The decomposition of triphenylimidazole-para-acetate follows specific base catalysis and can be conveniently followed by fluorescence

Article abstract of DOI:10.1002/bio.3600

The kinetics of the decomposition reaction of 4-(4,5-diphenyl-1H-imidazol-2-yl)phenyl acetate (1) in basic alcoholic media was investigated, using a simple fluorescence (FL) spectrophotometric procedure. The process was conveniently studied using FL, sinc

Full text of DOI:10.1002/bio.3600

Received: 5 November 2018
DOI: 10.1002/bio.3600
Revised: 17 December 2018
Accepted: 30 December 2018
R E S E A R C H A R T I C L E
The decomposition of triphenylimidazole‐para‐acetate follows
specific base catalysis and can be conveniently followed by
fluorescence
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Roberta Albino Reis
Diêgo Ulysses Melo
Andreia Boaro
Ronaldo Barros Orfão Jr.
Fernando Heering Bartoloni
Centro de Ciências Naturais e Humanas,
Abstract
Universidade Federal do ABC, Santo André,
SP, Brazil
The kinetics of the decomposition reaction of 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phe-
nyl acetate (1) in basic alcoholic media was investigated, using a simple fluorescence
(FL) spectrophotometric procedure. The process was conveniently studied using FL,
since the triphenylimidazole‐derived ester 1 and its reaction products (the corre-
sponding phenol 2 and phenolate 2−) are all highly fluorescent (ΦFL > 37%). By
carefully selecting excitation and emission wavelengths, observed rate constants k1
in the order of 10−3 to 10−2 s−1 were obtained from either reactant consumption
(λex = 300 nm, λem = 400 nm) or product formation (λex = 350 nm, λem = 475 nm);
these were shown to be kinetically equivalent. Intensity‐decay time profiles also gave
a residual FL intensity parameter, shown to be associated to the distribution of
produced species 2 and 2−, according to the basicity of the medium. Studying the
reaction in both methanol (MeOH) and isopropanol (iPrOH), upon addition of HO−,
provided evidence that the solvent's conjugate base is the active nucleophilic species.
When different bases were used (tBuO−, HO−, DBU and TEA), bimolecular rate
constants kbim ranging from 4.5 to 6.5 L mol−1 s−1 were obtained, which proved to
be non‐dependent on the base pKaH, suggesting specific base catalysis for the
decomposition of 1 in alcoholic media.
Correspondence
Fernando Heering Bartoloni, Centro de
Ciências Naturais e Humanas, Universidade
Federal do ABC, 09210–580, Santo André, SP,
Brazil.
Email: fernando.bartoloni@ufabc.edu.br
Funding information
Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior, Grant/Award Number: 001;
Fundação de Amparo à Pesquisa do Estado de
São Paulo, Grant/Award Numbers: 2012/
13807‐7, 2014/05813‐2 and 2016/10585‐4
KEYWORDS
imidazole, kineticslight emissionlophine, rate constant
transformation.[25–27] In part, the vast array of application of these
molecules, from FL to CL measurements, depends on the high FL
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1
INTRODUCTION
The triphenylimidazole structural framework has been intensely
studied throughout the years, by many different research groups,
due to its fluorescence (FL)[1–9] and chemiluminescence (CL)[10–22]
properties. The typical direct CL of 2,4,5‐triphenylimidazole (also
known as lophine) with oxygen in an alkaline media, first described
by Radziszewski in 1877,[23] was studied mainly to elucidate its reac-
tion mechanism.[11–13,18–22,24] However, lophine and its derivatives
were also applied in other CL systems, being used as activators
of the peroxyoxalate reaction[10] or enhancers of the luminol
quantum yields (ΦFL > 0.1) presented by them.[1–10,17,24–27]
In 1993, Kuroda et al. reported that triphenylimidazole‐para‐ace-
tate (1, IUPAC name 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenyl acetate),
as well as another three ester analogs based on pertinent fatty acids,
could be applied on the evaluation of lipase activity through FL
measurements.[8] Although ester 1 itself was not the most suitable for
interaction with the lipase, it was primarily used to define the method-
ology for activity determination. Briefly, the method consisted on
hydrolyzing the ester with the enzyme, followed by separation and FL
Luminescence. 2019;1–9.
wileyonlinelibrary.com/journal/bio
© 2019 John Wiley & Sons, Ltd.
1

2
REIS ET AL.
detection of the hydrolysate (i.e. 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phe-
nol, 2) by reversed‐phase high‐performance liquid chromatography.[8]
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2.2
Preparation of ester 1
The ester, 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenyl acetate (1), was
prepared and isolated according to an adapted procedure, based on
de Lucia et al.[33]
A 50 mL single neck round‐bottomed flask
containing 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenol (2, 1.00 mmol,
prepared according to the literature)[10] was mixed with acetic
anhydride (2.50 mmol) in PY (0.3 mL). After overnight stirring at room
temperature, the content was diluted with 20 mL of dichloromethane
and transferred to a separatory funnel. The organic phase was washed
with an aqueous ammonium chloride (NH4Cl) solution (pH 5–6,
3 × 20 mL) to remove the PY, followed by water (3 × 20 mL) and brine
(2 × 20 mL) washings, ultimately colleting the organic phase, dried
under magnesium sulfate (MgSO4). The solution was filtered and the
solvent removed by rotary evaporation, yielding a solid that was
purified by recrystallization from ethanol/hexane (0.25 g, 60% yield).
Elemental analysis calc. for C23H18N2O2: C, 78.0; H, 5.1; N, 7.9%;
found: C, 78.2; H, 5.3; N, 8.1%; IR (solid state) ν (cm−1) 3328, 3057,
1729, 1484, 1228; 1H‐NMR (500 MHz, CDCl3) δ 2.28 (s, 3H, OCH3),
7.08 (m, 2H), 7.24–7.31 (tdd, 6H), 7.49 (m, 4H), 7.88 (m, 2H);
13C‐NMR (125 MHz, CDCl3) δ 21.15, 121.89, 126.85, 127.59,
127.94, 128.51, 145.11, 151.04, 169.55.
Their report[8] was followed by others, where triphenylimidazole‐
derived esters and related compounds, such as acid chlorides and
amides, were applied as FL[5] or CL[24,25] probes, being of special
interest for enzyme activity determination[28] and drug‐binding site
characterization.[29] The fact that 1 and 2 are both markedly fluorescent
make them promising for studying ester decomposition pathways,[30]
especially using spectroscopic methods.[31,32] In order to avoid the
need of using chromatographic separation and quantification tech-
niques, we have explored the FL emission of both reactant and product
to study the decomposition of 1 in basic alcoholic media. Our method is
both useful and convenient, giving relevant aspects of the ester trans-
formation (i.e. rate constants, presence of an intermediate, products
distribution, solvent influence, and nature of base catalysis) directly
from the FL quartz cuvette containing the reaction mixture. Moreover,
our efforts aim at expanding the use of the triphenylimidazole
fluorescent framework in studying reaction mechanisms, particularly,
involving esters hydrolysis.
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2.3
Absorption and emission kinetic assays
UV‐visible absorption spectra were recorded on a Cary 60 UV‐Vis
spectrophotometer (Agilent Technologies, Santa Clara, California, USA)
with multicell holder thermostatized at 25°C by a Varian Cary PCB
1500 system. FL spectra were recorded on a Cary Eclipse fluorescence
spectrophotometer (Agilent Technologies, Santa Clara, California, USA)
with single‐cell holder thermostatized by a Varian Cary PCB 1500 system,
modifying the photomultiplier tube (PMT) voltage, as well as excitation
and emission slits, in order to record the highest possible signal intensity.
For the spectroscopic assays, the following method was applied.
Assays were conducted in a quartz cuvette for emission, already charged
with the ester (3.22 × 10−6 to 3.22 × 10−5 mol L−1 final concentration,
added as a 3.0 × 10−3 mol L−1 stock solution in MeOH). The FL intensity
versus time profile was immediately recorded after base addition (0.64 to
6.40 × 10−3 mol L−1 final concentration, added as a 0.2 mol L−1 stock
solution in MeOH), without causing significant changes to the final
3.0 mL solution volume. With 1 as limiting reactant, kinetic assays were
conducted at pseudo‐first‐order conditions, with the concentration of
base at least 10 times higher. Relative FL quantum yields (ΦFL) were
measured[3,10,34] by integration of the corrected emission spectra, using
anthracene in ethyl acetate as a standard (ΦFL = 0.28),[10] and correcting
for differences in the refractive indices of MeOH and ethyl acetate.
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2
EXPERIMENTAL SECTION
General materials and equipment
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2.1
Methanol (MeOH, Sigma‐Aldrich, St Louis, MO, USA, ACS reagent, ≥
99.5%), isopropanol (iPrOH, Sigma‐Aldrich, ≥ 99%), acetic anhydride
(Sigma‐Aldrich, ≥ 99%), dichloromethane (Sigma‐Aldrich, ≥ 99%, ACS
reagent, 99.5%) potassium tert‐butoxide (tBuOK, Sigma‐Aldrich, ≥
99%), triethylamine (TEA, Sigma‐Aldrich, ≥99.5%), potassium hydrox-
ide (KOH, Synth, analytical grade), 1,8‐diazabicyclo[5.4.0]undec‐7‐ene
(DBU, Sigma‐Aldrich, ≥ 99%), imidazole (IMZ, Sigma‐Aldrich, ≥ 99%),
pyridine (PY, Sigma‐Aldrich, ≥ 99%), 4‐dimethylaminopyridine, (DMAP,
Sigma‐Aldrich, ≥ 99%), and tetrabutylammonium hydroxide (TBAOH,
Sigma‐Aldrich, ≥ 99%) were used as received. Deionized water was
obtained through a Milli‐Q Millipore purifying system (conductivity
18.2 MΩ cm).
Infrared spectrum was recorded on
a Fourier transform
infrared (FT‐IR) Spectrum Two spectrometer (PerkinElmer, Waltham,
Massachusetts, USA) coupled to an UATR (universal attenuated total
reflectance) Two accessory, used for measurements of the sample in
the solid state. The compositions of C, H, N, were obtained in a Flash
EA 1112 (Thermo Scientific, Waltham, Massachusetts, USA), using
benzoic acid as standard. Nuclear magnetic resonance (NMR) spectra
were obtained at 25°C on a 500 MHz spectrometer (Varian, Palo Alto,
California, USA); chemical shifts (δ) are reported in parts per million
(ppm) relative to tetramethylsilane.
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2.4
Kinetic data fitting
Intensity‐decay time profiles were fitted using
a first‐order
exponential decay equation (Equation 1), to determine the observed
rate constant (k1) and the associated emission intensities (I0 and I1).
Equivalent parameters were obtained from intensity‐increase time
profiles with a first‐order exponential saturation equation (Equation 2).

REIS ET AL.
3
Every reported measurement for a kinetic parameter is a mean value
from three independent replicas per experimental condition.
1tÞ
IðtÞ ¼ I0 þ I1eð−k
(1)
(2)
ꢀ
ꢁ
IðtÞ ¼ I0 þ I1 1 − eð−k
1tÞ
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3
RESULTS
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3.1
Spectrophotometric data in MeOH
Both studied 2,4,5‐triphenylimidazole derivatives, 4‐(4,5‐diphenyl‐1H‐
imidazol‐2‐yl)phenyl acetate (1) and its corresponding leaving group
4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenol (2), show UV‐visible absorp-
tion and FL spectra (Supporting Information, Figure S1) with maxima
ca. 300 and 385 nm, respectively, and ΦFL above 37% (Table 1). There
is no substantial change on the absorbance spectra profile going from
phenol 2 to 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenolate (2−) upon the
addition of HO−, with a 15 nm bathochromic shift at the highest
base concentration (Figure 1). Furthermore, there is a larger difference
in FL, with phenolate 2− emitting with a 70 nm red‐shifted signal when
compared to its parent phenol 2 (Figure 1). At [HO−] = 0.64 mmol L−1
there is a major contribution of the phenol species in FL, with a shoul-
der at longer wavelengths due to 2− formation. With 2.63 mmol L−1
the emission signal of 2− becomes prominent, this being the only
detected emitting species at [HO−] = 6.45 mmol L−1 (Figure 1). A
similar trend is observed when 2− is generated by the hydrolysis of 1
in alkaline conditions (Figure 1).
FIGURE 1 Absorption and emission spectra (λex = 296 nm) for 2 (top)
and 1 (bottom) compared to the phenolate 2− (top and bottom), at
different HO− concentrations. In MeOH at 25°C; curves were
The absorption spectra of a solution containing 1, before and after
the addition of excess base (80 eq.), shows a broader signal, compatible
with the generation of 2− (Figure 2, top). Taking the difference between
absorption values (ΔABS) for all wavelengths, it can be observed that
the highest distinction between these species occurs at wavelengths
normalized according to their maxima; [1] and [2] = 3.22 × 10−5 mol L−1
;
using TBAOH as hydroxide source; [HO−] = 0.64 (A), 2.63 (B) and 6.45
(C) mmol L−1
to 1 (Figure S4). Thus, recording of the FL signal at 475 nm guarantees
that there is not a large contribution from the emission of 1 (Figure 2,
Figures S3 and S4).
>
340 nm. When excitation is promoted at 350 nm, only the
bathochromic‐shifted FL of 2− is observed, with maximum emission at
460 nm (Figure 2, bottom). Independently of base concentration and with
excitation at 350 nm, only the emission of phenolate 2− was observed,
and no shoulder on the spectra appeared, which could be attributed
3.2
Effect of HO− concentration on the
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TABLE 1 Maximum absorption wavelength (λabs), molar extinction
coefficient at λabs (ελ), maximum fluorescence wavelength (λFL) and
fluorescence quantum yield (ΦFL), for ester 1, phenol 2 and phenolate
2−a
decomposition of 1
Kinetic assays were performed in MeOH as solvent, following the con-
sumption of ester 1 (λex = 300 nm, λem = 400 nm) and observing
changes in the emission‐decay time profiles fitted by Equation 1, with
increasing concentrations of HO− (Figure 3, Figure S5). Three different
initial concentrations of 1 were used, as 3.30 × 10−6, 1.62 × 10−5 and
3.32 × 10−5 mol L−1, with corresponding data reported in Table 2, and
Supporting Information Tables S1 and S2. Kinetic assays keeping
constant the hydroxide concentration and changing [1] were also per-
formed, for two different [HO−] (2.63 and 12.5 mmol L−1, Table S3).
For both fixed base concentrations, changes on the I0 and I1 parame-
ters were observed, but not on the rate constant k1 (Table S3); as
expected, since 1 acts as limiting reactant.
b
ελ
Compound
λabs (nm)
(L mol−1 cm−1
)
λFL (nm)
ΦFL
c
1
304
294
309
28100 10
33400 300
39000 300
379
393
460
0.37 0.01
0.40 0.02
0.28 0.01
2
2−
aIn methanol (MeOH) at 25°C.
bObtained as the angular coefficient of λabs versus concentration linear
plots (Supporting Information, Figure S2), setting the linear coefficient as
zero; r > 0.9999 in all cases.
cDetermined using anthracene in ethyl acetate as fluorescence standard
(see Experimental section).

4
REIS ET AL.
TABLE 2 Residual intensity (I0), associated intensity change (I1) and
observed rate constant (k1) from the emission time profiles for the
consumption of ester 1a
[HO−] (mmol L−1
)
I0 (a.u.)
I1 (a.u.)
k1 (× 10−2 s−1
)
0.33
0.66
1.33
2.63
3.92
5.20
6.45
360 50
410 60
480 30
520 40
550 10
550 20
480 50
520 30
0.33 0.08
0.50 0.05
0.80 0.10
1.51 0.03
2.30 0.10
2.77 0.03
3.24 0.01
278
3
220 20
150
120
108
99
1
2
1
1
aIn methanol (MeOH) at 25°C, [1]
= 1.64 × ,
10−5 mol L−1
tetrabutylammonium hydroxide (TBAOH) was used as the hydroxide
source, λexc = 300 nm, λem = 400 nm, excitation and emission slits at
2.5 nm, photomultiplier tube (PMT) tension at 650 V.
Moreover, kinetic assays in MeOH were performed following the
consumption of 1 (λex = 300 nm, λem = 400 nm) or formation of
2− (λex = 350 nm, λem = 475 nm). Using another two different initial
concentrations of 1 (0.83 and 3.22 × 10−5 mol L−1), emission time
profiles were recorded for both possibilities (Figure S6). Kinetic time
profiles following ester decomposition and phenolate generation were
mathematically fitted (by Equation 1 and 2, respectively) for the
determination of observed rate constants (k1, Table 3). Similar k1
values were obtained for the same base concentration, using both
methodologies. Moreover, k1 did not seem to depend on the source
of HO−, once that comparable values were obtained with TBAOH
(Table S2) or KOH (Figure S7 and Table S4).
FIGURE 2 Absorption (top) and emission (bottom, λex = 350 nm)
spectra of a MeOH solution containing 1 (8.33 × 10−6 mol L−1),
before (full black line) and 5 min after the addition of HO−
(6.66 × 10−4 mol L−1, dashed line). The dotted line on the left figure
represents the difference between absorption values (ΔABS) for all
wavelengths. TBAOH was used as hydroxide source, but the same
behavior was observed with KOH (Figure S3)
TABLE 3 Observed rate constants (k1) obtained from emission time
profiles following the decomposition of ester 1a or formation of phe-
nolate 2−b
(i) 8.33 × 10−6
[1] (mol L−1
)
[HO−] (mmol L−1
)
k1 (× 10−2 s−1
)
k1 (× 10−2 s−1
)
a
b
0.17
0.33
0.66
1.33
1.96
2.60
3.22
ND
0.090 0.006
0.18 0.01
0.40 0.02
0.80 0.02
1.30 0.01
1.70 0.01
2.20 0.01
0.15 0.01
ND
0.70 0.02
1.10 0.02
1.40 0.01
1.80 0.01
(ii) 3.22 × 10−5
[1] (mol L−1
)
[HO−] (mmol L−1
)
k1 (× 10−2 s−1
)
k1 (× 10−2 s−1
)
a
b
0.33
0.66
1.32
2.63
3.92
5.19
6.45
0.40 0.01
0.50 0.05
0.80 0.08
1.50 0.03
2.20 0.03
2.70 0.03
3.00 0.01
0.10 0.01
0.29 0.01
0.87 0.03
1.70 0.01
2.00 0.01
3.20 0.01
3.50 0.01
Rate constant values for kinetics following the
FIGURE 3 Emission time profiles for the consumption of ester 1
(1.64 × 10−5 mol L−1, λexc = 300 nm, λem = 400 nm) at different HO−
concentrations. In MeOH at 25°C; TBAOH was used as the hydroxide
source; excitation and emission slits at 2.5 nm; PMT tension at 650 V
adecomposition of 1 (λex = 300 nm, λem = 400 nm) or
bformation of 2− (λex = 350 nm, λem = 475 nm). In methanol (MeOH) at
25°C; tetrabutylammonium hydroxide (TBAOH) was used as the hydroxide
source. ND, not determined.

REIS ET AL.
5
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3.3
Effect of reaction medium and catalysts on the
decomposition of 1
FL time profiles for the formation of 2− were recorded using iPrOH as
solvent, with different [HO−] ([1] = 8.33 × 10−6 mol L−1, λex = 350 nm,
λem = 475 nm, Figure S8) and kinetic data obtained using Equation 2
(Table S5). The effect of water addition on the kinetics in MeOH
was also assessed, keeping constant the concentrations of ester and
base ([1] = 8.33 × 10−6 mol L−1, [HO−] = 1.96 × 10−3 mol L−1
,
λex = 350 nm, λem = 475 nm, Table S6).
The formation of phenolate 2− from the base‐induced decomposi-
tion of ester 1 was also studied from the perspective of different basic
catalysts. IMZ, PY, DMAP, TEA, DBU and tBuOK were applied as
bases, in order to compare the obtained results with the ones with
HO− (using TBAOH). For IMZ, PY and DMAP, no changes were
observed in the absorbance and emission spectra, when these were
added to a solution of 1. The generation of excited species at
350 nm permits the exclusive observation of 2− FL above 450 nm
when DBU, KOH and tBuOK are used as bases (Figure S9), similarly
to what has been seen with TBAOH (Figure S4); FL of the phenolate
using TEA, without contribution from the emission of 2, was observed
only at the highest base concentration (0.09 mol L−1). Kinetic assays
were performed for the generation of phenolate 2− (λex = 350 nm,
λem = 475 nm) and observing changes in the emission time profiles
with increasing concentrations of DBU, tBuOK and TEA (Figure 4,
Table S7).
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4
DISCUSSION
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4.1
General aspects regarding ester 1
decomposition
The decomposition of 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐yl)phenyl ace-
tate (1) catalyzed by base generates 4‐(4,5‐diphenyl‐1H‐imidazol‐2‐
yl)phenol (2) as a leaving group or, depending on the basicity of the
medium, the corresponding phenolate 2− (Scheme 1), probably by a
BAC2 mechanism, passing through a tetrahedral intermediate.[35] Even
though the secondary products generated through basic hydrolysis or
solvolysis may differ in structure, still the important aspect is that
the main product of interest is phenol 2, or its conjugated base 2−
(Scheme 1).
FIGURE 4 Emission time profiles for the generation of phenolate 2−
(λexc = 350 nm, λem = 475 nm) at different concentrations of bases:
DBU (top), tBuOK (middle) and TEA (bottom). In MeOH at 25°C; initial
[1] = 3.23 × 10−5 mol L−1; excitation and emission slits at 2.5 nm; PMT
tension at 600 V
Depending on the concentration of base, FL spectra may contain
a large contribution from 2, but 2− is the main fluorescent species at
more basic conditions (Figure 1). When compared to phenol 2, the
emission of phenolate 2− is red‐shifted, suggesting that its excited
state is particularly stabilized by the extended π‐conjugated sys-
tem,[36] thus, reflecting on a smaller energy difference between funda-
mental and excited electronic states. Care must be taken when
analyzing intensity‐normalized spectra, as those from Figure 1 (also
at Figures S4 and S9), since an isosbestic point was, apparently, not
observed in all conditions. Inspection of non‐normalized emission
spectra shows that, for 1 and 2−, clearly there is no isosbestic point,
while one is obtained when the emission signals for 2 and 2− are in
perspective (Figure 5). This is in agreement with the nature of the
SCHEME 1 Simplified representation of the ester 1 hydrolysis/
solvolysis in MeOH, at the presence of a generic base as catalyst,
generating the imidazolyl‐derived leaving group (phenol 2 and/or
phenolate 2−) and an acetate derivative (acetate anion or methyl
acetate)

6
REIS ET AL.
correlation is observed (Figure S10), and values for ksolv and kbim are
taken from the linear and angular coefficients, respectively
(Table S8). The observed rate constants k1 were not expected to
depend upon the [1], since this reactant is not in excess. Nonetheless,
this observation implies that, within the studied concentration
interval of 10−6 to 10−5 mol L−1, there were no additional side
reactions for the consumption of 1 that would have been observed
at higher concentrations.[38,39] Average values 2.6 × 10−3 s−1 and
5.0 L mol−1 s−1 were obtained for ksolv and kbim, respectively, consider-
ing the three different initial concentrations of the ester (Table S8).
These ksolv and kbim values can be used to estimate how much each
pathway contributes to the general consumption of ester. For base
concentrations of 0.66 and 6.45 mmol L−1, it is obtained that
8.0 0.5% and 92 2% of ester 1 is consumed by the bimolecular
process, respectively, while the remaining is decomposed by the
solvent. It becomes clear that the contribution of neutral solvolysis
to the overall decomposition of the limiting reactant cannot be
ignored, particularly at low HO− concentrations.
v ¼ ksolv½1ꢀ þ kbim½1ꢀ½HO−ꢀ
(3a)
(3b)
(3)
v ¼ k1½1ꢀ
k1 ¼ ksolv þ kbim½HO−ꢀ
FIGURE 5 Non‐normalized fluorescence (FL) spectra (λex = 296 nm)
for 1 (top, black line) and 2 (bottom, black line), compared to 2− at
different HO− concentrations (colored lines). In MeOH at
25°C;[1,2] = 3.22 × 10−5 mol L−1; using KOH as hydroxide source;
[HO−] = 0.64 (A), 2.63 (B) and 6.45 (C) mmol L−1
|
4.3
Following reactant consumption or product
formation
With TBAOH as the hydroxide source, time profiles for the reaction
were obtained by following the consumption of 1 (λex = 300 nm,
λem = 400 nm) or the formation of 2− (λex = 350 nm, λem = 475 nm),
for completely independent experiments. The obtained absolute
values for k1 are not significantly different between them, for a given
concentration of 1 and HO− (Table 3). When a single k1 versus [HO−]
linear correlation (Equation 3) is taken including all data sets we have
found ksolv = (1.5 0.3) × 10−3 s−1 and kbim = 4.9 0.1 L mol−1 s−1
(Figure 6). These are in perfect agreement with the previously
obtained average values (Table S8), showing that the studied system
possess good reproducibility. When KOH was used as hydroxide
source (and not TBAOH) in assays where the formation of 2− was
followed (Table S4), the corresponding k1 versus [HO−] linear
correlation gave kbim = 5.9 0.6 L mol−1 s−1. This indicates that the
use of a soft (i.e. tetrabutylammonium) or hard (i.e. K+) counter cation
does not possess influence over the overall ester decomposition
process,[40] which could occur due to negative charge build‐up
attenuation.[41] Regarding the absence of an isosbestic point
(Figure 5)[35,37] and the observation that k1 is independent from the
way of following the conversion of 1 to 2− (Figure 6), it can be
rationalized that the process 1 ⟶ intermediate ⟶ 2− happens
with the first step as rate determining, as a BAC2 ester hydrolysis
mechanism indeed implies.[35]
transformation being studied: 2 is converted to 2− directly due to
proton loss and an isosbestic point should indeed be obtained[35,37]
;
since 1 can be converted to two possible species in equilibrium, 2
and 2−, an isosbestic point is not expected.[35]
|
4.2
Using HO− as the catalyst
The decomposition of ester 1 to phenol 2 (or phenolate 2−, Scheme 1)
was studied with HO− as catalyst, in MeOH (Figure 3). Parameters I0,
I1 and k1 were determined at different concentrations of HO− at
pseudo‐first‐order conditions (Table 2), while keeping 1 as limiting
reactant at constant initial concentrations (see Table 2, Tables S1
and S2). It can be assumed that two processes are taking place for
the decomposition of 1, these being considered in the overall rate
law (Equation 3a). One is an apparent unimolecular process for the
neutral solvolysis of 1, associated to a rate constant ksolv. The second
is a bimolecular reaction between ester and HO−, with an associated
rate constant kbim. Since 1 is the limiting reactant and the system is
in pseudo‐first‐order conditions (Equation 3b), the observed rate con-
stant k1 depends linearly on the concentration of base (Equation 3).
When the obtained k1 values are plotted against the [HO−], a linear

REIS ET AL.
7
taken with excitation occurring close to such wavelength (at 296 nm,
Figure 5) has shown that, at low [HO−], the phenol 2 species is the
one that mainly contributes to the emission signal, when compared
to its conjugate base. For higher [HO−], the FL of the phenolate 2−
becomes visible at longer wavelengths, and the contribution of 2 is
much lower since its concentration is, accordingly, reduced due to
higher base concentration. Reading the emission at 400 nm at low
[HO−] shows a signal corresponding to the phenol 2, while at higher
base concentrations the left‐end of the phenolate 2− emission signal
is being registered (Figure 5). Therefore, the non‐linear dependence
of I0 with [HO−] (Figure S11) furnishes the distribution of species 2
and 2− according to the basicity of the medium. This does not
affect the observed value for k1, since the rates associated with the
equilibrium involving 2 and 2− are likely higher than the one for 1
decomposition, thus, precisely reflecting the change on limiting
reactant concentration over time.
FIGURE 6 Single linear correlation between observed rate constants
(k1) and concentration of HO−, following the kinetics for the
adecomposition of 1 and bformation of 2−, with the ester at an initial
concentration of (i) 8.33 × 10−6 mol L−1 and (ii) 3.22 × 10−5 mol L−1
Data from Table 3; ksolv is the intercept and kbim is the angular
coefficient
.
|
4.5
Using different reaction medium and basic
catalysts
|
The influence of the solvent on the reaction kinetics was probed
using iPrOH instead of MeOH. Values for k1 were obtained
from the FL‐increase time profiles for the formation of 2−
([1] = 8.33 × 10−6 mol L−1, λex = 350 nm, λem = 475 nm) at different
[HO−] (Figure S8). The corresponding k1 versus [HO−] plot can only
be fitted linearly if the intercept is set as zero (Figure S12); if the
linear coefficient is not fixed as zero a negative value is obtained
with a large associated fitting error. Assuming that ksolv = 0 at
Equation 3 is equivalent to infer that the solvent iPrOH is not able
to perform a nucleophilic attack at ester 1 in the absence of base.
Given the increased steric hindrance that an iPrOH molecule has
when compared to MeOH, this may account for the absence of
reaction in neutral conditions.[43–45]
4.4
Additional information from the kinetic
parameters
When FL increase is followed over time, for the generation of pheno-
late 2− (e.g. Figures S6 and S7), the pre‐exponential factor of the
saturation equation (Equation 2) is the associated intensity change,
I1. This represents the total amount of product obtained at the end
of the transformation. For example, when the generation of 2− was
followed (Figure S7), the three highest used [HO−] show similar values
for the final emission intensity (I1 ca. 850 a.u.), indicating that the same
amount of product 2− has been obtained in the end. For the lowest
[HO−], although the registered final emission intensity is much smaller
(ca. 600 a.u.), the fitted pre‐exponential parameter furnishes a value
much closer to 850 a.u., indicating that the same end concentration
of 2− is obtained at a slower rate. Such information assures that an
equivalent amount of limiting reactant has been consumed at different
experimental conditions. From FL‐decay time profiles for the con-
sumption of ester 1 (e.g. Figures S5 and S6), the analogous I1 parame-
ter (Table 2) obtained with Equation 1, also reflects the amount of
consumed limiting reactant. When the concentration of HO− changed
step‐wisely from 0.33 to 6.45 mmol L−1, the obtained I1 values were
constant (Table 2, given the corresponding standard deviation), with
an average value of 500 50 a.u. This indicates that the quantity of
The obtained value of kbim = 4.0
0.1 L mol−1 s−1 in iPrOH
(Figure S12) indicates that the base‐induced decomposition of 1 is not
significantly different from the one in MeOH (ca. 5 to 6 L mol−1 s−1).
In basic conditions, both HO− and the alcohol conjugate base could
be acting as nucleophiles, generated by specific base catalysis (SBC).
The research group of Mayr observed that iPrO− in iPrOH is 25 times
more nucleophilic than MeO− in MeOH towards positively charged
electrophiles,[46] thus, it could be expected that kbim values in iPrOH
should be higher than in MeOH, if the corresponding conjugate bases
were acting as nucleophiles. Nevertheless, the same group reported
that this difference in reactivity is smaller at neutral SN2‐like condi-
tions.[45] Also, the Winsteins–Grunwald's ionizing power of the sol-
vent[45,47,48] strongly influences the reaction rates, and both decrease
going from MeOH to iPrOH.[49] Thus, iPrO− is a stronger nucleophile,
but MeOH has a larger ionizing power, compensating its reduced
nucleophilicity.[46] The acidity of the solvent must also be taken into
account, considering that its conjugate base is generated by
SBC.[46,50,51] Even though the nature of pKa is associated to the acidity
of a species in water, and not in an alcoholic environment as the one
used here, its values can be used to furnish a relative way of compar-
ison. Due to its higher pKa value of 16.5,[52] the equilibrium is less
consumed ester
1 was the same throughout this experiment,
validating the rationalization made on k1 dependence with [HO−]. No
significant reasoning can be performed upon observed rate constants
if different amounts of limiting reactant reacted at different experi-
mental conditions.[39,42]
From FL‐decay time profiles, a residual intensity (I0, Table 2)
was also obtained after fitting with Equation 1. It was observed that
I0 decreases with increasing [HO−], with a non‐linear dependence
(Figure S11). Interpretation for this parameter's implication goes as
follows. Initially, it should be recalled that, to follow 1 consumption,
λex = 300 nm and the emission signal is read at 400 nm. FL spectra

8
REIS ET AL.
shifted to the iPrO− generation in iPrOH, upon addition of HO−, con-
sidering that MeOH is more acidic (pKa = 15.5).[53] This is particularly
valid when small amounts of alcohols (up to 10% in volume) are added
to aqueous basic solutions, with at least a 10‐fold increase in reaction
rates observed with MeOH but not with other alcohols of lower
acidity.[46,50] Moreover, the nucleophilicity of iPrO− and the acidity
of its corresponding alcohol are, relatively to MeO− and MeOH,
two and twenty times lower, respectively, when in water‐free
environments.[52]
difference in pKaH values, Table 4). TEA is known to be a good
nucleophile,[59,60] and maybe the decomposition of ester 1 happens
through a nucleophilic TEA‐catalyzed reaction, which is apparently
not as efficient as the SBC process with tBuO−, HO− or DBU.
|
5
CONCLUSION
We have successfully applied simple FL kinetic measurements, at a
number of different experimental conditions, to assess the mechanism
When water was added to the reaction in MeOH, keeping con-
stant the used [HO−], a 20‐fold rise in [H2O] only increased k1 values
by a factor of 1.25 (Table S6). Thus, 1 preferably reacts with nucleo-
philes derived from the solvent itself, and the importance of the
relative contributions of the processes associated to ksolv and kbim
(Equation 3) just depend on the amount of generated MeO− upon base
addition.[46,50] To gather additional evidence supporting the plausibil-
ity of a SBC process, the formation of phenolate 2− from the decom-
position of ester 1 was studied with six different basic catalysts, in
addition to the already applied HO− species. When PY, IMZ, and
DMAP were used, the formation of phenolate 2− was not observed,
probably because these species are not basic enough (the pKaH values
for PY, IMZ, and DMAP are 5.25,[54] 6.95,[55] and 9.6,[56] respectively).
When stronger bases were used, in this case, TEA, DBU, and
tBuO−, the generation of phenolate 2− was recorded as intensity‐
increase time profiles (Figure 4), from which k1 values were obtained
(Table S7). Linear plots of k1 versus [base] gave values for ksolv and kbim
(Figure S13), which can be put into perspective with the ones for the
reaction with HO− (Table 4). There is absolutely no dependence
between the obtained log(kbim) values for tBuO−, HO− and DBU and
their corresponding pKaH constants (Table 4),[57,58] ruling out the
possibility of general base catalysis for the decomposition of 1 in
MeOH. Noteworthy, when TEA is used as base, the reaction rates
are severely reduced for the formation of 2−. Its kbim is more than
103 times smaller than the average for the three other bases, even
though it is only three times less basic than DBU (given a 0.5
of decomposition for ester
1 in basic alcoholic media. This
triphenylimidazole‐para‐acetate reacts by a classical BAC2 pathway
involving the solvent's conjugate base (i.e. MeO− or iPrO−) as nucleo-
phile, generated through SBC when HO−, tBuO− and DBU were used
as catalysts (kbim = 4.5–6.5 L mol−1 s−1). When TEA was used as base,
the obtained bimolecular rate constant is a 103 times smaller, account-
ing for a change in mechanism of reaction, probably going to a
nucleophilic‐catalyzed one. Also, a neutral solvolysis process can
decompose ester 1, which is particularly significant at low catalyst con-
centrations. Kinetic data was obtained accompanying the consumption
of ester 1 (through intensity‐decay time profiles) or formation of the
product(s) (through intensity‐increase time profiles), and have proved
to be essentially equivalent. Moreover, the associated intensity change
I1 was used to demonstrate that the same amount of limiting reactant
was consumed under different reaction conditions, enabling direct
comparison of rate constants. Also, the residual intensity I0 gives the
relative distribution of species generated as products (phenol 2 and
phenolate 2−), according to the concentration of base. Although it has
been a long time since 1 was applied as substrate for lipase activity
determination, we consider that new relevant data for enzymatic
systems could be obtained using this triphenylimidazole‐derived ester
and the basis of the methodological approach presented here.
ACKNOWLEDGEMENTS
The authors thank the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES, RAR, Support Code 001) and Fundação
de Amparo
à Pesquisa do Estado de São Paulo (FAPESP, AB
TABLE 4 Dissociation constants for the corresponding conjugate
acids (pKaH), and based‐induced bimolecular (kbim) rate constants for
the decomposition of ester 1 catalyzed by different bases, in methanol
(MeOH) at 25°C
2016/10585‐4, RBOJr 2014/05813‐2, FHB 2012/13807‐7) for
financial support, and the Multiuser Central Facilities (UFABC) for
the given experimental resources.
Base
pKaH
kbim (L mol−1 s−1
)
log(kbim)
d
tBuO−
17a
6.5 0.1
0.81 0.01
ORCID
e
HO−
15.7a
5.9 0.5
5.0 0.5
4.9 0.1
0.77 0.04
0.70 0.04
0.69 0.08
Roberta Albino Reis https://orcid.org/0000-0002-9014-2533
Andreia Boaro https://orcid.org/0000-0001-7141-0451
Ronaldo Barros Orfão Jr. https://orcid.org/0000-0002-2521-756X
Diêgo Ulysses Melo https://orcid.org/0000-0002-6860-6258
f
g
d
DBU
TEA
11.5b
4.5 0.2
0.65 0.02
c
11.0
(4.0 0.3) × 10−3 d
−2.40 0.03
Fernando Heering Bartoloni
https://orcid.org/0000-0001-7304-
apKaH values taken from reference 46.
bpKaH value taken from reference 57.
cpKaH value taken from reference 58.
dRate constants ksolv and kbim were determined as the linear and angular
coefficients for k1 versus [base] linear correlations, using data at different
conditions: tBuO−, DBU and TEA, 2− formation (Table S7);
0992
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DU, Bartoloni FH. The decomposition of triphenylimidazole‐
para‐acetate follows specific base catalysis and can be conve-
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