The Importance of Methyl Positioning and Tautomeric Equilibria for Imidazole Nucleophilicity

Article abstract of DOI:10.1002/chem.201602573

Imidazole (IMZ) rings catalyze many biological dephosphorylation processes. The methyl positioning effect on IMZs reactivity has long intrigued scientists and its full understanding comprises a promising tool for designing highly efficient IMZ-based catalysts. We evaluated all monosubstituted methylimidazoles (xMEI) in the reaction with diethyl 2,4-dinitrophenyl phosphate by kinetics studies, NMR analysis and DFT calculations. All xMEI showed remarkable rate enhancements, up to 1.9×10⁵fold, compared with spontaneous hydrolysis. Unexpectedly, the electron-donating methyl group acts to decrease the reactivity of the xMEI compared to IMZ, except for 4(5)methylimidazole, (4(5)MEI). This behavior was attributed to both electronic and steric effects. Moreover, reaction intermediates were monitored by NMR and surprisingly, the reactivity of the two different 4(5)MEI tautomers was distinguished.

Full text of DOI:10.1002/chem.201602573

DOI: 10.1002/chem.201602573
Full Paper
&
Tautomer Reactivity
The Importance of Methyl Positioning and Tautomeric Equilibria
for Imidazole Nucleophilicity
Renan B. Campos,[a, b, c] Leociley R. A. Menezes,[a] Andersson Barison,[a] Dean J. Tantillo,[b] and
Elisa S. Orth*[a]
Abstract: Imidazole (IMZ) rings catalyze many biological de-
phosphorylation processes. The methyl positioning effect on
IMZs reactivity has long intrigued scientists and its full un-
derstanding comprises a promising tool for designing highly
efficient IMZ-based catalysts. We evaluated all monosubsti-
tuted methylimidazoles (xMEI) in the reaction with diethyl
2,4-dinitrophenyl phosphate by kinetics studies, NMR analy-
sis and DFT calculations. All xMEI showed remarkable rate
enhancements, up to 1.9ꢀ105 fold, compared with sponta-
neous hydrolysis. Unexpectedly, the electron-donating
methyl group acts to decrease the reactivity of the xMEI
compared to IMZ, except for 4(5)methylimidazole, (4(5)MEI).
This behavior was attributed to both electronic and steric ef-
fects. Moreover, reaction intermediates were monitored by
NMR and surprisingly, the reactivity of the two different
4(5)MEI tautomers was distinguished.
Introduction
a promising starting scaffold for developing catalysts, since its
pKa of approximately 7 guarantees acidic, basic and nucleophil-
ic properties. Present in the histidine residue, IMZ plays essen-
tial roles in biological reactions involving phosphoryl group
transfer, such as the cleavage of RNA and signaling processes,
with the formation of phosphohistidines.[4] Interestingly, the
biological versatility and effectiveness of this group has justi-
fied pharmacological activities of some IMZ derivatives, yield-
ing the development of many drugs including antifungals, an-
tibiotics, antiseptics and even antineoplastics.[5] Nonetheless,
since the catalytic activity of IMZ in organisms is not fully un-
derstood, mutagenesis and other collateral effects may be
linked with the extended use of medicines from this class of
compounds.[6] Therefore, studies aimed at the elucidation of
detailed reaction mechanisms involving IMZ derivatives and
phosphate esters may provide not just support for organism-
drug interactions research, but also for the development of
novel and efficient catalysts for detoxification.
Phosphate esters are known for their high PꢀO bond stability
(e. g., resistance to hydrolysis), hence they exert multiple vital
biological roles.[1] In contrast, phosphotriesters are typically
toxic, and in environmental conditions leads to bioaccumula-
tion, which in turn is a serious concern in the development of
lethal substances like pesticides, insecticides and even chemi-
cal weapons.[2] Worldwide, the growth of food production has
been followed by a disproportionate increase in the use of
agrochemicals. For example, Brazil has become the greatest
consumer of pesticides in the world, and alarmingly, uses
some products that have been already banned elsewhere.[3]
Therefore, the search of new and efficient catalysts that target
the detoxification of organophosphorus pesticides (OPP) with
the potential to destroy large unused stocks (mostly prohibit-
ed) and also for monitoring abusive amounts of OPP in the en-
vironment and food, is assuredly important.
Consequently, mechanistic investigations on dephosphoryla-
tion have attracted increasing interest, especially those involv-
ing the design of optimum molecular architectures for catalyz-
ing dephosphorylation. In this context, imidazole (IMZ) is
Among IMZ derivatives, the monosubstituted methylimida-
zoles (xMEI) stand out since, despite their simple structures,
their reactivity has not been concisely correlated with methyl
positioning for various reactions (e.g. dephosphorylation, de-
acylation).[7] Indeed, the effects of methyl substitution have in-
trigued scientists for several years. Scheme 1 presents all xMEI
derivatives: 1-methylimidazole (1 MEI), 4(5)-methylimidazole
(4(5)MEI) and 2-methylimidazole (2 MEI). The known tautomeric
equilibrium in aqueous solution for IMZ is also present in
2 MEI and 4(5)MEI. However, due to symmetry, although for
IMZ and 2 MEI this effect results in an equilibrium between
two species with the same structure, for 4(5)MEI it involves
two different species: 4 MEI and 5 MEI.[8] 4(5)MEI tautomers
should have different properties, such as reactivity, and deter-
mining these is focus of our study.
[a] R. B. Campos, L. R. A. Menezes, Prof. Dr. A. Barison, Prof. Dr. E. S. Orth
Department of Chemistry, Universidade Federal do Paranꢀ
CP 19081, CEP 81531-990; Curitiba-PR (Brasil)
E-mail: elisaorth@ufpr.br
[b] R. B. Campos, Prof. Dr. D. J. Tantillo
Department of Chemistry, University of California-Davis
Davis, California 95616 (USA)
[c] R. B. Campos
current address: Departamento AcadÞmico de Quꢁmica e Biologia
Universidade Tecnolꢂgica Federal do Paranꢀ
Curitiba, PR, ZIP 81280-340 (Brazil)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201602573.
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Scheme 2. Proposed pathways for the reactions of DEDNPP with xMEI stud-
ied herein.
Scheme 1. xMEI structures and tautomeric equilibria.
Table 1. Kinetic parameters for the reaction of xMEI and IMZ with
DEDNPP.[a]
It is important to highlight that the mechanistic understand-
ing for dephosphorylation reactions with IMZ derivatives is
sometimes limited by the fact that the PꢀN bond is highly un-
stable,[4a] making intermediates difficult to monitor. Conse-
quently, the combination of experimental and computational
studies has allowed new insights into these processes.[7i,9]
Herein, we present mechanistic insight into the reactions of
1 MEI, 2 MEI and 4(5)MEI with diethyl 2,4-dinitrophenylphos-
phate (DEDNPP) in aqueous solution, based on various experi-
mental techniques: kinetics, 1D/2D nuclear magnetic reso-
nance (1H, 13C, 31P NMR) and theoretical calculations. Surpris-
ingly, phosphorylated intermediates for the 4(5)MEI tautomers
were distinguished by NMR. Furthermore, a comprehensive
theoretical study was carried out that sheds light on the mech-
anisms involved and the main factors that govern the relative
reactivity of xMEI species.
4(5)MEI
IMZ[4a]
1 MEI
8.0ꢀ10ꢀ6
2 MEI
k0 [sꢀ1
]
kOH [mꢀ1 sꢀ1
]
0.25
kN [10ꢀ3 mꢀ1 sꢀ1
pKa[11]
]
27.7 (0.29)
7.69
17.7 (0.21)
6.95
12.8 (0.09)
7.21
4.4 (0.08)
7.85
[a] Experimental data, equations and plots are given in the Supporting In-
formation. Errors are given in round parentheses.
respectively (Scheme 2). The term cxMEI refers to the molar frac-
tion of the neutral reactive species of each xMEI. Kinetic pa-
rameters are presented in Table 1.
kobs ¼ k0 þ kOH½OHꢀꢁ þ kN½xMEIꢁcxMEI
ð1Þ
The results show rate enhancements of 1.9ꢀ105, 8.9ꢀ104
and 3.0ꢀ104 for 4(5)MEI, 1 MEI and 2 MEI, respectively, com-
pared to the spontaneous hydrolysis of the triester. Interesting-
ly, this reactivity trend is not directly related to the xMEI pKa
values (for protonated species), shown in Table 1. In fact, 2 MEI
shows the lowest reaction rate compared to all derivatives, in
contrast to the highest pKa value, which would normally sug-
gest a more reactive nucleophile.[10] Finally, the most effective
catalyst for the cleavage of DEDNPP is 4(5)MEI, showing experi-
mental kN values 1.6-, 2.2- and 6.3-fold higher than IMZ, 1 MEI
and 2 MEI, respectively.
Results and Discussion
Kinetics
All xMEI showed remarkable catalytic activity in the dephos-
phorylation of DEDNPP, as depicted in the pH rate profiles
shown in Figure 1. The expected dependence of kobs with pH is
observed, confirming the reactivity of the neutral nucleophilic
species (plateau around the pKa). The fitting of experimental
results, given by the solid lines in Figure 1, is based on Equa-
tion 1, in which the terms correspond to the contribution of
hydrolysis (k0), alkaline hydrolysis (kOH) and xMEI reactions (kN),
To help assess the reaction pathway and to provide compari-
sons for calculations, thermodynamic parameters were ob-
tained (Table 2) through Eyring plots (given in the Supporting
Information). The largely negative values of entropies of activa-
tion obtained indicate that the reaction mechanism for all
cases is in fact most likely a bimolecular nucleophilic displace-
ment pathway.[4a]
Table 2. Activation parameters for the reactions of xMEI and IMZ (pH 8.5)
with DEDNPP.[a]
Cmpd.
DH° [kcal.molꢀ1
]
DS° [cal.Kꢀ1 molꢀ1
]
DG° [kcal.molꢀ1
]
4(5)MEI
2 MEI
1 MEI[12]
IMZ[12]
7.4
9.0
9.6
7.7
ꢀ42.7
ꢀ41.2
ꢀ37.0
ꢀ42.6
20.1
21.2
20.6
20.4
Figure 1. pH rate profiles for the reactions of xMEI and IMZ with DEDNPP,
0.5m at 258C (left axis). The spontaneous hydrolysis of DEDNPP (right axis)
and the IMZ profile are shown for comparisons.[4a] The fitting (solid lines) are
based on Equation 1.
[a] Experimental data, equations and plots are given in the Supporting In-
formation.
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Scheme 3. Mechanistic proposal for the dephosphorylation of DEDNPP in
which the intermediates labels (1 to 5-MIPI) are based on methyl positioning
in xMEI. The second step illustrates that xMEI are regenerated after the
xMIPI hydrolysis.
The rate enhancements and activation parameters observed
for the methyl nucleophiles are typical of nucleophilic attack
and, based on previous studies,[4a,12] we propose the mecha-
nism presented in Scheme 3, in which the nitrogen lone pair
of xMEI attacks the phosphorous atom of DEDNPP and displa-
ces 2,4-dinitrophenolate (DNP, pKa =4.1)[11] through a concerted
mechanism, leading to a phosphorylated intermediate that
should easily hydrolyze, regenerating the nucleophile.[13] It is
important to note that UV/Vis kinetic study follows the appear-
ance of DNP, hence, kN in Table 1, refers solely to the first step
of the mechanism. The decomposition of the phosphorylated
intermediates is expected to be fast, since they are knowingly
unstable.[4b] As will be discussed in following sections, NMR
analysis combined with DFT calculations support this overall
hypothesis and also provide important information on methyl
positioning effects on the reaction rates, not straightforward
by UV/Vis studies.
Figure 2. 31P{1H} NMR spectra for the reaction of DEDNPP (7.5ꢀ10ꢀ3 m) with
2 MEI (0.3m), pH 8.5, 258C.
mation, which are consistent with Scheme 3. Firstly, for the re-
action of DEDNPP with 2 MEI, 31P{1H} NMR spectra showed the
peaks at ꢀ7.5 and 0.8 ppm assigned to DEDNPP and diethyl-
phosphate (DEP), respectively, and an additional signal at
1
ꢀ4.9 ppm (Figure 2 and Scheme 3). Moreover, H NMR spectra
showed an additional set of signals, including a spin system at
7.32 and 7.02 ppm, as well as a signal at 2.53 ppm from
a methyl group, that were different than those observed for
2 MEI (Table 3). An additional spin system also was observed
1
for ethoxy groups at 3.92 and 1.25 ppm in the H NMR spec-
trum, different than initially observed for DEDNPP (Table 3). All
these hydrogen showed 1H–31P long-range NMR correlation
with the same phosphorous species at ꢀ4.9 ppm, supporting
the structure of the intermediate 2-MIPI (Scheme 3). Addition-
ally, a second aromatic spin system emerged in the 1H NMR
spectra during the course of reaction, with no 1H–31P long-
range NMR correlation, that were assigned to the phenolic
product DNP (Scheme 3, Table 3).
The 31P{1H} NMR spectra sequentially acquired directly from
the reaction medium with 2 MEI, Figure 2, showed the signal
at ꢀ7.5 ppm from DEDNPP decreasing and the simultaneous
increase of the signal at 0.8 ppm from DEP, according to reac-
Mechanistic investigation by NMR
For a more detailed mechanistic understanding, reactions of
DEDNPP with 2 MEI and 4(5)MEI (NMR data for IMZ and 1 MEI
has been reported previously)[4a,12] were investigated through
1H and 31P{1H} NMR spectroscopy. Indeed, a thorough analysis
of 1D NMR spectra and 2D NMR correlation maps lead to the
complete 1H, 13C, and 31P NMR chemical shift assignments
given in Table 3, Figures 2 and 3 and in the Supporting Infor-
Table 3. NMR data (600 MHz, H2O/D2O at pH 8.5 and 258C)[a] for the species detected in the reaction of DEDNPP with 2 MEI and 4(5)MEI.[b]
Cmpd.
dH
dP
2 MEI
7.09 (s, 2H, Ar), 2.45 (s, 3H, CH3)
4(5)MEI
DEDNPP
2-MIPI
4-MIPI
5-MIPI
DNP
7.80 (s, 1H, Ar), 6.87 (q, 1H, Ar), 2.23 (d, 3H, CH3)
8.99 (dd, 1H, Ar), 8.63 (dd, 1H, Ar), 7.80 (dd, 1H, Ar), 4.38 (dq, 4H, CH2), 1.39 (td, 6H, CH3)
7.32 (d, 1H, Ar), 7.02 (d, 1H, Ar), 4.27 (dq, 4H, CH2), 2.53 (s, 3H, CH3), 1.35 (td, 6H, CH3)
7.83 (Ar)[c], 6.88 (Ar)[c], 4.25 (dq, 4H, CH2), 2.33 (d, 3H, CH3), 1.33 (td, 6H, CH3)
7.91 (s, 1H, Ar), 7.05 (q, 1H, Ar), 4.22 (dq, 4H, CH2), 2.20 (d, 3H, CH3), 1.32 (td, 6H, CH3)
8.85 (dd, 1H, Ar), 8.09 (dd, 1H, Ar), 6.74 (dd, 1H, Ar)
ꢀ7.5
ꢀ4.9
ꢀ5.2
ꢀ5.6
DEP
3.92 (dq, 4H, CH2), 1.25 (td, 6H, CH3)
0.8
[a] Additional data given in the Supporting Information. [b] The assignments for DEDNPP, DNP and DEP are in agreement with data found in the literatur-
e.[4a,12] [c] Overlapping signal.
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tion progress. As expected, the signal at ꢀ4.9 ppm decreases
and disappears as the phosphorylated intermediate is hydro-
lyzed.
Surprisingly, for the reaction of DEDNPP with 4(5)MEI,
31P{1H} NMR spectra showed two new signals (ꢀ5.2 and
ꢀ5.6 ppm) in addition to those for DEDNPP and DEP (Figure 3
and Table 2). This indicates two phosphorylated intermediates,
which were attributed to separate reactions of the tautomers
1
4 MEI and 5 MEI (Scheme 3). The H NMR spectra also showed
Figure 4. Relative concentration vs time kinetic profiles for the species in-
volved in reactions of (A) 2 MEI and (B) 4(5)MEI with DEDNPP, detected by
31P NMR at 258C, according to Scheme 3. Residual plots are given in the
Supporting Information.
centration versus time with equations for consecutive first-
order reactions,[4a] and the results are summarized in Table 4.
Analysis of the data in Table 4 shows a reactivity trend for
the nucleophiles in agreement with the UV/Vis kinetic study
previously shown, hence 4(5)MEI>2 MEI. Thus, the reactivity of
4(5)MEI tautomers could be evaluated solely by 31P NMR kinet-
ic. 5 MEI shows higher reactivity than 4 MEI (31-fold) that
cannot be associated simply to the tautomer population distri-
bution. Indeed, the Boltzmann distribution from previously re-
ported computational results for 4(5)MEI, indicates a 40:60
ratio of 5 MEI/4 MEI (Scheme 1);[8] thus, the 4(5)MEI reaction
fits with a Curtin–Hammett scheme. In fact, 4 MEI showed only
Figure 3. 31P{1H} NMR spectra for the reaction of DEDNPP (7.5ꢀ10ꢀ3 m) with
4(5)MEI (0.04m), pH 8.5, 258C.
two additional sets of signals. The first set of hydrogen nuclei
at 7.91, 7.05 and 2.20 ppm from the IMZ moiety and those at
1
4.22 and 1.32 ppm from ethoxy moieties showed H–31P long-
range NMR correlation with the same phosphorous species at
ꢀ5.6 ppm. The second set of hydrogen nuclei at 7.83, 6.88 and
2.33 ppm from the IMZ moiety and those at 4.25 and
1.33 ppm from ethoxy moieties showed 1H–31P long-range
NMR correlation with the same phosphorous species at
ꢀ5.2 ppm. In addition, DNP signals again were observed in the
1H NMR spectra.
Consecutive 31P{1H} NMR spectra for the reaction of DEDNPP
with 4(5)MEI evidenced the consumption of DEDNPP and for-
mation of DEP (Figure 3), as observed previously. Simultane-
ously, the signals at ꢀ5.2 and ꢀ5.6 ppm from 4-MIPI and 5-
MIPI showed the typical increase followed by a decrease, with
the latter showing higher stability (i.e. a longer lifetime). Dis-
tinction of these phosphorylated isomers is a benchmark for
understanding overall reactivity of IMZ derivatives and their
stability was accessed by further kinetic analysis (vide infra).
Seeking a deeper understanding, kinetic profiles from the
31P NMR data were obtained and are shown in Figure 4. Rate
constants relative to the formation and hydrolysis of the inter-
mediates were calculated by fitting the profiles of relative con-
Table 4. Rate constants of formation (k1), cleavage (k2) and half-life of the
intermediates involved in reaction of 2 MEI and 4(5)MEI with DEDNPP, ob-
tained by fitting data in Figure 4.[a]
Intermediate k1 [mꢀ1 sꢀ1
]
k2 [sꢀ1
]
Half-life
[s]
15.2ꢀ10ꢀ3
6.33ꢀ10ꢀ4
1500
7380
7980
(ꢂ2.58ꢀ10ꢀ3
)
)
(ꢂ1.11ꢀ10ꢀ4
)
4-MIPI
2-MIPI
5-MIPI
8.17ꢀ10ꢀ3
(ꢂ4.08ꢀ10ꢀ4
9.35ꢀ10ꢀ5
(ꢂ4.77ꢀ10ꢀ6
)
8.63ꢀ10ꢀ5
(ꢂ1.25ꢀ10ꢀ5
0.47 (ꢂ0.127)
)
[a] Fitting was based on consecutive first-order reaction equations.[4a]
Errors are given in round parentheses.
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a marginally higher reactivity than 2 MEI (Table 4), which in
turn presents the lowest nucleophilicity toward the triester.
The relative stability of the phosphorylated intermediates
(Figure 4 and k2 in Table 4) can be attributed to their leaving
group (xMEI) basicity, however the pKa of protonated 4(5)MEI
(7.69)[11] is weighted by the contribution of both tautomers
5 MEI and 4 MEI and a direct analysis is not straightforward.
Nonetheless, the gas phase proton affinity is a reasonable ap-
proach to estimate basicity of a compound and, in fact, it is
predicted (vide infra) to be slightly (0.6 kcalmolꢀ1) higher for
5 MEI than for 4 MEI.[14] Although this difference is small, it is in
agreement with the lower lifetime of 4-MIPI relative to 5-MIPI
since it is expected that 4 MEI is a better leaving group. It is
important to note the solvent composition of the reaction
medium for NMR kinetic studies: 46 and 10% (in volume) of
acetonitrile and D2O, different from UV/Vis study. This large
amount of acetonitrile should affect more significantly k2, slow-
ing down this process.[15] Although it is important to assess the
half-life of the intermediates, the analysis herein are valid
solely under these conditions of solvents. Overall, the most im-
portant observation obtained from the NMR study is to con-
firm the intermediate structure. The kinetic profiles are shown
to complement the study but caution should be taken when
analyzing it. Firstly, the solvent composition is important and
secondly the apparent build-up of the intermediates is in
terms of relative concentration of the species detected by
31P NMR, hence, only phosphorylated species. If we consider
the DNP product, which was not detected by 31P NMR, this
built-up would not be as much (lower concentration).
pected that the presence of this substituent acts to enhance
the nucleophilicity of a molecule due to hyperconjugative don-
ation. The reactivity of 2 MEI towards DEDNPP is lower than
IMZ, 1 MEI and 4(5)MEI, indicating that a steric effect involving
the methyl group close to the nucleophilic nitrogen atom
likely inhibits nucleophilic attack on phosphorus. In rationaliz-
ing the higher reactivity of 4(5)MEI, it is important to note that,
in contrast to 1 MEI, the positioning of the methyl group in
2 MEI and 4(5)MEI allows its tautomerism, thus, reaction can
occur on both nitrogen atoms. For 2 MEI, these tautomers are
identical, but for 4(5)MEI, NMR results herein clearly show dif-
ferent reactivity for the 4 MEI and 5 MEI tautomers. Indeed, the
reactivities of 2 MEI and 4 MEI are similar (k1 in Table 4), which
is expected due to their similar steric hindrance—both have
a methyl group a to the nitrogen atom. Hence, the 5 MEI tau-
tomer is responsible for the increased effectiveness of 4(5)MEI
in the dephosphorylation reaction. DFT calculations were car-
ried out to explore these effects.
Firstly, we carried out calculations with the M06-2X[16] and
B3LYP[17] functionals combined with the 6-31+G(d,p) and 6-
311+ +G(d,p) basis sets to ascertain which method/basis set
provides DG° values in accord with the experimentally ob-
served reactivity trend. Unexpectedly, B3LYP calculations with
the smaller basis set best reproduced the experimental reactiv-
ity trend, 5 MEI>IMZ>1 MEI>4 MEIꢃ2 MEI, although barrier
heights were overestimated with this level of theory (Figure 5).
The same functional with a larger basis set 6-311+ +G(d,p)
predicted reversed barriers for 1 MEI and IMZ, whereas M06-
2X/6-31+G(d,p) predicted that IMZ is the best nucleophile in
the dephosphorylation reaction. When M06-2X is combined
with a larger basis set, 2 MEI actually is predicted to be the
more reactive derivative (which is the least reactive according
to experimental data; Figure 1), but furnishes a correct trend
for the other nucleophiles.[18] Thus, all further discussions are
focused on B3LYP/6-31+G(d,p) results. Relevant transition
state structures (TSS) for the xMEI reactions, obtained with
B3LYP/6-31+G(d,p), are shown in Figure 6, and activation bar-
riers and selected geometrical parameters are presented in
Table 5, Table 6 and Figure 7.
It is noteworthy that we already reported similar NMR kinet-
ics for the reactions of DEDNPP with IMZ and 1 MEI,[4a,12] for
which an analogous behavior was observed involving the for-
mation of phosphorylated intermediates. As pointed out previ-
ously, detection and most importantly understanding the reac-
tivity of IMZ-based phosphorylated intermediates is fundamen-
tal for biological purposes[4a] and we hope that the full set
NMR assignments and kinetic for the xMEI-based intermediates
presented furnishes an important database for future studies.
Undoubtedly, mechanistic investigation by NMR analysis was
crucial for confirming the proposed catalyzed nucleophilic re-
action between xMEI and DEDNPP (Scheme 3), and comple-
ments UV/Vis kinetic data. Additionally, discerning reactivity of
the tautomers 4 MEI and 5 MEI was possible, which is interest-
ing from the mechanistic point of view and will hopefully in-
spire further detailed studies involving nucleophiles with tau-
tomers. Nonetheless, in order to fully understand the methyl
positioning effect for IMZ derivatives, as well as to probe
whether the substitution reactions occur via concerted or step-
wise mechanisms, DFT calculations were pursued.
Although most geometric parameters of the TSSs do not
vary much, the C6-N5-P-O2 dihedral angle varies significantly
and tracks with the reaction barriers. For the less reactive nu-
cleophiles (2 MEI and 4 MEI) these dihedral angles are close to
Table 5. Activation barriers (experimental and theoretical (DG°/kcal.
molꢀ1), HOMO energy (nucleophiles) and imaginary frequencies of the
TSS for the reactions of xMEI and IMZ with DEDNPP.[a]
[c]
Cmpd.
Experimental
B3LYP
6-31+G(d,p)
TS imaginary
frequency (cmꢀ1
EHOMO
)
(Hartrees)
2 MEI
1 MEI
IMZ
5 MEI
4 MEI
21.3
20.6
26.4
24.2
24.0
23.3
26.3
ꢀ135.071
ꢀ138.317
ꢀ143.307
ꢀ138.358
ꢀ134.034
ꢀ0.2276
ꢀ0.2349
ꢀ0.2377
ꢀ0.2264
ꢀ0.2275
DFT calculations for understanding the reaction mechanism
and the methyl positioning effect
20.4
20.1[b]
20.1[b]
Our experimental results show that the effect of a methyl
group at most positions of the IMZ ring decreases its nucleo-
philicity towards DEDNPP, when compared to unsubstituted
IMZ. However, in the absence of steric effects, it is generally ex-
[a] Experimental data with xMEI 0.5m, pH 8.5, 258C. [b] 4(5)MEI. [c] Ob-
tained performing B3LYP/6-31+G(d,p) optimization.
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Figure 5. Experimental and calculated barriers DG° (kcalmolꢀ1) for the reaction of xMEI and IMZ with DEDNPP. The experimental data were obtained from
Eyring plots as given at the Supporting Information.
Table 6. Structural parameters[a,b] of the species involved in the reaction
of xMEI and IMZ with DEDNPP as shown in Scheme 3.
Structure
N5-P[c]
P-O1[c]
C6-N5-P-O2[c]
H(C7)- O2
TS2 MEI
TS4 MEI
TSIMZ
TS1 MEI
TS5 MEI
DEDNPP
2-MIPI
4-MIPI
IPI[d]
2.273
2.260
2.274
2.274
2.285
–
1.754
1.757
1.755
1.750
1.752
1.783
1.787
1.783
1.782
1.780
1.642
–
–
–
–
–
ꢀ56.75
ꢀ56.88
ꢀ21.13
ꢀ17.88
ꢀ12.10
–
37.70
40.59
15.67
18.06
13.39
–
4.096
2.734
2.757
2.737
–
–
4.302
2.747
2.765
2.725
1-MIPI
5-MIPI
[a] Additional data are given in the Supporting Information. [b] Obtained
performing B3LYP/6-31+G(d,p) calculations. [c] As labeled in Figure 6.
[d] Phosphorylated intermediate formed with the non-substituted IMZ.
Figure 6. SN2(P) transition states for the reaction of xMEI and IMZ with
DEDNPP obtained with B3LYP/6-31G+(d,p) and the general labelled TSS. All
Cartesian coordinates are given in the Supporting Information.
ꢀ578, whereas they are much lower for IMZ, 1 MEI and 5 MEI
(ꢀ21, ꢀ188 and ꢀ128, respectively). These results are consis-
tent with the nucleophilic attack by 2 MEI and 4 MEI involving
significant steric hindrance between the methyl group and the
O2 atom, that is, the rotation minimizes this effect. As ob-
served in the IRC plots shown in Figure 7, all reactions are con-
certed. Based on geometries, the formation of the N5ꢀP bond
and the cleavage of the PꢀO1 bond occur synchronously
along the reaction coordinate.
Finally, since the nucleophiles 5 MEI and 1 MEI do not pres-
ent steric hindrance from the methyl group, a different explan-
ation for their reactivity difference is required. We note that
differences in the HOMO energies for these species do corre-
late with reactivity, consistent with an electronic origin of the
reactivity difference. DFT calculations at the B3LYP/6-31+
G(d,p) level on xMEI structures show that 5 MEI and 4 MEI have
the highest energy HOMOs and IMZ and 1 MEI the lowest
(Table 5). Although the differences in energy are small, they are
consistent with the higher reactivity of 5 MEI compared to
1 MEI and IMZ toward DEDNPP. It is worth mentioning that
even if the HOMO energy of 2 MEI indicates a higher reactivity
compared to 1 MEI and IMZ, the steric effect is preponderant
and governs the reaction rates.
Figure 7. (A) Reaction coordinate diagram and (B) relative energies plots on
the IRC calculated with B3LYP/6-31+G(d,p).
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In conclusion, we report evidences for the antagonistic
methyl positioning effect observed for IMZ derivatives, which
shows that both electronic and steric effects contribute to
overall behavior. Interestingly, reactivity for the 4(5)MEI tauto-
mers was distinguished and their phosphorylated intermedi-
ates were detected by NMR. These results not only fill a gap in
the literature with regard to the understanding of mechanistic
details, they also provide guidance for designing optimal IMZ-
based catalysts for dephosphorylation reactions, with potential
applications in detoxification and biological processes.[19] Other
substituents on the IMZ ring are being studied, as are some
azole drugs.
Conclusion
Herein, dephosphorylation reactions involving methylated IMZ
derivatives (xMEI) and DEDNPP were thoroughly investigated
using a combination of experimental analysis and theoretical
calculations. Consistently, the methyl positioning in the IMZ
ring has direct influence on the reaction rates. Indeed, all xMEI
presented remarkable increases in the rate of cleavage of
DEDNPP (>3.0ꢀ104-fold), compared to the spontaneous hy-
drolysis, with the following nucleophilicity trend: 4(5)MEI>
IMZ>1 MEI>2 MEI. Interestingly, 4(5)MEI presented a higher
reactivity than IMZ, which can be attributed to the presence of
a pair of tautomers that react differently for 4(5)MEI, and NMR
analysis combined with DFT calculations revealed that the
5 MEI dominates its catalytic activity. The experimental moni-
toring of the different isomers formed during the reactions
with DEDNPP was crucial for understanding the methyl posi-
tioning effect. In addition, kinetics for the phosphorylated in-
termediates were followed by NMR and their half-lives were
shown to be related to the basicity of the leaving group.
In addition, for DFT calculations, the predicted barriers for
nucleophilic dephosphorylation reactions are sensitive to the
type of functional/basis set, suggesting that caution should be
taken when modeling similar reactions; the application of
more than one type of calculation may be essential. The trend
in calculated barriers at the B3LYP/6-31+G(d,p) level is in
good agreement with experimental data for the systems stud-
ied here. Computed geometries are consistent with steric re-
pulsion present in 2 MEI and 4 MEI, caused by the methyl
group adjacent to the nitrogen lone pair, being the key factor
in lowering their reactivity towards DEDNPP. In contrast, 5 MEI
and 1 MEI are free of steric hindrance, allowing an electronic
effect to increase the reactivity of 5 MEI (i.e., 5 MEI has
a higher HOMO energy). Calculations also indicate concerted
mechanisms with significant synchronism between bond
making and forming events for all reactions studied.
Experimental Section
Materials
DEDNPP was prepared as described in the literature[4a] and the
xMEI derivatives were obtained commercially.
Kinetics
Pseudo first-order conditions were maintained by adding 10 mL of
a stock solution of DEDNPP (7.5 mmolLꢀ1 in acetonitrile) into 3 mL
of a 0.5 mollꢀ1 aqueous solutions of xMEI. The reactions were
monitored at 400 nm by means of an UV/Vis spectrophotometer,
following the formation of 2,4-dinitrophenoxide (DNP, Scheme 3)
and the temperature was controlled with a thermostat-fitted cell
holder at 258C. The observed first-order rate constants (kobs) were
calculated with an iterative least-squares software from the plots
of absorbance against time and the correlation coefficients (r) were
higher than 0.99 for all curve fittings.
NMR analysis
NMR kinetics were evaluated by adding aliquots of a DEDNPP
stock solution (in acetonitrile) to 2 MEI or 4(5)MEI solutions in H2O,
directly into NMR tubes, and then 1H and 31P NMR spectra were
continuous acquired through reaction progress, respectively. Kinet-
ic profiles were obtained by the relative areas of the signals of the
Figure 8 summarizes the antagonistic methyl positioning
effect for the reactions of xMEI with DEDNPP, strongly support-
ed by the kinetic, NMR and DFT studies. The bottom box
shows 2 MEI and 4 MEI, the two species with the lowest reac-
tivity due to the steric hindrance caused by the methyl group,
whereas the reactivity difference between 1 MEI and 5 MEI can
be attributed to the highest HOMO energy of the latter, as
shown in the top box.
1
31P{1H} NMR spectra that were normalized. The H and 31P{1H} NMR
spectra were acquired at 298 K in H2O/acetonitrile (54%v/v) con-
taining some D2O (10%v/v) for lock and shimming, on a Bruker
1
AVANCE III 400 NMR spectrometer operating at 9.4 T, observing H
and 31P at 400.13 and 161.98 MHz, respectively, equipped with
a 5 mm direct detection multinuclear probe with z-gradient. One-
bond and long-range 1H–13C and 1H–31P NMR correlation experi-
ments were acquired on a Bruker AVANCE III 600 NMR spectrome-
1
ter operating at 14.1 T, observing H, 13C, and 31P at 600.13, 150.90,
and 242.94 MHz, respectively, equipped with a 5 mm inverse detec-
1
tion four channel (1H, 13C, 15N, and 31P) probe with z-gradient. H
and 13C NMR chemical shifts are given in ppm related to 3-(trime-
thylsilyl)-2,2’,3,3’-tetradeuteropropionic acid, sodium salt (TMSP-d4)
signal at 0.00 ppm as internal reference, whereas those of 31P NMR
are related to H3PO4 (85% in D2O) signal from an external refer-
ence.
DFT Calculations
All calculations were performed with Gaussian 09[20] and the water
environment was simulated implicitly by the Solvation Model Den-
Figure 8. The influence of steric hindrance and HOMO energy caused by the
methyl positioning on xMEI reactivity.
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sity (SMD) method.[21] Geometry optimizations were carried out
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31+G(d,p) or 6–311+ +G(d,p) basis sets. The obtained TS struc-
tures presented a single imaginary frequency and no imaginary fre-
quencies were found for minima. To confirm the connectivity of
the reaction pathways, IRC[22] calculations were computed with
B3LYP/6–31+G(d,p).
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Keywords: density functional calculations
derivatives · kinetics · NMR spectroscopy · tautomer reactivity
·
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Received: May 30, 2016
Published online on && &&, 0000
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FULL PAPER
Methyls on the move: The reaction of
all monosubstituted methylimidazoles
(xMEI) with diethyl 2,4-dinitrophenyl
phosphate has been evaluated by kinet-
ics studies, nuclear magnetic resonance
analysis and DFT calculations (see
&
Tautomer Reactivity
R. B. Campos, L. R. A. Menezes, A. Barison,
D. J. Tantillo, E. S. Orth*
&& – &&
The Importance of Methyl Positioning
and Tautomeric Equilibria for
Imidazole Nucleophilicity
figure). The electron-donating methyl
group acts to decrease the reactivity of
the xMEI, except for 4(5)methylimida-
zole. This behavior was attributed to
both electronic and steric effects. The
tautomer reactivity was distinguished.
Chem. Eur. J. 2016, 22, 1 – 9
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