Sites For Isomerization Research Articles

Influence of sulfating method on La–Ni–S2O82–/ZrO2–Al2O3 solid superacid catalyst for n-pentane isomerization

La–Ni–S2O82–/ZrO2–Al2O3 catalysts were successfully prepared by two different methods of sulfate impregnation, and the physico-chemical properties of the catalysts were characterized by X-ray diffraction, Brunauer–Emmett–Teller analysis, Fourier transform infrared spectroscopy, pyridine adsorption–infrared spectroscopy, and X-ray photoelectron spectroscopy techniques. Catalytic activities were evaluated in a fixed-bed flow reactor using n-pentane isomerization as the probe reaction. Compared with catalyst La–Ni–S2O82–/ZrO2–Al2O3-I, prepared by the traditional impregnation method, the catalyst La–Ni–S2O82–/ZrO2–Al2O3-W, prepared by the incipient-wetness impregnation method, possessed higher pore volume, pore size, sulfur content, and stronger Brønsted acid sites. The catalytic activity for La–Ni–S2O82–/ZrO2–Al2O3-W was maintained at around 56% within 3000 min with an isopentane selectivity of 88% which showed much greater stability than that of La–Ni–S2O82–/ZrO2–Al2O3-I. This can be attributed to the fact that (1) the large pore size and pore volume of La–Ni–S2O82–/ZrO2–Al2O3-W can largely suppress carbon deposition and (2) the more numerous and stronger Brønsted acid sites for La–Ni–S2O82–/ZrO2–Al2O3-W guaranteed to provide enough acid sites for isomerization during the reaction process.

Distribution of Effective Ferrierite Active Sites for Skeletal Isomerization of n‐Butene to Isobutene

AbstractIn this study, the effective active sites of aged ferrierite(FER) were confirmed to be located in the 10‐membered ring (10‐MR) pore mouths by modification with ammonium hexafluorosilicate((NH4)2SiF6 )and long‐term experiments. Analysis of SiO2/Al2O3 ratios showed that aluminum atoms were selectively removed from the FER surface after the modification with (NH4)2SiF6. Comparison of the catalytic properties before and after modification showed that the catalytic activity of aged FER depended only on the amount of acid sites located in the 10‐MR pore mouths. Monitoring of the change in acid amount with reaction time and the difference between aged and deactivated FER showed that aged FER retained only weak acid sites, and its deactivation was caused by carbon deposition on the 10‐MR pore mouth acid sites. Therefore, the catalytic performance of FER can be improved by increasing the number of 10‐MR pore mouths.

Location of the Active Sites for Ethylcyclohexane Hydroisomerization by Ring Contraction and Expansion in the EUO Zeolitic Framework

Identifying the location of the active sites in a zeolite is a current challenge, impeding the design of optimal catalysts. In this work, we identify the location of the most active sites of 1-ethylcyclohexene isomerization in the EUO framework (10 MR channels, 12 MR side pockets) thanks to DFT calculations corroborated by experiments. Skeletal isomerization of cycloalkenes is a crucial industrial reaction for the bifunctional isomerization of ethylbenzene. Ethylcyclohexene is protonated by framework protons into cyclic carbenium ions, which undergo ring contraction–expansion reactions through protonated cyclopropane (PCP) like transition states. Ab initio calculations clearly show that the acid sites located at the intersection between the channel and the pocket stabilize much less the cyclic carbenium ions involved in the reaction than 12 MR pockets and 10 MR channel sites due to stronger dispersion stabilizing interactions. This computational finding is fully confirmed experimentally by the comparison ...

Highly effective transformation of carbohydrates to 5-Hydroxymethylfurfural with Al- montmorillonite as catalyst

Aluminum ion-exchanged Ca-montmorillonite with different concentrations of aluminum ion were prepared and applied in the conversion of carbohydrates to 5-hydroxymethylfurfural (HMF). It was found that two types of acid sites were present in the catalyst, Lewis acid sites for isomerization of glucose and Brønsted acid sites for dehydration of fructose. With Al-mont-10 as catalyst, the yield of HMF converted from glucose was 80.4% in a biphase system (THF: NaCl solution) at 180 °C for 2.5 h. Further, the catalyst also shows excellent catalytic performance in the conversion of starch and inulin, and the yield of HMF reached 60.1% and 81.6% under identical conditions, respectively.

Coformulation of Broadly Neutralizing Antibodies 3BNC117 and PGT121: Analytical Challenges During Preformulation Characterization and Storage Stability Studies

In this study, we investigated analytical challenges associated with the formulation of 2 anti-HIV broadly neutralizing antibodies (bnAbs), 3BNC117 and PGT121, both separately at 100 mg/mL and together at 50 mg/mL each. The bnAb formulations were characterized for relative solubility and conformational stability followed by accelerated and real-time stability studies. Although the bnAbs were stable during 4°C storage, incubation at 40°C differentiated their stability profiles. Specific concentration-dependent aggregation rates at 30°C and 40°C were measured by size exclusion chromatography for the individual bnAbs with the mixture showing intermediate behavior. Interestingly, although the relative ratio of the 2 bnAbs remained constant at 4°C, the ratio of 3BNC117 to PGT121 increased in the dimer that formed during storage at 40°C. A mass spectrometry-based multiattribute method, identified and quantified differences in modifications of the Fab regions for each bnAb within the mixture including clipping, oxidation, deamidation, and isomerization sites. Each bnAb showed slight differences in the levels and sites of lysine residue glycations. Together, these data demonstrate the ability to differentiate degradation products from individual antibodies within the bnAb mixture, and that degradation rates are influenced not only by the individual bnAb concentrations but also by the mixture concentration.

Asp 58 modulates lens αA-crystallin oligomer formation and chaperone function.

Senile cataract onset is caused by insolubilization of lens proteins. The lens crystallin protein family correctly orders the formation of homo- or hetero-oligomers in lens fiber cells. Because lens fiber cells do not divide, covalent post-translational modifications, such as isomerization of aspartate residues, accumulate with aging. Although many isomerization sites of αA-crystallin have been reported, their structural and functional contributions have never been identified. In this study, αA-crystallin was extracted from aged human lens and separated into each oligomeric state by size exclusion chromatography and electrophoresis. The novel combination methodology of in-solution/gel tryptic digestion with liquid chromatography equipped with mass spectrometry (LC-MS/MS) was used to evaluate the isomerization of Asp 58. The contributions of isomerization to assembly, solubility, and chaperone functions of αA-crystallin were estimated using a series of mutations of Asp 58 in αA-crystallin. The results indicated that the isomerization of Asp 58 depended on the oligomer size and age of the lens. The substitution of Asp 58 for hydrophobic residues increased αA-crystallin oligomer size and decreased solubility. All substitutions decreased the chaperone function of αA-crystallin for aggregates of bovine βL-crystallin and alcohol dehydrogenase. The data indicated that Asp 58 in αA-crystallin was critical for intermolecular interactions in the lens. Our results also suggested that LC-MS/MS-based isomerization analyses of in-gel-digested products could be useful for investigating the isomerization of Asp residues in oligomeric states. This method could also be used to analyze d/l ratios of amino acid residues in soluble protein aggregates.

Identification of Isomeric Aspartate residues in βB2-crystallin from Aged Human Lens

Many post-translational modifications such as oxidation, deamidation and isomerization of amino acid residues occur in lens proteins with aging. One such modification, isomerization of aspartate in lens α-crystallin, has been well studied by amino acid enantiomer analysis and LC-MS/MS. LC-MS/MS can quickly and easily identify D- and L-amino acid-containing peptides without purification of lens protein mixtures. However, this method has a weak point in that isomeric peptides of major components are detected predominantly, while those from minor proteins such as β- and γ-crystallins have not been fully determined. Therefore, the isomerization of amino acid residues in β- and γ-crystallin families has been little studied. To solve those problems and detect the isomerization of Asp residues in lens βB2-crystallin, the main component of the β-crystallin family, here we have developed steps for sample fractionation before d/l analysis based on either LC-MS/MS or amino acid derivatization to diastereoisomers followed by RP-HPLC. To capture a small amount of peptide, a multiple reaction monitoring (MRM) method based on quadrupole MS/MS (Q-MS) was applied to the water-soluble fraction of whole lens. The d/l analysis based on both LC-MS/MS and diastereoisomer formation showed the presence of multiple isomerization sites, including Asp4, Asp83, Asp92 and Asp192, in βB2-crystallin in aged lens. These isomerization sites were confirmed to exist in an age-dependent manner by Q-MS. Synthetic peptides of βB2-crystallin containing different isomers of Asp showed differential elution profiles during RP-HPLC, indicating differences in the local structure or hydrophobicity of Asp-isomer-containing peptides. These results suggest that the isomerization sites are distributed on exposed regions of βB2-crystallin and thus likely to have an impact on crystallin subunit–subunit interactions, induce abnormal crystallin aggregation, and contribute to senile cataract formation in aged lens.

Conversion of glucose into lactic acid using silica-supported zinc oxide as solid acid catalyst

Abstract Zinc oxide (ZnO) has been proven to be highly effective in converting biomass into fine chemicals. It possesses several limitations, such as leaching in hydrothermal reactions and difficulty with regard to its recovery. Supporting ZnO on silica improves its recovery, stability and recyclability. In this study, we produced silica-supported ZnO by incipient wetness impregnation (IWI) method for the conversion of glucose into lactic acid. The presence of the ZnO provided active sites for isomerization to occur. The highest yield of lactic acid was 39.2% at 180 °C for 60 min. Prolonged reaction time and higher reaction temperature promoted further degradation of lactic acid into acetic acid. The yield of lactic acid decreased after the first cycle and decreased slightly for the nine consecutive cycles.

Insight into Aluminum Sulfate-Catalyzed Xylan Conversion into Furfural in a γ-Valerolactone/Water Biphasic Solvent under Microwave Conditions.

A simple and efficient biphasic system with an earth-abundant metal salt catalyst was used to produce furfural from xylan with a high yield of up to 87.8 % under microwave conditions. Strikingly, the metal salt Al2 (SO4 )3 exhibited excellent catalytic activity for xylan conversion, owing to a combination of Lewis and Brønsted acidity and its ability to promote good phase separation. The critical role of the SO42- anion was first analyzed, which resulted in the aforementioned characteristics when combined with the Al3+ cation. The mixed solvent system with γ-valerolactone (GVL) as the organic phase provided the highest furfural yield, resulting from its good dielectric properties and dissolving capacity, which facilitated the absorption of microwave energy and promoted mass transfer. Mechanistic studies suggested that the xylan-to-furfural conversion proceeded mainly through a hydrolysis-isomerization-dehydration pathway and the hexa-coordinated Lewis acidic [Al(OH)2 (aq)]+ species were the active sites for xylose-xylulose isomerization. Detailed kinetic studies of the subreaction for the xylan conversion revealed that GVL regulates the reaction rates and pathways by promoting the rates of the key steps involved for furfural production and suppressing the side reactions for humin production. Finally, the Al2 (SO4 )3 catalyst was used for the production of furfural from several lignocellulosic feedstocks, revealing its great potential for other biomass conversions.

Identification of Sequence Similarities among Isomerization Hotspots in Crystallin Proteins.

The eye lens crystallins represent an ideal target for studying the effects of aging on protein structure. Herein we examine separately the water-soluble (WS) and water-insoluble (WI) crystallin fractions and identify sites of isomerization and epimerization. Both collision-induced dissociation and radical-directed dissociation are needed for detection of these non-mass-shifting post-translational modifications. Isomerization levels differ significantly between the WS and the WI fractions from sheep, pig, and cow eye lenses. Residues that are most susceptible to isomerization are identified site-specifically and are found to reside in structurally disordered regions. However, isomerization in structured domains, although less common, often yields more dramatic effects on solubility. Numerous isomerization hotspots were also identified and occur in regions with aspartic acid and serine repeats. For example, 128KMEIVDDDVPSLW140 in βB3 crystallin contains three sequential aspartic acid residues and is isomerized heavily in the WI fractions, while it is not modified at all in the WS fractions. Potential causes for enhanced isomerization at sites with acidic residue repeats are presented. The importance of acidic residue repeats extends beyond the lens, as they are found in many other long-lived proteins associated with disease.

Controlled insertion of tin atoms into zeolite framework vacancies and consequences for glucose isomerization catalysis

The incorporation of tin heteroatoms within framework vacancy defects of dealuminated Beta zeolites via stannic chloride reflux in dichloromethane solvent (333K) enables preparing Sn-Beta zeolites comprising a wide range of Sn content (Si/Sn=30–144; 1.4–6.1wt% Sn) and >4× higher framework Sn densities than reported (Si/Sn>120) for reflux in isopropanol solvent (383K). Silanol defects form hydrogen bonds with isopropanol but not with dichloromethane under reflux conditions, evident in the evolution of adsorbed isopropanol (∼5× per vacancy) but not dichloromethane during temperature programmed desorption of dealuminated zeolites saturated with either solvent at ambient, and in IR features for perturbed hydroxyl stretches (∼3350cm−1) and CH stretches (∼2700–3000cm−1) that were retained after dealuminated zeolites were saturated with isopropanol and evacuated at 383K. Dichloromethane-assisted tin grafting provides a route to precisely control the density of framework Sn sites and residual vacancy defects in Sn-Beta, up to the point of grafting virtually every framework vacancy with Sn. Open Sn sites ((OSi)3Sn(OH), ν(CD3CN): 2316cm−1), quantified from IR spectra collected after CD3CN titration (303K), are preferentially incorporated over closed Sn sites (Sn(OSi)4, ν(CD3CN): 2308cm−1) at low Sn densities via grafting in dichloromethane solvent, suggesting that this preparation method can tune the open-to-closed framework Sn site ratio more systematically than hydrothermal synthesis methods. Open Sn sites are dominant active sites for aqueous-phase glucose-fructose isomerization; consequently, isomerization turnover rates (per total Sn) decrease systematically with increasing Sn content. Initial isomerization rates (per open Sn) are invariant (within ∼2×, 373K) among twelve Sn-Beta samples of varying Sn content (Si/Sn=30–144), the behavior expected of a single-site catalyst in which open framework Sn sites are the loci of catalytic reactivity. First-order isomerization rate constants are 15–50× lower (373K) when open Sn sites are confined within high-defect than within low-defect Beta micropores, consistent with previous reports that aqueous-phase sugar isomerization cycles turn over at faster rates within hydrophobic, low-defect micropores. These findings clarify the consequences of liquid-phase reflux procedures on the coordination of tin heteroatoms incorporated within zeolite framework vacancies, and underscore the requirement to quantify putative active site structures in order to rigorously normalize measured rate data prior to kinetic or mechanistic interpretation.

In vivo regulation of human CrkII by cyclophilin A and FK506-binding protein

Members of the Crk family of adaptor proteins are key players in signal transduction from a variety of cell surface receptors. CrkI and CrkII are two alternative-spliced forms of a single gene which possess an N-terminal SH2 domain and an SH3 domain that mediate interaction with other proteins. CrkII possesses an additional C-terminal linker region plus an extra SH3 domain, which does not interact with other proteins, but operates as regulatory moiety. Utilizing human Jurkat T cells, we demonstrate that CrkII-SH3N binding of C3G is inhibited by cyclosporin A (CsA) plus FK506 that inhibit the cyclophilin A (CypA) and FK506 binding protein (FKBP) peptidyl-prolyl cis-trans isomerases (PPIases; also termed immunophilins), respectively. Jurkat T cells were found to express ∼5-fold lower levels of CrkI protein compared to CrkII, but the efficiency of C3G binding by CrkI was ∼5-fold higher than that of CrkII, suggesting that the majority of cellular CrkII proteins adopt a conformation that is inaccessible for C3G. Treatment of Jurkat T cells with CsA plus FK506 led to a time-dependent conformational change in overexpressed human CrkII1-236 protein-containing FRET-based biosensor, supporting the accumulation of cis conformers of human CrkII1-236 in the presence of PPIase inhibitors. Our data suggest that the Gly219-Pro-Tyr motif in the human CrkII linker region serves as the recognition and isomerization site of PPIases, and raise the possibility that CsA and FK506 might interfere with selected effector T cell functions via a CrkII-, but not CrkI-dependent mechanisms.

Genesis of a bi-functional acid–base site on a Cr-supported layered double hydroxide catalyst surface for one-pot synthesis of furfurals from xylose with a solid acid catalyst

The cross boundary between Cr3+ oxide and Mg–Al LDH generates highly active bi-functional acid–base sites for xylose isomerization.

Identification of amino acid epimerization and isomerization in crystallin proteins by tandem LC-MS.

Post-translational modifications that do not result in a change in mass are particularly difficult to detect by mass spectrometry. For example, isomerization of aspartic acid or epimerization of any chiral residue within a peptide do not lead to mass shifts but can be identified by examination of independently acquired tandem mass spectra or by combination with another technique. For analysis of a biological sample, this means that liquid chromatography or some other type of separation must be used to first separate the isomers from one another. Furthermore, each specific m/z of interest must be sampled repeatedly to allow for comparison of the tandem mass spectra from each separated isomer, which contrasts with the traditional approach in proteomics where the goal is typically to avoid resampling the same m/z. We illustrate that isomerization and epimerization of peptides can be identified in this fashion by examination of long-lived crystallin proteins extracted from a sheep eye lens. Tandem mass spectrometry relying on a combination of radical directed dissociation (RDD) and collision induced dissociation (CID) following separation by liquid chromatography was used to identify modified peptides. Numerous sites of isomerization and epimerization, including several that have not been previously identified, were determined with peptide specificity. It is demonstrated that the specific sites of amino acid isomerization within each peptide can be identified by comparison with synthetic peptides. For α-crystallin proteins, the sites that undergo the greatest degree of isomerization correspond to disordered regions, which may have important implications on chaperone functionality within the context of aging.

Ion-pair reversed-phase high performance liquid chromatography method for the quantification of isoaspartic acid in a monoclonal antibody

Isomerization of aspartic acid residues is one of the major causes of chemical degradation during the shelf life of biological pharmaceuticals. Monoclonal antibody biopharmaceuticals are typically stored at mildly acidic pH conditions, which can lead to the isomerization reaction. The mechanism of this non-enzymatic chemical reaction has been studied in great detail. However, the identification and quantification of the isomerization sites in a given protein still remains a challenge. We developed an ion-pair reversed-phase HPLC method for the separation of an intact monoclonal antibody variant containing a single isoaspartic acid residue from its native counterpart. We identified and characterized the isomerization site using ion-pair reversed-phase HPLC mass spectrometry methods of the reduced and alkylated antibody and the enzymatically cleaved antibody. Lys-C followed by Asp-N digestion of the antibody was used for the identification of the isomerization site. Electron transfer dissociation (ETD) mass spectrometry was used to confirm the isomerization site at a DY motif at an aspartic acid residue in the CDR-H3 region of the antibody. Tyrosine at the C-terminus of an aspartic acid residue is typically not regarded as a hot spot for isomerization. Our findings suggest that it is not possible to predict isomerization sites in proteins with confidence and all aspartic acid residues located in the CDR regions of antibodies must be considered as potential isomerization site due to the solvent exposure or the flexibility of these regions of the molecule. Additionally, the effect of the pH on the isomerization rate was evaluated using the ion-pair reversed-phase HPLC method, showing that at a lower pH the isomerization rate is faster. Storage at 25°C for 6 months resulted in an increase of the amount of isoaspartic acid to 6.6% at pH 5.4, 6.0% at pH 5.8, and 5.6% at pH 6.2.

Site-Specific Characterization of d-Amino Acid Containing Peptide Epimers by Ion Mobility Spectrometry

Traditionally, the d-amino acid containing peptide (DAACP) candidate can be discovered by observing the differences of biological activity and chromatographic retention time between the synthetic peptides and naturally occurring peptides. However, it is difficult to determine the exact position of d-amino acid in the DAACP candidates. Herein, we developed a novel site-specific strategy to rapidly and precisely localize d-amino acids in peptides by ion mobility spectrometry (IMS) analysis of mass spectrometry (MS)-generated epimeric fragment ions. Briefly, the d/l-peptide epimers were separated by online reversed-phase liquid chromatography and fragmented by collision-induced dissociation (CID), followed by IMS analysis. The epimeric fragment ions resulting from d/l-peptide epimers exhibit conformational differences, thus showing different mobilities in IMS. The arrival time shift between the epimeric fragment ions was used as criteria to localize the d-amino acid substitution. The utility of this strategy was demonstrated by analysis of peptide epimers with different molecular sizes, [d-Trp]-melanocyte-stimulating hormone, [d-Ala]-deltorphin, [d-Phe]-achatin-I, and their counterparts that contain all-l amino acids. Furthermore, the crustacean hyperglycemia hormones (CHHs, 8.5 kDa) were isolated from the American lobster Homarus americanus and identified by integration of MS-based bottom-up and top-down sequencing approaches. The IMS data acquired using our novel site-specific strategy localized the site of isomerization of l- to d-Phe at the third residue of the CHHs from the N-terminus. Collectively, this study demonstrates a new method for discovery of DAACPs using IMS technique with the ability to localize d-amino acid residues.

Relating cumene hydrocracking activity to the acidic center of CrO3–ZrO2

The properties of CrO3–ZrO2 and Pt/CrO3–ZrO2 were studied by IR and ESR spectroscopy to elucidate the active sites in n-heptane isomerization and cumene cracking. Surface analyses showed the presence of cubic, monoclinic and tetragonal phases of ZrO2 with a BET specific surface area of about 150m2/g for both catalysts. 2,6-Lutidine adsorbed IR confirmed the presence of treble doublet bands at 1675+1660, 1650+1625 and 1640+1630cm−1 corresponding to Brønsted acid sites and quartet doublet bands at 1604+1580, 1595+1580, 1590+1580 and 1570+1560cm−1 corresponding to Lewis acid sites. The presence of Pt enhanced the activity of CrO3–ZrO2 in cumene hydrocracking, in which Pt facilitated the formation of protonic acid sites at 1675+1660 and 1650+1625cm−1 through a hydrogen spillover mechanism. The IR study verified that the Lewis acidic center at 1570+1560cm−1 was involved in the formation of protons by trapping electrons. Meanwhile, neither CrO3–ZrO2 nor Pt/CrO3–ZrO2 was active in n-heptane hydroisomerization.

Effects of crystallinity of ZSM-5 zeolite on para-selective tert-butylation of ethylbenzene

Highly crystalline ZSM-5 zeolites are important for para-selective alkylation of alkyl aromatics, because they carry few external acid sites for isomerization of p-dialkyl products. Such zeolites (Si/Al = 25, 50, and 75) were synthesized in a fluoride medium between pH 4 and 6. Their crystallinities, crystal sizes, and surface areas were higher than those of a commercial ZSM-5 zeolite. Their para selectivities in alkylation were tested for vapor-phase tert-butylation of ethylbenzene between 200 and 400 °C. As expected, all the catalysts showed more than 90% para selectivity. At 300 °C, ethylbenzene conversion decreased in the order ZSM-5(25, commercial) > ZSM-5(25) > ZSM-5(50) > ZSM-5(75). The catalysts had weak, medium, and strong acid sites, but all the acid sites of ZSM-5(75) were weaker than those of ZSM-5(25) and ZSM-5(50). The high activity of commercial ZSM-5 was caused by its strong acid sites being stronger than those of the synthesized zeolites. Although the activity of the commercial catalyst was higher than those of the present catalysts, the selectivity for 4-t-butylethylbenzene (4-t-BEB) was low. The optimum feed ratio (ethylbenzene:t-butyl alcohol) was 2:1 and the feed rate was 1.65 h−1 for high ethylbenzene conversion and 4-t-BEB selectivity. Time-on-stream studies showed slow catalyst deactivation. Highly crystalline ZSM-5 zeolites are therefore better than a commercial zeolite for para-selective alkylation of alkyl aromatics. They do not require much post-modification for high para selectivity. A fluoride medium is therefore better than an alkaline medium for obtaining highly crystalline para-selective ZSM-5 zeolites.

A large geometric distortion in the first photointermediate of rhodopsin, determined by double-quantum solid-state NMR

Double-quantum magic-angle-spinning NMR experiments were performed on 11,12-(13)C(2)-retinylidene-rhodopsin under illumination at low temperature, in order to characterize torsional angle changes at the C11-C12 photoisomerization site. The sample was illuminated in the NMR rotor at low temperature (~120 K) in order to trap the primary photointermediate, bathorhodopsin. The NMR data are consistent with a strong torsional twist of the HCCH moiety at the isomerization site. Although the HCCH torsional twist was determined to be at least 40°, it was not possible to quantify it more closely. The presence of a strong twist is in agreement with previous Raman observations. The energetic implications of this geometric distortion are discussed.

WO3 monolayer loaded on ZrO2: Property–activity relationship in n-butane isomerization evidenced by hydrogen adsorption and IR studies

The property–activity relationship of WO3 supported on ZrO2 (WZ) was evaluated in n-butane isomerization for a series of catalysts with WO3 loading ranging from 5 to 20wt% on ZrO2. The catalysts were prepared by incipient-wetness impregnation of Zr(OH)4 with an aqueous solution of (NH4)6[H2W12O40·nH2O], followed by drying and calcination at 1093K. The introduction of WO3 continuously increased the tetragonal phase of ZrO2, WO3 surface density and coverage. The specific surface area and total pore volume passed through a maximum of WO3 loading at 13wt%; this loading corresponds to 5.9WO3/nm2 and is near the theoretical monolayer-dispersed limit of WO3 on ZrO2. The IR results indicate that the presence of WO3 eroded the absorbance bands at 3738 and 3650cm−1 corresponding to bibridged and tribridged hydroxyl groups up to near the monolayer-dispersed limit of WO3. A new broad and weak band appeared, centered at 2930cm−1, indicating the presence of bulk crystalline WO3 for WO3 coverage exceeding the theoretical monolayer-dispersion limit. In addition to the band at 2930cm−1, two WO stretching bands were observed at about 1021 and 1014cm−1 for all WZ catalysts, confirming the existence of WO connected to coordinative unsaturated (cus) Zr4+ through O and to the other W through O, respectively. Pyridine adsorbed IR and NH3-TPD revealed that the presence of WO3 modified the nature and concentration of acidic sites. The highest acidity was observed with 13wt% loading WO3. The decrease in the intensity of peaks due to increasing WO3 loading was much higher on Lewis acid sites than on Brønsted acid sites. Hydrogen adsorption isotherms and the IR results for hydrogen adsorption on preadsorbed pyridine were used to evaluate the formation of active protonic acid sites from molecular hydrogen. The catalyst with 13wt% WO3 loading showed the maximum hydrogen uptake capacity and formation of protonic acid sites. These results show a direct correlation with the activity of WZ in n-butane isomerization at 573K in which 13wt% WO3 loading on ZrO2 yielded the highest amount of isobutane. It is suggested that the presence of strong Lewis acid sites on monolayer-dispersed WO3 facilitates the formation of protonic acid sites from hydrogen in the gas phase which act as active sites in n-butane isomerization. The presence of permanent Brønsted acid sites could not be directly associated with activity. In fact, no isomerization activity was observed in the absence of hydrogen.