DK161026B - Analogifremgangsmaade til fremstilling af antigenderivater - Google Patents

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Den foreliggende opfindelse angår en analogifremgangsmåde til fremstilling af antigenderivater.

Opfindelsen angår især en fremgangsmåde til fremstilling 5 af hidtil ukendte antigenderivater af et antigen og mindst ét muramylpeptid, der skal bindes covalent dertil, eventuelt over et broled. Disse nye antigenderivater kan f.eks. karakteriseres nærmere ved formlen A - [Z0_1 - KP]n (I) 10 hvori A er en rest af et antigen, Z et broled (spacer), MP en rest af et muramylpeptid og n et helt tal større end 0.

Derved forstås ved antigen et organisk stof, der af det fysiologiske medium, d.v.s. den menneskelige eller dyri-15 ske organisme, opfattes som immunologisk fremmed,eller under egnede forudsætninger kan opfattes som fremmed.

Til antigenerne hører i første række samtlige de stoffer, der forårsager specifik immunisering af en levende organisme mod infektiose sygdomsvækkere eller uønskede reak-20 tioner, såsom allergisk sensibilisering eller afstødning af transplanteret fremmedvæv. Herved skal især forstås antigener som indholdsstoffer i vacciner, I betragtning som vacciner kommer på den ene side sådanne, som indenfor rammerne af klassiske vaccinationsfrem-25 gangsmåder kan anvendes til immunologisk beskyttelse mod infektionssygdomme. På tale som antigener, der som aktive stoffer er indeholdt i sådanne vacciner, kommer svækkede levende eller døde, modificerede eller dekomponerede infektionssygdomsvækkere, de af disse sygdomsvækkere dannede toksoider 30 eller naturligt eller syntetisk fremstillede delkomponen-

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2 ter af sygdomsvækkere og toksoider. Som sygdomsvækkerklasser skal især nævnes vira, chlamydier, rickettsier, bakterier, protozoer og metazoiske parasitter. Foretrukne antigener er sådanne, der er egnede som bestanddele 5 af vacciner til behandling af f.eks. influenza A og B, parainfluenza 1-3? af respiratoriske syncytial-virus, rhinovira eller adenovira fremkaldte respiratoriske sygdomme, cytomegali, røde hunde, mæslinger, fåresyge, pertussis, poliomyelitis, herpes simplex 1 og 2, vari-10 celler og herpes zoster, rotavirus-sygdomme, hepatitis A, B og andre, rabies, mund- og klovsyge, trachom, caries, af meningokokker A, B og 0 forårsagede meningitiden, sygdomme som følge af pneumokokker, Ξ. influenza, streptokokker (især rheumatisk feber), Pseodomonas og Proteus, 15 tyfus, paratyfus og andre af enterobakterier fremkaldte diarrésygdomme, gonorrhoe, syfillis, malaria, trypano-somiaser (sovesyge og Chagas’ sygdom), leishmanioser, filarioser, schistosomiaser, ankylostomiaser og andre orme sygdomme.

20 På den anden side skal nævnes nye vacciner, som ikke er rettet mod infektions sygdoms vækkere, men derimod enten mod autologe bestanddele, d.v.s. normale og aberrante autoantigener eller mod sensibiliserende antigener fra omverdenen, d.v.s. allergener.

25 I ét tilfælde tilstræbes det ved immunisering mod normale eller afvigende autoantigener at afbryde funktionen af autologe molekyler (f.eks. af hormoner, af andre mediatorer og af humorale og cellulære receptorer) eller at ophæve udbredelsen eller persistensen af afvigende, navn-30 lig neoplastiske cellelinier. Foretrukne antigener som bestanddele af sådanne vacciner er f.eks. delsekvenser af humant choriongonadotropin eller bestanddele af spermatozoer til immunisering mod mediatorer for fertiliteten og dermed til immunologisk afbrydelse af reproduktions-35 funktionerne, mediatorer for erytemprocesser, især i

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5 renset form, derunder de af lymfocyter afsondrede lymfo-kiner, især ΜΙΡ (macrophage migration inhibitory factor) til immunologisk undertrykkelse af erytemsygdomme, immunologisk specifikke antigenreceptorer på lymfocyter og på 5 antistoffer (fraktionerede antigenspecifikke lymfocyter, fra disse lymfocyter ekstraherede eller secernerede antigenreceptorer, fraktionerede antigenspecifikke antistoffer eller antistoffragmenter) til antiidiotypeimmunise-ring (immunisering mod de for antigenreceptor-strukturer-10 ne karakteristiske autoantigener) med det formål at afbryde specifikke immunreaktioner til elimination af sygdomsfremkaldende immunprocesser, såsom autoimmunitet (f.eks. mod synovialantigener og immungiobuliner ved primær kronisk polyarthritis, mod myelinkomponenter ved 15 degenerative sygdomme i centralnervesystemet, mod TSH-re-ceptorer ved autoimmun thyreoiditis, mod acetylcholin-receptorer i den tværstribede muskulatur ved myasthenia gravis, mod ø-cellekomponenter ved juvenil diabetes etc.) eller allergi (f.eks. mod græspollen, støv eller medika-20 menter ved allergisk astma, allergisk rhinitis eller læge-middeloverfølsomhed) eller til undgåelse af i og for sig normale, men uønskede immunreaktioner (f.eks. induktion af immuntolerance til forhindring af afstødning af transplanterede fremmede organer og væv), autologe eller med 25 dem krydsreagerende homologe eller heterologe tumorceller, tumorcellefragmenter eller -membrankomponenter, herunder onkornaviruskodede glycoproteiner, såsom GP 70, til tumorspecifik immunisering indenfor rammerne af profylaksen og terapien af kræftsygdomme, 50 I et andet tilfælde forsøges det ved immunisering mod allergener i stedet for den patogenetisk relevante IgE-antistofreaktion overvejende eller i tilstrækkelig grad at inducere IgG- og IgÅ-antistoffer mod de sensibiliserende antigener fra omverdenen og derved at opfange aller-55 gener i cirkulationen og i sekreterne ved hjælp af specifikke antistoffer, før disse allergener reagerer med de

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4 til mastceller bundne IgE-antistoffer og dermed kan udløse frigørelsen af allergiske mediatorer, Yed denne antigenspecifikke desensibilisering er allergenerne selv,_ d.v,s. f.eks, græspollen, støv eller medikamenter, antigenbe-5 standdele i vacciner.

Som antigener anvendes fortrinsvis de aktive stoffer i en vaccine, der anvendes mod parasitter, bakterier, vira, tumorceller, svækkede eller dræbte former eller delkomponenter deraf, en vaccine mod autologe bestanddele, im-10 monulogiske genkendelsesstrukturer eller en vaccine, der er egnet til specifik desensibilisering ved allergier.

Som antigener anvendes især også sådanne aktive stoffer i en vaccine, der anvendes mod malaria, cholera, tyfus, meningitis, rheumatisk feber som følge af streptokok-15 infektion, influenza, hundegalskab, mund- og klovsyge, samt sådanne i en vaccine til forebyggelse af tumorsygdomme, til induktion af forplantningssystemet, til desensibilisering eller induktion af specifikke immuntolerancer mod allergier, til genoprettelse af tolerancen mod 20 autologe vævsbestanddele og cirkulerende molekyler.

Som antigener foretrækkes det også især at anvende malaria-merozoiter, typespecifikke meningokokpoly-saccharider A, B eller C, Μ-proteiner fra steptokokker, influenzahæmagglutininer, autologe tumorceller eller 25 tumorcellemembranbestanddele, partialsekvenser af humant choriongonadotropin, græspollenekstrakter, antigen-specifikke T-celle-lymfoblaster og deres receptorer for antigener eller antigenspecifikke immungiobuliner.

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5

Antigener kan, især når de er lavmolekylære, med fordel være fuldstændigt covalent bundet til en højmolekylær bærer, Som bærere skal især nævnes polymere af mælkesyre og dens derivater, som på kædeenden bærer en fri carboxyl-5 gruppe, såsom dens estere med glycolsyrer, polymælkesyre-amider eller mælkesyrepolyesteramider, som f.eks, er beskrevet i britisk patentskrift nr, 932.J82, endvidere alginsyre, polygalacturonsyre, pectinsyre, carboxymethyl-cellulose eller agarose. På tale som bærere kommer end-10 videre basiske, neutrale eller sure polyaminosyrer, som ikke selv er immunogene, såsom polyasparaginsyre, poly-glutaminsyre, polylysin eller polyornithin. I betragtning som bærere kommer i øvrigt også vilkårlige andre antigener af den ovenfor eller i de opførte eksempler angivne 15 art, for så vidt som en immunreaktion mod disse kan accepteres eller endda er ønsket. Således kan f.eks. et HCG-peptid være covalent bundet til tetanustoksoid som bærer.

Ved fremgangsmåden anvendelige muramylpeptider er især syntetisk fremstillede forbindelser med den almene formel ch2oe6 .-0 ζ VvOE1 R4o\å / V« 20 / 1 - Σ - E2 * / pi 3 R'* - CH (D) * \ E8 E10 E11 CON - CH - CON - CH - CH0CH - E12 l7 lq 2 E' (L) Ey (D) 6

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hvori X betyder en carbonyl-, carbonyloxy- eller sulfo-nylgruppe, E^ hydrogen, alkyl, eventuelt substitueret p benzyl eller acyl, E eventuelt substitueret alkyl eller n a carbocyclisk aryl, E og E uafhængigt af hinanden hydro- :z 5 gen, alkyl, eventuelt substitueret benzyl eller acyl, E^ 7 13 hydrogen eller alkyl, E' og E ^ hydrogen eller lavalkyl,

Q

E hydrogen, lavalkyl, frit, foresteret eller foretheret hydroxylavalkyl, frit, foresteret eller foretheret mer-capto-lavalkyl, frit eller acyleret aminolavalkyl, cyclo-10 alkyl med 5 eller 6 carbonatomer, cycloalkyl-lavalkyl, hvis cycloalkylgruppe indeholder 5 eller 6 carbonatomer, eventuelt substitueret aryl eller aralkyl, nitrogenhol- n o digt heterocyclyl- eller heterocyclyl-lavalkyl, E' og E sammen også alkylen med 3 eller 4 carbonatomer, E^ hydro-15 gen eller lavalkyl, grupperne E^, E^ og E^^ uafbængigt af hinanden en eventuelt foresteret eller amideret carboxylgruppe og Ε^ også hydrogen, eller e\ E^ og E^ er trilavalkylsilyl, især trimethylsilyl, eller også oligomere deraf, som f.eks. er beskrevet i Dt-OS nr.

20 2.45Ο.355, og som kan opnås ved isolering fra mikroorganismecellevægge .

De forskellige dele af de nye forbindelser er bundet covalent til hinanden, d.v.s. at antigenet med mindst én af dets funktionelle grupper er forbundet via en i pep-25 tidkemien almindelig binding med muramylpeptidresten, direkte eller via et broled (spacer).

Som broled (spacer) tjener især divalente rester af ali-phatiske forbindelser, f.eks. af sådanne, der har mindst to aminogrupper, mindst én aminogruppe og én carboxyl-30 eller thioearboxylgruppe, eller mindst to carboxyl- eller thiocarboxylgrupper, såsom aliphatiske diaminer, amino-thiocarboxylsyrer, neutrale, basiske eller sure aliphatiske aminosyrer, di- eller oligopeptider.

Som broled skal i første række nævnes a, -diaminoalkaner,

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7 især a, ω-diaminolavalkaner, såsom ethylendiamin, propylen-diamin, tetramethylendiamin, alkyldicarboxylsyrer, såsom ravsyre eller glutarsyre, α,β- eller γ-aminoalkanearboxyl-syrer, fortrinsvis α-amino-lavalkancarboxylsyrer, især de 5 naturlige α-amino-lavalkancarboxylsyrer, såsom glycin, β-alanin, L-alanin, α-amino-isosmørsyre, valin eller leuein.

Som covalente bindings element er skal især nævnes carboxyl-syreester-, carboxylsyreamid-, thiocarboxylsyreester- el-10 ler thi o c arb oxylsyre amidgrupper.

De nævnte nye forbindelser kan have flere covalente bindingselementer alt efter arten af de anvendte broled.

Dermed kan den ovenstående formel I f.eks. få den følgende mere udførlige form 15 Γγ i ' A Ί [χ'Ί — X'" - Ζ,Μ -X1V - ΜΡ (III) .it ^ “0,1 η

LL Μm J

Γς"1

τ J

0-1 hvori Α er antigenresten, T bæreresten, MP muramylpeptid-

1V

resten, Z1 og Z'" divalente broled og X1, X", X'" og Xx covalente bindingselementer, og m og n betyder hele tal større end 0.

20 Alkyl er ligekædet eller forgrenet, i vilkårlig stilling bundet alkyl med indtil 18 carbonatorner, i første række dog lavalkyl.

På tale som substituenter i den eventuelt substituerede alkylgruppe kommer i første række frie eller funktionelt 25 omdannede hydroxyl- eller mercaptogrupper, såsom forethe-

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8 rede eller foresterede hydroxyl- eller mercaptogrupper, f.eks, lavalkoxy- eller lavalkylmercaptogrupper, eller halogenatomer eller frie eller funktionelt omdannede carboxyl-, såsom carbo-lavalkoxy- eller carbamoylgrupper.

5 Derved kan den substituerede alkylgruppe, såsom lavalkyl-gruppen, bære en, to eller flere ens eller forskellige substituenter, især frie hydroxylgrupper eller halogenatomer.

Carbocycliske arylgrupper er især monocycliske samt bi-10 cycliske arylgrupper, i første række phenyl, men også naphthyl. De kan eventuelt være mono-, di- eller polysub-stituerede, f.eks, med lavalkylgrupper, frit, foretheret eller foresteret hydroxyl, f.eks. lavalkoxy eller lav-alkylendioxy, eller halogenatomer og/eller trifluormethyl-15 grupper.

Aralkyl er især aryl-lavalkyl, hvori aryl har den ovenfor angivne betydning, I første række står aryllavalkyl for benzyl eller phenylethyl, hvori phenylkernen kan være mono-, di- eller polysubstitueret.

20 Eventuelt substituerede benzylgrupper er især sådanne benzylgrupper, som i den aromatiske kerne eventuelt er mono-, di- eller polysubstituerede, f.eks. med lavalkyl, frie, foretherede eller foresterede hydroxyl- eller mercaptogrupper, f.eks. lavalkoxy eller lavalkylendioxy, 25 samt lavalkylmercapto- eller trifluormethylgrupper og/ eller halogenatomer.

iiitrogenholdigt heterocyclyl er især en rest af en 5- eller 6-leddet heterocyclisk forbindelse med 1 eller 2 nitrogenatomer i ringen. Det kan være mættet eller umæt-30 tet og kan f.eks. indeholde en anelleret phenylgruppe.

Som sådanne skal f.eks, nævnes en pyrrol-, indan-, pyridyl-eller imidazolring.

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9

En eventuelt foresteret eller amideret carboxylgruppe er i første række selve carboxylgruppen eller en med en lav-alkanol foresteret carboxylgruppe, eller også en carbam-oylgruppe, der er usubstitueret på nitrogenatomet eller 5 mono- eller disubstitueret med alkyl, især lavalkyl, aryl, i første række phenyl eller aralkyl, såsom benzyl. Carbam-oylgruppen kan dog også bære en alkylen-, såsom en tetra-eller pentamethylengruppe, Carbamoylgruppen kan også være substitueret på nitrogenatomet med en carbamoyl-10 methylgruppe.

Acyl er især en acylrest af en organisk syre, især af en organisk carboxylsyre. Således er acyl især alkanoyl, fremfor alt med 2 til 18 carbonatomer, i første række dog lavalkanoyl, eller også aroyl, såsom naphthoyl-1, naphth-15 oyl-2 og især benzoyl eller med halogen, lavalkyl, lav-alkoxy, trifluormethyl, hydroxy eller lavalkanoyloxy substitueret benzoyl eller naphthoyl, eller også en acylrest af en organisk sulfonsyre, f.eks, af en alkansulfon-syre, især af en lavalkansulfonsyre, eller af en aryl-20 sulfonsyre, især af en eventuelt lavalkyl- .eller halogensubstitueret phenylsulfonsyre, såsom benzensulfonsyre eller p-toluensulfonsyre, samt carbamoyl, f.eks. usubstitueret carbamoyl, lavalkylcarbamoyl eller arylcarbamoyl, såsom methyl- eller phenyl-carbamoyl.

25 Eoresteret eller foretheret hydroxyl er især lavalkoxy eller lavacyloxy, såsom lavalkanoyloxy.

Eoresteret eller foretheret mercapto er især lavalkylmer-capto- eller lavacyl-, såsom lavalkanoylmercapto,

Acyleret amino er især lavalkanoylamino eller carbamoyl-30 amino.

De i sammenhæng med den foreliggende beskrivelse og kravene med "lav" betegnede grupper og forbindelser indehol

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10 der fortrinsvis indtil 7 og i første række indtil 4 carbon-at orner, I det ovenstående og det efterfølgende kan de almene begreber have følgende betydning: 5 Lavalkyl er f.eks. n-propyl, n-butyl, isobutyl, sek,butyl eller tert.butyl, endvidere n-pentyl, n-hexyl, isohexyl eller n-heptyl og i første række methyl eller ethyl, I aryl-, cycloalkyl- eller heterocyclyllavalkyl er lavalkylgruppen især methyl eller ethyl, idet aryl-, cycloalkyl- eller 10 heterocyclylgruppen har den ovenfor angivne betydning,

Lavalkoxy er f.eks. n-propoxy, n-butoxy, isobutoxy, sek.butoxy eller tert.butoxy og i første række methoxy eller ethoxy.

Lavalkylmercapto er f.eks, n-propyl, n-butyl, isobutyl, 15 sek.butyl eller tert.butylmercapto og i første række methylmercapto eller etbylmercapto.

Lavalkylendioxy er især methylendioxy, ethylen- eller propylendioxy.

Halogen står for fluor eller brom, fortrinsvis dog for 20 chlor.

Lavalkanoyl er især propionyl eller butyryl, i første række dog acetyl.

De nye forbindelser ifølge den foreliggende opfindelse kan foreligge i form af blandinger af isomere eller af 25 rene isomere.

Det er kendt, at muramylpeptider er gode adjuvanser, som i egnet blanding med antigener formår at forøge immunogeni-teten af disse. Det har ganske vist vist sig, at de kun

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11 udøver en kortvarig virkning, da de relativt hurtigt udskilles fra den menneskelige eller dyriske organisme.

Fremfor alt er de kun under bestemte, til kliniske formål ikke egnede betingelser, nemlig i blanding med antigener 5 i en emulsion med mineralolie i stand til in vivo at inducere en celleformidlet immunitet mod opløselige antigener.

De ifølge den foreliggende opfindelse fremstillede nye forbindelser frembringer imidlertid en udpræget forøgelse af immunreaktionen på antigenet, især også en celleformidlet immuni-10 tet under klinisk acceptable administreringsbetingelser, hvilket kan vises ved hjælp af de i det følgende beskrevne forsøgsanordninger.

1, Potensering af den celleformidlede immunitet in vivo: Forøgelse af langsomt reagerende overfølsomhed mod 15 bovint serumalbumin (BSA) og mod fåreerythrocyter (SEBC) hos marsvin,

Pirbright marsvin immuniseres på dag O med 1 mg BSA eller med 1 mg SEBC-"Ghosts" (SEBCG) i komplet Freund1sk adju-vans ved injektion af 0,1 ml af en antigen-adjuvans-blan-20 ding i hver af bagpoterne, 3 uger senere udløses hudreaktioner ved intracutan injektion af 100 ug BSA eller 100 μg SEBCG i 0,1 ml pufferholdig fysiologisk saltopløsning, og disse reaktioner bestemmes kvantitativt 24 timer senere på grundlag af det ved hjælp af erytemfladen og hudtykkel-25 sesforøgelsen beregnede reaktionsvolumen. Den efter 24 timer (langsom reaktionstype) iagttagne antigenspecifikke forøgelse af reaktionsvolumenet er et mål for den celleformidlede immunitet. BSA og SEBCG er for svagt immonoge-ne til alene eller i en vand-olie-emulsion med ufuldstæn-30 dig Freund'sk adjuvans (10 dele BSA-opløsning respektive SEBCG-suspension i 0,9% Bad blandet med 8,5 dele "Bayol F" og 1,5 dele "Arlacel A") at inducere en reaktion af langsom type, men skal derimod til effektiv immunisering applikeres i komplet adjuvans, hvortil der er sat myco-35 bakterier (5 mg dræbte og lyofiliserede M, butyricum pr.

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12 10 ml "Bayol i"'/,,uArlacel A") · I stedet for mycobakterier applikeres de nye forbindelser, der som antigen indeholder BSA (1 mg BSA, 60 Ug MDP pr. dyr) eller SEBCG Cl m.1 SEBOG, 25 ug MDP pr. dyr) enten som 5 antigen-olie-blanding eller suspenderet i carboxymethyl-cellulose (CMC). De nye forbindelser inducerer under fraværelse af mycobakterier i den beskrevne forsøgsanordning reaktioner af langsom type.

En signifikant potensering af den langsomme reaktivitet 10 mod BSA og mod SEBCG kan også opnås ved applikation af de nye forbindelser ikke inkorporeret i ufuldstændigt Freund1sk adjuvans, men derimod suspenderet i CMC, I dette tilfælde viser intramuskulær applikation sig at være særligt virksom. Under disse omstændigheder er den samme 15 mængde frit muramylpeptid, der blandes i CMC-blandingen, væsentligt mindre aktiv end det nye virksomme stof. Dette viser, at de nye forbindelser under klinisk acceptable administreringsbetingelser, d.v.s. ved applikation med stofvenlige ledsagestoffer, formår at inducere cellefor-20 midlet immunitet, selv mod et opløseligt proteinantigen, 2, Potensering af den celleformidlede immunitet in vivo: Eorøgelse af langsomt reagerende overfølsomhed mod BSA og mod SEBCG hos mus, MAG-Hanmus Immuniseredes på dag 0 med trindelte doser af 25 med muramylpeptid ikke-konjugerede antigener (BSA-agarose eller SEBCG) eller af med muramylpeptid konjugerede antigener (BSA-agarose eller SEBCG). BSA-Agarose-præparaterne applikeres subcutant i en dosering på 0,1 til 100 ug (svarer ved de nye forbindelser med muranylpeptider til 30 en dosis virksomt stof på 0,0029-6 ug pr. dyr) i et volumen på 0,2 ml pufferholdig fysiologisk natriumchloridop-løsning. SEBCG-Præparaterne applikeres i en dosering på 0,01 til 3 mg (svarer ved de nye forbindelser med muramyl-

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13 peptider til en dosis virksomt stof på 0,25 - 75 ug pr. dyr) i et volumen på 0,5 ml i pufferholdig fysiologisk saltopløsning intraperitonealt eller 0,05 ml intradermalt respektive fordelt i 3 poter.

5 4 til 20 dage senere udløses reaktioner af langsom type ved injektion af 100 ug BSA respektive 10^ SEBO i 20 ul pufferlioldig fysiologisk saltopløsning- i den venstre bagpote, og disse reaktioner bestemmes kvantitativt på grundlag af det 24 og 48 timer senere ved hjælp af potens 10 opsvulmning konstaterede reaktionsvolumen. Den iagttagne 'antigenspecifikke forøgelse af potevolumenet er et mål for den celleformidlede immunitet.

På 14, dag efter immunisering med BSA-agarose-MDP-forbin-delser optræder der allerede ved meget lav dosering 15 (0,1 pg, svarer til 0,006 pg virksomt stof) udprægede reaktioner af langsom type. I modsætning dertil er frit BAS-agarose ikke i stand til at sensibilisere for reaktioner af langsom type. ligeledes: er SBBCG-MDP-forbindel-ser fremfor alt efter intradermal;, applikation i stand til 20 at inducere en langsom reaktivitet, der er signifikant mere udpræget end den, man kan ophå efter sensibilisering med SEBOG, som ikke er forbundet med muramylpeptid.

Dette viser ligeledes, at de nye forbindelser formår at forøge den cellulære immunitet væsentligt, 25 3· Potensering af den humorale immunitet in vivo:

Forøgelse af antistofproduktionen mod BSA hos mus.

MEI-Mus immuniseres ved 8 intraperitoneal injektion af 0,1 til 100 ug BSA forbundet med muramylpeptider (0,0014 til 6,0 ug virksomt stof) på dag 0. 10, 17 og 28 dage 30 senere udtages serumprøver, der undersøges for deres indhold af anti-BSA-antistoffer med en passiv bæmagglutina-tionsteknik, I den anvendte dosis er frit BSA subimmuno-

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14 gent for modtagerdyrene, d.v.s. at det kan udløse ingen eller kun en ganske ringe produktion af antistoffer. Forbindelser af BSA med et muramylpeptid muliggør en dobbelt til tredobbelt forøgelse af antistoftiteren i serum 5 (titersummen af log,-, titerdifferencerne på tre blødnings-dage). Bemærkelsesværdigt er det desuden, at de nye forbindelser, der desuden kobles til agarose som bærer, efter intraperitoneal eller subcutan administrering er endnu stærkere immonogene.

10 Ved hjælp af de skildrede forsøg vises det, at de omhandlede forbindelser også formår at forøge den humorale immunitet væsentligt.

De ifølge opfindelsen fremstillede nye antigenderivater 15 er kendte eller forbedrede kendte vacciner og tjener til nye vaccinationsmetoder eller til forenkling af almindelige vaccinationsmetoder, idet f.eks. det antal vaccinationer, der er nødvendige til opretholdelse af beskyttelsen over længere tidsrum, kan sænkes.

20 Opfindelsen angår især fremstillingen af nye antigenderivater, der indeholder antigen, eventuelt covalent bundet over broled til muramylpeptider med formlen II, hvori R1, r3, r4, r5 0g r7 betyder hydrogen, X carbonyl og r2 eventuelt med hydroxy eller lavalkoxy substitueret lavalkyl 25 eller eventuelt hydroxy, lavalkoxy, lavalkyl eller halogen substitueret phenyl og R®, R^, rIO, rH, r3-2 0g r13 har den ovenfor angivne betydning.

Opfindelsen angår navnlig fremstillingen af nye antigenderivater, der indeholder antigen, eventuelt covalent 30 bundet over broled til mutamylpeptider med formlen II, hvori Ri, R^, r6 Dg b7 betyder hydrogen, X carbonyl, R^ eventuelt med hydroxy eller lavalkoxy substitueret lavalkyl eller eventuelt med hydroxy, lavalkoxy, lavalkyl eller halogen substitueret phenyl og R^ methyl, og R^,

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15 R^, rIO^ rH, r!2 0g r13 har cien ovenfor angivne betydning.

Opfindelsen angår i første række fremstillingen af nye 5 antigenderivater, der indeholder antigener, eventuelt covalent bundet over broled til muramylpeptider med formlen II, hvori Ri, R^, r6 Qg R^-2 betyder hydrogen, X carbonyl, R2 eventuelt med hydroxy eller methoxy substitueret lavalkyl eller eventuelt med hydroxy, methoxy, 10 methyl, ethyl eller halogen substitueret phenyl, R2 hydrogen eller methyl, b7 og R^ hydrogen, R2 lavalkyl, lavalkylmercapto-lavalkyl, hydroxylavalkyl, benzyl, p-hydroxybenzyl eller phenyl og R12, R^, R^·2 carboxyl, lavalkoxycarbonyl eller carbamoyl og også hydrogen.

15 Opfindelsen angår fremfor alt fremstillingen af nye antigenderivater, der indeholder antigener, eventuelt covalent bundet over broled til muramylpeptider med formlen II, hvori Ri, R^, r6, r7 0g R^·2 betyder hydrogen, X carbonyl, R2 eventuelt med hydroxy eller methoxy 20 substitueret lavalkyl eller eventuelt med hydroxy, methoxy, mthyl, ethyl eller halogen substitueret phenyl, R2 og R^ hydrogen eller methyl, R2 methyl, ethyl, n-propyl, isopropyl, 2-methylpropyl, methylmercaptomethyl, hydroxymethyl, hydroxyethyl, phenyl, benzyl eller p-hydroxyben-25 zyl og R1^, R11 og R12 carboxy, lavalkoxycarbonyl eller carbamoyl og R11 også hydrogen.

Opfindelsen angår også fremstillingen af nye antigenderivater, der indeholder antigener, eventuelt covalent bundet over broled, til muramylpeptider med formlen II, 30 hvori R-*-, R^, R2 og R^·2 betyder hydrogen, X carbonyl, r7 og R2 sammen propylen eller butylen, og R2, R2, R2, R12, R1·1· og R·*·2 har den ovenfor angivne betydning.

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16

Broled i de ovenstående definitiuoner er især α,ω-di-aminolavalkaner, lavalkyl-dicarboxylsyrer, og naturligt forekommende a-amino-lavalkancarboxylsyrer.

5 Opfindelsen angår især de ifølge opfindelsen fremstillede, nye i eksemplerne beskrevne antigenderivater.

De nye forbindelser kan fremstilles ved i og for sig kendte metoder.

Således kan de opnås ved, at man kondenserer et eventuelt 10 med broled sammenknyttet antigen med eventuelt med broled s ammenknyttede muramylpeptider, idet den ene af de to dele indeholder frie amino-, hydroxy eller mercaptogrupper, og den anden carboxylsyregrupper, og om ønsket kondenserer den opnåede forbindelse med en eventuelt med broled for-15 bundet bærer.

Kondensationen sker derved f.eks. på den måde, at man omsætter den ene forbindelse i form af en aktiveret carboxylsyre med den anden forbindelse som fri amino-, hydroxyl-eller mercaptoforbindelse. Den aktiverede carboxylgruppe 20 kan f.eks. være et syreanhydrid, fortrinsvis et syreazid, et syreamid, såsom et imidazolid, eller isooxazolid, eller en aktiveret ester. Som aktiverede estere skal især.nævnes cyanmethylestere, carboxymethylestere, p-nitrophenylthio-estere, methoxyethylthioestere, acetylaminoethylthio-25 estere, p-nitrophenylestere, 2,4-,5-trichlorphenylestere, N-hydroxysuccinimidester, N-hydroxyphthalimidestere, 8-hydroxy^uinolinestere og N-hydroxypiperidinestere. Aktive estere kan eventuelt også opnås med et carbodiimid under tilsætning af K-hydr oxysue c inimid eller en usubsti-

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17 tueret eller f,eks, med halogen, methyl eller methoxy substitueret 1-hydroxybenzotriazol eller 3-hydroxy-zl—oxo-3,4~dihydro-benzo [d]-l, 2,3-triazin,

De ved denne kondensation anvendte fraspaltningsgrupper 5 må være ugiftige eller letfJernelige for at undgå, at de for det meste høgmolekylære forbindelser ved adsorption tilbageholder giftige bestanddele.

Man foretrækker derfor som aktive estere sådanne med ΪΓ-hydroxysuccinimid eller deres C-substitutionsprodukter, 10 såsom H-hydroxy-methyl- eller -dimethylsuccinimid, eller omsætning med carbodiimid, såsom carbodiimid selv eller 1-ethyl-3-(3-dimethylaminopropyl)-c arb odiimid♦

De ovenstående reaktioner gennemføres på sædvanlig måde under tilstedeværelse eller fraværelse af fortyndings-15 eller kondensationsmidler og/eller katalytiske midler, om nødvendigt ved nedsat eller forhøjet temperatur, .For ikke at ødelægge antigenerne arbejder man fortrinsvis i vandigt miljø og i et pH-område fra 6 til 9, i første række fra 7 til 8, 20 Opfindelsen angår også de udførelsesformer af fremgangsmåden, hvorved man går ud fra en på et eller andet trin af fremgangsmåden som mellemprodukt opnået forbindelse og gennemfører de manglende fremgangsmådetrin, eller hvorved man fremstiller udgangsstoffer under fremgangs-25 mådebetingelserne, eller hvorved man eventuelt anvender en reaktionskomponent i form af dens derivater, såsom dens salte og/eller i form af isomerblandinger eller rene isomere.

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18

Til gennemførelse af de omhandlede kondensationer anvender man hensigtsmæssigt sådanne udgangsstoffer, der fører til de ovenfor særligt omtalte grupper af slutprodukter og fremfor alt til de specielt beskrevne eller fremhævede 5 slutprodukter.

De anvendte udgangsstoffer er kendte eller kan, såfremt de er nye, fremstilles ifølge kendte metoder.

De ifølge opfindelsen fremstillede, antigenderivater kan anvendes i form af farmaceutiske præparater, som f.eks. er til enteral, såsom 10 oral eller rektal, samt parenteral administrering til varmblodede dyr eller mennesker. Præparaterne indeholder det farmakologisk virksomme stof alene eller sammen med et farmaceutisk anvendeligt bæremateriale.

De farmaceutiske præparater indeholder fra ca. 10 til 15 ca, 95%} fortrinsvis fra ca. ?0 til ca. 90% af det virksomme stof. De farmaceutiske præparater kan foreligge i enhedsdosisform, f.eks. som dragées, tabletter, kapsler, suppositorier eller ampuller.

De farmaceutiske præparater fremstilles på i og for sig kendt 20 måde, f.eks. ved konventionelle blandings- granulerings-, dragerings-, opløsnings- eller lyofiliseringsfremgangsmåder. Således kan farmaceutiske præparater til oral anvendelse opnås ved, at man kombinerer det virksomme stof med faste bærestoffer, eventuelt granulerer en opnået blanding, og for-25 arbejder blandingen eller granulatet, om ønsket eller nødvendigt efter tilsætning af egnede hjælpestoffer, til tabletter eller dragéekerner.

Egnede bærestoffer er især fyldstoffer, såsom sukker, f.eks. lactose, saccharose, mannitol eller sorbitol, cel-

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19 lulosepræparater og/eller calciumphosphater, f.eks. tri-calciumphosphat eller calciumhydrogenphosphat, endvidere bindemidler, såsom stivelsesklister, under anvendelse f.eks. af majs-, hvede-, ris- eller kartoffelstivelse, 5 gelatine, tragant, methylcellulose, hydroxypropyl-methylcellulose, natriumcarboxymethylcellulose og/eller polyvinylpyrrolidon, og/eller om ønsket sprængmidler, såsom de ovennævnte stivelser, endvidere carboxymethylsti-velse, tværbundet polyvinylpyrrolidon, agar, alginsyre 10 eller et salt deraf, såsom natriumalginat; hjælpemidler er i første række fluiditetsregulerings- og smøremidler, f.eks. kiselsyre, talk, stearinsyre eller salte deraf, såsom magnesium- eller calciumstearat og/eller poly-ethylenglycol. Dragéekerner forsynes med egnede, eventu-15 elt mavesaftresistente overtræk, hvorved man bl.a. anvender koncentrerede sukkeropløsninger, som eventuelt indeholder gummi arabicum, talk, polyvinylpyrrolidon, poly-ethylenglycol og/eller titandioxid, lakopløsninger i egnede organiske opløsningsmidler eller opløsningsmiddel-20 blandinger, eller til fremstilling af mavesaftresistente overtræk, opløsninger af egnede cellulosepræparater, såsom acetylcellulosephthalat eller hydroxypropylmethyl-cellulosephthalat. Til tabletterne eller dragéeovertrækkene kan sættes farvestoffer eller pigmenter, f.eks. til 25 identificering eller til kendetegning af forskellige virksomme stoffer.

Andre oralt anvendelige farmaceutiske præparater er stikkapsler af gelatine samt bløde lukkede kapsler af gelatine og en blødgører, såsom glycerol eller sorbitol.

30 Stikkapslerne kan indeholde det virksomme stof i form af et granulat, f.eks. i blanding med fyldstoffer, såsom lactose, bindemidler, såsom stivelser og/eller glidemid-ler, såsom talk eller magnesiumstearat og eventuelt stabilisatorer. I bløde kapsler er det virksomme stof for-35 trinsvis opløst eller suspenderet i egnede væsker, såsom fede olier, paraffinolie eller flydende polyethylengly-

i; DK 161026B

20 coler, idet der ligeledes kan være tilsat stabilisatorer.

Til parenteral administrering egner sig i første række vandige opløsninger af et virksomt stof i vandopløselig form, f.eks. af et vandopløseligt salt, endvidere suspen-5 sioner af det virksomme stof, såsom tilsvarende olieagti-ge injektionssuspensioner, hvorved man anvender egnede lipofile opløsningsmidler eller vehikler, såsom fede olier, f.eks. sesamolie, eller syntetiske fedtsyreestere, f.eks. ethyloleat eller triglycerider, eller vandige 10 injektionssuspensioner, som indeholder viskositetsfor-højende stoffer, f.eks. natriumcarboxymethylcellulose, sorbitol og/eller dextran og eventuelt stabilisatorer.

En foretrukket applikationsform består af en opløsning eller suspension af de nye antigenderivater indeholdende 15 fortrinsvis indtil 10 vægtprocent carboxymethylcellulose.

Doseringen af det virksomme stof afhænger af det varmblodede dyrs art, legemsvægt og alder og af den individuelle tilstand samt af applikationsmåden.

Derved applikeres de nye vacciner i analogi med de ved 20 kendte vaccinationsfremgangsmåder vedtagne og kendte doseringer i vægtenheder eller internationale enheder; man indgiver f.eks. lymfoblastholdige antigenderivater i en mængde, der pr. injektion indeholder 10-10x celleorganismer én til seks gange med mellemrum på 2 til 8 25 uger. Pr. mg protein skal indholdet af mur amylp ep tider derved fortrinsvis være 5 til 200 μg,

Til immunisering med opløselige forbindelser anvender man fortrinsvis den tilsvarende mængde antigenderivater i en

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21 saltopløsning (BSS), som består af 0,14 g calciumchlorid, 8,0 g natriumet!orid, 0,2 g magnesiumsulfat.72^5 0,2 g magnesiumchlorid.GH^O, 0,6 g kaliumdihydrogenphosphat og 0,24 g dinatriumhydrogenphosphat. 211^0 pr. 1 liter vand, 5 Såfremt en depotvirkning tilstræbes (f.eks, ved lokal, intradermal eller intramolekylær applikation) kan der til det således opløste antigenderivat yderligere sættes carboxymethylcellulose (slutkoncentration fortrinsvis 5°/°)-

Ikke-opløselige ma'kromolekylære antigenderivater applike-10 rer man fortrinsvis som suspension i BSS og carboxymethylcellulose som stabilisator (slutkoncentration fortrinsvis 5%)* Til fremstilling af en stabil suspension behandles den på is afkølede blanding derved fortrinsvis koncentreret med ultralyd, 15 Antigenderivater med celler applikeres i første række i et for den pågældende celletype særligt egnet vævskulturmedium (f.eks. for lymfocyter "EAGLE1 s high, amino acid medium") [gfr. Click et al. Cell. Immunol, vol, 3, Ρ» 264-276 (1972)].

20 De nye muramylpeptider med formlen II, der anvendes som udgangsstoffer, og hvori X betyder en carbonyl- gruppe, R^ hydrogen, alkyl, eventuelt substitueret 2 benzyl eller acyl, R eventuelt substitueret 4 6 alkyl eller carbocyclisk aryl, R og E uafhængigt af 25 hinanden hydrogen, alkyl, eventuelt substitueret benzyl eller acyl, B/ hydrogen eller alkyl, mindst en af grupperne R^, R^ og R1^ lavalkyl, i første række methyl, og de

O

andre hydrogen, R hydrogen, lavalkyl, frit, foresteret eller foretheret hydroxylavalkyl, frit, foresteret eller 30 foretheret mercaptolavalkyl, frit eller acyleret amino-lavalkyl, cycloalkyl med 5 eller 6 carbonatomer, cyclo-alkyl-lavalkyl, hvis cycloalkylgruppe indeholder 5 eller 6 carbonatomer, eventuelt substitueret aryl eller aralkyl, nitrogenholdigt heterocyclyl- eller heterocyclyllavalkyl,

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22 R7 og R8 sammen også alkylen med 3 ellen 4 carbonatomer og grupperne R^, R^1 og R12 uafhængigt af hinanden en eventuelt foresteret eller amideret carboxylgruppe og R11 også hydrogen, kan fremstilles ved at man på i og for sig 5 kendt måde kondenserer en forbindelse med formlen oh2or°6 / 0 " Εθ1 (IV)

Eo4(y|_Y

, / Έ - X - R2 RJ - OH (D) I -i ?

COOH

hvori X, R2, R3 og R"^ har den ovenfor angivne betydning og R°\ R0^ og R°8 står for grupperne r\ R^ eller R8, eller for en let fraspaltelig beskyttelsesgruppe, eller 10 et derivat deraf med en forbindelse med formlen R°8 -gOlO -gOll ΉΕ - OH - COH - CHCH2CH - R°12 (V) R7 (L) R9 hvori R°8, R°"^ og R0^^ samt R0^"2 har den for R8, R^"8, 11 1? R og R angivne betydning, med den bestemmelse., at i disse grupper tilstedeværende carboxyl- og om ønsket frie 15 hydroxylgrupper er beskyttet med letfraspaltelige beskyttelsesgrupper, og fraspalter eventuelt tilstedeværende beskyttelsesgrupper.

Kondensationen sker derved f.eks. på den måde, at man omsætter forbindelsen IV i form af den aktiverede carboxyl-20 syre med aminof orbindelsen V, eller at man omsætter syren IV med forbindelsen V, hvis aminogruppe foreligger

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23 i aktiveret form. Den aktiverede carboxylgruppe kan f.eks. være et syreanhydrid, fortrinsvis et blandet syreanhydrid, såsom et syreazid, et syreamid, såsom et imidazolid eller isoxazolid, eller en aktiveret ester. Som aktiverede este-5 re skal især nævnes cyanmethylester, carboxymethylester, p-nitrophenylthioester, p-nitroplienylester, 2,4,5-tri-chlorphenylester, pentachlorphenylester, IT- hydroxy sue c in-imidester, N-hydroxyphthalimidester, 8-hydroxyquinolin-ester, 2-hydroxy-l, 2-dihydro-l-carboethoxy-quinolinester, 10 N-hydroxypiperidinester eller enolester, der opnås med N-ethyl-5-phenyl~isoxazolium-3'-sulfonat. Aktiverede estere kan eventuelt også opnås med et carbodiimid under tilsætning af N-hydroxysuccinimid eller en usubstitueret eller f.eks. med halogen, methyl eller methoxy substitue-15 ret 1-hydroxybenzotriazol, 3-hydroxy-4-oxo-3,4-dihydro-benzo[d]-l,2,3-triazin,

Aminogruppen er f.eks. aktiveret ved reaktion med et phosphitamid.

Blandt metoderne til omsætning med aktiverede estere skal 20 især omtales dem med N-ethyl-5-phenylisoxazolium-3'-- sulfonat (Woodward Beagens E) eller 2-ethoxy-l,2-dihydro- 1-carboethoxy-quinolin eller carbodiimid.

Letfraspaltelige beskyttelsesgrupper er sådanne, der er kendt fra peptid- eller sukkerkemien. Som carboxylgrup-25 per skal især nævnes tert,butyl, benzyl eller benzhydryl, og som hydroxylgrupper især acylgrupper, f.eks. lavalk-anoylgrupper, såsom acetyl, aroylgrupper, såsom benzoyl og fremfor alt af kulsyre afledte grupper, såsom benzyl-oxycarbonyl eller lavalkoxycarbonyl, eller alkyl, især 30 tert.butyl, eventuelt med nitro, lavalkoxy eller halogen substitueret benzyl eller tetrahydropyranyl, eller eventuelt substituerede alkylidengrupper, som forbinder oxygenatomerne i 4- og 6-stilling, Sådanne alkylidengrupper er især en lavalkyliden-, i første række en

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24 ethyliden-, isopropyliden- eller propylidengruppe eller også en eventuelt substitueret, fortrinsvis i p-stilling substitueret benzylidengruppe.

Disse beskyttelsesgrupper kan fraspaltes på i og for sig 5 kendt måde. Således kan man fjerne dem hydrogenolytisk, f.eks. med bydrogen under tilstedeværelse af en ædelmetal-, såsom palladium- eller platinkatalysator, eller ved sur hydrolyse.

De anvendte udgangsstoffer er kendte eller kan fremstil-10 les på i og for sig kendt måde.

En anden fremgangsmåde til fremstilling af disse nye udgangsstoffer består i, at man på' i og for sig kendt måde kondenserer en fcy?bindelse med formlen CH2 - 0R°6 -0 ,/q \rv^O-R01 (VI) R°4 - o\| / o / Η - X - R2 R -CH fel3 \ f8

COE - CH - COOH

U

R' 15 hvori R2fR°\ R3, R°4, R0^, R*7 og R0^ bar den ovenfor angivne betydning, med en forbindelse med formlen

βο10 r°H

BH - is - CH20H - R012 (VII) r9 hvori R0^, R0^"3· og R0^2 bar den ovenfor angivne betydning, med den bestemmelse, at i grupperne R0^, R0*^,

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25 R0·^1 og R0·^ tilstedeværende carboxyl- og om ønsket frie hydroxylgrupper er beskyttet med le|;fraspaltelige beskyttelsesgrupper, og fraspalter eventuelt tilstedeværende beskyttelsesgrupper.

5 Kondensationen sker derved f.eks, på den måde, at man omsætter forbindelsen VI i form af den aktiverede carboxylsyre med aminoforbindelsen VII, eller at man omsætter syren VI med forbindelsen VII, hvis aminogrupper foreligger i aktiveret form. Den aktiverede carboxylgruppe kan 10 f.eks. være et syreanhydrid, fortrinsvis et blandet syreanhydrid, et syreamid eller en aktiveret ester. På tale som sådanne kommer især de ovennævnte syreanhydrider, amider eller estere. Aminogruppen er f.eks. aktiveret ved reaktion med et phosphitamid.

15 Også de letfraspaltelige beskyttelsesgrupper svarer til de allerede ovenfor omtalte. De kan fraspaltes på i og for sig kendt måde, f.eks, hydrogenolytisk, eksempelvis med hydrogen under tilstedeværelse af en ædelmetal-, såsom palladium- eller platinkatalysator eller ved sur hydrolyse.

20 Udgangsstofferne kan opnås på i og for sig kendt måde.

Således kan man f.eks. omsætte tilsvarende i J-stilling usubstituerede sukkere med en halogen-R3-acetamido-R° -eddikesyre, eller en forbindelse med formlen iv med en amino-R?^-eddikesyre, hvis carboxylgruppe er beskyttet på 25 den ovenfor viste måde, og fraspalte beskyttelsesgruppen.

En anden fremgangsmåde til udførelse af den i 3-stilling i sukkerresten siddende sidekæde består i, at man omsætter en forbindelse med formlen

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26 CH2 - O - E°6 /-° (oh /^° - r01 (VHI) E°4 _ o\_/ Ή - X - E2 R1? hvori X, E3 E°\ E0^, E0^ og E1^ har den ovenfor angivne betydning, og hvori eventuelt tilstedeværende hydroxyl-grupper er beskyttet med en letfraspaltelig beskyttelses-5 gruppe, med en forbindelse med formlen E°8 ^olO E0·*"1 Z - OH - OOHCH - COH - OH - 0H20H - E°12 (IX) É3· E7 É9 hvori Z er en reaktionsdygtigt foresteret hydroxylgruppe og R3, R7, R9, R°3, R°10/ R011 og R°12 har den ovenfor angivne betydning, og fraspalter eventuelt tilstedeværende 10 beskyttelsesgrupper.

En reaktionsdygtigt foresteret hydroxylgruppe er især en med en stærk uorganisk eller organisk syre foresteret hydroxylgruppe, i første række en sådan, der er foresteret med hydrogenhalogenider, såsom hydrogenchlorid-, -bromid 15 eller -iodid.

De letfraspaltelige beskyttelsesgrupper svarer til de allerede ovenfor omtalte. De kan fraspaltes på i og for sig kendt måde, f.eks. hydrogenolytisk, f.eks. med hydrogen under tilstedeværelse af en ædelmetal-, såsom palladium-20 eller platinkatalysator eller ved sur hydrolyse.

De nye, som udgangsstoffer anvendte muramylpeptider med formlen 14 6 II, hvori X betyder en carbonylgruppe, R , R og R trilav- 2 alkylsilyl, i første række trimethylsilyl, R eventuelt 3 27

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substitueret alkyl eller carbocyclisk aryl, R hydrogen eller alkyl, 7 13 8 R' og R J hydrogen eller lavalkyl, R hydrogen, lavalkyl, frit, foresteret eller foretlieret hydroxylavalkyl, frit, foresteret eller foretheret mercapto-lavalkyl, frit eller acyle-5 ret aminolavalkyl, cycloalkyl med 5 eller 6 carbonatomer, cycloalkyl-lavalkyl, hvis cycloalkylgruppe indeholder 5 eller 6 carbonatomer, eventuelt substitueret aryl eller aralkyl, nitrogenholdigt heterocyclyl- eller heterocyclyl- n o lavalkyl, E' og E sammen også alkylen med 3 eller 4 0 in 10 carbonatomer, E' hydrogen eller lavalkyl, og grupperne E , 11 io E og E uafhængigt af hinanden en eventuelt foresteret 11 eller amideret carboxylgruppe og E også hydrogen, kan fremstilles ved, at man på i og for sig kendt måde omsætter en forbindelse med formlen

GH20H

J_o

/ yvoH

15 l\p / / ' Ή - X - E2 (X) , / k1? - CH (D) \ E8 E10 E11 \ I il ip COH - CH - COH - CH - CH0CH - 1 η Iq 2 E7 (L) E9 med en reaktionsdygtig ester af en trilavalkylsilylforbin-delse.

Som reaktionsdygtige estere af en trilavalkylsilylforbin-delse skal især nævnes trilavalkylsilylhalogenider, især 20 -chlorider eller -bromider, bis-lavalkyl-silyl-acetamid eller -sulfamid.

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28

Omsætningen foretages fortrinsvis i et opløsningsmiddel, som ikke indeholder reaktionsdygtige hydroxyl- eller aminogrupper, såsom dimethylformamid, dioxan, tetrahydro-furan, dimethoxyethan eller chloroform.

5 De følgende eksempler belyser opfindelsen nærmere.

Eksempel 1.

Til en opløsning af 1 g okseserumalbumin i 100 ml af en 0,1 H natriumhydrogencarbonat-0,5 M natriumchloridopløs-ning sættes under omrøring 500 mg 2-acetamido-3-0-£[L-l-10 (D-l-carb amoyl-3-suecinimidoxyc arbonyl-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^ - 2- deoxy-D-glucose, Opløsningen omrøres i 4- timer ved stuetemperatur og sterilfiltreres derefter ("Mk-Milipore", 0,4-5 μτΒ. filter). Det konjugerede okseserumalbumin fraskilles i dialfiltreringsfremgangsmå-15 den ved hjælp af et "Amikon UM-10"-filter fra lavmolekylære reaktionsprodukter eller salte og frysetørres.

Den kvantitative bestemmelse af det til okseserumalbuminet bundne muramylpeptid sker ved Morgan-Elson-reaktionen ved modifikationen ifølge J. M. Ghuysen et al, [i "Methods in 20 Enzymology", 8, (1966),629]. Gennemsnitligt findes 60 μg muramylpeptid i 1 mg okseserumalbuminforbindelse.

Succinimodoesteren, der anvendes som udgangsstof, kan f.eks, fremstilles som følger: 1 mmol 2-acetamido-3-0->L-l-(D-l-carbamoyl-3-carboxy-25 propyl)-carbamoyl-ethylJ-c arbamoyl-methyl^-2-de oxy-D-glucose, 1 mmol dicyclohexylcarbodiimid og 1,1 mmol M-hydroxysuccinimid opløses i 3 ml absolut dimethylformamid og omrøres i en lukket beholder under vandudelukkelse i 20 timer. Det udfældede dicyclohexylurinstof skilles fra, 30 opløsningsmidlet afdampes i højvakuum og remanensen behandles med ether, hvorefter den suges fra og tørres. Den

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29 således opnåede suceinimidooxyester kan opbevares under fugtighedsudelukkelse, f.eks. i nitrogenampuller.

Eksempel 2,

Til kondensation af det i eksempel 1 opnåede antigenderi-5 vat på aktiveret agarose anvender man et kommercielt tilgængeligt agarosederivat fra BIO-EAD, "Affi-Gel 10", Denne indeholder på et agaroseskelet ethersidekæden ("spacer arms") af H-alkyl-succinamider, som er foresterede med H-hydroxysuec inimid,

10 Man opløser 120 mg af det ifølge eksempel 1 opnåede okse-serumalbumin-derivat i 12,5 ml 0,1 M phosphatpuffer, pH

7,5 ved 4°C. 500 mg "Affi-Gel 10" suspenderes derefter i opløsningen ved omrystning og omrystes i yderligere 4 timer ved 4°C, De endnu tilbageblivende aktive estergrupper 15 omsættes ved 3O minutters behandling med en 1 M ethanol-amin.HCl-0,1 M phosphatpufferopløsning (pH 7,3)· Derefter fyldes gelen i en søjle og vaskes først med 200 ml 0,1 M phosphatpuffer-1 M natriumchloridopløsning (pH 7,3)5 derefter med 20 ml fysiologisk natriumchloridopløsning, 20 Bestemmelsen af de med "Affi-Gel 10" kondenserede okse-serumalbumin-muramylpeptid-forbindelser sker ved kvantitativ analyse af repræsentative okseserumalbumin-aminosyrer i totalhydrolysater af definerede dele af gelen. Gennemsnitligt bindes 9-10 mg af okseserumalbumin-muramyl-25 peptidderivatet til 1 ml kvældet "Affi-Gel 10".

Eksempel 5.

Eåreerythrocytmembraner udvindes fra frisk fåreblod ved metoden ifølge Dodge, J, I, et al,, [Arch. Biochem.

Biophys. 100, (1963), side 114-180], Til en suspension af 30 5OO mg fåreerythrocytmembraner i 50 il 0,1 M natriumhydro-gencarbonat-0,1 M natriumchloridopløsning sættes under om-

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30 røring 400 mg 2-acetamido-3-0-^[L-l-(D-l-carbamoyl-3-suc cinimidooxycarb onyl-propyl)-carbamoyl-ethyl]-c arbamoyl-metbyl^-2-deoxy-D-grucose, og blandingen omrøres i 4 timer ved stuetemperatur. Derefter sedimenteres erythrocytmem-3 bran-kongugatet ved 1 times ultracentrifugering ved 90.000 g og 4°0. Sedimentet vaskes tre gange, hver gang ved resuspen-dering og ultracentrifugering, med phosphatpufferholdig natriumchloridopløsning og én gang med destilleret vand.

Det vaskede erythrocytmenbran-kong'ugat suspenderes i 100 ml 10 destilleret vand og frysetørres.

Den kvantitative bestemmelse af det til fåreerythrocyterne bundne muramylpeptid sker ved Morgan-Elson-reaktionen og viser et indhold på 23 muramyldipeptid pr. 1 mg ery-thr o cytmembr an.

15 Eksempel 4.

100 mg gruppe C polysaccharid fra Heisseria meningitidis og 110 mg (0,2 mmol) 2-acetamido-3-0- [[1-1-CD-I -carbamoyl- 3-N- amino ethyl- c arb amoyl-pr opyl) - carb amoyl- e thyl ] - c arb am-oyl-methyl^-2-deoxy-D-glucose-HCl opløses i 10 ml destil-20 leret vand, og opløsningen indstilles med fortyndet saltsyre til en pH på 5· 19,2 mg N-ethyl^'-(3-dimethylaminopropyl)-carbodiimid.HC1 sættes under omrøring til opløsningen. Blandingen omrøres i 1 time ved stuetemperatur, pH-værdien holdes på 5 under 25 tilsætning af fortyndet natriumhydroxidopløsning, og der tilsættes derefter 5 il 2 M natriumacetat-pufferopløsning, pH 5, og omrøres i yderligere 30 minutter. Derefter indstilles pH til 7 ned natriumhydroxidopløsning. Opløsningen sterilfiltreres og dialyseres ved 4°C med destilleret vand 30 og frysetørres derefter.

Den kvantitative bestemmelse af det til C polysaccharidet koblede desmethylmuramyldipeptid sker som beskrevet i

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31 eksempel 1 ved Morgan-Elson-reaktionen og viser et indhold på 80 μg desmethylmuramyldipeptid pr. 1 mg polysac-charid.

Det anvendte 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-H-5 aminoethyl-c arb amoyl-propy1)-carb amoyl-ethyl]-methyl^-2-desoxy-D-glucose-hydrochlorid kan f.eks. fremstilles som følger: 1 mmol 2-acetamino-3-0->[L-l-(D-l-carbamovl-3-carboxy-propyl)-c arb amoyl-ethyl]-carb amoyl-methyl<-2-de soxy-D-10 glucose og 9 mnLol N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimid.HOl opløses i 10 ml vand og pH indstilles til 5,0 med saltsyre. Derefter tilsættes en opløsning af 5 mmol ethylendiamindihydrochlorid i 10 ml vand. Chargen omrøres ved pH ^,0 og stuetemperatur i 6 timer. Blandingen 15 kommes på en svagt sur kationbyttersøjle "Amberlite CG 50 II", 2,5 x 45 cm og elueres med en lineær gradient fra 0,05 M pyridinacetat, pH 6 (3OO ml) til 0,5 M pyridin-acetat, pH 3,7 (300 ml). De fraktioner, som indeholder det opnåede 2-acetamino-3-0- £[1-1-(1)- 1-c arb amoyl-3-H-amino-20 ethyl-c arb amoyl-propyl)-c arb amoyl-ethyl]-c arb amoyl- methyl^-2-desoxy-D-glucose-aeetat, og som testes og karakteriseres med ninhydrin og høj spændingselektroforese frysetørres. Omdannelsen til hydrochloridformen sker på følgende måde: 25 Lyofilisatet opløses i 6 il 0,2 I HC1 og kromatograferes på en "Bio-Gel P2"-søjle, 2,5 x 90 cm, med vand som elue-ringsmiddel. De 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-H-amino ethyl-c arb amoyl-propyl)-c arb amoyl-ethyl]-c arb amoyl-me thyl ^-2-de s oxy-D-gluc o s e-hydro chloridhoIdige fraktioner 30 frysetørres. Præparatet er ensartet ifølge højspændings-elektroforese. Yed bestemmelsen af bestanddelene i total-hydrolysaterne findes molforholdet 1 muraminsyre:1 L-ala-nin:l D-glutaminsyre:1 ethylendiamin.

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32 På analog måde opnår man ud fra tilsvarende muramyldipep-tider de tilsvarende β1-aminoethylamid.HCl-forbindelser, nemlig: 2-acetamino-3-0- ^D-l- [L-l- (D-1- carbamoyl- 3-E- amino ethyl-3 carb amoyl-pr opyl)-c arb amoyl-ethyl]-carbamoyl-ethyl^-2-de s-oxy-D-glucose.HCl, 2-b enz oyl amino - 3- O- ^D-l- [ L-1- (D-1- c arb amoyl- ^-E- amino ethyl-c arb amoyl-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-desoxy-D-glucose.HCl, 10'. 2-b enzoyl amino- 3—0— ^ [ L-l- (D-l- c arb amoyl- 3-E- amino e thyl-carb amoyl-propyl)-c arb amoyl-ethyl]-carb amoyl-methyl^-2-descxy-Dr-glucose .HCl, 2-ac e tamino-3-0-^[L-1-(D-l-E-carbamoyl-methyl-carbamoyl-3-E-amino ethyl-c arb amoyl-propyl) - c arb amoyl-ethyl]-carbamoyl-15 methyl^-2-desoxy-D-glucose.HCl, 2-b enzoylamino-3-0-£[L-l-(D-l-carbamoyl-3-E-aminoetΗνία arb amoyl-pr opyl}- c arb amoyl-pr opyl ] - c arb amoyl-me thyl y- 2-desoxy-D-glucose.HCl, 2-propionylamino-3-0-^[L-l-(D-l-carbamoyl-3-E-aminoethyl-20 c arb amoyl-pr opyl) - c arb amoyl-ethyl ] - c arb amoy 1-me t hyl ^ - 2-desoxy-D-glucose.HCl, 2-acetylamino-3-0-^[L-l-(D-l-carbamoyl-3-H-aminoethyl-c arb amoyl-pr opyl) -c arb amoyl-pr opyl ] - c arb amoyl-me thyl ^-2-desoxy-D-glucose. HCl, 25 2-acetylamino-3-0-£[L-l-(D-l-carbamoyl-3-E-aminoethyl-carb amoyl-propyl)-carbamoyl-2-hydroxyethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose,HCl, 2-propionylamino-3-0-^[L-l-(D-l- (eller 3)-carboxy-3-(eller l)-E-amino ethyl-carb amoyl-propyl)-carbamoyl-ethyl]-c arb amoyl-me thyl ^ -2- de s oxy-D-gluc ose, HCl,

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33 2-benzoylamino-3-0- £ [L-l- (D-l-N-carbamoyl-methyl-carbam-oyl- 3-N- amino ethyl- c arb amoyl-pr opyl) - c arb amoyl- ethyl ] -carbamoyl-methyls-2-desoxy-D-glucose.HG1, 2-benzoylamino-3-0-^L-1-(D-l-c arb oxy-3-N-amino ethyl-c arb am-5 oyl-propyl)-c arb amoyl-ethyl)-carb amoylmethyl^-2-desoxy-D-glucose.HCl, 2-ac etamino-3-0-^[L-l-(D-1-carb amoyl-3-N-amino ethyl-carbamoyl-propyl)-carbamoyl-2’-methyl-propyl]-carbamoyl-methylj-2-desoxy-D-glucose,HC1, 10 2- ac et amino- 3- 0- ^D-1- [ L-l- (D-1- c arb amoyl- 3- (1-ΪΓ- amino-ethyl-c arb amoyl)- ethyl-c arb amoyl-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyls-2-de soxy-D-gluc ose.HCl, 2-acetamino-3-0-^[L-l-(D-l-carboxy-^-h-aminoethyl-carbam-oyl-propyl)-carbamoyl-2'-methyl-propyl]-c arb amoyl-methyl^-15 2-desoxy-D-glucose.HCl, 2-acetamino-3-0- -1-carbamoyl-3-N-aminoethyl- c arb amoyl-pr opyl) - c arb amoyl-N, N-1 etrarnethyl en ] - c arb amoyl-methyl^-2-desoxy-D-glucose.HC1, 2- a c e t amino - 3- 0- [ L-1 - ( D~ 1- c ar b amoyl - 3-N- amino e thyl-20 c arb amoyl-pr opyl) - c arb amoyl- e thyl ] -N-me thyl- c arb amoyl-methyl^-2-desoxy-D-glucose.H01 eller 2-benzoylamino-3-0-£[L-l-(D-l-carbamoyl-3-N-aminoethyl-carbamoyl-propyl)-carbamoyl-21-methyl-propyl]-carbamoyl-me thyl^-2-de s oxy-D-glue ose.HCl, 25 Disse forbindelser kondenseres som beskrevet ovenfor med gruppe-O-polysaccharid fra Neisseria meningitidis.

Eksempel 5.

Umiddelbart efter udvindingen suspenderes merozoiter fra malariaparasitten Plasmodium knowlesi, det samlede udbytte 30 fra blodet af en inficeret rhesusabe [metode til udvin-

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34 ding af merozoiter: se G·. H. Mitchel et al., (1975),

Immunology, 29, 397] i en opløsning af 100 mg (0,17 mmol) 2-acetamino-3-0-L-l-(D-l-carbamoyl-3-suecinimidooxy-carbonyl-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-des-5 oxy-D-glucose i 15 ml fysiologisk puf fer opløsning, pH· 7,2. Suspensionen inkuberes i 1 time ved 37°0. Derefter sedimenteres konjugaterne ved centrifugering. Sedimentet vaskes ved resuspendering i fysiologisk pufferopløsning og fornyet centrifugering. De vaskede merozoit-konjugater 10 suspenderes ifølge G, H. Mitchel (ref. se ovenfor) i 10%'s autologt rhesusabeserum og frysetørres.

Den kvantitative bestemmelse af det til merozoiterne koblede muramyldipeptid sker ved Morgan-Elson-reaktionen og giver 40-60 μg muramyldipeptid pr. 1 mg merozoiter.

15 Eksempel 6.

I en opløsning af 200 mg (0,34 mmol) 2-acetamino-3-0-^[L-1—(D—1-carb amoyl-3-sue cinimidooxy-c arbonyl-propyl)-c arb amoyl-ethyl]-carbamoyl-methyl^-2-de s oxy-D-glucose i 20 ml phosphatpufferholdig fysiologisk natriumchloridop-

Q

20 løsning, pH 7,2, suspenderer man 10y T-lyrnf oblast er fra CBA/J-mus. (T-Lymfoblasterne udvindes i en "mixed lymphocyte culture" mod G57BL/6-musestimulatorceller ifølge L. 0. Anderson et al., (1977), The Journal of Experimental Medicine, 146, 1124). Suspensionen inkuberes i 30 minutter 25 ved stuetemperatur. Derefter sedimenteres lymfoblast-konjugaterne ved centrifugering og vaskes ved resuspende-ring i phosphatpufferholdig fysiologisk natriumchloridopløsning og fornyet centrifugering.

Den kvantitative bestemmelse af det til T-lymfoblasterne 30 koblede muramyldipeptid sker ved Morgan-Elson-reaktionen (se eksempel 1) og viser et indhold på 60-70 pg muramyl-7 dipeptid pr. 101 T-lymfoblaster.

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35

Eksempel 7♦ På analog måde som i de ovenstående eksempler opnår man okseserumalbumin koblet med 2-acetamido-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 carbamoyl-ethyl]-carbamoyl-ethyl^-2-deoxy-D-glucose, 2-benzoylamino-3-0- -carb amoyl-3-c arb oxy- propyl)~carbamoyl-ethyl]-carbamoyl-ethyl^-2-deoxy-a,β-D-glucose, 2-b enz oylamino-3-0-£[L-l-(D-1-carb amoyl-3-c arb oxy-propyl)-10 carbamoyl-ethyl]-carbamoyl-methyl^-2-deoxy-a:, β-D-glucose, 2-acetamido-3-0-j>[L-l-(D-l-c arb amoylme thyl- carb amoyl- 3-c arb oxypr opyl) - c arb amoyle thyl ] - c arb amoylme thyl ^ -2-de oxy-D-glucose, 2- benzamido-2-deoxy-3-0-/> [L-l-(D-l-carbamoyl-3-carboxy- 13 propyl)-carbamoyl-propyl]-carbamoylmethyl^-D-glucopyranose, 3- 0-^[L-l-(D-l-carbamoyl-3-c arboxypropyl)-c arbamoylethyl]-c arb amoylmet hyl ^ - 2- de oxy- 2-propionamido-D- gluc ose, 2-acetamido-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoylpropyl]-carbamoylmethylj-2-deoxy-D-glucose, 20 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxypropyl)-c arb amoyl- 2-hydroxye thyl ] - c arb amoylme thyl j - 2- de s oxy-D-glucose, 2- propionylamino-3-0-£[L-l-(L-l,3-dicarboxy-propyl)-c arb amoylethyl]-c arb amoylmethylj-2-de soxy-D-gluc ose, 23 2-benzoylamino-3-0-£[L-l-(D-l-N-carbamoylmethyl-carbamoyl- 3- c arboxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^ - 2- de s-oxy-D-glucose, 2-acetamino-3- O- £ D-1- [ L-1- c arb amoyl-3-c arboxy-propyl)-carbamoyl-21-methyl-propyl]pcarbamoylethyl^-2-desoxy-D-

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36 glucose, 2-benzoylamino-3-0- ^ [1-1- (1-1,3-dicarboxy-propyl)-carbam-oylethyl]-carb amoylmethyl^-2-des oxy-1-glucose, 2-acetamino-3-0- [[1.-1-(11 -1-carbambyl-3-c arboxy-propyl)-3 carbamoyl-2‘-methyl-propyl]-carbamoylmethyl^-2-desoxy-l-glucose, 2-acetamiiio-3-0- £l-l-[l-l-(l-l-carb9in.oyl-3- (L-l-carboxy-ethyl)-carb amoyl-propyl)-c arbamoyl ethyl]-c arbamoylethyl^- 2-desoxy-l-gluco se, 10 2-acetamino-3-0-£[l-l-(1-1,3-dicarboxy-propyl)-carbamoyl-2 ‘ -methyl-propyl ]-carbamoyl-metbyl^-2-desoxy-!-glucose, 2-acetamino-3-0- [[1-1-(D-1 -carb amoyl-3-carboxy-propyl)-carb amoyl-N, N-t etr amethylen] -carb amoyl-methyl <-2-desoxy- 1- glucose, 15 2-acetamino-3-0- [[L-1-(B -1- c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl-methyl ] -N-methyl-carb amoyl-me thyl^-2-de s oxy-1-glucose eller 2- benzoylamino-3-0-^[l-l-(l-l-carbamoyl-3-carboxy-propyl)-carb amoyl-21 -methyl-propyl ]-c arb amoyl-methyl ^-2-de s oxy-1- 20 glucose»

Eksempel 8.

På analog måde som i eksempel 3 opnår man fåreerythrocyt-membraner koblet med 2-acetamino-3-0-^1-1-[L-l-(1-1-carbamoyl-3-carboxy-propyl)-25 carb amoyl-ethyl]-carbamoyl-ethyl^-2-des oxy-1-glucose, 2-benzoylamino-3-0-^1-1-[1-1-(l-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-carb amoyl-ethyl^-2-des oxy-1-glucose, 2-b enzoylamino-3-0-£[1-1-(1-1-c arbamoyl-3-c arboxy-propyl)- c arb amoyl-ethyl ] - c arb amoylmethyl ^-2-de s oxy- D- gluc ose, 37

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2-b enzoylamino~3-0- £ [ L-l- (D-l, 3-dic arb oxy-pr opyl) - carb am-oyle thyl]-c arb amoylme thyl^-2-de soxy-D-gluco s e, 2-propionylamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 c arb amoylethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose, 2-acetamino-3-0-^[L-l-(D-l-E-carbamoylmethylcarbamoyl-3-c arb oxy-propyl)-c arb amoyle thyl]-c arb amoylmethyl^-2-de soxy-D-glucose, 2-benzoylamino-3-0~£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-10 carbanoyl-propyl]-c arb amoylmethyl^-2-des oxy-D-gluc ose, 2-acetylamino-3-0-£[l-l-(D-l-carbamoyl-3-carb oxy-propyl)-c arb amoylpropyl]-c arbamoyl-methyl^-2-de s oxy-D-gluc ose, 2-acetamino-3-0-^[L-l-(D-l-carb amoyl-3-c arb oxypropyl)-carbamoyl-2-hydroxyethyl]-carbamoylmethyl^-2-desoxy-D-13 glucose, 2-propionylamino-3-0-£[L-l-(D-l,3-dicarboxy-propyl)-carbam-oylethyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2- b enz oylamino-3-0- [[L-1-(D -1-N-c arb amoylme thyl-c arb amoyl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^ - 2-20 desoxy-D-glucose, 2- ac et amino- 3- 0- £ [ L-l- (D-l- c arb amoyl- 3- c arb oxy-propyl) -c arb amoyl-2'-methyl-propyl]-c arb amoylmethyl^ -2-de s oxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-25 ethyl)-carbamoyl-propyl)-carbamoylethyl]-carbamoylethyl^- 2-desoxy-D-glucose, 2-acetamino-3-0->[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-2 ‘ -me thyl-pr opyl J - c arb amoyl-me thyl ^ - 2- de s oxy-D- gluc ose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-21 -methyl-propyl]-carbamoyl-methyl^-2-desoxy-I>- glucose, 38

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2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-E,l-tetramethylen]-carbamoyl-methyl<-2-desoxy-D-5 glucose, 2-ac e t amino - 3-0- £[1,-1-(1)-1 -c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-H-methyl-c arb amoyl-methyl^-2-de s oxy-D-glucose eller 2-acetamino-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-10 carbamoyl-21-methyl-propyl]-carbamoylethyl^-2-desoxy-D-glucose.

Eksempel 9.

På analog måde som i eksempel 5 opnår man merozoiter fra malariaparasitten Plasmodium knowlesi koblet med 15 2-acetamino~3-0-^D-l-[L-l-(D-l-carbamoyl-3-earboxy-propyl)-c arb amoyl-ethyl ]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0- ^D-1-[L-1- (D-l-carbamoyl-3-carboxy-propyl)-carb amoyl-ethyl]-c arbamoyl-ethyl^-2-de soxy-D-glucose, 20 2-benzoylamino-3-0-£[L-1-(D-1-carb amoyl-3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethylj-2-de s oxy-D-glucose, 2-benzoylamino-3-0- j> [L-l- (D-l, 3-dicarboxy-propyl)-carbam-oyle thyl) - c arb amoylme thyl ^ - 2- de s oxy-D- gluc ose, 2-propionylamino-3-0-£[L-l-(D-l-carbamoyL-3-carboxy-25 propyl)-carbamoylethyl]-carbamoylmethyls-2-desoxy-D-glueose, 2-acetamino-3-0-£[L-l-(D-l-h-carbamoylmethy1carb amoyl-3-c arb oxy-propyl)-c arbamoylethyl]-carbamoylmethyl^-2-des-oxy-D-glucose, 2-benzoylamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-propyl] - c arb amoylmethyl^-?-de soxy-D-gluc ose,

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39 2-acetylamino-3-0- [[L-1-(D-1 -c arb amoyl-3-c arb oxy-propyl)-c arb amoylpropyl]-c arb amoyl-me thyl^-2-de s oxy-D-gluc ose, 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxypropyl)-3 c arb amoyl- 2-h.y dr oxy e thyl ] - c arb amoylme thyl ^-2-de s oxy-D-glucose, 2-propionylamino-3-0-£[1-1-(D-l,3-dicarb oxy-propyl)-c arb am-oylethyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2-b enz oyl amino -3-0-£[L-l-(D-1-ϋ-c arb amoylme thyl- c arb am-10 oyl-3-carb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^- 2-desoxy-D-glucose, 2-ac et amino-3-0-£[L-l-(D-l-carbamoyl-3-c arboxy-propyl)-carbamoyl-21-methyl-propyl]-c arbamoylmethyl^-2-de s oxy-D-glucose, 15 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-ethyl)-carbamoyl-propyl)-carbamoylethyl]-carbamoylethyl^- 2-desoxy-D-glucose, 2- a c e t amino - 3- 0- £ [ L-1- ( D-1,3- di c arb oxy-p r opy 1) - carb amoyl-2'-methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glueose, 20 2-b enz oylamino-3-0-£[L-1-(D-1-carb amoyl- 3-c arb oxy-pr opyl)-c arb amoyl-21-methyl-propyl]-c arb amoyl-me t hyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-^[l-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-li, li-1 e t r arne thyl en ] - c arb amoyl-me t hyl < - 2- de s oxy-25 D-glucose, 2-acetamino-3- o-[[1-1-(1)-1 -carbamoyl-3-carboxy-propyl)-carb amoyl-ethyl]-ϋ-methyl-c arb amoyl-methyl^-2-de s oxy-D-glucose eller 2-ac et amino-3-0-^D-l-[L-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-21-methyl-propyl]-c arbamoylethyl^-2-desoxy-D-

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40 glucose.

Eksempel 10.

I en opløsning af 100 mg 2-acetamino-3-0-^D-l-[L-l-(D-l-c arb amoyl- 3- sue c inimido oxyc arbonyl-propyl) - c arb amoyl-5 ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose i 10 ml ph.ospb.at-pufferboldig fysiologisk natriumchlonidopløsning, pH 7?2,

Q

suspenderer man 10° mammacarcinomceller fra hund. [lumor-celler udvindes ifølge Η. H, Sedlacek, Ξ, Messmann og E. R. Seiler, Behring Inst, Mitt., nr, 35» 34-9-355 (1974-)]· 10 Suspensionen inkuberes i 90 minutter ved stuetemperatur.

Derefter sedimenteres tumorcelle-muramyldipeptid-konjugater-ne ved centrifugering og vaskes ved resuspendering i phos-phatpuff erholdig fysiologisk natrium chloridopløsning og fornyet centrifugering.

15 Den kvantitative bestemmelse af det til tumorcellerne koblede muramyldipeptid sker ved Morgan-Elson-reaktionen (se eksempel l) og viser et indhold på 60-80 μg muramyldipep-h tid pr. 10' mammacarcinomceller.

Eksempel 11.

20 På analog måde som i eksempel 10 opnår man mammacarcinomceller fra hund koblet med 2-acet amino-3-0-^D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-de s oxy-D-gluc ose, 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-25 propyl)-carb amoyl-ethyl]-carb amoyl-ethyl^-2-des oxy-D-glucose, 2-benzoylamino-3-0-£[L-l-(D-1-c arb amoyl-3-c arboxy-propyl)-c arb amoyl- ethyl ] - c arb amoylme thy 1 ^ - 2-de s oxy- D- gluc ose, 2-benzoylamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbam-30 oylethyl)-c arb amoylmethyl^ - 2- de s oxy-D-gluc ose, 2-propiontlamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy- propyl)-c arb amoyl e thyl ] - c arb amo ylme t hyl j-2-des oxy-D- glu cose,

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41 2- ac et amino- 3- O- £ [ L-1- ( D-l-U- c arb amoylme thylc arb amoyl- 3-3 c arb oxy-propyl) - c arb amoyl ethyl ] - c arb amoylme thyl j-2-des oxy-D-glucose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-pr opyl,] - c arb amoylme thyl ^-2-de s oxy- D- gluc ose, 2-acetylamino-3”0- -l-carbamoyl-3-carboxy-propyl)- 10 c arb amoylpropyl]-c arb amoyl-methyl^-2-de s oxy-D-gluc os e, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxypropyl)-c arb amoyl-2-hydroxyethyl]-c arb amoylmethyl^ -2- de s oxy-D-glucose, 2-propionylamino~3-0-^[L-l-(D-l,3-dicarboxy-propyl)-15 carbamoylethyl]-carbamoylmethyl^-2~desoxy-D-glucose, 2- b enz oylamino-3-0- £[1-1-(11-1 -H- c arb amoylme thyl- c arb amoyl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-desoxy-D-glucose, 2-ac etamino-3-0-^[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-20 carbamoyl-2'-methyl-propyl]-carb amoylmethyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-^[ll-l-[L-l-(D-l-carbamoyl-3- (1-1-carboxy-e thy1)-c arb amoyl-propyl)-c arb amoylethyl]-carb amoylethylj- 2-desoxy-D-glucose, 25 2-acetamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-2 ’-methyl-propyl]-c arb amoyl-methyl^-2-de s oxy-D-glu-cose, 2-b enz oylamino- 3- 0- £ [ L-1- (D-1- c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21-methyl-propyl]-carbamoyl-methyl^-2-desoxy-30 D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-N,H-tetramethylen]-carbamoyl-methylj-2-desoxy- D-glucose, 42

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2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 carbamoyl-ethyl ]-N-methyl-carbamoyl-methyl ^-2-desoxy-D-glucose eller 2-acetamirLO-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-2'-methyl-propyl]-c arb amoylethyl^-2-de s oxy-D-glucose.

10 Eksempel 12.

Til 10 ml af en suspension af mund- og klovsygekultur-vac-cine fra Beringwerke i phosphatpufferholdig fysiologisk natriumchloridopløsning sættes 100 mg 2-acetamino-3-0-£[1.-1-(D-l - c arb amo yl- 3- sue c inimido oxy- c arb onyl-pr opyl) -13 carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose. Suspensionen omrystes i 4 timer ved 4°0, Derefter sterilfiltreres virus-muramyldipeptid-konjugatet, fraskilles ved dialyse med vand fra lavmolekylære reaktionsprodukter og frysetørres. Den kvantitative bestemmelse af det virus-20 koblede muramyldipeptid sker ved Morgan-Elson-reaktionen (se eksempel l).

Eksempel 13.

På analog måde som i eksempel 12 opnår man mund- og klovsygevaccine koblet med 23 2-acet amino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-c arb oxy-propyl) -c arb amoyl- ethyl ] -carb amoyl-ethyl^ -2-desoxy-D-glucose, 2-b enz oylamino-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-de s oxy-D-30 gluc&se,

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2-b enz oylamino-3-0- [[l-l-CB-l -carbamoyl-3-carboxy-propyl)- c arb amoyl- ethyl ] - carb amoylme thyl^-2-de s oxy-D-gluc ose, 43 2-benzoylamino-3-0-£[L-l-(D-l,3-dicarboxy-propyl)-carbam-oylethyl)-c arb amoylmethyl^-2-de s oxy-D-gluc ose, 5 2-propionylamino-3-0- - c arb amoyl- 3- c arb oxy- pr opyl) - c arb amoyl e t hyl ] - c arb amoylmethyl j - 2- de s oxy-D-glu-cose, 2-ac et amino-3-0-£[L-1-(D-l-N-c arb amoylmethylc arb amoyl-3-c arb oxy-propyl)-c arb amoylethyl]-c arb amoylmethyl^-2-de s oxy-10 D-glucose, 2-b enz oylamino-3-O-^[L-l-(D-l-c arbamoyl-3-c arboxy-propyl)-carbamoyl-propyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2-ac etylamino-3-0-£[L-1-(D-1-carb amoyl-3-c arb oxy-propyl)-carbamoylpropyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 13 2-acetamino-3~0-^[L-l-(D-l-carbamoyl-3-carboxypropyl)-c arb amoyl-2-hydroxyethyl]-c arb amoylmethyl^-2-de soxy-D-glucose, 2- propionylamino-3-0- ,3-dic arb oxy-propyl)-c arb amoyl e thyl ] -c arb amoylmethyl j - 2-de s oxy-D- gluc ose., 20 2-benzoylamino-3-0- £ [L-l- ( D-1-1T-c arb amoylme thyl-carb amoyl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arbamoylmethyly-2-desoxy-D-glucose, 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2 *-methyl-propyl]-carbamoylmethyl^-2-desoxy-D-23 glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(1-1-carboxy-ethyl)-carb amoyl-propyl)-c arb amoylethyl]-c arb amoylethyl^- 2-desoxy-D-glucose, 2-acetamino-3-0- ,3-dicarboxy-propyl)-carbamoyl- 30 21-methyl-propyl]-carbamoyl-methylj-2-desoxy-D-glucose, 44

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2-b enz oyl amino- 3-0- £[L-l- (D-l- c arb amoyl- 3- c arb oxy-propyl) -c arb amoyl- 21 -me thyl-pr opyl ] - c arb amoyl-me thyl ^ - 2- de s oxy-D-glucose, 2-acetamino-3-0- £[1-1-(1) -l-carbamoyl-3-carboxy-propyl)-5 carb amoyl-K, N-tetr arne thyl en] -carb-amoyl-me thyl j -2-de s oxy-D-glucose, 2-acetamino-3-0-^[L-Ι-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-E-methyl-carbamoyl-methyl^-2-desoxy-D-glucose eller 10 2-acetamino-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21 -me thyl-pr opyl ] - c arb amoyl e thyl ^ - 2-de s oxy-D-glucose.

Eksempel 14.

På analog måde som i eksempel 6 opnår man T-lymfoblaster 15 fra OBA/J-mus koblet med 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propy]:)-c arb amoyl-ethyl]-c arb amoyl-ethyl^ - 2- de s oxy-D-glue ose, 2-b enz oylamino-3-0-^D-l-[1-1-(1-1-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-l-glu-20 cose, 2-benzoylamino-3-0-^[l-l-(l-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose, 2-benzoylamino-3-0- £[L-1-(D-1 ,3-dicarboxy-propyl)-carbamoyl ethyl)-carb amoylmethyl ^-2-de s oxy-D-gluc ose, 25 2-propionylamino-3-0- £[1-1-(1)-1 - c arb amoyl- 3- c arb oxy-pr opyl )-c arb amoyletbyl]-c arb amoylmethylj-2-desoxy-D-glucose, 2-ac etamino-3—0— ^[L-l-(D-l-K-c arb amoylmethylc arb amoyl-3-c arb oxy-pr opyl) - c arb amoyl e thyl ] - c arb amoylme thyl ^ -2-de s oxy-30 D-glucose, 45

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2-b enz oylamino-3-O- [[1-1-(D -1-c arbamoyl-3- c arb oxy-pr opyl) -c arb amoyl-propyl]-o arb amoylme thyl^-2-de s oxy-D-gluc ose, 2-acetylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arbamoylpropyl]- carb amoyl-methyl2-de s oxy-D-gluc ose, 5 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxypropyl)-c arb amoyl-2-hydroxye thyl ] - c arb amoylmethyl^ - 2-de s oxy-D-glucose, 2- propionylamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-c arb amoylethyl]-c arb amoylmethyl<-2-de soxy-D-gluc ose, 10 2-benzoylamino-3-0-^[L-l-(D-l-N-c arb amoylme thyl-carbamoyl- 3- carboxy-propy1)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-desoxy-D-glucose, 2-acetamino-3-0- [[D-1-(D -1-c arb amoyl-3-c arboxy-propyl)-carbamoyl-21-methyl-propyl]-carbamoylmethyl^-2-desoxy-D-15 glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-ethyl)-c arb amoyl-propyl)-c arb amoylethyl]-c arb amoylethyl^-2-desoxy-D-glucose, 2-ac e t amino-3-0-H L-l-(D-1,3-diearb oxy-propyl)-c arb amoyl-20 21-methyl-propylJ-carbamoyl-methyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-25 carbamoyl-N,N-tetramethylen]-carbamoyl-methyl<-2-desoxy-D-glucose, 2- ac et amino- 3- 0- ^ [ L-l- (D-l- c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl-ethyl j-N-methyl-carbamoyl·-methyl^-2-desoxy-D-glucose , 30 2-acetamino-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-

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46 carbamoyl-21-methyl-propyl]-carbamoylethyl^-2-desoxy-D-glucose.

Eksempel 15.

1 mg oks e serumalbumin- 2-acetamino-3-0-^[L-1-(D-1-c arb am-5 oyl- 3- c arb oxy-propyl) -c arb amoyl- ethyl ] - c arb amoyl-methyl ^ -2-desoxy-D-glucose-konjugat opløses i 0,1 ml phosphatpuff erholdig fysiologisk natriumchloridopløsning (PBS), I opløsninger blandes 0,1 ml af en 5%'s suspension af carboxymethylcellulose i PBS, Blandingen applikeres intra-1Q; muskulært til marsvin i to ens portioner.

Eksempel 16, 10^ T-lymf oblaster-2-acet amino-3-0-^[L-1-(D-c arb amoyl-3-c arb oxy-propyl) - c arb amoyl- ethyl ] - earb amoyl-methyl ^-2-des oxy-D- gluc o s e-konj ugat er (se eksempel 6) suspenderes i 15 0,5 ml phosphatpufferholdig fysiologisk natriumchloridop-løsning (PBS), I suspensionen blandes 0,5 ml af en 5%‘s suspension af carboxymethylcellulose i PBS, Pr, mus applikeres 0,1 ml af blandingen subcutant.

Eksempel 17, 20 Til 10 ml af en suspension af rabies-HDO-vaccine fra Behringwerke i phosphatpufferholdig fysiologisk natrium-chloridopløsning sættes 100 mg (0,17 mmol) 2-ac et amino-3- 0-£[1-1-(D-l-carb amoyl-3-sue c inimido oxy-c arb onyl-propyl)-c arb amoyl- ethyl ] -c arb amoyl-methyl <j-2-de s oxy-D-gluc ose, 25 Viruskoncentrationen er 2 x 10^ LD^/mus i 1 ml suspension, Suspensionen omrystes i 4 timer ved 4°G, Derefter sterilfiltreres muramyldipeptid-rabies-HDO-vaccine-konju-gatet, adskilles ved dialyse med vand fra lavmolekylære reaktionsprodukter og frysetørres.

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47

Eksempel 18.

På analog måde som i eksempel 17 opnår man rabies-HDC-vaccine fra Behringwerke koblet med 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-b enz oyl amino - 3- 0- ^D-1- [ L-1- (D-1- c arb amoyl- 3- c arb oxy-pr opyl) - c arb amoy1-ethyl]-c arbamoyl-ethyl^-2-de s oxy-D-glu-cose, 2-benzoylamino-3-0-£[L~l-(D-l-carbamoyl-3-carboxy-propyl)-10 c arb amoy1-ethyl]-c arb amoylmethy1^ - 2- de s oxy-D-gluc ose, 2-benzoylamino-3-0-£[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl ethyl )- c arb amoylme thyl ^ -2-de s oxy-D-gluc o s e , 2-propionylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoylethyl]-c arb amoylmethylj-2-desoxy-D-glu-13 cose, 2-acetamino-3-0- ¢[1-1-(1)-1 -N-c arb amoylmethylc arb amoyl-3-c arb oxy-pr opyl) - c arb amoyl ethyl ] - c.arb amoylme thyl ^ - 2-de s oxy-D-glucose, 2-benz oylamino-3-0-£[L-l-(D-l-c arbamoyl-3-c arb oxy-propyl)-20 carbamoyl-propyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2-ac etylamino-3-0-£[L-l-(D-l-c arbamoyl-3-c arb oxy-propyl)-c arb amoylpropyl]-c arb amoyl-methy1^-2-de soxy-D-gluc ose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxypropyl)-c arb amoyl-2-hydroxyethyl]-c arb amoylmethyl^-2-de s oxy-D-23 glucose, 2-propionylamino~3-0-£[L-l-(L-l,3-dicarboxy-propyl)- c arb amoylethyl]-c arb amoylmethyl<-2-de soxy-D-gluc ose, 2- benzoylamino-3-0-^[L-l-(D-l-E-carbamoylmethyl-carbamoyl- 3- carboxy-propyl)-carbamoyl-ethyl]-carbamoylmethyl^-2-

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48 desoxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21 -methyl-propyl]-carbamoylmethyl^-2-desoxy-D-glucose, 5 2-acetamino-3-0-£D-l-[l-l-(D-l-carbamoyl-3-(Ii-l-carboxy-e thyl) -c arb amoyl-pr opyl) - c arb amoyle thyl ] - c arb amoylethyl ^ -2-desoxy-D-glucose, 2-acetamino-3-0-/[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-2' -methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 10 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2 ’ -methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-E,E-tetramethylen]-carbamoyl-methyl^-2-desoxy-15 D-glucose, 2-acetamino-3-0-^[L-l-(L-carbamoyl-3-carboxy-propyl)-c arb amoyl- ethyl ] -E-methyl-c arb amoyl-methyl^ - 2-de s oxy-D-glucose eller 2-ac etamino-3-0-^D-l-[L-l-carbamoyl-3-c arboxy-propyl)-20 carbamoyl-21 -methyl-propyl]-carbamoylethyl^-2-desoxy-D-glucose.

Eksempel 19»

Til 10 ml af en suspension af ekstraherede influenza-virusantigener fra type A/Victoria/3/75 i phosphatpufferholdig 25 fysiologisk natriumchloridopløsning sættes 100 mg (0,17 mmol) 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-sue cinimido-oxy- c arb onyl-pr opyl) - c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ -2-desoxy-D-glucose. (Virus-antigenerne udvindes ved tween/ ether-spaltningsfremgangsmåden ifølge Behringwerke, Mar-30 burg, BED - analogt med fremstillingen af Begrivac S; koncentrationen er 4000 I.E. i 1 ml suspension). Suspensio- 49

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nen omrystes i 4 timer ved 4°C, Derefter adskilles virus-antigen-muramyldipeptid-konjugaterne ved dialyse med phos-phatpufferholdig fysiologisk natriumchloridopløsning fra lavmolekylære reaktionsprodukter. Den dialyserede suspen-5 sion indfryses og opbevares ved -20°C, indtil den skal bruges.

Eksempel 20.

På analog måde som i eksempel 19 opnår man influenza-virus-antigen koblet til 10 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-de s oxy-D-glucose, 2-benzoylamino-3-0-^D-l-[L-Ι-(D-l-carbamoyl-J-carboxy-pr opyl ) - c arb amoy 1- e thyl ] - c arb amoyl- e t hyl ^ - 2- de s oxy-D- glucose, 15 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose, 2-benzoylamino-3-0-^[L-l-(D~l, 3-dicarb oxy-propy 1) -.carb amoyl ethyl)-c arbamoylmethyl^-2-des oxy-D-gluco s e, 2-propionylamino-3-0-£[L-l-(D-l-carbamovl-3-carboxy-20 propyl)-c arb amoylethyl]-c arb amoylmethylj-2-de s oxy-D-glucose , 2- ac et amino- 3- 0- £ [ L-l- (D- 1-N- c arb amoylme thyl c arb amoyl- 3-c arb oxy-propyl)-c arb amoylethyl]-c arb amoylmethyl^-2-de s oxy-D-glucose, 25 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2-acetylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoylpropyl]-c arb amoyl-methyl^-2-de s oxy-D-gluc ose, 2-acetamino-3-0-£[L-1-(D-1-carbamoyl-3-carboxypropyl)- 50

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I c arb amoyl- 2-hydroxy ethyl ] - c arb amoylme tliyl ^-2-des oxy-D- glu cose, 2- propionylamino-3-0-^[L-l-(D-l,3-dic arb oxy-propyl)-c arb amoyl ethyl ]-c arb amoylme thyl^ - 2- de s oxy-D-glueo s e, 5 2-benzoylamino-3-0-£[L-l-(D-l-]ir-carbamoylmethyl-carbamoyl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-glue o s e, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carb amoyl-21-methyl-propyl]-c arb amoylmethyl^-2-de s oxy-D-10 glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-ethyl)-c arb amoyl-propyl)-c arb amoylethyl]-c arb amoylethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-15 21-methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-earboxy-propyl)-c arb amoyl-21-methyl-propyl]-c arb amoyl-methyl^-2-de soxy-D-glucose, 2-acetamino-3-0-£[1-1-(D-l-carbamoyl-3-carboxy-propyl)-20 c arb amoyl-E ,ΕΓ-tetrame thyl en ] - c arb amoyl-methyl ^-2-des oxy-D-glucose, 2-acet amino-3-0-£[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl- ethyl ] -E-methyl- c arb amoyl-methyl ^ -2-des oxy-D-glucose eller 25 2-acetamino-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21-methyl-propyl]-carbamoylethyl^-2-desoxy-D-glucose.

Eksempel 21.

Til 2 ml af en opløsning af tetanustoksoid i phosphatpuf-

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51 ferholdig fysiologisk natriumchloridopløsning sættes 10 mg 2-acetamido-3-0-£[L-l-(D-l-carbamoyl-3-succinimid-oxyc arb onyl-pr opyl)-c arb amoyl-ethyl]-c arb amoyl-me thyl^-2-desoxy-d-glucose, Toksoidkoncentrationen er 3 mg/ml. Opløs-5 ningen omrøres i 4 timer ved 4°C. Derefter adskilles tetanustoksoid-muramyldipeptid-konjugatet ved ultrafiltrering fra lavmolekylære reaktionsprodukter; den ultra-filtrerede opløsning indfryses og opbevares ved -20°0, indtil den skal bruges. Den kvantitative bestemmelse (Morgan-10 Elson-reaktionen) viser et indhold på ca, 50 μg muramyl-dipeptid pr. 1 mg tetanustoksoid-muramyldipeptid-konjugat.

Eksempel 22, På analog måde som i eksempel 21 opnår man tetanustoksoid koblet til 15 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glu-cose, 2-b enz oyl amino- 3- 0- ^D-l- [L-l- (D-l-c arb amoyl- 3- c arb oxy-propyl)-c arbamoyl-ethyl]-c arbamoyl-ethyl^-2-de s oxy-D-glu-20 cose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de soxy-D-gluco s e, 2-benzoylamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbam-oylethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose, 25 2-propionylamino-3-0-^[L-l-(D-l-carbamovl-3-carboxy-propyl)-c arb amoylethyl]-c arb amoylmethyl<-2-de s oxy-D-glu-cose, 2-acetamino-3-0-^[L-l-(D-l-N-carbamoylmethylcarbamoyl-methyl-3-c arb oxy-propyl)-c arb amoylethyl]-c arb amoylmethylj-30 2-desoxy-D-glucose, i ! 52

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2-benzoylamino-3-0- £[L-1-(D-1 -c arbamoyl-3-carb oxy-propyl)-c arb amoyl-pr opyl]-carb amoylmethyl^-2-des oxy-D-glucose, 2-acetylamino-3-0-£[L-l-(D-l-carbamoyl-3--carboxy-propyl)-c arb amoylpropyl ] - c arb amoyl-methyl ^-2-de s oxy-D- gluc ose, 5 2-acetamino-3-0-^[L-l-(D-l-carbaraoyl-3-carboxypropyl)-c arb amoyl- 2-hydroxyethyl ] -c arb amoylmetbyl ^-2-des oxy-D-glucose, 2- propionylamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbamoylethyl ]-carbamoylmethyl j-2-desoxy-D-glucose, 10 2-b enz oylamino-3-0-^[L-l-(D-l-N-carbamoylmetbyl-carbamoyl- 3- carb oxy-propyl)-carbamoyl-ethyl]-carbamoylmetbyl^ - 2-des oxy-D-glucose, 2- ac et amino-3-0-^[L-l-(L-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2' -me tbyl-pr opyl ] - c arb amoylme tbyl ^ - 2- de s oxy-D-15 glucose, 2-acetamino-3-0-^L-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-etbyl) - c arb amoyl-propyl)-c arbamoyletbyl]-c arb amoylethyl^-2-desoxy-D-glucose, 2-acetamino-3-0->[L-l-(L-l,3-dicarboxy-propyl)-carbamoyl-20 2 r-methyl-propyl]-carbamoyl-methyl^-2-desoxy-L-glucose, 2-benzoylamino-3-0-£[L-l-(D-1-carbamoyl-3-c arboxy-propyl)-carbamoyl-2 * -methyl-propyl]-carbamoyl-methyl^-2-desoxy-L-glucose, 2-acet amino-3-0-£[L-l-(D-1-carb amoyl-3-c arb oxy-propyl)-25 c arb amoyl-H, ΪΤ-t et ramethylen] - c arb amoyl-methyl ^-2-desoxy-D-glucose, 2-acetamino-3-0-£[L-l-(L-l-carbamoyl-3-carboxy-propyl)-c arb amoyl- ethyl ] -ΪΓ-me thyl-c arb amoyl-methyl ^ - 2-de s oxy-L-glucose eller 30 2-acetamino-3-0- j>D-l- [L-l-carb amoyl- 3- c arb oxy-propyl) - 53

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carbamoyl-21 -methyl-propyl ]-carbamoyletliyl^-2-desoxy-D-glucose.

Eksempel 23.

Til 10 ml af en opløsning af koleratoksoid fra Vibrio 5 cholerae i phosphatpufferholdig fysiologisk natriumchlorid-opløsning sættes 50 mg 2-acetanido-3-0->[L-l-(D-l-carbam-oyl-3-succinimidoxycarbonyl-propyl)-carbamoyl-etkyl]-carbamoyl-methylj-2-desoxy-D-glucose. Proteinkoncentrationen i opløsningen er 0,6 mg/ml. Opløsningen omrøres i 10 4 timer ved 4°0. Derefter adskilles koleratoksoid-muramyl-dipeptid-konjugatet ved ultrafiltrering fra lavmolekylære reaktionsprodukter. Den ultrafiltrerede opløsning indfryses og opbevares ved -20°C, indtil den skal bruges.

Den kvantitative bestemmelse (Morgan-Elson-reaktionen) 15 viser et indhold på 4-0-50 ug muramyldipeptid pr. 1 mg koleratoksoid-muramyldipeptid-konjugat.

Eksempel 24.

På analog måde som i eksempel 23 opnår man choleratoksoid koblet til 20 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D-25 glucose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoylmethyl^-2-desoxy-D-glucose, 2-benzoylamino~3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbam-oylethy1)-c arb amoylme thyl^-2-de s oxy-D-gluc ose 30 2-propionylamino-3-0- -1-c arb amoyl- 3- c arb oxy- 54 DK 161026 B ; propyl) - c art) amoyl e thyl ] - c art) amoylme thyl ^ - 2- de s oxy-D- glucose, ; 2-ac et amino- 3-O- £ [L-l- (D- 1-ΪΓ- c art> amoylme thylc art) amoyl- 3-c art> oxy-pr opyl) - c art) amoyl e tliyl ] - c arb amoylme thyl ^ - 2- de s- i 5 oxy-D-glucose, 2-b enz oyl amino- 3- O- £ [ L-1- (D-l- c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl-propyl]-c arb amoylmetbyl^ - 2- de s oxy-D-glucose, 2-acetylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoylpr opyl ] - c arb amoyl-me thyl ^-2-des oxy-D-gluc ose, 10 2-ac et amino-3-0-£[L-l-(D-l-c arb amoyl-3-carboxypropyl)-c arb amoyl- 2-hydroxyethyl ] -c arb amoylme thyl ^-2-de s oxy-D-glucose , 2- propionylamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-carbamoylethyl]-c arb amoylmethyl-2-de s oxy-D-glucose, 15 2-benzoylamino-3-0-^ [l-l-(D-l-N-carb amoylmeth.yl-carbam.oyl- 3- c arb oxy-pr opyl) - c arb amoyl- ethyl ] - c arb amoylme thyl ^-2-desoxy-D-glucose, 2-acetamino-3-0- £[L-1-(D -1-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-2 ‘ -me thyl-pr opyl ]-c arb amoylme thyl ^ -2-de s oxy-D-20 glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(l-l-carbpxy-ethyl)-carb amoyl-propyl)-c arb amoylethyl]-c arb amoylethyl^-2-desoxy-D-glucose, 2-acetamino-3-0->[l-l-(D-l,3-dicarboxy-propyl)-carbamoyl-25 21-methyl-propylJ-carbamoyl-methyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0- [[L-l-CD -1-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21 -methyl-propyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0- £[L-1-(D-1 -c arb amoyl-3-c arb oxy-propyl)-30 carbamoyl-N,IT-tetramethylen]-carbamoyl-methyl^-2-desoxy-

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2-acetamino-3-0- £[L-1-(D -1-c arb amoyl-3-c arb oxy-propyl)- c arb amoyl- e thyl ] -H-me thyl-c arb amoyl-me thyl j - 2-de s oxy-D- glucose eller 55 D-glucose, 3 2-acetamino-3-0-^D-l-[L-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-2’-methyl-propyl]-c arb amoylethyl^-2-de s oxy-D-glucose.

Eksempel 23.

20 mg af et syntetisk eicosapeptid, som i den C-terminale 10 sekvens er identisk med humant choriongonadotropin (HCG) [jfr. 0. H, Schneider, E. Blaser, Oh. Pfeuti og E.Gruden,

Bebs Letters, JjjO, 272 (1975)] °S 30 mg 2-acetamido-3-0-£[L-1-(D-l-c arb amoyl-3-sue cinimidoxy-c arb onyl-propyl)-carbamoyl-ethylJ-carbamoyl-methyl^-2-desoxy-D-glucose op-13 løses i 2 ml natriumphosphatpufferopløsning, pH 7,2. Opløsningen omrøres i 6 timer ved stuetemperatur. Eicosa-peptid-muramyldipeptid-konjugatet isoleres derefter ved kromatografi på en "Sephadex G-p5"-søjle (1,4- x 40 cm) med vand som eluat og frysetørres. Den kvantitative amino-20 syreanalyse viser, at 1-muramyldipeptidmolekylet er bundet til 1-eicosapeptidmolekylet.

Eksempel 26.

På analog måde som i eksempel 23 opnår man det O-terminale eicosapeptid-fragment fra humant choriongonadotropin kob-23 let til 2- ac e t amino- 3- 0- ^D-1-[L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arbamoyl-ethyl^-2-des oxy-D-glucose, 2-b enz oyl amino- 3- O- ^D-1- [ L-1- ( D-1- c arb amoyl- 3- c arb oxy- 30 propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D- glucose,

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56 2-b enz oylamino-3-0-^[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-I carbamoyl-ethyl ]-carbamoylmethyl^-2-desoxy-D-glncose, 2-benzoylamino-3-0-^[L-1-(D-1,3~ die arb oxy-propy1)-carb amoyl -ethyl)-carb amoylmethyl^ - 2- de s oxy-D-gine ose, 5 2-propionylamino-3-0-^[L-l-(D-l-carbamoyl-3-propoxy-propyl)-carbamoylethyl]-carbamoylmethylj-2-desoxy-D-glu-cose, 2- ac et amino- 3- 0- £ [ L-l- (D-1-N- c arb amoylme thyl c arb amoyl- 3-c arb oxy-propyl)-c arb amoylethyl]-carb amoylmethyl^ - 2- de s -10 oxy-D-glucose, 2-benzoylamino-3-0- £[L-1-(D-1 -c arb amoyl-3-carboxy-propyl)-carbamoyl-propyl ]-carbamoylmethyl^-2-desoxy-D-glucose, 2-acetylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoylpropyl]-carbamoyl-methyl^-2-de s oxy-D-glucose, 15 2-ac etamino-3-0-£[L-l-(D-carbamoyl-3-carboxypropyl)-c arb amoyl- 2-hydr oxy e t hy 1 ] - c arb amoylme t hyl ^ - 2-de s oxy-D-glncose, 2-propionylamino-3-0-^[L-1-(1)-1,3-dicarboxy-propyl)-c arb amoyl e thyl ] - c arb amoylme t hyl < - 2- de s oxy-D- gine ose, 20 2-benzoylamino-3-0-^[L-l-(D-l-N-carbamoylmethyl-carbam-i oyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^- 2-desoxy-D-glucose, 2-ac etamino-3-0-£[L-l-(D-l-c arb amoyl-3-c arboxy-propyl)-carbamoyl-21-methyl-propyl]-carbamoylmethyl^-2-desoxy-D-25 glucose, 2-acet amino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-(L-l-carboxy-ethyl)-carbamoyl-propyl)-carbamoylethyl]-carbamoylethyl^-2-desoxy-D-glucose, 2-acetamino-3-0->[L-l-(D-l,3-dicarboxy-propyl)-carbamoyl-3° 2,-methyl-propylJ-carbamoyl-methyl^-2-desoxy-D-glncose, 2-b enz oylamino- 3- O- ^ [ L-1- ( D-1- c arb amoyl-3-c arb oxy-propyl)- carbamoyl-2'-methyl-propyl]-carbamoyl-methyl^-2-desoxy-D- glucose, 57

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2-ac et amino-3-O-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-5 c arb amoyl-ΒΓ, ΪΓ-1 e trame thy1en ] - c arb amoyl-me thyl j - 2-de s oxy-D-glucose, 2-acetamino-3-O- £[L-1-(D -1-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-Η-methyl-carb amoyl-methyl^-2-de soxy-D-glncose eller 10 2- ac e t amino- 3-O- ^D- 1- [L-1- c arb amoyl- 3- c arb oxy-propyl) -carbamoyl-21 -methyl-propyl ]-carbamoylethyl^-2-desoxy-D-glucose*

Eksempel 27.

På analog måde som i eksempel 1 opnår man okseserumalbumin 13 koblet med 2-ac et amino-3-0-^[(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-me thyl J-IT-me thyl-c arb amoyl-me thyl ^-2-de s oxy-P-gluc o s e, 2-b enz oylamino-3-0-£[(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-me t hyl ]-N-me thyl-c arb amoyl-me t hyl^-2-desoxy-D-20 glucose, 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl)-H-ethyl-c arb amoyl-methyl^-2-de soxy-D-glucose, 2- ac e t amino- 3- O- > [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl) -25 carbamoyl-propylJ-F-methyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3--0-?[L-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-propyl]-1-ethyl-c arb amoyl-methyl^-2-de s oxy-D- glucose, 2-benzamido-3-O-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-propyl ] -E- e thyl- c arb amoyl-me t hyl ^ 2- de s oxy-D- glucose, 58

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2-acetamino-3-0-^[li-l-(D-l-carbamoyl~3-carboxy-propyl)-5 carbamoyl-ethyl]-E-propyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-O-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-E ,E-pent arne thyl en ] - c arb amoyl-me thyl j - 2- de s oxy-D-glucose, 10 2-benzoylamino-3-0- [[1-1-(1) -1-carbamoyl-3-c arboxy-propyl)-c arb amoyl-E, E-p ent ame thyl en ] - c arb amoyl-me thy 1 ^-2-des oxy-D-glucose, 2-acetamino-3-0- [[1-1-(1-1 -carb amoyl-3-c arb oxy-propyl)-E-methyl-c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ -2-de s oxy-D-15 glucose, 2-acet amino-3-0-^D-l-[(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-me thyl]-E-methyl-c arb amoyl-ethyl^-2-de s oxy-D-glucose, 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)-20 c arb amoyl-me thyl ] -E-me thyl- c arb amoyl- ethyl ^ - 2-de s oxy-D-glucose, 2- ac et amino- 3- 0- ^D-1- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl) -c arb amoyl- e thyl) -E- ethyl- c arb amoyl- ethyl ^ - 2- de s oxy-D-glucose, 25 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-propyl]-E-methyl-c arbamoyl-ethyl^-2-desoxy-D-glucose, 2- ac e t amino- 3- 0- ^D-l- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl ) - c arb amoyl-pr opyl ] -E- ethyl- c arb amoyl-me thyl ^ - 2-de s -30 oxy-D-glueose, 59

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2-b enz amido- 3- O- ^D-1- [ L-1- (D-l-c arb amoyl- 3- c arb oxy-propyl)-carbamoyl-propyl]-N-ethyl-carbamoyl-ethylj-2-de s oxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-3 c arb amoyl-ethyl]-N-propyl-c arbamoyl-ethyl<-2-de s oxy-D-glucose, 2-ac e t amino-3-0-[ L-l-(N-l-c arb amoyl-3-c arb oxy-pr opyl) - c arb amoyl-N, N-p ent ame thyl en ] - c arb amoyl- e thyl ^-2-desoxy-D-glucose, 10 2-benzoylamino-3-0-^D-l-[l-l-(D-l-carbamoyl-3-carboxy-pr opyl)-c arb amoyl-N, N-p ent amethyl en ]-c arb amoyl- ethyl j-2-de s oxy-D-gluc ose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-N-methyl-c arb amoyl-ethyl]-c arb amoyl-ethylj-2-13 desoxy-D-glucose, 2-ac etamino-3-0-£[1-1-(D-l-carb amoyl-3-c arb oxy-methyl-phenyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^[l-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl]-c arb amoyl-methyl^-2-de s oxy-D-glucose, 20 2-b enz amino-3-0-^[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-c arb amoyl-2-methyl-mercapto-ethyl]-c arb amoyl-methylj-2-de s oxy-D-gluc ose, 2-b enz amino-3-0-£[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-chlorethyl]-c arb amoyl-methyl^-2-de soxy-D-23 glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3,3-d.icarboxy-propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D-glu-cose, 2-(p-carbomethoxy-succinamido)-3-0-^[L-l-(D-l-carbamoyl- 30 3-carb oxy-propyl)-c arb amoyl-ethy1]-c arb amoyl-methyl^-2- desoxy-D-glucose, 60

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i 2-(p-carbomethoxy-succinamido)-3-0-£D-l-[L-l-(D-l-carbam- '! oyl- 3- c 3Tb oxy-propyl) - c arb amoyl-ethyl }-c arb amoyl-ethyl ^ - 2-desoxy-D-glucose, 2-benzami.no-3-0- £[l-i-(d -1-carb amoyl-3-trimethylsilyl-5 c arb oxy-propyl) - c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ - 2-desoxy-1,4,6-tris-trimethylsilyl-D-glucose. (Ved kontakt med vand hydrolyseres trimethylsilylestergruppen hurtigt), 2-acetamino-2-desoxy-3-0-^[L-Ι-(D-l-carbamoyl-3-trimethyl-s ilyl c arb oxy-pr opyl) - c arb amoyl-e thyl ] - c arb amoylme thy 1 ^-10 1,4,6-tris-trimethylsilyl-D-glucose, 2- ac et amino- 3-0- ^D-l-[L-l- (D-l-c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl-pr opyl ] -c arb amoyl- e thyl^ -2-de s oxy-D-gluc ose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl) -c arb amoyl-2 ’ -hydroxy-propyl ]-c arb amoyl- ethyl <j -2-15 desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-oarbamoyl-3-carboxy-propyl)-carbamoyl-2’-(p-hydroxy-phenyl)-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acet amino-3-0-^D-l-[L-l-(D-l-o arb amoyl-3-c arb oxy-20 propyl) - c arb amoyl-h, ΪΓ-1 e tr arne thylen] - c arb amoyl-ethyl ^-2- de s oxy-D-gluc ose, i 2-glycolyl amino-3-O-^D-l-[L-l-(L-l-carb amoyl-3-c arb oxy-pr opyl )-c arb amoyl-ethyl]-carb amoyl-ethyl^ - 2- de s oxy-D-glucose, 25 2-glyc olyl amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl ]- c arb amoyl-me thyl ^-2-des oxy-D-glucos e, 2- (H-methyl-ac et amino )-3-0- ^ [L-l- (D-l- c arb amoyl-3-c arb oxy-pr opyl) -c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ - 2-desoxy-D-glucose, 30 2- ( ΪΓ-me thyl- ac e t amino )-3-0-^D-l-[L-l-(D-l-c arb amoyl- 3- carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-des- 2-acetamino-3-0-^D-l-[L-1-(D-l-carb amoyl-3-c arb oxy- propyl)-c arb amoyl-2’-phenylethy 1]-carbamoyl-ethyl< - 2- desoxy-D-glucose, 61

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oxy-D-glucose, 5 2-acetamino-3~0- £[L-1-(D-1 -c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21-(p-hydroxyphenyl)-ethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose, 2- acetamino-3-0-£[L-l-(D-l-[l-l-carboxy-ethyl]-carbamoyl- 3- b enzylcarboxy-propy1)-c arb amoyl-ethyl]-c arb amoylmethyl^-10 2-desoxy-D-glucose eller 2- a c e t amino - 3- O- ^ [ L-1- ( D-1, 3-di-carboxy-propyl)-E-methyl-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose.

Eksempel 28.

På analog måde som i eksempel 3 opnår man fåreerythrocyt-13 membraner koblet til 2-aceta.mino-3-0-^[ (D-l-carbamoyl-3-carboxy-propyl)-carbam-oyl-methyl ] -E-me thyl- c arb amoyl-me thyl ^ -2-des oxy-D-g'luc os e, 2-b enz oylamino-3-0-^[(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-methyl]-N-methyl-c arb amoyl-methyl^-2- des oxy-D-20 glucose, 2-acetamino-3-0~^[L-l-(D-l~carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl)-E-ethyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-ac et amino-3-O->[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-25 carbamoyl-propylj-E-methyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acet amino-3-0->[1-1-(D~1-c arb amoyl-3-c arb oxy-propy1)- c arb amoyl-propyl]-E-ethyl-c arb amoyl-methyl^-2-de s oxy-D- glucose, 2-b enz amido- 3-O->[1-1-(D-1-c arb amoyl- 3- c arb oxy-pr opyl) - c arb amoyl-pr opyl ] -K- e thyl- c arb amoyl-me thyl ^-2-de s oxy-D- glucose, 62

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2-acetamino-3-0- £[L-l-(r -1-carbamoyl-3-carboxy-propyl)-5 c arb amoyl-ethyl]-K-propyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carb amoyl-K, N-pent arne thylen] -c arbamoyl-methyl j-2-desoxy-D-glucose, 10 2-benzoylamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-K, K-pent ane thyl en] - c arb amoyl-me thyl ^ -2-de s oxy-D-glucose, 2-ac etamino-3-0-^[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-ΚΙ 5 methyl-c arb amoyl-ethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose , 2-acetamino-3-0-/D-l-[D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-methyl]-K-methyl-c arb amoyl-ethyl^-2-de soxy-D-glucose, 20 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-me thyl ] -K-methyl- c arb amoyl- ethyl ^-2-de s oxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- · propyl)-c arb amoyl-ethyl)-K-ethyl-c arb amoyl-ethyl^-2-25 desoxy-D-glucose, 2- ac et amino- 3-0- ^D-l- [ L-1- (D-l-c arb amoyl- 3- c arb oxy-pr opyl )-c arbamoyl-pr opyl]-N-methyl-carb amoyl-ethyl^-2-de s oxy-D-glucose, 2- ac et amino- 3-0- ^D-l- [ L-l- (D-l-c arb amoyl- 3- c arb oxy-30 propyl) -c arb amoyl-propyl ] -K- ethyl- c arb amoyl-methyl ^ -2-de s oxy-D-gluc ose,

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63 2-benzamido-3-0-^D-l-[l-l-(D~l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl]-H-ethyl-carbamoyl-ethylj-2-desoxy-D-glucose, 2-ac et amino-3-0-^D-l-[L-1-(D-l-c arb amoyl-3-c arb oxy-5 propyl) - c arb amoyl- e tiiyl ] -K-pr opyl- c arb amoyl- e thyl j - 2-desoxy-D-glucose, 2-acetamino-3-0-^D-1-[L-1-(D-l-c arbamoyl-3-c arb oxy-pr opyl) - c arb amoy 1-N, U-p ent arnethy1 en ] - c arb amoyl- e t hyl ^ -2-desoxy-D-glucose, 10 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl) - carb amoyl-N,N-pentarnethylen]-c arb amoyl-ethyl<-2-de s oxy-D-glucose, 2- ac et amino- 3-0- ^D-l- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl)-K-methyl-c arb amoyl-ethyl]-c arb amoyl-ethyl< - 2-15 desoxy-D-glucose, 2- ac et amino- 3- 0- £ [ L-1- (D-1- c arb amoyl- 3- c arb oxy-me thyl-phenyl)-c arb amoyl-metby1^ - 2- de s oxy-D-gluc ose, 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl]-c arb amoyl-methylj-2-de soxy-D-gluc os e, 20 2-b enz amino-3-0-£[L-1-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-methyl-merc apto-ethyl]-c arb amoyl-methyly-;2-desoxy-D-glucose, 2-b enz amino-3-0-£[L-1-(D-1-carb amoyl-3-c arb oxy-propyl)-carbamoyl-2-chlorethyl]-carbamoyl-methyl^-2-desoxy-D-25 glucose, 2-acetamino-3-0-£D-l-[L-l-(D-l-carbamoyl-3,3-<licarboxy-propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-(p-carbomethoxy-succinamido)-3-0-^[L-l-(D-l-carbamoyl-30 3“carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-de s oxy-D-gluc ose, l 2-(p-carbometh.oxy-STiccinamido)-3-0-^I)-l-[L-l-(I)-l-carbam- oyl-3-c arb oxy-propyl) - c arb amoyl-ethyl]-carbamoyl-ethyl·^- 2-desoxy-D-glucose, 64

DK 161026 B

2-benzamino-3-0-£L-l-(l)-l-carbamoyl-3--trimethylsilyl-3 carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-l,4,6-tris-trimethylsilyl-D-glucose. (Yed kontakt med vand hydro lys er es trimetbylsilylestergrnppen hurtigt) , 2-ac etamino-2-des oxy-3—0— ^[L-l-(L-l-carb amoyl-3-trimethyl-s ilylcarboxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-10 1,4,6-tris-trimethylsilyl-b-glucose, 2-acetamino-3-0-/D-l-[l-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propylJ-carbamoyl-ethyl^-2-desoxy-D-glncose, 2-acetamino-3-0-^b-1-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21-hydroxy-propyl]-c arb amoyl-ethyl^ - 2-15 desoxy-D-glucose, 2-acetamino-3-0-^b-l-[L-l-(b-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2' - (p-hydroxy-phenyl)-ethyl ]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(L-l-carbamoyl-3-carboxy-20 propyl) - c arb amoyl-N, h-1 etrarnethyl en] - c arb amoyl- ethyl ^-2-j desoxy-L-glucose, 2-glycolylamino-3-0-£b-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carb amoyl-ethyl]-c arbamoyl-ethyl^-2-de s oxy-D-glucose, 25 2-glyc olylamino-3-0-^[L-l-(D-l-carbamoyl-3-c arb oxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-(N-me thyl- ac etamino)-3-0->[L-l-(L-l-c arb amoyl-3-c arb-oxy-pr opyl) - c arb amoyl- ethyl J - c arb amoyl-me thyl i - 2-desoxy-D-glucose, 30 2- (li-methyl-ac et amino )-3-0- ^L-l- [L-l- (L-l-carbamoyl-3- carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-des- 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-carbamoyl-21-phenylethyl]-carbamoyl-ethylj-2-des- oxy-D-glucose, 65

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oxy-D-glucose, 5 2-acetamino-3-O-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-21-(p-hydroxyphenyl)-ethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose, 2- acetamino-3-0-£[L-l-(D-l-[L-l-carboxy-ethyl]-c arb amoyl- 3- b enzylc arb oxy-propyl)-c arb amoyl-ethyl]-carb amoylmethyl^-10 2-desoxy-D-glucose eller 2-acetamino-3-0- £[L-l-(D -1,3-dicarboxy-propyl)-N-me thyl-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose.

Eksempel 29.

På analog måde som i eksempel 4- opnår man gruppe C-poly-13 saccharid fra Neisseria meningitidis koblet med 2-ac etamino-3-0-£[(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-me thyl ]-N-methyl-c arb amoyl-methyl^-2-de s oxy-D-gluc os e, 2-b enz oylamino- 3- 0- £ [ (D-1- c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-methyl]-N-methyl-c arb amoyl-methyl^-2-des oxy-D-20 glucose, 2-acetamino-3-0- £[1-1-(1)-1 -carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl)-N-ethyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-ac et amino-3-0-/[L-l-(D-1-carb amoyl-3-c arb oxy-propyl)-25 c arb amoyl-propylJ-N-metbyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-HL-l-(D-l-c arb amoyl-3-carboxy-propyl)- c arb amoyl-propyl]-N-ethyl-c arb amoyl-methyl^-2-des oxy-D- glucose, 2-b enz amido-3-0->[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)- c arb amoyl-pr opyl ] -BT- ethyl- c arb amoyl-me tbyl ^ - 2- de s oxy-D- glucose, 66

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2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-3 c arbamoyl-ethyl ]-Bf-propyl-carb amoyl-me thyl^-2-desoxy-D-glucose, 2-acet amino-3-0- [[I-l-CD -1-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-BT,BT-p ent arne thylen] -c arbamoyl-methylj-2-desoxy-D-glncose, 10 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-BT, BT-p ent ame thyl en ] - c arb amoyl-me thyl ^ - 2-de s oxy-D-glucose, 2-acetamino-3-0- £[L-1-(D -l-carbamoyl-3-carboxy-propyl)-Bf-me thyl- c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ -2-de s oxy-D-15 glucose, 2-ac e t amino-3-0-XD-1-[ (D-l-c arb amoy^.-3-c arb oxy-pr opyl)-carbamoyl methyl j-Ii-methyl-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)-20 carb amoyl-me thyl ] -Bi-me thyl- c arb amoyl- ethyl ^ - 2-de s oxy-D-glucose, 2-acetamino-3--0- ^D-l- [L-l- (D-l-carbamoyl-3-carboxy-pr opyl) -c arb amoyl- ethyl) -ΒΓ-e thyl- c arb amoyl- ethyl )-2-desoxy-D-glucose, 25 2- ac e t amino- 3-0- ^D-1- [L-1- (D-l- c arb amoyl- 3- c arb oxy-pr opyl) - c arb amoyl-pr opyl ] -ΒΓ-me t]jyl- c arb amoyl- ethyl ^-2-desoxy-D-glucose, 2-acetamino-3-0- j>D-1- [ L-1- (D-1- c arb, amoyl- 3- c arb oxy-pr opy 1) - c arb amoyl-pr opyl ] -li- ethyl- c arb amoyl-me thyl ^-2-30 desoxy-D-glucose, 2-benzamido-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-c arb amoyl-propyl]-Ή-ethyl-c arb amoyl-ethyl^-2-de s- oxy-D-glucose, 67

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2- ac et amino- 3-0- j>D-l- [ L-l- (D-l-c arb amoyl- 3-c arb oxy-5 propyl)-carbamoyl-ethyl]-ir-propyl-carbamoyl-ethyl^-2-des-oxy-D-glucose, 2-acetamino-3-0-^D-l-[l-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-N,N-pentamethylen]-c arb amoyl-ethylj-2-desoxy-D-glucose, 10 2-b enz oylamino-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arboxy-pr opyl) -c arbamoyl-N, N-p ent ame thylen ] - c arb amoyl- ethyl j -2-de s oxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl) -U-me thyl- c arb amoyl- e thyl ] - c arb amoyl- e thyl y-,2-13 desoxy- D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl]-c arb amoyl-methyl^ - 2- de s oxy-D-gluc ose, 2-benzoylamino-3-0-^[l/-l-(D-l-carbamoyl· " ^arboxy-methyl-phenyl]-carbamoyl-methyl^-2-desoxy-D-g se, 20 2-b enz amino-3-0-£[L-l-(D-l-c arbamoyl-3-c arb oxy-propyl)-carbamoyl-2-methyl-mercapto-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-b enz amino-3-0-£[L-1-(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-chlorethyl]-c arb amoyl-methyl^-2-de s oxy-D-25 glucose, 2-acetamino-3-0-^D-l-[li-l-(D-l-carbamoyl-3,3-cl.icarboxy- propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D-glu- cose, 2-(6-carbornethoxy-sue cinamido)-3-0-^[L-l-(D-l-c arb amoyl-30 3-c arboxy-propyl)-c arb amoyl-ethyl]-carbamoyl-methyl^ -2-de s oxy-D-glucose, 2-(β-carb orne thoxy-sue cinamido)-3-0-^D-l-[L-l-(D-l-c arb am oyl- 3-c arb oxy-propyl)-c arb amoyl- ethyl]-c arb amoyl-e thyl^- 2-desoxy-D-glucose, 68

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2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-trimetbylsilyl-3 carboxy-propyl)-carbamoyl-etbyl]-carbamoyl-metliyl^-2-desoxy-l,4,6-tris-trimethylsilyl-D-glucose. (Ved kontakt I med vand hydrolyseres trimethylsilylestergruppen hurtigt), i 2-acetamino-2-desoxy-3-0-^[L-l-(D-l-carbamoyl-3-trimethyl- s ilylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-10 1,4,6-tris-trimethylsilyl-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-ac e t amino - 3-0-1-[ L-1-( D-1-c arb amoy 1-3-c arb oxy-13 propyl)-carbamoyl-2’-hydroxy-propyl]-carbamoyl-etbyl^-2-de s oxy-D-gluc ose, 2-acet amino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21-(p-hydroxy-phenyl)-ethyl]-carbamoyl-ethylj-2-desoxy-D-glucose, 20 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-N,N-tetramethylen]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-glycolylamino- 3-0- ^ D-l-[L-l- (D-l-carb amoyl- 3-c arb oxy-propyl)-g arb amoyl-ethyl]-carb amoyl-ethyl^-2-de s oxy-D-23 glucose, 2-glycolylamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-methyls-2-de s oxy-D-glucose, 2- (H-methyl- ac et amino )-3-0-^[L-l-(D-l-c arb amoyl- 3- c arb oxy- 30 propyl)-carbamoyl-ethyl]-carbamoyl-metbyl^-2-desoxy-D- glucose, DK 161026 8 2-(1-methyl-ac et amino)-J-O-^D-l-[L-l-(D~l-c arbamoyl-J- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2- desoxy-D-glucose, 69 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 carbamoyl-2' -phenylethyl]-carbamoyl-ethyl^-2-desoxy-I)-glucose, 2- ac etamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-(p-hydroxyphenyl)-ethyl]-c arbamoyl-methyl^ - 2-desoxy-D-glucose, 10 2-ac etamino-3—0— ^[L-l-(D-1- [L-l-c arb oxy-ethyl]-c arb amoyl- 3- b enzylcarb oxy-propyl)-carbamoyl-ethyl]-carb amoylmethyl^-2-desoxy-D-glucose eller 2-acetamino-3-0-£[L-l-(D-l,3-dicarboxy-propyl)-l-methyl-carbamoyl-etby1]-carbamoylmethyl^-2-desoxy-D-glucose, 15 Eksempel 50« På analog måde som i eksempel 5 opnår man merozoiter fra malariaparasitten Plasmodium knowlesi koblet til 2-acetamino-3- 0-£[(d -1-c arb amoyl-3-c arboxy-propyl)-carb am-oyl-methyl]-1-methyl-carb amoyl-methyl^-2-de soxy-D-gluc ose, 20 2-benzoylamino-3-0-^[(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-methyl]-1-methyl-c arb amoyl-methyl^-2-de soxy-D-glucose, 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl- ethyl) -IT- ethyl- c arb amoyl-me thyl ^ - 2- de s oxy-D-25 glucose, 2-acetamino-3-0->[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)-carbamoyl-propylJ-N-methyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3~carboxy-propyl)-

DK 161026 B

70 c arb amoyl-propyl ] -ΪΓ- ethyl-c arb amoyl-methyl ^ - 2-de s oxy-D-glucose, 2-b enz amido- 3-0- > [L-l- (D-l- carb amoyl- 3- c arb oxy-propyl) - ! V ) carb amoyl-pr opyl J -N-ethyl-carb amoyl-me thyl ^ -2-des oxy-D- 5 glucose, 2- ac e t amino- 3-0- ^ [1-1- (D-l-c arb amoyl- 3-c arb oxy-propyl) -carbamoyl-ethyl ]-iT-propyl-earbamoyl-methyl^-2-desoxy-D-glucose, 2-acet amino-3—0— ^[L-l-(D-l-c arb amoyl-3-c arboxy-propyl)-10 c arb amoyl-ΪΓ, N-p ent ame thyl en- c arb amoyl-me thyl ^ - 2- de s oxy-D-glucose, 2-b enzoylamino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carb amoyl-F, N-pentamethyl en]-carb amoyl-me thyl <j-2-des oxy-D-glucose, 13 2-ac et amino-3-0-£[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-N-methyl-c arb amoyl-ethyl]-c arb amoyl-methyl^-2-des oxy-D-glucose, 2-acetamino-3-0->D-l-[(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-methyl]-D-methyl-c arb amoyl-ethyl<j -2-des oxy-D-.20 glucose, 2-b enz oylamino-3-0-^D-l-[(D-l-carb amoyl-3-c arboxy-propyl)-c arb amoyl-me thyl ] -ΕΓ-me thyl- c arb amoyl- ethyl ^ - 2-de s oxy-D-glucose, 2- ac e t amino- 3- 0- ^D-l- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-25 propyl)-c arb amoyl-ethyl)-D-ethyl-c arb amoyl-ethyl^-2-des-oxy-D-glucose, 2-acetamino-3-0-/D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-pr opyl ] -N-me thyl- c arb amoyl- e thyl y- 2- de s oxy-D-glucose, 30 2-acetamino-3-0-^D-1-[L-1-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-pr opyl ] -N- ethyl- c arb amoyl-me thy 1 <| - 2- de s oxy-D- 2--1) enz amido- 3-0- ^D-l- [ L-1- (D-1- c arb amoyl- 3-c arb oxy- propyl)-carbamoyl-propyl]-N-ethyl-carbamoyl-ethylj-2-des- oxy-D-glucose, 71

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glucose, 5 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- pr opyl) - c arb amoy 1- ethyl ] -ΪΓ-pr opyl- c arb amoyl- e thyl ^-2-de s-oxy-D-glucose, 2-ac et amino-3-0-^D-l-[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-carbamoyl-N,lT-pentamethylen]-carbamoyl-ethyl^-2-10 desoxy-D-glucose, 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-pr opyl) - c arb amoyl-N, h-pent arne t hyl en] - c arb amoyl- e thy 1 j - 2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-15 propyl)-H-methyl-c arb amoyl-ethyl]-c arb amoyl-ethylj-2-desoxy-D-glucose, 2-acetamino-3-0-£[L-l-(D-l-carbamoyl~3-carboxy-methyl-phenyl]-c arb amoyl-methyl^-2-de s oxy-D-gluc ose, 2-b enz oylamino-3-0-£[L-l-(D-1-carb amoyl-3-c arb oxy-methyl-20 phenyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-benzamino-3-0- £[L-1-(D-1 -c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-methyl-merc apt o-ethyl]-c arb amoyl-me thylj - 2-desoxy-D-glucose, 2-b enz amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-25 carbamoyl-2-chlorethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3,3-åicarboxy-propyl)-carb amoylethyl]-c arb amoyl-ethyl^-2-des oxy-D-glucose, 30 2-(β-c arb ornethoxy-sue c inamido)-3-O-£[L-1-(D-1-c arbamoyl-

DK 161026B

72 3-c arboxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl^ -2-i desoxy-D-glucose, 2- C β - c arb orne thoxy- sue c inamido )-3-0- ^D-l-[L-l- (D-l-c arb am-oyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-5 2-desoxy-D-glucose, s 2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-trimethylsilyl- ; c arb oxy-pr opyl) - c arb amoyl- ethyl ] - c arb amoyl-me thyl ^ - 2- desoxy-l,4,6-tris-trimethylsilyl-D-glucose. (Ved kontakt j med vand hydrolyseres trimethylsilylestergruppen hurtigt), i 10 2-acetamino-2-desoxy-3-0-j>[l-l-(D-l-carbanLoyl-3-trimethyl-s ilylc arb oxy-propyl)-c arb amoyl-ethyl]-carb amoylmethyl^- 1,4,6-tris-trimethylsilyl-D-glucose, 2- ae et amino-3-0- ^D-l-[ L-l- (D-l-c arb amoyl-3- c arb oxy-pr opyl)-c arb amoyl-propyl]-carb amoyl-ethyl^-2-desoxy-D-15 glucose, 2-acetamino-3-0-^1-1-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21-hydroxy-propyl]-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-ac et amino-3-0- ^D-1-[L-1- (D-l-carbamoyl-3-carboxy-20 propyl)-carbamoyl-2,-(p-hydroxy-phenyl)-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2- ace t amino- 3-O- £d-1-[L-1- (D-l-carb amoyl-3-c arb oxy-pr opyl) -carb amoyl-E, E-tetramethylen]-c arb amoyl- ethyl ^ -2-desoxy-D-glucose, 25 2-glycolylamino-3-0- £d-1- [L-l- (D-1-c arb amo yl- 3- c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-de s oxy-D-glucose, 2-glycolylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl^-2-de s oxy-D-gluc ose, 30 2-(E-methyl-ac et amino)-3-0-£[L-l-(D-l-c arb amoyl-3-carboxy-pr opyl) -carb amoyl- ethyl ] - carb amoyl-me thyl ^ -2-de s oxy-D- 2-(N-methyl-acetamino)-3-0-^D-l-[L-l-(D-l-carbamoyl-3- carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-des- oxy-D-glucose, 73

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glucose, 5 2- ac et amino- 3-0- ^D-l- [ L-l- (D-l-c arb amoyl- 3- c arb oxy-pr opyl) -carbamoyl-2'-phenylethyl]-c arb amoyl-ethyl^-2-de s oxy-D-glucose, 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21-(p-hydroxyphenyl)-ethyl]-carbamoyl-methyl^-10 2-desoxy-D-glucose, 2- acetamino-3-0-£[L-l-(D-l-[L-l-carboxy-ethyl]-carbamoyl- 3- b enzylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-desoxy-D-glucose eller 2-acetamino-3-0-£[L-l-(D-l,3-dicarboxy-propyl)-N-methyl-15 carbamoyl-ethyl]-carbamoylmethyl^-2-desoxy-D-glucose.

Eksempel 31» På analog måde som i eksempel 6 opnår man T-lymfoblaster fra CBA/J-mus koblet til 2-ac et amino-3-0-£[(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb am-20 oyl-methyl]-N-methyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0-£[(D-1-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-me thyl]-1-methyl-c arb amoyl-methyl^-2-de s oxy-D-glucose , 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-25 c arb amoyl- ethyl ] -ΪΓ- ethyl- c arb amoyl-me thyl ^ - 2-de s oxy-D-glucose , 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)- c arb amoyl-propyl]-N-methyl-c arb amoyl-methyl^-2-de s oxy-D- glucose, 2-acet amino-3-0->[L-l-(D-1-c arbamoyl-3-c arb oxy-pr opyl)- c arb amoyl-pr opyl J -ΕΓ-ethyl- c arb amoyl-me thyl ^ - 2-de s oxy-D- glucose, 74

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i i 2-benzamido-3-0->[L-l-(D-l-carbamoyl-3-carboxy-propyl)-5 c arb amoyl-propylJ-Ei- ethyl-carb amoyl-methyl^-2-des oxy-D-i glucose, 2- ac et amino- 3-0-l·-!- (D-l-c arb amoyl- 3- c arb oxy-propyl) -c arb amoyl- ethyl ] -ΕΓ-pr opyl- c arb amoyl-me thyl ^ - 2-de s oxy-D-| glucose, 10 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-U, U-pentame thyl en]-c arb amoyl-me thyl j-2-de s oxy-D-glucose, 2-b enz oylamino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ΕΓ, ET-p ent ame thyl en ] - c arb amoyl-me thyl ^ -2-de s oxy-15 D-glucose, 2-acetamino-3-0- - c arb amoyl- 3- c arb oxy-pr opyl) -El me thyl-carbamoyl-ethyl ]-c arb amoyl-methyl ^-2-des oxy-D-glucose, 2-acetamino-3-0->D-1-[(D-l-carbamoyl-3-carboxy-propyl)-; 20 c arb amoyl-me thyl ] -EF-me t hyl- c arb amoyl- e thyl ^ - 2- de s oxy-D- I glucose, 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-me thyl ]-ET-methyl- c arb amoyl- ethyl ^ -2-des oxy-D-glucose, 25 2- ac et amino- 3-0- ^D-l- [ L-l- (D-l-c arb amoyl- 3- c arb oxy- pr opyl ) - c arb amoyl- ethyl) -ET- ethyl- c arb amoyl-e thyl ^ - 2- de s -oxy-D-glucose, 2-ac et amino-3-0-^D-1-[L-l-(D-l-c arb amoyl-3-c arb oxy-pr opyl ) - c arb amoyl-pr opyl ] -ΕΓ-me thyl- c arb amoyl- ethyl ^-2-30 desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l~(D-l-carbamoyl-3-carboxy- propyl) - c arb amoyl-propyl ] -IT- ethyl-c arb amoyl-methyl ^-2- desoxy-D-glucose,

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-7 5 2-benz amido-3-0-j>D-l- [L-1-(D-1- c arb amoyl- 3- c arb oxy-3 propyl)-carbamoyl-propyl]-iT-ethyl-carbamoyl-ethylj-2-des-oxy-D-glucose, 2- ac e t amino- 3-0-£D-1-[L-1-(D-l-c arb amoyl- 3- c arb oxy-propyl)-carbamoyl-ethyl]-ir-propyl-carbamoyl-ethylj-2-des-oxy-D-glucose, 10 2-acetamino-3-0-^D-l-[l-l-(D-l-carbamoyl-3-carboxy- propyl)-c arb amoyl-N,D-pent arnetbylen]-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^D-l-[L-l-(D-lr-carbamoyl-3-carboxy-pr opyl) - c arb amoyl-N, IT-p ent arne tiiyl en ] - c arb amoyl-e thyl j - 2-15 desoxy-D-glucose, 2-ac et amino-3- 0- £ D-1- [ L-1- ( D-1- carb amoyl-3-c arb oxy-propyl)-N-me thy1-c arb amoyl-ethyl]-c arbamoyl-ethyl^-2-des oxy-D-glucose, 2-ac et amino-3- 0- £ [ L-1- ( D-1- c arb amoyl-3-c arb oxy-methyl-20 phenyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0-£[L-l-(D-l-c arb amoyl-3-c arboxy-methyl-pheny1]-c arb amoy1-methyl^ - 2- de s oxy-D-glucose, 2-b enz amino- 3-0- L-1- ( D-l- c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl- 2-me thyl-mer c apt o- ethyl ] - c arb amoy 1-me thyl ^-2-25 desoxy-D-glucose, 2-b enz amino-3-0-£[L-1-(D-1-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-chlorethyl]-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-£d-1-[L-1-(D-l-carbamoyl-3,3-dicarboxy-30 propyl)-c arb amoylethyl]-carb amoyl-ethyl^-2-des oxy-D-glucose,

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76 2-(β-carbomethoxy-succinamido)_ 3-O-£[L-l-(D-l-c arb amoyl-3~c arb oxy-propyl) -c arb amoyl- ethyl ] - c arb amoyl-methyl y- 2-desoxy-D-glucose, 2-(β-carb omethoxy-suecinamido)-3-0-^D-l-[L-l-(D-l-carb am-| 5 oyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^- ' 2-desoxy-D-glucose, 2-b enz amino- 3-0- [[L-1-(D -1-c arb amoyl-3-trimethylsilyl-c arb oxy-propyl) - c arb amoyl- ethyl ] - c arb amoyl-me thyl ^-2-desoxy-l,4,6-tris-trimethylsilyl-D-glucose. (Ved kontakt 10 med vand hydrolyseres trimethylsilylestergruppen hurtigt), 2-acetamino-2-desoxy-3-0-£[L-l-(D-l-c arb amoyl-3-tri-: me thy 1- silyl c arb oxy-propyl) - c arb amoyl- ethyl ] - c arb amoyl- methyl^-1,4,6-tris-trimethylsilyl-D-glucose, 2-acetamino-3-0->D-L-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-| 15 carbamoyl-propylj-carbamoyl-ethyl^-2-desoxy-D-glucosev - 2- ae e t amino-3-0- ^D-1- [ L-1- (D-1- c arb amoyl- 3- c arb oxy-: propyl)-carbamoyl-2’-hydroxy-propyl]-carbamoyl-ethyl^-2- ! desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-20 propyl)-carbamoyl-2 ’ -(p-hydroxy-ph.enyl)-eth.yl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2- ac et amino- 3-0- j>D-l- [ L-1- (D-l-c arb amoyl- 3- c arb oxy-propyl )-c arb amoyl-N,IT-1 etrarnethylen]-c arb amoyl-ethyl^-2-desoxy-D-glucose, 25 2- gly c o lyl amino- 3-O- ^D-1- [ L-1- ( D-1- c arb amoyl- 3- c arb oxy-propyl )-c arb amoyl-e thyl]-c arb amoyl-ethyl^-2-des oxy-D-glucose, 2-glyc olylamino- 3-0-[L-l-(D-l-carb amoyl-3-earb oxy- pr opyl ) - c arb amoyl-ethyl]-c arb amoyl-methyl^-2-des oxy-D- 30 glucose, 2- ( -me thyl- ac e t amido ) - 3- 0- £ [ L-1- ( D~ 1- c arb amoyl- 3- c arb oxy- propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl^-2-desoxy-D- glucose, 77

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2-(U-me tliyl-ac e t amino ) - 3-0-^D-l-[ L-l-( D-l-c arb amoyl-3-5 c arb oxy-pr opyl) - c arb amoyl- ethyl ]- c arb amoyl- ethyl ^-2-desoxy-D-glucose, 2-ac etamino-3-0-^D-l-[1-1-(D-l-c arbamoyl-3-c arb oxy-propyl)-carbamoyl-21-phenylethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 10 2-acetamino-3-0-£[l-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-21-(p-hydroxypheny1)-ethyl]-c arb amoyl-methyl^ - 2-desoxy-D-glucose, 2- acetamino-3-0- £[L-l-(D-l-[L-l -c arboxy-ethyl]-c arb arnoyl- 3- b enzylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethylj-15 2-desoxy-D-glucose eller 2-acetamino-3-0-£[l-l-(D-l,3-dicarboxy-propyl)-l-methyl-c arb amoyl-ethyl]-c arb amoylmethyl^-2-des oxy-D-glucose,

Eksempel 32, På analog måde som i eksempel 12 opnår man mund- og klov-20 sygekultur-vaccine (Behringwerke) koblet til 2-acetamino-3-0-£[(D-l-carbamoyl-3-carboxy-propyl)-carbam-oyl-methyl]-N-methyl-c arb amoyl-methyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0-^[(D-l-c arb amoyl-3-c arboxy-propyl)-c arb amoyl-me thyl ] -N-methyl-c arb amoyl-me thyl ^ - 2-de s oxy-D- 25 glucose, 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl- ethyl) -N- ethyl- c arb amoyl-me thyl -j-2-des oxy-D-glucose, 2-ac etamino-3-0-^[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)- 78

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carb amoyl-pr opyl ] -N-methyl-carb amoyl-methyl ^ -2-des oxy-D-! glucose, | ! 2-aeet amino-3-0-£[L-l-(D-l-c arh amoyl-3-c arh oxy-propyl)- carbamoyl~propyl]-K-ethyl-carbamoyl-methyl^-2-desoxy-D-I 5 glucose, 2-1> enz amido-3-0-> [l-l-(D-l-carh amoyl-3-c arh oxy-pr opyl)-c art) amoyl-pr opyl ] -ΪΓ-ethyl- c arh amoyl-me thyl ^ - 2-des oxy-D-glucose, 2- ac et amino- 3-0-^[ L-l- (D-l-c arh amoyl- 3- c arb oxy-pr opyl) -10 c arh amoyl-ethyl]-N-propyl-c arh amoyl-methylj-2-de s oxy-D-glucose, 2-ac et amino-3-0- ^ [L-l- (D-l-c arh amoyl- 3-c arh oxy-pr opyl) -c arh amoyl-if, K-p ent amethylen] -carh amoyl-me thyl j -2-des oxy-D-glucose, 15 2-b enz oylamino-3-0-^[L-l-(L-l-c arhamoyl-3-c arboxy-propyl)-carh amo yl-N ,ϊΓ-ρ ent arne thyl en ] - c arh amoyl-methyl y- 2- d e s oxy-D-glucose, 2-ac et amino-3-0-£[L-l-(D-l-carh amoyl-3-carhoxy-propyl)-N-methyl-carbamoyl-ethyl]-c arh amoyl-methyl^-2-des oxy-D- 20.glucose, 2-acetamino-3-0-^D-l-[(D-l-carbamoyl-3-earboxy-propyl)-c arh amoyl-me thyl ] -H-me thyl- c arh amoyl- ethyl ^ - 2-de s oxy-D-glucose, 2-b enz oylamino-3-0-^D-l-[(D-l-carh amoyl-3-c arb oxy-propyl)-25 c arb amoyl-methyl]-H-methyl-carhamoyl-ethyl^-2-desoxy-D-glucose, 2-ac e t amino-3-0-^ D-1-[ L-1-(D-1-c arh amoy 1-3-c arh oxy-pr opyl) - c arh amoyl- ethyl) -IT- ethyl- c arh amoyl- ethyl ^ - 2-des-oxy-D-glucose, 30 2- ac e t amino- 3- 0-^D-1-[L-1-(D-l-c arh amoyl- 3- c arh oxy-pr opyl) - c arh amoyl-pr opyl ] -N-methyl- c arh amoyl- ethyl j-2-desoxy-D- 2-acetamino-3-0-?D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-propyl]-N-ethyl-c arb amoyl-me thyl^-2-de s oxy-D- glucose, 79

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glucose, 5 2-b enz amido- 3-0-^D-1-[L-1-(D-1-c arb amoy 1- 3- c arb oxy- propyl) - c arb amoyl -propyl ] -N-ethyl-carb amoyl - ethyl j-2-des-oxy-D-glucose, 2-ac etamino-3-0-^D-l-[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-Π-propyl-c arb amoyl-ethylj-2-des-10 oxy-D-glucose, 2- ac et amino- 3-0- ^D-l- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl)-c arb amoyl-N,Π-pentarnethylen]-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-b enzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-13 propyl)-carbamoyl-N,N-pentamethylen]-carbamoyl-ethylj-2-desoxy-D-glucose, 2- ac et amino- 3-0- ^D-l- [ L-l- (D-l-c arb amoyl- 3- c arb oxv-propyl)-N-me thyl-c arb amoyl-ethyl]-c arb amoyl-ethylj-2-desoxy-D-glucose, 20 2- ac et amino- 3-0- ^ [ L-l- (D-l- c arb amoyl- 3- c arb oxy-methyl-phenyl]-c arb amoyl-methyl^ - 2- de s oxy-D-gluc ose, 2-b enz oylamino-3-0-£[L-1-(D-1-c arb amoyl-3-c arb oxy-methyl-pheny1]- carb amoyl-methyl^-2-de s oxy-D-gluc ose, 2-b enazmino-3-0-£[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-25 c arb amoyl-2-methyl-mercapt o-ethyl]-c arbamoyl-methylj-2-desoxy-D-glucose, 2-b enz amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-2-chlorethyl]-c arb amoyl-methyl^-2-de s oxy-D-glucose, 30 2-ac etamino-3-O-^D-l-[L-l-(D-l-c arb amoyl-3,3-dicarboxy- ( 80

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propyl) - c arb amoyl ethyl ] - c arb amoyl- ethyl ^-2-des oxy-D- glucose , 2- (β-carb ornethoxy-suecinamido)-3-0-£[L-l-(D-l-c arb amoyl- 3- c arb oxy-pr opyl) - c arb amoyl-e thyl ]-c arb amoyl-me thyl ^ - 2-5 desoxy-D-glucose, 2-(β-carbomethoxy-succinamido)-3-0-^D-1-[L-1-(D-l-carbam-oyl-3-c arboxy-propyl)-carb amoyl-ethyl]-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-benzamino-3-0-£[L-l-(D-1-c arb amoyl-3-1rimethyls ilyl-10 carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-des-oxy-l,4,6-tris-trimethylsilyl-D-glucose*(Ved kontakt med vand hydrolyseres trimethylsilylestergruppen hurtigt), 2-acetamino-2-desoxy-3-0-^[L-l-(D-l-carbamoyl-3-trimethyl-s ilylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-15 1,4,6-tris-trimethylsilyl-D-glucose, 2-acetamino-3-0-?D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-propyl]-carhamoyl-ethyl^-2-desoxy-D-glucose, 2-ac et amino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-c arb oxy-propyl)-carbamoyl-2'-hydroxy-propyl]-c arb amoyl-ethyl^-2-de s oxy-D-20 glucose, 2-acet amino-3-0-£D-1-[L-1-(D-1-carbamoyl-3-carboxy-propyl)-c arb amoyl-21 - (p-hydr oxy-phenyl) - ethyl ] - c arb amoyl- ethyl ^ -2-desoxy-D-glucose, 2-ac etamino-3-0-£D-l-[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-25 carb amoyl-N, N-t etr amethylen] -c arb amoyl- ethyl ^ -2-de s oxy-D-glucose, 2-glycolylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carb amoyl-ethyl ]-c arb amoyl-ethyl^-2-des oxy-D-glucose, 30 2-glycolylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-ethyl]-c arb amoyl-methyl^ - 2-de s oxy-D-gluc ose, 81

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2- (ϊΓ-me thy 1- ac et amino ) - 3-0- / [ L-1- ( D-l- c arb amo vi- 3- c arb-oxy-propyl)-c arbamoyl-ethyl] - c arb amoyl-methylj-2-de soxy-D-glucose, 2-(N-methyl-acetamino)-3-0-^D-l-[L-l-(D-l-carbamoyl-3-5 c arb oxy-propyl)-c arb amoyl-ethyl]-carbamoyl-ethy1^-2-des-oxy-D-glucose, 2- ac et amino- 3-0- ^D-1- [ L-1- (D-1- c arb amoyl- 3- c arb oxy-propyl)-carbamoyl-2'-phenylethyl]-c arb amoyl-ethyl^2-desoxy-D-glucose, 10 2-ac etamino-3-0-^[L-l-(D-l-c arbamoyl-3-carb oxy-propyl)-c arb amoyl-2'-(p-hydroxyphenyl)-ethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose, 2- ac e t amino-3-0-^ [ L-1-(D-1-[ L-1-c arb oxy-e thyl ] - c arb amoyl- 3- b enzylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-13 2-desoxy-D-glucose eller 2-acetamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-N-methyΙο arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluc ose,

Eksempel 35» På analog måde som i eksempel 17 opnår man rabies-HDC-20 vaccine (Beringwerke) koblet til 2-acetamino-3-0-^[(D-1-carbamoyl-3-carboxy-propyl)-carbam-oyl-methyl]-N-methyl-carb amoyl-methyl^-2-de s oxy-D-gluc ose, 2-b enzoylamino-3-0- £[(D-1 -carb amoyl-3-c arb oxy-propyl)-c arb amoyl-methyl]-N-methyl-c arb amoyl-methyl^-2-de s oxy-D-25 glucose, 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-ethyl)-N-ethyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-acetamino-3—0— ^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-

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82 o arb amoyl-pr opyl ] —IT—methyl- c arb amoyl-me thyl ) -2-de s oxy-D-glucose, 2-ac etamino-3—0— ^ ΠL-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-propylJ-E-ethyl-c arb amoyl-methyl^-2-de soxy-D-5 glucose, 2-b enz amido- 3-0- H L-l- ( D-l- c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl-propyl]-E-ethyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-acetamino-3-0- £[L-l-(D-l - c arb amoyl- 3- c arb oxy-pr opyl) -10 c arb amoyl- ethyl ] -E-propyl-c arb amoyl-me thyl ^-2-de s oxy-D-glucose, 2-ac etamino-3-0-^[L-l-(D-l-c arb amoyl-3-carboxy-propyl)-c arb amoyl-E, E-p ent ame thyl en] - c arb amoyl-me thyl j - 2-des oxy-D-glucose, : 15 2-benzoylamino-3“0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-E,E-pentamethylen]-carbamoyl-methyl)-2-desoxy- i D-glucose, I 2-ac etamino-3-0-[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)- E-methyl- c arb amoyl- ethyl ] - c arb amoyl-me thyl^ - 2-des oxy-D-20 glucose, 2-ac etamino-3-0->D-1-(D-l-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-methyl ]-E-methyl-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0- ^D-l- [ (D-l-c arb amoyl- 3-c arb oxy-25 propyl) -carb amoyl-methyl ]-E-methyl-c arb amoyl- ethyl )-2-de s oxy-D-gluc o s e, 2- ac e t amino- 3-0- D-1- [ L-1-( D-l-c arb amoyl- 3- c arb oxy-pr opyl) -c arb amoyl- ethyl) -E- ethyl- c arb amoyl- ethyl ^ - 2-desoxy-D-glucose, 30 2-acetamino-3-0->D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carb amoyl-pr opyl J-E-methyl-c arb amoyl-ethyl ^ -2-desoxy-D- 2-ac et amino-3-0-^D-l-[L-l-(D-l-carb amoyl-3-c arb oxy- propy1)-c arb amoyl-propyl]-H-ethyl-c arb amoyl-methyl^-2- desoxy-D-glucose, 83

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glucose, 3 2-b enz amido-3-0-^D-1-[L-l-(D-l-carb amoyl-3-c arb oxy- pr opyl) - c arb amoyl-pr opyl ] -ΪΓ- ethyl- c arb amoyl- ethyl ^-2-de s-oxy-D-glucose, 2-ac et amino-3-0-^D-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-N-propyl-c arb amoyl-ethyl^-2-de s oxy-D-10 glucose, 2- ac e t amino- 3- 0- j>D-1- [ L-1- (D-l- c arb amoyl- 3- c arb oxy-propyl) - carbamoyl-N,N-pentamethylen]-carbamoyl-ethyl^-2-desoxy- D-glucose, 2-b enz oylamino-3-0-^D-l-[L-l-(D-l-c arbamoyl-3-c arb oxy-15 propyl)-c arb amoyl-N,N-pent amethylen]-c arb amoyl-ethylj-2-desoxy-D-glucose, 2-ac etamino-3-0-^D-1-[L-l-(D-l-c arb amoyl-3-carb oxy-propyl )-N-methyl-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-des-oxy-D-glucose, 20 2-acetamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl]-c arb amoyl-methylj-2-de s oxy-D-gluc ose, 2-b enz oylamino-3- o-£[l-i-(d -1-c arbamoyl-3-c arboxy-methyl-phenyl]-c arb amoyl-methyl^-2-des oxy-D-gluc ose, 2-b enz amino- 3-0- j* [L-l- (D-l-c arb amoyl- 3- c arb oxy-pr op vi) -25 carbamoyl-2-methyl-mercapto-ethyl]-carbamoyl-methylj-2-desoxy-D-glucose, 2-b enz amino-3-0-£[L-l-(D-l-carb amoyl-3-c arboxy-propyl)-c arb amoyl-2-chiorethyl]-c arb amoyl-methyl^-2-de soxy-D-glucose, 30 2-ac et amino-3-0-^D-l-[L-l-(D-l-c arbamoyl-3 , 3-hic arb oxy- 84

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propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D-glu-| cose, I 2-(0-carb ome thoxy-succ inamido)-3-O-£[L-l-(D-1-carb amoyl- | 3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl^-2- | 5 desoxy-D-glucose, j 2-(β-carb omethoxy-sue cinamido) - 3-0- ^ D-1- [ 1-1- ( D-1- c arb am-; oyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^- I 2-desoxy-D-glucose, I 2-b enz amino-3-0-£l-1-(D-l-carb amoyl-3-trimethylsilyl- I 10 c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl^ - 2- S desoxy-l,4-,6-tris-trimethylsilyl-D-glucose. (Ved kontakt ! med vand hydrolyseres trimethylsilylestergruppen hurtigt), i ' 2-acetamino-2-desoxy-3-0-^[L-l-(D-l-carbamoyl-3-trimethyl-s ilyl c arb oxy-pr opyl) - c arb amoyl- ethyl ] - c arb amoylme thyl ^ -15 1,4-,6-tris-trimethylsilyl-D-glucose, i 2-ac et amino-3-0- ^D-1- [ L-1- ( D-1- carb amoyl-3-c arb oxy- j propyl) -c arb amoyl-propyl]-c arb amoyl- etbyl <-2-desoxy-D- glucose, i .

2- ae e t amino- 3-0-?D-1-[L-1-(D-1-c arb amoyl- 3-c arb oxy-20 propyl)-carbamoyl-21 -hydr oxy-pr opyl ] - c arb amoyl- e thyl ^ - 2-de s oxy-D-gluc ose, 2- ae et amino- 3-0- ^D-1- [ 1-1- (D-l- c arb amoyl- 3- c arb oxy-pr opyl) -carbamoyl-2' - ( p-hydroxy-phenyl) - e thyl ] - c arb amoyl- e thyl ^ -2-desoxy-D-glucose, 25 2-acetamino-3-0-^D-l-[L-l~(D-l-carbamoyl-3-carboxy- propyl)-carbamoyl-B’,]ii-tetramethylen]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-glycolylamino-3-0-^D-1- [ L-1- ( D-1- c arb amoyl- 3- c arb oxy- pr opyl )-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-des oxy-D- 30 glucose,

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2- glyc o lyl amino- 3-0-^[L-1-(D-1-c arb amoyl- 3- c arb oxy- propyl)-c arb amoyl-ethyl]-c arb amoyl-methylj-2-desoxy-D- glucose, 85 2-(N-metbyl-acetamino)-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-3 propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-(N-methyl-ac et amino)-3-0-£D-l-[L-1-(D-1-carb amoyl-3-c arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoyl-ethyl^-2-des-oxy-D-glucose, 10 2- ac et amino- 3-0- ^D-l- [ L-1- ( D-1- c arb amoyl- 3- c arb οχτ-pr opyl)-c arb amoyl-2 '-phenylethyl]-c arb amoy1-ethyl^ - 2-desoxy-D-glucose, 2-ac etamino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21-(p-hydroxyphenyl)-ethyl]-c arb amoyl-methyl^-15 2-desoxy-D-glucose, 2-ac et amino-3-0- £[L-1-(D -1-carboxy-ethyl]-carbamoyl-3-b enzylcarb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-desoxy-D-glucose eller 2-ac et amino-3-0-£[L-l-(D-l,3-dic arboxy-propyl)-N-methyl-20 carbamoyl-ethyl]-carbamoylmethyl^-2-desoxy-D-glucose.

Eksempel 34.

På analog måde som i eksempel 19 opnår man infTuenza-virus-antigener af typen A/Victoria/3/75 koblet til 2-acetamino-3-0- -1-c arb amoyl-3-c arb oxy-propyl)- 25 c arb amoyl-methyl]-N-methyl-c arb amoyl-methylj-2-de s oxy-D-glucose, 2-b enz oylamino-3-0-^[(D-l-c arb amoyl-3-c arb oxy-propyl)- carbamoyl-methyl]-h-methyl-c arb amoyl-methylj-2-desoxy-D- glucose, 2-ac etamino-3-0-£[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)- c arb amoyl- ethyl) -IT- ethyl- c arb amoyl-methyl ^-2-de s oxy-D- glucose, 86

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2-ac etamino-3-0->[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)-j 5 carbamoyl-propylj-N-methyl-carbamoyl-methyl^-2-desoxy-D- ! glucose, I 2-acetamino-3-0-HL-l-(D-l-carbamoyl-3-carboxy-propyl)- c arb amo yl-pr opyl J-K- ethyl-c arb amoyl-methyl ^ -2-de s oxy-D-glucose, 10 2-benz amido-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-c arb amoyl-propyl]-Ή-ethyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, j 2-ac et amino-3-0->[L-l-(D-l-carb amoyl-3-c arb oxy-propyl)- j c arb amoyl-ethylJ-D-propyl-c arb amoyl-methylj 2-des oxy-D- | 15 glucose, 2- a c e t amino - 3- 0- £ [ L-1- ( D-l-c arb amoyl- 3- c arb oxv-pr opyl) -c arb amoyl-U, IT-pent arnethyl en ]-c arb amoyl-methyl ^-2-de s oxy-D-glucose, I 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- 20 c arb amoyl-D, ΪΓ-pentamethylen] -u arb amoyl-methyl ^ -2-des oxy-D-glucose, 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-N-methyl-carb amoyl- ethyl ]-carb amoyl-methyl ^-2-des oxy-D-glucose, 25 2-acetamino-3-0->[(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-methyl J-JT-methyl-c arb amoyl- ethyl ^-2-des oxy-D-glucose, 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)- c arb amoyl-methyl ] -ΪΓ-me t hy 1- carb amoyl- ethyl ^ -2-des oxy-D- 30 glucose, 2- ac e t amino- 3-0- £ D-1- [ L-1- (D-1- c arb amoyl- 3- c arb oxy-pr opyl) - c arb amoyl-ethyl)-N-ethyl-c arb amoyl-ethj1^-2-des oxy-D- glucose, 87

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2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-5 propyl)-c arb amoyl-propyl]-N-methyl-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[li-l-(D-l-carbamoyl-3-carboxy-pr opyl) -c arb amoyl-propyl ] -li- ethyl-c arb amoyl-me tbyl ^ - 2-desoxy-D-glucose, 10 2-benzamido-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-pr opyl) - c arb amoyl-propyl j-Sf-ethyl-c arb amoyl- ethyl j-2-desoxy-D-glucose, 2- ac e t amino - 3- 0- ^D-1- [ L-1- (D-1- c arb amoyl- 3- c arb oxy-propyl)-carbamoyl-ethyl]-N-propyl-carbamoyl-ethyl<-2-15 desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-N,N-p ent arnethylen]-c arb amoyl-euhyl^-2-desoxy-D-glucose, 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-20 propyl)-c arb amoyl-U,N-pent arnethylen]-c arb amoyl-ethylj-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[li-l-(D-l-carbamoyl-3-carboxy-propyl)-N-methyl-carbamoyl-ethyl]-carbamoyl-ethylj-2-de s oxy-D-gluc ose, 25 2-acetamino-3-0- £[1-1-(1 -1-c arb amoyl-3-c arb oxy-methyl-phenyl]-c arb amoyl-methyl^-2-des oxy-D-glucose, 2-benzoylamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl]-c arb amoyl-methylj-2-des oxy-D-gluc ose, 2-benzamino-3-0-^[l'-l-(D-l-carbamoyl-3-carboxy-propyl)- 30 c arb amoyl- 2-me thyl-mer c apt o- ethyl ] - c arb amoyl-me thyl j- 2- desoxy-D-glucose, 2-benz amino-3-0- j>[L-l- (D-l-carbamoyl~3-carboxy-propyl)- c arb amoyl-2- chlorethyl ] - c arb amoyl-methyl^ -2- de s oxy-D- glu cose, 88

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2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3,3-dicarboxy-j 5 propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D- ! glucose, 2- ( β - c arb ometlioxy-sue c inamido ) - 3-0-^ [ 1-1-( D-l-c arb amoyl- 3- c arb oxy-propyl)-c arb amoyl-etbyl]-c arb amoy 1-me thyl^-2- j desoxy-D-glucose, I 10 2-(β-carbometboxy-succinamido)-3-0-^D-l-[L-l-(D-l-carbam- ! oyl-3-c arb oxy-propyl)-c arb amoyl-etbyl]-c arb amoyl-ethyl^- 2-desoxy-I)-glucose, ; 2-benzamino-3-0- [[L-1-(D-1 —carbamoyl—3-trimetbylsilyl— carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2- 15 desoxy-l,4,6-tris-trimethylsilyl-D-glucose. (Ved kontakt med vand bydrolyseres trimethylsilylestergruppen hurtigt), j 2-acetamino-2-desoxy-3-0-^[L-l-(D-l-c arb amoyl-3-trimetbyl- s ilylc arb oxy-propyl)-c arb amoyl-etbyl]-c arbamoylmetbyl^- 1,4,6-tris-trimethylsilyl-I)-glucose, 20 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-propyl]-carb amoyl-etbyl^-2-des oxy-D-glucose, 2-ac et amino-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-21-hydroxy-propyl]-carbamoyl-ethyl<|-2-desoxy-D-25 glucose, 2-ac et amino-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carb amoyl-2' - (p-bydroxy-pbenyl)-etbyl ]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- 30 propyl)-carbamoyl-N,N-tetramethylen]-carbamoyl-etbyl^-2- desoxy-D-glucose, 2-glycolylamino-3-0-£D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-D- glucose, 89

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2-glyc olylamino- 3- O- ^ [ L-1- ( D-1- c arb amoyl-3-carb oxy-5 propyl)-c arb amoyl-ethyl]-c arb amoyl-methyl\-2-de s oxy-D-glucose, 2-(E-methyl-acetamino)-3-0-£[L-1-(D-l-c arb amoyl-3-c arboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 10 2- (E-me thy 1- ac et amino ) - 3-0- £ D-l- [ L-1- (D-1- c arb amoyl- 3-c arb oxy-pr opyl) - c arb amoyl- ethyl ] - c arb amoyl- ethyl ^-2-desoxy-D-glucose, 2-ac etamino-3-0-^D-1-[L-1-(D-l-carb amoyl-3-c arb oxx-propyl)-carbamoyl-2'-phenylethyl]-carbamoyl-ethylj-2-15 desoxy-D-glucose, 2-acetamino-3-0-j>[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-(p-hydroxyphenyl)-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 2-ac et amino-3-0-^[L-l-(D-l-[L-l-c arb oxy-ethyl]-c arb amoyl-20 3-benzylcarboxy-propyl)-carbamoyl-ethyl]-c arb amoylmethyl ^-2-desoxy-D-glucose eller 2-acetamino-3-0-^[L-l-(D-l,3-dicarboxy-propyl)-N-methyl-c arb amoyl-ethyl]-c arb amoylmethyl^-2-de s oxy-D-gluco s e.

Eksempel 35.

25 På analog måde som i eksempel 21 opnår man tetanustoksoid koblet til 2-acetamino-3-0-^[(D-l-carbamoyl-3-carboxy-propyl)-carbam-oyl-me thyl]-E-methyl-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-b enz oylamino-3-0-£[(D-l-carb amoyl-3-c arb oxy-propyl)- 30 c arb amoyl-me thyl ] -E-methyl-c arb amoyl-methyl ^ -2-des oxy-D- 90

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glucose, 2-acetamino-3-0-£[L-l-(l>-l-carbamoyl-3-carboxy-propyl)-carb amoyl-ethyl) -H- ethyl-c arb amoyl-me thyl ^-2-des oxy-D- ! glucose, | | 5 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)- ! carbamoyl-propyl]-H-methyl-carbamoyl-methyl^-2-desoxy-I)- j glucose, 2-acetamino-3-0->[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl]-H-ethyl-carbamoyl-methyl^-2-desoxy-I)-10 glucose, i 2-b enz amido-3-0->[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)- ! c arb amoyl-propyl j -li- ethyl-c arb amoyl-me thyl 2-desoxy-D- ! glucose, j 2-acetamiuo-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- 15 c arb amoyl-ethyl]-H-propyl-carb amoyl-methyl^-2-des oxy-D- glucose, 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-H, H-p ent arne thyl en ] - c arb amoyl-me thyl < -2-de s oxy-D-glucose, 20 2-benzoylamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-H, Π-pent amethylen] -c arb amoyl-me thyl ^ -2-de s oxy-D-glucose, 2-ac et amino-3-0-^[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-Π-methyl-c arb amoyl-ethyl]-c arb amoyl-methyl^-2-de s oxy-D-25 glucose, 2-acetamino-3-0->D-1-[(D-l-c arbamoyl-3-c arb oxy-propyl)-c arb amoyl-me thyl ] -Π-methyl- c arb amoyl- ethyl^- 2-des oxy-D-glucose, 2-b enz oylamino-3-0-^D-l-[(D-l-carb amoyl-3-c arb oxy-propyl)- 30 c arb amoyl-methyl]-Π-methyl-c arb amoyl-ethyl^-2-de s oxy-D- glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-c arb amoyl-ethyl)-N-ethyl-carb amoyl-ethyl^-2- desoxy-D-glucose, 91

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2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-3 propyl)-c arb amoyl-propyl]-N-methyl-c arb amoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-propyl]-D-ethyl-c arb amoyl-methyl^-2-desoxy-D-glucose, 10 2-b enz amido-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arb oxv- propyl)-c arb amoyl-propyl]-N-ethyl-c arb amoyl-ethyl^-2-de s-oxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-N-propyl-carbamoyl-ethyl^-2-15 desoxy-D-glucose, 2-ac et amino-3-0-^D-l-[L-l-(D-l-c arb amoyl-3-c arb oxy-propyl)-carbamoyl-N,N-pentarnethylen]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0-^E-l-[L-l-(D-l-carb amoyl-3-c arb oxv-20 propyl)-c arb amoyl-if,D-pentamethylen]-c arb amoyl-ethylj-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-N-methyl-c arb amoyl-ethyl]-c arb amoyl-ethylj-2-desoxy-D-glucose, 25 2-ac et amino-3-0-£[L-l-(D-l-carb amoyl-3-c arb oxy-methyl-phenyl]-c arb amoyl-methyl^-2-de s oxy-D-glucose, 2-b enz oylamino-3-0-^[L-l-(D-l-carb amoyl-3-c arb oxy-methyl-phenyl]-c arb amoyl-methyl^-2-des oxy-D-gluc ose, 2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- 30 carbamoyl-2-methyl-mercapto-ethyl]-carbamoyl-methylj-2- desoxy-D-glucose, | 2-benzamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-2-chlorethyl]-carbamoyl-methyl^-2-desoxy-D- glucose, 92

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2-acetamino-3-0- ^D-1-[L-1- (D-l-carbamoyl-3,3-dicarboxy-5 propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2- ( β - carb owethoxy- succinamido )-3-0-^ [L-l- (D-l-carb amoyl- 3- carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-D-glucose, 10 2-(p-carbomethoxy-succinamido)-3-0-£D-l-[L-l-(D-l-carbam~ oyl-3-c arb oxy-pr opyl^ - c arb amoyl-e tnyl ]-c arb amoyl-e thyl ^-2-desoxy-L-glucose, 2-b ehz amino- 3-D-^ [L-l- (L-l- g arb amoyl- 3-trimethyl s ilyl-j c arb oxy-propyl) - c arb amoyl- ethyl ]-carb amoyl-me thyl \-2- i * / ! 15 desoxy-l,4-,6-tris-trimethylsilyl-D-glucose. [Ved kontakt med vand hydrolyseres trimethylsilylestergruppen hurtigt), 2-acetamino-2-desoxy-3- o-£[l-i-Cd-i -carbamoyl-3-trimethyl-silylc arb oxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-i 1,4-, 6-tris-trimethylsilyl-D-glucose, 20 2-acetamino-3-0-^1)-1-[L-l-(b-l-carbamoyl-3-carboxy-propyl)-c arb amoyl-pr opyl]-c arb amoyl-ethyl^-2-de s oxy-D-glucose, 2-acetamino-3-0-^D-1-[L-1-(L-l-carbamoyl-3-carboxy-propyl)-carb amoyl-21-hydroxy-propyl]-c arb amoyl-ethyl^-2-de s oxy-25 D-glucose, 2-acetamino-3-0-^L-l-[L-l-(L-l-carbamoyl-3-carboxy-propyl)-carbamoyl-21 - (p-hydroxy-phenyl)-ethyl]-carb amoyl-ethyl<j-2-desoxy-L-glucose, 2- acet amino- 3-0- j>D-l- [L-l- (L-l- c arb amoyl- 3-c arboxy-30 propyl)-carbamoyl-F,li-tetramethylen]-carbamoyl-ethyl^-2-desoxy-1-glucose, 2-glycolylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy- propyl)-c arb amoyl-ethyl]-c arbamoyl-ethyl^-2-de soxy-D- glucose, 93

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2-glycolylamino-3-0-£[L-l-{D-l-carDamoyl-3-carboxy-5 propyl)-c arb amoyl-ethyl]-c arb amoyl-methylj-2-de s oxy-D-glucose, 2- (iT-me thyl- ac e t amino} - 3-0- £ [ L-1- (D-1- c arb amoyl- 3- c arb oxy- propyl)-carbamoyl-ethyl]-carbamoyl-methylj-2-desoxy-D- glucose, 10 2- (ϊΓ-methyl- ac e t amino )-3-0- ^D-l- [ L- (D-1-c arb amoyl- 3-carboxy-propyl_.-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-P.-glucose, 2-acetamino-3-0-^D-l-LL-l-(h-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2t-phenylethyl]-carbamoyl-ethylj-2-15 desoxy-D-glucose, 2- a c e t amino - 3-0- j* [L-l- (D-l-c arb amoyl- 3- c arb oxy-pr opyl) -carbamoyl-2'-(p-hydroxyphenyl)-ethyl]-c arb amoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0- -1-c arb oxy-ethyl]-c arb amoyl- 20 3-b enzyloxy-propyl)-c arb amoyl-ethyl]-c arb amoylmethyl^-2-desoxy-D-glucose eller 2-acetamino-3-0- -1,3-dicarboxy-propyl)-N-methyl- c arb amoyl-ethyl]-c arb amoylmethyl^-2-de soxy-D-glucose.

Eksempel 36.

25 På analog måde som i eksempel 23 opnår man koleratoksoid fra Vibrio cholerae koblet til 2-acetamino-3-0-^[(D-l-carbamoyl-3-carboxy-propyl)-carbam-oyl-methyl]-N-methyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-b enz oylamino-3-0- £[p- 1-carbamoyl-3-c arb oxy-propyl)- c arb amoyl-methyl ] -ΪΓ-me thyl- c arh amoyl-me thyl ^-2-des oxy-D- glucose,

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94 2-ac et amino-3-0-£[L-1-(D-l-c arh amoyl-3-c arhoxy-propyl)-carb amoyl-ethyl) -ΪΓ- ethyl-c arbamoyl-methyl ^-2-desoxy-D-| 5 glncose, i i I 2-acetamino-3-0->[L-l-(D-l-carbamoyl-3-carhoxy-propyl)- I c arh amoyl-pr opyl J -ΪΓ-me thyl-c arh amoyl-methyl ^ -2-de s oxy- i D-glucose, | 2-acetamino-3-0->[L-l-(D-l-carbamoyl-3-carboxy-propyl)- i 10 c arb amoyl-pr opyl J-ΪΓ- ethyl-c arb amoyl-methyl ^-2-desoxy-D- i glucose, j 2-benzamido-3-0-/>[L-l-(D-l-carbamoyl-3-c.arboxy-propyl)- c arh amoyl-pr opyl ] -U- ethyl- c arh amoyl-me thyl ^-2-de soxy-D-glucose, j 15 2-acetamino-3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arh amoyl-ethyl ] -ΪΓ-propyl- c arh amoyl-me thyl ^-2-des oxy-D-glucose, 2-ac e t amino-3-0-^ [L-1-(D-1-c arb amoyl-3-c arb oxy-pr opyl)-: c arh amoyl-ΪΓ, N-p ent arne thyl en ] - c arh amoyl-methyl < - 2- de s- 20 oxy-D-glucose, 2-b enz oylamino- 3-0- £[L-1-(D -1-c arh amoyl-3-c arhoxy-propyl)-c arh amoyl-isr ,ΪΓ-pent arne thyl en ] - c arh amoyl-methyl ^-2-des oxy-D-glucose, 2- acet amino- 3-0- £ [L-l- (D-c arb amoyl- 3- c arhoxy-pr opyl) -N-25 methyl- c arh amoyl- ethyl ] - c arh amoyl-methyl ^ - 2-de s oxy-D-glucose, 2-acetamino-3-0-/D-l-[(D-l-c arh amoyl-3-c arboxy-propyl)-c arh amoyl-methyl J-E-methyl-carb amoyl- ethyl^-2-de soxy-D-glucose, 30 2-benzoylamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)- c arh amoyl-me thyl ] -U-me thyl- c arb amoyl- ethyl ^ - 2- de s oxy-D- 2-acetamino-3-0- ^ D-l-[ L-l- ( D-l-carbamoyl-3-carboxy- propyl)-carbamoyl-ethyl]-ET-ethyl-carbamoyl-ethyl^-2-des- oxy-D-glucose, 95

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glucose, 5 2-acetamino-3-0- ^ D-l-[ L-l-(D-l-carbamoyl-3-carboxy-propyl) -carbamoyl-propyl] -ET-me t hy 1 - c ar b amo y 1 - e thy 1 ^ -2-desoxy-D-glucose, 2-acetamino-3-0-^ D-l-[ L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-pr opyl] -ΕΓ-ethyl-carbamoyl-methyl^ -2-desoxy-D-10 glucose, 2-benzamido-3-0-^D-l-{ L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arbamoyl-pr opyl ]-ET-e thyl-c arbamoyl-e th.yl ^-2-de s oxy-D-glucose, 2-3οεΐ8Μχηο-3-0-(Β-1-[Ι.1-(Β-1-οβΛΜο71-3-ο8Λο^-ρΓορ71)-15 carbamoyl-ethyl]-ΕΓ-propyl-carbamoyl-ethyl^ -2-desoxy-D-glucose, 2-acetamino-3-0- i D-l-r L-l- ( D-l-carbamoyl-3-carboxy-propyl) -c arbamoyl-ΕΓ, Εί-pent arne thyl en] -carbamoyl-ethyl ^ -2-des oxy-D-glucose, 20 2-benzoylamino-3-0-^D-l-[ L-l- (D-l-carbamoyl-3-carboxy-propyl) -carbamoyl-N, ΕΓ-pentamethyl en] -carbamoyl-ethyl j -2-desoxy-D-glucose, 2-acetamino-3-0- ^ L-l-[ L-l- ( D-l-carbamoyl-^-ca^boxy-propyl) -N-me thy 1 -carbamoyl-e thy 1 ] -carbamoyl-ethyl^ -2-desoxy-D-25 glucose, 2-acetamino-3-0-^[ L-l- ( D-l-carbamoyl-3-carboxy-methyl-phenyl] -carbamoyl-methyl^ -2-desoxy-D-glucose, 2-benzoylamino-3-0-^[ L-l-(D-l-carbamoyl-3-carboxy-methyl-phenyl] -carbamoyl-methyl^ -2-desoxy-D-glucose, 30 2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)- • 96 carbamoyl-2-methyl-mercapto-ethyl] -carbamoyl-methyl^-2- desoxy-D-glucose, i

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i 2-benzamino-3-0-£[l-l-(D-l-carbamoyl-3-carbo2y-propyl)- | carbamoyl-2-chlorethyl]-carbamoyl-methyl^-2-deso:xy-D- | ! 5 glucose, i ( / j 2-acetamino-3-0-^D-l-[l-l-(D-l-carbamoyl-3,5--<li-carbo:xy- ! propyl)-carbamoylethyl]-carbamoyl-ethyl^-2-desoxy-D- glucose, 2- ( β-carbometboxy-succinamido )-3-0-^[ 1-1- ( D-l-carbamoyl-10 3-carboxy-propyl)-carbamoyl-etbyl] -carbamoyl-metbyl^ -2-desoxy-D-glucose, I 2-( p-carbomethoiny-succinamido )-3-0-^D-l-[ 1-1-( D-l-carbam- j oyl-3-carboxy-pr opyl) -carbamoyl-ethyl] -carbamoyl-etbyl ^ - I 2-desosy-D-glucose, i 1 ( ! 15 2-benzamino-3-0->[Ir-l-(D-l-carbamoyl-3-trimethylsilyl- : - v. ·) carbosy-propyl)-carbamoyl-ethyl] -c arbamoyl-me thyl ^-2- desoxy-l,4,6-tris-trimethylsilyl-D-glu.cose. (Ved kontakt med vand hydrolyseres trimethylsilylestergruppen hurtigt), 2-acetamino-2-desoxy-3-0-^[ l-l-(D-l-carbamoyl-3-trimethyl-20 silylcarboxy-propyl)-carbamoyl-ethyl]-carbamoylmethyl·^- 1,4,6-tris-trimethylsilyl-D-glucose, 2-acetamino-3-0- £ D-l-[ l-l-(l-l-carbam0yl-3-carboxy-propyl)-carbamoyl-propyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^ D-l-[1-1-(D-l-carbamoyl-3-carboxy-25 propyl)-carbamoyl-21 -hydroxy-propyl] -carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^D-l-[l-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-(p-hydroxy-phenyl)-ethyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 30 2-acetamino-3-0-^D-l-[ 1-1-(D-l-carbamoyl-3-carboxy-propyl)- 97

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carbamoyl-]tf,lT-tetramethylen]-carbamoyl-ethyl^-2-deso:xy-I>- glucose, 2-glycolamino-5-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl) -carbamoyl-ethyl] -carbamoyl-ethyl j -2-desoxy-D-5 glucose, 2-glycolamino-3-0- £[ L-l- ( D-l-carbamoyl-3-carboxy-propyl) -carbamoyl-ethyl] -carbamoyl-methyl ^ -2-desoxy-D-glucos e, 2- (N-methyl-ac et amino ) -3-0- L-l- ( D-l-carbamoyl-3-carboxy-pr opyl) -carbamoyl-ethyl] -carbamoyl-methyl ^ -2-desoxy-D-10 glucose, 2- (U-methyl-acetamino )-3-0- ^ D-l-[ L-l- ( D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-des-oxy-D-glucose, 2-ac etamino-3-0-^ D-l-[ L-l-(D-l-carbamoyl-3-carboxy-pr opyl)-15 carbamoyl-2' -phenylethyl] -carbamoyl-ethyl^-2-desoxy-D- glucose, 2-acetamino-3-0-^[Ir-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-(p-hydroxyphenyl)-ethyl]-carbamoy1-methyl^- 2- desoxy-D-glucose, 20 2-acetamino-3-0-^[L-l-(D-l-[L-l-carboxy-ethyl]-carbamoyl- 3- benzylcarboxy-pr opyl)-carbamoyl-ethyl]-carbamoylme thyl ^-2-desoxy-D-glucose eller 2-ac etamino-3-0-[ L-1-(D-1,3-dicarboxy-propyl)-N-methyl-carbamoyl-ethyl]-carbamoylmethyl^-2-desoxy-D-glucose.

25 Eksempel 37.

På analog måde som i eksempel 25 opnår man et syntetisk eicosapeptid, der er identisk med den C-terminale sekvens i det humane choriongonadotropin, koblet til 2-ac etamino-3-0-^[(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-methyl] -H-methyl-carbamoyl-methyl ^ -2-des oxy-D- ! glucose,

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98 | 2-benzoylamino-3-0-^[(D-1-carbamoyl-3-carboxy-pr opy1)- carl amoyl -methyl] -1-me tliy 1 - carbamoyl-me t by 1 ^-2-deso xy- D-5 glucose, : 2-acetamino-3-0-^[ 1-1-(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-ethyl)-N-ethyl-earbamoyl-methyl^-2-desoxy-I)-glucose, 2-acetamino-3-0-^[ 1-1- ( D-l-carbamoyl-3-c arboxy-propyl) - v \ 10 carbamoyl-propyl]-N-methyl-carbamoyl-methyl^^-desoxy-D-glucose, 2-acetamino-3-0-^[ l-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl] -N-ethyl-carbamoyl-methyl^-2-desoxy-D-glucose, 15 2-benzamido-3-0-^[l-l-(D-l-carbamoyl-3-carboxy-propyl)-c arbamoyl-propyl]-l-ethyl-carbamoyl-methyl^-2-desoxy-D-glucose, 2-acetamino-3-0-^[ l-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl] -N-propyl-carbamoyl-methyl ^ -2-desoxy-D-20 glucose, 2-acetamino-3-0-^[l-l-(D-l-carbamoyl-3-carboxy-propyl)- ^ \ carbamoyl-N,F-pentamethyleri.] -carbamoyl-methylj-2-desoxy- D-glucose, 2-b enzoylamino-3-0-1-1-(D-l-carbamoyl-3-carboxy-propyl)-25 carbamoyl-ϊί,N-pentamethylen] -carbamoyl-methyl -2-desoxy-D-glucose, 2-acetamino-3-0-^[1-1-(D-l-carbamoyl-3-c arboxy-propyl)-h-methyl-carbamoyl-ethyl ] -carbamoyl-metbyl ^ -2-desoxy-D-glucose, 30 2-acetamino-3-0-^D-l-[(D-l-carbamoyl-3-carboxy-propyl)- carbamoyl-metbyl]-ΪΓ-metbyl-carbamoyl-etbyl^-2-desoxy-D-

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2-benzoylamino-3-0-^I)-l-[ (D-l-carbamoyl-3-'Carbo:xy-propyl)- carbamoyl-methyl ] -h-methyl-carbamoyl-ethyl ^ -2-desoxy-D- glucose, 9¾ glucose, 5 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl] -II-ethyl-carbamoyl-ethyl^-2-deso:xy-D-glucose, 2-acetamino-3-0-^I)-l-[l-l-(D-l-carbamoyl-3-carbo:xy-propyl)~ carbamoyl-propyl]-H-methyl-carbamoyl-ethyl^~2-deso:xy-I)-10 glucose, 2-ac etamino-3-0-£D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl ] -h-ethyl-carbamoyl-methyl^-2-desoxy-I)-glucose, 2-benzamido-3-0-^D-l-[ L-l-(D-1-carb amoy 1 - 3- c arb oxy-pr opy1)-15 carbamoyl-propyl] -N-ethyl-carbamoyl-ethyl^-2-deso:xy-D-glucose, 2-acetamino-3-0-^ D-l-[L-l-(D-l-carbamoyl-3-carbo:Ky-propyl)-carbamoyl-ethyl] -IT-propyl-carbamoyl-eth.yl^-2-desoxy-D-glucose, 20 2-acetamino-3-0-j>D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)- v. Λ carbamoyl-N,K-pentamethylen]-carbamoyl-ethyl ^-2-desoxy~I)- glucose, 2-benzoylamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-pr opyl )-carbamoyl-N,lT-pentame thyl en]-carbamoyl-ethyl^-2-25 desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-ΪΤ-methyl-carbamoyl-ethyl] -carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetamino-3-0- L-l- ( D-l-carbamoyl-3-carboxy-methyl-30 phenyl)-carbamoyl-methyl^-2-desoxy-D-gluco se, 100

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i ( i 2-benzoylamino-3-0->[ L-l-(D-l-carbamoyl-3-carboxy-methyl- ! n. Λ phenyl] -carbamoyl-methyl ^ -2-desoxy-D-glucos e, 2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2-methyl-mercapto-ethyl] -carbamoyl-me thyl j -2-5 desoxy-D-glucose, 2-benzamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-i carbamoyl-2-chlorethyl]-carbamoyl-methyl^-2-desoxy-D- glucose, 2-acetamino-3-0-^D-l-[ L-l-(I)-l-carbamoyl-3,3-dicarboxy-10 propyl )-carbamoylethyl]-carbamoyl-ethyl^-2-deso:xy-I)-glu.cose, 2-( β-carbomethosy-succinamido )-3-0-L-l-( D-l-carbamoyl-3-c arb oxy-pr opyl)-carbamoyl-ethyl]-carbamoy1-methyl^-2-des-oxy-D-glucose, 2-(p-carbomethoxy-succinamido)-3-0-^D-l-[D-l-[L-1-(D-1-15 carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl] -carbamoyl-ethyl ^-2-desoxy-D-glucose, 2-benzamino-3-0-^[ L-l-(D-l-carbamoyl-3-trimethylsilylcarb-oxy-propyl) - carbamoyl-ethyl ] - c arbamoyl-me thyl ^ -2-desoxy- 1.4.6- tris-trimethylsilyl-D-glucose. (Ved kontakt med vand 20 hydrolyseres trimethylsilylestergruppen hurtigt), 2-ac etamino-2-de s oxy-3-O-^ [ L-l-( D-l-carbamoyl-3-tr imethyl-s ilylcarboxy-pr opyl) -carbamoyl-ethyl] -carbamoylmethyl ^ - 1.4.6- tris-trimethylsilyl-D-glucos e, 2-ac etamino-3-O-^L-l-[ L-l-(D-l-carbamoyl-3-carboxy-propyl)-25 carbamoyl-propyl] - c arbamoyl-ethyl ^ -2-de s oxy-L-gluco s e, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2' -hydroxy-propyl]-carbamoyl-ethyl ^-2-desoxy-I)-glucose, 2-ac etamino-3-O-£ D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)- 30 carbamoyl-2' -(p-hydroxy-phenyl)-ethyl]-carbamoyl-ethyl^-2-

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101 desoxy-D-glucose, 2-acetamino-3-0-^D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-H, E-tetrame thyl en]-carbamoyl-ethyl ^-2-desoxy-D-glucose, 5 2-glycolamino-3-0-^D-l-[ L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl] -c arbamoyl-ethyl j-2-de s oxy-D-gluc ose, 2-glycolamino-3-0-^[L-l-(D-l-carbamoyl-3-carboxy-propyl)-c arbamoyl-e thyl ] - c arbamoyl -methyl ^ -2-de s oxy-D-gluc o s e, 2- (N-methyl-ace tamino )-3-0- £ [ L-l- ( D-l-carbamoyl-3-carboxy-10 propyl)-carbamoyl-ethyl]-carbamoyl-methylj-2-desoxy-D-glucose, 2-(N-methyl-ac et amino)-3-0-^ D-l-[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl ]-carbamoyl-ethyl ^ -2-desoxy-D-glucose, 15 2-acetamino-3-0-^D-l-[ L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2' -phenyl ethyl] -carbamoyl-ethyl j-2-desoxy-D-glucose, 2-acetamino-3-0-^[ L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-2'-(p-hydroxyphenyl)-ethyl]-carbamoyl-methyl^-20 2-desoxy-D-glucose, 2- acetamino-3-0-^[ L-l-(D-l-[L-l-carboxy-ethyl]-carbamoyl- 3- benzylcarboxy-propyl]-carbamoyl-ethyl]-carbamoylmethyl 2-desoxy-D-glucose eller 2-acetamino-3-0-^[ L-l-(D-l,3-dicarboxy-propyl)-h-methyl-25 carbamoyl-ethyl] -carbamoylmethyl^-2-desoxy-D-glucose.

Forskellige af de ovennævnte muramylpeptider eller deres med spacere koblede former er nye. De kan f.eks. opnås som beskrevet i de følgende eksempler.

Eksempel 38.

102

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I C

En opløsning af 3?^ g benzyl-2-acetamino-3-0- D-l-^L-l-[D-l- ! carbamoyl-3- (L-l-carbosy-etbyl-carbamoyl) -propyl] -carbamoyl- ethyl ^-carbamoyl-ethyl -2-deso:ny-a-D-glucopyranosid-benzyl-: 5 ester i 100 ml methanol/destilleret vand (2:1) hydrogeneres under tilstede-værelse af 0,3 g 10%'s palladium på kul ved normaltryk og 45°C i 24 timer. Man frafiltrerer katalysatoren og inddamper filtratet. Remanensen opløses i 40 ml vand, og denne opløsning ekstraileres tre gange, hver gang med 10 40 ml vandmættet sek.butanol. De organiske faser vaskes endnu tre gange, hver gang med 40 ml sek,butanolmættet vand.

De vandige opløsninger samles og inddampes, og remanensen opløses i lidt destilleret vand og frysetørres. Man opnår således 2-acetamino-3-0- D-l-^L-l-[D-l-carbamoyl-3-(L-l-15 carboxy-ethyl-carbamoyl)-propyl] -carbamoyl-ethyl^-carbamoyl-! ethyl -2-desoxy-D-gluc os e som et hvidt pulver med [a] ^ = +9° ±i° (destilleret vand, c = 1,090).

Det anvendte udgangsmateriale fremstilles på følgende måde:

Til en opløsning af 6,1 g benzyl-2-acetamino-3-0-^D-l-[1-1-20 (D-l-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoyl-ethyl^-2-desoxy-a-D-glucopyranosid-monohydrat og 3,5 g D-alaninbenzylester-p-toluensulfonat i 30 nil E,E-dimethyl-formamid sættes 1,4 ml triethylamin, 1,1 g ΪΓ-hydroxy-succin-imid og 2,3 g dicyclohexy 1 carbodiimid, og blandingen omrø-25 res i 48 timer ved stuetemperatur. Det udkrystalliserede dicyc1ohexylurinstof suges fra og vaskes med 10 ml E,E-dimethylformamid, og filtratet inddampes til tørhed. Remanensen suspenderes i 100 ml vand og omrøres i en time ved 0°C, og de uopløselige bestanddele suges fra, vaskes med 30 lidt isvand og tørres. Produktet opløses nu i methanol, fældes med den dobbelte mængde ethylacetat, suges fra, vaskes med lidt etbylacetat og tørres: [oc]^ = +72° -1° (methanol, c = 0,998).

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103 På analog måde opnår man ud fra benzyl-2-acetamino-3-0-£[ L-l-(D-l-carbamoyl-3-carbo:xypropyl)-carbamoyl-ethyl] -carbamoyl-methyl^-2-desoxy-a-D-glucopyranosid 2-acetamino- 3-0- ^Ir-l-(D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl)-5 propyl ]-carbamoyl-ethyl^-carbamoyl-me thyl -2-desoxy-D-glucose.

Ud fra benzyl-3-0-£d-1-[ L-l-(D-l-carbamoyl-3-carboxy- V. Λ propyl) -carbamoyl-ethyl] -carbamoyl-ethyl j -2-desoxy-2-propionamino-a-D-glucopyranosid og glycinbenzylester-p-10 toluensulfonat fremstilles 3-0- D-l-^L-l-[D-l-carbamoyl-3-( carboxy-methyl-carbamoyl) -propyl] -carbamoyl-ethyl^ -carbamoyl-ethyl -2- de s oxy-2-propi onamino-D-gluco s e.

Analogt fremstilles: 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(carboxy-methyl-15 carbamoyl)-propyl]-carbamoyl-ethyl ^-carbamoyl-methyl -2-desoxy-D-glucose, 2- butyroylamino-3-0- D-l-^L-l-[l-l-carbamoyl-3-(L-l-carb-oxy-ethyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl-ethyl -2-deso.xy-D-glucose, 20 2-butyroylamino-3-0- ^L-l-[D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl)-propyl]-carbamoyl-ethylj-carbamoyl-methyl -2-desoxy-D-glucose, 3- 0- ^L-l-[D-l-carbamoyl-3-(carboxy-methyl-carbamoyl)-propyl]-carbamoyl-ethyl ^-carbamoyl-methyl -2-desoxy-2- 25 propionamino-D-glucose, 2-isobutyroylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(I<-l-carbo:xy-e thyl-carbamoyl) -propyl] -carbamoyl-propyl ^-carbamoyl-methyl -2-desoxy-D-glucose, 2-isobutyroylamino-3-0- D-l-^L-l-CD-l-carbamoyl-3-(L-l- 30 carboxy-ethyl-carbamoyl) -propyl ] - c arb amoy 1-pr opy 1 ^ - c arbam- oyl-ethyl -2-desoxy-D-glucose,

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104 I ( j 2-isobutyroylamino-3-0- £L-l-[ D-l-carbamoyl-3-(D-l-carboxy- ! ethyl-carbamoyl) -propyl] -earbamoyl-2-methylpropyl ^ -carbam- ! oyl-methyl -2-desoxy-D-glucose, j 2-isobutyroylami:n.o-3-0- D-l-£]>l-[D-l-carbamoyl-3-(L-l- ! V. "\ 5 carboxy-ethyl-carbamoyl)-propyl] -carbamoyl-2-methylpropyl^-| carbamoyl-ethyl -2-desoxy-D-glucose, i ( \ 2-isobutyroylamino-3-0- ^l-l-[D-l-carbamovl-3-(carboxy- I methyl-carbamoyl)-propyl] -carbamoyl-ethyl j -carbamoyl-methyl - ! 2-desoxy-D-glucose, ί 10 2-isobutyroylamino-3-0- ^L-l-[D-l-carbamoyl-3-(carboxy- ! methyl-carbamoyl) -propyl] -carbamoyl-ethyl -carbamoyl- ! methyl -2-desoxy-D-glucose, i 2-isobutyroylamirLO-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(carboxy- | methyl-carbamoyl)-propyl] -carbamoyl-ethyl^-carbamoylethyl - ! 15 2-desoxy-D-glucose, ! ( 2-isobutyroylamino-3-0- £l-l-[D-l-carbamoyl-3-(L-l-carboxy- I ’ ethyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl-methyl - 2-desoxy-D-glucose, 2-isobutyroylamino-3-0- D-l-^i-l-[D-l-carbamoyl-3-(L-l-20 carboxy-ethyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl-ethyl -2-desoxy-D-glueose, 2-isobutyroylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(ti-l-carboxy-propyl-carbamoyl) -propyl] -carbamoyl-ethyl j-carbamoyl-methyl -2-desoxy-D-glucose, 25 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(carboxy-methyl-carbamoyl) -propyl] -carbamoyl-2-methylpr opyl^ -carbamoyl-methyl -2-desoxy-D-glu.cose, 2-acetamino-3-0- D-l-^L-l-[D-l-carbamoyl-3-(carboxy-me thyl-carbamoyl) -propyl] -carbamoyl-2-me thylpr opyl j -earbam-30 oyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(D-l-carboxy-ethyl- carbamoyl)-propyl]-carbamoyl-2-methylpropyl^-carbamoyl- methyl -2-desoxy-D-glucose, 105

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2-acetamino-3-0- D-1-^L-1~[ D-l-carbainoyl-3-(Ir-l-car'boxy-3 ethyl-carbamoyl)-propyl]-carbamoyl-2-methylpropyl^-carbamoyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(L-l-carbo:xy-propyl-carbamoyl)-propyl]-carbamoyl-2-methyl-propyl^-carbamoyl-metbyl -2-desoxy-D-glucose, 10 2-butyroylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carboxy-butyl-carbamoyl) -propyl] -carbamoyl-ethyl^ -carbamoyl-methyl -2-desoxy-D-glucose, 2-acetamino-3-0- D-l-^L-l-[D-l-carbamoyl-3-(L~l-carboxy-ethyl-carbamoyl)-propyl] -carbamoyl-propyl^-carbamoyl-ethyl -15 2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(D-l-carboxy-ethyl-carbamoyl) -propyl ] -carbamoyl-propyl j -carbamoyl-methyl -2-desoxy-D-glucose, 2-acetamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(carboxy-methyl-20 carbamoyl)-propyl] -carbamoyl-propyl^-carbamoyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(carboxy-methyl-carbamoyl) -propyl] -carbamoyl-propyly-carbamoyl-methyl -2-desoxy-glucose, 25 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(D-l-carboxy-propyl-carbamoyl)-propyl] -carbamoyl-ethyl ^-carbamoyl-methyl -2-desoxy-D-glucose, 2-acetamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(D-l-carboxy- propyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl- 30 ethyl -2-desoxy-D-glucose, i 106 DK 1610261 2-acetamino-3-0- £lr-l-[ D-l-carbamoyl-3-(li-l-carboxy-2- i ^ \ j methylpropyl-carbamoyl) -propyl ]-carbamoyl-ethyl ^-carbamoyl- methyl -2-desoxy-D-glucose, 2-acetamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(Ii-l-carboxy-5 2-methyl-pr opyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbam-oyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-*0- ^L-l-[ D-l-carbamoyl-3-(L-l-carboxy-butyl-: carbamoyl)-propyl] -carbamoyl-etliyl^-carbamoyl-methyl -2- desoxy-D-glucose, ; 10 2-butyroylamino-3-0- ^ L-1-[ D-1 - carbamoyl - 3- ( c arb oxy-me thyl- I carbamoyl) -propyl] -carbamoyl-ethyl ^ -carbamoyl-methyl -2- I desoxy-D-glucose, 2-butyroylamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(D-l-carb-j o xy-pr o py 1 - c arb amoy 1) -propyl] -carbamoyl-ethyl ^ -carbamoyl- 15 ethyl -2-desoxy-D-glucose, 2-butyroylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(D-l-carboxy-propyl-carbamoyl)-propyl] -carbamoyl-ethyl^-carbamoyl-methyl -2-desoxy-D-glucose, ^ 2-butyroylamiiio-3-0- D-l-£l-l-[D-l-carbamoyl-3-(b-l-carb- j 20 oxy-2-methyl-propyl-carbamoyl)-propyl]-carbamoyl-ethyl<- carbamoyl-ethyl -2-desoxy-D-glucose, 2-butyroylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(l-l-carboxy-2-methyl-propyl- carbamoyl) -propyl ] -c arbamoyl- ethyl ^ -carbamoyl-methyl -2-desoxy-D-glucose, 25 2-benzoylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(l-l-carboxy-ethyl-carbamoyl) -propyl] -carbamoyl-ethyl ^ -carbamoyl-methyl -2-desoxy-D-glucose, 2-benzoylamino-3-0- D-l-^l-l-CD-l-carbamoyl-3-(carboxy- me thyl-carbamoyl) -propyl] -carbamoyl-ethyl j-carbamoyl- 30 ethyl -2-desoxy-D-glucose, 2-benzoylamino-3-0- ^L-l-[ D-l-carbamoyl-3KL-l“Carboxy- ethyl-carbamoyl)-propyl!-carbamoyl-propyl^-carbamoyl- methyl -2-desoxy-D-glucose, 107

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2-benzoylamiiio-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(carboxy-3 methyl-carbamoyl)-propyl] -carbamoyl-propyl^-carbamoyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl)-propyl]-carbamoyl-Ν,Ν-tetramethylenj-carbamoyl-methyl -2-desoxy-D-glucose, 10 2-acetamino-3-0- D-l-^L-l-CD-l-carbamoyl-3-(D-l-carboxy-ethyl-carbamoyl)-propyl]-carbamoyl-H,H-tetramethylen^-carbamoyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(carbox^-methyl-carbamoyl) -propyl] -carbamoyl-ΪΓ, N-t etr amethylen j -carbamoyl-15 methyl -2-desoxy-D-glucose, 2-acetamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(carboxy-methyl-carbamoyl) -propyl] -carbamoyl-N,N-tetramethylen^ -carbamoyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(I<-l-carboxy-ethyl-20 carbamoyl)-propyl]-carbamoyl-2-hydroxy-ethyl^-carbamoyl-methyl -2-desoxy-D-glucose, 2-acetamino-3-O- D-l-^L-l-[ D-l-carbamoyl-3-(I<-l--carboxy-ethyl-carbamoyl) -propyl] -carbamoyl-2-hydroxy-ethyl ^ -carbamoyl-ethyl -2-desoxy-D-glucose, 25 2-benzoylamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carboxy-ethyl-carbamoyl) -propyl ] -carbamoyl-2-hydroxy-ethyl ^ -carbamoyl-methyl -2-desoxy-D-glucose, 2-benzoylamino-3-0- D-l-^L-l-[ D-l-carbamoyl-3-(L-l-carboxy~ ethyl-carbamoyl) -propyl] -carbamoyl-2-hydroxy-ethyl ^ -carbam- 30 oyl-ethyl -2-desoxy-D-glucose, 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carbo:xy-methyl)- carbamoyl-propyl] -carbamoyl-me thylj-carbamoyl-methyl -2- desoxy-D-glucose, 108

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; 2-acetamino-3-0- D-l-^L-l-[, D-l-carbamoyl-3-(l-carboxy- ! 5 methyl-carbamoyl)-propyl] -carbamoyl-methyl^-carbamoyl-ethyl - ' 2-desosy-D-glucose, I 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carbo:xy-metbyl- I carbamoyl)-propyl]-carbamoyl-methyl^-carbamoyl-methyl -2- ! desoxy-D-glucose, i 10 2-acetamino-3-0- D-l-^L-l-[D-l-carbamoyl-3-(Ir-l-carboxy-methyl-carbamoyl)-propyl] -carbamoyl-me thyl ^ -carbamoyl-ethyl-2-deso:xy-D--glucose og 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(L-l-carboxy-methyl-! carbamoyl) -propyl] -carbamoyl-methyl^-carbamoyl-methyl -2- 15 de s oxy-D-gluc ose.

Eksempel 39»

En opløsning af 4,2 g benzyl-2-acetamino-3-0- ^L-l-[D-l-carbamoyl-3-(h-5-benzylozycarbonylamino-5-carbamoyl-pentyl-carbamoyl)-propyl] -carbamoyl-ethyl^-carbamoyl-methyl -2-20 des oxy-a-D-glucopyranosid i 100 ml methanol/vand (2:1) hydrogeneres under tilstedeværelse af 0,5 g 10%'s palladium på kul ved normaltryk og stuetemperatur. Derved holdes reaktionsblandingens pH på 6 ved tilsætning af 1 N saltsyre.

Efter endt hydrogenoptagelse frafiltrerer man katalysatoren 25 og inddamper filtratet til tørhed. Remanensen opløses i lidt destilleret vand og frysetørres. Man opnår således 2-acetamino-3-0- ^L-l-[ D-l-carbamoyl-3-(Ii-5-amino-5-earbam-oyl-pentyl-carbamoyl) -propyl] -carbamoyl-ethyl ^-carbamoyl-methyl -2-desoxy-D-glucose-hydrochlorid som et hvidt pulver.

30 Det anvendte udgangsmateriale kan fremstilles som følger:

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109

En opløsning af 15 g oc-carbobenzoxy--tert.butoxycarbonyl-L- lysin-methyl ester i 100 ml af en mættet methanolisk ammoniakopløsning henstilles i 48 timer ved stuetemperatur og inddampes til tørhed. Produktet, α-carbobenzoyl- -tert.butoxy-5 carbonyl-L-lysinamid omkrystalliseres fra methanol/ether, smp. 142°C, [a]^° = -3° ±1° (methanol, c = 1,018).

En til 0°0 afkølet opløsning af 3 S a-carbobenzosy- -tert. butoxycarbonyl-Ir-lysinamid i 25 ni trifluoreddikesyre omrøres i en time og inddampes derefter til tørhed. Til remanen-10 sen sættes 20 ml mættet natriumchloridopløsning og is, og blandingen gøres alkalisk med koncentreret ammoniakopløsning og ekstraheres med ethylacetat. Den organiske fase vaskes endnu en gang med mættet natriumchloridopløsning, tørres over natriumsulfat og inddampes til tørhed. Man opnår 15 således α-carbobenzoxy-L-lysinamid som et hvidt skum.

Til en opløsning af 5?6 g benzyl-2-acetamino-3-0- ^L-1-(D-1-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl^-carbamoyl-methyl -2-desoxy-a-D-glucopyranosid og 2,8 g a-carbobenz-oxy-L-lysinamid i 30 ml Ν,ϊΤ-dimethylformamid sættes 1,1 g 20 E-hydroxysuccinimid og 2,2 g dicyclohexylcarbodiimid, og blandingen omrøres i 40 timer ved stuetemperatur. Det udkrystalliserede dicyclohexylurinstof suges fra og vaskes med 10 ml IT,E-dimethylformamid. Filtratet inddampes til tørhed, og remanensen ekstraheres i 30 minutter med 100 ml destil-25 leret vand. De uopløste bestanddele suges fra, vaskes med vand og tørres og er benzyl-2-acetamino-3-0- £L-1-[D-1-c arbamoyl-3~(L-5~benzyloxycarbonylamino-5-carbamoyl-pentyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl-methyl -2-desoxy-ct-D-glucopyranosid. Produktet omkrystalliseres fra 30 methanol/ethylacetat.

Analogt fremstilles: 2-acetamino~3-0- £l<-l-[ D-l-carbamoyl-3-(5-amino-L-l-carbam- ^ Λ oyl-pentyl-carbamoyl)-propyl]-carbamoyl-ethyl^-carbamoyl- 110

DK 161026 E

methyl -2-deso:xy-D-glucose-hydrochlorid.

Eksempel 40.

i

Ecl opløsning af 2,8 g benzyl-3-0-^[1-1-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-propyl ]-carbamoyl-methyl ^ -2-des-5 oxy-2-isobutyroylamino-a-D-glucopyranosid i 80 ml methanol/ destilleret vand (1:1) hydrogeneres under tilstedeværelse i af 0,3 g 10^'s palladium på kul ved normaltryk og 45°C. Ef- i I ter oparbejdning og frysetørring af remanensen opnår man i 3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl- ' 10 propyl]-carbamoyl-methyl ^-2-desoxy-2-isobutyroylamino-D- ; glucose som et hvidt pulver.

i

Det anvendte udgangsmateriale fremstilles som følger: I En blanding af opløsningerne af 21,0 g benzyl-2-amino-2- ] deso2y-4,6-0-isopropyliden-a-D-glucQpyranosid i 150 ml 15 chloroform og 9,0 g kaliumhydrogencarbonat i 150 ml destilleret vand afkøles under omrøring til 0°C, og der tilsættes dråbevis 8,5 ml isosmørsyrechlorid. Efter 1 times omrøring ved stuetemperatur skilles den organiske fase fra, vaskes med iskold 0,5 I saltsyre, vand, en mættet natriumhydrogen- i I 20 carbonatopløsning og igen med vand, tørres og inddampes.

Produktet, b enzy 1 -2 - de s oxy-2-i s obuty lamino -4, 6-0-isopropyl-iden-a-D-glucopyranosid krystalliseres fra 150 ml ether, smp. 82°0, [a]^ = +109° -1° (chloroform, c = 1,017).

fil en opløsning af 15,1 g benzyl-2-desoxy-2-isobutyroyl-25 amino-4,6-0-isopropyliden-a-D-glucopyranosid i 150 ml absolut acetonitril sættes i en nitrogenatmosfære under fugtig-hedsudelukkelse og omrøring 1,9 g natriumhydrid (Pluka, pract.), og der omrøres i 1 1/2 time ved 40°C. Derefter afkøles reaktionsblandingen til -10°C, og der tilsættes 30 5,6 ml bromeddikesyremethylester. Han omrører i endnu 15 minutter i isbad og i 2 timer ved stuetemperatur. Efter oparbejdning opnår man benzyl-2-desoxy-2-isobutylamino-zi-,6- i

Jll

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O-isopropyliden-3-O-methoxycarbonyl-methyl-a-D-glucopyrano-sid, som omkrystalliseres fra ether/petroleumsether, smp. 119-120°C, [a]^° = +152° ±1° (chloroform, c = 0,963).

En opløsning af 3,16 g benzyl-2-deso2^-2-isobutyroylamino-5 4, 6-0-isopropyliden-3-0-methoxycarbonylmethyl-a-D-gluco-pyranosid i 30 ml methanol og 10 ml 1 N natriumhydroxidopløsning henstilles ved stuetemperatur i 1 time. Man tilsætter 3 ml 1 N saltsyre og inddamper opløsningen til tørhed. Remanensen opløses i 50 ml N, ΪΓ-dimethy 1 formamid, der til-10 sættes 2,26 g L-a-aminobutyroyl-D-isoglutamin-tert .butyl-ester-hydrochlorid og 1,75 g EEDQ, og blandingen henstilles i 24 timer ved stuetemperatur. Man tilsætter endnu 0,2 g EEDQ og lader blandingen stå i yderligere 24 timer. Opløsningsmidlet afdestilleres, og remanensen opløses i ethyl-15 acetat/vand. Den organiske fase skilles fra og vaskes med iskold 1 35Γ saltsyre, vand, en mættet natriumhydrogencarbonat-opløsning og vand, tørres over natriumsulfat og inddampes til tørhed. Man opnår således benzyl-3-0-^[L-l-(D-l-carbam-oyl- 3-carboxy-pr opyl) -carbamoyl-pr opyl] -carbamoyl-me thyl -20 2-desoxy-2-isobutylamino-4,6-0-isopropyliden-a-D-gluco-pyranosid-tert.butylester som et hvidt skum.

Dette produkt opløses i 60 ml iseddike, der tilsættes 60 ml vand under omrøring, og blandingen henstilles i 24 timer ved stuetemperatur. Denne opløsning inddampes i vandstråle- 25 vakuum til tørhed, og remanensen opløses i ethanol. Man filtrerer med aktivt kul og inddamper igen til tørhed. Man opnår således benzyl-3-0-^[L-l-[ D-l-carbamoyl-3-carboxy- pr opyl) -carbamoyl-propyl ] -carbamoyl-methyl ^ -2-de soxy-2- isobutyroylamino-a-D-glucopyranosid-tert.butylester som et 20 30 hvidt amorft materiale med [a]^ = +74° ^1° (methanol, c = 0,950).

En til 0°0 afkølet opløsning af 3,9 g af denne tert.butyl-ester i 40 ml 98%'s trifluoreddikesyre omrøres i 1 time ved 0°C og laældes på 500 ml absolut ether. Det udkrystalli-

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112 serede produkt suges fra, vaskes med ether og tørres i vakuum. Det opnåede pulver opløses i 4-0 ml vand/tetrahydro-furan (1:1) og omrøres i 30 minutter med 50 ml ionbytter-harpiks "Dowex 3" (acetatform). Ionbytteren frafiltreres og j 5 vaskes med 500 ml tetrahydrofuran/1 É eddikesyre. Filtratet inddampes til tørhed, og produktet krystalliseres fra methanol/ether, smp. 205-206°C.

Eksempel 41, I En. opløsning af 3»1 g benzyl-3-0- L-l- (D-l-c arbamoyl-3- 10 carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-des-I oxy-2-isobutyroylamino-a-D-glucopyranosid i 40 ml methanol/ vand (1:1) hydrogeneres under tilstedeværelse af 10%' s palladium på kul ved normaltryk og 4-5°C. Efter oparbejdning ! opnår man 3-0-£[L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbam-

| V. ’N

I 15 oyl-ethyl]-carbamoyl-metbyl<-2-deso:xy-2-isobutyroylamino- ! D-glucose som et hvidt pulver ved frysetørring.

Det anvendte udgangsmateriale fremstilles analogt med eksem-pel 40.

i

Ved kondensation af benzyl-3-0-carboxy-methyl-2-desoxy-2- 20 isobutyroylamino-4,6-0-isopropyliden-a-D-glucopyranosid- natriumsalt med L-alanyl-D-isoglutamin-tert.butylester- hydrochlorid opstår benzyl-3-0-j>[ L-l-(D-l-carbamoyl-3- carboxy-propyl) -carbamoyl-ethyl] -carbamoyl-methyl^ -2-des- oxy-2-is obutyr oylamino-4,6-0-is opr opyliden-a-D-glucopyrano- 25 sid-tert.butylester som et hvidt skum. Hydrolyse af 4,6-0- is opropylidengruppen giver benzyl-3-0-£[L-l-(D-l-carbamoyl-

v. S

3-carboxypropyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-des-oxy-2-isobutyroylamino-a-D-glucopyranosid-tert.butylester med [a]^ = +71° -1° (methanol, c = 0,956).

30 Tert.butylesteren spaltes med trifluoreddikesyre. Han opnår således benzyl-3-0-I-l-(D-l-carbamoyl-3-carbosy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl^-2-desoxy-2-isobutyroyl-

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113 amino-a-D-glucopyranosid som et hvidt amorft produkt.

Eksempel 42.

En opløsning af 4,2 g benzyl-2-acetyl-E-methylamino-3-0-[ 1-1- ( D-l-carbamoyl-3-carbo:xy-propyl) -carbamoyl-ethyl] -5 carbamoyl-methyl j-2-deso:xy-D-glucopyranosid i 75 ml methanol/ vand (1:1) hydrogeneres under tilstedeværelse af 0,5 s 10%'s palladium på kul ved normaltryk og 45°C. Katalysatoren frafiltreres, og filtratet inddampes til tørhed. Remanensen opløses i lidt destilleret vand og frysetørres. Man opnår 10 således 2-acetyl-N-methyl-amino-3-0-^[L-l-CD-l-carbamoyl^-carboxy-propyl )-carbamoylethyl ]-carbamoylmethyl ^ -2-des oxy-D-glucose som et hvidt pulver.

Det anvendte udgangsmateriale fremstilles som følger:

Til en opløsning af 8,5 g benzyl-2-acetamino-2-desoxy-4,6-15 O-isopropyliden-3-0-methoxycarbonylmethyl-a-D-glucopyrano-sid i 80 ml absolut acetonitril sættes i en nitrogenatmosfære under omrøring og fugtighedsudelukkelse 0,75 S natriumhydrid (Eluka, pract.), og der omrøres i 1 time ved 40°C. Reaktionsblandingen afkøles nu til stuetemperatur, og 20 der tilsættes dråbevis i løbet af 4 timer en opløsning af 6,0 g methyliodid i 50 ml absolut acetonitril. Efter yderligere 3 timer filtreres reaktionsblandingen, og filtratet inddampes til tørhed. Remanensen opløses i ethylacetat, og denne opløsning vaskes med vand, tørres over natriumsulfat 25 og inddampes til tørhed. Man opnår således benzyl-2-acetyl-N-methylamino~2-desoxy-4,6-0-isopropyliden-3-0-methoxy-carbonyl-methyl-α-D-glucopyranosid som en gul olie med Rf-værdien 0,45 på kiselgeltyndtlagsplader i systemet methylenchlorid/ethylacetat (85:15).

30 En opløsning af 4,4 g benzyl-2-acetyl-liT-methylamino-2-des-oxy-4,6-0-isopropyliden-3-0-methoxycarbonyl-methyl-a-D-glucopyranosid i 60 ml methanol og 15 ml 1 ΪΓ natrium- 114

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hydroxidopløsning henstår i 1 time ved stuetemperatur, og der tilsættes 5 ml 1 N saltsyre og inddampes til tørhed.

Det opnåede benzyl-2-acetyl-N-methylamino-3-0-carbo:xymethyl-2-desoxy-4, 6-0-isopropyliden-a-D-glucopyranosid-natrlumsalt ΐ 5 opløses i 50 ml ΪΓ, 1-dime thylformamid og kondenseres med 3,2 g 1-alanyl-D-isoglutamin-tert.butylestér-hydrochlorid I under tilstedeværelse af 2,5 g EEDQ. Opløsningen inddampes ! i vakuum til tørhed, og remanensen opløses i ethylacetat.

I Han vasker denne opløsning med vand, iskold 1 N saltsyre, I 10 vand, en mættet natriumhydrogencarhonatopløsning og vand, i tørrer over magnesiumsulfat og inddamper til tørhed. Man opnår således henzyl-2-acetyl-lii-methylamino-3-0-^[ L-1-(D- 1- carbamoyl-3-carboxy-propyl ) -carbamoylethyl] -carbamoyl- I methyl ^ -2-desoxy-4,6-0-isopropyliden-a-D-glucopyranosid- 15 tert.hutylester som et gulligt skum. Dette produkt opløses ' i 45 ml 95%’ s trifluoreddikesyre, som forinden er afkølet til 0°0, og omrøres i 1 time ved 0°0. Reaktionsblandingen I hældes på 400 ml absolut ether, og det udfældede produkt suges fra, vaskes med ether og tørres. Ved behandling af 20 dette stof med ionbytterharpiksen "Dowex 3” i acetatform ; opnår man det trifluoreddikesyrefrie benzyl-2-acetyl-E- me thyl amino-3-0- j>[ L-l- ( D-l-carbamoyl-3-carboxy-pr opyl) -carbamoyl-ethyl] -carbamoyl-methyl ^ -2-desoxy-a-D-glucopyrano-sid som et hvidt pulver.

25 På analog måde fremstilles: 2- ac etyl-H-methyl-amino-3-0- ^ D-l-[ 1-1- ( D-l-carbamoyl- 3-carboxy-propyl) -carbamoyl-ethyl ] -carbamoyl-ethyl ^ -2-des-oxy-D-glucose, 2-ac etyl-H-methyl-amino-3-0- D-l-[ 1-1- ( D-l-carbamoyl-3-30 carboxy-propyl)-carbamoyl-propyl]-carbamoyl-ethyl^-2-desoxy-D-glucose, 2-acetyl-E~-methyl-amino-3-0- l-l-(D-l-carbamoyl-3-carboxy-propyl )-carbamoyl-propyl] -carbamoyl-methyl ^ -2-desoxy-D-glucose, v

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115 2-acetyl-N-methyl-amino-3-0-£[L-l-(I)-l-carbamoyl-3-carboxy-propyl) -carbamoyl-2-methyl-propyl] -carbamoyl-methyl^ -2-de s oxy-D-glucose, 2- acetyl-N-methyl-amino-3-O- ^ D-l-[ L-l- ( D-l-carbamoyl-3-5 carboxy-propyl)-carbamoyl-2'-methyl-propyl]-carbamoyl- ethyl^-2-desoxy-D-glucose, 3- 0-L-l-(D-l-carbamoyl-3-carboxy-propyl)-carbamoyl-ethyl]-carbamoyl-methyl ^-2-desoxy-2-propionyl-H-methyl-amino-D-glucose og 10 2-butyryl-N-methyl-amino-3-0-^[L-l-(D-l~carbamoyl-3-carboxy-pr opyl)-carbamoyl-ethyl]-carbamoyl-methyl ^-2-desoxy-D-glucose.

Eksempel 43.

Trimethylsilyletheren kan. f.eks. fremstilles som følger: 15 0,3 g 2-benzamido-2-desoxy-3-0-^[L-l-(D-l-carbamoyl-3-carboxypropyl) -carbamoyl-etbyl] -carbamoyl-methyl^-D-glucose opløses i 3 ml dimethylformamid, og man tilsætter 0,4- ml bis-trimethylsilylacetamid. Efter 5 timer ved 35°C inddamper man i vakuum til en sirup, hvorfra man ved opløsning i 20 absolut ether eller absolut ethylacetat kan fraskille det dannede acetamid. Efter fornyet inddampning opnår man en farveløs sirup med [a]^ = +15° (dioxan, c = 0,8).

Analogt opnår man sirupagtigt 2-acetamino-2-desoxy-3-0-£[L-l-(D-l-carbamoyl-3-trimethylsilyl-carboxy-propyl)-25 carbamoylethyl] -carbamoylmethyl^-1,4-, 6-tris-trimethylsilyl-D-glucose med [a]^ = -10° (dioxan, c = 0,91).

Ved kontakt med vand hydrolyseres trimethylsilylestergrup-pen hurtigt, således at disse derivater også er egnede til kobling.

116 DK 1610261

Eksempel 44.

En 5%' s opløsning af benzyl-2-acetamido-3-0- ^L-l-[ D-1-(L-1-carboxy-ethyl)-carbamoyl-3-carboxypropyl]-carbamoyl-ethyl^-carbamoyl-methyl -2-desoxy-oc-D-glucopyranosid i tetrahydro-5 furan/vand (2;1) hydrogeneres under tilstedeværelse af 10%' s palladium på kul Ted normaltryk og stuetemperatur. Efter optagelse af den teoretiske mængde hydrogen frafiltrer es katalysatoren og filtratet frysetørres. Man opnår således 2-acetamido-3-0- ^L-l-[. D-l-(L-l-carboxy-ethyl) -carbamoyl-3-10 carboxypropyll-carbamoyl-ethyl^-carbamoyl-methyl -2-desoxy-D-glucose som et hvidt pulver. Bf-værdi i tyndtlagskromato-gram = 0,21 (ethylacetat/n-hutanol/pyridin/eddikesyre/vand I 42:21:21:6:10).

i På analog måde opnår man derivatet med ægte muraminsyre.

i 13 Udgangsmaterialet kan fremstilles som følger: 5,68 g E-tert.butoxycarbonyl-L-alanyl-D-Y-benzyl-glutamyl-L-alaninbenzylester opløses i en blanding af 5 ml trifluor-eddikesyre og 5 ml 1,2-dichlorethan og henstilles under fugtighedsudelukkelse i 16 timer ved stuetemperatur. Man 20 fortynder denne opløsning med 50 ml tetrahydrofuran, afkøler i isbad og neutraliserer med triethylamin. Efter tilsætning af en opløsning af 3,7 g benzyl-2-acetamido-3-carboxymethyl-2-desoxy-a-D-glucopyranosid og 1,38 ml triethylamin i 100 ml tetrahydrofuran tilsættes 2,6 g 2-ethoxy-B-ethoxycarbonyl-25 1,2- dihy dr o quino 1 in, og blandingen henstilles i 24 timer ved stuetemperatur. Efter afdampning af opløsningsmidlet opløses remanensen i chloroform/methanol (9:1), vaskes med vand, iskold 2 Ή saltsyre, vand, en mættet natriumhydrogen-carbonatopløsning og vand, filtreres og befries for opløs-30 ningsmiddel. Man opnår således benzyl-2-acetamido-3-0- ^L-l-[ D-l-(L-l-carboxy-ethyl)-carbamoyl-3-carboxy-propyl] -carbam-oylethyl ^-carbamoyl-methyl -2-desoxy-a-D-glucopyranosid som et farveløst pulver, Bf-værdi = 0,48 (i et identisk system).

DK 161026 B

117

Det anvendte udgangsmateriale kan fremstilles som følger: 7,15 S H-tert.butoxycarbonyl-L-alanyl-D-glutaminsyre-γ-benzylester og 6,15 g L-alaninbenzylester-p-toluensulfonat opløses i 100 ml vandfrit dime thylf ormamid. Man afkøler i 5 isbad og tilsætter under omrøring successivt 4,03 g R-hydr oxysuc c inimi d, 3? 61 g di cy c 1 o hexyl c ar b o di imi d og sluttelig 1,95 md N-methylmorpholin. Efter 6 timers omrøring ved 0°C og 15 timer ved stuetemperatur afkøles suspensionen, bundfaldet ( di cy c 1 ohexy lur ins t o f og morpholinhydrochlorid) 10 frafiltreres, og filtratet inddampes. Remanensen optages i ethylacetat, vaskes flere gange med vand, 1 H citronsyre og 1 ΪΤ natriumhydrogencarbonatopløsning og igen med vand.

Ethylacetat opløsningen tørres og inddampes, og den krystallinske remanens omkrystalliseres fra ethylacetat/petroleums-15 ether (1:1), smp. 135-136°C, [a]^° = -9° ±1° (methanol, c = 1), Rf-værdi = 0,82 (i det ovenstående system) og 0,86 (acetonitril/vand 3 ·* 1) · I stedet for med alanin kan det beskyttede dipeptid forlænges med andre naturlige L-aminosyrer på analog måde.

20 Eksempel 45«

O

7,5 x 10 dræbte Trypanosoma cruzi parasitter (forårsager Chagas' sygdom) suspenderes i en opløsning af 50 mg 2-acet-amino-3-0-^[ L-l-(D-carbamoyl-3-carboxypropyl)-carbamoyl-ethyl]-carbamoylmethyl^-2-desoxy-D-glucose-R-hydroxysuccin-25 imidester i 6 ml fysiologisk pufferopløsning. Suspensionen inkuberes i 2 timer ved 37°6. Derefter sedimenteres de med muramyldipeptidet konjugerede parasitter ved centrifugering. Sedimentet vaskes ved resuspension i fysiologisk pufferopløsning og fornyet centrifugering. De vaskede parasit-30 muramyldipeptid-konjugater suspenderes i fysiologisk pufferopløsning og anvendes til immunisering.

Den kvantitative bestemmelse af det til trypanosornerne kob-

DK 161026 E

118 lede muramyldipeptid sker som angivet i eksempel 1 med Morgan-Elson-reaktionen og viser et indhold på 50-70 mg i muramyldipeptid pr. mg trypanosomer.

i i i

Claims (16)

1. Analogifremgangsmåde til fremstilling af antigenderivater af et antigen og mindst éi dermed, even- 5 tuelt over et broled, covalent forbundet muramylpeptid samt eventuelt en eventuelt over et broled tilkondenseret basrer, kendetegnet ved, at man kondenserer et eventuelt med broled sammenknyttet antigen med eventuelt med broled sammenknyttede muramylpeptider, idet den ene af de to 10 dele indeholder mindst en fri amino-, hydroxy- eller mercaptogruppe og den anden mindst en carboxylsyregruppe, fortrinsvis ved at omsætte den ene forbindelse i form af en aktiveret carboxylsyre med den anden forbindelse som fri amino-, hydroxy- eller mercaptoforbindelse og om 15 ønsket tilkondenserer de fremkomne forbindelser til en eventuelt med broled forbunden bærer.

2. Fremgangsmåde ifølge krav 1, kendetegnet ved, at man omsætter den ene reaktant i form af en aktiveret carboxylsyre med den anden reaktant som fri amino-, hydr- 20 oxy- eller mercaptoforbindelse.

3. Fremgangsmåde ifølge et af kravene 1 eller 2, kendetegnet ved, at man vælger udgangsstofferne således, at man får et antigenderivat med formlen Γ A Ί Μ Γ Γ Ί I - X'" - Z"' - X1V - MP L Z’J L J 0,1 n |_ I 0-1 Jm L "* Γ ς«ί m L o-i DK 161026 E hvori A er en antigenrest, T en bærerrest, MP en muramyl-peptidrest, Z' og Z"' divalente broled og X', X", X"' og I Xiv covalente forbindelseselementer, og m og n betyder 5 hele tal større end 0.

4. Fremgangsmåde ifølge krav 1, 2 eller 3, kendetegnet ved, at man vælger udgangsstofferne således, at man fremstiller et antigenderivat med formlen A- 20-1-101 (I) n 10 hvori A er en rest af et antigen, Z et broled, MP en rest af et muramylpeptid og n et helt tal større end 0.

5. Fremgangsmåde ifølge et af kravene 1 til 4, kendetegnet ved, at man som antigen anvender et aktivt stof i en vaccine mod parasitter, bakterier, vira, tumorcel- 15 ler, svækkede eller dræbte former eller delkomponenter deraf, i en vaccine mod autologe bestanddele eller immunologiske identificeringsstrukturer, eller i en vaccine, som egner sig til specifik desensibilisering ved allergier.

6. Fremgangsmåde ifølge et af kravene 1 til 4, ken detegnet ved, at man som antigen anvender et aktivt stof i en vaccine mod malaria, kolera, tyfus, meningitis, rheumatisk feber som følge af streptokokinfektioner, influenza, rabies, mund- og klovsyge, samt i en vaccine 25 til profylakse og terapi af tumorsygdomme, til induktion af immunitet mod komponenter i forplantningssystemet, til desensibilisering eller induktion af specifikke immunto-

121 DK 161026 B lerancer mod allergier, til genoprettelse af tolerancen mod autologe vævsbestanddele og cirkulerende molekyler.

7. Fremgangsmåde ifølge et af kravene 1 til 4, ken-5 detegnet ved, at man som antigen anvender malariamero- zoiter, typespecifikke meningokokpolysaccharider A, B eller C, M-proteiner fra streptokokker, influenzevirus-hæmagglutininer, autologe tumorceller eller tumorcelle-membranbestanddele, partialsekvenser af human choriongon-10 adotropin, græspollenekstrakter, antigenspecifikke T-cel-le-iymfoblaster og deres receptorer for antigener, eller antigenspecifikke immungiobuliner.

8. Fremgangsmåde ifølge et af kravene 1 til 7, kendetegnet ved, at man som muramylpeptid anvender et med 15 formlen CH20R6 K4oN-^ / N - X - R R3 - CH (D) R13 (II) \ R8 R10 R11 \ 1 I I 12 CON - CH - CON - CH - CHgCH - R^ R7 (1) R9 (D) hvori X er en carbonyl-, carbonyloxy- eller sulfonylgrup-pe, Ri hydrogen, alkyl, eventuelt substitueret benzyl eller acyl, R^ eventuelt substitueret alkyl eller carb-20 ocyclisk aryl, og R^ uafhængigt af hinanden hydrogen, alkyl, eventuelt substitueret benzyl eller acyl, r3 hydrogen eller alkyl, R? og r!3 hydrogen eller lavalkyl, R® hydrogen, lavalkyl, frit, forestret eller forethret DK 1610261 mercapto-lavalkyl, frit eller acyleret aminolavalkyl, cycloalkyl med 5 eller 6 carbonatomer, cycloalkyllav-alkyl, hvis cycloalkylgruppe indeholder 5 eller 6 carbon-5 atomer, eventuelt substitueret aryl eller aralkyl, nitro-genholdigt heterocyclyl- eller heterocyclyllavalkyl, R? i og r8 sammen også alkylen med 3 eller 4 carbonatomer, r9 hydrogen eller lavalkyl og grupperne R^O, R^ og R^2 uafhængigt af hinanden en eventuelt forestret eller I 10 amideret carboxylgruppe og R^ også hydrogen, idet R4, i R4 og R^ dog også kan betyde trilavalkylsilyl, især tri- methylsilyl, eller oligomere deraf, som f.eks. er beskrevet i tysk offentliggørelsesskrift nr. 2.450.355 og kan ! udvindes ved isolering fra mikroorganismecellevægge.

9. Fremgangsmåde ifølge et af kravenen 1-3 og 5-8, j kendetegnet ved, at antigenet er fikseret til en højmole- j kylær bærer, idet man som bærer fortrinsvis anvender en ! polymer af mælkesyre og dens derivater, som på kædeenden bærer en fri carboxylgruppe, alginsyre, polygalacturon-| 20 syre, pectiner, carboxymethylcellulose, agarose eller en I basisk, neutral eller sur polyaminosyre. i ί 10. Fremgangsmåde ifølge et af kravene 1 til 9, kendetegnet ved, at man vælger udgangsstofferne således, at antigenderivaterne som covalente forbindelseselementer 25 indeholder carboxylsyreester-, carboxylsyreamid-, thio-carboxylsyreester- eller thiocarboxylsyreamidgrupper.

11. Fremgangsmåde ifølge et af kravene 1 til 10, kendetegnet ved, at man vælger udgangsstofferne således, at antigenet er adskilt fra muramylpeptidet med et bro- 30 led (spacer).

12. Fremgangsmåde ifølge et af kravene 1 til 11, kendetegnet ved, at man som muramylpeptid anvender et med den i krav 8 viste formel II, hvori R4, R^, R4, r6 og b7 hydrogen, X carbonyl og R^ eventuelt med hydroxy eller DK 161026 B lavalkoxy substitueret lavalkyl eller eventuelt med hydroxy, lavalkoxy, lavalkyl eller halogen substitueret phenyl.

13. Fremgangsmåde ifølge et af kravene 1 til 11, kendetegnet ved, at man som muramylpeptid anvender et med den i krav 8 viste formel II, hvori R^, R4, r6 og R2 betyder hydrogen, X carbonyl, R2 eventuelt med hydroxy eller lavalkoxy substitueret lavalkyl eller eventuelt med 10 hydroxy, lavalkoxy, lavalkyl eller halogen substitueret phenyl og R3 methyl.

14. Fremgangsmåde ifølge et af kravene 1 til 11, kendetegnet ved, at man som muramylpeptid anvender et med den i krav 8 viste formel II, hvori R1, R4, R3 og R^-3 15 betyder hydrogen, X carbonyl, R2 eventuelt med hydroxy eller methoxy substitueret lavalkyl eller eventuelt med hydroxy, methoxy, methyl, ethyl eller halogen substitueret phenyl, R3 hydrogen eller methyl, R? og R^ hydrogen, r8 lavalkyl, lavalkylmercapto-lavalkyl, hydroxylavalkyl, 20 benzyl, p-hydroxybenzyl eller phenyl, r!0, R^ og R^2 carboxy, lavalkoxycarbonyl eller carbamoyl og R11 også hydrogen og R13 hydrogen.

15. Fremgangsmåde ifølge et af kravene 1 til 11, kendetegnet ved, at man som muramylpeptid anvender et med 25 den i krav 8 viste formel II, hvori R^, R4 og R^ betyder hydrogen, X carbonyl, R2 eventuelt med hydroxy eller methoxy substitueret lavalkyl eller eventuelt med hydroxy, methoxy, methyl, ethyl eller halogen substitueret phenyl, R3 og r9 hydrogen eller methyl, b7 hydrogen eller 30 lavalkyl, R3 methyl, ethyl, n-propyl, isopropyl, 2-meth-ylpropyl, methylmercaptomethyl, hydroxymethyl, hydroxy-ethyl, phenyl, benzyl eller p-hydroxybenzyl, R43, r11 og R12 carboxyl, lavalkoxycarbonyl eller carbamoyl og R^1 også hydrogen og R^3 hydrogen. DK 16102

16. Fremgangsmåde ifølge et af kravene 1 til 11, kendetegnet ved, at man som muramylpeptid anvender et med formlen II, hvori R^·, R^ og R^ betyder hydrogen, X carbonyl og R? og R® sammen propylen eller butylen. i