CN114410717A - Phosphatidyl glucoside and preparation method and application thereof - Google Patents
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Abstract
The invention discloses phosphatidyl glucoside and a preparation method and application thereof, belonging to the technical field of phospholipid synthesis and modification. The phosphatidyl glucoside comprises phosphatidyl glucose, phosphatidyl glucosamine and phosphatidyl-N-acetylglucosamine, and can be prepared by the following method: phosphatidyl choline and glucoside are subjected to transphosphatidylation in a two-phase reaction system under catalysis of phospholipase D to synthesize phosphatidyl glucoside; the phospholipase D is selected from PLDr34. The application of the phosphatidyl glucoside as an active substance in the preparation of liposome and the drug delivery is provided. The phosphatidyl glucosamine and the phosphatidyl-N-acetylglucosamine have the activity and the function of phospholipid and glucoside, and have the function of the phosphatidyl glucosamine, and the phosphatidyl glucoside has the active functionThe method has wider application potential in the aspects of evaluation, liposome preparation, drug delivery and the like.
Description
Technical Field
The invention relates to phosphatidyl glucoside and a preparation method and application thereof, belonging to the technical field of phospholipid synthesis and modification.
Background
Phospholipids (PLs) are a generic name for a class of phosphorus-containing lipids, mostly natural products, and a few obtained by artificial synthesis. PLs contain both polar and non-polar parts, are important natural surfactants, and are widely applied in the fields of food, medicine, cosmetics and the like. The polar head-based portion of PLs is one of the criteria for phospholipid classification, and the fat chain length and saturation of the non-polar portion serve to partition the phospholipid sub-components. The fatty acid composition difference of the phospholipids is closely related to the source, the sn-1 and sn-2 positions of the soybean lecithin are mixed coordination of saturated acid and unsaturated fatty acid, the sn-1 position of the yolk phospholipid is almost all saturated fatty acid phase, the sn-2 position is usually unsaturated fatty acid, 58% of phospholipids of Antarctic krill oil contain n-3PUFAs in at least one position, and 10% of phospholipids contain n-3PUFAs in two positions. The raw materials can be used for modification and modification research of phospholipid.
In recent years, a phosphatidyl glycoside having a glycosyl group as a phospholipid head group has been widely paid attention, and among them, phosphatidyl glucoside (PtdGlc) has been studied most sufficiently, and phosphatidyl glucose is a natural phosphatidyl glycoside and can be isolated from a living body. The synthesis of other configurations of phosphatidylglycosides, such as phosphatidylsucrose, phosphatidylfructose and phosphatidylraffinose, requires transesterification by phospholipase D. Phosphatidylglucose and its derivatives have also been shown to play an important role in glial cell development and differentiation. In addition, many studies have shown that liposomes prepared from phospholipids can reduce the side effects of the encapsulated species on the organism as carriers for targeted delivery, and that phospholipid encapsulation can increase the ability of the encapsulated species to permeate the cell membrane. In addition, a large number of hydroxyl groups in glycosyl group interact to form hydrogen bonds, so that the liposome prepared from the phosphatidyl glucoside has better stability, and has more important application prospects in the aspects of targeted drug delivery and slow release. The kind of glycosyl can affect the liposome size, aggregation degree, dehydration and rehydration stability, for example, the stability of phosphatidyl sucrose glucoside is better than that of phosphatidyl glucose glucoside and phosphatidyl gossypol glucoside.
At present, most of research on phosphatidyl glycoside focuses on glucose, fructose, mannose and raffinose, and the synthesis of glucosamine and N-acetylglucosamine is not reported.
Glucosamine (GlcN) and N-acetyl-D-Glucosamine (GlcNAc) are derivatives of glucose, and the hydroxyl group at the 2-position of glucose is structurally substituted with an amino group or an acetylamino group. GlcN is a natural amino monosaccharide and is also an important component of the cartilage matrix necessary for the synthesis of proteoglycans. GlcNAc is widely present in nature and is also the main monomer of chitin and chitosan. GlcNAc as an important functional monosaccharide has anti-tumor and immunoregulation activities, and participates in liver and kidney detoxification agaro-oligosaccharide. Therefore, compared with the synthesis of the phosphatidyl glucose, the synthesis of the phosphatidyl glucosamine and the phosphatidyl-N-acetylglucosamine has more application prospect.
Disclosure of Invention
Aiming at the prior art, the invention provides two novel phosphatidyl glucoside compounds, namely phosphatidyl glucosamine and phosphatidyl-N-acetylglucosamine, and also provides a preparation method and application of the two phosphatidyl glucoside compounds. The preparation method has the advantages of simple steps, high yield, single structure and no toxic chemicals involved in the synthesis process.
The invention is realized by the following technical scheme:
the phosphatidyl glucoside (comprising phosphatidyl glucose, phosphatidyl glucosamine and phosphatidyl-N-acetylglucosamine) is prepared by the following method: phosphatidyl choline and glucoside are subjected to transphosphatidylation in a two-phase reaction system under catalysis of phospholipase D to synthesize phosphatidyl glucoside; the glucoside is selected from glucose, glucosamine and N-acetylglucosamine; the phospholipase D is selected from PLDr34。
Further, the fatty acid chains on the phosphatidylcholine are both C18:2 linoleic acid, or C16:0 palmitic acid and C18:2 linoleic acid, respectively, or phospholipid with other coordinated fatty acids on the basis of the fatty acid chains.
Further, the phosphatidylcholine is dissolved in an organic solvent to serve as an organic phase; the organic solvent is selected from cyclopentyl methyl ether; the concentration of the phosphatidylcholine in the organic solvent is 10-100 mg/mL.
Further, the glucoside is dissolved in an aqueous solution as an aqueous phase; the aqueous solution is selected from citric acid-sodium citrate buffer solution with pH value of 6.0.
Further, in the two-phase reaction system, the molar ratio of phosphatidylcholine to glucoside is 1: 1-100, preferably 1: 60; the volume ratio of the organic phase to the aqueous phase is 1: 1; the enzyme adding amount of the phospholipase D is 0.2-2.0U, and 1.0U is preferred.
Further, the specific reaction conditions for catalyzing transphosphatidylation are as follows: the reaction system is subjected to water bath reaction at 37-42 ℃ for 4-24 h, preferably 12 h; and after the reaction is finished, centrifuging to take an upper organic phase, and dissolving the product in the upper organic phase to obtain the phosphatidyl glucoside.
The prepared phosphatidyl glucosamine and phosphatidyl-N-acetylglucosamine are used as active substances in the preparation of liposome and the drug delivery.
The phosphatidyl glucosamine and the phosphatidyl-N-acetylglucosamine are two novel phosphatidyl glucosides, have the activities and functions of phospholipid and glucoside, and can generate new performances which are not possessed by common configuration phospholipid, glucoside and phosphatidyl glucosamine, so that the phosphatidyl glucoside has a wider application potential in the aspects of activity function evaluation, liposome preparation, drug delivery and the like.
The invention explores the occurrence condition of the transphosphatidylation of phosphatidylcholine and glucoside, and connects glucosamine and N-acetylglucosamine to the phosphatidylcholine through the transphosphatidylation, so as to synthesize two novel phosphatidylglucosides, namely, phosphatidylglucosamine and phosphatidyl-N-acetylglucosamine.
The various terms and phrases used herein have the ordinary meaning as is well known to those skilled in the art.
Drawings
FIG. 1: TLC results profile.
FIG. 2: HPLC-ELSD result map.
FIG. 3: and (4) carrying out MS analysis on the transesterification product.
FIG. 4: chemical structure schematic diagram of phosphatidyl glucose.
FIG. 5: chemical structure schematic diagram of phosphatidylglucosamine.
FIG. 6: chemical structure schematic diagram of phosphatidyl-N-acetylglucosamine.
Detailed Description
The present invention will be further described with reference to the following examples. However, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention.
The instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like in the prior art and are commercially available in a normal manner unless otherwise specified. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.
The phosphatidylcholine used in the present invention is Soy PC (95%) (SPC, 441601G-50G-I-175, Avanti Polar Lipids) (the phosphatidylcholine, the fatty acid chain of which is mainly two C18:2 linoleic acids, followed by one C16:0 palmitic acid and one C18:2 linoleic acid).
The glucose used in the present invention was purchased from national medicine group chemical reagents, Inc., and glucosamine hydrochloride and N-acetyl-D-glucosamine were purchased from Beijing Solaibao science and technology, Inc.
The phospholipase D used in the invention is PLDr34(Accession Number: MN604233) (this enzyme is described in CN 110564708A, another patent application filed by the applicant of the present invention, namely CN 110564708A, SEQ ID NO. 1), the amino acid sequence of which is shown below (SEQ ID NO. 1).
Amino acid sequence of PLDr 34:
MIISFRLSRPARAALICALALTVLPASPATAADAATPHLDAVERTLREVSPGLEGEVWERTAGNRLDAGADDPAGWLLQTPGCWGDAGCRDRVGTRRLLAKMTENISRATRTVDISTLAPFPNGAFQDAIVAGLKSSAARGNKLTVRVLVGAAPIYHMNVLPSKYRDELVAKLGADARNVDLNVASMTTSKTSFSWNHSKLLVVDGQSVITGGINDWKDDYLETAHPVADVDLALRGPAAASAGRYLDELWSWTCQNRNNIAGVWFASSNGTACMPAMAKDTAPAAPPAAPGDVPAIAVGGLGVGIKRSDPSSAFRPTLPSAADTKCVVGLHDNTNADRDYDTVNPEESALRTLISSAKGHIEISQQDVNATCPPLPRYDIRVYDALAARMAAGVKVRIVVSDPANRGAVGSGGYSQIKSLSEISDTLRDRLALLTGDQGAAKATMCSNLQLATFRSSKSPTWADGHPYAQHHKVVSVDDSAFYIGSKNLYPAWLQDFGYIVESPGAAQQLDAQLLSPQWTHSKETATVDYERGLCHI。
experiment 1: synthesis preference of phosphatidyl glucoside compounds
The method comprises the following steps:
phospholipase D catalyzed transesterification
(1) The organic phase of the transesterification reaction was a solution of cyclopentyl methyl ether containing 10mg/mL phosphatidylcholine.
(2) The aqueous phase of the transesterification reaction was a 0.1M citric acid-sodium citrate buffer solution (pH 6.0) containing 153mg/mL glucose and 1.0U/mL phospholipase D.
(3) The transesterification reaction is carried out in a two-phase reaction system, namely, the organic phase and the water phase prepared in the above are mixed according to the volume ratio of 1:1 and are placed in a rubber-mouth brown finger-shaped bottle.
(4) And (3) placing the sealed brown finger-shaped bottle in a water bath shaker, and reacting for 12 hours at 40 ℃ at 200 r/min.
(5) After the reaction, the mixture was centrifuged (12000r, 2min) to obtain supernatant A.
And (3) replacing the glucose in the step (2) with 167mg/mL glucosamine hydrochloride and 171mg/mL N-acetyl-D-glucosamine to obtain a supernatant B and a supernatant C.
(II) performing thin-layer chromatography analysis on the prepared supernatant A, B and C
(1) On the silica gel precast slab, a proper amount of sample is sampled by a sample sampling capillary tube with the specification of 0.3 multiplied by 100, an organic solvent is dried by a blower after each sample sampling, and the sample sampling is carried out for 3 times.
(2) And (3) placing the silica gel precast slab subjected to sample application in a chromatographic cylinder containing a developing agent, wherein the developing agent is chloroform: methanol: glacial acetic acid: water-50: 25:6:2 (v: v: v: v).
(3) Taking out the silica gel plate after about 20min, blowing the spreading agent on the surface of the prefabricated plate by a blower, and placing the prefabricated plate into iodine steam for developing for about 10 min.
(III) performing high performance liquid chromatography analysis on the prepared supernatant A, the supernatant B and the supernatant C
(1) And (4) passing the supernatant A, the supernatant B and the supernatant C through high-flow-rate nitrogen, and quickly evaporating the organic solvent.
(2) The residue from step (1) was diluted to about 1mg/mL with a reconstitution solution (n-hexane: isopropanol: 81:17) and filtered through a 0.22 μ M organic filter.
(3) Performing high performance liquid chromatography with evaporative light scattering detector (HPLC-ELSD). The chromatographic column is silica gel column YMC DIOL (5 μm, 250 mm. times.4.6 mm), the sample injection amount is 10 μ L, the total flow rate is 1mL/min, and the column temperature is 55 ℃. The yield was determined by measuring the peak area of the corresponding compound.
From the results of thin layer chromatography and high performance liquid chromatography (as shown in fig. 1 and fig. 2), it can be seen that, in the transesterification process using different glycosyl groups as raw materials, the phosphatidyl glucoside (ptglc) has extremely high yield, and the substrate phosphatidyl choline is almost completely degraded. Yield of phosphatidyl-N-acetylglucosamine (ptdcglcnac) secondly, the transesterification process is accompanied by the production of Phosphatidic Acid (PA), a hydrolysis by-product. The yield of the phosphatidyl amidoglucoside (PtdGlcN) is the lowest, and the transesterification catalysis process is accompanied by brown stain of glucosamine, so that the yield is lower.
Example 1: synthesis of phosphatidyl glucoside by PLD (pulsed laser deposition) catalytic enzyme method
The method comprises the following steps:
(1) the organic phase of the transesterification reaction was a solution of cyclopentyl methyl ether containing 10mg/mL phosphatidylcholine.
(2) The aqueous phase of the transesterification reaction was a 0.1M citric acid-sodium citrate buffer solution (pH 6.0) containing 153mg/mL glucose and 1.0U/mL phospholipase D.
(3) The transesterification reaction is carried out in a two-phase reaction system, namely, the organic phase and the water phase prepared in the above are mixed according to the volume ratio of 1:1 and are placed in a rubber-mouth brown finger-shaped bottle.
(4) And (3) placing the sealed brown finger-shaped bottle in a water bath shaker, and reacting for 12 hours at 40 ℃ at 200 r/min.
(5) After the reaction, the mixture was centrifuged (12000r, 2min) to obtain supernatant A.
And (3) replacing the glucose in the step (2) with 167mg/mL glucosamine hydrochloride and 171mg/mL N-acetyl-D-glucosamine to obtain a supernatant B and a supernatant C.
The relative molecular mass of the three phosphatidylglucosides is determined by a Mass Spectrometry (MS) method, the result is shown in figure 3, and according to the analysis of the result, the three phosphatidylglucosides mainly use glycosyl as a hydrophilic head group, and two linoleic acid residues (C18:2) as hydrophobic tail ends. I.e. PtdGlc [ M-H ]]-The corresponding molecular weight is 857.51891; PtdGlcN [ M-H ]]-The corresponding molecular weight is 856.53510; PtdGlcNAc [ M-H ]]-The corresponding molecular weight is 898.54623. Mass spectrometry and liquid phase results show that target phosphatidyl glycoside is generated after transesterification, so that phosphatidyl glucoside, phosphatidyl glucosaminide and phosphatidyl-N-acetylglucosamine are determined to be prepared, and the structures are shown in figure 3.
Experiment 2: key parameter of transphosphatidylation preparation process
The parameters critical to the phospholipid acylation reaction include: in the transesterification, the time of the transesterification reaction (4, 6, 8, 10, 12, 14, 24h) and the molar ratio of substrate PC to saccharide were determined for PLD enzyme addition (0.2, 0.4, 0.6, 0.8, 1.0, 1.5, 2.0U) in the reaction system (n (PC: Glu) ═ 1:1, 1:5, 1:10, 1:15, 1:20, 1:40, 1:50, 1:60, 1:80, 1: 100; n (PC: glcn1: 0.5, 1:1, 1:2.5, 1:5, 1:7.5, 1:10, 1:15, 1: sd 20, 1:30, 1:40, 1: 60; n (PC: GlcNAc) ═ 1:1, 1:2.5, 1:5, 1:7.5, 1:10, 1:15, 1:20, 1:30, 1:40, 1: 60; n (PC: GlcNAc): 1, 1:5, 1:7.5, 1:10, 1:20, 1:1, 1:40, 1: 100; 100) of the yield of the phosphatidylglucoside, 100, and the like were calculated by HPLC method, finally, the optimal synthesis conditions of the phosphatidyl glucoside compounds are determined. When the transesterification reaction time was 12 hours, the amount of PLD added was 1U when the molar ratio of substrate PC to saccharide was 1: 60. Under this optimal reaction condition, the yield of ptglcc was as high as 97.7%, the yield of ptglcnac was secondly 47.9%, and the yield of ptglcnc was 5.4%.
The above examples are provided to those of ordinary skill in the art to fully disclose and describe how to make and use the claimed embodiments, and are not intended to limit the scope of the disclosure herein. Modifications apparent to those skilled in the art are intended to be within the scope of the appended claims.
Sequence listing
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<120> phosphatidyl glucoside, preparation method and application thereof
<141> 2022-03-07
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Asp Ala Ala Thr Pro His Leu Asp Ala Val Glu Arg Thr Leu Arg Glu
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Val Ser Pro Gly Leu Glu Gly Glu Val Trp Glu Arg Thr Ala Gly Asn
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Arg Leu Asp Ala Gly Ala Asp Asp Pro Ala Gly Trp Leu Leu Gln Thr
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Pro Gly Cys Trp Gly Asp Ala Gly Cys Arg Asp Arg Val Gly Thr Arg
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Arg Leu Leu Ala Lys Met Thr Glu Asn Ile Ser Arg Ala Thr Arg Thr
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Val Asp Ile Ser Thr Leu Ala Pro Phe Pro Asn Gly Ala Phe Gln Asp
115 120 125
Ala Ile Val Ala Gly Leu Lys Ser Ser Ala Ala Arg Gly Asn Lys Leu
130 135 140
Thr Val Arg Val Leu Val Gly Ala Ala Pro Ile Tyr His Met Asn Val
145 150 155 160
Leu Pro Ser Lys Tyr Arg Asp Glu Leu Val Ala Lys Leu Gly Ala Asp
165 170 175
Ala Arg Asn Val Asp Leu Asn Val Ala Ser Met Thr Thr Ser Lys Thr
180 185 190
Ser Phe Ser Trp Asn His Ser Lys Leu Leu Val Val Asp Gly Gln Ser
195 200 205
Val Ile Thr Gly Gly Ile Asn Asp Trp Lys Asp Asp Tyr Leu Glu Thr
210 215 220
Ala His Pro Val Ala Asp Val Asp Leu Ala Leu Arg Gly Pro Ala Ala
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Ala Ser Ala Gly Arg Tyr Leu Asp Glu Leu Trp Ser Trp Thr Cys Gln
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Asn Arg Asn Asn Ile Ala Gly Val Trp Phe Ala Ser Ser Asn Gly Thr
260 265 270
Ala Cys Met Pro Ala Met Ala Lys Asp Thr Ala Pro Ala Ala Pro Pro
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Ala Ala Pro Gly Asp Val Pro Ala Ile Ala Val Gly Gly Leu Gly Val
290 295 300
Gly Ile Lys Arg Ser Asp Pro Ser Ser Ala Phe Arg Pro Thr Leu Pro
305 310 315 320
Ser Ala Ala Asp Thr Lys Cys Val Val Gly Leu His Asp Asn Thr Asn
325 330 335
Ala Asp Arg Asp Tyr Asp Thr Val Asn Pro Glu Glu Ser Ala Leu Arg
340 345 350
Thr Leu Ile Ser Ser Ala Lys Gly His Ile Glu Ile Ser Gln Gln Asp
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Val Asn Ala Thr Cys Pro Pro Leu Pro Arg Tyr Asp Ile Arg Val Tyr
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Asp Ala Leu Ala Ala Arg Met Ala Ala Gly Val Lys Val Arg Ile Val
385 390 395 400
Val Ser Asp Pro Ala Asn Arg Gly Ala Val Gly Ser Gly Gly Tyr Ser
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Gln Ile Lys Ser Leu Ser Glu Ile Ser Asp Thr Leu Arg Asp Arg Leu
420 425 430
Ala Leu Leu Thr Gly Asp Gln Gly Ala Ala Lys Ala Thr Met Cys Ser
435 440 445
Asn Leu Gln Leu Ala Thr Phe Arg Ser Ser Lys Ser Pro Thr Trp Ala
450 455 460
Asp Gly His Pro Tyr Ala Gln His His Lys Val Val Ser Val Asp Asp
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Ser Ala Phe Tyr Ile Gly Ser Lys Asn Leu Tyr Pro Ala Trp Leu Gln
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Asp Phe Gly Tyr Ile Val Glu Ser Pro Gly Ala Ala Gln Gln Leu Asp
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Ala Gln Leu Leu Ser Pro Gln Trp Thr His Ser Lys Glu Thr Ala Thr
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Val Asp Tyr Glu Arg Gly Leu Cys His Ile
530 535
Claims (10)
1. The preparation method of the phosphatidyl glucoside is characterized by comprising the following steps: phosphatidyl choline and glucoside are subjected to transphosphatidylation in a two-phase reaction system under catalysis of phospholipase D to synthesize phosphatidyl glucoside; the glucoside is selected from glucose, glucosamine and N-acetylglucosamine; the phospholipase D is selected from PLDr34。
2. The method for producing a phosphatidylglucoside of claim 1, wherein: the fatty acid chain on the phosphatidylcholine is two linoleic acids of C18:2, or is palmitic acid of C16:0 and linoleic acid of C18:2 respectively.
3. The method for producing a phosphatidylglucoside of claim 1, wherein: dissolving the phosphatidylcholine in an organic solvent to obtain an organic phase; the organic solvent is selected from cyclopentyl methyl ether;
the glucoside is dissolved in an aqueous solution to be used as an aqueous phase; the aqueous solution is selected from citric acid-sodium citrate buffer solution.
4. The method for producing a phosphatidylglucoside of claim 3, wherein: the organic solvent is selected from cyclopentyl methyl ether; the concentration of the phosphatidylcholine in the organic solvent is 10-100 mg/mL.
5. The method for producing a phosphatidylglucoside of claim 1, wherein: in the two-phase reaction system, the molar ratio of phosphatidylcholine to glucoside is 1: 1-100; the volume ratio of the organic phase to the aqueous phase is 1: 1; the enzyme adding amount of the phospholipase D is 0.2-2.0U.
6. The method for producing a phosphatidylglucoside of claim 5, wherein: in the two-phase reaction system, the molar ratio of phosphatidylcholine to glucoside is 1: 60; the volume ratio of the organic phase to the aqueous phase is 1: 1; the enzyme adding amount of the phospholipase D is 1.0U.
7. The method for producing a phosphatidylglucoside of claim 1, wherein: the specific reaction conditions for catalyzing the transphosphatidylation are as follows: the reaction system is subjected to water bath reaction at 37-42 ℃ for 4-24 h.
8. A phosphatidylglucosamine or phosphatidyln-acetylglucosamine, characterized in that: is a compound generated by transphosphatidylation of phosphatidylcholine and glucoside, wherein the glucoside is selected from glucosamine and N-acetylglucosamine.
9. The phosphatidylglucosamine or phosphatidyln-acetylglucosamine of claim 8, wherein: is prepared by the preparation method of any one of claims 1 to 7.
10. Use of the phosphatidylglucosamine or phosphatidyln-acetylglucosamine of claim 8 or 9 as an active substance, for the preparation of liposomes, for drug delivery.
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| CN110564708A (en) * | 2019-10-19 | 2019-12-13 | 中国海洋大学 | Recombinant phospholipase D and application thereof in synthesis of phosphatidylserine or other phospholipids |
| CN111514152A (en) * | 2020-05-18 | 2020-08-11 | 中国海洋大学 | Application of n-3PUFA phosphatidyl glycoside in preparation for improving blood brain barrier damage |
| CN111621535A (en) * | 2020-05-18 | 2020-09-04 | 中国海洋大学 | Preparation method and application of phosphatidyl glycoside |
| CN113151377A (en) * | 2021-04-08 | 2021-07-23 | 江苏澳新生物工程有限公司 | Enzymatic preparation method for preparing glucosamine from glucose and enzyme application |
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