WO2009056344A1 - Macromolécules noyau-enveloppe pour une coloration spécifique du noyau de cellules et/ou de la matrice de cellules - Google Patents

Macromolécules noyau-enveloppe pour une coloration spécifique du noyau de cellules et/ou de la matrice de cellules Download PDF

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WO2009056344A1
WO2009056344A1 PCT/EP2008/009224 EP2008009224W WO2009056344A1 WO 2009056344 A1 WO2009056344 A1 WO 2009056344A1 EP 2008009224 W EP2008009224 W EP 2008009224W WO 2009056344 A1 WO2009056344 A1 WO 2009056344A1
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macromolecule
shell
tetraphenoxy
macromolecules
pdi
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Meizen Yin
Klaus MÜLLEN
Tanja Weil
Jie Shen
Gert O. Pflugfelder
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MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V.
Johannes Gutenberg Universität Mainz
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F290/00—Macromolecular compounds obtained by polymerising monomers on to polymers modified by introduction of aliphatic unsaturated end or side groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F257/00—Macromolecular compounds obtained by polymerising monomers on to polymers of aromatic monomers as defined in group C08F12/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F277/00—Macromolecular compounds obtained by polymerising monomers on to polymers of carbocyclic or heterocyclic monomers as defined respectively in group C08F32/00 or in group C08F34/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F285/00—Macromolecular compounds obtained by polymerising monomers on to preformed graft polymers
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F289/00—Macromolecular compounds obtained by polymerising monomers on to macromolecular compounds not provided for in groups C08F251/00 - C08F287/00
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L51/00—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers
    • C08L51/003—Compositions of graft polymers in which the grafted component is obtained by reactions only involving carbon-to-carbon unsaturated bonds; Compositions of derivatives of such polymers grafted on to macromolecular compounds obtained by reactions only involving unsaturated carbon-to-carbon bonds
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08F—MACROMOLECULAR COMPOUNDS OBTAINED BY REACTIONS ONLY INVOLVING CARBON-TO-CARBON UNSATURATED BONDS
    • C08F2438/00—Living radical polymerisation
    • C08F2438/01—Atom Transfer Radical Polymerization [ATRP] or reverse ATRP

Definitions

  • Fluorescence is ideally suited for observing the location of molecules in cells as it is non-invasive and can be detected with high sensitivity and signal specificity.
  • the spectroscopic properties of fluorescence can be exploited to obtain information not only on the location of labelled macromolecules but also on the immediate molecular environment.
  • An ideal fluorescent dye for cell staining should combine water solubility, sufficient fluorescence quantum yield, high chemical and photostability, low toxicity, good biocompatibility and scalable production.
  • the highly fluorescent perylene-SA ⁇ .IO-tetracarboxydiimide (PDI) chromophore is a popular dye and pigment because of its excellent chemical, thermal and photochemical stability. (Y. Nagao, T. Misono, Dyes Pigm. 1984, 5, 171-188; H. Zollinger, Color Chemistry, VCH, Weinheim, 1987; R. M. Christie, Polym. Int. 1994, 34, 351-361 ).
  • PDI chromophores Due to these outstanding properties, there have been several successful applications of PDI chromophores in diverse fields, such as dye lasers, photovoltaic cells, organic light-emitting diodes, light-harvesting complexes, photoreactive thin films, and solar cells.
  • core shell macromolecules consisting of a non- fluorescent polyphenylene dendrimer core and a flexible polymer shell
  • V. Atanasov V. Sinigersky, M. Klapper and K. Mullen in Macromolecules 2005, 38, 1672-1683.
  • the authors obtained complex core shell structures with layers of different polarity and flexibility.
  • the stiff core - flexible shell nanoparticles are investigated as support for metallocenes catalysing the olefin polymerisation.
  • the described molecules are not fluorescent.
  • Core shell macromolecules with a tailored profile, i. e. high structural perfection, photostability, water-solubility, and biological specificity have not been demonstrated by the prior art.
  • a fluorescent dye fulfilling the above demands can be provided by a macromolecule consisting of a fluorescent core, an oligophenylene shell and a charged flexible polymer shell.
  • A is a chromophore comprising a polycyclic aromatic hydrocarbon
  • B x is a first polymer shell, wherein B is an oligo(phenylene)-based polymer comprising 1-
  • Cy is a second polymer shell, wherein C is a flexible polymer comprising 1 to 500 repeating units, and carrying at least one charged group; and y is an integer from 4-32.
  • the first oligo(phenylene)-based polymer shell B x is suitable to prevent the central chromophore A from aggregation in aqueous media, which usually leads to reduced fluorescence quantum yields.
  • the chromophore A preferably comprises 2 to 20 annelated phenylene rings. More preferred are polycyclic aromatic hydrocarbons comprising 4-11 or 5-8 annelated phenylene rings. Exemplary chromophores of the invention are based on polycyclic aromatic hydrocarbons selected from the group consisting of naphthalene, pyrene, perrylene, terrylene or quaterrylene and derivatives thereof, which may be substituted or unsubstituted. Mono- or diimide-derivatives thereof are particularly preferred.
  • the polycyclic aromatic hydrocarbon is selected from naphtalene, naphtalene-monoimide, naphtalene-diimide, pyrene, perylene, perylene-monoimide, perylene- diimide, terrylene and derivatives thereof.
  • the imide nitrogen is in each case independently bound to a radical R 1 .
  • R 1 is in each case independently an organic radical, in particular a secondary or tertiary aliphatic or aromatic, linear, branched chain or cyclic radical having typically 3-30 carbon atoms and optionally one or more heteroatoms which are preferably selected from N 1 O and/or S.
  • suitable radicals are in particular mono- or bicyclic aromatic or heteroaromatic radicals, for instance phenyl, pyridyl or naphthyl, which optionally bears one or more substituents.
  • R 2 examples include R 2 , CN, NO 2 , halogen (e.g. F, Cl, Br or I), OH, OR 2 , OCOR 2 , SH, SR 2 , SCOR 2 , SO 2 R 2 , CHO, COR 2 , COOH, COOR 2 , CONH 2 , CONHR 2 , CON(R 2 ) 2 , SO 3 H, SO 3 M, SO 3 R 2 , NH 2 , NHR 2 or N(R 2 ) 2 , wherein M is a cation, e.g. an alkali metal ion such as sodium, potassium, etc., and R 2 is an optionally halogen-substituted linear or branched chain Ci-Ce-alkyl moiety.
  • halogen e.g. F, Cl, Br or I
  • OH e.g. F, Cl, Br or I
  • OR 2 , OCOR 2 , SH, SR 2 , SCOR 2 , SO 2 R
  • the chromophore A is selected from
  • TMI 1,6,9,14-tetraphenoxy-N-(2, 6-diisopropylphenyl)-terrylene-3,4-dicarboxy- imide
  • QDI i .e.H .I ⁇ -tetraphenoxy-N.N' ⁇ .e-diisopropylphenyOquaterrylene-S ⁇ .IS.M- tetracarboxdiimide
  • A is
  • the central chromophore A is surrounded by a first shell B x , comprising x oligo(phenylene)-based polymers B which may be the same or different.
  • B comprises 1-100 phenylene rings, preferably 1- 64 phenylene rings, more preferably 4-16 phenylene rings.
  • the oligo(phenylene)-based shell B x is a rigid dendrimer shell, capable to suppress aggregation of the chromophore in aqueous solution, particularly preferred are first and second generation dendrimers.
  • the oligo(phenylene)-based polymers B may be bound to the chromophore for instance via a direct bond or via a bifunctional linker such as for example Ci-C 6 alkylene, -O-, -S-, etc. Further examples of suitable linkers are given below.
  • the second polymer shell C y enables the introduction of different numbers of polar functionalities. Thus it is possible to achieve tailor made properties with regard to charges, charge densities and water solubility.
  • C y comprises y flexible polymers C 1 which may be the same or different, each comprising 1- 500 repeating units, preferably 2-200 repeating units, and carrying at least one charged group, y is an integer from 4-32, preferably 4-16.
  • One or more flexible polymers C may be covalently bound to B, in particular C may be bound via a direct bond or via a bifunctional linker.
  • each B is bound to at least one C.
  • Bifunktional linkers suitable for the purpose of the invention are for example linear or branched chain alkylene, aralkylene, arylene, which may optionally be substituted with suitable substituents as defined above, -O-, -CO-, -COO-, -OR-, -COR-, -COOR-, -ROR-, -RCOR-, -RCOOR-, -NH-CO- -NH-, etc., wherein R is for example linear or branched chain alkylene, aralkylene or arylene.
  • the flexible polymer is preferably selected from the group consisting of linear or branched chain, substituted or unsubstituted polyethylene, polypropylene, polyacrylate, polymetacrylate, polyacrylamide and/or polymetacrylamide. [Examples of suitable substituents are as given above.
  • a flexible polymer C carries at least one positively or negatively charged group.
  • the charged group is preferably a charged group, which is charged in neutral media, for example at pH 7.
  • Examples for negatively charged groups are carboxylic acid or sulfonic acid groups, -CO 2 H or -SO 3 H.
  • Positively charged groups are preferably amines, for instance an amino group or an ammonium group, in particular a quatemized ammonium group, or an alkylated heteroaromatic nitrogen atom, in particular an N- alkylpyridinium, N-alkylquinolinium or N-alkylisoquinolinium group, wherein the alkyl moiety preferably has up to 6 carbon atoms and may optionally be substituted as described above.
  • the charged group(s) can be located at the polymer main chain and/or at side chains.
  • Examples of flexible polymers according to the invention are: H poly(meth)acrylic acid ide
  • the charged flexible polymer shell also provides the macromolecules of the invention with improved binding properties that allow the application of the described compounds as colorants in cytochemistry and histochemistry.
  • a further embodiment of the invention relates to the preparation of the above-described charged core-shell macromolecules.
  • Subject matter of the invention is a method for preparing a macromolecule of formula (I) comprising the steps i) providing a chromophore A, ii) building up a first polymer shell B x around A, iii) building up a second polymer shell C 7 around B x .
  • Step (ii) preferably comprises one or more Diels-Alder [3+2] cycloaddition reactions and/or palladium(O) catalyzed coupling reactions such as Suzuki reactions.
  • a chromophore A may be used, which has one or more substituents providing reactive functionalities.
  • Reactive functionalities may for example be alkynyl groups.
  • the first polymer shell may be built up by means of a Diels-Alder reaction with e.g. cyclopentadienone or derivatives thereof. It is particularly preferred to use cyclopentadienone derivatives, which are substituted by one or more phenyl rings.
  • additional substituents may be present at the cyclopentadienone or at phenyl residues bound thereto, Preferably, such additional substituents provide suitable reactive groups, which can then be applied in step (iii) for creating a second polymer shell.
  • step (iii) flexible polymer chains are attached to the polyphenylene shell in order to build up an outer second polymer shell C ⁇ .
  • Step (iii) involves for instance living anionic polymerization reactions and/or controlled radical polymerization.
  • macromolecules bearing negatively charged groups are capable to specifically stain those compartments of the nucleus where positively charged histones are enriched along with the genomic DNA.
  • negatively charged groups e.g. carboxylic acid groups
  • chromatin a dense structure called chromatin.
  • Chromatin is largely made up of higher order arrangements of nucleosomes.
  • DNA is tightly wrapped around a core of histone proteins.
  • the protein complement of chromatin largely consists of histones, basic proteins which are positively charged at neutral pH, and non-histone chromosomal proteins which are predominantly acidic at neutral pH.
  • DNA-histone binding is attributed to charge-charge interactions as well as to hydrogen bonds and salt links between DNA-phosphate oxygen atoms and protein basic and hydroxyl side chain groups.
  • 15d The inventors found out, that these negatively charged water-soluble core-shell macromolecules potentially interact with histones by mechanisms similar to the DNA-histone interactions. It is assumed that the interaction is due to the presence of negatively charged groups such as -COOH groups, which are capable to interact with histones by electrostatic interaction as well as by hydrogen bonds and salt links.
  • macromolecules bearing positively charged groups are capable to specifically stain the extracellular matrix (ECM).
  • ECM All cells in animal tissue are surrounded by ECM or, in the case of epithelia, are bounded on one side by the basement membrane, a specialized ECM.
  • the ECM provides mechanical strength and elasticity, absorbs shock from extracellular change, retention of water, transports signals between cells, binds growth factors, and interacts with cell-surface receptors.
  • the ECM does not constitute an inert environment but is affected by and feeds back on cellular physiology.
  • the ECM in general is composed of highly hydrated proteins and carbohydrates. Main protein components are collagen and elastin, the glycoproteins fibronectin and laminin, and proteoglycans containing long glycosaminoglycan (GAG) side chains (Fig. 1B).
  • GAGs 1 which can also occur in free form in the ECM, are build from characteristic repeating disaccharide units and are highly negatively charged due to the presence of uronic acids and sulphate modifications.
  • the main GAGs are hyaluronan, chondrotin and dermatan sulfate, heparan sulfate, and keratan sulfate.
  • a positively charged core-shell macromolecule of the invention is able to bind to the highly negatively charged ECM, and, thus, is applicable to label ECM in animal tissue.
  • chromophore A and flexible polymer C are as defined above in context with macromolecules of formula (I).
  • Preferred examples of chromophore A and flexible polymer C are as described above.
  • C is covalently bound to the chromophore A via a direct bond or via a bifunctional linker as defined above.
  • Linear macromolecules comprising a chromophore A and a flexible polymer can be prepared by a method involving living anionic polymerization reactions and/or controlled radical polymerization (NMP, ATRP, RAFT).
  • Fig. 1 shows PDI labeled star polymers containing peripheral carboxylic acid (P1 and P2) and hydroxyl-groups (P3) groups.
  • Fig. 2 (A) Absorption of P1 (dash) and P2 (line) in water;
  • Fig. 5 A. Normalized emission spectra of histones (dotted line), P1 (dashed line) and mixture of P1 and histones (continuous line) in water;
  • FIG. 6 Staining of Drosophila larval tissues by P1 (A to E), P2 (F), P3 (G), the DNA-specific detection agent DAPI (H), and anti-OMB (I).
  • A-E P1 shows exclusively nuclear localization in the squamous epithelium of the wing (A) or leg imaginal disc (B), in the columnar epithelium of the antennal imaginal disc (C), in the fat body (D), in tracheal cells (E).
  • P2 localizes to nuclei but also to the cell membrane (*) of fat body cells (F).
  • Anti-OMB trachea, I).
  • FIG. 7 Confocal images of P1 staining of Drosophila wing columnar (A, C and D) and squamous epithelium (B).
  • A-D P1 (red) showed exclusively nuclear localization in the wing epithelium.
  • Cell membrane was marked by CD8-GFP (green) (A).
  • Microtubule web was marked by anti-Tubulin (green) (B).
  • C P1 (red) and Omb (green) showed a complementary nuclear staining pattern.
  • D P1 (red) and DAPI (blue) staining was essentially coincident. Single channels were separated in (D') and (D").
  • Fig. 8 Gel-electrophoresis of free DNA and the mixture of DNA and P1.
  • Fig. 9 (A). Diagram to illustrate the Drosophila wing epithelium.
  • C Disaccharide building blocks of the sulfated GAGs chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS) and keratan sulfate (KS).
  • Fig. 10 Structures of the PDI derivatives containing multiple amine groups (P4-P6) and toluidine blue (TB).
  • Fig. 11 Dot blot assay for P4-protein interaction.
  • P4 did not accumulate on the dot of collagen (A, under VIS light; A', under UV light). P4 accumulated on the dot of aggrecan (B, under VIS light; B', under UV light).
  • Fig. 12 Normalized emission spectra of aggrecan (dot), P4 (dash) and mixture of P4 and aggrecan in water.
  • Fig. 13 PDI labelled polymers specificcally stained the ECM in Drosophila wing imaginal discs.
  • A A VIS light picture in X-Z plane showed the outline of the epithelia by Toluidine blue staining.
  • B A electron microscope picture in X-Z plane which was positioned by a box in (A) showed the ECM surrounding the epithela.
  • C A fluorescence image in X-Z plane showed the ECM by P4 (red) staining and the cell membranes of the epithelia by GFP (green) antibody staining. Fluorescence images of ECM network stained by PDI labelled polymers P4 (D), P5 (E) and P6 (F) in X-Y planes.
  • Fig. 15 GPC curves of the initiator 4 and star polymers 14a, 14b, 14c.
  • Fig. 16 (A) The absorption of P4 (solid), P5 (dash) and P6 (dot) in water; (B) Normalized emission spectra of P4 (solid), P5 (dash) and P6 (dot) in water.
  • Fig. 17 VIS images of P5 (A) and P6 (B) accumulated on the dot of Aggrecan.
  • the dendritic core-shell macromolecules P1 and P2 consist of a central perylenediimide (PDI) chromophore, a polyphenylene dendrimer scaffold and a polymer shell with multiple functional groups.
  • PDI central perylenediimide
  • the high tendency of the PDI chromophore to form aggregates in aqueous media usually leads to a significant reduction of fluorescence quantum yields. Therefore a rigid polyphenylene dendrimer shell was introduced in the bay region of the PDI dye in order to suppress the aggregation of the central PDI chromophore (Scheme 1).
  • a synthetic strategy towards the introduction of a PDI chromophore into the centre of a polyphenylene dendrimer was reported before and it was based on a PDI chromophore carrying four ethynyl groups (1, Scheme 1).
  • free ethynyl groups of the PDI chromophore were reacted with four tetraphenylcyclopentadienones 3 each carrying two 2-bromo-2-methylpropionic ester groups which are suited as initiators for atom transfer radical polymerization (ATRP) for the build up of the hydrophilic polymer shell.
  • ATRP atom transfer radical polymerization
  • the reaction proceeded in a microwave reactor at 170 0 C for 2-3 hours to give the first-generation polyphenylene dendrimer 4 with eight 2-bromo-2-methylpropionic ester groups.
  • 11 The functionalized cyclopentadienone 3 survives the reaction conditions required for the Diels-Alder cycloaddition since decomposition reactions occurred only above 250 °C.
  • a second-generation polyphenylene dendrimer was achieved by reacting 1 with tri/sopropyisilylcyclopentadienone (Cp-Tips 2 ), subsequent deprotection of the tri/sopropylsilyl groups in the presence of fluoride ions to give a first-generation polyphenylene dendrimer 2 with eight ethynyl groups. 81 Then, 2 reacted with 3 thus yielding the multifunctional initiator 5 with sixteen 2-bromo-2-methylpropionic ester groups at the periphery.
  • Cp-Tips 2 tri/sopropyisilylcyclopentadienone
  • core-shell macromolecules 6 or 7, having precisely eight or sixteen arms was achieved via an atom transfer reaction polymerization (ATRP) of tert-butyl acrylate (tBA) by using 4 or 5 as macroinitiators (Scheme 2).
  • ATRP atom transfer reaction polymerization
  • tBA tert-butyl acrylate
  • Scheme 2 macroinitiators
  • Compound 6 is based on a first generation polyphenylene dendrimer with eight arms and compound 7 consists of a second generation polyphenylene dendrimer with sixteen arms. Both macromolecules are soluble in organic solvents such as dichloromethane and tetrahydrofurane.
  • a hydrodynamic radius of 4 nm was found for P1 based on a first-generation polyphenylene dendrimer with eight arms, and a R h of 9 nm for P2 based on a second-generation polyphenylene dendrimer with 16 arms (Table 2).
  • the UV/Vis absorption and emission spectra of P1 and P2 were measured in water (Fig. 2, Table 2).
  • the fluorescence quantum yields ( ⁇ f ) of P1 and P2 were obtained by using the quantum yield of the negatively charged PDI derivative 8 in water as reference.
  • P2 had higher extinction coefficients ( ⁇ ) and ⁇ f in water than P1.
  • P2 showed a slight bathochromic shift of the longest wavelength absorption maximum in the UV spectrum (4 nm) in comparison to P1 (Fig. 2A).
  • no differences were observed in the emission spectra with both P1 and P2 revealing an emission maximum at 619 nm (Fig. 2B).
  • Emission maxima above 600 nm are generally considered to be advantageous for biological imaging experiments since the background coming from the autofluorescence of living cells is negligible in this region.
  • ⁇ The intensities of the UV/Vis absorptions and the fluorescence bands of P1 and P2 in water remained almost unchanged after irradiation under UV light (350 nm, 40 W) for two days or exposure to sun light for two weeks, pointing to a good photostability of these novel fluorescent dyes.
  • the emission spectra of their mixtures was recorded.
  • the mixtures of histones and P1 or P2 showed a hypsochromic shift (10 nm) of the emission maxima in comparison to P1 or P2 alone.
  • P1 had a similar sensitivity and specificity as the conventional nuclear fluorescent stain DAPI (FigJD and Fig. 6H).
  • DAPI has a broad fluorescence emission spectrum (380-700nm), its quantum yield is 0.043 and it is easily bleached during scanning on fluorescence microscopy. 17
  • the emission of P1 is much more stable than that of DAPI and the narrower emission spectrum of P1 provides the opportunity for greater choice for combinations with other fluorescent dyes when performing double or triple staining.
  • the P1 staining method has the advantage of being much simpler and faster.
  • the fluorescent dyes for staining the cell nucleus by interaction with DNA no histone-specific fluorescent dyes have been available so far.
  • Carboxylated water-soluble PDI core-shell macromolecules with attractive optical properties were synthesized. They specifically stain the cell nucleus by interaction with positively charged chromatin histones. We attribute the interaction to the presence of multiple side chain -COOH groups, which could interact with histones by electrostatic interaction as well as by hydrogen bonds and salt links. Due to their ease of synthesis and their high fluorescence quantum yields, these compounds offer an attractive alternative to conventional expensive antibodies or chromophores displaying broad emission spectra such as DAPI.
  • HEMA 2-Hydroxyethyl methacrylate
  • the PtBA polymer (6 or 7, 100 mg) was placed in a Schlenk flask equipped with a magnetic stirring bar. Then, the flask was evacuated and backfilled with argon three times. Dichloromethane (20 ml) was added to dissolve the polymer. Then, trifluoroacetic acid (10 ml) was added to the solution, and the mixture was stirred at room temperature for 20 h. The solvent was removed from the resulting heterogeneous mixture, and the residual solid was washed with dichloromethane (10 ml, 3 times) followed by drying under vacuum at room temperature for 10 h to give P1 or P2 as a red solids.
  • the PDI derivative 4 bearing eight 2-bromo-2-methylpropionic ester groups was used as macroinitiator for ATRP of HEMA.
  • 2-butanone and propanol 70:30, v/v, 1 g HEMA/4 ml solvent
  • Rinse 4 x in PBT Wash for 0.5 h rotating at room temperature. 5. Incubate dissected larvae in 0.1% P1 solution for 1 h rotating at room temperature.
  • Rinse 4 x in PBT Wash for 1 h rotating at room temperature. 6. Mount in 50% glycerol, optionally containing 0.5 ⁇ g/ml DAPI.
  • a 0.8% agarose (w/v) gel in 1X TBE buffer was performed for 2 h at 4 V/cm.
  • 5 ⁇ l 1kb DNAIadder (PeqLab) was incubated with 5 ⁇ l 0.2% core-sheH macromolecules for 10 min before loading.
  • the gel was stained with 1 ⁇ g/ml ethidium bromide in IxTBE for 30 min after electrophoresis. The photograph was taken under UV light.
  • the fluorescent star polymers P4 and P5 consist of a central PDI chromophore, a stiff polyphenylene dendrimer scaffold, and a polymer shell with multiple amine groups.
  • P4 is based on a first-generation polyphenylene dendrimer with eight arms
  • P5 is based on a second-generation polyphenylene dendrimer with 16 arms.
  • the linear polymer P6 which is based on the perylenemonoimide (PMI) chromophore was tested as well.
  • the photochemical properties of P4, P5, and P6 are summarized in Table 3 and figure 16. The emission maxima of all fluorescent polymers are above 600 nm.
  • ⁇ f was measured at room temperature using the negatively charged PDI derivative 8 in water (the standard value is 0.58) as reference.
  • Tissue collagen occurs as big fibers composed of trimeric largely uncharged ⁇ -chains which are stabilized by cystine bonds (Sigma product description).
  • the primary sequence of the collagen ⁇ -chains consists of a repeating tri peptide made up of glycine, proline and hydroxyproline such that collagen has little net charge.
  • the Drosophila wing imaginal disc was selected as an example of epithelial tissue.
  • the wing imaginal discs consists of two layers of cells (squamous and columnar epithelia) shown in cross- section of a toluidin-blue stained preparation in Fig. 13A.
  • the two epithelial layers enclose a lumenal space.
  • the basal aspects of the imaginal disc cells are oriented toward the exterior and secrete the basal lamina.
  • the common characteristic of the three polymers is the high density of mono-functional -NH 2 groups which will be largely protonated in the neutral- buffered solution. It is likely that they are involved in electrostatic interaction and hydrogen bonds as well as salt links with the negatively charged GAG chain sulfate groups. Despite of their different size and shape, P4, P5 and P6 with similar specificity visualized the ECM network.
  • a synthetic strategy towards the introduction of a PDI chromophore into the centre of a polyphenylene dendrimer was reported before and it was based on a PDI chromophore carrying four ethynyl groups (1, Scheme 1 ).
  • free ethynyl groups of the PDI chromophore were reacted with four tetraphenylcyclopentadienones 3 each carrying two 2-bromo-2- methylpropionic ester groups which are suited as initiators for atom transfer radical polymerization (ATRP) for the build up of the hydrophilic polymer shell.
  • ATRP atom transfer radical polymerization
  • the reaction proceeded in a microwave reactor at 170 0 C for 2-3 hours to give the first-generation polyphenylene dendrimer 4 with eight 2-bromo-2- methylpropionic ester groups. It was described before that the functionalized cyclopentadienone 3 survives the reaction conditions required for the Diels- Alder cycloaddition since decomposition reactions occurred only above 250
  • a second-generation polyphenylene dendrimer was achieved by reacting 1 with tri/sopropylsilylcyclopentadienone (Cp-Tips 2 ), subsequent deprotection of the tri/sopropylsilyl groups in the presence of fluoride ions to give a first- generation polyphenylene dendrimer 2 with eight ethynyl groups. Then, 2 reacted with 3 thus yielding the multifunctional initiator 5 with sixteen 2- bromo-2-methylpropionic ester groups at the periphery.
  • Cp-Tips 2 tri/sopropylsilylcyclopentadienone
  • a molecular initiator based on a perylenemonoimide (PMI) chromophore was synthesized in a multi-step reaction (Scheme 4).
  • a Suzuki coupling reaction of compound 9 and 10 in toluene at 100 0 C gave PMI 11 carrying a phenoxy group at position 9.
  • Subsequent ether cleavage was achieved with boron tribromide to give the hydroxy-substituted PMI 12 which underwent a condensation reaction with 2-bromo-2-methylpropionyl bromide under caustic condition to give the PMI-initiator 13.
  • the polymerization conversion was controlled at conversions below 20 %, monitored by 1 H-NMR spectroscopy.
  • the molecular weights of the prepared core-shell macromolecules were determined using GPC and 1 H-NMR spectroscopy.
  • the theoretical molecular weights, calculated by the conversion of polymerization and estimated from the relative intensities of the signals obtained by 1 H NMR spectroscopy usually point towards higher molecular weights of the macromolecules in comparison to the results from GPC experiments.
  • Such systematic errors were already reported in previous work and were attributed to the difference in shape between the star polymers and the linear standards used in the GPC measurements. Nevertheless, monomodal distributions were found in all the GPC measurements except for the star polymers with 80 repeat units of the polymers shell.
  • the UV/Vis absorption and emission spectra of P4, P5 and P6 were measured in water.
  • the fluorescence quantum yields ( ⁇ f ) of P4, P5 and P6 were obtained by using the quantum yield of the negatively charged PDI derivative 5 in water as reference.
  • both P4 and P5 revealing an absorption maximum at 580 nm (Fig. 16A).
  • P5 shows a bathochromic shift of the largest wavelength emission maximum in the UV spectrum (5 nm) in comparison to P4 (Fig. 16B).
  • P5 has higher extinction coefficients ( ⁇ ) and ⁇ f in water than PA. In comparison with the core-shell macromolecules, the absorption and emission spectra of linear polymer P6 are much smaller.
  • MALDI-TOF mass spectrometry measurements were performed on VG ZAB2-SE-FPD Spectrofield, Bruker Reflex I (MALDI-ToF) and Bruker Reflex Il (MALDI-TOF) mass spectrometers.
  • JR: v (cm- i ): 3050, 3030, 2970, 2930, 1750, 1710, 1670, 1590, 1500, 1260, 1200, 1160, 1140, 1100, 1020, 1010,700, 758, 698, 577 cm 1 .
  • N-(2, 6-Diisopropylphenyl)-9-bromo-perylene-3, 4-dicarboxy-imide (9) (2.2 g, 3.9 mmol) and 4-methoxyphenylboronic acid (10) (500 mg, 3.3 mmol) were dissolved in 200 ml_ toluene under argon atmosphere, then 30 mL of 1 M aqueous potassium carbonate solution and tetrakis(triphenylphosphine)palladium(0) (Pd(PPh 3 J 4 ) (380 mg, 0.33 mmol) added. The resultant mixture was heated under argon for 24 h at 100 0 C.
  • the monomer was placed in a Schlenk flask and dissolved in the 2- butanone. CuBr were then added and the mixture was degassed by three freeze-pump-thaw cycles.
  • the ligand 4,4'-di-tert-butyl-2 l 2'-bipyridine (DTB- bipy) was added under argon. And then the solution was stirred at 25 0 C for 10 min. Finally the initiator was added and the flask was sealed and placed in a thermostated oil bath at 7O 0 C to start the reaction. The polymerizations were stopped by cooling with liquid nitrogen after a defined reaction time. The monomer conversion was determined by 1 H NMR spectroscopy.
  • the reaction mixture was diluted and eluted through a column filled with neutral alumina to remove the copper complex. The solvent was removed under vacuum and the polymer was isolated by precipitation into methanol and drying under vacuum to constant weight.
  • Core-shell star polymer of 2-[(teff-butoxycarbonyl)amino]ethyl methacrylate (100 mg) was dissolved in CH 2 CI 2 (30 mL) in a 100-mL round- bottomed flask, and then phenol (3 M in CH 2 CI 2 , 5 mL) and chlorotrimethyl silane (1 M in CH 2 CI 2 , 5 mL) were added and the mixture was stirred for 2 h at room temperature. The solution was evaporated to dryness and the residue dissolved in methanol (10 mL). The product was precipitated upon addition of this solution to a mixture of diethyl ether and dried under vacuum.
  • BOC-AEMA 2-[(teff-butoxycarbonyl)amino]ethyl methacrylate

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  • Chemical Kinetics & Catalysis (AREA)
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  • Organic Chemistry (AREA)
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Abstract

Selon l'invention, des macromolécules noyau-enveloppe fluorescentes solubles dans l'eau contenant de multiples groupes acides carboxyliques ont été synthétisées par ATRP par application de dérivés de pérylènediimide comme matrice. Ces composés présentent des rendements quantiques de fluorescence élevés et une photostabilité élevée dans l'eau. Des expériences d'histochimie et de transfert en point montrent que ces macromolécules colorent de façon spécifique et rapide le noyau de cellules par interaction avec les histones. En raison de leurs propriétés spectrales, ces composés offrent une alternative attrayante aux procédés de coloration fluorescente existants.
PCT/EP2008/009224 2007-11-02 2008-10-31 Macromolécules noyau-enveloppe pour une coloration spécifique du noyau de cellules et/ou de la matrice de cellules WO2009056344A1 (fr)

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Cited By (4)

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CN103204999A (zh) * 2013-03-22 2013-07-17 北京化工大学 水溶性荧光树枝状大分子的合成方法及其应用
CN103999853A (zh) * 2014-06-11 2014-08-27 中国农业大学 荧光树枝状纳米大分子在制备药物载体中的应用
CN107033267A (zh) * 2017-03-15 2017-08-11 北京化工大学 一系列基于莱啉基化合物的水溶性聚合物的制备及其作为光热试剂在生物医学的应用
WO2021210377A1 (fr) * 2020-04-15 2021-10-21 コニカミノルタ株式会社 Composé luminescent, procédé de production de composé luminescent, composition luminescente, couche mince luminescente et particules luminescentes

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GB2407501A (en) * 2003-11-03 2005-05-04 Ist Superiore Sanita Nanoparticles for delivery of a pharmacologically active agent, comprising water insoluble (co)polymer core & hydrophilic acrylate-based (co)polymer shell
WO2006075328A1 (fr) * 2005-01-12 2006-07-20 Mempile Inc. Composes presentant un pouvoir d'absorption biphotonique ameliore pour des applications non lineaires
WO2006075327A1 (fr) * 2005-01-12 2006-07-20 Mempile Inc. Disques ameliores pour stockage de donnees
FR2909094A1 (fr) * 2006-11-28 2008-05-30 Arkema France Memoire optique 3d comprenant des particules multicouches comprenant un monomere photoactif porteur d'un groupement photoisomerisable.

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US3799779A (en) * 1971-09-13 1974-03-26 Minnesota Mining & Mfg Light-desensitizable imaging sheet
KR20030072658A (ko) * 2002-03-06 2003-09-19 한국전자통신연구원 블록 공중합체의 자기조립체 제조 방법 및 크로머포어가나노 캡슐화된 블록 공중합체의 자기조립체
GB2407501A (en) * 2003-11-03 2005-05-04 Ist Superiore Sanita Nanoparticles for delivery of a pharmacologically active agent, comprising water insoluble (co)polymer core & hydrophilic acrylate-based (co)polymer shell
WO2006075328A1 (fr) * 2005-01-12 2006-07-20 Mempile Inc. Composes presentant un pouvoir d'absorption biphotonique ameliore pour des applications non lineaires
WO2006075327A1 (fr) * 2005-01-12 2006-07-20 Mempile Inc. Disques ameliores pour stockage de donnees
FR2909094A1 (fr) * 2006-11-28 2008-05-30 Arkema France Memoire optique 3d comprenant des particules multicouches comprenant un monomere photoactif porteur d'un groupement photoisomerisable.

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103204999A (zh) * 2013-03-22 2013-07-17 北京化工大学 水溶性荧光树枝状大分子的合成方法及其应用
CN103999853A (zh) * 2014-06-11 2014-08-27 中国农业大学 荧光树枝状纳米大分子在制备药物载体中的应用
CN107033267A (zh) * 2017-03-15 2017-08-11 北京化工大学 一系列基于莱啉基化合物的水溶性聚合物的制备及其作为光热试剂在生物医学的应用
CN107033267B (zh) * 2017-03-15 2020-04-28 北京化工大学 一系列基于莱啉基化合物的水溶性聚合物的制备及其作为光热试剂在生物医学的应用
WO2021210377A1 (fr) * 2020-04-15 2021-10-21 コニカミノルタ株式会社 Composé luminescent, procédé de production de composé luminescent, composition luminescente, couche mince luminescente et particules luminescentes

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