Resolution of 1‐n‐Butyl‐3‐Methyl‐3‐Phospholene 1‐Oxide With TADDOL Derivatives and Calcium Salts of O,O'‐Dibenzoyl‐(2R,3R)‐ or O,O'‐di‐p‐Toluoyl‐(2R,3R)‐tartaric Acid

The resolution methods applying (?)‐(4R,5R)‐4,5‐bis(diphenylhydroxymethyl)‐2,2‐dimethyldioxolane (“TADDOL”), (?)‐(2R,3R)‐α,α,α',α'‐tetraphenyl‐1,4‐dioxaspiro[4.5]decan‐2,3‐dimethanol (“spiro‐TADDOL”), as well as the acidic and neutral Ca²⁺ salts of (?)‐O,O'‐dibenzoyl‐ and (?)‐O,O'‐di‐p‐toluoyl‐(2R,3R)‐tartaric acid were extended for the preparation of 1‐n‐butyl‐3‐methyl‐3‐phospholene 1‐oxide in optically active form. In one case, the intermediate diastereomeric complex could be identified by single‐crystal X‐ray analysis. The absolute P‐configuration of the enantiomers of the phospholene oxide was also determined by comparing the experimentally obtained and calculated CD spectra. Chirality 26:174–182, 2014. © 2014 Wiley Periodicals, Inc.     

Variable gene family usage of protective and non‐protective anti‐Vibrio cholerae O1 LPS antibody heavy chains

Vibrio cholerae causes cholera, an enteric disease of humans that is a worldwide problem. The O1 serogroup of Vibrio cholerae contains two predominant serotypes (Inaba and Ogawa) of LPS, a proven protective antigen for humans and experimental animals. We generated B‐cell hybridomas from mice immunized with either: (i) two doses of purified Inaba LPS; (ii) two doses of an Inaba hexasaccharide conjugate (terminal six perosamine bound to a protein carrier), (iii) four doses of purified Inaba LPS; or (iv) a low dose of purified Inaba LPS followed by a booster with the Inaba conjugate. We showed previously that the first and third immunization protocols induce vibriocidal antibodies, as does the fourth; the second protocol induces antibodies that bind Inaba and Ogawa LPS but are not vibriocidal. Anti‐LPS mAbs derived from hybridomas resulting from each immunization protocol were characterized for binding to Inaba and Ogawa LPS, their vibriocidal or protective capacity, and the variable heavy chain family they expressed. LPS immunogens selected different LPS‐specific B cells expressing six different Vh chain families. Protective and non‐protective mAbs could express variable regions from the same family. One mAb was specific for Inaba LPS, the other mAbs were cross‐reactive with both LPS serotypes. Sequence comparison suggests that the pairing of a specific light chain, somatic mutation, or the specific VDJ recombination can modulate the protective capacity of mAbs that express a common variable heavy chain family member.     

Separation of Enantiomers by Inclusion Gas Chromatography: On the Influence of Water in the Molecular Complexation of Methyl 2‐Chloropropanoate Enantiomers and the Modified γ‐Cyclodextrin Lipodex‐E

A profound influence of water has previously been detected in the complexation of the enantiomers of methyl 2‐chloropropanoate (MCP) and the chiral selector octakis(3‐O‐butanoyl‐2,6‐di‐O‐pentyl)‐γ‐cyclodextrin (Lipodex‐E) in NMR and sensor experiments. We therefore investigated the retention behavior of MCP enantiomers on Lipodex‐E by gas chromatography (GC) under hydrous conditions. Addition of water to the N₂ carrier gas modestly reduced the retention factors k of the enantiomers, notably for the second eluted enantiomer (S)‐MCP. This resulted in an overall decrease of enantioselectivity ‐Δ_S,R(ΔG) in the presence of water. The effect was fully reversible. Consequently, for a conditioned column in the absence of residual water, the determined thermodynamic data, i.e. Δ_S,R(ΔH) = –12.64 ± 0.08 kJ mol^‐1 and Δ_S,R(ΔS) = –28.18 ± 0.23 J K^‐1 mol^‐1, refer to a true 1:1 complexation process devoid of hydrophobic hydration. Chirality 28:124–131, 2015. © 2015 Wiley Periodicals, Inc.     

Life‐cycle and cost of goods assessment of fed‐batch and perfusion‐based manufacturing processes for mAbs

Life‐cycle assessment (LCA) is an environmental assessment tool that quantifies the environmental impact associated with a product or a process (e.g., water consumption, energy requirements, and solid waste generation). While LCA is a standard approach in many commercial industries, its application has not been exploited widely in the bioprocessing sector. To contribute toward the design of more cost‐efficient, robust and environmentally‐friendly manufacturing process for monoclonal antibodies (mAbs), a framework consisting of an LCA and economic analysis combined with a sensitivity analysis of manufacturing process parameters and a production scale‐up study is presented. The efficiency of the framework is demonstrated using a comparative study of the two most commonly used upstream configurations for mAb manufacture, namely fed‐batch (FB) and perfusion‐based processes. Results obtained by the framework are presented using a range of visualization tools, and indicate that a standard perfusion process (with a pooling duration of 4 days) has similar cost of goods than a FB process but a larger environmental footprint because it consumed 35% more water, demanded 17% more energy, and emitted 17% more CO₂ than the FB process. Water consumption was the most important impact category, especially when scaling‐up the processes, as energy was required to produce process water and water‐for‐injection, while CO₂ was emitted from energy generation. The sensitivity analysis revealed that the perfusion process can be made more environmentally‐friendly than the FB process if the pooling duration is extended to 8 days. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1324–1335, 2016     

A Simple Method for Resolution of Endo‐/Exo‐Monoesters of Trans‐Norborn‐5‐Ene‐2,3‐Dicarboxylic Acids Into Their Enantiomers

Separation of optical isomers obtainable from trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid methyl and tert‐butyl monoesters was performed by crystallization of the respective salts prepared with (R)‐ and (S)‐1‐phenylethylamine. Starting from racemic endo‐monomethyl ester of trans‐norborn‐5‐ene‐2,3‐dicarboxylic acid, prepared by partial hydrolysis of the cyclopentadiene‐dimethyl fumarate adduct, the corresponding (2R,3R)‐endo‐monoester was isolated in 97% enantiomeric excess (ee) yield after seven repeated crystallizations from tetrachloromethane. Starting from exo‐mono‐tert‐butyl ester of the same acid, prepared by alcoholysis of the cyclopentadiene‐maleic anhydride adduct followed by isomerization, (2R,3R)‐exo‐monoester was isolated in >98% ee yield after four repeated crystallizations from ethanol. Crystallization of the acids from the mother liquor containing (S)‐1‐phenylethylamine yielded products with inverse stereochemical configuration. Chirality 27:151–155, 2015. © 2014 Wiley Periodicals, Inc.     

Decadal changes in mean annual rainfall drive long‐term changes in bush‐encroached southern African savannas

Bush encroachment can have profound effects on the ability of savanna ecosystems to provide goods and services to society. It is therefore crucial to understand the key drivers of bush encroachment in savannas. In this study, we test whether decadal changes in mean annual rainfall significantly explain changes in the dominant patch size as well as the density of bush patches at six protected savanna sites located along a rainfall gradient in Zimbabwe. We first performed Maximal Overlap Discrete Wavelet transform within the intensity‐dominant scale theoretical framework on multi‐temporal aerial photographs and high spatial resolution satellite imagery to objectively detect changes in the dominant patch dimension as well as the intensity of bush cover over a 40‐year period at six test sites. We then pooled the data and performed regression analysis relating changes in dominant scale and intensity to decadal changes in mean annual rainfall in order to deduce a possible connection between dynamics of bush encroachment and rainfall variability. Our results indicate a significant nonlinear relationship between changes in the dominant scale and decadal changes in mean annual rainfall (R² = 0.85, F₁₃ = 35.96, P < 0.01). In contrast, the relationship between decadal changes in mean annual rainfall and changes in intensity was weak and not significant (R² = 0.29, F₁₃ = 2.69, P = 0.106). These results imply the importance of annual rainfall in explaining long‐term changes in the dominant scale of woody patches. However, mechanisms other than rainfall probably explain changes in the intensity of bush cover, and this needs further investigation.     

Development of methane conversion factor models for Zebu beef cattle fed low‐quality crop residues and by‐products in tropical regions

The enteric methane conversion factor (Y_m) is an important country‐specific value for the provision of precise enteric methane emissions inventory reports. The objectives of this meta‐analysis were to develop and evaluate the empirical Y_m models for the national level and the farm level for tropical developing countries according to the IPCC's categorization. We used datasets derived from 18 in vivo feeding experiments from 1999 to 2015 of Zebu beef cattle breeds fed low‐quality crop residues and by‐products. We found that the observed Y_m value was 8.2% gross energy (GE) intake (~120 g methane emission head^?1 day^?1) and ranged from 4.8% to 13.7% GE intake. The IPCC default model (tier 2, Y_m = 6.5% ± 1.0% GE intake) underestimated the Y_m values by up to 26.1% compared with its refinement of 8.4% ± 0.4% GE intake for the national‐level estimate. Both the IPCC default model and the refined model performed worse in predicting Y_m trends at the farm level (root mean square prediction error [MSPE] = 15.1%–23.1%, concordance correlation coefficient [CCC] = 0.16–0.18, R² = .32). Seven of the extant Y_m models based on a linear regression approach also showed inaccurately estimated Y_m values (root MSPE = 16.2%–36.0%, CCC = 0.02–0.27, R² < .37). However, one of the developed models, which related to the complexity of the energy use efficiencies of the diet consumed to Y_m, showed adequate accuracy at the farm level (root MSPE = 9.1%, CCC = 0.75, R² = .67). Our results thus suggest a new Y_m model and future challenges for estimating Zebu beef cattle production in tropical developing countries.     

Child‐Specific Thoracic Gas Volume Prediction Equations for Air‐Displacement Plethysmography

Objective: To develop child‐specific thoracic gas volume (TGV) prediction equations for use in air‐displacement plethysmography in 6‐ to 17‐year‐old children. Research Methods and Procedures: Study 1 developed TGV prediction equations using anthropometric variables after completing a measured TGV and air‐displacement plethysmography test in 224 healthy boys and girls (11.2 ± 3.2 years, 45.3 ± 18.7 kg, 149.9 ± 18.5 cm). Study 2 cross‐validated the prediction equations in a separate cohort of 62 healthy boys and girls (11.2 ± 3.4 years, 44.2 ± 15.3 kg, 149.4 ± 19.3 cm). Results: In Study 1 (development of TGV prediction equations), the quadratic relationship using height as the independent variable and the measured TGV as the dependent variable yielded the highest adjusted R² and the lowest SE of estimate in both genders, thus producing the following prediction equations: TGV = 0.00056 × H² ? 0.12422 × H + 8.15194 (boys) and TGV = 0.00044 × H² ? 0.09220 × H + 6.00305 (girls). In Study 2 (cross‐validation), no significant difference between the predicted and measured TGVs (?0.018 ± 0.377 liters) was observed. The regression between the measured TGV and the predicted TGV yielded a slope and intercept that did not significantly differ from the line of identity. Prediction accuracy was good as indicated by a high R² (0.862) and low SE of estimate (0.369 liters). Discussion: The new child‐specific TGV prediction equations accurately, precisely, and without bias estimated the actual TGV of 6‐ to 17‐year‐old children.     

Construction and characterization of two anti‐sweetener single chain antibodies using radioligand binding,fluorescence and circular dichroism spectroscopy

Two single‐chain antibodies (scFv) that bind the superpotent sweetener ligand, NC‐174, were generated from mouse monoclonal antibodies (mAb) NC6.8 (IgG, κ) and NC10.14 (IgG, λ). These scFv were constructed by cloning the variable region sequences of the mAb, connecting them in tandem with a 25‐amino‐acid polypeptide linker, and expressing them in E. coli using the pET‐11a system. The recombinant proteins were purified using Ni²⁺–NTA–agarose by virtue of a hexahistidine sequence introduced to the C‐terminus of the heavy chain variable region during the cloning process. The secondary structure and ligand binding properties of the two scFv, the parent mAbs and proteolytically derived Fab fragments were examined using radioligand binding, circular dichroism (CD) and fluorescence spectroscopy. The far‐UV CD spectra of both scFv possessed predominantly β character, as did those of the Fab, and the near‐UV CD spectral data for scFvNC10.14, NC6.8 and NC10.14 Fab indicated that chromophore perturbation occurred upon ligand binding. The affinity constants determined for the two scFv, Fab and mAb were nearly equivalent. Copyright © 1999 John Wiley & Sons, Ltd.     

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐ 2 ) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo‐keto reductase mutant KlAKR‐Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de_p > 99.5%) toward t‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐ 1 ). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co‐producer E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)‐ 1 at substrate loading size of up to 60.0 g/L (5R)‐ 1 in the absence of exogenous NADH, which was one‐fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)‐ 2 with productivity of 243.2 kg/m³ d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co‐expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235–1242, 2017     

Producing defucosylated antibodies with enhanced in vitro antibody‐dependent cellular cytotoxicity via FUT8 knockout CHO‐S cells

To engineer a host cell line that produces defucosylated mAbs with superior antibody‐dependent cellular cytotoxicity, we disrupted α‐1, 6 fucosyltransferase (FUT8 ) gene in CHO‐S (CHO is Chinese hamster ovary) cells by clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9. The gene knockout cell line was evaluated for growth, stability, and product quality. The growth profile of FUT8 gene knockout CHO‐S (FUT8 ^?/?) cells was comparable with wild type CHO‐S cells. FUT8 catalyzes the transfer of a fucose residue from GDP‐fucose to N‐glycans residue. Defucosylated IgG1 antibodies produced by FUT8 ^?/? cells showed increased binding affinities to human FcγRIIIa and higher activities in mediating antibody‐dependent cellular cytotoxicity, comparing with conventional fucosylated IgG1. Our results demonstrated the potential of using the clustered regularly interspaced short palindromic repeats‐CRISPR associated nuclease 9 technology in cell line engineering for biopharmaceutical industrial applications.     

Structural modeling and biochemical studies reveal insights into the molecular basis of the recognition of β‐2‐microglobulin by antibody BBM.1

Human β‐2‐microglobulin (β2m) is the light chain of human leucocyte antigen‐I (HLA‐I). It can disassociate from HLA‐I and accumulate to cause serious dialysis‐related amyloidosis (DRA) in long‐term hemodialysis patients. Monoclonal antibody (mAb) BBM.1 can recognize both free‐form and HLA‐I associated β2m. It can be used for specific elimination of β2m from serum and can induce apoptosis of several types of tumor cells, and thus has great therapeutic potential. In this study, we constructed structural models of the BBM.1 Fv (fragment of the variable domain) and the BBM.1 Fv‐β2m complex, followed by biochemical evaluation. Analysis of the optimal complex model reveals that the previously identified immunodominant residues Glu⁴⁴ and Arg⁴⁵ of β2m have direct interactions with BBM.1, while Asp³⁸ exerts its function mainly via stabilization of Arg⁴⁵. In addition, Arg⁸¹ of β2m is a newly identified immunodominant residue to have direct interaction with BBM.1. Further modeling study shows no steric conflict between the antibody and the HLA‐I heavy chain. These results provide insights into the molecular basis of the recognition of β2m by BBM.1 and explain why BBM.1 can bind both free‐form and HLA‐1 associated β2m. This information could be exploited in the engineering and improvement of BBM.1 and the development of other β2m‐targeting mAbs for therapeutic purposes. Copyright © 2009 John Wiley & Sons, Ltd.     

In vitro wound healing: Inhibition activity of insect‐derived mAb CO17‐1A in human colorectal cancer cell migration

The monoclonal antibody (mAb) CO17‐1A specifically binds to the tumor‐associated cell surface glycoprotein GA733 in colorectal cancer cells. Thus, mAb CO17‐1A has the potential to act as an immune therapeutic protein against colorectal cancer. Recently, it was shown that the baculovirus insect cell expression system produces anti‐colorectal cancer mAb CO17‐1A. In this study, the colorectal cancer antibody mAb CO17‐1A fused to the endoplasmic reticulum (ER) retention signal sequence (KDEL), and the (mAb CO17‐1AK) was expressed in Spodoptera frugiperda Sf9 insect cells. The yield, cell cytotoxicity, and in vitro anti‐tumor activity of mAb CO17‐1AK were verified. Western blotting was performed to confirm that both heavy and light chains of mAb CO17‐1A were expressed in Sf9 insect cells. The insect‐derived mAb (mAb^I) CO17‐1A was purified using a protein G affinity column. An in vitro wound healing assay was conducted to determine the inhibition activity of mAb CO17‐1A during tumor cell migration, showing that mAb^I CO17‐1AK was effective as mammalian‐derived mAb CO17‐1A (mAb^M CO17‐1A). These results suggest that the insect cell expression system can produce and properly assemble mAbs that inhibit tumor cell migration.     

Assessment of ATR‐NIR and ATR‐MIR spectroscopy as an analytical tool for the quantification of the total polyphenols in Dendrobium huoshanense

In this study, the potentiality of applying attenuated total reflectance near‐infrared (ATR‐NIR) and attenuated total reflectance mid‐infrared (ATR‐MIR) techniques combined with a partial least squares (PLS) regression technology to quantify the total polyphenols (TPs) in Dendrobium huoshanense (DHS) was investigated and compared. The real TP contents in the DHS samples were analysed using methods of reference. The capability of the two IR spectroscopic techniques to quantify the TPs in DHS was assessed by the root‐mean‐square error of calibration (RMSEC) and determination coefficients (R²). The results showed that both NIR and MIR might be used as a fast and simple tool to replace traditional chemical assays for the determination of the TP contents in DHS, and the best NIR model showed slightly better prediction performance [root‐mean‐square error of prediction (RMSEP): 0.307, R²: 0.9122, ratio performance deviation (RPD): 4.43] than the best MIR model (RMSEP: 0.440, R²: 0.9069, RPD: 3.09). Results from this study indicated that both the NIR and MIR models could be used to quantify the TP in DHS, and ATR‐NIR appeared to be the more predominant and more robust technique for the quantification of the TP in DHS.     

Enantiomeric assay of escitalopram S(+)‐enantiomer and its “in‐process impurities” using two different techniques

The enantiomeric purity of escitalopram oxalate ESC and its “in‐process impurities,” namely, ESC‐N‐oxide, ESC‐citadiol, and R(?)‐enantiomer were studied in drug substance and products using high‐performance liquid chromatography (HPLC)‐UV (Method I), synchronous fluorescence spectroscopy (SFS) (Method IIA), and first derivative SFS (Method IIB). Method I describes as an isocratic HPLC‐UV for the direct resolution and determination of enantiomeric purity of ESC and its “in‐process impurities.” The proposed method involved the use of α_l‐acid glycoprotein (AGP) chiral stationary phase. The regression plots revealed good linear relationships of concentration range of 0.25 to 100 and 0.25 to 10 μg mL^?1 for ESC and its impurities. The limits of detection and quantifications for ESC were 0.075 and 0.235 μg mL^?1, respectively. Method II involves the significant enhancement of the fluorescence intensities of ESC and its impurities through inclusion complexes formation with hydroxyl propyl‐β‐cyclodextrin as a chiral selector in Micliavain buffer. Method IIA describes SFS technique for assay of ESC at 225 nm in presence of its impurities: R(?)‐enantiomer, citadiol, and N‐oxide at ?λ of 100 nm. This method was extended to (Method IIB) to apply first derivative SFS for the simultaneous determination of ESC at 236 nm and its impurities: the R(?)‐enantiomer, citadiol, and N‐oxide at 308, 275, and 280 nm, respectively. Linearity ranges were found to be 0.01 to 1.0 μg mL^?1 for ESC and its impurities with lower detection and quantification limits of 0.033/0.011 and 0.038/0.013 μg mL^?1 for SFS and first derivative synchronous fluorescence spectra (FDSFS), respectively. The methods were used to investigate the enantiomeric purity of escitalopram.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.     

Synthesis and Chiral Recognition of Nickel(II) Macrocyclic Complex with (R)‐Naphthylethyleneamine Pendant Groups and its Self‐Assembled Framework

A novel nickel(II) hexaaza macrocyclic complex, [Ni(L^R,R)](ClO₄)₂ ( 1 ), containing chiral pendant groups was synthesized by an efficient one‐pot template condensation and characterized (L^R,R═1,8‐di((R)‐α‐methylnaphthyl)‐1,3,6,8,10,13‐hexaazacyclotetradecane). The crystal structure of compound 1 was determined by single‐crystal X‐ray analysis. The complex was found to have a square‐planar coordination environment for the nickel(II) ion. Open framework [Ni(L^R,R)]₃[C₆H₃(COO)₃]₂ ( 2 ) was constructed from the self‐assembly of compound 1 with deprotonated 1,3,5‐benzenetricarboxylic acid, BTC^3?. Chiral discrimination of rac‐1,1′‐bi‐2‐naphthol and rac‐2,2,2‐trifluoro‐1‐(9‐anthryl)ethanol was performed to determine the chiral recognition ability of the chiral complex ( 1 ) and its self‐assembled framework ( 2 ). Binaphthol showed a good chiral discrimination on the framework ( 2 ). The optimum experimental conditions for the chiral discrimination were examined by changing the weight ratio between the macrocyclic complex 1 or self‐assembled framework 2 and racemates. The detailed synthetic procedures, spectroscopic data including single‐crystal X‐ray analysis, and the results of the chiral recognition for the compounds are described. Chirality, 25:54‐58, 2013 © 2012 Wiley Periodicals, Inc.     

Successful selection of an infection‐protective anti‐Staphylococcus aureus monoclonal antibody and its protective activity in murine infection models

Recent clinical trials to develop anti‐methicillin‐resistant Staphylococcus aureus (MRSA) therapeutic antibodies have met unsuccessful sequels. To develop more effective antibodies against MRSA infection, a panel of mAbs against S. aureus cell wall was generated and then screened for the most protective mAb in mouse infection models. Twenty‐two anti‐S. aureus IgG mAbs were obtained from mice that had been immunized with alkali‐processed, deacetylated cell walls of S. aureus. One of these mAbs, ZBIA5H, exhibited life‐saving effects in mouse models of sepsis caused by community‐acquired MRSA strain MW2 and vancomycin‐resistant S. aureus strain VRS1. It also had a curative effect in a MW2‐caused pneumonia model. Curiously, the target of ZBIA5H was considered to be a conformational epitope of either the 1,4‐β‐linkage between N‐acetylmuramic acid and N‐acetyl‐D‐glucosamine or the peptidoglycan per se. Reactivity of ZBIA5H to S. aureus whole cells or purified peptidoglycan was weaker than that of most of the other mAbs generated in this study. However, the latter mAbs did not have the protective activities against S. aureus that ZBIA5H did. These data indicate that the epitopes that trigger production of high‐yield and/or high‐affinity antibodies may not be the most suitable epitopes for developing anti‐infective antibodies. ZBIA5H or its humanized form may find a future clinical application, and its target epitope may be used for the production of vaccines against S. aureus infection.     

The impact of β‐azido(or 1‐piperidinyl)methylamino acids in position 2 or 3 on biological activity and conformation of dermorphin analogues

The synthesis of new dermorphin analogues is described. The (R)‐alanine or phenylalanine residues of natural dermorphin were substituted by the corresponding α‐methyl‐β‐azidoalanine or α‐benzyl‐β‐azido(1‐piperidinyl)alanine residues. The potency and selectivity of the new analogues were evaluated by a competitive receptor binding assay in rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). The most active analogue in this series, Tyr‐(R)‐Ala‐(R)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ and its epimer were analysed by ¹H and ¹³C NMR spectroscopy and restrained molecular dynamics simulations. The dominant conformation of the investigated peptides depended on the absolute configuration around C^α in the α‐benzyl‐β‐azidoAla residue in position 3. The (R) configuration led to the formation of a type I β‐turn, whilst switching to the (S) configuration gave rise to an inverse β‐turn of type I′, followed by the formation of a very short β‐sheet. The selectivity of Tyr‐(R)‐Ala‐(R) and (S)‐α‐benzyl‐β‐azidoAla‐Gly‐Tyr‐Pro‐Ser‐NH₂ was shown to be very similar; nevertheless, the two analogues exhibited different conformational preferences. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.