Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D285/00—Heterocyclic compounds containing rings having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by groups C07D275/00 - C07D283/00
  • C07D285/15—Six-membered rings
  • C07D285/16—Thiadiazines; Hydrogenated thiadiazines
• • 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 invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleoside linkages of oligonucleotides.
• Oligonucleotides have become indispensable tools in modern molecular biology, being used in a wide variety of techniques, ranging from diagnostic probing methods to PCR to antisense inhibition of gene expression. Oligonucleotide phosphorothioates are of considerable interest in nucleic acid research and are among the analogues tested as oligonucleotide therapeutics. Oligonucleotides phosphorothioates contain internucleotide linkages in which one of the nonbridging oxygen atoms of the phosphate group is replaced by a sulfur atom. This widespread use of oligonucleotides has led to an increasing demand for rapid, inexpensive, and efficient methods for synthesizing oligonucleotides.
• oligonucleotides for antisense and diagnostic applications can now be routinely accomplished. See e.g., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs, pp. 165-189 (S. Agrawal, ed., Humana Press, 1993); Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, ed., 1991); and Uhlmann and Peyman, Chemical Reviews, 90: 543-584 (1990); Agrawal and Iyer, Curr. Op. in Biotech.
• Patent No. 5,149,798 (1992) discloses optimized synthesis of oligonucleotides by the H-phosphonate approach.
• Solid phase synthesis of oligonucleotides by each of the foregoing processes involves the same generalized protocol. Briefly, this approach comprises anchoring the 3'-most nucleoside to a solid support functionalized with amino and /or hydroxyl moieties and subsequently adding the additional nucleosides in stepwise fashion. Internucleoside linkages are formed between the 3' functional group of the incoming nucleoside and the 5' hydroxyl group of the 5'-most nucleoside of the nascent, support-bound oligonucleotide.
• the internucleoside linkage is a phosphite linkage
• H-phosphonate approach it is an H-phosphonate internucleoside linkage.
• the phosphite or H-phosphonate linkage must be oxidized by an appropriate sulfur transfer reagent.
• this sulfurization is carried out on all of the H-phosphonate linkages in a single step following the completion of oligonucleotide chain assembly, typically using elemental sulfur in a mixed solvent, such as CS 2 /pyridine.
• the phosphoramidite approach allows stepwise sulfurization to take place after each coupling, thereby providing the capability to control the state of each linkage in a site-specific manner.
• the phosphoramidite approach appears to offer advantages. Refinement of methodologies is still required, however, particularly when making a transition to large-scale synthesis (10 ⁇ mol to 1 mmol and higher). See Padmapriya et al., Antisense Res. Deo. 4: 185 (1994).
• Padmapriya et al., Antisense Res. Deo. 4: 185 (1994) Several modifications of the standard phosphoramidite processes have already been reported to facilitate the synthesis (Padmapriya et al, supra; Ravikumar et al.,
• Beaucage reagent has been widely used, however, its synthesis and stability are not optimal.
• the by-product formed by Beaucage reagent during sulfurization 3H-2,l-benzoxanthiolan-3-one-l-oxide, is a potential oxidizing agent that can lead to undesired phosphodiester linkages under certain conditions. Therefore, its application in large-scale synthesis of oligonucleotide phosphorothioates may not be particularly suitable.
• sulfur transfer reagents should be inexpensive to make, stable in storage, and highly efficient
• the invention provides new processes for sulfur transfer reagents in sulfurizing oligonucleotides.
• the processes according to the invention yield inexpensive to make, stable in storage, and highly efficient in sulfurization.
• the invention provides novel sulfur transfer reagents having the structure according to Formula I:
• X is R 1 , NR 2 R 3 , NR COR 5 , SR 6 , OR 7 ; each R l , R 2 , R 3 , R 4 , and R 5 , is indeperHently H or an alkyl or aromatic organic group; and each R 6 and R 7 is independently an alkyl or aromatic organic group.
• the invention relates to novel sulfur transfer reagents having the general structure according to Formula II: Formula II
• R s is H or an organic group
• R 9 is an organic group
• Another aspect of the invention provides for novel sulfur transfer reagents having the general structure according to Formula HI:
• R 10 and R u are each independently H or an organic group.
• the invention provides novel processes for adding a sulfur group to an internucleoside linkage of an oligonucleotide using the novel sulfur transfer reagents according to the, invention.
• the novel processes according to the invention comprise contacting an oligonucleotide having at least one sulfurizable internucleoside linkage'with a novel sulfur transfer reagent according to the invention for a time sufficient for sulfurization of the sulfurizable internucleoside linkage(s) to occur.
• Figure 1 shows the HPLC of the standard DMT protected T-T phosphothioate dimer.
• Figure 2 shows the HPLC of DMT protected T-T phosphothioate dimer synthesized with compound 1 as a sulfur transfer reagent
• Figure 2A represents sulfurization conditions with 4 equiv, 1 min.
• Figure 2B represents sulfurization conditions with 4 equiv, 5 min.
• Figure 2C represents sulfurization conditions with 12 equiv, 1 min.
• Figure 2D represents sulfurization conditions with 12 equiv, 5 min.
• Figure 3 is the HPLC of DMT protected T-T phosphothioate dimer prepared with compound 2 as a sulfur transfer reagent
• Figure 4 is ' the HPLC of DMT protected T-T phosphothioate dimer prepared using compound 3 as a sulfur transfer reagent.
• Figure 5 is the HPLC of DMT protected T-T phosphothioate dimer synthesized with compound 4 as a sulfur transfer reagent.
• Figure 6 shows the HPLC of SEQ ID NO:l synthesized with compound 1 as a sulfur transfer reagent.
• Figure 6A represents 1 min of sulfurization in each synthetic cycle.
• Figure 6B represents 2 min of sulfurization in each synthetic cycle.
• Figure 7 represents the CE of SEQ ID NO:l synthesized with compound
• Figure 8 represents the CE of SEQ ID NO: 1 synthesized with compound
• the invention relates to the chemical synthesis of oligonucleotides and to chemical entities useful in such synthesis. More particularly, the invention relates to sulfurization of the internucleoside linkages of oligonucleotides.
• the invention provides new sulfur transfer reagents and processes for their use in sulfurizing oligonucleotides.
• the sulfur transfer reagents according to the invention are inexpensive to make, stable in storage, and highly efficient in sulfurization.
• the invention provides novel sulfur transfer reagents having the structure according to Formula L
• X is R 1 , NR 2 R 3 , NR*COR 5 , SR 6 , OR 7 ; each R 1 , R 2 , R 3 , R 4 , and R 5 , is independently H or an alkyl or aromatic organic group; and each R* and R 7 is independently an alkyl or aromatic organic group.
• the sulfur transfer reagent having Formula I is 3-amino-1 ⁇ -dithiazole-5-thione (1), xanthane hydride (2), 3-N-acetyl-3- amino-l ⁇ 4-dithiazole-5-thione (3), 3-N-trimethylacetyl-3-amino-l,2,4-c thiazole- 5-thione (4), 3-N-benzoyl-3-amino-l,2,4-dithiazole-5-thione (5), or 3-N- benzenesulfonyl-3-amino-l ⁇ ,4-dithiazole-5-thione (6).
• the invention relates to novel sulfur transfer reagents having the general structure according to Formula II:
• R 8 is H or an organic group
• R 9 is an organic group
• Another aspect of the invention provides for novel sulfur transfer reagents having the general structure according to Formula IE:
• each R 10 and R n is independently H or an organic group. TrTa preferred embodiment, the sulfur transfer reagent having Formula
• HI is 3-ammo-l,2,4- ⁇ UtWazole-5-thione (l), xanthane hydride (2), 3-N-acetyl-3- amino-l ⁇ 4-dithiazole-5-thione (3), 3-N-trimethylacetyl-3-amino-l ⁇ ,4-dithiazole- 5-thione (4), 3-N-benzoyl-3-ammo-lA4-dithiazole-5-thione (5), or 3-N- benzenes ⁇ lfonyl-3-amino-lA4-dithiazole-5-thione (6).
• the compounds 1 and 2 3-ammo-l,2,4-ditt ⁇ azole-5-thione and xanthane hydride respectively, are commercially available from several chemical companies, including Lancaster, Crescent Chemicals, Maybridge, and TCI America.
• the purity of compounds 1 and 2 obtained is higher than 98%, and both compoimds can be directly used in oligonucleotide syntiiesis without any additional purification.
• These compounds have been used in the rubber industry as vulcanization reagents. However, these compounds, to our best knowledge, have not been applied in the synthesis of oligonucleotides.
• Derivatives of compound 1 may be easily synthesized. Starting from compound 1, 3-N-acetyl-3-a ⁇ mr 1 ⁇ -dithiazole-5-thione (3), 3-N- trimethylacetyl-3-amino-l ⁇ ,4-dithiazole-5-thione (4), 3-N-benzoyl-3-amino-l,2,4- dithiazole-5-thione (5), and 3-N-benzenesutfonyl-3-a ⁇ rtino-l,2,4-dithiazole-5- thione (6) were conveniently prepared by solid-phase phosphoramidite approach in a better than 70% yield according to Scheme 1.
• the reaction conditions included acetic anhydride/pyridine for 3 (96%); trimethyl acetic anhydride/EtjN for 4 (70%); benzoyl chloride/Et 3 N for 5 (70%); and benzene sulfonyl chloride/Et 3 N for 6 (76%).
• the final products can be purified by a simple precipitation.
• Parent compounds 1 and 2 can be dissolved in CH 3 CN to form a 0.01 M solution. Their solubility can be increased with addition of pyridine [0.02 M in pyridine-CH 3 CN (1:9); 0.5 M in neat pyridine]. Derivatization of the parent compounds also can improve the solubility in CH 3 CN (>0.02 M), e.g., compound 4 has a solubility better than 1 M in neat CH 3 CN.
• oligonucleotide phosphorothioates can be efficiently prepared by solid-phase phosphoramidite approach using 3-amino-l,2,4-dithiazole-5-thione (1), xanthane hydride (2), and their corresponding derivatives.
• Compounds 1 and 2 are commercially available and relatively inexpensive compared with currently used sulfurizing reagents, such as Beaucage reagent and EDITH. Due to its high efficiency and low cost, compounds 1 and 2, and their appropriately modified analogues can conclude . be considered as an advantageous alternative to Beaucage reagent, especially m large-scale preparation of oligonucleotide phosphorothioates.
• the invention provides novel processes for adding a sulfur group to an internucleoside linkage of an oligonucleotide using the novel sulfur transfer reagents according to the invention.
• the novel processes according to the invention comprise contacting an oligonucleotide having at least one sulfurizable internucleoside linkage with a novel sulfur transfer reagent according to the invention for a time sufficient for sulfurization of the sulfurizable internucleoside linkage(s) to occur.
• Each sulfurizable internucleoside linkage preferably contains a phosphorous (HI) atom.
• the sulfurizable internucleoside linkage is a phosphite, thiophosphite, H- phosphonate, thio-H-phosphonate, or alkylphosphite (especially methylphosphite) internucleoside linkage.
• the sulfurization reaction would be allowed to proceed to a sulfur transfer efficiency greater than that expected for the prior art compounds, as measured by 31 P-NMR. In typical synthesis conditions such efficiency is achieved within from about 1 to about 5 minutes reaction time with the novel transfer reagents. Typically, the reaction takes place in pyridine, THF, or mixtures thereof.
• oligonucleotide includes linear polymers of two or more natural deoxyribonucleotide, ribonucleotide, or 2'-0-substituted ribonucleotide monomers, or any combination thereof.
• the term oligonucleotide also encompasses such polymers having chemically modified bases or sugars and/or non-nucleosidic analogs linked by phosphodiester bonds or analogs thereof ranging in size from a few monomeric units, e.g., 2-3, to several hundred monomeric units and/or having additional substituents, including without limitation lipophilic groups, intercalating agents, diamines and adamantane.
• oligonudeotides may also indude non-natural oligomers having phosphorous-containing internudeoside linkages whose phosphorous (HI) precursors are amenable to sulfurization, (See, e.g., Takeshita et al., /. Biol.
• the term "2'-0-substituted" means substitution of the 2' position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, e.g., with halogen, hydroxy, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxy, or amino groups; or with a hydroxy, an amino or a halogen group, but not with a 2'-H group.
• Such oligonudeotides may indude any of the internucleoside linkages which are known in the art, induding without limitation phosphorothioate, phosphorodithioate, alkylphosphonate (espedally methylphosphonate), phosphoramidate, amide (PNA), carbamate, and alkylphosphonothioate linkages.
• the oligonudeotide is bound to a solid support, but such oligonudeotides may be sulfurized in solution phase as well.
• dT-CPG, 5'-DMT-thymidine cyanoethyl phosphoramidite, Cap A, Cap B, activator, oxidizing and deblock solutions were purchased from PerSeptive Biosystems, (Framingham, MA).
• Beaucage reagent 3 H-l,2-benzocUthiol-3-one-l,l-dioxide was purchased from R. I.
• NMR spectra (121.65 MHz) and l H NMR spectra (300 MHz) were recorded on a Varian UNITY 300 (the chemical shift was correlated to 85% H 3 P0 4 and tetramethylsilane, respectively).
• Dinudeotide and oligonudeotide syntheses were performed on an automated nudeic acid synthesizer (8909 ExpediteTM, Millipore, Bedford, MA).
• Reverse phase HPLC was performed on a Waters 600E pump with a Waters 440 absorbance detector, Waters 746 integrator, and a Nova-Pak C18 (3.9 X 150 mm) column, using a linear gradient of CH 3 CN- aqueous NH ⁇ OAc (0.1 M) (4:1) and 0.1 M aqueous NI- ⁇ OAc from 1:9 to 3:2 over a period of 40 min, flow rate 2 mL/min, detection at 260 ran.
• Capillary electrophoresis was performed on a Beckman P/ACE System 5010. Samples were injected for 5 seconds and analyzed for 40 min.
• the compound 3-am o-l,2,4-dithiazole-5-thione (1) was commerdally available at 98% purity from several chemical companies, induding Lancaster, Crescent Chemicals, and Maybridge, and can be directly used without further purification.
• the compound xanthane hydride (2) was commerdally available from
• Neat trimethylacetic anhydride (3.20 mL, 16 mmol) was added to the suspension of 3-ammo-l,24-ctithiazole-5-thione (compound 1, 2.0 g, 13 mmol) in the mixture of EtjN-CBjClz (1:5, 30 mL) in one portion. A dear yellow solution was obtained in 10 min, and stirred at 25°C for 2 h, and then concentrated under reduced pressure.
• Dimer was assembled on a PerSeptive DNA/RNA (Millipore 8909 ExpediteTM, Millipore, Bedford, MA) on a 0.1 ⁇ mol scale using the synthesis protocol "THIO 1 ⁇ mol" (Expedite software version 1.01), starting from DMT-T- Su-CPG (500 A, loading: 60 ⁇ mol/g).
• Sulfurizing reagents were prepared in either CHjCN or pyridine-CH 3 CN (1:9) at a concentration of 0.02 M unless specified. Sulfurization was carried out using 4 equiv or 12 equiv of sulfurizing reagents for 1 or 5 min reaction, respectively.
• the chain assembly was carried out using the same protocol as described in the dinudeotide phosphorothioate synthesis.
• Sulfurizing reagent (compound 1) was prepared at a concentration of 0.02 M in pyridine-CHjCN (1:9), and sulfurization was carried out using 12 equiv for 1 or 2 min. respectively. Ion exchange HPLC and CE analyses were performed to evaluate the synthesis of this oligomer.

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