EP2044035A1

EP2044035A1 - Process for the covalent coupling of two molecules by means of a diels-alder reaction with inverse electron requirement

Info

Publication number: EP2044035A1
Application number: EP07726066A
Authority: EP
Inventors: Manfred Wiessler; Eduard Müller; Peter Lorenz; Christian Kliem; Heinz Fleischhacker
Original assignee: Deutsches Krebsforschungszentrum DKFZ
Current assignee: Deutsches Krebsforschungszentrum DKFZ
Priority date: 2006-06-16
Filing date: 2007-06-18
Publication date: 2009-04-08
Also published as: JP5641735B2; JP2009539911A; US20100016545A1; EP1867638A1; WO2007144200A1; US8552183B2

Abstract

The present invention relates to a process for the coupling of two molecules by means of a Diels-Alder reaction with an inverse electron requirement, which comprises the following steps: reaction of (a) a triazine or tetrazine having one or more electron-pulling substituents on the ring as diene component, with the electron-pulling substituents being selected from among: -COOR, -C(O)NR2, -CX3 (X = halogen), -halogen, -CN, -SO2-R or SO3-R, -PR2, where R = H, alkyl, aryl, heterocycle, with these in turn being able, if appropriate, to be substituted by alkyl, -OH, -SH, halogen, aryl, heterocycle, nitro, carboxyamido or amino groups, heterocyclic rings having 1, 2 or 3 N, O or S atoms at a ring size of 5 or 6 ring atoms, with these being substituted by at least one carboxyl, sulfonic acid or phosphonic group, with (b) an isolated double bond or triple bond in a (hetero)carbocyclic ring or an isolated olefinic double bond or triple bond in a linear or branched hydrocarbon chain which may, if appropriate, contain heteroatoms as dienophile component.

Description

A process for covalently linking two molecules by means of a Diels-Alder reaction with inverse electron demand

The present invention relates to a process for covalently linking two molecules using the inverse electron demand Diels-Alder reaction.

In biological systems studies, molecular biologists and chemists are constantly coming up against the need to covalently link two molecular entities, such as an oligosaccharide with a peptide, a reporter molecule with a biopolymer, two biopolymers together, or a low molecular weight therapeutic with a biopolymer. Most of the compounds mentioned have a large number of chemical functions whose reaction behavior must be taken into account under the conditions of the ligation reaction. It follows that chemoselective ligation reactions should have a clear reaction pathway without attacking the other existing chemical functions or groups or actively intervening in the reaction process. This project can only be realized if two selectively interacting functional groups are involved in the ligation step. Furthermore, it would be desirable for such a ligation reaction to proceed without the use of protecting groups in each environment and at a pH adapted to the particular biopolymer.

One of the few chemical reactions that can fully satisfy all these conditions is cycloaddition, either of the 4 + 2 type known as the Diels-Alder reaction (Figure 1, J. Sauer, 1966, Angew Chem.78, 233 ), or the 3 + 2 type, known as 1, 3 dipolar cycloaddition. Based on this reaction, the Sharpless ligation was developed. (Lit). Another method that has been further developed in recent years is the Staudinger ligation (Review: Angew.Chem 2004, 116, 3168-3178).

In DE-A-100 41 221.1, the application of the classical Diels Alder reaction has been shown as a ligation reaction, wherein the diene with electron donating and the dienophile is provided with electron-withdrawing substituents. As dienes furan and its derivatives and as dienophiles substituted maleimides were used. This system was chosen because of the easy accessibility of the respective components and their simple chemistry. Many furans are easily produced from saccharides and are available in larger quantities. Like many chemical reactions, the Diels Alder reaction (hereafter referred to as "DAR") is reversible, especially at higher temperatures, and this reversibility is particularly pronounced in the furan / maleimide system, which is due to the high reactivity of the maleinimides for nucleophilic additions This can easily be seen from the use of maleimides to label peptides or their linkage, where the thiol group of the protein adds in a very fast, irreversible reaction to the double bond of the maleimide, even the small amounts of maleimide produced by the Reverse reaction of the DARs in equilibrium are intercepted by such an addition, shifting the equilibrium to the side of the starting materials, which is a real disadvantage of the DAR since it significantly minimizes the yield of the desired product.

The object of the present invention is therefore to provide a method with which complex compounds can be covalently and irreversibly linked to each other and which can also be used for the construction of substance libraries.

This object is solved by the subject matters of the claims.

The object is achieved by a Diels-Alder reaction with inverse electron demand, which comprises the following steps: reaction of a

(a) 1, 2,4-triazine or 1, 2,4,5-tetrazine or a 1, 2-diazine having one or more electron-withdrawing substituents on the ring as a diene component, wherein the electron-withdrawing substituents are selected from: - COOR - C (O) NR ₂ - CX ₃ (X = halogen)

- halogen -CN

-SO ₂ -R or SO ₃ -R

- PR ₂ with R = H, alkyl, aryl, heterocycle, which in turn may be optionally substituted with alkyl, OH, SH, halogen, aryl, heterocycle, nitro, carboxyamido, or amine Group.

- heterocyclic rings having 1, 2 or 3 N, O or S atoms in a ring size of 5 or 6 ring members, these being substituted with at least one carboxyl, sulfonic acid or phosphono group

With

(B) an isolated double bond or triple bond in a (hetero) carbocyclic ring or an isolated olefinic double bond or triple bond in a linear or branched hydrocarbon chain, which may optionally contain heteroatoms, as a dienophile component

The inventors have turned to such Diels-Alder reactions, which proceed with elimination of a moiety and thus completely shift the equilibrium of the reaction to the side of the product. If the cleaved part of the molecule is volatile, a back reaction is impossible. In addition to some special services within classical DAR, such as cyclopentadienone, this type of reaction is mainly represented by the Diels Alder reaction with inverse electron demand (Figure 2). This reaction has been well studied and has been used primarily in heterocyclic synthesis. In this type of DAR, the nature of the substituents in the diene and dienophile as defined by O. Diels and K. Alder are reversed or inverted. Now, the diene is provided with electron-withdrawing substituents and thus electron-poor, while the dienophile now becomes electron-rich by its substitution. Already the DAR of Tetrachlorocyclopentadiens with olefins is characterized as inverse electron demand DAR (hereinafter referred to as "DARinv"). As described by Sauer, such inverse electron demand Diels-Alder reactions can be completed at room temperature.

The criteria already defined at the beginning for the development of an effective ligation reaction are thus fulfilled by the inverse DAR in a nearly ideal manner. However, this requires the synthesis of suitably functionalized 1, 2,4,5-tetrazines, 1, 2,4-triazines and 1, 2-diazines as dienes and of olefins as dienophiles. Since the tailor-made for this purpose compounds are not yet known, the preparation of these syntheses for the particular purpose of the present application is based. Both possible variants of the DARinv as ligation reaction could be verified: first, the introduction of the diene into the target molecule, e.g. a peptide followed by reaction with the dienophile-saccharide, as well as the reverse procedure. Both the diene and the dienophile should have maximum reactivity with maximum stability in order to carry out the DARinv as possible in all solvents and at room temperature.

Diene component

The aim in the synthesis of suitable dienes for the functionalization of peptides, oligonucleotides, surfaces or therapeutics is the preparation of symmetrically or asymmetrically substituted 1, 2,4,5-tetrazines, 1, 2,4-triazines and 1, 2-diazines, which can be easily incorporated into the said bio-molecules. However, the already known 3,6-dicarboxylic acid ester of 1,2,4,5-tetrazine is not well suited as starting material for the preparation of suitably functionalized 1,2,4,5-tetrazines as dienes. On the one hand, this compound, which forms magnificent red crystals, does not have sufficient stability in alcoholic and aqueous solvents; on the other hand, nucleophilic substitution reactions on the ester groups are not possible, since under these conditions the attack of the nucleophile takes place on the tetrazine ring itself. (Kampchen T. et al 1982, Chem. Ber., 115, 683-694.) In the three-step synthesis of this tetrazine, starting with Diazoessigester but the stage of dihydro-1, 2,4,5-tetrazine dicarboxylic acid ester is run through. As the inventors found, nucleophilic substitution reactions, due to the high reactivity of the ester groups, can easily be carried out on this compound. Since the second nucleophilic substitution, especially with secondary amines, proceeds more slowly than the first, it is also possible to produce monoamides. By suitable reaction, the second nucleophilic substitution can be pushed back, so that dihydrotetrazine monoamides, such as the benzylamide, can be obtained purely by simple recrystallization. This is also an important step in the preparation of functionalized dihydrotetrazine diamides for the derivatization of peptides. It may be advantageous to first introduce the much more stable dihydro-tetrazines as later diene components. After completion of all reactions to be carried out then the oxidation to tetrazine with the immediately following DARinv. Further possibilities for the preparation of more stable monofunctionalized tetrazines based on diaryltetrazines are shown below.

A synthetic route for the preparation of triazines consists in the reaction of 1, 2-diketo derivatives with amiddrazones. In this way, the tricarboxylic acid ester of 1, 2,4-triazine can be prepared in large quantities according to literature. Here, the ester functions can be easily mixed with nucleophiles, e.g. Convert amines. So far, it has not been possible to discriminate between the ester functions in the reaction with amines. For a targeted introduction of the triazine residue in any molecules, an ester function is sufficient, but sufficient electronegative substituents should then be present to maintain the diene activity of the triazine.

Triazines-1, 2,4 are generally less reactive in the DARinv than the tetrazines, but their rate of reaction is still sufficient for ligation reactions, especially when reacted with a very reactive dienophile.

The 1,2-diazines, which are formed by oxidation of the dihydropyridazines formed in the DARinv of tetrazines with olefins, have even lower DARinv activity than dienes. They are formed directly by the DARinv of tetrazines with triple bonds or with enamines. This also offers the possibility in a sequence, starting from a tetrazine on the diazine by two consecutive DARinv reactions, interrupted by the oxidation of the dihydropyridazine to pyridazine, any two molecules containing a dienophilic anchor group in a predetermined manner to link together ,

Tetrazines, triazines and diazines which may be monosubstituted or polysubstituted with electron-withdrawing functional groups are suitable according to the invention as the diene component. These electron-withdrawing functional groups may be selected from:

- COOR (preferred: COOH)

- C (O) NR ₂

CX ₃ (X = halogen) (preferably CF ₃ )

- Halogen (F, Cl, Br or I) - CN

SO ₂ -R or SO ₃ -R - PR ₂

The functional groups R, which preferably provide functionality for attachment to further molecules (eg to peptides, saccharides or nucleic acids), may be selected from H, alkyl, aryl or heterocycle, where R may in turn optionally be substituted by alkyl, OH, SH, halogen, aryl, heterocycle, nitro, carboxyamido, or amine group. The said functional groups may also be linked directly to the tetrazine, triazine or diazine.

As electron-withdrawing substituents which may be bonded directly to the tetrazine, triazine or diazine, heterocyclic rings having 1, 2 or 3 N, O or S atoms in ring sizes with 5 or 6 ring members in question. At least one carboxyl group, sulfonic acid or phosphono group should be bonded to these rings for the purpose of connecting further functionalities.

"Alkyl" means Ci - C ₂ o, preferably methyl, ethyl, iso-propyl, tert-butyl, etc., The aryl or heterocycle substituents may be selected from: phenyl, - Thienyl, thiophenyl, furyl, furanyl, cyclopentadienyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiazolyl, oxazolyl, Indolyl, furazannyl, pyrrolinyl, imidazolinyl, pyrazolinyl, thiazolinyl, triazolyl, tetrazolyl, and the positional isomers of one or more heteroatoms which may comprise these groups, a group consisting of carbocyclic condensed rings, for example the naphthyl group or the phenanthrenyl group, a group consisting of fused heterocyclic rings, for example benzofuranyl, benzothienyl, benzimidazolyl, benzothiazolyl, naphtho [2,3-b] thienyl, thianthrenyl, I benzofuranyl, chromenyl, xanthenyl, phenoxathionyl, indolizinyl, isoindolyl, 3H-indolyl , Indolyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinolinyl, pteridinyl, carbazolyl, β-carbolinyl, cinnolinyl, acridinyl, phenazinyl, phenot hiazinyl, phenoxazinyl, indolinyl, isoindolinyl, imidazopyridyl, Imidazopyridmidinyl or the condensed polycyclic systems consisting of heterocyclic monocycles, such as those defined above, such as thionaphthenyl, furo [2,3-b] pyrrole or thieno [2,3-b] furan and in particular the phenyl, furyl groups, such as 2-furyl, imidazolyl, such as 2-imidazolyl, pyridyl, such as 2-pyridyl, 3-pyridyl, 4-pyridyl, pyrimidinyl, such as pyridmid-2-yl, thiazolyl, such as thiazole 2-yl, thiazolinyl such as thiazolin-2-yl, triazolyl such as triazolyl-2-yl, tetrazolyl such as tetrazol-2-yl, benzimidazolyl such as benzimidazol-2-yl, benzothiazolyl, benzothiazol-2-yl, purinyl, such as purine-7-yl, or quinolyl, such as 4-quinolyl.

The diene component may, however, also carry amino acid, peptide, saccharide, lipid or oligonucleotide or nucleic acid substituents at one or more positions. The diene component can also be coupled to all types of pharmaceutical actives, labels, dyes, complexes (e.g., carborane, ferrocene), quantum dots, chelating agents, diagnostics or therapeutics, and combinations thereof

Preferred diene components are all esters of the illustrated tetrazines, triazines and diazines and the compounds derived therefrom: e.g. For example, tetrazine monoamides, tetrazine diamides, tetrazine-3-trifluoromethyl-6-carboxamides, triazine tricarboxylic acid mono-, di- and triamides, 3-carboxyamide-5,6-bis-trifluoromethyl-triazine 1, 2, 4, 1, 2-diazines 3,6, diaryl 4,5-dicarboxylic acid amides. As well Of course, their homologues with ethyl or propyl group can be used instead of methyl. The mentioned tetrazines are relatively easy to prepare by oxidation from the corresponding dihydro compounds, which are accessible via the dihydro-tetrazine dicarboxylic acid ester, which in turn can be easily prepared in two stages from the commercial Diazoessigester

Dienophile component

As a dienophile for the DARinv is sufficient a terminal olefinic double bond without additional activation, a function that is not or rarely occurs in biological systems. A double-substituted double bond, as occurs in fatty acids or lipids, reacts only very slowly under normal conditions in the sense of DARinv. Even a methylene group such as in perilla alcohol reacts only very slowly at room temperature (60 hours) in the sense of DARinv to form the expected adduct. However, once a double bond is incorporated into a carbocyclic ring system, it reacts much faster than dienophile, decreasing reactivity as the ring size increases, starting with cyclopropene, and minimizing cyclohexene. In ring systems larger than seven-membered rings, triple bonds can also react as inverse dienophiles.

According to the invention, a carbocyclic ring is understood as meaning any mono-, bi- or tricyclic carbon ring. These rings may also contain heteratoms. (N, O, S, Si)

The dienophile component is also an isolated olefinic double bond or triple bond in a linear or branched hydrocarbon chain, which may optionally contain heteroatoms (N; O, S, Si) in question.

Since the reaction rates of different dienophiles differ from those of the same diene in the DARinv by powers of ten, targeted reactions can be carried out in the presence of two different dienophiles in a template. Sauer (Eur.J.Org.Chem 1998, 2885-2896) describes a difference in reactivity between cyclobutene and cyclopentene are about a factor of 12 compared to the unsubstituted tetrazine, while between cyclopentene and cyclohexene a factor of 1200 is observed. It follows that in the presence of a cyclobutene and a cyclohexene in the same molecule, a difference in the reaction rate can be observed by a factor of about 1200. In terms of yield, this means about a one per mille impurity. Of course, differently substituted tetrazines, triazines and diazines also have different reactivities to the same dienophile as dienes, so that it is possible to define reactivity scales of dienes and dienophiles which permit very specific and targeted multiple reactions. Extending this scheme to include the classical Diels Alder reaction, which is absolutely orthogonal to the electronic requirements of diene and dienophile for the inverse variant, results in a highly variable and efficient network of ligation reactions. Often, an upstream classic DAR first creates the dienophile for a DARinv. A very nice example of this sequence is the DAR between cyclopentadiene and MSA (maleic anhydride) to form the norbornene anhydride or the DAR between cyclooctatetraene and MSA to give an adduct that simultaneously contains a cyclobutene and a cyclohexene ring in the same molecule. Since both the norbornene anhydrides (exo and endo) and the bicyclic COT anhydride can be readily derivatized with amines, they can be readily converted to functional ligation-suitable molecules. The metathesis reaction also generates ring systems with double bonds. Cyclobutenes can also be generated as reactive dienophiles by photochemical cyclization reactions of cyclic 1,3-dienes, so that the DARinv-based ligation technique can be linked to photolithography.

It has been described in the literature that the surfaces of carbon (diamond, fullerenes, carbonanoropes), germanium and silicon behave like dienophiles in classical DAR. (Roucoules V. et al, 2005, Langmuir 21, 1412-1415). They react with cyclopentadiene to form the norbornene ring system, again forming a dienophile suitable for the DARinv. These surfaces can be easily functionalized by the DARinv. In connection with the photochemical activation by Cyclization of 1, 3-dienes to cyclobutenes results from the following: 1.) Covalent attachment of a cyclic 1,3-diene to a surface or a semiconductor 2.) Photochemical cyclization to cyclobutene, 3.) DARinv with a diene For example, a protein or a saccharide is covalently linked, completely new spatial functionalization possibilities.

Preferred dienophile components are acids and anhydrides, the functionalized imides and amides derived therefrom and their reduction products, as well as the associated esters and their substitution and reduction products containing a taut or terminal double bond, e.g. exo- or endo-norbornenedicarboxylic anhydride, the two norbornene monocarboxylic esters, cyclobutene monocarboxylic acid esters

Cyclobutene dicarboxylic acid anhydride, sym. Cyclopentenecarboxylic acid

Cyclohexenedicarboxylic anhydride., Sym. Cycloheptencarbonsäure, the easily accessible tricyclic COT-MSA adduct, or the corresponding monocarboxylic acid from COT and acrylic acid. , The readily accessible 2-allyl-2-propargyl malonate also has two different reactive dienophile groups. Other preferred dienophile components are dehydroproline, allyl-proline, allylmalonic esters, allylgalactose, allyl-silsesquioxane, and all compounds bearing an allyl, butenyl or pentenyl group. The dienophile component may optionally also be monosubstituted or polysubstituted with functional groups. The functional groups may be selected from, for example, alkyl chains (C ₂ - C ₂ o, methyl, ethyl, iso-propyl, tert-butyl, etc. preferably, optionally halogen-substituted), OH, SH, halogens, aryl, carboxyl , Carbonyl, nitro, carboxyamido, keto, sulfoxide, sulfone, sulfonic acid, sulfide, sulfate, phosphoric acid or amino groups which are bonded directly or via alkyl radicals. The dienophile component may also contain aromatic or heterocyclic radicals. These may be selected from: phenyl, - thienyl, thiophenyl, furyl, cyclopentadienyl, furanyl, pyranyl, pyrrolyl, imidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyrimidinyl, pyrazinyl, pyridazinyl -, thiazolyl, oxazolyl, indolyl, furazannyl, pyrrolinyl, imidazolinyl, pyrazolinyl, thiazolinyl, triazolyl, tetrazolyl group, as well as the position isomers of the heteroatom (s) that may comprise these groups, a radical consisting from carbocyclic condensed rings, for example the naphthyl group or the phenanthrenyl group, a group consisting of condensed heterocyclic groups Rings, for example, benzofuranyl, benzothienyl, benzimidazolyl, benzothiazolyl, naphtho [2,3-b] thienyl, thianthrenyl, isobenzofuranyl, chromenyl, xanthenyl, phenoxathionyl, indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl , Phthalinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinolinyl, pteridinyl, carbazolyl, β-carbolinyl, cinnolinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, indolinyl, isoindolinyl, imidazopyridyl, imidazopyridimidinyl, or else the condensed polycyclic systems consisting of heterocyclic monocycles such as above such as thionaphthenyl, furo [2,3-b] pyrrole or thieno [2,3-b] furan, and especially the phenyl, furyl groups such as 2-furyl, imidazolyl such as 2-imidazolyl, pyridyl, such as 2 Pyridyl, 3-pyridyl, 4-pyridyl, pyrimidinyl, such as pyridmid-2-yl, thiazolyl, such as thiazol-2-yl, thiazolinyl, such as thiazolin-2-yl, triazolyl, such as triazolyl-2-yl, tetrazolyl, as in Tetrazol-2-yl, benzimidazolyl such as benzimidazol-2-yl, benzothiazolyl, benzothiazol-2-yl, purinyl such as purin-7-yl, or quinolyl such as 4-quinolyl.

The variety of possible substitutions in the diene and dienophile allow a diene active in the DARinv to concurrently contain a diene structure or a dienophilic group of the classical DAR in the same molecule without any reaction occurring. The same naturally applies to the inverse dienophile. This results in a variety of targeted ligation options by simultaneously directed directed DAR and DARinv.

However, the dienophile component can also carry on one side amino acid, peptide, saccharide, lipid or oligonucleotide or nucleic acid substituents. The dienophile component can also be coupled to all types of pharmaceutical actives, labels, dyes, complexes (e.g., carborane, ferrocene), quantum dots, chelated compexators, diagnostics or therapeutics.

Orientational kinetic measurements of the DARinv for the prepared dienes and dienophiles show the expected broad reaction behavior.

Table 2 below shows rate constants k (s ^{"1 *} _mo f ^{1 *} l) for the reaction between the tetrazines (column 1) with the respective dienophiles (line Possible combinations with the classic DAR. Suitable, readily available compounds are named "dienophile linker systems." The same applies to the dienes, with the particular combination of tetrazine and triazine being of particular interest because of the different diene reactivity in the DARinv.

In the following, some important preferred aspects of the present invention will be highlighted, but they should not be construed as limiting the broad methodology.

In the following, therefore, we first describe some possible representations of dienes, then the dienophiles, and finally the Diels-Alder reaction with inverse electron demand.

Preparation of substituted triazines and tetrazines (diene component):

tetrazines:

Tetrazines have a high reactivity as dienes in the inverse DAR. The model compound for many studies is the readily available tetrazinodicarboxylic acid dimethyl ester. However, necessary for the purposes of the invention changes to this compound, such as nucleophilic substitutions, but lead to decomposition. In contrast, many reactions of the ester function with nucleophiles can be easily and quickly performed on the dihydro precursor. Since the rate of the first nucleophilic substitution with amines is usually greater than the rate of the second nucleophilic substitution, monosubstituted amides are easily prepared, thus opening the way to the preparation of unsymmetrical diamides. The subsequent oxidation of the dihydro compounds to the tetrazines can be carried out with a variety of oxidizing agents, such as Fe (III) Cl ₃ , nitrite, bromine or H ₂ O ₂ . A selection of the compounds shown are shown below and illustrate the possibilities of the synthesis concept. This thus makes it possible for every application to produce the analogous connections. It can the Oxidation to tetrazine are carried out at the very end of a reaction sequence because tetrazine dicarboxylic acid amides are much more stable than the Tetrazindicarbonsäureester itself.

Dihydro-tetrazine mono-methylamide

Dihydro-tetrazine mono-dimethylamides

F104M F117TM

Tetrazine dihydro monobenzylamide substituted

Tetrazine diamides-benzyl

Bis-trifluoromethyl-diaryl-tetrazine-mono-carboxylic acids

In addition to tetrazines with ester functions, many tetrazines with aromatic residues are known in the art. They have compared to the tetrazine-dicarboxylic acid dimethyl ester via a reduced diene activity with a significant increase in stability. The greater stability also allows reactions with nucleophiles without destroying the tetrazine ring. Methods are available for preparing unsymmetrically substituted compounds. The introduction of electron-withdrawing substituents such as fluorine, trifluoromethyl or heteroatoms in the phenyl rings leads to an increase in the diene activity. Here, the use of pyrimidine residues has proven to be extremely successful. In the presence of two pyrimidine residues, the monocarboxylic acid amides derived therefrom achieve the diene reactivity of the tetrazine-3,6-dicarboxylic acid ester. The removal of only one nitrogen atom leads to a significant loss of diene reactivity, so that with this series of diaryl monocarboxylic acids a speed range of at least a factor of 10,000 is covered. Again, the goal is the simple representation of monofunctional compounds for incorporation into peptides, oligonucleotides or their anchoring to surfaces. Although methods for the preparation of such tetrazines are known from the literature, mono-carboxylic acids of this type have not been described in the literature. The simplest representation of aromatic unsymmetrical compounds consists in the reaction of two amidines or imido esters with hydrazine or two nitriles with sulfur and hydrazine or with hydrazine alone. This simple, by the use of inexpensive starting compounds and practical synthesis route, is still unsatisfactory from the yield. It is interesting to observe that the dicarboxylic acids which can be obtained in very good yields by the same route of synthesis can be thermally decarboxylated to the monocarboxylic acids. The water-insoluble monocarboxylic acids of this type can be made more soluble by the conversion into the corresponding N-oxides. The introduction of hydrophilic radicals for this purpose is feasible.

triazines

In contrast to the tetrazines, the reactivity as a diene is markedly less pronounced for the triazines. But the chemical stability is clear better and is of the order of normal organic compounds. Here, the triazine tricarboxylic acid triethyl ester has been found to be a model compound. Disadvantage of the triazines is in addition to the lower reactivity, the formation of isomeric products in the DARinv. One is confronted with two necessities: on the one hand with the increase of the reactivity as diene and on the other hand the representation of defined monosubstituted compounds. Both can be achieved by the targeted replacement of the ester groups by trihalomethyl groups, preferably trifluoromethyl groups. Nucleophilic substitution on the ester groups of the triazines by amines is just as feasible as in the tetrazines. On the other hand, due to the lower differences in reactivity, unambiguous mono-substitution is not so easily possible. This can be achieved by the use of tert-butyl esters in the building blocks, resulting in the triazine two ester groups of different reactivity. Again, the goal is to introduce nucleophilic substitution with amines functional groups such as carboxy, hydroxy and amino. As with the tetrazines or dihydro-tetrazines, reporter molecules, therapeutics, peptides or oligonucleotides can then be introduced via these functions.

Triazine-tri-carboxylic acid amides

A preferred triazine is also the first-represented 3-carboxymethyl-5,6-bis-trifluoromethyl-triazine 1, 2,4. This triazine has only one function, through which a number of reactive groups can be introduced, which are suitable for the intended use. Through the two adjacent ones Trifluoromethyl groups tend these triazines for hydrate formation, a property that is known by many triazines. This hydrate formation may decrease the activity as a diene in the DARinv, but the DARinv takes place.

Bis-trifluoromethyl-triazine momo-carboxylic acids

Mono trifluormetyl-triazine-di-carboxylic acid ester

Already the introduction of only one trifluoromethyl group in the triazine ring is already sufficient to obtain a sufficient diene activity. 1437

diazines

The DARinv of terazines with a dienophile gives rise to dihydrodiazines, from which diazines are easily accessible by oxidation. If the dienophile is an enamine or a substituted acetylene, the diazines are formed directly. Representation of dienophiles

Dienophiles I

A number of known cyclic and bicyclic unsaturated anhydrides of different ring size can be easily reacted with substituted amines to the corresponding acid imides. Reference should be made explicitly to the exo- and endo-Norbornendicarbonsäureanhydrid, both of which are commercially available. Monocarboxylic acids, such as, for example, the cyclopentenecarboxylic acid, can also be attached via their acid chlorides to amino acids or diamine ligands. The derivatives of allyacetic acid can likewise be prepared in the same way. These can then be covalently linked by the DARinv to a tetrazine-bearing molecule, even on a surface. The Boc or Fmoc-lysine reactions yield peptide building blocks that can be targeted to any position in peptides. If dienophiles of different reactivity are incorporated into the same peptide chain, these peptides can be marked several times in a targeted manner. The same applies to oligonucleotides

NORBORNENIMIDE

These reactions are transferable to the exo-norbornene dicarboxylic acid anhydride, the cyclobutene dicarboxylic anhydride and the Cyclohexene-dicarboxylic anhydride or any other anhydride containing a strained or a terminal double bond.

Pentenes as dienophiles

Dienophile II

A special case is that of cyclooctatetraene, COT for short, and MSA readily accessible tricyclic anhydride. The DAR of the bicyclic form of the COT results in a molecule containing two different reactive dienophiles for the DARinv. Other functions can be introduced in the usual way via the anhydride ring, such as amino acids, amines or the coupling to a solid phase.

However, only the highly reactive cyclobutene ring is available for DARinv. The intrinsically less reactive cyclohexene ring can not be reacted as dienophile in this tricycle, a behavior that is due to the endo arrangement of the anhydride ring. This behavior has already been discussed for cyclobutene dicarboxylic anhydride. If the carbonyl groups are removed by reduction, the resulting amine with reactive dienes undergoes double DARinv. With a less reactive diene DARinv takes place only with the cyclobutene ring, by adding a more reactive diene then also the cyclohexene ring reacts. In this way, two completely different molecules, which carry tetrazines of different diene activity as anchor groups, can be specifically linked to one another in a predetermined manner.

Dienophiles based on cyclobutenes

-NH ₂ -OH

N- N-

The following amino acids can be purchased and all drφfi react as dienophiles in the DARinv. The three amino acids can be introduced at any position during the peptide synthesis and then new residues can be introduced via the DARinv at these positions; Such peptides can also be anchored to surfaces in a defined manner for detailed structural investigations. The enzymatic peptide synthesis also allows the introduction of amino acids with dienophilic anchor groups as the group of Bertozzi has shown.

Lysine derivatives

F 419 Fmoc-Lysine

F 416

F 417 F 415 endo

The building blocks for the peptide synthesis represented by the inventors are summarized above. The reverse principle, namely the incorporation of the diene into the peptide can also be carried out on the solid phase. As the last amino acid, a lysine is coupled, which is substituted at the amino group with a tetrazine. After cleavage from the solid phase, the peptide can be isolated as a pink solid. The implementation with the COT-Lys-EILDV Peptide leads to a coupled dipeptide. The same peptide is isolated when the DARinv is carried out immediately on the solid phase. The introduction of lysine building blocks of dihydrotetrazine diamides into peptides is also possible, followed by oxidation to tetrazine. This procedure is indicated when the tetrazine decomposes under the conditions of peptide synthesis. This shows that the coupling of biomolecules with the DARinv is possible and can be varied in many ways.

However, the peptides can also be labeled either on the solid phase or after cleavage by the DARinv with dyes or biotin.

F 109

A sequential double DARinv is shown below, which proceeds with two different reactive tetrazines on the bifunctional dienophile. Sequential DARinv of M 1476

All compounds that have a terminal double bond react as dienophiles in the DARinv. Below are implementations with purchasers Compounds listed. These are allyl malonate, allyl galactose and allyl silsesquioxane.

The scope of application of the ligation technology based on the DAR inv. Ranges from the functionalization of surfaces in the area of nanomaterials to the labeling of biopolymers with dyes or other reporter molecules. Also linking therapeutic agents to bioplymeres in drug-targeting, linking proteins with saccharides to improve pharmacokinetic parameters. In the following some applications are described in detail.

Applications I platinum complexes

The aim is to produce platinum complexes in which either the diamine ligand or the dicarboxylic acids used as the leaving group are formed as dienophile or as diene. The structures listed below are intended to illustrate this concept. This makes it possible to selectively the respective part of the Complex by the DARinv targeted change. This makes possible the construction of libraries of the original complex. The change in the leaving group, in this case the dicarboxylic acid, by the DARinv appears to be particularly interesting since this leaving group is split off during the intracellular activation of the Pt complexes and thus also the respective part attached by the DARinv. Thus, the leaving group could be targeted by the DARinv, for example by peptidic signal sequences, or other biomolecules that can mediate preferential uptake of these complexes in tumor cells. The expression of the diamine ligand as a dienophile allows, after covalent binding of the active platinum complex to the DNA to detect the localization of the resulting adduct by a DARinv with a reporter molecule .. Of course it is also possible both the amine ligands and the leaving group as dienophiles with different Dienophile activity, which allows simultaneous targeted ligation of both the amine ligand and the leaving group. To ensure that R ²⁺ does not react with olefinic double bonds during synthesis, the Pt complexes shown here were prepared ,

L 1772

L 1782

The ligands and leaving groups listed above are known from the literature.

Since the platinum complex of 1,2,3-triaminopropane shown is known, it can be bound to the tetrazine via its free amino group, thus providing another building block for the incorporation of Pt complexes into proteins, saccharides and other biomolecules and therapeutics.

Applications Il PET

Positron emission tomography is a radioactive, non-invasive, but very sensitive diagnostic method. The most commonly used positron emitter is F18, which decays into the element oxygen with a half-life of 18 min, releasing a positron. Because of the low Half life requires the preparation of appropriate F18-labeled compound in particular synthetic methods. They have to run fast and necessary cleaning procedures have to be easy. The most commonly used compound today is 2-fluoro18-2-deoxy-glucose. The ligation reaction based on the DARinv can be used very well for labeling peptides, oligonucleotides and saccharides with F18. Nucleophilic substitution reactions on aromatics are facilitated as the number of nitrogen atoms in the ring increases, such as in the series benzene, pyridine, pyrimidine and triazine. Thus, in 1, 2,4-triazine, a thiomethyl radical can easily be replaced by a number of nucleophiles, including halogens. However, since such triazines react as dienes in the DARinv, the prior introduction of F18 into such a triazine offers the elegant possibility to label the abovementioned biopolymers with F18 with the aid of the DARinv and thus make them accessible for detection by PET. But also the classical way of nucleophilic substitution on tosylates with fluoride can be labeled (see below).

Substances for PET

Also, the introduction of trifluoroacetyl groups labeled F18 via the amine function of the described tetrazines and triazines can be used here. Thus, the tetrazine can be trifluoacetylated in pyridine and directly in the Solution that DARinv perform. However, it is also possible to introduce the F18 at the dosi dihydrotetrazine stage and then to close the oxidation to tetrazine. Important for all of these reactions is that the DARinv allows very short reaction times and usually proceeds without the formation of by-products.

\

Applications Hl surfaces

As already described for the preparation of dienes, the dihydro-tetrazine dicarboxylic acid ester can be reacted with amines at RT with primary amines. This high reactivity can be used to build up reactive solid phases. The reaction sequences shown here can be carried out in yields between 70 and 90%. Thus, solid phases are available which carry either a diene or a dienophile for the DARinv. The reactivity in the DARinv is so high that the dienophile-bearing solid phase can be literally titrated with a tetrazine. The resulting applications range from the chip technology for oligonucleotides, proteins or saccharides to catalytic surfaces and solid phase reagents. However, it is also possible to use the illustrated trimethoxy-silyl compounds for anchoring to surfaces, in which case both the diene and the dienophile can be anchored to the surface. However, the acid chlorides of the diaryl-tetrazine monocarboxylic acids and of the dihydro-tetrazine-glycic acid chlorides are also available for this purpose.

Silica gel, polystyrene

Oxidate.

dienophiles

serving

Since the DARinv is a very fast reaction, two surfaces can be covalently bonded to each other in the sense of an adhesive. Surface reactions are usually slower than reactions in solution. At DARinv we have the observation made that the driving force of the reaction is so great that even in those cases in which the tetrazine is poorly soluble, such as in water, the reaction takes place at the surface of the tetrazine particles under visible evolution of nitrogen.

Applications IV DNA adducts

The analytical methods for the detection of DNA adducts are still not automatable. The P32 post-labeling method is still the most sensitive method, but so far no method is available that allows the simultaneous detection of different adduct types. Although the method of separation by capillary electrophoresis with subsequent fluorescence detection is very well suited for the detection of 5-methyl-cytosine, but missed the detection limit of the 32P-post.labeling method by at least a factor of 100. Here can also in the remedy the present invention described ligation reaction. The amine derived from norbornene can be coupled to the phosphate group of the nucleosides by a more recent method according to literature. Then any fluorescent dye linked to a tetrazine or triazine can be coupled via the DARinv and subsequently detected by means of capillary electrophoresis.

In addition to this general method, it is possible to detect directly those DNA adducts structurally containing a DARinv-capable double bond, for example the etheno adducts of dA and dC. These adducts play an important role in the assessment of oxidative stress.

With the help of the new ligation reaction, any substituents can be introduced into oligonucleotides obtained by synthesis. The required amidites have been prepared by and. In this way, a multiple mark is possible. Amidites for labeling oligonucleotides

Mass weak

For this purpose, it is sufficient to introduce a nucleotide during the synthesis which carries an alkyl radical having a terminal double bond. Applications V Photochemistry

The photochemical cyclization of 1,3-dienes to cyclobutenes proceeds with high quantum yield, generating a highly reactive inverse dienophile. Thus, the photolithography can be linked to the ligation by the DARinv. Because only where UV light is irradiated, the 2 + 2 cycloaddition takes place and only there can then proceed the DARinv. Thus, substituted tetrazines and triazines can also be added to surfaces here.

as acid chloride

hv

Also, the introduction of the 1, 3-dihydro-phthalic anhydride-5,6 by the reaction with the amino function is suitable here, the previously unknown 1,3-cycloheptadiene-6-carboxylic acid is suitable here.

Applications VI Construction of Dendrimers and Polymers The reaction of COT-MSA anhydrides is so rapid and unambiguous that reactions with polyamines give rise to dendritic structures, which can now be further modified by the DARinv reaction with any other dienes. The connections shown here are shown and characterized. The triple DARinv can be carried out easily. The amines produced after reduction of the carbonyl groups allow the introduction of two different molecules per bicyclic, since the four-membered ring and the six-membered ring have distinctly different dienophile activities. However, other polyfunctional molecules, such as inositol, are also readily allylated and thus can be used as a template for dendritic structures. These compounds include chitosan.

With such a technology, for example, peptides or saccharides for use as therapeutics, diagnostic agents or for the study of Interacting of peptides or saccharides with each other or with other biomolecules. The dihydro-tetrazinedicarboxylic acid can also be incorporated into polyamides during the condensation and, after oxidation to tetrazine, modified by DARinv. The diaryl-tetrazine-dicarboxylic acids are also suitable for incorporation in nylon / nylon-type polyamides. If tetrazines with different diene reactivity are incorporated in statistical distribution, they can be specifically modified in different ways. Oligomeric tetrazines or mixed oligomeric tetrazines / triazines can be prepared with the above-described building blocks and then selectively modified by DARinv. Again, the corresponding polymers are conceivable. Polymeric tetrazines

Applications VlI Quantum Dots

Quantum dots are nanoparticles made up of compounds such as CdS or CdSe and have special optical properties. Upon excitation with lasers they fluoresce very strongly depending on their size and therefore find more and more use in the diagnostic field, especially as they make the detection of single molecules possible. The prerequisite for this, however, is their doping with functional groups which proceeds via SH groups and permits a subsequent interaction with the molecules to be detected. Due to their special properties, gold nanoparticles are used for electron microscopic investigations of biomolecules. Again, the anchoring of molecules on the surface via SH groups accomplished. Again, the new ligation technique can be used by DARinv. For this purpose, SH-group-containing triazines and tetrazines were prepared, wherein first the disulfides were prepared and from this by reduction with dithiothreitol the mercapto compound. Analogously, SH-containing dienophiles of the norbornene type can also be prepared. The disulfides themselves were also anchored to gold surfaces.

Thus, both the dienes (tetrazines, triazines and diazines) and the dienophiles can be deposited via the disulfide group as to the surface of the quantum dots or other metals and are therefore accessible to the DARinv. Thus, for example, antibodies, peptides, saccharides or therapeutics can be anchored to the surface of the quantum dots for diagnostic or therapeutic purposes.

Disulfides for anchoring to surfaces

Applications VIII saccharides

The synthesis of complex saccharide structures requires a sophisticated protecting group strategy. The reducing end is often protected by an allyl group or a pentenoyl group. From natural sources isolated oligosaccharides can be easily provided with an allyl group or pentenoyl group at the reducing end. Thus, the conditions for anchoring these oligosaccharides to a solid phase or surface are given by the DARinv. The furan saccharide mimetics described in DE-A-100 41 221.1 can likewise be anchored either to biomolecules or surfaces with the aid of this DARinv technology. Prerequisite is the introduction of an allyl ether group or the use of linker molecules as described above.

F387 In this way, the compounds described in DE-A-100 41 221.1 can be used in the application described here.

These saccharide mimetics are also suitable as inverse dienophiles in the DARinv and can thus be introduced into any biomolecules.

Applications IX Therapeutics

The side effects of drug therapy, especially tumor therapy, are still part of everyday clinical practice. This is partly due to the fact that it has not been possible to inject therapeutically active substances targeted into the diseased cells. Thus, there is a need for simple ligation reactions that allow therapeutics to be linked to molecules that facilitate preferential uptake into the cell. With regard to tumor therapy, reference is made to EP-A-1 051 421, in which the allyl ether of a boron-containing tetracarborane is prepared, which has now proved to be an ideal dienophile for linking by means of the DARinv to peptides or saccharides. With this method, however, vitamin A, vitamin C, curcumin or other therapeutic agents can be coupled to proteins or surfaces and released there by hydrolysis or enzymatic cleavage. Another example of this application is the temozolomide used to treat brain tumors, which can be coupled via its acid function either to a tetrazine or to a dienohile. About the DARinv then the coupling to peptides is possible. Also, liposomes containing double bonds that are active in the DARinv can be easily modified with this technology, thereby improving targeting or modification

To achieve pharmacokinetics.

temozolomide

Coupling to tetrazine peptide

Coupling to COT-peptide

Applications X Oligonucleotides

The ligation reactions of oligonucleotides known hitherto in the literature with the aid of DAR use cyclic dienes and as dienophile maleimides (Tona R. and Häner Robert, 2005, Bioconjugate Chem 16, 837-842, Hill KW et al., 2001, JOC, 66, 5352 to 5358). In these systems, however, is the Reaction rate is not very high and is up to 7 days, or it must be used high excesses of reagents. Here the DARinv offers itself as a better, because more efficient ligation reaction. For this purpose, the two amidites were prepared to introduce at the end of the oligo synthesis an AIIyI or a pentenyl group at the 5 ^' end, which is active in the DARinv as dienophile.

Linker Systems of Dienophiles I

Cyclopentadiene and p-benzoquinone

butadiene

Both systems are suitable for the sequential coupling of molecules which carry either the same dienophile as L 1820 or different dienophiles such as L 1825. This type of compound is also suitable for anchoring to a solid phase by reaction with the carbonyl or the carbonyl group

Linker systems from dienophiles Il

Cyclopentadiene M 1452

The ketone dicyclopentadienone is an orthogonal dienophile that is accessible to both normal DAR and DARinv. By reaction with butadiene, it can also be converted into a double inverse dienophile (s. In addition to these linkers from dienophiles, linkers from dienes that allow DAR and DARinv at the same time are also available. The combination of tetrazine or triazine with a dienophile, such as a maleimide, for the classical DAR is possible. The structures are listed below.

EXO

endo

ENDO

That a DARinv can run alongside a classical DAR at the same time is shown below. In the presence of both Dienophile finds first the fast DArinv takes place, followed by the slower classic DAR. The products are characterized.

DAR and DARinv at the same time

Applications Xl substance libraries

Using the presently described, novel tetrazines, triazines and diazines as dienes in the DARinv can be with dienophiles, such as enamines, enol ethers, etc., build novel libraries of substances to search for new drugs. The diamides themselves are, as described, accessible in large numbers by successive reactions with various amines and subsequent oxidation. This reaction sequence can be automated. For the development of new therapeutics sufficient water solubility is an important prerequisite. One possible approach to improving water solubility and thus oral availability is the covalent attachment of drugs to saccharides to form conjugates. With the approach according to the invention, it is possible from the outset to consider the problem of a sufficient water solubility by the use of suitable amine building blocks in the DARinv using a combinatorial approach. This also has the additional advantage that these building blocks based on saccharides and their derivatives become part of the active ingredient and can thus contribute to strengthening the binding of the active ingredient to its target. These novel structures are both of their synthesis and their structure as peptidomimetics to call. The tetrazine-3,6-dicarboxylic acid ester and thus also its diamides and the other derivatives are derived from glycine.

libraries

N HN

The invention will be further described with reference to the following figures. Fig. 1: Diels-Alder reaction

Fig. 2: Diels-Alder reaction with inverse electron demand

The invention will be further described by way of examples.

example 1

Synthesis of

5,6-Bis-trifluoromethyl-1, 2,4-triazine-3-carboxylic acid ethyl ester

5 grams (25.7 mmol) of hexafluorobutene were dissolved under argon in 100 ml of DMF, placed in a 500 ml round bottom flask and the oxalamide hydrazone in 100 ml of DMF slowly added dropwise with cooling at 0 ° C (exothermic reaction!). After completion of the addition, the batch was further stirred overnight at room temperature. For workup, the DMF was removed under high vacuum at 50 ⁰ C and the remaining residue taken up in Essigester and washed with dilute hydrochloric acid and water and dried over sodium sulfate. After the ethyl acetate had evaporated, diisopropyl ether was recrystallized. By the same procedure also the methyl ester could be obtained. Both compounds are obtained as dihydrates, whereby one molecule of water is readily released, so that the monohydrate can also be obtained in crystalline form. Mass spectrum and 1 H-NMR prove the structures.

Similarly, the following triazenes could be obtained

Example 2:

Synthesis of

10 mmol of the tricyclic anhydride obtained from cyclooctatetraene and maleic anhydride were refluxed in 50 ml of methanol with 10 mmol of Boc-lysine for 3 hrs. The residue remaining after evaporation of the solvent could be recrystallized directly. If crystallization is unsuccessful, chromatography on silica gel may be used for purification, chloroform with 1% methanol. The product crystallizes on standing. Mass spectrum and NMR prove the structure.

The following compounds were prepared and characterized analogously.

Synthesis of

1 mmol of tris- (2-ethylamino) -amine were refluxed in 20 ml of methanol with 3 mmol of the tricyclic anhydride for 5 hours. Upon cooling, a precipitate forms, which is sucked off. Yield 80%. The mass spectrum shows the molecular peak at m / e 698 without appreciable fragmentation.

In an analogous manner, the following compound was prepared

Synthesis of

4 mmol (1, 36 gr.) Of obtained by reacting the tricyclic anhydride with N-Boc-ethylenediamine amide, mp. 135 ⁰ C is stirred in a mixture of 10 ml of methanol and 20 ml of 1 N hydrochloric acid overnight at room temperature, the Substance goes into solution. After lyophilization, the hydrochloride is obtained as a white residue in quantitative yield. The mass spectrum shows the molecular peak at 244 for the amine.

0.5 mmol of this hydrochloride was suspended in 10 ml of chloroform and then 0.5 mmol of dansyl chloride in solid form was added. While cooling at 0 ^° C., 0.28 ml of triethylamine, corresponding to 2 mmol, in 5 ml of chloroform were then added dropwise. The reaction was stirred overnight at room temperature. For workup, the organic phase was washed 3 times with 10 ml of water, dried over sodium sulfate and concentrated. For purification was chromatographed on silica gel with hexane / ethyl acetate 2: 1. 240 mg of solid product were isolated. a yield of 50%. The mass spectrum shows the molecular peak at 477 both in pos. as well as in neg. mode.

Dienophile modified peptides:

The peptide was synthesized on the synthesizer, with the previously described lysine derivative added with the tricyclic dienophile. This peptide was purified by HPLC and the structure confirmed by the mass spectrum. Molecular peak at m / e 899.

Diels-Alder reaction of the abovementioned peptide with the tetrazine-3,6-dicarboxylic acid dimethyl ester: 0.01 mmol (9 mg) of the peptide are dissolved in 0.5 ml of DMF and treated dropwise with a 0.01 molar solution of tetrazine. After each addition, the red color of the tetrazine disappears immediately and tetrazine is added until the red color just disappears. The DMF is evaporated in a high vacuum. The mass of the residue shows the molecular peak at m / e 1096, next to a small amount of unreacted peptide at m / e 899.

Example 3

Reaction of dihydro-tetrazine-3,6-dicarboxylic acid dimethyl ester with primary amines

Dihydro-tetrazine-3,6-dicarboxylic acid dimethyl ester 200 mg (1 mmol) are suspended in 5 ml of methanol and (2.5 mmol) glycine methyl ester in 5 ml of methanol - from 2.5 mmol glycine methyl ester hydrochloride and 2.5 mmol triethylamine prepared - added dropwise and stirred overnight at room temperature. The reddish solution has turned pale yellow and a yellow precipitate has formed. After cooling to -18 ° C, this precipitate is filtered off and recrystallized from methanol. Yield 55%. Mass spectrum and NMR confirm the structure.

Similarly, the following compounds were prepared and characterized:

A specification for the preparation of dihydro-tetrazine-carboxylic acid methyl ester monobenzylamide L1842

Nh

The suspension of dihydro-tetrazine-dicarboxylic acid dimethyl ester (5 mmol) in 30 ml of methanol is heated to 50 ⁰ C and at this temperature, the benzylamine (5.5 mmol) in 10 ml of methanol in the course of 2 hours were added dropwise. It is stirred for 2 hours at this temperature and then cooled to -20 ⁰ C overnight. there precipitates the monoamide, accompanied by some diamide .. In the subsequent recrystallization from methanol only the monoamide goes into solution and crystallized in yellow flakes. The yield is between 50 and 80%.

Oxidation of dihydrotetrazine to tetrazine

1 mmol (342 mg) of the dihydrotetrazine are dissolved in 10 ml of chloroform and treated with a small excess of isoamylnitrite. The solution turns intensely red and after 2 hours the chloroform turns red. The red residue is recrystallized from acetone. Mass spectrum and NMR confirm the structure of tetrazine.

Example 4

Reaction of dihydro-tetrazine-3,6-dicarboxylic acid dimethyl ester with glycine methyl ester in a ratio of 1: 1

Dimethyl dihydro-tetrazine-3,6-dicarboxylate 200 mg (1 mmol) is suspended in 10 ml of methanol and 1 mmol of glycine methyl ester as hydrochloride in solid form is added at 0 ° C. Then 1 mmol of triethylamine in 10 ml of methanol is slowly added dropwise. The mixture is stirred for 3 hours at this temperature, then the cooling is removed and allowed to come to room temperature overnight with stirring. The color of the solution is only light red and a yellow precipitate has formed. This is filtered off with suction after cooling to -20 ⁰ C, washed with cold methanol and with ether and dried. For further purification is recrystallized from methanol. Mass spectrum and 1 H-NMR clearly prove the structure. The following compounds were prepared in an analogous manner

Example 5

Method for the preparation of the aza-diaryl-tetrazine monocarboxylic acids. for example L1892

10 mmol of the corresponding nirile are heated together with the 5-fold excess in ethanol for 4 hours to boiling. After cooling, the precipitate formed is filtered off with suction and then boiled with acetone. The dihydrotetrazines without carboxyl group go into solution. The remaining residue is then oxidized with nitric acid in glacial acetic acid and the precipitated pink colored tetrazine mixture is filtered off. When boiling in DMF, the corresponding mono-carboxylic acid goes into solution, the dicarboxylic acids remain as a residue. The mono-carboxylic acids are thereby obtained for further syntheses in sufficiently pure form.

Reactions of dienes and dienophiles to inverse electron demand DARs

Reaction of the tricyclic anhydride with tetrazine-3,6-dicarboxylic acid dimethyl ester:

To a suspension of 1 mmol (198 mg) of the tetrazine-3,6-dicarboxylic acid dimethyl ester in 5 ml of tetrahydrofuran was slowly added the solution of the tricyclic anhydride in 5 ml of the same solvent while cooling. Under nitrogen evolution, a clear red solution formed, the color of which turned yellow with the last drops of added anhydride. It was concentrated and the remaining residue was recrystallized from ethyl acetate. Yield is quantitative, mp 165 ° C. The mass spectrum shows the molecular peak of the anhydride at m / e372.

Evidence of the Diels Alder reaction with inverse electron demand on the solid phase:

First, 1 g of amino-functionalized silica gel (according to the manufacturer's instructions 1 mmol of amino groups per gram of silica gel) in 10 ml of methanol by shaking 2 mmol of Dihydrotetrazins-3,6-dicarboxylic acid ester and shaken at 60 ⁰ C for 5 hrs. Shaken in a closed vessel. It was then filtered off, washed repeatedly with methanol and ether and dried. 900 mg of silica gel were obtained. By elemental analysis could be detected by the calculation of the C / N ratio about a 70% occupancy. Oxidation with isoamyl nitrite.

The resulting silica gel was suspended in ethyl acetate and shaken with a 5-fold excess of isoamyl nitrite for 5 hrs at room temperature. It was filtered, washed several times with ethyl acetate and ether and dried. The silica had now assumed a light pink color. The elemental analysis revealed only minor changes in the C / N ratio. The Diels Alder reaction was carried out with the already used tricyclic anhydride. 100 mg of the tetrazine-loaded silica gel were suspended in ethyl acetate and shaken with 0.3 mmol of the anhydride for 2 hours at room temperature. It was filtered again, washed 5 times with ethyl acetate and dried. The C / N ratio of the elemental analysis confirmed the original specific silica occupancy with dihydrotetrazine at about 70%.

Another Diels Alder reaction was carried out with the dansyl derivative, the synthesis of which has already been described under dienophiles. For this purpose, 50 mg of the tetrazine-loaded silica gel were suspended in ethyl acetate and reacted for one hour with 0.05 mmol of the dansyl tricycle. It was filtered off, washed 10 times and dried. The resulting silica gel showed a strong green fluorescence in UV light. For control, the dihydrotetrazine-loaded silica gel was reacted in the same manner, with no fluorescence observed on the silica gel.

Anchoring the tricycle as dienophile on the solid phase:

Amino-functionalized silica gel (1g.) Was suspended in 10 ml of ethanol, 2 mmol (400mg) of the tricyclic anhydride was added and shaken for 3 hrs. At 80 ⁰ C. It was filtered off with suction through a frit, washed several times with ethanol and finally with ether and dried. The determination of the C / N ratio by elemental analysis revealed a coverage of 70% of the available amino groups.

100 mg of the functionalized silica gel was suspended in tetrahydrofuran and titrated with a 0.5 molar solution of the tetrazine ester in THF. The decolorization of tetrazine occurs very quickly by the expiring Diels Alder reaction. From the consumption of tetrazine could turn to an originally about 70%. ige occupancy be closed.

Similarly, the following products were prepared, isolated, and characterized by Diels Alder's inverse electron demand reaction:

Example 6

Functionalization of triazines

Reaction of the S.e-bis-trifluoromethyl-triazine-S-carboxylic acid methyl ester with glycine methyl ester

Glycine methyl ester hydrochloride 3 mmol (375 mg) was suspended in 10 ml dioxane and then 3 mmol triethylamine (0.42 ml) was added. After 30 min., 2 mmol (550 mg) of the bis-trifluoromethyl-triazine-carboxylic acid ester in 10 ml of dioxane was then added and kept at 80 ^° C. for 5 hrs. It was concentrated and the residue was chromatographed on silica gel with hexane / ethyl acetate. 400 mg of the product were obtained. The mass confirms the structure, the compound apparently being present as a mono-hydrate.

The following compounds were prepared and characterized in an analogous manner:

Diels Alder reaction of triazines

The triazine tri-carboxylic acid methyl ester 1 mmol (255 mg) was dissolved in 2 ml THF and treated dropwise with a solution of 1 mmol (248 mg) of the tricyclic dicarboxylic acid dimethyl ester in 1 ml of THF. It is observed nitrogen evolution and a color lightening. After 2 hours at room temperature, the mixture is concentrated and the residue is chromatographed on silica gel with hexane / ethyl acetate 1: 1. 250 mg of the adduct are isolated corresponding to 50% yield. The mass spectrum confirms the structure, molecular peak at m / e 475.

Claims

claims

l. Method for linking two molecules by means of Diels-Alder reaction with inverse electron demand (DARinv), which comprises the following steps:

Implementation of a

(a) diazines, triazines or tetrazines having one or more electron-withdrawing substituents on the ring as the diene component, wherein the electron-withdrawing substituents are selected from:

- COOR

- C (O) NR ₂

- CX ₃ (X = halogen)

- halogen -CN

-SO ₂ -R or SO ₃ -R

With

(b) an isolated double bond or triple bond in a (hetero) carbocyclic ring or an isolated olefinic double bond or triple bond in a linear or branched hydrocarbon chain, which may optionally contain heteroatoms, as the dienophile component

2. The method of claim 1, wherein the diene and / or dienophile component one or more amino acid, peptide, saccharide, lipid or oligonucleotide or nucleic acid substituents, pharmaceutical agents, labels, dyes, complexes, Quantum dots, chelating / complexing agents, diagnostics or therapeutics.

3. The method of claim 1 or 2, wherein the diene and / or dienophile component contains one or more functionalities of different reactivity for carrying out the Diels-Alder reaction.

4. The method according to any one of claims 1 to 3 ,, wherein the diene is selected from: tetrazine monoamide, tetrazine diamine, tetrazine-3-trifluoromethyl-6-carboxamide, triazine tricarboxylic monoamide, triazine tricarboxylic diamide, triazine tricarboxylic acid triamide , or 1, 2-diazine-3,6-dicarboxylic acid amide.

5. The method of claim 4, wherein the diene is 3-carboxymethyl-5,6-bis-trifluoromethyl-triazine-1, 2,4. or 1, 2,4,5-tetrazine-dicarboxylic acid dimethyl ester.

6. The method of claim 1, 2 or 3, wherein the dienophile is selected from: exo- or endo-norbornendicarboxylic anhydride, cyclobutene dicarboxylic acid anhydride, cyclohexene dicarboxylic anhydride, dehydroproline, allyl proline, allyl malonate, allyl galactose, allyl silsesquioxane, or dicyclopentadienone.

7. The method of claim 4, wherein the tetrazine compounds are recovered from the corresponding dihydro compounds by subsequent oxidation.

8. The method according to any one of claims 1-7, wherein the diene and dienophile component are linked together via a linker.

9. The method according to any one of claims 1-8, wherein the Diels-Alder reaction with inverse electron demand in aqueous or alcoholic solution at 20-100 ⁰ C is performed.