A general method for the purification of synthetic oligodeoxyribonucleotides containing strong secondary structure by reversed-phase high-performance liquid chromatography on PRP-1 resin

Synthetic 5'-dimethoxytritylated oligodeoxyribonucleotides, which contained strong secondary structure, were satisfactorily denatured and purified by reversed-phase HPLC on PRP-1 columns when strongly alkaline conditions (0.05 M NaOH) were employed. This procedure was suitable for the purification of hairpin structures, e.g., d(CG)nT4(CG)n (n = 4, 5, 6), and oligo(dG) sequences, e.g., d(G)24, as well as oligodeoxyribonucleotide probes which contained degenerate base sites. Oligodeoxyribonucleotides as long as 50 bases in length were purified. Recovery of injected oligonucleotides was typically 90% or better. The high capacity of the PRP-1 resin also allowed purification to be performed on a preparative scale (2-8 mg per injection). Enzymatic degradation and HPLC analysis indicated that no modification of the heterocyclic bases occurred under the alkaline conditions described.     

H-phosphonate method in the synthesis of structural gene of human interleukin-4

A modified H-phosphonate method was used to synthesize 32 oligodeoxyribonucleotides ranging in length from 23 to 28, which were enzymatically joined together to give the human interleukin 4 gene. The high degree of the oligonucleotide purity, achieved through the application of anion-exchange and reverse phase HPLC, ensures the high percentage of the desired sequence (about 75%) in the cloned DNA.     

Ion-spray tandem mass spectrometry in peptide synthesis: structural characterization of minor by-products in the synthesis of ACP(65-74).

Ion-spray triple quadrupole mass spectrometry was used to investigate the products from the solid phase synthesis of the decapeptide (H)-Val-Gln-Ala-Ala-Ile-Asp-Tyr-Ile-Asn-Gly-(OH) [acyl carrier protein(65-74)]. The target sequence was assembled in stepwise fashion from the C-terminal using Boc chemistry on a Bly-OCH2-Pam-copoly(styrenedivinylbenzene) resin. The product was deprotected and cleaved from the resin by treatment with HF/p-cresol for 1 h at 0 degrees C. The crude product was analyzed by reverse-phase HPLC and contained a single major peptide component, one significant minor (late-eluting) component and several trace-level peptide by-products. The components were separated by HPLC and the fractions directly analyzed by mass spectrometry and tandem mass spectrometry. The major product was confirmed as the desired ACP(65-74). The significant minor component was apparently from incomplete deprotection of Asp70, an artifact of this particular experiment. The trace by-products were found to arise from succinimide formation at Asp70, succinimide formation at Asn73, acylation of the Tyr71 side chain phenolic hydroxyl leading to a branched heptadecapeptide, and tert-butylation of the decapeptide. The possible origins of these by-products are discussed in light of known peptide chemistry. Also notable was the absence, to very low detection levels, of by-products frequently reported to occur in peptide synthesis, illustrating the high degree of refinement and the accuracy of currently used synthetic methods.     

Production of L-phenylacetylcarbinol (L-PAC) from benzaldehyde using partially purified pyruvate decarboxylase (PDC)

Biotransformation of benzaldehyde to L-phenylacetylcarbinol (L-PAC) as a key intermediate for L-ephedrine synthesis has been evaluated using pyruvate decarboxylase (PDC) partially purified from Candida utilis. PDC activity was enhanced by controlled fermentative metabolism and pulse feeding of glucose prior to the enzyme purification. With partially purified PDC, several enzymatic reactions occurred simultaneously and gave rise to by-products (acetaldehyde and acetoin) as well as L-PAC production. Optimal reaction conditions were determined for temperature, pH, addition of ethanol, PDC activity, benzaldehyde, and pyruvate:benzaldehyde ratio to maximize L-PAC, and minimize by-products. The highest L-PAC concentration of 28.6 g/L (190.6 mM) was achieved at 7 U/mL PDC activity and 200 mM benzaldehyde with 2.0 molar ratio of pyruvate to benzaldehyde in 40 mM potassium phosphate buffer (pH 7.0) containing 2.0 M ethanol at 4 degrees C. (c) 1996 John Wiley & Sons, Inc.     

High-performance liquid chromatographic analysis of oligodeoxyribonucleotide base composition

A significantly improved method for base composition analysis of synthetic oligodeoxyribonucleotides is presented. This highly accurate and sensitive method used enzymatic digestion followed by high-resolution HPLC of the nucleosides to determine the empirical base composition of the parent compound. The enzymatic digestion reaction is quantitative and is not blocked by modified bases, thus allowing the degree of base deprotection and chemical modification to be assessed. Digestion data are presented for oligodeoxyribonucleotides which range from 18 to 150 bases in length with excellent agreement of experimental and theoretical composition. The method is also applicable to high-molecular-weight genomic DNA.     

alpha-DNA. VII. Solid phase synthesis of alpha-anomeric oligodeoxyribonucleotides.

An efficient procedure for the synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides is described. This solid-phase procedure is based on the use of alpha-nucleoside phosphoramidites and alpha-nucleoside derivatized solid supports corresponding to the four natural bases and allow rapid synthesis of oligonucleotides up to 20 alpha-deoxynucleotide units in length. After HPLC purification, a 15-mer: alpha-d(CCTCTCGTTCTTTAC) and a 20-mer: alpha-d(ATACTTGAGGAAGAGGTGTT) were obtained respectively in 27 and 29% overall yields. Their purity, nucleoside composition and primary structure were ascertained by HPLC and Maxam-Gilbert sequence analyses.     

Proteomics of Medicago sativa cell walls

A method for the sequential extraction and profiling by two-dimensional gel electrophoresis (2-DE) of Medicago sativa (alfalfa) stem cell wall proteins is described. Protein extraction included freezing, grinding in a sodium acetate buffer, separation by filtration of cell walls from cytosolic contents, and extensive washing. Cell wall proteins were then extracted sequentially with a solution containing 200 mM CaCl2 and 50 mM sodium acetate, followed by extraction with 3.0 M LiCl and 50 mM sodium acetate. Cell wall proteins from both the CaCl2 and LiCl fractions were profiled by 2-DE. Approximately 150 protein spots were extracted from these two gels, digested with trypsin, and analyzed using nanoscale HPLC coupled to a hybrid quadrupole time-of-flight (Q-tof) tandem mass spectrometer (LC/MS/MS). More than 100 proteins were identified and used in conjunction with the 2-DE profiles to generate proteomic reference maps for cell walls of this important legume. Identified proteins include classical cell wall proteins as well as proteins traditionally considered as non-secreted. Two unique extracellular proteins were also identified.     

Nonradioactive enzyme measurement by high-performance liquid chromatography of partially purified sugar-1-kinase (glucuronokinase) from pollen of Lilium longiflorum

Here we present a highly sensitive and simple high-performance liquid chromatography (HPLC) method that enables specific quantification of glucuronokinase activity in partially purified extracts from pollen of Lilium longiflorum without radioactive labeled substrates. This assay uses a recombinant UDP-sugar pyrophosphorylase with broad substrate specificity from Pisum sativum (PsUSP) or Arabidopsis thaliana (AtUSP) as a coupling enzyme. Glucuronokinase was partially purified on a DEAE-sepharose column. Kinase activity was measured by a nonradioactive coupled enzyme assay in which glucuronic acid-1-phosphate, produced in this reaction, is used by UDP-sugar pyrophosphorylase and further converted to UDP-glucuronic acid. This UDP-sugar, as well as different by-products, is detected by HPLC with either a strong anion exchange column or a reversed phase C18 column at a wavelength of 260 nm. This assay is adaptive to different kinases and sugars because of the broad substrate specificity of USP. The HPLC method is highly sensitive and allows measurement of kinase activity in the range of pmol min^-1. Furthermore, it can be used for determination of pure kinases as well as crude or partially purified enzyme solutions without any interfering background from ATPases or NADH oxidizing enzymes, known to cause trouble in different photometric assays.     

Distribution of the hallucinogens N,N-dimethyltryptamine and 5-methoxy-N,N-dimethyltryptamine in rat brain following intraperitoneal injection: application of a new solid-phase extraction LC–APcI–MS–MS–isotope dilution method

A method for the solid-phase extraction (SPE) and liquid chromatographic–atmospheric pressure chemical ionization–mass spectrometric–mass spectrometric–isotope dilution (LC–APcI–MS–MS–ID) analysis of the indole hallucinogens N,N-dimethyltryptamine (DMT) and 5-methoxy DMT (or O-methyl bufotenin, OMB) from rat brain tissue is reported. Rats were administered DMT or OMB by the intraperitoneal route at a dose of 5 mg/kg and sacrificed 15 min post treatment. Brains were dissected into discrete areas and analyzed by the methods described as a demonstration of the procedure’s applicability. The synthesis and use of two new deuterated internal standards for these purposes are also reported.     

T4 RNA ligase catalyzed synthesis of base analogue-containing oligodeoxyribonucleotides and a characterization of their thermal stabilities.

Self-complementary oligodeoxyribonucleotides containing the base analogues 2-aminopurine, 2,6-diaminopurine, N6-methyladenine, uracil, and 5-bromouracil were synthesized by a general method that allows incorporation of the analogues at specific positions. The method uses chemically synthesized partial sequences but circumvents the need for protected base analogues by incorporating their unprotected 3',5'-bisphosphate derivatives enzymatically. T4 RNA ligase was used to add the analogues to the oligodeoxyribonucleotides with yields from 54 to greater than 95 percent. Oligodeoxyribonucleotides were joined to the oligodeoxyribonucleotides containing the analogues at their 3'-termini in yields from 22 to 81 percent. The high yields obtained in these joinings suggest that RNA ligase should be of general use for the specific incorporation of other deoxyribonucleotide analogues into oligodeoxyribonucleotides. The oligodeoxyribonucleotides containing the base analogues were characterized by their mobilities during HPLC, nucleoside compositions, sequences, and thermal stabilities.     

Human serum biotinidase is a thiol-type enzyme

The effects of a disulfide reducing agent and sulfhydryl blocking agents on the biotinidase activity in human serum and on the purified biotinidase have been extensively studied by using a newly developed HPLC assay method. This HPLC method directly measures the product (p-aminobenzoate, PAB), and is not interfered with by sulfhydryl-reactive agents. Further, because the substrate solution of this HPLC assay method contains only substrate (biotin 4-amidobenzoate) and phosphate buffer, accurate studies on the effects of sulfhydryl blocking reagents on the purified enzyme could be performed. Biotinidase activities in human sera (n = 83) were always enhanced by 2-mercaptoethanol (ME). The optimum concentration was found to be 1 mM. The degree of activation was variable (100 to 400% of the original) depending on the serum sample. Sulfhydryl blocking reagents such as organic mercurials were tested on fresh serum and purified enzyme. Mercuric agents were found to inhibit the activity of fresh serum and purified enzyme at 0.05 and 0.005 mM, respectively. Sulfhydryl alkylating agents, N-ethylmaleimide (NEM) and dithiobis(2-nitro)benzoic acid (DTNB), inhibited 100 and 64% of the activity of the purified enzyme at 0.1 and 1.0 mM, respectively. However, lower concentrations (less than 5 nM) of organic mercurials and mercuric ion exhibited a slight enhancement (20-30%) of the activity of the purified enzyme. These results indicate the presence of an essential sulfhydryl residue at the active center. The enzyme contains 2.5 sulfhydryls per molecule, as determined by using Ellman's assay method. Serine protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF) and diisopropylfluorophosphate (DFP) did not inhibit the enzyme activity at 0.05 mM or higher concentration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation and transfer of novel synthetic 9-substituted sialic acids

In this report several NeuAc analogues differently modified at position C-9 were tested as substrates for CMP sialic acid synthase from bovine brain: the hydroxy group at C-9 was replaced by an amino, acetamido, benzamido, hexanoylamido and azido group. The synthase was partially purified by chromatography on CDP-hexanolamine--Sepharose. CMP-glycosides synthesized were measured by analytical HPLC at 275 nm. Each NeuAc analogue was activated to the respective CMP-glycoside: Km-values varied from 0.8 mM to 4.6 mM, the Km for NeuAc was 1.4 mM. Thus affinity of the enzyme was influenced only moderately by chemical modification at C-9. CMP-glycosides were synthesized on a preparative scale with good yield and characterized by analytical HPLC. In addition, 500-MHz 1H-NMR data of CMP-9-amino-NeuAc and CMP-9-acetamido-NeuAc were obtained. Each CMP-activated NeuAc analogue was a suitable donor substrate for Gal beta 1-4GlcNAc alpha 2,6-sialyltransferase from rat liver. Transfer was determined by the thiobarbituric acid method and by analytical HPLC at 200 nm. The results demonstrate that synthetic, not naturally occurring, non-labelled NeuAc analogues can be incorporated into glycoprotein with high yield.     

液相色谱-串联质谱分析化学合成多肽及其主要副产物(英文)

用与反相高效液相色谱偶联的串联质谱 (RPHPLC MS MS)分析一个固相化学合成的九肽H2 N Tyr Val Asn Val Asn Met Gly Leu Lys CONH2 及其 2个主要的副产物 .根据Fmoc化学 ,在Fmoc PAL PEG·PS树脂上从C端至N端手工操作逐步偶联合成九肽 .偶联完成后 ,用试剂R(90 %TFA ,5 %苯甲硫醚 ,3%二巯基乙烷 ,2 %苯甲醚 )室温 (2 0～ 2 5℃ )处理 2 5h ,进行多肽的去保护和切除树脂 .RP HPLC分析结果表明 ,合成粗品中含有 1个主要成分、2个次要成分及多个微量成分 .用RPHPLC MS MS分别对主要成分和 2个次要成分进行了鉴定 .结果证明 ,主要成分即为目标九肽 ;先于目标肽洗脱的副产物分子量比目标肽的分子量多 16 .MS MS证明 ,该副产物包括 2种九肽衍生物 :1种为其Tyr1氧化生成 ,另 1种为Met6氧化生成 ;后于目标肽洗脱的副产物为九肽的Tyr1残基增加 12所致 ,这种现象尚未见报道 .对副产物形成的可能机制进行了讨论     

Quantitation of free sphingosine in liver by high-performance liquid chromatography

Conditions were established for the extraction of free sphingosine from liver and the separation and quantitation of this and other long-chain (sphingoid) bases (e.g., sphingosine, sphinganine, phytosphingosine, and homologs) by reverse-phase high-performance liquid chromatography (HPLC). The long-chain bases were extracted with chloroform and methanol and then treated with base to remove interfering lipids. After preparation of the o-phthalaldehyde derivatives, the long-chain bases could be separated using C18 columns eluted isocratically with methanol:5 mM potassium phosphate, pH 7.0 (90:10). The HPLC analyses took 15 to 20 min per sample and had lower limits of detection in the picomole range. Quantitation was facilitated by using a 20-carbon long-chain base homolog as an internal standard. The utility of the method was demonstrated with rat liver, providing the first quantitation of free sphingosine in this tissue of approximately 7 nmol/g wet wt.     

Interaction of the recombinant human methylpurine-DNA glycosylase (MPG protein) with oligodeoxyribonucleotides containing either hypoxanthine or abasic sites.

Methylpurine-DNA glycosylases (MPG proteins, 3-methyladenine-DNA glycosylases) excise numerous damaged bases from DNA during the first step of base excision repair. The damaged bases removed by these proteins include those induced by both alkylating agents and/or oxidizing agents. The intrinsic kinetic parameters (k(cat) and K(m)) for the excision of hypoxanthine by the recombinant human MPG protein from a 39 bp oligodeoxyribonucleotide harboring a unique hypoxanthine were determined. Comparison with other reactions catalyzed by the human MPG protein suggests that the differences in specificity are primarily in product release and not binding. Analysis of MPG protein binding to the 39 bp oligodeoxyribonucleotide revealed that the apparent dissociation constant is of the same order of magnitude as the K(m) and that a 1:1 complex is formed. The MPG protein also forms a strong complex with the product of excision, an abasic site, as well as with a reduced abasic site. DNase I footprinting experiments with the MPG protein on an oligodeoxyribonucleotide with a unique hypoxanthine at a defined position indicate that the protein protects 11 bases on the strand with the hypoxanthine and 12 bases on the complementary strand. Competition experiments with different length, double-stranded, hypoxanthine-containing oligodeoxyribonucleotides show that the footprinted region is relatively small. Despite the small footprint, however, oligodeoxyribonucleotides comprising <15 bp with a hypoxanthine have a 10-fold reduced binding capacity compared with hypoxanthine-containing oligodeoxyribonucleotides >20 bp in length. These results provide a basis for other structural studies of the MPG protein with its targets.     

Base modification and cloning efficiency of oligodeoxyribonucleotides synthesized by the phosphoramidite method: methyl versus cyanoethyl phosphorus protection

We have found large oligodeoxyribonucleotides (50-120 bases) synthesized with N,N-dialkylmethylphosphoramidites to have considerably lower cloning efficiencies than biological DNA. As evidenced by HPLC analysis, these large fragments contain as much as 30% thymidine conversion to 3-methylthymidine. If cyanoethyl- instead of methyl-phosphorous protection is employed, as anticipated, no thymidine methylation is noted. More importantly, large fragments produced by N,N-diisopropylcyanoethyl-phosphoramidite coupling show cloning efficiencies indistinguishable from biological DNA.     

Inhibition of DNA chain elongation by a phosphotriester linkage in template oligodeoxyribonucleotides.

We synthesized two oligodeoxyribonucleotides bearing an isopropyl phosphotriester at defined positions using new phosphorobisamidite chemistry. Diastereomers were separated with HPLC and their template properties were analyzed. Priming oligodeoxyribonucleotides labeled with 32P at the 5'-end were annealed to the modified oligodeoxyribonucleotides and elongated with DNA polymerase I large fragment from Escherichia coli. Results show that the phosphotriester inhibits the DNA chain elongation partially and the extents of the inhibition are remarkably different between the two diastereomers.     

A single-step purification of assembled insulin on a resource RPC column

We have designed an efficient single step purification process using Resource RPC column in conjunction with triethyl amine/acetonitrile solvent system at similar pH at which the in vitro assembly of insulin is carried out from its two chains. The conditions have been optimized to separate native insulin from its single chain precursors as well as from other by-products. This method obviates the need of acidification, centrifugation, gel filtration, and concentration steps before HPLC purification of insulin from assembly mixture. The system offers wide pH stability, rapid regenerability, excellent pressure/flow characteristics, and high loading capacity. The authenticity and purity of human insulin purified by this procedure was evaluated by analytical reverse phase HPLC, peptide mapping, and isoelectric focusing.     

Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection.

Native and modified synthetic oligonucleotides were purified by reversed-phase HPLC using volatile ion-pairing mobile phases. Purification of 10-90 nmol of oligonucleotides in a single injection was demonstrated using a 4.6 x 75-mm HPLC column packed with porous 2.5 microm C18 sorbent. Separation of target products from N-1 failure fragments was achieved for oligonucleotides in the 4- to 60-mer size range. We employed a combination of absorbance and mass spectrometry detection to identify by-products of oligonucleotide synthesis. This method was also employed for analysis and purification of fluorescently labeled oligonucleotides.     

Interaction of endonuclease ecoRI with short specific and nonspecific oligonucleotides

The interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-step simplification in their complexity. Orthophosphate (KI = 31 mM), 2-deoxyribose 5-phosphate (KI = 4.6 mM) and different dNMPs (KI = 2.1-2.5 mM) were shown to be the minimal ligands of the enzyme. The lengthening of a nonspecific d(pN)n (n = 1-6) by one nucleotide unit resulted in the increase of their affinity by a factor of approximately 2.0. Weak nonspecific electrostatic contacts of EcoRI with internucleotide phosphate groups of ODNs can account for about 5 orders of magnitude in the ligand affinity, whereas the contribution of specific interactions between EcoRI and d(pN)n is no more than 2 orders of magnitude of a total ODN's affinity.