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I . J. Dmochowski et al. Oligonucleotide modifi cations enhance probe stability for single cell transcriptome in vivo analysis (TIVA)
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Synthesis and Immunomodulatory Activity of the Sulfated Tetrasaccharide Motif of Type B Ulvanobiuronic Acid 3-Sulfate† Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
Liangliang Zhang, Xiaotong Wang, Qingting Hua, Junchang Wang, Jianwen Liu*, and You Yang* Ulvan is a sulfated polysaccharide from green algae with potent antitumor, antiviral, and immunomodulatory activities. However, no chemical synthesis of ulvan saccharides has been reported to date. In this paper, we completed the first efficient synthesis of the unique sulfated tetrasaccharide motif of type B ulvanobiuronic acid 3-sulfate. Based on the gold(I)-catalyzed glycosylation with glycosyl ynenoates as donors, efficient construction of the challenging -(1→4)glycosidic bonds between the iduronic acid and the rhamnose building blocks was achieved to afford the tetrasaccharide skeleton in a stereospecific manner. The synthetic sulfated tetrasaccharide was found to significantly improve the phagocytic activity of macrophage RAW264.7 cells.
Introduction Ulvan is a water-soluble sulfated polysaccharide that exists in the 1 cell wall of marine green algae from Ulva and Enteromorpha. Among the structures isolated from ulvan, the two major repeating units are type A ulvanobiuronic acid 3-sulfate (A3S) [→4)--D-GlcA(1→4)--L-Rha3S-(1→] and type B ulvanobiuronic acid 3-sulfate (B3S) 1,2 [→4)--L-IdoA-(1→4)--L-Rha3S-(1→]. Although these sulfated ulvan polysaccharides remain largely underexploited, they have been found to possess strong antitumor, antiviral, antihyperlipidemic, anticoagulant, and immunomodulatory 1,3-5 activities. Considering the numerous potential application of ulvan in pharmaceutical field, well-defined and pure ulvan saccharides are highly required as molecular probes to explore their structure-activity relationships for the development of new carbohydrate-based drug candidates. Structurally, B3S consists of an unusual acidic disaccharide repeating unit which is connected by the -(1→4)-glycosidic bonds between the negatively charged 3-sulfated rhamnose residues and 2 the negatively charged iduronic acid residues. It is noteworthy that the iduronic acid residues and the sulfate groups in B3S, which are usually present in mammalian glycosaminoglycans such as heparin, heparin sulfates and dermatan sulfates, may have similar biological 6 functions to those of glycosaminoglycans. Although ulvan saccharides, especially B3S, have intriguing structural features and promising biological activities, chemical synthesis of ulvan 7 saccharides has never been reported. Recently, we developed a simple and versatile gold(I)-catalyzed glycosylation method with glycosyl ynenoates as donors for the effective synthesis of Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. E-mail: [email protected] (J. Liu); [email protected] (Y. Yang). † Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
Figure 1. The structure of the sulfated tetrasaccharide motif 1 of type B ulvanobiuronic acid 3-sulfate. 8
glycosides and polysaccharides. Here, we describe the first efficient synthesis of the unique sulfated tetrasaccharide 1 of B3S (Figure 1), where the challenging -(1→4)-glycosidic bonds between the iduronic acid residues and the rhamnose residues was efficiently constructed by the gold(I)-catalyzed glycosylation of glycosyl ynenoates in a stereospecific manner. Preliminary immunomodulatory evaluation showed that the target tetrasaccharide 1 from B3S significantly enhanced the phagocytic effect of macrophage RAW264.7 cells.
Results and discussion For the synthesis of the 3-sulfated rhamnose moieties in tetrasaccharide 1, we planned to install an orthogonally-protected p-methoxybenzyl (PMB) group at the 3-hydroxyl group of the rhamnose building block for the late-stage sulfation on the tetrasaccharide skeleton. Thus, regioselective protection of the 2,39 diol of the known odourless thiorhamnoside 2 with an isopropylidene acetal group using 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid in DMF afforded alcohol 3 in 97% yield (Scheme 1). Levulinoylation of alcohol 3 with the aid of DIC gave rhamnoside 4 in almost quantitative yield, which was treated
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Scheme 1. Preparation of the rhamnoside building block 8. with 80% aqueous acetic acid at 70 °C to produce diol 5 in an excellent 98% yield. The (Bu3Sn)2O-mediated regioselective protection of the 3-hydroxyl group of diol 5 with PMBCl in the presence of TBAI in toluene at 110 °C afforded alcohol 6 in 81% 10 yield. Treatment of 6 with benzoyl chloride and DMAP delivered compound 7 in 84% yield, after which the levulinyl (Lev) group was removed by NH2NH2‧HOAc in a mixture of pyridine and acetic acid gave rhamnoside acceptor 8 in 90% yield. Next, we turned to the gold(I)-catalyzed glycosylation of glycosyl 8 ynenoates for the stereoselective construction of the -(1→4)glycosidic bonds between the iduronic acid residues and the 11 rhamnose residues. Treatment of the iduronic acid thioglycoside 9 12 with (Z)-3-iodoacrylic acid under the activation of NIS and TfOH followed by Sonogashira coupling with 1-hexyne afforded -linked glycosyl ynenoate 10 as the single anomer in 77% yield over two steps (Scheme 2). Glycosylation of ynenoate 10 with rhamnoside acceptor 8 under the catalysis of Ph3PAuOTf (0.1 eq.) and TfOH (0.1 eq.) in dichloromethane at room temperature proceeded smoothly to provide exclusively the -(1→4)-linked disaccharide 11 in an excellent 91% yield. On one hand, the TBS group in 11 was removed with HF‧pyridine in THF to give disaccharide acceptor 12 (93%). On the other hand, compound 11 was converted into disaccharide ynenoate 13 via two steps similar to those for 9→10 (78% over two steps). The anomeric configuration of 13 was assigned by the coupling constants between C-1 and H-1 of the corresponding 1 iduronic acid and rhamnose residues ( JC1,H1 = 175.2 and 175.2 Hz, 13 respectively). The disaccharide-disaccharide coupling of ynenoate 13 with poorly nucleophilic uronate acceptor 12 under the activation of Ph3PAuOTf (0.3 eq.) and TfOH (0.1 eq.) in toluene at room temperature furnished the -(1→4)-linked tetrasaccharide 14 as the only anomer in 89% yield. It is worthy to note that almost no tetrasaccharide 14 was obtained when the above coupling was carried out in dichloromethane instead of toluene, indicating that the choice of toluene as solvent was very critical for this type of 14 gold(I)-catalyzed glycosylation reaction. However, the reason for the solvent effect of toluene remained unclear. To install an aminopentyl linker at the reducing end of tetrasaccharide skeleton, glycosylation of thioglycoside 14 with N15 benzyl-N-benzyloxycarbonyl pentyl linker 15 under the promotion of NIS and TfOH afforded -linked tetrasaccharide 16 in 85% yield (Scheme 3). Considering the difficult purification of highly polar sulfated product, we first replaced the TBS group in 16 with acetyl
Scheme 2. Synthesis of the tetrasaccharide skeleton 14. group to give compound 17 in 81% yield over two steps via removal of the TBS group with HF‧pyridine and subsequent acetylation with acetic anhydride. The anomeric configuration of tetrasaccharide 17 was assigned by the coupling constants between C-1 and H-1 of the 1 corresponding iduronic acid and rhamnose residues ( JC1,H1 = 171.6, 13 174.0, 175.2 and 172.2 Hz, respectively). Selective removal of the PMB group in 17 with DDQ in phosphate buffer (pH = 7.2) and dichloromethane afforded diol 18 in 95% yield. Sulfation of diol 18 with SO3‧pyridine in DMF, SO3‧pyridine in pyridine, or SO3‧NEt3 in DMF at room temperature or 55 °C led to no reaction or partial 16 sulfated products. To our delight, treatment of 18 with excess amount of SO3‧NEt3 (60 eq.) in pyridine at 55 °C provided fully sulfated tetrasaccharide 19 in almost quantitative yield. Finally, saponification of the ester groups in 19 using 1 M aqueous LiOH and
Scheme 3. Synthesis of the sulfated tetrasaccharide 1 of B3S.
2 | J. Name., 2013, 00, 1-3
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The authors Y.Y., J.L., L.Z., X.W. through East ChinaView University of Article Online Science and Technology have filed DOI: a patent application 10.1039/D0OB01852J 202010908994.2 on sulfated oligosaccharides containing the sulfated tetrasaccharide motif of type B ulvanobiuronic acid 3sulfate as immunomodulatory agents.
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Acknowledgements Figure 2. Proliferative activity (A) and phagocytic effect (B) of the RAW264.7 cells after stimulation with different concentrations of the sulfated tetrasaccharide 1. Each value is expressed by the mean ± standard deviation (SD) of three independent experiments. Significant differences with control group (RAW264.7 cells without treatment of 1) were labeled as *P
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Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: L. Zhang, X. Wang, Q. Hua, J. Wang, J. Liu and Y. Yang, Org. Biomol. Chem., 2020, DOI: 10.1039/D0OB01852J.
Organic & Biomolecular Chemistry rsc.li/obc
Volume 15 Number 47 21 December 2017 Pages 9945-10124
This is an Accepted Manuscript, which has been through the Royal Society of Chemistry peer review process and has been accepted for publication. Accepted Manuscripts are published online shortly after acceptance, before technical editing, formatting and proof reading. Using this free service, authors can make their results available to the community, in citable form, before we publish the edited article. We will replace this Accepted Manuscript with the edited and formatted Advance Article as soon as it is available. You can find more information about Accepted Manuscripts in the Information for Authors.
ISSN 1477-0520
PAPER
I . J. Dmochowski et al. Oligonucleotide modifi cations enhance probe stability for single cell transcriptome in vivo analysis (TIVA)
Please note that technical editing may introduce minor changes to the text and/or graphics, which may alter content. The journal’s standard Terms & Conditions and the Ethical guidelines still apply. In no event shall the Royal Society of Chemistry be held responsible for any errors or omissions in this Accepted Manuscript or any consequences arising from the use of any information it contains.
rsc.li/obc
Please&do not adjust margins Organic Biomolecular Chemistry
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Synthesis and Immunomodulatory Activity of the Sulfated Tetrasaccharide Motif of Type B Ulvanobiuronic Acid 3-Sulfate† Received 00th January 20xx, Accepted 00th January 20xx DOI: 10.1039/x0xx00000x www.rsc.org/
Liangliang Zhang, Xiaotong Wang, Qingting Hua, Junchang Wang, Jianwen Liu*, and You Yang* Ulvan is a sulfated polysaccharide from green algae with potent antitumor, antiviral, and immunomodulatory activities. However, no chemical synthesis of ulvan saccharides has been reported to date. In this paper, we completed the first efficient synthesis of the unique sulfated tetrasaccharide motif of type B ulvanobiuronic acid 3-sulfate. Based on the gold(I)-catalyzed glycosylation with glycosyl ynenoates as donors, efficient construction of the challenging -(1→4)glycosidic bonds between the iduronic acid and the rhamnose building blocks was achieved to afford the tetrasaccharide skeleton in a stereospecific manner. The synthetic sulfated tetrasaccharide was found to significantly improve the phagocytic activity of macrophage RAW264.7 cells.
Introduction Ulvan is a water-soluble sulfated polysaccharide that exists in the 1 cell wall of marine green algae from Ulva and Enteromorpha. Among the structures isolated from ulvan, the two major repeating units are type A ulvanobiuronic acid 3-sulfate (A3S) [→4)--D-GlcA(1→4)--L-Rha3S-(1→] and type B ulvanobiuronic acid 3-sulfate (B3S) 1,2 [→4)--L-IdoA-(1→4)--L-Rha3S-(1→]. Although these sulfated ulvan polysaccharides remain largely underexploited, they have been found to possess strong antitumor, antiviral, antihyperlipidemic, anticoagulant, and immunomodulatory 1,3-5 activities. Considering the numerous potential application of ulvan in pharmaceutical field, well-defined and pure ulvan saccharides are highly required as molecular probes to explore their structure-activity relationships for the development of new carbohydrate-based drug candidates. Structurally, B3S consists of an unusual acidic disaccharide repeating unit which is connected by the -(1→4)-glycosidic bonds between the negatively charged 3-sulfated rhamnose residues and 2 the negatively charged iduronic acid residues. It is noteworthy that the iduronic acid residues and the sulfate groups in B3S, which are usually present in mammalian glycosaminoglycans such as heparin, heparin sulfates and dermatan sulfates, may have similar biological 6 functions to those of glycosaminoglycans. Although ulvan saccharides, especially B3S, have intriguing structural features and promising biological activities, chemical synthesis of ulvan 7 saccharides has never been reported. Recently, we developed a simple and versatile gold(I)-catalyzed glycosylation method with glycosyl ynenoates as donors for the effective synthesis of Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, China. E-mail: [email protected] (J. Liu); [email protected] (Y. Yang). † Electronic Supplementary Information (ESI) available. See DOI: 10.1039/x0xx00000x
Figure 1. The structure of the sulfated tetrasaccharide motif 1 of type B ulvanobiuronic acid 3-sulfate. 8
glycosides and polysaccharides. Here, we describe the first efficient synthesis of the unique sulfated tetrasaccharide 1 of B3S (Figure 1), where the challenging -(1→4)-glycosidic bonds between the iduronic acid residues and the rhamnose residues was efficiently constructed by the gold(I)-catalyzed glycosylation of glycosyl ynenoates in a stereospecific manner. Preliminary immunomodulatory evaluation showed that the target tetrasaccharide 1 from B3S significantly enhanced the phagocytic effect of macrophage RAW264.7 cells.
Results and discussion For the synthesis of the 3-sulfated rhamnose moieties in tetrasaccharide 1, we planned to install an orthogonally-protected p-methoxybenzyl (PMB) group at the 3-hydroxyl group of the rhamnose building block for the late-stage sulfation on the tetrasaccharide skeleton. Thus, regioselective protection of the 2,39 diol of the known odourless thiorhamnoside 2 with an isopropylidene acetal group using 2,2-dimethoxypropane in the presence of p-toluenesulfonic acid in DMF afforded alcohol 3 in 97% yield (Scheme 1). Levulinoylation of alcohol 3 with the aid of DIC gave rhamnoside 4 in almost quantitative yield, which was treated
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
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Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/D0OB01852J
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Published on 24 September 2020. Downloaded by UNSW Library on 9/27/2020 12:42:31 PM.
Scheme 1. Preparation of the rhamnoside building block 8. with 80% aqueous acetic acid at 70 °C to produce diol 5 in an excellent 98% yield. The (Bu3Sn)2O-mediated regioselective protection of the 3-hydroxyl group of diol 5 with PMBCl in the presence of TBAI in toluene at 110 °C afforded alcohol 6 in 81% 10 yield. Treatment of 6 with benzoyl chloride and DMAP delivered compound 7 in 84% yield, after which the levulinyl (Lev) group was removed by NH2NH2‧HOAc in a mixture of pyridine and acetic acid gave rhamnoside acceptor 8 in 90% yield. Next, we turned to the gold(I)-catalyzed glycosylation of glycosyl 8 ynenoates for the stereoselective construction of the -(1→4)glycosidic bonds between the iduronic acid residues and the 11 rhamnose residues. Treatment of the iduronic acid thioglycoside 9 12 with (Z)-3-iodoacrylic acid under the activation of NIS and TfOH followed by Sonogashira coupling with 1-hexyne afforded -linked glycosyl ynenoate 10 as the single anomer in 77% yield over two steps (Scheme 2). Glycosylation of ynenoate 10 with rhamnoside acceptor 8 under the catalysis of Ph3PAuOTf (0.1 eq.) and TfOH (0.1 eq.) in dichloromethane at room temperature proceeded smoothly to provide exclusively the -(1→4)-linked disaccharide 11 in an excellent 91% yield. On one hand, the TBS group in 11 was removed with HF‧pyridine in THF to give disaccharide acceptor 12 (93%). On the other hand, compound 11 was converted into disaccharide ynenoate 13 via two steps similar to those for 9→10 (78% over two steps). The anomeric configuration of 13 was assigned by the coupling constants between C-1 and H-1 of the corresponding 1 iduronic acid and rhamnose residues ( JC1,H1 = 175.2 and 175.2 Hz, 13 respectively). The disaccharide-disaccharide coupling of ynenoate 13 with poorly nucleophilic uronate acceptor 12 under the activation of Ph3PAuOTf (0.3 eq.) and TfOH (0.1 eq.) in toluene at room temperature furnished the -(1→4)-linked tetrasaccharide 14 as the only anomer in 89% yield. It is worthy to note that almost no tetrasaccharide 14 was obtained when the above coupling was carried out in dichloromethane instead of toluene, indicating that the choice of toluene as solvent was very critical for this type of 14 gold(I)-catalyzed glycosylation reaction. However, the reason for the solvent effect of toluene remained unclear. To install an aminopentyl linker at the reducing end of tetrasaccharide skeleton, glycosylation of thioglycoside 14 with N15 benzyl-N-benzyloxycarbonyl pentyl linker 15 under the promotion of NIS and TfOH afforded -linked tetrasaccharide 16 in 85% yield (Scheme 3). Considering the difficult purification of highly polar sulfated product, we first replaced the TBS group in 16 with acetyl
Scheme 2. Synthesis of the tetrasaccharide skeleton 14. group to give compound 17 in 81% yield over two steps via removal of the TBS group with HF‧pyridine and subsequent acetylation with acetic anhydride. The anomeric configuration of tetrasaccharide 17 was assigned by the coupling constants between C-1 and H-1 of the 1 corresponding iduronic acid and rhamnose residues ( JC1,H1 = 171.6, 13 174.0, 175.2 and 172.2 Hz, respectively). Selective removal of the PMB group in 17 with DDQ in phosphate buffer (pH = 7.2) and dichloromethane afforded diol 18 in 95% yield. Sulfation of diol 18 with SO3‧pyridine in DMF, SO3‧pyridine in pyridine, or SO3‧NEt3 in DMF at room temperature or 55 °C led to no reaction or partial 16 sulfated products. To our delight, treatment of 18 with excess amount of SO3‧NEt3 (60 eq.) in pyridine at 55 °C provided fully sulfated tetrasaccharide 19 in almost quantitative yield. Finally, saponification of the ester groups in 19 using 1 M aqueous LiOH and
Scheme 3. Synthesis of the sulfated tetrasaccharide 1 of B3S.
2 | J. Name., 2013, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins
Organic & Biomolecular Chemistry Accepted Manuscript
DOI: 10.1039/D0OB01852J
Please&do not adjust margins Organic Biomolecular Chemistry COMMUNICATION
Journal Name
The authors Y.Y., J.L., L.Z., X.W. through East ChinaView University of Article Online Science and Technology have filed DOI: a patent application 10.1039/D0OB01852J 202010908994.2 on sulfated oligosaccharides containing the sulfated tetrasaccharide motif of type B ulvanobiuronic acid 3sulfate as immunomodulatory agents.
Published on 24 September 2020. Downloaded by UNSW Library on 9/27/2020 12:42:31 PM.
Acknowledgements Figure 2. Proliferative activity (A) and phagocytic effect (B) of the RAW264.7 cells after stimulation with different concentrations of the sulfated tetrasaccharide 1. Each value is expressed by the mean ± standard deviation (SD) of three independent experiments. Significant differences with control group (RAW264.7 cells without treatment of 1) were labeled as *P
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