Comprehensive analysis of insulin products complex disulfide bonds structure by high resolution mass spectrum / 药学学报

The correct pairing of disulfide bonds maintains the correct folding mode and high-level structure formation of peptides and protein drugs, which is crucial for the quality control of products. In order to ensure that the disulfide bonds are correctly paired, disulfide bond analysis is an essential part of peptides and protein drug characterization. Mass spectrometry can be used to analyze disulfide bonds. However, insulin and its analogues have two pairs of disulfide bonds without restriction enzyme cutting site. Conventional collision-induced dissociation (CID) and high-energy induced cleavage (HCD) cannot accurately locate the complex disulfide bond. In our study, three methods were used to localize the complex disulfide, including enzyme digestion combined with key peptide fragment in source decay (ISD) fragmentation method, enzyme digestion combined with partial reduction alkylation method, intact protein source ISD and electron transfer dissociation (ETD) cleavage method, The applicability of insulin aspart, insulin lispro and insulin glargine were also investigated. This study provides a new way for the quality control of disulfide bonding mode of insulin and its analogues, and also provides a reference for the disulfide bond localization of peptides or proteins containing this complex disulfide bond.

Synthesis and application of the methyl analogues of S-adenosyl-L-methionine / 生物工程学报

Methylation plays a vital role in biological systems. SAM (S-adenosyl-L-methionine), an abundant cofactor in life, acts as a methyl donor in most biological methylation reactions. SAM-dependent methyltransferases (MTase) transfer a methyl group from SAM to substrates, thereby altering their physicochemical properties or biological activities. In recent years, many SAM analogues with alternative methyl substituents have been synthesized and applied to methyltransferases that specifically transfer different groups to the substrates. These include functional groups for labeling experiments and novel alkyl modifications. This review summarizes the recent progress in the synthesis and application of SAM methyl analogues and prospects for future research directions in this field.

Synthesis and antifungal activity of new O-alkylamidoximes

The continuous prospection for molecules that may be useful in the development of new therapeutic agents is a highly relevant issue, mainly because the launch of new drugs on the market does not accompany the emergence of new resistant microorganisms. In this context, this work describes the synthesis of new O-alkylamidoximes and the evaluation of its antifungal activity. The new O-alkylamidoximes were prepared using easy synthetic protocols and tested against three Candida species using the broth microdilution method. The synthesized compounds were obtained in moderate to good yields in high purity and without any observable decomposition. All tested compounds shown moderate antifungal activity against at least one strain of Candida. Despite the moderate activity of the new compounds, this was the first report involving the antifungal activity of O-alkylamidoximes. In view of the low chemotherapy arsenal and the development of fungal strains resistant to traditional antifungal agents, the present study opens new possibilities for the preparation of a new class of more active antifungal agents.

DNA alkylation lesion repair: outcomes and implications in cancer chemotherapy / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Alkylated DNA lesions, induced by both exogenous chemical agents and endogenous metabolites, represent a major form of DNA damage in cells. The repair of alkylation damage is critical in all cells because such damage is cytotoxic and potentially mutagenic. Alkylation chemotherapy is a major therapeutic modality for many tumors, underscoring the importance of the repair pathways in cancer cells. Several different pathways exist for alkylation repair, including base excision and nucleotide excision repair, direct reversal by methyl-guanine methyltransferase (MGMT), and dealkylation by the AlkB homolog (ALKBH) protein family. However, maintaining a proper balance between these pathways is crucial for the favorable response of an organism to alkylating agents. Here, we summarize the progress in the field of DNA alkylation lesion repair and describe the implications for cancer chemotherapy.

Total synthesis of D-glycero-D-mannno-heptose 1β, 7-bisphosphate with 3-O-amyl amine linker and its monophosphate derivative / 中国天然药物

D-Glycero-D-mannno-heptose 1β, 7-bisphosphate (HBPβ) is an important intermediate for constructing the core structure of Gram-negative bacterial lipopolysaccharides and was reported as a pathogen-associated molecular pattern (PAMP) that regulates immune responses. HBPβ with 3-O-amyl amine linker and its monophosphate derivative D-glycero-D-mannno-heptose 7-phosphate (HP) with 1α-amyl amine linker have been synthesized as candidates for immunity study of HBPβ. The O3-amyl amine linker of heptose was installed by dibutyltin oxide-mediated regioselective alkylation under fine-tuned protecting condition. The stereoselective installation of 1β-phosphate ester was achieved by NIS-mediated phosphorylation at low temperature. The strategy for installation of 3-O-amyl amine linker onto HBP derivative can be expanded to the syntheses of other conjugation-ready carbohydrates bearing anomeric phosphoester.

Quantitation of DNA by nuclease P1 digestion and UPLC-MS/MS to assess binding efficiency of pyrrolobenzodiazepine / 药物分析学报

Accurate DNA quantitation is a prerequisite in many biomedical and pharmaceutical studies. Here we established a new DNA quantitation method by nuclease P1 digestion and UPLC-MS/MS analysis. DNA fragments can be efficiently hydrolyzed to single deoxyribonucleotides by nuclease P1 in a short time. The decent stabilities of all the four deoxyribonucleotides were confirmed under different conditions. Deoxyadenosine monophosphate (dAMP) was selected as the surrogate for DNA quantitation because dAMP showed the highest sensitivity among the four deoxyribonucleotides in the UPLC-MS/MS analysis. The linear range in DNA quantitation by this method is 1.2-5000 ng/mL. In the validation, the inter-day and intra-day accuracies were within 90％-110％, and the inter-day and intra-day precision were acceptable (RSD<10％). The validated method was successfully applied to quantitate DNA isolated from tumors and organs of a mouse xenograft model. Compared to the quantitation methods using UV absorbance, the reported method provides an enhanced sensitivity, and it allows for the accurate quantitation of isolated DNA with contamination of RNA and ribonucleotide.

Synthesis and anticonvulsant activity of 6-methyl-2-thioxo-2, 3-dihydropyrimidin-4(1H)-one acetamides

This research aimed at synthesizing new potential anticonvulsants in the series of 2-(4-methyl-6-oxo-1,6-dihydropyrimidin2-yl)thio-acetamides. An initial intermediate 6-methyl-2-thioxo-2,3-dihydro-pyrimidin-4(1Н)-one was obtained by thereaction of thiourea with an acetoacetic ester in the presence of sodium methoxide. The target 2-(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl) thioacetamides were synthesized by alkylation of initial 6-methyl-2-thiopyrimidin-4-one withcorresponding 2-chloroacetamides in Dimethylformamide (DMF) in the presence of potassium carbonate. The structureof compounds was confirmed by 1H Nuclear magnetic resonance (NMR)-spectroscopy, LCMS, and elemental analysis.A screening of anticonvulsant activity of synthesized compounds was carried out using the pentylenetetrazole- andmaximal electroshock-induced seizures models. In these studies, the highest anticonvulsant activity demonstrated acompound 5.5 2-[(4-methyl-6-oxo-1,6-dihydropyrimidin-2-yl)thio]-N-(4-bromophenyl)-acetamide, which decreased thelethality, the number and the severity of seizures, and increased their latent period. For these compound parameters ofЕD50, acute (LD50) and neurotoxicity (TD50), as well as therapeutic (TI) and protective (PI) indexes were determined.Logical structure analysis of anticonvulsant activity screening revealed some patterns of “structure–activity” relationship.

Syntheses of alkyl-de-sanguinarine-N5-methyl derivatives and evaluation of in vitro growth inhibitory activities against cancer cell lines / 药学学报

2,3:7,8-Bis(methylenedioxy)benzo[c]phenanthridine was synthesized in a strategy of converging synthesis with 6-bromo-2,3-dihydroxybenzaldehyde, 5-nitronaphthalene-2,3-diol, and dibromomethane, respectively, as starting materials. The reaction process included dioxy-de-dibromo nucleophilic substitution under alkaline condition, reduction reaction, Schiff base-forming reaction, and an arene radical cyclization step under the presence of Bu3SnH and AIBN as radical initiator, among others. The 2,3:7,8-bis(methylenedioxy)benzo[c] phenanthridine as intermediate was reacted with NaBH4 and different aliphatic acids as alkylation agent to afford 2,3:7,8-bis(methylenedioxy)-5,6-dihydro-N5-alkylbenzo[c]phenanthridines. These dihydro-type products were aromatized using DDQ as oxidant under alkaline condition, and then, salinized using HCl as source of equilibrium anion to yield the series of target alkyl-de-sanguinarine-N5-methyl derivatives. All the synthesized alkyl-de-sanguinarine-N5-methyl derivatives exhibited significantly improved in vitro growth inhibitory activities against cancer cell lines as compared with sanguinarine and the positive control. In pharmacological experiments targeting five cancer cell lines, the target compounds showed activities five-fold active than that of sanguinarine. The findings of this study indicated that the structure modification strategy of substituting n-alkyls for the N5-methyl of natural sanguinarine can be used to improve the growth inhibitory activities against cancer cell lines through increasing liposolubility and steric hindrance to protect the active 5,6-imine structure.

Therapeutic effect of tertiary butyl alcohol eyedrops on sodium selenite-induced cataract in rats / 第二军医大学学报

Objective: To study the therapeutic effect of tertiary butyl alcohol (TBA) eyedrops on selenite-induced rat cataract (simulating senile cataract). Methods: Rat cataract model was induced by sodium selenite. The right eyes of the rats were treated with 25, 50, or 75 mmol/L TBA eyedrops (1 drop/time, 3-5 times/day); the left eyes were taken as control. The eyes were observed everyday and were examined using slit lamp at the fifth and tenth day after treatment for nuclear plaques of the lens. The maximal diameter changes of the cataract plaques were detected using vernier cursor (with the minimum division value being 0.02 mm). Meanwhile, the right eyes of normal SD rats were stimulated with 100 mmol/L TBA eyedrops (1 drop/time, 3-5 times/day) to investigate the toxic effect of the eyedrops and other adverse reactions. Results: The diameter and degree of cataract plaques of the nuclear cataract in the right eyes were less than those in the left eyes, and the opacification degree in the right eyes was slighter than that in the left eyes. The TBA eyedrops had less toxicity and stimulation to the eye. Conclusion: TBA eyedrops have obvious therapeutic effect on selenite cataract.

Reactividad quíiica en la alquilación intramoleular de friedel-crafts de orto--alilanillnas tv-bencilo sustituidas / Chemical reaciviity in the intramolecular fridel-crafts alkylation of tv-bennzyl substituted ortho -allylanilfnes / Reatividade uímiica na alquilação intramoleclar do friedel-crafts de orto-aalilanilines v-bencil substituídas

En este trabajo se reporta el estudio experimental de la alquilación intramolecular de Friedel-Crafts de orto-alilanilinas N-bencilo sustituidas, que explica desde los puntos de vista cinético y termodinâmico la formación de dihidrodibenzo [b,e]azepinas y tetrahidrodibenzo[b, ƒ] azocinas. El seguimiento de los cambios en concentración resultantes del tratamiento en condiciones heterogéneas se llevó a cabo por Cromatografía de Gases-Detector de Ionización en Llama (CG-DILL), mientras que la espectroscopia Ultravioleta-Visible (UV-Vis) y el análisis quimiométrico con el Método Multivariante de Resolución de Curvas-Mínimos Cuadrados Alternados (MMRC-MCA) se usaron para examinar los efectos de las condiciones de reacción en fase homogénea e in situ. Con los resultados obtenidos se puede concluir que la supervisión de parámetros, tales como constantes de velocidad y energías de activación, hizo posible evidenciar los efectos de sustituyen-te, temperatura, velocidad de agitación y concentración, sobre la velocidad y re-gioselectividad de la reacción.

In this work, experimental studies of intramolecular Friedel-Crafts alkylation of N-benzyl sustituted ortho-allylanilines are reported; the results explain the formation of both dihydrodibenz[b,e]azepine and tetrahydrodibenz[b,ƒ]azocine isomers from kinetic and thermodynamic points of view. The concentration changes resulting from treatment under heterogeneous conditions were followed by Gas Chromatography-Flame Ionization Detector (GC-FID), while Ultraviolet-Visible (UV-Vis) spectroscopy with Multivariate Curve Resolution-Alternating Least Squares (MCR-ALS) chemometric analysis were used for analysis of the effects of reaction conditions in homogeneous phase and in-situ. With the obtained results could be conclude that the supervision of parameters such as rate constants and activation energies proved the effects of substituent, temperature, agitation speed and concentration on reaction velocity and regioselectivity.

No trabalho reporta-se o estudo experimental da alquilação intramolecular de Friedel-Crafts de orto-alil-anilines N-bencil substituídas o quais explicam desde a cinética e a termodinâmica, a produção de dihidrodibenzo[b,e]azepinas e tetrahidrodibenzo[b,ƒ]azocinas. O procedimento experimental nas condições erogén foi estabelecida mediante GC-FID e a espectroscopia UV-Vis com análise quimiométrico MCR-ALS foi usada para estabelecer as condições da reação na fase homogênea e in situ. Baseados nos resultados obtidos pode-se concluir como variação dos parâmetros constate de velocidade e energia de ativação fize possível evidenciar os efeitos do substituinte, temperatura, velocidade de agitação e concentração, sobra a velocidade e seletividade da reação.

Design and synthesis of a simplified analogue of salacinol / 中国药科大学学报

To investigate a key strategy of the total synthesis of salacinol. Methods: A simplified analogue of salacinol (1),1-(3-sulfooxypropyl)tetrahydrothiophenium inner salt (2) was designed and synthesized by a coupling reaction between tetrahydrothiophene (THT) and 3-iodopropanol (3-IPA) followed by the esterification of the resulting sulfonium with sulfur trioxide pyridine complex (SO3*Py). Replacement of the alkylating reagent (3-IPA) of THT led to the predominate formation of a undesirable cyclic compound,2,2-dioxo-1,3,2-dioxathiane (7). Resutls:This model experiments indicated that the tandem synthetic process leading to the sulfonium sulfate 2 could be applied for the total synthesis of salacinol (1) as an alternative method.

Preparation and charactarisition of a new type water soluble chitosan derivative / 中国海洋药物

Reaction of chitosan with glucose, galactose and lactose was performed in lactic acid MeOH to give corresponding schiff's base derivatives, the following reduction was carried out in the presence of sodium cyanoborohydride to prepare branched chain water soluble chitosan derivatives. The structure of derivatives were Confirmed by 1 HNMR and 13 CNMR.

Preparation and characterization of the N alkylated,O carboxymethyled chitosan derivative / 中国海洋药物

N alkylated Partially deacetylated chitin(DAC 85)was prepared via reductive N alkylation in aceques MeOH with lauraldehyde.Further,O carboxymethlation of N alkylatedchitosan was performed in 55% NaOH with carboxymethy chloride.A new type amphiphilic chitosan derivative was achieved.Elementary analysis and IR spectrume was performed to study the characterization of the chitosan derivative.

Purification and some properties of human DNA- O6-methylguanine methyltransferase.

DNA-O6-methylguanine methyltransferase was purified from the nuclear fraction of fresh human placenta using ammonium sulphate precipitation, gel filtration, affinity chromatography on DNA-cellulose and hydroxyapatite. The methyltransferase preparation was approximately 1-2% pure based on specific activity, and was free of nucleic acids. The protein reacts stoichiometrically with O6-methylguanine in DNA with apparent second-order kinetics. The human methyltransferase has a pH optimum of about 8·5, similar to that of the corresponding rat and mouse proteins. NaCl inhibits the reaction in a concentration-dependent fashion. The human protein, like the rodent and E. coli methyltransferases, needs no cofactor. While lmM MnCl2, lmM spermidine, 5mM MgCl2 and 10 mM EDTA individually do not significantly inhibit the initial rate of reaction, the protein is nearly completely inactive in 5 mM A1C13 or FeCl2 or 10 mM spermidine. The initial rate of reaction increases as a function of temperature at least up to 42°. The reaction is inhibited by DNA in a concentration-dependent manner, with singlestranded DNA being more inhibitory than duplex DNA.