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1.
Mol Pharm ; 16(4): 1507-1515, 2019 04 01.
Article in English | MEDLINE | ID: mdl-30865461

ABSTRACT

MicroRNAs (miRNAs) are endogenous, small, noncoding ribonucleic acids (RNAs) that bind to the 3' untranslated regions of messenger RNAs (mRNAs) and induce translational repression or mRNA degradation. Although numerous studies have reported that miRNAs are of potential use for disease diagnostics and gene therapy, little is known about their fates in vivo. This study elucidated the whole-body distributions and kinetics of intravenously administered miRNA-targeting molecules in vivo by positron emission tomography (PET) imaging. A 22-mer sequence targeting miR-15b was conjugated with three different chelators and labeled with gallium-68 (68Ga). These tracers were compared with a scrambled 22-mer sequence; 22-mer with two single base substitutions; anti-miR-34 22-mer; hexathymidylate (T6), a 6-mer sequence; and an unconjugated chelator. miR-15b was chosen as a target because it is important for bone remodeling. All three 68Ga-labeled anti-miR-15b molecules had similar biodistributions and kinetics, and they all accumulated in the bones, kidneys, and liver. The bone accumulation of these tracers was the highest in the epiphyses of long tubular bones, maxilla, and mandible. By contrast, the scrambled 22-mer sequence, the 6-mer, and the unconjugated chelator did not accumulate in bones. PET imaging successfully elucidated the distributions and kinetics of 68Ga-labeled chelated miRNA-targeting molecules in vivo. This approach is potentially useful to evaluate new miRNA-based drugs.


Subject(s)
Bone and Bones/diagnostic imaging , Kidney/diagnostic imaging , Liver/diagnostic imaging , MicroRNAs/pharmacokinetics , Positron-Emission Tomography/methods , RNA, Messenger/metabolism , Animals , Chelating Agents/chemistry , Female , Gallium Radioisotopes/chemistry , Kinetics , Male , RNA, Messenger/genetics , Rats , Rats, Sprague-Dawley , Tissue Distribution
2.
Bioorg Med Chem ; 22(24): 6806-13, 2014 Dec 15.
Article in English | MEDLINE | ID: mdl-25464879

ABSTRACT

(68)Ga labelled 2'-O-methyl oligoribonucleotides (anti-miR-15b) bearing one, three or seven d-galactopyranoside residues have been prepared and their distribution in healthy rats has been studied by positron emission tomography (PET). To obtain the heptavalent conjugate, an appropriately protected 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) precursor bearing a 4-[4-(4,4'-dimethoxytrityloxy)butoxy]phenyl side arm was first immobilized via a base labile linker to the support and the oligonucleotide was assembled on the detritylated hydroxyl function of this handle. A phosphoramidite building block bearing two phthaloyl protected aminooxy groups and one protected hydroxyl function was introduced into the 5'-terminus. One acetylated galactopyranoside was coupled as a phosphoramidite to the hydroxyl function, the phthaloyl protections were removed on-support and two trivalent galactopyranoside clusters were attached as aldehydes by on-support oximation. A two-step cleavage with aqueous alkali and ammonia released the conjugate in a fully deprotected form, allowing radiolabelling with (68)Ga in solution. The mono- and tri-galactose conjugates were obtained in a closely related manner. In vivo imaging in rats with PET showed remarkable galactose-dependent liver targeting of the conjugates.


Subject(s)
Oligoribonucleotides/chemistry , Radiopharmaceuticals/chemical synthesis , Animals , Female , Galactose/chemistry , Gallium Radioisotopes/chemistry , Heterocyclic Compounds/chemistry , Kidney/metabolism , Liver Diseases/diagnosis , Liver Diseases/metabolism , Male , Oligoribonucleotides/urine , Positron-Emission Tomography , Radiopharmaceuticals/urine , Rats , Rats, Sprague-Dawley
3.
J Org Chem ; 78(11): 5153-9, 2013 Jun 07.
Article in English | MEDLINE | ID: mdl-23638811

ABSTRACT

Peptide nucleic acid (PNA) building blocks, bearing a fluorine sensor at C-5 of the uracil base [viz. trifluoromethyl and 3,3-bis(trifluoromethyl)-4,4,4-trifluorobut-1-ynyl], were synthesized and incorporated to a PNA strand, and their applicability for the monitoring of different hybridization modes by (19)F NMR spectroscopy was studied. Both sensors gave unique (19)F resonance shifts in NMR when the PNA was targeted to a complementary antiparallel DNA, antiparallel RNA, parallel DNA, and parallel RNA. The 5-trifluoromethyl-derived sensor was additionally applied for the monitoring of interconversions from a parallel DNA/PNA complex to an antiparallel RNA/PNA complex and from a PNA/PNA complex to two DNA/PNA complexes (i.e., double-duplex invasion).


Subject(s)
DNA/analysis , Fluorine/chemistry , Oligonucleotides/analysis , Peptide Nucleic Acids/chemistry , RNA/analysis , Magnetic Resonance Spectroscopy , Molecular Structure , Peptide Nucleic Acids/chemical synthesis
4.
Bioconjug Chem ; 23(9): 1981-8, 2012 Sep 19.
Article in English | MEDLINE | ID: mdl-22871148

ABSTRACT

Esterified precursors of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA; 18) and 1,4,7-triazacyclononane-1,4,7-trisacetic acid (NOTA; 17,19) ligands bearing a dimethoxytritylated hydroxyl side arm were prepared and immobilized via an ester linkage to long chain alkyl amine derivatized controlled pore glass (LCAA-CPG). Oligonucleotide chains were then assembled on the hydroxyl function and conjugates were released and deprotected by a two-step cleavage with aqueous alkali and ammonia. The 3'-DOTA and 3'-NOTA conjugated oligonucleotides were converted to (68)Ga chelates by a brief treatment with [(68)Ga]Cl(3) at elevated temperature. Applicability of the conjugates for in vivo imaging with positron emission tomography (PET) was verified.


Subject(s)
Chelating Agents/chemistry , Oligonucleotides/chemistry , Magnetic Resonance Spectroscopy , Positron-Emission Tomography , Spectrometry, Mass, Electrospray Ionization
5.
Bioconjug Chem ; 22(8): 1559-66, 2011 Aug 17.
Article in English | MEDLINE | ID: mdl-21688836

ABSTRACT

The potential of aminoglycosides to induce RNA-invasion has been demonstrated. For this purpose, aminoglycoside-3'-conjugates of 2'-O-methyl oligoribonucleotides have been synthesized entirely on a solid phase. The synthesis includes an automated oligonucleotide chain elongation to solid-supported neomycin, ribostamycin, and methyl neobiosamine, and a two-step deprotection/release of the solid-supported conjugate, which allows exploitation of a simple protecting group scheme. Conjugates have been targeted to a (19)F labeled HIV-1 TAR RNA model (Trans Activation Response element of HIV), which allows monitoring of the invasion by (19)F NMR spectroscopy. A remarkably enhanced invasion, compared to that resulting from the corresponding unmodified 2'-O-methyl oligoribonucleotide (5'-CAGGCUCA-3'), has been obtained by the neomycin conjugate. The increased affinity results from a cooperative binding of the neomycin moiety and hybridization, though the invasion may also follow a mechanism, in which the first molar equivalent of the conjugate induces hybridization of the second.


Subject(s)
Aminoglycosides/chemistry , HIV Long Terminal Repeat , Molecular Targeted Therapy , Oligoribonucleotides/chemistry , Aminoglycosides/chemical synthesis , Fluorine , HIV-1 , Humans , Isotopes , Magnetic Resonance Spectroscopy/methods , Neomycin , Oligoribonucleotides/chemical synthesis
6.
Bioconjug Chem ; 22(6): 1249-55, 2011 Jun 15.
Article in English | MEDLINE | ID: mdl-21539388

ABSTRACT

2'-O-[(2-Bromoethoxy)methyl]cytidine and 2'-O-[(2-azidoethoxy)methyl]cytidine have been prepared and introduced as appropriately protected 3'-phosphoramidite (1) and 3'-(H-phosphonate) (2) building blocks, respectively, into 2'-O-methyl oligoribonucleotides. The support-bound oligonucleotides were subjected to two consecutive conjugations with alkynyl-functionalized monosaccharides. The first saccharide was introduced by a Cu(I) promoted click reaction with 2 and the second by azidation of the 2-bromoethoxy group of 1 followed by the click reaction. The influence of the 2'-glycoconjugations on hybridization with DNA and 2'-O-methyl RNA targets was studied. Two saccharide units within a 15-mer oligonucleotide had a barely noticeable effect on the duplex stability, while introduction of a third one moderately decreased the melting temperature.


Subject(s)
Azides/chemistry , Click Chemistry , Oligonucleotides/chemical synthesis , Carbohydrate Conformation , DNA/chemistry , Oligonucleotides/chemistry , RNA/chemistry
7.
Bioconjug Chem ; 21(10): 1890-901, 2010 Oct 20.
Article in English | MEDLINE | ID: mdl-20828203

ABSTRACT

4'-C-[N,N-Di(4-pentyn-1-yl)aminomethyl]thymidine and 4'-C-[N-methyl-N-(4-pentyn-1-yl)aminomethyl]thymidine 3'-(2-cyanoethyl-N,N-diisopropyl)phosphoramidites (1, 2) were synthesized, and one or two such monomers were incorporated into a 15-mer oligodeoxyribonucleotide. After chain assembly, azido-functionalized ligands, including appropriate derivatives of 1,4-phenylenedimethaneamine, mannose, paromamine, and neomycin, were conjugated to the alkynyl groups by the click chemistry on a solid support. The influence of the 4'-modifications on the melting temperature with DNA and 2'-O-methyl RNA targets was studied. Oligonucleotides containing one to four mannose ligands in the central part of the chain (up to two 4'-C-[N,N-di(4-pentyn-1-yl)aminomethyl]thymidine units) form equally stable duplexes with complementary 2'-OMe RNA as the corresponding unmodified DNA sequence. At high salt content, the mannose conjugation is even stabilizing. On using a DNA target, a modest destabilization occurs. All the amino group bearing conjugates stabilized the duplexes, the DNA·DNA duplexes more than the DNA·2'-O-methyl RNA duplexes.


Subject(s)
Alkynes/chemistry , Click Chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Ligands , Organophosphorus Compounds/chemistry , Thymidine/analogs & derivatives , Thymidine/chemistry , Transition Temperature
8.
J Am Chem Soc ; 132(25): 8560-2, 2010 Jun 30.
Article in English | MEDLINE | ID: mdl-20521791

ABSTRACT

(19)F NMR spectroscopy offers an efficient tool for monitoring RNA invasion. The invasion of 2'-O-methyl oligoribonucleotides into a (19)F-labeled HIV-1 TAR RNA model and the temperature-dependent behavior of the complexes obtained have been examined.


Subject(s)
Magnetic Resonance Spectroscopy/methods , RNA/chemistry , Base Sequence , Nucleic Acid Denaturation , Oligoribonucleotides/genetics , Oligoribonucleotides/metabolism , RNA/genetics , RNA/metabolism
9.
Bioconjug Chem ; 19(8): 1726-34, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18671421

ABSTRACT

4'-C-Azidomethylthymidine 3'-(H-phosphonate) monomer (10) was synthesized in high yield and three such monomers were incorporated by the H-phosphonate coupling into a 15-mer oligodeoxyribonucleotide. The unmodified 2'-deoxynucleosides could be coupled by either the H-phosphonate or phosphoramidite chemistry, indicating that the Staudinger reaction between the azido group and the phosphoramidite reagent severely hampered the coupling only when it took place intramolecularly. After chain assembly, three alkynyl group bearing ligands, viz., propargyl 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranoside (2), N-{4-[N-(trifluoroacetyl)aminomethyl]benzyl}-4-pentynamide (3) and N (1), N (3), N (2')-tris(trifluoroacetyl)-N (6')-(4-pentynoyl)neamine (4), were conjugated to the azido groups of the oligonucleotide by click chemistry both on a solid support and in solution. The products were deprotected by conventional ammonolysis and purified by HPLC chromatography. Melting temperature studies revealed that the mannose conjugated oligonucleotides formed more stable duplexes with 2'-O-methyl RNA than with DNA strand. With 2'-O-methyl RNA, a slight destabilization compared to an unmodified sequence was observed at low ionic strength, while at high salt content, the manno-conjugation was stabilizing.


Subject(s)
Oligodeoxyribonucleotides/chemistry , Thymidine/analogs & derivatives , Thymidine/chemistry , Base Sequence , Hydroxyl Radical/chemistry , Ligands , Nucleic Acid Denaturation , Oligodeoxyribonucleotides/genetics , Oligodeoxyribonucleotides/metabolism , Phosphates/chemistry , Quaternary Ammonium Compounds/chemistry , Solutions , Static Electricity , Thymidine/chemical synthesis , Transition Temperature
10.
J Am Chem Soc ; 126(35): 11040-5, 2004 Sep 08.
Article in English | MEDLINE | ID: mdl-15339190

ABSTRACT

Hydrolytic reactions of guanosyl-(3',3')-uridine and guanosyl-(3',3')-(2',5'-di-O-methyluridine) have been followed by RP HPLC over a wide pH range at 363.2 K in order to elucidate the role of the 2'-hydroxyl group as a hydrogen-bond donor upon departure of the 3'-uridine moiety. Under neutral and basic conditions, guanosyl-(3',3')-uridine undergoes hydroxide ion-catalyzed cleavage (first order in [OH(-)]) of the P-O3' bonds, giving uridine and guanosine 2',3'-cyclic monophosphates, which are subsequently hydrolyzed to a mixture of 2'- and 3'-monophosphates. This bond rupture is 23 times as fast as the corresponding cleavage of the P-O3' bond of guanosyl-(3',3')-(2',5'-di-O-methyluridine) to yield 2',5'-O-dimethyluridine and guanosine 2',3'-cyclic phosphate. Under acidic conditions, where the reactivity differences are smaller, depurination and isomerization compete with the cleavage. The effect of Zn(2+) on the cleavage of the P-O3' bonds of guanosyl-(3',3')-uridine is modest: about 6-fold acceleration was observed at [Zn(2+)] = 5 mmol L(-)(1) and pH 5.6. With guanosyl-(3',3')-(2',5'-di-O-methyluridine) the rate-acceleration effect is greater: a 37-fold acceleration was observed. The mechanisms of the partial reactions, in particular the effects of the 2'-hydroxyl group on the departure of the 3'-linked nucleoside, are discussed.


Subject(s)
Guanosine/analogs & derivatives , Uridine/analogs & derivatives , Guanosine/chemistry , Hydrogen Bonding , Hydrogen-Ion Concentration , Hydrolysis , Kinetics , Organophosphates/chemistry , Uridine/chemistry , Zinc/chemistry
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