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The combination of photo and copper catalysts has emerged as a novel paradigm in organic catalysis, which provides access to the acceleration of chemical synthesis. Herein, we describe an aminoalkylation of amino-dependent olefins with maleimides through a cooperative photo/copper catalytic system. In this report, the strategy allows the generation of a broad complex of functionalized nitrogenous molecules including oxazolidinones, 2-pyrrolidones, imidazolidinones, thiazolidinones, pyridines, and piperidines in the absence of an external photosensitizer and base. The approach is achieved through a photoinduced Cu(I)/Cu(II)/Cu(III) complex species of nitrogen nucleophiles, intermolecular radical addition, and hydrogen atom transfer (HAT) processes. The plausible mechanism is investigated by a series of control experiments and theoretical tests, including radical scavenging experiments, deuterium labeling experiments, ultraviolet-visible absorption, and cyclic voltammetry (CV) tests.

Methodologies employing LC-MS/MS have been increasingly used for characterization and identification of residual host cell proteins (HCPs) in biopharmaceutical products to ensure their consistent product quality and safety for patients. To improve the sensitivity and reliability for HCP detection, we developed a high pH-low pH two-dimensional reversed phase LC-MS/MS approach in conjunction with offline fraction concatenation. Proof-of -concept was established using a model in which seven proteins spanning a size range of 29-78 kDa are spiked into a purified antibody product to simulate the presence of low-level HCPs. By incorporating a tandem column configuration and a shallow gradient through the second-dimension, all seven proteins were consistently identified at 10 ppm with 100% success rate following LC-MS/MS analysis of six concatenated fractions across multiple analysts, column lots and injection loads. Using the more complex Universal Proteomic Standard 1 (UPS-1) as an HCP model, positive identification was consistently achieved for 19 of the 22 proteins in 8-12 ppm range (10 ppm ±20%). For the first time, we demonstrate an effective LC-MS/MS strategy that not only has high sensitivity but also high reliability for HCP detection. The method performance has high impact on pharmaceutical company practices in using advanced LC-MS/MS technology to ensure product quality and patient safety.

The bacterial acid-resistant chaperone HdeA is a "conditionally disordered" protein that functions at low pH when it undergoes a transition from a well-folded dimer to an unfolded monomer. The dimer dissociation and unfolding processes result in exposure of hydrophobic surfaces that allows binding to a broad range of client proteins. To fully elucidate the chaperone mechanism of HdeA, it is crucial to understand how the activated HdeA interacts with its native substrates during acid stress. Herein, we present a nuclear magnetic resonance study of the pH-dependent HdeA-substrate interactions. Our results show that the activation of HdeA is not only induced by acidification but also regulated by the presence of unfolded substrates. The variable extent of unfolding of substrates differentially regulates the HdeA-substrate interaction, and the binding further affects the HdeA conformation. Finally, we show that HdeA binds its substrates heterogeneously, and the "amphiphilic" model for HdeA-substrate interaction is discussed.

Twenty-two 2-aryl-9-methyl-3,4-dihydro-β-carbolin-2-ium bromides along with four 9-demethylated derivatives were synthesized and characterized by spectroscopic analysis. By using the mycelium growth rate method, the compounds were evaluated for antifungal activities in vitro against six plant pathogenic fungi, and structure-activity relationships (SAR) were derived. Almost all of the compounds showed obvious inhibition activity on each of the fungi at 150 μM. For all of the fungi, 10 of the compounds showed average inhibition rates of >80% at 150 μM, and most of their EC50 values were in the range of 2.0-30.0 μM. SAR analysis showed that the substitution pattern of the N-aryl ring significantly influences the activity; N9-alkylation improves the activity, whereas aromatization of ring-C reduces the activity. It was concluded that the present research provided a series of new 2-aryl-9-alkyl-3,4-dihydro-β-carbolin-2-iums with excellent antifungal potency and structure optimization design for the development of new carboline antifungal agents.

This paper reports a dramatic reduction in plasmon resonance line width of a single Au nanorod by coupling it to a whispering gallery cavity of a silica microfiber. With fiber diameter below 6 μm, strong coupling between the nanorod and the cavity occurs, leading to evident mode splitting and spectral narrowing. Using a 1.46-μm-diameter microfiber, we obtained single-band 2-nm-line-width plasmon resonance in an Au nanorod around a 655-nm-wavelength, with a quality factor up to 330 and extinction ratio of 30 dB. Compared to an uncoupled Au nanorod, the strongly coupled nanorod offers a 30-fold enhancement in the peak intensity of plasmonic resonant scattering.

Accurate and precise quantitative measurement of product-related variants of a therapeutic antibody is essential for product development and testing. Bispecific antibodies (bsAbs) are Abs composed of two different half antibody arms, each of which recognizes a distinct target, and recently they have attracted substantial therapeutic interest. Because of the increased complexity of its structure and its production process, as compared to a conventional monoclonal antibody, additional product-related variants, including covalent and noncovalent homodimers of half antibodies (hAbs), may be present in the bsAb product. Sufficient separation and reliable quantitation of these bsAb homodimers using liquid chromatography (LC) or capillary electrophoresis-based methods is challenging because these homodimer species and the bsAb often have similar physicochemical properties. Formation of noncovalent homodimers and heterodimers can also occur. In addition, since homodimers share common sequences with their corresponding halves and bsAb, it is not suitable to use peptides as surrogates for their quantitation. To tackle these analytical challenges, we developed a mass spectrometry-based quantitation method. Chip-based nanoflow LC-time-of-flight mass spectrometry coupled with a standard addition approach provided unbiased absolute quantitation of these drug-product-related variants. Two methods for the addition of known levels of standard (multi- or single-addition) were evaluated. Both methods demonstrated accurate and reproducible quantitation of homodimers at the 0.2% (w/w) level, with the single-addition method having the promise of higher analytical throughput.

Recombinant antibodies produced in Chinese hamster ovary (CHO) cells often exhibit a slight yellow-brown color, but the molecular basis for this color has remained elusive. We predicted that the color results from post-translational modifications on the antibody, because colored species were found to coelute with antibody products during size exclusion chromatography. Previously, modification by oxidation and advanced glycation end products (AGEs) had been shown to give rise to colored and fluorescent species whose spectral properties are in agreement with the spectral properties of CHO-derived recombinant antibodies. To test whether these modifications give rise to the color exhibited in our CHO-derived antibodies, we searched for 8 different oxidation and 28 different glycation and AGE modifications by mass spectrometry in a variety of samples exhibiting varying color intensities. Oxidation and glycation modifications correlated weakly with color in a subset of samples, but several AGEs exhibited a strong correlation with product color in all samples tested. This strong correlation with sample color was verified for a specific AGE, carboxymethyllysine, by ELISA, thus validating the mass spectrometry data. These data indicate that AGEs are at least partially responsible for the color seen in CHO-derived recombinant antibodies.

We report an efficient, high fidelity trypsin digestion method for peptide map analysis. This method minimizes artifacts caused by the sample preparation process, and we show its utility for the accurate determination of succinimide formation in a degraded monoclonal antibody product. A basic charge variant was detected by imaged capillary isoelectric focusing and was shown with reduced antigen binding and biological activity. Samples were reduced under denaturing conditions at pH 5.0, and digestion of the reduced protein with porcine trypsin was performed at pH 7.0 for 1 h. Following reversed phase high-performance liquid chromatography and online mass spectrometric analysis, succinimide formation was identified at Asp30 in the light chain. This result contrasts with the observation of only iso-Asp and Asp residues under conventional sample preparation conditions, which are therefore concluded to be artificially generated. The Asp30 residue is seen in the cocrystal structure model to participate in favorable charge interaction with an antigen molecule. Formation of succinimide and the resulting loss of negative charge are therefore hypothesized to be the degradation mechanism. After treatment of the degraded antibody sample to mildly alkaline pH conditions, we observed only Asp residue as the succinimide hydrolysis product and concurrent recovery of biological activity.

Translation errors in protein biosynthesis may result in low level amino acid misincorporation and contribute to product heterogeneity of recombinant protein therapeutics. We report the use of peptide map analysis by reversed-phase high-performance liquid chromatography and high-resolution mass spectrometry to detect and identify mistranslation events in recombinant monoclonal antibodies expressed in mammalian cell lines including Chinese hamster ovary (CHO) cells. Misincorporation of an asparagine residue at multiple serine positions was detected as earlier-eluting peptides with masses 27.01 Da higher than expected. The exact positions at which misincorporation occurred were identified by tandem mass spectrometry of the asparagine-containing variant peptides. The identified asparagine misincorporation sites correlated with the use of codon AGC but with none of the other five serine codons. The relative levels of misincorporation ranged from 0.01%-0.2% among multiple serine positions detected across three different antibodies by targeted analysis of expected and variant peptides. The low levels of misincorporation are consistent with published predictions for in vivo translation error rates. Our results demonstrate that state-of-the-art mass spectrometry with a combination of high sensitivity, accuracy, and dynamic range provides a new ability to discover and characterize low level protein variants that arise from mistranslation events.

Human testicular angiotensin-converting enzyme (tACE) is an extracellular protein that contains seven cysteine residues. The cysteines occur in a sequential distribution that is precisely mimicked in the tACE from rabbit and mouse, and in both domains of all known species of somatic ACE. One of the cysteines in human tACE, Cys496, is present in the reduced form as shown by labeling it with 5-[[2-(iodoacetyl)amino]ethylamino]naphthalene-1-sulfonic acid, isolating the fluorescent peptide from enzymatic digests by HPLC, and analyzing its sequence by matrix-assisted laser desorption ionization-mass spectrometry (MALDI-MS). Thiol reagents have no significant effect on the activity of tACE, indicating that this Cys is not involved in catalysis. The other six cysteines exist as three disulfides. Mass spectral analysis of cyanogen bromide peptides has established that the cystine connectivities follow a nearest-neighbor, aabbcc, pattern i.e., Cys152-Cys158, Cys352-Cys370, and Cys538-Cys550, in which the disulfides form three small loops of five, 17, and 11 residues, respectively. Although these disulfide loops constitute less than 5% of the total sequence of the protein, they contribute to the overall structural stabilization of tACE.

A schematic kinetic model is proposed for the hydroperoxide-mediated substrate reaction and cytochrome P450 inactivation. From the model, the relationships between the product concentration at infinite time (P infinity), the apparent rate constant of P450 inactivation (A), and the substrate concentrations are predicted, and the predictions were experimentally examined. The reciprocal of P infinity is proportional to the reciprocal of the substrate concentration in both CuOOH- and H2O2-supported substrate reactions. The reciprocal of P infinity is proportional to cumene hydroperoxide (CuOOH) concentration, but P infinity is independent of H2O2 concentration, indicating different effects of CuOOH and H2O2 in P450 inactivation. The apparent rate constant (A) is proportional to the reciprocal of the substrate concentrations, suggesting substrate protection of P450 from hydroperoxide inactivation. The model also suggests that a second CuOOH molecule may compete with substrate for binding to the P450 substrate binding site. Simulated kinetics of production formation vs time are quite consistent with the experimental kinetics.

A metastable intermediate was found in the course of the denaturation of purified cytochrome P450 2B1 by increasing concentrations of guanidine hydrochloride (GuHCl). The metastable intermediate has no or low absorbance at 450 nm in the reduced carbon monoxide difference spectrum and has no absorbance at 420 nm. The intermediate is easily converted to P420 by increasing concentrations of GuHCl. Before it becomes P420, the cytochrome can be completely reconverted to native P450 by dilution and incubation at 4 degrees C. Cytochrome P420 resulting from exposure to higher concentrations of GuHCl (> 3 M) failed to be reconverted to P450 by dilution. Denaturation of P450 2B1 by exposure to low concentrations of urea (< 2 M) is also completely reversible but no obvious intermediate is detectable. An intermediate is observed, however, when the urea denaturation is conducted in the presence of 1 M NaCl. As is the case with higher concentrations of GuHCl, cytochrome P450 denatured by exposure to 5 M or higher concentrations of urea is not reversible. The failure of reconversion of P420 denatured by exposure of cytochrome P450 to high concentrations of GuHCl or urea is probably attributable to the extensive unfolding of the apoprotein, which favors aggregation, rather than to heme loss. Our results also suggest that the active site is more sensitive to denaturants than other regions of the protein.