J. Am. Chem. Soc. 1996, 118, 913-914
A New Efficient Synthesis of Acetyltelluro- and
913
Acetylselenomethionine and Their Use in the
Biosynthesis of Heavy-Atom Protein Analogs
Wilhelm Karnbrock, Elisabeth Weyher, Nediljko Budisa,
Robert Huber, and Luis Moroder*
Max-Planck-Institut f u¨ r Biochemie
8
2152 Martinsried, Germany
ReceiVed September 20, 1995
It has recently been reported that both selenomethionine, Met-
Se), and telluromethionine, Met(Te), can be incorporated
1
2,3
(
into polypeptides in ViVo using bacterial strains auxotrophic for
Met, offering a new approach to solving the phase problem in
protein crystallography. The electron density of tellurium is,
in fact, sufficient to generate clear signals in the isomorphous
and anomalous difference Patterson maps at the commonly used
2
,3
Cu KR wavelengths, and therefore, biosynthetic replacement
of Met with Met(Te) represents a promising alternative to the
Figure 1. Synthetic scheme for L-Met(Te) and L-Met(Se).
4
soaking procedure for multiple isomorphous replacements. 3D
structures of Met(Se) proteins have also been resolved by the
method of multiwavelength anomalous diffraction;5 however,
this requires intensity measurements with monochromatic syn-
chroton radiation at a minimum of three different wavelengths.
good yields. As expected, reaction of N-acetyl-(R,S)-2-amino-
-butyrolactone with MeSeLi led to the corresponding Ac-DL-
Met(Se)-OH lithium salt in a similarly efficient manner (Figure
). Opening of the butyrolactone ring corresponds to an ester
,6
4
1
7
Various efficient syntheses of Met(Se) have been reported,
cleavage via an SN2 reaction, where the soft nucleophiles methyl
tellurolate and methyl selenolate are known to attack preferen-
but only two procedures have been described for Met(Te) so
far. One is a multistep synthesis based on the reaction of 5-(â-
bromoethyl)hydantoin with (methyltelluro)sodium (MeTeNa),
followed by alkaline ring opening to generate the racemic Met-
3
9,12
tially the soft sp center.
Since small amounts of a byproduct
were isolated and identified by FAB-MS and NMR as N-
acetylhomoserine after workup of both reaction mixtures, a
(
Te).8 Since this method proved to be exceedingly difficult to
2
partial attack of the soft nucleophiles at the sp center with
reproduce, an alternative synthesis was proposed by Silks et
formation of the related methyltelluro and methylseleno esters
9
al. It relies on ring opening of (S)-2-amino-4-butyrolactone
12d
cannot be excluded.
These esters are then apparently
hydrochloride by (methyltelluro)lithium (MeTeLi) to afford
directly L-Met(Te). Despite all the precautions adopted to
operate in dry and oxygen-free media, we were unable to obtain
the desired compound even by this procedure.
hydrolyzed to N-acetylhomoserine in the purification steps. The
observation that, despite identical reaction conditions, Ac-DL-
Met(Te)-OH was obtained in all syntheses in significantly higher
yields (80-90%) than the corresponding Met(Se) derivative
To allow for a more proper solvent choice, i.e., aprotic
solvents like THF instead of methanol, and to exclude the
presence of hydrochloride, we have examined the effect of
N-protection of the 2-amino-4-butyrolactone. In contrast to
what we observed with the unprotected lactone, reaction of
(
60-70%) is attributed to the softer nucleophile character of
the tellurolate compared to the selenolate.
Besides leading to a smooth nucleophilic ring opening of the
-amino-4-butyrolactone, N-acetylation allowed the enantiose-
2
lective enzymatic deacetylation of the Met analogs by amino-
10
11
N-acetyl-(R,S)-2-amino-4-butyrolactone with MeTeLi in THF
and in the presence of tetramethylethylenediamine (TMEDA)
produced the desired Ac-DL-Met(Te)-OH lithium salt in very
13
acylase-based procedures to generate the desired L-Met(Te)
and L-Met(Se). Monitoring of the enzymatic hydrolysis by
capillary electrophoresis (CE) proved to be very advantageous
(
Figure 2). Both amino acids showed chromatographic and
analytical properties identical with those of authentic probes
L-Met(Se), Sigma, M u¨ nchen, Germany; L-Met(Te), L. A. Silks,
*
Address for correspondence: Max-Planck-Institute of Biochemistry,
D-82152 Martinsried, Germany. Tel.: 49-89-8578-3905. Fax: 49-89-8578-
2
847.
(
(
1) Cowie, D. B.; Cohen, G. N. Biochim. Biophys. Acta 1957, 26, 252.
2) Boles, J. O.; Lewinski, K.; Kunke, M.; Odom, J. D.; Dunlap, B. R.;
Los Alamos National Laboratory, Los Alamos, NM).
(
Lebioda, L.; Hatada, M. Nat. Struct. Biol. 1994, 1, 283.
Dialkyltellurium(II) compounds oxidize rapidly under various
(
3) Budisa, N.; Steipe, B.; Demange, P.; Eckerskorn, C.; Kellermann,
14
conditions; therefore, L-Met(Te) and Ac-DL-Met(Te)-OH were
S.; Huber, R. Eur. J. Biochem. 1995, 230, 788.
analyzed for their stability in view of their application in the
biosynthesis of Met(Te) proteins. In non-degassed aqueous
solution at pH 7.0 and room temperature, Ac-DL-Met(Te)-OH
is markedly more stable (t1/2 ≈ 20 h) than L-Met(Te) (t1/2 ≈ 30
min) as monitored by CE. In both cases, air oxidation leads to
(
4) Green, D. W.; Ingram, V. M.; Perutz, M. F. Proc. R. Soc. London
954, A225, 287.
5) (a) Hendrickson, W. A.; Horton, J.; LeMaster, D. EMBO J. 1990, 9,
665. (b) Hendrickson, W. A. Science 1991, 254, 51.
1
(
1
(
6) Reinemer, P.; Prade, L.; Hof, P.; Neuefeind, T.; Huber, R.; Zettl, R.;
Palme, K.; Schell, J.; Koelln, I.; Bartunik, H. D.; Bieseler, B. J. Mol. Biol.,
submitted.
(
7) (a) Painter, E. P. J. Am. Chem. Soc. 1947, 69, 232. (b) Plieninger,
(12) (a) McMurray, J. Org. React. 1976, 24, 187. (b) Chen, J.; Zhou, X.
J. Synthesis 1987, 586. (c) Li, W.; Zou, X. J.; Ma, Q. Synth. Commun.
1995, 25, 553. (d) Liotta, D.; Sunay, U.; Santiesteban, H.; Markiewicz, W.
J. Org. Chem. 1981, 46, 2605.
(13) (a) Birnbaum, S. M.; Levintow, L.; Kingsley, R. B.; Greenstein, J.
P. J. Biol. Chem. 1952, 194, 455. (b) Greenstein, J. P. Methods Enzymol.
1957, 3, 554. (c) Chibata, I.; Tosa, T.; Sato, T.; Mori, T. Methods Enzymol.
1976, 44, 746. (d). Chenault, H. K.; Dahmer, J.; Whitesides, G. M. J. Am.
Chem. Soc. 1989, 111, 6354.
(14) (a) Reichel, L.; Kirschbaum, E. Liebigs Ann. Chem. 1936, 523, 211.
(b) Balfe, M. P.; Chaplin, C. A.; Phillips, H. J. Chem. Soc. 1938, 341. (c)
Balfe, M. P.; Nandl, K. N. J. Chem. Soc. 1941, 70. (d) Detty, M. R. J. Org.
Chem. 1980, 45, 274. (e) Kirsch, G.; Goodman, M. M.; Knapp, F. F.
Organometallics 1983, 2, 357.
H. Chem. Ber. 1950, 83, 265. (c) Zdansky, G. Ark. Kemi 1968, 29, 437. (d)
Barton, D. H. R.; Bridon, D.; Herv e´ , Y.; Potier, P.; Thierry, J.; Zard, S. Z.
Tetrahedron 1986, 42, 4983. (e) Krief, A.; Trabelsi, M. Synth. Commun.
989, 19, 1203. (f) Esaki, N.; Shimoi, H.; Soda, Y.-S. Biotechnol. Appl.
Biochem. 1989, 11, 312. (g) Koch, T.; Buchardt, O. Synthesis 1993, 1065.
1
(
8) Knapp, F. F. J. Org. Chem. 1979, 44, 1007.
(
9) Silks, L. A.; Boles, J. O.; Modi, B. P.; Dunlap, R. B.; Odom, J. D.
Synth. Commun. 1990, 20, 1555.
(10) (a) Snyder, H. R.; Andreen, J. H.; Cannon, G. W.; Peters, C. F. J.
Am. Chem. Soc. 1942, 64, 2082. (b) Fillman, J.; Albertson, N. J. Am. Chem.
Soc. 1948, 70, 171.
(
11) Evers, M. J.; Christiaens, L. E.; Renson, M. J. J. Org. Chem. 1986,
5
1, 5196.
0
002-7863/96/1518-0913$12.00/0 © 1996 American Chemical Society