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Journal ArticleDOI

Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

10 Nov 2008-Chemistry: A European Journal (WILEY-V C H VERLAG GMBH)-Vol. 14, Iss: 32, pp 10094-10100

TL;DR: Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
Abstract: The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His(6) tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His(6) tag does not affect AspB activity. The enzyme processes L-aspartic acid, but not D-aspartic acid, with a K(m) of approximately 15 mM and a k(cat) of approximately 40 s(-1). By using this recombinant enzyme in the reverse reaction, a set of four N-substituted aspartic acids were prepared by the Michael addition of hydroxylamine, hydrazine, methoxylamine, and methylamine to fumarate. Both hydroxylamine and hydrazine were found to be excellent substrates for AspB. The k(cat) values are comparable to those observed for the AspB-catalyzed addition of ammonia to fumarate ( approximately 90 s(-1)), whereas the K(m) values are only slightly higher. The products of the enzyme-catalyzed addition of hydrazine, methoxylamine, and methylamine to fumarate were isolated and characterized by NMR spectroscopy and HPLC analysis, which revealed that AspB catalyzes all the additions with excellent enantioselectivity (>97 % ee). Its broad nucleophile specificity and high catalytic activity make AspB an attractive enzyme for the enantioselective synthesis of N-substituted aspartic acids, which are interesting building blocks for peptide and pharmaceutical synthesis as well as for peptidomimetics.
Topics: Aspartate ammonia-lyase (57%), Methylamine (54%), Hydroxylamine (53%), Enzyme catalysis (51%), Michael reaction (51%)

Summary (1 min read)

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Summary

  • A hydrogen balloon was placed on top of the Schlenck flask and the solution stirred vigorously over night.
  • The mixture was filtered over celite and the filtrate was evaporated.
  • The crude aspartic acid was purified via IEC on cationic Dowex 50 (H + , 20-50 mesh, washed with water) by elution with 5% NH 3 -solution, and lyophilized (0.16 g, 0.80 mmol, 80%).
  • HPLC (C6 Crownpack, HClO 4, pH 2.0, flow 0.3 mL/min, 0°C) 4.33 min (D-Asp), 6.23 (L-Asp), 97% ee)).

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University of Groningen
Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate
Ammonia Lyase
Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.
Published in:
Chemistry
DOI:
10.1002/chem.200801407
IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from
it. Please check the document version below.
Document Version
Final author's version (accepted by publisher, after peer review)
Publication date:
2008
Link to publication in University of Groningen/UMCG research database
Citation for published version (APA):
Weiner, B., Poelarends, G. J., Janssen, D. B., & Feringa, B. L. (2008). Biocatalytic Enantioselective
Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase.
Chemistry
,
14
(32), 10094-10100.
https://doi.org/10.1002/chem.200801407
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Supporting Information
© Copyright Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, 2008

Biocatalytic enantioselective synthesis of N-substituted aspartic acids by
aspartate ammonia lyase
Barbara Weiner
[a]
, Gerrit J. Poelarends*
[b]
, Dick B. Janssen*
[c]
and Ben L. Feringa*
[a]
[a] Department of Organic and Molecular Inorganic Chemistry, Stratingh Institute of Chemistry,
University of Groningen, Nijenborgh 4, 9747AG Groningen, The Netherlands.
[b] Department of Pharmaceutical Biology, Institute of Pharmacy, University of Groningen, Antonius
Deusinglaan 1, 9713 AV Groningen, The Netherlands.
[c] Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747AG Groningen, The
Netherlands.

General methods
Reagents were purchased from Aldrich, Acros, Merck or Fluka and were used as
provided, unless stated otherwise. All solvents were reagent grade. Enzymes used for the
molecular biology procedures, DNA ladders, protein molecular weight standards,
deoxynucleotide triphosphates (dNTPs), the high pure plasmid isolation kit, the high pure
PCR product purification kit, and multipurpose agarose were purchased from F.
Hoffmann-La Roche, Ltd. Oligonucleotides for DNA amplification and sequencing were
synthesized by Sigma-Aldrich. All biocatalytic reactions were performed in 50 mL
Greiner tubes, which were shaken at ~100 rpm in a waterbath at 37°C. Buffer and stock
solutions of fumarate were prepared in distilled water and stored at 4°C. The pH of the
solutions was adjusted with a Professional Meter PP-15 pH-meter from Sartorius. All
moisture sensitive reactions were performed in round bottomed or modified Schlenk
flasks, previously heated with a heatgun under oilpump vacuum, which were fitted with
rubber septa under a positive pressure of nitrogen. Air- and moisture-sensitive liquids and
solutions were transferred via syringe. Organic solutions were concentrated by rotary
evaporation at 4060°C. Lyophilization was performed with a ALPHA 2-4 LD plus
freeze dryer from Christ. Flash column chromatography was performed as described by
Still et al.
[1]
As stationary phase, Silia-P flash silica gel from Silicycle, size 40-63 µm,
was used. For TLC analysis silica gel 60 from Merck (0.25 mm) impregnated with a
fluorescent indicator (254 nm) was used. TLC plates were visualized by exposure to
ninhydrin or phosphomolybdic acid (PMA) stain followed by brief heating with a
heatgun. Ion exchange chromatography was performed with either Dowex 50 (H
+
form)
activated with 1N HCl and rinsed with distilled water until a neutral pH was obtained (as
assessed with pH indicator paper), or Amberlite IRA 140 (Cl
form) activated with 1N
NaOH until chloride free and washed with distilled water until a neutral pH was obtained.
SPE SCX columns were purchased from IST. Optical rotations were recorded with a
Polartronic MH8 polarimeter from Schmidt + Haensch. The concentrations are given in
g/100 mL.
1
H and
13
C NMR spectra were recorded on a Varian VXR-300 (300 MHz) or a
Varian Mercury Plus (400 MHz) spectrometer. Chemical shifts for protons are reported
in parts per million scale (δ scale) downfield from tetramethylsilane and are referenced to
residual protium in the NMR solvents (CHCl
3
: δ = 7.25, H
2
O: δ = 4.67). Chemical shifts
for carbon are calibrated to the middle signal of the
13
C-triplet of the solvent CDCl
3
(δ =
77.0). HPLC spectra were obtained using a Shimadzu LC-20AD equipped with a
Chiralpak OD-H column. Reversed phase HPLC was performed on a Shimadzu LC-
10AD VP using either a C6 Crownpack column or an Astec CLC-L column. Kinetic data
were obtained on a Jasko V-550, V-560, or V-570 UV-spectrophotometer. Protein was
analyzed by polyacrylamide gel electrophoresis (PAGE) under either denaturing
conditions using sodium dodecyl sulfate (SDS) or native conditions on gels containing
12% polyacrylamide. The gels were stained with Coomassie brilliant blue. Protein
concentrations were measured using the Waddell method.
[2]
DNA sequencing was
performed by GATC Biotech.
[1] W. C. Still, M. Kahn, A. Mitra, J. Org. Chem. 1978, 43, 2923-2925.
[2] W. J. Waddell, J. Lab. Clin. Med. 1956, 48, 311-314.


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TL;DR: The structure of the apoenzyme has made it possible to identify some of the residues that are involved in binding the substrate, and their putative roles have been assigned.
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