Abstract
5-Hydroxyoxazole-4-carboxylic acid residues were advanced as substructures within the secondary bacterial metabolites precolibactins 969 and 795a. However, oxazoles containing both 5-hydroxy and 4-carboxy substituent are unprecedented. We have found these oxazoles are unstable toward hydrolytic ring-opening and decarboxylation. Comparison of reported and theoretical carbon-13 NMR chemical shifts between synthetic intermediates and the isolates revealed large discrepancies in the oxazole region. These results suggest that precolibactins 969 and 795a may not contain 5-hydroxyoxazole-4-carboxylic acid residues.
Graphical Abstract
Azlactones, or oxazolones, are an important class of five-membered ring heterocycles that can be employed in a variety of chemical transformations, as they contain both electrophilic and pronucleophilic sites.1 Their versatility arises from the facile deprotonation at C4 (pKa ≈ 9),2 which provides a stabilized enolate. The chemistry and reactivity of oxazolone scaffolds have been extensively investigated; for example, they have been employed as precursors to highly-substituted heterocycles and enantiomerically-enriched amino acids.1 Typically, oxazolones are synthesized by cyclization of N-acylated α-amino acids in the presence of dehydrating reagents, such as anhydrides or carbodiimides.1,3 It has been determined that azlac-tones exist primarily in their keto form, and a base, such as triethylamine, is required to promote tautomerization to the aromatic 5-hydroxyoxazole isomer (Fig. 1a).4 Pinhey and co-workers observed that acyl and phenyl substituents at the C4 and C2 positions, respectively, stabilize the enol tautomer of azlactones. Based on this finding, they developed a C4-arylation reaction of ethyl 5-hydroxy-2-phenyloxazole-4-carboxylate (1) using high-valent aryllead reagents (Fig. 1b).5 The 4-aryl- and 4-styryl oxazolone products 2 were reported to be moisture-sensitive and readily underwent hydrolysis and decarboxylation to provide a-arylglycines and a-styrylglycines 3.
Figure 1.
a. Structures of oxazolones (azlactones) and their 5-hydroxyoxazole tautomers. b. Arylation of ethyl 5-hydroxy-2-phenyloxazole-4-carboxylate (1) by Pinhey and co-workers. c. Proposed structures of precolibactin 969 (4) and precolibactin 795a (5), and the structure of the hydroxyoxazole 6.
Recently, a 2-thiazolyl-5-hydroxyoxazole-4-carboxylic acid residue was advanced as part of the genotoxic microbiome metabolite precolibactin 969 (4) and the related isolate precolibactin 795a (5, Fig. 1c).6 Because only 50 μg of precolibactin 969 (4) was obtained (from a 2000-L fermentation), its structure assignment was based largely on the spectroscopic data of precolibactin 795a (5).6 However, a literature search failed to reveal any known oxazoles containing both free 5-hydroxy and 4-carboxy substituents. Given the absence of strong precedent for this substructure we sought to synthesize this fragment to elucidate its reactivity and spectroscopic properties.
We targeted the thiazole–oxazole fragment 6 as it might be useful in an eventual synthesis of 4 or 5 (Fig. 1c). By analogy to the proposed biosynthetic pathway,6 we envisioned that the oxazole 6 could be obtained by the cyclodehydration of the aminomalonic acid derivative 7 (Scheme 1a). The requisite thiazole precursor 8 is accessible in two steps from commercial reagents.7 HATU-coupling of the carboxylic acid 8 with diethyl aminomalonate (9) provided the diester 10 (95%, Scheme 1b). Two-fold saponification (lithium hydroxide) formed the 1,3-diacid 7 (70%). The 1,3-diacid 7 was found to have a limited lifetime at room temperature or −20 °C and readily underwent decarboxylation. We attempted cyclodehydration of 7 using trifluoroacetic anhydride.5b Unfortunately, the desired product 6 was not observed; only the starting material 7 (83%) and its respective decarboxylation product 11 were isolated.
Scheme 1.
a. Retrosynthetic analysis of the 5-hydroxyoxazole 4-carboxylic acid 6. b. Synthesis of the 1,3-diacid 7 and attempted cyclodehydration. c. Synthesis of the 5-ethoxyoxazole 13.
We turned our attention to cyclization of the diester 10 as a means to avoid decarboxylation (Scheme 1c). Treatment of the 1,3-diester 10 with iodine and triphenylphosphine3 provided the protected oxazole 12 (84%). Saponification (lithium hydroxide) generated the carboxylic acid 13 (65%). The product 13 was stable toward aqueous workup and purification, presumably because the ethyl substituent prevents isomerization to a β-dicarbonyl.
We then evaluated enol protecting groups that could be removed under non-basic conditions, such as a tert-butyl or a benzyl substituent. Treatment of the tert-butyl-protected oxazole 14 (see Scheme S1a for synthesis) with trifluoroacetic acid or hydrochloric acid in dioxane for a short period of time (< 5 min) only provided the aminomalonic acid derivative 7, arising from two-fold deprotection and hydrolytic ring-opening (>99%, Scheme 2a).
Scheme 2.
a. Attempted deprotection of the oxazole 14 under acidic conditions. b. Hydrogenolysis of the carbonate 16.
The benzyloxycarbonyl (Cbz) derivative 16 was then prepared, with the expectation that the oxazole could be unmasked under neutral conditions (Scheme 2b). The β-carboxyester 15 was obtained by partial saponification of the diester 10 (potassium hydroxide, 50%). Treatment of 15 with benzyl chloroformate and N-methylmorpholine (NMM) then generated the Cbz-protected oxazole 16 (50%). Hydrogenolysis of 16 (palladium on carbon, dihydrogen) in tetrahydrofuran as solvent returned the β-carboxyester 15 (80%). The methyl ester 17 was obtained when the hydrogenolysis was conducted in methanol (>99%). The most plausible pathway for the conversion of 16 to 15 or 17 involves reductive removal of the benzyloxycarbonyl substituent, tautomerization to the azlactone, and nucleophilic cleavage of the β-ketoester intermediate by adventitious water or methanol. We also attempted to remove the Cbz and ethyl substituents from 16 under basic conditions. The carbonate substituent was rapidly (<1 h) removed when 16 was treated with lithium or potassium hydroxide at 23 °C. Prolonged heating was required to saponify the ester, and only the product 11, deriving from ring-opening and decarboxylation, could be detected (LC/MS analysis).
In a final attempt toward 6, we prepared the dibenzyl oxazole 18 (Scheme 3a; see Scheme S1b for synthesis). Surprisingly, the β-carboxyester 19 was obtained upon hydrogenolysis of 18 in tetrahydrofuran, indicating that the benzyl ether is removed faster than the benzyl ester and that the resulting 5-hydroxyoxazole readily undergoes hydrolytic ring-opening. As we knew that a C4 alkoxy substituent stabilizes the oxazole (vide supra), we prepared the carboxylic acid 20 by selective cleavage of the benzyl ester (lithium hydroxide, 72%, Scheme 3b). However, hydrogenolysis of 20 (palladium on carbon, tetrahydrofuran) provided the azlactone 21. We believe that the 5-hydroxyoxazole-4-carboxy intermediate 6 formed on deprotection equilibrates to the keto form 22, which can then undergo b-decarboxylation to azlactone 21. While the azlactone 21 is stable to aqueous work-up, purification on silica gel leads to further decomposition.
Scheme 3.
a. Hydrogenolysis of the enol ester 18. b. Hydrogenolysis of the enol acid 20.
In light of the apparent instability of the 5-hydroxyoxazole 4-carboxylate residue, we re-examined the carbon-13 NMR spectroscopic assignments for precolibactin 795a (5), which is believed to contain the same oxazole–thiazole found in precolibactin 969 (4). The carbon-13 NMR spectroscopic data for 13 were inconsistent with those reported for the oxazole region of precolibactin 795a (5, Table 1). In particular, large differences in chemical shifts were observed at C2, C6, and C7. The differences at C2 may be due to the presence of a 2-pyridone in 5 as compared to a carbamate in 13. However, the differences in the oxazole region (C6, C7, Δδ = 27.6 and 16.0 ppm, respectively) were more concerning.
Table 1.
Selected carbon-13 data for precolibactin 795a (5) and the synthetic oxazole 13.
 | |||
|---|---|---|---|
| position | δ C (ppm) precolibactin 795a (5) | δ C (ppm) 13 | |Δδ| |
| 1 | 44.4 | 46.1 | 1.7 |
| 2 | 164.1 | 172.8 | 8.7 |
| 3 | 120.0 | 120.6 | 0.6 |
| 4 | 142.9 | 141.5 | 1.4 |
| 5 | 164.0 | 163.5 | 0.5 |
| 6 | 133.0 | 160.6 | 27.6 |
| 7 | 123.9 | 107.9 | 16.0 |
| 8 | 160.9 | 155.8 | 5.1 |
Though we considered it unlikely that the presence of the ethoxy substituent in 13 was responsible for the differences at C6 and C7, we calculated the expected carbon-13 chemical shifts for the hydroxyoxazole residue in precolibactin 795a (5). To benchmark the calculations, we also calculated the expected carbon-13 chemical shifts for the 5-ethoxyoxazole 4-carboxylate 13 and the azlactone 21. All calculations were carried out using Spartan ‘18 according to the method of Hehre et al.8 We were particularly surprised to find that the theoretical carbon-13 shifts for the 5-hydroxyoxazole 4-carboxylate proposed within precolibactin 795 (5) were also in disagreement with experimental data (root-mean-square deviation (RMS) = 8.48, Table 2). The calculated carbon-13 chemical shifts for 13 and 21 were in agreement with the experimental values (RMS = 2.13, 2.17, respectively), indicating this computational method accurately estimates the carbon-13 chemical shifts of these structures.
Table 2.
Calculated vs. experimental carbon-13 data for precolibactin 795a (5), the oxazole 13 and the azlactone 21.
 | |||
|---|---|---|---|
| position | |Δδ| precolibactin 795a (5) |
|Δδ| 13 |
|Δδ| 21 |
| 1 | 1.7 | 1.5 | 1.5 |
| 2 | 5.6 | 4.1 | 5.6 |
| 3 | 2.1 | 1.8 | 0.5 |
| 4 | 1.4 | 0.3 | 0.8 |
| 5 | 1.1 | 2.8 | 5.2 |
| 6 | 33.6 | 1.9 | 1.3 |
| 7 | 14.9 | 2.6 | 0.3 |
| 8 | 7.5 | 2.1 | — |
| RMS | 8.48 | 2.13 | 2.17 |
The structure of precolibactin 969 (4) was determined by a combination of high-resolution mass spectrometry (HRMS) and NMR analysis.6 HRMS analysis indicated that precolibactin 969 (4) had a mass 83 Da greater than the known isolate precolibactin 886,9 the structure of which has been confirmed by chemical synthesis.10 It was determined that the mass difference corresponds to a C3HNO2 fragment and this was assigned as a C-terminal 5-hydroxyoxazole moiety based on NMR analysis of precolibactin 795a (5).6 Our data suggest this assignment is incorrect, and due to the high degree of un-saturation and substitution, alternative structural arrangements that are consistent with the most likely biosynthetic pathway are possible (Fig. S1). For these alternative structures, carbon-13 NMR shifts were predicted and compared to those of the isolate, but no structure showed a better agreement.
In conclusion, the experimental results outlined above indicate that the 5-hydroxyoxazole-4-carboxy derivatives are unstable toward hydrolytic ring-opening and decarboxylation. These stability issues, combined with the differences between the theoretical and experimental carbon-13 data, suggest precolibactins 795a (5) and 969 (4) may not contain 5-hydroxyoxazole-4-carboxylic acid residues.
Supplementary Material
ACKNOWLEDGMENT
Financial support from the Swiss National Science Foundation (P2EZP2_187928 to A.T.), the Chemistry Biology Interface Training Program (T32GM067543 to K.M.W.), the National Institutes of Health (R01CA215553 to S.B.H.) and Yale University is gratefully acknowledged.
Footnotes
Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
Detailed experimental procedures and characterization data for all new compounds (PDF)
The authors declare no competing financial interest.
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