CN102516208A - Cleavable connection unit, synthetic method thereof and use thereof - Google Patents
Cleavable connection unit, synthetic method thereof and use thereof Download PDFInfo
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Abstract
The invention discloses a cleavable connection unit, a synthetic method thereof and a use thereof. The structural formula of the cleavable connection unit is shown in the specification; and in the structural formula, n is 1 or 2, m is an arbitrary integer from 0 to 20, and k is an arbitrary integer from 1 to 20. The cleavable connection unit can be used for DNA sequencing by synthesis. Compared with the prior art, the synthetic method of the invention allows synthetic raw materials of compounds of the invention to be simple and easily obtained and the synthesis of a large amount of the compounds to be easily realized because the synthetic process is a routine chemical reaction; the compounds can be efficiently connected with nucleotides and fluoresceins; and results of researches on cracking performances of the compounds show that the compounds can be efficiently and completely cracked under mild conditions and also can be applied to the DNA sequencing.
Description
Technical field
The present invention relates to be used for the field of chemical synthesis of the synthetic compound that checks order of DNA, relate in particular to one type of novel cleavable connector element and compound method and purposes.
Background technology
The dna sequencing technology is one of means important in the modern biology research.After the Human Genome Project was accomplished, the dna sequencing technology had obtained developing rapidly.Dna sequencing (DNA sequencing) is meant the base sequence of analyzing specific DNA fragments, just the arrangement mode of VITAMIN B4 (A), thymus pyrimidine (T), cytosine(Cyt) (C) and guanine (G).Development accurately, high-throughput, the dna sequencing method has very important significance for biology, medical science etc. cheaply.
(Sequencing By Synthesis SBS) is one of dna sequencing technology of new generation in the synthesis method order-checking.The synthesis method sequence measurement is fixed through an a large amount of tested template DNA fragment, and hybridization combines general dna primer on immobilized dna sequencing template, controls four kinds of Nucleotide extensions on dna primer respectively.Through detecting the extension process or extending Nucleotide, realize the dna sequence dna detection of information of high-flux parallel.
In the synthesis method order-checking, at first want four kinds of Nucleotide raw materials of synthetic DNA chain extension, be " reversible terminal " (reversible terminator) again.This type Nucleotide is except requiring 3 ˊ-hydroxyl blocking-up, and in order not influence incorporating into and discern of next indication Nucleotide, the connector element that also requires to pass through a cleavable is Nucleotide and indication molecule, and for example resorcinolphthalein couples together.Then, before next one indication Nucleotide is incorporated into, under mild conditions, make this connector element fracture, continue the prolongation of DNA chain, thereby read the sequence of DNA base.This connector element to synthesis method order-checking read long and efficient has material impact, therefore, people also are devoted to develop new cleavable connector element always, improve the efficient of dna sequencing.The connector element of having reported at present has disulfide linkage, photodestruciton, Pd catalytic pyrolysis, fluorochemical cleavable etc.
Robi D. Mitra Jay; Shendure Jerzy Olejnik; Reported a kind of disulfide linkage connector element in Edyta-Krzymanska-Olejnik and George M. Churcha. Fluorescent in situ sequencing on polymerase colonies. Analytical Biochemistry 320 (2003) 55 – 65. document; Couple together resorcinolphthalein and deoxynucleotide through this connector element; This Nucleotide incorporate under the polysaccharase effect and to be detected after, under the effect of 2 mercapto ethanol, make disulfide bonds, realize the circulation of order-checking.Gerardo Turcatti, Anthony Romieu, Milan Fedurco and Ana-Paula Tairi. A new class of cleavable fluorescent nucleotides:synthesis and optimization as reversible terminators for DNA sequencing by synthesis. Nucleic Acids Research; 2008; Vol. 36, No. 4. and report as the cleavable connector element, has synthesized one type of new fluorescent mark Nucleotide as reversible terminal with disulfide linkage in the document; Be used for the synthetic order-checking of DNA; But, can only measure 10 nucleotide sequences, read long too short.But Pleasants, J. C.; Guo, W.; Rabenstein, D. L. J. Am. Chem. Soc. 1989,111,6553-6558 and Huyghues-Despointes, B. M. P.; Nelson, J. W. Biochemistry 1992, in two pieces of documents of 311476-1483 report under certain conditions, disulfide linkage is unstable, and the formation of disulfide linkage and fracture are reversible processes.
Jingyue Ju seminar is at document Zengmin Li, Xiaopeng Bai, Hameer Ruparel, Sobin Kim; Nicholas J. Turro, and Jingyue Ju. A photocleavable fluorescent nucleotide for DNA sequencing and analysis. PNAS January 21,2003, vol. 100; No. 2,414-419. Tae Seok Seo, Xiaopeng Bai; Hameer Ruparel, Zengmin Li, Nicholas J. Turro; And Jingyue Ju. Photocleavable fluorescent nucleotides for DNA sequencing on a chip constructed by site-specific coupling chemistry. PNAS April 13,2004, vol. 101; No. 15,5488-54936. Tae Seok Seo, Xiaopeng Bai; Dae Hyun Kim, Qinglin Meng, Shundi Shi; Hameer Ruparel, Zengmin Li, Nicholas J. Turro; And Jingyue Ju. Four-color DNA sequencing by synthesis on a chip using photocleavable fluorescent nucleotides. PNAS April 26,2005, Vol. 102; No. 17,5932-5937. Qinglin Meng, Dae Hyun Kim; Xiaopeng Bai, Lanrong Bi, Nicholas J. Turro; And Jingyue Ju. Design and Synthesis of a Photocleavable Fluorescent Nucleotide 3 '-O-Allyl-dGTP-PC-Bodipy-FL-510 as a Reversible Terminator for DNA Sequencing by Synthesis. J. Org. Chem. 2006,71 has reported a kind of smooth cleavable connector element among the 3248-3252; And with its as reversible terminal applies in synthetic order-checking, long too short but this connector element was read when being applied to check order, this has influenced the efficient of synthetic order-checking.
Jingyue Ju seminar is at document Lanrong Bi, Dae Hyun Kim, and Jingyue Ju. Design and Synthesis of a Chemically Cleavable Fluorescent Nucleotide; 3 '-O-Allyl-dGTP-allyl-Bodipy-FL-510, as a Reversible Terminator for DNA Sequencing by Synthesis. J. AM. CHEM. SOC. 2006,128; 2542-2543. Jingyue Ju, Dae Hyun Kim, Lanrong Bi; Qinglin Meng, Xiaopeng Bai, Zengmin Li; Xiaoxu Li, Mong Sano Marma, Shundi Shi; Jian Wu, John R. Edwards, Aireen Romu; And Nicholas J. Turro. Four-color DNA sequencing by synthesis using cleavable fluorescent nucleotide reversible terminators. PNAS December 26,2006, vol. 103 No. 52; Reported two kinds of Pd catalysis cleavable connector elements among 19635-19640 and the USP Patent No.US7883869 B2 (2011), this connector element derives according to 3 ˊ-hydroxyl allyl group blocking-up, when still being applied to check order; Cracking condition is gentle inadequately; Destroy other structure sometimes, read long falling short of simultaneously, the flux of the synthetic order-checking of influence.
Diana C. Knapp; Jennifer D ' Onofrio; And Joachim W. Engels. Fluorescent Labeling of (Oligo) Nucleotides by a New Fluoride Cleavable Linker Capable of Versatile Attachment Modes. Bioconjugate Chem. 2010; Reported a kind of connector element of fluorochemical cleavable in 21,1043 –, 1055. document.Diana C. Knapp, Saulius Serva, Jennifer D ' Onofrio; Angelika Keller, Arvydas Lubys, Ants Kurg; Maido Remm; And Joachim W.Engels. Fluoride-Cleavable, Fluorescently Labelled Reversible Terminators:Synthesis and Use in Primer Extension. Chem. Eur. J. 2011,17; The connector element that utilizes this fluorochemical cleavable of further having reported for work in 2903 –, 2915. document has synthesized four kinds of fluorescently-labeled Nucleotide, as the reversible terminal in the synthetic order-checking.But in order-checking, the time that the cracking of this connector element needs is longer, and this has influenced the efficient of order-checking.
The cleavable connector element to synthesis method order-checking read long and efficient has significant effects; Cracking condition is gentle inadequately, lysis efficiency is not high and existing connector element exists; Read shortcomings such as long too weak point when being used to check order; Therefore, design, synthetic new cleavable connector element, and explore suitable cracking condition and for the efficient that improves order-checking, the new sequence measurement of development very important meaning is arranged.
Summary of the invention
The objective of the invention is to overcome the deficiency of above-mentioned prior art, one type of novel cleavable connector element and compound method and purposes are provided.The present invention is based on the thought of hydroxyl protection, designed and synthesized one type of new cleavable connector element first, the synthetic raw material of such compound is simple and easy to, and building-up process is the conventional chemical reaction, be easy to realize a large amount of synthetic.This compounds can be realized high efficiency the connection with Nucleotide and resorcinolphthalein.Through studying the cracking performance of this compounds, find that this compounds can realize high efficiency complete cracking under the condition of gentleness, can be applicable to dna sequencing.
The objective of the invention is to realize through following technical scheme:
The present invention relates to a kind of cleavable connector element, its structural formula is:
Wherein, n is 1 or 2, and m is any integer in 0～20, and k is any integer in 1～20.
The invention still further relates to that a kind of said cleavable connector element connects Nucleotide and resorcinolphthalein according to the purposes of above-mentioned cleavable connector element in the synthetic order-checking of DNA, be used for the synthetic order-checking of DNA.
The invention still further relates to a kind of compound method according to above-mentioned cleavable connector element,
When n=1, comprise following synthesis step:
A, T-1's is synthetic: with L-L-glutamic acid is raw material, adds hydrochloric acid, under ice bath stirs, drips sodium nitrite solution, after dropwising, continues reaction 3～5h under ice bath, moves to then under the room temperature and spends the night; After reaction finished, pressure reducing and steaming water added acetic acid ethyl dissolution, filters, and filtrating is used anhydrous sodium sulfate drying, filters, and revolves dried solvent and gets T-1;
B, T-2's is synthetic: get T-1, (THF) makees solvent with THF, and nitrogen protection slowly drips borine/dimethyl sulphide solution under ice-water bath, after dropwising, continue at room temperature to react 4～5h; After reaction finishes, add methyl alcohol cancellation reaction, revolve and desolvate, add methyl alcohol again, revolve dried T-2;
C, T-3's is synthetic: get T-2, make solvent with methylene dichloride, add imidazoles, nitrogen protection is descended and dimethyl-tertiary butyl chloride silane at room temperature reacts 17～20 h; After reaction finishes, add the methylene dichloride dilution, use 2mol/L hydrochloric acid, water and saturated sodium bicarbonate washing reaction liquid respectively, anhydrous sodium sulfate drying revolves driedly, and column chromatography gets the pure article of T-3;
D, T-4's is synthetic: make solvent with methylene dichloride; T-3 reacts with diisobutyl aluminium hydride under nitrogen protection and cryosel bath, and TLC tracks to and reacts completely. after reaction finishes, and adding 0.2mol/L HCl cancellation reaction; Dichloromethane extraction; Organic phase is washed with saturated sodium bicarbonate aqueous solution, anhydrous sodium sulfate drying, rotary evaporation remove desolvate T-4;
E, T-5's is synthetic: make solvent with methylene dichloride, T-4 adds triethylamine (Et under nitrogen protection and cryosel bath
3N), then and methylsulfonyl chloride (MsCl
) Reaction 30～50 min, at 40～50 ℃ of 3～5h that reflux, after reaction finishes, the methylene dichloride dilution, washing, anhydrous sodium sulfate drying revolves driedly, and column chromatography gets T-5;
F, T-6's is synthetic: make solvent with methylene dichloride, T-5 is at tosic acid pyridinium salt (PPTS
)Effect is descended and raw material A is at room temperature reacted, and TLC tracks to and reacts completely, and reaction is revolved dried solvent after finishing; Add entry, use ethyl acetate extraction, saturated common salt water washing organic phase, anhydrous sodium sulfate drying; Filter, revolve driedly, column chromatography obtains the pure article of T-6 (comprising two kinds of isomer of T6-1 and T6-2); The structural formula of said raw material A is:
G, T-7's is synthetic: (THF) makees solvent with THF, under tetrabutyl fluoride amine (TBAF) effect, and T-6 dehydroxylation protection at room temperature; TLC tracks to and reacts completely; Reaction is revolved dried solvent after finishing, and column chromatography obtains T-7 (comprising two kinds of isomer of T7-1 and T7-2);
H, T-8's is synthetic: be dissolved in excess of ammonia water to T-7, react under the room temperature, TLC tracks to and reacts completely, and reaction adds ethanol after finishing, and decompression is revolved and desolvated, and gets T-8, and T-8 is said cleavable connector element (comprising two kinds of isomer of T8-1 and T8-2);
When n=2, comprise following synthesis step:
A, X-1's is synthetic: with N, dinethylformamide (DMF) is made solvent, 2-hydroxyl-3, and 4-dihydropyrane (X-0) is under the effect of NaH; At room temperature react with Benzyl Chloride, TLC tracks to and reacts completely, after reaction finishes; Drip water and remove unreacted NaH, use ethyl acetate extraction, organic phase is water, saturated common salt washing respectively; Anhydrous sodium sulfate drying, decompression is revolved and is desolvated, and column chromatography gets X-1;
B, X-2's is synthetic: as solvent, be dissolved in methylene dichloride to raw material A with methylene dichloride, under the effect of tosic acid pyridinium salt and X-1 at room temperature react; TLC tracks to and reacts completely, and after reaction finished, decompression was revolved and desolvated; Add entry afterwards, use ethyl acetate extraction, organic phase is water, saturated common salt washing respectively; Anhydrous sodium sulfate drying, decompression is revolved and is desolvated, and column chromatography gets X-2; The structural formula of said raw material A is:
; Wherein, M is any integer in 0～20, and k is any integer in 1～20;
C, X-3's is synthetic: make solvent with ETHYLE ACETATE, be dissolved in ETHYLE ACETATE to X-2, adding Pd/C also feeds hydrogen, reacts under the room temperature, and TLC tracks to and reacts completely, and after reaction finishes, filters, and decompression is revolved and desolvated, and gets X-3;
D, X-4's is synthetic: be dissolved in excess of ammonia water to X-3, react under the room temperature, TLC tracks to and reacts completely, and reaction adds ethanol after finishing, and decompression is revolved and desolvated, and gets X-4, and X-4 is said cleavable connector element.
Preferably, when n=1, hydrochloric acid described in the step a is 6 mol/L, and the mol ratio of said L-L-glutamic acid, hydrochloric acid and the Sodium Nitrite of adding is 1:2～2.5:1.5～2.
Preferably, when n=1, the said T-1 that adds among the step b and the mol ratio of borine are 1:1.1～1.5.
Preferably, when n=1, the mol ratio of the said T-2 that adds among the step c, imidazoles and dimethyl-tertiary butyl chloride silane is 1:1.1～1.5:1.1～1.5.
Preferably, when n=1, the said T-3 that adds in the steps d and the mol ratio of diisobutyl aluminium hydride are 1:1.2～1.5.
Preferably, when n=1, the said T-4, the Et that add among the step e
3The mol ratio of N and MsCl is 1:3～5:1.3～1.5.
Preferably, when n=1, the mol ratio of said T-5, raw material A and the PPTS that adds among the step f is 1:1.5～2:0.2～0.5.
Preferably, when n=1, the TBAF described in the step g is the tetrahydrofuran solution that contains 1mol/LTBAF, and the said T-6 of adding and the mol ratio of TBAF are 1:1.5～2.
Preferably, when n=2, the said 2-hydroxyl-3 that adds among the step a, the mol ratio of 4-dihydropyrane, sodium hydride and Benzyl Chloride is 1:3～5:1.5～2.
Preferably, when n=2, the mol ratio of the said X-1 that adds among the step b, raw material A and toluenesulphonic acids pyridinium salt is 1.5～2:1:0.2～0.5.
Preferably, when n=2, said X-2 that adds among the step c and the mass ratio of Pd/C are 1:10%～20%.
Compared with prior art; The present invention has following beneficial effect: when compound of the present invention is applied to dna sequencing, study it in differing temps, the cracking performance under the condition of different pH; Can know; Said compound (slightly acidic) under the condition of gentleness can be realized high efficiency complete cracking, can be applicable to dna sequencing, and its fragmentation pattern and performance and existing method all have essential distinction.
Description of drawings
Fig. 1 is the synthetic route synoptic diagram during n=1 in the cleavable connector element structural formula of the present invention;
Fig. 2 is the synthetic route synoptic diagram during n=2 in the cleavable connector element structural formula of the present invention;
Fig. 3 is the synthetic route synoptic diagram of the connector element X-6 that contains the ultraviolet group in the embodiment of the invention 3;
Fig. 4 is the cracking experiment reaction formula synoptic diagram of the connector element X-6 that contains the ultraviolet group in the embodiment of the invention 3;
Fig. 5 is the synthetic route synoptic diagram of the connector element T9 that contains resorcinolphthalein in the embodiment of the invention 4;
Fig. 6 is the cracking experiment reaction formula synoptic diagram of the connector element T9 that contains resorcinolphthalein in the embodiment of the invention 4;
Fig. 7 is the HPLC spectrogram of the reactant T9-1 of cracking experiment in the embodiment of the invention 4;
Fig. 8 is the HPLC spectrogram of the reactant T9-2 of cracking experiment in the embodiment of the invention 4;
Fig. 9 is the HPLC spectrogram of the product T9-A of cracking experiment in the embodiment of the invention 4;
The section H PLC figure of Figure 10 for testing when the T9-1 cracking of pH=2.60, the HPLC figure when wherein Figure 10 a is t=5min, the HPLC figure when Figure 10 b is t=7min, the HPLC figure when Figure 10 c is t=9min;
Figure 11 is the section H PLC figure of the T9-1 cracking experiment when the pH=2.25, the HPLC figure when wherein Figure 11 a is t=5min, and the HPLC figure when Figure 11 b is t=7min, the HPLC when Figure 11 c is t=9min schemes;
The section H PLC figure of Figure 12 for testing when the T9-2 cracking of pH=2.60, the HPLC figure when wherein Figure 12 a is t=5min, the HPLC figure when Figure 12 b is t=7min, the HPLC figure when Figure 12 c is t=9min;
Figure 13 is the section H PLC figure of the T9-2 cracking experiment when the pH=2.25, the HPLC figure when wherein Figure 13 a is t=5min, and the HPLC figure when Figure 13 b is t=7min, the HPLC when Figure 13 c is t=9min schemes.
Embodiment
Through embodiment the present invention is further set forth below, but protection scope of the present invention is not limited to following embodiment.
Used raw material, the reagent of the present invention is commercially available AR or CP level.
Gained intermediate product of the present invention and final product employing NMR etc. characterize.
The synthetic route synoptic diagram is as shown in Figure 1; Synthesis step is following:
(1) T-1's is synthetic:
Method one: in 500mL single port bottle, add 10g L-L-glutamic acid, add hydrochloric acid (the 11mL concentrated hydrochloric acid is dissolved in 22mL water), the solid dissolving.Under ice bath, stir 30min, continue under ice bath, to drip the solution (7g is dissolved in 30mL water) of Sodium Nitrite, have the cigarette of reddish-brown to produce in the dropping process.After dropwising, continue reaction 3h under ice bath, move to then under the room temperature and spend the night.White solid and pale yellow oily liquid body appear in pressure reducing and steaming water, add the 150mL acetic acid ethyl dissolution, the white solid that elimination is not allowed, and filtrating is used anhydrous sodium sulfate drying, filters, and revolves dried solvent and gets 10.12g pale yellow oily liquid body, directly is used for next step.
Method two: in 500ml single port bottle, add 20g L-L-glutamic acid, add hydrochloric acid (the 25ml concentrated hydrochloric acid is dissolved in 50ml water), the solid dissolving.Under ice bath, stir 30min, continue under ice bath, to drip the solution (17g is dissolved in the 70ml water) of Sodium Nitrite, have the cigarette of reddish-brown to produce in the dropping process.After dropwising, continue reaction 4h under ice bath, move to then under the room temperature and spend the night.White solid and pale yellow oily liquid body appear in pressure reducing and steaming water, add the 150ml acetic acid ethyl dissolution, the white solid that elimination is not allowed, and filtrating is used anhydrous sodium sulfate drying, filters, and revolves dried solvent and gets 22.22g pale yellow oily liquid body, directly is used for next step.
Method three: in 500ml single port bottle, add 20g L-L-glutamic acid, add hydrochloric acid (the 28ml concentrated hydrochloric acid is dissolved in 56ml water), the solid dissolving.Under ice bath, stir 30min, continue under ice bath, to drip the solution (19g is dissolved in the 80ml water) of Sodium Nitrite, have the cigarette of reddish-brown to produce in the dropping process.After dropwising, continue reaction 5h under ice bath, move to then under the room temperature and spend the night.White solid and pale yellow oily liquid body appear in pressure reducing and steaming water, add the 150ml acetic acid ethyl dissolution, the white solid that elimination is not allowed, and filtrating is used anhydrous sodium sulfate drying, filters, and revolves dried solvent and gets 23.22g pale yellow oily liquid body, directly is used for next step.
(2) T-2's is synthetic:
Method one: get the first step T-1 bullion 10.12g in the 500ml two-mouth bottle, vacuumize, nitrogen protection is injected into the anhydrous THF of 150ml, stirring and dissolving under the room temperature then.Under ice bath, slowly drip 9.6ml 10M borine/dimethyl sulphide solution in the 3h, after dropwising, continue at room temperature to react 5h, add 100ml methyl alcohol cancellation reaction.Revolve and desolvate, add 100ml methyl alcohol again, revolve dried 8.30g yellow oily liquid, directly be used for next step.
Method two: get the first step T-1 bullion 22.22g in the 500ml two-mouth bottle, vacuumize, nitrogen protection is injected into the anhydrous THF of 200ml, stirring and dissolving under the room temperature then.Under ice bath, slowly drip 23ml 10M borine/dimethyl sulphide solution in the 4h, after dropwising, continue at room temperature to react 6h, add 150ml methyl alcohol cancellation reaction.Revolve and desolvate, add 100ml methyl alcohol again, revolve dried 16.12g yellow oily liquid, directly be used for next step.
Method three: get the first step T-1 bullion 23.22 g in the 500mL two-mouth bottle, vacuumize, nitrogen protection is injected into the anhydrous THF of 200mL, stirring and dissolving under the room temperature then.Under ice-water bath, slowly drip 30mL 10M borine/dimethyl sulphide solution in the 4h, after dropwising, continue at room temperature to react 5h, add 150mL methyl alcohol cancellation reaction.Revolve and desolvate, add 100mL methyl alcohol again, revolve dried 16.87g yellow oily liquid, directly be used for next step.Take a morsel, silica gel dress post, DCM/MeOH=20:1 drip washing gets pure article and does
1H-NMR.
1H-NMR?(CDCl
3,?300M):
4.58～4.66(1H,?m)，3.89（1H,?dd，J
1?=?3.0,?J
2?=?12.6?HZ）,?3.63（1H，dd，J
1?=?4.5,?J
2?=?12.6Hz），2.46～2.68（2H,?m），2.11～2.29（2H，m）.
(3) T-3's is synthetic:
Method one: get T-2 bullion 8.30g in the 250ml two-mouth bottle, add the 5.3g imidazoles, vacuumize, the 100ml methylene dichloride is injected in nitrogen protection.Get dimethyl-tertiary butyl chloride silane (TBSCl) 11.8g and be dissolved in the 50ml methylene dichloride, be injected in the above-mentioned system stirring reaction 17h under the room temperature.Add the methylene dichloride dilution, use 2M HCl, water and saturated NaHCO respectively
3Washing reaction liquid, anhydrous sodium sulfate drying, revolve dried, 12.49g yellow oily liquid.Adorn post with silica gel, the 15:1 petrol ether/ethyl acetate is carried out the post separation, gets the pure article 4.32g of faint yellow T-3, and T-1 is 27.6%. to the T-3 total recovery
Method two: get T-2 bullion 16.12g in the 500ml two-mouth bottle, add the 11.3g imidazoles, vacuumize, the 150ml methylene dichloride is injected in nitrogen protection.Get TBSCl 25.2g and be dissolved in the 50ml methylene dichloride, be injected in the above-mentioned system, stirring reaction 19h under the room temperature.Add the methylene dichloride dilution, use 2M HCl, water and saturated NaHCO respectively
3Washing reaction liquid, anhydrous sodium sulfate drying, revolve dried, 22.48g yellow oily liquid.Adorn post with silica gel, the 15:1 petrol ether/ethyl acetate is carried out the post separation, gets the pure article 6.21g of faint yellow T-3, and T-1 is 20%. to the T-3 total recovery
Method three: get T-2 bullion 16.87g in two mouthfuls of 500ml put down, add the 14.8g imidazoles, vacuumize, the 150ml methylene dichloride is injected in nitrogen protection.Get TBSCl 32.9g and be dissolved in the 80ml methylene dichloride, be injected in the above-mentioned system, stirring reaction 20h under the room temperature.Add the methylene dichloride dilution, use 2M HCl, water and saturated NaHCO respectively
3Washing reaction liquid, anhydrous sodium sulfate drying, revolve dried, 26.49g yellow oily liquid.Adorn post with silica gel, the 15:1 petrol ether/ethyl acetate is carried out the post separation, gets the pure article 7.83g of faint yellow T-3, and T-1 is 25% to the T-3 total recovery.
1H-NMR?(CDCl
3,400M):
4.54～4.59?(1H，m)，3.84（1H，dd，J
1?=?3.2,?J
2?=?11.2Hz），3.67?(1H,?dd,?J
1?=?3.2,?J
2?=?11.2Hz)，2.40～2.63（2H，m），2.11～2.30（2H，m），0.87（9H，s）,?0.05?(6H,?d,?J?=?2.8Hz).
(4) T-4's is synthetic:
Method one: get T-3 3.03g in two mouthfuls of flasks, vacuumize N
2Protection is injected into the 30ml methylene dichloride, stirring and dissolving, and cryosel is bathed (15 ℃) down, slowly is injected into 15.8ml diisobutyl aluminium hydride (DIBAL-H) (1M in Toluene), and behind the stirring 30min, the some plate is found to have reacted.Stopped reaction adds 90ml 0.2M HCl cancellation reaction, dichloromethane extraction three times, and dichloromethane solution is used saturated NaHCO
3Solution is washed once, and anhydrous sodium sulfate drying revolves dried colourless liquid 2.55g, productive rate 83%.
Method two: get T-3 1.52g in two mouthfuls of flasks, vacuumize N
2Protection is injected into the 20ml methylene dichloride, stirring and dissolving, and cryosel is bathed (15 ℃) down, slowly is injected into 8.6ml DIBAL-H (1M in Toluene), and behind the stirring 30min, the some plate is found to have reacted.Stopped reaction adds 50ml 0.2M HCl cancellation reaction, dichloromethane extraction three times, and dichloromethane solution is used saturated NaHCO
3Solution is washed once, and anhydrous sodium sulfate drying revolves dried colourless liquid 1.30g, productive rate 85%.
Method three: get T3 0.85g in two mouthfuls of flasks, vacuumize N
2Protection is injected into 10 mL methylene dichloride, stirring and dissolving, cryosel is bathed (15 ℃) down, slowly is injected into 5.5 mL diisobutyl aluminium hydrides (DIBAL-H) (1M in Toluene), stir 30 min after, the some plate is found to have reacted.Stopped reaction adds 30 mL 0.2M HCl cancellation reaction, dichloromethane extraction three times, and dichloromethane solution is used saturated NaHCO
3Solution is washed once, and anhydrous sodium sulfate drying revolves dried colourless liquid 0.68 g, productive rate 79%.
1H-NMR（CDCl
3,?400M)：
5.38～5.56（1H，m），4.25～4.29（1H，m），3.80（1H，dd，J
1?=?2.8,?J
2?=?10.4?Hz），3.57（1H，dd，J
1?=?2.8,?J
2?=?10.8?Hz），2.16（2H,?s），1.92～1.96（2H，m），0.92（9H，s），0.11?(6H,?s).
(5) T-5's is synthetic:
Method one: get T-4 1g, vacuumize, N
2Protection is injected into the dissolving of 10ml methylene dichloride, and cryosel is bathed (15 ℃) and stirred half a hour down, is injected into 1.9ml Et
3N stirs 10min, continues to be injected into 0.43mlMsCl, stirs 30min, yellowing suspension-s, and 40 ℃ of backflow 3h leave standstill.Methylene dichloride dilution, washing, anhydrous sodium sulfate drying, revolve dried, brown oily liquids.With 100-200 purpose alkali alumina column chromatography, contain 2% Et
3The sherwood oil drip washing of N gets colourless transparent liquid 410mg, productive rate 44.5%.
Method two: get T-4 1.5g, vacuumize, N
2Protection is injected into the dissolving of 10ml methylene dichloride, and cryosel is bathed (15 ℃) and stirred half a hour down, is injected into 3.7mlEt
3N stirs 10min, continues to be injected into 0.7ml MsCl, stirs 40min, yellowing suspension-s, and 45 ℃ of backflow 4h leave standstill.Methylene dichloride dilution, washing, anhydrous sodium sulfate drying, revolve dried, brown oily liquids.With 100-200 purpose alkali alumina column chromatography, contain 2%Et
3The sherwood oil drip washing of N gets colourless transparent liquid 581mg, productive rate 42%.
Method three: get T-4 3.44g, vacuumize, N
2Protection is injected into the dissolving of 40 mL methylene dichloride, and cryosel is bathed (15 ℃) and stirred half a hour down, is injected into 10.6mlEt
3N stirs 10 min, continues to be injected into 1.7 mL MsCl, stirs 50 min, yellowing suspension-s, and 50 ℃ of backflow 5h leave standstill.Methylene dichloride dilution, washing, anhydrous sodium sulfate drying, revolve dried, brown oily liquids.With 100-200 purpose alkali alumina column chromatography, contain 2% Et
3The sherwood oil drip washing of N gets colourless transparent liquid 1.3g, productive rate 40.4%.
1H-NMR（CDCl
3,?400M）：
6.26～6.27（1H，m），4.84～4.84（1H,?m）,?4.56～4.63（1H，m）,?3.71（1H,?dd，J
1?=?6.0,?J
2?=?10.8Hz），3.61（1H,?dd，J
1?=?4.8,?J
2?=?10.4?Hz），2.60～2.67（1H,?m）,?2.36～2.42（1H,?m）,?0.90（9H,?s），0.07（6H,?d，J?=?2.0?Hz）.
(6) T-6's is synthetic:
Method one: in 50 ml single port bottles, add 245 mg2-bromoethanols (m=1, the raw material A of k=0 o'clock), 6 ml DCM, 66 mg PPTS stir 10 min under the room temperature, add 280 mg T-5 then.Put plate behind 5 min, find that raw material T-5 disappears, and produce two principal points, nuclear-magnetism finds that these two points are diastereomer.Stopped reaction revolves dried solvent, adds 30 ml water, and with ethyl acetate extraction three times, saturated common salt water washing organic phase, anhydrous sodium sulfate drying filters, and revolves dried 358 mg bullions.Use the silicagel column separation and purification, PE/EA=100/1 drip washing obtains little product T6-1 111 mg of polarity, productive rate 25.2%, and product T6-2 71 mg that polarity is big, productive rate 15.9% intersects 10 mg, total recovery 43.5%.T6-1 and T6-2 are a pair of diastereomers.
Method two: in 50 ml single port bottles, add 1.36g ethylene bromohyrin (m=1, the raw material A of k=0 o'clock), 6 ml DCM, 0.46g PPTS stirs 10 min under the room temperature, add 1.3g T-5 then.Put plate behind 10 min, find that raw material T-5 disappears, and produce two principal points, nuclear-magnetism finds that these two points are diastereomer.Stopped reaction revolves dried solvent, adds 30 ml water, and with ethyl acetate extraction three times, saturated common salt water washing organic phase, anhydrous sodium sulfate drying filters, and revolves dried 2.18g bullion.Use the silicagel column separation and purification, PE/EA=100/1 drip washing obtains little product T6-1 420 mg of polarity, productive rate 20%, product T6-2 210 mg that polarity is big, productive rate 10%, intersection 261mg, total recovery 43%.T6-1 and T6-2 are a pair of diastereomers.
Method three: in 50 ml single port bottles, add 1.17g2-bromoethanol (m=1, the raw material A of k=0 o'clock), 6 ml DCM, 0.59g PPTS stirs 10 min under the room temperature, add 1g T-5 then.Put plate behind 10 min, find that raw material T-5 disappears, and produce two principal points, nuclear-magnetism finds that these two points are diastereomer.Stopped reaction revolves dried solvent, adds 30 ml water, and with ethyl acetate extraction three times, saturated common salt water washing organic phase, anhydrous sodium sulfate drying filters, and revolves dried 1.8 g bullions.Use the silicagel column separation and purification, PE/EA=100/1 drip washing obtains little product T6-1 379 mg of polarity, productive rate 24%, and product T6-2 190 mg that polarity is big, productive rate 12% intersects 142 mg, total recovery 45%.T6-1 and T6-2 are a pair of diastereomers.
The T6-1 compound
1H-NMR (CDCl
3, 300M):
(5.18 1H, d, J=4.20 Hz), 4.15～4.24 (1H, m); 3.92～4.00 (1H, m), 3.71～3.79 (1H, m), 3.61 (2H; D, J=4.5Hz), 3.47～3.50 (2H, m), 1.65～2.10 (4H; M), 0.89 (9H, s), 0.06 (6H, s).
13C-NMR (CDCl
3, 100 MHz):
104.66,79.00,67.27,65.39,32.06,31.08,25.91,25.29,18.35 ,-5.27 ,-5.33. ESI-HRMS:calc for [C
13H
27O
3Si Br+Na] 361.0811, found 361.0835.
The T6-2 compound
1H-NMR (CDCl
3, 300M):
(5.13 1H, d, J=3.9 Hz), 4.09～4.18 (1H, m); 3.92～4.00 (1H, m), 3.68～3.77 (2H, m), 3.56～3.62 (1H; M), 3.43～3.51 (2H, m), 1.75～2.02 (4H, m); 0.91 (9H, s), 0.07 (6H, s).
13C-NMR (CDCl
3, 100 MHz):
104.36,81.26,67.21,67.19,32.82,30.97,29.69,26.24,25.93,18.36 ,-5.25 ,-5.28.ESI-HRMS:calc for [C
13H
27O
3Si Br+Na] 361.0811, found 361.0836.
(7) T-7's is synthetic:
A, T7-1's is synthetic
Method one: get the T6-1 80mg that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.35 ml 1M then.Stir 50min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains product T7-1 44mg, productive rate 84%.
Method two: get the T6-1 100mg that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.53 ml 1M then.Stir 60min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains product T7-1 54mg, productive rate 82%.
Method three: get the T6-1 110mg that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.65 ml 1M then.Stir 60min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains product T7-1 60mg, productive rate 83%.
1H-NMR（CDCl
3,?400M）：
5.19（1H,?d，J?=?4.8Hz）,?4.20～4.26（1H,?m）,?3.92～3.98（1H,?m）,?3.69～3.77（2H,?m）,?3.44～3.52（3H,?m）,?1.89～2.10（3H,?m），1.61～1.70（1H,?m）.
?13C-NMR（CDCl
3,?100?MHz）：
?104.59?,?78.62?,?67.34?,?64.78?,?32.41?,?30.94?,?24.87.ESI-HRMS：calc?for?[C
7?H
13?Br?O
3?+?Na]?246.9946，found?246.9929.
B, T7-2's is synthetic
Method one: get the T6-2 80mg that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.35 ml 1M then.Stir 40min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains product T7-2 45mg, productive rate 85%.
Method two: get the T6-2 220mg that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 1.3ml 1M then.Stir 60min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains colorless oil product T7-2 120mg, productive rate 83%.
Method three: get T6-2 70 mg that a step obtains, add 5ml THF, stir 10 min, add the TBAF solution (in THF) of 0.41 ml 1M then.Stir 40min under the room temperature, TLC finds that raw material disappears, directly revolves dry chromatography, adorns post with silica gel, and PE/EA=5/1 drip washing obtains product T7-2 42mg, productive rate 87.9%.
1H-NMR（CDCl
3,?400M）：
5.15（1H,?d，J?=?4.4?Hz）,?4.26～4.32（1H,?m）,?3.98～4.03（1H,?m）,?3.74～3.82（2H,?m）,?3.53～3.58（1H,?dd，J
1?=?5.2,?J
2?=?12.0?Hz）,3.48～3.51（2H,?t，J?=?6.0?Hz）,?1.91～2.07（4H,?m）.
13C-NMR（CDCl
3,?100?MHz）：
?104.735?,?81.554?,?67.93?,?65.61?,?33.25?,?30.79?,?24.33.ESI-HRMS：calc?for?[C
7?H
13?Br?O
3?+?Na]?246.9946，found?246.9938.
T7-1 and T7-2 are a pair of diastereomers.
(8) T-8's is synthetic:
A, T8-1's is synthetic
Method one: get the T7-1 40mg that a step obtains, be dissolved in 2ml ammoniacal liquor, room temperature (35 ℃) is reaction 3d down, and the some plate finds that raw material disappears basically.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-1 28mg, productive rate 97%.
Method two: get the T7-1 100mg that a step obtains, be dissolved in 5ml ammoniacal liquor, room temperature (35 ℃) is reaction 3d down, and the some plate finds that raw material disappears basically.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-1 70mg, productive rate 98%.
Method three: get the T7-1 185mg that a step obtains, be dissolved in 8ml ammoniacal liquor, room temperature (35 ℃) is reaction 3d down, and the some plate finds that raw material disappears basically.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-1 127mg, productive rate 96%.
1H-NMR（CD
3OD,?400M）：
?5.20（1H,?d，J?=?3Hz）,?4.17～4.23（1H,m）,?3.87～3.96（1H,?m）,?3.64～3.78（1H,?m）,?3.59（1H,?dd，J
1?=?4.0,?J
2?=?11.6?Hz）,?3.47～3.53（1H,?m）,?3.30～3.32（1H,?m），3.13～3.16（1H,?t，J?=?4.8?Hz）,?1.86～2.12（3H,?m），1.62～1.70（1H,?m）.
13C-NMR（CD
3OD,?100?MHz）：
?104.72?,?79.11?,?63.87?,?62.95?,?39.53?,?31.54?,?24.86.?ESI-HRMS：calc?for?[C
7?H
15?N?O
3?+?H]?162.1130，found?162.1135.
B, T8-2's is synthetic
Method one: get the T7-2 50mg that a step obtains, be dissolved in 3mL ammoniacal liquor, room temperature (35 ℃) is reaction 3d down.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-2 35mg, productive rate 97%.
Method two: get the T7-2 110mg that a step obtains, be dissolved in 5mL ammoniacal liquor, room temperature (35 ℃) is reaction 3d down.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-2 75mg, productive rate 96%.
Method three: get the T7-2 80mg that a step obtains, be dissolved in 4mL ammoniacal liquor, room temperature (35 ℃) is reaction 3d down.Add an amount of ethanol, decompression is revolved and is desolvated, and gets T8-2 55mg, productive rate 97%.
1H-NMR（CDCl
3,?400M）：
5.15（1H,?d，J?=?4.0?Hz）,?4.22～4.23（1H,?m）,?3.93～4.02（2H,?m）,?3.89（1H,?dd，J
1?=?2.4,?J
2?=?12.0?Hz）,?3.67（1H,?dd，J
1?=?4.8,?J
2?=?12.0?Hz）,?3.38～3.44（1H,?m）,?3.19～3.25（1H,?m），1.97～2.05（3H,?m），1.81～1.87（1H,?m）.
?13C-NMR（CD
3OD,?100?MHz）：
?105.02?,?81.38?,?64.01?,?63.49?,?39.78?,?32.68?,?24.20.ESI-HRMS：calc?for?[C
7?H
15?N?O
3?+?H]?162.1130，found?162.1128.
T8-1 and T8-2 are a pair of diastereomers.
The synthetic route synoptic diagram is as shown in Figure 2; Synthesis step is following:
(1) X-1's is synthetic:
Method one: 1g 2-hydroxyl-3,4-dihydropyrane (X-0) is dissolved in 4mL DMF, adds 0.6g NaH afterwards, stir 10 min after, add the 1.7g Benzyl Chloride be dissolved in 5 ml DMF through constant pressure funnel, 40 ℃ of complete reactions behind 19 h down.Drip water, the NaH that unreacted is intact reacts completely, and adds suitable quantity of water afterwards deposition is dissolved fully; Use ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-1 1.7g, productive rate 95%.
Method two: 2 g X-0 are dissolved in 6mL DMF, add 1.7g NaH afterwards, stir 10 min after, add the 4g Benzyl Chloride be dissolved in 5ml DMF, complete reaction behind 40 ℃ of following 21h through constant pressure funnel.Drip water, the NaH that unreacted is intact reacts completely, and adds suitable quantity of water afterwards deposition is dissolved fully; Use ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-1 3.3g, productive rate 92%.
Method three: 1.5g X-0 is dissolved in 5mL DMF, adds 1.5g NaH afterwards, behind the stirring 10min, adds 3.3 Benzyl Chlorides that are dissolved in 5ml DMF, complete reaction behind 40 ℃ of following 20h through constant pressure funnel.Drip water, the NaH that unreacted is intact reacts completely, and adds suitable quantity of water afterwards deposition is dissolved fully; Use ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-1 2.4g, productive rate 90%.
1H NMR (CDCl
3, 400M):
1.66～1.73 (m, 1H), 1.83～1.95 (m, 1H), 1.96～1.99 (m, 1H), 2.05～2.11 (m, 1H), 3.52 (dd, 1H,
J 1 =4.5 Hz,
J 2 =10.5 Hz), 3.59 (dd, 1H,
J 1 =6 Hz,
J 2 =11.5 Hz), 4.00～4.04 (m, 1H), 4.55～4.62 (m, 2H), 4.67～4.68 (m, 1H), 6.39 (d, 1H,
J=6.5 Hz, 6-CH), 7.26～7.30 (m, 1H), 7.32～7.36 (m, 4H).
(2) X-2's is synthetic:
Method one: the 0.5g ethylene bromohyrin is dissolved in the 5ml methylene dichloride, adds 0.4g PPTS afterwards, and behind the stirring 5min, adding is dissolved in 1.2g X-1, complete reaction behind the 6h under the room temperature.Decompression is revolved and is desolvated, and adds suitable quantity of water afterwards, uses ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-2 0.8g, productive rate 61%.
Method two: the 0.8g ethylene bromohyrin is dissolved in the 6ml methylene dichloride, adds 0.5gPPTS afterwards, and behind the stirring 5min, adding is dissolved in 1.53g X-1, complete reaction behind the 7h under the room temperature.Decompression is revolved and is desolvated, and adds suitable quantity of water afterwards, uses ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-2 1.2g, productive rate 58%.
Method three: the 0.6g ethylene bromohyrin is dissolved in the 5ml methylene dichloride, adds 0.25g PPTS afterwards, and behind the stirring 5min, adding is dissolved in 2g X-1, complete reaction behind the 6.5h under the room temperature.Decompression is revolved and is desolvated, and adds suitable quantity of water afterwards, uses ethyl acetate extraction; Organic phase is water, saturated common salt washing respectively, anhydrous sodium sulfate drying, and decompression is revolved and is desolvated; Column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid X-2 1g, productive rate 63 %.
1H?NMR?(CDCl
3,?400MHz):?
?1.38～1.43?(m,?1H),?1.45～1.64?(m,?2H),?1.65～1.74?(m,?1H),?1.82～1.91?(m,?1H),?3.49～3.57?(m,?3H),?3.77～3.85?(m,?1H),?3.96～4.04?(m,?1H),?4.56～4.59?(m,?2H),?4.93?(s,?1H),?7.25～7.29?(m,?1H),?7.33～7.37?(m,?4H).
(3) X-3's is synthetic:
Method one: 0.11g X-2 is dissolved in 4ml ETHYLE ACETATE, adds 11mg Pd/C afterwards and feeds hydrogen, complete reaction behind the 10min under the room temperature.Filter, decompression is revolved and is desolvated, and gets colourless oil liquid X-3 78mg, productive rate 97%.
Method two: 0.15g X-2 is dissolved in 5ml ETHYLE ACETATE, adds 23mg Pd/C afterwards and feeds hydrogen, complete reaction behind the 15min under the room temperature.Filter, decompression is revolved and is desolvated, and gets colourless oil liquid X-3 104mg, productive rate 96%.
Method three: 0.08g X-2 is dissolved in 4ml ETHYLE ACETATE, adds 16mg Pd/C afterwards and feeds hydrogen, complete reaction behind the 10min under the room temperature.Filter, decompression is revolved and is desolvated, and gets colourless oil liquid X-3 57mg, productive rate 98 %.
1H NMR (CDCl
3, 400M):
1.36～1.45 (m, 2H), 1.48～1.55 (m, 1H), 1.59～1.66 (m, 3H), 1.72～1.77 (m; 1H), 1.81～1.96 (m, 3H), 3.48～3.52 (m, 4H), 3.76～3.79 (m; 1H), 4.11～4.17 (m, 1H), 4.91 (d, 1H
J=2 Hz).
(4) X-4's is synthetic:
Method one: 70mg X-3 is dissolved in 3ml ammoniacal liquor, and reaction is 3 days under the room temperature.Add an amount of ethanol, revolve to steam to remove and desolvate, get colourless oil liquid X-4 49mg, productive rate 96%.
Method two: 100mg X-3 is dissolved in 5ml ammoniacal liquor, and reaction is 3 days under the room temperature.Add an amount of ethanol, revolve to steam to remove and desolvate, get colourless oil liquid X-4 71mg, productive rate 97%.
Method three: 50mg X-3 is dissolved in 3ml ammoniacal liquor, and reaction is 3 days under the room temperature.Add an amount of ethanol, revolve to steam to remove and desolvate, get colourless oil liquid X-4 36mg, productive rate 96%.
1H NMR (CD
3OD, 400M):
1.27～1.41 (m, 1H), 1.58～1.68 (m, 3H), 1.74～1.82 (m, 1H); 1.85～1.97 (m, 1H), 3.17～3.19 (m, 1H), 3.29～3.30 (m, 1H); 3.45～3.53 (m, 1H), 3.65～3.69 (m, 1H), 3.79～3.91 (m, 1H); 3.92～3.98 (m, 1H), 4.89 (d, 1H
J=2.5 Hz).
With n=2 described in the embodiment 2, m=1, the compound when k=0 (X-4) is an example, investigates its cracking performance.In order to analyze its cracking performance, at first need connect ultraviolet or fluorophor to this compound, so at first synthesized following compounds X-6, its synthetic route synoptic diagram is as shown in Figure 3, its structural formula is:
One, compounds X-6 is synthetic
(1) X-1 is synthetic
In ice-water bath, N
2Protection down, with 0.62g NaH (60%, 15.44mmol; 1.07eq) be dissolved among the 10 mL DMF, with 1.65g 2-methylol-3,4-dihydropyrane (14.47mmol; 1 eq) be added drop-wise to slowly that 30 min drip off in the reaction solution, 0 ℃ is stirred 1h down, with 2.64 g BnBr (15.44mmol; 1.07 eq) be added drop-wise in the reaction solution, remove ice-water bath, stir 8h.With the saturated NH of 10 mL
4Cl washing, use again (2 * 50mL) extracted with diethyl ether, organic phase are water, saturated common salt washing respectively, anhydrous sodium sulfate drying, decompression is revolved and is desolvated, column chromatography (sherwood oil: ETHYLE ACETATE=60:1) product colourless oil liquid 2.54g, productive rate 86%.
1H?NMR?(400MHz,?CDCl
3):?7.40～7.26?(m,?5H),?6.43(d,
?J?=?8.4?Hz,?1H),?4.73～4.68(m,?1H),?4.61?(dd,?
J 1? =?16.4?Hz,?
J 2 =?20.8?Hz,?2H),?4.08～4.01?(m,?1H),?3.61(dd,?
J 1? =?8?Hz,?
J 2? =?13.6?Hz,?1H),?3.54?(dd,?
J 1 =?5.6?Hz,?
J 2 ?=?13.6?Hz,1H),?2.18～2.05?(m,?1H),2.04-1.93?(m,?1H),?1.90～1.83?(m,?1H),1.78～1.64?(m,?1H).
(2) X-5 is synthetic
Under ice-water bath, 0.061g thanomin (1 mmol, 1 eq), 0.21g TsOH (1.1 mmol, 1.1 eq) are dissolved among the 2mL DCM, 0 ℃ is stirred 30min down, and reaction solution is a suspension liquid.0.2 GX-1 (1 mmol, 1 eq) is dissolved among the 1mL DCM, is added drop-wise in the reaction solution, 0 ℃ is stirred 2h down, and reaction solution becomes clarification.The pressure reducing and steaming solvent adds and contains 0.16 gK
2CO
3(1.2 mmol, 1.2 eq) acetonitrile 10ml, suction filtration a large amount of white solids appear, in vigorous stirring; Behind the pressure reducing and steaming solvent, column chromatography for separation, (eluent is respectively methylene dichloride: methyl alcohol=50:1,30:1 in gradient elution; 20:1 10:1), gets the faint yellow oily compound of 125 mg, productive rate 47%.
1H?NMR?(400MHz,?CDCl
3):?7.27～7.19?(m,?5H),?4.82(s,1H),?4.50?(dd,?
J 1? =?12?Hz,?
J 2? =?16.4?Hz,?2H),?3.91-3.86?(m,?1H),?3.72～3.67?(m,?1H),3.43～3.38?(m,?1H),?3.80～3.33?(m,?2H),2.85?(s,?2H),1.81～1.72?(m,?1H),1.65～1.59?(m,?1H),?1.57～1.48(m,?3H),1.38～1.27?(m,?1H).
(3) X-6 is synthetic
Under ice-water bath, 50mg p-Nitrobenzenecarboxylic acid (0.3mmol, 1.2 eq), 67 mg EDC (0.35mmol, 1.4 eq) are dissolved in 2mL DCM, solution clarification back adds 66mg X-5 (0.25 mmol, 1 eq), removes ice-water bath, stirs 3h under the room temperature.Use the saturated common salt water washing, anhydrous sodium sulfate drying, decompression is revolved and is desolvated, column chromatography (sherwood oil: ETHYLE ACETATE=3:1), get 67mg yellow oily liquid, productive rate 65%.
1H?NMR?(300MHz,?CDCl
3):?8.21～8.17(dd,?
J 1? =?1.5?Hz,?
J 2? =?8.7?Hz,?2H),?7.94～7.86(dd,?
J 1? =?9?Hz,?
J 2 =?12?Hz,2H),?7.64(s,?1H),?7.34～7.14(m,?5H),?4.89(s,?1H),?4.44～4.40(m,?2H),?4.16～4.10?(m,?1H),?4.00～3.90?(m,?1H),3.87～3.62(m,?2H),?3.58～3.43(m,?3H),?1.96～1.76(m,?2H),?1.76～1.64?(m,?1H),?1.64～1.50?(m,?2H),?1.50～1.30(m,?1H)?.
Two, investigate the cracking performance of compounds X-6
X-6 cracking experiment reaction formula is as shown in Figure 4, under different temperature and pH condition, qualitatively judges X-6 cracked situation through TLC.The result is following:
1. hydrochloric acid soln, < 1.63 have cleavage reaction to take place to pH, and reaction efficiency is lower, does not realize complete cracking.
2.TsOH/MeOH system, reaction 2h raw material disappears cracking fully.
3. the by product of cleavage reaction is more, but successfully isolates cleavage product, through
1H-NMR confirms that cleavage product is said compounds X-6-A, can clear and definite its fracture position.
1H?NMR?(300MHz,?DMSO-d
6):?8.56(s,?1H),?8.30(d,?
J?=?8.7?Hz,2H),?8.11(d,?
J?=?9?Hz,2H),?4.84(s,?1H),?3.58～3.52(m,?2H),?3.42～3.34(m,?2H).
4. temperature is to the influence less (25～50 ℃) of reaction
Conclusion: when n=2, said compound has by product to generate, but can rupture fully under acidic conditions, can be applicable in the synthetic order-checking of DNA.
With n=1 described in the embodiment 1, m=1, the compound when k=0 (T8-1 and T8-2) is an example, investigates its cracking performance.In order to use HPLC to analyze its cracking performance, at first need connect ultraviolet or fluorophor to this compound, so at first synthesized following compound T9, its synthetic route synoptic diagram is as shown in Figure 5.
One, compound T9's is synthetic
A, compound T9-1's is synthetic
In the single port bottle of 10 mL, add the 3mL dry DMF; Add 15mg (42
mol again) T8-1; Lucifuge; Stir under the room temperature; With 10 mg (19
mol) 5 (6)-TAMRA; SE (II) is dissolved in the 2mL dry DMF, add again 40
L (285
mol) triethylamine.Stirring reaction under the room temperature, TLC track to raw material and disappear.Stopped reaction is removed DMF under the decompression, makees developping agent with DCM/MeOH=2/1, and the big plate separation and purification of TLC gets pure compound 8mg, productive rate 73.4%.
1H-NMR (CD
3OD, 300M):
8.06～8.16 (m, 2H), 7.70 (s, 1H), 7.26 (d, 2H,
J=9.6 Hz), 7.00 (dd, 2H, J
1=2.1, J
2=9.6 Hz), 6.92 (d, 2H,
J=2.1 Hz), 5.17 (d, 1H,
J=3.0Hz), 4.07～4.11 (m, 1H), 3.76～3.84 (m, 1H), 3.40～3.65 (m, 5H), 3.28 (s, 12H), 1.83～2.03 (m, 3H), 1.60～1.63 (m, 1H). ESI-HRMS:calc for [C
32H
35N
3O
7+ H] 574.2553, found 574.2534; Calc for [C
32H
35N
3O
7+ Na] 596.2373, found 596.2363.
B, compound T9-2's is synthetic
In the single port bottle of 10mL, add the 2mL dry DMF; Add 30mg (84
mol again) T8-2; Lucifuge; Stir under the room temperature; With 20mg (38
mol) 5 (6)-TAMRA; SE (II) is dissolved in the 4mL dry DMF, add again 80
L (570
mol) triethylamine.Stirring reaction under the room temperature, TLC track to raw material and disappear.Stopped reaction is removed DMF under the decompression, makees developping agent with DCM/MeOH=3/1, and the big plate separation and purification of TLC gets pure compound 20mg, productive rate 96%.
1H-NMR (CD
3OD, 400M):
8.13 (d, 1H, J=8.0Hz), 8.08 (dd, 1H, J
1=1.6, J
2=8.0 Hz), 7.73 (d, 1H,
J=1.2 Hz), 7.25 (dd, 2H, J
1=1.6, J
2=9.6 Hz), 6.99 (dd, 2H, J
1=2.0, J
2=9.2 Hz), 6.89 (d, 2H,
J=2.4Hz), 5.10 (d, 1H,
J=1.6Hz), 4.07～4.11 (m, 1H), 3.78～3.85 (m, 1H), 3.46～3.61 (m, 5H), 3.26 (s, 12H), 1.87～1.95 (m, 3H), 1.68～1.76 (m, 1H) .ESI-HRMS:calc for [C
32H
35N
3O
7+ H] 574.2553, found 574.2531; Calc for [C
32H
35N
3O
7+ Na] 596.2373, found 596.2340.
Two, investigate the cracking performance of compound T9
The cracking experiment reaction formula of T9 is as shown in Figure 6.Concrete steps are following:
In 10mL single port bottle, add 3mg T9-1 solid, add the buffered soln of the Hydrocerol A/Sodium phosphate, dibasic of 1mL pH=2.60 (under 45 ℃ of oil bath constant temperature, recording), 45 ℃ of following stirring reactions of oil bath constant temperature.At 1 min, 3 min, 5 min; 7 min, 9 min, 11 min; 13 min; 15 min, 10
that take a sample respectively during 20 minL, be dissolved in cancellation reaction in the sodium hydrogen carbonate solution of 0.6 mL 1M.Relative content with raw material T9-1 and split product T9-A in the HPLC analysis gained sample.
In 10mL single port bottle, add 3mg T9-1 solid, add the buffered soln of the Hydrocerol A/Sodium phosphate, dibasic of 1mL pH=2.25 (under 45 ℃ of oil bath constant temperature, recording), 45 ℃ of following stirring reactions of oil bath constant temperature.At 1 min, 3 min, 5 min; 7 min, 9 min, 11 min; 13 min; 15 min, 10
that take a sample respectively during 20 minL, react with 1 mL, 1 M sodium hydrogen carbonate solution cancellation.Relative content with cracking stock T9-1 and split product T9-A in the HPLC analysis gained sample.
In 10mL single port bottle, add 3mg T9-2 solid, add the buffered soln of the Hydrocerol A/Sodium phosphate, dibasic of 1 mL pH=2.60 (under 45 ℃ of oil bath constant temperature, recording), 45 ℃ of following stirring reactions of oil bath constant temperature.At 1 min, 3 min, 5 min; 7 min, 9 min, 11 min; 13 min; 15 min, 10
that take a sample respectively during 20 minL, be dissolved in cancellation reaction in the sodium hydrogen carbonate solution of 1.2 mL 1M.Relative content with raw material T9-2 and split product T9-A in the HPLC analysis gained sample.
In 10mL single port bottle, add 3mg T9-2 solid, add the buffered soln of the Hydrocerol A/Sodium phosphate, dibasic of 1 mL pH=2.25 (under 45 ℃ of oil bath constant temperature, recording), 45 ℃ of following stirring reactions of oil bath constant temperature.At 1 min, 3 min, 5 min; 7 min, 9 min, 11 min; 13 min; 15 min, 8
that take a sample respectively during 20 minL, react with 1mL 1 M sodium hydrogen carbonate solution cancellation.Relative content with cracking stock T9-2 and split product T9-A in the HPLC analysis gained sample.
Split product T9-A characterizes with ESI-HRMS, and the result is following: calc for [C
27H
27N
3O
5+ H] 474.2029, found 474.2040; Calc for [C
27H
27N
3O
5+ Na] 496.1848, found 496.1863. result proves that T9-1 is identical with the structure of T9-2 split product, no matter promptly be T9-1 or T9-2, split product is T9-A.
Relative content with reaction raw materials T9 in the HPLC analytical pyrolysis response sample (comprising a pair of diastereomer T9-1 and T9-2) and their split product T9-A can be measured the cracked dynamic performance by law.Used HPLC condition is: pillar: C18; 5
m, 4.6 *, 250 mm; Flow velocity: 0.6 mL/min; Moving phase: water and methyl alcohol 0 min water, 5 min, 10% methyl alcohol, 30 min, 50% methyl alcohol, 50 min, 100% methyl alcohol; Fluorimetric detector: the 546-575 nm. HPLC spectrogram of the raw material T9-1 of scission reaction under the described conditions is as shown in Figure 7, and the HPLC spectrogram of the raw material T9-2 of scission reaction is as shown in Figure 8, and the HPLC spectrogram of split product T9-A is as shown in Figure 9.By Fig. 7,8,9 can find out, higher through raw material T9 (comprising T9-1 and the T9-2) purity of method of the present invention preparation, and raw material T9 (comprising T9-1 and T9-2) can be cracked into intending product T9-A fully, do not have other by product to generate.When pH=2.60, the section H PLC figure of one of scission reaction raw material T9-1 cracking experiment is shown in figure 10, and when pH=2.25, the section H PLC figure of T9-1 cracking experiment is shown in figure 11; When pH=2.60; The HPLC figure of one of scission reaction raw material T9-2 cracking experimental section is shown in figure 12; When pH=2.25; The section H PLC figure of T9-2 cracking experiment is shown in figure 13. and further can be known by Figure 10,11,12,13: raw material T9 (comprising T9-1 and the T9-2) purity through method preparation of the present invention is all higher, and raw material T9 (comprising T9-1 and T9-2) does not have other by product to generate in all cracking fully of different time sections.
The cracking experimental result is:
When pH=2.60, cracking experimental result such as following table 1:
Table 1
When pH=2.25, cracking experimental result such as following table 2:
Table 2
Can be known by above-mentioned table 1,2: the rate of cleavage of T9-1 and two kinds of diastereomers of T9-2 is variant, but is more or less the same.The both can realize high efficiency complete cracking under relatively mild condition, prove thus, and cleavable connector element T8 of the present invention (comprising two kinds of isomer of T8-1 and T8-2) can be applicable in the synthetic order-checking of DNA.
Claims (13)
1. a cleavable connector element is characterized in that, its structural formula is:
Wherein, n is 1 or 2, and m is any integer in 0～20, and k is any integer in 1～20.
2. the purposes of a cleavable connector element according to claim 1 in the synthetic order-checking of DNA is characterized in that said cleavable connector element connects Nucleotide and resorcinolphthalein, is used for the synthetic order-checking of DNA.
3. the compound method of a cleavable connector element according to claim 1 is characterized in that,
When n=1, comprise following synthesis step:
A, T-1's is synthetic: with L-L-glutamic acid is raw material, adds hydrochloric acid, under ice bath stirs, drips sodium nitrite solution, after dropwising, continues reaction 3～5h under ice bath, moves to then under the room temperature and spends the night; After reaction finished, pressure reducing and steaming water added acetic acid ethyl dissolution, filters, and filtrating is used anhydrous sodium sulfate drying, filters, and revolves dried solvent and gets T-1;
B, T-2's is synthetic: get T-1, make solvent with anhydrous tetrahydro furan, nitrogen protection slowly drips borine/dimethyl sulphide solution under ice-water bath, after dropwising, continue at room temperature to react 4～5h; After reaction finishes, add methyl alcohol cancellation reaction, revolve and desolvate, add methyl alcohol again, revolve dried T-2;
C, T-3's is synthetic: get T-2, make solvent with methylene dichloride, add imidazoles, nitrogen protection is descended and dimethyl-tertiary butyl chloride silane at room temperature reacts 17～20h; After reaction finishes, add the methylene dichloride dilution, use 2mol/L hydrochloric acid, water and saturated sodium bicarbonate washing reaction liquid respectively, anhydrous sodium sulfate drying revolves driedly, and column chromatography gets the pure article of T-3;
D, T-4's is synthetic: make solvent with methylene dichloride; T-3 reacts with diisobutyl aluminium hydride under nitrogen protection and cryosel bath, and TLC tracks to and reacts completely. after reaction finishes, and adding 0.2mol/L HCl cancellation reaction; Dichloromethane extraction; Organic phase is washed with saturated sodium bicarbonate aqueous solution, anhydrous sodium sulfate drying, rotary evaporation remove desolvate T-4;
E, T-5's is synthetic: make solvent with methylene dichloride, T-4 adds triethylamine under nitrogen protection and cryosel bath, then with methylsulfonyl chloride reaction 30～50min; At 40～50 ℃ of 3～5h that reflux, after reaction finishes, methylene dichloride dilution, washing; Anhydrous sodium sulfate drying revolves driedly, and column chromatography gets T-5;
F, T-6's is synthetic: make solvent with methylene dichloride, T-5 at room temperature reacts with raw material A under the effect of tosic acid pyridinium salt, and TLC tracks to and reacts completely; Reaction is revolved dried solvent after finishing, and adds entry; Use ethyl acetate extraction, saturated common salt water washing organic phase, anhydrous sodium sulfate drying; Filter, revolve driedly, column chromatography obtains the pure article of T-6; The structural formula of said raw material A is:
G, T-7's is synthetic: make solvent with THF, and under the effect of tetrabutyl fluoride amine, T-6 dehydroxylation protection at room temperature, TLC tracks to and reacts completely, and reaction is revolved dried solvent after finishing, and column chromatography obtains T-7;
H, T-8's is synthetic: be dissolved in excess of ammonia water to T-7, react under the room temperature, TLC tracks to and reacts completely, and reaction adds ethanol after finishing, and decompression is revolved and desolvated, and gets T-8, and T-8 is said cleavable connector element;
When n=2, comprise following synthesis step:
A, X-1's is synthetic: with N, dinethylformamide is made solvent, 2-hydroxyl-3, and the 4-dihydropyrane is under the effect of NaH; At room temperature react with Benzyl Chloride, TLC tracks to and reacts completely, after reaction finishes; Drip water and remove unreacted NaH, use ethyl acetate extraction, organic phase is water, saturated common salt washing respectively; Anhydrous sodium sulfate drying, decompression is revolved and is desolvated, and column chromatography gets X-1;
B, X-2's is synthetic: as solvent, be dissolved in methylene dichloride to raw material A with methylene dichloride, under the effect of tosic acid pyridinium salt and X-1 at room temperature react; TLC tracks to and reacts completely, and after reaction finished, decompression was revolved and desolvated; Add entry afterwards, use ethyl acetate extraction, organic phase is water, saturated common salt washing respectively; Anhydrous sodium sulfate drying, decompression is revolved and is desolvated, and column chromatography gets X-2; The structural formula of said raw material A is:
C, X-3's is synthetic: make solvent with ETHYLE ACETATE, be dissolved in ETHYLE ACETATE to X-2, adding Pd/C also feeds hydrogen, reacts under the room temperature, and TLC tracks to and reacts completely, and after reaction finishes, filters, and decompression is revolved and desolvated, and gets X-3;
D, X-4's is synthetic: be dissolved in excess of ammonia water to X-3, react under the room temperature, TLC tracks to and reacts completely, and reaction adds ethanol after finishing, and decompression is revolved and desolvated, and gets X-4, and X-4 is said cleavable connector element.
4. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, hydrochloric acid described in the step a is 6 mol/L, and the mol ratio of said L-L-glutamic acid, hydrochloric acid and the Sodium Nitrite of adding is 1:2～2.5:1.5～2.
5. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, the said T-1 that adds among the step b and the mol ratio of borine are 1:1.1～1.5.
6. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, the mol ratio of the said T-2 that adds among the step c, imidazoles and dimethyl-tertiary butyl chloride silane is 1:1.1～1.5:1.1～1.5.
7. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, the said T-3 that adds in the steps d and the mol ratio of diisobutyl aluminium hydride are 1:1.2～1.5.
8. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, the mol ratio of said T-4, triethylamine and the methylsulfonyl chloride that adds among the step e is 1:3～5:1.3～1.5.
9. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=1, the mol ratio of the said T-5 that adds among the step f, raw material A and tosic acid pyridinium salt is 1:1.5～2:0.2～0.5.
10. the compound method of cleavable connector element according to claim 3; It is characterized in that; When n=1, the tetrabutyl fluoride amine described in the step g is the tetrahydrofuran solution that contains 1mol/L tetrabutyl fluoride amine, and the said T-6 of adding and the mol ratio of tetrabutyl fluoride amine are 1:1.5～2.
11. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=2, and the said 2-hydroxyl-3 that adds among the step a, the mol ratio of 4-dihydropyrane, sodium hydride and Benzyl Chloride is 1:3～5:1.5～2.
12. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=2, the mol ratio of the said X-1 that adds among the step b, raw material A and toluenesulphonic acids pyridinium salt is 1.5～2:1:0.2～0.5.
13. the compound method of cleavable connector element according to claim 3 is characterized in that, when n=2, said X-2 that adds among the step c and the mass ratio of Pd/C are 1:10%～20%.
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| CN101935702A (en) * | 2010-08-10 | 2011-01-05 | 深圳华因康基因科技有限公司 | Nucleotide with removable mark, preparation method thereof of method for gene sequencing |
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| CN106588722A (en) * | 2015-10-20 | 2017-04-26 | 上海交通大学 | Synthesis of thioketal connecting unit and application thereof in DNA sequencing |
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