Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase

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Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase Afifa Ayu Koesoema a, Daron M. Standley b, Kotchakorn T.sriwong a, Mayumi Tamura a, Tomoko Matsuda a,⇑ a b

Department of Life Science and Technology, School of Life Science and Technology, Tokyo Institute of Technology, 4259 Nagatsuta-cho Midori-ku, Yokohama 226-8501, Japan Department of Genome Informatics, Genome Information Research Center, Research Institute of Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, Osaka 565-0871 Japan

a r t i c l e

i n f o

Article history: Received 20 December 2019 Revised 26 January 2020 Accepted 27 January 2020 Available online xxxx Keywords: Alcohol dehydrogenase Asymmetric reduction 2-Tetralol Drug intermediates

a b s t r a c t Both (S) and (R) forms of enantiomerically pure 2-tetralols, and their substituted analogs, are fundamental pharmaceutical intermediates. Here, we utilized the wild type and an engineered form of a highly enantioselective acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD) to produce (S)- and (R)-2-tetralols, and their substituted analogs. All mutations targeted residue Trp288, which has been shown to restrict substrate binding, but not play a direct role in catalysis. The wild type produced (S)-alcohols with excellent enantioselectivity, while the engineered forms produced either (S)or (R)- alcohols, depending on the substituent on the aromatic ring of the substrate, indicating that enantioselectivity can be rationally controlled. As a result, we were able to produce 6-hydroxy-2-tetralol, a potential antifungal drug intermediate, with 98% ee (S) and 81% ee (R) by wild type and Trp288Ser GcAPRD, respectively. To our knowledge, this is the first report of generating chiral 6-hydroxy-2-tetralol by rational enzyme design. Ó 2020 Elsevier Ltd. All rights reserved.

Introduction Enantiomerically pure 1-tetralols (1b), 2-tetralols (2b), and their substituted analogs are fundamental pharmaceutical intermediates [1]. Substituted 1-tetralol is the intermediate of sertraline, a widely used antidepressant [2], and halogen substituted (S)-2-tetralol is the intermediate of MK-0499, a cardiac arrhythmia drug [3–5]. 2-Tetralol and its hydroxy or alkoxy substituted analogs are intermediates of 2-aminotetralin, which can stimulate dopamine, serotonin, and melatonin receptors [6–8]. The analogs of 2-aminotetralin are also beneficial as central nervous system [9,10] and antifungal drug intermediates [11]. Since the biological activity of 2-aminotetralin is dependent on the substitution pattern on the aromatic ring as well as the chirality, enantiomerically pure (S)- and (R)-2-tetralols, and their substituted analogs are needed [10]. Non-enzymatic approaches, such as asymmetric hydrogenation [12–15], and enzymatic approaches using lipases [6], aminoacylases [16], or pancreatin [17], have been employed to produce chiral 2-tetralol and its substituted analogs. Use of alcohol dehydrogenases (ADHs) to asymmetrically reduce the correspond⇑ Corresponding author.

ing ketones is considered one of the most straightforward routes of production [4]. In recent years, both whole cell [18–21] and purified [22–24] ADHs have been employed to reduce 2-tetralones. ADH from Tricosphorum capitatum has been employed in the enantioselective reduction step of the synthesis of MK-0499 [21]. However, while both (S) and (R) forms are needed, most ADHs utilized to date have moderate or low ee, and are only (S)-selective [6]. Moreover, there are only a few ADHs [4,25] that can reduce wide varieties of substituted 2-tetralone analogs. For example, Thermoanaerobacter pseudoethanolicus ADH produces (S)- or (R)-2-tetralols and their substituted analogs depending on the substituents kind and position [4]. In consequence, several beneficial intermediates such as enantiomerically pure (R)-2-tetralol or (S)- and (R)-6-hydroxy-2-tetralol (a potential antifungal drug intermediate [11]) are still not accessible using ADH-mediated catalysis. Therefore, there is a need to find an ADH that can reduce substituted 2-tetralone analogs enantioselectively to their corresponding (S)- and (R)-alcohols. Our group has been studying an acetophenone reductase from Geotrichum candidum NBRC 4597 (GcAPRD), a robust ADH with excellent enantioselectivity of acetophenone and aliphatic ketone reduction to (S)-alcohols [26,27]. The crystal structure of GcAPRD [28] suggested that position Val153 is related to the coordination of the catalytic zinc with Glu67 [29] and position Trp288 restricts

E-mail address: [email protected] (T. Matsuda). https://doi.org/10.1016/j.tetlet.2020.151682 0040-4039/Ó 2020 Elsevier Ltd. All rights reserved.

Please cite this article as: A. A. Koesoema, D. M. Standley, K. T.sriwong et al., Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151682

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orientation of the ketone substrate [30]. Rational mutational studies have been conducted to alter the substrate specificity and enantioselectivity (Trp288) [30] or to modify the Zn2+ coordination dynamics (Val153) [29]. In this study, we investigated the reduction of substituted 2-tetralone analogs by GcAPRD wild type and Trp288 mutants (Fig. 1). The wild type was able to reduce substituted 2-tetralone analogs to their corresponding (S)-alcohols enantioselectively. Meanwhile, for the Trp288 mutants, the kind and position of substituents on the aromatic ring of 2-tetralone, which are relatively far from the reaction center, determined the enantioselectivity. With only a single point mutation, GcAPRD could produce beneficial substituted 2-tetralol analogs in both (S) and (R) forms with high enantioselectivity. We succeeded in reducing 6-hydroxy-2-tetralone (4a) to (S)-6-hydroxy-2-tetralol ((S)-4b)

1a

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GcAPRD wild type or Trp288 mutants

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2b-7b S up to >99% ee R up to 94% ee

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2a R = H 5a R = 6-OMe 3a R = 5-OMe 6a R = 6-Cl 7a R = 7-OMe 4a R = 6-OH Fig. 1. Asymmetric reduction of substituted tetralone analogs by GcAPRD wild type and Trp288 mutants.

a

with 98% ee by the wild type, which was the highest value reported by using ADHs. We also reduced 4a to (R)-4b by Trp288Ala, which is the first enantioselective synthesis reported by either non-enzymatic or enzymatic catalysis, to our knowledge.

Results and discussion Asymmetric reduction of 1a and 2a At first, we performed the asymmetric reduction of unsubstituted tetralones, 1a and 2a by using GcAPRD wild type and Trp288Ala (Fig. 2), since 1b and 2b are valuable intermediates for sertraline and 2-aminotetralin, respectively. As shown in Table S1, for the wild type, the relative activity of 2a was around 46% compared to that of acetophenone (8a), while for 1a, it was only 6% compared to that of 8a. As a result, the reduction of 2a proceeded much smoother than that of 1a (Fig. 2a). The wild type was able to reduce 1a and 2a with >99% ee (S) and 97% ee (S), respectively (Fig. 2c). For Trp288Ala (Fig. 2b), the reduction of 2a also proceeded much smoother than that of 1a. In terms of enantioselectivity, by only employing a single mutation, the enantioselectivity of 2a reduction was inverted from 97% ee (S) by the wild type to 94% ee (R) by Trp288Ala (Fig. 2d). To the best of our knowledge, 94% ee was the highest (R) enantioselectivity reported among ADHs [1,23] and chemical catalysts, with the highest reported ee of 90% (R), catalyzed by iridium reduction [31]. Trp288Ala showed a high preference to reduce bulkier substrates: 2a over 8a. As shown in Table S1, the relative activity of 2a reduction was around ten times that of 8a, while, for the wild type, the relative activity of 2a was around half of 8a. The preference to reduce 2a over 8a was also found in other Trp288 mutants (Trp288Phe, Trp288Met, Trp288Cys, Trp288Thr, Trp288Val, Trp288Ser, and Trp288Gly), and we observed that the larger the binding pocket of the enzyme active site, the higher the relative activity of 2a compared to 8a. The larger pocket accommodated

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ee (%)

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Fig. 2. Asymmetric reduction of substituted tetralone analogs (1a–7a) by GcAPRD wild type and Trp288Ala. Isolated yield of reduction by (a) wild type and (b) Trp288Ala. ee (%) of reduction by (c) wild type, and (d) Trp288Ala. Preparative scale reductions were done under the conditions described in experimental section. Positive and negative ee values indicate Prelog (S) and anti-Prelog (R) enantiopreference, respectively. N.d: not determined due to the low reduction activity.

Please cite this article as: A. A. Koesoema, D. M. Standley, K. T.sriwong et al., Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151682

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tivities. However, Trp288Ala reduced 3a with 72% ee (R), 5a with 44% ee (S), and 7a with 87% ee (S). Altogether, the results implied that the kind and position of substituent relatively far from the reaction center of the substrate could control the enantioselectivity of GcAPRD Trp288Ala. For example, 2a (no substitution), 3a (5-OMe), and 4a (6-OH) had (R)-enantioselectivity, while 5a (6-OMe), 6a (6-Cl), and 7a (7OMe) had (S)-enantioselectivity. One of the possible reasons for the difference of the enantioselectivity would be the presence of a clash or hydrophilic/hydrophobic interaction of these substituents with the enzyme binding pocket although they are remote from the reaction center, which would give different substrate orientations. Next, to examine our hypothesis, we performed docking simulations of the substrates and the enzymes using the wild type crystal structure [27,28] and the Trp288Ala model structure [30]. The docked ligand binding poses consistent with a given chirality can be seen in Fig. 3, while the binding energy values and details can be seen in Table S5. We found the pro-(S) poses with the phenyl ring of the substrates positioned inside the large pocket of the enzyme active sites for the 2a-wild type combination (Fig. 3a), and for the 7a-Trp288Ala combination (Fig. 3b). We also found the pro-(R) pose with the phenyl ring of the substrate positioned inside the engineered pocket with Ala288 for the 4a-Trp288Ala combination (Fig. 3c). Taken together, our hypothesis was supported by the docking simulations, and the mechanism underlying the enantioselectivity inversion in the Trp288Ala mutant appears to be similar to the previously proposed mechanism for the reduction of aliphatic ketones by the wild type and Trp288 mutants [30]. Flipping the positions of smaller and larger substituents of ketones

bulky 2a better also as shown in the reduction of various substituted 2a analogs by Ile86 and Trp110 mutants of TeSADH [4].

Asymmetric reduction of substituted 2-tetralone analogs (2a–7a) With the success of reduction of 2a, we proceeded with the reduction of various substituted 2-tetralone analogs (3a–7a). First, we performed the asymmetric reduction of 6a, for which the corresponding alcohol (6b) is beneficial as a MK-0499 derivative. Encouragingly, the wild type and Trp288Ala reduced 6a with good yield and an ee of 98% (S) and 98% (S), respectively. For Trp288Ala, a single substitution of hydrogen with a chloride at the 6-position, remote from the reaction center, inverted the enantioselectivity from 94% ee (R) (2a) to 98% ee (S) (6a). To understand the effect of the substituent at the 6-position, we compared the enantiopreference of 6a (6-Cl) with 4a (6-OH) and 5a (6-OMe). Independent of the substituent, the wild type reduced 4a, 5a, and 6a with satisfactory yields and excellent (S)-enantioselectivities. Meanwhile, for Trp288Ala, the reduction enantioselectivity shifted from (R) to (S) in the order of 4a (6-OH, 79% ee (R)), 5a (6-OMe, 44% ee (S)), and 6a (6-Cl, 98% ee (S)). By comparing GcAPRD to other ADHs (Table S2), we could deduce that both the wild type and Trp288Ala showed remarkable ability to reduce 4a to the corresponding 4b with 98% ee (S) and 79% ee (R), respectively, which was the first such report using ADHs [4,25]. We also compared the enantiopreference of 3a (5-OMe), 5a (6OMe), and 7a (7-OMe), all having methoxy substituent at different positions. Independent of the positions, the wild type reduced 3a, 5a, and 7a with satisfactory yields and excellent (S)-enantioseleca

b

S47

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A288 W288 c

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A288

Fig. 3. Binding poses of (a) 2a in the active site of GcAPRD wild type crystal structure (b) 7a in the active site of GcAPRD Trp288Ala model structure, and (c) 4a in the active site of GcAPRD Trp288Ala model structure. The CPK coloring was used except for the backbone of the receptor. The ligands 2a, 4a and 7a are shown in yellow, brown, and green stick, respectively. NADH are shown in purple stick. Catalytic zinc is shown in grey sphere, and the C4 of NADH with hydride to be transferred to the ketone is highlighted with white circle.

Please cite this article as: A. A. Koesoema, D. M. Standley, K. T.sriwong et al., Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151682

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(in this case, the sides without and with the phenyl ring) relative to the two binding pockets will cause inversion of enantioselectivity. Finally, we reduced substituted 2-tetralone analogs by using the Trp288 mutant library with various sizes of engineered pockets, from Trp288Phe to Trp288Gly (Fig. S1). Trp288Phe and Trp288Met had similar but lower (S)-enantioselectivity to wild type. Meanwhile, Trp288Cys, Trp288Thr, Trp288Val, Trp288Ser, and Trp288Gly had similar selectivity to Trp288Ala. 2a–4a were reduced to (R)-alcohol, and 5a and 7a were reduced to (S)-alcohol. By utilizing Trp288Ser we could obtain (R)-4b with a higher enantioselectivity of 81% ee (R). Every mutant tested had a slightly different enantioselectivity profile, indicating the effect of binding pocket size towards the enantioselectivity of 2-tetralone reduction. In the previous study, we were able to control the enantioselectivity of simple aliphatic ketone reduction by the substituent adjacent to the carbonyl carbon, reaction center [30]. In this study, by using the improved Trp288 mutant library, we were able to control the enantioselectivity of GcAPRD by the substituent remote from the reaction center.

Conclusions GcAPRD wild type and Trp288 mutants could produce the pharmaceutically important substituted 2-tetralol analogs in (S) and (R) forms with high enantioselectivity. We reported the highest enantioselectivity for the reduction of 4a to both (S)-4b and (R)-4b by using the wild type and Trp288Ser, respectively. Moreover, we were able to control the enantioselectivity of GcAPRD mutants by substituents far from the reaction center.

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments The authors acknowledged Professor Miki Senda and Professor Toshiya Senda from High Energy Accelerator Research Organization (KEK), Japan, for the help with the crystallization of enzymes and valuable discussions. This work was supported by the Japan Society for the Promotion of Science, Japan (grant number JP16K05864) to Tomoko Matsuda and the Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED, Japan (grant number 19am0101108j0003) to Daron M. Standley.

Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.tetlet.2020.151682.

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Please cite this article as: A. A. Koesoema, D. M. Standley, K. T.sriwong et al., Access to both enantiomers of substituted 2-tetralol analogs by a highly enantioselective reductase, Tetrahedron Letters, https://doi.org/10.1016/j.tetlet.2020.151682