HFIP Concentration Research Articles

The structure of aqueous solutions of hexafluoro-iso-propanol studied by neutron diffraction with hydrogen/deuterium isotope substitution and empirical potential structure refinement modeling.

Neutron diffraction measurements of H/D isotopic substitution are made at room temperature for seven H/D substituted hexafluoro-iso-propanol (HFIP; 1,1,1,3,3,3-hexafluoro-2-propanol)-water mixtures at 0.1, 0.2, and 0.4 HFIP mole fraction (xHFIP). The eight partial structure factors except for the H(CH)-H(CH) pair obtained are subjected to an empirical potential structure refinement (EPSR) method to derive all site-site pair correlation functions. It is found that with increasing HFIP concentration the ice-like network of water disappears between xHFIP = 0.1 and 0.2, followed by the formation of a chain-like water structure embedded in an intrinsic structure of HFIP evolved at xHFIP = 0.4. The hydroxyl group of HFIP forms hydrogen bonds with the surrounding water molecules at all HFIP mole fractions investigated. There is no evidence that the water structure is well defined around the CF3 groups of HFIP, but water molecules surround tangentially the CF3 groups of HFIP.

Examination of Interactions of Hexafluoro-2-propanol with Trp-Cage in Hexafluoro-2-propanol-Water by MD Simulations and Intermolecular Nuclear Overhauser Effects.

All-atom molecular dynamic simulations of the peptide Trp-cage in 30% hexafluoro-2-propanol- water (V/V) at 278 K have been carried out with the goal of exploring peptide hydrogen-solvent fluorine nuclear spin cross relaxation. Force field parameters for HFIP reported by Fioroni et al. along with the fluorine parameters of the TFE5 model reported by this lab were used. Water was represented by the TIP5P-Ew model. Peptide modeling used the AMBER99SB-ILDN force field. Translational diffusion coefficients of solution components at 278 K were predicted to within 35% of experimental values using these parameter sets. The simulations indicate that the solvent mixture is not homogeneous, with HFIP molecules clustered into aggregates as large as 53 fluoroalcohol molecules. The solvent environment of surface atoms of Trp-cage fluctuates between being HFIP-rich and more water-rich about every 10 ns. In accord with previous studies by other groups, the average concentration of HFIP near the surface of the peptide is significantly enhanced over the concentration of HFIP in the bulk solvent. In the simulations, ∼7% of the initial contacts between HFIP molecules and Trp-cage develop into peptide-fluoroalcohol interactions that persist for times as long as 8 ns. Most of the available experimental nuclear spin cross-relaxation rates (ΣHF) for hydrogens of the Trp-cage in 30% HFIP-water are reproduced from the MD trajectories to within uncertainties of the experimental data and the simulations. However, a few calculated ΣHF values for hydrogens of the Trp-cage do not agree with experiment. These tend to be situations where long-lived peptide-HFIP interactions are predicted. The disagreements between observed and calculated ΣHF in these instances signal defects in the modeling parameters and procedures that are presently unrecognized.

Fluoroalcohol – Induced coacervates for selective enrichment and extraction of hydrophobic proteins

As previously reported, fluoroalcohols can induce coacervation in aqueous solutions of amphiphilic compounds with subsequent formation of two-phase systems, where one phase is enriched in amphiphile and fluoroalcohol and the other is primarily an aqueous – rich phase. This study focuses on the use of simple coacervates made of a single component amphiphile induced by a fluoroalcohol for extraction and enrichment of proteins. 1,1,1,3,3,3-Hexafluoroisopropanol (HFIP) and 2,2,2-trifluoroethanol (TFE) were used to induce coacervation in the aqueous solutions of a cationic amphiphile, cetyltrimethylammonium bromide (CTAB) or tetra-n-butylammonium bromide (TBAB). Cationic amphiphiles (CTAB, TBAB) formed two-phase coacervate systems in a basic pH and/or sufficient ionic strength depending on the strength of coacervator (HFIP or TFE). The phase diagrams for TBAB paired with HFIP or TFE coacervates were created. By increasing the concentration of coacervator (HFIP or TFE) at a constant surfactant concentration, transition from a single liquid phase to a two or multiple phase mixture, and then eventually to a single liquid phase was observed. TBAB/HFIP mixture without additives showed a unique three-phase system before transitioning to a two-phase system upon increasing HFIP concentration. However, salt addition eliminated this three-phase region and expanded the region of two-phase formation. Select two-phase systems composed of TBAB and a perfluoroalcohol (HFIP or TFE) were utilized to extract model proteins of ranging hydrophobicity. All coacervate phases extracted bacteriorhodopsin, a membrane protein, and gramicidin, a very hydrophobic polypeptide ion channel. The most hydrophilic protein in the mixture, ribonuclease A, remained in aqueous phases. The coacervates formed from TBAB/TFE/200 mM NaCl mixture and TBAB/HFIP mixture exhibited the most selectivity in extracting proteins of high hydrophobicity. The partition coefficient (P) for each protein was calculated using the ratio of the protein concentration in the coacervate to that in the aqueous-rich phases. TBAB (50 mM)/HFIP (8%, v/v) coacervate showed remarkable selectivity and a high partition coefficient (>100) for both bacteriorhodopsin and gramicidin. Thus, this system may potentially be beneficial for facile fractionation of hydrophobic and membrane proteins in proteomics applications.

New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of 31P may be used to quantify these compounds at the level of 80μgL−1, while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3mgL−1. The limit of detection was in the range of 0.07 and 0.13mgL−1. Accuracy varied with concentration, but was in the range of 3%.

Molten globule-like partially folded state of Bacillus licheniformis α-amylase at low pH induced by 1,1,1,3,3,3-hexafluoroisopropanol.

Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) on acid-denatured Bacillus licheniformis α-amylase (BLA) at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE222 nm) up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0.

Simple assay of plasma sevoflurane and its metabolite hexafluoroisopropanol by headspace GC–MS

The anesthetic sevoflurane can now be delivered over periods of up to 48 h using a newly developed medical system, the AnaConDa (anesthetic conserving device). Lack of pharmacokinetic data on sevoflurane and its main metabolite (hexafluoroisopropanol, HFIP) in this indication prompted us to develop a headspace GC–MS method to quantify the two substances. The only previously published method for assaying the two substances could not be adapted to our study since it uses expensive and rarely employed system components together with toxic carbon disulfide as a dilution solvent. The method developed is straightforward and uses the relatively non-toxic solvent undecane as dilution solvent and chloroform as internal standard. The method is linear for a concentration range of 1–150 μg/ml, and presents high accuracy and precision. LOD and LOQ are 0.2 and 1 μg/ml, with a short analysis time (7.6 min for a single analysis). The method was applied to determine the plasma levels of sevoflurane and HFIP in six patients under 48-h anesthetic sedation delivered via the AnaConDa system. Average sevoflurane and HFIP concentrations plateaued at 75 and 4 μg/ml, respectively. Sevoflurane quickly tailed off after inhalation was stopped, and HFIP levels remained low.

Single electron transfer‐living radical polymerization of methyl methacrylate in fluoroalcohol: Dual control over molecular weight and tacticity

AbstractThe Cu(0)‐mediated single electron transfer‐living radical polymerization (SET‐LRP) of methyl methacrylate (MMA) using ethyl 2‐bromoisobutyrate (EBiB) as an initiator with Cu(0)/N,N,N′,N′′,N′′‐pentamethyldiethylenetriamine as a catalyst system in 1,1,1,3,3,3‐hexafluoro‐2‐propanol (HFIP) was studied. The polymerization showed some living features: the measured number‐average molecular weight (Mn,GPC) increased with monomer conversion and produced polymers with relatively low polydispersities. The increase of HFIP concentration improved the controllability over the polymerization with increased initiation efficiency and lowered polydispersity values. 1H NMR, MALDI‐TOF‐MS spectra, and chain extension reaction confirmed that the resultant polymer was end‐capped by EBiB species, and the polymer can be reactivated for chain extension. In contrast, in the cases of dimethyl sulfoxide or N,N‐dimethylformamide as reaction solvent, the polymerizations were uncontrolled. The different effects of the solvents on the polymerization indicated that the mechanism of SET‐LRP differed from that of atom transfer radical polymerization. Moreover, HFIP also facilitated the polymerization with control over stereoregularity of the polymers. Higher concentration of HFIP and lower reaction temperature produced higher syndiotactic ratio. The syndiotactic ratio can be reached to about 0.77 at 1/1.5 (v/v) of MMA/HFIP at −18 °C. In conclusion, using HFIP as SET‐LRP solvent, the dual control over the molecular weight and tacticity of PMMA was realized. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 6316–6327, 2009

Fluoroalcohols induced unfolding of Succinylated Con A: Native like β-structure in partially folded intermediate and α-helix in molten globule like state

Concanavalin A (Con A) exists in dimeric state at pH 5. In concentration range 20–60% (v/v) 2,2,2-trifluoroethanol (TFE) and 2–40% (v/v) 1,1,1,3,3,3-hexafluoroisopropanol (HFIP), Con A at pH 5.0 shows visible aggregation. However, when succinyl Con A was used, no aggregation was observed in the entire concentration range of fluoroalcohols (0–90% v/v TFE and HFIP) and resulted in stable α-helix formation. Temperature-induced concentration-dependent aggregation in Con A was also found to be prevented/reduced in succinylated form. Possible role of electrostatic repulsion among residues in the prevention of hydrophobically driven aggregation has been discussed. Results indicate that succinylation of a protein resulted in greater stability (in both β-sheet and α-helical forms) against alcohol-induced and temperature-induced concentration-dependent aggregation and this observation may play significant role in amyloid-forming proteins. Effect of TFE and HFIP on the conformation of a dimeric protein, Succinylated Con A, has been investigated by circular dichroism (CD), fluorescence emission spectroscopy, binding of hydrophobic dye ANS (8-anilinonaphthalene-1-sulfonic acid). Far UV-CD, a probe for secondary structure shows loss of native secondary structure in the presence of low concentration of both the alcohols, TFE (10% v/v) and HFIP (4% v/v). Upon addition of higher concentration of these alcohols, Succinylated Con A exhibited transformation from β-sheet to α-helical structure. Intrinsic tryptophan fluorescence studies, ANS binding and near UV-CD experiments indicate the protein is more expanded, have more exposed hydrophobic surfaces and highly disrupted tertiary structure at 60% (v/v) TFE and 30% (v/v) HFIP concentrations. Taken together, these results it might be concluded that TFE and HFIP induce two intermediate states at their low and high concentrations in Succinyl Con A.

NMR Investigation of the Dihydrogen‐Bonding and Proton‐Transfer Equilibria between the Hydrido Carbonyl Anion [HRe2(CO)9]− and Fluorinated Alcohols

The interaction of fluorinated alcohols with the anionic hydrido complex [HRe2(CO)9]- (1) has been investigated by NMR spectroscopy. According to the acidic strength of the alcohols, the interaction may result not only in the formation of dihydrogen-bonded ROH...[HRe2(CO)9]- adducts 2, but also in proton transfer to give the neutral species [H2Re2(CO)9] (3). With the weaker acid trifluoroethanol (TFE) evidence for the occurrence of the dihydrogen-bonding equilibrium was obtained by 2D 1H NOESY. The dependence of the hydride chemical shift on TFE concentration at different temperatures provided values for the constants of this equilibrium, from which the thermodynamic parameters were evaluated as deltaH(degrees) = -2.6(2) kcal mol(-1), deltaS(degrees) = -9.3(2) cal mol(-1) K(-1). This corresponds to a rather low basicity factor (E(j) = 0.64). Variable-temperature T1 measurements allowed the proton-hydride distance in adduct 2 a to be estimated (1.80 angstroms). In the presence of hexafluoroisopropyl alcohol (HFIP) simultaneous occurrence of both dihydrogen-bonding and proton-transfer equilibria was observed, and the equilibria shifted versus the protonated product 3 with increasing HFIP concentration and decreasing temperature. Reversible proton transfer between the alcohol and the hydrido complex occurs on the NMR timescale, as revealed by a 2D 1H EXSY experiment at 240 K. For the more acidic perfluoro-tert-butyl alcohol (PFTB) the protonation equilibrium was further shifted to the right. Thermal instability of 3 prevented the acquisition of accurate thermodynamic data for these equilibria. The occurrence of the proton-transfer processes (in spite of the unfavorable pK(a) values) can be explained by the formation of homoconjugated RO...HOR- pairs which stabilize the alcoholate anions.

Micellar Aggregates Formed Following the Addition of Hexafluoroisopropanol to Phospholipid Membranes

The addition of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) to aqueous phospholipid membranes leads to perturbation of the bilayer. In the case of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), calorimetric and small-angle X-ray scattering analyses indicate that effects are already apparent at bound molar HFIP/lipid ratios of less than 1:150, with a pronounced decrease in the temperature of the main (gel to liquid crystalline) phase transition and a decrease in the intensity of the first- and second-order scattering reflections. As the HFIP concentration is raised further, at bound molar HFIP/lipid ratios >2:1, uniform isotropic particulate structures are formed with higher intrinsic curvature than the parent liposomes. These observations are supported by the results of thin-film experiments and are consistent with the formation of DMPC/HFIP adducts that are detergent-like in nature. In the case of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) the effects are much less marked, with no blebbing observed over a comparable range of HFIP concentrations. Although HFIP interacts strongly with DOPC membranes, it appears that membrane rupture is not promoted as readily with this lipid. Data from electron microscopy, laser correlation spectroscopy, and marker release experiments suggest that some of the immediate (nonequilibrium) effects of HFIP on membranes are the consequence of microinhomogeneity in water/HFIP mixtures. On the basis of our observations, we propose a model for the interaction of HFIP with phospholipid membranes.

Kinetic Studies of Olefin Epoxidation with Hydrogen Peroxide in 1,1,1,3,3,3‐Hexafluoro‐2‐propanol Reveal a Crucial Catalytic Role for Solvent Clusters

AbstractThe epoxidation of cyclooctene and 1‐octene with hydrogen peroxide was studied kinetically in HFIP (1,1,1,3,3,3‐hexafluoro‐2‐propanol)/1,4‐dioxane mixtures. In the case of cyclooctene, no additional catalyst was applied, whereas the epoxidation of 1‐octene was run in the presence of phenylarsonic acid as catalyst. For both reaction types, two kinetic regimes can be distinguished: at low HFIP concentration (nHFIP/ntotal≤0.15), both the catalyzed and the uncatalyzed reaction show first order dependence on the HFIP concentration. This result is interpreted by the HFIP “charge template” for the uncatalyzed epoxidation (as postulated by Shaik and Neumann) and by the formation of an arsonic acid mono‐HFIP ester for the catalyzed reaction. At high HFIP concentration (nHFIP/ntotal≥0.5), both reaction types are up to ca. 5 orders of magnitude faster and show ca. 12th order dependence on the HFIP concentration. We propose that the dramatic accelerations observed at high HFIP concentrations are brought about by HFIP clusters. Based on literature data (Fioroni, Roccatano, Hong, Suhm), it is concluded that the epoxidation reactions studied here take place in a HFIP coordination sphere comprising ca. 12 HFIP molecules, and that this coordination sphere effects the enormous increases in epoxidation rates. This mechanistic model bears resemblance to enzymatic reactions taking place in and being catalyzed by a protein matrix.

Structure and dynamics of hexafluoroisopropanol-water mixtures by x-ray diffraction, small-angle neutron scattering, NMR spectroscopy, and mass spectrometry

The structure and dynamic properties of aqueous mixtures of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) have been investigated over the whole range of HFIP mole fraction (xHFIP) by large-angle x-ray scattering (LAXS), small-angle reutron scattering (SANS), F19-, C13-, and O-NMR17 chemical shifts, O-NMR17 relaxation, and mass spectrometry. The LAXS data have shown that structural transition of solvent clusters takes place at xHFIP∼0.1 from the tetrahedral-like hydrogen bonded network of water at xHFIP⩽∼0.1 to the structure of neat HFIP gradually formed with increasing HFIP concentration in the range of xHFIP⩾0.15. The Ornstein–Zernike plots of the SANS data have revealed a mesoscopic structural feature that the concentration fluctuations become largest at xHFIP∼0.06 with a correlation length of ∼9 Å, i.e., maximum in clustering and microhetrogeneities. The F19 and C13 chemical shifts of both CF3 and CH groups of HFIP against xHFIP have shown an inflection point at xHFIP∼0.08, implying that the environment of HFIP molecules changes due to the structural transition of HFIP clusters. The O17 relaxation data of water have shown that the rotational motion of water molecules is retarded rapidly upon addition of HFIP into water up to xHFIP∼0.1, moderately in the range of ∼0.1<xHFIP≲0.3, and almost constant at xHFIP≳0.3, reflecting the structural change in the solvent clusters at xHFIP∼0.1. The mass spectra of cluster fragments generated in vacuum from HFIP-water mixtures have shown that the predominant clusters are A1Wn (n<12, A=HFIP, W=water) and water clusters Wn (n=5–8) at xHFIP=0.09 and 0.20 and only HFIP oligomers in a water-rich region of xHFIP=0.005∼0.01. From all the information obtained in the present study, the models are proposed for the aggregation of HFIP and water molecules in HFIP-water mixtures.

MOLECULAR WEIGHT DISTRIBUTION OF POLYAMIDES BY GPC-VISCOMETRY. A COMPARISON BETWEEN HIGH TEMPERATURE AND LOW TEMPERATURE ELUANTS

Synthetic polyamides (PA) owe many outstanding properties to their semicristalline morphology and to the intermolecular hydrogen bonding of the amide groups. Although the strength of the Van der Waals network confers an exceptional solvent resistance to this class of polymers, it may lead to serious difficulties in their solution characterization, such as the determination of molecular weight distribution (MWD). Over the years, a large number of dissolution schemes have been developed for the GPC characterization of PA. A recent IUPAC round-robin test on the GPC of aliphatic PAs nevertheless showed large variations in the experimental data not only between laboratories employing different methods, but also between laboratories adopting similar protocols. The purpose of the present investigation is to understand the origins of these disparities by comparing GPC-Viscometry data obtained with some frequently used eluant systems: a. benzyl alcohol at high temperature; b. hexafluoro-isopropanol, pure or mixed with dichloromethane, at low temperature; c. common GPC solvents (THF, CH2Cl2,) after N-trifluoracetylation of the PA. Apart from pure HFIP, all the methods tested show consistent MW results when measured in the same laboratory. Viscometry data reveal, however, that the expected linear viscosity law is obeyed only in benzyl alcohol. With the other eluants, significant departure from the Mark-Houwink relation denotes presence of non-size exclusion effects, rendering data evaluation imprecise with the Universal Calibration. The problems are particularly acute for polyamides with short methylene sequences which can only be dissolved in mixtures containing a high concentration of HFIP. To improve accuracy, it is proposed that GPC calibration for this class of polymer be performed with absolute detection (on-line viscometer, light scattering) or with a broad MWD standard of identical chemical composition to the analyzed sample.

Kinetic Study of Hydrogen Bonded Exciplex Formation of N9-methyl Harmane

The kinetics of exciplex formation of N9-methyl-1-methyl-9H-pyrido[3,4-b]indole, MHN, in the presence of the proton donor hexafluoro2-propanol, HFIP, in cyclohexane has been studied by UV-vis, steady-state, and time-resolved fluorescence measurements. The results conclusively show the formation of a 1:2 ground-state proton-transfer hydrogen bonded complex, PTC, between the pyridinic nitrogen of the substrate and the proton donor. The formation of these complexes is a necessary prerequisite for the exciplex to be observed. Thus, upon excitation of PTC, an excited-state equilibrium is established between PTC* and a cation like exciplex, CL*, λem. 410 nm. This excited-state reaction is assisted by a proton donor molecule. From the analysis of the multiexponential decays, measured at different emission wavelengths and as a function of HFIP concentration, the excited-state kinetics of this phototautomeric process has been analyzed in detail.

Preparation of Cationic Cyclopentadienyl Diene Molybdenum Complexes through Hydride Abstraction Effected by Di- or Triarylmethyl Halides in the Presence of Hexafluoro-2-propanol

Hydride abstraction from the model system (η5-cyclopentadienyl)dicarbonyl[(1−3-η)-1-cyclohexen-3-yl]molybdenum (1) was effected using halides such as Ph3CCl (3b), Ph3CBr (3a), and (4-MeOC6H4)2CHBr (5), or their polymer-bound analogues, as carbocation precursors. The presence of hexafluoro-2-propanol (HFiP) as a cosolvent was critical for hydride abstraction to proceed. Good yields of (η5-cyclopentadienyl)dicarbonyl[(1−4-η)-cyclohexadiene]molybdenum halides 2a,b were obtained from soluble as well as polymer-supported hydride abstractors. The influence of HFiP concentration and carbocation precursor on reaction efficiency was investigated.

Thermochromism and solvatochromism of non-ionic polar polysilanes

Polar polysilanes bearing ethereal side groups [{CH 3Si(CH 2) m O(CH 2CH 2O) n CH 3} x , m=3–5 and n=0–3}] were prepared and were found to be soluble in a wide range of polar solvents. UV spectroscopic behavior is highly dependent on the nature of the solvents used but no simple relationship between λ max and specific solvent parameter is found. Small molar absorbance ( ε<10 000/SiSi bond) suggests that water-soluble polysilanes take highly entangled form in water. Solvatochromic redshift is induced by THF in aqueous solution of water-soluble polysilanes. This solvatochromism is ascribed to disentanglement the non-polar main chain by non-polar solvent. Another type of solvatochromic shift is also caused by (CF 3) 2CHOH (HFIP) in non-polar solvent (benzene, CH 2Cl 2) or even in (CH 3) 2CHOH solution, where higher HFIP concentration brings about longer wavelength absorption. This type of solvatochromism is originated by strong hydrogen bond formation between the ethereal side groups and HFIP. Because non-dissociative nature of HFIP-concerned hydrogen bonding, increased bulkiness of the side moieties brings about disentanglement. Polysilanes 1– 5, though having long side chains, show thermochromism of continuous spectral shift on cooling. The same λ max value in thermochromism and solvatochromism indicates that nearly the same degree of disentanglement is caused by HFIP and thermally.

Fluorinated alcohol, the third group of cosolvents that stabilize the molten-globule state relative to a highly denatured state of cytochrome c.

The effects of 1,1,1,3,3,3-hexafluoro-isopropanol (HFIP) on the conformation of cytochrome c (cyt c) at pH 1.9 were studied using a combination of spectroscopic and physical methods. Analysis varying the HFIP concentration showed that a compact denatured conformation (M(HF)) accumulates in a low concentration range of HFIP in the middle of structural transition from the highly unstructured acid-denatured state to the highly helical alcohol-denatured state of cyt c. This contrasts clearly with the effect of isopropanol (IP), in which no compact conformation accompanied with the transition. Analysis varying concentrations of HFIP and NaCl concurrently showed that the M(HF) state of cyt c is essentially identical to the salt-induced molten-globule (M(G)) state, and the M(G) state in the presence of salt was also stabilized by a low concentration of HFIP. Furthermore, 2,2,2-trifluoroethanol stabilized M(HF) similarly to HFIP, supporting the proposition that the specific effect observed for HFIP is caused by fluorination of alcohol. The mechanism stabilizing compact conformation by HFIP remains unclear, but is probably distinct from that of salts and polyols, which are also known to stabilize the M(G)-like state.

Separation of Scrapie Prion Infectivity from PrP Amyloid Polymers

The prion protein (PrP) undergoes a profound conformational change when the cellular isoform (PrP C) is converted into the scrapie form (PrP Sc). Limited proteolysis of PrP Scproduces 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 α-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 β-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 PrP Scconformation. The results also demonstrate that the specific β-sheet-rich structures required for prion infectivity can be differentiated from those needed for amyloid formation as determined by Congo red binding.

Biotransformation of sevoflurane.

Several characteristics of sevoflurane biotransformation are apparent from the preceding investigations. Metabolism is rapid, with fluoride and HFIP appearing in plasma within minutes after the start of sevoflurane administration (38-40,51). Peak plasma fluoride concentrations generally occur within approximately 1 h after the termination of sevoflurane administration in most patients, regardless of the dose or duration of exposure (ranging from 0.35-9.5 MAC-h) (39,48). Peak plasma inorganic fluoride concentrations are proportional to sevoflurane dose, measured in MAC-h (42-44). Inorganic fluoride concentrations decline rapidly after termination of sevoflurane administration, with concentrations well below peak levels by the first postoperative day. HFIP is rapidly conjugated, with more than 85% circulating in plasma as the glucuronide. Plasma HFIP concentrations peak later than fluoride concentrations, but both metabolites are eliminated at similar rates (52). Metabolism of sevoflurane does not contribute to the termination of clinical drug effect (52), unlike more extensively metabolized drugs such as halothane (55). Sevoflurane is metabolized by P-450 2E1, so pathophysiologic factors and drug interactions altering P-450 2E1 activity will also influence sevoflurane metabolism (52). The extent of metabolism of sevoflurane, 2% to 5%, is less than that of all other volatile anesthetics except isoflurane and desflurane. It has been proposed that the ideal anesthetic should resist biotransformation because anesthetic toxicity is related to anesthetic metabolism (67,68). Experience to date suggests that biotransformation of sevoflurane has not been causally related to either hepatic or renal toxicity. Sevoflurane does not result in formation of fluoroacetylated liver neoantigens or other reactive metabolites. Although both sevoflurane and methoxyflurane may produce plasma fluoride concentrations in excess of 50 microM, they have not produced the same nephrotoxic effects. Clearly, anesthetic metabolism and anesthetic toxicity can no longer be considered synonymous. The introduction of sevoflurane into clinical practice will hopefully stimulate new investigations into biochemical mechanisms of anesthetic toxicity and continued clinical investigations regarding the relationship between anesthetic metabolism and organ toxicity.

The β-Sheet Structure-Stabilizing Potential of Twenty Kinds of Amino Acid Residues in Protected Peptides

Abstract A host–guest approach was devised to investigate the β-sheet structure-stabilizing potential of the 20 common amino acid residues in protected peptides where functional groups in side chains were protected by suitable groups commonly used in peptide synthesis. The potential was evaluated using solvent-titration curves for the disruption of the β-sheet structure of cross-linked polystyrene resin-bound host–guest hexa- and octapeptides in CH2Cl2 by HFIP. The β-sheet structure of host–guest hexapeptides, Boc–Val–Ile–X2–Val–Ile-NHCH2–resins, in which X stands for guest amino acid residues, was disrupted in CH2Cl2 by adding increasing amounts of HFIP; the disrupted behaviors were strongly dependent on the nature of guest amino acid residues. A scale for the β-sheet structure-stabilizing potential of the 20 kinds of amino acid residues in CH2Cl2 could be derived at 0.25 M of HFIP concentration as follows: Ile, Val&amp;gt;&amp;gt;Asn&amp;gt;His(Tos) or His, Gln, Cys(Bzl), Tyr(Bzl)&amp;gt;Phe, Gly, Lys(Z), Arg(Tos), Leu&amp;gt;Thr(Bzl), Ala, Met(O), Glu(OBzl), Trp(CHO)&amp;gt;&amp;gt;Ser(Bzl), Asp(OBzl)&amp;gt;&amp;gt;Pro. To confirm the scale, their β-sheet structure-stabilizing potential was further examined using host–guest octapeptides, Boc–X2–Val–Ile–X2–Val–Ile–NHCH2–resins. The disrupted behaviors of the octapeptides having Gly, Ala, and Thr(Bzl) residues as guests were remarkably different from those of the corresponding hexapeptides; the β-sheet structure-stabilizing potential of the Gly, Ala, and Thr(Bzl) residues became significantly high with an increase in their ratios to host residues. The potential of the Gly, Ala, and Thr(Bzl) residues appears to reflect the ratios of guest amino acid residues to hosts, the repeated sequence, and/or neighbor interactions in the sequence. The significance of the present study in the design of synthetic routes for peptides and proteins is briefly discussed.