| Oligonucleotides and their analogs are an important class of drugs for the treatment of microbial infections and various genetic diseases.There are two main principles of treatment.First,Oligonucleotide drugs form mRNA-DNA double strands by hybridizing with target mRNA,thereby recruiting endogenous RNase to interact with target mRNA,triggering the destruction and degradation of target mRNA,inhibiting gene transcription and expression of diseased proteins.Second,oligonucleotide drugs can also combine with double-stranded DNA to form a triplet,hindering the interaction between functional groups involved in gene splicing and messenger RNA precursor(pre-mRNA),regulating or changing the messenger RNA precursor(pre-mRNA)-mRNA)gene splicing pattern,inducing the expression of new functional protein isoforms.The induced protein isoforms will not cause pathological changes to the body,so as to achieve the treatment of diseases.However,the application of oligonucleotide drugs has limitations.There are a large number of nucleases in the body,which can hydrolyze the phosphodiester bonds of oligonucleotide drugs,making them ineffective before reaching the target.The cell membrane permeability of nucleotide drugs is poor,and it is difficult to exert curative effect through the cell membrane.In order to solve the above defects,the covalent coupling of oligonucleotide drugs and cell-penetrating peptides can significantly improve the biochemical activity of oligonucleotide drugs.Currently,the solid-phase method is the most common method for preparing oligonucleotides and their derivatives.However,there are many disadvantages in the solid-phase method,the most important of which is that the traditional solid-phase synthesis of Fmoc polypeptides is not compatible with the solid-phase synthesis of oligonucleotides.Peptide synthesis removes side chain protecting groups from amino acids using trifluoroacetic acid,which breaks the glycosidic bonds of adenine and guanine on oligonucleotide fragments.To avoid this problem,scientists spliced peptides using Boc solid-phase synthesis with side-chain protected amino acids that can be removed in an alkaline environment.In this strategy,4-7 hours of ammonia treatment is required to completely remove the side chain protecting groups.However,this step has the following problems.On the one hand,the long-term ammonia water treatment will cause the peptide bond of the polypeptide to break.On the other hand,the guanidine group of arginine will undergo a deguanidinylation side reaction in ammonia water to form ornithine.So far,only a few amino acids can be introduced into peptide-oligonucleotide conjugates by solid-phase synthesis.The Fmoc solid-phase synthesis method is simple to operate and has high synthesis efficiency.It is currently the most commonly used method for peptide synthesis.However,the traditional Fmoc solid-phase synthesis method cannot prepare polypeptide-oligonucleotide conjugates containing functional amino acids such as arginine,histidine,tryptophan,aspartic acid,and glutamic acid.Therefore,it is still very necessary to develop a robust and reliable Fmoc solid-phase synthesis strategy for the preparation of polypeptide-oligonucleotide conjugates.In this thesis,for the first time,Fmoc solid-phase synthesis method was used to prepare polypeptide-oligonucleotide conjugates containing various functional amino acids,which solved the problems existing in the traditional solid-phase synthesis method.Specifically,firstly,amino acids whose side chains are protected by Boc/tBu are assembled on the resin by using Fmoc solid-phase peptide synthesis;then,oligonucleotide fragments are also assembled on the polypeptide chain by solid-phase synthesis;after sequence splicing is completed,Treat the solid phase support with ammonia water for 1 hour to cleave the polypeptide-oligonucleotide conjugate;finally,use 90℃ sodium borate solution to completely remove the amino acid side chain Boc/tBu protecting group.This method is suitable for the synthesis of peptideoligonucleotide conjugates containing arginine,histidine,tryptophan,lysine,aspartic acid and glutamic acid.Compared with previous methods,this technology has the following significant advantages.It does not use trifluoroacetic acid to remove the Boc/tBu protecting group of the side chain of the polypeptide,and avoids destroying the adenine and guanine of the oligonucleotide fragment.This method do not use longterm alkaline solvent treatment to avoid the side reaction of deguanidinization in the synthesis of arginine-containing polypeptide-oligonucleotide conjugates.This thesis also develops TBS-protected amino acids for the preparation of polypeptideoligonucleotide conjugates containing serine,threonine and tyrosine.The amino acid protected by TBS is compatible with all other amino acids,further expanding the amino acid types of the polypeptide-oligonucleotide conjugate.In addition,we directly use the solid-phase peptide chain cyclization to realize the Fmoc solid-phase synthesis method to prepare cyclic peptide-oligonucleotide conjugates.On the other hand,in addition to increasing the curative effect of oligonucleotide drugs,polypeptides themselves can also be directly used as therapeutic drugs.Compared with therapeutic protein drugs,peptide drugs have less immunogenicity,lower production costs,and better tissue penetration.Compared with small molecule drugs,the large size and larger surface of peptides make them the preferred molecule type for targeting protein-protein interactions.However,traditional peptide drugs have poor ability to penetrate cell membranes and are easily hydrolyzed by proteolytic enzymes in vivo.In the last part of the thesis,we improved the stability of polypeptide drugs through systematic chemical modification of mambalgin-1,tarantula toxin PcTx1,and somatostatin.Interestingly,we found a novel class of somatostatins,whose structure is different from natural somatostatin,but still maintain good biological activity.This provides an idea for the design of new somatostatin drugs. |
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