Albatredines A and B, a pair of epimers with unusual natural heterocyclic skeletons from edible mushroom Albatrellus confluens

A chemical study of the common species Albatrellus confluens present in Yunnan province, southwest China led to the identification of a pair of epimers named albatredines A (1) and B (2). They feature a natural unprecedented 1,2,4-oxadiazolidin-5-one skeleton. The acyl substitution pattern and complete configurational assignments were deduced from the comparison between experimental and theoretical 13C NMR and ECD data, respectively. Bioassay results showed that compound 1 exhibited a weak immunosuppressive activity against the concanavalin A-induced T lymphocyte cell proliferation (IC50 2.99 μM).


Introduction
Nitrogen-containing heterocyclic scaffolds are basic and prevalent structural units of many drugs, such as Sovaldi, Abilify, and Nexium. 1 Nitrogen-containing compounds originating from mushrooms are a large group of diverse secondary metabolites 2,3 (characterized by various chiral structures with different bioactivities), such as enantiomers and epimers. These metabolites are not equally effective in blocking the effect of some receptors and may have a large difference in their ability to cure some diseases, the reason for which is still not known. For instance, in the formation of the peptide for the origin of life, L-amino acids were chosen instead of D-amino acids and D-sugars were selected instead of L-sugars, which was an amazing natural choice. 4 However, non-crystallized natural products with multiple chiral centers, particularly the lower molecular weight compounds with more heteroatoms, dwarf the determination of their planar structures and absolute congurations to a large extent.
The edible mushroom Albatrellus conuens, which belongs to the family Albatrellaceae, is widely distributed in China. Although the phytochemical investigations on A. conuens have been extensively reported, [5][6][7][8][9][10][11][12] an interesting discovery has been made in the lower-polarity fraction of the extractions from the fruiting bodies of A. conuens collected in southwest China, Yunnan, which led to the isolation and structural elucidation of a pair of undescribed nitrogen-containing heterocyclic compounds albatredines A (1) and B (2) (Fig. 1). Structurally, albatredines A and B are quite unprecedented in nature, possessing ve-membered 1,2,4-oxadiazolidin-5-one heterocyclic skeletons. Although the efforts to cultivate single crystals of these compounds have failed, the absolute congurations of these compounds were unambiguously determined by ECD calculations as well as 13 C NMR calculations. 13 Bioassay results indicated that compound 1 exhibited inhibitory activity against concanavalin A-induced T lymphocyte cell proliferation, an important target in the treatment of immunosuppression.
Electronic circular dichroism (ECD) calculations were used to determine the absolute conguration of 1 by the time dependent density functional theory (TDDFT) method at the CAM-B3LYP/6-311+G (d,p) level in gas phase. Two stereoisomers, (4R,5S,2 0 S)-1 (1a) and (4S,5R,2 0 R)-1 (1f), exist on the basis of the relative conguration. Comparison of theoretically calculated and experimental ECD curves (Fig. 5) showed that the calculated curves of 1a were similar to the experimental data, indicating the absolute conguration of 1 as 4R,5S,2 0 S.
Compound 2 was isolated as a white powder. Its molecular formula was determined as C 11 H 21 N 2 O 3 by HRESIMS, which was the same as that of 1. The NMR data (Tables 1 and 2), UV and IR spectra of 2 and 1 had remarkable resemblances. In addition, the experimental ECD spectra of 1 and 2 also possessed mirror symmetry. All the abovementioned results      (Table S13 †) were also used to tackle the absolute congurations, which were conrmed as 4S,5S,2 0 S.
Albatredines A and B represent nor-peptides with unprecedented natural heterocyclic skeletons, which aroused our interest in their plausible biogenesis. Biosynthetically, this pair of epimers might possibly be traced back to L-isoleucine since they possessed this common side chain. The probable biogenetic pathways for 1 and 2 are proposed (Scheme 1). L-isoleucine was oxygenated at the amino site to give the key (S,E)-2-(hydroxyimino)-3methylpentanoic acid (C), which could induce a rearrangement reaction under acidic condition to afford D. Furthermore, the generation of D was also followed by an oxidation at the amino site to generate E, which proceeded with D to form the intermediate F involving the elimination of water. Correspondingly, a decarboxylative reaction of F took place to produce G, which could trigger an intramolecular SN 2 nucleophilic addition in basic condition to yield the intermediate H. Immunosuppressants are an important class of clinical drugs and an essential prerequisite for successful organ transplantation and treatment of various autoimmune diseases, such as psoriasis, pemphigus, myasthenia gravis, multiple sclerosis, systemic lupus and rheumatoid arthritis. 18 Thus, we have evaluated the immunosuppressive activity of 1 and 2 by a previously reported method. 19 In the experiment, cyclosporin A (CsA) was used as the positive control. Compound 1 exhibited weak activity against concanavalin A-induced T lymphocyte cell proliferation with an IC 50 value of 2.99 mM (Table 3). However, compound 2 did not show any activity, which could explain that there is a difference between the epimers to some extent.

Conclusions
In summary, chemical investigations on the famous edible mushroom A. conuens were conducted, which resulted in the isolation of a pair of epimers, albatredines A (1) and B (2), bearing a previously undescribed natural heterocyclic scaffold. Their structures were unambiguously determined through extensive spectroscopic analysis in combination with computational approaches. Moreover, immunosuppressive bioassay results suggested that the discovery of new types of heterocyclic compounds such as these from natural sources provides new possibilities to explore new immunosuppressants by the inhibition of T cell proliferation. Scheme 1 Proposed biosynthetic pathways for 1 and 2.  a SI was determined as the ratio of the concentration of the compound that reduced the cell viability to 50% (CC 50 ) to the concentration of the compound needed to inhibit the proliferation by 50% relative to the control value (IC 50 ). "-" stands for inactive.

Immunosuppressive activities assay
Live subject statement. The animal experiment was carried out in strict accordance with the institutional ethical guidelines on animal care and was approved by the Institute Animal Care and Use Committee (IACUC) at the Shanghai Institute of Materia Medica, Chinese Academy of Sciences (IACUC protocol# 2017-03-ZJP-62).
Preparation of spleen cells from mice. Female BALB/c mice were sacriced by cervical dislocation, and the spleens were removed aseptically. Mononuclear cell suspensions were prepared aer the cell debris and clumps were removed. Erythrocytes were depleted with an ammonium chloride buffer solution. Lymphocytes were washed and resuspended in the RPMI 1640 medium supplemented with 10% FBS, penicillin (100 U mL À1 ), and streptomycin (100 mg mL À1 ).
Cytotoxicity assay. Cytotoxicity was assessed with a Cell Counting Kit-8 (CCK-8) assay. Briey, fresh spleen cells were obtained from female BALB/c mice (Bagg albino) (18-20 g). Spleen cells (1 Â 10 6 cells) were cultured at 37 C for 48 h in 96-well at plates in the presence or absence of various concentrations of compounds in a humidied and 5% CO 2 -containing incubator. A certain amount of CCK-8 was added to each well at the nal 8-10 h of culture. Towards the end of the culture, we measured the OD values with a microplate reader (Bio Rad 650) at 450 nm. The cytotoxicity of each compound was expressed as the concentration of the compound that reduced the cell viability to 50% (CC50).
T cell and B cell function assay. Fresh spleen cells were obtained from female BALB/c mice (18-20 g). The 5 Â 10 5 spleen cells were cultured at the same conditions as those mentioned above. The cultures, in the presence or the absence of various concentrations of compounds, were stimulated with 5 mg mL À1 of concanavalin A (ConA) or 10 mg mL À1 of LPS (lipopolysaccharide) to induce T cells' or B cells' proliferative responses, respectively. Proliferation was assessed in terms of uptake of [ 3 H]-thymidine during 8 h of pulsing with 25 mL per well of [ 3 H]thymidine and then, the cells were harvested onto glass ber lters. The incorporated radioactivity was counted using a Beta scintillation counter (MicroBeta Trilux, PerkinElmer Life Sciences). The immunosuppressive activity of each compound was expressed as the concentration of the compound that inhibited the ConA-induced T cell proliferation or LPS-induced B cell proliferation to 50% (IC 50 ) of the control value.
Computational methods NMR calculation. The NMR calculations for compounds were performed using Gaussian 09. Conformation searches based on molecular mechanics with the MMFF94s force eld were performed for (4R,5S,2 0 S)-1 (1a), (4R,5R,2 0 S)-1 (1b), (4R,5R,2 0 R)-1 (1c) and (4R,5S,2 0 R)-1 (1d), which had 15, 15, 16 and 15 conformers with populations higher than 1%, respectively. 20,21 All these conformers were further optimized by the density functional theory method at the B3LYP/6-31G(d) level in Gaussian 09 program package, 22 which led to eleven (1a1 to 1a11), twelve (1b1 to 1b12), eleven (1c1 to 1c11) and ten (1d1 to 1d10) conformers within a 2.0 kcal mol À1 energy threshold from global minimum, respectively. These predominant conformers were subjected to NMR calculation. Gauge-Independent Atomic Orbital (GIAO) calculations of 13 C NMR of the conformers were accomplished by the density functional theory (DFT) at the mPW1PW91/6-311g(d,p) level in methanol with the PCM model. The 13 C NMR chemical shi of TMS was calculated in the same level and used as the reference. The calculated NMR data of these conformers were averaged according to the Boltzmann distribution theory and their relative Gibbs free energies. 13 The DP4+ calculations [15][16][17] were carried out using the Excel spreadsheet available for free at sarotti-NMR.weebly.com.

Conflicts of interest
There are no conicts to declare.