Synthesis and Properties of Functional Glycomimetics through Click Grafting of Fucose onto Chondroitin Sulfates

Article abstract of DOI:10.1021/acs.biomac.9b00878

Fucosylated chondroitin sulfate (fCS), a representative marine polysaccharide isolated from sea cucumber, possesses diverse biological functions especially as a promising anticoagulant. However, its supply suffers from the challenges of high-cost materials, different species, and batch-to-batch variability. In the present study, we designed a concise route for the synthesis of functional glycomimetics by natural fCS as a template. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride-mediated amidation was applied on chondroitin sulfates for site-selective alkynylation with controllable ratios between 0.15 and 0.78. A small library of 12 fCS glycomimetics with specific sulfation patterns and fucose branches was prepared through copper-catalyzed azide-alkyne cycloaddition, which was fully characterized by nuclear magnetic resonance spectroscopy and size-exclusion chromatography with multiangle light scattering and refractive index. Through screening of their biological activities, CSE-F1 and CSE-SF1 exhibited anticoagulant activities through intrinsic pathway and inhibition of factor Xa by antithrombin III. The concise approach developed herein supplies novel glycopolymers to mimic the distinct functions of natural polysaccharides and promote the development of marine carbohydrate-based drugs.

Full text of DOI:10.1021/acs.biomac.9b00878

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Article
Synthesis and properties of functional glycomimetics
through click grafting of fucose onto chondroitin sulfates
Fei Fan, Ping Zhang, Lihao Wang, Tiantian Sun, Chao Cai, and Guangli Yu
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.9b00878 • Publication Date (Web): 30 Jul 2019
Downloaded from pubs.acs.org on July 31, 2019
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Synthesis and properties of functional
glycomimetics through click grafting of fucose
onto chondroitin sulfates
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†
,§
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Fei Fan, Ping Zhang, Lihao Wang, Tiantian Sun, Chao Cai,*
Guangli
Yu,*,†,‡,§
†
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Key Laboratory of Marine Drugs, Ministry of Education, School of Medicine and
Pharmacy, Ocean University of China, Qingdao 266003, China.
‡
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Laboratory for Marine Drugs and Bioproducts, Pilot National Laboratory for Marine
Science and Technology (Qingdao), Qingdao 266003, China.
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Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, School
of Medicine and Pharmacy, Ocean University of China, Qingdao 266003, China.
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ABSTRACT:
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Fucosylated chondroitin sulfate (fCS), a representative marine polysaccharide isolated
from sea cucumber, possesses diverse biological functions especially as a promising
anticoagulant. However, its supply suffers from the challenges of high-cost materials,
17 different species and batch-to-batch variability. In the present study, we designed a
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concise route for the synthesis of functional glycomimetics by using natural fCS as
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template. DMTMM-mediated amidation was applied on chondroitin sulfates for
site-selective alkynylation with controllable ratios between 0.15~0.78. A small library
of 12 fCS glycomimetics with specific sulfation patterns and fucose branches was
prepared through copper-catalyzed azide–alkyne cycloaddition (CuAAC), which was
fully characterized by NMR and SEC-MALLS-RI. Through screening of their
biological activities, CSE-F1 and CSE-SF1 exhibited anticoagulant activities through
intrinsic pathway and inhibition of FXa by ATIII. The concise approach developed
herein supplies novel glycopolymers to mimic the distinct functions of natural
polysaccharides and promote the development of marine carbohydrate-based drugs.
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KEYWORDS: Fucosylated chondroitin sulfates (fCS), alkynylation, azide-alkyne
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click reaction, fucose, glycomimetics, anticoagulation.
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INTRODUCTION
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As a representative L-hexose, fucose is widely present in nature especially as a
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component of N-glycans1-3 involved in signal transduction and cell adhesion, as well
15 as being the fundamental building blocks of natural polysaccharides4-6 that exhibit
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antivirus, anticoagulant and antitumor activities. Fucosylated chondroitin sulfate (fCS)
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is exclusively isolated from sea cucumber, and the structural properties of fCS are
interesting with fucosylation at the O-3 position of D-glucuronic acid (GlcA)8, 9 or
19 O-6 position of N-acetyl-D-galactosamine (GalNAc)10-12 on the backbone of
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chondroitin sulfate (CS) (Figure 1A). fCS has anticoagulant and antithrombotic
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activity through inhibition of thrombin (FIIa) and factor Xa (FXa) mediated by
antithrombin (AT) and heparin cofactor II (HC-II),13-15 whereas the counterpart CS
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exhibits no parallel activity. It is noteworthy that fCS exhibits anticoagulant activity
even when it is administered orally, with low bleeding risk,17 promoting fCS
development as a potential drug candidate. The oligosaccharides depolymerized from
natural fCS exhibit selective inhibition of intrinsic tenase,18, 19 and a nonasaccharide
bearing a 2,4-di-O-sulfated fucosyl residue is the minimum fragment for intrinsic
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tenase inhibition. The anticoagulant activity of natural fCS is strongly related to its
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molecular weight, fucose branches, and sulfation patterns. Furthermore, fCS isolated
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from sea cucumber Holothuria Mexicana exhibited high affinity to fibroblast growth
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factor (FGF) 1 and 2 influenced by the specific linkage types. Chondroitin sulfates
12 are easily obtained with high purity by extraction21-23 and fermentation,24, 25 allowing
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them to be commercially available, while natural fCS is still rare and relatively
expensive with complex and diverse structures. Clinical studies of fCS
polysaccharides have been thoroughly performed in various countries,5, 16 but the
16 structure-activity relationships (SARs) are still unclear and must be clarified.
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Furthermore, potential contaminations including other types of GAGs and proteins
will also affect the purity and SAR studies of natural polysaccharides.
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To decipher the structure-activity relationship (SAR) of fCS, chemical synthesis is
an alternative way to obtain structurally well-defined fCS oligosaccharides for
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anticoagulant activity studies. To the best of our knowledge, although synthesis of the
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chondroitin sulfate backbone is increasingly reported, the chemical synthesis of fCS
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remains a significant challenge. Tamura et al. reported a total synthesis of the fCS
repeating trisaccharide via a stepwise coupling strategy that resulted in a
monosulfated trisaccharide. To study the effects of the sulfation patterns on the
bioactivity, Nifantiev et al. synthesized regioselectively sulfated fCS
oligosaccharides28 and analyzed their conformations for SAR.29 However,
bioactivities of the total synthesized fCS fragments were not evaluated. To circumvent
the tremendous effort and time consuming process of total synthesis, semi-synthetic
approaches have attracted attention to reduce glycosylation coupling steps and
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manipulations of protecting groups. Recently, Li et al. reported the synthesis of fCS
fragments by combining enzymatic degradation of chondroitin and chemical
12 fucosylation over 12 linear steps. The synthetic nonasaccharide exhibited selective
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intrinsic tenase inhibition, as previously reported.20 This approach proved that
semi-synthesis could serve as an efficient tool for mimicking natural fCS
polysaccharides.
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The synthesis of glycomimetics, which preserve the structural and biological
features of natural polysaccharides, has received considerable attention in recent years
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for assisting the SAR study of complex polysaccharides as well as for the
19 development of carbohydrate-derived drugs.31-33 As for fCS, Li et al. also reported
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the synthesis of glycoclusters as fCS mimetics through a click reaction by forming
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multivalent scaffolds from the trisaccharide motifs. The anticoagulant activities of the
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synthetic glycoclusters were relatively consistent with natural polysaccharides. These
synthetic glycoclusters with non-saccharide backbone had distinct structure from
natural fCS but exhibited a similar level of biological effects. In addition, the
semi-synthesis of fCS polysaccharides was reported by Bedini et al.35, 36 with direct
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chemical fucosylation on microbial-sourced chondroitin backbones. Through
orthogonal manipulation of protecting groups, semi-synthetic fCSs were achieved
with relatively defined structures that exhibited moderate bioactivities compared to
natural fCS. However, the structures of semi-synthetic fCS in their study were still
heterogeneous with linkage sites and sulfation patterns that differ from natural fCS.
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Structurally, fCS is composed of a CS backbone with sulfated or nonsulfated
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α-fucose branches linked to the O-3 position of D-glucuronic acid (GlcA) or O-6
12 position of N-acetyl-D-galactosamine (GalNAc). The branches are necessary for
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anticoagulant activity (Figure 1A). We hypothesize that preservation of the CS
backbone and fucose branches on glycomimetics could achieve the anticoagulant
activity of natural fCS. Therefore, novel fCS mimetics (Figure 1B) were designed and
16 synthesized through chemical grafting of fucose branches onto CS in this study.
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OR
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Figure 1. Chemical structures of (A) natural fucosylated chondroitin sulfates (fCS)
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and (B) designed fCS mimetics.
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Chondroitin sulfate (CS) contains hydroxyl, amine and carboxyl groups that could
serve as anchors for covalent modification. We chose the carboxyl groups for
coupling with the fucose branches to access the fCS mimetics, and their anticoagulant
activities were evaluated including APTT, TT, PT, and FXa assays.
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Experimental section
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Materials. Chondroitin sulfate A (CSA, M : 18.9 kDa, sulfate content 16.5%) was
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purchased from BeiErTe Biotechnology Co., Ltd (Qingdao, China). Chondroitin
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reported.37 Natural fucosylated chondroitin sulfate (fCS, M : 41.9 kDa, sulfate
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content 32.3%) was extracted from Holothuria Polli in our laboratory. Heparin (HP,
M : 12 kDa, sulfate content 29.8%) and low-molecular-weight heparin (LMWH)
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15 from porcine intestinal mucosa and tris(3-hydroxypropyltriazolylmethyl)amine
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N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide
(EDC,
98.0%),
N-hydroxysuccinimide (NHS, 98%), 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT,
97%), N-methylmorpholine (NMM, 99.5%), propargylamine (97%),
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2-(N-morpholino)ethanesulfonic acid (MES, 99%), sulfur trioxide pyridine complex
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(SO ·Py, 97%) and Amberlite® IR-120 cation exchange resin (Na form) were
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purchased from Aladdin and used without further purification. Other chemical
reagents were purchased from Energy Chemical and used directly. Deionized water
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with a resistivity of 18 MΩ·cm was used as solvent. Dialysis was performed using
cellulose membranes with a molecular weight cutoff of 3.5 kDa with water as solvent.
The activated partial thromboplastin time (APTT, F008-1) kit, prothrombin time (PT,
F007) kit and thrombin time (TT, F009) kit reagents were purchased from NanJing
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12 JianCheng Bioengineering Institute, NanJing, China. Sheep plasma was purchased
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from Jiulong Biological Co., Ltd, Shanghai, China. Antithrombin III (ATIII), bovine
coagulation factor Xa (FXa) FXa, human coagulation factor IIa (FIIa), chromogenic
substrate S-2765 and S-2238 were purchased from Adhoc International Technologies
16 Co., Ltd (Beijing, China).
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Characterization. Nuclear magnetic resonance (NMR) spectra were recorded on an
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Agilent DD2 spectrometer (500 MHz). The chemical shifts of all the NMR spectra
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spectra were referenced using CD OD ( H NMR δ = 3.31 ppm, C NMR δ = 49.00
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ppm), and D O ( H NMR δ = 4.79 ppm). The peak and coupling constant assignments
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are based on H NMR, H- H COSY, and H- C HSQC experiments. Multiplicities
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of the H NMR data are denoted as s (singlet), d (doublet), t (triplet), q (quartet), and
m (multiplet).
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Molecular weights (M ) were characterized by size exclusion chromatography with
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multi-angle light scattering and refractive index (SEC-MALLS-RI) using a high
performance liquid chromatography (HPLC, Agilent 1260) system equipped with two
OHpak water columns (SB-804 HQ, SB-803 HQ, Shodex), a light scattering detector
(miniDAWN, Wyatt Technology), and a refractive index detector (TREOS, Wyatt
Technology). The column temperature was set at 35 °C, and 0.1 M Na SO in H O
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was employed as an eluent at a flow rate of 0.6 mL·min .
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For Fourier transform infrared spectroscopy (FT-IR) spectra collection, the dried
12 sample was mixed with dried KBr and pressed to make transparent film for FT-IR
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measurements using a Nicolet Nexus 470 instrument (Thermo Electron Corp.,
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Madison, WI, USA) with a frequency resolution of 1 cm and 64 scans between 4000
and 500 cm−1.
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Sulfur content was determined by ion chromatography.38 Briefly, ~1.5 mg of
sample was hydrolyzed in ampoule with 1ꢀM HCl at 110ꢀ°C for 8ꢀh. The hydrolysate
was dried under vacuum before dissolved in deionized water (25ꢀmL). Subsequently,
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19 sulfur quantification was performed by using ion chromatography (CIC-100, Qingdao
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SH-AC-3 column and a suppressed conductivity detector. The column temperature
was set at 35ꢀ°C and 2 mM Na CO -8 mM NaHCO aqueous solutions was employed
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Synthesis
of
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
chloride (DMTMM). The reported procedure was applied to synthesize DMTMM.39
N-Methylmorpholine (NMM, 434.7 μL, 3.89 mmol) was added to a solution of
2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT, 751.4 mg, 4.28 mmol) in THF (15 mL)
at room temperature. A white solid appeared within several minutes. After stirring for
30 min at room temperature, the solid was collected through filtration and washed
with THF and dried to give DMTMM (1.03 g, 96%).
1
1
1
Synthesis of 1-azidoethyl-2,3,4-tri-O-sulfonato-α-L-fucopyranoside (F2). To a
4
0
12 solution of 1-azidoethyl-α-L-fucopyranoside (F1, Scheme 1) (40 mg, 0.1715 mmol)
1
1
1
3
4
5
in 600 μL DMF, SO ·Py (814.5 mg, 5.15 mmol) was added and stirred at 50 ºC for 8
3
h. Upon confirmation of complete conversion by TLC (CH Cl:MeOH:H O = 1:1:0.1,
3
2
R = 0.4), the reaction mixture was cooled to room temperature and neutralized by
f
16 adding saturated NaHCO (aq.). The solvent was evaporated under reduced pressure,
3
1
7
8
and the residue was redissolved in MeOH and filtrated. The filtration was
concentrated under reduced pressure and the crude product was purified by Sephadex
1
19 LH-20 gel filtration (MeOH). Subsequently, the crude product was passed through
+
2
0
Amberlite® IR-120 cation exchange resin (Na form) to give the desired product F2
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2
2
2
2
2
2
2
2
2
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3
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5
5
6
+
1
1
2
3
4
5
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8
9
0
as a white solid in Na form (95.9 mg, 72%). H NMR (500 MHz, CD OD): δ 5.21 (d,
3
J = 3.6 Hz, 1H, H1), 5.00 (d, J = 2.8 Hz, 1H, H4), 4.74 (dd, J = 10.7, 3.1 Hz, 1H, H3),
4.62 (dd, J = 10.6, 3.6 Hz, 1H, H2), 4.19 (q, J = 6.5 Hz, 1H, H5), 3.86 (ddd, J = 11.2,
7.9, 3.6 Hz, 1H, O-CH -CH -N ), 3.75 (ddd, J = 10.7, 5.2, 3.9 Hz, 1H,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
3
O-CH -CH -N ), 3.57 (ddd, J = 11.6, 7.8, 3.7 Hz, 1H, O-CH -CH -N ), 3.45 (ddd, J =
2
2
3
2
2
3
1
3
13.2, 5.1, 3.8 Hz, 1H, O-CH -CH -N ), 1.32 (d, J = 6.5 Hz, 3H, H6). C NMR (126
2
2
3
MHz, CD OD): δ 97.62 (C1), 78.35 (C4), 72.49 (C3), 71.88 (C2), 67.08
3
(O-CH -CH -N ), 65.89 (C5), 50.40 (O-CH -CH -N ), 15.65 (C6). HRMS: calcd for
2
2
3
2
2
3
2
-
2-
2-
[C H N NaO S ] 246.4695 ([M+Na] ), found 246.4691 ([M+Na] ) and 235.4782
8
12
3
14 3
2
-
1
1
([M+H] ).
+
1
Alkynylation of CS. The CS was transformed into H form before alkynylation.
+
+
12 Amberlite® IR-120 cation exchange resin (Na form) was transformed into H form
13
14
15
by eluted with 5% HCl and subsequent deionized water. CS (1.6 g) was dissolved in
40 mL deionized water and loaded on Amberlite® IR-120 cation exchange resin (H+
form) column. Afterward, the column was eluted by 2000 mL deionized water. The
+
16 eluent portions were collected and lyophilized to afford CS in H form. CS (1 g) was
1
7
8
dissolved in MES buffer (0.1 M, pH 5.0). The DMTMM (calculated based on the
disaccharide repeating units) was added and stirred at room temperature for 30 min.
1
19 The desired amount of propargylamine was mixed with MES buffer (pH 5.0) (v/v, 1/1)
2
0
1
and then added to the solution (based on disaccharide repeating units). MES buffer
(pH 5.0) was added to enable the desired final concentration of CS. The mixture was
2
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stirred at set temperature for 24 h and then cooled to room temperature. Subsequently,
NaCl was added to raise [NaCl] to 16% and stirred at room temperature for 30 min.
The mixture was precipitated by the addition of ethanol and the precipitate was
centrifuged at 4000 rpm. The residue was dialyzed against deionized water and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
lyophilized to afford the alkynylated CS as a white powder. The products were
1
characterized by H NMR and FT-IR. The degree of substitution of alkyne (DS ) was
y
1
calculated based on the disaccharide repeating units by H NMR integration between
4
1
the alkyne signals at 2.69 ppm and the CH signal of GalNAc at 2.05 ppm: DS =
3
y
[3×A(alkyne)]/A[CH3(G)]
.
1
0
1
Grafting fucose onto alkynylated CS by copper-catalysed azide–alkyne
cycloadditions (CuAAC).
1
12
Alkynylated CS coupling with nonsulfated fucose F1. In a 10 mL round bottomed
vessel, alkynylated CS (6 mg) and nonsulfated fucose F1 (3.0 eq., based on alkyne)
were dissolved in deionized water. Freshly prepared stock CuSO ·5H O solution (0.4
1
1
1
3
4
5
4
2
-1
eq., 25 mg·mL , based on alkyne) and stock Na-ascorbate solution (1.2 eq., 100
-1
16 mg·mL , based on alkyne) were added. Deionized water was added to adjust the final
-1
1
7
8
concentration of alkynylated CS to 10 mg·mL . The mixture was stirred at 60 ºC for
1
24 h and then cooled to room temperature. Subsequently, NaCl was added to raise
19 [NaCl] to 16% and stirred at room temperature for 30 min. The mixture was
precipitated by the addition of ethanol and the precipitate was centrifuged at 4000 rpm.
2
0
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2
2
2
2
2
2
2
2
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6
1
2
3
4
5
6
7
The residue was dialyzed against deionized water and lyophilized to afford fucose
grafted CS as white powder. The products were characterized by nuclear magnetic
resonance (NMR), FT-IR and size exclusion chromatography with multi-angle light
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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1
2
3
4
5
6
7
8
9
0
scattering and refractive index (SEC-MALLS-RI). The degree of substitution of
1
fucose (DS ) was calculated based on the disaccharide repeating units by H NMR
f
integration between the CH signal of fucose at 1.19 ppm and the CH signal of
3
3
GalNAc at 2.05 ppm: DS = A(F6)/A[CH3(G)]
.
f
8
9
0
1
Alkynylated CS coupling with trisulfated fucose F2. In a 10 mL round bottomed
vessel, alkynylated CS (6 mg) was dissolved in PBS buffer (pH 7.38).
Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA) (4 eq., based on alkyne) was
1
1
-1
mixed with freshly prepared CuSO ·5H O solution (0.4 eq., 25 mg·mL , based on
4
2
12 alkyne) in PBS buffer (pH 7.38) and added to the reaction solution. The reaction
1
1
1
3
4
5
mixture was placed under flow of nitrogen gas then to it was added freshly prepared
-1
Na-ascorbate solution (1.2 eq., 100 mg·mL , based on alkyne) in PBS buffer (pH
7.38). After stirring at room temperature for 30 min, trisulfated fucose F2 (3.0 eq.,
16 based on alkyne) in PBS buffer (pH 7.38) was added and PBS buffer (pH 7.38) was
-1
1
7
8
added to adjust the final concentration of alkynylated CS to 10 mg·mL . The mixture
1
was stirred at 60 ºC for 24 h and then cooled to room temperature. Subsequently,
19 NaCl was added to raise [NaCl] to 16% and stirred at room temperature for 30 min.
2
0
1
The mixture was precipitated by the addition of ethanol and the precipitate was
centrifuged at 4000 rpm. The residue was dialyzed against deionized water and
2
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lyophilized to afford fucose grafted CS as white powder. The products were
characterized by nuclear magnetic resonance (NMR), FT-IR and size exclusion
chromatography with multi-angle light scattering and refractive index
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(SEC-MALLS-RI). DS was calculated based on the disaccharide repeating units by
f
1
H NMR integration between the CH signal of fucose at 1.19 ppm and the CH signal
3
3
of GalNAc at 2.05 ppm: DS = A(F6)/A[CH3(G)].
f
7
8
9
0
1
Anticoagulant activity assay. For APTT assay, plasma (90 μL) was incubated at
37 °C with 10 μL of polysaccharide samples and APTT reagent (100 μL). After
−
2
−1
stirring 3 min, 2.5 × 10 mol L CaCl (100 μL) was added, and the clotting times
2
1
1
were measured by an automated coagulometer. For PT assay, plasma (90 μL) was
incubated at 37 °C with 10 μL of polysaccharide samples. After stirring 3 min, 200 μL
12 of PT reagent was added and the clotting times were measured by an automated
1
1
1
3
4
5
coagulometer. For TT assay, plasma (90 μL) was incubated at 37 °C with 10 μL of
polysaccharide samples. After stirring 3 min, 100 μL of TT reagent was added, and
the clotting times were measured by an automated coagulometer. Results were
16 expressed for each group (n = 3) as mean APTT, PT or TT (s) ± standard deviation of
1
7
8
the mean (SD).
1
Chromogenic Assays for the Measurement of Anti-FXa and anti-FIIa activity
19 in the presence of ATIII. The anti-FXa and anti-FIIa activities of fCS mimetics in
the presence of ATIII were estimated by following the previously published
2
0
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7
methods. Incubations were performed in 96-well plates and a mixture containing 20
μL sample and 20 μL of 0.5 IU/mL ATIII was incubated at 37 °C for 2 min. Then, 40
μL of 0.25 IU/mL FXa or 5 IU/mL FIIa was added. After incubation for 2 min, the
residual FXa or FIIa activity was measured by the addition of 50 μL of 1 mM FXa
chromogenic substrate S-2765 or FIIa chromogenic substrate S-2238. After
incubation for 1 min, the reaction was terminated by adding 80 μL of 30% acetic acid
and absorbance of the reaction mixture was recorded at 405 nm.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
8
RESULTS AND DISCUSSION
9
0
1
Commercially available chondroitin sulfate A (CSA) was chosen as starting
material for the preparation of fCS mimetics, and the carboxyl groups on CSA were
1
1
employed as sites for regioselective amidation. The carboxyl groups on CSA were
12 largely present in the Na form, leading to low conversion of the amidation reaction.43
+
+
1
1
1
3
4
5
Therefore, CSA was transformed into the proton (H ) form by pretreatment with a
+
40
cation exchange resin (H form) prior to amidation. Nonsulfated fucose F1 with
functionalized azide groups was utilized for grafting α-fucose onto CSA. After
16 catalytic hydrogenation, we attempted direct amidation between the free amine on
1
7
8
fucose and carboxyl groups on CSA with the conventional activators of
N-(3-(dimethylamino)propyl)-N’-ethylcarbodiimide (EDC) and
1
19 N-hydroxysuccinimide (NHS).44 Unfortunately, fucose branches were not
1
2
0
successfully grafted onto CSA as indicated by H NMR. We speculated that steric
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hindrance, especially for the carboxyl groups on the CSA, resulted in the failure of the
coupling reaction.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3
4
5
6
7
8
9
To overcome the steric hindrance during the coupling reaction, a two-step strategy
was adopted to graft fucose onto CSA through the copper-catalyzed azide-alkyne
cycloaddition (CuAAC)45 with lower sterically hindered molecule bearing alkyne
groups (Scheme 1). To this end, propargylamine was used for alkyne
functionalization of CSA, and the degree of substitution of the alkyne (DS ) could be
y
1
readily calculated based on the H NMR integration between the alkyne signals at
4
1
2.69 ppm and the CH signal of GalNAc at 2.05 ppm (Figure 2).
3
1
0
1
Scheme 1. Schematic Illustration of the Synthesis of fCS Mimetics with Various
Sulfation and Fucose Densities Through the Click Grafting of Azide-Fucose onto
1
12 Alkyne-Functionalized Chondroitin Sulfate
N
N
N
O
Click Reaction
Fucosylation
O
OR
OR
O
OR
O
OR
O
OH
O
NH
O
NH
O
RO
O
RO
O
RO
O
O
O
OR
O
RO
Alkynylation
O
O
O
HO
HO
HO
AcHN
AcHN
AcHN
OH
n
OH
n
OH
n
Chondroitin sulfate
(CS)
Alkynylated chondroitin sulfate
Fucosylated chondroitin
sulfate (fCS) mimetics
H2N
N3
O
Degree of alkynylation
CSA1: DSy 0.15
CSA2: DSy 0.54
CSA3: DSy 0.78
CSE1: DSy 0.19
CSE2: DSy 0.53
CSE3: DSy 0.72
O
OH
fCS mimetics
Fucose
Chondroitin sulfate
OH
HO
F1
OH
or
F1
F2
NaO3SO
CS
HOOC
O
O
N3
O
O
O
O
CSA1
CSA2
CSA3
CSE1
CSE2
CSE3
CSA-F1 CSA-SF1
CSA-F2 CSA-SF2
CSA-F3 CSA-SF3
CSE-F1 CSE-SF1
CSE-F2 CSE-SF2
CSE-F3 CSE-SF3
CSA HO
AcHN
OH
n
OSO3Na
OSO3Na
NaO3SO
F2
OSO3Na
O
NaO3SO
HOOC
O
O
O
CSE HO
AcHN
OH
n
1
3
1
4
5
Alkynylation of chondroitin sulfate A and chondroitin sulfate E.
1
Polysaccharides usually exhibit complex topological structures due to the strong
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1
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
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4
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0
1
hydrogen bonds between hydroxyl, carboxyl and amine groups, which dramatically
decrease the activation capability of the amidation reagents.46 The amidation of
polysaccharides with EDC/NHS for biomaterial application could result in a low
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
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0
efficiency,
as
previously
reported.47
Alternatively,
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM),48
an aqueous soluble activator, was shown to possess potent activation capabilities
when initially used in peptide synthesis.49 It had been applied for carboxyl
polysaccharides modification with a well-controlled degree of substitution since
2007.50-53 To the best of our knowledge, DMTMM mediated amidation of CS has not
been reported yet. In our study, DMTMM was employed for the controllable
alkynylation of CSA. To achieve a higher coupling efficiency, CSA was typically
1
1
12 activated in the presence of DMTMM before the addition of propargylamine. To
+
13
14
15
guarantee the H form of the carboxyl groups during the reaction, the amidation was
performed in a 2-(N-morpholino)ethanesulfonic acid (MES) buffer at pH 5.0 with the
premixing of propargylamine before addition. The alkynylation of CSA with
16 propargylamine mediated by DMTMM is summarized in Table 1. When the reaction
1
7
8
was conducted at room temperature at a carboxyl/DMTMM/propargylamine feed
1
1
ratio of 1:2:0.5, the alkyne signals were not observed in the H NMR spectrum (DS 0,
y
19 Table 1, entry 1). Changing the carboxyl/DMTMM/propargylamine feed ratio to 1:2:1,
20
the DS value was determined to be 0.15 (Table 1, entry 2).
y
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Table 1. Alkynylation of Chondroitin Sulfate A (CSA) with Propargylamine by
Using DMTMM as Condensation Reagent Under Various Conditions
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
OH
O
H2N
OH
O
HN
HO SO
HO SO
3
3
O
HOOC
MES Buffer (pH 5.0)
O
O
O
O
O
O
HO
HO
MeO
Me
N
N
AcHN
AcHN
OH
CSA
n
OH
n
N+
Cl-
O
Alkynylated CSA
N
MeO
DMTMM
3
[carboxyl]/[DMTMM]/[amine]a
Tb (ºC) CCSAc (mg/mL)
DSyd
producte
entry
1
2
3
4
5
6
7
8
9
1:2:0.5
1:2:1
1:4:1
1:4:5
1:4:10
1:4:15
1:4:2
1:4:3
1:4:4
1:4:5
R. T.
R. T.
R. T.
R. T.
R. T.
R. T.
37
20
20
20
20
20
20
10
10
10
10
0
-
0.15
0.06
0.54
0
CSA1
-
CSA2
-
0
-
0.69
0.78
0.75
0.69
-
37
CSA3
37
-
-
1
0
37
a
b
4
5
6
7
8
9
Molar ratio of [carboxyl group (carboxyl)]/[DMTMM]/[propargylamine (amine)]. R. T. = room
c
d
temperature. The final concentration of CSA in MES buffer (pH 5.0). Degree of substitution of
1
alkyne, based on the carboxyl group of CSA, determined by H NMR integration of the alkyne
e
signal (2.69 ppm) to the GalNAc acetyl signal (2.05 ppm). The alkynylated chondroitin sulfates
with DS were determined to be 0.15 (CSA1), 0.54 (CSA2), and 0.78 (CSA3) for further
y
fucosylation.
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2
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2
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8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
2
3
4
Figure 2. H NMR spectra of the alkynylation of CSA (Table 1) in D O. The degree
2
1
of substitution of the alkyne was determined by H-NMR integration of the alkyne
signal (2.69 ppm) to the GalNAc acetyl signal (2.05 ppm).
5
6
7
8
9
0
To enhance the DS value of the amidation on the CSA, the reaction conditions
y
were further optimized including the carboxyl/DMTMM/propargylamine feed ratio,
reaction temperature, and final concentration of CSA. When the feed ratio of
carboxyl/DMTMM/propargylamine was set up to 1:4:5, the DS value clearly
y
1
increased to 0.54, as shown by H NMR (Table 1, entry 4). Thus, the enhancement of
1
the loading of propargylamine and DMTMM dramatically improved DS . However,
y
11 the further increase of the carboxyl/DMTMM/propargylamine feed ratio at 1:4:10 and
1
2
3
1:4:15 (Table 1, entries 5 and 6) resulted in the reaction mixture becoming turbid at 0
1
of DS . The high loading of propargylamine induced the precipitation of the starting
y
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materials, retarding the amidation reaction. These results indicated that the
carboxyl/DMTMM/propargylamine feed ratio should be below 1:4:5 to prevent this
phenomenon.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
4
5
6
7
8
9
0
1
As previously reported, coupling reaction mediated by DMTMM can result in the
5
3
degree of HA substitution up to 0.76. Meanwhile, decreasing the viscosity of the
solution could also increase the derivatization efficiency of the polysaccharide. Thus,
the final concentration of CSA was decreased to 10 mg/mL for amidation, and the
reaction temperature was set at 37 ºC to avoid the decomposition of DMTMM (Table
1, entries 7-10). The DS values were determined to be 0.69, 0.78, 0.75, and 0.69 for
y
1
1
the reactions at carboxyl/DMTMM/propargylamine feed ratios of 1:4:2, 1:4:3, 1:4:4
and 1:4:5, respectively. The DS values were not significantly increased as the loading
y
12 of propargylamine increased. In particular, the carboxyl/DMTMM/propargylamine
1
1
1
3
4
5
feed ratio of 1:4:3 yielded the product with highest DS of 0.78 (Table 1, entry 8). As
y
shown in Table 1, alkynylated CSA with various DS values were obtained under
y
different reaction conditions. To study the influence of the free carboxyl on the
16 anticoagulant activity, we chose alkynylated CSA with DS values of 0.15 (CSA1),
y
1
7
8
0.54 (CSA2) and 0.78 (CSA3) for the next coupling with fucose.
1
Furthermore, to study the influence of different CS sulfation patterns on the
19 anticoagulant activity, the CSE (chondroitin sulfate E) in the triethylammonium salt
form obtained from our previous report37 was applied for fucose grafting and
2
0
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alkynylation through DMTMM mediated amidation with propargylamine.
Alkynylation of the CSE yielded CES1, CSE2 and CSE3 according to the
counterpart procedures for CSA1, CSA2 and CSA3 preparation (Table 2). The DSy
values of CES1, CSE2 and CSE3 were determined to be 0.19, 0.53, and 0.72,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
respectively, according to the H NMR integration of the alkyne signal (2.69 ppm) to
the CH signal of GalNAc (2.05 ppm) (Figure S1).
3
7
8
Table 2. Synthesis of Alkynylated Chondroitin Sulfate E (CSE) with Propargyl
Amine Using DMTMM as Condensation Reagent
OSO H
H2N
OSO H
3
HN
3
HO SO
HO SO
3
3
O
HOOC
O
MES Buffer (pH 5.0)
O
O
O
O
O
O
O
HO
HO
MeO
Me
N
AcHN
AcHN
OH
CSE
n
OH
n
N
N+
O
Alkynylated CSE
N
Cl-
MeO
DMTMM
Tb (ºC) CCSEc (mg/mL)
9
entry [carboxyl]/[DMTMM]/ [amine]a
DSyd
product
1
2
3
1:2:1
1:4:5
1:4:3
R. T.
R. T.
37
20
20
10
0.19
0.53
0.72
CSE1
CSE2
CSE3
a
b
10
1
2
Molar ratio of [carboxyl group (carboxyl)]/[DMTMM]/[propargylamine (amine)]. R. T. =
c
d
1
1
room temperature. The final concentration of CSE in MES buffer (pH 5.0). Degree of
1
substitution of the alkyne, based on the carboxyl group of CSE, determined by H NMR
13
integration of the alkyne signal (2.69 ppm) to GalNAc acetyl signal (2.05 ppm).
14
Coupling of azide-fucose with alkynylated chondroitin sulfate through
CuAAC. Upon obtaining alkynylated CSA and CSE, we next conducted the coupling
reaction of alkynylated CS with azide functionalized fucose. CuAAC, an efficient
click reaction to achieve molecular reagent, has been widely applied for the
1
1
1
5
6
7
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modification of natural polysaccharides. In our study, the CuAAC reaction in an
aqueous solvent was employed for the coupling between alkynylated CS and
azide-functionalized fucose.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The CuAAC reaction was conducted under conventional conditions55 for
4
5
6
7
8
9
0
1
condensation of alkynylated CSA (CSA1, CSA2 and CSA3) and non-sulfated fucose
F1 (Table 3, entries 1-3). The alkynylated CSA were first dissolved in deionized
water with the addition of non-sulfated fucose F1. The reaction was performed in the
presence of CuSO ·5H O and L-ascorbate at 60 ºC for 24 h. The formation of a
4
2
triazole ring and grafting of fucose branches produced representative signals at around
1
1
1
8.33, 1.19, and 2.69 ppm in the H NMR spectra with the disappearance of the alkyne
peaks. The degree of substitution of fucose (DS ) was calculated based on the
f
1
12 carboxyl groups by H NMR integration of the CH signal of fucose (1.19 ppm) to the
3
1
1
1
3
4
5
CH signal of GalNAc (2.05 ppm). The DS values were determined to be 0.15, 0.50,
3 f
and 0.71 for the products CSA-F1, CSA-F2 and CSA-F3 from CSA1, CSA2, and
CSA3, respectively.
16 Table 3. Synthesis of fCS Mimetics by Copper-Catalyzed Cycloadditions of
1
7
Azide-Fucose with Alkyne-Functionalized Chondroitin Sulfate
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2
2
2
2
2
2
2
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2
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3
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N
N
N
N3
O
O
O
O
OR3
OR1
OR3
OR1
NH
O
NH
O
R O
R O
2
OR3
2
O
OR3
O
R O
3
R O
O
3
O
O
O
O
O
HO
HO
0
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0
1
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9
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5
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8
9
0
AcHN
CuSO4·5H2O
Na-ascorbate
AcHN
OH
n
OH
n
Alkynylated CS
CSA-F1  CSA-F3, CSA-SF1  CSA-SF3
CSE-F1  CSE-F3, CSE-SF1  CSE-SF3
60 °C
1
Mwb
Mnb
PDIb
alkyne
DSfc
Entry CS unit solvent
R , R , R
3
product
1
2
a
conv. (%) (kDa) (kDa) (M /M )
w
n
-
1
CSA1 H O
H, SO , H
100
100
100
100
100
100
19.3 16.1 1.20
0.15
CSA-F1
CSA-F2
CSA-F3
CSA-SF1
CSA-SF2
CSA-SF3
CSE-F1
CSE-F2
CSE-F3
CSE-SF1
CSE-SF2
CSE-SF3
2
3
-
2
3
4
5
6
7
8
9
1
1
CSA2 H O
H, SO , H
22.1 19.1 1.16
24.3 18.5 1.31
19.6 16.2 1.21
26.2 22.8 1.15
29.8 26.8 1.11
9.4 9.3 1.01
10.2 8.4 1.21
11.1 8.2 1.35
9.8 7.9 1.24
11.6 11.0 1.05
0.50
0.71
0.14
0.52
0.70
0.14
0.51
0.72
0.13
0.49
0.71
2
3
-
CSA3 H O
H, SO , H
2
3
CSA1 Bufferd H, SO , SO
-
-
3
3
-
-
-
CSA2 Buffer H, SO , SO
3
3
3
-
CSA3 Buffer H, SO , SO
3
-
-
CSE1 H O
SO , SO , H 100
3 3
2
-
-
CSE2 H O
SO , SO , H 100
3 3
2
-
-
CSE3 H O
SO , SO , H 100
3 3
2
-
-
-
0
1
CSE1 Buffer SO , SO , SO 100
3 3 3
-
-
-
CSE2 Buffer SO , SO , SO 100
3
3
3
-
-
-
12
CSE3 Buffer SO , SO , SO 100
14.1 10.8 1.31
3
3
3
a
1
b
2
3
4
5
Determined by disappearance of the alkyne signal (2.69 ppm) on H NMR. Number average
molecular weight (M ), weight average molecular weight (M ), and polydispersity index (PDI)
n
w
c
were determined by SEC-MALLS-RI. Degree of substitution of fucose unit, based on the
d
carboxyl group of the CS. Buffer = PBS buffer (pH 7.38).
6
7
8
9
0
1
The fucose branches in natural fCS are generally sulfated and play an important
role in its anticoagulant activity. To mimic the sulfated branches in natural fCS,
persulfated fucose F2 with azide functionalization was synthesized from compound
5
6, 57
F1 in the presence of SO ·Py in DMF.
Compound F2 was finally obtained as the
3
1
1
triethylammonium salt after neutralization, and the structure of compound F2 was
1
13
1
1
1
13
well characterized by H NMR, C NMR, H- H COSY and H- C HSQC (Figure
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S20). Trisulfated fucose F2 was then applied to the CuAAC reaction under the same
1
reaction condition with non-sulfated fucose F1. However, the H NMR spectrum
showed no signals for the triazole ring or CH of fucose, which indicated that the
3
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
grafting was unsuccessful (Table S1, entry 1; Figure S2). We speculated that the
highly sulfated fucose with a negative charge could chelate the positive charged Cu(I)
through static interaction, impeding the catalytic process of Cu(I). However, the
reaction efficiency could be significantly accelerated in the presence of
Cu(I)-stabilizing ligands.58 The most commonly used Cu(I)-stabilizing ligands are
hydrophobic tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine (TBTA)59 and
hydrophilic tris(3-hydroxypropyltriazolylmethyl)amine (THPTA).60-62 Due to the
1
1
aqueous solubility of the starting materials, hydrophilic THPTA was adopted as a
1
12 Cu(I)-stabilizing ligand for the CuAAC reactions (Table S1, entry 2). The H NMR
13
14
15
spectrum still showed no signals for the triazole ring or CH of fucose, although the
3
signals of alkyne disappeared (Figure S2). During the process of the CuAAC reaction,
the alkyne groups first react with Cu(I) to form intermediates, after which they react
16 with the azide groups to form triazole rings (Scheme S1). This observation indicated
1
7
8
that THPTA indeed hindered the chelation between the sulfate groups and Cu(I), but
1
the intermediates did not react with trisulfated fucose F2. To this end, the trisulfated
+
19 fucose F2 was transformed into the Na form after treatment with a cation exchange
+
2
0
1
resin (Na form) in a PBS buffer (pH 7.38). In addition, the reaction was performed
2
under nitrogen gas to avoid oxidation of Cu(I) by O . A freshly prepared stock
2
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
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5
5
5
5
5
5
6
1
2
3
4
5
6
7
8
9
0
solution of CuSO ·5H O in a PBS buffer (pH 7.38) was premixed with THPTA and
4
2
added to the mixed solution with L-ascorbate in a PBS buffer (pH 7.38). The mixture
was stirred at room temperature for 30 min, and subsequently, trisulfated fucose F2 in
PBS buffer (pH 7.38) was added and stirred at 60 ºC for 24 h (Table 3, entry 4). The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
obtained product (CSA-SF1) showed representative signals for the triazole ring and
1
CH of fucose, and the DS was determined to be 0.14 (Figure S2) based on the H
3
f
NMR spectrum. According to the established protocol for the CuAAC reaction of
CSA1 with trisulfated fucose F2, the products CSA-SF2 with a DS of 0.52 and
f
CSA-SF3 with a DS of 0.70 were obtained from CSA2 and CSA3, respectively, with
f
1
1
an alkyne conversion up to 100% (Table 3, entries 5 and 6).
1
The CuAAC reaction of alkynylated CSE was performed according to the above
12 optimized procedure for the preparation of non- and tri-sulfated fucose grafted CSA.
13
14
15
The grafting products of CSE-F1, CSE-F2 and CSE-F3 were obtained with DSf
values of 0.14, 0.51, and 0.72, respectively (Table 3, entries 7-9). Under optimized
coupling reaction between alkynylated CSA and sulfated fucose F2, CSE-SF1,
16 CSE-SF2, and CSE-SF3 were obtained from CSE1, CSE2, and CSE3 with DSf
1
7
8
values of 0.13, 0.49, and 0.71, respectively (Table 3, entries 10-12).
1
In contrast to Bedini’s approach,35, 36 we developed an alternative modular route to
19 assemble fCS analogs without the need for protection/deprotection steps. 12 fCS
20
mimetics were obtained with various sulfation patterns and fucose densities (DSf
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values in the 0.13~0.72 range) through the concise grafting protocols developed in
this study (Table S4). The molecular weights of synthetic fCS mimetics were
characterized by SEC-MALLS-RI. The fCS mimetics with higher DS values
f
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
possessed higher molecular weights than those with lower DS values (Table 3). The
f
sulfate contents of CSA-F3, CSA-SF3, CSE-F3, and CSE-SF3 were consistent with
grafting of fucose onto natural CS (Table S5) and FT-IR spectra confirmed complete
1
reaction of alkyne with azide (Figure S21, S22). The integrated NMR analysis ( H
13
1
1
1
13
NMR, C NMR, H- H COSY and H- C HSQC) for the fCS mimetics with the
highest DS values of the fucose branches (CSA-F3, CSA-SF3, CSE-F3, and
f
1
1
CSE-SF3) also confirmed the successful grafting of fucose onto alkynylated CS
through observation of the triazole and CH of the fucose signals (Figure 3, S47-S49;
3
12 Table 4, S6-S8). Moreover, the anomeric proton signals of CS were around 4.5 ppm.
13
14
15
For the fucose branches, chemical shifts of the anomeric protons for the trisulfated
fucose were strongly shifted to 5.21 ppm compared with non-sulfated fucose at 4.83
ppm (CSA-SF3 and CSE-SF3 vs CSA-F3 and CSE-F3). Taking CSA-SF3 as an
16 example (Figure 3, Table 4), the signals at 5.21/96.27, 4.62/104.29, and 4.55/100.39
1
13
1
7
8
were assigned to anomeric H/ C of fucose, GlcA, and GalNAc, respectively,
1
13
1
according to the H- C HSQC. The signals for the fucose sugar ring were deduced
1
1
19 from the H- H COSY at 4.52/4.61/4.87 ppm for H-2/H-3/H-4. The crosslinking
1
13
2
0
1
signals of H-6/C-6 at 3.77/60.94 (GalNAc) were identified from the H- C HSQC
2
(blue circle) together with the signal of H-4/C-4 at 4.86/76.38 (GalNAc), which
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confirmed that sulfation occurred at the C-4 position of the backbone. The
H-2/H-3/H-4 signals at 3.40/3.67/3.86 ppm for GlcA were clearly observed from the
1
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H- H COSY, which demonstrated the linkage of GalNAc at the C-4 position of GlcA.
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13
In addition, another three H/ C signals at 4.73/50.24, 4.15/66.25, and 4.01/66.06
1
13
(blue circles) were attributed to CH residues of linkers based on the H- C HSQC
2
spectrum.
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1
13
Figure 3. 1D and 2D-NMR spectra of CSA-SF3: (A) H NMR, (B) C NMR, (C)
1
1
1
13
9
0
H- H COSY, (D) H- C HSQC. F, A, and G stand for fucose, GlcA and GalNAc,
1
1
respectively.
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13
1
Table 4. H and C NMR Chemical Shift Assignments of CSA-SF3 (500 MHz,
a
12 298 K, D O)
2
GlcA
GalNAc
Fucose
Linker
position
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1H
13C
1H
13C
1H
13C
1H
13C
1
2
3
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6
4.62
104.29 4.55
100.39 5.21
96.27
-
-
-
-
-
-
-
-
3.40
72.04
73.58
78.74
74.86
4.00
4.02
4.86
3.85
51.21
74.72
76.38
74.98
60.94
22.38
174.79
-
4.52
71.91
-
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0
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9
0
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9
0
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9
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9
0
3.67
4.61
72.35
-
3.86
4.87
78.73
-
3.74
3.59
66.06
-
-
-
-
-
-
-
-
169.03 3.77
1.20
15.69
-
CH CO
-
-
-
-
-
-
2.03
-
-
-
-
-
-
-
-
-
-
-
-
-
3
CH CO
-
-
-
-
-
-
-
3
n.d.b
triazole
8.29
4.01
4.15
4.73
fucose-CH CH
-
66.06
66.25
50.24
2
2
fucose-CH CH
-
2
2
CS-CONHCH2
-
a
b
1
Chemical shifts expressed in δ. Not determinable.
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1
Anticoagulant activities of fCS mimetics. To evaluate the anticoagulant activities
of synthetic fCS mimetics, preliminary activated partial thromboplastin time (APTT),
prothrombin time (PT), and thrombin time (TT) of plasma clotting assays, which are
frequently applied to evaluate the intrinsic, extrinsic, and common pathways of the
6
3
coagulation, were conducted as shown in Figure 4A-4C. Deionized water was used
as a negative control while heparin (HP) and low molecular weight heparin (LMWH)
were used as positive controls. The fCS mimetics with CSA backbones exhibited no
obvious anticoagulant activities, while CSE-F1 and CSE-SF1 with CSE backbones
indicated significant effects on their anticoagulant activities. They exhibited
significantly prolonged APTTs for more than 120 s, which were comparable to that of
1
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natural fCS extracted from Holothuria Polli. Meanwhile, all fCS mimetics exhibited
no significant capabilities for PT and TT prolongation. These preliminary results
indicated that fCS mimetics exhibited intrinsic anticoagulant activities, which is
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clearly consistent with the natural fCS reported in the literature before.8, 20, 34 The fCS
mimetics developed in our study possess moderate anticoagulant activity, which is
comparable to the substrates reported in Bedini’s work.35, 36 The degree of free
carboxyl groups, which was calculated by 1-DS , decreased with the increment of DS
f
f
values. Lower DS values (at ~0.20) for the fCS mimetics with CSE backbones
f
exhibited longer APTTs, which suggested that fucose branches were required for
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1
1
anticoagulant activity, even at low DS values, and free carboxyl groups were also
f
important for the anticoagulant activity. However, higher DS values resulted in the
f
12 elimination of their anticoagulation activities possibly due to the lower percentages of
13
14
15
free carboxyl groups. Accordingly, CSE-F1 and CSE-SF1 exhibited
anticoagulant activity due to highly reserved fCS structure properties including
fucose branches, specific sulfation and carboxyl groups, which are inevitable
16 for the biological activity of natural fCS. The fCS mimetic CSE-SF1 with higher
1
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sulfated fucose branches exhibited slightly better anticoagulant activity compared to
1
CSE-F1 with non-sulfated fucose branches, which revealed that the increase of
19 sulfation content on the fucose branches can improve the anticoagulant activities.
20
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Figure 4. The anticoagulant activities of synthetic fCS mimetics. Effect of synthetic
fCS mimetics on prolongation of (A) APTT, (B) TT, and (C) PT at a concentration of
500 μg/mL. APTT > 250s for HP and LMWH, and TT > 400s for HP and LMWH. (D)
Inhibition of ATIII-mediated FXa activity by synthetic fCS mimetics.
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To further understand the anticoagulant activity of CSE-F1 and CSE-SF1, the AT
III-dependent activity against factor Xa (Figure 4D) and IIa (Figure S4) were
1
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measured, and the IC values are summarized in Table 5. The IC values of the
50
50
1
1
anti-FXa activities for CSE-F1 and CSE-SF1 were determined to be 51.10 μg/mL
and 21.26 μg/mL, respectively, while anti-FIIa activities for CSE-F1 and CSE-SF1
12 were not detected. Meanwhile, CSE-SF1 also exhibited higher anti-Xa activities than
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