CN101967128A - Benzo uracil PNA (Peptide Nucleic Acid) monomer and intermediate thereof as well as preparation methods and applications of benzo uracil PNA and intermediate thereof - Google Patents

Benzo uracil PNA (Peptide Nucleic Acid) monomer and intermediate thereof as well as preparation methods and applications of benzo uracil PNA and intermediate thereof Download PDF

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CN101967128A
CN101967128A CN 201010224200 CN201010224200A CN101967128A CN 101967128 A CN101967128 A CN 101967128A CN 201010224200 CN201010224200 CN 201010224200 CN 201010224200 A CN201010224200 A CN 201010224200A CN 101967128 A CN101967128 A CN 101967128A
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nucleic acid
peptide nucleic
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姚建年
詹传郎
李鹏发
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Institute of Chemistry CAS
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Abstract

The invention discloses a benzo uracil PNA (Peptide Nucleic Acid) monomer and an intermediate thereof as well as preparation methods and applications of the benzo uracil PNA and the intermediate thereof. The intermediate is shown as a formula (I), and the PNA monomer is shown as a formula (III). The invention provides the PNA monomer in a novel structure and provides a new method for synthesizing the benzo uracil PNA monomer. The PNA monomer has different chemical structures with different electric performances so as to be used for constructing a nucleic acid bionic nano-material with a potential gradient and applied to the field of optoelectronics.

Description

Benzo uracil peptide nucleic acid monomer and intermediate thereof and their preparation method and application
Technical field
The invention belongs to nucleic acid material and biomimetic material field, relate to a kind of benzo uracil peptide nucleic acid monomer and intermediate thereof and their preparation method and application.
Background technology
(peptide nucleic acid, PNA see document: Nielsen, P.E. to peptide nucleic acid(PNA); Egholm, M.; Berg, R.H.; Buchardt, O., Sequence selective recognition of DNA by strand displacement with a thymine-substitute polyamide, Science, 1991,254,1498～1 500) be nucleic acid (DNA) the structural simulation molecule of a quasi-representative, be to be the phosphide chain that class peptide chain that the unit forms has been replaced nucleic acid with 2-aminoethyl-glycine, but the base structure that has kept DNA, base be the molecular structure of peptide nucleic acid(PNA) of thymine (T) suc as formula shown in a, formula b is the chemical structure of benzo uridylic.Therefore, pna molecule is an electric neutrality, and have good light, heat and a chemical stability, not only have similar molecular recognition of DNA and recognition sequence performance, its recognition sequence performance is stronger than DNA, has important application prospects at gene therapy, gene test, gene silencing aspects such as (silence), therefore, since 1991 by since people such as the Peter Nielsen invention, obtained chemical boundary and the experts and scholars' of molecular biosciences educational circles great attention, and obtained significant progress.
Figure BSA00000185341800011
(formula a)
Figure BSA00000185341800012
(formula b)
Figure BSA00000185341800013
(formula c)
It is the synthetic route of the peptide nucleic acid monomer of representative that formula c is depicted as with base T (1), on 1, insert the acetate unit and form compound 2, compound 2 forms compound 4 posthydrolysiss with compound 3 reactions and obtains peptide nucleic acid monomer 5,5th, can be directly used in the monomer of solid phase synthesis.Document (L.Kospkina, W.T.WeiWang and T.C.Liang, Tetrahedron Lett. are arranged, 1994,35,5173-5176 and A.R.Katritzky, T.Narindoshvili, Org.Biomol.Chem., 2008,6,3171-3176) report, compound 2 can obtain by single step reaction in the aqueous solution of potassium hydroxide (KOH) by compound 1 and bromoacetic acid, and productive rate is 50-80%.Other synthetic method comprises: at N, in the dinethylformamide (DMF), with salt of wormwood (K 2CO 3) be alkali, catalysis 1 and ethyl bromoacetate, and further the ethyl ester hydrolysis can be obtained product 2, productive rate is 30-70% (K.L.Dueholm, M.Egholm, C.Behrens, L.Christensen, H.F.Hansen, T.Vulpius, K.H.Petersen, R.H.Berg, P.E.Niesen, O, Buchardt, J.Org.Chem.1994,59,5767-5773).Above-mentioned test method is applicable to natural base T and is prepared peptide nucleic acid monomer as raw material.But for the base (formula b) of benzo uracil, aforesaid method can not obtain the peptide nucleic acid monomer of higher yields.
Summary of the invention
The purpose of this invention is to provide a kind of benzo uracil peptide nucleic acid monomer and intermediate thereof and their preparation method and application.
The intermediate of preparation benzo uracil peptide nucleic acid monomer provided by the invention is the described compound of formula I general structure,
Figure BSA00000185341800021
(formula I)
In the described formula I general structure, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3, preferred R is-F ,-Cl or-CH 3
The method for preparing above-claimed cpd provided by the invention comprises the steps:
1) benzo uracil derivative shown in the formula II is reacted with sodium hydroxide in organic solvent, reaction finishes and obtains reaction soln A;
Figure BSA00000185341800022
(formula II)
Among the described formula II, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3
2) bromoacetic acid is dissolved in obtains reaction soln B in the organic solvent, described reaction soln B and described reaction soln A are reacted, obtain compound shown in the formula I.
In the step 1) of this method, described organic solvent is selected from dimethyl sulfoxide (DMSO) and N, at least a in the dinethylformamide, preferred dimethyl sulfoxide (DMSO); The amount ratio of benzo uracil derivative, sodium hydroxide and described organic solvent shown in the described formula II is 1mmol: 2-10mmol: 2-10ml, specifically can be 1mmol: 5mmol: 5.3-5.9ml, 1mmol: 5mmol: 5.4-5.9ml or 1mmol: 5mmol: 5.3-5.4ml, preferred 1mmol: 3-6mmol: 5-6ml; Temperature is 20-80 ℃, specifically can be 20-70 ℃, 20-40 ℃ or 40-70 ℃, and preferred 20-70 ℃, the time is 10-300 minute, preferred 100-200 minute; Described step 2) in, described organic solvent is selected from dimethyl sulfoxide (DMSO) and N, at least a in the dinethylformamide, preferred dimethyl sulfoxide (DMSO); The amount ratio of described bromoacetic acid and described organic solvent is 1-1.5mmol: 1-10ml, preferred 1.2-1.4mmol: 2-5ml; In the described reactions steps, temperature is 20-80 ℃, and preferred 20-70 ℃, the time is 5-15 hour, preferred 10 hours.In described step 2) after reaction finishes, reaction system is carried out following purification process: with reaction system and ethyl acetate mixing, collect the precipitation obtain, described precipitation is soluble in water, and adjusting pH value to 2 obtains compound shown in the formula I behind the purifying.Wherein, ethyl acetate can determine that the consumption of water can be determined according to the resolution of precipitate situation according to the sedimentary situation of separating out.
In the described step 1), R is-CH 3,-F or-the formula II of Cl shown in the preparation method of benzo uracil derivative be respectively: R is-CH 3: with commercially available 2-amino-5 methyl-phenylformic acid (4.5g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described product; R is-F: with commercially available 2-amino-5-fluoro-phenylformic acid (4.65g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described product; R is-Cl: with commercially available 2-amino-5-fluoro-phenylformic acid (5.13g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described product.
Peptide nucleic acid monomer compound provided by the invention, its general structure shown in formula III,
Figure BSA00000185341800031
(formula III)
In the described formula III, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3
The method of the above-mentioned peptide nucleic acid monomer compound of preparation provided by the invention, comprise the steps: the described compound of formula I and benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (HBTU) is dissolved among the organic solvent a and N, after the N-diisopropylethylamine at room temperature reacts 10-30 minute, the organic solution that adds compound 3 is more at room temperature reacted after 60-240 minute and is obtained product a, described product a at room temperature reacted 30-120 minute with sodium hydroxide in organic liquid mixture after, regulate the pH value to 3-5, obtain described peptide nucleic acid monomer compound; The organic solution of N-tert.-butoxy-carbonyl-1 is N-tert.-butoxy-carbonyl-1 is dissolved among the organic solvent b and gets.
In this method, described organic solvent a and organic solvent b all are selected from N, at least a in dinethylformamide and the dimethyl sulfoxide (DMSO), preferred N, dinethylformamide; Described organic solvent b is selected from N, at least a in dinethylformamide and the dimethyl sulfoxide (DMSO), preferred N, dinethylformamide; Described organic liquid mixture is to be that 1: 1 methylene dichloride mixes with ethanol and gets by volume ratio;
The described compound of described formula I, benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate, organic solvent a, N, N-diisopropylethylamine, N-tert.-butoxy-carbonyl-1,2-diaminoethanes, organic solvent b: the amount ratio of sodium hydroxide is 1mmol: 1-1.2mmol: 0.5-1ml: 1-1.2mmol: 1-1.2mmol: 0.5-1ml: 1-1.2mmol, preferred 1mmol: 1.1mmol: 1ml: 1.1mmol: 1.1mmol: 1ml: 1.1mmol.Described room temperature is 10-25 ℃.
At described N-tert.-butoxy-carbonyl-1, after the organic solution of 2-diaminoethanes was at room temperature reacted 60-240 minute, before described and sodium hydroxide at room temperature react 30-120 minute, also reaction system is carried out following processing: reaction system is mixed with water, collecting precipitation, after ethyl acetate washing and extraction, to extract gained organic layer anhydrous magnesium sulfate drying, using by volume ratio is that the mixed solution that 10: 1 methylene dichloride and ethyl acetate are formed carries out silicagel column drip washing again, obtains described product a.
In addition, the compound of peptide nucleic acid monomer shown in the formula III has application in the nucleic acid bionic material of electric potential gradient in preparation, and is the peptide nucleic acid(PNA) that monomer prepares with the compound of peptide nucleic acid monomer shown in the formula III, also belongs to protection scope of the present invention.This peptide nucleic acid(PNA) is to be carried out condensation reaction preparation and got by the compound of peptide nucleic acid monomer shown in the formula III.Described nucleic acid bionic material with electric potential gradient is preferably biological organic photovoltaic battery.
The invention provides a kind of peptide nucleic acid monomer of novel texture, and a kind of novel method of synthetic this class benzo uracil peptide nucleic acid monomer is provided.Because this peptide nucleic acid monomer has different chemical structures, its electrical property is also different, thereby, can be used for making up nucleic acid bionic nano material with electric potential gradient, be applied to optoelectronic areas.
Description of drawings
Fig. 1 is the nucleus magnetic hydrogen spectrum and the carbon spectrum of embodiment 1 preparation gained intermediate.
Fig. 2 is the nucleus magnetic hydrogen spectrum and the carbon spectrum of embodiment 2 preparation gained intermediates.
Fig. 3 is the nucleus magnetic hydrogen spectrum and the carbon spectrum of embodiment 3 preparation gained intermediates.
Fig. 4 is the nucleus magnetic hydrogen spectrum and the carbon spectrum of embodiment 4 preparation gained peptide nucleic acid monomers.
Embodiment
The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.If no special instructions, described method is ordinary method in the following method.
The intermediate (compound 2) of the peptide nucleic acid monomer of benzo uracil shown in embodiment 1, the preparation formula I
1) under 20 ℃ heating condition, benzo uridylic (4g, 22.7mmol, compound 1) is dissolved among 120 milliliters the DMSO, (4.5g, 113.55mmol), under 40 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH.
2) (3.16g 22.71mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 40 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continued stirring reaction 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, the precipitation that collection obtains, precipitation is dissolved in 150 ml waters, with the pH value of 6N hydrochloric acid conditioning solution to pH=7, filtration obtains 1.5 gram (8.5mmol) compounds 1, regulate pH value of filtrate to 2 with 6N hydrochloric acid, obtain 1.1 gram (2.3mmol) product compounds 2, productive rate is 10%.
The nuclear-magnetism structural characterization data of this product are as follows:
1H-NMR(400MHz，DMSO-d 6)δ：12.99(s，1H，-OH)，11.49(s，1H，-NH)，7.74(s，1H，-phH)，7.53(d，1H，J＝8.0Hz，-phH)，7.13(d，1H，J＝8.0Hz，-phH)，4.55(s，2H，-CH 2)，2.35(s，3H，-CH 3)； 13C?NMR(DMSO-d 6，100MHz)δ：169.4，161.6，149.8，137.2，136.4，132.1，126.8，115.3，113.2，41.3，20.2；ESI-MS?m/z(％)：233.0(M-H -，100％).
The nucleus magnetic hydrogen spectrum of this product and carbon spectrum are as shown in Figure 1.As from the foregoing, this compound structure is correct, is the intermediate (compound 2) of the peptide nucleic acid monomer of benzo uracil shown in the formula I.
Wherein, the used reactant compound 1 of step 1) is to be prepared as follows and to get: with commercially available 2-amino-5 methyl-phenylformic acid (4.5g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtain described compound 1 (4.5g, 25.6mmol), productive rate 85%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.21 (s, 1H ,-NH), 11.05 (s, 1H ,-NH), 7.69 (s, 1H ,-phH), 7.47 (d, 1H, J=7.2Hz ,-phH), 7.08 (d, 1H, J=7.2Hz ,-phH), 2.32 (s, 3H ,-CH 3); 13CNMR (DMSO-d 6, 100MHz) δ: 162.8,150.3,138.7,135.9,131.5,126.5,115.3,114.2,20.2; ESI-MS m/z (%): 176.0 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 1.
The intermediate (compound 4) of the peptide nucleic acid monomer of benzo uracil shown in embodiment 2, the preparation formula I
Figure BSA00000185341800051
1) under 40 ℃ heating condition, benzo uridylic (4g, 22.2mmol, compound 3) is dissolved among 120 milliliters the DMSO, (4.44g, 111mmol), under 40 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH.
2) (3.1g 22.2mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 40 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, the precipitation that collection obtains, precipitation is dissolved in 150 ml waters, transfer the pH value of solution to pH=7 with 6N hydrochloric acid, filtration obtains 1.4 gram (7.8mmol) compounds 3, transfer pH value of filtrate to 2 with 6N hydrochloric acid, obtain 0.8 gram (3.4mmol) product compound 4, productive rate is 15%.
The nuclear-magnetism structural characterization data of this product are as follows:
1H-NMR(400MHz，DMSO-d 6)δ：13.04(s，1H，-OH)，11.66(s，1H，-NH)，7.65(m，2H，-phH)，7.27(m，1H，-phH)，4.55(s，2H，-CH 2)； 13C?NMR(DMSO-d 6，100MHz)δ：169.2，160.9，158.7，156.2，149.5，136.1，123.6，123.4，117.7，114.4，112.5，112.3，41.5；GCT-MS?m/z(％)：238(M，10％).
The nucleus magnetic hydrogen spectrum of this product and carbon spectrum are as shown in Figure 2.As from the foregoing, this compound structure is correct, is the intermediate (compound 4) of the peptide nucleic acid monomer of benzo uracil shown in the formula I.
Wherein, used reactant compound 3 is to be prepared as follows and to get in the described step 1): with commercially available 2-amino-5-fluoro-phenylformic acid (4.65g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtain described compound 3 (4.5g, 25mmol), productive rate 83%.
The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.29 (s, broad, 2H ,-NH), 7.60 (m, 2H ,-phH), 7.21 (s, 1H ,-phH); 13C NMR (DMSO-d 6, 100MHz) δ: 162.1,158.5,156.1,150.1,137.5,123.0,122.8,117.6,117.5,115.4,115.3,112.1,111.8; GCT-MSm/z (%): 180.0 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 3.
The intermediate (compound 6) of the peptide nucleic acid monomer of benzo uracil shown in embodiment 3, the preparation formula I
Figure BSA00000185341800061
1) under 70 ℃ heating condition, benzo uridylic (4g, 20.3mmol, compound 5) is dissolved among 120 milliliters the DMSO, (4.06g, 101.5mmol), under 40 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH.
2) (2.82g 20.3mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 40 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, the precipitation that collection obtains, precipitation is dissolved in 150 ml waters, transfer the pH value of solution to pH=7 with 6N hydrochloric acid, obtain 1.6 gram (8.2mmol) compounds 5, transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 0.7 gram (2.8mmol) product compound 6, productive rate is 14%.
The nuclear-magnetism structural characterization data of this product are as follows:
1H-NMR(400MHz，DMSO-d 6)δ：13.05(s，1H，-OH)，11.74(s，1H，-NH)，7.89(s，1H，-phH)，7.77(d，1H，J＝8.8Hz，-phH)，7.26(d，1H，J＝8.8Hz，-phH)，4.55(s，2H，-CH 2)； 13CNMR(DMSO-d 6，100MHz)δ：169.4，161.8，150.1，139.7，134.8，126.3，125.9，117.5，115.8，41.3；GCT-MS?m/z(％)：254.0(M，25％).
The nucleus magnetic hydrogen spectrum of this product and carbon spectrum are as shown in Figure 3.As from the foregoing, this compound structure is correct, is the intermediate (compound 6) of the peptide nucleic acid monomer of benzo uracil shown in the formula I.
Wherein, used reactant compound 5 is to be prepared as follows and to get in the described step 1): with commercially available 2-amino-5-fluoro-phenylformic acid (5.13g, 30mmol) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtain described compound 5 (5.0g, 25.5mmol), productive rate 85%.
The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.44 (s, 1H ,-NH), 11.27 (s, 1H ,-NH), 7.82 (s, 1H ,-phH), 7.70 (d, 1H, J=8.4Hz ,-phH), 7.19 (d, 1H, J=8.4Hz ,-phH); 13C NMR (DMSO-d 6, 100MHz) δ: 161.8,150.1,139.7,134.8,126.3,115.9,117.5,115.8; GCT-MS m/z (%): 196.0 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 5.
Benzo uracil peptide nucleic acid monomer (compound 9) shown in embodiment 4, the preparation formula III
Figure BSA00000185341800071
With embodiment 1 preparation gained midbody compound 2 (117mg, 0.5mmol), HBTU (170mg 0.5mmol) is mixed in 1 milliliter N, in the dinethylformamide (DMF), adds N, the N-diisopropyl ethyl amine (DIEA, 0.5mmol).Stir after 30 minutes, add compound 7N-tert.-butoxy-carbonyl-1,2-diaminoethanes (120mg, 0.5mmol) 1 milliliter of DMF solution, after the stirring at room 2 hours, pour in 20 ml waters, collecting precipitation, after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, (184mg, 0.41mmol), productive rate is 80% to obtain product compound 8.
It is in 1: 15 milliliters of the mixed solvents of being made up of methylene dichloride and ethanol that product compound 8 is dissolved in volume ratio, with the neutralization of 1N hydrochloric acid, the pH value of regulator solution is to 3-5 after 2 hours for the sodium hydroxide that adds 1 mmole, stirring at room, remove organic solvent and obtain compound 9, productive rate is 100%.
The nuclear-magnetism structural characterization data of this product are as follows:
1H-NMR(400MHz，DMSO-d 6)δ：12.99(s，1H，-OH)，11.49(s，1H，-NH)，7.74(s，1H，-phH)，7.53(d，1H，J＝8.0Hz，-phH)，7.13(d，1H，J＝8.0Hz，-phH)，6.7(s，1H，-NH)，4.55(s，2H，-CH 2)，3.0(d，2H，J＝5.6Hz，-CH 2)，2.5(m，3H，-CH 2)，2.35(s，3H，-CH 3)，2.1(s，1H，-CH 2)，1.37(s，9H，-Boc-H)； 13C?NMR(DMSO-d 6，100MHz)δ：169.3，169.1，160.7，155.4，150.2，137.7，136.7，132.8，127.6，125.3，114.2，80.5，54.2，52.8，49.8，42.3，28.6，20.2；ESI-MS?m/z(％)：433.0(M-H -，100％).
The nucleus magnetic hydrogen spectrum of this product and carbon spectrum are as shown in Figure 4.As from the foregoing, this compound structure is correct, is the uracil of benzo shown in formula III peptide nucleic acid monomer (compound 9).
Embodiment 5, has the peptide nucleic acid nano heterojunction of electric potential gradient with the preparation of embodiment 4 preparation gained peptide nucleic acid monomers: preparation Pc-R 2-CONH-AAAAA-CONH-R 1-PDI-R 1-CONH-T Py-8UTT φT Na-CONH 2(T φIn substituent R=-CH 3, T NaAnd T Py-8In substituent R be H) peptide nucleic acid nano heterojunction 1
1) is start element with 4-toluene hydrogen amine (mbha resin),, handles 3 times that each 3 minutes is that 1: 1 DMF/DCM washes 3 times with the 2mL mol ratio respectively then, after the 2mL pyridine is washed twice, is T with base with 2mL trifluoroacetic acid (TFA) with after the methylene dichloride swelling Py-8Peptide nucleic acid monomer activate with dehydration catalyst and diisopropyl ethyl amine after, with mbha resin in 125 μ L N, react in the dinethylformamide (DMF), use the DMF washed twice, with capping reagent (diacetyl oxide) handle and washing after, obtain MBHA-T Py-8Compound;
2) with described MBHA-T Py-8Compound and base are that the peptide nucleic acid monomer of U carries out dehydration reaction, obtain MBHA-T Py-8The U compound;
3) repeating said steps 2) (3) inferior, obtains MBHA-T Py-8UTT φT NaCompound;
4) PDI is activated with dehydration catalyst and diisopropyl ethyl amine after, with described MBHA-T Py-8UTT φT NaCompound reacts, and obtains MBHA-T Py-8UTT φT Na-R 1-PDI compound;
5) be A with base 1Peptide nucleic acid monomer activate with dehydration catalyst and diisopropyl ethyl amine after, with described MBHA-T Py-8UTT φT Na-R 1-PDI compound reacts, and obtains MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1Compound;
6) with described MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1Compound and base are A 2Peptide nucleic acid monomer carry out dehydration reaction, obtain MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1A 2Compound;
7) repeating said steps 6) 3 times, obtain MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1A 2A nCompound;
8) Pc is activated with dehydration catalyst and diisopropyl ethyl amine after, with described MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1A 2A nCompound reacts, and obtains MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1A 2A n-R 2-Pc;
9) with described MBHA-T Py-8UTT φT Na-R 1-PDI-R 1-A 1A 2A n-R 2-Pc and 5mL trifluoroacetic acid and 5mL TFMSA react respectively, and use the ethyl acetate post precipitation, and collecting precipitation obtains unpurified described peptide nucleic acid nano heterojunction molecule.
Above-mentioned unpurified peptide nucleic acid nano heterojunction molecule is dissolved among the 0.5mL DMF, is injected in the high pressure liquid chromatograph (HPLC), use TFA/H 2O (volume ratio 0.5%) and CH 3CN/H 2The mixed solvent wash-out of O (volume ratio 0.5%) is collected elutriant, determine required product with mass spectrum (TOF-MS) after, lyophilize, the gained solid detects with HPLC and TOF-MS again, is defined as target product, i.e. Pc-R 2-CONH-AAAAA-CONH-R 1-PDI-R 1-CONH-T Py-8UTT φT Na-CONH 2(T NaT φT Py-8In substituent R=-CH3), be the peptide nucleic acid(PNA) molecule that a covalent coupling has PDI and Pc;
10) being dissolved in by 50 μ L water and 50 μ L concentration by the peptide nucleic acid(PNA) molecule of PDI and Pc the covalent coupling that obtains in the described step 9) is in the mixed solution formed of the 200mM/L NaCl aqueous solution, place 37 ℃ incubator to cultivate 2 hours, obtain the described PNA of formula I and be-CONH-Tn-CONH 2Peptide nucleic acid nano heterojunction 1.
In described step 1)-step 9), described reaction is reaction with same mole, and in the step 1), described base is T as described 1The mol ratio of peptide nucleic acid monomer, dehydration catalyst, diisopropyl ethyl amine and 4-toluene hydrogen amine (MBHA) resin be 1: 1: 1: 1.In each reactions steps, the consumption of organic solvent is as the criterion with complete solubilizing reaction thing; Described dehydration catalyst is benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate.
This peptide nucleic acid nano heterojunction 1 can prepare biological organic photovoltaic battery according to ordinary method, its preparation method is as follows: handling on the clean ito glass surface, after spin coating one layer thickness is the PSS/PEDOT (the poly-p styrene sulfonic acid of poly-dioxoethyl thiophene) of 40nm, 150 ℃ of dryings after 0.5 hour, be spin-coated on this PSS/PEDOT layer in the mixture with described peptide nucleic acid nano heterojunction 1 and oil of mirbane composition, obtain the active coating (thickness is 100nm) of biological organic photovoltaic battery, after 1 hour, is 5 * 10 in vacuum tightness in 100 ℃ of dryings -5Under the condition of Pa, evaporation one layer thickness is the metal aluminium electrode of 150nm, obtains described biological organic photovoltaic battery.
In this method, described base is T φ(R is-CH 3) peptide nucleic acid(PNA) be compound 9 φ, be to be prepared as follows and to get:
1) with midbody compound 2 φ(117mg, 0.5mmol), HBTU (170mg 0.5mmol) is mixed in 1 milliliter N, in the dinethylformamide (DMF), adds N, and the N-diisopropyl ethyl amine (DIEA, 0.5mmol).Stir after 30 minutes, add compound 7 φN-tert.-butoxy-carbonyl-1,2-diaminoethanes (120mg, 0.5mmol) 1 milliliter of DMF solution, stirring at room was poured in 20 ml waters after 2 hours, collecting precipitation, after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, and obtains product compound 8 φ(184mg, 0.41mmol), productive rate is 80%.2) with product compound 8 φBe dissolved in volume ratio and be in 1: 15 milliliters of the mixed solvents of being made up of methylene dichloride and ethanol, add the sodium hydroxide of 1 mmole, stirring at room after 2 hours with the neutralization of 1N hydrochloric acid, remove organic solvent and obtain compound 9 to 3-5 by the pH value of regulator solution φ, productive rate is 100%.The nuclear-magnetism structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 12.99 (s, 1H ,-OH), 11.49 (s, 1H ,-NH), 7.74 (s, 1H ,-phH), 7.53 (d, 1H, J=8.0 Hz ,-phH), 7.13 (d, 1H, J=8.0Hz ,-phH), 6.7 (s, 1H ,-NH), 4.55 (s, 2H ,-CH 2), 3.0 (d, 2H, J=5.6Hz ,-CH 2), 2.5 (m, 3H ,-CH 2), 2.35 (s, 3H ,-CH 3), 2.1 (s, 1H ,-CH 2), 1.37 (s, 9H ,-Boc-H); 13C NMR (DMSO-d 6, 100MHz) δ: 169.3,169.1,160.7,155.4,150.2,137.7,136.7,132.8,127.6,125.3,114.2,80.5,54.2,52.8,49.8,42.3,28.6,20.2; ESI-MS m/z (%): 433.0 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the uracil of benzo shown in formula III peptide nucleic acid monomer (compound 9 φ).
Wherein, described midbody compound 2 φBe to be prepared as follows and to get:
Figure BSA00000185341800102
1) under 20 ℃ heating condition, with benzo uridylic (4g, 22.7mmol, compound 1 φ) be dissolved among 120 milliliters the DMSO, (4.5g, 113.55mmol), under 40 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH.2) (3.16g 22.71mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 40 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continued stirring reaction 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, collected the precipitation obtain, precipitation is dissolved in 150 ml waters, to pH=7, filter and obtain 1.5 and restrain (8.5mmol) compounds 1 with the pH value of 6N hydrochloric acid conditioning solution φ, transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 1.1 gram (2.3mmol) product compounds 2 φ, productive rate is 10%.The nuclear-magnetism structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 12.99 (s, 1H ,-OH), 11.49 (s, 1H ,-NH), 7.74 (s, 1H ,-phH), 7.53 (d, 1H, J=8.0Hz ,-phH), 7.13 (d, 1H, J=8.0Hz ,-phH), 4.55 (s, 2H ,-CH 2), 2.35 (s, 3H ,-CH 3); 13CNMR (DMSO-d 6, 100MHz) δ: 169.4,161.6,149.8,137.2,136.4,132.1,126.8,115.3,113.2,41.3,20.2; ESI-MS m/z (%): 233.0 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the intermediate (compound 2 of the peptide nucleic acid monomer of benzo uracil shown in the formula I φ).
Wherein, the used reactant compound 1 of step 1) φBe to be prepared as follows and to get: with commercially available 2-amino-5 methyl-phenylformic acid (4.5g, 30mmol) and urea (18g, 300mmol) be blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described compound 1 φ(4.5g, 25.6mmol), productive rate 85%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.21 (s, 1H ,-NH), 11.05 (s, 1H ,-NH), 7.69 (s, 1H ,-phH), 7.47 (d, 1H, J=7.2Hz ,-phH), 7.08 (d, 1H, J=7.2Hz ,-phH), 2.32 (s, 3H ,-CH 3); 13CNMR (DMSO-d 6, 100MHz) δ: 162.8,150.3,138.7,135.9,131.5,126.5,115.3,114.2,20.2; ESI-MS m/z (%): 176.0 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 1 φ
Described base is T NaThe peptide nucleic acid(PNA) of (R is H) is a compound 5 Na, be to be prepared as follows and to get:
Figure BSA00000185341800111
With midbody compound 2 Na(130mg, 0.5mmol) and HBTU (170mg 0.5mmol) is mixed in 1 milliliter N, in the dinethylformamide (DMF), add and the sec.-propyl ethylamine (DIEA, 0.5mmol).Stir after 30 minutes, add compound 3 Na(120mg, 0.5mmol) 1 milliliter of DMF solution, after the stirring at room 3 hours, pour in 20 ml waters, collecting precipitation is after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, and using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, and obtains product compound 4 Na(200mg, 0.41mmol), productive rate 80%.With compound 4 NaBe dissolved in volume ratio and be in 1: 15 milliliters of the mixed solvents of forming by methylene dichloride and ethanol, add the sodium hydroxide of 1 mmole, stirring at room 2 hours, the back is with the neutralization of 1N hydrochloric acid, and the pH value of transferring solution is removed organic solvent and is obtained compound 5 to 3-5 Na, productive rate is 100%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 13.04 (s, 1H ,-OH), 11.67 (s, 1H ,-NH), 8.71 (s, 1H ,-naph-H), 8.15 (d, 1H, J=8.4Hz ,-naph-H), 7.95 (d, 1H, J=8.4Hz,-naph-H), 7.63 (t, 1H, J=8.0Hz ,-naph-H), 7.59 (s, 1H ,-naph-H), 7.48 (t, 1H, J=8.0Hz ,-naph-H), 4.90 (s, 2H ,-CH 2), 3.0 (d, 2H, J=5.6Hz ,-CH 2), 2.5 (m, 3H ,-CH 2), 2.1 (s, 1H ,-CH 2), 1.37 (s, 9H ,-Boc-H); 13C NMR (DMSO-d 6, 100MHz) δ: 169.4,161.7,149.8,136.4,134.9,13 1.2,129.6,129.5,128.6,126.8,125.1,114.2,110.4,80.5,54.2,52.8,49.8,41.4,28.6,20.2; ESI-MSm/z (%): 469.2 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the uracil of naphtho-shown in formula III peptide nucleic acid monomer (compound 5 Na).
Wherein, midbody compound 2 NaBe to be prepared as follows and to get:
1) under 20 ℃ heating condition, with naphtho-uridylic (4.0g, 18.8mmol, compound 1 Na) be dissolved among 100 milliliters the DMSO, (3.76g, 94.0mmol), under 20 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH.2) (2.512g 18.8mmol) is dissolved among 4 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 20 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 1 hour.Reaction solution is poured in 200 milliliters of ethyl acetate, collected the precipitation obtain, precipitation is dissolved in 200 ml waters, the pH value of transferring solution with 6N hydrochloric acid is to pH=7, filters to obtain 1.2 and restrain (5.7mmol) compounds 1 Na, transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 0.91 gram (3.4mmol) compound 2 Na, productive rate 18%.The nuclear-magnetism structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 13.04 (s, 1H ,-OH), 11.67 (s, 1H ,-NH), 8.71 (s, 1H ,-naph-H), 8.15 (d, 1H, J=8.4Hz ,-naph-H), 7.95 (d, 1H, J=8.4Hz,-naph-H), 7.63 (t, 1H, J=8.0Hz ,-naph-H), 7.59 (s, 1H ,-naph-H), 7.48 (t, 1H, J=8.0Hz ,-naph-H), 4.90 (s, 2H ,-CH 2), 2.35 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 169.4,161.7,149.8,136.4,134.9,131.2,129.6,129.5,128.6,126.8,125.1,114.2,110.4,41.4; ESI-MS m/z (%): 269.1 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the intermediate (compound 2 of the peptide nucleic acid monomer of benzo uracil shown in the formula I Na).
Wherein, used reactant compound 1 in above-mentioned preparation method's step 1) NaBe to be prepared as follows and to get: with commercially available 2-amino-5 methyl-phenylformic acid (5.61g, 30mmol) and urea (18g, 300mmol) be blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described compound 1 Na(5.2g, 24.5mmol), productive rate 82%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.31 (s, 1H ,-NH), 11.20 (s, 1H,-NH), 8.64 (s, 1H ,-naph-H), 8.10 (d, 1H, J=8.4Hz ,-naph-H), 7.90 (d, 1H, J=8.4Hz ,-naph-H), 7.61 (t, 1H, J=8.0Hz,-naph-H), 7.52 (s, 1H ,-naph-H), 7.47 (t, 1H, J=8.0Hz ,-naph-H); 13C NMR (DMSO-d 6, 100MHz) δ: 162.9,150.3,136.5,136.3,129.5,129.2,129.0,128.5,126.7,124.9,115.2,110.2; GCT-MS m/z (%): 212.0 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 1 Na
Described base is T Py-8The peptide nucleic acid(PNA) of (R is H) is that compound 9 is to be prepared as follows to get:
Figure BSA00000185341800131
1) with intermediate (compound 3) (111mg, 0.5mmol), HBTU (170mg 0.5mmol) is mixed in 1 milliliter N, in the dinethylformamide (DMF), adds N, the N-diisopropyl ethyl amine (DIEA, 0.5mmol).Stir after 30 minutes, add compound 7 (120mg, 1 milliliter of DMF solution 0.5mmol), after the stirring at room 3 hours, pour in 20 ml waters, collecting precipitation is after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, and using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, and obtains product compound 8 (208mg, 0.46mmol), productive rate 90%.2) product compound 8 being dissolved in volume ratio is in 1: 15 milliliters of the mixed solvents of being made up of methylene dichloride and ethanol, adds the sodium hydroxide of 1 mmole, stirring at room 2 hours, the back neutralizes with 1N hydrochloric acid, the pH value of regulator solution is removed organic solvent and is obtained compound 9 to 3-5, and productive rate is 100%.It is as follows that the nuclear-magnetism of this compound 9 detects data: 1H-NMR (400MHz, DMSO-d 6) δ: 13.03 (s, 1H ,-OH), 11.46 (s, 1H ,-NH), 8.70 (d, 1H, J=8.0Hz ,-pyH), 8.30 (d, 1H, J=8.0Hz ,-pyH), 7.31 (q, 1H, J=4.8 and 6.8Hz ,-pyH), 6.5 (s, 1H ,-NH), 4.55 (s, 2H ,-CH 2), 3.85 (s, 2H ,-CH 2), 3.0 (m, 2H ,-CH 2), 2.5 (m, 2H ,-CH 2), 1.37 (s, 9H ,-Boc-H); 13C NMR (DMSO-d 6, 100MHz) δ: 169.3,168.1,162.2,160.7,154.8,151.3,150.4,135.7,118.5,114.2,80.5,54.2,52.8,49.8,42.3,28.6; ESI-MS m/z (%): 420.0 (M-H -, 100%.As from the foregoing, this compound structure is correct, is the uracil of benzo shown in formula III peptide nucleic acid monomer (compound 9).
Wherein, described midbody compound 3 is to be prepared as follows and to get:
Figure BSA00000185341800141
1) under 30 ℃ heating condition, (4g 24.5mmol) is dissolved among 120 milliliters the DMSO, and (2.3g, 56.8mmol), under 30 ℃ heating condition, stirring reaction 1 hour obtains reaction soln (A) to add NaOH with compound 2 pyridino-uracils.
2) (3.16g 22.71mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 30 ℃ the heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, the precipitation that collection obtains, precipitation is dissolved in 50 ml waters, transfer the pH value of solution to pH=7 with 6N hydrochloric acid, filtration obtains 1.5 gram (9.2mmol) compounds 2, transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 1.1 gram (5.0mmol) product compounds 3, productive rate 20%.It is as follows that the nuclear-magnetism of this product detects data: 1H-NMR (400MHz, DMSO-d 6) δ: 13.06 (s, 1H ,-NH), 11.48 (s, 1H ,-NH), 8.70 (d, 1H, J=8.0Hz ,-pyH), 8.26 (d, 1H, J=8.0Hz ,-pyH), 7.28 (q, 1H, J=4.8 and6.8Hz ,-pyH), 4.53 (s, 2H ,-CH 2), 3.30 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 169.4,162.2,152.5,151.8,149.3,135.7,126.5,117.2,41.2; GCT-MS m/z (%): 221.0 (M, 100%).As from the foregoing, this compound structure is correct, is the intermediate (compound 3) of the peptide nucleic acid monomer of benzo uracil shown in the formula I.Wherein, used reactant compound 2 pyridino-uracils are to be prepared as follows and to get in the aforesaid method step 1):
Figure BSA00000185341800142
With commercially available 2-amino-3-carboxyl-pyridine (4.14g, 30mmol, compound 1) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 50mL water, stir after 30 minutes, filter, collecting precipitation obtain compound 2 (2.5g, 15.33mmol), productive rate 51%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.64 (s, 1H ,-NH), 11.48 (s, 1H ,-NH), 8.60 (d, 1H, J=8.0Hz ,-pyH), 8.26 (d, 1H, J=8.0Hz ,-pyH), 7.26 (q, 1H, J=4.8and 6.8Hz ,-pyH), 3.30 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 162.2,154.8,151.3,150.4,135.7,126.5,110.2,20.2; GCT-MS m/z (%): 163.0 (M, 100%).As from the foregoing, this compound structure is correct, is compound 2 pyridino-uracils.
Embodiment 6, has the peptide nucleic acid nano heterojunction of electric potential gradient with the preparation of embodiment 4 preparation gained peptide nucleic acid monomers: preparation Pc-R 2-CONH-AAAAA-CONH-R 1-PDI-R 1-NHCO-T NaT φT φ 'T Py-8T Py-5-NHCO-R 2-Pc peptide nucleic acid nano heterojunction (wherein, base T φIn substituent R=-CH 3, T Na, T Py-5And T Py-8In substituent R be H, T φ 'In substituent R=-OCH 3)
1) with the mbha resin is start element,, handles 3 times that each 3 minutes is that 1: 1 DMF/DCM washes 3 times with the 2mL mol ratio respectively then, after the 2mL pyridine is washed twice, is T with base with 2mL trifluoroacetic acid (TFA) with after the methylene dichloride swelling 1Peptide nucleic acid monomer activate with dehydration catalyst and diisopropyl ethyl amine after, with mbha resin in 125 μ L N, react in the dinethylformamide (DMF), use the DMF washed twice, with capping reagent (diacetyl oxide) handle and washing after, obtain MBHA-T NaCompound;
2) with described MBHA-T NaCompound and base are T φPeptide nucleic acid monomer carry out dehydration reaction, obtain MBHA-T NaT φCompound;
3) repeating said steps 2) 3 times, obtain MBHA-T NaT φT φ 'T Py-8T Py-5Compound;
4) Pc is activated with dehydration catalyst and diisopropyl ethyl amine after, with described MBHA-T NaT φT φ 'T Py-8T Py-5Compound reacts, and obtains MBHA-T NaT φT φ 'T Py-8T Py-5-R 2-Pc compound;
5) with described MBHA-T NaT φT φ 'T Py-8T Py-5-R 2-Pc compound and 5mL trifluoroacetic acid and 5mL TFMSA react, and use the ethyl acetate post precipitation, collecting precipitation, and obtaining unpurified skeleton symbol is HOOC-T NaT φT φ 'T Py-8T Py-5-R 2The peptide nucleic acid(PNA) molecule A of-Pc;
With above-mentioned unpurified skeleton symbol is HOOC-T NaT φT φ 'T Py-8T Py-5-R 2The peptide nucleic acid(PNA) molecule A of-Pc is dissolved among the 2mLDMF, is injected in the high pressure liquid chromatograph (HPLC), uses TFA/H 2O (volume ratio 0.5%) and CH 3CN/H 2The mixed solvent wash-out of O (volume ratio 0.5%) is collected elutriant, determine required product with mass spectrum (TOF-MS) after, lyophilize, the gained solid detects with HPLC and TOF-MS again, determines that its structural formula is errorless, is HOOC-T NaT φT φ 'T Py-8T Py-5-R 2-Pc;
6) with the mbha resin be start element,, handle 3 times that each 3 minutes is that 1: 1 DMF/DCM washes 3 times with the 2mL mol ratio respectively then, after the 2mL pyridine is washed twice, is A with base with 2mL trifluoroacetic acid (TFA) with after the methylene dichloride swelling 1Peptide nucleic acid monomer activate with dehydration catalyst and diisopropyl ethyl amine after, with mbha resin in 125 μ L N, react in the dinethylformamide (DMF), use the DMF washed twice, with capping reagent (diacetyl oxide) handle and washing after, obtain MBHA-A 1Compound;
7) with described MBHA-A 1Compound and base are A 2Peptide nucleic acid monomer carry out dehydration reaction, obtain MBHA-A 1A 2Compound;
8) repeating said steps 7) 3 times, obtain MBHA-A 1A 2A nCompound;
9) Pc is activated with dehydration catalyst and diisopropyl ethyl amine after, with described MBHA-A 1A 2A nCompound reacts, and obtains MBHA-A 1A 2A n-R 2-Pc compound;
10) with described MBHA-A 1A 2A n-Pc compound and trifluoroacetic acid and TFMSA react, and use the ethyl acetate post precipitation, collecting precipitation, and obtaining skeleton symbol is HOOC-A 1A 2A n-R 2The peptide nucleic acid(PNA) molecule B of-Pc;
Above-mentioned unpurified peptide nucleic acid nano heterojunction molecule B is dissolved among the 2mL DMF, is injected in the high pressure liquid chromatograph (HPLC), use TFA/H 2O (volume ratio 0.5%) and CH 3CN/H 2The mixed solvent wash-out of O (volume ratio 0.5%) is collected elutriant, determine required product with mass spectrum (TOF-MS) after, lyophilize, the solid that obtains detects with HPLC and TOF-MS again, determines that its structural formula is errorless, is HOOC-A 1A 2A n-R 2-Pc.
11) be HOOC-T with described skeleton symbol NaT φT φ 'T Py-8T Py-5-R 2The peptide nucleic acid(PNA) molecule A of-Pc is dissolved among the DMF of 1mL, after the activation of dehydration catalyst and diisopropyl ethyl amine, in DMF, react 30 minutes with 1mL PDI after, adding is HOOC-A with dehydration catalyst and diisopropyl ethyl amine activatory skeleton symbol again 1A 2A n-R 2The peptide nucleic acid(PNA) molecule B reaction of-Pc is after 30 minutes, and HPLC separates, and obtains described Pc-R 2-CONH-AAAAA-CONH-R 1-PDI-R 1-NHCO-T NaT φT φ 'T Py-8T Py-5-NHCO-R 2-Pc has the peptide nucleic acid(PNA) molecule of PDI and Pc for another covalent coupling;
12) being dissolved in by 50 μ L water and 50 μ L concentration by the peptide nucleic acid(PNA) molecule of PDI and Pc the covalent coupling that obtains in the described step 11) is in the mixed solution formed of the 200mM/L NaCl aqueous solution, place 37 ℃ incubator to cultivate 2 hours, obtain the described PNA of formula I and be-NHCO-Tn-NHCO-R 2The peptide nucleic acid nano heterojunction 2 of-Pc.
In described step 1)-step 11), described reaction is reaction with same mole, and in the step 1), described base is T as described 1The mol ratio of peptide nucleic acid monomer, dehydration catalyst, diisopropyl ethyl amine and 4-toluene hydrogen amine (MBHA) resin be 1: 1: 1: 1.In each reactions steps, the consumption of organic solvent is as the criterion with complete solubilizing reaction thing; Described dehydration catalyst is benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate.
This peptide nucleic acid nano heterojunction 2 can prepare biological organic photovoltaic battery according to ordinary method, and its preparation method is as follows:
Handling on the clean ito glass surface, after spin coating one layer thickness is the PSS/PEDOT (the poly-p styrene sulfonic acid of poly-dioxoethyl thiophene) of 40nm, 150 ℃ of dryings after 0.5 hour, be spin-coated on this PSS/PEDOT layer in the mixture with described peptide nucleic acid nano heterojunction 2 and oil of mirbane composition, obtain the active coating (thickness is 100nm) of biological organic photovoltaic battery, after 1 hour, is 5 * 10 in vacuum tightness in 100 ℃ of dryings -5Under the condition of Pa, evaporation one layer thickness is the metal aluminium electrode of 150nm, obtains described biological organic photovoltaic battery.
Described base is T NaThe peptide nucleic acid(PNA) of (R is H) is to get according to the method preparation that embodiment 5 provides.
Described base is T φ(R=-CH 3) peptide nucleic acid(PNA) be the method preparation that provides according to embodiment 5 and getting.
Described base is T Py-8The peptide nucleic acid(PNA) of (R is H) is to get according to the method preparation that embodiment 5 provides.
Described base is T φ '(R=-CH 3) peptide nucleic acid(PNA) be to be prepared as follows and to get:
Figure BSA00000185341800171
With compound 2 φ '(140mg, 0.5mmol) and HBTU (170mg 0.5mmol) is mixed in 1 milliliter N, in the dinethylformamide (DMF), adds N, and (DIEA 0.5mmol) stirs after 30 minutes the N-diisopropyl ethyl amine, adds compound 3 φ '(120mg, 0.5mmol) 1 milliliter of DMF solution, after the stirring at room 3 hours, pour in 20 ml waters, collecting precipitation is after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, and using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, and obtains product compound 4 φ '(210mg, 0.42mmol), productive rate 81%.With compound 4 φ 'Being dissolved in by volume ratio is in 5 milliliters of the mixed solvents formed of 1: 1 methylene dichloride and ethanol, adds the sodium hydroxide of 1 mmole, stirring at room 2 hours, and the back is with the neutralization of 1N hydrochloric acid, and the pH value of transferring solution is removed organic solvent and is obtained compound 5 to 3-5 φ ', productive rate is 100%.This compound 5 φ 'Nuclear-magnetism to detect data as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 12.99 (s, 1H ,-OH), 11.49 (s, 1H ,-NH), 7.29 (s, 1H ,-ph-H), 6.71 (s, 1H ,-ph-H), 4.55 (s, 2H ,-CH 2), 3.85 (s, 3H ,-CH 3), 3.80 (s, 3H ,-CH 3), 3.0 (d, 2H, J=5.6Hz ,-CH 2), 2.5 (m, 3H ,-CH 2), 2.1 (s, 1H ,-CH 2), 1.37 (s, 9H ,-Boc-H); 13CNMR (DMSO-d 6, 100MHz) δ: 169.3,169.1,160.7,155.4,150.2,137.7,136.7,132.8,127.6,125.3,114.2,80.5,56.2,54.8,54.2,52.8,49.8,42.3,28.6; ESI-MS m/z (%): 479.0 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the uracil of benzo shown in formula III peptide nucleic acid monomer (compound 5 φ ').
Wherein, described compound 2 φ 'Be to be prepared as follows and to get:
Figure BSA00000185341800172
1) under 20 ℃ heating condition, with dimethoxy benzo uridylic (4g, 18mmol, compound 1 φ ') be dissolved among 90 milliliters the DMSO, (3.6g, 90mmol), under 20 ℃ heating condition, stirring reaction obtained reaction soln (A) after 1 hour to add NaOH.2) (2.5g 18mmol) is dissolved among 5 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 20 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 12 hours.Reaction solution is poured in 150 milliliters of ethyl acetate, collected the precipitation obtain, precipitation is dissolved in 100 ml waters, the pH value of transferring solution with 6N hydrochloric acid is to pH=7, filters to obtain 1.6 and restrain (7.2mmol) compounds 1 φ ', transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 0.81 gram (2.9mmol) product compound 2 φ ', productive rate 16%.The nuclear-magnetism structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 12.94 (s, 1H ,-OH), 11.36 (s, 1H ,-NH), 7.29 (s, 1H ,-ph-H), 6.71 (s, 1H ,-ph-H), 4.53 (s, 2H ,-CH 2), 3.85 (s, 3H ,-CH 3), 3.80 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 169.4,161.6,149.8,137.2,136.4,132.1,126.8,115.3,113.2,56.2,54.3,41.3; EI-MS m/z (%): 280.1 (M, 100%).As from the foregoing, this compound structure is correct, is the intermediate (compound 2 of the peptide nucleic acid monomer of benzo uracil shown in the formula I φ ').Wherein, used reactant compound 1 in the described step 1) φ 'Be to be prepared as follows and to get: with commercially available 2-amino-4,5-dimethoxy-phenylformic acid (5.92g, 30mmol) and urea (18g, 300mmol) be blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight cool to 100 ℃, add 150mL water, stir after 30 minutes, filter, collecting precipitation obtains described compound 1 φ '(5.8g, 26.1mmol), productive rate 87%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.36 (s, 1H ,-NH), 11.21 (s, 1H ,-NH), 7.29 (s, 1H ,-ph-H), 6.71 (s, 1H ,-ph-H), 3.85 (s, 3H ,-CH 3), 3.80 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 161.6,149.8,137.2,136.4,132.1,126.8,115.3,113.2,56.2,54.3; GCT-MS m/z (%): 222.2 (M, 100%).As from the foregoing, this compound structure is correct, is described compound 1 φ '
Described base is T Py-5The peptide nucleic acid(PNA) of (R is H) is a compound 11, is to be prepared as follows and to get:
Figure BSA00000185341800181
1) with intermediate (compound 6) (111mg, 0.5mmol), HBTU (224.4mg 0.66mmol) is mixed in 3 milliliters N, in the dinethylformamide (DMF), adds N, the N-diisopropyl ethyl amine (DIEA, 0.66mmol).Stir after 40 minutes, add compound 7 (144mg, 3 milliliters of DMF solution 0.66mmol), after the stirring at room 5 hours, pour in 30 ml waters, collecting precipitation is after ethyl acetate washing, extraction, the organic layer anhydrous magnesium sulfate drying, the oily matter of removing behind the organic solvent is crossed post with silica gel, and using by volume ratio is the mixed solution drip washing that 10: 1 methylene dichloride and ethyl acetate are formed, and obtains product compound 10 (188mg, 0.42mmol), productive rate 84%.2) product compound 10 being dissolved in volume ratio is in 1: 16 milliliters of the mixed solvents of being made up of methylene dichloride and ethanol, the sodium hydroxide that adds 1.2 mmoles, stirring at room 3 hours, the back neutralizes with 1N hydrochloric acid, the pH value of regulator solution is to 3-5, remove organic solvent and obtain compound 11, productive rate is 100%.It is as follows that the nuclear-magnetism of this compound 11 detects data: 1H-NMR (400MHz, DMSO-d 6) δ: 13.08 (s, 1H ,-OH), 11.49 (s, 1H ,-NH), 8.71 (d, 1H, J=8.0Hz ,-pyH), 8.28 (d, 1H, J=8.0Hz ,-pyH), 7.28 (q, 1H, J=4.8 and 6.8Hz ,-pyH), 6.5 (s, 1H ,-NH), 4.55 (s, 2H ,-CH 2), 3.85 (s, 2H ,-CH 2), 3.0 (m, 2H ,-CH 2), 2.5 (m, 2H ,-CH 2), 1.37 (s, 9H ,-Boc-H); 13C NMR (DMSO-d 6, 100MHz) δ: 169.3,168.5,160.7,154.4,152.2,148.7,138.7,135.2,117.6,115.3,80.5,54.2,52.8,49.8,42.3,28.6; ESI-MSm/z (%): 420.0 (M-H -, 100%).As from the foregoing, this compound structure is correct, is the uracil of benzo shown in formula III peptide nucleic acid monomer (compound 11).
Wherein, described midbody compound 6 is to be prepared as follows and to get:
Figure BSA00000185341800191
1) under 20 ℃ heating condition, pyridino-uracil (4g, 24.5mmol, compound 5) is dissolved among 100 milliliters the DMSO, (9.0g, 227.1mmol), under 20 ℃ heating condition, stirring reaction 3 hours obtains reaction soln (A) to add NaOH.2) (3.48g 24.9mmol) is dissolved among 3 milliliters of DMSO, obtains reaction soln (B) with bromoacetic acid.Under 20 ℃ heating condition, reaction soln B is joined among the reaction soln A, after adding, continue to stir 10 hours.Reaction solution is poured in 130 milliliters of ethyl acetate, the precipitation that collection obtains, precipitation is dissolved in 130 ml waters, transfer the pH value of solution to pH=7 with 6N hydrochloric acid, filtration obtains 1.5 gram (9.2mmol) compounds 5, transfer pH value of filtrate to 2 with 6N hydrochloric acid, filter and obtain 0.81 gram (3.7mmol) product compound 6, productive rate 15%.It is as follows that the nuclear-magnetism of this product detects data: 1H-NMR (400MHz, DMSO-d 6) δ: 13.04 (s, 1H ,-NH), 11.48 (s, 1H ,-NH), 8.60 (d, 1H, J=8.0Hz ,-pyH), 8.30 (d, 1H, J=8.0Hz ,-pyH), 7.3 1 (q, 1H, J=4.8 and 6.8Hz ,-pyH), 4.53 (s, 2H ,-CH 2), 3.30 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 169.4,161.9,154.8,151.3,149.4,135.7,118.5,114.2,41.2; GCT-MS m/z (%): 221.0 (M, 100%).As from the foregoing, this compound structure is correct, is the intermediate (compound 6) of the peptide nucleic acid monomer of benzo uracil shown in the formula I.Wherein, used reactant compound 5 pyridino-uracils are to be prepared as follows and to get in the aforesaid method step 1):
With commercially available 2-amino-3-carboxyl-pyridine (4.14g, 30mmol, compound 4) and urea (18g 300mmol) is blended in the round-bottomed flask of a 100mL, 160 ℃ of following heated overnight, cool to 100 ℃, add 50mL water, stir after 30 minutes, filter, collecting precipitation obtain compound 5 (3.5g, 21.5mmol), productive rate 71%.The structural characterization data of this product are as follows: 1H-NMR (400MHz, DMSO-d 6) δ: 11.64 (s, 1H ,-NH), 11.48 (s, 1H ,-NH), 8.60 (d, 1H, J=8.0Hz ,-pyH), 8.30 (d, 1H, J=8.0Hz ,-pyH), 7.31 (q, 1H, J=4.8and 6.8Hz ,-pyH), 3.30 (s, 3H ,-CH 3); 13C NMR (DMSO-d 6, 100MHz) δ: 162.2,154.8,151.3,150.4,135.7,126.5,110.2,20.2; GCT-MS m/z (%): 163.0 (M, 100%).As from the foregoing, this compound structure is correct, is compound 5.

Claims (11)

1. the described compound of formula I general structure,
Figure FSA00000185341700011
(formula I)
In the described formula I general structure, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3
2. according to the described compound of claim 1, it is characterized in that: described compound is to get according to the arbitrary described method preparation of claim 3-6.
3. a method for preparing claim 1 or 2 described compounds comprises the steps:
1) benzo uracil derivative shown in the formula II is reacted with sodium hydroxide in organic solvent, reaction finishes and obtains reaction soln A;
Figure FSA00000185341700012
(formula II)
Among the described formula II, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3
2) bromoacetic acid is dissolved in the organic solvent, obtains reaction soln B, described reaction soln B and described reaction soln A are reacted, obtain claim 1 or 2 described compounds.
4. method according to claim 3 is characterized in that: in the described step 1), described organic solvent is selected from dimethyl sulfoxide (DMSO) and N, at least a in the dinethylformamide, preferred dimethyl sulfoxide (DMSO); The amount ratio of benzo uracil derivative, sodium hydroxide and described organic solvent shown in the described formula II is 1mmol: 2-10mmol: 2-10ml, preferred 1mmol: 3-8mmol: 2-5ml;
Described step 2) in, described organic solvent is selected from dimethyl sulfoxide (DMSO) and N, at least a in the dinethylformamide, preferred dimethyl sulfoxide (DMSO); The amount ratio of described bromoacetic acid and described organic solvent is 1-1.5mmol: 0.1-1ml, preferred 1-1.4mmol: 0.1-0.5ml.
5. according to claim 4 or 5 described methods, it is characterized in that: in the described step 1), temperature is 20-80 ℃, and preferred 20-70 ℃, the time is 10-300 minute, preferred 100-200 minute;
Described step 2) in the reactions steps, temperature is 20-80 ℃, and preferred 20-70 ℃, the time is 5-15 hour, preferred 8-10 hour;
In described step 2) reaction finish after, reaction system is carried out following purification process:
With reaction system and ethyl acetate mixing, collect the precipitation obtain, described precipitation is soluble in water, regulate pH value to 2, obtain claim 1 or 2 described compounds behind the purifying.
6. the compound of peptide nucleic acid monomer shown in the formula III,
(formula III)
In the described formula III, R is-F ,-Cl ,-Br ,-I ,-H ,-CH 3,-CH 2CH 3Or-CH 2CH 2CH 3
7. compound according to claim 6 is characterized in that: described compound is according to claim 8 or the preparation of 9 described methods and get.
8. a method for preparing claim 6 or 7 described peptide nucleic acid monomer compounds comprises the steps:
With claim 1 or 2 described compound and benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate in organic solvent with N, after the N-diisopropylethylamine at room temperature reacts 10-30 minute, add N-tert.-butoxy-carbonyl-1 again, the organic solution of 2-diaminoethanes is at room temperature reacted after 60-240 minute and is obtained product a, described product a at room temperature reacted 30-120 minute with sodium hydroxide in organic liquid mixture after, regulate the pH value to 3-5, obtain claim 6 or 7 described peptide nucleic acid monomer compounds; The organic solution of described N-tert.-butoxy-carbonyl-1 is described N-tert.-butoxy-carbonyl-1 is dissolved among the organic solvent b and gets.
9. method according to claim 8 is characterized in that: described organic solvent a and organic solvent b all are selected from N, at least a in dinethylformamide and the dimethyl sulfoxide (DMSO), preferred N, dinethylformamide; Described organic liquid mixture is to be that 1: 1 methylene dichloride mixes with ethanol and gets by volume ratio;
Described claim 1 or 2 described compounds, benzotriazole-N, N, N ', N '-tetramethyl-urea hexafluorophosphate, organic solvent a, N, N-diisopropylethylamine, N-tert.-butoxy-carbonyl-1,2-diaminoethanes, organic solvent b: the amount ratio of sodium hydroxide is 1mmol: 1-1.2mmol: 0.5-1ml: 1-1.2mmol: 1-1.2mmol: 0.5-1ml: 1-1.2mmol, preferred 1mmol: 1.1mmol: 1ml: 1.1mmol: 1.1mmol: 1ml: 1.1mmol.
10. claim 6 or 7 described peptide nucleic acid monomer compounds have application in the nucleic acid bionic material of electric potential gradient in preparation.
11. with claim 6 or 7 described peptide nucleic acid monomer compounds is the peptide nucleic acid(PNA) that monomer prepares.
CN 201010224200 2010-07-12 2010-07-12 Benzo uracil PNA (Peptide Nucleic Acid) monomer and intermediate thereof as well as preparation methods and applications of benzo uracil PNA and intermediate thereof Pending CN101967128A (en)

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