CN102675262A - Cracking connecting unit for tetrahydrofuran ether derivatives and application of cracking connecting unit - Google Patents
Cracking connecting unit for tetrahydrofuran ether derivatives and application of cracking connecting unit Download PDFInfo
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Abstract
The invention discloses a cracking connecting unit for tetrahydrofuran ether derivatives and application of the cracking connecting unit. The cracking connecting unit has the following structural formula, wherein n is any integer of 0 to 44. The cis-tetrahydrofuran ether derivatives, the trans-tetrahydrofuran ether derivatives or the cis- and trans-tetrahydrofuran ether derivatives can be used for sequencing, and have the same effect; the synthesizing raw materials for the compounds are simple and readily available, the synthesizing process is conventional chemical reaction, and massive synthesis is easy to realize; the compounds can be efficiently connected with nucleotide and fluorescein; and by researching the cracking performance of the compounds, the compounds can realize efficient cracking under mild conditions, and can be applied to DNA sequencing.
Description
Technical field
The present invention relates to chemosynthesis and biochemical field, be specifically related to THF ether derivant cleavable connector element of the synthetic order-checking of a kind of DNA of can be used for and uses thereof.
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 4 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 except require 3 '-the hydroxyl blocking-up, 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, for example resorcinolphthalein couples together.Then, before next one indication Nucleotide is incorporated into, under the condition of gentleness, 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 significant effects, 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 reductive agent responsive (disulfide linkage, azo cpd); Photodestruciton (adjacent nitrobenzyl verivate, phenacyl-ester derivative and other light cleavable connector element); Electrophilic reagent/acid-sensitive sense (acid cleavage; Triazo-compound); Cracking under the metal function; Oxygenant sensitivity etc.Yet these connector elements all exist different separately shortcomings and limitation (Bioorganic&Medicinal Chemistry 2012,20,571-582).
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 that above-mentioned prior art exists, one type of new THF ether derivant cleavable connector element that can be used for the synthetic order-checking of DNA and uses thereof is provided.In synthetic order-checking; Promptly the check order chirality factor of effect and raw material of the fracture effect of such connector element is irrelevant; That is to say in synthetic THF ring derivatives, be that cis, trans or the two mixture all can be used for order-checking, and have identical effect.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 cracking under the condition of gentleness, have the value that is applied to dna sequencing.
The contriver has been developed a kind of cleavable connector element in previous work, such connector element comprises tetrahydropyranyl ethers and two kinds of (application numbers: 201110331659.1), can be used for the synthetic order-checking of DNA of THF ether.Compare with previous work, the present invention has further improved the compound method of T series compound, and has increased YZ and ZY series compound, in the improved compound method of T series compound, has avoided using hypertoxic chemical MsCl, has reduced single step reaction simultaneously.And reaction raw materials no longer is confined to L L-glutamic acid, also can be D type or DL type L-glutamic acid, and no matter institute's synthetic tetrahydrofuran derivatives is cis, and trans or the two mixture all can be used for the synthetic order-checking of DNA, and has similar cracking performance.
The objective of the invention is to realize through following technical scheme:
First aspect the present invention relates to a kind of THF ether derivant cleavable connector element, and its structural formula is suc as formula shown in (I):
(I); Wherein, n is any integer in 0～44.
Preferably, the unitary structural formula of said cleavable is suc as formula shown in (II):
Further, n is any integer in 21～44 in the said formula (I).
Further, said cleavable connector element is through the following steps synthetic:
A, T-1's is synthetic: D, and cyclization under hydrochloric acid and Sodium Nitrite effect, takes place and obtains T-1 in L or DL-L-glutamic acid;
B, T-2's is synthetic: reduction reaction takes place and gets T-2 in the ether solvent of borine in T-1;
C, T-3's is synthetic: get T-2, make solvent with methylene dichloride, add imidazoles, protection of inert gas is descended and dimethyl-tertiary butyl chloride silane reaction gets T-3;
D, T-4's is synthetic: make solvent with methylene dichloride, T-3 reacts with diisobutyl aluminium hydride under protection of inert gas and cryosel bath, gets T-4;
E, when n=0, T-6's is synthetic: get T-4, make solvent, add bromoethanol and Amberlyst A-15 (ion exchange resin A-15) with methylene dichloride, at 40～50 ℃ of backflow 1～3h, react T-6;
When n=1～44, T-6's ' is synthetic: get T-4, make solvent, add bromo three polyoxyethylene glycol and Amberlyst A-15 with methylene dichloride, at 40～50 ℃ of backflow 1～3h, react T-6 ';
F, T-7 or T-7's ' is synthetic: make solvent with THF, and under the tetrabutyl ammonium fluoride effect, T-6 or the protection of T-6 ' dehydroxylation, reaction obtains T-7 or T-7 ';
G, T-8 or T-8's ' is synthetic: substitution reaction takes place in T-7 or T-7 ' in ammoniacal liquor, gets T-8 or T-8 ', and T-8 or T-8 ' are said THF ether derivant cleavable connector element.
Preferably, the unitary structural formula of said cleavable is suc as formula shown in (III):
Further, said cleavable unit is through the following steps synthetic:
A, YZ1's is synthetic: ethyl malonate and 2-bromo-1, and the reaction of 1-glycol dimethyl ether obtains YZ1;
B, YZ2's is synthetic: reduction reaction takes place and obtains YZ2 under ether, Lithium Aluminium Hydride effect in YZ1;
C, YZ3's is synthetic: cyclization takes place and obtains YZ3 in YZ2 in camphorsulfonic acid pyridinium salt dichloromethane solution;
D, YZ4's is synthetic: YZ3 and bromotoluene reaction obtain YZ4;
E, YZ5's is synthetic: with 1, the reaction of 4-dioxane goes hydroxyl protecting group to obtain YZ5 to YZ4 under acidic conditions;
F, YZ6's is synthetic: YZ5 and bromoethanol reaction obtain YZ6;
G, YZ7's is synthetic: YZ6 is under the palladium-carbon catalyst effect, and debenzylation obtains YZ7;
H, YZ8's is synthetic: aminating reaction takes place and obtains YZ8 in YZ7 in ammoniacal liquor, YZ8 is described THF ether derivant cleavable connector element.
Preferably, the unitary structural formula of said cleavable is suc as formula shown in (IV):
Further, said cleavable connector element is through the following steps synthetic:
A, ZY1's is synthetic: gamma-butyrolactone and methyl-formiate in the presence of sodium hydride, react ZY1;
B, ZY2's is synthetic: reduction reaction takes place and obtains ZY2 under the effect of ether, methyl-formiate in ZY1;
C, ZY3's is synthetic: ZY2 and bromotoluene reaction obtain ZY3;
D, ZY4's is synthetic: with 1, the reaction of 4-dioxane goes hydroxyl protecting group to obtain ZY4 to ZY3 under acidic conditions;
E, ZY5's is synthetic: ZY4 and bromoethanol reaction obtain ZY5;
F, ZY6's is synthetic: ZY5 is under the effect of palladium-carbon catalyst, and debenzylation obtains ZY6;
G, ZY7's is synthetic: aminating reaction takes place and obtains ZY7 in ZY6 in ammoniacal liquor, ZY7 is described THF ether derivant cleavable connector element.
Second aspect the present invention relates to the purposes of aforesaid THF ether derivant cleavable connector element in the synthetic order-checking of DNA.
Preferably, said THF ether derivant cleavable connector element connects Nucleotide and resorcinolphthalein, is used for the synthetic order-checking of DNA.
Compared with prior art; The present invention has following beneficial effect: compound of the present invention all can be realized the high-level efficiency cracking under the condition of gentleness; And this connector element can be connected with Nucleotide and resorcinolphthalein effectively and obtains reversible terminal, can be applicable to dna sequencing.
Description of drawings
Fig. 1 is the building-up process synoptic diagram of embodiment 1;
Fig. 2 is the building-up process synoptic diagram of embodiment 2;
Fig. 3 is the building-up process synoptic diagram of embodiment 3;
Fig. 4 is the building-up process synoptic diagram of embodiment 4;
Fig. 5 is the building-up process synoptic diagram of embodiment 5;
Fig. 6 is the building-up process synoptic diagram of embodiment 6;
Fig. 7 is the building-up process synoptic diagram of compound 3 among the embodiment 7;
Fig. 8 is the building-up process synoptic diagram of compound 6 among the embodiment 7;
Fig. 9 is the building-up process synoptic diagram of compound 10 among the embodiment 7;
Figure 10 is the building-up process synoptic diagram of compound 11 among the embodiment 7;
Figure 11 is the building-up process synoptic diagram of compound 12 among the embodiment 7;
Figure 12 is the building-up process synoptic diagram of compound 13 among the embodiment 7;
Figure 13 is a scission reaction synoptic diagram among the embodiment 8.
Embodiment
Below in conjunction with accompanying drawing and specific embodiment the present invention is elaborated.Following examples will help those skilled in the art further to understand the present invention, but not limit the present invention in any form.Should be pointed out that to those skilled in the art, under the prerequisite that does not break away from the present invention's design, can also make some adjustment and improvement.These all belong to protection scope of the present invention.
Used raw material, the reagent of the present invention is commercially available AR, CP level.
Gained intermediate product of the present invention and final product employing NMR etc. characterize.
The synthetic route synoptic diagram of connector element T-8 (5-methylol-2-(2-amino ethoxy)-THF) is as shown in Figure 1, and synthesis step is following:
(1) T-1's is synthetic: D, L or DL-L-glutamic acid are in the presence of Sodium Nitrite, and reaction obtains T-1.
Said step is specially: in 500mL single port bottle, add 10g (68mmol) D, L or DL-L-glutamic acid, add hydrochloric acid (the 14mL concentrated hydrochloric acid is dissolved in 28mL water), the solid dissolving.Under ice bath, stir 30min, continue under ice bath, to drip the solution (7g, 100mmol are 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.
(2) T-2's is synthetic: reduction reaction takes place and gets T-2 in the ether solvent of borine in T-1.
Said step is specially: get the first step T-1 bullion 23.22g 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 18ml 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 16.87g yellow oily liquid, directly be used for next step.
1H-NMR(CDCl
3，300M)：δ4.61～4.63(1H，m)，3.89(1H，dd，J
1＝3.0，J
2＝12.6HZ)，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: the hydroxyl of T-2 is protected, and gets T-3.
Said step is specially: get T-2 bullion 16.87g in the 250ml two-mouth bottle, add 11.11g (163mmol) imidazoles, vacuumize, the 100ml methylene dichloride is injected in nitrogen protection.Get TBSCl24.68g (163mmol) and be dissolved in the 50ml methylene dichloride, be injected in the above-mentioned system, stirring reaction 20h under the room temperature.Add the methylene dichloride dilution, use 2MHCl, water and saturated NaHCO respectively
3Washing reaction liquid, anhydrous sodium sulfate drying, revolve dried, 27.48g yellow oily liquid.Adorn post with silica gel, petrol ether/ethyl acetate was carried out the post separation in 15: 1, got the pure article 6.21g of faint yellow T-3, and T-1 is 20% to the T-3 total recovery.
1H-NMR(CDCl
3，400M)：δ4.55～4.59(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)，2.44～2.59(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: reduction reaction takes place and gets T-4 in T-3.
Said step is specially: get T3 3.03g (13.2mmol) 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 17.1ml 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%.
1H-NMR(CDCl
3，400M)：δ5.38～5.56(1H，m)，4.25～4.29(1H，m)，3.80(1H，dd，J
1＝2.8，J
2＝10.4Hz)，3.57(1H，dd，J
1＝2.8，J
2＝10.8Hz)，2.16(2H，s)，1.92～1.96(2H，m)，0.92(9H，s)，0.11(6H，s)。
(5) T-6's is synthetic: T-4 and bromoethanol reaction get T-6.
Said step is specially: get 232mgT4 (1mmol), add the dissolving of 15mL methylene dichloride, add 250mg bromoethanol (2mmol), add 50mg Amberlyst A-15,50 ℃ add backflow 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing.Decompression steams solvent, gets 600mg pale yellow oily liquid body.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 5: 1, got the pure article 128mg of T-6, and productive rate is 30%.
The T-6-1 cis-isomeride
1H-NMR (CDCl
3, 300M): δ 5.18 (1H, d, J=4.20Hz), 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
3，100MHz)：δ104.66，79.00，67.27，65.39，32.06，31.08，25.91，25.29，18.35，-5.27，-5.33。ESI-HRMS：calc?for [C
13H
27O
3Si?Br+Na]361.0811，found?361.0835。
The T-6-2 trans-isomer(ide)
1H-NMR (CDCl
3, 300M): δ 5.13 (1H, d, J=3.9Hz), 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
3，100MHz)：δ104.36，81.26，67.21，67.19，32.82，30.97，29.69，26.24，25.93，18.36，-5.25，-5.28。ESI-HRMS：calc?for[C
13?H
27?O
3?Si?Br+Na]361.0811，found361.0836。
(6) T-7's is synthetic: the hydroxyl of T-6 goes protection, gets T-7.
Said step is specially: get the T-6-1110mg (0.32mmol) that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.65ml (0.65mmol) 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 T-7-160mg, productive rate 83.3%.
1H-NMR(CDCl
3，400M)：δ5.19(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)。
Get the T-6-270mg (0.21mmol) that a step obtains, add 5ml THF, stir 10min, add the TBAF solution (in THF) of 0.41ml (0.41mmol) 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 colorless oil product T-7-242mg, productive rate 87.9%.
1H-NMR(CDCl
3，400M)：δ5.15(1H，d，J＝4.40Hz)，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＝12.0Hz)，3.48～3.51(2H，t，J＝6.0Hz)，1.91～2.07(4H，m)。
T-7-1 and T-7-2 are a pair of diastereomers.
(7) T-8's is synthetic: substitution reaction takes place in T-7 in ammoniacal liquor, get T-8.
Said step is specially: get the T-7-140mg that a step obtains, be dissolved in 2ml ammoniacal liquor, react 40h under the room temperature, the some plate finds that raw material disappears basically.Add an amount of ethanol, decompression is revolved and is desolvated, and gets colourless oil liquid T-8-128mg, productive rate 97%.
1HNMR(CD
3OD，400M)：δ5.20(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＝11.6Hz)，3.47～3.53(1H，m)，3.30～3.32(1H，m)，3.13～3.16(1H，t，J＝4.8Hz)，1.86～2.12(3H，m)，1.62～1.70(1H，m)。
13C-NMR(CD
3OD，400M)：δ104.72，79.11，63.87，62.95，39.53，31.54，24.86。ESI-HRMS：calc?for[C
7H
16N?O
3+H]162.1130，found?162.1135。
Get the T-7-240mg that a step obtains, be dissolved in 2mL ammoniacal liquor, react 40h under the room temperature.Add an amount of ethanol, decompression is revolved and is desolvated, and gets colourless oil liquid T-8-228mg, productive rate 97%.
1H-NMR(CDCl
3，400M)：δ5.15(1H，d，J＝4.0Hz)，4.22～4.23(1H，m)，3.93～4.02(2H，m)，3.89(1H，dd，J
1＝2.4，J
2＝12.0Hz)，3.67(1H，dd，J
1＝4.8，J
2＝12.0Hz)，3.38～3.44(1H，m)，3.19～3.25(1H，m)，1.97～2.05(3H，m)，1.81～1.87(1H，m)。
T-8-1 and T-8-2 are a pair of diastereomers.
The synthetic route synoptic diagram is as shown in Figure 2, and synthesis step is following:
(1) T-1～T-4's is synthetic said identical with embodiment 1.
(2) T-6's ' is synthetic: T-4 and bromo trimerization glycol reaction get T-6 '.
Said step is specially: get 232mg T-4 (1mmol), add the dissolving of 15mL methylene dichloride, add the single bromo triethylene glycol (2mmol) of 426mg, add 50mg Amberlyst A-15,50 ℃ add backflow 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing.Decompression steams solvent, gets 600mg pale yellow oily liquid body.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 5: 1, got the pure article 128mg of light green T-6 ', and productive rate is 30%.Cis：
1H?NMR(CDCl
3，400M)：δ5.17(1H，d，J＝4.8Hz)，4.17～4.08(1H，m)，3.84～3.77(3H，m)，3.71～3.62(9H，m)，3.49～3.45(2H，m)，2.07～1.85(4H，m)，0.90(9H，s)，0.07(6H，s)；trans：5.12(1H，d，J＝4.4Hz)。Cis：
13C?NMR(CDCl
3，100MHz)：δ104.22，80.96，70.67，70.60，70.55，67.35，66.10，32.73，29.69，26.37，25.94，14.11，-5.31。trans：
13C?NMR(100MHz，CDCl
3)：δ104.59，78.74，71.24，70.57，70.54，66.32，65.47，31.99，30.26，25.91，25.45，18.35，-5.27。IR：3450，2928，2861，1640，1464，1354，1253，1189，1125，1057，1026，841，777，667。ESI-HRMS：calc?for[C
17H
36O
5?Si?Br+H]427.1515，found?427.1520。
(3) T-7's ' is synthetic: the hydroxyl of T-6 ' goes protection, gets T-7 '.
Said step is specially: get 213mg T-6 ' (1mmol), add the dissolving of 5mL THF, stir 10min, add 1mLTBAF solution (1M in THF).Stir 1h under the room temperature.Stopped reaction, decompression steams solvent, gets 430mg yellow oily liquid.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 4: 1, got the pure article 237mg of weak yellow liquid T-7 ', and productive rate is 76%.cis：
1H?NMR(CDCl
3，400M)：δ5.20(1H，d，J＝4.8Hz)，4.29～4.25(1H，m)，3.88～3.80(3H，m)，3.77～3.73(1H，m)，3.71～3.58(7H，m)，3.53～3.45(3H，m)，2.12～1.92(2H，m)，1.89～1.82(1H，m)，1.71～1.59(1H，m)；trans：5.09(1H，d，J＝4.8Hz)。Cis：
13C?NMR(CDCl
3，100MHz)δ104.22，81.44，71.39，70.71，70.59，66.82，64.59，33.36，31.92，29.35，23.57。trans：
13C?NMR(CDCl
3，100MHz)δ104.57，78.38，71.25，70.66，70.54，66.45，64.88，32.41，30.28，29.69，24.99。IR：3427，2943，1719，1640，1451，1361，1276，1198，1125，1084，1033，977，890，856，787，662，570，471。ESI-HRMS：calc?for[C
11H
22?O
5?Br+H]335.0469，found?335.0470。
(4) T-8's ' is synthetic: substitution reaction takes place in T-7 ' in ammoniacal liquor, get T-8 '.
Said step is specially: get 44mg T-7 ' (0.14mmol), add 5mL ammoniacal liquor, 40 ℃ are stirred 4d.Stopped reaction, decompression steams solvent, obtains 33mg liquid, and productive rate is 95%.
1H?NMR(CD
3OD，400M)：δ5.19(1H，d，J＝3.6Hz)，4.18～4.12(1H，m)，3.82～3.77(1H，m)，3.73(2H，t，J＝3.9Hz)，3.78～3.62(6H，m)，3.61～3.55(2H，m)，3.53～3.46(1H，m)，3.14(2H，t，J＝3.9Hz)，2.06～1.95(2H，m)，1.89～1.82(1H，m)，1.70～1.63(1H，m)。
13C?NMR(CD
3OD，100MHz)δ104.22，81.44，71.39，70.71，70.59，66.82，64.59，33.36，31.92，29.35，23.57。IR：3391，2942，1614，1452，1398，1125，1077，1026。ESI-HRMS：calc?for[C
11H
23?N?O
5+Na]272.1459，found?272.1474。
The synthetic route synoptic diagram is as shown in Figure 3, and synthesis step is following:
(1) T-1～T-4's is synthetic said identical with embodiment 1.
(2) T-6 " synthetic: T-4 and the reaction of bromo Macrogol 2000, must T-6 ".
Said step is specially: get 1mmol T-4, add the dissolving of 15mL methylene dichloride, add the single bromo Macrogol 2000 of 2mmol, add 50mg Amberlyst A-15,50 ℃ add backflow 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing.Decompression steams solvent, gets 600mg pale yellow oily liquid body.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 5: 1, got light green T-6 " pure article 213mg.
1H?NMR(CDCl
3，400M)：δ5.19(1H，d，J＝4.8Hz)，4.22～4.19(1H，m)，3.87～3.73(3H，m)，3.78～3.58(217H，m)，3.52～3.45(2H，m)，2.07～1.80(4H，m)，0.90(9H，s)，0.07(6H，s)。
(3) T-7 " synthetic: the hydroxyl of T-6 ' goes protection, T-7 "
Said step is specially: get 1mmol T-6 ", add the dissolving of 5mL THF, stir 10min, add 1mLTBAF solution (1M in THF).Stir 1h under the room temperature.Stopped reaction, decompression steams solvent, gets 470mg yellow oily liquid.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 4: 1, got weak yellow liquid T-7 " pure article 356mg.
1H?NMR(CDCl
3，400M)：δ5.13(1H，d，J＝4.8Hz)，4.29～4.35(1H，m)，3.88～3.73(4H，m)，3.71～3.58(215H，m)，3.53～3.45(3H，m)，2.12～1.92(2H，m)，1.89～1.82(1H，m)，1.71～1.59(1H，m)。
(4) T-8 " synthetic: T-7 " substitution reaction takes place in ammoniacal liquor, T-8 ".
Said step is specially: get 0.14mmol T-7 ", add 5mL ammoniacal liquor, 40 ℃ are stirred 4d.Stopped reaction, decompression steams solvent, obtains 57mg liquid.
1H?NMR(CD
3OD，400M)：δ5.19(1H，d，J＝4Hz)，4.17～4.12(1H，m)，3.85～3.78(1H，m)，3.73(2H，m)，3.81～3.57(214H，m)，3.57～3.47(3H，m)，3.14(2H，t，J＝3.9Hz)，2.05～1.95(2H，m)，1.89～1.83(1H，m)，1.71～1.63(1H，m)。
The synthetic route synoptic diagram is as shown in Figure 4, and synthesis step is following:
(1) T-1～T-4's is synthetic said identical with embodiment 1.
(2) T-6 " ' synthetic: T-4 and the reaction of bromo cetomacrogol 1000, must T-6 " '.
Said step is specially: get 1mmol T-4, add the dissolving of 15mL methylene dichloride, add the single bromo cetomacrogol 1000 of 2mmol, add 50mg Amberlyst A-15,50 ℃ add backflow 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing.Decompression steams solvent, gets 600mg pale yellow oily liquid body.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 5: 1, got light green T-6 " ' pure article 167mg.
1HNMR(CDCl
3，400M)：δ5.20(1H，d，J＝4.8Hz)，4.23～4.20(1H，m)，3.87～3.79(3H，m)，3.78～3.58(85H，m)，3.52～3.45(2H，m)，2.07～1.85(4H，m)，0.90(9H，s)，0.07(6H，s)。
(3) T-7 " hydroxyl of ' synthetic: T-6 " ' goes protection, T-7 " '
Said step is specially: get 1mmol T-6 " ', add the dissolving of 5mL THF, stir 10min, add 1mL TBAF solution (1M in THF).Stir 1h under the room temperature.Stopped reaction, decompression steams solvent, gets 470mg yellow oily liquid.Adorn post with silica gel, sherwood oil: ETHYLE ACETATE=post separated in 4: 1, got weak yellow liquid T-7 " ' pure article 356mg.
1H?NMR(CDCl
3，400M)：δ5.15(1H，d，J＝4.8Hz)，4.29～4.26(1H，m)，3.88～3.80(3H，m)，3.77～3.58(84H，m)，3.53～3.45(3H，m)，2.12～1.92(2H，m)，1.89～1.82(1H，m)，1.71～1.59(1H，m)。
(4) T-8 " substitution reaction takes place in ' synthetic: T-7 " ' in ammoniacal liquor, T-8 " '.
Said step is specially: get 0.14mmol T-7 " ', add 5mL ammoniacal liquor, 40 ℃ are stirred 4d.Stopped reaction, decompression steams solvent, obtains 57mg liquid.
1H?NMR(CD
3OD，400M)：δ5.16(1H，d，J＝4Hz)，4.19～4.15(1H，m)，3.85～3.78(1H，m)，3.73(2H，m)，3.81～3.60(82H，m)，3.60～3.47(3H，m)，3.16(2H，t，J＝3.9Hz)，2.05～1.95(2H，m)，1.89～1.83(1H，m)，1.71～1.63(1H，m)。
Embodiment 5, link unit ZY7's is synthetic
ZY linker series 3-methylol-2-(2-amino ethoxy)-THF, promptly the synthetic synoptic diagram of ZY7 is as shown in Figure 5, and synthesis step is following:
(1) ZY1's is synthetic: gamma-butyrolactone and methyl-formiate in the presence of sodium hydride, react ZY1.
Said step is specially: get 0.2g sodium hydride (in 60% kerosene), add the 10mL ether, vigorous stirring.Add the 0.6g methyl-formiate, add the 0.66g gamma-butyrolactone, stir 20h.Stopped reaction filters reactant normal hexane, ether washing leaching cake.In filter cake, add the 10mL dissolve with methanol, add 4mL hydrogenchloride/methanol solution, stir 1h.With the sodium hydroxide neutralization, remove by filter insolubles, will filtrate concentrates.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=5: 1), obtain colourless liquid 0.78g, productive rate 68%.trans-ZY1：
1HNMR(400MHz，CDCl
3)δ5.09(d，J＝7.1Hz，1H)，4.07～3.87(m，1H)，3.95～3.93(m，1H)，3.80(s，3H)，3.31(m，3H)，3.22～3.01(m，1H)，2.23～2.10(m，2H)。cis-ZY1：
1HNMR(400MHz，CDCl
3，distinctivepeaks)δ5.17(s，1H)，2.52～2.38(m，1H)。
(2) ZY2 is synthetic: reduction reaction takes place and obtains ZY2 in ZY1.
Said step is specially: get 0.4gZY1, add the 5mL ether, add the 1.5mL Lithium Aluminium Hydride.Reflux 2h.Add ETHYLE ACETATE cancellation reaction, add entry, repeatedly extract, use anhydrous magnesium sulfate drying with ETHYLE ACETATE.Decompression steams solvent and obtains colourless liquid 0.3g, productive rate 94%.trans-ZY2：
1HNMR(400MHz，CDCl
3)δ4.96(d，J＝6.8Hz，1H)，4.00～3.82(m，2H)，3.74～3.56(m，2H)，3.30(s，3H)，2.38～2.28(m，1H)，1.93(s，1H)，1.90～1.87(m，2H)。cis-ZY2：
1HNMR(400MHz，CDCl
3，distinctive?peaks)δ4.80(s，1H)，3.60～3.50(m，2H)，2.13～2.04(m，1H)，1.60～1.52(m，1H)。
(3) ZY3 is synthetic: ZY2 and bromotoluene reaction obtain ZY3.
Said step is specially: get 0.6g sodium hydride (in 60% kerosene), add 80mLDMF, add 1.8gZY2, add the 3.4mL cylite, stirring reaction 2h.Add 300mL water, use dichloromethane extraction, combined dichloromethane, anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=10: 1), obtain weak yellow liquid 2.7g, productive rate 78%.trans-ZY3：
1HNMR(400MHz，CDCl
3)δ7.38～7.26(m，5H)，4.99(d，J＝6.5Hz，1H)，4.63(s，2H)，4.42～3.83(m，2H)，3.80～3.64(m，1H)，3.52～3.47(m，1H)，3.33(s，3H)，2.53～2.40(m，1H)，2.16～1.98(m，1H)，1.60～1.50(m，1H)。cis-ZY3：
1HNMR(400MHz，CDCl
3，distinctive?peaks)δ4.85(s，1H)，3.38～3.34(m，2H)。
(4) ZY4 is synthetic: ZY3 goes hydroxyl protecting group to obtain ZY4 under acidic conditions.
Said step is specially: get 0.5g ZY3, add 10mL1, the 4-dioxane adds 20mL Hydrogen chloride (concentration is 0.1M).Reacted two days.Use the dichloromethane extraction reaction solution, anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=5: 1), obtain weak yellow liquid 0.31g, productive rate 77%.trans-ZY4：
1H-NMR(400MHz，CDCl
3)δ7.37～7.26(m，5H)，5.84(m，1H)，4.55(s，2H)，4.10(m，1H)，4.03(m，1H)，3.71(m，2H)，3.46(s，1H)，2.43～2.38(m，1H)，2.06～1.96(m，1H)，1.94～1.83(m，1H)。cis-ZY4：
1H-NMR(400MHz，CDCl
3，distinctivepeaks)δ5.37(s，1H)，4.53(s，2H)，4.03(m，1H)，3.92(m，1H)，3.42～3.34(m，2H)，3.10(s，1H)，2.52～2.44(m，1H)，1.66～1.58(m，1H)。
(5) ZY5 is synthetic: ZY4 and bromoethanol reaction obtain ZY5.
Said step is specially: get 104mg ZY4, add the 5mL methylene dichloride, add the 65mg2-bromoethanol, add 20mgAmberlystA-15, reaction 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing, and anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=10: 1), obtain weak yellow liquid 155mg, productive rate 80%.trans-ZY5：
1H-NMR(400MHz，CDCl
3)δ7.77～7.27(m，5H)，5.07(d，J＝0.4Hz，1H)，4.54(s，2H)，4.81(m，1H)，3.94～3.90(m，2H)，3.83～3.68(m，2H)，3.64(d，J＝2.8Hz，1H)，3.49～3.42(m，2H)，2.48～2.40(m，1H)，2.07～1.98(m，1H)，1.80～1.70(m，1H)。
13CNMR(100MHz，CDCl
3)δ138.20，128.37，127.68，127.58，103.26，73.31，70.88，67.26，66.82，44.73，30.89，26.70。cis-ZY5：
1H-NMR(400MHz，CDCl
3，distinctivepeaks)δ5.03(s，1H)，4.53(s，2H)，3.97～3.85(m，1H)，3.80～3.74(m，1H)，3.38(s，1H)，2.58～2.49(m，1H)，1.61～1.52(m，1H).
13C-NMR(100MHz，CDCl
3)δ138.55，128.43，127.66，127.58，106.42，73.06，69.17，67.45，66.59，45.74，31.06，26.85。IR：3454，3025，2862，1628，1452，1374，1085，1027，917，740，698。HRMS(ESI)m/z?calcdfor?C
14H
19O
3NaBr(M+Na)
+337.0421，found?337.0420。
(6) ZY6 is synthetic: ZY5 is under the effect of palladium-carbon catalyst, and debenzylation obtains ZY6.
Said step is specially: get 80mg ZY5, add 5mL methyl alcohol, add 10mg palladium carbon, vacuumize, logical hydrogen, reaction 2h.Stopped reaction removes by filter palladium carbon, and decompression steams solvent, obtains colourless liquid 54mg, productive rate 95%.trans-ZY5：
1HNMR(400MHz，CDCl
3)δ5.13(d，J＝4.8Hz，1H)，4.06～4.00(m，1H)，3.95～3.90(m，2H)，3.72～3.69(m，1H)，3.49～3.45(m，2H)，3.36(s，1H)，3.34(s，1H)，2.44～2.33(m，1H)，2.03～1.95(m，1H)，1.94～1.87(m，1H)。
13CNMR(100MHz，CDCl
3)δ104.01，69.24，67.14，64.72，39.17，35.80，31.04.cis-ZY5：
1HNMR(400MHz，CDCl
3，distinctivepeaks)δ5.02(s，1H)，3.98～3.84(m，1H)，3.80～3.73(m，1H)，3.61～3.57(m，1H)，3.54～3.50(m，1H)，2.16～2.07(m，1H)。
13CNMR(100MHz，CDCl
3)δ104.30，68.54，67.22，64.99，39.58，35.51，30.91。IR：3385，2951，2835，1598，1485，1389，1095，1019，917.HRMS(ESI)m/z?calcdfor?C
7H
13O
3NaBr(M+Na)
+246.9969，found?246.9975。
(7) ZY7 is synthetic: aminating reaction takes place and obtains ZY7 in ZY6 in ammoniacal liquor.
Said step is specially: get 100mg ZY6, add 10mL ammoniacal liquor, reaction 2h.Stopped reaction, decompression steams solvent, obtains colourless liquid 70mg, productive rate 98%.trans-ZY7：
1HNMR(400MHz，D
2O)δ5.12(d，J＝4.8Hz，1H)，4.05～4.01(m，1H)，4.00～3.90(m，2H)，3.76～3.68(m，1H)，3.33～3.31(m，2H)，3.23～3.16(m，2H)，2.43～2.36(m，1H)，2.05～2.01(m，1H)，1.75～1.65(m，1H)。
13CNMR(100MHz，CD
3OD)δ104.44，69.65，63.32，63.12，39.18，38.56，34.79。cis-ZY7：
1HNMR(400MHz，CDCl
3，distinctivepeaks)δ5.03(s，1H)，3.89～3.84(m，1H)，3.56～3.45(m，2H)，2.15～2.07(m，1H)。
13CNMR(100MHz，CD
3OD)δ104.71，69.33，63.40，63.25，39.37，38.39，34.54.IR：3438，2932，2886，1722，1453，1364，1282，1220，1102，1031，923。HRMS(ESI)m/z?calcdfor?C
7H
16NO3(M+H)
+162.1115，found162.1120。
YZ linker series 4-methylol-2-(2-amino ethoxy)-THF, promptly the synthetic synoptic diagram of YZ8 is as shown in Figure 6, and synthesis step is following:
(1) YZ1 is synthetic: ethyl malonate and 2-bromo-1, the reaction of 1-glycol dimethyl ether obtains YZ1.
Said step is specially: get 2g sodium hydride (in 60% kerosene), add 50mLDMF, vigorous stirring.Add the 9g ethyl malonate, add 9.39g 2-bromo-1, the 1-glycol dimethyl ether, 100 ℃ are stirred 12h.Stopped reaction adds 50mL water, uses the 100mL dichloromethane extraction.Organic layer is with the water washing of 100mL saturated common salt, and anhydrous sodium sulfate drying reduces pressure and steams solvent, obtains 12.8g pale yellow oily liquid body.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=5: 1), obtain colourless liquid 5.0g, productive rate 40%.
1HNMR(400MHz，CDCl3)δ4.42(t，J＝7.6Hz，1H)，4.20(q，J＝4.2Hz，4H)，3.48(t，＝5Hz，1H)，3.33(s，6H)，2.23～2.19(m，2H)，1.27(t，J＝7.2Hz，6H)。
(2) YZ2 is synthetic: reduction reaction takes place and obtains YZ2 in YZ1.
Said step is specially: get 2.2gYZ1, add the 10mL ether, add 40mL Lithium Aluminium Hydride (1.0M THF).Stirring at room 2h.Add ETHYLE ACETATE cancellation reaction, add entry, repeatedly extract, use anhydrous magnesium sulfate drying with ETHYLE ACETATE.Decompression steams solvent and obtains weak yellow liquid 1.42g, productive rate 70%.
1HNMR(400MHz，CDCl
3)δ4.81(t，1H)，3.97～3.65(m，4H)，3.56(s，6H)，2.86(s，2H)，1.93～1.86(m，1H)，1.71～1.68(m，2H)。
(3) YZ3 is synthetic: cyclization takes place and obtains YZ3 in YZ2 in camphorsulfonic acid pyridinium salt dichloromethane solution.
Said step is specially: get 272mgYZ2, add the dissolving of 3mL methylene dichloride, add 0.02M camphorsulfonic acid pyridinium salt dichloromethane solution.Stir 18h.Stopped reaction, decompression steams solvent.With 200-300 order silica gel column chromatography (methylene dichloride: methyl alcohol=20: 1), obtain weak yellow liquid 168mg, productive rate 85%.
1HNMR(400MHz，CDCl
3)δ5.39～5.07(m，0.23H)，5.42～4.98(m，0.77H)，4.05～3.92(m，1H)，3.71～3.49(m，3H)，3.33～3.30(m，3H)，2.62～2.54(m，0.77H)，2.52～2.46(m，0.23H)，2.21～2.13(m，1H)，2.00～1.95(m，0.77H)，1.75～1.70(m，0.77H)。
(4) YZ4 is synthetic: YZ3 obtains YZ4 with benzyl protection.
Said step is specially: get 0.47g sodium hydride (in 60% kerosene), add 80mLDMF, add 1.8gYZ3, add the 3.4mL cylite, stirring reaction 2h.Add 300mL water, with dichloromethane extraction twice, combined dichloromethane, anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=10: 1), obtain weak yellow liquid 2.3g, productive rate 76%.
1HNMR(400MHz，CDCl
3)δ7.77～7.30(m，5H)，5.02～5.00(m，1H)，4.50(s，2H)，4.37～3.96(m，1H)，3.75～3.63(m，1H)，3.56～3.45(m，1H)，3.42～3.35(m，1H)，3.32(m，3H)，2.74～2.67(m，0.5H)，2.66～2.25(m，0.5H)，2.24～2.13(m，0.5H)，2.02～1.96(m，0.5H)，1.74～1.68(m，0.5H)，1.66～1.60(m，0.5H).
13CNMR(100MHz，CDCl
3)δMajor：138.28，128.48，127.68，127.76，105.26，73.20，72.50，69.40，54.58，38.12，35.83.Minor：138.23，128.49，127.69，127.65，105.26，73.10，72.96，70.17，54.74，35.84.IR：3420，3030，2945，2862，1720，1605，1450，1364，1259，1203，1092，1020cm
-1.HRMS(ESI)calcd?for?C
13H
18O
3Na(M+Na)
+245.1148，found?245.1144。
(5) YZ5 is synthetic: YZ4 obtains YZ5 at the acidic conditions hydroxyl protecting group that goes down.
Said step is specially: get 0.5gYZ4, add 10mL1, the 4-dioxane adds 10mL Hydrogen chloride (concentration is 0.01M).Reacted two days.Use the dichloromethane extraction reaction solution, anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=5: 1), obtain weak yellow liquid 0.38g, productive rate 81%.
1HNMR(400MHz，CDCl
3)δ7.76～7.28(m，5H)，5.44～5.33(m，0.42H)，5.43～5.40(m，0.58H)，4.57(s，1.16H)，4.51(s，0.84H)，4.16～4.12(m，0.42H)，4.07～4.02(m，0.58H)，3.88～3.84(m，0.58H)，3.74～3.70(m，0.42H)，3.76～3.49(m，1.16H)，3.44～3.34(m，0.84H)，2.81～2.74(m，0.42H)，2.59～2.54(m，0.58H)，2.26～2.19(m，0.58H)，2.04～1.98(m，0.42H)，1.77～1.73(m，1H).
13CNMR(100MHz，CDCl
3)δMajor：137.31，128.60，127.94，127.66，104.24，98.39，73.58，71.61，69.44，37.38，37.05.Minor：138.18，128.45，128.05，127.70，104.23，98.54，73.12，72.23，69.86，37.30，37.61.IR：3456，3029，2948，1953，1739，1600，1449，1364，1260，1207，1097，1026cm
-1.HRMS(ESI)calcd?for?C
12H
16O
3Na(M+Na)
+231.0965，found?231.0966。
(6) YZ6 is synthetic: YZ5 and bromoethanol reaction obtain YZ6.
Said step is specially: get 104mgYZ5, add the 5mL methylene dichloride, add the 125mg2-bromoethanol, add 20mgAmberlystA-15, reaction 2h.Stopped reaction removes by filter A-15, uses saturated sodium bicarbonate, the saturated common salt water washing, and anhydrous magnesium sulfate drying, decompression steams solvent.With 200-300 order silica gel column chromatography (sherwood oil: ETHYLE ACETATE=10: 1), obtain weak yellow liquid 133mg, productive rate 85%.
1HNMR(400MHz，CDCl
3)δ7.37～7.28(m，5H)，5.16～5.15(m，1H)，4.51(s，2H)，4.07～4.00(m，1H)，3.96～3.88(m，1H)，3.77～3.69(m，2H)，3.59～3.32(m，4H)，2.77～2.68(m，0.58H)，2.61～2.53(m，0.42H)，2.19～2.12(m，0.42H)，2.08～2.02(m，0.58H)，1.75～1.70(m，1H).
13CNMR(100MHz，CDCl
3)δMajor：138.19，128.45，127.74，127.66，73.12，72.38，69.69，67.23，37.23，35.85.31.04.Minor：138.29，128.42，127.70，127.65，73.20，72.94，70.52，67.24，37.82，35.78，31.13.IR：3391，2943，1614，1452，1398，1125，1077，1022cm
-1.HRMS(ESI)calcd?for?C
14H
19O
3NaBr(M+Na)
+337.0405，found?337.0391。
(7) YZ7 is synthetic: YZ6 removes benzyl protecting group and obtains YZ7 under the palladium-carbon catalyst effect.
Said step is specially: get 80mgYZ6, add 5mL methyl alcohol, add 10mg10% palladium carbon, vacuumize, logical hydrogen, reaction 2h.Stopped reaction removes by filter palladium carbon, and decompression steams solvent, obtains colourless liquid 50mg, productive rate 90%.
1HNMR(400MHz，CDCl
3)δ5.18～5.16(m，0.4H)，5.09～5.07(m，0.6H)，4.05～3.93(m，0.4H)，3.90(t，J＝5.6Hz，2H)，3.81～3.63(m，2H)，3.53(m，J＝5.6Hz，2H)，3.50～3.41(m，1H)，3.33～3.21(m，0.6H)，2.64～2.56(m，0.6H)，2.53～2.48(m，0.4H)，2.19～1.92(m，2H)，1.77～1.61(m，1H).
13CNMR(100MHz，CDCl
3)δMajor：104.85，67.43，67.04，60.82，47.98，31.49，25.05.Minor：106.36，67.27，66.68，63.68，45.53，30.97，26.64.IR：3435，2961，1738，1639，1386，1261，1092，1022cm
-1.HRMS(ESI)calcd?for?C
7H
13O
3NaBr(M+Na)
+246.9940，found?246.9945。
(8) YZ8 is synthetic: aminating reaction takes place and obtains YZ8 in YZ7 in ammoniacal liquor.
Said step is specially: get 44mgYZ7, add 10mL ammoniacal liquor, reaction 2d.Stopped reaction, decompression steams solvent, obtains colourless liquid 29mg, productive rate 93%.
1HNMR(400MHz，D
2O)δ5.23～5.22(m，1H)，4.03～4.00(m，1H)，3.84～3.77(m，1H)，3.68～3.48(m，4H)，3.34～2.97(m，2H)，2.67～2.59(m，0.6H)，2.50～2.46(m，0.4H)，2.28～2.25(m，0.4H)，2.06～1.95(m，0.6H)，1.79～1.65(m，1H).
13CNMR(100MHz，D
2O)δMajor：103.43，67.33，62.11，60.16，45.64，39.37，26.22.Minor：106.49，67.11，63.42，62.26，47.26，39.32，25.57.IR：3443，2965，2860，1618，1451，1381，1269，1201，1098，1027cm
-1.HRMS(ESI)calcd?for?C
7H
16NO3(M+H)
+162.1113，found?162.1109。
The connector element of embodiment 1～6 all can couple together with resorcinolphthalein, Nucleotide effectively, forms the reversible terminal that can be used for checking order; The connector element that adopts in the present embodiment is the T-8-1 among the embodiment 1.
(1) compound 3 is synthetic: trifluoro-acetate and propargylamine react in organic solvent and obtain compound 3, and as shown in Figure 7, said step is specially:
Adding 15ml methyl alcohol in a single port bottle, ice-water bath is stirring down, and (30mmol, 1.65g), (39mmol, 4.99g), the water-bath of 10 minutes recession deicings was reacted 24 hours under the room temperature to stir slow adding trifluoro-acetate 1 after 20 minutes to add propargylamine 2.Reaction is monitored with the TLC plate.After reaction finished, the evaporated under reduced pressure solvent added the 30ml chloroform, saturated NaHCO
3Twice of solution washing (2 * 30ml), saturated NaCl solution washing (30ml), anhydrous sodium sulfate drying filters, the evaporated under reduced pressure solvent, underpressure distillation gets product 33.02g, productive rate 66.6%.
1H?NMR(300MHz，CDCl
3)：2.32(t，1H，J＝2.7Hz)，4.14(2H，dd，J
1＝2.7，J
2＝5.4Hz)，6.92(s，1H)。
(2) compound 6 is synthetic: linked reaction takes place with 5-iodo-2 '-deoxyuridine 4 and obtains compound 6 in compound 3 under the cuprous iodide effect, as shown in Figure 8, said step is specially:
Adding 5-iodo-2 '-deoxyuridine 4 in a single port bottle (0.7mmol, 247.9mg), 8mL DMF, stirring and dissolving, lucifuge.(0.14mmol, 26.7mg), nitrogen protection is stirred and cuprous iodide was fully dissolved in 20 minutes to add cuprous iodide.Add successively 0.25mL TEA, trifluoroacetyl propargylamine 3 (2.8mmol, 423.0mg), Pd (PPh
3)
4(0.07mmol, 80.9mg), reaction is spent the night under the room temperature.Reaction is monitored with the TLC plate.The evaporated under reduced pressure solvent, column chromatography, DCM: MeOH=20: 1 is eluent, gets product 6158mg, productive rate 60%.
1H?NMR(300MHz；DMSO-d6)：2.11(2H，t，J＝5.4Hz)，3.56～3.58(2H，m)，3.79(1H，m)，4.21(3H，d，J＝5.4Hz)，5.08(1H，t，J＝4.3Hz)，5.23(1H，d，J＝3.7Hz)，6.09(1H，t，J＝6.4Hz)，8.18(1H，s)，10.05(1H，t，J＝5.3Hz)，11.63(1H，s)。
(3) compound 10 is synthetic: compound 6 and tri-n-butylamine pyrophosphate salt 8,2-chloro-4H-1,3; 2-benzo dioxy phosphorus-4-ketone 7 is in the DMF solvent, and reaction obtained compound 9 under triethylamine and iodine existed, and went protection then; Obtain compound 10, as shown in Figure 9, said step is specially:
In glove box, take by weighing compound 6 (one) 60mg (0.16mmol), tri-n-butylamine pyrophosphate salt 8 (two) 150mg (0.32mmol), 2-chloro-4H-1,3,2-benzo dioxy phosphorus-4-ketone 7 (three) 66mg (0.32mmol) places three reaction tubess respectively.(1) is dissolved in the 0.5ml dry DMF, adds the new tri-n-butylamine that steams of 0.6ml again, stir half a hour.Be dissolved in (two) in the 0.5ml dry DMF, under high degree of agitation, be injected into (one) in (two), stir half a hour.Inject (three) to above-mentioned mixed solution, stir 1.5h.Add 5ml 3% iodine (py/H
2O, 9/1), the back adds 4ml water among the 15min, stirs 2h.Add 0.9ml3M NaCl solution, add the 30ml absolute ethyl alcohol again, subzero 20 ℃ of freeze overnight, centrifugal (3200r/min, 25 ℃) 20min.The supernatant that inclines is drained solvent.The TEAB solution that adds 1ml 1M again adds the 4ml strong aqua, stirred overnight at room temperature.White solid appears in the evaporated under reduced pressure solvent.Earlier analyze condition with analysis mode HPLC: pillar: C18,5 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 35min 20% ethanol; UV-detector: 260nm.Preparation HPLC separates, and gets the 26mg white solid, productive rate 18%.Pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 6mL/min; Moving phase: 20mM TEAAc and ethanol, 0min 20mM TEAAc, 40min 5% ethanol; UV-detector: 260nm.
1H?NMR(400MHz，D
2O)：1.27(Et
3N-CH
3，t，J＝8.0Hz)，2.33～2.48(2H，m)，3.18(Et
3N-CH
2，q，J＝8.0Hz)，4.03(2H，s)，4.20～4.26(3H，m)，4.58～4.64(1H，m)，6.27(1H，t，J＝8.0Hz)，8.38(s，1H)。
31P?NMR(162MHz，D
2O)：-22.22(1P)，-11.45(1P)，-9.90(1P)。
ESI-HRMS：calc?for[C
12?H
18?N
3?O
14?P
3+H]522.0080，found?522.0070；calc?for[C
12?H
18?N
3?O
14?P
3+Na]543.9899，found?543.9883。
(4) compound 11 is synthetic: T-8 and resorcinolphthalein generation substitution reaction, get compound 11, and shown in figure 10, said concrete steps are:
In the single port bottle of 10mL, add the 3mL dry DMF, add 15mg (42 μ mol) T-8-1 again, stir under the lucifuge, room temperature, with 5 (6)-TAMRA of 10mg (19 μ mol), SE (II) is dissolved in the 2mL dry DMF, adds the triethylamine of 40 μ L (285 μ mol) again.Stirring reaction under the room temperature, TLC track to raw material and disappear.Stopped reaction is removed DMF under the decompression, uses DCM/, and MeOH=3/1 makees developping agent, and the big plate separation and purification of TLC gets pure compound 118 mg, productive rate 73.4%.Trans-isomer(ide) T-9-1,
1H-NMR (CD
3OD, 300M): δ 8.06～8.16 (m, 2H), 7.70 (s, 1H), 7.26 (d, 2H, J=9.6Hz), 7.00 (dd, 2H, J
1=2.1, J
2=9.6Hz), 6.92 (d, 2H, J=2.1Hz), 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。
Cis-isomeride T-9-2, productive rate 96%.
1H-NMR(CD
3OD，400M)：δ8.13(d，1H，J＝8.0Hz)，8.08(dd，1H，J
1＝1.6，J
2＝8.0Hz)，7.73(d，1H，J＝1.2Hz)，7.25(dd，2H，J
1＝1.6，J
2＝9.6Hz)，6.99(dd，2H，J
1＝2.0，J
2＝9.2Hz)，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。
(5) compound 12 is synthetic: compound 11 and DSC under alkaline condition, react compound 12, shown in figure 11, said step is specially:
In a reaction flask, add compound 1110mg (0.017mmol), vacuumize, nitrogen protection is injected the 0.5ml anhydrous acetonitrile with syringe, adds 20 μ l triethylamines, stirs under the room temperature.In another reaction flask, add N, N-succinimidyl carbonate (DSC) 27mg (0.105mmol) vacuumizes, and stirring reaction under the room temperature is injected above-mentioned mixed solution in nitrogen protection.TLC follows the tracks of to react to raw material and disappears.After raw material disappeared, stopped reaction directly was used for next step.
(6) compound 13 is synthetic: substitution reaction takes place with compound 10 and gets compound 13 in compound 12 under alkaline condition, shown in figure 12, said step is specially:
(10mg 0.07mmol) is dissolved in 0.5ml NaHCO compound 10
3/ Na
2CO
3In the buffered soln of (pH is 8.73), add the reaction solution of compound 12 (starting material compound 117mg) in the buffered soln of compound 10 stirring reaction under the room temperature.At first separate unreacted compound 12 after reacting end, separate with preparation HPLC then, get compound 13 pure article 4.5mg, productive rate 33.3% with the big plate of TLC.TR is the 28.5min. condition: pillar: C18,5 μ m, 9.4 * 250mm; Flow velocity: 4mL/min; Moving phase: 20mM TEAAc and methyl alcohol, 0min 0% methyl alcohol, 10min 0% methyl alcohol, 30min 50% methyl alcohol, 50min 50% methyl alcohol; UV-detector: 546nm.Analysis tR is 32.8min.Condition: pillar: C18,10 μ m, 4.6 * 250mm; Flow velocity: 1mL/min; Moving phase: 20mM TEAAc and methyl alcohol, 0min 0% methyl alcohol, 10min 0% methyl alcohol, 30min 50% methyl alcohol, 50min 50% methyl alcohol; UV-detector: 260nm, 546nm.
1H?NMR(400MHz，D
2O)：δ8.13(d，1H，J＝4.0Hz)，8.00(d，1H，J＝8.0Hz)，7.95(s，1H)，7.87(s，1H)，7.18～7.26(m，2H)，6.93～7.01(m，2H)，6.81(s，1H)，6.76(s，1H)，6.14(t，1H，J＝4.0Hz)，5.25(s，1H)，4.45～4.56(m，2H)，4.29～4.38(m，2H)，4.13～4.27(m，4H)，4.64～4.80(m，5H)，3.30(s，12H)，3.22(Et
3N-CH
2，q)，2.26～2.90(m，1H)，2.09～2.11(m，3H)，1.67～1.72(m，2H)，1.31(Et
3N-CH
3，t)。
31P?NMR(162MHz，D
2O)：-20.63(1P)，-10.92(1P)，-8.32(1P)。
ESI-HRMS：calc?for[C
45H
51N
6O
22P
3-H]1119.2191，found?1119.2216；calc?for[C
45H
51N
6O
22P
3-PO
3H
2]
-1039.2528，found?1039.2551；calc?for[C
45H
51N
6O
22P
3+Na-2H]1141.2010，found?1141.1981。
It is following that cracking performance is investigated testing sequence:
One, get 1mL pH=2.5 SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution respectively, 30 ℃ of constant temperature 15min add 4mg 5-methylol-2-(2-bromine oxethyl)-THF respectively; Be T-7,3-methylol-2-(2-bromine oxethyl)-THF; Be ZY6,4-methylol-2-(2-bromine oxethyl)-THF, i.e. YZ7, sampling spot plate immediately; On same block of TLC plate, detect, developping agent is used methylene dichloride: methyl alcohol=20: 1.Every afterwards 5min point plate once to follow the tracks of reaction, is confirmed its cleavage product through HRMS.Rate of cleavage is relatively seen table 1:
The transformation efficiency of table 1 scission reaction (%)
| 0’ | 5’ | 11’ | 16’ | 21’ | 26’ | 29’ | 34’ | 39’ | 60’ | |||
| 1 | T7 | 3.7 |
0 | 50 | 75 | 90 | 95 | 100 | ||||
| 2 | ZY6 | 3.9 |
0 | 5 | 11 | 18 | 23 | 28 | 31 | 34 | 37 | 40 |
| 3 | YZ7 | 3.9 |
0 | 5 | 15 | 30 | 42 | 52 | 60 | 65 | 68 | 70 |
Can know by table 1: pH=2.5, T=30 ℃ of SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution, T7 ruptures at 26min fully, and ZY6, YZ7 are in 60min cracking 40% and 70% respectively.
Two, get 1mL pH=2.38 SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution respectively, 30 ℃ of constant temperature 15min add 4mg T-7, ZY6, YZ7 respectively, and the sampling spot plate detects on same block of TLC plate immediately, and developping agent is used methylene dichloride: methyl alcohol=20: 1.Every afterwards 5min point plate once to follow the tracks of reaction, is confirmed its cleavage product through HRMS.Rate of cleavage is relatively seen table 2:
The transformation efficiency of table 2 scission reaction (%)
Can know by table 2: pH=2.38, T=30 ℃ of SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution, T7 ruptures at 15min fully, and ZY6, YZ7 are in 50min cracking 50% and 95% respectively.
Under above-mentioned condition of different pH,, show that rate of cleavage is T7＞YZ7＞ZY6 to the investigation of three linker cracking performances.
The scission reaction synoptic diagram is shown in figure 13, confirms that through HRMS the cleavage product of T7, ZY6, three linker of YZ7 is respectively T, ZY, YZ, and the cleavage product that obtains has been verified the fracture position of three linker.
One, the split product of T-7 compound is investigated test
Said step is specially: method 1: in pH is 3.73 acetate and sodium acetate buffer solution; Temperature is under 30 ℃ the condition, and 8mg T-7-2 is added in this damping fluid, begins to observe tangible split product during 3min; During 40min on the plate raw material very light; The 50min raw material disappears, and changes split product fully into, and split product is clear.
Said step is specially: method 2: in pH is 2.73 acetic acid soln; Temperature is under 45 ℃ the condition, and 6mg T-7-2 is added in the solution, can be observed tangible split product during 1min; Feedstock conversion 90% on TLC plate during 7min; The 10min raw material disappears, and changes split product fully into, and split product is confirmed through HRMS.Calc.Mass C
5H
10O
3Na for 141.0528, actual measurement 141.0522.
Complete cracking takes place in T-7 under solutions of weak acidity, product point is very clear, does not have by product to generate.
Two, the split product of ZY6 compound is investigated test
Said step is specially: get 1mL pH=2.5 SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution respectively; 30 ℃ of constant temperature 15min add ZY6, sampling spot plate immediately; Developping agent is used methylene dichloride: methyl alcohol=20: 1, ammonium phosphomolybdate are baked plate detection reaction (with the amount of this plate as initial feed).Every afterwards 5min point plate detection reaction, detection reaction 1h, record experimental result.Confirm split product through HRMS.HRMS Calc.Mass C
5H
10O
3Na for 141.0528, actual measurement 141.0524.
Complete cracking takes place in ZY6 under solutions of weak acidity, product point is very clear, does not have by product to generate.
Three, the split product of YZ7 compound is investigated test
Said step is specially: get 1mL pH=2.5 SODIUM PHOSPHATE, MONOBASIC/citrate buffer solution respectively, 30 ℃ of constant temperature 15min add YZ7, sampling spot plate immediately, and developping agent is used methylene dichloride: methyl alcohol=20: 1, ammonium phosphomolybdate is baked the plate detection reaction.Every afterwards 5min point plate detection reaction, detection reaction 1h, record experimental result.Confirm split product through HRMS.HRMS Calc.Mass C
5H
10O
3Na for 141.0528, actual measurement 141.0529.
Complete cracking takes place in YZ7 under solutions of weak acidity, product point is very clear, does not have by product to generate.
Claims (10)
1. a THF ether derivant cleavable connector element is characterized in that, its structural formula is suc as formula shown in (I):
2. THF ether derivant cleavable connector element according to claim 1 is characterized in that, the unitary structural formula of said cleavable is suc as formula shown in (II):
3. THF ether derivant cleavable connector element according to claim 2 is characterized in that, n is any integer in 21～44 in the said formula (I).
4. THF ether derivant cleavable connector element according to claim 2 is characterized in that, said cleavable connector element is through the following steps synthetic:
A, T-1's is synthetic: get D, cyclization under hydrochloric acid and Sodium Nitrite effect, takes place and obtains T-1 in L or DL-L-glutamic acid;
B, T-2's is synthetic: reduction reaction takes place and gets T-2 in the ether solvent of borine in T-1;
C, T-3's is synthetic: get T-2, make solvent with methylene dichloride, add imidazoles, protection of inert gas is descended and dimethyl-tertiary butyl chloride silane reaction gets T-3;
D, T-4's is synthetic: make solvent with methylene dichloride, T-3 reacts with diisobutyl aluminium hydride under protection of inert gas and cryosel bath, gets T-4;
E, when n=0, T-6's is synthetic: get T-4, make solvent, add bromoethanol and ion exchange resin A-15 with methylene dichloride, at 40～50 ℃ of backflow 1～3h, react T-6;
When n=1～44, T-6's ' is synthetic: get T-4, make solvent, add bromo three polyoxyethylene glycol and Amberlyst A-15 with methylene dichloride, at 40～50 ℃ of backflow 1～3h, react T-6 ';
F, T-7 or T-7's ' is synthetic: make solvent with THF, and under the tetrabutyl ammonium fluoride effect, T-6 or the protection of T-6 ' dehydroxylation, reaction obtains T-7 or T-7 ';
G, T-8 or T-8's ' is synthetic: substitution reaction takes place in T-7 or T-7 ' in ammoniacal liquor, gets T-8 or T-8 ', and T-8 or T-8 ' are said THF ether derivant cleavable connector element.
5. THF ether derivant cleavable connector element according to claim 1 is characterized in that, the unitary structural formula of said cleavable is suc as formula shown in (III):
6. THF ether derivant cleavable connector element according to claim 5 is characterized in that, said cleavable unit is through the following steps synthetic:
A, YZ1's is synthetic: ethyl malonate and 2-bromo-1, and the reaction of 1-glycol dimethyl ether obtains YZ1;
B, YZ2's is synthetic: reduction reaction takes place and obtains YZ2 under ether, Lithium Aluminium Hydride effect in YZ1;
C, YZ3's is synthetic: cyclization takes place and obtains YZ3 in YZ2 in camphorsulfonic acid pyridinium salt dichloromethane solution;
D, YZ4's is synthetic: YZ3 and bromotoluene reaction obtain YZ4;
E, YZ5's is synthetic: with 1, the reaction of 4-dioxane goes hydroxyl protecting group to obtain YZ5 to YZ4 under acidic conditions;
F, YZ6's is synthetic: YZ5 and bromoethanol reaction obtain YZ6;
G, YZ7's is synthetic: YZ6 is under the palladium-carbon catalyst effect, and debenzylation obtains YZ7;
H, YZ8's is synthetic: aminating reaction takes place and obtains YZ8 in YZ7 in ammoniacal liquor, YZ8 is described THF ether derivant cleavable connector element.
7. THF ether derivant cleavable connector element according to claim 1 is characterized in that, the unitary structural formula of said cleavable is suc as formula shown in (IV):
8. THF ether derivant cleavable connector element according to claim 7 is characterized in that, said cleavable connector element is through the following steps synthetic:
A, ZY1's is synthetic: gamma-butyrolactone and methyl-formiate in the presence of sodium hydride, react ZY1;
B, ZY2's is synthetic: reduction reaction takes place and obtains ZY2 under the effect of ether, methyl-formiate in ZY1;
C, ZY3's is synthetic: ZY2 and bromotoluene reaction obtain ZY3;
D, ZY4's is synthetic: with 1, the reaction of 4-dioxane goes hydroxyl protecting group to obtain ZY4 to ZY3 under acidic conditions;
E, ZY5's is synthetic: ZY4 and bromoethanol reaction obtain ZY5;
F, ZY6's is synthetic: ZY5 is under the effect of palladium-carbon catalyst, and debenzylation obtains ZY6;
G, ZY7's is synthetic: aminating reaction takes place and obtains ZY7 in ZY6 in ammoniacal liquor, ZY7 is described THF ether derivant cleavable connector element.
9. a THF ether derivant cleavable connector element as claimed in claim 1 synthesizes the purposes in the order-checking at DNA.
10. purposes as claimed in claim 9 is characterized in that, comprises the steps: said THF ether derivant cleavable connector element is connected Nucleotide and resorcinolphthalein, is used for the synthetic order-checking of DNA.
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