CN1030424A - The preparation method of nucleic acid derivative and preparation thereof - Google Patents
The preparation method of nucleic acid derivative and preparation thereof Download PDFInfo
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- CN1030424A CN1030424A CN88104080A CN88104080A CN1030424A CN 1030424 A CN1030424 A CN 1030424A CN 88104080 A CN88104080 A CN 88104080A CN 88104080 A CN88104080 A CN 88104080A CN 1030424 A CN1030424 A CN 1030424A
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Abstract
The invention provides a kind of novel preparation method of nucleic acid derivative.This method mainly comprises: (a) make the classification of single-chain nucleic acid polymkeric substance, (or the sedimentation definite value is in the scope of 4S-13S) to make its molecular size be limited to 50-10,000 base pair; (b) even annealing is the process of single-chain nucleic acid polymer formation two strands also complimentary to one another.But the present invention's industrialization, and do not produce pollutions such as public hazards.Have very strong antitumous effect and other physiological activities by the prepared nucleic acid derivative of method of the present invention, and side effect is little, can be used as antineoplastic agent, antiviral agent etc.
Description
The present invention relates to a kind of novel preparation method of medical nucleic acid derivative.
Nucleic acid is that the sugar of ribose etc. is combined on purine skeleton or the pyrimidine ring,, and is linked to each other with chain and to form between between them by phosphoric acid.
RNA(ribonucleoside acid polymer in the nucleic acid) be chain-like macromolecule compound, it has ribose as sugar, and sugar moieties is connected with phosphodiester bond.In the purine skeleton or the pyrimidine ring part of the alkali (for example inosine, adenosine, cytidine, uridine etc.) that constitutes nucleic acid, two chains are linked to each other by hydrogen bond in so-called complementary mode and constitute spiral helicine three-dimensional arrangement.Because this nucleic acid that contains two chains can expect to have the useful physiological activity, therefore this has been carried out big quantity research [Biochemical and Biophysical Research Communications 58(1974) etc.] up to now.
In this manual, wherein the poly I-C derivative as synthetic two key RNA is called " poly IpolyC " derivative, and with its formation unit, promptly Polyinosinic acid and polycytidylic acid are called " poly I " and " polyC ".
In recent years, people knew already, and various natural or synthetic double-stranded RNAs have that Interferon, rabbit produces can (Off ィ-Le De ウ.Proc.Nat.Acad.Sci.U.S.58 1004（1967）。フィ-ルドウ。Proc.Nat.Acad.Sci.U.S.58 2102（1967）。フィ-ルドウ。Proc.Nat.Acad.Sci.U.S.61 340（1968）。ィテルウ。Proc.Nat.Acad.Sci.U.S.58 1719（1967）。フィ-ルドウ。J.Gen.Physiol.56 905（1970）。デクラ-クウ。Methods in Enzymology 78.291（1981）]。
In sum, now be summarized as follows:
As Interferon, rabbit, the nucleic acid derivative table look-up of inductor
(I) homopolymer homopolymer complex body (with the double-strandednucleic acid polymkeric substance of poly IpolyC) as matrix
(1) modification alkali
Polyinosinic acid poly (5-bromo cytidylic acid), Polyinosinic acid poly (2-sulfo-cytidylic acid), poly (7-denitrogenation mix t-inosinic acid) polycytidylic acid, poly (7-denitrogenation mix t-inosinic acid) poly (5-bromo cytidylic acid).
(2) modified sugars
Poly (2 '-the nitrine t-inosinic acid) polycytidylic acid.
(3) modified phosphate
The Polyinosinic acid poly [cytidine base (シ チ ジ Application)-5 '-thiophosphoric acid].
(II) checker multipolymer
Poly (adenylic acid (AMP)-uridylic acid).
(III) homopolymer multipolymer complex body
Polyinosinic acid poly (cytidine, uridylic acid); Polyinosinic acid poly (cytidylic acid, 4-thiourdine acid).
The complex body of (IV) nucleic acid and polycation
Poly I-C poly-l-lysine (being called " poly I CLC ").
(V) other
Polyinosinic acid poly (1-vinyl cytidylic acid).
As mentioned above, many relevant two key RNA have been delivered in recent years, particularly with the report of poly IpolyC as the derivative of matrix.And the someone has summarized relevant a series of nucleic acid derivatives and structure-activity [the デ Network ラ-Network ウ thereof that contains them.Texas Reports on Biology and Medicine 41 77（1982）]。
The inventor is based on prior art, find, poly IpolyC, and with its various derivative as matrix, when distributing as the sedimentation definite value with whole bulks of molecule, by this value being adjusted into 4S～13S(base number is 50-10, about 000), then they had both kept the physiologically active of the following stated, and its toxicity greatly reduces, this inventor has been proposed patent application [spy is willing to clear 62-167433 number, and states according to the domestic priority that this application proposes].
In above-mentioned research, in order to obtain the above-mentioned purpose thing expeditiously, the inventor has carried out various researchs for following operation:
(1) distribution with molecular size is unified in 50-10, and 0000 left and right sides base is counted the interior operation of scope, and [in this manual, the distribution adjustment operation within the specific limits with molecular size is called " classification ".In addition in the present invention, because with degraded phenomenon, so classification also means " short chainization ");
(2) operation that makes it into the double-strandednucleic acid polymkeric substance with two kinds of single-chain nucleic acid polymkeric substance (is called " annealing " (ア ニ-リ ニ グ) in this manual.
The base pair (or title " bp ") that the unit of expression nucleic acid molecule size is commonly used is to represent its bulk of molecule (10bp means the dichain polymer with 10 bases) by the base number of nucleic acid.In this manual, because also will handle nucleic acid polymers beyond the dichain polymer, so use the term of " base number " to replace bp(for example, so-called " 10 base number " then represents nucleic acid polymers with 10 bases).
In the time will making nucleic acid molecule big or small specific, then use common sedimentation definite value (S value) widely, the inventor is the group in contrast of the double-stranded DNA (M13 phage fragment) with known molecular size, adopt the high performance liquid chromatography (HPLC) or the electrophoretic method of following use gel-filtration column, by comparing conversion, can try to achieve above-mentioned base number with its control group.
In the past, when the high molecular molecular weight of expression nucleic acid, used the S value widely.Now commercially available nucleic acid polymer is also with the S value representation.But, because methods such as gel electrophoresis, gel filtration chromatography, ion-exchange chromatography have been adopted in the progress of experimental technique in recent years, having set up the more accurately means of determining molecular weight, can measure now its chain length.The problem that concerns that a S value representation and chain length represent is arranged, because during with the S value representation, each nucleic acid molecule is selected proper value for use here, thereby as the method for representing nucleic acid molecular weight, whether S value representation and chain length are represented can be correctly corresponding, on this angle, is not no problem.
In specification sheets of the present invention, when expression during molecular weight, the inventor has also put down in writing the S value representation according to the convention of so far nucleic acid chemistry.But, because the S value is that the nucleic acid polymer is carried out method for measuring as whole molecular grouping (perhaps molecular conformation), in view of be necessary from now on clearer and more definite he show the boundary of molecular weight distribution, so will also be put down in writing in the lump based on the method for expressing that chain length (in this manual for " base number ") is measured.
Be from poly IpolyC and with its so-called fractionated viewpoint above as the various derivatives of matrix, when preparation fractionated double-strandednucleic acid polymkeric substance, or adopt the method that makes already present double-strandednucleic acid polymer low-molecularization, that is employing makes its degraded method after the annealing of single-chain nucleic acid polymkeric substance.But in method in the past, classification needs spended time, thereby can not handle apace, so for prepare the object aspect with technical scale its weak point is arranged.And the people is satisfied from the yield angle.
On the one hand, after the classification, when being necessary to make the single-chain nucleic acid polymkeric substance to vulcanize, be with after the hydrogen sulfide sulfuration in the past, make the hydrogen sulfide effusion of from solvent, volatilizing.That is when from the reaction solution after the sulfuration, extracting pyridine with vacuum pump out, also hydrogen sulfide is removed together, but this method is because the volatility of hydrogen sulfide, implements such reaction with technical scale and will bring problem on the public hazards countermeasure.In addition, the water layer of removing behind the pyridine then enters dialyzer, the flowing water dialysis, obtain object thus, but whole operating process of this method need three days at least, and yield is to nearly about 80%, thereby have technical difficulty in all many-sides of yield, expense and required time.
In addition, classification technique itself also may not be no problem.
In this technical field, the degraded of nucleic acid polymers always is to adopt the method that heats with the methane amide coexistence, obtain the more suitable object of chain length by the time and the temperature of regulating this reaction, after this, by means of making reaction soln carry out means such as dialysis, removing degraded over-drastic does not need material.Yet, in this method in the past, owing to,, also can cause to have the classification phenomenon that different molecular weight distributes sometimes, thereby aspect circulation ratio, remain in problem even under identical reaction conditions as the character relation of the nucleic acid polymers of raw material.It is believed that this is that the size of raw molecule often can not keep the constant cause because modulate with enzyme reaction.In addition, be applicable to dialysis technology herein, on principle, can not remove the nucleic acid polymers longer, people's radical solving method that waits in expectation than the chain of object.
The inventor continues to do various investigations, 1. can obtain classified double-strandednucleic acid polymkeric substance with rapid operation as object, 2. its yield wants high, but 3. industrialization and do not produce pollutions such as public hazards, 4. can realize quantitatively that sequence of operations and reproducibility are high as the continuous result of study of purpose, just not at all easyly finish the present invention.
The objective of the invention is to:
1. before annealing, to make single-chain nucleic acid polymkeric substance classification separately earlier,
2. when the classification of above-mentioned (1), by using HPLC(gel-filtration high performance liquid chromatography) alternative present electrophoretic method, molecular size distribution is quantized, and the variation of molecular size distribution is known in utilization thus easily, and then to determine to rake in 4S～13S(base number and be about 50～10,000) method of the object in this molecular size distribution scope
3. establish following method, promptly in the processing after classification,, improve yield by adding this extremely simple operation that lower alcohols etc. handles the way that obtained object in the past with dialysis,
4. establish following method, promptly when being necessary after the classification to make the single-chain nucleic acid polymer sulfuration,, carried out this extremely simple operation of centrifugal treating by the interpolation aromatic alcohol and improved yield with making directly evaporable way from solvent of hydrogen sulfide after the hydrogen sulfide sulfuration in the past
5. the molecular weight distribution of the single-chain nucleic acid polymkeric substance after the conduct adjustment classification, and the method that its distribution is concentrated has been suitable for ion exchange resin.
Below will above-mentioned each point be explained.
Annealing is a kind of process that makes single-chain nucleic acid polymkeric substance complimentary to one another become two strands, is original operation with regard to carrying out easily.If after annealing, carry out classification, then can produce sulphidity fluctuation equal error, build quantitatively to obtain.So, expect that classification will carry out before annealing, and the result who carries out has like this obtained extremely good result.
There is substantial connection above-mentioned (1) with (2).Use the present means that predicted its molecular weight by electrophoretic method, subsistence level one night (swimming and dyeing) is difficult to accomplish rapid processing.The inventor just can extremely promptly know the reaction times of generation as the material (that is, be 4S～13S, the base number is about 50～10,000 material) of the molecular size distribution of purpose by the HPLC gel filtration method is used for wherein.
Among the present invention,, afterwards, add lower alcohols and handle even stop when predicting to react after fractional order reaction stops.The most handy ethanol in the lower alcohols.
When using ethanol precipitation, for example obtain the same meaning of L-poly C(classified following " L-" by polyC) reaction in, can obtain object with 93% high yield, obtaining to obtain object with 78% high yield in the reaction of L-poly I by poly I.
In the past, reaction soln is sent into dialysis tube do dialysis and handle, but yield at this moment was about 60%, can only expect to reach about 40% at the reaction soln that obtains L-poly I by poly I, and about three days of operational requirement.Adopt ethanol precipitation (in reaction soln, add the ethanol of its twice quantity, object is separated out, centrifugation afterwards, collecting precipitation thing, clean and exsiccant method), then can handle within an hour through stirring.
Above-mentioned (3) are the emphasis that constitutes main idea of the present invention.After the classification by the nitrogen-atoms in the nucleic acid in the sulphur atom displacement single-chain nucleic acid polymkeric substance (for example, in certain proportion by the amino of cytidine residue among the sulfydryl displacement polyC) and become the reaction (being referred to as " sulfuration " in this specification sheets) of other nucleic acid, the method for usually using when being synthetic copolymer.In addition, the present invention also aims in said heterophasic copolymer synthetic, add aromatic alcohol and handled achieve the goal [above-mentioned (4)].
Can use such as phenol as aromatic alcohol.
When using phenol, for example in the reaction soln that is mixed with pyridine, water and hydrogen sulfide, add the phenol of half quantity, through stirring and centrifugation, water layer and phenol layer clearly separate, and make the face bag of reaction soln and by product sulphur etc. also move to the phenol layer.Afterwards, separate water layer, handle making object separate out centrifugation again by salt solution, alcohol,, can obtain pure object by cleaning alcohol.
If adopt this method, because the hydrogen sulfide major part is included in the clear liquid on the precipitation of solution, so can easily remove.
For example, the method for the present invention of phenol is used in employing, operates within an hour to finish, and yield is almost 100, and can obtain object quantitatively.
To carry out classification be important before being implemented in annealing in the method for the uniqueness among above-mentioned the present invention (2)～(4), and be in order to use before the so-called annealing on the fractionated gimmick method that can use freely fully.
Below be another feature of the present invention, with the method and [above-mentioned (5)] of the size qualification between explanation classification and the annealing.
In this method, utilize ion exchange resin.As nucleic acid is that the example that has been suitable for ion exchange resin in the polymer has [BBA such as the DEAE-Mierocrystalline cellulose used at t-RNA, DEAE-dextrane gel, benzoylation DEAE-Mierocrystalline cellulose; 47; 193(1961), BBRC 10,200(1963), Biochem.6,3043(1967)].In this example, only in being about 80 low molecular nucleic acid refining, the base number uses ion exchange resin at the most.
The inventor has carried out various investigation results to the person's character at the adsorption charge that whether may answer during as index refining spent ion exchange resin to be had with the base number of high molecular weight nucleic acid polymkeric substance, has established the present invention.The present invention because its desired substance is exceedingly useful as medicine, has obvious improvement so be sure of the size qualification (also being referred to as " ion exchange method " in this specification sheets) of using this ion exchange resin.
In ion exchange method of the present invention, ion exchange resin only coexists with nucleic acid polymers and just can achieve the goal (batch process) in the same container, and uses column chromatography demarcate (post method) usually.Ion exchange resin is packed in post etc., the liquid that has dissolved nucleic acid polymers is flowed into, in case it is adsorbed in the ion exchange resin, on one side make the elutriant of salt-tris buffer etc. pass through post thereafter, make its common salt concn become linearity or segmentation on one side, obtain a certain amount of effluent liquid, then with the base number that similarly contained the nucleic acid polymers of various cuts in the past with HPLC gel filtration method handle, with the telltale is that index predicts, thereby can obtain the cut through wash-out of object.
In order to achieve the goal with above-mentioned ion exchange method, the kind of the ion exchange resin of filling in the post and the kind of elutriant become epochmaking factor.
For example, poly I is dissolved in hydrochloric acid-tris buffer, make it be adsorbed on the QAE(fourth stage amino-ethyl that the size that is used for poly I limits again) carrier and carry out the result of ion exchange method, even the salt concn of elutriant is extremely dense, also fail to obtain object.This is because as long as in the moderate eluent salt CONCENTRATION STATE of poly I in the QAE carrier, poly I itself is insoluble.Can structurally have more hydrophobicity explanation by t-inosinic acid as the component unit of poly I.Here, moderate eluent salt concentration is fixed with the polyC comparison.
In other experiments that the inventor did, in the damping fluid of the moderate elutriant common salt concn of poly I, dissolved poly I is the gonorrhoea shape in the QAE carrier, can observe and produce sedimentary phenomenon.Therefore, in the ion exchange method of the present invention, select the kind and the wash-out salt concn of ion exchange resin to become extremely important key element.
That is to say, under the situation of poly I, use the DEAE carrier, obtain very good result.Under poly I situation, no matter the QAE carrier, no matter the DEAE carrier can both obtain good result.Wash-out can be by adopting the linear concentration gradient or the sectional concentration gradient of salt, makes polymkeric substance boundary wash-out with the order of chain length.
For example, use the polyC(38 milligram, S
208.6), be adsorbed on 10 * 130 millimeters of DEAE-Toyopearl 650C(φ) in, with linear flow speed 1.32 centimeters/minute, 175/cut wash-out, A=OM Na Cl/10mM hydrochloric acid-tris buffer (pH7.0) B=0.5M Na Cl/10mM hydrochloric acid-tris buffer (pH7.0) are respectively with 100 milliliters, with B%0 → 100, adopt the linear concentration gradient result, by the order of following chain length wash-out clearly.
Cut base number
33 340
34 470
35 740
36 1000
37 1500
In addition, if same sample is carried out with the sectional concentration gradient, then when 0.3M Na Cl/10mM hydrochloric acid-tris buffer (pH7.0) (50 milliliters), can demarcate below the 500 base numbers, follow at 0.5M Na Cl/100mM hydrochloric acid-tris buffer (pH7.0) (50 milliliters), 500～1500 base numbers of can demarcating.
Equally, under identical conditions, carry out with linear concentration, then can be by following sequentially eluting poly I(7.8 milligram 1, S
207.3).
Cut base number
35 30
36 140
37 230
38 350
39 460
40 540
In addition, if same sample carries out with the sectional concentration gradient, then can demarcate below the 3000 base numbers at 0.3M Na Cl/10mM hydrochloric acid-tris buffer (pH7.0) (50 milliliters), follow at 0.5M Na Cl/10mM hydrochloric acid-tris buffer (pH0.7) (50 milliliters), 300～600 base numbers of demarcating.
Like this, by selecting common salt concn arbitrarily, the high molecular nucleic acid limited nucleic acid of chain length of all can demarcating.
Shown in two above-mentioned examples, the maximum purpose of ion exchange method of the present invention is to have the chain length distribution material (main component) that is fit to do the character of medicine in drug effect at random and on a large scale to tell from the nucleic acid macromolecule mixture that is made of various chain lengths.
, during as the medicine of injection, knew indispensable so-called operation of removing the pyrogen that in agent, cannot sneak into (pyrogen, known it constitute) already by polysaccharide in preparation.In the nucleic acid derivative relevant with the present invention, when it is delivered medicine to man-hour as injection, this also must become the problem that must solve.
The inventor has fortunately found to remove the method for this pyrogen by adopting above-mentioned ion exchange method.
The inventor proceeds experiment, and purpose is more carefully to verify above-mentioned phenomenon serendipitous, found that, to the single-chain nucleic acid derivative, no matter its chain length how, all available ion exchange method is removed thermal source, thereby finishes the present invention.
Therefore, this also is one of free-revving engine of the present invention.
The following table that the results are shown in by the endotoxic quantitative test of the test of single stranded RNA (commercially available poly C and the material that makes its short chainization).
In the table: on behalf of family, EU exempt from the unit [endotoxin unit: USP reference standard endotoxin(E.coli 0113)] of heat generation test.
1. represent distilled water for injection (blank), 2. represent poly(commercial goods-1), 3. represent polyC(commercial goods-2), 4. represent the material among the following embodiment (5-4).
Therefore the effect of the removal pyrogen of ion exchange method is tangible.
As medicine, the physiologically active of nucleic acid derivative of the present invention is exceedingly useful, and as described below, nucleic acid derivative of the present invention has strong antitumous effect.This effect only is a kind of in several physiologically actives of holding of nucleic acid derivative of the present invention.
Except being the physiologically active of nucleic acid derivative of the present invention of matrix with poly IpolyC, also can enumerate TNF produce can, Interferon, rabbit produce can, a leukin produces propagation interception in failing the cancer nude mice of energy, giant bacteriophage activation energy, the activation to the NK cell, tumor cell proliferation interception, human tumor cells, the lung metastasis inhibition effect of tumour cell etc.
Nucleic acid derivative of the present invention and the Interferon, rabbit of before poly IpolyC etc. spread out and lead thing and compare, and have high security.Therefore, compound of the present invention can be made disease-resistant plain agent, antineoplastic agent etc.
The physiological action of above-mentioned nucleic acid derivative of the present invention is described in detail in the specification sheets of above-mentioned patent application (application that requires domestic priority that special hope reaches based on it for clear 62-1676433 number).
Below disclosed relevant nucleic acid derivative preparation method's of the present invention embodiment, so that the present invention is described in more detail.
Embodiment
(1) the poly I of L-poly I(classificationization) preparation and purification
In 10 commercially available gram poly I, add 200 ml distilled waters, 250 milliliters of methane amides, and 500 milliliters 5M salt solution, heated 4 hours down at 80 ℃.
Employing has the high performance liquid chromatography of tsk gel G-DNA-PW post (7.88 millimeters (diameter) * 300 millimeter), and [elutriant is 50mM Tutofusin tris hydrochloride buffer (pH7.5), 0.3M salt, 2mM ETDA, flow velocity is 0.5 ml/min], gel-filtration, retention time reaches maximum value 21.86 ± 0.2(branch) time, termination reaction.
To the ethanol of 2 times of amounts of reaction solution adding, collect the throw out that is generated with centrifugation (3000 rev/mins, 4 ℃), after 70% ethanol was cleaned, vacuum-drying obtained 102 gram L-poly I thus.
In addition, all use the water and the aqueous solution of sterilizing state in the present embodiment, following examples together.
(2) the normalized polyC of L-poly C() preparation and purification
In 10 gram polyC, add the salt solution of 200 ml distilled waters, 250 milliliters of methane amides, 50 milliliters of 5M, heated about 4 hours down at 80 ℃.Adopt the high performance liquid chromatography gel-filtration means identical, confirm the terminal point (is 21.33 ± 0.2 timesharing) of reaction when keeping with above-mentioned (1).
To the ethanol of 2 times of amounts of reaction solution adding, by centrifugation (3000 rev/mins, 4 ℃) collecting precipitation thing, after 70% ethanol is cleaned, carry out vacuum-drying, promptly get the L-poly C of 9.5 grams thus.
(3) sulfuration of L-poly C
The 8.0 gram L-poly C of above-mentioned (2) gained are dissolved in 240 ml waters, place then in 500 milliliters of still kettles, under ice-cooled, add the pyridine solution (/ 120 milliliters of 12 grams) of hydrogen sulfide, behind the tube sealing, heated about 10 hours down at 50 ℃.After the cooling, add the saturated phenol of TE (200 milliliters), after the stirring, centrifugation (3000 rev/mins, 15 ℃, 5 minutes), in water layer, add 1/10(water layer amount 1/10) ethanol of the 5M salt solution of amount and 2 times (water layer amount 2 times) amount, produce precipitation immediately.Centrifugation (3000 rev/mins, 4 ℃, 10 minutes) after, clean with 70% ethanol, again through vacuum-drying, promptly obtain 8.0 gram L-poly(C
20S
4U) (mean in fractionated poly C have one in per 20 cytidylic acids) by the displacement of 4-sulfo-Methionin.
In addition, above-mentioned so-called TE ties up to adding EDTA in the 10mM Tutofusin tris hydrochloride buffer (pH7.5), makes latter's concentration reach 1mM, and the solution that is mixed with is called TE thus.
(4) annealing one example
Resultant L-poly(C from above-mentioned (3)
20, S
4U) take out 6.00 grams in, and take out 6.44 grams in above-mentioned (1) among the poly I of gained, respectively they are dissolved in 300 ml solns of being formed by 10mM Tutofusin tris hydrochloride buffer (pH7.5) and 50mM salt solution, mix then.The mixed solution of gained is heated to 70 ℃ in water-bath, is incubated after 10 minutes, intactly cool overnight.Through phenol handle and with ethanol sedimentation after, in the throw out that is generated, add water (about 200 milliliters), make its dissolving, dialysis in water, temperature is 4 ℃, dialyzate is concentrated into dried, promptly get 12.4 and restrains the annealed compounds.
(5) size with ion exchange method limits
Following ion exchange method can be undertaken by classification wash-out or linear gradient elution.If correctly select elution requirement, then under any circumstance, yield and almost can keep indefinite as the chain length of target.L-poly C and L-poly(C, S
4U) classification wash-out all uses 0.15M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0), 10MNa Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) to carry out continuously.
The classification wash-out of L-poly I can be with reference to following embodiment (5-1).
The linear gradient elution of L-poly I
With A[0M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0)], B[0.5M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) is an elutriant, and is to carry out wash-out under 0 to 100% the gradient condition at the percentage ratio of B liquid.L-poly C and L-poly(C, S
4U) linear gradient elution can reach (5-4) with reference to embodiment (5-2).
(5-1) size of L-poly I limits
210 milligrams of L-poly I of above-mentioned (1) gained are dissolved in 5 milliliters the 10mM Tutofusin tris hydrochloride buffer (pH7.0), make it be adsorbed on the DEAE-Toyopearl(registered trade mark then) on the 650C(φ 100mm * 130mm), be the elutriant of 0.03M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH 7.0(50 milliliter) and 0.5M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) (80 milliliters) then, carry out wash-out with the linear velocity of 1.30 centimeters/minute and the mode of classification wash-out with salt concn.Fraction when collecting with 0.5M Na Cl wash-out, and use the high performance liquid chromatography gel filteration determining retention time identical with above-mentioned (1), the value of gained is 21.90 ± 0.2 minutes.
Under 91% high yield, obtain a kind of L-poly I compound, wherein the base number as target is limited in the scope of 100-1000.
(5-2) size of L-poly C limits
610 milligrams of L-poly C of aforementioned (2) gained are dissolved in 10 milliliters the tris buffer (pH7.0), make it to be adsorbed on the QAE-Toyopearl(registered trade mark then) on the 550C(φ 10mm * 130mm), then under the linear velocity of 1.30 centimeters/minute, be 0.0M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) with 100 milliliters A[concentration respectively], B[concentration is 1.0M NaCl/10mM Tutofusin tris hydrochloride buffer (pH7.0)] elutriant, and be to carry out wash-out under 0 to 100 the linear gradient elution condition at the percentage ratio of B liquid.Collect the fraction in the main peak elution process, with high performance liquid chromatography gel filteration determining retention time, the value of gained is 21.35 ± 0.2 minutes.Under 93% high yield, obtain a kind of L-poly C compound, wherein the base number as target is limited in the scope of 100-1000.
(5-3) L-poly(C
12, size U) limits
With 19 milligrams of L-poly(C
12U) (mean that having one in per 12 cytidylic acids is replaced by uridylic acid) and (adopt the same high performance liquid chromatography, the retention time that records is 18.67 minutes) be dissolved in 5 milliliters of Tutofusin tris hydrochloride buffers (pH7.0), make it to be adsorbed on the DEAE-Toyopaerl(registered trade mark then) among the 650C(φ 10mm * 130mm), be 0.0M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) with 100 milliliters A[concentration respectively then], B[concentration is 0.5M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0)] elutriant, linear velocity from 1.30 centimeters/minute, and be under the condition of linear gradient elution of 0-100 at the percentage ratio of B liquid, carry out wash-out.Collect the fraction in the main peak elution process, and with high performance liquid chromatography gel filteration determining retention time, the numerical value of gained is 18.97 ± 0.2 minutes.Under 87% high yield, obtain a kind of L-poly(C
12, U), wherein the base number as target is limited in the scope of 100-1000.
(5-4) L-poly(C
20, S
4U) size limits
600 milligrams of L-poly(C with aforementioned (3) gained
20, S
4U) be dissolved in 10 milliliters of Tutofusin tris hydrochloride buffers (pH7.0), make it to be adsorbed on the QAE-Toyopearl(registered trade mark then) on the 550C(φ 10mm * 130mm), be 0.0M Na Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0) with 100 milliliters of A[concentration respectively then], B[1.0M Na Ca Cl/10mM Tutofusin tris hydrochloride buffer (pH7.0)] elutriant, with the linear velocity of 1.30 centimeters/minute, and be to carry out wash-out under the linear gradient elution condition of 0-100 at B liquid at percentage ratio.According to the identical high performance liquid chromatography gel filteration determining retention time described in above-mentioned (5-2), the numerical value of gained is 21.35 ± 0.2 minutes.
Under 90% high yield, obtain a kind of L-poly(C
20, S
4U), in the scope of the 100-1000 that wherein limits as the base number of target.
(6) annealing
(6-1) L-poly I and L-poly C
L-poly C3.0 gram that the size of gained in aforementioned (5-2) is limited and the L-poly I3.2 that the size of gained limits in (5-1) restrain and are dissolved in respectively in 150 milliliters of solution of being made up of Tutofusin tris hydrochloride buffer (pH7.5) and 50mM salt solution, mix then.The mixture of gained is heated to 70 ℃ in water-bath, is incubated after 10 minutes cool overnight under intact state.After phenol is handled, use ethanol sedimentation, in the throw out that is generated, add 400 ml waters approximately, make it dissolving, dialysis in water then, temperature is 4 ℃.With dialyzate be concentrated into do after, promptly obtain 6.2 gram annealing compounds.
(6-2) L-poly I and L-poly(C
20, S
4U)
By the described L-poly(C that handles the size qualification of gained in aforementioned (5-4) with quadrat method in above-mentioned (6-1)
20, S
4U) the poly I1.57 gram that the size of gained limits among 1.46 grams and aforementioned (5-1) obtains 3.0 gram annealing compounds thus.
Claims (5)
1, a kind of preparation method of nucleic acid derivative, it is characterized in that in preparation with RNA as matrix, and sedimentation definite value (being distributed as the sedimentation definite value with all bulks of molecule) is in the interior double chain acid derivative process of 4S-13S scope, makes the classification before annealing of each nucleic acid polymers.
2, a kind of preparation method of nucleic acid derivative, it is characterized in that in preparation with RNA as matrix, and the base number is 50-10, the molecule of 000(abundance maximum in whole molecular size distribution) in the double chain acid derivative process of scope, make the classification before annealing of each nucleic acid polymers.
3, a kind of preparation method of strand multipolymer is characterized in that the nucleic acid that constitutes the single-chain nucleic acid polymkeric substance being carried out in the sulfurized process after classification and before the annealing by claim 1 or 2 described methods, adds aryl alcohol and handles.
4, method according to claim 1 and 2 is characterized in that after the spent ion exchange resin processing classification and the double-strandednucleic acid polymkeric substance before the annealing, the distribution of its molecular size is concentrated within the specific limits, thereby size is limited.
5, the method for a kind of removal pyrogen (パ イ ロ ジ ェ Application) is characterized in that spent ion exchange resin is handled in the injection preparation process that with the nucleic acid derivative is main component.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP16743487 | 1987-07-03 | ||
| JP167434/87 | 1987-07-03 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1030424A true CN1030424A (en) | 1989-01-18 |
| CN1024556C CN1024556C (en) | 1994-05-18 |
Family
ID=15849635
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN88104080A Expired - Fee Related CN1024556C (en) | 1987-07-03 | 1988-06-29 | Nucleic acid derivatives and process for preparing them |
Country Status (19)
| Country | Link |
|---|---|
| KR (1) | KR890002226A (en) |
| CN (1) | CN1024556C (en) |
| AT (1) | AT397658B (en) |
| AU (1) | AU1864488A (en) |
| CH (1) | CH676123A5 (en) |
| DE (1) | DE3822406A1 (en) |
| DK (1) | DK369088A (en) |
| ES (1) | ES2007252A6 (en) |
| FI (1) | FI883163A (en) |
| FR (1) | FR2617403B1 (en) |
| GB (1) | GB2207138B (en) |
| HU (1) | HU202250B (en) |
| IL (1) | IL86884A0 (en) |
| IT (1) | IT1224504B (en) |
| NL (1) | NL8801664A (en) |
| NO (1) | NO882922L (en) |
| PT (1) | PT87903B (en) |
| SE (1) | SE8802480L (en) |
| ZA (1) | ZA884698B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117517551A (en) * | 2023-11-03 | 2024-02-06 | 成都迈科康生物科技有限公司 | Method for detecting polyinosinic cell content by adopting high performance liquid chromatography |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4963532A (en) * | 1987-11-25 | 1990-10-16 | Hem Research, Inc. | dsRNA-based prevention of viral escape |
| RU2222334C2 (en) | 1998-05-25 | 2004-01-27 | Ниппон Синяку Ко., Лтд. | Method for preparing nucleic acid-containing composite preparation |
| CN1194002C (en) * | 1999-02-15 | 2005-03-23 | 日本新药株式会社 | Shortened-chain polynucleotide and process for the preparation thereof |
| EP2930180B1 (en) * | 2012-12-06 | 2021-11-10 | Kyowa Hakko Bio Co., Ltd. | Double-stranded ribonucleic acid for adjuvants |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| NZ194880A (en) * | 1979-09-17 | 1983-07-15 | Merck & Co Inc | Interferon-inducing composition containing modified polyriboinosinic-polyribocytidylic acid |
-
1988
- 1988-06-27 GB GB8815262A patent/GB2207138B/en not_active Expired - Fee Related
- 1988-06-27 IL IL86884A patent/IL86884A0/en unknown
- 1988-06-29 CN CN88104080A patent/CN1024556C/en not_active Expired - Fee Related
- 1988-06-29 AT AT0169988A patent/AT397658B/en not_active IP Right Cessation
- 1988-06-29 CH CH2467/88A patent/CH676123A5/fr not_active IP Right Cessation
- 1988-06-30 NO NO88882922A patent/NO882922L/en unknown
- 1988-06-30 NL NL8801664A patent/NL8801664A/en not_active Application Discontinuation
- 1988-06-30 ZA ZA884698A patent/ZA884698B/en unknown
- 1988-07-01 IT IT8848147A patent/IT1224504B/en active
- 1988-07-01 FI FI883163A patent/FI883163A/en not_active IP Right Cessation
- 1988-07-01 HU HU883461A patent/HU202250B/en not_active IP Right Cessation
- 1988-07-01 FR FR8808938A patent/FR2617403B1/en not_active Expired - Fee Related
- 1988-07-01 DK DK369088A patent/DK369088A/en not_active Application Discontinuation
- 1988-07-01 PT PT87903A patent/PT87903B/en not_active IP Right Cessation
- 1988-07-01 ES ES8802078A patent/ES2007252A6/en not_active Expired
- 1988-07-01 SE SE8802480A patent/SE8802480L/en not_active Application Discontinuation
- 1988-07-01 AU AU18644/88A patent/AU1864488A/en not_active Abandoned
- 1988-07-01 DE DE3822406A patent/DE3822406A1/en not_active Withdrawn
- 1988-07-02 KR KR1019880008258A patent/KR890002226A/en not_active Application Discontinuation
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117517551A (en) * | 2023-11-03 | 2024-02-06 | 成都迈科康生物科技有限公司 | Method for detecting polyinosinic cell content by adopting high performance liquid chromatography |
Also Published As
| Publication number | Publication date |
|---|---|
| GB2207138B (en) | 1992-02-05 |
| IL86884A0 (en) | 1988-11-30 |
| HU202250B (en) | 1991-02-28 |
| DK369088A (en) | 1989-01-04 |
| AU1864488A (en) | 1989-04-27 |
| AT397658B (en) | 1994-06-27 |
| GB8815262D0 (en) | 1988-08-03 |
| NO882922L (en) | 1989-01-04 |
| PT87903B (en) | 1995-03-01 |
| ATA169988A (en) | 1993-10-15 |
| KR890002226A (en) | 1989-04-10 |
| FI883163A (en) | 1989-01-04 |
| CH676123A5 (en) | 1990-12-14 |
| SE8802480D0 (en) | 1988-07-01 |
| FI883163A0 (en) | 1988-07-01 |
| HUT48272A (en) | 1989-05-29 |
| IT1224504B (en) | 1990-10-04 |
| DK369088D0 (en) | 1988-07-01 |
| PT87903A (en) | 1989-06-30 |
| DE3822406A1 (en) | 1989-01-12 |
| CN1024556C (en) | 1994-05-18 |
| SE8802480L (en) | 1989-01-04 |
| NO882922D0 (en) | 1988-06-30 |
| FR2617403A1 (en) | 1989-01-06 |
| NL8801664A (en) | 1989-02-01 |
| FR2617403B1 (en) | 1993-05-07 |
| IT8848147A0 (en) | 1988-07-01 |
| ES2007252A6 (en) | 1989-06-01 |
| GB2207138A (en) | 1989-01-25 |
| ZA884698B (en) | 1989-03-29 |
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