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WO1998016540A1 - Activateurs de couplage ameliores pour la synthese d'oligonucleotides - Google Patents

Activateurs de couplage ameliores pour la synthese d'oligonucleotides Download PDF

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Publication number
WO1998016540A1
WO1998016540A1 PCT/US1997/015744 US9715744W WO9816540A1 WO 1998016540 A1 WO1998016540 A1 WO 1998016540A1 US 9715744 W US9715744 W US 9715744W WO 9816540 A1 WO9816540 A1 WO 9816540A1
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coupling
phosphoramidite
dci
phosphoramidites
protected
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PCT/US1997/015744
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English (en)
Inventor
Chandra Vargeese
Wolfgang Pieken
Jeffrey D. Carter
John Yegge
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Proligo Llc
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Priority to AU48001/97A priority Critical patent/AU4800197A/en
Publication of WO1998016540A1 publication Critical patent/WO1998016540A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom

Definitions

  • This invention is in the field of methodology for the synthesis of oligonucleotides.
  • Oligonucleotides have been proposed for use as therapeutics, such as ribozymes, antisense oligonucleotides, and compounds that bind to or interact with various target molecules.
  • Antisense oligonucleotides can bind certain coding regions in an organism to prevent the expression of proteins or to block various cell functions.
  • a process known as the SELEX process, or Systematic Evolution of Ligands for Exponential Enrichment allows one to identify and produce oligonucleotides that selectively bind to or interact with target molecules.
  • the SELEX process is described in United States Patent No. 5,270,163, entitled "Methods for Identifying Nucleic Acid Ligands," the contents of which are hereby incorporated by reference.
  • nucleases may rapidly degrade certain of these compounds in vivo.
  • One approach to minimizing the in vivo degradation has been to modify the nucleotides to impart in vivo and in vitro stability to endo and exonucleases, and/or to provide differences in the three dimensional structure of the resulting oligonucleotides.
  • nuclease stability has been demonstrated by incorporating 2'-modif ⁇ ed nucleotides, such as 2'-amino- and 2'-fluoro-2'-deoxypyrimidines, into oligonucleotides. (Pieken et al. (1991) Science 253:314-317; Paolella et al. (1992) EMBO J. 11:1913-1919).
  • Oligonucleotide synthesis is typically performed using the phosphoramidite method, shown below in Scheme 1. (Dahl et al. (1987) Nucleic Acids Res. 15:1729-1743; Beaucage and Iyer (1992) Tetrahedron 48:2223-2311; Zon and Geiser (1991) Anti-Cancer Drug Design 6:539-568; Matteucci and Caruthers (1981) J. Am. Chem. Soc. 103:3185-3191; and Scaringe et al. (1990) Nucleic Acids Res.
  • Activation of a phosphoramidite monomer for coupling of the phosphoramidite monomer to a nucleotide or oligonucleotide in automated solid phase oligonucleotide synthesis is usually achieved by addition of tetrazole.
  • the first step in activation is the fast protonation of the trivalent phosphorous by tetrazole, followed by the slow displacement of the N,N-diisopropylamine with tetrazolide.
  • a nucleophile such as the 5'-hydroxyl group of a protected, resin-bound oligonucleotide (HOR) to yield the phosphite triester product.
  • HOR resin-bound oligonucleotide
  • tetrazole acts both as an acid and as a nucleophilic catalyst.
  • Oligoribonucleotide syntheses have also been reported. (Scaringe et al. (1990) Nucleic Acids Res. 18:5433-5441, the contents of which are hereby incorporated by reference). These syntheses also are typically performed using the phosphoramidite method.
  • the phosphoramidite method appears to work well for preparing small scale batches of DNA oligonucleotides with a large excess of phosphoramidite monomer.
  • problems are observed when the chemistry is scaled- up to a multi-mmole level and/or a large excess of phosphoramidite monomer is not used.
  • coupling times are typically dramatically increased from the 45 second reaction time employed at the 1 ⁇ mole scale in research laboratories. These increased reaction times lead to undesired side reactions and result in a decreased overall yield.
  • the 5'-hydroxyl group on the growing oligonucleotide chain is protected at various stages during oligonucleotide synthesis.
  • One of the preferred protecting groups is a trityl ether.
  • An undesired side reaction when scaling up phosphoramidite coupling using tetrazole as a coupling activator is detritylation of the protected 5 '-hydroxyl group.
  • reaction times are relatively fast for the phosphoramidite coupling step, detritylation does not compete significantly with the desired coupling chemistry.
  • reaction times increase detritylation becomes a significant side reaction.
  • Scheme 2 depicts the tetrazole-mediated detritylation of 5'-dimethoxytrityl protected nucleosides, which occurs under the conditions typically employed to couple 5'- dimethoxytrityl protected nucleoside 3 '-phosphoramidite monomers to the hydroxyl group on a growing oligonucleotide chain.
  • Detritylation is an undesired side reaction because it results in unwanted side reactions and corresponding lower overall yields.
  • One of the major side reactions involves the coupling of detritylated monomers to other nucleoside phosphoramidite monomers in solution. This can lead to unwanted by-products that contain additional nucleotides which are difficult to separate from the desired product.
  • Detritylation is even more of a problem with oligoribonucleotide synthesis since oligoribonucleotide syntheses require longer reaction times than oligodeoxyribonucleotide syntheses. Detritylation also becomes limiting at larger scales, where process economics dictate performing coupling reactions at close to stoichiometric concentrations.
  • the present invention provides improved methods for coupling phosphoramidite monomers to nucleotides and oligonucleotides in oligonucleotide syntheses.
  • One embodiment of the present invention involves using a coupling activator that is less acidic and at least as nucleophilic as tetrazole and still provides a coupling efficiency that is as effective as tetrazole.
  • Another embodiment of the present invention involves using a combination of a coupling activator and a suitable buffer.
  • the present invention provides improved methods for coupling phosphoramidite monomers with nucleophiles, preferably for oligonucleotide syntheses.
  • One embodiment of the present invention involves using coupling activators that are less acidic and at least as nucleophilic as tetrazole and still provide a coupling efficiency that is at least as effective as tetrazole.
  • the pKa of the coupling activators of the present invention is between 5.0 and 6.0, preferably between 5.0 and 5.5, and, more preferably, between 5.1 and 5.3.
  • Any coupling activator can be used that has a pKa between 5.0 and 6.0 and that is at least as nucleophilic as tetrazole.
  • Means for determining the pKa and nucleophilicity of a coupling activator are well known to those of skill in the art.
  • Suitable coupling activators for use in the present invention include, but are not limited to, 4,5-dicyanoimidazole (DCI), 4-alkylthioimidazole, 2-alkylthioimidazole, 2-nitroimidazole, 4-nitroimidazole, 4,5- dihaloimidazole, 4-haloimidazole, 2-haloimidazole and 5-alkoxytetrazole.
  • DCI the most preferred coupling activator
  • pKa 5.2
  • DCI in the phosphoramidite oligonucleotide synthesis rather than tetrazole increases product yield at larger scales and increases the rate of the coupling reaction.
  • DCI is highly soluble in acetonitrile, a preferred solvent for phosphoramidite coupling. While commercially available technical grade DCI is effective in providing an improved coupling efficiency between a phosphoramidite monomer and a nucleophile, it was discovered that an even greater coupling efficiency is obtained by using a purified form of DCI.
  • an improved coupling method wherein detritylation is minimized by employing a combination of a coupling activator and a suitable buffer.
  • a coupling activator either the coupling activators of the present invention or more acidic coupling activators, such as tetrazole, may be used.
  • Preferred buffers of the present invention include, but are not limited to, tertiary amines, including both tertiary amines with three alkyl groups, and heterocyclic compounds in which one or more of the amines are tertiary amines.
  • the tertiary amine is less nucleophilic than DMAP, which is known to cause side reactions at the 6-position oxygen of guanosines due to its relatively high nucleophilicity.
  • N-methylimidazole (NMI) is the preferred tertiary amine.
  • Tertiary amines such as NMI can be added to coupling activator solutions to increase the coupling efficiency.
  • the basicity of NMI decreases the rate of the competing monomer detritylation.
  • the increase in reaction rate that is observed with the more nucleophilic, less acidic coupling activators such as DCI is not observed in this embodiment.
  • the preferred method for performing phosphoramidite coupling chemistry according to the present invention is to use the less acidic coupling activators described above.
  • Figure 1 is a graph from which the second order rate constants for detritylation of 5'-DMT-2',3'-isopropylidineuridine with tetrazole (dashed line) and 5-ethylthiotetrazole (solid line) are derived.
  • Figure 2 is an anion exchange chromatogram of the crude deprotected oligonucleotide NX288c05 prepared as described in Example 3 using tetrazole as the coupling activator.
  • Figure 3 is an anion exchange chromatogram of the deprotected oligonucleotide prepared as described in Example 6 using tetrazole as the coupling activator.
  • Figure 4 is an anion exchange chromatogram of the deprotected oligonucleotide prepared as described in Example 6 using tetrazole and 0.1 M N-methylimidazole as the coupling activator.
  • Figure 5 is an anion exchange chromatogram of the deprotected oligonucleotide prepared as described in Example 6 using DCI as the coupling activator.
  • Figure 6 is an overlay of the anion exchange chromatograms of the deprotected oligonucleotide NX 11702 prepared as described in Example 7 using tetrazole and tetrazole/NMI as the coupling activators.
  • Figure 7 is an anion exchange chromatogram of the oligonucleotide NX11702 as prepared in Example 7 using 1.0 M DCI as the coupling activator.
  • Figure 8 is an anion exchange chromatogram of the crude product, following cleavage from the support, of an oligonucleotide prepared as described in Example 8 using DCI as the coupling activator.
  • Figure 9 is an anion exchange chromatogram of the crude product, following cleavage from the support, of an oligonucleotide prepared as described in Example 8 using tetrazole as the coupling activator.
  • Figure 10 is an anion exchange chromatogram of the crude product, following cleavage from the support, of oligonucleotide NX28604 prepared as described in Example 9 using DCI as the coupling activator.
  • Figure 11 is a graph showing the optical density at 495 nm versus the trityl fraction for the synthesis of the RNA 41-mer in Example 10 using 0.5 M DCI as the coupling activator.
  • Figure 12 is a graph showing the optical density at 495 nm versus the trityl fraction for the synthesis of the RNA 41-mer in Example 10 using 1.0 M DCI as the coupling activator.
  • Figure 13 is an anion exchange chromatogram of the deprotected RNA 41-mer described in Example 10, synthesized using 0.5 M DCI as the coupling activator.
  • Figure 14 is an anion exchange chromatogram of the deprotected RNA 41-mer described in Example 10, synthesized using 1.0 M DCI as the coupling activator.
  • Figure 15 is a graph showing the optical density at 495 nm versus the trityl fraction for the synthesis of DNA 29-mers described in Example 10 using 1.0 M DCI as the coupling activator.
  • Figure 16 is a graph showing the optical density at 495 nm versus the trityl fraction for the synthesis of DNA 29-mers described in Example 10 using 0.5 M DCI as the coupling activator.
  • Figure 17 is an anion exchange chromatogram of the crude product, following deprotection and cleavage from the support, of the DNA 29-mer prepared as described in Example 10 using 1.0 M DCI as the coupling activator.
  • Figure 18 is an anion exchange chromatogram of the crude product, following deprotection and cleavage from the support, of the DNA 29-mer prepared as described in Example 10 using 0.5 M DCI as the coupling activator.
  • Figure 19 is an anion exchange chromatogram of the starting trimer described in Example 11.
  • Figure 20 is an anion exchange chromatogram of the tetramer prepared in solution phase as described in Example 11 , using 1.13 equivalents of DCI as the coupling activator.
  • Figure 21 is an overlay of the anion exchange chromatograms shown in Figures 19 and 20.
  • the present invention provides improved methods for preparing oligonucleotides by coupling phosphoramidite monomers to a nucleoside, preferably the 5'-hydroxyl group of a nucleotide or oligonucleotide.
  • the coupling chemistry is performed using a coupling activator that is less acidic and at least as nucleophilic as tetrazole and still provides a coupling efficiency that is at least as effective as tetrazole.
  • the coupling chemistry is performed using a coupling activator that can be as acidic as or even more acidic than tetrazole, in combination with a suitable buffer, wherein the buffer is less nucleophilic than DMAP. Definitions
  • Coupled activator is defined as a compound that effectively couples a phosphoramidite monomer and the 5'-OH group on a nucleotide, preferably according to the mechanism shown in Scheme 1.
  • the coupling chemistry is also intended to encompass the reaction of phosphoramidite monomers with other nucleophiles.
  • acidic coupling activator is defined as a coupling activator with a pKa of less than 4.85. Tetrazole is one example of an acidic coupling activator.
  • alkyl as used herein is defined as a C M 8 straight, branched, or cyclic alkyl group. Preferred alkyl groups are methyl, ethyl, propyl, isopropyl and n-butyl.
  • alkoxy as used herein is defined as a C M8 alkoxy group.
  • halo as used herein is defined as chloride, bromide, iodide or fluoride.
  • nucleotide as used herein is defined as a modified or naturally occurring deoxyribonucleotide or ribonucleotide. Nucleotides typically include purines and pyrimidines, which include thymidine, cytidine, guanosine, adenine and uridine.
  • oligonucleotide as used herein is defined as an oligomer of the nucleotides defined above.
  • phosphoramidite monomer as used herein is defined as a nucleotide with a protected 5'-hydroxy group, in which the 3'-hydroxy group is coupled to a trivalent phosphorous atom, which in turn is bonded to a suitable leaving group such as a cyanoethanol (CNEO) group and a suitable dialkylamine, such as diisopropylamine.
  • CNEO cyanoethanol
  • Any nucleoside phosphoramidite monomer that can be used in solid or liquid phase oligonucleotide synthesis can be used in the present invention, including protected dimer and trimer phosphoramidite monomers.
  • Suitable phosphoramidite monomers that can be coupled with the coupling activators of the present invention include, but are not limited to, protected 2'-deoxynucleoside 3'-phosphoramidites, protected 2'-tert- butyldimethylsiloxynucleoside 3'-phosphoramidites, protected 2'-deoxy-2'- trifiuoroacetamidonucleoside 3 '-phosphoramidites, protected 2'-deoxy-2'-fluoronucleoside 3'-phosphoramidites, and protected 2'-alkoxynucleoside 3 '-phosphoramidites.
  • nucleoside phosphoramidite monomers include those that are described in or can be prepared from nucleosides described in United States Patent No. 5,428,149, issued on June 27, 1995, entitled “Methods for Palladium Catalyzed Carbon-Carbon Coupling and Products”; co-pending United States Patent Application Serial No. 08/430,709, filed April 27, 1995, entitled “High Affinity Nucleic Acid Ligands Containing Modified Nucleotides”; United States Patent Application Serial No 08/264,029, filed June 22, 1994, entitled “Novel Method of Preparation of Known and Novel 2'-Modified Nucleosides by Intramolecular Nucleophilic Displacement”; United States Patent Application Serial No.
  • non-nucleoside phosphoramidite monomers such as dialkylglycerol (DAG) phosphoramidites and phosphitylating reagents such as 2- cyanoethyltetraisopropylphosphorodiamidite, which is used in the syntheses of nucleoside phosphoramidites from nucleosides, can also be coupled using the chemistry described herein.
  • DAG dialkylglycerol
  • phosphitylating reagents such as 2- cyanoethyltetraisopropylphosphorodiamidite
  • nucleophile is defined as an electron-rich compound that reacts with a phosphoramidite monomer.
  • Suitable nucleophiles for use in the present invention include, but are not limited to, hydroxyl groups, primary or secondary amines, halogens, carboxylate anions, thiols and other nucleophiles known by those of skill in the art to undergo S N 2 reactions at trivalent phosphorus.
  • solid support is defined as a solid support which can be covalently coupled to a nucleophile, such as a nucleotide or oligonucleotide, in such a fashion that the nucleophile can be further modified using the chemistry disclosed herein, and subsequently removed from the solid support using conventional chemistry.
  • Suitable solid supports for use in the present invention include, but are not limited to, controlled pore glass of various pore size and loading, polystyrene beads, and polystyrene-polyethylene glycol copolymer support (TentaGel).
  • the term "coupling efficiency" is defined as the percent coupling of each nucleophile, such as a nucleotide or oligonucleotide, with a phosphoramidite monomer.
  • the first step typically involves coupling a nucleotide or oligonucleotide having a trityl-protected hydroxyl group to a solid support, and then detritylating the hydroxyl group.
  • the first step typically involves deprotecting one of the hydroxyl groups on a diprotected nucleotide or oligonucleotide.
  • the subsequent steps typically involve coupling a phosphoramidite monomer to the nucleotide or oligonucleotide (the coupling and washing steps may be repeated), oxidizing the phosphorus atom with an oxidizing agent (i.e., iodine), and optionally capping unreacted hydroxyl groups with a capping agent (i.e., acetic anhydride). Between each of these steps, excess reagents are washed from the solid support.
  • an oxidizing agent i.e., iodine
  • a capping agent i.e., acetic anhydride
  • a range of from 2 to 12 equivalents of the coupling activator to the phosphoramidite monomer is used.
  • the coupling activators of the present invention are typically used in concentrations of between 0.1 and 1.2 M.
  • the oxidizing agent is typically used in a range of from 1.5 to 4 equivalents.
  • between 14 and 28 equivalents of the capping agent typically acetic anhydride
  • one embodiment of the present invention provides an improved coupling method wherein the reaction rate of the phosphoramidite coupling chemistry is increased by using coupling activators that are less acidic than and at least as nucleophilic as tetrazole.
  • the coupling activators of the present invention increase the overall reaction rate by increasing the rate of the nucleophilic displacement step in the coupling reaction.
  • the previously designed coupling activators utilized in the prior art methods are more acidic than tetrazole and thus increase the rate of the protonation step (see Scheme 1).
  • the phosphoramidite coupling is performed using a single nucleophilic coupling activator bearing an acidic proton in the desired pKa range.
  • the coupling activators used in the syntheses described herein are efficient nucleophilic catalysts and mild acids.
  • the pKa of the coupling activators is higher than that of tetrazole.
  • the coupling activators of the present invention have a pKa between 5.0 and 6.0 and are at least as nucleophilic as tetrazole.
  • the pKa of the coupling activators of the present invention is between 5.0 and 5.5, and, more preferably, between 5.1 and 5.3.
  • Suitable coupling activators for use in the present invention include, but are not limited to, 4,5-dicyanoimidazole (DCI), 4-alkylthioimidazole, 2-alkylthioimidazole, 2-nitroimidazole, 4-nitroimidazole, 4,5-dihaloimidazole, 4-haloimidazole, 2-haloimidazole and 5-alkoxytetrazole.
  • DCI is the preferred coupling activator.
  • DCI has a pKa of 5.2 and is highly soluble in acetonitrile, a preferred solvent for phosphoramidite coupling.
  • DCI is less acidic than tetrazole, increases product yield at larger scales and essentially eliminates detritylation of the phosphoramidite monomers, and increases the rate of the coupling reaction relative to when tetrazole is used as the coupling activator.
  • DCI is significantly more soluble in acetonitrile than tetrazole. With DCI, a 1.1 M solution in acetonitrile is easily prepared and remains a liquid at room temperature.
  • This high concentration of coupling activator allows for a higher effective concentration of activated phosphoramidite monomers during the coupling step.
  • This increased activated phosphoramidite monomer concentration in turn leads to increased reaction efficiency at decreased phosphoramidite monomer excess.
  • Using a higher effective phosphoramidite monomer concentration reduces the number of equivalents of phosphoramidite monomer needed and significantly improves process efficiency. Improving the process efficiency becomes increasingly important as the process scale increases.
  • the coupling efficiency is improved by minimizing detritylation of the phosphoramidite monomers. This is achieved by increasing the pH of the reaction medium by introducing a buffer component. The coupling efficiency may increase if this buffer component also is able to function as nucleophilic catalyst, thus speeding up the slow nucleophilic step.
  • the buffer is preferably a tertiary amine, including tertiary amines containing three alkyl groups, and heterocyclic compounds containing one or more tertiary amines.
  • Suitable trialkylamines include, but are not limited to, N- alkylimidazoles, N-alkyltriazoles, N-alkyltetrazoles, and dialkylaminopyridines.
  • the tertiary amine is less nucleophilic than DMAP, which is known to cause side reactions at the 6-position oxygen of guanosines due to its relatively high nucleophilicity.
  • N- methylimidazole (NMI) is the preferred tertiary amine.
  • the buffer is added to the coupling activator solutions to increase the coupling efficiency.
  • a solution of 0.5 M tetrazole and 0.5 M NMI in acetonitrile gives consistently high coupling efficiencies with the same wide variety of nucleoside phosphoramidite monomers described above.
  • the basicity of NMI also decreases the rate of monomer detritylation. Coupling reactions performed with this tetrazole/NMI mixture proceed at the same rate as is observed with tetrazole alone.
  • the increase in the reaction rate of the coupling chemistry that is observed with the more nucleophilic, less acidic coupling activators such as DCI is not observed in this embodiment.
  • the activation with DCI is also useful in the phosphitvlation of nucleosides with 2-cyanoethyltetraisopropylphosphorodiamidite.
  • this phosphitylating reagent is protonated by DCI, followed by displacement of the N,N- diisopropylamine with the 4,5-dicyanotetrazolide. This intermediate reacts with the 3'- hydroxyl group of the nucleoside to yield the nucleoside 3 '-phosphoramidite (Scheme 3).
  • 4,5-dicyanoimidazole and N-methylimidazole were obtained from Aldrich Chemical Co.
  • the 2',3'-O-isopropylidineuridine was obtained from Sigma Chemical Co. and converted to the 5'-DMT derivative by standard methods.
  • Deoxynucleoside phosphoramidites and oligonucleotide synthesis reagents were obtained from Applied Biosystems, Cruachem Inc., Glen Research, or PerSeptive Biosystems.
  • N- tert-butylphenoxyacetyl protected 2'-TBDMS protected ribonucleoside phosphoramidites were obtained from PerSeptive Biosystems.
  • Example 2 Detritylation of 5'-dimethoxytrityl-2',3'-O-isopropylidineuridine by tetrazole and 5-ethylthio-l-H-tetrazole.
  • a model study was conducted to establish the rate of detritylation of 5'- dimethoxytritylnucleosides by tetrazole and 5-ethylthiotetrazole in acetonitrile.
  • a stock solution of 0.2 M 5'-DMT-2',3'-isopropylidineuridine was prepared in dry acetonitrile.
  • To this solution was added an acetonitrile solution of the coupling activator solution at time zero.
  • the final concentration of the reaction mixture was adjusted to 0.1 M 5'-DMT-2',3'- isopropylidineuridine. An aliquot was removed at several time points and analyzed by reversed phase HPLC.
  • Rates were determined at three coupling activator concentrations (0.1 M, 0.2 M, 0.5 M).
  • Figure 1 shows the second order rate constants for detritylation by tetrazole and 5-ethylthiotetrazole. The rate constants correlate to the acidity of the tetrazoles. Thus, as the coupling times and the acidity of the coupling activators increases, detritylation and by-products associated with detritylation also increase.
  • the chromatogram was obtained on a 4.6 x 100 mm Gen-Pak Fax column (Waters) at 80°C eluting from 90% trizma ethylenediamine tetraacetate (pH 7.5, buffer A) : 10% trizma ethylenediamine tetraacetate + 1 M NaCl (pH 7.5, buffer B) to 90% buffer B over 30 minutes at 0.75 mL/min.
  • Example 4 Solid phase synthesis of (aU) 10 T oligonucleotides on a polystyrene support using tetrazole and tetrazole buffered with NMI.
  • a 2'-aminouridine oligonucleotide 5'-(aU) I0 T-3' (SEQ ID NO:2) was prepared at a 0.1 mmol scale on a Pharmacia OligoPilot IITM automated synthesizer using polystyrene support loaded at 26 mmol/g with 3'-succinylthymidine and using tetrazole or tetrazole buffered with NMI as the activator.
  • the activator formulation used is shown in Table 2.
  • the 2'-aminouridine phosphoramidites were coupled in two aliquots of three equivalents. The synthesis cycle is shown in Table 2.
  • the oligonucleotide was cleaved from support and deprotected by reaction in concentrated ammonia at 55 °C for 18 hours. The solution was then cooled, filtered and the filtrate was concentrated to dryness. As shown in Table 3, by adding 0.1 M NMI to 0.5 M tetrazole (8 eq) the stepwise average coupling efficiency for the preparation of a (aU) 10 -T oligonucleotide was increased from 96.4 % to 98.0 %. Adding more than 0.1 M of NMI resulted in decreased product yield, presumably due to the basicity of the medium.
  • Example 5 31 P NMR experiments demonstrating faster coupling rates in DCI reactions.
  • Example 6 Solid phase synthesis of an oligonucleotide on polystyrene support using tetrazole, tetrazole buffered with NMI and DCI.
  • Homopolymers of 2'-modified DNA were synthesized using tetrazole, tetrazole buffered with N-methylimidazole (NMI) and 4,5-dicyanoimidazole (DCI) as the coupling activators.
  • NMI N-methylimidazole
  • DCI 4,5-dicyanoimidazole
  • Oligodeoxynucleotide synthesis with 0.5 M tetrazole as the coupling activator.
  • the sequence synthesized in this example was 5'- aCaCaCaCaCaCaCaCaCaCaCaCdT-3'
  • the oligonucleotide was cleaved from the support and deprotected by reaction with concentrated aqueous ammonia at 80°C for 2 hours.
  • the purity of the final product as determined by anion exchange chromatography was 85.9%.
  • the average coupling efficiency was 98.3%.
  • An anion exchange chromatogram of the deprotected product is shown in Figure 3.
  • the sequence synthesized in this example was 5'- aCaCaCaCaCaCaCaCaCaCaCaCdT-3 1 (SEQ ID NO:3) where aC stands for 2'-aminocytidine and dT stands for thymidine.
  • the synthesis was performed in a Pharmacia OligoPilot IITM using a 12 mL axial flow Pharmacia column filled with 4 grams of Pharmacia polystyrene with 26 mmol dT/gram support.
  • the reagents used, equivalents used, and the exposure time for each step of the synthesis is the same as shown in Table 5.
  • the oligonucleotide was cleaved from the support and deprotected by reaction with concentrated aqueous ammonia at 80°C for 2 hours.
  • the purity of the final product as determined by anion exchange chromatography was 83.3%.
  • the average coupling efficiency was 98.0%.
  • An anion exchange chromatogram of the deprotected product is shown in Figure 4.
  • the sequence synthesized in this example was (5'- aCaCaCaCaCaCaCaCaCaCaCaCaCaCdT-3') (SEQ ID NO:3) where aC stands for 2'-aminocytidine and dT stands for thymidine.
  • the synthesis was performed in a Pharmacia OligoPilot IITM using a 48 mL axial flow Pharmacia column filled with 16 grams of Pharmacia polystyrene with 25 mmol dT/gram support.
  • the reagents used, equivalents used, and the exposure time for each step of the synthesis is described below in Table 6.
  • the oligonucleotide was cleaved from the support and deprotected by reaction with concentrated aqueous ammonia at 80°C for 2 hours.
  • the purity of the final product as determined by anion exchange chromatography was 89.9%.
  • the average coupling efficiency was 98.8%.
  • An anion exchange chromatogram of the deprotected product is shown in Figure 5. As the coupling efficiency approaches 99%, the yield and purity of the final product increases substantially relative to when the coupling efficiency is around 98%.
  • Example 7 Preparation of NX11702 with tetrazole, tetrazole buffered with NMI, and with DCI.
  • This example illustrates the effect of addition of NMI to the activator solution by the preparation of a 34mer 2'-fluoropyrimidine-ribopurine oligonucleotide.
  • the syntheses were performed at a 1 mmol scale with a 2-fold excess of phosphoramidite per coupling and a 20 minute or 30 minute coupling time for the 2'-fluoiOpyrimidine phosphoramidites or ribopurine phosphoramidites, respectively.
  • sequence prepared was 2'-fluoropyrimidine- ribopurine oligonucleotide of sequence 5'-rGrG-rArGfU-fCfUfU-rArG-fCrArG-fCrGfC- rGfUfU-fUfUfC-rGrArG-fCfUrA-fCfUfC-fC[3'-3'T]-3' (NX11702) (SEQ ID NO:4) where rG and rA stands for guanosine and adenosine respectively and fC and fU stands for 2'- deoxy-2'-fluorocytidine and 2'-fluorouridine, respectively, and [3'-3'] stands for a 3', 3'- internucleotidic linkage.
  • the synthesis was carried out at a 1 mmol scale on a Millipore 8800 automated synthesizer using 5'-DMT-2'-O-TBDMS-N 2 -tert-butyl- phenoxyacetylguanosine and 5'-DMT-2'-O-TBDMS-N 6 -tert-butylphenoxyacetyl-adenosine 3'-N,N-diisopropyl-(2-cyanoethyl) phosphoramidites and 2'-deoxy-2'-fluoro-5'-DMT-N' t - acetylcytidine and 2'-deoxy-2'-fluoro-5'-DMT-uridine 3'-N,N-diisopropyl-(2-cyanoethyl)- phosphoramidites.
  • Coupling activators DCI and tetrazole were compared by performing two syntheses under identical conditions at a 0.088 mmol scale.
  • the sequence synthesized for this comparison was 5'-[Ta]GGTAATGCAAATCGTGGAACATGACC[3'-3'T]-3' (SEQ ID NO:5) where Ta stands for 5-(hexylaminopropenamide)uridine, A stands for adenosine, T stands for thymine, C stands for cytidine, G stands for guanine, and [3'-3'T] stands for a thymidine, connected to the adjacent 5'-terminal base through a 3',3'-phosphodiester linkage.
  • a Pharmacia 6 mL axial flow column was filled with 1.66 grams of controlled pore glass support, that was derivatized with 5'-dimethoxytritylthymidine at a loading of 53 ⁇ mole/gram.
  • This column was connected to a Pharmacia OligoPilot IITM synthesizer and the instrument was programmed to deliver reagents per cycle as given in Table 8.
  • the resin was detritylated using 168 equivalents of 3% dichloroacetic acid in dichloromethane.
  • the [3'-p3'T] and [Ta] monomers used a double coupling and coupling wash cycle prior to the oxidation step.
  • the detritylation step was not performed in the last cycle. After the synthesis was completed, a sample of the product oligonucleotide was cleaved from the support and deprotected by reaction with concentrated aqueous ammonia at 70°C for 3 hours. Analytical results of the deprotected product are shown in Table 9.
  • the capillary electrophoresis chromatography provided baseline resolution, and is a more accurate technique for determining the purity of the final product than anion exchange chromatography.
  • Anion exchange chromatograms of the deprotected products are shown in Figures 8 and 9 for DCI and tetrazole, respectively.
  • Example 9 Preparation of NX28604, a 35mer oligonucleotide, with DCI. This example demonstrates that activation with DCI proved to be the most efficient way to assemble oligonucleotides containing 2'-deoxy-2'-aminopyrimidines.
  • the 35mer sequence synthesized was a 2'-aminopyrimidine oligonucleotide having the sequence 5'- TsTsTsTsmGmGaUrGaUrGaUrGrGmArArGmAaCrArGaCmGmGmGaUmGmGaUaUaCT sTsTsT-3' (NX28604) (SEQ ID NO: 6) where aU and aC stand for 2'-aminouridine and 2'-aminocytidine, mG and mA stand for 2'-O-methylguanosine and 2'-O-methyladenosine, rG and rA stand for guanosine and adenosine, T stands for thymidine, and s stands for a phosphorothioate internucleotide linkage.
  • This sequence was prepared at a 0.4 mmol scale on a Pharmacia OligoPilot IITM automated synthesizer using polystyrene support loaded at 26 mmol/g with 3'-succinylthymidine.
  • the synthesis cycle is shown in Table 10.
  • the oligonucleotide was cleaved from the resin and the base and phosphate protecting groups were removed by reaction with ammonia saturated ethanol at room temperature for 72 hours. The mixture was filtered and the supernatant was evaporated to dryness. The crude material was then subjected to 1 M TBAF in THF for 24 hours at room temperature. At that point the reaction was quenched with an equal volume of 1 M Tris*HCl (pH 7.5).
  • Figure 10 shows the anion exchange HPLC chromatogram of crude NX28604.
  • the chromatogram was obtained on a 4.6 x 100 mm Gen-Pak Fax column (Waters) at 80°C eluting from 90% trizma ethylenediamine tetraacetate (pH 7.5, buffer A) : 10% trizma ethylenediamine tetraacetate + 1 M NaCl (pH 7.5 , buffer B) to 90% buffer B over 30 minutes at 0.75 mL/min.
  • Example 10 Effect of DCI concentration on coupling efficiency.
  • RNA syntheses were 41-mers with mixed 2'-fluoro and 2'-TBDMS-O with a C 5 amino-modified dT on the 5' end.
  • Figures 11 and 12 reflect the coupling efficiency of these syntheses as calculated from the ratio of the first and last acidified DMT cation fraction raised to the 40 th root (or the number of coupling cycles).
  • the analytical HPLC chromatograms shown in Figures 13 and 14 are anion exchange analyses of the crude product, following deprotection and cleavage from the support (as well as subsequent desilylation of the 2' protecting groups).
  • the two DNA syntheses were 29-mers of mixed deoxy-ACGT with a C5-amino- modified dT on the 5' end.
  • one synthesis used 2 equivalents of phosphoramidite monomers and 1.0 M DCI, and the other used 2 equivalents of phosphoramidite monomers and 0.5 M DCI.
  • the graphs shown in Figures 15 and 16 reflect the coupling efficiency of these syntheses as calculated from acidified DMT
  • Example 11 Coupling a phosphoramidite monomer to an oligonucleotide using 1.13 equivalents of DCI.
  • a coupling and oxidation was performed with a 5'-DMT-thymidine-3'-(2-cyanoethyl)-diisopropylphosphoramidite (DMT-dT-OCEPA) and a 5'-HO-dG-T-3'-3'-T-5 * -OtBDPS trimer (5'-GTT trimer, tert-butyldiphenylsilyl protected) (SEQ ID NO:7), using 4,5-dicyanoimidazole (DCI) as the activator.
  • DMT-dT-OCEPA 5'-DMT-thymidine-3'-(2-cyanoethyl)-diisopropylphosphoramidite
  • DCI 4,5-dicyanoimidazole
  • a 0.2 M solution of the trimer (2:1 ACN:DCM) was prepared in a septum-sealed vial and stored over 4 A sieves over night. To 1.95 mL (0.39 mmols) of this solution, 0.44 mL (0.44 mmols) of a 1.0 M DCI solution was added. To this solution, 1.74 mL (0.47 mmols) of a 0.2 M DMT-T amidite solution in ACN was added.
  • Example 13 Conversion of 3',5'-O-diacetyl-2'-fluoro-2'-deoxyuridine to 2'-fluoro-2'- deoxycytidine by 4,5-dicyanoimidazole.
  • POCl 3 (0.85 mL, 9.1 mmol, 3 eq) was added to an ice cold solution of 4,5- dicyanoimidazole (3.6 g, 30.3 mmol, 10 eq) in dry CH 3 CN (10 mL). After stirring for 15 minutes, triethylamine 95.5 mL, 39.4 mmol, 13 eq) was added and the stirring was continued for another 20 minutes. A solution of the 2'-fluorouridine derivative (1.0 g, 3.03 mmol, 1 eq) in dry CH 3 CN (25 mL) was then added dropwise and the reaction was allowed to warm to room temperature over 2 hours.
  • reaction mixture was then concentrated under reduced pressure, redissolved in CH 2 C1 2 (50 mL), washed with 5 % NaHCO 3 (2 x 20 mL), water and brine and dried to afford 1.68 g of the crude N 4 -4,5-dicyanoimidazolide derivative.
  • Example 14 Conversion of 3',5'-O-diacetyl-2'-fluoro-2'-deoxyuridine to 2'-fluoro-2'- deoxycytidine by triazole.
  • nucleoside number 28 is an inverted orientation (3 '3' linkage) phosphorodiester linkage.
  • G is 2 ' -O-methylguanosine; A is 2 ' -O- methyladenosine; nucleotide numbers 1-5 and 31-35 are bound by a phosphorothioate linkage .

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Abstract

Procédé de couplage de monomères de phosphoramidite avec des nucléophiles, par exemple, le groupe 5'-hydroxyle sur la chaîne d'oligonucléotides en croissance, au moyen d'un activateur de couplage qui est moins acide que tétrazole et au moins aussi nucléophile que tétrazole, et qui permet d'obtenir une efficacité de couplage comparable ou supérieure à celle de tétrazole. Le pKa de cet activateur de couplage est situé entre 5 et 6, de préférence, entre 5 et 5,5, et, dans un mode de réalisation préféré, entre 5,1 et 5,3. Les activateurs de couplage appropriés comprennent 4,5-dicyanoimidazole (DCI), 4-alkylthioimidazole, 2-alkylthioimidazole, 2-nitroimidazole, 4-nitroimidazole, 4,5-dihaloimidazole, 4-haloimidazole, 2-haloimidazole et 5-alkoxytétrazole. DCI est l'activateur de couplage emportant la préférence. Dans un autre mode de réalisation, on peut utiliser une combinaison d'un activateur de couplage acide et un tampon approprié, tel qu'une amine tertiaire. Cette amine tertiaire peut être une amine tertiaire comportant trois groupes alkyle ou un composé hétérocyclique contenant une ou plusieurs amines tertiaires. Cette amine tertiaire est, de préférence, moins nucléophile que DMAP, dont on sait qu'il provoque des réactions secondaires au niveau de l'oxygène en position 6 de guanosines à cause de son caractère nucléophile relativement fort. N-méthylimidazole (NMI) est l'amine tertiaire préférée.
PCT/US1997/015744 1996-10-15 1997-10-08 Activateurs de couplage ameliores pour la synthese d'oligonucleotides WO1998016540A1 (fr)

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003006475A3 (fr) * 2001-07-12 2004-02-26 Santaris Pharma As Elaboration de phosphoramidites d'acide nucleique verrouille
EP1084134A4 (fr) * 1998-06-02 2004-05-26 Isis Pharmaceuticals Inc Activateurs pour la synthese d'oligonucleotides
US6894158B2 (en) 2002-02-22 2005-05-17 Honeywell International Inc. Methods of producing phosphitylated compounds
US6936472B2 (en) * 1999-04-28 2005-08-30 Agilent Technologies, Inc. Method for synthesizing a specific, surface-bound polymer uniformly over an element of a molecular array
US7153954B2 (en) 2001-07-12 2006-12-26 Santaris Pharma A/S Method for preparation of LNA phosphoramidites
US7501505B2 (en) * 2001-07-03 2009-03-10 Avecia Biotechnology, Inc. Activators for oligonucleotide synthesis
WO2019002237A1 (fr) 2017-06-28 2019-01-03 Roche Innovation Center Copenhagen A/S Procédé de couplage et d'oxydation multiples
CN116925050A (zh) * 2023-07-19 2023-10-24 安徽瑞拜药业有限公司 一种核酸合成用活化剂及其制备方法
CN119350418A (zh) * 2024-12-24 2025-01-24 中肽生化有限公司 一种寡核苷酸的合成方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5574146A (en) * 1994-08-30 1996-11-12 Beckman Instruments, Inc. Oligonucleotide synthesis with substituted aryl carboxylic acids as activators

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5574146A (en) * 1994-08-30 1996-11-12 Beckman Instruments, Inc. Oligonucleotide synthesis with substituted aryl carboxylic acids as activators

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1084134A4 (fr) * 1998-06-02 2004-05-26 Isis Pharmaceuticals Inc Activateurs pour la synthese d'oligonucleotides
US6846922B1 (en) 1998-06-02 2005-01-25 Isis Pharmaceuticals, Inc. Activators for oligonucleotide synthesis
US6936472B2 (en) * 1999-04-28 2005-08-30 Agilent Technologies, Inc. Method for synthesizing a specific, surface-bound polymer uniformly over an element of a molecular array
US7501505B2 (en) * 2001-07-03 2009-03-10 Avecia Biotechnology, Inc. Activators for oligonucleotide synthesis
WO2003006475A3 (fr) * 2001-07-12 2004-02-26 Santaris Pharma As Elaboration de phosphoramidites d'acide nucleique verrouille
US7153954B2 (en) 2001-07-12 2006-12-26 Santaris Pharma A/S Method for preparation of LNA phosphoramidites
US6894158B2 (en) 2002-02-22 2005-05-17 Honeywell International Inc. Methods of producing phosphitylated compounds
US7217814B2 (en) 2002-02-22 2007-05-15 Honeywell International Inc. Methods of producing phosphitylated compounds
WO2019002237A1 (fr) 2017-06-28 2019-01-03 Roche Innovation Center Copenhagen A/S Procédé de couplage et d'oxydation multiples
US11814407B2 (en) 2017-06-28 2023-11-14 Roche Innovation Center Copenhagen A/S Multiple coupling and oxidation method
CN116925050A (zh) * 2023-07-19 2023-10-24 安徽瑞拜药业有限公司 一种核酸合成用活化剂及其制备方法
CN119350418A (zh) * 2024-12-24 2025-01-24 中肽生化有限公司 一种寡核苷酸的合成方法

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