ABSTRACT
The design and synthesis of alanine-rich α-helical peptides constrained in a partially unfolded state by incorporation of the S,S-tetrazine phototrigger has been achieved, permitting, upon photochemical release, observation by 2D-IR spectroscopy of the subnanosecond conformational dynamics that govern the early steps associated with α-helix formation. Solid-phase peptide synthesis was employed to elaborate the requisite fragments, with full peptide construction via solution-phase fragment condensation. The fragment union tactic was also employed to construct (13)Câ(18)O isotopically edited amides to permit direct observation of conformational motion at or near specific peptide bonds.
Subject(s)
Alanine/chemistry , Peptides/chemical synthesis , Tetrazoles/chemistry , Molecular Structure , Peptides/chemistry , Photochemical ProcessesABSTRACT
Molecular ions undergoing ultrafast conformational changes on the same time scale of water motions are of significant importance in condensed phase dynamics. However, the characterization of systems with fast molecular motions has proven to be both experimentally and theoretically challenging. Here, we report the vibrational dynamics of the non-degenerate (C12,C13)-oxalate anion, an ultrafast rotor, in aqueous solution. The infrared absorption spectrum of the (C12,C13)-oxalate ion in solution reveals two vibrational transitions separated by approximately 40 cm(-1) in the 1500-1600 cm(-1) region. These two transitions are assigned to vibrational modes mainly localized in each of the carboxylate asymmetric stretch of the ion. Two-dimensional infrared spectra reveal the presence and growth of cross-peaks between these two transitions which are indicative of coupling and population transfer, respectively. A characteristic time of sub-picosecond cross-peaks growth is observed. Ultrafast pump-probe anisotropy studies reveal essentially the same characteristic time for the dipole reorientation. All the experimental data are well modeled in terms of a system undergoing ultrafast population transfer between localized states. Comparison of the experimental observations with simulations reveal a reasonable agreement, although a mechanism including only the fluctuations of the coupling caused by the changes in the dihedral angle of the rotor, is not sufficient to explain the observed ultrafast population transfer.
Subject(s)
Molecular Dynamics Simulation , Oxalates/chemistry , Rotation , Vibration , Absorption , Carboxylic Acids/chemistry , Molecular Conformation , Quantum TheoryABSTRACT
The relaxation of helical structures very close to equilibrium is observed via transient 2D IR spectroscopy. An initial distribution of synthetically distorted helices having an unnatural bridge linking the 10th and 12th residues of an alanine-rich α-helix is released to evolve into the equilibrium distribution of α-helix conformations. The bridge constrains the structure to be slightly displaced from the full α-helix equilibrium near these residues, yet the peptide is not unfolded completely. The release is accomplished by a subpicosecond pulse of UV irradiation. The resulting 2D IR signals are used to obtain snapshots of the â¼100-ps helical conformational reorganization of the distorted dihedral angle and distance between amide units at chemical bond length-scale resolution. The decay rates of the angle between the dipoles, dihedral angles, and distance autocorrelations obtained from molecular dynamics simulations support the experiments, providing evidence that the final helix collapse conforms to linear response theory.
Subject(s)
Molecular Dynamics Simulation , Peptides/chemistry , Protein Structure, Secondary , Spectrophotometry, Infrared/methods , Amino Acid Sequence , Kinetics , Models, Molecular , Molecular Sequence Data , Oligopeptides/chemistry , Spectroscopy, Fourier Transform InfraredABSTRACT
We describe the synthesis of the N-(2-seleninatoethyl) amide of N-Boc-phenylalanine, serving here as a peptide model, and its reductive coupling reactions under mild conditions with unprotected thiouridine and glutathione. Selenosulfide products such as these comprise reversibly conjugated bio-components, and can potentially find uses as probes of biological function, such as enzyme inhibitors, delivery systems, or structural mimics.
Subject(s)
Carboxylic Acids/chemistry , Organoselenium Compounds/chemistry , Phenylalanine/chemistry , Carboxylic Acids/chemical synthesis , Organoselenium Compounds/chemical synthesis , Oxidation-Reduction , Sulfhydryl Compounds/chemistry , Thiouridine/chemistryABSTRACT
The design, solid-phase synthesis, and photochemical validation of diverse peptide linchpins, containing the S,S-tetrazine phototrigger, have been achieved. Steady state irradiation or femtosecond laser pulses confirm their rapid photofragmentation. Attachment of peptides to the C- and N-termini will provide access to diverse constrained peptide constructs that hold the promise of providing information about early peptide/protein conformational dynamics upon photochemical release.
Subject(s)
Peptides/chemical synthesis , Tetrazoles/chemistry , Molecular Structure , Peptides/chemistry , Photochemical ProcessesABSTRACT
The tetrazine chromophore holds promise as an effective photochemical trigger to achieve structural release, directed at the determination of peptide/protein motions that occur early in the folding processes. The photochemistry of 3,6-di-cysteine-S,S-tetrazines was examined by femtosecond IR transient absorption spectroscopy. Excitation of the tetrazine chromophore by visible and near UV light in the end yields chemically inert, structurally unobtrusive photoproducts that are not expected to interfere with the conformational dynamics of peptides and proteins. Dicysteine S,S-tetrazine is suggested to undergo photocleavage via a photochemical pathway different than the parent molecule s-tetrazine, based on kinetic measurements that reveal a stepwise reaction pathway of photofragmentation, whereby the initial ring cleavage event occurs prior to the formation of the SCN groups.
ABSTRACT
Equimolar quantities of 2-ethoxyethaneseleninic acid and p-thiocresol react rapidly in dichloromethane solution to give the selenosulfide along with disulfide, diselenide, and two products oxidized at sulfur, the thiosulfonate and the selenosulfonate. The latter two are new for this sort of coupling; their formation may be the result of an early thioseleninate to selenosulfinate isomerization. A radical chain mechanism is proposed to account for all five products, as well as their relative amounts.
Subject(s)
Carboxylic Acids/chemistry , Organoselenium Compounds/chemistry , Sulfhydryl Compounds/chemistry , Sulfinic Acids/chemistry , Kinetics , Molecular Structure , Oxidation-ReductionABSTRACT
The title compound, which differs from the powerful O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline only at the chalcogen atom (Se for S), is a much weaker inhibitor in a direct OGA assay. In human cells, however, the selenazoline shows comparable ability to induce hyper-O-GlcNAc-ylation, and the two show similar reduction of insulin-stimulated translocation of glucose transporter 4 in differentiated 3T3 adipocytes.
Subject(s)
Enzyme Inhibitors/pharmacology , Glucosamine/analogs & derivatives , Thiazoles/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , 3T3-L1 Cells , Acetylglucosamine/analysis , Acetylglucosamine/metabolism , Adipocytes/metabolism , Amino Acid Sequence , Animals , Cell Differentiation/drug effects , Cell Line , Clostridium perfringens/enzymology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Glucosamine/chemical synthesis , Glucosamine/chemistry , Glucosamine/pharmacology , Glucose Transporter Type 4/metabolism , Glycosylation , HeLa Cells , Humans , Insulin/pharmacology , Mice , Models, Molecular , Molecular Sequence Data , Molecular Structure , Sequence Alignment , Structure-Activity Relationship , Thiazoles/chemical synthesis , Thiazoles/chemistryABSTRACT
2-Ethoxyethaneseleninic acid reacts with electron-rich aromatic substrates to deliver, by way of the selenoxides, the (2-ethoxyethyl)seleno ethers, which can in turn be transformed into a diverse set of aryl-selenylated products. Among these, a family of 5-uridinyl derivatives shows submicromolar inhibition of human and malarial orotate phosphoribosyltransferase.
Subject(s)
Organometallic Compounds/chemical synthesis , Selenium/chemistry , Humans , Malaria/enzymology , Molecular Structure , Organometallic Compounds/chemistry , Organometallic Compounds/pharmacology , Orotate Phosphoribosyltransferase/antagonists & inhibitors , StereoisomerismABSTRACT
The title compound, a powerful inhibitor of retaining N-acetylhexosaminidases, can move freely among three pyranose solution conformations of similar energy-two twist boats and the (4)C(1) chair-as revealed by NMR, calculational, and crystallographic studies. It binds in the enzyme active site only in the pseudo-(4)C(1) conformation, however, in which it most closely resembles the hypothetical bound substrate transition state, a (4)E sofa that is approximately trigonal bipyramidal at the anomeric carbon.
Subject(s)
Glucosamine/analogs & derivatives , Thiazoles/chemistry , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Glucosamine/chemistry , Magnetic Resonance Spectroscopy , Molecular Conformation , Thermodynamics , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/chemistryABSTRACT
A homotyrosine based seleninic acid irreversibly inhibits protein tyrosine phosphatases by forming a covalent selenosulfide linkage with the active site cysteine sulfhydryl specifically. The details of the event are revealed by model synthetic studies and by kinetic, mass spectrometric, and crystallographic characterization.
Subject(s)
Phosphates/chemistry , Protein Tyrosine Phosphatases/antagonists & inhibitors , Selenium Compounds/chemistry , Selenium Compounds/pharmacology , Apraxia, Ideomotor , Crystallography, X-Ray , Models, Molecular , Molecular Structure , Protein Tyrosine Phosphatases/metabolismABSTRACT
The synthesis of a variety of pyranose-, nucleoside-, (amino acid)-, and polyhydric-based seleninic and selenonic acids by DMDO oxidation of the corresponding selenoesters is reported, as well as some unusual coupling reactions of the seleninate and selenonate functionality with biological nucleophilic groups (sulfhydryl, indole, phenol, imidazole, carboxamide) that are found in proteins and enzyme active sites.
Subject(s)
Biomimetic Materials/chemical synthesis , Organoselenium Compounds/chemical synthesis , Amides/chemistry , Amino Acids/chemical synthesis , Biomimetic Materials/chemistry , Carboxylic Acids/chemical synthesis , Carboxylic Acids/chemistry , Imidazoles/chemistry , Indoles/chemistry , Nucleosides/chemical synthesis , Organoselenium Compounds/chemistry , Oxidation-Reduction , Phenols/chemistry , Pyrans/chemical synthesis , Sulfhydryl Compounds/chemistryABSTRACT
O-GlcNAcase (OGA) promotes O-GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O-GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown. We find that short OGA, which possesses O-GlcNAcase catalysis machinery like that of long OGA, exhibits comparative resistance to previously described potent inhibitors of long OGA and lysosomal hexosaminidases, including PUGNAc and NAG-thiazoline, suggesting a role for the HAT domain in O-GlcNAcase catalysis. We also find that alpha-GlcNAc thiolsulfonate (2) is the most potent inhibitor of short OGA yet described (Ki = 10 microM), and exhibits some degree of selectivity versus long OGA and lysosomal hexosaminidases. In contrast to its mode of inhibition of short OGA, 2 acts as a irreversible inhibitor of long OGA by covalently modifying the enzyme as an S-GlcNAc derivative. Covalent attachment of GlcNAc to the HAT domain of long OGA dramatically changes its properties with respect to enzymatic activity and caspase-3 cleavage.
Subject(s)
Enzyme Inhibitors/pharmacology , Thioglucosides/pharmacology , Tosyl Compounds/pharmacology , beta-N-Acetylhexosaminidases/antagonists & inhibitors , beta-N-Acetylhexosaminidases/metabolism , Enzyme Inhibitors/chemistry , Humans , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , Molecular Structure , Thioglucosides/chemistry , Tosyl Compounds/chemistryABSTRACT
The potent O-GlcNAcase (OGA) inhibitor GlcNAc-thiazoline has been modified by buffer- or acylation-induced imine-to-enamine conversion and then electrophile or radical addition (Xn = D3, F, N3, OH, SMe, COCF3, CF3). Several functionalized GlcNAc-thiazolines show highly selective inhibition of OGA vs human hexosaminidase and thus have promise as tools for targeted investigations of OGA, an enzyme linked to diabetes and neurodegeneration. A new radical addition/fragmentation reaction of the N-(trifluoroacetyl)enamine has been discovered.
