Synthesis of stereoarray isotope labeled (SAIL) lysine via the "head-to-tail" conversion of SAIL glutamic acid

Article abstract of DOI:10.1021/ol1026766

A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6- ²H₄;1,2,3,4,5,6-¹³C₆;2,6- ¹⁵N₂]lysine, was synthesized by the "head-to- tail" conversion of SAIL-Glu, (2S,3S,4R)-[3,4-²H ₂;1,2,3,4,5-¹³C₅;2-¹⁵N]glutamic acid, with high stereospecificities for all five chiral centers. With the SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons within the four methylene groups of the Lys side chains in proteins.

Full text of DOI:10.1021/ol1026766

ORGANIC
LETTERS
2011
Vol. 13, No. 1
161-163
Synthesis of Stereoarray Isotope
Labeled (SAIL) Lysine via the
“Head-to-Tail” Conversion of SAIL
Glutamic Acid
Tsutomu Terauchi,†,‡ Tomoe Kamikawai,‡ Maxim G. Vinogradov,§
Eugenia V. Starodubtseva,§ Mitsuhiro Takeda,¶ and Masatsune Kainosho*,†,¶
Center for Priority Areas, Tokyo Metropolitan UniVersity, 1-1 minami-ohsawa,
Hachioji, Tokyo, 192-0397, Japan, SAIL Technologies, Inc., 1-1-40 Suehiro-cho,
Tsurumi-ku, Yokohama, Kanagawa, 230-0045, Japan, N. D. Zelinsky Institute of
Organic Chemistry, Russian Academy of Sciences, Leninsky Prospect 47,
119991 Moscow, Russia, and Graduate School of Science, Structural Biology Research
Center, Nagoya UniVersity, Furo-cho, Chikusa-ku, 464-8601, Japan
kainosho@tmu.ac.jp
Received November 4, 2010
ABSTRACT
A stereoarray isotope labeled (SAIL) lysine, (2S,3R,4R,5S,6R)-[3,4,5,6-2H4;1,2,3,4,5,6-13C6;2,6-15N2]lysine, was synthesized by the “head-to-tail”
conversion of SAIL-Glu, (2S,3S,4R)-[3,4-2H2;1,2,3,4,5-13C5;2-15N]glutamic acid, with high stereospecificities for all five chiral centers. With the
SAIL-Lys in hand, the unambiguous simultaneous stereospecific assignments were able to be established for each of the prochiral protons
within the four methylene groups of the Lys side chains in proteins.
NMR spectroscopy has been widely used for determining
protein structures in solution, despite the serious limitation
of the molecular sizes for which the standard NMR meth-
odology can be successfully applied.
In the past decade, however, concordant developments in
sample preparation and spectroscopic methods have allevi-
ated various bottlenecks, such as excessive signal overlapping
and line broadening, and thus facilitated the structural studies
of larger proteins that could not be analyzed by conventional
methods.1 Unfortunately, most of these new advances have
extended the molecular size limit by sacrificing the structural
precision, since they use the methyl and backbone amide
signals as the exclusive source of the structural information,
which can only be obtained by eliminating all of the other
signals by extensive deuteration.2
We have recently developed a completely different approach,
the stereoarray isotope labeling (SAIL) method, in which all
redundant NMR signals are systematically eliminated by
extensive site- and stereospecific isotope labeling.3 One of the
(1) (a) Gardner, K. H.; Kay, L. E. In Biological Magnetic Resonance;
Krishna, R., Berliner, L. J., Eds.; Kluwer Academic Press: New York, 2001;
Vol. 16, pp 27-74. (b) Oki, S.; Kainosho, M. Prog. Magn. Reson. Spectrosc.
2008, 53, 208–226. (c) Pervushin, K.; Riek, R.; Wider, G.; Wu¨thrich, K.
Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 12366–12371.
(2) (a) Sprangers, R.; Kay, L. E. Nature. 2007, 445, 618–622. (b)
Tugarinov, V.; Choy, W. Y.; Orekhov, V. Y.; Kay, L. E. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 622–627.
† Graduate School of Science, Tokyo Metropolitan University.
‡ SAIL Technologies, Inc.
(3) (a) Kainosho, M.; Torizawa, T.; Iwashita., Y.; Terauchi, T.; Ono,
A.; Gu¨ntert, P. Nature 2006, 440, 52–57. (b) Takeda, M.; Ikeya, T.; Gu¨ntert,
P.; Kainosho, M. Nat. Protoc. 2007, 2, 2896–2902. (c) Kainosho, M.;
Gu¨ntert, P. Q. ReV. Biophys. 2009, 42, 247–300.
§ N. D. Zelinsky Institute of Organic Chemistry.
¶ Graduate School of Science, Structural Biology Research Center,
Nagoya University.
10.1021/ol1026766  2011 American Chemical Society
Published on Web 12/01/2010

most crucial elements in the SAIL method is the synthesis of
highly specific, isotope-labeled amino acids.4 It is not trivial to
synthesize, for example, a SAIL amino acid with a long aliphatic
side chain, such as Lys, Arg, or Pro, in which each one of the
prochiral methylene protons has to be deuterated at the high
stereospecificity required for NMR applications. This is due to
the fact that the chiral center of these amino acids cannot
effectively control the stereocenter of the deuteration for the
remote methylenes. We circumvented this problem by using a
novel “head-to-tail” conversion method for SAIL-Glu 1,
(2S,3S,4R)-[3,4-2H2;1,2,3,4,5-13C5;2-15N]glutamic acid, to syn-
thesize SAIL-Lys 2, (2S,3R,4R,5S,6R)-[3,4,5,6-2H4;1,2,3,4,5,6-
13C6;2,6-15N2]lysine, which was otherwise very difficult to
synthesize. Although a few synthetic methods exist for stereo-
selective lysine deuteration, no attempt to synthesize a lysine
where each one of the four prochiral methylene protons is
simultaneously and stereospecifically deuterated, has been
reported thus far.5
Scheme 1. Preparation of
(2S,3S,4R)-[3,4-2H2;1,2,3,4,5-13C5;2-15N]Glutamic Acid
(RuCl3 + NaIO4) into 7, followed by acid hydrolysis in 1 M
HCl at 110 °C to afford SAIL-Glu 1 in a 35% total yield.7
One of the most versatile approaches to synthesize amino
acids labeled with deuterium at the ꢀ-position is the
asymmetric catalytic hydrogenation (or deuteration) of the
corresponding R,ꢀ-dehydroamino acid (enamide) precursors.
In the presence of some phosphine rhodium(I) or ruthe-
nium(II) chiral complexes, as well as palladium- or platinum-
containing chiral complexes, the asymmetric reduction
proceeds in a highly stereoselective manner, with the syn
addition of H2 (D2) to the C-C double bonds almost
invariably occurring.8
Keeping this process in mind, we chose the enamide
intermediate for the SAIL-Lys synthesis, which can be
derived from SAIL-Glu 1, as shown in Scheme 2. The
conversion of 1 into the (2R,3S,4R)-[2,3,4-2H3;1,2,3,4-13C4;4-
15N]4-aminobutyric acid (GABA) 8 was achieved almost
quantitatively by the stereospecific decarboxylation of the
R carboxyl group, by enzymatic decarboxylation with
glutamic acid decarboxylase in D2O.9
We chose (2S,3S,4R)-[3,4-2H2;1,2,3,4,5-13C5;2-15N]glutamic
acid, SAIL-Glu, as our starting material, since SAIL-Glu can
be prepared by the catalytic deuteration of the dehydroglutamic
acid derivative prepared from L-[UL-13C;15N]glutamic acid. In
general, one might adapt the single carbon elongation reaction
to the terminal carboxyl group of glutamic acid, in order to
synthesize stereoselectively deuterated lysines from the glutamic
acid precursors. Obviously, it is not easy to synthesize lysines
with stereoselectively deuterated methylenes at the C5 (δ) and/
or C6 (ε) positions, under the stereocontrol of the remote chiral
center at C2 (R). Therefore, we used a completely different
scheme to deuterate the prochiral methylenes at the C5 and C6
carbons of lysine, in which the glutamic acid unit is converted
into the lysine building block in the “head-to-tail” manner. With
this new synthetic scheme, the C2 (R), C3 (ꢀ), C4 (γ), and C5
(δ) of Glu are converted into the C6 (ε), C5 (δ), C4 (γ), and
C3 (ꢀ) of Lys, respectively, and the R-amino group of Glu is
converted into the ε-amino group of Lys.
(2S,3S,4R)-[3,4-2H2;1,2,3,4,5-13C5;2-15N]Glutamic acid, SAIL-
Glu 1, was synthesized from commercially available L-[UL-
13C,15N]glutamic acid (Scheme 1). L-[UL-13C,15N]Glutamic acid
3 was converted to 4,6 and then the olefin 4 was subjected to
catalytic deuteration at the C3 and C4 positions to afford the
(2S,3S,4R)-[3,4,5-2H3;1,2,3,4,5-13C5;2-15N]pyrrolidone derivative
5. After removal of the TBDMS protecting group by treatment
with acetic acid, the intermediate 6 was oxidized with RuO4
After protecting the 4-amino group, the carboxyl group of 9
was converted, after derivatization into the carboxyl chloride
with thionyl chloride, into the deuterated aldehyde 10, by
reductive deuteration with tributyltin deuteride in the presence
of a catalytic amount of tetrakis(triphenylphosphine)palladium.
The aldehyde intermediate 10 was converted exclusively into
(7) Oba, M.; Miyakawa, A.; Nishiyama, K.; Terauchi, T.; Kainosho,
M. J. Org. Chem. 1999, 64, 9275–9278.
(8) (a) Detellier, C.; Gelbard, G.; Kagan, H. G. J. Am. Chem. Soc. 1978,
100, 7556–7561. (b) Koenig, K. E.; Knowles, W. S. J. Am. Chem. Soc.
1978, 100, 7561–7564. (c) Burk, M. J.; Feaster, J. E.; Nugent, W. A.;
Harlow, R. L. J. Am. Chem. Soc. 1993, 115, 10125–10138. (d) Baldwin,
J. E.; Merritt, K. D.; Schofield, C. J. Tetrahedron Lett. 1993, 34, 3919–
3920. (e) Oba, M.; Ueno, R.; Fukuoka (nee Yoshida), M.; Kainosho, M.;
Nishiyama, K. J. Chem. Soc., Perkin Trans. 1 1995, 1603–1609. (f) Oba,
M.; Terauchi, T.; Owari, Y.; Imai, Y.; Motoyama, I.; Nishiyama, K. J. Chem.
Soc., Perkin Trans. 1 1998, 1275–1281. (g) Okuma, K.; Ono, A. M.;
Tsuchiya, S.; Oba, M.; Nishiyama, K.; Kainosho, M.; Terauchi, T.
Tetrahedron Lett. 2009, 50, 1482–1484.
(4) Terauchi, T.; Kobayashi, K.; Okuma, K.; Oba, M.; Nishiyama, K.;
Kainosho, M. Org. Lett. 2008, 10, 2785–2787.
(5) (a) Aberhart, D. J.; Lin, H.-J.; Weiller, B. H. J. Am. Chem. Soc.
1981, 103, 6750–6752. (b) Asada, Y.; Tanizawa, K.; Sawada, S.; Suzuki,
T.; Misono, H.; Soda, K. Biochemistry 1981, 20, 6881–6886. (c) Aberhart,
D. J.; Gould, S. J.; Lin, H.-J.; Thiruvengadam, T. K.; Weiller, B. H. J. Am.
Chem. Soc. 1983, 105, 5461–5470.
(9) (a) Mandeles, S.; Koppelman, R.; Hanke, M. E. J. Biol. Chem. 1954,
209, 327–336. (b) Yamada, H.; O’Leary, M. H. J. Am. Chem. Soc. 1977,
99, 1660–1661. (c) Yamada, H.; O’Leary, M. H. Biochemistry 1978, 17,
669–672.
(6) Ohfune, Y.; Tomita, M. J. Am. Chem. Soc. 1982, 104, 3511–3513.
162
Org. Lett., Vol. 13, No. 1, 2011

SAIL-Lys or [UL-13C]Lys, and 19 other unlabeled amino
acids (1 mM each), was used for cell-free expression. A
comparison of their 2D-HCCH-TOCSY spectra12 clearly
shows that the connectivities for all 11 Lys residues in EPPIb
are fully traceable along the SAIL-Lys labeled side chains,
while they are not obvious for the protein labeled with [UL-
13C]Lys, as illustrated for Lys-62, highlighted in red circles
in Figure 1. It is worth noting that the stereospecific
Scheme 2. Preparation of
(2S,3R,4R,5S,6R)-[3,4,5,6-2H4;1,2,3,4,5,6-13C6;2,6-15N2]Lysine
the Z-isomer of the enamide precursor 11, after condensation
with the phosphoryl glycine derivative prepared from [1,2-13C2;
2-15N]Gly. After an extensive search for the proper conditions,
the asymmetric hydrogenation with (+)-1,2-bis[(2S,5S)-2,5-
diethylphospholano]benzene(cyclooctadiene)rhodium(I) tri-
fluoromethanesulfonate ((S,S)-Et-DuPHOS-Rh) in benzene
was found to be optimal. After the deprotection of 12 by
refluxing in 2 M HCl, followed by the H2NNH2 treatment,
(2S,3R,4R,5S,6R)-[3,4,5,6-2H4;1,2,3,4,5,6-13C6;2,6-
15N2]lysine, SAIL-Lys 2, was obtained in a total yield of
56% from SAIL-Glu 1, after purification by ion-exchange
column chromatography (DOWEX 50WX8). The enantiopu-
rity based on the R-position of 2 was found to be 99% ee by
an HPLC analysis.
1
Figure 1
.
2D HCCH-TOCSY NMR spectra (600 MHz for H at
37 °C) of EPPIb labeled with SAIL-Lys (a) and with [UL-13C]Lys
(b). The sample concentrations were both 0.7 mM (280 µL, Shigemi
microtube). Deuterium decoupling was applied during the chemical
shift encoding on carbon atoms. The connectivities of Lys-62,
starting from the Hδ to Hε and the Hδ to Hγ, Hꢀ, and HR, are
shown by red horizontal lines, although most of the cross peaks
are missing in part b.
assignments for all of the prochiral methylene protons are
automatically implemented in the case of SAIL-Lys.
SAIL-Lys 2 is extremely useful for NMR studies of
proteins.10 The 18.2 kDa protein, E. coli peptidyl prolyl
cis-trans isomerase b (EPPIb), labeled with either [UL-
13C]Lys or SAIL-Lys, was prepared by the E. coli cell-free
Acknowledgment. This work was partially supported by
CREST of JST, the University Start-Ups Creation Support
System of MEXT, and also by TPRP of MEXT. We thank
Prof. Makoto Oba (Tokai University) for the elemental
analysis.
protein expression system, as described previously.11
A
mixture of amino acids (70 mg), containing 2 mg of either
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.
(10) (a) Takeda, M.; Sugimori, N.; Torizawa, T.; Terauchi, T.; Ono,
A. M.; Yagi, H.; Yamaguchi, Y.; Kato, K.; Ikeya, T.; Jee, J. G.; Gu¨ntert,
P.; Aceti, D. J.; Markley, J. L.; Kainosho, M. FEBS J. 2008, 275, 5873–
5884. (b) Takeda, M.; Chang, C. K.; Ikeya, T.; Gu¨ntert, P.; Chang, Y. H.;
Hsu, Y. L.; Huang, T. H.; Kainosho, M. J. Mol. Biol. 2008, 380, 608–622.
(11) (a) Torizawa, T.; Shimizu, M.; Taoka, M.; Miyano, H.; Kainosho,
M. J. Biomol. NMR 2004, 30, 311–325. (b) Takeda, M.; Ono, A. M.;
Terauchi, T.; Kainosho, M. J. Biomol. NMR 2010, 46, 45–49.
OL1026766
(12) Kay, L. E.; Xu, G. Y.; Singer, A. U.; Muhandiram, D. R.; Forman-
Kay, J. D. J. Magn. Reson., Ser. B 1993, 101, 333–337.
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