CN104293859A - Enzymatic synthesis method of galactose ligand molecule - Google Patents
Enzymatic synthesis method of galactose ligand molecule Download PDFInfo
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Abstract
The invention relates to an enzymatic synthesis method of a galactose ligand molecule. The ligand molecule is synthesized by adopting biological enzyme catalysis through the method, and the enzymatic synthesis method can overcome the disadvantages of previous chemical methods in the synthesis of similar galactose ligand molecules, and has the advantages of high efficiency, high selectivity, high yield and low toxicity. The galactose ligand molecule prepared in the invention is a carrier raw material capable of modifying liposome, and can be used for coating a drug to form a liver targeting liposome compound, so galactose ligand molecule mediation related drugs target at hepatocytes, the toxicity to normal tissues is reduced, and the hepatopathy prevention and treatment effects of drugs are improved.
Description
Technical field
The invention belongs to medical art, specifically relate to a kind of small-molecular-weight semi-lactosi part, its enzymic synthesis and the Liver targeting liposome utilizing this semi-lactosi part to prepare and hepatic targeting drug.
Background technology
In new drug development, how allowing activeconstituents effectively be targeted in target cell or tissue is one of important research topic.Such as, no matter how good the pharmacological action of certain medicine is, if the tissue that need treat can not be arrived, as lesions position, the pharmacological effect of expectation can not be reached.Except local administration method, usually, active pharmaceutical ingredient (medicine), need through mode administration that is oral or injection, medicine reaches after site of action through blood transportation and plays its pharmacological action.If medicine effectively can not arrive treated tissue, just need the dosage strengthening medicine, but Side effects of pharmaceutical drugs increase thereupon.In order to improve the gathering of medicine at target spot position, need to be positioned target tissue, target organ, target cell or intracellular structure for helping carrier, part or antibody optionally to be concentrated by gi tract or systemic blood circulation by medicine, we claim the administering mode of this orientation to be targeting drug delivery system.
Asialoglycoprotein receptor (ASGPR) is the endocytosis acceptor being positioned at hepatic parenchymal cells surface, can single-minded identification end with the glycoprotein of irreducibility galactose residue or micromolecular compound.Because ASGPR is the peculiar acceptor of hepatic parenchymal cells, be single-mindedly present in hepatic parenchymal cells surface, therefore ASGPR becomes the best acceptor of liver orientation transfer, for the treatment of liver cancer, the orientation expression of hepatic gene and Liver function grade provide good Research approach.Utilize the characteristic of ASGPR, a kind of micromolecular compound containing galactose moiety can be designed, its covalent coupling is obtained liver target liposome in surface of liposome, guide effect is played for drug loading, selectivity improves drug level in hepatic parenchymal cells, extend act on liver fraction medicine timeliness, reduce dosage simultaneously, reduce drug toxicity, improve the therapeutic index of medicine.
Wherein can by the end of ASGPR specific recognition with the glycoprotein of irreducibility galactose residue or micromolecular compound, we are referred to as semi-lactosi part.The synthesis of current semi-lactosi ligand molecular adopts chemical synthesis more.Chemical synthesis technique is more complicated, and synthesis step is many, and productive rate is lower, and agents useful for same is many containing certain toxicity.Such as, (the Wang S N such as Shao-ning, Deng Y H, Xu H, et al.Synthesis of a novel galactosylated lipid and its application to the hepatocyte-selective targeting of liposomal doxorubicin [J] .Eur J Pharm Biopharm, 2006, 62 (1): 32-38.) by cholesterol, calcium lactobionate, quadrol, succinyl oxides etc. are raw material, divide three steps synthesis target products: lactobionic acid dehydration is formed lactone by the first step, then 1-N-[O-β-D-Galactopyranosyl-(1 is generated with quadrol acidylate, 4)-D-Gluconamide]-2-N`-methylamine (LA-ED), second step cholesterol, succinyl oxide and N-hydroxy-succinamide generate N-Hydroxysuccinimidly5-Cholesten-3-yloxy Succinate (CHS-NHS), 3rd step LA-ED and CHS-NHS generates target product (5-Cholesten-3-yl) 4-oxo-4-[2-(lactobionyl amido) ethylamido] butanoate (CHS-ED-LA), if take calcium lactobionate as starting raw material calculated yield, final yield is 10.8%, and previous reaction process can vide infra listed reaction formula:
Again such as, (the Sliedregt L A such as Leo, Rensen P C, Rump E T, et al.Design and synthesis of novel amphiphilic dendritic galactosides for selective targeting of liposomes to the hepatic asialoglycoprotein receptor [J] .J Med Chem, 1999, 42 (4): 609-618.) with a kind of band of chemical method synthesis three galactose units ligand moleculars, synthesis step reaches more than 6 steps, ultimate yield is about 0.1%, this combined coefficient far can not meet actual production demand.
Sum up the preparation method in the past disclosed about semi-lactosi ligand molecular, still have the following disadvantages:
The preparation method of the semi-lactosi ligand molecular 1, recorded in document adopts chemical method, agents useful for same and reaction medium how toxic, and be difficult to eliminate after reaction, product is difficult to the specification of quality meeting injection formulations.
2, chemical synthesis step is more, and by product is many, and purifying products difficulty, can only meet laboratory and prepare requirement on a small quantity, inapplicable suitability for industrialized production.
3, synthetic yield is low, adopts at present the most high yield pulp1 of chemical synthesis to be only about 10%.
4, the reagent that chemical synthesis is used and the by product that reaction produces generally are difficult to recycling, cause certain pollution to environment.
In addition, the semi-lactosi ligand molecular structure of above-mentioned existing method synthesis, can not extend arbitrarily the length of carbon bridge easily to obtain the ligand molecular of different chain length.And in molecule galactosyl and liposome top layer space length can remarkably influenced ASGPR to the recognition capability of liposome, in general, distance longer, the recognition capability of ASGPR to galactosylated liposome is higher.Therefore the ligand molecular of existing method synthesis is difficult to adjust the ability identified by ASGPR.Meanwhile, the semi-lactosi ligand molecular that back of the body technology is mentioned is positive polarity, with the phosphatide of negatively charged be coupled prepare liposome time, can reduce liposome ZETA current potential, cause obtaining liposome instability, when making to prepare liver target liposome, yield is low.
Summary of the invention
In order to solve the problems of the technologies described above, one of the object of the invention is to provide a kind of novel semi-lactosi ligand molecular, its have at least with the semi-lactosi ligand molecular of prior art quite or better characteristic, be convenient to be coupled with liposome prepare Liver targeting liposome.
Semi-lactosi ligand molecular of the present invention, it comprises three parts: galactosyl, long-chain carbon bridge and sterol base, and its chemical structural formula (I) is:
Wherein n is the integer of 0 to 99, is preferably 1-50, is more preferred from 5-20;
Wherein R is sterol base, is selected from the one in following structure:
Above-mentioned sterol base derives from vegetable seed sterol, cholesterol, ergosterol, sitosterol and stigmasterol respectively.
R ' is galactosyl, and reaction site is C-6 position, is selected from the one in following structure:
Described galactosyl derives from Saccharum lactis, semi-lactosi and lactose respectively.
Said n can between value 0-99, by selecting the ester group compound of different chain length as carbon bridge, be connected between galactosyl and sterol base, the length that can extend arbitrarily carbon bridge to obtain the ligand molecular of different chain length, to increase the recognition capability of ASGPR to carrier.Semi-lactosi ligand molecular of the present invention is almost in neutral molecule or slightly negative electricity, after being coupled, can not reduce the ZETA current potential of liposome, therefore liposome stability is better with electronegative liposome.
The galactose residue that can be identified by asialoglycoprotein receptor (ASGPR) is contained in one end of semi-lactosi ligand molecular of the present invention, and the other end contains sterol base, can be used for being coupled playing Liver targeting effect at surface of liposome.
Another object of the present invention is to provide a kind of new semi-lactosi ligand molecular synthetic method, can avoid using toxic chemical and raw material, meets the specification of quality making injection type targeted drug, environmentally safe, and synthesis step is simple, by product is few, and purifying is easy.
Semi-lactosi ligand molecular of the present invention be adopt sterol, Saccharum lactis (or lactose or semi-lactosi) forms at the catalytic esterification of enzyme with divinyl ester.Particularly, synthetic method of the present invention is in nonaqueous phase, and after using biological enzyme esterification in two steps, adopt recrystallization method and silica gel column chromatography purifying and get final product, described step comprises:
Step 1: sterol base connects carbon bridge step:
After a certain amount of divinyl ester, sterol being dissolved in dehydrated organic solvent, add appropriate enzyme catalyst, temperature 35 DEG C ~ 55 DEG C, after reaction 0 ~ 48h, filter, filtrate is concentrated flings to organic solvent, and namely concentrated solution recrystallization obtains intermediate product (formula II); Reaction is expressed as:
Wherein n=0 ~ 99, are preferably 1-50, are more preferred from 5-20;
Wherein ROH is vegetable seed sterol, cholesterol, ergosterol, sitosterol or stigmasterol, and R is sterol base, and R is selected from least one of following 5 kinds of groups:
Wherein, the molar ratio of sterol and divinyl ester is 1: 1 ~ 20: 1; More preferably 5: 1 ~ 10: 1.
Wherein, described dehydrated organic solvent is wherein at least one or the combination several arbitrarily of octane-iso, normal hexane, hexanaphthene, sherwood oil, acetone; For the consideration of the follow-up targeting vector as medicine, better employing low toxicity low boiling point solvent.
Wherein adopted enzyme catalyst is for being selected from the one in RCL (deriving from Candida rugosa), Chirazyme L-2 lipase (deriving from C.Antarctica type B), Chirazyme L 1 lipase (deriving from Ps.cepacia) and Lipase QLM [purchased from Meito Sangyo Co.Ltd. (Aich, Japan)].
Wherein, the consumption of step 1 enzyme is 1 ~ 20mg/mL reaction system, more preferably 5 ~ 10mg/mL reaction system.
Step 2: galactosyl connects carbon bridge step: after intermediate product formula II step 1 obtained and Saccharum lactis or semi-lactosi or lactose R ' OH dissolve in dehydrated organic solvent, add appropriate another kind of enzyme catalyst and appropriate molecular sieve, be placed in constant temperature 20 ~ 60 DEG C, reaction 0.5 ~ 48h, suction filtration, filter vacuum cryoconcentration flings to organic solvent, concentrated solution chromatography purification, obtain semi-lactosi ligand molecular formula I, above-mentioned reaction process is expressed as follows:
Wherein R ' OH is Saccharum lactis, semi-lactosi or lactose, and R ' is galactosyl, and reaction site is C-6 position, and R ' is selected from the one in following structure:
Wherein, the molar ratio of intermediate product formula II and R ' OH that step 1 obtains is 1: 1 ~ 20: 1; More preferably 3: 1 ~ 10: 1.
In step 2, that selected is candida antarctica lipase B (Candida Antarctica lipases-B, CAL-B), or its commercialization immobilized enzyme Novozym435.
Enzyme catalyst consumption is: 1 ~ 50mg/mL reaction system, more preferably 5 ~ 20mg/mL reaction system.
Wherein, reaction solvent used is selected from the one in pyridine, pyridine+acetone, pyridine+THF, pyridine+tertiary amyl alcohol, pyridine+glycol dimethyl ether, pyridine+DMF and pyridine+DMSO.
In the selection of solvent, because Saccharum lactis is insoluble in non-polar solvent, and enzyme activity in non-polar solvent is higher, and a kind of suitable reaction solvent therefore will be selected to take into account the solubleness of substrate and the activity of enzyme simultaneously.Single solvent is more difficult meets above-mentioned requirements.Therefore wherein optimal selection is acetone: pyridine (1: 1 ~ 1: 10), can comparatively good dissolving substrate and maintenance enzyme live.
As above, semi-lactosi ligand molecular synthetic method of the present invention completes in two steps: the first step, sterol and divinyl ester generation transesterification reaction, generate sterol list vinyl acetate, second step, No. 6 further transesterification reactions of position C of sterol list vinyl acetate and Saccharum lactis or lactose or semi-lactosi, obtain target product.
The order of this two-step reaction also can be put upside down, the i.e. the first step, first generates diacid lactose monoesters, second step by No. 6 position C of Saccharum lactis or lactose or semi-lactosi and divinyl ester generation transesterification reaction, there is transesterify in vinyl ester and the sterol of the diacid lactose monoesters the other end, generates target product again.But find through experiment, due to, if Saccharum lactis first reacts with diacid divinyl ester, easily generate binary sugar ester, and the productive rate of unitary sugar ester is lower; And if diacid divinyl ester first reacts with sterol, by controlling feed ratio, be easy to obtain mono-ester product.Therefore, after the present invention preferentially makes diacid divinyl ester and sterol be obtained by reacting intermediate product, then react with intermediate product and Saccharum lactis, to synthesize target product semi-lactosi ligand molecular.
3rd object of the present invention is to provide a kind of Liver targeting part liposome containing aforementioned semi-lactosi part, and it is formed in surface of liposome by above-mentioned semi-lactosi part covalent coupling.
Liposome is a kind of artificial rust.In water, phospholipid molecule hydrophilic head inserts in water, and liposome hydrophobic tail stretches to air, forms the spherical liposomes of double-deck fat molecule after stirring.Liposome can be used for transgenosis, or preparation medicine, utilize liposome can with the feature of cell membrane fusion, medicine is sent into cell interior.C/PL is the basic substance jointly forming cytolemma and liposome.
Liver targeting part liposome of the present invention is the liposome be made up of aforementioned semi-lactosi part and phosphatide and cholesterol, wherein, each component proportion relation is as follows: the mol ratio of cholesterol and phosphatide is 1: 1 ~ 10, and the molar content of semi-lactosi part is 0.5 ~ 50% of the total mole number of cholesterol and phosphatide.
According to said components proportion relation, membrane process, injection method and reverse evaporation three kinds of methods can be adopted to prepare Liver targeting part liposome, can prepare particle diameter and the stable Liver targeting part liposome of zeta current potential, encapsulation rate is 80% ~ 95%.Preparation method about Liver targeting liposome is existing method, does not describe in detail at this.
Preferably, above-mentioned phosphatide adopts formulation art to be usually used in the lipid types preparing liposome.Particularly, the phosphatide adopted when preparing Liver targeting liposome is neutral phospholipid, negative charge phosphatide or positive charge matrix material.
The present invention also comprises a kind of hepatic targeting drug, and it uses above-mentioned Liver targeting part liposome as encapsulated layer, will be used for the treatment of any one encapsulating mixture in the inner in the gene of hepatopathy, albumen, polypeptide or small-molecule drug.
Technique effect of the present invention is:
(1), semi-lactosi ligand molecular of the present invention and liposome form Liver targeting part liposome, the hydrophilic protective layer of one deck is formed at liposome skin because ligand molecular has hydrophilic galactosyl, add the stability of liposome, cause medicine not easily to leak; Adding of ligand molecular also makes the Zeta potential absolute value of part liposome increase than conventional liposome, semi-lactosi ligand molecular because synthesis be neutrality and slightly negative electricity substantially, current potential absolute value can be increased with after the liposome of negative charge equally if join, increase the stability of part liposome.By the liver target confirmatory experiment of semi-lactosi part liposome, prove that the liposome containing semi-lactosi ligand molecular has obvious liver target.
(2), the present invention synthesis semi-lactosi ligand molecular all adopt ester bond to connect, degradable is corresponding monomer component, and therefore whole carrier system has good biocompatibility and biodegradability.
(3), the present invention adopts the mode of enzyme' s catalysis, complete synthesis semi-lactosi ligand molecular in nonaqueous phase first.The present invention's synthesis method used can overcome many deficiencies of chemical synthesis, agents useful for same and reaction medium nontoxicity, make purifying simple, can be the follow-up carrier as medical injection and favourable condition is provided, enzymatic method synthesis step of the present invention is simple, and by product is few, yield is high, can be applicable to suitability for industrialized production.Because galactosyl is associated with the recognition capability size of ASGPR acceptor with the distance size of surface of liposome, by selecting the divinyl ester of different chain length as carbon bridge, the distance between sterol base and galactosyl can be increased easily, to regulate part liposome by the ability of liver ASGPR Receptor recognition as required.
(4), semi-lactosi ligand molecular of the present invention has amphipathic, can be mixed with liposome as liposome membrane material and phosphatide, improves the liver target of bag medicine carrying thing, reduces medicine systemic side effects.Mediation related drugs is realized (as small molecules hepatosis treating medicine by semi-lactosi part, macromole hepatopathy protective agents comprises albumen, polypeptide and gene) be targeted to liver cell, reduce the toxicity of its normal tissue, improve the hepatopathy prevention effect of medicine.
Accompanying drawing explanation
Fig. 1 is the target product semi-lactosi ligand molecular of embodiment 1
13c NMR schemes.
Fig. 2 is the high-efficient liquid phase chromatogram of the intermediate product (formula II) that embodiment 1 step 1 obtains.
Fig. 3 is the high-efficient liquid phase chromatogram of the target product semi-lactosi ligand molecular (formula I) that the step 2 of embodiment 1 obtains.
Fig. 4 is the distribution plan of blank conventional liposome (below) and semi-lactosi part liposome (top) Zeta potential value.
Fig. 5 is the liposome (GAL10-FL, GAL13-FL, GAL15-FL) of semi-lactosi ligand molecular modification and the comparison of computational results figure of conventional liposome FL and Cell binding rate of different carbon bridge length.
Fig. 6 is the DOC semi-lactosi part liposome (GAL-DOC-L, top) that obtains of the method (1) of embodiment 7 and the distribution plan of the Zeta potential value of DOC conventional liposome (DOC-L, below).
Fig. 7 is that the Plasma Concentration c (μ g/mL) of CL and the GAL-DOC-LP of DOC-LP is to the variation relation figure of time t (min).
Embodiment
In order to enable auditor understand the present invention in detail, and verifying the synthetic method of semi-lactosi ligand molecular of the present invention, the Advantageous Effects of semi-lactosi ligand molecular, below enumerating specific embodiment and being explained.
The method of enzyme' s catalysis semi-lactosi ligand molecular of the present invention, is divided into two steps, the first step, sterol and diacid divinyl ester generation transesterification reaction, generate sterol list vinyl acetate, second step, No. 6 further esterifications of C of sterol list vinyl acetate and Saccharum lactis or lactose or semi-lactosi, obtain target product.The order of this two-step reaction also can be put upside down, i.e. the first step, and No. 6 C of Saccharum lactis or lactose or semi-lactosi and diacid divinyl ester react and generate diacid lactose monoesters, second step, the other end vinyl ester of diacid lactose monoesters and sterol esterification, generates target product.Following examples 1-embodiment 5 prepares the specific embodiment of semi-lactosi ligand molecular.
Embodiment 1: reaction raw materials: cholesterol, sebacic acid divinyl ester, Saccharum lactis
Step 1: get 10mL band plug Erlenmeyer flask, add sebacic acid divinyl ester 0.224mmol, cholesterol 0.025mmol, with 5mL dehydration octane-iso ultrasonic dissolution, be placed in airbath isothermal vibration device jolting 30min, add enzyme RCL7.7mg/mL reaction system, start reaction, temperature of reaction 35 DEG C, after reaction 11.47h terminates, filtered and recycled enzyme, filtrate cryogenic vacuum removing octane-iso, then 20 times amount methyl alcohol ultrasonic dissolutions are used, 24h is left standstill at being placed in 0 DEG C, in time having a large amount of crystal to separate out, low temperature rapid filtration under suction, obtain white powdered solid intermediate product (II), productive rate 92%.Reaction process is as shown in below:
The structural characterization of intermediate product (II):
MS:ESI source (+) pattern; Spray voltage 3kV; Capillary temperature is 350 DEG C.MS spectrogram display [M+Na]+peak: 619.5, disclosing product relative molecular mass is 596.5, matches with target compound relative molecular mass 596.2.
NMR data are as follows: 13C-NMR (126MHz, CDCl3) δ: 173.57 (C-28), 171.15 (C-37), 141.50 (C-38), 140.02 (C-6), 122.90 (C-9), 97.76 (C-39), 74.01 (C-2), 56.99 (C-14), 56.43 (C-15), 50.32 (C-7), 42.61 (C-13), 40.03 (C-12), 39.82 (C-22), 38.46 (C-4), 37.30 (C-3), 36.90 (C-5), 36.48 (C-20), 36.09 (C-18), 34.97 (C-29), 34.22 (C-36), 32.21 (C-10), 32.16 (C-8), 29.34 (C-31), 29.32 (C-32, C-33), 29.26 (C-34), 28.53 (C-16), 28.32 (C-23), 28.12 (C-1), 25.30 (C-30), 24.85 (C-35), 24.58 (C-17), 24.13 (C-21), 23.12 (C-25), 22.86 (C-24), 21.33 (C-11), 19.63 (C-27), 19.01 (C-19), 12.16 (C-26).
Step 2: get 10mL band plug Erlenmeyer flask, add intermediate product (II) 0.15mmol, Saccharum lactis 0.04mmol, add 2.1mL dehydrated solvent (pyridine: acetone, 2: 1), mix, be placed in (55 DEG C, 250r/min) preheating 30min in airbath constant temperature oscillator, add 22.8mg lipase CAL-B, molecular sieve 100mg, temperature of reaction 55 DEG C, reaction times 31.14h.After reaction terminates, reaction solution vacuum filtration reclaims enzyme, and filtrate cryogenic vacuum revolves steaming removing organic solvent and obtains white powder.Gained solid silicone column chromatography, column chromatography condition is: chromatographic silica gel (100-200 order), wet method filling chromatographic column, post height 40cm, post footpath 2cm, eluent: hexanaphthene: ethyl acetate (95: 5), obtains white solid, productive rate 94%.Reaction process is expressed as follows:
Target product structural characterization:
ESI-MS?m/z：920[M+Na]+.
13CNMR：173.45(C-28),172.80(C-37),139.81(C-6),122.55(C-9),106.23(C-44),84.68(C-40),76.80(C-48),74.96(C-46),73.61(C-2),72.74(C-42),72.67(C-45),71.46(C-39),70.15(C-41),69.62(C-47),66.50(C-38),63.63(C-43),61.59(C-49),56.55(C-14),56.13(C-15),50.02(C-7),42.25(C-4),39.70(C-13),39.49(C-12),38.36(C-22),37.01(C-3),36.58(C-5),36.25(C-20),35.78(C-18),34.50(C-29),34.12(C-36),31.90(C-10),31.80(C-8),29.08(C-31,C-34),29.03(C-32,C-33),28.25(C-1),27.98(C-16),27.95(C-23),25.10(C-30),24.93(C-35),24.24(C-17),23.90(C-21),22.68(C-25),22.44(C-24),21.03(C-11),19.13(C-27),18.71(C-19),11.75(C-26).
1H?NMR：δ5.42(d,J＝4.2Hz,1H),5.15(d,J＝7.8Hz,1H),4.91-4.82(m,2H),4.81-4.62(m,4H),4.59(t,1H),4.54-4.28(m,7H),4.10(dd,J＝9.4,3.3Hz,1H),4.04(t,J＝6.2Hz,1H),2.59-2.44(m,2H),2.35(dt,J＝33.7,7.5Hz,4H),2.06-1.78(m,4H),1.75-1.34(m,14H),1.33-1.02(m,20H),0.99(d,J＝6.4Hz,3H),0.91(d,J＝6.6Hz,6H),0.69(s,3H)。
Target product semi-lactosi ligand molecular
13c NMR figure is shown in Fig. 1.Wherein, MS (Thermo Fisher Scientific Inc.): ESI source (+) pattern, spray voltage 3KV, capillary temperature: 350 DEG C.1H,
13c NMR (Avance III400MHz, Switzerland), solvent: deuterated pyridine, 1H,
13c NMR resolving power is respectively: 400MHz, 101MHz.
The total recovery stating two steps is 86.48%; Much larger than the yield of existing chemical synthesis process.The raw material wherein used and reagent toxicity low, the carbon bridge length between sterol base and galactosyl is directly determined by the chain length of diene ester; Therefore, it is possible to by adopting the distance between two diene acid esters increase sterol bases of different chain length and galactosyl, promote the recognition capability of liver specificity receptor for ligand carrier.
Embodiment 2: completely according to the method for embodiment 1, just change the sebacic acid divinyl ester used into H
2c=CH-OOC-(CH2)
11-COO-CH=CH
2, the enzyme in step 1 changes into and adopts Chirazyme L-2 lipase, and consumption is about 5mg/mL, and solvent is changed to acetone; And the enzyme in step 2 changes employing commercialization immobilized enzyme Novozym435 into, consumption is about 30mg, and solvent is acetone: pyridine 1: 5; The product shape obtained is white waxy solid, and total recovery is 83.26%, and structure is:
The structural characterization of target product is:
MS：[M+Na]+：961.3；
13C?NMR(101MHz,Pyridine-d5)δ173.06,172.40,139.40,122.16,105.88,84.38,79.08,76.42,74.57,73.21,72.35,72.28,71.08,69.76,69.23,66.11,63.25,61.20,56.15,55.74,49.62,41.85,39.31,39.09,37.97,36.61,36.19,35.85,35.39,34.15,33.75,31.50,31.40,29.14,29.05,28.90,28.74,27.85,27.58,24.77,24.59,23.85,23.51,22.29,22.05,20.64,18.74,18.31,11.36.
1H?NMR(400MHz,Pyr)δ5.41(s,1H),5.14(d,J＝7.8Hz,1H),5.12-4.46(m,9H),4.48(d,J＝2.9Hz,1H),4.52-4.28(m,3H),4.16-3.96(m,1H),2.46(dd,J＝27.3,10.6Hz,2H),2.33(dd,J＝24.2,16.7Hz,2H),1.98(s,1H),1.93(d,J＝16.9Hz,1H),1.91-1.65(m,4H),1.91-1.37(m,11H),1.36-0.65(m,26H),1.08-0.95(m,6H),0.95(ddd,J＝117.5,54.7,47.0Hz,14H),0.90(d,J＝6.5Hz,6H),0.68(s,2H)。
Embodiment 3: completely according to method and the condition of embodiment 1, just change the sebacic acid divinyl ester used into H
2c=CH-OOC-(CH
2)
13-COO-CH=CH
2, the enzyme in step 1 changes into and adopts Li Pazi QLM (Lipase QLM, the one of lipase), and consumption is about 10mg/mL, and solvent is changed to sherwood oil; And the enzyme in step 2 changes employing commercialization immobilized enzyme Novozym435 into, consumption is about 35mg, and solvent is pyridine+THF (1: 1); The product shape obtained is white waxy solid, and total recovery is 80.59%, and structure is:
Target product structural characterization:
MS:[M+Na]+peak: 990.1,
13c NMR (101MHz, Pyridine-d5) δ 173.09,172.43,139.40,122.17,105.88,84.36,76.44,74.59,73.22,72.37,71.10,69.77,69.24,66.13,63.26,61.21,56.14,55.73,49.62,41.85,39.30,39.09,37.97,36.60,36.19,35.85,35.38,34.16,33.76,31.50,31.40,29.23,29.10,28.93,28.75,27.85,27.57,24.78,24.60,23.84,23.50,22.28,22.04,20.63,18.73,18.30,11.35.
1h NMR (400MHz, Pyr) δ 5.41 (s, 1H), 5.22-4.48 (m, 13H), 4.81-4.48 (m, 4H), 4.84-4.48 (m, 4H), 4.48-4.15 (m, 3H), 4.15-3.92 (m, 1H), 3.59 (d, J=5.0Hz, 1H), 2.52 (s, 1H), 2.43 (s, 1H), 2.33 (s, 2H), 2.19-1.71 (m, 5H), 1.80 (d, J=9.5Hz, 2H), 1.89-1.71 (m, 3H), 1.73 (s, 1H), 1.63 (s, 1H), 1.60 (s, 3H), 1.52 (dd, J=45.0, 38.5Hz, 8H), 1.37-0.57 (m, 30H), 0.90 (d, J=6.0Hz, 5H), 0.90 (d, J=6.0Hz, 5H), 0.68 (d, J=5.4Hz, 2H), 0.68 (d, J=5.4Hz, 2H).
Embodiment 4: completely according to method and the condition of embodiment 1, the cholesterol just step 1 used changes sitosterol into, and divinyl ester is still sebacic acid divinyl ester, and the Saccharum lactis of step 2 changes lactose into; Enzyme in step 1 changes Chirazyme L-1 lipase into, and consumption is about 6mg/mL, and solvent is changed to sherwood oil; The product shape obtained is white waxy solid, and total recovery is 83.23%, and structure is:
Embodiment 5: completely according to method and the condition of embodiment 1, the cholesterol just step 1 used changes stigmasterol into, and divinyl ester is still sebacic acid divinyl ester, and the Saccharum lactis of step 2 changes semi-lactosi into; Enzyme in step 1 changes Chirazyme L-2 lipase into, and consumption is about 16mg/mL reaction system, and solvent is changed to normal hexane; The product shape obtained is white waxy solid, and total recovery is 87.61%, and structure is:
Below respectively with cholesterol, sebacic acid divinyl ester, Saccharum lactis; The diacid divinyl ester of cholesterol, 13 or 15 carbon, Saccharum lactis; Sitosterol, sebacic acid divinyl ester, lactose are raw material; Stigmasterol, sebacic acid divinyl ester, semi-lactosi are raw material is that example is illustrated synthetic method.After measured, the corresponding semi-lactosi part obtained respectively all has substantially identical particle diameter, the physico-chemical property such as electrical, at this no longer exclusive list.
Be illustrated in figure 1 the semi-lactosi part that embodiment 1 is synthesized
13c NMR spectrogram.At Saccharum lactis
13on C NMR spectrogram, δ 61.4,63.3,63.8 and 72.4ppm belongs to three primary hydroxyls (C-6 ', C-1, C-6) and secondary hydroxyl (C-5) respectively.On the spectrogram of esterification products, δ C-6 chemical shift moves to 66.5ppm to low field, and another two primary hydroxyl chemical shifts do not change, and the C-5 adjacent with C-6 position moves to 71.4ppm to High-Field, and in description of step 2, esterified positions only occurs on C-6 position.Because the esterification selectivity of poly-hydroxy carbohydrate mainly affects by sterically hindered around alcoholic extract hydroxyl group, and enzyme can distinguish these small differences significantly.Saccharum lactis three primary hydroxyls (C-6 ', C-1, C-6) all have higher by enzymatic activity, but the selective catalysis C-6 that lipase CAL-B can be special, instead of other positions.
Prove thus; the enzymic synthesis of semi-lactosi ligand molecular of the present invention;: enzyme catalyst has the regioselectivity of high conservative, during the esterification of particularly catalysis polyhydroxy-sugar, have to certain single monohydroxy acylate relative to chemical synthesis significant advantage.
Again see Fig. 2 and Fig. 3, be respectively the high-efficient liquid phase chromatogram of intermediate product (formula II) that embodiment 1 step 1 obtains and the target product semi-lactosi ligand molecular (formula I) that step 2 obtains.The step 1 of Example 1 and step 2 have been reacted rear unpurified preparation stoste and have been entered high-efficient liquid phase analysis, record color atlas respectively, as shown in Figures 2 and 3.As can be seen from the chromatographic peak of color atlas record, product peak is single, and peak shape is intact, and around inclusion-free peak occurs, the efficient of Enzyme catalyzed synthesis method is described, high specificity, almost occurs without side reaction.Two step enzymic synthesis cumulative yields adopt the yield of the yield × second step semi-lactosi ligand molecular (1) of the intermediate product (formula II) of step 1, calculate result is more than 80%, and in the chemical synthesis of the relevant semi-lactosi ligand molecular of the bibliographical information of existing chemosynthesis, cumulative yield is not all more than 10%.
Prove that the enzymic synthesis of semi-lactosi ligand molecular provided by the present invention relative to another significant advantage of chemical synthesis is: high yield, low by product thus.
Sum up thus, semi-lactosi ligand molecular enzymatic clarification technique of the present invention can overcome the deficiency of the chemical method synthesis technique in the past having bibliographical information, and concrete manifestation is as follows: (1), the present invention biological enzyme agent used is a kind of green catalyst efficiently.Chemical synthesis, for during containing the esterification of polyhydroxy-sugar compounds, is difficult to obtain the single esterification products of certain monohydroxy; And biological enzyme has high regioselectivity, reaction site high conservative, therefore reaction product is single, almost without side reaction.(2), the present invention's biological enzyme synthesis method used, synthesis step is few, only needs two steps; Productive rate is high, and the accumulative productive rate of two step synthesis reaches more than 80%; Reaction conditions is gentle, energy-conserving and environment-protective.(3), the present invention's purifying process used is simple, and isolated reaction substrate can recycle, and greatly reduces production cost.(4), the present invention's related raw material used and reagent is nontoxic or pole hypotoxicity, after simple purification, all easily removes, ensures human administration's security of product.(5), the present invention synthesis semi-lactosi ligand molecular all adopt ester bond to connect, degradable is corresponding monomer component, and therefore whole carrier system has good biocompatibility and biodegradability.
Next, further preparation-obtained for embodiment 1-5 semi-lactosi ligand molecular can be coupled at surface of liposome, form the Liver targeting liposome vectors mediated by semi-lactosi, it is that the semi-lactosi ligand molecular of preparation and cholesterol and phosphatide are made by embodiment 1-5, wherein cholesterol also can replace to Cholesterol sulfate sodium, and phosphatide can be natural phospholipid, also can be synthetic phospholipid, wherein, each component proportion relation is as follows: be 1: 1 ~ 10 by the mol ratio of cholesterol and phosphatide, the molar content of semi-lactosi ligand molecular is Liver targeting liposome prepared by the proportion relation of 0.5 ~ 50% of the total mole number of cholesterol and phosphatide.Further preferably, the molar content of semi-lactosi ligand molecular is 5 ~ 50% of the total mole number of cholesterol and phosphatide.
Particularly, liposome can adopt membrane process, injection method and reverse evaporation to prepare Liver targeting liposome, is below the general introduction of several preparation method:
1, membrane process:
A, take semi-lactosi ligand molecular, cholesterol and phosphatide be placed in right amount revolve steam bottle, add chloroform, ether or chloroform-methanol double solvents and make it to dissolve completely;
B, rotary evaporation in vacuo obtain liposome membrane, dry;
C, in liposome membrane, add glucose solution, PBS, HEPS (4-hydroxyethyl piperazine ethanesulfonic acid) or purified water aquation obtain Liver targeting blank liposomes liquid solution;
D, gene comprised uncorrected gene expression, the plasmid vector carrying gene or virus vector, positive polarity albumen or positive polarity polypeptide and Liver targeting blank liposomes liquid solution are hatched, and to obtain final product.
If the Liver targeting liposome of preparation containing small-molecule drug, adds with matrix material common film forming, or is first dissolved in the aquation solvent in step c in a step.
If the Liver targeting liposome of non-positive polarity albumen or polypeptide is carried in preparation, then first non-positive polarity albumen or polypeptide are dissolved in the aquation solvent in step c.
2, injection method
A, to take semi-lactosi ligand molecular, cholesterol and phosphatide appropriate, and ether or ethanol make it to dissolve completely;
B, injection glucose solution, PBS, HEPS or purification of aqueous solutions, rotary evaporation in vacuo eliminates organic solvent, obtains Liver targeting blank liposomes liquid solution;
C, gene comprised uncorrected gene expression, the plasmid vector carrying gene or virus vector, positive polarity albumen or positive polarity polypeptide and Liver targeting blank liposomes liquid solution are hatched, and to obtain final product.
If the Liver targeting liposome of preparation containing small-molecule drug, adds and dissolves altogether with matrix material, or be first dissolved in the aqueous phase solvent in b step in a step.
If the Liver targeting liposome of non-positive polarity albumen or polypeptide is carried in preparation, then first non-positive polarity albumen or polypeptide are dissolved in the aqueous phase solvent in b step.
3, reverse evaporation
A, to take semi-lactosi ligand molecular, cholesterol and phosphatide appropriate, and chloroform or ether make it to dissolve completely;
B, add the aqueous solution, carry out ultrasonic in short-term, until form stable w/o type emulsion;
C, reduction vaporization organic solvent, after reaching colloidal state, add the solution such as glucose solution, PBS, HEPS, rotates and the gel on wall is come off, and under reduced pressure continues evaporation, eliminate organic solvent, obtain Liver targeting blank liposomes liquid solution;
D, gene comprised uncorrected gene expression, the plasmid vector carrying gene or virus vector, positive polarity albumen or positive polarity polypeptide and Liver targeting blank liposomes liquid solution are hatched, and to obtain final product.
If the Liver targeting liposome of preparation containing small-molecule drug, adds and dissolves altogether with matrix material, or be first dissolved in the aqueous solution in b step in a step.
If the Liver targeting liposome of gene (comprising uncorrected gene expression, the plasmid vector carrying gene or virus vector), albumen or polypeptide (comprising various electrical albumen or polypeptide) is carried in preparation, then first gene, albumen or polypeptide are dissolved in the aqueous solution in b step.
Embodiment 6: the preparation of blank liposome and Property comparison:
1, the preparation method of blank part liposome and conventional liposome:
The formula of A, blank semi-lactosi ligand fluorescence liposome (GAL-FL): EPC: cholesterol CHOL: fluorescent phospholipid NBD-PC: ligand molecular (CHS-SE10-LA that embodiment 1 obtains) (4: 1: 0.04: 0.5, mass ratio);
The formula of B, blank common fluorescent liposome (FL): EPC: CHOL: NBD-PC (4: 1: 0.04); Preparation method: more than precision weighing respectively fill a prescription, dissolve with appropriate chloroform, move in 250mL eggplant-shape bottle, vacuum is revolved steaming and is flung to chloroform, until form even adipose membrane at bottle wall, continue vacuum-drying 24h, after add 5mL PBS (pH7.4) aquation 1 hour, educate and incubate liposome hydrating fluid Probe Ultrasonic Searching 15min (ultrasonic power 200W) after 30min, then 0.45,0.22,0.1 μm of millipore filtration is passed through 3 times successively, namely obtain the present invention and be not loaded with the semi-lactosi part liposome of medicine and blank conventional liposome, fill nitrogen in 4 DEG C and preserve, for subsequent use.
2, the outward appearance of blank common fluorescent liposome and semi-lactosi ligand fluorescence liposome, particle diameter and Zeta potential pH-value determination pH, the results are shown in Table shown in 1 and Fig. 4.
Table 1: conventional liposome compares with part liposome
Adding of ligand molecular, particle diameter and Zeta potential can be made to have slight increase, and size distribution controls within 70nm, size is moderate, meets the requirement of Liposomal formulation correlated quality, and the semi-lactosi ligand molecular added is little to grain diameter influence; Measure again its zeta current potential as Fig. 4 institute, result shows: mix semi-lactosi ligand molecular little on the impact of liposome zeta current potential, illustrates that semi-lactosi ligand molecular of the present invention is neutrality electronegativity slightly partially.
3, the target checking of semi-lactosi part liposome:
Again according to the preparation method of above-mentioned blank liposome, the blank semi-lactosi ligand fluorescence liposome (respectively called after GAL13-FL, GAL15-FL) that 13 carbon chain lengths obtained with embodiment 2 and embodiment 3 again and the semi-lactosi part of 15 carbon chain lengths prepare and blank common fluorescent liposome FL (not containing semi-lactosi part), being diluted to containing phospholipid concentration with serum-free medium is 1mmol/L, for subsequent use.Take the logarithm the cell (HepG2) of growth cycle through digestion, and cell counting, is diluted to concentration for (1.0 ~ 5.0) × 10
4the cell suspension of cells/mL, and be inoculated in 24 orifice plates by 1mL/ hole, in incubator, adherent preculture 24h, changes liquid, washes away not adherent cell with PBS.Getting previously prepared fluorescence labeled fatty acid body (GAL10-FL, GAL13-FL, GAL15-FL and blank conventional liposome FL) adds in Tissue Culture Plate, every hole 0.5mL, with after cell co-culture certain hour by careful for nutrient solution sucking-off, cell is rinsed gently with the PBS of precooling, with removing not with the liposome of cell mortise, totally 3 times, each 0.5mL.Add the PBS solution of 1%TriotnX-100,0.5mL/ hole, jolting that normal temperature is slight mixing 30min, with dissolved destruction cytolemma, discharge and dissolve the NBD-PC fluorescence labeled fatty acid body with Cell binding, measure the fluorescence intensity level (F) of cell pyrolysis liquid, measure the concentration (mg/L) of cell protein in this liquid with BCA protein determination kit.Be that index evaluates the uptake ratio of HepG2 to liposome with the concentration proportion of F value and cell protein, the multiple hole of every increment product parallel testing three, get mean value computation, result is shown in Figure 5.
The wherein blank liposome of dark column representative containing semi-lactosi part, the cylinder of grey represents common blank liposome, by can obviously observe in Fig. 5 semi-lactosi modify blank liposome and Cell binding rate far above conventional liposome, illustrate that semi-lactosi part can guide liposome to enter in cell through ASGPR mediated pathways.
4, the lateral comparison of blank semi-lactosi part lipid for preparing of semi-lactosi part CHS-SE10-LA, CHS-SE13-LA or CHS-SE15-LA of different carbon bridge length
Shown in Figure 5, test us by this and find, the top layer space length of galactosyl and liposome can affect the recognition capability of ASGPR to semi-lactosi part, and its recognition capability size is C-15 > C-13 > C-10.The liposome (GAL10-FL, GAL13-FL, GAL15-FL) of the semi-lactosi ligand molecular modification of different carbon bridge length.
Separately in the experiment of Cell binding rate, also can add lactose at cell culture fluid in advance, investigate its restraining effect, result shows, as adding of ASGPR acceptor inhibitor lactose, the uptake ratio of remarkable reduction cell double Lactose-modified liposome, and conventional liposome is not affected, these results show, semi-lactosi ligand molecular prepared by the present invention is after modified liposome, be situated between under different effect by liver cell ASGPR, have obvious liver target.
After paper examines liposome carries semi-lactosi ligand molecular in the present embodiment, HepG2 cell absorbs situation to it.Consider that DOC can produce killing action to cell, make experimental result produce deviation, therefore we select blank liposome as investigation object.But when liposome is as target medicine carrier, still needing to understand package has difference between the conventional liposome of medicine and semi-lactosi part liposome.
Embodiment 7: the preparation of drug-loaded liposome and comparing:
A: the preparation example of the preparation method that three kinds of medicine carrying semi-lactosi part liposome are different:
(1): membrane process prepares DOC semi-lactosi part liposome (GAL-DOC-L)
Precision weighing phosphatide, cholesterol, Docetaxel DOC, DSPE-PEG2000 (4: 1: 0.3: 0.3), add the 10% semi-lactosi ligand molecular obtained by embodiment 1, dissolve with anhydrous chloroform, move in eggplant-shape bottle, vacuum is spin-dried for bottle wall and forms even adipose membrane.Vacuum-drying 24h, adds pH7.4PBS solution in 45 DEG C of aquations 1 hour, educates and incubate 30min, Probe Ultrasonic Searching (power 200W) 15min, crushed 0.45,0.22,0.1 μm of millipore filtration, inflated with nitrogen, in 4 DEG C of sealings, obtains the Liver targeting liposome of Docetaxel.
(2): injection method prepares DOC semi-lactosi part liposome (GAL-DOC-L)
Precision weighing phosphatide, cholesterol, fluorescent phospholipid (NBD-PC) (4: 1: 0.05), add the 10% semi-lactosi ligand molecular obtained by embodiment 1, with anhydrous alcohol solution, inject pH7.4PBS solution, rotary evaporation in vacuo eliminates ethanol, educates and incubates 30min, Probe Ultrasonic Searching (power 200W) 15min, crushed 0.45,0.22,0.1 μm of millipore filtration, inflated with nitrogen, in 4 DEG C of sealings, obtains fluorescently-labeled Liver targeting liposome.
(3): reverse evaporation prepares DOC semi-lactosi part liposome (GAL-DOC-L)
Precision weighing phosphatide, cholesterol, Docetaxel, DSPE-PEG2000 (4: 1: 0.3: 0.3), add the 10% semi-lactosi ligand molecular obtained by embodiment 1, dissolve with chloroform, add the aqueous solution, carry out ultrasonic in short-term, until form stable w/o type emulsion; Reduction vaporization organic solvent, after reaching colloidal state, add HEPS solution, rotate and the gel on wall is come off, under reduced pressure continue evaporation, eliminate residual chloroform, educate and incubate 30min, Probe Ultrasonic Searching (power 200W) 15min, crushed 0.45,0.22,0.1 μm of millipore filtration, inflated with nitrogen, in 4 DEG C of sealings, obtains the Liver targeting liposome of Docetaxel (DOC).
Research finds, semi-lactosi ligand molecular can affect the recognition capability of ASGPR to liposome in the density of surface of liposome, and density is larger, and recognition capability is stronger; But after the amount of ligand molecular that add exceedes 10% of liposome total amount, easily tackled by Kuffer cell recognition.Therefore semi-lactosi ligand molecular is in time preparing part liposome, and the amount that ligand molecular adds is 10% of liposome quality.
B: the preparation of medicine carrying conventional liposome:
Membrane process is adopted to prepare the conventional liposome (DOC-L) of DOC
Concrete preparation method is as follows: precision weighing EPC, CHOL, DOC, DSPE-PEG2000 (4: 1: 0.3: 0.3), dissolves, move in eggplant-shape bottle with anhydrous chloroform, and vacuum is spin-dried for bottle wall and forms even adipose membrane.Vacuum-drying 24h, adds pH7.4PBS solution in 45 DEG C of aquations 1 hour, educates and incubate 30min, Probe Ultrasonic Searching (power 200W) 15min, crushed 0.45,0.22,0.1 μm of millipore filtration, and inflated with nitrogen is preserved in 5 DEG C of sealings, for subsequent use.
C: medicine carrying semi-lactosi part liposome compares with the physical properties of medicine carrying conventional liposome:
Now obtained by the method (1) of embodiment 7, namely DOC semi-lactosi part liposome (GAL-DOC-L) for preparing of membrane process and DOC conventional liposome (DOC-L) mode of appearance, encapsulation rate, particle diameter and Zeta potential data are as shown in table 2 and Fig. 6.
The parameter of table 2:DOC-L (DOC conventional liposome) and GAL-DOC-L (DOC part liposome) compares
According to the result of upper table 2 and Fig. 6, the drug-loaded liposome that drug-loaded liposome and semi-lactosi ligand molecular are modified is respectively: 139.8nm and 155.6nm, size is moderate, meets the requirement of Liposomal formulation correlated quality, and the semi-lactosi ligand molecular added is little to grain diameter influence; Measure again its zeta current potential, result shows: mix semi-lactosi ligand molecular little on the impact of liposome zeta current potential, illustrates that semi-lactosi ligand molecular of the present invention is neutrality electronegativity slightly partially.
Meanwhile, as shown in table 2, measure its encapsulation rate, result shows: the pastille liposome that semi-lactosi ligand molecular is modified and pastille conventional liposome encapsulation rate are respectively 94.1% and 93.5%.Visible, illustrate that semi-lactosi ligand molecular of the present invention does not make significant difference to encapsulation rate, and adding due to semi-lactosi ligand molecular, encapsulation rate can be increased a little, the hydrophilic protective layer of one deck may be formed at liposome skin with the wetting ability galactosyl of ligand molecular, add the stability of liposome, cause medicine not easily to leak; Simultaneously the adding of ligand molecular, particle diameter is caused to increase to some extent; Adding of ligand molecular also makes the Zeta potential absolute value of part liposome increase than conventional liposome, because the CHS-SE-LA molecular band negative electricity of synthesis, can increase current potential absolute value after adding liposome.
D: medicine carrying semi-lactosi part liposome compares with the pharmacokinetics of medicine carrying conventional liposome
Get 12 SD rat (male and female half and half, 150 ~ 180g), often organize 6, difference according to dosage 10mg/kg tail vein injection DOC-LP (medicine carrying conventional liposome) and GAL-DOC-LP (medicine carrying semi-lactosi part liposome is obtained by the method (1) of embodiment 7).After administration respectively at 5,15,30,60,120,360min adjoins venous blood collection through large rathole, chromatographic condition measures DOC concentration, and with Plasma Concentration c (μ g/mL) to time t (min) mapping, its result is shown in Figure 7.
As shown in Figure 7, the CL value of DOC-LP is less than GAL-DOC-LP, and t1/2 value is greater than GAL-DOC-LP, illustrates that GAL-DOC-LP removes comparatively fast in blood, illustrates higher relevant to GAL-DOC-LP uptake ratio with liver cell.
English shortenings in the application
Claims (5)
1. a synthetic method for semi-lactosi ligand molecular, is characterized in that: described method is under the catalytic condition of enzyme, esterification and synthesizing, and specifically comprises the following steps:
Step 1: sterol base connects carbon bridge step: after a certain amount of divinyl ester, sterol ROH being dissolved in dehydrated organic solvent, add appropriate enzyme catalyst, temperature 35 DEG C ~ 55 DEG C, after reaction 0 ~ 48h, filter, filtrate is concentrated flings to organic solvent, and namely concentrated solution recrystallization obtains intermediate product formula II; Reaction is expressed as:
Wherein n=0 ~ 99;
Wherein ROH is sterol, and R is sterol base, and R is selected from least one of following 5 kinds of groups:
Wherein, the molar ratio of sterol and divinyl ester is 1: 1 ~ 20: 1;
Wherein, described dehydrated organic solvent is wherein at least one or the combination several arbitrarily of octane-iso, normal hexane, hexanaphthene, sherwood oil, acetone;
Wherein adopted enzyme catalyst is be selected from the one in RCL, Chirazyme L-2 lipase, Chirazyme L 1 lipase and Lipase QLM;
Step 2: galactosyl connects carbon bridge step: after intermediate product formula II step 1 obtained and Saccharum lactis or semi-lactosi or lactose R ' OH dissolve in dehydrated organic solvent, add appropriate another kind of enzyme catalyst and appropriate molecular sieve, be placed in constant temperature 20 ~ 60 DEG C, reaction 0.5 ~ 48h, suction filtration, filter vacuum cryoconcentration flings to organic solvent, concentrated solution chromatography purification, obtain semi-lactosi ligand molecular formula I, above-mentioned reaction process is expressed as follows:
Wherein R ' OH is Saccharum lactis, semi-lactosi or lactose, and R ' is galactosyl, and reaction site is C-6 position, and R ' is selected from the one in following structure:
Wherein, the molar ratio of intermediate product formula II and R ' OH that step 1 obtains is 1: 1 ~ 20: 1;
Wherein, selected is candida antarctica lipase B, or its commercialization immobilized enzyme Novozym435;
Wherein, reaction solvent used is selected from the one in pyridine, pyridine+acetone, pyridine+THF, pyridine+tertiary amyl alcohol, pyridine+glycol dimethyl ether, pyridine+DMF and pyridine+DMSO.
2. the synthetic method of semi-lactosi ligand molecular according to claim 1, is characterized in that: described step 1 can be put upside down mutually with the order of step 2.
3. the synthetic method of semi-lactosi ligand molecular according to claim 1 and 2, is characterized in that: the dehydrated organic solvent that step 2 adopts is acetone+pyridine, according to acetone: the mixing of pyridine 1: 1 ~ 1: 10 and forming.
4. the synthetic method of semi-lactosi ligand molecular according to claim 1 and 2, is characterized in that: the sterol ROH of step 1 and the molar ratio of divinyl ester are 5: 1 ~ 10: 1, and the consumption of step 1 enzyme is 1 ~ 20mg/mL reaction system.
5. the synthetic method of semi-lactosi ligand molecular according to claim 1 and 2, it is characterized in that: in step 2, the molar ratio of intermediate product formula II and lactose or semi-lactosi or Saccharum lactis R ' OH is 3: 1 ~ 10: 1, and the consumption of step 2 enzyme is: 1 ~ 50mg/mL reaction system.
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