Spectroscopic studies and PM5 semiempirical calculations of new Schiff bases of gossypol with polyoxaalkylamines

Three Schiff bases of racemic gossypol with polyoxaalkylamines were synthesized and studied by FTIR and (1)H-NMR spectroscopy, and their structures were calculated by the PM5 semiempirical method. These Schiff bases exist in the solid state and in solutions as enamine forms. An increasing length of the polyoxaalkyl chain causes the increase of the interaction of the oxygen atoms of this chain with the OH groups in the 6,6' positions. This interaction is very well evidenced in the FTIR and (1)H-NMR spectra. The structures of the Schiff bases and the hydrogen bonds within these structures are discussed.     

Multinuclear magnetic resonance, electrospray ionization-mass spectroscopy, and parametric method 5 studies of a new derivative of gossypol with 2-thiophenecarbohydrazide as well as its complexes with LI+, Na+, K+, RB+, and Cs+ cations

A new derivative of racemic gossypol with 2-thiophenecarbohydrazide (GHHT) and its complexes with monovalent cations have been synthesized and studied by electrospray ionization-mass spectroscopy (ESI-MS), multinuclear nuclear magnetic resonance (NMR), as well as by the Parametric Method 5 (PM5) methods. It is demonstrated that GHHT forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by three types of intramolecular hydrogen bonds. The spectroscopic methods have provided clear evidence that GHHT and its complexes exist in the DMSO-d6 solution in the N-imine-N-imine tautomeric forms. The structures of the GHHT and its complexes with Li+, Na+, K+, Rb+, and Cs+ cations are visualized and discussed in detail.     

Spectroscopic studies and PM3 semiempirical calculations of Schiff bases of gossypol with L-amino acid methyl esters

Three Schiff bases of racemic gossypol with L-amino acid methyl esters are synthesized and studied by FTIR and (1)H-NMR spectroscopy, and their structures are calculated by the PM3 semiempirical method. The Schiff bases in the study exist in the solid state and in solutions as enamine forms. The existence of diastereoisomers is very visible in the (1)H-NMR spectra. The amount of the diastereoisomers depends on the amount of time the solutions are rested in diffused light. The epimerization from D,L-isomer to L,L-isomer is very slow. The structures of the Schiff bases and the hydrogen bonds within these structures are discussed.     

Schiff base of gossypol with 3,6,9-trioxa-decylamine complexes with monovalent cations studied by mass spectrometry, (1)H-NMR, FTIR, and PM5 semiempirical methods

A new Schiff base of gossypol with 3,6,9-trioxo-decylamine (GSTB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization mass spectrometry, (1)H-NMR and FTIR spectroscopy, and the PM5 (parametric method 5) semiempirical method. It is found that GSTB forms 1 : 1 and 1 : 2 complexes with Li(+) and Na(+) and 1 : 1 complexes with K(+), Rb(+), or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of trioxo-alkyl chains. All other cations are strongly localized. In the complex of GSTB with two protons localized on the N atoms of the Schiff base, the imine-imine tautomeric form is realized. The complexes of the Schiff base with K(+), Rb(+), or Cs(+) cations are the 1 : 1 type with the oxygen atoms of the trioxo-alkyl chains, as well as the O(1)H or O(1')H group coordinating the cation. The structures of the complexes are calculated by the PM5 semiempirical method and discussed.     

The schiff base of gossypol with 3,6,9,12,15,18,21,24-octaoxa-pentacosylamine complexes and monovalent cations studied by electrospray ionization-mass spectrometry, (1)H nuclear magnetic resonance, Fourier transform infrared, as well as PM5 semiempirical methods

A Schiff base of gossypol with 3,6,9,12,15,18,21,24-octaoxa-pentacosylamine (GSOB) forms stable complexes with monovalent cations. This process of complex formation was studied by electrospray ionization-mass spectrometry, (1)H-NMR and Fourier transform infrared spectroscopy as well as by the PM5 semiempirical method. It was found that GSOB forms 1:6 complexes with Li(+) and Na(+), and 1:4 complexes with K(+) as well as 1:2 complexes with Rb(+) or Cs(+) cations and exists in all these complexes in the enamine-enamine tautomeric form. Moreover, within these complexes only Li(+) cations can fluctuate between the oxygen atoms of the octaoxaalkyl chains. The interactions of Li(+) cations with hydroxyl groups of the gossypol part is also possible. All other cations are much more localized. In the complex of GSOB with protons, a 1:2 stoichiometry is realized. The two protons are localized on the N atoms of the Schiff base, and the complex exists in the imine-imine tautomeric form. The structures of the complexes are calculated by PM5 semiempirical methods and discussed.     

Optical stability of gossypol

The optical stability of gossypol [1,1',6,6',7,7'-hexahydroxy-3,3'-dimetyl-5,5'-bis(1-methylethyl)-2,2'-binaphthalene-8,8'-dicarboxaldehyde], a natural product exhibiting profoundly enantiospecific antitumor and male antifertility action, was investigated by means of computational methods and thermal racemization experiments. The calculations on gossypol and several derivatives and model compounds were carried out using the MM2 force field; energies and geometries of minimum energy conformations, as well as structures along various inversion pathways, were calculated. According to the calculations, gossypol (the dialdehyde form) and its simple analogues are not thermally racemizable (energy barriers for rotational inversion above 50 kcal/mol). By contrast, the calculations suggest that the acetal tautomer of gossypol and its dehydration product (anhydrogossypol) are thermally racemizable, although the energy barriers are still relatively high (35–40 kcal/mol). Optically pure (+)-anhydrogossypol was prepared and characterized; its racemization became rapid only at high temperatures (180–200°C). When dehydration of gossypol was hindered (in aqueous solution), no racemization of gossypol was observed after prolonged heating at 90°C. © 1992 Wiley-Liss, Inc.     

In vitro inhibitory effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri

AIM: This study was conducted to evaluate the toxic effect of gossypol from gossypol-acetic acid, and (+)- and (-)-isomers of gossypol on the growth of Edwardsiella ictaluri. METHODS AND RESULTS: Inhibitory effect of various concentrations of gossypol on the growth of E. ictaluri was determined. Bacterial recovery was performed by preincubation of bacteria in medium containing various concentrations of gossypol and subsequent activation of bacteria by inoculating on gossypol-free plates. Concentrations of racemic gossypol, (+)-gossypol and (-)-gossypol of 1.5 microg ml(-1) or higher significantly reduced the number of bacterial colonies compared with that of the control. The growth of E. ictaluri was completely inhibited on agar plates supplemented with 3 microg ml(-1), regardless of the forms of gossypol. The inhibitory effect of (+)-gossypol was higher than that of (-)-gossypol or gossypol-acetic acid. Recovery of E. ictaluri was <50% for all three forms of gossypol at concentrations of 5 microg ml(-1). Bacterial recovery remained relatively constant (6.5%) at gossypol concentrations from 10 to 100 microg ml(-1). Complete killing of E. ictaluri was not reached at gossypol levels up to 100 microg ml(-1). CONCLUSION: Gossypol-acetic acid, and (+)- and (-)-optical isomers have anti-bacterial effect against E. ictaluri. The results suggest the action is bacteriostatic rather than bactericidal. SIGNIFICANCE AND IMPACT OF THE STUDY: The therapeutic effect of gossypol against E. ictaluri may be useful in controlling enteric septicaemia of catfish.     

Monensin A methyl ester complexes with Li+, Na+, and K+ cations studied by ESI-MS, 1H- and 13C-NMR, FTIR, as well as PM5 semiempirical method

Monensin A methyl ester (MON1) was synthesized by a new method and its ability to form complexes with Li+, Na+, and K+ cations was studied by electrospray ionization-mass spectroscopy (ESI-MS), 1H and 13C nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR), and PM5 semiempirical methods. It is shown that MON1 with monovalent metal cations forms stable complexes of 1:1 stoichiometry. The structures of the complexes are stabilized by intramolecular hydrogen bonds in which the OH groups are always involved. In the structure of MON1, the oxygen atom of the C=O ester group is involved in very weak bifurcated intramolecular hydrogen bonds with two hydroxyl groups, whereas in the complexes of MON1 with monovalent metal cations the C=O ester group is not engaged in any intramolecular hydrogen bonds. Furthermore, it is demonstrated that the strongest intramolecular hydrogen bonds are formed within the MON1-Li+ complex structure. The structures of the MON1 and its complexes with Li+, Na+, and K+ cations are visualized and discussed in detail.     

Antifungal activity of alkyl and heterocyclic aza-derivatives of gossypol as well as their complexes with NaClO4 against Fusarium oxysporum f. sp. lupini

Eight alkyl and six heterocyclic aza-derivatives of gossypol (2–15) have been synthesized using gossypol (1) extracted from Gossypium Herbaceum cottonseeds. The ability of gossypol aza-derivatives to form complexes with NaClO₄ has been investigated by electrospray ionisation (ESI) mass spectra recorded in the positive and negative ion detection modes. The gossypol aza-derivatives have been characterized by FT-IR, ¹H and ¹³C NMR spectroscopic methods and subsequently tested for their antifungal properties against Fusarium oxysporum. Four alkyl aza-derivatives (2–5), present in the enamine–enamine tautomeric form, have shown activity comparable or higher than that of gossypol against this fungus. To improve the antifungal activity the complexes of the most active compounds 2–5 with NaClO₄ were prepared. Complexes of 2 and 5 with NaClO₄ have shown antifungal activity higher than that of the uncomplexed compounds.     

Synthesis and anti-H5N1 activity of chiral gossypol derivatives and its analogs implicated by a viral entry blocking mechanism

A series of chiral gossypol derivatives and its analogs were synthesized and tested in vitro for their anti-H₅N₁ activity. Interestingly, (+)-gossypol derivatives and its analogs were more active against H₅N₁ than the corresponding (?)-gossypol derivatives and its analogs. Through a simple chemical modification with amino acids, less active chiral gossypol could be converted into more active derivatives, and most of chiral gossypol derivatives were more potent against H₅N₁ than 1-adamantylamine. With regard to the mechanism of action, chiral gossypol derivatives and its analogs might impair the virus entry step of cell infection, likely targeting to HA2 protein.     

Spectroscopic, mass spectrometry, and semiempirical investigation of a new ester of Monensin A with ethylene glycol and its complexes with monovalent metal cations

A new ester of Monensin A with ethylene glycol (MON2) has been synthesized by a new method and its ability to form complexes with Li+, Na+, and K+ cations has been studied by ESI MS, 1H and 13C NMR, FT-IR, and PM5 semiempirical methods. It is demonstrated that MON2 forms stable complexes of 1:1 stoichiometry with monovalent metal cations. The structures of the complexes are stabilized by intramolecular hydrogen bonds in which the OH groups are always involved. In the structure of MON2 the oxygen atom of the C=O ester group is involved in very weak bifurcated intramolecular hydrogen bonds with two hydroxyl groups, whereas in the complexes of MON2 with monovalent metal cations the C=O ester group is not engaged in any intramolecular hydrogen bonds. The structures of the MON2 and its complexes with Li+, Na+, and K+ cations are visualized and discussed in detail.     

Biodegradation of gossypol by the white oyster mushroom,Pleurotus florida,during culturing on rice straw growth substrate,supplemented with cottonseed powder

The capacity of the white oyster mushroom, Pleurotus florida to biodegrade gossypol was studied, when grown on rice straw supplemented with cottonseed powder. The mushroom fruiting bodies did not contain any residues of gossypol at concentrations of cottonseed powder 0.15–0.60% nitrogen contents of rice straw at the end of mycelial ramification. However, the cottonseed supplementation (at 0.30% N level itself) caused a doubling in the mushroom yield and its protein content, per unit weight straw substrate. The mushroom mycelium when grown on synthetic medium in liquid cultures was able to biodegrade gossypol. A pre-incubation period of 5 days before the addition of gossypol into the culture medium, an inoculum load 10 mg and an incubation period of 10 days at 25 °C caused the biodegradation of 100 g gossypol. Increased concentrations of gossypol required increased duration and increased inoculum levels to effect biodegradation. However, the effect was more pronounced with an increase in inoculum density. The fungal monoculture when grown in rice straw (powder) (5%) + glucose (1%) liquid culture medium, showed an increase in hexosamine content and laccase activity that produced an increased degradation of gossypol over an incubation period from 5 to 25 days. Enzymic extracts of the mycelial monoculture raised on the chopped rice straw substrate when incubated with 100 g of gossypol demonstrated its biodegradability; the increase in enzyme concentration showed enhanced gossypol degradation. This study adds to the world list of organic compounds that Pleurotus is able to biodegrade, and explains the cause of non-yellowing of the white oyster mushroom (P. florida) fruiting bodies, during culture on rice straw with supplementation of cottonseed powder for enhancing the mushroom yields.     

Synthesis and antiviral activities of novel gossypol derivatives

In this study, a series of novel gossypol derivatives were synthesized and screened in vitro for their anti-HIV-1 and anti-H(5)N(1) activities, respectively. Replacing the aldehyde groups of gossypol with some amino acids not only reduced the cytotoxicity but also enhanced the activities against HIV-1 and H(5)N(1). Compounds 13-17 showed more potent activities against HIV-1 and H(5)N(1) than the other gossypol derivatives. Meanwhile, these compounds also exhibited more potent activities against H(5)N(1) than 1-adamantylamine. The absence of the COONa group in gossypol derivatives resulted in a loss of anti-HIV-1 activity, suggesting that this group might play an important role in mediating the antiviral activity. Time-of-addition assays indicated that compounds 13-17 had the similar mechanism of anti-HIV-1 action with T20. Molecular modeling analysis demonstrated that compounds 13-17 could fit inside the gp41 hydrophobic pocket through hydrogen bonding network, hydrophobic contacts and strong electrostatic interactions.     

Ultrastructural, fertility, and spermicidal studies with isomers and derivatives of gossypol in male hamsters

The effects of fourteen new, orally administered synthetic analogs of gossypol on testicular ultrastructure and fertility in hamsters and the spermicidal properties of these compounds, as well as of the optical isomers of gossypol against hamster and human sperm in vitro, are reported in this study. Test compounds were administered to adult male hamsters by daily gavage for 9 weeks at doses ranging from 15 to 50 mg/kg. The results of this study have demonstrated that the fourteen new gossypol analogs evaluated herein are not effective as male antifertility agents and their in vitro activity or lack of activity as spermicides is unrelated to their in vivo contraceptive potential. In addition, the results of the study suggest that (1) the isopropyl moiety of the gossypol molecule, like the aldehyde group, is essential for its mechanism of action and (2) the pathognomonic defect in the mitochondrial sheath induced by gossypol appears to be related to its unique activity as a male antifertility agent. The significance of these findings is discussed.     

The in vitro effect of gossypol and its interaction with salts on conidial germination and viability of Fusarium oxysporum sp. vasinfectum isolates

AIMS: To assess the effect of different concentrations of gossypol (0, 2, 4, 10 and 20 mg l(-1)) in combination with NaCl and Na(2)SO(4) (20 mS cm(-1)) on the conidial germination and viability of Fusarium oxysporum f.sp. vasinfectum (Fov). METHODS AND RESULTS: A multinomial logistic model was developed to estimate the germination probability of Fov. The inhibitory effect was markedly evident at the two highest concentrations of gossypol; it varied among the isolates tested and with time, and it was attenuated by the presence of sodium salts. The inhibition was temporary as the germination probability increased after 8 h. Fluorescent staining revealed that gossypol either killed the conidia or retarded the elongation of the germ tubes. CONCLUSION: Fov showed the ability to overcome gossypol inhibition over time, and the inhibitory effect is reduced under saline conditions. Differential responses among Fov isolates to the presence of gossypol suggest that gossypol tolerance is genetically determined in the pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: This study suggests that selecting for high plant gossypol cultivars would have minimal effect on the overall Fov resistance of cotton. A new statistical model was developed to explore the statistical significance of plant-pathogen interactions.     

Isolation of chlorogenic acids and their derivatives from Stemona japonica by preparative HPLC and evaluation of their anti-AIV (H5N1) activity in vitro

Two chlorogenic acids and five chlorogenic acid derivatives were simultaneously separated and purified from Stemona japonica by preparative high-performance liquid chromatography. Five of the collected compounds were over 95% pure while the other two compounds were over 90% pure. Their structures were elucidated as 3-O-feruloylquinic acid (1), 4-O-feruloylquinic acid (2), methyl 3-O-feruloylquinate (3), methyl 5-O-caffeyolquinate (4), methyl 4-O-feruloylquinate (5), ethyl 3-O-feruloylquinate (6) and the new compound ethyl 4-O-feruloylquinate (7) by UV, NMR and ESI-MS. All compounds were obtained from Stemona species for the first time, however compounds 6 and 7 are believed to be artefacts from the ethanol extraction. The anti-AIV (H5N1) activities were evaluated by Neutral Red uptake assay. Compounds 3 and 4 exerted moderate inhibitory effect against AIV (H5N1) in vitro.     

Optically active gossypol as a circular dichroism probe of interactions with serum albumins

The (+)-enantiomer of the polyphenolic binaphthyl gossypol, has been shown to be a useful CD probe of interactions with human and bovine serum albumin. (+)-Gossypol binds to albumin with same affinity as recemic (±)-gossypol, as shown by fluorescence quenching, and also displaces bilirubin from its albumin binding site. The CD characteristics of bound gossypol are different in the case of the two proteins.     

The distal residue-CO interaction in carbonmonoxy myoglobins: a molecular dynamics study of two distal histidine tautomers.

Four independent 90 ps molecular dynamics simulations of sperm-whale wild-type carbonmonoxy myoglobin (MbCO) have been calculated using a new AMBER force field for the haem prosthetic group. Two trajectories have the distal 64N delta nitrogen protonated, and two have the 64N epsilon nitrogen protonated; all water molecules within 16 A of the carbonyl O are included. In three trajectories, the distal residue remains part of the haem pocket, with the protonated distal nitrogen pointing into the active site. This is in contrast with the neutron diffraction crystal structure, but is consistent with the solution phase CO stretching frequencies (upsilon CO) of MbCO and various of its mutants. There are significant differences in the "closed" pocket structures found for each tautomer: the 64N epsilon H trajectories both show stable distal-CO interactions, whereas the 64N delta H tautomer) has a weaker interaction resulting in a more mobile distal side chain. One trajectory (a 64N delta H tautomer) has the distal histidine moving out into the "solvent", leaving the pocket in an "open" structure, with a large unhindered entrance to the active site. These trajectories suggest that the three upsilon CO frequencies observed for wild-type MbCO in solution, rather than representing significantly different Fe-C-O geometries as such, arise from three different haem pocket structures, each with different electric fields at the ligand. Each pocket structure corresponds to a different distal histidine conformer: the A3 band to the 64N epsilon H tautomer, the A1,2 band to the 64N delta H tautomer, and the A0 band to the absence of any significant interaction with the distal side chain.     

棉叶中棉酚的快速提取与测定方法研究

棉酚(G)及其相关物甲氧基半棉酚(DHG)、半棉酚酮(HGQ)、半棉酚(HG)、杀实夜蛾素(H1-4)等是棉花中重要的抗虫性萜烯类次生物质.利用高效液相色谱法(HPLC)对棉酚及其相关物进行了分离,测定了棉叶中的棉酚含量,讨论了不同的提取方法和测定条件对结果的影响,给出了一套简便、快速的分析测试方法,同时与紫外-可见分光光度法(苯胺法)的结果进行了对比,认为对于棉花的抗虫性的研究来说,HPLC是比较适宜的方法.