A Practical High‐Throughput Screening System for Enantioselectivity by Using FTIR Spectroscopy

Tielmann, Patrick; Boese, Matthias; Luft, Martin; Reetz, Manfred T.

doi:10.1002/chem.200304885

Cited by 63 publications

(27 citation statements)

References 75 publications

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“…17 and 27). Select protocols for the most efficient ee assays based on MS (28,29), NMR spectroscopy (30), and IR spectroscopy (31) have been published which allow 1,000-10,000 samples to be evaluated per day (32). Selection (33) rather than screening is a goal for the future, phage display constituting one possibility (34).…”

Section: High-throughput Ee Assaysmentioning

confidence: 99%

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Reetz

2004

Proc. Natl. Acad. Sci. U.S.A.

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319

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A fundamentally new approach to asymmetric catalysis in organic chemistry is described based on the in vitro evolution of enantioselective enzymes. It comprises the appropriate combination of gene mutagenesis and expression coupled with an efficient high-throughput screening system for evaluating enantioselectivity (enantiomeric excess assay). Several such cycles lead to a ''Darwinistic'' process, which is independent of any knowledge concerning the structure or the mechanism of the enzyme being evolved. The challenge is to choose the optimal mutagenesis methods to navigate efficiently in protein sequence space. As a first example, the combination of error-prone mutagenesis, saturation mutagenesis, and DNA-shuffling led to a dramatic enhancement of enantioselectivity of a lipase acting as a catalyst in the kinetic resolution of a chiral ester. Mutations at positions remote from the catalytically active center were identified, a surprising finding, which was explained on the basis of a novel relay mechanism. The scope and limitations of the method are discussed, including the prospect of directed evolution of stereoselective hybrid catalysts composed of robust protein hosts in which transition metal centers have been implanted.

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Section: High-throughput Ee Assaysmentioning

confidence: 99%

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Reetz

2004

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

319

177

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“…[6][7][8][9] There, an enzyme-catalyzed reaction leads to a spectroscopic signal that is monitored in real time. The approach complements other emerging screens by using chiroptical techniques, [10] liquid crystalline arrays, [11] IR thermography, [12] mass, [13] NMR, [14] IR [15] and fluorescence spectroscopy.[16] The technique is sensitive (i.e. 10 nmol of product gives rise to DA 340 % 0.12 for a dehydrogenase reporting enzyme in a 500 mL aqueous volume), allowing one to get information on catalyst performance at relatively early conversions/short reaction times.…”

mentioning

confidence: 99%

“Cassette” In Situ Enzymatic Screening Identifies Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides

Dey¹,

Powell²,

Hu³

et al. 2007

Angew Chem Int Ed

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For combinatorial catalysis, [1,2] rapid and information-rich screening methods are very useful. Toward this end, we describe herein a new "cassette" in situ enzymatic screening (ISES) approach that allows the experimentalist to obtain a parallel readout on substrate specificity, as well as a sense and magnitude of enantioselectivity.[3] In its first iteration, cassette-ISES is used to "cherry pick" only those catalysts in the array that show high ISES ee-value readouts across both test substrates. Two such catalysts are then investigated further, yielding promising results.In our ISES approach, typically the reaction product [4] or by-product [5] diffuses from an organic layer into an aqueous layer containing the "reporting enzyme". [6][7][8][9] There, an enzyme-catalyzed reaction leads to a spectroscopic signal that is monitored in real time. The approach complements other emerging screens by using chiroptical techniques, [10] liquid crystalline arrays, [11] IR thermography, [12] mass, [13] NMR, [14] IR [15] and fluorescence spectroscopy.[16] The technique is sensitive (i.e. 10 nmol of product gives rise to DA 340 % 0.12 for a dehydrogenase reporting enzyme in a 500 mL aqueous volume), allowing one to get information on catalyst performance at relatively early conversions/short reaction times. Catalysts may be screened in parallel in a standard spectrophotometer with a multicell changer without the need to draw aliquots or work up the reaction. Moreover, the need to install a chromophore (adding steps and potentially altering substrate reactivity) is obviated.Herein, we describe a new pair of reporting enzymes (Scheme 1) that are capable of differentiating the 1,2-hexanediol antipodes (Lactobacillus kefir alcohol dehydrogenase (LKADH): highly S selective k S /k R % 20, and horseliver alcohol dehydrogenase (HLADH): modestly S selective k S /k R % 2.2). This allows one to obtain simultaneous enantioselectivity readouts on two distinct substrates for the Co IIIsalen-mediated (salen = (salicylidene)ethylenediamine) hydrolytic kinetic resolution (HKR) of epoxides, [17] presenting both "short" (propylene oxide; R = Me) [4] and long (hexene oxide: R = Bu) R groups. In this way, one can begin to address the question of substrate generality, which is so important in asymmetric catalysis today. [18,19] To demonstrate proof of principle for cassette-ISES, we employed a focused chiral salen array (Figure 1) that crosses chiral space variation in the constituent 1,2-diamines with considerable steric [20] and electronic variation in the "salicylaldehyde partners" (including the benzoylacetaldehyde (baen) precursor [21] ). Figure 2 illustrates the "four coordinate" structure-enantioselectivity relationship (SER) data that one obtains from such a cassette-ISES protocol. The x and y axes represent the two structural variables in the salen array. The directionality and length of the z vector provide the Scheme 1. A suitable pair of "reporting enzymes" for 1,2-hexanediol permits a cassette-ISES evaluation of HKR catalyst candidat...

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“…This method therefore obviates the necessity to conduct enzyme-directed evolution experiments with spatially segregated clones, which limits the number of clones to be screened to a maximum of several thousand. [16,17] Moreover, the presence of typically more than 30 000 esterase molecules on the surface of a single bacterial cell eliminates the stochastic uncertainty inherent in enzyme display on phages, on the surface of which only a few enzyme molecules are present per single replicating entity.…”

Section: Discussionmentioning

confidence: 99%

Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

et al. 2007

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We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.

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A Practical High‐Throughput Screening System for Enantioselectivity by Using FTIR Spectroscopy

Cited by 63 publications

References 75 publications

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

Controlling the enantioselectivity of enzymes by directed evolution: Practical and theoretical ramifications

“Cassette” In Situ Enzymatic Screening Identifies Complementary Chiral Scaffolds for Hydrolytic Kinetic Resolution Across a Range of Epoxides

Ultrahigh‐Throughput Screening to Identify E. coli Cells Expressing Functionally Active Enzymes on their Surface

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