Turning to the final step of the total synthesis of ()-enigmazole A (2.1), K2CO3 was used to remove the Fm protecting group on the phosphoric acid (Scheme 2.37) following the procedure outlined in the Molinski publication.9 Monitoring the reaction by LC-MS revealed complete conversion after 3 hours. NMR experiments of the reaction product were carried out after removal of Fm residue through extraction.
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Scheme 2.37. Deprotection of Fm Group, Construction of Dipotassium Phosphate of
Enigmazole A
Disappointingly, although our deprotection product 2.88 displayed a very similar 1H NMR spectrum to enigmazole A recorded in the isolation paper, two disagreements (e.g., 0.11 ppm) for H2 and H5 were observed (Table 2.9). Given that the correctness of all eight stereogenic centers of our advanced intermediates had been demonstrated in the experiments related to δ-lactone 2.79 (Scheme 2.31 in Chapter 2.8.1), we initially reasoned these spectral disagreements, particularly of the protons near to the phosphoric acid and carbonyl functional groups including of H2 and H5, arose due to the presence of different counter-ions on the phosphate group based on the suggestion in the Molinski’s publication.9 Thus ion-exchange experiments were carried out.
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Table 2.9. Comparison of 1H NMR Spectra of Potassium Salt of Enigmazole A 2.88 with
Natural Enigmazole A
Ion-exchange chromatography as well as further purification studies were carried out employing similar procedures as those recorded in the Molinski total synthesis publication. By employing reverse phase HPLC, using an aqueous solution buffered with NaClO4 furnished the desired enigmazole A ()-2.1 probably as a sodium salt. Despite the limited quality of our NMR sample due to the large amount of H2O co-crystallized with NaClO4, our synthetic enigmazole A ()-2.1 displayed 1H NMR spectral properties in excellent agreement with those recorded in the elucidation paper (Table 2.10).
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Table 2.10. Initial Comparisons of 1H NMR Spectra of Synthetic Enigmazole A with
Natural Enigmazole A
Although the 1H NMR spectra of our totally synthetic enigmazole A matched that of natural product, the real format of the isolated enigmazole A remained an issue. Despite utilizing exactly the same method employed in the final purification, Gustafson and Molinski came to a different conclusion related to the counter-ions of ()-enigmazole A in their publications. Gustafson et al. reported ()-enigmazole A as a free phosphoric acid,35 whereas Molinski et al. concluded that the synthetic natural product as a sodium salt.9 Neither group however provided further evidence to justify their solution structural assignments.
To obtain full characterization data of our final product, as well as to solve the question related the real format of the isolated natural product, further experiments were carried out. To avoid the involvement of unnecessary potassium ion and subsequent
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troublesome ion-exchange experiment, Na2CO3 was employed in the Fm deprotection reaction instead of K2CO3 (Scheme 2.38). Monitoring the reaction by C18 TLC revealed complete conversion after 24 hours, with the Fm residue removed via extraction with pentane. NMR experiments of the reaction product after solvent removal were carried out. To our surprise, the 1H NMR spectrum of the sodium salt 2.89 is exactly the same as that of the potassium salt 2.88, thus proving that the different counter ions (Na+ or K+) are not the main reason for the spectral disagreements for H2 and H5. We then reasoned these disagreements were due to the different levels of proton dissociation of the phosphoric acid.
Scheme 2.38. Deprotection of Fm Group and Construction of Disodium Phosphate of
Enigmazole A
To prove this hypothesis, proton exchange experiments were carried out in a 5 mm- diameter glass NMR tube (Scheme 2.39). Treatment of a solution of disodium phosphate
2.89 in CD3OD with 10 μL of TFA-d furnished the free phosphoric acid. The 1H NMR
spectrum of 2.90 changed significantly for the H2 and H5 signals compared with those of the sodium salt 2.89. However the spectrum was still not in full agreement with the
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enigmazole A spectrum recorded in the original elucidation paper. We next treated phosphoric acid 2.90 with 20 mg of solid NaHCO3 to furnish monosodium phosphate 2.91. To our surprise, the 1H NMR spectrum of 2.91 was now in excellent agreement with enigmazole A recorded in the isolation paper.
Scheme 2.39. Phosphoric Acid Dissociation Experiments
Based on these NMR experiments, we conclude that the natural product ()- enigmazole A was isolated as a monophosphate. The 1H NMR spectra of the different levels of phosphoric acid dissociation however presented no neglectable disagreements, whereas the different counter ions (Na+ or K+) does not make an observable difference in the NMR spectra (Table 2.11). The product of Fm removal reaction utilizing K2CO3 is a dipotassium phosphate rather than the monopotassium phosphate recorded in Molinski’s publication.9 The disagreements of NMR spectra after the subsequent HPLC ion- exchange experiment are not due to the cation exchange, but due to the change of the proton association on the phosphate.
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Table 2.11. Comparisons of 1H NMR Spectra of Different Acid Dissociation Levels of
Engimazole A
To synthesize all three phosphate formats of enigmazole A, we next employed acetic acid-d4 (CD3COOD) to quench the reaction mixture during the Fm deprotection reaction utilizing Na2CO3 (Scheme 2.40). After removal of the Fm residue by extraction with pentane, the reaction mixture was concentrated to give enigmazole A monophosphate (2.1) together with sodium acetate-d3. The product was then purified and
converted to phosphoric acid format 2.90 by employing reverse phase HPLC
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format 2.90, the synthetic enigmazole A was converted to the monophosphate format through the addition of saturated NaHCO3 Methanol-d4 solution. The resulting synthetic monophosphate ()-enigmazole A 2.1 displayed NMR spectral properties in excellent agreement with those recorded in the elucidation paper [i.e., 1H, 13C, 31P NMR (500, 125, 200 MHz, respectively), HRMS parent ion identification, and chiroptic properties]. The NMR spectra comparisons are summarized in Figure 2.9, Figure 2.10. Table 2.12 and
Table 2.13. Subsequent Treatment of monophosphate 2.1 with dilute NaOH Methanol-d4
solution then furnished the diphosphate format 2.91.
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Figure 2.9. Comparisons of 1H NMR Spectra of Synthetic Enigmazole A with Natural
Enigmazole A (Upper: Natural Enigmazole A, Lower: Synthetic Monophosphate)
Figure 2.10. Comparisons of 13C NMR Spectra of Synthetic Enigmazole A with Natural
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Table 2.12. Comparisons of 1H NMR Spectra of Synthetic Enigmazole A with Natural
Enigmazole A
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Table 2.13. Comparisons of 13C NMR Spectra of Synthetic Enigmazole A with Natural
89
Finally, we synthesized all three possible phosphate forms of enigmazole A. The comparisons of 1H NMR are summarized in Figure 2.11 and the detail full characters of all the three formats are attached in experimental information (Chapter 3). Moreover, enigmazole A could exist as a mixture of two different phosphate formats in some certain pH level. Interestingly, the 1H NMR spectra in that case does not demonstrate a mixture of two different compounds, but one single compound with average signals between the two formats, presumably due to the rapid proton exchange (e.g. Figure 2.12). We therefore conclude that the NMR specta of enigmazole A vary with the different pH levels.
Figure 2.11. Comparisons of 1H NMR Spectra of Three Phosphate Formats of
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Figure 2.11. 1H NMR Spectra of the Mixture of Monophosphate and Diphosphate
(Upper: Diphosphate, Middle: Mixture of Monophosphate and Diphosphate, Lower: Monophosphate)