Generation and characterization of the selenocysteinyl radical: Direct evidence from time-resolved UV/Vis, electron paramagnetic resonance, and fourier transform infrared spectroscopy

Article abstract of DOI:10.1021/jo050479j

The selenocysteinyl radical 1 has been generated for the first time by laser flash photolysis (λ_exc = 266 nm) of dimethyl bis(N-tert-butoxycarbonyl)-L-selenocystine 2 and of [(9-fluorenylideneamino)- oxycarbonyl]methyl(N-tert-butoxycarbonyl)-L-selenocysteine 3 in acetonitrile and characterized by time-resolved (TR) UV/Vis, Fourier transform infrared (FTIR), and electron paramagnetic spectroscopy in combination with theoretical methods. A detailed product study was conducted using gas chromatography and one- and two-dimensional NMR spectroscopy. In the case of [(9-fluorenylideneamino) oxycarbonyl]methyl(N-tert-butoxycarbonyl)-L-selenocysteine 3, the (9-fluorenylideneamino)oxycarbonyl moiety serves as a photolabile protection group providing a caged selenocysteinyl radical suitable for biophysical applications. Cleavage of the diselenide bridge or the selenium-carbonyl bond by irradiation is possible in high quantum yields. Because of the lack of a good IR chromophore in the mid-IR region, the selenocysteinyl radical 1 cannot be monitored directly by TR FTIR spectroscopy. TR UV/Vis spectroscopy revealed the formation of the selenocysteinyl radical 1 from both precursors. The selenocysteinyl radical 1 has a lifetime τ ≈ 63 μs and exhibits a strong band located at λ_max = 335 nm. Calculated UV absorptions of the selenocysteinyl radical (UB3LYP/6-311G(d,p)) are in good agreement with the experimental results. The use of TR UV/Vis spectroscopy permitted the determination of the decay rates of the selenocysteinyl radical in the presence of two quenchers. The product studies demonstrated the reversible photoreaction of dimethyl bis(N-tert-butoxycarbonyl) -L-selenocystine 2. Products of the photolysis of the caged selenocysteinyl radical precursor 3 are dimethyl bis(N-tert- butoxycarbonyl)-L-selenocystine 2, carbon dioxide, and some further smaller fragments. In addition, the photo-decomposition of the (9-fluorenylideneamino) oxycarbonyl moiety produced 9-fluorenone-oxime 4, 9-fluoren-imine 5, and 6 and 7 as products of the dimerization of two 9-fluorenoneiminoxy radicals 8.

Full text of DOI:10.1021/jo050479j

Generation and Characterization of the Selenocysteinyl Radical:
Direct Evidence from Time-Resolved UV/Vis, Electron
Paramagnetic Resonance, and Fourier Transform Infrared
Spectroscopy
Christoph Kolano,*,† Go¨tz Bucher,* Olaf Schade, Dirk Grote, and Wolfram Sander*
Lehrstuhl fu¨r Organische Chemie II der Ruhr-Universita¨t Bochum, Universita¨tsstrasse 150,
44801 Bochum, Germany
wolfram.sander@rub.de; goetz.bucher@rub.de; c.kolano@pci.unizh.ch
Received March 10, 2005
The selenocysteinyl radical 1 has been generated for the first time by laser flash photolysis (λexc
)
266 nm) of dimethyl bis(N-tert-butoxycarbonyl)-L-selenocystine 2 and of [(9-fluorenylideneamino)-
oxycarbonyl]methyl(N-tert-butoxycarbonyl)-L-selenocysteine 3 in acetonitrile and characterized by
time-resolved (TR) UV/Vis, Fourier transform infrared (FTIR), and electron paramagnetic
spectroscopy in combination with theoretical methods. A detailed product study was conducted
using gas chromatography and one- and two-dimensional NMR spectroscopy. In the case of [(9-
fluorenylideneamino)oxycarbonyl]methyl(N-tert-butoxycarbonyl)-L-selenocysteine 3, the (9-fluorenyl-
ideneamino)oxycarbonyl moiety serves as a photolabile protection group providing a “caged
selenocysteinyl radical” suitable for biophysical applications. Cleavage of the diselenide bridge or
the selenium-carbonyl bond by irradiation is possible in high quantum yields. Because of the lack
of a good IR chromophore in the mid-IR region, the selenocysteinyl radical 1 cannot be monitored
directly by TR FTIR spectroscopy. TR UV/Vis spectroscopy revealed the formation of the
selenocysteinyl radical 1 from both precursors. The selenocysteinyl radical 1 has a lifetime τ ≈ 63
µs and exhibits a strong band located at λmax ) 335 nm. Calculated UV absorptions of the
selenocysteinyl radical (UB3LYP/6-311G(d,p)) are in good agreement with the experimental results.
The use of TR UV/Vis spectroscopy permitted the determination of the decay rates of the
selenocysteinyl radical in the presence of two quenchers. The product studies demonstrated the
reversible photoreaction of dimethyl bis(N-tert-butoxycarbonyl)-L-selenocystine 2. Products of the
photolysis of the “caged selenocysteinyl radical” precursor 3 are dimethyl bis(N-tert-butoxycarbonyl)-
L-selenocystine 2, carbon dioxide, and some further smaller fragments. In addition, the photo-
decomposition of the (9-fluorenylideneamino)oxycarbonyl moiety produced 9-fluorenone-oxime 4,
9-fluoren-imine 5, and 6 and 7 as products of the dimerization of two 9-fluorenoneiminoxy radicals
8.
Introduction
element, on the other hand, it has found medical applica-
tions.2,3 Thus, selenium shows strong interactions with
heavy metals such as cadmium, silver, and mercury.
Because of the significant concentrations of these heavy
metals in marine food, supplements of selenium are
required to diminish their toxic effects.
Selenium is a janus-faced element. While it is highly
toxic, small doses of selenium are essential for the life of
mammals and bacteria on earth. The toxicity is of
relevance due to the use of selenium and selenium
compounds in semiconductor and pharmaceutical indus-
try as well as in synthetic organic chemistry.1 As a trace
(1) Ogawa, A. Main Group Met. Org. Synth. 2004, 2, 813-866.
(2) Liu, T. Chem. Eng. News 2003, 81, 94.
(3) Thomson, C. D. Eur. J. Clin. Nutr. 2004, 58, 391-402.
† Present address: Physikalisch-Chemisches Institut der Universita¨t
Zu¨rich, Winterthurerstr. 190, CH-8057 Zu¨rich, Switzerland.
10.1021/jo050479j CCC: $30.25 © 2005 American Chemical Society
Published on Web 07/20/2005
J. Org. Chem. 2005, 70, 6609-6615
6609

Kolano et al.
CHART 1. Established Selenyl Radicals
selenocysteinyl radical 1 using product studies (NMR
spectroscopy and gas chromatography (GC)), time-
resolved (TR) UV/Vis, Fourier transform infrared IR
(FTIR), and electron paramagentic resonance (EPR), and
theoretical methods giving detailed insights into the
reactivity of a selenium centered radical of biological
importance.
In biological systems, selenium occurs as a constituent
of amino acids and other organic compounds, and in the
last years the biological role of selenium amino acids has
been intensely studied. Selenocysteine (Sec), selenocys-
tine, and selenomethionine were found in the polypeptidic
chains of several selenium-containing enzymes, among
them glutathione peroxidase, selenoprotein P, glycine
reductase, and thioredoxin reductase. Selenocysteine
has been recognized as the 21st “natural” amino acid.4
Among the selenoproteins, glutathione peroxidase
is of major interest due to its ability to catalyze reduc-
tion of reactive oxygen species (ROS), namely, super-
oxide anion and hydroxyl radical, at expense of gluta-
thione.3,5-7
It has been reported that selenium compounds (seleno-
cystine and ebselen) are effective scavengers of highly
reactive intermediates in the human body.9 Thus, traces
of these compounds are widely used as therapeutics to
diminish the cytotoxic properties of the hydroxyl radical
and the superoxide anion. Recently, the effect of selenium-
containing therapeutics has been discussed controver-
sially in the literature. There is strong evidence that,
while selenium compounds may quench free radicals
effectively, they are also involved in a mechanism that
is responsible for a new secondary formation of this type
of radicals.7
So far, organic selenium-centered radicals have not
been much studied. Only five types of radicals have been
investigated, none of them in context with a possible
medical or biophysical application. Among them are the
monomethyl selenide (9),10 a series of para-substituted
arylselenyl radicals 10a-b,11,12 the (2-hydroxyprop-1-yl)
selenyl radical (11),13 and the 1-naphthylselenyl and
2-naphthylselenyl radicals (12a-b)14 (Chart 1).
Results and Discussion
Dimethyl Bis(N-tert-butoxycarbonyl)-L-selenocys-
tine 2. Laser flash photolysis (LFP) (λexc ) 266 nm) of 2
in degassed CH3CN led to the formation of a long-lived
transient species with a first-order decay kinetics (kobs
) 1.6 × 104 s-1, τ ) 63 µs) and an absorption maximum
λmax ) 335 nm. In addition, a short-lived transient species
was observed with λmax ) 450 nm and a lifetime τ of 18
µs (kobs ) 5.4 × 104 s-1) (Figures 1 and 2). None of the
transients could be quenched by purging the solution
with oxygen.
LFP studies had previously been carried out on naph-
thyl diselenides (1-NaphSe)2 and (2-NaphSe)2, which
yielded the 1-naphthylselenyl and 2-naphthylselenyl
radicals 12a-b upon excitation.14 These radicals exhibit
absorption maxima at 420 and 680 nm for the 1-naph-
thylselenyl and 490 and 680 nm for the 2-naphthylselenyl
radicals, respectively. Our results indicate that both the
diselenide bridge (Se-Se cleavage, pathway (I)) and a
C-Se bond (pathway (II)) are cleaved upon laser excita-
tion of 2, resulting in formation of the selenocysteinyl
radical 1, the diselenocysteinyl radical 13, and the
corresponding alkyl radical 14 (Scheme 1).
Important properties of chalcogen-atom-centered radi-
cals are their reactivity toward carbon-carbon mul-
tiple bonds and their high affinity toward heavy metals.
Here we describe the generation and characterization of
(4) Anon Eur. J. Biochem. 1999, 264, 607-609.
(5) Stryer, L. Biochemie; 4. Auflage ed.; Heidelberg, Berlin, Oxford,
1996.
(6) Gettins, P.; Crews, B. C. J. Biol. Chem. 1991, 266, 4804-4809.
(7) Okuno, T.; Kawai, H.; Haegawa, T.; Ueno, H.; Nakamuro, K. J.
Health Sci. 2001, 47, 240-247.
(8) Roussyn, I.; Briviba, K.; Masumoto, H.; Sies, H. Arch. Biochem.
Biophys. 1996, 330, 216-218.
(9) Tappel, A. L.; Caldwell, K. A. Science 1967, 345-361.
(10) BelBruno, J. J.; Spacek, J.; Christophy, E. J. Phys. Chem. 1991,
95, 6928-6932.
To confirm the assignment of the radical spectra, we
calculated the UV/Vis spectra of the model radicals 15
and 16 (corresponding to 1 and 13 without the C- and
N-terminal protecting groups) using time-dependent
density functional theory (DFT) (TD-UB3LYP/6-311G(d,p)//
UB3LYP/6-311G(d,p)). The calculated absorption maxima
(11) Holm, A. H.; Yusta, L.; Carlqvist, P.; Brinck, T.; Daasbjerg, K.
J. Am. Chem. Soc. 2003, 125, 2148-2157.
(12) Ito, O. J. Am. Chem. Soc. 1983, 105, 850-853.
(13) Barton, D. H. R.; Jacob, M.; Peralez, E. Tetrahedron Lett. 1999,
40, 9201-9204.
(14) Alam, M. M.; Ito, O.; Koga, Y.; Ouchi, A. Int. J. Chem. Kinet.
1998, 30, 193-200.
6610 J. Org. Chem., Vol. 70, No. 17, 2005

The Selenocysteinyl Radical
FIGURE 2. Transient decay traces, recorded at λ ) 340 nm
(black dots) and 450 nm (white dots), upon laser flash
photolysis (λexc ) 266 nm) of 2 in acetonitrile at ambient
temperature.
TABLE 1. Observed and Calculated UV/Vis Maxima of
the Selenocysteinyl Radical 15 and the Diselenocysteinyl
Radical 16 as Models for the Protected Radicals 1 and 13
calcd λ
(nm)a
15
calcd rel
int (%)a
15
calcd λ
(nm)a
16
calcd rel
int (%)a
16
exptl λ
(nm)
648.8
490.6
363.2
6.1
100.0
8.1
501.5
464.3
399.5
61.2
65.7
100.0
450
335
FIGURE 1. (Top) Transient absorption spectra following 266
nm laser excitation [traces recorded after 1 µs (black dots), 5
µs (white dots), 17 µs (black triangles), and 75 µs (white
triangles), spectral resolution 5 nm, cell size 1.0 cm] in argon-
purged acetonitrile displaying the photochemistry of 2. (Bot-
tom) Transient absorption spectra following 266 nm laser
excitation [traces recorded after 1 µs (black dots), 5 µs (white
dots), 17 µs (black triangles), and 75 µs (white triangles),
spectral resolution 5 nm, cell size 1.0 cm] in oxygen-purged
acetonitrile displaying the photochemistry of 2.
a UB3LYP/6-311G(d,p).
SCHEME 1. Fragmentation Pattern of 2
FIGURE 3. Decay rate of 1 in the presence of variable
concentrations of tetramethylallene.
tions.15-17 Therefore, quenching experiments of 1 with
tetramethylallene were performed (Figure 3). A rate
constant of 1.1 × 105 L‚mol-1‚s-1 was determined for this
reaction.
In a similar way, the rate constant for the reaction of
1 with ethylpropiolate was determined to be 3.0 × 105
L‚mol-1‚s-1 (Figure 4). The deviation from linearity of the
regressions is caused by the absorption of laser light by
the quencher at higher concentrations. The measure-
ments have been performed in static cells where ap-
propriate mixing of the solution is not guaranteed under
all conditions. Thus, intramolecular reactions might
influence the local concentrations of the quencher.
are in good agreement with the experimental results
(Table 1).
(15) Ogawa, A.; Doi, M.; Tsuchii, K.; Hirao, T. Tetrahedron Lett.
2001, 42, 2317-2319.
(16) Ogawa, A.; Yokoyama, K.; Yokoyama, H.; Sekiguchi, M.; Kambe,
N.; Sonoda, N. Tetrahedron Lett. 1990, 31, 5931-5934.
(17) Ogawa, A.; Yokoyama, H.; Yokoyama, K.; Masawaki, T.; Kambe,
N.; Sonoda, N. J. Org. Chem. 1991, 56, 5721-5723.
The phenylselenyl radical (10a) is known to react
quickly with double and triple bonds in organic reac-
J. Org. Chem, Vol. 70, No. 17, 2005 6611

Kolano et al.
SCHEME 2.
Syntheses of [(9-Fluorenylidene-
amino)oxycarbonyl]methyl(N-tert-butoxy-
carbonyl)-L-selenocysteine (3)
FIGURE 4. Decay rate of 1 in the presence of variable
concentrations of ethylpropiolate.
These rate constants are in good agreement with the
results obtained for the reaction of the phenyselenyl
radical 10a with allenes.15-17 The reactivity of 1 is slightly
higher than that of 10a. This can be rationalized by some
degree of stabilization of the phenylselenyl radical 10a
by the aromatic ring system.
Because of the lack of a good IR chromophore in the
mid-IR region, the selenocysteinyl radical 1 cannot
be monitored directly by TR FTIR. DFT calculations
((UB3LYP/6-311G(d,p)) on the model radicals 15 and 16
predict very weak stretching vibrations in the region
around 630 cm-1 (δC-Se ) 641.3 cm-1 of 15, δSe-Se ) 324.8
cm-1, and δC-Se ) 623.5 cm-1 of 16), located in a spectral
region not accessible to our TR FTIR spectrometer.
The synthesis of 3 is outlined in Scheme 2. The key
intermediate, dimethyl bis(N-tert-butoxycarbonyl)-L-
selenocystine 2, was synthesized via a published proce-
dure.20-22 Reduction of the diselenide bridge of seleno-
cystine 2 was achieved in the presence of sodium boro-
hydride at low temperature, followed by the addition
of 9-fluorenone-oxime-chloroformate 17. [(9-Fluorenyl-
ideneamino)oxycarbonyl]methyl(N-tert-butoxycarbonyl)-
L-selenocysteine 3 was obtained in good yields and shows
excellent thermal stability.
LFP (λexc ) 266 nm) of 3 in degassed CH3CN at
ambient temperature resulted in the bleaching of the 300
nm absorption, indicating dissociation of the precursor.
A transient with λmax ) 660 nm and a lifetime of 4.2 µs
was assigned to the 9-fluorenoneiminyl radical 18, in
agreement with published data.23 In addition, a transient
with λmax ) 340 nm overlapping with an absorption
growing in was observed (Figure 5). Neither transient
lifetimes nor absorption maxima were affected by purging
the solution with oxygen.
A more detailed evaluation of the trace at 340 nm
revealed a complex kinetic behavior (Figure 6). The
transient initially decays with approximated first-order
kinetics. At the same monitoring wavelength, a second-
order growth (λmax ) 360 nm) partially overlapping with
the decay is observed. A comparison between the initial
part of the decay trace monitored at λ ) 340 nm with
the kinetic results obtained from the LFP of 2 revealed
that both transients decay with the same kinetics (inset
of Figure 6). Thus, the 340 nm transient (decay) is
assigned to the selenocysteinyl radical 1.
The assignment of the second-order growth proved to
be more difficult, and structures 4-8, 19, and 20 were
considered. 9-Fluorenoneoxime 4, 9-fluorenoneketimine
5, and 9-fluorenone 19 can be excluded on the basis of
their known UV/Vis spectra (Table S1). Dimers of the
9-fluorenoneiminoxy radical 8 are another potential
source of the transient growth observed. In an earlier
study on the photochemistry of 9-fluorenone oxime car-
bamates, no spectroscopic evidence could be gained for
The formation of 1 as a major product of the photolysis
of 2 was confirmed by product studies at ambient
temperature. A degassed solution of 2 in CD3CN was
irradiated with the 266-nm light of a Nd:YAG laser.
1
Before and after photolysis H, 13C, 77Se, heteronuclear
multiple quantum coherence (HMQC), and total correla-
tion spectroscopy (TOCSY) NMR spectra were recorded.
All signals in the spectra after irradiation can be clearly
assigned to the starting material 2 (Table S3 and S4).
Comparison of the 77Se NMR spectra before and after
irradiation ruled out the formation of other fragments
or selenoorganic compounds (Figure S5). The apparent
absence of photochemistry of 2 under conditions (laser
irradiation) where fragmentation is observed in the LFP
experiments indicates the efficient recombination of the
fragment radicals 1 back to 2. This recombination is
obviously a quantitative reaction since no other products
are formed.
[(9-Fluorenylideneamino)oxycarbonyl]methyl(N-
tert-butoxy-carbonyl)-L-selenocysteine 3. It was re-
cently shown that molecules functionalized with the (9-
fluorenylideneamino)oxycarbonyl group are useful photo
precursors for the generation of radicals.18,19 Therefore,
we synthesized [(9-fluorenylideneamino)oxycarbonyl]-
methyl(N-tert-butoxycarbonyl)-L-selenocysteine 3 as an
independent precursor for the selenocysteinyl radical 1.
Compound 3 should also be suitable as a “caged seleno-
cysteinyl radical” in biophysical applications.
(20) Barton, D. H. R.; Bridon, D.; Herve, Y.; Potier, P.; Thierry, J.;
Zard, S. Z. Tetrahedron 1986, 42, 4983-4990.
(21) Hauge, S. Acta Chem. Scand. 1971, 25, 3081-3093.
(22) Gieselman, M. D.; Xie, L.; van der Donk, W. A. Org. Lett. 2001,
3, 1331-1334.
(18) Kolano, C.; Bucher, G.; Wenk, H. H.; Jager, M.; Schade, O.;
Sander, W. J. Phys. Org. Chem. 2004, 17, 207-214.
(19) Bucher, G.; Halupka, M.; Kolano, C.; Schade, O.; Sander, W.
Eur. J. Org. Chem. 2001, 545-552.
(23) Bucher, G.; Scaiano, J. C.; Sinta, R.; Barclay, G.; Cameron, J.
J. Am. Chem. Soc. 1995, 117, 3848-3855.
6612 J. Org. Chem., Vol. 70, No. 17, 2005

The Selenocysteinyl Radical
FIGURE 6. Transient decay trace, recorded at λ ) 340 nm,
upon LFP (λexc ) 266 nm) of 3 in acetonitrile at ambient
temperature. Inset: Comparison of the transient decay traces
of 2 and 3, recorded at λ ) 340 nm (red dots, 2; blue triangles,
3), covering the time range up to 10 µs.
FIGURE 5. Transient absorption spectra following 266 nm
laser excitation [traces recorded after 0.6 µs (black dots), 4 µs
(white dots), and 10 µs (black triangles), spectral resolution 5
nm, cell size 1.0 cm] in argon-purged acetonitrile displaying
the photochemistry of 3. Inset: Magnification of the 500-700
nm region.
radicals derived from benzophenone oxime.24 We calcu-
lated the UV/Vis spectra of the corresponding dimers 6,
7, and 20 using time-dependent DFT (TD-B3LYP/6-
31+G*//B3LYP/6-31G*, Table S1). According to these
calculations, 6 and 7 are two possible structures of the
growing transient, since only these two compounds show
strong absorptions between 340 and 360 nm necessary
for the LFP detection. The transient growth at 340 nm
is therefore tentatively assigned to the dimerization of 8
(which is not observed directly) yielding 6 or 7.
TR EPR and IR Experiments with 3. TR FTIR
measurements were performed using the STEP/SCAN
technique. LFP (λexc ) 266 nm) with step-scan detection
of 3 in acetonitrile (3.0 mM, argon purge) resulted in the
bleaching of the 1743 cm-1 absorption of 3 and the
N-O cleavage competing with C-O scission, whereas
product studies revealed the formation of fluorenone 19.23
The formation of 19 was explained by a dark reaction of
fluorenoneimine 5 with water. However, further work has
shown that hydrolysis of 5 cannot be the source of 19,
since no 18O-labeled 19 was detected as product in the
presence of H218O (Figure S7). It is therefore likely that
iminoxy radical 8 is the precursor of 1924 and indeed
plays a minor role in the photochemistry of derivatives
of 9-fluorenone oxime.
concomitant formation of CO2 (νCdO ) 2342 cm-1
)
(Figure 7). Within the time range of 20aµs s covered by the
experiments, no further transient signals were detected.
A partial recovery of the signal after 20 µs indicates
the formation of the starting material 3 (1728 cm-1) and
the stable photoproduct 2 (1746 cm-1). Oxygen purging
of the solution did also not lead to detectable transient
signals.
LFP (λexc ) 308 nm) of a 6.8 mM solution of 3 in CCl4
(Ar purge) with TR EPR detection showed one transient
set of signals, which evolves during the laser pulse. The
1:1:1 triplet centered at 3484 G (JN ) 9.52 G, g ) 2.00331)
reached maximum intensity after 220 ns and is assigned
to the 9-fluorenoneiminyl radical 18 (Figure S8).18,25
The results from the STEP/SCAN and TR EPR mea-
surements reveal that on irradiation 3 is cleaved into
carbon dioxide, the selenocysteinyl radical 1, and the
9-fluorenoneiminyl radical 18. The fact that we were
unable to detect the 9-fluorenoneiminoxy radical 8 by TR
EPR (cf. g ) 2.0064, RN ) 30.85, and RH ) 2.7)26 indicates
that formation of 8 is not a major photochemical pathway.
This, however, does not rule out the formation of 8 in
minor amounts as mentioned above.
Ingold and co-workers earlier reported on the genera-
tion and characterization of stable dimers of iminoxy
(25) Forrester, A. R.; Gill, M.; Meyer, C. J.; Sadd, J. S.; Thomson,
R. H. J. Chem. Soc., Perkin Trans. 1 1979, 606-611.
(26) Gilbert, B. C.; Norman, R. O. C. J. Chem. Soc. B 1967, 981-
984.
(24) Brokenshire, J. L.; Roberts, J. R.; Ingold, K. U. J. Am. Chem.
Soc. 1972, 94, 7040-7049.
J. Org. Chem, Vol. 70, No. 17, 2005 6613

Kolano et al.
FIGURE 7. TR IR difference spectra (time resolution 500 ns, spectral resolution 6 cm-1) showing the photochemistry of 3 in
acetonitrile purged with argon. Bands appearing on irradiation (λexc ) 266 nm) are pointing upward; bands disappearing are
pointing downward. Red line, spectrum 500 ns after irradiation; blue line, spectrum after 20 µs.
Product Studies with 3. Product studies were carried
out with 3 under the same conditions as described above
its dimers 6 and 7 clearly indicate the presence of 8.
These results demonstrate that 3 is an excellent photo-
chemical precursor of the selenocysteinyl radical 1 that
could find broad applications in biophysical research as
“caged selenocysteinyl”.
1
for 2. After irradiation of a CD3CN solution of 3, the H
and 13C NMR spectra show a variety of new signals which
could be assigned by using two-dimensional NMR spec-
troscopy (TOCSY, HMQC, and heteronuclear multiple-
bond correlation (HMBC)). Major constituents were
unreacted starting material 3 and dimethyl bis(N-tert-
butoxycarbonyl)-L-selenocystine 2 (Table S2). In addition,
carbon dioxide and compounds 4-7 were identified. This
further confirms that iminoxy radical 8 is an intermedi-
ate in the photochemistry of 3. 77Se NMR spectroscopy
is especially suitable to demonstrate the clean photo-
chemistry of 3. After irradiation of 3, the 77Se NMR
spectrum shows one new signal of 2 approximately 100
ppm shifted toward high field. This reveals that 3 is
converted directly into 2 via radical 1 without generating
any other selenium containing molecules (Figure S6).
Experimental Section
General Methods. Starting materials, reagents, and sol-
vents were purchased from commercial suppliers and used
without further purification. Solvents were dried by the usual
methods. Na2SO4 was used as a drying agent unless otherwise
reported. Chemical shifts (δH and δC) are reported in ppm with
respect to internal solvent. δSe is expressed relative to a 50%
(v/v) solution of Me2Se in internal solvent. The abbreviations
used are as follows: BOC, tert-butoxycarbonyl; MTBE, methyl
tertiary-butyl ether; SeCys, selenocysteine; Ser, serine.
TR FTIR Spectroscopy (STEP/SCAN). For TR measure-
ments, we used the STEP/SCAN setup that was described
previously.18,19,27 Excitation of the samples was carried out by
the 4th harmonic (266 nm) of an Nd:YAG laser with a
repetition rate of 10 Hz. To avoid thermal effects and shock
waves, the laser energy was attenuated to 3.8-4.4 mJ/pulse
by an external attenuator. All measurements were carried out
with 6 cm-1 resolution in a spectral range between 0 and 3160
cm-1, resulting in an interferogram containing 1060 points.
The signal was averaged 20 times per sampling position. In
all experiments, a time range of 20 µs was recorded with a
resolution of 25 ns. After FT, the average of 20 successive
spectra was calculated to achieve a better signal-to-noise ratio,
resulting in an effective time resolution of 500 ns. The samples
were dissolved (between 1.2 and 1.9 g‚L-1, depending on the
layer thickness of the cell) in acetonitrile (spectroscopic grade)
and purged with argon for at least 45 min.
Conclusion
The selenocysteinyl radical 1 was generated from two
independent precursors 2 and 3 and characterized by a
variety of spectroscopic techniques and theoretical meth-
ods. On irradiation, selenocystine 2 undergoes reversible
cleavage to produce the selenocysteinyl radical 1 as a
short-lived radical pair. The lifetime of 1 produced from
precursor 2 is limited by rapid thermal recombination of
the radical pair. In precursor 3, the C- and N-terminal
protected selenocysteinyl radical is functionalized with
the photolabile (9-fluorenylideneamino)oxycarbonyl group.
Precursor 3 shows a high thermal stability and is readily
cleaved by monochromatic 355 nm light. The photo-
chemical cleavage of 3 is irreversible (in contrast to the
cleavage of 2) and leads to 1, the 9-fluorenoneiminyl
radical 18, carbon dioxide, and small amounts of the
9-fluorenoneiminoxy radical 8. Secondary products formed
via thermal reactions of the primary radicals are 2,
9-fluorenone-oxime 4, and 9-fluoren-imine 5. Although
radical 8 could not be directly detected, the formation of
Nanosecond LFP. A standard LFP setup was used,
consisting of a Nd:YAG laser, operated at 1 Hz and 266 nm
(50 mJ/pulse, 8 ns pulse duration), a pulsed Xe arc lamp, a
monochromator coupled to a photoelectron multiplier tube, and
a digital oscilloscope. The entire setup was controlled from a
personal computer using LabView software. Data evaluation
was performed using SigmaPlot software. To avoid depletion
of the precursor and product build up, a flow cell was used.
(27) Kolano, C.; Sander, W. Eur. J. Org. Chem. 2003, 1074-1079.
6614 J. Org. Chem., Vol. 70, No. 17, 2005

The Selenocysteinyl Radical
Quenching experiments were performed in the same way,
except that the flow cell was replaced by a static cell. The
concentrations of 2 and 3 were adjusted so that an optical
density of 0.3 was achieved at the laser wavelength used for
excitation.
TR EPR. For TR EPR measurements, we used the setup
described previously.18 The concentration of 3 in tetrachloro-
methane was 6.83 mmol/L. The photoreaction was initiated
by an excimer laser at λexc ) 308 nm with a repetition rate of
10 Hz.
through a 20% aqueous sodium hydroxide solution for removal
of the phosgene. After filtration of the organic solution, the
solvent was removed and the yellow product was recrystallized
from anhydrous n-hexane (ca. 1600 mL) yielding 2.32 g (90%)
of pure 12. Mp 132.0 °C (literature value30 ) 110-112 °C). 1H
NMR (200.13 MHz, CDCl3) δ 8.17 (d, 1H); 7.85 (d, 1H); 7.60
(d, 1H); 7.58 (d, 1H); 7.51 (t, 1H); 7.46 (t, 1H); 7.35 (t, 1H);
7.30 (t, 1H). 13C NMR (50.3 MHz, CDCl3) δ 160.1; 148.1; 142.8;
141.5; 133.5; 132.5; 130.7; 129.4; 128.9; 128.6; 123.7; 120.5;
120.3. IR (KBr) cm-1 3385, 1806, 1632, 1607, 1594, 1535, 1495,
1450, 1317, 1204, 1155, 1105, 1037, 964, 857, 784, 732, 666,
645. EI-MS m/z 257, 178, 164, 151, 76; UV λmax/nm 214, 256,
300.
[(9-Fluorenylideneamino)oxycarbonyl]methyl(N-tert-
butoxycarbonyl)-L-selenocysteine (3). In a flame-dried
flask, dimethyl bis(N-tert-butoxycarbonyl)-L-selenocystine 2
(0.76 g, 1.33 mmol) was placed under Ar atmosphere. To this
was added freshly distilled and dried acetonitrile (30 mL).
NaBH4 (0.14 g, 3.55 mmol) was added portionwise to the ice-
cooled and vigorously stirred solution. Afterward the solution
was stirred at room temperature until the color disappeared
(approximately 15 min). The progress of the reduction can be
checked by thin-layer chromatography (TLC). After that the
reaction mixture was cooled again by an ice bath and a solution
consisting of 0.904 g (3.55 mmol) 17 in 40 mL freshly distilled
and dried acetonitrile was added dropwise. The solution was
vigorously stirred for another 4 h under ice cooling, while the
reaction was monitored by TLC. The solvent was evaporated
and the pale-yellow residue was purified by column chroma-
tography (hexane:MTBE 65:35). Yield: 0.805 g, (60.0%) of a
pale-yellow solid. TLC (hexane:MTBE 65:35) Rf 0.32. Mp 148.0
°C. 1H NMR (400.1 MHz, CD3CN) δ 8.19 (d, 1H); 7.74 (m, 3H);
7.55 (m, 2H); 7.38 (m, 2H); 5.79 (d, 1H); 4.49 (m, 1H); 3.73 (s,
3H); 3.44 (m, 1H); 3.19 (m, 1H); 1.40 (s, 9H). 13C NMR (100.6
MHz, CD3CN) δ 172.5; 171.7; 158.2; 156.4; 143.6; 142.4; 134.6;
134.5; 133.4; 131.1; 130.3; 130.0; 129.7; 123.6; 121.8; 121.8;
80.4; 54.5; 53.1; 28.6; 28.5. 77Se NMR (76.3 MHz, CD3CN) δ
406.20; IR (KBr) cm-1 3351, 2330, 1753, 1728, 1689, 1527,
1450, 1368, 1337, 1281, 1207, 1167, 1121, 1053, 950, 785, 733,
708, 643. FAB-MS in m/z (rel. %): 527.0 (78) [M + Na]+, 505.1
(20) [M + H]+, 196.0 (56), 154.0 (100), 136.0 (66). Anal. Calcd
for C29H24N2O6Se (503.42): C, 54.88; H, 4.81; N, 5.56; O, 19.07;
Se, 15.68. Found: C, 54.72; H, 4.73; N, 5.36.
Product Studies. A product study was performed in the
following manner: Samples of 2 and 3 (0.044 mol/L of 2 and
0.049 mol/L of 3) were dissolved in [D3]acetonitrile, transferred
in quartz NMR tubes, and degassed by the use of a supersonic
bath and the “freeze and pump” technique. The tubes were
fused, and 1H, 13C, 77Se, TOCSY, HMQC, and HMBC NMR
spectra were taken before and after the photolysis. Preparative
photolysis was carried out with 0.42 W‚s-1 for 1 h using a Nd:
YAG laser operating at λexc ) 266 nm with a repetition rate of
10 Hz. The irradiated NMR tubes were opened and the
samples examined by analytical gas chromatography (OV 1
column, 10 m, Øinner 0.1 mm).
Calculations. Calculations were performed with the Gauss-
ian98 Rev.A11 suite of programs.28 Stationary points, energies,
and vibrational and UV spectra were calculated using the
B3LYP29 functional with a 6-311++g(d,p) or 6-31+g* basis set.
The calculated vibrational frequencies given are unscaled.
Dimethyl Bis(N-tert-butoxycarbonyl)-L-selenocystine
(2). Several protocols for the synthesis of 2 have been
published in the literature.20-22 Mp 94.0 °C. 1H NMR (600.13
MHz, CD3CN) δ 5.76 (d, 2H); 4.42 (m, 2H); 3.70 (s, 6H); 3.38
(m, 2H); 3.21 (m, 2H); 1.42 (s, 18H). 13C NMR (150.9 MHz,
CD3CN) δ 172.4; 156.3; 80.4; 55.1; 53.0; 32.2; 28.5. 77Se NMR
(114.5 MHz, CDCl3) δ 305.46. IR (KBr) cm-1 3380, 2982, 2328,
1747, 1687, 1511, 1437, 1406, 1393, 1361, 1314, 1279, 1248,
1216, 1164, 1060, 1026, 1013, 980, 914, 856, 829, 781, 757,
652, 559, 386, 346, 203. Fast-atom bombardment mass spec-
troscopy (FAB-MS) in m/z (rel. %): 587.1 (54) [M + Na]+, 562.1
(60) [M + H]+, 507.1 (38), 408.9 (96), 146.0 (100), 57.1 (95).
Anal. Calcd for C18H32N2O8Se2 (562.38): C, 38.44; H, 5.74; N,
4.98; O, 22.76; Se, 28.08. Found: C, 38.95; H, 5.72; N, 4.93.
These data agree with literature values.22
9-Fluorenone-oxime-chloroformate (17).30,31 9-Fluo-
renone-oxime (1.95 g, 0.01mol) was suspended in 200 mL of
tetrachloromethane. A solution of phosgene in toluene (25 mL,
c ) 2 mol L-1, 0.05 mol) was added dropwise at -7 °C. The
suspension was stirred for 18 h. During that time, it was
allowed to warm gradually to ambient temperature. Excess
phosgene was then destroyed by purging the resulting solution
with argon. The gaseous phosgene-argon mixture was led
Acknowledgment. C.K. thanks the Deutsche For-
schungsgemeinschaft for a Postdoctoral Research Fel-
lowship. This work was financially supported by the
Deutsche Forschungsgemeinschaft (SFB 452) and the
Fonds der Chemischen Industrie. The authors thank Dr.
F. Scheidt for valuable advice with the GC analysis and
K. Gomann for his help with the TR EPR measurements
of 2 and 3.
(28) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
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Jr.; Stratmann, R. E.; Burant, J. C.; Dapprich, S.; Millam, J. M.;
Daniels, A. D.; Kudin, K. N.; Strain, M. C.; Farkas, O.; Tomasi, J.;
Barone, V.; Cossi, M.; Cammi, R.; Mennucci, B.; Pomelli, C.; Adamo,
C.; Clifford, S.; Ochterski, J.; Petersson, G. A.; Ayala, P. Y.; Cui, Q.;
Morokuma, K.; Salvador, P.; Dannenberg, J. J.; Malick, D. K.; Rabuck,
A. D.; Raghavachari, K.; Foresman, J. B.; Cioslowski, J.; Ortiz, J. V.;
Baboul, A. G.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.;
Komaromi, I.; Gomperts, R.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-
Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill,
P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Andres, J. L.; Gonzalez,
C.; Head-Gordon, M.; Replogle, E. S.; Pople, J. A. Gaussian98, revision
A.11.1; Gaussian, Inc.: Pittsburgh, PA, 2001.
Supporting Information Available: Instrumentation,
1
calculated and experimental UV/Vis absorptions, H and 13C
NMR data of the products of 3 after irradiation, GC data, 2D
NMR spectra of 3, 77Se NMR spectra of 2 and 3 before and
after irradiation, distribution of the products after photolysis,
calculated energies, geometries and vibrational modes of 15
and 16, optimized Cartesian coordinates for compounds arising
from recombination of 8, and TR EPR spectrum. This material
is free of charge via the Internet at http://pubs.acs.org.
(29) Becke, A. D. J. Chem. Phys. 1993, 98, 5648-5652.
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JO050479J
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