CN103145714A - Feather weight PNA (pentose nucleic acid) synthesis method - Google Patents

Abstract

The invention discloses a feather weight PNA (pentose nucleic acid) synthesis method. The method comprises the following steps: reacting ethylene diamine with chloroacetic acid, and recrystallizing with DMSO to obtain N-(2-aminoethyl)glycine; by taking the N-(2-aminoethyl)glycine as a reactant, adding raw materials, and reacting to obtain N-(2-Fmoc-aminoethyl)glycine methyl ester hydrochloride; by taking adenine as a reactant, adding raw materials, and reacting to obtain 9-ehtyl acetate adenine; by taking the 9-ehtyl acetate adenine and carbonyl diimidazole as reactants, adding raw materials, and reacting to obtain 6-N-(benzhydryloxycarbonyl)adenine-9-acetic acid; by taking the 6-N-(benzhydryloxycarbonyl)adenine-9-acetic acid as a reactant, adding raw materials, and reacting to obtain the PNA. The feather weight PNA synthesis method belongs to large-scale production technology, does material preparation for the large-scale research of the PNA, as well as technological preparation for the future large-scale application.

Description

The method of the synthetic PNA of feather weight

Technical field

The present invention relates to the method for synthetic PNA a kind of, be specifically related to the method for the synthetic PNA of a kind of feather weight.

Background technology

Oligonucleotide is widely used on medicine for treatment thing, molecular biology research instrument and diagnostic reagent.The main restriction that oligonucleotide is used is that it is easily degraded, hybridizes rear poor heat stability and can be with the non-specific binding such as albumen etc. by nuclease.Be physics and chemistry and the biological characteristics of transformation oligonucleotide, many oligonucleotide analogs are found out.Peptide nucleic acid(PNA) is outbalance a kind of in oligonucleotide mimetic.Nielsen in 1991 etc. have reported with N-(2-amino-ethyl) glycine skeleton and have replaced sugar-phosphoric acid ester skeleton as repeated structural unit, synthesized the oligonucleotide mimetic that connects with peptide bond, be called peptide nucleic acid(PNA) (Peptide nucleic acid, PNA).Although PNA structurally relative oligonucleotide has had significant change, but the base complementrity pair principle is still followed in the combination between PNA and complementary nucleic acid, even have higher affinity than natural nucleotide, the ability of stronger nuclease-resistant and proteolysis enzyme liberating is arranged.The scientific research personnel designs the PNA that has synthesized various structures, optimizing its biological stability and availability, target binding characteristic and pharmacokinetic properties, and PNA is applied to the aspects such as diagnosis and treatment, thereby has started a new research field.

Various structure modules are used to synthetic PNA and analogue thereof, comprise skeleton structure, connect chirality and achiral group on N-(2-amino-ethyl) glycine, the type of base etc.The skeleton monomer of classical PNA is N-(2-amino-ethyl) glycine, connects the derivative of base on the nitrogen of glycine.Usually first synthetic end group N has the amino-ethyl glycinate of protecting group, and then the base derivative is connected on not protected nitrogen.Method commonly used has: the reduction reaction of alkylated reaction, Schiff's base, Mitsunobu reaction.

Four kinds of bases are all that the alkylated reaction through amine forms the base acetogenin, then adopt the synthetic method of common polypeptide, connect the not protected nitrogen on base acetic acid and skeleton.The alkylated reaction of thymus pyrimidine does not need to use blocking group usually, therefore when with the bromacetate reaction again through saponification or directly react with bromoacetic acid and get final product to get thymus pyrimidine acetic acid.On its excess-three kind base, reactive group is arranged, need first to be protected.Reactive group on cytosine(Cyt) is the amino on 4; selectable protecting group has: carbobenzoxy-(Cbz) (benzyloxycarbonyl; Cbz), to tert.-butylbenzene formyl radical (4-tert-butylbenzoyl; 4-t-BuBz), benzoyl (benzoyl; Bz) and Mmt etc.; carry out alkylated reaction with bromacetate again, then saponification namely gets the derivative of cytosine(Cyt) acetic acid.Protection process and the cytosine(Cyt) of VITAMIN B4 are basic identical, and available protecting group has Cbz, Mmt, p-methoxyphenyl (anisoly, An).The protection more complicated of guanine need to avoid the alkylating side reaction of N7 to disturb in the alkylating process of N9.A kind of method commonly used is to use the 2-amido-6-chloropurine in alkylation; reflux under acidity or alkaline condition again after alkylation; the chlorine water solution is converted into carbonyl; or direct alkylation N2 is connected with the VITAMIN B4 of protecting group; two kinds of alkylates of chromatographic separation N7/N9, then saponification gets the derivative of guanine acetic acid.

At present domestic had part company can customize synthetic PNA series product, but theirs is synthetic small, and generally in the milligram rank, this situation has restricted application and the research work of this compound of PNA greatly.

Summary of the invention

For addressing the above problem, the purpose of this invention is to provide the method for the synthetic PNA of a kind of feather weight, by the method for column chromatography, do not produce in a large number highly purified PNA.

For realizing above-mentioned technical purpose, reach above-mentioned technique effect, the present invention is achieved through the following technical solutions:

The method of the synthetic PNA of feather weight comprises the following steps:

Step 1) 1 liter of there-necked flask, the 500 milliliters of quadrols of packing into, ice bath be cooled to 10 the degree, with 75 gram Mono Chloro Acetic Acids in batches the access control temperature 10 the degree below, every batch add after, after being completely consumed, observation adds again next batch, after reinforced complete, the ambient temperature overnight reaction is spin-dried for quadrol, reclaiming quadrol can recycle, with oil pump, quadrol is removed totally as far as possible, obtained the white powder crystal with the DMSO recrystallization, be N-(2-aminoethyl) glycine;

step 2) 50 gram N-(2-aminoethyl) glycine is dissolved in the mixing solutions of 850 milliliters of methylene dichloride and 50 milliliters of DMF, add 134 milliliters of (1.05mol) trimethylchlorosilanes, reaction mixture stirring at room 40 minutes, then be cooled to 5 degree, 134 gram Fmoc-OSu are added in top mixture, drip the NMM186 milliliter under 5 degree, dripped off in 30 minutes, stirred 2 hours at this temperature, then add 100 ml methanol, stirred again 20 minutes, add 1 liter of ethyl acetate, cross filter solid after 20 minutes, wash with ethyl acetate, the product powder is spin-dried for, making beating washing in product water, filter washing, drying obtains sterling, be N-(2-Fmoc-aminoethyl) glycine,

Step 3) 12 gram N-(2-Fmoc-aminoethyl) glycine is dissolved in the anhydrous methanol of 100 milliliters, 7.72 the milliliter thionyl chloride splashes under ice bath, continues reaction 15 minutes after dripping off at this temperature, then refluxes 2 hours, remove solvent, ether with 9/1 and dissolve with methanol, 0 degree crystallization filters, the ether washing, drying obtains sterling, is N-(2-Fmoc-aminoethyl) glycine methyl ester hydrochloride;

step 4) VITAMIN B4 of 389 gram dryings is suspended in the DMF of 6 liters, the sodium hydride that adds 40 grams, reaction solution stirs, there is hydrogen to overflow, stir after 15 minutes, add again 42 gram sodium hydrides, stir after 1 hour, there are a large amount of solids to separate out, stir difficulty, the DMF that adds again 1.5 liters, continue to stir 3 hours, the ethyl bromoacetate of 351 milliliters dropwised within half hour, there is ice bath to maintain the temperature in 20-30 degree scope in this process, then stirred overnight at room temperature, steam except volume, add 3 premium on currency, stirred one hour, cross filter solid, wash three times, solid suspension is in 2 liters of ethanol, stirring and refluxing 45 minutes, be as cold as room temperature, stirring is spent the night, suction filtration, get solid, washing with alcohol, dry, get sterling, be 9-ethyl acetate VITAMIN B4,

Step 5) 433 gram 9-ethyl acetate VITAMIN B4 and 478 gram carbonyl dimidazoles are suspended in the DMF of 4 liters, reaction solution slowly is heated to 105 degree, then stirs 2 hours at this temperature, reduce the temperature to 95 degree, add 541 gram benzhydrols, remove heating, reaction solution stirs and spends the night, and the water of 2.5 times adds reaction solution, rapid stirring 1 hour, solid is separated out, and filters, and washes three times, recrystallizing methanol, obtain sterling, be 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-ethyl acetate, pistac;

step 6) 6-N-(hexichol methoxycarbonyl) VITAMIN B4 of 300 grams-9-ethyl acetate is suspended in the mixed solvent of 1.5 liters of ethanol and 1.5 liters of acetonitriles, be heated to dissolving, add 1 premium on currency, there is solid to separate out, be chilled to below 10 degree, 294 gram lithium hydroxides are dissolved in 2.5 premium on currency, add top suspension liquid, temperature rises to 20 degree, continue to stir 6 minutes, temperature rises to 25 degree, the citric acid solution that the ratio that adds 3 liters, water in 1346 gram citric acids is prepared adds reaction solution, stirred 10 minutes, add 1 premium on currency, cryosel is bathed cooling, until a large amount of solids are separated out, continue to stir half hour, filter, washing, obtain sterling, be 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid,

step 7) 2 gram 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid is dissolved in the mixed solvent of 5 milliliters of acetonitriles and 5 milliliters of DMF, add 1.9 gram benzo three nitrogen-N, N, N, N '-tetramethyl-urea hexafluorophosphate, reaction solution is chilled to 0 degree, drip 1.6 milliliters of N-methylmorpholines at this temperature, stirring reaction once hour after, C with 1.896 grams under 0 degree once adds reaction solution, this reaction solution stirring at room one hour, rear room temperature reaction two hours, be cooled to 0 degree, add the layering of aquatic products thing, separatory washing three times, get organic phase, be spin-dried for organic solvent and obtain thick product 3.7 gram Fmoc-PNA-A (Bhoc)-OMe,

step 8) the thick product F moc-PNA-A (Bhoc) of 3.7 grams-OMe is dissolved in the tetrahydrofuran (THF) of 25 milliliters, be chilled to zero degree, it is the lithium hydroxide aqueous solution of 5mol/l with 10 ml concns, vigorous stirring slowly is added dropwise in reaction solution, temperature is controlled at about zero degree, there is solid to separate out, continue at this temperature to stir 20 minutes, TLC follows the tracks of and is as the criterion, react complete, add in advance in 6 gram citric acids and add the aqueous citric acid solution that the ratio of 10 ml waters prepares, be chilled to subzero crystallization, filter after stirring half hour, wash three times, wash low polar impurity off with the ether making beating after the solid drying that obtains, filtration drying, obtain sterling Fmoc-PNA-A (Bhoc)-OH, purity is 95%,

Process is as follows:

The invention has the beneficial effects as follows:

The invention belongs to large-scale production process, carry out material for the broad scale research of PNA and prepare, and carry out technological preparation for later large-scale application.

Above-mentioned explanation is only the general introduction of technical solution of the present invention, for can clearer understanding technique means of the present invention, and can be implemented according to the content of specification sheets, below is described in detail as follows with preferred embodiment of the present invention.The specific embodiment of the present invention is provided in detail by following examples.

Embodiment

Below in conjunction with embodiment, describe the present invention in detail.

The method of the synthetic PNA of feather weight comprises the following steps:

Step 1) 1 liter of there-necked flask, the 500 milliliters of quadrols of packing into, ice bath are cooled to 10 degree, with 75 gram Mono Chloro Acetic Acids in batches the access control temperature below 10 degree, every batch add after, observation adds next batch after being completely consumed again, reinforced complete after, the ambient temperature overnight reaction, quadrol is spin-dried for, reclaiming quadrol can recycle, and with oil pump, quadrol is removed totally as far as possible, obtains the white powder crystal with the DMSO recrystallization, be N-(2-aminoethyl) glycine, yield 40-50%;

step 2) 50 gram N-(2-aminoethyl) glycine is dissolved in the mixing solutions of 850 milliliters of methylene dichloride and 50 milliliters of DMF, add 134 milliliters of (1.05mol) trimethylchlorosilanes, reaction mixture stirring at room 40 minutes, then be cooled to 5 degree, 134 gram Fmoc-OSu are added in top mixture, drip the NMM186 milliliter under 5 degree, dripped off in 30 minutes, stirred 2 hours at this temperature, then add 100 ml methanol, stirred again 20 minutes, add 1 liter of ethyl acetate, cross filter solid after 20 minutes, wash with ethyl acetate, the product powder is spin-dried for, making beating washing in product water, filter washing, drying obtains sterling, be N-(2-Fmoc-aminoethyl) glycine, yield 60%,

Step 3) 12 gram N-(2-Fmoc-aminoethyl) glycine is dissolved in the anhydrous methanol of 100 milliliters, 7.72 milliliters of thionyl chlorides splash under ice bath, continue reaction 15 minutes after dripping off at this temperature, then refluxed 2 hours, remove solvent, the ether with 9/1 and dissolve with methanol, 0 degree crystallization, filter, the ether washing, drying obtains sterling, be N-(2-Fmoc-aminoethyl) glycine methyl ester hydrochloride, yield 90%;

step 4) VITAMIN B4 of 389 gram dryings is suspended in the DMF of 6 liters, the sodium hydride that adds 40 grams, reaction solution stirs, there is hydrogen to overflow, stir after 15 minutes, add again 42 gram sodium hydrides, stir after 1 hour, there are a large amount of solids to separate out, stir difficulty, the DMF that adds again 1.5 liters, continue to stir 3 hours, the ethyl bromoacetate of 351 milliliters dropwised within half hour, there is ice bath to maintain the temperature in 20-30 degree scope in this process, then stirred overnight at room temperature, steam except volume, add 3 premium on currency, stirred one hour, cross filter solid, wash three times, solid suspension is in 2 liters of ethanol, stirring and refluxing 45 minutes, be as cold as room temperature, stirring is spent the night, suction filtration, get solid, washing with alcohol, dry, get sterling, be 9-ethyl acetate VITAMIN B4, yield 68%,

Step 5) 433 gram 9-ethyl acetate VITAMIN B4 and 478 gram carbonyl dimidazoles are suspended in the DMF of 4 liters, reaction solution slowly is heated to 105 degree, then stirred 2 hours at this temperature, reduce the temperature to 95 degree, add 541 gram benzhydrols, remove heating, reaction solution stirs and spends the night, 2.5 water doubly adds reaction solution, rapid stirring 1 hour, solid is separated out, and filters, wash three times, recrystallizing methanol obtains sterling, is 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-ethyl acetate, pistac, yield 73%;

step 6) 6-N-(hexichol methoxycarbonyl) VITAMIN B4 of 300 grams-9-ethyl acetate is suspended in the mixed solvent of 1.5 liters of ethanol and 1.5 liters of acetonitriles, be heated to dissolving, add 1 premium on currency, there is solid to separate out, be chilled to below 10 degree, 294 gram lithium hydroxides are dissolved in 2.5 premium on currency, add top suspension liquid, temperature rises to 20 degree, continue to stir 6 minutes, temperature rises to 25 degree, the citric acid solution that the ratio that adds 3 liters, water in 1346 gram citric acids is prepared adds reaction solution, stirred 10 minutes, add 1 premium on currency, cryosel is bathed cooling, until a large amount of solids are separated out, continue to stir half hour, filter, washing, obtain sterling, be 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid, yield 85%,

step 7) 2 gram 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid is dissolved in the mixed solvent of 5 milliliters of acetonitriles and 5 milliliters of DMF, add 1.9 gram benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate, reaction solution is chilled to 0 degree, drip 1.6 milliliters of N-methylmorpholines at this temperature, stirring reaction once hour after, C with 1.896 grams under 0 degree once adds reaction solution, this reaction solution stirring at room one hour, rear room temperature reaction two hours, be cooled to 0 degree, add the layering of aquatic products thing, separatory washing three times, get organic phase, be spin-dried for organic solvent and obtain thick product 3.7 gram Fmoc-PNA-A (Bhoc)-OMe, be directly used in next step,

step 8) the thick product F moc-PNA-A (Bhoc) of 3.7 grams-OMe is dissolved in the tetrahydrofuran (THF) of 25 milliliters, be chilled to zero degree, it is the lithium hydroxide aqueous solution of 5mol/l with 10 ml concns, vigorous stirring slowly is added dropwise in reaction solution, temperature is controlled at about zero degree, there is solid to separate out, continue at this temperature to stir 20 minutes, TLC follows the tracks of and is as the criterion, react complete, add in advance in 6 gram citric acids and add the aqueous citric acid solution that the ratio of 10 ml waters prepares, be chilled to subzero crystallization, filter after stirring half hour, wash three times, wash low polar impurity off with the ether making beating after the solid drying that obtains, filtration drying, obtain sterling Fmoc-PNA-A (Bhoc)-OH, purity 95% left and right, two step total recoverys are 60%,

Process is as follows:

The above only for the preferred embodiment of invention, is not limited to the present invention, and for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.

Claims (1)

1. the method for the synthetic PNA of feather weight, is characterized in that, comprises the following steps:

Step 1) 1 liter of there-necked flask, the 500 milliliters of quadrols of packing into, ice bath be cooled to 10 the degree, with 75 gram Mono Chloro Acetic Acids in batches the access control temperature 10 the degree below, every batch add after, after being completely consumed, observation adds again next batch, after reinforced complete, the ambient temperature overnight reaction is spin-dried for quadrol, reclaiming quadrol can recycle, with oil pump, quadrol is removed totally as far as possible, obtained the white powder crystal with the DMSO recrystallization, be N-(2-aminoethyl) glycine;

step 2) 50 gram N-(2-aminoethyl) glycine is dissolved in the mixing solutions of 850 milliliters of methylene dichloride and 50 milliliters of DMF, add 134 milliliters of (1.05mol) trimethylchlorosilanes, reaction mixture stirring at room 40 minutes, then be cooled to 5 degree, 134 gram Fmoc-OSu are added in top mixture, drip the NMM186 milliliter under 5 degree, dripped off in 30 minutes, stirred 2 hours at this temperature, then add 100 ml methanol, stirred again 20 minutes, add 1 liter of ethyl acetate, cross filter solid after 20 minutes, wash with ethyl acetate, the product powder is spin-dried for, making beating washing in product water, filter washing, drying obtains sterling, be N-(2-Fmoc-aminoethyl) glycine,

Step 3) 12 gram N-(2-Fmoc-aminoethyl) glycine is dissolved in the anhydrous methanol of 100 milliliters, 7.72 the milliliter thionyl chloride splashes under ice bath, continues reaction 15 minutes after dripping off at this temperature, then refluxes 2 hours, remove solvent, ether with 9/1 and dissolve with methanol, 0 degree crystallization filters, the ether washing, drying obtains sterling, is N-(2-Fmoc-aminoethyl) glycine methyl ester hydrochloride;

step 4) VITAMIN B4 of 389 gram dryings is suspended in the DMF of 6 liters, the sodium hydride that adds 40 grams, reaction solution stirs, there is hydrogen to overflow, stir after 15 minutes, add again 42 gram sodium hydrides, stir after 1 hour, there are a large amount of solids to separate out, stir difficulty, the DMF that adds again 1.5 liters, continue to stir 3 hours, the ethyl bromoacetate of 351 milliliters dropwised within half hour, there is ice bath to maintain the temperature in 20-30 degree scope in this process, then stirred overnight at room temperature, steam except volume, add 3 premium on currency, stirred one hour, cross filter solid, wash three times, solid suspension is in 2 liters of ethanol, stirring and refluxing 45 minutes, be as cold as room temperature, stirring is spent the night, suction filtration, get solid, washing with alcohol, dry, get sterling, be 9-ethyl acetate VITAMIN B4,

Step 5) 433 gram 9-ethyl acetate VITAMIN B4 and 478 gram carbonyl dimidazoles are suspended in the DMF of 4 liters, reaction solution slowly is heated to 105 degree, then stirs 2 hours at this temperature, reduce the temperature to 95 degree, add 541 gram benzhydrols, remove heating, reaction solution stirs and spends the night, and the water of 2.5 times adds reaction solution, rapid stirring 1 hour, solid is separated out, and filters, and washes three times, recrystallizing methanol, obtain sterling, be 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-ethyl acetate, pistac;

step 6) 6-N-(hexichol methoxycarbonyl) VITAMIN B4 of 300 grams-9-ethyl acetate is suspended in the mixed solvent of 1.5 liters of ethanol and 1.5 liters of acetonitriles, be heated to dissolving, add 1 premium on currency, there is solid to separate out, be chilled to below 10 degree, 294 gram lithium hydroxides are dissolved in 2.5 premium on currency, add top suspension liquid, temperature rises to 20 degree, continue to stir 6 minutes, temperature rises to 25 degree, the citric acid solution that the ratio that adds 3 liters, water in 1346 gram citric acids is prepared adds reaction solution, stirred 10 minutes, add 1 premium on currency, cryosel is bathed cooling, until a large amount of solids are separated out, continue to stir half hour, filter, washing, obtain sterling, be 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid,

step 7) 2 gram 6-N-(hexichol methoxycarbonyl) VITAMIN B4-9-acetic acid is dissolved in the mixed solvent of 5 milliliters of acetonitriles and 5 milliliters of DMF, add 1.9 gram benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate, reaction solution is chilled to 0 degree, drip 1.6 milliliters of N-methylmorpholines at this temperature, stirring reaction once hour after, C with 1.896 grams under 0 degree once adds reaction solution, this reaction solution stirring at room one hour, rear room temperature reaction two hours, be cooled to 0 degree, add the layering of aquatic products thing, separatory washing three times, get organic phase, be spin-dried for organic solvent and obtain thick product 3.7 gram Fmoc-PNA-A (Bhoc)-OMe,

step 8) the thick product F moc-PNA-A (Bhoc) of 3.7 grams-OMe is dissolved in the tetrahydrofuran (THF) of 25 milliliters, be chilled to zero degree, it is the lithium hydroxide aqueous solution of 5mol/l with 10 ml concns, vigorous stirring slowly is added dropwise in reaction solution, temperature is controlled at about zero degree, there is solid to separate out, continue at this temperature to stir 20 minutes, TLC follows the tracks of and is as the criterion, react complete, add in advance in 6 gram citric acids and add the aqueous citric acid solution that the ratio of 10 ml waters prepares, be chilled to subzero crystallization, filter after stirring half hour, wash three times, wash low polar impurity off with the ether making beating after the solid drying that obtains, filtration drying, obtain sterling Fmoc-PNA-A (Bhoc)-OH, purity is 95%,

Process is as follows: