Semisynthesis of a shortened open-chain proinsulin

Tetrahedron Letters,Vol.23,No..18,pp Printed in Great Britain

1877-1880,.1982

0040-4039/82/181877-04$03.00/O

01982 Pergamon Press Ltd.

SEMISYNTHESIS OF A SHORTENED OPEN-CHAIN PROINSULIN

Erika E. Biillesbach Deutsches Wollforschungsinstitut an der RWTH Aachen Veltmanplatz 8, D-5100 Aachen FRG

was preTheopenchainproinsulinmcdel : B-chain-Arg-Arg-Alasly-Lys-Arg-A-chain pared frompartialprotectednativeinsulinchainsand syntheticpeptides.

SLImnary:

Al B30 Inthecrystalstructureof insulinthe a-aminogroupGly andthea-carbcucylgroupAla . are separatedby only 8 - 10 A'. Theanalogve structureof insulinwas found inthebiological precursorproinsulin'.In this moleculethe C-terminalamino acid of the insulinB-chainand 1 amino acid of the insulinA-chainare connectedwith a C-peptide(length:27 the N-termina to 35 amino acids).This sequencehas a high species-variability. The connectingpeptidefaci2,3. Hcxeverthis role cannotbe exlitatesthe formationof the correctdisulphidebridges plainedby its lengthand variability. Moreover , its structure-inducing functioncan be reproWA1 EB29 ducedbyalinkagebetween thetwoamino groups inGly via one of severaldiand Lys carbonicacid derivatives4. Proinsulinmodelswith shortenedconnectingpeptidesshouldhave reoxidation properties sB29 similarto GlyClAland Lys linkedinsulins4but, in contrastto these,modelswith a-peptide bonds can be preparedby gene technology. The presentaim is to synthesizeseveralproinsulinmodelswith connectingpeptidesdiffering inlengthand sequencefrcmthe nativeC-peptide.KullmsnnandGutte5prepareda proinsulinanalogue in which theB-chain sequenceB,_2g was connectedwithaaodifiedA-&ain se&ence by the solid phase nethcde. The present work describesthe semisynthesis of the open-chainproinsulinanalogue(B-chain)-Arg-Arg-Ala-Gly-Lys-Arg-(A-chain) as hexa-S-sulphonate. The most importantstepswere: i) the N-terminalelongationof the native insulinA-chainwith syntheticpeptides,ii) the modification of the B-chainto permitselectiveactivationof the a-car-boxy1 group, iii) the condensation of the both partiallyprotected,large fragments,iv) the deprotection of the open-chain proinsulinnrdel,and v) the isolationof the singlechain peptideas hexa-S-sul@onate. For N-termina 1 elongationof nativeA-chainwith syntheticpeptides,the azide math&

(with

its low rate of reaction)as well as the fastermixed anhydridemethod7have been used. l'hestartingmaterialforthe proinsulinnodelwas Arg-A+in*disulphidem==-prepared by a trypticcatalyzedreaction8.Furthercondensationwithsyntheticpeptideswere performed usuallyvia mixed anhydridereactions'.Carboxylvents

ware activatedby reactionwith

iscbutyloqcarbonylchloride in DMF for 2 min at -1O“C.For coupling,the amino cceponats were dissolvedin 0.2MN-methylmxpholine/HClbuffer inm/water4:1at 1877

"pH 7.2andreactedwitha

1878

5-foldexcessof carboxylcuqmnent for 2 min at FT. The reactionwas stoppedby additionof a IO to 50 fold excessof NH20H*Hclin DMF/water4:l at "pH 7.2" for 2 h. ~~.siblyform& Oacyl derivatives were cleavedunder these conditions.Theproteinwasisolatedafteraddition of aceticacid by gel-filtration on SephadexG-25 in 10% aceticacid and lyophilization. The TnzW.timl I.uc n-d-mllcul lx, NM?c.lnPtmnhnrulcic ~T+l ‘x-d -,7n iI‘d-c.-in~+ir.~ kt i-h,2 nzw,.z.,-l_ ..-..-.e.. .._I -s-v__-1 pb-WU"y.NIL"IL _..A U‘.. yL"Uy ..Lc*~LL'y'u~L"A‘"~ ULS _W~A~ techniquelo. was conto the Arg-A-chain. No BythesePro=du= s, the peptideHoc-Gly-Lys(Msc)OH free a-aminogrmpwas observedbyendgroupdeterminaticmlo. The liberationof the a-amino groupby treatment with

TFA (99%)was inccmplete. Nearly 10% iscbutylqcaxbonyl-Arg-A-chain

derivativewasseparatedbyionexchangechrmatographyon SP-Sephadexin 7Mureaand 1.5Macetic acid with a linearNaCl-gradient(0-0.15M). After desaltingand lyophilization Gly-Lys (Msc)-Arg-A-chain*was isolatedin a yield of 73.1%based on Arg-A-chain: The tripeptidyl-Achain was furtherelongatedwith the protectedtetrapeptide Hcc-Thr(But)-Arg-Arg-Ala+H~mosOH.

Altbxghtheamim

componentreactedinccepletly, the isolationof the protectedheptapeptidyl-

A-chainwas notnecessarybecauseaftertreatnentwithT!?A(99%)the chargedifferencebetween unreactedanYino ccqmnent, isobutylcxycarbonylatedamino aqxment7, and the deprotectedproduct allowedseparationby ion exchangechrcsatographr in the previoussystem (withexception that the salt gradientwas frm 0 to 0.4M NaCl).Thr-Arg-Arg-AlaGly-Lys(Msc)-Aq-A-chain*was isolatedin a yield of 47.5%based on tripeptidyl A-chain.In this case the N-terminalamino acid Tbr corresponded to the C-terminalamino acid of the humn B-chain. Par subsequentN-terminalelongationof this heptapeptidyl-A-chain derivative,des-B30-Bchain had to be activatedat the C-t&

us. In an earlierwork the mixed anhydrideactivation

ofanaminoprotectedB_chainderiMtiveha~beenreported~. Inthisexperimnttheselective reactirmof the cr-carboxyl group was not achievedand only low yieldsresulted7.For the selective activationof the C-texmina 1 carboxylgroup,protectionof side chain carboxylgroupswas II,12 The preparationof such a protectednativeB-chainderivativewas possibleby necessa!q . 11,12 arouteinvolvingseveralsteps . Di-S-sul@mnate-des-B30-B-chain has preparedby digestion with ca&xypeptidaseA. TheBmte saltg-roupswerereducedwith 2-mercaptoethano1,and the &_tiioi__> m has ,%qxiram -a.~_. oy gei-filtration on SephadexG-25 in 30 % aceticacid. Cyc72,13 lit disulphideB-chainderivativewas preparedby oxidationwith iodine followedby separationon SephadexG5Of in 10% aceticacid. The esterfication of the three -1

groups

(GluB13 and B21, Lys B29) of this derivativewas performadin methanolusing BE'?. Selective I5 cleavageof the C-terminalmethoxygroup was achievedby trypticdigestion at pH 5.7 in dioxane/water1:I, 6h at FZ. After reactionwith methylsulphonylethyl~ ylsuccinimide ester,both amino groups (PheBI and Lys B29) were protected.The resultingNCXI ,Ns2'-bis(m._ _" yl)-des-B30-B-chain-disulphide~y'5,OYL'ili-methyl ester was purithylsulphonylethyl~ fiedby ionexzhangechrmatographyandconvertedto thehydrcchloridebygel+ronetogi-a*y

on m

G25f in 10-4MXl.

Topreparethe shortenedproinsulinmdel,17.5 ~1

B-chain

derivatives

dissolvedin

0.75mlDMFand 4 equiMlentsN-~thylmorpholine~readded. Under these conditionsthe c+carboxylgroupandbothhistidine side chains~redeprotonated.~emixedanhydridewas formd by reactionwith 2 equivalentsis&utyl chlorofomateby -1O“C for 30 sec. 5 rynolheptapeptidyl-A-chainderivativewere dissolvedin 0.37ml0.2 M-nethylmxpholinein m/water 4:1 at "pH

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7.2", and

addedtothemixedandydride. Thecouplingreactionwas stoppedafter2minby

ad-

ditionof 0.5ml 1 M NH20H~HC1in BlF/water4:l at "pH 7.2". The ntixbre was allowedto stand 12h, then separatedbygel-filtrationonSephadexG50f in1Maceticacidandlyo@ilized (yieldof protectedopen-chainproinsulinnode1 was 29% based cm heptapaptidyl-A-chain). Innumerous experimentsthe reaction~tianswerevariedbutthedescribedyields

could

notbe @xoved. Twoequivalentsofiscbutylchlorofomtewerenecessary,because tworeactions of the carboxylcanponentwere possible:first the formationof the tied anhydrideand secondthe incanpleteformtionof

Nim-isobutyloxycmkmyl derivativeof the histidineresi-

dues. With higherexcessof isobutylchloroform& the fomtion of Ne-iscbutyl~yl derivativesof the aminoccqon~tdaninated. Byelectrophoreticstudiesof several coupling reactionsboth side reactionswere alwaysobserved,but to differentextents.The irreversible blockingof the amine cmponent~slowunder

the describedconditions. The formtionoffl-

isobutyloxycarbonyl side productwas reversibleby the treatmentwith NH20H12andinthealkaline msdim~'~during the cleavageof theMsc-andn~thylprotectinggroups. Thedeprctectionof theapen-chainproinsulinmdelwasperfomedin

O.lMNaOH for3Ominat

O°C underN2 at a proteinconcentratimof lOsy/ml.The reactimwes stoppedbyadditionof glacialaceticacid, and the deprotectedderivativewas purifiedby gel-filtration on Sephadex G50f in 1M acetica$d, lyophilizedand convertedto the hexa-S-sul&onatederivativeby oxidative sul@ktolysis . Thiswas plrifiedbyionexchange chrmatographyonCM-celluloseatpIi 4 in 7M urea, O.lM aceticacid sad elutedwith a NaCl-g-radient (O-O.lM). !Ihe shortened open-

1880

chain proinsulinderivativewas characterized by electrophoresis at pH 2.2 and 4.8, by amino IO acid analysis,end group determination, by trypticdigestionfollowedby electrophoresis (PaulipositivefragmentsA,_21,BI_22,BI_29 and B23_29),anddigestionwith trypsinand carboxypeptidase B18 followedby amino acid analysis (ILysand 3 Arg were determinated fran the "connecting peptide").All

fragmentswere also formedby the analoguedigestionof the hexa-S-

sulphonatebovineproinsulin. 7,8 and the C-terminalelongation In summarywith the N-terminalelongationof the A-chain 12 of the protectedB-chainderivative a semisynthetic math&iwas developedwhich csn be used to prepareseveralshortenedproinsulinderivativesfor systematicinvestigations of the reoxidationproperties.These experimentsshouldallow furtherelucidation of the role of the C-peptidein the biologicalprecursor,proinsulin. Acknowledgemants This mrk‘was financiallysuppxted by the DeutscheForschungsgemeinschaft. I am gratefulto Prof. Dr. H. Zahn and Dr. H.-G. Gattnerfor their constantencouragment duringthe courseof this work. I thank Dr. M. Casarettofor her help in the peptidesynthesis, Dr. J. Fijkles for the amino acid analysis,and Dr. D. Saundersfor the criticalreading of this tiuscript. I thankMrs.G.Ortrmnnfor her competentassistance. Abbreviations: Eoc = tert.butyloxycarbonyl-, But = tert.butyl-, CM = carboxymethyl-, DMF = SP = sulphodin-ethylformsmide, M.A. = mixed anhydride,Msc = mathylsulphonylethyloxycarbonyl-, acid propyl-,TFA = trifluoroacetic References Dodson,G.,Hodgkin,D.& Mercola,D.(1972)AdvancesProt. Chem. 2, 279-402. I. Blundell,T., & Clark,J.L(1968)Prcc.Natl.Acad.Sci.USA 60, 622-629. 2. Steiner,D.F. & Burck,P.J.(1981)Proc. 7thAmxPeptide Syrqzos. 3. Frank,B.H.,Pettee,J.M.,Zinmerman,R.E. (Rich,D.E. ed.) PierceChemicalCo. PockfordIll.,inpress . 4. Geiger,R.(1979)ChemdkerZeitungI&& Ill-129. 3. 899-902. 5. Kullmann.W.& Q&te.B. (1975)Bicchem.Scc.Trans. %, 192-196, 197-200, 201-204. 6. Aiba,H.a Shimmi&,Y. .(198b) Ehll.Chem.Sx.Jpn Fijhles,J. & Zahn,H. (1979)in "Peptides" Prcc. 7. Naithani,V.K., Gattner,H.G., Biillesbach,E.E., 6th Amer.PeptideSympos. (Gross,E.& IMeienhofer,J. eds.) pp 571-576,PierceChemicalCo; F&c&fordIll. * Dsnho,W.& Naithani,V.K.(1980)"Insulin"Proc. 2nd Int. Insulin Sympos. 8. Gattner,H.G., (Brandenburg,D & Wollmer,A.,eds.) pp 117-123Walterde GruyterBerlin-NewYork. 9. Wieland,T.& Bemhard,H. (1951) JustusLiebigsAnn.Chem.572, 190-194;Boissonnas,R.A. (1951) Helv.Chim.Acta 34, 874-879;Vaughan,J.R.& Osato,R.L.(1951)J.Amar.Chem.Soc. 73 3547. IO.Gray,W.R.(1967)MethcdsEhzyml. 11, 139-151 Il.Sdmitt, E.W. (1977)Doctoralthe& RWTH AachenNaithani,V.K. & Fohles,J.(1982)Prcc. 3rd 12.Bi.illesbach,E.E., Schmitt,E.W., Gattner,H.G., USSR-FPGSympos. on Chemistq of Peptidesand Proteins(Voelter,W., WUnsch,E.,Ivanov,V.T., - _ Ovchinnikov,Yu.A.,eds.) Walterde GruyterBerlin-NewYork,inpress. Schmitt,E.W. & Gattner,H.G.(1982)Int.J.Peptide ProteinPes.,inpress . 13.Biillesbach,E.E., Schmitt,E.W. & Naithani,V.K.(1975)Hoppe Seyler'sZ.Physiol.Cbem. 356, lI.Gattner,H.G., 1465-1467. Previero,E.& Zuckerkandl,J. (1969)J.Eibl.Biol. 39 493-501. IS.Coletti-Previero,M.A., ~~hansen,~.~. & Lundt,B.F.(1979)in "Peptides"Proc. 6thAmx.Peptide 16.Gronvald,F.C., eds.) pp 309-312,PierceChemicalCo. RcckfordIll.. Qmpos. (Gross,E.& Meienhofer,J., ;;.~~~J.;. & Cole,R.D.(1959)J.Biol.Chem. 234, 1733-1739. , ., Peterson,J.D. & Steiner,D.F.(1971)J.Biol.Chem. 246, 6786-6791. * The ccmpensating ion of the arginineside chain was not determinated. (Received in Germany 4 December 1981)