Development of a method for comprehensive and quantitative analysis of plant hormones by highly sensitive nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry

In recent plant hormone research, there is an increased demand for a highly sensitive and comprehensive analytical approach to elucidate the hormonal signaling networks, functions, and dynamics. We have demonstrated the high sensitivity of a comprehensive and quantitative analytical method developed with nanoflow liquid chromatography-electrospray ionization-ion trap mass spectrometry (LC-ESI-IT-MS/MS) under multiple-reaction monitoring (MRM) in plant hormone profiling. Unlabeled and deuterium-labeled isotopomers of four classes of plant hormones and their derivatives, auxins, cytokinins (CK), abscisic acid (ABA), and gibberellins (GA), were analyzed by this method. The optimized nanoflow-LC-ESI-IT-MS/MS method showed ca. 5-10-fold greater sensitivity than capillary-LC-ESI-IT-MS/MS, and the detection limits (S/N = 3) of several plant hormones were in the sub-fmol range. The results showed excellent linearity (R² values of 0.9937-1.0000) and reproducibility of elution times (relative standard deviations, RSDs, <1.1%) and peak areas (RSDs, <10.7%) for all target compounds. Further, sample purification using Oasis HLB and Oasis MCX cartridges significantly decreased the ion-suppressing effects of biological matrix as compared to the purification using only Oasis HLB cartridge. The optimized nanoflow-LC-ESI-IT-MS/MS method was successfully used to analyze endogenous plant hormones in Arabidopsis and tobacco samples. The samples used in this analysis were extracted from only 17 tobacco dry seeds (1 mg DW), indicating that the efficiency of analysis of endogenous plant hormones strongly depends on the detection sensitivity of the method. Our analytical approach will be useful for in-depth studies on complex plant hormonal metabolism.     

New approaches to the structural modification of olean-type pentacylic triterpenes via microbial oxidation and glycosylation

Microbial transformation of 4 olean-type pentacyclic triterpenes (OPTs), 3-oxo oleanolic acid (1), 3-acetyl oleanolic acid (2), oleanolic acid (3), and esculentoside A (4) was studied. After the screening of 12 strains of microbes, preparative biotransformation by two strains of Streptomyces griseus ATCC 13273 and Aspergillus ochraceus CICC 40330 resulted in the isolation of 10 metabolites. The microbial catalyzed high efficient regio-selective methyl oxidation and glycosylation were discovered, which could be provided as an alternative method to expand the structural diversity of OPTs. All the structures of the metabolites were elucidated unambiguously by ESI-MS, ¹H NMR, ¹³C NMR, and 2D-NMR spectroscopy.     

Novel organomagnesium reagents in synthesis. Catalytic cyclomagnesiation of allenes in the synthesis of N-, O-, and Si-substituted 1Z,5Z-dienes

An efficient method for the synthesis of valuable N-, O-, and Si-containing 1Z,5Z-diene compounds was developed. The method comprises Cp₂TiCl₂-catalyzed homo- and cross-cyclomagnesiation of 1,2-dienes by Grignard reagents (RMgR′) to give 2,5-dialkylidenemagnesacyclopentanes in up to 96% yield. This approach was successfully used in the synthesis of 5Z,9Z-dienoic acids, precursors of acetogenins and insect pheromones.     

Convergence of hormones and autoinducers at the host/pathogen interface

Most living organisms possess sophisticated cell-signaling networks in which lipid-based signals modulate biological effects such as cell differentiation, reproduction and immune responses. Acyl homoserine lactone (AHL) autoinducers are fatty acid-based signaling molecules synthesized by several Gram-negative bacteria that are used to coordinate gene expression in a process termed “quorum sensing” (QS). Recent evidence shows that autoinducers not only control gene expression in bacterial cells, but also alter gene expression in mammalian cells. These alterations include modulation of proinflammatory cytokines and induction of apoptosis. Some of these responses may have deleterious effects on the host’s immune response, thereby leading to increased bacterial pathogenesis. Prokaryotes and eukaryotes have cohabited for approximately two billion years, during which time they have been exposed to each others’ soluble signaling molecules. We postulate that organisms from the different kingdoms of nature have acquired mechanisms to sense and respond to each others signaling molecules, and we have named this process interkingdom signaling. We further propose that autoinducers, which exhibit structural and functional similarities to mammalian lipid-based hormones, are excellent candidates for mediating this interkingdom communication. Here we will compare and contrast bacterial QS systems with eukaryotic endocrine systems, and discuss the mechanisms by which autoinducers may exploit mammalian signal transduction pathways.     

A facile one-pot stereoselective synthesis of trisubstituted (E)-2-methylalk-2-enoic acids from unactivated Baylis-Hillman adducts and a simple access to some important insect pheromones

An efficient one-pot stereoselective synthesis of trisubstituted (E)-2-methylalk-2-enoic acids has been accomplished by treatment of unactivated Baylis-Hillman adducts, 3-hydroxy-2-methylenealkanoates, with Al-NiCl₂·6H₂O in methanol at room temperature followed by hydrolysis. The method has been applied to the synthesis of three important insect pheromones, (4S,2E)-2,4-dimethyl-2-hexenoic acid, (+)-(S)-manicone and (+)-(S)-normanicone.     

Synthesis of a proposed structure for the diffusible extracellular factor of Xanthomonas campestris pv. campestris

A proposed structure for the diffusible extracellular factor (DF) of Xanthomonas campestris pv. campestris (Xcc) has been synthesized. Its MS spectrum and biological activity, however, contradict those of the natural product previously reported, suggesting that the structure for DF of Xcc must be reexamined.     

Simultaneous sampling and analysis of indoor air infested with Cimex lectularius L. (Hemiptera: Cimicidae) by solid phase microextraction,thin film microextraction and needle trap device

Air in a room infested by Cimex lectularius L. (Hemiptera: Cimicidae) was sampled simultaneously by three different sampling devices including solid phase microextraction (SPME) fiber coatings, thin film microextraction (TFME) devices, and needle trap devices (NTDs) and then analyzed by gas chromatography–mass spectrometry (GC–MS). The main focus of this study was to fully characterize indoor air by identifying compounds extracted by three different microextraction formats and, therefore, perform both the device comparison and more complete characterization of C. lectularius pheromone. The NTD technique was capable of extracting both (E)-2-hexenal and (E)-2-octenal, which were previously identified as alarm pheromones of bedbugs, and superior NTD recoveries for these two components allowed reliable identification based on mass spectral library searching and linear temperature programmed retention index (LTPRI) technique. While the use of DVB/CAR/PDMS SPME fiber coatings provided complementary sample fingerprinting and profiling results, TFME sampling devices provided discriminative extraction coverage toward highly volatile analytes. In addition to two alarm pheromones, relative abundances of all other analytes were recorded for all three devices and aligned across all examined samples, namely, highly infested area, less infested area, and control samples which were characterized by different bedbug populations. The results presented in the current study illustrate comprehensive characterization of infested indoor air samples through the use of three different non-invasive SPME formats and identification of novel components comprising C. lectularius pheromone, therefore, promising future alternatives for use of potential synthetic pheromones for detection of infestations.     

GCxGC/TOF MS technique-A new tool in identification of insect pheromones: Analysis of the persimmon bark borer sex pheromone gland

Conventional gas chromatography with electroantennographic detection (GC-EAD) and two-dimensional (GC × GC) gas chromatography using a time-of-flight mass spectrometric detector (TOFMS), were combined to analyse the female sex pheromone gland extract of the persimmon bark borer, Euzophera batangensis. GC-EAD analysis produced two EAD responses in GC areas where no compounds were detected by FID detection. GC × GC/TOFMS analysis of this area indicated the presence of several chemicals, including (Z9,E12)-tetradeca-9,12-dien-1-ol and (Z9)-tetradec-9-en-1-ol, pheromone components of closely related Euzophera species. Spectral characteristics, retention behaviour and the ability to elicit GC-EAD responses imply that both identified unsaturated alcohols are candidates for E. batangensis sex pheromone components. GC × GC/TOFMS facilitated the analysis of complex matrices on a subnanogram level and was shown to have great potential as a powerful tool in the analysis of insect pheromones.     

Identification of the sex pheromone of the lesser date moth, Batrachedra amydraula, using sequential SPME auto-sampling

The identification of the sex pheromone of the lesser date moth, Batrachedra amydraula (Meyrick) was based on GC-MS analysis of volatiles released by virgin females using sequential SPME auto-sampling of headspace and by synthesis of the key component, (4Z,7Z)-4,7-decadien-1-yl acetate. Substantial capture of males in a date palm plantation using a bait consisting of the key component and 5Z-decen-1-yl acetate in a ratio of 1:2 indicated that these are the essential components of the sex pheromone. Addition of 4Z-decen-1-yl acetate and decan-1-yl acetate, which were also identified, did not affect trap-capture. The lesser date moth is the first member of the Batrachedridae whose sex pheromone has been identified. (4Z,7Z)-4,7-Decadien-1-yl acetate is a novel compound among moth sex pheromones.     

Acid dissociation constants of uridine-5′-diphosphate compounds determined by phosphorus nuclear magnetic resonance spectroscopy and internal pH referencing

The acid dissociation constant (pK_a) of small, biological molecules is an important physical property used for investigating enzyme mechanisms and inhibitor design. For phosphorus-containing molecules, the ³¹P nuclear magnetic resonance (NMR) chemical shift is sensitive to the local chemical environment, particularly to changes in the electronic state of the molecule. Taking advantage of this property, we present a ³¹P NMR approach that uses inorganic phosphate buffer as an internal pH reference to determine the pK_a values of the imide and second diphosphate of uridine-5′-diphosphate compounds, including the first reported values for UDP-GlcNAc and UDP-S-GlcNAc. New methods for using inorganic phosphate buffer as an internal pH reference, involving mathematical correction factors and careful control of the chemical shift reference sample, are illustrated. A comparison of the newly determined imide and diphosphate pK_a values of UDP, UDP-GlcNAc, and UDP-S-GlcNAc with other nucleotide phosphate and thio-analogs reveals the significance of the monosaccharide and sulfur position on the pK_a values.     

Regio- and stereoselective 12-O-demethylation of schizandrin into gomisin T, an important intermediate to gomisin A, by Mortierella sp. (TM-I1104)

A strain TM-I1104 identified as Mortierella sp. was discovered from soil as the most efficient fungus, which converted schizandrin into gomisin T in 91% regioselectivity by microbial 12-O-demethylation. Under optimum conditions, the yield of gomisin T reached around 80%. The faculty of 12-O-demethylation was specific on (+)-schizandrin (natural form) and the optical purity of gomisin T converted from (±)-schizandrin was 96% ee.     

Experimental and theoretical study of the structure of N,N-dimethyl-4-nitroaniline derivatives as model compounds for non-linear optical organic materials

Molecular and crystal structure of a series of derivatives of N,N-dimethyl-4-nitroaniline has been studied by both X-ray diffraction method and high-level ab initio calculations. According to these data, the dimethylamino groups were found to have a trigonal-pyramidal configuration and are considerably turned with respect to the ring plane in all molecules having a substituent in the ortho-position; on the contrary, this group is planar in the meta-substituted molecules. Topological analysis of the electron density function for all molecules studied within the framework of Bader's ‘atoms in molecules’ (AIM) theory revealed that introduction of a substituent into the ortho- or meta-position of the ring results in increasing of the contribution of the resonance forms different from the quinoid one. Contribution of the latter form is predominant for the structure of N,N-dimethyl-4-nitroaniline (1). Topological analysis of the electron density distribution was used to explain a decreasing of the molecular hyperpolarisabilites of the ortho- and meta-substituted compounds as compared with those for 1.     

Chemomicrobial synthesis of (R)- and (S)-lavandulol

(R)- and (S)-Lavandulol are important compounds in the cosmetics industry and in pheromone research. We have developed syntheses of (R)- and (S)-lavandulol from (S)- and (R)-limonene, respectively, by microbial Baeyer-Villiger oxidation of the intermediate unsaturated hydroxy ketone. It has been found that the same strain of Acremonium roseum can be successfully used in the key step of the synthesis of both enantiomers of lavandulol.     

Novel microporous zirconium silicate (K2ZrSi3O9·2H2O) from high temperature phase transformation

A new microporous zirconosilicate K₂ZrSi₃O₉·2H₂O (AV-15) has been prepared by high-temperature phase transformation at 910 °C. Its structure has been determined ab initio from powder X-ray diffraction data. The unit cell is orthorhombic, space group C222₁ (no. 20), Z=4 with cell dimensions: a=8.105(3), b=10.684(5), c=12.030(5) Å, V=1041.76(7) Å³. The framework connection of AV-15 is essentially the same as the previously reported sodium stannosilicate AV-10 while the locations of potassium and water molecules in the former are quite different from those of the sodium and water molecules in AV-10. In AV-10 sodium and water molecules form a sinucoidal chain, while potassium and water molecules build up a linear chain in AV-15. The water molecules in AV-15 are lost on heating with a typical zeolitic behaviour. SEM shows that the particle sizes and habits of AV-15 and parent umbite material are the same. The ²⁹Si MAS NMR spectrum of AV-15 displays two resonances at ca. −89.4 and −90.1 ppm in a 1:2 intensity ratio. Thermogravimetry analysis confirms the existence of water in this material.     

n-Hexane sorption in poly(ethylene-co-1-octene)s: Effect on phase composition and character

Diffusion of small-molecule penetrants in semi-crystalline polymers is retarded by two factors: penetrant detour bypassing impenetrable crystals and the constraining effect of the crystals on the amorphous component. Previous experiments have shown that the latter factor becomes much less important at higher penetrant concentration in the polymer. Structural changes in a series of poly(ethylene-co-1-octene)s occurring on saturation in n-hexane at 296 K, covering a wide range of crystallinity (17-75 wt.%), were studied by wide-angle X-ray scattering, Raman spectroscopy and NMR spectroscopy. Densification of the crystal unit cell and partial dissolution of the interfacial component on n-hexane sorption are the main experimental findings. The conclusion is that the penetrant molecules increase the mobility of the polymer chain segments adjacent to the crystal interface, enabling better packing of the crystal stems and importantly also causes a reduction in the constraining factor (β) for diffusion.     

Chemical synthesis of ComX pheromone and related peptides containing isoprenoidal tryptophan residues

The ComX pheromone is a post-translationally modified oligopeptide that stimulates natural genetic competence controlled by quorum sensing in Bacillus subtilis. Recently, the structure of the ComX_RO-E-2 pheromone produced by strain RO-E-2 was determined. Based on the NMR analysis, a geranyl group is bound to the tryptophan residue, which results in the formation of a tricyclic ring structure. It was proposed that one of the four possible stereochemical isomers was based on a conformational search for model compounds and the assumption that amino acid residues in the natural pheromone have the l-configuration. All possible modified tryptophan residues and the corresponding ComX_RO-E-2 peptides were synthesized to confirm the precise stereochemistry. Here, the synthesis of the modified tryptophan derivatives was reported in detail. It was succeeded in synthesizing four optically active modified tryptophan methyl esters from which the four diastereomeric ComX_RO-E-2 peptides were prepared. Since only one of the four diastereomers was spectroscopically identical to the natural pheromone and exhibited biological activity, the absolute structure of the ComX_RO-E-2 pheromone was able to be established unambiguously. Furthermore, it was noticed that two other bioactive pheromones were present in the culture broth that were co-purified with ComX_RO-E-2 pheromone. These pheromones were presumed to be the N-terminal truncated peptides of ComX_RO-E-2 pheromone, i.e., [2-6]ComX_RO-E-2 and [3-6]ComX_RO-E-2, by LC-MS and NMR analyses. Using Fmoc solid-phase peptide synthesis, ComX_RO-E-2 pheromone and the [2-6]ComX_RO-E-2 and [3-6]ComX_RO-E-2 peptides were prepared. The synthetic peptides were identical to the natural pheromones and also showed significant biological activity.     

Experimental and computational studies of molecules with close,non-bonded hydrogen–hydrogen contacts: common computational methods grossly overestimate some ‘through-space’ NMR scalar coupling constants

The NMR spin–spin scalar coupling constants (J_HH's) of closely contacting, but non-bonded hydrogen atoms in a series of highly strained molecules (including a new in,in-cyclophane made specifically for this study) have been examined both experimentally and computationally. The experimental J_HH's are invariably quite small (0.1–0.6 Hz), but common DFT methods with modest basis sets nearly always overestimate these values, by factors of 10–30, and even with quite large basis sets (up to cc-pVQZ) the J_HH's of two of the molecules are overestimated by a factor of 10 or more. Possible reasons for these discrepancies are discussed.     

Microsphere resin chromatography combined with microbial biotransformation for the separation and purification of salvianolic acid B in aqueous extract of roots of Salvia multiorrihza Bunge

Salvianolic acid B was separated and purified from Salvia miltiorrhiza Bunge (danshen) by microbial transformation together with chromatography of microsphere resin. The aqueous extract of danshen was transformed by Fusarium graminearum in a bioreactor containing phosphate buffer (PBS), in which rosmarinic acid was transformed into danshensu and caffeic acid and the yield of salvianolic acid B was higher than 85%. After biotransformation, salvianolic acid B was purified by microsphere resin. A parallel test for making a comparison of microsphere resin chromatography between elution by methanol water solution and water was done. The purity of salvianolic acid B was up to 95% at the yield of 62% when impurities and salvianolic acid B were eluted by 45% and 55% methanol solution respectively. The purity of salvianolic acid B was up to 99% at the yield of 90% when distilled water was used to elute the impurities and salvianolic acid B. The total yield of salvianolic acid B was up to 75% at the purity over 99% while biotransformation combined with microsphere resin chromatography by water elution. Microbial biotransformation together with water elution of microsphere resin supplied an efficient method to eliminate the micromolecular impurities and a possible method to purify water-soluble compounds in traditional Chinese medicine.     

Determination of organophosphorus aromatic nitro insecticides by using electric-field cell orientation in microbial suspensions

We examined the effect of the cellular metabolism of the organophosphorus aromatic nitro insecticides metaphos and sumithion on the electro-physical properties (EPPs) of Pseudomonas putida C-11, P. putida BA-11, and Acinetobacter calcoaceticum A-122 suspensions. We used the dependences of cell-suspension absorbance changes induced by electric-field orientation on the orienting-field frequency in the range 10-10,000 kHz. Substantial orientational-spectrum changes, caused by insecticide action, occurred at frequencies of 10-1000 kHz. The plots of electro-optical effect versus insecticide-concentration were linear over the following concentration ranges: 0.5-3.0 mM metaphos and 0.5-3.5 mM sumithion for P. putida C-11 and BA-11; and 0.5-2.0 mM metaphos and 0.5-2.5 mM sumithion for A. calcoaceticum A-122. We discuss the possibility of developing a biosensor-method based on the measurement of cell-suspension orientational spectra (OS).