Enzymatic synthesis of aza-L-tyrosines

Article abstract of DOI:10.1016/S0960-894X(01)00376-6

Tyrosine phenol-lyase from Citrobacter freundii synthesizes 2-aza-L-tyrosine and 3-aza-L-tyrosine from 3-hydroxypyridine and 2-hydroxypyridine, respectively, and ammonium pyruvate.

Full text of DOI:10.1016/S0960-894X(01)00376-6

Bioorganic & Medicinal Chemistry Letters 11 (2001) 2099–2100
Enzymatic Synthesis of Aza-L-tyrosines
a
b,
E. Blake Watkins and Robert S. Phillips *
aDepartment of Medicinal Chemistry, University of Mississippi, University, MS 38677, USA
bDepartment of Chemistry and Department of Biochemistry and Molecular Biology, University of Georgia,
Athens, GA 30602-2556, USA
Received 23 March 2001; accepted 15May 2001
Abstract—Tyrosine phenol-lyase from Citrobacter freundii synthesizes 2-aza-l-tyrosine and 3-aza-l-tyrosine from 3-hydroxy-
pyridine and 2-hydroxypyridine, respectively, and ammonium pyruvate. # 2001 Elsevier Science Ltd. All rights reserved.
A convenient method for obtaining optically pure a-
amino acids involves using enzymes to establish the
desired stereochemistry at the a-carbon. Tyrosine phe-
acid, and 20 mM 2- or 3-hydroxypyridine in a total
volume of 350 mL. The reactions were performed at pH
ꢀ
25 C for 2-azatyrosine. Synthesis of 3-azatyrosine at
25 C resulted in a 50% decrease in yield. Tyrosine
8.8–9.0 and 37 C for 3-azatyrosine and pH 8.0–8.2 and
ꢀ
0
nol-lyase (deaminating) [EC 4.1.99.2] is a pyridoxal-5 -
ꢀ
phosphate (PLP)-dependent enzyme that catalyzes the
b-elimination of l-tyrosine to phenol and ammonium
phenol-lyase (42 mg) was added to the reaction mixture
once the pH had been adjusted with concentrated
NH OH. After 5h the pH had to be re-adjusted with
1
,2
pyruvate, as shown in Scheme 1.
reversible, and hence TPL also catalyzes the reverse
This process is
4
reaction which synthesizes l-tyrosine from phenol and
2 N NH OH. The reactions were then incubated for 5
4
2
,3
ammonium pyruvate. In the present study, we were
interested in determining if 2-aza-l-tyrosine (1) and 3-
aza-l-tyrosine (2) could be synthesized using tyrosine
phenol-lyase. Nagasawa et al. reported the syntheses of
numerous optically pure ring-substituted tyrosine deri-
days at the appropriate temperature. Each day, the pH
was checked and adjusted as needed. On the third day,
an additional 4.2 mg of tyrosine phenol-lyase was
added, after the pH had been adjusted, to compensate
for denaturation of the enzyme that may occur during
incubation.
4
vatives using tyrosine phenol-lyase. However, under
the conditions used in their experiments, 2-hydroxy-
pyridine (which exists in solution predominantly as the
2
-oxo-1H tautomer) and 3-hydroxypyridine failed to
2
-Aza-L-tyrosine
give rise to the corresponding amino acids, 3-aza-l-tyr-
osine and 2-aza-l-tyrosine. One of the purposes for the
present study was to determine if changes in the reac-
tion conditions would facilitate the formation of the
aforementioned amino acids. We now report that 2-aza-
l-tyrosine and 3-aza-l-tyrosine can be synthesized using
recombinant tyrosine phenol-lyase from Citrobacter
freundii, following a series of experiments designed to
determine the optimum conditions necessary for pro-
duction of these amino acids (Scheme 2).
Following incubation of the reaction mixture with TPL
for 5days, the yellow solution was heated quickly to
100 C for 1 min to denature the remaining enzyme.
ꢀ
After cooling, the denatured enzyme was removed by
filtration through Celite. The filtrate was applied to a
+
Dowex 50W X8 (H form) column (2.5ꢁ25cm). The
5
column was washed with 1 L of water to remove
unreacted 3-hydroxypyridine and pyruvic acid, and the
azatyrosine was eluted with 0.75M NH OH. The sol-
4
vent was removed in vacuo. The residue was suspended
in MeOH and evaporated to dryness. A small amount
of MeOH was then added, and the mixture was filtered
All reactions were performed using 100 mM ammonium
0
chloride, 162 mM pyridoxal-5 -phosphate, 63mM pyruvic
1
to give pure 2-aza-l-tyrosine (45.3 mg, 12.5%).
H
NMR (D O) d 7.69 (bs, 1H), 6.99 (d, J=8.8 Hz, 1H),
2
6.89 (dd, J=2.0, 8.8 Hz, 1H), 3.42 (dd, J=5.4, 8.6 Hz,
1H), 2.89 (dd, J=5.4, 13.3 Hz, 1H), 2.63 (dd, 8.6,
*Corresponding author. Fax: +1-706-542-9454; e-mail: rsphillips@
chem.uga.edu
0
960-894X/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00376-6

2100
E. B. Watkins, R. S. Phillips / Bioorg. Med. Chem. Lett. 11 (2001) 2099–2100
Scheme 1. Reaction catalyzed by tyrosine phenol-lyase.
and UV properties of 3-aza-l-tyrosine were identical
with those of synthetic racemic 3-azatyrosine.
Aza analogues of l-tyrosine are of interest as anti-
metabolites of tyrosine and inhibitors of enzymes which
react with free l-tyrosine in solution or tyrosyl residues
in proteins. Thus, azatyrosines are potentially useful as
antibiotics or drugs. The antitumor activity of 2-aza-l-
Scheme 2. Structures of 2-aza-l-tyrosine (1) and 3-aza-l-tyrosine (2).
7
tyrosine, a natural product originally isolated from a
Streptomyces sp., has stimulated interest in the stereo-
selective synthesis of this compound. 3-Aza-l-tyrosine is
also a natural product isolated from a toxic mushroom,
+
1
3.3 Hz, 1H); MS (ESI) m/z 183 (MH ); UV (0.1 M
2
0
HCl), lmax=290 nm (log e=3.83); [a]D +51.6 (c 0.91,
.1 M HCl), lit., +55 (c 1.1, 1.0 M HCl). The NMR,
MS and UV properties of 2-aza-l-tyrosine were iden-
tical with those of synthetic racemic 2-azatyrosine.
6
8
0
Clitocybe acromelalga. A number of syntheses of 2-aza-
8
,9
l-tyrosine and 3-aza-l-tyrosine have been reported.
Although the yields are low, the new procedure reported
herein provides 2-aza-l-tyrosine and 3-aza-l-tyrosine in a
single step from readily available achiral starting materials.
3-Aza-L-tyrosine
References and Notes
Following incubation of the reaction mixture with TPL
ꢀ
for 5days, the yellow solution was heated to 100
C
1
. Kumagai, H.; Yamada, H.; Matsui, H.; Ohkishi, H.;
Ogata, K. J. Biol. Chem. 1970, 245, 1767.
. Kiick, D. M.; Phillips, R. S. Biochemistry 1988, 27, 7339.
. Enei, H.; Matsui, H.; Okumura, S.; Yamada, H. Agric.
Biol. Chem. 1972, 36, 1869.
. Nagasawa, T.; Utagawa, T.; Goto, J.; Kim, C.-J.; Tani, Y.;
Kumagai, H.; Yamada, H. Eur. J. Biochem. 1981, 117, 33.
5. Recombinant tyrosine phenol-lyase from C. freundii
(Antson, A. A.; Demidkina, T. V.; Gollnick, P.; Dauter, Z.;
Von Tersch, R. L.; Long, J.; Berezhnoy, S. N.; Phillips, R. S.;
Harutyunyan, E. H.; Wilson, K. S. Biochemistry 1993, 32, 419)
was prepared as previously described (Chen, H.; Gollnick, P.;
Phillips, R. S. Eur. J. Biochem. 1995, 229, 540).
. Inouye, S.; Shomura, T.; Tsuruoka, T.; Ogawa, Y.; Watanabe,
H.; Yoshida, J.; Niida, T. Chem. Pharm. Bull. 1975, 23, 2669.
. Shindo-Okada, N.; Nagahara, H.; Yamaizumi, Z.; Makabe,
quickly for 1 min to denature the remaining enzyme.
After cooling, the denatured enzyme was removed by
filtration through Celite. The filtrate was applied to a
2
3
+
Dowex 50W X8 (H form) column (2.5ꢁ25cm). The
column was washed with 1 L of water to remove
unreacted hydroxypyridine and pyruvic acid. The aza-
tyrosine was eluted with 0.75M NH OH. The solvent
4
4
was removed in vacuo, and the residue was dissolved in
a minimum amount of water. Acetone was added until
the solution just remained cloudy. After sitting at room
temperature overnight, 3-aza-l-tyrosine was collected
1
by filtration (36.5mg, 10%). H NMR (D O/NaOD) d
2
6
7
.65(dd, J=2.4, 9.0 Hz, 1H), 7.42 (d, J=2.4 Hz, 1H),
.63 (d, J=9.0 Hz, 1H), 3.89 (t, J=6.5Hz, 1H), 3.01 (t,
6
J=6.5Hz, 2H); MS (ESI) m/z 183 (MH ); UV (H O),
7
+
2
O.; Nishimura, S. Nucleic Acids Symp. Ser. 1988, 19, 129.
8. Yamano, K.; Shirahama, H. Tetrahedron 1992, 48, 1457.
9. Myers, A. G.; Gleason, J. L. J. Org. Chem. 1996, 61, 813.
lmax=230 nm (log e=3.99), lmax=300 nm (log
2
0
e=3.75); [a] +17.9 (c 0.10, H O). The NMR, MS,
D
2