ES2360366T3 - MONOMERAL LINKS AND USEFUL POLYMERS TO CONJUG BIOLOGICAL MOLECULES AND OTHER SUBSTANCES. - Google Patents

Abstract

A linear polymeric linker comprising at least two consecutive units chosen from: where - R1 and R2 are chosen from NH and O, - R3 is chosen from methyl, ethyl, propyl, CH2OCH2 and (CH2OCH2) 2, and where the linker It comprises between one and three ethyloxy groups repeated consecutively.

Description

The present invention relates to monomeric or polymeric linkers useful in biological and chemical applications, to their synthesis, and to the synthesis and use of conjugated linker derivatives with a variety of detectable labels and other substances. The linkers can be used, for example, together with fluorescent markers, nucleic acid or nucleic acid analog probes, and solid phase systems, and to improve the solubility of conjugated atoms, groups or molecules.

Background and summary of the invention

Large molecules and detectable markers such as fluorophores, matrices or solid phase beads, antibodies and hybridization probes, are frequently attached to other molecules indirectly through linkers, to avoid steric shocks that could decrease the affinity between those molecules and their targets. , thereby reducing its biological activity. A "linker" is a molecule that serves to bind other atoms, molecules or functional groups together, through covalent or non-covalent interactions.

Ideally, a linker can separate a fixed detectable label, from a target molecule, without affecting the useful properties of the detectable label, such as its signal strength, its binding affinity or its solubility. However, in practice, numerous linker molecules used in the art have a relatively short length, making it difficult to avoid steric shocks between the substances used for juxtaposition. For example, linkers such as 6-amino-hexanoic acid, succinimidyl4- (N-maleimidomethyl cyclohexane-1-carboxylate (SMCC) and N-γ-maleimidobutyryloxy succinimide ester (GMBS); are between 7 and 9 atoms in length, which would correspond to approximately 1 nm, if fully extended in solution. That length is only a small fraction of the average size of the biological macromolecule, such as a protein or an oligonucleotide, and may be insufficient to avoid potential steric collisions between atoms, the groups or molecules with which these linkers are conjugated Other examples of relatively short linkers used in the art include homobifunctional linkers, such as glutaric dialdehyde, divinylsulfone, hexane di-isocyanate, dimethylapimidate, 1,5-difluoro-2 , 4-dinitrobenzene, heterobifunctional linkers, such as N-gamma-maleimidobutyryloxy-succinimide ester, and cer-length linkers or, such as 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide.

Longer polyethylene glycol (PEG) based linker molecules are also available in the art. (See, for example, Discrete PEG (dPEG) ® modification reagents, available from Quanta Biodesign, Ltd., Powell, OH, or www.quantabiodesign.com; PEG-based reagents available from EMD Biosciences, Inc., San Diego, CA, described in the Novabiochem brochure, April 2004, "Product focus: PEG reagents-bifunctional amino-PEGacid spacers", available at www.novabiochem.com; and see Baumeister et al., Biopolymers, 71: 339 ( 2003); Kumar and AIdrich, Org. Lett., 5: 613 (2003). (See also, "Chemistry of Protein Conjugation and Cross-Linking" Shan

S. Wong, CRC Press, Boca Raton, Florida, USA, 1993; "BioConjugate Techniques" Greg T. Hermanson Academic Press, San Diego, California, USA, 1996; "Catalog of Polyethylene Glycol and Derivatives for Advanced PEGylation, 2004" Nektar Therapeutics Inc., Huntsville, Alabama, USA). Although PEG-based linkers have a longer length, for example, from 20 to more than 70 atoms, they may not always remain extended in solution, and instead unwanted tertiary structures may be added or formed. Such unwanted homoaggregation or heteroaggregation may adversely affect the activity of conjugated atoms, groups or molecules.

The present invention is defined by the claims.

One object described was to find a linker structure that could adopt a variety of lengths, including very long lengths, that may have a well defined molecular and structural formula, even with very long lengths, that would have less tendency to form unwanted or aggregate tertiary structures , which could be easily and efficiently polymerized if desired, and could be found in extended form in solution. The linkers described may allow different atoms, groups or conjugated molecules to separate with short distances or, for example, up to a few nanometers, in solution. The present linkers can also be designed so that they have well defined molecular and structural formulas, providing greater uniformity to the molecular entities that comprise them. In some of the present embodiments, even very long linkers according to the present invention, they remain surprisingly in an extended and flexible structure in aqueous solutions and have a slight tendency to fold over themselves, to aggregate with each other, or to form other tertiary structures that interfere with the functions of the atoms, groups or conjugated molecules that are carriers. Some of the present linkers form chains with little steric volume along their length, and thus, can easily access narrow molecular spaces to interact with solid phase surfaces or membranes with a high density. The present invention also comprises methods for synthesizing the present linkers and fixing them covalently to other molecules and functional groups with high yield.

The linkers of the present invention may be covalently or non-covalently bound to a variety of substances that include color markers, solid surface components, nucleic acids and nucleic acid analogs, proteins and protein substrates. The ability of some of the present linkers to remain extended and flexible in solution, can minimize steric interference between the linker and the conjugated atom, group or molecule, so that the conjugate signal or its availability to be recognized by the target molecules, is minimally disturbed by the linker. Thus, the present invention comprises a method for improving signal strength, activity or affinity of the binding of a molecule, such as a detectable label, which includes a probe, comprising conjugating the molecule with one or more of the Present linkers.

For example, when protein-nucleic acid (PNA) probes are conjugated to linkers, PNAs are less susceptible to aggregation and remain more available to bind to their intended target sequences. Furthermore, since the structure of the present linkers in solution can be long and flexible, the present linkers can be used to cross-link large molecules to each other, in a way that allows the molecules to adopt their preferred orientations, and thereby minimally disturb the structure and affinity of the molecular complex.

As another example, steric volume or tertiary structure near a fluorescent label can cause an extinction of fluorescence due to the absorbance of the signal by the surrounding molecular bonds. Interactions between two or several nearby fluorophores can also cause the extinction of their signals. This can be a problem when designing detectable markers comprising multiple conjugated fluorophores. For example, in protein detection, typically only about 3-4 fluorophores can be added, before reaching a plateau in signal strength. The addition of other fluorophores may even decrease the overall signal. Said extinction can be reduced using embodiments of the present invention. Accordingly, this invention includes a method for improving the signal of one or more fluorophores, which comprises combining one or more fluorophores with at least one of the present linkers. Because the extinction due to the binding of the linker of the present invention can be minimal, since the individual fluorophores can be conjugated so as not to significantly interfere with each other, the present linkers also allow the design of fluorophores linker derivatives. multiple that have predictable emission colors that are a combination of emissions from individual fluorophores with different emission spectra.

Another surprising feature of some of the present linkers is their high water solubility, despite the relatively limited number of polar or charged groups. The present linkers can improve the solubility of a variety of different atoms, groups or conjugated molecules. Thus, this invention also comprises a method for improving the solubility of an atom, a group or a conjugated molecule, comprising directly or indirectly conjugating by combining one or more atoms, groups or molecules with one or more of the present linkers.

Additional objects and advantages of the invention are described in the description that follows.

Brief description of the drawings

The accompanying drawings, which are incorporated and constitute a part of this specification, illustrate some of the embodiments of the invention and, together with the description, serve to explain some of the principles of the invention.

Figure 1 depicts a synthesis of an exemplary L15 linker unit, obtained from 3,9,12-trioxa-6,15-diaza-5-oxo-pentadecanoic acid, and the synthesis of an exemplary L30 linker in accordance with the present. invention.

Figure 2 depicts exemplary linker derivatives that can be used to detect proteins or nucleic acid targets in a system. Panel A shows an antibody (represented by an upside down Y) conjugated to three linker derivatives, each comprising multiple fluorophores (shaded circles). Multiple fluorophores can be used to detect the binding between the antibody and an antigen (shaded triangle). Panel B shows an exemplary linker derivative comprising PNA (thick line) and multiple fluorophores (shaded circles), which recognizes a target nucleic acid sequence.

Figure 3 represents an exemplary multilayer test. Binding between an antibody (Y upside down) and an antigen (shaded triangle) can be detected by first conjugating the antibody directly or indirectly with a first linker derivative comprising a nucleic acid or nucleic acid analog sequence, then conjugating the first linker derivative with a second linker derivative that is conjugated with dextran and with a complementary sequence of the nucleic acid or nucleic acid analog. Hybridization between the two nucleic acids or the two nucleic acid sequences is represented by the parallel bars in the center of the drawing. Dextran pearls are shown as dark circles at the top of the drawing.

Figure 4 represents the activation of Lys (betaala) -NH2 and Lys (NTA) -NH2.

Figure 5 depicts exemplary capture assays using the present linkers conjugated with probes.

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Figure 6 represents exemplary nucleic bases that are not natural.

Figure 7 represents sample nucleic bases, intermediate products and linker derivatives.

Figure 8 represents another exemplary method for preparing linkers according to the invention.

Figure 9 represents additional exemplary nucleic bases that are not natural and base pairs.

Detailed description of the invention

A. Linkers

The present "linker" is a molecule that can help bind other atoms, molecules or functional groups to each other, through chemical bonds. For example, one of the present linkers can be conjugated with a protein as well as with another group, such as a fluorophore, linking those substances to each other through the chemical bonds interspersed in the linker.

In some embodiments of the invention, the "linker" comprises a molecular entity obtained from the reaction of at least one amine, such as a diamine, and a dicarboxylic acid anhydride. In some embodiments, the amine does not comprise more than two ethyleneoxy units (CH2-CH2-O). For example, the present "linker" may comprise an alternating copolymer of such amine and units obtained from dicarboxylic acid anhydride. In some embodiments, the amine comprises a structural formula with no more than two methylene units in a row. For example, the amine may comprise a structural formula, such as for example: NH2CH2-CH2-O-CH2-CH2-O-CH2-CH2-NH2 or NH2-CH2-CH2-O-CH2-CH2-O-CH2- CH2-O-CH2-CH2-NH2, or other structural formulas in which a heteroatom is placed after every two methylene units.

The "linker" comprises at least two monomer units referred to herein as "L15". In other embodiments, the present "linker" comprises two or more L15 units arranged in a linear or branched manner. For example, in some embodiments, the linker is a linear polymer chain comprising two L15 units, referred to as "L30" herein. In still other embodiments, the linker comprises a longer linear polymer comprising several L15 units, and may be referred to, for example, "L45" or "L60", "L90" or "L120" or "L300," and so on in this memory, depending on the number of units L15 comprising the polymer chain. In some embodiments comprising more than one L15 unit, the L15 units can be covalently connected through one or more interleaved chemical bonds:

L15 comprises the unit shown below in Formula I.

Formula I

image 1

In Formula I, R1 and R2 may comprise NH or O, while R3 may comprise methyl, ethyl, propyl, CH2O-CH2, and (CH2-O-CH2) 2. In addition, when Formula I is conjugated to other groups, atoms or molecules and, for example, R1 and / or R2 is an oxygen, no more than three ethyleneoxy groups (CH2-CH2-O) that are repeated consecutively are present.

Therefore, some of the L15 units according to this invention may have the following molecular structures:

Formula Il Formula III

image 1

image 1

Formula IV

image 1

Formula V

image 1

Other possible L15 structures that are not shown above can be envisaged based on the choice of groups R1, R2 and R3.

In some embodiments, the present linkers comprise a linear polymer of L15 units wherein each

Unit L15 has the same molecular formula, for example, as that of structural formula II. In other embodiments, one or more L15 units may differ from the others in the linker chain. For example, a linker may comprise different units of formula II, and one or more units comprising one or more different structures. Such an L15 unit may be useful for fixing a specific atom, group or molecule in a defined position in the chain, for example. Since each unit can be added iteratively to the linker chain

In growth, such as by solid phase synthesis, a variety of mixed L15 polymers can be simply constructed in a controlled manner.

In some embodiments, one or more of the groups of heteroatoms in the linker comprise an additional substituent. That substituent may comprise a variety of atoms or functional groups, such as a simple hydrogen atom, a protecting group or a conjugation group that serves to fix the linker with an att

20 mo, a group or a molecule. In some embodiments, the substituent is a detectable label or other linker. Fixation through the heteroatom groups, with an additional linker, forms a branched linker polymer.

B. Design and synthesis of linkers

New methods for synthesizing an L15 linker and linear polymers of the L15 linker are described, with high

25 performance According to this synthesis method, long linkers of, for example, L120, L200, L300 or even longer, such as L600 or L990, can be constructed by consecutive additions of units of L15 or portions of units of L15, for example, On a solid phase. Alternatively, long linkers can be formed by joining together two intermediate length linkers. L30 or longer linkers, synthesized according to this method, may comprise individual L15 units with the same or different molecular structure.

The L15 units can be prepared by contacting a protected amine with a dicarboxylic acid anhydride and acetic anhydride, resulting in a cyclic imide terminal group, which can then be opened, once the linker reaches its desired length, for example , by basic aqueous hydrolysis in dioxane using a tertiary amine, such as diisopropylethyl amine. A molecule of 3,9,12-trioxa-6,15-diaza-5-oxopentadecanoic acid has been synthesized from 2,2 '- (ethylenedioxy) bis (ethylamine) in a yield of approximately 87-91% (see , Pieters et al., Bioorg. Med. Chem. Lett. 9: 161-6 (1999)). In contrast, controlled methods for synthesizing L15 units according to the present invention can provide yields of 96%, for example.

The synthesis can be performed in solution. The amine can be protected with (Boc) 2O, monomethoxytrityl (MMT), benzyloxycarbonyl (z) or fluorenylmethyloxycarbonyl (Fmoc), or other suitable protecting group. In some embodiments, the amine is 2,2'- (ethylenedioxy) bis (ethylamine) (structure 1 of Fig. 1). In some embodiments, the amine is present in excess over the dicarboxylic acid anhydride and acetic anhydride. The addition of the dicarboxylic acid anhydride can be performed over a wide range of temperatures. Suitable dicarboxylic acid anhydrides include those from diglycolic acid, 1,5-dipentanoic acid, 1,4-dibutyric acid, 1,3-dipropanoic acid and 3,6-dioxaoctane dioic acid, depending on the desired R3 group. The subsequent addition of acetic anhydride can force the product in the form of cyclic imide, with high yield. (Structure 2 of Fig. 1). The cyclic imide can be opened quantitatively, if desired, for example, in excess of dioxane and di-isopropylethylamine (DIPEA). To start the preparation of an L30 linker, the cyclic imide may react with another amine. (Structure 3 of Fig. 1). Then, the dicarboxylic acid anhydride and acetic anhydride can be added and the resulting cyclic imide can be opened. (Structure 4 of Fig. 1). In some embodiments, the terminal cyclic imide in the linker may be left unopened to serve as a reactive group for the formation of other bonds, such as with reactive groups or other linkages.

For example, an L15 monomer obtained from 3,9,12-trioxa-6,15-diaza-5-oxo-pentadecanoic acid can be prepared by protecting one of the two amino groups of 2,2 '- (ethylenedioxy) bis (ethylamine) with (Boc) 2O. (See Fig. 1, reaction scheme 1, part A). After dissolving the mono-protected 2,2'- (ethylenedioxy) bis (ethylamine) (structure 1 of Fig. 1) in pyridine, diglycolic acid and then acetic anhydride can be added, at elevated temperature, such as about 100 ° C, to form a cyclic L15 structure (structure 2 of Fig. 1). An excess of 2,2'- (ethylenedioxy) bis (ethylamine) reacts with the terminal cyclic imide to form structure 3, shown in Figure 1. To form an L30 linker, an additional diglycolic acid can be added, and the cyclic terminal end, by the addition of 1,4-dioxane and diisopropylethylamine (DIPEA) in aqueous solution.

In some embodiments of the invention in which one or both R1 and R2 are oxygen rather than amino groups, the amino alcohols or dialcohols may be substituted for the amine in the reactions described above. The conditions can also be adjusted to explain the different reactivities of alcohols and amines. For example, reactions can be carried out at higher temperatures when alcohols are coupled rather than amines.

Using the present methods, large batches, such as 100 g, of purely protected L30 linkers can be prepared, by extractive processing, using a one-liter laboratory equipment. The following examples provide a more detailed description of the synthesis of exemplary molecules comprising linkers L15 and L30, in accordance with this invention.

The same synthetic principles apply to the preparation of longer linker polymer chains, used in some embodiments of this invention. For example, linkers such as L45, L60, L90, L120, L150, L200, L300, or even longer, for example, comprising as many as 40 units of L15, can be prepared by repeating the steps described above in succession: first react the cyclic imide of the preceding unit L15 in the growing chain, with excess amine, and add acid anhydride and then acetic anhydride to add another unit L15. Once the desired length is achieved, the terminal ring can be opened. If desired, these successive steps can also be performed on a solid support, which may be convenient for preparing linkers longer than L30 or L60, for example. In fact, compounds with molecular weights of 8 to 15 kDa can be prepared with the methods of the present invention, because the successive stages of the synthesis are not inhibited by the growing chains of poly-L15. This is absolutely unusual in peptide chemistry.

Alternatively, to reduce the number of steps necessary to prepare longer linkers, intermediate length linkers, such as L30, for example, they can be directly combined with each other or indirectly conjugated through an intercalated chemical group, such as a reactive group or a detectable marker. For example, a single stage of chromatographic purification allows the preparation of Boc-L30 with high purity, which can be easily oligomerized to prepare longer linkers, such as L300, with high purity. Even linkers such as L600 and longer such as L1000 can be prepared by such methods, the only limitations being the performance of each stage and the amount of material available. In addition, two long linkers can be joined along their chains to form very long branched linkers comprising, for example, up to 10, 20, 40 or even 60 units of L15 in total.

Because it is possible to control the conditions of the synthesis, the present invention further comprises linkers and linker derivatives comprising a homogeneous molecular and structural formula. A homogeneous molecular formula means here that the molecular weight of the linker can be obtained by mass spectrometry, with less than 1% deviation from the expected molecular weight, based on the synthesis protocol used, in some embodiments less than 0.1 % or even less than 0.01%. This is possible even with very long linkers, such as L300, L600 or L990. A homogeneous structural formula means here that the structural composition of each L15 unit and its position within the linker chain is controlled by the synthesis protocol used to prepare the linker. In contrast, numerous PEG-based linkers comprise a mixture of lengths and structural formulas. Thus, substances comprising these mixed linkers are intrinsically heterogeneous, regardless of the purity or homogeneity of the molecules, groups or atoms with which the linkers were conjugated.

Many of the linkers currently used, such as certain nucleic acid linkers or linkers based on nucleic acid analogs, carry a charge on each chain monomer, for example, of a phosphate or phosphorothioate group of the main chain. Such charges can cause those linkers to aggregate with substances of opposite charge, including the walls of a plate or a container, or attract unwanted metal ions, or precipitate in the presence of salts. Instead, the present linkers can be designed so that they do not carry a load, or only one or a few loads at defined positions along the chain. Since the synthesis of the present linkers can be controlled, they can be designed to place loads only at specific locations or not to carry loads at all. This allows precise modulation of the position and amount of charge in a given linker and in a linker derivative and is an additional method to avoid unwanted aggregation or tertiary interactions.

However, in some embodiments, the present linkers can also be prepared using an uncontrolled polymerization reaction. For example, a synthesis in a single container can be performed based on the above reaction steps, such as mixing a diamine in solution with an excess of dicarboxylic anhydride. The synthesis can also be completed, for example, by adding a reagent that will form a terminal group, such as an amino acid or a fluorophore. Controlling the amount of said termination reagent can help establish the distribution of the actual length of the mixture. Such methods may be useful, for example, when the absolute length is unimportant, or to immediately generate linkers with a variety of different lengths. Products can be divided according to length, for example, by chromatography.

C. Linker derivatives

The linkers of the present invention can be covalently linked to another atom, group or molecule to form a "linker derivative." Linker derivatives may comprise only one linker conjugated to another atom, group or molecule, or may comprise multiple linkers, conjugated with more than one of the same atom, group or molecule, or conjugated with different atoms, groups or molecules. The union of the atom, group or molecule can occur at one or several free ends of the linker, or along its length, such as at one of its heteroatoms. Examples of atoms, groups or molecules that can be conjugated to linkers to form linker derivatives, include hydrogen atoms, terminal groups such as protecting groups, methyl groups or acetyl groups. Other examples include heavy or radioactive isotopes, leaving groups; fluorophores; chromophores; enzymatic substrates; antigens; antibodies; other proteins and peptides; amino acids and amino acid analogs; nucleic acids and nucleic acid analogs; components of a membrane or a solid support; and other small or large molecules. The linkers according to this invention can therefore be conjugated with different atoms, groups or molecules of the same type, such as multiple fluorophores, or they can be conjugated with different types of groups or molecules, such as PNA and a fluorophore or a protein and a solid surface, or an acetyl group and an antibody. The covalent union can be direct, without the intervention of bonds or atoms, or indirectly, with one or several intercalated bonds or atoms. As used herein, any atom, group or molecule that is covalently bound to a linker, both directly and indirectly, is "conjugated" with the linker. Therefore, "linker derivatives" include molecular entities called "conjugations." Atoms, groups or molecules may also be non-covalently attached to the present linkers or linker derivatives, for example, by adsorption to a surface, intercalation, hybridization of nucleic acids or complementary nucleic acid analogs, or other interactions.

Atoms, groups or molecules can be conjugated at one end of the linker, or the same or different atoms, groups or molecules can be placed at both ends. Alternatively, the atoms, groups or conjugated molecules can be placed between two linkers, for example, in a defined separation that prevents unwanted interactions between them. Therefore, derivatives of linkers such as X-L, L-X or X-L-X or L-X-L, can be prepared, where X represents a directly or indirectly conjugated atom, group or molecule and L represents a linker. Such linker derivatives also include different atoms, groups or molecules, for example, X1-L-X2 or X1-L1-X2-L2 or L1-X1-L2-X2. An atom, a group or a molecule can also be conjugated, for example, through one of the heteroatoms in the linker. In addition, different linkers can be conjugated with a linker derivative to form branched structures. Linkers conjugated to atoms, groups or multiple molecules, such as color markers or radioactive isotopes, are advantageous, for example, for amplifying signals in detection assays, as an alternative to amplification methods based on antibodies and radioactivity.

1. Indirect Conjugation

A linker can be conjugated with another group, atom or molecule indirectly through a group that is more reactive with the anticipated atom, group or molecule, than the linker itself. As used herein, such groups are called "conjugation groups." In some embodiments, linkers are conjugated to conjugation groups based on carbodiimide. Amino acids can also serve as conjugation groups. For example, amino acids with amino side chains can be used to conjugate, for example, fluorophores. Cysteine sulfhydryl is also useful as a conjugation group to form cross-linkages of diazufre with other substances. In some embodiments, the linkers are conjugated to Lys (Cys), which is a C-terminal lysinecarboxamide, with a Cysteine amino acid linked to the N of the lysine side chain, and which can serve as a conjugation group. The juxtaposed amine and sulfhydryl groups of this cysteine moiety unexpectedly provide significantly higher reactivity towards dextran than, for example, carbodiimide conjugation groups.

In some embodiments, the linker is conjugated to amino groups, such as a lysine amino acid, wherein N is derivatized with beta-alanine-N, N-diacetic acid. The lysine derivative can be quantitatively converted into its cyclic form of N-2,6-dioxo-morpholino anhydride. That form was very reactive with amino under slightly basic aqueous conditions, when compared to reactive amino groups, such as NHS esters. Therefore, it can also serve as a conjugation group. A person of ordinary skill in the art can readily provide other conjugation groups that can be conjugated with the present linkers, to aid in the conjugation of a variety of substances of the present linkers.

2. Detectable markers

In some embodiments, one or more detectable markers are conjugated to a linker. In accordance with this invention, a "detectable label" is any molecule or functional group that allows the detection of a biological or chemical characteristic or a change in a system, such as the presence of a target substance in the sample.

Examples of detectable markers that can be used in the invention include fluorophores, chromophores, electrochemiluminescent markers, bioluminescent markers, polymers, polymer particles, beads or other solid surfaces, gold or other metal particles or heavy atoms, rotation markers, radioisotopes. , enzyme substrates, haptens, antigens, quantum dots, aminohexyl, pyrene, nucleic acids or nucleic acid analogs, or proteins, such as receptors, peptide substrates or substrates, enzymes and antibodies (including antibody fragments).

Examples of polymer particle markers that can be used in the invention include microparticles, beads or polystyrene latex particles, PMMA or silica, which can be embedded with fluorescent dyes or polymer micelles or capsules containing dyes, enzymes or substrates. . Examples of metal particles that can be used in the invention include gold particles and gold-coated particles, which can be converted by staining with silver. Examples of haptens that can be conjugated in some embodiments are fluorophores, myc, nitrotyrosine, biotin, avidin, streptavidin, 2,4-dinitrophenyl, digoxigenin, bromodeoxy uridine, sulfonate, acetylaminoflurene, trinitrophenol mercury and estradiol.

Examples of enzymes that can be used in the invention include horseradish peroxidase (HRP), alkaline phosphatase (AP), beta-galactosidase (GAL), glucose-6-phosphate dehydrogenase, beta-N-acetylglucosaminidase, βglucuronidase, invertase, Xanthine oxidase, firefly luciferase and glucose oxidase (GO). Examples of general purpose substrates for horseradish peroxidase (HRP) include 3,3'-diaminobenzidine (DAB), nickel-enhanced diaminobenzidine, 3-amino-9-ethylcarbazole (AEC), benzidine dihydrochloride (BDHC) , Hanker-Yates (HYR) reagent, indophenol blue (IB), tetramethylbenzidine (TMB), 4-chloro-1-naphthol (CN), alpha-naphthol pyronine (αNP), o-dianisidine (OD), 5- Bromo-4-Chloro-3-Indolylphosphate (BCIP), Nitroazul Tetrazolium (NBT), 2- (Pyodophenyl) -3-P-Nitrophenyl-5-Phenyltetrazolium Chloride (INT), Blue Tetranitro Tetrazolium (TNBT), 5 -bromo-4-chloro-3-indoxy-beta-Dgalactoside / ferro-ferricyanide (BCIG / FF). Examples of commonly used substrates for alkaline phosphatase include naphthol-AS-B1-phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS- B1 phosphate / fast red TR (NABP / FR), naphthol-AS-MX-phosphate / fast red TR (NAMP / FR), naphthol-AS-B1-phosphate / fuchsin new (NABP / NF), bromochloroindolyl phosphate / tetrazolium nitro blue (BCIP / NBT), 5-bromo-4-chloro-3-indolyl-betadelta-galactopyranoside (BCIG).

Examples of luminescent markers that can be used in the invention include luminol, isoluminol, acridinium esters, 1,2-dioxetanes and pyridopyrididazines. Examples of electrochemiluminescent markers include ruthenium derivatives. Examples of radioactive markers that can be used in the invention include radioactive isotopes of iodide, cobalt, selenium, hydrogen, carbon, sulfur and phosphorous.

Some "detectable markers" according to this invention, comprise "color markers," where the change or biological event in the system can be tested for the presence of a color, or a color change. Examples of "color markers" are chromophores, fluorophores, chemiluminescent compounds, electrochemiluminescent markers, bioluminescent markers and enzymes that catalyze a color change in a substrate.

"Fluorophores" as described herein are molecules that emit a detectable electromagnetic radiation after excitation with electromagnetic radiation, in one or several wavelengths. A wide variety of fluorophores are known in the art and have been developed by chemists for use as detectable molecular markers and can be conjugated to the linkers of the present invention. Examples include fluorescein or its derivatives, such as fluorescein 5-isothiocyanate (FITC), 5- (and 6) carboxyfluorescein, 5- or 6-carboxyfluorescein, 6- (fluorescein) -5 (and 6) -hexanoic carboxyamide acid, fluorescein isothiocyanate, rhodamine or its derivatives, such as tetramethylrodamine and 5- (and 6) tetramethylrodamine isothiocyanate (TRITC). Other examples of fluorophores that could be conjugated with the present linkers include: coumarin dyes, such as (diethyl-amino) coumarin or 7-amino-4-methylcumarin-3-acetic acid, succinimidyl ester (AMCA); sulphodamine 101 sulfonyl chloride (TexasRed® or TexasRed® sulfonyl chloride; 5- (and 6) carboxylamine 101, succinimidyl ester, also known as 5- (and 6) -carboxy-X-rhodamine, succinimidyl ester (CXR) ; lisamine or lysamine derivatives such as lisamine rhodamine B sulfonyl chloride (LisR); 5- (and 6) carboxyfluorescein, succinimidyl ester (CFI); fluorescein 5-isothiocyanate (FITC); 7-diethylaminocumarin-3-carboxylic acid, ester of succinimidyl (DECCA); 5- (and 6) -carboxytetramethylrodamine, succinimidyl ester (CTMR); 7-hydroxycoumarin-3-carboxylic acid, succinimidyl ester (HCCA); 6-fluorescein-5- (and 6) - carboxamidohexanoic acid (FCHA); N- (4,4-difluoro-5,7-dimethyl-4-bora-3a, 4a-diaza-3-indacenopropionic acid, succinimidyl ester; also known as 5,7-dimethyl BODIPYT® acid propionic, succinimidyl ester (DMBP); "activated fluorescein derivative" (FAP), available from Probes, Inc .; eosin 5-isothiocyanate (EITC); 5-i erythrosine thiocyanate (ErITC); and Cascada® blue acetylazide (CBAA) (the O-acetylazide derivative of 1-hydroxy-3,6,8 pyrethrisulfonic acid). Still other potential fluorophores useful in this invention include fluorescent proteins such as green fluorescent protein and its analogues or derivatives, fluorescent amino acids such as tyrosine and tryptophan and their analogs, fluorescent nucleosides, and other fluorescent molecules such as Cy2, Cy3, Cy 3.5 , Cy5, Cy5.5, Cy 7, IR dyes, Dyomics dyes, phycoerythrin, Oregon green 488, Pacific blue, rhodamine green and Alexa dyes. Still other examples of fluorescent markers that can be used in the invention include R-phycoerythrin or allophycoerythrin conjugates, inorganic fluorescent markers, such as particles based on semiconductor material, such as coated CdSe nanocrystallites.

A variety of the above fluorophores, as well as others, are commercially available, from companies such as Probes, Inc. (Eugene, OR), Pierce Chemical Co. (Rockford, IL), or Sigma-Aldrich Co. (St. Louis, MO).

In some embodiments, the detection unit may comprise from 1 to 500 detectable marker molecules. In some embodiments, the detectable label is an enzyme, which may be conjugated to a polymer, so that the number of enzyme molecules conjugated to each polymer molecule is, for example, 1 to 200, 2 to 50 or 2 to 25 In some embodiments, the detectable label is a gold particle, a radioactive isotope or a color marker, e.g. eg, a low molecular weight fluorophore, and the number of detectable markers conjugated with each polymer molecule is, for example, 1 to 500, or for example, 2 to 200. In some embodiments, the detectable label is a protein fluorophore and the number of detectable markers, conjugated with each polymer molecule is 1-50, 2-20. In some embodiments, the number of detectable marker molecules, conjugated with each polymer is 1-200, 2-50, 2-25, or is 10-20, 5-10 or 1-5.

The detectable label can be detected by numerous methods, including, for example, reflectance, transmittance, light scattering, optical rotation and fluorescence or combinations thereof in the case of optical markers or by films, scintillation counting or phosphoforming of images, in the case of radioactive markers. See, p. eg, Larsson, 1988, Immunocytochemistry: Theory and Practice, (CRC Press, Boca Raton, FL); Methods in Molecular Biology, vol. 80 1998, John D. Pound (compiler) (Humana Press, Totowa, NJ). In some embodiments, more than one detectable marker is employed.

3. Probes

Some linker derivatives according to this invention may comprise a conjugated "probe" which, as used herein, is a functional group or a molecule that monitors events or changes in a system, for example, by joining, reacting with, or hybridizing with a target substance in the system, directly or indirectly. An example of a probe is a nucleic acid or a nucleic acid analog capable of binding or hybridizing with a specific target sequence. Other examples include antibodies, such as primary antibodies.

or secondary, antigens, enzyme substrates and protein ligands, haptens, proteins and peptides.

4. Antibodies

In some embodiments, derivatives of the present linkers comprise, for example, at least one polyclonal, monoclonal, monospecific, polyspecific, humanized, single stranded, chimeric, synthetic, recombinant, hybrid, mutated and CDR grafted antibody. Different methods for producing antibodies and preparing recombinant antibody molecules are known in the art and have been described, see, p. eg, in Kohler and Milstein, (1975) Nature 256: 495; Harlow and Lane, Antibodies: a Laboratory Manual, (1988) (Cold Spring Harbor Press, Cold

5

10

fifteen

twenty

25

30

35

40

Spring Harbor, NY). The antibodies used in the invention can be obtained from any mammalian species, e.g. eg, rat, mouse, goat, guinea pig, donkey, rabbit, horse, llama, camel or any avian species, e.g. Eg chicken, duck. The origin of the antibody is defined by the genomic sequence, regardless of the method of production.

The antibody can have any isotype, e.g. eg, IgG, IgM, IgA, IgD, IgE or any subclass, e.g. eg, IgG1, lgG2, lgG3, lgG4. The skilled technician will appreciate that antibodies produced recombinantly, or by other means, for use in the invention, include any antibody fragment that can still bind to the antigen, e.g. eg, Fab, F (ab) 2, Fv, scFv. In certain embodiments, the antibody, which includes an antibody fragment, can be recombinantly manipulated to include a hapten, e.g. eg, a peptide. In certain embodiments, the hapten may be a myc marker (see, Figure 6N). The inclusion of a hapten in an antibody or in an antibody fragment facilitates the subsequent binding of a ligation agent, a probe, or a label.

Certain embodiments employ linker derivatives comprising a primary antibody that contains an antigen binding region. Some embodiments employ linker derivatives comprising a secondary antibody that contains an antigen binding region that specifically binds to a primary antibody, e.g. eg, the constant region of the primary antibody. In certain embodiments, the linker derivative is further conjugated with a polymer. Some embodiments employ linker derivatives comprising a tertiary antibody that contains an antigen binding region that specifically binds to the secondary antibody, e.g. eg, a constant region of the secondary antibody, or a hapten bound to the secondary antibody or a polymer conjugated to the secondary antibody. In certain embodiments, the tertiary antibody is further conjugated with a polymer.

5. Nucleic acids and nucleic acid analogs

In some embodiments, the linker is conjugated to at least one nucleic acid and / or a nucleic acid analog. As used herein, a "nucleic acid" refers to a sequence of nucleic bases comprising any oligomer, polymer or polymer segment, having a backbone consisting only of RNA or DNA nucleosides and comprising only the bases adenine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), where an oligomer means a sequence of two or more nucleic bases. Nucleic acids can also be called nucleic acids.

Non-natural bases may include, for example, purine and pyrimidine-like molecules, such as those that can interact using pairing interactions of the Watson-Crick, oscillatory or Hoogsteen type. Examples generally include any nucleic base referred to elsewhere as "unnatural" or "analogous." Figures 6 and 9 represent some examples.

Examples include: halogen substituted bases, alkyl substituted bases, hydroxy substituted bases and thiol substituted bases, as well as 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, isoguanine, isocytosine, pseudoisocytosine, 4-thiouracil, 2-thiouracil and 2-thiothimine, inosine, 2-aminopurine, N9- (2-amino-6-chloropurine), N9 (2,6-diaminopurine), hypoxanthine, N9- (7-deaza- guanine), N9- (7-deaza-8-aza-guanine) and N8- (7-deaza-8-aza-adenine).

Still other examples include bases in which an amino group with a hydrogen is substituted with a halogen (lower case "h"), such as 2-amino-6- "h" -purines, 6-oxo-2- "h" -purines, 2-oxo-4- "h" -pyrimidines, 2-oxo-6- "h" purines, 4-oxo-2- "h" -pyrimidines. Those will form two pairs of hydrogen-bound bases with non-thiolated and thiolated bases; respectively, 2,4-dioxo and 4-oxo-2-thioxo pyrimidines, 2,4-dioxo and 2-oxo-4-thioxo pyrimidines, 4-amino-2oxo and 4-amino-2-thioxo pyrimidines, 6-oxo -2-amino and 6-thioxo-2-amino purines, 2-amino-4-oxo and 2-amino-4-thioxo pyrimidines, and 6-oxo-2-amino and 6-thioxo-2-amino purines.

For example, some specific embodiments of unnatural bases are the structures shown in Figure 6 with the following substituents: These and other unnatural bases are described further below and in the accompanying international application document, entitled "New Non-Natural Base Pairs. "

Base (Symbol)

R2 R3 R4 R5 R6

TO

H or CH3  H H

isoA

H or CH3  H H

D

H or CH3  H

G

H or CH3  H

Gs

H or CH3  H

I

H or CH3 H

OR

H or CH3

U2s

H or CH3

U4s

H or CH3

C

H or CH3  H

Py-2nd

H or CH3 H or CH3

Cs

H or CH3  H

isoG

H or CH3  H

isoGs

H or CH3  H

Pu-2nd

H or CH3 H

isoC

H H or CH3

isoCs

H H or CH3

Py-4th

H or CH3 H or CH3

TO

H or CH3  H CH3

isoA

H or CH3  H - CH3

D

H or CH3  CH3

G

H or CH3  CH3

Gs

H or CH3  CH3

I

H or CH3 CH3

OR

H or CH3

U2s

H or CH3

U4s

H or CH3

C

H or CH3  CH3

Py-2nd

H or CH3 H or CH3

Cs

H or CH3  CH3

isoG

H or CH3  CH3

isoGs

H or CH3  CH3

Pu-2nd

H or CH3 CH3

isoC

CH3 -CH3 or CH3

isoCs

CH3 CH3 or CH3

Py-4th

H or CH3 H or CH3

In other examples, one or more of H or CH3 are independently substituted with a halogen, such as Cl or

F. Examples of other bases and base pairs compatible with the invention are shown in Figure 9. R1 or "BB" in the structures of Figures 6 and 9, may serve as a point of attachment to a group of the chain main, such as PNA, DNA, RNA, etc.

In some embodiments, the following types of base pairs are used: one or more of Us: A, T: D, C: G, and P: Gs. In some embodiments, T: A and P: G are used. Still other examples are illustrated in Figures 2 (A) and 2 (B) of Buchardt et al. (Document US 6,357,163).

The nucleic acid analogs may also comprise monomer units in which natural bases A, C, G, T, and U or unnatural bases are connected to an unnatural unit of the main chain. Unnatural units of the main chain include, for example, those with a main chain other than ribose phosphate or deoxyribose phosphate. For example, in some embodiments, one or more phosphate oxygens can be replaced by another molecule, such as sulfur. In other embodiments, a different sugar or a sugar analog can be used, for example, one in which an oxygen in the sugar is substituted by hydrogen or an amine, or an O-methyl. In still other embodiments, nucleic acid analogs comprise synthetic molecules that can bind to a nucleic acid or a nucleic acid analog. For example, a nucleic acid analog may comprise peptide nucleic acids (PNAs), blocked nucleic acids (LNAs) or any derivatized form of a nucleic acid.

As used herein, "peptide nucleic acid" or "PNA" means any oligomer or polymer comprising at least one or several subunits of PNA (residues), which include, but are not limited to, any of the oligomer or polymer segments referred to or claimed as peptide nucleic acid in US Pat. No. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986 .053, 6.107.470 6.201.103,

6,228,982 and 6,357,163.

The term PNA also applies to any oligomer or polymer segment comprising one or more subunits of the nucleic acid mimetics described in the following publications: Lagriffoul et al., Bioorganic & Medicinal Chemistry Letters, 4: 1081-1082 (1994) ; Petersen et al., Bioorganic & Medicinal Chemistry Letters, 6: 793-796 (1996); Diederichsen and others, Test. Lett. 37: 475-478 (1996); Fujii and others, Bioorg. Med. Chem. Lett.

7: 637-627 (1997); Jordan and others, Bioorg. Med. Chem. Lett. 7: 687-690 (1997); Krotz and others, Test. Lett. 36: 69416944 (1995); Lagriffoul and others, Bioorg. Med. Chem. Lett. 4: 1081-1082 (1994); Diederichsen, U., Bioorganic & Medicinal Chemistry Letters, 7: 1743-1746 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 1, (1997) 1: 539-546; Lowe et al., J. Chem. Soc. Perkin Trans. 11: 547-554 (1997); Lowe et al., J. Chem. Soc. Perkin Trans. 11: 555-560 (1997); Howarth et al., J. Org. Chem. 62: 5441-5450 (1997); Altmann, K-H et al., Bioorganic & Medicinal Chemistry Letters, 7: 1119-1122 (1997); Diederichsen, U., Bioorganic & Med. Chem. Lett., 8: 165-168 (1998); Diederichsen and others, Angew. Chem. Int. Ed., 37: 302-305 (1998); Cantin and others, Tett. Lett., 38: 4211-4214 (1997); Ciapetti et al., Tetrahedron, 53: 1167-1176 (1997); Lagriffoule et al., Chem. Eur. J., 3: 912-919 (1997); Kumar et al., Organic Letters 3 (9): 1269-1272 (2001); and peptide-based nucleic acid mimetics (PENAMs) from Shah et al., as described in WO96 / 04000.

As used herein, the term "blocked nucleic acid" or "LNA" means an oligomer or a polymer comprising at least one or more subunits of LNA. As used herein, the term "LNA subunit" means a ribonucleotide that contains a methylene bridge that connects the 2 ′ oxygen of the ribose with the 4 ′ carbon. See generally, Kurreck, Eur. J. Biochem., 270: 1628-44 (2003).

5. Derivatives of linkers comprising fluorophores

In some embodiments, the linkers may be conjugated with one or more fluorophores. For example, two fluorophores can be conjugated to form a fluorophore-linker-Lys (fluorophore) -linker-X derivative. Similarly, longer derivatives can be prepared in which various fluorophores are separated with linker units with the same length or different lengths, such as the structure Fluorophore- [linkerLys (fluorophore)] N-linker-X, in which X is a conjugation group or other atom, group or molecule. In some embodiments, two fluorophores are separated from each other by at least four L15 units (L90), to ensure that the fluorophores are separated beyond the Foerster radius (typically, not more than 5 nm), so that extinction due to Fluorescence energy transfer by resonance is minimized or eliminated. In some embodiments, two fluorophores may be separated by four to ten L15 units (L90 to L150), while in others, by four to six L15 units (L90 to L120). In fact, in some embodiments, the fluoresceins separated by at least four L15 units (L90), showed an insignificant extinction in aqueous buffers, strongly indicating that the linker adopts an extended structure in water. In contrast, polyethylene glycol (PEG) based spacers can adopt a folded structure, helical in water.

Because the linkers of the present invention can allow separation of individual fluorophores, so that extinction is reduced, two or other different types of fluorophores can be conjugated to one or more linkers to produce amplified or only colored signals. For example, groups of 2, 3, 4 or 5 fluorophores groups can be conjugated to linkers, each being separated from each other by at least four L15 units, for example, separated from each other with between four and ten L15 or four units and six units L15. Some embodiments of this invention comprise Rhodamine-L-Lys (fluorescein) -LX, where L is of sufficient length to prevent extinction of rhodamine and fluorescein signals, for example, and where X is another atom, group or molecule, or a conjugation group. The optimum separation for a given fluorophore depends in part on its size, but can be easily determined by testing the intensity of the fluorescent signal, for example, in different separations.

In some embodiments, a molecule, such as a protein, can be conjugated with different linker derivatives, which are each conjugated with a detectable label, such as a fluorophore. (See Figure 2A). In conventional methods to fluoresce a protein without linkers or with short linkers, the size of the conjugated protein determines how far the fixed fluorophores can be placed, and therefore, the fluorescence intensity is determined first. The same principle applies to other molecules as well. Instead, embodiments of the present invention can weaken this size limitation because the present linkers: (1) can be conjugated with various fluorophores, (2) they can retain an extended structure in solution that minimizes unwanted interactions between individual fluorophores, and ( 3) can be conjugated with numerous types of proteins and other molecules. Thus, in some embodiments, this invention can greatly improve the fluorescence intensity of a molecule, and can eliminate the need for extra steps, generally used to enhance a signal in a system. For example, the binding of multiple linker derivatives each comprising multiple fluorophores, to an antibody, may allow direct detection of tiny amounts of an antigen, without the need for secondary procedures to amplify the antibody signal.

6. Derivatives of linkers with multiple conjugated substances

Some linker derivatives, according to this invention, include different types of conjugated detectable markers. In some embodiments, the linkers are conjugated with two different detectable markers in which one marker serves to monitor a change or an event in a system, by binding or hybridization with a target, while the other serves to detect that event of binding through a color change, radioactivity or other detectable signal. For example, a fluorophore can be combined with a different type of color marker. Derivatives such as DNP-linker-Lys (fluorophore) -linker-X have been prepared. DNP (dinitrophenol) can be recognized with an anti-DNP antibody, while fluorescent emission provides a second type of signal, thereby providing two alternative methods of labeling.

In addition, the embodiments may comprise other combinations of atoms, groups or molecules, such as a detectable label and a solid surface, a membrane, a protein or other large molecule. Fixations can be covalent as well as non-covalent. Embodiments with more than one type of conjugate atom, group or molecule are useful, for example, in immunoprecipitation or ELISA assays, in situ detection assays, multilayer assays, fluorescent microsphere assays and capture assays.

In some embodiments, linkers are conjugated with fluorophores, as well as with probes, such as nucleic acids and nucleic acid analogs. For example, a nucleic acid or a nucleic acid analog can be used to hybridize with a target sequence in a system, while the fluorophore conjugated to the probe through the linker provides a means to detect hybridization. Such derivatives of bifunctionally labeled linkers may be useful, for example, in in situ hybridization assays, such as FISH.

Other linker derivatives comprise multiple conjugates of nucleic acid or nucleic acid analogs, optionally further conjugated with other types of detectable labels. For example, a single linker derivative molecule may comprise different nucleic acids or nucleic acid analogs, each separated from the others by segments of one or more L15 units. In some embodiments, the different nucleic acids or nucleic acid analogs are separated from each other by separations of L120 to L300, L150 to L300, or L300.

In some embodiments, the linkers are conjugated with fluorophores or other color-based markers, as well as with PNA or LNA probes. Derivatives of linkers containing PNA, prepared in accordance with the present invention, include, for example, derivatives of linkers comprising XL-PNA-L-Lys (Cys), wherein X may be a terminal group, such as acetyl, a conjugation group, or a color marker, such as a fluorophore or an enzyme substrate, such as DNP. The fixation of a fluorophore such as fluorescein may allow a simpler purification and analysis of the derivative. A separation of L30 or greater between the group of X and a PNA or an oligonucleotide is generally a sufficient distance to avoid a steric hindrance between the linker and the hybridization target, however a longer separation can be used, for example for increase entropy to further decrease unwanted tertiary interactions or aggregation. For example, the distance between the PNA or the oligonucleotide and a Lys (Cys) group can be, for example, up to L300, including from L120 to L300 or from L150 to L300. The separations between the PNA and other conjugates can be, for example, from L120 to L300, from L150 to L300, or L300. Other derivatives of exemplary linkers comprising PNA, according to the invention, also include multiple fixations of fluorophores or color markers. For example, derivative [(Flu-L60) 2Lys] 2-Lys-L30-PNA was prepared and contains four fluorescein linker derivatives (Flu). In other embodiments, a linear derivative of the linker can be prepared, conjugated to PNA and multiple fluorophores. The embodiment Lys (Flu) -L30-PNA-L30-Lys (Flu) -L90-Lys (Flu) -L90-Lys (FIu) was also prepared and similarly contains four fluoresceins. Although fluorophores conjugated with the same linker derivative can be added more likely in solution than fluorophores conjugated with different linker derivatives, surprisingly, linker derivatives comprising a PNA probe and different fluorophores separated by L90 linkers, not only provided signals boosted over others containing isolated fluorophores, but also showed reduced background noise, indicating that the high entropy due to the flexible, extended structure of each L90 linker compensated for any increased tendency to interact with fluorophores. In some embodiments, linker derivatives can also form branched structures or can specifically hybridize with each other, through conjugated PNAs, so that the signal is amplified.

7. Cross-linking agents

The linkers described can also be used as protein crosslinkers. For example, the present linkers can be conjugated with a protein conjugation group, such as maleimide, and / or with an antigen, a ligand or a substrate for a particular protein or antibody, and optionally also with a detectable marker to visualize. crosslinking For example, maleimide-linker-X groups have been prepared, in which X represents a reactive group such as Lys (beta-alanine-N-diacetic acid) or a ligand, an antigen or a specific substrate of the protein of interest. Said linker can be used, for example, to bind to a specific protein or antibody through the group X, and for the cross-linking of other proteins in the vicinity of that specific protein, through the maleimide linker. A detectable label can also be conjugated to the linker derivative to help detect crosslinking. Even relatively short linker segments, such as L30 between a marker and a protein ligand, can allow detection of the label without significant interference by the extinction of a fluorescent label, for example.

The extended and flexible structure of some of the present linkers allows them to aid in the crosslinking of even two large molecules, such as a fluorescent protein (eg, RPE or a green fluorescent protein) and an antibody. For example, long linkers according to the present invention may have solution lengths of, for example, 9 nm in the case of L90, or even 30 nm in the case of L300, or even longer. That length provides significant freedom even for very large proteins within a crosslinked complex, to adopt their preferred orientations, independently of others within the complex. Thus, the present linkers allow a more accurate identification of the interacting proteins because the complex is minimally disturbed by the presence of the linker.

8. Conjugation with a surface or a polymer

In some embodiments, the present linkers may serve to covalently or non-covalently fix a detectable label or other atom, group or molecule to a polymer or a surface such as dextran or a membrane or a cluster or a plate. The present linkers take up little space in the longitudinal direction. Therefore, they can be useful for coating such surfaces with high density because they can access relatively narrow spaces or pores on such surfaces. In addition, long linkers can be used to maximize the distance between the surface and other system components, so that the system is minimally disturbed. For example, linker derivatives can be prepared for conjugation with dextran together with nucleic acids or nucleic acid analogs, such as PNA, as well as color markers, such as HRP and one or more fluorophores. Other linker derivatives can be prepared for conjugation with combinations of dextran, PNA and antibodies. (See Figure 3, for example.)

Derivatives of linkers with multiple atoms, groups or conjugated molecules can be used to amplify signals in a variety of ways. For example, tiny amounts of antibodies or antigens can be detected, by fixing the antibody or antigen to a linker derivative comprising multiple fluorophores. (See Figure 2A, for example.) {Note: this has already been mentioned above.} In addition, two potentially cross-linked antigens can be distinguished by fixing the antigens to linker derivatives comprising different types of fluorophores. Multilayer systems can also be constructed such that certain atoms, groups or interacting molecules, such as nucleic acids or nucleic acid analogs or a complementary protein and ligand, are placed between another detectable marker, such as a color marker, and a surface or a molecule. (See Figure 3.)

9.

 Improved solubility of conjugated substances

Another surprising feature of some of the present linkers is a relatively high solubility despite the presence of relatively few polar or charged groups. Therefore, linkers, for example, those comprising two or L15 units in some derivatives, or four or more L15 units in other derivatives, can also be used to improve the solubility of an atom, of a group or of a molecule in aqueous solution. For example, an acetyl-LX group can be used to improve the solubility of the atom, group or molecule X. Surprisingly, PNA probes with multiple fluorophores, separated with at least four L15 units, showed a higher water solubility, as well as less aggregation and less non-specific binding, than probes without conjugated linkers and with only one fluorophore. In addition, the polylinker derivatives of the present invention conjugated to even 50 to 100 PNA bases, as well as fluorophores, were not added and showed high water solubility.

10.

 Other exemplary uses of linker derivatives

In still other embodiments, the present linkers can be conjugated with a drug compound, for example, to increase its retention, its solubility or to link it effectively with another group, to direct it towards a target in a particular area of the body. For example, in some embodiments, at least one linker of homogeneous molecular and structural formula is conjugated to at least one drug compound.

The present linkers are also useful in a variety of biological and chemical assays. For example, in capture trials, steric barriers can be relaxed, in fact supporting a probe from a surface, so that it is free in the solution to recognize its target. (See Figure 5.) If the binding rate between the probe and the target is slow, in relation to the diffusion rate, a shorter linker can be used. However, when the binding rate is fast compared to the diffusion rate in the system, a long linker can be advantageous. Because the present linkers can be prepared with precise short or long lengths, as well as in mixtures of different lengths, depending on the synthesis method used, the present linkers provide a uniform system around which such assays can be designed.

The present invention is further illustrated by the following non-limiting examples.

Examples

Example 1: Synthesis of linkers L30 and L60

Part A:

A solution of 146 ml of 2,2'- (ethylenedioxy) bis (ethylamine) (98%) in 360 ml of tetrahydrofuran (THF) was cooled in an ice bath. To this, a solution of 65 g of di-tert-butyl dicarbonate (97%) in 260 ml of THF was added dropwise over 1 h. The solvent was evaporated on a rotary evaporator. The product was washed with 300 ml of water and the solvent was removed again.

The product was added to 300 ml of water and extracted with 300 ml of dichloromethane (DCM), then extracted further with 2x 150 ml of DCM. The collected organic phase was washed with 50 ml of water before evaporating to about half the volume.

The DCM was then extracted with 400 ml solution of 1M NaCitrate solution (pH 4.5), then extracted again with another 50 ml of NaCitrate solution. The resulting two aqueous phases were washed with 50 ml of DCM before cooling in an ice bath. Under stirring, 100 ml of 10 M NaOH was added until the pH of the solution was> 13.

In a separatory funnel, the product separated by itself. It was stirred with 300 ml of DCM and 50 ml of water. The organic phase was then evaporated, providing a white oil.

Yield 48.9 g = 65.7%. MS calc. For C11H24N2O4 248.3, found 248.5. (See structure 1, Fig. 1).

Part B: Preparation of Boc-L15-imide

76.2 g of structure 1 (Fig. 1), prepared, for example, as described in part A above, were dissolved in 155 ml of pyridine. 54.0 g of diglycolic anhydride (90%) was added and the solution was stirred for 15 min. Next, 117 ml of acetic anhydride (min. 98%) was added and stirred continuously at 95 ° C for 1 h. The solvents were evaporated in a rotary evaporator.

117 ml of water were added and the solution was stirred for 15 minutes before adding another 272 ml of water and 193 ml of DCM. The organic layer was extracted twice with 193 ml of Na2CO3 (1 M) and then twice with a mixture of 72 ml of 4M HCI and 289 ml of 1M sodium citrate, pH 4.5. After each extraction, the aqueous phase was washed with a little DCM. The collected organic phase was washed with 150 ml of water. The solvent was evaporated leaving the product (structure 2 of Fig. 1) as a pale oil.

Yield 100.3 g (0.29 mol) = 94%. MS calc. for C15H26N2O7 346, found 346.7.

Part C:

100.3 g of structure 2 (Fig. 1) was dissolved in 100 ml of THF and added dropwise to 169.4 ml of 2.2 ′ (ethylenedioxy) bis (ethylamine) at 60 ° C for 1 h. The amine was distilled from the reaction mixture at 75-80 ° C / 0.3 mBar.

The residue from distillation was added to a mixture of 88 ml of 4M HCI and 350 ml of 1M NaCitrate, pH 4.5 and extracted three times with 175 ml of DCM. The aqueous phase was cooled in an ice bath and 105 ml of 10 M NaOH was added.

In a separatory funnel, the product slowly separated from the solution. When separated, the product was distributed between 100 ml of water and 950 ml of DCM. The aqueous phase was further extracted four times with 150 ml of DCM and the combined DCM phases were reduced to an oil. The oily residue was dehydrated by evaporation in toluene to provide a yellow oil.

Yield 115.48 g = 81%. MS calc. for C21H42N4O9. 494.6, found 494.8. (See structure 3, Fig. 1).

Part D: Preparation of Boc-L30-imide

115.5 g of structure 3 (Fig. 1) was dissolved in 115 ml of pyridine. 40.6 g of diglycolic anhydride (90%) was added. After stirring for 15 minutes, 97 ml of acetic anhydride (min. 98%) was added, and the solution was stirred at 95 ° C for 1 h. The solvent was evaporated under reduced pressure.

The reaction mixture was cooled slightly and 80 ml of water was added. The reaction mixture was stirred for 15 minutes, then 200 ml of water and 150 ml of DCM were added. The organic layer was extracted twice with 150 ml of Na2CO3 (1 M) and then twice with a mixture of 53 ml of HCI (4 M) and 213 ml of NaCitrate (1 M). After each extraction, the aqueous phase was washed with a little DCM.

The collected organic phase was washed with 150 ml of water. The solvent was evaporated. The oily residue was dehydrated by evaporation in toluene to provide a yellow oil.

Yield 125 g = 92%. MS calc. for C25H44N4O12 592.6, found 592.5. (See structure 4 of Fig 1).

A further purification of the product is performed on a silica column, with a gradient of 5-10% methanol in DCM. Alternatively, the product is used for other chemical reactions. Yield 69%, as white oil.

Part E: Opening the ring to prepare Boc-L30 free acid

12.4 g of structure 4 (Figure 1) was dissolved in 12 ml of water, 12 ml of 1,4-dioxane and 6 ml of DIPEA and refluxed for 30 minutes, then cooled, evaporated. The oily residue was dehydrated by evaporation in toluene to provide a yellow oil. MS calc. for C25H46N4O14 610.6, found 610.8. (See structure 5, Fig. 1).

Part F: Preparation of an L-60 linker from Boc-L30-imide

To prepare Boc-L60-imide from two shorter linkers, Boc-L30-imide, 10 g, were dissolved in 100 ml of trifluoroacetic acid (TFA). After 10 minutes, the TFA was removed by evaporation under reduced pressure, to leave 20 g of a free amine adduct-L30-imide: TFA. This was dissolved in 100 ml of acetonitrile and neutralized with 15 ml of triethylamine.

To this solution was added 10.3 g of Boc-L30-activated free acid with 6.2 g of HATU (N-oxide N-oxide hexafluorophosphate -1- (dimethylamino) -1H-1,2,3-triazolo [4, 5-b] pyridi-1-ylmethylene] -N-methylmethanamine) and 4.5 ml of diisopropylethylamine in 50 ml of acetonitrile, for 5 min.

After 1 hour, the solvents were removed by evaporation, the product was added to 500 ml of dichloromethane and extracted with 150 ml of 1 M sodium carbonate. The dichloromethane was removed by evaporation and the product was purified on silica with 15% methanol. % in dichloromethane. Yield: 12.3 g of Boc-L60-imide, or 67%.

To prepare Boc-L60-free acid, 12.3 g of Boc-L60-imide was dissolved in 20 ml of water, 20 ml of dioxane and 20 ml of diisopropylethylamine and boiled for 1 h. The solvents were evaporated off to leave 14.3 g of the free diisopropyl-ethyl amine salt of Boc-L60-acid. An analysis by TLC showed that the reaction was quantitative.

Example 2: Synthesis of linkers and longer linker derivatives - General procedures

Longer linker derivatives were prepared by solid phase synthesis. Initially, the resin "was discharged" for solid phase syntheses. Since many linker derivatives reach molecular weights of, for example, 10 kDa, a corresponding amount of resin must be discharged. For example, in general, the loading in mol / g multiplied by the molecular weight of the desired product in g / mol should not exceed 1, which corresponds to a formal raw yield of 1 product / 1g of resin.

The general procedures for the preparation of components of linker derivatives are also described in this example.

Linker Synthesis

Resin discharge procedure

3 g of MBHA resin (01-64-0042, NovaBiochem) were expanded in NMP (N-methyl-pyrrolidone) overnight. They were washed with pyridine (2x), then with NMP and drained.

0.15 mmol / g of the Boc-Lys (Fmoc) -OH resin (04-12-0063, NovaBiochem), 0.45 mmol, 211 mg and 162 mg of 0.95 equi. of HATU (GEN076525, Applied Biosystems) and 156 μl of 2 equi. of DIPEA (diisopropylethylamine, 550043, Aldrich), were dissolved in 7 ml of NMP, activated for 2 minutes, and then applied to the resin. The reaction was allowed to proceed for 1 h with gentle shaking. The reaction mixture was drained and the resin was repeatedly washed with NMP. The remaining amino groups that had not reacted in the resin were acetylated with NMP / pyridine / acetic anhydride (2: 2: 1), for not less than 1 h, and until a qualitative Kaiser test failed to show free amino groups . The resin was then washed repeatedly with NMP, followed by repeated washing with DCM, and finally dried under vacuum. It was designated as "discharged resin".

5

fifteen

25

35

Four. Five

A sample of the resin was deprotected with TFA (trifluoroacetic acid) / mCresol, 19: 1 for 2 x 5 min, washed repeatedly with DCM, and dried under vacuum. This sample was subjected to a quantitative Kaiser test to determine the load. Result = 0.085 mmol / g amino groups of resin.

Solid Phase Synthesis Procedure

Conventional procedures for solid phase synthesis of Boc were as follows:

First, the resin was deprotected with TFA (trifluoroacetic acid) / mCresol, 19: 1 for 2 x 5 min, washed repeatedly with DCM, then with NMP, then with pyridine. Second, Boc-L30-OH, 0.26 M in NMP, was mixed with equal volumes of 0.234 M HATU in NMP and 0.5 M DIPEA in pyridine and activation was allowed for 2 min before adding to the resin . A fair enough volume was used to cover the resin and the coupling reaction was allowed to take place for 20 min. Generally, such a single coupling would be sufficient to obtain a negative Kaiser test, however when coupled to a residue of αNH2Lys (εFmoc), a double coupling is performed.

To avoid traces of deleted products, an encapsulation step with acetic anhydride / NMP / pyridine 2:49:49 was used, for 2 min, after each coupling. The resin was washed with NMP and then with DCM for the preparation of the next deprotection step.

Boc protected amino acids and PNA monomers were introduced using conventional conditions. Lysine residues were introduced to allow deprotection of the side chain in the resin, as well as the introduction, for example, of fluorophores.

Fmoc protection groups were removed with piperidine / NMP 1: 4 for 2 x 5 min. Staining and protection groups were removed with 3% hydrazine in NMP for 2 x 5 min.

The syntheses were also performed in an ABI 433A peptide synthesizer, according to the manufacturer's instructions (Applied Biosystems, Inc., Foster City, CA).

In general, it was observed that Boc-L30-OH fitted well, especially on itself. Apart from the residues mentioned αNH2-Lys (εFmoc), double couplings had to be made, only if a branched structure was prepared, with simultaneous coupling at different free amino terminal ends of the same compound.

A notable feature of resins with longer bound linker segments, was an extraordinary extinction, especially during deprotection with TFA. For example, when starting from 1 g of discharged resin, 3 ml of TFA would be sufficient to extinguish and cover the resin. However, after 10 couplings of Boc-L30-OH (the resin now with Boc-L-300 attached), more than 10 ml of TFA was required to extinguish and cover the resin. On the other hand, it did not seem that even said Boc-L300 segment in the Nα of the C-terminal lysine gave rise to any problem in performing the chemical reaction in the Nε of the lysine residue. For example, an Fmoc group was easily cleaved from this position, and subsequent couplings in that position seemed to be unaffected by the length of the linker.

Solid phase resin removal

Once the desired product on the resin had been obtained, the resin was removed by cleavage, using trifluoromethanesulfonic acid / TFA / M-Cresol, 7: 2: 1, releasing the product as a C-terminal carboxamide. The product precipitated in diethyl ether and dried in a stream of nitrogen. The crude products were dissolved in 15% acetonitrile in water, and purification was performed by RP-HPLC. A gradient of 15% to 40% acetonitrile in 0.02% aqueous TFA was typically used. It was observed that the derivatives of longer linkers were slightly acid labile. Prior to lyophilization, the HPLC elution buffer was typically neutralized with an appropriate base, such as sodium salt of MES (2- [N-morpholino] ethanesulfonic acid). For example, an isolated product in 5 ml of 0.02% TFA contains 1 μl of TFA, or 12 μmol, and was neutralized to pH 6 with 25 μl of 1 M MES sodium salt. This procedure was used for derivatives of linkers that were to be used later in water. Pyridine or DIPEA was used for neutralization when the products were to be used in an organic solvent, or when inorganic salts had presented a problem of solubility in aqueous solutions.

Synthesis of solid phase PNA

In a peptide synthesizer (ABI 433A, Applied Biosystems), PNA monomers were coupled to the resin, using conventional procedures for amino acid coupling and conventional PNA chemical reactions. The resin is then handled in a glass container to remove the protective groups and to label with other amino acids or fluorophores.

The elimination of the indicated protective groups is achieved with the following conditions:

Boc: TFA / m-cresol (in a ratio of 95/5) 2x5 min. Fmoc: 20% piperidine in DMF 2x5 min.

Dde: 3% hydrazine in DMF 2x5 min.

When the synthesis is finished, the PNA is cleaved from the resin with TFA / TFMSA / m-cresol / thioanisole (in a ratio of 6/2/1/1). The PNA is then precipitated with ether and purified by HPLC. MALDI-TOF mass spectrometry is used to determine the molecular weight of the product. {NOTE - this comes from the Funny bases of Example 2}.

Preparation of unnatural bases and PNA monomers

Pyrimidinone monomer (method 1)

one.

 In the dry equipment, 4.6 g of solid Na were added in small pieces to 400 ml of ethanol (99.9%), and dissolved with stirring. Hydroxypyrimidine hydrochloride, 13.2 g, was added and the mixture was refluxed for 10 minutes. Then 12.2 ml of ethyl bromoacetate (98%) was added and the mixture was refluxed for an hour and a half. The reaction continued using thin layer chromatography (TLC). The ethanol was evaporated leaving a white compound, which was dissolved in a mixture of 80 ml of 1M NaCitrate (pH 4.5) and 40 ml of 2M NaOH. This solution was extracted four times with 100 ml of dichloromethane (DCM) . The DCM phases were combined and washed with 10 ml of a NaCitrate / NaOH mixture. The washed DCM phases were evaporated under reduced pressure and resulted in 17.2 g of crude solid product. This crude solid product was recrystallized from ethyl acetate to provide a yellow powder. The yield of this stage was 11.45 g (63%).

2.

 The yellow powder, 12.45 g, from above, was hydrolyzed by refluxing a mixture of 36 ml of DIPEA, 72 ml of water and 72 ml of dioxane overnight. The solvent was evaporated and the water was removed from the residue by evaporation in toluene. The yield of this stage was 100%.

3.

 OBS Pyrimidinone acetic acid (10.5 g), 16.8 g of PNA main chain ethyl ester, 12.3 g of DHBT-OH, 19 ml of triethylamine, were dissolved in 50 ml of N, N-dimethylformamide (DMF). DIPIDIC (11.8 ml) was added and the mixture was stirred overnight at room temperature. The product was added to 100 ml of DCM and extracted three times with 100 ml of dilute aqueous NaHCO3. The organic phase was extracted twice with a mixture of 80 ml of 1M NaCitrate and 20 ml of 4M HCI. Since TLC showed that some material was in the citrate phase, it was extracted twice with DCM. The organic phases were combined and evaporated. Because there was a precipitation of urea, the product was dissolved in DCM, and the urea was removed by filtration. The subsequent evaporation left an orange oil. The purification of the orange oil was performed on a silica column with 10% methanol in DCM. The fractions were collected and evaporated to provide a yellow foam. The yield of this stage was 7.0 g (26.8%).

Four.

 The yellow foam (8.0 g) was refluxed overnight in 11 ml of DIPEA, 22 ml of water and 22 ml of dioxane. The solvent was evaporated and the oil was dehydrated by evaporation in toluene, leaving an orange foam. The yield of this stage was 100%.

Pyrimidinone monomer (method 2)

Step 1. In a dry machine, 9.2 g of solid Na were dissolved in small pieces in 400 ml of ethanol (99.9%), with stirring. Hydroxypyrimidine hydrochloride, 26.5 g, was added and the mixture was stirred for 10 minutes at 50 ° C. Then 24.4 ml of ethyl bromoacetate (98%) was added and the mixture was stirred at 50 ° C for 1 hour. The reaction continued using thin layer chromatography (TLC).

The ethanol was evaporated leaving a white compound, which had dissolved in 70 ml of water and extracted with 20 ml of DCM. Another 30 ml of water was added to the aqueous phase, which was extracted with 3x100 ml of DCM. The DCM phase from the first extraction contains a lot of product, but also some impurities, so this phase was extracted twice with water. These two aqueous phases were then extracted with DCM.

The combined DCM phases were combined and washed with 10 ml of water. The washed DCM phases were evaporated under reduced pressure and resulted in 25.1 g of yellow powder. The yield of this stage was 25.1 g = 69%. Maldi-Tof: 181.7 (calc. 182).

Step 2. 34.86 g of the above yellow powder was dissolved in 144 ml of 2M NaOH. After stirring for 10 minutes at room temperature, the mixture was cooled in an ice bath. Then 72 ml of 4M HCI (cold) was added. The product precipitated. After stirring for 5 minutes, the precipitated material was filtered and washed thoroughly with ice water. Drying in a desiccator under reduced pressure provided 18.98 g of yellow powder. The yield of this stage was 18.98 g = 64%.

Step 3. 11.1 g of pyrimidone acetic acid and 12.5 ml of triethylamine were dissolved in 24 ml of N, N-dimethylformamide (DMF), 26.2 g of HBTU plus 6 ml extra DMF were added. After 2 minutes, a solution of 14.7 g of PNA-main chain ethyl ester dissolved in 15 ml of DMF was added. The reaction mixture was stirred at room temperature and then using TLC. After half an hour a precipitate formed. This was filtered off.

The product was added to 100 ml of DCM and extracted with 2 x 100 ml of dilute aqueous NaHCO3. Both aqueous phases were washed with a little DCM. The organic phases were combined and evaporated. The evaporation left an orange oil. Purification of the product was performed on a silica column with 10-20% methanol in ethyl acetate. The fractions were collected and evaporated, providing a yellow oil. The oil was dissolved and evaporated twice in ethanol. The yield of this stage was 20.68 g = 90%.

Step 4. The yellow oil (18.75 g) was dissolved in 368 ml of 0.2 M Ba (OH) 2. It was stirred for 10 minutes before adding 333 ml of 0.221 M H2SO4. Precipitation was performed immediately. It was filtered through celite, which had been washed with water. The solvent was evaporated. Before evaporation ended, the product was centrifuged to remove the rest of the precipitation. Evaporation of the solvent again gave a yellow oil. The yield of this stage was 13.56 g = 78%.

Step 5. To perform a test on the P-monomer, 3 consecutive P were coupled to the Boc-L300Lys resin (Fmoc), following a normal conventional PNA procedure. The product was cleaved from the resin and also precipitated following conventional procedures: HPPP-L300-Lys (Fmoc). Maldi-Tof of the crude product: 6000 (calc. 6000) showing only small impurities.

Thioguanine monomer

1,6-Loroguanin (4.93 g) and 10.05 g of K2CO3 were stirred with 40 ml of DMF for 10 minutes at room temperature. The reaction mixture was placed in a water bath at room temperature and 3.55 ml of ethyl bromoacetate was added. The mixture was stirred in a water bath until TLC (20% methanol / DCM) showed that the reaction was over. The precipitated carbonate was filtered off and washed twice with 10 ml of DMF. The solution, which was a bit cloudy, was added to 300 ml of water, which made it clear. In an ice bath, the target compound precipitated slowly. After filtration, the crystals were washed with cold ethanol and dried in a desiccator. The yield of this stage was 3.3 g (44.3%) of ethyl chloroguanine acetate.

2.

 Ethyl chloroguanin acetate (3.3 g) was dissolved under reflux in 50 ml of absolute ethanol. Thiourea (1.08 g) was added. After refluxing for a short period of time, a precipitate began to form. According to TLC (20% methanol / DCM) the reaction ended in 45 minutes. After completion, the mixture was cooled in an ice bath. The precipitated material was then filtered and dried overnight in a desiccator. The yield of this stage was 2.0 g (60%) of ethyl thioguanine acetate.

3.

 Ethyl thioguanine acetate (3.57 g) was dissolved in 42 ml of DMF. Then, benzyl bromide (2.46 ml) was added and the mixture was stirred in an oil bath at 45 ° C. The reaction continued using TLC (25% methanol / DCM). After 3 hours, all the basic material had been consumed. The target compound of step 3 precipitated after evaporation under reduced pressure and elevated temperature. The precipitated material was recrystallized from absolute ethanol, filtered and then dried in a desiccator. The yield of this step was 3.88 g (82%) of methyl benzyl thioguanine ethyl ester.

Four.

 The methyl benzyl thioguanine ethyl ester (5.68 g) was dissolved in 12.4 ml of 2M NaOH and 40 ml of THF, and then stirred for 10 minutes. THF evaporated. This was repeated. The material was dissolved in water and then 6.2 ml of 4M HCl was added, while the target product precipitated. It was filtered and dried in a desiccator. The yield of this stage was 4.02 g (77%).

5.

 The product of step 4 (4.02 g), 3.45 g of ethyl ester of the main chain material, 9 ml of DMF, 3 ml of pyridine, 2.1 ml of triethylamine and 7.28 g of PyBop they were mixed and then stirred at room temperature. After 90 minutes, a solid precipitation formed. The product was added to 125 ml of DCM and 25 ml of methanol. This solution was then extracted, first with a mixture of 80 ml of 1M NaCitrate and 20 ml of 4M HCI, and then with 100 ml of aqueous and diluted NaHCO3. Evaporation of the organic phase provided a solid material. The material was dissolved in 175 ml of boiling ethanol. The volume of the solution was reduced to approximately 100 ml, boiling. After cooling in an ice bath, the target product precipitates. The crystals were filtered, washed with cold ethanol and then dried in a desiccator. The yield of this stage was 6.0 g (86%).

6.

 The product of step 5 (6.0 g) was dissolved in 80 ml of THF, 7.5 ml of 2M NaOH and 25 ml of water. The solution became clear after ten minutes of stirring. THF evaporated. Water (50 ml) was added to the mixture. When the pH was adjusted by the addition of 3.75 ml of 4M HCI, the thioguanine monomer precipitated. It was then filtered, washed with water and dried in a desiccator. The yield of this stage was 5.15 g (91%).

Ethyl ester of diaminopurin-acetic acid

1. Diaminopurine (10 g) and 40 g of K2CO3 were added to 85 ml of DMF and stirred for 30 minutes. The mixture was cooled in a water bath at 15 ° C. Ethyl bromoacetate (3 ml) was added three times at 20 minute intervals between each addition. This mixture was then stirred for 20 minutes at 15 ° C. The mixture was left in the water bath for another 75 minutes, and the temperature was increased to 18 ° C. The DMF was removed by filtration and the remaining K2CO3 was added to 100 ml of ethanol and refluxed for 5 minutes. The K2CO3 was filtered and repeatedly refluxed in 50 ml of ethanol. The combined ethanol phases were placed in a freezer, after which crystals formed. These crystals were filtered, washed with cold ethanol, filtered again and then dried in a desiccator overnight. The yield of this stage was d 12 g (76%).

Other general procedures

Amino acid coupling

The Boc protecting group is removed from the resin with TFA / m-cresol (in a ratio of 95/5) 2x5 min. The resin was then washed with DCM, pyridine and DMF, before coupling with the amino acid, which is dissolved in NMP at a concentration between 0.2 and 0.4 M and activated with 0.95 eq. of HATU and 2 eq. of DIPEA, for 2 minutes. The coupling is completed when the Kaiser test is negative. Cover formation occurs by exposing the resin for 3 minutes at (Ac) 2O / pyridine / NMP (in a 1/2/2 ratio). The resin is then washed with DMF and DCM.

Boc-L300-Lys Resin (Fmoc)

To the resin loaded with Boc-Lys (Fmoc), an L30 linker with a concentration of 0.26 M was coupled, using a conventional amino acid coupling. This was done 10 times providing the Boc-L300Lys resin (Fmoc).

Fluorescein marking

5 (6) -carboxy fluorescein was dissolved in NMP until it had a concentration of 0.2 M. Activation was performed with 0.9 eq. of HATU and 1 eq. of DIPEA for 2 minutes, before coupling for at least 2x20 minutes or until the Kaiser test was negative.

Synthesis of Fmoc-L30

Fmoc-L30 is suitable for preparing conjugates with acid-labile substituents. 23 g of Boc-L30-acid were dissolved in 200 ml of TFA and the nitrogen bubbled through the solution for 15 min. The TFA was removed as much as possible in a rotary evaporator leaving 43 g, (approximately 19 g of amino acid L30, 24 g of TFA). 130 ml of acetonitrile and then 42 ml of triethylamine (1.5 equivalents relative to residual TFA) were added. The mixture was cooled to room temperature and 16 g of Fmoc-NHS ester was added. After 30 minutes, the reaction was complete, and the solvents were evaporated again as much as possible. The residue was added to 300 ml of DCM and 200 ml of water, in a separatory funnel. The pH of the emulsion was carefully adjusted with 2M NaOH, up to about 8, and a clear aqueous phase formed very slowly over the emulsion. After several hours, the aqueous phase was collected, and the remaining emulsion was extracted again with 200 ml of water, again adding a little extra NaOH until it had a pH 8. Again a slow separation followed and the combined aqueous phases They were collected. They were placed back in a separatory funnel, together with 300 ml of DCM, and with 4M HCI, the pH of the aqueous phase was adjusted to 3. After a certain time, a clear DCM solution was separated with the product. , and the aqueous phase was extracted with an additional 200 ml of DCM. The combined DCM phases were evaporated to dryness to provide 16 g of Fmoc-L30-acid.

Nomenclature of representative products

A strict notion of peptide is used to describe exemplary products below. Thus, the products are all written with the N-terminal end on the left, the C-terminal end on the right, indicating a "H" on the far left, a free amino terminal, indicating a "NH2" on the far right a C-terminal carboxamide. When lysine residues are substituted in the amino group of the side chain, this is indicated by placing the substituent in parentheses, indicating (NH2) a free amino group; (Flu) 5/6-carboxyfluorescein; (Cys) cysteine, (betaala) beta-alanine-N, N-diacetic acid. PNA residues are indicated as follows: A = adenine; C = cytosine; D = diaminopurine; G = guanine; Gs = thioguanine; l = inosine; P = 2-oxo-pyrimidine (pyrimidinone); T = thymine; U2s = 2-thiouracil.

Figures 6 and 7 represent representative structures of natural and non-natural nucleic bases, PNA monomers, intermediates and linker derivatives.

Example 3: Preparation of derivatives of exemplary linkers 1-13

1: Ac-L30-Lys (betaala) -NH2.

The unloaded Boc-Lys resin (Fmoc), 0.5 g, was deprotected in Fmoc and subjected to a coupling with beta-alanine-N, benzyl N-diacetate. The Boc protective group was then removed, and the resin was subjected to a coupling with L30. The amino terminal group protected with Boc was deprotected and acetylated for 5 minutes with 2% acetic anhydride in NMP / pyridine. The product was cleaved from the resin, precipitated and purified by RPHPLC, then neutralized with pyridine and lyophilized to form a colorless oil. The yield was 25 mg. MS calc. for C35H62N8O17 it was 866.923.

2: Ac-L60-Lys (betaala) -NH2.

The unloaded Boc-Lys resin (Fmoc), 0.5 g, was deprotected in Fmoc and subjected to a coupling with beta-alanine-N, benzyl N-diacetate. The Boc protective group was then removed, and the resin was subjected to two couplings with L30. The amino terminal group protected with Boc was deprotected and acetylated for 5 minutes with 2% acetic anhydride in NMP / pyridine. The product was cleaved from the resin, precipitated and purified by RP-HPLC, then neutralized with pyridine and lyophilized to form a colorless oil. The yield was 62 mg. MS calc. for C55H98N12O27 it was 1359,452.

3: Ac-L90-Lys (betaala) -NH2.

The unloaded Boc-Lys resin (Fmoc), 0.5 g, was deprotected in Fmoc and subjected to a coupling with beta-alanine-N, benzyl N-diacetate. The Boc protective group was then removed, and the resin was subjected to three couplings with L30. The amino terminal group protected with Boc was deprotected and acetylated for 5 minutes with 2% acetic anhydride in NMP / pyridine. The product was cleaved from the resin, precipitated and purified by RP-HPLC, then neutralized with pyridine and lyophilized to form a colorless oil. The yield was 90 mg. MS calc. for C75H134N16O37 it was 1851,980.

4: MCC-L150-Lys (beta) -NH2

The unloaded resin Boc-L150-Lys (Fmoc), 0.25 g, was deprotected in Fmoc and subjected to a coupling with beta-alanine-N, benzyl N-diacetate. The Boc protecting group was then removed and the product was cleaved from the resin, and precipitated. The crude product was resuspended in 1 ml of NMP and 0.25 ml of DIPEA and 14 mg of succinimidyl-14- (N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce 22360) were added. The reaction was completed after 1 h. The product was precipitated in diethyl ether and purified by RP-HPLC, neutralized with DIPEA and lyophilized to form a colorless oil. The yield was 35 mg. MS calc. for C125H217N25O59 it was 3014,239.

5: Flu-L120-Lys (NTA) -NH2

The unloaded Boc-Lys resin (2-CI-Z), 500 mg, was deprotected in Boc and subjected to two couplings with BocL60-OH. The Boc group was removed and the carboxyfluorescein, 150 mg, activated with HBTU, 136 mg, and added to the DIPEA resin, 70 µl, in 2 ml of NMP. After 1 h of coupling, the resin was washed and treated with 20% piperidine in DMF for 5 min. The resin was washed with TFA and the intermediate (FIu-L120-Lys (NH2) -NH2) was cleaved from the resin and precipitated in diethyl ether. The product was resuspended in 500 µl of NMP and 100 µl of DIPEA and NTA-diterbutyl ester, 9 mg, activated with succinimidyl-N hexafluorophosphate-N, N, N ', N'tetramethyluronium, 10 mg, and 10 µl of DIPEA in 50 μl of NMP. After 10 minutes of reaction, 50 µl of piperidine was added, and after another 5 minutes, the product was precipitated in diethyl ether. The product was dissolved in 500 µl of TFA, 50 µl of TFMSA, 50 µl of m-cresol, and after 10 minutes, precipitated again and purified by RP-HPLC. The yield was 26 mg. MS calc. for C113H176N20O it was 2646,752, and the measured MS was 2646.

6: DNP-L120-Lys (Rho) -L120-Lys Lys (NH2) -NH2

The unloaded Boc-Lys resin (2-CI-Z), 55 mg, was deprotected in Boc and subjected to two couplings with BocL60-OH. Boc-Lys (Fmoc) was then coupled, followed by two other couplings with Boc-L60-OH. The Fmoc group was removed and tetramethylrodamin carboxylic acid, 17 mg, was activated with succinimidyl N, N, N ', N'-tetramethyluronium hexafluorophosphate, 14 mg, and DIPEA, 13 μl, in 400 μl of NMP was added to the resin . After 30 minutes, the resin was washed and deprotected in Boc, followed by treatment with 2,4-dinitrofluorobenzene, 19 mg, and DIPEA, 35 μl, in 400 μl of NMP. The product was cleaved from the resin, precipitated in diethyl ether and purified by RPHPLC. Yield 6.5 mg. MS calc. for C203H336N41O90 it was 4791,135, while the MS found was 4778.3. (DNP loses oxygen in MS)

7: FIu-L120-Lys (Flu) -L120-Lys (NTA) -NH2

The unloaded Boc-Lys resin (2-CI-Z), 200 mg, was deprotected in Boc and subjected to two couplings with BocL60-OH. Boc-Lys (Fmoc) was then coupled, followed by two other couplings with Boc-L60-OH. The Boc and Fmoc groups and the carboxyfluorescein, 75 mg, were activated with HBTU, 68 mg, and DlPEA, 35 μl, in 1 ml of NMP, was added to the resin. After 1 h of coupling, the resin was washed and treated with 20% piperidine in DMF for 5 min. The resin was washed with TFA and the intermediate (FIu-L120-Lys (Flu) -L120-Lys (NH2) -NH2) was cleaved from the resin and precipitated in diethyl ether. The product was resuspended in 500 µl of NMP and 100 µl of DIPEA and NTA-di tert-butyl ester, 9 mg, activated with succinimidyl hexafluorophosphate-N, N, N ', N'-tetramethyluronium, 10 mg, and 10 were added μl of DIPEA in 50 μl of NMP. After 10 minutes of reaction, 50 µl of piperidine was added, and after another 5 minutes, the product precipitated in diethyl ether. The product was dissolved in 500 µl of TFA, 50 µl of TFMSA, 50 µl of m-cresol, and after 10 minutes, precipitated again and purified by RP-HPLC. The yield was 38 mg. MS calc. for C220H342N38O99 it was 5103,347, while the measured MS was 5094.

8: Rho-L120-Lys (Flu) -L120-Lys (betaala) Lys (betaala) -NH2

The unloaded Boc-Lys (Fmoc) resin, 100 mg, was deprotected in Boc and subjected to a coupling with BocLys (Fmoc) -OH. Two Fmoc groups were removed and subjected to a coupling with beta-alanine-N, benzyl N-diacetate, coupled to the two amino groups of the side chain. The synthesis continued with 4 x Boc-L30-OH, BocLys (Fmoc) -OH and then 4 x Boc-L30-OH. The Fmoc group and carboxyfluorescein, 37 mg, activated with HBTU, 34 mg, and DIPEA, 17 µl, in 0.5 ml of NMP were removed, added to the resin for 30 min. The terminal Boc group was then removed and 17 mg, activated with succinimidyl-N, N, N ', N'-tetramethyluronium hexafluorophosphate, 14 mg, and DIPEA 13 μl, in 400 μl was added to the resin of NMP ,. After 30 minutes of coupling, the resin was treated with 20% piperidine in NMP. The resin was washed with DCM, then with TFA, and the product was cleaved from the resin, precipitated in diethyl ether, and purified by RP-HPLC. The yield was 8 mg. MS calc. for C238H375N43O103 it was 5486,838, while the MS found was 5478.3.

9: Flu-L90-Lys (Flu) -L90-Lys (Flu) -L60-Lys (betalala) -Lys (betaala) -NH2

The unloaded Boc-Lys resin (Fmoc), 250 mg, was deprotected in Boc and subjected to a coupling with BocLys (Fmoc) -OH. The two groups Fmoc and beta-alanine-N, benzyl N-diacetate were removed, coupled to the two amino groups of the side chain. The synthesis continued with 2 x Boc-L30-OH, Boc-Lys (Fmoc) -OH, 3 x Boc-L30-OH, Boc-Lys (Fmoc) -OH and finally with 3 x Boc-L30-OH. The two Fmoc groups were removed, then the terminal Boc group. Carboxyfluorescein, 75 mg, activated with HBTU, 68 mg, and DIPEA, 35 μl, in 1 ml of NMP, was added to the resin twice for 30 minutes, followed by 5 min of 20% piperidine treatment in NMP. The resin was washed with DCM, then with TFA, and the product was separated from the resin by cleavage, precipitated in diethyl ether and purified by RP-HPLC. The yield was 25 mg. MS calc. for C261H387N43O112 it was 5919,183, while the MS found was 5030.

10: FIu-L120-Lys (Flu) -L120-Lys (Flu) -L120-Lys (betalala) -Lys (betaala) -NH2

The unloaded Boc-Lys resin (Fmoc), 250 mg, was deprotected in Boc and subjected to a coupling with BocLys (Fmoc) -OH. The two Fmoc groups were removed and beta-alanine-N, benzyl N-diacetate was coupled to the two amino groups of the side chain. The synthesis continued with 4x Boc-L30-OH, Boc-Lys (Fmoc) -OH, 4x Boc-L30-OH, Boc-Lys (Fmoc) -OH and finally with 4x Boc-L30-OH. The two Fmoc groups were removed, then the terminal Boc group. Carboxyfluorescein, 75 mg, activated with HBTU, 68 mg, and DIPEA, 35 μl, in 1 ml of NMP was added to the resin twice for 30 minutes, followed by 5 minutes of treatment with 20% piperidine in NMP. The resin was washed with DCM, then with TFA and the product was separated from the resin by cleavage, precipitated in diethyl ether and purified by RP-HPLC. The yield was 34 mg. MS calc. for C341H531N59O152 it was 7889,296, while the MS found was 7885.

11: FIu-L120-Lys (FIu) -L120-Lys (Flu) -L120-Lys (Flu) -L120-Lys (betalala) -Lys (betaala) -NH2

The unloaded Boc-Lys resin (Fmoc), 250 mg, was deprotected in Boc and subjected to a coupling with BocLys (Fmoc) -OH. The two groups Fmoc and beta-alanine-N, benzyl N-diacetate were removed, coupled to the two amino groups of the side chain. The synthesis continued with 4 x Boc-L30-OH, Boc-Lys (Fmoc) -OH, 4 x Boc-L30-OH, Boc-Lys (Fmoc) -OH, 4 x Boc-L30-OH, Boc-Lys ( Fmoc) -OH and finally with 4 x Boc-L30-OH. The three Fmoc groups were removed, then the terminal Boc group. Carboxyfluorescein, 75 mg, activated with HBTU, 68 mg, and DIPEA, 35 μl, in 1 ml of NMP was added to the resin twice for 30 minutes, followed by 5 minutes of treatment with 20% piperidine in NMP. The resin was washed with DCM, then with TFA and the product was separated by cleavage of the resin, precipitated in diethyl ether and purified by RP-HPLC. The yield was 25 mg. MS calc. for C448H697N77O199 it was 10345.89, while the MS found was 10336.

12: FIu-L120-Lys (FIu) -L120-Lys (Flu) -L120-Lys (Flu) -L120-Lys (Flu) -L120-Lys (beta) -NH2

The unloaded Boc-Lys resin (Fmoc) was deprotected in Fmoc and the benzyl beta-alanine-N, N-diacetate was coupled to the amino group of the side chain. Fifty mg of this resin were used for automated solid phase synthesis, the sequence coupling 4 x Boc-L30-OH, then Boc-Lys (Fmoc) -OH four times and finally 4 x Boc-L30-OH. The four Fmoc groups and the terminal Boc group were removed, and the resin was subjected to a carboxyfluorescein labeling for 3 x 1 h. The resin was treated with 20% piperidine in DMF, washed with NMP, DCM, TFA and the product was separated by cleavage. After precipitation with diethyl ether, it was purified by RP-HPLC. The yield was 7 mg. MS calc. for C542H942N92O240 it was 12487.16, while the MS found was 12463.

13: (betaala) -L60-Lys (Flu) -L240-TGTACCTTGA-NH2

The unloaded Boc-PNA-A 50 mg resin was used for automated synthesis in a 433A peptide synthesizer. First, the appropriate 9 PNA monomers were coupled, then 8 x Boc-L30-OH, then BocLys (Fmoc) -OH and finally again 2 x Boc-L30-OH. The resin was then deprotected in Boc manually, coupled with beta-alanine-N, benzyl N-diacetate, then deprotected in Fmoc and labeled with carboxyfluorescein on the lysine side chain. The product was separated from the resin by cleavage, precipitated in diethyl ether and purified by RP-HPLC. The yield was 4.5 mg. MS calc. for C342H528N98O144 it was 8316,547, while the MS found was 8318.

Example 4: Derivatives of linkers 14-19 and their fluorescence intensities

The following 6 PNAs with the same sequence but with different linker, and artificial fluorescein structures at one or both ends of the PNA, were prepared by conventional solid phase synthesis.

14: Ac-Lys (Flu) -L30-AACGGGATAACTGCACCT-L30-Lys (FIu) -L90-Lys (FIu) -NH2. PM calc. for C376H488N134O123 8852,852; Found 8855.

15: Ac-Lys (Flu) -L30-AACGGGATAACTGCACCT-L30-Lys (Flu) -L90-Lys (Flu) -L90-Lys (Flu) -NH2. PM cal. for C463H618N148O160 10816.92; found 10819.

16: H-Lys (Flu) -L30-AACGGGATAACTGCACCT-L30-Lys (Flu) -L90-Lys (Flu) -L90-Lys (Flu) -NH2. PM calc. for C461H615N148O159 10773.87; found 10777.

17: (Flu-L60) 2-Lys-L30-AACGGGATAACTGCACCT-NH2. PM calc. for C341H452N130O114 8196,159; Found 8197.

18: Flu-L30-AACGGGATAACTGCACCT-NH2. PM calc. for C234H286N112O67 5739,564; found 5741.9.

19: ((Flu-L60) 2-Lys-L60) 2-Lys-L30-AACGGGATAACTGCACCT-NH2. PM calc. for C555H784N166O208 13109.35; found 13113.6.

The PNA sequence was directed towards a repetitive DNA sequence in the centromere of human chromosome 17, allowing the comparison of PNAs by FISH in metaphases. To eliminate the variation of the specimen, each of the PNAs was mixed with a fluorescent red PNA directed to the same region of the centromere on chromosome 17, and the intensity of the red and green signals was visually compared and quantified with the “ Measurement. Intensity ”in the Leica QFISH program.

The intensity of the green signal obtained from different fluorescein-labeled PNAs increased with the number of fluoresceins in the PNA, indicating little or no extinction of fluorophores. Derivatives 15, 16 and 19, each comprising 4 fluorophores, gave more intense signals. The most intense of the three probes was derivative 16, indicating that its free extra N-terminal amino group, in relation to derivative 15, increased its affinity towards DNA. Derivative 16 was also more intense than derivative 19, indicating that the linear design of 16 gave the probe easier access to DNA than the design similar to a branched dendrimer of 19.

The fluorescence intensity of the PNA conjugates, according to this invention, was also compared with that of conventionally labeled PNAs, without linkers. Conventionally, the size of the PNA sequence limits the number of fluorophores that can be fixed. For example, only two fluorophores can typically be fixed to a 20-mer PNA, since extinction becomes a problem with a high number of fluorophores. In addition, guanine bases extinguish nearby fluorophores, so that the intensity of an individually labeled PNA probe typically drops to about 20-60% of the fluorophore intensity only in solution. Instead, the present linker derivatives mitigate those limitations. Linker derivatives comprising PNAs can be labeled with four fluorescent labels, for example, providing 200% of the intensity of a free fluorescent probe in solution.

Example 5: Linker derivatives 20-23

Protein conjugates and their fluorescent intensities

Linker derivatives 9, 10 and 11 were used for streptavidin coupling. The residues of beta-alanine-N, N-diacetic acid on the linkers were converted into amino-reactive cyclic anhydrides by treatment with diisopropyl carbodiimide. We found that this activation method was particularly suitable for conjugation in slightly basic aqueous media, and was superior to activation with NHS-ester and with other cyclic anhydrides, such as nitrilotriacetic acid (NTA). (See Figure 4 for a representation of the activation of Lys (betaala).) Not only were cyclic anhydrides of betaalanine-N, N-diacetic acid quantitatively formed in 30 minutes, they also showed a high selectivity to react with amino groups on proteins , more than to be hydrolyzed in water.

Typical procedure: The 10.1 micromolar linker derivative was dissolved in 86 µl of NMP and 4 µl of pyridine. Once the solution was complete, 10 µl of diisopropyl carbodiimide was added and the mixture was allowed to stand for 30 minutes at room temperature. To confirm that the conversion to cyclic anhydride had taken place, 1 µl of the mixture was reacted with a large excess of primary amine (see, eg, structure 3 of Figure 1, 493 Da). MALDI-TOF mass spectroscopy confirmed that the desired activation had taken place. The activated linker precipitated in diethyl ether, dried briefly in a stream of nitrogen, and was dissolved again in a solution of 2.6 mg streptavidin (50 nmol) in 100 µl of 150 mM sodium hydrogen carbonate, pH 8.0 . The reaction was completed after 10 min. The product was purified by size exclusion chromatography on a 200 column of SUPERDEX®. Mass spectroscopy and UV measurements showed that 4 molecules comprising linker with a total of 12 fluoresceins, had been fixed to streptavidin.

Using the above procedure, the following 4 conjugates were prepared: 20: Streptavidin with 4 linker derivatives, each comprising the above derivative 9. The conjugate comprised a total of 12 fluoresceins with L90 spacers between fluorophores.

21: Streptavidin with 8 linker derivatives, each comprising derivative 9. In total, the conjugate comprised 24 L90 spacer fluoresceins between fluorophores.

5 22: Streptavidin with 4 linker derivatives, each comprising the above derivative 10. In total, the conjugate comprised 12 fluoresceins with L120 spacers between fluorophores.

23: Streptavidin with 4 linker derivatives, each comprising the above derivative 11. In total, the conjugate comprised 16 fluoresceins with L120 spacers between fluorophores.

The fluorescence of the three linker derivatives 9, 10 and 11, the 4 streptavidin conjugates was determined

10 20, 21, 22 and 23, and a streptavidin conjugate conventionally labeled with FITC, with two fixed fluoresceins (Dako F422 with 2 FITC / streptavidin). All compounds were prepared as total solutions of 100 nm fluorescein in 150 mM carbonate pH 8, using 100 nM free carboxy fluorescein as a reference for normalization of relative intensities. The solutions were excited at 488 nm, and the fluorescence intensity was read at 520 nm. The results are presented in the following table:

Compound

Intensity at 520 nm (arbitrary units) Relative intensity for each fluorescein Total intensity for a compound = relative intensity x number of fluoresceins

carboxy fluorescein

884 1,000 (reference) 1,000 (reference)

9

517 0.58 1.75

10

 592 0.67 2.01

eleven

 535 0.61 2.42

twenty

 233 0.26 3.16

twenty-one

 161 0.29 3.53

22

 260 0.18 4.38

2. 3

 240 0.27 4.34

F422

 411 0.46 0.93

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As can be seen, the linker derivatives of the present invention are up to several times more intense than streptavidin conventionally labeled with F422. In addition, as expected, the longer the linkers are and, therefore, the greater the separation between the individual fluorophores, the less extinction was observed. In fact, slight extinction was observed in free linker derivatives 9, 10 and 11, indi

20 strongly that the linkers had extended completely in water.

The more linkers incorporated into the same conjugate, the extinction was evidently increased, since the linkers were immobilized on streptavidin. (Compare 20 to 21, for example). However, the absolute number of fluoresceins that could be conjugated through linker derivatives readily compensated for this, allowing conjugate preparation with 4-5 times the fluorescent intensity used nor

Badly in conventional methods, of the prior art.

Example 6: Linker derivatives comprising PNAs with unnatural bases

Part A: Exemplary embodiments of PNA sequences

All prepared by conventional PNA procedures (see Example 2)

NAME OF THE SEQUENCE

SEQUENCES OF PNA1 N-TERMINAL C-TERMINAL MOLECULAR WEIGHT

I KNOW THAT. AA

TCD-DGSGS-TAC-A  FLU-L30- -LYS (CYS) 8805

I KNOW THAT. AB

USGUS-DPP-TTG-D FLU-L30- -LYS (CYS) 8727

I KNOW THAT. AC

CUSGS-GSDD-TUSD-GSDC FLU-L30- -LYS (CYS) 9413

I KNOW THAT. AD

GTP-TAA-TTP-PAG FLU-L30- -LYS (CYS) 9203

I KNOW THAT. AE

DGST-CGSD-DGSG-USCUS  FLU-L30- -LYS (CYS) 9413

I KNOW THAT. AF

AGA-CPT-TPG-APT FLU-L30- -LYS (CYS) 9187

I KNOW THAT. AG

TCD-DI I-TAC-A FLU-L30- -LYS (CYS) 8742

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1 FIu is fluorescein; T is thiamine; C is cytosine; D is diaminopurine; GS is thioguanine; A is adenine; US is 2/4 thiouracil; G is guanine; P is pyrimidinone; I is inosine.

Part B - Three PNAs with the L30 linker with different amino acids at the C-terminal end

BA: Flu-L30-DGT-DTC-GTD-CCG-Lys (acetyl)

BB: Flu-L30-DGT-DTC-GTD-CCG-Lys (Cys)

BC: Flu-L30-DGT-DTC-GTD-CCG-Lys (Lys) 3

Part C - Synthesis of Flu-L90-Lys (Flu) -L90-Lys (Cys)

Using the unloaded resin procedure, provided in Example 2, an MBHA resin was loaded with BocLys (Dde) -OH. Using a peptide synthesizer, the amino acids were coupled according to the PNA solid phase procedure, provided in Example 2, providing Boc-L90-Lys (Fmoc) -L30-Lys (Dde). The Boc and Fmoc protecting groups were removed and the amino groups were labeled with fluorescein using the procedure in Example 2. The protective group Dde was then removed and 0.4 M cysteine was added, according to the procedure of Example 2. The PNA was cleaved from the resin, precipitated in ether and purified by HPLC according to Example 2. The product was found to have a molecular weight of 3062 using MALDI-TOF mass spectrometry; The calculated molecular weight was 3061.

Example 7: Synthesis of dextran conjugates

Conjugate prepared from the AA sequence of Example 6, DexVS70, and Flu (10)

Dextran (with a molecular weight of 70 kDa) was activated with divinylsulfone to a level of 92 reactive groups / dextran polymer; This product is designated DexVS70.

280 μl of DexVS70 20 nmol 66 μl of FIu2Cys 160 nmol (prepared from Example 8) 25 μl of 0.8 M NaHCO3 pH = 9.5 29 μl of H2O

The previous four compounds were mixed. The mixture was placed in a water bath at 30 ° C for 16 hours. The mixture was added to 50 nmol of lyophilized PNA (AA sequence). The mixture was placed in a water bath at 30 ° C for 30 minutes. The conjugation reaction was quenched rapidly with 50 µl of 500 mM cysteine for 30 minutes at 30 ° C. Product purification was performed using FPLC: SUPERDEX®-200 column, 10 mM Hepes buffer, 100 mM NaCl, method 7 bank 2, 1 ml loop. Two fractions were collected: one with the product and one with the residue. The relative absorbance FIu2 (ε500 nm = 146000 M-1, ε260 nm = 43350 M-1) and PNA (ε500 nm = 73000 M-1, ε260 nm = 104000 M-1) was used to calculate average conjugate ratio of FIu2 , PNA and DexVS70. The average ratio of conjugation between FIu2 and DexVS70 was 9.4. The ratio of conjugation between PNA (sequence AA) and DexVS70 was 1.2.

HRP-DexVS70-Sec. AA

Using the procedure for conventional synthesis of the HRP-DexVS70-PNA conjugate, the HRP-DexVS70-Sec conjugate was prepared. AA The ratio between HRP and DexVS70 is 12.2; The ratio between Sec. AA and Dex70 is 1.2.

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GaM-DexVS70-Sec. AB

The synthesis of GaM-DexVS70-Sec. AB was performed using the procedure of Example 17 with the following changes as indicated. Dextran (molecular weight 70 kDa) is activated with divinylsulfone to a level of 92 reactive groups / polymer of

dextran

105.0 μl of DexVS70 57.0 μl of goat anti-mouse immunoglobulin (GAM-Ig) 8.9 μl of 4 M NaCI 10.6 μl of 0.8 M NaHCO3 (pH = 9.5) 144.5 μl of H2O

     7.5 nmol 15 nmol

The previous five components were mixed and placed in a water bath at 30 ° C for 40 minutes. Two hundred and ninety µl were taken from the mixture and added to 100 nmol of Sec. AB, which had previously dissolved in 80 µl of H2O. Then, 20 μl of 0.8 M NaHCO3 (pH 9.5) was added and the mixture was placed in a 30 ° C water bath for 1 hour. Extinction was performed by adding 39 µl of 500 mM cysteine and allowing the resulting mixture to stand for 30 minutes at 30 ° C.

Purification of the product by FPLC: SUPERDEX® -200 column, 10 mM Hepes buffer, 100 mM NaCI, method 7 bank 2, 1 ml loop. Two fractions were collected: one with the product and one with the residue. The relative absorbance of PNA (FIu) (ε500 nm = 73000 M-1) and GAM (ε278 nm = 213000 M-1) (correction factor for PNA at 278 nm is due to the specific PNA and is calculated: 278/500 nm ) was used to calculate average proportion of conjugation of PNA, GAM and DexVS70. The ratio between PNA and DexVS70 was 5.3 and the ratio between GAM and DexVS70 was 0.8.

Exemplary embodiments of the PNA1-DexVS-PNA2 conjugates

Conjugate designation

proportion PNA1  PNA1 nmol PNA1 vs. DexVS PNA2 PNA2 nmol PNA2 vs. DexVS DexVS

Conj. AC

1: 9 Sec. AA 12.5 1.02 Sec. AD 100 8.2 DexVS70

Conj. CB

1: 6 Sec. AC 40 1.5 Sec. AB 200 7.4 DexVS70

Conj. DC

1:16 Sec. AC 13.3 0.84  Sec. AB 200 12.7 DexVS150

Conj. CD

1: 6 Sec. AC 40 2.3 Sec. AB 200 11.5 DexVS150

All conjugates were prepared by conventional conjugation procedures, described for the synthesis of PNA1-DexVS70-PNA2.

BCL2 anti-human-DexVS70-PNA

Dextran (molecular weight 70 kDa) was activated with divinylsulfone to a level of 92 reactive groups / dextran polymer, and was called DexVS70. The anti-human BCL2 antibody was designated AHB.

105 μl of DexVS70 7.5 nmol 800 μl of AHB conc. (2.9 g / l) 15.1 nmol 25 μl of 4 M NaCl 32 μl of 0.8 M NaHCO3 (pH = 9.5)

The previous four compounds were mixed and placed in a 30 ° C water bath for 65 minutes. From this mixture, 875 μl was taken and added to the indicated number of nmol of PNA in the table below; before the addition, the PNA had been dissolved in the μl of H2O indicated in the table below. Next, the μl number of 0.8 M NaHCO3 (pH 9.5) was added, according to the table below. The resulting mixture was placed in a 30 ° C water bath for 70 minutes. Extinction was performed by adding 6 mg of solid cysteine (0.05 M) to the mixture and allowing it to stand for 30 minutes at 30 ° C.

Purification of the product by FPLC: SUPERDEX®-200 column, 10 mM Hepes buffer, 100 mM NaCI, method 7 bank 2, 1 ml loop. Two fractions were collected: one with the product and one with the residue. The relative absorbance of PNA (FIu) (ε500 nm = 73000 M-1) and AHB (ε278 nm = 213000 M-1) (the correction factor for PNA at 278 nm is due to the specific PNA and is calculated: 278/500 nm) was used to calculate the average conjugation ratio of PNA, AHB and DexVS70.

Conjugates with different proportions of PNA

Conjugate designation

nmol of PNA added μl of H2O added μl of 0.8 M NaHCO3 (pH 9.5) added PNA vs. DexVS70 AHB vs. DexVS70

Conj. GIVES

100 75 25 9.5 1.6

Conj. DB

33  30  10 2.9 1.2

Conj. DC

67  60  twenty 5.6 1.1

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Example 7: Solid phase synthesis and purification of Lys (Flu) -L30-chr 17: 14-L30-Lys (FIu) -L90-Lys (Flu) -L90-Lys (Flu)

All conventional procedures are described in Example 2.

one.

 An MBHA resin was loaded with Boc-L30-Lys (Fmoc) -L90-Lys (Fmoc) -L90-Lys (Fmoc) using a conventional loading procedure for a loading of 0.084 mmol / g.

2.

 To this resin, Boc-Lys (Fmoc) -L30-AAC-GGG-ATA-ACT-GCA-CCT- was coupled using the peptide synthesizer, after a conventional solid phase chemical reaction of PNA. Fmoc protecting groups were removed and amino groups were labeled with fluorescein. After cleaving and precipitating, the PNA dissolved in TFA. The precipitated material was washed with ether. The precipitated material was dissolved in 200 µl of NMP. To this solution, 6 mg of Fmoc-Osu was added and dissolved. Next, DIPEA (9 µl) was added and the reaction was monitored using MALDI-TOF mass spectrometry. After 30 minutes, the reaction was terminated and the PNA precipitated and washed with ether.

HPLC after dissolving the PNA in 30% CH3CN and 10% TFA / H2O, provided three pure fractions. The fractions were pooled and lyophilized. The lyophilized PNA was then dissolved in 192 µl of NMP. Piperidine (4 μl) and 4 μl of DBU were added to this solution that was allowed to stand for 30 minutes. Mass spectrometry analysis of MALDI-TOF provided a molecular weight of 10777.

The precipitated material was washed with ether and then dissolved in 100 µl of TFA. The precipitated material was washed with ether and then dried using N2 gas.

Example 8: Conventional synthesis of the HRP-DexVS70-PNA conjugate

Dextran (molecular weight 70 kDa) is activated with divinylsulfone to a level of 92 reactive groups / polymer of

dextran

192 μl of DexVS70 255 μl of horseradish peroxidase (HRP) 15 μl of 4 M NaCI 19 μl of 0.8 M NaHCO3 pH = 9.5 119 μl of H2O

    13.7 nmol 602 nmol

The five previous components were mixed together, and placed in a water bath at 30 ° C for 16 hours. Five hundred microliters of this mixture was added to 50 nmol PNA, which had previously dissolved in 40 µl of H2O. Next, 10 µl of 0.8 M NaHCO3 (pH 9.5) was added. The mixture was then placed in a water bath at 30 ° C, for 2 hours. Extinction is performed by adding 55 μl of 110 mM cysteine and allowing the resulting mixture to stand for 30 minutes at 30 ° C.

The product is purified by FPLC: SUPERDEX®-200 column, 10 mM Hepes buffer, 100 mM NaCI, method 7 bank 2, 1 ml loop.

Two fractions were collected: one with the product and one with the residue. The relative absorbance of HRP (ε404 nm = 83000 M-1, ε500 nm = 9630 M-1) and PNA (Flu) (ε500 nm = 73000 M-1) was used to calculate the average conjugate ratio of HRP, PNA and DexVS70.

Example 9: Additional methods of synthesis of AP-DexVS70-PNA

Conjugate

The alkaline phosphatase ("AP") (from calf intestine, EIA quality) was dialyzed overnight against 2mM HEPES, pH 7.2; 0.1 M NaCI; 0.02 mM ZnCI2. Dextran (molecular weight 70 kDa) was activated with divinylsulfone to a level of 92 reactive groups per dextran polymer (DexVS70).

The three components below were mixed together and placed in a water bath at 40 ° C for 30 minutes.

192.0 μl of DexVS70 13.7 nmol 41.0 μl of PNA PNA 41 mmol dissolved in H2O 6.0 μl of 1 M NaHCO3

108.0 µl of the DexVS70-PNA conjugate was collected and added to a mixture of:

160.0 μl of AP 43.4 nmol 7.7 μl of 1 M NaHCO3 30.6 μl of 20 mM Hepes, pH 7.2; 1 M NaCI; 500 mM MgCl2; 1 mM ZnCI2

The mixture was placed in a water bath at 40 ° C for 3 hours. Extinction was performed by adding 30.6 μl of 0.1 M ethanolamine and allowing the mixture to stand for 30 minutes in a water bath at 40 ° C. The product was purified by FPLC with: SUPERDEX®-200 column, buffer: 200 mM Hepes, pH 7.2; 0.1 M NaCI; 5 mM MgCl2; 0.1 mM ZnCI2. Two fractions were collected: one with the product and one with the residue.

In comparison to the experiment described above, another conjugate was prepared with an extended conjugation time. The three components below were mixed together and placed in a 40 ° C water bath for 30 minutes.

192.0 μl of DexVS70 13.7 nmol 41.0 μl of PNA PNA 41 mmol dissolved in H2O 6.0 μl of 1 M NaHCO3

108.0 μl of the DexVS70-PNA conjugate were removed and added to a mixture of:

160.0 μl of AP 43.4 nmol 7.7 μl of 1 M NaHCO3 30.6 μl of 20 mM Hepes, pH 7.2; 1 M NaCI; 50 mM MgCl2; 1 mM ZnCI2

The mixture was placed in a water bath at 40 ° C for 5 hours. Extinction was performed by adding 30.6 μl of 0.1 M ethanolamine and allowing the mixture to stand for 30 minutes in a water bath at 40 ° C. Purification of the product in FPLC: SUPERDEX®-200 column, buffer: 2 mM Hepes, pH 7.2; 0.1 M NaCI; 5 mM MgCl2; 0.1 mM ZnCI2. Two fractions were collected: one with the product and one with the residue.

The relative absorbance of PNA (FIu) (ε500 nm = 73000 M-1) and AP (ε278 nm = 140000 M-1. Corrected for the absorbance of PNA at 278 nm, this correction factor is due to the specific PNA and is calculated : 278/500 nm) was used to calculate the average conjugation ratio of PNA, AP and DexVS70.

AP-DexVS70-PNA, 3 hours:

PNA / DexVS70: 1.8 AP / DexVS70: 1.8

AP-DexVS70-PNA, 5 hours:

PNA / DexVS70: 2.0 AP / DexVS70: 2.4

Due to these results, it is recommended to follow a procedure in which the conjugation time (AP + DexVS70-PNA) is 5 hours.

Example 10: Conventional synthesis of the GAM-DexVS70-PNA conjugate

Dextran (molecular weight 70 kDa) is activated with divinylsulfone to a level of 92 reactive groups / polymer dextran (DexVS70).

105.0 μl of DexVS70 7.5 nmol 57.0 μl of goat anti-mouse immunoglobulin (GAM) 15 nmol 8.9 μl of 4 M NaCI 10.6 μl of 0.8 M NaHCO3 (pH = 9.5 ) 144.5 μl of H2O

The previous five components were mixed and placed in a water bath at 30 ° C for 40 minutes. Two hundred and ninety μl were removed from the mixture and added to 50 nmol of PNA, which had previously dissolved in 40 μl of H2O. Next, 10 µl of 0.8 M NaHCO3 (pH 9.5) was added and the mixture was placed in a 30 ° C water bath for 1 hour. Extinction was performed by adding 34 μl of 500 mM cysteine and allowing the resulting mixture to stand for 30 minutes at 30 ° C.

Purification of the product by FPLC: SUPERDEX®-200 column, 10 mM Hepes buffer, 100 mM NaCI, method 7 bank 2, 1 ml loop. Two fractions were collected: one with the product and one with the residue. The relative absorbance of PNA (FIu) (ε500 nm = 73000 M-1) and GAM (ε278 nm = 213000 M-1. The correction factor for PNA at 278 nm is due to the specific PNA and is calculated: 278/500 nm ) was used to calculate the average conjugation ratio of PNA, GAM and DexVS70.

Example 11: Conventional Synthesis of PNA1-DexVS70-PNA2

Dextran (molecular weight 70 kDa) is activated with divinylsulfone to a level of 92 reactive groups / polymer dextran. PNA1 (100 nmol) is dissolved in 140 μl of DexVS70 (10 nmol). To this mixture, 12.5 µl of PNA2 (12.5 nmol) dissolved in H2O is added, and then 30 µl of NaHCO3 (pH 9.5) is added and the solution is mixed. The resulting mixture is placed in a water bath at 30 ° C for 35 minutes. Extinction was performed by adding 18.3 μl of 500 mM cysteine in Hepes and allowing this mixture to stand for 30 minutes at 30 ° C.

Purification of the product by FPLC: SUPERDEX®-200 column, 10 mM Hepes buffer, 100 mM NaCI, method 7 bank 2, 1 ml loop. Two fractions were collected: one with the product and one with the residue. The relative absorbance of PNA (FIu) (ε500 nm = 73000 M-1) and the ratio between the two PNAs was used to calculate the average conjugation ratio of PNA, PNA and DexVS70.

Example 12: Synthesis of the Boc-PNA-1 (O-Bz) monomer.

6-benzyloxypurine. Sodium hydride (60% dispersion in mineral oil; 3.23 g; 80 mmol) was added slowly to benzyl alcohol (30 ml; 34.7 mmol). After the addition of more benzyl alcohol (10 ml) and 6-chloropurine (5.36 g), the reaction mixture was heated at 100 ° C for 4 hours. When the reaction mixture had reached room temperature, water (1 ml) was added slowly. The 6-benzyloxypurine precipitated by the addition of acetic acid (4.6 ml) and diethyl ether (550 ml). The precipitated material was filtered off (11.72 g). Recrystallization from ether provided (4.78 g; 65.4%). Melting point 175-177 ° C (litt. 170-172 ° C) [Ramazaeva N., 1989 No. 473] 1H-NMR (DMSO-d6): 8.53 (1H, s); 8.39 (1H, s); 7.54-7.35 (5H, m); 5.62 (2H, s).

(6- (Benzyloxy) purin-9-yl-) methyl acetate. 6-Benzyloxyipurine (4.18 g; 18.5 mmol) was added to a suspension of potassium carbonate (3.1 g; 22.4 mmol) in DMF (100 ml). After 15 min, bromoacetic acid methyl ester (1.93 ml; 20.4 mmol) was added. The reaction was monitored with TLC in butanol: acetic acid: water 4: 1: 1. After completion, the reaction mixture was partitioned between water (600 ml) and ethyl acetate (600 ml). The organic phase was dried over magnesium sulfate and evaporated to a volume of ~ 10 ml and precipitated in pet ether. The two products were separated by column chromatography, using ethyl acetate as solvent. The products precipitated in pet ether. Yield: 2.36 g (43%). Melting point: 111.5-115 ° C. λ max UV = 250 nm (9-alkylated); λ max = 260 nm (7-alkylated). 1H-NMR (DMSO-d6): 8.60 (1H, s); 8.43 (1, H, s); 7.6-7.35 (5H, m); 5.69 (2H, s); 5.26 (2H, s); 3.75 (3H, s).

(6- (benzyloxy) purin-9-yl) acetic acid. (6- (Benzyloxy) purin-9-yl) -methyl acetate (2.10 g; 7.0 mmol) was dissolved in methanol (70 ml) and 0.1 M NaOH (85 ml) was added. After 15 min, the pH of the reaction mixture was decreased by the addition of 0.1 M HCI (~ 80 ml) to pH 3. The precipitated material was separated from the mixture by filtration and washed with water and ether. Yield: 1.80 g (90.2%). 1H-NMR (DMSO-d6): 8.55 (1H, s); 8.37 (1H, s); 7.55-7.30 (5H, m); 5.64 (2H, s); 5.09 (2H, s).

N- (6- (benzyloxy) purin-9-yl) acetyl) -N- (2-Boc-aminoethyl) glycine. N- (2-Boc-aminoethyl) ethyl glycinate (0.285 g; 1.15 mmol), (6- (benzyloxy) purin-9-yl) acetic acid (0.284 g; 1.0 mmol) and 3-hydroxy- 1,2,3-benzotriazin-4 (3H) -one (0.188; 1.1 mmol), was dissolved in dichloromethane / dimethylformamide 1: 1 (10 ml). After the addition of dicyclohexylcarbodiimide (0.248 g; 1.2 mmol) the reaction was allowed to stand overnight. The precipitated material was removed by filtration. The organic phase was extracted twice with saturated sodium bicarbonate, dried over magnesium sulfate and evaporated to form an oil. Purification of the column on silica using dichloromethane with 0-5% methanol as eluent gave the ester of the monomer that was dissolved in methanol (10 ml). 0.1 M sodium hydroxide (12 ml) was added. After 30 minutes, the reaction was filtered and the pH adjusted with saturated KHSO4 / water (1: 3) to 2.7. The aqueous phase was extracted twice with ethyl acetate (2 x 100). The combined organic phases were dried over magnesium sulfate and evaporated to a volume of 10 ml. With precipitation with pet ether. the monomer was obtained (0.15 g; 31%). 1H-NMR (DMSO-d6): 8.51 (1H, s); 8.23 (1H, s); 7.6-7.3 (5H, m); 5.64 (2H, s); 5.31 (ma.) + 5.13 (mi.) (2H, s); 4.23 (mi. + 3.98 (ma.) (2H, s); 3.55-3.00 (4H, m); 1.36 (9H, s).

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The synthesis of the hypoxanthine PNA monomer, (i) BnOH, NaH (ii) K2CO3, BrCH2CO2CH3 (iii) OH- (iv) DCC, Dhbt-OH, Boc-aeg-OEt (v) OH-

The Boc-PNA-diaminopurine- (N6-Z) monomer was prepared according to Gerald Haaima, Henrik F. Hansen, Leif Christensen, Otto Dahl and Peter E. Nielsen; Nucleic Acids Research, 1997, voI. 25, number 22 4639-4643

The monomer Boc-PNA-2-thiouracil- (S-4-MeOBz) was prepared according to Jesper Lohse, Otto Dahl and Peter E. Nielsen; “Proceedings of the National Academy of Science of the United States of America”, 1999, voI. 96, number 21, 1180411808.

The Boc-PNA-Adenine- (Z) monomer was from the catalog GEN063011 of PE Biosystems.

The Boc-PNA-Cytosine- (Z) monomer was from cat. GEN063013 from PE Biosystems.

The Boc-PNA-Guanine- (Z) monomer was from cat. GEN063012 from PE Biosystems.

The Boc-PNA-Timina monomer was from cat. GEN063010 from PE Biosystems.

IsoAdenine (2-aminopurine) can be prepared as a PNA monomer by 9-N alkylation with methyl bromoacetate, protection of the amino group with benzyl chloroformate, methyl ester hydrolysis, carbodiimide-mediated coupling, with (2- Boc-aminoethyl) methyl glycinate, and finally hydrolysis of the methyl ester.

4-Thiouracil can be prepared as a PNA monomer by S-protection with 4-methoxy-benzylchloride, 1-N alkylation with methyl bromoacetate, methyl ester hydrolysis, carbodiimide-mediated coupling, with (2-Boc-aminoethyl ) -methyl glycinate, and finally hydrolysis of the methyl ester.

Thiocytosine can be prepared as a PNA monomer by treating the methyl ester of the Boc-PNAcytosine (Z) monomer with the Lawessons reagent, followed by hydrolysis of the methyl ester.

A variety of halogenated bases are commercially available, and can be converted into NAP monomers analogously to non-halogenated bases. These include the guanine analog, 8-bromo-guanine, the adenine analogs, 8-bromo-adenine and 2-fluoro-adenine, the isoadenine analog, 2-amino-6-chloro-purine, the

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analogue of 4-thiouracil, 5-fluoro-4-thiouracil, and the analogue of 2-thiouracil, 5-chloro-2-thiouracil.

The Boc-PNA-Uracilo monomers were first described in "Uracil og 5-bromouracil I PNA", a degree project by Kristine Kilså Jensen, University of Copenhagen 1992. Example 13: PNA with positive and negative charges To prepare conjugations better simultaneously, providing PNA, a load was attempted. Both PNAs are

prepared by conventional PNA procedures (see Example 2).

one.

 Flu-L30-Glu-TCA-AGG-TAC-A-Glu-L30-Lys (Cys) GIu = glutamate has negative charges and for simplicity the PNA is designated -A4

2.

 Flu-L30-Lys (Me) 2-TGT-ACC-TTG-A-Lys (Me) 2-L30-Lys (Cys) Lys (Me) 2 = Boc-Lys (Me) 2-OH has positive charges and the PNA is designated + T +

Name

 number HRP GaM equiv. HRP / Dex Gam / Dex PNA / Dex

-A4-

D 13041 D 13050 9 12.3 0.13

-A4-

D 13041 D 13060 7 0.94 0.66

+ T4 +

D 13042 D 13058 9 13.5 0.19

+ T4 +

D 13042 D 13056 7 1.42 0.45

Example 14 - Derivatives of additional linkers

100 mg of MBHA resin was discharged to a loading of 0.1 mmol / g with Boc-Lys (Fmoc) and subjected to repeated cycles of coupling with Boc-L30-free acid. Each coupling was performed with 48 mg of free Boc-L30 activated acid with 23 mg of HATU and 14 mg of DIPEA in 0.9 ml of NMP. After 20 cycles, a small sample of the resin was cleaved, precipitated in diethyl ether, and subjected to mass spectroscopy.

This confirmed the expected structure:

H-L600-Lys (Fmoc) -NH2 calc. for C421H744N83O203 10217.00, found 10214.63

The resin was subjected to another 8 coupling cycles with Boc-L30-free acid. A small sample was again cleaved from the resin for analysis. Again the expected structure was confirmed:

H-L840-Lys (Fmoc) -NH2 calc. for C581H1032N115O283 14157.23, found 14153.4

The resin was subjected to another 5 coupling cycles with Boc-L30-free acid. A small sample was again cleaved from the resin for analysis. Again, the expected structure was confirmed:

H-L990-Lys (Fmoc) -NH2 calc. for C681H1212N135O333 16619.87, found 16618.88

Fifty mg of the resin was deprotected in Boc and labeled with carboxy fluorescein. The Fmoc group on lysine was removed, and the product was removed by cleavage of the resin and precipitated in diethyl ether. It was resuspended in TFA and precipitated again. The product was dissolved in water and the absorbance at 500 nm was used to quantify the yield of the full length product labeled with fluorescein. The yield was 1470 nm, 24 mg, corresponding to a surplus of 29% over the entire yield, of the 33 couplings with L30, which corresponded to an average coupling yield of 96.3%. The structure was confirmed by mass spectroscopy, and there was no evidence of any substantial impurity.

Flu-L990-Lys (NH2) -NH2 calc. for C687H1213N135O337 16756.94, found 16752.49.

Example 15: Alternative linker synthesis method

Synthesis of Diimide

See Figure 8, structure 6. 2.2 '(ethylenedioxy) bis (ethylamine), 2.96 g, 20 mmol, in pyridine, 10 ml, reacted with 5.8 g, 50 mmol, of diglycol anhydride for 5 minutes at 80 ° C. 5 ml of acetic anhydride was added and the mixture was stirred for an additional 1 h at 100 ° C. The solvents were evaporated and the residue was stirred with 20 ml of water for 30 min. The product was extracted in 100 ml of DCM, and washed with 100 ml of 1 M sodium citrate, pH 4.5, 100 ml of 1 M sodium hydrogen carbonate and then finally water. The DCM was evaporated to provide 5.3 g of a colorless, 77% oil, which solidified into a white mass over time.

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Polymerization of Diimide and an excess of 2,2 '(ethylenedioxy) bis (ethylamine)

344 mg of diimide, 1 mmol, and 160 mg of 2.2 '(ethylenedioxy) bis (ethylamine) 1.08 mmol were dissolved in 0.5 ml of NMP. The mixture was heated at 95 ° C for 90 min. Additionally, 148 mg of 2.2 '(ethylenedioxy) bis (ethylamine) 1 mmol was added to the heated mixture for an additional 60 min. The crude reaction mixture was analyzed by MALDI-TOF mass spectroscopy. This showed that a mixture of cyclic and linear products had formed. There was evidence of cycle-L30 (m = 0) and cycle L60 (m = 1), but not of cyclic oligomers with a higher number. Linear oligomers ranged from n = 1 to n = 9. See Figure 8 and the structures below:

Cyclic:

m = 0; calc. for C20H36N4O10 492.5282, found 492.23 m = 1; calc. for C40H72N8O20 985.0564, found 985.039 linear n = 1; calc. for C26H52N6O12 640.7334, found 640.23 n = 2; calc. for C46H88N10O22 1133.26, found 1132.02 n = 3; calc. for C66H124N14O32 1625.29, found 1624.80 n = 4; calc. for C86H160N18O42 2118.3, found 2117.6 n = 5; calc. for C106H196N22O52 2610.84, found 2609.53 n = 6; calc. for C126H232N26O62 3103.37, found 3101.48 n = 7; calc. for C146H268N30O72 3595.90, found 3593.54 n = 8; calc. for C166H304N34O82 4088.42, found 4086.74 n = 9; calc. for C186H340N38O92 4580.95, found 4580.02.

Example 16. Method of synthesis of mono- and 2,4-diamino-pyrimidin-5-yl PNA monomers.

2,4-Diamino-pyrimidin-5-yl can be introduced into DNA oligomers by methods known in the art (e.g.,

S.A. Benner et al., Nucleic Acid Research 24 (7): 1308-1313 (1996)). A corresponding PNA oligomer is prepared by treating pyrimidin-5-acetic acid with chlorine to obtain 2-chloro-pyrimidin-5-acetic acid, 4-chloro-pyrimidin-5-acetic acid and 2,4-dichloro-pyrimidin-5 acid -acetic. The separation of the isomers, followed by a treatment at elevated temperature and pressure with ammonia, provides the three corresponding aminopyrimidine derivatives (see Figure 9). The amino-pyrimidine derivatives are separated and the amino are protected, then coupled with a protected ester of the PNA main chain. Hydrolysis of the ester results in PNA monomers for the production of PNA oligomers containing 2-amino bases; 4-amino; and / or 2,4-diaminopyrimidin-5-yl.

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Example 17. Synthesis of xanthine and thioxanthin monomers coupled with PNA.

Xanthine, 2-thioxanthine and 6-thioxanthin are commercially available, for example, from ScienceLab.com. In addition, S.A. Benner et al., Nucleic Acid Research 24 (7): 1308-1313 (1996) describe the preparation of a xanthosine-DNA monomer, which includes a less acidic and preferable 7-deaza analogue, and notes the preferred protection of both oxygens. during solid phase synthesis.

The xanthine and PNA monomers, as well as the 2-thio and 4-thioxanthin monomers, are prepared:

one.

 Protecting both oxygens or oxygen and sulfur with appropriate protecting groups, such as benzyl (possibly substituted).

2.

 Renting in N-9 with ethyl bromoacetate. (Separating the by-product rented in N-7.)

5 3. Hydrolyzing the ethyl ester.

Four.

 By coupling with the mediation of HBTU or Carbodiimide the nucleic acid bases with 2-Boc-aminoethylglycinate.

5.

 Hydrolyzing the ester of the resulting monomer in the monomer with free acid.

6. Being able to use the resulting monomers in Merrifield solid phase synthesis of PNAs containing 10 xanthine, 2-thioxanthin and 6-thioxanthin.

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Example 18. Use of specific IHC detection units with different linker length.

Objective: Use 2-layer detection with horseradish peroxidase (HRP) to test and compare recognition units with different linker length (L150, L300, L540).

Unit No.

Linker Length Specific score Background noise score

209-033

 L150 2.5+ 1+

209-029

 L300 2.5+ 1+

195-147

 L540 2+ 0

Experimental stages: The primary mouse antibody M3515 (Dako) that was directed to the CK target was diluted to 1: 200 in S2022 (Dako) and applied to a multisection of tissue. After a 10 minute incubation at room temperature, the section was washed 5 minutes using S3006 diluted 10 times (Dako).

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Four. Five

GaM-dex-PNA1 (goat anti-mouse-dextran-PNA1) (209-033, 209-029 or 195-147) was diluted to a final concentration of 0.08 □ M / dex in BP-HEPES buffer (1 , 5% BSA, 3% PEG, 0.15 M NaCI, 10 mM HEPES, pH 7.2) and was applied. After 10 minutes of incubation at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako). The sections were rinsed in deionized water. After 10 minutes of incubation in 0.5% glutaraldehyde at room temperature, the sections were rinsed in deionized water and washed 5 minutes using S3006 diluted 10x (Dako).

PNA2-dex-HRP (209-041) was diluted to a final concentration of 0.05 □ M / dex in BAP-HEPES buffer (1.5% BSA, 3% PEG, 0.15 M NaCI, 0 , 05% of 4-aminoantipyrine, 10 mM HEPES, pH 7.2) and was applied. After 10 minutes of incubation at room temperature, the sections were washed 5 minutes using 10x diluted S3006 (Dako).

A working solution of DAB + (an aqueous imidazole buffer with hydrogen peroxide and DAB; K3468 Dako) was applied. After 10 minutes of incubation, the sections were washed 5 minutes with deionized water. Finally the sections were stained for counting for 5 minutes using hematoxylin S3301 (Dako), rinsed in deionized water, washed 3 minutes in wash buffer, and mounted on Faramount S3025 (Dako).

Result: In the brown final product of the HRP reaction, a specific cytokeratin staining of the epithelial cells is visualized and does not show a significant difference in the performance when recognition units having different linker length are compared.

Example 19. IHC test for the detection of units with different number of fixed-PNA linker.

Objective: to use 2-layer HRP detection to test and compare the detection units with different amounts of PNA per dextran (0.8 PNA / dex and 1.5 PNA / dex).

Unit No.

PNA / dex Specific score Background noise score

195-051

 0.8 3+ 0.5+

D15008

 1.5 3+ 0.5+

Experimental stages: The primary mouse antibody M3515 (Dako) that was directed to the CK target, was diluted until finally having 1: 200 in S2022 (Dako) and applied on a multisection of tissue. After incubating 10 minutes at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako).

GaM-dex-PNA1 (195-047) diluted to a final concentration of 0.08 □ / dex, was applied in BP-HEPES buffer (1.5% BSA, 3% PEG, 0.15 M NaCI, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako). The sections were rinsed in deionized water. After a 10 minute incubation in 0.5% glutaraldehyde at room temperature, the sections were rinsed in deionized water and washed 5 minutes using S3006 diluted 10x (Dako).

PNA2-dex-HRP (HRP = horseradish peroxidase) (195-051 or D15008) diluted to a final concentration of 0.05 □ M / dex, was applied in BAP-HEPES buffer (1.5% BSA, 3% PEG, 0.15 M NaCl, 0.05% 4-aminoantipyrine, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the sections were washed 5 minutes using 10x diluted S3006 (Dako).

A working solution of DAB + (K3468 Dako) was applied. After 10 minutes of incubation, the sections were washed 5 minutes with deionized water. Finally the sections were stained for counting for 5 minutes using hematoxylin S3301 (Dako), rinsed in deionized water, washed 3 minutes in wash buffer, and mounted on Faramount S3025 (Dako).

Result: In the brown final product of the HRP reaction, the specific cytokeratin staining of the epithelial cells is visualized and does not show a significant difference in the performance when the detection units having different amounts of PNA / dex were compared.

Example 20. IHC assay comparing recognition and detection units that have "PNA linker-linker" or "linker-linker-PNA linker" attached.

Objective: Use 2-layer detection to test and compare the recognition and detection units that have PNA sequences without and with "linker tail".

Unit No. GaM-dex-PNA

tail  unit number PNA-dex-HRP tail specific score background noise score

218-113

 no 218-021 no 3+ 1+

D16074

 yes 218-021 no 3+ 0.5+

218-113

 no D16076 yes 3+ 0.5+

D16074

 yes D16076 yes 4+ 1.5+

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Experimental stages: primary mouse antibody M3515 (Dako) that targets the CK target, was diluted until finally having 1: 200 in S2022 (Dako) and applied on a multisection of tissue. After incubating 10 minutes at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako). GaM-dex-PNA1 (218-113 or D16074) diluted to a final concentration of 0.08 □ / dex, was applied in BP-HEPES buffer (1.5% BSA, 3% PEG, NaCI 0.15 M, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako). The sections were rinsed in deionized water. After a 10 minute incubation in 0.5% glutaraldehyde at room temperature, the sections were rinsed in deionized water and washed 5 minutes using S3006 diluted 10x (Dako).

PNA2-dex-HRP (218-021 or D16076) diluted to a final concentration of 0.05 □ M / dex, was applied in BAP-HEPES buffer (1.5% BSA, 3% PEG, NaCI 0, 15 M, 0.05% 4-aminoantipyrine, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the sections were washed 5 minutes using 10x diluted S3006 (Dako).

A working solution of DAB + (K3468 Dako) was applied. After 10 minutes of incubation, the sections were washed 5 minutes with deionized water. Finally the sections were stained for counting for 5 minutes using hematoxylin S3301 (Dako), rinsed in deionized water, washed 3 minutes in wash buffer, and mounted on Faramount S3025 (Dako).

Result: In the brown final product of the HRP reaction, the specific cytokeratin staining of epithelial cells is visualized and shows that the embodiment depends on the presence of a "linker tail" in the PNA sequence. A "linker tail" in the PNA sequence can influence the specific score and the background noise score.

Example 21. IHC test comparing recognition and detection units that have "PNA-linker" or "PNA-linker-bound" attached.

Objective: Use 2-layer detection with HRP to test and compare the recognition and detection units that have PNA sequences with and without loading.

Unit No. GaM-dex-PNA

tail  unit number PNA-dex-HRP load specific score background noise score

D15078

 no D15069 no 0.5+ 0

209-149

 yes 209-157 yes 3+ 1+

Experimental stages: the primary mouse antibody M3515 (Dako) that targets the CK target, was diluted until finally having 1: 200 in S2022 (Dako) and applied on a multisection of tissue. After incubating 10 minutes at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako).

GaM-dex-PNA1 (2D15078 or 209-149) diluted to a final concentration of 0.08 □ / dex, was applied in BP-HEPES buffer (1.5% BSA, 3% PEG, NaCI 0.15 M, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the section was washed 5 minutes using S3006 diluted 10x (Dako). The sections were rinsed in deionized water. After a 10 minute incubation in 0.5% glutaraldehyde at room temperature, the sections were rinsed in deionized water and washed 5 minutes using S3006 diluted 10x (Dako).

PNA-dex-HRP (D15069 or 209-157) diluted to a final concentration of 0.05 □ M / dex, was applied in BAP-HEPES buffer (1.5% BSA, 3% PEG, NaCI 0, 15 M, 0.05% 4-aminoantipyrine, 10 mM HEPES, pH 7.2). After 10 minutes of incubation at room temperature, the sections were washed 5 minutes using 10x diluted S3006 (Dako).

A working solution of DAB + (K3468 Dako) was applied. After 10 minutes of incubation, the sections were washed 5 minutes with deionized water. Finally the sections were stained for counting for 5 minutes using hematoxylin S3301 (Dako), rinsed in deionized water, washed 3 minutes in wash buffer, and mounted on Faramount S3025 (Dako).

Result: In the brown final product of the HRP reaction, the specific cytokeratin staining of the epithelial cells is visualized and shows that the embodiment depends on the presence of charge in the PNA sequence. The load on the PNA sequences within a PNA pair can influence the specific score and the background noise score.

Example 22. Synthesis and IHC assay of an Antibody-PNA conjugate.

Part A. Assay of different linker to antibody ratios

Materials: Antibody: CD45 dialyzed overnight against 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC: molecular weight of succinimidyl-4 cyclohexane-1-carboxylate (N-maleimidomethyl). 334.33. PNA: acetyl-L30-GTP-TAA-TTP-PAG-L150-Lys (Cys)

Essay 1

10 nmol CD45 was dissolved in 161 μl of 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC 250 nmol was dissolved in 8 μl of NMP. The above components were mixed and placed in a 30 ° C water bath for 60 minutes. The mixture was purified on a mini-prep column (Sephadex G-25) with 0.01 M Hepes, 0.1 M NaCl pH = 7.2 as eluent. 0.3 ml fractions. By measuring the absorbance at 278 nm, three fractions containing the product (58%) were identified. These three fractions were added to 100 nmol of a lyophilized PNA. Then, 1 µl of 5% disodium EDTA / water was added and the solution was mixed until dissolved and placed in a 30 ° C water bath for 30 minutes. Extinction was performed by adding 2 mg of cysteine. Water bath at 30 ° C for 30 minutes.

The product was purified by FPLC: SUPERDEX®-75 column, 0.01 M Hepes buffer, 0.1 M NaCl pH 7.2. The fraction was collected with the product. The relative absorbance between PNA (ε260 nm) and the antibody (ε278 nm) was used to calculate the average proportion of conjugation between PNA and the antibody. PNA / CD45: 5.2. Yield 39% based on antibody.

Essay 2

10 nmol CD45 was dissolved in 161 μl of 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC 150 nmol was dissolved in 5 μl of NMP. The above components were mixed and placed in a 30 ° C water bath for 60 minutes. The mixture was purified on a mini-prep column (Sephadex® G-25) with 0.01 M Hepes, 0.1 M NaCl pH = 7.2 as eluent. 0.3 ml fractions. By measuring the absorbance at 278 nm, three fractions containing the product (74%) were identified.

These three fractions were added to 100 nmol of a lyophilized PNA. Then, 1 µl of 5% disodium EDTA / water was added and the solution was mixed until dissolved and placed in a 30 ° C water bath for 30 minutes. Extinction was performed by adding 2 mg of cysteine. Water bath at 30 ° C for 30 minutes.

The product was purified by FPLC: SUPERDEX®-75 column, 0.01 M Hepes buffer, 0.1 M NaCl pH 7.2. The fraction was collected with the product. The relative absorbance between PNA (ε260 nm) and the antibody (ε278 nm) was used to calculate the average proportion of conjugation between PNA and the antibody. PNA / CD45: 3.4. Yield 55% based on antibody.

IHC assay of conjugates

A subsequent IHC trial showed that PNA / CD45 in trial 2 gave a higher score than in trial 1. This led us to the conclusion that the ratio between CD45 / SMCC / PNA should be 10/150/100.

Part B. Assay of different conjugation times - antibody and linker

Materials: Antibody: GAM (goat anti-mouse) dialyzed overnight against 0.1 M NaCl. SMCC: molecular weight of succinimidyl-4-cyclohexane-1-carboxylate (N-maleimidomethyl). 334.33. Flu-linker: Flu-L90-Lys (Flu) -L30Lys (Cys)

Essay 1

20 nmol GAM was dissolved in 402 μl of 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC 400 nmol was dissolved in 13 μl of NMP. The above components were mixed and placed in a 30 ° C water bath for 1 hour. 207 μl of the mixture was purified on a mini-prep column (Sephadex® G-25) with 0.01 M Hepes, 0.1 M NaCl pH = 7.2 as eluent. 0.3 ml fractions were taken. By measuring absorbance at 278 nm, three fractions containing the product (79%) were identified. These three fractions were added to 200 nmol of a lyophilized Flu-linker. Then, 1 µl of 5% disodium EDTA / water was added and the solution was mixed until dissolved and placed in a water bath at 30 ° C overnight. Extinction was performed by adding 2 mg of cysteine. Water bath at 30 ° C for 30 minutes.

The product was purified by FPLC: SUPERDEX®-75 column, 0.01 M Hepes buffer, 0.1 M NaCl pH 7.2. The fraction was collected with the product. The relative absorbance between Flu-linker (ε498 nm) and the antibody (ε278 nm) was used to calculate the average proportion of conjugation between PNA and the antibody. Flu-linker / GAM: 7.1. Yield 55% based on antibody.

 Essay 2

20 nmol GAM was dissolved with 402 µl of 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC 400 nmol was dissolved in 13 μl of NMP. The above components were mixed and placed in a 30 ° C water bath for 2 hours. The rest of the synthesis and purification was performed with exactly the same procedure as for 1 hour. There was a 64% yield of GAM / SMCC before adding the Flu-linker. Flu-linker / GAM: 8.7. 33% yield based on antibody.

Part C. Test of different conjugation times - Fluorophore and linker

Materials: Antibody: GAM dialyzed overnight against 0.1 M NaCl. SMCC: molecular weight of succinimidyl-4 cyclohexan-carboxylate (N-maleimidomethyl). 334.33. Flu-linker: Flu-L90-Lys (Flu) -L30-Lys (Cys)

Essay 1

20 nmol GAM was dissolved in 378 μl of 0.01 M Hepes, 0.1 M NaCl pH = 7.2. SMCC 400 nmol was dissolved in 13 μl of NMP. The above components were mixed and placed in a 30 ° C water bath for 1 hour. The mixture was divided in two and purified on two mini-prep columns (Sephadex® G-25) with 0.01 M Hepes, 0.1 M NaCl pH = 7.2 as eluent. 0.3 ml fractions were taken. By measuring the absorbance at 278 nm, three fractions of each column containing the product were identified (76% in all). These six fractions were combined, divided into two and each added to 200 nmol of a lyophilized Flu-linker. Next, 1 µl of 5% disodium EDTA / water was added and the solution was mixed until dissolved and placed in a water bath at 30 ° C, one for 30 minutes, the other for 60 minutes. Extinction was performed by adding 2 mg of cysteine. Water bath at 30 ° C for 30 minutes.

The product was purified by FPLC: SUPERDEX®-75 column, 0.01 M Hepes buffer, 0.1 M NaCl pH 7.2. Fractions were collected with the product of each purification. The relative absorbance between Flu-linker (ε498 nm) and the antibody (ε278 nm) was used to calculate the average proportion of conjugation between PNA and the antibody. Thirty minutes of Flu-linker / GAM conjugation: 7.0 gave a 55% yield based on antibody. Sixty minutes of Flu-linker / GAM conjugation: 6.9 gave a 52% yield based on antibody. The above results show that 30 minutes of conjugation between GAM / SMCC and Flu-linker are sufficient.

Other exemplary embodiments include linker derivatives with the following structures:

to.

 Lys (PNA1) -L30-PNA2-L30-Lys (PNA1) -L30-Lys (PNA1) -L30-Lys (PNA1)

b.

 PNA1-linker- (PNA1-linker) n-PNA1-linker-PNA2

C.

 PNA1-linker- (PNA1-linker) n-PNA1-linker-PNA2-linker-PNA1-linker- (PNA1-linker) n-PNA1

d.

 PNA1-linker- (Lys (linker-PNA1) -linker) n-Lys (linker-PNA1) -linker-PNA2

and.

 PNA1-linker- (Lys (linker-PNA1) -linker) n-linker-PNA2- (linker-Lys (linker-PNA1)) n.

Other examples include linker derivatives with the general structure: ([L15] n-PNA) m, in which the linker derivative is additionally covalently or non-covalently coupled with an antibody and / or with dextran, and where nym are whole numbers equal to or greater than 1.

Still other examples include linker derivatives with the general structure: ([L15] nF) m wherein the linker derivative is covalently or non-covalently bound in addition to an antibody and / or dextran, and wherein F is a ynym fluorophore are whole numbers equal to or greater than 1.

Still other examples include:

Fluorescent microspheres comprising conjugated linkers with lisamine, and / or with color markers, such as DNP or fluorescent dyes, such as fluorescein or rhodamine or its derivatives, and / or with PNA.

Systems such as: antibody: dextran: (L-PNA) n; antibody: dextran: (L-F) n; PNA-L: dextran: (L-F) n; PNA-L: dextran: (HRP enzyme); PNA1-L: dextran (L-PNA2) n; wherein L represents a linker of at least one unit L-15, F represents a fluorophore, n represents an integer equal to or greater than 1, ":" represents a covalent or non-covalent fixation, and "-" represents a covalent fixation .

Claims (27)

1.-A linear polymeric linker comprising at least two consecutive units chosen from: image 1 5 where • R1 and R2 are chosen between NH and O, • R3 is chosen from methyl, ethyl, propyl, CH2OCH2 and (CH2OCH2) 2, and wherein the linker comprises between one and three ethyloxy groups repeated consecutively. 2. The linker according to claim 1, comprising at least one unit of Formula I in the 10 that R1 and R2 are NH, and R3 is CH2OCH2. 3. The linker according to claim 1, comprising two units of Formula I, of formula: image 1 4. The linker according to claim 1, comprising three units of Formula I, of formula: image 1 The linker according to claim 1, which comprises four units of Formula I, of formula: 6. The linker according to claim 1, which comprises six units of Formula I, of formula: image 1 image 1 7. The linker according to claims 3-6, wherein each R1 and R2 are individually selected from NH and O, and wherein each R3 is individually selected from methyl, ethyl, propyl, CH2OCH2 and (CH2OCH2) 2. 8. A linker derivative comprising a linker according to any one of claims 1-7, conjugated with at least one atom, a functional group or a molecule. 9. The linker derivative according to claim 8, wherein at least one atom, a functional group or a molecule comprises at least one detectable label or at least one probe. 10. The linker derivative according to claim 9, wherein at least one atom, a functional group or a molecule comprises at least one detectable marker and wherein at least one detectable marker comprises at least one color marker. 11. The linker derivative according to claim 10, wherein at least one color marker comprises at least one fluorophore. 12. The linker derivative according to claim 11, comprising two or more fluorophores with the same wavelength or with different and distinguishable emission wavelengths. 13. The linker derivative according to claim 10, which comprises at least one nucleic acid or at least one nucleic acid analog. 14-The linker derivative according to claim 13, wherein at least one nucleic acid analog comprises PNA. 15-The linker derivative according to claim 8, wherein the linker is additionally fixed covalently or non-covalently to at least one solid surface. 16-The linker derivative according to claim 15, wherein at least one solid surface is dextran. 17. The linker derivative according to claim 8, which comprises a detectable label, a probe, a nucleic acid or a nucleic acid analog, and / or a solid surface separated from each other by linkers comprising between four and ten consecutive units of Formula I. 18. A method for improving the signal strength, activity, binding affinity or aqueous solubility of a molecule, comprising conjugating the molecule with a linker according to any one of claims 1-7. 19. The method according to claim 18, wherein the molecule comprises at least one detectable label or probe. 20. The method according to claim 19, wherein the molecule comprises at least one detectable marker and wherein at least one detectable marker comprises at least one color marker. 21. The method according to claim 20, wherein at least one color marker comprises a fluorophore. 22. The method according to claim 21, wherein the molecule comprises at least one nucleic acid or a nucleic acid analog. 23. The method according to claim 22, wherein at least one nucleic acid analog comprises the PNA. 24. The method for amplifying a signal from a change in a molecular system, comprising detecting said change with at least one linker derivative according to claim 8. 25. The method according to claim 24, wherein the linker derivative comprises at least one color marker. 26. The method according to claim 25, wherein at least one color marker comprises a fluorophore. 27. A method according to claim 26, wherein the linker derivative comprises at least two fluorophores with the same wavelength or with different and distinguishable emission wavelengths, wherein each fluorophore is separated from the other fluorophores of the linker derivative by a linker comprising between four and ten consecutive units of Formula I. 28. A system comprising at least two linker derivatives, wherein each linker is a linker according to any one of claims 1-7, and wherein the first linker derivative comprises at least one fluorophore and one nucleic acid. or a nucleic acid analog; and wherein the second linker derivative comprises a protein or a solid surface and a nucleic acid or a nucleic acid analog. 29. The system according to claim 28, wherein the nucleic acid analog is PNA. 30. The system according to claim 29, wherein the nucleic acid or nucleic acid analogs of the first and second linker derivatives are capable of hybridizing with each other. 31. The system according to claim 30, wherein the first linker derivative is conjugated with at least one fluorophore and one PNA; and wherein the protein or solid surface of the second linker derivative is an antibody, an antigen and / or dextran; and wherein the PNA of the first linker derivative and the PNA of the second linker derivative are capable of hybridizing with each other. 32. The system according to claim 28, comprising three linker derivatives. 33. The system according to claim 29, comprising teres derived from linkers. 34. The system according to claim 29, wherein the first and second linker derivatives are not capable of hybridizing with each other, and wherein the third linker derivative comprises at least one PNA that recognizes PNA sequences on each one of the first and second derivatives of linkers.