Discovery of Coracanols A and B, Two Biological Nitrification Inhibition Diterpenoids from Finger Millet

Abstract

Natural products with biological nitrification inhibition (BNI) activity released from plant roots show potential for increasing the nitrogen-use efficiency and decreasing the extent of environmental pollution. We isolated two diterpenoids, coracanols A (1) and B (2), from finger millet grown under low-nitrogen conditions. Both compounds showed BNI activity against nitrifying bacterium Nitrosomonas europaea. The novel skeletons (1 and 2) featuring a tetracyclo­[10.2.2.0^1,10.0^2,7]­hexadecane core were elucidated by spectroscopic and X-ray crystal structure analyses utilizing the crystalline sponge method.

Low nitrogen-use efficiency (NUE) in agriculture is a serious problem that requires correction via genetic interventions and improvements in the agronomic production environment. This low efficiency occurs because crops use only about 30–50% of the nitrogen (N) contained in ammonia (NH₃/NH₄ ⁺)-based fertilizers. The remaining nitrogen is lost through microbial nitrification and denitrification processes (related to soil microbial activities) and can cause NO₃ ^– pollution of water bodies and N₂O emissions that cause global warming. Insufficient nitrification control poses serious risks to ecosystems and contributes to climate change. Therefore, an innovative strategy to optimize agricultural production practices is urgently needed.

Natural products with biological nitrification inhibition (BNI) activity released from root systems offer a sustainable strategy to improve NUE and reduce the environmental footprint of agricultural systems (Figure S1a). Recently, BNI-enabled wheat was developed and gave yields with low levels of nitrogen fertilization that were similar to those obtained with high levels of nitrogen input. Furthermore, this crop suppressed NO₃ ^– and N₂O production in the soil. In previous studies on maize, we identified several compounds with BNI activity against Nitrosomonas europaea (Figure S1b). ,

Wheat and maize are crucial staple crops for feeding the world’s population, which is projected to increase to 10 billion by 2050. However, the supply of these cereals is threatened by low resistance to environmental stresses and price fluctuation risks with world events. Millet is a promising nutritional crop that can adapt to low-input agriculture. − In recognition of this, 2023 was declared the International Year of Millets and policies were launched in India to promote millet production and consumption and emphasize nutritional, human health, climate resilience, and biodiversity benefits.

Finger millet [Eleusine coracana (L.) Gaertn.] is an annual crop in the Poaceae family grown with low-N inputs and is traditionally cultivated in eastern Africa and southern Asia, including India and Nepal. It forms a finger-like branching spike bearing small seeds that are used for food and beverage production and are rich in protein, vitamins, dietary fiber, and minerals, particularly calcium (Figure S2a,b). Finger millet is a climate-resilient crop, tolerating semiarid stresses and growing well in low-fertility soils with minimal chemical fertilizer input. Whole plant acclimation responses to low nitrogen stress have been documented in finger millet. Because of the resilience of finger millet to poor soils, we hypothesize that BNI functions to maintain more ammonia in the rhizosphere. Finger millet is an orphan (minor) crop, and information about its secondary metabolism remains limited, although some genomic and physiological data have been reported. ,

Herein, we describe the isolation and structural elucidation of two novel skeletal diterpenoids (Figure ), coracanols A (1) and B (2), from finger millet, that exhibit BNI activity.

1.

Structures of coracanols A (1) and B (2).

We cultivated finger millet for 40 days in potted soil with very low-N application (10 ppm of N as ammonium sulfate) (Figure S2). The experiment was replicated four times (Figure S2). Root exudates (10 mg from each of 300 plants) were collected by washing the roots with CH₂Cl₂ (1% AcOH). To measure the BNI activity, a recombinant strain of N. europaea (pHLUX20) expressing luciferase genes was used (Figure S3). On the basis of bioassay results, we fractionated root exudates by reversed-phase HPLC (Figure a and Figure S4) to isolate compound 1 (0.10 mg) and compound 2 (0.05 mg).

2.

HPLC profile of root exudates of finger millet and changes in the biosynthesis of 1 and 2 with growth stage in roots and leaves. (a) LC-MS chromatogram of root exudates. (b) Representative LC-MS chromatograms of CH₂Cl₂ extracts and photographs of finger millet at the corresponding growth stages. (c) Concentrations of 1 and 2 in CH₂Cl₂ extracts determined by HPLC. Error bars indicate the standard deviation (n = 2).

When finger millet was grown under fully N-fertilized conditions (100 ppm of N) for 40 days, compounds 1 and 2 were not detected in root exudates and in root tissue. Thus, all subsequent experiments and cultivation of finger millet were conducted under low-N conditions (10 ppm of N applied to the soil) with four replicates. Plants were grown in pots for up to 70 days and sampled from 10 days after sowing to 70 DAS. Fresh shoot and root tissues were extracted with CH₂Cl₂ and analyzed for the production of 1 and 2. Compounds 1 and 2 were detected in both leaf and root tissue extracts (Figure b,c and Table S1). The concentrations of BNI compounds (1 and 2) in plant tissues increased from 20 to 70 DAS. The plants reached the reproductive stage at 70 DAS.

Given these results, we cultivated finger millet (100 plants) without application of N fertilizer for 60 days and successfully isolated 1 (3.0 mg) and 2 (5.0 mg) from a CH₂Cl₂ extract (90 mg) (Supporting Information).

Compound 1 was isolated as a colorless oil. The molecular formula was elucidated to be C₂₁H₃₀O₃ based on HR-ESI-MS, which appeared at m/z 331.2268 [M + H]⁺ (calcd 331.2273 for C₂₁H₃₁O₃), indicating seven degrees of unsaturation. IR peaks at 3420, 1705, 1653, and 1623 cm^–1 showed the presence of a hydroxyl group, carbonyl group, and olefins. Analysis of the ¹H NMR, ¹³C NMR, DEPT, and HSQC spectra of 1 revealed 21 carbon resonances, comprising of two methyls, an oxygenated methyl (δ_H 3.689 and δ_C 51.3), eight sp³ methylenes, an exomethylene (δ_H 4.994 and 4.716 and δ_C 102.7), two sp³ methines, two quaternary carbons (δ_C 38.3 and 36.5), an oxygenated carbon (δ_C 72.1), three sp² carbons (δ_C 152.8, 145.7, and 124.5), and an ester carbon (δ_C 170.2) (Table ).

1. ¹³C NMR (201 MHz) and ¹H NMR (800 MHz) Data for 1 in CDCl₃ (δ in parts per million).

	δC, type	δH, mult. (J (Hz))
1a	28.8, CH2	1.461, ddd (12.5, 6.1, 1.6)
1b		1.360, m
2a	24.4, CH2	2.332, ddd (17.8, 6.1, 1.6)
2b		2.185, m
3	124.5, C
4	145.7, C
5	43.6, CH	2.241, brd (13.3)
6a	23.7, CH2	1.886, dq (13.5, 4.0)
6b		1.183, qd (13.2, 4.0)
7a	32.5, CH2	1.766, dq (13.5, 4.0)
7b		1.380, td (13.5, 4.0)
8	34.9, CH	2.074, m
9	38.3, C
10	36.5, C
11a	22.3, CH2	1.973, m
11b		1.578, overlap
12a	34.2, CH2	1.675, dd (11.5, 6.2)
12b		1.573, overlap
13	72.1, C
14a	42.9, CH2	1.840, qd (12.0, 3.5)
14b		1.217, dd (12.0, 5.4)
15	152.8, C
16a	36.4, CH2	2.434, dt (17.0, 2.1)
16b		2.285, dq (17.0, 2.4)
17a	102.7, CH2	4.994, td (2.4, 1.1)
17b		4.716, dd (2.1, 1.1)
18	170.2, C
19	18.6, CH3	1.863, brs
20	14.6, CH3	0.821, s
21	51.3, CH3	3.689, s

The tetracyclic (ABCD) skeleton of 1 was revealed by a detailed analysis of 1D and 2D NMR spectra. The partial structure of 1 (AB ring, C-1–C-10 and C-18–C-20) was afforded by ¹H–¹H COSY correlations of H₂-1/H₂-2, H-5/H₂-6, H₂-6/H₂-7, and H₂-7/H-8, together with HMBC correlations of H₃-19/C-3, C-4, C-5; H₃-20/C-1, C-5, C-9, C-10; H₂-1/C-3, C-5, C-9, C-10; H₂-2/C-3, C-4, C-10; H₂-6/C-4, C-8, C-10; H₂-7/C-5, C-9; and H-5/C-3 (Figure a). The HMBC correlations from H-2a to C-18 and from H₃-21 to C-18 indicated that a carboxylic acid methyl ester was attached to C-3, which was supported by reference data. Another moiety of 1 (CD ring) was yielded by ¹H–¹H COSY correlations of H-8/H₂-14 and H₂-11/H₂-12, along with HMBC correlations as follows: H₂-16/C-8, C-9, C-11, C-13, C-15; H₂-14/C-9, C-12, C-13, C-15; H₂-11/C-13, C-16; H₂-12/C-9, C-13, C-15; H₂-17/C-13, C-15, C-16; H-8/C-9, C-11, C-13; and OH (in pyridine-d ₅)/C-12, C-14, C-15 (Figure a, Figure S22a, and Table S3). Characteristic W coupling of H-8/H-11b, H-11a/H-16b, and H-12b/H-14a in the COSY experiment strongly suggested 1 contained a rigid bicyclo[2.2.2]­octane. HMBC correlations of H₂-7/C-14 and H₂-16/C-10 suggested that both C-8 and C-9 were located at the position connecting the AB ring to the CD ring. Thus, the planar structure of 1 possessing a tetracyclo­[10.2.2.0^1,10.0^2,7]­hexadecane core was determined.

3.

Identification and structural elucidation of compound 1. (a) Key COSY, TOCSY, and HMBC correlations and (b) key NOESY correlations of 1. (c) Thermal ellipsoid plot of encapsulated 1 inside APF-1 (drawn at the 50% probability level). (d) Structures, 2F _o – F _c electron density (1.5 e^– Å^–3 level), and hydrogen bond interactions of encapsulated 1 inside APF-1. Atom coloring scheme: purple for Co, gray for C, blue for N, red for O, and white for H. The green mesh shows the 2F _o – F _c electron density map.

To determine the relative configuration of 1, NOESY was measured (Figure b). NOESY correlations of H₃-20/H-6b, H-8, H-16b; H-8/H-14a; H-11b/H-16a; H-12a/H-14b; and H-11a/H-12a indicated 9S* and 13S* positions. The 5S*, 8S*, and 10S* positions of 1 were suggested by NOESY correlations of H₃-20/H-1a, 2b; H-1a/H-16a; and H-5/H-7b, H-11a. Consequently, the relative configuration of the entire molecule was (5S*,8S*,9S*,10S*,13S*)-1.

The absolute structure of (5S,8S,9S,10S,13S)-1 was independently elucidated by the crystalline sponge method (Figure c,d). Structural analysis of 1 was enabled by a reported metal–organic framework, denoted as APF-1, that in recent atomic-resolution crystal structures was shown to encapsulate various guests. , An acetone solution containing 20 μg of 1 was dispensed into a glass vial and dried under air. The residue was redissolved in a mixture of n-hexane and acetone. Crystals of APF-1 were then added to the vial, and soaking with the guest was performed at 40 °C for 2 days. The 1-soaked APF-1 crystals were picked up from the vial and sent to a synchrotron facility for single-crystal X-ray diffraction analysis.

Compound 2 was isolated as a white powder. Its molecular formula was determined to be C₂₀H₃₀O₂ by HR-ESI-MS corresponding to six degrees of unsaturation. An IR absorption band at 1685 cm^–1 and a weak UV band at 279 nm (n−π* transition) revealed the existence of a ketone. The 1D NMR data of 2 were similar to those of 1, except for the A ring moiety (Tables S3 and S4). The ¹H–¹H COSY correlation of H₂-1/H₂-2 and HMBC correlations of H₂-1/C-3, C-5, C-9, C-10, C-20; H₂-2/C-3, C-10; H-5/C-4, C-7, C-10, C-18, C-19, C-20; H₃-18/C-3, C-4, C-5, C-19; and H₃-19/C-3, C-4, C-5, and C-18 suggested that 2,2-dimethylcyclohexanone was present in ring A of 2 (Figure a). The remaining partial structure of 2 (BCD ring) was afforded in detail by 2D NMR experiments (Figure a and Table S4). Based on these data, the planar structure of 2 was established.

4.

Identification and structural elucidation of compound 2. (a) Key COSY and HMBC correlations of compound 2. (b) Key NOESY correlations of 2. (c) Calculated and observed ECD/UV spectra of 2.

NOESY experiments with 2 indicated that H-1a, H-2b, H-5, H-6a, H-7b, H-14b, and H₃-19 were α-oriented while H-1b, H-2a, H-6b, H-8, H-14a, H₃-18, and H₃-20 were β-oriented (Figure b and Table S3). NOESY correlations of H-5/H-7b; H-7b/H-11a, H-14b; H₂-16/H-1b, H-11b, H₃-20; H-12a/H-14b; and H-11a/H-12a indicated 9S* and 13S* positions. Based on these data, the relative configuration was elucidated as (5R*,8S*,9S*,10S*,13S*)-2.

The ECD spectrum of 2 was in accordance with the DFT-predicted spectrum for 5R,8S,9S,10S,13S (Figure c). Therefore, the absolute configuration of 2 was determined to be 5R,8S,9S,10S,13S.

Compounds 1 and 2 showed inhibitory activity against the bioluminescence of N. europaea (pHLUX20) with ED₅₀ values of 12 and 21 μM, respectively (Figure S6). We then examined each compound for suppression of the nitrification activity of N. europaea. Compound 1 (100 μM) inhibited the production of NO₂ ^– from NH₄ ⁺ with an inhibition rate of >99% 72 h after inoculation (Figure S7a). This inhibition suppressed the growth of N. europaea for 7 days (Figure S7b). The inhibitory activity of compound 1 was approximately 3 times that of compound 2.

A possible biosynthetic pathway for 1 and 2 is shown in Scheme . Initially, geranylgeranyl diphosphate is cyclized to syn-copalyl diphosphate (class II diterpene synthase). Subsequently, syn-copalyl diphosphate is further cyclized to 9β-pimara-15-en-8-yl⁺ (class I diterpene synthase), , followed by a series of rearrangement steps to give the coracane skeleton. In the first step, a 1,2-hydride shift occurs from C-9 to C-8. The second step involves intramolecular alkene attack of the Δ¹⁵⁽¹⁶⁾ olefin on C-9 to form bicyclo[2.2.2]­octane, which is accompanied by the Wagner–Meerwein rearrangement of C-17. In the third step, a 1,2-hydride shift occurs from C-15 to C-13. The fourth step is deprotonation at C-17. Precoracanol, a C-13-hydroxylated derivative of coracane subsequently undergoes an 18(4→3) methyl shift, carboxylation (cytochrome P450 monooxygenases), and methylation (carboxyl O-methyltransferase) to give 1. In parallel, oxidation at C-3 of precoracanol produces 2.

1. Proposed Biosynthetic Pathway of the Coracanols.

In summary, we identified two diterpenoids (1 and 2) with a BNI function produced in finger millet. These compounds are biosynthesized exclusively under low-N conditions in both roots and shoots. The production of compounds 1 and 2 in both root and leaf tissues raises the possibility that these metabolites have functions beyond BNI activity, including antiherbivore and antimicrobial defense against plant pathogens. In the rhizosphere, they are secreted from the roots as exudates and contribute to the BNI function of finger millet.

The data underlying this study are available in the published article and its Supporting Information.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.orglett.6c00300.

• Detailed experimental procedures and full spectroscopic data sets (figures, tables, and HRESIMS, IR, UV, ECD, NMR, and computational data) for compounds 1 and 2 (PDF)

The authors declare no competing financial interest.