EP0436582A1 - Phosphorothioate et oligodesoxynucleotides normaux a liaison d'acridine en position 5' - Google Patents

Phosphorothioate et oligodesoxynucleotides normaux a liaison d'acridine en position 5'

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Publication number
EP0436582A1
EP0436582A1 EP19890910516 EP89910516A EP0436582A1 EP 0436582 A1 EP0436582 A1 EP 0436582A1 EP 19890910516 EP19890910516 EP 19890910516 EP 89910516 A EP89910516 A EP 89910516A EP 0436582 A1 EP0436582 A1 EP 0436582A1
Authority
EP
European Patent Office
Prior art keywords
acridine
oligodeoxynucleotides
oligodeoxynucleotide
linked
normal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP19890910516
Other languages
German (de)
English (en)
Other versions
EP0436582A4 (en
Inventor
Jack S. Cohen
Kenya C/O Yamasu Shoyu Company Ltd. Mori
Shee Loong Loke
Xianghong Zhang
Leonard M. Neckers
Cy A. Stein
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
US Department of Health and Human Services
US Department of Commerce
Original Assignee
US Department of Health and Human Services
US Department of Commerce
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by US Department of Health and Human Services, US Department of Commerce filed Critical US Department of Health and Human Services
Publication of EP0436582A1 publication Critical patent/EP0436582A1/fr
Publication of EP0436582A4 publication Critical patent/EP0436582A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • the present invention relates to an automated synthesis method of 5'-acridine linked oligonucleotides using phosphoramidite-1inked acridine. These compounds are useful for inhibiting gene expression, and enable the kinetics of cellular uptake to be determined using fluorescence cell sorting. Background of the Invention
  • Oligodeoxynucleotides which are complementary to certain gene messages or viral sequences, are referred to as "anti-sense” compounds. These compounds have also been reported to have inhibitory effects against Rous sarcoma virus and human immunodeficiency virus, also referred to as HIV.
  • oligonucleotides bonded to intercalation groups for cleavage of RNA and DNA, for prevention of replication or development of viruses, or for detection and purification of particular DNA and RNA sequences.
  • These oligonucleotides are prepared by fusing an intercalation group by a covalent bond.
  • the compounds are prepared by known methods, particularly by a phosphotriester synthesis. In the process disclosed, the chain of nucleotides is first prepared, the groups not entering into the reaction being protected during the reaction, after which the protecting groups are eliminated to obtain the final products. For example, a 3 • -phosphodiester nucleotide is coupled with the hydroxyl derivative of the intercalating agent. Unfortunately, this synthetic method is rather complicated, and is not easily adaptable to the standard automated synthesizer processes.
  • Sproat et al., PCT WO 87/07611 disclose a process for labelling oligonucleotides using fluorescent dyes.
  • the labelling is effected by converting the oligonucleotide into its 5'-[HS-(Y)z]-derivative, and the latter is reacted with a derivative of a fluorescent dye, which together with the 5-derivative forms an oligonucleotide of a fluorphore oligonucleotide.
  • Yamane et al., European patent 0 251 283 disclose a method for preparing a poly-labelled oligonucleotide derivative by using the sequence used for forming the basic skeleton of the compound and/or introduction of substituents thereon.
  • One method includes synthesizing an aminoalkylated oligonucleotide, then introducing a polylysine into the compounds and labelling the polylysine with a labelling substance.
  • the aminoalkylated oligonucleotide can be bonded with a polylysine previously labelled with a labelling substance.
  • European patent 0 235 301 disclose a method for forming pyridopyrimidine oligonucleotides which are fluorescent and can form a base pair with guanidine or adenine.
  • the compounds can be synthesized by standard methods. All of the compounds are said to be fluorescent and display the common characteristics of the natural pyrimidine bases.
  • the synthesis is usually carried out either on a solid- phase substrate or in solution.
  • Solid-phase synthesis relies on sequential addition of ononucleotides to a growing chain attached at one end to the substrate.
  • the solid phase allow easy separation of the reactants, but the method requires excess quantities of reactants and usually provides only small quantities, less than 1 mg, or the desired sequence.
  • Solution phase synthesis while it requires lesser amounts of the expensive reagents and can provide larger quantities of the product sequence, requires isolation and purification of the intermediate product after every addition. Virtually all automated polynucleotide systems rely on solid-phase synthesis.
  • the present invention provides a phosphoramidite synthesis involving a simple, automated, high yield method for fluorescently tagging the 5' end of an oligodeoxynucleotide.
  • the reaction scheme is shown in Figure 1.
  • the 5'-acridine linked molecules, both PO and PS, are readily purified by reverse phase chromatography. Two major peaks are observed, each containing acridine, but it can be shown by H and 31 P NMR that the desired compound invariably elutes at longer retention time. The nature of the faster-eluting component is not clear at this time.
  • HL60 cells take up homo-oligodeoxynucleotides of thymidine as monitored by flow cytometric analysis. Thus, it appears that there is an energy-dependent transport mechanism for the oligodeoxynucleotides. Thus, these compounds are useful as markers and inhibitors of gene expression.
  • Figure 1 shows a scheme for the synthesis of 5'- linked acridine oligodeoxynucleotides using a linked phosphoramidite in an automatic synthesizer.
  • Figure 2 shows a proton NMR spectrum at 400 MHz and 65°C of the aromatic region of 5'-acridine-dT 7 .
  • Figure 3 shows the difference in melting temperatures between normal and phosphorothioate 14-m3 oligodeoxynucleotides with different GC content.
  • Figure 4 shows the effect of temperature on cellular uptake of free acridine and 5'-acridine-dT 12 cells.
  • Figure 5A and 5B show the cellular uptake of normal dT and S-dT oligodeoxynucleotides.
  • the 5'-acridine linked oligodeoxynucleotides can be synthesized in an automated synthesizer using phosphoramidite acridine. These compounds are important, because certain phosphorothioate oligodeoxynucleotide analogs, unlike their normal congeners, have been found to exhibit significant anti-HIV activity, as reported by Matsukura et al, Proc. Natl. Acad. Sci. USA 84: 7706-7710 (1987) .
  • the melting temperatures for acridine-thymidine base pairs of phosphorothioate oligodeoxynucleotides are significantly depressed relative to normal oligodeoxynucleotides, while GC-containing phosphorothioate oligodeoxynucleotides show much less depression in the melting temperature.
  • the melting temperatures of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers.
  • the 3-aminopropanol, 5-aminopentanol, and 6,9- dichloro-2-methoxy acridine were purchased from Aldrich Chemical Co.
  • the 6-chloro-2-methoxy(hydroxyalkylamino)- 9-acridine was prepared via the method of Asseline et al. in Proc. Natl. Acad. Sci. USA, op. cit.
  • the phosphoramidite was prepared by a modification of the procedure of Connally.
  • 6-chloro-2-methoxy-9-(3- hydroxypropyl)amino acridine (318 mg, 1 mmol) or the 5- hydroxypentylamino derivative (346 mg, 1 mmol) or the 5- hydroxypentylamino derivative (346 mg, 1 mmol) was dissolved in 2 ml of CH 2 C1 2 .
  • N-ethyl-diisopropylamine (380 microL, 2 mmol) was then added.
  • N,N-diisopropyl- methyl-phosphonamidic chloride (194 microL, 1 mmol) was added over a period of about five minutes.
  • oligodeoxyribonucleotides 5-methylcytidine 0-cyanoethyl phosphoramidite was obtained from Glen Research (Herndon, VA) . All of the normal oligodeoxynucleotides were synthesized on the Applied Biosystems 380B DNA Synthesizer, and were purified by HPLC reverse phase chromatography (PRP-1) column. An extra round of synthesis was carried out using a 100 itiM solution of the acridinyl phosphoramidites in acetonitrile.
  • methyl phosphate protecting groups were removed with thiophenol (total thirty minute exposure) , and cleavage from the resin was effected with concentrated aqueous ammonia.
  • the automated synthesis of the phosphorothioate oligodeoxynucleotides was accomplished by modification of the method of Stec. , as reported by Stein et al., Nucl. Acids Research, 1988, in press.
  • the standard iodine oxidation is substituted by a sulfurization step by using a 10% solution of elemental sulfur in CSg/pyridine/triethylamine (45:45:10) .
  • the column is washed repeatedly with a 1:1 solution of carbon disulfide and pyridine to remove any residual sulfur.
  • the phosphorothioate oligodeoxynucleotides were purified by reverse phase HPLC as above, although the percent organic phase (acetonitrile) is higher. Samples were detritylated at room temperature in 3% acetic acid, extracted with ethyl acetate, and lyophilized. Melting Temperatures
  • Poly-rA and poly-rl were obtained from Pharmacia. All optical measurements were made on a Shimadzu-UV-160 recording spectrophotometer coupled to a CPS Controller thermostat. Values of absorbance were recorded at 260 nm in 10 mM sodium cacodylate/140 mM NaCl buffer, pH 7.0. All duplexes were formed in 1:1 mixtures of a strand with its complement. All of the samples were pre-melted at 75-98°C to destroy secondary structures, and then were allowed to equilibrate thermally. Each melt curve is composed of a minimum of twenty individual temperature points.
  • NMR Measurements NMR spectra were recorded on a Varian XL-400 spectrometer at 400 MHz for X H and 162 MHz for 31 P at 22°C. Chemical shifts were measured with respect to internal TMS and external TMP, respectively. The recycle times were 2-3 seconds and the number of scans was 64-200 for K and up to 3000 for 31 P. Integration was performed using the Varian program.
  • SI nuclease and PI nuclease were obtained from BRL.
  • Bovine spleen phosphodiesterase and snake venom phosphodiesterase were obtained from Pharmacia. All of the reactions were run in a total volume of one ml at 37°C. The value of absorbance was measured at _ ⁇ ma ⁇ .
  • SI nuclease 1000 microns/ml was diluted 1:10 in reaction buffer, which consisted of 30 mM sodium acetate (pH 4.6)., 50 mM NaCl, 1 mM zinc acetate, and 5% (v/v) glycerol. The final concentration of enzyme was 100 u/ml.
  • PI nuclease (40 u/ml) was diluted 1:10 in reaction buffer, which consisted of 50 mM sodium acetate (pH 5.3) . The final enzyme concentration was 4 u/ml.
  • Bovine spleen phosphodiesterase was dissolved in water (0.04 u/ ⁇ L) , and added (1 u/ml) to a solution containing 125 mM succinate- HC1, pH 6.5.
  • Snake venom phosphodiesterase (46 u/mg solid) was dissolved in 500 ⁇ L water. One ⁇ L of this solution was added to the reaction mixture, which contained 100 mM Tris-HCl, pH 8.9, 100 mM NaCl, and 14 mM MgC 12 .
  • OD(T) (e) protest (l + K) + e b /(l-K)
  • 0D(T) optical density at any temperature T
  • ea and eb are the maximum and minimum values of the absorption
  • K exp[ H(T-Tm)RTm 2 ) ]
  • H van't Hoff enthalpy
  • Intracellular acridine was excited by the 488 nm line of an argon laser set at 300 mW, and the resultant emitted fluorescence of individual cells was recorded. Since the log amplified fluorescence of the populations was unimodal, the data are expressed as the median fluorescence of a population calculated using Consort 30 software supplied with the Facstar. Characterization of Acridine Containing Oligomers
  • S-5Me-dC28 42 80 Melting temperatures of duplexes of poly-rA and oligo-dT of various lengths containing the 5* linked acridine derivative (normal and PS) were compared to unmodified phophorothioate oligodeoxynucleotides of identical length, as shown in Table I. In each case, following the precedent of Asseline et al (op. cit. , 1984b) , the link between the intercalator and the oligo contained either three or five methylene groups. The melting temperatures for poly-rA duplexes with S-oligos with n ⁇ 12 could not be obtained because of their very low melting temperatures.
  • Nuclease Susceptibilities 5'-acridine-dT 15 , normal, m 3, was studied with regard to DNase sensitivity, as reported in Table III. DNases employed were the predominantly endonuclease SI, the exo- and endonuclease PI, snake venom phosphodiesterase (SVP) , and bovine spleen phosphodiesterase (BSP) , which required a free 5'-OH group.
  • SVP snake venom phosphodiesterase
  • BSP bovine spleen phosphodiesterase
  • the phosphoramidite synthesis of the present invention provides a simple, automated, high yield method for fluorescently tagging the 5' end of an oligodeoxynucleotide. Because when the molecule is subjected to base-deblocking conditions, e.g., aqueous ammonia, 60°C, for 10 hours, the acridine is cleaved from the oligodeoxynucleotide. Therefore, this method is primarily suited for homo-oligos of thy idine. The replacement of the ring chloride by thiophenol at the 6 position under mild conditions is novel, and appears to be aided by the presence of thymidine in the mixture.
  • base-deblocking conditions e.g., aqueous ammonia, 60°C
  • the 5'-acridine linked molecules both PO and PS, are readily purified by reverse phase chromatography. Two major peaks are observed, each containing acridine, but it can be shown by X H and 31 P NMR that the desired compound invariably elutes at longer retention times.
  • the difference in melting temperature between the normal and S-oligo decreases as the GC-content is increased, as shown in Figure 3, to a minimum at about 50% GC content. Additionally, the most active anti-HIV S-oligos so far identified have a high GC content (cf. Matsukura et al. , op_. cit.) .
  • the synthesis method of the present invention can be applied to all four bases.
  • the thiophosphate side reactions can be avoided by using other substituted acridines.
  • oligodeoxynucleotides are highly nuclease resistant. Capping the 5' end of an oligodeoxynucleotide with a modified acridine greatly modifies its sensitivity towards the 5'exonuclease bovine spleen phosphodiesterase, as expected, but does not change sensitivity towards SI and PI nucleases, or towards snake venom phosphodiesterase. Homo-oligos of thymidine are taken up by HL60 cells as monitored by flow cytometric analysis.
  • the fluorescent group on the oligodeoxynucleotide enables one to determine the kinetics of cellular uptake using fluorescence cell sorting. Additionally, this fluorescent group makes it possible to monitor the inhibitory effects of other substances on cellular uptake, metabolism effects, and release of oligos, related nucleotide derivatives such as plasmids, and naked RNA molecules.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Molecular Biology (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Saccharide Compounds (AREA)

Abstract

Cette invention concerne un procédé de synthèse automatique d'oligonucléotides à liasion acridine en position 5' utilisant de l'acridine à liaison phosphoramidite. Ces composés sont utiles pour l'inhibition de l'expression des gènes, et permettent de déterminer la cinétique de la fixation cellulaire par triage de cellules de fluorescence.
EP19890910516 1988-09-20 1989-09-19 Phosphorothioate and normal oligodeoxynucleotides with 5'-linked acridine Withdrawn EP0436582A4 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US24668888A 1988-09-20 1988-09-20
US246688 1994-05-20

Publications (2)

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EP0436582A1 true EP0436582A1 (fr) 1991-07-17
EP0436582A4 EP0436582A4 (en) 1992-06-17

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EP (1) EP0436582A4 (fr)
JP (1) JPH04503403A (fr)
AU (1) AU618414B2 (fr)
IL (1) IL91673A0 (fr)
WO (1) WO1990003383A1 (fr)

Families Citing this family (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04500679A (ja) * 1989-04-18 1992-02-06 アメリカ合衆国 オリゴデオキシヌクレオチド化合物およびその製造方法
EP0602524A1 (fr) * 1992-12-15 1994-06-22 Hoechst Aktiengesellschaft Sondes marqués par chemiluminescence et leur utilisation dans épreuves des sondes
DE4326466A1 (de) * 1993-08-06 1995-02-09 Boehringer Mannheim Gmbh Infrarot-Farbstoff-markierte Nucleotide und ihre Verwendung in der Nucleinsäure-Detektion
US5508289A (en) * 1994-03-14 1996-04-16 The United States America As Represented By The Department Of Health And Human Services Bis-acridone chemotherapeutic derivatives
US5766550A (en) * 1995-03-15 1998-06-16 City Of Hope Disposable reagent storage and delivery cartridge
US7101999B2 (en) 2000-09-06 2006-09-05 Evotec Ag Oxazine derivatives
DE10046215B4 (de) * 2000-09-19 2004-04-15 Institut für Chemo- und Biosensorik Münster e.V. i.Ins. Fluorochrome und deren Verwendung

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4835263A (en) * 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use

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JP2509574B2 (ja) * 1985-08-15 1996-06-19 アモコ・コーポレーション 標識付けした核酸

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4835263A (en) * 1983-01-27 1989-05-30 Centre National De La Recherche Scientifique Novel compounds containing an oligonucleotide sequence bonded to an intercalating agent, a process for their synthesis and their use

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
EMBO/INSERM WORKSHOP ON "REGULATION OF GENE EXPRESSION BY RNA STRUCTURE AND ANTI-MESSENGERS", Les Arcs, Savoie, 28th February - 4th March 1988, & GENE, vol. 72, 1988, pages 333-341; C.A. STEIN et al.: "Phosphorothioate and normal oligodeoxyribonucleotides with 5'-linked acridine: characterization and preliminary kinetics of cellular uptake" (Cat. L,P,X,Y) *
See also references of WO9003383A1 *

Also Published As

Publication number Publication date
JPH04503403A (ja) 1992-06-18
AU618414B2 (en) 1991-12-19
IL91673A0 (en) 1990-04-29
AU4317989A (en) 1990-04-18
WO1990003383A1 (fr) 1990-04-05
EP0436582A4 (en) 1992-06-17

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