Red-hair-inspired chromogenic system based on a proton-switched dehydrogenative free-radical coupling

Article abstract of DOI:10.1021/ol402161j

In the presence of micromolar peroxides or biometals (Fe(III), Cu(II), V(V) salts), and following a strong acid input, the stable 3-phenyl-2H-1,4- benzothiazine is efficiently converted to a green-blue Δ ^2,2′-bi(2H-1,4-benzothiazine) chromophor

Full text of DOI:10.1021/ol402161j

ORGANIC
LETTERS
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Vol. XX, No. XX
000–000
Red-Hair-Inspired Chromogenic System
Based on a Proton-Switched
Dehydrogenative Free-Radical Coupling
Loredana Leone,† Orlando Crescenzi,† Riccardo Amorati,‡ Luca Valgimigli,‡
Alessandra Napolitano,*,† Vincenzo Barone,§ and Marco d’Ischia†
Department of Chemical Sciences, University of Naples “Federico II”, Via Cinthia 4,
I-80126 Naples, Italy, Department of Chemistry “Ciamician”, University of Bologna,
Via S. Giacomo 11, I-40126 Bologna, Italy, and Scuola Normale Superiore, Piazza dei
Cavalieri 7, I-56126 Pisa, Italy
alesnapo@unina.it
Received July 30, 2013
ABSTRACT
In the presence of micromolar peroxides or biometals (Fe(III), Cu(II), V(V) salts), and following a strong acid input, the stable 3-phenyl-2H-1,
4-benzothiazine is efficiently converted to a green-blue Δ2,2 -bi(2H-1,4-benzothiazine) chromophore via dehydrogenative coupling of a 1,4-
benzothiazinyl radical. The new system is of potential practical interest for colorimetric peroxide and redox biometal detection.
0
0
Δ2,2 -Bi(2H-1,4-benzothiazine) is a unique photochro-
mic and acidichromic four-state system which provides the
system arises by a mutation-related deviation of the path-
way of melanogenesis involving the oxidative cyclization
of cysteinyldopas followed by self-coupling of the resulting
highly unstable 2H-1,4-benzothiazine intermediates.1d,e
Despite the growing interest in nature-inspired functional
systems for biomedical and technological applications, the
detailed mechanism of the key self-coupling step leading to
core π-structure of red human hair pheomelanin and tricho-
0
chrome pigments.1 In vivo, the Δ2,2 -bi(2H-1,4-benzothiazine)
† University of Naples “Federico II”.
‡ University of Bologna.
§ Scuola Normale Superiore.
2,20
the formation of the Δ -bi(2H-1,4-benzothiazine) system
(1) (a) Simon, J. D.; Peles, D. N. Acc. Chem. Res. 2010, 43, 1452. (b)
Ito, S.; Wakamatsu, K.; d’Ischia, M.; Napolitano, A.; Pezzella, A. In
Melanins and Melanosomes: Biosynthesis, Biogenesis, Physiological, and
Pathological Functions; Riley, P.A., Borovansky, J., Eds.; Wiley-VCH:
Weinheim, Germany, 2011; pp 167ꢀ185. (c) Greco, G.; Panzella, L.;
Verotta, L.; d’Ischia, M.; Napolitano, A. J. Nat. Prod. 2011, 74, 675.
(d) Napolitano, A.; Di Donato, P.; Prota, G. J. Org. Chem. 2001, 66,
6958. (e) Napolitano, A.; Memoli, S.; Crescenzi, O.; Prota, G. J. Org.
Chem. 1996, 61, 598.
has remained unknown, due to the high instability of the
2H-1,4-benzothiazine precursors.2 Herein, we report the
results of an integrated experimental and computational
approach to this issue using 3-phenyl-2H-1,4-benzothiazine
(1) as a model compound because of its unusual stability and
facile access in relatively large amounts.3
r
10.1021/ol402161j
XXXX American Chemical Society

In organic solvents (e.g., methanol) or in a neutral
aqueous medium, 1 was fairly stable to a broad range of
chemical and enzymatic oxidants, including potassium
ferricyanide, hydrogen peroxide, or, notably, peroxidase/
H2O2, remaining virtually unchanged over prolonged
periods of time up to several days.
The mechanism by which H2O2 mediates conversion of 1
to 3 under acid conditions was then investigated. Figure 1
shows the spectrophotometric course of the oxidation of 1
with acidic H2O2 (left panel) leading to formation of the
green chromophore. Data showed the relatively fast con-
version of 1 to the final product following the addition of
acid. The chromophore of dimers 2 was closely similar to
that of 1. The conversion of dimers 2a/b to 3 was therefore
separately followed (right panel). The pseudo-first-order
rate constant of 0.089 ( 0.005 minꢀ1 was determined with
1 at 50 μM and H2O2 at 500 μM based on the formation of
3 at 598 nm and decay of 2 at 320 nm (SI). Oxygen proved
Remarkably, however, exposure of 1 to H2O2 in a
strongly acidic medium (i.e., methanol/conc aq HCl 3:1
at room temperature) resulted in a fast and efficient
reaction leading to a stable blue-green chromophore
(λmax 598 nm) in a few minutes. In the absence of H2O2,
no detectable chromophore formation was observed over
the time scale of 1 h. HPLC analysisofthe reaction mixture
revealed the very rapid accumulation in the initial stages of
the reaction of colorless intermediates that were eventually
converted to the green chromophore, with no other
detectable intermediate/reaction product (see Support-
ing Information (SI)). This was also confirmed by
proton NMR monitoring of the reaction course (SI).
Workup of the mixtures allowed isolation and spectral
characterization of the colorless intermediates as the
meso/DL pair of diastereoisomers of the single-bonded
dimers 2a/b which were amply investigated by compu-
tational analysis (SI), whereas the chromophoric spe-
0
cies proved to be a mixture of Δ2,2 -bi(3-phenyl-2H-1,4-
benzothiazine) Z/E isomers 3a/b (Scheme 1) in an
approximate ratio of 3:1 (SI).
Assignment of the Z-configuration to the major compo-
nent of the mixture (3a) was made possible by comparison
of the experimental and computed 1H NMR spectra (SI).
For the mixture of isomeric 3, an overall ε598 = 5700 (
45 Mꢀ1 cmꢀ1 in methanol/36% HCl 3:1 v/v was determined,
which suggested a potential application for colorimetric
determination of H2O2.
Figure 1. Monitoring of chromophore formation by oxidation
of 1(left) and dimers 2(right) at50μM in air-equilibrated methanol/
36% HCl 3:1 v/v solutions by 500 μM H2O2 at 5 min intervals
over 30 min.
to be critical for chromophore formation from both 1 and
2a/b since no significant reaction occurred under an argon
atmosphere.
In another series of experiments, the ability of a series of
acid-compatible oxidants to bring about conversion of 1 to
3 was investigated. Besides H2O2, other peroxides such as
t-BuOOH, m-chloroperbenzoic acid (MCPBA), and ben-
zoyl peroxide also induced acid-dependent chromophore
formation, whereas persulfate at the same concentration
proved ineffective.
Scheme 1. Structures of Dimeric Oxidation Products of 1
In a screening aimed at identifying other oxidants cap-
able of promoting the oxidative coupling reaction, it was
found that some redox-active transition metal ions (e.g.,
Fe(III), V(V), and Cu(II)) were also able to generate the
chromophore (Figure 2), whereas Fe(II) was inactive even
in the presence of peroxides (i.e., under Fenton-type con-
ditions, both at neutral and acidic pH; see also Figure 2).
A possible mechanism accounting for the reported ob-
servations is given in Scheme 2. In this scheme, peroxides
or metal ions induce conversion of protonated 1 to the
resonance-stabilized benzothiazinyl radical 1. This conver-
sion may be the result of two alternative, not mutually
exclusive, routes, that is, direct H-atom abstraction and
electron transfer with concomitant/sequential proton
transfer. The former path is likely to be mediated mainly
(2) (a) Napolitano, A.; Panzella, L.; Leone, L.; d’Ischia, M. Acc.
Chem. Res. 2013, 46, 519. (b) Leone, L.; Crescenzi, O.; Napolitano, A.;
Barone, V.; d’Ischia, M. Eur. J. Org. Chem. 2012, 2012, 5136. (c) Becker,
R. S.; Natarajan, L. V. Chem. Phys. Lett. 1986, 132, 141. (d) Ciminale,
F.; Liso, G.; Trapani, G. Tetrahedron Lett. 1981, 22, 1455.
(3) (a) Santacroce, C.; Sica, D.; Nicolaus, R. A. Gazz. Chim. Ital.
1968, 98, 85. (b) Prota, G.; Giordano, F.; Mazzarella, L.; Santacroce, C.;
Sica, D. J. Chem. Soc. C 1971, 15, 2610.
B
Org. Lett., Vol. XX, No. XX, XXXX

spectra for the neutral and protonated radical form, using
calculated (B3LYP5/EPR-II6//B3LYP/N07D7) hyperfine
constants further optimized with the Monte Carlo
method.8
EPR spectra consistently gave good agreement with
those calculated for protonated 1• but not for the neutral
species, for which large coupling (∼10 G) with (C2)H is
predicted as a consequence of the larger spin density in
position 2 (SI).
Figure 2. (a) Kinetic analysis of chromophore formation at
598 nm by reaction of 50 μM 1 with 100 μM V5þ (triangles),
Fe3þ (squares), Cu2þ (circles), and H2O2 (crosses) in methanol/
36% HCl 3:1 v/v solutions at 25 °C.
Scheme 2. Proposed Mechanism of Formation of Blue-Green
Dimers 3 by Oxidation of 1 in Acidic Medium (All Formula
Numbers Refer to Protonated Products)
Figure 3. EPR spectrum of a solution of 1 in acidic MeOH and
simulated spectra of the neutral and protonated free-radical 1•.
The role of acids in triggering the H-atom abstraction
from 1 was then investigated at the PBE09/6-31þG(d,p)
level. The unrestricted formulation was used to describe
radicals, and for each species, different conformers were
explored. Computations were performed either in vacuo or
by adoption of a polarizable continuum medium.10 Pre-
dicted variations in free-energy changes associated with
H-atom abstraction from 1 and, for comparison, from the
parent 2H-1,4-benzothiazine are reported in Table 1.
Consistent with the experimental observations, calcula-
tions predicted that protonation results in a marked de-
crease in free-energy changes for H-atom abstraction from
both 1 and the parent benzothiazine. This effect would
reflect a potentiation of the pushꢀpull stabilization of
the carbon-centered free radical at C2 induced by proto-
nation at the imine group. The role of oxygen in the
proposed free-radical coupling/dehydrogenation scheme
deserves further investigation. Since product analysis did
not reveal formation of other products besides 2 and 3, and
the mass balance was completely accounted for by these
species, it follows that any oxygenated intermediate, such
as a peroxyl radical, must eventually be converted to the
final products with loss of oxygen.
by oxygenated species on the protonated benzothiazine,
while the latter would occur only on the enamine tautomer
of 1 and would be prevalent in the presence of metal ions
alone. The equilibrium of the imine and enamine forms of1
in an acidic medium should be considered in this regard
(SI). Further work is necessary to clarify this issue. Self-
coupling of 1 would then give dimer 2, which would
eventually be converted to 3 by acid-assisted dehydrogena-
tion. Spectrophotometric and HPLC analyses concurred
to indicate that conversion of 2 to 3 represents the rate
determining step of the process.
In support of the proposed scheme, the acid-promoted
(3 M HCl) oxidation of 1 in the presence of H2O2 and
atmospheric oxygen in MeOH was monitored by electron
paramagnetic resonance (EPR) spectroscopy, which re-
vealed the generation of a signal (Figure 3) attributed to a
C/N-centered radical (g = 2.0051) with hyperfine features
fully consistent with the structure of protonated radical
1•.4 Assignment was unambiguously confirmed by com-
parison of the experimental data with simulated EPR
(6) Barone, V. In Recent Advances in Density Functional Methods,
Part I; Chong, D. P., Ed.; World Scientific: Singapore, 1996.
(7) Barone, V.; Cimino, P.; Stendardo, E. J. Chem. Theory Comput.
2008, 4, 751.
(8) Amorati, R.; Pedulli, G. F.; Valgimigli, L.; Johansson, H.; Engman,
L. Org. Lett. 2010, 12, 2326.
(9) Adamo, C.; Barone, V. J. Chem. Phys. 1999, 110, 6158.
(10) (a) Miertus, S.; Scrocco, E.; Tomasi, J. J. Chem. Phys. 1981, 55, 117.
(b) Cossi, M.; Scalmani, G.; Rega, N.; Barone, V. J. Chem. Phys. 2002, 117,
43. (c) Tomasi, J.; Mennucci, B.; Cammi, R. Chem. Rev. 2005, 105, 2999.
(4) Sealy, R. C.; Hyde, J. S.; Felix, C. C.; Menon, I. A.; Prota, G.;
Swartz, H. M.; Persad, S.; Haberman, H. F. Proc. Natl. Acad. Sci. U.S.
A. 1982, 79, 2885.
(5) (a) Becke, A. D. J. Chem. Phys. 1993, 98, 5648. (b) Stephens, P. J.;
Devlin, F. J.; Chabalowski, C. F.; Frisch, M. J. J. Phys. Chem. 1994, 98,
11623.
Org. Lett., Vol. XX, No. XX, XXXX
C

Table 1. Computed Free Energy (kcal molꢀ1) of Radical
Formation from 1a
product/conditions
ΔΔrxnG°
benzothiazine, neutral form, in vacuo
benzothiazine, neutral form, MeOH
benzothiazine, monoprotonated form, MeOH
1, neutral form, in vacuo
0.0
0.5
ꢀ7.2
2.5
1, neutral form, MeOH
3.1
1, monoprotonated form, MeOH
ꢀ2.8
a All figures refer to the computed free energy of C2 radical forma-
tion from the parent neutral benzothiazine in vacuo, taken as zero. For
each species, only the most stable tautomer/conformer was considered.
Figure 5. Proton switched “OR” logic gating with peroxide and
biometal inputs and colometric output based on the oxidation of 1.
reduction of Fe(III) to Fe(II), slowing down rust genera-
tion or passivating the metal surface with Fe(II) oxides.11
An experimental protocol was also developed for detec-
tion of peroxide or metal-containing samples involving
addition of the solutions to be tested to 200 μM 1 in
methanol followed by the immediate addition of concen-
tratedHCl. Aftercareful mixing, spectrophotometricread-
ing at 598 nm after 10 min at room temperature against the
blank solution in the absence of added oxidant revealed the
presence of peroxides or metal salts. Minimal detection
limits with this protocol, as set by a ΔA598 of 0.05, were
50 μMforH2O2, 100 μMfort-BuOOH, 100 μMforMCPBA,
20 μM for benzoylperoxide, and 30ꢀ50 μM for metal ions.
From the above data, a two-input logic gate “OR”
system can be represented with the extra value of the
proton switch (Figure 5). Biologically and technologically
relevant inputs such as peroxides and redox-active biome-
tals lead to a colorimetric output at 598 nm, completing the
Hþ-switched OR logic gate. We plot the “truth table” for
the response of these inputs in which output “1” is a ΔA598
of at least 0.05 and “0” is no change in A598 (SI).
In conclusion, we have disclosed a unique acid-induced
free-radical coupling pathway inspired by red hair pigment
synthesis, which has a potential for routine colorimetric
detection of micromolar peroxides as well as of redox-active
metal ions. The recognition process involves color changes
from yellow to green that are clearly visible to the naked eye.
To our knowledge, this is the first probe system for both
peroxides and Fe(III), Cu(II), and V(V) detection regulated
by a proton switch. These results would expand the scope of
benzothiazine chemistry beyond main current applications
as photoinitiator dyes12 and in medicinal chemistry.13
Figure 4. (A) Development of the chromophore of 3on addition of
200 μM 1 in 3:1 methanol/36% HCl to an ethyl ether sample kept 1
month in air on the laboratory bench at a 3:1 v/v ratio before (left
vial) and after (right vial) passage through a basic alumina column.
(B) Rusty iron nails before (bottom) and after (middle) immersion
in 36% HCl overnight and in the presence of 6 mM 1 (top).
Based on these results, the scope of the new chromogenic
system was briefly assessed. Interestingly, 1 proved to be
useful for the visual detection of peroxides in aged ethereal
solvents such as THF, ethyl ether, dioxane (Figure 4A).
Typically, the solvent to be tested was mixed with a
solution of 200 μM 1 in 3:1 methanol/36% HCl (3:1 v/v),
and the absorbance at 598 nm was measured spectro-
photometrically. The method can be conveniently used
for routine peroxide quantitations also on a visual basis.
As a curious aside to these experiments, it was noticed that
addition of rusty iron objects to the typical mixture promoted
oxidation of as high as 6 mM 1 in a very fast and efficient
manner. Under these conditions, 1 was found to serve as an
efficient inhibitor against corrosion of the rusty iron objects
induced by concentrated HCl (Figure 4B). On average, 1could
decrease weight loss from rusty iron nails or staples immersed
in concentrated HCl by ∼50% over 24 h. Though the
mechanism by which 1 inhibits corrosion is at present unclear,
it is conceivable that the effect is related in some way to the
Acknowledgment. This work was supported by Italian
MIUR, PRIN 2010-11 (PROxi project).
Supporting Information Available. Experimental pro-
cedures, spectral and computational data for 1ꢀ3, com-
plete computational and EPR data for 3-phenyl-1,4-
benzothiazinyl radical. This material is available free of
charge via the Internet at http://pubs.acs.org.
(11) Nagahama, J.; Yumoto, H. Surf. Coat. Technol. 2003, 169 ꢀ 170,
658–661.
(12) (a) Podsiadzy, R. J. Photochem. Photobiol., A 2009, 202, 115. (b)
Podsiadzy, R.; Michalski, R.; Marcinek, A.; Sokozowska, J. Int. J.
Photoenergy 2012, 1, 497620.
(13) Qiao, Z.; Liu, H.; Xiao, X.; Fu, Y.; Wei, J.; Li, Y.; Jiang, X. Org.
Lett. 2013, 15, 2594.
The authors declare no competing financial interest.
D
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