ABSTRACT
[structure: see text] A novel, selective labeling of oligonucleotides with two different reporter groups is described. The oligonucleotide is synthesized using a stable 2-(4-methoxybenzamido)ethyl protection for a selected internucleosidic thiophosphate (PS) and a labile 2-(N-isopropyl-4-methoxybenzamido)ethyl for the 3'-terminal PS and internucleosidic phosphates. The latter group and the base protection are removed, and the 3'-terminal PS is labeled. The former protection is then cleaved by a prolonged ammonolysis, and the second reporter is introduced at the internucleosidic PS.
Subject(s)
Fluorescent Dyes/chemistry , Oligonucleotides/chemical synthesis , Organophosphorus Compounds/chemistryABSTRACT
The conjugation of oligonucleotide phosphorothioates with antennapedia peptide was studied in detail to allow efficient preparation of the conjugates on up to 15 mumol scale. Under optimized conditions, the use of oligonucleotides and the peptide in an equimolecular ratio gave the desired conjugates in more than 60% isolated yield.
Subject(s)
Oligonucleotides/chemical synthesis , Peptides/chemical synthesis , Oligonucleotides/chemistry , Peptides/chemistry , Thionucleotides/chemistryABSTRACT
The utility of 2-(N-isopropyl-N-anisoylamino)ethyl group for protection of internucleosidic phosphate linkages in oligonucleotide synthesis was studied. The group demonstrated high coupling yields, favorable deprotection kinetics and a high hydrolytic stability of phosphoramidite building blocks. The mechanism of deprotection was established using a model phosphate triester.
Subject(s)
Oligonucleotides/chemical synthesis , Organophosphates/chemistry , Thionucleotides/chemical synthesis , Organophosphorus Compounds/chemical synthesisABSTRACT
Novel oligonucleotide analogs that bear phosphodiester and bioreversible S-pivaloyl 2-mercaptoethyl (SPME) phosphate triester internucleosidic linkages are described. Their synthesis employs a novel methodology of oligonucleotide deprotection under mild, non-aqueous conditions.
Subject(s)
Oligonucleotides, Antisense/chemical synthesis , Organophosphates/chemical synthesis , Organophosphates/chemistry , Organophosphorus Compounds/chemical synthesis , Organophosphorus Compounds/chemistry , Organothiophosphorus Compounds/chemistry , Pentanoic Acids/chemistryABSTRACT
A number of 5'-O-(4,4'-dimethoxytrityl)thymidine N,N-diisopropylamino phosphoramidites protected at P(III) with derivatives of 2-benzamidoethanol were synthesized and incorporated into synthetic oligonucleotides. Depending on substitution patterns at the alkyl chain, amido group, and phenyl ring, the time required for removal of these protecting groups using concentrated ammonium hydroxide varied from 48 h at 55 degrees C to 25 min at 25 degrees C. Of the 11 groups studied, 2-[N-isopropyl-N- (4-methoxybenzoyl)amino]ethyl- (H) and omega-(thionobenzoylamino)alkyl protections (I and K) were most easily removed. Derivatives of the 2-[N-methyl-N-benzoylamino]ethyl group (E-G) demonstrated moderate stability, but those of the 2-(N-benzoylamino)ethyl group (A-C) were the most stable. For the most reactive group, H, a phosphitylating reagent, bisamidite 60, was synthesized and used in the preparation of four deoxynucleoside phosphoramidites 28 and 65-67, plus the 2'-O-(2-methoxyethyl)-5-methyluridine phosphoramidite 68. All of these novel building blocks were successfully tested in the preparation of natural, 20-mer oligonucleotides and their phosphorothioate analogues. With the model phosphotriester 37, the mechanism of deprotection was studied and revealed, in the case of group H, a pH-independent formation of the 2-oxazolinium cation 47. Under aqueous conditions, 47 gave 54, which in turn was converted in the presence of ammonia to a number of identified products. It is important to note that none of the products formed was reactive toward the oligonucleotide backbone or nucleic bases. Thus, a general strategy for protection of internucleosidic phosphodiester groups is described, which may also find application in synthetic organic chemistry of phosphorus(III) and (V).
Subject(s)
Benzamides , Oligonucleotides/chemical synthesis , Phosphates/chemistry , Base Sequence , Magnetic Resonance Spectroscopy , Oligonucleotides/chemistryABSTRACT
The coupling of 2-cyanoethyl thymidine phosphoramidite to solid-support-bound, phosphate-unprotected oligothymidylates and their phosphorothioate analogues was studied. The yield of the coupling reaction depended on the pK(BH)()+ values of protonated nitrogen bases that served as counterions to the phosphodiester functions of oligonucleotides. To maximize the coupling efficiency, the oligonucleotides were detritylated and washed with a mixture of 0.1 M DMAP and 0.1 M 1H-tetrazole, which resulted in a 98+% coupling efficiency. The utility of the results was demonstrated in the preparation of oligonucleotides with a mixed backbone that required the successive use of H-phosphonate and phosphoramidite methods of synthesis. Using this approach, 20-mer antisense oligonucleotides containing 2'-O-(2-methoxyethyl) ribonucleoside residues and phosphorothioate and phosphoramidate internucleosidic linkages were synthesized in high yield.
Subject(s)
Nucleosides/chemistry , Organophosphorus Compounds/chemistry , Thionucleotides/chemistry , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Phosphorus IsotopesABSTRACT
[reaction: see text] H-Phosphonate monomers of 2'-O-(2-methoxyethyl) ribonucleosides have been synthesized. Oxidation of oligonucleotide H-phosphonates has been optimized to allow the synthesis of oligonucleotides containing either 2'-deoxy or 2'-O-(2-methoxyethyl) ribonucleoside residues combined with three different phosphate modifications in the backbone, i.e., phosphodiester (PO), phosphorothioate (PS), and phosphoramidate (PN). Phosphodiester linkages were introduced by oxidation with a cocktail of 0.1 M Et(3)N in CCl(4)/Pyr/H(2)O (5:9:1) without affecting phosphorothioate or phosphoramidate linkages. For the synthesis of phosphoramidate-modified oligonucleotides, N(4)-acetyl deoxycytidine-3'-H-phosphonate monomers were used to avoid transamination during the oxidation step.
Subject(s)
Oligonucleotides, Antisense/chemical synthesis , Oligonucleotides/chemical synthesis , Oligonucleotides/chemistry , Oligonucleotides, Antisense/chemistry , Organothiophosphorus Compounds/chemical synthesis , Organothiophosphorus Compounds/chemistry , Thionucleotides/chemistryABSTRACT
Several new disulfide-tethered solid supports (S1-S5) were synthesized, and their resistance against ammonolysis was tested. Among these supports, only the one bearing an N-[15-[(4, 4'-dimethoxytrityl)oxy]-12,13-dithiapentadecanoyl] linker (S4b) tolerated ammonolysis and exhibited properties compatible with the oligonucleotide synthesis by phosphoramidite strategy. The applicability of this disulfide linker structure in postsynthetic oligonucleotide labeling on the support was demonstrated by introduction of two photoluminescent lanthanide chelates or two dansyl groups to the N4-(6-aminohexyl) amino-modified cytosine residues at the 5' end of the oligonucleotide sequence. Subsequent release of the resulting conjugates as their 3'-phosphates was achieved by reductive cleavage of the disulfide bond and precipitation of the conjugate from the solution with ethanol. The fluorescently tagged oligomer obtained showed hybridization properties similar to those of oligonucleotides labeled in solution.
Subject(s)
Disulfides/chemical synthesis , Fluorescence , Oligonucleotides/chemical synthesis , Chelating Agents/chemical synthesis , Europium/chemistry , Metals, Rare Earth/chemical synthesis , Molecular Structure , Nucleic Acid Hybridization , Polystyrenes/chemistry , Resins, Plant/chemistryABSTRACT
A novel solid-phase synthesis of 5'-radiolabeled oligonucleotides is described. The labeling reaction is carried out by the phosphoramidite method with the aid of [4,6-di-14C]-5'-dimethoxytritylthymidine building block 1. The feasibility of the method is demonstrated by preparation of 3'-phosphorylated dodecathymidylate phosphorothioate containing radiolabeled nucleoside at the 5'-terminus.
Subject(s)
Oligonucleotides/chemical synthesis , Organophosphorus Compounds , Thymidine/analogs & derivatives , Carbon Radioisotopes , Indicators and Reagents , Isotope Labeling/methods , Models, Molecular , Molecular Conformation , Molecular Structure , Oligonucleotides/chemistryABSTRACT
A novel method for the preparation of oligonucleotides conjugated with nucleophilic ligands is described. A new phosphoramidite building block derived from N-chloroacetyl-6-aminohexanol is attached at the 5'-terminus on the last step of oligonucleotide synthesis. Postsynthetic treatment of support-bound modified oligonucleotide with a variety of amines and mercaptans affords conjugates in high yield.
Subject(s)
Oligonucleotides/chemistry , Organophosphorus Compounds/chemistry , Thionucleotides/chemistry , Amines/chemistry , Chromatography, High Pressure Liquid , Indicators and Reagents/chemistry , Magnetic Resonance Spectroscopy , Sulfhydryl Compounds/chemistrySubject(s)
Peptides/chemistry , Ribonucleases/chemical synthesis , Sulfhydryl Compounds/chemistry , Catalysis , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Cysteine/chemistry , Hydrolysis , Norleucine/chemistry , Recombinant Fusion Proteins/chemical synthesis , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Ribonucleases/chemistry , Ribonucleases/metabolism , Substrate SpecificityABSTRACT
Synthesis of new non-nucleosidic phosphoramidites 1 and 2 derived from achiral precursors containing two reporter groups or amino functions is described. Among them, only phosphoramidites 1a-c allow multiple derivatization of synthetic oligonucleotides. The usefulness of building blocks 1a-c is demonstrated by introduction of several amino functions or fluorescent dansyl reporters at the 3'- and 5'-termini of synthetic oligonucleotides and their phosphorothioate analogues. It is demonstrated that multilabeled non-nucleosidic tether has no or only minor influence on the hybridization properties of the oligonucleotides.
Subject(s)
Oligodeoxyribonucleotides/chemical synthesis , Organophosphorus Compounds/chemical synthesis , Chromatography, High Pressure Liquid , Magnetic Resonance Spectroscopy , Molecular Structure , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/chemistry , Organophosphorus Compounds/chemistry , TemperatureABSTRACT
Synthesis of an artificial gene encoding the signal peptide of the Yersinia pestis capsule antigen (Caf1) was accomplished. A set of plasmids coding for hybrid proteins in which a modified sequence of the Caf1 signal peptide is connected to the amino acid sequence of the mature granulocyte-macrophage colony stimulating factor (GM-CSF) were constructed. Topography of the cleavage site of signal proteases was studied. The presence of an arginine residue within the N-terminal part of the mature human GM-CSF was shown to hinder the proper processing and translocation of the precursor through periplasmic membrane. A number of E. coli strains secreting biologically active mutants of human GM-CSF were obtained.
Subject(s)
Granulocyte-Macrophage Colony-Stimulating Factor/metabolism , Protein Sorting Signals/metabolism , Amino Acid Sequence , Base Sequence , Escherichia coli/genetics , Escherichia coli/metabolism , Granulocyte-Macrophage Colony-Stimulating Factor/genetics , Humans , Molecular Sequence Data , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Complexing of looped and circular oligonucleotides, composed of either 2'-deoxyribo- or 2'-O-methylribonucleoside units, with completely matching or partially mismatching complementary DNA sequences was studied. Melting experiments revealed considerable differences among the stabilities of these hybrid complexes. Maximum stability and selectivity was displayed by oligomers 2 and 5. It was concluded that a linear stretch, attached to 1'-O- of 3'-deoxypsicothymidine unit (Z) increases the selectivity of hybridisation and stability of the complex as a whole. This allows one to aim the target DNA very precisely at its polyadenine part as well as at adjacent sequence simultaneously. Experiments on termination of primer extension catalysed by different DNA-polymerases--Sequenase, Klenow fragment and Tth--have demonstrated that looped oligomer 5, composed of 2'-O-methylribonucleosides appears to be a highly selective and potent inhibitor of replication in vitro. Features of looped oligonucleotides, composed of 2'-O-methylribonucleosides seem to be useful for design of highly specific antigene oligonucleotides.
Subject(s)
DNA Replication , DNA, Single-Stranded/metabolism , Nucleic Acid Conformation , Oligonucleotides/metabolism , Base Sequence , DNA, Circular/metabolism , DNA-Directed DNA Polymerase/metabolism , Molecular Sequence Data , Nucleic Acid Denaturation , Nucleic Acid Hybridization , Oligodeoxyribonucleotides/metabolism , Oligoribonucleotides/metabolism , ThermodynamicsABSTRACT
Several new branched (1, 2), circular (9) and looped oligonucleotides (14-17) were synthesized. 3'-Deoxypsicothymidine was employed to create the site of branching when required. The circular and looped structures were obtained by oxidative disulfide bond formation between mercaptoalkyl tether groups. All the oligonucleotides prepared contained two T11 sequences, and the branched and looped oligomers an additional alternating CT sequence. The melting experiments revealed that the branched oligonucleotides form relatively weak hybrid (double/triple helix) complexes with the single-stranded oligodeoxyribonucleotide, showing a considerable destabilizing effect produced by the structure at the point of branching. The data obtained with looped oligonucleotides demonstrated considerable stabilization of the hybrid (double/triple helix) complexes with the complement. The data reported may be useful in attempting to design new antisense or antigene oligonucleotides capable of forming selective and stable bimolecular hybrid complexes with nucleic acids.
Subject(s)
DNA, Single-Stranded/chemistry , Oligodeoxyribonucleotides/chemistry , Base Sequence , Binding Sites , DNA, Single-Stranded/genetics , In Vitro Techniques , Molecular Sequence Data , Molecular Structure , Nucleic Acid Conformation , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/geneticsABSTRACT
Several analogues of the standard M13 sequencing primer that contain up to five 3'-deoxypsicothymidines, or one or two such units labeled with fluorescein at the 1'-position, have been prepared. All these oligonucleotides have been shown to prime the DNA-polymerase-catalyzed synthesis of DNA.