Differentiation of peptide isomers by excited-state photodissociation and ion-molecule interactions.

Solvochromatic effects are most frequently associated with solution-phase phenomena. However, in the gas phase, the absence of solvent leads to intramolecular solvation that can be driven by strong forces including hydrogen bonds and ion-dipole interactions. Here we examine whether isomerization of a single residue in a peptide results in structural changes sufficient to shift the absorption of light by an appended chromophore. By carrying out the experiments inside a mass spectrometer, we can easily monitor photodissociation yield as a readout for chromophore excitation. A series of peptides of different lengths, charge states, and position and identity of the isomerized residue were examined by excitation with both 266 and 213 nm light. The results reveal that differences in intramolecular solvation do lead to solvochromatic shifts in many cases. In addition, the primary product following photoexcitation is a radical. Ion-molecule reactions with this radical and adventitious oxygen were monitored and also found to vary as a function of isomeric state. In this case, differences in intramolecular solvation alter the availability of the reactive radical. Overall, the results reveal that small changes in a single amino acid can influence the overall structural ensemble sufficient to alter the efficiency of multiple gas-phase reactions.

Radionuclide Molecular Imaging of EpCAM Expression in Triple-Negative Breast Cancer Using the Scaffold Protein DARPin Ec1.

Efficient treatment of disseminated triple-negative breast cancer (TNBC) remains an unmet clinical need. The epithelial cell adhesion molecule (EpCAM) is often overexpressed on the surface of TNBC cells, which makes EpCAM a potential therapeutic target. Radionuclide molecular imaging of EpCAM expression might permit selection of patients for EpCAM-targeting therapies. In this study, we evaluated a scaffold protein, designed ankyrin repeat protein (DARPin) Ec1, for imaging of EpCAM in TNBC. DARPin Ec1 was labeled with a non-residualizing [125I]I-para-iodobenzoate (PIB) label and a residualizing [99mTc]Tc(CO)3 label. Both imaging probes retained high binding specificity and affinity to EpCAM-expressing MDA-MB-468 TNBC cells after labeling. Internalization studies showed that Ec1 was retained on the surface of MDA-MB-468 cells to a high degree up to 24 h. Biodistribution in Balb/c nu/nu mice bearing MDA-MB-468 xenografts demonstrated specific uptake of both [125I]I-PIB-Ec1 and [99mTc]Tc(CO)3-Ec1 in TNBC tumors. [125I]I-PIB-Ec1 had appreciably lower uptake in normal organs compared with [99mTc]Tc(CO)3-Ec1, which resulted in significantly (p < 0.05) higher tumor-to-organ ratios. The biodistribution data were confirmed by micro-Single-Photon Emission Computed Tomography/Computed Tomography (microSPECT/CT) imaging. In conclusion, an indirectly radioiodinated Ec1 is the preferable probe for imaging of EpCAM in TNBC.

An Activatable and Switchable Nanoaggregate Probe for Detecting H2 S and Its Application in Mice Brains.

Employing a sequentially activated probe design method, an activatable, switchable and dual-mode probe was designed. This nanoprobe, HSDPP, could be effectively activated by H2 S to form H-type aggregates with green emission; subsequently, the aggregates could bind to mtDNA to form monomers and the emIssion color switched from green to deep-red. We exploited HSDPP to image exogenous and endogenous H2 S in living cells. Of note, for the first time, this novel nanoprobe with an optimal partition coefficient value (LogP=1.269) was successfully applied for tracking the endogenous H2 S upregulation stimulated by cystathionase activator S-adenosyl-L-methionine (SAM) in mice brains. Finally, our work provides an invaluable chemical tool for probing endogenous H2 S upregulation in vitro/vivo and, importantly, affords a prospective design strategy for developing switchable chemosensors to unveil the relationship between biomolecules and DNA in mitochondria in many promising areas.

Physicochemical evaluation of new tetrahydroacridine and iodobenzoic acid hybrids as the next step in the design of potential drugs for treating Alzheimer's disease.

Tacrine derivatives containing iodobenzoic acid were developed as a novel multitarget-directed ligand and find potential application in the treatment of Alzheimer's disease. The aim of this study is to perform a physicochemical profile of this series. Experimental log P and pKa values were determined and compared with those already calculated. The results indicated better values of the tested compounds than the values predicted using computer software. The stability report was obtained using the developed HPLC method. The stability assay in different environment conditions provided information about the photosensitivity of these compounds and a proper method for the storage of this series of compounds.

Analysis of Glutamine Deamidation: Products, Pathways, and Kinetics.

Spontaneous chemical modifications play an important role in human disease and aging at the molecular level. Deamidation and isomerization are known to be among the most prevalent chemical modifications in long-lived human proteins and are implicated in a growing list of human pathologies, but the relatively minor chemical change associated with these processes has presented a long standing analytical challenge. Although the adoption of high-resolution mass spectrometry has greatly aided the identification of deamidation sites in proteomic studies, isomerization (and the isomeric products of deamidation) remain exceptionally challenging to characterize. Herein, we present a liquid chromatography/mass spectrometry-based approach for rapidly characterizing the isomeric products of Gln deamidation using diagnostic fragments that are abundantly produced and capable of unambiguously identifying both Glu and isoGlu. Importantly, the informative fragment ions are produced through orthogonal fragmentation pathways, thereby enabling the simultaneous detection of both isomeric forms while retaining compatibility with shotgun proteomics. Furthermore, the diagnostic fragments associated with isoGlu pinpoint the location of the modified residue. The utility of this technique is demonstrated by characterizing the isomeric products generated during in vitro aging of a series of glutamine-containing peptides. Sequence-dependent product profiles are obtained, and the abundance of deamidation-linked racemization is examined. Finally, comparisons are made between Gln deamidation, which is relatively poorly understood, and asparagine deamidation, which has been more thoroughly studied.

Synthesis, structure and in vitro cytotoxicity testing of some 1,3,4-oxadiazoline derivatives from 2-hydroxy-5-iodobenzoic acid.

The syntheses of nine new 5-iodosalicylic acid-based 1,3,4-oxadiazoline derivatives starting from methyl salicylate are described. These compounds are 2-[4-acetyl-5-methyl-5-(3-nitrophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6a), 2-[4-acetyl-5-methyl-5-(4-nitrophenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6b), 2-(4-acetyl-5-methyl-5-phenyl-4,5-dihydro-1,3,4-oxadiazol-2-yl)-4-iodophenyl acetate, C19H17IN2O4 (6c), 2-[4-acetyl-5-(4-fluorophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate, C19H16FIN2O4 (6d), 2-[4-acetyl-5-(4-chlorophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate, C19H16ClIN2O4 (6e), 2-[4-acetyl-5-(3-bromophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6f), 2-[4-acetyl-5-(4-bromophenyl)-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6g), 2-[4-acetyl-5-methyl-5-(4-methylphenyl)-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6h) and 2-[5-(4-acetamidophenyl)-4-acetyl-5-methyl-4,5-dihydro-1,3,4-oxadiazol-2-yl]-4-iodophenyl acetate (6i). The compounds were characterized by mass, 1H NMR and 13C NMR spectroscopies. Single-crystal X-ray diffraction studies were also carried out for 6c, 6d and 6e. Compounds 6c and 6d are isomorphous, with the 1,3,4-oxadiazoline ring having an envelope conformation, where the disubstituted C atom is the flap. The packing is determined by C-H...O, C-H...π and I...π interactions. For 6e, the 1,3,4-oxadiazoline ring is almost planar. In the packing, Cl...π interactions are observed, while the I atom is not involved in short interactions. Compounds 6d, 6e, 6f and 6h show good inhibiting abilities on the human cancer cell lines KB and Hep-G2, with IC50 values of 0.9-4.5 µM.

Novel tetrahydroacridine derivatives with iodobenzoic acid moiety as multifunctional acetylcholinesterase inhibitors.

New synthesized series of 9-amino-1,2,3,4-tetrahydroacridine derivatives with iodobenzoic acid moiety were studied for their inhibitory activity toward cholinesterase and against ß-amyloid aggregation. All novel molecules 3a-3i interacted with both cholinesterases-acetylcholinesterase and butyrylcholinesterase-delivered nanomolar IC50 values. The structure-activity relationship showed that N-butyl moiety derivatives are stronger inhibitors toward AChE and BuChE than N-ethyl and N-propyl moieties compounds. The most potent compound toward acetylcholinesterase was inhibitor 3f (IC50  = 31.2 nm), and it was more active than reference drug, tacrine (IC50  = 100.2 nm). Compound 3f showed strong inhibition of butyrylcholinesterase (IC50  = 8.0 nm), also higher than tacrine (IC50  = 16.3 nm). In the kinetic studies, compound 3f revealed mixed type of acetylcholinesterase inhibition. The computer modeling was carried out. The most active compound 3f was confirmed as peripheral anionic site inhibitor of acetylcholinesterase. Moreover, molecule 3f inhibited ß-amyloid aggregation (at the concentration 10 µm-24.96% of inhibition, 25 µm-72%, 50 µm-78.44%, and 100 µm-84.92%). Therefore, among all examined, compound 3f is the most promising molecule for further, more detailed research of novel multifunctional agents in the therapy of Alzheimer's disease.

Crystal structures of serum albumins from domesticated ruminants and their complexes with 3,5-diiodosalicylic acid.

Serum albumin (SA) is the most abundant protein in plasma and is the main transporter of molecules in the circulatory system of all vertebrates, with applications in medicine, the pharmaceutical industry and molecular biology. It is known that albumins from different organisms vary in sequence; thus, it is important to know the impact of the amino-acid sequence on the three-dimensional structure and ligand-binding properties. Here, crystal structures of ovine (OSA) and caprine (CSA) serum albumins, isolated from sheep and goat blood, are described, as well those of their complexes with 3,5-diiodosalicylic acid (DIS): OSA-DIS (2.20 Šresolution) and CSA-DIS (1.78 Šresolution). The ligand-free OSA structure was determined in the trigonal space group P3221 at 2.30 Šresolution, while that of CSA in the orthorhombic space group P212121 was determined at 1.94 Šresolution. Both albumins are also capable of crystallizing in the triclinic space group P1, giving isostructural crystals that diffract to around 2.5 Šresolution. A comparison of OSA and CSA with the closely related bovine serum albumin (BSA) shows both similarities and differences in the distribution of DIS binding sites. The investigated serum albumins from domesticated ruminants in their complexes with DIS are also compared with the analogous structures of equine and human serum albumins (ESA-DIS and HSA-DIS). Surprisingly, despite 98% sequence similarity, OSA binds only two molecules of DIS, whereas CSA binds six molecules of this ligand. Moreover, the binding of DIS to OSA and CSA introduced changes in the overall architecture of the proteins, causing not only different conformations of the amino-acid side chains in the binding pockets, but also a significant shift of the whole helices, changing the volume of the binding cavities.

Chemoenzymatic Synthesis and α-Glucosidase Inhibitory Activity of Dimeric Neolignans Inspired by Magnolol.

A chemoenzymatic synthesis of a small library of dimeric neolignans inspired by magnolol (1) is reported. The 2-iodoxybenzoic acid (IBX)-mediated regioselective ortho-hydroxylation of magnolol is described, affording the bisphenols 6 and 7. Further magnolol analogues (12, 13, 15-17, 19-23) were obtained from eugenol (3), tyrosol (4), and homovanillic alcohol (5), through horseradish peroxidase (HRP)-mediated oxidative coupling and regioselective ortho-hydroxylation or ortho-demethylation in the presence of IBX, followed by reductive treatment with Na2S2O4. A chemoselective protection/deprotection of the alcoholic group of 4 and 5 was carried out by lipase-mediated acetylation/deacetylation. The dimeric neolignans, together with 1 and honokiol (2), were evaluated as inhibitors of yeast α-glucosidase, in view of their possible utilization and optimization as antidiabetic drugs. The synthetic analogues of magnolol showed a strong inhibitory activity with IC50 values in the range 0.15-4.1 µM, much lower than those of honokiol and the reference compounds quercetin and acarbose. In particular, a very potent inhibitory activity, with an IC50 of 0.15 µM, was observed for 1,1'-dityrosol-8,8'-diacetate (15), and comparable inhibitory activities were also shown by bisphenols 6 (0.49 µM), 13 (0.50 µM), and 22 (0.86 µM). A kinetic study showed that 15 acts as a competitive inhibitor, with a Ki value of 0.86 µM.

Chemical Genetics Uncovers Novel Inhibitors of Lignification, Including p-Iodobenzoic Acid Targeting CINNAMATE-4-HYDROXYLASE.

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization.

Design and Synthesis of Biaryl DNA-Encoded Libraries.

DNA-encoded library technology (ELT) is a powerful tool for the discovery of new small-molecule ligands to various protein targets. Here we report the design and synthesis of biaryl DNA-encoded libraries based on the scaffold of 5-formyl 3-iodobenzoic acid. Three reactions on DNA template, acylation, Suzuki-Miyaura coupling and reductive amination, were applied in the library synthesis. The three cycle library of 3.5 million diversity has delivered potent hits for phosphoinositide 3-kinase α (PI3Kα).

Photolytic determination of charge state for large proteins and fragments in an ion trap mass spectrometer.

RATIONALE: One of the major shortcomings of linear ion trap mass spectrometers is poor resolution. Failure to resolve isotopic peaks makes charge state determination for large proteins very difficult, hindering the ability to perform top-down proteomics. METHODS: Peptides, proteins and corresponding fragments modified with para-iodobenzoate were trapped and irradiated with 266 nm light from an Nd:YAG laser. Loss of iodine due to photodissociation was then used to assign charge states by measuring the corresponding m/z shifts. RESULTS: Initial experiments on small peptides illustrate the feasibility of the method. Further studies performed on larger proteins in higher charge states yielded similar results, revealing that fragment ions over a significant mass range either remain in or are quickly cooled to the laser overlap region of the ion trap. CONCLUSIONS: Rapid charge state assignment for both whole molecules and collision-induced dissociation (CID) fragments can be obtained by photoactivation of chromophores with labile carbon-iodine bonds.

Synthesis and Structure of Hypervalent Iodine(III) Reagents Containing Phthalimidate and Application to Oxidative Amination Reactions.

A new class of hypervalent iodine reagents containing phthalimidate was synthesized, and structurally characterized by X-ray analysis. The benziodoxole-based reagent displays satisfactory solubility in common organic solvents and is reasonably stable in solution as well as in the solid state. The reagent was used for the oxidative amination of the C(sp(3))-H bond of N,N-dimethylanilines. In addition, the reagent was also applicable to oxidative amination with rearrangement of trialkylamines as well as enamines that were prepared in situ from secondary amines and aldehydes.

Modular synthesis of multisubstituted furans through palladium-catalyzed three-component condensation of alkynylbenziodoxoles, carboxylic acids, and imines.

Mild and regiocontrolled synthesis of a multisubstituted furan is achieved through Pd(OAc)2 -catalyzed room-temperature condensation of an alkynylbenziodoxole, a carboxylic acid, and an enolizable ketimine, which contribute to C1, CO, and C2 fragments, respectively, to the furan skeleton. The reaction tolerates a broad range of functional groups in each of the reaction components, and enables highly modular and flexible synthesis of variously substituted furans. The reaction is particularly effective for the rapid generation of tri- and tetraarylfurans and furan-containing oligoarylenes without relying on conventional cross-coupling chemistry.

Platinum-catalyzed domino reaction with benziodoxole reagents for accessing benzene-alkynylated indoles.

Indoles are omnipresent in natural products, bioactive molecules, and organic materials. Consequently, their synthesis or functionalization are important fields of research in organic chemistry. Most works focus on installation or modification of the pyrrole ring. To access benzene-ring-functionalized indoles with an unsubstituted pyrrole ring remains more challenging. Reported herein is a platinum-catalyzed cyclization/alkynylation domino process to selectively obtain C5- or C6-functionalized indoles starting from easily available pyrroles. The work combines, for the first time, a platinum catalyst with ethynylbenziodoxole hypervalent iodine reagents in a domino process for the synthesis of polyfunctionalized arene rings and gives access to important building blocks for the synthesis of bioactive compounds and organic materials.

Longitudinal monitoring adipose-derived stem cell survival by PET imaging hexadecyl-4-¹²4I-iodobenzoate in rat myocardial infarction model.

This study aims to monitor how the change of cell survival of transplanted adipose-derived stem cells (ADSCs) responds to myocardial infarction (MI) via the hexadecyl-4-(124)I-iodobenzoate ((124)I-HIB) mediated direct labeling method in vivo. Stem cells have shown the potential to improve cardiac function after MI. However, monitoring of the fate of transplanted stem cells at target sites is still unclear. Rat ADSCs were labeled with (124)I-HIB, and radiolabeled ADSCs were transplanted into the myocardium of normal and MI model. In the group of (124)I-HIB-labeled ADSC transplantation, in vivo imaging was performed using small-animal positron emission tomography (PET)/computed tomography (CT) for 9 days. Twenty-one days post-transplantation, histopathological analysis and apoptosis assay were performed. ADSC viability and differentiation were not affected by (124)I-HIB labeling. In vivo tracking of the (124)I-HIB-labeled ADSCs was possible for 9 and 3 days in normal and MI model, respectively. Apoptosis of transplanted cells increased in the MI model compared than that in normal model. We developed a direct labeling agent, (124)I-HIB, and first tried to longitudinally monitor transplanted stem cell to MI. This approach may provide new insights on the roles of stem cell monitoring in living bodies for stem cell therapy from pre-clinical studies to clinical trials.

Conformational analysis, spectroscopic study (FT-IR, FT-Raman, UV, 1H and 13C NMR), molecular orbital energy and NLO properties of 5-iodosalicylic acid.

In this study, 5-iodosalicylic acid (5-ISA, C7H5IO3) is structurally characterized by FT-IR, FT-Raman, NMR and UV spectroscopies. There are eight conformers, Cn, n=1-8 for this molecule therefore the molecular geometry for these eight conformers in the ground state are calculated by using the ab-initio density functional theory (DFT) B3LYP method approach with the aug-cc-pVDZ-PP basis set for iodine and the aug-cc-pVDZ basis set for the other elements. The computational results identified that the most stable conformer of 5-ISA is the C1 form. The vibrational spectra are calculated DFT method invoking the same basis sets and fundamental vibrations are assigned on the basis of the total energy distribution (TED) of the vibrational modes, calculated with scaled quantum mechanics (SQM) method with PQS program. Total density of state (TDOS) and partial density of state (PDOS) and also overlap population density of state (COOP or OPDOS) diagrams analysis for C1 conformer were calculated using the same method. The energy and oscillator strength are calculated by time-dependent density functional theory (TD-DFT) results complement with the experimental findings. Besides, charge transfer occurring in the molecule between HOMO and LUMO energies, frontier energy gap, molecular electrostatic potential (MEP) are calculated and presented. The NMR chemical shifts ((1)H and (13)C) spectra are recorded and calculated using the gauge independent atomic orbital (GIAO) method. Mulliken atomic charges of the title molecule are also calculated, interpreted and compared with salicylic acid. The optimized bond lengths, bond angles and calculated NMR and UV, vibrational wavenumbers showed the best agreement with the experimental results.

Room temperature alkynylation of H-phosphi(na)tes and secondary phosphine oxides with ethynylbenziodoxolone (EBX) reagents.

Highly efficient protocols for the alkynylation of H-phosphi(na)tes and secondary phosphine oxides with silyl, aryl and alkyl ethynyl-benziodoxolone (EBX) reagents are reported. Alkynyl phosphorus compounds were obtained in 69-93% yield without the need for a transition metal catalyst at room temperature under open flask conditions.

A theoretical study on diastereoselective oxidative dearomatization by iodoxybenzoic acid.

The reaction mechanism of diastereoselective oxidative dearomatization by iodoxybenzoic acid of key compounds involved in the total synthesis of epicocconone analogs, which are efficient fluorophores with a wide range of applications in protein staining and separation, was studied using density functional theory. In particular, the conformational space was investigated, as was the role of the so-called hypervalent twist move, which is thought to be the rate-determining step. Both kinetic and thermodynamical aspects of the mechanism were considered from static and dynamic viewpoints, including solvent effects. The results were then rationalized using conceptual density functional theory and Bader's atoms-in-molecules framework, which demonstrated how complementary these two approaches are when studying organic chemistry reactions theoretically.

Comparison of succinimidyl [(125)I]iodobenzoate with iodogen iodination methods to study pharmacokinetics and ADME of biotherapeutics.

PURPOSE: To assess the application of succinimidyl iodobenzoate (SIB) iodination method in labeling biotherapeutics to study their pharmacokinetics (PK) and biodistribution. METHOD: An IgG molecule (protein-01) and a 40 KDa protein (protein-02) were evaluated. Pharmacokinetics (PK) and biodistribution of the radiolabeled IgG ((125)I-protein-01) in mice compared parameters from SIB and Iodogen protein iodination labeling methods. In addition, PK of radiolabeled 40 KDa protein ((125)I-protein-02) using SIB was compared with non-labeled protein-02 analyzed by ligand binding assay (LBA). RESULTS: Up to 72 h following a single IP injection to mice, the percentage of "free-label" determined by the soluble counts after TCA precipitation to total radioactivity in serum samples was 2.8-49.4% vs. <1.0% for (125)I-protein-01 iodinated via Iodogen or SIB methods, respectively, suggesting a higher in vivo stability of (125)I-protein-01 labeled via the SIB method. The serum exposure of (125)I-protein-01 was two-fold higher, and correspondingly, the tissue exposure was also higher (averaging 3.6 fold at 168 h) when using SIB protein labeling method than when using the Iodogen method. In addition, following subcutaneous (SC) administration to mice, the serum exposure of (125)I-protein-02 labeled via SIB method was similar to protein-02 measure by LBA. CONCLUSION: In this study, iodination of proteins using SIB methodology has overcome the dehalogenation problem in vivo which is inherent in Iodogen method, and PK parameters of a protein iodinated via SIB were comparable to the un-labeled protein measured by LBA. The SIB iodination method is an improved labeling approach for biotherapeutics used in studying PK and biodistribution characteristics.