EP1313750A1

EP1313750A1 - Method for producing water-soluble saccharide conjugates and saccharide mimetics by diels-alder reaction

Info

Publication number: EP1313750A1
Application number: EP01962674A
Authority: EP
Inventors: Manfred Weissler; Hans-Christian Kliem; Bernd Sauerbrei; Birgit Schmauser
Original assignee: Deutsches Krebsforschungszentrum DKFZ
Current assignee: Deutsches Krebsforschungszentrum DKFZ
Priority date: 2000-08-22
Filing date: 2001-08-22
Publication date: 2003-05-28
Also published as: US20040059101A1; US6958395B2; DE10041221A1; WO2002016378A1

Abstract

The invention relates to a method for producing saccharide compounds in a very simple manner. The inventive method comprises the following steps: (a) attaching at least one saccharide to a cyclic or acyclic diene, (b) reacting the saccharide-containing diene obtained in step (a) or a commercially available saccharide-containing diene with a dienophile by Diels-Alder reaction.

Description

METHOD FOR THE PRODUCTION OF WATER-SOLUBLE SACCHARIDE CONJUGATES AND SACCHARIDMIMETICS BY DIELS-ALDER REACTION

The present invention relates - ^'a method for producing substance libraries on the basis of naturally occurring saccharides and saccharide mimetics.

The interactions between proteins and natural or synthetic oligosaccharides are known to be highly specific. These interactions essentially involve hydrogen bonds, mutually present donors and acceptors, and hydrophobic interactions. The binding constants achieved are several orders of magnitude lower than those measured for antigen-antibody interactions. In order to be able to optimize the interactions mentioned on the basis of saccharides and to allow all types of interactions, a large number of substituted saccharides and saccharide mimetics must be tried out and synthesized. The basic idea is the development of effective therapeutics based on saccharides. A major problem in the synthesis of saccharide (e) -containing compounds, however, is the complicated, multistage process procedure, which additionally requires a pronounced protective group chemistry. This applies in particular if several saccharides are to be synthesized in or on a compound or if saccharide libraries are to be built up. There is therefore an urgent need for one Process with which complex saccharide (e) -containing compounds can be synthesized in a simple manner. In particular, the process is intended to be able to use the process to build saccharide clusters that are suitable as therapeutic agents because they interact with receptors in or on cells or organs. Furthermore, saccharide libraries and saccharide-containing compound libraries are to be built up with it. The method should also be suitable for linking saccharides with peptides, nucleic acids and / or lipids, it also being possible to link the polymers to one another.

The object of the present invention is to provide a method with which complex saccharide compounds or libraries, as mentioned above, can also be built up.

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

The inventors developed a process for the synthesis of saccharide compounds, which comprises the following steps:

(a) attachment of at least one saccharide to a cyclic or acylic diene,

(b) Reacting the saccharide-containing diene formed in step (a) or a commercially available saccharide-containing diene with a dienophile by means of the Diels-Alder reaction.

The Diels-Alder reaction is a reaction in which a diene reacts with an olefin, passing through a transition state involving 6π electrons. This transition state gives the Diels-Alder reactions, in Compared to the usual organic reactions ^ - relatively low activation energies, so that these reactions can take place at room temperature or slightly elevated temperatures. Diels-Alder reactions can be accelerated by high pressure. A number of catalysts have become known in recent years which are capable of effectively catalyzing Diels-Alder reactions under mild conditions (K. Pindur et al., Chem Rev. 1993, 93, pp. 741-761; Kündig et al., Angew: Chem. 1999, 111, pp. 1298-1301). Since Diels-Alder reactions are fundamentally reversible, this type of reaction is also suitable for dynamic combinatorial chemistry. The formation of exo and endo isomers can be controlled via the temperature and also the catalyst. With regard to the yields, the Diels-Alder reaction offers great advantages, since it proceeds without further by-products and with an almost quantitative yield. The Diels-Alder reaction is therefore used by the inventors to build complex saccharide compounds and libraries from saccharide-containing compounds or saccharide libraries. With skillful substitution of the two starting compounds (diene and dienophile) with functional groups or residues, molecules are accessible that can contain three or even four different residues.

According to the invention, furan, fulvene, furfural, cyclopentadiene, cyclohexadiene, pyrrole, 1,3-ozazole, 1,2-oxazole, pyrazole, thiophene and 1,3-dienes as acyclic compounds are preferably suitable as cyclic diene component in step (a) (eg trans-trans hexadiene-2, 4-1, 6-diol), which can be substituted one or more times with functional groups. The functional groups can be selected from, for example, alkyl chains (C ₂ -C ₂₀ , preferably methyl, ethyl, isopropyl, tert-butyl, etc.), OH, SH, halogens, aryl , Carboxyl, carbonyl, nitro, carboxyamido, .. keto, sulfoxide, sulfone, sulfonic acid, phosphoric acid or amino groups, which are bonded directly or via alkyl radicals. However, the diene component can also carry amino acid, peptide, lipid or oligonucleotide or nucleic acid substituents. All types of active pharmaceutical ingredients, markings, dyes or complexes (for example carborane, ferrocene) can also be coupled to the diene component.

Preferred diene components are: Bishydroxyalkylfurans, such as 2,3-bishydroxymethylfuran, 3,4-bishydroxymethylfuran, 2,5-bishydroxymethylfuran, hydroxymethylfurfural, α-GMF (α-glycosylmethyl-furfural; or with, for example, NaBH reduced aldehyde function). Of course, their homologs with ethyl or propyl groups can also be used instead of methyl. The diene components are commercially available, for example from Aldrich (furfural = Aldrich # 27,886-6; hydroxymethylfurfural = Aldrich # 4,080-7; 3-hydroxymethyl-furan = Aldrich # 19,639- 8) or from Südzucker AG (α-GMF) , In general, 2,5-disubstituted furans in Diels-Alder reactions have a lower reactivity than 3,4-disubstituted ones. This graded reactivity can be used synthetically. It is advantageous here that the reactivity of hydroxymethyl groups differs in the individual positions of the furan, so that sequential substitution is possible. Different saccharides can thus be introduced very easily into the furan (see FIG. 1). Cyclopentadiene and its substituted S-derivatives are also very suitable as dienes in the Diels-Alder reaction. Aldehyde derivatives of cyclopentadiene have been known for a long time (Chem. Ber. 1964, 97, p. 2066) and can be converted step by step into the hydroxymethyl compounds which are reacted with saccharides according to the Imid.at method and then as Services are available. Carboxylic acid derivatives of cyclopentadiene are also known, such as esters, a ide or nitriles (J. Chem. Soc. 1966, p. 1641). Cyclopentadiene-tetra-carboxylic acid esters can also be prepared from the corresponding cyclopentane precursors by dehydrogenation. These precursors are easily accessible from the Diels-Alder adducts of cyclopentadiene and maleic anhydride. Cyclopentadienes can also be prepared by cyclization reactions of 1,3-diacarbonyl compounds (J. Chem. Soc. 1952, p. 1127). Naturally occurring iridoid glucosides such as Catapol and Aucubin (Liebigs Ann. Chem. 1990, p. 715) can be regarded as precursors for ssacharide-substituted cyclopentadienes (THL 1997, 38, p. 6433). In addition, the glucosides mentioned can also be used as dienophiles in the generation of substance libraries to search for active substances.

In step (a) of the process according to the invention, a saccharide is linked to a diene by reacting the diene with an imidate component which is substituted with a saccharide. The reaction conditions for this reaction have been described, for example, by RR Schmidt, in: Glycosciences, ed. HJ Gabius, S. Gabius, pp. 31-53. The saccharide can also be linked to the diene component by means of other reactions (for example by means of the known Königs-Knorr reaction). The term “saccharide” encompasses all types of saccharides, in particular mono-, di-, oligo- or polysaccharides (eg mono-, di-, ultimate, and dendritic saccharides) in all stereoisomeric and enantiomeric forms. These can be pentoses or hexoses, which are in the L or D form. Particularly suitable monosaccharides are glucose, very particularly α- and β-D-glucose, fructose, galactose, mannose, arabinose, Xylose, fucose, rhamnose, digitoxose and derivatives thereof are preferred. Particularly suitable disaccharides are sucrose, maltose, lactose or gentobiose, either 1,4- or 1,6-linked, and derivatives thereof. Saccharides here also include sugar alcohols, polyols, inositol and derivatives thereof, very particularly cis-inositol, epi-inositol, allo-inositol, myo-inositol, muco-inositol, chiro-inositol, neo-inositol, scyllo-inositol, pinpollitol, Streptamine, quercitol, quinic acid, shikimic acid, conduritol A or B, validatol and quebrachitol, e.g. from galactinols, both from plant sources, such as sugar beets (obtainable from them: hydroxymethyl furfural; FW Lichtenthaler, Mod. Synth. Meth. 1993, 6, p. 273-376), as well as from milk products, or compounds obtained by enzymatic separation of enantiomers. Saccharides which can be used according to the invention are also glycoconjugates. These can be conjugates of eg saccharides with peptides, lipids, acids (-> esters), alkyl residues (>

Ether), heterocycles or other carbohydrates. An example of glycoconjugates is Z1-Z10, a mixture of 10 glycoconjugates. The compounds Z1-Z10 are naturally occurring glycopeptides, glycoproteins and lipopolysaccharides. Derivatives of the saccharides mentioned are, for example, saccharides protected with protective groups (for example benzoyl, silyl, dirnethoxytrityl) and / or with functional groups such as amino, nitro, carboxy, carboxy ido, keto, sulfoxide, sulfone , Sulfonic acid, phosphoric acid, phosphonic acid, mono / di / trilalkylamide groups or halide groups, modified saccharides. The above saccharides can occur naturally or can be made synthetically. The imidate preferably has only one saccharide, but a number of 2, 3, 4, 5 and 6 saccharide components is also conceivable if the imidate is selected accordingly. The saccharides can be the same or different from one another. Preferred saccharide-substituted imidate components are tri-O-benzoyl-fucoseimidate or tetra-O-benzoyl-galactoseimidate. The saccharide-substituted imidate components are, for example, according to H. Paulsen et al., 1992, Liebigs Ann. Chem. 747-750.

In order to obtain a diene completely modified with saccharides in step (a), the above-described reaction of diene with the saccharide-substituted imidate can take place several times in succession (see Example 1) or the imidate is added in excess, after which a reaction occurs - and multiple saccharide-modified diene should be separated from one another in order to arrive at uniform products in the following step (b). The compounds which can be prepared in step (a) from the dienes and saccharide-substituted imidates mentioned above as preferred are, for example, 3,4-bis (fucosyloxymethyl) furan, 3,4-bis (galactosyloxymethyl) furan, 3- fucosyloxymethyl-4-galactosyloxymethylfuran, 2, 5-bis-galactosyloxymethyl-furan, 2,5-bis-fucosyloxymethyl-furan, 2-fucosyloxymethyl-5-galactosyloxymethylfuran and alcohols derived from furan-2, 5-di-ß- propionic acid or - derived from furan-2,5-di-acetic acid. If the compounds contain protective groups (preferably: benzoyl protective group) they can e.g. be split off with sodium methoxide solution according to standard procedures.

Suitable dienophiles in step (b) are maleic acid (anhydride) derivatives, fumaric acid (anhydride) derivatives, maleimide derivatives (preferably: N-substituted. Maleimide), acrylic acid derivatives, acetylene derivatives, butynedicarboxylic acid or derivatives thereof or enol ether. Below ^j derivatives are those that, after substitution de: Compounds mentioned alkyl chains (C ₂ - C ₂ o), _-_- OH, SH, halogens, aryl, carboxy, carbonyl, nitro, carboxyamido, keto, sulfoxide, sulfonic, sulfonic, phosphoric acid -, Amino, phosphonic acid or mono / di / trialkyla id groups. Furthermore, the dienophile component can be substituted with saccharides as defined above. However, the dienophile component can also carry amino acid, peptide, lipid or oligonucleotide or nucleic acid substituents. All types of active pharmaceutical ingredients, labels, dyes or complexes can also be coupled to the dienophile component. Preferred dienophiles are tris (2-Maleini idoethyDamin (TMEA), ^• N-phenyl, N-ethyl, Maleini idolysin or Conduritol.

The diene component and the dienophile component can individually or both also contain aromatic or heterocyclic radicals. These can be selected from: phenyl, thienyl, thiophenyl, furyl, furanyl, pyrany.l, pyrrolyl, irαidazolyl, pyrazolyl, pyridyl, pyrazinyl, pyridinyl, pyrazinyl, pyridazinyl , Thiazolyl, oxazolyl, indolyl, furazannyl, pyrrolinyl, imidazolinyl, pyrazolinyl, thiazolinyl, triazolyl, tetrazolyl group, and the positional isomers of the hetero atom or atoms which these groups can comprise, a radical consisting of carbocyclic fused rings, for example the naphthyl group or the phenanthrenyl group, a radical ^" consisting of fused heterocyclic rings, for example benzofuranyl, benzothienyl, benzimidazolyl, benzot iazolyl, naphtho [2,3-b] thienyl, thianthrenyl, isobenzofuranyl, phenyl, xenyl, xenyl, phenyl Indolizinyl, isoindolyl, 3H-indolyl, indolyl, indazolyl, purinyl, quinolizinyl, isoquinolyl, quinolyl, phthalzinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, quinolinyl, pteridinyl, Carbazolyl, ß-carbolinyl, cinnolinyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, indolinyl, isoindolinyl, imidazopyridyl, imidazopyridmidinyl or also the condensed polycyclic systems consisting of heterocyclic monocycles, as defined for example above, such as thionaphthenyl, furo .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, pyridinyl such as pyridmid-2-yl, thiazolyl such as thiazol-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 purin-7-yl, or quinolyl, such as 4-quinolyl.

The Diels-Alder reaction is a standard method in organic chemistry and the reaction conditions are well known to a person skilled in the art or can be looked up in relevant textbooks. In the context dej _j present invention, the Diels-Alder reaction is preferably performed in any solvents between 20 ° C and 100 ° C. Preferred solvents are water or alcohols, such as methanol or ethanol. The formation of exo and endo compounds, as observed in this type of Diels-Alder reaction, can be controlled by the experimental conditions and increases the number of compounds to be obtained by a factor of two. This variation in experimental conditions is within the skill of one of ordinary skill in the art.

All Diels-Alder reactions are equilibrium reactions in which, depending on the temperature, the components are always present and can therefore react with other partners out of equilibrium. The System_ described above is therefore also suitable for the controlled release of the dienophile component, while at the same time anchoring the diene component to a polymer support or vice versa. If the dienophile carries, for example, a peptide or a therapeutic agent, a system is available that can be used for the controlled and controllable release of drugs from a solid phase. In extension of the reactions described above, the method according to the invention provides a possibility of linking peptides with saccharides, peptides with nucleic acids, saccharides with nucleic acids and the respective component with itself, provided that one component has a diene and the other one Dienophile would be connected. The resulting Diels-Alder adducts can themselves be modified, for example by ring opening of the anhydride ring or by hydrogenation of the double bond or by addition reactions to this double bond.

The oxabicycloheptane ring system can be opened under acidic conditions to form a cyclohexane ring system. A splitting of the Diels-Alder adduct into the components is no longer possible and a new structure type arises, an inositol derivative that certainly has other pharmacological properties, since it no longer has the rigidity of the bicyclus.

In order to build up larger clusters or libraries, steps a) and / or b) of the method according to the invention have to be carried out several times in succession.

In the context of the present invention, step a) can of course be omitted if a saccharide-substituted diene is already used. Some important preferred aspects of the present invention are to be emphasized below, which should not be interpreted as a limitation of the broad process concept.

In the Diels-Alder reaction (Scheme 1), a diene 1 is reacted with a dienophile 2.

Scheme 1

The diene 1 (here: furan with the substituents R 1 to R ₄ ) is reacted in an aqueous solution directly with the dienophile 2 (here: maleinid with the substituent R 5). This creates a product mix of endo- and exo-Diels-Alder product 3. The composition of the product mixture can be controlled. In addition, it is possible to separate the endo and exo compounds from one another by chromatography. (Meaning of R 1 to R ₄ and R ₅ : one or more substituents and in all conceivable combinations can be incorporated. For the definition of the radicals: see above)

In the following, some options for the presentation of dienes (type 1, scheme 1) and their saccharide conjugation, then the dienophile (type 2) and finally the Diels-Alder reaction are described.

Presentation of the dienes (preferably Fürande ivate) According to the concept according to the invention, saccharide-modified furan derivatives are produced. Suitable compounds are therefore furan derivatives which contain a hydroxyl group (directly or by spacer from the ring) which allows covalent, glycosidic linkage with a saccharide molecule (for example Ri = CH ₂ -OH). The synthesis possibilities for this are numerous and mostly known from the literature.

The following reaction schemes (FIGS. 5 (a) - (f)) are representative of the easy accessibility of the required furan derivatives and the performance of the synthetic routes.

In addition to the synthetic routes presented here, there are other variants already known from the literature. The services described here are listed as examples. In addition, there is another number of dienes that were used by the inventors in Diels-Alder reactions after conjugation with saccharides. These are tabulated below.

Synthesis of saccharide-containing dienes

The covalent linkage of the furan derivatives with saccharides is preferably carried out using the imidate method (Scheme 3, see FIG. 6). In the first step of reaction (A), simple galactosidation is achieved, although one equivalent of furan derivative is reacted with one equivalent of galactose idate. The mono-galactosidated compound is purified by chromatography. In a second step of the reaction (B), 1 equivalent of a further glycosylimidate is then added. In a double reaction with gal-idate, a connection with two galactose units is obtained after the protective groups have been split off. You sit down if, on the other hand, the fucose imidate is present in step B, then a mixed glycosidated furan derivative is obtained after the protective groups have been split off. The entire reaction can also be carried out by the Königs-Knorr reaction or other methods known in the literature.

Combinatorics with furans αcharides

Reaction of 2, 3,4-trishydroxyfuran (or other "polyhydroxylated" furan derivatives) with three times the molar amount of the mixture of 10 different imidates in the same proportions. Deprotection and Diels-Alder reaction with a labeled maleimide in excess. Isolation of the labeled Library and test for fixed cells: The optimally effective structures can be determined by varying the saccharide imidates, adding and omitting them and their concentrations.

If a furan which is substituted twice with sugar residues is used as the diene and an N-substituted maleimide is used as the dienophile which carries a saccharide over a spacer of any length, trisaccharides are formed in the Diels-Alder reaction. According to this scheme, when using e.g. ten different saccharide imidates generate a library with almost 1000 different trisaccharides. Since the protective groups of both the diene and the dienophile can be removed beforehand, the Diels-Alder reaction can be carried out in water, which has a favorable effect on the reaction rate and yield (see FIG. 2).

The method according to the invention can be used to produce substances / libraries which are suitable for inhibiting the interaction of lectins with proteins. Such Substances can be used in cancer therapy to avoid metastasis or as an anti-inflammatory. When using spacers, which can be inserted both in the diene and in the dienophile, or of rare or with saccharides provided with unusual functional groups, the diversity of the libraries increases. The multiantenarity, which can be important for the interaction of saccharides with lectins, can also be produced with the aid of the method according to the invention. Such molecules are easily accessible through the double or triple Diels-Alder reaction with a corresponding dienophile (see FIG. 3).

The method according to the invention can also be used to generate compounds / libraries which, in addition to the saccharides, also contain other pharmacophoric groups in order to make new lead structures for therapeutics accessible in general. Such groups can be heterocycles, aromatics or peptide structures, which are contained either in the diene or dienophile. These diverse combination options increase the diversity of the libraries considerably. The often low conformative mobility of the resulting Diels-Alder products can also be beneficial in this context, since such structures attach better to receptors. Due to the presence of the saccharide residues in the products, they are correspondingly water-soluble.

The Diels-Alder reaction of dienes with dienophiles (eg substituted maleimides) can also be used to connect proteins with saccharides and saccharides with nucleic acids. Here it is useful to have a lysine residue in the protein which allows the addition of the maleinimide residue via the free amino group. It it is also conceivable that a lysine residue is added to the inoterminal end. With these neo-glycoproteins that are accessible in this way, the influence of different ones can be

Examine (oligo) saccharides for the effectiveness of the proteins or their pharmacokinetics. In the same way, labeling of saccharides with biotin can be carried out without any problems, which would be important for a possible diagnosis based on lectin-saccharide interactions

(Fig. 2).

The Diels-Alder reaction of dienes (e.g. substituted furans) with dienophiles (e.g. substituted maleinides) also leads to the doping of surfaces with saccharides, lipids, peptides or (oligo) nucleotides. Both the dienophile and the diene can be immobilized on the surface. An application in chip technology is therefore conceivable. The increase in the loading density on the surface can also be achieved by using structures as in FIG. 2.

Since the Diels-Alder reaction with these components can be carried out very well in water and generally requires no catalysis, all components can be reacted with one another without protective groups. This eliminates the final splitting off of protective groups, a process that often creates great difficulties.

Since all Diels-Alder reactions are reversible and the equilibria are established even at room temperature, the reversal of adduct formation can also be used for the controlled release of the components of the Diels-Alder reaction that are loaded with active ingredients. As already explained in the introduction, the natural binding constants between proteins and (oligo) saccharides are not very high. Apparently there was no need for further optimization of these binding constants in the course of evolution. Based on fundamental considerations, it should be possible, by authorizing all types of interactions, including ionic interactions, on the basis of saccharides and saccharide mimetics to represent effective agonists and antagonists of receptors in analogy to peptides. The use of substituted saccharides, whereby all types of functional groups can be used, even those that have not previously been found in saccharides, is certainly a prerequisite for this optimization.

With the help of combinatorial chemistry, it is possible to identify very effective agonists and antagonists based on peptides, lipids or nucleic acids. In order to develop therapeutic agents, especially for oral administration, substances that are available orally are required. Sufficient water solubility is a necessary requirement for this. A possible approach to improve water solubility and thus oral availability is the covalent linkage of active ingredients with saccharides to form conjugates. With the approach according to the invention it is possible, from the outset, to take into account the problem of sufficient water solubility by using suitable building blocks using a combinatorial approach. This also has the 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. The inventors have also thought of moving the method towards the solid phase transferred, whereby the binding to the solid phase takes place via the saccharide part.

The invention is further described with reference to the following figures.

Fig. 1: Introduction of 2 saccharide residues in a furan system. Fig. 2: Reaction of a doubly substituted furan with an N-substituted maleimide. Fig. 3: Multiple Diels-Alder reaction

Fig. 4: Exemplary building blocks for the Diels-Alder reaction

Fig. 5 (a) - (f): Representation of saccharides conjugate for other ivaten Fig. 6: Glycosidation of 2,5-bis-hydroxymethylfuran

The invention is further illustrated by the following examples.

Example 1: Synthesis of glycosidated hydroxymethyl furans

4 5 20 mmol of 3,4-bis-hydroxymethylfuran 1 and 21 mmol of tri-O-benzoyl-fucose-imidate 2 are dissolved in 300 ml of dichloromethane. The solution is cooled to -40DC and a few drops of triflate are added. The reaction solution is then stirred in an ice bath for 1 hour, the organic phase is shaken with dilute bicarbonate solution and then with water, dried over sodium sulfate and evaporated down i. concentrated. The reaction product 3 is obtained after column chromatography on silica gel with petroleum ether / ethyl acetate (2/1) in a yield of about 60%.

6 mmol of monofucosylated furan 3 is reacted with 6.3 mmol of imidate 4 as described above. The purification is carried out by column chromatography on silica gel with petroleum ether / ethyl acetate (2/1). The product 5 is obtained in a yield of approximately 55%.

For deprotection (removal of the benzoyl groups), the product 5 was reacted with sodium methoxide solution. Yields: approx. 60%.

In order to produce the corresponding 2,5-products, instead of 3,4-bis-hydroxymethylfuran the 2,5-bis-hydroxymethylfuran is used in the first step and the procedure is analogous to that described above.

Example 2: Glycosidation of maleimide or derivatives

5 mmol of suitably derivatized maleimide 1 (maleimide + spacer + OH) are dissolved with 4.99 mmol of galactosylidate 2 in 100 ml of dichloromethane and 10 drops of triflate are added at -40DC. It is dormant at ODC. ^" After the reaction is complete, the reaction solution is shaken out with dilute bicarbonate solution and water. The solvent is dried over sodium sulfate and evaporated down in vacuo. The residue is chromatographed on silica gel with petroleum ether / ethyl acetate (2/1). Yield of product 3: 54% ,

To split off the benzoyl groups, the reaction is carried out with sodium methoxide or a mixture of methanol / water / triethylamine.

Example 3: Diels-Alder reaction

a) Biotinylation

3

50umol glycosidated furan 1 (see Example 1) and 50 μmol biotin derivative 2 (from Pierce Kat.-No. 2 1900 ZZ) are dissolved in 1 ml water. The solution is stirred at room temperature. The progress of the reaction is monitored by HPLC. When the reaction has ended, the reaction solution is freeze-dried. Product 3 (endo / exo mixture) is isolated by means of preparative HPLC. Yield 50-60%.

The following reactions were also carried out under the same conditions, the Df-en component in each case being prepared according to Example 1 and the dienophile component according to K. Wakisaka et al., J. Med. Chem. 1997, 40, p. 2643- 2652 was manufactured.

Introduction of a lysine residue

c) reaction of 2, 5 and 3, 4-glycosyl-hydroxymethylated furans

Example 4: Representation of Glycol Clusters Using Diels-

Alder reaction

A solution of 50 μmol tris (-2-maleimidoethyl) amine 1 (TMEA) and 190 μmol bis-galactosylfuran 2 in 1 ml water is stirred for 50 hours at room temperature. The product mixture 3 (combination of endo and exo products) is obtained in approximately 30% yield by means of HPLC.

Example 5: Crossed Diels-Alder reaction

This is the simultaneous reaction of

2,5- and 3,4-furans with a dienophile (-> combinatorics)

50 μmol of a mixture of the glycosidated 3,4- and 2,5-furans 1, 2 (see Example 1) and 50 μmol N-ethyl-maleimide 3 are dissolved in 1 ml water. The solution is stirred at room temperature. The progress of the reaction is monitored by HPLC. When the reaction has ended, the reaction solution is freeze-dried. Products 4 and 5 are isolated using preparative HPLC. Yield 50-60%. Example 6: Diels-Alder reaction on the solid phase

1.0 g of tris ₍ 2-aminoethyl) amine polymer (Aldrich # 47,210-7 ⁾ is left to swell in 20 ml of water for 1 hour. It is then slurried in 20 ml of saturated NaHC0 ₃ solution and mixed with 1080 mg of methoxycarbonylmaleimide at ODC and then stirred at room temperature for 16 hours. It is adjusted to pH 3-4 with H ₂ S0 ₄ , extracted with ethyl acetate and dichloromethane. The aqueous phase is dried. 108 mg of glycosylated furan derivative (see Example 1 ^{) are added} to 100 mg of the product Given 3 ml of water. It is stirred at room temperature. After 16 hours, the water is removed and the product is purified by HPLC.

Example 7 Preparation of trisaccharides

5 mmol of suitably derivatized maleimide 1 (maleimide + spacer + saccharide) are dissolved in 4.9 ml of diglycosylated furan 2 in 100 ml of dichloromethane and mixed with 10 drops of triflate at -40DC. It is dormant at ODC. After the reaction is complete, the reaction solution is shaken out with dilute bicarbonate solution and water. The solvent is dried over sodium sulfate and evaporated down i. concentrated. The residue is chromatographed on silica gel using petroleum ether / ethyl acetate (2/1). Yield of product 3: 54%. Example 8: Glycosidation of furan derivatives with hydroxy functions

(a) Method A: Monoglycosidation:

2, 5-Bishydroxymethylfuran (2 mmol, 265 mg) and 2,3,4-Tri-O-Benzoylfucoseimidat (2 mmol, 1.28g) are mixed in 50 ml dichloromethane at -40 ° C with 5 drops of triflate and then at 0 ° C stirred for one hour. The reaction solution is stopped by adding dilute aqueous bicarbonate solution. The organic phase is washed with 20 ml of water and then concentrated in vacuo after drying over sodium sulfate. The residue is chromatographed on silica gel (petroleum ether / ethyl acetate: 2/1). Yield: 700mg (60%); ESI-MS: [M + H ⁺ ]: 58611 This compound can now be reacted again in a second reaction with an equivalent of any saccharide imidate.

(b) Method B: multiple glycosidation

2, 5-Bishydroxymethylfuran (2 mmol, 265 mg) and 2,3,4, 6-tetra-O-benzoylgalctoseimidate (4 mmol, 1.48 g) in 50 ml dichloromethane at -40 ° C with 5 drops of triflate and then stirred at 0 ° C for two hours. The reaction solution is stopped by adding dilute aqueous bicarbonate solution. The organic phase is washed with 20 ml of water and then concentrated in vacuo after drying over sodium sulfate. The residue is chromatographed on silica gel (petroleum ether / ethyl acetate: 2/1). Yield: 1.4 g (55%); ESI-MS: [M + H ⁺ ]: 1284.3

To remove the protective groups, the compounds are reacted in methanolic solution with sodium methoxide. The saponification takes place quantitatively.

If the furan derivative contains several glycosidatable hydroxyl functions, step A is used for step-by-step glycosidation. In contrast, if only one carbohydrate species is to be conjugated to all available hydroxyl functions, method B is used.

The following furan derivatives were produced in this way:

3-Hydroxymethylfuranglycosid

Ri

glucose 2, 3-Bishydroxymethylfuranglycoside

2, 4-Bishydroxymethylfuranglycoside

3, 4-Bishydroxymethylfuranglycoside

2, 5-Bishydroxym.ethylfuranglycoside

2, 3, 4-trishydroxymethylfuranglycosides

2,3,5-trishydroxymethylfuranglycoside

Other:

Example 9: Representation of dienophiles

Numerous maleimide-type dienophiles have been synthesized. However, some are also commercially available.

General Maleimide Syntheses:

Synthesis of N- (ethoxycarbonyl) -maleini ids: 0. Keller, J. Rudinger, Hei. Chim. Acta 1975, 58, 531541.

J.T. Elliott, G.D. Prestwich, Bioconjugate Chemistry 2000,

11, 832-841.

S. Kaigutkar, B.C. Crews, L.J. Marnett, J. Med. Chem. 1996,

39, 1692-1703.

M. Dörr, R. Zentel, R. Dietrich, K. Meerholz, C. Bräuchle, J.

Wichern, S. Zippel, P. Boldt, Macromolecules 1998, 31, 1454-

1465th - N-dodecyl-maleimide

300 mg (1.62 mmol) dodecylamine are dissolved in 10 ml CHC13 and cooled to 0 ° C. N-methoxycarbonylmaleimide (NMM, 507 mg, 3.24 mmol) and tetrabutylammonium hydrogen sulfate (503 mg, 1.48 mmol) are added. Triethylamine (0.3 ml, 2.16 mmol) is slowly added and the mixture is stirred at 0 ° C. for a further 10 min. The ice bath is removed and 20 ml of saturated NaHC03 solution are added. After 3 h at room temperature, the reaction mixture is extracted with ethyl acetate (3 x 50 ml). The combined organic extracts are washed once with sat. Washed NaCl solution. The organic phase is dried with magnesium sulfate and the solvent i. Vak. away. The N-dodecylamine is then purified by chromatography on silica gel (petroleum ether / EtOAc 10: 1) as a colorless solid.

Yield: 365 mg (85%)

1H NMR (250 MHz, CDC13): δ = 0.88 (t, J = 6.6 Hz, 3H), 1.25 (br. S., 16H), 1.54-1.60 (, 4 H), 3.50 (t, J = 7.3 Hz , 2H), 6.67 (s, 2H).

- N-stearyl malinimide

500 mg (1.85 mmol) of stearylamine are dissolved in 10 ml of CHC13 and cooled to 0 ° C. N-methoxycarbonylamine imide (NMM, 580 mg, 3.71 mmol) and tetrabutylammonium hydrogen sulfate (574 mg, 1.69 mmol) are added. Triethylamine (0.34 ml, 2.46 mmol) is slowly added and a further 10 are left Stir min at 0 ° C. The ice bath is removed and 20 ml of saturated NaHC03 solution are added. After 3 h at room temperature, the reaction mixture is extracted with ethyl acetate (3 x 50 ml). The combined organic extracts are washed once with sat. Washed NaCl solution. The organic phase is dried with magnesium sulfate and the solvent i. Vak. away. The N-stearylamine is then purified by chromatography on silica gel (petroleum ether / EtOAc 10: 1) as a colorless solid.

Yield: 515 g (80%)

1H NMR (250 MHz, CDC13): δ = 0.88 (t, J = 6.6 Hz, 3H), 1.25

(br. s., 28H), 1.50-1.60 (m, 4H), 3.50 (t, J = 7.3 Hz, 2H),

6.67 (s, 2H).

ESI-MS: [M + H +] 350.0

- N-cholesteryl-maleimide

Cholesterylamine (1.0 g, 2.58 mmol) is dissolved in CHC13 (50 ml) and mixed with maleic anhydride (253 mg, 2.58 mmol). The mixture is stirred overnight and the solvent is then removed i. Vac. The residue is taken up in acetic anhydride (30 ml) and sodium acetate (300 mg) is added. The reaction mixture is stirred at 100 ° C. for 4 h and then poured onto 100 ml of ice water. It is extracted with ethyl acetate (3 x 100 ml) and the organic phase with sat. Washed NaCl solution (1 x 100 ml). The organic phase is dried with magnesium sulfate and the solvent is evaporated down i. Vak. away. The residue is purified by chromatography on silica gel (petroleum ether / EtOac 7: 1) cleaned up. The N-cholesteryl-maleimide is obtained as a colorless oil.

ESI-MS: [M + H +] 468.3

Synthesis of cholesterylamine: R. Krieg, R. Wyrwa, U. Möllemann, H. Görls, B. Schönecker, Steroids, 1998, "63, 531-541; M. Hasan, N. Rashid, KM Khan, G. Snatzke, H. Duddeck, W. Voelter, Liebigs Ann. 1995, 889-896.

Bis-maleimide

300 mg (1.49 mmol) 1,2 diaminododecane are dissolved in 10 ml CHC13 and cooled to 0 ° C. N-ethoxycarbonyl-maleimide (NMM, 702 mg, 4.49 mmol) and tetrabutylammonium hydrogen sulfate (508 mg, 1.49 mmol) are added. Triethylamine (0.5 ml, 3.97 mmol) is slowly added and the mixture is stirred at 0 ° C. for a further 10 min. The ice bath is removed and 20 ml of saturated NaHC03 solution are added. After 3 h at room temperature, the reaction mixture is extracted with ethyl acetate (3 x 50 ml). The combined organic extracts are washed once with sat. Washed NaCl solution. The organic phase is dried with magnesium sulfate and the solvent i. Vak. away. The desired bis-maleimide is then purified by chromatography on silica gel (petroleum ether / EtOAc 10: 1) and obtained as a colorless solid.

Yield: 420 mg (78%) 1H NMR (250 MHz, CDC13): δ = 1.25 (br. S., 16H), 1. 54-1-59 (m, 4H), 3. 50 (t, J = 7.3 Hz, 4H), 6. 68 (s, 2H).

- Tris-maleimide

Tris- (2-aminoethyl) amine (100 mg, 0.68 mmol) is sat in 5 ml. NaHC03 solution / THF (1: 1) dissolved. At 0 ° C, N- (methoxycarbonyl) aleinimide (641 mg, 4.13 mmol) is added in portions. After every hour, 20 ml is sat. NaHC03 solution / THF (1: 1) added. After 4 h at 0 ° C, extract with ethyl acetate (3 x 100 ml) and wash the organic phase with sat. NaCl solution (1 x 100 ml). The organic phase is dried with magnesium sulfate and the solvent is evaporated down i. Vak. away. The desired compound is obtained as a light yellow solid.

Yield: 140 mg

1H NMR (250 MHz, CDC13): δ = 2.71 (t, J = 6.6 Hz, 6H), 3.52 (t, J = 6.6 Hz, 6H), 6.70 (s, 6H). ESI-MS: [M + H +] 386.9

Synthesis of tris-maleimide: J.C. Cheronis, E.T. Whalley, K.T. Nguyen, S.R. Eubanks, L.G. Allen, M.J. Duggan, S.D, Loy, K.A. Bonham, J.K. Blodgett, J. Med. Chem. 1992, 35, 1563-1572.

- tetra-maleimide

Polypropylenimine tetraa in Dendri er (DAB-Am-4, 90 mg, 0.28 mmol) is sat in 5 ml. NaHC03 solution / THF (1: 1) dissolved. At 0 ° C., N- (methoxycarbonyl) maleimide (356 mg, 2.27 mmol) is added in portions. After every hour, 20 ml is sat. NaHC03 / THF solution (1: 1) added. After 4 h at 0 ° C., the mixture is extracted with ethyl acetate (3 × 100 ml) and the organic phase is washed with sat. NaCl solution (1 x 100 ml). The organic phase is dried with magnesium sulfate and the solvent is evaporated down i. Vak. away. The desired compound is obtained as a light yellow solid.

Yield: 120 mg

1H NMR (250 MHz, CDC13): δ = 1.42-1.43 (m, 4H), 1.69 (dt, J =

7.2, J = 7.2 Hz, 8H), 2.37-2.40 (m, 4H), 2.40 (t, J = 7.0 Hz,

8H), 3.55 (t, J = 7.4 Hz, 8H), 6.68 (s, 8H).

13C NMR (63 MHz): δ = 24.76, 26.16, 36.31, 51.24, 53.68,

134.04, 170.76.

ESI-MS: [M + H +] 637.2

- N-maleimido-butanoic acid

δ-aminocarboxylic acid (500 mg, 4.84 mmol) is sat in 20 ml. NaHC03 solution / THF (1: 1) dissolved. At 0 ° C., N- (methoxycarbonyl) maleimide (910 mg, 5.81 mmol). After 10 min at 0 ° C., the mixture is allowed to warm to room temperature and 20 ml of sat. NaHC03 / THF solution (1: 1) too. After 3 h, the mixture is extracted with ethyl acetate (3 × 100 ml) and the organic phase is washed with sat. NaCl solution (1 x 100 ml). The organic phase is dried with magnesium sulfate and the solvent is evaporated down i. Vak. away. Chromatography on silica gel (EtOAc) provides the desired compound as a colorless solid.

Yield: 798 mg (90%)

- 5-Maleiιrtido-fluorescein

5-aminofluorescein (500 mg, 1.44 mmol) is dissolved in 50 ml acetic acid / chloroform (1: 1) (possibly suspension). At room temperature, maleic anhydride (141 mg, 1.43 mmol) is added and the mixture is stirred overnight. The solvent is then removed i. Vak. And takes up the residue in acetic anhydride (30 ml). Sodium acetate (200 mg) is added and the mixture is heated at 100 ° C. for 4 h. The reaction mixture is poured onto 100 ml of ice water and extracted with ethyl acetate (3 x 100 ml). The organic phase is once with sat. Washed NaCl solution and dried with magnesium sulfate. The solvent is i. Vac .. removed and the residue chromatographed on silica gel (EtOAc / hexane 3: 1).

Yield: 590 mg ESI-MS: [M + H +] 512.0 N, N-bis (2-chloroethγl) -4-maleimidoaniline

N, N-Bis (2-chloroethyl) -4-nitroaniline (1.8 g, 6.87 mmol) is dissolved in methanol (40 ml) and 10% Pd / C (200 mg) is added. The reaction mixture is stirred under an H2 atmosphere at normal pressure for 4 h at room temperature. The solution is then filtered and the solvent i. Vak. away. The residue is sat in 30 ml. NahC03 / THF solution (1: 1) was added and N-ethoxycarbonyl) -maleinimd (1.61 g, 10.0 mmol) was added in portions at 0 ° C. After 10 min. the ice bath is removed and the mixture is stirred at room temperature for 3 h. After every hour, 20 ml of sat. NaHC03 / THF solution (1: 1) too. The reaction mixture is extracted with ethyl acetate (3 x 100 ml) and the combined organic phases with sat. NaCl solution (1 x 100 ml) washed. The solvent is i. Vak. removed and the residue chromatographed on silica gel with hexane / EtOAc (1: 1). The desired compound is obtained as an orange solid.

Yield: 600 mg (28%)

1H NMR (250 MHz, CDC13): δ = 3.59-3.77 (m, 8H), 6.73 (d, J =

9.2 Hz, 2H), 6.80 (s, 2H), 7.17 (d, J = 9.2 Hz, 2H).

13C NMR (63 MHz): δ = 40.24, 53.49, 112.22, 120.93, 127.84,

134.09, 145.86, 169.91.

ESI-MS: was not possible Synthesis of N, N-bis (2-chloroethyl) -4-nitroaniline: BD Palmer, WR Wilson, SM Pullen, WA Denny, J. Med. Chem. 1990, 33, 112-121.

- Synthesis of a cis-platinum maleimide derivative

2,3-diaminopropionic acid methyl ester dihydrochloride

2,3-Diaminopropionic acid monohydochloride (2.0 g, 14.3 mmol) is suspended in dry methanol (80 ml) and cooled to 0 ° C. Dry HCl gas is passed into the solution for 30 min. The mixture is stirred at room temperature for 48 h and then the solvent is removed i. Vak. The desired compound is obtained as a colorless solid and used directly for the next reaction.

Synthesis of the 2,3-diaminopropionic acid methyl ester dihydrochloride: P. Jones, G. B. Villeneuve, C. Fei, J. DeMarte, A. J. Haggarty, K. T. Nwe, D. A. Martin, A.-M. Lebuis, J.M. Finkelstein, B.J. Gour-Salin, T.H. Chan, B.R. Leyland-Jones, J. Med. Chem. 1998, 41, 3062-3077.

- N, N-Di-tert-butoxycarbonyl-2,3-diaminopropionic acid methyl ester

2, 3-Diaminopropionic acid methyl ester dihydrochloride (1.2 q, 6.31 mmol) is dissolved in 1, 4-dixane / water (40 ml, 1: 1) and triethylamine (4.4 ml, 31.61 mmol) was added. Then di-tert-butyl dicarbonate (3.0 g, 13.78 mmol) is added and the mixture is stirred overnight. Ethyl acetate (100 ml) and 1N HCl (100 ml) are added to the reaction mixture. The organic phase is saturated. Washed NaCl solution (2 x 100) and dried with magnesium sulfate. The solvent is removed and the residue is purified by chromatography on silica gel (EtOAc / hexane 1: 3). The desired compound is obtained as a colorless solid.

Yield: 1.83 g (91%)

1H NMR (250 MHz, D6-DMSO): δ = 1.36 (br. S, 18H, Boc-H),

3.20-3.25 (m, 2H), 3.59 (s, 3H, OCH3), 4.03 (t, J = 7.0 Hz,

CH), 6.78 (br. T, NH), 6.98 (d, J = 7.6 Hz, NH).

13C NMR (63 MHz, D6-DMSO): δ = 28.10, 40.97, 52.39, 61.24,

77.52, 77.58, 155.15, 155.84.

ESI-MS: [M + Na +] 341.1; [M + H +] 319.1

Method: E. B. van der Toi, H. J. van Ramesdonk, J. W. Verhoeven, F. J. Steemers, E. G. Kerver, W. Verboom, D. N. Reinhoudt, Chem. Eur. J. 1998, 4, 2315-2323.

- N, N-di-tert-butoxycarbonyl-2,3-diaminopropanol

N, N-Di-tert-butoxycarbonyl-2,3-diaminopropionic acid methyl ester (1.5 g, 4.70 mmol) is dissolved in dry THF (30 ml). At 0 ° C., an excess of lithium aluminum hydride (150 mg) is added in portions and the mixture is stirred at room temperature for 2 h. The reaction mixture is cooled to 0 ° C. and hydrolyzed by adding water. It is extracted with ethyl acetate (3 x 100 ml) and washed with sat. NaCl solution (1 x 100 ml). The organic phase is dried with magnesium sulfate and the residue is chromatographed on silica gel with hexane / EtOAc (1: 1). The alcohol is obtained as a colorless solid.

Yield: 966 mg (70%)

1H NMR (250 MHz, D6-DMS0): δ = 1.37 (br. S, 18H, Boc-H) _#

2.94-3.06 (, 2H), 3.30 (dd, J = 5.5 Hz, J = 9.5 Hz, 2H),

3.40-3.46 (m, 1H, CH), 4.50 (t, J = 5.6 Hz, 1H, OH), 6.23

(br. d, 1H, NH), 6.57 (br. t, 1H, NH).

13C NMR (63 MHz, D6-DMSO): δ = 28.10, 40.97, 52.39, 61.24,

77.52, 77.58, 155.15, 155.84.

ESI-MS: [M + Na +] 312.9; [M + H +] 290.9

- N, N-di-tert-butoxycarbonyl-2,3-diaminopropyl-maleimide

N, N-Di-tert-butoxycarbonyl-2, 3-diaminopropanol (400 mg, 1.38 mmol) is dissolved in dry THF and triphenylphosphine (398 mg, 1.52 mmol) and maleimide (148 mg, 1.52 mmol) are added. DEAD (0.26 ml, 1.67 mmol) is then added dropwise and the mixture is stirred at room temperature for 24 h. The solvent is i. Vak. removed and the residue chromatographed twice on silica gel with hexane / EtOAc 2: 1. The desired compound is obtained as a colorless solid, but not in pure form. A separation by means of HPLC also did not bring the desired success.

Yield: 50 mg

ESI-MS: [2M + Na +] 761.2; [M + Na +] 392.0; [M + H +] 370.1. IH NMR (250 MHz, CDC13): δ = 1.39 (br. S, 9H, Boc-H) ^. 1.44

(br. s, 9H, Boc-H), 3.22 (t, J = 5.9 Hz, 2H), 3.61 (d, J =

6.6 Hz, 2H), 3.80-3.88 (m, IH), 5.00-5.15 (m, 2H, NH), 6.72 (s, 2H).

Mitsunobu reactions with maleinimide:

M. A. Walker, Tetrahedron Lett. 1994, 35, 665-668.

M. A. Walker, J. Org. Chem. 1995, 60, 5352-5355.

M. A. Walker, Tetrahedron 1997, 53, 14591-14598.

K.I. Booker-Milburn, C.E. Anson, C. Clissold, N.J. Costin,

R.F. Dainty, M. Murray, D. Patel, A. Sharpe, Eur. J. Org.

Chem. 2001, 1473-1482.

Mitsunobu reactions with Scavenger reagents:

L. D. Arnold, H. I. Assil, J. C. Verderas, J. Am. Chem. Soc.

1989, 111, 3973-3976.

Commercially available maleimide derivatives that have been used successfully in the Diels-Alder reaction:

N-ethylmaleimide

Example 10: Diels-Alder reactions

All compounds shown were prepared according to the general instructions and purified by HPLC. They were characterized at least by ESI mass spectra, often additionally by NMR (4.- and ¹³ C).

General rule:

The furan derivative is dissolved with the corresponding maleimide derivative in water or, if necessary, in a mixture of THF / water (5: 2) and stirred at 50 ° C. for several (2-4) days. Then the solvent i. Vak. removed and the residue on silica gel (CH ₂ Cl ₂ / MeOH 5: 1) purified. The corresponding Diels-Alder adducts were obtained as mixtures of the exo- / endo- and some of them could be separated. - Synthesized Diels-Alder products with N-ethylmaleinimide as dienophile:

Ri / R ₂ : fucose / fucose, galactose / galactose and fucose / galactose

Ri / R ₂ : H / H; H / galactose; H / fucose; Fucose / fucose, galactose / galactose and fucose / galactose, lactose / lactose

Ri / R ₂ :; Fucose / fucose, galactose / galactose

Ri / R ₂ / R ₃ :; Fucose / fucose / fucose

- Synthesized Diels-Alder products with biotin-maleimide as dienophile:

Ri / R ₂ : fucose / fucose, galactose / galactose and fucose / galactose

Compounds with biotin-maleimide are particularly suitable for the elucidation of cellular surface structures and therefore for diagnostics and therapy.

- Other synthesized Diels-Alder products:

Acrylamide as a dienophile

Ri / R ₂ : fucose / fucose - Introduction of carborane-derivatized furan and N-ethylmaleinimide

- Diels-Alder reaction with nucleosides (as an example for Diels-Alder reaction on oligonucleotides, DNA)

Trimethoxyphenylmaleimide (potential active substances, P53)

Bromophenyl maleimide (potential active ingredients, P53)

gl

This reaction was also successfully carried out with double fucosylated or galactosylated 3,4- and 2,5-bishydroxymethylfurans.

A combinatorial approach was used for both of the above compounds. Thus, by combining differently glycosylated furans with differently substituted maleimides, smaller libraries of new, potential drugs, e.g. as modulators from / for p53.

- Diels-Alder product with maleic anhydride

Rι / R ₂ : fucose / fucose

And a derivative of it:

In the following four reactions, 2-glucosylmethyl-5-hydroxymethlfuran was used as the furan derivative.

- with cholesterol

Approach: 240 mg (0.82 mmol) furan

530 mg (1.13 mmol) N-cholesteryl-fαaleinimd Yield: 250 mg (44%) ESI-MS: [M + C1-] 792.7

- dodecane (lipophilic residue)

Batch: 200 mg (0.69 mmol) furan

365 mg (1.38 mmol) N-dodecyl-maleimide Yield: 210 mg (55%) ESI-MS: [M + Na +] 578.0

- steryl residue (lipophilic residue)

Batch: 200 mg (0.69 mmol) furan

300 mg (0.86 mmol) N-stearyl-maleimide Yield: 230 mg (52%) ESI-MS: [M + C1-] 674.5 Aniline (analog Glu-IPM)

Approach: 100 mg (0.34 mmol) furan

215 mg (0.68 mmol) aniline-N-mustard ₇ irιaleinimid Yield: <50 mg ESI-MS: [M + Na +] 625.0

Example 11: Diels-Alder reaction with an amino acid (lysine)

2-tert-Butoxycarbonylamino-6- [3, -dioxo-8, 9-bis- (3,4,5-trihydroxy-6-methyl-tetrahydro-pyran-2-yloxymethyl) -10-oxa-4-aza- tricyclo [5.2.1.02, 6] dec-4-yl] -hexanoic acid (3): To a solution of 0.042 g (0.1 mmol) 1 in 3 ml water, which is adjusted to pH = 6.9, 0.033 g (0.1 mmol) 2 (prepared according to instructions [1]) are added and the mixture is then stirred at 50 ° C. for 4 days. The solvent is removed by freeze-drying and the white crude product (0.075 g) is purified by preparative HPLC. 12 mg and 16 mg of two conformers are obtained. Overall yield 40%. ESI-MS: 647.2 [(MH-Boc) + H] +, 669.1 [(MH-Boc) + Na] +, 769.2 [M + Na] +, 1293., 3 [2 (MH -Boc) + H] +, 1315.4 [2 (MH-Boc) + Na] +

HPLC: Abimed. : analytical: Merck Lichrospher-RPl8 (E) 5μ, (250x4) mm,

Water: CH3CN (100: 0)% grade 40 min 100% CH3CN, 1 ml / min flow, detection at 210 n preparative: Merck Lichrospher-RPlδ (E) 5μ, (250x25) mm,

Water: CH3CN (100: 0)% grade 45 min 25% CH3CN, 10 ml / min flow,

Detection at 210 nm

Claims

claims

1. A method for the synthesis of saccharide compounds, comprising the following steps:

(a) linking at least one saccharide to a cyclic or acyclic diene,

(b) Reaction of the saccharide-containing diene formed in steps (a) or of a commercially available saccharide-containing diene with a dienophile by means of the Diels-Alder reaction.

2. The method according to claim 1, wherein the diene is a substituted furan, fulvene, furfural, pyrrole, pyrazole, oxazole, thiophene, cyclopentadiene, cyclohexadiene or acyclic 1,3-diene.

3. The method according to claim 1 or 2, wherein the dienophile is a maleic acid (anhydride) derivative, fumaric acid (anhydride) derivative, maleimide derivative, acrylic acid derivative, acetylene derivative or an enol ether.

4. The method according to any one of claims 1 to 3, wherein the diene and / or dienophile as substituents alkyl chains, OH, SH, halogens, aryl, carboxyl, nitro, carboxyamido, keto, sulfoxide, sulfonic, sulfonic acid -, phosphoric acid, amino groups, amino acid or peptide substituents, oligonucleotide or nucleic acid substituents, lipid substituents, saccharides, active pharmaceutical ingredients, labels, complexes or dyes.

5. The method according to any one of claims 1 to 4, wherein in step (a) the saccharide with the cyclic diene is linked by serving with a. _^,. I idat component is implemented, which is substituted with a saccharide.

6. The method according to any one of claims 1 to 5, wherein the diene is a cyclic diene selected from 2,3-bishydroxymethylfuran, 3, 4-bishydroxymethylfuran, 2,5-bishydroxymethylfuran or a derivative of α-GMF with modified aldehyde function.

7. The method according to any one of claims 1 to 6, wherein the saccharide-modified imidate is tri-O-benzoyl-fucoseimidate or tetra-O-benzoyl-galactoseimidate.

8. The method according to any one of claims 1 to 7, wherein the product obtained in step a) 3, 4-bis (fucosyl-oxymethyl) furan, 3, 4-bis (galactosyl-oxymethyl) furan, 3-galactosyl -hydroxymethyl-4-fucosyl-hydroxymethylfuran, 3-fucosyl-4-fucosyl-3, 4-bis-hydroxymethylfuran, 3-fucosyl-4-galactosyl-3, 4-bis-hydroxymethylfuran, 2-galactosyl-5-galactosyl-2 , 5-bis-hydroxymethylfuran or 2, 5-bis (galactosyloxymethyl) furan.

9. The method according to claim 4, wherein the substituent is a biotin residue or a lysine residue.