Optimize Sonogashira Reaction As A Potential Bioorthogonal Reaction And Design And Synthesize Nucleotide Probes Containing Bioorthogonal Groups

Objective:Bioorthogonal reaction refers to a kind of chemical reaction that have high selective in the organism and not react with the biological molecules.It is widely used to detect and label biomacromolecules due to its mild reaction conditions,high selectivity and good biological compatibility.The Sonogashira reaction refers to the cross-coupling reaction palladium-catalyzed between terminal alkylation and the halides of carbon sp2.With the continuous exploration in recent years,the reaction conditions become more mild and even can be carried out efficiently in the aqueous phase system.Non-natural nucleoside,such as BrdU,5-bromodeoxyuracil nucleoside,can be inserted into newly synthesized DNA during the S phase of cell proliferation,and then proliferated cells are detected by antibodies.However,the specificity of antibody detection in this method is not high,requires repeated elution,and the operation is complex,and cells need to be denatured with hydrochloric acid,so it cannot be stained together with other methods,we want to explore a now methods to replace this method.BrdU is a type sp2 carbon halide and a potential substrate for the Sonogashira reaction.Therefore,we hope to explore a Bioorthogonal Sonogashira reaction condition to achieve the combination of BrdU and fluorescence probe containing terminal alkynes to directly label BrdU,so as to detect cell proliferation instead of an antibody reaction.Methods:(1)Synthesis of fluorescent dyes:Fluorescence probes with alkynes terminal were synthesized for Sonogashira reaction with BrdU.Two kinds of probes,rhodamine and coumarin probes,were synthesized,respectively,with the terminal terminal of aromatic alkynes and aliphatic alkynes,the fluorescence spectra were measured.(2)Optimization of the Sonogashira reaction:In order to make the Sonogashira reaction an orthogonal biological reaction,we need to optimize it so that it can be carried out efficiently in the mild water phase.The rhodamine type terminal aryne probe,probe 1 reacting with BrdU,was used as the template reaction to optimize the base and catalytic system to obtain the best reaction conditions.(3)Probes reaction with BrdU:Verify the applicability of the best conditions.The best reaction conditions were used for the Sonogashira reaction between other probes and BrdU.The relationship between reaction yield and structure was compared.Results:(1)Synthesis of fluorescent dyes:Four fluorescent probes were synthesized:rhodamine probe 1 which the end is the alkynes,rhodamine probe 3 which the end is the alkynes,coumarin probe 4 which the end is the alkynes,and coumarin probe 5 which the end is the alkynes.By measuring the fluorescence spectra of four kinds of fluorescent dyes,it was found that the maximum excitation and emission wavelength of rhodamine type were all above 500 nm,and the maximum excitation and emission wavelength of coumarin type probe were all above 400 nm.Moreover,the alkyne probes containing aromatic terminal alkynes have longer maximum generating wavelength than the alkyne probes containing fatty terminal alkynes.Under ultraviolet light,rhodamine probes are pink and coumarins are blue-green.(2)Optimization of Sonogashira reaction:the rotamine terminal aryne probe,namely probe 1 reacting with BrdU,was used as the template reaction.The required base was screened first,and it was found that the water-soluble base n-Bu4N+OH+ had the highest reactivity.The catalytic system was screened to find the ligand SS-Phos,which could be complexated with K2PdCl4 to achieve better catalysis.The best reaction conditions were as follows:0.2eq K2PdCl4 as the target catalyst,0.2eq SS-Phos as the ligand,the solvent was ethanol:water=1:1,and 1.5eq sodium ascorbate was added to prevent oxidation,free of CuI.(3)Probe reaction with BrdU:Other probes reacted with BrdU under the best conditions obtained by screening.The results show that this reaction condition has wide applicability.Comparing the activity of rhodamine probe and coumarin probe,rhodamine probe was better than coumarin probe.The reactivity of aromatic terminal alkynes of rhodamine is better than that of aliphatic terminal alkynes,but the reactivity of aromatic terminal alkynes of coumarin is not as good as that of aliphatic terminal alkynes,which may be related to the poor solubility of aromatic terminal alkynes of coumarin.Conclusion:The optimized Sonogashira reaction condition is:0.2 eq K2PdCl4 as the target catalyst,0.2 eq SS-Phos as the ligand,the solvent was ethanol:water=1:1,and 1.5 eq sodium ascorbate antioxidant was added,free of CuI.This reaction can be used as a bioorthogonal reaction to identify the fluorescence probe containing alkyne group and BrdUObjective:Small molecule probes are widely used for labeling and separation of biomacromolecules.The labeling of Biotin is widely used because it can be detected and separated by its reaction with the affinity.Biotin-16-UTP is an industrial reagent for gene capture,which can be inserted into a specific sequence and paired with the target sequence,and then the target gene fragment can be extracted by binding with biotin by magnetic beads whose surface were attached with the avidin.However,the difficulty in synthesis of nucleotides containing triphosphate groups is well known in the field of organic synthesis,and the modified nucleoside of Biotin-16-UTP contains super-large chain hydrocarbons and biotins,resulting in the extremely poor solubility of this molecule,and the difficulty in synthesis is self-evident.In addition,the problem of low specificity between biotin and avidin affects its popularization.Bioorthogonal reaction refers to a kind of chemical reaction that have high selective in the organism and not react with the biological molecules.It is widely used to detect and label biomacromolecules due to its mild reaction conditions,high selectivity and good biological compatibility.We hope to use Bioorthogonal reaction to replace the combination of biotin and avidin and improve specificity.Moreover,Bioorthogonal reactiongroups are less difficult to synthesize than Biotin-16-UTP because the groups of Bioorthogonal reactions are generally smaller and easier to be inserted into the target sequence with high biological compatibility.Methods:(1)The synthesis of Biotin-16-UTP:We expect to obtain a reasonable and feasible synthesis method of Biotin-16-UTP,and the explored synthesis route can also guide the synthesis of probes containing Bioorthogonal groups in the next step.(2)Synthesis of UTP containing Bioorthogonal groups:According to the route of synthesis of Biotin-16-UTP,various uridine containing terminal alkynes/azide Bioorthogonal groups of different lengths were designed and synthesized.(3)Explore triphosphorylation method:We want to explore a triphosphorylation method with cheap raw materials and high reaction yield,which is used for triphosphorylation of Biotin-16-uridine and uridine with Bioorthogonal groups.Results:(1)Synthesis of Biotin-16-UTP:two synthesis routes were designed.The first route was to take uridine triphosphate as the starting material,and after iodization,Heck reaction with allyl amine protected by trifluoroacetyl,and after detrifluoacetyl reaction,it was condensed into biotin-16-utp with the long chain of activated ester containing Biotin.However,this method did not get the target product,so route 2 was designed.Iodinuridine was used as the raw material,conduct Heck reaction with allyl amine protected by trifluoroacetyl.Hydroxyl group was protected by TBS and then further reduced the polarity.In this way,triphosphorylation is introduced finally to increase the stability of the intermediates,greatly reduce the polarity of the intermediates and facilitate purification and separation.(2)Synthesis of UTP containing the Bioorthogonal group:According to the method of synthesizing Biotin-16-UTP route 2,acrylaminidine with hydroxyl protected by TBS was obtained as the key intermediate,and then it was directly condensed with carboxylic acid containing terminal alkynyl or azide of different lengths.The terminal azide uridine with the connecting arm length of 8 and 10 and the terminal alkynyl uridine with the connecting arm length of 8,10 and 15 were obtained.(3)Exploration of triphosphate method:the triphosphorylation of Biotin-16-uridine and the alkynyl uridine terminal with the connecting arm length of 10 was successfully achieved by salicylphosphine chloride and pyrophosphate.Because pyrophosphate is expensive and unstable,in order to popularize the triphosphorylation method,we made PPN to replace pyrophosphate and successfully achieved triphosphorylation.Conclusion:By first synthesizing Biotin-16-uridine and then triphosphorylation,the synthesis difficulty of Biotin-16-UTP can be greatly reduced,and a variety of UTP probes containing Bioorthogonal groups are synthesized by the same method.PPN can replace pyrophosphate and achieve better triphosphorylation with salicylphosphatidyl chloride.