High Throughput Enzymatic Enantiomeric Excess : Quick-ee

There is a growing need for enzymatic and multienzymatic cascade processes in the chemical and pharmaceutical industries to replace chemical steps with steps that incorporate green chemistry principles. When enantiomerically pure products are required, enzymes are the catalysts of choice for enantio-, diastereoand site-selective transformations, due to their specificity in discriminating prochiral centres and faces. Currently, tailored enzymatic performance is achieved using genetic engineering to modify catalytic sites using several techniques, including site-directed evolution. The transformation process requires modification of the DNA fragment encoding the enzyme, which is inserted into a vector (plasmid, phosmid or virus) and introduced into host cells, producing several clones with different specificities. Sorting these clones leads to highly specific enzymes and products with high enantiomeric excesses (ee) and yields. Such transformations can be further improved to broaden their catalytic applications by generating enzymes that accept several substrates (substrate promiscuity). These attributes enhance the application of this biotechnology in industry, where molecular engineering and green chemistry work together to add value to products. These biocatalyst issues (ee, enantiomeric ratio (E), etc.) require access to fast and sensitive methodologies, such as high throughput screening (HTS). Methodologies to rapidly obtain the enantiomeric ratio require chiral substrates and initial rate monitoring (V0) for each enantiomer. 24,25 These HTS techniques usually employ chromogenic or fluorogenic probes, allowing for the simultaneous evaluation of 6, 24, 96, or 384 reactions. Based on this idea, Kazlauskas and co-workers introduced Quick-E for hydrolases through the application of chiral chromogenic probes. A chromogenic competitor was also introduced into the experiment, which behaved as the enantiomer, a statement not exactly true because they are different compounds, thus producing a good E evaluation. Similar methodology was proposed by Reymond et al. However, this methodology does not use a competitor and is based on fluorogenic probes, which are usually more sensitive (approximately 10). The lack of enantiomeric competition for the enzyme active site results in large deviations from true E values. In this study, we applied both concepts, exploiting both the sensitivity of fluorogenic probes and competition, for the HTS evaluation of ee, which is referred to here as Quick-ee.


Introduction
[8][9][10] Currently, tailored enzymatic performance is achieved using genetic engineering to modify catalytic sites using several techniques, including site-directed evolution. 11,12he transformation process requires modification of the DNA fragment encoding the enzyme, which is inserted into a vector (plasmid, phosmid or virus) and introduced into host cells, producing several clones with different specificities.Sorting these clones leads to highly specific enzymes and products with high enantiomeric excesses (ee) and yields. 13uch transformations can be further improved to broaden their catalytic applications by generating enzymes that accept several substrates (substrate promiscuity). 14,15These attributes enhance the application of this biotechnology in industry, where molecular engineering and green chemistry work together to add value to products. 16,17These biocatalyst issues (ee, enantiomeric ratio (E), etc.) require access to fast and sensitive methodologies, [18][19][20][21] such as high throughput screening (HTS). 22,23Methodologies to rapidly obtain the enantiomeric ratio require chiral substrates and initial rate monitoring (V 0 ) for each enantiomer. 24,257][28] Based on this idea, Kazlauskas and co-workers introduced Quick-E for hydrolases through the application of chiral chromogenic probes.A chromogenic competitor was also introduced into the experiment, which behaved as the enantiomer, a statement not exactly true because they are different compounds, thus producing a good E evaluation. 26imilar methodology was proposed by Reymond et al. [27][28][29] However, this methodology does not use a competitor and is based on fluorogenic probes, which are usually more sensitive (approximately 10 3 ). 29The lack of enantiomeric competition for the enzyme active site results in large deviations from true E values.
In this study, we applied both concepts, exploiting both the sensitivity of fluorogenic probes and competition, for the HTS evaluation of ee, which is referred to here as Quick-ee.

Cultivation of microorganisms
Bacteria were inoculated into nutrient broth.Yeasts and fungi were inoculated into yeast extract-malt and cultivated for 16 h at 28 °C with stirring at 200 rpm.The cells were then transferred to a Petri dish containing nutrient agar (NA) or yeast extract-malt extract agar (YMA) and incubated at 30 °C for an additional 16 h period.
The assays (enzymatic assay, negative control and positive control) were monitored for 24 h at 28 °C, simultaneously.

Biotransformations
To the implementation of the Quick-ee methodology, the racemic fluorogenic probe 1 (10 µL, 2.0 mmol L −1 in H 2 O/MeCN, 1:1) were added to the cell suspension (190 µL, 2.0 g L −1 ) to reach a final concentration of 100 mmol L −1 .The diol enantiomeric excess (ee) was determined by high performance liquid chromatography (HPLC) using a CHIRALPAK-IC (Daicel: 25 cm × 0.46 cm) column, which contains a chiral stationary phase, with 6:4 ethanol:hexane as the eluent, an injection volume of 20 μL, a flow rate of 1.0 mL min −1 and l abs = 320 nm.

Calculations E estimated and Quick-E
The E value determined in assays without competitor 2 (E estimated ) and E value determined in assays with competitor 2 (Quick-E) were determined from the initial reaction rates (V 0 ) for each enantiomer (equation 1), which were obtained from the slope of the curve of reactant concentration versus time (t) at t = 0 (V 0 ).
ee with competition (Quick-ee) and without competition (ee estimated ) The enantiomeric excess values were calculated (equation 2) for each point using the fluorescence signal measurement for each assay (relative fluorescence unit, RFU).

Quick-ee
Assay conversions (c) and enantiomeric excess (ee) values at a specific time are parameters of fundamental importance in the kinetic characterization of an enzyme.Therefore, it is interesting to investigate new methodologies to obtain these parameters using high throughput screening.
Two methodologies employing fluorogenic probes were developed.The first is based on Reymond's methodology 26 and calculates E from the ratio between the initial rates of each enantiomer, which are evaluated separately.However, this methodology does not take into consideration the enantiomeric competition for the active site, which can produce large deviations from the real ee.Kazlauskas' methodology 27 of using chromogenic probes introduces competition to minimize effects resulting from the lack of competition.However, both methodologies do not reveal the ee and conversions at a particular reaction time.To overcome previous limitations, we fused the Reymond's and Kazlauskas's 26,27 methodologies by implementing the assay with fluorogenic probe 1 and a nonfluorogenic competitor 2 to obtain ee and conversion (Figures 2 and 3).

Choosing the nonfluorogenic competitor
The nonfluorogenic competitor 2 was selected by taking into consideration its structural similarity to the probe 1 (Scheme 1).Thus (R)-1 and 2 (or (S)-1 and 2) can compete for the hydrolase enzymatic site producing diols (R)-3 and (S)-3 and alcohol 4, respectively.However diols (R)-3 and (S)-3 are cleaved by the periodate present in the reaction mixture, producing aldehyde 5, which undergoes β-elimination, catalysed by BSA, producing a fluorescent signal (Scheme 1).The fluorescent signal intensity is concentration dependent and reveals the amount of umbelliferone produced. 27The same enzymatic cascade does not occur with alcohol 4, which is not oxidized, does not undergo β-elimination and, consequently, does not produce a fluorescent signal (Scheme 1).
E values were obtained from the initial speed ratios of each enantiomer in each assay.E values improved in the presence of competitors (Quick-E) (Table 2) This is assigned to the competition of both fluorogenic probes ((R)-1 and (S)-1) and its competitor (2) to the enzyme active site.

Conclusions
The Quick-ee was validated for enzymes and microorganisms and the results in the presence of a competitor were closer to real ee for low E reactions.This methodology provides an easy access evaluation of numerous samples, such as libraries of mutants and clones.        .

Figure 5 .
Figure 5. Enantiomeric excess and conversions with Aspergillus lipase (Sigma-Aldrich 84205) (a) without competitor 2 and (b) with competitor 2 (Quick-ee).Just like in Pseudomonas fluorescens (CCT-7393) whole cells (c) without competitor 2 and (d) with competitor 2 (Quick-ee).In both cases, minor ee values are observed in experiments with the competitor 2 for the same conversion level.

Table 1 .
ee values for assays without competition (ee estimated ), with competition (Quick-ee), and the real ee (ee real )

Table 2 .
E values from experiments with and without competitors