Laser-induced in vitro isomerization of urocanic acid in UVA region and the origin of excited triplet state

Article abstract of DOI:10.1016/S0040-4039(02)02210-4

We demonstrate here unambiguously that (E)-urocanic acid, an epidermal and photobiologically important chromophore, undergoes isomerization to the (Z) isomer upon excitation with UVA (320-400 nm) light using monochromatic 355 and 340 nm laser radiation. Additionally, chemical evidence is presented that supports the isomerization coming from the excited singlet state, thus proving unequivocally that the previously observed triplet state is populated from the excited singlet manifold via intersystem crossing rather a weak S₀→T₁ transition.

Full text of DOI:10.1016/S0040-4039(02)02210-4

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 8897–8900
Laser-induced in vitro isomerization of urocanic acid in UVA
region and the origin of excited triplet state
Taj Mohammad*
Department of Chemistry, Purdue University, West Lafayette, IN 47907-1393, USA
Received 5 August 2002; accepted 2 October 2002
Abstract—We demonstrate here unambiguously that (E)-urocanic acid, an epidermal and photobiologically important chro-
mophore, undergoes isomerization to the (Z) isomer upon excitation with UVA (320–400 nm) light using monochromatic 355 and
3
40 nm laser radiation. Additionally, chemical evidence is presented that supports the isomerization coming from the excited
singlet state, thus proving unequivocally that the previously observed triplet state is populated from the excited singlet manifold
via intersystem crossing rather a weak S “T transition. © 2002 Elsevier Science Ltd. All rights reserved.
0
1
Urocanic acid (2-propenoic acid, 3-[1H-imidazol-4(5)-
yl], UA) continues to attract interest of biologists,
environmentalists, photochemists, photobiologists,
medicinal chemists, and (photo)immunologists, primar-
ily due to its presence in the upper layer of the mam-
malian skin and its excellent light absorbing
1
,2
properties. It is synthesized in vivo from the essential
amino acid histidine by the action of the enzyme histi-
dase, a histidine-ammonia-lyase (EC 4.3.1.3), and accu-
mulates in the epidermis in high concentration (ca. 30
(1)
2
mg/cm ). Upon exposure to UV light, the naturally
occurring isomer, trans, i.e. (E)-urocanic acid [(E)-UA],
undergoes isomerization to produce cis, i.e. (Z)-uro-
canic acid [(Z)-UA]. The latter is of current immense
dependent chemistry. However, there are recent reports
describing UA photoreactivity in the UVA (320–400
nm) region, where it shows minimum absorption. This
spectral region has become increasingly more important
since more UVA radiation penetrates from the sun to
the earth due to constant depletion of the stratospheric
(
photo)biological interest because of its immunosup-
2
pressive properties in several animal models.
Until recently, the photochemistry and photobiology of
UA has been restricted to the UVB (280–320 nm) and
UVC (200–280 nm) region of the electromagnetic spec-
trum due to its significant absorption in these regions.
The most efficient photochemical reaction is (E)/(Z)
isomerization (Eq. (1)), which shows a wavelength
6
7,8
2,9
ozone layer. Direct as well as sensitized photoiso-
merization has been observed upon excitation of UA
with the UVA-I (340–400 nm) and UVA-II (320–340
nm) light. Since in vivo photoisomerization could also
result from the sensitization of UA by the endogenous
9
8,9
3
4
5
chromophores, and there are contradictory reports
dependency. Both spectroscopic and theoretical data
support the presence of multiple electronic transitions
within the broad structureless absorption band centered
ca. 275 nm, and these account for the wavelength-
on the UVA-induced in vitro isomerization (perhaps
due to broadband light sources used in these studies),
we demonstrate here unequivocally that excitation of
UA with the 355 and 340 nm monochromatic laser light
leads to isomerization of (E)-UA to (Z)-UA.
Keywords: isomerization; laser excitation; singlet oxygen; urocanic
acid; UVA absorption.
Recently, Hanson and Simon observed for the first time
that 351 nm laser-excitation of UA leads to the sensi-
tization of singlet oxygen via triplet energy transfer
*
Present address: Department of Forestry and Natural Resources,
Purdue University, West Lafayette, IN 47907-1159, USA. Tel.:
10
7
65-494-3569; fax: 765-496-2422; e-mail: taj@purdue.edu
mechanism. They have used sensitive techniques of
0
040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)02210-4

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T. Mohammad / Tetrahedron Letters 43 (2002) 8897–8900
emission spectroscopy and photoacoustic calorimetry to
establish the production of singlet oxygen and excited
triplet state of UA, respectively. However, it was not
established if the excited triplet state arrives via inter-
system crossing from the excited singlet state or it is
medium-pressure mercury vapor lamp. Degassed dupli-
cate solution of (E)-UA was irradiated at 8°C for 24 h.
The reversed-phase high-performance liquid chro-
14
matography (RP-HPLC) analysis indicated a 10.9%
(
±1.8%) formation of (Z)-UA. A second set of dupli-
10
cate solution was irradiated under identical conditions
directly populated from the ground singlet state. We
present here chemical evidence in favor of the former
scenario. This is reminiscent that often times the chem-
ical methodology has proven an invaluable tool to
but saturated with oxygen, when a 10.5% (±1.9%)
15
formation of (Z)-UA was observed. The lack of
quenching of isomerization by oxygen suggests the
involvement of excited singlet state in this process since
oxygen quenches the UA triplet state via energy trans-
1
1
distinguish between such dichotomy.
10
fer mechanism. The amount of (Z)-UA increased
linearly with the light dose concomitant to the disap-
pearance of the (E) isomer in a time-dependent manner.
The mass balance during photoisomerization was excel-
lent since no new product besides (Z)-UA was
observed. No isomerization was observed in the dark
when analyzed after 24, 48 and 72 h (data not shown).
The electronic absorption spectrum of a 49.2 mM solu-
tion of (E)-UA in 50 mM phosphate buffer (pH 7.0) is
presented in Fig. 1. The structureless absorption maxi-
mum in the UVB region centers at 277 nm and there is
negligible absorbance in the UVA region. For compari-
12
son the unpublished absorption spectrum of (Z)-UA
in the UVA region at equal molar concentration is also
included in Fig. 1, and it has absorption band similar to
the (E) isomer, but is accompanied by 5 nm hyp-
sochromic shift with reduced absorptivity. The absorp-
tion spectra of UA isomers in concentrated solutions
Since the medium-pressure mercury vapor lamp has a
continuum of wavelengths ranging from UV into IR,
the 366-nm light tested in the UA photoisomerization
could be contaminated with other wavelengths. To rule
out the possibility for the leakage of some short wave-
length(s), we utilized the monochromatic 355-nm line
from the Nd:YAG laser to confirm UA isomerization.
Indeed, irradiation of a magnetically stirred and unde-
gassed (E)-UA solution at ambient temperature with
the third harmonic of a Continuum Nd:YAG laser
(
3.0 mM) used in the photoisomerization studies are
presented as inset in Fig. 1. Both the spectra are
remarkably similar and are associated with tailing in
the UVA region. The absorbance values at the excita-
tion wavelength of 355 nm are 0.014 and 0.012 for (E)-
and (Z)-UA, respectively. The absorbance value for
(
E)-UA is comparable with the reported value in the
(u =355 nm) produced a 3.6% (Z)-UA after 60 min.
irr
1
0
this region.
As observed above in the 366-nm experiment, the pho-
toisomerization with the laser radiation was also dose
dependent, and data for a time course irradiation are
presented in Fig. 2. There is a linear formation of
Early experiments for UA photoisomerization with the
1
3
UVA light employed 366 nm radiation from a
Figure 1. Electronic absorption spectrum of 49.2 mM (E)-UA and (Z)-UA in 50 mM sodium phosphate buffer (pH 7.0) at room
temperature. The corresponding spectra of concentrated solutions (3.0 mM) in the UVA region are depicted in the inset.

T. Mohammad / Tetrahedron Letters 43 (2002) 8897–8900
8899
Figure 2. A time course photoisomerization of (E)-UA solu-
tion with the 355-nm laser radiation at room temperature. At
specified intervals, the photolysates were analyzed by RP-
HPLC.
Figure 3. Effect of argon and oxygen on irradiation of (E)-
UA with the 355 and 340 nm laser radiation at room temper-
ature; (a) unirradiated, (b) irradiation at 355 nm under argon,
(
c) irradiation at 355 nm under oxygen, and (d) irradiation at
(
Z)-UA concomitant to the disappearance of (E)-UA
340 nm under oxygen. The run time was 25 min for each
chromatogram.
with the light dose. Thus, despite the low extinction
coefficient of UA in the UVA region, we have con-
firmed that UA isomerizes in vitro in the absence of a
photosensitizer.
arguments and conclusions are depicted in Scheme 1. If
3
path ‘a’ for direct population of (E)-UA is valid and
responsible for singlet oxygen sensitization, we should
3
The issue of the nature of the excited state responsible
for the UA chemistry in the UVA region was intrigu-
ing, especially the excited state that produces reactive
oxygen species. Hanson and Simon have observed the
population of excited triplet state of UA upon excita-
tion with the 351-nm laser radiation. The excited triplet
not have observed formation of (Z)-UA since (E)-UA
1
does not isomerize. This is supported from our data
that oxygen does not quench UA isomerization, albeit
quenches the excited triplet state. Alternatively, the
1
0
(
E)-UA is excited to the singlet manifold (path ‘b’),
which can decay either via isomerization (path ‘c’) or
crosses over to the previously observed triplet state
(path ‘d’). We have observed both reactions corre-
state was quenched by ground triplet state molecular
1
oxygen leading to the generation of singlet oxygen ( O ,
2
15
1
sponding to path ‘c’ and ‘d’, thus unequivocally prov-
ing that UA is initially populated to the excited singlet
state which is responsible for the population of previ-
D ). The authors confirmed this by observing singlet
g
1
3
oxygen emission due to D “ S decay. We have stud-
g
g
ied simultaneously the effect of argon and oxygen on
the rate of UA photoisomerization. The (E)-UA solu-
tions were irradiated with the 355-nm laser line for 4 h.
The RP-HPLC results are presented in Fig. 3, and
indicate comparable formation of (Z)-UA, i.e. 11.9 and
10
ously observed UA excited triplet state. These conclu-
sions are in good agreement with the most recent
femtosecond time-resolved spectroscopic data identify-
ing a short-lived (ca. 1.0 ps) excited singlet state of UA,
1
3.0% under argon and oxygen, respectively. Since oxy-
gen did not inhibit UA isomerization, supporting that
the excitation of UA initially populates the singlet state
that undergoes rapid isomerization (in competition with
intersystem crossing to the excited triplet state). The
action spectrum for the formation of UA triplet has
been observed at 340 nm, we employed this wave-
length to test the UA isomerization. The data from
irradiation of an oxygen-saturated solution of (E)-UA
with this wavelength are included in Fig. 3. One notes
that this wavelength is equally effective in converting
10
(
E)-UA to (Z)-UA, and in reality it seems more
efficient (38.6% isomerization after 2.5 h) than 355-nm
light because of the increased absorption of UA at
shorter wavelengths. Since UA triplet state is quenched
by oxygen and in our hands oxygen did not intercept
isomerization, we conclude that the unimolecular UA
reaction comes from the excited singlet state. These
1
0
Scheme 1.

8
900
T. Mohammad / Tetrahedron Letters 43 (2002) 8897–8900
which populates excited triplet state via intersystem
7. Webber, L. J.; Whang, E.; De Fabo, E. C. Photochem.
Photobiol. 1997, 66, 484–492.
8. Kammeyer, A.; Teunissen, M. B. M.; Pavel, S.; De Rie,
M. A.; Bos, A. D. Br. J. Dermatol. 1995, 132, 884–891.
1
6
crossing. Theoretical calculations on the UA con-
formers also support the presence of such excited state
1
7
transitions in the UVA region.
9
. (a) Gibbs, N. K.; Torr, G.; Johnson, B. E. J. Photochem.
Photobiol. B: Biol. 1996, 34, 63–66; (b) Gibbs, N. K.;
Norval, M.; Traynor, N. J.; Wolf, M.; Johnson, B. E.;
Crosby, J. Photochem. Photobiol. 1993, 57, 584–590. Cor-
rection, Photochem. Photobiol. 1993, 58, 769.
In conclusion, we have established that the monochro-
matic 355 and 340 nm laser radiation of the UVA
region are effective in the in vitro UA isomerization,
and this reaction initiates from the excited singlet man-
ifold. The UA triplet state responsible for the sensitiza-
tion of singlet oxygen is derived from the excited singlet
state. Future studies will be focused on the chemical
reactivity of these excited states with biological
molecules in understanding the consequences of UA/
UVA chemistry in photocarcinogenesis and photoaging
of the skin.
1
1
1
1
0. Hanson, K. M.; Simon, J. D. Proc. Natl. Acad. Sci. USA
998, 95, 10576–10578.
1. Mohammad, T.; Morrison, H. J. Am. Chem. Soc. 1996,
18, 1221–1222.
2. Mohammad, T.; Morrison, H. Org. Prep. Proceed. Int.
000, 32, 581–584.
1
1
2
3. For the photoisomerization experiments, a 2.0 mL solu-
tion of (E)-UA (3.0 mM) in 50 mM phosphate buffer
(
pH 7.0) was irradiated with one of the following light
Acknowledgements
sources: (a) Canrad-Hanovia 450 W medium-pressure
mercury vapor lamp surrounded by a cylindrical uranium
yellow glass filter that transmits wavelengths >330 nm
Financial support from the Purdue Gerontology Pro-
gram is gratefully acknowledged. The assistance of Dr.
Hartmut Hedderich of the Chemistry Department
Laser Facility, and Jason Clark of Professor Edward
Grant’s group in the use of 355 and 340 nm laser setup,
respectively, is also acknowledged. Dr. Harry Morrison
provided the laboratory facilities. We appreciate
encouragement from Professor Dennis Le Master.
(
uirr=366 nm); (b) third harmonic of a Continuum
Nd:YAG laser (u =355 nm) operating at 10 Hz with a
Q-switch delay of 265 ms with an output power of 90
mW; (c) dye-pumped excimer laser (u =340 nm) operat-
ing at 10 Hz with an output power of 50 mW. Prior to
irradiation, the solutions were either degassed with argon
or saturated with oxygen for 15 min, and sealed with
septum and parafilm.
irr
irr
14. The analysis and separation of the UA isomers were
carried on a Rainin Microsorb-MV C8 analytical column
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