Structure of a Putative Fluorinated Natural Product from - American

Communication pubs.acs.org/jnp

Structure of a Putative Fluorinated Natural Product from Streptomyces sp. TC1 Hülya Aldemir,†,§ Stefanie V. Kohlhepp,‡,§ Tanja Gulder,*,‡ and Tobias A. M. Gulder*,† †

Biosystems Chemistry, Department Chemie and Center for Integrated Protein Science Munich (CIPSM), and ‡Department Chemie, Technische Universität München, Lichtenbergstraße 4, 85747 Garching, Germany S Supporting Information *

ABSTRACT: Fluorine-containing natural products are extremely rare. The recent report on the isolation and biological activity of the bacterial secondary metabolite 3(3,5-di-tert-butyl-4-fluorophenyl)propionic acid was thus highly remarkable. The compound contained the first aromatic fluorine substituent known to date in any natural product. The promise to discover an enzyme capable of aromatic fluorination in the producing strain Streptomyces sp. TC1 prompted our immediate interest. A close inspection of the originally reported analytical data of the fluoro metabolite revealed inconsistencies that triggered us to validate the reported structure. The results of these efforts are presented in this communication.

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Marimuthu et al.,13 where they reported the isolation, characterization, and structure elucidation of 3-(3,5-di-tertbutyl-4-hydroxyphenyl)propionic acid (1, Figure 1) from

econdary metabolites containing carbon−halogen bonds form a constantly growing group of structurally, biosynthetically, and biomedically interesting small molecules. More than 5000 halogenated natural products have been reported in the literature.1 Nature has evolved an intriguing set of halogenating enzymes introducing chlorine, bromine, and iodine into organic molecules, including heme- and vanadiumdependent haloperoxidases and FADH2- and O2-dependent, as well as non-heme iron- and O2-dependent, halogenases.2 The basic halogenation principle of all these biocatalysts is the oxidative generation of a reactive halogen species, either a halogen radical or cation. The unique electrochemical properties of fluorine, however, preclude such an oxidative halogenation mechanism. The installation of fluorine into secondary metabolites is thus a biochemically challenging task, as evident from the fact that only a handful of fluorinated natural products are known to date.3,4 Streptomyces cattleya was shown to produce fluoro acetate and 4-fluorothreonine. Pioneering work by O’Hagan and co-workers identified a unique fluorinase (flA) that facilitates the nucleophilic attack of a desolvated fluoride on S-adenosyl-L-methionine (SAM) to install a C−F bond yielding 5′-fluorodeoxyadenosine (5′-FDA), which is subsequently metabolized into fluoro acetate and 4fluorothreonine.5−7 Interestingly, the marine bacterium Salinispora tropica utilizes a similar enzymatic process for chlorine incorporation into salinosporamide A by the chlorinase SalL via the analogous 5′-ClDA derivative.8−10 This allowed the straightforward engineering of fluoro salinosporamide by replacing the chlorinase gene salL with the fluorinase gene f lA.11 More recently, the site-selective incorporation of fluoro acetate into polyketides was demonstrated using excised modules of the erythromycin biosynthetic machinery.12 These two examples clearly show the potential of the fluorinase flA to be used to generate novel C(sp3)−F bond containing natural products. No natural product containing an sp2-bound fluorine had been published prior to the most recent paper by © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. (A) Structure of 1 as originally reported; (B) 1H (black numbers) and 13C NMR (blue numbers) chemical shifts of 1 (in ppm).

Streptomyces sp. TC1. This intriguingly suggested the existence of an unprecedented biocatalytic system that achieves aromatic fluorination, a finding challenged by O’Hagan and co-workers, who recently synthesized 1 and showed that the spectroscopic data of natural and synthetic 1 did not match.14 The actual structure of the initially isolated metabolite, however, was not further investigated in this work. In parallel to O’Hagan’s group, Received: August 13, 2014

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dx.doi.org/10.1021/np500643g | J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 2. Copy of the originally reported 1H NMR spectrum of 1.13

functionalities. The integration of the signals was calibrated by Marimuthu et al. by setting the signal of b1 to 2.00. When simply looking at the numbers of the resulting integrations of all other signals, this leads to the reported outcome of one proton for a1 (0.9609), one for a2 (0.9360), two for c1 (not clearly readable in the original spectrum), and eight to nine for d1 and d2 (8.4096 and 8.9212, respectively). However, this does not match the graphical integrals shown in red in the spectrum itself. The distance of the two lower dashed lines (double arrow 1) shows the length of the integral of signal b1, consequently corresponding to two protons. This is also expectedly true for c1. Despite the reported numbers below the spectrum, the integral representing a1 is of equal height (→ also two protons, not 0.9609), while the one for a2 has only half this height (corresponding to one proton). Similar inconsistencies can be found for signals d1 and d2, where the integral of d2 shows a length depicted by double arrow 2, while the integral of d1 exceeds the frame of the original presentation of the 1H NMR spectrum (despite the fact that the reported integrals by number are even smaller for d1 when compared to d2; see above). This clearly shows not only that the 1H NMR data of 1 have been misinterpreted but that integration of the individual signals is incorrect. Given the distribution of protons as tentatively established by us, we suspected that the NMR spectrum is in fact derived from a 2:1 mixture of two compounds, 2 and 3, with similar substructures. Compound 2 was expected to contain the protons corresponding to signals a1, b1, c1, and d1, with 3 consequently harboring protons a2 and d2. A likely candidate for 2, which would contain the side chain located at C-4 in the original structure 1, would thereby be a compound that could match the X-ray crystallographic data collected for 1. We thus

we set out to elucidate the true identity of the original compound, triggered by major inconsistencies in the spectroscopic data of Marimuthu et al.13 Excited by the yet unprecedented structure of 1 (Figure 1A) presented in the work of Marimuthu et al.,13 we had a close look at the reported analytical data of this secondary metabolite. While the structure of 1 at first sight seemed to be firmly established by X-ray structure analysis, we were puzzled by the reported NMR data. Unexpectedly, six carbon chemical shifts were given for the aromatic system, in addition to two aromatic proton resonances as well as two full sets of signals for the chemically and magnetically equivalent tert-butyl groups (see Figure 1B). This is clearly not explainable, as the molecular symmetry of 1 should lead to only a single resonance for C-2 and C-6, C/H-3 and C/H-5, C-1′ and C-1‴, and C/H2′ and C/H-2‴. Furthermore, no F−C or F−H coupling constants were reported. In addition, the mass spectrometric data (FAB in positive ion mode) deposited in the Supporting Information13 did not match the expected value for 1: while a molecular ion of [M + H]+ = 281.1 was expected, the spectrum showed a peak at 280.18 instead. Equally disturbing was the provided 19F NMR spectrum of 1, with an extremely broad signal (approximately 40 ppm), which is not consistent with the common sharp signals of low molecular weight organofluorines.14 The summation of these inconsistencies led us to have a careful look at the original 1H NMR spectrum (Figure 2). At first sight the NMR data reported in Table 1 of the original manuscript seemed to match the provided spectrum (not the elucidated structure), with the two aromatic protons a1 and a2, the two signals b1 and c1 representing the methylene units, and two signals for the methyl groups at two tert-butyl B



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Figure 3. 1H NMR spectra (CDCl3) of (A) 3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionic acid (2, red) and 2,6-di-tert-butylcyclohexa-2,5-diene-1,4dione (3, blue) and (B) a 2:1 mixture of 2 and 3; (C) copy of the originally reported 1H spectrum of 1.

strongly indicating the expected interaction of the sp2-bound proton in 3 with the “southern” keto functionality. Concerning the reported antioxidative properties of 1, it is interesting to note that compounds 2 and 3 are degradation products of the commercial compounds Stabiliff15 and Irganox 1010,16 which are used in the fragrance and plastic industry, respectively, to prevent oxidative degradation. Work by Dreher et al. furthermore showed that quinone 3 can be easily produced by oxidation of the methyl ester of 2.17 Leaching of fenozan (2) and 3 from plastic materials, such as PVC-free polyolefins, into water or water/organic solvent mixtures has also been reported in the literature.18 It is thus likely that the authors of the original study isolated components of their laboratory plastics, rather than a natural product from Streptomyces sp. TC1.

assumed that the corresponding hydroxy derivative of 1, fenozan (2) (Figure 3A), is a good candidate. Indeed, when we measured a 1H NMR spectrum of 2 it showed a perfect match to the signal series a1−d1 of the original spectrum. The phenolic proton signal was additionally found at 5.09 ppm, a signal also present in the original spectrum (Figure 2 at 5.0867 ppm), but not taken into consideration before. The minor constituent of the mixture was expected to be structurally highly related to 2, but without the C-4 side chain. In addition, the system was not expected to be aromatic because of the obvious upfield shift of the protons a2 and d2. In consequence, benzoquinone 3 seemed to perfectly fulfill these requirements, as proven by the perfect match of the corresponding 1H NMR when compared to a2 and d2 (Figure 3A). To further corroborate our results, we made a 2:1 mixture of compounds 2 and 3 that was likewise analyzed by 1H NMR spectroscopy. The resulting spectrum (Figure 3B) perfectly matched the one reported for the natural product 1 (Figure 3C), thus clearly evidencing the initial NMR spectrum not to originate from 1, but rather from a 2:1 mixture of 2 and 3 (see also Table S1). It is important to note the APT (attached proton test)-derived 13 C chemical shifts reported for 1 do not contain all signals expected from a mixture of compounds 2 and 3. However, the APT spectrum reported in the original article is of general low quality. At least all 13C signals of 2 can be identified in the initially provided list of NMR signals (see also Table S2). In addition, the HMBC (heteronuclear multiple bond correlation) spectrum reported in the Supporting Information of the paper by Marimuthu et al.13 shows a strong cross-peak between proton a2 and a carbon chemical shift of >185 ppma 13C chemical shift not listed in the published NMR table for 1

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ASSOCIATED CONTENT

* Supporting Information S

Original copies of the 1H and 13C NMR spectra of 2 and 3 as well as a tabular summary of these data in comparison with the originally reported data of 1 are provided. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*(T. Gulder) Tel: +49-89-228-14591. E-mail: tanja.gulder@ tum.de. *(T. A. M. Gulder) Tel: +49-89-228-13833. E-mail: tobias. [email protected]. Author Contributions §

H. Aldemir and S. V. Kohlhepp contributed equally.

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Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS H.A. thanks the Fonds der Chemischen Industrie (FCI) for her Ph.D. scholarship. Research in the laboratories of T.G. and T.A.M.G. is generously funded by the Fonds of the Chemical Industry (FCI) as well as the German Research Foundation (DFG) through the Emmy Noether Program (T.G.: GU 1134/ 3-1, T.A.M.G.: GU 1233/1-1/2) and the Cluster of Excellence Center for Integrated Protein Science Munich (CIPSM).

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REFERENCES

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Structure of a Putative Fluorinated Natural Product from - American

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