Stability of porphyrin-calix[4]arene complexes analyzed by electrospray ionization mass spectrometry

The stability of some porphyrin-calix[4]arene sodium-ion complexes were determined by a collision-activated decomposition (CAD) method utilizing electrospray ionization mass spectrometry (ESI-MS). Comparing the values of E(1/2), the collision energy at which the relative intensity of the complex ion is 0.5, we found that the porphyrin-calix[4]arene complex with the higher value of E(1/2) corresponded to that with the larger association constant (Kass), as measured by 1H-NMR in CDCl3. Both our ESI-MS and NMR studies proved that the number of hydrogen bonds and the rigidity of the calix[4]arene stabilized the complex. The ESI-MS technique could be successful in screening the binding affinity in host-guest systems with a small amount of sample.     

Depression of the apparent chiral recognition ability obtained in the host-guest complexation systems by electrospray and nano-electrospray ionization mass spectrometry

Chiral recognition in the host-guest complexation systems of chiral crown ether hosts and amino ester guests was thoroughly examined using the electrospray ionization (ESI) mass spectrometry/enantiomer labeled (EL)-guest method. In this method, the mass spectra of a mixture of three components in a solution, a chiral host (H), an equal amount of an (S)-enantiomer guest labeled with deuterium atoms (G(S-dn)(+)) and an unlabeled (R)-enantiomer guest (G(R)+), were measured and the relative peak intensity value [I(H + G(R))(+) / I(H + G(S-dn))(+) = IRIS] of the host-guest complex ions, observed with an excess guest concentration, was taken to provide the chiral recognition ability of the host. In our earlier report (1996), we demonstrated that the apparent chiral recognition abilities using a mass spectrometer with a homemade ESI interface were depressed by about one tenth compared with the corresponding abilities obtained by fast-atom bombardment (FAB) MS. In the present study, the enantioselective complexation behaviors of various combinations of chiral crown hosts with chiral guests were further investigated in detail mainly using a modern commercial ESI/ion trap (IT) mass spectrometer. Consequently, it was found that the apparent IRIS values from the ESI-MS/EL-guest method changed significantly, depending upon the instrument used, and in particular, upon the ESI interfaces. Moreover, under the specific measuring conditions in ESI-IT-MS, the degrees of depression of the apparent chiral recognition abilities are roughly grouped into three classes, depending upon the number (or probably the type) of the hydrophobic substituents of the hosts. Representing the degrees by the slopes when plotting the apparent IRIS values in ESI-MS versus those in FAB-MS, the slopes for the three classes are (1) 1.0, (2) 0.7 and (3) 0.3; the higher the hydrophobicity of the hosts (and then, the host-guest complex ions), the lower the slope (the apparent enantioselectivity). Strengthening the degree of depression may be caused by an increase in the local concentration of the host close to the surface of the droplets produced during the electrospary ionization process. The chiral recognition ability (K(R )/ K(S)) in an equilibrated solution agrees quite well with the IRIS value in FAB-MS rather than that in ESI-MS.     

Measurement of inclusion complex formation between cyclophane and biological relevant amino acids using electrospray ionization, cold-spray ionization and fast atom bombardment mass spectrometry

The investigation of the host-guest complex formations between cyclophane (TGDMAP) (1) as a host and L-acidic amino acids such as L-glutamic acid (Glu) and L-aspartic acid (Asp) as guests was carried out using fast atom bombardment (FAB), electrospray ionization (ESI) and cold-spray ionization (CSI) mass spectrometry (MS). The stability constant (K(s)) values obtained by the three different MS methods almost agreed. However, the complex ion peaks of a novel cyclophane (CPCn) (2) with Glu and Asp were not observed in FAB-MS. Then, these host-guest complex formations by use of CSI-MS and ESI-MS was examined, as the results, these complex ion peaks were observed clearly and the measurement values by the two MS methods are mostly in agreement. It was concluded that ESI-MS and CSI-MS are available for the determination of K(s) value as well as FAB-MS.     

电喷雾质谱在手性识别和分析中的应用

近年来电喷雾质谱以其快速、灵敏、简便、样品用量少等特点被越来越多地应用于手性分析研究中。本文综述了目前电喷雾质谱法在手性识别和分析中的应用,包括常用的分析方法及常用的质谱手性选择剂,并对其发展进行了展望。     

Formation and recognition of G-quadruplex in promoter of c-myb oncogene by electrospray ionization mass spectrometry

In this study, electrospray ionization mass spectrometry (ESI-MS) is used to study the formation of G-quadruplex by d(GGAGGAGGAGGA) which locates at the promoter region of c-myb gene. In addition, a natural small molecule, dehydrocorydaline from a Chinese herb, is found to have the highest binding affinity with the G-quadruplex in nine natural small molecules studied, and the binding selectivity of this natural molecule toward the c-myb G-quadruplex with respect to corresponding duplex DNA is significantly higher than that of the broad-spectrum G-quadruplex-ligand TMPyP4. The result from ESI-MS indicates that the gas-phase kinetic stability of the G-quadruplex can be enhanced by binding of dehydrocorydaline. To further investigate the binding properties of dehydrocorydaline to the G-quadruplex, Autodock3 is used to calculate the docked sites and docked energies of small molecules binding to the G-quadruplex and the result shows that the docked energy of dehydrocorydaline is the biggest in the nine small molecules used, consistent with the result from ESI-MS.     

Mass Activated Droplet Sorting (MADS) Enables High-Throughput Screening of Enzymatic Reactions at Nanoliter Scale

Microfluidic droplet sorting enables the high-throughput screening and selection of water-in-oil microreactors at speeds and volumes unparalleled by traditional well-plate approaches. Most such systems sort using fluorescent reporters on modified substrates or reactions that are rarely industrially relevant. We describe a microfluidic system for high-throughput sorting of nanoliter droplets based on direct detection using electrospray ionization mass spectrometry (ESI-MS). Droplets are split, one portion is analyzed by ESI-MS, and the second portion is sorted based on the MS result. Throughput of 0.7 samples s⁻¹ is achieved with 98 % accuracy using a self-correcting and adaptive sorting algorithm. We use the system to screen ≈15 000 samples in 6 h and demonstrate its utility by sorting 25 nL droplets containing transaminase expressed in vitro. Label-free ESI-MS droplet screening expands the toolbox for droplet detection and recovery, improving the applicability of droplet sorting to protein engineering, drug discovery, and diagnostic workflows.     

Binding affinities of host-guest, protein-ligand, and protein-transition-state complexes

The affinities of hosts-ranging from small synthetic cavitands to large proteins-for organic molecules are well documented. The average association constants for the binding of organic molecules by cyclodextrins, synthetic hosts, and albumins in water, as well as of catalytic antibodies or enzymes for substrates are 10(3.5+/-2.5) M(-1). Binding affinities are elevated to 10(8+/-2) M(-1) for the complexation of transition states and biological antigens by antibodies or inhibitors by enzymes, and to 10(16+/-4) M(-1) for transition states with enzymes. The origins of the distributions of association constants observed for the broad range of host-guest systems are explored in this Review, and typical approaches to compute and analyze host-guest binding in solution are discussed. In many classes of complexes a rough correlation is found between the binding affinity and the surface area that is buried upon complexation. Enzymes transcend this effect and achieve transition-state binding much greater than is expected from the surface areas.     

Gas-Phase Binding of Noncovalent Complexes Between α-cyclodextrin and Amino Acids Investigated by Mass Spectrometry

Noncovalent complexes between cyclodextrins and small molecules have been extensively studied recently because of their widespread application in the pharmaceutical industry for chiral and molecular recognition. To date, gas phase noncovalent binding affinities between α-cyclodextrin and amino acids have not been widely investigated. In this study, gas-phase binding of noncovalent complexes between α-CD and amino acids was investigated by electrospray ionization mass spectrometry (ESI-MS), demonstrating the formation of 1:1 stoichiometric noncovalent complexes. The binding of the complexes were further confirmed by collision-induced dissociation by tandem mass spectrometry. Mass spectrometric titrations between α-cyclodextrin and phenylalanine, glutamic acid, and arginine were performed to provide binding constants (lgK_a) as references for competitive ESI-MS. Calibration curves for the complexes of α-cyclodextrin with phenylalanine, glutamic acid, and arginine were plotted. Through competitive ESI-MS, the lgK_a for the complexes of α-CD with aspartic acid, lysine, proline, glycine, alanine, asparagine, cystine, glutamine, histidine, leucine, isoleucine, methionine, serine, threonine, and valine were measured directly. By comparison, it is seen that the measured binding constants for the complexes of α-cyclodextrin with basic amino acids such as arginine and lysine are lower than those for most complexes of neutral amino acids. The chiral selectivity of α-cyclodextrin for L- and D-isomers of methionine, threonine, asparagine, and phenylalanine determined by ESI-MS revealed its application as a chiral selector.     

Characterization of noncovalent complexes formed between minor groove binding molecules and duplex DNA by electrospray ionization-mass spectrometry

The noncovalent complex formed in solution between minor groove binding molecules and an oligonucleotide duplex was investigated by electrospray ionization-mass spectrometry (ESI-MS). The oligonucleotide duplex formed between two sequence-specific 14-base pair oligonucleotides was observed intact by ESI-MS and in relatively high abundance compared to the individual single-stranded components. Only sequence-specific A:B duplexes were observed, with no evidence of random nonspecific aggregation (i.e., A:A or B:B) occurring under the conditions utilized. Due to the different molecular weights of the two 14-base pair oligonucleotides, unambiguous determination of each oligonucleotide and the sequence-specific duplex was confirmed through their detection at unique mass-to-charge ratios. The noncovalent complexes formed between the self-complementary 5′-dCGCAAATTTGCG-3′ oligonucleotide and three minor groove binding molecules (distamycin A, pentamidine, and Hoechst 33258) were also observed. Variation of several electrospray ionization interface parameters as well as collision-induced dissociation methods were utilized to characterize the nature and stability of the noncovalent complexes. The noncovalent complexes upon collisional activation dissociated into single-stranded oligonucleotides and single-stranded oligonucleotides associated with a minor groove binding molecule. ESI-MS shows potential for the study of small molecule-oligonucleotide duplex interactions and determination of small molecule binding stoichiometry.     

Characterization of host-guest complexes of cucurbit[n]uril (n = 6, 7) by electrospray ionization mass spectrometry

When an intramolecular cavity exists in a molecule, it can trap another chemical species to form a host-guest complex. We examine the formation of such an inclusion complex with cucurbit[n]uril (CBn, n = 6, 7) as the host to trap alkali metal or ammonium ions as the guest, by electrospray ionization mass spectrometry (ESI-MS). The results show that the inclusion complexes are formed between the three-dimensional cylinder of CBn hosts and the guest cations. Selectivity of the complex formation is dependent both on (1) ion-dipole interactions between the cylindrical portal of the CBn hosts and the guest cations and (2) the hydrophobic interactions at the inner cavity of CBn.     

Qualitative and quantitative determination of ten major saponins in Platycodi Radix by high performance liquid chromatography with evaporative light scattering detection and mass spectrometry

Saponins in Platycodi Radix (platycosides) exhibit potent biological activities in mammalian systems, including several beneficial effects such as anti-inflammatory, immunomodulatory and anti-obesity activities. In this study, we developed a new HPLC separation coupled with evaporative light scattering detector (ELSD) for the simultaneous quantitative determination of ten major saponins in Platycodi Radix. Simultaneous separation of these saponins was achieved on a C18 analytical column. The mobile phase consisted of a gradient of aqueous acetonitrile. The method was validated for linearity, precision, accuracy, limit of detection and quantification. Electrospray ionization mass spectrometry (ESI-MS) and liquid chromatography coupled with on-line mass spectrometry (LC-ESI MS/MS) were applied to identify platycosides in the purified fractions and in the crude extract. Under ESI-MS/MS conditions, the fragmentation patterns of [M-H]- ions exclusively show signals corresponding to cleavage of the glycosidic bonds, thus allowing a rapid identification of saponins in the crude extract of Platycodi Radix. The validated HPLC method provides a new basis of overall assessment on quality of Platycodi Radix, and ESI-MS/MS and LC-ESI MS/MS approaches offers analytical tools for a rapid screening of platycosides in the crude extract.     

Fluorine-NMR experiments for high-throughput screening: theoretical aspects,practical considerations,and range of applicability

Competition ligand-based NMR screening experiments have recently been introduced to overcome most of the problems associated with traditional ligand-based NMR screening. Molecules with marginal solubility and high affinity for a given target can be easily identified in a high-throughput manner by screening chemical mixtures against the target in the presence of a weak- to medium-affinity ligand of known binding constant. While the original competition-based approaches utilized (1)H detection, significant advantages are obtained using (19)F detection. The absence of spectral overlap permits the screening of large chemical mixtures and allows for automated analysis of the spectra, even in the presence of protonated buffers, solvents, and detergents. The large chemical shift anisotropy of fluorine and the significant exchange contribution allow for the selection of a weak-affinity spy molecule, thus resulting in a lower binding affinity threshold for the identified NMR hits. The method, labeled FAXS (fluorine chemical shift anisotropy and exchange for screening) is rapid and requires only a limited amount of protein and, therefore, compares favorably with the other established non-NMR techniques used in high-throughput screening. Herein the theoretical aspects of this powerful (19)F-based approach are presented and discussed in detail. The experimental conditions together with the detection limits and binding constant measurements are investigated using human serum albumin as the target.     

Studying aminoglycoside antibiotic binding to HIV-1 TAR RNA by electrospray ionization mass spectrometry

The recognition of the aminoglycosides neomycin and streptomycin by HIV-1 TAR RNA was studied by electrospray ionization mass spectrometry (ESI-MS). Members of the aminoglycoside family of antibiotics are known to target a wide variety of RNA molecules. Neomycin and streptomycin inhibit the formation of the Tat protein–TAR RNA complex, an assembly that is believed to be necessary for HIV replication. The noncovalent complexes formed by the binding of aminoglycosides to TAR RNA and the Tat–TAR complex were detected by ESI-MS. Neomycin has a maximum binding stoichiometry of three and two to TAR RNA and to the Tat–TAR complex, respectively. Data from the ESI-MS experiments suggest that a high affinity binding site of neomycin is located near the three-nucleotide bulge region of TAR RNA. This is consistent with previous solution phase footprinting measurements [H.-Y. Mei et al., Biochemistry 37 (1998) 14204]. Neomycin has a higher affinity toward TAR RNA than streptomycin, as measured by ESI-MS competition binding experiments. A noncovalent complex formed between a small molecule inhibitor of TAR RNA, which has a similar solution binding affinity as the aminoglycosides, and TAR RNA is much less stable than the RNA–aminoglycoside complexes to collisional dissociation in the gas phase. It is believed that the small molecule inhibitor interacts with TAR RNA via hydrophobic interactions, whereas the aminoglycosides bind to RNAs through electrostatic forces. This difference in gas phase stabilities may prove useful for discerning the types of noncovalent forces holding complexes together.     

Ligand design by a combinatorial approach based on modeling and experiment: application to HLA-DR4

Combinatorial synthesis and large scale screening methods are being used increasingly in drug discovery, particularly for finding novel lead compounds. Although these "random" methods sample larger areas of chemical space than traditional synthetic approaches, only a relatively small percentage of all possible compounds are practically accessible. It is therefore helpful to select regions of chemical space that have greater likelihood of yielding useful leads. When three-dimensional structural data are available for the target molecule this can be achieved by applying structure-based computational design methods to focus the combinatorial library. This is advantageous over the standard usage of computational methods to design a small number of specific novel ligands, because here computation is employed as part of the combinatorial design process and so is required only to determine a propensity for binding of certain chemical moieties in regions of the target molecule. This paper describes the application of the Multiple Copy Simultaneous Search (MCSS) method, an active site mapping and de novo structure-based design tool, to design a focused combinatorial library for the class II MHC protein HLA-DR4. Methods for the synthesizing and screening the computationally designed library are presented; evidence is provided to show that binding was achieved. Although the structure of the protein-ligand complex could not be determined, experimental results including cross-exclusion of a known HLA-DR4 peptide ligand (HA) by a compound from the library. Computational model building suggest that at least one of the ligands designed and identified by the methods described binds in a mode similar to that of native peptides.     

Measurement of Enantiomeric Excess by Kinetic Resolution and Mass Spectrometry

New applications for your mass spectrometer —use it to measure enantiomeric excess! The enantiomeric content of very small quantities of chiral alcohols and amines has been determined by derivatization with chiral acylating agents in which mass is correlated to absolute configuration. The resultant esters and amides were then analyzed by electrospray ionization mass spectrometry (ESI-MS; shown schematically). The technique requires surprisingly low levels of diastereoselectivity in the acylation step, and is therefore generally applicable.     

Free Energy Peturbation and Molecular Dynamics Simulation Studies on the Enantiomeric Discrimination of Amines by Dimethyldiketopyridino-18-Crown-6

Abstract

Discrimination of chiral amines by dimethyldiketopyridino-18-crown-6 (1) is studied by free energy peturbation (FEP) and molecular dynamics (MD) methods. 1 has two (S)-chiral centers and discriminates chiral amines through host-guest interactions. The optically active amines in this study are α-(1-naphthyl)ethylamine, methylbenzylamine, cyclohexylethylamine, and sec-butylamine. The trends in binding free energy differences obtained from FEP calculations were in excellent agreement with experimental results obtained in the gas phase. In order to explain the enantioselectivity of the host in terms of the host-guest interactions at the molecular level, we analyzed the structures generated by 10-ns MD simulations of host-guest complexes. The suggested chiral discrimination mechanism, the π-π interaction and the steric repulsion between the guest and the host, was verified by our MD simulation analysis.     

手性金属卟啉配合物的性能研究

本文综述了近几年来手性金属卟啉配合物在主客体化学、立体催化及光电化学领域的性能研究成果。分析了在主客体化学中手性卟啉作为分子识别反应主体所具有的结构特征;介绍了模拟生物大分子的功能时手性卟啉所反映出的识别机制;评述了手性卟啉对苯乙烯及其衍生物的环丙烷化、羟基化及氨化等催化反应中所展示出的对映选择性和催化稳定性;概括了手性卟啉配合物在模拟植物光合成反应和制作新型光学材料方面所具有的光电性能。最后总结了手性卟啉研究成果的应用范围,并展望了手性卟啉在催化氧化方面的研究和发展方向。     

Identification of face-to-face inclusion complex formation of cyclodextrin bearing an azobenzene group by electrospray ionization mass spectrometry

The solution-based self-assembly of native and permethylated cyclodextrins (CD) bearing an azobenzene substituent has been studied by electrospray ionization mass spectrometry (ESI-MS). The results revealed that the CD molecules form either a contact or a face-to-face inclusion complex depending on the interaction of their substituents. The mass spectrometric study further demonstrated that the inclusion complex is formed through the interaction between the host CD cavity and the guest-substituent and that a contact complex is formed by hydrogen-bonding of the hydroxyl functions at the rims of the CD molecule. We also found that in order to detect the face-to-face inclusion complex by ESI-MS, the following conditions have to be met: (1) The CD moieties must be permethylated to avoid formation of the contact complex, (2) they must possess a guest-substituent of suitable length, such as an azobenzene moiety, and (3) they must possess an NH(2) or OH group at the substituent terminals for protonation and for detection as cations by ESI-MS. Formation of the inclusion complexes was further confirmed by the synthesis of a capped inclusion dimer and a capped monomer. Collision-induced dissociation (CID) experiments have been carried out for the contact, the host-guest inclusion, and the capped inclusion dimers, and the contact complexes are found to be the most stable among them.     

Matrix Assisted Ionization in Vacuum, a Sensitive and Widely Applicable Ionization Method for Mass Spectrometry

An astonishingly simple new method to produce gas-phase ions of small molecules as well as proteins from the solid state under cold vacuum conditions is described. This matrix assisted ionization vacuum (MAIV) mass spectrometry (MS) method produces multiply charged ions similar to those that typify electrospray ionization (ESI) and uses sample preparation methods that are nearly identical to matrix-assisted laser desorption/ionization (MALDI). Unlike these established methods, MAIV does not require a laser or voltage for ionization, and unlike the recently introduced matrix assisted ionization inlet method, does not require added heat. MAIV-MS requires only introduction of a crystalline mixture of the analyte incorporated with a suitable small molecule matrix compound such as 3-nitrobenzonitrile directly to the vacuum of the mass spectrometer. Vacuum intermediate pressure MALDI sources and modified ESI sources successfully produce ions for analysis by MS with this method. As in ESI-MS, ion formation is continuous and, without a laser, little chemical background is observed. MAIV, operating from a surface offers the possibility of significantly improved sensitivity relative to atmospheric pressure ionization because ions are produced in the vacuum region of the mass spectrometer eliminating losses associated with ion transfer from atmospheric pressure to vacuum. Mechanistic aspects and potential applications for this new ionization method are discussed.