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Total Synthesis Of Mambalgins Of Analgesic Effect By Hydrazide-based Native Chemical Ligation

Posted on:2017-08-21Degree:MasterType:Thesis
Country:ChinaCandidate:H LanFull Text:PDF
GTID:2321330536958943Subject:Chemistry
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Mambalgins are a class of 57-residue polypeptide toxins isolated from the venom of the African mamba and have potent analgesic effects by inhibiting the acid-sensing ion channels(ASICs)with fewer side effects than traditional opioid drugs.Classified as a member of the family of three-finger toxins(3FTxs),mambalgin contains four pairs of disulfide bridges which help to stabilize the three-dimensional scaffold.To study the mechanism of the mambalgins inhibiting the ASICs,it is necessary to acquire sufficient amounts of high-quality mamba peptide for the structure-function relationship studies.However it is still laborious and difficult to obtain homogeneous mambalgins from mamba snake venom extractions.Chemical synthesis provides an alternative source to access chemically well-defined mambalgins and their analogues.Protein chemical synthesis provides an alternative method for acquiring peptide or proteins which can't be obtained by biological technologies.Native chemical ligation(NCL)developed by Kent in 1994 has dramatically expanded the application of protein synthesis.The principle of NCL is based on the reaction of a C-terminal peptide thioester with the other peptide bearing a N-terminal Cys,which a Cys for the ligation is a must.Toxins usually contain 10~80 amino acids and rich disulfide bridges,making them ideal targets of native chemical ligation.Based on the native chemical ligation,we can first prepare the segments of protein and then conduct the final ligation which overcomes the limit of SPPS that ~ 50 amino acids long peptide can be stepwise assembled.Later methods based on native chemical ligation were developed and the hydrazide-based native chemical ligation developed by our lab has become popular due to its high efficiency in preparation of peptide thioester surrogate.In this thesis,we demonstrated the successful chemical synthesis of mambalgin-1/2/3 by using one-step two-segment hydrazide-based chemical ligation combined with the microwave-assisted solid-phase peptide synthesis.Patch-clamp electrophysiological analysis demonstrated ASICs inhibition activities of three in vitro folded mambalgins similar to previously reported data.Compared with previous three-segment ligation strategies,our two-segment ligation approach offers a simpler and faster protocol for chemical synthesis of mambalgins,which should enable to produce enough quantity toxins for structure analysis.
Keywords/Search Tags:mambalgins, hydrazide-based native chemical ligation, microwave-assisted solid phase peptide synthesis, acid-sensing ion channels, analgesic activity
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