Liposomal Circular Dichroism. Assignment of Remote Stereocenters in Plakinic Acids K and L from a Plakortis -Xestospongia Sponge Association

Abstract

Two new ω-phenyl polyketide peroxides, plakinic acids K and L, were isolated from a two-sponge association of Plakortis halichondroides and Xestospongia deweerdtae. The absolute configurations of the remote dimethyl-branched stereocenters in plakinic acid K were assigned by degradation of plakinic acid K to a long-chain naphthamide and analysis by liposomal circular dichroism (L-CD) and comparison with synthetic standards.

The marine sponges of the genera Plakortis and Plakinastrella¹ are prolific producers of cyclic polyketide peroxides that exhibit a broad spectrum of biological properties, including antifungal-¹, antimalarial-², antiprotozoal-³ and immunosuppressant⁴ activities. Sponge-derived cyclic peroxides may occur as 1,2-dioxane or 1,2-dioxolane ring systems.⁵ We recently reported the stereochemical assignments of two ω-phenyl polyketide peroxides, plakinic acids I (1) and J (2) from the symbiotic two-sponge association, Plakortis halichondroides–Xestospongia deweerdtae Lehnert & van Soest, 1999,⁶ which showed differential inhibition against haplodefficient lag1Δ/LAG1 strains of Saccharomyces cerevisiae.⁷ The remote methyl-branched C8 stereocenters of 1 and 2 were solved by the application of liposomal circular dichroism (L-CD)⁸ – a very sensitive technique (limit of detection ~16 nmol) that amplifies CD Cotton effects (CEs) through ordering of the long-chain lipids in highly-uniform, unilamellar liposomes.⁷. Here, we show a remarkable long-range perturbation of naphthamide chromophores by a single methyl branch, over eight bonds away, that discriminates between spectroscopically indistinguishable diastereomers. L-CD was used to assign configurations of both proximal C8 and distal C12 methyl-branched centers in two new ω-phenyl polyketide peroxides, plakinic acids K (3) and L (4).

P. halichondroides–X. deweerdtae, collected from the Bahamas, was extracted with MeOH:CH₂Cl₂ (1:1) to yield a dark-brown residue which was sequentially extracted with n-hexane, chloroform, n-butanol and water. Antifungal bioassay-guided fractionation revealed that the CHCl₃-soluble extract exhibited potent activity against S. cerevisiae lag1Δ/LAG1 strain. This fraction was subjected to flash chromatography (silica, 0–100% MeOH-CHCl₃, stepped gradient) and the active fraction further purified by HPLC (RP-C₁₈ CH₃CN/H₂O) to yield known compounds 1, 2,⁷ new methyl-branched plakinic acids K (3), L (4) and plakinic acid M, a linear homolog of 1.⁹

Plakinic acid K (3) was isolated as an optically active colorless oil (4.74 × 10⁻³ % yield, w/w, wet weight), [α]_D −137 (c 1.31, CHCl₃) of molecular formula C₂₆H₄₄O₄ as determined by HREIMS [M⁺] m/z 432.3239, with 14 mass units greater than 1. The UV, IR, ¹H and ¹³C NMR spectra of 3 were almost identical to those of 1, except for an additional methyl group (δ_H 0.82, d, J = 6.4 Hz; δ_C 19.9, CH₃). Inspection of the COSY and HMBC spectra established the position of the second methyl branch at C12 (see Supporting Information). Using a similar analysis, plakinic acid L (4, 7.5 × 10⁻³ % yield w/w wet weight, [α]_D −26.2 (c 1.90, CHCl₃) C₂₅H₄₀O₄; HREIMS [M⁺] m/z 404.2918) was shown to be a homolog of 2.

The common origin of 1–4, similar specific rotations and ¹H NMR suggest that the respective stereocenters at C3, C4 and C6 have the same configurations as those of 1 and 2.¹⁰ The configurations of the proximal C8/C7 stereocenters in 1 and 2 were assigned as R by interpretation of the intense CEs in L-CD of the derived 6-methoxy-2-naphthamide (in contrast, CD of the naphthamides in isotropic media, such as methanol solutions, showed only baseline).⁷ We had proposed⁷ that the strong, bi-signate CEs displayed in L-CD spectra arise from pair-wise intramolecular exciton coupling of naphthamides within the liposomal bilayers. Phospholipid bilayers, comprised of saturated long chains, promote hexagonal close packing of extended CH₂ groups.

The dimensions of the problem expand significantly for assignment of the remote C12/C11 methyl-branched stereocenters in 3 and 4. We surmised the remote methyl branch in 3 and 4 may alter the chain packing and influence the magnitude and form of the naphthamide CEs in L-CD. This would constitute net transmission of stereochemical information from the remote C12 center to the naphthamide group, however, it was not certain the effect would be large enough to be readily observable in the CD spectrum.

In order to test this hypothesis, 3¹¹ was degraded (Scheme 1) by cleavage of the C6–C7 bond using Fe(II)-promoted reductive fragmentation (FeCl₂, aq. CH₃CN-H₂O, N₂-sparged) to give primary alkyl chloride 5¹², which was subsequently transformed by a three-step sequence⁷ to give naphthamide 7.

Scheme 1.

Degradation of 3 and conversion to 7

Compound 7 was formulated into a highly-uniform, unilamellar liposomes of distearoyl-sn-glycero-3-phosphocholine (DSPC) as previously described,¹³ (phospholipid: 7 ratio = 20:1) prior to measurement of the CD spectrum. Remarkably, no signal appeared in the CD spectrum of 7 (Figure 2a) immediately after formulation in liposomes, but over 24 hours intense, complex CEs gradually appeared that were stable (>30 days) and reproducible [λ 196 nm (Δε −11.6), 213 (+37.4), 226 (− 6.5), 255 (+5.6)]. Fitting the time-dependence of the CE at λ = 226 nm in L-CD spectrum of 7 (Figure 2c) to an exponential function gave t_1/2 = 385 minutes. Evidently, the kinetics of lipid chain reorganization of liposomal 7 at room temperature are considerably slower than the corresponding naphthamides derived from 1 and 2 in which less complex CEs appeared essentially immediately.¹⁴ The assignment of configuration in 7 was completed by comparison with synthetic model compounds (2S,6R)-8 and (2R,6R)-9 which were prepared as follows.

Figure 2.

Liposomal circular dichroism (L-CD) spectra (T= 23 °C). (a) 7 from t = 0 to 48 hrs after formulation of liposomes (H₂O, diastearoyl-sn-3-glycero-phosphocholine, 2 mg/mL; mole ratio of phospholipid:7 = 20:1). (b) 7, c = 2.24 × 10⁻⁴ M and (2S,6R)-8, c = 2.25 × 10⁻⁴ M. (c) Time-dependence of the Cotton effect of 7 (λ = 226nm), post-formulation. (d) (2R,6R)-9, c = 2.47 × 10⁻⁴ M and ent-9 (calculated).

Tosylation of (S)-(−)-citronellol¹⁵ 10 gave 11¹⁶ in 87% yield (Scheme 2), which upon treatment with lithium phenylacetylide (reflux, THF), afforded enyne 12 in 92% yield.

Scheme 2.

Synthesis of an epimeric mixture of acids 17.

Two-step Johnson-Lemieux oxidation¹⁷ of 12 provided aldehyde 14 (Scheme 2), which was subjected to Horner-Wadsworth-Emmons olefination to give α,β-unsaturated ester 15 as an inseparable 1:1 mixture of E/Z isomers. Hydrogenation of enyne 15 (Pd/C) afforded saturated ester 16, which was saponified (LiOH, THF/water) to give acid 17 as a 1:1 mixture of diastereomers, epimeric at C2. Acid 17 was coupled with (S)-phenethylamine (HATU, i-Pr₂NEt) (Scheme 3) to provide a mixture of diastereomeric amides (2S,6R)-18 and (2R,6R)-19, which were separated by silica gel chromatography.¹⁸ Amides 18 and 19 were individually treated with BH₃•THF to afford secondary amines 20 and 21. Hydrogenolysis of 20 and 21 (Pd/C, CF₃CH₂OH) provided primary amines 22 and 23 in 92% and 72% yield, respectively. Separate acylation of 22 and 23 (6-methoxy-2-naphthoyl chloride, Et₃N, DMAP) gave naphthamides (2S,6R)-8 and (2R,6R)-9.

Scheme 3.

Synthesis of standards, naphthamides 8 and 9.

Comparisons of the L-CD spectra of three stereoisomers are shown in Figure 2: 7 derived from 3 or 4, and synthetic compounds (2S,6R)-8 and (2R,6R)-9. The signs and magnitudes of the CEs are primarily dominated by perturbation of the naphthamide chromophore by the proximal stereocenter C2 within the first sphere of asymmetry. It is also evident that longer range perturbation from the remote methyl branch alters the forms of L-CD spectra of (2S,6R)-8 and (2S,6S)-9) as a function of the C6 configuration (Figures 2b and d). Whereas the diastereomers (2S,6R)-8 and (2R,6R)-9 were indistinguishable by NMR,¹⁹ they were clearly differentiated by the CEs and fine structure of their L-CD spectra. In order to evaluate the effect of the distal C6 stereocenter, the L-CD of (2S,6R)-8 was compared with the inverted L-CD of (2R,6R)-9 (Figure 2d) which corresponds to (2S,6S)-9. The CEs in 8 [λ_max 196 (Δε +11.0), 213 (−34.0), 226 (+27.3), 258 (−4.9)] were changed in 9, particularly λ 217 nm (Δε +17.5) and λ 256 nm (Δε −6.2) (see Table S4 for complete data).

The L-CD of 7 did not match either enantiomer of 9: it was equal in magnitude and form, but opposite in sign to that of (2S,6R)-8 (Figure 2b). Therefore, 7 and (2S,6R)-8 are enantiomers and the complete configurations of 3 and 4 are 3S,4S,6R,8R,12S and 3R,5R,7R,11S, respectively.

Compounds 1–4 were assayed for antifungal activity against strains of Candida albicans, C. glabrata, C. krusei and Cryptococcus neoformans (Table 1). All compounds were exceedingly potent antifungal agents (MICs ≤ 0.5 μg/mL) against all seven strains. The monomethyl-branched ω-phenyl polyketide peroxides 1 and 2 were about twice as active as plakinic acids K (3) and L (4). Compound 2 showed the most potent activity (MIC< 0.12 μg/mL) against C. albicans 96–489, C. albicans UCD-FRI and C. glabrata suggesting that the 1,2-dioxolane ring and a monomethyl-branch in the ω-phenylalkyl side-chain are key determinants of antifungal activity.

Table 1.

Antifungal activities of plakinic acids I-L (1–4). Minimum inhibitory concentration, MIC (μg/mL)^a.

	1	2	3	4
C. albicans, ATCC 14503	0.25	0.25	0.50	0.50
C. albicans 96–489b	0.06	<0.03	0.12	0.12
C. albicans UCD-FR1b	0.25	0.12	0.50	0.50
C. glabrata	0.12	0.06	0.12	0.12
C. krusei	0.06	0.12	0.12	0.12
Cryptococcus neoformans var. gattii	0.12	0.25	0.12	0.50
C. neoformans var. grubii	0.12	0.25	0.12	0.50

^aThe in vitro susceptibilities were determined by the microbroth dilution method according to the guidelines of the National Committee for Clinical Laboratory Standards (NCCLS).

^bFluconazole-resistant (MIC >64 (μg/mL).

In conclusion, liposomal CD differentiates long-chain methyl-branched naphthamides, with epimeric configurations at a stereocenter eight bonds removed from the chromophore. Comparison of the L-CD spectrum of the naphthamide, obtained by degradation of plakinic acid K (3), with those of diastereomeric synthetic models, allowed assignment of C8/C7 and C12/C11 of plakinic acids K (3) and L (4), respectively. This remarkable long-range propagation of stereochemical information is made possible by amplification of the Cotton effects through ordering of lipid chains within the liposomal bilayer. The current work also provides CD references for interrogation of remote double methyl-branched polyketides by L-CD. Additional applications of L-CD are the subject of ongoing investigations in our laboratories.

Figure 1.

Structures of plakinic acids I (1), J (2), K (3) and L (4).