An efficient method for conjugation of a lipopolysaccharide from Salmonella enterica sv. Minnesota with probes bearing hydrazine or amino functional groups

A conjugation method for coupling probes bearing hydrazine or primary amino groups to a lipopolysaccharide (LPS) is described. LPS is modified through the hydroxyl groups present in its O-antigen moiety by activation with cyanogen bromide in aqueous acetone using triethylamine to enhance the electrophilicity of CNBr. The method yields conjugates with good labeling ratios, preserving the endotoxic activity of the lipid A moiety, which in blood exerts pleiotropic effects on many tissues and organs, resulting in multiple-organ damage, circulatory collapse, and death. Conjugation of smooth-form LPS from Salmonella enterica sv. Minnesota to dansyl hydrazine yielded a labeling ratio of 330 nmol dansyl/mg LPS, with nearly no loss of the original endotoxic activity. In the case of horseradish peroxidase, in which a spacer was introduced, the ratio was 28 nmol HRP/mg of LPS, preserving 65% of the original endotoxic activity. This work shows that under these conditions of CNBr activation, the labeling process has practically no effect on the endotoxic behavior of LPS. The method can be used effectively for the conjugation of LPS to probes bearing primary amino, hydrazine, or hydrazide functional groups.     

4-vinyl-substituted pyrimidine nucleosides exhibit the efficient and selective formation of interstrand cross-links with RNA and duplex DNA

The formation of interstrand cross-links in nucleic acids can have a strong impact on biological function of nucleic acids; therefore, many cross-linking agents have been developed for biological applications. Despite numerous studies, there remains a need for cross-linking agents that exhibit both efficiency and selectivity. In this study, a 4-vinyl-substituted analog of thymidine (T-vinyl derivative) was designed as a new cross-linking agent, in which the vinyl group is oriented towards the Watson–Crick face to react with the amino group of an adenine base. The interstrand cross-link formed rapidly and selectively with a uridine on the RNA substrate at the site opposite to the T-vinyl derivative. A detailed analysis of cross-link formation while varying the flanking bases of the RNA substrates indicated that interstrand cross-link formation is preferential for the adenine base on the 5′-side of the opposing uridine. In the absence of a 5′-adenine, a uridine at the opposite position underwent cross-linking. The oligodeoxynucleotides probe incorporating the T-vinyl derivative efficiently formed interstrand cross-links in parallel-type triplex DNA with high selectivity for dA in the homopurine strand. The efficiency and selectivity of the T-vinyl derivative illustrate its potential use as a unique tool in biological and materials research.     

Synthesis of 5-(pyridinyl and pyridiniumyl)-2'-deoxyuridine nucleosides: reversible electron traps for DNA

The desire to produce reversible electron traps for direct, room temperature studies of excess electron transport in DNA duplexes and hairpins motivated our efforts first to link pyridines to 2'-deoxyuridine (pyridinyl-dU) and then to convert these new conjugates into pyridiniumyl-dU nucleosides. Base sensitivity studies presented here rule out general use of bipyridinediiumyl compounds, but show that pyridiniumyl compounds are suitable for use under the strand cleavage and base deprotection procedures required for automated solid-phase oligonucleotide synthesis. This paper presents the synthesis of four 5'-O-DMT-protected 5-(N-methylpyridiniumyl)-dU conjugates using either ethynyl or ethylenyl linkers to join the pyridiniumyl and dU subunits.     

Sequence walkers: a graphical method to display how binding proteins interact with DNA or RNA sequences.

A graphical method is presented for displaying how binding proteins and other macromolecules interact with individual bases of nucleotide sequences. Characters representing the sequence are either oriented normally and placed above a line indicating favorable contact, or upside-down and placed below the line indicating unfavorable contact. The positive or negative height of each letter shows the contribution of that base to the average sequence conservation of the binding site, as represented by a sequence logo. These sequence 'walkers' can be stepped along raw sequence data to visually search for binding sites. Many walkers, for the same or different proteins, can be simultaneously placed next to a sequence to create a quantitative map of a complex genetic region. One can alter the sequence to quantitatively engineer binding sites. Database anomalies can be visualized by placing a walker at the recorded positions of a binding molecule and by comparing this to locations found by scanning the nearby sequences. The sequence can also be altered to predict whether a change is a polymorphism or a mutation for the recognizer being modeled.     

Keto-fluorothiopyranosyl nucleosides: a convenient synthesis of 2- and 4-keto-3-fluoro-5-thioxylopyranosyl thymine analogs

A novel series of fluorinated keto-β-d-5-thioxylopyranonucleosides bearing thymine as the heterocyclic base have been designed and synthesized. Deprotection of 3-deoxy-3-fluoro-5-S-acetyl-5-thio-d-xylofuranose (1) and selective acetalation gave the desired isopropylidene 5-thioxylopyranose precursor 3. Acetylation and isopropylidene removal followed by benzoylation led to 3-deoxy-3-fluoro-1,2-di-Ο-benzoyl-4-O-acetyl-5′-thio-d-xylopyranose (6). This was condensed with silylated thymine and selectively deacetylated to afford 1-(2′-Ο-benzoyl-3′-deoxy-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (8). Oxidation of the free hydroxyl group in the 4′-position of the sugar led to the formation of the target 4′-keto compound together with the concomitant displacement of the benzoyl group by an acetyl affording, 1-(2′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-4′-ulose)thymine (9). Benzoylation of 3 and removal of the isopropylidene group followed by acetylation, furnished 3-deoxy-3-fluoro-1,2-di-Ο-acetyl-4-O-benzoyl-5′-thio-d-xylopyranose (12). Condensation of thiosugar 12 with silylated thymine followed by selective deacetylation led to the 1-(4′-Ο-benzoyl-3′-fluoro-5′-thio-β-d-xylopyranosyl)thymine (14). Oxidation of the free hydroxyl group in the 2′-position and concomitant displacement of the benzoyl group by an acetyl gave target 1-(4′-O-acetyl-3′-deoxy-3′-fluoro-β-d-xylopyranosyl-2′-ulose)thymine (15).     

A general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.     

An efficient synthesis of 3-fluoro-5-thio-xylofuranosyl nucleosides of thymine, uracil, and 5-fluorouracil as potential antitumor or/and antiviral agents

1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose by the sequence of mild oxidation, reduction, fluorination, periodate oxidation, borohydride reduction, and sulfonylation gave 3-deoxy-3-fluoro-1,2-O-isopropylidene-5-O-p-toluenesulfonyl-alpha-D-xylofuranose (5). Tosylate 5 was converted to thioacetate derivative 6, which after acetolysis gave 1,2-di-O-acetyl-5-S-acetyl-3-deoxy-3-fluoro-5-thio-D-xylofuranose (7). Condensation of 7 with silylated thymine, uracil, and 5-fluorouracil afforded nucleosides 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) thymine (8), 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) uracil (9), and 1-(5-S-acetyl-3-deoxy-3-fluoro-5-thio-beta-D-xylofuranosyl) 5-fluorouracil (10). Compounds 8, 9, and 10 are biologically active against rotavirus infection and the growth of tumor cells.     

Site-directed conjugation of nonpeptide groups to peptides and proteins via periodate oxidation of a 2-amino alcohol. Application to modification at N-terminal serine.

The 2-amino alcohol structure -CH(NH2)CH(OH)- exists in proteins and peptides in N-terminal Ser or Thr and in hydroxylysine. Its very rapid oxidation by periodate at pH 7 generates an aldehyde in the peptide and is the first step in a method for site-directed labeling with biotin or a fluorescent reporter. The modifying group is a hydrazide, RCONHNH2, which reacts with the new aldehyde to form a hydrazone-peptide conjugate, RCONHN = CH-peptide. Experiments with two synthetic peptides, Ser-Ile-Gly-Ser-Leu-Ala-Lys and Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly, and with recombinant murine interleukin-1 alpha (an 18-kDa cytokine with N-terminal Ser) demonstrated this method of peptide tagging. The use of a low molar ratio of periodate to peptide minimized the potential for side reactions during the oxidation, and the desired oxidation was rapid and highly specific. The hydrazones formed were stable at pH 6-8 for at least 12 h at 22 degrees C, but were labile at more acidic pH values. Potential uses of this method include the attachment of biotin, reporter groups, metal chelating groups, imaging agents, and cytotoxic drugs to peptides.     

Haloacetyl groups as reversible protection of the amino function: cleavage with 2-aminothiophenol.

Haloacetylamino acids and haloacetyl peptides react rapidly with 2-aminothiophenol in weakly alkaline media to yield 2-aminothiophenoxyacetyl derivatives. These intermediates are subject to acidolysis under mild conditions with release of free amino acids or peptides. With this mild method for removal of the haloacetyl group N-haloacetoxysuccinimide derivatives, which rapidly and specifically acylate amino groups of polypeptides in aqueous solutions, become promising reagents for the reversible protection of amino groups. The chloroacetylation of amino groups in lima bean trypsin inhibitor and the quantitative removal of the chloroacetyl groups demonstrate the applicability of the method for polypeptides. The haloacetyl group also serves an analytical function in that treatment of a completely or partially haloacetylated polypeptide with cysteine forms one carboxymethylcysteine residue per haloacetyl group in the polypeptide derivative. Carboxymethylcysteine is readily measured by amino acid analysis of acid hydrolysates. Approaches to further improvement of conditions for removal of haloacetyl groups are discussed and potential applications of the general chemistry of 2-haloacids to modern polypeptide chemistry are outlined.     

Unusual sequences,homologous to 5S RNA,in ribosomal DNA repeats of the nematodeMeloidogyne arenaria

Summary There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematodeMeloidogyne arenaria. This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA. Previously, only prokaryotes and certain lower eukaryotes (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat. This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes.Evidence is presented for rearrangements and deletions withinMeloidogyne rDNA. The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences. The 5S RNA sequences inMeloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.     

Detection of chlorinated DNA and RNA nucleosides by HPLC coupled to tandem mass spectrometry as potential biomarkers of inflammation

Upon inflammation, activated neutrophils secrete myeloperoxidase, an enzyme able to generate hypochlorous acid (HOCl) from hydrogen peroxide and chloride ions. An analytical method, involving HPLC coupled to electrospray tandem mass spectrometry, has been set-up to detect low levels of HOCl-induced nucleic acids lesions, including both ribo and 2'-deoxyribonucleoside derivatives of 8-chloroguanine, 8-chloroadenine and 5-chlorocytosine. Validation of the developed method was achieved using isolated cells treated with HOCl. The method was found to be sensitive enough to allow the measurement of background levels of 5-chloro-2'-deoxycytidine in the DNA of human white blood cells isolated from 7 mL of blood.     

Avian retroviral RNA encapsidation: reexamination of functional 5' RNA sequences and the role of nucleocapsid Cys-His motifs.

RNA packaging signals (psi) from the 5' ends of murine and avian retroviral genomes have previously been shown to direct encapsidation of heterologous mRNA into the retroviral virion. The avian 5' packaging region has now been further characterized, and we have defined a 270-nucleotide sequence, A psi, which is sufficient to direct packaging of heterologous RNA. Identification of the A psi sequence suggests that several retroviral cis-acting sequences contained in psi+ (the primer binding site, the putative dimer linkage sequence, and the splice donor site) are dispensable for specific RNA encapsidation. Subgenomic env mRNA is not efficiently encapsidated into particles, even though the A psi sequence is present in this RNA. In contrast, spliced heterologous psi-containing RNA is packaged into virions as efficiently as unspliced species; thus splicing per se is not responsible for the failure of env mRNA to be encapsidated. We also found that an avian retroviral mutant deleted for both nucleocapsid Cys-His boxes retains the capacity to encapsidate RNA containing psi sequences, although this RNA is unstable and is thus difficult to detect in mature particles. Electron microscopy reveals that virions produced by this mutant lack a condensed core, which may allow the RNA to be accessible to nucleases.     

Detection by RNA blot hybridization of RNA sequences homologous to the BglII-N fragment of herpes simplex virus type 2 DNA

RNA species, extracted at the time of peak synthesis of the alpha, beta, and gamma classes of herpes simplex virus polypeptides from lytically infected Vero cells, were examined for homology to the BglII-N fragment (map units 0.58 to 0.63) of herpes simplex virus type 2 DNA. By using northern blot analysis, two major and several minor polyadenylated RNA species showed homology to the BglII-N fragment at times corresponding to the maximum synthesis of the beta (7 h postinfection) and gamma (12 h postinfection) herpes simplex virus polypeptides. No alpha RNA homologous to the BglII-N fragment was detected.     

Role of modified nucleosides in tRNA: effect of modification of the 2-thiouridine derivative located at the 5''-end of the anticodon of yeast transfer RNA Lys2.

Yeast tRNA Lys2 codes preferentially for AAA and contains a 2-thiouridine derivative (U) at the 5'-position of the anticodon. Removal of the 2-thio group from U by treatment with CNBr did not affect the amino acid accepting activity of the modified tRNA Lys2. CNBr treated tRNA Lys2 was active in protein synthesis but with a much reduced efficiency. Although the modified tRNA Lys2 was recognized by elongation factor (EF) T, the EFT dependent binding to ribosomes to tRNA Lys2 (CNBr) was markedly decreased.     

Chemical modification of pig liver initiation factor eIF-2 with N-ethylmaleimide. Amino acid sequences around the N-ethylmaleimide-reactive sulfhydryl groups and the effect of GDP on the modification

The activity of eukaryotic initiation factor eIF-2 as to the formation of the ternary complex, eIF-2 GTP Met-tRNA(f), is inhibited by N-ethylmaleimide. Our preparation of pig liver eIF-2 contained alpha and gamma subunits and was inhibited by more than 90% by N-ethylmaleimide. Using our eIF-2, we determined the sequences around the N-ethylmaleimide-reactive sulfhydryl groups, studied the effect of GDP on the sulfhydryl modification and that of NEM on the [3H]GDP binding, and examined the protective effect of GTP against the inhibition of ternary complex formation by N-ethylmaleimide. Both subunits of native eIF-2 contained [14C]N-ethylmaleimide-reactive sulfhydryl groups. One N-ethylmaleimide-reactive sulfhydryl group was in the alpha subunit and 4 were in the gamma subunit. The sequence of the peptide of the alpha subunit was determined to be: Ala-Gly-Leu-Asn-Cys-Ser-Thr-Glu-Thr-Met-Pro-Ile. Two of the four [14C]N-ethylmaleimide-reactive sulfhydryl groups in the gamma subunit were highly reactive, their sequences being: Ile-Val-Leu-Thr-Asn-Pro-Val-Cys-Thr-Glu-Val-Gly-Glu-Lys (gamma 1); Ser-Cys-Gly-Ser-Ser-Thr-Pro-Asp-Glu-Phe-Pro-Thr-Asp-Ile-Pro-Gly-Thr-Lys (gamma 3a). Peptide gamma 3a contained the consensus sequence element (AspXaaXaaGly) of GTP-binding proteins. With preincubation of eIF-2 with GDP, the incorporation of [14C]N-ethylmaleimide into the gamma subunit was reduced to 40% of the control level, but the 14C-incorporation into the alpha subunit did not change.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolonging the action of protein and peptide drugs by a novel approach of reversible polyethylene glycol modification

Polyethylene glycol (PEG)-conjugated therapeutic peptides/proteins have been shown to exhibit clinical properties superior to those of their corresponding unmodified parent molecules. However, the desirable pharmacological features gained by protein PEGylation become irrelevant if conjugates are inactivated or cannot reach their target tissues. Here we describe the design and synthesis of MAL-FMS-OSU. This bifunctional agent enables PEG chains to be linked to peptides and proteins through a slowly hydrolysable chemical bond. PEG-FMS-peptide/protein conjugates thus formed undergo spontaneous hydrolysis at a slow rate upon incubation at pH 8.5, 37 degrees C with a t(1/2) value of 8-14 +/- 2 h, generating the unmodified parent molecule. The validity of this approach was studied with exendin-4 and human growth hormone. A single subcutaneous administration of PEG(40,000)-FMS-exendin-4 facilitated a prolonged and stable reduction in glucose levels in mice (t(1/2) = 30 +/- 2 h) and exceeded the effect obtained by the same dose of the native hormone by 7-8 times.