formal intramolecular hydride transfer regioselective biocatalytic self-sufficient … · s1...
TRANSCRIPT
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Regioselective Biocatalytic Self-Sufficient Tishchenko-Type Reaction via Formal Intramolecular Hydride Transfer
Erika Tassano,[a] Kemal Merusic,[a] Isa Buljubasic,[a] Olivia Laggner,[a] Tamara Reiter,[a] Andreas Vogel,[b] and Mélanie Hall*[a]
[a] Dr. Erika Tassano, Kemal Merusic, Isa Buljubasic, Olivia Laggner, Tamara Reiter, Dr. Mélanie Hall
Department of Chemistry, University of Graz, Heinrichstrasse 28, 8010 Graz, Austria
E-mail: [email protected]
[b] Dr. Andreas Vogel
c-LEcta GmbH, Perlickstrasse 5, 04103 Leipzig, Germany
*Corresponding authors: [email protected]
ELECTRONIC SUPPORTING INFORMATION
Table of contentGeneral remarks ...................................................................................................................................2Enzymes ...............................................................................................................................................2Standard biocatalytic procedure ...........................................................................................................3Supplementary tables ...........................................................................................................................4Supplementary figures........................................................................................................................10Supplementary schemes .....................................................................................................................12Synthetic procedures ..........................................................................................................................13GC analyses........................................................................................................................................21NMR spectra ......................................................................................................................................30References ..........................................................................................................................................51
Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020
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General remarksChemical reagents employed in the synthesis of starting and reference materials were purchased from Alfa Aesar, Sigma-Aldrich/Merck and Fluorochem. TLC analyses were performed on Merck Silica Gel 60 F254 precoated plates and visualized under UV light (λ = 254 nm), stained with Hanessian’s stain [dipping into a solution of (NH4)6Mo7O24·4H2O (21 g) and Ce(SO4)2·4H2O (1 g) in H2SO4 (31 mL) and H2O (469 mL) and warming] and with bromocresol green stain (solution of 0.1 g bromocresol green in 500 mL ethanol and 5 mL 0.1 N NaOH). Petroleum ether (40–60 °C) is abbreviated PE. Organic extracts were always dried with anhydrous Na2SO4 and filtered before evaporation of the solvent under reduced pressure. Column chromatography was done with the flash methodology1 by using 220–400 mesh silica. NMR spectra were measured on a Bruker Avance III 300 MHz NMR spectrometer. Chemical shifts are reported in ppm relative to TMS (δ = 0.00 ppm) and the coupling constants (J) in Hertz (Hz). GC-MS analyses were performed on an Agilent 7890A GC system, equipped with an Agilent 5973 mass selective detector and an Agilent HP-5MS column (30 m x 0.25 mm x 0.25 µm).
EnzymesADH-A (from R. ruber) and HLADH (from E. caballus) were cloned and overexpressed as previously reported.2, 3
ADH-107, 109, 110, 111, 114, 117, 118, 132, 170, 171, 172, 173, 174, 175 and 182 were obtained from c-LEcta as lyophilized cell-free extracts and are all NADH dependent. The ADH enzyme collection consists of variants obtained by enzyme engineering.4 The enzymes were expressed in E. coli and cell-free extracts were obtained after cell lysis, centrifugation and freeze-drying of the supernatants.
The N-terminal strep II-tagged Lk-ADHmut (mutations with respect to the wild type from L. kefir: L17H; D25T; E29T; H40R; A64V; T71G; A80T; V87L; A94G; V95Y; S96R; N131C; E145L; F147Q; T152A; L153T; N157S; D173L; Y190P; V196M; L199M; E200P; I266V)5 was cloned in pASK-IBA5plus vector and overexpressed in E. coli BL21(DE3). 250 mL LB medium supplemented with ampicillin (100 μg mL-1) and 1 mM MgCl2 were inoculated with 2 mL overnight culture (starter culture, 15 mL LB medium supplemented with ampicillin) and incubated at 37 °C and 120 rpm until an OD600 of 0.6 was reached. Then AHTC (anhydrotetracycline, 0.4 μM) was added for induction and the cells left overnight at 20 °C and 120 rpm. The next day the cells were harvested by centrifugation (20 min, 4000 rpm, 4 °C). To obtain the cell-free extract, the cell pellet (4 g from 500 mL culture) was resuspended in buffer W (100 mM TRIS·HCl, 150 mM NaCl, pH = 8.0; 15 mL) and lysed on ice by sonication (time: 2.30 min, pulse ON: 1 s, pulse OFF: 4 s, amplitude: 40%). Afterwards the cell debris were removed by centrifugation (20 min, 15000 rpm, 4 °C) and the cell-free extract was directly applied to Strep-Tactin XT Superflow gravity flow column (5 mL); the purification was performed according to the protocol provided by the supplier (IBA). Fractions containing the purified enzyme (see SDS PAGE below) were collected and concentrated to 2.5 mL; then buffer was exchanged to KPi (50 mM, pH=7.0; supplemented with 2 mM MgCl2); two fractions were collected, frozen in liquid nitrogen and stored at -20 °C.
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SDS PAGE: 1, 10, 15: standard (PageRulerTM, 10 to 180 kDa); 2: supernatant after sonication; 3: flow-through; 4-9: washing fraction; 11-19: elution fractions
Standard biocatalytic procedureStandard enzymatic Tishchenko-type reaction was run as follows: an aliquot of purified enzyme (1 mg/mL final concentration, unless otherwise stated) was added to a phosphate buffer solution (50 mM, pH 7.0, supplemented with 2 mM MgCl2) containing the substrate (10 mM, from a 200 mM stock solution in CH3CN, final co-solvent concentration 5 vol%) and the nicotinamide cofactor (0.5 mM). The reaction mixture (500 µL total volume) was incubated at 30 °C and 120 rpm. After 24 h, the product was extracted from the reaction mixture with EtOAc (2 x 250 µL, spiked with 10 mM (R)-limonene as internal standard). The combined organic fractions were dried over anhydrous Na2SO4 and analyzed by GC. All reactions were run in duplicates.
Acetonitrile was identified as suitable co-solvent to solubilize the substrate during the reaction, while preserving the enzyme activity in most cases. While the actual effect of the co-solvent on the activity of each enzyme was not studied individually, ADHs are usually robust toward the presence of organic solvents at such low concentration (5 vol%). The resulting mixture can be seen as a microemulsion but the state of the solution was not studied in detail, since in most cases the selected condition was found satisfactory with at least several biocatalysts. It cannot be excluded that variation in yields may be related to various degrees of acceptance of the co-solvent.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20kDa
180130
100
70
55
40
35
25
15
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Supplementary tablesTable S1. Conversion of 1a to 1b through ADH-catalyzed reduction-oxidation sequence[a]
Entry Enzyme[b] Conv. (%) TONNAD(P)H[c]
1 ADH-107 57 11.4
2 ADH-110 63 12.6
3 ADH-111 74 14.8
4 ADH-114 <1 n.a.
5 ADH-132 59 11.8
6 ADH-170 10 2
7 ADH-172 74 14.8
8 ADH-173 <1 n.a.
9 ADH-174 60 12
10 ADH-175 1 2
11 HLADH 0 n.a.
12 ADH-A 0 n.a.
13 Lk-ADHmut 86 17.2
[a] 10 mM 1a, 0.5 mM NAD(P)H, 1 mg/mL enzyme, KPi buffer (50 mM, pH 7.0, supplemented with 2 mM MgCl2 in entry 1-10 and 13), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h. [b] ADH 107, 110,111, 114, 132, 170, 172-175 were obtained from c-LEcta; HLADH and ADH-A were employed as purified enzyme and obtained as reported;2, 3 Lk-ADHmut was used as purified enzyme and obtained as described herein. [c] TONNAD(P)H calculated based on product formation (1 turnover corresponds to two half-reactions). n.a. not applicable.
Table S2. Substrate and cofactor feeding experiment performed with 1a and ADH-132.[a]
Entry 1a (mM) NADH (mM) Reaction time (h) 1b (mM)
1 5 0.5 3 2.7
2 10 (5+5)[b] 0.5 6 6.7
3 10 (5+5)[b] 1.0 (0.5+0.5)[b] 6 7.0
4 10 0.5 3 6.1
5 20 (10+10)[b] 0.5 6 10.1
6 20 (10+10)[b] 1.0 (0.5+0.5)[b] 6 14.1
[a] Reaction conditions: 1 mg/mL ADH-132, KPi (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm. [b] Added in two equal aliquots, second aliquot added after 3 h.
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Table S3. Conversion of 2a and 3a to 2b-2c and 3b-3c, respectively, using a panel of ADHs according to Scheme 2.[a]
Entry ADH Conv. (%)to 2b-2c[b]
Ratio2b/2c[c]
Conv. (%)to 3b-3c[b]
Ratio3b/3c[d]
1 107 13 23:77 23 62:38
2 109 n.d. n.a. 42 76:24
3 110 n.d. n.a. 70 87:13
4 111 n.d. n.a. 55 79:21
5 114 2 >99:1 56 99:1
6 114[e] n.t. n.a. 67 >99:1
7 117 n.d n.a. 43 77:23
8 118 n.d n.a. 56 88:12
9 132 n.d. n.a. 55 83:17
10 170 3 >99:1 24 41:59
11 171 27 70:30 13 54:46
12 172 2 >99:1 67 86:14
13 173 3 >99:1 <2 n.a
14 174 15 20:80 22 63:37
15 175 3 >99:1 <2 n.a.
16 Lk-ADHmut 52 90:10 48 73:27
17 Lk-ADHmut 81[f] 90:10 69[e] 61:39
[a] Reaction conditions: 10 mM of 2a/3a, 1 mg/mL ADH, 0.5 mM NAD(P)H, KPi buffer (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h; entries 1-15: ADHs from c-LEcta available as lyophilized cell lysates. [b] Conversion based on total formation of lactone products. [c] Quantification of 2b and 2c using commercial standards. [d] Quantification of 3b based on calibration curve obtained from synthesized reference material (see the Supporting Information); quantification of 3c calculated assuming that 3b and 3c have similar response factor on GC-FID. [e] After 5 h, additional 10 mM of 3a and 0.5 mM NADH were added, total reaction time 24 h. [f] 20 mM of 2a. n.a. not applicable, n.d. not detected, n.t. not tested.
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Table S4. Conversion of 4a to 4b through ADH-catalyzed reduction-oxidation sequence[a]
Entry ADH [4a] (mM) Conv. (%)
1 107 10 2
2 109 10 62
3 110 10 7
4 111 10 40
5 114 10 15
6 117 10 59
7 118 10 43
8 132 10 15
9 170 10 3
10 171 10 15
11 172 10 9
12 173 10 1
13 174 10 2
14 175 10 1
15 Lk-ADHmut 5 90
16 Lk-ADHmut 10 84
17 Lk-ADHmut 15 82
18 Lk-ADHmut 20 26
19 Lk-ADHmut 50 9
[a] Reaction conditions: 1 mg/mL enzyme, 0.5 mM NAD(P)H, KPi (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h.
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Table S5. Conversion of 5a-7a to 5b-7b and 5c-7b through ADH-catalyzed reduction-oxidation sequence[a]
Entry ADHConv. (%)
to 5b[b]
Conv. (%)
to 6b/6c
Ratio
6b/6c
Conv. (%)
to 7b/7c
Ratio
7b/7c
1 Lk-ADHmut 5 75 97:3 98 14:86
2 99 n.c. 6 64:36 11 6:94
3 107 n.c. 7 56:44 43 13:87
4 109 1 23 72:28 67 9:91
5 110 8 32 94:6 58 10:90
6 111 1 24 73:27 67 9:91
7 114 n.c. 7 70:30 11 8:92
8 117 1 40 77:24 69 10:90
9 132 32 61 72:28 62 23:77
10 170 n.c. 5 65:35 16 8:92
11 171 n.c. 6 55:45 51 13:87
12 172 1 20 94:6 78 10:90
13 173 n.c. 2 65:35 4 14:86
14 174 n.c. 8 71:29 38 15:85
15 175 n.c. 2 68:32 2 18:82
16 182 n.c. 3 50:50 15 5:95
[a] Reaction conditions: 10 mM substrate, 1 mg/mL ADH, 0.5 mM NAD(P)H, KPi (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 24 h. [b] no trace of 5c was detected. n.c. no conversion.
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Table S6. Conversion of 8a-10a through ADH-catalyzed reduction-oxidation sequence[a]
Entry ADH Conv. (%) to 8c[b] Conv. (%) to 9c[b] Conv. (%) to 10c
1 Lk-ADHmut 4 46 90
2 99 <1 19 14
3 107 <1 3 >99
4 109 1 5 92
5 110 14 15 84
6 111 2 5 90
7 114 4 2 58
8 117 1 6 94
9 118 13 52 n.t.
10 132 57 6 >99
11 170 <1 3 29
12 171 <1 4 93
13 172 5 27 81
14 173 <1 <1 <2
15 174 <1 3 69
16 175 <1 2 4
17 182 <1 2 10
[a] Reaction conditions: 10 mM substrate, 1 mg/mL ADH, 0.5 mM NAD(P)H, KPi (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h. [b] no trace of 8b/9b was detected. n.t. not tested.
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Table S7. Effect of additional EWG on conversion of 8a-10a (compared to mono-substituted 5a-7a)
Entry ADHConv. (%)
from 5b to 8c
Conv. (%)
from 6b to 9c
Conv. (%)
from 7c to 10c
1 Lk-ADHmut -20 -39 -8
2 99 n.a. +217 +27
3 107 n.a. -57 +133
4 109 0 -78 +37
5 110 +75 -53 +45
6 111 +100 -79 +34
7 114 n.a. -71 +427
8 117 0 -85 +36
9 132 +78 -97 +63
10 170 n.a. -40 +81
11 171 n.a. -33 +82
12 172 +400 +35 +4
13 173 n.a. n.a. n.a.
14 174 n.a. -63 +82
15 175 n.a. 0 +100
16 182 n.a. -33 -33
[a] Reaction conditions: 1 mg/mL ADH, 0.5 mM NAD(P)H, KPi (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h. n.a. not applicable.
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Supplementary figures
Figure S1. GC-MS analysis of the reaction of 1a with ADH-170 (corresponding to entry 6 in Table S1) showing accumulation of intermediate lactol. MS fragmentation is of the lactol (peak at 6.0 min) and compatible with the one reported in literature.6
0 250 500 750 1000 1250 15000
2
4
6
8
time (min)
1b (m
M)
Figure S2. Time study of the conversion of 1a to 1b by ADH-172. 10 mM 1a, 0.5 mM NADH, 1 mg/mL enzyme, KPi buffer (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm.
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Peak at 7 min: EI MS (70 eV, m/z (%)): 150.0 (M+, 3), 132.1 (13), 104.1 (100), 103.1 (27), 78.1 (22).
Peak at 7.5 min: EI MS (70 eV, m/z (%)): 146.0 (M+, 61), 118.1 (100), 90.1 (67), 63.0 (30).
Figure S3. GC-MS traces of biotransformations (A, using ADH-110; B, using ADH-174) and blank reaction (C, no enzyme) using compound 2a as substrate. 10 mM 2a, 0.5 mM NADH, 1 mg/mL ADH, KPi buffer (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm, 16 h.
0 250 500 750 1000 1250 15000
1
2
3
4
5
6
7
time (min)
3b (m
M)
Figure S4. Time study of the conversion of 3a to 3b by ADH-114 (3c not detected). 10 mM 3a, 0.5 mM NADH, 1 mg/mL enzyme, KPi buffer (50 mM, pH 7.0, 2 mM MgCl2), 5 vol% CH3CN, 30 °C, 120 rpm.
Trace A
Trace B
Trace C
O
OH
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Figure S5. 1H-NMR of lactone 3b obtained from scale-up biotransformation of 3a with ADH-114 (200 mg scale, see 'synthetic procedures' section). Only one regioisomer is detected.
Supplementary schemes
O
OH
OH
O
OH
O
OH
OH
O
O
O
NAD(P)HNAD(P)+
ADH
ADHO
O
OH
O
O
OH
m/z: 146
m/z: 150
O
O
m/z: 148
-H2OH2O
2a
2a
Scheme S1. Possible pathways in the conversion of 2a leading to depletion of substrate and formation of side-products in a redox-neutral manner, as detected in Figure S3.
O
ADHO
O
8c-9c8a-9a
EWG
O
ADHO
ONADPH
7c7a
O
O
ADHO
O NADPH
5b-6b5a-6a
EDG EDG EWGEWG
O
O
NADPH
EDGEDG
EWG
Scheme S2. Effect of the electronic properties of the substitution on the regioselectivity of ADH in the conversion of substituted [1,1'-biphenyl]-2,2'-dicarbaldehyde substrates 5a-9a (EDG: electron-donating group; EWG: electron-withdrawing group).
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Synthetic procedures
Synthesis of 2-(2-oxoethyl)benzaldehyde 2a
11 12
OH
OH
Method A (76%)Method B (30%)
2a
CHO
CHOH2O/THF
NaIO4
Method A: in a sealed glass vial indene 11 (250 mg, 2.6 mmol) was dissolved in a mixture of THF/H2O/tBuOH 5:1:1 (total volume: 2.5 mL), then K2OsO4 (8 mg, 0.02 mmol) and N-methylmorpholine (328 mg, 2.8 mmol) were added, and the mixture was stirred at RT overnight. The reaction was quenched by addition of a 10% Na2SO3 solution, stirred 30 minutes and then extracted with EtOAc; the collected organic layers were dried over Na2SO4 and solvent was removed under vacuum. The crude was purified by FC (EtOAc), affording the cis-diol 12 in 76% yield (247 mg, 1.64 mmol).
Method B: a mixture of indene 11 (350 mg, 3.0 mmol), NaIO4 (193 mg, 0.9 mmol) and LiBr (50 mg, 0.6 mmol) was dissolved in acetic acid (5 mL) and heated at 95 °C overnight. After cooling, the reaction was diluted with water and a 10% Na2S2O5 solution and extracted with EtOAc; the collected organic fractions were washed with NaHCO3 sat., brine and the solvent was evaporated. The crude was dissolved in MeOH (20 mL) and K2CO3 (620 mg, 4.5 mmol) was added; the mixture was stirred at 40 °C overnight. Then the solvent was removed, the residue was solubilized in NH4Cl sat. and extracted with EtOAc; the collected organic layers were dried over Na2SO4 and solvent was removed under vacuum. The crude was purified by FC (CyHex/EtOAc 4:6), affording the cis-diol 12 in 30% yield (135 mg, 0.9 mmol). Spectroscopic data agree with those reported in literature.71H NMR (300 MHz, CDCl3) δ = 7.46 – 7.34 (m, 1H), 7.31 – 7.18 (m, 3H), 4.95 (d, J = 5.0 Hz, 1H), 4.52 – 4.37 (m, 1H), 3.09 (dd, J = 16.4, 5.7 Hz, 1H), 2.92 (dd, J = 16.4, 3.5 Hz, 1H), 2.68 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 142.1, 140.3, 129.0, 127.3, 125.5, 125.2, 76.1, 73.6, 38.7.
A solution of NaIO4 (240 mg, 1.1 mmol) in H2O (11 mL) was added to a solution of the diol 12 in THF (4.8 mL) and the mixture was stirred at RT for 60 minutes. The reaction was extracted with CH2Cl2 the collected organic fractions were washed with brine, dried over Na2SO4 and solvent was removed under vacuum, yielding 2-(2-oxoethyl)benzaldehyde 2a in 87% yield (122 mg, 0.84 mmol), as an unstable oil. Spectroscopic data agree with those reported in the literature.8 1H NMR (300 MHz, CDCl3) δ = 10.05 (s, 1H), 9.80 (s, 1H), 7.85 (dd, J = 7.2, 1.8 Hz, 1H), 7.70 – 7.45 (m, 2H), 7.36 – 7.15 (m, 1H), 4.15 (s, 2H). 13C NMR (75 MHz, CDCl3) δ 198.5, 193.5, 135.8, 134.3, 134.2, 132.9, 128.3, 48.4.
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Synthesis of 2-(3-oxopropyl)benzaldehyde 3a
NaIO4
CH2Cl2/H2O
CHOOH
OH
CHO
1) NaIO4/LiBrAcOH,
2) K2CO3, MeOH
13 14 3a
A mixture of 1,2-dihydronaphtalene 13 (800 mg, 6.1 mmol), NaIO4 (394 mg, 1.84 mmol) and LiBr (106 mg, 1.22 mmol) was dissolved in acetic acid (10 mL) and heated at 95 °C overnight. After cooling, the reaction was diluted with water and a 10% Na2S2O5 solution and extracted with EtOAc; the collected organic fractions were washed with NaHCO3 sat., brine and the solvent was evaporated. The crude was dissolved in MeOH (30 mL) and K2CO3 (1.26 g , 9.1 mmol) was added; the mixture was stirred at room temperature overnight. Then the solvent was removed, the residue was solubilized in NH4Cl sat. and extracted with EtOAc; the collected organic layers were dried over Na2SO4 and solvent was removed under vacuum. The crude was purified by FC (CyHex/EtOAc 7:3), affording the cis-diol 14 in 30% yield (302 mg, 1.8 mmol). Spectroscopic data agree with those reported in literature.91H NMR (300 MHz, CDCl3) δ = 7.47 – 7.40 (m, 1H), 7.25 – 7.20 (m, 2H), 7.16 – 7.10 (m, 1H), 4.70 (s, 1H), 4.09 – 3.96 (m, 1H), 3.04 – 2.91 (m, 1H), 2.86 – 2.70 (m, 1H), 2.37 (s, 1H), 2.12 – 1.76 (m, 1H). 13C NMR (75 MHz, CDCl3) δ 136.5, 136.3, 130.0, 128.8, 128.3, 126.6, 70.1, 69.7, 27.1, 26.4.
A solution of NaIO4 (472 mg, 0.5 mmol) in H2O (3.6 mL) was added to a solution of the diol 14 in CH2Cl2 (18 mL) and the mixture was stirred at RT for 60 minutes. The two phases were then separated and the organic fraction was dried over Na2SO4 and solvent was removed under vacuum, yielding 2-(3-oxopropyl)benzaldehyde in 91% yield (271 mg, 1.67 mmol). Spectroscopic data agree with those reported in the literature.10
1H NMR (300 MHz, CDCl3) δ = 10.17 (s, 1H), 9.83 (t, J = 1.3 Hz, 1H), 7.81 (dd, J = 7.5, 1.6 Hz, 1H), 7.53 (td, J = 7.5, 1.5 Hz, 1H), 7.44 (td, J = 7.5, 1.4 Hz, 1H), 7.33 (dd, J = 7.6, 1.3 Hz, 1H), 3.36 (t, J = 7.4, 2H), 2.80 (td, J = 7.5, 1.3 Hz, 2H) . 13C NMR (75 MHz, CDCl3) δ 201.4, 193.2, 142.9, 134.0, 133.9, 131.2, 127.2, 45.2, 25.9.
Synthesis of 4,5-dihydrobenzo[c]oxepin-1(3H)-one 3b
CHO
CHO cat. NADH
O
O
3a 3b
ADH-114
An aliquot of purified enzyme (30 mg, 1 mg/mL final concentration) was added to a phosphate buffer solution (50 mM, pH 7.0, supplemented with 2 mM MgCl2 and 5 vol% CH3CN) containing the substrate (100 mg, 10 mM final concentration) and the nicotinamide cofactor (0.5 mM). The reaction mixture was incubated at 30 °C and 120 rpm. After 5 h, additional 10 mM of 3a and 0.5 mM of NADH were added; total reaction time 24 h. After incubation, the mixture was extracted with EtOAc, and the
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CHO
CHO
CHO
CHOMeO
crude was purified by FC (CyHex:EtOAc 7:3), yielding 105 mg (0.65 mmol) of lactone 3b (53% isolated yield from 200 mg of starting material).1H NMR (300 MHz, CDCl3) δ = 7.72 (d, J = 7.6 Hz, 1H), 7.49 (t, J = 7.4 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.22 (d, J = 7.5 Hz, 1H), 4.16 (t, J = 6.3 Hz, 2H), 2.91 (t, J = 7.2 Hz, 2H), 2.13 (p, J = 6.8 Hz, 2H). Spectroscopic data agree with literature.11
Synthesis of substituted 1,1´-[biphenyl]-2,2´-dicarbaldehydes 4-10a
Pd cat.
Na2CO3THF/H2O CHO
CHOCHO
Br
BOH
OH
CHO
+
R1R2
R1
R2
4-10a
4a: R1= H; R2 = H5a: R1= 3-OMe; R2 = H6a: R1= 5-OMe; R2 = H7a: R1= 3-F; R2 = H8a: R1= 3'-OMe; R2 = 3-F9a: R1= 5'-OMe; R2 = 3-F10a: R1= 3'-F; R2 = 3-F
X Y
General procedure: a mixture of the bromobenzaldehyde X, Pd catalyst, ligand, Na2CO3 and the formylphenylboronic acid Y were dissolved in THF and H2O (10 %v/v); the reaction was stirred at 60 °C until completion. After cooling, the reaction was diluted with 0.5 N HCl and extracted with EtOAc; the collected organic layers were dried over Na2SO4 and the solvent was removed under vacuum. The crude was purified by FC affording the desired product.
[1,1´-biphenyl]-2,2´-dicarbaldehyde (4a)
Reaction conditions: 2-bromobenzaldehyde (200 mg, 1.08 mmol), 2-formylphenylboronic acid (211 mg, 1.41 mmol), Pd(AcO)2 (12 mg, 0.05 mmol), PPh3 (43 mg, 0.16 mmol), Na2CO3 (229 mg, 2.16 mmol) in THF (5.40 mL THF) and H2O (0.54 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 88% (200 mg, 0.95 mmol).1H NMR (300 MHz, CDCl3) δ = 9.85 (s, 2H), 8.07 (dd, J = 7.6, 1.6 Hz, 2H), 7.71 – 7.64 (m, 2H), 7.61 (t, J = 7.5 Hz, 2H), 7.36 (dd, J = 7.4, 1.4 Hz, 2H). HRMS: 211.0756 (calc. 211.0753). Spectroscopic data agree with literature.12
3-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde (5a)
Reaction conditions: 2-bromo-6-methoxybenzaldehyde (200 mg, 0.93 mmol), 2-formylphenylboronic acid (181 mg, 1.21 mmol), Pd(AcO)2 (10 mg, 0.05 mmol), PPh3 (37 mg, 0.15 mmol), Na2CO3 (197 mg, 1.86 mmol) in THF (4.65 mL THF) and H2O (0.47 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 147 mg (0.61 mmol, 66%). 1H NMR (300 MHz, CDCl3) δ = 10.36 (s, 1H), 9.79 (d, J = 0.7 Hz, 1H), 7.99 (dd, J = 7.7, 1.5 Hz, 1H), 7.66 – 7.46 (m, 3H), 7.22 (dd, J = 7.4, 1.3 Hz, 1H), 7.10 (d, J = 8.5 Hz, 1H), 6.84 (d, J = 7.5 Hz, 1H), 3.99 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 191.6, 190.4, 162.1, 143.8, 141.0, 134.4, 134.1, 133.4, 130.4, 128.1, 128.0, 124.3, 123.8, 111.7, 56.8. HRMS: [M+] 241.0858 (calc. 241.0859).
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CHO
CHO
OMe
CHOOHC
F
OMe
CHO
CHOF
5-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde (6a)
Reaction conditions: 2-bromo-4-methoxybenzaldehyde (200 mg, 0.93 mmol), 2-formylphenylboronic acid (181 mg, 1.21 mmol), Pd(AcO)2 (10 mg, 0.05 mmol), PPh3 (37 mg, 0.15 mmol), Na2CO3 (197 mg, 1.86 mmol) in THF (4.65 mL THF) and H2O (0.47 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 129 mg (0.54 mmol, 58%).1H NMR (300 MHz, CDCl3) δ = 9.85 (s, 1H), 9.67 (s, 1H), 8.06 (dd, J = 7.6, 1.6 Hz, 1H), 8.04 (d, J = 8.7 Hz, 1H), 7.67 (td, J = 7.5, 1.6 Hz, 1H), 7.63 – 7.54 (m, 1H), 7.37 (dd, J = 7.4, 1.2 Hz, 1H), 7.12 – 7.06 (m, 1H), 6.81 (d, J = 2.5 Hz, 1H), 3.91 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 191.2, 189.8, 163.5, 143.8, 141.4, 134.7, 133.6, 131.5, 131.2, 129.0, 128.5, 128.3, 116.8, 114.7, 55.9. HRMS: [M+] 241.0860 (calc. 241.0859). Spectroscopic data agrees with literature.13
3-fluoro-[1,1´-biphenyl]-2,2´-dicarbaldehyde (7a)
Reaction conditions: 2-bromo-6-fluoro-benzaldehyde (200 mg, 0.98 mmol), 2-formylphenylboronic acid (192 mg, 1.28 mmol), Pd(AcO)2 (11 mg, 0.05 mmol), PPh3 (39 mg, 0.15 mmol), Na2CO3 (208 mg, 2.16 mmol) in THF (4.90 mL THF) and H2O (0.49 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 161 mg (0.71 mmol, 72%).1H NMR (300 MHz, CDCl3) δ = 10.17 (s, 1H), 9.82 (s, 1H), 8.06 – 7.92 (m, 1H), 7.76 – 7.49 (m, 3H), 7.37 – 7.18 (m, 2H), 7.09 (d, J = 7.6 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ = 191.1, 187.8 (d, JCF = 6.7 Hz), 164.4 (d, JCF = 261.3 Hz), 141.9 (d, JCF = 1.4 Hz), 141.4 (d, JCF = 2.4 Hz), 134.9 (d, JCF = 10.4 Hz), 134.2, 133.6, 130.8, 129.3, 128.8, 127.7 (d, JCF = 3.6 Hz), 123.2 (d, JCF = 7.1 Hz), 116.7 (d, JCF = 21.5 Hz). HRMS: [M+] 229.0662 (calc. 229.0659).
3-fluoro-3´-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde (8a)
Reaction conditions: 2-bromo-6-methoxybenzaldehyde (225 mg, 1.04 mmol), 3-fluoro-2-formylphenylboronic acid (260 mg, 1.55 mmol), Pd(dppf)Cl2 (20 mg, 0.03 mmol, 0.02 eq.), Na2CO3 (254 mg, 2.40 mmol) in THF (6.00 mL THF) and H2O (0.60 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 244 mg (0.94 mmol, 91%). 1H NMR (300 MHz, CDCl3) δ = 10.38 (d, J = 0.6 Hz, 1H), 10.11 (s, 1H), 7.59 – 7.49 (m, 2H), 7.18 (ddd, J = 10.6, 8.4, 1.1 Hz, 1H), 7.07 (dd, J = 8.5, 0.9 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.74 (d, J = 7.6 Hz, 1H), 3.98 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 190.3, 188.1 (d, JCF = 6.6 Hz), 164.1 (d, JCF = 259.8 Hz), 162.3, 144.5 (d, JCF = 1.6 Hz), 141.1 (d, JCF = 2.4 Hz), 134.7 (d, JCF = 10.4 Hz), 134.6, 126.09 (d, JCF = 3.5 Hz), 123.1, 123.1, 122.6 (d, JCF = 7.3 Hz), 115.8 (d, JCF = 21.5 Hz), 111.6, 56.1. HRMS: 259.0767 (calc. 259.0765).
S17
CHO
CHOF
F
CHO
CHOF
OMe
3-fluoro-5´-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde (9a)
Reaction conditions: 2-bromo-4-methoxybenzaldehyde (200 mg, 0.93 mmol), 3-fluoro-2-formylphenylboronic acid (235 mg, 1.40 mmol), Pd(dppf)Cl2 (16 mg, 0.02 mmol, 0.02 eq.), Na2CO3 (229 mg, 2.16 mmol) in THF (5.40 mL THF) and H2O (0.54 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 197 mg (0.76 mmol, 82%). 1H NMR (300 MHz, CDCl3) δ = 10.14 (s, 1H), 9.66 (d, J = 0.6 Hz, 1H), 7.97 (d, J = 8.7 Hz, 1H), 7.62 (ddd, J = 8.4, 7.6, 5.5 Hz, 1H), 7.32 – 7.21 (m, 1H), 7.14 – 6.94 (m, 2H), 6.72 (d, J = 2.5 Hz, 1H), 3.89 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 189.7, 187.7 (d, JCF = 5.9 Hz), 164.0 (d, JCF = 261.6 Hz), 163.6, 143.7 (d, JCF = 2.4 Hz), 142.0 (d, JCF = 1.4 Hz), 134.8 (d, JCF = 10.4 Hz), 132.0, 127.8, 127.3 (d, JCF = 3.6 Hz), 123.2 (d, JCF = 7.1 Hz), 116.8 (d, JCF = 21.4 Hz), 116.2, 114.1, 55.8. HRMS: 259.0770 (calc. 259.0765).
3,3’-difluoro-[1,1´-biphenyl]-2,2´-dicarbaldehyde (10a)
Reaction conditions: 2-bromo-6-fluorobenzaldehyde (200 mg, 0.98 mmol), 3-fluoro-2-formylphenylboronic acid (215 mg, 1.28 mmol), Pd(AcO)2 (11 mg, 0.05 mmol), PPh3 (39 mg, 0.15 mmol), Na2CO3 (200 mg, 1.96 mmol) in THF (4.90 mL THF) and H2O (0.49 mL). The crude was purified by FC (Cyhex/EtOAc 8:2). Yield: 225 mg (0.91 mmol, 93%).1H NMR (300 MHz, CDCl3) δ = 10.22 (s, 1H), 7.64 – 7.55 (m, 2H), 7.30 – 7.18 (m, 2H), 6.98 (d, J = 7.6 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 187.6 (d, JCF = 8.4 Hz), 164.9 (d, JCF = 259.4 Hz), 142.0 (d, JCF = 2.1 Hz), 135.1 (d, JCF = 10.4 Hz), 126.4 (d, JCF = 3.6 Hz), 122.6 (d, JCF = 7.3 Hz), 116.4 (d, JCF = 21.7 Hz). HRMS: 247.0565 (calc. 247.0565).
Synthesis of substituted dibenzo[c,e]oxepin-5(7H)-ones
ADHcat. NAD(P)H R3
R1O
R3
R1
R3
R1O
O
O
4a-10a 4c-10c4b-10b
R1
HOMeHFFFF
R2
HHOMeHHHH
R3
HHHHOMeHF
4a-c5a-c6a-c7a-c8a-c9a-c10a-c
R2 R2R2
O
O
R4 R4 R4R4
HHHHHOMeH
General procedure: an aliquot of enzyme (final concentration 1 mg/mL) was added to phosphate buffer solution (50 mM, pH 7.0, supplemented with 2 mM MgCl2 and 5 vol% CH3CN) containing 10 mM substrate and the nicotinamide cofactor (0.5 mM). The reaction mixture was incubated at 30 °C and 120 rpm, 16 h. The reaction mixture was then extracted with EtOAc; the collected organic fractions
S18
OOMeO
O
MeO
O
OOF
O
OMeO
F
were dried over Na2SO4 and the solvent was removed under vacuum. The crude was purified by FC affording the desired product.
4-methoxydibenzo[c,e]oxepin-5(7H)-one (5b)
Starting from 48 mg (0.2 mmol) of 5-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde 5a, using ADH-132 and NADH. The crude was purified by FC (toluene/EtOAc 12:1). Yield: 14 mg (0.058 mmol, 29%).1H NMR (300 MHz, CDCl3) δ = 7.67 (d, J = 7.3 Hz, 1H), 7.72 – 7.64 (m, 1H), 7.59 – 7.45 (m, 2H), 7.45 – 7.36 (m, 2H), 7.14 (dd, J = 7.8, 0.9 Hz, 1H), 7.06 (dd, J = 8.4, 0.9 Hz, 1H), 5.07 (d, J = 12.1 Hz, 1H), 4.90 (d, J = 12.1 Hz, 1H), 3.95 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 166.7, 158.6, 138.9, 138.6, 135.4, 132.3, 130.2, 128.8, 128.7, 128.6, 120.7, 120.4, 111.4, 68.8, 56.5. HRMS: [M+] 241.0859 (calc. 241.0859).
2-methoxydibenzo[c,e]oxepin-5(7H)-one (6b)
Starting from 24 mg (0.1 mmol) of 3-methoxy-[1,1´-biphenyl]-2,2´-dicarbaldehyde 6a, using Lk-ADHmut and NADPH. The crude was purified by FC (toluene/EtOAc 10:1). Yield: 16 mg (0.067 mmol, 66%).1H NMR (300 MHz, CDCl3) δ = 7.98 (d, J = 8.6 Hz, 1H), 7.68 – 7.60 (m, 1H), 7.58 – 7.50 (m, 1H), 7.49 – 7.40 (m, 2H), 7.11 – 6.95 (m, 2H), 5.06 (bs, 1H), 4.97 (bs, 1H), 3.93 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 170.1, 162.7, 139.4, 139.0, 135.0, 134.4, 130.1, 128.8, 128.6, 123.1, 114.1, 113, 69.1, 55.6. HRMS: [M+] 241.0855 (calc. 241.0859). Spectroscopic data agree with literature.14
8-fluorodibenzo[c,e]oxepin-5(7H)-one (7c)
Starting from 23 mg (0.1 mmol) 3-fluoro-[1,1´-biphenyl]-2,2´-dicarbaldehyde 7a, using Lk-ADHmut and NADPH. The crude was purified by FC (CyHex/EtOAc 8:2). Yield: 17 mg (0.074 mmol, 74%).1H NMR (300 MHz, CDCl3) δ = 8.00 (dd, J = 7.7, 1.2 Hz, 1H), 7.76 – 7.37 (m, 5H), 7.23 – 7.11 (m, 1H), 5.51 (s, 1H), 4.80 (s, 1H). 13C NMR (75 MHz, CDCl3): δ = 170.2, 159.6 (d, JCF = 249.7 Hz), 141.6 (d, JCF = 3.0 Hz), 136.3 (d, JCF = 3.0 Hz), 132.8, 132.2, 131.3 (d, JCF = 9.0 Hz), 130.8, 129.2 , 128.9, 124.3 (d, JCF = 3.5 Hz), 122.6 (d, JCF = 15.8 Hz), 115.5 (d, JCF = 22.3 Hz), 60.6 (d, JCF = 5.9 Hz). HRMS: [M+] 229.0660 (calc. 229.0659).
8-fluoro-4-methoxydibenzo[c,e]oxepin-5(7H)-one (8c)
Starting from 77 mg (0.30 mmol) of 3-fluoro-3'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 8a, enzyme: ADH132 (Cfin: 2 mg/mL), NADH. The crude was purified by FC (toluene/EtOAc 12:1). Yield: 22 mg (0.85 mmol, 28%).
S19
OO
F
F
OO F
MeO
1H NMR (300 MHz, CDCl3) δ = 7.56 (t, J = 8.1 Hz, 1H), 7.49 – 7.41 (m, 2H), 7.18 – 7.05 (m, 3H), 5.43 (d, J = 12.5 Hz, 1H), 4.79 (d, J = 12.5 Hz, 1H), 3.95 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 166.6, 159.7 (d, JCF = 249.5 Hz), 158.6, 141.3 (d, JCF = 3.0 Hz), 137.5 (d, JCF = 2.7 Hz), 132.5, 131.2 (d, JCF = 9.0 Hz), 124.2 (d, J = 3.4 Hz), 123.0 (d, JCF = 16.0 Hz), 120.7, 120.2, 115.5 (d, JCF = 22.3 Hz), 111.9, 60.1 (d, JCF = 5.7 Hz), 56.5. HRMS: [M+] 259.0764 (calc. 259.0764).
8-fluoro-2-methoxydibenzo[c,e]oxepin-5(7H)-one (9c)
Starting from 52 mg (0.2 mmol) of 3-fluoro-5'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 9a using Lk-ADHmut and NADPH. The crude was purified by FC (toluene/EtOAc 13:1). Yield: 30 mg (0.12 mmol; 58 %).1H NMR (300 MHz, CDCl3) δ = 7.99 (d, J = 8.8 Hz, 1H), 7.58 – 7.37 (m, 2H), 7.18 (t, J = 8.4 Hz, 1H), 7.07 (s, 2H), 5.46 (s, 1H), 4.83 (s, 1H), 3.94 (s, 3H). 13C NMR (75 MHz, CDCl3): δ = 170.1, 162.8, 159.6 (d, JCF = 249.6 Hz), 141.5 (d, JCF = 3.1 Hz), 138.4 (d, JCF = 3.0 Hz), 134.7, 131.2 (d, JCF = 8.9 Hz), 124.3 (d, JCF = 3.4 Hz), 123.1, 122.7 (d, JCF = 15.9 Hz), 115.7 (d, JCF = 22.2 Hz), 114.6, 114.1, 60.5 (d, JCF = 5.7 Hz), 55.8. HRMS: [M+] 259.0766 (calc. 259.0764).
4,8-difluorodibenzo[c,e]oxepin-5(7H)-one (10c)
Starting from 100 mg (0.41 mmol) of 3,3’-difluoro-[1,1´-biphenyl]-2,2´-dicarbaldehyde 10a, using Lk-ADHmut and NADPH. The crude was purified by FC (CyHex/EtOAc 8:2). Yield: 57 mg (0.23 mmol, 57%).1H NMR (300 MHz, CDCl3) δ = 7.63 (td, J = 8.1, 5.4 Hz, 1H), 7.56 – 7.42 (m, 2H), 7.39 (d, J = 7.8 Hz, 1H), 7.35 – 7.23 (m, 1H), 7.27 – 7.14 (m, 1H), 5.50 (d, J = 12.6 Hz, 1H), 4.82 (d, J = 12.6 Hz, 1H). 13C NMR (75 MHz, CDCl3): δ = 161.3 (d, JCF = 258.0 Hz), 159.7 (d, JCF = 250.1 Hz), 140.4 (d, JCF = 2.6 Hz), 138.0 (d, JCF = 2.8 Hz), 133.3 (d, JCF = 9.6 Hz), 131.6 (d, JCF = 8.9 Hz), 124.4 (d, JCF = 6.1 Hz), 124.3 (d, JCF = 6.1 Hz), 122.7 (d, JCF = 16.1 Hz), 119.5 (d, JCF = 12.9 Hz), 116.9 (d, JCF = 22.2 Hz), 116.1 (d, JCF = 22.3 Hz), 60.4 (d, JCF = 5.8 Hz). HRMS: [M+] 247.0564 (calc. 247.0565).
One-pot synthesis of dibenzo[c,e]oxepin-5(7H)-one (4b)
ADHcat. NAD(P)H
OO
O4a 4b
Br
O O
B(OH)2+
Pd(dppf)Cl2
Na2CO3, H2O90 °C
KPi buffer50 mM, pH 7.0
30 °C
dilution and pH adjustment
O
S20
2-Bromobenzaldehyde (50 mg, 0.27 mmol), Pd(dppf)Cl2 (4 mg, 0.05mmol, 0.02 eq.), Na2CO3 (85 mg, 0.81 mmol, 3 eq.) and 2-formylphenylboronic acid (45 mg, 0.30 mmol, 1.1 eq.) were dissolved in degassed H2O; the reaction was stirred at 90 °C until completion (checked by GC-MS). After cooling, the reaction was diluted with phosphate buffer (50 mM, pH 7.0, 2 mM MgCl2) to a final volume of 18 mL and supplemented with 5% vol. CH3CN as cosolvent; the enzyme (Lk-ADHmut, final concentration: 1 mg/mL) and the cofactor (NADPH, final concentration 0.5 mM) were added and the reaction was incubated at 30 °C, 120 rpm for 18 hours. The biotransformation was extracted with EtOAc; the collected organic layers were dried over Na2SO4 and the solvent was removed under vacuum. The crude was purified by FC (Cyhex/EtOAc 8:2) affording 27 mg of the desired product (0.13 mmol, 48% isolated yield).1H NMR (300 MHz, CDCl3) δ = 8.01 (d, J = 8.2 Hz, 1H), 7.74 – 7.61 (m, 3H), 7.60 – 7.52 (m, 2H), 7.51 – 7.42 (m, 2H), 5.05 (d, J = 15.8 Hz, 2H). 13C NMR (75 MHz, CDCl3): δ = 170.4, 139.1, 137.4, 134.9, 132.7, 132.1, 130.8, 130.3, 128.8, 128.8, 128.7, 128.7, 128.6, 69.3. Spectroscopic data agree with literature.15
S21
GC analysesGC analyses were carried out on an Agilent 7890A, with FID detector, equipped with an Agilent HP5 column (30 m x 0.25 mm x 0.25 m; method 1) or an Agilent DB-1701 column (30 m x 0.25 mm x 0.25 m; method B and C), using H2 as carrier gas; injector temperature 250 °C, detector temperature 250 °C.
Method A: flow 1.4 mL/min, injection volume 5 µL, split ratio 50:1; temperature program: 100 °C, hold 1 min; 10 °C/min to 150 °C, hold 0 min, 20 °C/min to 300 °C, hold 1 min.
Method B: flow 1.0 mL/min, injection volume 5 µL, split ratio 20:1; temperature program: 100 °C, hold 5 min; 10 °C/min to 230 °C, hold 0 min, 20 °C/min to 280 °C, hold 2 min.
Method C: flow 1.0 mL/min, injection volume 5 µL, split ratio 20:1; temperature program: 100 °C, hold 1 min; 10 °C/min to 280 °C, hold 5 min.
Compounds 1a-b
Retention times (method A): 4.9 min 1a; 6.2 min 1b.
o-phthalaldehyde 1a (purchased):
Phthalide 1b (purchased):
Internal
standard
Internal
standard
S22
Biotransformation:
Compounds 2a-c
Retention times (method B): 13.7 min 2a; 16.2 min 2b; 16.5 min 2c.
2-(2-oxoethyl)benzaldehyde 2a:
Isochroman-1-one 2b (purchased):
Isochroman-3-one 2c (purchased):
Internal
standard
S23
Biotransformation:
Compounds 3a-c
Retention times (method B): 15.1 min 3a; 16.8 min 3b; 18.4 min 3c.
2-(3-oxopropyl)benzaldehyde 3a:
4,5-dihydrobenzo[c]oxepin-1(3H)-one 3b:
Biotransformation:
S24
Compounds 4a-b
Retention times (method C): 16.6 min 4a; 19.3 min 4b.
[1,1'-biphenyl]-2,2'-dicarbaldehyde 4a:
dibenzo[c,e]oxepin-5(7H)-one 4b (purchased):
Biotransformation:
Compounds 5a-c
Retention times (method C): 16.7 min 5a; 18.2 min 5b.
S25
3-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 5a:
4-methoxydibenzo[c,e]oxepin-5(7H)-one 5b:
Biotransformation:
Compounds 6a-b
Retention times (method C): 16.7 min 6a; 18.6 min 6c, 18.9 min 6b.
5-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 6a:
S26
2-methoxydibenzo[c,e]oxepin-5(7H)-one 6b: 18.9 min
Biotransformation:
Compounds 7a-c
Retention times (method C): 13.7 min 7a; 15.8 min 7c; 16.7 min 7b.
3-fluoro-[1,1'-biphenyl]-2,2'-dicarbaldehyde 7a:
8-fluorodibenzo[c,e]oxepin-5(7H)-one 7c:
S27
Biotransformation:
Compounds 8a-c
Retention times (method C): 16.0 min 8a; 17.8 min 8c.
3-fluoro-3'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 8a:
8-fluoro-4-methoxydibenzo[c,e]oxepin-5(7H)-one 8c:
Biotransformation:
S28
Compounds 9a-c
Retention times (method C): 16.5 min 9a; 18.5 min 9c.
3-fluoro-5'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 9a:
8-fluoro-2-methoxydibenzo[c,e]oxepin-5(7H)-one 9c:
Biotransformation:
Compounds 10a-c
Retention times (method C): 12.9 min 10a; 16.3 min 10c.
S29
3,3‘-difluoro-[1,1'-biphenyl]-2,2'-dicarbaldehyde 10a:
4,8-difluorodibenzo[c,e]oxepin-5(7H)-one 10c:
Biotransformation:
S30
NMR spectra2-(2-oxoethyl)benzaldehyde 2a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)
2.00
2.46
2.31
1.09
0.92
0.96
4.1
5
7.5
27.5
27.5
47.5
57.5
77.5
77.5
77.5
87.5
97.6
07.6
27.8
37.8
47.8
67.8
67.8
6
9.8
1
10.0
5
102030405060708090100110120130140150160170180190200210f1 (ppm)
48.3
128.2
132.8
134.0
134.1
135.6
193.3
198.3
S31
2-(3-oxopropyl)benzaldehyde 3a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)
0.1
91.9
5
2.0
7
1.0
11.0
11.0
01.0
0
0.8
7
0.8
8
2.6
92.7
12.7
42.7
72.7
72.7
92.8
02.8
22.8
23.3
33.3
63.3
8
7.3
17.3
27.3
47.3
47.4
17.4
27.4
47.4
47.4
67.4
77.5
07.5
17.5
37.5
37.5
57.5
67.8
07.8
07.8
27.8
39.8
29.8
39.8
310.1
710.2
1
0102030405060708090100110120130140150160170180190200210f1 (ppm)
25.9
45.2
127.2
131.5
133.9
134.0
134.7
142.9
193.2
201.4
S32
4,5-dihydrobenzo[c]oxepin-1(3H)-one 3b
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
2.0
8
2.0
1
2.0
2
0.9
70.9
81.0
00.9
6
2.1
12.1
32.1
52.1
72.2
0
2.9
02.9
32.9
5
4.1
64.1
84.2
0
7.2
37.2
57.3
67.3
97.4
17.4
87.4
87.5
17.5
37.7
37.7
5
[1,1'-biphenyl]-2,2'-dicarbaldehyde 4a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
2.1
42.2
52.3
02.1
4
2.0
2
7.3
77.3
77.3
97.4
07.6
07.6
37.6
57.6
77.6
87.7
07.7
07.7
27.7
38.0
88.0
88.1
08.1
1
9.8
6
S33
dibenzo[c,e]oxepin-5(7H)-one 4b
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
2.0
3
1.9
91.9
83.0
00.9
8
5.0
25.0
7
7.4
37.4
57.4
87.4
87.5
07.5
27.5
37.5
57.5
77.5
97.5
97.6
27.6
47.6
67.6
77.6
87.6
97.6
97.7
07.7
27.7
38.0
08.0
3
0102030405060708090100110120130140150160170180190f1 (ppm)
69.4
128.6
128.7
128.8
128.8
128.9
130.3
130.8
132.1
132.8
135.0
137.4
139.1
170.4
S34
3-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 5a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)
3.2
9
1.0
11.1
01.0
13.2
7
1.0
2
0.8
7
0.7
4
3.9
9
6.8
26.8
57.0
97.1
17.2
07.2
17.2
37.2
37.4
97.5
17.5
47.5
67.5
67.5
77.5
97.6
07.6
27.6
27.9
87.9
88.0
08.0
08.0
19.7
99.7
9
10.3
6
0102030405060708090100110120130140150160170180190200f1 (ppm)
56.2
111.7
123.8
124.3
128.0
128.1
130.4
133.4
134.1
134.4
141.0
143.8
162.1
190.4
191.6
S35
4-methoxydibenzo[c,e]oxepin-5(7H)-one 5b
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
3.0
3
1.0
21.0
0
1.0
21.0
32.2
62.0
21.0
2
3.9
54.8
84.9
25.0
55.0
97.0
47.0
47.0
77.0
77.1
27.1
37.1
57.1
57.3
97.4
07.4
07.4
17.4
27.4
27.4
37.4
37.4
57.4
97.5
07.5
17.5
17.5
27.5
27.5
37.5
47.5
57.5
77.6
57.6
8
0102030405060708090100110120130140150160170180190200f1 (ppm)
56.5
68.9
111.4
120.4
120.8
128.6
128.7
128.8
130.2
132.3
135.4
138.6
138.9
158.6
166.8
S36
3.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.6f2 (ppm)
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
f1 (
ppm
)
{7.43,5.05}{7.43,4.91}
{7.07,3.95} {7.04,3.95}
2D NOESY
3.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0f2 (ppm)
50
60
70
80
90
100
110
120
130
140
150
160
170
f1 (
ppm
)
{4.87,167.08}{7.15,166.92}
{5.05,166.80}
{4.91,166.80}{7.04,166.75}
{7.07,166.66} {5.09,166.64}
{7.53,158.78} {3.95,158.54}
{4.94,138.95}
{7.15,138.91}
{7.65,138.90}
{5.05,138.88} {4.91,138.83}
{5.09,138.71}
{7.43,135.44}
{7.66,128.95} {4.88,128.82} {4.93,128.68}
{7.06,120.73}{7.14,120.29}
{7.14,111.33}
{7.43,68.38}
HMBC
S37
5-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 6a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)
3.2
9
1.0
11.1
01.0
13.2
7
1.0
2
0.8
7
0.7
4
3.9
9
6.8
26.8
57.0
97.1
17.2
07.2
17.2
37.2
37.4
97.5
17.5
47.5
67.5
67.5
77.5
97.6
07.6
27.6
27.9
87.9
88.0
08.0
08.0
19.7
99.7
9
10.3
6
0102030405060708090100110120130140150160170180190f1 (ppm)
56.2
111.7
123.8
124.3
128.0
128.1
130.4
133.4
134.1
134.4
141.0
143.8
162.1
190.4
191.6
S38
4-methoxydibenzo[c,e]oxepin-5(7H)-one 6b
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0f1 (ppm)
-2000
-1000
0
1000
2000
3000
4000
5000
6000
7000
8000
9000
10000
11000
12000
13000
14000
15000
16000
3.1
7
2.0
9
2.0
0
1.9
01.0
21.0
80.9
7
3.9
54.9
95.0
87.0
47.0
57.0
77.0
87.0
97.1
07.4
57.4
57.4
77.4
77.4
87.4
97.5
37.5
47.5
57.5
57.5
67.5
67.5
77.5
87.6
47.6
57.6
57.6
77.9
88.0
1
102030405060708090100110120130140150160170180190f1 (ppm)
55.8
69.3
113.9
114.2
123.2
128.7
128.7
128.9
130.2
134.6
135.1
139.1
139.5
162.8
170.3
S39
6.16.26.36.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.98.08.18.28.38.4f2 (ppm)
70
80
90
100
110
120
130
140
150
160
170
f1 (
ppm
)
{7.961,170.317}
{7.063,163.057} {7.021,162.764}{7.976,162.719}
{7.088,162.585}
{7.624,139.472}
{7.976,139.366}
{7.088,139.016}
{7.516,138.968}
{7.642,134.981} {7.454,134.961}
{7.449,129.566}
{7.011,123.282}
{7.077,123.071}
{7.993,114.248} {7.058,114.059}{7.962,113.714}
{7.459,69.144}
HMBC
6.46.56.66.76.86.97.07.17.27.37.47.57.67.77.87.98.08.18.28.3f2 (ppm)
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
f1 (
ppm
)
{7.628,7.073}{7.997,7.035}
{7.454,4.970}
{7.074,3.928}
2D NOESY
S40
3-fluoro-[1,1'-biphenyl]-2,2'-dicarbaldehyde 7a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.512.0f1 (ppm)
1.0
02.3
03.1
91.0
0
0.9
5
0.9
4
7.0
87.1
07.2
47.2
47.2
47.2
57.2
67.2
77.2
77.2
87.2
87.3
17.3
17.5
57.5
67.5
67.5
67.5
87.5
87.5
87.5
97.6
07.6
17.6
17.6
17.6
27.6
27.6
37.6
37.6
47.6
57.6
67.6
77.6
77.9
98.0
08.0
18.0
28.0
29.8
29.8
210.1
7
0102030405060708090100110120130140150160170180190200f1 (ppm)
116.6
116.9
123.1
123.2
127.7
127.7
128.8
129.4
130.8
133.6
134.2
134.8
135.0
141.4
141.4
141.9
142.0
162.6
166.1
187.7
187.8
191.2
S41
8-fluorodibenzo[c,e]oxepin-5(7H)-one 7c
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
0.9
9
0.9
8
0.9
9
5.1
8
1.0
0
4.8
07.1
87.2
17.2
17.4
17.4
27.4
47.4
47.4
77.4
87.4
97.5
17.5
37.5
47.5
67.5
67.5
87.5
97.5
97.6
07.6
27.6
27.6
67.6
77.6
87.6
97.9
87.9
98.0
18.0
1
0102030405060708090100110120130140150160170180f1 (ppm)
60.6
60.6
115.4
115.7
122.5
122.7
124.3
124.3
128.9
129.1
130.8
131.3
131.4
132.2
132.8
136.3
136.4
141.5
141.6
158.0
161.3
170.2
S42
3-fluoro-3'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 8a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)
3.1
4
1.0
11.0
11.0
61.0
72.1
6
0.9
7
0.9
8
3.9
86.7
36.7
66.9
36.9
67.0
67.0
67.0
97.0
97.1
47.1
57.1
77.1
87.1
87.1
87.2
17.2
17.5
07.5
27.5
27.5
37.5
37.5
47.5
57.5
57.5
67.5
77.5
810.1
110.3
810.3
8
0102030405060708090100110120130140150160170180190f1 (ppm)
56.1
111.6
115.6
115.9
122.6
122.7
123.1
123.1
126.1
126.1
134.6
134.6
134.8
141.1
141.2
144.5
144.5
162.3
162.4
165.8
188.0
188.1
190.3
S43
8-fluoro-4-methoxydibenzo[c,e]oxepin-5(7H)-one 8c
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
3.0
2
1.0
0
1.0
2
3.0
11.9
51.1
0
3.8
8
4.6
94.7
04.7
34.7
4
5.3
45.3
87.0
07.0
07.0
27.0
37.0
47.0
57.0
67.0
77.0
77.0
87.0
87.1
07.1
17.3
57.3
77.3
77.3
87.3
87.3
97.4
17.4
67.4
87.5
1
0102030405060708090100110120130140150160170180190f1 (ppm)
56.5
60.1
60.2
111.9
115.4
115.7
120.2
120.7
122.9
123.1
124.2
124.2
131.1
131.3
132.5
137.5
137.5
141.3
141.3
158.0
158.6
161.3
166.6
S44
3.54.04.55.05.56.06.57.07.5f2 (ppm)
50
60
70
80
90
100
110
120
130
140
150
160
170
180
190
f1 (
ppm
)
{7.069,166.915} {5.430,166.729} {4.811,166.522}{5.412,161.486}{7.444,161.364} {4.806,161.231}
{5.440,141.241} {4.811,141.206}
{4.769,123.113}
{5.453,122.786}
HMBC
3.43.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.88.0f2 (ppm)
3.0
3.5
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
f1 (
ppm
)
{7.096,3.962} {7.066,3.958}
2D NOESY
S45
3-fluoro-5'-methoxy-[1,1'-biphenyl]-2,2'-dicarbaldehyde 9a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5f1 (ppm)
3.1
6
1.0
22.0
81.7
91.0
91.0
4
0.9
9
0.9
9
3.9
1
6.7
46.7
47.0
67.0
77.0
97.1
07.1
07.1
37.2
57.2
67.2
87.2
97.3
27.3
27.6
07.6
17.6
27.6
27.6
47.6
47.6
57.6
77.9
78.0
09.6
8
10.1
6
-100102030405060708090100110120130140150160170180190200210f1 (ppm)
55.8
77.6
114.1
116.2
116.6
116.9
123.2
123.3
127.3
127.3
127.8
132.0
134.8
134.9
142.0
142.0
143.7
143.7
162.3
163.6
165.8
187.7
187.8
189.7
S46
8-fluoro-2-methoxydibenzo[c,e]oxepin-5(7H)-one 9c
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
-500
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
3.0
7
1.1
4
1.1
7
1.9
91.1
21.1
01.1
4
1.1
0
3.9
6
4.8
5
5.4
8
7.0
97.1
77.2
07.2
37.4
27.4
57.4
87.5
07.5
27.5
57.9
98.0
2
0102030405060708090100110120130140150160170180190200f1 (ppm)
55.8
60.5
60.5
114.1
114.6
115.5
115.8
122.6
122.8
123.1
124.3
124.3
131.2
131.3
134.7
138.4
138.4
141.5
141.5
158.0
161.3
162.9
170.1
S47
3,3‘-difluoro-[1,1'-biphenyl]-2,2'-dicarbaldehyde 10a
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0f1 (ppm)
2.0
12.6
82.3
7
1.9
1
6.9
66.9
97.2
17.2
17.2
47.2
47.2
57.2
57.2
67.2
77.2
87.5
67.5
87.5
87.5
97.6
07.6
17.6
17.6
3
10.2
2
0102030405060708090100110120130140150160170180190200f1 (ppm)
116.3
116.6
122.5
122.6
126.4
126.5
135.0
135.2
142.0
142.0
163.2
166.6
187.5
187.6
S48
4,8-difluorodibenzo[c,e]oxepin-5(7H)-one 10c
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.5f1 (ppm)
1.0
5
1.0
4
1.0
31.3
31.0
32.1
41.0
7
4.8
04.8
55.4
87.2
07.2
07.2
17.2
67.2
67.2
97.2
97.2
97.3
27.3
27.3
77.3
77.3
87.4
07.4
07.4
07.4
47.4
67.4
77.4
87.5
07.5
17.5
27.5
97.6
17.6
27.6
3
0102030405060708090100110120130140150160170180f1 (ppm)
60.4
60.4
116.0
116.3
116.7
117.0
119.4
119.6
122.6
122.8
124.3
124.3
124.4
124.4
131.5
131.6
133.2
133.4
138.0
138.1
140.4
140.4
158.1
159.6
161.4
163.0
S49
cis-2,3-dihydro-1H-indene-1,2-diol 12
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.0f1 (ppm)
2.9
02.9
02.9
12.9
12.9
62.9
73.0
23.0
63.0
73.0
83.0
93.1
23.1
23.1
43.1
44.4
34.4
5
4.9
44.9
6
7.2
57.2
57.2
67.2
67.2
67.2
77.2
87.2
97.2
97.3
07.4
17.4
27.4
37.4
4
0102030405060708090100110120130140150160170180190f1 (ppm)
38.5
6
73.4
675.9
6
125.0
6125.3
6127.1
7128.8
1
140.1
5141.9
4
S50
cis-1,2,3,4-tetrahydronaphthalene-1,2-diol 14
0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)
2.1
4
1.9
1
1.0
51.0
5
1.0
2
1.0
2
1.0
01.9
11.0
1
1.6
31.9
01.9
11.9
21.9
21.9
31.9
31.9
31.9
41.9
41.9
41.9
51.9
51.9
51.9
61.9
61.9
71.9
71.9
82.0
02.0
12.0
32.0
42.0
62.0
62.0
82.3
72.7
32.7
92.8
12.8
22.8
42.9
32.9
42.9
62.9
83.0
04.0
04.0
24.0
44.7
07.1
17.1
37.1
37.1
47.2
27.2
37.2
37.2
47.2
57.4
27.4
37.4
47.4
47.4
47.4
5
0102030405060708090100110120130140150160170180190200f1 (ppm)
26.4
27.1
69.7
70.1
126.6
128.3
128.7
130.0
136.3
136.5
S51
References1. W. C. Still, M. Kahn and A. Mitra, J. Org. Chem., 1978, 43, 2923.2. E. Tassano, K. Faber and M. Hall, Adv. Synth. Catal., 2018, 360, 2742.3. C. Wuensch, H. Lechner, S. M. Glueck, K. Zangger, M. Hall and K. Faber, ChemCatChem,
2013, 5, 1744.4. Germany Pat., US 20140199735, 2015.5. US Pat., US 20140199735, 2014.6. I. Constantinides and R. S. Macomber, J. Org. Chem., 1992, 57, 6063.7. V. Laserna, G. Fiorani, C. J. Whiteoak, E. Martin, E. Escudero-Adan and A. W. Kleij, Angew.
Chem. Int. Ed., 2014, 53, 10416.8. H. Mang, J. Gross, M. Lara, C. Goessler, H. E. Schoemaker, G. M. Guebitz and W. Kroutil,
Angew. Chem. Int. Ed., 2006, 45, 5201.9. L. Emmanuel, T. M. A. Shaikh and A. Sudalai, Org. Lett., 2005, 7, 5071.10. X. Baucherel, J. Uziel and S. Juge, J. Org. Chem., 2001, 66, 4504.11. T. Morimoto, M. Fujioka, K. Fuji, K. Tsutsumi and K. Kakiuchi, J. Organomet. Chem., 2007,
692, 625.12. Y. A. Chen and C. Y. Liu, Rsc Advances, 2015, 5, 74180.13. Y. Xia, Z. X. Liu, Q. Xiao, P. Y. Qu, R. Ge, Y. Zhang and J. B. Wang, Angew. Chem. Int. Ed.,
2012, 51, 5714.14. X. S. Zhang, Y. F. Zhang, Z. W. Li, F. X. Lao and Z. J. Shi, Angew. Chem. Int. Ed., 2015, 54,
5478.15. R. A. Smith, G. Y. Fu, O. McAteer, M. Z. Xu and W. R. Gutekunst, J. Am. Chem. Soc., 2019,
141, 1446.