CN106083676A - Sulfur connects unit and synthetic method, purposes for ketal - Google Patents

Sulfur connects unit and synthetic method, purposes for ketal Download PDF

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CN106083676A
CN106083676A CN201610402708.9A CN201610402708A CN106083676A CN 106083676 A CN106083676 A CN 106083676A CN 201610402708 A CN201610402708 A CN 201610402708A CN 106083676 A CN106083676 A CN 106083676A
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ketal
sulfur
unit
compound
oxidation
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CN106083676B (en
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沈玉梅
王雪里
邵志峰
龚兵
谭连江
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Shanghai Jiaotong University
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    • C07C323/51—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and carboxyl groups bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
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    • C07C323/23—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton
    • C07C323/24—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton
    • C07C323/25—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atoms of the thio groups bound to acyclic carbon atoms of the carbon skeleton the carbon skeleton being acyclic and saturated
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Abstract

The open a kind of sulfur of the present invention connects unit and synthetic method, purposes for ketal；Described sulfur connects the structure of unit as shown in formula I for ketal:Wherein, R=OH or COOH；R1With R2The one met in following condition: (1) R1=R2=methyl；Or, (2) R1=phenyl, p-methoxyphenyl, 2,4,6 trimethoxyphenyls, naphthyl or to methoxyl group naphthyl, R2=H；Or, (3) R1、R2Collectively form cyclohexyl or cyclopenta.This unit is connected the fluorescein labeled nucleotide obtained and can be used for DNA sequencing with nucleotide and fluorescein.Compared with prior art, the present invention connects the reversible terminator of unit when DNA sequencing, and it extends efficiency close to 100%, and when template is continuous multiple identical base, the most only extends a reversible terminator；Its synthesis needed raw material is simple and easy to get, and building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.

Description

Sulfur connects unit and synthetic method, purposes for ketal
Technical field
The invention belongs to chemosynthesis and biochemical field, relate to the connection unit that can be used for DNA sequencing, be specifically related to A kind of sulfur connects unit and synthetic method, purposes for ketal.
Background technology
DNA sequencing technology is one of means important in modern biology research.After the Human Genome Project completes, DNA Sequencing technologies is developed rapidly.DNA sequencing (DNA sequencing) refers to analyze the base sequence of specific DNA fragments, The namely arrangement mode of adenine (A), thymus pyrimidine (T), cytosine (C) and guanine (G).Development accurately, high flux, low The DNA sequencing method of cost has very important significance for biological, medical science.
Synthetic method order-checking (Sequencing By Synthesis, SBS) is one of a new generation's DNA sequencing technology.Synthetic method Sequence measurement is by being fixed template DNA fragment the most to be measured, and hybridization combines in immobilized DNA sequencing template General DNA primer, controls the extension on DNA primer of 4 kinds of nucleotide respectively.By detection extension process or extension core Thuja acid, it is achieved the detection of the DNA sequence information of high-flux parallel.
In synthetic method checks order, first have to four kinds of nucleotide material of synthetic DNA chain elongation, be again " reversible terminator " (reversible terminator).This kind of nucleotide is in addition to requiring 3-hydroxyl and blocking, in order to not affect next labelling Being incorporated to and identifying of nucleotide, also requires by the connection unit of a cleavable nucleotide and indication molecule, such as fluorescence Element, couples together.Then, before next labeled nucleotide is incorporated to, make this connect unit fracture, so in a mild condition Rear continuation DNA extends, and completes once sequencing circulation, thus reads the base sequence of DNA.And connect unit and synthetic method is checked order Reading length and efficiency have an extremely important impact, therefore, people are devoted to develop new cleavable the most always and connect unit, to carry The efficiency of high DNA order-checking.The connection unit reported at present has reducing agent sensitive (disulfide bond, azo-compound)；Photodestruciton is (adjacent Nitrobenzyl derivatives, phenacyl ester derivant and other light cleavable connect unit)；(the acid of electrophilic reagent/acid-sensitive Cracking；Azido compound)；Cracking under metal function；Oxidant is sensitive.But sulfur does not the most always have for ketal connection unit For DNA sequencing.
Cleavable connects the character of unit has important impact to reading length and the efficiency of DNA sequencing, and existing connection is single Unit exists that cracking condition is gentle not, lysis efficiency is the highest, reads the long shortcoming such as the shortest when being used for checking order, therefore, design, synthesize new Cleavable connect unit, and explore suitable cracking condition for improving the efficiency of order-checking, developing new sequence measurement and have non- The most important meaning.Inventor in previous work, developed one class acid-sensitive connect unit (201110331659.1, 201210132695.X, 201310015235.3,201410186697.6,201410203327.9,201510401611.1) with And reduction sensitivity connection unit (201310462509.3,201310401580.0,201410692943.5, 201510031230.9,201510388164.0) application in DNA sequencing.
Reversible terminator based on acid-sensitive connection unit is when DNA sequencing, the most right DNA causes damage, remains in the molecule trace on DNA relatively big after the reversible terminator fracture of azo-based connection unit, Have impact on the extension efficiency of next reversible terminator to a certain extent；And based on sulfur for the connection unit of ketal structure necessarily Disadvantages mentioned above can be overcome, so being expected to bring new opportunity in DNA sequencing field in degree.
Summary of the invention
It is an object of the invention to provide a kind of sulfur and connect the synthesis of unit and the application in DNA sequencing thereof for ketal. The present invention designs the sulfur of synthesis and connects unit and reversible terminator for ketal, and such compou nd synthesis raw material is simple and easy to get, synthesis Process is conventional chemical reaction, it is easy to accomplish synthesize in a large number；This compounds can realize high efficiency with nucleotide and fluorescein Connection.By studying the cracking performance of this compounds, find that this compounds can realize efficiently under mild conditions The cracking of rate, has the value being applied to DNA sequencing.
Compared with previous work, the present invention be based on sulfur for ketal structure oxidation-sensitive cleavable connect unit, at oxygen Under agent existence condition, sulfur connects unit for ketal and acetal and all can quick and complete rupture.Such connects the fracture mechanism of unit The fracture being connected unit with condition with acid-sensitive is entirely different, so, exist for the connection unit of ketal and ethylidene ether structure based on sulfur DNA sequencing system is expected to overcome document reported that other connects the unit deficiency for DNA sequencing.Such as: acid-sensitive can Inverse terminator needs in acid condition, is cut away by the fluorescein of labelling, thus realizes next reversible terminator on DNA Extension；And the sulfur of the present invention for ketal structure connect unit have only under the conditions of weak oxidant, by the fluorescence of labelling Element cuts away, it is to avoid the use of acid condition disadvantageous to DNA, contributes to overcoming acid-sensitive to connect the unit deficiency for order-checking And weakness.
It is an object of the invention to be achieved through the following technical solutions:
First aspect, the present invention relates to a kind of oxidation-sensitive sulfur and connects unit for ketal, as shown in formula I:
Wherein, R=-OH or-COOH；
R1With R2The one met in following condition:
(1)R1=R2=methyl；
Or, (2) R1=phenyl, p-methoxyphenyl, 2,4,6-trimethoxyphenyls, naphthyl or to methoxyl group naphthyl, R2= H；
Or, (3) R1、R2Collectively form cyclohexyl or cyclopenta.
Second aspect, the present invention provides a kind of described oxidation-sensitive sulfur to connect the synthetic method of unit for ketal, including such as Under:
(1) as R=-COOH:
S11, under protective gas, solvent condition, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, 1, 4,6-TMB, naphthaldehyde or to methoxyl group naphthaldehyde, with 2-sulfydryl trifluoroacetamideAnd 3-sulfydryl third Acid methyl esterReaction, obtains sulfur for ketal compound
S12, described sulfur are converted into sulfur for ketal carboxylic acid compound for ketal compound in buffer solution
S13, remove the described sulfur amino protecting group for ketal carboxylic acid compound, obtain described oxidation-sensitive sulfur for ketal even Order unit
(2) as R=-OH:
S21, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, 1,4,6-TMB, naphthalene Aldehyde or to methoxyl group naphthaldehyde, with 2-sulfydryl trifluoroacetamideAnd the 2 mercapto ethanol reaction of hydroxyl protection group, Obtain sulfur for ketal compound
S22, described sulfur remove hydroxyl protecting group for ketal compound in alkaline solution, obtain compound
S23, remove compoundAmino protecting group, obtain described oxidation-sensitive sulfur generation Ketal connects unit
Preferably, preparing of described 2-sulfydryl trifluoroacetamide is as follows: under protective gas, solvent condition, trifluoroacetic acid Ethyl esterWith 2 mercapto ethanol hydrochlorateReact under triethylamine effect, obtain 2-sulfydryl trifluoroacetyl Amine.
It is further preferred that described protective gas is nitrogen, described solvent is methanol；Described Trifluoroacetic Acid Ethyl Ester and 2-mercapto Base ethylate hydrochlorate, the mol ratio of triethylamine are 1:(1～1.5): (1.5～2.5).More preferably 1:1.05:2；Described reaction It is after 0 DEG C of reaction 2.5～3.5h, at room temperature continues reaction 10～14h.
Preferably, the preparation of described 2-sulfydryl trifluoroacetamide specifically includes: under nitrogen protection, 2 mercapto ethanol hydrochloric acid Salt and triethylamine are dissolved in methanol to obtain mixed liquor, are cooled to 0 DEG C；The most under agitation dropping Trifluoroacetic Acid Ethyl Ester is in described mixed Close in liquid and react.
Preferably, the preparation of described 2-sulfydryl trifluoroacetamide also includes: after reaction terminates, and vacuum backspin removes solvent, residual Stay thing add acetic acid ethyl dissolution, with water and saturated aqueous common salt wash successively, organic phase with sodium sulfate is dried, column chromatography purification, Obtain described 2-sulfydryl trifluoroacetamide.
Preferably, in S11 step, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, Isosorbide-5-Nitrae, 6-front three Epoxide benzaldehyde, naphthaldehyde or to methoxyl group naphthaldehyde, with the mol ratio of 2-sulfydryl trifluoroacetamide and 3-mercapto-propionate be (0.8～1.2): 1:(1～1.5).More preferably 1.03:1:1.12.
Preferably, in S11 step, described protective gas includes the noble gas such as nitrogen, argon, solvent include acetonitrile, four Hydrogen furan isopolarity or non-polar organic solvent, the condition of described reaction is: 0 DEG C, 1～3h.
Preferably, described S11 step specifically includes: nitrogen protection under, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, to first Epoxide benzaldehyde, Isosorbide-5-Nitrae, 6-TMB, naphthaldehyde or to methoxyl group naphthaldehyde, with 2-sulfydryl trifluoroacetamide and 3-mercapto Base methyl propionate is dissolved in acetonitrile, adds boron trifluoride diethyl etherate after being cooled to 0 DEG C；Reaction 1～3h is continued at 0 DEG C.
Preferably, described S11 step also includes: after reaction terminates, and adds 15% sodium carbonate liquor, and ethyl acetate extracts (100mL × 3), organic facies is washed with 5% sodium carbonate liquor, is dried, revolves except solvent, isolated and purified with silicagel column, obtains described Sulfur is for ketal compound.
Preferably, in S12 step, described buffer includes PBS, triethylamine acetate buffer etc..
Preferably, in S12 step, described conversion is carried out under conditions of acetone and Pig Liver Esterase exist.
It is further preferred that described Pig Liver Esterase and sulfur are (0.1～0.3) for the mass ratio of ketal compound: 1, more excellent Elect 0.2:1 as；The volume ratio of described acetone and buffer is (0.05～0.15): 1, more preferably 0.1:1.
Preferably, in S12 step, every 1ml buffer adds described sulfur for the amount of ketal compound be 0.05～ 0.1mmol, more preferably 0.095mmol.
Preferably, S12 step specifically includes: sulfur is dissolved in acetone for ketal compound, is then dissolved in PBS again Add Pig Liver Esterase, obtain mixture；Mixed liquor is stirred at room temperature, as pH<when 6.0, dropping 2M NaOH solution is until pH> 7.0；After reaction 6h, add saturated aqueous common salt in the reactive mixture, be extracted with ethyl acetate, isolated from organic facies Unreacted sulfur is transferred to 2 for ketal compound, the pH of aqueous phase, is extracted with ethyl acetate, the colourless liquid of isolated from organic facies Body, obtains sulfur for ketal carboxylic acid compound.
The specific embodiment of R=-COOH is shown in embodiment 1-8.
Preferably, in S21 step, described protection group includes Pentamethylene oxide., oxolane, tert-butyldimethyl silyl etc..
Preferably, in S22 step, described alkaline solution includes tetrabutyl ammonium fluoride solution；Described acid solution includes dilute Hydrochloric acid or dilute sulfuric acid.
The specific embodiment of R=-OH is shown in embodiment 9.
The third aspect, the present invention provides a kind of described oxidation-sensitive sulfur to connect unit purposes in DNA sequencing for ketal, Refer specifically to: described oxidation-sensitive sulfur connects unit for ketal and is connected with nucleotide and fluorescein and to obtain reversible terminator, described can Inverse terminator can be used for DNA synthesis order-checking.
Fourth aspect, the present invention provides a kind of reversible terminator connecting unit based on described oxidation-sensitive sulfur for ketal, Connected unit by described oxidation-sensitive sulfur for ketal to be connected with nucleotide and fluorescein and obtain.
5th aspect, the present invention relates to a kind of reversible terminator, and described reversible terminator is by described oxidation-sensitive sulfur generation Ketal connects unit and is connected with nucleotide and fluorescein and obtains.
Preferably, described oxidation-sensitive sulfur includes walking as follows for the connection of ketal connection unit with nucleotide and fluorescein Rapid:
A1, with dry DMF as solvent, TEA exist under conditions of, described oxidation-sensitive sulfur for ketal connect unit with glimmering Light element reacts, and obtains compound G
A2, TEA exist under conditions of, compound G and DSC react, obtain intermediate H This intermediate H need not isolated and purified directly react with dNTP (AP3), obtains compound II The most described reversible terminator.
Preferably, in step A1, described fluorescein, oxidation-sensitive sulfur connect rubbing of unit and TEA (triethylamine) for ketal That ratio is 1:(1～3): (3～10)；Wherein, described fluorescein selected from BODIPY, rhodamine, coumarin, ton, cyanine, pyrene, Phthalocyanine, alexa, squarene dyestuff, the combination producing energy transfer dye and its derivant.
Preferably, in step A2, the mol ratio of described compound G, DSC, TEA and dNTP (AP3) is 1:(5～12): (6 ～15): (2～4).
Compared with prior art, there is advantages that
1) present invention has synthesized the new cleavable of a class and has connected unit, and is used for having synthesized based on such connection unit this Reversible terminator；And such reversible terminator is successfully used to DNA sequencing；Such sulfur is for the reversible terminator of ketal and acid The sensitive reversible terminator of ketal is compared, it is not necessary to will connect unit fracture in acid condition, thus avoid acid to DNA Damage, the reading contributing to extending order-checking is long.It is simultaneously based on the sulfur connection unit for ketal structure for 3 '-OH unprotected reversible end Only agent, when participating in DNA and extending, strict guarantee can the most only extend a reversible terminator.Meanwhile, its synthesis needed raw material Simple and easy to get, building-up process is conventional chemical reaction, can be used for large-scale promotion and uses.
2) Y012 is comparedConnecting unit, connection unit of the present invention is that sulfur is for ketal Connect unit, first its synthetic method entirely different；Secondly such reaction mechanism connected when unit ruptures is different, agents useful for same The most entirely different.Rareer is the connection unit of the present invention, remains Y012 this kind of connection unit and is participating in DNA extension Time, it is possible to control well the most only to extend a reversible terminator, and extend efficiency and be almost 100%.It is to say, should When class is connected to DNA sequencing, both remained acid-sensitive and connected unitDeng participate in DNA During extension, the most only extend one and extend efficiency and be almost the advantageous property of 100%, turn avoid sour environment pair simultaneously The damage of DNA.
Accompanying drawing explanation
The detailed description with reference to the following drawings, non-limiting embodiments made by reading, other features of the present invention, mesh And advantage can become more apparent upon:
Fig. 1 is the synthesis schematic diagram that the oxidation-sensitive sulfur of embodiment 1 connects unit for ketal；
Fig. 2 is the synthesis schematic diagram that the oxidation-sensitive sulfur of embodiment 8 connects unit for ketal；
Fig. 3 is synthesis schematic diagram based on terminator reversible described in embodiment 9；
Fig. 4 is that the DNA of the reversible terminator described in embodiment 10 extends glue figure.
Detailed description of the invention
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.Following example will assist in this area Technical staff be further appreciated by the present invention, but limit the present invention the most in any form.It should be pointed out that, general to this area For logical technical staff, without departing from the inventive concept of the premise, it is also possible to make certain adjustments and improvements.These broadly fall into Protection scope of the present invention.
Raw material used by the present invention, reagent are commercially available AR, CP level.
Gained intermediate product of the present invention and end product use NMR etc. to characterize.
Embodiment 1, work as R 1 =R 2 =Me, R=-COOH, such connects the synthesis of unit
The oxidation-sensitive sulfur of the present embodiment connects the synthesis schematic diagram of unit as it is shown in figure 1, specifically comprise the following steps that for ketal
The first step, the synthesis of compound 5, see below formula:
Under nitrogen protection, 2 mercapto ethanol hydrochlorate (10g, 88mmol) and triethylamine (24mL, 0.17mol) are dissolved in In methanol (0.25L), mixed liquor is cooled to 0 DEG C, the most under agitation drips Trifluoroacetic Acid Ethyl Ester (10mL, 84mmol) in reaction In mixed liquor, after 0 DEG C of reaction 3h, continue reaction 12h at room temperature；Removing solvent with Rotary Evaporators, residue adds acetic acid Ethyl ester (0.10L) dissolves, and washs successively with water (50mL) and saturated aqueous common salt (50mL), and organic phase with sodium sulfate is dried, post layer Analysis purification obtains compound 5 (7.9g, yield 54%).1H NMR(400MHz,CDCl3, TMS): δ 1.48 (1H, t, J= 8.6Hz), 2.75 (2H, dt, J=8.6,6.6Hz), 3.55 (2H, q, J=6.5Hz), 7.44 (1H, br s).13C NMR (100MHz,CDCl3): δ 23.5,42.7,115.8 (q, J=285Hz), 157.7 (q, J=37Hz).
Concurrently separate by-product (3.2g, yield 21%).1H NMR(400MHz,CDCl3,TMS):δ2.91(4H,t, J=6.4Hz), 3.72 (4H, q, J=6.2Hz), 6.59 (1H, br s).
Second step, the synthesis of compound 6, see below formula:
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And acetone (2.0g, 34mmol) is dissolved in acetonitrile (50mL), reactant mixture adds boron trifluoride diethyl etherate after being cooled to 0 DEG C (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor (0.30L), second in the reactive mixture Acetoacetic ester extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except solvent, use silicagel column Isolated and purified, must expect that the product sulfur is for ketal compound 6 (yield 4.2g, 37%).1H NMR(300MHz,CDCl3,TMS):δ 1.61 (6H, s), 2.63 (2H, t, J=7.1Hz), 2.88 (4H, overlapped m), 3.60 (2H, q, J=6.4Hz), 3.71 (3H,s),7.53(1H,br s).13C NMR(75.5MHz,CDCl3):δ25.1,29.2,30.8,33.5,39.2,51.9, 56.3,115.9 (q, J=288Hz), 157.4 (q, J=37Hz), 172.6.MS (ESI): m/z=356.0579, calcd.for C11H18F3NNaO3S2 +m/z(M+Na)+=356.0573.
Obtain two by-products simultaneously:
Sulfur is for ketal 9 (yield 2.2g, 23%).1H NMR(400MHz,CDCl3,TMS):δ1.60(6H,s),2.62 (4H, t, J=7.4Hz), 2.87 (4H, t, J=7.4Hz), 3.70 (6H, s).13C NMR(100MHz,CDCl3):δ25.3, 30.9,34.4,51.9,56.4,172.5.MS (ESI): m/z=303.0708, calcd.forC11H20NaO4S2 +m/z(M+Na)+ =303.0695.
Sulfur is for ketal 8 (yield 3.5g, 26%).1H NMR(400MHz,CDCl3,TMS):δ1.63(6H,s),2.85 (4H, t, J=6.7Hz), 3.60 (4H, q, J=6.4Hz), 6.80 (2H, br s).13C NMR(100MHz,CDCl3):δ29.5, 31.1,39.5,56.6,115.9 (q, J=287Hz), 157.6 (q, J=37Hz) .MS (ESI): m/z=409.0450, calcd.for C11H16F6N2NaO2S2 +m/z(M+Na)+=409.0450.
3rd step, the synthesis of compound 10, see below formula:
Sulfur is dissolved in acetone (0.20mL) for ketal 6 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) until pH > 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6 (10mg, 16%), and the pH of aqueous phase is transferred to 2, It is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid (yield 44mg, 73%) from organic facies.
1H NMR(400MHz,CDCl3, TMS): δ 1.61 (6H, s), 2.67 (2H, t, J=7.1Hz), 2.86and 2.89 (1H, overlapped t and t, J=6.7Hz and 7.0Hz, correspondingly), 3.59 (2H, q, J= 6.4Hz),3.71(3H,s),7.27(1H,br s),9.27(1H,br s).13C NMR(100MHz,CDCl3):δ25.0, (29.3,30.9,33.7,39.3,56.5,115.9 q, J=286Hz), 157.5 (q, J=37Hz), 176.9.MS (ESI): m/z =342.0419, calcd.for C10H16F3NNaO3S2 +m/z(M+Na)+=342.0416.
Compound 10 deprotects and i.e. obtains sulfur and connect unit for ketal.
Embodiment 2, work as R 1 With R 2 Collectively forming cyclohexyl, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-1:
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And Ketohexamethylene (34mmol) is dissolved in acetonitrile (50mL), reactant mixture adds boron trifluoride diethyl etherate after being cooled to 0 DEG C (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor (0.30L), second in the reactive mixture Acetoacetic ester extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except solvent, use silicagel column Isolated and purified, must expect that the product sulfur is for ketal 6-1.
3rd step, the synthesis of compound 10-1:
Sulfur is dissolved in acetone (0.20mL) for ketal 6-1 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-1 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-1 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 3, work as R 1 With R 2 Collectively forming cyclopenta, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-2:
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And Ketocyclopentane (34mmol) is dissolved in acetonitrile (50mL), reactant mixture adds boron trifluoride diethyl etherate after being cooled to 0 DEG C (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor (0.30L), second in the reactive mixture Acetoacetic ester extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except solvent, use silicagel column Isolated and purified, must expect that the product sulfur is for ketal 6-2.
3rd step, the synthesis of compound 10-2:
Sulfur is dissolved in acetone (0.20mL) for ketal 6-2 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-2 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-2 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 4, work as R 2 For-H, R 1 For phenyl, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-3, see below formula:
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And benzaldehyde (34mmol) is dissolved in acetonitrile (50mL), reactant mixture adds boron trifluoride diethyl etherate after being cooled to 0 DEG C (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor (0.30L), second in the reactive mixture Acetoacetic ester extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except solvent, use silicagel column Isolated and purified, must expect that the product sulfur is for ketal 6-3.
3rd step, the synthesis of compound 10-3:
Sulfur is dissolved in acetone (0.20mL) for ketal 6-3 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-3 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-3 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 5, work as R 2 For-H, R 1 For p-methoxyphenyl, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-4, see below formula:
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And P-methoxybenzal-dehyde (34mmol) is dissolved in acetonitrile (50mL), reactant mixture adds boron trifluoride after being cooled to 0 DEG C Ether (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor in the reactive mixture (0.30L), ethyl acetate extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except molten Agent, isolated and purified with silicagel column, must expect that the product sulfur is for ketal 6-4.
3rd step, the synthesis of compound 10-4:
Sulfur is dissolved in acetone (0.20mL) for ketal 6-4 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-4 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-4 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 6, work as R 2 For-H, R 1 Being 2,4,6-trimethoxyphenyls, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-5
Under nitrogen protection, 2-sulfydryl trifluoroacetamide 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) And 2,4,6-TMB (34mmol) are dissolved in acetonitrile (50mL), and reactant mixture adds three after being cooled to 0 DEG C Boron fluoride ether (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor in the reactive mixture (0.30L), ethyl acetate extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except molten Agent, isolated and purified with silicagel column, must expect that the product sulfur is for ketal 6-5.
3rd step, the synthesis of compound 10-5
Sulfur is dissolved in acetone (0.20mL) for ketal 6-5 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-5 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-5 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 7, work as R 2 For-H, R 1 For to methoxyl group naphthyl, during R=-COOH, such connects the synthesis of unit
The first step, the synthesis of compound 5: with embodiment 1.
Second step, the synthesis of compound 6-6:
Under nitrogen protection, thiol derivative 5 (5.7g, 33mmol), 3-mercapto-propionate (4.4g, 37mmol) and right Methoxy naphthyl aldehyde (34mmol) is dissolved in acetonitrile (50mL), and reactant mixture adds boron trifluoride diethyl etherate after being cooled to 0 DEG C (13mL, 0.11mol), after 0 DEG C is continued reaction 2h, adds 15% sodium carbonate liquor (0.30L), second in the reactive mixture Acetoacetic ester extraction (100mL × 3), organic facies is washed (0.30L) with 5% sodium carbonate liquor, is dried, revolves except solvent, use silicagel column Isolated and purified, must expect that the product sulfur is for ketal 6-6.
3rd step, the synthesis of compound 10-6:
Sulfur is dissolved in acetone (0.20mL) for ketal 6-6 (63mg, 0.19mmol), is then dissolved in PBS (2.0mL) adding PLE (Pig Liver Esterase) (13mg,>=195units), mixed liquor is stirred at room temperature, when pH<when 6.0, drips Add 2M NaOH solution (10 μ L) and know pH 7.0, after reaction 6h, add saturated aqueous common salt (3.0mL) in the reactive mixture, Being extracted with ethyl acetate, from organic facies, the unreacted sulfur of isolated is for ketal 6-6 (10mg, 16%), and the pH of aqueous phase is transferred to 2, it is extracted with ethyl acetate (5.0mL × 7), isolated colourless liquid from organic facies, compound 10-6 deprotects and i.e. obtains sulfur Unit is connected for ketal.
Embodiment 8, work as R 1 =R 2 When=Me, R=-OH, such connects the synthesis of unit
The first step, the synthesis of T1, see below formula:
Weigh tert-butyl chloro-silicane (10.00g, 66.34mmol) and imidazoles (4.73g, 69.74mmol) in single port In Ping, after adding dichloromethane stirring and dissolving, add 2 mercapto ethanol (4.32g, 55.29mmol), be stirred overnight under room temperature.Instead After should terminating, rotation, except dissolving, is dissolved residue, column chromatography (PE:EA=5:1) with methanol, is obtained T1 8.25g, productivity 77%.
Second step, the synthesis of TS-1
Compound T1 (8.25g, 42.89mmol) and T2 (8.99g, 51.91mmol) is dissolved in 120ml anhydrous tetrahydro furan, Adding p-methyl benzenesulfonic acid (0.73g, 4.23mmol), stirring is lower adds dried 5A molecular sieve (34.16g), stirs under room temperature After 30 minutes, add 2-methoxyl group propylene (2.81g, 38.99mmol), under room temperature, stir 48h.After reaction terminates, it is centrifuged off Molecular sieve, rotation is evaporated off oxolane, and acetic acid ethyl dissolution washs three times to neutrality, anhydrous slufuric acid with saturated sodium bicarbonate solution Sodium revolves after drying except ethyl acetate, and thick product obtains TS-1 1.67g by silica gel column chromatography.
3rd step, the synthesis of TS-2
TS-1 (1.46g, 3.60mmol) is dissolved in 50ml oxolane, add tetrabutyl ammonium fluoride (TBAF) (1.88g, 7.19mmol), reactant mixture is stirred at room temperature reaction 4h.Decompression rotation except solvent, thick product by silica gel column chromatography (PE: EA=2:1) TS-2 0.94g, productivity 89% are obtained.
4th step, the synthesis of TS-3
Weigh TS-2 (0.94g, 3.23mmol), add 20ml ammonia, be stirred overnight under room temperature.After reaction terminates, rotation removes Ammonia, silica gel plate separates to obtain TS-3 434mg, productivity 69%.
Embodiment 9, the synthesis of Reversible terminal based on such connection unit
The Reversible terminal of the present embodiment is that the synthesis of cleavable based on embodiment 1 connection unit obtains, and its synthesis is shown It is intended to (work as X=CH such as Fig. 22Time) shown in, specifically comprise the following steps that
The first step, the synthesis of compound M1:
By F1 (10mg, 0.061mmol) as in single port bottle, add the TAMRA (5/6) being dissolved in 1.5ml dry DMF (20mg, 0.038mmol), adds TEA (anhydrous triethylamine) 80uL and stirs 3.5h at room temperature, and rotation is except using analytical type after solvent HPLC is analyzed: pillar: C18,5 μm, 4.6 × 250mm；Flow velocity: 1mL/min；Flowing phase: A, 0.1%TEA aqueous solution and B, CH3OH, gradient wash, 30%～60%CH3OH (20min), 60%～80%CH3OH (20min), it is seen that photodetector: 546nm.There is when t=22.8min product peak to generate, prepare HPCL isolated 15mg, productivity 69%.
Second step, the synthesis of compound M2:
Weigh M1 (9mg, 0.0156mmol) in single port bottle, add 1.5ml MeCN (acetonitrile), and triethylamine 22uL, stir Mix, add DSC (N, N'-bis-succinimidyl carbonate) (26mg, 0.102mmol) evacuation nitrogen afterwards protection stirring 4h After, obtain intermediate
DUTP (AP3) (16mg, 0.031mmol) is dissolved in 1.5mL Na2CO3/NaHCO3Buffer joins in intermediate Reaction stirring 2h, is analyzed with analytical type HPLC: pillar: C18,5 μm, 4.6 × 250mm；Flow velocity: 1mL/min；Flowing phase: A, 0.1%TEA aqueous solution and B, CH3OH, gradient wash, 0%～20%CH3OH (35min), it is seen that photodetector: 546nm.? Having product peak to generate during t=27.9min, preparation HPLC separates to obtain compound M2 2.8mg. productivity 16.1%.
The present embodiment nucleotide dUTP (AP3) is known compound.
Base described in the present embodiment can also be C, A, G；Described fluorescein selected from BODIPY, rhodamine, coumarin, Ton, cyanine, pyrene, phthalocyanine, alexa, squarene dyestuff, the combination producing energy transfer dye and its derivant.Work as base For C, when A, G, described reversible terminator, i.e. compound II Except inclusion compound M2, (fluorescein in wherein said reversible terminator can be replaced also to comprise the compound of following structure Become other structure, be not only limited in fluorescein described in following structure):
Embodiment 10, the biological assessment of reversible terminator to synthesis
In order to detect whether the Reversible terminal synthesized by the present invention can apply to DNA sequencing, the present embodiment have detected reality Execute the characteristic of the reversible terminator of example 9: whether can be identified by archaeal dna polymerase, thus the substrate as archaeal dna polymerase participates in The extension of DNA and when there being continuous multiple identical base in template, if once can only extend a reversible termination Agent；
The experimental procedure of extension: the primer of base pair complementarity and template are dissolved in Tris-EDTA buffer (TE, PH 7.5) in, anneal according to following condition: 95 DEG C are incubated 3 minutes, are then cooled to 4 DEG C with the speed of 0.1 DEG C/sec and are incubated. Annealed primer and template form double-strand, can be used for DNA extension.PCR pipe is sequentially added into: 2 μ L 10x Klenow reaction buffer, 1.5 μ L sodium chloride solution (1M), 1.5 primers annealed for μ L and template (1 μ g/ μ L), 5 μ L DUTP-ketal connects unit-TAMRA (1mM), 0.4 μ L (2U) of Klenow Fragment exo-Archaeal dna polymerase, 9.6 μ L Aquesterilisa, forms the reaction system that cumulative volume is 20 μ L.After mix homogeneously, this reaction system is incubated 15 minutes at 37 DEG C, Then heat to 72 DEG C and be incubated 10 minutes, finally naturally cool to 16 DEG C.Precipitate through phenol chloroform and ethanol, extended Product.
Implementation result:
1) Fig. 3 be template be the extension products sequencing gel figure of and continuous two A, from left to right, be followed successively by Lane 1, Lane 2、Lane 3.As shown in Figure 3 when template is continuous two A, once can only extend a reversible terminator.
Lane 1:24nt；
Lane 3:oligo 2-3, dUTP-sulfur is for ketal-TAMRA, extension products；
Lane 3:oligo 2-4, dUTP-sulfur is for ketal-TAMRA, extension products；
5’-GAGGAAAGGGAAGGGAAAGGAAGG Oligo 2(5’-Dylight 800)(SEQ ID NO.1)
3’-CTCCTTTCCCTTCCCTTTCCTTCCATCGATCGCCATGTCG Oilgo 3(SEQ ID NO.2)
3’-CTCCTTTCCCTTCCCTTTCCTTCCAACGATCGCCATGTCG Oilgo 4(SEQ ID NO.3)
Conclusion: when template is continuous multiple identical base, when compound participates in DNA extension under polymerase effect The most all can only extend a sulfur for ketal modified nucleotide, illustrate that 3 '-unprotected reversible terminator still can once only Extend one, and extend efficiency close to 100%.
Above the specific embodiment of the present invention is described.It is to be appreciated that the invention is not limited in above-mentioned Particular implementation, those skilled in the art can make various deformation or amendment within the scope of the claims, this not shadow Ring the flesh and blood of the present invention.

Claims (10)

1. an oxidation-sensitive sulfur connects unit for ketal, it is characterised in that as shown in formula I:
Wherein, R=-OH or-COOH；
R1With R2The one met in following condition:
(1)R1=R2=methyl；
Or, (2) R1=phenyl, p-methoxyphenyl, 2,4,6-trimethoxyphenyls, naphthyl or to methoxyl group naphthyl, R2=H；
Or, (3) R1、R2Collectively form cyclohexyl or cyclopenta.
2. an oxidation-sensitive sulfur according to claim 1 connects the synthetic method of unit for ketal, it is characterised in that bag Include as follows:
(1) as R=-COOH:
S11, under protective gas, solvent condition, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, Isosorbide-5-Nitrae, 6- TMB, naphthaldehyde or to methoxyl group naphthaldehyde, with 2-sulfydryl trifluoroacetamideAnd 3-mercaptopropionic acid first EsterReaction, obtains sulfur for ketal compound
S12, described sulfur are converted into sulfur for ketal carboxylic acid compound for ketal compound in buffer solution
S13, remove the described sulfur amino protecting group for ketal carboxylic acid compound, obtain described oxidation-sensitive sulfur and connect for ketal single Unit
(2) as R=-OH:
S21, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, 1,4,6-TMB, naphthaldehyde or To methoxyl group naphthaldehyde, with 2-sulfydryl trifluoroacetamideAnd the 2 mercapto ethanol reaction of hydroxyl protection group, obtain Sulfur is for ketal compound
S22, described sulfur remove hydroxyl protecting group for ketal compound in acid solution or alkaline solution, obtain compound
S23, remove compoundAmino protecting group, obtain described oxidation-sensitive sulfur for ketal Connect unit
Oxidation-sensitive sulfur the most according to claim 2 connects the synthetic method of unit for ketal, it is characterised in that described 2- Preparing of sulfydryl trifluoroacetamide is as follows: under protective gas, solvent condition, Trifluoroacetic Acid Ethyl EsterWith 2-sulfydryl second Alcohol hydrochlorideReact under triethylamine effect, obtain 2-sulfydryl trifluoroacetamide.
4. connect the synthetic method of unit for ketal according to the oxidation-sensitive sulfur described in claim 2, it is characterised in that S11 step In, acetone, Ketocyclopentane, Ketohexamethylene, benzaldehyde, P-methoxybenzal-dehyde, Isosorbide-5-Nitrae, 6-TMB, naphthaldehyde or to methoxy Base naphthaldehyde, is (0.8～1.2): 1:(1～1.5 with the mol ratio of 2-sulfydryl trifluoroacetamide and 3-mercapto-propionate)；
Described protective gas is noble gas, and described solvent includes for polarity or non-polar organic solvent；
The condition of described reaction is: 0 DEG C, 1～3h.
5. connect the synthetic method of unit for ketal according to the oxidation-sensitive sulfur described in claim 2, it is characterised in that S12 step In, described buffer includes PBS, triethylamine acetate buffer；Described conversion exists at acetone and Pig Liver Esterase Under the conditions of carry out.
6. connect the synthetic method of unit for ketal according to the oxidation-sensitive sulfur described in claim 2, it is characterised in that S21 step In, described protection group includes Pentamethylene oxide., oxolane, tert-butyldimethyl silyl；Described alkaline solution includes that the tetrabutyl is fluorinated Ammonium salt solution；Described acid solution includes dilute hydrochloric acid or dilute sulfuric acid.
7. oxidation-sensitive sulfur according to claim 1 connects a unit purposes in DNA sequencing, its feature for ketal It is, refers specifically to: described oxidation-sensitive sulfur connects unit for ketal and is connected with nucleotide and fluorescein and to obtain reversible terminator, institute State reversible terminator and can be used for DNA synthesis order-checking.
8. the reversible terminator connecting unit based on the oxidation-sensitive sulfur described in claim 1 for ketal, it is characterised in that Connected unit by described oxidation-sensitive sulfur for ketal to be connected with nucleotide and fluorescein and obtain.
9. the synthetic method of the most reversible terminator, it is characterised in that described method includes walking as follows Rapid: to be connected unit by described oxidation-sensitive sulfur for ketal and be connected with nucleotide and fluorescein and obtain.
The synthetic method of the most reversible terminator, it is characterised in that comprise the steps:
A1, with dry DMF as solvent, under conditions of TEA exists, described oxidation-sensitive sulfur connects unit and fluorescein for ketal Reaction, obtains compound G
A2, TEA exist under conditions of, compound G and DSC react, obtain intermediate H This intermediate H need not isolated and purified directly react with dNTP, obtains compound II The most described reversible terminator.
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