WO2011018496A2

WO2011018496A2 - Method for modifying and functionalizing saccharides

Info

Publication number: WO2011018496A2
Application number: PCT/EP2010/061742
Authority: WO
Inventors: Mikhail Tsurkan; Uwe Freudenberg; Carsten Werner
Original assignee: Leibniz-Institut für Polymerforschung Dresden e. V.
Priority date: 2009-08-13
Filing date: 2010-08-12
Publication date: 2011-02-17
Also published as: WO2011018496A3; EP2464669A2; DE102009028526A1

Abstract

The invention relates to a method for modifying and functionalizing saccharides, comprising the process steps: a) activating the carboxyl groups of the saccharides by a primarily reacting each of the carboxyl groups with a carbodiimide or a triazole as an activating reagent, and subsequently reacting with N-hydroxysuccinimide (NHS) or one of the derivatives thereof, b) reacting the activating carboxyl groups with primary amino groups of amine-functionalized, optically or biologically active molecules, forming an amide bond.

Description

Method for modifying and functionalizing

saccharides

The invention relates to a process for the modification and functionalization of saccharides.

In particular, modifications to saccharides are made to attach chromophoric markers to the saccharides for use of various analytical methods, both in in vivo and in v / fro techniques. In particular, fluorescence marker molecules for microscopy can be bound to the saccharides in this way. The labeling of functional OH groups of the saccharides is often carried out in organic solvents due to the low reactivity of the OH group in aqueous solutions. Thus, these general labeling techniques are used in organic solvents under "non-natural" conditions, accompanied by possible irreversible structural changes of the polysaccharide target due to the harsh conditions. In addition, many naturally occurring polysaccharides, such as glycosaminoglycans, are poorly soluble in organic solvents and could not be modified under such conditions.

There are several publications on the labeling of glycosaminoglycans and other polysaccharides in aqueous solutions. These approaches typically employ three different techniques, such as reductive amination, the reaction of free amines with NHS esters, and the reaction of carbodiimides with free carboxyl groups. The current state of scientific knowledge in the development and application of these techniques is well described in Bioorganic & Medicinal Chemistry 14 (2006) 2300- 2313 and Carbohydrate Research 341 (2006) 1253-1265. The reductive amination as a labeling technique is usually used for the product the cleavage of glycosaminoglycans with nitrous acid applied. It follows the reaction with a marker containing a primary amine within its structure. The final step is reduction with sodium cyanoborohydride or a similar reducing agent. This process has been studied in detail in the labeling of glycosaminoglycans with 2-aminopyridine [J. Biochem. 95 (1984), 197-203; J. Biochem 110 (1991) 132-135]. The main disadvantages are low yields of polysaccharides with higher molecular weights and in the complexity of this method, which precludes its broad application. An alternative method is to react the free amines of the polysaccharides by selectively binding the target molecules to the amine groups via hydroxysuccinimide (NHS) ester, which is disclosed in Bioorganic & Medicinal Chemistry 14 (2006) 2300-2313. This highly selective technique is subject to extensive limitations. In particular, this technique can only be applied to polysaccharides containing free amino groups. Another essential limitation is that the molecules that are to be chemically bound to a polysaccharide need an NHS ester group or a functional group that can be converted into an NHS ester, which in turn, in turn, the possible functional residues of the to be stored molecules in that no amino groups or similar nucleophilic groups should be present, which would be able to react even with the NHS ester. Thus, the choice of molecules applicable to the chemical modification of polysaccharides (according to the previously described method) as well as the biological or physical versatility of the final products is significantly limited. More specifically, this technique is limited primarily to fluorescein, coumarin, dansyl and biotin, which do not contain free amino groups. Labeling of polysaccharides by the family of common chromophoric labels, such as Alexa ™ Flour, HiLyte ™ Flour, DyLight ™ Flour and others, does not allow this chemistry to be used. The use of carbodiimide chemistry to form a covalent bond between the free carboxyl groups of the target molecule and any amino group of a label is another method of functionalizing polysaccharides. The standard method involves the use of ethyl 3- (3-dimethylaminopropyl) carbodiimide (EDC) or similar activating reagents for the modification of a polysaccharide in admixture with a marker in a one-step mechanism. Hydroxysuccinimide (NHS) or hydroxysulfosuccinimide (sulfo-NHS) is used as a catalyst to increase the yield in this reaction by forming a more stable intermediate, the NHS or s-NHS ester of the carboxyl groups. However, the average yield in the methods described so far is in the range of 30 to 50%, which is unprofitable for the use of expensive markers such as Alexa ™ Flour. Furthermore, this method is due to the structure of the markers and functional molecules used only limited. For example, no carboxyl functions should be present to prevent intermolecular crosslinking.

The object underlying the invention is to provide a method for carrying out modifications to saccharides having a multiplicity of markers or functional molecules which can be carried out inexpensively and in high yields even under aqueous conditions. Such a method should be capable of covalently attaching a variety of physically or biologically active molecules to the saccharide units for use in tissue engineering and drug development. In this context, the process should include the use of a variety of mutable molecules for modern in vitro and in vivo

Tests with polysaccharides, including glycosaminoglycans and their

Derivatives allow. The solution of this problem according to the invention consists in a process for the modification and functionalization of saccharides, comprising the individual process steps:

a) activation of the carboxyl groups of the saccharides by a primary reaction of the respective carboxyl group with a carbodiimide or a triazole as activating reagent and a subsequent reaction with N-hydroxysuccinimide (NHS) or one of its derivatives,

b) Reaction of the activated carboxyl groups with primary amino groups of amine-functionalized, optically or biologically active molecules to form an amide bond.

According to the concept of the invention, this covalent modification of the saccharides in aqueous solutions is carried out in a two-step mechanism to avoid unwanted side reactions. In a first step, N HS esters of the free carboxyl groups are formed on the saccharide. In a second step, the conversion then takes place by the reaction of the NHS-activated saccharide carboxyl groups with primary amino groups of a marker molecule or of another functional molecule.

The proposed method can be used to functionalize a variety of saccharides by forming an amide linkage with a variety of other molecules (eg, signal molecules and markers). The peculiarity of this method is that almost all molecules which are soluble in aqueous solutions and contain a free amino group can be covalently attached to saccharides. For example, almost all proteins fulfill this requirement. The possibility of covalent attachment in aqueous solution reduces interfering side reactions caused by other functional groups within the marker molecule or the functional molecule, respectively. This method allows the To control the reaction between a saccharide and a marker or similar functional molecule for the formation of a covalently-labeled product in the form that yields greater than 90% are achieved. Often, yields of up to 95% (based on the conversion of the carboxyl groups on the saccharides) are achieved with the technique according to the invention. Therefore, when using the method according to the invention, the use of equimolar amounts of saccharide and on marker molecules or biofunctional molecules is possible, so that modifications even with costly markers, such as fluorescent dyes, or tailor-made proteins are economically justifiable.

With the method according to the invention, the labeling or modification is achieved by the covalent attachment of a primary amino group of a marker or a functional molecule to an NHS-activated carboxyl group of the saccharides. The reaction takes place in aqueous solutions, which allows the functionalization of very hydrophilic saccharides, such as glycosaminoglycans. As saccharides or polysaccharides in the process according to the invention thus polyglucosaminoglycans, in particular heparin or hyaluronic acid, can be modified and functionalized.

As activating reagents which are used in the activation step of the activation of carboxyl groups in the primary activation step, in particular compounds from the class of carbodiimides are provided, preferably 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC), but also similar compounds, such as For example, dicyclohexylcarbodiimide (DCC) or diisopropylcarbodiimide (DIC) can be used. As an alternative to the use of carbodiimides, compounds from the class of triazoles are also possible as activating reagents for the primary reaction of the activation step a). These include 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7-aza-benzotriazole (HOAt), but also other similar compounds. In a particularly preferred embodiment of the process according to the invention, N-hydroxysulfosuccinimide (s-NHS) is used as the N-hydroxysuccinimide derivative for the activation of the carboxyl groups. In particular, with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) in the primary reaction and with N-hydroxysulfosuccinimide (s-NHS) in the subsequent reaction, activated carboxyl groups of saccharides react with the amino groups of amine functionalized functional molecules or chromophoric markers. This allows the formation of modified or labeled saccharides with a wide range of optically and biologically active moieties or functions, which properties are useful in biomaterial studies as well as in biological and medical investigations. In this embodiment of the process, in the first so-called activation step, the carboxyl groups of the respective saccharide or polysaccharide are converted into highly reactive sulfo-NHS esters which are suitable for the subsequent stoichiometric reaction of approximately 1: 1 with primary amino groups which belong to the respective functional molecule or chromophoric marker.

The peculiarity of the method according to the invention is that the special connection of a chromophoric marker, for example a marker with a strong fluorescence, of a multiplicity of optically active molecules, also in the presence of carboxyl groups, is to be bound within the zone

Molecules themselves is possible. The same applies to biologically active molecules to be attached to the carboxyl groups of the saccharides, such as biologically active peptides and proteins. In addition, this allows

Process the covalent attachment of markers / functional molecules

Saccharide molecules in aqueous solution, without complicated

To have to apply labeling techniques in organic solvents, as they are necessary, for example, in the implementation (labeling) of hydroxyl groups (OH-) under absolutely anhydrous conditions. Therefore, it is also possible to label highly charged glycosaminoglycans such as heparin, which can not be modified in organic solvents due to their poor solubility. In general, this method allows a "gentler" mark. For this reason, bioactive molecules can be labeled without losing their biological functionality or their structural integrity. In addition to the economic benefits derived from the fact that the greater than 95% reaction yields avoid waste from expensive reagents and make this reaction very favorable for industrial use, the process allows for precise control of the stoichiometry between the saccharides and the labels, which is very important to precisely adjust the properties of the conjugate formed.

Further details, features and advantages of the invention will become apparent from the following description of exemplary embodiments with reference to the accompanying drawings. Show it:

Fig. 1a: a UV-Vis spectrum of azure A;

Fig. 1b: a UV-Vis spectrum of heparin;

Fig. 1c: superimposed normalized UV-Vis spectra of heparin and heparin azure A;

Fig. 2a: a high pressure size exclusion chromatogram (HPSEC) of

Heparin when using an 8 μm column PL-Aquagel-OH

Mixture and 100 mM phosphate buffer of pH 7;

Fig. 2b: a high pressure size exclusion chromatogram (HPSEC) of heparin azure A product using a 8 μm column PL-Aq uagel-OH mixture and 100 mM phosphate buffer of pH 7; FIG. 3a: an HPSEC chromatogram of heparin using an 8 μm column of PL-Aquagel-OH mixture and 10 mM phosphate buffer of pH 7; FIG. 3b: an HPSEC chromatogram of a Heparin-Alexa ™ Flour 488. FIG

Reaction mixture using an 8 μm column of PL-Aquagel-OH mixture and 100 mM phosphate buffer of pH 7;

FIG. 3 c: an HPSEC chromatogram of purified heparin Alexa ™ Flour 488 product using an 8 μm column PL-Aquagel

OH mixture and 100 mM phosphate buffer of pH 7;

FIG. 4a: an HPSEC chromatogram of Alexa ™ Flour 488 at

Use of an 8 μm column PL-Aquagel-OH mixture and 100 mM phosphate buffer of pH 7;

Fig. 4b: a UV-Vis spectrum of Alexa ™ Flour 488 in pH 7 phosphate buffer; Fig. 4c: a UV-Vis spectrum of heparin Alexa ™ Flour 488 in

Phosphate buffer of pH 7;

Fig. 5a: Emission spectra of Alexa ™ Flour 488, excitation at 488 nm in phosphate buffer of pH 7;

Fig. 5b: Emission spectra of Heparin-Alexa ™ Flour 488, excitation at 488 nm in phosphate buffer of pH 7; 6a: HPSEC chromatogram of hyaluronic acid using an 8 μm column of PL-Aquagel-OH mixture and 10 mM phosphate buffer of pH 7; Fig. 6b: HPSEC chromatogram of Alexa ™ Flour 488 using an 8 μm column of PL-Aquagel-OH mixture and 10 mM phosphate buffer of pH 7;

FIG. 6 c: HPSEC chromatogram of Alexa ™ -Flour-488-hyaluronic acid product using an 8 μm column PL-Aquagel-OH

Mixture and 100 mM phosphate buffer of pH 7;

Fig. 7a: HPSEC chromatogram of low hyaluronic acid

Molecular weight using a Phenomenex® BioSep S-2000 column and 100 mM pH 7 phosphate buffer;

Fig. 7b: HPSEC chromatogram of Alexa ™ hyaluronic acid product at

Using a Phenomenex® BioSep S-2000 column and

100 mM phosphate buffer of pH 7;

Fig. 8a: HPSEC chromatogram of heparin using a

8 μm column of PL-Aquagel-OH mixture and 100 mM

Phosphate buffer of pH 7; 8b: HPSEC chromatogram of cRGD using an 8 μm column of PL-Aquagel-OH mixture and 100 mM phosphate buffer of pH 7; 9a: HPSEC chromatogram of the reaction mixture of heparin cRGD using an 8 μm column of PL-Aquagel-OH mixture and 100 mM phosphate buffer of pH 7; Fig. 9b: HPSEC chromatogram of the purified heparin cRGD at

Use of an 8 μm column of PL-Aquagel-OH mixture and 100 mM phosphate buffer of pH 7;

Fig. 10a: a UV-Vis spectrum of unmodified heparin;

Fig. 10b: a UV-Vis spectrum of heparin c-RGD product and

Fig. 10c: a UV-Vis spectrum of c-RGD peptide. Embodiment I:

Embodiment I relates to the labeling of heparin with Azur A. Heparin is often used as a drug. More recently, heparin has also been used as an active component of some bio-hybrid materials that have great potential in both tissue engineering applications and as a drug delivery medium Heparin is a natural polyglucosaminoglycan molecule has a very high negative charge density due to the high degree of sulfation This characteristic of heparin is crucial for its biological activity, therefore the application of the reductive amination to labeling is disadvantageous because of a possible desulfation of its amino groups.

Heparin has no characteristic absorption pattern in

UV-Vis spectrum, but labeling with a strong dye allows easy observation and quantitation of heparin in tissues or Biohybrid materials. The low solubility in organic solvents and high sensitivity to hydrolysis limits the chemistry for the modification. Heparin varies in its molecular weight. A commercially available heparin has an average molecular weight of about 14 kDa and about 28 carboxyl groups per molecule which react with dyes containing a primary amino group. Azur A has been shown to react with heparin through the formation of amide bonds.

The procedure is performed as follows: 0655 mg EDC [10-fold excess] and 0.45 mg NHS [5-fold excess] were added to a solution of 5 mg heparin in borate buffer (pH = 8). The reaction mixture was kept at room temperature for 10 minutes. Next, the equimolar solution Azur A in borate buffer (pH = 8) was added. The total volume of the reaction mixture was 100 μl. The reaction was continued at 40 ^° C. overnight. Purification of the heparin-Azur-A product was carried out by dialysis against 800 ml of 1 M sodium chloride solution followed by dialysis three times against 800 ml of deionized water using a membrane with a molecular weight exclusion quantity (MWCO) of 1000 Da. Unreacted azure A should be removed quantitatively. Thereafter, the product was freeze-dried and 4.8 mg of amorphous blue residue were collected.

Results of Embodiment I:

Heparin shows no absorption pattern in the UV-Vis spectrum and therefore also in accordance with FIG. 1b no absorption at 600 nm, whereas the azure-A molecule according to FIG. 1a shows a characteristic absorption at 290 nm and at 600 nm. The UV-Vis spectra of the purified heparin-azure-A conjugate show the characteristic absorbances at 290 nm and 600 nm as shown in Fig. 1c. The difference between the UV-Vis spectra of the heparin-azure-A conjugate and heparin can be clearly seen:

The purity of the product is confirmed by high performance size exclusion chromatography (HPSEC). Heparin elutes as shown in Figure 2a as a peak in the HPSE chromatogram with an elution time of 17 minutes, as evidenced by the appearance of absorbance at 210 nm. The HPSE chromatogram of the purified product showed, as seen in Figure 2b, only one peak at 17 minutes characteristic of heparin. The product causes a strong absorption at 600 nm, as can be seen in Figures 1a and 1c, which clearly indicates the presence of azure A within the structure of the product.

Exemplary embodiment II:

Exemplary embodiment II relates, by way of example, to the use of the method of the invention for the labeling of heparin with unmodified Alexa ™ flour 488 dye using a two-step procedure based on carbodiimide chemistry. Alexa ™ is a US molecular probe brand. The aliphatic amino group in the structure of the dye is used for the covalent attachment of this dye to a carboxyl group of the heparin by forming an amide bond, the yield being more than 90%.

The procedure is performed as follows: 30 mg EDC [3.5-fold excess] and 0.17 mg NHS [2.0-fold excess] were added to a solution of 5.4 mg heparin (3.85 * 10 ^-4 mmol) in borate buffer of pH = 8. The reaction mixture was held at 5 ^° C. for 15 minutes. Next, 0.25 mg Alexa Flour ™ were ^(* 10-4 3.9 mmol) was added in borate buffer, pH = 8 488th The total volume of the reaction mixture was 50 μl. The reaction was continued overnight at room temperature. The Heparin-Alexa ™ 488 product was purified by dialysis against 800 ml of 1 M sodium chloride solution to unreacted Alexa ™ Flour 488 quantitatively, using a membrane with a mean molecular weight exclusion size of 1000 Da. This was followed by three dialysis sessions against 800 ml of water. Thereafter, the product was freeze-dried and 5.35 mg of amorphous orange residue was collected, which corresponds to a yield of 95%.

Results of Embodiment II:

The strong absorption of the heparin Alexa ™ product at 488 nm according to

Figure 3c and its absence with pure heparin, as shown in Figure 3a, allows easy monitoring of the reaction by high pressure size exclusion chromatography (HPSEC). The elution time of heparin is 17 minutes, which can be seen by monitoring the absorption at 230 nm in the HPSE chromatogram in Fig. 3a. The reaction mixture provides, as shown in Figure 3b, several peaks in the HPSE chromatogram at 17, 19 and 20 minutes corresponding to heparin (peak at 17 minutes), unreacted Alexa ™ flour (peak at 19 minutes) and low molecular weight by-product of EDC and NHS (peak at 20 minutes). The HPSE chromatogram of the purified product should show only one peak at 17 minutes, which is characteristic of heparin when monitoring absorbance at 230 nm. However, the product of Figure 3c also has a strong 488 nm absorption, which indicates the presence of Alexa ™ Flour 488 in the structure of the product.

Alexa ™ Flour 488 has a strong absorption at 488 nm, which is visible in UV-Vis spectra and, as shown in FIG.

Size exclusion chromatography (HPSEC) can be monitored. The Heparin Alexa ™ Flour 488 product was expected to be a similar

Absorbent behavior like Alexa ™ Flour. Apparently, that delivers

Heparin Alexa ™ Flour 488 product almost identical UV Vis spectra as Alexa ™ Flour 488, which can be seen by comparing Figures 4b and 4c. Similarly, when comparing the aqueous solution of Alexa ™ Flour 488 shown in Figure 5a and the heparin Alexa ™ Flour 488 product shown in Figure 5b. The reaction product provides, as shown in Figure 5b, a strong emission at 530 nm due to the emission of the Alexa ™ dye unaltered in its functionality during the conjugation reaction.

Embodiment III:

Embodiment III relates to the labeling of hyaluronic acid by Alexa ™ Flour 488. Hyaluronic acid, also known as hyaluronan, is a non-sulfated glycosaminoglycan that is widely used as a drug and in cosmetics. Like most polysaccharides, hyaluronic acid does not have a characteristic absorption pattern in the UV-Vis spectrum, therefore labeling with fluorescent dyes is necessary to be detectable by this method. Each disaccharide residue of hyaluronic acid contains a carboxylic acid group which can be modified with dyes each having a primary amino group. Alexa ™ Flour 488 reacts with hyaluronic acid (17 kDa) to form an amide bond. The procedure is carried out as follows: Similar to Working Example II, 0.14 mg EDC [2.0-fold excess] and 0.086 mg NHS [1-fold excess (= equimolar amount)] of a solution of 3.03 mg hyaluronic acid, which correspond to 3.85 ^* 10 ^{~ 4} mmol with a molecular weight of = 17,000 Da, added in borate buffer (pH = 8). The reaction mixture was held at 5 ° C for 15 minutes. Next, 0.25 mg Alexa Flour ™ 488, 10 ^"correspond to 3.9 ^* ⁴ mmol, added in borate buffer (pH = 8). The total volume of the reaction mixture was 50 ul. The reaction was continued overnight at room temperature. The Alexa ™ Flour 488-hyaluronic acid product was purified by dialysis against 800 ml of 1 M sodium chloride solution using a membrane with an average molecular mass exclusion size of 1,000 Da to quantitatively remove unreacted Alexa ™ Flour 488; This was followed by a three-time dialysis against 800 ml of water. Thereafter, the product was freeze-dried. There was obtained 3.0 mg of an amorphous orange residue, which corresponds to a yield of 95.6%.

Results of Embodiment III:

The strong absorption of the Alexa ™ hyaluronic acid product at 488 nm and its absence for hyaluronic acid allows easy monitoring of the reaction by high pressure size exclusion chromatography (HPSEC). The unreacted hyaluronic acid gives two peaks in the HPSE chromatogram, with the majority of each peak eluting at 18 minutes. The small proportion in the HPSE chromatogram of the hyaluronic acid according to FIG. 6a can be attributed to low molecular weight saccharides. Under similar conditions, Alexa ™ Flour 488 elutes at 19 minutes as shown in Figure 6b by both absorbance at 210 nm and absorbance at 488 nm. The purified conjugate of hyaluronic acid with Alexa ™ Flour 488, as shown in Figure 6c, shows several peaks in elution, with the majority of peaks occurring at 18 minutes, which is similar to the behavior of unreacted hyaluronic acid. However, all of the components in the product's HPSE chromatogram also show strong absorption at 488 nm, clearly indicating the presence of Alexa ™ Flour 488 within the structure of the product.

Exemplary embodiment IV:

The embodiment IV relates to the labeling of low molecular weight

Hyaluronic acid with Alexa ™ Flour 488. The example shows the labeling of low molecular weight hyaluronic acid (4.7 kDa), only a few Contains disaccharide residues. Similar to Embodiment III, Alexa ™ Flour 488 shows the formation of an amide with the carboxyl group of hyaluronic acid using standard carbodiimide chemistry. The procedure was carried out as follows: Similarly as in Embodiment III, 0.14 mg EDC [2.0-fold excess] and 0.086 mg NHS [the equimolar amount] of a solution of 0.84 mg hyaluronic acid, which was 3.85 ^* 10 ^"4 mmol at a molecular weight of 4,700 Da correspond added in borate buffer pH 8. the reaction mixture was maintained at 5 ⁰ C for 15 minutes. Thereafter, 0.25 mg Alexa Flour ™ 488, the 3.9 ^* ^{10 4} mmol The total volume of the reaction mixture was 50 μl and the reaction was continued overnight at room temperature The Alexa ™ flour 488-hyaluronic acid product was dialyzed using a membrane of average molecular weight exclusion size of 1000 Da purified to 800 ml of 1 M sodium chloride solution to quantitatively remove unreacted Alexa ™ Flour 488. Three times dialysis to 800 ml of water was then carried out, Next, the product was freeze-dried 0.8 mg of amorphous orange residue, corresponding to a yield of 84.4%.

Results of Embodiment IV:

Similarly to Embodiment III, the unreacted low molecular weight hyaluronic acid in the HPSEC elutes in the form of two peaks, with the first peak of Fig. 7a being very broad and having a maximum at 16 minutes. The purified conjugate of low molecular weight hyaluronic acid with Alexa ™ Flour 488 shows, according to FIG. 7b, a very similar pattern of peaks in the HPSE chromatogram which, in contrast to the starting component, also give a very strong absorption at 488 nm. This clearly indicates the presence of Alexa ™ Flour 488 within the structure of the product. Embodiment V:

Embodiment V relates to the functionalization of heparin by a cRGD peptide. The modification of saccharides with biologically active molecules, such as the cell-adhesive peptide sequences, are of great scientific and technological interest. By way of example, the following embodiment describes the modification of the polysaccharide heparin with the integrin-binding cyclic RGD peptide, which is generally known as a frequently used cell-adhesive ligand in biomaterials research. The peptide sequence is KYRGD in cyclic form (cRGD). The side-chain amino group of lysine is utilized for the covalent coupling of cRGD to heparin carboxyl groups to form an amide bond, respectively. Due to the fact that this peptide contains both carboxylic acids and primary amine groups within its structure, covalent attachment to polysaccharides by carbodiimide chemistry poses a challenge because of a one-step mechanism of side reactions between the carboxyl groups of the peptides and the amine. Groups of other peptide molecules would produce unwanted aggregation products. In order to avoid these side reactions and to accurately control the amount of the peptide bound to a polysaccharide, the two-stage conjugation method of the present invention was used. Specifically, the stoichiometric coupling of cRGD to heparin was demonstrated by formation of amide bonds between the activated carboxyl groups of heparin and the amino groups of lysine molecules. Standard EDC, Sulfo-N HS chemistry was used with 2-fold excess of crosslinking reagents. The amount of peptide within the heparin-cRGD conjugate proved to be regulatable by the chemistry used.

The procedure was carried out as follows: 2.71 mg EDC [1.75-fold excess] and 1.75 mg NHS [1-fold excess (equimolar amount)] became the solutions of both 29 mg and 19 mg heparin (3.85 * 1CT ⁴ mmol) in pH 8 borate buffer. The reaction mixture was held at 5 ^° C. for 20 minutes. Next, 2.5 mg of cRGD in pH 8 borate buffer was added to each reaction mixture, corresponding to twice or three times the excess of cRGD. The total volume of the reaction mixtures was about 100 μl. The reactions were continued overnight at room temperature. To remove any unreacted cRGD, the cRGD heparin product was purified by dialysis against 1 M sodium chloride solution. This was followed by the triple dialysis against water. Thereafter, the product was freeze-dried, and it was 28 mg, which corresponds to a yield of 88.9%, or 18 mg, which corresponds to a yield of 87.2%, of the amorphous white residue.

Results of Embodiment V:

The tyrosine amino acid residue within the cRGD structure has a characteristic absorption at 274 nm with an extension coefficient of 1405. This absorption of the cRGD heparin product at 274 nm allows monitoring of the reaction by high pressure size exclusion chromatography (HPSEC). , The HPSE chromatogram of heparin, as shown in Figure 8a, has only one peak at 17 minutes which has no substantial absorption at 274 nm. The cRGD elutes at 21 minutes under similar conditions, as shown in Figure 8b, but gives a relatively strong absorption at 274 nm. The reaction mixtures, as shown in Figure 9a, provide three main peaks in the HPSE chromatogram at 17, 19 and 21 minutes, respectively each can be assigned to heparin (17 minutes), a low molecular weight by-product of EDC and NHS (19 minutes) and unreacted cRGD peptide (21 minutes). After purification, both reaction mixtures showed only one major peak in the HPSE chromatogram according to FIG. 9b with the elution time of 17 minutes, which corresponds to the chromatogram of similar to unreacted heparin. However, unlike heparin, these products provided greater absorption at 274 nm, suggesting the presence of cRGD within their structures. FIG. 10a shows a UV-Vis spectrum of unmodified heparin, FIG. 10b a UV-Vis spectrum of the heparin c-RGD product, and FIG. 10c a UV-Vis spectrum of c-RGD peptide. The content of cRGD peptide within the cRGD modified heparin (heparin cRGD) is determined by comparing the absorbance of equal molarity solutions of unmodified heparin in Fig. 10a on the one hand and the cRGD heparin product of Fig. 10b on the other hand at 274 nm , The amount of cRGD peptide in the heparin cRGD product (conjugate) is determined using the extinction coefficient of 1405 at 274 nm of tyrosine. By comparing the calculated cRGD peptide molarity and the total mass of the heparin cRGD product, the content of the cRGD peptide within the cRGD heparin product is determined.

Both cRGD-heparin samples, synthesized with two- or three-fold excess of the cRGD peptide, showed a strong absorption at 274 nm. The calculation of the cRGD content showed that both samples each contain about one or two molecules of cRGD per molecule of heparin ,

LIST OF ABBREVIATIONS

DCC dicyclohexylcarbodiimide

DIC diisopropylcarbodiimide

EDC ethyl 3- (3-dimethylaminopropyl) carbodiimide

HOAt 1 -hydroxy-7-aza-benzotriazole

HOBt 1-hydroxybenzotriazole (HOBt)

HPSEC high pressure size exclusion chromatography

NHS hydroxysuccinimide

s-NHS hydroxysulfosuccinimide

RGD stands for the amino acid sequence arginine, glycine and

aspartic acid

cRGD cyclic RGD

Claims

claims

1. A process for the modification and functionalization of saccharides, comprising the process steps:

2. The method according to claim 1, characterized in that are used as saccharides Polyglukosaminoglykane, in particular heparin, or hyaluronic acid.

3. The method according to claim 1 or 2, characterized in that provided as carbodiimide activating reagents for the primary reaction of the activation step a) ethyl 3- (3-dimethylaminopropyl) carbodiimide (EDC), dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are.

4. The method according to claim 1 or 2, characterized in that as triazole activating reagents for the primary reaction of the activation step a) 1-hydroxy-benzotriazole (HOBt) and 1-hydroxy-7 aza-benzotriazole (HOAt) are provided.

5. The method according to any one of claims 1 to 4, characterized in that as N-hydroxysuccinimide derivative for the activation of the carboxyl groups N-hydroxysulfosuccinimide (s-NHS) is used.

6. The method according to any one of claims 1 to 5, characterized in that the amine-functional molecule is a chromophoric marker containing a primary amino group.

7. The method according to any one of claims 1 to 5, characterized in that the amine-functional molecule is a cyclic RGD peptide.