Publications by authors named "Leon Zhou"

21 Publications

  • Page 1 of 1

Prognostic implications and outcomes of cardiac arrest among contemporary patients with STEMI treated with PCI.

Resusc Plus 2021 Sep 15;7:100149. Epub 2021 Jul 15.

Heart and Vascular Institute, Cleveland Clinic Foundation, Cleveland, OH United States.

Background: Cardiac arrest (CA) complicating ST-elevation myocardial infarction (STEMI) is associated with a disproportionately higher risk of mortality. We described the contemporary presentation, management, and outcomes of CA patients in the era of primary percutaneous coronary intervention (PCI).

Methods: We reviewed 1,272 consecutive STEMI patients who underwent PCI between 1/1/2011-12/31/2016 and compared characteristics and outcomes between non-CA (N = 1,124) and CA patients (N = 148), defined per NCDR definitions as pulseless arrest requiring cardiopulmonary resuscitation and/or defibrillation within 24-hr of PCI.

Results: Male gender, cerebrovascular disease, chronic kidney disease, in-hospital STEMI, left main or left anterior descending culprit vessel, and initial TIMI 0 or 1 flow were independent predictors for CA. CA patients had longer door-to-balloon-time (106 [83,139] vs. 97 [74,121] minutes, p = 0.003) and greater incidence of cardiogenic shock (48.0% vs. 5.9%, p < 0.001), major bleeding (25.0% vs. 9.4%, p < 0.001), and 30-day mortality (16.2% vs. 4.1%, p < 0.001). Risk score for 30-day mortality based on presenting characteristics provided excellent prognostic accuracy (area under the curve = 0.902). However, over long-term follow-up of 4.5 ± 2.4 years among hospital survivors, CA did not portend any additional mortality risk (HR: 1.01, 95% CI: 0.56-1.82, p = 0.97).

Conclusions: In a contemporary cohort of STEMI patients undergoing primary PCI, CA occurs in >10% of patients and is an important mechanism of mortality in patients with in-hospital STEMI. While CA is associated with adverse outcomes, it carries no additional risk of long-term mortality among survivors highlighting the need for strategies to improve the in-hospital care of STEMI patients with CA.
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http://dx.doi.org/10.1016/j.resplu.2021.100149DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8319445PMC
September 2021

Effect of High-Density Lipoprotein Cholesterol Levels on Overall Survival and Major Adverse Cardiovascular and Cerebrovascular Events.

Am J Cardiol 2021 05 31;146:8-14. Epub 2021 Jan 31.

Robert and Suzanne Tomsich Department of Cardiovascular Medicine, Heart, Vascular and Thoracic Institute, Cleveland Clinic, Cleveland, Ohio. Electronic address:

Several studies designed to augment high density lipoprotein (HDL) levels have so far been unsuccessful in reducing rates of major adverse cardiovascular and cerebrovascular events (MACCE). In this study, we report the effect of HDL-C levels on overall survival outcomes and rates of MACCE following percutaneous coronary intervention (PCI). We reviewed patients who underwent PCI at the Cleveland Clinic from 2005 to 2017 and followed them through the end of 2018. Restricted cubic splines incorporated into Cox proportional hazard regression models were used to assess the outcomes. The HDL-C level associated with the lowest mortality was used as a reference value.15,633 patients underwent PCI during the study period, of which 70% were male, 81% were white, and 73% were on statins. The mean age at the time of procedure was 65.8 ± 11.8 years. After adjusting for demographics, co-morbidities, lipid profile, statin use, and date of procedure, our model demonstrated a U-shaped association between HDL-C and overall mortality, with HDL-C levels of 30-50 mg/dl associated with the most favorable outcomes, and HDL-C levels < 30 mg/dl or > 50 mg/dl associated with worse outcomes. A sensitivity analysis in men yielded a similar U-shaped association. In conclusion, our study shows that both low and high levels of HDL-C are associated with worse overall survival, with no effect on rates of MACCE in PCI patients. Further studies are required to understand the mechanism of this association between elevated HDL-C levels with increased overall mortality in patients with atherosclerotic cardiovascular disease (ASCVD).
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http://dx.doi.org/10.1016/j.amjcard.2021.01.014DOI Listing
May 2021

Implementation of a Comprehensive ST-Elevation Myocardial Infarction Protocol Improves Mortality Among Patients With ST-Elevation Myocardial Infarction and Cardiogenic Shock.

Am J Cardiol 2020 11 15;134:1-7. Epub 2020 Aug 15.

Heart and Vascular Institute, Cleveland Clinic Foundation, Cleveland, Ohio. Electronic address:

Mortality in patients with STEMI-associated cardiogenic shock (CS) is increasing. Whether a comprehensive ST-elevation myocardial infarction (STEMI) protocol (CSP) can improve their care delivery and mortality is unknown. We evaluated the impact of a CSP on incidence and outcomes in patients with STEMI-associated CS. We implemented a 4-step CSP including: (1) Emergency Department catheterization lab activation; (2) STEMI Safe Handoff Checklist; (3) immediate catheterization lab transfer; (4) and radial-first percutaneous coronary intervention (PCI). We studied 1,272 consecutive STEMI patients who underwent PCI and assessed for CS incidence per National Cardiovascular Data Registry definitions within 24-hours of PCI, care delivery, and mortality before (January 1, 2011, to July 14, 2014; n = 723) and after (July 15, 2014, to December 31, 2016; n = 549) CSP implementation. Following CSP implementation, CS incidence was reduced (13.0% vs 7.8%, p = 0.003). Of 137 CS patients, 43 (31.4%) were in the CSP group. CSP patients had greater IABP-Shock II risk scores (1.9 ± 1.8 vs 2.8 ± 2.2, p = 0.014) with otherwise similar hemodynamic and baseline characteristics, cardiac arrest incidence, and mechanical circulatory support use. Administration of guideline-directed medical therapy was similar (89.4% vs 97.7%, p = 0.172) with significant improvements in trans-radial PCI (9.6% vs 44.2%, p < 0.001) and door-to-balloon time (129.0 [89:160] vs 95.0 [81:116] minutes, p = 0.001) in the CSP group, translating to improvements in infarct size (CK-MB 220.9 ± 156.0 vs 151.5 ± 98.5 ng/ml, p = 0.005), ejection fraction (40.8 ± 14.5% vs 46.7 ± 14.6%, p = 0.037), and in-hospital mortality (30.9% vs 14.0%, p = 0.037). In conclusion, CSP implementation was associated with improvements in CS incidence, infarct size, ejection fraction, and in-hospital mortality in patients with STEMI-associated CS. This strategy offers a potential solution to bridging the historically elusive gap in their care.
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http://dx.doi.org/10.1016/j.amjcard.2020.08.012DOI Listing
November 2020

Combining solid dispersion-based spray drying with cyclodextrin to improve the functionality and mitigate the beany odor of pea protein isolate.

Carbohydr Polym 2020 Oct 5;245:116546. Epub 2020 Jun 5.

Department of Plant Sciences, North Dakota State University, Fargo, ND, 58108, USA. Electronic address:

The beany flavor of pea protein limits its application in the food industry. This study aimed at addressing this problem by combining the advantages of solid-based spray drying technique and the ability of cyclodextrins (CD) to entrap volatiles. Pea protein isolates (PPI) was extracted by alkaline extraction-isoelectric precipitation, followed by co-spray drying with CD. The resulted PPI-CD showed no major structure changes. HS-SPME-GC-MS coupled to untargeted metabolomics successfully identified 23 aroma compounds that represent the different odorants among PPI-control, physically mixed PPI-CD, and co-spray dried PPI-CD samples. Heat map analysis also showed a remarkable beany odor mitigation effect upon the addition of CD, which was further proved to be due to CD entrapping aroma compounds during spray drying. In the meantime, the functional attributes of PPI-CD were not adversely impacted by the addition of CD.
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http://dx.doi.org/10.1016/j.carbpol.2020.116546DOI Listing
October 2020

Chemistry Behind Rare Sugars and Bioprocessing.

J Agric Food Chem 2018 12 13;66(51):13343-13345. Epub 2018 Dec 13.

State Key Laboratory of Food Science and Technology , Jiangnan University , Wuxi , Jiangsu 214122 , People's Republic of China.

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http://dx.doi.org/10.1021/acs.jafc.8b06293DOI Listing
December 2018

Improving the Thermostability and Catalytic Efficiency of the d-Psicose 3-Epimerase from Clostridium bolteae ATCC BAA-613 Using Site-Directed Mutagenesis.

J Agric Food Chem 2016 May 22;64(17):3386-93. Epub 2016 Apr 22.

Roquette America , 1003 Commercial Street, Keokuk, Iowa 52632, United States.

d-Psicose is a highly valuable rare sugar because of its excellent physiological properties and commercial potential. d-Psicose 3-epimerase (DPEase) is the key enzyme catalyzing the isomerization of d-fructose to d-psicose. However, the poor thermostability and low catalytic efficiency are serious constraints on industrial application. To address these issues, site-directed mutagenesis of Tyr68 and Gly109 of the Clostridium bolteae DPEase was performed. Compared with the wild-type enzyme, the Y68I variant displayed the highest substrate-binding affinity and catalytic efficiency, and the G109P variant showed the highest thermostability. Furthermore, the double-site Y68I/G109P variant was generated and exhibited excellent enzyme characteristics. The Km value decreased by 17.9%; the kcat/Km increased by 1.2-fold; the t1/2 increased from 156 to 260 min; and the melting temperature (Tm) increased by 2.4 °C. Moreover, Co(2+) enhanced the thermostability significantly, including the t1/2 and Tm values. All of these indicated that the Y68I/G109P variant would be appropriate for the industrial production of d-psicose.
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http://dx.doi.org/10.1021/acs.jafc.6b01058DOI Listing
May 2016

Engineering of Alicyclobacillus hesperidum L-arabinose isomerase for improved catalytic activity and reduced pH optimum using random and site-directed mutagenesis.

Appl Biochem Biotechnol 2015 Dec 3;177(7):1480-92. Epub 2015 Sep 3.

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu, 214122, China.

A mutation, D478N, was obtained by an error-prone polymerase chain reaction using the L-arabinose isomerase (L-AI) gene from Alicyclobacillus hesperidum URH17-3-68 as the template. The mutated isomerase showed higher activity for D-galactose isomerization. The mutation site obtained from random mutagenesis was then introduced as a single-site mutation using site-directed mutagenesis. Single-site variants, D478N, D478Q, D478A, D478K, and D478R, were constructed. The optimum temperatures were all higher than 60 °C. D478A, D478N, and D478Q retained more than 80 % of the maximum relative activity of the wild-type L-AI at 75 °C. With the exception of the D478A variant, all variants showed decreased optimum pH values in the acidic range (6.0-6.5). All of the variant L-AIs could be significantly activated by the addition of Co(2+) and Mn(2+). D478N and D478Q showed higher catalytic efficiencies (k cat/K m) toward D-galactose than that of wild-type L-AI. In addition, the D478N and D478Q variants exhibited a much higher conversion ratio of D-galactose to D-tagatose at 6.0 than the wild-type L-AI. According to the molecular model, residue D478 was located on the surface of the enzyme and distant from the active site. It was supposed that the charged state of residue 478 may influence the optimum pH for substrate binding or isomerization.
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http://dx.doi.org/10.1007/s12010-015-1828-3DOI Listing
December 2015

Taste Masking of Griseofulvin and Caffeine Anhydrous Using Kleptose Linecaps DE17 by Hot Melt Extrusion.

AAPS PharmSciTech 2016 Feb 20;17(1):99-105. Epub 2015 Aug 20.

Department of Pharmaceutics and Drug Delivery, University of Mississippi, University, Mississippi, 38677, USA.

The objective of this project was to investigate the potential of Kleptose Linecaps DE17 (KLD) in masking the unpleasant/bitter taste of therapeutic agents by hot melt extrusion (HME). Griseofulvin (GRI) and caffeine anhydrous (CA) were used as a bitter active pharmaceutical ingredient (API) model drugs. Thermogravimetric studies confirmed the stability of GRI, CA, and KLD at the employed extrusion temperatures. The differential scanning calorimetry (DSC) studies revealed a characteristic melting endotherm of GRI at 218-220°C and CA at 230-232°C in the physical mixtures as well as in all extrudates over the period of study, indicating the crystalline nature of drug. HME of KLD was achieved only in the presence of plasticizer. Among the several plasticizers investigated, xylitol showed improved processability of KLD at 15% w/w concentration. Dissolution studies of HME extrudates using simulated salivary medium exhibited ∼threefold less release compared to physical mixture at the end of 5 min (the lesser drug release, better the taste masking efficiency). Furthermore, the results from the sensory evaluation of products in human panel demonstrated strong bitter taste in the case of physical mixture compared to the HME formulation, suggesting the potential of Kleptose Linecaps DE17 as taste masking polymer in melt extruded form.
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http://dx.doi.org/10.1208/s12249-015-0374-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4766133PMC
February 2016

Characterization of a metal-dependent D-psicose 3-epimerase from a novel strain, Desmospora sp. 8437.

J Agric Food Chem 2013 Nov 15;61(47):11468-76. Epub 2013 Nov 15.

State Key Laboratory of Food Science and Technology, Jiangnan University , 214122 Wuxi, People's Republic of China.

The rare sugar d-psicose is an ideal sucrose substitute for food products, due to having 70% of the relative sweetness but 0.3% of the energy of sucrose. It also shows important physiological functions. d-Tagatose 3-epimerase (DTEase) family enzymes can produce d-psicose from d-fructose. In this paper, a new member of the DTEase family of enzymes was characterized from Desmospora sp. 8437 (GenBank accession no. WP_009711885 ) and was named Desmospora sp. d-psicose 3-epimerase (DPEase) due to its highest substrate specificity toward d-psicose. Desmospora sp. DPEase was strictly metal-dependent and displayed maximum activity in the presence of Co(2+). The optimum pH and temperature were 7.5 and 60 °C, respectively. The enzyme was relatively thermostable below 50 °C, but easily lost initial activity when preincubated at 60 °C. The thermostability property was almost not affected by the addition of Co(2+). Desmospora sp. DPEase had relatively high catalysis efficiency for the substrates d-psicose and d-fructose, which were measured to be 327 and 116 mM(-1) min(-1), respectively. The equilibrium ratio between d-psicose and d-fructose of Desmospora sp. DPEase was 30:70. The enzyme could produce 142.5 g/L d-psicose from 500 g/L of d-fructose, suggesting that the enzyme is a potential d-psicose producer for industrial production.
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http://dx.doi.org/10.1021/jf4035817DOI Listing
November 2013

Stability of benzocaine formulated in commercial oral disintegrating tablet platforms.

AAPS PharmSciTech 2013 Dec 30;14(4):1333-40. Epub 2013 Aug 30.

Department of Biopharmaceutical Sciences, University of Illinois, Chicago, Illinois, 60612-7231, USA.

Pharmaceutical excipients contain reactive groups and impurities due to manufacturing processes that can cause decomposition of active drug compounds. The aim of this investigation was to determine if commercially available oral disintegrating tablet (ODT) platforms induce active pharmaceutical ingredient (API) degradation. Benzocaine was selected as the model API due to known degradation through ester and primary amino groups. Benzocaine was either compressed at a constant pressure, 20 kN, or at pressure necessary to produce a set hardness, i.e., where a series of tablets were produced at different compression forces until an average hardness of approximately 100 N was achieved. Tablets were then stored for 6 months under International Conference on Harmonization recommended conditions, 25°C and 60% relative humidity (RH), or under accelerated conditions, 40°C and 75% RH. Benzocaine degradation was monitored by liquid chromatography-mass spectrometry. Regardless of the ODT platform, no degradation of benzocaine was observed in tablets that were kept for 6 months at 25°C and 60% RH. After storage for 30 days under accelerated conditions, benzocaine degradation was observed in a single platform. Qualitative differences in ODT platform behavior were observed in physical appearance of the tablets after storage under different temperature and humidity conditions.
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http://dx.doi.org/10.1208/s12249-013-0015-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3840786PMC
December 2013

Characterization of a D-psicose-producing enzyme, D-psicose 3-epimerase, from Clostridium sp.

Biotechnol Lett 2013 Sep 10;35(9):1481-6. Epub 2013 May 10.

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.

The gene coding for D-psicose 3-epimerase (DPEase) from Clostridium sp. BNL1100 was cloned and expressed in Escherichia coli. The recombinant enzyme was purified by Ni-affinity chromatography. It was a metal-dependent enzyme and required Co(2+) as optimum cofactor. It displayed catalytic activity maximally at pH 8.0 and 65 °C (as measured over 5 min). The optimum substrate was D-psicose, and the K m, turnover number (k cat), and catalytic efficiency (k cat/K m) for D-psicose were 227 mM, 32,185 min(-1), and 141 min(-1 )mM(-1), respectively. At pH 8.0 and 55 °C, 120 g D-psicose l(-1) was produced from 500 g D-fructose l(-1) after 5 h.
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http://dx.doi.org/10.1007/s10529-013-1230-6DOI Listing
September 2013

Characterization of a novel metal-dependent D-psicose 3-epimerase from Clostridium scindens 35704.

PLoS One 2013 30;8(4):e62987. Epub 2013 Apr 30.

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, People's Republic of China.

The noncharacterized protein CLOSCI_02528 from Clostridium scindens ATCC 35704 was characterized as D-psicose 3-epimerase. The enzyme showed maximum activity at pH 7.5 and 60°C. The half-life of the enzyme at 50°C was 108 min, suggesting the enzyme was relatively thermostable. It was strictly metal-dependent and required Mn²⁺ as optimum cofactor for activity. In addition, Mn²⁺ improved the structural stability during both heat- and urea-induced unfolding. Using circular dichroism measurements, the apparent melting temperature (T m) and the urea midtransition concentration (C m) of metal-free enzyme were 64.4°C and 2.68 M. By comparison, the Mn²⁺-bound enzyme showed higher T m and C m with 67.3°C and 5.09 M. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) values for substrate D-psicose were estimated to be 28.3 mM, 1826.8 s⁻¹, and 64.5 mM⁻¹ s⁻¹, respectively. The enzyme could effectively produce D-psicose from D-fructose with the turnover ratio of 28%.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0062987PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3639893PMC
November 2013

Recent advances on applications and biotechnological production of D-psicose.

Appl Microbiol Biotechnol 2012 Jun 10;94(6):1461-7. Epub 2012 May 10.

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China.

D-Psicose is a hexoketose monosaccharide sweetener, which is a C-3 epimer of D-fructose and is rarely found in nature. It has 70 % relative sweetness but 0.3 % energy of sucrose, and is suggested as an ideal sucrose substitute for food products. It shows important physiological functions, such as blood glucose suppressive effect, reactive oxygen species scavenging activity, and neuroprotective effect. It also improves the gelling behavior and produces good flavor during food process. This article presents a review of recent studies on the properties, physiological functions, and food application of D-psicose. In addition, the biochemical properties of D-tagatose 3-epimerase family enzymes and the D-psicose-producing enzyme are compared, and the biotechnological production of D-psicose from D-fructose is reviewed.
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http://dx.doi.org/10.1007/s00253-012-4093-1DOI Listing
June 2012

Cloning, expression, and characterization of a D-psicose 3-epimerase from Clostridium cellulolyticum H10.

J Agric Food Chem 2011 Jul 23;59(14):7785-92. Epub 2011 Jun 23.

Jiangnan University, Jiangsu, People's Republic of China.

The noncharacterized protein ACL75304 encoded by the gene Ccel_0941 from Clostridium cellulolyticum H10 (ATCC 35319), previously proposed as the xylose isomerase domain protein TIM barrel, was cloned and expressed in Escherichia coli . The expressed enzyme was purified by nickel-affinity chromatography with electrophoretic homogeneity and then characterized as d-psicose 3-epimerase. The enzyme was strictly metal-dependent and showed a maximal activity in the presence of Co(2+). The optimum pH and temperature for enzyme activity were 55 °C and pH 8.0. The half-lives for the enzyme at 60 °C were 6.8 h and 10 min when incubated with and without Co(2+), respectively, suggesting that this enzyme was extremely thermostable in the presence of Co(2+) but readily inactivated without metal ion. The Michaelis-Menten constant (K(m)), turnover number (k(cat)), and catalytic efficiency (k(cat)/K(m)) values of the enzyme for substrate d-psicose were estimated to be 17.4 mM, 3243.4 min(-1), and 186.4 mM min(-1), respectively. The enzyme carried out the epimerization of d-fructose to d-psicose with a conversion yield of 32% under optimal conditions, suggesting that the enzyme is a potential d-psicose producer.
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http://dx.doi.org/10.1021/jf201356qDOI Listing
July 2011

Purification and characterization of inulin fructotransferase (DFA III-forming) from Arthrobacter aurescens SK 8.001.

Bioresour Technol 2011 Jan 8;102(2):1757-64. Epub 2010 Oct 8.

State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi 214122, Jiangsu, PR China.

The soil bacterium Arthrobacter aurescens SK 8.001 produces inulin fructotransferase (IFTase), and liquid chromatography-mass spectrometry (LC-MS) and carbon-13 nuclear magnetic resonance (13C NMR) analysis demonstrated that the main product of the enzyme was difructose anhydride III (DFA III). The IFTase was purified by ethanol precipitation, DEAE Sepharose Fast Flow, and Superdex 200 10/300 GL gel chromatography. Its molecular mass was estimated to be 40 kDa by SDS-PAGE and 35 kDa by gel filtration. The enzyme showed maximum activity at pH 5.5 and 60-70 °C, and retained 86.5% of its initial activity after incubation at 60 °C for 4 h. Chemical modification results suggested that a tryptophan residue is essential to enzyme activity. The N-terminal amino acid sequence was determined as AEGAKASPLNSPNVYDVT. The kinetic values, Km and Vmax, were estimated to be 0.52 mM and 0.3 μmol/ml min. Nystose was observed to be the smallest substrate for the produced IFTase. This IFTase provides a promising way to utilize inulin for the production of DFA III.
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http://dx.doi.org/10.1016/j.biortech.2010.08.093DOI Listing
January 2011

LC-MS/MS and density functional theory study of copper(II) and nickel(II) chelating complexes of elesclomol (a novel anticancer agent).

J Pharm Biomed Anal 2011 Jan 15;54(2):331-6. Epub 2010 Sep 15.

Analytical Sciences, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.

Elesclomol (N-malonyl-bis(N'-methyl-N'-thiobenzoylhydrazide)), which is a novel anticancer agent, can form chelating complexes with Fe(II), Co(II), Ni(II), Cu(II), and Zn(II) in the gas phase during electrospray ionization (ESI) mass spectrometry. In the solution phase with acidic medium during chromatographic separation, however, only Cu(II) and Ni(II) to a lesser degree favor the formation of chelating complexes with elesclomol. The Cu(II)-chelating complex [Cu(II)+elesclomol-H]+· exhibits more complicated MS/MS fragmentation pathways than the Ni(II)-chelating complex [Ni(II)+elesclomol-H]+. One significant difference is the ready occurrence of the electron transfer upon collision-induced dissociation (CID) of [Cu(II)+elesclomol-H]+·. This leads to the reduction of Cu(II) to Cu(I). However, such phenomenon was not observed upon CID of [Ni(II)+elesclomol-H]+. On the basis of the density functional theory (DFT) calculations at the B3LYP/6-31+G(d)/LANL2DZ level, the Cu(II)- and Ni(II)-chelating complexes of elesclomol exist in the keto-form with tetra-coordinated trapezoid geometry in the gas phase but at different levels of distortion. As compared to the Ni(II)-elesclomol complex, the Cu(II)-elesclomol complex is more stable (by -55.25 kcal/mol). This relative stability of the chelating complexes of elesclomol is consistent with the Irving-Williams series of bindings to ligands.
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http://dx.doi.org/10.1016/j.jpba.2010.09.007DOI Listing
January 2011

Identification and control of metal-chelating chromatographic artifacts in the analysis of a malonohydrazide derivative drug compound.

J Pharm Biomed Anal 2010 Nov 24;53(3):371-5. Epub 2010 Apr 24.

Product Development Group, Pharmaceutical Development, GlaxoSmithKline, USA.

Two unusual chromatographic artifact peaks were detected in the HPLC analysis for content of a malonohydrazide derivative drug and drug-related impurities. The artifacts were identified as the copper(II) chelating complexes with the drug compound and one of the process impurities. Our investigations suggested that built-up of Cu(2+) contamination in the HPLC system was the primary source for formation of the chelating artifacts. A rinse procedure using diluted EDTA solution was developed, and demonstrated to effectively purge trace level of heavy metals including Cu(2+) from the system, and therefore inhibited the formation of both chelates. Furthermore, the rinse was shown to introduce no detrimental impact on the response accuracy of the active drug compound and related impurities.
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http://dx.doi.org/10.1016/j.jpba.2010.04.021DOI Listing
November 2010

Gas-phase Smiles rearrangement in structural analysis of a pseudo-oxidative impurity generated in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid.

J Pharm Biomed Anal 2010 Feb 24;51(3):577-82. Epub 2009 Sep 24.

Analytical Sciences, Chemical Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA 19406, USA.

Several mass spectrometry (MS) techniques including accurate MS and MS/MS, as well as hydrogen/deuterium (H/D) exchange, were utilized to characterize a pseudo-oxidative reaction by-product (impurity I) in the pharmaceutical synthesis of S-(thiobenzoyl)thioglycolic acid. The negative ion MS/MS data provided complementary structural information to the positive ion MS/MS data. An understanding of the gas-phase Smiles rearrangement upon collision-induced dissociation (CID) in the negative ion MS/MS mode played an important role in structural elucidation of impurity I. The theoretical calculations by density functional theory (DFT) at the B3LYP/6-311G(d,p) level provided insights into the thermochemistry of the Smiles rearrangement reaction. This pseudo-oxidative impurity is proposed to be generated via the base-catalyzed hydrolysis in solution.
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http://dx.doi.org/10.1016/j.jpba.2009.09.019DOI Listing
February 2010

Glutamate 636 of the Escherichia coli pyruvate dehydrogenase-E1 participates in active center communication and behaves as an engineered acetolactate synthase with unusual stereoselectivity.

J Biol Chem 2005 Jun 31;280(22):21473-82. Epub 2005 Mar 31.

Department of Chemistry, Rutgers University, Newark, New Jersey 07102, USA.

The residue Glu636 is located near the thiamine diphosphate (ThDP) binding site of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), and to probe its function two variants, E636A and E636Q were created with specific activities of 2.5 and 26% compared with parental PDHc-E1. According to both fluorescence binding and kinetic assays, the E636A variant behaved according to half-of-the-sites mechanism with respect to ThDP. In contrast, with the E636Q variant a K(d,ThDP) = 4.34 microM and K(m,ThDP) = 11 microM were obtained with behavior more reminiscent of the parental enzyme. The CD spectra of both variants gave evidence for formation of the 1',4'-iminopyrimidine tautomer on binding of phosphonolactylthiamine diphosphate, a stable analog of the substrate-ThDP covalent complex. Rapid formation of optically active (R)-acetolactate by both variants, but not by the parental enzyme, was observed by CD and NMR spectroscopy. The acetolactate configuration produced by the Glu636 variants is opposite that produced by the enzyme acetolactate synthase and the Asp28-substituted variants of yeast pyruvate decarboxylase, suggesting that the active centers of the two sets of enzymes exhibit different facial selectivity (re or si) vis à vis pyruvate. The tryptic peptide map (mass spectral analysis) revealed that the Glu636 substitution changed the mobility of a loop comprising amino acid residues from the ThDP binding fold. Apparently, the residue Glu636 has important functions both in active center communication and in protecting the active center from undesirable "carboligase" side reactions.
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http://dx.doi.org/10.1074/jbc.M502691200DOI Listing
June 2005

Evidence for dramatic acceleration of a C-H bond ionization rate in thiamin diphosphate enzymes by the protein environment.

Biochemistry 2005 Feb;44(7):2237-43

Department of Chemistry, Rutgers, The State University of New Jersey, Newark, New Jersey 07102, USA.

The hypothesis that thiamin diphosphate-dependent enzymes achieve a significant fraction of their catalytic rate acceleration by providing a protein environment that helps to stabilize unstable zwitterionic/dipolar intermediates (including the enamine/C2alpha-carbanion present on all such enzymes) was tested experimentally using the intermediate C2alpha-hydroxyethylthiamin diphosphate (HEThDP) with the Escherichia coli pyruvate dehydrogenase complex and its E1 subunit (PDHc-E1). Using pre-steady-state and steady-state methods, it was shown that HEThDP is a substrate for this enzyme after ionization of the C2alpha-H bond. An experiment was then carried out to measure the PDHc-E1 catalyzed pre-steady-state rate constant for the D --> H exchange from the C2alpha position of HEThDP-d(4), as an indicator of the formation of the enamine. Importantly, the enzyme accelerates the rate of ionization of this bond by a factor of 10(7), corresponding to a 10 kcal/mol stabilization of the enamine intermediate by the enzyme. This finding is likely a general feature of thiamin diphosphate enzymes.
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http://dx.doi.org/10.1021/bi047696jDOI Listing
February 2005

Amino-terminal residues 1-45 of the Escherichia coli pyruvate dehydrogenase complex E1 subunit interact with the E2 subunit and are required for activity of the complex but not for reductive acetylation of the E2 subunit.

Biochemistry 2004 Nov;43(44):14037-46

Department of Chemistry, Rutgers, the State University of New Jersey, Newark, New Jersey 07102, USA.

While N-terminal amino acids 1-55 are not seen in the structure of the Escherichia coli pyruvate dehydrogenase complex E1 subunit (PDHc-E1), mass spectrometric analysis indicated that this amino-terminal region of PDHc-E1 was protected by PDHc-E2. Hence, five deletion constructs of PDHc-E1 were created, Delta6-15, Delta16-25, Delta26-35, Delta36-45, and Delta46-55, along with single-site substitutions at Asp7, Asp9, Pro10, Ile11, Glu12, Thr13, Arg14, and Asp15. The decarboxylation of pyruvate and the ability of PDHc-E1 to dimerize are not affected by any of the deletions or substitutions. While Delta46-55 and the Pro10Ala, Ile11Ala, and Thr13Ala variants could form a complex with PDHc-E2, and produced NADH in the overall assay, Delta16-25, Delta26-35, and Delta36-45 and the Asp7Ala, Asp9Ala, Glu12Gln, Glu12Asp, Arg14Ala, and Asp15Ala variants failed in both respects. Remarkably, all constructs of PDHc-E1 from E. coli, as well as PDHc-E1 from Mycobacterium tuberculosis, could carry out reductive acetylation of the E. coli lipoyl domain, but only constructs of the E. coli PDHc-E1 could reductively acetylate E. coli PDHc-E2. It was concluded that there are at least two loci of interaction between the PDHc-E1 and PDHc-E2 subunits: (1) the thiamin diphosphate-bound substrate on PDHc-E1 and the lipoylamide of PDHc-E2, as reflected by the ability to reductively acetylate the latter; and (2) amino terminal residues 1-45 of PDHc-E1 with regions of PDHc-E2 (so far undefined for the E. coli complex), as reflected by the overall activity of the entire complex. These studies add important information regarding recognition within this multienzyme complex class with an alpha(2) E1 assembly.
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http://dx.doi.org/10.1021/bi049027bDOI Listing
November 2004
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