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1.
J Am Chem Soc ; 143(37): 15039-15044, 2021 09 22.
Artigo em Inglês | MEDLINE | ID: mdl-34516087

RESUMO

Peptides constrained by intramolecular cross-links, especially stapled α-helices, have emerged as versatile scaffolds for drug development. However, there are fewer examples of similarly constrained scaffolds for other secondary structures. Here, we used a novel computational strategy to identify an optimal staple for antiparallel ß-strands, and then we incorporated that staple within a ß-hairpin peptide. The hairpin uses 4-mercaptoproline as a novel staple component, which contributes to a unique, kinked structure. The stapled hairpins show a high degree of structure in aqueous solution, excellent resistance to degradation in cell lysates, and cytosolic penetration at micromolar concentrations. They also overlay with a unique subset of kinked hairpin motifs at protein-protein interaction interfaces. Thus, these scaffolds represent promising starting points for developing inhibitors of cellular protein-protein interactions.


Assuntos
Peptídeos/síntese química , Prolina/análogos & derivados , Sequência de Aminoácidos , Modelos Moleculares , Peptídeos/química , Prolina/química , Estrutura Secundária de Proteína
2.
J Org Chem ; 86(24): 18287-18291, 2021 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-34851645

RESUMO

Thioamide substitution of backbone peptide bonds can probe interactions along the main chain of proteins. Despite theoretical predictions of the enhanced hydrogen bonding propensities of thioamides, previous studies often do not consider the geometric constraints imposed by folded peptide secondary structure. This work addresses drawbacks in previous studies that ignored the geometry dependence and local dielectric properties of thioamide hydrogen bonding and identifies cases where thioamides may be either stronger or weaker hydrogen-bonding partners than amides.


Assuntos
Peptídeos , Tioamidas , Amidas , Ligação de Hidrogênio , Estrutura Secundária de Proteína
3.
Chemistry ; 26(35): 7881-7888, 2020 Jun 23.
Artigo em Inglês | MEDLINE | ID: mdl-32315472

RESUMO

Fast magic-angle spinning (MAS), frequency selective (FS) heteronuclear multiple quantum coherence (HMQC) experiments which function in an analogous manner to solution SOFAST HMQC NMR experiments, are demonstrated. Fast MAS enables efficient FS excitation of 1 H solid-state NMR signals. Selective excitation and observation preserves 1 H magnetization, leading to a significant shortening of the optimal inter-scan delay. Dipolar and scalar 1 H{14 N} FS HMQC solid-state NMR experiments routinely provide 4- to 9-fold reductions in experiment times as compared to conventional 1 H{14 N} HMQC solid-state NMR experiments. 1 H{14 N} FS resonance-echo saturation-pulse double-resonance (RESPDOR) allowed dipolar dephasing curves to be obtained in minutes, enabling the rapid determination of NH dipolar coupling constants and internuclear distances. 1 H{14 N} FS RESPDOR was used to assign multicomponent active pharmaceutical ingredients (APIs) as salts or cocrystals. FS HMQC also provided enhanced sensitivity for 1 H{17 O} and 1 H{35 Cl} HMQC experiments on 17 O-labeled Fmoc-alanine and histidine hydrochloride monohydrate, respectively. FS HMQC and FS RESPDOR experiments will provide access to valuable structural constraints from materials that are challenging to study due to unfavorable relaxation times or dilution of the nuclei of interest.


Assuntos
Histidina/química , Núcleo Celular/química , Espectroscopia de Ressonância Magnética , Prótons
4.
Angew Chem Int Ed Engl ; 59(43): 19275-19281, 2020 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-33448542

RESUMO

For many years, Clar's aromatic sextet theory has served as a qualitative method for assessing the aromatic character of polycyclic aromatic hydrocarbons. A new approach, based on the calculation of isotropic magnetic shielding (IMS) contour plots, is shown to provide a feature-rich picture of aromaticity that is both quantitative yet still easily interpreted. Chemists are visual creatures who are adept at discerning reactivity and chemical behavior from molecular structures. To quote Roald Hoffmann, "People like pictures. Chemists live off them." Thus, the detailed image analysis we present simultaneously provides quantitative assessment of electronic structure, which is still easy-to-understand through visual inspection, embedded in an aesthetically appealing and intuitive picture that draws the reader in. We provide novel computed IMS contour plots for a representative selection of aromatic molecules. Where Clar's static drawings capture only a partial sketch of the electronic properties of a molecule, IMS contour plots present a detailed, global landscape of a molecule that sums all possible resonance structures. This novel analysis allows us to correct certain drawbacks of Clar's analysis with respect to polycyclic aromatics and quantitatively assess the bonding and electronic structure of acene hydrocarbons.

5.
J Am Chem Soc ; 141(1): 441-450, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30525547

RESUMO

The ubiquity of oxygen in organic, inorganic, and biological systems has stimulated the application and development of 17O solid-state NMR spectroscopy as a probe of molecular structure and dynamics. Unfortunately, 17O solid-state NMR experiments are often hindered by a combination of broad NMR signals and low sensitivity. Here, it is demonstrated that fast MAS and proton detection with the D-RINEPT pulse sequence can be generally applied to enhance the sensitivity and resolution of 17O solid-state NMR experiments. Complete 2D 17O → 1H D-RINEPT correlation NMR spectra were typically obtained in less than 10 h from less than 10 mg of material, with low to moderate 17O enrichment (less than 20%). Two-dimensional 1H-17O correlation solid-state NMR spectra allow overlapping oxygen sites to be resolved on the basis of proton chemical shifts or by varying the mixing time used for 1H-17O magnetization transfer. In addition, J-resolved or separated local field (SLF) blocks can be incorporated into the D-RINEPT pulse sequence to allow the direct measurement of one-bond 1H-17O scalar coupling constants (1 JOH) or 1H-17O dipolar couplings ( DOH), respectively, the latter of which can be used to infer 1H-17O bond lengths. 1 JOH and DOH calculated from plane-wave density functional theory (DFT) show very good agreement with experimental values. Therefore, the 2D 1H-17O correlation experiments, 1H-17O scalar and dipolar couplings, and plane-wave DFT calculations provide a method to precisely determine proton positions relative to oxygen atoms. This capability opens new opportunities to probe interactions between oxygen and hydrogen in a variety of chemical systems.

6.
J Org Chem ; 84(4): 2346-2350, 2019 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-30681336

RESUMO

A strategy to control the oxidation potential of catechol using borinic acids is presented. Borinic acids reversibly bind catechol to form boron "ate" complexes (BACs) that alter the electron density on the oxygen atoms of catechol and, in turn, the propensity of the catechol toward electrochemical oxidation. The effect of different substituents on the borinic acid are investigated to determine their efficacy in tuning the electron density within the BAC and the resulting oxidation potential.

7.
Org Biomol Chem ; 17(5): 1076-1080, 2019 01 31.
Artigo em Inglês | MEDLINE | ID: mdl-30534794

RESUMO

Excited-state intramolecular proton transfer involves a photochemical isomerization and creates the opportunity for the emission of two distinct wavelengths of light from a single fluorophore. The selectivity between these two wavelengths of emission is dependent on the environment around the fluorophore and suggests the possibility for ratiometric monitoring of protein microenvironments. Unfortunately, nonspecific binding of ESIPT fluorophores does not often lead to dramatic changes in the ratio between the two wavelengths of emission. A protein binding pocket was designed to selectively discriminate between the two channels of emission available to an ESIPT fluorophore. This work is significant because it demonstrates that specific interactions between the protein and the fluorophore are essential to realize strong ratiometric differences between the two possible wavelengths of emission. The design strategies proposed here lead to an ESIPT fluorophore that can discern subtle differences in the interface between two proteins.


Assuntos
Proteínas/química , Prótons , Sítios de Ligação , Fluorescência , Corantes Fluorescentes/química , Ligação de Hidrogênio , Ligação Proteica , Espectrometria de Fluorescência
8.
Phys Chem Chem Phys ; 21(22): 11608-11614, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31070652

RESUMO

The aromatic character of an arene is proposed to switch from aromatic in the ground state (S0) to antiaromatic in the S1 and T1 excited states. This behavior is known as Baird's rule and has been invoked to explain excited-state properties, primarily in the triplet state, whereas rationalization of antiaromaticity in the singlet state is less developed. This work demonstrates the first application of Baird's rule to rationalize previously unexplained experimental behavior of the singlet state process known as excited-state intramolecular proton transfer (ESIPT). Further, by analyzing the variations in isotropic magnetic shielding around the base arenes (benzene and naphthalene) of ESIPT fluorophores in the S0 and S1 electronic states, different shielding distributions indicate a complementarity to Baird's rule: greater aromaticity in S0 leads to greater antiaromaticity in S1 and vice versa. These findings have immediate application in the design of functional ESIPT fluorophores and, more generally, for photochemical reactions that are driven by the relief of antiaromaticity in the excited state. Notably, a tenet of traditional chromophore design states that expansion of conjugation generally leads to a red-shift in absorbance and emission wavelengths. The results of this study show that ESIPT fluorophores run contrary to those conventional design principles and this behavior can only be rationalized by considering Baird's rule.

9.
J Org Chem ; 83(17): 9850-9857, 2018 09 07.
Artigo em Inglês | MEDLINE | ID: mdl-30024169

RESUMO

The delocalization of electron density upon tautomerization of a proton across a conjugated bridge can alter the strength of hydrogen bonds. This effect has been dubbed resonance-assisted hydrogen bonding (RAHB) and plays a major role in the energetics of the tautomeric equilibrium. The goal of this work was to investigate the role that π-delocalization plays in the stability of RAHBs by engaging other isomerization processes. Similarly, acid-base chemistry has received little experimental attention in studies of RAHB, and we address the role that acid-base effects play in the tautomeric equilibrium. We find that π-delocalization and the disruption of adjacent aromatic rings is the dominant effect in determining the stability of a RAHB.

10.
Chem Sci ; 2024 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-39430930

RESUMO

Self-labeling proteins are powerful tools for exploring biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye. HaloTag7 is the most popular self-labeling protein due to its broad utility, its bio-orthogonality, and the simplicity of its chloroalkane ligand. However, reaction rates of HaloTag7 with different chloroalkane-containing substrates are highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we use yeast surface display to produce a HaloTag variant, BenzoTag, with improved performance with a fluorogenic benzothiadiazole dye. Molecular evolution improved conjugation kinetics and increased the signal from the dye-protein complex, allowing for robust, no-wash fluorescence labeling in live cells. The new BenzoTag-benzothiadiazole system has improved performance compared to the best existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. The BenzoTag system was also found to be sufficiently orthogonal to the HaloTag7-silicon rhodamine system to enable multiplexed no-wash labeling in live cells. The BenzoTag system will be immediately useful for a large variety of cell-based assays monitoring biological processes and drug action in real time.

11.
bioRxiv ; 2024 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-38617361

RESUMO

Self-labeling proteins are powerful tools in chemical biology as they enable the precise cellular localization of a synthetic molecule, often a fluorescent dye, with the genetic specificity of a protein fusion. HaloTag7 is the most popular self-labeling protein due to its fast labeling kinetics and the simplicity of its chloroalkane ligand. Reaction rates of HaloTag7 with different chloroalkane-containing substrates is highly variable and rates are only very fast for rhodamine-based dyes. This is a major limitation for the HaloTag system because fast labeling rates are critical for live-cell assays. Here, we report a molecular evolution system for HaloTag using yeast surface display that enables the screening of libraries up to 108 variants to improve reaction rates with any substrate of interest. We applied this method to produce a HaloTag variant, BenzoHTag, which has improved performance with a fluorogenic benzothiadiazole dye. The resulting system has improved brightness and conjugation kinetics, allowing for robust, no-wash fluorescent labeling in live cells. The new BenzoHTag-benzothiadiazole system has improved performance in live-cell assays compared to the existing HaloTag7-silicon rhodamine system, including saturation of intracellular enzyme in under 100 seconds and robust labeling at dye concentrations as low as 7 nM. It was also found to be orthogonal to the silicon HaloTag7-rhodamine system, enabling multiplexed no-wash labeling in live cells. The BenzoHTag system, and the ability to optimize HaloTag for a broader collection of substrates using molecular evolution, will be very useful for the development of cell-based assays for chemical biology and drug development.

12.
Chem Commun (Camb) ; 60(2): 200-203, 2023 Dec 21.
Artigo em Inglês | MEDLINE | ID: mdl-38048049

RESUMO

Recent years have seen dramatic improvements in the design of organic fluorophores based on limiting non-radiative decay pathways. We sought to extend this understanding to benzothiadiazoles that have been used as turn-on fluorescent substrates for the self-labeling protein HaloTag. When conjugated to HaloTag, the benzothiadiazoles reside in a narrow tunnel that precludes twisted internal charge transfer, which allowed us to explore steric and electronic effects on other non-radiative decay pathways. By minimizing both non-radiative decay and nonspecific interactions with cellular components, we produced improved turn-on dyes with 136-fold increase in fluorescence over background in cells.


Assuntos
Hidrolases , Proteínas , Corantes Fluorescentes/metabolismo , Microscopia de Fluorescência , Ligantes
13.
Org Lett ; 22(1): 270-273, 2020 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-31846343

RESUMO

An unexpected nucleophilic aromatic substitution lead to a novel benzothiadiazole scaffold that bore the functional group pattern associated with benzyl-type photocleavable protecting groups. The new molecules display efficient photochemical release of leaving groups with blue light. The performance of both ortho- and meta-substituted derivatives was probed through both structural manipulation and computational metrics to improve performance.

14.
Org Lett ; 21(17): 7015-7018, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31403302

RESUMO

Thioamides are useful biophysical probes for the study of peptide structure and folding. The α-C stereochemistry of thioamide amino acids, however, is easily epimerized during solid-phase peptide synthesis (SPPS), which limits the sequence space that is available to thioamide incorporation. This work demonstrates that the α-C stereochemistry of thioamides can be reversibly protected in a manner that is compatible with the standard methodology of SPPS to enable the facile implementation of thioamide probes.


Assuntos
Peptídeos/síntese química , Técnicas de Síntese em Fase Sólida , Tioamidas/química , Estrutura Molecular , Peptídeos/química , Estereoisomerismo
15.
Org Lett ; 21(10): 3817-3821, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31038316

RESUMO

A new class of push-pull dyes based on the reactive isobenzofuran core have been synthesized. The new dyes have a smaller HOMO-LUMO gap than a related class of dyes based on benzofurazan and allow for isolation of structural factors that contribute to environmental sensitivity. Experimental and theoretical evidence implicate different photophysical processes are responsible for a reversal of emissive behavior that is observed between isobenzofuran and benzofurazan analogues.

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