Stereoselective synthesis of triply isotope-labeled Ser, Cys, and Ala: Amino acids for stereoarray isotope labeling technology

Article abstract of DOI:10.1021/ol800970t

(Chemical Equation Presented) Efficient access to highly enantioselective isotope-labeled serine, cysteine, and alanine for stereoarray isotope labeling (SAIL) is described.

Full text of DOI:10.1021/ol800970t

ORGANIC
LETTERS
2008
Vol. 10, No. 13
2785-2787
Stereoselective Synthesis of Triply
Isotope-Labeled Ser, Cys, and Ala:
Amino Acids for Stereoarray Isotope
Labeling Technology
Tsutomu Terauchi,† Kuniko Kobayashi,‡ Kosuke Okuma,†,§ Makoto Oba,§
Kozaburo Nishiyama,§ and Masatsune Kainosho*,‡,|
SAIL Technologies, Inc., 1-1-40, Suehiro-cho, Tsurumi-ku, Yokohama, Kanagawa,
230-0045, Japan, Graduate School of Science, Tokyo Metropolitan UniVersity, 1-1
minami-osawa, Hachioji, Tokyo 192-0397, Japan, Department of Materials Chemistry,
Tokai UniVersity, 317 Nishino, Numazu, Shizuoka 410-0395, Japan, and Graduate
School of Science, Structural Biology Research Center, Nagoya UniVersity, Furo-cho,
Chikusa-ku, 464-8601, Japan
kainosho@NMR.chem.metro-u.ac.jp
Received April 28, 2008
ABSTRACT
Efficient access to highly enantioselective isotope-labeled serine, cysteine, and alanine for stereoarray isotope labeling (SAIL) is described.
The sequencing of the human genome has been completed.
In the postgenomic era, attention is now being focused on
understanding the functions of human genes. One of the main
areas of interest is high-throughput protein structure deter-
mination by nuclear magnetic resonance1 (NMR) or X-ray
crystallography. Unlike X-ray crystallography, NMR has a
great advantage, in that it can be applied to proteins in
solution and the natural environment. However, it is currently
difficult to determine the NMR structures of proteins larger
than 30 kDa because their spectra are complicated by many
complex overlapping signals and significant line broadening,
even when higher-field NMR spectrometers equipped with
cryoprobes are used.
isotope labeling (SAIL) technology.2 SAIL technology
enables researchers to carry out an NMR structural analysis
rapidly and with high accuracy, such as of the detailed
structure of an active site, even for a 50 kDa protein. In
addition, isotope labeled amino acids are useful for the
estimation of the stereochemical courses of enzyme-mediated
processes performed with the participation of amino acids.3
In this paper, we describe the chemical syntheses of SAIL
serine (1), SAIL cysteine (2), and SAIL alanine (3), which
have been designed to have optimal isotope labeling for the
NMR structure determination of proteins.
As shown in Figure 1, SAIL serine (1) and SAIL cysteine
(2) should be stereoselectively labeled with deuterium at the
ꢀ-position, and all of the carbon and nitrogen atoms should
be labeled with 13C and 15N, respectively. This labeling
pattern would increase the intensity of the 1H NMR signals
by decreasing the dipole-dipole relaxation and would enable
To overcome this problem, we have developed a new
method for protein NMR spectroscopy, called stereoarray
† SAIL Technologies, Inc.
‡ Graduate School of Science, Tokyo Metropolitan University.
§ Department of Materials Chemistry, Tokai University.
| Graduate School of Science, Structural Biology Research Center,
Nagoya University.
(2) Kainosho, M.; Torizawa, T.; Iwashita, Y.; Terauchi, T.; Ono, A.;
Guntert, P. Nature 2006, 440, 52, and references cited therein.
(3) Young, D. W. Top. Stereochem. 1994, 21, 381.
(1) Wuthrich, K. NMR of Proteins and Nucleic Aacids; John Wiley &
Sons: NewYork, 1986.
10.1021/ol800970t CCC: $40.75
Published on Web 06/07/2008
2008 American Chemical Society

Figure 1. Structures of SAIL-Ser (1), -Cys (2), and -Ala (3).
the stereospecific assignment of a diastereotopic ꢀ-proton,
1
based on the 13C-13C and H-13C correlations.
There are many other examples of methods used for the
synthesis of isotope-labeled serine, cysteine, and alanine. The
synthesis of a racemic mixture of serine labeled with
deuterium at the ꢀ-position, via the reduction of the dehy-
droserine derivative, was reported by M. Kainosho4 and D. J.
Aberhart5 more than 20 years ago. For the enantioselective
synthesis of deuterium-labeled serine, Gani and co-workers
reported using Baeyer-Villiger oxidation as a key reaction;6
however, the enantiopurity of the deuterated serine is
insufficient for the SAIL method. Therefore, we planned to
use catalytic asymmetric hydrogenation in this experiment,
for the chiral introduction of a deuterium atom at the
ꢀ-position of serine, using the more easily accessible [1,2-
13C2;15N;2,2-2H2]glycine and ethyl [13C,2H]formate as our
starting materials for the synthesis of SAIL serine.
Figure 2
[1H]serine.
.
1H NMR Spectra of (2S,3R)-[2,3-2H2]serine and
lowed by the silylation of the enolate 5, to give exclusively
the Z isomer of the dehydroserine derivative 6 in 64% yield.
The obtained derivative 6 was examined using asymmetric
catalytic hydrogenation, followed by deprotection to give
SAIL serine 1 (Scheme 1). The enantiopurity based on the
Scheme 1
Before starting the synthesis of SAIL serine, we sought
to optimize the hydrogenation of the dehydroserine deriva-
tives using various nonlabeled dehydroserine derivatives, in
the presence of (+)-1,2-bis((2S,5S)-2,5-diethylphospholano)-
benzene(cyclooctadiene)rhodium(I) tetrafluoroborate ((S,S)-
Et-DuPHOS-Rh)7 as the catalyst. The optical yield of the
R-position during the hydrogenation was determined from
serine. When the acyl group was used to protect the enol
group, the deoxygenation occurred at the ꢀ-position, and the
optical yields at the R-position were not sufficient. To inhibit
the deoxygenation, silyl enol ether was applied to the
substrate. The t-butyldiphenylsilyl (TBDPS) and thexyldim-
ethylsilyl (TDS) derivatives of the protective group did not
exhibit deoxygenation; however, TBDMS gave rise to a
complex mixture, due to its instability. The optical purities
based on the positions of the TBDPS and TDS derivatives
were determined to be 99% ee and 92% ee, respectively.
Therefore, to check the enantiopurity of the ꢀ-position by
deuteration, we examined the catalytic deuteration of the
TBDPS derivatives using deuterium gas instead of hydrogen
gas, followed by deprotection, to give R,ꢀ-dideuterium-
labeled serine 1b. The 300 MHz 1H NMR spectrum of serine
1b is shown in Figure 2. As shown in the spectrum, the
signals for the R-proton and the 3S proton have disappeared,
due to the cis addition to the re face, indicating that the
stereochemistry at the ꢀ-position is an R configuration.
Our synthesis began with the condensation of ethyl [1,2,3-
13C;15N;3-2H]hippurate (4) with ethyl [13C;2H]formate, fol-
R-position was determined to be 95% ee by an HPLC
analysis using a chiral stationary column (SUMICHIRAL
OA-6100). We are not sure why 1 had a slightly low optical
yield, but it probably resulted from racemization at the
hydrolysis step,8 since the NMR experiment revealed that
the NMR signal of the ꢀ-proton of (2S,3R) or (2R,3S)-7 was
not detected; however, that of 1 was detected.9
The technique of stereoselective labeling with deuterium
at the ꢀ-position of cysteine was developed by Axelesson et
al., starting with the enzymatic reaction of fumaric acid to
produce malic acid, which would form (2S,3S)-[3-2H]cys-
tine.10 Our latest paper also dealt with the preparation of
(4) Kainosho, M.; Ajisaka, K. J. Am. Chem. Soc. 1975, 97, 5630.
(5) Aberhart, D. J.; Russell, D. J. J. Am. Chem. Soc. 1984, 106, 4902.
(6) Gani, D.; Young, D. W. J. Chem. Soc., Perkin Trans. 1983, 1, 2393.
(7) (a) Burk, M. J.; Kalberg, C. S.; Pizzano, A. J. Am. Chem. Soc. 1998,
120, 4345. (b) Burk, M. J. J. Am. Chem. Soc. 1991, 113, 8515.
(8) Manning, J. M. J. Am. Chem. Soc. 1970, 92, 7449.
(9) See Supporting Information.
(10) Axelesson, B. S.; O‘Toole, K. J.; Spencer, P. A.; Young, D. W.
J. Chem. Soc., Chem. Commun. 1991, 1085.
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Org. Lett., Vol. 10, No. 13, 2008

(2S,3R)-[3-2H]serine and (2R,2′R,3S,3′S)-[3,3′-2H2]cystine
starting from (2R)-serine.11 Although these methods have
the advantage of being sufficiently affordable for application
to both SAIL serine and SAIL cysteine with minor variations,
it could not be applied to the SAIL method because of
difficulties in the procurement of materials and low yield.
We have developed the synthesis of SAIL cysteine, using
SAIL serine 2,6,7-trioxabicyclo[2,2,2]octyl ester.6 However,
this method suffers from poor reproducibility, and similar
to SAIL serine starting from [1,2,3-13C;15N]serine, it is
difficult to increase the scale of the production capacity.
Therefore, we planned to use the abovementioned SAIL
serine and convert the hydroxyl group of serine into a thiol
group, by an SN2-type displacement, to yield SAIL cysteine.
For the synthesis of SAIL cysteine, we followed the scheme
developed by Arnold et al.12 As a result, we accomplished
the synthesis of SAIL cystine ((2R,2′R,3S,3S)-[1,1′,2,2′,3,3′-
13C6,2,2′-15N2;3,3′-2H2]cystine) with a 35% overall yield,
although potentially risky chemicals, such as dimethyl
azodicarboxylate, were used. To avoid potentially hazardous
reaction materials, we also examined a more easily accessible
means to synthesize the stereoisomer at the ꢀ-position of
SAIL cysteine (Scheme 2). First, we selected the (2S,3R)-
potassium thioacetate, to yield the deuterium-labeled cysteine
derivative 10 (Scheme 2). The deprotection of compound 10
was accomplished by refluxing with 1 M HCl, and the resulting
SAIL cysteine hydrochloride 2 ((2R,3R)-[1,2,3-13C3;2-15N;3′-
2H] cysteine) was purified by ion-exchange column chroma-
tography on an DOWEX 50W-X8 column with a 43% yield.
The enantiopurity based on the R-position of amino acid 2 was
determined to be 95% ee, by HPLC analysis using a chiral
stationary column (DAICEL CROWNPAK CR+).
SAIL alanine 3 should be dideuterated at the ꢀ-position,
to increase the signal intensity. In particular, this labeling
pattern could be more effective for larger proteins than for
smaller ones. We employed deoxygenation of the hydroxyl
group of the SAIL serine derivative 8 (Scheme 3). Derivative
Scheme 3
Scheme 2
8 was treated with polymer-supported triphenylphosphine and
carbon tetrabromide for conversion to the bromoalanine
derivative 11, after which derivative 11 was reduced with
tributyltin deuteride, to give derivative 12. The deprotection
of compound 12 was carried out by refluxing with 1 M HCl,
and the resulting alanine hydrochloride was subjected to ion-
exchange column chromatography, using a DOWEX 50W-
X8 column, to give SAIL alanine 3 ((2S)-[1,2,3-13C3;2-
15N;3,3-2H2]alanine) with a 63% yield from derivative 8. The
enatiopurity based on the R-position was determined to be
93% ee by an HPLC analysis using a chiral stationary column
(SUMICHIRAL OA-6100).
In conclusion, we have developed the efficient syntheses
of isotope-labeled serine, cysteine, and alanine. The starting
materials uniformly labeled with 13C and 15N are now
commercially available. This approach proved more ame-
nable to scale-up, enabling the preparation of significant
quantities of these amino acids. Structural determinations of
proteins using the 20 SAIL amino acids are underway in
our laboratory. Details of the syntheses of other SAIL amino
acids will be reported in due course.
N-benzoyl serine ethyl ester 7, which was converted to
tosylate and then treated with potassium thioacetate to give
the (2R,3S)-cysteine derivative. Although the displacement
at the ꢀ-position by the thioacetate anion proceeded with
inversion, unfortunately, the yield was low, due to byproduct
formation. We attempted to exchange the protection with
the t-butoxycarbonyl group, to prevent the byproduct forma-
tion. Thus, derivative 8, derived from SAIL serine 1, was
then subjected to tosylation followed by thioacetylation by
Acknowledgment. This work has been supported by the
Core Research for Evolutional Science and Technology (CREST)
of the Japan Science and Technology Agency (JST).
(11) Oba, M.; Iwasaki, A.; Hitokawa, H.; Ikegami, T.; Banba, H.; Ura,
K.; Takamura, T.; Nishiyama, K. Tetrahedron: Asymmetry 2006, 17, 1890.
(12) (a) Arnold, L. D.; Kalantar, T. H.; Vederas, J. C. J. Am. Chem.
Soc. 1985, 107, 7105. (b) Arnold, L. D.; May, R. G.; Vederas, J. C. J. Am.
Chem. Soc. 1988, 110, 2237. (c) Ramer, S. E.; Moore, R. N.; Vederas,
J. C. Can. J. Chem 1986, 64, 706. (d) Williams, R. M. Synthesis of Optically
ActiVe R-Amino Acids, Organic Chemistry Series, J. E. Baldwin, Pergamon
Press: Oxford, 1989; p 134.
Supporting Information Available: Experimental details
and spectral data for all key compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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