hello everyonewelcome to my presentation today i amyoungi'm the lead scientist from chemicaldevelopment department from fairingpharmaceuticals indenmark the title of my presentationtoday is the peptide set reactions anddegradationsand part of the contents from mypresentation today iscited in my book site reactions inpeptide synthesisif you're interested you'll recommend itrefer to the book for further readingi my presentation today will be splitinto two parts the part one is aboutside reactions in peptide synthesis andpart twois about the peptide instability anddegradationsso the first part is also categorizingto two groupsthe first part is about the sidereactions in the peptide assemblyand the second part is the set reactionsin the peptide cleavage and globaldeprotectionthe first categories of the sidereactions in the peptideassembly is about endo impurities soendo impurities meansin the peptide assembly some certainamino acids is incorporated twiceso for example this peptide it has two xa m minus two residues so we call thisimpurities and x a and minus twothe reason of this impurities uh hassomecourses the first one is the f mark xaxa a die peptide impurities in thestarting materialor the unprotectedxaa impurities english starting materialora the exhibition ranging after the fmark xaacoupling which is can also introduce theendo impuritiesthe fourth categories of the reason isthe premature f marked blocking so thisanime animation isillustrating this mechanism during theassembly of the xaait's coupled to the peptide howeverunder whatever reasonsthe f mark could be prematurely cleavedoff during or before thecouplings and if this unprotected aminoacid is coupled to the peptideresins its free amino groups can allow afurther coupling of the anothermolecules making these endo impuritiesthe reason of this premature f markblocking could be thepresence of some basic motives like thelysine amino epsilon inducedf marked blocking or the uh n alphaamino groups from the proline or the dmfsome dmf can contain levels ofdimethylamine which can cleave f markduring the couplingsor the rescue preparingcan also remove the f mark if thepreparing is notthoroughly washed away after f markedblockinga ubiquitous anal annual impurities istheendo xaac terminal is normally addressedctc resinthe mechanism is that when thepenultimate amino acid is activated bydic and further by hovtthe formed hobd ester is addedto the amino acid resins togive the target dipeptidehowever even though hobt is usingthe catalyst in the reactions it's inexcessand the excess hovt can cleavethe first amine acid off the resinsso the stability of the amino acidsis much weaker than the full sequencessoit could be partially cleaved by thehobtand release to the reaction mixturesand the released amino acids can reactwith the amino acid resins to form thedipeptideand in the presence of the huel excessof thepenultimate amino acidsthe endo impurity will be formedand this mode of the endo impurities ispredominantly address the ctcresins because of its high acidsensitivitiesand the sum of the amino acids like theproline or glycine when theyserve against the c-terminal residuesthey are more susceptible to suchpremature acidosis compared to the otheramino acidsand the mold of the activation of thepenultimate amino acids very criticalso the process parameters like theactivation temperaturesproactivation time and couplingtemperatures are very criticalfor the formation of the endo xaaimpuritiesthe second categories of the impuritiescommonly addressed peptide assembly iscalledxa impurities so this means someamino acids is deleted from thesequences and weform this tests for example xaa and -3impuritiesthe reason of this death computer isnormally because of the incompletecoupling of the specific amino acidsso the potential solution to address itis through the recouplingand using stronger coupling reagentsenhance the equivalence of the reactantschange the solvents enhance the reactiontemperaturesuse catalysts like the d-map for thecouplingsand reduce the rising loading ratebecause of the low rate loading rate thecoupling kinetics could beenhanced or using a catatropicsalt like the potassium bromideor using the dipeptide building blocksto bypass the formations of thisxaa impurities another reason for thistest impurities is theinsufficient proceeding f multiblockingso the fmark is not fully removed of the resinsby preparating and therefore when youcouple the second amino acid of coursethesite is blocked and can impede thecoupling of that amino acidthe solution to address this course isto increase the basetreatment circles like normallythey treat by two times we can enhanceto three times orfull time as needed to completely removethe f markor to address the to use the highertemperatures for the f modblocking using stronger base like thedbuto remove the fthe third group of the set reactions inthe peptide assembly is called dicend capping in theindustry peptide assembly dic is thecoupling regionby default because of the cox advantagesthe amino acid is normallyactivated by the dic to thecorresponding dic estersand this is added to the peptide resinsto coupleand form the pipette bond however inexcess of thecoupling rate dic and if the couplingreactions kinetics is slowthe probability of the reactions betweenthe dicand the alpha amino groups on therussians is enhancedand thus forming this guanine dean saidreactions we call thisdic end capping it formsimpurities with 126 molecular weightincrementcorresponding to the targetedintermediatesin case of the depth of peptide theformed dic and capping periods canmediate the intramolecular cyclizationto form theimmunohedantoin impuritiesthe fourth categories of the sadreactions is calleddkp formations is the the ketopiparasiteis very common ubiquitous set reactionsin the peptide assemblythis rectangle because of thenucleophilicities of the alpha aminogroups it canattack the peptide bond of its ownmolecules to form this dkprings and chopping off the seed at theof the die peptide making this truncatedsequencesand we call this dkp formationsit could either be formed during themanufacturing processes orduring the formulations and storage isregarded as the degradations impuritiesand dkpset reactions is predominantly occurredunder basic conditions it prevails atthe f mark the blocking stepsand it's very strongly related to thetype of the secondary base likepreparating piperazineand solvents for the f mark removalit's high it's highly temperature andtime dependentand it has some significant scale-upeffects if they prolongeda case of the prolonged whole timebetween the coupling and the f multiblockingit could generate some enhanced degreesof the dkpformations and it could also occurin the api manufacturing austin storageeven asthe solid and during the formulationsit's verysequence dependent so the c miconfigurations promote the this dkpformationsfor example if it has the uh the naccu amino acids like the xa poolingpepper bondor the c alpha alpha diaculated aminoacids like the aibor the glycine is on thec to the n terminals like a glycineglycine is very highlydkp prone also when glycine is on thethirdposition from the n-terminus the dkp isenhancedhasting proline is prone to dkpformation as welland the alternating configuration of theamino acids like the land d o d and l are also very prone todkp formations so here is a question foryouso what if the dkp information isoccurredat the c terminus of the dipeptide onctc is rising so what's theconsequence will bethe aspartate information is alsonotoriously known as the side reactionsin the peptide thicknessesthis is because of the nucleophilicitiesof the atomite nitrogens it can attackthe carboxylate groups on the asparticside chains to form this aspartameintermediateswhich can energize the um glycophyllicattackeither by waters or preparation if atthethis site hydrolyzed by by waterit can um transform be transformed intothe aspartic aciduh l and d because of the rasterizationcan occurat the steps of the aspartame formationsif it's uh attacked by the preparationit will form the aspartic acid paradisewith m plus67 molecular weight increasementsand if this attackoccurs at the site b hereit will form the iso aspartic acid orthe iso aspartate gases preparationimpuritiesasparagus as part of malformation couldbe catalyzed by bothacid and base and its accumulativeformations it canoccur all the way along as long as theaspartic acid is introducedit could be taking place at the steps ofthe peptide synthesis formulationand storage it can also addressthe protected aspartic acid and theprotecting groups has very significantimpacts onaspartate gas inflammation for thepeptide assembly it's highly sequencedependentso when is the second amino acid glycineorserine throning ironing asparagineaspartic acidhasting or it has the alternatingconfigurations likel-aspartia d amino acid ord-aspartic acid l-amino acids theprobability ofaspartame formation is increasedglutamic acids can also undergo similarreactions but toa much lesser extent umit's need to be highlighted here thatsome of thepeptide modifications like the sidechain to side chaincircularizations could alsoinduce some aspartate aspartamideformationsand when the carboxylate groups onosbotic side chains has activatedcorresponding active asterit can react with the amino groups hereto form the targetside chain sighting cyclization but onthe other sidethe probability of the aspartic aspartinformationis also increased because of theactivations of thesecarboxylic groupsasparagine diamination is also a wereknown set reactions in peptidethicknesses sothe us purging capoximiniteside chains is transformed to thecorresponding carboxylate groupsand of course the m plus one impuritiesthe mechanism of the the ammunitioncouldgo by the aspartic acid aspart of my deformations to form thisaspartateintermediate first and when thehydrolysis is taking place athere or here it will generate theaspartic acid or the iso aspartateanswerthis set reaction is regarded as themost pronounced decompositions of thepeptide drugs and it can proceedeither in low ph within low phit could go viral directly hydrolysis orhigh ph neutral ph maybe go virally asuh as part of my formations and it'shighly sequence dependentas protein gly asparagine glycineor searing throttling histine lysinetryptophan aspartic acid glutamateare more moresusceptible to the applicationsit's a temperature iron ironic strengthsolvent and viscosity dependenceit could be occurring the peptidethicknesses purificationslocalizations formulations and storageand itneed to be noted that the side chains ofthe asparasparagine can also attack the backboneof the peptide toto form this succinamide peptideand give the fragmentation of thepeptidesso the second part of the side reactionsin the peptide synthesis is thesad reaction during the peptide cleavageand globalthe first one says acculations someamino acids like the uh tryptophan ishighly prone to activateacculations by the for example thetributary cations from the protectinggroups orit's a tfa ester to form the mutilatedtryptophan impurities umcysteine can also be susceptible to theacculations by the tubule to form thetributary cysteineon the mercaptan groups and some of thepeptide linkerresin linkers if it is apparentlycleaveduh like the at the power ratings or thering camera resinsor ring mbha resins if this is clippedat theside of the linker sideit can generate different types of thecationsand these contains could make thealkylations on somesome susceptible residues like thetryptophan here it can generatedifferentacculated computers with plus 202106 163 and265 impurities byanother types of the cell reactions inthe peptide cleavage and globalprotections is the adducts umtryptophan for example could be undergomodifications by thetfa and edt to the impuritiesof m plus 72and also the edt can modify the sustainto form the m plus 92impurities with their structuresum the existing acmprotecting groups could be uhprematurely cleavedby tfa in the presence of the scanmanagers like theedt and form thethe system and could be further modifiedby the edt to the correspondingimpuritiesand also searing could be modified bythepbf protecting groups from the arginineto form the sulfonated impurities with mplus80 molecular weight incrementoxidation and reductions can occurduring the peptide cleavage and globalprotections the histamine tryptophanterracingsustained methionine could be oxidizedto corresponding oxidized degradationsand also the dimerizations of thetryptophan for examples could also occurduring theoxidations to form the dimers of thetryptophansome amino acid residues which is mostlyaromatic one are like the terracinghastings tryptophanthromolanines fluoroalaninesand pyruvate ironings could besubjected to the alternations in thepresence of the t3and aldine corresponding alternatedimpuritiesreductions in the peptide assemblycleavageis not very predominant but the sum ofof the amino acids like the trip fan canbe reduced by the triathlon to thecorrespondingdegradations with n plus two molecularweight incrementsand methylated or ethylated tyrosine forexamples can besubjected to the d-methylationd-ethylations bythe uh cell anisoleto form this corresponding terracingimpurities with m plus 14molecular weight decreasements disulfidecan be reduced by tfaum actually in the tfa byselene to corresponding mercaptan pairnow we come to the second part of mypresentation is about the peptideinstability and the degradation ofcourse it's intertwined with the sidereactions in peptide thicknessesbut this part i will focus on theinherent properties of the peptidemoleculesand the degradations mostly in thepipette formulationsand peptide instability predictions isvery critical forpharmaceuticals because it is veryusefulfor drug discoveries and toassess which potential drug candidatescould beunstable it is very helpful forguide the api manufacturabilityassessment to understandwhich manufacturing processes candegrade the peptideit is helpful to predict the stabilityof the peptidon storageand to guide the development of thecorresponding analytical methods todetectcertain peptide gradients it's also veryusefulfor drug product manufacturabilityassessments as wellso the part two of the peptideinstability predictionsis grouped into six categoriespepto hydrolysis peptide rearrangementscirculation fragmentations pet peteliminationspeptide adducts peptide cross linkingandsolvent induced peptide degradationpeptide hydrolysis is very common in thepeptide degradations the first part isabout thens acetyl and acute peptide hydrolysisthis small disease is pretty prone tohydrolysislike the accelerated soccer thingsthe mechanism is that they canform this uh five member rings5 hydroxyl ox zodiumand chopping off the n-terminal aminoacids making the truncated sequenceswhen an aqib residues is located in theinternal sequence of the peptide it cantriggeracetolosis the mechanism is that itinitiates such a nucleophilic attackthrough the full formations of thesefive memory intermediatesand cleaving the peptide at the site ofthe aiband c terminals for example this cyclicpeptidebearing and a methyl aib residuesit's hydrolyzed at this siteby tfa to cleave thecyclic peptide into the correspondinglinear peptidethe third categories of the hydrolysisis on the cterminals where it has the n-methyl xaait is more prone to acidosisfor example this mode is with then-methylc-terminal residues and the m-bond hereis more labeled to cleaving off theamino acids to forming thispipette impurities the mechanism is thatthe carboxylate groups on thec terminals could attack the mi bondhere because of theadvantages conformations at this peptidebondand forming these five membraneactivities and rearrange to thecorrespondinganhydride and cleaved by the acids tothe corresponding peptideacidssearing or surrounding and the internalsequence of the peptidecould also also drive thehydrolysis because of the nuclearvelocitiesof the hydroxyl group here it can attackthe preceding pipette bondat this site to form the five membraneintermediateswhich rearrange to the correspondingesters and this esters is hydrolyzed tothe peptideat the site of the searingthe sink uh could also catalyze thehydrolysis of the hastingand searing sequences tolock the peptide into an advantageousconformations that promoting theum nucleophilic attackfrom the cell rings to form these fivememory intermediatesand cleaving off the peptide bond hereto form thehydrolysisaspartic acid can mediate peptide bondhydrolysisasp pro is a notoriously known sequencefor its highacidosis propensities the aspartic acidand proline peptide bond could becleaved in the presence of the acids toform the aspartic acidand proling fragments aspsearing sequence is also prone tohydrolysis due to the advantageshydrogen bond here which chopping offthepeptide bond at the side of theasparagus and serumsand also aspartate gases can attackthe amide bond preceding amide bonds toform the six membrane intermediateswhich rearrange to the anhydride andhydrolyzed to the two partshaste pro sequence is also prone tohydrolysis when it's located on then-terminus of the peptideto chop off this hast pro fragments andform the impurities withm plus two three four in molecularweightthe e imidazole side chains of thehistidinemight be involved in this process tocleave off the hispros dipeptide by means of the dkpand form the fragmentation of thepeptidewhen asparagine or isosparging islocated on the cterminals of the peptide they are proneto hydrolysisto form the corresponding ionic to theacid this might be due to thehydrogen bond between the side chain andbackbones of the peptideand promote the water attack on theatomized bond to form the correspondingacidan alternative mechanism is that thecarboxylate cterminals can attack the amidebound on the side chains to form thesuccinate anhydridewhich is hydrolyzed subsequently to thecorresponding aspartic acidscarbinolamine and carbonylamide are veryprone to hydrolysis even thoughsuch motives are sometimes present insome peptide moleculesum cabin is this structures it'ssynonymous tohemi amino and carbinolamide isthe amide form of the carbonyl domainand carbon domain is pretty prone tohydrolysis to the correspondingamine and aldehyde or alternativelyit could be dehydrated to the e meanand cabinet amount similarly it veryprone tohydrolysis to give the correspondingamideby releasing the aldehydeenamite and dehydroalamine are prone tohydrolysisthese are the structures of thedehydroalanineand enamite the edamitecould be hydrated to the correspondingcarbinolateand we know that carbinolamide are notstable and could be hydrolyzed to thecross bonding ionideand ketone this is a concrete exampleof the hydrolysis of the dehydroalanineit could be hydrated first to thecorrespondingcarbonylamide and carbonylamide could behydrolyzed to the correspondingiodide and here is the empirevial peptidethe next categories of the peptidedegradationis the rearrangement so the firstexamples is theno also migrations we know that as cytogroups can migratebetween the amino groups and the hydroxygroupfor example when we have the acetylserines on the peptide and terminalsthe acetyl groups can migrate betweenthese two motives depend on the phfrom the isomer of the osu or n-acylas isomer so pay attention to thesequence of then-accept accelerated seriesand also the depth of peptide when itappears atamino groups the acetyl groups canmigratefrom the uh hydroxyl group to the aminogroups to form the correspondingionic isomers so also pay attention tothe depth c peptide bearing alpha aminogroupand also the serine could be modified bythetfa to corresponding tfa esters and thisafter this uh trifluoroacetylgroups can also migrate from thehydroxyl group to the amino groupto form the sort of the irreversible umtfa acetylated impurities with molecularweight increasementof 96next we will talk about the end-to-endiso migrationssome amino acids like the diaminopropionic acidsdpr for short the dab whichis the diaminoburic acids or the otherthingsare prone to such a rearrangement forexamplesthe dpr is prone to accommodate theiso migrations from the backbone to theside chains while these five membraneintermediatesto form the corresponding isomersand once the dab is concerned it has thetwo probabilities of the migrations oneroot a oneroot b and root a it forms this isomersandroot b it's lead to the fragmentationsof theof the peptide and these twomigrations are very risky because itgenerates a isomers and sometimesif you don't have the reference of theisomers this kind of migrations canignore your detections we have thisexperience that sometimes when you don'tmake the reference of the impuritiesthis degradationis escaped from being detectedand also other things can lead to theelectronizations bythe similar degradation pathways to formthefragmentations of the peptide and ithink the nature selects the licenseoverother things the b and dpr has somereasonsand this might account for why thelysineis a selectednextly we talk about the end-to-endcopper moisture migrationscarbon molecule is sometimes in thepeptide drugsand carbon moisture can migrate from onevoltageto the other by thismechanism to form the thecopper mole migrations and aromatic ureafor examples if the middle groups hereis linked to aromatic residuesthis kind of the migrations is moreprobable to occuruh next we'll talk about the peptideeliminationsso the electron withdrawingsubstitutions on thebigger positions for example here is thebeta positionswhere it has electron withdrawing groupsthe probability of theelimination is high and this is reasonwhy we call it beta eliminationsand beta eliminations can occur in thebasic conditionsthrough the e1cb mechanismit forms the dehydral adeninesand corresponding adductsso beta eliminations can occuron the system residueswhen it has some protecting groups hereum and in presence of the preparationthis mobilities could be cleaved offand form the correspondingdehydroalanine degradantsfor examples in the assembly of thepeptide and when the systemis located on the c terminals and it'svery prone tobeta eliminations to form thecorrespondingand prepared then can react with thatby means of the microadditions to formthe correspondingpreparation impurities with m plus 51.disulfide is prone to beta eliminationas wellthis is the mechanism in the presence ofa basedisulfide and it goes beta eliminationsto form the dihydroalanine andpresulphideand prosulfite can be degraded to thesustain and sulfuror alternatively it could be hydrolyzedto the correspondingsulfatic acid and sulfatic acidcan be disproportionatedisproportionated intoa cysteine and safinic aciddisulfide can also accommodate betaeliminations to giverise to the formation of the erasmicdisulfidetrisulfide and lethaling or monosulphitethe two molecules of the disulfidescan undergo disulfide scrambling to givethedimers and dimers can further undergobeta eliminations uh giving two motivesbearing the dehydroalanine andprosulfite respectivelyand for the presultified peptideit can undergo trisulfide formations togive the trisulfidewhile rest for dehydroadenine it cangivelandfilling by the microadditions oralternativelythe um dehydrogenating canundergo hydrolysis and form thecorresponding and provide pyruvatepeptidedisulfide peptide can also undergobetter eliminations toform the dehydroalanine and prosulfiteand this can reshuffle to the disulfideby micro additions but give to therasterizationsso in total four types of thedegradations can be generated by thedisulfidebeta eliminationnext we will talk about peptide adductsthe first categories of the adducts isthe acculationsthe peptide could be modified by theformaldehydecorresponding intermediates withmolecular weightm plus 30 and this derivatives could befurther dehydrated to the correspondingshift base of the m plus 12.when these modifications occurs on thepeptide and terminalsthe formed sheath base could be cyclizedto the correspondingimidazolidinal also m plus 12.some amino acids like the cysteine ortryptophanor asparagines or histaminescould also undergoes different type ofthe cyclization to form thecorresponding and plus 12impurities also theformaldehyde and presence of theformic acids can accommodate isola clarkreactions to formthe acculations of the mineral groups oreven thedye alkylation of the amino groupsnext we will talk about the peptidesaturations peptide saturations could becatalyzed by acetic acid in the presenceof theethyl acetate to form the correspondingacetylated peptideand also histidine can catalyze theacetylations we know thatimidazole is a good catalyst for acidmigrationsand also the histidine can catalyze thiskind ofreactions to form the firstlyaccelerated histidineand the cyto groups can migrate from thehistidine to the corresponding aminogroups togenerate the acceleration's impuritieswe know that urea sometimescan be a fastly even fastly degradedcorresponding sciatic acidsand in the process of the carboxylategroups it forms the correspondingcarboxylic acidcarbomic acid mixed anhydride andonce this antiderive is formed it canmodifythe amino groups and to form thecorresponding and acetyl-amineimpuritiesand releasing the dicarbon dioxideammoniumthe next categories of the adducts isthe peptide coupled modulationsthe capacitance can be triggered by theureaas well and the urine can form the crossbonding isothermic acidand it can give the amino degraded tocrest bodyammonium the carbon dioxideand once these isosonic acids formedit can modify a varieties of the peptideincluding the mineral groupsthe cysteines the histidinesand carboxylate groups and pterosanesand arginines and phosphoric acidto form the corresponding carbomolationsimpuritiesnextly we talk about the peptidecrosslinking to form dimers or multimersthe first categories is thetransaminationsand we know that the transformations cantake place between the amino groupsand imi to form the transformed imideand amine and if this transformationtakes place between two moleculesone molecules compare contains aminogroups the other onehas a correspondingcarboxymite and this kind oftransformations willlead to the formations of the of thedimerwith molecular weight two times of themonomers minus17 because uh one molecules of ammoniumis kicked out oftherenextly we talk about aspartamide inducedcrosslinkingaspartame can take placeon a aspartic acid containing peptideand once theaspartamide is formed it could bealso attacked by the amino group fromanother peptide moleculesto form the dimers of the peptideimpuritiesdepending on the attack pathwaysalso the succeeding and dendrite caninduce cross-linking as wellif the the asparagine c-terminalresiduesis formed the correspondingcinnamic acid anhydride it could beattacked by the amino group from othermolecules to form thethe dimers for example the insulin canform the insulin dimersby this mechanismbeta eliminations can also lead to thecross-linking of the peptidefor examples if the beta elimination isaddressing a peptide it will form thecorresponding dehydrogen motifsand this could be attacked by anotherpeptide molecules by the microaddictionsto form thethe dimers herewe show that the dehydrogenating couldbe reacting withthe lysines with histamines or withsystems to form the different kind oftheof the diameter combinationsin disulfide is definitely a hotspot forcrosslinking disulfide could be degradedin the presence of the trace of thelinearsustain peptide impuritiesto form the dimer and dimer is already across linkingbut the dimer could further undergo bigeliminationsand give the corresponding peptides withcysteineand prosulfite and this can furtherundergo someintermolecular cross-linking to give thedifferent types of the cross-linkingimpuritiesalternatively this dimer itself can givehigh odors of oligomers through thefurther ethanoldisulfide exchangealternatively the dissolve fat itselfcan negotiate eliminations to give thepresulfireand the hydroaldening here andit can undergo desulphurizations to formthe system and this thing canundergo further cross-linkingdisulfide can also undergo alphaeliminations in present waters to formthemercaptans and cyphenic acidand suffering acids can undergoesfurther cross-linking by theta disulfideexchange etc to form the different typesof the cross-linking degradationsso it's very critical to control thelevels of the linearimpurities in a disulfide peptides forexamplesometimes of the peptide is very verysensitive tothe um to the captain impurities andthe uncontrolled organizations can takeplaceduring the formulations because of thismechanismpeptide oxidation can sometimes lead tocross-linkingfor example histidine could sometimes beoxidized to the corresponding forhydroxy to oxalyl histidineand this derivatives can react with alot of theamino acid abilities for exampleargininehistidine lysine andsustain and form the correspondingthe dimeric impurities um through thecross-linking between the hassling andthecorresponding nucleophilic amino acidsnext we will talk about uh thesolvent-induced peptide gradations thefirst one is the dichloromethanedichromating is relatively an inertorganic solvent but it's nottotally ignorant in the presence of thepreparing for examplesdichromate thing can react withreparating preparing to form this is thegerminal degradations which canbe further degraded to the correspondinge mean or ammonium salt and ammoniumsource can be hydrolyzedto release the formaldehydealso the dichromethanes can be slowlyhydrolyzedand by releasing the hcl to theintermediates which can be furtherdegradedand give the formic acid uh sorry forformaldehyde and format could bedisproportionated to the cross bondingformic acid and the methanolnext we talk about dmf dmf is a rathercomplicated scenario and dmf cangive to the formulations of the aminogroup directlyor alternatively dmf can functions andreactants we know that emfcan work as a catalyst sometimesbecause of these nucleophilicities andin the presence of the strongelectrophiles for examples thephosphoryl chloridebmf can react with phosphoryl chlorideby means of the fisma hack reactions togivethis phase my rate and the intermediatesand we know that is prettyreactive towards different nucleophilesand can givethe ammonium salt intermediates which ishydrolyzed to corresponding formulationsof the nucleophilesnot only the phosphoryl chloride butalso otherelectrophiles for example like hbtucan be reactive towards the emf and theycan slowlyproceed involve the phase my hackreactionsand give to the formulations of the ofthenucleophile so it's giving the end mplus 82 formulations impuritiesacetone notch well certain nitrogencould be oxidized to the correspondingradicals and these radicals can reactwith the oxygens to formthose intermediates and this can befurtherdegraded and by releasing the waterto give this the formal solenoid andformula cell nut can reactfor example with the amine to form theum the formulations of the mean bygiving releasing their solenoidthe mso dmso can oxidize the variety ofthepeptides like the mechanical ethertryptophantertiary immune tyrosine etc anddmf itself can undergo somedegradations by radicalmechanism to form those intermediatesand this can givethe methyl radicals and thecorrespondingmethane suffinic acid and methylradicals can be trapped by oxygens togive the methyl peroxide radicalsand they can form the dimer by theterminationsbut they can further undergo the rootcell terminationsand give rise to the formations of theformaldehydeand formaldehyde can further triggerpeptide degradationsmethanol and tributal the methanol canreact with the carboxylate groupsto form the corresponding methyl esterand the traditional can undergo radicaldegradations to form these radicals andthey can form the dimersor alternatively they can form this kindof radicals on the oxygensand they can degrade it by releasingthe acetones and form the correspondingmethyl radicals and we have introducedin the preceding slides like themethyl radicals can further react withoxygens and to be degraded to theformaldehyde so formaldehydeand acetones can trigger some peptidemodificationsthen is acetones so acetones can reactwith thehistory with the hastenings especiallywhen hasting is located on the nterminals of the peptideand they give these intermediates of theadductswhich is the further dehydrated throughthe correspondinguh enamine the molecular weight isincreased by40. also theacetone can react with the n-terminalamino groups to form the intermediatesof the immune salt andit can undergo cyclizationintramolecularlyto form the immune diazolidinol it alsohas amolecular weight increasement of thefullyso that's all my presentations for todayandmany thanks for your attentions

以下为机翻

大家好，欢迎来到我今天的演讲

我amyoungi 是来自fairingpharmaceuticals indenmark 化学开发部门的首席科学家

我今天演讲的标题是肽组反应和降解，

我今天演讲的部分内容引用在我的书中多肽合成中的现场反应

如果你感兴趣的话'我推荐它参考这本书进一步阅读

我今天的演讲将分为两部分

第一部分是关于肽合成中的副反应，第二部分是关于肽的不稳定性和降解，所以第一部分也分为两组，第一部分是关于肽中的副反应组装和第二部分是肽切割和全局去保护中的集合反应

肽组装中的第一类副反应是关于内切杂质

所以内切杂质是指在肽组装中某些某些氨基酸被掺入两次例如这个肽它有两个 xa m 减去两个残基所以我们称其为杂质和 xa 和负二这种杂质的原因 uh hassome course 第一个是 f 标记 xaxa 原料中的肽杂质或未受保护的xaa 杂质英文起始材料或展览范围在fmark xaacoupling 之后也可以引入endo 杂质第四类原因是过早的f 标记阻塞所以这个动画动画说明了xaait 组装过程中的这种机制s 与肽偶联，然而无论出于何种原因，f 标记可能在偶联期间或之前被过早切割，如果这种未受保护的氨基酸与肽树脂偶联，其游离氨基可以允许另一个分子进一步偶联，从而产生这些内切杂质。这种过早 f 标记阻断的原因可能是存在一些基本动机，如赖氨酸氨基ε诱导的显着阻断或脯氨酸或 dmf 中的 uhn α氨基基团 dmf 可能含有一定水平的二甲胺，在偶联过程中可以切割 f 标记，或者如果制备物没有彻底清洗，拯救制备也可以去除 f 标记f 标记阻断后远离肛门常年杂质istheendo xaac 末端通常被处理ctc 树脂机制是当倒数第二个氨基酸被dic 激活时并且进一步通过将形成的hobd酯添加到氨基酸树脂中以产生目标二肽，但是即使hobt在反应中使用催化剂s 过量和过量的 hovt 可以将第一个氨基酸从树脂上裂解下来，因此氨基酸的稳定性比完整序列弱得多，因此它可能被部分裂解并释放到反应混合物中，并且释放的氨基酸可以与氨基酸树脂反应形成二肽并在倒数第二个氨基酸的huel 过量的存在会形成内切杂质，这种内切杂质模式主要针对 ctcresins，因为它具有高酸敏感性，并且当它们对 c 末端残基起作用时，它们是氨基酸的总和，如脯氨酸或甘氨酸，它们更容易受到影响对于这种过早的酸中毒，与其他氨基酸相比，倒数第二个氨基酸的活化霉菌非常关键，因此活化温度、活化时间等工艺参数和偶联温度对于内型 xaa 杂质的形成非常关键。

第二类杂质通常被称为肽组装物，称为 xa 杂质，因此这意味着从这些序列中删除了一些氨基酸，我们进行了此测试，例如 xaa 和 -3 杂质，这台死亡计算机的原因通常是由于不完全偶联因此，通过重新偶联和使用更强的偶联剂来解决该问题的潜在解决方案提高反应物的等价性改变溶剂提高反应温度使用催化剂（如偶联的 d-map）并降低上升的负载率，因为低速率负载率偶联动力学可以改进或使用像溴化钾这样的变质盐或使用二肽构建块绕过这个xaa杂质的形成这个测试杂质的另一个原因是多嵌段的进行不够，所以fmark没有通过制备完全去除树脂，因此当你偶联第二个氨基酸时，当然该位点被阻塞并可能阻碍该氨基酸的偶联解决这个问题的解决方案是像往常一样将碱处理圈增加两倍，我们可以根据需要将其提高到三倍或全部时间，以完全去除 f 标记以解决肽组装在行业中被称为 dicend capping将相应的二酯添加到肽树脂中以偶联并形成移液管键，但是如果偶联率 dic 不超过，并且如果偶联反应动力学缓慢，则 dic 和 α-氨基之间的反应概率会提高，从而形成这种鸟嘌呤，我们称这种反应为此 dic 封端 它形成分子量增加 126 的杂质 对应于目标中间体 在肽深度的情况下 形成的 dic 和封端周期可以介导分子内环化形成免疫 己内酰脲杂质 第四类反应称为 dkp 形成是酮体寄生虫是肽组装中非常常见的普遍设置反应 this长方形，因为 α 氨基的亲核性，它可以攻击肽键其自身的分子形成这个 dkprings 并在肽的末端切掉种子，形成这个截短的序列，我们称之为 dkp 形成它可以在制造过程中形成，也可以在配方和储存过程中形成，被认为是降解杂质，dkpset 反应主要发生在碱性条件下在 f 标记的封闭步骤中占主导地位，它与二级碱基的类型密切相关，例如制备哌嗪和去除 f 标记的溶剂，它很高's 高度温度和时间依赖性，如果它们延长耦合和 f 多嵌段之间的整个时间延长的情况，它具有一些显着的放大效应，它可以产生一些增强的 dk 形成程度，它也可能发生在 api 制造奥斯汀存储中，即使是固体和在配制过程中's 非常依赖于序列，因此 c 错误配置促进了这种 dkp 形成，例如，如果它具有 uh naccu 氨基酸，如 xa poolingpepper bond 或 c alpha alpha diaculated 氨基酸，如 aibor，甘氨酸在 c 到 n 末端，如甘氨酸甘氨酸非常高 dkp当甘氨酸位于 n 末端的第三个位置时，dkp 也很容易发生 dkp 增强，脯氨酸也容易形成 dkp，并且像土地 dod 和 l 这样的氨基酸的交替构型也很容易形成 dkp，所以这里有一个问题，如果 dkp信息发生在二肽 onctc 的 c 末端，所以什么？结果将是天冬氨酸信息也被称为肽厚度的副反应这是众所周知的，这是由于原子氮的亲核性，它可以攻击天冬氨酸侧链上的羧酸酯基团以形成这种天冬氨酸中间体，如果在该位点水解，则可以通过水或制剂激活 um 甘氨酸攻击by waterit 可以 um 转化为天冬氨酸s uh 受到制剂的攻击，它会形成天冬氨酸天堂，分子量增加 m+67，如果这种攻击发生在此处的 b 位点，它将形成异天冬氨酸或异天冬氨酸气体制剂杂质芦笋作为畸形的一部分，可以被酸和碱催化及其累积形成只要引入天冬氨酸就可以一直发生它可以在肽合成配方和储存的步骤中发生它还可以处理受保护的天冬氨酸并且保护基团对肽组装的天冬氨酸气体炎症具有非常显着的影响s 高度序列依赖性因此第二个氨基酸甘氨酸或丝氨酸何时熨烫天冬酰胺天冬氨酸或它具有交替构型，如天冬氨酸 d 氨基酸或天冬氨酸 l 氨基酸 增加阿斯巴甜形成的可能性谷氨酸也可以经历类似的反应，但程度要小得多。这里需要强调的是，一些肽修饰，如侧链到侧链环化也可以诱导一些天冬氨酸天冬酰胺的形成，当羧酸基团在osbotic侧链上被激活时，相应的活性星形可以与氨基反应形成目标侧链瞄准环化但另一方面概率由于这些羧基的活化，天冬氨酸的含量也增加我形成这种天冬氨酸的变形首先是中间体，当水解是在那里或这里发生，它会产生天冬氨酸或异天冬氨酸，这种反应被认为是肽类药物最明显的分解反应，它可以在低 ph 值下进行我的形成和它的高度序列依赖性作为蛋白质甘氨酸天冬酰胺甘氨酸或灼热节流组氨酸赖氨酸色氨酸天冬氨酸谷氨酸更容易应用它'sa 温度 铁 熨斗 强度 溶剂和粘度依赖性 它可能会发生 肽厚度 纯化 定位 配方和储存 需要注意的是，天冬酰胺的侧链也可以攻击肽的主链以形成这种琥珀酰胺肽并导致肽的片段化，因此侧的第二部分肽合成中的反应是肽裂解和全球范围内的可悲反应，第一个说积累一些氨基酸如 uh 色氨酸很容易被例如来自保护基团的支流阳离子激活积累 orit'sa tfa 酯形成残缺的色氨酸杂质 umcysteine 也容易被小管积聚以形成支流半胱氨酸 硫醇基团和一些肽接头树脂接头 如果它像额定功率或thering camera 树脂或环形mbha 树脂一样明显裂解被剪辑在连接体一侧它可以产生不同类型的阳离子，这些包含可以使一些易感残基上的烷基化，如色氨酸在这里它可以通过肽切割和全局保护的其他类型的细胞反应产生不同的累积计算机，其中包括 202106 163 和 265 杂质。例如可以通过 tfa 和 edt 对 m plus 72 的杂质进行修改，并且 edt 可以修改持续音以形成 m plus92 杂质及其结构和现有的 acm 保护基团可以在像 theedt 这样的扫描管理器存在下被 tfa 过早裂解并形成系统，并且可以通过 edt 进一步修饰为相应的杂质，并且灼热可以被精氨酸的 pbf 保护基团修饰以形成磺化杂质随着 mplus80 分子量的增加，在肽裂解和全局保护过程中会发生氧化和还原，组胺色氨酸与持续的蛋氨酸会被氧化成相应的氧化降解，例如色氨酸的二聚化也可能在氧化过程中发生，形成色氨酸氨基酸残基的二聚体，这主要是芳香族的。 the terracinghastings 色氨酸 氟丙氨酸和丙酮酸在存在 t3 和醛的情况下，熨烫可能会发生变化，相应的交替杂质减少肽组装体中的切割不是很显着，但铁人三项可以将诸如 trip fan 的氨基酸总和减少到相应的降解，其中 n 加两个分子量增量和甲基化或乙基化酪氨酸例如可以通过 uh 细胞苯甲醚进行 d-甲基化-乙基化以形成这种相应的梯田杂质与 m 加上 14 分子量减少二硫化物可以通过 tfaum 实际上在 tfa 中通过硒到相应的硫醇对现在我们来到我的第二部分介绍是关于肽的不稳定性和当然它的退化's 与肽厚度的副反应交织在一起，但本部分我将重点关注肽分子的固有特性和主要在移液管配方中的降解和肽不稳定性预测对于药物非常关键，因为它对于药物发现和评估哪些潜在的候选药物可能不稳定非常有用。 api 可制造性评估以了解哪些制造过程可以降解肽有助于预测肽储存的稳定性并指导相应分析方法的开发以检测某些肽梯度s 对药物产品的可制造性评估也非常有用，因此肽不稳定性预测的第二部分分为六类肽水解肽重排循环片段化宠物肽肽加合物肽交联和溶剂诱导肽降解肽水解在肽降解中非常常见第一部分是关于乙酰和急性肽水解这个小疾病很容易发生水解，就像加速的足球一样，机制是它们可以形成这个 uh 五元环，5 羟基氧化钠，并切断 n 端氨基酸，从而截断序列它通过完整的形成引发这样的亲核攻击这五个记忆中间体和切割 aiband c 末端位点的肽，例如这个带有甲基 aib 残基的环状肽s 在该位点通过 tfa 水解，将环肽切割成相应的线性肽。第三类水解位于具有 n-甲基 xaa 的 c 端更容易发生酸中毒，例如这种模式是带有然后-甲基 c-末端残基和 m-此处的键被更多标记为切割氨基酸以形成该吸管杂质 其机制是 c 末端的羧酸酯基团可以攻击此处的 mi 键，因为该肽键的优势构象并形成这五种膜活性并重排为相应的酸酐并被酸裂解为相应的肽酸灼烧或周围和肽的内部序列也可以驱动水解，因为羟基的核速度在这里它可以攻击前面的移液器在该位点键合形成五种膜中间体，这些中间体重排成相应的酯，这些酯在灼热位点水解成肽，sink uh 还可以催化 Hasting 和灼热序列的水解，以将肽锁定成有利的构象，促进细胞环的亲核攻击形成这五个记忆中间体并切断肽键从而形成水解天冬氨酸可以介导肽键水解asp pro是一个众所周知的序列，因为它具有高酸中毒倾向天冬氨酸和脯氨酸肽键可以在酸的存在下被切割形成天冬氨酸和proling片段，因为它的序列也是容易水解，因为这里的氢键可以切断侧面的肽键芦笋和血清以及天冬氨酸气体可以攻击酰胺键之前的酰胺键，形成六种膜中间体，这些中间体重排为酸酐并水解为两部分。s 位于肽的 then 末端，以切断该 hast pro 片段并形成杂质，m 加上分子量中的二三四，组氨酸的 e 咪唑侧链可能参与该过程，通过 dkp 切割 hispro 二肽并形成片段化肽当天冬酰胺或异构体位于肽的 c 末端时，它们易于水解形成相应的酸离子，这可能是由于肽的侧链和主链之间的氢键，并促进水对原子化键的攻击形成相应的酸，另一种机制是羧酸酯末端可以攻击结合在侧链上的酰胺，形成琥珀酸酐，随后水解生成相应的天冬氨酸甲醇胺和羰酰胺非常容易水解，即使这种动机有时存在于某些肽分子舱中，是这种结构吗？同义的半氨基和甲醇酰胺是羰基结构域的酰胺形式，碳结构域很容易水解成相应的胺和醛，或者它可以脱水到平均和内阁的量，类似地它很容易水解产生相应的酰胺，通过释放醛烯酸和脱氢丙胺很容易水解这些是脱氢丙氨酸和烯酸的结构吗？ 依达米特可以水合成相应的甲醇盐，我们知道甲醇酰胺不稳定，可以水解成交联的离子和酮，这是脱氢丙氨酸水解的一个具体例子相应的碘化物，这是帝国肽的下一个类别肽降解是重排，所以第一个例子是没有迁移我们知道，由于细胞基团可以在氨基和羟基之间迁移，例如当我们在肽和末端有乙酰丝氨酸时，乙酰基可以在这两个动机之间迁移取决于 osu 异构体的 ph或n-酰基异构体所以要注意then-accept 加速系列的这些序列以及肽的深度当它出现在氨基时乙酰基可以从uh羟基迁移到氨基形成相应的离子异构体所以还要注意深度c肽轴承α氨基和丝氨酸可以被tfa修饰为相应的tfa酯，此后，uh三氟乙酰基也可以从羟基迁移到氨基形成那种不可逆的 umtfa 乙酰化杂质，分子量增加 96 接下来我们将讨论端到端的异迁移一些氨基酸，例如二氨基丙酸 dpr 简称 dab，即二氨基丁酸或其他容易发生这种重排的物质，例如 dpr 容易适应异迁移从主链到侧链，而这五个膜中间体形成相应的异构体，一旦涉及到 dab，它具有两种迁移概率：一个根 a 一个根 b 和根 a 它形成这种异构体和根 b 它导致肽的片段化，这两个迁移是非常危险，因为它会产生异构体，有时如果你不这样做'没有异构体的参考这种迁移可以忽略您的检测我们有这种经验，有时当您不这样做时tmake 杂质的参考，这种降解不会被检测到，还有其他东西可以通过类似的降解途径导致电子化，从而形成肽的碎片，我认为大自然选择许可而不是其他东西 b 和 dpr 有一些原因，这可能解释了为什么赖氨酸被选中接下来我们谈论端到端的铜水分迁移碳分子有时在肽药物中，碳水分可以通过这种机制从一个电压迁移到另一个电压，形成铜摩尔迁移和芳族脲，例如如果中间基团与芳族残基相连，这种迁移是接下来我们更有可能发生将讨论肽的消除，因此在较大位置上的吸电子取代例如这里是具有吸电子基团的β位置，消除的可能性很高，这就是为什么我们称之为β消除和β消除可以通过e1cb机制发生在基本条件下它形成脱水腺嘌呤和当系统残基具有一些保护基时，系统残基上可能会发生相应的加合物，因此在存在制剂的情况下，这种迁移率可能会被切断并形成相应的脱氢丙氨酸降解物，例如在肽的组装中以及当系统位于 c 末端时，它很容易发生β消除，形成相应的和制备的物质，然后可以通过微加成反应形成相应的制备杂质，m加51。二硫化物也很容易发生β消除，这也是在碱基二硫化物存在的情况下发生β消除形成二氢丙氨酸和前硫化物和亚硫酸氢盐可以被降解成持续和硫化，或者它可以被水解成相应的硫酸，并且硫酸可以被歧化成比例二硫化物也容易发生β消除，这是在碱性二硫化物存在下的机理，它发生β消除形成二氢丙氨酸，预硫化物和亚硫酸盐可以降解为持续和硫化物，或者它可以水解成相应的硫酸，并且硫酸可以按比例歧化。二硫化物也容易发生β消除，这是在碱性二硫化物存在下的机理，它发生β消除形成二氢丙氨酸，预硫化物和亚硫酸盐可以降解为持续和硫化物，或者它可以水解成相应的硫酸，并且硫酸可以按比例歧化。被合成半胱氨酸和丁香酸二硫化物也可以进行β消除，从而形成erasmic二硫化物三硫化物和致死性或单亚硫酸盐二硫化物的两个分子可以进行二硫化物加扰，产生二聚体，二聚体可以进一步进行β消除，嗯，产生两个动机，分别是脱氢丙氨酸和亚硫酸丙酯，以及预加的肽it可以进行三硫化物形成以产生三硫化物，而其余为脱氢腺嘌呤它可以通过微量加成进行填埋，或者脱氢可以进行水解并形成相应的丙酮酸肽并提供丙酮酸肽二硫化物肽也可以进行更好的消除以形成脱氢丙氨酸和亚硫酸丙酯，这可以通过微量添加重新洗牌为二硫化物，但总体上会产生光栅化sso四二硫化物β消除可以产生的降解类型接下来我们将讨论肽加合物的第一类加合物是加合物肽可以被甲醛修饰，相应的中间体分子量m加30，这种衍生物可以进一步脱水到m加12的相应移位碱基。这些修饰发生在肽和末端，形成的鞘碱基可以环化为相应的咪唑烷醛也 m 加 12。一些氨基酸，如半胱氨酸或色氨酸、天冬酰胺或组胺，也可以进行不同类型的环化形成相应的环化反应，再加上 12 种杂质，甲醛和甲酸的存在可以适应伊索克拉克反应，形成矿物基团的积累，甚至是氨基的染料烷基化。接下来我们将讨论关于肽饱和 肽饱和可以在乙酸乙酯存在下被乙酸催化形成相应的乙酰化肽，组氨酸也可以催化乙酰化我们知道咪唑是酸迁移的良好催化剂，组氨酸可以催化这种反应，首先形成加速组氨酸，细胞基团可以从组氨酸迁移到相应的氨基以产生加速's 杂质我们知道，尿素有时可以是一种快速甚至快速降解的相应坐骨酸，在羧酸酯基团的过程中形成相应的羧酸碳酸混合酸酐，一旦形成这种反衍生物，它可以修饰氨基并形成相应的乙酰胺杂质并释放二二氧化碳铵加合物的类别是肽耦合调制，电容也可以由尿素触发，尿液可以形成交叉键合等温酸，它可以给氨基降解的顶体铵二氧化碳，一旦这些异音酸形成，它可以修饰多种肽，包括矿物基团半胱氨酸、组氨酸和羧酸酯基团和翼糖和精氨酸和磷酸形成相应的carbomolation 杂质接下来我们讨论肽交联形成二聚体或多聚体第一类是氨基转移，我们知道氨基和 imi 之间可以发生转化以形成转化的酰亚胺和胺，如果这种转化发生在两个分子之间，一个分子比较含有氨基，另一个分子有相应的羧基这种转变会导致形成分子量是单体减17的两倍的二聚体，因为呃一个铵分子被踢出然后我们讨论天冬酰胺诱导的交联，天冬氨酸可以在含有天冬氨酸的肽上发生，一旦形成天冬酰胺，它也可能被攻击来自另一个肽分子的氨基形成二聚体取决于攻击途径的肽杂质也可以诱导交联，如果天冬酰胺c-末端残留物形成相应的肉桂酸酐，它可以被来自其他分子的氨基攻击形成二聚体，例如胰岛素可以通过这种机制形成胰岛素二聚体β消除也可以导致肽的交联，例如，如果 β 消除是针对肽，它将形成相应的脱氢基序，这可能会被另一个肽分子通过微吸附攻击形成二聚体，我们表明脱氢可以与赖氨酸与组胺或与系统反应在二硫化物中形成不同种类的直径组合绝对是二硫化物交联的热点在微量线性持续肽杂质的存在下降解形成二聚体，二聚体已经交叉连接，但二聚体可以进一步进行双消除并产生相应的肽与半胱氨酸和亚硫酸氢盐，这可以进一步进行一些分子间交联以产生不同类型的交联得到这些加合物的中间体，通过相应的烯胺进一步脱水，分子量增加40。丙酮也可以与n-末端氨基反应形成免疫盐的中间体，它可以在分子内进行环化形成免疫二唑烷醇它还具有完全的分子量增加所以这就是我今天的所有演讲，非常感谢您的关注