Total Synthesis and Prediction of Ulodione Natural Products Guided by DFT Calculations

Abstract A biomimetic synthetic strategy has resulted in a two‐step total synthesis of (±)‐ulodione A and the prediction of two potential natural products, (±)‐ulodiones C and D. This work was guided by computational investigations into the selectivity of a proposed biosynthetic Diels–Alder dimerization, which was then utilized in the chemical synthesis. This work highlights how biosynthetic considerations can both guide the design of efficient synthetic strategies and lead to the anticipation of new natural products.

1.1 Overview of previous total synthesis of ulodione A 1 • 9 steps • 5.2-5.5% overall yield (9-pot approach), 4.5-4.7% overall yield (7-pot approach) • 2.6 mg prepared 1 Alcohol 3 (20 mg, 167 µmol) was dissolved in EtOAc (0.5 mL) in a sample vial equipped with a magnetic stirring bar. The vial was sealed and the contents were stirred at room temperature for 7 days, after which time the solvent was removed under reduced pressure and the crude reaction mixture was redissolved in CDCl3 and analysed by 1 H NMR spectroscopy. No change was observed.

Subjecting alcohol 3 to aqueous reaction conditions
Alcohol 3 (20 mg, 167 µmol) was dissolved in deionised water (0.5 mL) in a sample vial equipped with a magnetic stirring bar. The vial was sealed and the contents were heated to 65 ℃ for 7 days, after which time the solvent was removed under reduced pressure and the crude reaction mixture was redissolved in CDCl3 and analysed by 1 H NMR spectroscopy. No change was observed.

Subjecting ulodione B (2) to conditions which mimic the isolation conditions 3
Ulodione B (20 mg, 120 µmol) was dissolved in EtOAc (0.5 mL) in a sample vial equipped with a magnetic stirring bar. The vial was sealed and the contents were stirred at room temperature for 7 days, after which time the solvent was removed under reduced pressure and the crude reaction mixture was redissolved in CDCl3 and analysed by 1 H NMR spectroscopy. No change was observed.

Subjecting ulodione B (2) to aqueous reaction conditions
Ulodione B (20 mg, 120 µmol) was dissolved in deionised water (0.5 mL) in a sample vial equipped with a magnetic stirring bar. The vial was sealed and the contents were heated to 65 ℃ for 7 days, after which time the solvent was removed under reduced pressure and the crude reaction mixture was redissolved in CDCl3 and analysed by 1 H NMR spectroscopy. The composition of the crude reaction mixture was approximately 65% ulodione B (2), 32% alcohol 3, 1.5% ulodione A (1) and 0.7% ulodione D (9). See 1 H NMR spectrum below ( Figure S1) and see section 4.5 for spectroscopic data of ulodione A (1) and ulodione D (9).
Figure S1: 1 H NMR spectrum (600 MHz, CDCl3) of crude reaction mixture obtained when ulodione B (2) was subjected to aqueous reaction conditions (65 ℃ for 7 days). Representative signals used to determine the quantity of ulodione D (9), A (1) and B (2) and alcohol 3 are shown.

Synthesis of ulodione A using 2,4-dinitrobenzenesulfenyl chloride
Based on conditions reported by Reich and co-workers. 4 To a stirred solution of alcohol 3 (200 mg, 1.59 mmol, 1 equiv.) in dichloroethane (2 mL

Synthesis of ulodione A using PEP-K
Based on conditions reported by Kanai and co-workers. 5 To a stirred solution of alcohol 3 (20 mg, 160 µmol, 1 equiv.) and PEP-K (147 mg, 711 µmol, 4.4 equiv.) in anhydrous DMF (4 mL) was added TBAHS (35 mg, 95 µmol, 0.6 equiv.) and the reaction was stirred at 100 ℃ for 21 h. The reaction mixture was concentrated under reduced pressure to give an orange oil. The crude mixture was analysed by 1 H NMR spectroscopy, using benzyl benzoate as an internal standard. Ulodione A was formed in an NMR yield of 22%.
See page 11 for spectroscopic data for ulodione A (1).  Table S1 -Comparative NMR data for synthetic and natural ulodione A (1), 3 and assignments of synthetic ulodione C (8) and D (9).

Procedure 2: Large scale synthesis of ulodione A using T3P with optimised purification
To a stirred solution of alcohol 3 (1.00 g, 7.93 mmol, 1 equiv.) and i-Pr2NEt (2.00 mL, 11.5 mmol, 1.5 equiv.) in EtOAc (6 mL) at 0 o C, was added T3P (50% w/w in EtOAc, 6.00 mL, 10.  To a stirred solution of a mixture of ulodione A (1), ulodione C (8) and ulodione D (9) (50 mg, 0.23 mmol, 1 equiv.) in MeCN-d3 (1.8 mL), in a 5 mL round bottom flask, was added durene (17.2 mg, 0.13 mmol). An aliquot from this mixture was analysed by 1 H NMR spectroscopy (400 MHz, t = 0). The aliquot was returned to the reaction mixture and DBU (60 mg, 0.39 mmol, 1.7 equiv.) was added in a single portion. The resulting solution was heated at 80 o C for 1 hour. A second aliquot was then analysed by 1 H NMR spectroscopy (400 MHz, t = 1 h). Consumption of ulodione D (9) is evident by the decrease in the integral for the peak at 5.77 ppm, while the formation of bis-enone 10 (d.r. 4:1) is evident by the emergence of peaks at 6.05 ppm and 6.01 ppm, representing the major and minor diastereomers of 10, respectively.

Computational methods
All density functional theory (DFT) calculations were performed using the PW6B95D3 functional, [1] which includes the empirical D3 dispersion correction [2] and was demonstrated to be among the most accurate hybrid functionals for main group thermochemistry, kinetics and non-covalent interactions. [3] Initial calculations were primarily focused on obtaining the minimum-energy paths for the key expected products with the nudged elastic band (NEB) approach. [4] NEB calculations were performed using the def2-SVP basis set, the geometrical counterpoise correction gCP [5] to account for the basis set superposition error, and the chain of sphere (RIJCOSX) approximation for two-electron integrals, [6] as implemented in the Orca 4.2.1 program. [7,8] The highest energy geometries along each path were then used as starting points for geometry optimizations of transition state (TS) structures. All the TS geometries as well as the structures of substrate complexes and products were optimized using the PW6B95D3 functional and the def2-TZVPP basis set. The character of all stationary points was confirmed by analytical calculation of the Hessian, and for each transition state we identified only one imaginary frequency associated with the expected reaction coordinate. Gibbs free energy differences were calculated assuming the rigid rotor and harmonic oscillator approximations and the temperature of 298.15 K. All values reported in the main article were obtained in the gas phase which should be representative of the experimental conditions, which included a non-polar solvent, namely ethylacetate. Solvation effects exerted by bulk water were calculated with the conductor-like continuum solvation model CPCM for TSA, TSA/G and TSB/D and the corresponding substrate complexes, to estimate the influence of polar solvent on energy barriers. Energies including the effect of CPCM were calculated for the geometries previously optimized in the gas phase. The distortion interaction analysis was performed using the protocol proposed by Ess and Houk. [9,10] Quasi classical molecular dynamics simulations (MD) initiated from the optimized structure of TSB/D were performed employing the Atom-Centered Density Matrix Propagation (ADMP) formalism [11] and the PW6B95D3/def2-TZVP level of theory. For this purpose the geometry of TSB/D was reoptimized with the slightly smaller def2-TZVP basis set and used as the starting point for the MD simulations. 70 initial conditions for the MD simulations were generated based on randomized velocity vectors for all atoms for which the initial total nuclear kinetic energy was set to the zero-point vibrational energy obtained from the frequency calculations. Temperature was kept constant during the simulations (353 K) by applying the velocity rescaling scheme with the rescaling occurring every five steps. The time step for the integration of Newton's equations of motion for the nuclei was set to 1 fs and Kohn-Sham self-consistent field was converged for each step during the dynamics. All trajectories were propagated for 200 fs. All 70 trajectories were then analysed to identify the products. The optimizations of stationary points and MD simulations were performed with the Revision C.01 of the Gaussian 16 program. [12] The analysis of noncovalent interactions between the two reacting dienone molecules was performed for all investigated [4+2] transition states using the NCIPlot program. [13,14] Intrinsic bond orbital analysis was carried out using the IboView program. [15] 6.2 Bifurcating reaction that could enable the formation of products B and I TSB/I is another bifurcating ambimodal TS that could lead to the formation of cyclohexene B and dihydropyran I. This transition state has a slightly higher ΔG ‡ (13.1 kcal mol -1 ) than the primary transition states presented and discussed in the main article. Considering that TSB/D is associated with a barrier lower by merely 0.9 kcal mol -1 , TSB/I could be an alternative route for the formation of product B. Similar to TSB/D, the second forming bond leading to product B is shorter that the second forming bond leading to product I (2.81 vs 3.02 Å), thus indicating strong dynamic control leading to cyclohexene B as the major/sole product. However, even if dihydropyran I forms, it can undergo the reverse reaction to dienone 4 (23.4 kcal mol -1 ). Cyclohexene B could also form through [3,3]-Claisen rearrangement of product I, which has a barrier of 14.1 kcal mol -1 .

Figure S5
-Calculated (PW6B95D3/def2-TZVPP) free energy profile diagram and transition state structures for the formation of products B and I. 6.3 Minimum energy path for the formation of ulodione A via TS A and analysis of intrinsic bond orbitals. Fig. S6 shows a minimum energy path calculated using the NEB approach (see the Computational methods section) with single point energies calculated at the PW6B95D3/def2-TZVPP level of theory. The insets show intrinsic bond orbitals (IBOs) for the most important geometries along the path, which allow one to analyse the flow of valence electrons involved in the formation of new bonds. The selected valence orbitals correspond to the three double bonds involved in the [4+2] reaction. The IBOs plotted for the TSA geometry demonstrate a symmetric flow of electrons from both reacting terminal α-alkene carbon atoms of the approaching dienone 4 monomers. This indicates that the formation of the first C-C single bond involves one electron from each of the involved π (green and blue) orbitals. Once the first C-C bond is formed (first structure after TSA), both α carbon atoms possess an equally sized fragment of an IBO (green), which is then involved in the formation of the second C-C single bond (red/orange; also with symmetric flow of electrons) and the C=C π bond (green). This indicates that once the first C-C single bond is formed, the system adopts biradical character leading to barrierless formation of the second C-C single bond. Figure S6 -The minimum energy path for the most energetically favorable reaction path leading to ulodione A. The structures above include the analysis of intrinsic bond orbitals for the most crucial structures along the reaction path.
We base our reasoning on the fact that TSA is C2-symmetric and the electronic density right after the formation of the first C-C bond (green) is evenly distributed between the two α carbon atoms, which would not be the case if the mechanism had ionic character. In particular, an ionic-type mechanism would involve movement of whole electron pairs, localization of the electronic density only on one of the two α carbon atoms and most likely an asymmetric geometry of the corresponding transition state. Furthermore, a concerted mechanism with radical-like character, albeit completely synchronous, has been proposed for the [4+2] cycloaddition of ethylene and 1,3-butadiene. [16] Our analysis demonstrates that despite the highly asynchronous nature of the [4+2] cycloaddition of dienone 4, the basic electronic characteristics of the reaction are analogous to that for the [4+2] cycloaddition of ethylene and butadiene.

Effects of polar solvent on Gibbs free energy barriers.
Since the biosynthesis of ulodione A could occur in an aqueous environment we additionally considered how bulk water could affect the ΔG ‡ of TSA, TSA/G and TSB/D. For this purpose, we used the substrate complexes and transition state structures optimized in the gas phase and recalculated the potential energies including the conductor-like continuum solvation model C-PCM with the epsilon value for bulk water. In particular we see that polar environment stabilizes TSB/D by 1.9 kcal mol -1 (the resulting ΔG ‡ amounts to 10.3 kcal mol -1 ), which indicates that cyclohexene B might also be formed alongside ulodione A in aqueous environments. In contrast, TSA and TSA/G are stabilized by 0.6 and 0.7 kcal mol -1 (with the resulting ΔG ‡ of 7.9 and 10.4 kcal mol -1 ), respectively. Even though these differences are far below the accuracy of the method, it is clear that all of the primary products could readily form in polar environments via the proposed mechanisms and with analogous kinetics.

Analysis of noncovalent interactions
To rationalize the difference in Gibbs free energy barriers for all the considered transition states that could enable the formation of the most stable products (i.e., cyclohexenes A, B, C or D) we performed an analysis of non-covalent interactions with the NCIPlot program. [13,14] Figure S7 shows a comprehensive analysis, part of which was shown in the main article in Scheme 3. The surfaces plotted between the reacting monomers qualitatively demonstrate the extent of attractive interactions, which correlates with the calculated interaction energies. The green colour of these surfaces indicates that these transition states are primarily stabilized by dispersion interactions. The strongest stabilization can be observed for TSA and TSB/D, which enable the formation of the two experimentally observed [4+2] products, namely cyclohexenes A and B. In contrast, weakest stabilization by dispersion can be observed for transition states for which the dienophile is in the β-alkene of the dienone, namely for TSC, TSC' and TSD. These three latter transition states are also characterized by more pronounced structural distortion from the equilibrium geometries, which results in high Gibbs free energy barriers.