Chemoenzymatic Total Synthesis of the Proposed ... - ACS Publications

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Chemoenzymatic Total Synthesis of the Proposed Structures of Putaminoxins B and D Carolin Bisterfeld,†,§ Claudia Holec,†,§ Dietrich Böse,† Patrick Marx,‡ and Jörg Pietruszka*,†,‡ †

Institut für Bioorganische Chemie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Stetternicher Forst, Geb. 15.8, 52426 Jülich, Germany ‡ Institut für Bio- und Geowissenschaften (IBG-1: Biotechnologie), Forschungszentrum Jülich, 52425 Jülich, Germany S Supporting Information *

ABSTRACT: Different enzymatic and nonenzymatic approaches were tested and compared to afford enantiopure homoallylic and allylic alcohols as building blocks in a total synthesis showcase. Thereby, highly enantioselective alcohol dehydrogenases and the P450 BM3 monooxygenase variant A74G L188Q were compared to classical asymmetric reagentcontrolled allyl additions. Thus, the first total syntheses of the proposed structures for putaminoxins B/D and their respective enantiomers were accomplished. Detailed spectroscopic analysis of the newly synthesized compounds unraveled a discrepancy with respect to the reported structures of putaminoxins B/D. Furthermore, it was demonstrated that total synthesis is generally required for unequivocal assignment of configuration, because purely comparative NMR studies and judgment by analogy can lead to false predictions.

I

show and compare several routes toward common building blocks such as homoallylic and allylic alcohols (Scheme 1) in the context of a natural product synthesis. For the synthetic preparation of homoallylic alcohol 1, three different approaches are depicted in Scheme 1. Enzymatically, an ADH can be applied in either an asymmetric reduction of ketone 2 or an oxidative kinetic resolution of the racemic alcohol rac-1. The so obtained results will be compared to a classical chemical asymmetric allylation of hexanal (3) with the Brown and Leighton reagents, respectively. The synthesis of the second building block, allylic alcohol 4, will be performed enzymatically by reducing either ketone 5 with ADHs or by a P450 BM3 monooxygenase variant catalyzing an allylic hydroxylation of olefin 6. Here, we utilize the shown building blocks 1 and 4 to showcase the total synthesis of the proposed structures of the natural products putaminoxins B/D (7, Figure 1).20,21 The compounds 7 are C-9 epimers, but the C-9 configuration was not established for either proposed epimer. Despite the fact that these compounds present an interesting structural pattern, they have not been synthesized before, resulting in an unassigned stereogenic center at C-9. The (S)-configuration of C-5 was assigned by comparison of the coupling constants with putaminoxin and was never confirmed experimentally.20,21 Moreover, no optical rotation was determined for these compounds. Overall, the insufficient data make these

n the past decade, enzymatic catalysis has been successfully established as an alternative to conventional chemical transformations.1−5 By considering it more a supplement than a replacement to classic synthetic transformations, a treasure trove of new options became available, resulting in elegant routes toward complex compounds. Thus, looking at a target, enzymatic catalysis should not be neglected in the retrosynthetic analysis.6 Chiral homoallylic alcohols are wellknown motifs for a variety of natural products and can be synthesized by “chemical” allyl addition to an aldehyde or by enzymatic transformations in terms of asymmetric reduction of a prochiral ketone or kinetic resolution of the respective racemic alcohol.7−12 A decision in favor of one route over the other in terms of practicability, efficiency, and convenience is often not obvious. Chemical methods for the synthesis of chiral homoallylic alcohols often rely on complex ligands.13−15 Additionally, excellent enantioselectivity is still not easy to achieve when dealing with linear alkyl-substituted aldehydes (i.e., hexanal).14,16 In previous studies, we presented alcohol dehydrogenases (ADHs) as convenient biocatalysts for the synthesis of chiral alcohols (e.g., homoallylic and allylic alcohols) via oxidative kinetic resolutions (OKRs) as well as asymmetric reductions.17,18 In addition, aiming at the synthesis of chiral allylic alcohols, the P450 BM3 monooxygenase variant A74G L188Q was described for stereoselective hydroxylations of linear terminal olefins representing a short and elegant route toward these compounds.19 At the same time, known chemical methods for direct C−H activations are limited and often result in low selectivity. Here, in a methodical approach we want to © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 6, 2017 Published: April 26, 2017 1563

DOI: 10.1021/acs.jnatprod.7b00101 J. Nat. Prod. 2017, 80, 1563−1574

Journal of Natural Products

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Scheme 1. Synthetic Options to Obtain (A) Homoallylic and (B) Allylic Alcohols

can be afforded by a cross metathesis of the building blocks 12 and 13 (Scheme 2). To obtain these building blocks with high enantiomeric purity and in high yield, we investigated the aforementioned routes toward the building blocks by applying conventional chemical methods as well as enzyme catalysis.

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RESULTS AND DISCUSSION

Synthesis of Fragment A. Starting with fragment 12, different methods for the stereoselective allylation of aldehydes are known. In order to compare conventional chemical reactions with enzyme catalysis, asymmetric allylations according to Brown and Leighton were performed. The Ballyldiisopinocampheylboranes were introduced by Brown and co-workers in the 1980s for the allylation of various substrates with high enantioselectivities.16,28 Even though the reagent 14 can easily be purchased from a commercial source, the reaction protocol is challenging. The allylation was performed at −100 °C and under strictly anhydrous conditions.14 Starting with hexanal (3) and either (−)- or (+)-14, we obtained the (R)and (S)-alcohol 1 with 31−39% yield and an enantiomeric excess (ee) of 90% in both cases (Scheme 3A, Table 1, entries 1, 2). For the asymmetric allylation according to Leighton and coworkers, reagent 15 was employed.13 After several trials with the commercially purchased reagent, we were not able to observe any product formation. Thus, the reagent was freshly prepared and, by carefully keeping it under an argon atmosphere, directly used for the allyl addition.13 After a reaction time of 7 days, (R)-alcohol 1 was isolated in 24% yield with a good ee of 93% (Scheme 3B, Table 1, entry 3). Repeatedly, the allylation with hexanal gave a surprisingly low

Figure 1. Proposed structures for putaminoxins B/D (7) and related nonenolides.

compounds challenging targets for our synthesis. Putaminoxins B/D are nonenolides, secondary metabolites with a 10membered macrolactone core structure. The disubstituted putaminoxins were isolated in the 1990s from the fungus Phoma putaminum, a pathogen causing leaf necrosis of the weed Erigeron annuus.20−22 In structure−activity studies, the phytotoxicity of these compounds has been reported.23 Syntheses of the macrolide core of putaminoxins B/D or the structurally closely related nonenolides putaminoxin (8), hypocreolide A (9), and putaminoxin E (10) were achieved with either a ring closing metathesis or a macrolactonization as a key step (Figure 1).24−27 In this study a macrolactonization strategy was envisioned to complete the total synthesis of the proposed structures of putaminoxins B/D (7) and their respective C-5 epimers.27 Hence, retrosynthetic analysis resulted in fragment 11, which

Scheme 2. Retrosynthetic Analysis of the Macrolide 7 (PG = Suitable Protecting Group)

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Scheme 3. Asymmetric Allyl Addition to Aldehyde 3 with (A) the Brown Reagent 14 and (B) the Leighton Reagent 15 to Afford Homoallylic Alcohol 1a

a

Reaction conditions: (a) (+)-14 or (−)-14, Et2O, −100 °C, 3 h; (b) (R,R)-15, CH2Cl2, −10 °C, 7 d.

TbADH (Prelog-ADH) and an 2-propanol-coupled NADP+regeneration system was performed, and the desired (R)alcohol 1 was obtained in 68% yield and with an excellent ee of >99% (Scheme 4A; Table 1, entry 4). As the ADH from Lactobacillus brevis (LbADH) is known to give opposite selectivity relative to the TbADH (anti-Prelog),30,31 it was chosen for the synthesis of the (S)-enantiomer of compound 1. Unfortunately, with substrate 2 an ee of only 42% was observed in favor of alcohol (S)-1 (Table 1, entry 5). However, we recently applied the TbADH in an oxidative kinetic resolution of substrate rac-1 using the P450 BM3 monooxygenase variant F87A as a NAD(P)H-oxidase for regeneration of the nicotinamide cofactor. Here, we exploited its high enantioselectivity for the synthesis of alcohol (S)-1 (Scheme 4B).17 As the TbADH oxidizes preferentially the (R)-enantiomer, the desired (S)-alcohol (S)-1 was isolated with a yield of 41% and an ee of 93−96% (Scheme 4B; Table 1, entry 6). Obviously, the enantiomeric purity of alcohol (S)-1 is varying slightly from batch to batch, as the ee is strongly dependent on the exact conversion of the reaction. By applying TbADH in either an asymmetric reduction or an oxidative kinetic resolution, both enantiomers of the homoallylic alcohols (R)-1 and (S)-1 are accessible with high ee’s (Scheme 4).

Table 1. Overview of the Different Methods Applied to Obtain Homoallylic Alcohol 1 entry

method/reagent

substrate

yield [%]

1 2 3 4 5 6

Brown allyl addition Brown allyl addition Leighton allyl addition asymmetric reduction, TbADH asymmetric reduction, LbADH oxidative kinetic resolution, TbADH

3 3 3 2 2 rac-1

31 39 24 68a n.d. 41

a

ee [%] 90 90 93 >99 42 93−96

(R) (S) (R) (R) (S) (S)

A total yield of 61% from commercially available rac-1.

yield, as much higher values were described for these reactions with similar substrates.13,16 Next, we focused on the enzyme-catalyzed synthesis of homoallylic alcohol 1 (Scheme 4). First, we envisaged an ADHcatalyzed asymmetric reduction of ketone 2 to obtain the corresponding alcohol 1 enantioselectively. Therefore, the alcohol rac-1 (commercially available) was first oxidized to ketone 2 using the Dess−Martin periodinane (yield 90%, not shown).29 A previously performed screening of different ADHs revealed the enzyme from Thermoanaerobacter brockii (TbADH) as an enantioselective catalyst leading to (R)configured alcohol 1.17 A preparative-scale reaction with

Scheme 4. (A) Asymmetric Reduction and (B) Oxidative Kinetic Resolution Applying TbADHa

Reaction conditions for A: TbADH, 2-propanol, NADP+, buffer (50 mM, pH 7, 1 mM MgCl2), 30 °C, 120 rpm, 5 h. Reaction conditions for B: TbADH, P450 BM3 variant F87A, NADP+, catalase, buffer (100 mM, pH 7, 1 mM MgCl2), O2, 30 °C, 120 rpm, 8 h.

a

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Scheme 5. (A) Enzymatic Asymmetric Reduction with Alcohol Dehydrogenase and (B) Allylic Hydroxylation with P450 Monooxygenase to Afford Fragment B 13a

THP = tetrahydropyranyl. Reaction conditions for A: (a) LbADH or TbADH, 2-propanol, NADP+, buffer (50 mM, pH 7.0, 1 mM MgCl2), 30 °C, 120 rpm, 4.5 h; (b) PPTS, DHP, CH2Cl2, rt, 14 h. Reaction conditions for B: P450 BM3 variant A74G L188Q, activity buffer (30 vol%; 50 mM KPi, 50 mM Tris-HCl, 250 mM KCl, pH 8.0), buffer (50 mM KPi, pH 7.5), NADP+, DMSO, glucose dehydrogenase (GDH), glucose, catalase, O2, 30 °C, 24 h. a

Scheme 6. Acetylation and Dimerization of Homoallylic Alcohol 1a

Reaction conditions: (a) triethylamine, acetic anhydride, 4-(N,N-dimethylamino)pyridine, CH2Cl2, 0 °C to rt, 1 h; (b) Hoveyda−Grubbs secondgeneration catalyst, 2,3-dichloro-5,6-dicyano-1,4-benzochinone, CH2Cl2, 40 °C, 5 d. a

In summary, our results indicate that the enzymatic catalytic approach toward the synthesis of enantiomerically enriched substrate 1 represents a good alternative to the traditional reagent controlled allyl addition in terms of yield, enantioselectivity, and practicability. The products with the best enantiomeric excessesalcohol (R)-1, obtained from the asymmetric reduction, and alcohol (S)-1, obtained by the oxidative kinetic resolution with TbADHwere used for all further synthetic steps. Synthesis of Fragment B. For the synthesis of allylic alcohol 4, we first followed a reported three-step procedure from our group using ADHs for the asymmetric reduction of ketone 5, which was synthesized in two steps starting from ethyl 4-bromobutanoate (16).18 Reduction with TbADH yielded the alcohol (R)-4 in 87% yield, whereas the alcohol (S)-4 was obtained with LbADH in 92% yield. Both enantiomers were obtained with an excellent ee of >99% (Scheme 5A). Next to the application of ADHs for the synthesis of allylic alcohols, an elegant alternative is the P450 BM3 monooxygenase-catalyzed enantioselective allylic hydrox-

ylation of ethyl 6-heptenoate (6), where the best results were obtained with the P450 BM3 variant A74G L188Q.19 Ester 6 was synthesized by esterification of the acid 17 with EtOH with catalytic amounts of H2SO4 and microwave irradiation in a quantitative yield. The P450 BM3 A74G L188Q-catalyzed hydroxylation of ester 6 yielded allylic alcohol (S)-4 in 25% yield (40% conversion) and 91% ee (Scheme 4B). A possible explanation of the low conversion of substrate 6 could stem from the enzyme’s instability and decreased activity in purified samples.19 However, so far this monooxygenase strategy only gives access to the (S)-configured allylic alcohol 4. Coupling of Fragments A and B. Having obtained both fragments 1 and 4 in high enantiomeric purities, we used them for the synthesis of the proposed structures of putaminoxins B/ D (7) and the respective diastereomers via the presented cross metathesis/macrolactonization strategy. Before coupling, alcohol 1 was protected by acetylation.27 The products (S)-12 and (R)-12 were isolated in good yields of 99% and 83%, respectively. Both acetates were dimerized in a cross metathesis using the Hoveyda−Grubbs second-generation catalyst, yield1566



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Scheme 7. Final Synthetic Steps Towards All Four Diastereoisomers of 7a

Reaction conditions: (a) Grubbs second-generation catalyst, dichloroethane, microwave irradiation for 2 h (100 W, 100 °C); (b) 1. LiOH, THF:MeOH:H2O (2:1:1), rt, 24 h; 2. 2,4,6-trichlorobenzoyl chloride, trimethylamine, THF, rt, 50 min; 3. 4-(N,N-dimethylamino)pyridine (DMAP), toluene, reflux, 3 h; 4. PPTS, p-TsOH·H2O, EtOH, 40 °C, 14 h.

a

ing diacetate (S,S)-18 (73%) and (R,R)-18 (38%) (Scheme 6).27 The dimerization step was necessary in order to improve the yields of the following cross metathesis step; when applying the nondimerized homoallylic alcohol 1 (or acetate 12) the dimerization product was observed, but never its full conversion to the desired product in good yield. Both enantiomers of allylic alcohol 4 were protected by installing a THP group with 87% yield for (S)-13 and 70% yield for (R)-13 (Scheme 5).27 Next, a second cross metathesis applying the Grubbs second-generation catalyst was performed with each enantiomer of both fragments 18 and 13 (Scheme 7). All four diastereomers of compound 11 were isolated with yields ranging from 24 to 83%. The synthesis of nonenolides 7 was finalized by a saponification, Yamaguchi macrolactonization, and subsequent deprotection, without isolation of the intermediates following the protocol of Goetz et al. for the synthesis of hypocreolide A (9).27 Good yields from 82 to 88% were reached in all cases for this last step, finalizing the synthesis of all four diastereoisomers (Scheme 7). Structure Analysis. In the next step of our investigations, we began with the comparison of our synthesized compounds (with known absolute and relative configuration) to the isolated products putaminoxins B/D. While MS and IR data appear to be similar but not identical, we immediately found substantial

deviations in the NMR data. Surprisingly, comparison of the data of the synthesized compounds (5S,9R)-7 and (5S,9S)-7 with the published NMR data of the isolated natural compounds putaminoxins B/D revealed that neither of the diastereomers could be matched with the isolated products (Table 2; for detailed comparison of 1H and 13C NMR data, see Supporting Information Tables S4.1 and S4.2). As the proton NMR signals for the natural products were not always assigned and sometimes overlapping, we used the 13C NMR signals for further analyses. Even though some signals show just slight deviations, a difference analysis of all 13C NMR signals shows distinct deviations (Figure 2). One possible explanation might be that either the synthesized molecules 7 or the isolated natural compounds are (Z)-configured and not (E)-configured as proposed. However, the coupling constants of 15.5 Hz for the synthesized and 15.0 Hz for the isolated putaminoxin B are typical for (E)configured double bonds. The 5-O-acetyl derivative of putaminoxin D showed a coupling constant of 15.8 Hz, supporting the (E)-configuration of the double bond.20 Moreover, by comparison with analogues containing a (Z)double bond, strongly deviating chemical shifts and coupling constants oppose this option (for detailed information, see Table S4.3). Next, we investigated the 13C NMR data of our 1567



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Table 2. Comparison of 1H and 13C NMR Chemical Shifts of the Synthesized Diastereomers (5S,6E,9S)-7 and (5S,6E,9R)-7 with Chemical Shifts of Putaminoxins B21 and D20 from the Literaturea (5S,6E,9S)-7 position

a

δC, type

δH (J in Hz)

(5S,6E,9R)-7 δC, type

putaminoxin B

δH (J in Hz)

δC, type

176.7, C

δH (J in Hz)

172.5, C

putaminoxin D δC, type

δH (J in Hz)

1

176.0, C

2

35.7, CH2

(a) 2.44, m (b) 1.98−2.06, m

35.7, CH2

(a) 2.40−2.50, m (b) 2.00−2.15, m

32.6, CH2

(a) 2.35, m (b) 1.85, m

31.9, CH2

1.30−2.40, m

3

21.9, CH2

(a) 1.86−1.95, m (b) 1.45−1.70, m

17.8, CH2

(a) 2.00−2.15, m (b) 1.60−1.70, m

23.2, CH2

(b) 1.20−1.90, m (a) 2.28, m

24.6, CH2

1.30−2.40, m

4

38.8, CH2

(a) 1.98−2.06, m (b) 1.45−1.70, m

36.6, CH2

(a) 2.00−2.15, m (b) 1.49−1.60, m

34.3, CH2

(a) 1.82, m (b) 1.50, m

33.0, CH2

1.30−2.40, m

5

74.1, CH

4.01, ddd (10.0, 10.0, 3.4)

68.4, CH

4.44, br s

71.5, CH

4.11, ddd

69.2, CH

4.09, ddd (11.3, 8.7, 4.0)

6

137.1, CH

5.31, dd (15.4, 10.2)

136.6, CH 5.44, dd (15.5, 3.2)

135.5, CH

5.40, ddd

134.4, CH

5.47. m

7

131.7, CH

5.54, ddd (13.5, 10.6, 5.5)

126.3, CH 5.54, dddd (15.5, 10.3, 5.0, 2.3)

130.0, CH

5.50, ddd

133.0, CH

5.47. m

8

40.4, CH2

(a) 2.36, ddd (13.5, 5.5, 5.5) (b) 1.86−1.95, m

40.8, CH2

39.0, CH2

(a) 2.41, m

39.8, CH2

1.30−2.40, m

(a) 2.40−2.50, m (b) 1.97, ddd (11.0, 5.5, 5.0)

171.0, C

(b) 2.18, m

9

75.7, CH

5.01, m

76.8, CH

5.01, m

73.1, CH

5.10, m

71.0, CH

5.05, m

10

34.3, CH2

(a) 1.45−1.70, m (b) 1.45−1.70, m

34.2, CH2

(a) 1.60−1.70, m (b) 1.49−1.60, m

37.2, CH2

1.20−1.90, m

36.0, CH2

1.30−2.40, m

11

25.6, CH2

(a) 1.45−1.70, m (b) 1.26−1.34, m

25.6, CH2

1.22−1.44, m

24.0, CH2

1.70, m

27.6, CH2

1.30−2.40, m

12

31.6, CH2

1.26−1.34, m

31.6, CH2

1.22−1.44, m

30.0, CH2

1.57, m

27.6, CH2

1.30−2.40, m

13

22.7, CH2

1.26−1.34, m

22.7, CH2

1.22−1.44, m

18.8, CH2

1.36, m

16.5, CH2

1.30−2.40, m

14

14.1, CH3

0.88, t (6.8)

14.1, CH3

0.88, t (6.9)

13.9, CH3

0.95, t

12, CH3

0.91, t (7.3)

Each spectrum was measured in CDCl3.

diastereomer (5S,9S)-7 correspond well to the signals of the natural product aspinolide A (5R,9R)-(19) (Figure 3E,F), whereas the spectra of its diastereomer stagonolide F (5S,9R)19 do not (Figure 3G,H). Nevertheless, the absolute configurations of aspinolide A and stagonolide F were also confirmed by several syntheses.34−40 Comparing the specific rotations, it appears that the configuration on C-9 has a higher influence, resulting in a negative value in case of a (9R)configuration. The negative specific rotations of our synthesized compounds (5S,9R)- and (5R,9R)-7 are in line with all other (9R)-configured nonenolides. Because just two pairs of diastereomers are availablefor putaminoxins B/D and aspinolide A/stagonolide Fit is difficult to draw further conclusions. Overall, we were not able to gain further structural insights by solely comparing 13C NMR difference spectra or specific rotation values of different nonenolides. Nevertheless, the proposed structure for the isolated natural products putaminoxins B/D should be reconsidered. Without original spectra of the isolated material or the samples of the natural compounds themselves, it is currently not possible for us to

synthesized molecules (5S,9R)-7 and (5S,9S)-7 with other nonenolidesjust differing in the length of the C-9 side chainto further evaluate the method of 13C NMR difference analysis for structural insights. Here we focused on hypocreolide A27 (9) with a heptyl residue, putaminoxin22 (8) containing a propyl side chain, and aspinolide A32 [(5R,9R)-19] and its respective diastereomer stagonolide F33 [(5S,9R)-19] with a methyl side chain. We found that the NMR data of the diastereomer (5S,9R)-7 are in good agreement with the data of hypocreolide A (9), which is also (5S,9R)-configured (Figure 3A,B and for further information Tables S4.4 and S4.5). Only significant deviations at the side chain were observed, as expected. The configuration of hypocreolide A (9) was elucidated by Opatz and co-workers on the basis of the specific rotation as well as spectroscopic data, having the C-9 epimers and (E/Z)-diastereomers in hand.27 Unfortunately, the data mismatch with putaminoxin (5S,9R)-8 (Figure 3 C,D and for further information Table S4.6), whereas its total syntheses confirmed the (5S,9R)configuration.24,26,34 In addition, the data of the other 1568



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Figure 2. Comparison of 13C NMR chemical shifts of the synthesized diastereomers (5S,9R)-7 and (5S,9S)-7 with the respective chemical shifts from isolated putaminoxins B21 and D.20 Represented are the differences of the respective 13C NMR chemical shifts. n.d. = not determined.

25% yield of alcohol (S)-4 with an ee of only 91%. At last, we were able to embed both building blocks in a natural product synthesis showcase, aimed at the synthesis of the proposed structures of putaminoxins B/D (7) and their enantiomeric counterparts. Comparison of the analytical data (details in Supporting Information) of the synthesized compounds with literature data of putaminoxins B/D (7), putaminoxin (8), and stagonolide F (19) revealed substantial deviations in NMR data, whereas the absolute configurations of putaminoxin (8) and stagnolide F (19) were proven by total syntheses. However, the data fit well to the reported hypocreolide A (9) and aspinolide A (19), portending a necessary reanalysis of the proposed structures of putaminoxins B/D.

analyze the samples in more detail or to suggest alternative structures for the natural products.

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CONCLUSION In summary, we investigated and compared chemical and enzymatic approaches aimed at the synthesis of homoallylic and allylic alcohols as important building blocks for a variety of natural compounds. For the synthesis of homoallylic alcohol 1 in both enantiomeric forms, chemical allylations according to Brown and Leighton as well as an ADH-catalyzed synthesis were applied. The TbADH was used for the OKR of substrate rac-1 [41% yield, 93% (S)-ee] and the asymmetric reduction of ketone 2 [68% yield, >99 (R)-ee] representing alternatives compared to the traditional reagent controlled allyl addition (24−31% yield, 90−93% ee) in terms of yield, enantioselectivity, and practicability. For the synthesis of allylic alcohol 4 we presented a chemoenzymatic route with an ADH-catalyzed selective reduction of ketone 5 as well as a P450 BM3 monooxygenase-catalyzed hydroxylation of ethyl 6-heptenoate (6). By using stereocomplementary ADHs a direct access to both enantiomeric forms of alcohol 4 was achieved in 87−92% yield with excellent enantioselectivity (>99%). Furthermore, the stereoselective allylic hydroxylation with P450 BM3 monooxygenase variant A74G L188Q describes an elegant alternative for the synthesis of allylic alcohols, albeit resulting in

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured in CHCl3 using a PerkinElmer 341 polarimeter at the sodium D-line using a cell with 100 mm path length. Absorbance measurements were conducted using an UV-160 spectrophotometer. IR data were recorded on a PerkinElmer SpectrumOne instrument as thin film, and absorbance frequencies are reported in cm−1. 1H and 13C NMR spectra were recorded on an Avance/DRX 600 NMR spectrometer (Bruker) at ambient temperature in CDCl3 at 600 and 151 MHz, respectively. The chemical shifts are given in ppm relative to the solvent signal [1H: δ (CHCl3) = 7.26 ppm] and [13C: δ (CDCl3) = 77.16 ppm, for centerline of CDCl3 triplet]. NMR signals were assigned by means of COSY, HSQC, and HMBC experiments. GC/ 1569



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Figure 3. Comparison of 13C NMR chemical shifts of the synthesized diastereomers (5S,9S)-7 and (5S,9R)-7 with the respective chemical shifts from synthezised hypocreolide A27 (5S,9R)-9, isolated putaminoxin22 (5S,9R)-8, aspinolide A32 (5R,9R)-19, and stagonolide F33 (5S,9R)-19. Represented are the differences of the respective 13C NMR chemical shifts and the specific rotations. on a Trace GC Ultra gas chromatograph (Thermo Finnigan, Thermo Scientific) or a GC-17A gas chromatograph (Shimadzu) with a flame ionization detector (FID). The Trace GC Ultra gas chromatograph was equipped with a FS-Lipodex G column (25 m × 0.25 mm,

MS measurements were carried out using a Hewlett-Packard HP 6890 Series GC System/Hewlett-Packard 5973 mass selective detector. HRMS (ESI, positive ion) was performed by the analytical service of the Forschungszentrum Jülich (ZEA-3). GC analysis was performed 1570



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Macherey Nagel). The injector and detector were operated at 250 °C, and H2 was used as the carrier gas at a flow rate of 30 mL·min−1. The GC-17A gas chromatograph was equipped with a CP-Chirasil-Dex CB column (25 m × 0.25 mm, Varian), and He was used as the carrier gas at a flow rate of 1.3 mL·min−1. Substances were dissolved in MTBE and analyzed according to the given temperature protocols. Chiral stationary-phase HPLC measurements were performed on a Dionex system equipped with a pump with a gradient mixer and devolatilizer, including a WPS-3000TSL autosampler and a DAD-3000 UVdetector. A Chiralpak IA column (250 mm × 4.6 mm, Daicel) and a mixture of n-heptane/2-propanol (95:5) as solvent were used, applying a flow rate of 0.5 mL·min−1 at room temperature (rt). Flash column chromatography was performed on silica gel 60, particle size 40−63 μm (230−240 mesh). Chemicals. All reagents were used as purchased from commercial suppliers without further purification. The nicotinamide cofactor NADP+ was a generous gift from Codexis. Petroleum ether, diethyl ether, n-pentane, and EtOAc for column chromatography were distilled before usage. The synthesis of ethyl 5-oxo-6-heptenoate (5), ethyl (S)-5-hydroxyhept-6-enoate [(S)-4], and ethyl (R)-5-hydroxyhept-6-enoate [(R)-4] was carried out according to a published procedure by Fischer et al.,18 and (S)-non-1-en-4-ol [(S)-1] was synthesized as described previously.17 Enzyme Production. For heterologous protein expression, the Escherichia coli strain BL21 (DE3) was used. The P450 BM3 A74G L188Q (GQ) variant was obtained as described before.19 Expression, purification, and quantification of the monooxygenase, and production and activity measurements of LbADH and TbADH, were performed as described earlier.30,41 Asymmetric Reduction: (R)-Non-1-en-4-ol [(R)-1]. An Erlenmeyer flask was charged with substrate 2 (1.1 g, 7.85 mmol), 2propanol (5 vol %), TbADH (170 U), and NADP+ (300 μM) in KPi buffer (50 mM, pH 7, 1 mM MgCl2) to a final volume of 250 mL. The solution was stirred at 30 °C and 120 rpm on a shaker (Unimax 1010, Heidolph). After a reaction time of 5 h, the mixture was extracted with methyl tert-butyl ether (3 × 70 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Chromatography of the crude product on silica gel (npentane/Et2O, 95:5 → 90:10) afforded product (R)-1 as a colorless oil (758 mg, 5.33 mmol, 68%). The analytical data were in accordance with those reported in the literature.42,43 Rf 0.63 (n-pentane/EtOAc, 98:2); [α]20 D +7.8 (c 1.0, CHCl3); FT-IR ṽmax 3377, 2957, 2929, 2859, 1641, 1378, 1025, 994, 911 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.83 (1H, m, H-2), 5.15 (1H, m, H-1a), 5.13 (1H, m, H-1b), 3.65 (1H, m, H-4), 2.31 (1H, m, H-3a), 2.14 (1H, m, H-3b), 1.60−1.54 (1H, m, 4OH), 1.51−1.40 (3H, m, H-5a, H-6), 1.40−1.24 (5H, m, H-5b, H-7, H-8), 0.89 (3H, t, J = 6.9 Hz, H-9); 13C NMR (CDCl3, 151 MHz) δ 135.1 (C-2), 118.2 (C-1), 70−9 (C-4), 42.1 (C-3), 36.9 (C-6), 32.0 (C-7), 25.5 (C-5), 22.8 (C-8), 14.2 (C-9); EIMS m/z 142 (1), 124 (1), 101 (52), 83 (100), 71 (11), 67 (3), 57 (10), 55 (98). Brown Allylation: (R)-Non-1-en-4-ol [(R)-1]. To a stirred solution of (−)-B-allyldiisopinocampheylborane (5 mL, 5 mmol, 1 M in n-pentane) in dry Et2O (12 mL) was added at −100 °C a solution of hexanal (737 μL, 6 mmol) in dry Et2O (6 mL). The mixture was stirred at −100 °C for 3 h, MeOH (120 μL) was added and warmed to rt, and NaOH (1 M, 2.4 mL) and H2O2 [30% (w/w), 4.8 mL] were added to the mixture, which was then stirred for 3 h at 40 °C. The solution was washed with saturated aqueous solution of NH4Cl and extracted with Et2O (3 × 30 mL), and the combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 96:4) afforded product (R)-1 as a colorless oil (218 mg, 1.53 mmol, 31%, ee 90%). The analytical data were in accordance with those stated for compound (R)-1 (see above). Brown Allylation: (S)-Non-1-en-4-ol [(S)-1]. Alcohol (S)-1 was synthesized as described for (R)-1 starting from (+)-B-allyldiisopinocampheylborane (5 mL, 5 mmol, 1 M in n-pentane) and hexanal (737 μL, 6 mmol). Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 96:4) afforded the product (S)-1 (278 mg,

1.95 mmol, 39%, ee 90%) as a colorless oil. The analytical data were consistent with those stated for compound (R)-1 (see above). Leighton Allylation: (R)-Non-1-en-4-ol [(R)-1]. The Leighton allylation was performed with the reagent (R,R)-1513 under nitrogen atmosphere. The (R,R)-silane 15 (1.23 g, 2.21 mmol, 1.8 equiv) was dissolved in 8 mL of CH2Cl2 at −10 °C, hexanal (150 μL, 1.22 mmol) was added, and the mixture was stirred at −10 °C for 7 d. The reaction mixture was diluted with EtOAc and 1 M HCl. The aqueous phase was extracted with EtOAc. The organic phases were combined and dried over MgSO4. After the solvent was removed under reduced pressure, the crude product was purified by column chromatography (petroleum ether/EtOAc, 96:4), affording the alcohol (R)-1 (43 mg, 300 μmol, 24%, ee 93%). The analytical data were consistent with those stated for compound (R)-1 (see above). Non-1-en-4-one (2). To a solution of alcohol rac-1 (1.20 g, 8.72 mmol) in dry CH2Cl2 (45 mL) was added Dess−Martin periodinane (4.80 g, 11.3 mmol, DMP), and the mixture was stirred for 2 h. Saturated aqueous Na2S2O3 (25 mL) and NaHCO3 (20 mL) were added and stirred for 30 min, followed by extraction with CH2Cl2 (3 × 50 mL). The combined organic layer was dried over MgSO4, filtered, and concentrated under reduced pressure. Chromatography of the crude product on silica gel (n-pentane/Et2O, 98:2 → 95:5) afforded product 2 as a yellow solid (1.10 g, 7.85 mmol, 90%). The analytical data were in accordance with those reported in the literature.44 Rf 0.61 (petroleum ether/EtOAc, 90:10); FT-IR ṽmax 3081, 2957, 2931, 2873, 2361, 1716, 1638, 1459, 1408, 1364, 1135, 1052, 994, 918 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.92 (1H, ddd, J = 17.2, 10.2, 7.0 Hz, H2), 5.18 (1H, ddd, J = 10.2, 1.4, 1.3 Hz, H-1a), 5.13 (1H, ddd, J = 17.2, 1.6, 1.4 Hz, H-1b), 3.17 (2H, ddd, J = 7.0, 1.6, 1.3 Hz, H-3), 2.43 (2H, t, J = 7.5 Hz, H-5), 1.58 (2H, tt, J = 7.5, 7.4 Hz, H-6), 1.36−1.22 (4H, m, H-7, H-8), 0.89 (3H, t, J = 7.2 Hz, H-9); 13C NMR (CDCl3, 151 MHz) δ 209.1 (C-4), 130.9 (C-2), 118.8 (C-1), 47.8 (C-3), 42.5 (C5), 31.5 (C-7), 23.5 (C-6), 22.6 (C-8), 14.0 (C-9); EIMS m/z 140 (1), 125 (4), 99 (100), 84 (20), 71 (60), 69 (52), 55 (21). P450 BM3 A74G L188Q Catalyzed Biotransformation of Ethyl 6-Heptenoate: Ethyl (S)-5-Hydroxyhept-6-enoate [(S)-4]. A sterile, three-necked flask equipped with a cross-shaped magnetic stir bar was charged with purified P450 BM3 monooxygenase variant A74G L188Q (3 μM), activity buffer (30 vol %; 50 mM KPi, 50 mM Tris-HCl, 250 mM KCl, pH 8.0), NADP+ (100 μM), DMSO (2 vol %), glucose dehydrogenase (GDH, 15 U), glucose (400 mM; 3.0 M stock solution in distilled H2O, sterile filtered), catalase from Micrococcus lysodeikticus (36 kU), and KPi buffer (50 mM, pH 7.5) to a final volume of 120 mL. The solution was saturated with O2 by introducing the gas for 5 min with stirring, and the reaction was initialized by addition of ethyl 6-heptenoate (6) (187 mg, 1.2 mmol). The flask was immediately connected to a Metrohm 877 Titrino Plus pH stat, which continuously adjusted the reaction pH to 7.5 by addition of aqueous 1 M NaOH, and the oxidation proceeded at 30 °C within 24 h, whereas after reaction periods of 2, 4, 6, 8, and 10 h, further catalyst was added (0.6 μM). Additionally, after 8 h, GDH (15 U) and NADP+ (100 μM) were added. Progress was followed via the amount of base consumed over time, and the reaction was stopped by acidifying with aqueous 1 M HCl to pH 4 once the curve slope indicated saturation. The solution was saturated with ammonium sulfate, and the proteins were denaturated at 4 °C overnight. The reaction was extracted with methyl tert-butyl ether (5 × 40 mL), and the combined organic phases were dried with MgSO4 and concentrated under reduced pressure. Chromatography on silica gel with n-pentane/Et2O (80:20 → 60:40) afforded alcohol (S)-4 (52 mg, 300 μmol, 25%, ee 91%) as a colorless oil. The analytical data were consistent with those previously reported.18 Rf 0.29 (petroleum ether/ EtOAc, 70:30); FT-IR ṽmax 3445, 2981, 1732, 1724, 1373, 1240, 1161, 1119, 1030, 991, 920 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.87 (1H, ddd, J = 17.4, 10.4, 6.2 Hz, H-3), 5.24 (1H, ddd, J = 17.4, 1.4, 1.4 Hz, H-7a), 5.12 (1H, ddd, J = 10.4, 1.4, 1.2 Hz, H-7b), 4.13 (2H, q, J = 7.1 Hz, H-1′), 4.11 (1H, m, H-5), 2.34 (2H, t, J = 7.0 Hz, H-2), 1.79−1.67 (3H, m, H-3, 5-OH), 1.57 (2H, m, H-4), 1.25 (3H, t, J = 7.1 Hz, H2′); 13C NMR (CDCl3, 151 MHz) δ 173.6 (C-1), 140.9 (C-6), 114.9 1571



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dd, J = 4.2, 3.2 Hz, HA-2′), 4.65 (1H, dd, J = 3.0, 4.2 Hz, HB-2′), 4.12 (4H. q, J = 7.1 Hz, HA,B-1″), 3.91−3.85 (2H, m, HA,B-6′a), 3.54−3.43 (2H, m, HA,B-6′b), 2.37−2.28 (4H, m, HA,B-2), 1.90−1.48 (20H, m, HA,B-3, HA,B-4, HA,B-3′, HA,B-4′, HA,B-5′), 1.25 (6H, t, J = 7.1 Hz, HA,B2″); 13C NMR (CDCl3, 151 MHz) δ 173.6/173.5 (C-1), 139.4/138.3 (C-6), 117.5/115.2 (C-7), 97.8/95.1 (C-2′), 77.6/77.3 (C-5), 62.9/ 62.6 (C-6′), 60.3/60.2 (C-1″), 33.9/34.2 (C-2), 35.0/34.3/30.9/30.8/ 25.6/25.5/21.1/20.6/19.8/19.7 (C-4, C-3, C-5′, C-4′, C-3′), 14.3 (C2″); EIMS m/z 172 (1), 127 (22), 109 (25), 88 (100). Ethyl (5R)-5-(Tetrahydropyran-2′-yloxy)-hept-6-enoate [(R)13]. The THP-protected alcohol (R)-13 was synthesized as described for (S)-13 starting from alcohol (R)-4 (400 mg, 2.32 mmol). Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 90:10) afforded the product (R)-13 (280 mg, 1.62 mmol, 70%) as a colorless oil. The analytical data were consistent with those stated for compound (S)-13 (see above); however, a fraction containing a diastereomeric mixture with a dr (A:B) = 80:20 was isolated. [α]20 D +7.1 (c 1.0, CHCl3). (1R,6R)-1,6-Bis(acetoxy)hexadec-8-ene [(R,R)-18]. To a solution of compound (R)-12 (204 mg, 1.09 mmol) in dry CH2Cl2 (10 mL) were added Hoveyda−Grubbs second-generation catalyst (0.03 equiv) and 2,3-dichloro-5,6-dicyano-1,4-benzochinone (15 mg, 60 μmol, DDQ), and the mixture was stirred at 40 °C for 5 d. The mixture was then filtered over Celite and concentrated under reduced pressure. Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 96:4) afforded product (R,R)-18 as a light yellow oil (317 mg, 930 μmol, 38%). (E/Z)-ratio 4.6:1 (1H NMR); Rf 0.43 (petroleum ether/EtOAc, 90:10); [α]20 D +47.4 (c 1.0, CHCl3); FT-IR ṽmax 2931, 2861, 1735, 1372, 1233, 1021, 969, 727 cm−1; 1H NMR [CDCl3, 600 MHz, (E)-isomer] δ 5.41 (2H, m, H-3, H-4), 4.85 (2H, tt, J = 6.5, 6.0 Hz, H-1, H-6), 2.35−2.19 (4H, m, H-2, H-5), 2.02 (6H, s, COOCH3), 1.55−1.46 (4H, m, H-7, H-1′), 1.35−1.18 (12H, m, H-10, H-9, H-8, H-4′, H-3′, H-2′), 0.87 (6H, t, J = 6.8 Hz, 6.8 Hz, H-11, H-5′); 13C NMR [CDCl3, 151 MHz, (E)-isomer] δ 170.9 (COOCH3), 128.5 (C-3, C-4), 73.8 (C-1, C-6), 37.4 (C-2/C-5), 33.4 (C-7, C-1′), 31.8 (C-2/C-5), 31.8, 25.1, and, 22.7 (C-10, C-9, C-8, C4′, C-3′, C-2′), 21.4 (COOCH3), 14.1 (C-11, C-5′); EIMS m/z 340 (1), 220 (100), 110 (32), 96 (62); HRMS m/z 358.2956 (calcd for C20H40NO4, 358.2957). Characteristic data of the (Z)-isomer: 1H NMR [CDCl3, 600 MHz, (Z)-isomer] δ 5.45 (2H, m, H-3, H-4); 13C NMR [CDCl3, 151 MHz, (Z)-isomer] δ 127.3 (C-3, C-4), 33.8 (C-7, C-1′), 32.2 (C-2, C-5), 31.8 (C-8, C-2′). (1S,6S)-1,6-Bis(acetoxy)hexadec-8-ene [(S,S)-18]. Diacetate (S,S)-18 was synthesized as described for (R,R)-18 starting from acetate (S)-12 (360 mg, 1.95 mmol). Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 96:4) afforded product (S,S)-18 as a light yellow oil (483 mg, 1.42 mmol, 73%). The analytical data were in accordance with those stated for compound (R,R)-18 (see above) and in the literature.49 [α]20 D −44.7 (c 1.0, CHCl3). General Procedure A: Cross-Metathesis. A solution of the diacetate 18 (1.50 equiv) and THP-protected allylic alcohol (1.00 equiv) in dichloroethane (filtered over basic aluminum oxide prior usage) was treated with Grubbs second-generation catalyst (0.15 equiv) and irradiated with microwaves for 2 h (100 W, 100 °C, 1.7 bar, CEM Discovery). The reaction mixture was filtered through a pad of Celite and concentrated under reduced pressure, and the residue was purified via column chromatography (petroleum ether/EtOAc, 96:4 → 80:20). Ethyl (5R,6E,9S)-9-Acetoxy-5-(tetrahydropyran-2′-yloxy)-hexapent-6-enoate [(5R,9S)-11]. According to the general procedure A, compound (5R,9S)-11 was synthesized starting from (S,S)-diacetate (S,S)-18 (100 mg, 290 μmol) and (R)-13 (50 mg, 200 μmol), yielding product (5R,9S)-11 (29 mg, 70 μmol, 36%) as a light yellow oil. Compound (5R,9S)-11 was due to the THP-protecting group obtained as a diastereomeric mixture indicated as A and B; dr (A:B) = 1:1.4 (1H NMR). Rf 0.15 (petroleum ether/EtOAc, 90:10); [α]20 D +30.1 (c 1.0, CHCl3); FT-IR ṽmax 2935, 2861, 1733, 1372, 1239, 1114, 1020, 971, 732 cm−1; 1H NMR (CDCl3, 600 MHz) 5.60−5.50 (3H, m, HA,B-6, HA-7), 5.30 (1H, ddd, J = 13.7, 11.5, 8.6 Hz, HB-7), 4.87 (2H, pseudo-quin,J = 6.1 Hz, 2 H, HA,B-9), 4.66 (1H, dd, J = 3.7, 3.7

(C-7), 72.4 (C-5), 60.4 (C-1′), 36.3 (C-4), 34.1 (C-2), 20.8 (C-3), 14.3 (C-2′); EIMS m/z 172 (1), 127 (22), 109 (25), 88 (100). Ethyl 6-Heptenoate (6). Ethyl 6-heptenoate (6) was synthesized by dissolving 6-heptenoic acid 17 (500 mg, 3.90 mmol) in dry EtOH (5 mL) containing concentrated H2SO4 (100 μL, 2 vol %), and the resulting mixture was irradiated in a sealed vessel with microwaves (100 W, 100 °C, 1.5 bar, CEM Discovery) for 20 min. The reaction mixture was concentrated under reduced pressure, the residue diluted with CH2Cl2 (20 mL), and the product washed with saturated aqueous NaHCO3 (3 × 10 mL). The combined organic phases were successively washed with brine (10 mL), dried with MgSO4, and concentrated under reduced pressure to give substrate 6 (608 mg, 3.89 mmol, quant.) as a colorless oil. The analytical data were consistent with those reported in the literature.45 Rf 0.59 (petroleum ether/ EtOAc, 90:10); FT-IR ṽmax 2980, 2934, 1734, 1641, 1446, 1372, 1173, 1033, 993, 911, 747 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.79 (1H, ddt, J = 17.0, 10.2, 6.9 Hz, H-6), 4.98 (2H, m, H-7a, H-7b), 4.12 (2H, q, J = 7.2 Hz, H-1′), 2.30 (2H, t, J = 7.5 Hz, H-2), 2.06 (2H, dt, J = 7.6, 6.9 Hz, H-5), 1.64 (2H, tt, J = 7.7, 7.6 Hz, H-4), 1.42 (2H, tt, J = 7.7, 7.5 Hz, H-3), 1.25 (3H, t, J = 7.2 Hz, H-2′); 13C NMR (CDCl3, 151 MHz) δ 173.9 (C-1), 138.6 (C-6), 114.8 (C-7), 60.4 (C-1′), 34.4 (C2), 33.5 (C-5), 28.5 (C-3), 24.6 (C-4), 14.4 (C-2′); EIMS m/z 156 (2), 115 (3), 111 (33), 101 (14), 88 (100), 83 (44), 73 (25). (S)-Non-1-en-4-yl Acetate [(S)-12]. To a solution of alcohol (S)12 (313 mg, 2.20 mmol) in dry CH2Cl2 (10 mL) were added triethylamine (946 μL, 6.83 mmol), acetic anhydride (624 μL, 6.01 mmol), and 4-(N,N-dimethylamino)pyridine (27 mg, 220 μmol, DMAP) at 0 °C. The solution was allowed to warm to rt and was stirred for 1 h. Saturated aqueous NH4Cl (10 mL) was added, followed by extraction with CH2Cl2 (3 × 15 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated under reduced pressure. Chromatography of the crude product on silica gel (npentane/EtOAc, 98:2) afforded product (S)-12 as a colorless oil (405 mg, 2.20 mmol, 99%). The analytical data were in accordance with those reported in the literature.46,47 Rf 0.63 (n-pentane/EtOAc, 98:2); [α]20 D −31.2 (c 1.0, CHCl3); FT-IR ṽmax 2932, 1737, 1372, 1233, 1022, 914 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.75 (1H, ddt, J = 17.2, 10.2, 7.1 Hz, H-2), 5.09−5.03 (2H, m, H-1a, H-1b), 4.91 (1H, pseudoquin, J = 6.2 Hz, H-4), 2.35−2.24 (2H, m, H-3), 2.03 (3H, s, COOCH3), 1.56−1.50 (2H, m, H-5), 1.35−1.21 (6H, m, H-6, H-7, H8), 0.88 (3H, t, J = 6.8 Hz, H-9); 13C NMR (CDCl3, 151 MHz) δ 171.0 (COOCH3), 134.0 (C-2), 117.7 (C-1), 73.5 (C-4), 38.8 (C-3), 33.7 (C-5), 31.8 (C-7), 25.1 (C-6), 22.7 (C-8), 21.4 (COOCH3), 14.1 (C-9); EIMS m/z 184 (1), 143 (86), 113 (8), 83 (100); HRMS m/z 207.1356 (calcd for C11H20O2Na, 207.1361). (R)-Non-1-en-4-yl Acetate [(R)-12]. Acetate (R)-12 was synthesized as described for (S)-12 starting from alcohol (R)-1 (477 mg, 3.35 mmol). Chromatography of the crude product on silica gel (npentane/EtOAc, 98:2) afforded the product (R)-12 (512 mg, 2.78 mmol, 83%) as a colorless oil. The analytical data were consistent with those stated for (S)-12 (see above) and in the literature.48 [α]20 D +33.4 (c 1.0, CHCl3). Ethyl (5S)-5-(Tetrahydropyran-2′-yloxy)-hept-6-enoate [(S)13]. To a solution of alcohol (S)-4 (502 mg, 2.91 mmol.) in dry CH2Cl2 (38 mL) were added pyridinium p-toluenesulfonate (73.0 mg, 290 μmol, PPTS) and 3,4-dihydro-2H-pyran (680 μL, 7.45 mmol). The mixture was stirred at rt for 14 h and then quenched by addition of saturated aqueous NaHCO3 (120 mL) and extracted with Et2O (3 × 70 mL). The combined organic layer was dried over MgSO4, filtered over Celite, and concentrated under reduced pressure. Chromatography of the crude product on silica gel (petroleum ether/EtOAc, 90:10) afforded the product (S)-13 (646 mg, 2.52 mmol, 87%) as a colorless oil. The analytical data were in accordance with the literature.27 Compound (S)-13 was due to the THP-protecting group obtained as a diastereomeric mixture indicated as A and B; dr (A:B) = 50:50 (1H NMR). Rf 0.40 (petroleum ether/EtOAc, 90:10); [α]20 D −22.7 (c 1.02, CHCl3); FT-IR ṽmax 2934, 2862, 1733, 1372, 1238, 1162, 1130, 1115, 1076, 1020, 971, 868, 812 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.87 (1H, ddd, J = 17.2, 10.5, 6.6 Hz, HA-6), 5.62 (1H, m, HB-6), 5.25−5.10 (4H, m, HA,B-7a, HA,B-7b), 4.69 (1H, 1572



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Hz, HA-2′), 4.59 (1H, dd, J = 3.7, 3.7 Hz, HB-2′), 4.11 (4H, q, J = 7.1 Hz, HA,B-1″), 4.05 (1H, ddd, J = 6.5, 5.8, 5.8 Hz, HA-5), 3.99 (1H, ddd, J = 6.5, 5.8, 5.8 Hz, HB-5), 3.84 (2H, m, HA,B-6′a), 3.50−3.40 (2H, m, HA,B-6′b), 2.34−2.22 (8H, m, HA,B-2, HA,B-8), 2.01 (6H, s, COOCH3), 1.87−1.77 (2H, m, HA,B-4b), 1.77−1.44 (24H, m, HA,B-10, HA,B-4a, HA,B-3, HA,B-5′, HA,B-4′b, HA,B-3′), 1.37−1.16 (20H, m, HA,B-13, HA,B12, HA,B-11, HA,B-4′a, HA,B-2″), 0.86 (6H, t, J = 6.7, 6.7 Hz, HA,B-14); 13 C NMR (CDCl3, 151 MHz) δ 173.7/173.6 (C-1), 170.9/170.8 (COOCH3), 134.4/133.3 (C-7), 129.4/126.8 (C-6), 97.6/94.8 (C-2′), 77.0/75.4 (C-5), 73.7/73.5 (C-9), 62.6/62.44 (C-6′), 60.4/60.3 (C1″), 37.1/37.0 (C-8), 35.3/34.2/34.4/34.3/33.7/33.6 (C-4, C-2, C3′), 31.8 (C-13), 31.1/30.9 (C-10), 25.7/25.6 (C-5′), 25.1/25.0 (C11), 22.7 (C-12), 21.4/21.3 (COOCH3), 20.8 (C-3), 19.8/19.7 (C4′), 14.4 (C-2″), 14.1 (C-14; EIMS m/z 412 (1), 267 (4), 250 (51), 162 (31), 85 (100); HRMS m/z 435.2713 (calcd for C23H40NaO6, 435.2717). Ethyl (5S,6E,9R)-9-Acetoxy-5-(tetrahydropyran-2′-yloxy)-hexapent-6-enoate [(5S,9R)-11]. According to the general procedure A, compound (5S,9R)-11 was synthesized starting from (R,R)-diacetate (R,R)-18 (94 mg, 280 μmol) and (S)-13 (52 mg, 200 μmol), yielding product (5S,9R)-11 (70 mg, 170 μmol, 83%) as a light yellow oil. The analytical data were in accordance with those stated for compound (5R,9S)-11 (see above). [α]20 D −33.0 (c 1.0, CHCl3). Ethyl (5S,6E,9S)-9-Acetoxy-5-(tetrahydropyran-2′-yloxy)-hexapent-6-enoate [(5S,9S)-11]. According to the general procedure A, compound (5S,9S)-11 was synthesized starting from (S,S)-diacetate (S,S)-18 (94 mg, 280 μmol) and (S)-13 (47 mg, 180 μmol), yielding product (5S,9S)-11 (54 mg, 130 μmol, 72%) as a light yellow oil. Compound (5S,9S)-11 was due to the THP-protecting group obtained as a diastereomeric mixture indicated as A and B; dr (A:B) = 1:1.5 (1H NMR). Rf 0.15 (petroleum ether/EtOAc, 90:10); [α]20 D −23.1 (c 1.0, CHCl3); FT-IR ṽmax 2934, 2862, 1733, 1372, 1238, 1162, 1130, 1115, 1076, 1020, 971, 868, 812 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.61−5.50 (3H, m, HA,B-6, HA −7), 5.28 (1H, ddd, J = 15.4, 11.5, 8.2 Hz, HB-7), 4.93−4.81 (2H, m, HA,B-9), 4.65 (1H, dd, J = 3.8, 3.8 Hz, HA-2′), 4.59 (1H, dd, J = 3.8, 3.8 Hz, HB-2′), 4.11 (4H, q, J = 7.1 Hz, HA,B-1″), 4.05 (1H, ddd, J = 6.5, 5.8, 5.8 Hz, HA-5), 3.99 (1H, ddd, J = 6.5, 5.8, 5.8 Hz, HB-5), 3.84 (2H, ddd, J = 11.2, 8.0, 3.2 Hz, HA,B-6′a), 3.52−3.40 (2H, m, HA,B-6′b), 2.34−2.22 (8H, m, HA,B-2, HA,B-8), 2.01 (6H, s, COOCH3), 1.85−1.44 (26H, m, HA,B-10, HA,B-4, HA,B-3, HA,B3′, HA,B-4′, HA,B-5′), 1.35−1.18 (18H, m, HA,B-13, HA,B-12, HA,B-11, HA,B-2″), 0.87 (6H, t, J = 6.6 Hz, HA,B-14); 13C NMR (CDCl3, 151 MHz) 173.7/173.6 (C-1), 170.9/170.8 (COOCH3), 134.5/133.3 (C7), 129.5/126.9 (C-6), 97.8/94.8 (C-2′), 77.3/75.3 (C-5), 73.6/73.4 (C-9), 62.7/62.4 (C-6′), 60.4/60.3 (C-1″), 37.3/37.1 (C-8), 35.3/ 34.4/34.3/33.7/33.6 (C-4, C-2, C-3′), 31.8 (C-13), 31.0/30.9 (C-10), 25.7/25.6 (C-5′), 25.1/25.0 (C-11), 22.6 (C-12), 21.4/21.3 (COOCH3), 20.8 (C-3), 19.8/19.7 (C-4′), 14.4 (C-2″), 14.1 (C14); EIMS m/z 412 (1), 267 (3), 250 (42), 162 (36), 85 (100); HRMS m/z 435.2717 (calcd for C23H40NaO6, 435.2717). Ethyl (5R,6E,9R)-9-Acetoxy-5-(tetrahydropyran-2′-yloxy)-hexapent-6-enoate [(5R,9R)-11]. According to the general procedure A, compound (5R,9R)-11 was synthesized starting from (R,R)-diacetate (R,R)-18 (98 mg, 290 μmol) and (R)-13 (54 mg, 210 μmol), yielding product (5R,9R)-11 (21 mg, 50 μmol, 24%) as a light yellow oil. The analytical data were in accordance with those stated for compound (5S,9S)-11 (see above). [α]20 D +21.0 (c 1.0, CHCl3). General Procedure B: Proposed Structures of Putaminoxins B/D. A solution of the ester 11 (50 μmol, 1.0 equiv) was dissolved in a mixture of THF:MeOH:H2O (2:1:1, 5.1 mL), treated with LiOH (4.8 equiv), and stirred at rt for 24 h. The reaction mixture was diluted with Et2O (15 mL) and washed with saturated aqueous KH2PO4 (3 × 10 mL). The combined organic layers were washed with brine (10 mL), dried over MgSO4, and concentrated under reduced pressure. The residue was taken up in THF (6.1 mL), 2,4,6-trichlorobenzoyl chloride (5.60 equiv) and triethylamine (6.1 equiv) were added, and the mixture was stirred for 50 min at rt. The reaction mixture was diluted with toluene (12 mL), filtered over a pad of Celite, and added dropwise over a period of 2.5 h to a refluxing solution of DMAP (7.0 equiv) in toluene (36 mL). After being stirred for another 30 min, the

reaction mixture was allowed to cool to rt. The mixture was quenched with 1 M aqueous HCl (18 mL) and washed with saturated aqueous NaHCO3 (18 mL) and brine (15 mL). The combined organic layers were dried over MgSO4 and concentrated under reduced pressure. The residue was dissolved in EtOH (6 mL) and treated with PPTS (1.10 equiv) and p-TsOH·H2O (0.30 equiv). The solution was stirred for 14 h at 40 °C. The reaction mixture was quenched by addition of ice water (20 mL) and saturated aqueous NaHCO3 (20 mL). The mixture was extracted with EtOAc (3 × 10 mL), dried over MgSO4, filtered over Celite, and concentrated under reduced pressure. Chromatography of the crude product (petroleum ether/EtOAc, 90:10 → 80:20) yielded the final product. (5S,6E,9S)-5-Hydroxy-9-pentyl-6-nonen-9-olide [(5S,9S)-7]. According to the general procedure B, the proposed structure of putaminoxins B/D [(5S,9S)-7] was synthesized starting from ester (5S,9S)-11 (22 mg, 53 μmol, generated from (S)-1 with ee 96% and (S)-4 with ee > 99%), yielding product [(5S,9S)-7] (11 mg, 46 μmol, 87%) as light yellow oil. Rf 0.18 (petroleum ether/EtOAc, 90:10); [α]20 D +16.1 (c 1.0, CHCl3); FT-IR ṽmax 3398, 2929, 2859, 1721, 1713, 1443, 1249, 1176, 1155, 1008, 973, 754 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.54 (1H, ddd, J = 13.5, 10.6, 5.5 Hz, H-7), 5.31 (1H, dd, J = 15.4, 10.2 Hz, H-6), 5.01 (1H, m, H-9), 4.01 (1H, ddd, J = 10.0, 10.0, 3.4 Hz, H-5), 2.44 (1H, m, 1 H, H-2a), 2.36 (1H, ddd, J = 13.5, 5.5, 5.5 Hz, H-8a), 2.06−1.98 (2H, m, H-2b, H-4a), 1.95−1.86 (3H, m, OH, H-8b, H-3a), 1.70−1.45 (5H, m, H-4b, H-3b, H-10a, H-10b, H11a), 1.34−1.26 (5H, m, H-11b, H-12, H-13), 0.88 (3H, t, J = 6.8 Hz, H-14); 13C NMR (CDCl3, 151 MHz) δ 176.0 (C-1), 137.1 (C-6), 131.7 (C-7), 75.7 (C-9), 74.1 (C-5), 40.4 (C-8), 38.8 (C-4), 35.7 (C2), 34.3 (C-10), 31.6 (C-12), 25.6 (C-11), 22.7 (C-13), 21.9 (C-3), 14.1 (C-14); EIMS m/z 240 (1), 153 (12), 140 (54), 80 (100); HRMS m/z 263.1617 (calcd for C14H24NaO3, 263.1618). (5R,6E,9R)-5-Hydroxy-9-pentyl-6-nonen-9-olide [(5R,9R)-7]. According to the general procedure B, the proposed structure of putaminoxins B/D [(5R,9R)-7] was synthesized starting from ester (5R,9R)-11 (21 mg, 51 μmol, starting from (R)-1 with ee > 99% and (R)-4 with ee > 99%), yielding product (5R,9R)-7 (10 mg, 42 μmol, 82%) as a light yellow oil. [α]20 D −15.4 (c 1.0, CHCl3). The analytical data were in accordance with those stated for compound (5S,9S)-7 (see above). (5R,6E,9S)-5-Hydroxy-9-pentyl-6-nonen-9-olide [(5R,9S)-7]. According to the general procedure B, the proposed structure of putaminoxins B/D [(5R,9S)-7] was synthesized starting from ester (5S,9R)-11 (20 mg, 49 μmol, starting from (S)-1 with ee 95% and (R)4 with ee > 99%), yielding product (5R,9S)-7 (10 mg, 43 μmol, 88%) as a light yellow oil. Rf 0.18 (petroleum ether/EtOAc, 90:10); [α]20 D +28.0 (c 1.0, CHCl3); FT-IR ṽmax 3457, 2928, 2859, 1736, 1726, 1436, 1366, 1217, 1155, 1025, 973, 875, 716 cm−1; 1H NMR (CDCl3, 600 MHz) δ 5.54 (1H, dddd, J = 15.5, 10.3, 5.0, 2.3 Hz, H-7), 5.44 (1H, dd, J = 15.5, 3.2 Hz, H-6), 5.01 (1H, m, H-9), 4.44 (1H, br s, H-5), 2.50−2.40 (2H, m, H-2a, H-8a), 2.15−2.00 (3H, m, H-2b, H-3a, H4a), 1.97 (1H, ddd, J = 11.0, 5.5, 5.0 Hz, H-8b), 1.70−1.60 (2H, m, H3b, H-10a), 1.60−1.49 (2H, m, H-4b, H-10b), 1.44−1.22 (6H, m, H11, H-12, H-13), 0.88 (3H, t, J = 6.9 Hz, H-14); 13C NMR (CDCl3, 151 MHz) δ 176.7 (C-1), 136.6 (C-6), 126.3 (C-7), 76.8 (C-9), 68.4 (C-5), 40.8 (C-8), 35.7 (C-2), 36.6 (C-4), 34.2 (C-10), 31.6 (C-12), 25.6 (C-11), 22.7 (C-13), 17.8 (C-3), 14.1 (C-14); EIMS m/z 240 (1), 153 (17), 140 (74), 80 (100); HRMS m/z 263.1617 (calcd for C14H24NaO3, 263.1618). (5S,6E,9R)-5-Hydroxy-9-pentyl-6-nonen-9-olide [(5S,9R)-7]. According to the general procedure B, the proposed structure of putaminoxins B/D [(5S,9R)-7] was synthesized starting from ester (5R,9S)-11 (20 mg, 49 μmol, starting from (R)-1 with ee > 99% and (S)-4 with ee > 99%), yielding product (5S,9R)-7 (10 mg, 42 μmol, 86%) as a light yellow oil. [α]20 D −25.6 (c 1.0, CHCl3). The analytical data were in accordance with those stated for compound (5R,9S)-7 (see above). 1573



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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00101. General numbering of the natural compound, NMR data, GC and HPLC chromatograms, and comparison of NMR data with reported spectra (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Phone: 0049-2461-614158. Fax: 0049-2461-616196. E-mail: j. [email protected]. ORCID

Jörg Pietruszka: 0000-0002-9819-889X Author Contributions §

C.B. and C.H. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank the Fonds der chemischen Industrie (scholarship to C.H. and D.B.), the Ministry of Innovation, Science and Research of the German federal state of North Rhine-Westphalia, the Heinrich Heine University Düsseldorf, and the Forschungszentrum Jülich GmbH for their generous support of our projects. We gratefully acknowledge Birgit Henßen and Patrick Bongen for their ongoing assistance with HPLC measurements.

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REFERENCES

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