Multivalent Fluorinated Nanorings for On-Cell 19F NMR.

The design of imaging agents with a high fluorine content is necessary for overcoming the challenges of low sensitivity in 19F magnetic resonance imaging (MRI)-based molecular imaging. Chemically self-assembled nanorings (CSANs) provide a strategy to increase the fluorine content through multivalent display. We previously reported an 19F NMR-based imaging tracer, in which case a CSAN-compatible epidermal growth factor receptor (EGFR)-targeting protein E1-dimeric dihydrofolate (E1-DD) was bioconjugated to a highly fluorinated peptide. Despite good 19F NMR performance in aqueous solutions, a limited signal was observed in cell-based 19F NMR using this monomeric construct, motivating further design. Here, we design several new E1-DD proteins bioconjugated to peptides of different fluorine contents. Flow cytometry analysis was used to assess the effect of variable fluorinated peptide sequences on the cellular binding characteristics. Structure-optimized protein, RTC-3, displayed an optimal spectral performance with high affinity and specificity for EGFR-overexpressing cells. To further improve the fluorine content, we next engineered monomeric RTC-3 into CSAN, η-RTC-3. With an approximate eightfold increase in the fluorine content, multivalent η-RTC-3 maintained high cellular specificity and optimal 19F NMR spectral behavior. Importantly, the first cell-based 19F NMR spectra of η-RTC-3 were obtained bound to EGFR-expressing A431 cells, showing a significant amplification in the signal. This new design illustrated the potential of multivalent fluorinated CSANs for future 19F MRI molecular imaging applications.

Fluorinated Pharmaceutical and Pesticide Photolysis: Investigating Reactivity and Identifying Fluorinated Products by Combining Computational Chemistry,19F NMR, and Mass Spectrometry.

Fluorinated breakdown products from photolysis of pharmaceuticals and pesticides are of environmental concern due to their potential persistence and toxicity. While mass spectrometry workflows have been shown to be useful in identifying products, they fall short for fluorinated products and may miss up to 90% of products. Studies have shown that 19F NMR measurements assist in identifying and quantifying reaction products, but this protocol can be further developed by incorporating computations. Density functional theory was used to compute 19F NMR shifts for parent and product structures in photolysis reactions. Computations predicted NMR spectra of compounds with an R2 of 0.98. Computed shifts for several isolated product structures from LC-HRMS matched the experimental shifts with <0.7 ppm error. Multiple products including products that share the same shift that were not previously reported were identified and quantified using computational shifts, including aliphatic products in the range of -80 to -88 ppm. Thus, photolysis of fluorinated pharmaceuticals and pesticides can result in compounds that are polyfluorinated alkyl substances (PFAS), including aliphatic-CF3 or vinyl-CF2 products derived from heteroaromatic-CF3 groups. C-F bond-breaking enthalpies and electron densities around the fluorine motifs agreed well with the experimentally observed defluorination of CF3 groups. Combining experimental-computational 19F NMR allows quantification of products identified via LC-HRMS without the need for authentic standards. These results have applications for studies of environmental fate and analysis of fluorinated pharmaceuticals and pesticides in development.

Recent progress and structural analyses of domain-selective BET inhibitors.

Epigenetic mechanisms for controlling gene expression through heritable modifications to DNA, RNA, and proteins, are essential processes in maintaining cellular homeostasis. As a result of their central role in human diseases, the proteins responsible for adding, removing, or recognizing epigenetic modifications have emerged as viable drug targets. In the case of lysine-ε-N-acetylation (Kac ), bromodomains serve as recognition modules ("readers") of this activating epigenetic mark and competition of the bromodomain-Kac interaction with small-molecule inhibitors is an attractive strategy to control aberrant bromodomain-mediated gene expression. The bromodomain and extra-terminal (BET) family proteins contain eight similar bromodomains. These BET bromodomains are among the more commonly studied bromodomain classes with numerous pan-BET inhibitors showing promising anticancer and anti-inflammatory efficacy. However, these results have yet to translate into Food and Drug Administration-approved drugs, in part due to a high degree of on-target toxicities associated with pan-BET inhibition. Improved selectivity within the BET-family has been proposed to alleviate these concerns. In this review, we analyze the reported BET-domain selective inhibitors from a structural perspective. We highlight three essential characteristics of the reported molecules in generating domain selectivity, binding affinity, and mimicking Kac molecular recognition. In several cases, we provide insight into the design of molecules with improved specificity for individual BET-bromodomains. This review provides a perspective on the current state of the field as this exciting class of inhibitors continue to be evaluated in the clinic.

Fluorous Liquids for Magnetic Resonance-Based Thermometry with Enhanced Responsiveness and Environmental Degradation.

Accurate temperature measurement via magnetic resonance is valuable for both in vitro and in vivo analysis of local tissue for evaluating disease pathology and medical interventions. 1H MRI-based thermometry is used clinically but is susceptible to error from magnetic field drift and low sensitivity in fatty tissue and requires a reference for absolute temperature determination. As an alternative, perfluorotributylamine (PFTBA), a perfluorocarbon liquid for 19F MRI thermometry, is based on chemical shift responsiveness and approaches the sensitivity of 1H MRI thermometry agents; however, environmental persistence, greenhouse gas concerns, and multiple resonances which can lead to MRI artifacts indicate a need for alternative sensors. Using a 19F NMR-based structure-property study of synthetic organofluorine molecules, this research develops new organofluorine liquids with improved temperature responsiveness, high signal, and reduced nonmagnetically equivalent fluorine resonances. Environmental degradation analysis using reverse-phase HPLC and quantitative 19F NMR demonstrates a rapid degradation profile mediated via the aryl fluorine core of temperature sensors. Our findings show that our lead liquid temperature sensor, DD-1, can be made in high yield in a single step and possesses an improved responsiveness over our prior work and an 83% increase in aqueous thermal responsiveness over PFTBA. Degradation studies indicate robust degradation with half-lives of less than two hours under photolysis conditions for the parent compound and formation of other fluorinated products. The improved performance of DD-1 and its susceptibility to environmental degradation highlight a new lead fluorous liquid for thermometry applications.

Garcinolic Acid Distinguishes Between GACKIX Domains and Modulates Interaction Networks.

Natural products are often uniquely suited to modulate protein-protein interactions (PPIs) due to their architectural and functional group complexity relative to synthetic molecules. Here we demonstrate that the natural product garcinolic acid allosterically blocks the CBP/p300 KIX PPI network and displays excellent selectivity over related GACKIX motifs. It does so via a strong interaction (KD 1 µM) with a non-canonical binding site containing a structurally dynamic loop in CBP/p300 KIX. Garcinolic acid engages full-length CBP in the context of the proteome and in doing so effectively inhibits KIX-dependent transcription in a leukemia model. As the most potent small-molecule KIX inhibitor yet reported, garcinolic acid represents an important step forward in the therapeutic targeting of CBP/p300.

Design of Highly Fluorinated Peptides for Cell-based 19F NMR.

The design of imaging agents with high fluorine content is essential for overcoming the challenges associated with signal detection limits in 19F MRI-based molecular imaging. In addition to perfluorocarbon and fluorinated polymers, fluorinated peptides offer an additional strategy for creating sequence-defined 19F magnetic resonance imaging (MRI) imaging agents with a high fluorine signal. Our previously reported unstructured trifluoroacetyllysine-based peptides possessed good physiochemical properties and could be imaged at high magnetic field strength. However, the low detection limit motivated further improvements in the fluorine content of the peptides as well as removal of nonspecific cellular interactions. This research characterizes several new highly fluorinated synthetic peptides composed of highly fluorinated amino acids. 19F NMR analysis of peptides TB-1 and TB-9 led to highly overlapping, intense fluorine resonances and acceptable aqueous solubility. Flow cytometry analysis and fluorescence microscopy further showed nonspecific binding could be removed in the case of TB-9. As a preliminary experiment toward developing molecular imaging agents, a fluorinated EGFR-targeting peptide (KKKFFKK-ßA-YHWYGYTPENVI) and an EGFR-targeting protein complex E1-DD bioconjugated to TB-9 were prepared. Both bioconjugates maintained good 19F NMR performance in aqueous solution. While the E1-DD-based imaging agent will require further engineering, the success of cell-based 19F NMR of the EGFR-targeting peptide in A431 cells supports the potential use of fluorinated peptides for molecular imaging.

Wavelength-Dependent UV-LED Photolysis of Fluorinated Pesticides and Pharmaceuticals.

The wavelength dependence of photoproduct formation and quantum yields was evaluated for fluorinated pesticides and pharmaceuticals using UV-light emitting diodes (LEDs) with 255, 275, 308, 365, and 405 nm peak wavelengths. The fluorinated compounds chosen were saflufenacil, penoxsulam, sulfoxaflor, fluoxetine, 4-nitro-3-trifluoromethylphenol (TFM), florasulam, voriconazole, and favipiravir, covering key fluorine motifs (benzylic-CF3, heteroaromatic-CF3, aryl-F, and heteroaromatic-F). Quantum yields for the compounds were consistently higher for UV-C as compared to UV-A wavelengths and did not show the same trend as molar absorptivity. For all compounds except favipiravir and TFM, the fastest degradation was observed using 255 or 275 nm light, despite the low power of the LEDs. Using quantitative 19F NMR, fluoride, trifluoroacetate, and additional fluorinated byproducts were tracked and quantified. Trifluoroacetate was observed for both Ar-CF3 and Het-CF3 motifs and increased at longer wavelengths for Het-CF3. Fluoride formation from Het-CF3 was significantly lower as compared to other motifs. Ar-F and Het-F motifs readily formed fluoride at all wavelengths. For Het-CF3 and some Ar-CF3 motifs, 365 nm light produced either a greater number of or different major products. Aliphatic-CF2/CF3 products were stable under all wavelengths. These results assist in selecting the most efficient wavelengths for UV-LED degradation and informing future design of fluorinated compounds.

Alternative Mechanisms for DNA Engagement by BET Bromodomain-Containing Proteins.

Epigenetic reader domains regulate chromatin structure and modulate gene expression through the recognition of post-translational modifications on histones. Recently, reader domains have also been found to harbor double-stranded (ds) DNA-binding activity, which is as functionally critical as histone association. Here, we explore the dsDNA recognition of the N-terminal bromodomain of the bromodomain and extra-terminal (BET) protein, BRD4. Using protein-observed 19F NMR, 1H-15N HSQC NMR, electrophoretic mobility shift assays (EMSA), and competitive-inhibition assays, we establish the binding surface of dsDNA and find it to be largely overlapping with the acetylated histone (KAc)-binding site. Rather than engaging in electrostatic contacts, we find dsDNA to interact competitively within the KAc-binding pocket. These interactions are distinct from the highly homologous BET bromodomain, BRDT. Nine additional bromodomains have also been characterized for interacting with dsDNA, including tandem BET bromodomains. Together, these studies help establish a binding model for dsDNA interactions with BRD4 bromodomains and elucidate the chromatin-recognition mechanisms of the BRD4 protein for regulating gene expression.

Development of a Highly Responsive Organofluorine Temperature Sensor for 19F Magnetic Resonance Applications.

Temperature can affect many biological and chemical processes within a body. During in vivo measurements, varied temperature can impact the accurate quantification of additional abiotic factors such as oxygen. During magnetic resonance imaging (MRI) measurements, the temperature of the sample can increase with the absorption of radiofrequency energy, which needs to be well-regulated for thermal therapies and long exposure. To address this potentially confounding effect, temperature can be probed intentionally using reporter molecules to determine the temperature in vivo. This work describes a combined experimental and computational approach for the design of fluorinated molecular temperature sensors with the potential to improve the accuracy and sensitivity of 19F MRI-based temperature monitoring. These fluorinated sensors are being developed to overcome the temperature sensitivity and tissue limitations of the proton resonance frequency (10 × 10-3 ppm °C-1), a standard parameter used for temperature mapping in MRI. Here, we develop (perfluoro-[1,1'-biphenyl]-4,4'-diyl)bis((heptadecafluorodecyl)sulfane), which has a nearly 2-fold increase in temperature responsiveness, compared to the proton resonance frequency and the 19F MRI temperature sensor perfluorotributylamine, when tested under identical NMR conditions. While 19F MRI is in the early stages of translation into clinical practice, development of alternative sensors with improved diagnostic abilities will help advance the development and incorporation of fluorine magnetic resonance techniques for clinical use.

Endothelial p300 Promotes Portal Hypertension and Hepatic Fibrosis Through C-C Motif Chemokine Ligand 2-Mediated Angiocrine Signaling.

BACKGROUND AND AIMS: During liver fibrosis, liver sinusoidal endothelial cells (LSECs) release angiocrine signals to recruit inflammatory cells into the liver. p300, a master regulator of gene transcription, is associated with pathological inflammatory response. Therefore, we examined how endothelial p300 regulates angiocrine signaling and inflammation related to portal hypertension and fibrogenesis. APPROACH AND RESULTS: CCl4 or partial inferior vena cava ligation (pIVCL) was used to induce liver injury. Mice with LSEC-specific p300 deletion (p300LSECΔ/Δ ) or C-C motif chemokine ligand 2 (Ccl2) deficiency, nuclear factor kappa B (NFκB)-p50 knockout mice, and bromodomain containing 4 (BRD4) inhibitors in wild-type mice were used to investigate mechanisms of inflammation regulation. Leukocytes were analyzed by mass cytometry by time-of-flight. Epigenetic histone marks were modified by CRISPR endonuclease-deficient CRISPR-associated 9-fused with the Krüppel associated box domain (CRISPR-dCas9-KRAB)-mediated epigenome editing. Portal pressure and liver fibrosis were reduced in p300LSECΔ/Δ mice compared to p300fl/fl mice following liver injury. Accumulation of macrophages was also reduced in p300LSECΔ/Δ mouse livers. Ccl2 was the most up-regulated chemokine in injured LSECs, but its increase was abrogated in p300LSECΔ/Δ mice. While the macrophage accumulation was increased in NFκB-p50 knockout mice with enhanced NFκB activity, it was reduced in mice with LSEC-specific Ccl2 deficiency and mice treated with specific BRD4 inhibitors. In vitro, epigenome editing of CCL2 enhancer and promoter regions by CRISPR-dCas9-KRAB technology repressed TNFα-induced CCL2 transcription through H3K9 trimethylation. In contrast, TNFα activated CCL2 transcription by promoting p300 interaction with NFκB and BRD4, leading to histone H3 lysine 27 acetylation at CCL2 enhancer and promoter regions. CONCLUSIONS: In summary, endothelial p300 interaction with NFκB and BRD4 increases CCL2 expression, leading to macrophage accumulation, portal hypertension, and liver fibrosis. Inhibition of p300 and its binding partners might serve as therapy in the treatment of liver diseases.

Finding Fluorine: Photoproduct Formation during the Photolysis of Fluorinated Pesticides.

The photolysis of pesticides with different fluorine motifs was evaluated to quantify the formation of fluorinated products in buffered aqueous systems, advanced oxidation (AOP) and reduction processes (ARP), and river water. Simulated sunlight quantum yields at pH 7 were 0.0033, 0.0025, 0.0015, and 0.00012 for penoxsulam, florasulam, sulfoxaflor, and fluroxypyr, respectively. The bimolecular rate constants with hydroxyl radicals were 2 to 5.7 × 1010 M-1 s-1 and, with sulfate radicals, 1.6 to 2.6 × 108 M-1 s-1 for penoxsulam, florasulam, and fluroxypyr, respectively. The rate constants of sulfoxaflor were 100-fold lower. Using quantitative 19F-NMR, complete fluorine mass balances were obtained. The maximum fluoride formation was 53.4 and 87.4% for penoxsulam and florasulam under ARP conditions, and 6.1 and 100% for sulfoxaflor and fluroxypyr under AOP conditions. Heteroaromatic CF3 and aliphatic CF2 groups were retained in multiple fluorinated photoproducts. Aryl F and heteroaromatic F groups were readily defluorinated to fluoride. CF3 and CF2 groups formed trifluoroacetate and difluoroacetate, and yields increased under oxidizing conditions. 19F-NMR chemical shifts and coupling analysis provided information on hydrogen loss on adjacent bonds or changes in chirality. Mass spectrometry results were consistent with the observed 19F-NMR products. These results will assist in selecting treatment processes for specific fluorine motifs and in the design of agrochemicals to reduce byproduct formation.

Development of a single culture E. coli expression system for the enzymatic synthesis of fluorinated tyrosine and its incorporation into proteins.

Current experiments that rely on biosynthetic metabolic protein labeling with 19F often require fluorinated amino acids, which in the case of 2- and 3-fluorotyrosine can be expensive. However, using these amino acids has provided valuable insight into protein dynamics, structure, and function. Here, we develop a new in-cell method for fluorinated tyrosine generation from readily available substituted phenols and subsequent metabolic labeling of proteins in a single bacterial expression culture. This approach uses a dual-gene plasmid encoding for a model protein BRD4(D1) and a tyrosine phenol lyase from Citrobacter freundii, which catalyzes the formation of tyrosine from phenol, pyruvate, and ammonium. Our system demonstrated both enzymatic fluorotyrosine production and expression of 19F-labeled proteins as analyzed by 19F NMR and LC-MS methods. Further optimization of our system should provide a cost-effective alternative to a variety of traditional protein-labeling strategies.

Soluble Methane Monooxygenase Component Interactions Monitored by 19F NMR.

Soluble methane monooxygenase (sMMO) is a multicomponent metalloenzyme capable of catalyzing the fissure of the C-H bond of methane and the insertion of one atom of oxygen from O2 to yield methanol. Efficient multiple-turnover catalysis occurs only in the presence of all three sMMO protein components: hydroxylase (MMOH), reductase (MMOR), and regulatory protein (MMOB). The complex series of sMMO protein component interactions that regulate the formation and decay of sMMO reaction cycle intermediates is not fully understood. Here, the two tryptophan residues in MMOB and the single tryptophan residue in MMOR are converted to 5-fluorotryptophan (5FW) by expression in defined media containing 5-fluoroindole. In addition, the mechanistically significant N-terminal region of MMOB is 19F-labeled by reaction of the K15C variant with 3-bromo-1,1,1-trifluoroacetone (BTFA). The 5FW and BTFA modifications cause minimal structural perturbation, allowing detailed studies of the interactions with sMMOH using 19F NMR. Resonances from the 275 kDa complexes of sMMOH with 5FW-MMOB and BTFA-K15C-5FW-MMOB are readily detected at 5 µM labeled protein concentration. This approach shows directly that MMOR and MMOB competitively bind to sMMOH with similar KD values, independent of the oxidation state of the sMMOH diiron cluster. These findings suggest a new model for regulation in which the dynamic equilibration of MMOR and MMOB with sMMOH allows a transient formation of key reactive complexes that irreversibly pull the reaction cycle forward. The slow kinetics of exchange of the sMMOH:MMOB complex is proposed to prevent MMOR-mediated reductive quenching of the high-valent reaction cycle intermediate Q before it can react with methane.

Investigation of the Post-Synthetic Confinement of Fluorous Liquids Inside Mesoporous Silica Nanoparticles.

Perfluorocarbon (PFC) filled nanoparticles are increasingly being investigated for various biomedical applications. Common approaches for PFC liquid entrapment involve surfactant-based emulsification and Pickering emulsions. Alternatively, PFC liquids are capable of being entrapped inside hollow nanoparticles via a postsynthetic loading method (PSLM). While the methodology for the PSLM is straightforward, the effect each loading parameter has on the PFC entrapment has yet to be investigated. Previous work revealed incomplete filling of the hollow nanoparticles. Changing the loading parameters was expected to influence the ability of the PFC to fill the core of the nanoparticles. Hence, it would be possible to model the loading mechanism and determine the influence each factor has on PFC entrapment by tracking the change in loading yield and efficiency of PFC-filled nanoparticles. Herein, neat PFC liquid was loaded into silica nanoparticles and extracted into aqueous phases while varying the sonication time, concentration of nanoparticles, volume ratio between aqueous and fluorous phases, and pH of the extraction water. Loading yields and efficiency were determined via 19F nuclear magnetic resonance and N2 physisorption isotherms. Sonication time was indicated to have the strongest correlation to loading yield and efficiency; however, method validation revealed that the current model does not fully explain the loading capabilities of the PSLM. Confounding variables and more finely controlled parameters need to be considered to better predict the behavior and loading capacity by the PSLM and warrants further study.

Fragment-Based Ligand Discovery Using Protein-Observed 19F NMR: A Second Semester Organic Chemistry CURE Project.

Curriculum-based undergraduate research experiences (CUREs) have been shown to increase student retention in STEM fields and are starting to become more widely adopted in chemistry curricula. Here we describe a 10-week CURE that is suitable for a second-semester organic chemistry laboratory course. Students synthesize small molecules and use protein-observed 19F (PrOF) NMR to assess the small molecule's binding affinity to a target protein. The research project introduced students to multistep organic synthesis, structure-activity relationship studies, quantitative biophysical measurements (measuring Kd from PrOF NMR experiments), and scientific literacy. Docking experiments could be added to help students understand how changes in a ligand structure may affect binding to a protein. Assessment using the CURE survey indicates self-perceived skill gains from the course that exceed gains measured in a traditional and an inquiry-based laboratory experience. Given the speed of the binding experiment and the alignment of the synthetic methods with a second-semester organic chemistry laboratory course, a PrOF NMR fragment-based ligand discovery lab can be readily implemented in the undergraduate chemistry curriculum.

Site-Specific 5-Formyl Cytosine Mediated DNA-Histone Cross-Links: Synthesis and Polymerase Bypass by Human DNA Polymerase η.

DNA-protein cross-links (DPCs) between DNA epigenetic mark 5-formylC and lysine residues of histone proteins spontaneously form in human cells. Such conjugates are likely to influence chromatin structure and mediate DNA replication, transcription, and repair, but are challenging to study due to their reversible nature. Here we report the construction of site specific, hydrolytically stable DPCs between 5fdC in DNA and K4 of histone H3 and an investigation of their effects on DNA replication. Our approach employs oxime ligation, allowing for site-specific conjugation of histones to DNA under physiological conditions. Primer extension experiments revealed that histone H3-DNA crosslinks blocked DNA synthesis by hPol η polymerase, but were bypassed following proteolytic processing.

Selective N-Terminal BET Bromodomain Inhibitors by Targeting Non-Conserved Residues and Structured Water Displacement*.

Bromodomain and extra-terminal (BET) family proteins, BRD2-4 and T, are important drug targets; however, the biological functions of each bromodomain remain ill-defined. Chemical probes that selectively inhibit a single BET bromodomain are lacking, although pan inhibitors of the first (D1), and second (D2), bromodomain are known. Here, we develop selective BET D1 inhibitors with preferred binding to BRD4 D1. In competitive inhibition assays, we show that our lead compound is 9-33 fold selective for BRD4 D1 over the other BET bromodomains. X-ray crystallography supports a role for the selectivity based on reorganization of a non-conserved lysine and displacement of an additional structured water in the BRD4 D1 binding site relative to our prior lead. Whereas pan-D1 inhibitors displace BRD4 from MYC enhancers, BRD4 D1 inhibition in MM.1S cells is insufficient for stopping Myc expression and may lead to its upregulation. Future analysis of BRD4 D1 gene regulation may shed light on differential BET bromodomain functions.

NMR Analyses of Acetylated H2A.Z Isoforms Identify Differential Binding Interactions with the Bromodomain of the NURF Nucleosome Remodeling Complex.

Gene specific recruitment of bromodomain-containing proteins to chromatin is affected by post-translational acetylation of lysine on histones. Whereas interactions of the bromodomain with acetylation patterns of native histones (H2A, H2B, H3, and H4) have been well characterized, the motif for recognition for histone variants H2A.Z I and H2A.Z II by bromodomains has yet to be fully investigated. Elucidating these molecular mechanisms is crucial for understanding transcriptional regulation in cellular processes involved in both development and disease. Here, we have used protein-observed fluorine NMR to fully characterize the affinities of H2A.Z I and II acetylation patterns for BPTF's bromodomain and found the diacetylated mark of lysine 7 and 13 on H2A.Z II to have the strongest interaction with K7ac preferentially engaging the binding site. We further examined the selectivity of H2A.Z histones against a variety of bromodomains, revealing that the bromodomain of CECR2 binds with the highest affinity and specificity for acetylated H2A.Z I over isoform II. These results support a possible role for different H2A.Z transcriptional activation mechanisms that involve recruitment of chromatin remodeling complexes.

2-Fluorotyrosine is a valuable but understudied amino acid for protein-observed 19F NMR.

Incorporation of 19F into proteins allows for the study of their molecular interactions via NMR. The study of 19F labeled aromatic amino acids has largely focused on 4-,5-, or 6-fluorotryptophan, 4-fluorophenylalanine, (4,5, or 6FW) or 3-fluorotyrosine (3FY), whereas 2-fluorotyrosine (2FY) has remained largely understudied. Here we report a comparative analysis with different fluorinated amino acids. We first report the NMR chemical shift responsiveness of five aromatic amino acid mimics to changes in solvent polarity and find that the most responsive, a mimic of 3FY, has a 2.9-fold greater change in chemical shift compared to the other amino acid mimics in aprotic solvents including the 2FY mimic. We also probed the utility of 2FY for 19F NMR by measuring its NMR relaxation properties in solution and the chemical shift anisotropy (CSA) of a polycrystalline sample of the amino acid by magic angle spinning. Using protein-observed fluorine NMR (PrOF NMR), we compared the influence of 2FY and 3FY incorporation on stability and pKa perturbation when incorporated into the KIX domain of CBP/p300. Lastly, we investigated the 19F NMR response of both 2FY and 3FY-labeled proteins to a protein-protein interaction partner, MLL, and discovered that 2FY can report on allosteric interactions that are not observed with 3FY-labeling in this protein complex. The reduced perturbation to pKa and similar but reduced CSA of 2FY to 3FY supports 2FY as a suitable alternative amino acid for incorporation into large proteins for 19F NMR analysis.

Multidimensional Nanoparticle Characterization through Ion Mobility-Mass Spectrometry.

Multidimensional techniques that combine fully or partially orthogonal characterization methods in a single setup often provide a more comprehensive description of analytes. When applied to nanoparticles, they have the potential to reveal particle properties not accessible to more conventional 1D techniques. Herein, we apply recently developed 2D characterization techniques to nanoparticles using atmospheric-pressure ion mobility-mass spectrometry (IM-MS), and we demonstrate the analytical capability of this approach using ultraporous mesostructured silica nanoparticles (UMNs). We show that IM-MS yields a 2D particle size-mass distribution function, which in turn can be used to calculate not only important 1D distributions, i.e. particle size distributions, but also nanoparticle structural property distributions not accessible by other methods, including size-dependent particle porosity and the specific pore volume distribution function. IM-MS measurement accuracy was confirmed by measurement of NIST-certified polystyrene latex particle standards. For UMNs, comparison of IM-MS results with TEM and N2 physisorption yields quantitative agreement in particle size and qualitative agreement in average specific pore volume. IM-MS uniquely shows how within a single UMN population, porosity increases with increasing particle size, consistent with the proposed UMN growth mechanism. In total, we demonstrate the potential of IM-MS as a standard approach for the characterization of structurally complex nanoparticle populations, as it yields size-specific structural distribution functions.