WO1996008578A2 - Improved procedure for the solid phase synthesis of oligonucleotides - Google Patents
Improved procedure for the solid phase synthesis of oligonucleotides Download PDFInfo
- Publication number
- WO1996008578A2 WO1996008578A2 PCT/US1995/011302 US9511302W WO9608578A2 WO 1996008578 A2 WO1996008578 A2 WO 1996008578A2 US 9511302 W US9511302 W US 9511302W WO 9608578 A2 WO9608578 A2 WO 9608578A2
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- WIPO (PCT)
- Prior art keywords
- support
- bound
- oligonucleotide
- mononucleoside
- capping
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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
Definitions
- This invention relates to the field of solid phase synthesis of oligonucleotides. Description of the Related Art
- oligonucleotides in most instances, is carried out on a solid-phase matrix, such as controlled pore glass (CPG), using phosphoramidite chemistry. Beaucage and Iyer, Tetrahedron Report 48(12), 2223-2311 (1992).
- CPG controlled pore glass
- One of the first steps in oligonucleotide synthesis is the attachment of the first nucleoside to the solid support.
- oligonucleotide with a greater proportion of N-1 (i.e., oligonucleotide of one fewer nucleotide in length than the desired, or "N,” oligonucleotide) and other "failure sequences".
- N-1 oligonucleotide of one fewer nucleotide in length than the desired, or "N,” oligonucleotide
- Most failure sequences, except N-1 and N-2, can be separated easily from the desired N oligonucleotide by reverse-phase HPLC.
- the crude oligonucleotide is obtained in two steps: the first step involves the cleavage of the oligonucleotide from the support and the second step involves complete deprotection of the phosphate (e.g.
- CPG CPG protocol
- the present invention provides an improved method of solid phase synthesis of oligonucleotides.
- a second capping step is introduced after the first nucleoside has been bound to the solid support. We have unexpectedly found that this second capping step decreases the amount of N-1 oligonucleotide detected after synthesis is complete.
- the initial loading density of the first nucleoside can be substantially greater than previously thought.
- the prior art theorizes that growing oligonucleotides on highly loaded supports prevents diffusion of reactants to the growing chain ends, resulting in a greater number of failure sequences as compared to supports with lower loads.
- high loading densities when used in combination with the second capping step alone or in addition to the SUP protocol, results in a final product having a smaller N-1 content than when lower loading levels are used.
- Figure 1 displays a schematic representation of the generalized method of solid phase oligonucleotide synthesis.
- Figure 2 displays a schematic representation of solid support functionalization, loading, and capping.
- Figures 3A-B display ion-exchange profiles of dT 5 synthesized on a T-CPG column
- Figure 4 displays a histogram of the percentage N-1 content in a dT 5 homopolymer purified with the SUP protocol as a function of initial nucleoside loading and second capping of the CPG.
- Figure 5 displays a histogram of the percentage N-1 content in a dT 5 homopolymer purified with the CAP protocol as a function of initial nucleoside loading and second capping of the CPG.
- Figure 6 displays the capillary electrophoretic profile of a crude phosphorothioate oligonucleotide (25-mer) synthesized on a 1 mmol scale and worked up using the SUP protocol.
- Figure 7 displays the chemistry of adding the first nucleoside to the CPG.
- Figure 8 displays the chemistry of trityl exchange.
- Figures 9A-B display the capillary electrophoretic profile of two 25-mer phosphorothioate oligonucleotides using the SUP protocol with (top) and without
- an "N" oligonucleotide is the synthetic oligonucleotide of the desired length.
- An "N-1" oligonucleotide is 1 less nucleotide in length as compared to the N oligonucleotide.
- an "N-2" oligonucleotide is 2 less nucleotides in length as compared to the N oligonucleotide.
- fouling sequence generally refers to oligonucleotides synthesized on the solid support but having fewer nucleosides in length than the oligonucleotides of desired length.
- load refers to the amount of first nucleoside attached to the solid support, expressed in units of ( ⁇ mol nucleoside)/(g support material), or ⁇ mol/g.
- Figure 7 displays two illustrative methods by which a solid support may be loaded.
- N is an integer between 3 and 150: a) a first nucleoside is chemically linked to a solid support having reactive sites, forming a support-bound mononucleoside and a mononucleoside- bound support with reactive sites, b) the mononucleoside-bound support with reactive sites is contacted with a capping solution, forming a capped mononucleoside-bound support, c) a second nucleoside is chemically linked to the support-bound mononucleoside, forming a nascent oligonucleotide-bound support and a support-bound nascent oligonucleotide, d) the nascent oligonucleotide-bound support is contacted with a capping solution, e) another nucleoside is chemically linked to the support-bound nascent oligonucleotide, forming a nascent oligonucleotide-bound support and
- the term "reactive site” refers to terminal amino and hydroxyl groups capable of forming a chemical bond with incoming nucleosides during oligonucleotide synthesis.
- the term “nascent oligonucleotide” refers to any oligonucleotide of 2 or more nucleotides in length that must be subjected to further treatment to obtain the desired oligonucleotide.
- the "capping solution” is one or more solutions that combined comprise a reactive mixture capable of linking a blocking/protecting group to unreacted reactive sites. Contacting the capping solution with the support results in some or all of the unreacted reactive sites being chemically linked to a blocking/protecting group, thereby becoming “capped.”
- the general protocol for loading nucleoside and capping the non-functionalized hydroxyl groups is schematically displayed in Figure 2.
- the solid support initially has terminal hydroxyl groups that may be functionalized with a long chain amino-alkyl- triethoxy silane. Not all terminal hydroxyl groups become functionalized, however.
- the DMT protected nucleoside is attached to the solid support via the amino moiety of a long chain alkyl amine, resulting in a support-bound nucleoside and, conversely, a nucleoside-bound support. This is followed by capping any reactive sites on the solid support. Capping prevents subsequent nucleosides from attaching directly to the support rather than the growing oligonucleotide chain. Often the capped, nucleoside-bound support is stored for future use.
- a method of capping a mononucleoside-bound support comprising consecutively contacting the mononucleoside- bound support with same or different capping solutions two or more times.
- two cappings i.e., contacting the mononucleoside-bound support with a capping solution
- the term "consecutively" means that the mononucleoside-bound support is subjected to temporally separated capping procedures.
- a second aspect of this embodiment we provide an improved method of synthesizing an oligonucleotide, the improvement comprising contacting a capped mononucleoside-bound support with a capping solution before a second nucleoside is added.
- a second capping step is performed after the initial loading of nucleoside, but before addition of the second nucleoside.
- This second capping step can be by any means that renders the free hydroxyl and amino groups unreactive to incoming nucleoside.
- a number of such means are known in the art and may be used in the present invention. Kg., Methods in Molecular Biology, Vol 20: Protocols for Oligonucleotides and Analogs (S. Agrawal, Ed., Humana Press, 1993). Any non-acid labile capping group can be used in the present invention.
- the capping is accomplished by adding to the loaded support equal volumes of Cap A and Cap B reagents (acetic anhydride and N-methylimidazole, respectively, or acetic anhydride and DMAP, respectively), shaking or stirring for about two hours followed by filtering and washing with dichloromethane.
- the second capping step may use the same materials and protocol as the first step or use different materials and a different protocol.
- the second capping step may immediately follow the first capping step, or it may be employed some time later, such as after storage of the loaded support. In the most preferred embodiment, the second capping step is carried out just before the remaining nucleotides are to be added.
- a milder cleavage step is employed in combination with the second capping step to remove the full length oligonucleotide from the solid support.
- Any method of cleavage that detaches the full length oligonucleotide from the solid support without significantly or substantially altering one's ability to obtain the desired full length oligonucleotide can be employed.
- the cleavage step will preferably preferentially cleave the full length N oligonucleotide from the solid support without cleaving from the support N-1 oligonucleotides and other failure sequences.
- the cleavage step of the present invention will comprise any suitable method that is able to cleave preferentially the desired N oligonucleotide from the support-bound succinic acid without substantially effecting cleavage of N-1 oligonucleotides linked to the support via phosphoramidate linkages.
- N-1 failure sequences arise is that a trityl exchange reaction occurs.
- Figure 8 It is conceivable that the acetylated groups in the capped CPG matrix could participate in trityl exchange reaction through neighboring group participation, resulting in a tritylated amino group and a capped nucleoside. Detritylation of the amino group could then result in the synthesis of N-1 sequences.
- This aspect of the present invention comprises using what has herein been called the "SUP" protocol for cleavage of oligonucleotides from the solid support and deprotection of the base and sugar phosphate moieties.
- the SUP protocol comprises contacting the support-bound oligonucleotides with 5-30% ammomum hydroxide at ambient temperature for 30 minutes to 2 hours, followed by heating the supernatant in a closed tube for 8-16 hours at 55°C. Heating the supernatant is for the purpose of deprotecting the base and sugar moieties and does not affect the concentration of N-1 oligonucleotides obtained.
- Cleavage is accomplished by contacting the nascent oligonucleotide-bound support with a weak base.
- the oligonucleotide is cleaved from the support with from about 10% to about 30% (saturated) ammonium hydroxide for about 30 minutes to 2 hours. In the most preferred embodiment, about 28-30% ammonium hydroxide is used for about 1 to 2 hours. It is a routine matter to vary the conditions under which the N oligonucleotide is cleaved from the solid support to adjust yield and purity of the desired N oligonucleotide to an acceptable level. Harsher conditions and treatment for longer times will increase yield and decrease purity; less harsh conditions will decrease yield, but increase purity.
- the apparent yield of N oligonucleotide is increased. But, as mentioned before, this will also increase contamination from N-1 and other failure sequences.
- using lower temperatures and lower concentrations of ammonium hydroxide will result in lesser contamination by N-1 and other failure sequences as well as lesser yield.
- the period of heating the supernatant is for the purpose of cleaving the base and sugar phosphate protecting groups.
- decreased contamination from N-1, N-2, and other failure sequences is obtained by increasing the initial nucleoside loading density.
- loading refers to chemically linking the first nucleoside to the solid support.
- the prior art teaches that too high levels of loading are undesirable because the growing oligonucleotide chains prevent diffusion of reactants, thereby resulting in more failure sequences. Agrawal, Protocol in Molecular Biology, supra. We have unexpectedly found, however, that when used in combination with the second capping step and with or without mild cleavage conditions, higher loading densities actually result in lesser contamination by N-1 failure sequences.
- Initial loading densities are typically in the range of about 50 to 70 ⁇ mol/g. We have unexpectedly found that an initial loading density of 100 ⁇ mol/g resulted in a lesser amount of N-1 oligonucleotide content than oligonucleotides synthesized on supports having loading densities in the range of about 50 ⁇ mol/g.
- oligonucleotide capable of being synthesized on a solid support.
- oligonucleotides for which the present methods are suitable are RNA, 2'-0-methyl RNA, DNA or RNA/DNA hybrids, each of which can be unmodified or modified in any number of positions, including the sugar phosphate backbound and/or the nucleoside base.
- oligonucleotides examples include, but are not limited to, phosphodiesters, phosphorothioates, phosphorodithioates, phosphoroamidates, methyl- or other alkyl-phosphonates, carbonates, carbamates and oligonucleotides having modified bases. Modifications may occur in any number of nucleotides and may occur alone or in any combination.
- Oligonucleotide synthesis may be accomplished in any manner consistent with application of the presently disclosed methods. E.g., Methods in Molecular Biology, Vol. 20, supra; Uhlmann and Peyman, Chem Rev. 90, 543 (1990); Oligonucleotides and Analogues: A Practical Approach (REckstein, Ed., 1991).
- any unmodified base e.g., A, G, C, T, and U
- any suitable linking group that links the first nucleoside to the denvatized solid support via an ester bond may be used in the present invention.
- the terms "derivatized” and “functionalized” are used interchangeably and, when used in relation to a solid support, mean that the support has reactive hydroxyl and/or amino moieties suitable for oligonucleotide synthesis.
- a number of such supports are known in the art. Among these are low cross-linking polystyrene, polyamide, polyamide bonded silica gel, cellulose, silica gel, controlled pore glass, polystyrene/PEG “tentacle” copolymer, and high cross- linking polystyrene. See Methods of Molecular Biology, Vol 20, supra, and references cited therein.
- the solid support is capable of being used in phosphoramidate synthesis of oligonucleotides.
- the solid support is controlled pore glass (CPG).
- the CPG was treated with about 28-30% ammonia (1.5 ml for 1 ⁇ mol and 5 ml for 10 ⁇ mol scale) at ambient temperature for 30 to 120 minutes. The supernatant was removed and heated at 50-55°C for 8-10 hours.
- Ion exchange chromatography was carried out on a Waters 660 E high pressure liquid chromatograph (HPLC) equipped with a Waters 996 photodiode Array Detector.
- HPLC high pressure liquid chromatograph
- the sample was dissolved in 0.1 M ammonium acetate and analyzed on a GEN-PAK FAX column (4.6 X 100 mm) at 45°C using a linear gradient of 100% -> 50% buffer A over 90 minutes at a flow rate of 0.5 ml/minute.
- the following buffers were used:
- Buffer A 25 mM Tris HC1; pH 8.5, 10% CH 3 CN,
- Buffer B 25 mM Tris HC1, 1 M LiCl; pH 8.5, 10% CH 3 CN.
- Figure 3 shows the typical HPLC profile of a crude homopolymer dT 5 as analyzed on a
- Figure 4 displays the N-1 content of the 5-mer (depicted as a percentage of the area of the N peak) expressed as a function of initial loading of the nucleoside on the
- the N-1 content is less in oligonucleotides which were prepared using the SUP protocol with second capping (left column of each loading pair) as compared to those prepared using the SUP protocol without second capping (right column of each loading pair).
- Example 1 it is seen that second capping decreases the N-1 content irrespective of whether the CPG or SUP protocol is used. Furthermore, on comparison of these results with those from Example 1, it is seen that the combination of second capping and the
- SUP protocol yields less N-1 than does second capping with the CPG protocol.
- Figs. 3 - 5 are typical examples of the experimental data used to evaluate the Example 4. It was observed that the difference in N-1 content with and without second capping is 26% for 30 minutes of ammonia treatment and 20% for 2 hours of ammoma treatment. By comparison, the difference in N-2 content with and without second capping is 23% for 30 minutes of ammonia treatment and 14% for 2 hours of ammonia treatment. Thus, longer treatment with ammoma releases more N-1 and other failure sequences.
- the reduction in the amount of failure sequences was also tested on the 1 mmol scale with two 25-mer phosphorothioate oligonucleotides using the SUP protocol with and without second capping.
- the same experimental protocols as previously described were used.
- the capillaiy electrophoresis profiles are displayed in Figure 9A-B.
- the top chromatograms in Figures 9A and 9B display the capillary electrophoresis analysis of the each 25-mer phosphorothioate synthesized with second capping.
- the bottom chromatograms show the capillary electrophoresis analysis of the same 25-mers synthesized without second capping.
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Abstract
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Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU35469/95A AU3546995A (en) | 1994-09-06 | 1995-09-05 | Improved procedure for the solid phase synthesis of oligonucleotides |
EP95932418A EP0779892A2 (en) | 1994-09-06 | 1995-09-05 | Improved procedure for the solid phase synthesis of oligonucleotides |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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US30133794A | 1994-09-06 | 1994-09-06 | |
US08/301,337 | 1994-09-06 |
Publications (2)
Publication Number | Publication Date |
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WO1996008578A2 true WO1996008578A2 (en) | 1996-03-21 |
WO1996008578A3 WO1996008578A3 (en) | 1996-05-30 |
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Application Number | Title | Priority Date | Filing Date |
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PCT/US1995/011302 WO1996008578A2 (en) | 1994-09-06 | 1995-09-05 | Improved procedure for the solid phase synthesis of oligonucleotides |
Country Status (4)
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EP (1) | EP0779892A2 (en) |
AU (1) | AU3546995A (en) |
CA (1) | CA2199246A1 (en) |
WO (1) | WO1996008578A2 (en) |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0040099A1 (en) * | 1980-05-14 | 1981-11-18 | ens BIO LOGICALS INC. | Polynucleotide synthesis |
US4458066A (en) * | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
-
1995
- 1995-09-05 WO PCT/US1995/011302 patent/WO1996008578A2/en not_active Application Discontinuation
- 1995-09-05 CA CA 2199246 patent/CA2199246A1/en not_active Withdrawn
- 1995-09-05 AU AU35469/95A patent/AU3546995A/en not_active Abandoned
- 1995-09-05 EP EP95932418A patent/EP0779892A2/en not_active Withdrawn
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4458066A (en) * | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
EP0040099A1 (en) * | 1980-05-14 | 1981-11-18 | ens BIO LOGICALS INC. | Polynucleotide synthesis |
Non-Patent Citations (2)
Title |
---|
NUCLEIC ACIDS RES., vol. 18, 1990 pages 3813-21, M.J. DAMHA ET AL. 'An improved procedure for derivatization of controlled-pore glass beads for solid-phase oligonucleotide synthesis' * |
NUCLEOSIDES, NUCLEOTIDES, vol. 14, 1995 pages 1349-57, R.P. IYER AND S. AGRAWAL 'Improved procedure for the reduction of N-1 content in synthetic oligonucleotides' * |
Also Published As
Publication number | Publication date |
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AU3546995A (en) | 1996-03-29 |
EP0779892A2 (en) | 1997-06-25 |
WO1996008578A3 (en) | 1996-05-30 |
CA2199246A1 (en) | 1996-03-21 |
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