Introduction

Stereospecific reduction of a carbonyl group leading to enantiomerically pure alcohols is catalyzed by both the isolated enzymes and whole-cell systems of yeasts, filamentous fungi or plants (Goldberg et al. 2007a, b; Homann et al. 2004; Mączka and Mironowicz 2004). The majority of bioreductions are catalyzed by dehydrogenases that follow Prelog’s rule delivering the hydride ion from the re-face of a prochiral ketone which, in most cases, leads to (S)-alcohols. Bioreductions have a wide application in the production of chiral intermediates or final products used in pharmacy or agrochemistry (Rodrigues et al. 2004; Fardelone et al. 2011). Among them, hydroxylactones represent one of the most common group of biologically active compounds with cytotoxic, antifeedant and antifungal properties (Murcia et al. 2010; Gliszczyńska et al. 2011; Grudniewska et al. 2015). They can be easily accessed from the corresponding oxolactones by the reduction of a carbonyl group (Ribeiro et al. 2006, 2008).

The synthesis of β-secretase inhibitors that may be used in the therapy of Alzheimer’s disease has been presented (Sussman et al. 2011). The starting material was D-glucono-1,5-lactone which was transformed to the key chiral intermediate hydroxylactone, I. The latter was treated with methanesulfonylchloride to afford corresponding mesylate which was subsequently reacted with sodium azide. Stereoselective alkylation of lactone ring at C-2 with lithium diisopropylamide followed by treating with methyl iodide yielded azidolactone, II, (Scheme 1).

Scheme 1
scheme 1

Fragment of the synthesis of β-secretase inhibitors (Sussman et al. 2011) and hydroxylactone 3c as an alternative intermediate in the synthetic pathway

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In this paper we propose a modification of this synthetic pathway in which the intermediate is chiral α-methylhydroxylactone, 3c, obtained by yeast-mediated reduction of α-methyl-γ-(1-oxo-3-methylbutyl)-γ-butyrolactone (4). This approach was inspired by the results of Ribeiro et al. (2006, 2008). They used different fungal and yeast strains (Aspergillus niger, Geotrichum candidum and Hansenula sp.) for the stereospecific reduction of the carbonyl group in α-acetyl-γ-butyrolactones to produce chiral hydroxylactones used, that is as GHB receptor ligands.

Materials and methods

Chemical and biological materials

Racemic 5-methylhex-1-en-2-ol (1) was obtained by Chojnacka et al. (2007) as the product of reaction of acrolein with isobutyl magnesium bromide. Triethyl orthopropionate (97 %) and m-chloroperbenzoic acid (m-CPBA, 77 %) were purchased from Sigma-Aldrich. Other chemicals and reagents were of analytical grade. Yeast strains: Rhodotorula glutinis KCh 242, Rhodotorula rubra KCh 82, Rhodotorula marina KCh 77 and Saccharomyces cerevisiae KCh 464 came from the collection of the Department of Chemistry of Wrocław University of Environmental and Life Sciences. The strains were cultivated on a Sabouraud agar consisting of 5 g Aaminobac l−1, 5 g peptone K l−1, 40 g glucose l−1, and 15 g agar l−1 at 28 °C, pH 5.5.

Analysis

Gas chromatography was carried out using HP-5 column (cross-linked methyl silicone gum, 30 m × 0.32 mm × 0.25 μm) and an FID detector. The enantiomeric compositions of bioreduction products were determined on a Varian CP Chirasil-DEX CB column (25 m × 0.25 mm × 0.25 μm) using the program: injector 250 °C, detector (FID) 280 °C, column: 80–150 °C (25 °C min−1), 150 °C (25 min hold), 150–200 °C (30 °C min−1), 200 °C (2 min hold). Spectroscopic measurements were carried out for CDCl3 solutions on a Bruker Avance AMX 300 spectrometer. IR spectra were determined using Mattson IR 300 Thermo Nicolet spectrophotometer. Optical rotations were measured in CH2Cl2 on a Jasco P-2000-Na digital polarimeter with intelligent Remote Module (iRM) controller.

Synthesis of racemic (E)-ethyl 2,7-dimethylocta-4-enoate (2)

The alcohol 1 (2.1 g, 18 mmol) was subjected to the reaction with triethyl orthopropionate according to the standard protocol of Johnson–Claisen rearrangement. The crude product was purified by silica gel column chromatography (hexane/acetone, 19:1, v/v) to give ester 2 (3.3 g, 90 % isolated yield): 1H NMR δ: 0.85 (d, J = 6.6 Hz, 6H, (CH 3)2CH–), 1.13 (d, J = 7.0 Hz, 3H, CH3-2), 1.24 (t, J = 7.1 Hz, 3H, –OCH2CH 3), 1.57 (m, 1H, (CH3)2CH–), 1.86 (m, 2H, CH2-6), 2.12, 2.34 (two dt, J = 13.9, 7.0 Hz, 2H, CH2-3), 2.46 (m, H-2), 4.11 (q, J = 7.1 Hz, 2H, –OCH 2CH3), 5.32 (dt, J = 15.2, 7.0 Hz, 1H, H-4), 5.44 (dt, J = 15.2, 7.0 Hz, 1H, H-5), 13C NMR δ: 14.24 (–OCH2 CH3), 16.50 (CH3-2), 22.20 ((CH3)2CH–), 28.34 ((CH3)2 CH–), 36.76 (C-3), 39.78 (C-2), 41.91 (C-6), 60.11 (–OCH2CH3), 127.82 (C-4), 131.74 (C-5), 176.31 (C-1), IR (film, cm−1): 1737, 1453, 1375, 1177, 970.

Synthesis of hydroxylactones 3a,b

The ester 2 (3 g, 15 mmol) was epoxidized by m-CPBA in a standard manner. The resulting crude mixture of epoxyesters was stirred in the solution of THF/H2O/HClO4 (10:5:0.5 by vol.) for 72 h. The products were extracted with diethyl ether and the extracts were washed with NaHCO3, brine and dried. Pure lactones 3a and 3b were isolated by column chromatography (hexane/acetone/ethyl acetate/2-propanol/methylene chloride/diethyl ether, 45:0.2:0.2:3:0.2:0.2 by vol.).

(±)-cis-5-(1′-Hydroxy-3′-methylbutyl)-3-methyldihydrofuran-2-one (3a)

Yield 1.33 g (45 %), 1H NMR δ: 0.93, 0.96 (two d, J = 6.6 Hz, 6H, (CH 3)2CH–), 1.16 (ddd, J = 13.8, 9.0, 3.3 Hz, 1H, one of CH2-2′), 1.27 (d, J = 7.2 Hz, 3H, CH3-3), 1.37 (ddd, J = 13.8, 9.9, 5.1 Hz, 1H, one of CH2-2′), 1.80-1.87 (m, 2H, (CH3)2CH– and OH), 1.93 (td, J = 12.0, 10.2 Hz, 1H, one of CH2-4), 2.28 (ddd, J = 12.0, 9.0, 6.0 Hz, 1H, one of CH2-4), 2.69 (ddq, J = 12.0, 9.0, 7.2 Hz, 1H, H-3), 4.05 (dt, J = 9.9, 3.3 Hz, 1H, H-1′), 4.28 (ddd, J = 10.2, 6.0, 3.3 Hz, 1H, H-5), 13C NMR δ: 15.03 (CH3-3), 21.76, 23.46 (CH3)2CH–), 24.38 ((CH3)2 CH–), 29.51 (C-4), 35.46 (C-3), 40.32 (C-2′), 68.37 (C-1′), 80.81 (C-5), 179.47 (C-2), IR (film, cm−1): 3448, 1768, 1456, 1379, 1201, 1017.

(±)-trans-5-(1′-Hydroxy-3′-methylbutyl)-3-methyldihydrofuran-2-one (3b)

Yield 1.48 g (53 %), 1H NMR δ: 0.92, 0.96 (two d, J = 6.6 Hz, 6H, (CH 3 )2CH–), 1.16 (ddd, J = 13.8, 9.0, 3.3 Hz, 1H, one of CH2-2′), 1.27 (d, J = 7.8 Hz, 3H, CH3-3), 1.39 (ddd, J = 13.8, 10.2, 5.4 Hz, 1H, one of CH2-2′), 1.77–1.84 (m, 2H, (CH3)2CH– and one of CH2-4), 2.19 (s, 1H, –OH), 2.48 (ddd, J = 12.6, 9.6, 4.8 Hz, 1H, one of CH2-4), 2.80 (dq, J = 9.6, 7.8 Hz, 1H, H-3), 3.99 (dt, J = 10.2, 3.3 Hz, 1H, H-1′), 4.37 (ddd, J = 8.4, 4.8, 3.3 Hz, 1H, H-5), 13C NMR δ: 16.48 (CH3-3), 21.76, 23.48 (CH3)2CH–), 24.42 ((CH3)2 CH–), 29.36 (C-4), 34.54 (C-3), 40.94 (C-2′), 69.87 (C-1′), 80.92 (C-5), 180.61 (C-2), IR (film, cm−1): 3451 (s), 1765, 1456, 1381, 1204, 1014.

Oxidation of hydroxylactone 3b

Following a standard procedure, hydroxylactone 3b (1.4 g, 7.5 mmol) was oxidized by a solution of Na2Cr2O7 (1.96 g, 7.5 mmol) in H2SO4 to afford 1.27 g (92 % yield) of (±)-trans-5-(1′-oxo-3′-methylbutyl)-3-methyldihydrofuran-2-one (4): 1H NMR δ: 0.92, 0.93 (two d, J = 6.6 Hz, 6H, (CH 3)2CH–), 1.28 (d, J = 6.9 Hz, 3H, CH3-3), 2.04–2.24 (two m, 2H, one of CH2-4 and (CH3)2CH–), 2.40 (dd, J = 17.1, 6.6 Hz, 1H, one of CH2-2′), 2.50 (dd, J = 17.1, 6.9 Hz, 1H, one of CH2-2′), 2.49-2.62 (m, one of CH2-4 and H-3), 4.77 (dd, J = 9.3, 3.3 Hz, 1H, H-5), 13C NMR δ: 15.43 (CH3-3), 22.45 ((CH3)2CH–), 23.86 ((CH 3 )2 CH–), 32.49 (C-4), 32.93 (C-3), 47.77 (C-2′), 79.73 (C-5), 178.81 (C-2), 207.30 (C-1′), IR (film, cm−1): 1779, 1722, 1456, 1370, 1269, 1162, 1026.

Isolation of products obtained after biotransformation of oxolactone 4 catalyzed by R. glutinis KCh 242

After 7 days of transformation (Scheme 3) biomass was centrifuged and the products were extracted from the liquid medium with CH2Cl2 and separated by column chromatography (hexane/acetone/ethyl acetate/2-propanol/methylene chloride/diethyl ether, 60:0.2:0.2:3:0.2:0.2 by vol.) to afford pure products.

(−)-trans-(3R,5S,1′S)-5-(1′-Hydroxy-3′-methylbutyl)-3-methyldihydrofuran-2-one (3c)

Yield 0.069 g (43 %), e.e. = 48 %, \( [\alpha ]_{D}^{20} \) = −2.4 (c 3.9, CH2Cl2); 1H NMR δ: 0.92, 0.95 (two d, J = 6.9 Hz, 6H, (CH 3)2CH–), 1.24 (m, 1H, one of CH2-2′), 1.28 (d, J = 7.5 Hz, 3H, CH3-3), 1.54 (ddd, J = 13.8, 9.6, 4.8 Hz, 1H, one of CH2-2′), 1.77–1.88 (two m, 2H, (CH3)2CH– and OH), 1.95 (ddd, J = 12.9, 9.3, 7.5 Hz, 1H, one of CH2-4), 2.33 (ddd, J = 12.9, 9.3, 4.8 Hz, 1H, one of CH2-4), 2.80 (dq, J = 9.3, 7.5 Hz, 1H, H-3), 3.65 (dt, J = 9.6, 4.8 Hz, 1H, H-1′), 4.36 (dt, J = 9.3, 4.8 Hz, 1H, H-5), 13C NMR δ: 15.03 (CH3-3), 21.76, 23.46 (CH3)2CH–), 24.38 ((CH3)2 CH–), 29.51 (C-4), 35.46 (C-3), 40.32 (C-2′), 68.37 (C-1′), 80.81 (C-5), 179.47 (C-2), IR (film, cm−1): 3448, 1768, 1456, 1379, 1201, 1017.

(−)-trans-(3S,5R,1′S)-5-(1′-Hydroxy-3′-methylbutyl)-3-methyldihydrofuran-2-one (3d)

Yield 0.085 g (52 %), e.e. = 90 %, \( [\alpha ]_{D}^{20} \) = −6.2 (c 2.6, CH2Cl2); spectroscopic data of in accordance with those of racemic lactone 3b.

Results and discussion

The synthetic pathway leading to opically active isomers of trans-5-(1′-hydroxy-3′-methylbutyl)-3-methyldihydrofuran-2-one (Scheme 2) started with the Johnson–Claisen rearrangement of racemic allyl alcohol 1 to γ,δ-unsaturated ester 2 in the reaction of 1 with triethyl orthopropionate. Ester 2 was further transformed in two-step procedure into the mixture of diastereoisomeric hydroxylactones 3a and 3b involving epoxidation of double bond with m-CPBA followed by acidic lactonization. Isomer trans (3b) was separated from the products mixture by column chromatography and oxidized to the corresponding oxolactone 4 with sodium dichromate.

Scheme 2
scheme 2

Reagents: (a) EtC(OEt)3, H+, 140 °C (b) m-CPBA, CH2Cl2, rt, 24 h (c) THF/H2O/HClO4, rt, 72 h (d) Na2Cr2O7/H2SO4, Et2O, rt, 48 h

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Lactone 4 was subjected to biotransformation using four yeast strains from our culture collection. They are known for their ability to reduce chalcones, prophiophenones and flavanones (Janeczko et al. 2013, 2014; Janeczko and Kostrzewa-Susłow 2014). Two diastereoisomeric hydroxylactones (3c, 3d) were formed as a result of the reduction of the carbonyl group at C-1′. The substrate conversion and a composition of the products mixture depended on a strain. R. marina KCh 77 the reduced substrate only to the isomer 3d which started to accumulate after 4 days to reach 52 % on the 10th day (Fig. 1c). S. cerevisiae KCh 464 preferred the reduction of opposite enantiomer of substrate and after 10 days the conversion reached 30 % and isomer 3c made up 25 % of the products mixture (Fig. 1d). In the experiments with R. rubra KCh 82 and R. glutinis KCh 242 (Fig. 1a, b), the complete conversions of substrate 4 were achieved after 10 days. In both cases until 4th day isomer 3c predominated but in the case of R. glutinis KCh 242 from that time the opposite enantiomer of substrate was reduced faster and in 7th day of transformation hydroxylactone 3d was the major product (Fig. 1a). For R. rubra KCh 82 the tendency was maintained and after 10 days hydroxylactone 3c significantly prevailed over isomer 3d (78 vs. 22 %) (Fig. 1b).

Fig. 1
figure 1

Time course of biotransformation of oxolactone 4 in yeast cultures under the following conditions: Yeasts were cultivated for 3 days with shaking (150 rpm) at 25 °C in 50 ml medium (glucose 30 g l−1, peptone 10 g l−1), followed by addition of lactone 4 (in acetone 10 mg ml−1). For the analysis of reaction progress the samples were extracted with CH2Cl2, the extracts were dried over MgSO4, concentrated and analyzed by GC

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Since biotransformation of oxolactone 4 with R. glutinis KCh 242 afforded both hydroxylactones 3c and 3d in significant yields, in this experiment they were isolated, purified and identified (Scheme 3).

Scheme 3
scheme 3

Bioreduction of oxolactone 4 by R.glutinis KCh 242: substrate 160 mg, 25 °C, 150 rpm, 7 days

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Spectroscopic measurements confirmed the S configuration at C-1′ in both products as a result of stereospecific reduction of a carbonyl group following the Prelog’s rule. The same stereochemical preference by the strains of R. glutinis has been demonstrated in the course of reduction of different phenones (Homann et al. 2004; Zilbeyaz and Kurbanoglu 2010) and also during the reduction of propiophenone and dihydrochalcone catalyzed by R. glutinis KCh 242 studied herein (Janeczko et al. 2013; Janeczko and Kostrzewa-Susłow 2014). On the contrary, formation of two diastereoisomers (3c, 3d) was the result of different enantioselectivity of strains towards the substrate. Enantiomer 3R, 5S was reduced faster by R. rubra KCh 82 and S. cerevisiae KCh 464 to afford (3R,5S,1′S)-hydroxylactone 3c. R. marina KCh 77 and R. glutinis 242 favoured the reduction of enantiomer.

3S, 5R and (3S,5R,1′S)-hydroxylactone 3d as was the only or the major isomer produced (Fig. 1a c, respectively).

The presented chemoenzymatic strategy is a useful way to produce chiral hydroxylactones as key intermediates for the synthesis of β-secretase inhibitors. The application of orthopropionate in the Johnson–Claisen rearrangement of 1 avoids the methylation step in synthetic pathway presented in Scheme 1. The stereospecificity of yeast dehydrogenases and different enantioselectivity of reduction observed for particular strains expands the scope of synthesized stereoisomers. The starting materials, easily accessed by Grignard reaction, may be also the analogs of 5-methylhex-1-en-2-ol with various alkyl substituent at the hydroxy group which makes the presented methodology a general route to introduce a variety of alkyl side chain groups at P1/P1′ fragment of peptides which may be studied as potential β-secretase inhibitors.