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Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX
Synthesis and Structure Reassignment of Malylglutamate, a Recently Discovered Earthworm Metabolite Corey M. Griffith,†,§ Abigail Feceu,‡,§ Cynthia K. Larive,‡ and David B. C. Martin*,‡ †
Environmental Toxicology Graduate Program and ‡Department of Chemistry, University of California, Riverside, California 92521, United States
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ABSTRACT: Malylglutamate, a newly identified metabolite in earthworms, was synthesized using a traditional peptide coupling approach for assembling the amide from protected malate and glutamate precursors. The proposed structure (1) and a diastereomer were synthesized, but their NMR spectra did not match the natural sample. Further analysis of the natural sample using HMBC spectroscopy suggested an alternative attachment of the malyl moiety, and β-malylglutamate (2) diastereomers were synthesized, L,L-2 and D,D-2. NMR spectra were an excellent match with the natural sample, and chiral-phase chromatography was employed to identify (−)-β-Lmalyl-L-glutamate (2) as the isomer native to Eisenia fetida.
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alylglutamate (1) is a newly identified metabolite found in earthworm species Eisenia fetida.1 It is a relatively abundant metabolite, present at an average of 7 μg/mg in whole earthworms, and similar in structure to metabolites such as N-acetylaspartylglutamate (NAAG, 3) and β-citrylglutamate (4). Malylglutamate is prominent in the NMR metabolite profiles of whole earthworm, coelomic fluid, and coelomocyte extracts.1−3 Coelomic fluid is a biofluid that fills the body cavity of the worm and is critical for movement, excretion, metabolism, and nutrient storage, while coelomocytes are freemoving immune and liver-like cells in the coelomic fluid.4 The metabolite profiles of coelomic fluid and coelomocytes are primarily composed of amino acids, organic acids, and polyamines, including N-trimethylated amino acids (i.e., betaine analogues).1 Malylglutamate is a suggested anionic osmolyte and a charge-balance counterpart to betaine analogues in earthworms, which are known cationic osmolytes.1,5 Additionally, malylglutamate is postulated to be a store for glutamic acid and malic acid, and a chelator, due to its similarity in structure to β-citrylglutamate (4), a known chelator.1,6,7 Following up on the recent identification of malylglutamate by two of the authors,1 we sought to confirm the proposed structure of malylglutamate and fully assign the relative and absolute configuration. Due to complexity of 1H NMR spectra of earthworm extracts and the difficulty in isolating a pure sample for further studies, we turned our attention to synthesis using a traditional peptide coupling approach. This strategy allows the incorporation of either L- or D-isomers of both glutamic and malic acids to determine both relative and absolute configuration. As we describe below, these efforts have led to a reassignment of the structure of natural (−)-malylglutamate as the β-isomer L,L-2. Our synthesis strategy takes advantage of previous reports that describe the selective protection of either carboxylic acid of malic acid.8 Benzyl ester protecting groups were chosen to © XXXX American Chemical Society and American Society of Pharmacognosy
allow easy deprotection by hydrogenolysis in the final step. A Fischer esterification of L-glutamic acid with benzyl alcohol gave doubly protected intermediate L-6 as the tosylate salt (Scheme 1); D-6 was also synthesized in the same manner.9 For the malic acid fragment, the two carboxylic acids were differentiated using an acetonide protecting group, favoring the five-membered ring.8 Benzylation of the free acid provided acetonide intermediate L-8. Selective hydrolysis of the acetonide ester group gave monobenzyl ester L-9. Coupling of the two fragments was explored with a variety of common amide coupling reagents. Acceptable results were obtained using EDCI and HOBt in the presence of NEt3 to deprotonate the tosylate salt L-6, giving amide L,L-10 in moderate yield. Similarly, the coupling of protected D-glutamate gave amide L,D-10. Removal of the benzyl protecting groups using Received: December 21, 2018
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DOI: 10.1021/acs.jnatprod.8b01083 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Scheme 1. Synthesis of Both Diastereomers of the Originally Proposed Structure 1a
a a. BnOH, 1.22 equiv of p-TsOH, toluene, rt; b. 2,2-dimethoxypropane (DMP), 1 mol % p-TsOH, CH2Cl2; c. BnBr, Cs2CO3, DMF; d. AcOH, H2O, 60 °C; e. 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI), N-hydroxybenzotriazole (HOBt), NEt3, DMF; f. H2, 5 mol % Pd/C, EtOH.
hydrogenolysis gave the two diastereomeric products L,L-1 and L,D-1 in 73% and 71% yields, respectively. Comparison of the 1H NMR data for synthetic L,L-1 and L,D-1 in D 2 O with a naturally derived sample of malylglutamate showed that the compounds were not identical (Figure 1). In particular, the resonances for proton H-2 were
Figure 2. 1H−13C HMBC spectrum of an 80-worm pooled coelomocyte sample. The peaks of malylglutamate are annotated, and the H-3′a/C-4′ and H-3′b/C-4′ peaks suggest a reassignment of the structure to β-malylglutamate (2).
3, and H2-4 protons nicely show the correct assignment. Looking at the malyl moiety, equal intensities were observed between the carbonyls and H-2′ proton, which did not aid with the assignment, labeled as H-2′/C-1′ and H-2′/C-4′ peaks (Figure 2); however, a very intense correlation between the amide carbonyl (C-4′) and H2-3′ protons was observed, noted as H-3′a/C-4′ and H-3′b/C-4′ (Figure 2 and Scheme 2). This suggests that H2-3′ protons were positioned directly next to the amide carbonyl and indicates that the malyl moiety was attached to glutamate in the β position (i.e., β-malylglutamate 2). An alternative structure that would be consistent with the available MS fragmentation data, HMBC analysis, and the lower chemical shift of proton H-2′ was proposed. In the
Figure 1. 1H NMR spectrum of an earthworm coelomocyte sample (top) compared to L,L-1 and L,D-1 (Scheme 1).
slightly shifted to the left in both synthetic molecules, and proton H-2′ was significantly shifted to the left in both αmalylglutamate (1) isomers (Scheme 1). To ascertain whether the inconsistencies between the synthetic standards and native sample were due to matrix or other effects, heteronuclear multiple bond correlation spectroscopy (HMBC) was employed to explore long-range correlations between protons and carbons.10 Pooled earthworm coelomocyte samples were used to conduct the analyses (Figure 2). Correlations between both of glutamate’s carbonyls (C-1 and C-5) with the H-2, H2B
DOI: 10.1021/acs.jnatprod.8b01083 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Scheme 2. Synthesis of Both Diastereomers of the Revised Structure 2a
a
a. TFAA, BnOH; b. EDCI, HOBt, NEt3, DMF; c. H2, 10 mol % Pd/C, EtOH.
revised proposal, the β-carboxylic acid of malic acid forms the amide to glutamate rather than the α-carboxylic acid (i.e., 2). For comparison, both α-malylarginine and β-malylarginine isomers are found in apple and pear trees.11 This alternative structure was therefore synthesized using a different protecting group strategy. Selective benzylation of the desired α-carboxylic acid was achieved using a known procedure with trifluoroacetic anhydride to give ester L-11 (Scheme 2).12 Amide coupling of L-11 with L-glutamate derivative L-6 followed by reductive removal of all three benzyl groups gave the new target amide L,L-2. A similar sequence starting with D-malic acid gave diastereomer D,L-2 and enantiomer D,D-2. Purification of the protected intermediates was easier in these cases, and three of the four stereoisomers of β-malylglutamate were efficiently accessed for comparison to the natural product. Access to synthetic β-malylglutamate allowed comparison of the 1H NMR data in D2O to the natural sample. While the of D,L-2 isomer differed in J-coupling of the H2-4 protons (Figure 3), the L,L-2 isomer provided an excellent match in chemical shift and J-coupling in the earthworm extract, establishing the correct relative configuration.
standards (Figure 4), indicating that it is an isomer of 264.071 m/z. To identify the enantiomer of malylglutamate present in the natural sample, coelomic fluid, coelomocyte, and whole earthworm extracts were injected on the LC-MS, and the retention time and MS/MS spectra revealed L,L-2 as the correct diastereomer (Figure 4A). To confirm the assignment, L,L-2 and D,D-2 were spiked into separate coelomic fluid samples where an increase in the L,L-2 peak intensity was observed in the L,L-2-spiked sample (Figure 4B), while a new peak was observed in the D,D-2-spiked sample and the L,L-2 peak intensity stayed consistent with the original sample (Figure 4C). The specific rotation of synthetic L,L-2 was determined to be [α]25D −13.6 (c 1.0, MeOH), thereby establishing the natural product to be (−)-β-L-malyl-Lglutamate. In summary, we synthesized several diastereomers and constitutional isomers of the originally proposed structure of malylglutamate using a selective protecting group strategy. By comparison to natural malylglutamate derived from earthworms, we identified the correct structure to be the β-isomer L,L-2, resulting in the reassignment of the originally proposed structure. We also determined the absolute configuration, confirming that natural (−)-malylglutamate is derived from Lmalic acid and L-glutamic acid, providing support for the hypothesis that this new metabolite can serve as a store of these two essential α-hydroxy acid and α-amino acid derivatives.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotation was measured on a Rudolph Research Analytical Autopol IV automatic polarimeter. IR spectra were recorded on a Bruker Alpha FT-IR spectrometer. 1H and 13C NMR spectra of natural samples were recorded on a Bruker Avance 600 MHz NMR spectrometer equipped with a SmartProbe. 1H and 13C NMR spectra of synthetic samples were recorded on a Varian Inova 400 MHz spectrometer unless otherwise indicated and were internally referenced to residual solvent signal (note: D2O was referenced at 4.79 ppm for 1H and CDCl3 referenced at 7.26 ppm for 1H NMR and 77.16 ppm for 13C NMR, respectively). Data for 1H NMR are reported as follows: chemical shift (δ ppm), integration, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), and coupling constant (Hz). Data for 13C NMR are reported in terms of chemical shift, and no special nomenclature is used for equivalent carbons. High-resolution mass spectrometry data were recorded on an Agilent LCTOF instrument using direct injection of samples in dichloromethane into the electrospray source (ESI) with positive ionization. Chiral-phase
Figure 3. 1H NMR spectrum of an earthworm coelomocyte sample (top) compared to L,L-2 and D,L-2 (Scheme 2).
To finalize the configurational assignment of β-malylglutamate, we sought to determine the absolute configuration. A chiral-phase chromatography method was developed to separate L,L-2 and D,D-2 using liquid chromatography−mass spectrometry (LC-MS). Excellent separation was achieved, yielding retention times of 1.61 min for L,L-2 and 8.28 min for D,D-2 and identical MS/MS spectra (Figure 4). The D,D-2 standard contained a second peak at 3.14 min with identical m/z as our standards; however, the MS/MS spectrum of the unknown peak was inconsistent with L,L-2 and D,D-2 C
DOI: 10.1021/acs.jnatprod.8b01083 J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products
Note
Figure 4. Chiral-phase separation of L,L-2 and D,D-2 isomers using LC-MS at m/z 264.071. Chromatograms of (A) coelomic fluid sample; (B) coelomic fluid sample spiked with L,L-2; and (C) coelomic fluid sample spiked with D,D-2, where *denotes an unknown isomer of the [M + H]+ ion. Positive-ion ESI-MS and annotated MS/MS of malylglutamate (calculated m/z 264.0719 for [M + H]+ ion) are shown for each isomer: (D) MS1 of L,L-2 isomer and (E) its annotated MS/MS spectrum for the [M + H]+ ion; (F) MS1 of D,D-2 isomer and (G) its annotated MS/MS spectrum for the [M + H]+ ion; and (H) MS1 of the unknown isomer and (I) its annotated MS/MS spectrum for the [M + H]+ ion. separation was performed on a Waters UPLC coupled to a G2-XS QTOF. Earthworm Sample Preparation and Metabolite Extraction. Earthworm (Eisenia fetida) samples were collected by removing worms from a laboratory culture, the soil was rinsed off, and worms were patted dry.1−3,13 Whole earthworms were immediately flashfrozen, lyophilized, and homogenized by bead beating prior to extraction. Coelomic fluid and coelomocyte samples were extruded by placing the worm in 0.1% NaCl solution, and a voltage was applied 10 times for
