Abstract

Asymmetric reduction of the heteroaryl prochiral ketones to corresponding chiral alcohols by Daucus carota was studied. The study highlights selective bioreduction of different substituted heteroaryl ketones (1a - 1j) to their respective chiral alcohols (2a - 2j) using plant dehydrogenase enzymes present in Daucus carota in good yields (60% - 95%) and enantioselectivity (76% - 99%) with S-form configuration. The results obtained confirm that the membrane bound dehydrogenase enzyme has broad substrate specificity and selectivity in catalyzing both six and five membered heteroaryl methyl ketones. The present methodology demonstrates promising and alternative green route in the synthesis secondary chiral alcohols of biologically importance in a simple, inexpensive and eco-friendly process.

1. Introduction

Aromatic and aliphatic heterocyclic compounds are frequent structural motifs in nature’s molecules and in man–made chemical active substances such as pharmaceuticals and agrochemicals. Asymmetric reduction of prochiral ketones is one of the most important, fundamental and practical reactions for producing chiral alcohols. Enantiomerically pure secondary alcohols are important synthons/intermediates for the synthesis of numerous pharmaceuticals, agrochemicals, flavors, fragrances and industrial fine chemicals [1-3]. There are number of chemical and biological methodologies available to obtain chiral molecules; of these biocatalysts’ has proven to be useful supplementary technology, allowing in some cases reactions which are not easily conducted by classical organic synthesis or in other cases reactions which take several chemical steps, can be carried out in a single step using biocatalyst under highly selective and mild conditions. Thus the biocatalytic process (both whole cells and isolated enzymes) continues to remain an area of intensive research, with a desire to develop alternative green routes to the synthesis of fine chemicals [4-6]. Heterocyclic aromatic compounds containing nitrogen, oxygen or sulfur in the heterocyclic ring are important core groups found in natural and synthetic products of biological interest. Acetyl-pyridines (2 - 4) are known as aromatic components of foods, perfumes and smoking suppressants. Chiral heteroaryl alcohols have numerous applications as important intermediates in the synthesis of biological active molecules and also act as chiral ligands/auxiliaries in a number of asymmetric addition reactions [7-9]. There are many reports describing synthesis of chiral secondary alcohols using biocatalysts obtained from microbial/different parts of plant tissues [10-12].The asymmetric reductions of heteroaryl methyl ketones is a straight forward approach and a large number of chemical and biological methodologies (microbial and plant) are known to produce heterocyclic chiral alcohols of biological interest [13-15]. However, most of these processes have limitation in commercial application due to long incubation time, low substrate loading, poor isolated yields and enantioselectivity [16,17]. In continuation of our interest towards synthesis of chiral alcohols of biological importance using biocatalysts [18,19], we here in present, the process for production of chiral aromatic heterocyclic secondary alcohols by bioreduction of substituted heteroaryl ketones using D.carota.

2. Results and Discussion

Chiral reduction of substituted heteroaryl prochiral ketones (1a - 1j) Scheme 1 were studied using D carota. (carrot). The results obtained Table 1, high lights enan-

tioselective synthesis of heterocyclic secondary alcohols (2a - 2j) using D.carota (carrot). It was demonstrated that the enzyme dehydrogenases present in the D.carota selectively reduced substituted aromatic heterocyclic methyl ketones (1a - 1j) to the corresponding single chiral secondary alcohols (2a - 2j) in good isolated yields and high enantioselectivity (only single isomer was obtained), as conformed through HPLC using chiral column (no second isomer was present in the reaction medium, the un-reacted starting compound was recovered). It was observed that the enzymes responsible for chiral reduction of both six and five membered heteroaryl ketones (1a - 1j), has shown broad enzyme specificity and enantioselectivity. The results also highlights the enzyme dehydrogenase enantioselectivity towards six member heterocyclic N-containing aromatic compounds, when compared to five membered aromatic heterocyclic compounds containing “N”, “O” and “S” atoms. It is well known that the N-atom present in the heterocyclic aromatic ring increased the affinity of the substrate towards the enzyme when compared to other three compounds (1h - 1j), which show low selectivity and poor yields, this may be due to steric binding factors of the compounds towards enzyme active sight. [20,21]. When compared to earlier reports [22-25], the present study demonstrates that the dehydrogenase enzymes present in D.carota has reduced keto groups attached to five member heteroaryl compounds (1h - 1j) (pyrrole, furan and thiophene) with good enantio-selectivity. thus demonstrating the broad specificity of the dehydrogenase enzyme present in the carrot. The obtained optically pure chiral pyridyl alcohols (2a - 2h) show exclusively (S) configuration thus following common Prelog^, s rule [26]. Whereas biocatalytic reduction of thermodynamically unstable 2-acetyl furan and 2-acetyl thiophene (1i - 1j) gave respective chiral alcohols with (R) configuration [27]. The present process developed has the advantage over the currently known chemical and biological methodologies in obtaining value added chiral aromatic hetero alcohols in higher yields and enantioselectivity [28,29]. To study the steric-orientation of the enzyme active site in enantioselective reduction of the heteroaryl methyl ketones to respective chiral alcohols, it is planned to carry out chemical additive studies like allyl bromide and ethylenediamine tetra acetic acid (EDTA) that influence the stereoselectity in enzymatic bioreduction processes [30-32]. On addition of allyl bromide (0.2 mM solution) to the incubation medium the process of bio-reduction of pyridyl ketones to chiral alcohols using D.carota, was found increased by 20% - 40%, whereas no difference was observed in the enzyme dehydrogenase activity on addition of EDTA (0.5 mM) (results not presented). Thus, from the enzyme inhibition studies, it was confirmed that the cell membrane bound enzyme alcohol dehydrogenase has shown broad substrate specificity and enantioselectivity in bioreduction of substituted heteroaryl methyl ketones. The main advantage of the methodology developed is to obtain optically pure heterocyclic secondary alcohols with (S) configuretion in a mild, inexpensive and eco-friendly environment. Further commercial application of this methodology and characterization of the enzyme responsible for selective reduction process is in progress.

Values were mean of the three independent experiments. The specific activity of the enzyme dehydrogenase present in D.carota was 255 mmol min^–1 mg^–1 protein (protein—25 µg/ml). Spectral data for the products (2a - 2j) obtained: 2a. (S)-2-(1-hydroxyethyl) pyridine (C₇H₉NO), liquid; optical rotation [a] ²⁵_{D =} –30.2^˚ (c 1, methanol), 99% ee (HPLC, Diacel Chiralcel OJH) [lit.^{32 - 34}[a] ²⁵_D -29.6 (c7.7 CHCl₃) for 99% ee]; IR (cm^–1) 3395 (OH), 2923 (CH₃); ¹H NMR (200 MHz, CDCl₃): d 1.48 (d, 3H, J = 6.04 Hz),3.59(br s,1H), 4.84 (q, 1H, J = 6.04 Hz), 7.13 - 7.34 (m, 2H), 7.66 (t, 1H, J = 7.55 Hz), 8.53 (s, 1H); ¹³C NMR (400 MHz, CDCl₃): 21.4 (CH₃), 66.5 (C-OH), 121.4 `36.1, 142.3, 164.1 (C1); GC/MS: 123 M⁺. 2b. (S)-3-(1-hydroxyethyl)pyridine  (C₇H₉NO), liquid; optical rotation [a]²⁵_D  = –44.9^˚, (c1, methanol), 98% ee (HPLC, Diacel (Chiralcel OJH) [lit. ^{32 - 34}[a] ²⁵_D  –38.9 (c1.0 ethanol) or 96% ee]; IR(cm^–1) 3259 (OH), 2972 (CH3); ¹H NMR (200 MHz, CDCl₃): d 1.43 (d, 3H, J = 6.04 Hz), 3.46 (br s, 1H), 4.83 (q, 1H, J = 6.043 Hz), 7.19 (s, 1H), 7.65 (d, 1H, J = 7.554 Hz), 8.29 - 8.48 (m, 2H); ^{13C NMR (400 MHz, CDCl}₃): 23.2 (-CH₃) , 67.8 (C-OH), 134.5, 140.4, 122.5, 149.5; GC/MS- 123 M⁺. 2c. (S)-4-(1-hydroxyethyl)pyridine (C₇ H₉N O), solid, mp 64˚C - 65˚C; optical rotation [a]²⁵_D  = –58.0^˚, (c1, methanol) 92% ee (HPLC Diacel Chiralcel OJH) [lit. ^32-34[a] ²⁵_D  –43.0 (c5.0 methanol) for 93% ee]; IR (cm^-1) 3313 (OH), 2924(CH₃); ¹H NMR (200 MHz, CDCl₃): d 1.53 (d, 3H, J = 6.61 Hz), 3.06 (br s, 1H), 4.90 (q, 1H, J = 6.61 Hz), 7.28 - 7.40 (m, 2H), 8.44 - 8.55 (m, 2H); ^{13C NMR (400 MHz, CDCl}₃): d 24.9 (C4-CH₃), 68.2 (C-OH), 120.5, 149.0, 155.8 ppm; GC/MS: 123 M⁺. 2d.(S)-2-(1-hydroxyethyl) 6-chloro pyridine (C₇H₈ClNO), colorless oil, optical rotation [α]_D²⁰ -13.2(c1 chloroform), 89% ee (HPLC Dialcel Chiralcel OD); IR(cm^-1) 3350 (OH); ¹H-NMR (200 MHz): δ = 1.61(d, J = 6.6 Hz, 3H, CH₃), 3.59 (br s, 1H), 4.60 (d, q, J = 6.6, 3.6 Hz, 1H, CHOH), 7.33(d, J = 8.3 Hz, 1H; aromatic) 7.73 (dd, J = 8.4, 2.6Hz.1H; aromatic, H); ^{13C-NMR(400 MHz): δ24.8 (CH}₃), 66.8, 123.2,134.6, 140.2, 148.0, 152.1; EI-MS (m/z): 157 M⁺. 2e.(S)-2-(1-hydroxyethyl)6-bromo pyridine (C₇ H₈ BrNO), colorless oil; Optical rotation [α] _D ²⁵ –12.1 (c2, chloroform), 92% ee ( HPLC Diacel Chiralcel OJH); IR (cm^-1) (film ) 3390 (OH), 1742; ¹HNMR (200MHz) δ = 1.43 (d, J-6.6Hz,3H,CH3), 4.02 (br, d, J = 3.4Hz, 1H, OH), 4.94 (d, q, J = 6.6, 3.6 Hz, 1H, CHOH),7.43 (d, J = 8.2 Hz, 1H, aromatic), 8.44 (d,J=2.6Hz,1H, aromatic); ¹³C NMR (400 MHz) δ28.0 (CH₃), 67.2 (CH), 129 (CH), 136.4 (CH), 140.8 (C) 148.0 (CH); = MS (EI, 70 ev) = m/z = 203 M⁺.( lit.^38-39). 2f. (S)-2-(1-hydroxyethyl)4-methyl pyridine (C₈ H₁₁NO), color less liquid; optical rotation [a] ²⁵_D –60.2^˚ (c 1, chloroform), 96% ee (HPLC Diacel Chiralcel OJH) ; IR (cm^-1) 3416 (OH), 2923 (CH₃); ¹H NMR (200 MHz, CDCl₃): d 1.46 (d, 3H, J = 6.42 HzCH₃), 2.37 (s, CH3 at C-4), 4.78 (q, brs, 1H, J = 6.42 Hz), 6.98 (d, 1H, J = 5.66 Hz), 7.03 (s, 1H), 8.35 (d, 1H, J = 4.91 Hz); ¹³C NMR (400 MHz, CDCL₃) d 20.8 (C4-CH₃), 21.6 (C2-CH₃), 66.5 (C-OH), 119.2 (C3), 121.8, 140.4, 151.2, 164.7 (C1); GC/MS: 137 M⁺. 2g (S)-2-(1-hydroxypropyl)pyridine (C₈ H₁₂NO), colorless oil; optical rotation [α]_D²⁰ –56.5 (c2, methanol), 90%ee (HPLC Diacel Chiralcel OJH); IR (cm^-1) 3404 cm^-1 (OH);.¹H NMR (200MHz, CD Cl₃) δ = 0.93 (d, 3H, J = 6.42 Hz ,CH₃), 4.66 (1H, dd, J = 13.9, 7.2 Hz), 7.63 (1H, ddm, J = 7.6,4.6Hz), 8.32 (1H, dm J = 4.8 Hz); ¹³C NMR (400 MHz), CDCl₃) δ = 8.9 (CH₃),29.2 (CH₂), 73.6, 119, 123.1, 138.2, 150.1, 165.0; MS (FAB) m/z = 138. 2h. (S)-2-(1-hydroxyethyl)pyrrole (C₆H₉ NO) colorless oil; optical rotation [α] _D²⁵ –37.8 (c2, chloroform) 80% ee (HPLC, Diacel, Chiral ODH); IR (cm^-1) 3332 (OH), 2872 (CH₃); ¹H NMR (200 MHz, CDCl₃) δ = 1.41 (d, J = 6.4 Hz, 3H, CH₃), 4.66 (OH), 6.05 (OH) ,5.52 (dd, J = 3.3, 0.8 Hz, 1H), 5.64 (dt, J = 3.3, 0.7 Hz, 1H), 7.00( dd, J = 3.0, 0.6Hz, N-H); ¹³C NMR (400MHzCD Cl₃) δ 21.6 (CH₃), 69.8, 104.2, 109.5, 144.2 MS m/z 112. 2i. (R)-2-(1-hydroxyethyl)furan (C₆H₈O₂), colorless liquid; optical rotation [α] _D²⁵ +22.8 (c1.0,chloroform), 76% ee (HPLC Diacel, Chiralcel OJH) [lit.^36-37[a] ²⁵_D +23.2 (neat) for 96 % ee]; IR (cm^-1) 3334 (OH) ,2979 (CH₃); ¹H NMR (200 MHz) δ 1.43 (d, J = 6.6 Hz, 3H, Me), 3.06 (br.s, 1H, OH), 4.80(q, J = 6.6 Hz CHMe), 6.19 (dt, J = 3.3, 0.7 Hz 1H, 7.23 (dd, J = 3.3, 0.8 Hz, 1 H); ¹³C NMR (400MHzCD Cl₃) δ 22.5 (CH₃), 64.8, 104.2, 139.5, 156.2; MS m/z 112. 2j. (R)-2-(1-hydroxyethyl)thiophene (C₆H₈OS) colorless oil; optical rotation [α]_D²⁵ +22.1 (c3.0, chloroform), 78% ee (HPLC Diacel, Chiralcel OJH) [lit.^36-37[a] ²⁵_D +21.9 (neat) for 91% ee]; IR (cm-1) 3343 (OH), 2973 (CH₃), 842(C-S); ¹HNMR (200 MHz, CDCl₃) δ1.56 (d, J = 6.6Hz,3H, CH₃), 2.90 (br.s, 1H, OH) 5.12 (q, 1H J = 6.4Hz, CHCH₃); 6.92 - 7.1 (m, 2H, thienyl H3 and H5 and 7.15 - 7.20 (m, 1H thienyl H4 ppm); ¹³CNMR (400MHz, CDCl₃) δ 25.0 (CH₃), 64.0, 119.2, 124.3, 124.6, 149.0; MS m/z 128.

3. Experimental

Daucus carota were purchased from local super market. The heterocyclic compounds used in the study were obtained from M/S.Alkali Metals Ltd; Hyderabad India and Aldrich Chemicals USA, all other reagents and chemicals used in the study were of analytical grade obtained locally.Bioreduction of substituted heteroaryl prochiral ketones (1a - j) was carried using fresh Daucus carota. In brief, add 5 grams of cut pieces(1 cm - 1.5 cm long slices) of D.carota tubers roots, in 200 ml of 0.1 mM sodium phosphate buffer pH 6.8, except compounds 1i - 1j suspended in phosphate buffer pH 8.2, containing 0.2 mM MgCl_2. To this add 200 mg of different heteroaryl prochiral ketones (1a - 1j) and the reaction mixture was incubated under nitrogen atmosphere at 37˚C in an orbital shaker. The progress of the reaction was monitored at regular intervals by TLC/HPLC analysis (RP-18 column using mobile phase acetonitrile: water 80: 20, 254 nm). The products obtained were isolated, purified through silica gel column chromatography and the chiral alcohols obtained were confirmed by HPLC using chiral columns Daicel CHIRALCEL OD or OJH or ODH columns (25 cm - 4.6 mm I.D.) UV detector at 210 nm, varying the mobile phase conditions depending on the specific substrate/product. The structures of the products were determined by mass, infrared spectra, ¹H and ¹³C NMR spectroscopic studies and also confirmed by the spectral data of the products obtained through chemical synthesis of the compounds (1a - 1g) by NaBH₄ reduction and compared with literature values [33-40]. The enantiomeric excess (ee) and absolute configuration of chiral compounds 2a - 2j were determined by the sign of specific rotation or by HPLC using chiral columns. Protein concentrations of D.carota homogenates determined by Bradford method and were found to be 25 µg/ml (bovine serum albumin as a standard protein) [41].

4. Conclusions

In conclusion, this study highlights a practical and efficient process of bioreduction of heterocyclic prochiral ketones to respective chiral alcohols by alcohol dehydrogenase enzyme present in D.carota. The results confirm that the plant cell membrane bound enzyme alcohol dehydrogenase show broad substrate specificity and chiral selectivity. The bioreduction of different heteroaryl methyl ketones (pyridyls and pyrrole) to corresponding optically chiral alcohols has shown exclusively (S) configuration thus following common Prelog’s rule, whereas (furan and thiophene) has shown (R) configuration anti Prelog’s rule. Thus, this study demonstrates a simple, inexpensive approach in synthesis of optically pure (S)- heteroaryl secondary alcohols of biological importance in an eco friendly environment.

5. Acknowledgements

Authors wish to thank (late) Mr. Y. S. S. Murthy garu, Vice Chairman, Alkali Metals Ltd; Hyderabad, India for the support and the kind gift of the acetyl pyridines.

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