JP5885136B2 - Method for synthesizing highly sulfated chondroitin sulfates, highly sulfated chondroitin sulfates, and analytical reagents - Google Patents

Description

  The present invention relates to a method for synthesizing highly sulfated chondroitin sulfates, highly sulfated chondroitin sulfates, and a reagent for analysis.

  Chondroitin sulfate (CS) is one of glycosaminoglycans, which are linear polysaccharides having a repeating structure of an amino sugar derivative and a hexose derivative. Chondroitin sulfate is widely distributed in animal tissues as a proteoglycan bound to a core protein, and plays an important role in animal development, differentiation, growth and regeneration. Chondroitin sulfate, for example, as a major component in cartilage, contributes to hydration power and elasticity and is useful for cartilage tissue formation. In addition, chondroitin sulfate exhibits binding properties to various bioactive molecules, has the role of storing, stabilizing or masking bioactive molecules, and controls signal transduction mechanisms in cooperation with cell membrane receptors. Yes. In addition, chondroitin sulfate has an effect of promoting and inhibiting the axon extension of nerve cells in the nervous system, and is present in granules in immune system cells, and controls the accumulation and release of immune substances. In infections with malaria parasites and viruses, chondroitin sulfate is known to be a receptor and have an infection inhibitory effect. In many cases, it is known that chondroitin sulfate such as D structure and E structure described below exhibits particularly high physiological activity.

  Chondroitin sulfate has a linear polysaccharide structure with a molecular weight of several tens of thousands (20 to 400 sugar chains). This structure is based on a repeating disaccharide in which glucuronic acid (GlcA) and N-acetylgalactosamine (GalNAc) are alternately linked at β1-3 and β1-4. Chondroitin sulfate is a substance in which chondroitin (CH) is subjected to various sulfate group modifications as shown in FIG.

  Specifically, chondroitin sulfate (CS) is composed of a chondroitin (CH) disaccharide unit A structure (CSA) in which GalNAc residue 4 is sulfated (4S), and GalNAc residue 6 is sulfate. (6S) C structure (CSC), GlcA residue 2-position and GalNAc residue 6-position disaccharide unit D-structure (CSD) in which two positions are sulfated (2S, 6S), GalNAc residue E structure (CSE) in which the 4- and 6-positions are sulfated (4S, 6S), GlcA residue 2-position, GalNAc residue 4-position and 6-position in total 3 places are sulfated (2S, 4S, 6S) contains various modified disaccharide units, such as triS structures (CTriS or CtriS). In this specification, a structure in which the A structure and the C structure are mixed in one polymer is called an AC structure or an AC mixed structure (CSAC), and a structure in which the A structure and the D structure are mixed in one polymer. An AD structure or an AD mixed structure (CSAD) is called, and a structure in which a D structure and an E structure are mixed in one polymer is called a DE structure or a DE mixed structure (CSDE). Chondroitin sulfate has a very complex polysaccharide structure in which these sulfate group-modified structures are combined.

  Sulfate transferases that specifically sulfate the sugar chain skeleton of chondroitin are widely produced by animal cells, and there are separate enzymes specific to the hydroxyl position of the sugar residue that modifies the sulfate group ( Non-patent document 1). In humans, three isozymes of C4ST-1, C4ST-2, and C4ST-3 are known as chondroitin 4-sulfotransferases that sulfate the 4-position of GalNAc residues. There is also D4ST, an enzyme that sulfates the 4-position of the GalNAc residue of the dermatan sulfate skeleton (GlcA epimerized to IdoA). In humans, there are two isozymes, C6ST-1 and C6ST-2, as chondroitin 6 sulfate transferases that transfer sulfate groups to the 6-position of GalNAc residues. An enzyme that specifically modifies sulfate group at position 6 of the sulfated GalNAc (4S) residue to form an E structure is called GalNAc4S-6 sulfate group transferase (GalNAc4S-6ST). An enzyme that creates a D structure by modifying a sulfate group at position 2 of the GlcA residue on the non-reducing end side of the GalNAc (6S) residue is called uronic acid disulfate transferase (UST).

Trends in Glycoscience and Glycotechnology Vol. 12, no. 67 (2000) 307-319

  As described above, chondroitin sulfate having a D structure or an E structure has high physiological activity. Therefore, chondroitin sulfate sulfated with high frequency is required. However, it has been difficult to synthesize chondroitin sulfate highly sulfated to the required level by conventional sulfation methods.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a technique for synthesizing highly sulfated chondroitin sulfates.

  In order to solve the above-described problems, a method for synthesizing highly sulfated chondroitin sulfates according to an aspect of the present invention includes the step of causing chondroitin to act on sulfotransferase C4ST-1 to form an A structure of chondroitin sulfate (A1). ), A sulfate transfer enzyme GalNAc4S-6ST is allowed to act on the A structure to form an E structure of chondroitin sulfate, and a sulfotransferase UST is allowed to act on the E structure to produce a triS structure of chondroitin sulfate. Forming (A3).

  According to this embodiment, highly sulfated chondroitin sulfates having a highly sulfated structure such as a triS structure at a high frequency can be efficiently synthesized.

  In the present specification, “chondroitin sulfates” means chondroitin sulfate and its derivatives. In the present specification, “highly sulfated” means that at least 50% of a plurality of disaccharide units constituting chondroitin sulfates are disulfated (D structure, E structure, DE structure, etc.) or trisulfate. (TriS structure). In addition to or instead of this, “high sulfation” may mean that the “sulfation degree” described later is 100% or more.

  Another embodiment of the present invention is a method for synthesizing highly sulfated chondroitin sulfates. This method of synthesizing highly sulfated chondroitin sulfates comprises a step of forming a structure A and a structure C in a single molecule of chondroitin by allowing the sulfotransferase C4ST-1 and the sulfotransferase C6ST-1 to act on chondroitin. (B1), a step (B2) of forming a D structure by allowing a sulfotransferase UST to act on the C structure, and a step of forming a D structure by allowing the sulfate A structure GalNAc4S-6ST to act on the A structure; Forming a structure (B3).

  According to this embodiment, highly sulfated chondroitin sulfates having a highly sulfated structure such as a DE structure can be synthesized efficiently.

  Another embodiment of the present invention is highly sulfated chondroitin sulfates. The highly sulfated chondroitin sulfates are synthesized by the method for synthesizing highly sulfated chondroitin sulfates according to any of the above-described embodiments.

  According to this aspect, it is possible to provide highly sulfated chondroitin sulfates having a triS structure, a DE structure or the like at a high frequency.

  Another embodiment of the present invention is an analytical reagent. This analytical reagent contains highly sulfated chondroitin sulfates synthesized according to any of the above-described embodiments, and specifically interacts with physiologically active molecules or cell surface molecules and the highly sulfated chondroitin sulfates.

  According to this aspect, it is possible to provide an analytical reagent that has a triS structure, a DE structure, etc. at a high frequency and is useful for analyzing an interaction with a physiologically active molecule or a cell surface molecule.

  According to the present invention, highly sulfated chondroitin sulfates can be synthesized.

It is a figure which shows the main disaccharide unit structure of chondroitin sulfate. FIG. 2A is a diagram illustrating a synthesis method according to the first embodiment. FIG. 2B is a diagram illustrating a synthesis method according to the second embodiment.

  Hereinafter, embodiments for carrying out the present invention will be described in detail.

  The present inventor has developed a highly sulfated chondroitin sulfate in which sulfate groups are frequently bound to specific sugar hydroxyl positions by combining chondroitin (CH) or chondroitin sulfate (CS) with a sulfotransferase in a stepwise manner. The synthesis method which can synthesize was found. Chondroitin sulfate synthesized by this synthesis method is highly modified with a sulfate group that does not exist in nature. In addition, the chondroitin sulfate synthesized does not undergo random sulfation as when chondroitin or chondroitin sulfate is chemically sulfated. Therefore, it is possible to control the properties of synthesized chondroitin sulfate to some extent.

  FIG. 2A is a diagram illustrating a synthesis method according to the first embodiment. FIG. 2B is a diagram illustrating a synthesis method according to the second embodiment. Based on FIG. 1, FIG. 2 (A) and FIG. 2 (B), the synthesis route of highly sulfated chondroitin sulfates will be described.

(First embodiment)
The synthesis method (route A) of the first embodiment includes steps A1 to A3. Step A1 is a step in which sulfate group C4ST-1 is allowed to act on chondroitin to form the A structure (CSA) of chondroitin sulfate having an A structure [GlcA-GalNAc (4S)] almost completely. In step A2, chondroitin sulfate E structure (CSE), in which sulfate group transfer enzyme GalNAc4S-6ST is allowed to act on the A structure formed in step A1, and most of which is E structure [GlcA-GalNAc (4S, 6S)]. Is a step of forming. Step A3 is a high sulfate containing triS structure [GlcA (2S) -GalNAc (4S, 6S)], which is produced by allowing sulfotransferase UST to act on the E structure formed in Step A2 in nature. This is a step for forming a modified CtriS.

  By route A, highly sulfated chondroitin sulfates in which disaccharide units are frequently disulfated (E structure, etc.) or trisulfated (triS structure) are obtained. The resulting highly sulfated chondroitin sulfates have a structure in which at least 50% of disaccharide units are disulfated (such as E structure) or trisulfated (triS structure). Moreover, it has the structure whose below-mentioned sulfation degree is 100% or more.

(Second Embodiment)
The synthesis method (path B) of the second embodiment includes steps B1 to B3. In step B1, sulfated transferase C4ST-1 and sulfated transferase C6ST-1 are allowed to act on chondroitin simultaneously or in random order to form A structure [GlcA-GalNAc (4S)] and C structure [GlcA-GalNAc (6S). ] Is a step of forming CSAC mixed in one molecule. Step B2 is a step in which a sulfotransferase UST is allowed to act on the C structure in the CSAC formed in Step B1 to form a CSAD in which the C structure is a D structure [GlcA (2S) -GalNAc (6S)]. It is. In step B3, after the formation of the D structure, sulfate group transferase GalNAc4S-6ST is allowed to act on the A structure in the CSAD formed in step B2 to convert most of the A structure into the E structure, which is highly enriched in the DE structure. It is a step of forming a sulfated CSDE. In FIG. 2B, the CSAC in which the CSA is arranged on the left side and the CSC is arranged on the right side is illustrated, but the arrangement of the CSA and the CSC is not limited thereto. For example, the order of CSC and CSA may be reversed. Moreover, you may have the structure where CSA continues, or the structure where CSC continues. The same applies to CSAD and CSDE.

  In step B1, it is preferable to cause C4ST-1 and C6ST-1 to act on CH simultaneously. Thereby, CSAC which has A structure and C structure in one molecule | numerator in the ratio according to the amount of enzymes is compoundable by one step. Here, “acting simultaneously” means that C4ST-1 and C6ST-1 should be at least temporarily overlapped in the same system. That is, at least one of the start time and the end time of the action of C4ST-1 and C6ST-1 may be different.

  By route B, highly sulfated chondroitin sulfates in which disaccharide units are frequently disulfated (DE structure and the like) are obtained. The resulting highly sulfated chondroitin sulfates have a structure in which at least 50% of disaccharide units are disulfated (DE structure or the like). Moreover, it has the structure whose below-mentioned sulfation degree is 100% or more.

  In addition, the chondroitin before the sulfation reaction used in the first embodiment and the second embodiment can use commercially available chemically desulfated chondroitin as a raw material. In addition, chondroitin (CH) synthesized by allowing chondroitin polymerase (K4CP) derived from E. coli to act on chondroitin oligosaccharide (also referred to as oligoCH) can also be used (step 0). CHs obtained by this method are more preferable because chondroitin having an arbitrary sugar chain length can be synthesized. By using CHs synthesized in Step 0, highly sulfated chondroitin sulfates of pathway A and pathway B can be efficiently synthesized. However, Step 0 can be omitted if the already synthesized CH can be used. The chondroitin oligosaccharide used in Step 0 is not limited in sugar chain length as long as it is 2 or more, but is preferably a chondroitin oligosaccharide having 4 or more sugars (chondroitin tetrasaccharide: CH4), and 6 sugars (chondroitin hexasaccharides). : CH6) or more chondroitin oligosaccharide is more preferable. Chondroitin oligosaccharide can be obtained by limited degradation of chondroitin with a hydrolase. The chondroitin oligosaccharide may be labeled at the reducing end. Examples of such a label include a hexamethylenediamine group (HMDA), a pyridylamine group (PA), and a benzylamide group (AB).

  First, before describing the following Experimental Examples 1 to 8 in detail, the measurement method, the degree of sulfation, the preparation of CH, and the preparation of sulfate group transferase common to each experimental example and each comparative experimental example will be described.

(Fluorescent unsaturated disaccharide analysis method)
The chondroitin sulfates synthesized in each experimental example were quantitatively and compositionally analyzed by the following method. Synthetic chondroitin sulfates are completely decomposed with chondroitinase ABC or chondroitinase ACII (Seikagaku Corporation), and the decomposition product is separated with a Senshu PAK DOCOSIL column (4.6 × 150 mm, manufactured by Senshu Kagaku). Analysis was performed using a post-column microfluorescence disaccharide analytical HPLC system (Toypda H. et al. J. Biol. Chem. 275 (2000) 2269-2275) in which cyanoacetamide is reacted. Analyze unsaturated disaccharide standard products (manufactured by Seikagaku Corporation) with known concentrations, calculate the composition ratio of each disaccharide component from the comparison with the standard product, and calculate the original chondroitin sulfate from the total amount of disaccharide components The content of was calculated.

(Sulphation degree)
In the present specification, “sulfation degree” is defined as an index indicating the degree of sulfation. In the chondroitin disaccharide unit, 3 positions of GlcA residue 2 position, GalNAc residue 4 position and 6 position can be sulfated. The proportion of unsulfated disaccharide units is A (%), the proportion of disaccharide units that are sulfated is B (%), the proportion of disaccharide units that are sulfated is C (%), The degree of sulfation is defined by Equation 1 below, where D (%) (where A + B + C + D = 100%) is a trisulfated disaccharide unit.
Degree of sulfation = A × 0 + B × 1 + C × 2 + D × 3 (%) (Formula 1)

  When the synthesis method shown in the first embodiment or the second embodiment is used, the degree of sulfation is preferably 100% or more, more preferably 120% or more, and 140% or more. More preferably.

(Preparation of CHs (Step 0))
As substrates for synthesizing highly sulfated chondroitin sulfates, chondroitin hexasaccharide (CH6) not labeled with reducing end, reducing end hexamethylenediamine (HMDA) labeled chondroitin hexasaccharide (CH6-HMDA), reducing end pyridylamine Four (PA) group-labeled chondroitin hexasaccharide (CH6-PA) and reducing end benzylamide (AB) group-labeled chondroitin hexasaccharide (CH6-AB) were used. Chondroitin sulfates synthesized using oligoCH labeled with a reducing end as a raw material are useful in that they can be used for immobilization and detection as described below. Although chondroitin hexasaccharide is shown here as a substrate for synthesizing highly sulfated chondroitin sulfates, chondroitin oligosaccharide having a sugar chain length of 2 or more can be used as described above.

  CH6 and oligoCH were synthesized by limited degradation of chondroitin (manufactured by Seikagaku Corporation) obtained by chemically desulfating natural chondroitin sulfate with a sheep testicular-derived hyaluronidase (manufactured by Sigma) (N. Sugiura et al. Glycoconj. J. 25 (2008) 521-530).

CH6-HMDA was reacted overnight (19 hours) at 70 ° C. in an aqueous solution prepared by adjusting CH6 to 100-fold amount of HMDA and 50-fold amount of NaBH ₃ CN to pH 5, and gel filtration (Superdex 30 column, GE Healthcare). (Manufactured by Care).

CH6-PA was reacted overnight at 90 ° C. (20 hours) in an aqueous solution prepared by adjusting pH 6.2 with 100-fold amount of aminopyridine and 15-fold amount of NaBH ₃ CN in CH6, and subjected to gel filtration with a Superdex 30 column. It was synthesized by purification (N. Sugiura et al. Glycoconj. J. 25 (2008) 521-530).

CH6-AB was prepared by reacting CH6 with 100-fold amount of 2-aminobenzamide and 200-fold amount of NaBH ₃ CN in a 30% acetic acid-DMSO mixture at 65 ° C. overnight (16 hours), and using Superdex 30 column. It was synthesized by gel filtration purification (A. Kinoshita et al. Anal. Biochem. 269 (1999) 367-378).

CH6 (0.24 mg), CH6-HMDA (0.26 mg), CH6-PA (0.26 mg), and CH6-AB (0.27 mg) were each obtained from E. coli-derived chondroitin polymerase (K4CP: T. Ninomiya et al. J). Biol.Chem.277 (2002) 21567-21575) 0.5 μg, UDP-GlcA 6 nmol, and UDP-GalNAc 6 nmol, 50 mM Tris-HCl, pH 7.2, 0.15 M NaCl, 0.2 mM MnCl ₂ aqueous solution 3 . The reaction was carried out in 2 ml with shaking at 30 ° C. for 48 hours. After the reaction, the enzyme was inactivated by heating at 100 ° C. for 5 minutes, and gel filtration purification was performed with a Superdex 30 column to obtain CHs (CH or reducing end-labeled CH). Specifically, CH (10k), CH (10k) -HMDA, CH (10k) -PA, and CH (10k) -AB are obtained in 1.6 mg, 1.86 mg, 1.54 mg, and 1.72 mg, respectively. It was. The basic skeleton of the obtained CHs had a molecular weight of about 10,000 (10k: about 46 sugar chains).

  The molecular weight of chondroitin (CH) is not limited to 10,000 (10k), and may be about 1000 (1k) to about 1 million (1000k). These molecular weights can be adjusted by the enzyme amount, the base mass, the reaction time, and the like.

(Preparation of sulfate transferase)
C4ST1 (accession number: NM — 018713), which is the sulfotransferase used in step A1 and step B1, C6ST1 (accession number: NM — 004273) used in step B1, UST used in step A3 and step B2 ( (Accession number: NM_005715), GalNAc4S-6ST (Accession number: NM_0155892) used in Step A2 and Step B3 was prepared. These enzymes were expressed and purified by culturing animal cell stable expression strains as follows, but those expressed and purified in other expression systems such as E. coli, yeast, insect cells and cell-free systems were used. It may be used. Moreover, you may use the enzyme refine | purified from the natural product.

  As PCR templates, pFLAG-CMV3-DEST-C4ST1 for C4ST1, pFLAG-CMV3-DEST-C6ST1 for C6ST1, pFLAG-CMV3-DEST-UST for UST, pFLAG-CMV3-DEST-GalNAc4S for GalNAc4S-6ST -6ST (both provided by National Institute of Advanced Industrial Science and Technology, Glycomedical Engineering Research Center) was used. Using these as templates, the DNA fragment of the sulfotransferase gene containing the FLAG tag was amplified by PCR. The amplified DNA fragment was ligated to the MCS region of the pIRESpuro expression vector (Clontech), pIRESpuro-FLAG-C4ST1 for C4ST1, pIRESpuro-FLAG-C6ST1 for C6ST1, and pIRESpuro-FLAG-UST, GalNAc4 for UST PIRESpuro-FLAG-GalNAc4S-6ST plasmids were prepared for -6ST, respectively. These plasmid DNAs were transfected into the animal cell line HEK293T using Lipofectamine 2000 (Invitrogen). Passage cells were selected with puromycin to establish each stable sulfotransferase stable expression strain.

  C4ST1, C6ST1, UST, GalNAc4S-6ST stable and highly expressing HEK293T cell line was cultured in GlutaMax-DMEM liquid medium (manufactured by Invitrogen) containing 10% fetal bovine serum and 5 μg / ml puromycin at 37 ° C. for 7 days. The culture supernatant was passed through an anti-FLAG antibody-immobilized resin (manufactured by Sigma) column, washed with 10 mM Tris-HCl, 150 mM NaCl, 5% glycerol, 0.01% Tween 20, pH 7.4 buffer, and 10 mM. Elution was performed with Tris-HCl, 150 mM NaCl, 20% glycerol, 200 μg / ml FLAG peptide, pH 7.4 buffer to obtain a purified enzyme solution. C4ST1 was 980 pmol / min / ml, C6ST1 was 1580 pmol / min / ml, UST was 280 pmol / min / ml, and GalNAc4S-6ST was 950 pmol / min / ml.

[Experimental example]
In the following, Experimental Examples 1 to 4 show the route A and Experimental Examples 5 to 8 show the experimental example related to the route B. As CHs, in Experimental Examples 1 and 5, CH (10k) -HMDA, in Experimental Examples 2 and 6, CH (10k), in Experimental Examples 3 and 7, CSAC (10k) -PA, and in Experimental Examples 4 and 8 CSAC (10k) -AB was used respectively. The enzyme solution obtained by the above preparation was used as the enzyme solution.

[Experimental Example 1]
(Step A1)
CH (10k) -HMDA 0.5 mg, 50 mM TriS-HCl containing 1.8 ml of C4ST1 solution, 11.5 μmol of 3′-phosphoadenyl 5′-phosphosulfate (PAPS, manufactured by Sigma) and 2 mM dithiothreitol (DTT) , PH 6.8 buffer solution (3 ml) and reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.34 mg of CSA (10k) -HMDA in which 98% or more of GalNAc residues were sulfated at the 4-position.

(Step A2)
0.2 mg of the obtained CSA (10k) -HMDA was dissolved in 0.52 ml of GalNAc4S-6ST solution, 4.6 μmol of PAPS and 50 mM TriS-HCl, pH 6.8 buffer (1 ml) containing 2 mM DTT, and 37 After reacting at 24 ° C. for 24 hours, 0.26 ml of GalNAc4S-6ST and 2.3 μmol of PAPS were added, and further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column, whereby 92.5% of the disaccharide unit in which both the 4-position and 6-position of the GalNAc residue were sulfated, and the 4-saccharide sulfated disaccharide unit in 7. 0.22 mg of 5% CSE (10k) -HMDA was obtained.

(Step A3)
0.2 mg of the obtained CSE (10k) -HMDA was dissolved in 0.95 ml of UST solution, 4.6 mM PAPS and 50 mM TriS-HCl, pH 6.8 buffer (1.6 ml) containing 2 mM DTT, and 37 ° C. For 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 18% of the trisulfated disaccharide unit in which the 2-position of the GlcA residue and the 4-position and 6-position of the GalNAc residue were both sulfated, 0.14 mg of CtriS (10k) -HMDA having 75% disaccharide units sulfated at both positions 4 and 6 and 8% sulfated disaccharide units at position 4 was obtained.

  In Experimental Example 1, highly sulfated chondroitin sulfates having a structure in which about 92% of disaccharide units were at least disulfated (E structure, triS structure, etc.) could be synthesized. In this case, the degree of sulfation was 0% × 0 + 8% × 1 + 75% × 2 + 18% × 3 = about 212 (%).

[Experiment 2]
(Step A1)
0.4 mg of CH (10k) was dissolved in 1.6 ml of C4ST1 solution, 10 μmol of PAPS and 50 mM TriS-HCl, pH 6.8 buffer (3 ml) containing 2 mM DTT, and reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.36 mg of CSA (10k) in which 98% or more of GalNAc residues were sulfated at the 4-position.

(Step A2)
0.12 mg of the obtained CSA (10k) was dissolved in 0.36 ml of GalNAc4S-6ST solution, 50 mM TriS-HCl, pH 6.8 buffer (1 ml) containing PAPS 3.2 μmol and 2 mM DTT, and the solution was dissolved at 37 ° C. for 24 hours. After reacting for a period of time, 0.15 ml of GalNAc4S-6ST and 0.8 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, whereby 86% of the disaccharide units in which both the 4-position and 6-position of the GalNAc residue were sulfated, and 13% of the 4-position sulfated disaccharide units. 0.13 mg of (10k) was obtained.

(Step A3)
0.005 mg of the obtained CSE (10k) was dissolved in 50 ml Tris-HCl, pH 6.8 buffer solution (0.15 ml) containing 0.05 ml UST solution, 0.3 μmol PAPS and 2 mM DTT, and 24 ° C. at 24 ° C. Reacted for hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 51% of the trisulfated disaccharide unit in which the 2-position of the GlcA residue and the 4-position and 6-position of the GalNAc residue were both sulfated and the GalNAc residue As a result, 0.003 mg of CtriS (10k) having 39% disaccharide units both sulfated at the 4-position and 6-position and 8% sulfated disaccharide units at the 4-position was obtained.

  In Experimental Example 2, highly sulfated chondroitin sulfates having a structure in which about 90% of disaccharide units were at least disulfated (E structure, triS structure, etc.) could be synthesized. In this case, the degree of sulfation was 2% × 0 + 8% × 1 + 39% × 2 + 51% × 3 = about 239 (%).

[Experiment 3]
(Step A1)
0.1 mg of CH (10k) -PA was dissolved in 0.4 ml of C4ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer (1 ml) containing 2.5 μmol of PAPS and 2 mM DTT, and reacted at 37 ° C. for 24 hours. It was. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.08 mg of CSA (10k) -PA in which 98% or more of GalNAc residues were sulfated at the 4-position.

(Step A2)
0.03 mg of the obtained CSA (10k) -PA was dissolved in 0.09 ml of GalNAc4S-6ST solution, 50 mM TriS-HCl, pH 6.8 buffer (0.25 ml) containing PAPS 0.8 μmol and 2 mM DTT, After reacting at 37 ° C. for 24 hours, 0.03 ml of GalNAc4S-6ST and 0.2 μmol of PAPS were added, and further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that CSE having 68% of disaccharide units in which both the 4-position and 6-position of GalNAc residue were sulfated and 29% of 4-position sulfated disaccharide units. 0.04 mg of (10k) -PA was obtained.

(Step A3)
The obtained CSE (10k) -PA 0.005 mg was dissolved in 50 ml TriS-HCl, pH 6.8 buffer solution (1.8 ml) containing 0.8 ml of UST solution, 0.8 μmol of PAPS and 2 mM DTT, and 37 ° C. For 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 11% of the trisulfated disaccharide unit in which the 2-position of the GlcA residue and the 4-position and 6-position of the GalNAc residue were both sulfated and the GalNAc residue As a result, 0.003 mg of CtriS (10k) -PA having 54% disaccharide units both sulfated at the 4-position and 6-position and 28% 4-saccharide sulfated disaccharide units was obtained.

  In Experimental Example 3, highly sulfated chondroitin sulfates having a structure in which about 65% of disaccharide units were at least disulfated (E structure, triS structure, etc.) could be synthesized. In this case, the degree of sulfation was 7% × 0 + 28% × 1 + 54% × 2 + 11% × 3 = about 169 (%).

[Experimental Example 4]
(Step A1)
0.1 mg of CH (10k) -AB was dissolved in 0.4 ml of C4ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer (0.8 ml) containing 2.5 μmol of PAPS and 2 mM DTT, and incubated at 37 ° C. for 24 hours. Reacted. The reaction solution was subjected to gel filtration purification using a Superdex 30 column to obtain 0.08 mg of CSA (10k) -AB in which 98% or more of GalNAc residues were sulfated at the 4-position.

(Step A2)
0.02 mg of the obtained CSA (10k) -AB was dissolved in 0.06 ml of GalNAc4S-6ST solution, 0.52 μmol of PAPS and 50 mM TriS-HCl, pH 6.8 buffer (0.2 ml) containing 2 mM DTT, After reacting at 37 ° C. for 24 hours, 0.03 ml of GalNAc4S-6ST and 0.26 μmol of PAPS were added, and further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, whereby 57% of the disaccharide units in which both the 4-position and 6-position of the GalNAc residue were sulfated and 41% of the 4-position sulfated disaccharide units. 0.02 mg of (10k) -AB was obtained.

(Step A3)
The obtained CSE (10k) -AB 0.001 mg was dissolved in 50 mM TriS-HCl, pH 6.8 buffer solution (0.2 ml) containing 0.15 ml of UST solution, 0.9 μmol of PAPS and 2 mM DTT, and 37 ° C. For 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 8% of the trisulfated disaccharide units in which the 2-position of the GlcA residue and the 4-position and 6-position of the GalNAc residue were both sulfated were obtained. As a result, 0.0008 mg of CtriS (10k) -AB having 48% disaccharide units sulfated at both positions 4 and 6 and 40% of 4-saccharide sulfated disaccharide units was obtained.

  In Experimental Example 4, highly sulfated chondroitin sulfates having a structure in which about 56% of disaccharide units were at least disulfated (E structure, triS structure, etc.) could be synthesized. In this case, the degree of sulfation was 4% × 0 + 40% × 1 + 48% × 2 + 8% × 3 = about 160 (%).

[Experimental Example 5]
(Step B1)
CH (10k) -HMDA 0.3 mg was dissolved in 50 mM TriS-HCl, pH 6.8 buffer (2 ml) containing C6ST1 solution 0.66 ml, C6ST1 solution 0.27 ml, PAPS 6.9 μmol and 2 mM DTT, 37 The reaction was carried out at 24 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.19 mg of CSAC (10k) -HMDA in which the 4-position sulfation of the GalNAc residue was 48% and the 6-position sulfation was 51%.

(Step B2)
0.14 mg of the obtained CSAC (10k) -HMDA was dissolved in 1.13 ml of UST solution, 50 mM TriS-HCl, pH 6.8 buffer (2 ml) containing 3.3 μmol of PAPS and 2 mM DTT, and 24 ° C. at 24 ° C. After the reaction for 0.5 hour, 0.56 ml of UST and 1.6 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 48% of the disaccharide units in which the 2-position of the GlcA residue and the 6-position of the GalNAc residue were both sulfated were converted into the 4-saccharide sulfated disaccharide unit. 0.14 mg of CSAD (10k) -HMDA having 52% was obtained.

(Step B3)
0.1 mg of the obtained CSAD (10k) -HMDA was dissolved in 0.18 ml of GalNAc4S-6ST solution, 50 mM TriS-HCl, pH 6.8 buffer (0.3 ml) containing 2.3 μmol of PAPS and 2 mM DTT, The reaction was performed at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 20% of the disaccharide units were sulfated at both the 4-position and 6-position of the GalNAc residue, the 2-position of the GlcA residue and the 6-position of the GalNAc residue were As a result, 0.32 mg of CSDE (10k) -HMDA having 48% of disaccharide units both sulfated and 32% of 4-saccharide sulfated disaccharide units was obtained.

  In Experimental Example 5, highly sulfated chondroitin sulfates having a structure in which about 68% of disaccharide units were at least disulfated (such as a DE mixed structure) could be synthesized. In this case, the degree of sulfation was 0% × 0 + 32% × 1 + 68% × 2 + 0% × 3 = about 168 (%).

(Reference Experiment 1)
CH (10k) -HMDA 0.2 mg was dissolved in 50 ml TriS-HCl, pH 6.8 buffer solution (2 ml) containing 0.6 ml C6ST1 solution, 4.6 μmol PAPS and 2 mM DTT, and reacted at 37 ° C. for 24 hours. It was. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.14 mg of CSC (10k) -HMDA in which 98% or more of GalNAc residues were sulfated at the 6-position. CSAC (10k) -HMDA may be synthesized by combining Step A1 of Experimental Example 1 and the reaction of Reference Experiment 1 instead of Step B1 of Experimental Example 5.

[Experimental Example 6]
(Step B1)
0.2 mg of CH (10k) was dissolved in 0.44 ml of C4ST1 solution, 0.20 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer solution (2 ml) containing 4.5 μmol of PAPS and 2 mM DTT, and 37 ° C. For 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.14 mg of CSAC (10k) in which the 4-position sulfation of the GalNAc residue was 42% and the 6-position sulfation was 56%.

(Step B2)
0.07 mg of the obtained CSAC (10k) was dissolved in 1.1 ml of UST solution, 50 mM TriS-HCl, pH 6.8 buffer (2 ml) containing PAPS 3.2 μmol and 2 mM DTT, and reacted at 37 ° C. for 24 hours. Then, 0.55 ml of UST and 1.6 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 42% of disaccharide units in which 2-position of GlcA residue and 6-position of GalNAc residue were both sulfated were converted to 4-saccharide sulfated disaccharide units. As a result, 0.04 mg of CSAD (10k) having 44% and 8% of a 6-sulfated disaccharide unit was obtained.

(Step B3)
0.006 mg of the obtained CSAD (10 k) was dissolved in 0.03 ml of GalNAc4S-6ST solution, 0.3 mM mol of PAPS and 50 mM TriS-HCl, pH 6.8 buffer (0.15 ml) containing 2 mM DTT, and 37 ° C. For 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, whereby 19% of the disaccharide units in which both the 4-position and 6-position of the GalNAc residue were sulfated, the 2-position of the GlcA residue and the 6-position of the GalNAc residue were As a result, 0.007 mg of CSDE (10k) having 44% of disaccharide units that were both sulfated and 26% of 4-position sulfated disaccharide units and 8% of 6-position sulfated disaccharide units was obtained.

  In Experimental Example 6, it was possible to synthesize highly sulfated chondroitin sulfates having a structure (such as a DE mixed structure) in which about 63% of disaccharide units was at least disulfated. In this case, the degree of sulfation was 3% × 0 + 34% × 1 + 63% × 2 + 0% × 3 = about 160 (%).

(Reference Experiment 2)
0.2 mg of CH (10k) was dissolved in 0.5 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer solution (2 ml) containing 4.2 μmol of PAPS and 2 mM DTT, and reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.15 mg of CSC (10k) in which 98% or more of GalNAc residue was sulfated at the 6-position. CSAC (10k) may be synthesized by combining Step A1 of Experimental Example 2 and the reaction of Reference Experiment 2 instead of Step B1 of Experimental Example 6.

[Experimental Example 7]
(Step B1)
CH (10k) -PA 0.05 mg was dissolved in 0.14 ml of C4ST1 solution, 0.05 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer solution (0.5 ml) containing 1.1 μmol of PAPS and 2 mM DTT. , And reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.14 mg of CSAC (10k) -PA in which the 4-position sulfation of the GalNAc residue was 38% and the 6-position sulfation was 61%.

(Step B2)
The obtained CSAC (10k) -PA 0.02 mg was dissolved in USST solution 0.3 ml, PAPS 0.9 μmol and 50 mM TriS-HCl, pH 6.8 buffer (0.5 ml) containing 2 mM DTT, and 37 After the reaction at 24 ° C. for 24 hours, 0.15 ml of UST and 0.45 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 30% of the disaccharide units in which both the 2-position of the GlcA residue and the 6-position of the GalNAc residue were sulfated were converted to the 4-saccharide sulfated disaccharide unit. 0.011 mg of CSAD (10k) -PA having 38% and 31% of a 6-sulfated disaccharide unit was obtained.

(Step B3)
0.005 mg of the obtained CSAD (10k) -PA was dissolved in 50 mM TriS-HCl, pH 6.8 buffer (0.15 ml) containing 0.02 ml of GalNAc4S-6ST solution, 0.2 μmol of PAPS and 2 mM DTT, The reaction was performed at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 31% of the disaccharide units in which both the 4-position and 6-position of the GalNAc residue were sulfated, the 2-position of the GlcA residue and the 6-position of the GalNAc residue were 0.005 mg of CSDE (10k) -PA having 30% disaccharide units both sulfated and 7% 4-position sulfated disaccharide units and 28% 6-position sulfated disaccharide units was obtained. It was.

  In Experimental Example 7, it was possible to synthesize highly sulfated chondroitin sulfates having a structure (such as a DE mixed structure) in which about 61% of disaccharide units was at least disulfated. In this case, the degree of sulfation was 4% × 0 + 35% × 1 + 61% × 2 + 0% × 3 = about 157 (%).

(Reference Experiment 3)
0.1 mg of CH (10k) -PA is dissolved in 0.25 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer (1 ml) containing 2.1 μmol of PAPS and 2 mM DTT, and reacted at 37 ° C. for 24 hours. It was. The reaction solution was subjected to gel filtration purification using a Superdex 30 column to obtain 0.08 mg of CSC (10k) -PA in which 98% or more of GalNAc residue was sulfated at the 6-position. CSAC (10k) -PA may be synthesized by combining Step A1 of Experimental Example 3 and the reaction of Reference Experiment 3 instead of Step B1 of Experimental Example 7.

[Experimental Example 8]
(Step B1)
0.1 mg of CH (10k) -AB was dissolved in 0.22 ml of C4ST1 solution, 0.10 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer solution (0.8 ml) containing 2.1 μmol of PAPS and 2 mM DTT. , And reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column to obtain 0.07 mg of CSAC (10k) -AB in which the 4-position sulfation of the GalNAc residue was 39% and the 6-position sulfation was 59%.

(Step B2)
The obtained CSAC (10k) -AB (0.01 mg) was dissolved in UST solution (0.15 ml), PAPS (0.5 μmol) and 50 mM TriS-HCl, pH 6.8 buffer (0.3 ml) containing 2 mM DTT, and 37 ° C. Then, 0.09 ml of UST and 0.25 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 24% of the disaccharide units in which the 2-position of the GlcA residue and the 6-position of the GalNAc residue were both sulfated were converted to the 4-saccharide sulfated disaccharide unit. As a result, 0.008 mg of CSAD (10k) -AB having 35% of a disaccharide unit of 38% and 6-position sulfated was obtained.

(Step B3)
0.001 mg of the obtained CSAD (10k) -AB was dissolved in 0.05 ml of GalNAc4S-6ST solution, 0.5 mM mol of PAPS and 50 mM TriS-HCl, pH 6.8 buffer (0.1 ml) containing 2 mM DTT, The reaction was performed at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration on a Superdex 30 column, so that 26% of the disaccharide units were sulfated at both the 4-position and 6-position of the GalNAc residue, the 2-position of the GlcA residue and the 6-position of the GalNAc residue were 0.0007 mg of CSDE (10k) -AB having 24% of disaccharide units that are both sulfated and 12% of 4-position sulfated disaccharide units and 34% of 6-position sulfated disaccharide units is obtained. It was.

  In Experimental Example 8, highly sulfated chondroitin sulfates having a structure in which at least 50% of disaccharide units were disulfated (such as a DE mixed structure) could be synthesized. In this case, the degree of sulfation was 4% × 0 + 46% × 1 + 50% × 2 + 0% × 3 = about 146 (%).

(Reference Experiment 4)
0.1 mg of CH (10k) -AB is dissolved in 0.25 ml of C6ST1 solution, 50 mM TriS-HCl, pH 6.8 buffer solution (0.8 ml) containing 2.1 μmol of PAPS and 2 mM DTT, and incubated at 37 ° C. for 24 hours. Reacted. The reaction solution was subjected to gel filtration purification using a Superdex 30 column to obtain 0.08 mg of CSC (10k) -AB in which 98% or more of GalNAc residues were sulfated at the 6-position. CSAC (10k) -AB may be synthesized by combining Step A1 of Experimental Example 4 and the reaction of Reference Experiment 4 instead of Step B1 of Experimental Example 8.

[Comparative experiment example]
Next, the results of Comparative Experimental Example 1 and Comparative Experimental Example 2 in which three or more enzymes used in the reactions of Path A and Path B shown in FIG.

(Comparative Experimental Example 1)
Four enzymes C4ST1, C6ST1, UST, and GalNAc4S-6ST used for pathway B were reacted at the same time. Specifically, CH (10k) -HMDA 0.1 mg was added to C4ST1 solution 0.26 ml, C6ST1 solution 0.09 ml, UST solution 0.76 ml, GalNAc4S-6ST solution 0.18 ml, PAPS 2.3 μmol and 2 mM DTT. It was dissolved in 50 mM TriS-HCl, pH 6.8 buffer (1.5 ml), and reacted at 37 ° C. for 48 hours. The reaction solution was purified by gel filtration using a Superdex 30 column. The obtained sulfated sugar chain had a disaccharide unit composition of 66.3% for 4S and 12.0% for 6S, and a disulfated disaccharide unit could not be synthesized. In this case, the degree of sulfation was 21.7% × 0 + 78.3% × 1 + 0% × 2 + 0% × 3 = about 78.3 (%).

(Comparative Experiment Example 2)
Three enzymes C4ST1, UST, and GalNAc4S-6ST used in the route A were reacted at the same time. Specifically, 0.1 mg CH (10k) -HMDA, 0.33 ml C4ST1 solution, 0.76 ml UST solution, 0.18 ml GalNAc4S-6ST solution, 50 mM TriS-HCl containing PAPS 2.3 μmol and 2 mM DTT, It was dissolved in pH 6.8 buffer (1.5 ml) and reacted at 37 ° C. for 48 hours. The reaction solution was purified by gel filtration using a Superdex 30 column. The obtained sulfated sugar chain had a disaccharide unit composition with 4S of 30.5%, and a disulfated disaccharide unit and a trisulfated disaccharide unit could not be synthesized. In this case, the degree of sulfation was 69.5% × 0 + 30.5% × 1 + 0% × 2 + 0% × 3 = about 30.5 (%).

  When 3 or more enzymes were reacted simultaneously using CH (10k) -HMDA as a substrate, disulfated disaccharide units and trisulfated disaccharide units could not be synthesized. Therefore, it was revealed that in order to synthesize highly sulfated chondroitin sulfates, it is necessary to carry out the reaction in the order of route A or route B in FIG. Note that the synthesis efficiency can be maintained even when a plurality of enzymes (C4ST1 and C6ST1) are reacted at the same time only in Step B1 of Path B.

  Next, a result of examining whether the synthesis reaction from CSC to CSD proceeds in Step B2 of Path B in FIG.

(Comparative Experiment 3)
0.1 mg of CSC (10k) -HMDA obtained in Step B1 of Experimental Example 5 was added to 0.76 ml of UST solution, 50 mM TriS-HCl, pH 6.8 buffer solution (1.5 ml) containing PAPS 2.3 μmol and 2 mM DTT. ) And reacted at 37 ° C. for 24 hours, 0.38 ml of UST and 1.1 μmol of PAPS were added, and the mixture was further reacted at 37 ° C. for 24 hours. The reaction solution was purified by gel filtration using a Superdex 30 column. The obtained sulfated sugar chain has 4% of disaccharide units (D structure) in which the 2-position of the GlcA residue and the 6-position of the GalNAc residue are both sulfated, and the 6-position sulfated disaccharide. The unit remained 94%.

  In the absence of CSA, the synthesis efficiency from CSC (10k) -HMDA to CSD (10k) -HMDA was significantly reduced. From this, it became clear that in order to efficiently synthesize CSD (10k) -HMDA, the CSA structure must be present in the same molecule in addition to the CSC structure.

  As described above, in any of the substrates, CtriS in route A and CSDE in route B could be synthesized in high yield. On the other hand, when these pathways were not passed through (when three or more enzymes were reacted simultaneously), highly sulfated chondroitin sulfates could not be synthesized. In addition, when the amount of the substrate used for each reaction is increased, it is considered that the synthesis efficiency can be further improved for both CtriS of route A and CSDE of route B.

(Use)
The highly sulfated chondroitin sulfates synthesized by the synthesis method according to the present invention can be used for the following applications, for example.

  The highly sulfated chondroitin sulfates synthesized by the synthesis method of the present invention can be used for functional research reagents such as an interaction analysis between a sugar chain and a physiologically active molecule that specifically acts.

  The modified reduced-end HMDA synthesized by the synthesis method of the present invention (Experimental Example 1) can be covalently immobilized on a carrier such as a plate or chip activated with an active ester or epoxy. For this reason, highly sulfated chondroitin sulfates synthesized by the synthesis method of the present invention and modified at the reducing end with HMDA are useful for affinity analysis (intermolecular interaction analysis) with various bioactive molecules and cell surface molecules. It is.

  Highly sulfated chondroitin sulfates synthesized by the synthesis method of the present invention can be detected by fluorescence when the reducing end is modified with PA or AB (PA modification: Experimental example 3, AB modification: Experimental example 4). Therefore, even when a highly sulfated chondroitin sulfate having a reducing end modified by PA or AB synthesized by the synthesis method of the present invention is used in a very small amount of 1 pmol or less, the highly sulfated chondroitin sulfate can be traced. .

  The present invention has been described above with reference to the above-described embodiments and experimental examples. However, the present invention is not limited to the above-described embodiments and experimental examples, and the configurations of the embodiments and experimental examples are appropriately set. Combinations and substitutions are also included in the present invention. In addition, it is possible to appropriately change the combination of the combinations and steps in the embodiments and experimental examples based on the knowledge of those skilled in the art, and to make modifications such as various design changes to the embodiments. Modified embodiments and experimental examples can also be included in the scope of the present invention.

Claims (8)

A step (A1) of causing chondroitin to act on sulfotransferase C4ST-1 to form chondroitin sulfate A structure;
A step (A2) of forming a E structure of chondroitin sulfate by allowing a sulfotransferase GalNAc4S-6ST to act on the A structure;
The E configuration, by the action of sulfotransferase UST, the step (A3) for forming a triS structure of chondroitin sulfate, only including,
By steps (A1) to (A3), at least part of the plurality of disaccharide units constituting chondroitin sulfate is trisulfated, and at least 50% is disulfated or trisulfated,
A method for synthesizing highly sulfated chondroitin sulfates having a molecular weight of 1 kDa to 1000 kDa . A step (B1) of causing chondroitin to act in parallel with sulfotransferase C4ST-1 and sulfotransferase C6ST-1 to form A structure and C structure in one molecule of chondroitin;
A step (B2) of causing a sulfotransferase UST to act on the C structure to form a D structure;
After formation of the structure D, wherein A structure is reacted with sulfotransferase GalNAc4S-6ST, saw including a step (B3) to form a E structure, and
By steps (B1) to (B3), at least 50% of the plurality of disaccharide units constituting chondroitin sulfate is disulfated.
A method for synthesizing highly sulfated chondroitin sulfates having a molecular weight of 1 kDa to 1000 kDa . The synthesis method according to claim 1 or 2, wherein the highly sulfated chondroitin sulfate has a sulfation degree of 140% or more. The synthesis method according to any one of claims 1 to 3 , further comprising a step of synthesizing the chondroitin by causing chondroitin polymerase to act on the chondroitin oligosaccharide. Highly sulfated chondroitin sulfates synthesized by the synthesis method according to any one of claims 1 to 4 . An analytical reagent for specifically interacting a physiologically active molecule or a cell surface molecule with the highly sulfated chondroitin sulfates, comprising the highly sulfated chondroitin sulfates according to claim 5 . 7. The analytical reagent according to claim 6 , wherein the highly sulfated chondroitin sulfates are added with a terminal modification group for immobilizing a carrier. 7. The reagent for analysis according to claim 6 , wherein the highly sulfated chondroitin sulfates are added with a terminal modification group for fluorescence detection.