Proton-Sensitive Free-Radical Dimer Evolution Is a
Critical Control Point for the Synthesis of Δ2,2′-Bibenzothiazines
The mechanism of the acid-dependent interring dehydrogenation in the conversion of the single-bonded 3-phenyl-2H-1,4-benzothiazine dimer 2 to the Δ2,2′-bi(2H-1,4-benzothiazine) scaffold of red hair pigments is disclosed herein. Integrated chemical oxidation and oxygen consumption experiments, coupled with electron paramagnetic resonance (EPR) analyses and DFT calculations, allowed the identification of a key diprotonated free-radical intermediate, which was implicated in a remarkable oxygen-dependent chain process via peroxyl radical formation and evolution to give the Δ2,2′-bi(2H-1,4-benzothiazine) dimer 3 by interring dehydrogenation. The critical requirement for strongly acidic conditions was rationalized for the first time by the differential evolution channels of isomeric peroxyl radical intermediates at the 2- versus 3-positions. These results offer for the first time a rationale to expand the synthetic scope of the double interring dehydrogenation pathway for the preparation of novel symmetric double-bond bridged captodative heterocycles.
Introduction<!><!>Introduction<!>Chemical
Oxidation Experiments<!>EPR Spectroscopy<!><!>EPR Spectroscopy<!>Oxygen
Uptake Measurements<!><!>DFT Calculations<!>Proposed Formation and Evolution Pathways of the Free-Radical Dimer 2· in Acidic Methanol<!>DFT Calculations<!><!>DFT Calculations<!><!>DFT Calculations<!>Conclusions<!>General
Information<!><!>Computational Studies<!>EPR Spectroscopy<!>Oxygen Uptake
Measurements<!>
<p>The
Δ2,2′-bibenzothiazine system,
the core structure of trichochromes and related pigments found in
red hair and feathers, is characterized by an interring double bond,
which allows efficient push–pull interactions and π-electron
conjugation across the S–C=C–C=N–
systems (Figure 1).1−4 Because of the indigoid nature of the chromophore and the inherent
structural rigidity, Δ2,2′-bibenzothiazines
display redox properties and an intense absorption in the visible
region with a peculiar acidichromic behavior finely tunable by substituents,
which attract potential interest for various applications.5</p><!><p>Structures of red hair pigment trichochromes.</p><!><p>From the synthetic point of view, Δ2,2′-bibenzothiazines can be easily produced by the doubly dehydrogenative
dimerization of 2H-1,4-benzothiazine derivatives.
This process is spontaneous and remarkably facile in strongly acidic
media and in the presence of oxygen, but it is completely inhibited
under mild or nonacidic conditions (Scheme 1).1,4</p><p>The factors accounting for the tendency of two sp3 C–H
bonds in the benzothiazine ring to undergo doubly dehydrogenative
coupling are of mechanistic interest in the broad general context
of the C–H bond activation strategies.6−9 Recent evidence10 suggested that the marked activating effect of acids on
the dehydrogenative coupling of 3-phenyl-1,4-benzothiazine (1) to the corresponding Δ2,2′-bibenzothiazine (Scheme 1) is due to a decrease in the energy of the initial
H-atom abstraction step caused by N-protonation.</p><p>The process
evolves via captodatively stabilized10,11 free-radical
intermediates, which appear to dimerize rather than
to couple with oxygen, based on the lack of detectable oxygenated
products or intermediates.</p><p>The most puzzling issue in this process
concerns the role of acids
and oxygen in the desaturation step. Current evidence indicates that
the single-bonded dimer can be detected as an intermediate or isolated
at neutral pH.10 Its conversion to the
final double-bonded bibenzothiazine is promoted by hydrogen peroxide
and is critically dependent on (a) strong acids and (b) the presence
of oxygen. So far, the role of acids and the involvement and fate
of oxygen in the conversion of the interring single bond in 2 to the double bond in 3 have remained little
understood.</p><p>Herein, we report electron paramagnetic resonance
spectroscopy
(EPR), oxygen uptake experiments, and DFT calculations on the mechanism
of desaturation of 2 to 3 by H2O2. Specific aims of the study were (a) to elucidate the
role of oxygen and strong acids in the dehydrogenative conversion
of the interring single bond in 2 to the double bond
in 3 and (b) to identify and characterize free-radical
intermediates in the process, as yet still elusive.</p><!><p>Initial experiments
were directed to reassess the mechanism of formation of the unsaturated
Δ2,2′-bibenzothiazine system on
monomer 1 as a probe substrate under various oxidation
conditions.</p><p>It was thus confirmed that under strongly acidic
conditions, i.e., methanol/36% HCl 3:1, 1 reacts rapidly
with H2O2 in the presence of oxygen to give
dehydrogenated dimer 3,10 whereas
in neutral organic solvents, e.g., methanol, no reaction occurred
with H2O2 even over prolonged periods of time.</p><p>Reaction of 1 with free-radical species such as 2,2-diphenyl-1-picrylhydrazyl
(DPPH) or 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl (TEMPO) induced
a slow conversion to the single-bonded dimer 2a/b, without detectable 3a/b. Under
mildly acidic conditions, such as picric acid in ethanol, 1 was converted to dimers 2a/b, which accumulated,
and were not further oxidized to 3. When purified dimers 2a/b were exposed to oxidants like 2,3-dichloro-5,6-dicyanobenzoquinone
or chloranil or to an excess of DPPH or TEMPO in organic solvents,
no significant reaction occurred. On the other hand, peroxides and
hydroperoxides were shown to allow the conversion of 1 into 3(10) and 2 to 3 (Figure S1) though
hydrogen peroxide was found to be the most efficient and synthetically
convenient oxidant to promote the conversion.</p><p>The conversion
of dimers 2a/b to 3 in methanol
under acidic conditions in the presence of excess
H2O2 was next investigated by spectrophotometrically
monitoring the development of the green-blue chromophore of 3 at 598 nm after 30 min in methanol/aq. HCl 3:1, using different
concentrations of the acid (Figure S2).
Formation of 3 was apparent with 3 M or 2 M HCl, whereas
at acid concentration below 1 M, no chromophore was observed. Formation
of double-bonded dimer 3 was confirmed by HPLC analysis.</p><!><p>In our previous study in which the
reaction of 12.5–25 mM 1 in air-equilibrated MeOH
containing 3 M HCl was performed in the cavity of an electron paramagnetic
resonance (EPR) spectrometer, we observed that addition of H2O2 (final concentration, 0.2–0.4 equiv) to the
solution led to the slow buildup of a weak EPR signal centered at g = 2.0051 with a characteristic hyperfine structure,10 while no signal was observed in control experiments
in the absence of H2O2. The hyperfine structure
of the signal showed large coupling constants (ca. 6 Gauss) due to
two different protons (spin = 1/2), one relatively
large coupling constant (ca. 3 Gauss) with nitrogen (spin = 1) and
two smaller coupling constants with nonequivalent protons. Both the
measured g-factor and the hyperfine structure were
not compatible with peroxyl radicals (1-OO·) produced by coupling of oxygen with a carbon-centered radical (expected g ≈ 2.015)12 but suggested
rather a C/N-centered conjugated radical with spin delocalization
on heavier atoms like sulfur. In this connection, the radical cations
of protonated/methylated 1,4-diazines (lacking sulfur) exhibit g-factors in the range 2.0030–2.0033,13 similar to N,N-diphenylaminyl radical, g = 2.0032, while the structurally
related aminyl radical of phenothiazine exhibited g = 2.0046.14</p><p>DFT calculations of
the expected spin distribution and corresponding EPR coupling constants
for neutral and protonated radical 1· (Table S1) allowed to rule out neutral
species, unequivocally assigning the spectrum to the protonated radical.
Simulation of the EPR spectra and interactive fitting according to
the Monte Carlo method,15 using the DFT-calculated
coupling constants as an initial input, allowed to reproduce reasonably
well the experimental spectra despite the modest signal/noise ratio.
Match of simulated and experimental spectra was, however, not fully
satisfactory, in that the central portion of the experimental spectrum
(of the highest quality) showed three lines of approximately equal
intensity (Figure 2a), at variance with simulation predicting a less intense central
line (Figure S3). We hypothesized that
this was due to superimposition of the spectrum of radical 1· to that of other radical species formed in the
reaction mixture. The most reasonable candidate was the dimeric radical 2·, formed upon oxidation of 2 (see Scheme 1).</p><!><p>Experimental
EPR spectrum obtained with (a) 12.5 mM monomer 1 or (b)
dimer 2 in MeOH/36% HCl 3:1 upon reaction
with 4 mM H2O2 (black) and its computer simulation
(red) using the coupling constants (hccs) displayed
on the structure. Simulation of the spectrum shown for monomer 1 (panel a) was obtained with a radical mixture of 1· and 2· at a 10:6 ratio
matched with the experimental spectrum. For radical 2·, calculated hccs (see the Supporting Information) are reported in parentheses
(blue).</p><!><p>To confirm this hypothesis and
shed more light on the mechanism
of conversion of 1 to 3, we performed matched
EPR experiments using dimer 2 in place of phenylbenzothiazine 1. Upon addition of H2O2, an intense
spectrum was observed (Figure 2b). The measured g-factor was 2.0052, just
slightly higher than that of radical 1·, indicating a similar structure with increased spin delocalization
on heavy atoms (e.g., two sulfur atoms of the two benzothiazine moieties).
Spectral analysis and interactive simulation afforded coupling constants
in excellent agreement with those calculated for the diprotonated
radical 2· (Figure 2b and Table S2), while the agreement with those calculated for the monoprotonated
species were less satisfactory. Interestingly, no EPR signal attributable
to the corresponding peroxyl radical 2-OO·, expected at a much lower field, could be observed although experiments
were performed in air-saturated solutions, confirming a general instability
of peroxyl radical intermediates.12 This
is in keeping with the work of Pratt and Porter who showed that β-fragmentation
of an alkylperoxyl radical is facilitated by electron-withdrawing
substituents (like the iminium function in 2-OO·) that would destabilize it while stabilizing the C-centered radical
(e.g., 2·) due to hyperconjugative effects.9</p><p>With both sets of spectral parameters available,
simulations of
spectra due to the superimposition of those of protonated radical 1· and diprotonated radical 2· were fitted to the experimental EPR spectrum previously
assigned to radical 1· alone (see above).
The quality of matching in the central portion of the spectrum was
significantly improved (Figure 2a), confirming our hypothesis. This finding fully supports
a free-radical formation pathway for dimer 3.</p><!><p>Our previous investigation
showed that oxygen is necessary to the overall process of conversion
of phenylbenzothiazine 1 to colored dimer 3, as no color development is observed in the presence of various
oxidizing species in the absence of oxygen.10 On the other hand, from analysis of the products, it clearly appears
that oxygen is not incorporated in the reaction products, suggesting
that any oxygen consumed in the process must be eliminated, e.g.,
in the form of water or as hydrogen peroxide. EPR studies did not
reveal the formation of oxygen-centered radicals such as peroxyl radicals.
The role of oxygen in the conversion of 2a/b to 3 was then investigated by monitoring oxygen consumption
in a differential oxygen uptake apparatus.16,17</p><p>When dimer 2 was incubated in MeOH containing
3 M H2SO4 at 303 K, oxygen consumption was poor
until 1 Î¼mol of H2O2 was added to the
system, causing a rapid oxygen consumption, which stopped after approximately
0.2–0.3 equiv (with respect to the starting compound) had been
consumed. Reinjection of a second aliquot of H2O2 caused oxygen consumption to restart, and the phenomenon was observed
for subsequent additions of H2O2 until the reaction
was complete (Figure 3a, the fifth addition causes no further reaction). The exact stoichiometry
of oxygen uptake depended on the initial concentration of H2O2, ranging from 0.6 to 1.0 with respect to the starting
dimer 2. Indeed, addition of equimolar H2O2 as a single aliquot resulted in a lower oxygen consumption
with respect to that obtained by repeated addition of substoichiometric
amounts, and a large molar excess brought the reaction close to completion
with apparent lower overall O2 consumption (Figure 3b), likely as a result of acid-catalyzed
dismutation of H2O2 that partly restores the
oxygen consumed by the reaction. Indeed, control experiments where
a similar amount (10 Î¼mol) of H2O2 was
added to 3 M H2SO4 in MeOH, in the oxygen uptake
apparatus, in the absence of dimer 2 showed oxygen evolution
at a rate compatible with the "missing" oxygen consumption
recorded in the presence of dimer 2 (Figure S4). Incubating monomer 1 in place of
the dimer under similar settings, a similar behavior was observed,
with a higher oxygen consumption following repeated addition of H2O2 than that observed by adding the same overall
amount in a single bolus (Figure S5). On
the basis of these data, it can be concluded that in the present experiment,
hydrogen peroxide plays a key role in the generation of the free-radical
intermediates, while oxygen is involved in propagation steps. It may
also be noted that the need for an amount of H2O2 comparable to that of 1 or 2, albeit substoichiometric,
and the consumption of 0.6–1.0 equiv of oxygen suggest that
the chain reaction is poorly efficient (short chain), and other mechanistic
possibilities are likely coinvolved.</p><!><p>Oxygen uptake measured by incubating 5
Î¼mol of dimer 2 in 4 mL of MeOH containing 3 M
H2SO4 (final conc., 1.25 mM) at 303 K with (a)
addition of aliquots of
1 Î¼mol of H2O2 to the system at time points
indicated by an arrow or (b) by a single addition of 10 Î¼mol
of H2O2.</p><!><p>The results reported above indicated
that conversion of 2 to 3 requires strong
acids, to allow the generation of an EPR-detectable diprotonated free-radical
dimer, and is activated by addition of hydrogen peroxide triggering
oxygen consumption. However, the precise step underlying the critical
requirement for strong acids and the actual role of oxygen in promoting
desaturation of the diprotonated dimer remained unclear.</p><p>To
settle these issues, the influence of protonation on the various critical
steps of the most plausible reaction pathways was assessed by DFT
calculations. The PBE018 functional in
combination with a reasonably large basis set [6-31+G(d,p)] was used
for extensive structural explorations and for computation of vibrational–rotational
contributions to the free energy. The M06-2X19 functional with a much larger basis set [6-311++G(2d,2p)] was adopted
for single-point energy evaluations. Geometry optimizations were performed
either in vacuo or by adoption of a polarizable continuum medium (PCM)20 to account for the influence of the solution
environment. The M06-2X single-point calculations also included nonelectrostatic
contributions to the solvation free energy, employing radii and nonelectrostatic
terms of the SMD solvation model.21</p><p>The pH value in the methanol-containing medium adopted in the experiments
of this study was estimated by making a reference to substituted anilines,
for which pKa values in methanol were
reported.22 The extent of protonation of
the selected anilines was evaluated based on the shifts of the absorption
maxima of the protonated or free base forms in the 3:1 methanol/HCl
with the acid at the concentrations used (Figure S6). On this basis, the pH of the methanol/3 M HCl medium was
estimated to be below 0, whereas for the methanol/1 M HCl medium,
the estimated pH was higher than 1.</p><p>The reaction pathways considered
for this study are illustrated
in Scheme 2 for the
case of monoprotonated species. They involve the following key steps.</p><!><p>For each species
in the reaction
path, only the most stable tautomer of the monoprotonated form is
represented; however, depending on the specific pH of the reaction
medium, each species will populate several tautomers of both the mono-
and diprotonated forms. A detailed DFT characterization of such protonation
microstates is provided as Supporting Information.</p><!><p>(Step 1) H-atom abstraction
from the single-bonded
dimer 2 either by a hydroxyl radical (HO·), produced from hydrogen peroxide, or by the hydroperoxyl radical
(HOO·), produced from hydrogen peroxide and HO· (eq 1,
initiation) or during propagation steps (vide infra), to give dimer radical 2· existing
mainly as a resonance-stabilized captodative form as an enamine tautomer.1</p><p>(Step 2 (C2)/2
(C3)) Free-radical coupling of
the dimer radical 2· with oxygen to give
isomeric peroxyl radical intermediates at C2/C3 (2-(C2)-OO· and 2-(C3)-OO·,
respectively).</p><p>(Step 3 (C2)/3 (C3)) Intramolecular
H-atom abstraction
to generate the corresponding β-hydroperoxyalkyl/aminyl radicals, 2·-(C2)-OOH and 2·-(C3)-OOH.</p><p>The reaction proceeds with identical ease and outcomes either
in
the presence of 3 M HCl or of 3 M H2SO4, suggesting
that it is not promoted by a specific acid, but it requires a strongly
acidic medium.</p><p>For the present study, steps 1–3 were
investigated in methanol
as a solvent. All species involved were fully characterized in all
possible protonation states, including consideration of the different
tautomers and of conformational equilibria; pKa values for the two protonation steps were estimated by comparison
of the computed free-energy changes with those obtained at the same
theory level for a series of nitrogen bases for which experimental
data in methanol were available.22 Only
the most stable species are represented in the following schemes.
Further details of the computational aspects are provided in the Supporting Information (Tables S3–S8),
including energy data at different theory levels for the most stable
conformer of each species examined and computed pKa data. Representative formulae for the free-radical intermediates
in the diprotonated forms and relevant equilibria are provided in Scheme 3.</p><p>Simple inspection of Scheme 3 reveals an important
mechanistic clue: H atom abstraction
from the C2 position of the diprotonated form of 2 generates
a resonance-stabilized free radical localized on a single thiazine
ring due to disruption of captodative interring resonance effects,
with higher spin density on the 2- and N-positions. Oxygen coupling
at C3 is prevented unless a tautomerization step is considered, which,
however, leads to an unstable (ca. 8 kcal/mol) >NH2+ species.</p><p>Consistent with this view, computational analysis
of the regioisomeric
free-radical intermediates in Scheme 2 revealed the most noticeable difference in the relative
energies as a function of the protonation state. Whereas under neutral
or weakly acidic conditions, oxygen coupling proved to be more favorable
at C3 than at C2, as inferred by the greater stability of 2-(C3)-OO· over 2-(C2)-OO· (2.9
kcal/mol for the neutral forms and 4.3 for the monoprotonated forms),
the situation is reversed in the case of the diprotonated forms, with 2-(C2)-OO· more stable than 2-(C3)-OO· by 6.2 kcal/mol. At acidic pH, moreover, step
1 proved to be more exergonic than under neutral conditions (by 8.8
kcal/mol for the monoprotonated forms and by 3.2 kcal/mol for the
diprotonated forms). The pH effects (selected pH 3 and −1.5)
are summarized graphically in Figure 4, in which the free energy of each species has been
corrected to account for the coexistence of different protonation
forms, based on the computed pKa values
(Table S7).</p><!><p>Computed free-energy
diagram for reagents, products, and putative
intermediates in the reaction pathway leading from 2 to 3, under different pH conditions. (left panel) pH 3.0; (right
panel) pH −1.5.</p><!><p>It follows from the points
above that only a strongly acidic medium
can efficiently direct the reaction pathway toward the C2 coupling
route, which appears to be the privileged channel to interring dehydrogenation
and product formation compared to the C3 route. Conversion of peroxyl
radicals at C2 to the final Δ2,2′ dimeric product 3 may take different nonexclusive pathways,
namely, (a) intramolecular H-atom abstraction by −OO· from C–H in 2′ to form a hydroperoxide intermediate
bearing a C-centered radical that may cleave to release the hydroperoxyl
radical HOO· (the two steps, intramolecular H-abstraction
and loss of HOO· could be concerted) (Figure 4 for selected pH 3 and −1.5
and Figure S7 for other pH conditions explored)
or (b) intermolecular H-atom abstraction (from hydrogen peroxide generating
HOO· as a chain transporter) to give a hydroperoxide
intermediate, which would then undergo loss of H2O2 (Figure S8).</p><p>In a highly
acidic medium, the reaction path (a) resembles the
chemistry recently described by Pratt and coworkers to explain the
release of HOO· during the autoxidation of unsaturated
hydrocarbons and is likely to benefit from accelerated kinetics due
to quantum tunneling of the activation barrier.23,24 In a highly acidic medium, release of HOO· as the
last step also justifies the radical-chain nature of this reaction,
which requires less than stoichiometric amounts of initiating reactants
(e.g., H2O2) to proceed to completion.</p><p>On the other hand, isomeric peroxyl radicals at C3 may take convenient
reaction channels based, e.g., on cyclization leading to endoperoxides
(Scheme 4), as predicted
by DFT calculations, which would hardly evolve toward interring dehydrogenation
and formation of 3 (Figure 5).</p><!><p>Computed free-energy diagram for alternative
evolution pathways
of the peroxyl radicals from 2. The energy scale is the
same as in Figure 4. (left panel) pH 3.0; (right panel) pH −1.5.</p><!><p>ESI-MS analysis of the crude product mixture
arising from oxidation
of 2 to 3 did not reveal the formation of
signals attributable to endoperoxides although the ionization conditions
might have caused decomposition of such moderately stable products.
Although experimental data do not allow to unambiguously demonstrate
the unproductive outcome of coupling reactions at C3 and the putative
endoperoxy intermediates thereof, it is worth noting that the analogous
cyclization routes of peroxyl radicals on the 2-position are not favored
over intramolecular H-abstraction (Figure 5), whereby elimination/fragmentation with
loss of oxygen appears to be by far the best option possible for peroxy
species at C2. Additionally, elimination/fragmentation is in line
with the lack of recovery of oxygenated products and with the observations
by EPR spectroscopy.</p><p>A more detailed investigation of the reaction
pathway at the transition
state level was hindered by the highly demanding computational effort,
especially on account of the remarkable conformational freedom of
most species. Nonetheless, the clear pH-dependent trend of the energy
order for the matching series of intermediates along the competing
oxygenation pathways at C2 vs. C3 justifies reliance on Hammond's
postulate to put the main mechanistic conclusions of this study on
solid ground.</p><!><p>Altogether, chemical,
EPR, and oxygen consumption data coupled
with DFT calculations allowed to propose for the first time a consistent
mechanism accounting for the intriguing acid-promoted interring dehydrogenation
of single-bonded dimer 2 leading to the central bibenzothiazine
core of red hair pigments. In addition, it has been possible to identify
and characterize the transient free-radical dimer 2· in its diprotonated form by careful EPR experiments
coupled with computational analysis.</p><p>Besides shedding new light
on the chemistry of captodative free
radicals with oxygen, these results offer an improved rationale to
expand the synthetic scope of the double interring dehydrogenation
pathway for the preparation of novel symmetric double-bond bridged
captodative heterocycles.</p><!><p>All solvents and reagents were
obtained from commercial sources and used without further purification.
UV–vis absorption spectra were registered at room temperature
on a V-560 JASCO spectrophotometer using calibrated 2 mL quartz cuvettes.
LC–MS analyses were performed on an HPLC instrument Agilent
1100 Series MSD equipped with a UV–vis detector and an electrospray
ionization source in positive ion mode (ESI+). Detection
wavelength was set at 254 nm. The spray voltage was set at 3.5 kV.
Nitrogen was employed as both drying and nebulizer gas. Mass spectra
were registered with the cone and fragmentator voltage set at 4 kV
and 80 V, respectively. An octyl column (15 cm × 4.6 mm, 3 μm
particle size) was used. An acetonitrile/water gradient was used as
follows: 0–50 min, 50–70% acetonitrile and 50–60
min, 70% acetonitrile. The flow rate was set at of 0.7 mL/min.</p><p>2,2-Diphenyl-1-picrylhydrazyl (DPPH) or 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl
(TEMPO), 2,3-dichloro-5,6-dicyanobenzoquinone, sodium persulfate, m-chloroperbenzoic acid, iron(II) chloride, and chloranil
were purchased from Sigma-Aldrich. 3-Phenyl-1,4-benzothiazine (1) and 3,3′-diphenyl-2,2′-bi(1,4-benzothiazine)
(2) were prepared as previously described.10</p><!><p>Picric acid: Compound 1 at 13 mM in ethanol was treated
with equimolar picric acid, and
the mixture was left at reflux under vigorous stirring in air. The
reaction course was followed by HPLC analysis. After 30 min at complete
consumption of the starting material, two main products at RT of 45.2 and 47.6 min at a 1:2 ratio identified
as the single-bonded dimers 2a/b (meso/dl
pair diastereoisomers) were formed. The solid that separates from
the mixture was shown to consist of a single compound (RT = 47.6 min) by HPLC analysis. Under the same conditions,
dimers 2a/b were not appreciably consumed
as evidenced by HPLC analysis.</p><p>DDQ or Chloranil: The reaction was
carried out on 1 or 2 at rt as in (a) using
dioxane as the solvent and the oxidant at equimolar concentration.
No significant consumption of the starting compound was observed in
either case over at least 2 h.</p><p>TEMPO/DPPH: Compound 1 at 2 mM in methanol was treated
with equimolar TEMPO or DPPH, and
the mixture was left under vigorous stirring in air. The reaction
course was followed by HPLC showing the complete consumption of the
starting compound after 1 h with formation of dimers 2a/b. Treatment of the mixture with an additional molar
equivalent of TEMPO or DPPH did not result in any significant consumption
of 2a/b. Compound 2a/b was treated separately with either reagent under the conditions
described for 1. No appreciable consumption was observed
in either case by HPLC analysis.</p><p>HCl/H2O2: Compound 2a/b at 50 Î¼M in methanol/HCl at a 3:1
v/v ratio up to different concentrations of the acid in the range
0.25–3 M was treated with 10 molar equivalents of H2O2 according to the protocol already developed.10 Development of the absorbance at 598 nm for
dimer 3 was monitored over 30 min.</p><p>m-Chloroperbenzoic
acid or persulfate/iron: Compound 2a/b at
50 Î¼M in methanol/HCl at a 3:1 v/v ratio was treated with m-chloroperbenzoic acid (0.8 equiv) or sodium persulfate/Fe(II)
at a 1:1 molar ratio (1.5 equiv). Development of the absorbance at
598 nm for dimer 3 was monitored over 30 min.</p><!><p>All calculations
were performed
with the Gaussian package of programs.25 Structures were geometry-optimized at the DFT level, with a hybrid
functional (PBE0)18 and a reasonably large
basis set, 6-31+G(d,p). For radical species, the unrestricted formulation
was adopted. For each chemical species, all significant tautomers
in the neutral, monoprotonated, and diprotonated state were examined.
Extensive conformational explorations were carried out, separately
for each of the above conditions, based essentially on relaxed grid
searches in torsion angle space. In those cases where conformational
enantiomers exist, a single enantiomeric series has been examined.
Computations were performed either in vacuo (neutral forms only) or
by adoption of a polarizable continuum medium (PCM)20 (all neutral, monoprotonated, and diprotonated forms) to
account for the influence of the solution environment. In view of
the faster convergence, a scaled van der Waals cavity based on universal
force field (UFF) radii26 was used, and
polarization charges were modeled by spherical Gaussian functions;27 vibrational–rotational contributions
to the free energy were also computed (at 298.15 K, in the rigid rotor-harmonic
oscillator approximation). Nonelectrostatic contributions to the solvation
free energy were disregarded at this stage; these terms were accounted
for in single-point PCM calculations at the M06-2X19/6-311++G(2d,2p) level, employing radii and nonelectrostatic
terms of the SMD solvation model.21</p><p>For computation of EPR parameters, geometry optimizations were carried
out at the unrestricted DFT level, with the B3LYP functional28 and the N07D basis set, as optimized for B3LYP,29 either in vacuo or by adoption of a polarizable
continuum medium. Single-point calculations were then carried out
with the B3LYP functional and specifically tailored basis sets, namely,
EPR-II or EPR-III;30 the sets were completed
for the sulfur center with a 6-31+G(d) or 6-311++G(2d) basis, respectively.</p><!><p>X-Band EPR spectra were collected
at 298 K in a CW spectrometer equipped with a variable temperature
unit, after mixing a solution (12–25 mM) of dimer 2 in methanol containing 3 M HCl with H2O2 (0.1–0.4
equiv) in an open (presence of atmospheric oxygen) suprasil quartz
tube with 1 mm i.d. To increase the S/N ratio, up to eight spectra
were accumulated and digitally averaged. Blank experiments in the
absence of H2O2 did not produce any detectable
EPR signal even under continuous photolysis of the mixture in the
cavity of the spectrometer with a 500 W Hg lamp. The measured g-factor was corrected with respect to that of 2,4,6-tri-tert-butylphenoxyl radical (g = 2.0046).
Optimized hyperfine constants were obtained by interactive fitting
of the experimental spectrum with simulated ones, using the Monte
Carlo method.15 Simulations were performed
with WINESR software developed by Prof. Marco Lucarini (University
of Bologna). As an initial input for computer simulations, calculated
(B3LYP, see Tables S1 and S2) values were
used along with literature data for similar structures.</p><!><p>Oxygen consumption measurements
were performed in a two-channel oxygen uptake apparatus, based on
a Validyne DP 15 differential pressure transducer built in the laboratory.17 The oxygen consumption in the sample was measured
after calibration of the apparatus from the differential pressure
recorded with time between the two channels. Monomer 1 or dimer 2 was incubated in MeOH containing 3 M H2SO4 (4 mL) at 1–2 mM at 303 K; H2O2 was added either as a single addition up to 8 mM or
in aliquots (0.25 mM equivalent each) at time intervals during the
course of oxidation. The reaction was monitored up to 24 h following
addition of H2O2; the extent of conversion to 3 was judged by color changes during the reaction and by spectrophotometry
at the end of the reaction.</p><!><p>UV–vis analysis
of the oxidation of dimer 2 with peroxides and hydroperoxides
followed by development
of the chromophore of dimer 3, EPR spectrum and oxygen
uptake measurement of 3-phenyl-1,4-benzothiazine, UV–vis analysis
of substituted anilines, computed free-energy diagram for reagents,
products, and putative intermediates in the reaction pathway leading
from 2 to 3; DFT calculations and EPR coupling
constants for neutral and protonated radical 1·; computed pKa values (PDF)</p><p>jo0c01520_si_001.pdf</p><p>The authors declare no
competing financial interest.</p>