CN106366163A - Beta-1,2-D-oligomeric mannose peptide-protein conjugate and preparation method and application thereof - Google Patents
Beta-1,2-D-oligomeric mannose peptide-protein conjugate and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a beta-1,2-D-oligomeric mannose peptide-protein conjugate shown in the formula I and a preparation method and application thereof. In the formula, n is 0 or 1 or 2, and R is carrier protein KLH or HSA. Alpha-D-glucose and alanine resin serve as raw materials, beta-1,2-D-oligomeric mannose peptide is synthesized, the beta-1,2-D-oligomeric mannose peptide is coupled with the carrier protein, and the corresponding beta-1,2-D-oligomeric mannose peptide-protein conjugate is obtained. The prepared compound serves as an anti-candida albicans vaccine to immunize mice, the result shows that the compound can induce the organism to generate high immune response, immunogenicity of the beta-1,2-D-oligomeric mannose peptide-protein conjugate is highest, generated immune serum can be bonded with a candida albicans cell surface antigen, therefore, the effect of resisting candida albicans infection can be achieved, and the beta-1,2-D-oligomeric mannose peptide-protein conjugate has high application value.
Description
Technical field
The present invention relates to antifungal glycopeptide vaccines arts technical field, specifically, it is a kind of β -1,2-d- oligomerization manna
Glycopeptide-protein conjugate and preparation method thereof, such glycopeptide-protein conjugate as vaccine infection by Candida albicans prevention and
Application in treatment.
Background technology
At this stage, the fungal infection clinically being caused by Candida albicanss is in continuous ascendant trend, and pathogenic bacteria is directed to antimicrobial drug
Thing has produced serious drug resistance, studies new antifungal funguses vaccine and undoubtedly provides good treatment meanss and plan
Slightly.At present, the research target of such vaccine is concentrated mainly on the aspects such as functional protein and the polysaccharide of Candida albicanss cell surface.Grind
Study carefully discovery, the albumen of the number of different types containing in the cell wall of Candida albicanss and the function such as the intrusion of funguses, adhesion, phagocytosis
Closely related, and these albumen have certain immunogenicity, are used for the design of funguses vaccine as antigen.Phosphorylation
β -1,2- mannan is the t- cell dependent type polysaccharide antigen that there is Candida albicanss cell surface, generally by them with carrier egg
(bsa/tt) is coupled and forms glucoprotein vaccine in vain.But by research we have found that the design of above-mentioned vaccine all exist certain not
Foot: the sugar-protein compound combining to form β -1,2- oligomerization mannose only using cell surface protein or with carrier protein is as epidemic disease
Seedling immune mouse, the immunne response causing is limited.This study we plan synthesize the β -1,2- oligomerization mannose of phosphorylation with
Connect selected from the polypeptide of the n- terminal amino acid sequence of Candida albicanss cell surface protein als1 and form glycopeptide, and by they and carrier
Albumen keyhole worm Qi Xue azurin (klh) is coupled and forms glycopeptide-protein conjugate, is desirably to obtain the glycopeptide-egg of high immunogenicity
White vaccine.
Content of the invention
First purpose of the present invention is for deficiency of the prior art, provides a kind of β -1,2-d- oligomerization mannose
Peptide-protein conjugate.
Second object of the present invention is to provide β -1 as described above, the system of 2-d- oligomerization mannosazone-protein conjugate
Preparation Method.
Third object of the present invention is to provide β -1 as described above, the use of 2-d- oligomerization mannosazone-protein conjugate
On the way.
Fourth object of the present invention is to provide a kind of antifungal funguses vaccine of offer.
For realizing above-mentioned first purpose, the present invention adopts the technical scheme that:
A kind of β -1,2-d- oligomerization mannosazone-protein conjugate, described β -1,2-d- oligomerization mannosazone-Protein Conjugation
Thing general structure is:
Wherein, n is carrier protein klh or hsa for 0,1 or 2, r.
Further, the monosaccharide unit of described composition oligosaccharide is β -1,2-d- mannose.
Further, described peptide sequence is selected from the n- terminal amino acid sequence of Candida albicanss cell surface protein als1.
Further, using phosphoethanolamine base as β -1, the linking group of 2-d- oligomerization mannose and polypeptide.
Further, described β -1,2-d- oligomerization mannosazone-protein conjugate is by β -1 of phosphorylation, 2-d- oligomerization manna
Glycopeptide is obtained with carrier protein couplet.
Further, using glutaryl as β -1, the linking group of 2-d- oligomerization mannosazone and carrier protein.
For realizing above-mentioned second purpose, the present invention adopts the technical scheme that:
As above arbitrary described β -1, the preparation method of 2-d- oligomerization mannosazone-protein conjugate, comprise the steps:
The first step, synthesis β -1,2-d- mannobiose (receptor 7), β -1,2-d- mannotriose receptor (acceptor 10):
Second step, synthesizes phosphorylation β -1,2-d- oligomerization mannose 19a-c:
3rd step, synthesis polypeptide 21:
4th step, synthesizes β -1,2-d- oligomerization mannosazone-protein conjugate:
Further, described phosphorylation β -1,2-d- oligomerization mannose preparation method method is as follows:
(1) with alpha-d-glucose as raw material, preparation β -1 in the presence of reaction promoter, 2-d- manna monosaccharide receptor and 3,
4,6- tribenzyl -2- acetoxyl group glucose tri- chloroacetimidate donor；
(2) there is glycosylation system in the monosaccharide donor of above-mentioned preparation and monosaccharide receptor under catalyst (tmsotf) effect
Standby disaccharide, prepares β -1,2-d- mannobiose receptor by configuration reversal on 2 '-position of disaccharide；β -1,2-d- mannobiose is subject to
Body and monosaccharide donor prepare β -1,2-d- mannotriose receptor by preceding method；
(3) β -1,2-d- mannose receptor respectively with 6- tert-butyl diphenyl silicon substrate -3,4- dibenzyl -2- acetoxyl group Portugal
Grape sugar tri- chloroacetimidate donor prepares corresponding β -1,2-d- oligomerization mannose through glycosylation；Oligomerization mannose is non-reduced
React and phosphorylation β -1,2-d- oligomerization mannose is obtained with phosphorylation agent after tbdps protection is taken off in end 6- position.
Further, the preparation method of described polypeptide 21 is: with fmoc-l- alanine resin as raw material, using solid phase synthesis
Method synthesis polypeptide 21.
Further, described β -1,2-d- oligomerization mannosazone-protein conjugate preparation method comprises the steps:
(1) phosphorylation β -1,2-d- oligomerization mannose is obtained β -1,2-d- oligomerization mannose with polypeptide 21 through condensation reaction
Peptide；
(2) β -1,2-d- oligomerization mannosazone prepared by step 1 and hydroxyl Succinimidyl glutarate react be obtained β -
1,2-d- oligomerization mannosazone Acibenzolar；
(3) Acibenzolar prepared by step 2 under weak basic condition with carrier protein couplet, obtain β -1,2-d- oligomerization manna
Glycopeptide-protein conjugate.
For realizing above-mentioned 3rd purpose, the present invention adopts the technical scheme that:
As above arbitrary described β -1,2-d- oligomerization mannosazone-protein conjugate is in preparation prevention and/or treatment funguses
Application in the medicine of infection；Described funguses are Candida albicanss.
For realizing above-mentioned 4th purpose, the present invention adopts the technical scheme that:
A kind of antifungal funguses vaccine, including β -1 of therapeutically effective amount as described above, 2-d- oligomerization mannosazone -
Protein conjugate and pharmaceutically acceptable adjuvant or adjuvant.
The invention has the advantages that:
Klh- glycopeptide-the protein conjugate of present invention preparation, as antifungal vaccine subcutaneous inoculation mice, can induce body
Produce stronger immunne response, wherein β -1, the immunogenicity of 2-d- mannotriose peptide-protein conjugate is the strongest and exempting from of producing
Epidemic disease serum can be combined with Candida albicanss cell surface antigen, such that it is able to reach the effect of antifungal infection, this
Bright β -1,2-d- mannotriose peptide-protein conjugate has stronger using value.
Brief description
Accompanying drawing 1 is β -1,2-d- oligomerization mannosylglycoprotein conjugate formula.
Accompanying drawing 2 is β -1,2-d- mannobiose, β -1,2-d- mannotriose receptor synthetic route.Reaction condition: a)
Tmsotf, ch2cl2,Molecular sieve, -40 DEG C, 92.4%；b)ch3Ona, ch3Oh, 94.2%；C) i) dmso, ac2o；ii)l-
Selectride, thf, -78 DEG C, 79.5% (two step total recoverys)；D) tmsotf, ch2cl2,Molecular sieve, -40 DEG C,
90.3%；e)ch3Ona, ch3Oh, 92.7%；F) i) dmso, ac2o；Ii) l-selectride, thf, -78 DEG C, 77.6% (two
Step total recovery).
Accompanying drawing 3 is phosphorylation β -1,2-d- oligomerization mannose disaccharide, trisaccharide synthetic route.Reaction condition: a) tmsotf,
ch2cl2,Molecular sieve, -40 DEG C, (12a, 84.3%；12b, 89.5%；12c, 81.5%)；b)ch3Ona, ch3Oh, 91.3-
95.5%；C) i) dmso, ac2o；Ii) l-selectride, thf, -78 DEG C, 79.6-84.5% (two step total recoverys)；d)
Bnbr, nah, tbai, 0 DEG C, 83.7-89.4%；E) tbaf, thf, 83.4-88.2%；f)i)tetrazole,ch2cl2/
ch3cn；Ii) tert-buooh, 79.3-87.5% (two step total recoverys)；G) dbu, ch2cl2, 86.3-93.2%.
Accompanying drawing 4 is the solid phase synthesis process of polypeptide 21.Reaction condition: a) hbtu, hobt, dipea, dmf；b)
Piperidine/dmf=(1:4, volume ratio)；C) acoh/tfe/dcm (2:1:16, volume ratio).
Accompanying drawing 5 is the synthetic route of glycopeptide vaccine target compound.Reaction condition: a) edc, hobt, ch2cl2/nmp(2:
1, volume ratio), 0 DEG C, 78.9-83.2%；b)i)h2, pd (oh)2/ c, ch2cl2/ meoh (1:2, volume ratio), 24 hours；ii)
Tfa/tes/dcm (3:1:6, volume ratio), 2 hours, 78.4-82.7% (two step total recoverys)；C) dmf/pbs buffer (4:1,
Volume ratio), 4 hours；D) pbs buffer, 2.5 days.
Accompanying drawing 6 is total antibody titer in detection serum after compound 1a-c immune mouse, and what wherein+adj represented is to add
Adjuvant group.
Accompanying drawing 7 is the immunofluorescence that gained serum is combined with Candida albicanss cell after compound 1b third time immune mouse
Figure.
Accompanying drawing 8 is the fluidic cell that gained serum is combined with Candida albicanss cell after compound 1b third time immune mouse
Figure.
Specific embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate this
Bright rather than limit the scope of the present invention.In addition, it is to be understood that after having read the content of present invention record, art technology
Personnel can make various changes or modifications to the present invention, and these equivalent form of values equally fall within the application appended claims and limited
Fixed scope.
The preparation reaction road of β -1,2-d- oligomerization mannosazone-protein conjugate (general structure is as shown in Figure 1) of the present invention
Line (Fig. 2, Fig. 3, Fig. 4, Fig. 5):
The first step, synthesis β -1,2-d- mannobiose (receptor 7), β -1,2-d- mannotriose receptor (acceptor 10):
Second step, synthesizes phosphorylation β -1,2-d- oligomerization mannose 19a-c:
3rd step, synthesis polypeptide 21:
4th step, synthesizes β -1,2-d- oligomerization mannosazone-protein conjugate:
The synthesis of embodiment 1 compound 5
By monosaccharide receptor 3 (1.23g, 2.5mmol) (j.org.chem.2001,66,8411.), donor 4 (1.95g,
3.0mmol) (j.carbohydr.chem.1994,13,421.) and activationMolecular sieve (1.0g) is placed in 100ml round bottom
Flask is simultaneously vacuum dried half an hour, then dissolves mixture 50ml anhydrous methylene chloride.Suspension argon protection under in
Under room temperature stirring half an hour after be cooled to -40 DEG C, be then slowly added dropwise trimethylsilyl trifluoromethanesulfonate (35.0 μ l,
0.18mmol).After half an hour, the neutralization of reactant liquor triethylamine is quenched and is filtered with kieselguhr, and after concentration, gained head product is through silica gel
Column chromatography (n-hexane/ethyl acetate, 12:1, v/v) obtains white foam solid (2.23g, 92.4%).
1hnmr(400mhz,cdcl3)δ7.40-7.19(m,30h,ar),5.88(m,1h,och2ch=ch2),5.37(m,
1h,och2Ch=ch 2),5.24-5.20(m,1h,och2Ch=ch 2), 5.15 (dd, j=7.6hz, 9.6hz, 1h, h-2 '),
4.86-4.75(m,6h,h-1’,ch2ph),4.56-4.43(m,7h,ch2ph),4.42-4.39(m,1h,och 2Ch=ch2),
4.34 (s, 1h, h-1), 4.28 (d, j=3.2hz, 1h, h-2), 4.22-4.01 (m, 1h, och 2Ch=ch2),3.82-3.74
(m, 3h, h-3, h-6, h-6 '), 3.73-3.57 (m, 5h, h-4, h-4 ', h-5 ', h-6 '), 3.52 (d, j=3.2hz, h-3),
3.44(m,1h),1.97(s,3h,ac).esi-ms:calcd.for c59h64o12[m+na]+m/z,987.4；found,
987.9.
The synthesis of embodiment 2 compound 6
Compound 5 (1.85g, 2.0mmol) is used 100ml absolute methanol to dissolve, addition Feldalat NM (11.0mg,
0.2mmol) juxtaposition is stirred overnight at room temperature, adds ion exchange resin ir 120 (h+form) neutralization, mistake after completion of the reaction
Filter resin, after concentration, gained head product obtains white foam through silica gel column chromatography (n-hexane/ethyl acetate, 4:1, v/v)
Solid (1.74g, 94.2%).
1h nmr(400mhz,cdcl3)δ7.55-7.23(m,30h,ar),6.05-5.93(m,1h,och2ch=ch2),
5.40(m,1h,och2Ch=ch 2),5.29(m,1h,och2Ch=ch 2), 5.17 (dd, j=11.2hz, 1h, och2ph),
5.05-4.93(m,4h,och2), ph 4.90 (d, j=11.2hz, 1h, och2Ph), 4.81 (d, j=7.6hz, 1h, h-1 '),
4.72 (d, j=12.0hz, 1h, och2ph),4.67-4.46(m,10h,och2ph,och 2Ch=ch2, h-1), 4.36 (d, j=
3.2hz,1h,h-2),4.17-4.10(m,1h,och 2Ch=ch2), 4.01 (t, j=9.6hz, 1h, h-4), 3.88-3.71 (m,
6h,h-2’,h-3’,h-6a’,h-6b’,h-6a,h-6b),3.70-3.57(m,3h,h-3,h-4’,h-5’),3.53-3.42
(m,1h,h-5).13c nmr(100mhz,cdcl3)δ139.13,138.52,138.33,138.27,138.16,138.11,
133.52,128.78,128.56,128.45,128.41,128.36,128.23,128.17,128.12,128.03,127.98,
127.93,127.87,127.80,127.72,127.70,127.66,127.54,117.86,104.11,99.39,85.17,
80.33,77.30,75.70,75.46,75.34,75.11,74.78,74.63,74.37,73.48,70.39,69.97,
69.81,69.23.esi-ms:calcd.for c57h62o11[m+k]+m/z,962.2；found,930.5.
The synthesis of embodiment 3 compound 7
Compound 6 (1.38g, 1.5mmol) is placed in 100ml round-bottomed flask, is subsequently adding dimethyl sulfoxide (22.0ml)
With acetic anhydride (11.0ml).Reactant liquor is extracted with ethyl acetate after being stirred at room temperature 18 hours, respectively use saturated sodium carbonate and
Brine It, then uses anhydrous sodium sulfate drying, and the head product being concentrated to give after filtration toluene band revolves twice.Residue is used
40ml anhydrous tetrahydro furan dissolves and is cooled to -78 DEG C, is slowly added dropwise l-selectride (1 under argon protectionmthf,
7.5ml) and stir 15 minutes, then remove chiller juxtaposition and continue at room temperature to stir 15 minutes, methanol is quenched instead
Should after add 50ml dchloromethane, solution respectively use hydrogenperoxide steam generator (5%, 30ml), sodium hydroxide solution (1m,
30ml), hypo solution (5%, 30ml) and saturated aqueous common salt (30ml) washing, through anhydrous magnesium sulfate be dried, filter and
Pale yellow oily liquid after concentration, gained head product obtains white through silica gel column chromatography (n-hexane/ethyl acetate, 4:1, v/v)
Foaming solid (1.10g, 79.5%).
1h nmr(400mhz,cdcl3)δ7.55-7.23(m,30h,ar),5.99-5.86(m,1h,och2ch=ch2),
5.27(m,1h,och2Ch=ch 2),5.21(m,1h,och2Ch=ch 2), 5.03 (s, 1h, h-1 '), 5.02 (dd, j=
10.8hz,1h,och2), ph 4.99 (dd, j=11.2hz, 1h, och2), ph 4.94 (dd, j=10.8hz, 1h, och2ph),
4.89 (dd, j=10.8hz, 1h, och2), ph 4.69 (dd, j=10.8hz, 1h, och2Ph), 4.68 (dd, j=11.6hz,
1h,och2ph),4.64-4.59(m,2h,och2Ph), 4.57 (d, j=3.2hz, 1h, h-2), 4.56-4.48 (m, 4h,
och2ph),4.47(s,1h,h-1),4.46-4.43(m,1h,och 2Ch=ch2), 4.41 (d, j=3.2hz, 1h, h-2 '),
4.11-4.06(m,1h,och 2Ch=ch2), 3.98 (t, j=9.2hz, 1h, h-4 '), 3.91-3.70 (m, 5h, h-6a, h-4,
h-6a’,h-6b,h-6b’),3.67-3.54(m,3h,h-3’,h-3,h-5’),3.51-3.44(m,1h,h-5).13c nmr
(100mhz,cdcl3)δ138.44,138.31,138.18,133.83,128.38,128.27,128.16,128.01,
127.95,127.78,127.72,127.65,117.18,100.15,99.35,81.42,80.34,75.6,75.14,74.44,
74.07,73.50,73.39,70.85,70.75,70.07,69.40,67.70.esi-ms:calcd.for c57h62o11[m+
li]+m/z,930.1；found,930.5.
The synthesis of embodiment 4 compound 8
By two saccharide acceptors 7 (0.95g, 1.0mmol), monosaccharide donor 4 (0.78g, 1.2mmol) and activationMolecule
Sieve (0.25g) is placed in 50ml round-bottomed flask and is vacuum dried half an hour, then dissolves mixture 30ml anhydrous methylene chloride.
Suspension is cooled to -40 DEG C after stirring half an hour at room temperature under argon protection, is then slowly added dropwise trimethyl silicon substrate trifluoro
Methanesulfonates (14.0 μ l, 0.075mmol).After half an hour, the neutralization of reactant liquor triethylamine is quenched and is filtered with kieselguhr, concentrates
Afterwards gained head product through silica gel column chromatography (n-hexane/ethyl acetate, 12:1, v/v) white foam solid (1.26g,
90.3%).
1h nmr(600mhz,cdcl3)δ7.42-7.02(m,45h,ar),5.86(m,1h,och2ch=ch2),5.26
(d, j=8.0hz, 1h, h-1 "), and 5.20-5.17 (m, 2h, h-2 ", och2Ch=ch 2),5.11-5.07(m,1h,och2Ch=
ch 2),4.98-4.94(m,3h,och2ph),4.85-4.80(m,2h,och2ph),4.75-4.69(m,4h,h-1’,
och2), ph 4.63 (d, j=13.2hz, 1h, och2ph),4.56-4.47(m,9h,h-2’,och2), ph 4.42 (d, j=
12.0hz,1h,och2ph),4.41(s,1h,h-1),4.38-4.35(m,1h,och2Ch=ch2), 4.20 (d, j=3.0hz,
1h, h-2), 3.91 (t, j=8.4hz, 1h, h-3 "), and 3.82-3.66 (m, 8h, h-4 ", h-5 ', h-5 ", h-6a " and, h-6a, h-
6b, h-6a ', h-6b '), 3.62 (t, j=9.6hz, 1h, h-4 '), 3.54-3.46 (m, 3h, h-3, h-3 ', h-6a "), 3.37
(m,1h,h-5).maldi-tof-ms:calcd.for c86h92o17[m+na]+m/z,1419.6,found 1420.0.
The synthesis of embodiment 5 compound 9
Compound 8 (1.02g, 0.75mmol) is used 50ml absolute methanol to dissolve, addition Feldalat NM (11.0mg,
0.2mmol) juxtaposition is stirred overnight at room temperature, adds ion exchange resin ir 120 (h+form) neutralization, mistake after completion of the reaction
Filter resin, after concentration, gained head product obtains white foam through silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v/v)
Solid (0.94g, 92.7%).
1h nmr(400mhz,cdcl3)δ7.56-7.08(m,45h,ar),6.01-5.89(m,1h,och2ch=ch2),
5.29(m,1h,och2Ch=ch 2),5.24(m,1h,och2Ch=ch 2),5.16-5.02(m,6h,och2ph,h-1’),4.96
(d, j=10.8hz, 1h, och2Ph), 4.88 (d, j=8.0hz, 1h, h-1 "), 4.81-4.66 (m, 3h, och2ph),4.65-
4.41(m,13h,och2ph,h-2’,h-2,och 2Ch=ch2,h-1),4.15-4.07(m,1h,och 2Ch=ch2),4.01-
3.71(m,9h,h-2”,h-3”,h-4’,h-6b”,h-6a”,h-6a,h-6b,h-6a’,h-6b’),3.68-3.56(m,5h,
1h,h-5”,h-3’,h-4”,h-3,h-5),3.52-3.44(m,1h,h-5’).13c nmr(100mhz,cdcl3)δ139.21,
138.68,138.59,138.44,138.39,138.16,138.11,133.79,128.50,128.41,128.36,128.31,
128.25,128.20,128.11,128.06,127.92,127.86,127.83,127.73,127.69,127.64,127.59,
127.48,127.43,127.27,117.43,105.35,100.08,99.93,86.73,80.25,80.03,77.18,
75.52,75.44,75.31,75.25,74.99,74.83,74.72,74.62,74.09,73.66,73.46,73.33,
71.16,70.31,70.19,69.77,69.45.maldi-tof-ms:calcd.for c84h90o16[m+na]+m/z,
1378.617；found,1378.115.
The synthesis of embodiment 6 compound 10
Compound 9 (678.0mg, 0.5mmol) is placed in 50ml round-bottomed flask, is subsequently adding dimethyl sulfoxide (7.0ml)
With acetic anhydride (3.5ml).Reactant liquor is extracted with ethyl acetate after being stirred at room temperature 18 hours, uses saturated sodium carbonate and food respectively
Salt water washing, then uses anhydrous sodium sulfate drying, and the head product being concentrated to give after filtration toluene band revolves twice.Residue is used
20ml anhydrous tetrahydro furan dissolves and is cooled to -78 DEG C, is slowly added dropwise l-selectride (1 under argon protectionmthf,
2.45ml) and stir 15 minutes, then remove chiller juxtaposition and continue at room temperature to stir 15 minutes, methanol is quenched instead
Should after add 20ml dchloromethane, solution respectively use hydrogenperoxide steam generator (5%, 20ml), sodium hydroxide solution (1m,
20ml), hypo solution (5%, 20ml) and saturated aqueous common salt (20ml) washing, through anhydrous magnesium sulfate be dried, filter and
Pale yellow oily liquid after concentration, gained head product obtains white through silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v/v)
Foaming solid (526.3mg, 77.6%).
1h nmr(400mhz,cdcl3)δ7.66-6.94(m,45h,ar),5.95-5.84(m,1h,och2ch=ch2),
5.25(m,1h,och2Ch=ch 2),5.20(s,1h,h-1”),5.19(m,1h,och2Ch=ch 2),5.17(s,1h,h-1’),
5.06-4.96(m,4h,och2), ph 4.90 (d, j=10.8hz, 1h, och2ph),4.75(br s,1h,h-2’),4.71-
4.64(m,2h,och2ph,h-2),4.61-4.42(m,10h,och2ph,och 2Ch=ch2, h-1), 4.38 (d, j=
10.8hz,1h,och2ph),4.35(br s,1h,h-2”),4.28-4.21(m,1h,och2ph),4.10-3.89(m,4h,h-
4”,h-4’,och2ph),3.88-3.49(m,13h,h-6a”,h-6b”,h-5”,h-3”,h-6a’,h-6b’,h-5’,h-3’,
h-6a,h-6b,h-5,h-4,h-3),3.47-3.41(m,1h,h-5).13c nmr(100mhz,cdcl3)δ138.56,
138.48,138.26,138.20,138.15,138.10,138.02,137.88,133.62,129.16,128.44,128.38,
128.35,128.29,128.25,128.19,128.11,128.05,127.94,127.79,127.74,127.69,127.65,
127.60,127.51,127.45,127.34,127.18,117.42,100.69,100.06,83.12,80.48,80.35,
75.42,75.35,75.22,75.17,75.07,74.78,74.43,74.34,73.52,73.44,73.36,71.38,
70.34,70.09,70.03,69.95,69.80,69.67,69.49,69.42,68.93,67.29.maldi-tof-ms:
calcd.for c84h90o16[m+na]+m/z,1378.617；found,1379.306.
The synthesis of embodiment 7 compound 12a
By monosaccharide receptor 3 (245mg, 0.5mmol), donor 11 (471.0mg, 0.6mmol)
(eur.j.org.chem.1999,2523.) and activationMolecular sieve (200mg) is placed in 25ml round-bottomed flask and vacuum is done
Dry half an hour, then mixture 10ml anhydrous methylene chloride is dissolved.Suspension stirs half under argon protection at room temperature
It is cooled to -40 DEG C after hour, be then slowly added dropwise trimethylsilyl trifluoromethanesulfonate (6.35 μ l, 0.035mmol).Half an hour
The neutralization of reactant liquor triethylamine is quenched and is filtered with kieselguhr afterwards, and after concentration, gained head product is through silica gel column chromatography (normal hexane/second
Acetoacetic ester, 18:1, v/v) obtain white foam solid (478.5mg, 84.3%).
1h nmr(600mhz,cdcl3)δ7.71-7.01(m,35h,ar),5.99-5.91(m,1h,och2ch=ch2),
5.36(m,1h,och2Ch=ch 2),5.21(m,1h,och2Ch=ch 2), 5.16 (dd, j=9.0,8.0hz, 1h, h-2 '),
5.02 (d, j=8.0hz, 1h, h-1 '), 4.97-4.91 (m, 2h, och2ph),4.81-4.73(m,3h,och2ph),4.59(br
s,2h),4.49-4.41(m,5h,och2ph,och 2Ch=ch2, h-1), 4.38 (d, j=3.6hz, 1h, h-2), 4.08-4.01
(m,2h,och 2Ch=ch2, h-6a '), 3.65-3.60 (m, 1h, h-6b '), 3.79 (br d, j=9.6hz, 1h, h-6a),
3.75 (t, j=9.0hz, 1h, h-3 '), and 3.64-3.59 (m, 2h, h-6b, h-4), 3.56-3.49 (m, 4h, h-3, h-4 ', h-
5’),3.48-3.43(m,1h,h-5),1.95(s,3h),1.04(s,9h).13c nmr(150mhz,cdcl3)δ169.9,
138.5,138.4,137.9,137.7,135.7,135.6,133.9,133.4,133.3,129.7,129.6,128.3,
128.28,128.25,128.2,128.0,127.9,127.8,127.7,127.69,127.65,127.6,127.5,127.4,
116.5,100.9,99.8,83.2,79.8,77.9,76.6,75.5,75.2,74.8,74.7,74.6,73.5,73.2,71.7,
70.4,69.41,69.37,64.0,26.9,21.4,19.3.hrms(esi):calcd.for c68h76o12si[m+na]+m/z,
1135.5004；found,1135.4979.
The synthesis of embodiment 8 compound 12b
Prepared by compound 7 and compound 11, concrete operation step is with the synthesis of compound 12a, yield 89.5%.
1h nmr(600mhz,cdcl3)δ7.64-6.87(m,50h,ar),5.87-5.78(m,1h,och2ch=ch2),
5.36 (d, j=8.4hz, 1h, h-1 "), 5.21 (dd, j=9.0,8.4hz, 1h, h-2 "), 5.20 (br d, j=10.8hz,
1h,och2Ch=ch 2), 5.09 (br d, j=10.2hz, 1h, och2Ch=ch 2),4.98-4.89(m,3h,och2ph),4.85
(d, j=12.0hz, 1h, och2ph),4.77-4.72(m,2h,och2Ph, h-1 '), 4.68 (q, j=11.4hz, 2h,
och2), ph 4.63 (d, j=12.8hz, 1h, och2ph),4.59-4.44(m,8h,och2ph,h-2’),4.43-4.36(m,
3h,och2ph,och 2Ch=ch2, h-1), 4.23 (d, j=2.7hz, 1h, h-2), 4.02-3.95 (m, 3h, och 2Ch=ch2,
H-6a ', h-4), 3.91 (t, j=9.0hz, 1h, h-3 "), and 3.88-3.84 (m, 1h, h-6b "), 3.79-3.58 (m, 7h, h-
6a’,h-6b’,h-6a,h-6b,h-5’,h-4”,h-4’),3.54-3.45(m,3h,h-3’,h-3,h-5”),3.37-3.33
(m,1h,h-5),1.96(s,3h),1.02(s,9h).13c nmr(150mhz,cdcl3)δ170.6,138.6,138.5,
138.3,138.25,138.22,138.2,138.1,138.0,135.7,135.5,133.9,133.5,133.1,116.8,
102.2,100.8,100.1,83.9,80.4,79.7,77.9,76.6,75.4,75.2,74.9,74.7,74.6,74.4,
73.5,73.2,69.7,69.6,69.2,68.9,63.9,26.9,21.2,19.2.hrms(esi):calcd.for
c95h104o17si[m+na]+m/z,1567.6942；found,1567.6924.
The synthesis of embodiment 9 compound 12c
Prepared by compound 10 and compound 11, concrete operation step is with the synthesis of compound 12a, yield 81.5%.
1h nmr(600mhz,cdcl3)δ7.46-6.74(m,65h,ar),5.79-5.73(m,1h,och2ch=ch2),
5.56 (d, j=7.8hz, 1h, h-1 " '), 5.23 (t, j=9.0,7.8hz, 1h, h-2 " '), 5.16 (s, 1h, h-1 "), 5.16-
5.09(m,3h,och2Ch=ch 2,h-1”,h-1’),5.06(m,1h,och2Ch=ch 2), 5.02 (d, j=11.4hz, 2h,
och2), ph 4.95 (d, j=12.0hz, 1h, och2), ph 4.91 (d, j=10.8hz, 1h, och2), ph 4.85 (d, j=
10.8hz,1h,och2Ph), 4.83 (d, j=3.0hz, 1h, h-2 "), 4.80-4.73 (m, 3h, och2), ph 4.66 (q, j=
11.4hz,2h,och2ph),4.57-4.60(m,3h,och2ph,h-2’),4.52-4.36(m,10h,och2ph,h-2),
4.34-4.30(m,1h,och 2Ch=ch2),4.27-4.24(m,2h,och2ph,h-1),4.01-3.97(m,2h,h-4’,h-
4”’),3.90-3.85(m,2h,och 2Ch=ch2,h-3”’),3.82-3.60(m,14h),3.56-3.54(m,1h,h-3’),
3.44-3.38(m,2h,h-3,h-5’),3.31-3.28(m,1h,h-5),1.94(s,3h),0.94(s,9h).13c nmr
(150mhz,cdcl3)δ170.6,138.67,138.6,138.5,138.4,138.3,138.2,137.9,137.8,137.3,
135.64,135.62,133.5,133.1,132.8,129.4,129.3,128.4,128.3,128.3,128.22,128.20,
128.17,128.11,128.1,127.9,127.88,127.86,127.8,127.7,127.6,127.5,127.49,
127.44,127.40,127.36,127.33,127.2,126.9,117.2,101.8,100.7,100.5,100.4,84.0,
81.90,80.6,77.4,75.8,75.5,75.3,75.2,75.1,75.00,74.9,74.8,74.7,74.5,74.4,
73.65,73.4,73.3,73.1,72.7,71.2,70.6,70.2,69.6,69.4,69.3,69.0,68.1,62.8,26.9,
21.2,19.2.hrms(esi):calcd.for c122h132o22si[m+na]+m/z,1999.8877；found,1999.8931.
The synthesis of embodiment 10 compound 13a
Compound 12a (470mg, 4.22mmol) is used 15ml absolute methanol to dissolve, addition Feldalat NM (4.5mg,
0.84mmol) juxtaposition is stirred overnight at room temperature, adds ion exchange resin ir 120 (h+form) neutralization, mistake after completion of the reaction
Filter resin, after concentration, gained head product obtains white foam solid through silica gel column chromatography (n-hexane/acetone, 14:1, v/v)
(417.5g, 92.3%).
1h nmr(600mhz,cdcl3)δ7.77-7.06(m,35h,ar),6.02-5.95(m,1h,och2ch=ch2),
5.39(m,1h,och2Ch=ch 2),5.28(m,1h,och2Ch=ch 2), 5.13 (dd, j=11.4hz, 1h, och2ph),4.99
(d, j=10.8hz, 1h, och2), ph 4.95 (d, j=12.0hz, 1h, och2Ph), 4.90 (d, j=12.0hz, 1h,
och2Ph), 4.88 (d, j=7.6hz, 1h, h-1 '), 4.85 (d, j=11.4hz, 1h, och2Ph), 4.71 (d, j=12.0hz,
1h,och2ph),4.62-4.50(m,6h,och2ph,och 2Ch=ch2, h-1), 4.43 (d, j=3.6hz, 1h, h-2),
4.15-4.10(m,1h,och 2Ch=ch2), 4.06 (d, j=10.8hz, 1h, h-6a '), 4.02 (t, j=9.6hz, 1h, h-
4),3.96-3.92(m,1h,h-6b’),3.86-3.80(m,3h,h-6a,h-6b,h-2’),3.76-3.71(m,1h,h-3’),
3.65-3.52(m,3h,h-3,h-4’,h-5’),3.50-3.46(m,1h,h-5),1.09(s,9h).13c nmr(150mhz,
cdcl3)δ139.2,138.5,138.25,138.21,137.8,135.7,135.6,133.57,133.51,133.5,
129.64,129.60,128.4,128.34,128.29,128.1,128.0,127.99,127.8,127.73,127.70,
127.6,127.57,127.5,117.8,103.7,99.6,85.2,80.3,76.9,75.7,75.2,74.9,74.8,74.3,
73.9,73.5,70.1,70.0,69.1,63.9,26.9,19.3.hrms(esi):calcd.for c66h74o11si[m+na]+
m/z,1093.4898；found,1093.4884.
The synthesis of embodiment 11 compound 13b
By compound 12b and Feldalat NM preparation, concrete operation step is with the synthesis of compound 13a, yield 95.5%.
1h nmr(600mhz,cdcl3)δ7.71-6.93(m,50h,ar),5.92-5.86(m,1h,och2ch=ch2),
5.22(m,1h,och2Ch=ch 2),5.16(m,1h,och2Ch=ch 2), 5.04 (d, j=11.4hz, 1h, och2ph),4.95
(d, j=10.8hz, 1h, och2ph),4.98-4.91(m,4h,och2Ph, h-1 '), 4.84 (d, j=7.8hz, 1h, h-1 "),
4.78 (d, j=10.8hz, 1h, och2), ph 4.63 (d, j=12.6hz, 1h, och2Ph), 4.57 (d, j=11.4hz, 2h,
och2Ph), 4.54 (d, j=3.0hz, 1h, h-2 '), 4.51 (d, j=3.0hz, 1h, h-2), 4.49-4.35 (m, 9h,
och2ph,och 2Ch=ch2,h-1),4.12-4.02(m,3h,och 2Ch=ch2,h-6a”,h-4),3.93-3.87(m,1h,h-
6b”),3.83-3.72(m,5h,h-2”,h-4’,h-6a,h-6b,h-6a’),3.71-3.64(m,2h,h-6a’,h-3”),
3.62-3.58(m,1h,h-5’),3.57-3.39(m,5h,h-3’,h-3,h-5’,h-4”,h-5),0.99(s,9h).13c nmr
(150mhz,cdcl3)δ139.1,138.57,138.54,138.1,137.9,135.6,133.7,133.5,133.4,129.6,
128.8,128.4,128.3,128.29,128.23,128.22,128.17,128.13,128.01,128.0,127.77,
127.72,127.7,127.56,127.52,127.4,127.3,127.2,117.3,105.1,100.03,100.01,86.6,
80.2,79.5,75.5,75.4,75.2,74.9,74.8,74.6,74.5,74.1,73.5,73.4,71.0,70.2,70.1,
69.6,69.5,64.5,27.0,19.2.hrms(esi):calcd.for c93h102o16si[m+na]+m/z,1525.6835；
found,1525.6777.
The synthesis of embodiment 12 compound 13c
By compound 12c and Feldalat NM preparation, concrete operation step is with the synthesis of compound 13a, yield 91.3%.
1h nmr(600mhz,cdcl3)δ7.64-6.86(m,65h,ar),5.83-5.76(m,1h,och2ch=ch2),
5.31(s,1h,h-1”),5.15(m,1h,och2Ch=ch 2),5.09(m,1h,och2Ch=ch 2), 5.06 (d, j=
11.4hz,1h,och2), ph 5.01 (d, j=10.8hz, 1h, och2Ph), 4.98 (s, 1h, h-1 '), 4.97 (d, j=
10.8hz,1h,och2), ph 4.90 (d, j=11.4hz, 1h, och2Ph), 4.85 (d, j=3.0hz, 1h, h-2 '), 4.84-
4.78(m,3h,och2Ph), 4.72 (d, j=7.8hz, 1h, h-1 " '), 4.62 (d, j=12.0hz, 1h, och2ph),4.59
(d, j=11.4hz, 2h, och2ph),4.52-4.39(m,10h,och2ph,h-2’),4.37-4.32(m,3h,och2ph,
och 2Ch=ch2, h-1), 4.22 (d, j=12.0hz, 1h, och2), ph 4.18 (d, j=10.8hz, 1h, och2ph),4.02
(t, j=9.6hz, 1h, h-4 "), 3.95-3.88 (m, 4h, och 2Ch=ch2,h-4’,h-6a”’,h-6b”’),3.85-3.80
(m,2h,h-6a”,h-6b”),3.75-3.55(m,10h),3.54-3.46(m,4h,h-3,h-2,h-5”’,h-5”),3.33-
3.30(m,1h,h-5),0.96(s,9h).13c nmr(150mhz,cdcl3)δ139.1,138.8,138.6,138.5,
138.34,138.30,138.2,138.1,138.0,135.7,135.6,133.7,133.5,133.3,129.5,128.4,
128.4,128.3,128.26,128.24,128.2,128.14,128.09,128.0,127.97,127.93,127.8,
127.77,127.73,127.6,127.5,127.49,127.4,127.36,127.3,127.2,127.0,117.2,105.7,
101.4,100.3,99.7,86.2,81.0,80.3,79.8,76.6,75.8,75.7,75.6,75.5,75.4,75.34,
75.32,75.2,74.8,74.7,74.5,73.4,72.2,70.2,70.1,70.0,69.99,69.91,69.7,69.4,
69.2,63.9,26.9,19.3.hrms(esi):calcd.for c120h130o21si[m+na]+m/z,1957.8772；found,
1957.8856.
The synthesis of embodiment 13 compound 14a
Compound 13a (380.0mg, 0.36mmol) is placed in 25ml round-bottomed flask, is subsequently adding dimethyl sulfoxide
(5.0ml) with acetic anhydride (2.5ml).Reactant liquor is extracted with ethyl acetate after being stirred at room temperature 18 hours, uses saturated carbon respectively
Sour sodium and brine It, then use anhydrous sodium sulfate drying, and the head product being concentrated to give after filtration toluene band revolves twice.Residual
Stay thing 20ml anhydrous tetrahydro furan to dissolve and be cooled to -78 DEG C, under argon protection, be slowly added dropwise l-selectride (1m
Thf, 1.75ml) and stir 15 minutes, then remove chiller juxtaposition and continue at room temperature to stir 15 minutes, methanol is quenched
Go out and after reacting, add 20ml dchloromethane, solution uses hydrogenperoxide steam generator (5%, 20ml), sodium hydroxide solution respectively
(1m, 20ml), hypo solution (5%, 20ml) and saturated aqueous common salt (20ml) washing, through anhydrous magnesium sulfate be dried, mistake
Pale yellow oily liquid is obtained, head product obtains white through silica gel column chromatography (n-hexane/ethyl acetate, 6:1, v/v) after filter and concentration
Foaming solid (323.5mg, 84.5%).
1h nmr(600mhz,cdcl3)δ7.69-7.07(m,35h,ar),5.92-5.84(m,1h,och2ch=ch2),
5.26(m,1h,och2Ch=ch 2),5.16(m,1h,och2Ch=ch 2),5.12(br s,1h,h-1’),4.96-4.90(m,
2h,och2ph),4.88-4.82(m,2h,och2Ph), 4.69 (d, j=3.6hz, 1h, h-2), 4.67-4.62 (m, 2h,
och2Ph) 4.57 (d, j=12.0hz, 1h, och2ph),4.51-4.37(m,5h,och2ph,och 2Ch=ch2,h-1),4.36
(d, j=3.0hz, 1h, h-2 '), 4.09-4.03 (m, 2h, och 2Ch=ch2,h-6a’),3.91-3.79(m,4h,h-6b’,h-
4,h-4’,h-6a),3.76-3.72(m,1h,h-6b),3.64-3.57(m,2h,h-3’,h-3),3.51-3.43(m,2h,h-
5’,h-5),1.03(s,9h).13c nmr(150mhz,cdcl3)δ139.1,138.6,138.4,138.24,138.20,
133.9,128.8,128.49,128.42,128.4,128.2,128.12,128.10,127.8,127.76,127.72,
127.6,127.5,127.4,117.2,102.3,100.1,81.7,80.7,75.9,75.5,75.24,75.20,74.46,
74.42,74.3,73.8,73.6,73.4,70.8,70.4,70.0,69.3,62.5.hrms(esi):calcd.for
c66h74o11si[m+na]+m/z,1093.4898；found,1093.4882.
The synthesis of embodiment 14 compound 14b
Prepared through acetic anhydride/dimethylsulfoxide oxidation, l-selectride reduction reaction by compound 13b, concrete operation step
With the synthesis of compound 14a, yield 81.5%.
1h nmr(600mhz,cdcl3)δ7.65-6.89(m,50h,ar),5.88-5.82(m,1h,och2ch=ch2),
5.29(s,1h,h-1”),5.20(m,1h,och2Ch=ch 2),5.16(m,1h,och2Ch=ch 2),5.12(s,1h,h-1’),
4.94 (d, j=10.8hz, 2h, och2), ph 4.90 (d, j=12.0hz, 1h, och2Ph), 4.85 (d, j=10.8hz, 1h,
och2), ph 4.77 (d, j=10.8hz, 1h, och2Ph), 4.73 (d, j=3.0hz, 1h, h-2 '), 4.63 (d, j=3.0hz,
1h,h-2),4.51-4.42(m,6h,och2ph,h-1),4.41-4.37(m,3h,och2ph,och 2Ch=ch2),4.34(d,j
=10.8hz, 1h, och2Ph), 4.32 (d, j=2.4hz, 1h, h-2 "), 4.22 (d, j=10.8hz, 2h, och2ph),4.06-
3.97(m,3h,och2ph,och 2Ch=ch2,h-6a”),3.92-3.86(m,2h,h-4”,h-4’),3.80-3.72(m,2h,
h-6a’,h-6b’),3.69-3.55(m,5h,h-6a,h-6b,h-4,h-3’,h-3),3.54-3.47(m,3h,h-5”,h-5’,
h-3”),3.41-3.37(m,1h,h-5).13c nmr(150mhz,cdcl3)δ138.5,138.4,138.2,138.1,138.1,
138.07,138.03,137.9,135.63,135.61,133.6,133.5,133.4,129.5,128.9,128.5,128.3,
128.3,128.2,128.2,128.1,128.0,127.9,127.85,127.82,127.7,127.69,127.65,127.63,
127.5,127.4,127.4,127.2,127.1,117.3,100.6,100.3,99.6,83.1,80.4,79.9,76.6,
75.4,75.3,75.1,74.8,74.8,74.5,74.1,73.4,70.8,70.3,70.2,70.1,69.9,69.6,69.3,
69.0,67.5,64.1,26.9,19.3.hrms(esi):calcd.for c93h102o16si[m+na]+m/z,1525.6835；
found,1525.6805.
The synthesis of embodiment 15 compound 14c
Prepared through acetic anhydride/dimethylsulfoxide oxidation, l-selectride reduction reaction by compound 13c, concrete operation step
With the synthesis of compound 14a, yield 79.6%.
1h nmr(600mhz,cdcl3)δ7.69-6.95(m,65h,ar),5.85-5.78(m,1h,och2ch=ch2),
5.47(s,1h,h-1”’),5.26(s,1h,h-1”),5.19(m,1h,och2Ch=ch 2),5.11(m,1h,och2Ch=
ch 2), 5.01 (s, 1h, h-1 '), 4.98 (d, j=10.8hz, 1h, och2ph),4.94-4.91(m,2h,och2ph,h-2’),
4.94-4.91(m,4h,och2), ph 4.68 (d, j=12.0hz, 1h, och2ph),4.61-4.57(m,3h,och2ph,h-
2”’),4.50-4.39(m,7h,och2Ph), 4.38 (d, j=3.0hz, 1h, h-2 "), 4.36-4.31 (m, 4h, och2ph,h-
2,h-1,och 2Ch=ch2),4.24-4.21(m,3h,och2ph),4.09-4.01(m,5h,och2ph,h-6a”’,h-6b”’,
h-4”’,h-4”),3.95-3.92(m,1h,och 2Ch=ch2),3.62-3.60(m,10h),3.56-3.49(m,5h,h-3”,
h-6a,h-5’,h-5”,h-3),3.35-3.32(m,1h,h-5),1.03(s,9h).13c nmr(150mhz,cdcl3)δ
138.9,138.69,138.49,138.4,138.24,138.20,138.1,138.04,138.01,138.0,135.7,
135.6,133.7,133.6,133.4,129.48,129.5,129.0,128.4,128.33,128.32,128.30,128.27,
128.22,128.20,128.1,128.0,127.9,127.8,127.79,127.76,127.73,127.64,127.62,
127.5,127.45,127.42,127.3,127.2,127.1,127.0,117.1,101.8,100.4,100.2,99.8,
83.1,80.7,80.6,79.7,76.6,75.5,75.44,75.3,75.2,75.1,74.8,74.7,73.9,73.5,73.4,
72.2,72.0,70.3,70.0,69.9,69.88,69.8,69.7,69.1,69.0,68.7,67.6,63.9,26.9,
19.3.hrms(esi):calcd.for c120h130o21si[m+na]+m/z,1957.8772；found,1957.8750.
The synthesis of embodiment 16 compound 15a
Compound 14a (85.0mg, 0.08mmol) is dissolved in 25ml n ' n- dimethylformamide and adds tetrabutyl iodate
Ammonium (1.0mg, 0.003mmol) and cylite (19 μ l, 0.16mmol), reactant liquor be cooled to 0 DEG C after add sodium hydride (4.8mg,
0.12mmol) and continue reaction one hour under argon protection, methanol adds 50ml dchloromethane, then after reaction is quenched
With 30ml saturated common salt water washing, organic layer be dried through anhydrous magnesium sulfate, filter and concentrate after head product, head product is through silica gel
Column chromatography (n-hexane/acetone, 18:1, v/v) obtains white foam solid (77.0mg, 83.7%)
1h nmr(600mhz,cdcl3)δ7.79-7.10(m,40h,ar),6.02-5.93(m,1h,och2ch=ch2),
5.36(m,1h,och2Ch=ch 2),5.25(m,1h,och2Ch=ch 2), 5.24 (br s, 1h, h-1 '), 5.21 (d, j=
10.8hz,1h,och2ph),5.03-4.91(m,3h,och2Ph), 4.79 (d, j=3.0hz, 1h, h-2), 4.71-4.58 (m,
4h,och2ph,h-1),4.57-4.53(m,1h,och 2Ch=ch2),4.52-4.44(m,1h,och2), ph 4.30 (d, j=
3.0hz,1h,h-2’),4.18-4.13(m,2h,och 2Ch=ch2,h-6a’),4.07-4.03(m,1h,och 2Ch=ch2,h-
6b '), 3.95-3.84 (m, 3h, h-4, h-4 ', h-6a), 3.80-3.76 (m, 1h, h-6b), 3.73 (d, j=9.2,3.6hz,
1h, h-3), 3.67 (d, j=9.2,3.2hz, 1h, h-3 '), and 3.65-3.60 (m, 1h, h-5 '), 3.58-3.54 (m, 1h, h-
5),1.12(s,9h).13c nmr(150mhz,cdcl3)δ139.5,138.53,138.51,138.5,138.4,135.7,
135.6,133.9,133.7,133.5,129.6,129.58,128.7,128.6,128.37,128.35,128.3,128.2,
128.04,128.02,127.9,127.72,127.70,127.6,127.5,127.4,127.1,117.1,100.3,100.8,
82.3,80.5,77.4,75.6,75.2,75.1,75.0,74.2,74.1,73.7,73.5,70.7,70.6,70.2,69.6,
69.5,64.4,26.9,19.3.hrms(esi):calcd.for c73h80o11si[m+na]+m/z,1183.5368；found,
1183.5321.
The synthesis of embodiment 17 compound 15b
By compound 14b and cylite reaction preparation, concrete operation step is with the synthesis of compound 15a, yield
89.4%%.
1h nmr(600mhz,cdcl3)δ7.69-6.93(m,55h,ar),5.93-5.85(m,1h,och2ch=ch2),
5.41(s,1h,h-1”),5.24(m,1h,och2Ch=ch 2),5.21-5.15(m,3h,och2Ch=ch 2,och2ph,h-1’),
4.94 (d, j=11.4hz, 1h, och2), ph 4.96 (d, j=10.8hz, 1h, och2ph),4.90-4.84(m,4h,
och2ph),4.81-4.75(m,2h,och2Ph, h-2 '), 4.65 (d, j=3.0hz, 1h, h-2), 4.57 (d, j=11.4hz,
1h,och2), ph 4.52 (d, j=12.0hz, 1h, och2ph),4.47-4.30(m,9h,h-1,och2ph),4.20(br s,
1h,h-2”),4.17-4.12(m,1h,och2ph),4.08-3.98(m,4h,h-6a”,h-6b”,och 2Ch=ch2,
och2Ph), 3.91 (t, j=10.8,9.6hz, 1h, h-4 "), 3.84-3.76 (m, 2h, h-4 ', h-6a), 3.73-3.66 (m,
4h,h-4,h-6b,h-6a’,h-6b’),3.65-3.57(m,4h,h-3’,h-3,h-3”,h-5”),3.56-3.51(m,1h,h-
5’),3.45-3.41(m,1h,h-5),1.01(s,9h).13c nmr(150mhz,cdcl3)δ140.0,138.8,138.6,
138.53,138.50,138.3,138.2,138.1,137.9,135.7,133.8,133.7,133.6,129.4,128.6,
128.4,128.3,128.24,128.20,128.17,128.15,128.1,127.99,127.95,127.8,127.7,
127.66,127.60,127.56,127.54,127.51,127.46,127.3,127.2,127.0,126.8,117.2,
101.5,100.7,83.4,80.6,80.2,75.37,75.34,75.2,75.1,74.9,74.8,74.8,74.6,74.4,
74.3,73.4,73.3,70.8,70.5,70.2,70.18,69.9,69.7,69.1,68.9,64.4,26.9,19.3.hrms
(esi):calcd.for c100h108o16si[m+na]+m/z,1615.7340；found,1615.7274.
The synthesis of embodiment 18 compound 15c
By compound 14c and cylite reaction preparation, concrete operation step is with the synthesis of compound 15a, yield 87.9%.
1h nmr(600mhz,cdcl3)δ7.69-6.97(m,70h,ar),5.85-5.78(m,1h,och2ch=ch2),
5.49(s,1h,h-1”’),5.40(s,1h,h-1”),5.25(m,1h,och2Ch=ch 2),5.18(m,1h,och2Ch=
ch 2), 5.11 (d, j=10.8hz, 1h, och2Ph), 5.03 (s, 1h, h-1 '), 4.99 (d, j=11.4hz, 1h, och2ph),
4.94-4.91(m,2h,och2ph,h-2’),4.87-4.77(m,6h,och2Ph), 4.65 (d, j=3.0hz, 1h, h-2 " '),
4.59 (d, j=12.0hz, 1h, och2ph),4.54-4.34(m,13h,och2ph,och 2Ch=ch2,h-2,h-1),4.32
(d, j=10.8hz, 1h, och2Ph), 4.26 (d, j=3.0hz, 1h, h-2 "), 4.24 (d, j=10.8hz, 1h, och2ph),
4.13-4.17(m,2h,och2ph),4.10-4.04(m,4h,och2ph,h-6a”’,h-6b”’,h-4”),3.97-3.92(m,
1h,och2Ph), 3.86 (t, j=9.6hz, 1h, h-4 '), 3.80-3.59 (m, 11h), 3.58-3.48 (m, 4h, h-6a, h-
5”’,h-5”,h-3),3.37-3.33(m,1h,h-5),0.98(s,9h).13c nmr(150mhz,cdcl3)δ140.3,
138.9,138.9,138.7,138.6,138.4,138.3,138.2,138.1,138.0,137.98,137.92,135.74,
135.70,133.8,133.7,133.4,129.4,129.3,128.7,128.5,128.38,128.4,128.3,128.29,
128.27,128.24,128.20,128.0,127.96,127.92,127.90,127.87,127.86,127.77,127.73,
127.7,127.62,127.60,127.56,127.50,127.4,127.3,127.2,127.1,127.0,126.9,126.7,
117.2,101.8,100.4,100.3,83.5,80.9,80.7,79.5,75.4,75.39,75.33,75.2,75.0,74.9,
74.8,74.7,74.5,74.4,73.4,73.39,73.36,71.9,70.4,70.0,69.9,69.7,69.7,69.1,68.9,
68.7,64.0,26.9,19.3.hrms(esi):calcd.for c127h136o21si[m+na]+m/z,2047.9241；found,
2047.9266.
The synthesis of embodiment 19 compound 16a
Compound 15a (183.0mg, 0.16mmol) is dissolved in 5ml oxolane, is subsequently adding tetrabutyl ammonium fluoride
(0.63ml,1m), stirring under room temperature added 30ml dchloromethane after one day, then uses 30ml saturated common salt water washing, has
Machine layer through anhydrous magnesium sulfate be dried, filter and concentrate after head product, head product through silica gel column chromatography (n-hexane/acetone, 10:
1, v/v) obtain white foam solid (77.0mg, 83.4%)
1h nmr(600mhz,cdcl3)δ7.63-7.15(m,30h,ar),5.97-5.88(m,1h,och2ch=ch2),
5.28(m,1h,och2Ch=ch 2),5.21(m,1h,och2Ch=ch 2), 5.15 (d, j=12.0hz, 1h, och2ph),5.04-
4.96(m,5h,och2Ph, h-1 '), 4.93 (d, j=10.8hz, 1h, och2Ph), 4.71 (d, j=10.8hz, 1h,
och2ph),4.66-4.60(m,2h,och2ph),4.58-4.47(m,5h,och2Ph, h-1), 4.43 (d, j=12.0hz, 1h,
och2), ph 4.39 (d, j=3.0hz, 1h, h-2), 4.29 (d, j=3.0hz, 1h, h-2), 4.10-4.07 (m, 1h, och 2ch
=ch2), 4.06 (t, j=9.6hz, 1h, h-4 '), 3.95 (t, j=9.6hz, 1h, h-4), 3.92-3.84 (m, 2h, h-6a ',
H-6b '), 3.82 (d, j=10.8hz, 1h, h-6a), 3.76 (dd, j=10.8,5.0hz, 1h, h-6b), 3.66 (dd, j=
9.3,2.9hz, 1h, h-3), 3.59 (dd, j=9.6,3.0hz, 1h, h-3 '), 3.52-3.47 (m, 1h, h-5), 3.44-3.41
(m,1h,h-5’),13c nmr(150mhz,cdcl3)δ139.10,138.56,138.36,138.24,138.20,133.89,
128.78,128.49,128.42,128.35,128.18,128.12,128.10,127.83,127.76,127.72,127.61,
127.52,127.37,117.20,102.25,100.15,81.69,80.68,77.42,77.21,77.00,75.98,75.46,
75.24,75.20,74.46,74.42,74.33,73.80,73.56,73.43,70.81,70.42,70.00,69.30,
62.49.hrms(esi):calcd.for c57h62o11[m+na]+m/z,945.4190；found,945.4161.
The synthesis of embodiment 20 compound 16b
By compound 15b and tetrabutyl ammonium fluoride reaction preparation, concrete operation step is with the synthesis of compound 16a, yield
88.2%.
1h nmr(600mhz,cdcl3)δ7.51-6.95(m,45h,ar),5.92-5.84(m,1h,och2ch=ch2),
5.29(s,1h,h-1”),5.24(m,1h,och2Ch=ch 2),5.19-5.15(m,2h,och2Ch=ch 2,h-1’),5.07
(d, j=12.0hz, 1h, och2), ph 4.98 (d, j=11.4hz, 1h, och2Ph), 4.93 (d, j=10.8hz, 1h,
och2ph),4.89-4.84(m,3h,och2), ph 4.71 (d, j=12.0hz, 1h, och2Ph), 4.71 (d, j=3.0hz, 1h,
H-2 '), 4.65 (d, j=12.0hz, 1h, och2), ph 4.61 (d, j=3.0hz, 1h, h-2), 4.56 (d, j=10.8hz,
1h,och2ph),4.46-4.40(m,6h,och2ph,h-1,och 2Ch=ch2), 4.29 (d, j=10.8hz, 1h, och2ph),
4.15 (d, j=3.0hz, 1h, h-2 "), 4.13 (d, j=11.4hz, 1h, och2Ph), 4.07 (d, j=11.4hz, 1h,
och2ph),4.03-3.98(m,1h,och 2Ch=ch2), 3.94 (t, j=9.6hz, 1h, h-4 "), 3.86 (t, j=9.6hz,
1h,h-4’),3.76-3.80(m,2h,h-6a”,h-6a’),3.75-3.67(m,5h,h-6b”,h-4,h-6a,h-6b,h-
6b’),3.66-3.63(m,1h,h-3’),3.63-3.58(m,1h,h-3),3.55-3.51(m,2h,h-3”,h-5’),3.42-
3.35(m,2h,h-5,h-5”).13c nmr(150mhz,cdcl3)δ139.6,138.7,138.5,138.4,138.4,138.2,
138.1,138.0,137.7,133.6,128.6,128.41,128.40,128.4,128.3,128.29,128.23,128.2,
128.04,128.00,127.8,127.7,127.69,127.65,127.61,127.6,127.4,127.1,127.1,117.4,
101.6,100.7,100.4,83.0,80.7,80.6,75.5,75.4,75.2,75.1,74.7,74.66,74.62,74.6,
74.5,73.6,73.4,71.6,70.5,70.4,69.9,69.8,69.6,68.7,62.4.hrms(esi):calcd.for
c84h90o16[m+na]+m/z,1377.6127；found,1377.6112.
The synthesis of embodiment 21 compound 16c
By compound 15c and tetrabutyl ammonium fluoride reaction preparation, concrete operation step is with the synthesis of compound 16a, yield
87.2%.
1h nmr(500mhz,cdcl3)δ7.52-6.02(m,60h,ar),5.92-5.83(m,1h,och2ch=ch2),
5.53(s,1h,h-1”’),5.32(s,1h,h-1”),5.24(m,1h,och2Ch=ch 2),5.18(m,1h,och2Ch=
ch 2), 5.15 (s, 1h, h-1 '), 4.99 (d, j=12.0hz, 1h, och2ph),5.00-4.88(m,5h,och2ph,h-2’),
4.83 (d, j=10.8hz, 1h, och2), ph 4.75 (d, j=12.0hz, 1h, och2ph),4.87-4.77(m,15h,
och2ph,h-2,h-2”’,och 2Ch=ch2), 4.25 (d, j=3.0hz, 1h, h-2 "), 4.18-4.10 (m, 2h, och2ph),
4.05-3.96(m,3h,och 2Ch=ch2,och2ph),3.87-3.54(m,15h),3.48-3.40(m,2h,h-5,h-5”)
.13c nmr(125mhz,cdcl3)δ139.6,138.75,138.72,138.6,138.5,138.4,138.2,138.16,
138.1,137.9,137.8,133.6,128.62,128.60,128.5,128.45,128.4,128.3,128.32,128.3,
128.25,128.22,128.21,128.2,128.1,128.04,128.02,128.0,127.8,127.72,127.7,
127.6,127.5,127.4,127.3,127.1,127.0,117.3,101.7,101.2,100.7,100.2,82.9,81.8,
80.7,80.4,75.6,75.5,75.3,75.2,75.1,75.0,74.8,74.8,74.5,74.4,73.6,73.5,73.45,
72.6,70.5,70.5,70.3,70.1,69.8,69.6,69.6,69.5,69.3,68.8,62.4.maldi-tof-ms:
calcd.for c111h118o21[m+na]+m/z,1811.10；found,1810.76.
The synthesis of embodiment 22 compound 18a
By compound 16a (50.0mg, 0.06mmol) and phosphorylation agent 17 (138mg, 0.26mmol)
(synthesis.1992,1269.) is placed in 10ml round-bottomed flask and adds 3.0ml dichloromethane to dissolve, after solution is cooled to 0 DEG C
Deca 1h- tetrazolium solution (0.6ml, 0.45m), the lower reaction of argon protection is removed chiller after 15 minutes and is continued at room temperature
Stirring two hours, and Deca tert-Butanol peroxide after being subsequently cooled to -40 DEG C (0.07ml, 5.5m) solution, remove chiller and continue
After stirring three hours under room temperature, add 15ml 10% hypo solution that reaction is quenched, reactant liquor is extracted with 30ml dichloromethane
Take, then washed with saturated sodium bicarbonate solution (30ml) and saline solution (30ml) successively, organic layer through anhydrous magnesium sulfate be dried,
Head product is obtained, head product obtains diastereo-isomerism through silica gel column chromatography (n-hexane/ethyl acetate, 3:2, v/v) after filtering and concentrating
Mixture (66.7mg, 82.0%).
selected signals:1h nmr(500mhz,cdcl3)δ4.97(br s,1h),4.41(br s,1h).13c
nmr(125mhz,cdcl3)δ102.14,102.09,100.31,100.26.31p nmr(160mhz,cdcl3)δ-0.61,-
0.76.hrms(esi):calcd.for c81h84no16p[m+na]+m/z,1380.5425；found,1380.5381.
The synthesis of embodiment 23 compound 18b
Preparation is reacted by compound 16b and phosphorylation agent 17, concrete operation step is with the synthesis of compound 18a, yield
87.5%.
selected signals:1hnmr(500mhz,cdcl3)δ5.40(br s,1h),5.18(br s,1h),4.46
(br s,1h).13c nmr(125mhz,cdcl3)δ101.62,101.52,100.79,100.70,100.65,100.54.31p
nmr(160mhz,cdcl3)δ-0.50,-0.70.hrms(esi):calcd.for c108h112no21p[m+na]+m/z,
1812.7362；found,1812.7319.
The synthesis of embodiment 24 compound 18c
Preparation is reacted by compound 16c and phosphorylation agent 17, concrete operation step is with the synthesis of compound 18a, yield
79.3%.
nmr signals for 18c(isomer 1):1h nmr(600mhz,cdcl3)δ7.73-6.93(m,73h,
ar),5.85-5.78(m,1h,och2ch=ch2),5.48(s,1h,h-1”’),5.30(s,1h,h-1”),5.19(m,1h,
och2Ch=ch 2),5.14-5.10(m,1h,och2ph),5.09(s,1h,h-1’),5.05-5.00(m,1h,och2ph),
4.97-4.82(m,9h,och2Ph, h-2 '), 4.71 (d, j=12.0hz, 1h, och2Ph), 4.62 (d, j=12.0hz, 1h,
och2), ph 4.59 (d, j=12.0hz, 1h, och2), ph 4.56 (d, j=10.8hz, 1h, och2), ph 4.57 (d, j=
12.0hz,1h,och2ph),4.53-4.44(m,6h,och2Ph, h-1, h-2 " '), 4.41-4.32 (m, 8h), 4.29 (d, j=
3.6hz, 1h, h-2 "), 4.26 (d, j=10.8hz, 1h, och2ph),4.15-4.06(m,4h),4.03-3.93(m,4h),
3.92-3.75 (m, 5h), 3.72-3.58 (m, 8h), 3.55 (dd, j=9.6,3.6hz, 1h, och2ph),3.52-3.43(m,
2h),3.37-3.32(m,1h),3.26-3.18(m,1h),3.08-2.99(m,1h).13c nmr(150mhz,cdcl3)δ
156.18,144.08,143.92,141.16,141.12,139.49,138.64,138.52,138.43,138.34,138.21,
138.05,138.02,137.99,137.84,137.75,135.80,136.76,133.59,128.64,128.57,128.52,
128.45,128.32,128.28,128.26,128.22,128.18,128.16,128.12,128.04,128.02,127.97,
127.96,127.88,127.85,127.81,127.70,127.64,127.56,127.54,127.50,127.43,127.39,
127.24,127.11,127.06,126.97,126.95,125.21,125.12,119.77,117.31,102.08,101.31,
100.55,100.29,83.10,81.97,80.53,80.35,75.52,75.31,75.27,75.12,75.05,74.69,
74.58,74.24,74.20,73.61,73.56,73.41,73.21,70.92,70.54,70.19,70.18,69.69,
69.60,69.51,69.36,69.15,69.12,68.77,66.89,66.85,66.57,66.45,47.04,40.95.31p
nmr(160mhz,cdcl3)δ-0.67.maldi-tof-ms:calcd.for c108h112no21p[m+h]+m/z,2222.947；
found,2223.236.nmr signals for 18c(isomer 2):1h nmr(600mhz,cdcl3)δ7.73-6.93(m,
73h,ar),5.87-5.78(m,1h,och2ch=ch2),5.48(s,1h,h-1”’),5.32(s,1h,h-1”),5.19(m,
1h,och2Ch=ch 2),5.15-5.11(m,2h,och2ph),5.07(s,1h,h-1’),5.06-5.00(m,3h,och2ph),
4.97-4.82(m,8h,och2Ph, h-2 '), 4.74 (d, j=11.4hz, 1h, och2Ph), 4.69 (d, j=12.0hz, 1h,
och2), ph 4.66 (d, j=12.0hz, 1h, och2), ph 4.62 (d, j=10.8hz, 1h, och2), ph 4.57 (d, j=
12.0hz,1h,och2), ph 4.54 (d, j=12.0hz, 1h, och2), ph 4.51 (br s, 1h, h-1), 4.49 (d, j=
10.8hz,2h,och2ph),4.47-4.44(m,3h,och2ph,h-2”’),4.41-4.34(m,8h),4.27(m,2h),4.26
(d, j=3.0hz, 1h, h-2 "), 4.22 (d, j=10.8hz, 2h, och2ph),4.13-4.04(m,3h),4.03-3.93(m,
3h), 3.87-3.83 (m, 3h), 3.68-3.58 (m, 9h), 3.55 (dd, j=9.6,3.6hz, 1h, och2ph),3.52-3.47
(m,2h),3.37-3.34(m,1h),3.14-3.08(m,2h).13c nmr(150mhz,cdcl3)δ156.22,144.01,
143.97,141.19,139.59,138.68,138.60,138.33,138.26,138.11,138.08,138.04,137.88,
137.78,136.08,136.04,133.61,128.67,128.52,128.46,128.42,128.37,128.30,128.29,
128.23,128.22,128.17,128.09,128.05,128.01,127.98,127.96,127.92,127.79,127.72,
127.66,127.63,127.58,127.53,127.40,127.21,127.18,127.09,127.05,127.00,126.57,
125.16,119.81,117.27,101.97,101.47,100.52,100.27,83.12,81.73,80.62,80.31,
75.47,75.35,75.28,75.23,75.09,75.01,74.97,74.94,74.70,74.51,74.30,74.26,
73.60,73.53,73.40,73.37,72.86,71.25,70.43,70.15,70.12,69.70,69.63,69.59,
69.57,69.42,69.27,69.24,68.83,66.70,66.66,66.59,47.11,41.16,41.12.31p nmr
(160mhz,cdcl3)δ-0.52.hrms(esi):calcd.for c108h112no21p[m+na]+m/z,2244.9293；
found,2244.9475.
The synthesis of embodiment 25 compound 19a
Compound 18a (50.0mg, 0.037mmol) is placed in 5ml eggplant type bottle and adds 0.8ml dichloromethane to dissolve, room
Temperature lower stirring simultaneously Deca 51,8- diazabicylo 11 carbon -7- alkene (dbu), add 5 acetic acid neutralization reactions after two minutes,
Under low-temp low-pressure remove solvent after gained head product through silica gel column chromatography (methylene chloride/methanol, 30:1, v/v) diastereomeric different
Structure mixture (38.0mg, 90.4%).
selected signals:1h nmr(600mhz,cdcl3)δ4.96(br s,1h),4.40(br s,1h).13c
nmr(125mhz,cdcl3)δ101.84,101.69,100.14,99.86.hrms(esi):calcd.for c66h75no14p
[m+h]+m/z,1136.4920；found,1136.4925.
The synthesis of embodiment 26 compound 19b
Preparation is reacted by compound 18b and dbu, concrete operation step is with the synthesis of compound 19a, yield 93.2%.
selected signals:1h nmr(600mhz,cdcl3)δ5.31(s,1h),5.13(s,1h),4.44(s,
1h).13c nmr(150mhz,cdcl3)δ101.27,100.78,100.67,100.55,100.22,100,08.hrms(esi):
calcd.for c93h102no19p[m-h]-m/z,1566.6711；found,1566.6705.
The synthesis of embodiment 27 compound 19c
Preparation is reacted by compound 18c and dbu, concrete operation step is with the synthesis of compound 19a, yield 86.3%.
nmr signals for 19c(isomer 1):1h nmr(600mhz,cdcl3)δ7.50-6.92(m,65h,
ar),5.85-5.77(m,1h,och2ch=ch2),5.46(s,1h,h-1”’),5.31(s,1h,h-1”),5.18(m,1h,
och2Ch=ch 2),5.04(s,1h,h-1’),5.03-4.97(m,5h,och2ph),4.90-4.78(m,5h,och2ph),
4.67-4.60(m,3h),4.59-4.45(m,6h,och2ph),4.43-4.33(m,8h,och2ph,h-1),4.31-4.19(m,
3h), 4.09 (d, j=11.2hz, 1h, och2), ph 4.04 (d, j=11.2hz, 1h, och2ph),3.98-3.88(m,3h),
3.84-3.68 (m, 6h), 3.67-3.56 (m, 6h), 3.54 (dd, 1h, j=9.6,3.0hz), 3.51-3.45 (m, 1h),
3.44-3.40(m,1h),3.37-3.32(m,1h).13c nmr(150mhz,cdcl3)δ138.78,138.49,138.24,
138.16,138.11,138.05,137.99,137.87,137.71,133.59,128.76,128.56,128.50,128.42,
128.40,128.31,128.30,128.24,128.17,128.16,128.11,128.07,128.04,128.00,127.98,
127.90,127.85,127.76,127.73,127.69,127.64,127.51,127.40,127.20,127.15,127.10,
117.25,101.65,101.59,100.40,100.10,83.33,81.50,80.66,80.26,75.46,75.36,75.27,
75.23,75.03,74.99,74.95,74.73,74.59,74.35,73.58,73.51,73.38,70.25,70.08,
69.78,69.52,69.48,69.40,69.03,68.83,66.60.nmr signals for 19c(isomer 2):1h
nmr(600mhz,cdcl3)δ7.55-6.93(m,65h,ar),5.85-5.79(m,1h,och2ch=ch2),5.53(s,1h,h-
1”’),5.38(s,1h,h-1”),5.18(m,1h,och2Ch=ch 2),5.15-5.11(m,2h,och2ph,h-1’),5.10
(s,1h,h-1’),5.01-4.91(m,5h,och2), ph 4.89 (d, j=3.0hz, 1h), 4.85-4.78 (m, 3h), 4.66
(d, j=10.8hz, 1h, och2ph),4.61-4.57(m,2h),4.47-4.37(m,14h,och2ph,h-2,h-1),4.29
(d, j=10.8hz, 1h, och2), ph 4.25-4.14 (m, 3h), 4.07 (d, j=11.2hz, 1h, och2ph),3.98-3.75
(m,7h),3.71-3.55(m,9h),3.51-3.44(m,2h),3.39-3.34(m,2h).13c nmr(150mhz,cdcl3)δ
138.94,138.20,138.13,138.05,138.02,138.00,137.98,137.91,137.84,137.71,133.61,
129.19,128.93,128.62,128.56,128.52,128.46,128.37,128.34,128.30,128.25,128.21,
128.07,128.04,128.02,127.95,127.93,127.92,127.87,127.84,127.74,127.67,127.60,
127.55,127.50,127.35,127.24,126.92,117.26,101.32,101.15,100.46,99.78,82.15,
80.61,80.54,75.58,75.39,75.30,75.20,75.09,75.02,74.93,74.82,74.65,74.49,
73.61,73.48,73.43,71.13,70.38,70.30,70.12,70.04,69.33,69.16,69.09,69.05,
68.89,66.31,60.03.hrms(esi):calcd.for c120h131no24p[m+h]+m/z,2000.8793；found,
2000.8799.
The synthesis of embodiment 28 compound 21
With alanine resin 20 (0.13mmol/g) as initiation material, it is added to Solid phase peptide synthssis pipe, is subsequently adding and contains
The dmf solution of 20% piperidines, shakes 30 minutes under room temperature, removes reactant liquor, and resin washs 3 times with the mixed solution of dmf/dcm.
The aminoacid being activated with hbtu/hobt/dipea is added to solid phase synthesis pipe, adds concussion 1-2 under 5ml dmf room temperature little
When, after completion of the reaction, washed with the mixed solution of dmf/dcm 3 times, add the dmf solution containing 20% piperidines toward in gained resin,
Under room temperature, concussion removes reactant liquor in 30 minutes, and resin washs 3 times with the mixed solution of dmf/dcm.So repeat reaction repeatedly straight
Connect to all aminoacid and finish.It is little that the mixed solution of addition acoh/tfe/dcm (1:1:8, volume ratio) shakes 2 at room temperature
When, filter, filter cake wash with dcm after by the crude Compound after gained filtrate reduced in volume, must be pure after column chromatography and lh-20
Target compound 21.
hrms(esi):calcd.for c139h183n17o27[m-h]-m/z,2521.3391；found,2521.3341.
The synthesis of embodiment 29 compound 22a
By compound 19a (25.0mg, 0.022mmol), polypeptide fragment 21 (75.6mg, 0.03mmol) and hobt
(17.8mg, 0.13mmol) is placed in 10ml round-bottomed flask and adds dichloromethane and the mixed solution of n- methyl pyrrolidone
(3.0ml, 2:1) dissolves, and solution adds 1- ethyl-(3- dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate after being cooled to 0 DEG C
(edc.hcl, 26.0mg, 0.14mmol), removes chiller after reacting 15 minutes and continues at room temperature under argon protection
Stirring twenty four hours.After completion of the reaction, reactant liquor is extracted with 30ml dichloromethane, then uses saturated sodium bicarbonate solution successively
(30ml) wash with saline solution (30ml), organic layer obtains head product, head product warp after being dried through anhydrous magnesium sulfate, filter and concentrating
Silica gel column chromatography (methylene chloride/methanol, 40:1) obtains compound as white solid (63.2mg, 78.9%).
hrms(esi):calcd.for c205h255n18o40p[m+2na]2+m/z,1842.9003；found,1842.9056
The synthesis of embodiment 30 compound 22b
Preparation is reacted with polypeptide 21 by compound 19b, concrete operation step is with the synthesis of compound 22a, yield 82.4%.
hrms(esi):calcd.for c232h283n18o45p[m+2h]+m/z,2037.0146；found,2037.0198；
[m+3h]3+m/z,1358.3455；found,1358.3461.
The synthesis of embodiment 31 compound 22c
Preparation is reacted with polypeptide 21 by compound 19c, concrete operation step is with the synthesis of compound 22a, yield 83.2%.
hrms(esi):calcd.for c259h312n18o50p[m+h]+m/z,4506.2157；found,4506.1724；[m
+2h]2+m/z,2253.1115；found,2253.1270；[m+3h]3+m/z,1502.4100；found,1502.3901.
The synthesis of embodiment 32 compound 23a
Compound 22a (15.0mg, 0.004mmol) and hydroxide palladium carbon (10wt.%, 10.0mg) are dissolved in meoh/
ch2cl2(2ml, 2:1, v/v) mixed solution, is placed in 24 hours of hydriding reactor oscillating reactionss under room temperature, reactant liquor uses diatom
Native cake filtration is simultaneously washed with methanol, and residue after concentrating under reduced pressure for the filtrate is molten with tfa/tes/dcm (3:1:6) mixed liquor
Solution, stirring reaction two hours under room temperature, after completion of the reaction, and removal of solvent under reduced pressure, obtain just through lyophilization after deionized water extraction
Product, head product obtains compound as white solid (6.8mg, 80.5%) after gel resin g-25 filtration, lyophilization.
maldi-tof-ms:calcd.for c88h131n18o36p[m+h]+m/z,2049.087；found,2049.201.
The synthesis of embodiment 33 compound 23b
Prepared by the hydrogenated reduction reaction of compound 22b, concrete operation step is with the synthesis of compound 23a, yield
78.4%.
maldi-tof-ms:calcd.for c94h142n18o41p[m+h]+m/z,2211.228；found,2211.368.
The synthesis of embodiment 34 compound 23c
Prepared by the hydrogenated reduction reaction of compound 22c, concrete operation step is with the synthesis of compound 23a, yield
82.7%.
maldi-tof-ms:calcd.for c100h152n18o46p[m+h]+m/z,2373.369；found,2374.062.
The synthesis of embodiment 34 Acibenzolar 25a-c
Compound 23 and Succinimidyl glutarate 24 (dsg, 15equiv) are dissolved in dmf/pbs (4:1,0.1m
Pbs) mixed solution, stirs four hours under room temperature, removes solvent and revolves twice with toluene band, residue adds under low-temp low-pressure
Excess ethyl acetate repeatedly washs, and obtains Acibenzolar sterling after being dried.
The synthesis of embodiment 35 glycopeptides-protein conjugate (1a-c, 2a-c)
Compound 25 is dissolved in 0.1 according to mol ratio for 30:1 (Acibenzolar: carrier protein) with carrier protein klh or hasm
Pbs buffer solution, is stirred at room temperature two and half under argon protection.Reactant liquor is through biogela0.5 column chromatography (0.1mPbs buffers
Liquid, ph=7.8), after deionized water dialysis and lyophilization sterling glycopeptide-protein conjugate 1a-c and 2a-c.
Embodiment 36 the compounds of this invention immunocompetence is tested
First, experiment material and source
Test compound: prepared glycoprotein compound 1a-c in the embodiment of the present invention 34.
2nd, test method
(1) mouse immunization protocol
1) select 6-8 week female balb/c mice (being purchased from Chinese Academy of Sciences's Shanghai Experimental Animal Center), according to vaccine+assistant
Agent and being only grouped with Vaccination Protocols, matched group is only adjuvant group, every group of 6 mices；
2) by glycopeptide-proteinate 1a-c and its corresponding interpolation adjuvant group (being dissolved in pbs) suspension subcutaneous injection mice,
Glycopeptide-the albumen of immunity contains the glycopeptide of 10 μ g every time, uses same method booster immunization again every 2 weeks, altogether immunity 4 times.Respectively
Before immunity, latter 13 days of the 2nd immunity, latter 13 days of third time immunity and the 4th immunity take within latter 14 days blood, separate serum juxtaposition
Frozen to be measured in -80 DEG C of refrigerators.
(2) Serum Antibody titer determination before and after mouse immune
Each every mice pooled serum titre of experimental group is all detected using elisa method.
1) wrapper sheet: compound 2a-c is diluted to 5ug/ml with ph 9.6 carbonate buffer solution respectively and is coated in enzyme mark row's batten
On.37 DEG C of combinations 2 hours, use 4 DEG C of confining liquid (15% skim milk) 200ul/ hole overnight afterwards；
2) after pbst washs 3 times, every hole adds 100ul doubling dilution each group mice serum, and 37 DEG C are incubated 2 hours；
3), after pbst washs 5 times, rabbit anti-mouse igg bis- of the hrp labelling after 100ul 1:2000 dilution is added to resist
(invitrogen), 37 DEG C are incubated 45 minutes；
4), after pbst washs 5 times, after tmb colour developing, microplate reader (multiscanmk3, thermo) reads a450 numerical value, is higher than
The definition that 3 times of negative control is positive serum.
(3) after mouse immune taken serum and Candida albicanss cell combination
1) picking Candida albicanss sc5314 monoclonal bacterium colony ypd fluid medium increases bacterium 24 hours, now right for growing
The yeast like cell of number phase.Candida albicans hyphal phase abductive approach: logarithmic (log) phase Candida albicanss will be grown and utilize the resuspended washing 2 of pbs
Secondary, it is placed in containing 10% calf serum, 37 DEG C of quiescent culture of 1640 culture medium 90 minutes；
2) by the Candida albicanss of Hyphal form or Yeast Phase resuspended 2 times with pbs, 10 points are fixed for -20 DEG C with 4% paraformaldehyde
Clock, uses resuspended 3 times of pbs again after centrifugation；
3) by experimental group and matched group serum the pbs containing 10% calf serum press 1:100 dilution after respectively with fixation
Thalline afterwards is incubated 40 minutes altogether, thereafter with pbs centrifugation, resuspended 3 times；
4) be incubated 30 minutes altogether with the sheep anti mouse igg antibody of 488 labellings and thalline, thereafter with pbs centrifugation, resuspended 3 times；
5) resuspended bacterium is placed in 96 orifice plates and stands 10 minutes, using fluorescence microscope in 488 observed under fluorescent light；
6) resuspended bacterium is placed in direct flow cytometer detection after fluidic cell centrifuge tube, exciting light selects fitc, profit
Returned to zero with negative control Candida albicanss, again the bacterium after being incubated altogether with fluorescent antibody is carried out upper machine testing, each loading
Read 10000 cells.
3rd, experimental result
Klh- glycopeptide-protein conjugate 1a-c subcutaneous inoculation the mice of present invention preparation, can induce body to produce stronger
Immunne response, wherein β -1, the immunogenicity of 2-d- mannotriose peptide-protein conjugate is the strongest and immune serum that produce can
Combine with Candida albicanss cell surface antigen, experimental result is shown in accompanying drawing 6-8.
The above is only the preferred embodiment of the present invention it is noted that ordinary skill people for the art
Member, on the premise of without departing from the inventive method, can also make some improvement and supplement, these improve and supplement also should be regarded as
Protection scope of the present invention.
Claims (10)
1. a kind of β -1,2-d- oligomerization mannosazone-protein conjugate is it is characterised in that described β -1,2-d- oligomerization mannose
Peptide-protein conjugate general structure is:
Wherein, n is carrier protein klh or hsa for 0,1 or 2, r.
2. β -1 according to claim 1,2-d- oligomerization mannosazone-protein conjugate is it is characterised in that composition oligosaccharide
Monosaccharide unit is β -1,2-d- mannose；Described peptide sequence is selected from the n- terminal amino acid of Candida albicanss cell surface protein als1
Sequence.
3. β -1 according to claim 1,2-d- oligomerization mannosazone-protein conjugate is it is characterised in that with phosphoric acid ethanol
Amido is as the linking group of β -1,2-d- oligomerization mannose and polypeptide；Using glutaryl as β -1,2-d- oligomerization mannosazone
Linking group with carrier protein.
4. β -1 according to claim 1,2-d- oligomerization mannosazone-protein conjugate is it is characterised in that described β -1,2-
D- oligomerization mannosazone-protein conjugate is obtained with carrier protein couplet by the β -1,2-d- oligomerization mannosazone of phosphorylation.
5. β -1 described in claim 1, the preparation method of 2-d- oligomerization mannosazone-protein conjugate is it is characterised in that include
Following steps:
The first step, synthesis β -1,2-d- mannobiose (receptor 7), β -1,2-d- mannotriose receptor (acceptor 10):
Second step, synthesizes phosphorylation β -1,2-d- oligomerization mannose 19a-c:
3rd step, synthesis polypeptide 21:
4th step, synthesizes β -1,2-d- oligomerization mannosazone-protein conjugate:
6. β -1 according to claim 5, the preparation method of 2-d- oligomerization mannosazone-protein conjugate it is characterised in that
Described phosphorylation β -1,2-d- oligomerization mannose preparation method method is as follows:
(1) with alpha-d-glucose as raw material, β -1,2-d- manna monosaccharide receptor and 3,4,6- are prepared in the presence of reaction promoter
Tribenzyl -2- acetoxyl group glucose tri- chloroacetimidate donor；
(2) there is glycosylation preparation two in the monosaccharide donor of above-mentioned preparation and monosaccharide receptor under catalyst (tmsotf) effect
Sugar, prepares β -1,2-d- mannobiose receptor by configuration reversal on 2 '-position of disaccharide；β -1,2-d- mannobiose receptor and
Monosaccharide donor prepares β -1,2-d- mannotriose receptor by preceding method；
(3) β -1,2-d- mannose receptor respectively with 6- tert-butyl diphenyl silicon substrate -3,4- dibenzyl -2- acetoxyl group glucose
Tri- chloroacetimidate donor prepares corresponding β -1,2-d- oligomerization mannose through glycosylation；Oligomerization mannose non-reducing end 6-
React and phosphorylation β -1,2-d- oligomerization mannose is obtained with phosphorylation agent after tbdps protection is taken off in position.
7. β -1 according to claim 5, the preparation method of 2-d- oligomerization mannosazone-protein conjugate it is characterised in that
The preparation method of described polypeptide 21 is: with fmoc-l- alanine resin as raw material, using solid phase synthesis process synthesis polypeptide 21.
8. β -1 according to claim 5, the preparation method of 2-d- oligomerization mannosazone-protein conjugate it is characterised in that
Comprise the steps:
(1) phosphorylation β -1,2-d- oligomerization mannose is obtained β -1,2-d- oligomerization mannosazone with polypeptide 21 through condensation reaction；
(2) β -1,2-d- oligomerization mannosazone prepared by step 1 is reacted with hydroxyl Succinimidyl glutarate and β -1,2- is obtained
D- oligomerization mannosazone Acibenzolar；
(3) Acibenzolar prepared by step 2 under weak basic condition with carrier protein couplet, obtain β -1,2-d- oligomerization mannose
Peptide-protein conjugate.
9. the arbitrary described β -1,2-d- oligomerization mannosazone-protein conjugate of claim 1-6 in preparation prevention and/or is treated
Application in the medicine of fungal infection；Described funguses are Candida albicanss.
10. a kind of antifungal funguses vaccine, including arbitrary described β -1 of claim 1-6 of therapeutically effective amount, 2-d- is few
Poly- mannosazone-protein conjugate and pharmaceutically acceptable adjuvant or adjuvant.
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