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1.
Proc Natl Acad Sci U S A ; 119(2)2022 01 11.
Artículo en Inglés | MEDLINE | ID: mdl-34996869

RESUMEN

NMR-assisted crystallography-the integrated application of solid-state NMR, X-ray crystallography, and first-principles computational chemistry-holds significant promise for mechanistic enzymology: by providing atomic-resolution characterization of stable intermediates in enzyme active sites, including hydrogen atom locations and tautomeric equilibria, NMR crystallography offers insight into both structure and chemical dynamics. Here, this integrated approach is used to characterize the tryptophan synthase α-aminoacrylate intermediate, a defining species for pyridoxal-5'-phosphate-dependent enzymes that catalyze ß-elimination and replacement reactions. For this intermediate, NMR-assisted crystallography is able to identify the protonation states of the ionizable sites on the cofactor, substrate, and catalytic side chains as well as the location and orientation of crystallographic waters within the active site. Most notable is the water molecule immediately adjacent to the substrate ß-carbon, which serves as a hydrogen bond donor to the ε-amino group of the acid-base catalytic residue ßLys87. From this analysis, a detailed three-dimensional picture of structure and reactivity emerges, highlighting the fate of the L-serine hydroxyl leaving group and the reaction pathway back to the preceding transition state. Reaction of the α-aminoacrylate intermediate with benzimidazole, an isostere of the natural substrate indole, shows benzimidazole bound in the active site and poised for, but unable to initiate, the subsequent bond formation step. When modeled into the benzimidazole position, indole is positioned with C3 in contact with the α-aminoacrylate Cß and aligned for nucleophilic attack. Here, the chemically detailed, three-dimensional structure from NMR-assisted crystallography is key to understanding why benzimidazole does not react, while indole does.


Asunto(s)
Alanina/análogos & derivados , Dominio Catalítico , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia Magnética/métodos , Triptófano Sintasa/química , Catálisis , Indoles , Imagen por Resonancia Magnética , Resonancia Magnética Nuclear Biomolecular , Fosfato de Piridoxal/metabolismo , Triptófano Sintasa/metabolismo
2.
Phys Chem Chem Phys ; 26(13): 10183-10190, 2024 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-38497123

RESUMEN

Sequestration of small molecule guests in the cavity of a water-soluble deep cavitand host has a variety of effects on their NMR properties. The effects of encapsulation on the longitudinal (T1) and transverse (T2) relaxation times of the protons in variably sized guest molecules are analyzed here, using inversion recovery and spin-echo experiments. Sequestration of neutral organic species from the bulk solvent reduces the overall proton relaxation times, but the magnitude of this effect on different protons in the same molecule has a variety of contributors, from the motion of the guest when bound, to the position of the protons in the cavity and the magnetic anisotropy induced by the aromatic walls of the host. These subtle effects can have large consequences on the environment experienced by the bound guest, and this sheds light on the nature of small molecules in enclosed environments.

3.
J Chem Educ ; 101(2): 490-500, 2024 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-38370574

RESUMEN

In an ongoing effort to incorporate active learning and promote higher order learning outcomes in undergraduate organic chemistry, a hybrid ("flipped") classroom structure has been used to facilitate a series of collaborative activities in the first two courses of the lower division organic chemistry sequence. An observational study of seven classes over a five-year period reveals there is a strong correlation between performance on the in-class activities and performance on the final exam across all classes; however, a significant number of students in these courses continue to struggle on both the in-class activities and final exam. The Activity Engagement Survey (AcES) was administered in the most recent course offering included in this study, and these preliminary data suggest that students who achieved lower scores on the in-class activities had lower levels of emotional and behavioral/cognitive engagement and were less likely to work in collaborative groups. In total, these findings suggest that if students can be guided to engage more successfully with the in-class activities, they are likely to be more successful in carrying out the higher order learning required on the final exam. In addition to the analyses of student performance and engagement in the in-class activities, the implementation of the flipped classroom structure and suggestions for how student engagement in higher order learning might be improved in future iterations of the class are described herein.

4.
Acc Chem Res ; 55(7): 1035-1046, 2022 04 05.
Artículo en Inglés | MEDLINE | ID: mdl-35302733

RESUMEN

Simple macrocyclic water-soluble hosts such as cucurbiturils, cyclophanes, and calixarenes have long been used for biosensing via indicator displacement assays. Using multiple hosts and dyes in an arrayed format allows pattern recognition-based "chemical nose" sensing, which confers exquisite selectivity, even rivaling the abilities of biological recognition tools such as antibodies. However, a challenge in indicator displacement-based biosensing with macrocyclic hosts is that selectivity and scope are often inversely correlated: strong selectivity for a specific target can limit wide application, and broad scope sensing can suffer from a lack of selectivity between similar targets. This problem can be addressed by using water-soluble, self-folding deep cavitands as hosts. These flexible bowl-shaped receptors can be easily functionalized with different motifs at the upper and lower rim, and the large cavities can bind many different fluorescent dyes, causing either fluorescence enhancement or quenching upon binding.Cavity-based affinity is strongest for NMe3+ groups such as trimethyl-lysine, and we have exploited this for the site-selective recognition of post-translational lysine methylations in oligopeptides. The host recognizes the NMe3+ group, and by applying differently functionalized hosts in an arrayed format, discrimination between identical modifications at different positions on the oligopeptide is possible. Multiple recognition elements can be exploited for selectivity, including a defined, yet "breathable" cavity, and variable upper rim functions oriented toward the target.While the performance of the host/guest sensing system is impressive for lysine methylations, the most important advance is the use of multiple different sensing mechanisms that can target a broad range of different biorelevant species. The amphiphilic deep cavitands can both bind fluorescent dyes and interact with charged biomolecules. These non-cavity-based interactions, when paired with additives such as heavy metal ions, modulate fluorescence response in an indirect manner, and these different mechanisms allow selective recognition of serine phosphorylation, lysine acetylation, and arginine citrullination. Other targets include heavy metals, drugs of abuse, and protein isoforms. Furthermore, the hosts can be applied in supramolecular tandem assays of enzyme function: the broad scope allows analysis of such different enzymes as chromatin writers/erasers, kinases, and phosphatases, all from a single host scaffold. Finally, the indirect sensing concept allows application in sensing different oligonucleotide secondary structures, including G-quadruplexes, hairpins, triplexes, and i-motifs. Discrimination between DNA strands with highly similar structures such as G-quadruplex strands with bulges and vacancies can be achieved. Instead of relying on a single highly specific fluorescent probe, the synthetic hosts tune the fluorophore-DNA interaction, introducing multiple recognition equilibria that modulate the fluorescence signal. By applying machine learning algorithms, a classification model can be established that can accurately predict the folding state of unknown sequences. Overall, the unique recognition profile of self-folded deep cavitands provides a powerful, yet simple sensing platform, one that can be easily tuned for a wide scope of biorelevant targets, in complex biological media, without sacrificing selectivity in the recognition.


Asunto(s)
Éteres Cíclicos , G-Cuádruplex , Colorantes Fluorescentes/química , Resorcinoles
5.
Chemistry ; 29(11): e202203588, 2023 Feb 21.
Artículo en Inglés | MEDLINE | ID: mdl-36409525

RESUMEN

Appending functional groups to the exterior of Zn4 L4 self-assembled cages allows gated control of anion binding. While the unfunctionalized cages contain aryl groups in the ligand that can freely rotate, attaching inert functional groups creates a "doorstop", preventing rotation and slowing the guest exchange rate, even though the interiors of the host cavities are identically structured. The effects on anion exchange are subtle and depend on multiple factors, including anion size, the nature of the leaving anion, and the electron-withdrawing ability and steric bulk of the pendant groups. Multiple exchange mechanisms occur, and the nature of the external groups controls associative and dissociative exchange processes: these bulky groups affect both anion egress and ingress, introducing an extra layer of selectivity to the exchange. Small changes can have large effects: affinities for anions as similar as PF6 - and SbF6 - can vary by as much as 400-fold between identically sized cavities.

6.
Chemistry ; 29(63): e202302499, 2023 Nov 13.
Artículo en Inglés | MEDLINE | ID: mdl-37584901

RESUMEN

Spacious M4 L6 tetrahedra can act as catalytic inhibitors for base-mediated reactions. Upon adding only 5 % of a self-assembled Fe4 L6 cage complex, the conversion of the conjugate addition between ethylcyanoacetate and ß-nitrostyrene catalyzed by proton sponge can be reduced from 83 % after 75 mins at ambient temperature to <1 % under identical conditions. The mechanism of the catalytic inhibition is unusual: the octacationic Fe4 L6 cage increases the acidity of exogenous water in the acetonitrile reaction solvent by favorably binding the conjugate acid of the basic catalyst. The inhibition only occurs for Fe4 L6 hosts with spacious internal cavities: minimal inhibition is seen with smaller tetrahedra or Fe2 L3 helicates. The surprising tendency of the cationic cage to preferentially bind protonated, cationic ammonium guests is quantified via the comprehensive modeling of spectrophotometric titration datasets.

7.
Bioconjug Chem ; 33(12): 2245-2253, 2022 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-35362963

RESUMEN

Synthetic receptors are powerful tools for molecular recognition. They can bind to guests with high selectivity and affinity, and their structures are tunable and diversified. These features, plus the relatively low cost and high simplicity in synthesis and modification, support the feasibility of array-based molecular analysis with synthetic receptors for improved selectivity in the recognition of a wide range of targets. More attractively, host-guest interaction is reversible and guest displacement allows biocompatible and gentle release of the host-bound molecules, simplifying the stimulation designs needed to control analyte sensing, enrichment, and transportation. Here, we highlight a few recent advancements in using synthetic receptors for molecular analysis and manipulation, with the focus on macrocyclic receptors and their applications in displacement sensing, separation, imaging, and drug transport.


Asunto(s)
Receptores Artificiales , Sistemas de Liberación de Medicamentos
8.
Angew Chem Int Ed Engl ; 61(11): e202117011, 2022 03 07.
Artículo en Inglés | MEDLINE | ID: mdl-35030288

RESUMEN

A self-assembled FeII4 L6 cage was synthesized with 12 internal amines in the cavity. The cage forms as the dodeca-ammonium salt, despite the cage carrying an overall 8+ charge at the metal centers, extracting protons from displaced water in the reaction. Despite this, the basicity of the internal amines is lower than their counterparts in free solution. The 12 amines have a sliding scale of basicity, with a ≈6 pKa unit difference between the first and last protons to be removed. This moderation of side-chain basicity in an active site is a hallmark of enzymatic catalysis.


Asunto(s)
Aminas/química , Compuestos Ferrosos/síntesis química , Cationes/síntesis química , Cationes/química , Compuestos Ferrosos/química , Ligandos , Estructura Molecular
9.
J Am Chem Soc ; 143(32): 12791-12799, 2021 08 18.
Artículo en Inglés | MEDLINE | ID: mdl-34346209

RESUMEN

An arrayed host:guest fluorescence sensor system can discriminate among and classify multiple different noncanonical DNA structures by exploiting selective molecular recognition. The sensor is highly selective and can discriminate between folds as similar as native G-quadruplexes and those with bulges or vacancies. The host and guest can form heteroternary complexes with DNA strands, with the host acting as mediator between the DNA and dye, modulating the emission. By applying machine learning algorithms to the sensing data, prediction of the folding state of unknown DNA strands is possible with high fidelity.


Asunto(s)
ADN/química , Aprendizaje Automático , Motivos de Nucleótidos
10.
J Org Chem ; 86(18): 12862-12871, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34492175

RESUMEN

A self-assembled Fe4L6 cage with internally oriented carboxylic acid functions was shown to catalyze a variety of dissociative nucleophilic substitution reactions that proceed via oxocarbenium ion or carbocation intermediates. The catalytic behavior of the cage was compared to that of other small acid catalysts, which illustrated large differences in reactivity of the cage-catalyzed reactions, dependent on the structure of the substrate. For example, only a 5% cage confers a 1000-fold rate acceleration of the thioetherification of vinyldiphenylmethanol when compared to the rate with free carboxylic acid surrogates but only a 52-fold acceleration in the formation of small thioacetals. Multiple factors control the variable reactivity in the host, including substrate inhibition, binding affinity, and accessibility of reactive groups once bound. Simple effective concentration increases or the overall charge of the cage does not explain the variations in reactivity shown by highly similar reactants in the host: small differences in structure can have large effects on reactivity. Reaction of large spherical guests is highly dependent on substitution, whereas flat guests are almost unaffected by size and shape differences. The cage is a promiscuous catalyst but has strong selectivity for particular substrate shapes, reminiscent of enzymatic activity.


Asunto(s)
Ácidos Carboxílicos , Catálisis , Fenómenos Químicos Orgánicos
11.
Anal Bioanal Chem ; 412(24): 6189-6198, 2020 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-32064571

RESUMEN

Post-translational modifications (PTMs) greatly increase protein diversity and regulate their functions by changing the structures, properties, and molecular interactions of proteins. In peptide regions with high density of PTMs, PTMs can influence modification on residues in proximity or even at distal positions, adding another layer of regulation. Methods that can monitor the activities of PTM enzymes on peptides carrying multiple modifications are valuable tools for better understanding of PTM crosstalk. Herein, we developed a host-assisted capillary electrophoresis (CE) method to separate histone peptides with methylation and phosphorylation and applied it to monitor the crosstalk between serine phosphorylation and lysine methylation when they were added by Aurora B kinase and G9a lysine methyltransferase, respectively. A synthetic receptor molecule, 4-hexasulfonatocalix[6]arene (CX6), was included in the CE buffer to improve the resolution of the corresponding substrates and products. A linear polyacrylamide-coated capillary was employed to effectively reduce wall adsorption of the cationic histone peptides. The peptide substrates were labeled with fluorescein to enhance their detectability during CE separation. Our method successfully revealed that the activity of G9a methyltransferase was completely inhibited by the adjacent phosphorylation, while 25% reduction in the activity of Aurora B kinase was observed with the presence of dimethylation on the nearby residue. The PTM crosstalk was examined not only using a pure peptide substrate, but also in a competitive reaction environment, in which the modified and unmodified peptides were mixed and the enzyme actions on both peptides were monitored simultaneously. Our work demonstrates that host-assisted CE is an effective method for study of PTM crosstalk, which could offer the advantages of fast separation, high resolution, and low sample consumption. Graphical abstract.


Asunto(s)
Electroforesis Capilar/métodos , Histonas/metabolismo , Péptidos/metabolismo , Secuencia de Aminoácidos , Lisina/metabolismo , Metilación , Péptidos/química , Fosforilación , Procesamiento Proteico-Postraduccional
12.
Angew Chem Int Ed Engl ; 59(52): 23505-23509, 2020 12 21.
Artículo en Inglés | MEDLINE | ID: mdl-32914518

RESUMEN

An endohedrally functionalized self-assembled Fe4 L6 cage complex can catalyze oxa-Pictet-Spengler cyclizations of tryptophols and various aldehyde derivatives, showing strong rate accelerations and size-selectivity. Selective molecular recognition of substrates controls the reactivity, and the cage is capable of binding and activating multiple different species along the multistep reaction pathway. The combination of a functionalized active site, size-selective reactivity, and multistep activation, all from a single host molecule, illustrates the biomimetic nature of the catalysis.

13.
Chemistry ; 25(43): 10232-10238, 2019 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-31087791

RESUMEN

A self-assembled Fe4 L6 cage complex internally decorated with acid functions is capable of accelerating the thioetherification of activated alcohols, ethers and amines by up to 1000-fold. No product inhibition is seen, and effective supramolecular catalysis can occur with as little as 5 % cage. The substrates are bound in the host with up to micromolar affinities, whereas the products show binding that is an order of magnitude weaker. Most importantly, the cage host alters the molecularity of the reaction: whereas the reaction catalyzed by simple acids is a unimolecular, SN 1-type substitution process, the rate of the host-mediated process is dependent on the concentration of nucleophile. The molecularity of the cage-catalyzed reaction is substrate-dependent, and can be up to bimolecular. In addition, the catalysis can be prevented by a large excess of nucleophile, where substrate inhibition dominates, and the use of tritylated anilines as substrates causes a negative feedback loop, whereby the liberated product destroys the catalyst and stops the reaction.

14.
Chemistry ; 25(7): 1740-1745, 2019 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-30427566

RESUMEN

Arrayed complexes of a water-soluble deep cavitand and two fluorescent indicators show selective sensing of anabolic-androgenic steroids in aqueous environments. By combining the host-guest complexes with small amounts of heavy metal ions, discrimination between steroids that vary in structure by only a single π bond is possible. The sensing occurs through a triggered aggregation mechanism, which can be mediated by both the presence of metal ions and the steroids. The use of both "turn-on" and "turn-off" fluorophores is essential for good discrimination. As low as 10 µm steroid can be detected, and the discrimination is selective in steroid samples spiked into human urine.


Asunto(s)
Anabolizantes/orina , Espectrometría de Fluorescencia , Congéneres de la Testosterona/orina , Anabolizantes/química , Técnicas Biosensibles , Análisis Discriminante , Humanos , Metales Pesados/química , Congéneres de la Testosterona/química , Agua/química
15.
J Org Chem ; 84(18): 12000-12008, 2019 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-31449754

RESUMEN

A self-assembled Fe4L6 cage is capable of co-encapsulating multiple carboxylic acid containing guests in its cavity, and these acids can act as cofactors for cage-catalyzed nucleophilic substitutions. The kinetics of the substitution reaction depend on the size, shape, and binding affinity of each of the components, and small structural changes in guest size can have large effects on the reaction. The host is quite promiscuous and is capable of binding multiple guests with micromolar binding affinities while retaining the ability to effect turnover and catalysis. Substrate binding modes vary widely, from simple 1:1 complexes to 1:2 complexes that can show either negative or positive cooperativity, depending on the guest. The molecularity of the dissociative substitution reaction varies, depending on the electrophile leaving group, acid cofactor, and nucleophile size: small changes in the nature of substrate can have large effects on reaction kinetics, all controlled by selective molecular recognition in the cage interior.

16.
Proc Natl Acad Sci U S A ; 113(1): E41-50, 2016 Jan 05.
Artículo en Inglés | MEDLINE | ID: mdl-26607451

RESUMEN

The exocyst complex regulates the last steps of exocytosis, which is essential to organisms across kingdoms. In humans, its dysfunction is correlated with several significant diseases, such as diabetes and cancer progression. Investigation of the dynamic regulation of the evolutionarily conserved exocyst-related processes using mutants in genetically tractable organisms such as Arabidopsis thaliana is limited by the lethality or the severity of phenotypes. We discovered that the small molecule Endosidin2 (ES2) binds to the EXO70 (exocyst component of 70 kDa) subunit of the exocyst complex, resulting in inhibition of exocytosis and endosomal recycling in both plant and human cells and enhancement of plant vacuolar trafficking. An EXO70 protein with a C-terminal truncation results in dominant ES2 resistance, uncovering possible distinct regulatory roles for the N terminus of the protein. This study not only provides a valuable tool in studying exocytosis regulation but also offers a potentially new target for drugs aimed at addressing human disease.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endosomas/metabolismo , Exocitosis , Limoninas/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Membrana Celular/metabolismo , Secuencia Conservada , Evolución Molecular , Humanos , Estructura Secundaria de Proteína
17.
J Am Chem Soc ; 140(42): 13869-13877, 2018 10 24.
Artículo en Inglés | MEDLINE | ID: mdl-30269482

RESUMEN

Simple tuning of a host:guest pair allows selective sensing of different peptide modifications, exploiting orthogonal recognition mechanisms. Excellent selectivity for either lysine trimethylations or alcohol phosphorylations is possible by simply varying the fluorophore guest. The phosphorylation sensor can be modulated by the presence of small (µM) concentrations of metal ions, allowing array-based sensing. Phosphorylation at serine, threonine, and tyrosine can be selectively sensed via discriminant analysis. The phosphopeptide sensing is effective in the presence of small-molecule phosphates such as ATP, which in turn enables the sensor to be employed in continuous optical assays of both serine kinase and tyrosine phosphatase activity. The activity of multiple different kinases can be monitored, and the sensor is capable of detecting the phosphorylation of peptides containing multiple different modifications, including lysine methylations and acetylation. A single deep cavitand can be used as a "one size fits all" sensor that can selectively detect multiple different modifications to oligopeptides, as well as monitoring the function of their post-translational modification writer and eraser enzymes in complex systems.

18.
J Am Chem Soc ; 140(26): 8078-8081, 2018 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-29913069

RESUMEN

Self-assembly of a carboxylic acid-containing ligand into an Fe4L6 iminopyridine cage allows endohedral positioning of the acid groups while maintaining a robust cage structure. The cage is an effective supramolecular catalyst, providing up to 1000-fold rate enhancement of acetal solvolysis. This enhanced reactivity allows a tandem deprotection/cage-to-cage interconversion that cannot be achieved with other acid catalysts. The combination of rate enhancements and sequestration of the reactive function confers both activity and selectivity on the process, mimicking enzymatic behavior.


Asunto(s)
Ácidos Carboxílicos/química , Complejos de Coordinación/síntesis química , Piridinas/química , Catálisis , Complejos de Coordinación/química , Ligandos , Modelos Moleculares , Estructura Molecular
19.
Anal Chem ; 90(3): 1881-1888, 2018 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-29286640

RESUMEN

Lysine methylation in protein is one important epigenetic mechanism that regulates diverse biological processes but is challenging to study due to the large variability in methylation levels and sites. Here, we show that supramolecular hosts such as calixarenes and cucurbiturils can be applied in the background electrolyte (BGE) of capillary electrophoresis (CE) for highly effective separation of post-translationally methylated histone peptides. The molecular recognition event causes a shift in the electrophoretic mobility of the peptide, allowing affinity measurement for binding between the synthetic receptor and various methylated lysine species. Successful separation of the H3 peptides carrying different methylation levels at the K9 position can be achieved using CX4 and CX6 as the BGE additives in CE, enabling monitoring of the activity of the histone lysine demethylase JMJD2E. This reveals the power of combining high resolution CE with synthetic hosts for study of protein methylation, and the method should be capable of analyzing complex biological samples for better understanding of the functions of histone methylation.


Asunto(s)
Calixarenos/química , Electroforesis Capilar/métodos , Histonas/química , Compuestos Macrocíclicos/química , Péptidos/análisis , Electrólitos/química , Humanos , Metilación , Modelos Moleculares , Péptidos/aislamiento & purificación , Procesamiento Proteico-Postraduccional
20.
Inorg Chem ; 57(7): 4155-4163, 2018 Apr 02.
Artículo en Inglés | MEDLINE | ID: mdl-29561595

RESUMEN

A strained, "springloaded" Fe2L3 iminopyridine mesocate shows highly variable reactivity upon postassembly reaction with competitive diamines. The strained assembly is reactive toward transimination in minutes at ambient temperature and allows observation of kinetically trapped intermediates in the self-assembly pathway. When diamines are used that can only form less favored cage products upon full equilibration, trapped ML3 fragments with pendant, "hanging" NH2 groups are selectively formed instead. Slight variations in diamine structure have large effects on the product outcome: less rigid diamines convert the mesocate to more favored self-assembled cage complexes under mild conditions and allow observation of heterocomplex intermediates in the displacement pathway. The mesocate allows control of equilibrium processes and direction of product outcomes via small, iterative changes in added subcomponent structure and provides a method of accessing metal-ligand cage structures not normally observed in multicomponent Fe-iminopyridine self-assembly.

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