CN109111533B - Enzymatic chemical synthesis and application of heparin sulfate and FK506 conjugate - Google Patents

Enzymatic chemical synthesis and application of heparin sulfate and FK506 conjugate Download PDF

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CN109111533B
CN109111533B CN201810444478.1A CN201810444478A CN109111533B CN 109111533 B CN109111533 B CN 109111533B CN 201810444478 A CN201810444478 A CN 201810444478A CN 109111533 B CN109111533 B CN 109111533B
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heparin sulfate
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heparin
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CN109111533A (en
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周文
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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
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Abstract

The invention discloses an enzymatic chemical synthesis and application of a heparin sulfate and FK506 conjugate in the technical field of pharmaceutical chemicals; provides a small molecule tool or probe for researching the mechanism of heparin sulfate for regulating embryonic development and understanding the structure-activity relationship of heparin sulfate in cell nucleus, wherein the heparin sulfate refers to a hexuronic acid and glucosamine repeating disaccharide unit with determined and diversified structures and isotope14C-labeled radioactivity analysis shows that the membrane penetrating and nuclear entering capacity of the conjugate is greatly improved.

Description

Enzymatic chemical synthesis and application of heparin sulfate and FK506 conjugate
Technical Field
The invention belongs to the technical field of pharmaceutical chemicals, and particularly relates to enzymatic chemical synthesis and application of a heparin sulfate and FK506 conjugate.
Background
Heparin Sulfate (HS) is a linear mucopolysaccharide, consists of repeated disaccharide structures of hexuronic acid and glucosamine, is connected by α -1, 4-glycosidic bonds, has anticoagulant, antiviral, anti-inflammatory and antitumor effects, and regulates embryonic development, and particularly has remarkable effect of regulating embryonic development.A stem cell comprises a highly sulfated form of heparin sulfate, which is an exogenous substance essential for stem cell growth and is prevented from growing when being added or lacking, which indicates the importance of regulating embryonic development by the heparin sulfate in the nucleus.
Heparin sulfate is an endogenous functional biopolymer, biosynthesis is started by endoplasmic reticulum, the heparin sulfate is processed into heparin sulfate with different lengths and sulfation degrees through Golgi body extension, and finally the heparin sulfate is transported to a target site through different carriers to play a biological function, however, because the heparin sulfate has extremely high water solubility, the problems that how the heparin sulfate is transported into cell nucleus and structural fragments determining the strength and weakness of the transport function in the heparin sulfate are not clear and the like seriously restrict the centi-definition for understanding the nuclear biological action mechanism and the structure-activity relationship (the control of the nuclear heparin sulfate and the cell function thereof, stroma biology, 2014, 35, 56-59). Therefore, the stable, orderly and safe membrane penetration of exogenous heparin sulfate into the nucleus is one of the new solutions for researching the biological function of the heparin sulfate in the nucleus.
The literature search of the prior art shows that the clinical immunosuppressant FK506(tacrolimus) is a macrocyclic lactone, is usually used for preventing allograft rejection after organ transplantation (including liver, kidney, heart and pancreas), has the characteristics of high specific targeting FKP12, strong lipid solubility, good biocompatibility, stable metabolism and the like (synthesis ligand control signal transduction, science, 1993,262, 1019 and 1024.), can be used as an in vivo transport carrier, but is easy to cause immunosuppressive side effects. The double bond in the exocyclic propenyl group of FK506 is a functional structural fragment for generating immunosuppressive effects, and the disruption of the allylic double bond abolishes FK506 immunosuppressive activity (inhibition of T cell signaling by immunophilin-ligand complex is associated with loss of calmodulase activity, biochemistry, 1992, 31,3896-3901.), but it has not been reported to use FK506 to carry heparin sulfate to penetrate membrane, nor to use the exocyclic double bond of FK506 as a linking site to conjugate heparin sulfate to guide its penetration membrane into nucleus. The invention takes FK506 as a carrier, is conjugated with heparin sulfate with different structures, carries exogenous heparin sulfate into nucleus, is a more convenient method for researching the regulation and control of the biological function and the structure-activity relationship of cells by the heparin sulfate in the nucleus, and has not been reported so far. However, because the source of the heparin sulfate is difficult, natural extraction and separation are often mixtures, the steps of chemically synthesized heparin sulfate are more, and the yield is low.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides enzymatic chemical synthesis and application of a heparin sulfate and FK506 conjugate; in particular to heparin sulfate with different structures, which is prepared by an immunosuppressant drug FK506 conjugated enzyme with target specificity FKP12 and is applied to research on biological functions and structure-effect relationship of the heparin sulfate in a nucleus. The conjugate provided by the invention is unique in design, mild in synthesis reaction conditions and easy to obtain reaction reagents, can carry heparin sulfate to penetrate into the nucleus through a membrane, and can be used as a small molecule or a probe for researching the relation between the biological function and the structure activity of the heparin sulfate in the nucleus.
The heparin sulfate-FK 506 conjugate is a compound represented by a structural formula I, wherein R2, R3 and R4 are hydrogen or Sulfonyl (SO)3 -) R1 is hydrogen, acetyl (Ac-) or Sulfonyl (SO)3 -) N is 1-6, Y is different aromatic hydrocarbon or 3-10 carbon saturated or unsaturated aliphatic hydrocarbon, or the aromatic hydrocarbon and the saturated or unsaturated aliphatic hydrocarbon are covalently and alternately linked, X is oxygen atom (-O-) or sulfur atom (-S-) or amido bond (-CONH)2-) or ester linkage (-COO-), which can be used for exploring the action mechanism and structure-activity relationship of the nuclear heparin sulfate or treating diseases caused by abnormal nuclear heparin sulfate.
Figure BDA0001656849080000021
Specifically, the purpose of the invention is realized by the following technical scheme:
in a first aspect, the present invention relates to heparin sulfate-FK 506 conjugates of formula I:
Figure BDA0001656849080000031
i, wherein R2, R3 and R4 are hydrogen or Sulfonyl (SO)3 -) R1 is hydrogen, acetyl (Ac-) or Sulfonyl (SO)3 -) N is 1-6; y is aromatic hydrocarbon, saturated or unsaturated aliphatic hydrocarbon of C3-10, or a group formed by covalent alternate linkage of aromatic hydrocarbon and saturated or unsaturated aliphatic hydrocarbon of C3-10, and X is oxygen atom (-O-), sulfur atom (-S-), amide bond (-CONH)2-) or an ester linkage (-COO-).
Preferably, the aromatic hydrocarbon comprises a benzene ring or a heteroaromatic ring. The heteroaromatic ring includes pyridine, pyrimidine, furan, thiophene, indole, or purine.
Preferably, the C3-10 saturated aliphatic hydrocarbon is
Figure RE-GDA0001874270800000032
Wherein m is 3-10, R5 is H, -CH3or-CH2CH3(ii) a C3-10 unsaturated aliphatic hydrocarbon is
Figure RE-GDA0001874270800000033
Wherein I is 3-10, R6, R7 and R8 are respectively selected from H and-CH3、-CH2CH3。
In the second aspect, the invention relates to an enzymatic chemical synthesis method of a heparin sulfate-FK 506 conjugate, which takes p-nitrophenyl β -D-glucuronide as a starting material to synthesize heparin sulfate under the action of a series of recombinant enzymes for heparin sulfate biosynthesis
Figure BDA0001656849080000034
The nitro group on the benzene ring is hydrogenated to form amino group by catalysis, and then the amino group is conjugated with F506 activated by exocyclic double bond to obtain the compound represented by the structural formula I.
Preferably, the series of recombinant enzymes for heparin sulfate biosynthesis are selected from pmSH2,KfiA,NST,NDST C5-Epi,2-OST,6-OST,3-OST。
Preferably, the synthesis method specifically comprises the following steps:
s1, at 30-37 ℃, p-nitrophenyl- β -D-glucuronide is used as a starting material, and then the mixture is added into HS polymerase heparin synthase-2from Pasteurella multocida (pmSH)2) Or alternately reacting with UDP-activated acetylglucosamine (UDP-Glc NAc) or UDP-activated trifluoroacetylglucosamine (UDP-Glc NTFA) or UDP-activated glucuronic acid (UDP-glcA) under the catalysis of KfiA to extend to pentose; then according to the requirements of target structure making it pass through N-sulfonyltransferase (N-deacetylase/N-sulfotransferase, NDST), 2-O-sulfonyltransferase (2-O-sulfonyltransferase, 2-OST) and C-sulfonyltransferase5-isomerase (C)5-epimerase,C5-Epi), 6-O-sulfonyltransferase (6-O-sulfonyltransferase, 6-OST), 3-O-sulfonyltransferaseCatalyzing by one or more of transferase (6-O-transferase, 3-OST) to obtain the repetitive disaccharide unit heparin sulfate
Figure BDA0001656849080000041
S2, under the protection of hydrogen, dissolving the repetitive disaccharide unit heparin sulfate into water, carrying out catalytic hydrogenation by using 10% Pd/C, and stirring for half an hour at normal temperature to obtain the heparin sulfate containing aromatic amine
Figure BDA0001656849080000042
S3, under the protection of nitrogen, taking anhydrous tetrahydrofuran as a solvent, specifically activating the exocyclic double bond of FK506, and then reacting with linkers (linkers) with different lengths to obtain the FK506 derivative containing the Linker
Figure BDA0001656849080000043
S4, under the protection of nitrogen, mixing the aromatic amine-containing heparin sulfate and FK506 derivatives containing linkers into 1, 4-dioxane and 20-150 mu M sodium acetate buffer solution, and fully stirring for 3-5 hours at normal temperature to obtain the heparin sulfate-FK 506 conjugate.
Preferably, in step S2, the hydrogen source used for the catalytic hydrogenation is selected from one or more of hydrogen gas and ammonium formate.
Preferably, in step S3, the reaction with the linker after specifically activating the exocyclic double bond of FK506 includes the following steps:
a1, FK506 and compound using anhydrous dichloromethane as solvent
Figure BDA0001656849080000051
Reacting with benzildimethylketal in an amount of 1:1.05 to 1.2:0.01 to 0.02 to produce a compound 6
Figure BDA0001656849080000052
A2, taking anhydrous dichloromethane as solvent, and carrying out mixed reaction on the compound 6, Y-H and dicyclohexyl diimine according to the mass ratio of 1:1.05-1.2:1.1-2 to generate the FK506 derivative containing the linker.
Preferably, in step S4, the pH of the sodium acetate buffer is 5.0 to 6.0; the volume ratio of the sodium acetate buffer solution to the 1, 4-dioxane is 1:1-1: 4. The sodium acetate buffer solution is formed by mixing sodium acetate and acetic acid.
In a third aspect, the invention relates to the use of the heparin sulfate-FK 506 conjugate as a tool small molecule or probe for researching the biological function and/or structure-activity relationship of the nuclear heparin sulfate.
In a fourth aspect, the invention relates to the use of said heparin sulfate-FK 506 conjugate in the preparation of a medicament for the treatment of a disease caused by an abnormality of intranuclear heparin sulfate.
Compared with the prior art, the invention has the following beneficial effects:
1. the method can covalently couple two substances with large difference in polarity, one is heparin sulfate with a structure with extremely high water solubility and the other is FK506 derivatives with extremely high fat solubility, and the heparin sulfate-FK 506 conjugate I is obtained with high yield;
2. the activation of the ring of the exocyclic double bond of FK506 related by the method adopts reactions compatible with other functional groups of FK506, such as click reaction and the like, avoids fussy protection and deprotection steps, ensures the yield of FK506 activation, and is used for preparing a subsequent heparin sulfate-FK 506 conjugate;
3. to which the method of the invention relates14The C isotope heparin sulfate is firstly marked by adopting an enzyme catalysis method, the condition is mild, the yield is high, and the C isotope heparin sulfate is catalyzed by the enzyme14C isotope labeling and radioactivity intensity determination and analysis show that the obtained conjugate can greatly improve the membrane penetration and nucleus penetration capability of the heparin sulfate, and other prior art is difficult to complete and cannot be compared with the existing technology;
4. the heparin sulfate-FK 506 conjugate prepared by the synthetic method can be used as a small molecular tool or a probe for exploring the biological function and the specific action mechanism of the nuclear heparin sulfate after penetrating into a nucleus, and the structure-activity relationship of the nuclear heparin sulfate is excavated and used for treating and diagnosing diseases caused by the change of the nuclear heparin sulfate. Meanwhile, the method can be used as a biological function and action mechanism exploration model of other physiological substances with large water solubility in the nucleus.
Drawings
Other features, objects and advantages of the present invention will become more apparent upon reading of the detailed description of the non-limiting embodiments with reference to the following drawings:
FIG. 1 is a synthetic scheme of the heparin sulfate-FK 506 conjugate of the present invention;
FIG. 2 is an exemplary roadmap for activation of FK 506;
FIG. 3 is a synthetic scheme of FK 506- heparin sulfate conjugates 10, 13.
Detailed Description
The present invention will be described in detail with reference to examples. The following examples are given by way of illustration of the present invention, and the detailed embodiments and specific procedures are given by using heparin sulfate tetrasaccharide and heparin sulfate decadisaccharide as representative structures of heparin sulfate and covalent coupling of FK506 with thioether bond, which will help those skilled in the art to further understand the present invention, but will not limit the present invention in any way. It should be noted that it would be apparent to those skilled in the art that several modifications and improvements can be made without departing from the inventive concept. All falling within the scope of the present invention.
Example 1 Synthesis of heparin sulfate-FK 506 conjugate
This example specifically relates to the synthesis of heparin sulfate tetrasaccharide-FK 506 conjugate (10) and heparin sulfate decadisaccharide-FK 506 conjugate (13), the reaction scheme is shown in FIG. 1.
Synthesis of linker-containing FK506 derivatives
As shown in FIG. 2, FK506 derivatives containing Linker are obtained by activating exocyclic double bond of FK506 specifically via different reactions, and reacting with Linker (Linker) activated in different lengths.
1.1 Synthesis of Compound 6
FK506(0.404g,0.5mmol), and 2-mercaptoacetic acid (39.5mg,0.52mmol), benzil dimethyl ketal (6)4mg,25.0nmol) was dissolved in 8mL of anhydrous dichloromethane, stirred under 365nm uv irradiation for 15 minutes and purified directly on a silica gel column (ethyl acetate/acetone ═ 1:1, V/V) to give 443.5mg of white solid in 99% yield. (Rf ═ 0.51).1HNMR(400MHz,CDCl3):5.32-5.19(d,J＝52.0Hz,1H),5.07-4.98(m,2H), 4.59-4.40(m,1H),3.99-3.54(m,3H),3.41-3.26(m,10H),3.21-2.19(d,J＝8Hz,2H) 3.05-2.99(m,2H),2.88-2.36(m,11H),2.35-2.24(m,2H),2.19-1.22(m,28H),1.06-0.72(m, 11H).ESI-MS(m/z),C42H73NO14S(M):[M-H]-Calculated 894.4679, found 894.4685.
1.2 Synthesis of Compound 7
Under nitrogen protection, compound 6(0.447g,0.5mmol), and N-hydroxysuccinimide (59.8mg,0.52mmol), dicyclohexyldiimine (DDC) (111.24mg,0.54mmol) were dissolved in 20mL of anhydrous tetrahydrofuran, respectively, stirred at room temperature for 2 hours, precipitated by adding petroleum ether, filtered, concentrated under reduced pressure, and the crude product was purified by silica gel column (ethyl acetate/acetone ═ 1:1, V/V) to give 446.5mg of a white solid in 90% yield. Rf is 0.53.1H NMR(400MHz, CDCl3):5.32-5.18(d,J＝56.0Hz,1H),5.08-4.99(m,1H),4.57-4.33(m,2H),4.01-3.53(m, 3H),3.42-3.26(m,11H),3.05-2.98(m,2H),2.86(d,J＝16Hz,2H),2.69(d,J＝16Hz,2H), 2.37-1.86(m,14H),1.78-1.56(m,15H),1.45-1.32(m,10H),1.20-1.06(m,5H),1.03-0.86(m, 8H).13H NMR(100MHz,CDCl3):213.1,196.5,172.3,169.2,169.1,168.9,166.1,165.8, 164.5,157.1,138.9,132.8,132.6,129.6,129.4,122.8,97.1,84.2,75.1,93.6,72.8,70.3,56.9, 56.4,51.9,49.3,48.4,43.0,39.9,39.3,34.9,34.7,33.8,32.6,32.5,31.2,30.5,29.5,27.5,26.2,25.6,25.5,25.4,24.9,24.5,20.5,16.3,15.9,14.3,9.6.ESI-MS(m/z)C50H76N2O16S(M): [M+Na+]Calculated 1015.4808, found 1015.4815.
2, synthesis of FK 506-heparin sulfate conjugates 10 and 13, the synthetic route is shown in FIG. 3.
2.1 Synthesis of p-nitrophenyl- β - (2-N-sulfonylglucose- α -1, 4-glucuronic acid- β -1,4- (2-N-sulfonylglucose) - α -1, 4-glucuronic acid) glycoside (8)
315mg of p-nitrophenyl- β -D-glucuronide (1mmoL) is dissolved in 20mL of Tris (25mM, pH 7.2) buffer solution, the concentration of manganese chloride in the reaction solution is 5mM, then 1.25mmoL of UDP-GlcNTFA and 1.05mg of polymerase pmHS2 are added, respectively, the reaction is carried out overnight at 30 ℃, the reaction progress is monitored by Poly II-HPLC, a C18 chromatographic column (1.5X 70cm, Biotage, 0-100% aqueous methanol (containing 0.1% trifluoroacetic acid) is used as a mobile phase, the flow rate is 2mL/min) to purify the disaccharide, the white powder is dried by centrifugation and concentrated to be directly dissolved in 20mL of Tris buffer solution containing 5mM manganese chloride (25mM, pH 7.2), UDP-GlcA is added to 1.25mmoL and reacted with the polymerase 2, 37 ℃ after purification by C18 column (as ESI and purification method), the procedure is repeated, the steps of elongation of the sugar is prepared, the glucose is purified by UDP-GlcA-GlcNAcifyl transferase, the reaction is carried out after the reaction is carried out on a 5mM sulfuric acid-N-PBS buffer solution, the supernatant, the reaction solution is purified by adding 0.82, the supernatant of 1.7.2% of acetyl sulfonyl transferase, the supernatant of heparin sulfonylurea, the reaction solution, the reaction is carried out after the reaction is carried out, the reaction is carried out overnight, the reaction is carried out, the reaction progress is carried out, the reaction progress is carried out, the reaction is carried out, the purification is carried.1H NMR(600MHz,CDCl3): 8.23(d,J＝12Hz,2H),7.21(d,J＝12Hz,2H),5.63(d,J＝3.6Hz,1H)，5.59(d,J＝3.6Hz,1H)， 5.31(d,J＝7.2Hz,1H)，4.56(d,J＝7.2Hz,1H),4.13(d,J＝7.2Hz,1H),3.98-3.20(m,18H). ESI-MS,m/z:C30H43N3O29S2Calculated 973.14, found 973.46.
2.2 Synthesis of p-aminophenyl-O- β - (2-N-sulfonyl-glucose- α -1, 4-glucuronic acid- β -1,4- (2-N-sulfonyl-glucose) - α -1, 4-glucuronic acid) glycoside (9)
195mg of compound 8 solution was added to double distilled water with 20mg of 10% Pd/C, hydrogen was introduced to catalyze and quantitatively reduce nitro to amino, and the mixture was filtered through celite to obtain 188mg of compound 9. ESI-MS, calculated value of m/z C30H45N3O27S2943.17, found 943.56.
2.3 Synthesis of p-nitrophenyl β -heparin sulfate dodecaglycoside (11)
315mg of p-nitrophenyl- β -D-glucuronide (1mmoL) is dissolved in 20mL of Tris (25mM, pH 7.2) buffer solution, the concentration of manganese chloride in the reaction solution is 5mM, then 1.25mmoL of UDP-GlcNTFA and 1.05mg of polymerase pmHS2 are added in sequence, the reaction is carried out overnight at 30 ℃, the reaction progress is monitored by Poly II-HPLC, C18 column chromatography (1.5X 70cm, Biotage, 0-100% methanol aqueous solution (containing 0.1% trifluoroacetic acid) is used as a mobile phase, the flow rate is 2mL/min) is purified to obtain disaccharide, the disaccharide is purified by centrifugation, concentration and drying to obtain white powder, the white powder is directly dissolved in 20mL of Tris buffer solution (25mM, pH 7.2) containing 5mM of manganese chloride, UDP-GlcA is added in sequence with 1.25mmoL of UDP-GlcA and the polymerase pmHS2 is reacted at 37 ℃, the white powder is purified by C18 column (as the disaccharide monitoring and purification method), the production yield of the disaccharide is monitored by alternately repeating the steps 9, 9 mg of heparin-D- β% of heparin-sulfuric acid (the same as the yield).1H NMR(600MHz,CDCl3):8.21(d,J＝12Hz, 2H),7.18(d,J＝12Hz,2H),5.34(m,6H)，5.24(d,J＝5.2Hz,1H)，4.47(m,5H)，4.03(d,J＝6.0 Hz,1H),3.89-3.24(m,60H),2.02(s,18H).ESI-MS,m/z:C90H131N7O69Calculated 2413.69, found 2416.92.
2.4 Synthesis of p-aminophenyl β -heparin sulfate dodecaglycoside (12)
48.3mg of compound 11 solution was added to double distilled water, 5mg of 10% Pd/C was added, hydrogen was introduced to catalyze and reduce nitro to amino, and after filtration through celite, 45.0mg of compound 12 was obtained by centrifugation and concentration, with a yield of 94.3%. ESI-MS, m/z: C90H133N7O67Calculated 2383.72, found 2384.92.
2.5 Synthesis of heparin sulfate tetrasaccharide-FK 506 conjugate (10)
11.4mg of Compound 9(0.012mmoL) were dissolved in 5mL of 50. mu.M sodium acetate buffer solution (pH 5.5) and 1, 4-dioxane (V: 1), and then a1, 4-dioxane solution containing 9.93mg of Compound 7(0.01mmoL) was droppedAddition, dropwise addition over 5 minutes, stirring for 4 hours, monitoring the progress of the reaction by HPLC, C18HPLC preparative liquid phase purification to 12.4 mg. (UV-254 nM, mobile phase 0% -100% aqueous methanol (0.1% trifluoroacetic acid), 68% yield ESI-MS, m/z: C76H116N4O40S3Calculated 1820.63, found 1821.21.
2.6 Synthesis of heparin sulfate decadisaccharide FK506 conjugate (13)
28.6mg of Compound 12(0.12mmoL) are dissolved in 5mL of sodium acetate buffer pH 5.550 mM and 1, 4-dioxane (V: 1), followed by dropwise addition of 9.93mg of Compound 7(0.01mmoL) to the 1, 4-dioxane solution within 5 minutes, stirring for 5 hours, monitoring the progress of the reaction by HPLC, C18HPLC preparative liquid phase purification to give 15.7mg of the target compound. (UV-254 nM, mobile phase 0% to 100 aqueous methanol with 0.1% trifluoroacetic acid) in 48% yield. ESI-MS, m/z: C136H204N8O80S calculated 3261.18, found 3263.41.
Example 2 FK 506-heparin sulfate conjugates 10,13 determination of the ability to penetrate membranes into the nucleus
Isotope of carbon monoxide14C labeling method and measurement of nuclear radioactivity: UDP-substituted under the action of polymerase pmSH214CGlc A was introduced to heparin sulfate and FK506 conjugates 10 and 13 to give isotopes14C labeling of the conjugate of the non-reducing end of heparin sulfate, C18HPLC detection of the progress of the reaction, separation and purification by preparative HPLC, concentration and drying14Incubating the C-labeled conjugate with cells for several hours, breaking membranes, centrifuging to separate cell nuclei, and measuring the radioactivity of the cell nuclei by a liquid scintillation counter to obtain14C label unconjugated heparin sulfate compound 9,12 as control.
The method comprises the following specific operations: will be 1x105Planting HEK293T cells in 6-well plate, culturing until the cells reach exponential growth phase and the concentration reaches 60% -80%, adding 5000cpma compound to be tested, repeating 3 wells for each sample, and culturing in incubator (37 deg.C, 5% CO)2) After the incubation is finished, carefully washing the cells with the medium for 3 times to remove the radioactive substances outside the cell membranesThe cell membrane was broken with 0.2% Triton X-100 ice, the nuclei were collected by centrifugation at 15,000 Xg, and the radioactivity was measured by liquid scintillation counter.
The measurement results are shown in the following table 1, which shows that the ability of the heparin sulfate-FK 506 conjugate to penetrate into the membrane and the core is obviously enhanced, and the strength is more than 10 times.
TABLE 1 results of the transmembrane-nuclear assay of conjugates 10,13
Figure BDA0001656849080000091
Note that a represents the average of the measurement results of three independent experiments.
The foregoing description of specific embodiments of the present invention has been presented. It is to be understood that the present invention is not limited to the specific embodiments described above, and that various changes and modifications may be made by those skilled in the art within the scope of the appended claims without departing from the spirit of the invention.

Claims (8)

1. A heparin sulfate-FK 506 conjugate has a structural formula shown in formula I:
Figure FDA0002322219260000011
i, wherein R1H, -Ac, or-SO3,R2＝R3＝R4H, or-HSO3And n is 1 to 6.
2. An enzymatic chemical synthesis method of a heparin sulfate-FK 506 conjugate is characterized in that p-nitrophenyl- β -D-glucuronide is used as a starting material, and the heparin sulfate is synthesized under the action of a series of recombinant enzymes for heparin sulfate biosynthesis
Figure FDA0002322219260000012
Carrying out Pd/C catalytic hydrogenation on nitro on a benzene ring to obtain amino to obtain aromatic amine-containing heparin sulfate, and then reacting with FK506 activated by an exocyclic double bond to prepare the heparin sulfate-FK 506 conjugate;
the structural formula of the FK506 is as follows:
Figure FDA0002322219260000013
the structural formula of the heparin sulfate-FK 506 conjugate is shown as a formula I:
Figure FDA0002322219260000021
i, wherein R1H, or-Ac, or-HSO3,R2＝R3＝R4H, or-HSO3And n is 1 to 6.
3. The method of the enzymatic chemical synthesis of heparin sulfate-FK 506 conjugate according to claim 2, wherein the method comprises the steps of:
s1, at 30-37 deg.C, using p-nitrophenyl- β -D-glucuronide as initial material, in HS polymerase pmSH2Or alternately reacting with UDP-activated acetylglucosamine or UDP-activated trifluoroacetylglucosamine and UDP-activated glucuronic acid under the catalysis of KfiA to prolong the reaction time to pentose; then according to the requirements of target structure making it pass through N-sulfonyltransferase, 2-O-sulfonyltransferase and C5Catalyzing by one or more of-isomerase, 6-O-sulfonyltransferase and 3-O-sulfonyltransferase to obtain repeated disaccharide unit heparin sulfate
Figure FDA0002322219260000022
S2, under the protection of hydrogen, dissolving the repetitive disaccharide unit heparin sulfate into water, carrying out catalytic hydrogenation by using 10% Pd/C, and stirring for half an hour at normal temperature to obtain the heparin sulfate containing aromatic amine
Figure FDA0002322219260000023
S3, under the protection of nitrogen, taking anhydrous tetrahydrofuran as a solvent, specifically activating the exocyclic double bond of FK506, and then reacting with a linker to obtain the FK506 derivative containing the linkerBiological organisms
Figure FDA0002322219260000024
S4, under the protection of nitrogen, mixing and dissolving the aromatic amine-containing heparin sulfate and FK506 derivatives containing the linker in 1, 4-dioxane and 20-150 mu M sodium acetate buffer solution, and fully stirring for 3-5 hours at normal temperature to obtain the heparin sulfate-FK 506 conjugate.
4. The method for the enzymatic chemical synthesis of heparin sulfate-FK 506 conjugate as set forth in claim 3, wherein the hydrogen source used in the catalytic hydrogenation is one or more selected from hydrogen and ammonium formate in step S2.
5. The method of claim 3, wherein the step of reacting with linker after the specific activation of exocyclic double bond of FK506 in step S3 comprises the steps of:
a1, FK506 and compound using anhydrous dichloromethane as solvent
Figure FDA0002322219260000031
Reacting with benzildimethylketal according to the mass ratio of 1:1.05-1.2: 0.01-0.02 to generate a compound 6
Figure FDA0002322219260000032
A2, taking anhydrous dichloromethane as solvent, and mixing the compound 6 and the compound
Figure FDA0002322219260000033
And dicyclohexyldiimine at a mass ratio of 1:1.05-1.2:1.1-2 to form the FK506 derivative containing a linker.
6. The method for the enzymatic chemical synthesis of heparin sulfate-FK 506 conjugate as set forth in claim 3, wherein the pH of said sodium acetate buffer solution is 5.0-6.0 in step S4; the volume ratio of the sodium acetate buffer solution to the 1, 4-dioxane is 1:1-1: 4.
7. Use of the heparin sulfate-FK 506 conjugate of claim 1 as a small tool molecule or probe for studying the biological function and/or structure-activity relationship of heparin sulfate in the nucleus.
8. Use of the heparin sulfate-FK 506 conjugate of claim 1 in the preparation of a medicament for treating a disease caused by an abnormality of heparin sulfate in the nucleus.
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