Einbauversuche mit (3H und 14C)-doppelmarkiertem Farnesol in Cantharidin. 5. Mitteilung zur Biosynthese des Cantharidins

Incorporation experiments with (³H and ¹⁴C) doubly labelled farnesols into cantharidin After injection of 11′, 12-[³H]-7-[¹⁴C]-farnesol or 11′, 12-[³H]-5,6-[¹⁴C]-farnesol, the ³H-label is located specifically in the C(9)-methyl-group of cantharidin, whereas the ¹⁴C-labelling pattern follows an incorporation via acetic acid (Scheme 4). C-Atoms 5, 6 and 7 from the middle part of the farnesol molecule are utilized for cantharidin biosynthesis to an extent that is about 2.1–11% of the incorporation rate of the methyl groups C(11′) and C(12), depending on the position of the ¹⁴C-label in farnesol. These results confirm our earlier hypothesis [1] that the C₁₀-molecule cantharidin is biosynthesized from the C₁₅-precursor farnesol which is cleaved between C(1)–C(2), C(4)–C(5), and C(7)–C(8). The synthesis of 7-[¹⁴C]-farnesol and of 5,6-[¹⁴C]-farnesol is described.     

Stereochemical applications of mass spectrometry. 3—Energy dependence of the fragmentation of stereoisomeric methylcyclohexanols

Breakdown graphs have been constructed from charge exchange data for the epimeric 2-methyl-, 3-methyl- and 4-methyl-cyclohexanols. Although the breakdown graphs for epimeric pairs are essentially identical above ～12 eV recombination energy, significant differences are observed for the epimeric 2-methyl- and 4-methyl-cyclohexanols at low internal energies. For the 2-methylcyclohexanols the ratio ([M? H₂O]^+·/[M]^+·)_cis/([M? H₂O]^+·/[M]^+·)_trans is 3.2 in the [C₆F₆]^+· charge exchange mass spectra. This is attributed to both energetic and conformational effects which favour the stereospecific cis-1,4-H₂O elimination for the cis epimer. The breakdown graph for trans-4-methylcyclohexanol shows a sharp peak in the abundance of the [M? H₂O]^+· ion at ～10 eV recombination energy which is absent from the breakdown graph for the cis epimer. This peak is attributed to the stereospecific cis-1,4-elimination of water from the molecular ion of the trans isomer; the reaction appears to have a low critical energy but a very unfavourable frequency factor, and alternative modes of water loss common to both epimers are observed at higher energies. As a result, in the [C₆F₆]^+· charge exchange mass spectra the ([M? H₂O]^+·/[M]^+·)_trans/([M? H₂O]^+·/[M]^+·)_cis ratio is ～24, compared to the value of 13 observed in the 70 eV EI mass spectra. No differences are observed in either the metastable ion abundances or the associated kinetic energy releases for epimeric molecules.     

On the incorporation of geraniol and farnesol into cantharidin (author's transl)

On the incorporation of geraniol and farnesol into cantharidin Earlier investigations [1] have shown that cantharidin (1) is biosynthesized by the male Lytta vesicatoria L. (Meloidae, Coleoptera) from the common terpenoid precursors mevalonate and farnesol (3) . To prove if geraniol (2) is incorporated via farnesol (3) into cantharidin (1) the following geraniols have been synthesized and injected into either larvae or male adult Lytta vesicatoria, partly in a mixture with synthetic 11′, 12-[³H]-farnesol as an internal standard: 2-[¹⁴C]-, 7-[¹⁴C]-, 7′, 8-[¹⁴C]-, 7′, 8-[³H]-geraniol. Unexpectedly, geraniol (2) was not specifically incorporated into cantharidin (1) perhaps due to its higher toxicity or its faster degradation relative to the other precursors before incorporation. The incorporation of U-[¹⁴C]-leucine, U-[¹⁴C]-isoleucine and 1-[¹⁴C]-glucose into cantharidin (1) via their metabolites is evident by degradation studies, whereas 1-[¹⁴C]- and 2-[¹⁴C]-glycine do not serve as precursors for cantharidin (1) .     

Divergent Melanophore-Dispersing and Tyrosinase-Stimulating Activity of Synthetic Leucine9-α-melanotropin

Leucine⁹-α-melanotropin ([Leu⁹]α-MSH) was synthesized in homogeneous solution by a fragment condensation approach, and it was assayed for its melanophore-dispersing and its tyrosinase-stimulating activity with a reflectometric in vitro frog skin assay and with cultured mouse melanoma cells, respectively. In both assay systems, parallel log dose-response curves were obtained for ([Leu⁹)]α-MSH and α-MSH; however, in the frog skin assay the activity of the title compound was 1 middot; 10¹⁰ Units/mmol, i.e. 25% of the activity of α-MSH, whereas its tyrosinasestimulating potency was only 1% compared to α-MSH (EC₅₀= 2.5 · 10^?7M ). This indicates a major difference in the recognition/stimulation process of the receptors of the two cell types.     

Synthesis and biological properties of p-azidophenylalanine13-β-melanotropin,a potent photoaffinity label for MSH receptors

p-Azidophenylalanine¹³-α-melantropin ([Pap¹³]-α-MSH) was synthesized in homogeneous solution by the fragment condensation method, and its biological activity was determined in three different assay systems. The pigment-dispersing activity relative to α-MSH was 65%, measured with melanophores of Rana pipiens or of Xenopus laevis tadpoles. The tyrosinase-stimulating activity was 50%, determined with cultured mouse melanoma cells. UV. irradiation of solutions containing ≤10^?4_M[Pap¹³]-α-MSH at 338 nm (intensity: 10^?3 W · cm^?2) led to complete photolysis of the photolabel within <20 min. Under these conditions [Pap¹³]-α-MSH was covalently inserted into MSH-receptors which produced a longlasting pigment dispersion in Xenopus melanphores (see [3]). The extent of this prolonged stimulation depended on the hormone concentration used during photolysis. 1.8·10^?9_M [Pap¹³]-α-MSH which produced a full initial response failed to prolong the effect, whereas 1.2·10^?8_M hormone caused irreversible stimulation. It appears that only about 10% of the initially occupied receptors were covalently labelled because the log dose response curve was shifted to ～ 10x higher concentration after a 200 min wash period: EC₅₀ immediately after photolysis was 6 · 10^?10_M; after 200 min EC₅₀ increased to ～8·10^?9_M.     

Reactions of lanthanoid metals with 3,5-diphenylpyrazole at elevated temperatures: synthesis and structures of both homoleptic, [Ln3(Ph2pz)9] (Ln = La, Nd), [Ln2(Ph2pz)6] (Ln = Er, Lu), and heteroleptic, [Ln(Ph2pz)3(Ph2pzH)2] (Ln = La, Nd, Gd, Tb, Er or Yb), pyrazolate complexes

The direct reaction of lanthanoid metals with 3,5-diphenylpyrazole (Ph2pzH) at 300 degrees C under vacuum in the presence of mercury gives the structurally characterized [Ln3(Ph2pz)9] (Ln = La or Nd), [Ln2(Ph2pz)6] (Ln = Er or Lu). Similar reactions provided heteroleptic [Ln(Ph2pz)3(Ph2pzH)2] (Ln = La, Nd, Gd, Tb, Er and Y). The last was obtained only from impure Ph2pzH, but was subsequently prepared by treatment of [Yb(Ph2pz)3(thf)2] with Ph2pzH. Reactions of Yb with Ph2pzH at 200 degrees C gave a poorly soluble divalent species which was converted by 1,2-dimethoxyethane into [Yb(Ph2pz)2(dme)2]. Single crystal X-ray structures established a bowed trinuclear pyrazolate-bridged structure for [Ln3(Ph2pz)9] (Ln = La or Nd), Ln...Ln...Ln being 135.94(1) degrees (La) and 137.41(1) degrees(Nd). There are two eta2-Ph2pz ligands on the terminal Ln atoms and one on the central metal with adjacent Ln atoms linked by one mu-eta2:eta2 and one mu-eta5 (to terminal Ln):eta2 pyrazolate group. Thus the terminal Ln atoms are formally nine-coordinate and the central Ln, ten-coordinate. By contrast, [Ln2(Ph2pz)6] (Ln = Er or Lu) complexes are dimeric with two terminal (eta2) and two bridging (mu-eta2:eta2) pyrazolates and eight-coordinate lanthanoids. All six heteroleptic complexes [Ln(Ph2pz)3(Ph2pzH)2] (Ln = La, Nd, Gd, Tb, Er or Yb) are isomorphous with three equatorial eta2-Ph2pz groups, transoid(N-Ln-N 158.18(6)-161.43(9) degrees) eta1-pyrazole ligands, and eight-coordinate Ln throughout.     

Orthogonalization of channel spaces in the quark resonating group model

A recently developed coupled-channel resonating group method with orthogonalized function spaces is tested. As test example a six-quark three-channel resonating group model is used. The channels are theNN-, - andCC- (hidden colour) channels. In earlier calculations without orthogonalization of channel spaces the relative motion function of theNN-channel had a node in theS-wave at approximately 0.5 fm. In the new treatment, in which the - andCC-channels are only orthogonal corrections to theNN-channel, this node is no longer present. The newNN wave function is very similar to the one of the single-channel approximation. Thus, in the one- and three-channel approximation, we consistently find that the six-quark resonating group model does not support the notion of a short-distance node in theNN wave function.     

Analysis and quantification of ether lipids by chromatographic methods.

Chromatographic methods, especially thin-layer chromatography (TLC) and gas-liquid chromatography (GLC) are widely used in investigations of the occurrence, molecular structure and metabolism of ether lipids. The application of such techniques to structural analysis and quantification, in combination with methods for the degradation and derivatization of ether lipids, is discussed.     

One-, two-, and three-photon absorption induced fluorescence of a novel chromophore in chloroform solution

One-, two-, and three-photon absorption induced fluorescence intensities of a novel nonlinear optical chromophore have been measured by using a tunable femtosecond pulsed laser as the excitation. Four resonance peaks are observed as the excitation wavelength is tuned from 600 to 2000 nm. These peaks correspond to the one-, two- and three-photon fluorescence resonance. Except for intensity difference, the lifetime and the fluorescence spectrum are found to be the same for the one-, two-, or three-photon resonance, hence suggesting that the same excited energy level is involved in emitting the fluorescence intensity. A three-level model is developed to account for the incident excitation laser intensity dependence of the one-photon and multiphoton fluorescence intensity. The model allows the multiphoton absorption cross sections to be extracted; it can also account for the deviation observed in the linear, square, and cubic intensity dependence of the one-, two-, and three-photon fluorescence intensity, respectively. To determine the absorption cross sections, the present method does not require the fluorescence quantum efficiency data, needed in the low intensity technique.     

MO-Studies of enzyme reaction mechanisms. I. Model molecular orbital study of the cleavage of peptides by carboxypeptidase A

Ab initio and semiempiridal (AM1) molecular orbital theory has been used to model the cleavage of formamide at the active site of carboxypeptidase A. The model active site consists of a zinc dication coordinated to two imidazoles, an acetate, a water with a hydrogen-bonded formate, and a formamide molecule as model substrate. AM1 has been compared with ab initio theory for the coordination of water and formamide to Zn⁺⁺ and found to give excellent energetic results. The course of the amide cleavage was therefore calculated with AM1. The first step of the reaction is the dissociation of the zinc-coordinated water to give an active ZnOH⁺ species. The remote formate acts as proton acceptor. This process has an activation energy of only 4.6 kcal mol^?1. The next and rate-determining step is the concerted addition of the ZnOH⁺ moiety to the formamide C?O bond. The Zn? O distance in the transition state is more than 3 Å. In four further steps, the amide nitrogen is protonated and the C? N bond cleaved. The net activation energy for the entire process is 15.5 kcal mol^?1 relative to the active site model and 19.6 kcal mol^?1 relative to the most stable point on the calculated reaction profile.