Two ambuic acid dimers derived from homo-endo Diels-Alder reaction from Pestalotiopsis versicolor

Abstract Two new dimers of ambuic acid, pestaloversicoloric acids A (1) and B (2), and a known derivative, 13(S)-hydroxyambuic acid (3), were isolated from the static fermentation product of Pestalotiopsis versicolor. The structural identification was accomplished via analyses on the data of HR-MS, 1 D and 2 D NMR, and ECD. Different from the well-known exo-type dimer, torreyanic acid, compounds 1 and 2 represent the first example of endo-type product derived from the intermolecular Diels-Alder reaction of two molecules of ambuic acid derivative with the identical absolute stereochemistry.  


Introduction
Isoprenylated epoxyquinols are the representative polyketides generated by the fungi of the genus Pestalotiopsis, exemplified by the pentaketide jesterone [1], and the heptaketides ambuic acid [2] and cytosporin D [3]. These polyketide monomers undergo oxidation/electrocyclization to afford two considerably reactive epimeric 2H-pyran intermediates which are readily subjected to intermolecular [4 þ 2] Diels-Alder dimerization reaction. This reaction usually occurs in three patterns: (1) the dienophile (the alkyl at the epimeric carbon is anti to the epoxy group) approaches the epimeric diene (the alkyl is cis to the epoxy group) from the direction opposite to the epoxy group, to yield the hetero-exo-type dimer, (2) the homo-dimerization between two identical 2H-pyran monomers (the alkyl at the epimeric carbon is cis to the epoxy group) results in the homo-endo-type product, and (3) the recently reported homoexo-type product, eutyscoparol J [4], derived from the homo-dimerization of two cisalkyl/epoxy monomers. The collective stereochemical selectivity for these three patterns is that the dienophile approaches the cis-alkyl/epoxy diene anti to the epoxy group, and the alkyls at the epimeric carbon are usually anti to each other except for eutyscoparol J [4]. The hetero-exo, homo-endo, and homo-exo dimers of the heptaketide, cytosporin D, have been reported [3,4], while, only the hetero-exo-type dimers of ambuic acid, torreyanic acid derivatives, were found in the separation [5,6] and synthetic [7] investigations. During our efforts for the polyketide metabolites of Ascotricha [8,9] and Pestalotiopsis genera [10], two homo-endo dimers of ambuic acid, pestaloversicoloric acids A (1) and B (2), were firstly isolated from the static fermentation product of Pestalotiopsis versicolor, together with a known biogeneticallyrelated derivative, 13(S)-hydroxyambuic acid (3) (Figure 1). The structural elucidation of compounds 1 and 2, especially the stereoselectivity of the Diels-Alder reaction and the conformational analysis, is described in this work.
The conformation (Figure 4) of this 1S,8S,7 0 S,8 0 S-isomer was in great accordance with the NOESY data of 1, and the allylic coupling of H-1 0 /H-3 0 could be reasonably associated with their dihedral angle (90 ). While, it was noteworthy that the conformation of the C-9 0 -involved 2H-pyran was different from that of the homo-endo-type dimer, epoxyquinol B (CCDC No. 188062) [14], for that the NOESY correlation of H-9/H-9 0 of 1 was observed, instead of that of H-8/H-10 0 in epoxyquinol B. Additionally, the dihedral angle of H-8 0 /H-9 0 of epoxyquinol B at about 180 resulted in the coupling constant of 6.6 Hz [15], while H-8 0 of pestaloversicoloric acid A (1) was observed as a singlet. This difference could be attributed to the steric hindrance between the two pentyls at C-9 and C-9 0 of 1 which was more crowded than that between the two methyls of epoxyquinol B.
The peak at m/z 693.2928 [M À H] À in the HRESIMS experiment assigned compound 2 a molecular formula of C 38 H 46 O 12 with one more hydrogen deficiency than 1. The 1 H and 13 C NMR data of 2 greatly resembled those of 1, expect for the replacement of the characteristic aliphatic proton and carbon signals of the epoxyquinol moiety of the dienophile by an enol proton at d H 11.12 (br. s), two olefinic carbons at d C 158.0 and 119.7, and a carbonyl at d C 180.1, as well as the significant down-field shift of H-1 0 (from d H 6.17 to 7.34) and C-1 0 (from d C 140.4 to 152.8). The aforementioned variation, together with the HMBC correlation ( Figure 3) between H-1 0 and the carbonyl at d C 180.1, and those between the methylene protons of the 3-carboxy-3-methyl-allyl and the two carbons at d C 158.0 and 119.7, revealed the existence of a carbonyl at C-3 0 and an enol between C-4 0 /C-5 0 , similar to the oxidative derivative of ambuic acid, pestallic acid A [16]. The absolute configuration of 3 R/4R/5R was established by J H-3/4 at 2.8 Hz, the NOESY ( Figure 5) experiment, and the biogenetic relevance to compounds 1 and 3. Furthermore, the key NOESY correlations of H-8/H-9 0 , H-9/H-8 0 , H-9/H-9 0 , and H-1 0 /H-3 clarified the stereo-selectivity of the Diels-Alder reaction and the consistency of the absolute configurations of C-1/ C-8/C-9/C-7 0 /C-8 0 /C-9 0 between 1 and pestaloversicoloric acid B (2).
Despite the co-isolation of the exo-and endo-type dimers of epoxyquinol from some fungi, e.g. pestaloquinols A and B [3] and epoxyquinols A and B [13,15], the heptaketide, ambuic acid, tends to be the substrate of a Diels-Alder reaction catalyticase with relatively higher stereo-selectivity. Torreyanic acid arises from the   hetero-exo-dimerization, while, pestaloversicoloric acids A and B (1 and 2) represent the first example of the homo-endo-dimerization product derived from ambuic acid. Although the exo-type dimer was reported to be the major product of both the biological [3,13,15] and chemical [14,17] workshops, it is still necessary for the synthetic chemists to re-examine the minor by-products accompanying with torreyanic acid analogues to check the existence of homo-or endo-type dimers. Additionally, the isolation of the fourth type, hetero-endo stereoisomer of prenylated epoxyquinol challenging the ability of the enzyme to overcome the steric hindrance between alkyls, from the nature, intrigues our curiosity and is greatly anticipated.

Strain and cultivation
The Erlenmeyer flasks (500 ml) of rice media prepared from rice (80 g) and distilled water of 120 ml by autoclaving. Static cultivation for another 60 days was carried out at room temperature.