Abstract
Fucoidan, a sulfated glycan derived from brown algae, has garnered significant attention for its anticoagulant properties. However, the structural complexity and heterogeneity of naturally extracted fucoidan have hindered a comprehensive understanding of its structure-activity relationship, limiting the development of fucoidan-based anticoagulant drugs. To address this challenge, we synthesize a diverse library of 58 distinct fucoidans with multiple contiguous 1,2-cis glycosidic bonds, ranging from disaccharides to dodecasaccharides, using a highly efficient preactivation-based one-pot glycosylation strategy. This library includes compounds with various sulfation patterns (2,3-O-di-, 3,4-O-di-, and 2,3,4-O-tri-sulfation) encompassing nearly all possible fucoidan structures. In vitro anticoagulant assays demonstrate that both molecular size and degree of sulfation play crucial roles in anticoagulant potency. Notably, compounds 29, 30, 37, and 58 significantly prolong human plasma activated partial thromboplastin time (APTT), comparable to the effect of enoxaparin, without affecting prothrombin time (PT) or thrombin time (TT). This selective inhibition of the intrinsic coagulation pathway suggests a reduced risk of bleeding, highlighting the therapeutic potential of these fucoidans as safer anticoagulant agents.
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Introduction
Thrombosis, the formation of blood clots within blood vessels, is the main underlying pathology of cardiovascular diseases, which remain the leading global cause of mortality1. Anticoagulants are widely used in both medical and surgical settings to manage thrombotic events2,3. Beyond the classical drugs such as heparin and warfarin, a range of heparin derivatives (e.g., enoxaparin and fondaparinux) and direct oral anticoagulants (e.g., dabigatran, rivaroxaban, apixaban, and edoxaban) have been developed. However, all the currently available anticoagulants target coagulation factor Xa (FXa) and/or thrombin (FIIa), which are located in the common pathway, downstream of the coagulation cascade, and are essential to hemostasis function. Despite their efficacy and widespread use, these anticoagulants are associated with a high risk of bleeding4. This prompts researchers to search for safer alternatives. Particularly, emerging evidence underscores the critical role of the intrinsic coagulation pathway in coagulation propagation and thrombosis, while having a limited contribution to hemostasis. Therefore, targeting intrinsic coagulation factors is recognized as a promising strategy for achieving safer and more effective antithrombotic therapies.
Fucoidan, a class of fucose-rich sulfated glycans, are predominantly found in the extracellular matrix of brown algae5. These glycans exhibit a wide range of biological activities, including anticoagulant5, antitumor6, anti-inflammatory7, and antiviral effects8, with anticoagulant activity being one of the most extensively studied9,10,11,12. However, the efficacy of fucoidan in anticoagulants varies significantly across different brown algae species, owing to their highly species-specific structures13. Currently, most studies on the anticoagulant properties of fucoidan have focused on naturally extracted compounds, which are structurally complex and heterogeneous14. This heterogeneity has limited the ability to fully understand the structure-activity relationship (SAR) of fucoidan, as access to structurally well-defined fucoidan is challenging. To address this gap and advance the emerging field of fucoidan-based therapeutics, it is crucial to develop a comprehensive collection of structurally well-defined fucoidans with diverse linkages, molecular sizes, and sulfation patterns. Such a library would enable systematic investigation into how structural variations influence biological activities15,16,17,18,19. Over the past two decades, several glycan libraries have been established to explore their functions20,21,22,23,24,25. Furthermore, strategies have been employed to synthesize fucoidan15,17,26,27, and recent advances in the synthesis of complex polysaccharides have greatly improved our ability to accomplish glycan synthesis28,29,30,31,32,33,34. Nevertheless, synthesizing fucoidan with multiple contiguous 1,2-cis glycosidic bonds35, coupled with selective O-sulfation modifications23, has continued to pose a considerable challenge.
Herein, we report the development of a fucoidan library utilizing a preactivation-based one-pot glycosylation strategy36. This strategy streamlines synthesis by eliminating the need for intricate leaving group design and laborious intermediate purifications, thus significantly enhancing synthetic efficiency36,37,38,39. Through the application of selective O-sulfation modifications to the 22 synthesized glycans, ranging from disaccharides to dodecasaccharides, we have constructed a comprehensive library of 58 sulfated fucoidans. This collection encompasses nearly all conceivable fucoidan structures from disaccharides to dodecasaccharides. The comprehensive nature of this library facilitates a systematic investigation of how anticoagulant activity correlates with glycan sizes, linkage types, and sulfation patterns. Notably, unlike enoxaparin, most compounds in the library significantly prolonged activated partial thromboplastin time (APTT) without altering prothrombin time (PT) or thrombin time (TT). These findings indicate that fucoidans selectively inhibit the intrinsic coagulation pathway while sparing the extrinsic and common pathways, potentially reducing the risk of bleeding40. As a result, these fucoidans hold the potential as novel anticoagulant agents.
Results
Chemical structures of fucoidans and the general synthetic strategy
The chemical structures of fucoidans vary depending on algal species, cultivation conditions, and extraction methods. Natural fucoidans can be structurally categorized into two primary types (Type A and Type B, Supplementary Fig. 1). Type A, predominantly isolated from Cladosiphon okamuranus and Chorda filum, features backbones composed of repeating α-(1,3)-linked L-fucopyranosyl residues. In contrast, Type B, primarily derived from Ascophyllum nodosum and Fucus vesiculosus, consists of backbones with alternating α-(1,3)-linked and α-(1,4)-linked L-fucopyranosyl residues. Although methods such as chemical and enzymatic hydrolysis have been employed to extract fucoidan fragments from natural sources41, obtaining pure and structurally well-defined fucoidans remains a difficult task. While some fucoidan oligosaccharides have been synthesized in recent decades15,17,26,27, their number and diversity are insufficient to establish a comprehensive structure-activity relationship. To address this issue, we designed a comprehensive fucoidan library comprising 58 compounds, ranging from disaccharides to dodecasaccharides (Fig. 1a). The fucoidans can be classified into two types: Type A (1-30), with repeating α-(1,3)-linked L-fucopyranosyl residues, and Type B (31-58), with alternating α-(1,3)-linked and α-(1,4)-linked L-fucopyranosyl residues. Importantly, each type of fucoidans undergoes the late-stage selective O-sulfation. Specifically, both Type A and Type B compounds are modified with three sulfation patterns: 2,3-O-di-, 3,4-O-di-, and 2,3,4-O-tri-sulfation. Consequently, the library encompasses most sulfation patterns, spanning from disaccharides to dodecasaccharides.
a Structures of the diverse fucoidan library containing 58 compounds. b General synthetic strategy and late-stage sulfate modification steps. Tol, p-methylphenyl; PG, protecting groups; Bz, benzoyl; Bn, benzyl; TBS, tert-butyldimethylsilyl; PMB, p-methoxybenzyl.
To facilitate library synthesis (Fig. 1b), we designed several monosaccharide building blocks with a p-methylphenylthio (STol) anomeric leaving group, known for its stability and high reactivity in glycosylation42,43,44. Since all glycosidic linkages constructed in the library are in the 1,2-cis configuration, the benzoyl (Bz) group was installed at the 4-O position of monosaccharide building blocks to control the anomeric α-stereoselectivity during glycosylation through long-range participation45. Super-armed temporary protecting groups, including benzyl (Bn), tert-butyldimethylsilyl (TBS), and p-methoxybenzyl (PMB), were installed at the 2-O and 3-O positions to enhance the monosaccharide reactivity and improve the overall glycosylation yield46. Furthermore, these orthogonal temporary protecting groups enable the selective removal to expose the desired hydroxyl position for O-sulfation.
To efficiently construct the diverse fucoidans, we convergently synthesized two types (Type A and Type B) of glycan backbones (22 protected glycans) from the monosaccharide and oligosaccharide building blocks using the preactivation-based one-pot glycosylation protocol, which maximizes glycan assembly efficiency and reduces the need for tedious intermediate purification steps. The most challenging aspects are the selective deprotection and late-stage sulfation modifications (260 steps for 58 compounds), which involve labor-intensive, multi-step purification of highly polar, charged compounds using silica gel and size-exclusion chromatography. After optimizing sulfation conditions and removing protecting groups, 58 sulfated glycans can be synthesized from the 22 protected glycans by adjusting the order of protecting group removal and sulfation modifications.
Synthesis of monosaccharide and oligosaccharide building blocks
Commercially available L-fucose was selected as the starting material to synthesize all the monosaccharide building blocks (Fig. 2a). Building block 59 was generated through six-step functional group manipulations including successive acetylation, installation of the STol leaving group, deacetylation, selective protection of 3-O and 4-O with trimethyl orthobenzoate, installation of the Bn group on 2-O, and selective partial deprotection of the orthoester. The Bz group at the 4-O position of 59 serves both as a temporary protecting group and a stereoselective control element for its long-range participation effect. To obtain the highly reactive glycosyl donor 60, we installed a TBS group at the 3-O position, as the TBS group can improve the reactivity of the glycosyl donor in the glycosylation process, and its protection and deprotection can be achieved in high yields46. To prevent palladium catalyst poisoning during benzyl group deprotection and the anomeric hemiacetal hydroxyl group isomerization of the final product, we converted the STol group of 60 into an OMe group, followed by removal of the TBS group to generate the monosaccharide acceptor 61. For the synthesis of alternating α-(1,3)- and α-(1,4)-linked (Type B) compounds, we employed the PMB group to temporarily protect the hydroxyl group at the 3-O position. To achieve this, we first deprotected the 4-O Bz group of 59, and then selectively installed a PMB group at the 3-O position using dibutyltin oxide, followed by treatment with PMBCl to obtain the monosaccharide 62. The same protocol was applied to synthesize the monosaccharide 63 from 61
a Synthesis of monosaccharides 59-63. b Synthesis of disaccharides 64, 65, and 67. c Synthesis of disaccharide acceptors 69 and 71. d Synthesis of tetrasaccharides 72 and 73. e Synthesis of tetrasaccharides 74, 75, and 77. Reagents and conditions: (1) (a) Ac2O, HClO4, 0 °C, 1 h; (b) p-TolSH, BF3-OEt2, DCM, 0 °C to rt; (c) NaOCH3, CH3OH, rt, 1 h; (d) PhC(OCH3)3, CSA, MeCN, rt, 3 h; (e) BnBr, NaH, DMF, 0 °C, 3 h; (f) 1 N HCl, rt, 1 h; (2) TBSCl, imidazole, DMF, 70 °C, 4 h; (3) (a) CH3OH, 4 Å MS, p-TolSCl, AgOTf, TTBP, DCM,− 78 °C, 1 h; (b) HF-pyridine, THF, 50 °C, 4 h; (4) (a) NaOCH3, CH3OH, 50 °C, 1 h; (b) Bu2SnO, toluene, 120 °C, 6 h; (c) PMBCl, CsF, DMF, 70 °C, 4 h; (5) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 59, − 78 °C to rt, 1.5 h; (6) HF-pyridine, THF, 50 °C, 4 h; (7) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 61, − 78 °C to rt, 1.5 h; (8) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 62, − 72 °C to rt, 1.5 h; (9) HF-pyridine, THF, 45 °C, 6 h; (10) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 63, − 72 °C to rt, 1.5 h; (11) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 59, − 78 °C to rt, 1.5 h; (12) p-TolSCl, AgOTf, then 59, − 78 °C to rt, 1.5 h; (13) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 62, − 72 °C to rt, 1.5 h; (14) p-TolSCl, AgOTf, then 59, − 72 °C to rt, 1.5 h; (15) p-TolSCl, AgOTf, then 62 or 63, − 72 °C to rt, 1.5 h. Tol, p-methylphenyl; Bz, benzoyl; Bn, benzyl; TBS, tert-butyldimethylsilyl; PMB, p-methoxybenzyl; TTBP, 2,4,6-tri-tert-butylpyrimidine; MS, molecular sieves.
With all the monosaccharides in hand, we began synthesis of the necessary oligosaccharide building blocks to construct the glycan library (Fig. 2b). After optimization of glycosylation conditions (Supplementary Fig. 2), we eventually chose p-TolSOTf, generated in situ by p-TolSCl and AgOTf, as the promoter to construct the fucosyl α-glycosidic bond. Given that the TBS group in 60 is sensitive to the trifluoromethanesulfonic acid (TfOH) byproduct that is produced during the glycosylation, the sterically-hindered acid scavenger 2,4,6-tri-tert-butylpyrimidine (TTBP) was added into the reaction mixture. Glycosyl donor 60 was first preactivated with p-TolSCl/AgOTf in the presence of TTBP, and then reacted with acceptor 59, resulting in disaccharide 64 in 91% isolated yield. The long-range participation of the 4-O Bz group led to excellent α-selectivity, which was confirmed by 1H-NMR (JH1-H2 = 3.0 Hz) and HMBC (JH1-C1 = 168 Hz) (Supplementary Fig. 3). Removal of the TBS group from the 3-O position of disaccharide 64 using HF-pyridine produced disaccharide acceptor 65 in excellent yield. The same protocol was used to synthesize disaccharide 66 by the coupling of glycosyl donor 60 with acceptor 61 in 93% yield, followed by subsequent deprotection of TBS, which afforded the disaccharide acceptor 67 in high yield. For Type B fucoidan synthesis, two α-(1,4)-linked disaccharide building blocks (69 and 71) were prepared by a similar approach (Fig. 2c). We found that the formation of the α-(1,4) linkage was slightly more challenging than the α-(1,3) linkage; therefore, we increased the temperature of the glycosylation from − 78 °C to − 72 °C. Due to the presence of the PMB group and the increased glycosylation temperature, the equivalents of TTBP used were increased from 1.2 to 1.3 to mitigate PMB group instability during glycosylation. Eventually, α-(1,4) disaccharides 68 and 70 were successfully synthesized by coupling of 60 with 62 and 63, respectively, again utilizing the p-TolSCl/AgOTf promoter system. Lastly, the two disaccharide acceptors 69 and 71 were generated by deprotection of the TBS groups at 3-O position of 68 and 70 using HF-pyridine. The anomeric α-configuration of the newly formed 1,4-linkage was confirmed by its 1H-NMR (JH1-H2 = 3.3 Hz) and HMBC (JH1-C1 = 167 Hz) (Supplementary Fig. 4).
To synthesize tetrasaccharide building blocks, we prioritized the preactivation-based one-pot glycosylation strategy. This approach minimizes purification steps and is independent of the reactivity of the glycosylation building blocks, thereby significantly reducing workload and improving overall synthesis efficiency. This strategy has been successful in complex glycan synthesis, including in the construction of 1080-mer glycans31. For the synthesis of tetrasaccharide 72 (Fig. 2d), we screened several different promoters including p-TolSCl/AgOTf36, NIS/AgOTf47, NIS/TfOH48, Ph2SO/Tf2O49, benzenesulfinyl piperidine (BSP)/Tf2O50, and benzenesulfinyl morpholine BSM/Tf2O51. Among these, p-TolSCl/AgOTf again proved to be the most effective for conducting the preactivation-based one-pot, four-component glycosylation (Supplementary Fig. 5). Following further optimization of the solvent and reaction temperature, eventually the tetrasaccharide 72 was successfully synthesized by one-pot four-component [1 + 1 + 1 + 1] glycosylation in 73% overall yield starting from monosaccharide building blocks 60 and 59 (three subsequent additions), with the usage of p-TolSCl/AgOTf as the promoter and dichloromethane as the solvent at − 78 °C. The α-glycosidic configurations were again confirmed by the coupling constants between H-1 and H-2 (JH1-H2), which were found to be 3.1 Hz, 3.2 Hz, and 3.5 Hz. The TBS group at 3-O position of 72 was deprotected using HF-pyridine, yielding tetrasaccharide acceptor 73 in 85% yield. For the synthesis of the alternating α-(1,3)-linked and α-(1,4)-linked tetrasaccharide 74 (Fig. 2e), the similar protocol enabled the rapid assembly of tetrasaccharide 74 using p-TolSCl/AgOTf in a one-pot four-component [1 + 1 + 1 + 1] reaction, achieving a 60% overall yield. A similar procedure was employed to synthesize tetrasaccharide 76 in 65% yield. Deprotection of the TBS group at the 3-O position of tetrasaccharides 74 and 76 produced tetrasaccharide glycosyl acceptors 75 and 77, respectively.
One-pot assembly of protected Type A and Type B glycans
Equipped with a comprehensive set of monosaccharides and oligosaccharides, we then aimed to construct a diverse fucoidan library with high efficiency using the preactivation-based one-pot glycosylation strategy (Fig. 3). In the synthesis of Type A compounds (Fig. 3a), we utilized monosaccharides 60, 59, and 61. Trisaccharide 78 was synthesized in 81% overall yield via a one-pot three-component [1 + 1 + 1] glycosylation, promoted by p-TolSCl/AgOTf. Subsequent removal of the TBS group at 3-O position using HF-pyridine yielded trisaccharide acceptor 79 in 91% yield. Similarly, tetrasaccharide 80 was obtained in 72% yield through a one-pot four-component [1 + 1 + 1 + 1] glycosylation. The TBS group deprotection at 3-O position provided tetrasaccharide 81 in 90% yield. For pentasaccharide 82, a one-pot three-component [2 + 1 + 2] glycosylation was performed using disaccharide 64, monosaccharide 59, and disaccharide acceptor 67, achieving an 81% overall yield. Following TBS removal, pentasaccharide acceptor 83 was obtained in 88% yield. Hexasaccharide 84 was assembled via a one-pot three-component [2 + 2 + 2] glycosylation utilizing disaccharide donor 64, acceptors 65 and 67, resulting in an 80% overall yield. Deprotection with HF-pyridine produced hexasaccharide 85 in 95% yield. Heptasaccharide 86 was synthesized through a one-pot three-component [4 + 1 + 2] glycosylation, starting from tetrasaccharide 72, monosaccharide 59, and disaccharide 67, in a 76% overall yield. The subsequent TBS deprotection yielded heptasaccharide 87 in 87% yield. Octasaccharide 88 was prepared via a one-pot four-component [2 + 2 + 2 + 2] glycosylation with disaccharides 64, 65 (two additions) and 67, achieving a 65% yield. TBS deprotection afforded octasaccharide 89 in 92% yield. Nonasaccharide 90 was synthesized in 62% yield using a one-pot four-component [4 + 2 + 2 + 1] glycosylation, starting from tetrasaccharide 72, disaccharide 65 (two additions), and monosaccharide 61. Removal of the TBS group produced nonasaccharide 91 in 92% yield. Decasaccharide 92 was constructed via a one-pot four-component [4 + 2 + 2 + 2] glycosylation, utilizing tetrasaccharide 72, disaccharide 65 (two additions), and disaccharide 67, resulting in a 64% overall yield. The TBS deprotection gave decasaccharide 93 in 90% yield. Undecasaccharide 94 was synthesized through a one-pot four-component [4 + 4 + 2 + 1] glycosylation in 63% yield from tetrasaccharide 72, tetrasaccharide 73, disaccharide 65, and monosaccharide 61. The TBS group removal produced undecasaccharide 95 in 86% yield. Finally, dodecasaccharide 96 was synthesized using a one-pot four-component [4 + 4 + 2 + 2] glycosylation, starting from tetrasaccharide donor 72, tetrasaccharide acceptor 73, disaccharide acceptors 65 then 67, achieving a 60% overall yield. Deprotection of the TBS group at the 3-O position afforded dodecasaccharide 97 in 88% yield.
a Synthesis of Type A glycans. b Synthesis of Type B glycans. (1) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 59, − 78 °C to rt, 1.5 h; (2) p-TolSCl, AgOTf, then 61, − 78 °C to rt, 1.5 h; (3) HF-pyridine, THF, 50 °C, 4 h; (4) p-TolSCl, AgOTf, then 59, − 78 °C to rt, 1.5 h; (5) p-TolSCl, AgOTf, then 67, − 78 °C to rt, 1.5 h; (6) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 65, − 78 °C to rt, 1.5 h; (7) p-TolSCl, AgOTf, then 65, − 78 °C to rt, 1.5 h; (8) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 73, − 78 °C to rt, 1.5 h; (9) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 59, − 72 °C to rt, 1.5 h; (10) p-TolSCl, AgOTf, 63, − 72 °C to rt, 1.5 h; (11) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 62, − 72 °C to rt, 1.5 h; (12) p-TolSCl, AgOTf, then 59, − 72 °C to rt, 1.5 h; (13) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 69, − 72 °C to rt, 1.5 h; (14) p-TolSCl, AgOTf, then 71, − 72 °C to rt, 1.5 h; (15) p-TolSCl, AgOTf, then 69, − 72 °C to rt, 1.5 h; (16) p-TolSCl, AgOTf, then 77, − 72 °C to rt, 1.5 h; (17) TTBP, 4 Å MS, DCM, p-TolSCl, AgOTf, then 75, − 72 °C to rt, 1.5 h. Tol, p-methylphenyl; Bz, benzoyl; Bn, benzyl; TBS, tert-butyldimethylsilyl; PMB, p-methoxybenzyl; TTBP, 2,4,6-tri-tert-butylpyrimidine; MS, molecular sieves.
For the synthesis of Type B compounds (Fig. 3b), we again employed the preactivation-based one-pot glycosylation strategy, which we have already successfully utilized in the synthesis of Type A compounds. Our preliminary studies revealed that the efficiency of the α-(1,4) glycosylation reaction was significantly diminished compared to α-(1,3)-glycosylation, especially when the molecular size of the glycosyl donors and acceptors was increased. To address this issue, we designed our synthetic route such that the α-(1,4) linkages would be formed with higher efficiency at the disaccharide level, and then we would use these intermediates to further construct the subsequent α-(1,3) linkages. Throughout the synthesis of the 10 fully protected glycans, ranging from trisaccharide to dodecasaccharide, p-TolSCl/AgOTf was consistently identified as the optimal promoter for all glycosylation reactions. Specifically, trisaccharide 98 was synthesized via a one-pot three-component [1 + 1 + 1] glycosylation, achieving an 80% overall yield from monosaccharide donor 60 and monosaccharide acceptors 59 then 63. Tetrasaccharide 76 was produced through a one-pot four-component [1 + 1 + 1 + 1] glycosylation from monosaccharide donor 60 and monosaccharide acceptors 62, 59, then 63. Pentasaccharide 100 was assembled in 80% yield by a one-pot three-component [1 + 2 + 2] approach from monosaccharide 60 and disaccharides 69 then 71. Hexasaccharide 102 was synthesized by a one-pot three-component [2 + 2 + 2] glycosylation, with an overall yield of 69% from disaccharides 68, 69, and 71. Heptasaccharide 104 was assembled using a one-pot four-component [1 + 2 + 2 + 2] strategy, achieving a 70% overall yield from monosaccharide donor 60, and disaccharide acceptors 69 then 71. Octasaccharide 106 was synthesized via a one-pot three-component [4 + 2 + 2] approach in 75% overall yield from tetrasaccharide donor 74, and disaccharide acceptors 69 then 71. Nonasaccharide 108 was constructed by a one-pot four-component [1 + 2 + 2 + 4] glycosylation, in 64% overall yield from monosaccharide donor 60, disaccharide acceptor 69, and lastly tetrasaccharide acceptor 77. Decasaccharide 110 was assembled through a one-pot three-component [4 + 2 + 4] glycosylation, achieving a 77% overall yield from tetrasaccharide donor 74, disaccharide acceptor 69, then tetrasaccharide acceptor 77. Undecasaccharide 112 was synthesized using a one-pot four-component [1 + 4 + 2 + 4] strategy in 62% overall yield from monosaccharide donor 60, tetrasaccharide acceptor 75, disaccharide acceptor 69, and tetrasaccharide acceptor 77. Finally, dodecasaccharide 114 was assembled by a one-pot three-component [4 + 4 + 4] glycosylation, with a 68% overall yield from tetrasaccharide donor 74, and tetrasaccharide acceptors 75 then 77. Lastly, the TBS group at the 3-O position of all assembled glycans, from trisaccharide 98 to dodecasaccharide 114, was effectively removed using HF-pyridine to afford the corresponding oligosaccharides 99 to 115, achieving yields ranging from 85% to 95%.
Divergent synthesis of the fucoidan library
To investigate the impact of sulfation patterns on the anticoagulant activity of fucoidan, we modified both Type A and Type B glycans with three distinct sulfation patterns: 2,3-O-di-, 3,4-O-di-, and 2,3,4-O-tri-sulfation. Glycans 67 and 79–97, synthesized by a preactivation-based one-pot glycosylation strategy, served as the precursors for constructing the differentially sulfated Type A fucoidans. To prepare the 2,3-O-di-sulfated compounds (Fig. 4), referring to the reported orthogonal deprotection strategy15,17, the protected glycans first underwent hydrogenolysis using palladium hydroxide under a 0.4 MPa H2 atmosphere to remove all Bn groups at the 2-O position, yielding intermediates 116–126 in considerable yields. Initially, a mixed solvent of ethyl acetate and methanol in a 1:1 ratio was employed for hydrogenolysis across glycans ranging from the trisaccharide to the heptasaccharide. However, this solvent proved to be inadequate for solubilizing the octasaccharide and larger glycans. After optimization of the reaction conditions, we found that a slightly more polar mixed solvent of ethyl acetate and methanol in a 1:1.5 ratio facilitated more efficient reactions for larger glycans. After acquiring the intermediates 116–126, we then focused on the O-sulfation modification. Given the high polarity of the products and the sensitivity of the sulfation reaction to trace amounts of moisture, real-time monitoring via TLC was not feasible. Instead, we employed NMR and HRMS to trace the purified sulfation products. Initially, we selected sulfur trioxide triethylamine complex (SO3-NEt3) as the sulfation reagent to achieve 2,3-O-di-sulfation. However, certain hydroxyl groups proved difficult to sulfate, leading to incomplete sulfation in many cases. After optimizing the sulfation conditions, we utilized 15 equivalents per hydroxyl group of the less sterically hindered sulfur trioxide trimethylamine complex (SO3-NMe3) as the sulfation reagent to achieve complete sulfation. Following purification by size exclusion chromatography, the sulfated intermediates 127–137 were treated with sodium hydroxide solution to deprotect the Bz groups. Simultaneously, the products in the form of ammonium salts were converted to the products in the form of sodium salts, resulting in 2,3-O-di-sulfated fucoidans 1–11 in quantitative yields.
Conditions for deprotection of Bz group: KOH, THF/MeOH = 3:1; or 3 M NaOH or NaOCH3, CH3OH; Conditions for deprotection of Bn group: 0.4 MPa H2, Pd(OH)2/C, ethyl acetate/MeOH = 1:1; Conditions for O-sulfation: SO3-NMe3, DMF; or SO3-pyridine, DMF. Bz, benzoyl; Bn, benzyl.
To achieve the synthesis of 3,4-O-di-sulfated fucoidans, we initially treated the protected Type A glycans (67 and 79–97) with sodium methoxide in methanol to remove the Bz groups. However, the poor solubility of the relatively non-polar protected glycans in methanol impeded the complete removal of the Bz groups, even when employing a mixed solvent of tetrahydrofuran and methanol. After optimizing the conditions, we successfully dissolved the glycans in a mixed solvent of tetrahydrofuran and methanol (3:1) and deprotected the Bz groups using potassium hydroxide. This approach ensured quantitative removal of Bz groups, resulting in intermediates 138–148. Following the sulfation protocol described above for 2,3-O-di-sulfation, sulfate groups were installed at the 3,4-O positions of 138–148 to afford sulfated intermediates 149–159. Lastly, hydrogenolysis with palladium hydroxide under a 0.4 MPa H2 atmosphere was accomplished to remove the Bn groups, which was followed by treatment with 3 M NaOH, yielding the 3,4-O-di-sulfated fucoidans 12–22.
To synthesize 2,3,4-O-tri-sulfated fucoidans, intermediates 116–124 were utilized as starting materials. The Bz groups on these intermediates were first deprotected via treatment with sodium methoxide in methanol, yielding fully unprotected glycans 160–167 in quantitative yields. The primary challenge in this series of compounds was the subsequent sulfation of all hydroxyl groups present due to both the increased steric hindrance and the increased electrostatic repulsion accumulating throughout the sulfation process, compared to single-site sulfation. In addition, when exposed to prolonged reaction time, we observed partial decomposition of these fucoidans. Ultimately, SO3-pyridine was chosen as the sulfation reagent, with DMF as the solvent. The reaction was conducted at room temperature for 14 h, successfully yielding fully sulfated fucoidans. However, achieving complete sulfation remained challenging for glycans larger than decasaccharide, despite increased reaction temperatures and extended reaction times. After purification of the fully sulfated fucoidans, the products were treated with sodium hydroxide to produce the sodium salts 23–30.
The protected Type B glycans were initially subjected to TfOH in dichloromethane for the rapid deprotection of PMB groups, providing compounds 168–178 in 74–91% yields (Fig. 5). Subsequently, removal of the Bn groups by hydrogenolysis under palladium hydroxide in a 0.4 MPa H2 atmosphere afforded intermediates 179–189. During the hydrogenolysis process, a mixed solvent (ethyl acetate and methanol in a 1:1 ratio) ensured the solubility of substrates up to nonasaccharide. For larger glycans, the solvent ratio was slightly altered (ethyl acetate and methanol 1:1.5). The 2,3-O-di-sulfation modification commenced with the treatment of intermediates 179–189 using SO3-pyridine. After purification via size exclusion chromatography, intermediates 190–200 were obtained, which were treated with sodium hydroxide solution to remove the Bz groups and convert the pyridinium sulfate salts into the sodium salts, resulting in the formation of 2,3-O-di-sulfated products 31–41. To construct 3,4-O-di-sulfated glycans, protected glycans 168–178 were used as starting materials. Treatment with potassium hydroxide facilitated the removal of the Bz groups present, affording intermediates 201–211 in quantitative yields, which were followed by sulfation with SO3-pyridine, affording compounds 212–222. After acquiring sulfated intermediates, hydrogenolysis of all the Bn groups at 2-O position under palladium hydroxide generated 3,4-O-di-sulfated pyridinium salts, which, after treatment with sodium hydroxide solution, yielded 3,4-O-di-sulfated sodium salts 42–52. The most challenging aspect of constructing the Type B fucoidan compounds was the synthesis of 2,3,4-O-tri-sulfated products. After obtaining fully unprotected glycans 223–228 in quantitative yield from 179–184 via treatment with NaOMe/MeOH, we meticulously optimized parameters critical to achieving complete sulfation, including the choice of sulfation reagent, temperature, reaction duration, solvent, and concentration. Ultimately, we found that SO3-pyridine was the most effective reagent for sulfation of glycans up to heptasaccharide, yielding 2,3,4-O-tri-sulfated products 53–58. For larger glycans, achieving complete sulfation proved difficult due to frequent incomplete sulfation and glycan decomposition, thereby hindering the synthesis of the larger desired 2,3,4-O-tri-sulfated products.
Conditions for deprotection of PMB group: TfOH/DCM = 1:10; conditions for deprotection of Bz group: KOH, THF/MeOH = 3:1; or 3 M NaOH; or NaOCH3, MeOH; conditions for deprotection of Bn group: 0.4 MPa H2, Pd(OH)2/C, ethyl acetate/MeOH=1:1 or ethyl acetate/MeOH = 1:1.5 or H2O/MeOH = 1:1; conditions for O-sulfation: SO3-NMe3, DMF; or SO3-pyridine, DMF; Bz, benzoyl; Bn, benzyl; PMB, p-methoxybenzyl.
Evaluation of anticoagulant activity
Having synthesized a structurally homogeneous and well-defined library of fucoidan sulfates, we aimed to evaluate their anticoagulant activity and investigate the structure-activity relationships. The 58 precisely synthesized fucoidan sulfate compounds were evaluated for their effects on activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) using human plasma, reflecting their influence on the intrinsic, extrinsic, and common coagulation pathways, respectively (Fig. 6a and Supplementary Tables S1–S3). The results revealed that at a concentration of 128 μg/mL, compounds 25, 26, 27, 28, 29, 30, 35, 36, 37, 38, 39, 40, 41, 56, 57, and 58 significantly prolonged APTT to at least twice that of the control group. Notably, compounds 28, 29, 30, 37, and 58 demonstrated the most potent efficacy, prolonging APTT to over 12 folds compared to the control group, indicating potent inhibition of the intrinsic coagulation pathway. In contrast, at the same concentration, none of the compounds 1–58 exhibited significant effects on prolonging either PT or TT, suggesting that these fucoidans exert negligible inhibitory activity towards the extrinsic coagulation pathway and common coagulation pathway. Whereas, the positive control, enoxaparin at 4 μg/mL extended TT to 3.5 folds compared to the control group. Taken together, these findings underscore that certain chemically synthesized fucoidans have a distinct anticoagulant mechanism, they selectively inhibit the intrinsic coagulation pathway, with minimal effects on the extrinsic and common pathways.
a Effects of the fucoidans on the prolongation of APTT, TT, and PT. Fucoidans were all at 128 μg/mL, Enox was at 4, 16, and 100 μg/mL for TT, APTT, and PT, respectively. Experiments were conducted in triplicate, and mean values were presented. b EC2.0× values of active fucoidans, assessing their inhibition of the intrinsic coagulation pathway by APTT. c Effects of the most potent compounds (29, 30, 37, and 58) on intrinsic coagulation factor Xase (iFXase) activity. Experiments were conducted in duplicate, and data were presented as mean. d IC50 values of 29, 30, 37, and 58 against iFXase and their effects on FXa and FIIa in the presence of antithrombin. APTT, activated partial thromboplastin time; PT, prothrombin time; TT, thrombin time.
To further investigate the inhibitory effects of the fucoidan sulfates on intrinsic coagulation, the concentrations of compounds required for doubling APTT (EC2.0×) were determined (Fig. 6b and Supplementary Fig. 6). Compounds 29, 30, 37, and 58 exhibited potent activity with EC2.0× values of 13.6 μg/mL, 14.5 μg/mL, 16.4 μg/mL, and 12.9 μg/mL, respectively, which are comparable to that of enoxaparin (EC2.0× value of 7.0 μg/mL). By analyzing the structures and APTT-prolonging anticoagulant activities of the 58 fucoidan sulfate compounds, we found that for Type A compounds, the 2,3,4-O-tri-sulfation modification significantly enhanced anticoagulant activity. For Type B compounds, both 2,3-O-di-sulfation and 2,3,4-O-tri-sulfation modifications exhibited superior anticoagulant effects. These findings suggest that while the degree of sulfation significantly contributes to the anticoagulant efficacy of fucoidan sulfates, the connectivity of the glycosidic bonds also plays a role. In addition, we observed that, for 2,3,4-O-tri-sulfation modification, the anticoagulant activity was enhanced with the increase of molecular size.
We conducted a detailed investigation into the anticoagulant mechanism of the most promising compounds 29, 30, 37, and 58. Specifically, we assessed their effects on intrinsic coagulation factor Xase (iFXase) activity, a critical rate-limiting enzyme in the intrinsic pathway52. In addition, we evaluated their impact on the activities of factor Xa (FXa) and thrombin (factor IIa, FIIa) in the presence of antithrombin (AT), given that heparin-like anticoagulants exert their effects by AT-mediated inhibition of FXa and FIIa. Compounds 29, 30, 37, and 58 demonstrated potent, concentration-dependent inhibition of iFXase within the range of 4–4096 ng/mL. Notably, compound 29 exhibited an IC50 value of 7.4 ng/mL, closely aligning with that of enoxaparin (IC50 = 6.7 ng/mL). Moreover, compounds 30, 37, and 58 demonstrated considerable anti-iFXase activity, with IC50 values of 24.4 ng/mL, 28.8 ng/mL, and 27.1 ng/mL, respectively (Fig. 6c). In the presence of AT, these compounds did not inhibit FXa or FIIa activities, with IC50 values all exceeding 5000 ng/mL. In contrast, the positive control, enoxaparin, exhibited IC50 values of 24.7 ng/mL for FXa and 27.6 ng/mL for FIIa (Fig. 6d and Supplementary Figs. 7, 8). These findings suggest that compounds 29, 30, 37, and 58 inhibited the intrinsic coagulation pathway by selectively targeting iFXase in an AT-independent manner. This indicates that their anticoagulant mechanism is distinct from those of heparin-like molecules. In addition, taking compound 37 as an example, its superior anticoagulant activity could be explained by molecular docking simulations that simulated the interactions between FIXa (the key enzyme of iFXase complex) and the compound (Supplementary Figs. 9–12). To evaluate the safety profiles of the four compounds, we also tested the cytotoxicity of these compounds on two cell lines, RAW246.7 and NIH 3T3. The results showed that the compounds exhibited negligible cytotoxicity even at a high concentration of 128 µg/mL (Supplementary Figs. 13, 14). This indicates that these compounds are likely to be safe for their intended applications.
The fucoidan sulfates we have developed selectively inhibit the intrinsic coagulation pathway without interfering with the extrinsic and common pathways. By specifically targeting iFXase, the rate-limiting enzyme complex in the intrinsic coagulation pathway, our compounds enable safe and effective antithrombosis. This is particularly crucial for patients requiring long-term anticoagulant therapy, such as those with chronic conditions like venous thromboembolism. This targeted approach is particularly beneficial for patients who may have contraindications or intolerances to extrinsic pathway inhibitors like warfarin. Thus, selective inhibition of the intrinsic pathway provides a valuable alternative for patients who need tailored anticoagulant therapy.
Discussion
By utilizing a preactivation-based one-pot glycosylation strategy, we have efficiently and expeditiously assembled a comprehensive library of sulfated fucoidan compounds. This library comprises 58 distinct molecules, ranging from disaccharides to dodecasaccharides, incorporating three diverse sulfation patterns. To our knowledge, this library stands as the most comprehensive and systematic array of sulfated fucoidans reported to date. Our detailed analysis of the structural and anticoagulant properties of these molecules has revealed key insights into previously poorly understood structure-activity relationships. Each parameter investigated, including molecular size, glycosidic bond connectivity, and specific sulfation pattern, was shown to play a crucial role in anticoagulant efficacy. For Type A compounds, the modification of 2,3,4-O-tri-sulfation significantly enhanced anticoagulant activity. Similarly, in Type B compounds, both 2,3-O-di-sulfation and 2,3,4-O-tri-sulfation modifications yielded superior anticoagulant effects, with efficacy improving as molecular size increases. Fucoidans 29, 30, 37, and 58 exhibited exceptional anticoagulant properties, rivaling commercially available options such as enoxaparin. Notably, these compounds demonstrate a unique mechanism of anticoagulation by selectively modulating the intrinsic coagulation pathway. This targeted approach will provide enhanced precision in anticoagulant therapy, promising a safer and more controllable therapeutic profile. Such specificity not only mitigates the risks of bleeding but also offers potential advantages for tailored treatment in particular patient populations and clinical situations. While conventional anticoagulants provide broad-spectrum inhibition, the molecules developed in this study offer promising alternatives that could pave the way for more personalized and adaptable treatment strategies. This research underscores the potential for marine-derived molecules to inspire novel pharmaceuticals, thereby expanding the horizons of drug discovery and therapeutic innovation.
Methods
General
The experimental details and compound characterization data can be found in Supplementary Information. For the NMR and MS data of the compounds in the article, see Supplementary Information.
General procedure for the preactivation-based one-pot glycosylation reaction
A mixture of glycosyl donor, TTBP, and freshly activated 4 Å molecular sieves in anhydrous CH2Cl2 under argon atmosphere was stirred for 20 min at room temperature and then cooled to − 78 °C (or − 72 °C). After 5 min, AgOTf (dissolved in anhydrous toluene) was added to the mixture, followed by the addition of the stoichiometric amount of p-TolSCl. After TLC indicated the disappearance of the donor, a solution of glycosyl acceptor in anhydrous CH2Cl2 was slowly added. The resulting mixture was slowly warmed to room temperature within 2 h, stirred for another 20 min, and then cooled back to − 78 °C (or − 72 °C). The glycosylation operation mentioned above was repeated until the generation of the desired product.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
All data supporting the results and conclusions are available within the article, its Supplementary Information, and from the corresponding authors upon request. Source data are provided in this paper.
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Acknowledgements
This work was financially supported by the grants from the National Natural Science Foundation of China (22237001 (X.-S.Y.), 22227801 (X.-S.Y.), 81930097 (M.W.), 82473811 (M.W.)), the National Key Research and Development Program of China (2022YFC3400800 (X.-S.Y.)), Yunnan Fundamental Research Projects (202201AS070073 (M.W.), 202301AV070008 (M.W.), 202302AA310012 (M.W.)), and Postdoctoral Directional Training Foundation of Yunnan Province (L.L.). We thank Dr. Yuan Wang and Dr. Fen Liu at Peking University Health Science Center for their assistance in analysis of glycan structures. We thank Chandler Wells and Rohan P. Patel at the University of Texas at Austin for language editing.
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X.-S.Y. conceived the research. X.-S.Y., S.-C.C., and M.W. designed the experiments. S.-C.C. performed most of the synthetic experiments. N.X. and L.L. performed biological evaluation experiments. Y.W., Q.L., and D.S. performed partial chemistry experiments. X.Q., S.-C.C., X.-S.Y., and M.W. analyzed the data. X.Q. and X.-S.Y. wrote the manuscript. D.-C.X. and C.E.C. discussed the data analysis and revised the manuscript. M.W. guided the biological assay. X.-S.Y. supervised the project.
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X.-S.Y., S.-C.C., and M.W. are applying for a Chinese patent filed by Peking University and Kunming Institute of Botany, Chinese Academy of Sciences. The other authors declare no competing interests.
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Chen, SC., Qin, X., Xiong, N. et al. Comprehensive synthesis and anticoagulant evaluation of a diverse fucoidan library. Nat Commun 16, 4364 (2025). https://doi.org/10.1038/s41467-025-59632-2
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DOI: https://doi.org/10.1038/s41467-025-59632-2
