CN112679575A - Perylene bisimide-glycopeptide self-assembly anti-freezing compound and preparation method and application thereof - Google Patents
Perylene bisimide-glycopeptide self-assembly anti-freezing compound and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a perylene bisimide-glycopeptide self-assembly anti-freezing compound and a preparation method and application thereof. The preparation method comprises the steps of taking perylene anhydride as a raw material, and preparing perylene imide compounds PBI-Man, PBI-Gal and PBI-Glu modified by mannose, galactose or glucose through condensation reaction, click reaction and deacetylation reaction; the compound has an inhibiting effect on the growth of ice crystals; wherein, MGA value of the inhibiting effect of the mannose and glucose modified perylene imide-glycopeptide molecules on the ice crystals under the concentration of 40mg/mL is 38.0 percent of sodium chloride-potassium chloride buffer solution; the freezing survival rate of the perylene imide glycopeptide molecules modified by mannose, galactose and glucose to human cervical carcinoma cells HeLa is improved from 18.62 percent to 44.24 percent, 38.08 percent and 22.20 percent.
Description
Technical Field
The invention relates to the technical field of antifreeze materials, in particular to a perylene bisimide glycopeptide self-assembly antifreeze compound and a preparation method and application thereof.
Background
Cryopreservation techniques have wide applications in the fields of medicine, biology, pharmacy, food science and the like, and are the basis of modern biology and medical development, particularly in cryopreservation of cells, tissues, organs and the like. Among them, a cryopreservation agent, i.e., an antifreeze material, is a key to the application of cryopreservation technology. Currently, the commonly used cryopreservative agents are mainly dimethyl sulfoxide (DMSO), glycerol and the like. However, the addition of DMSO and glycerol can cause cell damage to different degrees, for example, the rate of the embryonic stem cell reviving in 5% DMSO solution is only about 5%. In addition, the existing low-temperature preservative has the problems of large dosage, toxicity, short preservation time, low survival rate and the like. Therefore, the development of new antifreeze materials has become a focus of research by scientists in various countries around the world.
The natural antifreeze protein and the antifreeze glycoprotein can effectively protect animals and plants from death caused by freezing of water in vivo. However, the low content of antifreeze protein and antifreeze glycoprotein, and the imperfect synthesis technology of the current genetic engineering, limit the application thereof. Biomimetic synthesis of antifreeze materials provides strong power for the research of antifreeze molecules, and mainly comprises polypeptides, saccharides and glycopeptides, polyvinyl alcohol and derivatives thereof, polyelectrolytes, self-assembled dyes, two-dimensional and three-dimensional nano materials and the like. Typical research efforts include the poly-proline antifreeze material studied by Gibson et al, which increases the cell recovery rate from 20% to over 50%. Ben et al found that the type of sugar affects the growth of the ice crystals. Drori and Kahr et al found that assemblies of the organic dye safranin O had good anti-freeze effects. The Wangjiaojun researchers at the national institute of Chinese academy of sciences have made systematic and prominent work in the research aspect of antifreeze materials, such as in the aspects of supercharged polypeptides, carbon nitride quantum dots, silver materials, graphene oxide, polyelectrolytes, polyamine and the like, the understanding of the molecular mechanism of ice crystal formation is improved, the ionic effect of ice heterogeneous nucleation is discovered, the Janus mechanism of protein regulation and control of ice nucleus formation is disclosed, and the like. Although the research on novel anti-freezing materials has achieved great results, most of them are in the research stage and are difficult to be applied practically, so that the preparation of anti-freezing materials which can be used for practical production is still a challenge and has important research significance.
Disclosure of Invention
The invention aims to provide a perylene bisimide glycopeptide self-assembly antifreeze compound and a preparation method and application thereof, thereby providing a novel antifreeze material with good application effect for inhibiting ice crystal formation and cell cryopreservation.
The technical scheme adopted by the invention is as follows: a perylene bisimide-glycopeptide self-assembly antifreeze compound has a structural general formula as follows:
wherein R is one of formula (1), formula (2) and formula (3),
the preparation method of the perylene bisimide-glycopeptide self-assembled antifreeze compound comprises the following steps:
a. adding perylene anhydride serving as a raw material into a pyridine solvent, adding zinc acetate and alanyl-phenylamido-phenylalanine modified alkynyl derivative 1, heating the mixed solution to 110-120 ℃, and reacting for 14-18 hours; stopping the reaction, cooling the reaction liquid to room temperature, then evaporating to remove pyridine, dissolving the solid with ethyl acetate, simultaneously adding hydrochloric acid for washing, then removing water with anhydrous sodium sulfate, filtering, concentrating ethyl acetate, and finally separating by using a silica gel column to obtain a dark red solid intermediate 2;
b. dissolving the intermediate 2 and the saccharide azide intermediate in a tetrahydrofuran solvent, wherein the saccharide azide intermediate is a mannose azide intermediate Man-N3Galactose azide intermediate Gal-N3Or glucose azide intermediate Glu-N3(ii) a Then adding a copper sulfate aqueous solution and an sodium ascorbate aqueous solution, heating to 50-60 ℃, and reacting for 10-14 hours; stopping reaction, cooling to room temperature, evaporating to remove tetrahydrofuran, adding dichloromethane for liquid separation, drying, and separating by column chromatography to obtain red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu;
c. dissolving red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu and sodium methoxide in absolute methanol, and reacting at room temperature for 10-14 hours; stopping the reaction, cooling to room temperature, and dialyzing in water for 1-3 days by using a dialysis bag to obtain black red solid PBI-Man, PBI-Gal or PBI-Glu.
In the step a, the molar ratio of the perylene anhydride to the zinc acetate to the alkynyl derivative 1 is 1: 2-4.
In step a, the eluent for silica gel column separation is 70/1 volume ratio of dichloromethane/methanol.
In the step b, the molar ratio of the intermediate 2, the saccharide azide intermediate, the copper sulfate and the sodium ascorbate is 1: 6-8: 3-4.
In step b, the eluent for column chromatography separation is dichloromethane/methanol with the volume ratio of 25/1.
In the step c, the molar ratio of the red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu to the sodium methoxide is 1: 10-20.
The reaction formula of the preparation method is as follows:
wherein, a: zn (OAc)2,Pyridine,115○C；b：CuSO4 .5H2O,L-ascorbic acid sodium salt,THF/H2O,
55○C,Man-N3、Gal-N3Or Glu-N3；c：CH3OH,CH3ONa,r.t。
The perylene bisimide-glycopeptide self-assembled antifreeze compound is applied to the aspect of inhibiting the formation of ice crystals and the aspect of freezing and storing cells.
The compound PBI-Man, PBI-Gal or PBI-Glu prepared by the method is applied to the aspects of inhibiting ice crystal formation and cell cryopreservation.
The perylene imide-glycopeptide compound prepared by the invention has the characteristics of polyhydroxy molecules, aromatic conjugated molecules, peptide structures, capability of forming self-assembly bodies and the like. It forms a supermolecule assembly body through self-assembly, thereby having an inhibiting effect on the growth of ice crystals; in the compounds PBI-Man, PBI-Gal and PBI-Glu, MGA value of inhibitory effect of mannose and glucose modified perylene imide-glycopeptide molecules PBI-Man and PBI-Glu on ice crystals at the concentration of 40mg/mL is 38.0 percent of sodium chloride-potassium chloride buffer solution; the freeze-preservation reactivation rate of the perylene imide glycopeptide molecules PBI-Man, PBI-Gal and PBI-Glu modified by mannose, galactose and glucose to human cervical carcinoma cells HeLa is improved from 18.62% (10% DMSO) to 44.24%, 38.08% and 22.20%, wherein the perylene imide glycopeptide molecules modified by mannose and galactose have good synergistic effect.
Drawings
FIG. 1: UV-Vis spectra of compounds PBI-Man, PBI-Gal and PBI-Glu in aqueous solution.
FIG. 2: circular dichroism chromatogram of compound PBI-Man, PBI-Gal and PBI-Glu in water solution.
FIG. 3: dynamic light scattering patterns of the compounds PBI-Man, PBI-Gal and PBI-Glu in aqueous solution.
FIG. 4: ice crystal pattern of sodium chloride-potassium chloride buffer solution set.
FIG. 5: ice crystal patterns of different concentrations of PBI-Man, PBI-Gal and PBI-Glu.
FIG. 6: MGA profiles of ice crystal inhibition for different concentrations of PBI-Man, PBI-Gal and PBI-Glu compared to sodium chloride-potassium chloride buffer solution groups.
FIG. 7: the blank group DMSO, and the test group (PBI-Man, PBI-Gal and PBI-Glu) for the freezing experiment of HeLa cells cell recovery rate.
Detailed Description
The present invention is described in detail below with reference to specific examples, wherein reagents and procedures not mentioned in the examples are all performed according to the routine procedures in the art.
EXAMPLE 1 Synthesis of intermediate 2
Perylene anhydride (218mg, 0.56mmol), zinc acetate (308mg, 1.68mmol), propylamino-phenylalanyl-phenylalanine modified alkynyl compound 1(1.00g, 1.68mmol) was added to 200mL of pyridine solvent at room temperature;
the reaction was heated to 115 ℃ and maintained for 16 hours. The reaction was stopped, cooled to room temperature, the pyridine solvent was evaporated down on a rotary evaporator, the solid was dissolved in 20mL of ethyl acetate, washed twice with 5mL of 1mol/L hydrochloric acid, and the water was removed with anhydrous sodium sulfate, filtered, concentrated in ethyl acetate, and separated on a silica gel column with eluent dichloromethane/methanol at a volume ratio of 70/1 to give 191mg of the product as a red solid, intermediate 2, in 21.9% yield.
The intermediate 2 was a red solid and was,1H NMR(CDCl3,600MHz):δ(ppm)1.57(d,6H,J＝6.6Hz,-CH3),2.32(s,6H,-C≡CH),3.08-3.18(m,4H),3.23-3.28(m,4H),3.71(q,12H,J＝9.6Hz,-CH2),4.03(q,12H,J＝16.2Hz,-CH2),4.63(q,2H,J＝7.2Hz),4.76(q,2H,J＝6.0Hz),5.60(q,2H,J＝6.6Hz),6.20(s,2H),6.24(d,2H,J＝0.6Hz),7.10(d,4H,J＝6.6Hz),7.19-7.24(m,8H),7.27-7.31(m,10H),8.51(s,4H,perylene-H),8.63(d,4H,J＝7.2Hz,perylene-H)；13C NMR(CDCl3,150MHz):δ(ppm)14.23,29.72,36.58,37.71,50.54,53.93,55.63,58.63,59.42,68.28,74.54,79.71,122.80,123.55,126.12,126.84,127.08,128.90,129.11,129.26,129.41,132.17,134.74,136.31,137.52,163.00,169.37,170.62,170.64；HRMS:calcd.for C92H85N8O16,1557.6078,found 1567.6047.
EXAMPLE 2 Synthesis of intermediates PBI-AcMan, PBI-AcGal and PBI-AcGlu
Intermediate 2(100mg, 0.064mmol) was separately reacted with mannose azide intermediate Man-N at room temperature3(192mg, 0.46mmol), galactose azide intermediate Gal-N3(192mg, 0.46mmol) and glucose Azide intermediate Glu-N3(192mg, 0.46mmol) in 10mL tetrahydrofuran solvent; copper sulfate pentahydrate (58mg, 0.23mmol, dissolved in 1mL of aqueous solution) and sodium ascorbate (46mg, 0.23mmol, dissolved in 1mL of aqueous solution) were then added separately;
the reaction was heated to 55 ℃ and kept for 12 hours. The reaction was stopped, cooled to room temperature, and the tetrahydrofuran was removed by rotary evaporator, separated by addition of dichloromethane, dried, and separated by column chromatography eluting with 25/1 vol% dichloromethane/methanol to give PBI-AcMan, PBI-AcGal, and PBI-AcGlu products as 160.0mg, 162.1mg, and 173.0mg, in 61.5%, 62.3%, and 66.5% yields, respectively.
PBI-AcMan: a red solid.1H NMR(CDCl3,600MHz):δ(ppm)1.60(d,6H,J＝10.2Hz,-CH3),2.01(s,18H,-COCH3),2.06(s,18H,-COCH3),2.11(s,18H,-COCH3),2.16(s,18H,-COCH3),3.00-3.11(m,4H),3.18(dd,2H,J＝13.2Hz,7.8Hz),3.33(dd,2H,J＝9.0Hz,12.6Hz),3.65-3.72(12H),3.89-3.93(m,6H),4.05-4.13(12H),4.25(dd,6H,J＝7.8Hz,10.8Hz),4.50(s,12H),4.55-4.69(14H),4.83(s,6H),5.23-5.31(18H),5.64(dd,2H,J＝9.6Hz,10.2Hz),6.31(d,1H,J＝10.2Hz),6.59(s,1H),7.06-7.13(11H),7.22-7.29(9H),7.53(d,2H,J＝12.6Hz),7.75(s,6H,Triaz-H),6.71(d,4H,J＝12.0Hz,perylene-H),8.87(d,4H,J＝12.0Hz,perylene-H)；13C NMR(CDCl3,100MHz):δ(ppm)14.09,20.66,20.69,20.72,20.82,29.68,36.39,37.42,49.43,50.32,54.13,55.36,59.87,62.15,64.61,65.66,66.25,68.57,68.84,68.90,69.14,97.51,122.70,123.87,126.41,126.65,126.89,128.39,128.68,128.97,129.36,132.21,135.15,136.40,137.59,145.06,163.08,169.66,169.95,170.60,170.85,170.86；HRMS:calcd.for C188H223N26O76,4060.4378,found 4060.4337.
PBI-AcGal: a red solid.1H NMR(CDCl3,600MHz):δ(ppm)1.57(d,6H,J＝6.6Hz,-CH3),1.90(s,18H,-COCH3),1.96(s,18H,-COCH3),2.02(s,18H,-COCH3),2.13(s,18H,-COCH3),2.99-3.07(m,4H),3.16(dd,2H,J＝9.6Hz,13.8Hz),3.29(dd,2H,J＝5.4Hz,9.0Hz),3.69(d,6H,J＝9.6Hz),3.75(d,6H,J＝9.0Hz),3.90-3.92(m,12H),4.08-4.15(m,12H),4.20-4.23(m,6H),4.42-4.59(32H),4.65(dd,2H,J＝8.4Hz,6.0Hz),5.00(dd,6H,J＝3.6Hz,7.2Hz),5.16(dd,6H,J＝8.4Hz,1.8Hz),5.37(d,6H,J＝3.0Hz),5.61(dd,2H,J＝6.6Hz,7.2Hz),6.34(d,2H,J＝4.8Hz),6.60(d,2H,J＝12.6Hz),7.03-7.10(m,10H),7.18(t,2H,J＝7.2Hz),7.23(t,4H,J＝7.2Hz),7.28(2H),7.54(d,2H,J＝8.4Hz),7.61(s,6H,Triaz-H),8.68(d,4H,J＝7.8Hz),8.84(d,4H,J＝7.8Hz)；13C NMR(CDCl3,100MHz):δ(ppm)14.17,20.56,20.64,20.66,22.68,29.34,29.68,31.91,36.36,37.51,49.82,50.40,53.48,54.30,55.56,59.86,61.17,64.67,66.95,67.59,68.45,68.76,70.59,70.81,100.93,122.73,123.93,124.02,126.32,126.69,128.43,128.65,129.05,129.26,129.43,132.18,135.07,136.59,137.58,144.66,163.09,169.51,169.56,170.05,170.88,170.94；HRMS:calcd.for C188H222N26NaO76,4083.4237,found 4083.5924.
PBI-AcGlu: a red solid.1H NMR(CDCl3,600MHz):δ(ppm)1.57(d,6H,J＝6.6Hz,-CH3),1.89(s,18H,-COCH3),1.98(s,18H,-COCH3),2.01(s,18H,-COCH3),2.06(s,18H,-COCH3),300-3.07(m,4H),3.16(dd,2H,J＝9.0Hz,4.8Hz),3.30(dd,2H,J＝5.4Hz,8.4Hz),3.67-3.70(12H),3.75(d,6H,J＝9.6Hz),4.12(dd,6H,J＝1.8Hz,10.2Hz),4.41-4.56(32H),4.64(dd,2H,J＝8.4Hz,6.0Hz),4.95(t,6H,J＝7.8Hz),5.05(t,6H,J＝10.2Hz),5.17(t,6H,J＝9.0Hz),5.60(dd,2H,J＝6.6Hz,7.2Hz),6.36(d,2H,J＝4.8Hz),6.60(s,2H),7.03-7.11(10H),7.18-7.25(8H),7.28(2H),7.53(d,2H,J＝8.4Hz),7.60(s,6H,Triaz-H),8.68(d,4H,J＝7.8Hz,perylene-H),8.84(d,4H,J＝8.4Hz,perylene-H)；13C NMR(CDCl3,100MHz):δ(ppm)14.20,22.73,29.73,31.96,36.34,37.48,49.79,50.40,54.27,55.63,59.81,61.77,64.68,67.78,68.24,68.76,70.84,71.89,72.46,100.54,122.78,123.96,126.39,126.73,126.88,128.46,128.68,129.07,129.33,129.44,132.24,135.14,136.60,137.63,144.71,163.13,169.40,169.48,169.56,170.19,170.67,170.91,171.98；HRMS:calcd.for C188H223N26O76,4060.4378,found 4060.4521.
EXAMPLE 3 Synthesis of the Compounds PBI-Man, PBI-Gal and PBI-Glu
The compounds PBI-AcMan (118mg, 0.029mmol), PBI-AcGal (118mg, 0.029mmol) and PBI-AcGlu (118mg, 0.029mmol) were dissolved in 10mL of anhydrous methanol at room temperature with sodium methoxide (9.2mg, 0.58mmol), respectively;
the reaction system was reacted at room temperature for 12 hours, the reaction was stopped, cooled to room temperature, dialyzed with dialysis bag in 2L of water for 2 days, and the aqueous solution was lyophilized to give PBI-Man, PBI-Gal and PBI-Glu products of 85.0mg, 80.0mg and 83.0mg, respectively, in yields of 95.9%, 90.3% and 93.8%, respectively.
PBI-Man: brick red solid.1H NMR(DMSO-d6+D2O,600MHz):δ(ppm)1.60(s,6H),2.61-2.63(m,2H),2.79-2.80(4H),2.99(d,2H,J＝10.2Hz),3.13-3.16(m,6H),3.35-3.43(18H),3.53(6H),3.59-3.64(18H),3.76-3.78(m,6H),3.92-3.94(m,6H),4.40-4.55(26H),4.81(s,6H),4.81(dd,1H,J＝3.6Hz,9.6Hz),5.38(d,2H,J＝6.6Hz),6.97-7.07(10h),7.22-7.29(10H),7.99(s,6H,Triaz-H),8.36(s,4H,perylene-H),8.74(s,4H,perylene-H)；13C NMR(DMSO-d6+D2O,100MHz):δ(ppm)14.40,22.58,29.19,36.35,49.70,54.55,54.75,60.27,61.60,64.59,65.32,67.20,68.32,70.53,71.27,74.60,100.27,123.21,124.22,124.60,125.53,126.35,128.26,128.48,128.76,129.36,129.81,131.14,134.08,138.19,138.58,144.35,162.64,169.68,171.59,171.78；HRMS:calcd.for C140H174N26NaO52,3074.1662,found 3074.1640.
PBI-Gal：1H NMR(DMSO-d6+D2O,600MHz):δ(ppm)1.60(s,6H,-CH3),2.59-2.61(m,2H),2.78(m,4H),2.98(d,2H,J＝10.2Hz),3.27-3.28(m,12H),3.33-3.35(m,6H),3.47-3.50(12H),3.83-3.85(m,6H),4.02-4.04(m,6H),4.14(d,6H,J＝6.6Hz),4.38-4.51(32H),4.69-4.78(m,2H),4.99(d,1H,J＝4.2Hz),5.36(d,2H,J＝7.2Hz),6.95-6.96(m,8H),7.03-7.04(m,2H),7.21-7.22(m,2H),7.26-7.29(m,8H),8.06(s,6H,Triaz-H),8.31(s,4H,perylene-H),8.67(s,4H,perylene-H)；13C NMR(DMSO-d6+D2O,100MHz):δ(ppm)14.40,22.59,29.51,36.36,38.24,50.06,54.54,60.30,60.91,64.62,67.63,68.36,68.58,70.87,73.78,75.83,103.93,123.19,124.23,125.08,126.33,126.71,128.25,128.48,129.38,129.83,130.13,131.08,134.06,138.22,138.58,144.23,144.53,162.62,169.62,171.64,171.75；HRMS:calcd.for C140H175N26O52,3052.1843,found 3052.1724.
PBI-Glu：1H NMR(DMSO-d6+D2O,600MHz):δ(ppm)1.59(s,6H,-CH3),2.58-2.61(m,2H),2.76-2.78(m,4H),2.94(t,6H,J＝8.4Hz),3.01(t,6H,J＝9.0Hz),3.10-3.15(12H),3.40(dd,6H,J＝6.0Hz,5.4Hz),3.60-3.64(10H),3.85-3.88(m,6H),4.03-4.05(m,6H),4.20(d,6H,J＝7.8Hz),4.37-4.50(30H),4.67-4.69(m,1H),5.04(t,1H,J＝7.8Hz),5.17(d,1H,J＝4.8Hz),5.35(d,2H,J＝6.6Hz),6.94-7.04(10H),7.21-7.28(10H),8.05(s,6H,Triaz-H),8.31(s,4H,perylene-H),8.66(s,4H,perylene-H)；13C NMR(DMSO-d6+D2O,100MHz):δ(ppm)14.41,22.59,29.51,36.36,38.24,50.05,54.53,60.31,61.52,63.53,64.61,67.76,68.36,70.44,72.97,7375,77.04,77.42,103.32,123.15,124.20,125.13,126.34,126.72,128.25,128.48,129.37,129.83,131.06,134.00,138.20,138.58,144.21,144.50,162.60,169.64,171.63,171.76；HRMS:calcd.for C140H175N26O52,3052.1843,found 3052.1836.
EXAMPLE 4 Compounds PBI-Man, PBI-Gal and PBI-Glu form supramolecular assemblies in aqueous solution
Respectively mixing 1 × 10-5The compounds PBI-Man, PBI-Gal and PBI-Glu of M are dissolved in water, and have strong absorption peaks between 400 and 600nm as can be seen by UV-Vis spectra (figure 1), wherein the absorption peak at 503nm is the largest, and the absorption peak at 540nm is weaker in intensity, and the spectral characteristics indicate that PBI-Man, PBI-Gal and PBI-Glu form assemblies in the water solution. In addition, the self-assembly behavior of PBI-Man, PBI-Gal and PBI-Glu in aqueous solution was also studied by Circular Dichroism (CD) (FIG. 2), which was found to show positive-then-negative Cotton effect from low wavelength to long wavelength between 400-650 nm, indicating that PBI-Man, PBI-Gal and PBI-Glu form right-handed supramolecular assemblies in aqueous solution. Further, the particle size of the assembly was characterized by Dynamic Light Scattering (DLS) experiments, and as shown in FIG. 3, the particle sizes of PBI-Man, PBI-Gal and PBI-Glu were 111.6, 183.8 and 158.6, respectively.
From the above, the perylene bisimide-glycopeptide compounds PBI-Man, PBI-Gal and PBI-Glu form a supramolecular assembly with right-handed characteristics in aqueous solution. The ultraviolet absorption characteristics and circular dichroism characteristics of PBI-Man, PBI-Gal and PBI-Glu are similar, but the self-assembly particle sizes of PBI-Man are different, the self-assembly particle size formed by the mannose-modified perylene diimide molecules PBI-Man is the largest, and the self-assembly particle size formed by the galactose-modified perylene diimide molecules PBI-Gal is the smallest.
EXAMPLE 5 Ice Crystal growth inhibition assays for Compounds PBI-Man, PBI-Gal and PBI-Glu
The experiments were divided into a control group (sodium chloride-potassium chloride buffer solution group), a low concentration group (10mg/mL), a medium concentration group (20mg/mL) and a high concentration group (40mg/mL), and were performed on a polarizing microscope and a cold and hot stage. The experimental procedure was as follows: (1) placing the glass slide on a cold and hot table, cooling to-90 ℃ under the action of liquid nitrogen, dripping 10 mu L of a control group sample or samples with different concentrations onto the cooled glass slide from the height of 1.5 m, gradually heating to-9 ℃ at the speed of 15 ℃/min, keeping the temperature at-9 ℃ for 45 min, and observing the appearance and the size of the ice crystal through a polarizing microscope.
Randomly selecting the central area and three edge areas of the ice crystals of the sample on the glass slide for photographing, counting the number of the ice crystals in the visual field under the 5 times of the microscope, and calculating the average number of the ice crystals of the sodium chloride-potassium chloride buffer solution (figure 4) to be 132.
In the low concentration group (FIG. 5, 10mg/mL), the central region and three edge regions of the ice crystals of the samples on the slide were randomly selected, and the number of ice crystals in the 5-fold under-the-lens field was counted, and the ice crystal numbers of PBI-Man, PBI-Gal and PBI-Glu were 156, 183 and 196, respectively, and the ice crystal growth inhibitory activities were 84.9%, 72.5% and 67.6%, respectively, as represented by MGA (mean grain area), as compared with the sodium chloride-potassium chloride buffer control group. The result shows that in the low concentration group, the mannose modified perylene imide glycopeptide molecule PBI-Man has weak effect of inhibiting the growth of ice crystals, while the galactose and glucose modified perylene imide glycopeptide molecules PBI-Gal and PBI-Glu have certain effect of inhibiting the growth of ice crystals, and the PBI-Glu has the best effect.
In the middle concentration group (FIG. 5, 20mg/mL), the central region and three edge regions of the ice crystals of the samples on the slide were randomly selected, and the number of ice crystals in the 5-fold under-lens field was counted, and the ice crystal numbers of PBI-Man, PBI-Gal and PBI-Glu were 213, 212 and 244, respectively, and the activities of inhibiting the ice crystal growth were 63.1%, 62.6% and 54.3%, respectively, as expressed by MGA (mean grain area), as compared with the sodium chloride-potassium chloride buffer control group. The results show that in the medium concentration group, the glucose modified perylene imide glycopeptide molecule PBI-Glu has good effect of inhibiting the growth of ice crystals, the MGA value of inhibiting the ice crystals reaches 54.3 percent, and the ice crystal growth inhibition effect is enhanced along with the increase of the concentration of the galactose and glucose modified perylene imide glycopeptide molecules PBI-Gal and PBI-Gal.
In the high concentration group (FIG. 5, 40mg/mL), the central region and three edge regions of the ice crystals of the samples on the slide were randomly selected, and the number of ice crystals in the 5-fold under-lens field was counted, and the ice crystal numbers of PBI-Man, PBI-Gal and PBI-Glu were 367, 350 and 376, respectively, and the ice crystal growth inhibitory activities were expressed as 36.1%, 37.9% and 35.3% by MGA (mean grain area), respectively, as compared with the sodium chloride-potassium chloride buffer control group. The results show that in the high concentration group, the mannose-and glucose-modified perylene imide glycopeptide molecules PBI-Man and PBI-Glu have good ice crystal growth inhibition effect, and the MGA value for inhibiting ice crystals is about 36.0%, wherein the MGA value for inhibiting ice crystal growth of the glucose-modified perylene imide glycopeptide molecules PBI-Glu is 35.3%.
From the above, the ice crystal growth inhibition effect of the perylene imide-glycopeptide compounds PBI-Man, PBI-Gal and PBI-Glu is related to the concentration, the ice crystal growth inhibition effect is weaker at low concentration, the ice crystal growth inhibition effect is achieved at high concentration, and the MGA value of the ice crystal growth inhibition effect is lower than 40%; particularly, the MGA value of the glucose modified perylene imide-glycopeptide molecule PBI-Glu on the growth inhibition of ice crystals is about 35.3%.
Example 6 application of the Compounds PBI-Man, PBI-Gal and PBI-Glu to cryopreservation of HeLa cells of cervical carcinoma
HeLa cells in 5% CO2Culturing in 37 deg.C culture flask in DMEM containing 10% fetal calf serum, digesting cells with 0.25% trypsin during cell log increment, centrifuging at 1200rpm for 3min, and collecting cells (concentration of 2 × 10)5mL-1) Transferring to a freezing tube, wherein the blank group is frozen under the following conditions: 80% medium, 10% serum and 10% DMSO; the cryopreservation conditions for the test groups were: 80% medium, 10% serum and 10% DMSO, and 1.0mg/mL of PBI-Man, PBI-Gal and PBI-Glu, respectively, was added. The freezing and storing process comprises the following steps: balancing in a refrigerator at-4 deg.C for 0.5h, pre-freezing in a refrigerator at-20 deg.C for 1h, and freezing in a low-temperature refrigerator at-80 deg.C for 24 h. After 24 hours, the cells were thawed, stored in a 37 ℃ water bath, returned to 37 ℃ and equilibrated for 0.5 hours, and the revived cells were seeded in culture flasks at 5% CO2Culturing in a 37 deg.C culture bottle with 10% DMEM of fetal calf serum, culturing for 24 hr, washing dead cells, digesting adherent cells with 0.25% trypsin, centrifuging at 1200rpm for 3min, and collecting cellsCounting by a counter and calculating the cell recovery rate. Blank and experimental groups 5 replicates were set and cell count results were rounded off to maximum and minimum values.
The experimental results show that: the recovery rate of the blank group of HeLa cells is 18.62%, and the recovery rate of the test group of PBI-Man, PBI-Gal and PBI-Glu cells is respectively improved to 44.24%, 38.08% and 22.20%. Wherein, the perylene imide glycopeptide molecule modified by mannose and galactose has better cell cryopreservation effect.
Claims (10)
1. A perylene bisimide-glycopeptide self-assembly anti-freezing compound is characterized in that the structural general formula of the compound is as follows:
wherein R is one of formula (1), formula (2) and formula (3),
2. the preparation method of the perylene imide glycopeptide self-assembled antifreeze compound as claimed in claim 1, which is characterized by comprising the following steps:
a. adding perylene anhydride serving as a raw material into a pyridine solvent, adding zinc acetate and alanyl-phenylamido-phenylalanine modified alkynyl derivative 1, heating the mixed solution to 110-120 ℃, and reacting for 14-18 hours; stopping the reaction, cooling the reaction liquid to room temperature, then evaporating to remove pyridine, dissolving the solid with ethyl acetate, simultaneously adding hydrochloric acid for washing, then removing water with anhydrous sodium sulfate, filtering, concentrating ethyl acetate, and finally separating by using a silica gel column to obtain a dark red solid intermediate 2;
b. dissolving the intermediate 2 and the saccharide azide intermediate in a tetrahydrofuran solvent, wherein the saccharide azide intermediate is a mannose azide intermediate Man-N3Galactose azide intermediate Gal-N3Or glucose azide intermediate Glu-N3(ii) a Then adding a copper sulfate aqueous solution and an sodium ascorbate aqueous solution, heating to 50-60 ℃, and reacting for 10-14 hours; stopping reaction, cooling to room temperature, evaporating to remove tetrahydrofuran, adding dichloromethane for liquid separation, drying, and separating by column chromatography to obtain red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu;
c. dissolving red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu and sodium methoxide in absolute methanol, and reacting at room temperature for 10-14 hours; stopping the reaction, cooling to room temperature, and dialyzing in water for 1-3 days by using a dialysis bag to obtain black red solid PBI-Man, PBI-Gal or PBI-Glu.
3. The preparation method of claim 2, wherein in step a, the molar ratio of the perylene anhydride to the zinc acetate to the alkynyl derivative 1 is 1: 2 to 4.
4. The process of claim 2, wherein the eluent for the silica gel column separation in step a is 70/1 volume ratio of dichloromethane/methanol.
5. The preparation method of claim 2, wherein in the step b, the molar ratio of the intermediate 2, the saccharide azide intermediate, the copper sulfate and the sodium ascorbate is 1: 6-8: 3-4.
6. The process of claim 2, wherein in step b, the eluent for column chromatography is 25/1 volume ratio of dichloromethane/methanol.
7. The method according to claim 2, wherein in step c, the molar ratio of the red solid PBI-AcMan, PBI-AcGal or PBI-AcGlu to sodium methoxide is 1: 10-20.
8. The process according to claim 2, wherein the reaction formula of the process is as follows:
9. the use of the perylene imide glycopeptide self-assembling antifreeze compound of claim 1 for inhibiting ice crystal formation and cell cryopreservation.
10. Use of a compound PBI-Man, PBI-Gal or PBI-Glu produced by the process of any one of claims 2 to 8 for inhibiting ice crystal formation and cell cryopreservation.
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| US20150240027A1 (en) * | 2008-03-26 | 2015-08-27 | Yeda Research And Development Co., Ltd. | Supramolecular polymers derived from perylene-diimides |
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| US20150240027A1 (en) * | 2008-03-26 | 2015-08-27 | Yeda Research And Development Co., Ltd. | Supramolecular polymers derived from perylene-diimides |
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