Development of a High-Affinity Antibody-Binding Peptide for Site-Specific Modification

Immunoglobulin G (IgG)-binding peptides such as 15-IgBP are convenient tools for the site-specific modification of antibodies and the preparation of homogeneous antibody–drug conjugates. A peptide such as 15-IgBP can be selectively crosslinked to the fragment crystallizable region of human IgG in an affinity-dependent manner via the ϵ-amino group of Lys8. Previously, we found that the peptide 15-Lys8Leu has a high affinity (K_d=8.19 nM) due to the presence of the γ-dimethyl group in Leu8. The primary amino group required for the crosslinking to the antibodies has, however, been lost. Here, we report the design and synthesis of a novel unnatural amino acid, 4-(2-aminoethylcarbamoyl)leucine (Aecl), which possesses both the γ-dimethyl fragment and a primary amino group. A peptide containing Aecl8 (15-Lys8Aecl) was synthesized and showed a binding affinity ten times higher (K_d=24.3 nM) than that of 15-IgBP (K_d=267 nM). Fluorescein isothiocyanate (FITC)-labeled 15-Lys8Aecl with an N-hydroxy succinimide ester at the side chain of Aecl8 (FITC-15-Lys8Aecl(OSu)) successfully labeled an antibody (trastuzumab, Herceptin^®) with the fluorophore. This peptide scaffold has both strong binding affinity and crosslinking capability, and could be a useful tool for the selective chemical modification of antibodies with molecules of interest such as drugs.     

Platinum(II) as Bifunctional Linker in Antibody–Drug Conjugate Formation: Coupling of a 4‐Nitrobenzo‐2‐oxa‐1,3‐diazole Fluorophore to Trastuzumab as a Model

The potential of platinum(II) as a bifunctional linker in the coordination of small molecules, such as imaging agents or (cytotoxic) drugs, to monoclonal antibodies (mAbs) was investigated with a 4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD) fluorophore and trastuzumab (Herceptin?) as a model antibody. The effect of ligand and reaction conditions on conjugation efficiency was explored for [Pt(en)(L‐NBD)Cl](NO₃) (en=ethylenediamine), with L=N‐heteroaromatic, N‐alkyl amine, or thioether. Conjugation proceeded most efficiently at pH 8.0 in the presence of NaClO₄ or Na₂SO₄ in tricine or HEPES buffer. Reaction of N‐coordinated complexes (20 equiv) with trastuzumab at 37 °C for 2 h, followed by removal of weakly bound complexes with excess thiourea, afforded conjugates with an NBD/mAb ratio of 1.5–2.9 that were stable in phosphate‐buffered saline at room temperature for at least 48 h. In contrast, thioether‐coordinated complexes afforded unstable conjugates. Finally, surface plasmon resonance analysis showed no loss in binding affinity of trastuzumab after conjugation.     

Use of chitosan for removal of bisphenol A and bisphenol derivatives through tyrosinase‐catalyzed quinone oxidation

In this study, the availability of chitosan was systematically investigated for removal of bisphenol A (BPA, 2,2‐bis(hydroxyphenyl)propane) through the tyrosinase‐catalyzed quinone oxidation and subsequent quinone adsorption on chitosan beads. In particular, the process parameters, such as the hydrogen peroxide (H₂O₂)‐to‐BPA ratio, pH value, temperature, and tyrosinase dose, were discussed in detail for the enzymatic quinone oxidation. Tyrosinase‐catalyzed quinone oxidation of BPA was effectively enhanced by adding H₂O₂ and the optimum conditions for BPA at 0.3 mM were determined to be pH 7.0 and 40°C in the presence of H₂O₂ at 0.3 mM ([H₂O₂]/[BPA] = 1.0). Removal of BPA from aqueous solutions was accomplished by adsorption of enzymatically generated quinone derivatives on chitosan beads. The use of chitosan in the form of beads was found to be more effective because heterogeneous removal of BPA with chitosan beads was much faster than homogeneous removal of BPA with chitosan solutions, and the removal efficiency was enhanced by increasing the amount of chitosan beads dispersed in the BPA solutions and BPA was completely removed by quinone adsorption in the presence of chitosan beads more than 0.10 cm³/cm³. In addition, a variety of bisphenol derivatives were completely or effectively removed by the procedure constructed in this study, although the enzyme dose or the amount of chitosan beads was further increased as necessary for some of the bisphenol derivatives used. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Antibody Epitope of Human α‐Galactosidase A Revealed by Affinity Mass Spectrometry: A Basis for Reversing Immunoreactivity in Enzyme Replacement Therapy of Fabry Disease

α‐Galactosidase (αGal) is a lysosomal enzyme that hydrolyses the terminal α‐galactosyl moiety from glycosphingolipids. Mutations in the encoding genes for αGal lead to defective or misfolded enzyme, which results in substrate accumulation and subsequent organ dysfunction. The metabolic disease caused by a deficiency of human α‐galactosidase A is known as Fabry disease or Fabry–Anderson disease, and it belongs to a larger group known as lysosomal storage diseases. An effective treatment for Fabry disease has been developed by enzyme replacement therapy (ERT), which involves infusions of purified recombinant enzyme in order to increase enzyme levels and decrease the amounts of accumulated substrate. However, immunoreactivity and IgG antibody formation are major, therapy‐limiting, and eventually life‐threatening complications of ERT. The present study focused on the epitope determination of human α‐galactosidase A against its antibody formed. Here we report the identification of the epitope of human αGal(309–332) recognized by a human monoclonal anti‐αGal antibody, using a combination of proteolytic excision of the immobilized immune complex and surface plasmon resonance biosensing mass spectrometry. The epitope peptide, αGal(309–332), was synthesized by solid‐phase peptide synthesis. Determination of its affinity by surface plasmon resonance analysis revealed a high binding affinity for the antibody (K_D=39×10^?9 m ), which is nearly identical to that of the full‐length enzyme (K_D=16×10^?9 m ). The proteolytic excision affinity mass spectrometry method is shown here to be an efficient tool for epitope identification of an immunogenic lysosomal enzyme. Because the full‐length αGal and the antibody epitope showed similar binding affinities, this provides a basis for reversing immunogenicity upon ERT by: 1) treatment of patients with the epitope peptide to neutralize antibodies, or 2) removal of antibodies by apheresis, and thus significantly improving the response to ERT.     

Performance study of modified ZSM‐5 as support for bimetallic chromium–copper catalysts for VOC combustion

The catalytic performance of bimetallic chromium–copper supported over untreated ZSM‐5 (Cr? Cu/Z), ZSM‐5 treated with silicon tetrachloride (Cr–Cu/SiCl₄‐Z) and ZSM‐5 treated with steam (Cr–Cu/H₂O‐Z) is reported. The activity is based on the combustion of ethyl ethanoate and benzene at a feed concentration of 2000 ppm and a gas hourly space velocity (GHSV) of 32 000 h^?1. Due to higher reactivity and larger molecular size compared with that of water molecules, SiCl₄ reacted at the external surface of ZSM‐5 crystals. Cr–Cu/SiCl₄‐Z and Cr–Cu/H₂O‐Z both gave slightly lower conversion and carbon dioxide yield compared with Cr–Cu/Z. This was attributed to larger active metal crystallites formed in the mesopores and narrowing pore mouth and pore intersection by extraframework species. Cr–Cu/SiCl₄‐Z and Cr–Cu/H₂O‐Z both had reduced concentration and strength of acid sites, thus making them less susceptible to deactivation by coking. The coke accumulated by these two catalysts was relatively softer and more easily decomposed in oxygen during catalyst regeneration. Copyright © 2004 Society of Chemical Industry     

Non‐covalent interactions of pyrene end‐labeled star poly(ɛ‐caprolactone)s with fullerene

Pyrene end‐labeled star poly(?‐caprolactone)s (PCLs) with polyhedral oligomeric silsesquioxane (POSS) core were prepared by combination of copper(I)‐catalyzed azide–alkyne cycloaddition (CuAAC) click chemistry and ring‐opening polymerization techniques. First, ?‐caprolactone (?‐CL) is polymerized by using 1‐pyrene methanol as initiator and stannous octoate as catalyst to obtain α‐pyrene‐ω‐hydroxyl telechelic PCL with different chain lengths. Then, its hydroxyl group is converted to acetylene functionality by esterification reaction with propargyl chloroformate. Finally, the CuAAC click reaction of α‐pyrene‐ω‐acetylene telechelic PCL with POSS‐(N₃)₈ leads to corresponding pyrene end‐labeled star‐shaped PCLs. The successful synthesis of pyrene end‐labeled star polymers is clearly confirmed by ¹H‐nuclear magnetic resonance, Fourier transform infrared, gel permeation chromatograph, differential scanning calorimeter, and thermogravimetric analysis. Furthermore, non‐covalent interactions of obtained star polymers with fullerene are investigated in liquid media. Based on Raman spectroscopy and visual investigations, the star polymer having shorter chain length exhibited better and more stable dispersion with fullerene. The amount of pyrene units present per polymer chains can directly influence the dispersion stability of fullerene. © 2018 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 46520.     

A Versatile and Convenient Synthesis of 34S‐Labeled Phosphorothioate Oligonucleotides

A synthetic protocol for ³⁴S‐labeled phosphorothioate oligonucleotides (PS ONs) was developed to facilitate MS‐based assay analysis. This was enabled by a highly efficient, two‐step, one‐pot synthesis of ³⁴S‐labeled phenylacetyl disulfide (³⁴S‐PADS), starting from ³⁴S‐enriched elemental sulfur (³⁴S₈). ³⁴S‐PADS was subsequently used for stable isotope labeling (SIL) of oligonucleotides containing a phosphorothioate backbone. The ³⁴S‐SIL PS ONs are shown to retain the same melting temperature, antisense activity, and secondary structure as those of the corresponding unlabeled ³²S PS ONs.     

Direct synthesis of surface molecularly imprinted polymers based on vinyl–SiO2 nanospheres for recognition of bisphenol A

Molecularly imprinted polymers (MIP) with high performance in selectively recognizing bisphenol A (BPA) were prepared by using a novel and facile surface molecular‐imprinting technique. Vinyl‐functionalized, monodispersed silica spheres were synthesized by a one‐step emulsion reaction in aqueous solution. Then, BPA surface molecularly imprinted polymers (SMIP) were prepared by polymerization with 4‐vinylpyridine as the functional monomer and ethylene glycol dimethacrylate as the crosslinker. Maximal sorption capacity (Q_max) of the resulting SMIP was up to 600 μmol g^?1, while that of nonimprinted polymers was only 314.68 μmol g^?1. Kinetic binding study showed that sorption capacity reached 70% of Q_max in 20 min and sorption equilibrium at 80 min. SMIP had excellent accessibility and affinity toward BPA, for the selectivity coefficients of SMIP for BPA in respect to phenol, p‐tert‐butylphenol, and o‐phenylphenol were 3.39, 3.35, and 3.02, respectively. The reusage process verified the SMIP owning admirably stable adsorption capacity toward BPA for eight times. © 2012 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013     

Performance of H‐ZSM‐5‐supported bimetallic catalysts for the combustion of polluting volatile organic compounds in air

The performance of H‐ZSM‐5‐supported bimetallic catalysts with chromium as the base metal in the combustion of ethyl acetate and benzene is reported. A reactor operated from 100 to 500 °C at a gas hourly space velocity (GHSV) of 32 000 h^?1 was used for study of the activity. A combination of 1.0 wt% chromium and 0.5 wt% copper yielded a catalyst (Cr_1.0Cu_0.5/Z) with improved conversion and carbon dioxide yield. Cr₂O₃ (Cr³⁺) and CuO (Cu²⁺) were the predominant metal species in the catalyst. In agreement with the Mars–van Krevelen model, improved reducibility of Cr³⁺ in the presence of Cu²⁺ led to an improvement in activity. The copper content in Cr_1.0Cu_0.5/Z also favored the formation of deep combustion products. Condensation and subsequent growth of coke precursors in the catalyst pores led to the formation of a softer and less aromatic coke fraction while dehydrogenation activity on acid sites formed a harder and more aromatic coke fraction. The use of Cr_1.0Cu_0.5/Z favored the formation of lower molecular weight intermediates, leading to reduction in formation of softer coke. Copyright © 2005 Society of Chemical Industry     

A Single-Framework Synthetic Antibody Library Containing a Combination of Canonical and Variable Complementarity-Determining Regions

Synthetic antibody libraries have been used to generate antibodies with favorable biophysical and pharmacological properties. Here, we describe the design, construction, and validation of a phage-displayed antigen-binding fragment (Fab) library built on a modified trastuzumab framework with four fixed and two diversified complementarity-determining regions (CDRs). CDRs L1, L2, H1, and H2 were fixed to preserve the most commonly observed “canonical” CDR conformation preferred by the modified trastuzumab Fab framework. The library diversity was engineered within CDRs L3 and H3 by use of custom-designed trinucleotide phosphoramidite mixes and biased towards human antibody CDR sequences. The library contained ≈7.6 billion unique Fabs, and >95 % of the library correctly encoded both diversified CDR sequences. We used this library to conduct selections against the human epidermal growth factor receptor-3 extracellular domain (HER3-ECD) and compared the CDR diversity of the naïve library and the anti-HER3 selection pool by use of next-generation sequencing. The most commonly observed CDR combination isolated, named Her3-3, was overexpressed and purified in Fab and immunoglobulin G (IgG) formats. Fab HER3-3 bound to HER3-ECD with a K_D value of 2.14 nm and recognized cell-surface HER3. Although HER3-3 IgG bound to cell-surface HER3, it did not inhibit the proliferation of HER3-positive cells. Near-infrared imaging showed that Fab HER3-3 selectively accumulated in a murine HER3-postive xenograft, thus providing a lead for the development of HER3 imaging probes.     

Phospha‐Michael Addition as a New Click Reaction for Protein Functionalization

A new type of click reaction between an alkyl phosphine and acrylamide was developed and applied for site‐specific protein labeling in vitro and in live cells. Acrylamide is a small electrophilic olefin that readily undergoes phospha‐Michael addition with an alkyl phosphine. Our kinetic study indicated a second‐order rate constant of 0.07 m ^?1 s^?1 for the reaction between tris(2‐carboxyethyl)phosphine and acrylamide at pH 7.4. To demonstrate its application in protein functionalization, we used a dansyl–phosphine conjugate to successfully label proteins that were site‐specifically installed with N^?‐acryloyl‐l ‐lysine and employed a biotin–phosphine conjugate to selectively probe human proteins that were metabolically labeled with N‐acryloyl‐galactosamine.     

Rational Design of Domain‐Swapping‐Based c‐Type Cytochrome Heterodimers by Using Chimeric Proteins

The design of protein oligomers with multiple active sites has been gaining interest, owing to their potential use for biomaterials, which has encouraged researchers to develop a new design method. Three‐dimensional domain swapping is the unique phenomenon in which protein molecules exchange the same structural region between each other. Herein, to construct oligomeric heme proteins with different active sites by utilizing domain swapping, two c‐type cytochrome‐based chimeric proteins have been constructed and the domains swapped. According to X‐ray crystallographic analysis, the two chimeric proteins formed a domain‐swapped dimer with two His/Met coordinated hemes. By mutating the heme coordination structure of one of the two chimeric proteins, a domainswapped heterodimer with His/Met and His/H₂O coordinated hemes was formed. Binding of an oxygen molecule to the His/H₂O site of the heterodimer was confirmed by resonance Raman spectroscopy, in which the Fe?O₂ stretching band was observed at 580 cm^?1 for the reduced/oxygenated heterodimer (at 554 cm^?1 under an ¹⁸O₂ atmosphere). These results show that domain swapping is a useful method to design multiheme proteins.     

Synthesis,Characterization, and Initial Biological Evaluation of [99mTc]Tc‐Tricarbonyl‐labeled DPA‐α‐MSH Peptide Derivatives for Potential Melanoma Imaging

α‐Melanocyte stimulating hormone (α‐MSH) derivatives target the melanocortin‐1 receptor (MC1R) specifically and selectively. In this study, the α‐MSH‐derived peptide NAP‐NS1 (Nle‐Asp‐His‐d ‐Phe‐Arg‐Trp‐Gly‐NH₂) with and without linkers was conjugated with 5‐(bis(pyridin‐2‐ylmethyl)amino)pentanoic acid (DPA‐COOH) and labeled with [^99mTc]Tc‐tricarbonyl by two methods. With the one‐pot method the labeling was faster than with the two‐pot method, while obtaining similarly high yields. Negligible trans‐chelation and high stability in physiological solutions was determined for the [^99mTc]Tc‐tricarbonyl–peptide conjugates. Coupling an ethylene glycol (EG)‐based linker increased the hydrophilicity. The peptide derivatives displayed high binding affinity in murine B16F10 melanoma cells as well as in human MeWo and TXM13 melanoma cell homogenates. Preliminary in vivo studies with one of the [^99mTc]Tc‐tricarbonyl–peptide conjugates showed good stability in blood and both renal and hepatobiliary excretion. Biodistribution was performed on healthy rats to gain initial insight into the potential relevance of the ^99mTc‐labeled peptides for in vivo imaging.     

Thermal Properties and Phase Transition of 2ZrO2●P2O5 Studied by In Situ Synchrotron X‐ray Diffraction

High‐temperature in situ synchrotron X‐ray powder diffraction experiments were carried out to investigate the phase transition mechanism of Zr₂P₂O₉ (2ZrO₂·P₂O₅ or “Z₂P”). Linear thermal expansion coefficients were calculated for the low‐temperature phase (α‐Z₂P) and the high‐temperature phase (β‐Z₂P) from temperature‐dependent changes in lattice parameters. The crystal structures of α‐ and β‐Z₂P were determined as a function of temperature by performing Rietveld crystal structure refinements. The structural changes at the phase transition are accompanied by an increase in the average atomic distance between neighboring Zr atoms. The occurrence of shorter metal–metal distances in α‐Z₂P is interpreted to result from stronger interactions between partly occupied valence orbitals of the d⁰ metal atoms. The bond valence method was used to calculate the valence sums of the atoms of α‐ and β‐Z₂P, respectively, considering also contributions resulting from covalently bonded atoms. As the bond strength between the metal atoms in Z₂P decreases with the transition into the high‐temperature phase, notably, the metal–metal interactions are regarded to constitute a prerequisite for the stabilization of the α‐phase over the energetically favored β‐phase.     

CuI and H2O2 Inactivate and FeII Inhibits [Fe]‐Hydrogenase at Very Low Concentrations

[Fe]‐Hydrogenase (Hmd) catalyzes reversible hydride transfer from H₂. It harbors an iron‐guanylylpyridinol as a cofactor with an Fe^II that is ligated to one thiolate, two COs, one acyl‐C, one pyridinol‐N, and solvent. Here, we report that Cu^I and H₂O₂ inactivate Hmd (half‐maximal rates at 1 μM Cu^I and 20 μM H₂O₂) and that Fe^II inhibits the enzyme with very high affinity (K_i=40 nM ). Infrared and EPR studies together with competitive inhibition studies with isocyanide indicated that Cu^I exerts its inhibitory effect most probably by binding to the active site iron‐thiolate ligand. Using the same methods, it was found that H₂O₂ binds to the active‐site iron at the solvent‐binding site and oxidizes Fe^II to Fe^III. Also it was shown that Fe^II reversibly binds away from the active site iron, with binding being competitive to the organic hydride acceptor; this inhibition is specific for Fe^II and is reminiscent of that for the [FeFe]‐hydrogenase second iron, which specifically interacts with H₂.     

Study of third‐order optical nonlinearities of substituted hydrazones in PMMA

We investigated the third‐order nonlinear optical properties of donor–acceptor substituted hydrazones doped into PMMA matrix using single beam Z‐scan technique at 532 nm. The magnitude of third‐order susceptibility, χ⁽³⁾ is of the order of 10⁻¹³esu. The nonlinear refractive index, n₂ and the molecular second order hyperpolarisability, γ_h are of the order of 10⁻¹¹esu and 10⁻³²esu, respectively. The compounds exhibit larger third‐order NLO properties in PMMA host when compared to the pure compounds. The study reveals that these hydrazones are a good candidate of nonlinear optical materials for photonic applications. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Two‐Step Protein Labeling by Using Lipoic Acid Ligase with Norbornene Substrates and Subsequent Inverse‐Electron Demand Diels–Alder Reaction

Inverse‐electron‐demand Diels–Alder cycloaddition (DA_inv) between strained alkenes and tetrazines is a highly bio‐orthogonal reaction that has been applied in the specific labeling of biomolecules. In this work we present a two‐step labeling protocol for the site‐specific labeling of proteins based on attachment of a highly stable norbornene derivative to a specific peptide sequence by using a mutant of the enzyme lipoic acid ligase A (LplA^W37V), followed by the covalent attachment of tetrazine‐modified fluorophores to the norbornene moiety through the bio‐orthogonal DA_inv . We investigated 15 different norbornene derivatives for their selective enzymatic attachment to a 13‐residue lipoic acid acceptor peptide (LAP) by using a standardized HPLC protocol. Finally, we used this two‐step labeling strategy to label proteins in cell lysates in a site‐specific manner and performed cell‐surface labeling on living cells.     

Asymmetric Sharpless epoxidation of 13S‐hydroxy‐ 9Z, 11E‐octadecadienoic acid (13S‐HODE)

The asymmetric Sharpless epoxidation of methyl 13S‐hydroxy‐9Z, 11E‐octadeca‐dienoate (13S‐HODE, 1 ) with tert‐butyl hydroperoxide (TBHP) catalysed by titanium tetraisopropoxide {Ti(ⁱOPr)₄} in the presence of L(+)‐diisopropyl tartrate (L‐DIPT) gave methyl 13S‐hydroxy‐11S, 12S‐epoxy‐9Z‐octadecenoate 2 (erythro isomer) in 84% diastereomeric excess (de). The epoxidation of 1 with TBHP catalysed by Ti(ⁱOPr)₄ in the presence of D(‐)‐DIPT yielded methyl 13S‐hydroxy‐11RR12R‐epoxy‐9Z‐octadecenoate (threo isomer) 3 in 76% de.     

Insight into C35 terpene Biosyntheses by Nonpathogenic Mycobacterium Species: Functional Analyses of Three Z‐Prenyltransferases and Identification of Dehydroheptaprenylcyclines

Nonpathogenic Mycobacterium species produce rare cyclic C₃₅ terpenes that are biosynthesized by cyclization of Z‐type C₃₅ polyprenyl diphosphate. To provide deeper insight into the biosynthesis of C₃₅ terpenes, we carried out functional analyses of three Z‐prenyltransferase homologues in M. vanbaalenii identified by genomic analysis. Mvan_3822, a novel bifunctional Z‐prenyltransferase, biosynthesizes C₃₅‐heptaprenyl diphosphate as a main product from (E,E)‐farnesyl diphosphate (E,E‐FPP) and (E,E,E)‐geranylgeranyl diphosphate (E,E,E‐GGPP), but produces a C₅₀‐decaprenyl diphosphate from geranyl diphosphate. Mvan_1705 is a novel Z,E,E‐GGPP synthase. In addition, novel cyclic C₃₅ terpenes, (14E)‐ and (14Z)‐dehydroheptaprenylcycline, were identified as minor metabolites in nonpathogenic Mycobacterium cells. C₃₅ terpenes could be biosynthesized by two routes, in which E and Z geometric isomers of heptaprenyl diphosphate are produced from E,E‐FPP and E,E,E‐GGPP, and the prenylreductase responsible for the biosynthesis of C₃₅ terpenes could reduce both E and Z prenyl residues.     

Genetic Incorporation of Nε‐Formyllysine,a New Histone Post‐translational Modification

Lysine formylation is a newly discovered post‐translational modification (PTM) in histones and other nuclear proteins; it has a well‐recognized but poorly defined role in chromatin conformation modulation and gene expression. To date, there is no general method to site‐specifically incorporate N^ε‐formyllysine at a defined site of a protein. Here we report the highly efficient genetic incorporation of the unnatural amino acid N^ε‐formyllysine into proteins produced in Escherichia coli and mammalian cells, by using an orthogonal N^ε‐formyllysine tRNAsynthetase/tRNA_CUA pair. This technique can be applied to study the role of lysine formylation in epigenetic regulation.