A Novel Versatile Phosphoramidite Building Block for the Synthesis of 5′- and 3′-Hydrazide Modified Oligonucleotides

We introduce a novel versatile phosphoramidite building block for the modification of oligonucleotides (ONs) with acyl hydrazides on the 5′- or 3′-terminus, or both. The reaction of these hydrazide functionalized ONs with 4-methoxyphenylaldehyde is demonstrated for solution derivatization. Hydrazides are considered nowadays as promising reactants, which show enhanced reactivity at neutral and slightly acidic conditions and higher stability of yielding products as compared to the aliphatic amines, which are broadly used for ONs derivatization.

Our method to introduce hydrazides into ONs employs a phosphoramidite modifier designed to split, during ammonia or lithium hydroxide treatment, into two hydrazides via β-elimination of a central bis-2-carbonylethoxysulfone unit. It allows the creation of ONs derivatized with a hydrazide moiety at the 5′-, 3′- and both 5′- and 3′-termini, as well as two different hydrazide containing ONs at the same time, viz. in one sequence on the same solid support. In latter case one can, for example, synthesize two hydrazide containing ONs, where one is 5′-modified and second one is 3′-modified.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Kinetic model studies on the chemical ligation of oligonucleotides via hydrazone formation

We report on the suitability of hydrazone formation for activator-free ligation of oligonucleotides. 5'-Acyl hydrazides were synthesized using a previously described phosphoramidite modifier, whereas 3'-hydrazides resulted from a hydrazinolysis of an ester group serving as a linker to the solid support. Aromatic aldehydes could be directly introduced on the 5'-terminus via the respective phosphoramidates. Aliphatic aldehydes were generated by periodate cleavage of the corresponding 3'- and 5'-modified diol precursors. Ligation of a 3'-hydrazide-modified oligonucleotide with oligonucleotides bearing an aromatic aldehyde in 5'-position showed a fast reaction kinetics (k(1) about 10(-1) M(-1)s(-1)) [corrected] and irreversible hydrazone formation. The ligation of a 5'-hydrazide-modified oligonucleotide and a 3'-ribobisaldehyde appeared to proceed reversibly at the beginning, but became irreversible with increasing reaction time. Hydrazide-modified oligonucleotides were found to be somewhat unstable in aqueous solutions.     

2'-modified nucleosides for site-specific labeling of oligonucleotides.

We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Spectrophotometric determination of hydrazine, hydrazides, and their mixtures with trinitrobenzenesulfonic acid

A method has been developed to measure hydrazine, hydrazides, and their mixtures using a modification of the trinitrobenzenesulfonic acid method [T. Okuyama and K. Satake (1960) J. Biochem. (Tokyo) 47, 654-660]. After incubation of the sample containing hydrazine and hydrazide with trinitrobenzenesulfonate at pH 8.5 at room temperature for 40 min, the reaction mixture was diluted with a Na2CO3-NaHCO3 buffer (0.1 M, pH 10.8) rather than with 0.5 M HCl. Different chromogens were produced from the reaction of hydrazine (lambda max = 570 nm) and hydrazides (lambda max = 385 and 500 nm) with trinitrobenzenesulfonic acid. The method allowed simultaneous determination of hydrazine (5 to 60 nmol) with hydrazide (10 to 120 nmol) in a mixture with a standard deviation of less than 5%. The presence of amino compounds (except for amino sugars) did not interfere with the measurement of hydrazine or hydrazides. Interference by amino sugars in the determination of hydrazine or hydrazides was eliminated by pretreatment of the sample with NaBH4 to reduce the amino sugars to 2-amino-2-deoxy-hexitols.     

Nonradioactive hybridization probes prepared by the reaction of biotin hydrazide with DNA

A novel one-step chemical method has been developed for the introduction of biotin into nucleic acids for non-isotopic hybridization. The method is based on the interaction of biotin hydrazide with unpaired cytosine residues. The interaction is catalyzed by sodium bisulfite with an optimum at a buffered pH of about 4.5. The reaction reached its maximum after 24 h incubation at a biotin hydrazide concentration of 10 mg/ml. Using streptavidin-alkaline phosphatase conjugates, the limits for detecting the biotinylated probe, either adsorbed directly to nitrocellulose or hybridized to filter-bound target DNA, were 0.3 and 0.9 pg, respectively. The salience of the approach described here over previously used biotin derivatives is that it is quick (one-step), simple and does not involve any enzymatic or instrument-mediated step to introduce the reporter moiety. In addition, other low- and high-molecular-weight hydrazides (e.g. fluorescent or enzyme hydrazides) can serve as the reporter group. The same procedure may be employed for the single-step biotinylation of free cytidine.     

Semisynthesis of 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite and its use in the synthesis of fluorescently labeled oligonucleotides

An efficient enzymatic synthesis of 6-chloropurine-2'-deoxyriboside from the reaction of 6-chloropurine with 2'-deoxycytidine catalyzed by nucleoside-2'-deoxyribosyltransferase (E.C. 2.4.2.6) followed by chemical conversion into the 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite derivative is described. The phosphoramidite derivative was incorporated site-specifically into an oligonucleotide and used for the introduction of a tethered tetramethylrhodamine-cadaverine conjugate. The availability of an efficient route to 6-chloropurine-2'-deoxyriboside 5'-dimethoxytrityl 3'-(2-cyanoethyl-N,N-diisopropylamino)phosphoramidite enables the facile synthesis of oligonucleotides containing a range of functional groups tethered to deoxyadenosine residues.     

Fluorescent hydrazides for the high-performance liquid chromatographic determination of biological carbonyls

Methods for the determination of carbonyl compounds of biological origin by high-performance liquid chromatography were improved by the use of new fluorescent derivatizing agents. Eight fluorescent hydrazides were either synthesized or obtained commercially and compared to dansyl hydrazine (1-dimethylaminonaphthalene-5-sulfonylohydrazide). Four of the compounds yielded carbonyl hydrazones with a higher relative fluorescence quantum yield than dansyl hydrazine in acetonitrile:water mixtures. Darpsyl hydrazide [(3-phenylpyrazoline-1-yl)-4-phenylsulfonylohydrazide] and apmayl hydrazide [N-(2-aminophenyl-6-methylbenzthiazole)-acetylohydrazide] both yielded an increase of greater than 20-fold in sensitivity over dansyl hydrazine in determinations of abscisic acid and jasmonic acid from plant tissues. Different hydrazides and derivatizing conditions were found to be optimum for the determination of different carbonyl compounds. Also, a simple method for precolumn purification of the hydrazones of acidic carbonyls was developed to remove contaminants arising during derivatization and from the tissue source.     

Analysis of oxidative stress-induced protein carbonylation using fluorescent hydrazides

Protein carbonyl detection has been commonly used to analyze the degree of damage to proteins under oxidative stress conditions. Most laboratories rely on derivatization of carbonyl groups with dinitrophenylhydrazine followed by Western blot analysis using antibodies against the dinitrophenyl moiety. This paper describes a protein carbonyl detection method based on fluorescent Bodipy, Cy3 and Cy5 hydrazides. Using this approach, Western blot and immunodetection are no longer needed, shortening the procedure and increasing accuracy. Combination of Cy3 and Cy5 hydrazides allows multiplexing analyses in a single two-dimensional gel. Derivatization with Bodipy hydrazide allows easy matching of the spots of interest and those obtained by general fluorescent protein staining methods, which facilitates excising target proteins from the gels and identifying them. This method is effective for detecting protein carbonylation in samples of proteins submitted to metal-catalyzed oxidation "in vitro" and assessing the effect of hydrogen peroxide and chronological aging on protein oxidative damage in yeast cells.     

Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion

We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2′-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2′-methylseleno cytidine phosphoramidite and improved the accessibility of the 2′-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2′-selenium derivatization had no effect on the stability of the duplexes with the 3′-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3′-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2′-Se-C and 2′-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2′-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography.     

Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged internucleotide 5''-phosphorothioate linkage.

A self complementary oligodeoxynucleotide dodecamer containing an achiral bridged 5'-phosphorothioate linkage 3'-O-P-S-5' has been prepared using the solid phase phosphoramidite procedure. For the incorporation of the phosphorothioate link we have used a 5'-(S-trityl)-mercapto-5'-deoxythymidine-3'-phosphoramidite. After coupling this building block the S-trityl group was removed by silver ions. The free thiol moiety was then coupled with a standard phosphoramidite in the presence of tetrazole. After oxidation of the 5'-phosphorothioite with iodine/water the synthesis was continued with standard building blocks up to the desired dodecamer. This backbone modified dodecamer can be cleaved selectively and quantitatively at the P-S bond by silver or mercuric ions under very mild conditions.     

Oligonucleotides containing fluorescent 2'-deoxyisoinosine: solid-phase synthesis and duplex stability.

The fluorescent nucleoside 2'-deoxyisoinosine (2, isoId) has been incorporated into oligonucleotides. For this purpose the phosphonate 3a and the phosphoramidite 3b, as well as the polymer-linked 3d, have been synthesized and oligonucleotides were prepared by P(III) solid-phase chemistry. One or two isoId-residues were introduced into the oligomer d(T12), replacing dT either in the middle or at the 3'- and 5'-ends. The isoId-containing oligomers were hybridized with a modified d(A)12 containing the conventional nucleosides (dA, dT, dG and dC) opposite to isoId. The replacement of one dT by isoId in the centre of the duplex reduced the Tm value by approximately 15 degrees C and a decrease of approximately 25 degrees C was found when two isoId residues were incorporated. Thermodynamic data were determined from the melting curves. The destabilization was almost independent of the four naturally occurring nucleosides located opposite to isoId. The isoId (2) seems to be stacked in the duplex when dT-dA base pairs are the nearest neighbours; an internal loop is formed in the case of oligomers containing two consecutive isold residues.     

Synthesis of (2'S)- and (2'R)-2'-deoxy-2'-[(2-methoxyethoxy)amino] pyrimidine nucleosides and oligonucleotides

Syntheses of specified 2'-modified nucleosides were achieved: a) via oximation of the 5',3'-blocked 2'-oxocytidine, followed by reduction, or b) by intramolecular nucleophilic addition of 3'-(2-methoxyethoxy)carbamate to the 2'-position with opening of O(2),2'-anhydrouridine. For the first time, 3'-phosphoroamidites of these 2'-modified nucleosides were successfully incorporated into oligonucleotides by solid-phase synthesis. Incorporation of 2'-modified nucleotides into oligodeoxyribonucleotides had a negative effect on the duplex T(m) values with the DNA or RNA complements. Nevertheless, modified nucleotides have shown good target recognition; the (S)-isomer binds preferably to RNA and the (R)-isomer to DNA. Both modified nucleosides significantly increased nuclease resistance of the oligodeoxyribonucleotides.     

Synthesis, nucleic acid hybridization properties and molecular modelling studies of conformationally restricted 3'-O,4'-C-methylene-linked alpha-L-ribonucleotides

Nucleotides with conformationally restricted carbohydrate rings such as locked nucleic acid (LNA), alpha-L-LNA or 2',5'-linked 3'-O,4'-C-methyleribonucleotides exhibit significant potential as building blocks for antigene and antisense strategies. 2',5'-Linked alpha-L-ribo configured monomer X (termed alpha-L-ONA) was designed as a potential structural mimic of alpha-L-LNA. The corresponding phosphoramidite building block of monomer X was obtained in five steps (10% overall yield) from the easily obtainable thymine derivative 1. Incorporation of monomer X into oligodeoxyribonucleotides (ONs) results in dramatically decreased thermal stabilities with DNA/RNA complements (DeltaTm/mod=-11.5 to -17.0 degrees C) compared to unmodified reference ONs. Less pronounced decreases (DeltaTm/mod=-4.5 to -8.5 degrees C) are observed when monomer X is incorporated into triplex forming ONs and targeted against double-stranded DNA (parallel orientation, pyrimidine motif). This biophysical data, together with modelling studies, suggest that 2',5'-linked alpha-L-ONA is a poor structural mimic of alpha-L-LNA.     

Hydrazinolysis of heparin and other glycosaminoglycans.

Heparin, carboxy-group-reduced heparin, several sulphated monosaccharides and disaccharides formed from heparin, and a tetrasaccharide prepared from chondroitin sulphate were treated at 100 degrees C with hydrazine containing 1% hydrazine sulphate for periods sufficient to cause complete N-deacetylation of the N-acetylhexosamine residues. Under these hydrazinolysis conditions both the N-sulphate and the O-sulphate substituents on these compounds were completely stable. However, the uronic acid residues were converted into their hydrazide derivatives at rates that depended on the uronic acid structures. Unsubstituted L-iduronic acid residues reacted much more slowly than did unsubstituted D-glucuronic acid or 2-O-sulphated L-iduronic acid residues. The chemical modification of the carboxy groups resulted in a low rate of C-5 epimerization of the uronic acid residues. The hydrazinolysis reaction also caused a partial depolymerization of heparin but not of carboxy-group-reduced heparin. Treatment of the hydrazinolysis products with HNO2 at either pH 4 or pH 1.5 or with HIO3 converted the uronic acid hydrazides back into uronic acid residues. The use of the hydrazinolysis reaction in studies of the structures of uronic acid-containing polymers and the implications of the uronic acid hydrazide formation are discussed.     

Synthesis and evaluation of thymidine-5'-O-monophosphate analogues as inhibitors of Mycobacterium tuberculosis thymidylate kinase

A number of 2'- and 3'-modified thymidine 5'-O-monophosphate analogues were synthesized as potential leads for new anti-mycobacterial drugs. Evaluation of their affinity for Mycobacterium tuberculosis thymidine monophosphate kinase showed that a 2'-halogeno substituent and a 3'-azido function are the most favorable leads for further development of potent inhibitors of this enzyme.     

Synthesis of 2'-modified oligonucleotides containing aldehyde or ethylenediamine groups

Oligonucleotides carrying 2'-aldehyde groups were synthesized and coupled to peptides containing an N-terminal cysteine, aminooxy or hydrazide group to give peptide-oligonucleotide conjugates in good yield. The synthesis of a novel phosphoramidite reagent for the incorporation of 2'-O-(2,3-diaminopropyl)uridine into oligonucleotides was also described. Resultant 2'-diaminooligonucleotides may be useful intermediates in further peptide conjugation studies.     

Human Ape2 protein has a 3'-5' exonuclease activity that acts preferentially on mismatched base pairs

DNA damage, such as abasic sites and DNA strand breaks with 3'-phosphate and 3'-phosphoglycolate termini present cytotoxic and mutagenic threats to the cell. Class II AP endonucleases play a major role in the repair of abasic sites as well as of 3'-modified termini. Human cells contain two class II AP endonucleases, the Ape1 and Ape2 proteins. Ape1 possesses a strong AP-endonuclease activity and weak 3'-phosphodiesterase and 3'-5' exonuclease activities, and it is considered to be the major AP endonuclease in human cells. Much less is known about Ape2, but its importance is emphasized by the growth retardation and dyshematopoiesis accompanied by G2/M arrest phenotype of the APE2-null mice. Here, we describe the biochemical characteristics of human Ape2. We find that Ape2 exhibits strong 3'-5' exonuclease and 3'-phosphodiesterase activities and has only a very weak AP-endonuclease activity. Mutation of the active-site residue Asp 277 to Ala in Ape2 inactivates all these activities. We also demonstrate that Ape2 preferentially acts at mismatched deoxyribonucleotides at the recessed 3'-termini of a partial DNA duplex. Based on these results we suggest a novel role for human Ape2 as a 3'-5' exonuclease.     

Synthesis and antituberculosis activity of the derivatives of glycoside steviolbioside from the plant Stevia rebaudiana and diterpenoid isosteviol containing hydrazone, hydrazide and pyridinoyl moieties

Conjugates of antitubercular drug Isoniazid (hydrazide of isonicotinic acid), nicotinic and alpha-picolinic acid hydrazides and glycoside steviolbioside from the plant Stevia rebaudiana as well as the product of its acid hydrolysis, diterpenoid isosteviol, were synthesized. Besides, isosteviol hydrazide and hydrazone derivatives as well as conjugates containing two isosteviol moieties connected by dihydrazide linker were also obtained. Both initial compounds and their synthetic derivatives inhibit the growth of Mycobacterium tuberculosis (H37Rv in vitro). The minimum concentration at which the growth of M. tuberculosis was inhibited by 100% (MIC) for stevioside and steviolbioside equals 7.5 and 3.8 microg/mL, respectively. MIC values for conjugates of the hydrazides of pyridine carbonic acids and steviolbioside as well as isosteviol are in the ranges 5-10 and 10-20 microg/mL, respectively. Maximum inhibitory effect against M. tuberculosis showed the conjugates of isosteviol and adipic acid dihydrazide (MIC values ranged from 1.7 to 3.1 microg/mL). Antitubercular activity of the compounds studied is higher than the activity of antitubercular drug Pyrizanamide (MIC = 12.5-20 microg/mL) but lower than the activity of antitubercular drug Isoniazid (MIC = 0.02-0.04 microg/mL).