Prolinimines: N-Amino-l-Pro-methyl Ester (Hydrazine) Schiff Bases

Nov 26, 2017 - HRESIMS, UV−vis, [α]D) identical to those isolated from. Table 1. NMR (Methanol-d4) Data for Prolinimines A (1), B (2), and C (3). 1...
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Letter Cite This: Org. Lett. 2018, 20, 377−380

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Prolinimines: N‑Amino‑L‑Pro-methyl Ester (Hydrazine) Schiff Bases from a Fish Gastrointestinal Tract-Derived Fungus, Trichoderma sp. CMB-F563 Osama G. Mohamed, Zeinab G. Khalil, and Robert J. Capon* Institute for Molecular Bioscience, The University of Queensland, St Lucia, Queensland 4072, Australia S Supporting Information *

ABSTRACT: A rice cultivation of a fish gastrointestinal tractderived fungus, Trichoderma sp. CMB-F563, yielded natural products incorporating a rare hydrazine moiety, embedded within a Schiff base. Structures inclusive of absolute configurations were assigned to prolinimines A−D (1−4) on the basis of detailed spectroscopic and C3 Marfey’s analysis, as well as biosynthetic considerations, biomimetic total synthesis, and chemical transformations. Of note, monomeric 1 proved to be acid labile and, during isolation, underwent quantitative transformation to dimeric 3 and trimeric 4. Prolinimines are only the second reported natural products incorporating an N-amino-Pro residue, the first to include L-Pro, the first to occur as Schiff bases, and the first to be isolated from a microorganism.

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n an effort to enhance investigations into the secondary metabolites of Australian marine-derived fungi, we speculated that commercial fish species, those with a feeding preference for detritus and algae, may act as natural myco-accumulators. We further hypothesized that such fish may be a readily accessible source of fungal taxonomic, genetic, and chemical diversity. To test these hypotheses, three Mugil mullet acquired from a local fish market were used to assemble a library of ∼500 gastrointestinal tract-derived (GIT) fungal isolates. UPLCDAD profiling of the crude extracts obtained from agar plate cultivations facilitated dereplication and revealed a remarkable level of chemical productivity. This report provides an account of our investigations into this fish GIT-derived resource, commencing with Trichoderma sp. CMB-F563. Following cultivation trials on an array of media, UPLC-DAD profiling revealed extracts obtained from rice grain cultivations of Trichoderma sp. CMB-F563 as particularly rich in a diverse array of secondary metabolites. To explore this potential, the EtOAc extract obtained from a 45 d rice grain cultivation was subjected to sequential solvent partitioning and trituration, followed by gel and reversed-phase chromatography to yield known and new phenolic bisanthraquinones, alkyl nucleosides, and aryl glycosides (to be reported later). More significantly, this fractionation also yielded an unprecedented family of hydrazinyl Schiff bases, prolinimines B−D (2−4, Figure 1), the structure elucidation of which was secured by spectroscopic and C3 Marfey’s analysis, as well as total biomimetic synthesis and chemical transformations, as summarized below. HRESI(+)MS analysis of 2 afforded a sodium adduct ion attributed to a molecular formula (C18H24N4O5, Δmmu −0.6) requiring 9 double bond equivalents (DBE). The 1D NMR (methanol-d4) data for 2 (Table 1) revealed symmetry (i.e., an © 2018 American Chemical Society

Figure 1. Prolinimines A−D (1−4).

axis). Further analysis identified a CO2CH3 moiety (δH 3.73, CO2CH3; δC 52.7, CO2CH3; 175.8, CO2CH3) which 2D NMR correlations (Figure 2) linked to an N-substituted proline methyl ester residue, and after allowing for an axis of symmetry accounted for 4 DBE. This conclusion was confirmed by a C3 Marfey’s analysis,1 which also established the L-Pro configuration (Supporting Information). Consideration of NMR resonances for the remaining elements of (C3HN)2, namely, two deshielded Received: November 26, 2017 Published: January 9, 2018 377

DOI: 10.1021/acs.orglett.7b03666 Org. Lett. 2018, 20, 377−380

Letter

Organic Letters Table 1. NMR (Methanol-d4) Data for Prolinimines A (1), B (2), and C (3) 1 Pos. furanyl F1 1 2 3 4 5 6 prolinyl P1 1′ 2′ 3′ 4′ 5′ CO2Me

δH, m (J in Hz) 7.14, s 6.37, d (3.3) 6.32, d (3.3) 4.50, s

4.28, dd (8.9, 3.1) a 2.00, m b 2.26, m 2.05, m a 3.24, dd (16.9, 7.7) b 3.46, m 3.72, s

2 δC 125.9 153.1 109.5 110.4 155.7 57.6 175.8 66.1 29.4 23.5 49.9 52.7

δH, m (J in Hz) 7.12, s

3 δC 125.4 152.9 110.6

6.45, s

δH, m (J in Hz) 7.12, s

126.3 152.7 109.9 109.8 152.4 28.3

6.36, d (3.3) 6.15, d (3.3) 4.02, s

4.30, dd (8.9, 3.2) a. 2.02, m b. 2.27, m 2.07, m a. 3.26, dd (16.9, 7.7) b. 3.48, m 3.73, s

175.8 66.1 29.5 23.5 49.9 52.7

δC

4.26, dd (8.9, 3.2) a. 2.01, m b. 2.26, m 2.06, m a. 3.23, dd (16.9, 7.7) b. 3.46, m 3.72, s

175.9 66.1 29.4 23.5 50.0 52.7

prolinimine C (3) was assigned as shown, with the L-Pro absolute configuration assigned on the basis of biosynthetic considerations (cometabolite with 2) and total synthesis from L-Pro (see below). HRESI(+)MS analysis of 4 afforded a sodium adduct ion attributed to a molecular formula (C35H42N6O9, Δmmu −2.2), requiring 18 DBE. Although the quantity of 4 isolated from CMB-F563 extracts did not allow for the acquisition of 1D and 2D NMR (DMSO-d6) data sufficient for unambiguous structure assignment, the available data did nevertheless indicate the presence of an asymmetric molecule containing three each of furanyl, imine, and N-substituted-Pro methyl ester residues, accounting for all DBE (Table 2). To solve the structure of 4 required total synthesis. To confirm structures assigned to 2−3 and assign a structure to 4, a convergent biomimetic synthesis was designed around the putative biosynthetic precursors N-amino-L-Pro methyl ester (5), 2,5-furandicarboxaldehyde (6), and 5-hydroxymethylfurfural (7) (Scheme 1). In a two-step process, L-Pro (8) was converted to its N-nitroso methyl ester 9, which was in turn reduced to 5.2,3 Exposure of 5 to 6 readily yielded the bis-Schiff base prolinimine B (2), whereas exposure of 5 to 7 yielded the mono-Schiff base prolinimine A (1) whose structure, including imine configuration, was assigned by detailed spectroscopic analysis (Figure 2) and comparisons to 2−4. Significantly, exposure of 1 to slightly acidic conditions yielded 3, with accompanying yields of prolinimine D (4). In summary, prolinimines A−D (1−4) were prepared in nonoptimized yields of 36, 50, 3, and 1%, respectively, from 8. A plausible mechanism for the acid-mediated transformation of 1 to 3−4 involves dehydration to form an activated furanyl oxonium species, primed for dimerization and the loss of formaldehyde to yield 3 and trimerization to 4 (Scheme 2). By contrast, as 2 lacks an acid sensitive allylic 1°-OH moiety, it is incapable of generating a furanyl oxonium and is inert to acidmediated transformation. Although acid modifiers were not used during the isolation of prolinimines, fractions were nevertheless acidic due to high concentrations of accompanying phenolic bisanthraquinone cometabolites. Significantly, synthetic samples of 2−4 were chemically (UPLC-DAD) and spectroscopically (1D and 2D NMR, HRESIMS, UV−vis, [α]D) identical to those isolated from

Figure 2. Diagnostic NMR correlations for 1−4.

sp2 methines (δH 7.12, δC 125.4; δH 6.45, δC 110.6) and one sp2 quaternary carbon (δC 152.9) as well as 5 unattributed DBE and additional 2D NMR correlations (Figure 2), supported the structure for prolinimine B (2) as indicated. The E imine configuration was assigned on the basis of diagnostic ROESY correlations between H-1 and H2-5′ (Figure 2). HRESI(+)MS analysis of 3 afforded a sodium adduct ion attributed to a molecular formula (C23H28N4O6, Δmmu −0.4), requiring 12 DBE. As with 2, the 1D NMR (methanol-d4) data for 3 (Table 1) indicated symmetry (i.e., an axis), with selected resonances and correlations (Figure 2) readily attributed to Nsubstituted Pro methyl ester residues linked through an imine to a bis-furanyl moiety. Identical chemical shifts (δH 7.12) for H-1 in 3 and 2 supported a common E imine configuration, further confirmed by diagnostic ROESY correlations between H-1 and H2-5′. The 2,5-disubstituted bis-furanyl moiety in 3 was evidenced by diagnostic 2D NMR correlations to and from C6/H2-6 at the axis of symmetry (Figure 2). Thus, the structure for 378

DOI: 10.1021/acs.orglett.7b03666 Org. Lett. 2018, 20, 377−380

Letter

Organic Letters Table 2. NMR (DMSO-d6) Data for Prolinimine D (4) Pos. furanyl F1 1 2 3 4 5 6 prolinyl P1 1′ 2′ 3′ 4′ 5′ CO2Me A−G

δH, m (J Hz)

δC

7.06, s

4.00, s

123.6 150.9 108.2A 108.7 150.4 26.9

4.22, dd (8.9, 2.3) a. 1.91, mB b. 2.18, mC 1.97, mD a. 3.14, mE b. 3.36, mF 3.63, sG

173.3B 64.3C 27.8D 22.0E 48.6F 51.7G

6.34, d (3.3) 6.19, d (3.3)

A

Pos. furanyl F2 7 8 9 10 11 prolinyl P2 1″ 2″ 3″ 4″ 5″ CO2Me

δH, m (J Hz)

δC

6.07, s 7.10, s

150.0 110.4 118.8 146.3 123.2

4.22, dd (8.9, 2.3) a. 1.91, mB b. 2.18, mC 1.97, mD a. 3.14, mE b. 3.36, mF 3.62, s

A

173.3B 64.5 27.7 22.0E 48.5 51.8

Pos. furanyl F3 12 13 14 15 16 17 prolinyl P3 1‴ 2‴ 3‴ 4‴ 5‴ CO2Me

δH, m (J Hz)

δC

3.87, br s 6.07, d (3.3) 6.31, d (3.3) 7.05, s

4.22, dd (8.9, 2.3) a. 1.91, mB b. 2.18, mC 1.97, mD a. 3.14, mE b. 3.36, mF 3.63, sG

23.4 153.1 107.8 108.2A 150.6 123.9

A

173.3B 64.3C 27.8D 22.0E 48.6F 51.7G

δH and δC assignments with the same superscript may be interchanged.

CMB-F563. With synthetic 4 in hand, we acquired higher quality NMR (DMSO-d6) data (Supporting Information), revealing diagnostic 2D NMR correlations, and in particular ROESY correlations from H-11 to H2-12 (Figure 2), which established the C-9 to C-12 substitution and provided an unambiguous structure assignment for prolinimine D (4). HPLCMS analyses of crude EtOAc extracts derived from CMB-F563 rice cultivations successfully detected 1 and 2, but not 3 nor 4. From these observations, we conclude that 1 is a natural product and cometabolite of 2, albeit one that is acid labile and in need of careful handling during extraction and fractionation. Furthermore, although isolated from the crude EtOAc extract, 3 and 4 are in fact acid-mediated handling artifacts. Of note, the acid-mediated biomimetic transformation of 1 to 3 and 4 reveals a promising process for regio-controlled

Scheme 1. Synthesis of 1−4

Scheme 2. Plausible Mechanism for the Acid-Mediated Interconversion of 1 to 3 and 4

379

DOI: 10.1021/acs.orglett.7b03666 Org. Lett. 2018, 20, 377−380

Letter

Organic Letters

(4) Klaiklay, S.; Rukachaisirikul, V.; Phongpaichit, S.; Buatong, J.; Preedanon, S.; Sakayaroj, J. Nat. Prod. Res. 2013, 27 (19), 1722. (5) Li, H.; Saravanamurugan, S.; Yang, S.; Riisager, A. ACS Sustainable Chem. Eng. 2015, 3 (12), 3274. (6) Le Goff, G.; Ouazzani, J. Bioorg. Med. Chem. 2014, 22 (23), 6529. (7) Mayengbam, S.; Yang, H.; Barthet, V.; Aliani, M.; House, J. D. J. Agric. Food Chem. 2014, 62 (2), 419.

assembly of methylene interrupted 2,5-disubstituted furans. While such furans are exceptionally rare among natural products, being limited to flavodonfuran isolated from the mangrove endophytic fungus Flavodon flavus PSU-MA201,4 they are of considerable interest in the catalytic transformation of biomassderived carbohydrates to biodiesel.5 Naturally occurring hydrazines are also extremely rare, with only a handful of nonacylated examples reported to date.6 A particularly interesting and relevant example of an acylated hydrazine (i.e., a hydrazide) is the flaxseed toxin linatine, which is an amide conjugate of glutamic acid and the hydrazine N-amino2 D-Pro. In chickens reared on linseed meal (i.e., flaxseed) linatine undergoes in vivo proteolysis to N-amino-D-Pro, which then forms a Schiff base with and sequesters the aldehyde pyridoxine, leading to vitamin B6 deficiency syndrome,7 rendering linseed meal unsuitable as animal feed. The fungal prolinimines represent only the second reported occurrence of a naturally occurring metabolite incorporating an N-amino-Pro residue. As many fungi are known to infect stored grains, should any such strains produce hydrazine-based vitamin B6 antagonists (i.e., N-amino-Pro), or closely related pro-drugs (i.e., hydrazides or Schiff bases), they could render grain supplies toxic. Forewarned is forearmed, and if recognized early, such hydrazine intoxication could be treated with vitamin supplements.7 Our discovery of the fungal origin, structures, and chemistry of the hydrazinyl Schiff base prolinimines represents a timely opportunity to raise awareness of the potential contamination of foodstuffs by fungi-derived N-amino-L-Pro (or related prodrugs).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.orglett.7b03666. General experimental procedures, microbial taxonomy and cultivation, purification, characterization, synthesis, chemical interconversion, and spectroscopic data (tabulated and spectra) for 1−4 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +61-7-3346-2979. Fax: +61-73346-2090. ORCID

Robert J. Capon: 0000-0002-8341-7754 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank P. Abdala for assistance in fungal isolations. O.G.M. acknowledges the provision of a University of Queensland, UQ International Scholarship. This work was funded in part by the Institute for Molecular Bioscience and the University of Queensland.

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REFERENCES

(1) Vijayasarathy, S.; Prasad, P.; Fremlin, L. J.; Ratnayake, R.; Salim, A. A.; Khalil, Z.; Capon, R. J. J. Nat. Prod. 2016, 79 (2), 421. (2) Klosterman, H. J.; Lamoureux, G. L.; Parsons, J. L. Biochemistry 1967, 6 (1), 170. (3) Waibel, M.; Hasserodt, J. J. Org. Chem. 2008, 73 (16), 6119. 380

DOI: 10.1021/acs.orglett.7b03666 Org. Lett. 2018, 20, 377−380