Pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], a rare sterol detected in the opportunistic pathogen Pneumocystis carinii hominis: structural identity and chemical synthesis

Abstract

Pneumocystis carinii pneumonia (PcP) remains among the most prevalent opportunistic infections among AIDS patients. Currently, drugs used clinically for deep mycosis act by binding ergosterol or disrupting its biosynthesis. Although classified as a fungus, P. carinii lacks ergosterol. Instead, the pathogen synthesizes a number of distinct Delta7, 24-alkylsterols, despite the abundance of cholesterol, which it can scavenge from the lung alveolus. Thus, the pathogen-specific sterols appear vital for organism survival and proliferation. In the present study, high concentrations of a C32 sterol were found in human-derived P. carinii hominis. The definitive structural identities of two C-24 alkylated lanosterol compounds, previously not reported for rat-derived P. carinii carinii, were determined by using GLC, MS, and NMR spectroscopy together with the chemical syntheses of authentic standards. The C31 and C32 sterols were identified as euphorbol (24-methylenelanost-8-en-3beta-ol) and pneumocysterol [(24Z)-ethylidenelanost-8-en-3beta-ol], respectively. The identification of these and other 24-alkylsterols in P. carinii hominis suggests that (i) sterol C-24 methyltransferase activities are extraordinarily high in this organism, (ii) 24-alkylsterols are important components of the pathogen's membranes, because the addition of these side groups onto the sterol side chain requires substantial ATP equivalents, and (iii) the inefficacy of azole drugs against P. carinii can be explained by the ability of this organism to form 24-alkysterols before demethylation of the lanosterol nucleus. Because mammals cannot form 24-alkylsterols, their biosyntheses in P. carinii are attractive targets for the development of chemotherapeutic strategies against this opportunistic infection.

Figure 1

Chemical synthesis of authentic 24Z- and 24E-ethylidenelanost-8-en-3β-ol. I, lanosterol; II, lanosteryl acetate; III, 3β-acetoxylanost-8-en-24-one (24-ketolanosteryl acetate); IV, lanost-8-en-3β-ol-24-one; V, 24E-ethylidenelanost-8-en-3β-ol; VI, 24Z-ethylidenelanost-8-en-3β-ol (numbering is according to ref. 31).

Figure 2

GLC analyses. (A) Authentic 24Z- and 24E-ethylidenelanost-8-en-3β-ol mixture analyzed by using GLC with flame ionization detection. The major component (24Z isomer) comprised 85% and the minor component (24E isomer) comprised 15% of the chemically synthesized product (a). Co-chromatography of the authentic standard mix and GLC-purified P. carinii hominis pneumocysterol. The minor 24E-isomer peak was unchanged, whereas the major 24Z-isomer peak was enhanced (b). (B) Effects of formalin fixation on rat PcP-lung free sterols. Formalin-fixed rat PcP lungs stored for 12 weeks at room temperature (a); freshly isolated rat PcP lungs (b); and purified P. carinii carinii organisms (c). (C) Pneumocystis carinii hominis sterols. Total sterols from a formalin-fixed PcP lung (a); total sterols from organisms isolated from a frozen PcP lung (b); total sterols of BALF from a PcP patient (c); free sterol fraction from a formalin-fixed PcP lung (d); and steryl ester sterols from a formalin-fixed PcP lung (e). The ratios of peak 24 (pneumocysterol) to peak 13 (fungisterol) were: a, 21.9; b, 2.5; c, 2.2; d, 21.8; and e, 0.3.

Figure 3

GLC–MS analyses of P. carinii hominis C₃₁ and C₃₂ sterols. (A) Mass spectrum of the P. carinii hominis sterol in peak 23. (B) The mass spectrum of authentic euphorbol (24-methylenelanost-8-en-3β-ol) identified peak 23 as 24-methylenelanost-8-en-3β-ol. (C) Mass spectrum of the P. carinii hominis sterol in peak 24. (D) Mass spectrum of authentic pneumocysterol (24Z-ethylidenelanost-8-en-3β-ol) identified peak 24 as 24Z-ethylidenelanost-8-en-3β-ol.

Figure 4

NMR analyses. (A) The NOESY spectrum of the synthetic mixture at 400 MHz. The “normal” one-dimensional spectrum lies along the diagonal, and cross peaks are observed off the diagonal at the chemical shifts of two protons that are in close proximity with each other. The expected cross peaks between the methine proton at 2.818 ppm and the C-29 methyl group at 1.521 ppm (A) and the C-26 and C-27 methyl groups at 0.916 ppm (B) of the 24Z-isomer are indicated. (B) The expanded region of the NOESY spectrum of the synthetic mixture (Upper) and of authentic fucosterol (Lower). Only one cross peak is observed between the vinyl proton at 5.097 ppm and the C-29 methyl groups of the major synthetic product at 1.521 ppm (labeled A), whereas two cross peaks are observed between the vinyl proton at 5.122 ppm and the C-29 methyl group at 1.521 ppm (labeled B) and the C-26 and C-27 methyl groups at 0.916 ppm (labeled C) for both the minor product and fucosterol. (C) The 400-MHz ¹H spectra of pneumocysterol isolated from PcP lung (Upper trace) and the synthetic mixture (Lower trace). The chemical shifts of the vinyl proton on the C-28 carbon and the methine proton on the C-25 carbon are at 5.097 ppm and 2.818 ppm, respectively, for the 24Z isomer, similar to that of pneumocysterol isolated from PcP lungs.