Systematic identification of engineered methionines and oxaziridines for efficient, stable, and site-specific antibody bioconjugation.

The field of chemical modification of proteins has been dominated by random modification of lysines or more site-specific labeling of cysteines, each with attendant challenges. Recently, we have developed oxaziridine chemistry for highly selective modification of methionine called redox-activated chemical tagging (ReACT) but have not broadly tested the molecular parameters for efficient and stable protein modification. Here we systematically scanned methionines throughout one of the most popular antibody scaffolds, trastuzumab, used for antibody engineering and drug conjugation. We tested the expression, reactivities, and stabilities of 123 single engineered methionines distributed over the surface of the antibody when reacted with oxaziridine. We found uniformly high expression for these mutants and excellent reaction efficiencies with a panel of oxaziridines. Remarkably, the stability to hydrolysis of the sulfimide varied more than 10-fold depending on temperature and the site of the engineered methionine. Interestingly, the most stable and reactive sites were those that were partially buried, presumably because of their reduced access to water. There was also a 10-fold variation in stability depending on the nature of the oxaziridine, which was determined to be inversely correlated with the electrophilic nature of the sulfimide. Importantly, the stabilities of the best analogs were sufficient to support their use as antibody drug conjugates and potent in a breast cancer mouse xenograft model over a month. These studies provide key parameters for broad application of ReACT for efficient, stable, and site-specific antibody and protein bioconjugation to native or engineered methionines.

Structural and conformational analysis of 1-oxaspiro[2.5]octane and 1-oxa-2-azaspiro[2.5]octane derivatives by (1) H, (13) C, and (15) N NMR.

A structural and conformational analysis of 1-oxaspiro[2.5]octane and 1-oxa-2-azaspiro[2.5]octane derivatives was performed using (1) H, (13) C, and (15) N NMR spectroscopy. The relative configuration and preferred conformations were determined by analyzing the homonuclear coupling constants and chemical shifts of the protons and carbon atoms in the aliphatic rings. These parameters directly reflected the steric and electronic effects of the substituent bonded to the aliphatic six-membered ring or to C3 or N2. The parameters also were sensitive to the anisotropic positions of these atoms in the three-atom ring. The preferred orientation of the exocyclic substituents directed the oxidative attack.

Fast and fully automated analytical method for the screening of residues of aziridine and 2-chloroethylamine in pharmaceutical active principles.

A simple, fast and fully automated method for the screening of aziridine (AZD) and 2-chloroethylamine (CEA) in active pharmaceutical ingredients (API) has been developed. The method is based on the in-fiber derivatization of the amines extracted from the sample headspace (previously dissolved or suspended in alkaline water) with 2,3,4,5,6-pentafluorobenzoyl chloride (PFBCl) previously adsorbed in the PDMS/DVB solid phase microextraction (SPME) fiber. The derivatives formed are further desorbed and analyzed in a gas chromatograph with negative ion chemical ionization mass spectrometry (GC-NCI-MS) using methane as reagent gas. The different operational parameters of the procedure have been optimized to get highest sensitivity. The validation of the method, however, revealed a poor repeatability, particularly evident in water-soluble APIs (RSD>20% for AZD). In spite of that, the low detection limits (1-3ngg(-1) for AZD and CEA), speed (44min total analysis time) and automation make that this method can be satisfactorily used as screening tool to accept or reject API batches attending to their volatile amine content and a critical specified value derived from the 1.5µg/day Threshold of Toxicological Concern (TTC) and maxima daily dosages. This was shown by analyzing seventy-five fluvoxamine maleate samples containing known levels of AZD and CEA (between 0.05 and 1.05µgg(-1)) in intermediate reproducibility conditions to get reliable estimations of precision and linearity. From these data, acceptance, rejection and non-conclusive areas of response are defined for both analytes at different confidence and replication levels using normal statistics. The method was satisfactorily applied to real fluvoxamine maleate samples.

Purity profile of the indoloquinone anticancer agent EO-9 and chemical stability of EO-9 freeze dried with 2-hydroxypropyl-beta-cyclodextrin.

Two new bladder instillations of the investigational anticancer agent EO-9 containing 2-hydroxypropyl-beta-cyclodextrin (HP beta CD) and the alkalizers sodium bicarbonate (NaHCO3) and tri(hydroxymethyl)aminomethane (Tris) were developed. During the stability study of these freeze-dried products, formation of new degradation products was seen. We have characterized these products by high performance liquid chromatography in combination with photodiode array detection and mass spectrometry. In total, five new degradation products were identified of which three were detected in both freeze-dried products and two only in the freeze-dried product composed of EO-9/HP beta CD/NaHCO3. Furthermore, the purity profile of two lots of EO-9 drug substance was investigated. Five, probably synthetic intermediates were found. However, the amount of total impurities was very small for both lots of drug substance and below acceptable international limits for pharmaceutical use.

Electron and electrospray ionization mass spectrometry in the characterization of some 1-allyl-3-phenylaziridines.

A series of cis- and trans-isomeric aziridines has been studied under electron impact (EI) and electrospray ionization (ESI) conditions. The fragmentation patterns of the examined compounds have been elucidated by means of sequential product ion fragmentation experiments (MS(n)) performed using an ion trap mass spectrometer. Particular attention has been paid to isomer characterization in these precursors of azetidinones, that in turn are precursors of new beta-lactam antibiotics.

Searches for new interstellar molecules, including a tentative detection of aziridine and a possible detection of propenal.

Rotational spectroscopy at millimeter wavelengths is a powerful means of investigating the chemistry of dense interstellar clouds. These regions can exhibit an interesting complement of gas phase molecules, including relatively complex organics. Here we report the tentative first astronomical detection of aziridine (ethylenimine), the possible detection of propenal (acrolein), and upper limits on the abundances of cyclopropenone, furan, hydroxyethanal (glycolaldehyde), thiohydroxylamine (NH2SH), and ethenol (vinyl alcohol) in various interstellar clouds.

GC-MS analysis of methamphetamine impurities: reactivity of (+)- or (-)-chloroephedrine and cis- or trans-1,2-dimethyl-3-phenylaziridine.

S-(+)-Methamphetamine is frequently found as the only isomer in urine specimens from methamphetamine abuseres. Enantiomerically pure S-(+)-methamphetamine can be synthesized from ephedrine or pseudoephedrine via chloroephedrine intermediates. These intermediates are unstable and capable of cyclizing to form cis- and trans-1,2-dimethyl-3-phenyl aziridine. Studies were done to determine if these intermediates could be detected when using a common gas chromatographic-mass spectrometric analytical method (derivatization with heptafluorobutyric anhydride, HFBA) for toxicological screening of methamphetamine. Analysis of (+)- or (-)-chloroephedrine after extraction into hexane and derivatization with HFBA indicated that both pseudoephedrine and ephedrine were the major compounds detected. Direct derivatization of a hexane solution of cis-1,2-dimethyl-3-phenyl aziridine yielded only the derivatives of ephedrine and pseudoephedrine, indicating that the aziridine intermediate is responsible for the formation of the ephedrine or pseudoephedrine. These studies indicate that the aziridine intermediates would not be detected in methamphetamine samples following HFBA derivatization.

Determination of N,N-dimethylaminoethyl chloride and the dimethylaziridinium ion at sub-ppm levels in diltiazem hydrochloride by LC-MS with electrospray ionisation.

An LC-MS method is described for the determination of the synthetic reagent N,N-dimethylaminoethyl chloride (DMC) in the drug substance diltiazem hydrochloride, for which the permissible limit is not more than 1 ppm (microgram g-1). The N,N-dimethylaziridinium ion (DMA), the reactive intermediate formed by cyclisation of DMC, is also detected. A column switching arrangement is used: diltiazem hydrochloride is trapped on a reversed-phase HPLC column, and the polar analytes are separated by ion exchange chromatography. Ionisation is effected by positive-ion electrospray, and the quadrupole filter mass spectrometer is operated in the selected ion recording mode. The detection limit (peak height-to-baseline noise ratio = 3) for DMC varies from day to day in the range < 0.05 to 0.1 ppm. The response for DMC is linear (r > 0.999) over the concentration range 0.2-10 ppm, and the repeatability is better than 7% (relative standard deviation) at 1.0 ppm. Concentrations of DMC in diltiazem hydrochloride from the manufacturing facility under study ranged from undetectable to about 0.07 ppm. An indirect TLC method has been published for the determination of DMC in mepyramine maleate, but it lacks the necessary sensitivity and specificity. The LC-MS method presented is direct, straightforward and suitable for routine use.

Sensitive isocratic high-performance liquid chromatographic determination of a novel indoloquinone cytotoxic drug (EO9) in human plasma and urine.

A reversed-phase isocratic high-performance liquid chromatographic method is described for the simultaneous determination of EO9, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta- en-alpha-ol (I), and its ring-opened aziridine analogue EO5A (II), employing ultraviolet detection. Solid-phase sample extraction was used without addition of an internal standard. Plots of peak heights and areas of I and II were linear in the range 5-10,000 ng/ml. The lower limit of detection of both I and II in plasma was 2 ng/ml. The between-day variation of I was 13.9% at 5 ng/ml and lower than 6.2% for concentrations > or = 10 ng/ml. The between-day variation of II at 5 ng/ml was 13.8% and lower than 4.5% for concentrations > or = 10 ng/ml. The assay was developed to enable pharmacological guiding of a phase I study of I in solid tumour cancer patients.

Gradient high-performance liquid chromatographic assay for the determination of the novel indoloquinone antitumour agent E09 in biological specimens.

A high-performance liquid chromatographic method for the determination of the novel indoloquinone antitumour agent E09, 3-hydroxymethyl-5-aziridinyl-1-methyl-2-(1H-indole-4,7-dione)prop-beta-e n-alpha - ol, in mouse plasma and urine is described. Following protein precipitation by means of methanol (2 volumes), separation and quantification of parent drug, metabolites and internal standard E012 (5-morpholine substituted analogue) were achieved on a 5-microns Resolve C18 Rad-Pak with a 15-min linear gradient of 10-30% acetonitrile in a 0.02 M pH 7.4 sodium phosphate buffer with UV detection at 280 and 310 nm. The utility of the assay is also demonstrated for the aziridine ring-opened analogue E05A. 3-hydroxymethyl-5-beta-hydroxyethylamino-2-(1H-indole-4,7-dione)pr op-beta-en- alpha-ol. Plots of area ratios of analytes versus internal standard were linear in the range 50-15,000 ng/ml. The detection limit for indoloquinones in plasma was ca. 30 ng/ml. The within-assay and day-to-day variation were consistently lower than 12.5%. The assay was applied in preliminary pharmacokinetic investigations. One minor metabolite of E09 could be identified; further metabolites were characterized by ultraviolet-visible spectra.

Determination of 2-methylaziridine in workplace atmospheres.

A sampling and analytical procedure was developed for the monitoring of airborne 2-methylaziridine (MA). The analyte is collected by drawing air through a solution of 2,4,6-trinitrobenzenesulphonic acid (TNBS). In situ derivatisation of MA with TNBS during sample collection provides stability to the highly reactive analyte and makes it amenable to a sensitive high-performance liquid chromatographic determination with ultraviolet detection. The purified synthetic derivative of MA with TNBS is more suitable as a calibration standard than commercially available MA.

Supercritical fluid chromatography/chemical ionization/mass spectrometry of some anticancer drugs in a thermospray ion source.

A packed-column supercritical-fluid chromatograph was interfaced with a mass spectrometer via a modification of a thermospray probe. This modification allowed a capillary restrictor for the supercritical fluid (CO2) and reagent gas for chemical ionization to be introduced directly into a thermospray source. Chemical ionization conditions were observed when either the filament or discharge electrode was used and the source pressure was above 0.5 torr. The discharge electrode produced more efficient ionization, resulting in approximately a tenfold larger signal than that observed in the filament mode. The usefulness of this instrumentation was demonstrated on several anticancer drugs. Methanol positive ion chemical ionization (PICI) spectra were recorded for cyclophosphamide, diaziquone, mitomycin C and thiotepa. Methane PICI spectra of thiotepa were obtained in the absence of methanol as a mobile-phase modifier. A 50 ng on-column injection of diaziquone produced approximately a 6:1 signal to noise ratio in the scanning mode.

Contrasting cytotoxic mechanisms of similar antitumour diaziridinylbenzoquinones.

The mechanisms of cytotoxicity of the antitumour diaziridinylbenzoquinones, AZQ and BZQ, have been investigated. HPLC analysis has been used to study the products as well as the rate of decomposition of acid-assisted ring-opening in aqueous medium as a function of pH. Microconcentrators with a molecular weight cutoff of 30 kDa were utilised to study the covalent binding of both compounds to calf thymus DNA. Radical production of both compounds in K562 cell incubations was followed using ESR and their uptake into K562 cells was monitored using radiolabelled compounds. The results show that these two diaziridinylbenzoquinones, although very similar in structure, have diverse mechanisms of cytotoxicity. The implications of these findings are discussed in the light of antitumor action.

Dynamic mechanical thermal analysis of dental polymers. II. Bis-phenol A-related resins.

Dynamic mechanical analysis of bis-phenol A-related resins for dental usage provided loss tangent maxima at their transitions and an Arrhenius-type dependence of transition temperature and frequency. A constant maximum in the loss tangent curves, over the frequency range for the unfilled resins below their glass transition temperature, was thought to be caused by their high degree of unsaturation. The calculated activation energies showed the polycarbonate denture base resin required considerably greater energy for segmental motion than the other tested materials. In contrast, the ethylene imine-based temporary crown and bridge material exhibited the lowest activation energy.

Mechanism for the reductive activation of diaziquone.

In order to understand the redox properties of diaziquone (AZQ) and the reductive alkylation role of its one-electron reduced free radical anion AZQ- in biological systems, we investigated the electrochemical and structural properties of AZQ and its reduced species by cyclic voltammetry, controlled potential electrolysis, optical absorbance spectroscopy and 1H NMR. This study was carried out in aqueous media as well as in Me2SO. In aqueous media AZQ can be reduced by 2 electrons to the dianion AZQ2- which is oxidized back to AZQ- and AZQ. In Me2SO, 1 - e- reduction or oxidation steps are possible. This allowed the characterization of AZQ and its 1 or 2 - e- reduced species by UV-visible absorbance spectroscopy. The redox properties of the aziridine rings were observed by cycle voltammetry. Using 1H NMR, it was possible to follow the structural dependence of AZQ on the nature of the medium. Protonation of the aziridine rings at low pH may facilitate the opening of the ring leading to the carbonium ion, the required species for alkylation. Cyclic voltammetry data indicate that reduction of the quinone facilitates the aziridine ring opening.

Method for the quantitation and characterization of the cholinergic neurotoxin, monoethylcholine mustard aziridinium ion (AF64A).

Monoethylcholine aziridinium ion (AF64A), which is generated from the precursor, acetylcholine mustard hydrochloride, exerts selective neurotoxic effects against brain cholinergic neurons when injected intracerebrally. Conditions associated with optimal generation of AF64A were examined. The results indicate that hydrolysis and cyclization of the precursor are optimal at 25 degrees C with the former occurring at pH 9.0 and the latter at pH 7.3. The aziridinium ion is best stabilized at pH 7.3 at 4 degrees C.

Pathways of decomposition of propylbenzilylcholine mustard in neutral and alkaline solution.

High-voltage electrophoresis has been used to follow the decomposition of propylbenzilylcholine mustard (PrBCM) in aqueous solution. Dilute solutions of PrBCM in 10 mM phosphate buffer, pH 7.5, or Krebs-Henseleit solution allowed to stand for 1 h at room temperature (22-24 degrees C) contain mainly the aziridinium ion derivative. At pH 7.5 the concentration of this ion declines slowly, giving rise first to the N-hydroxyethyl derivative and then ultimately, following hydrolysis of the ester bond, to NN-bis(2-hydroxyethyl)propylamine and benzilic acid. In contrast, in 5 mM NaOH the ester bond undergoes rapid hydrolysis, so that the major species present after 15 min at room temperature is the N-hydroxyethylaziridinium ion. This ion then undergoes slow reaction with hydroxy ion to yield the same final decomposition product, NN-bis(2-hydroxyethyl)propylamine, as is observed at pH 7.5.

2,5-Diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone I: Kinetics in aqueous solutions by high-performance liquid chromatography.

The application of a rapid, selective, and sensitive reversed-phase high-performance liquid chromatographic method to the analysis of 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone (I) and its degradation products is described. The method was used to study the kinetics of degradation of I over pharmaceutically useful pH ranges. The overall reaction rate followed pseudo-first-order kinetics. The pH-rate profile demonstrated optimal stability between pH 6.0 and 6.5. The degradation behavior suggests the existence of multiple pathways. The temperature dependence of th disappearance of I also was evaluated from the regression equation derived from the Arrhenius plot.

2,5-Diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone II: Isolation and characterization of degradation products.

A direct high-performance liquid chromatographic (HPLC) method was applied to monitor 2,5-diaziridinyl-3,6-bis(carboethoxyamino)-1,4-benzoquinone (I) and its degradation products in pharmaceutical vehicles at 25 +/- 0.1 degrees. At the optimal pH for stability of I, an increase in buffer concentration [phosphate and tris(hydroxymethyl)-aminomethane] or ionic strength accelerated degradation. The reaction rate in the solutions studied followed pseudo-first-order kinetics. Degradation products were characterized by mass spectrometry after isolation by semipreparative HPLC. Different degradation pathways prevailed in acidic and basic media. The acid-catalyzed reaction resulted in consecutive aziridine ring opening, while the base-catalyzed reaction led to nucleophilic displacement of thie aziridine ring(s).