Marfey's reagent: Past, present, and future uses of 1‐fluoro‐2,4‐dinitrophenyl‐5‐L‐alanine amide

B’Hymer, Clayton; Montes-Bayón, Marı́a; Caruso, Joseph A.

doi:10.1002/jssc.200390019

Cited by 94 publications

(56 citation statements)

References 89 publications

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“…To measure amino donor activities for isopropylamine, reaction conditions were 20 mM isopropylamine and 20 mM pyruvic acid. L-Alanine was analyzed by chiral HPLC after derivatization with Marfey's reagent (33,34).…”

Section: Chemicalsmentioning

confidence: 99%

“…UV detection was done at 254 nm. Quantitative chiral analyses of alanine, norvaline, and norleucine were carried out using a Crownpak CR(Ϫ) column (Daicel Co., Japan) or a Sunfire C 18 column after chiral derivatization with Marfey's reagent (33,34). ⌲eto acids were analyzed using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) with isocratic elution of a 5 mM H 2 SO 4 solution at 0.5 ml/min.…”

Section: Chemicalsmentioning

confidence: 99%

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Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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-Transaminase (-TA) is a promising enzyme for use in the production of unnatural amino acids from keto acids using cheap amino donors such as isopropylamine. The small substrate-binding pocket of most -TAs permits entry of substituents no larger than an ethyl group, which presents a significant challenge to the preparation of structurally diverse unnatural amino acids. Here we report on the engineering of an (S)-selective -TA from Ochrobactrum anthropi (OATA) to reduce the steric constraint and thereby allow the small pocket to readily accept bulky substituents. On the basis of a docking model in which L-alanine was used as a ligand, nine active-site residues were selected for alanine scanning mutagenesis. Among the resulting variants, an L57A variant showed dramatic activity improvements in activity for ␣-keto acids and ␣-amino acids carrying substituents whose bulk is up to that of an n-butyl substituent (e.g., 48-and 56-fold increases in activity for 2-oxopentanoic acid and L-norvaline, respectively). An L57G mutation also relieved the steric constraint but did so much less than the L57A mutation did. In contrast, an L57V substitution failed to induce the improvements in activity for bulky substrates. Molecular modeling suggested that the alanine substitution of L57, located in a large pocket, induces an altered binding orientation of an ␣-carboxyl group and thereby provides more room to the small pocket. The synthetic utility of the L57A variant was demonstrated by carrying out the production of optically pure L-and D-norvaline (i.e., enantiomeric excess [ee] > 99%) by asymmetric amination of 2-oxopantanoic acid and kinetic resolution of racemic norvaline, respectively. U nnatural amino acids are widely used as essential chiral building blocks for diverse pharmaceutical drugs, agrochemicals, and chiral ligands (1-3). In contrast to natural amino acids, a fermentative method for the production of unnatural amino acids is not yet commercially available (4,5). This has driven the development of various biocatalytic approaches to afford scalable processes for preparing enantiopure unnatural amino acids. These processes include kinetic resolution of racemic amino acids using acylase (6, 7), amidase (8, 9), hydantoinase (10, 11), and amino acid oxidase (12, 13) and asymmetric reductive amination of keto acids using dehydrogenase (14, 15) and transaminase (16,17). Asymmetric amination is usually favored over kinetic resolution because the former affords a 100% yield without racemization of an unwanted enantiomer.In contrast to a mandatory requirement for the supply and regeneration of an expensive external cofactor for the dehydrogenase reactions, transaminase catalyzes the transfer of an amino group from an amino donor to an acceptor using pyridoxal 5=-phosphate (PLP) as a prosthetic group (18). Nevertheless, industrial implementation of the transaminase-mediated synthesis of unnatural amino acids has lagged behind because of low equilibrium constants, usually close to unity, for the reactions between keto acids a...

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Section: Chemicalsmentioning

confidence: 99%

Section: Chemicalsmentioning

confidence: 99%

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Han

Park

Dong

et al. 2015

Appl Environ Microbiol

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“…Quantitative chiral analyses of alanine and amines were carried out using aC rownpak CR(À)c olumn (Daicel Co., Japan) or aS unfire C18 column after chiral derivatizationw ith Marfeys reagent. [22] Details of the HPLC analysis are described in the Supporting Information.…”

mentioning

confidence: 99%

Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

et al. 2015

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Asymmetricr eductivea mination of ketones using w-transaminases (w-TAs)offers apromising alternative to the chemocatalytic synthesiso f chiral amines.O ne fundamental challenge to the biocatalytic strategyi st he very low enzyme activities for most ketones compared with native substrates (i.e., <1% relativet op yruvate). Here we have demonstrated that as ingle point mutation in the active site of the (S)-selective w-TAf rom Ochrobactrum anthropi could induce ar emarkable acceleration of the amination reactionw ithout anyl oss in stereoselectivity and enzyme stability.M olecular modeling of quinonoid intermediates,a lanine scanning mutagenesis and kinetic analysis revealedthat the W58 residue acted as as tericb arrier to binding and catalytic turnover of ketone substrates. Removal of the steric strain by W58L substitution, which was selected by partials aturation mutagenesis,l ed to dramatica ctivity improvements for structurally diverse ketones (e.g.,3 40-fold increase in k cat /K M for acetophenone). TheW 58Lm utant afforded an efficient synthesis of enantiopure amines (i.e., >99% ee)u sing isopropylamine as an amino donor.

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“…Brückner and Keller-Hoehl (1990) synthesized FDNP-L-Phe-NH 2 , FDNP-L-Val-NH 2 and FDNP-L-Pro-NH 2 as the structural variants of MR and used them for HPLC separation of α-amino acids. The comprehensive literature reports (B'Hymer et al, 2003;Bhushan and Brückner, 2004) reveal a wide scope for application of MR and its chiral variants for indirect enantiomeric resolution of a variety of chiral drugs. Recently, MR and its structural variants have been used for indirect separation of enantiomers of penicillamine (Bhushan et al, 2007a) and amino acids (Bhushan et al, 2007b) in this laboratory.…”

Section: Introductionmentioning

confidence: 99%

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed‐phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

Bhushan

Kumar

2008

Biomedical Chromatography

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A high-performance liquid chromatographic (HPLC) method was developed for chiral assay of baclofen enantiomers in pharmaceutical formulations using an indirect approach. Baclofen enantiomers were derivatized with Marfey's reagent (FDNP-L-Ala-NH2) and its structural variants FDNP-L-Phe-NH2, FDNP-L-Val-NH2, FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2. The resultant diastereomers were separated on RP-TLC [triethylammonium phosphate buffer (pH 4.0, 50 mm)-acetonitrile, 50:50] and on a C18 column using a linear gradient (45 min) of acetonitrile and 0.01% aqueous trifluoroacetic acid (TFA) with UV detection at 340 nm. The differences in the retention times (Delta t R) of diastereomers due to the five chiral reagents were compared. The maximum and minimum difference in retention times between separated diastereomers was for FDNP-L-Leu-NH2 and FDNP-L-Pro-NH2, respectively. The effect of flow rate, acetonitrile content and TFA concentration on resolution was studied. The method was validated for linearity, repeatability, limit of detection and limit of quantification.

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Marfey's reagent: Past, present, and future uses of 1‐fluoro‐2,4‐dinitrophenyl‐5‐L‐alanine amide

Cited by 94 publications

References 89 publications

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Active-Site Engineering of ω-Transaminase for Production of Unnatural Amino Acids Carrying a Side Chain Bulkier than an Ethyl Substituent

Mechanism‐Guided Engineering of ω‐Transaminase to Accelerate Reductive Amination of Ketones

Indirect resolution of baclofen enantiomers from pharmaceutical dosage form by reversed‐phase liquid chromatography after derivatization with Marfey's reagent and its structural variants

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