hexafluoroacetone and Acetone

Among numerous studies on synthetic approaches to and the biological activities of vitamin D analogues, we herein focused on falecalcitriol, an analogue of calcitriol (1α,25-dihydroxyvitamin D

Topics: Acetone; Calcitriol; Fluorocarbons; Propanols

Topics: Acetone; Amino Acids; Depsipeptides; Fluorocarbons; Hydroxy Acids; Peptides; Sulfhydryl Compounds

A new method for the preparation of N-methylamino acids and some of their derivatives starting from hexafluoroacetone protected amino acids is described. The new concept results in saving of steps compared to conventional protection/activation techniques. Protection and deprotection proceed without recemization.

Topics: Acetone; Amino Acids; Amino Acids, Sulfur; Fluorocarbons; Glutamic Acid; Phosphorus

Fluorine-containing monomers form the basis for production of a large number of commercially important polymers. Most of the polymerization occurs as gas-phase reactions, hence the hazards associated with the monomers arises primarily from inhalation. The chemicals covered in this review include bromotrifluoroethylene (BTFE), chlorotrifluoroethylene (CTFE), hexafluoroacetone (HFA), hexafluoroisobutylene (HFIB), hexafluoropropylene (HFP), perfluorobutylene (PFBE), tetrafluoroethylene (TFE), trichloropropene (TFP), vinyl fluoride (VF), and vinylidene fluoride (VF2). The amount of toxicologic information available on the compounds is relatively small and for certain of these the information consists is short-term or acute, hence the current need to make predictions of biologic activity based on analogy or chemical reactivity is great. In animal models and in man, these monomers may be absorbed into the body at varying rates and the metabolism ranges from extensive to little in a species, dose, and chemical specific fashion. The major toxicologic target of these materials is the kidney, and the degree of involvement depends greatly on the excretion patterns and metabolic profiles of the monomers. However, other target sites exist, such as the reproductive system for HFA, making the use of structure-activity relationships difficult.

Topics: Acetone; Animals; Chlorofluorocarbons; Fluorocarbons; Humans; Hydrocarbons, Fluorinated; Hydrocarbons, Halogenated; Vinyl Compounds

An unprecedented photoredox-catalyzed phosphine-mediated deoxygenation of hexafluoroacetone hydrate was established to accomplish the hydroxylpolyfluoroalkylation of electron-deficient alkenes. A range of bis(trifluoromethyl)carbinols were facilely accessed by using readily available hexafluoroacetone hydrate, instead of toxic gaseous hexafluoroacetone. A range of electron-deficient alkenes are tolerated, giving the corresponding hydro-hydroxylpolyfluoroalkylated products in moderate to high yields. Remarkable features of this synthetic strategy include operational simplicity, mild reaction conditions, excellent regioselectivity, and broad functional group tolerance. The success of this strategy relies on the delicate utilization of aldehyde/ketone-gem-diol intrinsic equilibrium, which offers an innovated open-shell pathway for the assembly of synthetically challenging polyfluoroalkylated scaffolds.

Topics: Acetone; Alkenes; Catalysis; Fluorocarbons

Bombyx mori (B. mori) silk sericin is a protein with features desirable as a biomaterial, such as increased hydrophilicity and biodegradation, as well as resistance to oxidation, bacteria, and ultraviolet light. In contrast to other widely studied B. mori silk proteins such as fibroin, sericin is still unexplored as a building block for fabricating biomaterial, and thus a facile technique of processing it into a material is needed. Here, electrospinning technology was used to fabricate it into biomaterials from two forms of B. mori silk sericin with different molecular weights, one is a low (12.0 kDa) molecular sericin (LS) form and another is a high (66.0 kDa) molecular weight sericin (HS) form. Circular dichroism (CD) spectra showed that LS in hexafluoroacetone (HFA) solvent adopted a predominantly random coil conformation, whereas HS tended to form a β-sheet structure along with a large content of random coils. In addition, LS and HS in HFA solvent were found to form cylinder-like smaller nanoparticles and larger irregular aggregates before electrospinning, respectively. As a result, biomaterials based on microparticles and nanofibers were successfully fabricated by electrospinning of LS and HS dissolved in HFA, respectively. The cell viability and differentiation assay indicated that nanofibers and microparticles improved cell adhesion, growth, and differentiation, proving that the scaffolds electrospun from sericin are biocompatible regardless of its molecular weight. The microparticles, not common in electrospinning of silk proteins reported previously, were found to promote the osteogenic differentiation of mesenchymal stem cells in comparison to the nanofibers. This study suggested that molecular weight of sericin mediates its secondary structure and assembly structure, which in turn leads to a control of final morphology of the electrospun materials. The microparticles and nanofibers of sericin can be potentially used as building blocks for fabricating the scaffolds for tissue engineering.

Topics: Acetone; Animals; Biocompatible Materials; Bombyx; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cell Survival; Chromatography, Gel; Circular Dichroism; Fluorocarbons; HEK293 Cells; Humans; Molecular Weight; Nanofibers; Nanoparticles; Particle Size; Sericins; Solutions; Solvents; Spectroscopy, Fourier Transform Infrared

A feasible nucleophilic trifluoromethylating protocol has been developed using trifluoroacetaldehyde hydrate as an atom-economical trifluoromethyl source. The reaction was found to be applicable to the nucleophilic trifluoromethylation of a broad spectrum of carbonyl compounds with satisfactory yields in general. DFT calculations have been performed to provide mechanistic insight into the present and related reactions employing 2,2,2-trifluoro-1-methoxyethanol and hexafluoroacetone hydrate.

Topics: Acetaldehyde; Acetone; Ethylene Glycols; Fluorocarbons; Methylation; Molecular Structure

A powerful, new reagent, an amidinate salt of hexafluoroacetone hydrate, is an air-stable salt that can be used for the preparation of fluorinated organic molecules. Nucleophilic trifluoromethylation reactions are demonstrated following the base-promoted release of trifluoroacetate. This reagent is soluble in many polar organic solvents and produces fluoroform, following the release of trifluoroacetate. Reactions with this reagent and common electrophiles provide excellent yields of trifluoromethylated products.

Topics: Acetone; Amidines; Catalysis; Combinatorial Chemistry Techniques; Fluorocarbons; Hydrocarbons, Fluorinated; Indicators and Reagents; Molecular Structure; Salts; Trifluoroacetic Acid

Synthetic N-glycosylated CSF114(Glc) and related peptides were proved to be able to recognize specific and high-affinity autoantibodies circulating in blood of relapsing-remitting multiple sclerosis (MS) patients and correlating with disease activity. The effect of these peptides has been linked to the β-turn structure around the minimal epitope Asn(Glc). In this work we performed Hamiltonian replica exchange molecular dynamics simulations on the central heptapeptide fragment of a CSF114(Glc)-derived peptide in water and in a water/hexafluoroacetone mixture, confirming a significant incidence of β-turn structures in both solvents. The structural similarity of the glycosylated and unglycosylated forms in all environments proves that the conformation of the heptapeptide is only marginally affected by the presence of the sugar. Moreover, the presence of a significant amount of bioactive hairpin-like conformations in the water environment suggests a possible use not only in the diagnosis but also in the treatment of MS.

Topics: Acetone; Amino Acid Sequence; Autoantibodies; Fluorocarbons; Glycopeptides; Glycosylation; Molecular Dynamics Simulation; Multiple Sclerosis; Oligopeptides; Peptide Fragments; Protein Conformation; Protein Folding; Solvents; Water

Within the perspective of the current and next space missions to Mars (MSL 2011 and Exomars 2016-2018), the detection and enantioselective separation of building blocks such as the amino acids are important subjects which are becoming fundamental for the search for traces of life on the surface and subsurface of Mars. In this work, we have developed and optimized a method adapted to space experimentation to derivatize and analyze amino acids, using hexafluoroacetone as the derivatizing agent. The temperature, duration of the derivative transfer to the analyser, and chromatographic separation parameters have been optimized to meet the instrument design constraints imposed on devices for extraterrestrial experiments. The work presented in this rationale has established that hexafluoroacetone, in addition to its intrinsic qualities, such as the production of light-weight derivatives (no racemization) and great resistance to the drastic operating conditions, has indeed facilitated simple and fast derivatization that appears to be suitable for in situ analysis in space. By using hexafluoroacetone as the derivatizing agent, we successfully identified, 21 amino acids including 12 of the 20 proteinic amino acids without stirring or extraction steps. Ten of these derivatized amino acids were enantioselectively separated. The precision and accuracy measurements for the D/L ratio showed that the proposed method was also suitable for the determination of both enantioselective forms of most of the tested amino acids. The limits of detection obtained were lower than the ppb level of organic molecules detected in Martian meteorites.

Topics: Acetone; Amino Acids; Extraterrestrial Environment; Fluorocarbons; Gas Chromatography-Mass Spectrometry; Humans

We present measurements of the pressure dependence of stabilized Criegee intermediate (SCI) formation utilizing a hexafluoroacetone scavenger. SCI yields in the ozonolysis of 2,3-dimethyl-2-butene (TME) were measured in a high pressure flow reactor within a range of 50-710 Torr. Within this pressure range, SCI yields increase linearly with pressure. A zero pressure intercept of about 15% indicates that a significant fraction of CI are formed below the barrier to isomerization. By comparison of our results of the pressure dependence of SCI formation and both prompt and long-time OH yields, our results indicate that OH formation from ozonolysis proceeds via at least two intermediates, the SCI and presumably a vinylhydroperoxide (VHP).

Topics: Acetone; Alkenes; Fluorocarbons; Free Radical Scavengers; Halogenation; Heterocyclic Compounds; Hydrogen Peroxide; Ozone; Pressure

Two silklike proteins, [TGRGDSPAGG(GAGAGS)3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8) were designed to demonstrate the superior performance as biomaterials of silklike proteins. The former protein consists of the crystalline domain sequence, (GAGAGS)n from Bombyx mori silk fibroin and cell-adhesive sequence TGRGDSPA coming from fibronectin-containing RGD triplet. The additional sequence (GVPGV)n from elastin was included in the latter protein. The considerably higher cell-adhesion activities of these proteins for NHDF and VERO cells were observed by comparing with those of silklike materials without RGD sequences and also the crystalline fraction of B. mori silk fibroin. This tendency was independent of the treatments, 4.5M LiClO4 or formic acid (FA), on silklike proteins. Their activities are also higher than those of commercial Fibronectin F for NHDF cell. Their structural characterization was studied using 13C solid-state NMR. Although the overlapped peaks in usual 13C CP/MAS NMR spectra make the detailed structural analysis difficult, the methyl resonance regions observed using dipolar dephasing NMR were very useful for the analysis. The presence of both random coil and beta-sheet structures was observed in these proteins clearly. The content of beta-sheet structure in both proteins increases after FA treatment when compared with the lyophilized samples. The production of electrospun nanofibers from their hexafluoroacetone solution was also tried. The silklike protein FES8 could prepare nonwoven silk fibers although FS5 could not.

Topics: Acetone; Animals; Base Sequence; Biocompatible Materials; Bombyx; Cell Adhesion; Chlorocebus aethiops; Elastin; Escherichia coli; Fibroins; Fibronectins; Fluorocarbons; Magnetic Resonance Spectroscopy; Microscopy, Electron, Scanning; Molecular Sequence Data; Nanotubes; Protein Structure, Secondary; Recombinant Proteins; Silk; Tissue Scaffolds; Vero Cells

beta-Hydroxy acids were reacted with hexafluoroacetone and carbodiimides to give carboxy-activated six-membered lactones in good yields. On reaction with amines, the corresponding amides were obtained. We demonstrate the following applications of this protecting/activating strategy: preparation of carboxamides in solution and on solid phase (both normal and reverse mode); recovery and reuse of the excess material in solid-phase synthesis; and convergent solid-phase peptide synthesis (CSPPS) with peptide segments bearing C-terminal Ser or Thr with very low levels of epimerization (<1%, HPLC).

Topics: Acetone; Amides; Amino Acids; Carbodiimides; Fluorenes; Fluorocarbons; Hydroxy Acids; Lactones; Malates; Peptides

Low-temperature 1H and 13C NMR spectra of formic acid (1) showed separate signals for the E and Z conformations in solvents containing a hydrogen bond acceptor, dimethyl ether. The population of E-1 (6.2% in 3:1:1 CHClF2/CHCl2F/(CH3)2O) was larger than that for 13C-labeled methyl formate in the same solvent (0.2%), which indicated that the relative populations are not determined by steric effects. The free-energy difference between the E and Z conformations of 1 was 0.9 kcal/mol. In a 1:3 CD2Cl2/(CH3)2O solvent mixture, peaks for E and Z conformations were found at low temperatures by 1H and 13C NMR for both formic acid and an adduct with hexafluoroacetone, HCO2C(CF3)2OH (2). The population of E-1 in this solvent mixture was 4.3% by 13C NMR. The carbon spectrum showed two peaks in the carbonyl carbon region of nearly equal intensities at -151.6 degrees C, with E-2 (48%) absorbing downfield of the major Z-2 (52%). The large population of E-2 confirms that electron-withdrawing groups R' in RCO2R' enhance the populations of the E-isomers. The free-energy barriers for 2 of 6.24 (E-to-Z) and 6.26 kcal/mol (Z-to-E) were determined from rate constants obtained by line shape analysis at -143.2 degrees C.

Topics: Acetone; Fluorocarbons; Formates; Magnetic Resonance Spectroscopy; Molecular Conformation; Solvents; Stereoisomerism; Thermodynamics

Chlordecone (CD) and mirex (M) differ by a single carbonyl group in CD in place of two chlorines in M. Although both compounds are lipophilic, their tissue distributions differ markedly: CD concentrations are highest in liver; M concentrations are highest in fat. We used tissue time course data in rats from our laboratory for CD and M and literature data from monkeys to develop PBPK models to study differences in liver and fat partitioning. The PK model for M had partitioning in tissue without specific hepatic binding. The CD model had partitioning similar to M, and also included liver binding: the maximal binding (B(max)) and binding affinity constant (Kd) required to describe the rat data were 370 nmol/g liver and 100 nM, respectively. To see if other ketones with electron withdrawing constituents at the alpha carbon were also preferentially distributed to liver, we developed a PBPK description for tissue distribution of hexafluoroacetone (HFA). Compared to acetone, HFA is known to be preferentially sequestered in liver and more slowly excreted unchanged from the body. Acetone is more equally distributed to tissues. HFA distribution was evaluated with a PBPK model that included hepatic binding. B(max) and Kd were 1.58 micromol/g liver and 301 microM. In summary, liver sequestration of CD and HFA most likely represents relatively high-affinity but reversible binding of activated carbonyls in these compounds (activated by the presence of electron withdrawing substituents on the alpha-carbons) with glutathione and glutathione transferases, that are present at much higher concentrations in liver than in other tissues. Strong, but reversible hemithioketal formation with active sulfhydryls may also be associated with the toxic responses to CD and HFA.

Topics: Acetone; Administration, Oral; Algorithms; Animals; Chlordecone; Drug Evaluation, Preclinical; Female; Fluorocarbons; Hydrophobic and Hydrophilic Interactions; Injections, Intravenous; Insecticides; Lipid Metabolism; Liver; Macaca mulatta; Male; Mirex; Models, Biological; Molecular Conformation; Rats; Rats, Sprague-Dawley; Tissue Distribution

Employing high-resolution (13)C solution NMR and circular dichroism (CD) spectroscopic techniques, the distinctive influence of two intimately related hexafluoro solvents, 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) and hexafluoroacetone trihydrate (HFA), on the structural characteristics of Bombyx mori (B. mori) silk fibroin, the chymotrypsin precipitate (C(p)) fraction, and two synthetic peptides, (AGSGAG)(5) and (AG)(15), is described. The observed (13)C solution NMR and CD spectra of these polypeptides in HFIP and HFA revealed a distinctive influence on their conformational characteristics. The (13)C NMR spectra, as analyzed from the unique chemical shifts of C(alpha) and C(beta) resonances of constituent residues revealed that fibroin largely assumes helical conformation(s) in both solvents. However, the peak shifts were greater for the samples in HFIP, indicating that the types of helical structure(s) may be different from the one populated in HFA. Similar structural tendencies of these polypeptides were reflected in CD spectra. The observed CD patterns, i.e., a strong positive band at approximately 190 nm and negative bands at approximately 206 and 222 nm, have been attributed to the preponderance of helical structures. Of the two prevalent helical structures, alpha-helix and 3(10)-helix, the evidence emerged for the fibroin protein in favor of 3(10)-helical structure stabilization in HFIP and its significant disruption in HFA, as deduced from the characteristic R1 (=[theta](190)/[theta](202)) and R2 (=[theta](222)/[theta](206)) ratios, determined from the CD data. Conversely, the native polypeptides and synthetic peptide fragments derived from highly crystalline regions of the silk fibroin protein sustained predominantly an unordered structure in HFA solvent.

Topics: Acetone; Animals; Bombyx; Chymotrypsin; Circular Dichroism; Fibroins; Fluorocarbons; Magnetic Resonance Spectroscopy; Peptide Fragments; Propanols; Protein Conformation; Solvents

Cecropin A (1-8)-Melittin (1-18) is a synthetic cecropin A-melittin hybrid peptide with leishmanicidal activity. The primary sequence of the peptide is as follows: KWKLPKKIGIGAVLKVLTTGLPALIS-NH2. 1H and 13C 2D NMR techniques were used to deduce the conformational parameters of chemical shift, 3JNHalpha coupling constants, temperature coefficients of NH chemical shifts and the pattern of intra and inter-residue nOe's. NMR studies were carried out in water (pH 6.0) and hexafluoroacetone (HFA). The peptide was found in a beta-pleated structure in water, and in HFA it adopts a right-handed alpha-helix conformation. Solution structures generated using restrained molecular dynamics simulations were refined by Mardigras to R factors ranging from 0.5 to 0.6.

Topics: Acetone; Amino Acid Sequence; Anti-Bacterial Agents; Antimicrobial Cationic Peptides; Fluorocarbons; Hydrogen Bonding; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Melitten; Molecular Sequence Data; Molecular Structure; Protein Structure, Secondary; Water

Intermolecular (1)H[(19)F] and (1)H[(1)H] nuclear Overhauser effects have been used to explore interaction of solvent components with melittin dissolved in 50% hexafluoroacetone trihydrate (HFA)/water. Standard nuclear Overhauser effect experiments and an analysis of C(alpha)H proton chemical shifts confirm that the conformation of the peptide in this solvent is alpha-helical from residues Ala4 to Thr11 and from Leu13 to Arg24. The two helical regions are not collinear; the interhelix angle (144 +/- 20 degrees ) found in this work is near that observed in the solid state and previous NMR studies. Intermolecular NOEs arising from interactions between spins of the solvent and the solute indicate that both fluoroalcohol and water molecules are strongly enough bound to the peptide that solvent-solute complexes persist for > or =2 ns. Preferential interactions of HFA with many hydrophobic side chains of the peptide are apparent while water molecules appear to be localized near hydrophilic side chains. These results indicate that interactions of both HFA and water are qualitatively different from those present when the peptide is dissolved in 35% hexafluoro-2-propanol/water, a chemically similar helix-supporting solvent system.

Topics: Acetone; Fluorocarbons; Magnetic Resonance Spectroscopy; Melitten; Protein Structure, Tertiary; Solvents; Water

Hexafluoroacetone was applied as a bidentate protecting and activating agent for the syntheses of RGD-peptide mimetics starting from iminodiacetic acid in solution and on solid phase.

Topics: Acetone; Biochemistry; Fluorocarbons; Imino Acids; Indicators and Reagents; Integrins; Molecular Mimicry; Oligopeptides; Peptide Library; Peptides

CCK-15, a peptide derived from the 115-membered CCK preprohormone, was the object of a comparative conformational analysis by NMR spectroscopy and molecular modeling methods. NMR data in several solvents demonstrate that the propensity of the peptide to fold into a helical conformation is intrinsic, not merely a consequence of the interaction with phosphatidylcholine micelles or with a putative receptor, as suggested by a previous study on CCK-8 (Pellegrini, M.; Mierke, D. Biochemistry 1999, 38, 14775-14783.). The prevailing CCK-15 conformer in a mixture 1,1,1,3,3,3-hexafluoroacetone/water reveals that the residues common to CCK-15 and CCK-8 assume very similar conformations. Our CCK-15 structure is consistent with the model of receptor interaction proposed by Pellegrini and Mierke and discloses possible novel interactions that involve a larger area of the putative receptor. The consensus structure between CCK-15 and CCK-8 shows a good superposition of the side chains of residues 12-14 with crucial moieties of two non-peptidic CCK-A antagonists.

Topics: Acetone; Cholecystokinin; Circular Dichroism; Dimethyl Sulfoxide; Fluorocarbons; Magnetic Resonance Spectroscopy; Micelles; Models, Molecular; Peptide Fragments; Protein Structure, Secondary; Receptor, Cholecystokinin A; Receptors, Cholecystokinin; Solutions; Solvents; Water

The N-terminal portion of HIV-1 Tat covering residues 1-9 is a competitive inhibitor of dipeptidyl peptidase IV (DP IV). We have used 1H NMR techniques, coupled with molecular dynamics methods, to determine the conformation of this peptide in the three diverse media: DMSO-d6, water (pH 2.7) and 40% HFA solution. The results indicate that in both DMSO-d6 and HFA the peptide has a tendency to acquire a type I beta-turn around the segment Asp5-Pro6-Asn7-IIe8. The N-terminal end is seen to be as a random coil. In water, the structure is best described as a left-handed polyproline type II (PPII) helix for the mid segment region Asp2 to Pro6. The structures obtained in this study have been compared with an earlier report on Tat (1-9).

Topics: Acetone; Computer Simulation; Dimethyl Sulfoxide; Dipeptidyl Peptidase 4; Enzyme Inhibitors; Fluorocarbons; Gene Products, tat; Magnetic Resonance Spectroscopy; Models, Molecular; Peptide Fragments; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Software; tat Gene Products, Human Immunodeficiency Virus; Water

A convenient synthetic method which could allow flexible modification at C-23 of 26,26,26,27,27,27-hexafluoro-1alpha,25-dihydroxyvitamin D3 (3) has been developed. An effective construction of hexafluoroacetone (HFA) aldol part on the side chain of 10 was achieved by aldol reaction with HFA gas. This route is also attractive as an approach to diverse 26,27-modified vitamin D3 analogues. The preliminary biological activities of 23-modifed 26,27-F6 vitamin D3 analogues are evaluated. The potency of VDR affinities of the C-23-substituted analogues (keto group (4); OH group (5a,5b); fluorine atom (6a,6b); and oxetane ring (7a,7b)) was found to vary depending upon both the nature and stereochemistry of the substituents. In contrast, the HL-60 cell differentiation property was less varied than VDR affinity, and depended upon the nature rather than the stereochemistry of the substituents.

Topics: Acetone; Animals; Binding, Competitive; Chickens; Cholecalciferol; Fluorine; Fluorocarbons; HL-60 Cells; Humans; Intestines; Molecular Conformation; Molecular Structure; Protein Binding; Receptors, Calcitriol; Receptors, Cytoplasmic and Nuclear; Stereoisomerism; Structure-Activity Relationship; Thermodynamics

The mechanism of protein folding has been the subject of extensive investigation during the last decade, both because of its academic challenge and because of its relation to many diseases which are known to occur due to misfolding of proteins. In this context, we report here a systematic investigation on the step-wise formation of a helical structure by the addition of hexafluoroacetone, in a 14-residue peptide derived from a part of the scorpion neurotoxin protein. The NMR and circular dichroism results indicate that the peptide has an inherent propensity for helix formation and this is limited to the internal few residues in aqueous solution. With the addition of the fluorosolvent, the helical content progressively increases and spans the whole sequence. This is accompanied by concomitant packing of the side chains. These results provide support to the so-called hierarchic model of protein folding which dictates that the local sequence determines the secondary structures in the protein and the side chains play an important role in this process.

Topics: Acetone; Amino Acid Sequence; Animals; Chemical Phenomena; Chemistry, Physical; Circular Dichroism; Fluorocarbons; Magnetic Resonance Spectroscopy; Models, Molecular; Neurotoxins; Peptide Fragments; Protein Folding; Protein Structure, Secondary; Scorpion Venoms; Solvents

The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH approximately 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 M (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of approximately 8 M. Temperature dependent CD experiments support a 'cold denaturation' of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 M HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH/NHi+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10-G11-T12. The helical structure of melittin obtained at 3.6 M HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.

Topics: Acetone; Alcohols; Amino Acid Sequence; Circular Dichroism; Fluorine; Fluorocarbons; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Melitten; Molecular Sequence Data; Peptides; Proteins; Spectrometry, Fluorescence

Riboflavin carrier protein (RCP) plays an important role in transporting vitamin B2 across placental membranes, a process critical for maintenance of pregnancy. Association of the vitamin with the carrier protein ensures optimal bioavailability, facilitating transport. The conformations of three antigenic peptide fragments encompassing residues 4-23 (N21), 170-186 (R18), and 200-219 (Y21) from RCP, which have earlier been studied as potential leads toward a synthetic peptide-based contraceptive vaccine, have been investigated using CD and NMR spectroscopy in aqueous solution and in the presence of the structure-stabilizing cosolvent hexafluoroacetone trihydrate (HFA). In aqueous solution at pH 3.0, all three peptides are largely unstructured, with limited helical population for the peptides R18 and Y21. The percentage of helicity estimated from CD experiments is 10% for both the peptides. A dramatic structural transition from an unstructured state to a helical state is achieved with addition of HFA, as evidenced by intensification of CD bands at 222 nm and 208 nm for Y21 and R18. The structural transition is completed at 50% HFA (v/v) with 40% and 35% helicity for R18 and Y21, respectively. No structural change is evident for the peptide N21, even in the presence of HFA. NMR analysis of the three peptides in 50% HFA confirms a helical conformation of R18 and Y21, as is evident from upfield shifts of CalphaH resonances and the presence of many sequential NH/NH NOEs with many medium-range NOEs. The helical conformation is well established at the center of the sequence, with substantial fraying at the termini for both the peptides. An extended conformation is suggested for the N21 peptide from NMR studies. The helical region of both the peptides (R18, Y21) comprises the core epitopic sequence recognized by the respective monoclonal antibodies. These results shed some light on the issue of structure and folding of antigenic peptides.

Topics: Acetone; Amino Acid Sequence; Antibodies, Monoclonal; Carrier Proteins; Circular Dichroism; Fluorocarbons; Magnetic Resonance Spectroscopy; Membrane Transport Proteins; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Peptide Fragments; Protein Folding; Protein Structure, Secondary

Three synthetic peptides corresponding to distinct segments of the subunit interface of the dimeric enzyme thymidylate synthase (residues 17-38, N 22; residues 174-190, M 17; and residues 201-220, C 20) have been investigated for their ability to function as inhibitors by modifying the quaternary structure of the enzyme. A dramatic reduction of enzyme activity is observed following incubation of TS with the C 20 peptide. The N 22 and M 17 peptides were unable to cause any loss of enzymatic activity. Addition of the C 20 peptide results in a loss of fluorescence of TS labeled with a dansyl group at Cys 198, following aggregation and precipitation of the protein. The effects are not observed for the N 22 or M 17 peptides. Loss of enzymatic activity is related to the ability of C 20 to promote protein aggregation. The conformations of the peptides have been studied using CD and NMR in order to correlate the observed function with solution structures. Peptides N 22 and M 17 are largely unstructured in aqueous solution. A population of nascent helical structures or multiple turn conformations has been detected for the C 20 peptide in aqueous solution by NMR. Addition of 50% (v/v) hexafluoroacetone trihydrate (HFA), a structure-stabilizing cosolvent, stabilizes the helical conformation in the C 20 peptide. Under similar conditions, N 22 and M 17 remain largely extended with observations of local beta-turn conformations. Interestingly, the C 20 peptide is a beta-hairpin in the native structure, whereas the other two peptides are individual strand components of a beta-sheet.

Topics: Acetone; Amino Acid Sequence; Catalysis; Circular Dichroism; Dimerization; Enzyme Inhibitors; Fluorocarbons; Lacticaseibacillus casei; Models, Molecular; Molecular Sequence Data; Nuclear Magnetic Resonance, Biomolecular; Peptide Fragments; Protein Conformation; Solutions; Thymidylate Synthase; Water

A molten globule-like state of hen egg-white lysozyme has been characterized in 25% aqueous hexafluoroacetone hydrate (HFA) by CD, fluorescence, NMR, and H/D exchange experiments. The far UV CD spectra of lysozyme in 25% HFA supports retention of native-like secondary structure while the loss of near UV CD bands are indicative of the overall collapse of the tertiary structure. The intermediate state in 25% HFA exhibits an enhanced affinity towards the hydrophobic dye, ANS, and a native-like tryptophan fluorescence quenching. 1-D NMR spectra indicates loss of native-like tertiary fold as evident from the absence of ring current-shifted 1H resonances. CD, fluorescence, and NMR suggest that the transition from the native state to a molten globule state in 25% HFA is a cooperative process. A second structural transition from this compact molten globule-like state to an "open" helical state is observed at higher concentrations of HFA (> or = 50%). This transition is characterized by a dramatic loss of ANS binding with a concomitant increase in far UV CD bands. The thermal unfolding of the molten globule state in 25% HFA is sharply cooperative, indicating a predominant role of side-chain-side-chain interactions in the stability of the partially folded state. H/D exchange experiments yield higher protection factors for many of the backbone amide protons from the four alpha-helices along with the C-terminal 3(10) helix, whereas little or no protection is observed for most of the amide protons from the triple-stranded antiparallel beta-sheet domain. This equilibrium molten globule-like state of lysozyme in 25% HFA is remarkably similar to the molten globule state observed for alpha-lactalbumin and also with the molten globule state transiently observed in the kinetic refolding experiments of hen lysozyme. These results suggest that HFA may prove generally useful as a structure modifier in proteins.

Topics: Acetone; Animals; Chickens; Circular Dichroism; Female; Fluorocarbons; Magnetic Resonance Spectroscopy; Muramidase; Protein Conformation; Protein Denaturation; Protein Folding; Protein Structure, Secondary; Protein Structure, Tertiary; Tryptophan; Water

Topics: Acetone; Amino Acid Sequence; Animals; Carrier Proteins; Chickens; Drug Stability; Epitopes; Fluorocarbons; Membrane Transport Proteins; Models, Structural; Molecular Sequence Data; Peptides; Polytetrafluoroethylene; Protein Structure, Secondary; Riboflavin

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrPC) is converted into the scrapie form (PrPSc). Limited proteolysis of PrPsc produces PrP 27-30 which readily polymerizes into amyloid. To study the structure of PrP amyloid, we employed organic solvents that perturb protein conformation. Hexafluoro-2-propanol (HFIP), which promotes alpha-helix formation, modified the ultrastructure of rod-shaped PrP amyloids; flattened ribbons with a more regular substructure were found. As the concentration of HFIP was increased, the beta-sheet content and proteinase K resistance of PrP 27-30 as well as prion infectivity diminished. HFIP reversibly decreased the binding of Congo red dye to the rods while inactivation of prion infectivity was irreversible. In contrast to 10% HFIP, 1,1,1-trifluoro-2-propanol (TFIP) did not inactivate prion infectivity but like HFIP, TFIP did alter the morphology of the rods and abolish Congo red binding. This study separates prion infectivity from the amyloid properties of PrP 27-30 and underscores the dependence of prion infectivity on PrPSc conformation. The results also demonstrate that the specific beta-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

Topics: 1-Propanol; Acetone; Alcohols; Animals; Congo Red; Cricetinae; Electrophoresis, Polyacrylamide Gel; Endopeptidase K; Female; Fluorocarbons; Glycerol; Microscopy, Electron; Propanols; Protein Conformation; Protein Structure, Secondary; PrP 27-30 Protein; PrPC Proteins; Scrapie; Serine Endopeptidases; Solubility; Solvents; Spectrophotometry; Spectroscopy, Fourier Transform Infrared; Sucrose

The biotransformation of the aerosol propellant 1,1,1,2,3,3,3-heptafluoropropane (HFA-227) was investigated in rats in vivo and in rat and human liver microsomes. In the urine of rats exposed to 5000 ppm HFA-227 for 6 hr, very small amounts of hexafluoroacetone trihydrate were identified as an HFA-227 metabolite by 19F-NMR. Fluoride concentrations in the urine samples (0-48 hr after the end of the exposure) from exposed animals were not significantly different from those found in samples from nonexposed rats. In rat and human liver microsomes, fluoride and hexafluoroacetone trihydrate formation from HFA-227 was detected in very low levels only in liver microsomes from pyridine-treated rats and in two of eight human liver microsome samples, which exhibited the highest cytochrome P4502E1 activities. Because some aldehydes may covalently bind to proteins and the formation of fluorinated protein adducts has been implicated in immune-mediated hepatitis induced by halothane, the binding of hexafluoroacetone trihydrate to proteins was also investigated. Hexafluoroacetone trihydrate also gave only a very small resonance in fluorine NMR experiments when binding to human serum albumin was studied in comparison with the acylating agent S-ethyltrifluoroacetate. Moreover, no fluorine-containing products were formed by the reaction of hexafluoroacetone trihydrate with N alpha-acetyl-L-lysine, and hexafluoroacetone trihydrate was not metabolized to fluorine-containing metabolites or inorganic fluoride in rats. Comparative studies in human liver microsomes demonstrated that a halothane metabolite may covalently bind to proteins; in contrast, metabolism and covalent binding of HFA-227 could not be demonstrated. In summary, these data indicate that HFA-227 is biotransformed at very low rates to hexafluoroacetone trihydrate but irreversible binding of hexafluoroacetone trihydrate cannot be demonstrated, even with the application of very sensitive methods, and is considered unlikely, based on the combination of the results obtained.

Topics: Acetone; Aerosols; Animals; Biotransformation; Fluorine; Fluorocarbons; Humans; Hydrocarbons, Fluorinated; Magnetic Resonance Spectroscopy; Male; Microsomes, Liver; Protein Binding; Rats; Rats, Sprague-Dawley; Subcellular Fractions

Rats were exposed to 0, 0.1, 1, and 12 ppm of hexafluoroacetone (HFA) for 6 h/day, 5 days/week for 90 days. The exposed rats were killed after 30 or 90 days exposure, and 28 or 84 days post-exposure (PE). There were no exposure-related pathological lesions in the rats exposed to 0.1 or 1.0 HFA for 90 days. After 30 days exposure to 12 ppm HFA, rats showed lower body weight gain, testicular atrophy, and oligospermia or aspermia in the epididymal tubules. At 30 days exposure, the atrophic testes had marked depletion of round spermatids in spermatogenic stages I-VIII and elongated spermatids in spermatogenic stages IX-XIV, but mature spermatids appeared only slightly decreased. Numerous spermatocytes in meiotic division in spermatogenic stage XIV were necrotic. At 90 days exposure, the testes showed severe atrophy with almost all seminiferous tubules affected and both immature and mature spermatids had disappeared from the seminiferous tubules. The epididymal tubules were devoid of spermatozoa. After 28 days PE, regeneration of atrophic testes was evident but varied markedly among the exposed rats. The number of seminiferous tubules producing elongated and mature spermatids was significantly lower than that of normal testes. Many seminiferous tubules had not regained normal spermatogenesis and the epididymal tubules showed marked oligospermia. After 84 days PE, normal spermatogenesis was still only partially restored to the atrophic testes, with many of the regenerating tubules still devoid of normal spermatogenesis.

Topics: Acetone; Administration, Inhalation; Animals; Atrophy; Body Weight; Epididymis; Female; Fluorocarbons; Male; Oligospermia; Organ Size; Rats; Sertoli Cells; Spermatids; Spermatogenesis; Testis

Steroidogenesis was investigated in Leydig cell-enriched fractions isolated from the testes of rats dosed dermally with 130 mg/kg/day hexafluoroacetone (HFA) for 14 days and from pair-fed control rats. Compared to controls, Leydig cells from HFA-treated rats exhibited decreased incorporation of [14C]acetate (78%) and [3H]mevalonate (41%) into sterols and steroids. Testosterone was decreased 50% in the testes of HFA-treated rats. Incubation of Leydig cells from untreated rats with 1.0 mM HFA did not affect steroidogenesis. HFA treatment led to the development of histopathological lesions in the testes within 24 hr after a single dose; with daily dosing the lesions became progressively more severe. Despite the development of severe lesions, HFA treatment did not affect the blood levels of luteinizing hormone or testosterone; however, follicle-stimulating hormone was slightly elevated (48%) after 14 days of treatment. The data indicate that steroidogenesis is inhibited in Leydig cells of HFA-treated rats; the inhibition is not due to a direct or immediate effect of HFA, nor does it appear to be hormonally mediated.

Topics: Acetates; Acetone; Animals; Cholesterol; Fluorocarbons; Follicle Stimulating Hormone; In Vitro Techniques; Leydig Cells; Luteinizing Hormone; Male; Mevalonic Acid; Rats; Spermatogenesis; Steroids; Testosterone

Testicular atrophy was induced in rats by dermal application of hexafluoroacetone (HFA) at 39 or 130 mg/kg/day for 14 days, but not at a dosage of 13 mg/kg/day. Affected germ cells were mostly spermatids and to a much lesser extent spermatocytes; spermatogonia were unaffected. Late spermatids were retained in Sertoli cells and showed degenerative changes. Sertoli cells exhibited cytoplasmic vacuolation, distended endoplasmic reticulum, and a marked increase in lipid droplets. Leydig cells exhibited a slight increase in lipid droplets, fewer mitochondria, and diminution and segregation of the agranular endoplasmic reticulum from mitochondria. A correlation between ultrastructural and biochemical changes in HFA-induced testicular atrophy is presented.

Topics: Acetone; Animals; Fluorocarbons; Leydig Cells; Lipid Metabolism; Male; Rats; Sertoli Cells; Spermatogenesis; Testicular Diseases

The disposition and metabolism of [14C]hexafluoroacetone (HFA) were studied in the rat as part of an investigation of the mechanism of HFA-induced testicular atrophy. After sc injection of 13 mg/kg [14C]HFA, radioactivity was eliminated in a biphasic manner from the blood; the half-life of the initial phase was 22.6 hr and that of the elimination phase 75.1 hr. Following injection of 130 mg/kg [14C]HFA, the elimination of radioactivity was initially zero order, but with time it became first order and biphasic with half-lives of the initial and terminal elimination phases being 23.0 and 59.9 hr, respectively. The primary route of elimination was via the urine and all of the [14C]HFA was excreted unmetabolized. [14C]HFA was uniformly distributed throughout the major organs of the body with the exception of the liver which contained disproportionately higher levels of [14C]HFA. The hepatic binding of [14C]HFA was noncovalent and capacity limited. Notably, the testes, the target organ of HFA-induced toxicity, did not exhibit any unusual accumulation or retention of [14C]HFA.

Topics: Acetone; Animals; Fluorocarbons; Half-Life; Kinetics; Liver; Male; Rats; Testis; Tissue Distribution

Rats dosed dermally with 39 or 130 mg/kg/day hexafluoroacetone sesquihydrate (HFA) for 14 days developed moderate or severe testicular atrophy, respectively; rats dosed with 13 mg/kg/day HFA for 14 days did not. Histologic evaluation of the testes revealed that spermatids, followed by spermatocytes, were the germ cells most affected by HFA; spermatogonia and Sertoli cells appeared to be less vulnerable. Lipogenesis from [3H]acetate and[14C]glucose was investigated in vitro in testes from HFA-treated and pair-fed control rats. Triacylglycerol and phospholipid synthesis was increased whereas sterol synthesis was decreased in testes from HFA-treated rats. Vitamin A and zinc were measured in the testes of control and HFA-treated rats; no differences in the levels of these nutrients were observed between the two groups. The data support the hypothesis that altered lipid metabolism, in particular sterol metabolism, is associated with the development of HFA-induced testicular atrophy.

Topics: Acetone; Animals; Atrophy; Fluorocarbons; Glucose; Lipid Metabolism; Male; Rats; Testis