HRP20010833A2 - Short-chain peptide dye conjugates used as contrast agents for optical diagnostics - Google Patents
Short-chain peptide dye conjugates used as contrast agents for optical diagnostics Download PDFInfo
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- HRP20010833A2 HRP20010833A2 HR20010833A HRP20010833A HRP20010833A2 HR P20010833 A2 HRP20010833 A2 HR P20010833A2 HR 20010833 A HR20010833 A HR 20010833A HR P20010833 A HRP20010833 A HR P20010833A HR P20010833 A2 HRP20010833 A2 HR P20010833A2
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- dye
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- OSWULUXZFOQIRU-UHFFFAOYSA-N tert-butyl 2-aminoacetate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)CN OSWULUXZFOQIRU-UHFFFAOYSA-N 0.000 description 1
- ZJXHVYSDMUKUCA-UHFFFAOYSA-N tert-butyl 3-aminopropanoate Chemical compound CC(C)(C)OC(=O)CCN ZJXHVYSDMUKUCA-UHFFFAOYSA-N 0.000 description 1
- DOMTZTVJNZKUNX-UHFFFAOYSA-N tert-butyl 3-aminopropanoate;hydrochloride Chemical compound Cl.CC(C)(C)OC(=O)CCN DOMTZTVJNZKUNX-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- ZGYICYBLPGRURT-UHFFFAOYSA-N tri(propan-2-yl)silicon Chemical compound CC(C)[Si](C(C)C)C(C)C ZGYICYBLPGRURT-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 230000036326 tumor accumulation Effects 0.000 description 1
- 230000002227 vasoactive effect Effects 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 238000010626 work up procedure Methods 0.000 description 1
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Abstract
Description
Izum se odnosi na spojeve, koji se primjenjuju u tumorskoj dijagnostici, a sastoje se od konjugata boja s kratkolančanim peptidima, koji su izvedeni od vazoaktivnih intestinalnih peptida, od somatostatina ili od neurotenzina, primjenu tih spojeva kao optičkih dijagnostika i dijagnostičkih sredstava, koja sadrže takve spojeve. The invention relates to compounds, which are used in tumor diagnostics, and consist of dye conjugates with short-chain peptides, which are derived from vasoactive intestinal peptides, from somatostatin or from neurotensin, the use of these compounds as optical diagnostics and diagnostic agents, which contain such connections.
Promjene uzrokovane oboljenjima, odražavaju se na celularnoj razini često kao jedno stanje suprotno od normalnog, a manifestira se u izmjenjenoj raspodjeli receptora ili opetovanoj ekspresiji. Te razlike mogu biti kako kvantitativne prirode (npr. količina transferin receptora na proliferirajuće stanice) tako i kvalitativne prirode (npr. ekspresija vaskularnog endotelijskog faktora rasta, VEGF). Do sada se upotreba u prikazu jedne patološke receptorne ekspresije ili raspodjele, zbog potrebne senzitivnosti postupka detekcije, koristila isključivo u radiodijagnostici. Changes caused by diseases are reflected at the cellular level, often as a state opposite to normal, and manifests itself in a changed distribution of receptors or repeated expression. These differences can be both of a quantitative nature (eg the amount of transferrin receptors on proliferating cells) and of a qualitative nature (eg the expression of vascular endothelial growth factor, VEGF). Until now, the use in the display of one pathological receptor expression or distribution, due to the necessary sensitivity of the detection procedure, was used exclusively in radiodiagnostics.
Heptahelikalni receptori su ciljne molekule za mnoge farmakološke djelotvorne tvari (npr. ß-blokeri, H2 blokeri kiselina, antihistaminici). Pored farmaceutske primjene, isključivo su u upotrebi radioaktivno markirani agonistički ligandi tih receptora, koji se koriste dijagnostički za tzv. receptornu scintigrafiju za in-vivo detekciju i lokalizaciju tumora. Pri tom se koristi mehanizam receptor-posredujuća endocitoza, npr. preko somatostatin receptora, koji je pojačano eksprimiran na neuroendokrinim tumorima. Klinički dopušteno za Scintigrafsku rutinsku dijagnostiku je somatostatin analog 111In-DTPA-pentetreotid (Octreoscan®); Heptahelical receptors are target molecules for many pharmacologically active substances (eg ß-blockers, H2 acid blockers, antihistamines). In addition to pharmaceutical application, only radioactively labeled agonistic ligands of these receptors are used, which are used diagnostically for the so-called receptor scintigraphy for in-vivo detection and localization of tumors. In doing so, the mechanism of receptor-mediated endocytosis is used, for example via the somatostatin receptor, which is strongly expressed on neuroendocrine tumors. Clinically approved for Scintigraphic routine diagnostics is the somatostatin analog 111In-DTPA-pentetreotide (Octreoscan®);
Literatura: J. Steroid. Biochem. Mol. Biol. 37, 1079-82, 1990, J. Nucl. Med. 32, 1184-9, 1991.; J. Nucl. Med. 33, 652-8, 1992; Digestion 3, 54-9, 1994., J. Clin. Invest. 93, 1321-5, 1994, Metabolism 45, 21-3, 1996. Literature: J. Steroid. Biochem. Mole. Biol. 37, 1079-82, 1990, J. Nucl. Honey. 32, 1184-9, 1991; J. Nucl. Honey. 33, 652-8, 1992; Digestion 3, 54-9, 1994, J. Clin. Invest. 93, 1321-5, 1994, Metabolism 45, 21-3, 1996.
Drugi način korištenja radioaktivno markiranih VIP i VIP-analoga, koji se vežu na VIP-receptor. VIP-receptor će biti od širokog spektra tumora pojačano eksprimiran (između ostalog od adenokarcinoma). Another way of using radioactively labeled VIP and VIP-analogues, which bind to the VIP-receptor. The VIP-receptor will be overexpressed in a wide range of tumors (including adenocarcinoma).
WO 96/30055 opisuje radiodijagnostičke i radioterapeutske reagense, posebice VIP-receptorvezujuće peptide, koji su radioaktivno markirani i mogu se koristiti za radio dijagnostiku i terapiju. S posebnom prednošću su opisani VIP-receptorvezujući, s Tc-99m markirani peptidi za scintigrafiju. WO 96/30055 describes radiodiagnostic and radiotherapeutic reagents, in particular VIP-receptor binding peptides, which are radioactively labeled and can be used for radiodiagnosis and therapy. VIP-receptor-binding, Tc-99m-labeled peptides for scintigraphy have been described with particular advantage.
Daljnja literatura: Cancer Research 54, 690-700, 1994; Endocrinology 136, 2662-80, 1994, J. Nucl. Med. 40, 353-361, 1999. Further reading: Cancer Research 54, 690-700, 1994; Endocrinology 136, 2662-80, 1994, J. Nucl. Honey. 40, 353-361, 1999.
Svi opisani dijagnostički načini primjene, koji se baziraju na somatostatin-receptoru, su radiodijagnostički načini primjene (scintigrafija s 123I, 125I, 111In ili 99mTc-markiranim peptidima). All described diagnostic methods of application, which are based on the somatostatin receptor, are radiodiagnostic methods of application (scintigraphy with 123I, 125I, 111In or 99mTc-labeled peptides).
Literatura: EP 588754, US 5650134; US 6520675; US 5225180; WO 96/23527; J. Steriod. Biochem. Mol. Biol. 37, 1083-87, 1990, Lancet 242-4, 1989, J. Nucl. Med. 39, 1913-17,1998. Literature: EP 588754, US 5650134; US 6520675; US 5225180; WO 96/23527; J. Steriod. Biochem. Mole. Biol. 37, 1083-87, 1990, Lancet 242-4, 1989, J. Nucl. Honey. 39, 1913-17, 1998.
Do sada nisu bili poznati fluorescentno markirani peptidi, koji su konjugirani s bojama, koje omogućuju jednu in-vivo-fluorescenc detekciju tumora (Photochem. Photobiol. 68, 603-632, 1998.). Until now, fluorescently labeled peptides, which are conjugated with dyes, have not been known, which enable an in-vivo-fluorescence detection of tumors (Photochem. Photobiol. 68, 603-632, 1998).
Izum ima zadaću, predstaviti nove spojeve, koji omogućuju senzitivnu dijagnozu tumora, kroz detekciju fluorescentnog zračenja, zu korištenje receptor-specifičnog spajanja spojeva na ciljne organe. Pri tom trebaju specijalne molekule boje, koje su pripojene na biomolekule, dati jedan visokosenzitivno detektirajući fluorescirajući signal. The purpose of the invention is to present new compounds that enable sensitive diagnosis of tumors, through the detection of fluorescent radiation, through the use of receptor-specific binding of compounds to target organs. In doing so, special dye molecules, which are attached to biomolecules, should give a highly sensitive fluorescent signal.
Zadaća će biti riješena pripremom spojeva, koji su kovalentno vezani na kratkolančane peptide. Ti konjugati posjeduju visoki afinitet prema spajanju sa heptahelikalnim receptorima, specijalno somatostatin receptor, VIP-receptor (vazoaktivni intestinalni receptor) i neurotenzin receptor i biti će isto tako preko receptor-posredovane endocitoze intracelularno primljen. Izumom predočeni spojevi su primjereni za tehnički jednostavnu, bezopasnu optičku dijagnostiku tumorskih stanica i tumorskog tkiva, koja somatostatin receptore, VIP-receptore ili neurotenzin receptore u usporedbi sa zdravim stanicama jače eksprimiraju. Posebice su za to primjereni spojevi za fluorescentnu dijagnostiku, a od posebne prednosti su za fluorescentno-endoskopsku dijagnostiku utrobnih organa, kao ezofagus, cerviks, kolon i bronhije različitih tipova tumora, kao npr. adenokarcinom, neuroendokrini tumori ili duktalni pankreatski tumori. The task will be solved by preparing compounds, which are covalently bound to short-chain peptides. These conjugates have a high affinity for binding to heptahelical receptors, especially somatostatin receptor, VIP-receptor (vasoactive intestinal receptor) and neurotensin receptor and will also be received intracellularly via receptor-mediated endocytosis. The compounds presented by the invention are suitable for technically simple, harmless optical diagnostics of tumor cells and tumor tissue, which express somatostatin receptors, VIP-receptors or neurotensin receptors more strongly compared to healthy cells. Compounds for fluorescent diagnostics are especially suitable for this, and they are of particular advantage for fluorescent-endoscopic diagnostics of internal organs, such as the esophagus, cervix, colon and bronchi of various types of tumors, such as adenocarcinoma, neuroendocrine tumors or ductal pancreatic tumors.
Posebno se prednost daje bojama, koje su specifične po tome, da ispunjavaju posebne fotofizikalne i kemijske zahtjeve. S foto-fizikalnog aspekta, moraju boje imati visoki koeficijent apsorpcije, da bi moge dati efektivan signal, čak i kod malih koncentracija tkiva. Apsorpcijski maksimum moraju, slobodnim odabirom, jedno široko spektralno područje pokrivati. Tako je za detekciju u dubljim slojevima tkiva (više centimetara ispod površine kože) esencijalno spektralno područje između 600 i 900 nm, dok su za površinsku detekciju dovoljne apsorbcijske valne duljine od 400 do 600 nm. S kemijskog aspekta moraju boje posjedovati visoki fotosenzibilitet i nikakve pojave raspadanja (photobleaching) za vrijeme poticaja (snimanja). Boje moraju biti uklopive, kao gradivi elementi u postupku sinteze sintetičke čvrste faze peptida i time biti stabilni u normalnim uvjetima sinteze, da bi time bio zadovoljen jednostavan, cijenom pristupačan postupak proizvodnje strukturno definiranih konjugata peptida i boja s čvrstim stehiometrijskim odnosom između boje i peptida. Postavljeni zahtjevi su najbolje ispunjeni kod polimetin boje, posebice cijanin, merocijanin, oksonol i skvarilij. Particular preference is given to colors that are specific in that they meet special photophysical and chemical requirements. From the photo-physical aspect, dyes must have a high absorption coefficient, in order to be able to give an effective signal, even at low tissue concentrations. Absorption maxima must, by free selection, cover a wide spectral range. Thus, for detection in deeper tissue layers (several centimeters below the surface of the skin), the spectral range between 600 and 900 nm is essential, while absorption wavelengths from 400 to 600 nm are sufficient for surface detection. From a chemical point of view, the colors must have high photosensitivity and no phenomena of decay (photobleaching) during stimulation (recording). Dyes must be compatible, as building blocks in the synthetic solid phase synthesis process of peptides and thus be stable under normal synthesis conditions, in order to satisfy a simple, affordable production process of structurally defined conjugates of peptides and dyes with a solid stoichiometric relationship between dye and peptide. The set requirements are best met with polymethine dyes, especially cyanine, merocyanine, oxonol and squarilium.
Predmet izuma su stoga The subject of the invention are therefore
Konjugati peptid-polimetin-boja opće formule (I) Peptide-polymethine-dye conjugates of the general formula (I)
A1 – (X)m – A2 (I) A1 – (X)m – A2 (I)
pri čemu whereby
X za α, β ili γ-aminokiseline ili D ili L-konfiguraciju i X for α, β or γ-amino acids or D or L-configuration i
m za broj od 5 do 30 stoji, m stands for the number from 5 to 30,
pri čemu rezultirajuća amino sekvenca (X)m je ravnolančane naravi ili preko disulfidne veze između cisteina ili homocisteina ili amidno, između N i C-terminusa ciklizirana i za aminosekvencu vazoaktivnog intestinalnog peptida (VIP), somatostatina ili neurotenzina ili za fragmente, djelomične sekvenece, derivate ili analoge VIP, somatostatina ili neurotenzina stoji, where the resulting amino sequence (X)m is straight-chain or via a disulfide bond between cysteine or homocysteine or amido, between the N and C-terminus cyclized and for the amino sequence of vasoactive intestinal peptide (VIP), somatostatin or neurotensin or for fragments, partial sequences, derivatives or analogues of VIP, somatostatin or neurotensin stands,
A1 za jedan atom vodika, jedan acetilni ostatak ili jedan alkilni ostatak s do 10 C-atoma, koji isto tako mogu biti supstituirani s 1 do 3 karboksilne skupine i/ili 1 do 6 hidroksi-skupine ili jedan poli(oksietilen)ostatak s 2 do 30 -CH2CH2O-jedinica ili jedna molekula boje iz klase polimetinske boje, koja pokazuje jedno apsorpcijsko područje od 380 do 1200 nm, A1 for one hydrogen atom, one acetyl residue or one alkyl residue with up to 10 C-atoms, which can also be substituted with 1 to 3 carboxyl groups and/or 1 to 6 hydroxy groups or one poly(oxyethylene) residue with 2 up to 30 -CH2CH2O-units or one dye molecule from the polymethine dye class, which shows one absorption region from 380 to 1200 nm,
A2 za hidroksi skupinu, jednu amino skupinu ili jednu molekulu boje iz klase polimetinske boje, koja pokazuje najmanje jedan apsorcijski maksimum u području od 380 do 1200 nm, stoji A2 for a hydroxy group, one amino group or one dye molecule from the polymethine dye class, which shows at least one absorption maximum in the range from 380 to 1200 nm, reads
pod uvjetom, da najmanje jedan od ostataka A1 ili A2 predstavlja jednu molekulu boje iz klase polimetinskih boja, koje najmanje jedan apsorpcijski maksimum u području od 380 do 1200 nm pokazuju, provided that at least one of the residues A1 or A2 represents one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm,
pri čemu, za slučaj, da A1 i/ili A2 jedna molekula boje iz klase polimetinskih boja, koje najmanje jedan apsorpcijski maksimum, u području od 380 do 1200 nm pokazuju, predstavlja, A1 je povezana na jednu N-terminalnu amino skupinu, a A2 na jednu amino skupinu aminokiseline lizin ili na jednu hidroksi skupinu aminokiseline serin na bilo kojoj poziciji unutar sekvence aminokiselina (X)m, where, in the event that A1 and/or A2 one dye molecule from the class of polymethine dyes, which show at least one absorption maximum in the range from 380 to 1200 nm, represents, A1 is connected to one N-terminal amino group, and A2 to one amino group of the amino acid lysine or to one hydroxy group of the amino acid serine at any position within the amino acid sequence (X)m,
i njihove fiziološki podnošljive soli. and their physiologically tolerable salts.
Fragmenti, dijelovi sekvenci, derivati ili analozi navedenih peptida stoje između ostalog za skraćene sekvence aminokiselina, izmjene pojedinih ili više aminokiselina nasuprot odgovarajućih D-aminokiselina, invertiranih sekvenca i kombinacija navedenih oznaka. Fragments, parts of the sequences, derivatives or analogs of the mentioned peptides stand, among other things, for shortened sequences of amino acids, changes of individual or several amino acids against the corresponding D-amino acids, inverted sequences and combinations of the mentioned labels.
Fragmenti, djelovi sekvenci, derivati ili analozi navedenih peptida mogu isto tako sadržavati neprirodne aminokiseline, kao npr. naftalanin, cikloheksilalanin, norleucin, norvalin, α-aminoadipinsku kiselinu, α-amino maslačnu kiselinu, β-alanin, β-cikloheksilalanin, ornitin, sarcozin ili бhidroksilizin. Fragments, parts of the sequences, derivatives or analogs of the mentioned peptides can also contain unnatural amino acids, such as naphthalanine, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-amino butyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or bhydroxylysine.
Posebno preferirani izvedeni oblici izuma su spojevi opće formule I, koji se time odlikuju, da molekula boje A1 i/ili A2 stoji za jedan cijanin, skvarilij, krokonij, merocijanin ili oksonol. Te boje pripadaju klasi polimetinskih boja i imaju gore navedene prednosti. Particularly preferred derivatives of the invention are compounds of the general formula I, characterized by the fact that the color molecule A1 and/or A2 stands for one cyanine, squarilium, croconium, merocyanine or oxonol. These colors belong to the class of polymethine colors and have the above-mentioned advantages.
Daljnji preferirani, izumom predočeni spojevi opće formule I odlikuju se time da, Further preferred compounds of the general formula I presented by the invention are characterized in that,
je molekula boje A1 i/ili A2 za jedan cijanin odnosno skvarilij (boja) opće formule II is a molecule of color A1 and/or A2 for one cyanine or squarilium (color) of the general formula II
[image] [image]
pri čemu whereby
D za jedan fragment koji odgovara općoj formuli III do VI, pri čemu pozicija označena zvjezdicom označava povezanost sa B, stoji, D for one fragment corresponding to the general formula III to VI, wherein the asterisked position indicates linkage to B, reads,
[image] [image]
B za fragment odgovarajuć općoj formuli VII do XII B for the fragment corresponding to the general formula VII to XII
[image] [image]
R1 i R2 za E1, R3 za jedan fluor, klor, brom, jod- atom ili jednu nitro skupinu ili za jedan ostatak –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, SO3E1, -SO2NHE1, -E1, R1 and R2 for E1, R3 for one fluorine, chlorine, bromine, iodine atom or one nitro group or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, SO3E1, - SO2NHE1, -E1,
pri čemu E1 i E2 neovisno međusobno za jedan atom vodika, jedan C1-C4-sulfoalkilni lanac, jednu zasićenu ili nezasićenu, razgranjen ili nerazgranjen C1-C50-alkilni lanac, pri čemu lanac ili dijelovi lanca isto tako jedan ili više aromatične ili zasićene ciklične C5-C6- ili biciklične jedinice tvoriti wherein E1 and E2 independently of each other for one hydrogen atom, one C1-C4-sulfoalkyl chain, one saturated or unsaturated, branched or unbranched C1-C50-alkyl chain, wherein the chain or parts of the chain also have one or more aromatic or saturated cyclic C5-C6- or bicyclic units to form
mogu, stoje i pri tom C1-C50-alkilni lanac od 0 do 15 atoma kisika i/ili od 0 do 3 karbonilne skupine su prekinute i/ili su s 0 do 5 hidroksilnih skupina supstituirani, stoje, can, provided that the C1-C50 alkyl chain of 0 to 15 oxygen atoms and/or 0 to 3 carbonyl groups are interrupted and/or substituted with 0 to 5 hydroxyl groups, provided that
R4 za jedan atom vodika, za jedan fluor, klor, brom, jod-atom ili za jedan razgranjeni ili ravnolančani C1-C10-alkilni lanac, stoji, R4 for one hydrogen atom, for one fluorine, chlorine, bromine, iodine atom or for one branched or straight-chain C1-C10-alkyl chain, it says,
b jedan broj 2 ili 3 označava, b one number 2 or 3 means,
X i Y neovisno međusobno O, S, Se, -CH=CH- ili C(CH3)2 X and Y are independently O, S, Se, -CH=CH- or C(CH3)2
označuje, denotes,
L za jednu skupinu odgovarajuće slijedeće formule L for one group corresponding to the following formula
[image] [image]
pri čemu n označava jedan broj od 1 do 10, where n denotes a number from 1 to 10,
stoji. stands.
(Kod gore navedene formule označava u dijelu molekule lijevo naznačena provučena linija vezu prema osnovnom kosturu boje, a isprekidana linija u dijelu molekule desno, vezu prema peptidu.) (In the above formula, the solid line indicated in the part of the molecule on the left indicates the connection to the basic skeleton of the dye, and the broken line in the part of the molecule on the right, the connection to the peptide.)
Iz klase polimetinskih boja su cijanin boje, npr. one koje se baziraju na indol-strukturi, indokarbo-, indodikarbo- i indotrikarbcijanin su posebno od prednosti. Takve strukture su karakteristične po tome što pokazuju visoku kemijsku i fotokemijsku stabilnost. Preko povoljnu sintezu se mogu dobiti derivati, koji proizvoljno apsorbiraju između 400 i 1000 nm i fluoresciraju, kroz supstituciju sa sličnim linkovima i funkcionalnim skupinama, primjerice s karboksilnim skupinama, mogu biti pripojeni na peptide i posjeduju visoku topivost, primjerice preko sulfonatnih skupina. Nasuprot u literaturi poznatih cijanin boja, posjeduju izumom predočeni, primjenjivani spojevi samo jednu reaktivnu skupinu, koja omogućuje jedno stehiometrijski definirano spajanje na peptid u tijeku sinteze smole konjugata. From the class of polymethine dyes are cyanine dyes, for example those based on the indole structure, indocarbo-, indodicarbo- and indotricarbacyanine are particularly advantageous. Such structures are characterized by high chemical and photochemical stability. Derivatives can be obtained via favorable synthesis, which arbitrarily absorb between 400 and 1000 nm and fluoresce, through substitution with similar links and functional groups, for example with carboxyl groups, can be attached to peptides and have high solubility, for example via sulfonate groups. In contrast to the cyanine dyes known in the literature, the compounds presented by the invention have only one reactive group, which enables one stoichiometrically defined connection to the peptide during the synthesis of the conjugate resin.
Posebno cijenjeni izvedbeni oblici izumom predočenih spojeva opće formule I odlikuju se time da, Particularly valued embodiments of the compounds of the general formula I presented by the invention are characterized by the fact that,
- molekula boje A1 i/ili A2 za jedan indokarbocijanin-, jedan indodikarbocijanin- ili jednu indotrikarbocijanin boju stoje, - dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye stand,
- molekula boje A1 i/li A2 za jedan indokarbocijanin-, jedan indodikarbocijanin- ili jedan indotrikarbocijaninsku boju opće formule XIII ili XIV - dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye of the general formula XIII or XIV
[image] [image]
[image] [image]
pri čemu whereby
p za 1,2 ili 3 p for 1,2 or 3
n za jedan broj od 1,2,3,4 ili 10 stoji, n stands for a number of 1,2,3,4 or 10,
R1 i R2 neovisno međusobno za jedan 4-Sulfobutil-, 3-sulfopropil-, 2-sulfoetil-, 3-metil-3-sulfopropil-, metil-, etil- ili propil ostatak stoji R1 and R2 independently of each other for one 4-Sulfobutyl-, 3-sulfopropyl-, 2-sulfoethyl-, 3-methyl-3-sulfopropyl-, methyl-, ethyl- or propyl residue is
I AND
R3 za vodik, jedan klor, brom, jod atom ili jednu nitroskupinu ili za jedan ostatak –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, ---SO2NHE1, R3 for hydrogen, one chlorine, bromine, iodine atom or one nitro group or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, ---SO2NHE1,
pri čemu E1 i E2, međusobno neovisno, za jedan atom vodika ili za jedan metil, etil ili jedan C3-C6-alkil ostatak, koji od 0 do 2 atoma kisika i/ili od 0 do 1 karbonilne skupine je prekinut i/ili s 0 do 5 hidroksilnih skupina supstituiran, ili za jedan poli(oksietilen)glikol ostatak s 2 do 30 –CH2CH2O-jedinica stoji, wherein E1 and E2, mutually independent, for one hydrogen atom or for one methyl, ethyl or one C3-C6-alkyl residue, which from 0 to 2 oxygen atoms and/or from 0 to 1 carbonyl group is interrupted and/or with 0 to 5 hydroxyl groups substituted, or for one poly(oxyethylene)glycol residue with 2 to 30 –CH2CH2O-units it stands,
stoji, stands,
- da molekula boje A1 i/ili A2 za jedan indokarbocijanin-, jedan indodikarbocijanin- ili jedan indotrikarbocijanin boju opće formule XIII ili XIV - that the dye molecule A1 and/or A2 for one indocarbocyanine-, one indodicarbocyanine- or one indotricarbocyanine dye of the general formula XIII or XIV
[image] [image]
[image] [image]
pri čemu whereby
p za 1, 2 ili3 p for 1, 2 or 3
n za 1, 2 ili 4, n for 1, 2 or 4,
R1 i R2 neovisno međusobno za jedan 4-sulfobutil- ili 3-sulfopropil ostatak, R1 and R2 independently of each other for one 4-sulfobutyl- or 3-sulfopropyl residue,
R3 za atom vodika ili za jedan –COOE1 ostatak ili -CONHE1, R3 for a hydrogen atom or for one –COOE1 residue or -CONHE1,
pri čemu E1 jedan atom vodika ili jedan metil-, etil- ili jedan C3-C6-alkil ostatak, koji je od 0 do 2 atoma kisika i/ili od 0 do 1 karboksilne skupine prekinut i/ili s 0 do 5 hidroksilnih skupina supstituitran, ima značenje, where E1 is one hydrogen atom or one methyl-, ethyl- or one C3-C6-alkyl residue, which is terminated from 0 to 2 oxygen atoms and/or from 0 to 1 carboxyl group and/or substituted with 0 to 5 hydroxyl groups , has the meaning,
stoji, stands,
- da jedna molekula boje A1 i/ili A2 za jednu indotrikarbocijanin boju opće formule XV ili XVI stoji: - that one molecule of dye A1 and/or A2 for one indotricarbocyanine dye of the general formula XV or XVI reads:
[image] [image]
[image] [image]
pri čemu whereby
n za 2 ili 3 stoji, n stands for 2 or 3,
R1 i R2 neovisno međusobno jedan 4-sulfobutil-, 3-sulfopropil- ili 2-sulfoetilostatak predstavlja, R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,
R3 za jedan ostatak –CONH-peptid, -CONH-(CH2)m-CONH-peptid, -CONH-(CH2)n-NH-CS-NH-peptid ili -CONH-(CH2)n-NHCO-CH2-peptid s m=1 do 10 i n=2 ili 3,stoji, R3 for one residue –CONH-peptide, -CONH-(CH2)m-CONH-peptide, -CONH-(CH2)n-NH-CS-NH-peptide or -CONH-(CH2)n-NHCO-CH2-peptide with m=1 to 10 and n=2 or 3, it stands,
ili slijedeću skupinu predstavlja: or the following group is represented by:
[image] [image]
R4 i R5 neovisno međusobno za jedan atom vodika, jedan metilni ostatak ili jedan hidroksilirani alkilni ostatak kao npr. 2-hidroksietil, 3-hidroksipropil, 2,3-dihidroksipropil, 1,3-dihidroksi-2-propil, 2,3,4-trihidroksibutil, 1,3,4-trihidroksi-2-butil, 2,3,4,5,6-pentahidroksiheksil, stoji R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3,4 -trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl, stands
R6 za jednu od slijedećih skupina stoji: R6 stands for one of the following groups:
-(CH2)m-CONH-peptid s m=0 do 2, -(CH2)m-CONH-peptide with m=0 to 2,
-(CH2)m-NH-CS-NH-peptid s m=0 do 2, -(CH2)m-NH-CS-NH-peptide with m=0 to 2,
i X za jedan atom kisika ili jedan atom sumpora stoji; and X stands for one oxygen atom or one sulfur atom;
- da molekula boje A1 i/ili A2 za jednu indotrikarbocijanin boju opće formule XVII stoji: - that the dye molecule A1 and/or A2 for one indotricarbocyanine dye of the general formula XVII reads:
[image] [image]
pri čemu whereby
R1 i R2 neovisno međusobno jedan 4-sulfobutil- ili 3-sulfopropil ostatak predstavljaju, R1 and R2 independently represent one 4-sulfobutyl- or 3-sulfopropyl residue,
R3 za jedan ostatak –CONH-peptid, CONH-(CH2)m-CONH-peptid, -CONH-(CH2)n-NH-CS-NH-peptid ili –CONH-(CH2)n-NHCO-CH2-peptid s m=1 do 10 i n=2 ili 3, stoji R3 for one residue –CONH-peptide, CONH-(CH2)m-CONH-peptide, -CONH-(CH2)n-NH-CS-NH-peptide or –CONH-(CH2)n-NHCO-CH2-peptide with m =1 to 10 and n=2 or 3, it says
ili slijedeću skupinu predstavlja: or the following group is represented by:
[image] [image]
i R4 i R5 neovisno međusobno za jedan atom vodika ili jedan metilni ostatak ili jedan hidroksilirani alkilni ostatak, kao npr. 2-hidroksietil, 3- hidroksipropil, 2,3-dihidroksipropil, 1,3-dihidroksi-2-propil, 2,3,4-trihidroksibutil, 1,3,4-trihidroksi-2-butil, 2,3,4,5,6-pentahidroksiheksil, stoji. and R4 and R5 independently of each other for one hydrogen atom or one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3 ,4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl, it says.
Primjena antitijela markiranih bojom pri detekciji tumora je poznata u literaturi (J. Cell. Pharmacol. 3, 141-145, 1992; Cancer Immunol. Immunother. 41, 257-63,1995; Cancer Research 54, 2643-9, 1994, Biotechnol. Prog. 13, 649-658, 1997). The use of color-labeled antibodies in tumor detection is known in the literature (J. Cell. Pharmacol. 3, 141-145, 1992; Cancer Immunol. Immunother. 41, 257-63, 1995; Cancer Research 54, 2643-9, 1994, Biotechnol. Prog. 13, 649-658, 1997).
U suprotnosti s tim sadrže izumom predočeni spojevi kao biomolekule niskomolekularne peptide i derivate peptida, koji prednosti antitijela, kao visoka mogućnost spajanja na ciljane molekule, pokazuju, bez da je pri tom dijagnostički potencijal preko jedne nepovoljne farmakokinetike (dugo vrijeme sedimentacije krvi, alergijski nepoželjni učinci - imunogenitet) ograničen. In contrast to this, the compounds presented by the invention contain as biomolecules low-molecular peptides and peptide derivatives, which demonstrate the advantages of antibodies, such as a high possibility of binding to target molecules, without the diagnostic potential exceeding unfavorable pharmacokinetics (long time of blood sedimentation, allergic adverse effects - immunogenicity) limited.
Biološki i farmakološki zahtjevi na peptidne sekvence su tome odgovarajuće zadovoljavajući plazmabilitet kod bržeg sakupljanja u ciljanom tkivu i istovremeno brza eliminacija iz ostatka tijela, primjerice preko renalnog puta izlučivanja. Biological and pharmacological requirements for peptide sequences are correspondingly satisfactory plasmability with faster collection in the target tissue and at the same time rapid elimination from the rest of the body, for example via the renal excretion route.
Iznenađujuće je da je pri tom otkriveno, da peptidna sekvenca, koja sadrži najmanje 5 aminokiselina na strani C-teminusa VIP-sekvence i na kojoj je povezana boja za fluorescentnu dijagnostiku, posjeduje jednu s nativnim VIP usporedivim snimkama u tumorskih stanica. Obzirom na to otkriveno je, da preko ugradnje najmanje jedne D-aminokiseline ili izmjenom preko najmanje jedne D-aminokiseline, stabilnost plazme može biti značajno povećan. Na primjeru jedne potpune izmjene svih L- s D-aminokiselinama u jednom VIP-vezujućem peptidnom konjugatu boja, moglo se dokazati, da je odlika vezivanja i prodiranja u stanice ostala neizmjenjena. Surprisingly, it was discovered that the peptide sequence, which contains at least 5 amino acids on the C-terminus side of the VIP-sequence and to which a dye for fluorescent diagnostics is attached, has one with native VIP comparable recordings in tumor cells. In view of this, it was discovered that through the incorporation of at least one D-amino acid or by changing at least one D-amino acid, plasma stability can be significantly increased. On the example of a complete exchange of all L- with D-amino acids in one VIP-binding peptide dye conjugate, it could be proven that the property of binding and penetration into cells remained unchanged.
Daljnje posebnosti izumom predočenih spojeva opće formule I, kojima se daje prednost, odlikuju se time, Further particularities of the compounds of general formula I presented by the invention, which are preferred, are characterized by,
da (X)m za sekvencu aminokiselina nativnog, u prirodi poznatog, humanog vazoaktivnog intestinalnog peptida, odgovara that (X)m for the amino acid sequence of the native, known in nature, human vasoactive intestinal peptide, corresponds to
[image] [image]
ili za fragmente, dijelove sekvenci, derivate ili analoge vazoaktivnih intestinalnih peptida, koji se sastoje od 5 do 30 aminokiselina, or for fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides, consisting of 5 to 30 amino acids,
stoje, What is it,
- da (X)m za aminosekvencu somatostatinu odgovarajuću - yes (X)m for the amino sequence corresponding to somatostatin
[image] [image]
ili za fragmente, dijelove sekvenci, derivate ili analoge somatostatina, koji se sastoje od 5 do 20 aminokiselina or for fragments, parts of sequences, derivatives or analogs of somatostatin, consisting of 5 to 20 amino acids
stoje, What is it,
- da (X)m za sekvencu aminokiselina odgovarajuću neotenzinu - yes (X)m for the amino acid sequence corresponding to neotensin
piroglutaminska kiselina-LYENKPRPYIL pyroglutamic acid-LYENKPRPYIL
ili za fragmente, dijelove sekvenci, derivate ili analoge neurotenzina, koji se sastoje od 5 do 20 aminokiselina, or for fragments, parts of sequences, derivatives or analogs of neurotensin, consisting of 5 to 20 amino acids,
stoje, What is it,
- da kao fragmenti, dijelovi sekvenci, derivati ili analoga vazoaktivnog intestinalnog peptida (VIP) slijedeća sekvenca aminokiselina je odabrana: - that as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptide (VIP) the following sequence of amino acids was selected:
[image] [image]
- da kao analoga VIP je odabrana slijedeća sekvenca: - that the following sequence was selected as a VIP analogue:
FSDAVFTDNY TRLRKQMAVK KYLNSILN FSDAVFTDNY TRLRKQMAVK KYLNSILN
[image] [image]
[image] [image]
[image] [image]
- da kao analog VIP-u je jedan spoj, prema slijedećoj formuli odabran: - that as an analogue of VIP, one compound was selected according to the following formula:
[image] , [image]
pri čemu X1, X2 i X3 može predstavljati bilo koju aminokiselinu, where X1, X2 and X3 can represent any amino acid,
da 2 do m aminokislina neovisno međusobno, nasuprot njene bilo aminokiseline ili nasuprot druge L- ili D-aminokiseline može biti izmjenjena, pri čemu ista ima gore navedeno značenje, that 2 to m amino acids independently of each other, opposite any amino acid or opposite another L- or D-amino acid can be changed, whereby the same has the above-mentioned meaning,
- da najmanje jedna aminokiselina (X)m neovisno međusobno, za drugu, neprirodnu aminokiselinu ili derivat aminokiseline može biti zamjenjena, - that at least one amino acid (X)m can be independently substituted for another, unnatural amino acid or amino acid derivative,
- da najmanje jedna aminokiselina (X)m neovisno međusobno, za drugu, neprirodnu aminokiselinu ili derivat aminokiseline, kao. npr. naftalanin, cikloheksilalanin, norleucin, norvalin, α-aminoadipinska kiselina, α-aminomaslačna kiselina, β-alanin, β-cikloheksilalanin, ornitin, sarkozin ili б-hidroksilizin, može biti zamjenjena - that at least one amino acid (X) m independently of each other, for another, unnatural amino acid or amino acid derivative, as. eg naphthalene, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-aminobutyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or b-hydroxylysine, may be substituted
- da kao analog VIP-u, jedan spoj, prema slijedećoj formuli, je odabran: - that as an analogue of VIP, one compound, according to the following formula, was selected:
[image] [image]
pri čemu X1, X2, X3 i X4 nisu prirodne aminokiseline ili derivati aminokiselina, kao npr. naftalanin, cikloheksilalanin, norleucin, norvalin, α-aminoadipinska kiselina, α-aminomaslačna kiselina, β-alanin, β-cikloheksilalanin, ornitin, sarkozin ili б-hidroksilizin, može predstavljati, where X1, X2, X3 and X4 are not natural amino acids or amino acid derivatives, such as naphthalanine, cyclohexylalanine, norleucine, norvaline, α-aminoadipic acid, α-aminobutyric acid, β-alanine, β-cyclohexylalanine, ornithine, sarcosine or b -hydroxylysine, can represent,
- da su neke aminokiseline (X)m za njihove određene oblike D-aminokiselina zamjenjene - that some amino acids (X)m are substituted for their specific D-amino acid forms
- da kao fragmenti, dijelovi sekvenci, derivati ili analoga vazoaktivnih intestinalnih peptida retrosintetičkih sekvenci aminokiselina, kod kojih 2 do m aminokiselina su izmjnenjene za neke D-aminokiseline, - that as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides of retrosynthetic amino acid sequences, in which 2 to m amino acids are changed for some D-amino acids,
su odabrane, pri čemu imaju gore navedeno značenje, are selected, having the above meaning,
- da kao fragmenti, dijelovi sekvenci, derivati ili analoga vazoaktivnih intestinalnih peptida su odabrane slijedeće sekvence aminokiselina: - that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogues of vasoactive intestinal peptides:
[image] [image]
- da kao fragmenti, dijelovi sekvenci, derivati ili analoga somatostatina su slijedeće sekvence aminokiselina odabrane: - that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogs of somatostatin:
[image] [image]
- da kao fragmenti, dijelovi sekvenci, derivati ili analoga neurotenzina su slijedeće sekvence aminokiselina odabrane: - that the following amino acid sequences were selected as fragments, parts of sequences, derivatives or analogues of neurotensin:
[image] [image]
Terminologija opće formule I sadrži uobičajeni način označavanja sekvanca aminokiselina. N-terminus je stalno lijevo, a C-terminus desno (nesupstituiran odgovara H-(X)m-OH ili, u slučaju jednog amida, H-(X)m-NH2. Upotrebljavane kratice od jednog slova aminokiselina mogu biti očitane u M. Bodanszky, Peptide chemistry – A practical textbook, 2nd edition, Springer-Verlag Heidelberg 1993, str 3. velika slova označuju aminokiseline s L-konfiguracijom (prirodne aminokiseline), mala slova označuju D-aminokiseline, disulfidne veze (ciklički peptidi) su preko crtica koje označuju veze između odgovarajućih slova (C=cistein ili homocistein) označen. Kao retrosintetičke označuju se sekvence, kod kojih pri sintezi slijed aminokiselina u usporedbi prema danoj nativnoj sekvenci, je invertan, što znači da sinteza započinje s početnom N-terminalnom aminokiselinom i proizvodi sekvencu prema prvobitnoj C-terminalnoj aminokiselini. The terminology of general formula I contains the usual way of notation of amino acid sequences. The N-terminus is always on the left and the C-terminus on the right (unsubstituted corresponds to H-(X)m-OH or, in the case of an amide, H-(X)m-NH2. The one-letter abbreviations used for amino acids can be read in M .Bodanszky, Peptide chemistry – A practical textbook, 2nd edition, Springer-Verlag Heidelberg 1993, p 3. uppercase letters denote amino acids with L-configuration (natural amino acids), lowercase letters denote D-amino acids, disulfide bonds (cyclic peptides) are over dashes indicating the connections between the corresponding letters (C = cysteine or homocysteine) marked Sequences are marked as retrosynthetic, in which, during synthesis, the sequence of amino acids compared to the given native sequence is inverted, which means that the synthesis begins with the initial N-terminal amino acid and produces a sequence according to the original C-terminal amino acid.
Analoga VIP-a biti će pomoću supstitutivne analize pokazan (pogledaj primjer 43). Posebno prednost imaju slijedeći analoga VIP-a: An analogue of VIP will be shown by substitution analysis (see example 43). The following analogues of VIP have a special advantage:
[image] [image]
[image] [image]
Izumom predočene supstance imaju različite prednosti u odnosu na radioaktivno markirane supstance. Fluorescentne boje mogu se bezbrojno često navesti na fluorescentnu emisiju. Ne leži nikakav kontinuirani signal, koji bi doveo do raspada s odgovarajućim vremenom poluraspada, koji leži u suštini. Odgovarajuće tome je vrijeme dijagnoze moguće prema želji odabirati i isto tako prema želji često ponavljati i nije limitirano kroz poluvrijeme jednog izotopa. Pacijent nije nikakvom ionizirajućem zračenju izložen, primjenjeni snop svijetla je u primjenjenim količinama neškodljiv. Optička detekcijska tehnika omogućuje visokosenzitivnu detekciju, manje fotona i prema tome je u odnosu na senzitivitet pomoću radiodijagnostike usporediva. The substances presented by the invention have different advantages compared to radioactively labeled substances. Fluorescent dyes can be referred to fluorescent emission countless times. There does not lie any continuous signal, which would lead to a decay with a corresponding half-life, which lies in the essence. Correspondingly, the diagnosis time can be selected as desired and also repeated as often as desired and is not limited by the half-life of one isotope. The patient is not exposed to any ionizing radiation, the applied light beam is harmless in the applied amounts. The optical detection technique enables high-sensitivity detection, fewer photons, and is therefore comparable to the sensitivity of radiodiagnostics.
Jedan postupak za dijagnostiku pomoću blizog infracrvenog snopa svijetla (NIR-snop svjetla) zu primjenu konjugata boja biomolekula već je opisano (WO 96/17628). Kao posebna prednost pokazalo se u slučaju predočenog izuma, da su konjugati peptida u povoljnijoj količini i višoj čistoći, kroz postupak automatizirane sinteze čvrste faze, predstavljivi. Iznnađujuće je da je pri tom otkriveno, da različiti spojevi indolcijanina, koji nose karboksilne skupine, kao analoga karboksilnim kiselinama, mogu biti korištena i kao N-terminus, kao što mogu biti na aminoskupinu lizina, na čvrstoj komponenti (smola) nadovezana. One method for diagnostics using a near-infrared light beam (NIR-light beam) using dye conjugates of biomolecules has already been described (WO 96/17628). As a special advantage, in the case of the presented invention, it was shown that peptide conjugates in a more favorable quantity and higher purity, through the process of automated synthesis of the solid phase, are presentable. It is surprising that it was discovered that various compounds of indolcyanine, which carry carboxylic groups, as analogues of carboxylic acids, can be used as N-terminus, as they can be attached to the amino group of lysine, on a solid component (resin).
Nakon odcjepljenja od smole i kromatografskog pročišćavanja, dobiju se konjugati boje i peptida, stupnja čistoće > 95%. Jedan novi način vezivanja dopušta vezivanje spojeva halogenacetil-boja na aminokiseline cistein i homocistein, koje mogu zamjeniti svaku željenu aminokiselinu nativne VIP-sekvence. After separation from the resin and chromatographic purification, dye and peptide conjugates are obtained, purity level > 95%. One new mode of attachment allows the attachment of halogenacetyl dye compounds to the amino acids cysteine and homocysteine, which can replace any desired amino acid of the native VIP sequence.
Značajniji problem kod upotrebe svjetlosti za poticanje fluorescencije je ograničena dubina prodiranja svijetlosti, koja u VIS području leži u okviru submilimetra, a u NIR području može iznositi i centimetar. S obzirom na dubinu prodiranja neproblematični su postupci detekcije u površinskom sloju oboljelog tkiva, kao i mekih tkiva. A more significant problem with the use of light to stimulate fluorescence is the limited depth of light penetration, which in the VIS range is within a sub-millimeter, and in the NIR range can be as much as a centimeter. Considering the depth of penetration, detection procedures in the surface layer of diseased tissue, as well as soft tissues, are unproblematic.
Predmet izuma su stoga mamografski postupci, endoskopski postupci i intraoperativni postupci, kod kojih pod primjenom izumom predočenih spojeva, oboljela područja tkiva, kroz detekciju na principu fluorescencije ili neapsorbirajućih zračenja, mogu biti dijagnosticirana. Poseban predmet izuma je primjena izumom predočenih spojeva u endoskopskim postupcima, kao npr. koloskopija, bronhoskopija, ezofagijalna endoskopija, kod kojih se vrši dijagnosticiranje promjena na površinskim slojevima tkiva. Primjena bijelog svijetla s direktnom vizuelnom procjenom je vrlo raširena u endoskopskoj dijagnostici. Izumom predočeni spojevi pridonose, preko izazivanja signala specifičnih za pojedina tkiva jednom značajnom poboljšanju postupka, posebice kod dijagnosticiranja ranih, vizuelno nevidljivih promjena na tkivima (npr. displazije kolona). The subject of the invention is therefore mammographic procedures, endoscopic procedures and intraoperative procedures, in which, under the application of the compounds presented by the invention, diseased tissue areas can be diagnosed through detection based on the principle of fluorescence or non-absorbing radiation. A special subject of the invention is the application of the compounds presented by the invention in endoscopic procedures, such as colonoscopy, bronchoscopy, esophageal endoscopy, in which changes in the surface layers of tissue are diagnosed. The use of white light with direct visual assessment is very widespread in endoscopic diagnostics. The compounds presented by the invention contribute, by inducing signals specific for individual tissues, to a significant improvement of the procedure, especially when diagnosing early, visually invisible changes in tissues (eg colonic dysplasia).
Otkriveno je, da se nakon raspršivanja izumom predočenih spojeva kemijski povezanih s bojama, u crijevu štakora s kemijski induciranim displazijama i karcinomom kolona, nastavnim ispiranjem i provođenjem jedne endoskopske fluorescentne dijagnostike (inicijacija 740 nm, detekcija 760 nm), mogu dokazati bolesni dijelovi tkiva kolona pomoću povišene fluorescencije. It was discovered that after dispersing the compounds presented by the invention chemically related to dyes, in the intestines of rats with chemically induced dysplasias and colon cancer, by washing and performing an endoscopic fluorescent diagnosis (initiation 740 nm, detection 760 nm), diseased parts of the colon tissue can be proven. using increased fluorescence.
Predmet izuma je stoga isto tako i postupak za endoskopsku dijagnostiku, posebice gastrointestinalnog trakta, uz primjenu izumom predočenih spojeva. Pri tom se u tkiva jedna ili više supstanci, prije svega intravenozno ili topički raspršivanjem spreja impliciraju, a nakon toga se s pomoću svijetla iz odgovarajućeg spektralnog područja vrši elektronička inicijacija (podražaj) i obasjavanje primjenjene obojane supstance. Reflektirajuća ili emitirana fluorescencija obojene supstance se registrira. Prednost se pri tom daje metodama, kod kojih se tkiva na velikim površinama izlažu svjetlosnom pordražaju, a fluorescencija je lokalno izazvana, koja se snima jednom CCD-kamerom ili se areali tkiva koji su u fazi razgradnje s pomoću jednog svjetlosnog prijenosnika mogu snimiti u rasteru i dobiveni signali se računalnim putem mogu prenijeti u jednu sintetičku sliku. Pri tom se fluorescencija može spektralno i/ili fazno selektivno, kao i stacionarno i/ili vremenski izdvojeno detektirati i interpretirati. Dobivene fluorescentne slike mogu se simultano s pomoću slika dobivenih bijelim svijetlom, proizvesti i za interpretaciju podataka u bilo kojem obliku međusobno koristiti. The subject of the invention is therefore also a procedure for endoscopic diagnostics, especially of the gastrointestinal tract, with the use of the compounds presented by the invention. In doing so, one or more substances are injected into the tissues, first of all intravenously or topically by spraying, and then with the help of light from the appropriate spectral range, electronic initiation (stimulus) and illumination of the applied colored substance is performed. The reflected or emitted fluorescence of the colored substance is registered. Preference is given to methods in which tissues on large surfaces are exposed to a light substimulus, and fluorescence is locally induced, which is recorded with a single CCD camera, or tissue areas that are in the stage of decomposition can be recorded in a raster and the received signals can be transferred by computer into one synthetic image. At the same time, fluorescence can be detected and interpreted spectrally and/or phase selectively, as well as stationary and/or separated in time. The fluorescent images obtained can be produced simultaneously with the images obtained with white light and used for data interpretation in any form.
Sinteza izumom predočenih spojeva slijedi oslanjajući se na metode već poznate u literaturi. Peptidi se spravljaju sintezom na čvrstoj fazi, na polimernoj smoli. Pojedinosti su u struci već poznate. The synthesis of the compounds presented by the invention follows relying on methods already known in the literature. Peptides are made by synthesis on solid phase, on polymer resin. The details are already known in the field.
Literatura: Peptide chemistry – A practical textbook (M. Bodanszky), 2nd edition, Springer-Verlag Heidelberg 1993; Anti-Cancer Drug Design 12, 145-167, 1997; J. Am. Chem. Soc. 117, 11821-2, 1995. Literature: Peptide chemistry – A practical textbook (M. Bodanszky), 2nd edition, Springer-Verlag Heidelberg 1993; Anti-Cancer Drug Design 12, 145-167, 1997; J. Am. Chem. Soc. 117, 11821-2, 1995.
Bojane supstance se spravljaju odvojeno i zatim se tijekom sintetičog spravljanja čvrste faze peptida na iste spajaju, a izumom predočeni spojevi se nakon odcjepljenja smole i pročišćavanja dobiju kao visoko čisti spojevi. Prednost pri tom imaju takve obojane tvari, koje sadrže karboksilne skupine, a one se nakon aktiviranja s pomoću reagencija s aminoskupinama peptida, posebice ε-amino skupine lizina ili N-terminalne peptid-amino skupine, budu spojene. Nadalje, prednost se daje spojevima boja s halogenalkil- ili halogenacetil ostacima, koji na tiolnim skupinama peptida, posebice na aminokiselini cisteinu ili homocisteinu, budu povezane. Colored substances are prepared separately and then, during the synthetic preparation, the solid phase of the peptide is connected to it, and the compounds presented by the invention are obtained as highly pure compounds after separation of the resin and purification. Such colored substances, which contain carboxyl groups, are preferred, and after activation with the help of reagents, they are joined with amino groups of peptides, especially ε-amino groups of lysine or N-terminal peptide-amino groups. Furthermore, preference is given to dye compounds with haloalkyl or haloacetyl residues, which are linked to the thiol groups of the peptide, especially to the amino acid cysteine or homocysteine.
Literatura za sintezu polimetinskih boja: Bioconjugate Chem. 4, 105-111, 1993; Bioconjugate Chem. 7, 356-62, 1996; Bioconjugate Chem. 8, 751-56, 1997; Cytometry 10, 11-19, 1989 i 11, 418-30, 1990; J. Heterocycl. Chem. 33, 1871-6, 1966; J. Org. Chem. 60, 2391-5, 1995; Dyes and Pigments 17, 19-27, 1991, Dyes and Pigments 21, 227-34, 1993; J. Fluoresc. 3, 153-155, 1993; Anal. Biochem. 217, 197-204, 1994; US 4981977; US 5688966; US 5808044; WO97/42976; WO 97/42978; WO98/22146; WO 98/26077; EP 0800831. Literature for the synthesis of polymethine dyes: Bioconjugate Chem. 4, 105-111, 1993; Bioconjugate Chem. 7, 356-62, 1996; Bioconjugate Chem. 8, 751-56, 1997; Cytometry 10, 11-19, 1989 and 11, 418-30, 1990; J. Heterocycl. Chem. 33, 1871-6, 1966; J. Org. Chem. 60, 2391-5, 1995; Dyes and Pigments 17, 19-27, 1991, Dyes and Pigments 21, 227-34, 1993; J. Fluoresc. 3, 153-155, 1993; Anal. Biochem. 217, 197-204, 1994; US 4981977; US 5688966; US 5808044; WO97/42976; WO 97/42978; WO98/22146; WO 98/26077; EP 0800831.
Posebice su prikladni za spajanje s peptidima u čvrstoj fazi, su boje, koje točno jednu karboksilnu skupinu sadrže, s posebnom prednošću cijaninskih boja opće formule XVIII Dyes that contain exactly one carboxyl group, with a particular advantage of cyanine dyes of the general formula XVIII, are especially suitable for coupling with peptides in the solid phase.
[image] [image]
pri čemu whereby
p za 1,2 ili 3, p for 1,2 or 3,
n za 1,2,3,4 ili 10 stoji, n stands for 1,2,3,4 or 10,
R1 i R2 su međusobno neovisni za jedan 4-sulfobutil-3-sulfopropil-, 2-sulfoetil-, 3-metil-3-sulfopropil-, metil-, etil-, ili propil ostatak stoje i R1 and R2 are mutually independent for one 4-sulfobutyl-3-sulfopropyl-, 2-sulfoethyl-, 3-methyl-3-sulfopropyl-, methyl-, ethyl-, or propyl residue and
R3 za vodik ili za jedan ostatak –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, -SO2NHE1, R3 for hydrogen or for one residue –COOE1, -CONE1E2, -NHCOE1, -NHCONHE1, -NE1E2, -OE1, -OSO3E1, -SO3E1, -SO2NHE1,
pri čemu E1 i E2 međusobno neovisno za jedan atom vodika where E1 and E2 are mutually independent for one hydrogen atom
ili za jednu metil-, etil-, ili jedan C3-C6-alkilni ostatak, koji od 0 do 2 atoma kisika i/ili 0 do 5 hidroksilnih skupina je supstituiran, stoji or for one methyl-, ethyl-, or one C3-C6-alkyl residue, which is substituted by 0 to 2 oxygen atoms and/or 0 to 5 hydroxyl groups, it says
ili cijaninske boje opće formule XIX ili XX or cyanine dyes of the general formula XIX or XX
[image] [image]
[image] [image]
pri čemu whereby
n za 2 ili 3 stoji n stands for 2 or 3
R1 i R2 neovisno međusobno jedan 4-sulfobutil-, 3-sulfopropil- ili 2-sulfoetil ostatak predstavlja, R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,
R3 za jednu –COOH-skupinu ili jedan od slijedećih ostataka stoji: R3 for one –COOH-group or one of the following residues is:
-CONH-(CH2)n-COOH s n=2 ili 3, -CONH-(CH2)n-COOH with n=2 or 3,
-CONH-(CH2)n-NCS s n=2 ili 3, -CONH-(CH2)n-NCS with n=2 or 3,
-CONH-(CH2)n-NHCO-CH2-X1 s n=2 ili 3 i X1 = Cl, Br, J -CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3 and X1 = Cl, Br, J
[image] [image]
R4 i R5 neovisno međusobno za jedan atom vodika, jedan metilni ostatak ili jedan hidroksilirani alkilni ostatak, kao npr. 2-hidroksietil, 3-hidroksipropil, 2,3-dihidroksipropil, 1,3-dihidroksi-2-propil, 2,3,4-trihidroksibutil, 1,3,4-trihidroksi-2-butil, 2,3,4,5,6-pentahidroksiheksil stoji, R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3, 4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stands,
R6 za jednu od slijedećih skupina stoji: R6 stands for one of the following groups:
-(CH2)m-COOH s m= 0 do 2, -(CH2)m-COOH with m= 0 to 2,
-(CH2)m-NCS s m= 0 do 2 -(CH2)m-NCS with m= 0 to 2
i X za jedan atom kisika ili jedan atom sumpora stoji; and X stands for one oxygen atom or one sulfur atom;
ili cijaninske boje opće formule XXI or cyanine dyes of the general formula XXI
[image] [image]
pri čemu whereby
R1 i R2 neovisno međusobno jedan 4-sulfobutil-, 3-sulfopropil- ili 2-sulfoetil ostatak predstavljaju, R1 and R2 independently represent one 4-sulfobutyl-, 3-sulfopropyl- or 2-sulfoethyl residue,
R3 za jednu –COOH-skupinu ili jedan od slijedećih ostataka stoji: R3 for one –COOH-group or one of the following residues is:
-CONH-(CH2)n-COOH s n=2 ili 3, -CONH-(CH2)n-COOH with n=2 or 3,
-CONH-(CH2)n-NCS s n=2 ili 3, -CONH-(CH2)n-NCS with n=2 or 3,
-CONH-(CH2)n-NHCO-CH2-X1 s n=2 ili 3 i X1 = Cl, Br, J -CONH-(CH2)n-NHCO-CH2-X1 with n=2 or 3 and X1 = Cl, Br, J
[image] [image]
R4 i R5 neovisno međusobno za jedan atom vodika, jedan metilni ostatak ili jedan hidroksilirani alkilni ostatak, kao npr. 2-hidroksietil, 3-hidroksipropil, 2,3-dihidroksipropil, 1,3-dihidroksi-2-propil, 2,3,4-trihidroksibutil, 1,3,4-trihidroksi-2-butil, 2,3,4,5,6-pentahidroksiheksil stoji. R4 and R5 independently of each other for one hydrogen atom, one methyl residue or one hydroxylated alkyl residue, such as, for example, 2-hydroxyethyl, 3-hydroxypropyl, 2,3-dihydroxypropyl, 1,3-dihydroxy-2-propyl, 2,3, 4-trihydroxybutyl, 1,3,4-trihydroxy-2-butyl, 2,3,4,5,6-pentahydroxyhexyl stands.
Prednost samo jedne skupine koja se može aktivirati, kao npr. jedne karboksilne skupine ili jedne već aktivirane skupine, kao npr. jednog izotiocijanata, jedne halogenalkilne skupine ili jedne halogenacetilne skupine, sastoji se u tome, da može uslijediti jedno kemijski jedninstveno povezivanje. Halogenacetilna skupina ima jednu posebnu prednost, da jedno kemijski jedinstveno povezivanje na merkapto skupinu cisteina ili homocisteina slijedi. Takvo vezivanje može u otopini na nevezani i od zaštitnih skupina oslobođeni peptid uslijediti. Preko aktiviranih skupina je jedno povezivanje na peptid moguće, bez da posljedične reakcije uslijede. Za povišenje topljivosti u vodi pokazuju konjugati peptid-boja na boji jedan povećani broj hidroksi skupina. Preko pozicije linkera na indol sistemu boje, može dodatno jedna zadovoljavajuća hidrofilija, preko ostataka na sulfonatnim skupinama na atomima kisika indolnog sustava biti proizvedene. Tako može jedna strukturno jedinstvena reakcija povezivanja s peptidima (vidi primjer 4 do 38 i 44 do 49) biti provedena. The advantage of only one group that can be activated, such as one carboxyl group or one already activated group, such as one isothiocyanate, one haloalkyl group or one halogenacetyl group, consists in the fact that one chemically unique connection can follow. The haloacetyl group has one special advantage, that a chemically unique connection to the mercapto group of cysteine or homocysteine follows. Such binding can take place in solution on the unbound and deprotected peptide. Through the activated groups, one connection to the peptide is possible, without the consequent reactions following. To increase solubility in water, peptide-dye conjugates show an increased number of hydroxy groups on the dye. Through the position of the linker on the indole system of the dye, an additional satisfactory hydrophilicity can be produced through the residues on the sulfonate groups on the oxygen atoms of the indole system. Thus, a structurally unique coupling reaction with peptides (see examples 4 to 38 and 44 to 49) can be carried out.
Jedan daljnji predmet izuma je jedna optička dijagnostika za in-vivo-dijagnosticiranje oboljelih dijelova tkiva, koji se odlikuje time, da najmanje jedan spoj opće formule I zajedno s uobičajenim pomoćnim i/ili nosivim tvarima kao i sredstvo za razrijeđivanje sadrži. A further object of the invention is an optical diagnostic for in-vivo diagnosis of diseased tissue parts, which is characterized by the fact that it contains at least one compound of the general formula I together with the usual auxiliary and/or carrier substances as well as a diluting agent.
Slijedeći primjeri objašnjavaju izum: The following examples illustrate the invention:
Primjeri 1 do 3: Examples 1 to 3:
Sinteza indocijanin boje 1-3 za sintetičko povezivanje čvrstih faza na amino skupinama (N-terminal ili ε-lizin) peptida Synthesis of indocyanine dye 1-3 for synthetic connection of solid phases on amino groups (N-terminal or ε-lysine) of peptides
Sinteza slijedi generalno polazeći od 1-(4-sulfobutil)-2,3,3-trimetil-3H-indolena i 1-(4-Sulfobutil)-2,3,3-trimetil-5-karboksi-3H-indolena (Cytometry 10, 11-19, 1989, Talanta 39, 505-510, 1992). The synthesis follows generally starting from 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolene and 1-(4-Sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolene (Cytometry 10, 11-19, 1989, Talanta 39, 505-510, 1992).
Primjer 1: Example 1:
Sinteza 1,1`-bi(4-sulfobutil)indokarbocijanin-5-karbonske kiseline, natrijeva sol (1) Synthesis of 1,1`-bi(4-sulfobutyl)indocarbocyanine-5-carboxylic acid, sodium salt (1)
0,8 g (4,0 mmol) N,N-difenilformamidina biti će u 15 ml anhidrida octene kiseline položeno i pri sobnoj temp. u manjim porcijama s 1,4 g (4,2 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-5-karboksi-3H-indolenina pomiješano, 30 min, pri sobnoj temp. ohlađeno. Nastavno će biti 1,2 g (4,1 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-3H-indolenina, 1,2 g (14,6 mmol) suhog natijacetata, 15 ml anhidrida octene kiseline i 6 ml octene kiselina dodano. Reakcijska mješavina se zatim zagrijava 1 sat na 120ºC, tamno crvena otopina se ohladi i pomiješa sa 100 ml etera. Istaložena čvrsta tvar se odfiltrira. Slijedi jedno kromatografsko pročišćavanje na RP-Kieselgelu EUROPREP 60-30 C18, 60A, 20-45 m (eluent: voda/MeOH, stepenasti gradijent od 0% do 70% MeOH). Frakcije koje sadrže produkt biti će najprije u rotacijskom otparivaču oslobođene metanola i nastavno liofilizirane, iskoristivost: 1,5 g (58%), crveni liofilizat. 0.8 g (4.0 mmol) of N,N-diphenylformamidine will be placed in 15 ml of acetic anhydride at room temperature. in smaller portions with 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine mixed, 30 min, at room temp. chilled. Next will be 1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride acid and 6 ml of acetic acid added. The reaction mixture is then heated for 1 hour at 120ºC, the dark red solution is cooled and mixed with 100 ml of ether. The precipitated solid is filtered off. This is followed by one chromatographic purification on RP-Kieselgel EUROPREP 60-30 C18, 60A, 20-45 m (eluent: water/MeOH, step gradient from 0% to 70% MeOH). Fractions containing the product will first be freed from methanol in a rotary evaporator and subsequently lyophilized, yield: 1.5 g (58%), red lyophilizate.
Primjer 2: Example 2:
Sinteza 1,1`-bi(4-sulfobutil)indodikarbocijanina-5-karbonske kiseline, natrijeva sol (2) Synthesis of 1,1`-bi(4-sulfobutyl)indodicarbocyanine-5-carboxylic acid, sodium salt (2)
1,2 g (4,1 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-3H-indolenina i 1,0 g (3,9 mmol) malonaldehida-bi-fenilimin-hidroklorida biti će u 15 ml anhidrida octene kiseline 30 min, pri 120ºC miješano i tada u jednoj vodenoj kupelji do sobne temp. ohlađeno. Nastavno će biti jedno nakon drugog 1,4 g (4,2 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-5-karboksi-3H-indolenina, 1,2 g (14,6 mmol) suhog natrijacetata, 15 ml anhidrida octene kiseline i 6 ml octene kiseline dodano. Reakcijska mješavina se zagrijava 1 sat na 120ºC, a tada se plava otopina ohladi i sa 100 ml etera pomiješa. Dorada i pročišćavanje slijedi kao šro je opisano u primjeru 1, iskoristivost: 1,8 g (66%), plavi liofilizat. 1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.0 g (3.9 mmol) of malonaldehyde-bi-phenylimine-hydrochloride will be in 15 ml of acetic anhydride for 30 min, mixed at 120ºC and then in a water bath until room temp. chilled. Next will be one after the other 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride and 6 ml of acetic acid added. The reaction mixture is heated for 1 hour at 120ºC, and then the blue solution is cooled and mixed with 100 ml of ether. Work-up and purification follows as described in example 1, yield: 1.8 g (66%), blue lyophilisate.
Primjer 3: Example 3:
Sinteza 1,1`-bi(4-sulfobutil)indotrikarbocijanina-5-karbonske kiseline, natrijeva sol (3) Synthesis of 1,1`-bi(4-sulfobutyl)indotricarbocyanine-5-carboxylic acid, sodium salt (3)
1,2 g (4,1 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-3H-indolenina i 1,1 g (3,9 mmol) glutakonaldehid-dianilhidroklorida biti će u 15 ml anhidrida octene kiseline 30 min pri 120ºC miješano i tada u jednoj vodenoj kupelji na sobnu temp. ohlađeno. Nastavno se 1,4 g (4,2 mmol) 1-(4-sulfobutil)-2,3,3-trimetil-5-karboksi-3H-indolenina, 1,2 g (14,6 mmol) suhog natrijacetata, 15 ml anhidrida octene kiseline i 6 ml octene kiseline dodano. Reakcijska mješavina će se zagrijavati 1 sat na 120ºC, plava otopina ohladiti i sa 100 ml etera pomiješati. Obrada i pročišavanje slijedi kao u primjeru 1 opisano, iakoristivost: 1,8 g (60%), plavi liofilizat. 1.2 g (4.1 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-3H-indolenine and 1.1 g (3.9 mmol) of glutaconaldehyde-dianylhydrochloride will be in 15 ml of acetic anhydride acid for 30 min at 120ºC, mixed and then in a water bath at room temperature. chilled. Next, 1.4 g (4.2 mmol) of 1-(4-sulfobutyl)-2,3,3-trimethyl-5-carboxy-3H-indolenine, 1.2 g (14.6 mmol) of dry sodium acetate, 15 ml of acetic anhydride and 6 ml of acetic acid added. The reaction mixture will be heated for 1 hour at 120ºC, the blue solution will be cooled and mixed with 100 ml of ether. Processing and purification follows as described in example 1, yield: 1.8 g (60%), blue lyophilisate.
Strukture spojeva iz primjera 1-3 su u opisu 1 prikazane. The structures of compounds from examples 1-3 are shown in description 1.
Primjeri 4 do 6: Examples 4 to 6:
Sinteza indocijanin boja 4-6 iz 1-3 do sintetičkog povezivanja čvrste faze na amino skupine (N-terminus ili ε-lizin) peptida Synthesis of indocyanine dyes 4-6 from 1-3 to the synthetic connection of the solid phase to the amino groups (N-terminus or ε-lysine) of the peptide
Sinteza slijedi preko amidiranja boja 1-3 s β-alanin-t-butilestera i “kiselog” odcjepljenja grupe t-butilestera. The synthesis follows through amidation of dyes 1-3 with β-alanine-t-butylester and "acidic" cleavage of the t-butylester group.
Jedna otopina od 0,5 mmol boje 1-3 i 0,1 g (1,0 mmol) trietilamina u 20 ml dimetilformamida biti će na 0ºC s 0,5 mmol TBTU u 10 ml dimetilformamida pomiješano i 15 min pri 0ºC miješano. Nastavno će biti jedna otopina od 0,11 g (0,6 mmol) β-alanin-t-butilester-hidroklorida i 0,6 mmol trietilamina u 5 ml dimetilformamida dokapano te će reakcijska mješavina biti mješana 2 sata pri sobnoj temp. Nakon dodatka 100 ml dietiletera, istaložena čvrsta tvar će biti otfiltrirana, u 20 ml diklormetana otopljena, sa 10 ml trifluoroctene kiseline pomiješana i pri sobnoj temp. 24 sata miješana. Mješavina će biti u vakumu reducirana, a ostatak kao u primjeru 1 opisano, kromatografski pročišćen i liofiliziran: iskoristivost: 0,21 g (58%) 4, 0,29 g (76%) 5, 0,28 g (72%) 6. One solution of 0.5 mmol of dye 1-3 and 0.1 g (1.0 mmol) of triethylamine in 20 ml of dimethylformamide will be mixed at 0ºC with 0.5 mmol of TBTU in 10 ml of dimethylformamide and stirred for 15 min at 0ºC. Next, a solution of 0.11 g (0.6 mmol) of β-alanine-t-butyl ester hydrochloride and 0.6 mmol of triethylamine in 5 ml of dimethylformamide will be added dropwise, and the reaction mixture will be stirred for 2 hours at room temperature. After adding 100 ml of diethyl ether, the precipitated solid will be filtered, dissolved in 20 ml of dichloromethane, mixed with 10 ml of trifluoroacetic acid and at room temperature. 24 hours mixed. The mixture will be reduced in a vacuum, and the residue as described in example 1, chromatographically purified and lyophilized: utilization: 0.21 g (58%) 4, 0.29 g (76%) 5, 0.28 g (72%) 6.
Primjeri 7 do 9: Examples 7 to 9:
Sinteza indocijanin boja 7-9 iz 1-3 do sintetičkog povezivanja čvrste faze na amino skupine (N-terminus ili ε-lizin) peptida Synthesis of indocyanine dyes 7-9 from 1-3 to the synthetic connection of the solid phase to the amino groups (N-terminus or ε-lysine) of the peptide
Priprema slijedi analogno primjerima 4-6 zu primjenu 0,1 g (0,6 mmol) glicin-t-butilester-hidroklorida, iskoristivost: 0,25 g (68%) 4, 0,30 g (80%) 5, 0,32 g (83%) 6. The preparation follows analogously to examples 4-6 with the use of 0.1 g (0.6 mmol) glycine-t-butyl ester hydrochloride, utilization: 0.25 g (68%) 4, 0.30 g (80%) 5, 0 ,32 g (83%) 6.
Primjeri 10 do 12: Examples 10 to 12:
Sinteza indocijanin boja 10-12 iz 1-3 do sintetičkog povezivanja čvrste faze na peptide preko vezivanja na tiolne skupine cisteina Synthesis of indocyanine dyes 10-12 from 1-3 to synthetic coupling of the solid phase to peptides via binding to cysteine thiol groups
Sinteza slijedi preko amidiranja boja 1-3 s 3-aminopropanolom i nastavnim prevođenjem alkoholnih skupina u jedan bromid. The synthesis follows the amidation of dyes 1-3 with 3-aminopropanol and the conversion of alcohol groups into one bromide.
Jedna otopina iz 0,5 mmol boje 1-3 i 0,1 g (1,0 mmol) trietilamina u 20 ml dimetilformamida biti će pri 0ºC s 0,5 mmol TBTU u 10 ml pomiješana i 15 min na 0ºC miješana. Nastavno će biti jedna otopina od 850 mg (1,2 mmol) 3-aminopropanola i 1,2 mmol trietilamina u 5 ml dimetilformamida dokapano te će reakcijska mješavina biti mješana 6 sati pri sobnoj temp. Nakon dodatka 100 ml dietiletera, istaložena čvrsta tvar će biti otfiltrirana i kao u primjeru 1 opisano, kromatografski pročišćen i liofiliziran. One solution of 0.5 mmol of dye 1-3 and 0.1 g (1.0 mmol) of triethylamine in 20 ml of dimethylformamide will be mixed at 0ºC with 0.5 mmol of TBTU in 10 ml and stirred for 15 min at 0ºC. Next, a solution of 850 mg (1.2 mmol) of 3-aminopropanol and 1.2 mmol of triethylamine in 5 ml of dimethylformamide will be added dropwise, and the reaction mixture will be stirred for 6 hours at room temperature. After the addition of 100 ml of diethyl ether, the precipitated solid substance will be filtered and, as described in example 1, chromatographically purified and lyophilized.
Prevođenje do bromida 10-12 slijedi miješanjem 0,3 mmol međuprodukta sa 55 mg (0,5 mmol) N-bromsukcinida i 130 mg (0,5 mmol) trifenilfosfina u mješavini od 4 ml diklormetana i 4 ml dimetilformamida za 48 sati na 4ºC. Dodatkom 3 ml etera produkti se istalože, odfiltriraju i kao sirovina u povezivanje s peptidom dodaju. Conversion to bromide 10-12 follows by mixing 0.3 mmol of the intermediate with 55 mg (0.5 mmol) of N-bromosuccinide and 130 mg (0.5 mmol) of triphenylphosphine in a mixture of 4 ml of dichloromethane and 4 ml of dimethylformamide for 48 hours at 4ºC. . With the addition of 3 ml of ether, the products are precipitated, filtered and added as a raw material for connection with the peptide.
Primjeri 14-16 i 18-27: Examples 14-16 and 18-27:
Sinteza smole peptidnih konjugata iz VIP-receptor vezujućih peptida i boja 1-9 i 13. Synthesis of resin peptide conjugates from VIP-receptor binding peptides and dyes 1-9 and 13.
a) Sinteza čvrste faze: Na 50 µmola TentaGel-Sram-smole (Rapp Polymere, Tubingen) biti će peptidi prema Fmoc-strategiji, analogno Standard-Fmoc-Maschinen-Protokoll (Pept. Res., 36 (1990) 225) zu primjenu reagensa za povezivanje PyBOP (benzotriazol-1-iloksi-tris-pirolidionofosfonij heksafluorofosfat) i N-metilmorfina, sintetiziran. Slijedeće postrane zaštitne skupine se pri tom primjenjuju: tritil za Cys, His, Asn, Gln: t-butil za Asp, Glu, Ser i Thr; t-butiloksikarbonil za Lys i Trp te pentametilkloramsulfonil za Arg. a) Solid phase synthesis: On 50 µmol of TentaGel-Sram-resin (Rapp Polymere, Tubingen) there will be peptides according to the Fmoc-strategy, analogous to the Standard-Fmoc-Maschinen-Protokoll (Pept. Res., 36 (1990) 225) for application of reagents for linking PyBOP (benzotriazol-1-yloxy-tris-pyrrolidionophosphonium hexafluorophosphate) and N-methylmorphine, synthesized. The following secondary protective groups are used: trityl for Cys, His, Asn, Gln: t-butyl for Asp, Glu, Ser and Thr; t-butyloxycarbonyl for Lys and Trp and pentamethylchloramsulfonyl for Arg.
Vezivanje na ε-amino skupine jednog lizina (primjer 24) biti će preko ortogonalne tehnike zaštitnih skupina postignuto. Kao gradivni element lizina biti će korišten Fmoc-Lys(Dde)-OH, a peptid kao gore opisano sintetiziran. Nakon acetiliranja N-terminusa slijedi odcjepljenje Dde-zaštitne skupine selektivno, preko 3% hidracinhidrata. Binding to the ε-amino group of one lysine (example 24) will be achieved through the orthogonal technique of protecting groups. Fmoc-Lys(Dde)-OH will be used as the building block of lysine, and the peptide will be synthesized as described above. After the acetylation of the N-terminus, the removal of the Dde-protecting group is followed selectively, via 3% hydrazine hydrate.
b) Vezivanje boje: Na peptide koji su još vezani na čvrstu fazu biti će boje na N-terminus odnosno na lizin vezane. Za to će biti 75 µmola određene boje 1-9 i 13 (1,5 eq) u 600 µmola dimetilformamida otopljeno i sa 83 µmola TBTU (2-(1Hbenzotriazol-1-il)-1,1,3,3-tetrametiluronija-tetrafluorborata) kao i 150 µmola N,N-diizopropiletilamina pomiješano. Nakon 2 minute biti će ista reakcijska mješavina na pripadajuću smolu koja može nositi peptid, dodana i ostavljena preko noći u reakciji. Zaključno će biti smola 5x sa dimetilformamidom i 3x sa diklormetanom isprana i na zraku osušena. b) Binding of the dye: The peptides that are still attached to the solid phase will have the dye attached to the N-terminus or to the lysine. For this, 75 µmol of the specified dye 1-9 and 13 (1.5 eq) will be dissolved in 600 µmol of dimethylformamide and with 83 µmol of TBTU (2-(1Hbenzotriazol-1-yl)-1,1,3,3-tetramethyluronium- tetrafluoroborate) as well as 150 µmol of N,N-diisopropylethylamine mixed. After 2 minutes, the same reaction mixture will be added to the corresponding resin that can carry the peptide, and left overnight in the reaction. Finally, the resin will be washed 5x with dimethylformamide and 3x with dichloromethane and air-dried.
c) Odcjepljenje zaštitnih skupina i odvajanje konjugata boja-peptid: 1,5 ml mješavine koja se sastoji od 750 mg fenola, 250 µl etanditiola, 500 µl tioanizola i 500 µl vode u 10 ml trifluorne octene kiseline biti će na smolu koja može nositi peptidne konjugate dodano i ostavljeno da djeluje 4 sata. Konjugati boje i peptida biti će sa hladnim t-butilmetileterom istaloženi, 6x sa hladnim dietileterom isprani, u 5% octenoj kiselini otopljeni i osušeni suhim zamrzavanjem. Analiza čistoće i pročišćavanje konjugata slijedi s pomoću RP-HPLC (reverzno fazna visokotlačna tekuća kromatografija) na jednoj Vydac-C18-koloni (gradijent: voda +0,05% TFA/acetonitril, 5% na 60% acetonitrila u 20 min, detekcija: 214 i 750 nm.) c) Removal of protective groups and separation of the dye-peptide conjugate: 1.5 ml of a mixture consisting of 750 mg of phenol, 250 µl of ethanedithiol, 500 µl of thioanisole and 500 µl of water in 10 ml of trifluoroacetic acid will be on a resin that can carry peptide conjugate added and allowed to act for 4 hours. The dye and peptide conjugates will be precipitated with cold t-butyl methyl ether, washed 6 times with cold diethyl ether, dissolved in 5% acetic acid and dried by freeze-drying. Purity analysis and purification of the conjugate follows by means of RP-HPLC (reversed phase high-pressure liquid chromatography) on a Vydac-C18-column (gradient: water +0.05% TFA/acetonitrile, 5% to 60% acetonitrile in 20 min, detection: 214 and 750 nm.)
Strukture sintetiziranih konjugata boja i peptida su u slijedećem pregledu objedinjene: The structures of the synthesized conjugates of dyes and peptides are summarized in the following review:
Konjugati boje sa VIP-receptor-vezujućim peptidom I Dye conjugates with VIP-receptor-binding peptide I
Primjer 14 do 16: Example 14 to 16:
konjugati boja 1-3 sa VIP (1-28) color conjugates 1-3 with VIP (1-28)
[image] [image]
n = 1: indokarbocijanin-VIP(1-28)-konjugat 14 n = 1: indocarbocyanine-VIP(1-28)-conjugate 14
n = 2: indodikarbocijanin-VIP(1-28)-konjugat 15 n = 2: indodicarbocyanine-VIP(1-28)-conjugate 15
n = 3: indotrikarbocijanin-VIP(1-28)-konjugat 16 n = 3: indotricarbocyanine-VIP(1-28)-conjugate 16
Primjer 17: Example 17:
Konjugat boje 12 sa Cys17-VIP(1-28) Conjugate of dye 12 with Cys17-VIP(1-28)
[image] [image]
Indotrikarbocijanin-Cys17-VIP(1-28)-konjugat 17 Indotricarbocyanine-Cys17-VIP(1-28)-conjugate 17
Primjer 18 do 20: Example 18 to 20:
Konjugati boje 1-3 sa VIP(14-28) Conjugate colors 1-3 with VIP(14-28)
[image] [image]
n = 1: indokarbocijanin-VIP(14-28)-konjugat 18 n = 1: indocarbocyanine-VIP(14-28)-conjugate 18
n = 2: indodikarbocijanin-VIP(14-28)-konjugat 19 n = 2: indodicarbocyanine-VIP(14-28)-conjugate 19
n = 3: indotrikarbocijanin-VIP(14-28)-konjugat 20 n = 3: indotricarbocyanine-VIP(14-28)-conjugate 20
Primjer 21 do 23: Example 21 to 23:
Konjugati boje 4-6 sa VIP(14-24) Conjugate colors 4-6 with VIP(14-24)
[image] [image]
n = 1: indokarbocijanin-β-alanin-VIP(14-28)-konjugat 21 n = 1: indocarbocyanine-β-alanine-VIP(14-28)-conjugate 21
n = 2: indodikarbocijanin-β-alanin-VIP(14-28)-konjugat 22 n = 2: indodicarbocyanine-β-alanine-VIP(14-28)-conjugate 22
n = 3: indotrikarbocijanin-β-alanin-VIP(14-28)-konjugat 23 n = 3: indotricarbocyanine-β-alanine-VIP(14-28)-conjugate 23
Primjer 17: Example 17:
Sinteza smole peptidnih konjugata koji se sastoje od VIP-receptor vezujućih peptida i boja 10-12. Synthesis of resin peptide conjugates consisting of VIP-receptor binding peptides and dyes 10-12.
Da bi se boje 10-12, koje nose brom, kemoselektivno preko tioeterne veze vezale na peptid, mora u peptid jedan cistein ili homocistein sa ortogonalnom zaštitnom skupinom biti ugrađen. Sinteza čvrste faze peptida biti će kao u primjerima 14-16/18-27 opisano, provedena i gradivi element Fmoc-cys(Mmt)-OH primješan. Monometoksitritil skupina (Mmt) je zu dobitak uvriježenih postranih zaštitnih skupina, preko 1% TFA/5% triizobutilsilan u diklormetan, moguće odcijepiti. Za to će smola 3x sa po 1 ml gornje otopine , 10 min, biti inkubirana. Nakon ispiranja smole sa diklormetanom (3x), DMF (5x) i etanol (3x) biti će smola 2 min s jednom 20%-om otopinom cezijkarbonata (1 ml) inkubirana i zaključno sa vodom (2x), etanolom (2x) i DMF-om (2x) isprana. Zaključno će biti 75 µmola određene boje 10-12 (1,5 eq) u 600 µl dimetilformamida otopljeno i na određenu smolu dodano, a postupak će biti ponovljen nakon 30 min. Zaključno će smola 5x sa dimetilformamidom te sa diklormetanom (2x) isprana. Nakon sušenja smole na zraku, slijedi odcjepljivanje zaštitnih skupina i odvajanje od “nosača”, kao što je gore opisano. In order for dyes 10-12, which carry bromine, to chemoselectively bind to the peptide through a thioether bond, one cysteine or homocysteine with an orthogonal protective group must be incorporated into the peptide. The synthesis of the solid phase of the peptide will be carried out as described in examples 14-16/18-27, and the building element Fmoc-cys(Mmt)-OH will be added. The monomethoxytrityl group (Mmt) can be cleaved off via 1% TFA/5% triisobutylsilane in dichloromethane as a result of the established side protective groups. For this, the resin will be incubated 3 times with 1 ml of the above solution for 10 minutes. After washing the resin with dichloromethane (3x), DMF (5x) and ethanol (3x), the resin will be incubated for 2 min with a 20% solution of cesium carbonate (1 ml) and finally with water (2x), ethanol (2x) and DMF - (2x) washed. In conclusion, 75 µmol of a specific color 10-12 (1.5 eq) will be dissolved in 600 µl of dimethylformamide and added to the specific resin, and the procedure will be repeated after 30 min. Finally, the resin will be washed 5x with dimethylformamide and with dichloromethane (2x). After drying the resin in air, the protective groups are removed and separated from the "support", as described above.
Strukture daljnjih sintetiziranih konjugata peptida i boja su u slijedećem pregledu sažete: The structures of further synthesized conjugates of peptides and dyes are summarized in the following review:
Konjugati boja sa VIP-receptor-vezujućim peptidima II Dye conjugates with VIP-receptor-binding peptides II
Primjer 24: Example 24:
Konjugat boje 6 sa Lys25-VIP(14-25) Conjugate of dye 6 with Lys25-VIP(14-25)
[image] [image]
(ε-amino-indotrikarbocijanin)-Lys25-VIP(14-25)-konjugat 24 (ε-amino-indotricarbocyanine)-Lys25-VIP(14-25)-conjugate 24
Primjer 25: Example 25:
Konjugat boje 3 sa D-VIP(14-24) Color 3 conjugate with D-VIP(14-24)
[image] [image]
Indotrikarbocijanin-D-VIP(14-24)-konjugat 25 Indotricarbocyanine-D-VIP(14-24)-conjugate 25
Primjer 26: Example 26:
Konjugat boje 13 sa D-VIP(14-24) Conjugate of color 13 with D-VIP(14-24)
[image] [image]
Indotrikarbocijanin-5-glukamid karbonske kiseline-D-VIP(14-24)-konjugat 26 Indotricarbocyanin-5-glucamide of carboxylic acid-D-VIP(14-24)-conjugate 26
Primjer 27: Example 27:
Konjugat boje 13 sa retro-D-VIP(14-24) Conjugate of color 13 with retro-D-VIP(14-24)
[image] [image]
Indotrikarbocijanin-5-glukamid karbonske kiseline-retro-D-VIP(14-24)-konjugat 27 Indotricarbocyanine-5-glucamide of carboxylic acid-retro-D-VIP(14-24)-conjugate 27
Primjer 28 do 32: Example 28 to 32:
Sinteza smole konjugata peptida koja se sastoji od somatostatinvezujućih peptida i boja 1-9 i 13 Synthesis of peptide conjugate resin consisting of somatostatin-binding peptides and dyes 1-9 and 13
Opće odredbe: General provisions:
Sinteza će biti provedena na 500 mg TCP-Thr(But)-fmoc-smole (firma Pepchem Tubingen) sa jednim dopunjavanjem (specifičnim skupinama ) od 0,49 mmol/g. Peptid će biti “korak po korak” zu primjenu slijedećih temporarnih zaštitnih skupina sintetiziran: terc.-butil za Thr i Ser, tritil za Cys i Asp, Boc za trp i Lys. Kao reagens za kondenzaciju biti će korišten HBTU. The synthesis will be carried out on 500 mg of TCP-Thr(But)-fmoc-resin (Pepchem Tubingen) with one addition (specific groups) of 0.49 mmol/g. The peptide will be synthesized "step by step" using the following temporary protecting groups: tert.-butyl for Thr and Ser, trityl for Cys and Asp, Boc for trp and Lys. HBTU will be used as the condensation reagent.
Nakon odcjepljenja N-terminusa Fmoc-zaštitne skupine boja 1-9, 13 će biti u jednom posebnom koraku sinteze kondenzirana. Za to će biti 255 mg smole u ca. 2 ml DMF suspendirano i sa 0,5 mmol boje, 0,5 mmol HBTU i 0,17 ml DIEA pomiješano. Smjesa će biti narednih 18 sati na sobnoj temp. miješana, smola će nakon toka biti odstranjena, ostatak sa diklormetanom ispran i osušen. Odcjepljenje konjugata boja-peptid od smole slijedi sa 95% trikloroctenom kiselinom, zu dodatak triizopropilsilana i nastavnim liofiliziranjem iz 10% octene kiseline. Sirovi proizvod biti će pomoću aktivnog ugljena cikliziran i kromatografski pročišćen (50x300 mm VYDAC RP-18, gradijent: voda/acetonitril). After the separation of the N-terminus of the Fmoc-protecting group of dyes 1-9, 13 will be condensed in a special synthesis step. For that there will be 255 mg of resin in ca. 2 ml DMF suspended and with 0.5 mmol dye, 0.5 mmol HBTU and 0.17 ml DIEA mixed. The mixture will be at room temperature for the next 18 hours. mixed, the resin will be removed after the flow, the residue washed with dichloromethane and dried. Separation of the dye-peptide conjugate from the resin is followed by 95% trichloroacetic acid, with the addition of triisopropylsilane, and subsequent lyophilization from 10% acetic acid. The crude product will be cyclized using activated carbon and chromatographically purified (50x300 mm VYDAC RP-18, gradient: water/acetonitrile).
Strukture sintetiziranih konjugata boja-peptid su u slijedećem pregledu sažete: The structures of synthesized dye-peptide conjugates are summarized in the following review:
Konjugati boja sa somatostatin vezujućim peptidima Dye conjugates with somatostatin binding peptides
Primjeri 28 do 30: Examples 28 to 30:
Konjugat boje 1-3 sa pentetreotidom Conjugate dye 1-3 with pentetreotide
[image] [image]
n = 1: Indokarbocijanin-pentetreotid 28 n = 1: Indocarbocyanine-pentetreotide 28
n = 2: Indodikarbocijanin-pentetreotid 29 n = 2: Indodicarbocyanine-pentetreotide 29
n = 3: Indotrikarbocijanin-pentetreotid 30 n = 3: Indotricarbocyanine-pentetreotide 30
Primjer 31: Example 31:
Konjugat boje 3 sa somatostatinom-14 Dye 3 conjugate with somatostatin-14
[image] [image]
Indotrikarbocijanin-somatostatin-14-konjugat 31 Indotricarbocyanin-somatostatin-14-conjugate 31
Primjer 32: Example 32:
Konjugat boje 13 sa somatostatinom-14 Conjugate of dye 13 with somatostatin-14
[image] [image]
Indotrikarbocijanin-5-glutamid karbonske kiseline-somatostatin-14-konjugat 32 Carbonic acid indotricarbocyanin-5-glutamide-somatostatin-14-conjugate 32
Primjeri 33 do 38: Examples 33 to 38:
Sinteza konjugata peptida iz neurotenzin-peptida i boje 3 Synthesis of peptide conjugates from neurotensin-peptide and dye 3
Sinteza supstanci slijedi analogno kao što je opisano za primjere 14-27, opći protokol. Synthesis of substances follows analogously as described for Examples 14-27, general protocol.
Strukture sintetiziranih konjugata peptida i boja su u slijedećem pregledu sažete: The structures of synthesized conjugates of peptides and dyes are summarized in the following review:
Konjugati boja sa neurotenzin vezujućim peptidima Dye conjugates with neurotensin binding peptides
Primjeri 33 do 35: Examples 33 to 35:
Konjugat boja 7-9 sa D-Tyr11-neurotenzinom (7-13) Conjugate dye 7-9 with D-Tyr11-neurotensin (7-13)
[image] [image]
n = 1: Indokarbocijanin-D-Tyr11-neurotenzin (7-13)-konjugat 33 n = 1: Indocarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 33
n = 2: Indodikarbocijanin-D-Tyr11-neurotenzin (7-13)-konjugat 34 n = 2: Indodicarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 34
n = 3: Indotrikarbocijanin-D-Tyr11-neurotenzin (7-13)-konjugat 35 n = 3: Indotricarbocyanine-D-Tyr11-neurotensin (7-13)-conjugate 35
Primjer 36: Example 36:
Konjugat boje 2 sa D-Tyr11-neurotenzinom Conjugate of dye 2 with D-Tyr11-neurotensin
[image] [image]
(ε-amino-Lys6-indodi-karbocijanin)-D-Tyr11-neurotenzin-konjugat 36 (ε-amino-Lys6-indodi-carbocyanine)-D-Tyr11-neurotensin-conjugate 36
Primjer 37 do 38: Example 37 to 38:
Konjugat boje 10-11 sa D-Tyr11-neurotenzin (5-13) - Cys Conjugate of color 10-11 with D-Tyr11-neurotensin (5-13) - Cys
[image] [image]
n = 1: Indokarbocijanin-D-Tyr11-neurotenzin (5-13)-Cys-konjugat 37 n = 1: Indocarbocyanin-D-Tyr11-neurotensin (5-13)-Cys-conjugate 37
n = 2: Indodikarbocijanin-D-Tyr11-neurotenzin (5-13)-Cys-konjugat 38 n = 2: Indodicarbocyanine-D-Tyr11-neurotensin (5-13)-Cys-conjugate 38
Primjer 39: Example 39:
Svojstva apsorpcije i fluorescencije sintetiziranih konjugata boja i peptida Absorption and fluorescence properties of synthesized dye and peptide conjugates
Apsorpcijski maksimumi i keoficijenti specifičnih valnih dužina karakterističnih za navedene spojeve (Extinktionskoeffizient) bili su određivani u PBS-i i plazmi goveda (uređaj Perkin Elmer Lambda 2). Fluorescencijski emisijski spektar bio je nakon poticaja na kratkovalnoj strani (ca. 40 nm od apsorpcijskog maksimuma) dobiven (SPEX Fluorolog, R928 PMT) Absorption maxima and coefficients of specific wavelengths characteristic of the mentioned compounds (Extinktionskoeffizient) were determined in PBS and bovine plasma (Perkin Elmer Lambda 2 device). The fluorescence emission spectrum was obtained after excitation on the short-wave side (ca. 40 nm from the absorption maximum) (SPEX Fluorolog, R928 PMT)
Apsorpcijski i podaci o fluorescenciji su u tabeli 4 objedinjeni. U tabeli 5 su tipični apsorpcijski i fluorescentno-emisijski spektri na primjerima pokazani. Absorption and fluorescence data are combined in Table 4. Table 5 shows typical absorption and fluorescence-emission spectra on examples.
Primjer 40: Example 40:
Utvrđivanje snimaka stanice pomoću fluorescentne mikroskopije Determination of cell images using fluorescence microscopy
Vezivanje i preuzimanje (apsorpcija u tkiva) izumom predočenih spojeva bili su in vitro na humanim tumorskim stanicama ispitivani, koje receptore za vazoaktivni intestinalni peptid i/ili somatostatin i/ili neurotenzin eksprimiraju. The binding and uptake (absorption into tissues) of the compounds presented by the invention were investigated in vitro on human tumor cells, which receptors for vasoactive intestinal peptide and/or somatostatin and/or neurotensin are expressed.
Za to je 5x105 tumorskih stanica u 1,5 ml medija inkubirano, koji sadrži test supstance. Za to su različite koncentracije test supstanci (10 nM - 10 µM) korištene, vrijeme inkubacije je varirano (1 min – 24 sata). Nakon inkubacije, stanice su bile fiksirane i pripremljeni mikroskopski preparati. Pregled je uslijedio na jednom Axiovert 135-fluorescentnom mikroskopu, koji je bio opremljen sa jednim Cy 7-(Exciter HQ 710/70nm, Emitter 810/90nm, Beamsplitter (razpršivač snopa zraka) 750nm LP), Cy5 -(Exciter 575-625 nm, Emitter 660-710nm BP, Beamsplitter (razpršivač snopa zraka) 645nm) i Cy3- skupinom filtera (Exciter 546/12 nm, Emitter 590 nm LP, Beamsplitter (razpršivač snopa zraka) 580 nm). Od svih preparata bile su snimljene fotografije pomoću bijelog izvora svijetla i fluorescencije, s pomoću jedne CCD-kamere (Visitron RTE/CCD – 576), i digitalno pohranjene. For this, 5x105 tumor cells were incubated in 1.5 ml of medium containing the test substance. For this, different concentrations of test substances (10 nM - 10 µM) were used, the incubation time was varied (1 min - 24 hours). After incubation, the cells were fixed and microscopic preparations were prepared. Examination was performed on an Axiovert 135 fluorescence microscope, which was equipped with one Cy 7-(Exciter HQ 710/70nm, Emitter 810/90nm, Beamsplitter 750nm LP), Cy5-(Exciter 575-625 nm , Emitter 660-710nm BP, Beamsplitter 645nm) and Cy3- filter group (Exciter 546/12 nm, Emitter 590 nm LP, Beamsplitter 580 nm). Photographs of all preparations were taken using a white light source and fluorescence, using one CCD-camera (Visitron RTE/CCD – 576), and digitally stored.
Izdvojeni rezultati su u slijedećem opisani: The selected results are described below:
Napravljene su mikroskopske snimke sa bijelim svijetlom i fluorescentnim izvorom (Cy7-filteri) stanica HT29, nakon 30 minutne inkubacije s 10 µM Indotrikarbocijanin-VIP(1-28)-konjugatom 16. Na “fluorescentnim” smimkama su nadalje homogena, preko stanica raspodjeljena fluorescencija detektirana. Dodatno su vidljivi areali s povećanim intenzitetom signala, koji mogu asocirati sa vezikularnim kompartimentima. Stanice na “fluorescentnim” snimkama koreliraju po svom prostornom rasporedu sa onima snimljenim s pomoću izvora bijele svijetlosti. Microscopic images were taken with white light and a fluorescent source (Cy7-filters) of HT29 cells, after 30 minutes of incubation with 10 µM Indotricarbocyanine-VIP(1-28)-conjugate 16. On the "fluorescent" images, fluorescence was further homogeneously distributed over the cells. detected. Additionally, areas with increased signal intensity are visible, which may be associated with vesicular compartments. The cells on the "fluorescent" images correlate in their spatial arrangement with those recorded using a white light source.
Sa slijedećim spojevima su “bijele” i ”fluorescentne” snimke (Cy7-filteri) u analognom slijedu dobivene: With the following compounds, "white" and "fluorescent" recordings (Cy7-filters) were obtained in an analog sequence:
Indotrikarbocijanin-VIP(14-28)-konjugat 20, 10 µM, HT29-stanice. Homogeno preko stanica rasprostranjena fluorescencija, dobra korelacija sa “bijelim”-slikama. Indotricarbocyanine-VIP(14-28)-conjugate 20, 10 µM, HT29-cells. Homogeneously distributed fluorescence over the cells, good correlation with "white" images.
Indotrikarbocijanin-retro-D-VIP(24-14) 25, 10 µM, HT29-stanice. Homogeno preko stanica rasprostranjena fluorescencija, dobra korelacija sa “bijelim”-slikama. Indotricarbocyanine-retro-D-VIP(24-14) 25, 10 µM, HT29-cells. Homogeneously distributed fluorescence over the cells, good correlation with "white" images.
Indotrikarbocijanin-pentetreotid-konjugat 30, 10 µM, RIN38-VIP1-stanice. Fluorescentni areali sa vezikularnim uzorkom u području stanične membrane, dobra korelacija sa “bijelim”-slikama. Indotricarbocyanine-pentetreotide-conjugate 30, 10 µM, RIN38-VIP1-cells. Fluorescent areas with a vesicular pattern in the area of the cell membrane, good correlation with "white" images.
Nadalje su fluorescentni snimci u analognoj izvedbi sa slijedećim spojevima dobiveni: Furthermore, fluorescent images were obtained in an analog version with the following compounds:
(Amino-indotrikarbocijanin-Lys25-VIP(14-25)-konjugat 24, 10 µM, RIN38-VIP1-stanica, Cy7-filteri. Fluorescentna slika pokazuje jednu homogenu, intracelularnu fluorescenciju, u blizini stanične jezgre. (Amino-indotricarbocyanine-Lys25-VIP(14-25)-conjugate 24, 10 µM, RIN38-VIP1-cell, Cy7-filters. The fluorescence image shows one homogeneous, intracellular fluorescence, near the cell nucleus.
Indokarbocijanin-VIP(1-28)-konjugat 15, 10 µM, RIN38-VIP1-stanica, Cy5-filteri. Fluorescentna slika pokazuje intracelularne fluorescentne areale sa vezikularnim uzorkom u području stanične membrane. Indocarbocyanine-VIP(1-28)-conjugate 15, 10 µM, RIN38-VIP1-cell, Cy5-filters. The fluorescent image shows intracellular fluorescent areas with a vesicular pattern in the area of the cell membrane.
Indokarbocijanin-VIP(1-28)-konjugat 14, 10 µM, RIN38-VIP1-stanica, Cy3-filteri. Fluorescentna slika pokazuje intracelularne fluorescentne areale sa vezikularnim uzorkom u području stanične membrane. Indocarbocyanine-VIP(1-28)-conjugate 14, 10 µM, RIN38-VIP1-cell, Cy3-filters. The fluorescent image shows intracellular fluorescent areas with a vesicular pattern in the area of the cell membrane.
Primjer 41: Example 41:
Ispitivanje akumuliranja tumora pomoću in-vivo-fluorescentnih fotografija na miševima oboljelim od tumora Examination of tumor accumulation using in-vivo-fluorescence photographs in tumor-bearing mice
Svojstva koja daju sliku o izumom predočenim spojevima bila su ispitana in-vivo, nakon injekcije miševima (bez dlake), nositeljima tumora. Za to je 0,1 µmol/kg do 2 µmol/kg supstance intravenozno aplicirano, te je nakupljanje u području tumora u vremenskom intervalu od 0 do 48 sati praćeno. Fluorescencija supstance bila je preko ozračivanja životinja s blizim infracrvenim svijetlom valne dužine 640 nm (indodikarbocijanini), odnosno 740 nm (indotrikarbocijanini), koje je s pomoću jednog Nd:YAG lasera proizvedeno, izazvana. Fluorescentno isijavanje je kod valne dužine od >700 nm, odnosno >800 nm, preko jedne intenzivirane CCD-kamere, detektirano, a fluorescentne snimke su digitalno pohranjene. The properties that give an image of the compounds presented by the invention were tested in-vivo, after injection in mice (hairless), tumor carriers. For this, 0.1 µmol/kg to 2 µmol/kg of the substance was administered intravenously, and the accumulation in the tumor area was monitored in the time interval from 0 to 48 hours. The fluorescence of the substance was induced by irradiating the animals with near-infrared light of wavelength 640 nm (indodicarbocyanins) or 740 nm (indotricarbocyanins), which was produced using a Nd:YAG laser. Fluorescent emission at a wavelength of >700 nm or >800 nm was detected by an intensified CCD-camera, and the fluorescent images were digitally stored.
Izdvojene rezultati su u slijedećem opisani: The selected results are described below:
Od jednog “golog” miša, nositelja tumorskog oboljenja (HT29-tumor u desnoj zadnjoj slabini) bile su fluorescentne snimke cijelog tijela prije i 1 sat nakon aplikacije 0,1 µmol/kg indotrikarbocijanin-VIP(14-28)-konjugata 20, snimljene. Intenzitet fluorescencije prije aplikacije je zanemariv (slaba autofluorescencija). 1 sat nakon aplikacije rezultiralo je jednim 2-strukim intenzitetom signala u tumoru, relativno prema kontralateralnoj slabini, kod inače homogene preko ostatka tijela zaspodjeljene fluorescentne emisije. Fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg indotricarbocyanine-VIP(14-28)-conjugate 20 were taken from one "naked" mouse, the carrier of a tumor disease (HT29-tumor in the right hind loin) . Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 2-fold signal intensity in the tumor, relative to the contralateral loin, in an otherwise homogeneous distributed fluorescence emission over the rest of the body.
Od jednog miša nositelja tumorskog oboljenja (RIN38-SSTR2-tumor u desnoj stražnjoj slabini) bile su fluorescentne snimke cijelog tijela prije i 1 sat nakon aplikacije 0,1 µmol/kg indotrikarbocijanin-pentetreotid-konjugata 30, snimljene. Intenzitet fluorescencije prije aplikacije je zanemariv (slaba autofluorescencija). 1 sat nakon aplikacije rezultiralo je jednim 3-strukim intenzitetom signala u tumoru, relativno prema kontralateralnoj slabini, kod inače homogene preko ostatka tijela zaspodjeljene fluorescentne emisije. Fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg indotricarbocyanine-pentetreotide-conjugate 30 were taken from one tumor-bearing mouse (RIN38-SSTR2-tumor in the right posterior loin). Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 3-fold signal intensity in the tumor, relative to the contralateral loin, in an otherwise homogeneous distributed fluorescence emission over the rest of the body.
Od jednog miša nositelja tumorskog oboljenja (HT29-tumor u desnoj stražnjoj slabini) bile su fluorescentne snimke cijelog tijela prije i 1 sat nakon aplikacije 0,1 µmol/kg (ε-amino-indotrikarbocijanin)-Lys25-VIP(14-25)-konjugata 25, snimljene. Intenzitet fluorescencije prije aplikacije je zanemariv (slaba autofluorescencija). 1 sat nakon aplikacije rezultiralo je jednim 1,5-strukim intenzitetom signala u tumoru, relativno prema kontralateralnoj slabini.Dodatno je jedan povišeni fluorescentni signal u bubrezima detektiran. From one mouse bearing a tumor disease (HT29-tumor in the right back loin) there were fluorescence images of the whole body before and 1 hour after the application of 0.1 µmol/kg (ε-amino-indotricarbocyanine)-Lys25-VIP(14-25)- of conjugate 25, recorded. Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after the application resulted in a 1.5-fold signal intensity in the tumor, relative to the contralateral loin. Additionally, an increased fluorescent signal was detected in the kidneys.
Od jednog miša nositelja tumorskog oboljenja (HT29-tumor u desnoj stražnjoj slabini) bile su fluorescentne snimke cijelog tijela prije i 5 min nakon aplikacije 0,2 µmol/kg indodikarbocijanin)-VIP(1-28)-konjugata 15, snimljene. Intenzitet fluorescencije prije aplikacije je zanemariv (slaba autofluorescencija). 1 sat nakon aplikacije rezultiralo je jednim 1,4-strukim intenzitetom signala u tumoru, prema kontralateralnoj slabini.Dodatno je jedan povišeni fluorescentni signal u bubrezima detektiran. Fluorescence images of the whole body before and 5 min after the application of 0.2 µmol/kg indodicarbocyanine)-VIP(1-28)-conjugate 15 were recorded from one mouse bearing a tumor disease (HT29-tumor in the right posterior loin). Fluorescence intensity before application is negligible (weak autofluorescence). 1 hour after application resulted in a 1.4-fold signal intensity in the tumor, towards the contralateral flank. In addition, an increased fluorescent signal was detected in the kidneys.
Primjer 42: Example 42:
Istraživanje stabilnosti konjugata peptida i boja u plazmi goveda Investigation of the stability of peptide and dye conjugates in bovine plasma
Kemijska stabilnost izumom predočenih spojeva u plazmi bio je in vitro, u ovisnosti o vremenu, pomoću HPLC, ispitivan. Za ti je bilo 1mM otopine peptida u PBS-u u plazmi goveda (firma Graeber, zamrznuta, za analizu heparina), koncentracije 30µM pipetirano, a otopina je pri 37ºC inkubirana. The chemical stability of the compounds presented by the invention in plasma was investigated in vitro, depending on the time, by means of HPLC. For this, a 1mM peptide solution in PBS was added to bovine plasma (Graeber company, frozen, for heparin analysis), a concentration of 30µM was pipetted, and the solution was incubated at 37ºC.
U različitim vremenskim intervalima (0,5; 1; 2; 4; 6; 24 sata) uslijedila je prerada proba, pri kojoj je 1 ml otopine plazme pomiješan sa 1 ml MeOH, a istaložene bjelančevine su bile otcentrifugirane. At different time intervals (0.5; 1; 2; 4; 6; 24 hours) the samples were processed, during which 1 ml of the plasma solution was mixed with 1 ml of MeOH, and the precipitated proteins were centrifuged.
Analiza preostalog tekućeg dijela uslijedila je pomoću HPLC-a, preko određivanja količine pri 750 nm, koji se odnosi na sadržaj nakon 1 minute inkubacije pri 0ºC (kontrolna proba). The analysis of the remaining liquid part followed by means of HPLC, through the determination of the amount at 750 nm, which refers to the content after 1 minute of incubation at 0ºC (control test).
HPLC: Beckmann, diodno”array”-detektor TIDAS (firma J&M) 350-1000 nm; HPLC: Beckmann, diode array detector TIDAS (J&M company) 350-1000 nm;
Kolona: Chromasil 5µ, 250 mm x 4,5 mm Column: Chromasil 5µ, 250 mm x 4.5 mm
Eluent: A: 90% H2O(+0,5 % TFA) / 10% MeOH Eluent: A: 90% H2O(+0.5% TFA) / 10% MeOH
B: 10% H2O(+0,5 % TFA) / 90% MeOH B: 10% H2O(+0.5% TFA) / 90% MeOH
Gradijent: 10% B do 100% B u vremenu od 20 min Gradient: 10% B to 100% B in 20 min
Primjeri su u tabeli 6 sažeti. Examples are summarized in Table 6.
Primjer 43: Example 43:
Supstitucijska analiza VIP-a pomiću “spot”-sinteze Substitution analysis of VIP moves "spot" syntheses
1. Sinteza peptida na celulozi 1. Peptide synthesis on cellulose
Sinteza peptida na celulozi (spot-sinteza) bila je najprije 1988. Od R. Franka i R. Doringa objavljena, a 1992. Detaljno od R. Franka opisana. Ovdje je bila u AG Schneider-Mergener etablirana metoda, upotrijebljena. Peptide synthesis on cellulose (spot-synthesis) was first published in 1988 by R. Frank and R. Doring, and described in detail by R. Frank in 1992. Here was the method established at AG Schneider-Mergener, used.
Celulozna membrana bila je kemijski modificirana, da bi prikladne funkcije ankera za slijedeću sintezu peptida bila pripremljena. Pri tom je na jednu amino-funkcionaliziranu celuloznu membranu (CAPE-membran) jedna merkapto-funkcija uvedena. Na tu merkaptofunkciju mogla se prva aminokiselina u obliku jednog brompropilestera nadovezati. Nastavno su sve aminokiseline peptida sukcesivno prema Fmoc-strategiji nadograđene. Na kraju je indodikarbojanin-boja N-terminusa na peptid vezana i nastavno su sve postrane zaštitne skupine bile odcijepljene. The cellulose membrane was chemically modified in order to prepare suitable anchor functions for the subsequent peptide synthesis. In doing so, one mercapto-function was introduced on one amino-functionalized cellulose membrane (CAPE-membrane). The first amino acid in the form of a bromopropyl ester could be attached to this mercaptofunction. Subsequently, all amino acids of the peptide were successively upgraded according to the Fmoc strategy. At the end, the indodicarboyanin-dye of the N-terminus was attached to the peptide, and then all side protective groups were cleaved off.
Da bi se peptidi mogli testirati kao receptorni spojevi, peptid je morao biti odcijepljen od celuloze. Za to je razvijena jedna metoda, kod koje je prvi puta uspijelo, peptide sa autentičnim C-terminusom od celuloze odcijepiti. In order for the peptides to be tested as receptor compounds, the peptide had to be cleaved from the cellulose. For this, a method was developed, which for the first time succeeded in cleaving peptides with an authentic C-terminus from cellulose.
1a. Modifikacija celulozne membrane 1a. Cellulose membrane modification
Jedna 20x30 cm velika celulozna membrana (Whatman 50) bila je na 2 min. s metanolom/1,2 % perkloroctenom kiselinom inkubirana i nastavno osušena. One 20x30 cm cellulose membrane (Whatman 50) was on 2 min. incubated with methanol/1.2% perchloroacetic acid and further dried.
Nakon 3-satne inkubacije sa 10% epibromhidrinom u dioxanu/1,2% perklornoj kiselini ostavljena je 30 min. u reakciji sa metanolom, a nakon toga je 2x isprana metanolom. After a 3-hour incubation with 10% epibromohydrin in dioxane/1.2% perchloric acid, it was left for 30 min. in the reaction with methanol, and after that it was washed twice with methanol.
Nastavno je 3x sa dimetilformamidom (DMF) isprana i preko noći sa 50% 1,3-dijaminopropanom (v/v) u DMF-u inkubirana. Nakon toga je na slijedeći način isprana: 3xDMF, 2x etanol, 2x aqua dest., 2x etanol 15 min sa 5M natrijmetanolatom, 3x matanolom, 4x aqua dest., 3x etanol, 1x dietileter. It was subsequently washed 3x with dimethylformamide (DMF) and incubated overnight with 50% 1,3-diaminopropane (v/v) in DMF. After that, it was washed as follows: 3x DMF, 2x ethanol, 2x aqua dest., 2x ethanol for 15 min with 5M sodium methanolate, 3x methanol, 4x aqua dest., 3x ethanol, 1x diethyl ether.
1b. Definicija “spots”-a 1b. Definition of "spots".
Za definiranje spotsa bilo je 1,3 µl 0,6 M Fmoc-ß-alanin-Opfp-otopine sa auto-spot robotom 222 XL (Abimed, Langenfeld) pri reakcijskom vremenu od 15 min na određene točke celulozne membrane dvostruku pipetirano. To define the spots, 1.3 µl of 0.6 M Fmoc-ß-alanine-Opfp-solution was double-pipetted with an auto-spot robot 222 XL (Abimed, Langenfeld) at a reaction time of 15 min to certain points of the cellulose membrane.
Membrana je 2 minute sa 2% acetanhidrid otopinom i 30 min sa 20% acetanhidridom/10% diizopropiletilaminom acetilirana. The membrane was acetylated for 2 minutes with 2% acetic anhydride solution and 30 minutes with 20% acetic anhydride/10% diisopropylethylamine.
Za razdvajanje Fmoc-zaštitnih skupina bila je zatim membrana 3x sa DMF-om isprana, 2x 10 min. sa 20% piperidin otopinom inkubirana, 5x sa DMF-om i 1x sa etanolom isprana. Slobodne aminoskupine mogle su s pomoću bromfenoli plavetnilom biti vidljive. Nakon ponovljenog pranja sa etanolom, membrana je bila osušena. To separate the Fmoc-protecting groups, the membrane was then washed 3x with DMF, 2x for 10 min. incubated with 20% piperidine solution, washed 5x with DMF and 1x with ethanol. Free amino groups could be visualized with bromphenol blue. After repeated washing with ethanol, the membrane was dried.
1c. Vezivanje Mmt-merkaptopropionske kiseline i brompropil estera 1c. Binding of Mmt-mercaptopropionic acid and bromopropyl ester
0,6 M merkaptopropionske kiseline bilo je u reakcijiakom vremenu od 15 min. dvostruko na definirani spot pipetirano. Nastavno je bila 3x sa DMF-om i 3x sa diklormetanom (DCM) isprana. 0.6 M mercaptopropionic acid was in the reaction time of 15 min. double on the defined spot pipetted. It was then washed 3 times with DMF and 3 times with dichloromethane (DCM).
Odcjepljenje Mmt-zaštitnih skupina uslijedilo je inkubacijom od 2 min. sa 10% dikloroctenom kiselinom/0,5 % trifluoroctenom kiselinom i 3x5 min. sa 10% dikloroctenom kiselinom/0,5 % trifluoroctenom kiselinom/ 5% triizobutilsilan. Slijedeći koraci ispiranja bili su provedeni: Separation of the Mmt-protecting groups was followed by incubation for 2 min. with 10% dichloroacetic acid/0.5% trifluoroacetic acid and 3x5 min. with 10% dichloroacetic acid/0.5% trifluoroacetic acid/5% triisobutylsilane. The following washing steps were performed:
1x DCM, 2x etanol, 1x aqua dest., 1-2 min. sa 10% cezijkarbonata, 1x aqua dest., 2x etanol, 1x dietileter. 1x DCM, 2x ethanol, 1x aqua dest., 1-2 min. with 10% cesium carbonate, 1x aqua dest., 2x ethanol, 1x diethylether.
Određeni Fmoc-brompropil-esteri amino kiselina bili su 3x, pri koncentraciji od 0,6 M i reakcijskom vremenu od 15 min. povezani. Odcjepljenje Fmoc-zaštitnih skupina bilo je u 1b provedeno. The determined Fmoc-bromopropyl-esters of amino acids were 3x, at a concentration of 0.6 M and a reaction time of 15 min. connected. Removal of the Fmoc-protecting groups was carried out in 1b.
1d. Povezivanje aminokiselina 1 d. Linking of amino acids
Peptidi su preko ponavljanih pipetiranja od 0,6 M otopine aminokiselina u N-metilpirolidonu na spotove i zaključnog odcjepljenja Fmoc-zaštitnih skupina, izgrađeni. Peptides were built through repeated pipetting of a 0.6 M solution of amino acids in N-methylpyrrolidone onto the spots and final removal of the Fmoc-protecting groups.
1e. Povezivanje indodikarbocijanin boje 1e. Binding of indodicarbocyanine dye
Jedna otopina od 0,3 M indodikarbocijanin boja sa 0,3 M TBTU i 0,6 M diizopropiletilamina bila je aktivirana i 4x pri jednom reakcijskom vremenu od 15 min na spotove pipetirana. One solution of 0.3 M indodicarbocyanine dye with 0.3 M TBTU and 0.6 M diisopropylethylamine was activated and pipetted 4 times with a reaction time of 15 min onto the spots.
1f. Odcjepljenje postranih zaštitnih skupina 1 f. Cleavage of side protecting groups
Odcjepljenje postranih zaštitnih skupina slijedi preko tretmana membrane u pravilnim slijedovima sa 90% trifluor octenom kiselinom/3% triizobutilsilanom/2% aqua. dest./1% fenol od 30 min te sa 50% trifluoroctenom kiselinom/3% triizobutilsilanom/2% aqua.dest./1% fenola za 2,5 sati. Nastavno je 4x sa diklormetanom, 3x sa DMF-om i 1x sa etanolom isprano. Removal of side protective groups follows through membrane treatment in regular sequences with 90% trifluoroacetic acid/3% triisobutylsilane/2% aqua. dest./1% phenol for 30 min and with 50% trifluoroacetic acid/3% triisobutylsilane/2% aqua.dest./1% phenol for 2.5 hours. It was subsequently washed 4x with dichloromethane, 3x with DMF and 1x with ethanol.
2. Odcjepljenje peptida od celulozne membrane 2. Separation of the peptide from the cellulose membrane
Spotovi su bili izbušeni te s metanolom isprani. Za odcjepljenje bilo je 30 min, sa 70 mM natrijmetanolatom u metanolu inkubirano. PH je bio dodatkom 37% solne kiseline korigiran. Nastavno su peptidi u jednoj Speed-Vac (liofilizator) osušeni. Spots were drilled and washed with methanol. For separation, it was incubated for 30 min with 70 mM sodium methanolate in methanol. The pH was corrected by adding 37% hydrochloric acid. Next, the peptides were dried in a Speed-Vac (lyophilizer).
Nakon sušenja peptidi su otopljeni u aqua dest. te pomoću RP-HPLC i MALDI-TOF analizirani. After drying, the peptides were dissolved in aqua dest. and analyzed by RP-HPLC and MALDI-TOF.
3. Stanični assay i protočna citometrija 3. Cell assay and flow cytometry
Koncentracija VIP-derivata bila je fotometrijski preko boje očitavana. U staničnom assay-u peptidi su bili upotrebljeni u konc. od 150 mM. Pri tom je bilo 1x105 RIN38(VAC1)-stanica sa VIP-derivatima jedan sat, pri 37ºC u veznom puferu (50 mM Tris/HCl, pH 7,5, 5nM MgCl2, 1mM CaCl2, 100 mM NaCl, 4% BSA) inkubirano. The concentration of VIP-derivatives was read photometrically via color. In the cell assay, the peptides were used in conc. of 150 mM. In doing so, 1x105 RIN38(VAC1)-cells with VIP-derivatives were incubated for one hour at 37ºC in binding buffer (50 mM Tris/HCl, pH 7.5, 5nM MgCl2, 1mM CaCl2, 100mM NaCl, 4% BSA). .
Nastavno su stanice bile 2x sa PBS-om ispirane, u FACS-reagencijske posudice prenesene te 5 min. na 377 g centrifugirane. Stanični talog bio je u 300 µl staničnog fiksirnog sredstva resuspendiran, na FACS-Calibur (Bacton Dickinson) sa FL4-Optik izmjeren. Subsequently, the cells were washed twice with PBS, transferred to FACS-reagent containers and incubated for 5 min. on 377 g centrifuged. The cell sediment was resuspended in 300 µl of cell fixative, measured on a FACS-Calibur (Bacton Dickinson) with FL4-Optik.
4. Analiza rezultata 4. Analysis of results
Intenzitet fluorescencije u protočnoj citometriji mjeren nativno, prirodno dolazeći, humanih VIP-peptida bio je 100% korišten. Standardna odstupanja od 28 nativnih VIP-peptida iznosila su 11%. Daljnji VIP-peptidi bili su s takvim 100% ujednačeni. Fluorescence intensity in flow cytometry measured by native, naturally occurring, human VIP-peptides was 100% used. The standard deviations of 28 native VIP-peptides were 11%. Further VIP-peptides were 100% uniform with such.
Prikaz 7 pokazuje relativni intenzitet fluorescencije od RIN38 VPAC1-stanica nakon inkubacije u prisutnosti 150 nM peptida markiranih bojama za 1 sat, pri 37ºC. Podaci u postocima, odnose se na nativni peptid određenog reda. Figure 7 shows the relative fluorescence intensity of RIN38 VPAC1-cells after incubation in the presence of 150 nM dye-labeled peptides for 1 hour, at 37ºC. Data in percentages refer to the native peptide of a certain order.
Literatura za spot-sintezu: Literature for spot-synthesis:
1. Frank, R. (1992) Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedon 48, 9217-9232 1. Frank, R. (1992) Spot synthesis: an easy technique for the positionally addressable, parallel chemical synthesis on a membrane support. Tetrahedron 48, 9217-9232
2. Kramer, A., Schneider-Mergener, J. (1998) Synthesisi and screening of peptide libraries on continuoscellulose membrane supports. Methods in Molecular Biology 87, 25-39 2. Kramer, A., Schneider-Mergener, J. (1998) Synthesis and screening of peptide libraries on continuous cellulose membrane supports. Methods in Molecular Biology 87, 25-39
3. Volkmer-Engert, R., Hoffmann, B., Schneider.Mergener, J. (1997) Tetrahedron Lett. 38, 1029-1032 3. Volkmer-Engert, R., Hoffmann, B., Schneider, Mergener, J. (1997) Tetrahedron Lett. 38, 1029-1032
4. Licha, K., Bhargava, S:, Rheinländer, C., Becker, A., Schneider-Mergener, J., Volkmer-Engert, R. (in press) Highly paralles Nano-synthesis of cleavable peptide-dye conjugates on cellulose membranes. Tetrahedron Lett. 4. Licha, K., Bhargava, S:, Rheinländer, C., Becker, A., Schneider-Mergener, J., Volkmer-Engert, R. (in press) Highly parallel Nano-synthesis of cleavable peptide-dye conjugates on cellulose membranes. Tetrahedron Lett.
Primjer 44 Example 44
a) 5-N-(2,3-dihidroksipropil)aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin a) 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
0,9 g (2,6 mmol) 5-carboksi-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin (Anal. Biochem. 217, 197, 1994) biti će u 30 ml apsolutnog N,N-dimetilformamida i 3 ml piridin položen i sa 1,35 g (5,3 mmol) disukcinimidilkarbonata pomiješano. Nakon 3 sata doda se 0,965 g (10,6 mmol) 2,3-dihidroksipropilamina. Sve se izmiješa pri sobnoj temp., mješavina se otpari do suhog, a ostatak se pomiješa sa dietileterom. Čvrsta tvar će biti odsisana, a pročišćavanje će biti izvršen na RP-materijalu, kromatografski. 0.9 g (2.6 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine (Anal. Biochem. 217, 197, 1994) will be in 30 ml of absolute N,N-dimethylformamide and 3 ml of pyridine were added and mixed with 1.35 g (5.3 mmol) of disuccinimidyl carbonate. After 3 hours, 0.965 g (10.6 mmol) of 2,3-dihydroxypropylamine was added. Everything is mixed at room temperature, the mixture is evaporated to dryness, and the residue is mixed with diethyl ether. The solid substance will be sucked off, and purification will be performed on RP-material, chromatographically.
Iskoristivost: 0,82 g (76% d. Th.) Yield: 0.82 g (76% d. Th.)
Analiza (odnosi se na supstance slobodne od otapala). Analysis (refers to substances free from solvents).
[image] [image]
b) 4-[2-[4-klor-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5–heptatrien-1-il]-5-N(dihidroksipropil)aminokarbonil–3,3-dimetil(3H)indolio]butansulfonat, natrijeva sol b) 4-[2-[4-chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane -1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt
Jedna otopina od 360 mg (1 mmol) N-[5-anilino-3-klor-2,4-(propan-1,3-diil) 2,4-pentadien-1-yliden]anilinum-klorid, 825 mg (2 mmol) 5-N-(2,3-dihidroksipropil) aminokarbonil -1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin (primjer 44a) i 330 mg (4 mmol suhog natrij acetata u 30 ml etanola biti će 2 sata, pod argonom, na povratnom toku, kuhano. Nastavno, oddestilira se etanol, a ostatak se pročisti kromatografski. One solution of 360 mg (1 mmol) N-[5-anilino-3-chloro-2,4-(propane-1,3-diyl) 2,4-pentadien-1-ylidene]anilinum chloride, 825 mg ( 2 mmol) of 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Example 44a) and 330 mg (4 mmol of dry sodium acetate in 30 ml of ethanol will be boiled for 2 hours, under argon, on the return flow.
Iskoristivost: 0,58 g (59% d. Th.) Yield: 0.58 g (59% d. Th.)
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
c) 4-[2-[4-(4-(2-karboksietil)feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5–heptatrien-1-il]-5-N(dihidroksipropil)aminokarbonil–3,3-dimetil(3H)indolio]butansulfonat, natrijeva sol, N-hidroksisukcinimidester c) 4-[2-[4-(4-(2-carboxyethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indoline-2- ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt, N-hydroxysuccinimide ester
225 mg (1,4 mmol) 3-(4-hidroksifenil)propionske kiseline bilo je u 10 ml suhog N,N-dimetilformamida, pod zaštitnim plinom, otopljeno te sa 65 mg (2,7 mmol) natrij hidrida (60% u ulju) pomiješano. Nakon 30 min. dodano je 138 mg (0,14 mmol) 4-[2-[4-klor-7-[5-N-(dihidroksipropil) aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil) indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5–heptatrien-1 il]-5-N (dihidroksipropil) aminokarbonil –3,3-dimetil (3H) indolio] butansulfonata, natrijeva sol (primjer 44b) te se miješa narednih 30 min. Nastavno se reakcijska mješavina pomiješa sa suhim ledom te do suhog otpari. Ostatak se pročisti preko jedne preparativne HPLC. Za spravljanje aktivnog estera otopi se 14 mg (120 µmol) N-hidroksisukcinimida i 2 mg (2,4 µmol) karbonske kiseline u 200 µl N,N-dimetilformamida. Nakon 10 min. doda se 24 mg (120 µmol) dicikloheksilkarbodiimida i miješa preko noći pri sobnoj temp. Aktivni ester će biti nadalje bez dodatne obrade u slijedećem stupnju korišten. 225 mg (1.4 mmol) of 3-(4-hydroxyphenyl)propionic acid was dissolved in 10 ml of dry N,N-dimethylformamide, under a protective gas, and with 65 mg (2.7 mmol) of sodium hydride (60% in oil) mixed. After 30 min. 138 mg (0.14 mmol) of 4-[2-[4-chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene] were added ]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1 yl]-5-N (dihydroxypropyl)aminocarbonyl-3,3-dimethyl (3H)indolio]butanesulfonate, sodium salt (example 44b) and mixed for the next 30 min. The reaction mixture is then mixed with dry ice and evaporated to dryness. The residue is purified via a preparative HPLC. To prepare the active ester, 14 mg (120 µmol) of N-hydroxysuccinimide and 2 mg (2.4 µmol) of carboxylic acid are dissolved in 200 µl of N,N-dimethylformamide. After 10 min. 24 mg (120 µmol) of dicyclohexylcarbodiimide is added and stirred overnight at room temperature. The active ester will be further used in the next stage without further processing.
Analogno sintezi prema primjeru 14-16 i 18-27a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFWDNY TRLRKQMAVK KYLNSILN na čvrstu fazu sintetiziran. Boja 4-[2-[4-(4-(2-karboksietil)feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5–heptatrien-1il]-5-N (dihidroksipropil) aminokarbonil –3,3-dimetil (3H) indolio] butansulfonat, natrijeva sol, Analogous to the synthesis according to Example 14-16 and 18-27a (solid phase peptide synthesis) the solid phase VIP analog HSDAVFWDNY TRLRKQMAVK KYLNSILN will be synthesized. Dye 4-[2-[4-(4-(2-carboxyethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)–1,3,5–heptatrien-1yl]-5-N (dihydroxypropyl) aminocarbonyl –3,3-dimethyl (3H) indolio] butanesulfonate, sodium salt,
biti će prema primjeru 14-16 i 18-27 b (povezivanje boja) na peptid povezano i prema primjeru 14-16 i 18-27 c (odcjepljenje zaštitnih skupina i odvajanje konjugata peptid-boja) taj konjugat izoliran i pročišćen. it will be according to example 14-16 and 18-27 b (binding of dyes) linked to the peptide and according to example 14-16 and 18-27 c (detachment of protective groups and separation of the peptide-dye conjugate) that conjugate is isolated and purified.
d) 4-[2-[4-(4-(2-izotiocijanatoetil)feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5–heptatrien-1-il]-5-N(dihidroksipropil)aminokarbonil–3,3-dimetil(3H)indolio]butansulfonat, natrijeva sol d) 4-[2-[4-(4-(2-isothiocyanatoethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2- ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt
Suspenziji od 28 mg (0,6 mmol) natrij hidrida (60%-ni u ulju) u 4 ml suhog N,N-dimetilformamida doda se, pri 0ºC, 116 mg (0,6 mmol) 4-[2-[4-klor-7-[5-N-(dihidroksipropil) aminokarbonil -3,3- dimetil -1-(4-sulfonatobutil) indolin-2-yliden]-3,5-(propan-1,3-diil) –1,3,5 – heptatrien-1 il]-5-N (dihidroksipropil) aminokarbonil –3,3-dimetil (3H) indolio] butansulfonata, natrijeva sol (primjer 44b). Smjesa se miješa preko noći, pri sobnoj temp. i pomiješa nastavno sa suhim ledom. Na rotacionom otparivaču se otpari do suhog i ostatak pročisti preparativno na HPLC-u. To a suspension of 28 mg (0.6 mmol) of sodium hydride (60% in oil) in 4 ml of dry N,N-dimethylformamide, 116 mg (0.6 mmol) of 4-[2-[4 -chloro-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1 ,3,5-heptatrien-1 yl]-5-N (dihydroxypropyl)aminocarbonyl-3,3-dimethyl (3H)indolio]butanesulfonate, sodium salt (Example 44b). The mixture is stirred overnight at room temperature. and mixed with dry ice. It is evaporated to dryness on a rotary evaporator and the residue is preparatively purified on HPLC.
Iskoristivost: 85 mg (54% d. Th). Usability: 85 mg (54% d. Th).
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFTDNY TRLRFQMAVK KYLNSILN na čvrstu fazu sintetiziran. Boja 4-[2-[4-(4-(2-izotiocijanatoetil)feniloksi)-7-[5-N-(dihidroksipropil) aminokarbonil -3,3- dimetil-1-(4-sulfonatobutil)indolin-2-yliden]-3,5-(propan-1,3-diil)–1,3,5-heptatrien-1il]-5-N (dihidroksipropil) aminokarbonil –3,3-dimetil (3H) indolio] butansulfonat, natrijeva sol, biti će na peptidni N-terminus pripojena prema primjeru 14-16 i 18-27 b (povezivanje boja) na peptid povezano i prema primjeru 14-16 i 18-27 c (odcjepljenje zaštitnih skupina i odvajanje konjugata peptid-boja) taj konjugat izoliran i pročišćen. Analogous to the synthesis according to Example 14-16 and 18-27a (solid phase peptide synthesis) the solid phase VIP analog HSDAVFTDNY TRLRFQMAVK KYLNSILN will be synthesized. Color 4-[2-[4-(4-(2-isothiocyanatoethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)–1,3,5-heptatrien-1yl]-5-N (dihydroxypropyl) aminocarbonyl –3,3-dimethyl (3H) indolio] butanesulfonate, sodium salt, it will be attached to the peptide N-terminus according to example 14-16 and 18-27 b (binding of dyes) to the peptide connected and according to example 14-16 and 18-27 c (detachment of protective groups and separation of the peptide-dye conjugate) that conjugate is isolated and purified.
Na analogan način mogu daljnje sintetičke hidrofilne boje iz slijedećih gradivnih elemenata biti izgrađeni: In an analogous way, further synthetic hydrophilic paints can be constructed from the following building elements:
Hidrofilni indolenin-derivati sa hidroksialkilnim supstituentima: (spravljeno prema primjeru 44a Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44a
a) 5-N-(2,3-dihidroksipropil)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
b) 5-N-(dihidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin b) 5-N-(dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
c) 5-N-(2,3-dihidroksipropil)-N-(hidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
d) 5-N,N-(bi-dihidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H) indolenin d) 5-N,N-(bi-dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine
e) 5-N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin e) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine
f) 5-N-(1,3,4-trihidroksibutil-2-il)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine
Primjer 45 Example 45
A) 5-N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin A) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine
0,6 g (1,8 mmol) 5-karboksi-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin (Anal. Biochem. 217, 197, 1994) biti će u 20 ml apsolutnog N,N-dimetilformamid i 2 ml piridin stavljeno i sa 0,95 g (3,6 mmol) disukcinimidilkarbonata pomiješano. Nakon 2 sata doda se 1,4 ml(10 mmol) trietilamina i 322 mg (1,8 mmol) glukamina. Sve se miješa preko noći, pri sobnoj temp., otpari do suhog, a ostatak pomiješa sa dietileterom. Čvrsta tvar se odsiše te se pročisti na RP-materijalu, kromatografski. 0.6 g (1.8 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Anal. Biochem. 217, 197, 1994) will be in 20 ml of absolute N,N-dimethylformamide and 2 ml of pyridine were added and mixed with 0.95 g (3.6 mmol) of disuccinimidyl carbonate. After 2 hours, 1.4 ml (10 mmol) of triethylamine and 322 mg (1.8 mmol) of glucamine are added. Everything is mixed overnight at room temperature, evaporated to dryness, and the residue is mixed with diethyl ether. The solid substance is sucked off and purified on RP-material, chromatographically.
Iskoristivost: 0,67 g (74% d.Th.). Yield: 0.67 g (74% d.Th.).
Analiza (odnosi se na supstance bez otapala): Analysis (refers to substances without solvents):
[image] [image]
B) 4-[2-[4-klor-7-[5-N-(2,3,4,5,6-pentahidroksiheksil)aminokarbonil-3,3-dimetil-1-(4sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil5-[N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil](3H)indolio]butansulfonat, natrijeva sol B) 4-[2-[4-chloro-7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene ]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4,5,6-pentahydroxyhexyl )-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt
Otopina od 180 mg (0,5 mmol) N-[5-anilino-3-klor-2,4(propan-1,3-diil)-2,4-pentadien-1-iliden]anilin-klorid, 503 mg (1 mmol) 5-N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin (primjer 45a) i 165 mg (2 mmol) suhog natrijacetata u 10 ml etanola biti će 2 sata, pod argonom na povratnom hladilu kuhano. Nastavno se oddestilira etanol i pročisti ostatak kromatografski. Solution of 180 mg (0.5 mmol) N-[5-anilino-3-chloro-2,4(propane-1,3-diyl)-2,4-pentadien-1-ylidene]aniline chloride, 503 mg (1 mmol) 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Example 45a) and 165 mg (2 mmol) of dry sodium acetate in 10 ml of ethanol will be boiled for 2 hours under argon on a reflux condenser. Ethanol is further distilled off and the residue is purified by chromatography.
Iskoristivost: 0,31 g (53% d.Th.) Usability: 0.31 g (53% d.Th.)
Analiza (odnosi se na supstancu bez otapala): Analysis (refers to substance without solvent):
[image] [image]
C) 4-[2-[4-(4-izotiocijanatotiofeniloksi)-7-[5-N-(2,3,4,5,6-pentahidroksiheksil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil5-[N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil](3H)indolio]butansulfonat, natrijeva sol C) 4-[2-[4-(4-isothiocyanatothiophenyloxy)-7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) )indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4 ,5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt
54 mg (0,4 mmol) 4-aminotiofenola biti će u 10 ml apsolutnog N,N-dimetilformamida pod atmosferom zasićenom argonom, otopljeno i pri sobnoj temperaturi sa 165 mg (0,14 mmol) 4-[2-[4-klor-7-[5-N-(2,3,4,5,6-pentahidroksiheksil)aminokarbonil-3,3-dimetil-1-(4sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil5-[N-(2,3,4,5,6-pentahidroksihe-ksil)-aminokarbonil](3H)indolio]butansulfonat, natrijeva sol (primjer 45b). nakon 10 minuta biti će reakcijska mješavina sa suhim ledom pomiješana i sa 210 mg (1 mmol) tiokarbonildiimidazola dodano. Nakon 45 min biti će boja sa dietieterom precipitirana, a čvrsta tvar centrifugiranjem izolirana. Za pročišćavanje može se na RP-materijalu kromatografirati. 54 mg (0.4 mmol) of 4-aminothiophenol will be in 10 ml of absolute N,N-dimethylformamide under an atmosphere saturated with argon, dissolved at room temperature with 165 mg (0.14 mmol) of 4-[2-[4-chloro -7-[5-N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1, 3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio] butanesulfonate, sodium salt (example 45b). after 10 minutes, the reaction mixture will be mixed with dry ice and 210 mg (1 mmol) of thiocarbonyldiimidazole will be added. After 45 minutes, the dye will be precipitated with dietether, and the solid substance will be isolated by centrifugation. For purification, RP-material can be chromatographed.
Iskoristivost: 78 mg (43% d.Th.) Usability: 78 mg (43% d.Th.)
Analiza (odnosi se na supstance bez otapala): Analysis (refers to substances without solvents):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27 a (sinteza čvrste faze peptida) biti će VIP-analog HSWAVFTDNY TRLRKQMAVK KYLNSILN na čvrstoj fazi sintetiziran. Boja 4- [2- [4- (4-izotiocijanatotiofeniloksi) -7- [5- N- (2,3,4,5,6 -pentahidroksiheksil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil5-[N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil](3H)indolio]butansulfonat, natrijeva sol biti će na peptid, N-terminus povezana i prema primjeru 14-16 i 18-27 c (odcjepljenje zaštitnih skupina i odvajanje konjugata boje i peptida) biti će taj konjugat izoliran i pročišćen. Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSWAVFTDNY TRLRKQMAVK KYLNSILN on the solid phase will be synthesized. Dye 4-[2-[4-(4-isothiocyanatothiophenyloxy)-7-[5- N-(2,3,4,5,6-pentahydroxyhexyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl5-[N-(2,3,4, 5,6-pentahydroxyhexyl)-aminocarbonyl](3H)indolio]butanesulfonate, the sodium salt will be attached to the peptide, the N-terminus and according to examples 14-16 and 18-27 c (removal of protective groups and separation of the conjugate of dye and peptide) will be that conjugate will be isolated and purified.
Na analogan način mogu daljnje simetrične hidrofilne boje na slijedećim gradivnim elementima biti nadograđeni: In an analogous way, further symmetrical hydrophilic paints can be upgraded on the following building elements:
Hidrofilni indolenin-derivati sa hidroksialkilnimsupstituentima: (spravljeni prema primjeru 44 a). Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44 a).
a) 5-N-(2,3-dihidroksipropil)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
b) 5-N-(hidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin b) 5-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
c) 5-N-(2,3-dihidroksipropil)-N-(hidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
d) 5-N,N-(bi-hidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin d) 5-N,N-(bi-hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
e) 5-N,N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin e) 5-N,N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
f) 5-N-(1,3,4-trihidroksibutil-2-il)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
Primjer 46 Example 46
7-[5-N-(2,3-dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden] -1,3,5- heptatrien -1-il] -3,3 -dimetil -5-karboksi (3H) aminokarbonil] (3H) indolio] butansulfonat, natrijeva sol 7-[5-N-(2,3-dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-1,3,5- heptatrien-1-yl]-3 ,3-dimethyl-5-carboxy (3H) aminocarbonyl] (3H) indolio] butanesulfonate, sodium salt
Otopina od 2,35 g (5,71 mmol) 5-N-(2,3-dihidroksipropil)aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin (primjer 44a) i 1,57 g (5,5 mmol) glutakonaldehiddianilid-hidroklorid u 25 ml anhidrida ostene kiselina, biti će 30 min miješana, pri 120ºC. Nastavno se doda 2,4 g (7,1 mmol) 5-karboksi-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin, 1,71 g natrijacetata, 22 ml anhidrida octene kiseline i 8,6 ml octene kiseline. Reakcijska mješavina se miješa 1 sat, pri 120ºC, ohladi na sobnu temperaturu i precipitira produkt sa dietilnim eterom. Sirovina se kromatografira preko RP-materijala. A solution of 2.35 g (5.71 mmol) of 5-N-(2,3-dihydroxypropyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolene (Example 44a) and 1 57 g (5.5 mmol) of glutaconaldehyde dianilide hydrochloride in 25 ml of acetic anhydride will be mixed for 30 minutes at 120ºC. 2.4 g (7.1 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine, 1.71 g of sodium acetate, 22 ml of acetic anhydride and 8 .6 ml of acetic acid. The reaction mixture is stirred for 1 hour at 120ºC, cooled to room temperature and the product is precipitated with diethyl ether. The raw material is chromatographed over RP-material.
Iskoristivost: 9,1 g (40% d.Th.) Usability: 9.1 g (40% d.Th.)
Analiza (odnosi se na supstance bez otapala): Analysis (refers to substances without solvents):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27 a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFTDNY TRLRKKMAVK KYLNSILN na čvrstoj fazi sintetiziran. Boja 7- [5- N- (2,3-dihidroksipropil) aminokarbonil -3,3-dimetil -1- (4-sulfonatobutil) indolin-2-iliden]-1,3,5-heptatrien-1-il]-3,3-dimetil-5-karboksi (3H)indolio]butansulfonat, natrijeva sol biti će na peptid, N-terminus povezana i prema primjeru 14-16 i 18-27 c (odcjepljenje zaštitnih skupina i odvajanje konjugata boje i peptida) biti će taj konjugat izoliran i pročišćen pomoću HPLC. Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSDAVFTDNY TRLRKKMAVK KYLNSILN on the solid phase will be synthesized. Dye 7- [5- N - (2,3-dihydroxypropyl) aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-1,3,5-heptatrien-1-yl]- 3,3-dimethyl-5-carboxy (3H)indolio]butanesulfonate, the sodium salt will be attached to the peptide, the N-terminus and according to examples 14-16 and 18-27 c (removal of protective groups and separation of the conjugate of dye and peptide) will be this conjugate will be isolated and purified by HPLC.
Na analogan način mogu daljnje nesimetrične hidrofilne boje na slijedećim gradivnim elementima biti nadograđeni: In an analogous way, further asymmetric hydrophilic paints can be upgraded on the following building elements:
Hidrofilni indolenin-derivati sa hidroksialkilnim supstituentima: (spravljeni prema primjeru 44 a). Hydrophilic indolenine derivatives with hydroxyalkyl substituents: (prepared according to example 44 a).
a) 5-N-(2,3-dihidroksipropil)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin a) 5-N-(2,3-dihydroxypropyl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
b) 5-N-(dihidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin b) 5-N-(dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
c) 5-N-(2,3-dihidroksipropil)-N-(hidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin c) 5-N-(2,3-dihydroxypropyl)-N-(hydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
d) 5-N,N-(bi-dihidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin d) 5-N,N-(bi-dihydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
e) 5-N,N-(2,3,4,5,6-pentahidroksietil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin e) 5-N,N-(2,3,4,5,6-pentahydroxyethyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
f) 5-N-(1,3,4-trihidroksibutil-2-il)-N-metilaminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin f) 5-N-(1,3,4-trihydroxybutyl-2-yl)-N-methylaminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
Indolenin derivati sa karboksilnim skupinama: Indolenine derivatives with carboxyl groups:
a) 5-karboksi-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin a) 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine
b) 5-karboksimetil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin b) 5-carboxymethyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H) indolenine
c) 5-karboksi-1-(3-sulfopropil)-2,3,3-trimetil (3H) indolenin c) 5-carboxy-1-(3-sulfopropyl)-2,3,3-trimethyl (3H) indolenine
d) 5-karboksimetil-1-(3-sulfopropil)-2,3,3-trimetil (3H) indolenin d) 5-carboxymethyl-1-(3-sulfopropyl)-2,3,3-trimethyl (3H)indolenine
e) 5-karboksi-1-(2-sulfoetil)-2,3,3-trimetil (3H) indolenin e) 5-carboxy-1-(2-sulfoethyl)-2,3,3-trimethyl (3H) indolenine
f) 5-karboksimetil-1-(2-sulfoetil)-2,3,3-trimetil (3H) indolenin f) 5-carboxymethyl-1-(2-sulfoethyl)-2,3,3-trimethyl (3H) indolenine
Dialin derivati za reakcije sa o. g. indoleninima zu stvaranje mono-, di- ili trikarbocijanina: Dialin derivatives for reactions with the aforementioned indolenines to form mono-, di- or tricarbocyanine:
a) glutakonaldehiddianilid-hidroklorid a) glutaconaldehydedianilide hydrochloride
b) malonaldehid-bi-fenilimin-hidroklorid b) malonaldehyde-bi-phenylimine-hydrochloride
c) N,N-difenilformamid c) N,N-diphenylformamide
d) N-[5-anilino-2,4-(propan-1,3-diil)-2,4-pentadien-1-iliden]anilinium-klorid d) N-[5-anilino-2,4-(propane-1,3-diyl)-2,4-pentadien-1-ylidene]anilinium chloride
e) N-[5-anilino-2,4-(etan-1,2-diil)-2,4-pentadien-1-iliden]anilinium-klorid e) N-[5-anilino-2,4-(ethane-1,2-diyl)-2,4-pentadien-1-ylidene]anilinium chloride
f) N-[5-anilino-3-klor-2,4-(propan-1,3-diil)-2,4-pentadien-1-iliden]anilinium-klorid f) N-[5-anilino-3-chloro-2,4-(propane-1,3-diyl)-2,4-pentadien-1-ylidene]anilinium chloride
g) N-[5-anilino-3-klor-2,4-(etan-1,2-diil)-2,4-pentadien-1-iliden]anilinium-klorid g) N-[5-anilino-3-chloro-2,4-(ethane-1,2-diyl)-2,4-pentadien-1-ylidene]anilinium chloride
Primjer 47 Example 47
7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-1,3,5-heptatrien-1-il]-3,3–dimetil-5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil](3H)indolio]butansulfonat, natrijeva sol, 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-1,3,5-heptatriene- 1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H)indolio]butanesulfonate, sodium salt,
N-hidroksicukcinimidester N-hydroxysuccinimide ester
a) 4-[2-[4-klor-7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4sulfonatobutil)indolin-2-iliden]-3,5-[2-(metoksikarbonil)propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil-5-[N-(1,3,4-trihidroksibut-2-il)-aminokarbonil](3H)indolio]butansulfonat, natrijeva sol a) 4-[2-[4-chloro-7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4sulfonatobutyl)indolin-2-ylidene ]-3,5-[2-(methoxycarbonyl)propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-[N-(1,3, 4-trihydroxybut-2-yl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt
Otopini od 0,8 g (5 mmol) 4-(metoksikarbonil)-cikloheksanon u 5 ml diklormetan, doda se 5,5 ml (10 mmol) fosforoksiklorida i 6 ml N,N-dimetilformamida, pri 0ºC. Nastavno se zagrijava 1 sat u povratnom toku. Diklormetan se destilira k tome i doda, pri max. 5ºC anilin i 10 ml metanol. Reakcijska mjeüavina se izlije na led, doda se 5 ml koncentrirane solne kiseline i ostavi da međuprodukt 5 sati, pri 0ºC, iskristalizira. Kristali se odvoje i upotrijebe, bez daljnjeg pročišćavanja u slijedeću reakciju. Za to se otope kristali u suhom metanolu i doda se 4,4 g (10 mmol) 5-N-(2,3,4,5,6-pentahidroksiheksil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin (vidi primjer 45) i 0,8 g suhog natrij acetata. Sve se zagrijava 1 sat na povratnom hladilu, odfiltrira čvrsta tvar te otpari filtrat do suhog. Ostatak se za pročišćavanje kromatografira. To a solution of 0.8 g (5 mmol) of 4-(methoxycarbonyl)-cyclohexanone in 5 ml of dichloromethane, 5.5 ml (10 mmol) of phosphorus oxychloride and 6 ml of N,N-dimethylformamide were added at 0ºC. It is continuously heated for 1 hour in the return flow. Dichloromethane is distilled to it and added, at max. 5ºC aniline and 10 ml methanol. The reaction mixture is poured onto ice, 5 ml of concentrated hydrochloric acid is added and the intermediate product is left to crystallize for 5 hours at 0ºC. The crystals are separated and used without further purification in the next reaction. For this, the crystals are dissolved in dry methanol and 4.4 g (10 mmol) of 5-N-(2,3,4,5,6-pentahydroxyhexyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3 ,3-trimethyl(3H)indolenine (see Example 45) and 0.8 g of dry sodium acetate. Everything is heated for 1 hour on a reflux condenser, the solid substance is filtered off and the filtrate is evaporated to dryness. The residue is chromatographed for purification.
Iskoristivost: 3,25 g (58% d.Th.) Usability: 3.25 g (58% d.Th.)
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
b) 7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]–3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3–dimetil-5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil](3H)indolio]butansulfonat, natrijeva sol b) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio]butanesulfonate, sodium salt
Jedna mješavina od 10 mg (0,4 mmol) natrijhidrida i 80 mg (1,3 mmol) etantiola u 10 ml suhog N,N-dimetilformamida biti će, pod dušikom, 30 minuta pri sobnoj temp., miješano. Nastavno se doda 112 mg (0,1 mmol) 4-[2-[4-klor-7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4sulfonatobutil)indolin-2-iliden]-3,5-[2-(metoksikarbonil)propan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil-5-[N-(1,3,4-trihidroksibut-2-il)-aminokarbonil](3H)indolio] butansulfonat, natrijeva sol (primjer 47 a) u 3 ml N,N-dimetilformamida. Reakcijska mješavina će biti 2 sata , pri 100ºC zagrijavana i nakon što se ohladi na sobnu temperaturu, pomiješana sa ugljičnim dioksidom. Smjesa će biti otparena na rotacionom otparivaču do suha, a ostatak će biti ekstrahiran sa vrućim etanolom. Ekstrakt će biti otparen do suha i kromatografiran. A mixture of 10 mg (0.4 mmol) of sodium hydride and 80 mg (1.3 mmol) of ethanethiol in 10 ml of dry N,N-dimethylformamide will be stirred, under nitrogen, for 30 minutes at room temp. 112 mg (0.1 mmol) of 4-[2-[4-chloro-7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1 -(4sulfonatobutyl)indolin-2-ylidene]-3,5-[2-(methoxycarbonyl)propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5 -[N-(1,3,4-trihydroxybut-2-yl)-aminocarbonyl](3H)indolio]butanesulfonate, sodium salt (Example 47 a) in 3 ml of N,N-dimethylformamide. The reaction mixture will be heated for 2 hours at 100ºC and after cooling to room temperature, mixed with carbon dioxide. The mixture will be evaporated to dryness on a rotary evaporator, and the residue will be extracted with hot ethanol. The extract will be evaporated to dryness and chromatographed.
Iskoristivost: 75 mg (67% d.Th.) Usability: 75 mg (67% d.Th.)
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
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c) 7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]–3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3–dimetil-5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil](3H)indolio] butansulfonat-N-hidroksisukcinimidester, natrijeva sol c) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio]butanesulfonate-N-hydroxysuccinimide ester, sodium salt
Za spravljanje aktivnog estera 14 mg (0,12 mmol) N-hidroksisukcinimida i 64 mg (0,06 mmol) karbonske kiseline u 2 ml N,N-dimetilformamida. Nakon 15 min. doda se 25 mg (0,12 mmol) dicikloheksilkarbodiimid te se miješa preko noći pri sobnoj temp. Aktivni ester bio je pomoću preparativnog HPLC-a pročišćen. To prepare the active ester, 14 mg (0.12 mmol) of N-hydroxysuccinimide and 64 mg (0.06 mmol) of carboxylic acid in 2 ml of N,N-dimethylformamide. After 15 min. 25 mg (0.12 mmol) of dicyclohexylcarbodiimide is added and stirred overnight at room temperature. The active ester was purified by preparative HPLC.
Iskoristivost: 60 mg (86% d. Th.) Usability: 60 mg (86% d. Th.)
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27 a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFTDNY TRLRKAMAVK KYLNSILN na čvrstoj fazi sintetiziran. Boja 7- [5- N- (2,3-dihidroksipropil) aminokarbonil -3,3-dimetil -1- (4-sulfonatobutil) indolin-2-iliden]-3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil-5-N-1,3,4-trihidroksibutil-2-il)-aminokarbonil (3H)indolio]butansulfonat, natrijeva sol biti će prema primjeru 14-16 i 18-27 c (povezivanje boje) na peptid N-terminus i prema primjeru 14-16 i 18-27 c (odcjepljenje zaštitnih skupina i odvajanje konjugata boja-peptid) biti će taj konjugat izoliran i pročišćen. Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP analog HSDAVFTDNY TRLRKAMAVK KYLNSILN on the solid phase will be synthesized. Dye 7- [5- N - (2,3-dihydroxypropyl) aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(2-carboxypropane-1,3- diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl (3H)indolio]butanesulfonate, sodium salt be will according to example 14-16 and 18-27 c (binding of dye) to the peptide N-terminus and according to example 14-16 and 18-27 c (removal of protective groups and separation of the dye-peptide conjugate) that conjugate will be isolated and purified.
Primjer 48 Example 48
7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]–3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3–dimetil-5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil](3H)indolio] butansulfonat, natrijeva sol 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2-carboxypropane -1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H)indolio ] butanesulfonate, sodium salt
a) 5-N-(11-aminoundecil)aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin a) 5-N-(11-aminoundecyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine
340 mg (1 mmol) 5-karboksi-1-(4-sulfobutil)-2,3,3-trimetil(3H)indolenin (Anal. Biochem. 217,197, 1994) biti će u 5 ml apsolutnog N,N-dimetilformamida i 1 ml piridina položeno i sa 0,5 g (2 mmol) disukcinimidilkarbonata pomiješano. Nakon 3 sata doda se 0,805 g (4 mmol) 11-aminoundekanske kiseline. Miješa se preko noći pri sobnoj temp., otpari se do suha i miješa ostatak sa dietileterom. Čvrsta tvar se odstrani i pročisti kromatografski na RP-materijalu. 340 mg (1 mmol) of 5-carboxy-1-(4-sulfobutyl)-2,3,3-trimethyl(3H)indolenine (Anal. Biochem. 217,197, 1994) will be in 5 ml of absolute N,N-dimethylformamide and 1 ml of pyridine was added and mixed with 0.5 g (2 mmol) of disuccinimidyl carbonate. After 3 hours, 0.805 g (4 mmol) of 11-aminoundecanoic acid was added. It is stirred overnight at room temperature, evaporated to dryness and the residue mixed with diethyl ether. The solid substance is removed and purified by chromatography on RP-material.
Iskoristivost: 0,37 g (71% d. Th.) Yield: 0.37 g (71% d. Th.)
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
b) 7-[5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]–3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3–dimetil-5-N-(1,3,4-trihidroksibut-2-il)aminokarbonil](3H)indolio] butansulfonat, natrijeva sol b) 7-[5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]–3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-(1,3,4-trihydroxybut-2-yl)aminocarbonyl](3H )indolio] butanesulfonate, sodium salt
Jedna mješavina od 0,35 g (0,67 mmol) 5-N-(11-aminoundecila)aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H) indolenin (primjer 48a) i 0,18 g (0,645 mmol) glutakonaldehiddianila u 3 ml anhidrida octene kiseline biti će 20 min, pri 110ºC miješano. Nastavno doda se 344 mg (0,83 mmol) 5-N-(2,3-dihidroksipropil)-aminokarbonil-1-(4-sulfobutil)-2,3,3-trimetil (3H)indolenin (primjer 44a), 0,2 g natrijacetata, 3 ml anhidrida octene kiseline u 1 ml octene kiseline. Reakcijska mješavina miješa se pri 110ºC, 2 sata, ohladi na sobnu temp. i precipitira produkt sa dietileterom. Sirovi produkt se kromatografira preko RP-materijala. One mixture of 0.35 g (0.67 mmol) of 5-N-(11-aminoundecyl)aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolene (Example 48a) and 0, 18 g (0.645 mmol) of glutaconaldehydedianil in 3 ml of acetic anhydride will be stirred for 20 min at 110ºC. 344 mg (0.83 mmol) of 5-N-(2,3-dihydroxypropyl)-aminocarbonyl-1-(4-sulfobutyl)-2,3,3-trimethyl (3H)indolenine (example 44a), 0 ,2 g of sodium acetate, 3 ml of acetic anhydride in 1 ml of acetic acid. The reaction mixture is stirred at 110ºC for 2 hours, cooled to room temperature. and precipitate the product with diethyl ether. The crude product is chromatographed over RP-material.
Iskoristivost: 0,41 g (60% d. Th.). Yield: 0.41 g (60% d. Th.).
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27 a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFTDNY TRLRKQMVK KYLNSILN na čvrstoj fazi sintetiziran. Boja 7- [5- N- (1,3,4-trihidroksibutil-2-il)-aminokarbonil -3,3-dimetil -1- (4-sulfonatobutil) indolin-2-iliden]-3,5-(2-karboksipropan-1,3-diil)-1,3,5-heptatrien-1-il]-3,3-dimetil-5-N-1,3,4-trihidroksibutil-2-il)-aminokarbonil (3H)indolio]butansulfonat, natrijeva sol biti će prema primjeru 14-16 i 18-27 c (povezivanje boje) na peptid N-terminus i prema primjeru 14-16 i 18-27 c (povezivanje boje) na peptid N-terminus, povezan prema primjeru 14-16 i 18-27c (odcjepljenje zaštitnih skupina i odvajanje konjugata boja-peptid) biti će taj konjugat izoliran i pročišćen. Analogously to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP-analog HSDAVFTDNY TRLRKQMVK KYLNSILN on the solid phase will be synthesized. Color 7- [5- N - (1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl -3,3-dimethyl -1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(2 -carboxypropane-1,3-diyl)-1,3,5-heptatrien-1-yl]-3,3-dimethyl-5-N-1,3,4-trihydroxybutyl-2-yl)-aminocarbonyl (3H) indolio]butanesulfonate, the sodium salt will be according to example 14-16 and 18-27 c (linkage of dye) to the peptide N-terminus and according to example 14-16 and 18-27 c (linkage of dye) to the peptide N-terminus, linked according to example 14-16 and 18-27c (removal of protecting groups and separation of dye-peptide conjugate), that conjugate will be isolated and purified.
Daljnje izumom predočene boje mogu prema primjeru 44a i b biti spravljene, pri čemu se na mjestu 11-aminoundekanske kiseline u primjeru 48a slijedeće aminokiseline i u primjeru 46 navedene indoleninderivate sa hidroksialkilsupstituenti a)-f) primjenjuju: Furthermore, the colors presented by the invention can be prepared according to example 44a and b, whereby in place of 11-aminoundecanoic acid in example 48a, the following amino acids and in example 46 mentioned indolene derivatives with hydroxyalkyl substituents a)-f) are used:
i). glicin and). glycine
ii). alanin ii). alanine
iii). β-alanin iii). β-Alanine
iv). 4-aminobutanska kiselina iv). 4-aminobutanoic acid
v). 6-aminoheksanska kiselina c). 6-aminohexanoic acid
vi). H2N-(CH2CH2O)3CH2COOH (TH 53, 20, 6977) you). H2N-(CH2CH2O)3CH2COOH (TH 53, 20, 6977)
vii). H2N-(CH2CH2O)4CH2COOH (JOC 63, 5, 1728, 1998) vii). H2N-(CH2CH2O)4CH2COOH (JOC 63, 5, 1728, 1998)
viii). H2N-CH2CH2COO(CH2CH2O)4-CO-CH2CH2COOH (Let. Pept. Sci. 6, 135, 1999) viii). H2N-CH2CH2COO(CH2CH2O)4-CO-CH2CH2COOH (Let. Pept. Sci. 6, 135, 1999)
ix). HCl*H2N-PEG-COOH (MW 3400 g/mol; Shearwater Polymers Inc., USA) ix). HCl*H2N-PEG-COOH (MW 3400 g/mol; Shearwater Polymers Inc., USA)
Primjer 49 Example 49
4-[2-[4-(4-(N-(4-aza-6-brom-5-oksoheksil)aminokarbonil-etil)feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil(3H) indolio]butansulfonat, natrijeva sol 4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxohexyl)aminocarbonyl-ethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3- dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-(dihydroxypropyl) aminocarbonyl-3,3-dimethyl(3H) indolio]butanesulfonate, sodium salt
a) [3-[N-(terc.-butoksikarbonil)amino]propil]-N'-(bromacetil)-amid a) [3-[N-(tert-butoxycarbonyl)amino]propyl]-N'-(bromoacetyl)-amide
2,5 g (14,4 mmol) [3-[N-(terc.-butoksikarbonil)amino]propil-amida biti će u 15 ml dioksana otopljeno i nakon dodatka 4,4 ml trietilamina, pri 0ºC, sa 3,2 g (16 mmol) bromacetilbromida pomiješano. Smjesa se miješa preko noći, pri sobnoj temperaturi, a zatim se doda daljnjih 320 mg bromacetilbromida. Nakon 2 sata, pri sobnoj temp., talog će biti odstranjen, otopina koncentrirana, a ostatak u etilnom esteru octene kiseline otopljen. Otopina se ispire sa vodom, a organska faza se suši preko natrijeva sulfata. 2.5 g (14.4 mmol) of [3-[N-(tert.-butoxycarbonyl)amino]propyl-amide will be dissolved in 15 ml of dioxane and after the addition of 4.4 ml of triethylamine, at 0ºC, with 3.2 g (16 mmol) of bromoacetyl bromide mixed. The mixture is stirred overnight at room temperature and then a further 320 mg of bromoacetyl bromide is added. After 2 hours, at room temperature, the precipitate will be removed, the solution will be concentrated, and the residue will be dissolved in the ethyl ester of acetic acid. The solution is washed with water, and the organic phase is dried over sodium sulfate.
Iskoristivost: 3,2 g (75% d. Th.). Yield: 3.2 g (75% d. Th.).
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
b) [3-[N-(terc.-bromacetil)amino]propil] amin, hidroklorid b) [3-[N-(tert.-bromoacetyl)amino]propyl] amine, hydrochloride
3,1 g (10,5 mmol) [3-[N-(terc.-butoksikarbonil)amino]-N`-(bromacetil)-amida (primjer 49a) biti će sa 50 mmol 1M solne kiseline u etilnom eteru octene kiseline za 5 sati pomiješano, pri sobnoj temp. Talog/produkt će biti odstranjen, a ostatak u etilnom esteru octene kiseline ispran. 3.1 g (10.5 mmol) of [3-[N-(tert-butoxycarbonyl)amino]-N`-(bromoacetyl)-amide (Example 49a) will be with 50 mmol of 1M hydrochloric acid in ethyl acetic acid ether for 5 hours mixed, at room temp. The precipitate/product will be removed and the residue washed in ethyl acetate.
Iskoristivost: 2,3 g (95% d. Th.). Yield: 2.3 g (95% d. Th.).
Analiza (odnosi se na supstance slobodne od otapala): Analysis (refers to solvent-free substances):
[image] [image]
c) 4-[2-[4-(4-(N-(4-aza-6-brom-5-okso-heksil)aminokarbonil-etil)feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil(3H) indolio]butansulfonat, natrijeva sol c) 4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxo-hexyl)aminocarbonyl-ethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl- 3,3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N -(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt
121 mg (0,1 mmol) 4-[2-[4-(4-(2-karboksietilfeniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil(3H) indolio]butansulfonat, natrijeva sol, N-hidroksisukcinimidester (primjer 44c) biti će u 0,5 ml N,N-dimetilformamida otopljeno, sa 0,06 ml trietilamina i 70 mg (0,3 mmol) [3-[N-(bromacetil)amino]propil]amina, hidroklorida (primjer 49b) pomiješano. 121 mg (0.1 mmol) 4-[2-[4-(4-(2-carboxyethylphenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl-1-(4-sulfonatobutyl) indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-(dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H ) indolio]butanesulfonate, sodium salt, N-hydroxysuccinimide ester (example 44c) will be dissolved in 0.5 ml of N,N-dimethylformamide, with 0.06 ml of triethylamine and 70 mg (0.3 mmol) [3-[N- (bromoacetyl)amino]propyl]amine, hydrochloride (Example 49b) mixed.
Smjesa se miješa 4 sata, pri 60ºC, ohladi nastavno na sobnu temperaturu, a produkt se istaloži sa dietileterom. Čvrsti ostatak se odstrani i obilno ispere sa dietileterom. The mixture is stirred for 4 hours at 60ºC, cooled continuously to room temperature, and the product is precipitated with diethyl ether. The solid residue is removed and washed abundantly with diethyl ether.
Iskoristivost: 0,11 mg (80% d. Th.) Usability: 0.11 mg (80% d. Th.)
Analiza (odnosi se na supstance bez otapala): Analysis (refers to substances without solvents):
[image] [image]
Analogno sintezi prema primjeru 14-16 i 18-27 a (sinteza čvrste faze peptida) biti će VIP-analog HSDAVFTDNY TRLRKQCAVK KYLNSLLN na čvrstoj fazi sinteteiziran Analogous to the synthesis according to example 14-16 and 18-27 a (solid phase peptide synthesis) the VIP-analog HSDAVFTDNY TRLRKQCAVK KYLNSLLN on the solid phase will be synthesized
Bromidna boja 4-[2-[4-(4-(N-(4-aza-6-brom-5-okso-heksil) aminokarboniletil) feniloksi)-7-[5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil-1-(4-sulfonatobutil)indolin-2-iliden]-3,5-(propan-1,3-diil)-1,3,5-heptatrien-1-il]-5-N-(dihidroksipropil)aminokarbonil-3,3-dimetil(3H) indolio]butansulfonat, natrijeva sol, biti će prema primjeru 17 na peptidni N-terminus nadovezan, a prema primjeru 14-16 i 18-27 c (odcjepljivanje zaštitnih skupina i odvajanje konjugata peptid-boja) taj konjugat izoliran i pročišćen. Bromide dye 4-[2-[4-(4-(N-(4-aza-6-bromo-5-oxo-hexyl)aminocarbonylethyl)phenyloxy)-7-[5-N-(dihydroxypropyl)aminocarbonyl-3, 3-dimethyl-1-(4-sulfonatobutyl)indolin-2-ylidene]-3,5-(propane-1,3-diyl)-1,3,5-heptatrien-1-yl]-5-N-( dihydroxypropyl)aminocarbonyl-3,3-dimethyl(3H)indolio]butanesulfonate, sodium salt, will be attached to the peptide N-terminus according to example 17, and according to examples 14-16 and 18-27 c (detachment of protective groups and separation of peptide conjugate -color) that conjugate isolated and purified.
LISTA SEKVENCI LIST OF SEQUENCES
OPĆE INFORMACIJE: GENERAL INFORMATION:
Podnositelj zahtjeva: Applicant:
(A) IME: Institut fur Diagnostikforschung GMBH (A) NAME: Institut fur Diagnostikforschung GMBH
an der freien Universitat Berlin an der freien Universitat Berlin
ULICA: Spandauer Damm 130 STREET: Spandauer Damm 130
GRAD: Berlin CITY: Berlin
(E) ZEMLJA: Njemačka (E) COUNTRY: Germany
BROJ POŠTE (ZIP): D-14050 POSTAL CODE (ZIP): D-14050
TELEFON: (030)-30390412 TELEPHONE: (030)-30390412
TELEFAX: (030)-30390499 TELEFAX: (030)-30390499
NASLOV PRIJAVKA: Kratkolančani konjugati peptida i boja kao kontrastna sredstva u optičkoj dijagnostici APPLICATION TITLE: Short-chain conjugates of peptides and dyes as contrast agents in optical diagnostics
BROJ SEKVENCI: 8 NUMBER OF SEQUENCES: 8
KOMP. ČITLJIVI OBLIK: COMP. READABLE FORM:
MEDIJSKI OBLIK: Floppy disk MEDIA FORM: Floppy disk
KOMPJUTOR: IBM PC kompatibilan COMPUTER: IBM PC compatible
OPERATIVNI SUSTAV: PC-DOS/MS-DOS OPERATING SYSTEM: PC-DOS/MS-DOS
SOFTVER: Patentin Release #1.0, Verzija #1.25 (EPO) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO)
(v) SADAŠNJI APLICIRANI PODACI: (v) CURRENT APPLIED DATA:
APLIKACIJSKI BROJ: APPLICATION NUMBER:
(vi) STARIJI APLICIRANI PODACI (vi) OLDER APPLICATION DATA
APLIKACIJSKI BROJ APPLICATION NUMBER
REGISTRIRANI PODACI REGISTERED DATA
(2) INFORMACIJE O SEQ ID BR.: 1 (2) INFORMATION ABOUT SEQ ID NO: 1
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: Synthetic
(xi) OPIS SEKVENCE: SEQ ID BR: 1 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1
His-Trp-Asp-Ala-Val-Phe-Trp-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Trp-Asp-Ala-Val-Phe-Trp-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(3) INFORMACIJA ZA SEQ ID NO: 2 (3) INFORMATION FOR SEQ ID NO: 2
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 2 SEQUENCE DESCRIPTION: SEQ ID NO: 2
His-Ser-Asp-Ala-Val-Phe-Thr-Phe-Asn-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Phe-Asn-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile- Leu-Asn
(4) INFORMACIJA ZA SEQ ID NO: 3 (4) INFORMATION FOR SEQ ID NO: 3
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 3 SEQUENCE DESCRIPTION: SEQ ID NO: 3
His-Ser-Asp-Ala-Val-Phe-Thr-Lys-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Lys-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(5) INFORMACIJA ZA SEQ ID NO: 4 (5) INFORMATION FOR SEQ ID NO: 4
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 4 SEQUENCE DESCRIPTION: SEQ ID NO: 4
His-Ser-Asp-Ala-Val-Phe-Thr-Gln-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Gln-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(6) INFORMACIJA ZA SEQ ID NO: 5 (6) INFORMATION FOR SEQ ID NO: 5
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 5 SEQUENCE DESCRIPTION: SEQ ID NO: 5
His-Ser-Asp-Ala-Val-Phe-Thr-Arg-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Arg-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(7) INFORMACIJA ZA SEQ ID NO: 6 (7) INFORMATION FOR SEQ ID NO: 6
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 6 SEQUENCE DESCRIPTION: SEQ ID NO: 6
His-Ser-Asp-Ala-Val-Phe-Thr-Trp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Trp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(8) INFORMACIJA ZA SEQ ID NO: 7 (8) INFORMATION FOR SEQ ID NO: 7
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 7 SEQUENCE DESCRIPTION: SEQ ID NO: 7
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Arg-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Arg-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
(9) INFORMACIJA ZA SEQ ID NO: 8 (9) INFORMATION FOR SEQ ID NO: 8
(i) KARAKTERISTIKE SEKVENCI: (i) SEQUENCE CHARACTERISTICS:
DUŽINA: 28 amino kiselina LENGTH: 28 amino acids
TIP: peptidi TYPE: peptides
LANČANOST: pojedinačni CHAINING: single
TOPOLOGIJA: TOPOLOGY:
(ii) TIP MOLEKULA: peptidi (ii) TYPE OF MOLECULES: peptides
(iii) HIPETETSKE: NE (iii) HYPETHETICAL: NO
(iv) ORIGINALNI IZVOR: sintetičke (iv) ORIGINAL SOURCE: synthetic
OPIS SEKVENCE: SEQ ID BR: 8 SEQUENCE DESCRIPTION: SEQ ID NO: 8
His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Tyr-Arg-Leu-Arg-Lys-Gln-Met-Arg-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Tyr-Arg-Leu-Arg-Lys-Gln-Met-Arg-Val-Lys-Lys-Tyr-Leu-Asn-Ser- Ile-Leu-Assn
Claims (33)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE19917713A DE19917713A1 (en) | 1999-04-09 | 1999-04-09 | Short-chain peptide-dye conjugates as contrast agents for optical diagnostics |
PCT/EP2000/002697 WO2000061194A2 (en) | 1999-04-09 | 2000-03-28 | Short-chain peptide dye conjugates used as contrast agents for optical diagnostics |
Publications (1)
Publication Number | Publication Date |
---|---|
HRP20010833A2 true HRP20010833A2 (en) | 2004-08-31 |
Family
ID=7905136
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
HR20010833A HRP20010833A2 (en) | 1999-04-09 | 2001-11-09 | Short-chain peptide dye conjugates used as contrast agents for optical diagnostics |
Country Status (28)
Country | Link |
---|---|
EP (2) | EP1176987B1 (en) |
JP (1) | JP2002541219A (en) |
KR (1) | KR20020000555A (en) |
CN (1) | CN1351505A (en) |
AT (2) | ATE421341T1 (en) |
AU (1) | AU769392B2 (en) |
BG (1) | BG105988A (en) |
BR (1) | BR0009658A (en) |
CA (1) | CA2368490A1 (en) |
CZ (1) | CZ20013562A3 (en) |
DE (3) | DE19917713A1 (en) |
DK (1) | DK1176987T3 (en) |
EE (1) | EE200100521A (en) |
ES (2) | ES2321586T3 (en) |
HK (1) | HK1046867A1 (en) |
HR (1) | HRP20010833A2 (en) |
HU (1) | HUP0202990A3 (en) |
IL (1) | IL145596A0 (en) |
MX (1) | MXPA01010174A (en) |
NO (1) | NO20014911L (en) |
NZ (2) | NZ522136A (en) |
PL (1) | PL351766A1 (en) |
PT (1) | PT1176987E (en) |
RU (1) | RU2001129707A (en) |
SK (1) | SK14152001A3 (en) |
WO (1) | WO2000061194A2 (en) |
YU (1) | YU70501A (en) |
ZA (1) | ZA200109238B (en) |
Families Citing this family (19)
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JP2004514697A (en) | 2000-11-28 | 2004-05-20 | モンドバイオテック・ソシエテ・アノニム | Compounds with vasoactive intestinal peptide biological activity for the treatment of pulmonary and arteriolar hypertension |
KR100406460B1 (en) * | 2001-09-29 | 2003-11-19 | 한국과학기술연구원 | Coupled Styrylcyanine Dyes and Their Synthesis |
IL148921A0 (en) | 2002-03-26 | 2002-09-12 | Peptor Ltd | Photo active backbone cyclized somatostatin analogs for optical imaging and photodynamic therapy |
JP4599948B2 (en) * | 2003-09-18 | 2010-12-15 | チッソ株式会社 | α-Fluoroacrylate compound, composition and polymer thereof |
GB0328060D0 (en) * | 2003-12-04 | 2004-01-07 | Sod Conseils Rech Applic | Botulinum toxin treatment |
NO20035682D0 (en) * | 2003-12-18 | 2003-12-18 | Amersham Health As | Optical imaging of oesophageal cancer and Barrett's oesophagus |
NO20035681D0 (en) * | 2003-12-18 | 2003-12-18 | Amersham Health As | Optical imaging of lung cancer |
NO20035683D0 (en) * | 2003-12-18 | 2003-12-18 | Amersham Health As | Optical imaging of prostate cancer |
EP1679082A1 (en) * | 2005-01-07 | 2006-07-12 | Schering AG | Use of cyanine dyes for the diagnosis of proliferative diseases |
CN101098916A (en) | 2005-01-13 | 2008-01-02 | 金文申有限公司 | Composite materials containing carbon nanoparticles |
KR100716840B1 (en) * | 2005-08-29 | 2007-05-09 | 삼성전기주식회사 | Auto opening and closing device for a note book |
GB0615211D0 (en) * | 2006-07-31 | 2006-09-06 | Ge Healthcare Uk Ltd | Asymmetric flouro-substituted polymethine dyes |
GB0718957D0 (en) * | 2007-09-28 | 2007-11-07 | Ge Healthcare Ltd | Optical imaging agents |
ES2715633T3 (en) | 2008-05-20 | 2019-06-05 | Univ Health Network | Device and method for imaging and fluorescence monitoring |
CZ304948B6 (en) * | 2013-01-02 | 2015-02-04 | Vysoká škola chemicko-technologická v Praze | Use of polymethine salts as sensors for tumor markers |
US10438356B2 (en) | 2014-07-24 | 2019-10-08 | University Health Network | Collection and analysis of data for diagnostic purposes |
JP2020066637A (en) * | 2017-02-27 | 2020-04-30 | 富士フイルム株式会社 | Dyeing agent for pathological diagnosis, cell nucleus dyeing method, manufacturing method of pathological specimen, and dye |
EP4168492A1 (en) * | 2020-06-23 | 2023-04-26 | Bracco Imaging SPA | Near-infrared cyanine dyes and conjugates thereof |
CN118048050B (en) * | 2024-04-16 | 2024-06-25 | 南京诺源医疗器械有限公司 | Heptamethine cyanine near-infrared fluorescent dye and preparation method and application thereof |
Family Cites Families (17)
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US5627027A (en) * | 1986-04-18 | 1997-05-06 | Carnegie Mellon University | Cyanine dyes as labeling reagents for detection of biological and other materials by luminescence methods |
JPS6488537A (en) * | 1987-09-30 | 1989-04-03 | Fuji Photo Film Co Ltd | Silver halide photographic sensitive material |
JPS6491134A (en) * | 1987-10-02 | 1989-04-10 | Fuji Photo Film Co Ltd | Silver halide photographic sensitive material |
MY106120A (en) * | 1988-12-05 | 1995-03-31 | Novartis Ag | Peptide derivatives. |
US5382654A (en) * | 1992-02-05 | 1995-01-17 | Mallinckrodt Medical, Inc. | Radiolabelled peptide compounds |
US5849261A (en) * | 1991-02-08 | 1998-12-15 | Diatide, Inc. | Radiolabeled vasoactive intestinal peptides for diagnosis and therapy |
JP3137349B2 (en) * | 1991-03-27 | 2001-02-19 | 生化学工業株式会社 | Peptide derivative |
EP0591820A1 (en) * | 1992-10-05 | 1994-04-13 | E.I. Du Pont De Nemours And Company | Near-infrared absorbing dyes prepared from Stenhouse salts |
JPH06202260A (en) * | 1993-01-07 | 1994-07-22 | Konica Corp | Silver halide photographic sensitive material and its processing method |
DE4445065A1 (en) * | 1994-12-07 | 1996-06-13 | Diagnostikforschung Inst | Methods for in-vivo diagnostics using NIR radiation |
US5824772A (en) * | 1995-04-04 | 1998-10-20 | Advanced Bioconcept, Inc. | Fluorescent somatostatin |
EP0898596B1 (en) * | 1996-04-19 | 2001-08-29 | Amersham Pharmacia Biotech UK Limited | Squarate dyes and their use in fluorescent sequencing method |
DE19649971A1 (en) * | 1996-11-19 | 1998-05-28 | Diagnostikforschung Inst | Optical diagnostics for the diagnosis of neurodegenerative diseases using near-infrared radiation (NIR radiation) |
DE19717904A1 (en) * | 1997-04-23 | 1998-10-29 | Diagnostikforschung Inst | Acid-labile and enzymatically cleavable dye constructs for diagnostics with near infrared light and for therapy |
GB9712524D0 (en) * | 1997-06-16 | 1997-08-20 | Nycomed Imaging As | Method |
DE19817517A1 (en) * | 1998-04-09 | 1999-10-14 | Diagnostikforschung Inst | New benzylguanidine derivatives useful for diagnosis or therapy, especially of neuroblastoma |
US6284223B1 (en) * | 1998-10-15 | 2001-09-04 | Fluoroprobe, Inc. | Method for viewing tumor tissue located within a body cavity |
-
1999
- 1999-04-09 DE DE19917713A patent/DE19917713A1/en not_active Ceased
-
2000
- 2000-03-28 WO PCT/EP2000/002697 patent/WO2000061194A2/en not_active Application Discontinuation
- 2000-03-28 JP JP2000610526A patent/JP2002541219A/en active Pending
- 2000-03-28 IL IL14559600A patent/IL145596A0/en unknown
- 2000-03-28 ES ES02090268T patent/ES2321586T3/en not_active Expired - Lifetime
- 2000-03-28 BR BR0009658-0A patent/BR0009658A/en not_active IP Right Cessation
- 2000-03-28 PL PL00351766A patent/PL351766A1/en not_active Application Discontinuation
- 2000-03-28 EE EEP200100521A patent/EE200100521A/en unknown
- 2000-03-28 DK DK00922560T patent/DK1176987T3/en active
- 2000-03-28 EP EP00922560A patent/EP1176987B1/en not_active Expired - Lifetime
- 2000-03-28 ES ES00922560T patent/ES2215641T3/en not_active Expired - Lifetime
- 2000-03-28 SK SK1415-2001A patent/SK14152001A3/en unknown
- 2000-03-28 MX MXPA01010174A patent/MXPA01010174A/en unknown
- 2000-03-28 AT AT02090268T patent/ATE421341T1/en not_active IP Right Cessation
- 2000-03-28 CN CN00806087A patent/CN1351505A/en active Pending
- 2000-03-28 YU YU70501A patent/YU70501A/en unknown
- 2000-03-28 AU AU42911/00A patent/AU769392B2/en not_active Ceased
- 2000-03-28 NZ NZ522136A patent/NZ522136A/en unknown
- 2000-03-28 CA CA002368490A patent/CA2368490A1/en not_active Abandoned
- 2000-03-28 KR KR1020017012861A patent/KR20020000555A/en not_active Application Discontinuation
- 2000-03-28 DE DE50005261T patent/DE50005261D1/en not_active Expired - Fee Related
- 2000-03-28 DE DE50015530T patent/DE50015530D1/en not_active Expired - Fee Related
- 2000-03-28 AT AT00922560T patent/ATE259246T1/en not_active IP Right Cessation
- 2000-03-28 HU HU0202990A patent/HUP0202990A3/en unknown
- 2000-03-28 PT PT00922560T patent/PT1176987E/en unknown
- 2000-03-28 CZ CZ20013562A patent/CZ20013562A3/en unknown
- 2000-03-28 NZ NZ514533A patent/NZ514533A/en unknown
- 2000-03-28 RU RU2001129707/04A patent/RU2001129707A/en not_active Application Discontinuation
- 2000-03-28 EP EP02090268A patent/EP1281405B1/en not_active Expired - Lifetime
-
2001
- 2001-10-08 BG BG105988A patent/BG105988A/en unknown
- 2001-10-09 NO NO20014911A patent/NO20014911L/en not_active Application Discontinuation
- 2001-11-08 ZA ZA200109238A patent/ZA200109238B/en unknown
- 2001-11-09 HR HR20010833A patent/HRP20010833A2/en not_active Application Discontinuation
-
2002
- 2002-11-26 HK HK02108542.4A patent/HK1046867A1/en unknown
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