JPS6379898A - Control of deprotection reaction in polynucleotide synthesis reaction - Google Patents

Abstract

PURPOSE:To produce the titled compound in high yield, by controlling the contacting time with a deprotecting agent within a specific range according to the kind of base bonded to a terminal hydroxyl-containing deoxyribose of a polynucleotide in the synthesis of a polynucleotide with a synthesis apparatus. CONSTITUTION:A polynucleotide is synthesized by phosphite process using a polynucleotide synthesis apparatus constructed to feed the reaction raw materials 7-10, reagents, solvents 2-6, etc., necessary in the polynucleotide synthesis reaction to a reactor 11 with pressure of nitrogen gas supplied from a bomb 1 in prescribed order of feeding controlled by a controlling means 12 and discharge the raw materials, etc., from the reactor. The objective polynucleotide can be produced in high yield by adjusting the contacting time of a polynucleotide with a deprotecting agent to 0.8-1.2 for a polynucleotide wherein the base bonded to deoxyribose containing terminal hydroxyl group of polynucleotide is thymine (T), 0.8-1.2 for cytosine (C) and 0.4-0.8 for guanine (G) taking the contacting time for adenine (A) as 1.

Description

Detailed Description of the Invention Technical Field of the Invention The present invention relates to a polynucleotide synthesis reaction using a phosphite method using a polynucleotide synthesizer, in which a protecting group that protects the terminal hydroxyl group of a polynucleotide is brought into contact with a deprotecting group. It concerns the method of controlling the removal of alcohol when making zazete and removal [8].

Technical background of the invention and its problems In recent years, molecular biology, represented by JQ Engineering, has made rapid progress. ) There have been many attempts to synthesize the vorinucle Aji1~ constituting the following.

Polynucleotides are made up of multiple nucleotides in which a phosphate is bound to a deoxyribose small nucleoside, which is a combination of four types of nucleobases: adenine, guanine, cytosine, or thymine, and dixyribose. To synthesize this polynucleotide, the phosphotriester method has conventionally been adopted. However, since this phosphoric acid triester method uses 51 tfti of phosphoric acid, there is a problem that the activity is low.

For this reason, the phosphite 1 method, which uses trivalent phosphorous triester rich in reactive oils, has come to be widely adopted in the synthesis of polystyrene. To synthesize polyscleotide using the phosphite method, the following four steps (a) to (d) are usually required.
is necessary.

(a) The tri-1-yl group, which is a protecting group at the 5'-position of the nucleoside bonded to the support, is cleaved with an acid, and the 5-position of the nucleoside is converted into hydroxyl viscosity through the I-stage (deprotection). A-group process>

(b) Next, to this hydroxyl group, an amigate bonded with a base of either adenine, guanine, cy[hesine or L-mine] such as N,N-cyisobrobilbosphoramidite activated with tetrazole or the like is added. A step of reacting to form a phosphite (coupling step).

(C) Coupling] A step (oxidation step) of oxidizing the phosphite obtained from the two culms using a reagent consisting of iodine, tetrabitrofuran, 2,6-lutidine, water, etc.

(d) I+W (capping step) 1° in which the 5'-position hydroxyl group of the nucleotide that was not present in the coupling step is retained using a rubbing solution consisting of acetic anhydride, dimethylaminopyridine, etc. The above four steps may be performed in one order of deprotecting group step, coupling step, acclimatization step, and capping step, but depending on the case, C
This refers to the 1st step of the process, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step, the 1st step in the usage order.
In the polyscleotide synthesis process, which consists of the four steps described above, there is usually a cleaning process with a cleaning solution such as acetonitrile between each of the above steps.
Each step is carried out in the absence of impurities.

Since the synthesis of polynucleotides like this is too cumbersome to carry out at Sensaku Kuwa, the reaction raw materials, reagents, solution W, etc. necessary for each of the above steps are automatically supplied to the reactor in sequence. A polyscleotide synthesizer has been proposed that is configured to discharge the polystyrene.

However, when attempting to synthesize polyscleotide using such a 4c polynucleotide synthesis method, there was a problem in that it was difficult to synthesize a long polyscleotide chain with more than 10-0 nucleotides. .

In order to solve these problems, the Ministry of Invention and others have
According to JI research, the reason why it is not possible to synthesize a long chain polynucleotide 1"f-do in which more than 100 g of nucleotides are linked is because the protecting group that protects the terminal hydroxyl group of polynucleotide is brought into contact with the lQ protecting group ?111. When avoiding the pre-reaction, this vorinucle; d:z, l.
: If you want to bond to deoxyribose containing a hydrating group, and the rate of the elimination reaction is different depending on the type of the four groups, if all the bases are contacted with the deprotecting base for the same time. This is because when the L'lli group is guanine, side reactions occur in addition to the de-locking group reaction, reducing the yield of polyscleotide obtained in the subsequent reaction (b I discovered that.

Based on these findings, further research by the Ministry of the Invention et al. (3) found that by changing the contact time between the polynucleotide and the depolymerizing base depending on the type of the base, nucleoyl- 'is more than 100.

We discovered that it is possible to synthesize long chain polynucleotides using a polynucleotide synthesizer.

OBJECTS OF THE INVENTION The present invention is directed to solving the problems associated with the prior art as described above.
Synthesis of borinucleotide (-) by synthesis thickness fr (5) by the phosphoric acid method using r''j, the 4-group of the borinucleotide is removed by contacting with the 1-4 base. In this case, this legal evasion lawsuit kt reaction (!-system'tQD
The present invention aims to provide a polynucleotide synthesizer capable of synthesizing long-chain polynucleotides with high yield.

Summary of the Invention A method for controlling the elimination group reaction in a polynucleotide synthesis reaction using a polynucleotide synthesis apparatus according to the present invention is to prepare reaction materials, reagents, solvents, etc. necessary for a polynucleotide synthesis reaction in a predetermined order. 1) to the reactor, and then discharge it to the reactor, 1) using the constructed polynucleotide synthesizer, carry out a polynucleotide synthesis reaction according to the methods of 1 to 1), -1
;1111! by contacting the protective group that protects the hydroxyl group with the fB'2 protecting base. When stirring, the contact time between the polynucleotide and the base when the base bound to the deoxyribose containing the 71-hydroxyl group at the end of the polynucleotide is adenine (△) is set to 1, In the above story, when the 11 groups are ``f-mine (T), cytosine (C), or guanine (G), the above-mentioned contact time is 111 times as shown below, respectively, which is considered to be a ``t:j'' characteristic.

(1) Thymine (C): 0.8-1.2 cm
): 0.8 to 1.2 Guanine (G): 0.4 to 0.8, and (II) the ratio of the contact time, G, /A, G, ·'T, G/C, is 0. .8 or less.

According to the present invention, J5 is used in the polynucleotide synthesis reaction by the phosphite method using a polynucleotide synthesizer.
C. The contact between the polynucleotide and the filler, the end of the polynucleotide (changes are avoided depending on the type of base bonded to deoxyribose, which contains a weak hydroxyl group, so it can be avoided) - Through subsequent steps, polynucleotides with extended chain length can be obtained in good yield.
Therefore, it is possible to easily synthesize long-chain polynucleotides.

DETAILED DESCRIPTION OF THE INVENTION Hereinafter, the present invention (1.
I will explain in detail how to do this.

First, an example of the polynucleotide synthesis apparatus used in the present invention will be described with reference to the drawings. In the drawing, reference numeral 1 is an inert gas (nitrogen gas) cylinder, 2 is a solvent bottle, 3 to 6 are reagent bottles, 7 to 10 are raw material bottles, 11 is a reactor, and 12 is a gate control means.

The two gases filled in the cylinder 1 pass through the pressure regulating valve 13 and the distributor 14a to the solvent bottle 2 and the reagent bottle 3.
~・6, Kasumipo Ttobin 7~101 were sent, tA S
2 fJsu(7) ri',)) nir')? ? 4
i″i'l, reagent, Ij<'t''l crab 4th road 14b
(consisting of a thin tube of about imm0) and sent to the reactor 11. In addition, N2 dregs pass through A Lee Noise 15, Ryugo't<, 171 b, and anti-1. IS container 11 (varnished. A refill 15 is the N2 scum). The harvest is in Song Dynasty. N2 scum is 1 pile, leakage: ~
] It is used after being recycled in a desiccant such as Lasieves and filtered. In addition to N2 scum, helium, argon, etc. can be used as the inert gas.

) 8 Reagent bottle 2 contains solvents such as acetonitrile, and reagent bottle 3 contains trichloroacetic acid and dichloroacetic acid ili! 2, an oxidizing agent consisting of iodine, tetrahydrofuran, 2,6-luzidine, and water in reagent bottle 4, and a mixture of acetic anhydride and dimedylaminopyridine in reagent bottle 5.・Tupping agent, reagent bottle 6 contains tetrazole,
Each is filled with an activator such as tetrahydrofuran or acetonitrile. In addition, raw material bin 7, raw material bin 8.1 Kyo 1 mo [bin 9.1 Kyo 1 mo] O, adenine (A), guanine (G), cy 1 to syn (C
) J puyo and thymine (T> as salt ↓) Small spores (nucleoti 1 to sample mulberry) and '+8 AI and I) X
It is filled with Solu IIQ.

Check valves 16 are installed on each side of the reagent bottles 3, 4, and 5.
．． 17 and 18 are provided to prevent the hot air mist containing the 5 base, excretion, and rubbing from flowing backward through the flow path 14b and into other bottles. ing.

A solenoid valve 19 is provided in the flow path 14b, and a solenoid valve 19 is provided in the flow path 14b.
The outlet side of the bottles 2 to 10 is equipped with electric power valves 20 to 2, respectively.
B is provided, and electromagnetic valves 29 to 32 are provided at the bottom and top sides of the reactor 11 in the flow path 14b, respectively. These electromagnetic valves 19 to 32 are controlled by a control means (programmable controller) 12 to open and close.

The solenoid valve 19 opens when energized at 2154t, and the solenoid valve 2
0 to 30 are three-way valves, and the dotted line portion communicates with the solid line portion when energized, and the electric Ei valves 31 and 32 are three-way valves that switch from the solid line state to the dotted line state when energized.

Next, to explain the general operation method of such a polynucleotide synthesis apparatus, in the reaction operation, first, a porous glass having, for example, thymine nucleoside (T) bonded thereon is supported in the reactor 11. Filling the body (support) and then operating the control means 12 causes the control means 1 to
Solenoid valves 19 to 3 according to the contents of the program set in 2.
2/))lii'i Next, it is opened and closed, and the line barcier culm → washing process - deprotection group process → washing process → coupling process - washing process → oxidation process → washing process → capping culm - washing process is performed.

These are the four main features of a polynucleotide synthesizer.

In the polynucleotide synthesis reaction using the bosphite method using such a polynucleotide synthesis apparatus, the end of the polynucleotide: 1 of the protecting group that retains the WM 5
To explain the protective group process, first, the electric limit i'21
．． 29, from a certain test bottle 3, add 2 liters of a protective agent such as trichloroacetic acid into the line leading to the reactor 11. The opening/closing interval of the solenoid valve 21 is set so that the opening/closing interval of the solenoid valve 21 is satisfied to about %.

Next, the electric water valve 19 is turned on, and the 11)2 base material in the line is supplied to the reactor 11, and bubbling is performed in that state. - After this, a deprotecting base is further supplied to the reactor in the same manner and bubbling is performed. Thereafter, a deprotecting base is further supplied to the reactor in the same manner and bubbling is performed.

After contacting the deprotecting base with the polynucleotide supported on the carrier, the reactor 1 is closed by closing solenoid valves 19 and 29 while listening to solenoid valve 30.32.31.
The deprotecting base present in 1 is discharged.

At this time, in the present invention, the contact time between the polynucleotide and the deactivation dk agent is set to vary depending on the type of base bonded to the deoxyribose containing the terminal hydroxyl group of the vorinucle Δ-dide. For example, the last nine of a polynucleotide: ; When the contact time with the salt bonded to deoxyribose containing a hydroxyl group is 1, the polynucleotide when A> is A> and the contact time with the base agent is 1. If the salt is thymine (T), the contact time is 0.8.
~1.2, and when the base is cytosine (C), the contact time is 0083~1.2, and when the base is guanine (G), the contact time is 80
．． The ratio C is set to be 4 to 0.8. G/A is 0.
Set it so that it is 8 or less, and similarly set G/T and G/C
The value of is also set to be 0, 8 or less.

Specifically, the base is adenine (A), thymine (T)
, i? if it is cytosine (C)? i'I? J! The contact time is preferably 30 to 80 seconds, and when the base is guanine, the contact time is preferably 15 to 65 seconds.

In this way, if the contact time between the polynucleotide and the n)2 protective base is avoided to change as the base bonded to the deoxyribose containing the hydroxyl group of the polynucleotide is avoided, the δ base can be removed. There is less possibility that side reactions will occur in the reaction, and therefore the chain length will be extended by the steps following the U11m2 protecting group step, resulting in a higher yield of the polynucleotide. C) For this reason, long-chain polynucleotides in which 1 to 100 or more nucleotides are linked can be synthesized with high yield.

Na cj >> 3 is the polynucleusian chi 1-no! l, M:: Keep the water leak 4Jru keep it as early as (Jyu,
1 to rityl, monomethoxytrityl group, dimethoxytrityl group, etc. are used. Further, as the removal agent, acidic compounds such as monochloroacetic acid, trichloroacetic acid, benzenesulfonic acid, and 1-lyfluoroacetic acid are used.

Effects of the Invention According to the present invention, iJ is used in the polynucleotide synthesis reaction by the phosphite method using a polynucleotide synthesizer.
3, the contact gap between the polyscleotide and the decoupling base is changed depending on the group (Φ) bonded to the deoxyribose containing the terminal hydroxyl group of the polyscleotide, so that the decoupling base is Through subsequent steps, polynucleotides with extended chain length can be obtained in high yield.
Therefore, long-chain polynucleotides Δdi1- can be synthesized with high yield.

The present invention will be explained below with reference to examples, but the present invention is not limited to these examples.

Example 1 Using a polynucleotide synthesizer as shown in the drawing,
Each nucleotide is 51.80i, 130 weeks consecutive 4
The polynucleotide was synthesized.

At this time, the contact time with the polyscleotide (2) tongue-holding base was 60 seconds when the salt bonded to the deoxyribose containing the terminal hydroxyl group of polyscleotide was adenine (A), The case is 58 seconds, the case of cytosine (C) is 58 seconds, and the case of guanine (G) is 58 seconds.
The time was set to 30 seconds.

A polynucleotide was synthesized in this way, and the 5' end of the synthesized polynucleotide was labeled with [γ32-pEATP] using vorinucle A dokylyase.
It was developed on a polyacrylamide gel, electrophoresed, and then analyzed by autoradiography, and its presence was determined to be 'MfL 3+' by the absence of the main hand.

The results are shown in Table 1. .

In Example 1, the base, azodine (△), amine (
The following steps were carried out in the same manner as in Example 1.
C. Synthesis of polyscleotide was performed.

The results are not shown in Table 1.

Leaving: 50mer-80mer i 130
a+er: From this Table 1, the contact time of the polynucleotide and the protective base to the polynucleotide 1;
By changing the base bonded to the deoxylipose containing a hydroxyl group, it is possible to obtain a polynucleotide with an extended chain length in high yield through various steps subsequent to the initial step. Therefore, it can be seen that long-chain polynucleotides 1 to 1 can be synthesized with good yield.

[Brief explanation of the drawing]

FIG. 1 is an explanatory diagram of a polynucleotide synthesis apparatus used in the present invention. 1...Cylinder 2-6...Solvent, reagent bottle 7-
10... Raw material bottle 11... Reactor 12... Control means

Claims (1)

[Claims] 1) A host using a polynucleotide synthesizer configured to supply reaction materials, reagents, solvents, etc. necessary for a polynucleotide synthesis reaction to a reactor in a predetermined order and then discharge them. In the polynucleotide synthesis reaction by the phyto method, when the protective group that protects the terminal hydroxyl group of the polynucleotide is removed by contacting it with a deprotecting base, the base bound to the deoxyribose containing the terminal hydroxyl group of the polynucleotide is removed. When the contact time of the polynucleotide and the deprotection base is 1 when is adenine (A), the contact time when the base is thymine (T), cytosine (C) or guanine (G) A method for controlling the deprotection group reaction in a polynucleotide synthesis reaction in a polynucleotide synthesizer, which is characterized by controlling the following, respectively: (I) Thymine (T): 0.8 to 1.2 Cytosine ( C): 0.8 to 1.2 Guanine (G): 0.4 to 0.8, and (II) the ratio of the contact time, G/A, G/T, G/C, is 0.8. The following shall apply.