JPS5862199A - Production of purified 5'-terminally triphosphorylated oligonucleotide - Google Patents

Abstract

PURPOSE:A 5'-terminally triphosphorylated oligonucleotide, which shows a plurality of peaks in the high-speed liquid chromatography, is treated with ammonia to produce the titled oligonucleotide. CONSTITUTION:5'-Terminally monophosphorylated oligonucleotide and a pyrophosphate salt are subjected to reaction in the presence of a condensation reaction agent such as 1,1-carbonyldiimidazole to give a 5'-terminally phosphorylated oligonucleotide, which shows a plurality of peaks in the high-speed liquid chromatography and is represented by formulaI(formula II is a nucleotide skelton containing B as a base; symbols}{show that O-H and P-O bonds existing between them are not limited to 2'-terminal, and 3'-terminal in ribose; n is 0 or integer). Then, the product is dissolved in conc. aqueous ammonia and stood at room temperature to give the objective oligomucleotide.

Description

DETAILED DESCRIPTION OF THE INVENTION [1] BACKGROUND OF THE INVENTION Technical Field The present invention relates to a method for producing 5'-terminal triphosphates of purified oligonucleotides. In other words, the present invention relates to a method for purifying terminally triphosphorylated orinonucleotides-5'.

The 5'-ends of DNA and RNA are sometimes in the triphosphate form, and triphosphorylation (sometimes referred to as TP conversion) is essential for their total and partial synthesis.

In addition, oligonucleotides that have protein synthesis inhibitory activity 1 and are considered to be used as antiviral agents, antitumor agents, and anticancer agents, such as 2-5A (i.e., 2'-5'
Ori! TP formation also plays an important role in the synthesis of adenylic acid).

Prior art triphosphorylation of oligonucleotide P was carried out in the presence of inorganic phosphoric acid as a condensing agent DCC, but handling oligonucleotide 'i with DCC for a long time poses a risk of rearrangement of internucleotide bonds. It wasn't the right method.

In recent years, a method of reacting a pyrophosphate with the 5'-phosphate group □ of orinonucleotide r under condensation conditions has been widely used. In that case, car-nyldiimidazole, morpholine, or a phosphorylating agent containing a methylthio group (for example 0-2°4-dichlorophenyl-8-
methyl phosphorochloride thioate), etc. are used.

Among these, the method using car-nyldiimidazole allows the reaction to proceed at room temperature, and is more efficient than the method using morpholine.
It is considered suitable for TP conversion of nucleotides.

On the other hand, the separation of the target TP compound from the TP reaction product is as follows:
Usually, desalting is carried out using activated carbon or the like, followed by ion exchange using DEAE cellulose or the like. Thereafter, it was confirmed whether or not it was a single entity by paper chromatography, electrophoresis, or enzymatic ice thawing.

However, with the advancement of analytical technology and the use of HPLC (C-18 column), the present inventors have succeeded in performing conventional TP analysis using pyrophosphate using car-nyldiimidazole and the like.
2-5A, which was synthesized by the chemical method and confirmed to be a single product, was found to be a mixture with more than one peak.

Furthermore, the peaks after these two are oligonucleotide-
It was found to be based on the Cape 1 product produced by the condensing agent used in TPing 5'-monophosphorus oxide.

As is clear from the fact that the conventional triphosphate preparation synthesized from pyrophosphate using a condensing agent was considered to be a pure product, this impurity has almost no difference in physical properties from the target product. It is difficult to remove from the target object. Separation is possible using HPLC (c-ls column), but it is time-consuming to separate the target product in large quantities, and for this reason, it is necessary to prepare M sufficiently before mass fractionation using DEAE cellulose. It is desirable to leave it there.

(11. Overview of the invention) The present invention aims to solve the above-mentioned problems, and attempts to achieve this object by an extremely simple process called ammonia treatment.

Therefore, the method for producing purified 5'-terminally triphosphorylated oligonucleotides according to the present invention is as follows:
'-terminal triphosphorylated orinonucleotide r
- is subjected to ammonia treatment.

In addition, another purified dar-terminal triphosphorylated oligonucleotide according to the present invention! Nucleotide manufacturing method
) or after step (d).

(al) A 5'-terminal monoline-bearing orinonucleotide of the following formula is reacted with pyrophosphate in the presence of a condensing agent,
Triphosphorylate the 5'-end.

(b) Collecting the nucleotide fraction from the triphosphorylation reaction product.

(c) The nucleotide fraction is subjected to ammonia treatment.

The symbols ) and (indicate that the oxygen-hydrogen bond and oxygen-phosphorus bond existing between them are not limited to the bonding position with the τ-terminus and 3'-terminus of the +7/-su moiety; (n is 0 or an integer) Effect: Since the second and subsequent peaks in HPLC substantially disappear due to ammonia treatment, this treatment converts impurities to the target 5'-terminal triphosphorylated orinonucleotide. It is presumed that the present invention is not bound by such a presumption.

Although it is unknown whether the impurities found in HPLC are examples, it is undesirable for biologically active substances such as 2-5A to contain such impurities in terms of decreased activity or toxicity. Needless to say, yes. Therefore, it is extremely advantageous that a pure product can be obtained by a simple purification method such as ammonia treatment (specifically, for example, dissolving the synthesized triphosphorus oxide in concentrated aqueous ammonia and leaving it at room temperature). If impurities are converted to the target triphosphate, this will also lead to an increase in the yield of the target product.

1) Definition The 5'-terminal triphosphorylated oryza nucleotide to be used in the present invention is a compound represented by the above formula (2).

In the formula, B specifically represents adenine, guanine, cytosine,
It is a base such as thymine or uracil, and n is O or an integer (eg, up to 15, preferably 1 to 6).

As is clear from the definition of formula (2) above, the 5'-terminal triphosphorylated orino nucleotide 1'
(The same applies to compound (1)) when n is 1 or more, I-
The phosphate ester bond connecting the base portions may be a 2', 5' bond, a 3/-s/ bond, or a combination or mixture of both bonds.

One specific example of the compound targeted by the present invention is a 2'-5' oligonucleotide in which B is adenine and n is an integer, particularly 1 to 6, especially 2. Gochuryo Nanashi is impure in the sense that it shows multiple peaks on HPLC. The HPLC used to define the target compound in this case is as follows.

HPLC measurement conditions
pak) C-18J (Japan Waters Limited)
Eluent 050mM-IQ (2P04 PH7,26
■H20-MeOH (1:1) Gradient ■0/20ch Flow rate for 10 minutes 2.9*e/min Chart speed 1.03/min b) Column [Micro〆〆〇〉C-18J (Japan Waters・Limited) #Spray ■1. /15M ICH2PO4-1/'15
M hl 2HPO4 pH 7,5 (I H2O-MeOH (1:t) Gradient ■0/20ch Flow rate for 10 minutes 2.Oyxl/min Chart speed 9.5ct/minC) Column "Radial pak"
C-18J (Japan Waters Limited) eluent
■1/15M KH2PO4-1/15M N12HP
O4 pH 7,5 ■H20-MeOH (1:1) Gradient ■0/20ch I minute flow rate 2. Omj/min chart speed 1. On/min~2) Synthesis P! 11 The compound of formula (2) can be synthesized by triphosphorylating the 5'-monophosphoric acid group of the compound of formula (1).

Triphosphorylation of the 5'-end of the compound of formula (1) is known, and this reaction can be carried out in the present invention by any suitable method. Specifically, for example, a suitable condensing agent, in particular carbonyl di-ludiimidazole (CDIm
), carzenyl ditriazole, caryl ditetrazole, especially CDIm, in the presence of alkali gold I! pyrophosphate salts, especially pyrophosphoric acid! i4 salt or trialkyl (about 01-08) amine salt, especially tri-n-butylamine salt, in a suitable solvent, first in dimethylformamide (D
MF ) with a compound of formula (1). 5'-
In order to convert the terminal into a salt form to facilitate condensation, triethylamine or tri-n-octylamine, in the case of a trialkylamine, can be added to the condensation reaction system.

The TP compound synthesized in this way is usually recovered from the TP reaction product by activated carbon column treatment, etc., and subjected to ion exchange chromatography such as DEAE cellulose column treatment to fractionate the desired TP compound. . Compound (2) thus isolated, ie, the conventional product, showed multiple peaks on HPLC as described above.

The present invention can be carried out not only on the thus isolated compound * (2) or a commercially available product, but also at a stage subsequent to the synthesis stage in the above-mentioned synthesis process (see postscript).

3) Contents of by-product impurities Although it is unknown what the by-product impurities are, compound (1)
(PA2'p5'A2'P5'A) The reaction solution was separated for each of the following steps of triphosphorylation, and each was subjected to HPL.
Analysis by C revealed that by-products were formed immediately upon addition of the car-nyldiimidazole condensing agent.

■ Compound (1) (2-5A) ■ One hour after adding carzenyldiimidazole ■ M
1 hour after adding eOH ■ 1 hour after adding pyrroline Compound (II) with the 2-terminus protected with an azide group was similarly triphosphorylated, and the separated sample was
When analyzed by LC, two or more peaks appeared in the case of compound (1'), but one peak appeared in the case of compound (1'). This revealed that the condensing agent had some effect on the -OH terminal of the lysase moiety.

Furthermore, compound (1) (pA”1)”A”p”A)
”+ Triphosphorylated and fractionated TP body in the same way as HP
Analysis by LC (C-18) revealed two or more 2-wires (see attached Figure 1, measurement conditions a)). Commercially available 2-5A (PL Chemicals) was similarly analyzed by HPLC.
(C-18) (Fig. 2, measurement conditions a)) and compared the positions of the peaks. Among the multiple peaks, the leftmost peak coincided with 2-5A.

In addition, the above-mentioned TP bodies were l-suit in conventional analytical methods ((--arm chromatography, '-area electrophoresis, and enzyme hydrolysis).

The ammonia treatment described in 1) is preferably carried out in the presence of a solvent for compound (2) or both compound (2) and ammonia. The solvent may be of various types that provide the necessary bath dissolution ability, but typical examples include water, lower alcohols, or mixtures thereof.

A preferred ammonia treatment method is to leave compound (2) dissolved in concentrated ammonia water with or without stirring, or to blow ammonia into a solution or dispersion of compound 5(2). Song titles are generally preferred.

The processing temperature is usually Nucleochi 1? This is the temperature used for the treatment, and it is desirable to sieve at 40'C or less.

This is because if the width is greater than that, there is a risk that the nucleotide skeleton itself may be destroyed.

When treated in concentrated NH4OH at room temperature, approximately 21 hours are required.

After the treatment, the purified compound (2) can be obtained by removing the solvent together with the ammonia present in the bath (for example, by distilling it off).

2) Timing of implementation Ammonia treatment can be performed at any stage after triphosphorylation, but before desalting, the treatment becomes cumbersome, and after Dli:AE cellulose column preparative separation,
Since it may be necessary to perform preparative separation on DEAN cellulose columns, it is preferable to use DEAE after desalting.
It is desirable to carry out this procedure before separating the cellulose into five fractions.

Note that the desalting treatment and the ion exchange chromatography treatment using DEAE cellulose chromatography and the like are publicly known as described above, and even if the purification process of the present invention is involved, changes may be made as necessary. It can be carried out first.

3. Experimental Example 1) Compound (1) has a tetrahydrofuranyl group of 3′-
〇As a holding group for H group [synthesized pA2'p5'A2'
P”A (patent application biennial lθ month 9th).

Reference Example 1 (1) Reagent compound (1) (PA2'P"A2'p"A) (
Approximately 5800. D, 17jotel) triethylamine
(20 μl) tri-n-octylamine
(10 μl 1.1-carbonyldiimidazole (17~, 105 μmol) Sodium pyrophosphate (Nishikiri, 197 μmol)
2) l) Tri-n-butyramii (0,3 translation l) DMF (Synthesis of 21TP compound and its purification Compound (1) (it is above) was mixed with pyridine three times, toluene four times, and DMF once. After boiling, DMF
(Generally understood as 0.5g (shi). Add triethylamine (the above) and tri-n-octylamine (t is above), and then add 1,1-car-7yldiimidazole (t is above) 1- and react with stirring at 35°C.Sodium pyrophosphate (t is above) is dissolved in water (2 volumes) and [
DOWEX 50WX2J50-100 mesh 7,
Py+a! ! (it is above) and bathed with 50% pyridine water (40 txl). The solvent was evaporated, trilobutyl (It is described above) was added to the residue, and the solvent was evaporated. Scrape the residue 3 times with lysine, 4 times with toluene, and rinse with K.
After azeotroping once with DMF, DMF (0.3tnl
) is commonly understood as This was mixed with the above reaction solution (after 3 hours, with NaoOH (
Add 6.5 μl) and react with stirring for an additional hour. After 16 hours, water (1 smz) was added,
0. The pH was adjusted to 4 with IN-HCl, and the pH was adjusted to 4 using an activated carbon column (7c
c) inject concentrated NH4OH:EtOH:H2O (5
:50 :45. 100 ml). Concentrate the eluate and chromatograph on DEAE cellulose column CH.
CO3-type, φ1.6 x 18,5 (Jl, 37C
1:, 0.05M TEAE buffer, /'0.4M
TEAB buffer solution] Pickling solution is 7 lactation! ~V is obtained (see Figure 3). .

Conventionally, the fraction ■ (or ■ and ■) is divided into 2-
It was set to 5A.

7 lactation - yield l
(Fraction number 83-85) 460. D. 8%
1 (102-111) 650. D, 11%V (
125-132) 490. D.

(Analysis by 33HPLC (C-18 column) K The HPLC measurement results for the 7-lactones I, I, y and Y in Figure 4 are shown in Figures 4.5.6 and 7 (measurement conditions b), respectively).

There are two or more peaks in each case, and it is clear that by-products are produced.

Example 1 (1) Reagent compound %r(1)(PA2'p"A2'p"A) (6
000,D, 17,6Amol) triethylamine
(2[) μl) tri-n-octylamine
(1() μl) 1.1-L2Nyldiimidazole (18,7〃L, 115 μmol) + Umbrella) 13P207- ・n-Bu 3NH (198p n
o1)-NH40H('X)IRg)DMF* This was derived from sodium pyrophosphate, and the method was described in (2) of Reference Example 1 above.

(Synthesis of 21TP compound and its M!!! compound (1) (
If is above) was azeotropically distilled three times with pyridine, four times with toluene, and once with DMF, and then dissolved in DMF (0.5 ml). triethylamine (t above) and trin
- Add octylamine (t as above). Furthermore, 1.
Add 1′-carbonyldiimidazole (t is above),
The reaction is carried out at 30'C with stirring. After 1 hour, the disappearance of the raw material was confirmed by electrophoresis, and viK MeOH (8μ
1) and further reacted at 30'C for 1 hour with stirring. Add H3P207-/n-Bu3NH (amount above) to this and react with stirring at 30"C.18
Add water (15 wrl) after 0.0 hours. IN-M
Adjust the pH to 4 with CI, inject into an activated carbon column (TOCC), wash with water (200*t), and then add EtOH: H2
O: concentrated NH, i0H (50: 45: 5 200
elute with ynl). The solvent was distilled off, and the residue was diluted with concentrated NH4.
Add OH (t as above) and leave at n°C for 21 hours.

The solvent was distilled off, and DEAE cellulose column chromatography [
HCO3-type, φ1,6X213.0.05 M-T
EABli/'0.4 M TEAB buffer] fractions IV are obtained (see Figure 8).

Let the fraction ■ be 2-5A.

fraction yield yield l
(Fraction number 84-94) 570. D.1
oqk1 (100-111) 470. D.
8 *■ (112-118) 740. D.

■(119-131) 2660. D. 44
Chi V (132-134) 150. D.

(3) Analysis by T (PLC (C-18 column)) The HPLC measurement results of the 7-lactation W shown in FIG. 8 are shown in FIG. 9 (measurement conditions b)).

It can be seen that there is only one peak of 2-5A.

2) Compound (1) converts the O-nitrobenzyl group into 3'-O
pA2'p5'A2' synthesized as a protective group for H etc.
Reference Example 2 (1) Donation Same as (1) of Reference Example 1 when p5' person (%1 No. 1984-46059-46064).

(2) Synthesis of TP body and the same operation as in (2) of Reference Example 1 made of M to separate the TP body.

(3) Analysis by HPLC (C-18 column) Figure 10 shows the HPLC measurement results of the collected TP body (measurement conditions c)
)It was shown to.

There are three peaks, and it is clear that by-products are produced. Also, the target 2-5A is the leftmost peak (
(Compare with Figure 11).

Example 2 (1) Sample TP body” (200,D, )II
INH40H (10iLt) **In Reference Example 2, the TP form which was converted into TP and separated by DEAE cellulose column chromatography (has 3 peaks according to HPLC measurement) (2) The ammonia-treated TP form (the amount is above) was concentrated. It is taken up in ammonia (t as above) and left under stirring at room temperature. After 21 hours, the solvent is distilled off and the sample is dissolved in water to obtain an ammonia-treated sample.

(3) Analysis by HPLC (C-18 column) K HPLC measurement results of the above sample are shown in Figure 11 (611 constant conditions
))It was shown to.

One peak appeared, indicating that 2-5A was obtained in a pure manner. It is also clear that the two by-products (see figure @10) contained in the sample before ammonia treatment have all been converted to 2-5A shown in the leftmost peak.
It was revealed.

3) HPLC pattern and chromatography The conditions for high performance liquid chromatography and cellulose column chromatography mentioned in this experimental example were as follows.

fl) HPLC measurement conditions 1) Column p-Bondapak C-18 (Wats
rs) Age syneresis ■50 orchid-KH2PO4pH7,26
(g) H2O-MeOH (1:1) gradient ■O
,,'21)% 10 minute flow rate 2.0 Shoulder t/min Chart speed 1Qcx/min b) Column μm Bondapak C-1) 1 (Wa
ters) Eluent ■1Δ5M KH2PO4-17
5MN 2HPO4pH7,5■H20-MeOH(1
: 1) Gradient ■0/addition 10 minute flow rate 2.9 m67 minute chart speed Q, 5 cit/min C) Column Ra
dial Pak C-18 (Waters) Eluent ■1/15M KH2PO4-1/15M N12
HPO4 pH 7, 5 ■H20-MeOH (1:, 1) Gradient ■0/addition I minute flow rate 2. OR17 min chart speed l, Qcm/min (2) Purification conditions using DEAE cellulose column Column DEAE cellulose [HCO3-type] column gel φ1,6 X 18,5 clI'-137
cc gradient 0.05M TEAB buffer (75ON
/) 10.4M T Island BI! Bond liquid (750 machine tons) Fraction amount 8,8*t/7,25 minutes/fraction

[Brief Description of the Drawings] Figures 1, 2, 4, 5, 6, 7, 9, 10 and l1 are
All of them show HPLC patterns (reproductions) of various compounds. Figures 3 and 8 both show DEAE cellulose column chromatograms (reproductions) of the reaction products. Applicant's agent Inomata Sei Procedural Amendment (formality) 1.・Il: Indication of the matter Patent Application No. 161439 of 1981 Process for producing orinonucleotide 3, Person making the amendment Relation to the case Patent applicant Drawing 8, Contents of the amendment Engraving of the drawing (no change in content)

Claims (1)

[Claims] 1. High performance liquid chromatography (HPLC)
characterized by subjecting a 5'-terminal triphosphorylated oligo-J nucleotide of the following formula showing a peak to ammonia treatment,
Purified 5' showing essentially a single peak on HPLC
- A method for producing a terminally triphosphorylated oligonucleotide. tibb θ represents the skeleton of P, and the symbols ) and ( represent the oxygen-hydrogen bond and acid-phosphorus bond existing between them, and there are limitations regarding the bonding position with the r-terminus and 3'-terminus of the d-su moiety. 2. B is adenine and n is 0 or 1 to 15, and n is 0 or 1 to 15, and n is 0 or an extra number). Claim 1 ← 艷
4. A method according to claim 3, which comprises bubbling ammonia into the solution. 6. Perform the following steps (a), (b) and (d) in this order, and perform step (e) between steps (a) and (b), between steps (b) and (d), or between steps ( A method for producing a purified 5'-terminally triphosphorylated oligonucleotide P (aJ), which is carried out after dl. Let it react,
Triphosphorylate the 5'-end. (b) Collecting the nucleotide fraction from the triphosphorylation reaction product. (,) One fraction of nucleotides is subjected to ammonia treatment. (d) The nucleotide fraction was subjected to fractionation using ion-exchange chromatography to remove 5'-triphosphorylated oligonucleotides.
Collect Nucleochi P. The symbols ) and (indicate that the oxygen-water collecting bond and oxygen-phosphorus bond existing between them are not limited to the bonding position with the !-terminus and 3'-terminus of the iJ case moiety, and n indicates 0 or an integer). 7. The order of steps is (a) - (b) - (e) - a)
The method according to claim 6, wherein: 8. The method according to claim 6, wherein the order of steps is (a) (c) = (b) - (d). 9. The order of steps is (a) - (b) - (a) - (C
), and the method according to claim 6, further carrying out additional step (d) as necessary. 10. The method according to claim 6, wherein the condensing agent is 1,1-carzenyldiimidazole. 11.) Claims 6 to 1, characterized in that the nucleotide fraction is obtained by subjecting the IJ phosphorylation reaction product to activated carbon column treatment.
The method according to any of item 0. 12. The method according to any one of claims 6 to 11, wherein the ion exchange chromatography is performed using a DEAR cellulose column (HC03e type). 13. The method according to any one of claims 6 to 12, wherein the ammonia treatment is performed in the presence of a solvent for the nucleotide fraction. 14. The method according to claim 13, wherein the ammonia treatment comprises dissolving the nucleotide fraction in aqueous ammonia for a predetermined period of time. 15. Claim 13, wherein the ammonia treatment consists of bubbling ammonia into the solution of the nucleotide fraction.
The method described in section.