JP5365005B2 - Neutral and cleavable resin for nucleic acid synthesis - Google Patents

Description

  The present invention relates to solid phase synthesis of nucleic acids including DNA and RNA.

  Nucleic acid solid phase (automated) synthesis has been performed for over 20 years, and automatic synthesizers were also on sale at that time. Even now, nucleic acid raw materials (amidides and resins) that can obtain nucleic acids under mild conditions for modification of antisense DNA, siRNA, and fluorescent labels of nucleic acids are being improved.

Conventional linkers are often undesirable because they cause degradation or the like depending on the type of nucleic acid used. As a linker capable of recovering a nucleic acid from a solid phase resin capable of alleviating this problem, the structure shown in FIG. 1 has already been reported (for example, see Non-Patent Document 1). However, in this case, deprotection of the allyl carbamate group with Pd is necessary in advance, which is not suitable for practical use. In the present specification, CPG means a resin (specifically, Controlled Pore Glass).
Nucleic Acids Research, 1996, 24, p. 2793

  An object of the present invention is to provide a resin for nucleic acid synthesis using a novel linker. Still other objects and advantages of the present invention will become apparent from the following description. The nucleic acid in this case includes natural products and synthetic products, and further includes natural products and modified products.

  According to one embodiment of the present invention, there is provided a resin for nucleic acid synthesis in which a nucleic acid is bound to a resin via a linker, and the linker can be cleaved under neutral conditions.

  According to the embodiment of the present invention, a resin for nucleic acid synthesis using a linker that can be cleaved under conditions that do not cause degradation of nucleic acids or cause degradation or the like is obtained.

The linker structure preferably has a —SS— (CH ₂ ) _n —COO— bond, and a nucleic acid is preferably bound to the COO— side. The S—S bond can be easily cleaved and the COO-side bonded nucleic acid can be released by cyclization.

In other words, regardless of the structure of the linker bonded to the nucleic acid and the resin, the structure after cleavage of the linker has an HS— (CH ₂ ) _n —COO— bond, and the nucleic acid is present on the COO− side. It can be said that the thing which produces the intermediate body which has couple | bonded is preferable.

  In addition, a nucleic acid synthesis resin having a linker having such a structure has not been known so far.

  For the cleavage of the linker, it is preferable to use a reducing agent such as dithiothreitol or TCEP {Tris (2-carboxyethyl) phosphine}. When these reducing agents are used, the reduction can be performed under neutral conditions.

  According to the present invention, a resin for nucleic acid synthesis using a novel linker can be obtained. In addition, a resin for nucleic acid synthesis using a linker that can be cleaved under conditions that do not cause degradation of nucleic acids or cause little degradation or the like can be obtained.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings, formulas, examples and the like. In addition, these figures, formulas, examples, etc., and explanations illustrate the present invention, and do not limit the scope of the present invention. It goes without saying that other embodiments may belong to the category of the present invention as long as they match the gist of the present invention.

A resin for nucleic acid synthesis in which a nucleic acid is bonded to the resin through a novel linker shown below has been developed. This resin can cleave the linker under neutral conditions.
No linker is known so far that is cleavable under neutral conditions.

  A nucleic acid synthesis resin in which a nucleic acid is bound to the resin via this “linker that can be cleaved under neutral conditions” causes degradation or the like even if the nucleic acid is weak in basicity or acidity, such as modified DNA. Is less likely to occur or decompose. For this reason, automatic composition becomes possible. In this case, the “neutral conditions” are not limited to merely indicating a pH of 7.0, but may vary depending on the basicity or acidity of the nucleic acid used. Can think. For example, it may be in the range of pH 6.0 to 8.0.

  In the above, there is no restriction | limiting in particular in the kind of resin and nucleic acid which a linker couple | bonds, It can select suitably from a well-known thing. As described above, it is preferable to use a nucleic acid that is acidic or basic and easily decomposes.

The new linker that has been developed is characterized by having a —SS— (CH ₂ ) _n —COO— linkage, where n is 3 or 4. Nucleic acids bind to the COO- side. Therefore, the resin binds to the -SS side.

  There is no particular limitation on the mode of binding between the linker and the nucleic acid other than those described above, and known binding can be used. The easiest is the binding by the reaction between the hydroxyl group of the nucleic acid, the nucleic acid and the COOH group of the linker precursor (ie, the linker before binding to the nucleic acid).

  There is no particular limitation on the mode of bonding between the linker and the resin, other than the above, and a known bond can be used. For the convenience of synthesis, a mode in which a methylene group, an amino group, an ester bond and the like are further arranged on the —S—S bond of the linker, and the resin and the ester bond are bonded at the terminal can be exemplified.

When using a nucleic acid synthesis resin (where n is 3 or 4) in which the linker has a —S—S— (CH ₂ ) _n —COO— bond, and a nucleic acid is bound to the COO— side. The elimination of the moiety containing the nucleic acid from the resin due to the cleavage of the linker is considered to be caused by the cleavage of the bond between SS. Any means may be used for this cleavage, but a reducing agent can be used, which is preferable. This cleavage may be used under basic conditions or acidic conditions other than neutral conditions, but if it can be cleaved under neutral conditions, the condition “cleave the linker under neutral conditions” is satisfied. Therefore, even a nucleic acid that is weak in basicity or acidity, such as modified DNA, does not cause degradation or the like, and is less likely to cause degradation.

It is possible to obtain an intermediate having an HS— (CH ₂ ) _n —COO— bond and a nucleic acid bonded to the COO— side by cleavage of the bond between S—S. It was confirmed by obtaining a stable substance in which S of HS was bonded to maleimide by the reaction with. From this, the above-mentioned novel resin for nucleic acid synthesis has an HS— (CH ₂ ) _n —COO— bond when the linker is cleaved, and an intermediate in which the nucleic acid is bound to the COO− side. It is also possible to grasp as a resin for nucleic acid synthesis that yields (where n is 3 or 4). Also in this case, cutting is possible under neutral conditions. If the nucleic acid is cleaved under neutral conditions, even a nucleic acid that is weak in basicity or acidity, such as modified DNA, will not cause degradation or decrease.

It is considered that the nucleic acid is cleaved when the obtained intermediate having an HS— (CH ₂ ) _n —COO— bond and having the nucleic acid bonded to the COO— side cyclizes. n is 3 or 4 because a 5-membered or 6-membered ring is stable as a structure. There may be a case where n is 3 and n is 4.

  In the case where the COO bond is directly linked to the hydroxyl group of the nucleic acid, the developed linker was linked to the 3 'hydroxyl group of deoxyribonucleic acid or ribonucleic acid by an ester bond with 4-mercaptobutyric acid or 5-mercaptovaleric acid. It will be cleaved via an intermediate. When such a resin for nucleic acid synthesis is used, the nucleic acid is considered to be cleaved when the butyric acid binding moiety or valeric acid binding moiety having a mercapto group at the terminal cyclizes.

  In any case, “O” at the right end of COO— may be considered as a part of the linker or a part of the nucleic acid. In general, “O” at the right end of COO− is considered to be a part of nucleic acid.

  The reducing agent used can be appropriately selected from known ones, and specifically, DTT (dithiothreitol) or TCEP {tris (2-carboxyethyl) phosphine} is preferable. In any case, the reduction can be performed under neutral conditions.

  Next, examples and comparative examples of the above aspects will be described in detail.

[Example 1]
In the route shown in FIG. 2, a nucleic acid synthesis resin in which a nucleic acid is bonded to the resin via a linker that can be cleaved under neutral conditions was synthesized.

(1) Synthesis from compound represented by formula (1) to compound represented by formula (4) 4.77 g (20 mmol) of 3,3′-dithiodibutyric acid was suspended in 100 mL of dehydrated dichloromethane, and oxalyl dichloride was suspended. 5.14 mL and 62 μL of dimethylformamide were added, and the mixture was stirred at room temperature for 2 hours. The solution was concentrated under reduced pressure, dehydrated toluene was added to the residue, and the mixture was concentrated under reduced pressure.

  The residue was dissolved in 100 mL of dehydrated dichloromethane, added with 3.35 g of triazole and 8.71 mL of diisopropylethylamine, and stirred at room temperature for 10 minutes. At this stage, it is considered that the compound represented by the formula (1) was obtained.

  Next, 5.49 g (10 mmol) of 5 'dimethoxytritylthymidine was added to this solution and stirred for 6 hours. At this stage, it is considered that the compound represented by the formula (2) was obtained.

  To this solution, 4.60 g of N-hydroxysuccinimide was added and stirred for 15 minutes. At this stage, it is considered that the compound represented by the formula (3) was obtained.

  Thereafter, 4.69 g of 6-hydroxyhexylamine was added and stirred for 3 hours. The solution was diluted with dichloromethane and washed with water. The dichloromethane solution was concentrated under reduced pressure, and the residue was purified by medium pressure chromatography (ethyl acetate-ethanol 1: 0 → 19: 1) to obtain 5.14 g (58%) of the compound represented by the formula (4). The structure was confirmed from the NMR chart shown in FIG.

(2) Synthesis from the compound represented by the formula (5) to the structure represented by the formula (6) 439 mg of the compound (0.5 mmol) represented by the formula (4) was dissolved in dehydrated acetonitrile, and the pressure was reduced. The operation of concentrating was performed 3 times. The residue was dissolved in 5 mL of dehydrated acetonitrile, 99 μL of diisopropylethylamine, 50 mg of succinic anhydride and 6.1 mg of dimethylaminopyridine were added, and the mixture was stirred at room temperature for 1 day. At this stage, it is considered that the compound represented by the formula (5) was obtained.

Thereafter, genoglass-PG-200-γ-amino resin 0.5 g (NH ₂ , 10 μmol), dehydration condensing agent HCTU {1- [bis- (dimethylamino) -methylene] -5-chloro-1H-benzotriazolium Hexafluorophosphate 3-oxide} 207 mg and diisopropylethylamine 207 μL were added and gently stirred at room temperature for 2 hours. The resin was collected by filtration, washed with methanol and dichloromethane, and dried to obtain a structure represented by the formula (6). In FIG. 2, it goes without saying that deoxyadenosine derivatives, deoxycytidine derivatives, deoxyguanosine derivatives, uridine derivatives, adenosine derivatives, cytidine derivatives, and guanosine derivatives can be used instead of thymidine.

[Example 2]
Using a resin prepared according to the method of Example 1 and a dA, dG, dC amidite (lower left in FIG. 9) synthesized by the route shown in FIG. Was synthesized. Synthesis was possible with a yield of 98% or more on average synthesis efficiency.

  To the synthesized dCGAT resin, 5 mL of 0.01 M DBU (diazabicycloundecene) -acetonitrile solution was allowed to flow over 1 hour to remove the protecting group.

  Then, after washing with acetonitrile and water, 0.1 M TCEP {Tris (2-carboxyethyl) phosphine} -tris (pH = 7.0, 250 μL) was allowed to flow for 4 hours, whereby the S—S bond was obtained. The solution was recovered by cleaving by reduction. The reducing atmosphere had a pH of 7.0, which was a neutral condition.

  The solution immediately after collection over 4 hours was analyzed by HPLC (high pressure liquid chromatography). The results are shown in FIG. 4 and the HPLC analysis results of the solution which was allowed to stand at room temperature for 24 hours. Moreover, the analysis result of dCGAT synthesized according to a conventional method is shown in FIG. As a result, it was found that the main product after standing for 24 hours coincided with dCGAT synthesized according to a conventional method. That is, FIG. 5 is considered to correspond to the product of FIG.

  Further, a solution in which maleimide was added immediately after reduction by TCEP (that is, after 4 hours) was analyzed using HPLC. The results are shown in FIG. In FIG. 7, the first two large peaks correspond to the reaction product of maleimide and TCEP, and the subsequent sharp peak corresponds to the reaction product of compound (B) and maleimide. Since the mobility of this peak is different from the mobility of the peak in FIG. 6, it is understood that this reactant is a substance different from the main product (C). Since maleimide specifically binds to -SH, cyclization of a butyric acid-binding moiety having a mercapto group at the end can be prevented. From this, FIG. 4 is considered to correspond to the product of FIG. 3B, that is, an intermediate. The solution of this reaction product ((c ′) in FIG. 3) was allowed to stand at room temperature for 24 hours, but no change was observed.

  As a result, it is considered that DNA excision occurs by the mechanism shown in FIG.

  In FIG. 3, T represents thymidine. Of course, deoxyadenosine derivatives, deoxycytidine derivatives, deoxyguanosine derivatives, uridine derivatives, adenosine derivatives, cytidine derivatives, guanosine derivatives can be used in place of thymidine, and other nucleic acid sequences can be used in place of dCGA. Yes.

It is a figure which shows the example of the conventional linker. It is a figure which shows the synthetic pathway of the resin for nucleic acid synthesis | combination which the nucleic acid couple | bonded with resin through the linker of the said embodiment. It is a figure which shows the presumed cutting | disconnection mechanism of a linker. It is a figure which shows the HPLC result of the solution immediately after collection | recovery of TCEP process. It is a figure which shows the HPLC result of the solution after leaving still at room temperature for 24 hours after collection | recovery of TCEP process. It is a figure which shows the HPLC result of dCGAT synthesize | combined according to the usual method. It is a figure which shows the HPLC result of the solution which added maleimide immediately after collection | recovery of TCEP process. It is NMR of the compound represented by Formula (4) of FIG. Synthesis route of dA, dG, dC amidide

Claims (4)

A resin for nucleic acid synthesis which is bound to nucleic acid resin via the linker, the linker _{-S-S- (CH 2) n} -COO- have binding, nucleic acid bound to the COO- side (Wherein n is 3 or 4) , the -SS-bond can be cleaved under neutral conditions, and as a result, a hydroxyl group-resin can be generated under neutral conditions .   The resin for nucleic acid synthesis according to claim 1, wherein the reaction for generating the nucleic acid having a hydroxyl group end under neutral conditions is spontaneous. If the linker is _cleaved, HS- _(CH 2) have a n -COO- bond, its COO- side resulting intermediates nucleic acid is bound to, nucleic acid synthesis according to claim 1 or 2 Resin for use (where n is 3 or 4). Cleavage of the linker can be carried out by a reducing agent, nucleic acid synthesis resin according to any one of claims 1-3.

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