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Synthesis and Bioassay of Neurogenically Potent Ganglioside DSG-A, Hp-s1 and Their Analogues Ganesh B. Shelke, Yu-Hsuan Lih, Ying-Ju Liao, ChihWu Liang, Tzer-Min Kuo, Ying-Chin Ko, and Shun-Yuan Luo ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00055 • Publication Date (Web): 20 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018
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ACS Chemical Neuroscience
Synthesis and Bioassay of Neurogenically Potent Ganglioside DSG-A, Hp-s1 and Their Analogues Ganesh B. Shelke,1 Yu-Hsuan Lih,1 Ying-Ju Liao,1 Chih-Wu Liang,1 Tzer-Min Kuo,2 Ying-Chin Ko,2 and Shun-Yuan Luo*,1 1 2
Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan Environment-Omics-Disease Research Centre, China Medical University Hospital, Taichung 404, Taiwan
Supporting Information Placeholder
ABSTRACT: In the search of a potent candidate for neurotherapy, we designed and synthesized various analogs of ganglioside Hp-s1. The modification includes the change in hydrophobicity by varying the carbon chain length, altering the number of hydrogen bonds, and replacing the anomeric atom. The chemical synthesis was carried out by using various methods and discussed in details. The neuritogenic activities of these analogs are confirmed in a human neuroblastoma cell line SH-SY5Y. A higher activity of ganglioside Hp-s1 analogue on IL-17A transcript up-regulation than ganglioside Hp-s1 was found. Keywords: Ganglioside Hp-s1 and analogs, Hydrophobicity, neurogenic activity, SH-SY5Y cells, IL-17A Introduction: Gangliosides are a glycosphingolipids with one or more N-acetylneuraminic acid(s) linked to the non-reducing end of the sugar chain. They are mainly found in tissues, body fluids, brain, nervous system,1 and membrane constituents of vertebrate cells.2 Gangliosides possesses the functions in cell-cell recognition,3 as toxin receptor,4 the formation of raft domains,5 intracellular and intranuclear calcium homeostasis,6 as markers in stem
cells,7 processes in the nervous system.8 Gangliosides are also involved in the pathology of diseases such as Guillain-Barré syndrome,9 in influenza viral infection,10 Alzheimer’s disease,11 epilepsy syndrome,12 multiple sclerosis,13 Graves’ disease,14 and many more. Several studies proposed the pivotal role of gangliosides in the development and the functions of the nervous system.15 Interestingly, some gangliosides extracted from echinoderms such as sea cucumbers, starfish, sea urchins, and feather stars16 show neuritogenic activity on cell line PC-12 in the presence of NGF.17,18 Among these gangliosides, SJG-2, LLG-3, GAA-7, LLG-5, PNG-2A, DSG-A (1), and Hp-s1 (2) were more potent than the mammalian ganglioside GM1, and are considered as lead compounds for the carbohydrate-based drug development in the treatment of neurodegenerative diseases. As extracting sufficient quantity of these EGs is tedious and time consuming task hence many research groups, including our group19 are synthesizing and investigating the potent neuritogenic activity exerted by echinodermatous gangliosides (EGs). Ganglioside DSG-A (1) and Hp-s1 (2), isolated from the ovary of the sea urchin Diadema setosum20 and later prepared by chemical methods.21 Herein, we wish to present the synthesis and the neurite outgrowth (the struc-
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ture activity relationship) of echinodermatous gangliosides (EGs) analogs 2a-2h for neurotherapeutic study. C16H33
R5 O
OH
O
CO2H
HO AcHN
R3 OH
O
HO HO
HO
R4
HN
O
O
X
CnH2n+1
HO HO
R
1
R2
1, n = 13, R1 = R2 = R4 = H, R3 = OH, R5 = Me, X = O, DSG-A, 2, n = 13, R1 = R2 = R3 = R4 = R5= H, X = O, Hp-s1, 2a, n = 3, R1 = R2 = R3 = R4 = R5= H, X = O, 2b, n = 7, R1 = R2 = R3 = R4 = R5= H, X = O, 2c, n = 12, R1 = R2 = R3 = R4 = R5= H, X = O, 2d, n = 15, R1 = R2 = R3 = R4 = R5= H, X = O, 2e, n = 13, R1 = OH, R2 = R3 = R4 = R5= H, X = O, 2f, n = 13, R1 = H, R2 = OH, R3 = R4 = R5= H, X = O, 2g, n = 13, R1 = R2 = R3 = R4 = R5= H, X = S, 2h, n = 13, R1 = R2 = R4 = H, R3 = OH, R5 = Me, X = S
Figure 1. Ganglioside DSG-A 1, Hp-s1 2 and their analogs 2a-2h.
Result and Discussion We devised the important synthons to achieve the synthesis of the target molecule and assembled them subsequently. The heterogeneity of gangliosides is maintained by using varying chain length, substituent pattern and anomeric linkage. By following the literature procedure, acceptors 3a-3e22 were prepared with differing the carbon chain length. We chose the imidate donor 423 for incorporation of ceramide to glycan by glycosylation reaction of 1-OH of acceptors 3a-3e. The acceptors 3a-3d were coupled with donor 4 by using TMSOTf as promotor in DCM/ACN solvents which provides solvent effect in favor of βisomers. The resulting products were hydrolyzed with sodium methoxide to get the second list of acceptors 5a-5d respectively (Scheme 1). CnH2n+1
N3 HO
O
O
BnO BnO
O
1. TMSOTf, 3 A MS DCM/ACN(1/2) -30 oC to rt
OAc O
BnO
CCl3 NH
BnO BnO
2. NaOMe, MeOH
4
3a, n = 3 3b, n = 7 3c, n = 12 3d, n = 15 3e, n = 13
OH O
CnH2n+1
N3 O
O
BnO
O
5a, n = 3, 75%, α/β = 1/3.1 5b, n = 7, 79%, α/β = 1/3.4 5c, n = 12, 66%, α/β = 1/2.2 5d, n = 15, 51%, α/β = 1/2.0
Scheme 1. Synthesis of varying chain length acceptors 5a-5d. We intended to design the analog with C5 hydroxyl group, which may enhance the interaction with the receptors to produce higher potency of analog. For this purpose, synthesis of 5-substituted ceramide acceptors 6a, 6b was carried out.24 Which were subsequently gave intermediate products 7a and 7b after coupling with donor 4 using TMSOTf as a promotor followed by removal of O6 acetyl group using sodium methoxide (Scheme 2).
N3
1. TMSOTf, 3A MS DCM/ACN =1/2 -30 oC to rt
OBn
HO BnO
4
2
R1 R C11H23
2. NaOMe, MeOH
6a, R1 = OBn, R2 = H, 6b, R1 = H, R2 = OBn
O 1. PPh3 , Pyr., THF, 60 oC 2. C17H35COOH, EDC, HOBt, NEt3, DCM
O BnO 1
OH O
N3
NH
OBn
O
The intermediates 7a and 7b were put under the Staudinger reaction condition to obtain the free amines which were taken for next step after trituration. Stearic acid was coupled with the above intermediates using EDC, HOBt reagents to obtain products 8a and 8b respectively. The E/Z isomers were not separated at this stage and taken forward together. The double bond in 8a and 8b were reduced under 1 atm hydrogen pressure using Pd/C as catalyst to get the compounds 9a and 9b in 82% and 81% respectively (Scheme 2). For the synthesis of 2’ ceramide substituted ganglioside analogs 2g and 2h, the building block 12 was synthesized by using the commercially available 2,3-Isopropylidene glyceraldehyde 10 and Wittig salt 11 in presence of n-BuLi. The E/Z isomers were taken forward without separation. The compound 12 was reduced to alkane using Pd/C, H2 balloon pressure followed by 2,3Isopropylidene deprotection in acidic condition to get the diol 13. We tried to oxidize the primary alcohol of 13 selectively25 but failed to get the selectivity. To overcome this difficulty, we masked the diol as benzylidene acetal 14 and subsequently opened the ring by using regioselective oxidative cleavage strategy26 to get the compound 15 (Scheme 3). O H O
O
n-BuLi BrPh3P(CH2)14CH3 11
BnO
C14H29 HO
OH 13
2 R1 R C11H23
O
EtOAc BnO R1 R2 C11H23
O BnO
C17H35 NH
O
OBn C13H27
2 1 BnO R R
2
8a, R = OBn, R = H, 62% 8b, R1 = H, R2 = OBn, 65%
12
PhCH(OMe)2, CSA DCM, ACN 85%
2. AcOH, EtOH, H2O, reflux 67% in 2 steps
C15H31 O
RuCl3 xH2O, NaIO4,
O
14
ACN, CCl4, H2O, rt, 57%
O HO
C15H31 OBz 15
To confirm the inevitability and/or effect of anomeric O-linkage between glucosyl part and ceramide, we designed the two molecules 2g, 2h by replacing anomeric O-linkage with S-linkage. To achieve this, the synthesis of S-linked acceptor 21 was carried out as shown in scheme 4. The 1-OH of phytosphingosine derivative 3e was substituted with Iodine to get compound 16. The donor 1727 was coupled with 16 in basic condition to obtain the compound 18. Attempts to hydrolyze O-6 acetyl group was failed. Hence subsequently we moved to stepwise deprotectionprotection approach. The peracetylated glucosyl 18 was hydrolyzed by sodium methoxide and O-6 hydroxyl group was protected with TBDPS group to get 19. For the benzyl protection of 2,3,4-hydroxyl groups in 19, we fine-tuned the reaction conditions using the sodium hydride as a base in DMF solvent to reach the compound 20. The required free primary alcohol 21 was obtained by unmasking the O-6 TBDPS group using TBAF in THF (Scheme 4).
OBn
OH BnO BnO
O
1. Pd/C, H2 balloon, EtOAc, 24 h
Scheme 3. Synthesis of fatty acid 15.
7a, 59% in 2 steps, α/β = 1/3.4 7b, 39% in 2 steps, α/β = 1/2.7
Pd/C, H2
C14H29
O THF, -30 o C 97%, E/Z=2.5/1
10
O
BnO
C17H35
OH BnO BnO
BnO BnO
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9a, R1 = OBn, R2 = H, 82% 9b, R1 = H, R2 = OBn, 81%
Scheme 2. Synthesis of acceptors 9a-9b.
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ACS Chemical Neuroscience Scheme 4. Synthesis of S-linked acceptor 21. (a) 1. NaOMe, MeOH (92%), 2. TBDPSCl, Im, DCM (84%), Two steps; (b) 1. NaOMe, MeOH 2. TBDPSCl, Im, DCM (42%, one-pot) After all appropriate acceptors 5a-5d, 9a-9b, and 21 in hand, we moved to link sialic acid derivatives 22, 23 to accomplish the precursors of desired analogs. The S-linked analogs were prepared by the route presented in Scheme 5. The sialic acid derivative donor 22, 23 were pre-activated28 with AgOTf, p-TolSCl reagents followed by addition of acceptor 21 at -78 oC to obtain the compounds 24, 25 in 74%, 52% yield respectively with α-selectivity. The 4,5-oxazolidinone of intermediates 24, 25 were acetylated using acetyl chloride to get the compounds 26, 27 in 90% and 86% yields respectively. The azido group of 26, 27 were reduced by Staudinger reaction followed by acids 15, 15a coupling using EDC, HOBt reagents to achieve the precursors 28, 29 of analogs 2g and 2h in 45%, 38% respectively over two steps (Scheme 5). RO
OAc
AgOTf, p-TolSCl, ACN/DCM (1/5), -78 oC, 15 min;
STol AcO HN
O
CO2Me
O
RO
O O
O
O 22, R= Ac, 23,R = Me
RO
CO2Me O O O
O
BnO BnO
OAc O O O
O
OBn
O
HN
O
BnO BnO
O
O
2. EDC, HOBt, DCM C15H31
O
O HO R2
R3
15, R2= OH, R3= H, 15a, R2= R3= H
O
CO2Me
C13H27
S
OBn
C15H31 R
S
OBn O 28, R= Ac, R2= R3= H, 45%, 2 3 29,R = Me, R = OH, R = H, 38%
2
O
STol
O O
32a-32b
30
AcO
1. PPh3, THF, H2O, 50 oC
OAc CO2 Me
AcO AcN
2. 15a, EDC, HOBt, DCM
AcO
31a-32d
DCM -40 o C
9a-9b
O
BnO BnO
O O
O
O
HN
O
C17H35 CnH2n+1
O
O
BnO
33a, n = 3, 39% 33b, n = 7, 54% 33c, n = 12, 54% 33d, n = 15, 51%
O
OAc CO2Me
AcO AcN
O O
O
AcO
O O
BnO BnO
31a, n = 3, 76%, αβ/ββ = 3.7/1 31b, n = 7, 82%, αβ/ββ = 4.2/1 31c, n = 12, 71%, αβ/ββ = 3.3/1 31d, n = 15, 73%, αβ/ββ = 4.1/1
CnH2n+1
N3 O
O
BnO
O
OAc CO2Me
AcO AcN
O
O
O BnO BnO
O BnO
C17H35 NH
O
32a, R1 = OBn, R2 = H, 91%, αβ/ββ = 11.1/1 32b, R1 = H, R2 = OBn, 77%, αβ/ββ = 11.1/1
OBn
C13 H27 1 R2 BnO R
C13H27
S
1. PPh3, THF, H2O N3
O
26, R= Ac, 90% 27,R = Me, 86%
AcO AcN
O
OAc
AcO AcN
DCM, 0 oC
RO
N3
O
BnO BnO
24, R= Ac, 74%, α only 25,R = Me, 52%, α only
AcCl, DIPEA
AcO AcN
NIS, TfOH, 4 ÅMS
5a-5d
CO2Me
O
CO2Me
acceptor 21
OAc
O
OAc
AcO HN
AcO
R3 C13H27 O
Scheme 5. Pre-activation based approach to assemble of S-linked, fully protected precursors 28, 29 of analog 2g and 2h. The N-acetyl-5-N,4-O-carbonyl-protected p-tolyl thiosialoside donor 3029 was glycosylated with acceptors 5a-d and 9a-b by using NIS, TfOH reagents to get the compounds 31a-d and 32a-b successively (Scheme 6). The glycosylated products 31a-d were reduced to amine by using Staudinger reagents and subsequently coupled with acid 15a using EDC, HOBt reagents to access the fully protected precursors 33a-33d. Subsequently, all the protected analogs 33a-33d, 32a-b, 28, and 29 were purified with column chromatography to get the desired protected analogs.
Scheme 6. Synthesis of fully protected phytosphingosine modified analogues. The β isomers collected through column chromatography, which were major in ratio are taken forward for deprotection reactions. After getting required precursors 33a-d, 32a-b, 28, and 29 with sufficient amount in hand, we started the deprotection using one pot strategy as demonstrated in Scheme 7. The Acetyl, benzoyl, ester, and oxazolidinone groups were dislodged by using 1N NaOH in MeOH solvent at 50 oC. The reaction solvent was removed and MeOH/DCM, Pd(OH)2, and AcOH were added followed by H2 pressure in still bomb hydrogenator and subsequently purified by column chromatography using MeOH, CHCl3, and few drops of H2O as mobile phase to access the final analogs 2a2h as shown in scheme 7. 33a, 33b, 33c, 33d, 32a, 32b, 28, 29
1. 1N NaOH, MeOH, 50 oC 2. Pd(OH)2, 60 psi H2, AcOH, MeOH/DCM (1/3), 5 h
2a, 60% 2b, 45% 2c, 73% 2d, 56% 2e, 69% 2f, 82% 2g, 33% 2h, 48%
Scheme 7. Final one pot unmasking to achieve analogs 2a-2h.
Biological activity of Hp-s1 analogues It is demonstrated that stimulation of gangliosides increased the neuritogenic activity including neurite-bearing cells and branch point count in a human neuroblastoma cells SH-SY5Y.25 To further evaluate the neuritogenic activities of ganglioside Hp-s1 analogues, neurite-bearing cells and branch point count were measured in SH-SY5Y cells treated with 10 µM 1 and its analogues 2a-2f for 72 h. Analogues 2a, 2b, 2c and 2f significantly increased neurite-bearing cells in SH-SY5Y cells while their effects on branch-point count tended to up-regulation (Fig. 3A and Fig. 3B). Higher effects of 1 were observed in both neurite-bearing cells and branch-point count than other analogues except 2b. A selected cytokines antibody-array analysis on the 1-stimulated SH-SY5Y cells was performed, which revealed an inductive effect of 1 on inflammatory cytokine IL-17A secreted protein (Fig. 2-B left panel). IL-17A promotes neuronal differentiation in cultured neural precursor cells (ref). A neuroprotective role of IL17A during acute neuroinflammation is suggested (ref). Nevertheless, the death effect of IL-17 stimulation in SH-SY5Y cells is reported (ref). The significantly higher activity of analogue 2c
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than 1 on IL-17A transcripts was measured by real time RT-PCR analysis (Fig. 2-B right panel). The results implicated that IL-17A is involved in the neuritogenic effect of Hp-s1 and its analogues in SH-SY5Y cells, suggested the possibility of ganglioside Hp-s1 and its analogues in medical applications after further exploration.
logs which will help to compare the synthetic methods for further research. These results validate the rationality of gangliosides Hps1 based molecules for further discovery. These results may support for the development of the ganglioside based neurotherapeutic drugs.
Experimental Section Neurite outgrowth and IL-17A mRNA measurement: Human neuroblastoma SH-SY5Y cells (ATCC CRL-2266) were obtained from American type cell culture (ATCC, Manassas, VA) and maintained in DMEM:Ham's F12 (1:1) medium supplemented with 10% fetal bovine serum at 37 °C in 5% CO2. For analysis of neurite outgrowth, cells (1×104) were seeded into each well of sixwell plate and then incubated with indicated compounds (10 µM) for 72 hours. Neurite-bearing cells and neurite branch point were counted as described.17 Triplicate wells for each tested compound and three random fields in each well were measured (40 cells/field). One-way analysis of variance (ANOVA) and Tukey’s multiple comparison post hoc test was used for statistical analysis. For determination of Hp-s1 effects on cytokines, the cultured medium from Hp-s1(5µM; 48h)-treated SH-SY5Y cells was analyzed by a Bio-Plex Pro™ Human cytokine 27-plex assay kit (Bio-Rad; Hercules, CA). For determination of Hp-s1 effects on IL-17A transcripts, the RNAs and cDNA templates for RT-qPCR analysis were prepared as previously described.17 The qPCR analysis with IL-17A specific primer pairs was performed using Power SYBR® Green PCR Master Mix (Life Technologies, Carlsbad, CA, USA). IL-17A primer sequences were: forward: 5’GTCAACCTGAACATCCATAACCG3’; reverse: 5’ACTTTGCCTCCCAGATCACAG3’.
ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website in PDF format. Author Contributions S.Y.L. and Y. C. K. designed the research. G. B. S., Y. H. L., Y. J. L. and C. W. L. prepared the compounds 2a−2h. T. M. K. analyzed the data. G. B. S. and T. M. K. wrote the manuscript.
Corresponding Author E-Mail:
[email protected] Present Addresses Department of Chemistry, National Chung Hsing University, Taichung 402, Taiwan
Author Contributions Notes The authors declare no competing financial interests.
ACKNOWLEDGMENT The authors thank the Ministry of Science and Technology (MOST) in Taiwan (MOST 105-2113-M-005-007 and 1062113-M-005-007) and National Chung Hsing University for financial support.
REFERENCES
Figure 2. Effects of ganglioside Hp-s1 and its analogues on neurite outgrowth and levels of IL-17A mRNAin SH-SY5Y cells. SH-SY5Y cells were treated with control DMSO or 10 µM ganglioside Hp-s1 and its analogues for 72 h. The percentage of (A) neurite-bearing cells (upper panel) and branch point count (lower panel) were rated in each compound-treated cell. (B) The expressions of secreted IL-17A proteins were determined by cytokine assay with specific antibodies (left panel). The inductive effect of each compound on IL-17A mRNA was measured by real time RT-qPCR (right panel). (∗) p
