Absolute stereochemistry of fungal metabolites: icterinoidins A1 and B1, and atrovirins B1 and B2 #

The absolute stereochemistry at the C 3 (and C 3', where appropriate) chiral centre(s) in the coupled dihydroanthracenones, the icterinoidins A 1 and B 1 and atrovirin B 2 (from Dermocybe icterinoides ), is deduced by application of the ' syn-anti rule', which relies on an empirical relationship between the sign of the Cotton effect couplet centred close to 275 nm in the CD spectrum and the chemical shift of the enantiotopic methylene protons at C 4 in the 1 H NMR spectrum of these pre-anthraquinones. The conclusions also allow assignment of central stereochemistry to atrovirin B 1 (from Cortinarius atrovirens ). In addition, we have applied Steglich's kinetic resolution method to confirm the ( P )-axial configuration of icterinoidin B 1 , previously deduced by using Nakanishi's 'exiton chirality' method.


Introduction
In an earlier paper in this series 1 we described, inter alia, the isolation and structural elucidation of two new atropisomeric 5,5'-coupled dihydroanthracenone-anthraquinones, the icterinoidins A 1 (1) and B 1 (2), atrovirin B 2 (3), a diastereoisomer of the known atrovirin B 1 (4) 2 (no central stereochemistry yet implied), and the well known orange pigment (P)-(+)-skyrin (5) 3 from the ethanolic extracts of the pale-green capped toadstool Dermocybe icterinoides, first described by Horak 4 and examined chromatographically by Keller et al., 5 which was gathered by us in native forest on the South Island of New Zealand.The axial stereochemistry of the natural products 1, 2 and 3 was evident by inspection of the sign of the long and short wavelength maxima and minima of the intense Cotton effect doublet close to 275 nm resulting from 'exciton coupling' between the two extended naphthalene chromophores in molecules of this type.2b,6-9 However, at that time, the stereochemistry at the C 3 and C 3' chiral centres in the icterinoidins A 1 (1)  and B 1 (2) and in atrovirin B 2 (3) [and in the known atrovirin B 1 (4)] 2 was not known.Nevertheless, the CD spectra of the icterinoidins 1 and 2 reveal that they are atropisomers, and consequently, that both compounds must have the same chirality at the C 3 and C 3' stereogenic centres.Similarly, while it was plain that the atrovirins B 2 (3) (from D. icterinoides) 1 and B 1 (4) (from Cortinarius atrovirens) 2, 9 have near super imposable B-type CD curves, 1 distinct differences in the respective 1 H NMR spectra established that these pigments too must be diastereoisomers.We describe herein the application of the 'syn-anti rule', 2b,9 which exploits the empirical relationship between the respective CD and 1 H NMR spectra of individual pre-anthraquinones such as 1, 2, 3, and 4, which allows the determination of the absolute central stereochemistry in all four of these complex natural products.

OH
The axial stereochemistry of icterinoidin B 2 (2), previously defined by the 'exciton chirality' method, 1 is confirmed by application of Steglich's kinetic resolution method. 9

Results and Discussion
Details of the isolation and purification of the icterinoidins A 1 (1), B 1 (2) and atrovirin B 2 (3) from Dermocybe icterinoides are described in detail elsewhere 1 and need not be repeated here.
The axial chirality of dimeric pre-anthraquinones of the type discussed here is conveniently determined by inspection of the CD spectrum in which the sign of an intense (∆ε ≈100) Cotton effect couplet centred near 275 nm can be directly correlated with the helical twist between the asymmetric chromophores. 6-9Thus, a compound exhibiting a negative Cotton effect at longer wavelength and a positive one at shorter wavelength (an 'A-type' curve according to Steglich) 10 is consonant with 'negative chirality' (an anticlockwise twist between the aromatic chromophores), 6 while a compound showing the mirror image Cotton effect couplet (a 'B-type' curve) 10 corresponds to 'positive chirality' (a clockwise aromatic helical twist). 6In the case of the icterinoidins and atrovirins this leads, according to the Prelog-Helmchen rules, 11 to the (P)-axial stereochemistry for icterinoidin B 1 (2) and atrovirin B 2 (3) and the (M)-axial chirality for icterinoidin A 1 (1). 1  A far more demanding task in coupled pre-anthraquinones of this type is the determination of the absolute configuration at the chiral centres.Although chemical methods have been developed in certain cases, 9,12 (vide infra), an empirical relationship between the axial configuration, evident from the CD spectrum, and the chemical shift difference (∆δ) in the 1 H NMR signals of the diastereotopic C 4 methylene protons can be reliably translated into the absolute stereochemistry at the adjacent C 3 chiral centre(s).][15] This relationship, which was pioneered by Oertel and Steglich, 2b notes the difference in the magnitude of anisotropic influence of one half of the pre-anthraquinone dimer on the C 4 protons of the other.Thus, the pseudo-axial and pseuedo-equatorial protons at C 4 in the monomer system, torosachrysone (6) and its derivatives, resonate in the 1 H NMR spectrum, near coincidentally, between δ 3.02 and 3.10. 10,16This is also the case in 7,7'-linked dimers belonging to the flavommanin group of torosachrysone dimers, 10 in which the biaryl linkage is remote and therefore the C 4 protons are relatively unperturbed by any anisotropic influence from the second aromatic ring.However, in the spectra of 5,5'-(atrovirin), 2 5,10'-(pseudophlegmacin), 17 7,10'-(phlegmacin) 18 and 10,10'-(tricolorin) 19 dimers, in which the C 4 methylene protons are in the zone of influence of the adjacent biaryl ring system, differential shielding can be translated in streochemical terms. 20The method is particularly effective in those cases where more than one diastereoisomer of a biaryl system is known as a natural product. 20 plausible rationale for these spectroscopic observations is illustrated here by using the simplified model systems that are shown in Figures 1 and 2. Thus, in a dimer with the (3S,M)-[or the (3R,P)]-stereochemistry [Figure 1, (a) and (b)], the C 4 methylene protons are shielded, more or less equally so, by the appended C 5 naphthalene ring system, and signals from both protons are near-coincident or show only a small ∆δ that is typically ≤0.08 ppm.Since the hydroxyl group at C 3 in the dihydroanthracenone ring occupies an axial configuration, it follows that the relative stereochemistry between C 3 and the biaryl axis in this case must be (3S*,M*).This relative disposition of the naphthalene rings and the C 3 hydroxyl was termed 'anti' by Oertel.Turning now to the natural products 1-4, the 1 H NMR spectroscopic data for the C 4 methylene protons of the icterinoidins A 1 (1) and B 1 (2), and the atrovirins B 2 (3) 1 and B 1 (4) 2 are collected in Table 1.The spectrum of icterinoidin A 1 (1) contains an AB quartet with components centred at δ 2.75 and 2.90 (∆δ = 0.15 ppm).This relatively large shift difference categorizes 1 as belonging in the syn model (Figure 2) and, since the pigment exhibits an A-type CD Cotton effect curve, it follows that the absolute stereochemistry of icterinoidin A 1 ( 1) is (3R,M).In contrast, the C 4 methylene protons in the 1 H NMR spectrum of icterinoidin B 1 (2) resonate closer together at δ 2.90 and 2.85 (∆δ = 0.05 ppm) corresponding to the anti model (Figure 1) and therefore 2 has the (3R,P)-absolute configuration.

OH
The signals from H ax 4,4' and H eq 4,4' in the 1 H NMR spectrum of atrovirin B 2 (3) appear together as a broad, two-proton singlet at δ 2.87.This is in accord with the anti model (Figure 1) and, when coupled with a B-type CD curve, leads to the (3R,P,3'R) absolute configuration for 3.It is likely that atrovirin B 2 is a biogenetic precursor of icterinoidin B 1 in Dermocybe icterinoides (see Scheme 3).
Finally, the

Taxonomic notes and possible biogenetic relationships
Dermocybe icterinoides has been placed taxonomically close to another green capped Australasian species, D. austroveneta by Keller. 26Some time ago, we studied the chemistry of D. austroveneta, 27,28 Fruit bodies of D. austroveneta, when attacked by predators or upon decay turn to a rich, red-purple colour.From the fresh fruit bodies of D. austroveneta extracted under 'normal conditions' we isolated (P)-(+)-skyrin ( 5) and the purple pigment hypericin (Scheme 3). 27hen the fungus was frozen in liquid N 2 in the field and subsequentially extracted under N 2 in the absence of air and light we were able to isolate the purple protohypericin and the labile green pigment austrovenetin (Scheme 3). 28The absolute stereochemistry at C presence of the atrovirins (3) and/or (4), skyrin (5), and probably hypericin. 26Our results 29 provide further support for the suggestion that section Pauperae of subgenus Icterinula should be grouped with subsection Atrovirentes, section Scauri Fr. of subgenus Icterinoides. 5wavelength UV light.Commercial deuteriochloroform (Cambridge Isotope Laboratories) was washed with water, dried (K 2 CO 3 ), distilled, and stored in the dark.All other solvents and reagents were purified before use by published procedures.

HO
Equipment 1 H-and 13 C-nmr spectra were recorded on a JEOL JNM GX-400 spectrometer operating at 399.65 MHz ( 1 H) and 100.4MHz ( 13 C) using Varian Unity Plus Version 5.1 software.Chemical shifts (δ) are quoted in ppm from tetramethylsilane as internal standard and using deuteriochloroform as the solvent unless stated otherwise.UV-vis.spectra were recorded on a Varian SuperScan 3 spectrophotometer using ethanol as the solvent; log ε is quoted in parentheses after each absorption maximum.Electron impact (EI) mass spectra were recorded with either VG Micromass 7070F or JEOL JMS-AX505HF instruments operating at 70 eV unless stated otherwise.The results of accurate mass measurements are presented as a molecular formula in parentheses.Specific rotations were measured on a Perkin-Elmer 241 MC polarimeter at the room temperature; the solvent used is quoted in parenthesise with concentration (c) measured in g/100 mL.Melting points were determined on a Köfler micro hot stage apparatus and are uncorrected.Combustion analysis was performed by Chemical and Micro Analytical Services Pty Ltd, North Essendon, Victoria.

9 M
3 and C 3' in atrovirin pigment B 2 (2) and in the other coupled pigments from Dermocybe icterinoides and D. austroveneta that are shown in Scheme 3 is (R) and is (P) at the axis.This points to a close biogenetic relationship between these members of, what we here dub, 'the atrovirin B 2 cascade'.The pigments of D. icterinoides and D. austroveneta fall logically into the biogenetic pattern shown in Scheme 3. Chemically, they have affinities, either actual or artefactual, 28 not only with other members of the section Pauperae of the subgenus Icterinula but also to subsection Atrovirantes of section Scauri Fr. of subgenus Phlegmacium, which is characterized by the Compound Specific rotation [α] D of residual dinitrodiphenic acid [α] D of residual dinitro-diphenic acid according to Steglich
icterinoides All reactions were carried out under atmosphere of dry N 2 .PTLC was performed on 20 x 20 cm glass plates coated with 1.0 mm of silica gel (Merck Kieselgel GF 254 applied as a suspension in water) Plates were activated at 110 °C for 1.5 h prior to use.TLC was performed on Macherey-Nagel precoated aluminium plates (0.25 mm, Macherey-Nagel SIL G-25 UV 254 ) and visualised both in daylight and under short (254 nm) and long (360 nm) OHScheme 3. Possible biosynthetic relationships between members of the 'atrovirin B 2 cascade'.