Compound based on cascade sulfydryl/bromomaleimide Michael addition and application thereof
阅读说明：本技术 基于级联巯基/溴代马来酰亚胺迈克尔加成的化合物及其应用 (Compound based on cascade sulfydryl/bromomaleimide Michael addition and application thereof ) 是由 黄智豪 施秋楠 刘宝磊 张正彪 于 2020-07-06 设计创作，主要内容包括：本发明涉及一种基于级联巯基/溴代马来酰亚胺迈克尔加成的化合物及其应用。本发明的数字编码大分子,其结构式如式(I)所示：<Image he="240" wi="700" file="DDA0002571403940000011.GIF" imgContent="drawing" imgFormat="GIF" orientation="portrait" inline="no"></Image>其具有单一的分子量,精确的序列结构,通过迭代序列指数增长策略合成所得。以上化合物可利用串联质谱实现简单、快速的测序,能够广泛应用于信息存储和防伪领域。(The invention relates to a compound based on cascade sulfydryl/bromomaleimide Michael addition and application thereof. The structural formula of the digital coding macromolecule is shown as the formula (I): the molecular weight-variable peptide has a single molecular weight and an accurate sequence structure, and is synthesized by an iterative sequence exponential growth strategy. The compounds can realize simple and rapid sequencing by utilizing tandem mass spectrometry, and can be widely applied to the fields of information storage and anti-counterfeiting.)
1. A digitally encoded macromolecule having the structural formula shown in formula (I):
wherein R in each repeating unit is independently selected from substituted or unsubstituted C6-C14Aryl radical, C2-C12Alkyl or C3-C6An amide group obtained by opening a ring of cysteine thiolactone with a primary amino group; wherein the substituents on the substituted aryl are selected from C1-C8Alkyl radical, C1-C8Alkoxy or halogen;
n＝2m-1, wherein m is 2 or 3.
2. The digitally encoded macromolecule of claim 1, wherein: r in each repeating unit is independently selected from benzyl, hexyl, p-isopropylphenyl and
3. a method of synthesizing a digitally encoded macromolecule of claim 1 or 2, comprising the steps of:
synthesizing the digital coding macromolecule shown in the formula (I) by using an iterative exponential growth method, wherein the iteration number is m, and the method comprises the following steps:
the first generation reaction comprises the steps of respectively removing furyl from monomers shown in a formula (1) and carrying out sulfhydrylation to respectively obtain a compound shown in a formula (2) and a compound shown in a formula (3), carrying out first sulfydryl/bromomaleimide Michael addition reaction on the compounds shown in the formula (2) and the formula (3), and then carrying out second sulfydryl/bromomaleimide Michael addition reaction on the compounds with small molecular compounds containing sulfydryl to obtain a first generation product;
the mth generation reaction is that the mth-1 generation product is subjected to furyl removal and sulfhydrylation respectively, then is subjected to first sulfydryl/bromomaleimide Michael addition reaction, and then is subjected to second sulfydryl/bromomaleimide Michael addition reaction with a sulfydryl-containing small molecular compound;
the m generation product is a digital coding macromolecule shown in formula (I), and the polymerization degree n is 2m-1, wherein m is 2 or 3;
wherein the thiol-group containing small molecule compounds used in each generation of reaction are independently selected from compounds of formula RSH, wherein R is as defined in claim 1 or 2; the structural formulas of formulas (1) to (3) are as follows in sequence:
4. the method of claim 3, wherein: in each generation of reaction, in the first sulfydryl/bromomaleimide Michael addition reaction process, the molar ratio of sulfydryl to bromomaleimide in reactants is 1.05-1.3: 1.
5. The method of claim 3, wherein: the reaction for removing furyl is carried out in an organic solvent at the temperature of 110-120 ℃.
6. The method of claim 3, wherein: the reaction for effecting the thiolation is carried out in an acidic organic solvent at 55-65 ℃.
7. The method of claim 3, wherein: the first mercapto/bromomaleimide Michael addition reaction is carried out in an organic solvent containing triethylamine, and the reaction temperature is 20-30 ℃; the molar ratio of the reactants for removing furyl to triethylamine is 1: 3.0-4.0.
8. The method of claim 7, wherein: the second sulfydryl/bromomaleimide Michael addition reaction is carried out in an organic solvent containing triethylamine, and the reaction temperature is 20-30 ℃; the molar ratio of the product of the second sulfydryl/bromomaleimide Michael addition reaction to triethylamine is 1: 4.7-6.0.
9. Use of a digitally encoded macromolecule as claimed in claim 1 or claim 2 for anti-counterfeiting or as a molecular scale information storage carrier.
10. Use according to claim 9, characterized in that: when applied, the method comprises a step of establishing a standard and a step of reading information,
the step of establishing the standard comprises the steps of establishing a reference standard corresponding to the mass spectrum spectrogram of the known anti-counterfeiting information or the known information to be expressed and the known digital coding macromolecules;
and the step of reading the information comprises sequencing unknown digital coding macromolecules through a tandem mass spectrometry, comparing an obtained mass spectrum spectrogram with the reference standard, and carrying out anti-counterfeiting identification or obtaining the information expressed by the unknown digital coding macromolecules according to the read information.
Technical Field
The invention relates to the technical field of compound synthesis, in particular to a compound based on cascade sulfydryl/bromomaleimide Michael addition and application thereof.
Background
The sequence arrangement of different monomers in the polymer can be used not only for information storage and transmission (such as DNA, RNA), but also has a decisive influence on the topology and performance of the polymer (such as different structures and functions of proteins). Manually and precisely controlling the sequence distribution in a polymer chain, and chemically synthesizing a digital coding macromolecule, which is an important research direction for precisely synthesizing macromolecules. The method is characterized in that a more complex and precise monodisperse sequence control polymer, namely a digital coding macromolecule, such as biological macromolecules (DNA, RNA, protein and the like) is synthesized by a chemical method, and the monodisperse sequence control polymer is applied to the fields of information storage, catalysis, drug research and development and the like, still has great challenges and wide development space, and is an important research field which is very worth paying attention.
The digital coding macromolecule is a macromolecule which codes different specific chemical structures into different specific numbers (0,1 …), and by means of specific chemical synthesis, digital information for checking the realization that a sequence precise macromolecule is written into a macromolecule chain, and digital information can be read by using a certain method (such as mass spectrometry). The storage process of the digital coding macromolecule as an information storage carrier comprises the following steps: obtaining single molecular weight high molecular weight with accurate sequence through a high molecular precision synthesis strategy so as to realize information writing; furthermore, the sequence of the macromolecule is analyzed by means of modern macromolecule sequencing means to obtain the information represented by the sequence, so as to read the information. Wherein, the synthesis strategy of the digital coding macromolecule comprises solid phase carrier synthesis, liquid phase carrier synthesis, multi-component reaction and the like; the macromolecular sequencing means is mainly a tandem mass spectrometry technology. At present, the digital coding macromolecules developed are polytriazole amides, polyphosphodiesters, poly (alkoxyamine amides), poly (alkoxyamine phosphodiesters) and polyurethanes, among others.
However, digitally encoded macromolecules have thus far remained at the theoretical level of research and have not been generated with true molecular-scale information storage devices. The reasons for this are two: (1) the synthesis process is limited. The synthesis of the digital coding macromolecule mainly adopts a step-by-step growth mode represented by solid-phase synthesis, and although the monomer sequence is easy to control, the growth speed is slow and the scale is difficult. Although the exponential growth mode is easy to scale up, has few synthesis steps and fast monomer growth, the sequence structure is difficult to control. (2) Sequencing is difficult. Tandem mass spectrometry is the main sequencing means for the synthesis of macromolecules, however, sequencing of tandem mass spectrometry of most macromolecules with single molecular weight is difficult. Because the breakage of chemical bonds in molecules has no selectivity, the tandem mass spectrograms are often disordered and cannot realize simple and rapid sequencing.
The documents "Combining organic Chain-End purification and thio-maleic electrochemical Coupling" Engineering copolymer by an Iterative purification growth, Angew. chem. int. Ed.2017,56, 13612-13617 "report an oligomer and its synthesis. By using the thiol-maleimide coupling combined iterative exponential growth strategy reported in the text, the precise macromolecule with high repeating unit number can be rapidly constructed in a large scale. However, the method can only prepare precise macromolecules with single molecular weight, the sequence control is relatively weak, the breaking selectivity of chemical bonds in the obtained structure is low, and the simple and rapid sequencing is difficult through tandem mass spectrometry. The document "Binary tree-amplified digital dendrimer" reports a polymer with a topological structure, and a digital coding macromolecule with a complex dendritic topological structure can be obtained by using a divergent propagation method in the document, but sequence information in the document can be read only by means of a matrix and computer-aided calculation, and cannot meet the requirement of information suggestion rapid reading.
Therefore, how to prepare compounds capable of efficient information writing (synthesis) and easy information reading (sequencing) is currently the focus of research.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a compound based on cascade sulfydryl/bromomaleimide Michael addition and application thereof.
It is a first object of the present invention to disclose a compound of formula (I) which is useful as a digitally encoded macromolecule:
wherein R in each repeating unit is independently selected from substituted or unsubstituted C6-C14Aryl radical, C2-C12Alkyl or C3-C6An amide group obtained by opening a ring of cysteine thiolactone with a primary amino group; wherein the substituents on the substituted aryl are selected from C1-C8Alkyl radical, C1-C8Alkoxy or halogen;
n＝2m-1, wherein m is 2 or 3.
n represents the number of repeating units.
Preferably, substituted C6-C14Aryl is C1-C8Alkyl-substituted phenyl.
Preferably, R in each repeating unit is independently selected from benzyl, hexyl, p-isopropylphenyl andsince n is 3 or 7, the R groups in each repeating unit in formula (I) may be different.
Preferably, the structure of the digitally encoded macromolecule is as shown in 4mer-I (encoding 010), 4mer-II (encoding 101), 8mer-I (encoding 0101010), 8mer-II (encoding 1012101) or 8mer-III (encoding 0103010):
it is a second object of the invention to provide a method of synthesizing a digitally encoded macromolecule of formula (I), comprising the steps of:
synthesizing the digital coding macromolecule shown in the formula (I) by using an iterative exponential growth method, wherein the iteration number is m, and the method comprises the following steps:
the first generation reaction comprises the steps of respectively removing furyl from monomers shown in a formula (1) and carrying out sulfhydrylation to respectively obtain a compound shown in a formula (2) and a compound shown in a formula (3), carrying out first sulfydryl/bromomaleimide Michael addition reaction on the compounds shown in the formula (2) and the formula (3), and then carrying out second sulfydryl/bromomaleimide Michael addition reaction on the compounds with small molecular compounds containing sulfydryl to obtain a first generation product;
the mth generation reaction is that the mth-1 generation product is subjected to furyl removal and sulfhydrylation respectively, then is subjected to first sulfydryl/bromomaleimide Michael addition reaction, and then is subjected to second sulfydryl/bromomaleimide Michael addition reaction with a sulfydryl-containing small molecular compound;
the m generation product is a digital coding macromolecule shown in formula (I), and the polymerization degree n is 2m-1, m is 2 or 3;
wherein, the small molecule compound containing sulfhydryl group used in each generation of reaction is independently selected from the compound with the molecular formula of RSH, wherein R is selected from benzyl, hexyl, p-isopropylphenyl or
the structural formulas of formulas (1) to (3) are as follows in sequence:
the reaction route for the first generation reaction is as follows:
preferably, the first generation reaction gives a dimer 2mer-I (code 0) or 2mer-II (code 1) of the following formulae:
furthermore, in each generation of reaction, in the first sulfydryl/bromomaleimide Michael addition reaction process, the molar ratio of sulfydryl to bromomaleimide in the reactant is 1.05-1.3: 1.
Further, the reaction for removing furyl group is carried out in an organic solvent at 110-120 ℃. Preferably, the organic solvent is toluene.
Further, the reaction for effecting the thiolation is carried out in an acidic organic solvent at 55 to 65 ℃. Preferably, the organic solvent is methanol; the reaction is carried out in an inert atmosphere. Preferably, the acidic organic solvent contains concentrated hydrochloric acid.
Further, carrying out the first sulfydryl/bromomaleimide Michael addition reaction in an organic solvent containing triethylamine, wherein the reaction temperature is 20-30 ℃; the molar ratio of the reactants for removing furyl to triethylamine is 1: 3.0-4.0.
Further, carrying out a second sulfydryl/bromomaleimide Michael addition reaction in an organic solvent containing triethylamine, wherein the reaction temperature is 20-30 ℃; the molar ratio of the product of the first thiol/bromomaleimide Michael addition reaction to triethylamine is 1: 4.7-6.0.
The two mercapto/bromomaleimide Michael addition reactions of the present invention are all carried out in an inert atmosphere.
The third purpose of the invention is to disclose the application of the digital coding macromolecule shown in the formula (I) in anti-counterfeiting or as a molecular information storage carrier.
Further, when the method is applied, the method comprises the steps of establishing a standard and reading information,
the step of establishing the standard comprises the steps of establishing a reference standard corresponding to the mass spectrum spectrogram of the known anti-counterfeiting information or the known information to be expressed and the known digital coding macromolecules;
and the step of reading the information comprises sequencing unknown digital coding macromolecules through a tandem mass spectrometry, comparing an obtained mass spectrum spectrogram with the reference standard, and carrying out anti-counterfeiting identification or obtaining the information expressed by the unknown digital coding macromolecules according to the read information.
By the scheme, the invention at least has the following advantages:
the digital coding macromolecule provided by the invention utilizes the Michael addition cascade reaction of sulfydryl/bromomaleimide in the preparation process, the preparation process is simple, the synthesis process is efficient, no metal catalysis is adopted, the exponential growth of a monomer and the accurate control of a side chain sequence can be realized simultaneously, the selectable range of a side chain group is large, and the whole compound has a single molecular weight.
The digital coding macromolecule can be quickly constructed with accurate sequence by combining the orthogonal deprotection reaction of sulfydryl and bromomaleimide in the preparation process and utilizing an iterative sequence exponential growth mode.
The digital coding macromolecule contains a succinimide disulfide structure formed by a sulfydryl/maleimide cascade reaction, and the succinimide disulfide structure enables the digital coding macromolecule to be subjected to simple and rapid tandem mass spectrometry sequencing. Sequencing results show that the digital coding macromolecules can generate clear and predictable tandem mass spectrum fragmentation patterns, and simple and quick information reading is realized. By utilizing the structural characteristics of the digital coding macromolecules, the anti-counterfeiting liquid can be applied to the anti-counterfeiting field or can be used as a molecular information storage carrier.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is a nuclear magnetic spectrum of a monomer prepared according to the present invention;
FIG. 2 is a nuclear magnetic spectrum of 2mer-I in example 1 of the present invention;
FIG. 3 is a nuclear magnetic spectrum of 2mer-II in example 1 of the present invention;
FIG. 4 is a nuclear magnetic spectrum of 4mer-I in example 2 of the present invention;
FIG. 5 is a macromolecular mass spectrum and size exclusion chromatogram of 4mer-I in example 2 of the present invention, wherein (a) is an SEC spectrum and (b) is a MALDI-TOF MS spectrum;
FIG. 6 is a nuclear magnetic spectrum of 4mer-II in example 2 of the present invention;
FIG. 7 is a macromolecular mass spectrum and size exclusion chromatogram of 4mer-II in example 2 of the present invention, wherein (a) is an SEC spectrum and (b) is a MALDI-TOF MS spectrum;
FIG. 8 shows the result of tandem mass spectrometry of 4mer-I in example 2 of the present invention;
FIG. 9 shows the result of tandem mass spectrometry of 4mer-II in example 2 of the present invention;
FIG. 10 is a nuclear magnetic spectrum of 8mer-I in example 3 of the present invention;
FIG. 11 is a macromolecular mass spectrum and size exclusion chromatogram of 8mer-I in example 3 of the present invention, wherein (a) is an SEC spectrum and (b) is a MALDI-TOF MS spectrum;
FIG. 12 is a nuclear magnetic spectrum of 8mer-II in example 3 of the present invention;
FIG. 13 is a macromolecular mass spectrum and size exclusion chromatogram of 8mer-II in example 3 of the present invention, wherein (a) is an SEC spectrum and (b) is a MALDI-TOF MS spectrum;
FIG. 14 is a nuclear magnetic spectrum of 8mer-III in example 3 of the present invention;
FIG. 15 is a macromolecular mass spectrum and size exclusion chromatogram of 8mer-III in example 3 of the present invention, wherein (a) is an SEC spectrum and (b) is a MALDI-TOF MS spectrum;
FIG. 16 shows the result of tandem mass spectrometry of 8mer-I in example 3 of the present invention;
FIG. 17 shows the result of tandem mass spectrometry of 8mer-II in example 3 of the present invention;
FIG. 18 shows the results of tandem mass spectrometry of 8mer-III in example 3 of the present invention;
FIG. 19 is a fragmentation mechanism in tandem mass spectrometry for examples 2-3 of the present invention;
FIG. 20 shows the fragment naming rules in tandem mass spectrometry according to examples 2-3 of the present invention.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
The raw materials used in the invention are all known products and are obtained by purchasing commercial products.
The starting monomer structure for synthesizing the digital coding macromolecule is shown as a formula (1), and the reaction route is as follows:
the specific synthesis steps are as follows:
1. preparation of Small molecule M-1
To a 1.0L three necked round bottom flask equipped with a condenser was added maleimide (30.0g, 0.31mol) and 500mL CCL4. The mixture was stirred at room temperature under argon atmosphere. Bromine (18.0mL, 0.35mol) was then added and the mixture was refluxed at 78 ℃ for about 1 h. After cooling to room temperature, the crude product was crystallized and filtered. The filter cake was washed with 2X 100mL of Petroleum Ether (PE) and dried under vacuum at 25 deg.C overnight to give crude M-1 as yellow crystals without further purification (73.1g, 92.1% yield).
2. Preparation of Small molecule M-2
M-1(73.0g, 0.28mol) was dissolved in dry THF (700mL) and cooled to 0 ℃ under argon. Triethylamine (TEA, 43.4mL, 0.31mol) was dissolved in anhydrous THF (200mL) and slowly added dropwise to the reaction system at 0 ℃ for 15 minutes or more. Subsequently, the mixture was allowed to warm to room temperature for reaction. After completion of the reaction, the filter cake was filtered, washed with 3X 200mL of Ethyl Acetate (EA), and the combined filtrates were concentrated. The residue was redissolved in 500mL EA, then washed with 3X 250mL brine. Anhydrous Na for organic phase2SO4Drying, concentration and drying of the crystals in a vacuum oven gave M-2 as a yellow solid (41.8g, 83.6% yield).
3. Preparation of Small molecule M-3
A350 mL thick walled pressure flask was charged with M-2(12.0g, 68.2mmol), furan (46.4g, 0.68mol) and dehydrated ether (18.0 mL). The flask was sealed and the mixture was stirred at 78 ℃ for 4 days. After cooling to room temperature, the mixture was filtered. The filter cake was washed with 3X 50mL of Petroleum Ether (PE) and the product was oven dried overnight in vacuo at 25 ℃ to afford M-3 as a light colored powder (14.2g, 85.3% yield).
4. Preparation of Small molecule S-1
6-bromo-1-hexanol (26.0g, 0.14mol) was dissolved in 500mL of DMF. Potassium thioacetate (32.8g, 0.29mol) was then added and stirred at room temperature overnight. The reaction mixture was diluted with 500mL EA and saturated NH 3X 500mL4Aqueous Cl is washed to remove DMF. The upper phase was collected and washed with anhydrous Na2SO4And (5) drying. Evaporation of the solvent gave S-1 as a colorless oily liquid (25.0g, 98.8% yield).
5. Preparation of a monomer represented by the formula (1)
To a 1.0L three-necked round bottom flask was added S-1(25.0g, 0.14mol), triphenylphosphine (55.8g, 0.21mol) and 800mL anhydrous THF. M-3(45.0g, 0.18mol) was then added to the above solution at-10 ℃. Diisopropyl azodicarboxylate (DIAD, 43.0g, 0.21mol) was slowly added dropwise to the mixture at-10 ℃ under an argon atmosphere. The mixture was stirred at room temperature for a further 2 hours, after which the solvent was evaporated. The residue was purified by column chromatography, eluting with PE/EA (v/v-6/1 to 3/1), and recrystallized to give the monomer as a white solid (40.8g, yield 71.5%). The nuclear magnetic spectrum is shown in figure 1.
The monomer represented by formula (1) was characterized as follows:
1H NMR(300MHz,CDCl3,ppm):6.65(s,2H),5.38–5.18(m,2H),3.53(t,J＝9.7Hz,2H),2.92–2.77(m,2H),2.32(s,3H),1.56(dt,J＝19.6,8.9Hz,4H),1.33(s,4H).13C NMR(75MHz,CDCl3,ppm):195.90,173.29,173.21,136.53,136.49,83.09,82.52,55.73,55.22,39.58,30.64,29.26,28.90,28.12,27.15,25.96.