P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation

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P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation of 6-Iodo-tetralone: Preparative-Scale Synthesis of a Key Intermediate for Pd-Catalyzed Transformations Adriana Ilie, Klaus Harms, and Manfred T. Reetz J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b02878 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 13, 2018

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The Journal of Organic Chemistry

P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation of 6-Iodo-tetralone: Preparative-Scale Synthesis of a Key Intermediate for Pd-Catalyzed Transformations Adriana Ilie,†,‡ Klaus Harms‡ and Manfred T. Reetz†,‡* †

Max-Planck-Institut für Kohlenforschung, Kaiser-Wilhelm-Platz 1, 45470 Mülheim an der Ruhr, Germany

‡

Department of Chemistry, Philipps-Universität Marburg, Hans-Meerwein Str. 4, 35032 Marburg, Germany

Supporting Information ABSTRACT: Chiral alcohols are important building blocks for the production of pharmaceuticals, catalytic access being possible by the use of enzymes, transition metal catalysts or organocatalysts. Herein, we report the use of cytochrome P450-BM3 mutants for the oxidative hydroxylation of 6-iodotetralone regio- and enantioselectively at the C4-position with formation of the (R)-alcohol. This CH-activation is not possible using modern man-made catalysts. The synthetic utility of this valuable synthon was explored in palladium catalyzed coupling reactions which occur in the absence of undesired racemization.

Biocatalytic regio- and stereoselective oxidative hydroxylation of structurally simple and complex organic compounds, 1 catalyzed by cytochrome P450 monooxygenases (CYPs), 2 complements modern CH-activating chemical catalysis. Since wildtype (WT) CYPs generally do not show acceptable levels of selectivity that the organic chemist may envision, 3 directed evolution has been employed to solve this funda4 mental problem. For example, using the self-sufficient P4501,4 BM3 we have previously evolved mutants that catalyze the hydroxylation of 1-tetralone and several derivatives thereof regioselectively at the desired position C4 with high enantioselectivity in favor of the (S)- or (R)-alcohol depending on 5 the substitution pattern. In that study a semi-rational approach was taken by focusing NNK-based site-saturation mutagenesis at residue A328 lining the binding pocket of P450-BM3, a “hot spot” identified in several earlier studies 4 using structurally completely other substrates. Several single-mutants proved to be highly regio- and stereoselective, e.g., A328P for the parent tetralone and A328F for 6methoxy-tetralone. In the present study we focused on 6iodo-tetralone (1) (Scheme 1), hoping that the product (R)- or (S)-2 could serve as a synthetically useful chiral intermediate in subsequent elaborating Pd-catalyzed transformations such as Suzuki arylation and carbonylative arylation as well as ester-forming carbonylation. This plan appeared risky due to the possibility of Pd-catalyzed racemization at the chiral benzylic alcohol position. As shown below, a highly regioand (R)-selective mutant was indeed obtained, but surprisingly not by mutating the favored residue A328. This set the stage for the three types of subsequent Pd-catalyzed cross-

coupling reactions under conditions that avoid undesired racemization. We first tested six previously evolved single mutants A328F, A328K, A328R, A328Y, A328W, A328H that had enabled pronounced regio- and enantioselectivity at position C4 of 1-tetralone and several of its derivatives, in contrast to WT 5 (typically 33% ee). Unfortunately, none of these mutants led to acceptable levels of selectivity in the case of the new substrate 1. Using cell lysate and the standard reaction time of 20 hours, only ~3% conversion and poor regio- and stereoselectivity were observed, which means that the compound is essentially not accepted by the enzyme. Thus, 6-iodotetralone, unlike 6-methoxy-tetralone, appears to be a special substrate. In contrast, upon testing WT P450-BM3, we were surprised once more to discover that (R)-2 is formed with 99% regioselectivity and 98.7% ee (Table 1, entry 1). This result means nearly perfect selectivity, but it was not planned.

Scheme 1. P450-BM3 catalyzed oxidative hydroxylation of 6-iodo-tetralone (1).

We then tested 14 mutants at residue F87 that we had 5,6 evolved earlier for other relatively small compounds, and discovered that single mutants with amino acid exchanges at position 87 are even more efficient (Table 1, entries 4-8).

Table 1. P450-BM3 catalyzed oxidative hydroxylation of ketone 1 with preferential formation of (R)-2. [a]

Entry

P450-BM3

%-Regio.

%-Enantio.

%-Conv.

1

WT

99

98.7 (R)

42

2

F87L

99

98 (R)

59

3

F87G

88

rac

21

4

F87V

99

99.9 (R)

68

5

F87S

93

99.6 (R)

39

6

F87R

99

99.2 (R)

38

ACS Paragon Plus Environment

The Journal of Organic Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

7

F87A

98

99.7 (R)

54

8

F87T

91

99.7 (R)

20

[a]

Values obtained from average of at least three independent + experiments performed using NADP (100 µM), substrate 1 (8 mM), at 25°C, 200 rpm, 20h. The conversion was determined by GC analysis and it is based on the amount of converted substrate. Indeed, residue 87 is known to be a “hot spot” in protein engineering of P450-BM3, the phenyl moiety in phenylalanine in the WT partially blocking access to the catalytically active high-spin heme-Fe=O (Cpd I), while smaller amino acids at this position enable high activity and broader sub1,4 strate scope. It can be seen that further improvements in regio- and enantioselectivity as well as conversion under the operating conditions were achieved, but reversal of stereoselectivity was not observed. Interestingly, mutant F87G leads to a racemic product with low conversion. We then conducted scale-up experiments using ketone 1 and what appeared to be the best mutant F87V. Several rounds of biotransformation reactions were made (0.40 mmol, 108 mg of substrate 1), resulting in 68% conversion and 36% yield of isolated (R)2 after column chromatography (see Experimental Section). With pure enantiomer (R)-2 in hand, we proceeded to the three types of Pd-catalyzed cross-coupling reactions using in all cases PdCl2(PPh3)2 as pre-catalyst (Scheme 2). As noted in the introduction, a potential problem was a possible undesired Pd-catalyzed racemization at the benzylic alcohol position of the compounds. Indeed, upon subjecting (R)-2 to Suzuki cross-coupling using PhB(OH)2 under conditions o utilizing 5 mol% Pd(OAc)2 [Na2CO3, PEG-H2O, 50 C, 1 h], 96% conversion was observed, but product 3 proved to be largely racemic. Thereafter, we turned to PdCl2(PPh3)2 as (pre)catalyst. Fortunately, the results point to complete retention of configuration for the products (R)-3, (R)-4 and (R)-5 (in all cases ≥99% ee) (Scheme 2). The reasons for the distinct difference are currently unclear, but this observation may be of significance in other future Pd-catalyzed reactions of halogen compounds which contain racemization-prone benzylic or allylic alcohol moieties. It is also of interest in those cases in which racemization is desired, as in lipase7 catalyzed dynamic kinetic resolution.

Scheme 2. Utilization of key intermediate (R)-2 in Pdcatalyzed cross coupling reactions: i) Suzuki cross coupling using PhB(OH)2, PdCl2(PPh3)2 3 mol%, K2CO3, dry anisole, 90°C, 1h, 96% yield. ii) Carbonylation using PdCl2(PPh3)2 3 mol%, NEt3, MeOH, COballoon, 50°C, 24h, 94% yield. iii) Carbonylative Suzuki cross coupling using PhB(OH)2, PdCl2(PPh3)2 3 mol%, K2CO3, dry anisole, CO-balloon, 90°C, 5h, 85% yield.

By GC-MS and HPLC-MS analyses (Fig. 1), we have identified all products arising from the (bio)catalytic oxidative hydroxylation and coupling reactions. In order to determine the absolute configuration of products 3-5, a racemic sample of 2 was first prepared, using dioxolan 7 as intermediate (Scheme 3). Then, rac-2 was subjected to coupling reactions under the same conditions as in the synthesis of pure enantiomers (R)-3, (R)-4 and (R)-5 to afford the corresponding racemic samples rac-3, rac-4 and rac-5, respectively. Evidence for an (R)-enantiomer for 3-5 was then gained by the Mosher derivatization reaction, i.e., by employing (R)Mosher’s chloride as chiral inducer (see SI). To prove that this protocol does in fact lead to the target (R)diastereoisomer, we performed two control reactions using (R)-2 and rac-2, the absolute configuration of (R)-2 being inferred from the X-Ray diffraction studies (Fig. 2). Thereafter, the optical rotation signs of 3-5 were compared with the optical rotation sign of (R)-2.

Scheme 3. Racemic synthesis of compound 2: i) CH(OEt)3, TBATB, ethylene glycol, r.t. 15h. ii) Rh2(cap)4, NaHCO3, tert-BuOOH, DCE, 40°C, 16h. iii) NaBH4, MeOH, r.t., 0.5 h. iv) I2, acetone, r.t., 0.5 h.

Suitable crystals for single crystal diffraction studies were obtained for compounds 1, 2 and 4, by keeping them at 4°C in a concentrated sample in ethyl acetate. The crystal structures of 1, 2 and 4 show that all molecules exhibit a slightly distorted envelope conformation with the benzene ring fused with the six-membered ketone ring. A closer inspection of the structure of molecule 2 reveals that the absolute configuration of 2 is (R), in line with the Mosher analysis. An inspection of the crystal packing in 2 and 4 shows nonbonding interactions between the oxygen atom in the ketone and the adjacent hydroxyl group, O···H-O, distances being in the 8 range 1.97-2.10 Å, cf. ΣrvdW(O,H) 2.7 Å]. These intermolecular interactions lead to the formation of a polymeric chain in 2, while in 4 a tetramer is formed (see SI). In summary we have developed an enantioselective biocatalytic route to (R)-4-hydroxy-6-iodo-tetralone (2) in a CH-


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The Journal of Organic Chemistry activating oxidative regio- and enantioselective selective hydroxylation reaction catalyzed by wildtype and mutants of P450-BM3. In contrast, this product, which would be difficult to prepare from starting material 1 using modern oxidation catalysts or reagents in a selective manner, is a key synthon which can be further used as a building block in subsequent C-C bond-forming transformations. The parent compound 4hydroxy-3,4-dihydronaphthalen-1(2H)-one has been prepared by synthetic methods using different starting materi9 als. Thus, the initial CH-activating transformation is another example of the complementarity of biocatalysis and transition metal catalysis and organocatalysis. In previous protein engineering studies of P450-BM3 as catalyst in regio- and stereoselective oxidative hydroxylation of halogencontaining substrates, subsequent Pd-catalyzed transfor5,6 mations were not tested. As a proof of principle, we performed three different palladium catalyzed C-C coupling reactions using the enantiopure synthon 2 as substrate under conditions which prevent undesired racemization. Such compounds have potential pharmaceutical applications. For example, glucosides incorporating the 4-hydroxy-1-tetralone skeleton have been used in Chinese folk medicine as anti10 tumor and anti-inflammatory medicines. Figure 1. Chiral HPLC profiles of rac-3 and (R)-3 (left), rac-4 and (R)-4 (middle) and rac-5 and (R)-5 (right.) O

O OH (R)-5

(R)-3

rac-3

3

4

5

6

7

8

(R)-4

rac-4

9

10 min 5

6

7

8

9

10

11

12

13

(R)-5

rac-5

14

15 min 8

Figure 2. Single crystal X-Ray structures of 1 (left), (R)-2 (middle) and (R)-4 (right)


9

10

11

12

13

14min


EXPERIMENTAL SECTION Molecular Biology Materials: E. coli BOU730 cells used are described else11 where. Electro-competent cells were prepared in-house 12 according to standard protocols. Biohydroxylation procedure using P450 mutants was performed as described previ13 ously. Small scale biohydroxylation using P450 WT or mutants: An Erlenmeyer flask (100 mL) containing LB (20 mL) and kan (50 µg/mL) was inoculated with a colony from BOU730 cells expressing P450 and incubated 6h (37°C, 220 rpm). This pre-culture was then inoculated into TB (400 mL) containing kan (50 µg/mL) and allowed to grow at 37°C until O.D. was 0.8-0.9. IPTG was further added to a final concentration of 0.2 mM and the culture was grown 16 h (30°C, 130 rpm). Cells were collected by dividing the culture in 2 mL aliquots, which were centrifuged (15 min, 5000 rpm), supernatant discarded and pellets stored at -20°C for subsequent use. For biotransformation, aliquots were resuspended in 480 µL lysis buffer containing phosphate buffer (pH 7.4, 100 mM), lysozyme (14 mg/mL), and DNAse I (6 U/mL), and incubated at 37°C (45 min, 700 rpm). Then, tubes were centrifuged (10000 rpm, 15 min), the supernatant (445 µL) was + transferred into a new 1.5 mL tube, and 5 µL NADP (100 µM final concentration), 50 µL glucose (100 mM final concentration) and substrate 1 (8 mM final concentration) were added. Samples were incubated at 25°C for 20 h at 700 rpm. After 20h, the organic phase was extracted with 500 µL ethyl acetate, and samples were analysed by GC. High scale reactions using P450-BM3 Biotransformation reaction of 6-iodo-tetralone (1) using P450-BM3 mutant F87V 14 was up-scaled according to the published protocol. An Erlenmeyer flask (100 mL) containing LB (20 mL) and kan (50 µg/mL) was inoculated with a colony from BOU730 cells expressing P450 F87V mutant and incubated 6h (37°C, 220 rpm). This pre-culture was then inoculated into TB (400 mL) containing kan (50 µg/mL) and allowed to grow at 37°C until O.D. was 0.8-0.9. IPTG was further added to a final concentration of 0.2 mM and the culture was grown 16 h (30°C, 130 rpm). Cells were pelleted by centrifugation, washed once with pH=7.4 potassium phosphate buffer (100 mM) and then resuspended in pH=7.4 potassium phosphate buffer (45 mL, 100 mM). Cells were then transferred to a 50 mL Erlenmeyer flask and 1.4 mg/mL lysozyme (70 mg), 6U DNase I (50 µL) were added, and incubated at 37°C (45 min, 700 rpm). Cells were centrifuged (10000 rpm, 15 min), the supernatant (45 mL) was transferred into a new 50 mL Erlenmeyer flask, then + 5 mL glucose (100 mM) and 100 µL NADP (100 mM) were added. In continuation, substrate (108 mg, 0.40 mmol) dissolved in 500 µL MeCN were added. Bioconversion was carried out at 25°C, 220 rpm. Reaction completion was monitored by GC. The organic phase was extracted with ethyl acetate (3 x 100 mL) and crude reaction product was purified using column chromatography. Chemistry General remarks: 6-Amino-3,4-dihydronaphthalen1(2H)-one, phenylboronic acid, triethyl amine, 4dimethylaminopyridine, (R)-(−)-α-methoxy-α(trifluoromethyl)phenylacetyl chloride (MTPACl-Mosher chloride), PdCl2(PPh3)2, anhydrous CH2Cl2, and polyethylene glycol (PEG 3350) were purchased from Alfa Aesar and Sig-

ma-Aldrich, and used without further purification. NMR 1 spectra were recorded on a Bruker Avance 300 ( H: 300 MHz, 13 C: 75 MHz) spectrometer using TMS as internal standard (d=0). Analytical thin layer chromatography was performed on Merck silica gel 60 F254q while for column chromatography Merck silica gel 60 was used. High resolution mass spectra recorded in EI mode were performed on an AccuTOF GCv (JEOL) spectrometer, using a TOF analyzer or in ESI mode on a Finnigan LTQ-FT spectrometer using an ion trap LTQ analyzer. Conversion and enantiomeric excess were determined by achiral gas chromatography and chiral HPLC. Optical rotation measurements were performed on a Krüss Optronic P8000 Digital Automatic High-Speed Polarimeter at 22°C. Absolute configuration was assigned by X-Ray diffraction studies for 2 and after NMR analysis of derivatized alcohols 2-5 with Mosher chloride and also comparison of the optical rotation signs of 3-5 with the optical rotation sign of 2. All palladium catalyzed reactions were performed starting from the enantiopure (R)-2 (ee 99.9%). Chiral HPLC conditions for rac-2 and (R)-2: 150 mm Chiralcel OD-3, 4.6 mm i.D., n-Heptane/Ethanol =98:2, 1 ml/min, 6.5 MPa, 298 K, UV, 266 nm; for rac-3 and (R)-3: 150 mm Chiralpak IB-3, 4.6 mm i.D., n-Heptane/Ethanol = 90:10, 1.0 mL/min, 8.8 MPa, 298 K, UV 266 nm; for rac-4 and (R)-4: 150 mm Chiralpak IC-3, 4.6 mm i.D., n-Heptane/2-Propanol = 80:20, 1.0 mL/min, 8.8 MPa, 298 K, UV 266 nm; for rac-5 and (R)-5: 150 mm Chiralpak IG-3, 4.6 mm i.D., n-Heptane/2-Propanol = 80:20, 1.0 mL/min, 10.1 MPa, 298 K, UV 266 nm. CCDC numbers for the single crystal X-Ray structure of 1 (1581091), for 2 (1581092) and for 4 (1581093). 6-Iodo-3,4-dihydro-2H-naphthalen-1-one (1): Compound 1 15 was prepared according to a published procedure. The crude reaction product was purified by column chromatography (EA:PE=1:9, Rf=0.45) to afford 1 as red crystalline solid, 1 (1.39 g, yield=82%). H NMR (300 MHz, CDCl3) δ 7.74–7.62 3 3 (m, 3H), 2.91 (t, J=6.0 Hz, 2H), 2.63 (t, J=6.0 Hz, 2H), 2.17– 13 2.06 (m, 2H). C NMR (75 MHz, CDCl3) δ 197.8 (s), 146.1 (s), 137.9 (s), 136.2 (s), 132.1 (s), 128.8 (s), 101.8 (s), 39.0 (s), 29.4 + (s), 23.2 (s). HRMS (ESI+) calcd for C10H9IONa [M] : 294.9596; found: 294.9594. (R)-(−)-4-Hydroxy-6-iodo-3,4-dihydronaphthalen-1(2H)-one (2): Compound 2 was prepared according to the biohydroxilation procedure described above. After extraction and concentration using a rotavap, the crude reaction product was purified by column chromatography (EA:PE=1:1, Rf=0.18) to afford (R)-2 as an orange crystalline solid, (37.7 mg, 1 yield=36%). H NMR (300 MHz, CDCl3) δ 8.02 (s, 1H), 7.77 (dd, J = 8.3, 1.5 Hz, 1H), 7.68 (d, J = 8.2 Hz, 1H), 4.93 (dd, J = 8.5, 4.0 Hz, 1H), 2.95–2.85 (m, 1H), 2.64–2.53 (m, 1H), 2.44– 13 2.34 (m, 2H), 2.21–2.09 (m, 2H). C NMR (75 MHz, CDCl3) δ 196.9 (s), 146.9 (s), 137.8 (s), 136.3 (s), 130.5 (s), 128.7 (s), 102.6 (s), 67.5 (s), 35.4 (s), 32.3 (s). HRMS (ESI+) calcd for C10H9IO2 + 22 [M] : 288.9719; found: 288.9720. [α]D = -84.99 (chloroform, c = 1, ee =99%) (rac)-4-Hydroxy-6-iodo-3,4-dihydronaphthalen-1(2H)-one (2): Compound rac-2 was prepared according to a similar 16 procedure. A 10 mL round-bottom flask was charged with 1 (500 mg, 0.84 mmol), triethyl orthoformate (300 mg, 2.02 mmol), 1,2-ethanediol (456.24 mg, 7.35 mmol), TBATB (8.86 mg, 0.18 mmol). The mixture was stirred at room temperature for 15 h, and then sat. NaHCO3 was added. The organic layer was separated, dried over MgSO4, concentrated and passed through a plug of silica gel. (GC indicated 99% con-


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Page 5 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

The Journal of Organic Chemistry version, orange oil, 580 mg, 1.83 mmol, 99% yield). The crude product was further transferred to a 25 mL round-bottom flask, then dry DCE (8 mL), NaHCO3 (77mg, 0.9 mmol) and Rh2(cap)4 (12 mg, 0.018 mmol, 1 mol%) were added. The flask was sealed with a septum and an empty balloon was installed to trap the oxygen. Then, TBHP (1.27 mL, 9.17 mmol) was injected. The reaction mixture was stirred for 16h at 40°C, and then filtered over silica gel. The organic layer was further concentrated and the reaction crude was purified using column chromatography (EA:PE 1:4) to afford ketone 7 as a red 1 solid (533 mg, yield=88%). H NMR (300 MHz, CDCl3) δ 8.30 3 3 3 (d, J=1.9 Hz, 1H), 7.89 (dd, J = 8.2, 1.9 Hz, 1H), 7.29 (d, J=8.2 Hz, 1H), 4.20–4.09 (m, 4H), 2.88–2.84 (m, 2H), 2.31–2.27 (m, 13 2H). C NMR (75 MHz, CDCl3) δ 195.7 (s), 142.6 (s), 142.3 (s), 136.0 (s), 133.4 (s), 127.1 (s), 105.6 (s), 95.2 (s), 65.6 (s), 35.8 (s), 33.0 (s). In the next step, the ketone 7 was reduced as follows: to a solution of ketone (314 mg, 0.951 mmol) in MeOH (20 mL), NaBH4 (43.18 mg, 1.14 mmol) is added. The mixture is stirred for 30 min, then solvent is removed using rotavap, ethyl acetate is added and the excess of NaBH4 is neutralized with 1M HCl. The organic phase is washed with H2O, dried over MgSO4, and solvent is removed in vacuum. To the resulting orange oil, acetone (10 mL) and I2 (24 mg, 0.094 mmol) are added, and then the reaction mixture was stirred for 30 min. Acetone is removed in vacuum, ethyl acetate is added, the organic phase is washed with 5% Na2S2O3, H2O and brine and then the solvent is removed to afford rac-2 as 1 an orange solid. H NMR (300 MHz, CDCl3) δ 8.03 (s, 1H), 7.79 (dd, J = 8.2, 1.3 Hz, 1H), 7.70 (d, J = 8.2 Hz, 1H), 4.94 (dd, J = 8.5, 4.0 Hz, 1H), 2.96-2.86 (m, 1H), 2.65-2.54 (m, 1H), 2.4513 2.36 (m, 1H), 2.22-2.10 (m, 1H). C NMR (75 MHz, CDCl3) δ 196.8 (s), 146.9 (s), 137.8 (s), 136.3 (s), 130.6 (s), 128.8 (s), 102.6 (s), 67.6 (s), 35.4 (s), 32.3 (s). (R)-(−)-4-Hydroxy-6-phenyl-3,4-dihydronaphthalen-1(2H)one (3): PdCl2(PPh3)2 (3.65 mg, 3 mol%), substrate 2 (50 mg, 0.17 mmol), phenylboronic acid (23.75 mg, 0.19 mmol), K2CO3 (71.96 mg, 0.52 mmol) were added to a Schlenk flask fitted with septum inlet. After standard cycles of evacuation and backfilling with dry and pure argon the flask was charged with dry anisole (3 mL). The mixture was stirred under pressure at 90°C for 8 h. When the reaction reached completion (detected by GC), the mixture was cooled to room temperature. After extraction with ethyl acetate, the mixture was dried over MgSO4, filtered and concentrated using a rotavap, the crude reaction product was purified by column chromatography (EA:PE=1:1, Rf= 0.5) to afford compound (R)-3 as an 1 orange solid, (39.6 mg, yield=96%). H NMR (300 MHz, CDCl3) δ 8.09 (d, J = 8.2 Hz, 1H), 7.84 (s, 1H), 7.68–7.60 (m, 3 3H), 7.49–7.38 (m, 3H), 5.04 (dd, J = 8.1, 3.9 Hz, 1H), 3.01–2.91 (m, 1H), 2.69–2.58 (m, 1H), 2.49–2.40 (m, 1H), 2.27–2.16 (m, 13 1H). C NMR (75 MHz, CDCl3) δ 197.2 (s), 146.9 (s), 146.0 (s), 139.9 (s), 130.1 (s), 129.1 (s), 128.5 (s), 128.0 (s), 127.4 (s), 127.2 (s), 125.7 (s), 68.2 (s), 35.4 (s), 32.4 (s). HRMS (ESI+) calcd for + 22 C16H14O2 [M] : 238.0994; found: 238.0988. [α]D = -111.71 (chloroform, c = 1, ee =99.4%) (R)-(−)-4-Hydroxy-6-carboxylate-3,4-dihydronaphthalen1(2H)-one (4): PdCl2(PPh3)2 (3.65 mg, 3 mol%), substrate 2 (50 mg, 0.17 mmol) and NEt3 (0.72 mL, 0.5 mmol) were added to a Schlenk flask fitted with septum inlet. After standard cycles of evacuation and backfilling with dry and pure carbon monoxide the flask was charged with dry MeOH (3 mL). The mixture was stirred under pressure at 50°C for 24 h (GC control). The product was extracted with ethyl acetate and

dried over MgSO4. The crude reaction product was purified by column chromatography (EA:PE=1:1, Rf=0.32) to afford compound 4 as an light red crystalline solid (35.9 mg, 1 yield=94 %). H NMR (300 MHz, CDCl3) δ 8.25–8.23 (m, 1H), 3 8.03–7.94 (m, 2H), 4.99 (dd, J=8.1, 4.0 Hz, 1H), 3.90 (s, 3H), 2.99–2.89 (m, 2H), 2.66–2.56 (m, 1H), 2.45–2.36 (m, 1H), 2.25– 13 2.13 (m, 1H). C NMR (75 MHz, CDCl3) δ 197.1 (s), 166.3 (s), 145.7 (s), 134.6 (s), 134.1 (s), 129.1 (s), 128.7 (s), 127.3 (s), 67.6 (s), 52.6 (s), 35.3 (s), 31.9 (s). HRMS (ESI+) calcd for C12H12O4 + 22 [M] : 220.0736; found: 220.0733. [α]D = -53.58 (chloroform, c = 1, ee =99.1%) (R)-(−)-4-Hydroxy-6-benzoyl-3,4-dihydronaphthalen-1(2H)one (5): PdCl2(PPh3)2 (3.65 mg, 3 mol%), substrate 2 (50 mg, 0.17 mmol), phenylboronic acid (23.28 mg, 0.19 mmol) and K2CO3 (71.96 mg, 0.52 mmol) were added to a Schlenk flask fitted with septum inlet. After standard cycles of evacuation and backfilling with dry and pure carbon monoxide the flask was charged with solvent (dry anisole 3 mL). The mixture was stirred at 90°C for 5h under an atmospheric pressure of carbon monoxide. When the reaction reached completion (detected by GC), the mixture was cooled to room temperature. The product was extracted with ethyl acetate and dried over MgSO4. The crude reaction product was purified by column chromatography (EA:PE=3:1, Rf=0.6) to afford com1 pound 5 as an orange oil (39.4 mg, yield=85%). H NMR (300 MHz, CDCl3) δ 8.11–8.02 (m, 2H), 7.80–7.73 (m, 3H), 7.64– 3 3 7.55 (m, 1H), 7.49 (t, J=7.6 Hz, 2H), 5.04 (dd, J=8.1, 3.9 Hz, 1H), 3.04–2.95 (m, 1H), 2.72–2.61 (m, 1H), 2.50–2.41 (m, 1H), 13 2.29–2.17 (m, 1H). C NMR (75 MHz, CDCl3) δ 196.9 (s), 196.2 (s), 145.7 (s), 142.4 (s), 137.0 (s), 133.6 (s), 133.2 (s), 130.3 (s), 129.4 (s), 128.7 (s), 127.3 (s), 67.8 (s), 35.4 (s), 32.1 (s). HRMS + (ESI+) calcd for C17H14O3 [M] : 266.0943; found: 266.0942. 22 [α]D = -59.39 (chloroform, c = 1, ee =99%).

ASSOCIATED CONTENT Supporting Information Selected NMR spectra, X-Ray data, GC and HPLC chromatograms. This material is available free of charge via the Internet at http://pubs.acs.org/.

AUTHOR INFORMATION Corresponding Author * [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT Financial support by the Max-Planck-Society and the Arthur C. Cope Fund is gratefully acknowledged. We thank Dr. R. Agudo for library construction.

REFERENCES (1) (a) Ortiz de Montellano, P. R. Chem. Rev. 2010, 110, 932−948. (b) Isin, E. M.; Guengerich, F. P. Biochim. Biophys. Acta, Gen. Subj. 2007, 1770, 314−329. (c) Munro, A. W.; Girvan, H. M.; McLean, K. J. Nat. Prod. Rep. 2007, 24, 585−609. (d) Shaik, S.; Cohen, S.; Wang, Y.; Chen, H.; Kumar, D.; Thiel, W. Chem. Rev. 2010, 110, 949−1017. (e) Whitehouse, C. J. C.; Bell, S. G.; Wong, L.-L. Chem. Soc. Rev. 2012, 41, 1218−1260. (f) Urlacher, V. B.; Girhard, M. Trends Biotechnol. 2012, 30, 26-36. (2) Reviews and key studies featuring CH-activating hydroxylation using man-made reagents or catalysts: (a) Newhouse, T.; Baran, P. S.



Angew. Chem. Int. Ed. Engl. 2011, 50, 3362−3374. (b) White, M. C. Science. 2012, 335, 807−809. (c) Neufeldt, S. R.; Sanford, M. S. Acc. Chem. Res. 2012, 45, 936−946. (d) Roduner, E.; Kaim, W.; Sarkar, B.; Urlacher, V. B.; Pleiss, J.; Gläser, R.; Einicke, W. D.; Sprenger, G. A.; Beifuß, U.; Klemm, E.; Liebner, C.; Hieronymus, H.; Hsu, S. F.; Plietker, B.; Laschat, S. ChemCatChem 2013, 5, 82−112. (e) Nanjo, T.; de Lucca, E. C. Jr.; White, M. C. J. Am. Chem. Soc. 2017, 139, 14586−14591. (3) Reviews of directed evolution: (a) Reetz, M. T. Directed Evolution of Selective Enzymes: Catalysts for Organic Chemistry and Biotechnology, Wiley-VCH: Weinheim, 2016. (b) Bommarius, A. S. Annu. Rev. Chem. Biomol. Eng. 2015, 6, 319−345. (c) Denard, C. A.; Ren, H.; Zhao, H. Curr. Opin. Chem. Biol. 2015, 25, 55−64. (d) Brustad, E. M.; Arnold, F. H. Curr. Opin. Chem. Biol. 2011, 15, 201−210. (e) Jäckel, C.; Hilvert, D. Curr. Opin. Biotechnol. 2010, 21, 753−759. (f) Turner, N. J. Nat. Chem. Biol. 2009, 5, 567−574. (g) Lutz, S., Bornscheuer, U. T. Eds. Protein Engineering Handbook, Volume 1 & Volume 2, Wiley-VCH: Weinheim, Germany, 2009. (4) Reviews and selected key papers of directed evolution of CYPs with emphasis on oxidative hydroxylation:1e (a) Kumar, S.; Halpert, J. R. Biochem. Biophys. Res. Commun. 2005, 338, 456−464. (b) Grogan, G. Curr. Opin. Chem. Biol. 2011, 15, 241−248. (c) Roiban, G.-D.; Reetz, M. T. Chem. Commun. 2015, 51, 2208−2224. (d) Zhang, K.; Shafer, B. M.; Demars, M. D.; Stern, H. A.; Fasan, R. J. Am. Chem. Soc. 2012, 134, 18695−18704. (e) Kolev, J. N.; O’Dwyer, K. M.; Jordan, C. T.; Fasan, R. ACS Chem. Biol. 2014, 9, 164−173. (f) Loskot, S. A.; Romney, D. K.; Arnold, F. H.; Stoltz, B. M. J. Am. Chem. Soc. 2017, 139, 10196−10199. (g) Rühlmann, A.; Antovic, D.; Müller, T. J. J.; Urlacher, V. A. Adv. Synth. Catal. 2017, 359, 984−994. (h) Dennig, A.; Lülsdorf, N.; Liu, H.; Schwaneberg, U. Angew. Chem. Int. Ed. 2013, 53, 8459−8462. (i) Eichler, A.; Gricman, L.; Herter, S.; Kelly, P. P.; Turner, N. J.; Pleiss, J.; Flitsch, S. L. ChemBioChem 2016, 17, 426−432. (5) Roiban, G.-D.; Agudo, R.; Ilie, A.; Lonsdale, R.; Reetz, M. T. Chem. Commun. 2014, 50, 14310−14313. (6) Agudo, R.; Roiban, G.-D.; Lonsdale, R.; Ilie, A.; Reetz, M. T. J. Org. Chem. 2015, 80, 950−956 (7) (a) Martin-Matute, B.; Bäckvall, J.-E. Dynamic Kinetic Resolutions. In Asymmetric Organic Synthesis with Enzymes; Gotor, V., Alfonso, I., Garcia-Urdiales, E., Eds.; Wiley-VCH: Weinheim, 2008; pp 89−113. (b) Kim, M.-J.; Ahn, Y.; Park, J. Curr. Opin. Biotechnol. 2002, 13, 578–587. (8) J. Emsley, Die Elemente, Walter de Gruyter, Berlin, 1994 (9) (a) Ferreira, E. M.; Stoltz, B. M. J. Am. Chem. Soc. 2001, 123, 7725–7726. (b) Suzuki, T.; Ghozati, K.; Katoh, T.; Sasai, H. Org. Lett. 2009, 11, 4286–4288. (10) (a) Liu, L.; Li, W.; Koike, K.; Zhang, S.; Nikaido, T. Chem. Pharm. Bull., 2004, 52, 566−569. (b) Ito, H.; Okuda, T.; Fukuda, T.; Hatano, T.; Yoshida, T. J. Agric. Food Chem. 2007, 55, 672−679. (c) Li, F.-S.; Shen, J.; Tan, G.-S. Chin. Tradit. Pat. Med. 2007, 29, 1490. (d) Liu, Q.; Zhao, P.; Li, X.-C.; Jacob, M. R.; Yang, C.-R.; Zhang, Y.-J. Helv. Chim. Acta. 2010, 93, 265−271. (11) Roiban, G.-D.; Agudo, R.; Reetz, M. T. Tetrahedron, 2013, 69, 5306−5311. (12) Sambrook, J.; Russell, D. W. The Condensed Protocols From Molecular Cloning: a Laboratory Manual, CSHL: New York, 2006. (13) Roiban, G.-D.; Agudo, R.; Reetz, M. T. Angew. Chem. Int. Ed. 2014, 53, 8659−8663. (14) Agudo, R.; Roiban, G.-D.; Reetz, M. T. ChemBioChem. 2012, 13, 1465−1473. (15) Murineddu, G.; Ruiu, S.; Mussinu, J. M.; Loriga, G.; Grella, G. E.; Carai, M. A.; Lazzari, P.; Pani, L.; Pinna, G. A. Bioorg. Med. Chem. 2005, 13, 3309−3320. (16) Catino, A. J.; Nichols, J. M.; Choi, H.; Gottipamula, S.; Doyle, M. P. Org. Lett. 2005, 7, 5167−5170.


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The Journal of Organic Chemistry


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P450-Catalyzed Regio- and Stereoselective Oxidative Hydroxylation

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