Electrospray‐Ionization Mass Spectrometry for Screening the Specificity and Stability of Single‐Stranded‐DNA Templated Self‐Assemblies

Supramolecular complexes consisting of a single‐stranded oligothymine ( dTn ) as the host template and an array of guest molecules equipped with a complementary diaminotriazine hydrogen‐bonding unit have been studied with electrospray‐ionization mass spectrometry (ESI‐MS). In this hybrid construct, a supramolecular stack of guest molecules is hydrogen bonded to dTn . By changing the hydrogen‐bonding motif of the DNA host template or the guest molecules, selective hydrogen bonding was proven. We were able to detect single‐stranded‐DNA (ssDNA)–guest complexes for strands with lengths of up to 20 bases, in which the highest complex mass detected was 15 kDa; these complexes constitute 20‐component self‐assembled objects. Gas‐phase breakdown experiments on single‐ and multiple‐guest–DNA assemblies gave qualitative information on the fragmentation pathways and the relative complex stabilities. We found that the guest molecules are removed from the template one by one in a highly controlled way. The stabilities of the complexes depend mainly on the molecular weight of the guest molecules, a fact suggesting that the complexes collapse in the gas phase. By mixing two different guests with the ssDNA template, a multicomponent dynamic library can be created. Our results demonstrate that ESI‐MS is a powerful tool to analyze supramolecular ssDNA complexes in great detail.     

Crystal‐Packing‐Driven Enrichment of Atropoisomers

Crystal‐packing forces can have a significant impact on the relative stabilities of different molecules and their conformations. The magnitude of such effects is, however, not yet well understood. Herein we show, that crystal packing can completely overrule the relative stabilities of different stereoisomers in solution. Heating of atropoisomers (i.e. “frozen‐out” conformational isomers) in solution leads to complex mixtures. In contrast, solid‐state heating selectively amplifies minor (<25 mole %) components of these solution‐phase mixtures. We show that this heating strategy is successful for compounds with up to four rotationally hindered σ bonds, for which a single stereoisomer out of seven can be amplified selectively. Our results demonstrate that common supramolecular interactions—for example, [methyl⋅⋅⋅π] coordination and [C−H⋅⋅⋅O] hydrogen bonding—can readily invert the relative thermodynamic stabilities of different molecular conformations. These findings open up potential new avenues to control the folding of macromolecules.     

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.     

Electrospray ionization of nucleic acid aptamer/small molecule complexes for screening aptamer selectivity

Molecular recognition of small molecule ligands by the nucleic acid aptamers for tobramycin, ATP, and FMN has been examined using electrospray ionization mass spectrometry (ESI-MS). Mass spectrometric data for binding stoichiometry and relative binding affinity correlated well with solution data for tobramycin aptamer complexes, in which aptamer/ligand interactions are mediated by hydrogen bonds. For the ATP and FMN aptamers, where ligand interactions involve both hydrogen bonding and significant pi-stacking, the relative binding affinities determined by MS did not fully correlate with results obtained from solution experiments. Some high-affinity aptamer/ligand complexes appeared to be destabilized in the gas phase by internal Coulombic repulsion. In CAD experiments, complexes with a greater number of intermolecular hydrogen bonds exhibited greater gas-phase stability even in cases when solution binding affinities were equivalent. These results indicate that in at least some cases, mass spectrometric data on aptamer/ligand binding affinities should be used in conjunction with complementary techniques to fully assess aptamer molecular recognition properties.     

Electrospray ionization mass spectrometry: a major tool to investigate reaction mechanisms in both solution and the gas phase

Electrospray ionization mass spectrometry (ESI-MS), in conjunction with its tandem version ESI-MS/MS, is now established as a major tool to study reaction mechanisms in solution. This suitability results mainly from the ability of ESI to "fish" ions directly from solution to the gas phase environment of mass spectrometers. In this review, we summarize recent studies from our laboratory on the use of on-line monitoring by ESI-MS ion fishing of several types of reactions that permitted us to follow how these reactions progress as a function of both time and conditions using the ultra-high sensitivity and speed of ESI-MS to detect and even characterize transient reaction intermediates. We also show that the intrinsic reactivity of each key gaseous species fished by ESI can be further investigated via ESI-tandem mass spectrometry experiments, searching for the most active species via gas-phase ion/molecule reactions. In the gas-phase, solvent and counter-ion effects are absent. These studies often permit a detailed overview of major steps via the interception, characterization and reactivity investigation of key reaction players.     

The mechanism of the Stille reaction investigated by electrospray ionization mass spectrometry

On-line monitoring of Stille reactions was performed via direct infusion electrospray ionization mass spectrometry (ESI-MS) and its tandem version (ESI-MS/MS). When operated in the positive ion mode, ESI(+)-MS was able to transfer, directly from solution to the gas phase, the species involved in all main steps of a Stille reaction, that is, the catalytically active palladium species Pd(PPh3)2, in its molecular ion form as well as the key cationic Pd(II) intermediates, including cyclic IPd-(CH2CH)Sn species. When searching for anionic species, ESI(-)-MS monitoring showed I- as the only anion detectable in the reaction medium. A detailed catalytic cycle for a Stille reaction was elaborated in which reaction intermediates and the previously elusive catalytically active Pd(0) species are shown in association with the respective ionic species intercepted by ESI-MS and further characterized by ESI-MS/MS.     

Crystal-Packing-Driven Enrichment of Atropoisomers

Crystal-packing forces can have a significant impact on the relative stabilities of different molecules and their conformations. The magnitude of such effects is, however, not yet well understood. Herein we show, that crystal packing can completely overrule the relative stabilities of different stereoisomers in solution. Heating of atropoisomers (i.e. “frozen-out” conformational isomers) in solution leads to complex mixtures. In contrast, solid-state heating selectively amplifies minor (<25 mole %) components of these solution-phase mixtures. We show that this heating strategy is successful for compounds with up to four rotationally hindered σ bonds, for which a single stereoisomer out of seven can be amplified selectively. Our results demonstrate that common supramolecular interactions—for example, [methyl⋅⋅⋅π] coordination and [C−H⋅⋅⋅O] hydrogen bonding—can readily invert the relative thermodynamic stabilities of different molecular conformations. These findings open up potential new avenues to control the folding of macromolecules.     

Chelation-controlled radical chain reactions studied by electrospray ionization mass spectrometry

Electrospray ionization mass spectrometry (ESI-MS) is a novel tool for the investigation of chemical reactions in solution and for the direct detection and identification of reactive intermediates. The tributyltin hydride mediated addition of tert-butyl iodide to dimethyl 2-cyclohexyl-4-methyleneglutarate (2) in the presence of Lewis acids was investigated by ESI-MS using a microreactor coupled on-line to an ESI mass spectrometer. For the first time we have been able to show that transient radicals in radical chain reactions can be detected unambiguously under steady-state conditions in the reaction solution and can be characterized by ESI-MS/MS and accurate mass determination. The detection of different heterodimer radical complexes by ESI-MS/MS has provided new insights into the mechanism of Lewis acid controlled radical chain reactions. Dimeric chelate complexes of glutarates, such as 2 and 3, and Lewis acids, like Sc(OTf)3, MgBr2OEt2 and LiClO4, were observed as well as higher aggregates with additional equivalents of Lewis acid. Evidence for a dynamic equilibrium of the complexes in solution was found by NMR spectroscopy. The ESI-MS investigation of the chelation of glutarate 2 with various Lewis acids has led to the conclusion that the tendency for Lewis acids to form dimeric chelate complexes and higher aggregates has an important effect on the stereoselective outcome of the radical reactions.     

Mass spectrometry of protein-ligand complexes: Enhanced gas-phase stability of ribonuclease-nucleotide complexes

Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS). Ligand binding stoichiometry can be determined easily by the ESI-MS method. The ability to detect noncovalent protein-ligand complexes depends, however, on the stability of the complexes in the gas-phase environment. Solution binding affinities may or may not be accurate predictors of their stability in vacuo. Complexes composed of cytidine nucleotides bound to ribonuclease A (RNase A) and ribonuclease S (RNase S) were detected by ESI-MS and were further analyzed by MS/MS. RNase A and RNase S share similar structures and biological activity. Subtilisin-cleavage of RNase A yields an S-peptide and an S-protein; the S-peptide and S-protein interact through hydrophobic interactions with a solution binding constant in the nanomolar range to generate an active RNase S. Cytidine nucleotides bind to the ribonucleases through electrostatic interactions with a solution binding constant in the micromolar range. Collisionally activated dissociation (CAD) of the 1:1 RNase A-CDP and CTP complexes yields cleavage of the covalent phosphate bonds of the nucleotide ligands, releasing CMP from the complex. CAD of the RNase S-CDP and CTP complexes dissociates the S-peptide from the remaining S-protein/nucleotide complex; further dissociation of the S-protein/nucleotide complex fragments a covalent phosphate bond of the nucleotide with subsequent release of CMP. Despite a solution binding constant favoring the S-protein/S-peptide complex, CDP/CTP remains electrostatically bound to the S-protein in the gas-phase dissociation experiment. This study highlights the intrinsic stability of electrostatic interactions in the gas phase and the significant differences in solution and gas-phase stabilities of noncovalent complexes that can result.     

Synthesis of novel chiral bis-N-substituted-hydrazinecarboxamide receptors and probing their solution-phase recognition to chiral carboxylic guests by ESI-TOF/MS and tandem ESI-MS

Seven novel bis-N-substituted-hydrazinecarboxamide receptors were synthesized in good to excellent yields by reacting chiral dicarbohydrazides, obtained from commercially available tartaric acid, with substituted aromatic isocyanates. The newly synthesized hydrazinecarboxamides formed structurally unique supramolecular aggregates, which have been confirmed by ESI-TOF/MS and tandem ESI-MS. They also showed molecular recognition to a selection of chiral carboxylic guests and oligopeptides, which mimic the backbone structure of the bacterial cell wall. The structures of the novel compounds were verified by various spectroscopic techniques including FTIR, ¹H NMR, ¹³C NMR, ESI-TOF/MS, tandem ESI-MS, 2D ROESY NMR, and CD spectroscopy.     

Advantages and drawbacks of nanospray for studying noncovalent protein-DNA complexes by mass spectrometry

The noncovalent complexes between the BlaI protein dimer (wild-type and GM2 mutant) and its double-stranded DNA operator were studied by nanospray mass spectrometry and tandem mass spectrometry (MS/MS). Reproducibility problems in the nanospray single-stage mass spectra are emphasized. The relative intensities depend greatly on the shape of the capillary tip and on the capillary-cone distance. This results in difficulties in assessing the relative stabilities of the complexes simply from MS(1) spectra of protein-DNA mixtures. Competition experiments using MS/MS are a better approach to determine relative binding affinities. A competition between histidine-tagged BlaIWT (BlaIWTHis) and the GM2 mutant revealed that the two proteins have similar affinities for the DNA operator, and that they co-dimerize to form heterocomplexes. The low sample consumption of nanospray allows MS/MS spectra to be recorded at different collision energies for different charge states with 1 microL of sample. The MS/MS experiments on the dimers reveal that the GM2 dimer is more kinetically stable in the gas phase than the wild-type dimer. The MS/MS experiments on the complexes shows that the two proteins require the same collision energy to dissociate from the complex. This indicates that the rate-limiting step in the monomer loss from the protein-DNA complex arises from the breaking of the protein-DNA interface rather than the protein-protein interface. The dissociation of the protein-DNA complex proceeds by the loss of a highly charged monomer (carrying about two-thirds of the total charge and one-third of the total mass). MS/MS experiments on a heterocomplex also show that the two proteins BlaIWTHis and BlaIGM2 have slightly different charge distributions in the fragments. This emphasizes the need for better understanding the dissociation mechanisms of biomolecular complexes.     

Structural characterization and differentiation of modified isomeric tryptophans

Different mass spectrometry (MS) techniques have been applied to the study of modified tryptophan isomers obtained by photochemical reactions. The gas phase behavior of the molecular ions and the most abundant fragment ions produced under electron ionization has been selectively studied by MS/MS experiments. Both the fragmentation reactions occurring in the ion source, as well as those produced under collision-induced dissociation conditions have allowed to characterize and differentiate each isomer from the others. Investigation of a bisubstituted derivative has been useful in the rationalization of the gas phase behavior of this series of modified tryptophans. This study has allowed the evaluation of the role played by the substituents and their positions at the indolic ring on the gas phase decompositions that are distinctive and selective for each isomer. The occurrence of regiospecific reactions suggests that isomerization phenomena do not occur either in the molecular ions or in the main fragment ions in the gas phase.     

Microwave-assisted synthesis of novel cyclodextrin–cucurbituril complexes

Microwave irradiation was successfully used in order to obtain stable supramolecular aggregates between cyclodextrins and cucurbiturils, without the participation of any long-chain common ‘molecular thread’ guest. These aggregates were characterised by means of various different techniques, namely NMR, thermogravimetry, polarimetry and ESI-MS. Cross-analysis of experimental data allowed us to obtain insights on the stoichiometries of the composites and their thermal stabilities. The possible structures of the composites are briefly discussed, as well as the actual nature of their intrinsic stability.     

Counterion-dependent proton-driven self-assembly of linear supramolecular oligomers based on amino-calix[5]arene building blocks

Self-assembly of a calix[5]arene bearing a 12-aminododecyl pendant group on the lower rim into supramolecular oligomers through intermolecular iterative inclusion events is readily triggered by contact with acid solutions and is reversed to the amino monomer precursor by treatment with a base. 1H NMR data are consistent with the formation of head-to-tail assemblies derived from endo-cavity inclusion of the alkylammonium moiety. Diffusion NMR and light-scattering studies provide evidence for the presence of oligomers in solution and show that different counterions and concentrations result in different oligomer sizes, whereas ESI-MS and SEM investigations, respectively, indicate that self-assembly also takes place in the gas phase and in the solid state. The growth of these supramolecular oligomers is concentration-dependent; however, as a consequence of the saline nature of the monomer, it also shows a distinct counterion-dependence owing to ion-pairing/solvation effects.     

Restricted guest tumbling in phosphorylated self-assembled capsules

ABii diphosphonatocavitands self-assemble in chloroform solution to form dimeric molecular capsules. The molecular capsules can incarcerate an N-methylpyridinium or N-methylpicolinium guest. We have demonstrated that the supramolecular assembly acts as a molecular rotor as a result of the restricted motion of the guest inside the molecular cavity. In the solid state, X-ray diffraction analysis of the free host showed that two cavitands interact through strong hydrogen bonds to give the supramolecular self-assembled capsule. The solid-state structure of the N-methylpicolinium complex is comparable to that of the free host and indicates that the guest is not a prerequisite for the formation of the capsule. DOSY NMR studies provided a definitive argument for the formation of the free and complexed supramolecular capsule in CDCl(3) solution. In solution, the tumbling of the N-methylpyridinium and N-methylpicolinium guests about the equatorial axes of the host can be frozen and differs by the respective energy barriers, with the larger picolinium substrate having a larger value (ΔG(++) = 69.7 kJ mol(-1)) than the shorter pyridinium guest (ΔG(++) = 44.8 kJ mol(-1)). This behavior corresponds to the restricted rotation of a rotator in a supramolecular rotor.     

质子化4,4’-联吡啶盐与二苯并-24-冠-8的包合行为

通过1H NMR, ESI-MS, 紫外-可见光谱等分析手段研究了两个新的质子化联吡啶盐客体与冠醚二苯并-24-冠-8在溶液中和气态下的包合行为. 研究结果表明, 质子化的联吡啶盐与冠醚主要形成1∶1的超分子包合物, 且存在夹心式的几何结构, 双质子化的联吡啶盐比单质子化的联吡啶盐具有更强的结合冠醚的能力, 其缔合常数分别为200, 116 L&#8226;mol－1.     

Mass spectrometric characterization and gas-phase chemistry of self-assembling supramolecular squares and triangles

A detailed mass spectrometric characterization of self-assembling polynuclear metal complexes is described. The complexes can only be ionized as intact species under a surprisingly narrow range of conditions by electrospray ionization. Comparison with the results from NMR experiments shows that several solution-phase features of these squares and triangles (such as trends in bond energies, ligand-exchange reactions, or square-triangle equilibria) are qualitatively reflected in the gas-phase data. Consequently, mass spectrometry represents a valuable method for the characterization of these compounds. Nevertheless, the formation of unspecific aggregates during the ionization process occurs and its implications are discussed. Beyond the chemistry in solution, the fragmentation pathways of these complexes in the gas phase have been studied by infrared multiphoton dissociation (IRMPD) experiments. The results of IRMPD studies allow us to draw conclusions with respect to the structure and energetics of fragmentation products. In this tandem MS experiment, reaction pathways can be observed directly which can hardly be analyzed in solution. According to these results, the equilibration of triangles and squares involves the supramolecular analogue of a neighboring-group effect.     

Analysis of metal complex azo dyes by high-performance liquid chromatography/electrospray ionization mass spectrometry and multistage mass spectrometry

Five metal complex azo compounds were analyzed using negative-ion electrospray ionization mass spectrometry (ESI-MS). Mass spectra of all compounds yield intense peaks corresponding to [M - H](-) ions without any fragmentation, where M denotes the neutral compound with a proton as the counterion. Under collision induced dissociation (CID) conditions, structurally important fragment ions were studied using the ion trap analyzer with a multistage mass spectrometry (MS(n) facility. Synthesized compounds with (15)N atoms in the azo group facilitated the fragmentation pattern recognition. A reversed-phase high-performance liquid chromatography (HPLC) method using 5 mM ammonium acetate in 70% aqueous acetonitrile as mobile phase was developed making possible the separation of all complex compounds tested. The lower detection limits of the ESI-MS method are in the range 10-20 ng of each compound. The HPLC/ESI-MS method makes possible the monitoring of ligand exchange in aqueous solutions of metal complex azo dyes, and also investigation of the stabilities of the complexes in solution. Copyright 2000 John Wiley & Sons, Ltd.     

Dynamic covalent chemistry

Dynamic covalent chemistry relates to chemical reactions carried out reversibly under conditions of equilibrium control. The reversible nature of the reactions introduces the prospects of "error checking" and "proof-reading" into synthetic processes where dynamic covalent chemistry operates. Since the formation of products occurs under thermodynamic control, product distributions depend only on the relative stabilities of the final products. In kinetically controlled reactions, however, it is the free energy differences between the transition states leading to the products that determines their relative proportions. Supramolecular chemistry has had a huge impact on synthesis at two levels: one is noncovalent synthesis, or strict self-assembly, and the other is supramolecular assistance to molecular synthesis, also referred to as self-assembly followed by covalent modification. Noncovalent synthesis has given us access to finite supermolecules and infinite supramolecular arrays. Supramolecular assistance to covalent synthesis has been exploited in the construction of more-complex systems, such as interlocked molecular compounds (for example, catenanes and rotaxanes) as well as container molecules (molecular capsules). The appealing prospect of also synthesizing these types of compounds with complex molecular architectures using reversible covalent bond forming chemistry has led to the development of dynamic covalent chemistry. Historically, dynamic covalent chemistry has played a central role in the development of conformational analysis by opening up the possibility to be able to equilibrate configurational isomers, sometimes with base (for example, esters) and sometimes with acid (for example, acetals). These stereochemical "balancing acts" revealed another major advantage that dynamic covalent chemistry offers the chemist, which is not so easily accessible in the kinetically controlled regime: the ability to re-adjust the product distribution of a reaction, even once the initial products have been formed, by changing the reaction's environment (for example, concentration, temperature, presence or absence of a template). This highly transparent, yet tremendously subtle, characteristic of dynamic covalent chemistry has led to key discoveries in polymer chemistry. In this review, some recent examples where dynamic covalent chemistry has been demonstrated are shown to emphasise the basic concepts of this area of science.     

Spontaneous formation of hexameric resorcinarene capsule in chloroform solution as detected by diffusion NMR

NMR diffusion measurements provide unequivocal proof that the resorcinarene 1b self-assembles spontaneously into a stable hexamer capsule in chloroform solution by encapsulating several chloroform molecules, which occupy different chemical positions. Although the affinity of tetrahexylammonium bromide (2a) toward the cavity of the hexamer is much higher than that of the chloroform molecules, it was found that the same amount of DMSO is needed to disrupt the two hexamers, thus suggesting similar stabilities for these two supramolecular capsules.