Kinetic study on the aromatic nucleophilic substitution reaction of 3,6-dichloro-1,2,4,5-tetrazine by biothiols

Article abstract of DOI:10.1002/poc.3316

The aromatic nucleophilic substitution reaction of 3,6-dichloro-1,2,4,5- tetrazine (DCT) with a series of biothiols RSH: (cysteine, homocysteine, cysteinyl-glycine, N-acetylcysteine, and glutathione) is subjected to a kinetic investigation. The reactions are studied by following spectrophotometrically the disappearance of DCT at 370nm. In the case of an excess of N-acetylcysteine and glutathione, clean pseudo first-order rate constants (k_obs1) are found. However, for cysteine, homocysteine and cysteinyl-glycine, two consecutive reactions are observed. The first one is the nucleophilic aromatic substitution of the chlorine by the sulfhydryl group of these biothiols (RSH) and the second one is the intramolecular and intermolecular nucleophilic aromatic substitutions of their alkylthio with the amine group of RSH to give the di-substituted compound. Therefore, in these cases, two pseudo first-order rate constants (k_obs1 and k_obs2, respectively) are found under biothiol excess. Plots of k_obs1 versus free thiol concentration at constant pH are linear, with the slope (k_N) independent of pH (from 6.8 to 7.4). The kinetic data analysis (Bronsted-type plot and activation parameters) is consistent with an addition-elimination mechanism with the nucleophilic attack as the rate-determining step. Copyright

Full text of DOI:10.1002/poc.3316

Research Article
Received: 3 February 2014,
Revised: 1 April 2014,
Accepted: 18 April 2014,
Published online in Wiley Online Library: 19 May 2014
(wileyonlinelibrary.com) DOI: 10.1002/poc.3316
Kinetic study on the aromatic nucleophilic
substitution reaction of 3,6-dichloro-1,2,4,
5-tetrazine by biothiols
Daniela Andrade-Acuñaa, José G. Santosa, William Tiznadob,
Álvaro Cañetea* and Margarita E. Aliagaa*
The aromatic nucleophilic substitution reaction of 3,6-dichloro-1,2,4,5-tetrazine (DCT) with a series of biothiols RSH: (cysteine,
homocysteine, cysteinyl–glycine, N-acetylcysteine, and glutathione) is subjected to a kinetic investigation. The reactions are
studied by following spectrophotometrically the disappearance of DCT at 370 nm. In the case of an excess of N-acetylcysteine
and glutathione, clean pseudo first-order rate constants (kobs1) are found. However, for cysteine, homocysteine and cysteinyl–
glycine, two consecutive reactions are observed. The first one is the nucleophilic aromatic substitution of the chlorine by the
sulfhydryl group of these biothiols (RSH) and the second one is the intramolecular and intermolecular nucleophilic aromatic
substitutions of their alkylthio with the amine group of RSH to give the di-substituted compound. Therefore, in these cases, two
pseudo first-order rate constants (kobs1 and kobs2, respectively) are found under biothiol excess. Plots of kobs1 versus free thiol
concentration at constant pH are linear, with the slope (kN) independent of pH (from 6.8 to 7.4). The kinetic data analysis
(Brønsted-type plot and activation parameters) is consistent with an addition–elimination mechanism with the nucleophilic attack
as the rate-determining step. Copyright © 2014 John Wiley & Sons, Ltd.
Keywords: aromatic nucleophilic substitution; biothiolysis; inter/intramolecular displacement; kinetic study; tetrazine
have also been reported.[4] However, there are only a few works
INTRODUCTION
related to the ability of DCT to react with sulfhydryl groups of
amino acids.[13] In this context, it is important to note its reac-
The s-tetrazines are relatively old molecules, their discovery dated
tion with cysteine, because it may be used for introducing a
tetrazine group capable of undergoing photofragmentation
into a protein structure with the aim of subsequent conforma-
tional analysis of peptides.[13,14] On the other hand, the devel-
opment of selective thiol-reactive linkers and the reactions of
biothiols with alternative probes is a subject of much current
interest.[15–22]
Therefore, with the aim to shed some light on the mechanisms
of these reactions and to propose a strategy for thiol detection
based on nucleophilic aromatic substitution, this work under-
takes a kinetic investigation on the reactions of biothiols with
DCT. The studied biothiols are as follows: cysteine (Cys), homo-
cysteine (Hcy), cysteinyl–glycine (Cys–Gly), N-acetylcysteine
(NAC) and glutathione (GSH), and the conventional thiols 2-
mercaptoethanol (2-ME) and 1-propanethiol (1-PT).
back to the end of the 19th century. The first synthesis was
reported by Pinner who performed the reaction of equimolar
quantities of hydrazine and benzonitrile. Upon further oxidation
of the dihydrotetrazine intermediate, the deep red 3,6-diphenyl-
s-tetrazine was isolated.[1,2]
Tetrazines are unique heterocyclic compounds containing
the maximum number of nitrogen atoms in the aromatic
ring. Because of the presence of four electron-withdrawing
heteroatoms in the tetrazine core,[3] these compounds ex-
hibit particular properties among which highlights a high
electrophilicity.
Regarding the latter, the introduction of fragments at
positions 3 and 6 capable of leaving as anions, opens a possible
pathway for the modification of 1,2,4,5-tetrazines using aromatic
nucleophilic substitution. These reactions enable the preparation
of new symmetrically and asymmetrically substituted deriva-
tives, including products containing heteroatoms. The most
common groups in tetrazines capable of leaving as anions are
halogen atoms,[4] the methylsulfanyl group,[5,6] and the 3,5-
dimethylpyrazolyl fragment.[3]
* Correspondence to: Álvaro Cañete and Margarita E. Aliaga, Facultad de
Química, Pontificia Universidad Católica de Chile, Casilla 306, Santiago
6094411, Chile.
E-mail: acanetem@uc.cl; mealiaga@uc.cl
One excellent example of what is mentioned earlier is the
modification of 3,6-dichloro-1,2,4,5-tetrazine (DCT) by the
action of N-nucleophiles[4,7] and O-nucleophiles,[8–12], which
are often used to obtain tetrazine derivatives symmetrically
and asymmetrically substituted with heteroatoms. In addition,
reactions involving nucleophilic substitution of the halogen
atoms in this tetrazine by the action of isobutyl mercaptan
a D. Andrade-Acuña, J. G. Santos, Á. Cañete, M. E. Aliaga
Facultad de Química, Pontificia Universidad Católica de Chile, Casilla 306,
Santiago 6094411, Chile
b W. Tiznado
Departamento de Ciencias Químicas, Facultad de Ciencias Exactas,
Universidad Andrés Bello, Avenida República 275, Piso 3, Santiago, Chile
J. Phys. Org. Chem. 2014, 27 670–675
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AROMATIC NUCLEOPHILIC SUBSTITUTION REACTIONS
using the model thiol 2-mercaptoethanol in the same experimental
conditions. In the case of high-performance liquid chromatography–mass
spectrometry (HPLC-MS), the analysis was carried out by direct insertion
of the sample in an electrospray ionization (ESI) source. The low mass
EI-MS experiments were performed on a MAT 95XP Thermo-Finnigann
spectrometer by direct inlet probe (Thermo Fisher Scientific). HPLC-MS
experiments were performed on an Exactive Plus Orbitrap MS Thermo
Scientific (Thermo Fisher Scientific). The accurate mass measurements were
performed at a resolution of 140.000. On the other hand, 1H-NMR spectra
were acquired on a Bruker AM-400 instrument using tetramethylsilane as
internal reference.
EXPERIMENTAL
Materials
The reagents were purchased from Sigma-Aldrich (St. Louis, MO) and
used as received. Unless indicated, all solutions employed in this study
were prepared in Chelex-100-treated HEPES buffer (20 mM; pH 7.4).
The compound DCT was prepared as reported by Hiskey et al.[23,24] This
modified Pinner synthesis involves four steps (see Scheme 1). First,
triaminoguanidine monohydrochloride (2) was obtained by mixing (1) with
hydrazine in dioxane. Compound 2 was then condensed with two equiva-
lents of 2,4-pentanedione to yield dihydrotetrazine (3), which was oxidized
using NaNO2 to form dipirazol tetrazine (4). Finally, the latter compound
was treated with hydrazine and chlorine gas to obtain DCT.
Computational methods
This compound was identified by its spectroscopic properties. 13C-NMR
To ensure the consistence of our theoretical predictions, the molecular
geometries of the ground state structures were optimized using the
hybrid PBE0 and the range-separated hybrid ωB97X-D functionals. Both
functionals have been recently proposed as adequate to model reaction
profiles and their stationary points (reagents, transition state, and prod-
uct) in thiol-Michael additions.[25]
(100 MHz, CDCl3)
δ (ppm): 167.42 and by MS (low resolution),
m/z (I %) = 150.13 (30), 87.09 (85), 61.03 (100), (Fig. S1).
Kinetic measurements
These measurements were carried out by either an HP-8453 diode
array spectrophotometer or an Applied Photophysics DX17MV stopped-
flow spectrophotometer in aqueous solution at different temperatures
(10–40 0.1 °C) under an ionic strength of 0.2 M (KCl). HEPES buffers were
used in all reactions. The reactions studied in this work, under excess of
the biothiols over the substrate, were initiated with an injection of a sub-
strate stock solution in DMSO (10 μL) into the biothiols aqueous solution
(2.5 mL in the spectrophotometric cell). The initial DCT concentration was
about 2 × 10ꢀ4 M.
The calculations employed a triple-ζ (6-311++G(d,p))[26–28a] and double-
ζ (6-31 + G(d)).[28b,28c] Solvent effects (in water) were included using the po-
larizable continuum model.[29] The nature of the stationary points was
checked by means of frequency calculations. All calculations were
performed using the Gaussian 09, Revision C.01, suite of programs.[30]
RESULTS AND DISCUSSION
The reactions in the stopped-flow spectrophotometer were carried out
with unequal mixing. The DCT dissolved in dry DMSO was placed in a
small syringe (0.1 mL), and a larger syringe (2.5 mL) was filled with the
thiol aqueous solution. Pseudo first-order rate constants (kobs1) were
found for all reactions. These were determined through the spectropho-
tometer kinetic software, by least-squares fitting of the experimental ab-
sorbance data to the single-exponential curve At = A0 exp(ꢀkobs1 t) + C,
where At and A0 are the absorbances at time t and 0, respectively. The
experimental conditions of the reactions and the kobs1 values are shown
in Table S1.
From temperature-dependent rate constants, the Arrhenius and
Eyring equations were plotted as follows: log (kN) versus 1/T and log
(kN/T) versus 1/T, respectively. From these plots, energy (Ea) and the
enthalpy (ΔH≠) and entropy (ΔS≠) of activation were determined for the
reaction of DCT with biothiols.
Spectrophotometric study
The compound DCT provides an important optical signal
(compound highly colored). Figure 1 shows the UV/visible spec-
trum of DCT, which exhibits two characteristic bands (λ1 and λ2).
As reported previously,[7] the first transition is attributed to that
of n → π* transition of the s-tetrazine with a small molar extinc-
tion coefficient. The second band is attributed to a π → π* transi-
tion and is more intense. In the presence of 10 equiv. of GSH,
both absorption bands decrease (Fig. 1). Similar spectral behav-
iors were observed when other biothiols including Cys (Fig. S2),
Hcy, Cys–Gly, and NAC were used. Thus, the reaction between
DCT and the series of biothiols was followed spectrophotometri-
cally at 370 nm. The insert in Fig. 1 depicts the kinetic profile at
this wavelength obtained for the reaction between DCT and
GSH. For this reaction, only a fast decrease of absorbance due
to the disappearance of DCT was observed. Similar results were
Product studies
For the reaction between DCT and RSH, it was possible to identify the
final product of each reaction and also to compare them with others
Figure 1. Absorption spectra of DCT (200 μM) in the presence of gluta-
thione (GSH, 10 equiv.) in aqueous solution (20 mM HEPES buffer, pH 7.4,
1% DMSO) at different times. Insert shows the kinetic profile, at 370 nm,
for this reaction
Scheme 1. Synthetic route to 3,6-dichloro-1,2,4,5-tetrazine (DCT)
J. Phys. Org. Chem. 2014, 27 670–675
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D. ANDRADE-ACUÑA ET AL.
obtained for NAC and other conventional thiols, such as 2-
mercaptoethanol (2-ME) and 1-propanethiol (1-PT), (Fig. S3).
However, in some cases (i.e., Cys, Hcy, and Cys–Gly), a fast absor-
bance decrease, followed by a slower decrease at 370 nm was
also observed (for Cys, see insert in Fig. S2).
reaction could also be an intermolecular process (see succeeding
text). The kinetic values for kobs1 and kobs2 are summarized in
Table S1. The second-order rate constants (kN1 and kN2) were
obtained as the slopes of plots (kobs1) and (kobs2) against free
thiol concentration or free amine concentration, respectively.
Figure S4 shows an example of such plots.
The absence of a consecutive reaction for 1-PT (Fig. S3) and
NAC is due to the lack of an amine group responsible to undergo
the aforementioned second reaction, and thus, only the first
reaction is observed. However, in the case of GSH, the apparent
absence of the second reaction (insert in Fig. 1) is in line with the
recently reported inability of the amine group present in GSH to
undergo an intramolecular re-arrangement.[33]
Kinetic results
For the reactions of DCT with GSH, NAC, 2-ME, and 1-PT, pseudo
first-order rate constants (kobs1) were obtained under nucleo-
phile (thiol) excess. These values were determined by means of
the spectrophotometer kinetic software for first-order reactions.
The values of kobs1 for these reactions are shown in Table S1.
The values of kobs1 versus free nucleophile concentration obey
Eqn. (1), where k0 and kN1 are the rate constants for solvolysis
and thiolysis of the substrate (DCT). The k0 and kN1 values were
obtained as the intercept and slope, respectively, from linear
plots of kobs1 against free nucleophile concentration at constant
pH. The values of k0 and kN1 show no dependence on pH within
the pH range employed (from 6.8 to 7.4).
The values of kN1 found in this work, together with the pKa
values of the sulfhydryl group of the tested thiols,[15,35,36] are
summarized in Table 1 showing the following reactivity order
for the series of the studied thiols toward DCT: Cys–Gly < Cys <
Hcy < GSH <2-ME < NAC < 1-PT. Consequently, the reactivity
increases with the basicity of the sulfhydryl group of the corre-
sponding thiol. With the pKa and kN1 values in Table 1, the
Brønsted type plot for the thiolysis studied was obtained. As
shown in Fig. 2, this plot is linear with a slope (β) of 0.33. The
value of β found for the reactions of biothiols with DCT is in
agreement with the β values found for stepwise reactions when
the formation of the intermediate is the rate-limiting step.[36]
To gain further understanding on the mechanism of the
reaction of DCT with biothiols, the activation parameters were
calculated from the rate constants measured at five different tem-
peratures (10–40 °C). These thermodynamic activation parameters
are summarized in Table 2. The large negative ΔS# values obtained
indicate that the transition states are highly ordered compared
with reactants; this is consistent with the fact that the translational
degree of freedom are restricted because of a condensation
reaction or great participation of water molecules in the activated
complex. We find it interesting that the values obtained for ΔH≠
and ΔS≠ are in line with a bimolecular reaction similar to that
commonly accepted for aromatic bimolecular two-stage process
in which the formation of a Meisenheimer complex (intermediate
MC) is the rate-determining step, with a rapid decomposition of
this intermediate into product.[37]
kobs1 ¼ k0 þ kN1½free nucleophileꢁ
(1)
For these reactions, the k0 values were much smaller than the
kN1 [free nucleophile] term in Eqn. (1). Table 1 summarizes the
values of kN1 found, together with those for the pKa of the series
of nucleophiles employed. As shown, all reactions are associated
with large rate constant values (>37 sꢀ1 Mꢀ1). Considering the
high values of kN1 obtained in this study, DCT could be proposed
as an efficient electrophile toward these biothiols. In fact, as
suggested by Gao et al.,[31] DCT is a powerful electrophile, similar
in reactivity toward other nucleophiles, as picryl fluoride.
In contrast, the biothiols Cys, Hcy, and Cys–Gly showed toward
DCT a consecutive reaction model. This behavior can be
explained considering the recently reported ability of Cys and
Hcy to react via nucleophilic substitution by its sulfhydryl group
to form an intermediate thioether.[32–34] Subsequently, this inter-
mediate undergoes further intramolecular aromatic substitution
reaction that causes the displacement of the thio group by the
amine group present in the thiol.
Therefore, we propose that the first decrease observed at
370 nm (Fig. S2) is attributed to the thiol-halogen nucleophilic ar-
omatic substitution of DCT by thiolate of biothiol (kinetic values
for the fast process, kobs1) and the second slow decrease could
be due to the intramolecular nucleophilic aromatic substitution
of alkylthio with the amine group of RSH (kobs2). This latter
On the basis of the kinetic results and by comparing the
reactions under investigation between each other and with
other similar processes, we propose a bimolecular reaction type:
Table 1. Values of pKa for the sulfhydryl group of tested
thiols (RSH) and kN1 values for the reactions of the RSH with
DCT
kN1/sꢀ1 Mꢀ1
a
Thiols (RSH)
pKa
Cys–Gly
Cys
Hcy
7.00
8.10
8.25
8.72
9.70
9.90
10.7
37.7 0.9
98
102
167
193
3
6
5
8
GSH
2-ME
NAC
1-PT
207 12
1160 30
apKa data in water were taken from[15,35,36]
Figure 2. Brønsted-type plot for the thiolysis of DCT, in aqueous solu-
tion, at 25.0 °C and ionic strength 0.2 M (KCl)
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AROMATIC NUCLEOPHILIC SUBSTITUTION REACTIONS
relationship,[40] which suggests that all kN1 values for the studied
reactions correspond to the same rate-limiting step.
Table 2. Thermodynamic activation parameters for the reac-
tions of biothiols (RSH) with DCT
Also, it is interesting to mention that the rate constants (kobs2
,
Fig. S4) obtained for the decomposition of the intermediate
product (P1 in Scheme 3) show a linear dependence on the
corresponding free amine (N) concentration. Therefore, we do
not only suggest an intramolecular substitution reaction but also
an intermolecular one, as shown in Scheme 3.
DCT
Biothiols
Ea/kJ molꢀ1 ΔH≠/kJ molꢀ1 ΔS≠/J molꢀ1 Kꢀ1
Cys–Gly
Cys
Hcy
GSH
NAC
56
54
47
38
21
3
4
3
2
2
54
51
45
36
18
9
2
2
3
3
ꢀ33
ꢀ34
ꢀ53
ꢀ83
ꢀ138
2
1
4
5
Theoretical calculations
4
The present study is complemented with theoretical calculations
of the Gibbs free energy (ΔG) of the two possible products
proposed (P1 and P2, see Scheme 3).
The calculation of the Gibbs free energies, allow us to predict
that the product P2, obtained from the nucleophilic aromatic
substitution of DCT by the amine group of Cys, is thermodynam-
ically preferred by ~10 kcal molꢀ1 in comparison with the forma-
tion of P1, as shown in Fig. 4.
Similar results were obtained when Hcy was used instead of
Cys. It is important to remark that different structural possibilities
have been tested as starting geometries for the minimization of
each isomer. The most stable geometry at ωB97X-D/6-311++G**
level for the reaction of DCT with Cys (n = 1) is presented in Fig. 4.
The optimized structural parameters (distances and angles) are
addition–elimination (SNAr) for the nucleophilic attack of the
series of thiols to DCT (as suggested in Scheme 2).
On the other hand, it is known that when a series of structur-
ally related substrates undergo the same general reaction or
when the reaction conditions for a single substrate are changed
in a systematic way, the enthalpies and entropies sometimes
satisfy a linear isokinetic relationship, as written in Eqn. (2).
ΔH≠i ¼ α þ βΔS≠i
(2)
In this equation, α and β are constants, where β is the so-called
‘compensation temperature’ and α has energy dimension.
Figure 3 shows that a plot of ΔH≠ versus ΔS≠ for the tested reactions
gives a straight line. This behavior is related to the enthalpy–
entropy compensation effect,[38,39] also known as the isokinetic
Scheme 3. Proposed mechanism for the reactions of DCT with Cys
(n = 1) and Hcy (n = 2)
Scheme 2. Proposed mechanism for the reaction between DCT and
biothiols (RSH)
Figure 4. Computed relative Gibbs free energies (ΔG) in kcal molꢀ1 at
the PBE(0) and ωB97X-D with the 6-311++G** basis set level of theory.
The values without and in parenthesis are those that include solvent
effects (read the computational details) and in vacuum, respectively.
P1 and P2 represent the products for the reaction of DCT with cysteine
as nucleophile
Figure 3. Correlation between activation enthalpy (ΔH≠) and activation
entropy (ΔS≠) for the reaction of biothiols with DCT
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D. ANDRADE-ACUÑA ET AL.
almost the same when PBE(0) or ωB97X-D is used in the calcu-
lations. In addition, it is observed that the use of a small base
set (6-31 + G*) in vacuum is enough to provide similar descrip-
tion about the thermodynamic preference of the P2 against P1
(see Tables S2–S3).
Therefore, considering the kinetic profiles and theoretical
results presented, we propose that for the biothiols Cys, Hcy,
and Cys–Gly, the kinetic product P1 is formed at short times.
However, under long reaction times, the thermodynamic prod-
uct P2 is favored.
different intensities. The more important signals correspond to
m/z = 200, which was assigned as [M-Cys-CO2 + H]+, m/z = 157 is
attributed to [M-Cys]+, and the molecular ion with m/z = 319
corresponds to [M-H]+. It is noteworthy that the latter involves
the formation of a di-substituted compound. However, it is not
possible to decide if this compound corresponds at P1 or P2, due
to their identical molecular ions. A similar result was also attained
for Hcy (Fig. S5) and 2-ME (Fig. S6), using the same technique.
1
In the H-NMR spectrum, obtained after the addition of DCT
(in DMSO-d6) on Cys (in D2O, Figs. 6 and S7), we assigned a broad
signal at ∼7.8 ppm and a signal at 2.4 ppm to the exchangeable
protons of the amine group and SH group, respectively. With
the aim to confirm this exchange, we compared the spectra
obtained by reacting DCT with an authentic amino-substituted
compound, such as n-butylamine (BA) and a model compound
of amino-free thiol, such as NAC. For the former (Fig. S8), we first
observed that the resonance signals of the protons of amine
group of BA were shifted to lower field due to the nucleophilic
substitution of chlorine atoms in DCT by
the amine group. Second, it is possible to
observe that the signal at 2.7 ppm, corre-
sponding to the proton of alpha carbon of
the BA, is shifted to 3.3 ppm for the product
between DCT and BA. A similar effect was
observed for the alpha carbon of Cys after
its reaction with DCT (shifted by
∼ 0.2 ppm). In the case of NAC, we observed
the disappearance of the signal at 2.4 ppm
associated with the proton of its sulfhydryl
group. We find it interesting that Fig. 6(A)
shows the existence of the aforementioned
signal at 2.4 ppm.
Product analysis
Finally, with the objective to assess the products formed by inter-
action between each biothiol and DCT, we performed a product
analysis using mass spectrometry (HPLC-ESI-MS) and 1H-NMR.
The mass spectrum obtained after the addition of an excess of
Cys on DCT (Fig. 5) depicts a series of mass fragments with
Thus, results obtained from NMR spectra
suggest that Cys is attached to DCT
through its amine group and this is in con-
cordance with those described in other
study for a process based on a similar intra-
molecular displacement.[33] Therefore, this
information is crucial to corroborate the
final product P2 previously proposed in
Scheme 3, as the product thermodynami-
cally favored, and the P1 as the product
kinetically favored.
Figure 5. Mass spectrum obtained after the addition of DCT to cysteine
CONCLUSIONS
On the basis of the overall evidence avail-
able, we have proposed that the nucleo-
philic reactivity of the tested sulfhydryl
groups toward DCT increases in the se-
quence Cys–Gly < Cys < Hcy < GSH <2-ME
NAC <1-PT. By comparing the reactions
under investigation between each other
and with other similar processes, we
propose a bimolecular reaction type: addi-
tion–elimination (SNAr) for the nucleophilic
attack of the series of thiols to DCT, in
which the formation of the Meisenheimer-
type complex (intermediate MC) is the
Figure 6. Changes in the Cys 1H-NMR spectrum induced by the addition of DCT. Spectra of Cys
in the presence of DCT (A) or in the absence of DCT (B)
rate-determining step of the reaction,
followed by a fast substitution of the
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AROMATIC NUCLEOPHILIC SUBSTITUTION REACTIONS
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This work was supported by FONDECYT with grant #1130062 and
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spectrophotometer) in the Centro Singular de Investigación en
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