CN103554198B - Influenza virus sialyloligosaccharide functional receptor and synthetic method thereof - Google Patents

Abstract

The invention discloses functional receptor of a kind of influenza virus sialyloligosaccharide and preparation method thereof. The functional receptor of influenza virus sialyloligosaccharide provided by the present invention is compound shown in compound shown in formula X-1 or formula X-2, and this receptor can be received on gold nano grain jointly with other linking arm, the sialic acid trisaccharide-golden nanometer particle of acquisition. This sialic acid trisaccharide-golden nanometer particle can be for detection of HA albumen and the strain of the A type influenza virus of the hypotypes such as H1, H3, H5 and H7, the receptor-specific of difference influenza virus, by visually observing the receptor-specific that can judge fast influenza virus to be measured, can obtain quickly and easily the interaction information between influenza virus and host cell, the prevention and control of infected by influenza are significant.

Description

Influenza virus sialyloligosaccharide functional receptor and synthetic method thereof

Technical field

The present invention relates to influenza virus sialyloligosaccharide functional receptor in organic chemistry filed and preparation method thereof.

Background technology

Influenza (influenza) is the infectious diseases common to human beings and animals being caused by influenza virus, its host involve people, pig,The many animals such as bird, horse and dolphin. Studies confirm that, influenza virus surface glycoprotein hemagglutinin (hemagglutinin, HA) is specialThe sialic acid sugar chain acceptor of identification host cell surface is influenza infection host and then the biology that copies and continue propagationBasis. Influenza virus is to sialic identification and be combined with preference type, and avian influenza virus tends in conjunction with Sia α 2,3Gal acceptor,Human influenza virus is mainly in conjunction with Sia α 2,6Gal acceptor. The enteron aisle of bird mainly contains Sia α 2,3Gal acceptor, and the mankind's is upperAirway epithelial cell mainly contains Sia α 2,6Gal acceptor. Human influenza virus is combined closely with people's tracheal epithelial cell, and fowlInfluenza virus is combined very weak with people's tracheal epithelial cell. The existing Sia α 2 of pig, 3Gal acceptor also has Sia α 2,6Gal acceptor, pig pairAll susceptibles of human influenza virus and avian influenza virus. Influenza virus affects it to the identification desalivation acid of bonding of sialic acid sugar chain acceptorOutward, its internal sugar chain structure (as sulphation, fucosylated etc.) also has a significant impact. Therefore, synthesize and there are different inner sugar chainsThe sialyloligosaccharide of structure, is connected it (containing sulfydryl) with functional connection chain, can be fixed in easily carrier (coreSheet, golden nanometer particle, quantum dot etc.) on study flu virus and the interaction of sialic acid sugar chain acceptor.

Summary of the invention

The object of this invention is to provide functional receptor of influenza virus sialyloligosaccharide and preparation method thereof.

The functional receptor of influenza virus sialyloligosaccharide provided by the present invention is compound shown in formula X-1 or formula X-2Shown in compound;

Wherein, the integer that described x is 1-3, the integer that described m is 0-6, the integer that described n is 0-1, described k is 0-11Integer, described p is 0 or 2, described R is S or H; Described Ac is acetyl group, described R¹For hydroxyl or acetamido, described R²For hydrogenOr L-fucose, described R³For hydrogen or sulfuric ester, (sulfuric acid becomes ester, SO with the hydroxyl on sugar ring₃H), described R⁴For hydroxyl or acetylAmido. Preferably, the integer that described m is 3-6; Described k is the integer of 5-11. Preferred, described m is 3 or 6, and described n is 1Or 0, described k is 5 or 11, described p is 2 or 0.

Shown in preparation formula provided by the present invention X-1, the method for compound shown in compound or formula X-2, comprises following stepRapid:

1) glycosyl of formula X-11 is reacted with propilolic alcohol, make it reducing end with alkynyl;

2) synthetic one end is with sulfydryl, and the other end is with the short connection chain in formula X-12 of azido;

3) formula X-11 compound and the step 2 that step 1) are obtained) formula X-12 compound " click " that obtain are connectedTo formula X-13 compound;

4) under the effect of sialyltransferase, by synthetic to compound shown in formula X-13 and CMP-NeuAcFor compound shown in compound shown in formula X-1 or formula X-2.

Shown in preparation formula provided by the present invention X-1, the another kind of method of compound shown in compound or formula X-2, comprisesFollowing steps:

1) lactose is reacted with the compound shown in formula X-14, make alkynyl on lactose reducing end band;

2), under the effect of sialyltransferase, the compound that step 1) is obtained and CMP-NeuAc are syntheticFor compound shown in compound shown in formula X-15 or formula X-16;

3) by step 2) length shown in compound shown in compound shown in formula X-15 that obtain or formula X-16 and formula X-16Connection chain carries out " click " reaction, obtains compound shown in compound shown in formula X-1 or formula X-2.

In above-mentioned two kinds of methods provided by the present invention, to substituent definition in each general formula with above-mentioned to shown in formula X-1The definition of compound shown in compound or formula X-2. Two kinds of methods, described sialyltransferase is α 2,3-sialyltransferaseTime, what obtain is compound shown in formula X-1; Described sialyltransferase is α 2, and when 6-sialyltransferase, what obtain is formulaCompound shown in X-2. The condition that described " click " connects is: glycosyl propine glycosides and connection chain are mixed with mole 1:1.5 ratioClose, in the mixed solution of DMF (DMF) and methyl alcohol (MeOH) (volume ratio is 1:1), with cuprous iodide(CuI) be catalyst, ambient temperature overnight reaction under nitrogen protection.

The present invention by α 2, the 3 sialic acid trisaccharides of being combined with influenza virus HA protein-specific and α 2,6 sialic acid trisaccharides withSpecific linking arm is connected, and forms the functional receptor of influenza virus sialyloligosaccharide, and this receptor can be total to other linking armWith receiving on gold nano grain, the sialic acid trisaccharide-golden nanometer particle of acquisition. This sialic acid trisaccharide-golden nanometer particle can be usedIn HA albumen and the strain of A type influenza virus that detects the hypotypes such as H1, H3, H5 and H7, the receptor-specific of difference influenza virus,By visually observing the receptor-specific that can judge fast influenza virus to be measured, can obtain quickly and easily influenza virus withInteraction information between host cell, the prevention and control of infected by influenza are significant.

Brief description of the drawings

Fig. 1 is the structural representation of 16 kinds of sialyloligosaccharide-golden nanometer particles of the present invention. Wherein, the left figure of the first row is α 2,3 sialic acid trisaccharides-long connection chain and long connection chain are fixed on sialyloligosaccharide-Jenner grain of rice that golden nanometer particle surface formsSon; The right figure of the first row is that α 2,6 sialic acid trisaccharides-long connection chain and long connection chain are fixed on the saliva that golden nanometer particle surface formsLiquid acid oligosaccharides-golden nanometer particle; The left figure of the second row is that α 2,3 sialic acid trisaccharides-short connection chain and short connection chain are fixed on gold nanoSialyloligosaccharide-golden nanometer particle that particle surface forms; The right figure of the second row is α 2,6 sialic acid trisaccharides-short connection chain and short companyChain link is fixed on sialyloligosaccharide-golden nanometer particle that golden nanometer particle surface forms, and each figure is according to α 2,3 or α 2,6 salivasThe ratio of liquid acid trisaccharide-length/short connection chain and length/short connection chain is different is divided into again 4 kinds, totally 16 kinds.

Fig. 2 is the qualitative inspection of sialyloligosaccharide-golden nanometer particle of the present invention (3 ' SLs-AuNP5:1 or 6 ' SLs-AuNP5:1)Survey the result that visually observes of 5 kinds of influenza virus HA albumen.

Fig. 3 is 5 kinds of influenza virus HA of sialyloligosaccharide-golden nanometer particle of the present invention (3 ' SLs-AuNP5:1) qualitative detectionThe UV-Vis spectrum change of albumen.

Fig. 4 is 5 kinds of influenza virus HA of sialyloligosaccharide-golden nanometer particle of the present invention (6 ' SLs-AuNP5:1) qualitative detectionThe UV-Vis spectrum change of albumen.

Fig. 5 is 5 kinds of influenza virus HA of sialyloligosaccharide-golden nanometer particle of the present invention (3 ' SLs-AuNP5:1) qualitative detectionThe DLS of albumen changes.

Fig. 6 is 5 kinds of influenza virus HA of sialyloligosaccharide-golden nanometer particle of the present invention (6 ' SLs-AuNP5:1) qualitative detectionThe DLS of albumen changes.

Fig. 7 is the qualitative inspection of sialyloligosaccharide-golden nanometer particle of the present invention (3 ' SLs-AuNP5:1 or 6 ' SLs-AuNP5:1)Survey the TEM image section result of 5 kinds of influenza virus HA albumen. In figure, scale represents 200nm.

Fig. 8 is that sialyloligosaccharide-golden nanometer particle of the present invention (6 ' SLs/6 ' SLNs/Su-6 ' SLNs-AuNP5:1) is quantitativeAssess 9 kinds of influenza virus HA protein receptor specificitys.

Fig. 9 is sialyloligosaccharide-golden nanometer particle of the present invention (3 ' SLs/3 ' SLNs/Su-3 ' SLNs/SLe^X-AuNP5:1）9 kinds of influenza virus HA protein receptor specificitys of qualitative assessment.

Figure 10 is sialyloligosaccharide-golden nanometer particle of the present invention (6 ' SLs/6 ' SLNs/Su-6 ' SLNs/3 ' SLs/3 'SLNs/Su-3’SLNs/SLe^X-AuNP5:1) 3 kinds of influenza virus strain receptor-specifics of qualitative assessment.

Figure 11 is different from inspection from the mol ratio of length/short connection chain for visually observing α 2,3 sialic acid trisaccharide-length/short connection chainsSurvey the impact of result.

Figure 12 is the influenza virus qinH5 that sialyloligosaccharide-golden nanometer particle (3 ' SLs-AuNP5:1) detects variable concentrationsHA albumen visually observe result.

Figure 13 is the influenza virus qinH5 that sialyloligosaccharide-golden nanometer particle (3 ' SLs-AuNP5:1) detects variable concentrationsThe UV-Vis spectrum change result of HA albumen.

Figure 14 is the influenza virus 68H3 that sialyloligosaccharide-golden nanometer particle (6 ' SLs-AuNP5:1) detects variable concentrationsHA albumen visually observe result.

Figure 15 is the influenza virus 68H3 that sialyloligosaccharide-golden nanometer particle (6 ' SLs-AuNP5:1) detects variable concentrationsThe UV-Vis spectrum change of HA albumen.

Detailed description of the invention

The experimental technique using in following embodiment if no special instructions, is conventional method.

Material, reagent etc. used in following embodiment, if no special instructions, all can obtain from commercial channels.

In following embodiment, the solvent of Tris-HCl buffer solution (100mM, pH8.0) used is water, and solute is Tris, instituteStating the concentration of solute Tris in described Tris-HCl buffer solution is 12.1g/L; The pH value of described Tris-HCl buffer solution is8.0。

The solvent of PBS buffer solution (10mM, pH7.4) used is water, and solute is NaH₂PO₄、Na₂HPO₄, KCl and NaCl,Described solute NaH₂PO₄、Na₂HPO₄, KCl and the concentration of NaCl in described PBS buffer solution be respectively 0.24g/L, 1.42g/L,0.2g/L and 8.0g/L; The pH value of described PBS buffer solution is 7.4.

The preparation of embodiment 1, sialic acid trisaccharide-short connection chain

1, the preparation of short connection chain

Get compound shown in 1.68 grams of formula III (4.96mmol, its synthetic method is shown in document: K.Kim, H.Yang, S.Jon,E.Kim, J.Kwak, J.Am.Chem.Soc.2004,126,15368 – 15369.) be dissolved in 20 milliliters of carrene (DCM) chamberUnder temperature, add 1 milliliter of triethylamine (Et₃N, 7.5mmol) and 780 microlitre mesyl chlorides (MsCl, 10mmol); Reaction system is existedUnder room temperature, stir 1 hour. Reaction process is monitored by TLC, after raw material disappears, reactant liquor is by diatomite filtration, orangely concentratedYellow slurry. Concentrate is placed in to 20 milliliters of acetonitrile (CH₃CN), in, add 3.25 grams of sodium azide (NaN₃, 50mmol), returnStream stirs and spends the night. TLC detection reaction is complete, by solution under reduced pressure concentrated steam desolventize after, separate with silica gel chromatographic columnIt (is chemical combination shown in formula IV that purified mixture (leacheate is benzinum: ethyl acetate=3:1) obtains buff syrup shape compoundThing, 1.46 grams, productive rate 81%).

The structural characterization data of compound shown in formula IV prepared by the present embodiment are as follows:

¹HNMR(400MHz,CDCl₃):δ4.23(2H,t,J4.7Hz,COOCH₂),3.71–3.66(8H,m,CH₂OCH₂),3.59–3.52(1H,m,S–SCH),3.38(2H,t,J5.0Hz,CH₂N₃),3.16–3.12(2H,m,S–SCH₂),2.46–2.44(1H,m),2.35(2H,t,J7.4Hz,CH₂COO),1.94–1.85(1H,m,),1.70–1.61(4H,m),1.47–1.45(2H,m);¹³CNMR(150MHz,CDCl₃):δ173.5(CO),71.5,70.2,69.3,63.5,56.4,50.7,42.8,42.3,38.5,34.6,34.0,28.8,24.7;HRESIMS:m/z386.11726[M+Na]⁺(calcdforC₁₄H₂₅N₃O₄S₂:386.11787[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

2, the preparation of the short connection chain of lactose

Get 76 milligrams of lactose propine glycosides shown in formula I-1 (0.2mmol, its synthetic method is shown in document: F.Lutz,U.B.Tietze, Chem.Eur.J.1998,4,1179 – 1183.) with 108.9 milligrams of short connection chains (shown in IV,0.3mmol) be dissolved in the mixed solution of 10 milliliters of DMFs (DMF) and methyl alcohol (MeOH) (volume ratio is 1:1),Then add 19 milligrams of cuprous iodides (CuI, 0.1mmol), reaction overnight under nitrogen protection. TLC monitoring raw material disappears, and will reactLiquid filters, with G-15 gel column purifying, final that white solid is compound shown in formula II-1-1 after freeze drying is concentrated(118.9 milligrams, productive rate 80%).

The structural characterization data of compound shown in formula II-1-1 prepared by the present embodiment are as follows:

¹HNMR(400MHz,D₂O):δ8.13(1H,s,C=CHN₃),5.01(1H,d,J12.6Hz,H1^I),4.88(1H,d,J12.6Hz,H1^II),4.65(2H,t,J5.0Hz),4.59(1H,d,J7.5Hz),4.45(1H,d,J7.8Hz),4.23(2H,t,J4.4Hz),3.98(3H,t,J5.1Hz),3.93(2H,d,J3.1Hz),3.85–3.54(14H,m),3.35(2H,t,J8.4Hz),3.26–3.17(2H,m),2.5–2.47(1H,m),2.41(2H,t,J7.2Hz),2.0–1.94(1H,m),1.79–1.57(4H,m),1.47–1.39(2H,m);¹³CNMR(150MHz,D₂O):δ176.3(CO),143.6(C=CHN₃),125.8(C=CHN₃),103.1(C1^II),101.5(C1^I),78.5,75.5,74.9,74.5,72.8,72.7,71.1,69.8,69.7,68.9,68.6,63.9,62.1,61.1,60.2,56.6,50.2,40.4,38.3,33.9,33.7,28.1,24.2;HRESIMS:m/z766.24890[M+Na]⁺(calcdforC₂₉H₄₉N₃O₁₅S₂:766.24973[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

3, the preparation of sialic acid trisaccharide-short connection chain

Get 14.87 milligrams of short connection chains of lactose (formula II-1-1, final concentration is 10mM), and 14.72 milligrams of CMP-N-acetylNeuraminic acid (CMP-Neu5Ac, final concentration is 15mM) is dissolved in the Tris-HCl(pH8.0 of 2 milliliters) in buffer solution after, will reactSystem temperature rises to 37 DEG C, then adds 100 microlitres to kill p pestic α 2 more, 3-sialyltransferase (PasteurellaMultocida α 2,3-sialyltransferase, is called for short Pm2,3-ST, 0.2U, purchased from the still limited public affairs of moral medicine edge science and technology of TianjinDepartment), after 2-3 hour, thin-layer chromatography (TLC) detects raw material and disappears completely, and reactant liquor is removed to zymoprotein by milipore filter, coldFreeze-drying dry concentrated after after gel column G-15 purifying white solid be that sialic acid trisaccharide (shown in formula X-1-1,17.9 milligrams, producesRate 86.9%)

Shown in formula X-1-1 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.15(1H,s,C=CHN₃),5.02(1H,d,J12.6Hz,H1I),4.90(1H,d,J12.5Hz,H1^II),4.67(2H,t,J4.8Hz),4.60(1H,d,J8.0Hz),4.55(1H,d,J7.7Hz),4.25(2H,t,J3.6Hz),4.16(1H,d,J9.7Hz),4.02–3.99(5H,m),3.91–3.85(6H,m),3.78–3.58(14H,m),3.36(1H,t,J8.1Hz),3.29–3.18(2H,m),2.77(1H,dd,J4.4,12.4Hz,H3eq-Sia),2.53–2.48(1H,m),2.42(2H,t,J7.1Hz),2.09–1.98(5H,m),1.87(1H,t,J7.1Hz,H3ax-Sia),1.83–1.65(5H,m),1.48–1.42(2H,m);¹³CNMR(150MHz,D₂O):δ176.9,175.2,174.1,143.7(C=CHN₃),125.9(C=CHN₃),102.9(C1^II),101.5(C1^I),100.0(C1^III),78.4,75.7,75.4,75.1,74.6,73.1,72.9,72.0,69.8,69.6,69.0,68.7,68.3,67.7,64.0,62.8,62.2,61.6,61.2,60.3,59.6,56.7,51.9,50.3,40.5,39.9,38.3,33.9,33.8,28.1,24.2,22.3;HRESIMS:m/z1057.34256[M+Na]⁺(calcdforC₄₀H₆₆N₄O₂₃S₂:1057.34515[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 2, sialic acid trisaccharide-short connection chain

The preparation of sialic acid trisaccharide-short connection chain

Get the short connection chain of lactose in 14.87 milligrams of embodiment 1 (formula II-1-1, final concentration is 10mM), and 14.72 milligramsCMP-NeuAc (CMP-Neu5Ac, final concentration is 15mM) is dissolved in the Tris-HCl(pH8.0 of 2 milliliters) in buffer solutionAfter, temperature of reaction system is risen to 37 DEG C, then add 100 microlitre Mermaid luminous bacillus α 2,6-sialyltransferase(Photobacteriumdamsela α 2,6-sialyltransferase are called for short Pd2,6-ST, and 0.2U, purchased from Tianjin Shang DeYao Yuan Science and Technology Ltd.), after 2-3 hour, thin-layer chromatography (TLC) detect raw material disappear completely, by reactant liquor by ultrafiltrationFilm is removed zymoprotein, through gel column G-15 purifying, final that white solid is saliva shown in formula X-2-1 after freeze drying is concentratedLiquid acid trisaccharide-15.5 milligrams of short connection chains, productive rate 74.8%.

Shown in formula X-2-1 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.16(1H,s,C=CHN₃),5.03(1H,d,J12.5Hz,H1^I),4.91(1H,d,J12.5Hz,H1^II),4.67(2H,t,J4.9Hz),4.62(1H,d,J8.0Hz),4.45(1H,d,J7.7Hz),4.25(2H,t,J4.3Hz),4.01–3.95(6H,m),3.93–3.82(7H,m),3.76–3.54(12H,m),3.39(1H,t,J8.3Hz),3.29–3.18(2H,m),2.73(1H,dd,J4.5,12.4Hz,H3eq-Sia),2.55–2.49(1H,m),2.43(2H,t,J7.2Hz),2.14–1.98(5H,m),1.79–1.61(5H,m),1.48–1.41(2H,m);¹³CNMR(150MHz,D₂O):δ176.8,175.1,173.7,143.7(C=CHN₃),125.9(C=CHN₃),103.4(C1^II),101.4(C1^I),100.5(C1^III),79.8,74.9,74.8,73.9,72.8,72.7,72.5,72.0,71.0,69.9,69.7,68.9,68.7,68.7,68.5,64.0,63.7,62.8,62.1,60.4,59.5,56.7,52.0,50.3,40.5,40.3,38.2,33.8,33.8,28.0,24.2,22.2;HRESIMS:m/z1057.33547[M+Na]⁺(calcdforC₄₀H₆₆N₄O₂₃S₂:1057.34515[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 3, sialic acid trisaccharide-short connection chain

1, the short connection chain of lactose amine is synthetic

Get 60 milligrams of lactose amine propine glycosides shown in formula I-2 (0.14mmol, its synthetic method is shown in document: R.Daly,G.Vaz, A.M.Davies, M.O.Senge, E.M.Scanlan, Chem.Eur.J.2012,18,14671 – 14679.) with77.63 milligrams of short connection chains (as shown in embodiment 1 formula IV, 0.214mmol) are dissolved in 7 milliliters of DMFs (DMF)And in the mixed solution of methyl alcohol (MeOH) (volume ratio is 1:1), then add 13.3 milligrams of cuprous iodides (CuI, 0.07mmol),Reaction overnight under nitrogen protection. TLC monitoring raw material disappears, by reacting liquid filtering, pure with G-15 gel column after freeze drying is concentratedChange, final that white solid is compound shown in formula II-1-2 (65.88 milligrams, productive rate 60%). Concrete reaction equation is as follows:

The structural characterization data of compound shown in formula II-1-2 prepared by the present embodiment are as follows:

¹HNMR(400MHz,D₂O):δ8.05(1H,s,C=CHN₃),4.91(1H,d,J12.6Hz,H1^I),4.89–4.79(1H,overlappedbysolventpeak,H1^II),4.64–4.59(3H,m),4.44(1H,d,J8.0Hz),4.21–4.19(2H,m),3.98–3.94(3H,m),3.82(1H,dd,J3.6,12.2Hz),3.75–3.58(17H,m),3.51(1H,t,J9.2Hz),3.24–3.12(2H,m),2.49–2.41(1H,m),2.38(2H,t,J7.2Hz),1.99–1.86(4H,m),1.73–1.66(1H,m),1.64–1.53(3H,m),1.43–1.35(2H,m);¹³CNMR(150MHz,D₂O):δ174.3,174.0,143.7(C=CHN₃),125.7(C=CHN₃),103.0(C1^II),100.2(C1^I),78.6,75.5,75.0,72.7,72.5,71.1,70.0,69.7,69.0,68.9,68.7,63.9,62.1,61.1,60.2,56.6,55.1,50.2,40.4,38.3,33.9,33.8,28.1,24.2,22.2;HRESIMS:m/z785.29652[M+H]⁺(calcdforC₃₁H₅₃N₄O₁₅S₂:785.29433[M+H]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

2, the preparation of sialic acid trisaccharide-short connection chain

(formula II-1-2, final concentration is 10mM, and 16.92 milligrams of CMP-N-acetyl god to get 18 milligrams of short connection chains of lactose amineBe dissolved in the Tris-HCl(pH8.0 of 2.3 milliliters through propylhomoserin (CMP-Neu5Ac, final concentration is 15mM)) in buffer solution after, will reactSystem temperature rises to 37 DEG C, then adds 115 microlitre Pm2,3-ST(0.23U), after 2-3 hour, thin-layer chromatography (TLC) detects formerMaterial disappears completely, and reactant liquor is removed to zymoprotein by milipore filter, final through gel column G-15 purifying after freeze drying is concentratedObtain white solid and be 17 milligrams of compounds shown in formula X-1-2, productive rate 68.9%.

Shown in formula X-1-2 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(500MHz,D₂O):δ8.06(1H,s,C=CHN₃),4.92(1H,d,J12.8Hz,H1^I),4.91-4.64(1H,overlappedbysolventpeak,H1^II),4.63(2H,t,J4.8Hz),4.59(1H,d,J8.1Hz),4.54(1H,d,J7.9Hz),4.21(2H,t,J4.2Hz),4.10(1H,dd,J2.8,9.9Hz),4.00–3.48(21H,m),3.25–3.14(2H,m),2.74(1H,dd,J4.4,12.4Hz,H3eq-Sia),2.50–2.43(1H,m),2.39(2H,t,J7.2Hz),2.02(3H,s,Ac-Sia),2.00–1.94(1H,m),1.91(3H,s,Ac-I),1.79(1H,t,J12.1Hz,H3ax-Sia),1.74–1.69(1H,m),1.67–1.57(3H,m),1.43–1.37(2H,m);¹³CNMR(125MHz,D₂O):δ176.7,175.1,174.3,173.9,143.6(C=CHN₃),125.7(C=CHN₃),102.6(C1^II),100.2(C1^I),99.9(C1^III),78.4,75.5,75.2,74.9,72.9,72.3,71.8,69.7,69.6,69.4,68.8,68.5,68.4,68.1,67.5,63.8,62.6,62.0,61.0,60.1,59.4,56.6,55.0,51.7,50.1,42.3,40.3,38.1,33.7,33.6,27.9,24.1,22.1;HRESIMS:m/z1076.39183[M+H]⁺(calcdforC₄₂H₇₀N₅O₂₃S₂:1076.38975[M+H]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 4, sialic acid trisaccharide-short connection chain

The preparation of sialic acid trisaccharide-short connection chain

Get the short connection chain of 18 milligrams of embodiment, 3 lactose amine (formula II-1-2, final concentration is 10mM), and 16.92 milligrams of CMP-N-acetyl-neuraminate (CMP-Neu5Ac, final concentration is 15mM) is dissolved in the Tris-HCl(pH8.0 of 2.3 milliliters) in buffer solutionAfter, temperature of reaction system is risen to 37 DEG C, then add 115 microlitre Pd2,6-ST(0.23U), after 2-3 hour, thin-layer chromatography(TLC) detect raw material disappears completely, reactant liquor is removed to zymoprotein by milipore filter, freeze drying concentrate after through gel column G-15 purifying are final that white solid is 15.9 milligrams of compounds shown in formula X-2-2, productive rate 64.4%.

Shown in formula X-2-2 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(500MHz,D₂O):δ8.06(1H,s,C=CHN₃),4.93(1H,d,J12.9Hz,H1^I),4.80(1H,d,J11.0Hz,H1^II),4.64–4.63(3H,m),4.43(1H,d,J7.9Hz),4.20(1H,d,J4.4Hz),4.00–3.49(29H,m),3.25–3.14(2H,m),2.64(1H,dd,J4.4,12.3Hz,H3eq-Sia),2.50–2.44(1H,m),2.39(2H,t,J7.1Hz),2.05–1.80(7H,m),1.76–1.55(5H,m),1.43–1.37(2H,m);¹³CNMR(150MHz,D₂O):δ176.6,174.9,174.3,173.5,143.6(C=CHN₃),125.6(C=CHN₃),103.5(C1^II),100.1(C1^I),99.9(C1^III),80.6,74.5,73.7,72.5,72.4,71.7,70.7,69.7,69.5,68.7,68.5,68.4,68.2,63.8,63.3,62.6,61.9,61.2,60.3,59.4,56.5,54.8,51.9,50.0,40.3,40.1,38.0,33.6,33.6,27.8,24.0,22.2,22.0;HRESIMS:m/z1076.38864[M+H]⁺(calcdforC₄₂H₇₀N₅O₂₃S₂:1076.38975[M+H]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 5, sialic acid trisaccharide-short connection chain

1, the short connection chain of lactose amine sulfuric ester is synthetic

Get in 50 milligrams of lactose amine sulfuric ester propine glycosides (0.1mmol) shown in formula I-3 and 54.37 milligrams of embodiment 1Short connection chain (shown in IV, 0.15mmol) is dissolved in 5 milliliters of DMFs (DMF) and methyl alcohol (MeOH) (volumeThan for 1:1) mixed solution in, then add 9.5 milligrams of cuprous iodides (CuI, 0.05mmol), under nitrogen protection, spend the night anti-Should. TLC monitoring raw material disappears, by reacting liquid filtering, after freeze drying is concentrated with G-15 gel column purifying, final white solidBe compound shown in formula II-1-3 (60.47 milligrams, productive rate 70%).

The structural characterization data of compound shown in formula II-1-3 prepared by the present embodiment are as follows:

¹HNMR(400MHz,D₂O):δ8.10(1H,s,C=CHN₃),4.92(1H,d,J12.9Hz,H1^I),4.84(1H,d,J12.9Hz,H1^II),4.65(2H,t,J4.3Hz),4.53(1H,d,J7.8Hz),4.41(1H,d,J9.9Hz),4.31(1H,dd,J4.1,11.0Hz),4.24–4.22(2H,m),3.98(3H,t,J5.0Hz),3.93(1H,d,J3.8Hz),3.85–3.67(12H,m),3.53(1H,t,J8.9Hz),3.27–3.15(2H,m),2.52–2.45(1H,m),2.41(2H,t,J7.2Hz),2.10–1.97(1H,m),1.95(3H,s),1.78–1.68(1H,m),1.66–1.56(3H,m),1.46–1.38(2H,m);¹³CNMR(150MHz,D₂O):δ176.8,174.6,143.9(C=CHN₃),126.0(C=CHN₃),102.8(C1^II),100.6(C1^I),77.8,75.6,72.8,72.5,71.3,70.0,69.8,68.9,68.7,66.6,64.1,62.5,61.3,59.6,56.8,55.3,50.3,40.5,38.4,34.0,33.9,28.1,24.3,22.4;HRESIMS:m/z909.21743[M+2Na]⁺(calcdforC₃₁H₅₁N₄Na₂O₁₈S₃:909.21599[M+2Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

Wherein, the building-up process of the lactose amine sulfuric ester propine glycosides shown in formula I-3 is as follows:

Get 708 milligrams of compound 13(1.0mmol, its synthetic method is shown in document: R.Daly, and G.Vaz, A.M.Davies,M.O.Senge, E.M.Scanlan, Chem.Eur.J.2012,18,14671 – 14679.) be dissolved in 10 milliliters of pyridine/acetic anhydrides（Py/Ac₂O, 1:1) in solution. Reactant liquor at room temperature stirs to spend the night and then concentrates, and separates (benzinum/acetic acid second through silicagel columnEster 1:1) obtain compound amorphous solid 14(730 milligram, productive rate 98%).

The structural characterization data of compound 14 prepared by the present embodiment are as follows:

¹HNMR(400MHz,CDCl₃):δ5.62(1H,d,J₂,N9.6Hz,NH),5.34(1H,d,J2.8Hz,H4^II),5.08(1H,d,J_3,48.0Hz,J_2,310.4Hz,H3^I),5.03(1H,t,J8.8Hz,H2^II),4.95(1H,dd,J_3,42.8Hz,J_2,310.4Hz,H3^II),4.63(1H,d,J_1,28.0Hz,H1^II),4.61(1H,d,J_1,27.6Hz,H1^I),4.32(2H,d,J2.4Hz,CH₂C≡CH),4.00–4.12(3H,m),3.88–3.95(2H,m),3.80–3.88(2H,m),3.34–3.36(1H,m),2.43(1H,t,J2.4Hz,C≡CH),1.95–2.13(18H,m,6×Ac),0.91,0.09,0.08(15H,3s,5×CH₃ofTBS);¹³CNMR(125MHz,CDCl₃):δ171.1,170.4,170.3,170.2,170.1,169.2,100.4,98.4,78.7,75.7,75.2,73.9,72.6,71.1,70.8,69.3,67.0,61.2,61.1,55.2,53.0,25.9,23.4,20.9,20.8,20.7,20.6,18.3,-5.0,-5.2;MALDI-TOFMS:m/z768.4[M+Na]⁺(calcdforC₃₃H₅₁NO₁₆Si:745.3[M]⁺).

By 746 milligrams of compound 14(1.0mmol) be dissolved in 10 milliliters of carrene (DCM), be cooled to 0 DEG C and add again0.38 milliliter of BFEE (BF₃·Et₂O, 3.0mmol). Reactant liquor stirs after 1 hour and adds sodium acid carbonate (NaHCO₃）Solution, with carrene (DCM) extraction three times, collects organic phase concentrated. Concentrate is dissolved in to DMF(DMF), add 954mg sulfur trioxide-pyridine complex (SO₃Pycomplex, 6.0mmol). This reactant liquor at room temperatureStir 8 hours, add 1 milliliter of triethylamine (Et₃N) concentrated after neutralization. Mixture obtains amorphous after silicagel column separates (EtOAc)Solid 15(512 milligram, productive rate 72%).

The structural characterization data of compound 15 prepared by the present embodiment are as follows:

¹HNMR(400MHz,MeOD):δ5.36(1H,d,J3.2Hz,H4^II),5.09–5.13(2H,m,H2^II,H3^II),4.97–5.02(1H,dd,J_3,48.0Hz,J_2,310.4Hz,H3^I),4.88(1H,d,J_1,27.6Hz,H1^I),4.73(1H,d,J_{1, 2}8.4Hz,H1^II),4.34(2H,d,J2.4Hz,CH₂C≡CH),4.25–4.30(2H,m),4.09–4.17(3H,m),3.82–3.88(2H,m),3.62–3.65(1H,m),2.86(1H,t,J2.4Hz,C≡CH),1.91–2.12(18H,m,6×Ac);¹³CNMR(125MHz,MeOD):δ172.1,170.8,170.7,170.3,170.0,100.1,98.6,78.4,75.1,73.4,73.0,71.3,70.4,69.3,67.5,65.2,61.0,55.4,53.7,21.4,19.8,19.5,19.3,19.1;MALDI-TOFMS:m/z734.4[M+Na]⁺(calcdforC₂₇H₃₇NO₁₉S:711.2[M]⁺).

By 712 milligrams of compound 15(1.0mmol) be dissolved in 10 ml methanol/carrene (MeOH/DCM, 1:1) solutionIn, add sodium methoxide/methyl alcohol (NaOMe/MeOH, 1M) adjust its pH be 9. reactant liquors at room temperature stir within 3 hours, add ion hand overChange resin (Dowex-50 (H⁺)) neutralization. Filtrate is concentrated after filtering. Mixture separates and obtains through gel column (Bio-GelP2)Amorphous solid 3(formula I-3,705 milligrams, productive rate 99%).

Compound 3(formula I-3 prepared by the present embodiment) structural characterization data as follows:

¹HNMR(400MHz,MeOD):δ4.61(1H,d,J8.0Hz,H1^I),4.34–4.39(3H,m,H1^II,CH₂C≡CH),3.90(1H,dd,J2.4Hz,12.0Hz),3.82–3.87(2H,m),3.59–3.78(7H,m),3.50–3.54(2H,m),3.40-3.42(1H,m),3.38–3.41(1H,m),2.87(1H,t,J2.4Hz,C≡CH),2.00(3H,s,Ac);¹³CNMR(125MHz,MeOD):δ172.5,103.8,99.1,79.6,78.6,75.8,75.3,75.0,73.5,72.9,71.3,69.1,61.2,60.6,55.3,55.1,21.6;ESIMS:m/z524.1[M+Na]⁺,540.1[M+K]⁺(calcdforC₁₇H₂₇NO₁₄S:501.1[M]⁺). from above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is targetCompound.

2, the short connection chain of sialic acid trisaccharide is synthetic

Get 23 milligrams of short connection chains of lactose amine sulfuric ester (formula II-1-3, final concentration is 10mM), and 19.87 milligrams of CMP-N-N acetylneuraminic acid n (CMP-Neu5Ac, final concentration is 15mM) is dissolved in the Tris-HCl(pH8.0 of 2.7 milliliters) in buffer solution after,Temperature of reaction system is risen to 37 DEG C, then adds 135 microlitre Pm2,3-ST(0.27U), after 2-3 hour, thin-layer chromatography (TLC)Detect raw material and disappear completely, reactant liquor is removed to zymoprotein by milipore filter, pure through gel column G-15 after freeze drying is concentratedChange final that white solid is 19 milligrams of compounds shown in formula X-1-3, productive rate 61.8%.

Shown in formula X-1-3 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.09(1H,s,C=CHN₃),4.91(1H,d,J13.0Hz,H1^I),4.83(1H,d,J12.7Hz,H1^II),4.65–4.59(3H,m),4.41(1H,d,J10.4Hz),4.30(1H,dd,J3.8,10.7Hz),4.23–4.20(2H,m),4.11(1H,dd,J3.1,9.9Hz),3.98–3.58(25H,m),3.56–3.52(1H,m),3.26–3.14(2H,m),2.74(1H,dd,J4.6,12.3Hz,H3eq-Sia),2.51–2.43(1H,m),2.40(2H,t,J7.2Hz),2.02(3H,s,Ac-Sia),1.98(1H,t,J6.6Hz),1.93(3H,s,Ac-I),1.80(1H,t,J12.1Hz,H3ax-Sia),1.75–1.68(1H,m),1.66–1.55(3H,m),1.45–1.37(2H,m);¹³CNMR(100MHz,D₂O):δ179.5,177.8,177.2,173.7,146.6(C=CHN₃),128.7(C=CHN₃),105.1(C1^II),103.2(C1^I),102.7(C1^III),80.2,78.2,78.0,77.1,75.7,75.6,75.1,74.4,72.6,72.4,72.3,71.6,71.4,71.0,70.6,69.2,66.7,65.4,65.1,63.9,62.3,59.4,57.8,54.6,52.9,43.2,42.5,41.0,36.5,30.7,26.9,25.0,24.9;HRESIMS:m/z1178.32610[M+Na]⁺(calcdforC₄₂H₆₉N₅NaO₂₆S₃:1178.32851[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 6, sialic acid trisaccharide-short connection chain

(formula II-1-3, final concentration is 10mM, and 19.87 milligrams to get the short connection chain of 23 milligrams of embodiment, 5 lactose amine sulfuric esterCMP-NeuAc (CMP-Neu5Ac, final concentration is 15mM) is dissolved in the Tris-HCl(pH8.0 of 2.7 milliliters) buffer solutionIn after, temperature of reaction system is risen to 37 DEG C, then add 135 microlitre Pd2,6-ST(0.27U), after 2-3 hour, thin-layer chromatography(TLC) detect raw material disappears completely, reactant liquor is removed to zymoprotein by milipore filter, freeze drying concentrate after through gel column G-15 purifying are final that white solid is 18.5 milligrams of compounds shown in formula X-2-3, productive rate 60%.

Shown in formula X-2-3 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.10(1H,s,C=CHN₃),4.91(1H,d,J13.4Hz,H1^I),4.84(1H,d,J13.2Hz,H1^II),4.67–4.63(3H,m),4.47–4.42(2H,m),4.27–4.23(1H,m),4.22–4.20(2H,m),4.00–3.49(20H,m),3.26–3.14(2H,m),2.65(1H,dd,J4.4,12.2Hz,H3eq-Sia),2.51–2.43(1H,m),2.39(2H,t,J7.3Hz),2.05–1.93(7H,m),1.77–1.55(5H,m),1.44–1.37(2H,m);¹³CNMR(100MHz,D₂O):δ179.5,177.8,177.3,176.4,146.5(C=CHN₃),128.7(C=CHN₃),106.3(C1^II),103.1(C1^I),102.2(C1^III),83.2,76.5,75.4,75.3,75.3,74.6,73.6,72.6,72.4,71.6,71.4,71.3,71.0,69.6,66.7,66.0,65.5,65.1,59.4,57.6,54.8,52.9,43.2,43.0,40.8,36.5,30.7,26.9,25.1,24.9;HRESIMS:m/z1178.32537[M+Na]⁺(calcdforC₄₂H₆₉N₅NaO₂₆S₃:1178.32851[M+Na]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 7, sialic acid tetrose-short connection chain

1、Le^XSynthesizing of short connection chain

Get 40 milligrams of Le shown in formula I-4^XTrisaccharide propine glycosides (0.07mmol) and 38.41 milligrams of short connection chains are (suc as formula IVShown in, 0.106mmol) be dissolved in the mixing of 5 milliliters of DMFs (DMF) and methyl alcohol (MeOH) (volume ratio is 1:1)In solution, then add 6.7 milligrams of cuprous iodides (CuI, 0.035mmol), reaction overnight under nitrogen protection. TLC monitors raw materialDisappear, by reacting liquid filtering, with G-15 gel column purifying, final that white solid is formula II-1-4 institute after freeze drying is concentratedShow compound (32.8 milligrams, productive rate 50%).

The structural characterization data of compound shown in formula II-1-4 prepared by the present embodiment are as follows:

¹HNMR(400MHz,D₂O):δ8.09(1H,s,C=CHN₃),5.09(1H,d,J4.0Hz,H1^III),4.95(1H,d,J12.8Hz,H1^I),4.83(1H,d,J12.9Hz,H1^II),4.67(2H,t,J4.8Hz),4.48(1H,d,J7.8Hz),4.26–4.24(2H,m),4.01–3.99(3H,m),3.96–3.88(7H,m),3.81(1H,d,J2.8Hz),3.76–3.61(15H,m),3.54–3.50(1H,m),3.29–3.17(2H,m),2.54–2.46(1H,m),2.43(2H,t,J7.2Hz),2.05–1.96(1H,m),1.94(3H,s,Ac),1.80–1.72(1H,m),1.70–1.58(2H,m),1.48–1.40(2H,m),1.19(3H,d,J8.0Hz,H6^III);¹³CNMR(125MHz,D₂O):δ176.2,174.0,143.5(C=CHN₃),125.6(C=CHN₃),101.8(C1^II),99.9(C1^I),98.6(C1^III)75.4,74.9,74.8,73.3,72.4,71.9,71.0,69.7,69.2,68.7,68.5,68.3,67.7,66.7,63.7,61.9,61.4,59.8,56.5,55.7,50.0,40.3,38.1,33.8,33.6,28.0,24.1,22.3,22.1,15.3;HRESIMS:m/z931.35143[M+H]⁺(calcdforC₃₇H₆₃N₄O₁₉S₂:931.35224[M+H]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

Wherein, the Le shown in formula I-4^XThe building-up process of trisaccharide propine glycosides is as follows:

By 373 milligrams of compound 17(1.0mmol, it synthesizes sees document: R.Daly, G.Vaz, and A.M.Davies,M.O.Senge, E.M.Scanlan, Chem.Eur.J.2012,18,14671 – 14679.) and molecular sieve (4) be dissolved in 20 millilitersIn anhydrous methylene chloride (DCM), be chilled in advance 0 DEG C, under nitrogen protection, add 20 microlitre TFMS trimethyl silicone grease(TMSOTf, 0.11mmol) and 383 milligrams of N-N-iodosuccinimides (NIS, 1.7mmol). Reactant liquor stirred after 10 minutes, willBe dissolved in 757 milligrams of compound 16(1.1mmol of 20 milliliters of carrene (DCM), it synthesizes sees document: A.Fekete, A.Borbá s, S.Antus, A.Lipt á k, Carbohydr.Res.2009,344,1434 – 1441.) drip wherein. Continue to stir 30 minutesAfter, reactant liquor neutralizes with triethylamine, and saturated sodium thiosulfate solution cleans, and organic phase is concentrated after separating, collect, being dried. MixThing obtains white solid compound 18(685 milligram, productive rate after silica gel column chromatography separates (petrol ether/ethyl acetate 2:1)72%）。

The structural characterization data that the present embodiment is prepared compound 18 are as follows:

¹HNMR(400MHz,CDCl₃):δ7.22–8.08(20H,m,PhH),5.98(1H,d,J3.2Hz,H4^II),5.82(1H,dd,J_1,28.0Hz,J_2,310.4Hz,H2^II),5.56(1H,dd,J_3,43.6Hz,J_2,310.8Hz,H3^II),5.82(1H,d,J_2,N8.4Hz,NH),5.01(1H,d,J_1,28.0Hz,H1^I),4.82(1H,d,J_1,28.0Hz,H1^II),4.59–4.63(1H,m,H6^II),4.46–4.51(1H,m),4.38–4.38(2H,m),4.24–4.32(3H,m),4.06(1H,t,J9.6Hz),3.74(1H,t,J9.2Hz),3.50–3.60(1H,m),3.32–3.34(1H,m),2.76(1H,s,OH),2.39(1H,t,J2.4Hz,C≡CH),2.00(3H,s,Ac),0.84,0.07,0.06(15H,3s,5×CH₃ofTBS);¹³CNMR(125MHz,CDCl₃):δ170.6,166.2,165.6,165.5,165.1,133.8,133.6,133.5,133.4,131.0,130.1,130.0,129.8,129.8,129.3,128.9,128.9,128.8,128.7,128.6,128.4,101.7,97.8,80.8,78.9,75.0,72.2,71.8,69.7,68.0,65.6,62.3,61.3,57.9,56.6,55.3,26.0,25.9,18.3,-5.1,-5.3;MALDI-TOFMS:m/z974.4[M+Na]⁺(calcdforC₅₁H₅₇NO₁₅Si:951.4[M]⁺).

By 952 milligrams of compound 18(1.0mmol) and 442 milligrams of compounds 19 (1.2mmol, it is synthetic sees document:A.Mukherjee, M.M.Palcic, O.Hindsgaul, Carbohydr.Res.2000,326,1 – 21.) be dissolved in 10 milliliters of nothingsWater-toluene (PhCH₃) in. Under 0 DEG C of condition that is connected with nitrogen, add 405 milligrams of N-N-iodosuccinimides (NIS, 1.8mmol)With 22 microlitre TFMS trimethyl silicone grease (TMSOTf, 0.12mmol). When thin-layer chromatography (TLC, petrol ether/ethyl acetateSo 1:1) show that when initiation material disappears, reaction stops, reactant liquor neutralizes with triethylamine, after saturated sodium thiosulfate cleans, hasMachine is dried mutually and concentrates. Mixture obtains oily compound 20 after silica gel column chromatography (petrol ether/ethyl acetate 2:1) separates(931 milligrams, productive rate 74%).

The structural characterization data that the present embodiment is prepared compound 20 are as follows:

¹HNMR(400MHz,CDCl₃):δ6.82–8.09(24H,m,PhH),5.98(1H,d,J3.2Hz,H4^II),5.95(1H,d,J_2,N8.4Hz,NH),5.75(1H,dd,J_1,28.0Hz,J_2,310.4Hz,H2^II),5.59(1H,dd,J_3,43.6Hz,J_{2, 3}10.4Hz,H3^II),5.26(1H,d,J_1,23.6Hz,H1^III),5.12(1H,d,J_1,28.0Hz,H1^I),4.70–4.75(2H,m,H1^II,H6^II),4.52–4.63(3H,m),4.50(1H,dd,J7.6,11.6Hz),4.36(1H,dd,J5.6,8.0Hz),4.24(1H,t,J6.8Hz),4.14–4.17(4H,m),3.83(1H,dd,J2.8,11.6Hz),3.77(3H,s,OCH₃),3.55–3.61(2H,m),3.18–3.20(1H,m),2.26(1H,t,J2.4Hz,C≡CH),1.95(3H,s,Ac),1.44(3H,d,J6.8Hz,H5^III),1.40,1.26(3H,s,2×CH₃),0.89,0.07,0.01(15H,3s,5×CH₃ofTBS);¹³CNMR(125MHz,CDCl₃):δ170.2,166.0,165.8,165.5,165.4,133.8,133.6,133.4,133.3,130.7,130.0,130.0,129.8,129.7,129.5,129.1,129.0,128.9,128.8,128.7,128.6,128.5,128.4,113.9,108.6,100.0,98.1,96.3,79.0,77.3,76.7,76.5,75.7,74.7,74.4,74.3,72.3,71.8,71.6,70.1,68.4,63.7,61.8,61.4,63.7,61.8,61.4,55.3,55.2,28.4,26.2,25.9,23.6,18.3,16.5,-5.0,-5.3;MALDI-TOFMS:m/z1280.4[M+Na]⁺(calcdforC₆₈H₇₉NO₂₀Si:1257.5[M]⁺).

By 630 milligram of three sugar compounds 20(0.5mmol) be dissolved in 15 milliliters of carrene/water (DCM/H₂O, 18:1) solutionIn, adding 227 milligram 2,3-bis-is chloro-5,6-dicyan 1,4-benzoquinone (DDQ, 1.0mmol). Reactant liquor at room temperature stirred after 7 hoursAdd 10 milliliters of hypo solution (Na₂S₂O₃, 20%). Mixture is with ethyl acetate extraction three times, and organic phase is collected, is driedAnd concentrated, be then suspended from (AcOH, 80%) in 20 milliliters of acetic acid solutions. Reactant liquor is dense altogether with toluene after 30 minutes 60 DEG C of reactionsContracting. Concentrate joins in pyridine/solution of acetic anhydride and carries out acetylization reaction according to the method for compound 14. Through silica gel column chromatography(petrol ether/ethyl acetate 1:2) separates and obtains oily three sugar compounds 21(392 milligrams, productive rate 68%).

The structural characterization data that the present embodiment is prepared compound 21 are as follows:

¹HNMR(400MHz,CDCl₃):δ7.22–8.06(20H,m,PhH),5.98(1H,d,J3.2Hz,H4^II),5.77(1H,d,J₂,N9.2Hz,NH),5.69(1H,dd,J_1,28.0Hz,J_2,310.4Hz,H2^II),5.59(1H,dd,J_3,43.6Hz,J_2,310.4Hz,H3^II),5.26(1H,d,J_1,24.0Hz,H1^III),5.34(1H,s,H4^III),5.32(1H,dd,J3.2,9.2Hz,H3^III),5.10(1H,dd,J4.0,9.6Hz,H2^III),4.90(1H,dd,J7.2,11.6Hz),4.78(1H,d,J8.0Hz,H1^I),4.72–4.76(1H,m),4.66(1H,dd,J9.6,11.6Hz),4.61(1H,d,J6.4Hz,H1II),4.44(1H,dd,J4.0,12.0Hz),4.27–4.32(2H,m,CH₂C≡CH),4.09–4.19(3H,m),3.91–3.97(2H,m),3.51–3.54(1H,m),2.32(1H,t,J2.4Hz,C≡CH),1.91–2.17(15H,m,5×Ac),1.24(3H,d,J6.8Hz,H5^III);¹³CNMR(125MHz,CDCl₃):δ171.1,170.5,170.4,169.9,166.2,166.0,165.4,165.3,133.7,133.6,133.4,129.9,129.8,129.7,129.6,129.3,128.9,128.7,128.4,100.9,98.1,95.1,78.7,75.1,74.5,72.9,72.8,72.4,71.6,71.4,69.8,68.2,68.0,67.9,65.0,62.4,61.7,55.7,23.5,21.1,20.9,20.8,20.7,16.0;MALDI-TOFMS:m/z1174.4[M+Na]⁺(calcdforC₅₉H₆₁NO₁₃:1151.4[M]⁺).

By 115 milligrams of compound 21(0.1mmol) be dissolved in 10 ml methanol/carrene (MeOH/DCM, 1:1) solutionIn, add sodium methoxide/methyl alcohol (NaOMe/MeOH, 1M) adjust its pH be 9. reactant liquors at room temperature stir within 3 hours, add ion hand overChange resin (Dowex-50 (H⁺)) neutralization. Filtrate is concentrated after filtering. Mixture separates and obtains through gel column (Bio-GelP2)Amorphous solid 4(formula I-4,57 milligrams, productive rate 99%).

Compound 4(formula I-4 prepared by the present embodiment) structural characterization data as follows:

¹HNMR(400MHz,D₂O):δ5.05(1H,d,J4.0Hz,H1^III),4.77(1H,q,J7.8Hz,H5^III),4.69(1H,d,J8.0Hz,H1^I),4.39(1H,d,J7.6Hz,H1^II),4.34(2H,d,2.8Hz,CH₂C≡CH),3.94(1H,dd,J2.4Hz,12.4Hz),3.83–3.90(5H,m),3.80(1H,dd,J4.8Hz,12.4Hz),3.73(1H,d,J2.8Hz,H4^II),3.64–3.69(2H,m),3.58–3.62(2H,m),3.52–3.56(2H,m),3.44(1H,dd,J8.0Hz,10.0Hz),2.86(1H,t,J2.4Hz,C≡CH),1.98(3H,s,Ac),1.12(3H,d,J6.8Hz,H6^III);¹³CNMR(125MHz,D₂O):δ174.6,102.0,99.4,98.7,76.3,75.6,75.0,75.0,73.5,72.6,72.1,71.2,69.4,68.5,67.9,66.9,61.6,59.9,56.9,55.7,22.4,15.4;MALDI-TOFMS:m/z590.2[M+Na]⁺,606.2[M+K]⁺(calcdforC₅₉H₆₁NO₁₃:567.2[M]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

2, the short connection chain of sialic acid tetrose is synthetic

Get 9 milligrams of Le^XThe short connection chain of trisaccharide (formula II-1-4, final concentration is 10mM), and 7.14 milligrams of CMP-N-acetyl godBe dissolved in the Tris-HCl(pH8.0 of 1 milliliter through propylhomoserin (CMP-Neu5Ac, final concentration is 15mM)) in buffer solution after, will react bodyBe that temperature rises to 37 DEG C, then add 50 microlitre Pm2,3-ST(0.1U), after 2-3 hour, thin-layer chromatography (TLC) has detected raw materialDisappear completely, reactant liquor is removed to zymoprotein by milipore filter, final in vain through gel column G-15 purifying after freeze drying is concentratedLook solid is 2 milligrams of compounds shown in formula X-1-4, productive rate 16.9%.

Shown in formula X-1-3 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(500MHz,D₂O):δ8.06(1H,s,C=CHN₃),5.04(1H,d,J3.9Hz,H1^III),4.91(1H,d,J13.1Hz,H1^I),4.79-4.73(1H,overlappedbysolventpeak,H1^II),4.62(2H,dd,J6.2,10.9Hz),4.51(1H,d,J7.8Hz),4.21–4.20(1H,m),4.10(1H,dd,J9.9Hz),4.01–3.85(16H,m),3.75–3.49(23H,m),3.22–3.16(1H,m),2.75(1H,dd,J4.2,12.4Hz,H3eq-Sia),2.48–2.44(1H,m),2.38(1H,t,J7.1Hz),2.02(3H,s,Ac-Sia),1.98–1.94(1H,m),1.89(3H,s,Ac-I),1.71–1.58(2H,m),1.43–1.38(1H,m),1.14(3H,d,J6.4Hz,H6^III);¹³CNMR(150MHz,D₂O):δ180.4,176.6,175.0,174.1,143.5(C=CHN₃),125.6(C=CHN₃),101.6(C1^II),100.0(C1^I),99.5(C1^IV),98.5(C1^III),75.6,75.3,74.8,73.3,72.9,71.9,71.6,70.3,70.2,69.7,69.2,68.7,68.5,68.2,68.1,67.7,67.3,67.0,66.6,63.8,63.2,62.6,61.9,61.4,60.3,56.5,55.7,52.1,51.7,50.0,40.3,39.7,39.1,33.6,27.8,24.0,22.0,15.2;HRESIMS:m/z1222.44473[M+H]⁺(calcdforC₄₈H₇₉N₅O₂₇S₂:1222.44766[M+H]⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation (formula X-1-5) of embodiment 8, sialic acid trisaccharide-long connection chain

1, the preparation of sialic acid trisaccharide

Get 76 milligrams of lactose propine glycosides (formula I-1, final concentration is 10mM, its synthetic method is shown in document: F.Lutz,U.B.Tietze, Chem.Eur.J.1998,4,1179 – 1183.) and 197.5 milligrams of CMP-NeuAc (CMP-Neu5Ac, final concentration is 15mM) be dissolved in the Tris-HCl(pH8.0 of 20 milliliters) in buffer solution after, temperature of reaction system is risen to37 DEG C, then add 500 microlitre Pm2,3-ST(1U), after 2-3 hour, thin-layer chromatography (TLC) detects raw material and disappears completely, willReactant liquor is removed zymoprotein by milipore filter, and after freeze drying is concentrated, after gel column G-15 purifying, obtaining white solid is sialic acidTrisaccharide (shown in formula II-1-5,127.5 milligrams, productive rate 95%). Concrete reaction equation is as follows:

2, the long connection chain of sialic acid trisaccharide is synthetic

Get 67 milligrams of sialic acid trisaccharides (0.1mmol) shown in formula II-1-5 and the long connection chain shown in 80.4 milligrams of formula V(0.15mmol, its synthetic method is shown in document: A.G.Barrientos, J.M.delaFuente, T.C.Rojas,A.Fernandez, S.Penades, Chem.Eur.J.2003,9,1909 – 1921.) be dissolved in 5 milliliters of DMFs(DMF) and in the mixed solution of methyl alcohol (MeOH) (volume ratio is 1:1), then add 9.5 milligrams of cuprous iodides (CuI,0.05mmol), in order further to remove the acetyl group on chain, then add 2 ml methanol (MeOH) and appropriate sodium methoxide (MeONa)Solid makes pH reach 10-11, reaction overnight under nitrogen protection. TLC monitoring raw material disappears, by reacting liquid filtering, freezingWith G-15 gel column purifying, final that white solid is 83 milligrams of compounds shown in formula X-1-5, productive rate after dry concentrating80%。

Formula V:

Shown in formula X-1-5 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.15(1H,s,C=CHN₃),5.01(1H,d,J12.48Hz,H1^I),4.87(1H,d,J12.68Hz,H1^II),4.64(2H,t,J4.86Hz),4.59–4.53(1H,m),4.16(1H,d,J9.88Hz),3.98–3.62(41H,m),3.48(2H,t,J6.36Hz),3.36(1H,t,J8.12Hz),2.77–2.72(3H,m),2.05(3H,s),1.90(1H,t,J12.48Hz,H3ax-Sia),1.71–1.59(4H,m),1.43-1.34(13H,m);¹³CNMR(150MHz,D₂O):δ175.1,174.9,143.6(C=CHN₃),125.7(C=CHN₃),103.1(C1^II),101.5(C1^I),95.5(C1^III),78.5,75.6,75.5,75.1,74.9,74.5,73.2,72.9,72.7,71.2,70.5,70.3,70.0,69.8,69.7,68.9,68.7,68.4,67.9,67.7,66.8,63.3,63.0,62.0,61.1,61.0,60.2,52.2,51.8,51.9,50.2,39.0,30.4,29.8,29.6,29.5,29.3,29.0,28.9,26.1,22.2;HRESI-MS:m/z1164.54939[2M+H]²⁺(calcdforC₉₈H₁₇₆N₈O₅₀S₂:1164.54529[2M+H]²⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 9, sialic acid trisaccharide-long connection chain

1, the preparation of sialic acid trisaccharide

Get 76 milligrams of lactose propine glycosides (formula I-1, final concentration is 10mM, its synthetic method is shown in document: F.Lutz,U.B.Tietze, Chem.Eur.J.1998,4,1179 – 1183.) and 197.5 milligrams of CMP-NeuAc (CMP-Neu5Ac, final concentration is 15mM) be dissolved in the Tris-HCl(pH8.0 of 20 milliliters) in buffer solution after, temperature of reaction system is risen to37 DEG C, then add 500 microlitre Pd2,6-ST(1U), after 2-3 hour, thin-layer chromatography (TLC) detects raw material and disappears completely, willReactant liquor is removed zymoprotein by milipore filter, and after freeze drying is concentrated, after gel column G-15 purifying, obtaining white solid is sialic acidTrisaccharide (shown in formula II-1-6,124.8 milligrams, productive rate 93%).

2, the long connection chain of sialic acid trisaccharide is synthetic

Get 67 milligrams of sialic acid trisaccharides (0.1mmol) shown in formula II-1-6 and the long connection chain shown in 80.4 milligrams of formula V(0.15mmol) be dissolved in the mixed solution of 5 milliliters of DMFs (DMF) and methyl alcohol (MeOH) (volume ratio is 1:1)In, then add 9.5 milligrams of cuprous iodides (CuI, 0.05mmol), in order further to remove the acetyl group on chain, then add 2 millisLiter methyl alcohol (MeOH) and appropriate sodium methoxide (MeONa) solid make pH reach 10-11, reaction overnight under nitrogen protection. TLCMonitoring raw material disappears, by reacting liquid filtering, with G-15 gel column purifying, final that white solid is formula after freeze drying is concentratedCompound 70 shown in X-2-5 milligram, productive rate 60%.

Shown in formula X-2-5 prepared by the present embodiment, the structural characterization data of compound are as follows:

¹HNMR(400MHz,D₂O):δ8.17(1H,s,C=CHN₃),5.02(1H,d,J12.96Hz,H1^I),4.88(1H,d,J12.64Hz,H1^II),4.66(2H,t,J4.28Hz),4.61(1H,d,J7.88Hz),4.44(1H,t,J7.54Hz),4.02–3.57(42H,m),3.50(2H,t,J6.14Hz),3.39(1H,t,J8.22Hz),2.73–2.71(3H,m),2.05(3H,s),1.85(1H,t,J12.26Hz,H3ax-Sia),1.73–1.60(4H,m),1.45–1.35(12H,m);¹³CNMR(150MHz,D₂O):δ175.0,173.6,143.6(C=CHN₃),125.8(C=CHN₃),103.4(C1^II),101.4(C1^I),100.4(C1^III),79.7,74.8,73.7,72.8,72.6,72.5,71.9,71.2,70.9,69.9,69.8,69.8,69.7,68.9,68.6,68.5,68.4,63.6,62.7,62.0,61.5,60.4,59.4,56.7,51.9,50.1,40.2,30.4,30.0,29.7,29.7,29.6,29.4,29.2,29.0,28.9,26.1,22.2;HRESI-MS:m/z1164.54939[2M+H]²⁺(calcdforC₉₈H₁₇₆N₈O₅₀S₂:1164.54529[2M+H]²⁺).

From above-mentioned characterization data, the structure of the compound that the present embodiment obtains is correct, is target compound.

The preparation of embodiment 10, sialic acid trisaccharide-golden nanometer particle

1.5 milliliters of golden nanometer particles (average grain diameter is 13nm, and final concentration is 10nM) are dissolved in 9 milliliters of ultra-pure waters, thenAdd any embodiment of 0.6-0.7 milligram 1-9 preparations sialic acid trisaccharide-length/short connection chain (make sialic acid trisaccharide-length/The mol ratio of short connection chain and golden nanometer particle is 40000:1), and different proportion with sialic acid trisaccharide-length/short connection chain inThe identical length/short connection chain of connection chain (mol ratio of sialic acid trisaccharide-length/short connection chain and length/short connection chain is respectively2:1,5:1,10:1 and 1:0) mixture, stirring at room temperature 24 hours, obtains 36 kinds of sialic acid trisaccharide-golden nanometer particles. Reaction knotShu Hou, with ultrapure washing twice, PBS buffer solution (10mM, pH7.4) is washed one time (14000rpm, 30min) respectively, is finally dissolved in 1In milliliter PBS buffer solution (10mM, pH7.4), obtain 36 kinds of solution containing sialic acid trisaccharide-golden nanometer particle, 4 DEG C of preservations.

Describing according to the preceding paragraph the sialic acid trisaccharide-length/short connection chain obtaining with embodiment 1-2 and embodiment 8-9 (is total to16 kinds, be called for short Sia-Lac-AuNPs, its structural formula schematic diagram is as shown in Figure 1) for example describes, concrete reaction equation is as follows:

Sialic acid trisaccharide-golden nanometer particle that embodiment 11, embodiment 10 obtain detects the host specificity of influenza virus

The strain information of the influenza virus that the present embodiment uses is as follows:

Strain A/California/04/2009(H1N1): be called for short 09H1, document: W.Zhang, J.Qi, Y.Shi, Q.Li,F.Gao,Y.Sun,X.Lu,Q.Lu,C.J.Vavricka,D.Liu,J.Yan,G.F.Gao，ProteinCell2010,1,The amino acid sequence of 459 – 467. its HA albumen be for No. Genbank ACP41105.1; Being subject to of known this virus HA albumen identificationBody is sialic acid α 2,6 oligosaccharides of host cell secretion.

Strain A/SouthCarolina/1/1918(H1N1): be called for short 18H1, document: J.Stevens, O.Blixt,L.Glaser,J.K.Taubenberger,P.Palese,J.C.Paulson,I.A.Wilson,J.Mol.Biol.2006,The amino acid sequence of 355,1143 – 1155. its HA albumen be for No. Genbank AAD17229.1; Known this virus HA albumen is knownOther acceptor is sialic acid α 2,6 oligosaccharides of host cell secretion.

Strain A/SouthCarolina/1/1918(H1N1) (mutant strain of D225G): be called for short 18H1mut, document:J.Stevens,O.Blixt,L.Glaser,J.K.Taubenberger,P.Palese,J.C.Paulson,I.A.Wilson,J.Mol.Biol.2006, the amino acid sequence of 355,1143 – 1155. its HA albumen be for No. Genbank AAD17229.1; ?The acceptor of knowing the identification of this virus HA albumen is sialic acid α 2,3 or α 2,6 oligosaccharides of host cell secretion.

Strain A/USSR/90/77(H1N1): be called for short 77H1, document: A.S.Gambaryan, V.P.Marinina,A.B.Tuzikov,N.V.Bovin,I.A.Rudneva,B.V.Sinitsyn,A.A.Shilov,M.N.Matrosovich,Virology, the amino acid sequence of 1998,247,170 – 177. its HA albumen be for No. Genbank ABD95350.1; Known being somebody's turn to doThe acceptor of virus HA albumen identification is sialic acid α 2,3 or α 2,6 oligosaccharides of host cell secretion.

Strain A/WSN/1933(H1N1): be called for short WSNH1, document: W.-C.Liu, S.-C.Lin, Y.-L.Yu, C.-L.Chu, S.-C.Wu, J.Virol.2010, is for No. Genbank of the amino acid sequence of 84,12011 – 12017. its HA albumenACF54598.1; The acceptor of known this virus HA albumen identification is sialic acid α 2,3 or α 2,6 oligosaccharides of host cell secretion.

Strain A/PR8/8/34(H1N1): be called for short PR8, document: C-T.Guoetal.Glycobiology2007,17,713 – 724, in known experiment, the acceptor of strain identification used is sialic acid α 2,3 oligosaccharides of host cell secretion.

Strain A/Aichi/2/1968(H3N2): be called for short 68H3, document: N.K.Sauter, J.E.Hanson,G.D.Glick,J.H.Brown,R.L.Crowther,S.-J.Park,J.J.Skehel,D.C.Wiley,Biochemistry1992, the amino acid sequence of 31,9609 – 9621. its HA albumen be for No. Genbank BAF37221.1; ?The acceptor of knowing the identification of this virus HA albumen is sialic acid α 2,6 oligosaccharides of host cell secretion.

Strain A/Bar-headedGoose/Qinghai/1/2005(H5N1): be called for short qinH5, document: Z.Li,Z.Liu,C.Ma,L.Zhang,Y.Su,G.F.Gao,Z.Li,L.Cui,W.He,Arch.Virol.2011,156,1803–The amino acid sequence of 1812. its HA albumen be for No. Genbank ABA29447.1; Known this virus HA albumen and strain identification thereofAcceptor be sialic acid α 2,3 oligosaccharides of host cell secretion.

Strain A/VietNam/1203/2004(H5N1): be called for short vieH5, document: J.Stevens, O.Blixt,T.M.Tumpey, J.K.Taubenberger, J.C.Paulson, I.A.Wilson, Science2006,312,404 – 410. itsThe amino acid sequence of HA albumen be for No. Genbank ABW90135.1; The acceptor of known this virus HA albumen identification is that host is thinSialic acid α 2,3 oligosaccharides of intracrine.

Strain A/Shenzhen/1/2011(H5N1): be called for short SheH5, document: C.Wu, X.Lu, X.Wang, T.Jin,X.Cheng, S.Fang, X.Wang, H.Ma, R.Zhang, J.Cheng, J.Med.Virol.2013,85,760 – 768. its HA eggsCasamino acid sequence is as shown in sequence table sequence 1. The acceptor of known this virus HA albumen identification is the saliva of host cell secretionAcid α 2,3 oligosaccharides.

Strain A/Anhui/1/2013(H7N9): be called for short AnhH7, document: D.Liu, W.Shi, Y.Shi, D.Wang,H.Xiao,W.Li,Y.Bi,Y.Wu,X.Li,J.Yan,W.Liu,G.Zhao,W.Yang,Y.Wang,J.Ma,Y.Shu,F.Lei,G.F.Gao, TheLancet, the amino acid sequence of 2013,381,1926 – 1932. its HA albumen as 2 of sequence table sequencesShow. The acceptor of known this virus HA albumen and strain identification thereof is sialic acid α 2,3 or α 2,6 oligosaccharides of host cell secretion.

Strain A/Shanghai/1/2013(H7N9): be called for short ShaH7, document: D.Liu, W.Shi, Y.Shi, D.Wang,H.Xiao,W.Li,Y.Bi,Y.Wu,X.Li,J.Yan,W.Liu,G.Zhao,W.Yang,Y.Wang,J.Ma,Y.Shu,F.Lei,G.F.Gao, TheLancet, the amino acid sequence of 2013,381,1926 – 1932. its HA albumen as 3 of sequence table sequencesShow. The acceptor of known this virus HA albumen identification is sialic acid α 2,3 oligosaccharides of host cell secretion, and the acceptor of strain identification isSialic acid α 2,3 or α 2,6 oligosaccharides of host cell secretion.

Below, α 2,3 sialic acid trisaccharides-short connection chain embodiment 1 being obtained and short connection chain are solid taking mol ratio as 5:1Sialic acid trisaccharide-the golden nanometer particle forming due to golden nanometer particle surface is referred to as 3 ' SLs-AuNP5:1;

α 2,6 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 2 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as 6 ' SLs-AuNP5:1;

α 2,3 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 3 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as 3 ' SLNs-AuNP5:1;

α 2,6 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 4 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as 6 ' SLNs-AuNP5:1;

α 2,3 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 5 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as Su-3 ' SLNs-AuNP5:1;

α 2,6 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 6 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as Su-6 ' SLNs-AuNP5:1;

α 2,3 sialic acid trisaccharides-short connection chain and short connection chain that embodiment 7 is obtained are fixed on gold taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as SLe^Xs-AuNP5:1；

α 2,3 sialic acid trisaccharides-long connection chain that embodiment 8 is obtained is fixed on gold with long connection chain taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as 3 ' SLl-AuNP5:1;

α 2,6 sialic acid trisaccharides-long connection chain that embodiment 9 is obtained is fixed on gold with long connection chain taking mol ratio as 5:1Sialic acid trisaccharide-golden nanometer particle that nanoparticle surface forms is referred to as 6 ' SLl-AuNP5:1.

One, host specificity qualitative detection

1, HA protein level

In 1.5mL centrifuge tube respectively by PBS buffer solution for 3 ' SLs-AuNP5:1 and 6 ' SLs-AuNP5:1 (10mM,PH7.4) being diluted to 2.5nM(is OD_522nmValue be 0.2) obtain dilution, connect every Kong Zhongjia in PCR pipes in 96 orifice plates or 8Enter the above-mentioned dilution of 150uL, then add 2.5uL influenza virus strain HA albumen to make its final concentration in reaction system beDescribed in 16.7ug/mL(, the ratio of sialyloligosaccharide-golden nanometer particle and influenza virus strain HA albumen to be measured is 2.8pmol:16.7ug), to add PBS buffer solution (10mM, pH7.4) as blank, under room temperature, hatch, in 40min, visually observe colorChange, and UV-Vis spectrum, dynamic light scattering (DLS) and TEM image for reactant liquor are detected with the result.

Macroscopic result is as shown in Figure 2:

Compared with not containing the blank of HA albumen, in 3 ' SLs-AuNP5:1, add after the HA albumen of various viruses,All there is obvious variation (by red stain purple) in qinH5,18H1mut and SheH5 sample, illustrate 3 ' SLs-AuNP5:1 withThere is combination largely in the HA albumen of qinH5,18H1mut and SheH5; It is little (by red that 77H1 amplitude of variation is comparedShoal red), the HA protein combination that 3 ' SLs-AuNP5:1 and 77H1 be described a little less than; 68H3 color does not change (still for red), saysThe HA albumen of bright 3 ' SLs-AuNP5:1 and 68H3 is without combination;

Compared with not containing the blank of HA albumen, in 6 ' SLs-AuNP5:1, add after the HA albumen of various viruses 68H3All there is obvious variation (by red stain purple) with 77H1 sample, the HA egg of 6 ' SLs-AuNP5:1 and 68H3 and 77H1 has been describedWhite in conjunction with strong; The color of other strain does not change (still for red), illustrate 6 ' SLs-AuNP5:1 and qinH5,18H1mut andThe HA albumen of SheH5 is without combination.

Ultraviolet-visible absorption spectroscopy (UV-Vis spectrum) changes as shown in Figure 3 and Figure 4. The change color of sample and spectrum becomeChange corresponding. When occur more by force in conjunction with time, sample not only can reduce at the maximum absorption band of about 522nm, and absorption maximumWavelength also can be moved, as the 68H3 in the qinH5 in Fig. 3, SheH5 and 18H1mut and Fig. 4; Only have when weakThe reduction of large absworption peak, as the 77H1 in 77H1 and Fig. 4 in Fig. 3; Not in conjunction with time, absworption peak is substantially constant, in Fig. 3PBS(blank), PBS(blank in 68H3 and Fig. 4), qinH5, SheH5 and 18H1mut.

Dynamic light scattering (DLS) changes as shown in Figure 5 and Figure 6. DLS survey be sample particle size, when sialic acid trisaccharide-Golden nanometer particle and HA albumen have while combination very by force, and particle flocks together and forms larger group, and the particle diameter of measuring is larger, as68H3 in qinH5, SheH5 and 18H1mut and Fig. 6 in Fig. 5; When weak, particle concentration class is lower, the particle diameter of measuringLess, as the 77H1 in 77H1 and Fig. 6 in Fig. 5; Not in conjunction with time, without assembling, particle diameter is constant, as the PBS in Fig. 5 and 68H3And PBS, qinH5, SheH5 and 18H1mut in Fig. 6.

TEM image as shown in Figure 7. Change correspondingly with DLS, can observe intuitively the aggregation extent of particle, result alsoConsistent with DLS result.

In addition, the following strain HA albumen result of detection is as follows according to the method described above:

AnhH7: identification α 2,3 and 2,6 two kinds of sialyloligosaccharides of α. Similar to SZH5 to 3 ' SLs-AuNP5:1 combination degree;Similar to 77H1 to the combination degree of 6 ' SLs-AuNP5:1.

ShaH7: only identify α 2,3 sialyloligosaccharides, and ShaH7 and 3 ' SLs-AuNP5:1 combination degree similar in appearance to 77H1 withThe combination degree of 6 ' SLs-AuNP5:1.

WSNH1: identification α 2,3 and 2,6 two kinds of sialyloligosaccharides of α. Slightly strong compared with 77H1 with 3 ' SLs-AuNP5:1 combination degree;Similar to 77H1 to the combination degree of 6 ' SLs-AuNP5:1.

09H1: only identify α 2,6 sialyloligosaccharides. Similar to 68H3 to the combination degree of 6 ' SLs-AuNP5:1.

18H1: only identify α 2,6 sialyloligosaccharides. With the combination degree of 6 ' SLs-AuNP5:1 compared with 68H3 slightly a little less than.

VieH5: only identify α 2,3 sialyloligosaccharides. Similar to qinH5 to 3 ' SLs-AuNP5:1 combination degree.

2, influenza virus level

In 1.5mL centrifuge tube respectively by PBS buffer solution for 3 ' SLs-AuNP5:1 and 6 ' SLs-AuNP5:1 (10mM,PH7.4) being diluted to 2nM(is OD_522nmValue be 0.16) obtain dilution, connect in PCR pipes and add in every hole in 96 orifice plates or 8The above-mentioned dilution of 88uL, then add 12uL influenza virus strain, making its final concentration in reaction system is 10⁸Individual/mL(ratio of described sialyloligosaccharide-golden nanometer particle and influenza virus strain to be measured is 2.5pmol:10⁸Individual), to add PBSBuffer solution (10mM, pH7.4) is blank, hatches for 4 DEG C, visually observes change color in 30min, and to reactant liquor UV-Vis spectrum, dynamic light scattering (DLS) and TEM image detect with the result. Result in result and step 1 is without significantlyDifference.

In step 1, test 1 and 2 result and show, can judge intuitively multiple stream by the change color of reaction systemThe receptor-specific of Influenza Virus strain and HA albumen thereof, UV-Vis, DLS and TEM image have further confirmed to be observed visuallyExperimental result, and this result is consistent with the corresponding known identification receptor type of virus. Whole system is under room temperature or 4 DEG C of low temperatureCarry out, without any instrument, naked eyes can be read experimental result, simple and fast.

Two, host specificity quantitatively detects

1, HA protein level

The short connection chain of sialyloligosaccharide/short connection chain 5:1 ratio of respectively being prepared by embodiment 10 in 1.5mL centrifuge tubeGolden nanometer particle to be diluted to 3.75nM(with PBS buffer solution (10mM, pH7.4) be that the value of OD522nm is 0.3) obtain dilutionLiquid adds the above-mentioned dilution of 150uL in the 96 every holes of orifice plate, then add 2.5uL influenza virus strain HA albumen make itsFinal concentration in reaction system is sialyloligosaccharide-golden nanometer particle and influenza virus strain HA egg to be measured described in 16.7ug/mL(White ratio is 2.8pmol:16.7ug), to add PBS buffer solution (10mM, pH7.4) as blank, under room temperature, hatch, withUV-Vis spectrum A when 40min₆₈₀/A₅₂₂The power that ratio qualitative assessment HA albumen is combined with sugar chain.

By Fig. 8,9 can find out, 09H1, and 18H1,68H3 is only in conjunction with α 2,6 sialyloligosaccharides, vieH5, qinH5, SheH5,ShaH7 is only in conjunction with α 2,3 sialyloligosaccharides, and 18H1mut and AnhH7 be in conjunction with two kinds of sugar chains, and this is complete with qualitative detection beforeCoincide. In addition,, for the different sugar chain of internal structure, various HA albumen also has different affinity. As inner sugar chain isLactose amine (LN) can increase the adhesion to humanized HA albumen; And for SheH5, inner sugar chain is Le^XAfterwards, its parentGreatly improve with power. These qualitative assessment result and reported in literature that are similar to finger-print are basically identical.

2, influenza virus level

The short connection chain of sialyloligosaccharide/short connection chain 5:1 ratio of respectively being prepared by embodiment 10 in 1.5mL centrifuge tubeGolden nanometer particle with PBS buffer solution (10mM, pH7.4), to be diluted to 6.25nM(be OD_522nmValue be 0.5) obtain dilution,In 96 orifice plates, in every hole, add the above-mentioned dilution of 88uL, then add 12uL influenza virus strain, make it in reaction systemIn final concentration be 10⁸Described in individual/mL(, the ratio of sialyloligosaccharide-golden nanometer particle and influenza virus strain to be measured is2.5pmol：10⁸Individual), to add PBS buffer solution (10mM, pH7.4) as blank, hatch UV-Vis during with 30min for 4 DEG CSpectrum A₆₈₀/A₅₂₂The power that ratio qualitative assessment influenza virus is combined with sugar chain.

As shown in figure 10, strain PR8 is only in conjunction with α 2,3 sialyloligosaccharides, and strain Ah-H7N9 and Sh-H7N9 are in conjunction with two kindsThe sialyloligosaccharide of type, this and reported in literature are basically identical. With protide seemingly, influenza virus strain is for different inside sugar chainsAlso there is its special identification activity.

In step 2, test 1 and 2 result and show, this reaction system not only can judge qualitatively influenza virus protein orCan person's strain be combined with certain sugar chain, can also assess quantitatively the relatively strong and weak of this combination, and for fine structureAs its adhesion power of the derivatization of inner sugar chain also can distinguish. Can obtain quickly and easily influenza by this systemInteraction information between virus and host cell, particularly in the time that new type influenza breaks out, can be at different hosts to itBetween propagate make in time, effectively prediction, therefore significant to the prevention and control of influenza.

Sialic acid trisaccharide-long the connection chain that uses embodiment 8 to obtain is fixed on taking mol ratio as 5:1 with long connection chain respectivelySialic acid trisaccharide-golden nanometer particle that golden nanometer particle surface forms is respectively according to the method influenza virus poison of above-mentioned steps 1 and 2Sialic acid trisaccharide-short connection chain that the result of strain HA albumen and strain and use embodiment 1 obtain and short connection chain are with mol ratioSialic acid trisaccharide-the golden nanometer particle that is fixed on the formation of golden nanometer particle surface for 5:1 detects above-mentioned influenza virus strain HA eggWhite result is without significant difference.

Sialic acid trisaccharide-long the connection chain that uses embodiment 9 to obtain is fixed on taking mol ratio as 5:1 with long connection chain respectivelySialic acid trisaccharide-golden nanometer particle that golden nanometer particle surface forms is respectively according to the method influenza virus poison of above-mentioned steps 1 and 2Sialic acid trisaccharide-short connection chain that the result of strain HA albumen and strain and use embodiment 2 obtain and short connection chain are with mol ratioSialic acid trisaccharide-the golden nanometer particle that is fixed on the formation of golden nanometer particle surface for 5:1 detects above-mentioned influenza virus strain HA eggWhite result is without significant difference.

Three, sensitivity detects

1, sialic acid trisaccharide-short connection chain is different from the mol ratio of short connection chain

In 36 kinds of sialyloligosaccharide-golden nanometer particles that embodiment 10 prepares, the length of connection chain is to sensitivityImpact is little, and the sensitivity of the mol ratio of sialic acid trisaccharide-length/short connection chain and length/short connection chain during with 5:1 is the highest, and shortChain is synthetic easy, and the mol ratio that therefore uses sialic acid trisaccharide-short connection chain and short connection chain is carried out experiment effect for 5:1Good. α 2, the 3 sialic acid trisaccharides-short connection chain and the short connection chain that obtain with embodiment 1 are below fixed on Jenner with different mol ratio(referred to as 3 ' SLs-AuNP, different mol ratio is respectively 3 ' SLs-to sialic acid trisaccharide-golden nanometer particle that rice corpuscles surface formsAuNP2:1,3 ' SLs-AuNP5:1,3 ' SLs-AuNP10:1,3 ' SLs-AuNP1:0) and α 2,3 sialic acids that obtain of embodiment 8Trisaccharide-long connection chain is fixed on different mol ratio sialic acid trisaccharide-Jenner that golden nanometer particle surface forms with long connection chain(referred to as 3 ' SLl-AuNP, different mol ratio is respectively 3 ' SLl-AuNP2:1,3 ' SLl-AuNP5:1,3 ' SLl-to rice corpusclesAuNP10:1,3 ' SLl-AuNP1:0) describe for example:

In 1.5mL centrifuge tube by 3 ' SLs-AuNP2:1,3 ' SLs-AuNP5:1,3 ' SLs-AuNP10:1,3 ' SLs-AuNP1:0,3 ' SLl-AuNP2:1,3 ' SLl-AuNP5:1,3 ' SLl-AuNP10:1 or 3 ' SLl-AuNP1:0 PBS buffer solution(10mM, pH7.4) is diluted to 7.5nM(is OD_522nmValue be 0.6) obtain dilution, connect in PCR pipes every in 96 orifice plates or 8In hole, add the above-mentioned dilution of 150uL, then add the HA albumen of 2.5uL influenza virus strain qinH5 to make it in reaction systemFinal concentration be 16.7ug/mL, to add PBS buffer solution (10mM, pH7.4) as blank, to add the solidifying of same concentrationsCollect plain RCA₁₂₀Negative contrast, hatches under room temperature, visually observes change color in 40min, result as shown in figure 11, and surveyDetermine the light absorption value under 522nm, calculate percentage light absorption value, result is as shown in table 1.

Percentage light absorption value=(A_d-A_a）/A_d；A_dFor the light absorption value of blank under 522nm; A_aFor reactant liquor is under 522nmLight absorption value.

The impact of the mol ratio of table 1, different sialic acid three sugar chains and length/short connection chain on testing result (percentage light absorption value)

In Figure 11, with respect to blank, color shoals, and degree is descending to be followed successively by: 5:1,10:1,2:1,1:0, the moonProperty contrast is identical with blank color, is redness. "-" represents not carry out.

As can be seen from Table 1, percentage light absorption value is descending is followed successively by: 5:1,10:1,2:1,1:0.

2, the HA protein content difference of virus stain

1) in 1.5mL centrifuge tube, 3 ' SLs-AuNP5:1 is diluted to 2.25nM with PBS buffer solution (10mM, pH7.4)(be OD_522nmValue be 0.18) obtain dilution, connect in PCR pipes and add the above-mentioned dilution of 150uL in every hole in 96 orifice plates or 8Liquid, then add the HA albumen of the influenza virus strain qinH5 of 2.5uL gradient dilution, to add PBS buffer solution (10mM, pH7.4)For blank, under room temperature, hatch, in 40min, visually observe change color (result is as shown in figure 12), and to reactant liquor UV-Vis spectral detection (result as shown in figure 13) is with the result, and detectability is to visually observe change color as boundary.

The macroscopic result of Figure 12 shows: the degree that when the HA protein concentration of qinH5 is less than 2.5nM, color shoals is not clearAobvious; The result of the UV-Vis of Figure 13 shows: when the HA protein concentration of qinH5 is less than 2.5nM, the variation of spectrum is not remarkable yet. I.e. inspectionSurvey is limited to 2.5nM.

2) in 1.5mL centrifuge tube, 6 ' SLs-AuNP5:1 is diluted to 2nM(with PBS buffer solution (10mM, pH7.4)OD_522nmValue be 0.16) obtain dilution, connect in PCR pipes and add the above-mentioned dilution of 150uL in every hole in 96 orifice plates or 8, thenAdd the HA albumen of the influenza virus strain 68H3 of 2.5uL gradient dilution, to add PBS buffer solution (10mM, pH7.4) as blankContrast, hatches under room temperature, visually observes change color (result is as shown in figure 14) in 40min, and to reactant liquor UV-Vis lightSpectrum detects (result as shown in figure 15) with the result, and detectability is to visually observe change color as boundary.

The macroscopic result of Figure 14 shows: the degree that when the HA protein concentration of 68H3 is less than 2.5nM, color shoals is not clearAobvious; The result of the UV-Vis of Figure 15 shows: when the HA protein concentration of 68H3 is less than 2.5nM, spectrum change is not remarkable yet. DetectBe limited to 2.5nM.

Claims (7)

1. compound shown in compound shown in formula X-1 or formula X-2;

Wherein, described x is 1, the integer that described m is 0-6, and the integer that described n is 0-1, the integer that described k is 0-11, described p is0 or 2, described R is H; Described Ac is acetyl group, described R¹For hydroxyl or acetamido, described R²For hydrogen, described R³For hydrogen or sulphurAcid esters, described R⁴For hydroxyl or acetamido.

2. compound according to claim 1, is characterized in that: the integer that described m is 3-6; Described k is the whole of 5-11Number.

3. compound according to claim 2, is characterized in that: described m is 3 or 6, and described n is 1 or 0, described k be 5 or11, described p is 2 or 0.

4. a compound, is characterized in that: described compound be formula X-1-1, formula X-2-1, formula X-1-2, formula X-2-2,Any in formula X-1-3, formula X-2-3 and X-1-4;

5. a method of preparing the arbitrary described compound of claim 1-3, comprises the following steps:

1) prepare the compound shown in formula X-14 by lactose, make alkynyl on lactose reducing end band;

2) under the effect of sialyltransferase, by step 1) compound and the CMP-NeuAc that obtain synthesize formulaCompound shown in compound shown in X-15 or formula X-16;

3) by step 2) the long connection chain shown in compound shown in compound shown in formula X-15 that obtain or formula X-16 and formula VCarry out " click " reaction, then add methyl alcohol and sodium methoxide solid to be hydrolyzed, remove acetyl group, obtain claim 1～3In compound shown in compound shown in arbitrary described formula X-1 or formula X-2.

6. method according to claim 5, is characterized in that: described sialyltransferase is α 2,3-sialyltransferaseTime, what obtain is compound shown in arbitrary described formula X-1 in claim 1～3; Described sialyltransferase is α 2,6-salivaWhen acid transferase, what obtain is compound shown in formula X-2 described in claim 1～3.

7. according to the method described in claim 5 or 6, it is characterized in that: the condition that described " click " connects is: glycosyl propineGlycosides mixes with mole 1:1.5 ratio with connection chain, and the DMF that is 1:1 in volume ratio and the mixing of methyl alcohol are moltenIn liquid, taking cuprous iodide as catalyst, ambient temperature overnight reaction under nitrogen protection.