Capillary electrophoretic studies of acid-base properties of sanguinarine and chelerythrine alkaloids

Capillary zone electrophoresis with UV detection was used for determination of dissociation constants of alkaloids sanguinarine and chelerythrine. Despite the limited solubility of the uncharged forms of the alkaloids resulting in insufficient analytical signal at higher pH the reliable dissociation constants were obtained when acidified samples containing low amount of the alkaloid were injected into the capillary. The precipitation of the alkaloid in the capillary induced by injecting sample of low pH into the background electrolyte of higher pH does not affect the mobility of the alkaloid if its concentration injected exceeds the solubility only to a small extent. Dissociation constants (pK(R+)) of sanguinarine and chelerythrine calculated to 8.3 +/- 0.1 and 9.2 +/- 0.1, respectively, are relevant to Good buffers of ionic strength of 30 mM.     

Effects of the limited analyte solubility on its mobility and zone shape: electrophoretic behavior of sanguinarine and chelerythrine around pH 7

Electrophoretic mobilities and shapes of zones of sanguinarine and chelerythrine in aqueous media around pH 7 are affected by limited solubility of their uncharged forms and by the pH-dependent chemical equilibrium between cationic and uncharged forms of these alkaloids. The sanguinarine solubility in sodium MOPS of pH 7.4 was estimated at 50 micromol x L(-1). Sanguinarine zones in this buffer have the shape of tailed peak with concentration-independent mobility if the injected sanguinarine concentration exceeds this solubility limit only slightly. The chelerythrine solubility is higher because of lower dissociation constants of its cations. Precipitation of sanguinarine and chelerythrine with the phosphate anions decelerates their electrophoretic transport in phosphate buffer. Sanguinarine solubility is 5 micromol x L(-1) at the most in 13 mmol x L(-1) sodium phosphate buffer of pH 7.4. Acidifying of the sample up to pH 3 decreases the tailing of the peaks of sanguinarine and chelerythrine and contributes to the rise of sharp maxima of their migrating zones. Any capillary coating deteriorates the peak shape.     

A simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence detection for the analysis of chelerythrine and sanguinarine in Chinese herbal medicines

Laser-induced fluorescence (LIF) is a highly sensitive detection method for capillary electrophoresis (CE). However, it usually requires analyte to be derivatized, unless the wavelength of native fluorescence of analyte matches the laser's. That limits its application in drug analysis. In this work, we introduced a rapid, simple and sensitive method of nonaqueous capillary electrophoresis with laser-induced native fluorescence (NACE-LIF) detection for the analysis of chelerythrine and sanguinarine for the first time. As these two alkaloids have some native fluorescence, they were directly detected using a commercially available Ar⁺ laser without troublesome fluorescent derivatization. The fluorescence was enhanced by nonaqueous media. Compared with previously reported UV detection method, lower limit of detection (LOD) is achieved thanks to the high sensitivity of LIF detection (2.0 ng/mL for chelerythrine and 6.3 ng/mL for sanguinarine). Moreover, with NACE, the baseline separation of these alkaloids is finished within 3.5 min. This method is successfully applied to determine the contents of chelerythrine and sanguinarine in Macleaya cordata (Willd.) R. Br. and Chelidonium majus L.     

A spectral study of the interaction between chelerythrine chloride and cytidine

To study the possible anticancer mechanisms of chelerythrine (CHE), and its interactions with cytidine were investigated by UV-vis spectrophotometric and spectrofluorimetric measurements and by thermodynamic calculations. The binding of CHE to cytidine could be characterized by the hypochromic and bathochromic effects in the absorption bands, and the quenching of fluorescence intensity. The spectral data were fit by linear analysis, yielding a binding constant of 2.49×104 L mol-1 at 25℃of CHE and cytidine, and a van't Hoff enthalpy of -20.02 kJ/mol for the exothermic interaction in the standard state. In addition, with     

Entropy driven binding of the alkaloid chelerythrine to polyadenylic acid leads to spontaneous self-assembled structure formation

The binding thermodynamics and interaction of the putative anticancer alkaloid chelerythrine with polyadenylic acid were investigated by isothermal titration calorimetry, absorption and fluorescence spectroscopy, circular dichroism, differential scanning calorimetry and thermal melting experiments. The equilibrium binding constant was evaluated to be of the order of 10⁷ M⁻¹. Strong positive entropic and favorable enthalpic contributions to the binding were revealed. The binding affinity was enhanced within (10 to 100) mM Na⁺ concentration. Circular dichroism spectra confirmed that the increase in entropy change was caused by a strong conformational change in the RNA polynucleotide. Absorption and circular dichroism melting studies revealed that chelerythrine binding induced self-assembled duplex structure formation in poly(A) molecules resulting in a cooperative melting profile. This was further confirmed from differential scanning calorimetry data. The intercalation binding of the alkaloid involved strong energy transfer from the polynucleotide bases to the bound alkaloid molecules. The remarkably high entropy driven binding of the alkaloid induced spontaneous self-assembled structure formation in poly(A) and the associated binding affinity is the highest so far reported for a small molecule binding to poly(A).     

Capillary zone electrophoretic studies of interactions of some quaternary isoquinoline alkaloids with DNA constituents and DNA

Capillary zone electrophoresis was applied for the investigation of interactions of some quaternary isoquinoline alkaloids, namely sanguinarine, chelerythrine, berberine, and jatrorrhizine, with DNA constituents and with DNA. None of these alkaloids attach covalently to nucleotides or to the whole DNA under physiological conditions. The interaction with DNA constituents is a noncovalent complexation based on weak intermolecular forces. Electrostatic attraction participates in the interaction but other types of intermolecular forces are involved as well. Cations were identified as the most probable interacting forms of the alkaloids. The interaction with compounds derived from purine was always stronger than those derived from pyrimidine. All alkaloids behaved analogously and similarly to ethidium bromide, the classic DNA intercalator. Stability constants K (in l.mol(-1)) for sanguinarine and chelerythrine in phosphate buffer of pH 7.4 (I(S) = 30 mM) ranged from tens to hundreds.     

Fluorescence of sanguinarine: fundamental characteristics and analysis of interconversion between various forms

The quaternary isoquinoline alkaloid, sanguinarine (SG) plays an important role in both traditional and modern medicine, exhibiting a wide range of biological activities. Under physiological conditions, there is an equilibrium between the quaternary cation (SG⁺) and a pseudobase (SGOH) forms of SG. In the gastrointestinal tract, SG is converted to dihydrosanguinarine (DHSG). All forms exhibit bright fluorescence. However, their spectra overlap, which limited the use of powerful techniques based on fluorescence spectroscopy/microscopy. Our experiments using a combination of steady-state and time-resolved techniques enabled the separation of individual components. The results revealed that (a) the equilibrium constant between SG⁺ and SGOH is pK _a = 8.06, while fluorescence of DHSG exhibited no changes in the pH range 5–12, (b) the SGOH has excitation/emission spectra with maxima at 327/418 nm and excited-state lifetime 3.2 ns, the spectra of the SG⁺ have maxima at 475/590 nm and excited-state lifetime 2.4 ns. The DHSG spectra have maxima at 327/446 nm and 2-exponential decay with components 4.2 and 2.0 ns, (c) NADH is able to convert SG to DHSG, while there is no apparent interaction between NADH and DHSG. These techniques are applicable for monitoring the SG to DHSG conversion in hepatocytes.     

High-Performance Liquid Chromatography with Electrospray Mass Spectrometry for Rapid and Sensitive Determination of Sanguinarine and Chelerythrine in Exogenously Contaminated Honey

A simple, rapid, and sensitive high-performance liquid chromatographic (HPLC) method coupled with electrospray mass spectrometry (ESI-MS) has been used to determine sanguinarine and chelerythrine in exogenously contaminated honey. Sample extracts were separated on a C₈ reversed-phase HPLC column with acetonitrile–acetate buffer (40:60) as mobile phase. After ESI the abundance of protonated molecules was recorded by selected-ion recording (SIR) of m/z 332.5, 348.5, and 356.5 for sanguinarine, chelerythrine, and the internal standard, tetrahydropalmatine, respectively. The internal standard technique was used to construct calibration plots for quantitation of sanguinarine and chelerythrine; the linear ranges were 5.25–1050 and 3.75–750 ng mL^–1, respectively, with correlation coefficients of 0.9993 and 0.9989, respectively. The limits of detection for sanguinarine and chelerythrine were 1.60 and 1.11 ng mL^–1, respectively.     

2-Aminopurine hairpin probes for the detection of ultraviolet-induced DNA damage

Nucleic acid exposure to radiation and chemical insults leads to damage and disease. Thus, detection and understanding DNA damage is important for elucidating molecular mechanisms of disease. However, current methods of DNA damage detection are either time-consuming, destroy the sample, or are too specific to be used for generic detection of damage. In this paper, we develop a fluorescence sensor of 2-aminopurine (2AP), a fluorescent analogue of adenine, incorporated in the loop of a hairpin probe for the quantification of ultraviolet (UV) C-induced nucleic acid damage. Our results show that the selectivity of the 2AP hairpin probe to UV-induced nucleic acid damage is comparable to molecular beacon (MB) probes of DNA damage. The calibration curve for the 2AP hairpin probe shows good linearity (R² = 0.98) with a limit of detection of 17.2 nM. This probe is a simple, fast and economic fluorescence sensor for the quantification of UV-induced damage in DNA.     

Binding of hairpin polyamides to DNA studied by fluorescence correlation spectroscopy for DNA nanoarchitectures

We have recently constructed a “DNA strut” consisting of two DNA-binding hairpin polyamides of Dervan-type connected via a long flexible linker and were able to show that this strut can be used to sequence-selectively connect DNA helices. This approach provides a second structural element (besides the Watson–Crick base pairing) for the assembly of higher-order DNA nanoarchitectures from smaller DNA building blocks. Since none of the existing analytical techniques for studying this kind of system were found suitable for detection and quantification of the formation of the resulting complexes, we chose fluorescence correlation spectroscopy (FCS). In the present study we show that FCS allowed us in a versatile and fast way to investigate the binding of Dervan polyamides to DNA. In particular it also shows its power in the quantitative detection of the formation of multimeric complexes and the in investigation of binding under nonphysiological conditions. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Recognition of hairpin DNA from coil DNA by electrospray mass spectrometry with annealing strategy

This research presented an annealing strategy to identify hairpin DNA from coil DNA with the same base composition but different arrangements using electrospray mass spectrometry(ESI-MS).A series of single-stranded DNA were annealed with their complementary sequences,respectively.All the five pairs of hairpin DNA and coil DNA were unambiguously distinguished by ESIMS with annealing strategy.This research offers a potential method to probe the DNA structure by comparing with mass spectral characteristics.     

类黄酮化合物对G-四链体及双链DNA选择性的电喷雾质谱研究

利用电喷雾质谱(ESI-MS)研究了4种常见的类黄酮化合物芦丁、 槲皮素、 葛根素和柚皮苷与2种不同形态结构的G-四链体DNA和3种双链DNA的非共价相互作用, 比较了这些小分子化合物与不同形态结构DNA结合的强弱及形成复合物的化学计量. 结果表明, 芦丁和槲皮素对G-四链体DNA具有一定的选择性, 同时它们对双链DNA的选择性也较高; 而葛根素和柚皮苷对G-四链体DNA仅显示了较低的选择性.     

Formation of Human Telomeric G-quadruplex Structures Induced by the Quaternary Benzophenanthridine Alkaloids:Sanguinarine,Nitidine,and Chelerythrine

The ligands which can facilitate the formation and stabilize G‐quadruplex structures have attracted enormous attention due to their potential ability of inhibiting the telomerase activity and halting tumor cell proliferation. It is noteworthy that the abilities of the quaternary benzophenanthridine alkaloids (QBAs), the very important G‐quadruplex binders, in inducing the formation of human telomeric DNA G‐quadruplex structures, have not been reported. Herein, the interaction between single‐strand human telomeric DNA and three QBAs: Sanguinarine (San), Nitidine (Nit) and Chelerythrine (Che), has been investigated. Although these molecules are very similar in structure, they exhibit significantly different abilities in inducing oligonucleotide d(TTAGGG)₄ (HT4) to specific G‐quadruplex structures. Our experimental results indicated that the best ligand San could convert HT4 into antiparallel G‐quadruplex structure completely, followed by Nit, which could transform to mixed‐type or hybrid G‐quadruplex structure partially, whereas Che could only transform to antiparallel G‐quadruplex structure in small quantities. The relative QBAs' inducing abilities as indicated by the CD data are in the order of San>Nit>Che. Further investigation revealed that the G‐quadruplex structures from HT4 induced by QBAs are of intramolecular motif. And only sequences with certain length could be induced by QBAs because of their positive charges which could not attract short chain DNA molecules to close to each other and form intermolecular G‐quadruplex. In addition, the factors that affect the interaction between HT4 and QBAs were discussed. It is proposed that the thickness of the molecular frame and the steric hindrance are the primary reasons why the subtle differences in QBAs' structure lead to their remarkable differences in inducing the formation of the G‐quadruplex structures.     

Fluorescent detection of single nucleotide polymorphism utilizing a hairpin DNA containing a nucleotide base analog pyrrolo-deoxycytidine as a fluorescent probe

A novel fluorescent method for the detection of single nucleotide polymorphism (SNP) was developed using a hairpin DNA containing nucleotide base analog pyrrolo-deoxycytidine (P-dC) as a fluorescent probe. This fluorescent probe was designed by incorporating a fluorescent P-dC into a stem of the hairpin DNA, whose sequence of the loop moiety complemented the target single strand DNA (ss-DNA). In the absence of the target ss-DNA, the fluorescent probe stays a closed configuration in which the P-dC is located in the double strand stem of the fluorescent probe, such that there is weak fluorescence, attributed to a more efficient stacking and collisional quenching of neighboring bases. In the presence of target ss-DNA, upon hybridizing the ss-DNA to the loop moiety, a stem-loop of the fluorescent probe is opened and the P-dC is located in the ss-DNA, thus resulting in strong fluorescence. The effective discrimination of the SNP, including single base mismatch ss-DNA (A, T, G) and double mismatch DNA (C, C), against perfect complementary ss-DNA was achieved by increased fluorescence intensity, and verified by thermal denaturation and circular dichroism spectroscopy. Relative fluorescence intensity had a linear relationship with the concentration of perfect complementary ss-DNA and ranged from 50 nM to 3.0 μM. The linear regression equation was F/F₀ = 2.73 C (μM) + 1.14 (R = 0.9961) and the detection limit of perfect complementary ss-DNA was 16 nM (S/N = 3). This study demonstrates that a hairpin DNA containing nucleotide base analog P-dC is a promising fluorescent probe for the effective discrimination of SNP and for highly sensitive detection of perfect complementary DNA.     

Synthesis of pyrene—acridine bis-intercalators and effects of binding to DNA

A bis-intercalating compound containing pyrene and 9-aminoacridine chromophores (N-(5-(1-pyrenyl)-pentyl)-6-(9-acridinylamino) hexylamide, I), was prepared and its interaction with double-stranded DNA was investigated. Homologous compounds in which the two chromophores were connected by a linear carbon chain (pentamethylene (II), tetramethylene (III) and methylene (IV)) were also prepared. In acetonitrile solutions of the free ligands, the presence of the proximal pyrene results in reduced acridine fluorescence relative to 9methylaminoacridine (9-MAA), and the degree of quenching increases with decreasing chain length. The quenching process is assigned to exothermic electron transfer from pyrene to the excited 9-aminoacridine (9-AA) chromophore. In the presence of DNA, the relative quenching order is reversed, and I and IV are quenched more strongly than II and III. From linear dichroism experiments, it is concluded that I binds by bis-intercalation of the pyrene and acridine moieties, III and IV undergo intercalation of the acridine chromophore and II binds by partial bis-intercalation at two contiguous sites.     

High performance liquid chromatography and capillary electrophoresis determination of sanguinarine in biological matrices

HPLC and CE methods were employed to determine the quaternary benzo[c]phenanthridine alkaloid sanguinarine in biological matrices (rat hepatocytes, human gingival fibroblasts, feed, porcine faeces, body fluids and tissues). HPLC was carried out on a C₁₈ column using gradient elution and ion pairing techniques with 1‐heptanesulfonic acid as ion pairing agent under acidic conditions. The detection limit for fluorimetric detection at λ_ex = 327 nm and λ_em = 577 nm was 3 nM sanguinarine. CE analyses were performed in 50 mM phosphate‐Na buffer pH 2.5, with 150 mM SDS used for pre‐concentration by the sweeping effect. This experimental configuration allows injecting the total capillary length with sanguinarine sample. The detection limit for UV detection at 285 nm was 12 nM. Both methods are suitable for analysing submicromolar quantities of sanguinarine in biological materials. The HPLC method is more sensitive than CE because it uses fluorescence detection.     

电喷雾质谱法研究防己诺林碱与G-四链体的相互作用

采用电喷雾质谱法研究了防己诺林碱与双链核酸及G-四链体的相互作用. 结果表明, 防己诺林碱可选择性地与G-四链体结合. 利用串联质谱技术对防己诺林碱与核酸的结合模式进行了研究, 结果表明, 防己诺林碱可能通过末端堆积作用与G-四链体结合, 而通过插入作用与双链核酸结合. 结合模式的差异导致防己诺林碱选择性地与G-四链体结合.     

DNA与苯胺红T的相互作用与荧光定量检测

荧光染料作为分子探针用于核酸的定量测定和构象分析一直备受关注 [1] .已有数十种荧光染料被应用[2 ,3 ] ,如溴化乙锭早期常用作 DNA探针[4 ,5] ,因其有很强的致癌性 ,目前已很少采用 ,而那些无毒性或含有可被衍生的活性基团的探针分子越来越受到青睐 [6～ 8] .苯胺红 T(ST)是一种阳离子型荧光染料 ,它既可很容易地与 DNA双螺旋结构发生插入作用 ,又可由带正电的吩嗪环与 DNA上带负电的磷酸基发生强烈的静电吸附作用 ,还可利用其氨基与 DNA或其它生物分子进行交联 .He等 [9] 曾研究了它与小牛胸腺 DNA之间的作用 ,得到的结合位点数…     

Investigation of the interaction between HIV-1 DNA and cyclic peptides by electrospray ionization mass spectrometry

The interaction between HIV-I DNA and five cyclic peptides (CPI-CP5) was investigated using electrospray ionization mass spectrometry (ESI-MS). It revealed that CPI [c(Ala-Tyr-Leu-Ala-Gly)] and CP4 [c(Pro-D-Tyr-Leu-D-Ala-Gly)] have the higher binding affinity with the duplex DNA among the five cyclic peptides.     

毛细管电泳结合电喷雾质谱分析人类癌基因c-myb四链体核酸与活性天然产物的相互作用

药物与靶点间的作用关系直接影响到药理和药效。药物-靶点结合能力、结合计量关系等信息是药物研发过程中必需的表征数据。人类癌基因c-myb在结直肠癌等多种癌症组织中存在过度表达,目前已成为结直肠癌、白血病等癌症疾病潜在的治疗靶点。位于癌基因c-myb启动子区的一段富含鸟嘌呤(G)的DNA序列,通过阳离子的诱导可自发折叠形成分子内G-四链体,而小分子的特异性识别可以稳定该G-四链体,进而调节基因的转录和表达过程。该文采用压力辅助毛细管电泳前沿分析(PACE-FA)结合电喷雾质谱(ESI-MS)研究人类癌基因c-myb启动子G-四链体(G4)与天然产物分子间的相互作用。PACE-FA法在毛细管电泳前沿分析(CE-FA)过程中施加一个与分析物迁移同向的压力,在保证结果准确度的前提下,能够大大加快分析速度。同时结合ESI-MS,可快速解析结合分子与靶点的亲合力和化学计量关系。首先,利用ESI-MS快速筛选出3种有亲合力的天然产物,亲合力大小依次为:土荆皮乙酸>丁溴东莨菪碱>荷叶碱。考虑到溶液相中存在特异性与非特异性结合,接着用PACE-FA法准确分析溶液相中结合的特异性和结合常数。结果...