafini
F.I.S. Fabbrica Italiana Sintetici, Viale Milano 26, I-36041 Alte di Montecchio
Maggiore (VI), Italy
J. Phys. Org. Chem. 2011, 24 122–128
Copyright ß 2010 John Wiley & Sons, Ltd.

COOPERATIVITY IN BENZOTRIAZOLE–AMINE COMPLEXES
temperature. At ca. 60 8C, the formation of colourless crystals
began. The precipitate is filtered obtaining the DCH ꢀ PHE ꢀ BTZ
complex (36.2 g, 86% yield).
DCH ꢀ BTZ2 synthesis: DCH (8.76 ml, 0.0441 mol) was added
dropwise into a solution of BTZ (10.0 g, 0.0839 mol) in
isopropylacetate (100 ml). The mixture was shortly heated to
reflux and then slowly cooled and stirred at 20 8C for 2 h. The
precipitate was filtered obtaining the DCH ꢀ BTZ2 complex
(15.7 g, 0.0374 mol, 89.2% yield).
Continuous variation method (Job Plot): The continuous
variation method (Job plot) was performed based on the
differences in the chemical shift of BTZ in the presence of
the three AMM, Ddobs ¼ d ꢁ d0, where d represents the shift in
the presence at different concentrations, and d0 represents the
shift attributed to the free compounds. BTZ solutions and AMM
solutions in CDCl3 were prepared and mixed, keeping the total
molar concentrations of BTZ and AMM in each measurement.
Scheme 1. 2D representation of the network of hydrogen bonds present
in the crystal structures. The full crystallographic analysis is reported
elsewhere[53]
1
The H NMR spectrum of each sample was then recorded, and
these spectra were used to produce a graph of (~d ꢂ x) against x
shown as the Job plot. The maximum, or the minimum, on the
plot corresponds to the stoichiometry of the two species in the
complex.
precipitate. By NMR analysis of the redissolved crystal, this was
shown to be an assemblage of three components: two BTZs and
one DCH. To understand the unexpected formation of the
precipitate, the attention was focused on the capability of BTZ to
act as an amphoteric reagent by virtue of its acidic proton and
basic site.[54] The crystallographic analysis revealed, as a common
feature of all complexes, that the amines are protonated by BTZ
to produce ammonium benzotriazolates (AMMR ꢀ BTZS) and
that the side nitrogen of BTZS opposite to AMMR acts as a
second hydrogen-bond acceptor (Scheme 1).
Four BTZ–amine complexes were characterized by single
crystal X-ray diffraction, displaying interesting trends of donor–
acceptor hydrogen bond distances between corresponding
molecular entities.[53] Cooperativity mediated by induction was
considered to be responsible for the variation of the hydro-
gen-bond distances among the various complexes in the solid
state. These complexes are noteworthy because, being consti-
tuted by several units, they represent proper substrates to
investigate cooperativity also in solution.
NMR dilution and titration experiments
1H NMR Dilution Experiments. A sample of known concentration
(of the order of 100 mM) in CDCl3 was prepared. Aliquots of this
solution were added successively to an NMR tube containing
0.5 ml of CDCl3, the tube was shaken to mix the two solutions
and the 1H NMR spectrum was recorded after each addition. The
changes in chemical shift for each signal were recorded and
analysed.
1H NMR Titration Experiments. A 3.0 ml sample of host of known
concentration (of the order of 10 mM) was prepared in CDCl3. A
portion (0.5 ml) of this solution was removed and a 1H NMR
spectrum was recorded. An accurately weighted sample of the
guest was then dissolved using 2.2 ml of the remaining host
solution. This allows that the concentration of the host remained
constant during the course of the titration. Aliquots of this
solution were added successively to an NMR tube containing the
guest, the tube was shaken to mix the two solutions and the 1H
NMR spectra were recorded after each addition. The changes in
chemical shift for each signal were recorded and analysed.
Determination of stability constants. The procedure summar-
ized here has been described elsewhere.[14,24,55] The program
NMRDil_Dimer, kindly provided by Professor C. A. Hunter
(University of Sheffield), was used to get the dimerization
constant of BTZ and the chemical shifts of BTZ free and BTZ2
dimer. NMRTit_HGHHGG, NMRTit_HGGHHGG or an on-purpose
written routine using Scientist (MicroMath Scientific Software)
were used to fit signals for the 1:1, 1:2 and 1:3 binding isotherms
allowing for dimerization of the guest. These software use a
nonlinear curve fitting procedure to fit the data to the
appropriate binding model to yield the optimum solutions for
the association constants and the bound chemical shifts. In the
models, dimerization is taken into account using the dimerization
constant and limiting chemical shifts obtained from dilution
experiments. Two to six signals were followed in each exp-
eriment, and the value quoted for the association constant is the
weighted average based on the observed changes in chemical
shifts.
Here we report on the study in solution of these complexes
aimed at detecting and confirming their formation in solution
and also aimed at furnishing quantitative information about the
thermodynamic contribution of cooperativity to the binding
1
process. The analysis, conducted by means of H NMR titration
on the above-mentioned BTZ–amines complexes, led to (i) the
disclosure of hitherto unobserved aggregative complexes with
different stoichiometries among the components, (ii) confir-
mation of the formation of complexes in solution previously
observed in the crystals, and a comparison of the stoichiometries
in the two states, (iii) provide new information about the
contribution of cooperativity in the complexes and (iv) provide
evidence of a uniform complexation behaviour between BTZ and
amines (AMM).
EXPERIMENTAL SECTION
DCH ꢀ PHE ꢀ BTZ synthesis: In a solution of phenol PHE (10.0 g,
0.106 mol), dicyclohexylamines DCH (19.3 g, 0.106 mol) in
isopropylacetate (100 ml) BTZ (12.6 g, 0.106 mol) is added. To
the white slurry obtained, isopropylacetate was added (100 ml)
and the solution heated to reflux. After complete solubilization
of the precipitate, the mixture was slowly cooled to room
J. Phys. Org. Chem. 2011, 24 122–128
Copyright ß 2010 John Wiley & Sons, Ltd.
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C. ZONTA ET AL.
Table 1. Screening of different amines with one equivalent of BTZa
Entry
Amine
ml (eq.)a
T (8C)
Yield (g)b
Stoich.c
1
2
3
4
5
6
7
8
Dicyclohexylamine DCH
Diisopropylamine DIP
Diisobutylamine
Pyrrolydine
8.3 (1.0)
6.0 (1.0)
7.7 (1.0)
1.7 (0.5)
3.7 (1.0)
2.7 (0.5)
1.8 (0.5)
1.5 (0.5)
2.4 (0.5)
4.5 (1.0)
1.3 (0.5)
0
20
0
0
0
2.0
4.1
2.5
3.6
6.4
2.4
3.0
2.8
4.6
6.3
4.3
2:1
1:1
2:1
1:1
1:1
1:1
2:1
3:1
1:1
1:1
2:1
Morfoline
(ꢁ)-S-Phenylethylamine
0
Isopropylamine
20
ꢁ10
20
0
Cyclopropylamine CYP
Cyclohexylamine
Benzylamine
9
10
11
Monoethanolamine
20
a Each experiment was performed with a solution of BTZ (5 g, 0.042 mol) in IPAC (50 ml). The solid was recrystallized in 25 ml of
solvent.
b Grams of recrystallized solid obtained.
1
c Stoichiometry BTZ:amine has been obtained measuring H NMR integrals of the redissolved crystals.
complexes with BTZ were maintained in solution (Fig. 1).[60,61]
Solutions for the Job plot experiments (method of continuous
RESULTS AND DISCUSSION
Complex formation
BTZ ¼ 0.1 M and x (mole fraction) values of the two components
A large variety of amines were added to an IPAC solution of BTZ.
variations) were prepared with the restriction that AMM þ
were varied. Despite the fact that no interpolation between the
different complexes could be unravelled, an interesting obser-
vation on the BTZ complexes with DCH and CYP emerged.
Some of them furnished colourless precipitates and the 1H NMR
analysis of the redissolved crystals revealed a wide assortment of
BTZ ꢀ AMM stoichiometries though the 1:1 ratio was often
The analysis of the DCH solution confirms the DCH ꢀ BTZ2
stoichiometry observed in the crystal (Fig. 1a DCH signal), but, at
higher amine concentrations, the plot displays a second maxi-
mum, corresponding to a DCH2 ꢀ BTZ complex. This observation
reveals that, at high DCH concentration, a replacement of the
second BTZ by DCH takes place in consistency with the ability of
DCH to act as a hydrogen bond donor (Scheme 2). A similar
situation is also observed for the CYP ꢀ BTZ complex. The Job plot
analysis is coherent with the presence in solution of a 1:1 complex
(Fig. 1b CYP trace). However, the BTZ signal clearly displays the
presence of the CYP2 ꢀ BTZ and CYP ꢀ BTZ2 complexes. As
shown in Fig. 1c no further equilibria were detected for the DIP
complex. In this case, the plot displays only one maximum in
correspondence to a CYP ꢀ BTZ stoichiometry in line with the
results observed in the solid state.
prevailing (Table 1).[56] Noticeably, aromatic amines did not form
precipitates in solution, possibly because of their low basic
nature. However, basicity seems not to be the only factor
responsible for the observed effects. As example, in the case of
a series of secondary amines, despite their similar pKas, an
unpredictable diversified behaviour was observed. Our attent-
ion was mainly directed towards diisopropylamine (DIP) and
DCH complexes because, besides the similar pKas and steric
hindrance of the amines, the two crystals showed different
stoichiometries, as measured by 1H NMR integration of the
redissolved precipitate.[57,58] Furthermore, the high stoichio-
metric ratio observed in the cyclopropylamine (CYP) complex
was intriguing and deserved a rationale.
X-ray analysis
In summary, the stoichiometric analysis highlights the fact
that in solution both the amines and the BTZ, in the range of
concentration observed, have the possibility to act as a hydrogen
bond donor or acceptor giving rise to complexes displaying at
least 1:1, 2:1, 1:2 BTZ ꢀ AMM stoichiometries. The complexity of
binding possibilities already demonstrated in solution by the
binary systems has prevented us to carry over an analysis of the
ternary mixture BTZ ꢀ DCH ꢀ PHE.
The crystallographic analysis has been treated by us else-
where.[53] The 2D representation of the crystal structures is
summarized in Scheme 1. In addition, the published crystal-
lographic structure of BTZ highlights a complex H-bonding
network, in which each BTZ molecule simultaneously acts both as
H-bond donor and acceptor.[59] Many factors contribute to the
final displacement and stoichiometry in the solid state (e.g.
solubility and packing effects). With the aim of gaining more
insights in these systems, an analysis in solution was started.
Binding constant determination
The knowledge of the multiple equilibria present in solution was
used to set up the complexation experiments. In fact, in order to
promote the formation of the BTZ ꢀ AMM, followed by the
complexation of other BTZ molecules, the amine is kept at
constant concentration around 0.05 M, and a solution of BTZ is
added reaching a final concentration around 0.5 M. To evaluate
Stoichiometry in solution
1
A method of continuous variations’ analysis (Job plot) using H
NMR chemical shifts in deuterochloroform was performed to
verify if the stoichiometries of the DIP, DCH and CYP solid
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J. Phys. Org. Chem. 2011, 24 122–128

COOPERATIVITY IN BENZOTRIAZOLE–AMINE COMPLEXES
Figure 1. 1H NMR Job plots analysis for the complexes in deuterochloroform solutions. The curves are not representative of any interpolation.[60,61] The
maximum, or the minimum, on the plot corresponds to the stoichiometry of the complex. (a) BTZ þ DCH. The DCH plot (left) shows two maxima
corresponding to the formation of a 2:1 complex and 1:2 complex. A minima corresponding to a 1:2 complex is observed in the BTZ trace (right). (b)
BTZ þ CYP. The plot shows a maximum corresponding to a 1:1 complex in the CYP trace (left). 2:1 and 1:2 stoichiometry complexes are represented in
the BTZ trace (right). (c) BTZ þ DIP. The two plots show a maximum, DIP trace (left), and a minimum, BTZ trace (right), corresponding to a 1:1 complex
the energies involved, the complexes have been analysed in
solution by means of NMR binding isotherm determinat-
ion experiments.[24] At the outset, the analysis of BTZ itself in
solution was undertaken, followed by the determination of the
binding constants in the solution of the DIP ꢀ BTZ, DCH ꢀ BTZ,
CYP ꢀ BTZ complexes.
complex. The binding constant value (Kdim ¼ 6.0 Mꢁ1) is in line
with the formation of a single hydrogen bond.[3] The three
binding studies’ experiment were carried out at millimolar
concentrations using the amine as a host. Under these conditions,
the formation of AMM2 ꢀ BTZ complexes is minimized. The
titration data were fitted with a model that allowed for the
dimerization of BTZ. The model used the dimerization constants
predetermined in the previously described experiments, limiting
dimerization-induced changes in chemical shift, but ignored the
possibility of the formation of AMM2 ꢀ BTZ complexes.
1H NMR dilution experiments were used to investigate the
extent of BTZ dimerization.[55] A dilution experiment was per-
formed in deuterochloroform and the observed data were
analysed fitting a binding isotherm for a dimerization process
(Table 2).[62] Besides small variations of the aromatic protons
chemical shifts, the variation of the NH chemical shift pro-
vides evidence of the formation of the hydrogen-bonded
Due to a non-conclusive response of the Job plot analysis,
the data for all titrations were fitted with a 1:1, 1:2 and 1:3
(AMM ꢀ BTZ) binding isotherm.
J. Phys. Org. Chem. 2011, 24 122–128
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View this article online at wileyonlinelibrary.com

C. ZONTA ET AL.
considerations can be outlined for the DCH ꢀ BTZ complex. In this
case, the 1:2 stoichiometry observed in the stoichiometry analysis
was confirmed and it was possible to fit the data from the binding
constant experiment (Table 2). A difference was observed for the
CYP ꢀ BTZ complex. The Job plot determination did not offer
unique results for the stoichiometry in solution. For this reason
1:1, 1:2 and 1:3 stoichiometries were used to fit the data acquired
during the binding constant titration experiment. Noteworthy,
the 1:3 fitting isotherm allowing for the dimerization of BTZ gave
the best fitting for the observed data (Table 2).
All of the complexes display a similar behaviour in solution. The
formation of the first AMM ꢀ BTZ 1:1 complex and the following
1:2 AMM ꢀ BTZ2 complex is accompanied in all the entries by
similar binding constant energies and similar variation in
chemical shifts. The first binding event is related to the formation
of the ion-pair, and the following complexation is associated with
the formation of the ternary complex. As expected, the chemical
shifts’ variations observed are small for all the binding
events.[63,64] As shown by the crystals, the aromatic rings of
BTZ, which are the only functional groups owning a strong
magnetic anisotropy, are distant from the protons of the amine.
The small variations in chemical shifts observed for the second
complexation are in line with a process taking place far from the
amine.
Scheme 2. Species in solution in excess of DCH or in excess of BTZ
The data of the observed binding constants in Table 2 offer us
the possibility to make some considerations on how these systems
are affected by cooperative induction effects. Our measure of the
cooperative effect is related to the dimerization of the BTZ. More in
detail, we are analysing the variation of the dimerization of BTZ
exerted by the presence of the amines. Indeed, the second binding
constant as shown in Table 2 for all the complexes can be seen as
the formation of a BTZ dimer in which one of the two BTZ
molecules is involved in an ‘extremely polarized’ version of an
hydrogen bond, which is sometimes referred to as zwitterionic
hydrogen bond (Scheme 4).[65–67] The second series of binding
constants, corresponding to the binding of BTZ to the
benzotriazolate–ammonium complex, show some variation
between the three titrations, with an average value of K2 ¼
66 ꢃ 8 Mꢁ1. As described previously, this binding event can be
seen as the dimerization of BTZ modulated by the presence of an
amine (Scheme 5). The magnitude of the cooperativity observed
in the ternary complex can thus be determined by comparing this
Scheme 3. Proton labelling scheme for complexes in Table 2
The association constants and the complexation-induced
changes in chemical shift, corresponding to the stoichiometry
which allows the best interpolation, are reported in Table 2.
The DIP ꢀ BTZ complex, which displayed a 1:1 stoichiometry
both in the Job plot and in the crystal, showed at least two
processes taking place in solution during the titration exper-
iment. Indeed, the fitting of the chemical shifts’ variation using a
1:1 stoichiometry, allowing for the dimerization of BTZ, failed to
fit the values observed in the titration experiment. Instead, it was
possible to fit the binding data with a 1:2 stoichiometry taking
into consideration the dimerization of BTZ (Table 2). Similar
Table 2. Association constants and limiting complexation-induced changes in chemical shift in chloroform at 295 Ka
Complex
K (Mꢁ1
)
Ab
0.1
Bb
0.2
cb
db
eb
fb
BTZ R BTZ Ð BTZ2
6.0 ꢃ 0.3
47 ꢃ 4
—
0.4
0.3
0.6
0.2
0.1
0.0
0.1
—
0.3
0.0
—
—
—
—
—
—
0.2
ꢁ0.3
—
—
—
DIP R BTZ Ð DIP ꢀ BTZ
ꢁ0.1
ꢁ0.2
DIP ꢀ BTZ R BTZ Ð DIP ꢀ BTZ2
BTZ R DCHvBTZ ꢀ DCH
63 ꢃ 16
46 ꢃ 2
0.2
0.0
ꢁ0.1
0.2
ꢁ0.2
0.0
0.3
ꢁ0.1
0.4
ꢁ0.2
BTZ ꢀ DCH R BTZ Ð DCH ꢀ BTZ2
CYP R BTZ ÐCYP ꢀ BTZ
60 ꢃ 1
49 ꢃ 3
ꢁ0.1
ꢁ0.1
0.1
0.0
0.2
0.0
CYP ꢀ BTZ R BTZ ÐCYP ꢀ BTZ2
CYP ꢀ BTZ2 R BTZ Ð CYP ꢀ BTZ3
74 ꢃ 8
0.1
0.0
0.1
ꢁ0.1
6.9 ꢃ 1.4
0.0
0.0
a Average values from at least two separate experiments. Titration data for three to six different signals were used to determine the
association constant.
b Limiting complexation-induced changes in chemical shift for the formation of the complexes in ppm. Refer Scheme 3 for the proton
labelling scheme. NH signals were not considered due to proton exchange between BTZ and the amines.
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J. Phys. Org. Chem. 2011, 24 122–128

COOPERATIVITY IN BENZOTRIAZOLE–AMINE COMPLEXES
CONCLUSIONS
The described experiments show that BTZ and amines, due to the
presence of multiple hydrogen bond donor and acceptor sites,
form complexes with various stoichiometries in the chloroform
solution. A tuning of the experimental conditions allowed for the
study of complexes reproducing the situation observed in the
crystals. Indeed, the binding constant values and the chemical
shifts’ variations observed for these processes indicate that
these complexes are similar among them. The first binding
event is related to the formation of the ion-pair and results in
similar values for all of the amines. This is followed by a second
association event. The constants for the formation of the ternary
complexes, which can be regarded as the dimerization of BTZ
modulated by the amine, are significantly larger than that
expected based on the stabilities of the BTZ dimer (the
increase of a factor of 10 is equivalent to around 6 kJ molꢁ1).
This enhanced stability is explained by the polarization of the
hydrogen-bonding groups resulting in increased strengths of the
individual hydrogen-bonding interactions. This stabilization
value is higher than the values previously observed by Hunter
(3 kJ molꢁ1) for hydrogen bond of amides. Indeed, our study deals
with an extreme case of hydrogen bond in which the proton is
transferred to the basic site. This can be seen as an upper limit of
allosteric tuning in hydrogen bond.
Scheme 4. Representation of the evolution of the polarization in the
case of hydrogen-bond of BTZ with an amine
The 1:3 stoichiometry of the CYP ꢀ BTZ3 complex theoretically
offered us the possibility of studying the consequences of
cooperativity in a more remote molecule. In fact, the third
binding constant event is responsible in the crystal structure for a
sensible variation of the bond distances. However, due to
limitations in the method for the determination of the binding
constants, it has not been possible to observe sensible variations
in the binding energies for the quaternary complex of CYP.
Scheme 5. Dimerization of BTZ modulated by the prescence of an
amine
Acknowledgements
This work was co-founded by MIUR (Rome) within the national
PRIN framework.
association constant with the individual bimolecular association
of the BTZ.
K2
66
6
REFERENCES
Cooperativityfactor ¼
¼
¼ 11
(1)
Kdim
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In other words, cooperative interactions in the ternary complex
make it 11 times more stable than expected, based on the
strengths of the interactions previously measured in the BTZ
complex. This stabilization is equivalent to 6 kJ molꢁ1, and we
attribute the additional interaction energy to induction effects
caused by the polarization of the benzotriazolate ion and thus
enhanced hydrogen-bonding interactions in the ternary complex.
In the case of CYP, it is possible to observe the formation of a
quaternary complex. In comparison with the two previous cases,
it is possible to study how another BTZ molecule modifies the
thermodynamics of the whole system. However, as we have
already commented, the first and the second binding constants
do not seem to be largely affected by the presence of the third
BTZ molecules. In the same manner, the third binding event
seems to be slightly affected by the polarization present in the
CYP ꢀ BTZ2.[68]
J. Phys. Org. Chem. 2011, 24 122–128
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J. Phys. Org. Chem. 2011, 24 122–128