Antiproliferative Properties of Vinyl Dipeptides: Synthesis and MCF-7 Cell Line Testing

Peptide mimics derived with close structure to peptide have vast utility because they are expected to interfere with biological targets while having superior drug-like properties if compared to peptides. In this work, novel vinyl dipeptides which are different in a double bond between the α-carbon of peptide and C1 of its side chain. Added to that, suitable substituents were selected to harness drug-like properties. The compounds were found to have moderate activities when tested against MCF-7 breast cancer cell line. For instance, the adamantyl analogue 2-(benzoylamino)-3-(2-furyl)-N-(1-adamantyl) propenamide (1c) and the heterocyclic analogue 2-(Benzoylamino)-3-(2-furyl)-N-[2-(5-cyanothia-zol-2-yl)] propenamide (1o) exhibited inhibition potency at 27.4 and 37.8 μM, respectively.

Proline-based Amino Pyridine Dipeptides as Efficient Organocatalysts for Direct Aldol Reaction

A series of proline-based amino pyridine dipeptide organocatalysts was synthesized and applied in direct asymmetric intermolecular aldol reaction. These catalysts showed good solubility in organic solvents, good yields （73%--97%） and enantioselectivitives（74%--94%）. Among them, dipeptide organocatalyst（2） was found to be the most efficient one for the asymmetric aldol reaction between cyclohexanone and 4-nitrobeznaldehyde. After optimizing the catalytic reaction conditions, we found that the catalyst showed high yield（97%）, enantioselectivity（e.e., up to 92%） and anti-diastcreoselectivity（up to 95：5） at mild room temperature without any additives.

Systematic screening and structural characterization of dipeptides using offline 2D LC-LTQ-Orbitrap MS:A case study of Cordyceps sinensis

Cordyceps sinensis(C.sinensis)is a widely used and highly valuable traditional Chinese medicine.Several dipeptides have been detected in C.sinensis,but current scientific knowledge of its chemical makeup remains limited.In this study,an improved approach that integrates offline two-dimensional liquid chromatography(2D LC)separation,precursor ion list,library screening,and diagnostic ion filtering was established to systematically screen and characterize dipeptides in C.sinensis.Offline 2D LC integrating hydrophilic interaction LC and reverse phase separations was established to eliminate interference and identify the target dipeptides.A library containing the potential 400 dipeptides was created,and a precursor ion list with all theoretical precursor ions was adopted to trigger the MS/MS scan with high sensitivity.To identify dipeptides,the type and connection sequence of amino acids were determined according to the product ions.Ile and Leu residues were differentiated for the first time according to the characteristic ion at m/z 69.07.Ultimately,170 dipeptides were identified or tentatively characterized from C.sinensis,and most are reported for the first time in this species herein.In addition,the identified dipeptides were also applied for discrimination among the three Cordyceps species,and 11 markers were identified.The obtained results provide a deeper understanding of the chemical basis of C.sinensis.

A New Metal Tag for Highly Selective and Sensitive Analyses of Amino Acids and Dipeptides by HPLC/ICP-MS

We have developed a novel metal tag, bis(ethylenediamine)-4'-methyl-4-carboxybipyridine-ruthenium N-succinimidyl ester (ECRS) for sensitive analysis of amino acids using high performance liquid chromatography/inductively coupled plasma mass spectrometry (HPLC/ICP-MS). ECRS is a functional reagent, containing an ester group at one end that can be activated to bind to amino group and a chelated ruthenium at the other. The activated ester was reacted briefly with amino groups under weakly alkaline conditions. The ruthenium was detected sensitively by ICP-MS. ECRS was reacted with 17 proteinogenic amino acids in borate buffer. The derivatives were separated by reversed phase HPLC and identified by quadrupole-based ICP-MS. ECRS was suitable for speciation;low molecular weight compounds containing amino groups. We have thus established a quantitative analytical method for amino acids and dipeptides. The detection limits of branched amino acids (signal-to-noise ratio of 3) were 1.5 nmol·L-1 in the standard solution (100 amol per injection).

DETERM INATION OF THE STABILITY CONSTANTS OF COMPLEXES OF HISTIDINE,SOME DIPEPTIDES AND TRIGLYCINE WITH Zn(Ⅱ)AND Cd(Ⅱ)USING SMALL-SCALE PH-METRIC TITRATIONS

A potentiometric study on the complexes of His,Gly-His,ALa-His,Gly-Gly and Gly—Gly—Gly with Zn(Ⅱ)and Cd(Ⅱ)has been reported.Small-scale potentiometric titrations were car- ried out to determine stabil ity constants of complexes at 25℃ with I=0.10 mol dm^(-3)(KNO_3).The com- puter programs SUPERQUAD were applied for data treatment with satisfactory results.

环二肽生物活性的研究进展

环二肽是两个氨基酸通过肽键缩合而成的化合物。环二肽具有独特的结构赋予其两个氢供体与氢受体的特征,它与不同靶标物质结合后能产生诸多效应,从而表现出多种生物活性。环二肽来源广泛,随着现代科技的发展,可以从真菌和细菌中提取分离出环二肽,也可以通过生物或化学方法合成环二肽。除了在抗生素、抗肿瘤药物研发、抗氧化、降血糖等方面具有巨大的潜力外,在调节能量代谢、调控食欲、改善神经系统和降低心脏传导速率等方面也有很广阔的前景。因此,本文对近5年来发现的环二肽进行综述,主要介绍了环二肽的来源、结构与理化性质,并对其生物活性展开重点阐述,并提出了建议和展望。

转染DPP-4 siRNA或(和)加入SP600125的小鼠肺泡巨噬细胞极化及JNK/AP-1信号通路激活情况观察

目的观察转染二肽基肽酶-4(DPP-4)siRNA或(和)加入c-Jun N-末端激酶(JNK)抑制剂(SP600125)的小鼠肺泡巨噬细胞极化情况、JNK/AP-1信号通路激活情况。方法体外培养小鼠肺泡巨噬细胞(MH-S),并随机分组:Control组转染空载siRNA、siDPP-4组转染DPP-4 siRNA、LPS组转染空载siRNA+LPS、siDPP-4+LPS组转染DPP-4 siRNA+LPS、LPS+SP600125组转染空载siRNA+LPS联合SP600125、siDPP-4+LPS+SP600125组转染DPP-4 siRNA+LPS联合SP600125。采用Western boltting法检测巨噬细胞中M1型和M2型极化标志物CD86、CD206蛋白及JNK、激活蛋白-1(AP-1)转录蛋白(c-Jun、c-Fos)磷酸化,RT-qPCR法检测巨噬细胞中M1型标志物(CD86、TNF-α、iNOS、IL-1β)和M2型标志物[CD206、精氨酸激酶-1(ARG-1)、IL-4、IL-10]mRNA,一氧化氮(NO)测定试剂盒、免疫荧光检测巨噬细胞上清液中M1型促炎因子NO和细胞内活性氧(ROS)生成情况。结果与Control组比较,LPS组M1型促炎型因子NO、ROS生成含量及M1型极化标志物(CD86蛋白及CD86、TNF-α、iNOS、IL-1βmRNA)表达升高,M2型极化标志物(CD206蛋白及CD206、ARG-1、IL-4、IL-10 mRNA)表达降低,P均<0.05。与LPS组比较,siDPP-4+LPS组M1型促炎型因子NO、ROS生成含量及M1型极化标志物(CD86蛋白及CD86、TNF-α、iNOS、IL-1βmRNA)表达升高,M2型极化标志物(CD206蛋白及CD206、ARG-1、IL-4、IL-10 mRNA)表达降低,P均<0.05。与LPS组比较,siDPP-4+LPS组p-JNK/JNK、p-c-Jun/c-Jun、p-c-Fos/c-Fos蛋白磷酸化相对表达量升高,P均<0.05。与siDPP-4+LPS组比较,siDPP-4+LPS+SP600125组p-JNK/JNK、p-c-Jun/c-Jun、p-c-Fos/c-Fos蛋白磷酸化相对表达量降低,P均<0.05。结论转染DPP-4 siRNA可促进LPS诱导的小鼠肺泡巨噬细胞M1型极化,抑制肺泡巨噬细胞M2型极化,并增加JNK、c-Jun、c-Fos蛋白磷酸化表达;加入JNK抑制剂后,可降低由转染DPP-4 siRNA引起的JNK、c-Jun、c-Fos蛋白磷酸化表达升高。转染DPP-4 siRNA促进LPS诱导的小鼠肺泡巨噬细胞M1型极化的作用机制可能与激活JNK/AP-1信号通路有关。

两株湛江红树林放线菌次级代谢产物及其细胞毒活性研究

目的研究两株来源于广东湛江红树林自然保护区的红树林放线菌Nesterenkonia sp.N267和Micromonospora echinofuscaT DSM 43913的化学成分。方法综合运用开放硅胶柱层析、薄层色谱(TLC)、高效液相色谱(HPLC)等色谱学方法,对菌株的发酵粗提取物进行分离和纯化,通过核磁共振波谱(NMR)、质谱(MS)等现代波谱法,并结合相关文献数据对获得的单体化合物进行结构鉴定。结果从两株放线菌菌株中分离鉴定出10个化合物,其中化合物1-6为环二肽类:环-(l-脯氨酸-l-亮氨酸)(1)、环-(l-脯氨酸-l-缬氨酸)(2)、环-(l-脯氨酸-l-苯丙氨酸)(3)、环-(d-脯氨酸-d-苯丙氨酸)(4)、环-(甘氨酸-l-苯丙氨酸)(5)、环-(l-脯氨酸-l-酪氨酸)(6)、phenylalanine(7)、pyridoxine(8)、3,3-di(1H-indol-3-yl)propane-1,2-diol(9)、pyrocatechol(10)。对分离得到的化合物1~6和9进行多种肿瘤细胞毒活性评价,结果显示环二肽类化合物(1~6)在50μmol/L浓度下对肿瘤未表现出明显的细胞毒活性;化合物9表现出中等细胞毒活性,其IC50范围为(42.32±3.64)~(48.35±5.28)μmol/L。结论化合物1~5为首次从涅斯捷连科氏菌属中分离得到,化合物4和6~10为首次从小单胞菌属中分离得到,化合物9具有一定的抗肿瘤活性。

Simultaneous determination of doxorubicin and its dipeptide prodrug in mice plasma by HPLC with fluorescence detection

A simple and sensitive high performance liquid chromatography with fluorescence detection （HPLC-FD） has been developed for simultaneous quantification of doxorubicin （DOX） and its dipeptide conjugate prodrug （PDOX） in mice plasma. The chromatographic separation was carried out on an Amethyst C18-H column with gradient mobile phase of 0.1% formic acid and 0.1% formic acid in acetonitrile at a flow rate of 1.0 mL/min. The excitation and emission wavelengths were set at 490 and 550 nm, respectively. The method was comprehensively validated. The limits of detection were low up to 5.0 ng/mL for DOX and 25.0 ng/mL for PDOX. And the limits of quantification were low up to 12.5 ng/mL for DOX and 50 ng/mL for PDOX, which were lower than those for most of the current methods. The calibration curves showed good linearity （R2 〉 0.999） over the concentration ranges. The extraction recoveries ranged from 84.0% to 88.2% for DOX and from 85.4% to 89.2% for PDOX. Satisfactory intra-day and inter-day precisions were achieved with RSDs less than 9.1%. The results show that the developed HPLC-FD method is accurate, reliable and will be helpful for preclinical pharmacokinetic study of DOX and PDOX.

The Effect of Glycyl-Glutamine Dipeptide Concentration on Enzyme Activity, Cell Proliferation and Apoptosis of Jejunal Tissues from Weaned Piglets

An experiment was conducted in a singly factorial design to study the effect of glycyl-glutamine dipeptide on enzyme activity, cell proliferation and apoptosis of jejunal tissues from weaned piglets at different glycyl-glutamine concentration levels of 2, 4, 10, 20, and 30 mmol L-1, respectively. The glutaminase activity, diamine oxidase （DAO） activity, cell peoliferation, apoptosis, and perotein metabolism were measured by the tissue culture method in vitro using jejunal tissues. The immunohistochemical method was used to study the cell proliferation and apoptosis of jejunal tissues. The results showed that compared to the blank control, the percentage and MOD value of BrdU-positicve cells incubated with glycyl-glutamine dipeptide solution were significantly （P0.05） increased. Accordingly, the percentage and MOD value of caspase-3-positive cells from tissue incubated with glycyl-glutamine dipeptide were notably lower （P0.05） than that from the control treatment. The glycyl-glutamine dipeptide increased the glutaminase activity, DAO activity and protein content of jejunal tissues, as the dipeptide concentration was on the rise （P0.05）. These results indicated that glycyl-glutamine dipeptide affected the jejunum development and adaptation of weaned piglets, and the function might be fulfilled by enhancing the glutamine-related enzyme activity, thereby increasing the consumption of glutamine, and then improving the jejunal cell proliferation and suppressing cell apoptosis. The effects of glycyl-glutamine dipeptide relied in a dose-dependent manner, and the maximum effect was achieved at 20-30 mmol L-1 glycyl-glutamine dipeptide.

Glutamine dipeptide for parenteral nutrition in abdominal surgery:A meta-analysis of randomized controlled trials

AIM: To assess the clinical and economical validity of glutamine dipeptide supplemented to parenteral nutrition (PN) in patients undergoing abdominal surgery. METHODS: A meta-analysis of all the relevant randomized controlled trials (RCTs) was performed. The trials compared the standard PN and PN supplemented with glutamine dipeptide in abdominal surgery. RCTs were identified from the following electronic databases: the Cochrane Library, MEDLINE, EMBASE and ISI web of knowledge (SCI). The search was undertaken in April 2006. Literature references were checked by computer or hand at the same time. Clinical trials were extracted and evaluated by two reviewers independently. Statistical analysis was performed by RevMan4.2 software from Cochrane Collaboration. A P value of < 0.05 was considered statistically significant. RESULTS: Nine RCTs involving 373 patients were included. The combined results showed that glutamine dipeptide has a positive effect in improving postoperative cumulative nitrogen balance (weighted mean difference (WMD = 8.35, 95% CI [2.98, 13.71], P = 0.002), decreasing postoperative infectious morbidity (OR = 0.24, 95% CI [0.06, 0.93], P = 0.04), shortening the length of hospital stay (WMD= -3.55, 95% CI [-5.26, -1.84], P < 0.00001). No serious adverse effects were found. CONCLUSION: Postoperative PN supplemented with glutamine dipeptide is effective and safe to decrease the infectious rate, reduce the length of hospital stay and improve nitrogen balance in patients undergoing abdominal surgery. Further high quality trials in children and severe patients are required, and mortality and hospital cost should be considered in future RCTs with sufficient size and rigorous design.

Synthesis and Biological Activities of N-Phosphoryl Branched Peptides

A series of N-phosphoryl branched peptides were synthesized by coupling of various N-phosphoryl amino acids to L-Lysine methyl ester, and their structures were confirmed by 31P NMR, 1H NMR, MS and elemental analysis. The results of cell biological tests indicated that compound 1d and 1e obviously inhibited the growth of both K562 and A2780 cells.

Theoretical Study on the Activity of a-COOH and b-COOH of N-Phosphoryl Aspartic Acids

The bio-mimic reactions of N-phosphoryl amino acids are very important in the study of many biochemical processes. The difference of reactivity between a-COOH and b-COOH in phosphoryl aspartic acid was studied by theoretical study (Hartree-Fock and Density Functional methods) in this paper. The intermediates II containing five-membered ring were more stable than III with six-membered ring. While for intermediates III, the isomers with six-membered ring in apical-equatorial spanning arrangement were more stable than those with di-equatorial spanning arrangement. At B3LYP/6-31G** level, it was shown that transition states IV and V involving a-COOH or b-COOH group had energy barriers of DE = 58.67 kJmol-1 and 103.94 kJmol-1, respectively. These results were in agreement with the experimental data. So the a-COOH group was involved in form of the intramolecular penta-coordinate phosphoric-carboxylic mixed anhydride intermediates, but not b-COOH group.

Synthesis of Muramyl dipeptide Analogs by Incorporation of 3,3,3-Trifluoroalanine

Carbobenzoxy-L-3, 3, 3-trifluoroalanine was synthesized and it was incorporated into MDP for replacement of L-alanine.

Dipeptide transport: an active process with energy-and proton-dependence in the human intestinal cell line,Caco-2

Objective: To determine the active process on dipeptide transport with proton-and energy-dependence in Caco-2 cells.Methods: A human intestinal cell monolayer(Caco-2) was used as the in vitro model of human small intestine and cephalexin as the model substrate for dipeptide transporter(PepT1).Caco-2 cells grown on multiwell dishes(24 wells) and Transwell membrane filters were incubated in the culture medium. The transport and uptake experiments of cephalexin across apical membranes were then conducted with different temperature and different pH values.Uptake of cephalexin in Caco-2 cells gsown on multiple well dishes with addition of energy inhibitors(sodium azide,SA and 2,4-dinitrophenol,DNP) were then measured. Results: The accumulation of cephalexin into Caco-2 monolayers increased with the duration of culture.The uptake from the apical surface was markedly influenced by the pH of the apical medium,and the maximal uptake was achieved at pH 5.5;further acidification of the incubation medium may decrease transport of cephalexin despite an increase inward H+ gradient.Cephalexin uptake was linear over the concentration range when the cells were incubated at 4℃ while the uptake rate was enhanced and tended to be saturated as the cephalexin concentration increased when the cells were incubated at 37℃.The kinetic parameters for the cephalexin transport carrier were determined to be: Vmax of(22.173±1.9) nmol/min per mg protein,Km of(2.069±0.9)mol/L,the Kd was estimated to be(0.07±0.02) nmol/min per mg protein per mmol/L.Uptake of cephalexin was markedly inhibited by sodium azide(SA) and 2,4-dinitrophenol(DNP). Conclusion: Cephalexin was transported actively across Caco-2 cells,and the transport process was proton-and energy-dependent.In addition, Caco-2 cells taked up cephalexin by dipeptide transporters that closely resembled the transporters present in the intestine.Caco-2 cells represented an ideal cellular model for future studies of the dipeptide transporter.

SYNTHESIS OF DIPEPTIDE THROUGH A TWO-LIQUID-PHASE ENZYME MEMBANE REACTOR

The synthesis of dipeptide AcPheLeuNH2 catalyzed by immobilized pancreatic lipase was carried out in a two- liquid-phase hollow-fiber membrane reactor, operated in a batch mode. Kinetic properties of free and immobilized enzyme, partition behavior between aqueous buffer phase and organic solvent phase, and effective diffusion coefficients of substrates and products through the membrane were investigated respectively. Based on the preliminary experimental results, the performance of the enzyme membrane reactor, which is evaluated by the purity and the yield, is discussed.

Design and Solid-Phase Synthesis of Multiple Muramyl Dipeptide (MMD)

As a non-specific modulator of macrophage, multiplied muramyl dipeptide (MMD) is solid-phase synthesized by application of standard Fmoc chemistry strategy. Tarn's multiple antigen system (MAS) is used as our four branched-linker on Lysine.

Hydrolysis of Dipeptide Heptyl Esters with Newlase F

Newlase F is a rude enzyme which contains triacylglycerol lipase and acid protease. Hydrolysis of dipeptide heptyl esters with Newlase F was studied in phosphate buffer-organic solvent by HPLC. When the Newlase F's level reached 5 mg/mL under mild condition (PH 7.0, 30 degreesC), the lipase had the highest activity. The reaction was also affected greatly by organic solvents and their concentrations. It is found that protease in Newlase F does not hydrolyze amide bond under this condition (pH 7.0, r.t.).