Internally stabilized selenocysteine derivatives: Syntheses, 77Se NMR and biomimetic studies

Article abstract of DOI:10.1039/b505299h

Selenocystine ([Sec]₂) and aryl-substituted selenocysteine (Sec) derivatives are synthesized, starting from commercially available amino acid L-serine. These compounds are characterized by a number of analytical techniques such as NMR (¹H, ¹³C and ⁷⁷Se) and TOF mass spectroscopy. This study reveals that the introduction of amino/imino substituents capable of interacting with selenium may stabilize the Sec derivatives. This study further suggests that the oxidation-elimination reactions in Sec derivatives could be used for the generation of biologically active selenols having internally stabilizing substituents. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b505299h

View Article Online / Journal Homepage / Table of Contents for this issue
A R T I C L E
Internally stabilized selenocysteine derivatives: syntheses, 77Se NMR
and biomimetic studies†
Prasad P. Phadnis and G. Mugesh*
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore,
560 012, India. E-mail: mugesh@ipc.iisc.ernet.in; Fax: +91 80 2360 1552/2360 0683
Received 15th April 2005, Accepted 11th May 2005
First published as an Advance Article on the web 9th June 2005
Selenocystine ([Sec]2) and aryl-substituted selenocysteine (Sec) derivatives are synthesized, starting from
commercially available amino acid L-serine. These compounds are characterized by a number of analytical
techniques such as NMR (1H, 13C and 77Se) and TOF mass spectroscopy. This study reveals that the introduction of
amino/imino substituents capable of interacting with selenium may stabilize the Sec derivatives. This study further
suggests that the oxidation–elimination reactions in Sec derivatives could be used for the generation of biologically
active selenols having internally stabilizing substituents.
derivatives poses a major problem in synthetic and purification
procedures. To overcome this difficulty we employed an aryl
selenium moiety having internally chelating substituents, which
would increase the stability by Se · · · N non-covalent interac-
tions. These substituents may also modulate the reactivity of
selenols that are generated in situ by elimination reactions. In
this article, we report the first examples of Sec derivatives having
basic amino/imino groups in the close proximity to selenium.
Introduction
Selenium, an essential trace element,1 exerts its biological
effect through several selenoenzymes, which include glutathione
peroxidase (GPx),2,3 iodothyronine deiodinase (ID)4 and thiore-
doxin reductase (TrxR).5 Although these enzymes have seleno-
cysteine (Sec), the 21st amino acid, in their active site,6–10 their
substrate specificity and cofactor systems are strikingly different.
GPx is an antioxidant selenoenzyme11 that protects various
organisms from oxidative stress by catalyzing the reduction of
hydroperoxides at the expense of thiols.12 ID, particularly the
type I enzyme (ID-I), on the other hand, is responsible for
the activation of thyroid hormones by deiodination reactions.
The role of TrxR is to reduce thioredoxin (Trx) by NADPH,
which is important for a variety of biological functions such
as DNA synthesis.5,13 However, the synthetic and biological
studies on Sec are hampered by instability of Sec derivatives
as compared with the cysteine (Cys) analogues. In general,
Sec derivatives undergo fast and mild oxidative elimination to
produce dehydroalanines.14
Results and discussion
The synthesis of selenocysteine derivatives was approached with
protected L-serine. Our initial route to these derivatives focused
on modification to literature preparations of selenocysteine and
the corresponding phenyl derivative. Boc-protected serine ester
(1) was converted into bromoalanine methyl ester (3), which
was treated with Li2Se2 to afford the protected selenocystine (4)
(Scheme1).
Initial attempts to synthesize Sec derivatives in the laboratory
met with limited success. Soda et al. synthesized selenocys-
teine by treating b-L-chloroalanine with disodium diselenide15
whereas Pete Silks et al. approached the synthesis from tosylated
N-Boc-serine methylester. The conversion of this serine ester
into an iodide or a bromide derivative followed by treatment with
dilithium diselenide and then deprotection with TFA afforded
the required Sec.16,17 Walther and coworkers reported one of
the first syntheses of optically pure Sec derivatives.18 A key
step in the synthesis involved the nucleophilic displacement
of an O-tosylated L-serine derivative. An alternative approach
has been reported by Shirahama and coworkers, in which
diphenyl diselenide was first reduced with sodium metal and
the resulting selenolate was treated with tert-butyloxy carbonyl
(Boc) protected serine b-lactone.19 Recently, this approach has
been modified by performing an in situ reduction of diphenyl dis-
elenide with sodium trimethoxyborohydride [NaBH(OMe)3].14,20
More recently, a modified synthetic route to Sec derivatives using
Fmoc strategy has also been reported.21
Scheme 1 Synthetic route to diselenide 4.
Although this reaction afforded the expected compound in
low yield, our initial attempts towards scale up did not work.
The 77Se NMR spectrum showed a number of signals which
were probably due to different diastereomers. The activation of
serine –OH in 1 with phenylmethanesulfonyl fluoride (PMSF)
or tosyl chloride (TsCl) followed by reaction with NaHSe and
subsequent oxidation also did not look promising. The major
problem in these reactions lies in the tosylation step, which leads
to the formation of dehydroalanine derivatives. In view of these
results, we decided to find an alternative synthetic approach to
4 that does not involve the tosylation and would be amenable to
scale up. Scheme 2 depicts the conversion of the protected serine
into selenocystine, in which the –OH group can be converted
directly to a bromide without involving the tosylation step.23
Recent studies on aryl substituted Sec conjugates show that
the oxidative elimination reactions could lead to the generation
of catalytically active selenols.22 Therefore, the fast and mild
oxidativeeliminationofselenocysteine derivatives can beutilized
for further catalytic reactions. However, the instability of such
† Electronic supplementary information (ESI) available: 77Se NMR
data for selected compounds. See http://www.rsc.org/suppdata/ob/
b5/b505299h/
Scheme 2 Modified route for the synthesis of 4.
2 4 7 6
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1
T h i s j o u r n a l i s
T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 5
©

View Article Online
According to this method, compound 1 was treated with
triphenyl phosphine, bromine and imidazole in CH2Cl2 to
convert the –OH function into the corresponding bromide. After
this step, the bromide was added to a THF solution of Li2Se2 to
afford the protected selenocystine (4). Deprotection of ester and
Boc groups in 4 afforded the free amino acid in the diselenide
form. All analytical data obtained for this compound are in
accordance with the data reported in the literature. From these
results, it is clear that the N-Boc-L-Serine methyl ester may be
an ideal starting material for the preparation of substituted
selenocysteine derivatives. By employing the above mentioned
methodology, Boc–Ser–OMe (1) was transformed into the
corresponding bromide, and subsequent reaction with benzene
selenolate (PhSe−Li+), generated in situ by reducing diphenyl
diselenide (PhSeSePh) by superhydride, proved to be an effective
way to produce phenyl substituted selenocysteine (5). It is
noteworthy to mention that competing side products stemming
from elimination or ester saponification were suppressed by the
use of triethylamine along with the bromide (Scheme 3).
compounds, possibly by non-covalent Se · · · N interactions. The
variable temperature 1H NMR spectra of compound 7 show that
the Se · · · N interactions do exist in solution as evidenced by the
AB pattern observed for the benzylic protons.
By following the above method the oxazoline based com-
pound (10) was synthesized (Scheme 3). The deprotected deriva-
tives were also synthesized by following similar procedures.
These compounds were also found to be stable in solution,
as proved by 77Se NMR spectroscopy. However, during the
deprotection of the Boc group by TFA, cleavage of oxazoline
ring leads to the formation of acid 14.
One of the important objectives of this work was to study
these compounds by 77Se NMR spectroscopy. Although a few
methods have been described in the literature for the synthesis of
selenocysteine derivatives, only a small amount of information
is available regarding the 77Se NMR chemical shifts. The 77Se
nucleus has a very wide chemical shift range (over 3000 ppm) and
is extremely sensitive to changes in bonding and conformation.
The relatively large chemical shift anisotropy (CSA) of 77Se
provides a very efficient relaxation mechanism and consequently
broad 77Se resonances in the NMR spectra of biomolecules. It
is, therefore, very difficult to detect sharp 77Se NMR signals
for biomolecules due to the relatively low percentage of the
77Se isotope in elemental selenium. 77Se NMR data for D-L-
selenocystine in the solution phase were reported by Pan and
Fackler.25 However, the 77Se NMR studies on compounds in the
present study show that it is possible to obtain good 77Se NMR
spectra at appropriate concentrations. Table 1 summarizes the
77Se NMR chemical shift for Sec and related derivatives (see
electronic supplementary information (ESI) for spectra†).
All compounds exhibited 77Se NMR signals in the range
∼200–300 ppm with respect to Me2Se. The diselenide (4)
exhibited a signal at 294 ppm, which is greatly shifted upfield
compared with that of aromatic diselenides, but it is comparable
with other dialkyl diselenides.26 The phenyl derivative (5)
exhibited a signal at 252 ppm. This signal appears to be shifted
upfield as compared with those of PhSeR (R = n-alkyl), which
normally occur around 290 ppm. Although compound 5 can
be readily synthesized from 3, this compound was found to
be unstable in solution as evidenced by the appearance of a
new signal at 460 ppm for diphenyl diselenide. This indicates
that compound 5, similar to the parent [Sec]2, undergoes b-
elimination reactions.
Scheme 3 Synthetic routes to internally chelated Sec derivatives.
The improvement of yield in this reaction by the addition of
NEt3 led to an assumption that a selenium moiety containing
an internal amino/imino group would provide an interesting
strategy to synthesize selenocysteine derivatives containing het-
eroatoms in close proximity to selenium. It should be mentioned
that basic amino/imino groups in close proximity to selenium
have been shown to play a significant role in modulating the
biological activity of natural and synthetic selenium compounds.
In light of this we approached the synthesis of 7 starting from
diselenide 13.24 As in the case of phenyl derivative, reduction
of diselenide 13 by super hydride, followed by reaction with the
bromo derivative 3, afforded the expected selenocysteine deriva-
tive 7. Although this reaction produced the expected compound,
the yield was found to be very poor. Therefore we approached the
synthesis starting from N,N-dimethyl benzylamine. It is known
that the heteroatom directed metallation of N,N-dimethyl ben-
zylamine with n-BuLi, followed by selenium insertion, affords
the corresponding lithium selenolate. Treatment of the lithium
selenolate with 3 at low temperatures afforded compound 7 in
good yield. Deprotection of the ester group by aq. NaOH and
the removal of Boc group by TFA afforded compounds 8 and
9, respectively. It is worth mentioning that the stability of these
derivatives was found to be much higher than those derived from
unsubstituted phenyl derivatives. This indicates that the presence
of an amino group near selenium enhances the stability of these
The benzylamine derivative, on the other hand, exhibited a
signal at 216 ppm, which is shifted significantly upfield as com-
pared with that of the phenyl derivatives. The signal at 216 ppm
disappeared completely upon treatment with aq. NaOH and
a new signal appeared at 212 ppm for the corresponding
Table 1 77Se NMR data of Sec derivatives
No.
Compound
77Se NMR chemical shift (ppm)
Solvent
1
2
3
4
5
6
7
8
9
4
5
295
252
240
216
212
191
273
258
249
CDCl3
CDCl3
D2O
CDCl3
D2O
6
7
8
9
D2O
10
11
12
CDCl3
CDCl3
D2O
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1
2 4 7 7

View Article Online
acid. Furthermore, the signal also disappeared completely upon
treatment with TFA, exhibiting a new signal at 191 ppm for
the completely deprotected derivative 9. The oxazoline based
selenocysteine derivative (10) exhibited a signal at 273 ppm,
which is shifted downfield as compared with that of phenyl and
benzylamine derivatives. The deprotected derivative exhibited a
signal at 249 ppm, which is also shifted downfield as compared
with the corresponding phenyl and benzylamine derivatives.
However, the chemical shifts for the protected compounds
cannot be directly compared with those of the deprotected
derivatives because the 77Se NMR spectra for the protected and
deprotected derivatives were recorded in different solvents and
the solvent shift can be substantial for 77Se measurements. The
benzylamine and oxazoline based compounds were found to be
more stable than the corresponding phenyl derivatives due to
the presence of heteroatoms in the close proximity to selenium.
Because the selenocysteine derivatives can undergo fast
oxidation–elimination reactions under mild conditions,22,27 these
compounds could be used to generate catalytically active
selenols. Therefore, compound 7 was treated with H2O2 and
the progress of the reaction was followed by 77Se NMR spec-
troscopy. Interestingly, compound 7 underwent facile oxidation–
elimination reactions to produce the corresponding dehydroala-
nine derivative and seleninic acid (15) (Scheme 4). This suggests
the possibility that the substituted selenocysteine derivatives
could be used as procatalysts for GPx-like activity. It has
been shown previously that the diselenide (13) derived from
N,N-dimethyl benzylamine reduces hydroperoxides with thiols,
mimicking the selenoenzyme GPx in vitro. Therefore, we studied
the GPx-like catalytic activity by using PhSH as a thiol.
The conversion of PhSH to PhSSPh was followed by reverse-
phase HPLC, using methanol–water as the eluent (Fig. 1). The
activities were compared with ebselen, a well known GPx mimic,
and are summarized in Table 2.
The GPx activity of the phenyl derivative was only marginally
higher than ebselen. The benzylamine derivative, however, exhib-
ited an almost 16-fold increase in the GPx activity as compared
with ebselen. This indicates that the selenenic acid generated
during the reaction with H2O2 may be reduced by PhSH, exerting
a catalytic cycle. The addition of H2O2 to compound 7 produced
a signal at 1018 ppm in the 77Se NMR, which could be ascribed
to the selenenic acid 15. Further reaction of this species with
one equivalent of PhSH produced a signal at 565 ppm for
the corresponding selenenyl sulfide 16, which is confirmed by
comparison with the chemical shift of an authentic sample.
The treatment of 16 with an excess amount of PhSH then
produced the corresponding selenol 17 along with the oxidized
product 13. Addition of an excess amount of H2O2 to 7 produced
another signal at 1181 ppm, which disappeared upon addition
of PhSH. The 77Se NMR chemical shift observed at 216 ppm for
compound 7 has never been detected during the entire process,
indicating that the selenide 7 acts only as a procatalyst. Although
compound 17 has been previously shown to be a catalyst in the
reduction of H2O2,28 capping it on a biogenic moiety such as Sec
would increase its biological applications.
Conclusions
Aryl-substituted Sec derivatives containing basic amino/imino
groups in close proximity to selenium could be synthesized
from commercially available starting materials. The presence
of a heteroatom near selenium increases the stability of Sec
derivatives by intramolecular Se · · · N interactions. These inter-
actions appear to modulate the reactivity of selenium. This study
further suggests that the internally chelated aryl-substituted Sec
derivates could be used as procatalyst for generating catalytically
active selenols. The fast and mild oxidative elimination of Sec
derivatives to produce dehydroalanines, therefore, may give easy
access to biologically active selenium compounds.
Experimental
Scheme 4 Oxidation–elimination reaction of 7.
Materials and methods
The commercially available Boc-L-serine, di-tert-butyl dicar-
bonate [(Boc)2O], N, N-dimethylbenzylamine, 4,4-dimethyl-2-
oxazoline and n-BuLi, LiBEt3H (super hydride) were purchased
from Aldrich/Sigma Chemicals. All reactions were carried out
under a nitrogen atmosphere by using standard vacuum line
techniques. Solvents were purified by standard procedures29
and were freshly distilled prior to use. The products were
purified by column chromatography on silica gel 60/120 mesh
size washed with 2% Et3N in order to avoid the cleavage of
Boc group under mild acidic conditions. The compounds were
characterized by NMR spectroscopy on a Bruker AVENCE
400 MHz spectrometer operating at 400 MHz (1H), 100.56 MHz
(13C) and 76.29 MHz (77Se). 1H and 13C chemical shifts are
cited with respect to SiMe4 as an internal standard. The 77Se
NMR spectra were recorded using diphenyl diselenide as an
external standard and are shown in the ESI†. Chemical shifts are
reported relative to dimethyl selenide (0 ppm) by assuming that
the resonance of the standard is at 461.0 ppm. IR spectra were
recorded on Perkin Elmer Spectrum One FTIR Spectrometer.
Mass spectral studies were carried out on a Q-TOF Micro Mass
Spectrometer with an ESI MS mode of analysis. In the case of
isotopic patterns, the value given is for the most intense peak.
Fig. 1 Rates of formation of PhSSPh from oxidation of PhSH (5 mM)
with H2O2 (7 mM) in the presence of catalysts ebselen and 7 (0.47 mM)
in CH2Cl2–MeOH (95 : 5) at 23 ◦C.
Table 2 Initial Rate (v0) and activity for the reduction of H2O2 (7 mM)
by PhSH (5 mM) in the presence of selenium catalysts (0.47 mM) at
23 ◦
C
Compound
v0 /lM min−1
Activity
Ebselen
5
7
0.59 × 10−3
1.10 × 10−3
9.59 × 10−3
1.0
1.8
16.1
Synthesis of N-Boc-L-serine methyl ester (1)30. To a solution
of L-serine (10.029 g, 95.44 mmol) in 1 N NaOH (200 ml, 8.020 g,
200.50 mmol), a solution of di-tert-butyl dicarbonate [(Boc)2O]
2 4 7 8
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1

View Article Online
(22.0 ml, 20.900 g, 95.76 mmol) in 1,4-dioxane (100 ml) was
added at 0 ◦C. The resulting turbid two phase solution was
stirred at ∼5 ◦C for 30 min, allowed to warm up to room
temperature and stirred for an additional 4 h. The mixture was
concentrated and then acidified to pH 2–3 by addition of 1
N KHSO4 (200 ml, 27.245 g, 200.09 mmol). The product was
extracted with ethyl acetate (4 × 150 ml) and then the combined
extracts were dried (Na2SO4), filtered and concentrated to give
N-Boc-L-serine as a colorless oil. The essentially pure N-Boc-L-
serine was used for further reactions without any purification.
Yield: 18.429 g (94.3%), 1H NMR (CDCl3): d 1.46 (s, 9H, CMe3);
3.79–3.86 (dd, J 22.4, 9.6 Hz) (1H), 4.01–4.08 (dd, J 14.8,
10.0 Hz) (1H) (CH2); 4.20 (s, 1H, CH); 4.38 (s, 1H, OH); 5.86
(d, J 7.6 Hz, 1H, NH); 7.13 (s, 1H, OH); 7.65 (br, 1H, COOH).
To a solution of N-Boc-L-serine (18.429 g, 89.81 mmol) in
DMF (200 ml), solid K2CO3 (13.099 g, 94.78 mmol) was added
at 0 ◦C. The resulting white suspension was stirred for 15 min and
then methyl iodide (18.0 ml, 40.950 g, 288.56 mmol) was added.
The reaction mixture was stirred for ∼20 h and the progress of
the reaction was monitored by TLC. The reaction mixture was
filtered through celite and the filtrate was partitioned between
ethyl acetate (4 × 150 ml) and distilled water (400 ml). The ethyl
acetate extracts were combined and dried (Na2SO4), filtered and
concentrated in vacuo to give a pale amber viscous oil, which
was purified by column chromatography using ethyl acetate–
further purified by column chromatography using ethyl acetate
and petroleum ether (1 : 2). Yield: 0.601 g (48%), IR (KBr): t =
=
=
2962 (NH); 2925 (NH) (C O, ester); 2874 (s); 2855 (s); (C O,
Boc); 1495 (s); 1465 (s); 1456 (s); MS (TOF MS ES+) m/z (%):
587 (100), 585 (87.0), 583 (51.3) [M+Na].
O-Phenyl-N-Boc-L-selenocysteine methyl ester (5)27. This
compound was synthesized by following the method used for
the synthesis of diselenide 4. The bromo derivative was added
dropwise to a solution of PhSeLi (0.235 g, 1.44 mmol) (prepared
separately by treatment of diphenyldiselenide (Ph2Se2) (0.225 g,
0.72 mmol) with LiBEt3H (super hydride) (1.4 ml, 0.067 g,
1.4 mmol)) in dry THF at 0 ◦C. The reaction mixture was
stirred for 20 h at this temperature. The solvent was evaporated in
vacuo and the product was purified by column chromatography
using ethyl acetate–petroleum ether (1 : 25) to give the desired
compound. Yield: 0.235 g (45%), IR (KBr): t = 2979 (NH),
1
=
=
1752 (C O, ester); 1715 (C O, Boc); 1500 (s); 1162 (s); H NMR
(CDCl3): d 1.42 (s, 9H, CMe3); 3.33–3.48 (d, J 4.8 Hz, 2H, CH2);
3.48 (s, 3H, OCH3); 4.67 (m, 1H, CH); 5.36–5.38 (d, J 8.0 Hz,
1H, NH) 7.46–7.70 (m, 5H, Ph). 13C NMR (CDCl3): dC = 28.3
(CMe3); 30.7 (CH2); 52.3 (OCH3); 53.3 (CH); 80.1 (3 ◦C); 127.6
=
(HC-3, 4, 5); 129.2 (C of Ph); 133.8 (HC-2, 6); 156.0 (HNC O);
+
=
171.1 (O C–O). MS (TOF MS ES ) m/z (%): 382 (100), 380
(48), 378 (18.9) [M+Na]; 398 (100), 396 (48), 394 (18.9) [M+K].
petroleum ether (1 : 2). Yield: 15 g (72.3%), IR (KBr): t = 3436
O-N,N-Dimethylbenzyl-N-Boc-L-selenocysteine methyl ester
(7). This compound was synthesized by following the method
used for the synthesis of diselenide 4. The bromo derivative was
added dropwise to a solution of lithium selenolate (0.731 g,
3.32 mmol) generated in situ from N, N-dimethylbenzylamine
in ether at 0 ◦C. The resulting yellowish brown suspension was
allowed to warm up to room temperature and the stirring was
continued for an additional 20 h, leading to an orange yellow
solution. The compound, after evaporation of the solvent,
was purified by column chromatography using ethyl acetate–
petroleum ether (1 : 20) to give the desired compound. Yield:
1
=
=
(O–H); 2980 (NH); 1746 (C O ester); 1705 (C O, Boc); H
NMR (CDCl3): d 1.44 (s, 9H, CMe3); 3.2 (s, 1H, OH); 3.78 (s, 3H,
OCH3); 3.92 (d, J 17.6 Hz, 2H, CH2); 4.37 (s, 1H, CH); 5.6 (d,
1H, NH). 13C NMR (CDCl3): d 28.3 (CMe3); 52.6 (OCH3); 55.7
◦
=
=
(CH); 63.4 (CH2); 80.3 (3 C); 155.8 (NHC O); 171.5 (O C–O).
Synthesis of methyl-[2-(tert-butoxycarbonyl)amino]-3-bromo-
propionate (3). To a solution of PPh3 (0.646 g, 2.47 mmol) in
CH2Cl2 (15 ml), bromine (0.13 ml, 0.399 g, 2.50 mmol) was
added dropwise and the resulting orange–yellow solution was
stirred for 15 min. To this was added a solution of N-Boc-L-
serine methyl ester (0.269 g, 1.23 mmol) and imidazole (0.169 g,
2.48 mmol) in 20 ml CH2Cl2 at 0–5 ◦C. This resulted in an
immediate formation of a white precipitate. The reaction mixture
was stirred for 3 h, the precipitate was filtered off and the filtrate
was concentrated in vacuo to give a viscous pale yellow oil. This
product was further purified by silica gel column using ethyl
acetate–petroleum ether (1 : 3) to give the expected product as
a colorless oil. Yield = 0.135 g (39%). The NMR data are in
accordance with the literature values.
=
0.676 g (49%), IR (KBr): t = 2983 (NH); 1754 (C O, ester);
1
=
1708 (C O, Boc); H NMR (CDCl3): d 1.37 (s, 9H, CMe3); 2.25
(s, 6H, NMe2); 3.22 (dd, J 13, 4.8 Hz, 1H), 3.33 (d, J 12.4 Hz,
1H), 3.46 (dd, J 13.2, 4.8 Hz, 1H) (CH2), 3.55 (s, 3H, COOCH3);
3.63 (d, J 12 Hz, 2H, CH2); 4.4 (m, 1H, CH); 6.5 (d, J 8.0 Hz,
1H, NH); 7.19–7.57 (m, 4H, Ph). 13C NMR (CDCl3): d 28.3
(CMe3); 30.5 (SeCH2); 45.0 (NMe2); 52.3 (OCH3); 52.8 (CH);
64.8 (NCH2); 79.7 (3 ◦C); 126.9, 128.2, 130.4, 132.2, 134.2; 140.8;
+
=
=
155.4 (HNC O); 171.5 (OC O). MS (TOF MS ES ) m/z (%):
417 (100), 415 (48.0), 418 (20.2) [M+Na].
Synthesis of N-Boc-L-selenocystine methyl ester-diselenide
(4)16. To a suspension of selenium powder (0.349 g, 4.43 mmol)
in THF (15 ml) was added carefully a solution of LiBEt3H (super
hydride) (4.90 ml, 0.234 g, 4.89 mmol) in THF at 0 ◦C. This
resulted in the formation of a dark brown suspension of Li2Se2,
which was boiled at reflux for 30 min and then allowed to cool
to room temperature. This solution was used for the next step
without any manipulation.
In another flask, methyl-[2-(tert-butoxycarbonyl)amino]-3-
bromo-propionate (3) (0.626 g, 2.22 mmol) was prepared by
treatment of N-Boc-L-serine methyl ester (0.540 g, 2.46 mmol)
and imidazole (0.335 g, 4.93 mmol) mixture with PPh3 (1.292 g,
4.92 mmol) and bromine (0.25 ml, 0.800 g, 5.00 mmol) in CH2Cl2
at 0–5 ◦C as previously mentioned. The bromo compound was
dissolved in dry THF, cooled to −78 ◦C and treated with Li2Se2
solution (0.381 g, 2.22 mmol). The reaction mixture was allowed
to warm to room temperature and the stirring was continued for
additional 3 h. The resulting suspension was filtered through
celite. The clear yellow solution was concentrated in vacuo to
give yellow oil. All attempts to directly purify the product by
chromatographic techniques were unsuccessful. However, the
expected diselenide was obtained in pure form when the crude
product was treated with I2 in CH2Cl2 at room temperature.
This resulted in the formation of a brown solid, which was
4,4-Dimethyl-2-oxazoline-N-Boc-L-selenocysteine methyl es-
ter (10).
Method A. This compound was synthesized by following
the method used for the synthesis of diselenide 4. The bromo
derivative was added dropwise to a solution of lithium selenolate
(0.502 g, 1.93 mmol), generated in situ from 4,4-dimethyl-2-
oxazoline, in ether at 0 ◦C. The resulting yellowish brown
suspension was stirred for 18 h and then concentrated to give the
expected product as an orange oil, which was further purified
by column chromatography using ethyl acetate–petroleum ether
(1 : 20). Yield: 0.410 g (47%), IR (KBr): t = 2975 (NH); 1748
1
=
=
(s), 1720 (C O, ester); 1715 (s); 1647 (w) (C O, Boc). H NMR
(CDCl3): d 1.42 (s, 9H, CMe3); 1.58 (s, 6H, NCMe2); 3.28 (m,
2H, SeCH2); 3.68 (s, 3H, OCH3); 4.09 (s, 2H, CH2); 4.69 (m,
1H, CH); 5.52 (d, J 8.0 Hz, 1H, NH); 7.2–7.75 (m, 4H, Ph).
MS (TOF MS ES+) m/z (%): 457 (100), 455 (50.8), 454 (21.9)
[M+Na].
Method B. The title compound can also be synthesized from
the diselenide31 derived from 4,4-dimethyl-2-oxazoline. In this
method, the oxazoline diselenide (0.206 g, 0.41 mmol) was
reduced by LiBEt3H (0.82 ml, 0.039 g, 0.82 mmol) in dry THF
at 0 ◦C to give the corresponding lithium selenolate (0.106 g,
0.41 mmol) which upon treatment with the bromo derivative
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1
2 4 7 9

View Article Online
at −78 ◦C afforded the expected compound in low yield. The
product was purified by column chromatography using ethyl
acetate–petroleum ether (1 : 20).
were calculated from the first 5–10% of the reaction to avoid any
reverse reaction from the products.
Acknowledgements
Deprotection of O-phenyl-N-Boc-L-selenocysteine methyl ester
(5). To a stirred solution of O-phenyl-N-Boc-L-selenocysteine
methyl ester (0.150 g) in MeOH (25 ml) at 0–5 ◦C, 1 N NaOHaq
(∼20 ml) solution was added dropwise. The reaction mixture
was stirred for 30 min at this temperature and neutralized
with aqueous 1 N KHSO4 solution (∼20 ml). The product was
extracted with ethyl acetate and the combined organic layer was
dried (Na2SO4), filtered and concentrated to give the desired
acid-deprotected derivative as a pale yellow oil. MS (TOF MS
ES+) m/z 343 (M+H−). This compound was dissolved in CH2Cl2
and treated with TFA (2 ml). After stirring for 2 h at room
temperature, the solvent was evaporated completely. The residue
was washed with CH2Cl2 and petroleum ether to remove Ph2Se2
that resulted from the elimination reactions. The resulting water
soluble off-white solid was dried in vacuo to yield the desired
This study was supported by the Department of Science and
Technology (DST), New Delhi, India.
References
1 K. Schwarz and C. M. Foltz, J. Am. Chem. Soc., 1957, 79, 3292.
2 L. Flohe´, E. A. Gu¨nzler and H. H. Schock, FEBS Lett., 1973, 32,
132.
3 J. T. Rotruck, A. L. Pope, H. E. Ganther, A. B. Swanson, D. G.
Hafeman and W. G. Hoekstra, Science, 1973, 179, 588.
4 (a) J. L. Leonard and T. J. Visser, Biochemistry of Deiodination, in
Thyroid Hormone Metabolism, ed. G. Hennemann, Marcel Dekker,
New York, 1986, p. 189; (b) M. J. Berry, L. Banu and P. R. Larsen,
Nature, 1991, 349, 438; (c) J. L. Leonard and J. Ko¨hrle, Intracel-
lular Pathways of Iodothyronine Metabolism, in The Thyroid, ed.
L. E. Braverman and R. D. Utiger, Lippincott-Raven, Philadelphia,
1996, p. 144; (d) D. L. St Germain and V. A. Galton, Thyroid, 1997,
7, 655.
5 (a) T. C. Stadtman, Annu. Rev. Biochem., 1996, 65, 83; (b) J. Ko¨hrle,
R. Brigelius-Flohe´, A. Bo¨ck, R. Gartner, O. Meyer and L. Flohe´,
Biol. Chem., 2000, 381, 849; (c) E. S. J. Arne´r and A. Holmgren,
Eur. J. Biochem., 2000, 267, 6102; (d) J. M. May, C. E. Cobb, S.
Mendiratta, K. E. Hill and R. F. Burk, J. Biol. Chem., 1998, 273,
23039; (e) G. E. Arteel, K. Briviba and H. Sies, Chem. Res. Toxicol.,
1999, 12, 264; (f) K. Becker, S. Gromer, R. H. Schirmer and S. Mu¨ller,
Eur. J. Biochem., 2000, 267, 6118; (g) D. Mustacich and G. Powis,
Biochem. J., 2000, 346, 1.
=
compound 6. IR (KBr): t = 3433 (OH); 2980 (NH); 1689 (C O,
=
ester); 1578 (C O, Boc); 1427 (m); 1210 (s); 1188 (s); 1140 (s)
1H NMR (D2O): d 1.76 (s, 2H, NH2); 2.98–3.03 (m, 1H, CH2),
3.11–3.15 (m, 1H, CH2); 3.32(s, br, 1H, CH); 7.25 (m, 3H); 7.47
(m, 2H) (Ph). 13C NMR (D2O): d 23.2 (CH2), 56.5 (CH); 127.1
=
(HC-3, 4, 5); 129.3 (C of Ph); 132.6 (HC-2, 6); 181.5(O C–O).
Deprotection of O-N,N-dimethylbenzyl-N-Boc-L-selenocysteine
methyl ester (7). To a solution of O-N,N-dimethylbenzyl-N-
Boc-L-selenocysteine methyl ester (0.140 g) in MeOH (∼25 ml)
was added 1 N NaOH solution (∼20 ml) at 0–5 ◦C. The reaction
progress was monitored by TLC. The mixture was partitioned
between distilled water and ethyl acetate. The organic phase was
separated and the aqueous phase was further extracted with
CH2Cl2 to remove any organic impurities. The aqueous phase
was filtered and concentrated to give yellowish green solid. This
solid was dissolved in MeOH (20 ml) and TFA (10 ml) was
6 W. A. Gu¨nzler, G. J. Stefens, A. Grosman, S. M. Kim, F. Oeting, A.
Wendel and L. Flohe´, Hoppe Seyler’s Z. Physiol. Chem., 1984, 353,
195.
7 F. Zinoni, A. Birkmann, T. C. Stadtman and A. Bo¨ck, Proc. Natl.
Acad. Sci., USA, 1986, 83, 4650.
8 A. Bo¨ck, K. Forchhamer, J. Heider and C. Baron, Trends Biochemical
Sci., 1991, 16, 463.
9 (a) R. Wilting, S. Schorling, B. C. Persson and A. Bo¨ck, J. Mol.
Biol., 1977, 266, 637; (b) E. Garcin, X. Vernede, E. C. Hatchikian,
A. Volbeda, M. Frey and J. C. Fontecilla-Camps, Structure, 1999, 7,
557; (c) M. Pfeiffer, R. Bingemann and A. Klein, Eur. J. Biochem.,
1998, 256, 447.
10 A. Bo¨ck, K. Forchhammer, J. Heider, W. Leinfelder, G. Sawers, B.
Veprek and F. Zinoni, Mol. Microbiol., 1991, 5, 515.
11 (a) G. Mugesh and H. B. Singh, Chem. Soc. Rev., 2000, 29, 347; (b) L.
Flohe´, G. Loschen, W. A. Gu¨nzler and E. Eichele, Hoppe Seyler’s
Z. Physiol. Chem., 1972, 353, 987; (c) O. Epp, R. Ladenstein and
A. Wendel, Eur. J. Biochem., 1983, 133, 51; (d) Selenium in Biology
and Human Health,ed. R. F. Burk, Springer Verlag, New York, 1994;
(e) M. Maiorino, K.-D. Aumann, R. Brigelius-Flohe´, D. Doria, J.
van den Heuvel, J. McCarthy, A. Rovery, F. Ursini and L. Flohe´,
Biol. Chem. Hoppe-Seyler, 1995, 376, 651.
12 (a) Review:T. C. Stadtman, J. Biol. Chem., 1991, 266, 16257;
(b) Review: F. Ursini, In Oxidative Processes and Antioxidants, ed.
R. Paoletti, Raven Press, New York, 1994, p. 25.
13 G. Waksman, T. S. R. Krishna, R. M. Sweet, C. H. Williams, Jr. and
J. Kuriyan, J. Mol. Biol., 1994, 236, 800.
14 M. D. Gieselman, Y. Zhu, H. Zhou, D. Galonic and W. A. van der
Donk, ChemBioChem., 2002, 3, 709.
15 P. Chocat, N. Easaki, H. Tanaka and K. Soda, Anal. Biochem., 1985,
148, 485.
◦
added dropwise at 0–5 C. The stirring was continued for 2 h
and then the solution was concentrated in vacuo to afford 9. IR
(KBr): t = 2977; 2354; 1739; 1470; 1182; 1110. 1H NMR (D2O):
d 2.57 (s, 2H, NH2); 2.74 (s, 6H, NMe2); 3.31 (d, J 3.6 Hz, 2H,
CH2); 4.09 (s, 1H, CH); 4.44 (m, 2H, SeCH2); 7.32–7.4, 7.1–7.73
(m, 4H, Ph). 13C NMR (D2O): d 28.3 (SeCH2); 42.4 (NMe2);
52.5 (H2NCH); 61.2 (NCH2); 129.9 (C3–Ph); 130.1 (C4–Ph);
131.2 (C5–Ph); 132.4 (Se–C1–Ph); 132.7 (C2–Ph);137.1 (C6–
+
=
Ph); 170.5 (C O). MS (TOF MS ES ) m/z (%): 303 (100), 301
(47.9), 299 (18.9) [M+H].
Deprotection of 4,4-dimethyl-2-oxazoline-N-Boc-L-selenocy-
steine methyl ester (10). 4,4-Dimethyl-2-oxazoline-N-Boc-L-
selenocysteine methyl ester was deprotected by following a
similar method to that employed for the phenyl derivative.
During the deprotection, the oxazoline ring is hydrolyzed to
1
give the corresponding acid derivative 4. [13C{ H} in D2O: d
=
=
56.3 (SeCH2); 60.2 (OC CHNH); 111.9 (SeC ); 114.8 (C4–Ph);
117.7 (C5–Ph); 120.6 (C2–Ph); 162.5 (C3–Ph); 162.8 (C6–Ph);
=
=
163.1 (O COH attached to Ph ring): 163.5 (O COH)].
16 L. A. Pete Silks, III, Phosphorus, Sulfur and Silicon, 1998, 136, 137
& 138, 611.
17 E. M. Stocking, J. N. Schwarz, H. Senn, M. Salzmann and L. A.
Silks, J. Chem. Soc., Perkin Trans.1, 1997, 2443.
18 J. Roy, W. Gordon, I. L. Schwartz and R. Walter, J. Org. Chem., 1970,
35, 510.
19 S. Mitsuru, H. Kimiko and S. Harushisa, Heterocycles, 1997, 44, 319.
20 N. M. Okeley, Y. Zhu and W. A. van der Donk, Org. Lett., 2000, 2,
3603.
21 (a) M. D. Gieselman, L. Xie and W. A. van der Donk, Org. Lett.,
2001, 3, 1331; (b) Y. Zhu, M. D. Gieselman, H. Zhou, O. Averin and
W. A. van der Donk, Org. Biomol. Chem., 2003, 1, 3304.
22 M. Rooseboom, N. P. E. Vermeulen, I. Andreadou and J. N. M.
Commandeur, J. Pharmacol. Exp. Ther., 2000, 294, 762.
23 A. H. G. Siebum, W. S. Woo, J. Raap and J. Lugtenburg, Eur. J. Org.
Chem., 2004, 2905.
HPLC Assay. The GPx-like activity of ebselen and Sec
derivatives was studied with H2O2 as a substrate and PhSH as
thiol co-substrate by following a method reported by Back et al.
for BnSH.32 In this assay, we employed a mixture containing
a 1 : 1.2 molar ratio of PhSH and H2O2 in dichloromethane–
methanol (95 : 5) at room temperature as our model system.
Runs with and without 10 mol% of added catalyst were carried
out under the same conditions and the column was eluted
with a methanol–water (90 : 10) mixture. Periodically, aliquots
were removed and the concentrations of the product diphenyl
disulfide (PhSSPh) were determined from the detector response,
using pure PhSSPh as an external standard. The initial rates
2 4 8 0
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1

View Article Online
24 R. Kaur, H. B. Singh and R. P. Patel, J. Chem. Soc., Dalton Trans.,
1996, 2719.
25 W.-H. Pan and J. P. Fackler, Jr., J. Am. Chem. Soc., 1978, 100, 5783.
26 (a) H. Duddeck, Prog. Magn. Reson. Spectros., 1995, 27, 1; (b) T. M.
Klapo¨tke and M. Broschag, Compilation of Reported 77Se NMR
Chemical Shifts, Wiley, Chichester, 1996.
28 S. R. Wilson, P. A. Zucker, R.-R. C. Huang and A. Spector, J. Am.
Chem. Soc., 1989, 111, 5936.
29 D. D. Perrin, W. L. F. Armarego and D. R. Perrin, Purification of
Laboratory Chemicals, Pergamon, Oxford, 2nd edn., 1980.
30 P. Garner and J. M. Park, Org. Synth., 1991, 70, 18.
31 G. Mugesh, A. Panda, H. B. Singh, N. S. Punekar and R. J. Butcher,
J. Am. Chem. Soc., 2001, 123, 839.
27 I. Andreadou, W. M. P. B. Menge, J. N. M. Commandeur, E. A.
Worthington and N. P. E. Vermeulen, J. Med. Chem., 1996, 39, 2040.
32 T. G. Back and B. P. Dyck, J. Am. Chem. Soc., 1997, 119, 2079.
O r g . B i o m o l . C h e m . , 2 0 0 5 , 3 , 2 4 7 6 – 2 4 8 1
2 4 8 1