ABSTRACT
Due to its unique role as a hydrogen-bond donor and its positive charge, the guanidine group is an important pharmacophoric group and often used in synthetic ligands. The chemical modification of the guanidine group is often considered to destroy its function. Herein, we show that the N-methylation, N-alkylation, or N-acylation of the guanidine group can be used to modify the receptor subtype specificity of the integrin ligand cilengitide. Using the αvß6/α5ß1-biselective ligand c(isoDGRkphg) and the αvß6-specific ligand c(FRGDLAFp(NMe)K(Ac) as examples, we show that the binding affinities of the ligands can be fine-tuned by this method to enhance the selectivity for αvß6. Furthermore, we describe a new strategy for the functionalization of integrin ligands. By introducing longer N-alkylguanidine and N-acylguanidine groups, we are able to simultaneously identify a hitherto unknown anchoring point and enhance the subtype selectivity of the ligand.
Subject(s)
Guanidine/chemistry , Integrins/chemistry , Oligopeptides/chemistry , Binding SitesABSTRACT
The αvß6 integrin binds the RGD-containing peptide of the foot and mouth disease virus with high selectivity. In this study, the long binding helix of this ligand was downsized to an enzymatically stable cyclic peptide endowed with sub-nanomolar binding affinity toward the αvß6 receptor and remarkable selectivity against other integrins. Computational studies were performed to disclose the molecular bases underlying the high binding affinity and receptor subtype selectivity of this peptide. Finally, the utility of the ligand for use in biomedical studies was also demonstrated here.
Subject(s)
Antigens, Neoplasm/chemistry , Integrins/chemistry , Peptides/chemistry , Amino Acid Sequence , Cell Line, Tumor , Humans , Ligands , Magnetic Resonance Spectroscopy , Molecular Dynamics SimulationABSTRACT
The chemical complexity of oxyluciferin (OxyLH2), the light-emitting molecule in the bioluminescence of fireflies, originates from the possibility of keto/enol tautomerism and single or double deprotonation. Herein, we present detailed infrared spectroscopic analysis of OxyLH2 and several of its chemical isomers and isotopomers. To facilitate the future characterization of its biogenic forms, we provide accurate assignments of the solid-state and solution FTIR spectra of OxyLH2 based on comparison to six isotopically labeled variants ([2-(13)C]-OxyLH2, [3-(15)N]-OxyLH2, [4-(13)C]-OxyLH2, [5-(13)C]-OxyLH2, [2'-(13)C]-OxyLH2, [3'-(15)N]-OxyLH2), five closely related structural analogues, and theoretically computed spectra. The computed DFT harmonic vibrational force fields (B3LYP and M06 functionals with basis sets of varying flexibility up to 6-311++G**) reproduce well the observed shifts in the IR spectra of both isotopically labeled and structurally related analogues.
Subject(s)
Indoles/chemistry , Pyrazines/chemistry , Acetonitriles/chemistry , Animals , Fireflies/chemistry , Fireflies/metabolism , Isomerism , Isotope Labeling , Spectrophotometry, InfraredABSTRACT
The chemistry of firefly bioluminescence is important for numerous applications in biochemistry and analytical chemistry. The emitter of this bioluminescent system, firefly oxyluciferin, is difficult to handle. The cause of its lability was clarified while its synthesis was reinvestigated. A side product was identified and characterized by NMR spectroscopy and X-ray crystallography. The reason for the lability of oxyluciferin is now ascribed to autodimerization of the coexisting enol and keto forms in a Mannich-type reaction.
Subject(s)
Fireflies/chemistry , Indoles/chemistry , Pyrazines/chemistry , Animals , Crystallography, X-Ray , Dimerization , Fireflies/metabolism , Ketones/chemistry , Mannich Bases/chemistry , Molecular Conformation , Time FactorsABSTRACT
A complete understanding of the physics underlying the varied colors of firefly bioluminescence remains elusive because it is difficult to disentangle different enzyme-lumophore interactions. Experiments on isolated ions are useful to establish a proper reference when there are no microenvironmental perturbations. Here, we use action spectroscopy to compare the absorption by the firefly oxyluciferin lumophore isolated in vacuo and complexed with a single water molecule. While the process relevant to bioluminescence within the luciferase cavity is light emission, the absorption data presented here provide a unique insight into how the electronic states of oxyluciferin are altered by microenvironmental perturbations. For the bare ion we observe broad absorption with a maximum at 548 ± 10 nm, and addition of a water molecule is found to blue-shift the absorption by approximately 50 nm (0.23 eV). Test calculations at various levels of theory uniformly predict a blue-shift in absorption caused by a single water molecule, but are only qualitatively in agreement with experiment highlighting limitations in what can be expected from methods commonly used in studies on oxyluciferin. Combined molecular dynamics simulations and time-dependent density functional theory calculations closely reproduce the broad experimental peaks and also indicate that the preferred binding site for the water molecule is the phenolate oxygen of the anion. Predicting the effects of microenvironmental interactions on the electronic structure of the oxyluciferin anion with high accuracy is a nontrivial task for theory, and our experimental results therefore serve as important benchmarks for future calculations.
Subject(s)
Fireflies/metabolism , Indoles/chemistry , Pyrazines/chemistry , Water/chemistry , Animals , Anions , Color , Electrochemistry , Enzyme-Linked Immunosorbent Assay , Luminescence , Mass Spectrometry , Models, Chemical , Models, Molecular , Molecular Dynamics Simulation , Spectrometry, Mass, Electrospray Ionization , StereoisomerismABSTRACT
The deactivation pathways of Jørgensen-Hayashi-type organocatalysts modified with an ionic liquid fragment in asymmetric Michael reactions of α,ß-enals with C- (nitromethane, dimethylmalonate) or N-nucleophiles (N-carbobenzyloxyhydroxylamine) that involved an iminium-ion formation step were studied for the first time by the electrospray ionization mass spectrometry (ESI-MS). "Parasitic" side reactions and undesirable cation intermediates that poisoned the catalysts were identified in accordance with their m/z values as well as their relation to the reported mechanisms of Michael reactions in the presence of O-TMS-α,α-diarylprolinol (TMS=trimethylsilyl) derivatives. The proposed approach may be useful for the study of transformations of other types of organocatalysts modified with ionic groups in various organocatalytic reactions and for the development of novel robust catalysts and processes that would be suitable for large-scale industrial applications.
ABSTRACT
Prefilled syringes (PFS) constitute a widely used medical device for drug delivery particularly for the drugs of biological origin. Interactions between the product contents and the components of the PFS play a critical role in determining the suitability of selected PFS. A diluent (with benzyl alcohol/BzOH as a preservative) containing PFS used for reconstitution of the lyophilized product revealed a systematic decrease in the BzOH content during accelerated and stress stability program. Investigation was carried out to understand and identify the underlying causes of this phenomenon. BzOH has a varying propensity to bind to the rubber components (stopper and tip-cap) of the PFS. Vapor permeation behavior across the tip-cap of the PFS was studied via headspace-gas chromatography-mass spectroscopy (HS-GC-MS) enabled analysis. Depending on the properties of the rubber components, BzOH can not only bind but also traverse across them, resulting in a systematic loss during the course of the stability. PFS can allow not only water vapor permeation across the tip-cap as shown in previous studies, but also molecules like benzyl alcohol. This phenomenon stresses the need for careful selection of the components of the primary packaging and also provides an opportunity to deploy novel tools like HS-GC-MS in the early selection of the optimal primary packaging configuration.
Subject(s)
Glass/chemistry , Pharmaceutical Preparations/chemistry , Rubber/chemistry , Chromatography, Gas/methods , Drug Delivery Systems/methods , Drug Packaging/methods , Excipients/chemistry , Mass Spectrometry/methods , SyringesABSTRACT
Integrins play important roles in physiological and pathophysiological processes. Among the RGD-recognizing integrin subtypes, the αvß8 receptor is emerging as an attractive target because of its involvement in various illnesses, such as autoimmune diseases, viral infections, and cancer. However, its functions have, so far, not been investigated in living subjects mainly because of the lack of a selective αvß8 ligand. Here, we report the design and potential medical applications of a cyclic octapeptide as the first highly selective small-molecule ligand for αvß8. Remarkably, this compound displays low nanomolar αvß8 binding affinity and a strong discriminating power of at least 2 orders of magnitude versus other RGD-recognizing integrins. Peptide functionalization with fluorescent or radioactive labels enables the selective imaging of αvß8-positive cells and tissues. This new probe will pave the way for detailed characterization of the distinct (patho)physiological role of this relatively unexplored integrin, providing a basis to fully exploit the potential of αvß8 as a target for molecular diagnostics and personalized therapy regimens.
Subject(s)
Integrins/metabolism , Peptides, Cyclic/pharmacology , Boron Compounds/metabolism , Boron Compounds/pharmacology , Cell Line, Tumor , Drug Design , Fluorescent Dyes/metabolism , Fluorescent Dyes/pharmacology , Gallium Radioisotopes , Humans , Microscopy, Fluorescence , Molecular Docking Simulation , Molecular Dynamics Simulation , Peptides, Cyclic/metabolism , Proof of Concept Study , Radiopharmaceuticals/metabolism , Radiopharmaceuticals/pharmacologyABSTRACT
In this work, we used firefly oxyluciferin (OxyLH2) and its polarity-dependent fluorescence mechanism as a sensitive tool to monitor biomolecular interactions. The chromophores, OxyLH2, and its two analogues, 4-MeOxyLH and 4,6'-DMeOxyL, were modified trough carboxylic functionalization and then coupled to the N-terminus part of Tat and NCp7 peptides of human immunodeficiency virus type-1 (HIV-1). The photophysical properties of the labeled peptides were studied in live cells as well as in complex with different oligonucleotides in solution. By monitoring the emission properties of these derivatives we were able, for the first time, to study in vitro biomolecular interactions using oxyluciferin as a sensor. As an additional application, cyclopropyl-oxyluciferin (5,5-Cpr-OxyLH) was site-specifically conjugated to the thiol group (Cys-232) of the human protein α-1 antytripsin to investigate its interaction with porcine pancreatic elastase. Our data demonstrate that OxyLH2 and its derivatives can be used as fluorescence reporters for monitoring biomolecular interactions.
Subject(s)
Fluorescent Dyes/chemistry , Indoles/chemistry , Oligodeoxyribonucleotides/metabolism , Pyrazines/chemistry , gag Gene Products, Human Immunodeficiency Virus/metabolism , tat Gene Products, Human Immunodeficiency Virus/metabolism , Animals , Fireflies , Fluorescent Dyes/chemical synthesis , HIV-1 , HeLa Cells , Humans , Hydrogen-Ion Concentration , Indoles/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Pancreatic Elastase/chemistry , Pancreatic Elastase/metabolism , Protein Binding , Pyrazines/chemical synthesis , Swine , alpha 1-Antitrypsin/chemistry , alpha 1-Antitrypsin/metabolism , gag Gene Products, Human Immunodeficiency Virus/chemistry , tat Gene Products, Human Immunodeficiency Virus/chemistryABSTRACT
Expression of the cellular transmembrane receptor αvß6 integrin is essentially restricted to malignant epithelial cells in carcinomas of a broad variety of lineages, whereas it is virtually absent in normal adult tissues. Thus, it is a highly attractive target for tumor imaging and therapy. Furthermore, αvß6 integrin plays an important role for the epithelial-mesenchymal interaction and the development of fibrosis. Methods: On the basis of the 68Ga chelators TRAP (triazacyclononane-triphosphinate) and NODAGA, we synthesized mono-, di-, and trimeric conjugates of the αvß6 integrin-selective peptide cyclo(FRGDLAFp(NMe)K) via click chemistry. These were labeled with 68Ga and screened regarding their suitability for in vivo imaging of αvß6 integrin expression by PET and ex vivo biodistribution in severe combined immunodeficiency mice bearing H2009 tumor (human lung adenocarcinoma) xenografts. For these, αvß6 integrin expression in tumor and other tissues was determined by ß6 immunohistochemistry. Results: Despite the multimers showing higher αvß6 integrin affinities (23-120 pM) than the monomers (260 pM), the best results-that is, low background uptake and excellent tumor delineation-were obtained with the TRAP-based monomer 68Ga-avebehexin. This compound showed the most favorable pharmacokinetics because of its high polarity (log D = -3.7) and presence of additional negative charges (carboxylates) on the chelator, promoting renal clearance. Although tumor uptake was low (0.65% ± 0.04% injected dose per gram tissue [%ID/g]), it was still higher than in all other organs except the kidneys, ranging from a maximum for the stomach (0.52 ± 0.04 %ID/g) to almost negligible for the pancreas (0.07 ± 0.01 %ID/g). A low but significant target expression in tumor, lung, and stomach was confirmed by immunohistochemistry. Conclusion: Because of highly sensitive PET imaging even of tissues with low αvß6 integrin expression density, we anticipate clinical applicability of 68Ga-avebehexin for imaging of αvß6 tumors and fibrosis by PET.
Subject(s)
Adenocarcinoma/diagnostic imaging , Antigens, Neoplasm/metabolism , Integrins/metabolism , Lung Neoplasms/diagnostic imaging , Oligopeptides/chemistry , Peptides, Cyclic/chemistry , Positron-Emission Tomography , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma of Lung , Amino Acid Sequence , Animals , Catalysis , Cell Line, Tumor , Copper/chemistry , Female , Humans , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mice , Oligopeptides/pharmacokinetics , Tissue DistributionABSTRACT
Integrins, a diverse class of heterodimeric cell surface receptors, are key regulators of cell structure and behaviour, affecting cell morphology, proliferation, survival and differentiation. Consequently, mutations in specific integrins, or their deregulated expression, are associated with a variety of diseases. In the last decades, many integrin-specific ligands have been developed and used for modulation of integrin function in medical as well as biophysical studies. The IC50-values reported for these ligands strongly vary and are measured using different cell-based and cell-free systems. A systematic comparison of these values is of high importance for selecting the optimal ligands for given applications. In this study, we evaluate a wide range of ligands for their binding affinity towards the RGD-binding integrins αvß3, αvß5, αvß6, αvß8, α5ß1, αIIbß3, using homogenous ELISA-like solid phase binding assay.
Subject(s)
Integrins/metabolism , Ligands , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Biological Therapy , Enzyme-Linked Immunosorbent Assay , Gene Expression Regulation , Humans , Immunomodulation , Inhibitory Concentration 50 , Integrins/agonists , Protein BindingABSTRACT
Fireflies generate flashes of visible light via luciferase-catalyzed chemiexcitation of the substrate (luciferin) to the first excited state of the emitter (oxyluciferin). Microenvironment effects are often invoked to explain the effects of the luciferase active pocket on the emission; however, the exceedingly complex spectrochemistry and synthetic burdens have precluded elucidation of the nature of these interactions. To decipher the effects of microenvironment on the light emission, here the hydrophobic interior of cucurbit[7]uril (CB7) is used to mimic the nonpolar active pocket of luciferase. The hydrophobic interior of CB7 induces shifts of the ground-state pKas by 1.9-2.5 units to higher values. Upon sequestration, the emission maxima of neutral firefly oxyluciferin and its conjugate monodeprotonated base are blue-shifted by 40 and 39 nm, respectively, resulting in visual color changes of the emitted light.
Subject(s)
Indoles/chemistry , Photochemical Processes , Pyrazines/chemistry , Animals , Bridged-Ring Compounds/chemistry , Bridged-Ring Compounds/metabolism , Fireflies , Firefly Luciferin/chemistry , Firefly Luciferin/metabolism , Hydrogen-Ion Concentration , Hydrophobic and Hydrophilic Interactions , Imidazoles/chemistry , Imidazoles/metabolism , Indoles/metabolism , Pyrazines/metabolism , Solutions/chemistry , Spectrum AnalysisABSTRACT
The first systematic steady-state and time-resolved emission study of firefly oxyluciferin (emitter in firefly bioluminescence) and its analogues in aqueous buffers provided the individual emission spectra of all chemical forms of the emitter and the excited-state equilibrium constants in strongly polar environment with strong hydrogen bonding potential. The results confirmed the earlier hypothesis that excited-state proton transfer from the enol group is favored over proton transfer from the phenol group. In water, the phenol-keto form is the strongest photoacid among the isomers and its conjugate base (phenolate-keto) has the lowest emission energy (634 nm). Furthermore, for the first time we observed green emission (525 nm) from a neutral phenol-keto isomer constrained to the keto form by cyclopropyl substitution. The order of emission energies indicates that in aqueous solution a second deprotonation at the phenol group after the enol group had dissociated (that is, deprotonation of the phenol-enolate) does not occur in the first excited state. The pH-dependent emission spectra and the time-resolved fluorescence parameters revealed that the keto-enol tautomerism reaction, which can occur in a nonpolar environment (toluene) in the presence of a base, is not favored in water.
Subject(s)
Fireflies , Indoles/chemistry , Luminescence , Pyrazines/chemistry , Water/chemistry , Absorption, Physicochemical , Animals , Hydrogen-Ion ConcentrationABSTRACT
Palladium-catalyzed dehydrosulfurative Liebeskind-Srogl coupling of terminal alkynes with 2-mercapto-1,3-pyrimidine-5-carbaldehyde under base-free conditions provides 2-(alkynyl)-1,3-pyrimidine-5-carbaldehydes, which are substrates for autocatalytic amplification of chirality according to Soai et al. The mercapto aldehyde acceptor is obtained by condensation of Arnold's vinamidinium salt with thiourea.