Efficient Stereoselective Biotransformation of Prochiral Carbonyls by Endophytic Fungi from Handroanthus impetiginosus

Abstract

Endophytic microorganisms are promising sources for new biocatalysts as they must deal with their host plants’ chemicals by developing adaptative strategies, such as enzymatic pathways. As part of our efforts in selecting endophytic strains as biocatalysts, this study describes the screening of endophytic fungi isolated from Handroanthus impetiginosus leaves for selective bioreduction of Acetophenone. The bioreductions were monitored by chiral gas chromatography and conducted to the selection of the endophyte Talaromyces sp. H4 as capable of reducing acetophenone to (S)-1-phenylethanol in excellent conversion and enantiomeric excess rates. The influence of seven parameters on the stereoselective bioreduction of acetophenone by Talaromyces sp. H4 was studied: reaction time, inoculum charge, shaking, pH, temperature, substrate concentration, and co-solvent. The optimal conditions were then used to reduce substituted acetophenones and Acetophenone scale-up, which furnished (S)-1-Phenylethanol in 73% yield and 96% ee. The results highlight the endophytic fungus Talaromyces sp. H4 as an excellent biocatalyst for stereoselective reduction of prochiral carbonyls.

1. Introduction

Chemical reductions play a crucial role in organic synthesis, as they can produce multiple chiral centers and new functional groups in valuable starting materials for the pharmaceutical and fine chemical industries [1]. Although chiral pharmaceuticals are traditionally sold as the racemate, specialized studies have shown that cell receptors discriminate between the two enantiomers, and their physiochemical and biochemical properties differ significantly [2]. Building blocks with chiral hydroxyl moieties are vital intermediates of multiple drug candidates [3]. Conventional synthetic methods for their preparation include asymmetric hydrogenation or carbonyl reduction using metal hydrides, such as sodium borohydride, which requires long reaction times. Achieving chiral alcohols through carbonyl reduction by biocatalysis approach has been raised as a helpful technology due to the selectivity [4]. Several efforts to provide powerful tools for achieving chiral secondary alcohols at high enantiomeric excesses have been undertaken [5], highlighting the importance of work in modern synthetic organic chemistry and biochemical methods, complementary for sustainably developing promising results. Ketoreductases or carbonyl reductases are naturally ubiquitous enzymes that catalyze reductions at room temperature (25–30 °C) and atmospheric pressure and operate at near-neutral pH; the use of mild conditions may avoid problems such as isomerization, racemization, epimerization, and rearrangement of products [6]. Ketoreductases from fungi have been widely investigated to provide stereo- and enantioselective reduction of ketones to afford (S)- or (R)-alcohols. Frequently, the bioreduction of acetophenone and its derivatives have been employed as predictive models to discover new fungal strains able to provide various chiral aromatic alcohols with excellent enantiomeric purity and high yield [7,8,9]. Biocatalysis is an essential branch of industries, since the reactions mediated by bioprocesses display high levels of stereoselectivity, minimizing the formation of by-products, reducing energy consumption, and having a low environmental impact, fitting perfectly the rules of Green Chemistry [10]. Bioreduction of Acetophenone (1) to chiral products has high industrial application potential, since these compounds can be used as versatile intermediates for synthesizing cosmetics, pharmaceuticals, and agrochemicals. Bioreduction is one of the most common reactions evaluated when a new biocatalyst is being studied [11]. In the aroma industry, (S)-1-Phenylethanol displays a gardenia aroma with strawberry nuances, while the (R)-enantiomer has a floral, earthy-green honeysuckle odor [12]. Moreover, (R)-1-Phenylethanol is used as a chiral building block in fine chemicals and the pharmaceutical industry [13].

Fungi whole cells are an acceptable source for several enzymes because they can convert a wide range of substrates through specific chemical reactions concomitant with efficient cofactor regeneration [14]. Despite some limitations, mainly related to the toxicities of some substrates and products to the fungal cell, fungi play a crucial role in developing new approaches for providing several chemical reactions that are challenging to obtain in synthetic chemistry processes [15].

The choice of enzyme source that will be employed is crucial for the success of biotransformation, and accessing enzymatic systems of fungi from uncommon habitats favors the discovery of new catalytic systems. Endophytic microorganisms naturally occur in vegetal tissue without causing notable damage to the host plants [16]. Plants biosynthesize secondary metabolites that are genuine chemical weapons to deal with predators, and endophytic microorganisms have adaptive strategies to deal with the chemical arsenal of the host plant [17]. Consequently, they have developed enzymatic ways to convert or degrade toxic compounds. These microorganisms are then promising niches for the discovery of new biocatalysts. In addition, the host–endophytic coevolution favors the diversification of the endophytic enzymatic system, since the genes may be changed during the evolutionary steps [18].

The study reported herein focused on screening endophytic fungi isolated from Handroanthus impetiginosus leaves for selective bioreduction of prochiral carbonyls. Biotransformations of acetophenone by the endophytic fungi were monitored by chiral gas chromatography and conducted to select the Talaromyces sp. H4 strain as able to selectively reduce the prochiral carbonyl at high yield. Finally, we developed an approach for achieving chiral alcohol building blocks, which can be an excellent starting point for synthesizing novel compounds combining biotransformation and other chemical processes.

2. Results

2.1. Screening of Endophytic Fungi

Endophytic fungi isolated from Handroanthus impetiginosus leaves were assayed for their abilities in the stereoselective biotransformation of acetophenone (1) prochiral carbonyl to 1-Phenylethanol (1a). Conversions and enantiomeric excess (ee) were determined by gas chromatography employing a chiral column (chiral GC-FID) (Figures S21–S26, Supplementary Material). The best achieved results are summarized in

Table 1. Results of conversion and enantiomeric excess (ee) rates of the biotransformation of acetophenone (1) into S- or R-1-phenylethanol (S-1a or R-1a) by endophytic fungi isolated from Handroanthus impetiginosus leaves.

Entry	Endophytic fungus	Conversion (%)	ee (%)	Main product
1	H2	6.3	89.8	S-1a
2	H3	>99.9	76.2	S-1a
3	H4	>99.9	82.9	S-1a
4	H6	47.0	87.2	S-1a
5	H7	71.4	90.0	S-1a
6	H8	2.2	21.6	R-1a

, where it can be seen that, among all evaluated strains, six carried out the reduction of 1 at different conversion and stereoselectivity grades.

The most promising result at our initial screening was achieved at assay with the strain initially named H4, which was identified through molecular techniques. The phylogenetic tree generated by the Neighbor-Joining approach based on ITS sequences of H4 and related species is shown in Figure S64 (Supplementary Material). The endophyte H4 clustered with sequences of Talaromyces species and was identified as Talaromyces sp. H4. Additionally, the identification of the strain was confirmed by analysis of its morphology (Figure S65 Supplementary Material). Figure 1 depicts the chromatogram of bioreduction of acetophenone (1) by Talaromyces sp. H4. The stereochemistry of the main enantiomer was determined by measuring the specific rotation of the mixture obtained after the bioreduction step and comparing it with the specific rotation value reported in the literature for (R)-1a [19].

2.2. Optimization of the Bioreduction of Acetophenone (1) by Talaromyces sp. H4

Biotransformation parameters exert a critical effect on the performance of any biocatalytic processes, with a fine regulation of these parameters being necessary to identify the optimal working conditions of a biocatalytic system [20]. In this study, our efforts have been devoted to using Talaromyces sp. H4 as a biocatalyst in reducing acetophenone (1) and its derivatives (2–5). Therefore, 1 was chosen as a model substrate to evaluate the kinetic parameters. The first series of bioreductions was carried out using different organic co-solvents for the substrate solubilization, with assays conducted using nine different co-solvents at a final concentration of 50 µL acetophenone/300 µL co-solvent (v/v). As a control, a reaction without co-solvent was carried out. Samples were taken at the end of the reaction and analyzed by chiral GC-FID.

Table 2. Effect of co-solvents in the bioreduction of acetophenone (1) by Talaromyces sp. H4. All reactions led to the formation of S-1a as the main product.

Entry	Co-Solvent	Conversion (%)	ee (%)
1	None	99.4	89.2
2	dimethyl sulfoxide	>99.9	82.9
3	Ethanol	98.5	95.3
4	methanol	99,1	85.2
5	isopropanol	99.4	93.9
6	Glycerol	99.7	92.5
7	Butanol	29.8	67.0
8	cyclohexanol	9.06	>99.9
9	tetrahydrofuran	99.0	91.9
10	Acetone	99.5	80.1

shows the co-solvent effect on the performance of the bioreduction, which had a diverse impact on the percentage conversion and S-1a ee.

Additionally, the influence of six other parameters on the stereoselective bioreduction of 1 by Talaromyces sp. H4 was studied: (i) reaction time (1, 2, 3, 4, and 5 days); (ii) inoculum charge (0.619, 1.186, 1.816, and 2.732 g of mycelium/mmol acetophenone); (iii) shaking speed (80, 120, and 140 rpm); (iv) culture medium pH (5.0, 6.0, 7.0, 8.0, and 9.0); (v) acetophenone concentration (0.22, 0.44, and 0.88 mmol acetophenone/100 mL reaction medium); and (vi) temperature (26, 28, and 30 °C). Samples were taken at the end of the reaction and analyzed by chiral GC-FID.

Table 3. Influence of reaction time, inoculum charge, shaking speed, pH, acetophenone concentration, and temperature on the bioreduction of acetophenone (1) by Talaromyces sp. H4. All reactions led to the formation of S-1a as the main product.

Entry		Conversion (%)	ee (%)
	Time (days)
1	1	11.1	72.2
2	2	18.6	66.9
3	3	>99.9	82.9
4	4	77.0	84.6
5	5	90.0	86.0
	Inoculum charge a
6	0.619	97.9	93.6
7	1.186	99.3	92.5
8	1.816	>99.9	82.9
9	2.732	99.6	84.9
	Shaking speed (rpm)
10	80	98.7	93.1
11	120	>99.9	82.9
12	160	82.3	45.4
	pH
13	5.0	99.6	20.8
14	6.0	98.9	59.6
15	7.0	>99.9	82.9
16	8.0	99.7	87.0
17	9.0	99.5	86.6
	Acetophenone concentration b
18	0.22	78.7	72.6
19	0.44	99.9	82.9
20	0.88	58.2	48.3
	Temperature (°C)
21	26	>99.9	87.6
22	28	>99.9	82.9
23	30	97.1	73.9

^a mycelium (g)/mmol acetophenone; ^b acetophenone (mmol)/100 mL reaction medium.

presents the results achieved by varying the parameters, which had a diverse impact on the percentage conversion and S-1a ee.

2.3. Bioreduction of Substituted Acetophenones by Talaromyces sp. H4

We selected four acetophenone derivatives to evaluate how the structure of the substrate influences the stereochemical course of bioreductions catalyzed by Talaromyces sp. H4. For this, 4′-Nitroacetophenone (2), 4′-Methoxyacetophenone (3), 4′-Chloroacetophenone (4), and 2-chloroacetophenone (5) were chosen to verify the influence of the relative steric volumes of the alkyl and aryl groups linked to the carbonyl acetophenones. The successfully optimized conditions for the bioreduction of acetophenone (1) were used to reduce the four substituted acetophenones (2–5), i.e., 80 rpm speed, 26 °C, three days’ incubation, 0.44 mmol substrate concentration, 1.184 g mycelium/mmol substrate, ethanol as cosolvent, and pH8. The results are shown in

Table 4. Results of the biotransformation of Acetophenone (1), 4′-Nitroacetophenone (2), 4′-Methoxyacetophenone (3), 4′-Chloroacetophenone (4), and 2-Chloroacetophenone (5) into 1-Phenylethanol (1a), 1-(4′-Nitrophenyl)ethanol (2a), 1-(4′-Methoxyphenyl)ethanol (3a), 1-(4′-Chlorophenyl)ethanol (4a), and 1-(2-Chlorophenyl)ethanol (5a), respectively, by Talaromyces sp. H4 under the optimized conditions.

Entry	R1	R2	Conversion (%)	ee (%)	Major product
1	-H	-CH3	97.0	96.0	S-1a
2	-NO2	-CH3	52.2	29.3	S-2a
3	-OCH3	-CH3	40.0	90.9	S-3a
4	-Cl	-CH3	32.3	26.9	S-4a
5	-H	-CH2Cl	11.7	61.4	R-5a a

^a The configuration inversion is due to the priority inversion of the groups linked to the stereogenic center, according to the Cahn–Ingold–Prelog priority rule.

.

2.4. Preparative Scale Bioreduction of Acetophenone (1)

A significant-scale reaction was carried out using the same optimal conditions as determined in our previous studies. Therefore, 516 mg of acetophenone (1) was converted into 382 mg of (S)-1-Phenylethanol (S-1a) in 73% of yield and 96% ee.

3. Discussion

In general, the biotransformation of prochiral ketones by microorganisms follows the Prelog model [21], i.e., enzymes have large and small pockets that form the active site in which the substrate binds and controls the stereoselectivity of the product based on the geometry of the substrate and coenzyme NAD(P)H (Figure 2). Within this context, the stereospecific alcohol dehydrogenases (ADHs) transfer the hydride ion (H⁻) of NAD(P)H to the Re-face of prochiral ketones, and they are usually transformed to (S)-chiral alcohols. However, it should be noted that the R/S definition, according to the Cahn–Ingold–Prelog priority rule, is conversely exhibited by the substituted group containing higher priority atoms at its chiral center, for example, -CH₂Cl. Many ketone-reducing enzymes follow this rule and give Prelog chiral alcohols, such as (S)-1-Phenylethanol from Acetophenone (Table 1, entries 1–5) [22]. In contrast, dehydrogenases/reductases, such as H8 ADHs, in which the hydride ion (H⁻) of NAD(P)H is transferred from the Si-face of ketones and yields anti-Prelog chiral alcohols, such as (R)-1-Phenylethanol from acetophenone, are rare in nature (Table 1, entry 6).

Our results showed that, among all evaluated endophytic strains for reducing Acetophenone (1), only the H4 strain showed very good performance (Table 1, entry 3) with very high conversion percentages (higher than 99%) and good enantiomeric excess (82.9% for the S isomer). Sequencing ITS from H4 led to its identification as Talaromyces sp. H4. Previously, the Talaromyces genus has been assayed for the bioreduction of pro-chiral ketones [23]. Talaromyces flavus was employed to prepare enantioenriched heteroarylethanols and aryl heteroarylmethanols. Asymmetric biocatalytic reduction of corresponding ketones produced the desired heteroaryl S-alcohols in high conversions and enantiomeric excess [24]

Right away, the influence of seven parameters on the stereoselective bioreduction of 1 by Talaromyces sp. H4 was studied. The results are summarized in Table 2. It was found that all conditions led to products with (S)-configuration preference, as predicted by the Prelog model. After analyzing the effect of the co-solvent, two of them (butanol and cyclohexanol; Table 2, entries 7 and 8) had a fully inhibitory effect on the reaction at the concentration used. On the other hand, three co-solvents (DMSO, glycerol, and acetone; Table 2, entries 2, 6, and 10) positively affected the conversion compared with the reaction without co-solvent (Table 2, entry 1). Furthermore, the stereoselectivity improved with ethanol, methanol, isopropanol, glycerol, and tetrahydrofuran (Table 2, entries 3, 4, 5, 6, and 9). Finally, for the pursuit of our studies, ethanol was selected as the best co-solvent considering the high conversion and ee rates, added to the fact that it is the solvent with the lowest environmental impact.

Regarding the time of reaction effect, the best conversion and ee rates were achieved at three and five days, respectively (Table 3, entries 3 and 5). The reductase enzyme of Talaromyces sp. H4 probably has its maximum production at three days after the start of biotransformation, and on the fourth day, some oxidases may also be secreted. Because of this, we selected three days to follow our assays.

The bioreduction reactions were also studied using different fungal cell concentrations related to acetophenone (1.0 mmol). The results showed the variation of the ee percentage with different cell concentrations. Conversion yields were barely affected by the change in inoculum charge. It can be seen that 1 was almost wholly converted and with 92.5% ee in favor of the isomer S-1a (Table 3, entry 7) at the cell concentration of 1.186 g mycelium/mmol substrate.

The effect of three different shaking speeds (80, 120, and 160 rpm) on the reduction efficiency was investigated to select a suitable level. Table 3 shows that the ee increased when the shaking speed was 80 rpm (entry 10). On the other hand, the conversion rate was high (>98.7%) for 80 and 120 rpm (entries 10 and 11). Therefore, the shaking speed of 80 rpm was selected as the best for our bioreduction system.

In order to determine the optimal pH, assays were carried out at 5.0, 6.0, 7.0, 8.0, and 9.0. Table 3 shows that the ee increased from 59.6% to 86.6% when the culture medium pH increased from 6.0 to 8.0 (entries 14 and 16), revealing that the stereoselectivity of the enzyme complex present in Talaromyces sp. H4 is affected by the pH changes. The yield of the conversion was kept constant across pH variations. Considering such results, the optimal pH value for achieving S-1a was 8.0 (99.7% conversion).

Table 3 shows that substrate concentration notably affected the reductive process. Comparing the conversions and ee values obtained with increasing concentration of 1, the best result was achieved when 0.44 mmol of acetophenone was employed (entry 19). The higher load of acetophenone (0.88 mmol—entry 20) inhibits the reaction, probably due to increased toxicity to the microorganism. Therefore, the best substrate concentration for the bioreduction was 0.44 mmol/100 mL of reaction medium.

To determine the optimal temperature for the production of (S)-1a, assays were performed at 26, 28, and 30 °C. Table 3 shows that when the temperature was increased from 26 to 30 °C (entries 21 and 23), the conversion rate decreased, with the yield falling from 99.9% to 97.7%. When the temperature was increased from 26 to 28 °C (entries 21 and 22), the ee decreased from 87.6% to 82.7%. From these results, 26 °C was chosen as the most suitable temperature for the reductive process catalyzed by Talaromyces sp. H4.

To complete our endophyte bioreduction assay, the influence of the chemical structure of the substrate (nature and position of the substituents) on the stereochemical course was evaluated. Table 4 shows different effects on the bioreduction of 1 or its derivatives catalyzed by Talaromyces sp. H4. Any substituent group (donor or acceptor electron groups) had a significant negative impact at both conversion and ee rates. Important observations relating to the relative position of the substituents concerning the carbonyl group can be extracted from Table 4. Substituents at the 2- and 4-positions had a negative effect on the percentage reduction compared to Acetophenone (1), with slightly lower conversion observed with 2-Chloroacetophenone (5a) (Table 4, entry 5), compared to 4′-Nitroacetophenone (2a) (Table 2, entry 2). Assays with other α- substituted acetophenone are necessary to clarify this bioreduction result. Previously, no steric hindrance had been shown in the bioreduction of α-bromoacetophenone, p-bromo-α-bromoacetophenone, and p-nitro-α-bromoacetophenone by whole cells of the marine fungus Aspergillus sydowii Ce19 [25].

In addition, it is interesting to note that the low enantiomeric excesses observed in assays with substituted acetophenones can be attributed to enzymes with opposing stereoselectivity (pro-R or pro-S) operating simultaneously and competitively. It is possible to obtain a difference in their affinity depending on the spatial conformation in which the carbonyl group is located with respect to the substituent [26]. Several enzymes may be present in Talaromyces sp. H4, and this might explain the different enantioselectivities.

4. Material and Methods

4.1. General Procedures and Chromatographic Analysis

Acetophenone and substituted acetophenones were purchased from Sigma-Aldrich (St. Louis, MO, USA). NMR spectra were recorded at 500 MHz with a Varian Inova spectrometer. Chemical shifts (δ) were referenced to the residual deuterated methanol (CD₃OD) or deuterated chloroform (CDCl₃) peak at δ_H 7.27 for ¹H. Optical rotations were measured in a PerkinElmer (Wellesley, MA, USA) Model 343 polarimeter using a sodium D-line. Infrared (IR) spectra were recorded in KBr on Shimadzu FT-IR IRAffinity-1 (Kyoto, Japan). The bands were assigned in cm⁻¹. The biotransformation products were identified by comparing their optical rotations, ¹H NMR, and IR spectra with literature data.

To establish the chromatographic analytical method, acetophenones were reduced with sodium borohydride (Sigma-Aldrich) in methanol (Synth, São Paulo, Brazil) to obtain a racemic mixture of the 1-phenylethanol. The crude reactions were extracted with ethyl acetate, the organic solutions evaporated, and the products were purified using a column with silica gel (70–230 mesh) using hexane-ethyl acetate 80:20 as the eluent. All chemical structures of products have been confirmed through NMR and IR data analysis, along with a comparison with reported data.

Gas chromatography (GC) analyses were carried out on Agilent Technologies (Waldbronn, Germany), model 7820A, with a flame ionization detector (FID). The GC separations were performed on a chiral stationary phase Cyclosyl B 30% heptakis(2,3-di-O-methyl-6-O-tert-butyl-dimethylsilicon)-β-cyclodextrin (28.5 m, 0.25 mm, 0.25 μm) with GC general conditions of split, 18.113 mL min⁻¹; injector, 220 °C; detector FID, 220 °C; carrier gas, N₂ (25 mL min⁻¹); and head pressure, 11.161 psi. The conversion percentages of each biotransformation were determined by using normalized peak areas without a correction factor. The identities of the peaks in the chromatogram were obtained using acetophenone (1) and (R/S)-1-Phenyl ethanol (1a) as a reference standard: GC Rt Acetophenone (1): 12.01 min, Rt (R)-1-Phenylethanol (R-1a): 18.21 min, and Rt (S)-1-Phenyl ethanol (S-1a): 18.70 min.

4.2. Screening for Stereoselective Bioreduction of Acetophenone by Endophytic Fungi

Seventy-two endophytic fungi (coded as H1-H72) were isolated from aerial parts of Handroanthus impetiginosus (Mart. Ex DC.) Mattos, and the endophytic fungi with outstanding features in the biotransformation screening were identified by DNA sequencing, as previously described by our research group [27,28].

For the initial biotransformation screening, endophytic strains were cultured in Petri dishes containing potato dextrose agar (PDA, Kasvi, Curitiba, Brazil) at 28 °C for seven days. The resulting cultures were used in the biotransformation assays. To this end, an initial inoculum of fifteen 6 mm disks containing mycelia and agar (780 mg) was added to 250 mL Erlenmeyer flasks holding 100 mL of a fermentative medium consisting of 0.18% glucose (Synth), 0.06% peptone (Merck, Darmstadt, Germany), and 0.04% yeast extract (Acumedia, Baltimore, MD, USA), pH 6.5. Then, acetophenone (50 μL—0.43 mmol) was added to the Erlenmeyer flask as a solution in 300 μL dimethyl sulfoxide (DMSO, Synth), used as a co-solvent. Three controls were used. (i) One consisted of the culture medium, DMSO, and fungus, with no substrate; (ii) another control consisted of the culture medium and substrate but no fungus; and (iii) the third control consisted of culture medium only. The biotransformation experiments were carried out at 28 °C for three days under stirring at 120 rpm (Tecnal TE-420, Piracicaba, São Paulo, Brazil). All the experiments were performed in triplicate. Samples were taken daily; the mycelia were separated by filtration, and the fermentation broths were extracted with ethyl acetate (Synth). The solvent was evaporated under reduced pressure to yield crude extracts, which were analyzed by chiral GC–FID at the concentration of 1 mg mL⁻¹.

Acetophenone (1). GC conditions: T₀ = 80 °C (5 min), ΔT₁ = 2 °C min⁻¹, T₁ = 100 °C, ΔT₂ = 5 °C min⁻¹, T₂ = 220 °C. GC Rt acetophenone (1): 12.01 min.

¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.47 (s, 3H, CH₃), 7.00–7.05 (m, 3H, ArH), 7.31–7.35 (m, 2H, ArH). IR (cm⁻¹) 3062.96 (C-H), 1681.93 (C=O), 759.95 and 586.36 (Ar_C-H)

1-Phenylethanol (1a). GC conditions: T₀ = 80 °C (5 min), ΔT₁ = 2 °C min⁻¹, T₁ = 100 °C, ΔT₂ = 5 °C min⁻¹, T₂ = 220 °C. GC Rt (+)-(R)-1-phenylethanol (R-1a): 18.21 min, and Rt (-)-(S)-1-phenylethanol (S-1a): 18.70 min. Unpurified product optical rotation: α = −17.0° (MeOH), ([α]²⁵_D = +44.5 (c 1.0, MeOH) for R enantiomer [19]). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.47 (d, 3H, CH₃, J = 6.4 Hz), 2.70 (s, 1H, OH), 4.84 (q, 1H, CH, J = 6.4 Hz), 7.30–7.37 (m, 5H, ArH) [29]. IR (cm⁻¹) 3360.0 (O-H), 2877.59 (C-H), 1099.43 (C-O), 2974.23 (C-H), 759.95 and 698.23 (ArC-H) [29].

4.3. Optimization of the Methodology for Stereoselective Reduction of Acetophenone

The influence of seven parameters on the stereoselective bioreduction of acetophenone by Talaromyces sp. H4 was studied: (i) reaction time (1, 2, 3, 4, and 5 days); (ii) inoculum charge (0.619, 1.186, 1.816, and 2.732 g of mycelium/mmol acetophenone); (iii) shaking speed (80, 120, and 140 rpm); (iv) culture medium pH (5.0, 6.0, 7.0, 8.0, and 9.0); (v) acetophenone concentration (0.22, 0.44, and 0.88 mmol acetophenone/100 mL reaction medium); (vi) temperature (26, 28, and 30 °C); and (vii) co-solvent (ethanol, methanol, butanol, cyclohexanol, glycerol, dimethylsulfoxide, isopropanol, tetrahydrofuran, and acetone). The results are shown as an average of triplicates.

4.4. Bioreduction of Substituted Acetophenones by Talaromyces sp. H4

The optimal conditions selected in the bioreduction study of Acetophenone (1) were used to reduce substituted acetophenones: 4′-Nitroacetophenone (2), 4′-Methoxyacetophenone (3), 4′-Chloroacetophenone (4), and 2-Chloroacetophenone (5). Three Erlenmeyer flasks holding 100 mL of a fermentative medium have been preparate for each substituted acetophenone. Assays were carried out with fifteen 6 mm disks containing mycelia and agar (510 mg), substrate (0.43 mmol) dissolved in 350 μL ethanol, at 28 °C for three days under stirring at 120 rpm (Tecnal TE-420). Then, the solutions were gathered, and the product was extracted with ethyl acetate. The pure extract was analyzed by chiral GC-FID at the concentration of 1 mg mL⁻¹

1-(4′-Nitrophenyl)ethanol (2a). GC conditions: T₀ = 80 °C (5 min), ΔT₁ = 1 °C min⁻¹, T₁ = 185 °C. GC Rt 4′-Nitroacetophenone (2): 63.84 min, Rt (+)-(R)-1-(4-Nitrophenyl)etanol (R-2a): 85.34 min, and Rt (-)-(S)-1-(4′-Nitrophenyl)ethanol (S-2a): 86.26 min. Unpurified product optical rotation: α = −9.0° (MeOH), ([α]²¹_D = +31.0 (c 1.23, MeOH) for R enantiomer [30]). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.45 (d, 3H, CH₃; J = 6.4 Hz), 3.20 (s, 1H, OH), 4.95 (q, 1H, CH; J = 6.4 Hz), 7.47 (d, 2H, ArH; 8.7 Hz), 8.08 (d, 2H, ArH; J = 8.7 Hz) [31]. IR (cm⁻¹) 3390.86 (OH), 2870.08 (C-H), 1109.07 (C-O), 854.47 (Ar_C-H), 1519.91 and 1346.31 (N-O_NO2) [31].

1-(4′-Methoxyphenyl)ethanol (3a). GC conditions: T₀ = 80 °C (5 min), ΔT₁ = 5 °C min⁻¹, T₁ = 100 °C, ΔT₂ = 0.5 °C min⁻¹, T₂ = 130 °C, ΔT₃ = 45 °C min⁻¹, T₃ = 220 °C. GC Rt 4′-Methoxyacetophenone (3): 33.85 min, Rt (+)-(R)-1-(4′-Methoxyphenyl)ethanol (R-3a): 39.39 min, and Rt (-)-(S)-1-(4′-Methoxyphenyl)ethanol (S-3a): 40.15 min. Unpurified product optical rotation: α = −7.0° (MeOH), ([α]²¹_D = −51.9 (c 0.72, MeOH) for S enantiomer [32]). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.47 (d, 3H, CH₃; J = 6.4 Hz), 2.28 (s, 1H, OH), 3.80 (s, 3H, OCH₃), 4.83 (q, 1H, CH; J = 6.4 Hz), 6.88 (d, 2H, ArH; J = 8.7 Hz), 7.30 (d, 2H, ArH; J = 8.7 Hz) [33]. IR (cm⁻¹) 3360.00 (O-H), 2835.00 (C-H), 1246.02 and 1033.85 (C-O_ether), 1087.75 (C-OH), 833.25 (Ar_C-H) [20]

1-(4′-Chlorophenyl)ethanol (4a). GC conditions: T₀ = 80 °C (5 min), ΔT₁ = 5 °C min⁻¹, T₁ = 100 °C, ΔT₂ = 0.5 °C min⁻¹, T₂ = 130 °C, ΔT₃ = 45 °C min⁻¹, T₃ = 220 °C. GC Rt 4′-Chloroacetophenone (4): 20.64 min, Rt (+)-(R)-1-(4′-Chlorophenyl)ethanol (R-4a): 37.77 min and Rt (-)-(S)-1-(4′-Chlorophenyl)ethanol (S-4a): 39.54 min. Unpurified product optical rotation: α = −15.2° (MeOH), ([α]²⁵_D = + 39.8 (c 1.06, MeOH) for R enantiomer [30]). ¹H NMR (500 MHz, CDCl₃) δ (ppm) 1.46 (d, 3H, CH₃, J = 6.4 Hz), 2.26 (s, 1H, OH), 4.85 (q, 1H, CH, J = 6.4 Hz), 7.30–7.35 (m, 5H, ArH) [20]. IR (cm⁻¹) 3348.42 (O-H), 2885.51 (C-H), 1102.36 (C-O), 829.39 (ArC-H) [20]

2-Chloro-1-phenylethanol (5a). GC conditions: T₀ = 80 °C (2 min), ΔT₁ = 2 °C min⁻¹, T₁ = 100 °C, ΔT₂ = 2 °C min⁻¹, T₂ = 220 °C. GC Rt 2-Chloroacetophenone (5): 23.87 min, Rt (-)-(S)-2-Chloro-1-phenylethanol (S-5a): 25.86 min and Rt (+)-(R)-2-Chloro-1-phenylethanol (R-5a): 26.11 min. Unpurified product optical rotation: α = −11.5° (cyclohexane), ([α]²⁵ _D = +49.6 (c 2.8, cyclohexane) for S enantiomer [34]). ¹H NMR (500 MHz, CDCl₃) δ (ppm) = 2.66 (s, 1H, OH), 3.66 (dd, 1H, CH₂, J = 8.7 and 11.2 Hz), 3.74 (dd, 1H, CH₂, J = 3.5 and 11.2 Hz), 4.90 (dd, 1H, CH, J = 3.5 and 8.7 Hz), 7.33–7.40 (m, 5H, ArH) [35]. IR (cm⁻¹) 3390.86 (O-H), 1454.33 (C-H (CH₂)), 1104.71 (C-O), 767.67 and 698.38 ((ArC-H), 613.36 (C-Cl) [35].

4.5. Scale-Up of Biotransformation of Acetophenone by Talaromyces sp. H4

The ideal conditions for bioreduction employing Talaromyces sp. H4 were used to scale up. Acetophenone (1) was chosen as a substrate for scaling up due to the good results already obtained. This experiment was carried out using 10 Erlenmeyer flasks. In each Erlenmeyer flask holding 100 mL of a fermentative medium, an initial inoculum of fifteen 6 mm disks containing mycelia and agar (510 mg) was added. Then, acetophenone (50 μL—0.43 mmol) was added to each Erlenmeyer flask dissolved in 350 μL ethanol. The biotransformation experiments were carried out at 28 °C for three days under stirring at 120 rpm (Tecnal TE-420). Then, the solutions were gathered, and the product was extracted with ethyl acetate and purified using a column with silica gel (70–230 mesh) using hexane-ethyl acetate 80:20 as the eluent. The solvent was evaporated under reduced pressure to yield 0.3822g of S-1a in 73% yield and 96% ee. The pure extract was analyzed by chiral GC-FID at the concentration of 1 mg mL⁻¹ (Figure S63, Supplementary Information).

S-1a experimental optical rotation: [α]_D ²⁵ = −37.5° (c 1.0, MeOH); R-1a literature optical rotation: [α]_D ²⁵ = +44.5° (c 1.0, MeOH), 98.4% ee, [19].

5. Conclusions

The broad enzymatic potential of fungi has guaranteed them a key role in biotechnology studies focused on developing new selective chemical transformations. The results presented in this paper demonstrated our preliminary screening results of endophytic fungi isolated from aerial parts of Handroanthus impetiginosus (Mart. ex DC.) Mattos. Talaromyces sp. H4 displayed high enantioselective reductase activity for producing (S)-1-Phenylethanol (S-1a).

Endophyte community composition significantly differs between distinct geographic areas. Specific environmental characteristics may act as ecological filters in selecting the endophytic strains that are better adapted to local conditions. Studies on endophytes from a particular geographic area contribute significantly to the planning of forthcoming scientific studies and also to the species preservation of natural occurrence in that area. The present study employed endophytic fungi isolated from typical vegetation from the southeast region of Brazil. This clearly showed the emergence of an opportunity to establish new applications for Brazilian biodiversity.

From our assays, we selected the endophytic strain Talaromyces sp. H4 as a biocatalyst that provides new strategies for reducing prochiral ketones in organic synthesis, thus avoiding the use of expensive and non-renewable reducing metal-based agents. We carefully evaluated the influence of seven parameters (reaction time, inoculum charge, shaking speed, culture medium pH, acetophenone concentration, temperature, and co-solvent) in the stereoselective bioreduction of acetophenone and compared with achieved results with substituted acetophenones. Our final bioreduction (using the optimal determined conditions) reached the conversion of acetophenone into (S)-1-Phenylethanol at 73% of yield and 96% ee.

Although traditional methods for the asymmetric synthesis of chiral alcohols are widely reported, biocatalytic routes remain extremely attractive due to their selectivity and environmentally friendly approach.