Cutting Long Syntheses Short: Access to Non-Natural Tyrosine Derivatives
by centrifugation at 48C and 15000 rpm for 20 min and the
1H, Ar), 6.72–6.79 (m, 1H, Ar), 3.80–3.84 (m, 1H, Ar-CH2-
supernatant was lyophilised to give the cell-free extract.
CH), 3.02–3.08 (m, 1HA, Ar-CHAHB), 2.90–2.95 (m, 1HB, Ar-
CHAHB), 2.09 (s, 3H, CH3); MS (EI): m/z=57, 73, 100, 117,
147, 193, 218 (100%), 294, 324, 368, 396 (trimethylsilyl deriv-
ative); ee >97% (HPLC).
General Procedure for Activity Testing
l-3-Methoxytyrosine (1c): yield: 38 mg (60%), 1H NMR
(300 MHz, D2O, TMS): d=6.76–6.79 (m, 2H, Ar), 6.63–
6.65 m, 1H, Ar), 3.78–3.82 (m, 1H, Ar-CH2-CH), 3.70 (s,
3H, OCH3), 2.99–3.06 (m, 1HA, Ar-CHAHB), 2.88–2.95 (m,
1HB, Ar-CHAHB); MS (EI): m/z=73, 100, 133, 146, 179, 209,
218 (100%) (trimethylsilyl derivative); ee >97% (HPLC).
l-3-Fluorotyrosine (1e): yield: 50 mg (83%), 1H NMR
(300 MHz, D2O, TMS): d=6.84–6.96 (m, 3H, Ar), 3.79–3.83
(m, 1H, Ar-CH2-CH), 3.00–3.06 (m, 1HA, Ar-CHAHB), 2.94–
2.96 (m, 1HB, Ar-CHAHB); MS (EI): m/z=73, 100, 130, 147,
197, 218 (100%), 298 (trimethylsilyl derivative); ee >97%
(HPLC).
Lyophilised cell-free extract (2 mg) was rehydrated in a po-
tassium phosphate buffer [640 mL, 50 mM, pH 8, 100 mM
NH4Cl, 40 mM (NH4)2SO4, 0.04 mM PLP] for 30 min at
308C and 120 rpm. Then phenol (derivative) stock (16 mmol
in acetonitrile, final concentration 23 mM) and pyruvate (32
mmol, final concentration 46 mM) were added and the
sample was incubated at 308C and 120 rpm for 24 h and
worked up for cation exchange HPLC measurements.
Variation of Substrate Concentration
Lyophilised cell-free extract (2 mg) was rehydrated in potas-
sium phosphate buffer [50 mM, pH 8, 100 mM NH4Cl,
40 mM (NH4)2SO4, 0.04 mM PLP] for 30 min at 308C and
120 rpm. Then phenol derivative (1 equiv.) and pyruvate (2
equiv.) were added. The concentration of phenol derivative
was varied between 23 mM and 237 mM, the concentration
of cosolvent (acetonitrile, diethyl ether) was kept at 10%
l-3-Chlorotyrosine (1f): yield: 17 mg (26%), 1H NMR
(300 MHz, D2O, TMS): d=7.14 (s, 1H, Ar), 6.86–6.93 (m,
2H, Ar), 3.76–3.79 (m, 1H, Ar-CH2-CH), 2.97–3.03 (m,
1HA, Ar-CHAHB), 2.86–2.93 (m, 1HB, Ar-CHAHB); MS (EI):
m/z=73, 147, 207, 218 (100%) (trimethylsilyl derivative); ee
>97% (HPLC).
ACHTUNGTRENNUNG(v/v).The samples were incubated at 308C and 120 rpm for
2 h using the recombinant wild-type enzyme and 4 h for the
variant M379V. Then the samples were prepared for cation
exchange HPLC measurements. For detailed information
see the Supporting Information.
Acknowledgements
Financial support by FFG and Province of Styria and SFG is
gratefully acknowledged. Christoph Gçbl (University of
Graz) is thanked for measuring CD spectra. Heidi Ehammer
and Peter Macheroux (University of Technology, Graz) are
acknowledged for their support concerning molecular biology
issues.
Preparative Biocatalytic Synthesis of Tyrosine
Derivatives
Pyruvate (0.85 mmol, final concentration 120 mM), and
lyophilised cell-free extract (20 mg, recombinant wild-type
or variant) were mixed with potassium phosphate buffer
[6.8 mL, 50 mM, pH 8, 100 mM NH4Cl, 40 mM (NH4)2SO4,
0.04 mM PLP] in a Sarstedt tube (15 mL). Then phenol (de- References
rivative) (0.3 mmol in diethyl ether, final concentration
43 mM) was added in two portions during 2 h to the reac-
tion solution The concentration of the cosolvent was kept at
10%
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ACHTUNGTRENNUNG(v/v). The reaction was incubated at 308C and 120 rpm for
24 h until no starting material could be detected anymore.
Phosphoric acid was added (2 mL, 85%) to the reaction
mixture and mixed well. Then the solution was filtered and
the amino acid was purified via flash chromatography using
the cationic exchanger Lewatit MonoPlus SP-112 (column
size: Ø 3 cm, height: 10 cm; ordering number: Fluka 62102).
The column was washed prior to use with HCl (6M, 2 vol-
umes), NaOH (6M, 2 volumes) and water (2 column vol-
umes). The reaction solution was acidified (pH <3) using
phosphoric acid (85%) and applied onto the column. After
washing with water the amino acid was eluted with aqueous
NH4OH (8%). The fractions showing a positive ninhydrin
reaction were combined and lyophilised yielding the tyro-
sine derivative with remaining salts. The absolute amount of
amino acid was determined using the standard ninhydrin
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1
assay.[20] The resulting amino acid was analysed by H NMR
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tical purity was determined by HPLC on a chiral stationary
phase (see Supporting Information).
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(300 MHz, D2O, TMS): d=6.98 (s, 1H, Ar), 6.89–6.90 (m,
Adv. Synth. Catal. 2010, 352, 731 – 736
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735