High Recovery Chromatographic Purification of mRNA at Room Temperature and Neutral pH.

Messenger RNA (mRNA) is becoming an increasingly important therapeutic modality due to its potential for fast development and platform production. New emerging RNA modalities, such as circular RNA, drive the need for the development of non-affinity purification approaches. Recently, the highly efficient chromatographic purification of mRNA was demonstrated with multimodal monolithic chromatography media (CIM® PrimaS), where efficient mRNA elution was achieved with an ascending pH gradient approach at pH 10.5. Here, we report that a newly developed chromatographic material enables the elution of mRNA at neutral pH and room temperature. This material demonstrates weak anion-exchanging properties and an isoelectric point of 5.3. It enables the baseline separation of mRNA (at least up to 10,000 nucleotides (nt) in size) from parental plasmid DNA (regardless of isoform composition) with both a NaCl gradient and ascending pH gradient approach, while mRNA elution is achieved in a pH range of 5-7. In addition, the basic structure of the novel material is a chromatographic monolith, enabling convection-assisted mass transfer of large RNA molecules to and from the active surface. This facilitates the elution of mRNA in 3-7 column volumes with more than 80% elution recovery and uncompromised integrity. This is demonstrated by the purification of a model mRNA (size 995 nt) from an in vitro transcription reaction mixture. The purified mRNA is stable for at least 34 days, stored in purified H2O at room temperature.

Removal of Flotation Collector O-Isopropyl-N-ethylthionocarbamate from Wastewater.

Flotation collector O-isopropyl N-ethylthionocarbamate (IPETC) is widely used for separation of sulfide ores. Its removal from water by several oxidation processes was studied. Photocatalytic oxidation with air in the presence of iron salts, utilizing solar irradiation or artificial UV-A light is very efficient. Oxidation leads through the formation of O-isopropyl N-ethylcarbamate and several other reaction intermediates to total decomposition of organic compound in the final stage in 1 day. Similar results were obtained with a Fenton type oxidation with hydrogen peroxide and iron salts. Treatment with sodium hypochlorite yields mainly O-isopropyl N-ethylcarbamate. The formation of this compound in wastewaters can be of concern, since simple alkyl carbamates are cancer suspect agents.

Transformation of avobenzone in conditions of aquatic chlorination and UV-irradiation.

Emerging contaminants represent a wide group of the most different compounds. They appear in the environment at trace levels due to human activity. Most of these compounds are not yet regulated. Sunscreen UV-filters play an important role among these emerging contaminants. In the present research the reactions of 4-tert-butyl-4'-methoxydibenzoylmethane (avobenzone), the most common UV filter in the formulation of sunscreens, were studied under the combined influence of active chlorine and UV-irradiation. Twenty five compounds were identified by GC/MS as transformation products of avobenzone in reactions of aquatic UV-irradiation and chlorination with sodium hypochlorite. A complete scheme of transformation of avobenzone covering all the semivolatile products is proposed. The identification of the two primary chlorination products (2-chloro-1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)-1,3-propanedione and 2,2-dichloro-1-(4-tert-butylphenyl)-3-(4-methoxyphenyl)-1,3-propanedione) was confirmed by their synthesis and GC/MS and NMR analysis. Although the toxicities of the majority of these products remain unknown substituted chlorinated phenols and acetophenones are known to be rather toxic. Combined action of active chlorine and UV-irradiation results in the formation of some products (chloroanhydrides, chlorophenols) not forming in conditions of separate application of these disinfection methods. Therefore caring for people «well-being¼ it is of great importance to apply the most appropriate disinfection method. Since the primary transformation products partially resist powerful UV-C irradiation they may be treated as stable and persistent pollutants.

Functionalization of epoxy esters with alcohols as stoichiometric reagents.

Glycidyl esters, frequently employed as reactive groups on polymeric supports, were functionalized with alcohols as stoichiometric reagents, yielding ß-alkoxyalcohols. Among the solvents studied, best results were obtained in ethers in the presence of a strong proton acid as a catalyst. Alcohols include simple alkanols, diols, protected polyols, 3-butyn-1-ol 3-hydroxypropanenitrile and cholesterol. This protocol represents a convenient way for introduction of various functionalities onto epoxy-functionalized polymers. Under the reaction conditions, some side reactions take place, mostly due to the reactive ester group and water present in the reaction mixture.

Daphnid life cycle responses to the insecticide chlorantraniliprole and its transformation products.

Chlorantraniliprole (CAP) is a newly developed, widely applied insecticide. In the aquatic environment, several transformation products are formed under natural conditions, one by dehydration and others by photoinduced degradation. Data on aquatic ecotoxicity of CAP can mainly be found in registration and regulatory evaluation reports. Moreover, the toxicity of its transformation products and especially effects upon chronic exposure remain completely unknown. Hence, our aim was to investigate the acute and chronic toxicity of CAP and its transformation products to the daphnid Daphnia magna. The results showed that CAP is extremely toxic to D. magna, with an acute and chronic LC50 of 9.4 and 3.7 µg/L, respectively. No effects on daphnid reproduction were observed, but the impact on daphnid survival also affected population growth rate, with an EC50 of 3.5 µg/L. In contrast, no negative effects of the two main degradation products were observed. The present study demonstrated a high sensitivity of nontarget microcrustaceans to CAP. However, the actual risk of CAP in water diminishes with its spontaneous or light-induced degradation into two transformation products, showing no toxicity to the daphnids in the present study.

Chemical and photochemical degradation of chlorantraniliprole and characterization of its transformation products.

This study aimed at assessing the photodegradation of the insecticide chlorantraniliprole (CAP) in deionized water and in tap water amended with humic acids and nitrate. Photolysis was carried out under simulated solar or UV-A light. CAP (39 µM) photodegradation was slightly faster in tap water than in deionized water with half lives of 4.1 and 5.1 days, respectively. Photodegradation rate of CAP was hardly affected by humic acids (up to 100 mg L(-1)) and nitrate. Photodegradation pattern was different in slightly acidic (pH=6.1) deionized water compared to basic (pH=8.0) tap water. Four main degradation products have been isolated and characterized spectroscopically, and crystal structure was recorded for the first two photodegradation products. CAP also degraded in the dark controls, but only at basic pH (23% loss at pH 8.0 in tap water after 6 days), resulting in the formation of one single degradation product. Our study shows that the degradation of chlorantraniliprole in water is a combination of chemical and photochemical reactions, which are highly dependent on the pH of the solution.

Stability and toxicity of selected chlorinated benzophenone-type UV filters in waters.

In our study, the transformation of two most widely used UV filters, benzophenone-3 (BP3) and benzophenone-4 (BP4), in chlorinated water with disinfection reagents sodium hypochlorite (NaClO) and trichloroisocyanuric acid (TCCA) was studied. Based on the HPLC/MS and UV-Vis analysis the formation of two different chlorinated products (5-chloro-2-hydroxy-4-methoxybenzophenone and 3,5-dichloro-2-hydroxy-4-methoxybenzophenone) was established. Identity of chlorinated products was confirmed by means of comparison of retention times with independently synthesized standards. Photostability study showed that dichloro-derivative in water is less stable then parent compounds, which is not the case for monochloro-derivatives. Toxicity of chlorinated compounds tested by Vibrio fischeri was found to be in the same range as that of the starting compounds. Preliminary testing of real water samples from swimming pools and sea swimming areas confirmed the presence of BP3 and its 3,5-dichloro derivative.

Photolytic and photocatalytic degradation of 6-chloronicotinic acid.

This work describes for the first time the photolytic and photocatalytic degradation of 6-chloronicotinic acid (6CNA) in double deionised water, which is a degradation product of neonicotinoid insecticides imidacloprid and acetamiprid, and it is known to appear in different environmental matrices. Photolytic experiments were performed with three UVA (ultraviolet A) polychromatic fluorescent lamps with broad maximum at 355 nm, while photocatalytic experiments were performed using immobilised titanium dioxide (TiO2) on six glass slides in the spinning basket inside a photocatalytic quartz cell under similar irradiation conditions. Photolytic degradation revealed no change in concentration of 6CNA within 120 min of irradiation, while the photocatalytic degradation within 120 min, obeyed first-order kinetics. The observed disappearance rate constant was k=0.011 ± 0.001 min⁻¹ and t½ was 63.1 ± 5.5 min. Mineralisation rate was estimated through total organic carbon (TOC) and measurements revealed no carbon removal in case of photolysis after 120 min of exposure. However in photocatalytic experiments 46 ± 7% mineralisation was achieved within 120 min of irradiation. Nevertheless, the removal of total nitrogen (TN) was not observed across all experiments. Ion chromatographic analyses indicated transformation of chlorine atoms to chloride and increase of nitrate(V) ions only via photocatalytic experiments. Efficiency of selected advanced oxidation process (AOP) was investigated through toxicity assessment with Vibrio fischeri luminescent bacteria and revealed higher adverse effects of treated samples on bacteria following photocatalytic degradation in spite of the fact that higher mineralisation was achieved. New hydroxylated product generated in photocatalytic experiments with TiO2, was confirmed with liquid chromatography-electro spray ionisation mass spectrometry (LC-ESI-MS/MS) analyses, gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance spectroscopy (¹H NMR).

Complexation of molybdenum(V) with glycolic acid: an unusual orientation of glycolato ligand in {Mo2O4}2+ complexes.

(PyH)5[Mo(V)OCl4(H2O)]3Cl2 and (PyH)n[Mo(V)OBr4]n reacted with glycolic acid (H2glyc) or its half-neutralized ion (Hglyc(-)) to afford a series of novel glycolato complexes based on the {Mo(V)2O4}2+ structural core: (PyH)3[Mo2O4Cl4(Hglyc)]. (1)/ 2CH 3CN (1), (PyH) 3[Mo 2O 4Br 4(Hglyc)].Pr(i)OH(2), (PyH)2[Mo2O4(glyc) 2Py 2] (3), (PyH) 4[Mo 4O 8Cl 4(glyc) 2].2EtOH (4), and [Mo 4O 8(glyc) 2Py 4] (5) (Py = pyridine, C 5H 5N; PyH(+) = pyridinium cation, C 5H 5NH (+) and glyc (2-) = a doubly ionized glycolate, (-)OCH 2COO (-)). The compounds were fully characterized by X-ray crystallography and infrared spectroscopy. The Hglyc (-) ion binds to the {Mo 2O 4} (2+) core through a carboxylate end in a bidentate bridging manner, whereas the glyc (2-) ion adopts a chelating bidentate coordination through a deprotonated hydroxyl group and a monodentate carboxylate. The orientations of glyc (2-) ions in 3- 5 are such that the alkoxyl oxygen atoms occupy the sites opposite the multiply bonded oxides. {(C6H5) 4P}[Mo(VI)O 2(glyc)(Hglyc)] ( 6), an oxidized complex, features a reversed orientation of the glyc(2-) ion. The theoretical DFT calculations on the [Mo(V)2O4(glyc) 2Py 2](2-) and [Mo(VI)O2(glyc)2](2-) ions confirm that binding of glycolate with the alkoxyl oxygen to the site opposite the MoO bond is energetically more favorable in {Mo(V)2O4}(2+) species, whereas a reversed orientation of the ligand is preferred in Mo(VI) complexes. An explanation based on the orbital analysis is put forward.

Autoxidation of hydrazones. Some new insights.

Autoxidation of hydrazones is a generally occurring reaction, leading mostly to the formation of alpha-azohydroperoxides. All structural kinds of hydrazones, having at least one hydrogen atom on nitrogen, are prone to autoxidation; however, there are marked differences in the rate of the reaction. Hydrazones of aliphatic ketones are 1-2 orders of magnitude more reactive than analogous derivatives of aromatic ketones. Even less reactive are the hydrazones of chalcones, which function also as efficient inhibitors of autoxidation of other hydrazones. These differences can be attributed to the reduction of the rate of the addition of oxygen to a hydrazonyl radical, which is a reversible reaction. In the case of conjugated ketones, it becomes endothermic, making this elementary step slow down and the chain termination reactions become important. Substituents influence the stability of hydrazonyl radicals and, consequently, the bond dissociation energies of the N-H bonds. In acetophenone phenylhydrazones, the substituents placed on the ring of hydrazine moiety exhibit a higher effect (Hammett rho = -2.8) than those on the ketone moiety (rho = -0.82), which denotes higher importance of the structure with spin density concentrated on nitrogen in delocalized hydrazonyl radical. Electronic effects of the substituents also affect the transition state for the abstraction of hydrogen atom by electrophilic peroxy radicals; NBO analysis display a negative charge transfer of about 0.4 eu from hydrazone to a peroxy radical in the transition state.

Abstraction of iodine from aromatic iodides by alkyl radicals: steric and electronic effects.

Abstraction of the iodine atom from aryl iodides by alkyl radicals takes place in some cases very efficiently despite the unfavorable difference in bond dissociation energies of C-I bonds in alkyl and aryl iodides. The abstraction is most efficient in iodobenzenes, ortho-substituted with bulky groups. The ease of abstraction can be explained by the release of steric strain during the elimination of the iodine atom. The rate of abstraction correlates fairly well with the strain energy, calculated by density functional theory (DFT) and Hartree-Fock (HF) methods as a difference in the total energy of ortho and para isomers. However, besides the steric bulk, the presence of some other functional groups in an ortho substituent also influences the rate. The stabilization of the transition state, resembling a 9-I-2 iodanyl radical, by electron-withdrawing groups seems to explain a positive sign of the Hammett rho value in the radical abstraction of halogen atoms.

Direct conversion of bromohydrins to ketones by a free radical elimination of hydrogen bromide.

Secondary beta-bromo alcohols can be transformed directly to ketones in very good yields by a free radical process. Tertiary beta-bromo alcohols do not react while the primary ones are transformed to aldehydes in lower yields. The reaction involves an abstraction of a hydrogen atom alpha to an OH group, followed by elimination of the bromine atom and subsequent tautomerization of an enol to a ketone.