Selenotyrosine and related phenylalanine derivatives

Article abstract of DOI:10.1016/S0968-0896(01)00052-9

A new series of Se-substituted phenylalanine derivatives has been synthesized having the para position of the phenyl ring substituted by selenocyanate (-SeCN), seleninic acid (-SeO₂ H), or selenol (-SeH) functional groups. The starting material for synthesis was 4′-aminophenylalanine which is readly available in DL- or L-forms. Selenium was incorporated into the ring by reacting the unprotected amino acid with nitrous acid, followed by reaction of the diazotized aromatic amine with potassium selenocyanate at pH 4-5 to give phenylalanine slenocyanate. The selenocyanate derivative was converted to the selenol directly by reduction with sodium borohydride, or oxidized to the seleninic acid, which was then reduced to the selenol. Alkylation of the selenol gave the diselenide. Mild oxidation of the selenoether 4′-(MeSe)Phe with peroxide gave the selenoxide derivative, 4′- [Se(O)Me]. Because of their stability and useful redox properties, aromatic selenoamino acids can be used as synthetic analogues to increase chemical functionality in protiens or peptides, and have potential pharmaceutical or nutritional applications. The possibility that aromatic selenoamino acids could be formed metabolically through reactions of reactive selenium intermediates with aromatic amino acid residues is discussed. Copyright

Full text of DOI:10.1016/S0968-0896(01)00052-9

Bioorganic & Medicinal Chemistry 9 (2001) 1459–1466
Selenotyrosine and Related Phenylalanine Derivatives
Howard E. Ganther*
Department of Nutritional Sciences, 1415 Linden Drive, University of WI Madison, Madison, WI 53706, USA
Received 4 December 2000; accepted 15 January 2001
Abstract—Anew series of Se-substituted phenylalanine derivatives has been synthesized having the para position of the phenyl ring
substituted by selenocyanate (-SeCN), seleninic acid (-SeO2H), or selenol (-SeH) functional groups. The starting material for
synthesis was 40-aminophenylalanine, which is readily available in dl- or l- forms. Selenium was incorporated into the ring by
reacting the unprotected amino acid with nitrous acid, followed by reaction of the diazotized aromatic amine with potassium sele-
nocyanate at pH 4–5 to give phenylalanine selenocyanate. The selenocyanate derivative was converted to the selenol directly by
reduction with sodium borohydride, or oxidized to the seleninic acid, which was then reduced to the selenol. Alkylation of the
selenol (‘selenotyrosine’) gave the selenoether derivatives of phenylalanine [(Phe-SeR), R=methyl or allyl], and air oxidation of the
selenol gave the diselenide. Mild oxidation of the selenoether 40-(MeSe)Phe with peroxide gave the selenoxide derivative, 40-
[Se(O)Me]. Because of their stability and useful redox properties, aromatic selenoamino acids can be used as synthetic analogues to
increase chemical functionality in proteins or peptides, and have potential pharmaceutical or nutritional applications. The possibi-
lity that aromatic selenoamino acids could be formed metabolically through reactions of reactive selenium intermediates with aro-
matic amino acid residues is discussed. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
other analogues of non-protein sulfur amino acids are
found in plants and selenized garlic.6 Se-methylseleno-
cysteine and Se-allylselenocysteine are more active than
selenomethionine in cancer chemoprevention studies.7
The nutritional roles of selenium (Se) at sub-ppm diet-
ary levels are well established.1 Cancer chemopreventive
activity demonstrated with supranutritional levels of Se
in animals2 and Se supplements in humans3 has drawn
much attention. The increasing use of long-term Se
supplementation by large numbers of persons increases
the need for a fuller understanding of the chemistry of
Se relevant to its biological activities. Selenium com-
pounds undergo extensive metabolism and form highly
reactive intermediates. Generation of hydrogen selenide
and increased formation of methylated selenide deriva-
tives is associated with supranutritional levels of Se
intake.4 Minute quantities of hydrogen selenide also
provide Se needed for specific co-translational synthesis
of selenoproteins containing selenocysteine; the func-
tions of Se as an essential trace element at normal diet-
ary levels are known to involve at least a dozen
selenoproteins.5 Translational incorporation of seleno-
methionine in place of methionine occurs non-
specifically, reflecting the relative abundance of these
amino acids. No other selenoamino acids are known to
occur in proteins, but Se-methylselenocysteine and some
Aromatic selenoamino acids offer interesting possibi-
lities for study, but very few have been synthesized and
none are known to occur naturally. In general, aromatic
Se compounds are more stable than aliphatic Se com-
pounds.8 Aromatic selenoamino acids would be useful
synthetic analogues for studies of protein or peptide
chemistry, and possibly could have pharmaceutical or
nutraceutical applications. Thiotyrosine, an analogue of
tyrosine having the phenolic oxygen replaced by sulfur,
was synthesized almost a century ago.9 The present
article describes the synthesis and some properties of
selenotyrosine and related phenylalanine derivatives.
This group of aromatic selenoamino acids was chosen
for their chemical simplicity and stability. The corre-
sponding sulfur derivatives of phenylalanine already are
known.10,11 The starting material for synthesis was 40-
aminophenylalanine, which is readily available in dl- or
l- forms from commercial sources or by synthesis.12
Upon reaction with nitrous acid, selective diazotization
of the p-amino group on the aromatic ring is possible,
so that the a-amino group of the amino acid is unaf-
fected.13 The reaction of potassium selenocyanate with a
diazotized aromatic amine is a well-established method
*Tel.: +1-608-262-2727; fax: +1-608-262-5860; e-mail: hganther@
facstaff.wisc.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00052-9

1460
H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
for attaching Se to an aromatic ring.8 By diazotization
of the p-amino group of 40-aminophenylalanine and
reaction with potassium selenocyanate, the selenocya-
nate derivative of phenylalanine was obtained, from
which selenotyrosine and other derivatives were synthe-
sized (Scheme 1).
reaction was carried out at pH 4. Optimization of reac-
tion conditions such as reagent concentrations and pH
to increase yield might be worthwhile in the case of iso-
tope labeling reactions, but was not explored. The pur-
ification method employed a polymeric XAD-4 column
under conditions that retain aromatic aminoacids.14
The strategy was to isolate the selenocyanate inter-
mediate in high purity (at the expense of yield) using
conditions as mild as possible, thus minimizing side
reactions that might complicate subsequent reactions.
The filtered reaction mixture was applied to a large
XAD column to adsorb the aromatic selenocyanate.
After washing with water and 20% methanol, elution
with 40% methanol gave a ninhydrin-positive product
having an Rf higher than the parent 40-aminophenylala-
nine and similar to phenylalanine. The UV spectrum
(Fig. 1) showed features similar to phenylselenocyanate
and phenylthiocyanate.15 HPLC analysis showed a
major peak of 40-(SeCN)Phe 1 (identified by mass spec-
troscopy) plus small amounts of more polar impurities.
Amore polar ninhydrin-positive impurity observed by
TLC of the 40% methanol eluate showed a Se peak at
m/z=434.0 corresponding to [M+H]+ [80Se] for
Results
Selenenylation of phenylalanine ring
The 40-amino group of 40-aminophenylalanine was dia-
zotized using the optimal conditions described by Gram
et al.,13 then reacted with KSeCN (Scheme 2). The sele-
nylation reaction uses KSeCN, an inexpensive Se
reagent; it is readily prepared from elemental Se,8 thus
KSeCN can be labeled with 75Se or other isotopes of Se
for synthesis of isotopically labeled selenoamino acid
derivatives if desired. Under the strongly acidic conditions
needed for diazotization, KSeCN is unstable and releases
elemental Se; for this reason the selenocyanylation
Scheme 1.

H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
1461
C19H19N3O4Se, as well as a Se-containing peak at m/
z=217.5 for the doubly-charged molecule. These prop-
erties are consistent with a biphenyl derivative that
would be formed by further coupling of diazotized
aminophenylalanine with 40-(SeCN)Phe, the initial
product in the diazotization reaction.
material remained at the top of the column and the
nearly colorless eluate of pure 40-(SeCN)Phe 1 was col-
lected. This work up procedure gave good purification
but left considerable amounts of 40-(SeCN)Phe on the
column, as shown by subsequent analysis. Alternative
procedures such as Fluorosil column chromatography
gave good purification but resulted in deposition of a
glassy solid on the walls of the glass fraction collection
tubes. Polymerization of organic selenocyanates may be
catalyzed by trace metals present in Fluorosil. Dilute
aqueous methanol solutions of the purified selenocya-
nate were stable when stored in a refrigerator, and this
stock solution of 1 was used for the synthesis of other
derivatives.
In order to remove dimeric or polymeric by-products
(expected to be more cationic), the XAD eluate was
passed through a Dowex 50 (NH+4 ) cation exchange
column (the sample was first adjusted to neutral pH so
that the amino acid would be in its isoelectric form to
minimize retention on the column). Abrown band of
Synthesis of DL-40-methylselanylphenylalanine: [40-
(MeSe)Phe] (2)
The reduction/methylation of selenocyanate 1 was car-
ried out in one step under nitrogen (Scheme 1, top),
using sodium borohydride16 and excess methyl iodide.
The pH was maintained between 6 and 7 so that the
intermediate selenol was in the unprotonated (seleno-
late) form and the amino group remained protonated.
TLC analysis of the reaction mixture showed a single
ninhydrin-positive spot (Rf=0.77) having a higher
mobility than that of the parent compound (Rf=0.56)
or a phenylalanine standard (Rf=0.64). HPLC analysis
with diode array detection showed a major peak (reten-
tion time 8.3 min) having a distinctive UV spectrum
with maxima at 251 and 266 nm (Fig. 2). This peak was
identified as 40-(MeSe)Phe 2 on the basis of mass spec-
troscopy and other methods. Alternatively, selenoether
2 was prepared conveniently in two successive reactions
from phenylalanine seleninic acid 3 (see below). After
reduction with sodium borohydride to give the
Scheme 2.
Figure 2. Absorption spectrum of 40-(methylselenanyl)phenylalanine
2. The crystalline compound was dissolved in 65% methanol and
scanned against a solvent reference solution. The sample concentration
was 0.167 mM, determined by Se analysis.
Figure 1. Absorption spectrum of 40-(selenocyanato)phenylalanine 1
(0.0928 mM in 65% methanol).

1462
H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
intermediate selenol 4 (selenotyrosine), the solution
(maintained at pH 6, under nitrogen) was treated with a
methanolic solution of methyl iodide to give 40-(MeSe)-
Phe 2, identified by its characteristic UV spectrum using
HPLC diode array analysis.
244 nm; these spectral properties are similar to those of
benzene selenolate (lmax 271 nm, emM=13.7) and ben-
zene selenol.18 Upon spontaneous oxidation of the bor-
ohydride-treated sample, a broad absorption band
corresponding to the diselenide 6 was formed (lmax 248
and 329 nm) (Fig. 3b); the spectrum is similar to that of
diphenyl diselenide.18 Electrospray mass spectroscopy
of the sample confirmed the formation of the diselenide
6. The presence of a Cl8Na9 ion at m/z=488.8 precluded
observation of the diselenide [M+H]+ [80Se] at m/z=
489.0 calculated for C18H20N2O4Se2, but the expected
The stability of 40-(MeSe)Phe was investigated by stor-
ing pure peaks collected offꢀthe HPLC column in 65%
methanol/0.025% TFAat 4 under air, with or without
1 mM DTT. Slow formation of more polar substances
(about 5%) was observed by HPLC analysis over a
period of 30 days, and this was partly prevented by
DTT.
Synthesis of DL-40-seleninylphenylalanine: [40-(SeO2 ꢁ)Phe]
(3)
Many seleninic acids have been prepared by oxidation
of aromatic selenocyanates or diselenides with con-
centrated hydrogen peroxide, nitric acid, or peracetic
acid.8 Such aggressive conditions seemed unlikely to be
suitable for amino acids, but 3% hydrogen peroxide has
been used for selective oxidation of the sulfur in sulfur
amino acids and therefore was chosen for oxidation of
40-(SeCN)Phe 1 to the seleninic acid 3. Using UV diode
array HPLC to monitor the reaction, no oxidation was
seen at room temperature over 2 h, but oxidation began
to occur at 40ꢀ and rapid oxidation ensued upon
warming to 80ꢀ. The 40-(SeCN)Phe peak was lost within
15 min, with concurrent formation of a new peak eluted
at 5.3 min having a UV absorption peak at 224 nm (not
shown). Since seleninic acids are weak acids (pKa 4–5),17
anion exchange chromatography was employed for
purification of the reaction mixture. The seleninic acid
was eluted with 10 mM HCl in 20% methanol (59%
yield). Yields were lower when heating at 80ꢀ exceeded
15 min, and considerable amounts of strongly retained
selenium (not eluted with 0.01 N HCl but eluted with 1
N HCl) were formed, indicating that over-oxidation
may have occurred with the longer heating times.
The pooled sample of 3 showed a single ninhydrin- and
starch/iodide-positive spot (Rf=0.52 upon TLC in
acidic system 1, 0.11 in basic system 2), and anionic
mobility upon TLE at pH 7. The charged properties and
ninhydrin reactivity confirmed that the product was an
amino acid having an additional weak anion moiety; the
presence of the seleninic acid functional group was
confirmed by the starch/iodide test, which measures
oxidation of iodide to iodine and is characteristically
given by seleninic acids. At pH 1.6, TLE mobility was
cationic, confirming that the anionic groups had been
protonated [this weak acid behavior showed that the
product is a seleninic acid rather than a selenonic
(strong) acid]. Mass spectroscopy and NMR analysis
confirmed the structure.
Figure 3. Absorption spectra of selenotyrosine 4. A NaBH4 (50 mL 0.5
M) was added to 0.123 mM 40-(SeO2ꢁ)Phe in N2-sparged water (final
volume 2 mL). The spectrum of the borohydride-generated selenotyr-
osine sample was recorded immediately (dashed line, selenolate) or
after acidification to pH 2.2 with 40 mL l M HCl (solid line, selenol). B
Spectrum of selenotyrosine diselenide 6 recorded 16 h after sample (a)
was opened to air.
Properties of phenylalanine seleninic acid 3
Reduction of 3 with sodium borohydride (Fig. 3a) gen-
erated the spectrum of the selenolate form (lmax 272
nm, emM=16.5) of 40-(SeH)Phe (‘selenotyrosine’) 4.
Upon acidification of the sample the UV peak shifted to

H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
1463
isotope pattern was seen for the doubly-charged
diselenide [M+2H]++ ion at m/z 245.1.
was synthesized previously by a different procedure19
using addition of methyl Grignard reagent to diethyl
acetamido(cyanoselenobenzyl)malonate and subsequent
hydrolysis to give the amino acid. The method described
here introduces Se into the preformed phenylalanine
structure under mild conditions in aqueous solution,
avoiding the difficulties associated with Grignard
methodology.
Addition of methyl iodide to a borohydride-generated
sample of 40-(SeH)Phe gave a new HPLC peak having a
UV spectrum and retention time corresponding to the
alkylated product 2. Treatment of a parallel sample of 4
with allyl chloride gave a new HPLC peak (retention
time 11.6 min) having properties [UV spectrum and
mobility on TLC (Rf=0.79, system 1)] consistent with
the Se-allyl derivative of phenylalanine. Mass spectro-
metry showed a Se-isotope peak at m/z=285.9, corre-
sponding to [M+H]+ [80Se] (m/z=286.0) calculated for
(Se-allyl)Phe, C12H15NO2Se.
Organic selenocyanates and seleninic acids are well
known classes of organoselenium compounds with use-
ful properties. The phenylalanine selenocyanate or sele-
ninic acid derivatives described here are stable, water-
soluble amino acids with minimal odor. They are sui-
table for direct use in biological studies, or for intro-
duction of a reactive Se functional group in relatively
stable form into peptides or proteins. The phenylalanine
seleninic acid is especially convenient as a precursor that
can be treated with sodium borohydride or other sui-
table reducing agent for generating the selenol deriva-
tive of phenylalanine, selenotyrosine. The Se-
methylphenylalanine selenoether derivative is readily
oxidized to give the selenoxide derivative. Selenoxides
are far more reactive than their sulfoxide counterparts,
and react readily with thiols, leading to possible redox
cycling.20,21 Many aliphatic selenoxides are unstable,
whereas alkylaryl selenoxides have good handling
properties,21 thus a phenylalanine-based selenoxide is
expected to have superior stability compared to one
formed from Se-methylselenocysteine or similar type of
aliphatic selenoether.
Synthesis
of
DL-40-methylseleninylphenylalanine:
[40(MeSe=O)Phe] (5)
Controlled oxidation of 40-(MeSe)Phe 2 at 0ꢀ using
dilute hydrogen peroxide (H2O2:Se=3.3) gave the
selenoxide 5. The reaction was monitored by HPLC,
using the disappearance of the 8–9 min peak of 2 as an
endpoint; as this peak disappeared a new peak having a
maximum at 223 nm was eluted at 5.1 min, along with a
trace of hydrogen peroxide. Treatment of an aliquot of
this oxidized sample with a slight excess of DTT resul-
ted in restoration of the characteristic peak of 40-
(MeSe)Phe and nearly complete disappearance of the
5.1 min peak, showing that the oxidation was reversible
(data not shown). This is consistent with the oxidation
product being a selenoxide, rather than some other
product such as seleninic acid 3. Similar results were
obtained using oxidation at 4ꢀ with an excess of hydro-
gen peroxide (H2O2:Se=100), followed by addition of
excess DTT.
Use of phenylalanine analogues in biochemistry and
therapeutics
Numerous ring-substituted phenylalanine derivatives
have been synthesized chemically or occur naturally.
Nitro, amino, sulfo, and halogen derivatives were syn-
thesized in the l9th century by Erlenmeyer and other
chemists.10 Other phenylalanine derivatives containing
arsenic or sulfur substituents were synthesized for use as
antimetabolites.22,23 The thio analogue of tyrosine
(‘thiotyrosine’) was first synthesized in l912.9 Escher et
al.24 have described methods for protecting thiotyrosine
and other sulfur-containing phenylalanine derivatives
for peptide synthesis, and reported some biological
activities of angiotensin II analogues containing these
amino acids. More recently, aminothiotyrosine disulfide
derivatives undergoing facile homolytic cleavage have
been used as optical triggers in studies of protein folding
kinetics.25
Positive ion mass spectrometry showed one major com-
ponent corresponding to the protonated molecular ion
of the selenoxide 5. NMR analysis confirmed the pre-
sence of a methyl group at 2.84 ppm, compared to
2.36 ppm for the unoxidized parent compound 2. TLC
of the oxidized sample showed a single ninhydrin-posi-
tive spot having a low Rf (0.37) compared to the parent
selenide (0.77); upon TLE it behaved as a neutral amino
acid at pH 7 and had cathodic mobility slightly greater
than phenylalanine at pH 1.6. The electrophoretic
behavior under acidic conditions is consistent with the
slightly basic properties of selenoxides.8
Discussion
Anew series of Se-substituted phenylalanine derivatives
has been synthesized having the para position of the
phenyl ring substituted by selenocyanate (-SeCN), sele-
ninic acid (-SeO2H), or selenol (-SeH) functional
groups. Alkylation of the selenol (‘selenotyrosine’) gave
the selenoether derivatives of phenylalanine [(Phe-SeR),
R=methyl or allyl], and air oxidation of the selenol
gave the diselenide. Mild oxidation of the selenoether 40-
(MeSe)Phe with peroxide gave the selenoxide derivative,
40-[Se(O)Me]. Six of these compounds were previously
unknown; the Se-methyl selenoether 40-(MeSe)Phe 2
Aromatic selenoamino acids have useful properties for a
wide range of biochemical applications. The p-seleno-
cyanate derivative of phenylalanine is readily prepared
and converted to other stable derivatives such as the
seleninic acid with standard oxidizing agents. Reduction
of phenylalanine-40-seleninic acid is a convenient means
of generating selenotyrosine 4. Because of the facile
oxidation of selenotyrosine, isolation of the free selenol
4 was not attempted, but it can be generated easily for
study of redox properties. Alkylation of the freshly-
formed selenol using alkyl halides allows synthesis of

1464
H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
Se-substituted phenylalanine analogues. It is hoped that
the present study will facilitate further investigations
with aromatic selenoamino acids to explore their chem-
istry and biological significance.
onto the tyrosine ring was suggested to modulate the
redox potential of the free radical site in the enzyme.34
The thioether apparently is attached to tyrosine in the
ortho position, but isolation of the thioether bridged
dipeptide by chemical methods has not been reported.
Phenols undergo facile electrophilic substitution and it
is likely that tyrosine would undergo selenylation pre-
ferentially compared to phenylalanine. However, sub-
stituted phenylalanine derivatives may be more stable
than substituted tyrosine derivatives because phenols
having Se constituents can undergo oxidative reactions
leading to formation of quinones and related products.
Naturally occurring phenylalanine derivatives
Besides tyrosine, the best known naturally-occurring
phenylalanine derivatives are the various iodinated tyr-
osines and thyronines, and bromo-substituted aromatic
amino acids found in marine organisms. The formation
of mono- and dibromotyrosine derivatives also occurs
in humans through reaction of tyrosine residues with
the eosinophil peroxidase that preferentially utilizes
bromide or thiocyanate in peroxidative reactions with
H2O2.26 It is not known whether analogous metabolic
activation reactions might occur with Se, either in
intermediary metabolism of Se4 or in association with
its biological function as a component of glutathione
peroxidases and other selenoproteins. One purpose of
this work was to develop a basis for such studies and
synthesize stable model compounds.
Conclusion
The Se-containing phenylalanine derivatives described
here can be used in free form, or incorporated into
peptides and engineered proteins to increase chemical
functionality. The various functional groups are rela-
tively stable because of direct Se bonding to the phenyl
ring, and confer physical properties such as polar neu-
tral (-SeCN), hydrophilic anionic (-SeO2 ꢁ), or hydro-
phobic, as in the case of the selenoethers (-SeR). Other
modifications of the selenoether derivatives are possible,
such as alkylation to give positive-charged selenonium
derivatives (-Se+R1R2), or reversible oxidation to rela-
tively stable alkylphenyl selenoxides that undergo redox
cycling.21 The selenol group found in selenotyrosine has
high reactivity in redox reactions or in nucleophilic
reactions. Alkylation of the freshly-formed selenol using
alkyl halides allows synthesis of a variety of Se-sub-
stituted phenylalanine analogues. Photolability of aro-
matic selenoamino acid selenocyanates or facile
reactions of other Se-containing aromatic selenoamino
acids leading to radical formation also could be useful.
Anionic Se-substituted analogues of tyrosine are of
interest as possible surrogates for phosphotyrosine.
Metabolic activation of Se and adduct formation with
proteins is known to occur. Methylseleninic acid and
inorganic sodium selenite can react with thiol groups of
proteins to give adducts containing selenenylsulfide (SꢁSe)
or selenotrisulfide (SꢁSeꢁS) bonds.27 These adducts are
cleaved by reducing agents to release Se from the pro-
tein. However, there is evidence from radioactive tracer
studies28,29 that proteins may incorporate unidentified
forms of Se that are resistant to reductive removal, in
addition to selenocysteine and selenomethionine. A
mechanism for forming stable Se adducts might be the
generation of highly reactive Se intermediates within
suitable domains and further reaction with nearby
amino acid residues. The successive reaction of two
thiol groups with inorganic selenite forms RS-Se(=O)-
SR, a very reactive electrophilic intermediate.30,31 In
proteins containing a cluster of two or more proximal
cysteine residues, it is conceivable that initial reaction of
selenite with two cysteines would be followed by a
reaction of this reactive intermediate [RS-Se(=O)-SR]
with a nearby residue, forming an aromatic selenoamino
acid adduct in the protein. When additional thiol
groups are available, further reaction of the RS-
Se(=O)-SR intermediate may give the selenotrisulfide
RS-Se-SR, plus disulfide, as a competing reaction. A4:1
thiol/selenite reaction stoichiometry was demonstrated
for reduced ribonuclease, a model protein having 8
sulfhydryl groups.32 In a protein having cysteine resi-
dues located in a relatively inaccessible hydrophobic
domain, it is possible that the reaction would be con-
strained to the initial phase that generates electrophilic
intermediates from the reaction of only one or two thiol
groups, followed by electrophilic substitution of Se onto
the ring of tyrosine or phenylalanine.
Experimental
Materials and methods
Reagents were obtained from Sigma-Aldrich (St. Louis,
MI). Mass spectra were acquired on a SciexAPI365 tri-
ple quadrupole mass spectrometer at the University of
Wisconsin Biotechnology Center. The instrument was
operated in positive ion mode using minimal energy
conditions in order to prevent fragmentation of the Se
compounds. Samples were injected after acidification
with 0.5% acetic acid, or directly injected for samples
eluted from ion exchange columns with 0.01 N HCl.
1
The H NMR spectra were obtained at the National
Magnetic Resonance Facility in Madison using a Bru-
ker DMX 500 spectrometer. Chemical shifts are repor-
ted in parts per million (ppm, d) relative to DSS [3-
(TMS)-1-propanesulfonic acid, sodium salt] as internal
standard. Ultraviolet spectra were obtained on a Shi-
madzu Model 1601 spectrophotometer using 1 cm
pathlength quartz cuvettes.
Some evidence has been obtained for the occurrence of
thiolated derivatives of tyrosine in proteins. The active
site of galactose oxidase has a novel thioether bond
linking Cys 228 and Tyr 272, as shown by crystal-
lographic studies.33 Substitution by the cysteine sulfur

H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
1465
For HPLC a polymer-based reversed phase column
(Hamilton PRP-1, 1ꢂ10 cm, fitted with a guard column)
was operated at 25ꢀ using isocratic elution (1 mL/min)
with methanol/water (65:35) containing 0.025% tri-
fluoroacetic acid, pH 2.5. Aphotodiode array detector
(Waters Model 991) was used to monitor the UV spec-
tra of eluted compounds. TLC was done on precoated
cellulose plastic sheets using system 1 (n-butanol/acetic
acid:water, 5:2:3, v/v/v) or system 2 (n-butanol/pyr-
idine:water, 7:8:6, v/v/v). Ninhydrin (0.2% in acetone)
or starch/iodide (1% soluble starch plus 1% KI) were
used as detection reagents. Thin layer electrophoresis
(TLE) was carried out on 20ꢂ20 cm cellulose-coated
glass plates at 10ꢀ, 350 volts. The buffers used were pH
1.6 (formic acid/acetic acid/water, 150:100:750), pH 5.2
(pyridine/acetic acid/water, 10:2.5:988), or pH 7 (0.05 M
HEPES). N-DNP-ethanolamine was used as a neutral
marker. Phenylalanine (net charge +1 at pH 1.6) was
used as a cationic marker and N-DNP-taurine (net
charge -1) as an anionic standard. Selenium was assayed
by a fluorometric procedure following acid digestion of
duplicate aliquots.35 Iodometric analysis was done by
standard procedures.
phenylalanine and one-half that of 40-aminophenylala-
nine; pH 5.2, single spot, same mobility as phenylala-
nine. H NMR (D2O) d 7.76 (d, 2H), 7.37 (d, 2H), 3.99
(m, 1H), 3.14–3.31 (m, 2H).
1
DL-40-(methylselenanyl)phenylalanine (2). Asolution of
40-(SeCN)Phe 1 (550 mL, 0.47 mmol) was placed in a 3-
necked flask fitted with a pH electrode. Nitrogen was
swept through the stirred solution, then a solution of
sodium borohydride (2.4 mmol) was added over 30 min,
maintaining the pH at 5 to 7 by addition of 1 N HCl.
Iodomethane (total of 3.6 mmol) then was added in
several portions and the mixture left to react for 22 h at
25ꢀ in the dark under N2. The reaction mixture was
concentrated two-fold by rotary evaporation to lower
the methanol concentration, then applied to an XAD
column. After washing with water followed by 20%
methanol to remove salts and other impurities, 40-(Me-
Se)Phe was eluted with 65% methanol, as shown by
HPLC analysis of the collected fractions. The leading
edge of the peak contained traces of a more polar sub-
stance; these fractions were excluded from the final pool
of 40-(MeSe)Phe 2 (55% yield). One half of the pool of
pure 40-(Me-Se)Phe 2 was concentrated to remove the
methanol. Aportion was lyophilized and dissolved in
CD3OD plus D2O for NMR analysis. The remainder of
the aqueous concentrate, stored at 4ꢀ, slowly crystal-
lized. A65% methanol solution of the crystalline mate-
rial was scanned to obtain the UV spectrum. UV (65%
MeOH): Shoulder 220 nm, emM10.4; lmax 251 and
266 nm, lmin 260 nm; emM 7.61, 7.32, and 7.09, respec-
tively. MS: m/z 259.9, [M+H]+ [80Se] calc 260.0. HPLC
retention time 8.3 min. TLC: (system 1) single ninhy-
drin-positive spot (Rf=0.77). TLE: pH 1.6, single nin-
Synthesis of compounds. Note: Certain forms of selenium
and cyanide are volatile and highly toxic, or have
offensive odors. The reactions described should be carried
out using apparatus designed to minimize the release of
volatile compounds, within a good quality fume hood
DL-40-(selenocyanato)phenylalanine (1). dl-40-aminophe-
nylalanine (10 mmol) dissolved in 4.5 mL 6 M HCl was
chilled in an ice bath and 5.9 mM NaNO2 in ice-cold
water was added in small portions with stirring until
starch/iodide test paper gave a positive test (total of
10 mmol NaNO2). The pH was adjusted to 4 by addi-
tion of 5 M sodium acetate, and a chilled solution of
KSeCN (10.1 mmol in 4 mL water) was added in small
portions over 15 min. After evolution of N2 ceased, the
solution (pH about 5) was stirred at room temperature
for 19 h. (During this reaction and subsequent opera-
tions, care was taken to to protect the sample from
light). TLC analysis of the orange-red solution showed
loss of the starting compound and the presence of a
ninhydrin-positive spot of higher Rf. After dilution and
filtration to remove elemental Se, the sample was
applied to a large XAD column (230 mL bed vol) pre-
viously cleaned as described.14 After washing with 1 bed
vol of water and 100 mL of 20% methanol, 40%
methanol was used to elute the nearly pure 40-(SeCN)-
Phe (2.31 mmol, 23% yield). The light-golden solution
was adjusted to pH 7.4 and passed through a
2.5ꢂ9.5 cm column of Dowex 50 (NH4 +) to remove
small amounts of impurities, giving an almost colorless
solution of pure 40-(SeCN)Phe 1 in 40% methanol. This
solution was stored at 4ꢀ until used for synthesis of the
remaining compounds. Aportion was lyophilized to
dryness and dissolved in D2O for NMR. UV: lmax
236 nm (emM=7.18), with a shoulder from 250–300 nm.
MS: m/z 270.9, [M+H]+ [80Se] calculated 271.0. HPLC
retention time 6.3 min. TLC: (system 1) single ninhy-
drin-positive spot (Rf=0.7). TLE: (pH 1.6), single nin-
hydrin-positive cationic spot, mobility similar to
1
hydrin-positive cationic spot. H NMR (CD3OD, D2O)
d 7.45 (d, 2H), 7.22 (d, 2H), 3.87 (m, 1H), 2.97–3.23 (m,
2H), 2.36 (s, 3H).
DL-40-(seleninyl)phenylalanine (3). 40-(SeCN)Phe 1 from
the 65% methanol pool off Dowex 50 (20 mL,
0.041 mmol) was concentrated under nitrogen to 1/3
vol. After addition of hydrogen peroxide (3.2 mmol)
and warming to 80–85ꢀ, HPLC analysis showed com-
plete disappearance of the starting compound within 15
min and formation of a new peak eluted at 5.3 min. The
solution was cooled on ice to halt further oxidation. The
sample (pH 3.9) was adjusted to pH 7.5 with NH4OH
and applied to a 1.5ꢂ7.5 cm Dowex 1 (Clꢁ) column
previously washed with 3% H2O2 and methanol and
equilibrated with water. The column was washed with 2
bed vol of water until a negative starch/iodide test was
obtained, then elution was begun with 10 mM HCl in
20% methanol. Amajor UV and starch/iodide positive
peak was eluted, followed by a minor second compo-
nent. The pure fractions containing the main peak of 3
were pooled and analyzed (24 mmols, 59% yield, based
on Se analysis and confirmed by iodometric analysis). A
2 mL aliquot was lyophilized and dissolved in 0.5 mL
D2O for NMR. UV (20% MeOH, 0.01 M HCl): lmax
224 nm, emM=13.8, with a shoulder from 250 to 280
nm. Mass spectrum: m/z 277.9, [M+H]+ [80Se] calcu-
lated 278.0. HPLC retention time 5.3 min. TLC: single
ninhydrin- and starch/iodide-positive spot, Rf=0.46

1466
H. E. Ganther / Bioorg. Med. Chem. 9 (2001) 1459–1466
(system 1); Rf=0.11 (system 2). TLE: pH 7, single
anionic spot; pH 1.6, single cationic spot. 1H NMR
(D2O) d 7.87 (d, 2H), 7.58 (d, 2H), 4.30 (m, 1H), 3.29–
3.46 (m, 2H).
4. Ganther, H. E.; Lawrence, J. R. Tetrahedron 1997, 53,
12299.
5. Stadtman, T. C. Ann. Rev. Biochem. 1996, 65, 83.
6. Ip, C.; Birringer, M.; Block, E.; Kotrebai, M.; Tyson, J. F.;
Uden, P. C.; Lisk, D. J. J. Agric. Food Chem. 2000, 48, 2062.
7. Ip, C.; Zhu, Z.; Thompson, H. J.; Lisk, D.; Ganther, H. E.
Anticancer Res. 1999, 19, 2875.
8. Klayman, D. L.; Gunther, W. H. H. Organoselenium Com-
pounds: Their Chemistry and Biology; Wiley-Interscience, New
York, 1973.
9. Johnson, T. B.; Brautlecht, C. A. J. Biol. Chem. 1912, 12,
175.
10. Greenstein, J. P.; Winitz, M. Chemistry of the Amino
Acids; Wiley, New York, 1961, Vol. 3.
11. Colescott, R. L.; Herr, R. R.; Dailey, J. P. J. Am. Chem.
Soc. 1957, 79, 4232.
12. Bergmann, E. D. J. Am. Chem. Soc. 1952, 74, 4947.
13. Gram, H. F.; Mosher, C. W.; Baker, B. R. J. Am. Chem.
Soc. 1959, 81, 3103.
14. Mills, G. C. J. Chromatog. 1986, 355, 193.
15. Bordwell, F. G.; Boutan, P. J. J. Am. Chem. Soc. 1956, 78,
854.
16. Krief, A.; Delmotte, C.; Dumont, W. Tetrahedron 1997,
53, 12147.
DL-40-(methylseleninyl)phenylalanine (5). An aliquot of
40-MeSePhe 4 (10 mmol) was concentrated three fold to
remove methanol, then cooled on ice and treated with
small portions of H2O2 (total of 33 mmol). After 2 h,
HPLC showed that the 40-MeSePhe had completely
disappeared, forming a new peak eluted at 5.1 min
having an UV maximum at 223 nm. Treatment of an
aliquot of the reaction mixture (0.083 mmol Se) with
DTT (0.5 mmol) caused loss of the 5.1 min peak, with
reappearance of the peak of 40-MeSePhe, followed by a
peak of oxidized DTT (lmax 282 nm) at 11.1 min. An
aliquot of the H2O2-oxidized 40-(MeSe)Phe sample was
lyophilized to dryness and dissolved in D2O to give a
solution of 5 for NMR analysis. UV (65% MeOH,
0.025% TFA): lmax 223 nm, shoulder 255–280 nm. MS:
m/z 275.9, [M+H]+ [80Se] calc 276.0. HPLC retention
time 5.1 min. TLC: (system 1) single ninhydrin- and
starch/iodide-positive spot (Rf=0.37). TLE: pH 1.6,
single ninhydrin- and starch/iodide-positive cationic
spot, slightly higher mobility than phenylalanine; pH 7,
single neutral spot (same mobility as phenylalanine). 1H
NMR (D2O) d 7.77 (d, 2H), 7.54 (d, 2H), 4.02 (t, 1H),
3.22–3.34 (m, 2H), 2.85 (s, 3H).
17. Gould, E. S.; McCullough, J. D. J. Am. Chem. Soc. 1951,
73, 1109.
18. Ergman, L.; Andersson, C.; Morgenstern, R.; Cotgreave,
I.; Andersson, C.-M.; Hallberg, A. Tetrahedron 1994, 50, 2929.
19. Loeschorn, C. A.; Kelley, C. J.; Hanson, R. N.; Davis,
M. A. Tetrahedron Lett. 1984, 25, 3387.
20. Goeger, D. E.; Ganther, H. E. Arch. Biochem. Biophys.
1994, 310, 448.
21. Chen, G.-P.; Ziegler, D. M. Arch. Biochem. Biophys. 1994,
312, 566.
Acknowledgements
22. Elliot, D. F.; Harington, C. J. Chem. Soc. 1949, 1949,
1374.
23. Bressler, W. L.; Ciereszko, L. S. J. Am. Chem. Soc. 1955,
77, 2330.
24. Escher, E.; Bernier, M.; Parent, P. Helvetica Chim. Acta
1983, 66, 1355.
25. Lu, H. S. M.; Volk, M.; Kholodenko, Y.; Gooding, E.;
Hochstrasser, R. M.; DeGrado, W. F. J. Am. Chem. Soc.
1997, 119, 7173.
This work was supported by grants DK 14184 and CA
45164 from the National Institutes of Health. The
author thanks Robert Lawrence for technical assistance.
The assistance of Dr. Amy Harms with the electrospray
ionization mass spectrometry studies at the University
of Wisconsin-Madison Biotechnology Center is appre-
ciated. The National Magnetic Resonance Facility at
Madison is an NIH-funded shared instrumentation
laboratory.
26. Wu, W.; Chen, Y.; d’Avignon, A.; Hazen, S. L. Biochem-
istry 1999, 38, 3538.
27. Ganther, H. E. Carcinogenesis 1999, 20, 1657.
28. McConnell, K. P.; Wabnitz, C. H. J. Biol. Chem. 1957,
226, 765.
29. Beilstein, M. A.; Whanger, P. D. J. Nutr. 1986, 116, 1711.
30. Kice, J. L.; Lee, T. W. S.; Pan, S.-T. J. Am. Chem. Soc.
1980, 102, 4448.
References
31. Kice, J. L.; Lee, T. W. S. J. Am. Chem. Soc. 1978, 100,
5094.
32. Ganther, H. E.; Corcoran, C. Biochemistry 1969, 8, 2557.
33. Ito, N.; Phillips, S. E. V.; Yadav, K. D. S.; Knowles, P. F.
J. Mol. Biol. 1994, 238, 794.
1. Levander, O. A. In Trace Elements in Human and Animal
Nutrition; Mertz, W., Ed.; Academic Press: Orlando, 1986;
Vol. 2, pp 209–279.
2. Ip, C. J. Nutr. 1998, 128, 1845.
3. Clark, L. C.; Combs, G. F., Jr.; Turnbull, B. W.; Slate,
E. H.; Chalker, D. K.; Chow, J.; Davis, L. S.; Glover, R. A.;
Graham, G. R.; Gross, E. G.; Krongrad, A.; Lesher, J. L., Jr.;
Park, H. K.; Sanders, B. B., Jr.; Smith, C. L.; Taylor, J. R. J.
Am. Med. Assoc. 1996, 276, 1957.
34. Babcock, G. T.; El-Deeb, M. K.; Sandusky, P. O.; Whit-
taker, M. M.; Whittaker, J. W. J. Am. Chem. Soc. 1992, 114,
3727.
35. Oh, S.-H.; Ganther, H. E.; Hoekstra, W. G. Biochemistry
1974, 13, 1825.