TECHNICAL BULLETIN. Pioneer Peptide Synthesis System. Monitoring Peptide Synthesis. Page 1. Introduction * Detector PUMP

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1 Peer Pepde Syhe Sye Mrg Pepde Syhe Irdu * he Peer Pepde Syhe Sye ulze a V deer eaure he abrbae f lu flwg hrugh he ye. h V daa a prvde valuable dag ad qualave fra abu he yhe pre. he Peer fware prvde everal he fr feedbak rl baed he V daa a pepde aebly. Mrg ued ju wh her aalyal ehque (.e., PLC, MS) pree a effeve ea f eaurg he ue f a yhe. h ehal Bulle eded a he ulza f h V rg l. h lude uderadg he far ha a erfere wh he erprea f he deer daa a well a ehd redue hee erferee, he ue f he feedbak rg p, ad rublehg p. Deer gure llurae he a pe f he Peer Pepde Syhe Sye. he Peer a a varable peed pup ha delver reage ad lve hrugh he ye. A ere f valve dre he flud r ga hrugh he lu wae durg wah r deblk ep, he ube fr dlu f a ad, r arud a reyle lp (delvery fr ad he ube) fr uplg f a ad. he flw a al bypa he lu fr ep vlvg prg flud le r preava ad xg f a ad ad avar. he p-lu V deer r he abrbae a hee lu flw hrugh he ye. he deer ue a fxed wavelegh (365 ), gle bea p ye wh a erury lap. he efflue fr eah lu pae hrugh a 0.5 quarz apllary flw ell. he gal fr he l phdde deer e he CP he rue a ed erval. here are everal p fr vewg he deer daa: he deer value, gve a aalg vlage, he rue au ree (fr he Ma eu, pre Sa he Sy). A har rerder eed he rue wh he pal har rerder able (GE6038). he Irue wdw he Wrka (he deer rae ave fr a MPS yhe r whle he rue dle). WASE EAGES V DEEC A ypal deer rae (fr a har rerder r he Wrka) fr e yhe yle hw gure. he appearae f he deer rae ay vary depedg he yhe ale, he yle, he ava ehd, ad he daa aplg rae. he equee f ep a ypal yhe yle are (he ep rrepd he label gure ): A) eve he -eral preg grup wh deblk lu (ypally 0% DM). he area f he PMP V l Prbe Clu pael e () A B C D E gure. Peer ye pe. gure. Deer rae. * Abbreva: DIEA,,-dprpylehylae; DMA,,-dehylaeade; DKP, dkepperaze; DM,,-dehylfrade;, flureylehylxyarbyl; A, -(7-azabezrazl--yl)-,,3,3- eraehyluru hexaflurphphae; B, -(bezrazl--yl)-,,3,3-eraehyluru hexaflurphphae; A, -hydrxy-7- azabezrazle; B, -hydrxybezrazle hydrae; PLC, hgh perfrae lqud hragraphy; MS, a perery;, aeer; MP, -ehyl-pyrrlde; Pfp, peaflurpheyl; PyAP, 7- azabezrazl-ylxyr(pyrrld)phphu hexaflurphphae; PyBP, bezrazl-ylxyr(pyrrld)phphu hexaflurphphae; B, -(bezrazl--yl)-,,3,3-eraehyluru eraflurbrae;, eraehyl-flurfradu hexaflurphphae; V, ulravle. peak ay be prpral he au f he grup reved (ee he fllwg e fr a dealed du f deblk rg). Wah he lu reve he deblk lu. B) Dlve ad avae he g a ad. he harp, lely paed peak are aaed wh ar flwg hrugh he deer durg bubblg f he a ad lu he dlu ep. E: h ep perfred befre ep A a DKP yle. C) Cuple he avaed a ad he uppr by reylg he lu hrugh he lu ad prbe. he llag paer he avaed a ad lu flwg hrugh he lu Page

2 repeaedly. I ay ae h wll appear a a fla le a he axu deer readg due aura f he deer gal. D) Wah he prbe. he harp, lely paed peak are equaed ar flwg hrugh he deer durg he prbe wah. E) Wah he uppr. he exe reage are wahed fr he lu ad he deer reur a baele readg. Deblk Mrg he rea fr he reval f he grup fr a pepde-re ug hw gure 3. he dbezfulvee- addu (I) lberaed fr he uppr ad red a 365 gve a peak he deer rae (ee gure ). Ideally, he egraed area f he peak prpral he quay f reved fr he pepde uppr C gure 3. eval f -preg grup. dbezfulvee Dbezfulvee- addu I ad he uplg effey f he prevu a ad a be alulaed. wever, here are everal far ha redue he auray f h relahp. he rrela bewee he era ad pal dey DM rly lear. I DM, he flureylehyl hrphre ha a rg flureee e bad a 3 ha a erfere wh aurae V dee. he ex effe f he dbezfulvee- addu rgly depede he era he lu. he lal era f a he addu elued a vary wh a gle yhe. If he deblk preed a ad uppr arbaae Deblked a ad uppr rea fa, he he addu elued a he leadg edge f he reage ad xed wh he DM he lu. I ra, a lwer deblk rea reul he addu elued a re eraed lu f. Al, ued he rea by add he dbezfulvee. he ex effe f he dbezfulvee- addu wa eaured varu era f DM. he ex effe wa abu e greaer a 5% lu DM ha a 0% lu DM. herefre equal peak area fr ueve deblk rea a ly be expeed uder rly equvale expereal d. Alhugh her by-prdu abrb a 365 fr reval, her reage ued durg he uplg are deeed a h wavelegh. I parular, razle-baed uplg reage (.e., B, A, A, B, B, PyAP, PyBP) bd he ld uppr ad ay be elued durg he wah. I dfful rre fr flureee effe ad ex effe vara a -aalyal rue. Alhugh hee far affe he pre f peak quafa, he gfa rbu he erferee f he razle-baed reage. Mr Wah A yhe perfred whu a razle-baed avar r addve (.e., ava r pre-avaed a ad-pfp eer whu added B) exhb rg prble a a reul f uppr bud reage. he ae equee yhezed ug razle-baed avar r addve exhb peak wh area 0 e hgher. he peak fllwg a pr uplg a be very large, dag ha he razle-baed reage al bd he free a grup ha were aylaed by he g -preed a ad. gure 4 heaally llurae he bdg phee ad he reulg erferee he dee f he peak. -preed a ad ad a razle-baed avar are added he upreed pepde uppr. he uplg rea ur ad he exe reage are reved wh a wah lve. I e ae, a adard DM wah ued; h de reve all he bud razle-baed reage. I he her ae, a addal bae wah ued whh reve he bud razle-baed reage. he grup reved wh he deblk lu. I add revg he grup, ay bud razle reage al reved by he ba lu. he reulg peak fr he yhe whu he bae wah uh hgher ha he yhe wh he bae wah due he addal abrbae f he razle reage. A varey f ehd were eplyed elae he erferee f he bud reage wh he dee f he peak (ee able ). he bud razle reage wa reved effevely by a eraed bae (DIEA) wah afer he uplg. lu f DIEA DM were eed abve 0% beaue re eraed lu wuld eparae w layer. reae f he uppr wh ea DIEA (00%) fr wa he effe wah d reve he bud razle reage. Page

3 peak Cuple DM wah ly DM ad bae wahe Deblk Deblk peak blked a ad-uppr -preed a ad Avar (razle-baed) gure 4. Bdg f reage. able. eval f razle-baed reage. Expere e a dffere wavelegh dee he peak. e MP r DMA a a wah lve afer he uplg. Ireae fal DM wah fr 4 0. Add a ehal wah afer he uplg. Add a 4 bae wah (5% DIEA DM) afer he uplg. Add a bae wah (0% DIEA DM) afer he uplg. Add a bae wah (00% DIEA) afer he uplg. eul uable wavelegh. he abrbae prfle f razlebaed reage ad he dbezfulvee- addu verlap gfaly. Lle prvee revg bud reage. Bud reage ally reved. prvee revg bud reage. Bud reage ally reved. Bud reage ally reved. Bud reage reved. he grup reved a a lw rae ( / ~ 0 h) by DIEA 3. hu, a e ue expure f he -preed uppr ea DIEA wll reve le ha 0.% f he grup. Preaure reval -prblea gle uplg prl e he grup reved by he deblk reage fllwg he uplg ep. I duble uplg prl, preaure reval fllwg he fr add f a ad reul a udered addal rpra f h redue he equee. avd h udered rea, he bae wah auaally kpped whe he ex yle a reuplg yle. he adard Peer eplae prl pefe he Aux ble p fr he DIEA r wah. Pure DIEA (GE90308) huld be ued fr h wah avd ay udered de rea (he 900 L ble f dlled DIEA aahe drely he Aux p he Peer). he r wah ly perfred f e f he feedbak rg p eleed fr he yhe (ee eedbak Mrg e fr re deal). A DIEA wah requred uefully yheze a pure pepde he Peer. I ueful fr re ragh-frward erprea f deer daa ad requred fr feedbak rg. wever, he expee f he added reage ad addal yle e ay be warraed fr he aebly f hr r le plex equee. gra he daa fr he hgra are geeraed fr he deer rae daa ad are a egra f he peak prdued durg he deblk ep. he deblk pr f he eplae prl (a yle) hw gure 5. gure 5. Deblk prl ep. Le ) Le ) Le 3) Le 4) Le 5) luh le wh Deblk: Pre he flud le ad fll he deer wh deblk lu. Zer deer: Iru he fware ue he urre deer value a he baele fr he hgra alula. Sar daa lle: Iru he fware ae daa lle fr he deer. he fllwg alula perfred every ed: hgra value = deer value - baele value he dvdual hgra value are auulaed ul he fware rued p daa lle a le 6. Deblk lu: Delver Deblk lu he lu reve he grup fr he pepde uppr. luh lu wh Wah: Wahe he lu wh DM reve he deblk lu. Page 3

4 Le 6) Sp daa lle: Iru he fware p lleg he hgra daa. he u f he value dplayed a a bar he hgra. gure 6 llurae a ypal hgra fr he yhe (wh ad whu DIEA bae wah) f a 5 a ad ag equee whu bae wah yle uber gure 6. ypal hgra wh bae wah yle uber he fr bar abu he ae hegh bh wh ad whu he bae wah (e: he Y-ax ale he hgra are dffere). Se uplg ha urred befre he deblk, he uppr ha bee exped he razle-baed reage ad here erferee durg he deblk. he bar red upward he yhe whu he bae wah beaue, geeral, a he pepde grw here re bdg f he razle-baed reage he uppr. Addally, he value fr he bar he yhe whu he bae wah are gfaly larger ha he f he yhe wh he bae wah due he rbu f he razle-baed reage wahu he peak area. he yhe wh he bae wah hw ral vara bar hegh. Durg a ueful yhe he bar ed aa a a level r red lghly dwward durg he yhe. gure 7 a exaple f a hgra f a yhe wh a pr uplg durg he ed yle (wh ad whu bae wah) whu bae wah yle uber gure 7. gra f pr uplg 5000 wh bae wah yle uber here a large bar a he hrd yle e he pr uplg he ed yle lef free a grup exped whh bd he razlebaed reage. he ae yhe wh he bae wah, hwever, hw a all bar fr he hrd yle e le a ad wa aahed he ld uppr ad here wa erferee fr razle-baed reage bdg. Alhugh bh hgra dae ha a prble urred durg pepde aebly, he e wh he bae wah gve a re aurae derp. eedbak Mrg eedbak rg a ehd ha dfe rea e baed he effey f he reval. A lw reval ay be arbued pr aebly he grwg pepde uppr ad hu he ubeque aahe f he a ad ay be dfful. Wh lw depre, a reae he deblk ad/r uplg e ay drve he rea ple ad prdue a purer prdu. he Peer Pepde Syhe Sye ffer he uer everal level f feedbak rl a he yhe f he pepde. he fllwg p derbe he deblk rg relaed pera:. he deblk reage puped hrugh lu ae he reval f he grup fr he ld uppr.. he deblk ep ued ul eher: he ep er ha ued dw zer (Au Exed Sep = ) r he ep er ha ued dw zer ad he hage he deer value ver a perd f e fall belw he lpe far value r he axu deblk e reahed (Au Exed Sep = Dblk r Cupl). 3. he ubeque uplg ep ued ul eher: he ed f he uplg ep exeued (Au Exed Sep = r Dblk) r he uplg e he prl ulpled by a far equal he ra f he exeded deblk e he deblk e he prl (Au Exed Sep = Cupl). r exaple, f he deblk e wa exeded we he e pefed he prl, he he ubeque uplg e dubled. 4. he lu wahed wh DIEA fr he Aux ble reve ay rae f avar bud he ld uppr ha ay erfere he ubeque deblk ep (Au Exed Sep = Dblk r Cupl). ae hee feedbak rg p eher: Chage he Au exed p he Irue Prfle eu. r Chage he Mr p he drp-dw l bxe he Prpere f he Wrka Irue wdw, a derbed belw. he defaul axu deblk e 0 ad a be haged ay value bewee 0 ad 99. Ireag he axu deblk e reae he axu uplg e e he far ued ulply he prl uplg e a be greaer. r exaple, he defaul axu deblk e 0 ad he deblk e he prl 5. herefre, he axu uplg far 0/5 =. If he axu deblk e reaed 0 he he axu uplg far 0/5 = 4. he defaul lpe far 5 ad a be haged ay value bewee 0 ad 99. he lpe far a eaure f he ably f he deer gal. A large lpe far dae ha he gal a vary re durg a e e perd wherea a all lpe far dae here a be lle vara deer gal ad he deblk re lkely be exeded. Page 4

5 rublehg Cau huld alway be exered ludg fr he hgra ha a yhe ha faled. he deer rae huld be exaed ee f here are ay aale ha a expla uexpeed hgra daa. able derbe e feaure f he deer rae ha a be a da f era prble he ye. able. Deer rublehg berva la le fr ere rae Sharp pke appear durg reyle May harp pke (where ar upped be rdued) Lw gal durg reyle y baele Pble Caue ardware prble (eler, flw ell raked) Bad lap Ar rdued durg reyle due a valve faul Ar leakg le (ga valve r fg leak) eage() depleed a ad val Clgged prbe fr Wrg avar eleed ebk Lw lap eergy Dry flw ell he hgra a be al ueful dagg prble. able 3 derbe e addal feaure f he hgra ha a be a da f era prble. able 3. gra rublehg berva he fr hgra bar larger r aller ha he her bar. All hgra bar are g Se bar g A large bar fr he la yle whe ug a yle Pble Caue he deer wa uffely wared up. If he rue dle fr h, he deer whe ff erve he lap lfe ad de ur ul a key preed he Peer keypad r a yhe ubed fr he Wrka. If he deer able durg he eablhe f a baele value fr he hgra alula, he he value fr he fr bar wll be aurae. he ye ad/r lu wa prperly pred ad he baele value wa deered wh ar he flw ell. he uppr de a a eral grup. repve deer due hardware prble (.e., fauly eler, dry r raked flw ell, bad lap) Chery prble (.e., pr uplg, depleed r bad reage). Baele value wa deered wh ar he flw ell. he fal yle ue a exeded deblk ha ay lead hgher al alulaed hgra value. Clu he Peer V dee ye prvde a ueful l fr rg he pepde yhe pre. Several far ha ay pre he pre f he deer daa were defed. M gfaly, razle-baed reage bd he pepde-ld uppr epeally whe here are free a grup. he bud reage erfere wh he dee ad quafa f he peak. A hr DIEA wah a be ued afer he uplg ep reve he bud reage ad reae he auray f he ubeque peak eauree. Alhugh he deer daa a prvde ueful dag ad qualave fra, huld be ued exluvely eaure he ue f he yhe r replae her aalyal ehque (.e., PLC, MS). We are alway ereed hearg yur e ad ag wh ay que yu ay have. Yur e help u ually prve ur prdu. Pleae dre que ad repr ay ue e f ur ffe led belw. ed Sae ad Caada elephe: (800) r (508) AX: (508) E-al: Peer@pb. ed Kgd elephe: AX: rae elephe: AX: Geray ad all her Eurpea ure elephe: AX: Japa elephe: AX: r furher du abu V rg uu flw pepde yhezer ee Aher, E. ad Sheppard,.C., Sld Phae Pepde Syhe- A Praal Apprah (989), IL Pre, pp Drylad, A. ad Sheppard,.C., J. Che. S. Perk ra. I, (986), pp eld, G.B. ad ble,.l., I. J. Pepde Pre e., 33 (989), p. Page 5