Access to any site-directed isotopomer of methionine, selenomethionine, cysteine, and selenocysteine - Use of simple, efficient modular synthetic reaction schemes for isotope incorporation

Article abstract of DOI:10.1002/ejoc.200400063

Simple modular reaction schemes that allow access to any isotopomer of protected serine and homoserine have been worked out. These systems could be simply converted into cysteine, selenocysteine, homocysteine, homoselenocysteine, the essential amino acid methionine, and selenomethionine by Mitsunobu chemistry. These sulfur- and selenium-containing amino acids fulfil many essential roles in the living organism. In addition, homoserine could be converted in a few steps into optically active L-vinylglycine. As well as the stable isotopes ¹³C, ¹⁵N, ¹⁷O, and ¹⁸O, the radioactive isotopes of sulfur, selenium and carbon can also be easily introduced in a site-directed fashion. In view of the wide scope of the Mitsunobu reaction, we feel that many more important systems with the carbon skeleton of serine and homoserine should be preparable through this basic chemistry in any site-directed isotopically labeled form. Wiley-VCH Verlag GmbH & Co, KGaA, 69451 Weinheim, Germany, 2004.

Full text of DOI:10.1002/ejoc.200400063

FULL PAPER
Access to Any Site-Directed Isotopomer of Methionine, Selenomethionine,
Cysteine, and Selenocysteine ؊ Use of Simple, Efficient Modular Synthetic
Reaction Schemes for Isotope Incorporation
Arjan H. G. Siebum,[a] Wei Sein Woo,[a] Jan Raap,[a] and Johan Lugtenburg*[a]
Keywords: Amino acids / Chirality / Cystathionine / Homocysteine / Isotope labels / Selenohomocysteine / Site-directed
isotopomers / Vinylglycine
Simple modular reaction schemes that allow access to any
isotopomer of protected serine and homoserine have been
worked out. These systems could be simply converted into
cysteine, selenocysteine, homocysteine, homoselenocysteine,
the essential amino acid methionine, and selenomethionine
by Mitsunobu chemistry. These sulfur- and selenium-con-
taining amino acids fulfil many essential roles in the living
organism. In addition, homoserine could be converted in a
stable isotopes 13C, 15N, 17O, and 18O, the radioactive iso-
topes of sulfur, selenium and carbon can also be easily intro-
duced in a site-directed fashion. In view of the wide scope of
the Mitsunobu reaction, we feel that many more important
systems with the carbon skeleton of serine and homoserine
should be preparable through this basic chemistry in any
site-directed isotopically labeled form.
( Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2004)
few steps into optically active L-vinylglycine. As well as the
Introduction
Cysteines are involved in the disulfide bridges that consti-
tute an important element in protein structure and peptide
hormones such as insulin. Cys also coordinates to metal
ions in metaloproteins, forms covalent bonds with the haem
group in the active site of the protein part of cytochrome
C, and is the basis of the active site of hydrolytic enzymes
such as papaine.[3]
A special case is selenocysteine, the 21st proteinogenic
amino acid, which is translationally incorporated into pro-
teins by a special mechanism.[4] Most human selenoproteins
have homologues in which Sec has been replaced by Cys.
These sulfur proteins, though, are poor catalysts in com-
parison with selenoproteins.[5] In the meantime, 18 different
human selenoproteins have been described. Recently, it has
been established that the human selenoproteonome consists
of 25 selenoproteins.[6] Interestingly, the genome sequences
of H. influenzae and M. jannaschii indicate that their
genomes code for Sec-containing enzymes involved in the
biosyntheses of selenocysteine.[7,8] Dietary selenium is an
Methionine (Met), cysteine (Cys), and selenocysteine
(Sec) are the three proteinogenic amino acids possessing
high-mass chalcogen atoms (S and Se) in their side chains
(Figure 1). These amino acid residues are responsible for
important protein functions that cannot be achieved by
other amino acid residues. The sulfur atoms in methionine
and cysteine, for example, coordinate to metal ions involved
in many redox and catalytic processes of metaloproteins.[1,2]
Figure 1. The structures of -methionine (1), -selenomethionine essential element in human nutrition, playing important
(2), -homocysteine (3), - homoselenocysteine (4), -cysteine (5),
roles in cancer prevention, immunology, aging, male repro-
and -selenocysteine (6); the letters a, b, etc. indicate the source of
duction, and other physiological processes.[6,9] Seleno-
proteins are thought to be responsible for most of the bio-
medical effects of dietary selenium, which is essential to
mammals. The reduction of incidence of cancer by
dietary supplementation with selenomethionine via Se-
(methyl)selenocysteine has been reported.[10] Among the
sulfur-containing amino acids, besides the effects discussed
above, methionine deserves special mention. One of the
nine essential amino acids that should be present in suf-
the 13C enriched starting materials: the ‘‘a’’ and ‘‘c’’ carbon atoms
are derived from the carboxylic group of acetic acid and ‘‘b’’ and
‘‘d’’ from the methyl group of acetic acid, ‘‘e’’ originates from
methyl iodide, and ‘‘f’’ from paraformaldehyde, while the source of
15N, indicated by ‘‘n’’, is 15NH3
[a]
Leiden Institute of Chemistry, Gorleaus Laboratories, Leiden
University,
P. O. Box 9502, 2300 RA Leiden, The Netherlands
Fax: (internat.) ϩ 31-71-5274488
E-mail: Lugtenbu@chem.leidenuniv.nl
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ficient quantity in food,[1] methionine reacts with ATP with pomers of a system, the optimal method is to develop a
formation of S-(adenosyl)methionine (SAM), which serves modular synthetic approach involving a minimal number of
as the methyl donor in biological methylation. After the steps in convergent fashion, each of them preferably in a
transfer of the methyl group, S-(adenosyl)homocysteine is high yield. These conditions and the use of relatively inex-
formed, and hydrolyses to homocysteine, the essential re- pensive isotopically enriched starting materials should pro-
agent in the biosynthesis of cysteine.[1] Homocysteine can vide the molecule in question in such a fashion that each
be reconverted into methionine by methylation by N5- atom derives in a unique way from the enriched starting
methyl tetrafolate. Impairment of this regeneration process material. Finally, the isotope-enriched -amino acids should
results in elevated plasma levels of homocysteine. It has be prepared in the highest optical purity. Recently we pub-
been found that plasma levels of homocysteine are a causal lished a synthetic scheme, based on the O’Donnell strat-
risk factor for cardiovascular diseases of importance equal egy,[25] to allow access to any site-directed isotopomer of -
to that of hypercholesterimia, hypertension, and smok- leucine. A protected glycine derivative is treated with a pri-
ing.[11,12] Selenomethionine mimics almost all the roles of mary alkyl halide in the presence of a chiral phase-transfer
methionine. [75Se]-Selenomethionine has a central place in catalyst to form the -form of leucine in high enantiomeric
medical diagnosis and therapy as a radiotracer in which excess. For that study we also worked out a scheme to syn-
temporal and spatial aspects of the interactions of various thesize the protected glycine derivative in all its possible iso-
organs and tumors can be followed. Recently, the potential topomers. It is clear that for the preparation of the full set
of [73Se]-selenomethionine as a short-lived positron emitter of isotopomers of methionine and selenomethionine we
for in vivo positron emission tomography (PET) has been need to prepare the full set of isotopomers of homoserine,
discussed. This technique is the method of choice for meas- which can subsequently be converted into Met and SeMet
urement of the rate of protein synthesis in the brain as an via homocysteine and homoselenocysteine, respectively, fol-
important tool to understand the function of the human lowed by methylation with isotopically labeled methyl iod-
brain.[13] A similar role for [11C]-methionine has also been ide. Similarly, the precursor for Cys and Sec, protected ser-
discussed.[14] The substitution of Met residues by selenome- ine, would be prepared by the O’Donnell method. In this
thionine, constituting an isomorphous substitution of S by paper these schemes have been optimized for the prep-
the heavy Se, is of great importance in X-ray crystallogra- aration of Cys, Sec, Met, and SeMet with synthons in natu-
phy of proteins.[15,16] Besides its properties as heavy atom in ral isotopic abundance. The starting materials on which
X-ray structural analysis, natural Se also contains 7.6% of these conversions are based are commercially available in
the 77Se isotope, a spin 1/2 nucleus with excellent properties any isotopically enriched form. In our schemes all atoms
for 77Se spectroscopy.[17] In view of the importance of Cys, derive from well defined source. This approach has the ad-
Sec, Met, and SeMet in various vital biological processes, vantage of a modular and efficient method, resulting in
access to the full set of 13C and 15N isotopomers of these schemes in which any isotopomer is accessible, in high yield
amino acids is of crucial urgency. Their roles in nutrition and in high enantiomeric excess (ee).
can be followed in exquisite detail by mass spectrometry of
these isotopomers, by analysis of the serum after adminis-
Results and Discussion
tration in food. Site-directed incorporation into proteins
can elucidate the structural factors relating to each carbon,
hydrogen, nitrogen, and selenium atom with atomic resolu-
tion through 1H, 13C, 15N and 77Se NMR spectroscopy. Vi-
Synthesis
The central synthon in the preparation of -methionine
brational spectroscopy of a set of different site-directed iso- (1), -selenomethionine (2), -homocysteine (3), and -
topomers should provide the force fields of the various homoselenocysteine (4) is the protected homoserine 10
chemical bonds in the desired residue in a protein. Even (Scheme 1). The reactions shown in Scheme 1 and all
more detailed knowledge should be obtainable by compari- further schemes were optimized with synthons of natural
son of pairs of proteins in which a sulfur atom has been isotopic abundance. The starting materials Ϫ tert-butyl
replaced by a selenium atom. Quantitative biosynthetic re- benzophenoneimine glycinate (7), dimethylallyl bromide
placement has been achieved by use of methionine auxotro- (8), and the chiral cinchona-derived phase-transfer cata-
phe E. coli strains.[16] In the meantime, the chemical incor- lyst[26,27] Ϫ are all commercially available. In order to pre-
poration of cysteine, methionine (via homocysteine), selen- pare homoserine 10, 7 was dissolved in a toluene/chloro-
ocysteine, and selenomethionine (via homoselenocysteine) form mixture and treated with 8 in the presence of 0.01
has also been achieved.[18Ϫ21] Two approaches for the syn- equiv. chiral phase-transfer catalyst (PTC) and base (50%
thesis of -selenomethionine have been published,[22,23] and KOH) to yield the alkylated species in 90% yield.
recently the preparation of -selenocysteine and -[77Se]-se-
Before treatment of the double bond of the side chain
lenocysteine has been published.[24] The syntheses for the with O3, the O3-sensitive benzophenoneimine protecting
sulfur analogues mentioned above cannot serve our pur- group had to be removed with citric acid, and the resulting
pose for the synthesis of the site-directed isotopomers, be- free amine was reprotected with the N-Boc group, which
cause they all start from starting materials with preformed is stable to ozonolysis. The Boc group was introduced by
carbon skeletons not amenable to simple stable 13C and 15
N
treatment of the free amine with Boc-anhydride in the pres-
incorporation. For the preparation of the whole set of isoto- ence of triethylamine to give 9 in 92% yield over two steps.
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Site-Directed Isotopomers of Methionine, Selenomethionine, Cysteine, and Selenocysteine
FULL PAPER
(DIAD) in dry THF gave 11 in 86% yield. Treatment of an
ethanolic solution of 11 with aqueous NaOH in the pres-
ence of MeI gave immediate saponification of the thioester,
followed by methyl thioether formation to give the required
protected methionine 12 in near quantitative yield. Sub-
sequent treatment of 12 with a 10% solution of trifluoro-
acetic acid in dichloromethane gave pure Met (1), which
showed all the analytical characteristics of an authentic
sample of commercial methionine after recrystallization.
Carrying out the described reaction sequence with 13C-en-
riched methyl iodide should also give incorporation of a
stable 13C isotope label in the SCH3 group of methionine.
Treatment of 11 with NaOH in the absence of CH3I gave
the protected homocysteine, which upon deprotection with
trifluoroacetic acid gave homocysteine (3), with all the
characteristics of the authentic material. For the prep-
aration of selenomethionine (2), 10 was treated with tri-
phenylphosphane, iodine, and imidazole dissolved in dry di-
chloromethane to convert the alcohol function into the cor-
responding iodide in 86% yield. After this, the iodide was
added to a DMF solution containing a mixture of elemen-
tal selenium, hydrazine monohydrate, and sodium hydrox-
ide. The reaction between selenium and hydrazine in the
presence of NaOH gives Na2Se2, which reacted twice with
the iodide to give the N-Boc-protected homoselenocystine
13. Treatment of 13 with NaBH4 in the presence of methyl
iodide resulted in the reduction of the diselenide bond, fol-
lowed by methylation of the resulting selenide to give the
protected selenomethionine. Deprotection with trifluoro-
acetic acid gave selenomethionine (2), while deprotection of
13 with trifluoroacetic and subsequent reduction with
NaBH4 gave homoselenocysteine. Both products show all
the analytical characteristics as described in the litera-
ture.[29,30]
Scheme 1. Preparation of the protected homoserine 10 from the
protected glycine derivative 7 and dimethylallyl bromide (8); the
letters a, b, c, d, and n indicate the 13C or 15N source (see legend
of Figure 1)
Subsequently, 9 was dissolved in dry dichloromethane and
treated with ozone at Ϫ40 °C. The resulting ozonide could
be reduced with dimethyl sulfide to provide the correspond-
ing aldehyde, which was further reduced with NaBH4 to
give homoserine 10 in 78% yield. The preparation of all
13C,15N isotopomers of 7 as well as the full set of 13C isoto-
pomers of 8 is possible and has been reported by us in an
earlier publication.[28] This means that all isotopomers of
protected homoserine 10 are now accessible in a few steps,
each in a high yield. We have also prepared 10 through al-
kylation of 7 with crotyl bromide, followed by the same
conversions as described in Scheme 1. Compound 10 was
obtained in good yield, but the synthesis and purification
of dimethylallyl bromide (8) and its isotopomers proved to
be more efficient than the preparation of isotopically en-
riched crotyl bromide. Scheme 2 describes the conversion of
From the schemes presented above it is clear that the N-
the protected homoserine into either Met (1) or SeMet (2). Boc-serine tert-butyl ester (17) is the ideal starting material
Mitsunobu treatment of 10 with thioacetic acid, tri- for the preparation of the full set of isotopomers of cysteine
phenylphosphane, and diisopropyl diazodicarboxylate and selenocysteine. For their preparations, the synthesis of
Scheme 2. Preparation of -methionine (1) and -selenomethionine (2) from -homoserine (10)
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A. H. G. Siebum, W. S. Woo, J. Raap, J. Lugtenburg
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Scheme 3. A short and efficient route to protected serine 17
-serine was optimized first (Scheme 3).[31] To prepare pro- irreversible inhibitor for pyridoxal phosphate-dependent
tected serine 17, glycinate 7 was treated with benzyl chloro- aminotransferases.[33] Again, the full set of isotopomers
methyl ether under chiral phase-transfer conditions to give would be necessary for investigation of these vital biological
16 in 70% yield. Benzyl chloromethyl ether can be prepared processes at atomic resolution. Oxidation of the protected
in good yield in a one-pot reaction from paraformaldehyde, selenomethionine 14 with hydrogen peroxide at low tem-
benzyl alcohol, and HCl.[32] By this approach, 18O and 17O perature and subsequent CH3Se(O)H elimination should
can be incorporated from isotopically enriched water and give the protected -vinylglycine derivative. Unfortunately,
13C from isotopically enriched paraformaldehyde.[32]
this reaction gives 21 in only 20% yield. It is known from
The O-benzyl protection has to be removed by catalytic the literature, however, that formation of a terminal double
reduction (Pd/C and hydrogen), but the benzophenoneim- bond by selenium oxide elimination can be accomplished
ine protecting group is not stable under reducing con- efficiently in high yield by use of an o-nitrophenyl selen-
ditions. It was therefore replaced with the N-Boc protecting ide.[34,35] By this approach, 20 was prepared in 82% yield
group, by treatment of 16 first with citric acid, and then by treatment of 10 with (Bu)3P in dry THF (Scheme 5),
with Boc-anhydride and triethylamine to give N-Boc, O- followed by addition of o-nitrophenyl selenocyanate. Sub-
benzylserine tert-butyl ester, which was finally converted sequent treatment of 20 with aqueous H2O2 at Ϫ5 °C
into N-Boc-serine tert-butyl ester (17) by catalytic hydro- yielded the protected vinylglycine 21 in 79% yield. Depro-
genation.
tection with trifluoroacetic acid gave -vinylglycine 21 with
Scheme 4 depicts the conversion of the protected serine the same analytical characteristics as given in the literature.
into Cys (5) and Sec (6). These reactions are essentially the Protected vinylglycine can also serve as a starting material
same as discussed for Scheme 2. The Cys (5) and Sec (6) to form important novel chiral derivatives.
obtained in this way both show, after recrystallization, the
same analytical characteristics as the authentic materials.
In this way access is gained to the full set of 13C and 15N
isotopomers for both Cys and Sec.
Discussion
From the Mitsunobu reaction of the phenyl selenocyan-
-Vinylglycine (21) is a potent inhibitor of β-cystathion- ate shown in Scheme 5 it is clear that reagents of this type
ase in the methionine biosynthetic pathway. It is also an can easily convert primary alcohols into the corresponding
Scheme 4. Preparation of cysteine and selenocysteine
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UV-light, spraying with KMnO4 solution, ninhydrin-staining (0.2%
in ethanol) or staining with a mixture of 4,4Ј-methylenebis(N,N-
dimethylaniline) and ninhydrin (TDM staining). Chiral HPLC was
performed on a Chiralcel ODH column (25 cm) with hexane and
2-propanol (iPrOH) as the solvent system or on a Daicel crown-
pack CR(ϩ) column (150 mm) with a HClO4 solution as the sol-
vent system. Dry diethyl ether (ether, Et2O) was obtained by distil-
lation from P2O5. Dry petroleum ether (PE) 40Ϫ60 and dry di-
chloromethane (DCM) were obtained by distillation from CaH2.
Dry tetrahydrofuran (THF) was obtained by drying over sodium
benzophenone. Deaereated solvents were obtained by bubbling of
a steady stream of argon or dry nitrogen through the liquid for one
hour. All reagents were purchased from Aldrich Chemical Co.,
Acros Chimica, or Fluka.
Scheme 5. Preparation of vinylglycine starting from protected
homoserine
tert-Butyl 2-(Benzhydrylideneamino)-5-methylhex-4-enoate: Di-
methylallyl bromide (8) (0.55 g, 1.1 equiv.), catalyst (102 mg, 0.05
equiv.), and aqueous KOH (50%, 10 equiv.) were added at 0 °C to
a solution of 7 (1 g, 3.4 mmol) in toluene (10 mL). The mixture
was stirred vigorously at 0 °C for 6 h and at room temp. overnight.
It was then diluted with water (15 mL) and extracted with ethyl
acetate (3 ϫ 20 mL). After evaporation the crude product was puri-
fied by column chromatography (PE/Et2O, 80:20). After the prod-
uct had been collected, the column was eluted with DCM to re-
cover the catalyst. The yield was 90% (1.11 g), ee Ͼ 90% (Chiralcel
seleno and thio diethyl ethers. We feel that there should be
no barrier to extension of these reactions to the preparation
of the corresponding tellurium systems.[24] Our schemes
also provide the building blocks for the preparation of cys-
tathionine, an intermediate in the biosynthesis of cysteine
(it is the thioether with the same carbon skeleton as Cys)
and lanthionine (the thioether made of two Cys carbon
skeletons), which occur in proteins in the skin[36Ϫ38] and are ODH, 25 cm, hexane/iPrOH, 99.9:0.1 v/v, flow 0.8 mL/min. Reten-
tion time (min): 15.26 (S), 16.47 (R). 1H NMR (300 MHz): δ ϭ
formed during the biosynthesis of certain antimicrobial
4
compounds.[39,40]
1.44 (s, 9 H, tBu), 1.56 (s, 3 H, H6), 1.64 (d, JH,H ϭ 0.75 Hz, 3
3
3
H, H6), 2.57 (m, 2 H, H3), 3.98 (1 H, dd JH,H ϭ 5.62, JH,H
ϭ
3
4
7.54 Hz, H4), 5.05 (1 H, tq JH,H ϭ 7.5, JH,H ϭ 0.75 Hz, H4),
7.70Ϫ7.13 (m, 10 H, arom) ppm. 13C NMR (75.5 MHz): δ ϭ 17.74
(CH3), 25.60 (CH3), 27.85 (tBu), 32.24 (C3), 66.22 (C2), 80.42 (Cq),
120.11 (C4), 127Ϫ140 (C, Phe), 136.55 (C5), 169.33 (CN), 171.03
(C1) ppm.
Conclusion
In this paper we describe a modular and convergent strat-
egy for the preparation of -Ser, Cys, Sec, Met, SeMet, and
vinylglycine in optically pure forms, in such a fashion that
all the stable 13C, 15N, 17O, 18O, 77Se and the radioactive
isotopomers can be easily introduced in a site-directed man-
ner, through the alkylation of a protected glycine derivative
with alkyl halides in the presence of a chiral active phase-
transfer catalyst. After the reaction, the optically active
catalyst can be easily isolated, ready to be reused. By this
method no difficult separation of the product from an op-
tically active scaffold is needed.[41] The central step for the
tert-Butyl 2-Amino-5-methylhex-4-enoate: The alkylated glycinate
(1.11 g, 3.05 mmol) was dissolved in THF (10 mL) and stirred with
citric acid solution (10%, 10 mL) for one night. TLC showed com-
plete disappearance of the starting material, and the mixture was
extracted with diethyl ether. The water layer was brought to pH 12
and extracted twice with ethyl acetate (30 mL) to give the product
in 98% yield (0.6 g, 3.0 mmol) after evaporation. 1H NMR
(300 MHz): δ ϭ 1.46 (s, 9 H, tBu), 1.64 (s, 3 H, H6c), 1.72 (3 H,
d 4JH,H ϭ 1.0 Hz, H6t), 2.35 (m, 2 H, H3), 3.37 (dd, 3JH,H ϭ 5.49,
3JH,H ϭ 6.87 Hz, 1 H, H2), 5.10 (tq, 3JH,H ϭ 6.87, 4JH,H ϭ 1.0 Hz,
incorporation of S and Se is an efficient Mitsunobu reac- 1 H, H4) ppm. 13C NMR (75.5 MHz): δ ϭ 17.19 (CH3), 25.60
(CH3), 27.73 (tBu), 33.33 (C3), 54.76 (C2), 80.36 (tBu), 119.00
(C4), 124.62 (C5), 174.61 (C1) ppm.
tion. From the scope of the Mitsunobu reaction it is to be
expected that many important optically active α-amino ac-
ids with the serine and homoserine carbon skeleton should
become easily available.
tert-Butyl 2-tert-Butoxycarbonylamino-5-methylhex-4-enoate (9):
The amino ester prepared above was dissolved in DMF (5 mL),
triethylamine (0.3 g, 1 equiv., 0.42 mL) was added, followed by di-
tert-butyl dicarbonate (0.71 mL, 1.1 equiv.), and the mixture was
stirred until TLC showed completion of the reaction. The DMF
was evaporated and ethyl acetate (25 mL) was added. The organic
layer was washed thrice with KHSO4 solution (pH 2, 25 mL),
Experimental Section
1
General Remarks: H NMR spectra were recorded with Jeol FX-
200 and Bruker DPX-300 spectrometers, with tetramethylsilane water, and brine, and was dried with MgSO4. The crude product
(TMS: δ ϭ 0 ppm) or water (H2O: δ ϭ 4.8 ppm) as internal stand-
ards. 13C noise-decoupled NMR spectra were recorded on a Jeol
FX-200 at 50.1 MHz or on a Bruker DPX-300 spectrometer at
75.5 MHz, with CDCl3 (δ ϭ 7 ppm), (CD3)2CO (δ ϭ 206 ppm), or
was purified over a short silica column (PE/Et2O 80:20). The yield
was 94% (0.85 g). 1H NMR (300 MHz): δ ϭ 1.44 (s, 9 H, tBu),
1.45 (s, 9 H, tBu), 1.61 (s, 3 H, H6c), 1.70 (d, 4JH,H ϭ 1.0 Hz, 3 H,
H6t), 2.45 (m, 2 H, H3), 4.21 (dd, 3JH,H ϭ 5.6, 3JH,H ϭ 13.5 Hz, 1
TSP (δ ϭ 0 ppm) as internal standards. All spectra were recorded H, H2), 5.04 (m, 1 H, H4), 5.04 (s, 1 H, NH), ppm. 13C NMR
in CDCl3, except where noted otherwise. Column chromatography
was performed on Merck silica gel 60 (0.040Ϫ0.063 mm 230Ϫ400
(75.5 MHz): δ ϭ 18.64 (CH3), 26.58 (CH3), 28.69 (tBu), 29.05
(tBu), 31.94 (C3), 54.57 (C2), 80.14 (tBu), 85.81 (tBu), 118.80 (C4),
mesh), and spots on thin-layer chromatography were detected with 136.17 (C5), 155.91 (CO), 172.19 (C1) ppm.
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N-Boc-homoserine tert-Butyl Ester (10): A solution of 9 (0.84 g,
2.8 mmol) in DCM (150 mL) in a three-necked, round-bottomed
10%) was added. The mixture was stirred at room temp. for 1 night
and then extracted with water to give (after evaporation) the meth-
flask fitted with a dropping funnel containing dimethyl sulfide ionine.TFA salt in 82% yield from 12 (135 mg, 0.51 mmol). Recrys-
(0.37 mL, 1.5 equiv.) in DCM (10 mL) was cooled to Ϫ50 °C in an
acetone/dry ice bath. Ozone was passed through the solution until
the liquid had a persistent blue color. The excess ozone was purged
from the solution with a soft nitrogen stream, and subsequently the
tallization of -methionine from a water/ethanol mixture (pH 3.9)
gave -methionine in Ͼ 98% ee (HClO4 (pH 1.5), 15% MeOH, flow
0.6 mL/min. Retention time (min): 3.31 (R), 6.39 (S). 1H NMR
(300 MHz, D2O): δ ϭ 2.13 (s, 3 H, H5), 2.20 (m, 2 H, H3), 2.70
3
3
3
dimethyl sulfide was added dropwise while the temperature was (t, JH,H ϭ 7.38 Hz, 2 H, H4), 4.20 (dd, JH,H ϭ 6.3, JH,H ϭ 6.5
kept below Ϫ40 °C. The reaction mixture was warmed to room
temp. over two hours and the solvents were evaporated (careful,
stench) in vacuo. The crude product was taken up in ethanol
(60 mL) and the solution was cooled to 0 °C. NaBH4 (0.21 g, 2
equiv.) was added, and the mixture was stirred for 4 h. Workup
was accomplished by addition of HCl solution (1 , 10 mL), fol-
lowed by evaporation of the bulk of the ethanol. Diethyl ether
(40 mL) was added, and the organic layer was extracted with
NaHCO3 solution, water, and brine, and the organic layers were
subsequently dried with MgSO4. The crude product was purified
by column chromatography (gradient 20% to 40% Et2O in PE).
Hz, 1 H, H2) ppm. 13C NMR (75.5 MHz D2O): δ ϭ 14.69 (C5),
29.39 (C4), 29.76 (C3), 52.79 (C2), 172.79 (C1) ppm.
tert-Butyl 2-tert-Butoxycarbonylamino-4-iodobutyrate: Triphenyl-
phosphane (1.04 g, 4.0 mmol, 2 equiv.) was dissolved in dry di-
chloromethane (15 mL), and the solution was cooled to 0 °C and
stirred under dry nitrogen while iodine (0.99 g, 1.99 equiv.) was
added in portions. After 30 minutes, a mixture of 10 (0.55 g,
2.0 mmol) and imidazole (0.28 g, 2.05 equiv.) was slowly added to
the dark purple solution. After one minute a white solid became
visible. The reaction mixture was stirred for another two hours,
while the temperature was maintained at 0 °C. Subsequently, the
solids were filtered off and the solvent was evaporated. The re-
sulting solid was redissolved in DCM (1 mL) and purified by col-
umn chromatography (PE/Et2O, 90:10) to give the iodide in 86%
1
The yield was 78% (611 mg, 2.2 mmol). H NMR (300 MHz): δ ϭ
1.49 (s, 18 H, 2 ϫ tBu), 2.04 (m, 2 H, H3), 3.58 (m, 2 H, H4), 4.26
3
(m, 1 H, H2), 5.38 (d, JH,H ϭ 7.8 Hz, 1 H, NH) ppm. 13C NMR
(75.5 MHz): δ ϭ 28.17 (tBu), 27.87 (tBu), 36.16 (C3), 50.93 (C2),
58.14 (C3), 80.12 (tBu), 82.11 (tBu), 156.40 (CO), 171.72 (C1) ppm.
1
yield (0.66 g, 1.74 mmol). H NMR (300 MHz): δ ϭ 1.45 (s, 9 H,
tBu) 1.48 (s, 9 H, tBu), 2.17 (m, 1 H, H3), 2.35 (m, 1 H, H3), 3.43
S-Acetyl-N-boc-homocysteine tert-Butyl Ester (11): DIAD
(0.317 mL, 2 equiv.) was added at 0 °C under dry nitrogen to a
solution of triphenylphosphane (418 mg, 1.6 mmol, 2 equiv.) in dry
THF (10 mL) in a flame-dried round-bottomed flask, and the mix-
ture was stirred until a white solid appeared. Stirring was continued
for 10 min at 0 °C, after which 10 (0.22 g, 0.8 mmol) in dry THF
(2 mL) was added. After the mixture had been stirred for 45 min,
thioacetic acid (0.114 mL, 1.6 mmol) was added and stirring was
continued for 3 h. Diethyl ether (30 mL) was added, and the mix-
ture was extracted with water (25 mL), followed by drying of the
organic layer over MgSO4. After evaporation of the solvents, the
resulting solid was taken up in DCM (3 mL) and flashed over silica,
which was then rinsed with diethyl ether (50 mL). The product was
concentrated in vacuo and was further purified by chromatography
(PE/Et2O, 85:15). The yield was 86% (0.23 g, 0.68 mmol). 1H NMR
(300 MHz): δ ϭ 1.46 (s, 9 H, tBu), 1.48 (s, 9 H, tBu), 1.88 (m, 1
H, H3), 2.04 (m, 1 H, H3), 2.33 (s, 3 H, CH3), 2.90 (m, 2 H, H4),
4.22 (m, 1 H, H2), 5.14 (d, 1 H, NH) ppm. 13C NMR (75.5 MHz):
δ ϭ 25.04 (C5), 27.93 (tBu), 28.29 (tBu), 30.54 (C4), 33.07 (C3),
53.31 (C2), 80.20 (tBu), 82.28 (tBu), 155.40 (CO), 171.09 (C1),
196.20 (SCO) ppm.
(m, 2 H, H4), 4.24 (m, 1 H, H2), 5.13 (1 H, broad s, NH) ppm.
N-Boc-homoselenocystine Di-tert-Butyl Ester (13): N2H4·H2O (100
µL, 1.1 equiv.) was added to a suspension of Se (0.15 g, 1.87 mmol,
1.1 equiv.) and NaOH (0.1 g) in DMF (5 mL). The mixture was
stirred at 60 °C under argon for 3 h. Subsequently, the iodide pre-
pared above (0.65 g, 1.70 mmol) was added dropwise, and the mix-
ture was stirred for 3 h. TLC indicated complete disappearance of
the starting material, and the solvent was evaporated in vacuo. The
resulting oil was dissolved in diethyl ether (25 mL) and extracted
with water (5 mL) and brine (5 mL), and was dried with MgSO4
to give 13, which was obtained in 71% yield (0.41 g) after column
1
chromatography (PE/Et2O, 85:15). H NMR (300 MHz): δ ϭ 1.45
(s, 18 H, 2 ϫ tBu) 1.48 (s, 18 H, 2 ϫ tBu), 1.96Ϫ2.32 (4 H, 2 ϫ
H3), 2.89 (m, 4 H, 2 ϫ H4), 4.19 (m, 2 H, H1), 5.14 (2 H, broad
s, NH) ppm.
N-Boc-selenomethionine tert-Butyl Ester (14): Diselenide 13 (0.41 g,
0.60 mmol) was dissolved in EtOH (10 mL), the mixture was co-
oled to 0 °C, and NaBH4 (50 mg, 1.33 mmol) was added. The mix-
ture was stirred at 0 °C for 15 minutes, and CH3I (0.18 mL, 2.9
mol) was added. After 30 minutes, HCl (1 , 2 mL) was added,
followed by water (20 mL), and the mixture was extracted thrice
with EtOAc (25 mL). The collected organic fractions were dried
with MgSO4 and the solvent was evaporated in vacuo. The resulting
oil was purified by column chromatography to give the title com-
N-Boc-methionine tert-Butyl Ester (12): Compound 11 (0.23 g,
0.68 mmol) was redissolved in ethanol (10 mL), and NaOH solu-
tion (2 , 5 mL) was added to the stirred solution, which was kept
under argon. The hydrolysis was followed by TLC. After 3 h, CH3I
(90 µL, 2 equiv.) was added, and the solution was stirred overnight.
The solution was concentrated in vacuo to 4 mL, and ethyl acetate
(50 mL) was added. The organic layer was washed with water
(10 mL) and brine, and dried with MgSO4. After evaporation the
product was purified over a silica column (PE/Et2O, 80:20), giving
1
pound in 90% yield (0.38 g). H NMR (300 MHz): δ ϭ 1.44 (s, 9
H, tBu), 1.47 (s, 9 H, tBu), 1.97 (m, 1 H, H3) 1.99 (s, 3 H, H5),
2.16 (m, 1 H, H3), 2.53 (m, 2 H, H4), 4.25 (m, 1 H, H2), 5.19 (1
H, broad s, NH) ppm. 13C NMR (75.5 MHz): δ ϭ 4.06 (C5), 20.26
(C4), 28.35 (6 ϫ CH3), 33.56 (C3), 54.13 (C2), 79.69 (tBu), 82.62
(cq), 155.24 (CO), 171.79 (C1) ppm.
1
a 91% yield (190 mg). H NMR (300 MHz): δ ϭ 1.45 (s, 18 H, 2
ϫ tBu), 1.99 (m, 1 H, H3), 2.11 (s, 3 H, H5), 2.15 (m, 1 H, H3),
2.58 (m, 2 H, H4), 4.45 (1 H, H2), 5.25 (1 H, NH) ppm. 13C NMR
(75.5 MHz): δ ϭ 15.36 (C5), 28.28 (6 ϫ CH3), 29.95 (C4), 31.72
(C3), 52.63 (C2), 80.35 (tBu), 82.60 (tBu), 155.58 (CO), 170.80
(C1) ppm.
Trifluoroacetate of Selenomethionine (2): The protected selenome-
thionine 14 was dissolved in TFA (10%, 15 mL) in dry DCM and
the mixture was stirred for 3 h. When TLC indicated complete
deprotection, the mixture was extracted twice with water (15 mL),
and the collected water layers were evaporated under reduced press-
ure to give selenomethionine trifluoroacetic acid salt (0.31 g, 91%
L-Methionine Trifluoroacetate (1): The protected methionine 12
1
(190 mg) was dissolved in DCM, and trifluoroacetic acid (TFA,
yield). H NMR (200 MHz, D2O, pH ϭ 7): δ ϭ 2.0 (s, 3 H, H5),
2910
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
Eur. J. Org. Chem. 2004, 2905Ϫ2913

Site-Directed Isotopomers of Methionine, Selenomethionine, Cysteine, and Selenocysteine
FULL PAPER
3
2.1Ϫ2.3 (m, 2 H, H3), 2.6 (t, JH,H ϭ 7.14 Hz, 2 H, H4), 3.8 (m, over a short silica column (PE/Et2O, 85:15). The yield was 82%
1 H, H2) ppm.
(128 mg). 1H NMR (200 MHz): δ ϭ 1.5 (s, 18 H, 2 ϫ tBu), 3.7 (m,
1 H, Hβ), 3.9 (m, 1 H, Hβ), 4.3 (m, 1 H, Hα), 4.4Ϫ4.6 (m, 2 H,
OCH2,), 5.4 (broad s, 1 H, NH), 7.2Ϫ7.4 (m, 5 H, Phe) ppm.
L-Homocysteine Trifluoroacetate (3): Compound 11 (0.5 mmol,
167 mg) was redissolved in deaereated ethanol (5 mL), and deaere-
ated NaOH solution (1 , 3 mL) was added to the stirred solution, N-Boc-serine tert-Butyl Ester (17): The N-Boc-protected serine de-
which was subsequently kept under argon. After 6 h, the mixture
was extracted twice with deaereated DCM (30 mL) and the col-
lected organic layers were dried with MgSO4 in an Erlenmeyer
rivative (128 mg, 0.36 mmol) was dissolved in a mixture of deaere-
ated methanol and acetic acid (97:3 v/v, 10 mL), to which a cata-
lytic amount of 10% Pd/C was added. Hydrogen was bubbled
filled with argon. The solvent was then evaporated, and the product through at normal pressure for 18 h. The solution was flushed with
was redissolved in deareated TFA in DCM solution (10%, 10mL).
After 12 h, the solvent and acid were extracted with deaereated
water (2 ϫ 10 mL), and the water layers were evaporated under
reduced pressure. The yield of 3 (as the trifluoroacetate) was 73%
nitrogen gas, and water (10 mL) and ethyl acetate (50 mL) were
added. The organic layer was extracted twice with water (5 mL)
and was evaporated in vacuo. The crude product was purified by
column chromatography (PE/Et2O, 80:20), giving 17 in a yield of
1
1
(90 mg). H NMR (200 MHz, D2O): δ ϭ 2.2 (m, 2 H, H3) 2.6 (m,
79% (75 mg, 0.29 mmol). H NMR (200 MHz): δ ϭ 1.45 (s, 9 H,
2 H, H4), 3.9 (m, 1 H, H2) ppm.
tBu), 1.49 (s, 9 H, tBu), 3.9 (m, 2 H, H3), 4.3 (1 H, broad s, H2),
5.4 (1 H, broad s, NH) ppm.
L-Homoselenocystine Trifluoroacetate (4): The diselenide 13 (0.3 g,
0.44 mmol) was dissolved in TFA in DCM solution (10%, 10 mL),
and the mixture was stirred for 18 h without stopper. The solvent
and TFA were evaporated in vacuo. The resulting oily solid was
S-Acetyl-N-boc-cysteine tert-Butyl Ester (18): DIAD (0.317 mL, 2
equiv.) was added at 0 °C under a dry nitrogen to a solution of
triphenylphosphane (0.44 g, 2.0 equiv.) in dry THF (10 mL) in a
taken up in water (5 mL) and washed twice with DCM (5 mL) to flame-dried round-bottomed flask, and the mixture was stirred un-
give, after evaporation of the water layer, the title compound in
til a white solid appeared. Stirring was continued for 10 min at 0
°C, after which 17 (0.22 g 0.84 mmol) in dry THF (2 mL) was ad-
79% yield (208 mg). 1H NMR (300 MHz, D2O): δ ϭ 1.72Ϫ1.91
(m, 2 ϫ 2 H, H3), 2.42 (m, 2 ϫ 2 H, H4), 3.62 (d, 3JH,H ϭ 6.45 Hz, ded. After 45 min stirring, thioacetic acid (0.120 mL, 1.68 mmol)
2 ϫ 1 H, H1) ppm.
was added, and stirring was continued for 3 h. Diethyl ether
(30 mL) was added to the reaction mixture, the mixture was ex-
tracted with water (15 mL), and the organic layer was dried with
MgSO4. After evaporation of the solvents, the resulting solid was
taken up in DCM (5 mL) and flashed over a glass filter filled with
silica, which was then rinsed with diethyl ether (50 mL). After evap-
oration of the ether, the product was further purified by chromatog-
raphy (PE/Et2O, 85:15). The yield was 76% (0.2 g). 1H NMR
(300 MHz): δ ϭ 1.44 (s, 9 H, tBu), 1.47 (s, 9 H, tBu), 2.35 (s, 3 H,
H4), 3.34 (m, 2 H, H3), 4.50 (broad s, 1 H, H2), 5.21 (1 H, broad
s, NH) ppm.
tert-Butyl 2-(Benzhydrylidene)amino-3-(benzyloxy)propionate (16):
tert-butyl
N-(Benzophenoneimine)glycinate
(7)
(200 mg,
0.67 mmol) was dissolved in dry DCM (3 mL). The solution was
cooled to Ϫ5 °C, and benzyl chloromethyl ether (1.5 equiv., 70µL),
O-allyl N-methylanthracenyl cinchonidium bromide (20%, 80 mg),
and BEMP (2-tert-butylimino-2-diethylamino-1,3-dimethyl-per-
hydro-1,3,2-diazaphosphorine, 0.58 mL, 3 equiv.) were added. The
mixture was stirred vigorously at this temperature for 18 h. TLC
indicated almost complete disappearance of the starting material,
and water (5 mL) and DCM (20 mL) were added. The organic layer
was washed with water (5 mL) and dried with MgSO4, and the
solvents were evaporated. The crude product was purified over a
silica column (PE/ethyl acetate, 95:5), giving 16 in 70% yield
(193 mg). ee Ͼ 81% (Chiralcel ODH, 25 cm, hexane/iPrOH,
L-Cysteine (5): Compound 18 (0.2 g) was dissolved in deaereated
ethanol (10 mL), and deaereated NaOH solution (5 mL) was added
to the solution, which was stirred under argon. The hydrolysis was
followed by TLC. After 5 h the bulk of the ethanol was evaporated
99.5:0.5 v/v, flow 0.8 mL/min. Retention times (min): 16.45 (R), in vacuo (care was taken to keep the product under argon as much
1
18.81 (S). H NMR (300 MHz): δ ϭ 1.36 (s, 9 H, tBu), 3.79 (dd,
as possible) and the resulting solution was extracted twice with
2JH,H ϭ 9.64, JH,H ϭ 7.27 Hz, 1 H, H-3), 3.90 (dd, JH,H ϭ 9.64, deaereated DCM (25 mL). The organic layers were collected and
3
2
3JH,H ϭ 4.76 Hz, 1 H, H-3), 4.23 (dd, JH,H ϭ 7.27, JH,H
ϭ
dried with MgSO4, and the solution was filtered without vacuum
3
3
2
4.76 Hz, 1 H, H-2), 4.51 (d, JH,H ϭ 12.34 Hz, 1 H, OCH2), 4.52 suction, but with an argon blanket, created by a steady stream of
2
(d, JH,H ϭ 12.34 Hz, 1 H, OCH2), 7.21Ϫ7.66 (m, 15 H, 3 ϫ Phe) argon coming from an inverted funnel suspended over the glass
ppm. 13C NMR (75.5 MHz): δ ϭ 28.01 (tBu), 66.51 (C2), 71.55
(C3), 73.18 (C4), 81.23 (tBu), 125.64Ϫ139.68 (arom.), 169.42 (Cϭ
N), 171.22 (C1) ppm.
filter, covering the solution. TFA (5 mL) was added to the collected
organic layers, and the mixture was allowed to stir under argon
overnight. The reaction mixture was extracted twice with deaere-
ated water (10 mL) to give cysteine TFA salt (0.133 g, 89%,
0.57 mmol) after evaporation of the water. 1H NMR (300 MHz):
O-Benzyl-N-boc-serine tert-Butyl Ester: The O-benzyl-protected
serine ester 16 was dissolved in THF (5 mL), and the mixture was
stirred with citric acid solution (10%, 5 mL) for one night. TLC
showed complete disappearance of the starting material, and the
mixture was washed twice with diethyl ether (15 mL). The water
layer was brought to pH 13 and extracted twice with ethyl acetate
(30 mL) to give the product in 95% yield (112 mg) after evapor-
ation. The amino ester prepared above was dissolved in DMF
(5 mL), and triethylamine (136 µL, 2 equiv.) was added, followed
by di-tert-butyl dicarbonate (153 µL, 1.5 equiv.), and the mixture
was stirred until TLC showed completion of the reaction. The
DMF was evaporated, and ethyl acetate (25 mL) was added. The
δ ϭ 2.58 (m, 2 H, H3), 3.78 (t, 3JH,H ϭ 4.8 Hz, 1 H, H2) ppm. 13
C
NMR (75.5 MHz): δ ϭ 24.14 (C3), 54.70 (C2), 170.17 (C1) ppm.
N-Boc-selenocystine tert-Butyl Ester (19): Triphenylphosphane
(0.44 g, 2 equiv.) was dissolved in dry dichloromethane (5 mL), and
the solution was cooled to 0 °C and stirred under dry nitrogen
while bromine (86 µL) dissolved in DCM (2 mL) was added drop-
wise. After 20 min, a mixture of 17 (0.22 g, 0.84 mmol) and imidaz-
ole (0.12 g, 2 equiv.) dissolved in DCM (5 mL) was slowly added
to the pale yellow solution. After one minute a white solid became
visible. The reaction mixture was stirred for another two hours,
organic layer was washed with KHSO4 solution (pH 2, 2 ϫ 15 mL) while the temperature was maintained at 0 °C. Subsequently, the
and water, and dried with MgSO4. The crude product was purified solids were filtered off and the solvent was evaporated. The re-
Eur. J. Org. Chem. 2004, 2905Ϫ2913
www.eurjoc.org
 2004 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2911

A. H. G. Siebum, W. S. Woo, J. Raap, J. Lugtenburg
FULL PAPER
sulting solid was redissolved in DCM (1 mL) and purified by col-
umn chromatography (PE/Et2O, 90:10) to give the bromide in 81%
yield (0.22 g, 0.68 mmol). H NMR (200 MHz): δ ϭ 1.45 (s, 9 H,
(48 mg). 1H NMR (200 MHz): δ ϭ 4.6 (d,3JH,H ϭ 6 Hz, 1 H, H2),
5.6 (m, 2 H, H4), 6.1 (m, 1 H, H3) ppm.
1
tBu) 1.47 (s, 9 H, tBu), 3.5 (m, 1 H, H3), 3. 6 (m, 1 H, H3), 4.5
(m, 1 H, H2), 5.2 (1 H, broad s, NH) ppm. A N2H4·H2O solution
(40 µL) was added to a suspension of Se (62 mg, 1.1 equiv.) and
pulverized NaOH (0.1 g) in DMF. The mixture was stirred under
argon for 3 h at 60 °C. Subsequently the bromide (0.22 g,
0.68 mmol) was added dropwise and the mixture was stirred for 3
h. TLC indicated complete disappearance of the starting material,
and the mixture was poured into HCl (1 , 5 mL) and subsequently
extracted with DCM (3 ϫ 15 mL), after which the system was dried
with MgSO4 and the solvents were evaporated in vacuo to give 19
Acknowledgments
We gratefully acknowledge Mr. Rian van den Nieuwenberg for his
help with the determination of the optical activities of the synthe-
sized amino acids by chiral HPLC techniques.
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L. Stryer, Biochemistry, 3rd ed., W. H. Freeman and Company,
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Ed. 2003, 42, 4742Ϫ4758.
1
in 74% yield (161 mg, 0.25 mmol). H NMR (300 MHz): δ ϭ 1.44
(s, 9 H, tBu), 1.47 (s, 9 H, tBu), 3.34 (m, 2 H, H3), 4.50 (broad s,
1 H, H2), 5.21 (broad s, 1 H, NH) ppm.
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S. C. Low, M. J. Berry, Trends Biochem. Sci. 1996, 21, 203Ϫ208.
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A. Hüttenhofer, A. Böck, in RNA Structure and Function, Cold
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D. L. Hatfield, V. N. Gladyshev, Mol. Cell. Biol. 2002, 22,
L-Selenocysteine (6): NaBH4 (30 mg, 3 equiv.) was added at 0 °C
3565Ϫ3578.
[6]
to a solution of the protected selenocystine 19 (0.26 g, 0.25 mmol)
dissolved in ethanol (4 mL), and the reaction was allowed to pro-
ceed for 30 minutes at 0 °C. The solution was then concentrated to
1 mL in vacuo, deaereated water (5 mL) was added, and the solu-
tion was extracted twice with deaereated DCM (20 mL). The or-
ganic layers were collected and dried with MgSO4, and the solution
was filtered without vacuum suction, but with an argon blanket.
TFA (2 mL) was added to the solution, and the whole was stirred
overnight. The bulk of the acid and solvent were distilled off by
use of a high vacuum pump equipped with a cold-trap, and the oily
product was taken up in deaereated water (10 mL). Evaporation of
the water in vacuo gave the selenocystine.TFA salt as an off-white
solid in 85% yield (70 mg, 0.42 mmol). 1H NMR (300 MHz, D2O):
δ ϭ 3.05 (dd, 3JH,H ϭ 5.55, 2JH,H ϭ 14.17 Hz, 1 H, H-3), 3.14 (dd,
G. V. Kryukov, S. Castello, S. V. Novoselov, A. V. Lobanov, O.
´
Zehtab, R. Guigo, V. N. Gladyshev, Science 2003, 300,
1439Ϫ1443.
[7]
[8]
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R. D. Fleischmann, M. D. Adams, O. White, R. A. Clayton,
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Dougherty, J. M. Merrick, K. McKenney, G. Sutton, W.
Fitzhugh, C. Fields, J. D. Gocayne, J. Scott, R. Shirley, L. I.
Liu, A. Glodek, J. M. Kelley, J. F. Weidman, C. A. Phillips, T.
Spriggs, E. Hedblom, M. D. Cotton, T. R. Utterback, M. C.
Hanna, D. T. Nguyen, D. M. Saudek, R. C. Brandon, L. D.
Fine, J. L. Fritchman, J. L. Fuhrmann, N. S. M. Geoghagen,
C. L. Gnehm, L. A. McDonald, K. V. Small, C. M. Fraser, H.
O. Smith, J. C. Venter, Science 1995, 269, 496Ϫ512.
C. J. Bult, O. White, G. J. Olsen, L. X. Zhou, R. D. Fleisch-
mann, G. G. Sutton, J. A. Blake, L. M. FitzGerald, R. A. Clay-
ton, J. D. Gocayne, A. R. Kerlavage, B. A. Dougherty, J. F.
Tomb, M. D. Adams, C. I. Reich, R. Overbeek, E. F. Kirkness,
K. G. Weinstock, J. M. Merrick, A. Glodek, J. L. Scott, N. S.
M Geoghagen, J. F. Weidman, J. L. Fuhrmann, D. Nguyen, T.
R. Utterback, J. M. Kelley, J. D. Peterson, P. W. Sadow, M. C.
Hanna, M. D. Cotton, K. M. Roberts, M. A. Hurst, B. P.
Kaine, M. Borodovsky, H. P. Klenk, C. M. Fraser, H. O. Smith,
C. R. Woese, J. C. Venter, Science 1996, 273, 1058Ϫ1072.
L. C. Clark, G. F. Combs, B. W. Turnbull, E. H. Slate, D. K.
Chalker, J. Chow, L. S. Davis, R. A. Glover, G. F. Graham, E.
G. Gross, A. Krongrad, J. L. Lesher, H. K. Park, B. B. Sanders,
C. L. Smith, J. R. Taylor, J. Am. Med. Assoc. 1996, 276,
1957Ϫ1996.
2
3
3JH,H ϭ 4.41, JH,H ϭ 14.17 Hz, 1 H, H-3), 4.41 (dd, JH,H
ϭ
5.55 Hz, JHH ϭ 4.41 Hz, 1 H, H-21) ppm. 13C NMR (75.5 MHz,
3
D2O): δ ϭ 15.64 (C3), 54.14 (C2), 170.23 (C1) ppm.
tert-Butyl 2-tert-Butoxycarbonylamino-4-(2-nitrophenylselenyl)but-
anoate (20): Bu3P (95%, 93 mg, 1.2 equiv., 113 µL) dissolved in dry
THF (3 mL) was added dropwise under dry nitrogen to a solution
of 10 (100 mg, 0.36 mmol) and 2-nitrophenyl selenocyanate (0.25 g,
3 equiv.) in dry THF (5 mL) . After 45 minutes TLC indicated
almost complete disappearance of the starting material, and the
mixture was concentrated in vacuo and purified by column chro-
matography (PE/Et2O, 90:10) to give 20 in 82% yield (137 mg,
[10]
[11]
[12]
[13]
J. E. Spallholz, B. J. Shriver, T. W. Reid, Nutr. Cancer. 2001,
1
0.30 mmol). H NMR (300 MHz): δ ϭ 1.45 (s, 9 H, tBu), 1.47 (s,
40, 34Ϫ41.
9 H, tBu), 2.05 (m, 1 H, H3), 2.20 (m, 1 H, H3), 2.93 (m, 2 H, H4)
4.27 (broad s, 1 H, H2), 5.19 (broad s, 1 H, NH) 7.32Ϫ8.30 (m, 4
H, Phe) ppm.
˚
O. NygArd, S. E. Vollset, H. Refsum, L. Brattström, P. M.
Ueland, J. Int. Med. 1999, 246, 425Ϫ454.
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16, 383Ϫ389.
N-Boc-vinylglycine tert-Butyl Ester: Compound 20 (137 mg) was
dissolved in THF (10 mL), cooled to Ϫ5 °C, and treated with H2O2
(30%, 3 mL). After 30 minutes the solution was extracted thrice
with diethyl ether (50 mL), and the collected organic layers were
concentrated in vacuo. The crude product was purified by column
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