Coniothyrione: Anatomy of a Structure Revision

Gary E. Martin1, Alexei V. Buevich2, Mikhail Reibarkh3, Sheo B. Singh4, John G. Ondeyka4, and R. Thomas Williamson3*

1Merck Research Laboratories Discovery and Preclinical Sciences – Global Chemistry Process and Analytical Chemistry, Structure Elucidation Group Summit, NJ 07901 USA 2Merck Research Laboratories Discovery and Preclinical Sciences – Global Chemistry Process and Analytical Chemistry, Structure Elucidation Group Kenilworth, NJ 07033 USA 3Merck Research Laboratories Discovery and Preclinical Sciences – Global Chemistry Process and Analytical Chemistry, Structure Elucidation Group Rahway, NJ 07065 USA 4Merck Research Laboratories Discovery and Preclinical Sciences – Global Chemistry Medicinal Chemistry Rahway, NJ 07065 USA *To whom inquiries should be addressed Merck Research Laboratories Discovery and Preclinical Sciences Global Chemistry - Structure Elucidation Group 128 E. Scott St. Rahway, NJ 07901 robert.williamson2@merck.com 732-594-1743 732-594-9456 (fax) Abstract

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Coniothyrione is a xanthone-derived antibiotic reported several years ago by researchers at Merck & Co. Inc.. Revision of the position of the chloro substitution was recently proposed based on empirical re-interpretation of the carbon chemical shift data and a hypothetical biosynthetic argument without the acquisition of any new spectral data to support the postulated change in substituent location. The originally published GHMBC data leads to an equivocal assignment of the structure and does not provide a solid basis of support for either structure. Neural network 13C chemical shift calculations and DFT calculations also led to undifferentiated structures. Definitive confirmation of the structure of coniothyrione based on the acquisition and interpretation of 1,1-ADEQUATE and inverted 1JCC 1,n-ADEQUATE data is now reported.

3

INTRODUCTION

Coniothyrione is a xanthone-derived antibiotic discovered by an antisense screening approach approximately a decade ago.[1] Recently, a revision of the chloro-substitution in the structure of coniothyrione based on the analysis of previously published data was reported (Figure 1).[2] The reported investigation and interpretation piqued our interest and led us to rigorously explore the basis of this new structure proposal. It was our goal to both explore the validity of the proposed structural arguments and to provide new NMR data that supports an unequivocal assignment of the compound in question. A secondary goal of the present work was to highlight the capabilities of newly available NMR cryoprobe technology and to showcase recently developed NMR experiments and processing methods that can be used to address structural challenges such as this. These methods are shown to be effective even in cases of proton-deficient molecules with limited sample availability like coniothyrione. As can be seen below (Figure 1), the deceptively simple structural proposals differ by the regiochemical placement of a single chlorine atom. The original structure, 1, was chosen based on the absence of a key 3JCH HMBC correlation from the proton assigned as H4 to C1.[1] As pointed out in the work of Kong et al,[2] the bond angle between these two atoms is ~70o. This dihedral angle suggests that the 3JCH coupling would be expected to be small based on a Karplus-like angular dependence on the coupling constant thus leaving the utility of this negative evidence questionable.

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Figure 1. Structure of coniothyrione originally proposed (1)[1] and the revised structure proposal (2)[2]. One of the main tenets of the updated structural proposal was an argument based on an empirical analysis of the 13C chemical shifts reported in the original communication.[2] Upon further investigation, we discovered that analysis of the 13C NMR chemical shifts of this structure class was not straightforward and that an empirical approach to their analysis for the regiochemical assignment of the structure of coniothyrione may not be reliable. Secondary evidence for the revised structure included the contribution of a hypothetical biosynthetic intermediate and suggested that the biosynthesis of 1 should derive from a 3-chloro xanthone precursor. The authors went on to state in support of structure 2 that none of these examples have been reported in the scientific literature and that only 4-chloro xanthones were known to occur in nature.2 In fact, there are a number of well-known 3-chloro xanthone-derived metabolites isolated from microbial sources.[3, 4] In particular, the biosynthetic origin of xantholipin (3)[5] and lysolipin (4).[6] has been studied in detail and the entire 52 kb biosynthetic gene cluster of xantholiopin has been characterized and designated as xan.[5] This study confirmed the chlorine at the C3 position of

5

xantholipin was installed by an enzyme known as XanH, which has been fully characterized and found to be the only putative halogenase in the entire xan gene cluster. In addition, the XanH protein showed 70% homology to LlpH, which is a non-heme, flavin-dependent halogenase implicated in the chlorination of lysolipin (4).[7] Enzymes in this class catalyze the formation of carbon-halogen bonds at electron-rich positions, using FADH2, a halide ion (usually Cl), and molecular oxygen.[8] These results support the conclusion that the presumed 3-chloro xanthone intermediate for 1 has a solid biosynthetic precedent, which leaves the structure of coniothyrione still in question.

Figure 2. Structures of xantholipin (3) and lysolipin (4). Fortuitously, a 10 year old ~1.2 mg sample of coniothyrione was located in the Merck sample repository and found to be reasonably intact. We subjected this sample to a full suite of state-of-the-art suite of NMR experiments using 1.7 mm MicroCryoProbe™ technology at 600 MHz to unambiguously assign the molecular constitution and substitution arrangements of this compound.

6

RESULTS AND DISCUSSION

Our first step in the reinvestigation of this structure problem was to evaluate the 13C chemical shift assignments of both structural proposals using two orthogonal approaches. First, we compared results from the ACD Labs Chemical Shift prediction database (Spectrus™, CNMR v14.0) to those reported in the original work[1] and in the recently published structure revision[2]. These results (Table 1) indicated poor agreement for the 3- and 4-positions in both structural proposals with errors exceeding 11 ppm in both cases for one or the other of the primary carbons of interest (C3 and C4). The chemical shifts derived from initial quantum mechanical DFT calculations at the B3LYP/6-311+G(d,p) level of theory[9] also showed insubstantial similarity with either structure; NMR 13C chemical shift errors exceeding 5.5 ppm were observed for either C3 or C4 in both cases. There were also significant disparities between the observed and DFT calculated carbon chemical shifts for the 14-position and the methyl ester carbonyl. Only when we used a DFT function specifically optimized for the calculation of carbon NMR chemical shifts in chlorinated compounds (WC04)[10] was the accuracy of the quantum mechanical calculations of the C3 and C4 carbon shifts high enough to suggest structure 2 over 1 (Table 1). Root mean square deviations for the C3 and C4 carbon chemical shifts (RMSD2) were 2.8 ppm for 2 and 20.6 ppm for 1. In summary, comparison of the reported 13C chemical shifts to both the ACD Labs chemical shift prediction database and standard quantum mechanical calculations at the B3LYP/6-311+G(d,p) level demonstrated that the actual chemical shifts of the compound allowed ambiguous interpretation for both choices. These results clearly underscore the danger of

7

relying on calculated 13C chemical shift arguments as a basis for discriminating between closely similar, isomeric structures. Position Original

Report (1)

ACD Labs

CNMR v14.0

(1)

ACD Labs CNMR v14.0

(2)

DFT (B3LYP)

(1)

DFT (B3LYP)

(2)

DFT (WC04)

(1)

DFT (WC04)

(2)

1 168.7 172.7 173.3 180.4 181.9 172.1 173.72 79.8 77.8 78.3 88.9 85.9 74.2 71.13 127.2* 139.7 128.4 174.8 152.1 166.5 145.94 143.1* 121.3 129.4 130.6 146.2 119.1 132.85 164.5 165.7 167.2 176.7 173.0 171.8 167.77 155.7 155.2 156 164.3 164.4 157.8 157.98 108.1 110.5 111.1 110.7 111.2 98.5 98.99 135.8 134.6 134.9 140.3 140.7 136.8 137.3

10 112.7 113.2 113.3 119.1 119.2 106.3 106.311 160.6 160.8 160.9 172.1 172 167.1 166.912 110.7 112.1 112.1 117.3 117.2 104.6 104.213 176 176.7 176.2 181.1 182.3 178.8 179.714 120.8 113.6 110.4 128.6 130.4 112.2 11515 52.9 52.6 53.0 57.0 56.8 50.5 50.3

RMSD {14} 7.15 5.16 15.14 9.24 13.42 5.47RMSD {2} 12.56 7.43 22.01 9.40 20.59 2.80

R 0.9790 0.9893 0.9436 0.9949 0.9422 0.9952*These chemical shifts are switched in structure 2[2].

Table 1. Experimental, predicted, and calculated 13C NMR chemical shifts for 1 and 2. Chemical shifts calculated for 1 and 2 using ACD Spectrus CNMR program v.14.0 were calculated using the neural network method. RMSD, root mean square deviation (Σ(δexp – δcalc)2/n)1/2 were calculated for all fourteen carbons (RMSD {14}) and for C3 and C4 carbons only (RMSD {2}), R, the correlation coefficient.

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HMBC Following the acquisition of an initial HSQC spectrum, a pair of HMBC spectra were acquired for the sample with the long-range delay optimized for 3 and 8 Hz. The proton singlet resonating at 7.25 ppm, which can be readily assigned to the isolated sp2 proton on the five-membered ring, gave a weak correlation to the carbonyl resonating at 168.0 ppm (C1) in the 8 Hz optimized spectrum and a significantly stronger correlation in the 3 Hz optimized experiment (Figure 3 and Table 5). The proton resonating at 7.25 ppm also exhibited a correlation to the carbonyl resonating at 176.1 ppm that can be assigned as C13 in both spectra. Hence, there is a degree of ambiguity inherent to the HMBC data, particularly when angular considerations are taken into account, insofar as where the proton singlet might be located in the structure at either the 3- or 4-position. These data would be consistent with the presence of two 4-bond HMBC correlations for structure 1 and one 4-bond correlation in 2, neither of which is unreasonable. Due to sample availability constraints and instrumental limitations when the structure was originally determined and reported, only three HMBC correlations were observed in the original work. In that case, only nJCH couplings from the methine proton at 7.20 to C2, C5, and C14 were visualized. The annotated aromatic region of 3 and 8 Hz optimized GHMBC spectra are shown in Figure 3.

9

F2 Chemical Shift (ppm)7.50 7.25 7.00

F1 C

hem

ical

Shi

ft (p

pm)

80

100

120

140

160

F2 Chemical Shift (ppm)7.75 7.50 7.25 7.00

F1 C

hem

ical

Shi

ft (p

pm)

80

100

120

140

160

A B

2

1210

8

144

93

117

51

13

H9H3

H10

H8

Figure 3. A.) GHMBC spectrum of coniothyrione optimized for 3 Hz showing correlations for the aromatic region of the molecule. B.) Corresponding region of the 8 Hz optimized GHMBC spectrum of coniothyrione. Data were acquired as 4096 x 200 points with accumulation times of 1 h 47 m and 51 m, respectively. The 3 Hz optimized spectrum was acquired with 16 transients/t1 increment vs. 8 for the 8 Hz experiment to compensate for signal losses during the length of the long-range delay in the 3 Hz optimized experiment. Both experiments were linear predicted to 512 points and zero-filled prior to Fourier transformation to afford the 2K x 1K spectra from which the segments shown were extracted. Data were subjected to apodization using a sin2 function phase-shifted 90o prior to both Fourier transforms.

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HSQMBC In an effort to better understand the long-range nJCH coupling pathways present in this molecule, and to further constrain the atomic arrangement of coniothyrione, we acquired a long-range 1H-13C correlation data set utilizing the G-BIRDx,y-CPMG-HSQMBC pulse sequence.[11] We utilized DFT calculations at the B3LYP/6-311+G(d,p) level of theory to calculate and optimize nJCH.[9] The two step algorithm of calculating J-couplings was employed in accordance with the previously described protocol.[12] Experimental and calculated nJCH couplings for coniothyrione are summarized in Table 2. Overall, nJCH couplings predicted for structure 2 have a much better correlation with experimental values as compared with those for structure 1 (Figure 4). Root mean square deviation (RMSD) of calculated nJCH couplings for structure 2 was 0.76 Hz and 2.86 Hz for 1. It is noteworthy, that the largest contribution into RMSD of 1 was associated with two couplings: 3JH4,C5 and 3JH4,C2 as seen as two outliers in the correlation plot in Figure 4a. The RMSD calculated for these two J-couplings (RMSD2) is 8.51 Hz for 1 and 0.5 Hz for 2. Such large difference in the errors of the two coupling constants provides further convincing support for the contention that the correct structure of coniothyrione is 2. Interestingly, the coupling constant from the singlet proton at 7.25 to C1 was predicted to be only 1.3 Hz in structure 2. This vanishingly small value provides an explanation as to why this key correlation was missing in the original lower sensitivity HMBC data.

11

Position Original

Report (1) δC (ppm)

HSQMBC(8 Hz)

δH (ppm), multiplicity

HSQMBC (8 Hz) nJCH

Calc’d for 1 Calc’d for 2

1 168.7 7.25, s < 2.0 Hz 0.1 1.3 3.64, s 3.9 Hz 4.2 4.2

2 79.8 7.25,s 3.5 Hz 9.0 3.9 3 127.2 --- --- 4 143.1 --- --- 5 164.5 7.24, d 12.1 Hz 1.4 12.7 7 155.7 7.71 11.3 Hz 12.4 12.4 7.24 wk 4.5 Hz -3.2 -3.2 6.92 2.1 Hz -1.4 -1.4

8 108.1 7.71 1.8 Hz 1.9 1.9 6.92 7.8 Hz 8.3 8.3

9 135.8 --- --- 10 112.7 7.71 < 2 Hz 1.7 1.8

7.24 9.4 Hz 7.6 7.6 11 160.6 7.71 11.2 Hz 10.8 10.9

7.24 < 2.0 Hz -1.1 -1.1 6.92 < 2.0 Hz -2.0 -2.0

12 110.7 7.71 1.6 Hz -1.3 -1.3 7.24 4.0 Hz 4.6 4.7 --- 5.3 Hz 5.6 5.6

13 176.0 7.25 ovlp --- ovlp 6.92, wk < 2.0 Hz 0.9 0.9

14 120.8 7.25 4.4 Hz 4.6 4.1 15 52.9 --- ---

RMSD, Hz 2.9 0.8 Table 2. Experimental and calculated nJCH NMR coupling constants for coniothyrione.

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A

B

Figure 4. Experimental and calculated nJCH NMR coupling constants for coniothyrione. Panel A shows the plot for 1 whereas panel B shows the data for 2.

-6

-4

-2

0

2

4

6

8

10

12

14

16

-6 -4 -2 0 2 4 6 8 10 12 14 16DFT

Calc

ulat

ed n J

HC, H

z

Experimental nJHC, Hz

-6

-4

-2

0

2

4

6

8

10

12

14

16

-6 -4 -2 0 2 4 6 8 10 12 14 16DFT

Calc

ulat

ed n J

HC, H

z

Experimental nJHC, Hz

13

ROESY A strong correlation in the 2D ROESY data was observed from CH3-15 to the proton resonating at 7.25. On casual inspection, these data would suggest that only structure 2 could be consistent with these data. However, careful analysis of energy-minimized structures for each regioisomer (Figure 5) reveals that this correlation could conceivably be present in either case. Therefore, the discriminating power of the dipolar coupling data for a single isomer is dubious and cannot distinguish between structures 1 and 2 unambiguously. Distances between H4/H3(7.25 ppm) and the closest proton from the CH3-15 group of 5.8 Å in 1 and 4.6 Å in 2, respectively, suggest a higher probability to find ROESY cross-peak for 2 rather than for 1. However, considering overall isolation of the CH3-15 methyl from other protons, an investigator could expect that some ROE could be also observed in case of structure 1. Figure 5. Lowest energy conformations of 1 and 2 as determined by DFT calculations.

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1,1-ADEQUATE While 2D NMR experiments such as COSY and GHMBC have an inherent degree of ambiguity associated with them insofar as the “length” of an observed correlation, the same is not true of experiments specifically designed to probe the identity of directly adjacent carbon resonances, i.e. via 2JCH. The 2J-3J HMBC[13], H2BC[14], and HAT-H2BC[15] experiments have been developed for this purpose and operate with adjacent, protonated carbons. Unfortunately, these experiments do not operate with adjacent non-protonated carbons and hence have no utility in the differentiation of 1 from 2. In contrast, the 1,1-ADEQUATE experiment, originally developed in 1996[16,17], which operates via 1JCC couplings, is capable of unequivocally identifying both protonated and non-protonated adjacent carbons. The family of ADEQUATE experiments and the applications of them that have been reported, have recently been reviewed.[18] Utilizing the quantum mechanical DFT calculations of the 1JCC couplings as a basis for optimization, a 1,1-ADEQUATE experiment optimized for 1JCC = 75 Hz was acquired as 2048 x 80 points to unequivocally probe the identity of the carbons adjacent to the proton singlet resonating at 7.25 ppm. These data showed a correlation to the quaternary aliphatic carbon resonating at ~80 ppm, which irrefutably locates the proton on the five-membered ring at the 3-position, establishing 2 as the correct structure of coniothyrione (Figure 6). 1,1-ADEQUATE, while an extremely useful experiment, is considerably lower in sensitivity than the HMBC experiment. While the HMBC data for the 1.2 mg

15

sample of coniothyrione could be acquired in ~1 h, in contrast, acquisition of the 1,1-ADEQUATE data required overnight acquisition of ~17 h.

7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9F2 Chemical Shift (ppm)

80

100

120

140

160

F1 C

hem

ical

Shi

ft (p

pm)

H9

H3

H10

H8

39

414

812

10

2

13

5

711

1

C3,C2

C9,C8

C8,C9

C10,C11

C10,C9C3,C4

C8,C7

C9,C10

Figure 6. Expansion of the 75 Hz optimized 1,1-ADEQUATE spectrum of 2 showing correlations in the aromatic region of the spectrum. The data were acquired as 2048 x 80 points and were linear predicted to 192 points in the second frequency domain and zero-filled to afford the 2K x 512 point spectrum, an excerpt of which is shown above. The data were apodized using sin2 multiplication phase-shifted 90o prior to both Fourier transforms. The data were acquired in 16 h 25 m. The 75 Hz optimization of the experiment was based on the DFT calculations of the carbon-carbon coupling constants. The (Cx,Cy) labels on the spectrum above designate associated 1JCC carbon pairs leading to adjacent correlation.

16

Inverted 1JCC 1,n-ADEQUATE

The acquisition of the 1,1-ADEQUATE experiment was followed by the acquisition of an inverted 1Jcc 1,n –ADEQUATE spectrum (Supplemental Figure S1).[19] The asymmetric delays in the experiment are used to selectively invert the 1Jcc correlations that inadvertently “leak” into a 1,n-ADEQUATE experiment and were optimized for 1Jcc = 60 Hz and nJcc= 6 Hz. As shown by the calculated amplitude transfer functions in Figure 7, this optimization gives inversion or nulling of the 1Jcc correlations for commonly encountered 1JCH coupling constants accompanied by good positive intensity across the range of nJcc couplings normally encountered in a small molecule.[18,20] 1JCC Edited HSQC-1,n-ADEQUATE

Subjecting the inverted 1JCC 1,n-ADEQUATE spectrum and a non-edited HSQC spectrum to generalized indirect covariance[23] processing followed by calculation of the matrix square root afforded the 1JCC edited HSQC-1,n-ADEQUATE spectrum of 2 shown in Figure 8.[21] In addition to the improved spectral dispersion afforded by the covariance visualization of the correlation data, which is carbon-carbon vs. carbon-proton for the inverted 1JCC 1,n-ADEQUATE spectrum used in the calculation, there is also an improvement in the signal-to-noise ratio following the covariance calculation.[24] The spectrum visualizes and differentiates 1JCC correlations that are inverted and plotted in red from nJCC carbon-carbon correlations that are positive and plotted in black as described in Figure 8. The 1JCC correlation from C3 to C2 in Figure 8 (red boxed correlation) has what would appear to be an anomalous phase.

17

However, the positive phase of this response is consistent with expectation based on the amplitude transfer curves (Figure 7) and the measured 1JCC coupling constant between C3 and C2 of 40.4 Hz (see discussion on J-modulated ADEQUATE below and Supplemental Figure S3).

18

Figure 7. Caption continues on the following page.

19

Figure 7. Top panel) Amplitude transfer function across the range of 30-100 Hz for an inverted 1JCC 1,n-ADEQUATE experiment optimized for 1JCC = 60 Hz and nJCC = 6 Hz.[19] Bottom panel) Amplitude transfer function for nJCC couplings across the range from 0-16 Hz for an experiment optimized for 1JCC = 60 Hz and nJCC = 6 Hz. Amplitude transfer curves calculated for the following pairings (1JCC/ nJCC): 60/6, 70/5, and 72/6 Hz are compared in Supplemental Figure S2.

20

F2 Chemical Shift (ppm)176 168 160 152 144 136 128 120 112 104 96 88 80

F1 C

hem

ical

Shi

ft (p

pm)

48

56

64

72

80

88

96

104

112

120

128

136

1443

9

14

812

10

2

4

15

21012

8144

9311 75113

XX

X

XXX X

X

X X

1,15 2,15

14,8

12,10

12,9

14,3

13,8

13,10

13,3

5,37,911,3

7,8

7,10

11,85,8

2,3

11,10

8,10

10,89,8

8,9

9,10

10,9 Figure 8. 1JCC-edited HSQC-1,n-ADEQUATE spectrum[21] calculated from the inverted 1JCC 1,n-ADEQUATE spectrum shown in Supplemental Figure S1 and the GHSQC spectrum (not shown) using the generalized indirect covariance method[23] followed by the calculation of the square root of the matrix. The spectrum visualizes carbon-carbon correlations in the following manner: pairs of adjacent (correlated via

1JCC) protonated carbons are diagonally symmetic and inverted, e.g. C8-C9. These correlations are plotted and labeled in red. Adjacent protonated and non-protonated carbons are diagonally asymmetric and are plotted and labeled in red , e.g. C10-C11. Pairs of protonated long-range correlated carbons (correlation via nJCC, where n = 2-5) have positive intensity, are diagonally symmetric, and plotted and

21

labeled in black, e.g. C8-C10. Long-range coupled (via nJCC) protonated and non-protonated carbons are diagonally asymmetric, have positive intensity, and are plotted and labeled in black, e.g. C2-C15. The 1JCC correlation from C3 to C2 (red boxed correlation) has what would appear to be anomalous phase, although the positive phase of this response is consistent with expectation based on the amplitude transfer curves (Figure 7) and the measured 1JCC coupling constant between C3 and C2 of 40.4 Hz (see discussion on J-modulated ADEQUATE below and Supplemental Figure S3).

22

J-Modulated ADEQUATE A J-modulated ADEQUATE[25] spectrum of 2 was also acquired to measure the 1JCC couplings between protonated carbon pairs and between protonated carbons and adjacent non-protonated neighbors for comparison to the DFT calculated values for the carbon-carbon couplings. The couplings between C3-C2 and C3-C4 are critical for a rigorous analysis of the inverted 1JCC HSQC-1,n-ADEQUATE spectrum shown in Figure 8. The 1JCC coupling for C3-C2 was measured as 40.4 Hz. In contrast, the C3-C4 coupling was significantly larger with a measured value of 81.7 Hz (Supplemental Figure S3). Returning to the modulation curves shown in Figure 7, for an inverted 1JCC 1,n-ADEQUATE spectrum optimized for 1JCC = 60 Hz and nJCC = 6 Hz, a carbon with a 1JCC coupling of 40.4 Hz would be expected to give a positive correlation. Consistent with this expectation, the C3-C2 correlation is indeed positive. The balance of the 1JCC couplings extractable from the J-modulated ADEQUATE spectrum are summarized in Table 3, accompanied by the DFT calculated 1JCC coupling constants. As can be seen in Figure 9, the primary outlier for the experimental vs. calculated 1JCC coupling constants is the C3-C2 bond in 1 providing even more support for structure 2. Correlations observed in the HMBC, 1,1-ADEQUATE, and 1JCC edited HSQC-1,n-ADEQUATE spectrum are summarized in Table 4.

23

Coupled resonant pair

Measured 1JCC (Hz) for 2

from the J-modulated ADEQUATE spectrum

1JCC Calculated1

1JCC Calculated2

C3-C2

40.4 65.3* 45.1

C3-C4

81.7 86 86.4

C8-C7

71.9 75 75

C8-C9 C9-C8

57.9 59.9 60.3 60.2

C9-C10

60.6 61 60.9

C10-C11

67.3

71 71.1 * in 1 that coupling is related to the C4-C5 pair of carbons. Table 3. 1JCC Coupling constants for carbons adjacent to a protonated carbon that were extracted from the J-modulated ADEQUATE spectrum. DFT calculated 1JCC couplings for 1 and 2 are shown for comparison. The carbon from which the measurement was made is listed first in the column of coupled pairs.

24

Figure 9. Plot of DFT calculated vs. experimental 1JCC NMR coupling constants for coniothyrione structures 1 and 2. The coupling constant for C4-C5 of 1 varies significantly from the degree of agreement for the other

1JCC couplings measured with the J-modulated ADEQUATE experiment.

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100

DFT

Calc

ulat

ed 1 J

CC, H

z

Experimental 1JCC, Hz

Structure 1

Structure 2

25

Position

δ 1H (ppm,

multiplicity)a

δ 13C

(ppm)

3 Hz

GHMBC H→C

8 Hz

GHMBC H→C

1,1-

ADEQUATE C→C

Inverted 1JCC 1,n-

ADEQUATEC→C

(1Jcc, nJCC)b

1 --- 168.7 --- --- --- ---2 --- 78.9 --- --- --- ---3 7.25, s 127.2 C1, C2, C14 C1 wk, C2, C14 C2, C4 C2, C5, C7, C13, C14 4 --- 143.1 --- --- --- ---5 --- 164.5 --- --- --- ---7 --- 160.6 --- --- --- ---8 6.92, d 108.1 C7, C10, C11, C13 C7, C10, C11 wk, C12 C7, C9 C7, C9, C10, C11, C13

9 7.71, dd 135.8 C7, C8 wk, C10, C11, C12 C7, C11, C12 wk C8, C10 C8, C10, C1110 7.24, d 112.7 C7 wk, C8, C11, C12, C13 C5, C7, C10, C11, C12 C9, C11 C5, C7, C8, C9, C11, C13, C1411 --- 155.7 --- --- --- ---12 --- 110.7 --- --- --- ---13 --- 176.0 --- --- --- ---14 --- 120.8 --- --- --- ---15 --- 52.9 --- --- --- --- a Multiplicity: s = singlet; d = doublet; dd = doubled doublet. b 1JCC Edited HSQC-1,n-ADEQUATE: inverted 1JCC correlations have negative phase and are noted in red; nJCC correlations have positive phase and are noted in black; 1JCC correlations with "anomalous" positive phase are noted in blue.

Table 4. Proton and carbon chemical shift assignments and heteronuclear correlations observed for 2 in the various hetero- and homonuclear shift correlation experiments performed on 2.

CONCLUSIONS Although the recent publication of Kong et al.[2] correctly revised the structure of 2 from that previously reported[1] the revision of the structure was based on empirical chemical shift arguments and a speculative biosynthetic pathway rather than on new experimental data. As we have attempted to demonstrate in this report, there is considerable reason to be extremely cautious of using such an approach. Chemical shift arguments based on neural network or HOSE-code calculations, and even DFT calculations not optimized to deal with chlorine-containing compounds, are prone to potentially inaccurate prediction of chemical shifts in a molecule such as 2. It was only through the use of the WC04 function specifically optimized for DFT calculations of Cl-containing molecules that reasonably accurate prediction of the 13C chemical shifts of 2 were finally obtained (see Table 1). Even experiments such as HMBC that has been used in the elucidation of thousands of chemical structures, as seen in the original report, can lead to erroneous structural assignments when small or proton-deficient samples are encountered or when there are potential problems with the angular orientation of the long-range coupled systems. Furthermore, HMBC spectra do not allow the differentiation of 2JCH from 3JCH correlations. In contrast, experiments such as 1,1-ADEQUATE yield unequivocal identification of adjacent protonated and non-protonated carbons via 1JCC couplings that can be used to reliably assign complex structures and to differentiate between closely related regioisomers. While ADEQUATE experiments are of considerably lower sensitivity than experiments such as HMBC, they can still be performed in a reasonable period of time on samples

27

of approximately 1 mg when an investigator has access to a 1.7 mm Micro CryoProbe™ or comparable technology. More sophisticated experiments such as inverted 1JCC 1,n-ADEQUATE[19] can also be acquired on samples of the size used in the present study although these generally require a weekend acquisition. Subjecting those data to unsymmetrical[22] or generalized indirect covariance[23] processing with an unedited HSQC spectrum to afford a 1JCC edited HSQC-1,n-ADEQUATE[21] spectrum provides the means of beneficially visualizing correlations in a carbon-carbon format in addition to enhancing the overall s/n ratio of the data thereby affording viable data even when dealing with limited sample availability.[18,24] In concert, these techniques have been utilized in the present study to unequivocally confirm the speculative reassignment[2] of the structure of coniothyrione as 2.

28

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Supporting Information

F2 Chemical Shift (ppm)7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9

80

88

96

104

112

120

128

136

144

152

160

168

176

3

9

4

14

812

10

2

13

5

711

1

H9 H10

H3H8

Figure S1. Inverted 1JCC 1,n-ADEQUATE spectrum acquried with 1JCC = 60 Hz and

nJCC = 6 Hz. The data were acquried as 2K x128 points and were linear predicted in the F1 to 384 points before zero-filling and Fourier transform to afford the final 2K x 1K spectrum shown above. The data were processed with sin2 apodization phase-shifted 90o prior to both Fourier transforms. 1Jcc correlations are inverted and plotted in red; nJCC correlations (n = 2-5) have positive phase and are plotted in black. The data were acquired in 91 h 46 m over a long weekend.

32

Figure S2. Top panel) Amplitude modulation curves for 1JCC ; Bottom panel.) nJCC responses in the inverted 1JCC 1,n-ADEQUATE experiment. The curves were calculated with the following values (1JCC/ nJCC): 60/6 (current parameters, red); 70/5 (green); 72/6 (blue).

-1

-0.5

0

0.5

1

30 40 50 60 70 80 90 100

-1

-0.5

0

0.5

1

0 2 4 6 8 10 12 14 16

33

FIGURE S3. J-modulated ADEQUATE spectrum of 2. The data were acquired using a scaling factor of 15 as 2048 x 200 points with 320 transients accumulated/t1 increment; the data were acquired in 57 h 50 min. The data were processed with linear prediction to 512 points in the second frequency domain followed by zero-filling to afford the 2K x 2K spectrum, a segment of which is shown. The data were apodized with sin2 multiplication phase-shifted 90o prior to both Fourier transforms. The 1JCC couplings, C3-C2 and C3-C4, were 40.4 and 81.7 Hz, respectively.