Nitrile Reductase from Geobacillus kaustophilus: A Potential Catalyst
1
1
15.24 (-CN), 130.82 (C-6), 145.85 (C-2), 148.56 (C-7a),
57.07 (C-4).
formation. In the case of the natural substrate preQ , addi-
0
tionally, a synthetic reference material was used to verify
the HPLC-MS results. For all screening reactions, including
blank reactions, multiple parallel determinations were run.
2
-Amino-4-chloro-5-cyano-7H-pyrrolo
dine: 2-Amino-4-chloro-5-cyano-7H-pyrrolo
dine was prepared according to a modified literature proce-
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
[18]
dure.
PreQ , 1, (2.00 g, 11.42 mmol) was suspended in
0
12 mL acetonitrile. Triethylamine (3.2 mL, 23 mmol) was
added and the suspension was heated to 958C. Phosphoryl
chloride (2.2 mL, 24 mmol) was slowly added to the warm
reaction mixture. The reaction mixture was allowed to stir
at 958C overnight. The reaction mixture was then allowed
to cool to 658C and an additional 10 mL acetonitrile, phos-
phoryl chloride (2.2 mL, 24 mmol), and triethylamine
Acknowledgements
This study was performed within the Austrian Centre of In-
dustrial Biotechnology (ACIB GmbH). This work has been
supported by the Federal Ministry of Economy, Family and
Youth (BMWFJ), the Federal Ministry of Traffic, Innovation
and Technology (bmvit), the Styrian Business Promotion
Agency SFG, the Standortagentur Tirol and ZIT-Technology
Agency of the City of Vienna through the COMET-Funding
Program managed by the Austrian Research Promotion
Agency FFG. We gratefully acknowledge Christoph Reising-
er, Margaretha Schiller, Elena Loncar, Torsten Bachler and
Hannelore Mandl for technical support and Prof. Bernd Ni-
detzky and Sigrid Egger for helpful advice.
(
3.2 mL, 23 mmol) were added. The reaction was allowed to
stir at 658C for additional 48 h at which time HPLC-MS
analysis showed full conversion. The reaction mixture was
cooled to room temperature and ice was slowly added. The
mixture was then heated to 758C for 15 min and subse-
quently the precipitate was filtered off. The pH value of the
filtrate was adjusted to pH 2 using aqueous ammonia solu-
tion. The filtrate was then cooled to 08C. The product pre-
cipitated from the solution and was isolated by vacuum fil-
tration. After washing with water and drying the product
was isolated as a brown solid; yield: 807.7 mg (37%).
1
H NMR (DMSO-d ): d=6.96 (s, 2H, NH ), 8.12 (s, 1H, H-
6
2
13
References
6
1
1
); C NMR (DMSO-d ): d=83.05 (C-5), 106.10 (C-4a),
6
15.05 (CN), 134.16 (C-6), 151.09 (C-7a), 154.49 (C-4),
60.38 (C-2).
[
1] a) S. G. Van Lanen, J. S. Reader, M. A. Swairjo, V. de
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2
-Amino-5-cyano-7H-pyrrolo
Amino-4-chloro-5-cyano-7H-pyrrolo
650 mg, 3.36 mmol) was suspended in 80 mL ethanol. 10%
ACHTUNGTRENNUNG
AHCTUNGTRENNUNG
(
1
2844–12854.
palladium on charcoal catalyst (65 mg) and sodium bicar-
bonate (33.8 mg, 4.02 mmol) were added. The suspension
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trate ws concentrated under vacuum until dryness. 2-Amino-
[
[
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5-cyano-7H-pyrrolo ACHTUNGTRENNUNG[ 2,3-d]pyrimidine was obtained as brown
solid; yield: 440 mg (82%). Anal. calcd. for C H N C 52.83,
7
5
5
1
H 3.17, N 44.01: found: C 54.30, H 4.23, N 41.47; H NMR
1
987, 52, 2301–2303; d) E. H. R. Walker, Chem. Soc.
Rev. 1976, 5, 23–50; e) W. Huber, J. Am. Chem. Soc.
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(
DMSO-d ): d=6.55 (s, 2H, NH ), 8.02 (s, 1H, H-6), 8.63 (s,
6
2
13
1
4
1
H, H-4); C NMR (DMSO-d ): d=82.83 (C-5), 108.96 (C-
6
1
a), 115.45 (CN), 133.01 (C-6), 149.93 (C-4), 153.55 (C-7a),
61.13 (C-2).
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Screening reactions were run in 96-deep well plates using
the following conditions and concentrations: nitrile reduc-
tase (55 mM, purified by heat precipitation, purity between
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6
1
0% and 85%) in buffer (100 mM Tris, 50 mM KCl,
.15 mM TCEP, pH 7.5 adjusted with conc. HCl), substrate
in DMSO (2 mM, 10% v/v DMSO), NADPH (0.25 mM in
buffer), total volume 200 mL. Blank reactions contained
buffer, substrate in DMSO (2 mM, 10% v/v DMSO), and
NADPH (0.25 mM in buffer). Reactions were started by ad-
dition of NADPH, and NADPH depletion was monitored at
3
4
08C on a plate reader for 14 h. Subsequently, additional
mM of NADPH were added to each well and the screen-
ing reactions were placed on a thermomixer at 308C and
00 rpm for 24 h to allow full conversion. Samples were
then analyzed with HPLC-MS to observe possible product
5
Adv. Synth. Catal. 2012, 354, 2191 – 2198
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2197