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Importance and Difficulties in the Use of Chiroptical Methods to Assign the Absolute Configuration of Natural Products: The Case of Phytotoxic Pyrones and Furanones Produced by Diplodia corticola Giuseppe Mazzeo,† Alessio Cimmino,‡ Marco Masi,‡ Giovanna Longhi,† Lucia Maddau,§ Maurizio Memo,† Antonio Evidente,‡ and Sergio Abbate*,† †

Dipartimento di Medicina Molecolare e Traslazionale, Università degli Studi di Brescia, Viale Europa 11, 25123 Brescia, Italy Dipartimento di Scienze Chimiche, Università di Napoli Federico II, Complesso Universitario Monte S. Angelo, Via Cintia 4, 80126 Napoli, Italy § Dipartimento di Agraria, Sezione di Patologia Vegetale ed Entomologia, Università degli Studi di Sassari, Viale Italia 39, 07100 Sassari, Italy ‡

S Supporting Information *

ABSTRACT: α-Pyrones and furanones are metabolites produced by Diplodia corticola, a pathogen of cork oak. Previously, the absolute configuration (AC) of diplopyrone was defined by chiroptical methods and Mosher’s method. Using Xray and chiroptical methods, the AC of sapinofuranone C was assigned, while that of the (4S,5S)-enantiomer of sapinofuranone B was established by enantioselective total synthesis. Diplofuranone A and diplobifuranylones A−C ACs are still unassigned. Here electronic and vibrational circular dichroism (ECD and VCD) and optical rotatory dispersion (ORD) spectra are reported and compared with density functional theory computations. The AC of the (4S,5S)-enantiomer of sapinofuranone B and sapinofuranone C is checked for completeness. The AC of diplobifuranylones A−C is assigned as (2S,2′S,5′S,6′S), (2S,2′R,5′S,6′R), and (2S,2′S,5′R,6′R), respectively, with the Mosher’s method applied to define the absolute configuration of the carbinol stereogenic carbon. The AC assignment of sapinofuranones is problematic: while diplofuranone A is (4S,9R), sapinofuranones B and C are (4S,5S) according to ORD and VCD, but not to ECD. To eliminate these ambiguities, ECD and VCD spectra of a di-p-bromobenzoate derivative of sapinofuranone C are measured and calculated. For phytotoxicity studies, it is relevant that all six compounds share the S configuration for the stereogenic carbon atom of the lactone moiety.

C

diplobifuranylones A−C (1−3), together with the well-known sphaeropsidins A and C.4−6 The AC of sapinofuranone C (6) was determined by X-ray analysis combined with the advanced Mosher’s method.6 From the same fungus also a methyl 4,5dihydroxydeca-6,8-dienoate, sapinofuranone D, being close to sapinofuranones, was isolated together with sphaeropsidins A and C.6 Several studies were carried out on fungal phytotoxins with potential application in agriculture7,8 and the relation between their AC and the biological activity.9,10 Diplofuranones and diplobifuranylones differ from sapinofuranones, which were first isolated as phytotoxins from Sphaeropsis sapinea obtained from infected cypress trees,11,12 the first for the different functionalization of the 1-hydroxyhexadienyl side chain at C-5 of the dihyrofuranone ring and the other one for its

ork oak trees largely spread on the island of Sardinia have been threatened by several phytopathogenic fungi: among others Biscognauxia mediterranea, Diplodia corticola, Diplodia mutila and Diplodia quercina have caused heavy economic losses.1 These fungi species are well known as phytotoxin producers. In fact, in vitro these fungi produce a plethora of secondary bioactive metabolites, some of which with strong activity on host and nonhost plants.1 Among them, the first was a tetrahydropyran[3,2-b]pyran-2-one, diplopyrone, isolated from D. mutila,2 whose absolute configuration (AC) was assigned through analysis of its ECD spectrum and ab initio calculation of its optical rotation (OR). The AC of its hydroxyethyl side chain was assigned via the advanced Mosher’s method.3 D. corticola produces several metabolites: α-pyrones such as diplopyrone and diplopyrone B, furanones such as sapinofuranone B, its (4S,5S)-enatiomer (5, Scheme 1), sapinofuranone C (6), diplofuranones A (4) and B, and © 2017 American Chemical Society and American Society of Pharmacognosy

Received: February 10, 2017 Published: September 13, 2017 2406

DOI: 10.1021/acs.jnatprod.7b00119 J. Nat. Prod. 2017, 80, 2406−2415

Journal of Natural Products

Article

Scheme 1. Structures of Diplobifuranylones A−C (1−3), Diplofuranone A (4), (4S,5S)-Enantiomer of Sapinofuranone B (5), and Sapinofuranone C (6)

Figure 1. Top: Superimposed experimental VCD and ECD spectra of diplofuranylone A (1), diplofuranylone B (2), and diplofuranylone C (3). Lower: Superimposed experimental IR and UV spectra and ORD curves for the same compounds with the same color coding.

documented evidence13−17 that the combination of several independent chiroptical methods is beneficial and sometimes crucial for the AC assignment and 3D-structure determination. For this reason we employed an almost full set of chiroptical spectroscopies, excluding the Raman optical activity (ROA) technique, the role of which has been advocated as important, together with VCD, in the field of natural products.18−20 However, the problem of natural products may be even more difficult than that of pharma molecules,21,22 and success has been met in just a small number of cases;18,23 for this reason we will also critically discuss our results, especially when success will not be unambiguous. We are of the opinion that the main positive aspect of this work is that several compounds, with quite similar structures and produced by the same fungus, permit the judgment of results not for individual compounds but collectively.

rearrangement into the corresponding dihydrofuran unit. Thus, it is plausible to hypothesize that sapinofuranones are common biosynthetic precursors of both diplofuranones and diplobifuranylones.5 The (4S,5S)-enantiomer of sapinofuranone B, produced by D. corticola, is identical to the enantiomer isolated from Acremonium strictum and whose AC was determined by stereoselective total synthesis.13 Herein we wish to confirm the assignment. However, among the α-pyrones and furanones reported above, only the AC of diplopyrone was assigned using chiroptical methods. Thus, this work aims at assigning the AC of the two groups of furanones and in particular the (4S,5S)-enantiomer of sapinofuranone B, sapinofuranone C, and diplofuranone A as well diplobifuranylones A−C (1−6) by electronic and vibrational circular dichroism (ECD and VCD) and optical rotatory dispersion (ORD) spectra and subsequent comparison with density functional theory (DFT) and timedependent DFT (TDDFT) calculated spectra. There is 2407



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Figure 2. Comparison of experimental and computed VCD spectra for the different ACs for diplofuranylone A (1), left, diplofuranylone B (2), center, and diplofuranylone C (3), right, in the region 1950−950 cm−1.

Figure 3. Comparison of experimental and computed IR spectra for the different ACs for diplofuranylone A (1), left, diplofuranylone B (2), center, and diplofuranylone C (3), right, in the region 1950−950 cm−1.

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RESULTS AND DISCUSSION The (4S,5S)-enantiomers of sapinofuranone B, sapinofuranone C, and diplofuranone A as well diplobifuranylones A−C (1−6, Scheme 1) were obtained as homogeneous compounds from the fractionation of the organic extract of D. corticola culture filtrates as reported in the Experimental Section.6 They were identified by comparing their physical and spectroscopic properties with reported data.6 In order to carry out a comparative analysis of chiroptical results and to check whether common features may be identified for compounds 1−3, in Figure 1 all experimental chiroptical spectra are superimposed. The VCD spectra exhibit the following features common to all three molecules: the CO stretching region contains negative VCD bands for all three molecules (left panel in Figure 1), and a triplet of bands with signs (−,−,+) in order of increasing frequency is found in the vicinity of 1150 cm−1, with differences in the central frequency for the three molecules of up to 50 cm−1: the triplet feature corresponds to an isolated medium-intense IR peak. All ECD spectra show a broad intense negative Cotton effect between 205 and 210 nm, with a weak positive Cotton effect at ca. 190 nm or below. All ORD curves are negative and rather strong, ranging from 100 to 600 units (please note that “ORD curves” should more appropriately be defined as lines

connecting OR values; for discussion of this important aspect, see refs 23−27). This unbiased identification of chiroptical experimental characteristics common to the three molecules shows that the AC of one of the stereogenic centers might be common to the three molecules. Figure 2 depicts a comparison of experimental and calculated VCD spectra (1950−950 cm−1 range) for the four possible configurations of compounds 1−3, while Figure 3 shows the analogous comparison of their IR spectra. These calculations were done using the configuration determined by previous studies as a starting point.4,28 In particular, for 1 the absolute configuration at C-6′ was established as S by applying the advanced Mosher’s method, while from the NMR data the stereogenic C-5′ and C-2′ were established to possess the same AC and thus to be either (2′S,5′S) or (2′R,5′R). For compounds 2 and 3, similarly, the C-6′ absolute configuration should be R, while C-2′ and C-5′ are either (2′R,5′S) or (2′S,5′R). By visual inspection of VCD data a tentative choice of AC could be proposed; however a safer assignment is possible on the basis of the similarity indices S.I. and Sim_NN of experimental and calculated VCD spectra. The first index, defined in refs 15 and 29 varies between −1 (reversed AC assignment should be made) and +1 (perfect AC assignment has been made) without intensity sensitivity; the second 2408



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index,15,30 instead, is sensitive also to intensity. The first index was used previously:22 Empirically it was concluded that a very good AC assignment is reached when S.I. ≥ 0.5. Instead the second index was used for the first time. In Table 1 the values

of S.I. and Sim_NN for compounds 1−6 are presented, as well as the S.I. values for ECD spectra. The largest S.I. and Sim_NN values are found for the (2S, 2′S,5′S,6′S) configuration of 1, for the (2S,2′R,5′S,6′R) configuration for 2, and for (2S,2′S,5′R,6′R) for 3. Interestingly the same choice is suggested by considering the comparison of the experimental and calculated ECD spectra (Figure 4). The IR spectra shown in Figure 3, reported for completeness, are not helpful. The only problem with the chiroptical data is with the ORD curves. While the prediction of sign and trend is excellent for 1 and 3, for 2 the only wrong prediction of sign is for (2S, 2′S,5′S,6′S), the configuration chosen on the basis of VCD and ECD, and this even against the Kronig−Kramers relation relating ECD and ORD:31 On the basis of the latter observation the ORD data are ignored and the proposed configuration assignment is accepted. A more detailed investigation of the results in Figures 2 and 4 and in Table 1 shows that, for all proposed configurations, the predicted sign of the CO stretching band is negative. In a number of other lactones22 the negative sign of the CO stretching band corresponds to an S-stereogenic carbon within the lactone moiety, though not in the same position as in the present case. The AC assignment also ensures acceptable, not excellent, reproduction of the wavenumber region centered at 1150 cm−1, due to the C−O stretching modes of the lactone (coupled to a myriad of other normal modes) and hosting the (−,−,+) triplet of VCD bands. Examination of the experimental and calculated ECD spectra shows that for 1 and 2 there is a clear indication that the negative broad and intense Cotton effect is accounted for by the calculated spectra of stereoisomers with 5′S configuration located in the furan moiety, where the electronic transition takes place. For 3 the Cotton effects in the observed ECD spectrum are weaker and broader and the AC assignment for that center is not as clear-cut: indeed some interference from the electronic transition in the unsaturated lactone moiety is expected. Concluding this part, the assignment (2S,2′S,5′S,6′S) for 1, (2S,2′R,5′S,6′R) for 2, and (2S,2′S,5′R,6′R) for 3 is thought to be reliable, in particular the (2S) assignment for the lactone

Table 1. Values of Similarity Indices S.I. and Sim_NN of Computed and Experimental VCD and Similarity Indices S.I. for ECD Spectra for Compounds 1−7 Examined in This Worka S.I.

Sim_NM VCD

S.I.

compound

isomer

(0.98)

(0.98)

ECD

1

(2R,2′R,5′R,6′S) (2S,2′R,5′R,6′S) (2R,2′S,5′S,6′S) (2S,2′S,5′S,6′S) (2R,2′S,5′R,6′R) (2S,2′S,5′R,6′R) (2R,2′R,5′S,6′R) (2S,2′R,5′S,6′R) (2R,2′S,5′R,6′R) (2S,2′S,5′R,6′R) (2R,2′R,5′S,6′R) (2S,2′R,5′S,6′R) (4R,9R) (4S,9R) (4S,5S) (4R,5S) (4S,5S) (4R,5S) (4S,5S) (4R,5S)

−0.34 0.16 −0.13 0.34 −0.04 0.32 −0.19 0.26 −0.01 0.55 −0.32 0.33 −0.23 0.006 0.47 −0.28 0.38 −0.08 0.39 0.40

−0.14 0.05 −0.04 0.17 −0.016 0.16 −0.09 0.12 −0.004 0.33 −0.18 0.15 −0.13 0.003 0.26 −0.16 0.24 −0.04 0.11 0.11

−0.75 −0.8 0.8 0.77 −0.54 0.11 0.85 0.91 0.13 0.52 −0.44 −0.09 −0.62 0.87 −0.57 −0.39 −0.47 −0.32 0.73 0.62

2

3

4 5 6 7

VCD

choice (see text)

X

X X

X X X X

a For the definition of S.I. and Sim_NN, see refs 15, 29, and 30 and Section 1 of the Supporting Information. In the last column the X symbol denotes the AC assignment suggested in the present work.

Figure 4. Comparison of experimental and computed ECD spectra and ORD curves for the different ACs of diplofuranylone A (1), left, diplofuranylone B (2), center, and diplofuranylone C (3), right. 2409



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Figure 5. Top: Superimposed experimental VCD and ECD spectra of diplofuranone A (4), (4S,5S)-sapinofuranone B (5), and sapinofuranone C (6). Lower: Superimposed experimental IR and UV spectra and ORD curves for the same compounds with the same color coding.

Figure 6. Comparison of experimental and computed VCD spectra for the different ACs for diplofuranone A (4), left, (4S,5S)-sapinofuranone B (5), center, and sapinofuranone C (6), right, in the 1500−950 cm−1 region.

similarity index S.I. between the experimental VCD data: one has an S.I. of +0.60 for 5 and 6, +0.29 for 4 and 5, and +0.13 for 4 and 6. Thus, 5 and 6 are indeed more similar to each other than to 4, even though they do not differ much from 4 either. The ECD spectra of compounds 4−6 contain a weak negative Cotton effect centered at ca. 230 nm; compound 4 though has a markedly different ECD spectrum, exhibiting a positive Cotton effect at ca. 215 nm; 5 and 6 possibly possess a weak and broad positive Cotton effect at shorter wavelength, 5 and 6 being similar also from this point of view. ORD curves are all positive and weak, with the specific rotation values ranging from +25 to +80. From this analysis it is hard to draw firm conclusions about the AC of any stereogenic center, except for some resemblance between chiroptical data of 5 and 6, differing to some extent in either case from the corresponding data of 4.

moiety (either saturated or unsaturated) in all three cases. The latter conclusion in particular is concordant with the unbiased consideration of the common experimental features (at least in sign) among the three compounds. The second family of compounds 4−6 possesses a conjugated hexadienyl substituent in either E,E (4) or E,Z configuration (5 and 6). For these three molecules no appreciable VCD signal was recorded in the CO stretching region, and, thus, these parts of the VCD and IR spectra are not reported. On the basis of the strongest IR peak at ca. 1175 cm−1 a clear VCD triplet (−,−,+) in order of increasing wavenumbers is observed for 5; for 4 and 6 the existence of such a triplet is questionable, but still possible. However, the VCD spectra of 5 and 6 are more similar to each other than they are to 4. The latter aspect was investigated by calculating the 2410



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Figure 7. Comparison of experimental and computed IR spectra for the different ACs for diplofuranone A (4), left, (4S,5S)-sapinofuranone B (5), center, and sapinofuranone C (6), right, in the 1500−950 cm−1 region.

Figure 8. Comparison of experimental and computed ECD and ORD spectra for the different ACs for diplofuranone A (4), left, (4S,5S)sapinofuranone B (5), center, and sapinofuranone C (6), right.

(and for Sim_NN as well). Calculated IR spectra are different for different AC assignments; however, these are not reliable and do not permit AC assignment upon comparing with experimental data. The calculated versus experimental ECD spectra (Figure 8) confirm the (4S,9R) configurational assignment for 4 (positive S.I. from Table 1), but none of the possible choices for 5 and 6 have a positive S.I. Finally, the (4S,9R) assignment for 4 and the VCD assignments for 5 and 6 are acceptable on the basis of ORD. The ECD spectra appear to be simple, and the reversal required by comparison of the calculated and experimental spectra (Figure 8) is rather disappointing. It calls for either a better level of theory in TDDFT calculations or reversing the assignments just proposed. Thus, if one accepts that an opposite AC is possible, the (4S,9S) assignment for 4 would have S.I. values equal to +0.23 for VCD and +0.62 for ECD. The (4R,9S) assignment for 5 (and 6) would have S.I. values of +0.24 (and +0.066) for VCD and +0.39 (and +0.32) for ECD. However, the latter assignment would be conflicting with the

This may not be necessarily due to the configuration of the stereogenic carbon atoms, but to the E/Z isomerism of double bonds dangling outside the lactone moiety. A solution to this puzzling situation is expected from DFT calculations: In Figures 6 and 7 the experimental and calculated VCD and IR spectra, respectively, are compared for the three compounds with the two possible configurations, which could not be assigned by the advanced Mosher’s method and NMR investigation.4,28 The AC of 5 should be (4S,5S) since it has the same sign for the OR as the one isolated by Kumar and coauthors from Acremonium strictum.12 These authors suggest a configuration of (4S,5S), thus opposite to that isolated from Sphaeropsis sapinea,11 exhibiting opposite OR (see also refs 32 and 33). This was confirmed by X-ray diffraction methods.6 Using the data of Table 1, it may be concluded that for 4 a (4S,9R) absolute configuration is preferable to (4R,9R), since the S.I. is positive in the first case and negative in the second. For 5 and 6 the (4S,5S) configuration is definitely favored, for the same reason, but with larger positive numbers for the S.I. 2411



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Table 2. Conformational Analysis of the (4S,5S)- and (4R,5S)-Diastereomers of the Di-p-bromobenzoate of Sapinofuranone C (7)a and Chemical Structure of the Di-p-bromobenzoate of Sapinofuranone C (Right)

a

Population factors are calculated according to ΔG at the DFT/B3LYP/TZVP/IEF-PCM(CHCl3) level.

Comparison of the calculated and experimental VCD and ECD spectra is shown in Figure 9. Both diastereomers show acceptable VCD and ECD predicted spectra as compared to the experimental spectra, even if the similarity indices S.I. and Sim_NN do not favor the (4S,5S) over the (4R,5S) configuration. For the ECD spectra the S.I. of the (4S,5S)diastereomer is slightly better than that for the (4R,5S)analogue. On the basis of the study of this compound, it may be concluded that the Mosher analysis is indeed correct. These results and previous analysis of compound 6 confirmed its (4S,5S) configuration. The present work based on the combined use of VCD, ECD, and ORD techniques has led to the AC assignments of diplobifuranylone A (1) as (2S,2′S,5′S,6′S), diplobifuranylone B (2) as (2S,2′R,5′S,6′R), diplobifuranylone C (3) as (2S,2′S,5′R,6′R), and diplofuranone A (4) as (4S,9R); summary of the assignment proposed here in provided in Figure SI. Regarding sapinofuranones B (5) and C (6) the assignment of the (4S,5S) configuration from the current chiroptical data is more problematic, probably due to the conformational mobility of the systems and to the mismatching results of VCD and ECD.34,35 Thus, a more in-depth analysis of the sapinofuranone C derivative 7 was undertaken. This provided more confidence in the (5S) configuration assignment of 6,6 previously assigned via the Mosher’s method.28,36 However, ECD calculations of 5 and 6 are not satisfactory for the (5S) configuration established in previous work. Owing to the high conformational freedom of these compounds leading to a large number of conformers, screening of functionals and basis sets should be undertaken, but goes beyond the scope of this paper. Additionally, investigation of basis set and functional dependence on one of the compounds did not give satisfactory results. Finally, it should be noted that for the six compounds extracted from D. corticola the stereogenic center in the lactone moiety is S configured. While sapinofuranones A and B, with (4R) configurations, showed phytotoxic activity on host and nonhost plants,11 the other furanones, except the (4S,5S)-enantiomer of sapinofuranone B, which was not tested, did not show phytotoxicity. This is probably due to the (4S) configuration and/or to the structural modification of the 1-hydroxy-2,4hexadienyl side chain, as was reported for diplobifuranylones A and B4 and for sapinofuranone C.6 The same structural modification could be invoked for the absence of phytotoxicity of diplofuranones A and B, for which a biosynthetic pathway starting from sapinofuranones A and B was hypothesized.5 On this basis it is possible that the biosynthetic pathway could also generate diplobifuranylones, inasmuch as hydroxylation of the

prediction of ORD calculations, which would be opposite with respect to those reported in Figure 8. Thus, for compounds 4− 6, the ECD data set turns out to be at odds with any of the two possible AC assignments, indicating a limitation either in the level of theory in the ECD and/or ORD calculations or in the use of the Mosher’s method and NMR data. It may be concluded that both calculations and unbiased consideration of experimental chiroptical data show intrinsic differences between compound 4 and compounds 5 and 6, while the latter two have similar chiroptical properties. To overcome the problem posed by the ECD spectra, the sapinofuranone C di-p-bromobenzoate (7)8 was investigated. The VCD and ECD spectra of the (5S) diastereomer were recorded in CDCl3 and CH3CN, respectively, followed by DFT calculations for the (4S,5S) and (4R,5S) diastereomers. The conformational analysis data of the two diastereomers are shown in Table 2, the presence of the bulky p-bromobenzoate obviously influencing conformational flexibility (Table 3). Table 3. Number of Conformers Predicted by Molecular Mechanics, MM, and DFT Calculations for Compounds 1−6

Diplofuranylone A (2R,2′R,5′R,6′S) (2S,2′R,5′R,6′S) (2R,2′S,5′S,6′S) (2S,2′S,5′S,6′S) Diplofuranylone B (2R,2′S,5′R,6′R) (2S,2′S,5′R,6′R) (2R,2′R,5′S,6′R) (2S,2′R,5′S,6′R) Diplofuranylone C (2R,2′S,5′R,6′R) (2S,2′S,5′R,6′R) (2R,2′R,5′S,6′R) (2S,2′R,5′S,6′R) Diplofuranone A (4R,9R) (4S,9R) S,S-Sapinofuranone B (4S,5S) (4R,5S) Sapinofuranone C (4S,5S) (4R,5S)

no. conf MM

no. conf 90% PCM

46 46 46 44

17 23 13 9

45 45 44 44

11 8 13 18

23 22 22 23

7 6 13 9

40 42

4 5

41 40

5 5

154 155

9 22 2412



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Figure 9. Left: Comparison of the experimental and computed VCD spectra of the (4S,5S)- and (4R,5S)-diastereomers of 7. Right: Comparison of the experimental and computed ECD spectra of the (4S,5S)- and (4R,5S)-diastereomers of 7. solvent at the same wavelength was carried out. The experimental data at two adjacent wavelengths were connected through a straight line. Computational Details. Prior to DFT calculations, a molecular mechanics (MM) search of conformers within 10 kcal/mol was performed by means of the Spartan0237 program with MMFF94s as the force field and through a systematic search method. The conformers were fully optimized at the DFT level by means of the Gaussian09 program,38 with B3LYP/TZVP as functional/basis set. Besides better definition of the conformers previously found at the MM level and reduction of the number of the allowed conformers, DFT allows one to generate IR and VCD spectra from calculated frequencies and dipole and rotational strengths, by assigning a 4 to 10 cm−1 bandwidth Lorentzian band shape to each vibrational transition with a program resident in the Jasco VCD software package. A scaling factor of 0.98 was applied to all calculated vibrational transitions, but several others were tried (vide infra). Dipole and rotational strengths for the first 30 excited states in the UV range were calculated by means of TDDFT at the CAM-B3LYP/TZVP level of theory. Calculated ECD spectra were obtained from excitation energies and rotational strengths as averages weighed on Boltzmann conformer relative populations as a sum of Gaussian functions centered at the wavelength of each transition with a width of the band at half-height of 0.2 eV using SpecDis v1.64.39 ORD calculations were performed at the CAMB3LYP/6-311++G(d,p) level; all DFT and TD-DFT calculations were performed in the PCM solvent approximation.40 CHCl3 was used for geometry optimizations, as well as for VCD and ORD calculations. PCM approximation in CH3CN was used for geometry optimizations, frequencies, and ECD spectra calculations. Conformational Search. Since the AC of C-6′ in 1−3 was assigned via the advanced Mosher’s method28 and the relative configurations of the stereocenters of the dihydrofuran moiety were assigned by NMR studies,4,6 conformational searches on 1−3 were carried out for the (2R,2′R,5′R,6′S), (2S,2′R,5′R,6′S), (2R,2′S,5′S,6′S), and (2S,2′S,5′S,6′S) for 1 and (2R,2′S,5′R,6′R), (2S,2′S,5′R,6′R), (2R,2′R,5′S,6′R), and (2S,2′R,5′S,6′R) diastereomers of 2 and 3. The conformational search at the MM level provided the number of conformers given in Table 1; this number is considerably reduced by the DFT/PCM approach.40 The advanced Mosher’s method was also applied to compounds 3, 4, and 6, resulting in assignment of the (9R) absolute configuration for 4 and (9S) for 5 and 6.5,28 Thus, the analyses for the (4R,9R)- and (4S,9R)-diastereomers of 4 and (4S,5S)- and (4R,5S)-diastereomers for 5 and 6 were carried out. The number of conformers in the MM and DFT cases is given in Table 1.

methyl group of the (4S,5S)-enantiomer of sapinofuranone B (5) yields sapinofuranone C (6). Thus, the configuration at C-4 and/or the functionalization of the side chain appear to be essential structural features imparting phytotoxic activity.

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EXPERIMENTAL SECTION

Fungal Strains. The D. corticola strain used in this study was originally isolated from stems of infected cork oak trees (Quercus suber) collected in Caprera Island, a small island belonging to the National Park of La Maddalena Archipelago (northeast Sardinia, Italy). Pure cultures were maintained on potato-dextrose agar (PDA) (Fluka, Sigma-Aldrich Chemic GmbH, Buchs, Switzerland) in the collection of Dipartimento di Agraria, Universitá di Sassari, Italy, N. PVS 114S.5 Extraction and Purification of Diplodia corticola Secondary Metabolites. The fungus was grown in 1 L Roux flasks containing 170 mL of Czapek medium amended with 2% corn meal (pH 5.7). The metabolites were isolated and purified as reported earlier.6 The identification of the (4S,5S)-enantiomer of sapinofuranone B, sapinofuranone C, diplofuranone A, and diplobifuranylones A−C (1−6, Scheme 1) was carried out by comparison of their OR, 1H NMR, and ESIMS data with reported data.6 Chiroptical Spectroscopies. All VCD/IR spectra were recorded on a Jasco FVS6000 FTIR spectrometer equipped with a VCD module, comprising a wire-grid linear polarizer, a ZnSe photo elastic modulator to produce 50 kHz modulated circularly polarized radiation over a rather wide range (from 3000 to 800 cm−1), and a liquid N2cooled MCT detector. The spectra were recorded in CDCl3 solutions in a 200 μm BaF2 cell. Concentrations were 0.18 M for 1 and 2, 0.16 M for 3, 0.12 M for 4, 0.25 M for 5, and 0.20 M for 6 in the 950−1500 cm−1 range. In the carbonyl stretching range for compounds 1−3 concentrations of 0.08, 0.09, and 0.07 M, respectively, were used. Five thousand scans were used for each spectrum, and subtractions of IR and VCD spectra from the solvent were performed. ECD/UV spectra were recorded with a Jasco 815SE spectrometer in 0.1 mm quartz cells for acetonitrile solutions at concentrations of 0.0051 M for 1, 0.0065 M for 2, 0.0061 M for 3, 0.0053 M for 4, 0.011 M for 5, and 0.0050 M for 6. The ORD measurements were carried out with a Jasco P-2000 polarimeter. A 10 cm micro SiO2 cuvette was employed in all cases with CHCl3 solutions for concentrations of 0.16 to 0.18 g/100 mL. Solutions were studied at 25 °C, and four wavelengths were considered for optical rotations: 589 nm (Na lamp), 546, 435, and 405 nm (Hg lamp). OR data were obtained with 10 measurements at each wavelength, and proper subtraction of the OR data from the 2413



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(16) Polavarapu, P. L. Chirality 2008, 20, 664−672. (17) Scafato, P.; Caprioli, F.; Pisani, L.; Padula, D.; Santoro, F.; Mazzeo, G.; Abbate, S.; Lebon, F.; Longhi, G. Tetrahedron 2013, 69, 10752−10762. (18) Batista, J. M., Jr.; Blanch, E. W.; da Silva Bolzani, V. Nat. Prod. Rep. 2015, 32, 1280−1302. (19) Nakahashi, A.; Yaguchi, Y.; Miura, N.; Emura, M.; Monde, K. J. Nat. Prod. 2011, 74, 707−711. (20) Bergueño-Tapia, E.; Joseph-Nathan, P. Nat. Prod. Commun. 2015, 10, 1785−1795. (21) Rossi, D.; Nasti, R.; Marra, A.; Meneghini, S.; Mazzeo, G.; Longhi, G.; Memo, M.; Cosimelli, B.; Greco, G.; Novellino, E.; Da Settimo, F.; Martini, C.; Taliani, S.; Abbate, S.; Collina, S. Chirality 2016, 28, 434−440. (22) Rossi, D.; Nasti, R.; Collina, S.; Mazzeo, G.; Ghidinelli, S.; Longhi, G.; Abbate, S. J. Pharm. Biomed. Anal. 2017, 144, 41−51. (23) Polavarapu, P. L.; Donahue, E. A.; Shanmugam, G.; Scalmani, G.; Hawkins, E. K.; Rizzo, C.; Ibnusaud, I.; Thomas, G.; Habel, D.; Sebastian, D. J. Phys. Chem. A 2011, 115, 5665−5673. (24) Djerassi, C. Optical Rotatory Dispersion: Applications to Organic Chemistry; McGraw-Hill: New York, 1960. (25) Crabbé, P. Applications de la Dispersion Rotatoire Optique et du ̈ Dichroisme Circulaire Optique en Chemie Organique; Gauthier-Villars: Paris, 1968. (26) Castiglioni, E.; Abbate, S.; Longhi, G. Chirality 2011, 23, 711− 716. (27) Giorgio, E.; Viglione, R. G.; Zanasi, R.; Rosini, C. J. Am. Chem. Soc. 2004, 126, 12968−12976. (28) Ohtani, I.; Kusumi, T.; Kashman, Y.; Kakisawa, H. J. Am. Chem. Soc. 1991, 113, 4092−4096. (29) Kuppens, T.; Langenaeker, W.; Tollenaere, J. P.; Bultinck, P. J. Phys. Chem. A 2003, 107, 542−553. (30) Shen, J.; Zhu, C.; Reiling, S.; Vaz, R. Spectrochim. Acta, Part A 2010, 76, 418−422. (31) Polavarapu, P. L.; Petrovic, A. G.; Zhang, P. Chirality 2006, 18, 723−732. (32) Yadav, J. S.; Mandal, S. S.; Reddy, J. S. S.; Srihani, P. Tetrahedron 2011, 67, 4260−4267. (33) Clough, S.; Raggatt, M. E.; Simpson, T. J.; Willis, C. L.; Whitney, A.; Wrigley, S. J. Chem. Soc., Perkin Trans. 1 2000, 2475− 2481. (34) Polavarapu, P. L.; Donahue, E. A.; Shanmugam, G.; Scalmani, G.; Hawkins, E. K.; Rizzo, C.; Ibnusaud, I.; Thomas, G.; Habel, D.; Sebastian, D. J. Phys. Chem. A 2011, 115, 5665−5673. (35) Santoro, E.; Mazzeo, G.; Petrovic, A. G.; Cimmino, A.; Koshoubu, J.; Evidente, A.; Berova, N.; Superchi, S. Phytochemistry 2015, 116, 359−366. (36) Cimmino, A.; Masi, M.; Evidente, M.; Superchi, S.; Evidente, A. J. Pharm. Biomed. Anal. 2017, 144, 59. (37) SPARTAN 02; Wavefunction, Inc.: Irvine, CA. (38) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09, Revision A. 02; Gaussian, Inc.: Wallingford, CT, 2009. (39) Bruhn, T.; Schaumlöffel, A.; Hemberger, Y.; Bringman, G. Chirality 2013, 25, 243−249.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00119. Definition of parameters S.I. and Sim_NN; tables with free energy values, population percentages, and calculated OR values for all conformers of all diastereomers of molecules 1−6, summary scheme of molecular structures with proposed configuration (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 030 3717415. E-mail: [email protected]. ORCID

Giuseppe Mazzeo: 0000-0002-3819-6438 Giovanna Longhi: 0000-0002-0011-5946 Maurizio Memo: 0000-0002-7543-0289 Antonio Evidente: 0000-0001-9110-1656 Sergio Abbate: 0000-0001-9359-1214 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the use of computer and software facilities at CINECA-Bologna, Italy, and Regione Lombardia for the LISA grant “LI08p_ChiPhyto”, HPL13POZE1. We thank also Cariplo Foundation, Milan, Italy, and AGROFOOD Lab, University of Brescia, for financial support to this research.

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REFERENCES

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