d‑Glucuronate and d‑Glucuronate Glycal Acceptors for the Scalable Synthesis of d‑GlcN-α-1,4‑d‑GlcA Disaccharides and Modular Assembly of Heparan Sulfate

Reported herein is a scalable chemical synthesis of disaccharide building blocks for heparan sulfate (HS) oligosaccharide assembly. The use of d-glucuronate-based acceptors for dehydrative glycosylation with d-glucosamine partners is explored, enabling diastereoselective synthesis of appropriately protected HS disaccharide building blocks (d-GlcN-α-1,4-d-GlcA) on a multigram scale. Isolation and characterization of key donor (1,2 glycal)- and acceptor (ortho-ester, anhydro)-derived side products ensure methodology improvements to reduce their formation; protecting the d-glucuronate acceptor at the anomeric position with a para-methoxyphenyl unit proves optimal. We also introduce glycal uronate acceptors, showing them to be comparative in reactivity to their pyranuronate counterparts. Taken together, this gram-scale access offers the capability to explore the iterative assembly of defined HS sequences containing the d-GlcN-α-1,4-d-GlcA repeat, highlighted by completing this for two tetrasaccharide syntheses

d‑Glucuronate and d‑Glucuronate Glycal Acceptors for the Scalable Synthesis of d‑GlcN-α-1,4‑d‑GlcA Disaccharides and Modular Assembly of Heparan Sulfate

Reported herein is a scalable chemical synthesis of disaccharide building blocks for heparan sulfate (HS) oligosaccharide assembly. The use of d-glucuronate-based acceptors for dehydrative glycosylation with d-glucosamine partners is explored, enabling diastereoselective synthesis of appropriately protected HS disaccharide building blocks (d-GlcN-α-1,4-d-GlcA) on a multigram scale. Isolation and characterization of key donor (1,2 glycal)- and acceptor (ortho-ester, anhydro)-derived side products ensure methodology improvements to reduce their formation; protecting the d-glucuronate acceptor at the anomeric position with a para-methoxyphenyl unit proves optimal. We also introduce glycal uronate acceptors, showing them to be comparative in reactivity to their pyranuronate counterparts. Taken together, this gram-scale access offers the capability to explore the iterative assembly of defined HS sequences containing the d-GlcN-α-1,4-d-GlcA repeat, highlighted by completing this for two tetrasaccharide syntheses

An Updated Synthesis of the Diazo-Transfer Reagent Imidazole-1-sulfonyl Azide Hydrogen Sulfate

Imidazole-1-sulfonyl azide and salts thereof are valuable reagents for diazo-transfer reactions, most particularly conversion of primary amines to azides. The parent reagent and its HCl salt present stability and detonation risks, but the hydrogen sulfate salt is significantly more stable. An updated procedure for the large-scale synthesis of this salt avoids isolation or concentration of the parent compound or HCl salt and will facilitate the use of hydrogen sulfate salt as the reagent of choice for diazo transfer

First Gram-Scale Synthesis of a Heparin-Related Dodecasaccharide

The first example of a gram-scale synthesis of a structurally defined, heparin-related dodecasaccharide is reported. An iterative 14-step process using an iduronate donor disaccharide delivers >1g quantities of the dodecasaccharide sequence [GlcNS-IdoA2S]₆-OMe in 15% overall yield from the reducing terminal disaccharide, a 2 orders of magnitude increase in scale for access to synthetic heparanoid dodecasaccharide mimetics. The synthesis also delivers multigram amounts of the protected oligosaccharides from tetra- through to dodecasaccharide

Site-selectively 6-<i>O</i>-sulfated dodecasaccharides.

<p>Schematic representation of completely non-6-<i>O</i>-sulfated ([NSIS]₆), site-selectively mono-6-<i>O</i>-sulfated ([NSIS6S]-[NSIS]₅) and fully 6-<i>O</i>-sulfated ([NS6SIS]₆) dodecasaccharides. [NSIS]₆ refers to a dodecasaccharide that consists of 6 alternate <i>N</i>-sulfated glucosamine (NS)-Iduronic acid 2-<i>O</i>-sulfate (IS) disaccharides. [NSIS6S]-[NSIS]₅ refers to a dodecasaccharide that contains a 6S moiety in glucosamine of the first disaccharide at the non-reducing end. [NSIS6S]₆ refers to a dodecasaccharide sulfated at every NSIS disaccharide.</p

<i>In vitro</i> inhibitory potential of site-selectively 6-<i>O</i>-sulfated dodecasaccharides.

<p>A, dodecasaccharides were tested for effects on FGF2- and VEGF₁₆₅-induced HUVEC proliferation. The treatments were performed for 5 days. Stimulation of proliferation in response to FGF2 (20 ng/ml) and VEGF₁₆₅ (20 ng/ml) is expressed as 100%. Dodecasaccharides were used at 50 μg/ml concentration. The mean ± SEM (n = 3) is shown. †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05. B to C, inhibition of HUVEC FGF2- and VEGF₁₆₅-induced migration by dodecasaccharides was tested in wound healing assay. Wounds were created in confluent monolayers of serum-starved HUVEC and FGF2 (B) or VEGF₁₆₅ (C) was added to promote cell migration into the wounds. Dodecasaccharides were used at a range of concentrations starting from 0.1 μg/ml for FGF2 and 1 μg/ml for VEGF₁₆₅ with the highest concentration being 50 μg/ml in all assays. The wound area was measured at the beginning and 24 hours after the treatment. The area repopulated after 24 hours in response to growth factor alone is expressed as 100% (control). The effect of dodecasaccharides is expressed as a percentage of repopulated area induced by a growth factor alone. All experiments were performed three times in triplicates. The mean ± SEM (n = 3) is shown. *, <i>P</i> < 0.001; †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05. D, HUVEC spheroids were embedded in fibrin gels that were overlaid with either EBM-2 media lacking FGF2 and VEGF₁₆₅, EBM-2 media supplemented with FGF2 (5 ng/ml) or VEGF₁₆₅ (2.5 ng/ml) and EBM-2 media supplemented with FGF2 or VEGF₁₆₅ and dodecasaccharides (50 μg/ml). The treatment was performed for 24 hours. Scale bars represent 200 μm. E, sprouting area in each spheroid was evaluated using Metamorph software where the area of outgrowing sprouts was derived by subtracting the area of a spheroid without sprouts from the total area of a spheroid. The increase of sprouting area after stimulation with FGF2 or VEGF₁₆₅ is expressed as 100% (control). The ability of oligosaccharides to reduce FGF2 and VEGF₁₆₅-induced endothelial cell sprouting is expressed as a percentage of control. 20–30 spheroids were analysed per each experiment and three independent experiments were performed. The values are shown as mean ± SEM (n = 3). *, <i>P</i> < 0.001; †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05. F, the effect of oligosaccharides on endothelial tube formation was evaluated in three-dimensional fibrin gel bead assay. FGF2 and VEGF₁₆₅ were used at 5 ng/ml concentration. No endothelial tubes developed in the absence of FGF2 and VEGF₁₆₅. Dosing was performed for 5 days. The ratio of average number of endothelial tubes per bead is shown. Three independent experiments were performed each in triplicate. The values are expressed as mean ± SEM (n = 3). *, <i>P</i> < 0.001; †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05.</p

Impact of dodecasaccharides on FGF2/FGFR1 and VEGF/VEGFR2 complex formation.

<p>A, levels of FGF2 bound to FGFR1 IIIc-Fc–coated plates were measured by ELISA. Binding of FGF2 to FGFR1 is expressed as 1 (control). Fold change in FGF2 binding to FGFR1-coated plates in the presence of HS or specific dodecasaccharides at increasing concentrations as compared to control is shown. Each experiment was performed twice in triplicate. The data are presented as the mean ± SD (n = 2). *, <i>P</i> < 0.001; †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05. B, the ability of [NSIS]₆ and [NSIS6S]-[NSIS]₅ to compete with HS (1 μg/ml) for binding of FGF2 to FGFR1. FGF2 was premixed with HS alone (1 μg/ml), HS and [NSIS]₆ or [NSIS6S]-[NSIS]₅ (50 and 100 μg/ml) or dodecasaccharides alone (50 μg/ml). FGFR1-bound FGF2 was detected by ELISA. FGF2 binding to FGFR1 in the absence of HS and dodecasaccharides is expressed as 1 (control). The data were derived from two independent experiments performed in triplicate and shown as the mean ± SD. *, <i>P</i> < 0.001; †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.05. C, A745 CHO flg-1A cells were added to the wild type CHO-K1 monolayers in serum-free medium containing FGF2 in the absence or presence of indicated dodecasaccharides. Cells adherent to the monolayer were counted following incubation for 2 hours. The data are presented as a percentage of cell binding in the absence of dodecasaccharides (100%). Each point is the mean ± SD of three independent experiments performed in triplicate. D, serum-starved HUVEC were stimulated with FGF2 (20 ng/ml) for 10 min in the absence or presence of indicated dodecasaccharides. Phosphorylated FRS2 and ERK were detected by Western blotting. Total protein loading levels were visualized by probing with the anti-GAPDH antibody. Stimulation with FGF2 alone is expressed as 1. Normalized fold change in the intensities of bands as compared to FGF2 stimulation alone is shown below each blot as determined by densitometric analysis. E, densitometric evaluation of the intensities of bands combined from an independent experiment performed as in D and an experiment shown in D. Fold change in phosphorylated FRS2 and ERK levels in response to FGF2 stimulation in the absence and presence of oligosaccharides is shown. Values represent the mean ± SD (n = 2). *, <i>P</i> <0.0001 †, <i>P</i> < 0.01. F, levels of VEGF₁₆₅ bound to VEGFR2-Fc were measured using VEGF-specific ELISA. VEGF₁₆₅ binding to VEGFR2-coated plate in the absence of HS or dodecasaccharides is expressed as 1. Two independent experiments were performed in triplicate and the data are presented as the mean ± SD. †, <i>P</i> < 0.01; ‡, <i>P</i> < 0.0001. G, inhibition of VEGF₁₆₅-induced phosphorylation of VEGFR2 and ERK. Serum-starved HUVEC were stimulated with VEGF₁₆₅ (20 ng/ml) for 5 min in the absence or presence of dodecasaccharides at indicated concentrations. Phospho-VEGFR2 (Y1214 and Y1175) and phospho-ERK were detected by immunoblotting with the respective antibodies. GAPDH levels show equal total protein levels. The intensities of bands for phospho-VEGFR2, phospho-ERK and GAPDH were analysed by densitometry. Phospho-VEGFR2 and phospho-ERK levels were normalized to GAPDH levels. Stimulation with VEGF₁₆₅ alone is expressed as 1. Fold change in the intensities of phosphorylated VEGFR2 and ERK upon each treatment is shown below the blots. H, average values of band intensities generated by densitometric analysis of bands shown in G and those from another independent experiment are shown. Numbers on the horizontal axis show different concentrations of each oligosaccharide. The mean ± SD (n = 2) is shown. †, <i>P</i> <0.01 ‡, <i>P</i> < 0.05.</p

[NSIS6S]-[NSIS]₅ inhibits FGF2 secreted from cancer cells.

<p>A to B, FGF2 concentration in HEC-1-B and FGF2-B9 cell lysates and conditioned medium was determined by ELISA. Two independent experiments were performed and the data are expressed as the mean ± SD. C, angiogenic potential of conditioned media collected from HEC-1-B and exogenous FGF2 overexpressing HEC-1-B cell line FGF2-B9 was evaluated in three dimensional fibrin gel HUVEC bead assay. Conditioned medium collected from HEC-1-B cells induced minimal endothelial tube outgrowth insensitive to blocking anti-FGF2 antibody or treatment with anti-angiogenic dodecasaccharides [NSIS]₆ and [NSIS6S]-[NSIS]₅ (upper panel). Treatment with FGF2-B9 conditioned medium resulted in increased outgrowth of endothelial tubes which was reduced by treatment with anti-FGF2 antibody, [NSIS]₆ and [NSIS6S]-[NSIS]₅. Scale bars represent 100 μm. D, quantification of a number of endothelial tubes per bead as shown in C. The number of tubes per bead induced by HEC-1-B conditioned medium is expressed as 1 (control). The effect of FGF2-B9 conditioned medium and all treatments is shown as fold change compared to the control. Two independent experiments were performed in triplicate. The data are expressed as the mean ± SD (n = 2). *, <i>P</i> < 0.001; ‡, <i>P</i> < 0.05.</p

[NSIS6S]-[NSIS]₅ inhibits FGF2-induced tumor blood vessel formation.

<p>A, tumor xenografts were established subcutaneously from HEC-1-B and FGF2-B9 cell lines in female Balb/c-NUDE mice (n = 9) and allowed to grow to a volume of approximately 50 mm³ before starting the dosing. HEC-1-B tumor-bearing mice were left untreated, while FGF2-B9 tumor-bearing mice were treated with saline (daily), [NSIS6S]-[NSIS]₅ (160 mg/kg b.i.d) or sunitinib (40 mg/kg daily) for 9 days. Tumor volume was monitored every day for 9 days. B, two hours after the last dose, animals were sacrificed and tumor sections were processed for immunofluorescence staining with anti-mouse CD31 antibody to visualize tumor vasculature (red). Nuclei were visualized with Hoechst staining (blue). Scale bars represent 250 μm. C, number of vessels in each tumor section was evaluated using ImageJ software where each vessel represents a fluorescent object visualized by anti-CD31 staining. The number of vessels was normalized per area of non-necrotic tumor tissue. Five tumor sections from each of the nine tumors from each treatment group were analyzed. The data are presented as the mean ± SEM (n = 9). D, average vessel size was derived from the analysis of a size of CD31-stained blood vessels in each tumor section using Image J programme. Sections from each treatment group consisting of 9 tumors were analyzed. The data are shown as the mean ± SEM (n = 9). ‡, <i>P</i> < 0.05. E, phospho-FRS2 in blood vessels was visualized through immunofluorescence staining of tumor xenograft sections with antibodies against phospho-FRS2 and CD31. White arrows indicate blood vessels that are positive for phospho-FRS2 staining. Arrowheads show blood vessels that are negative for phospho-FRS2 staining. Scale bars represent 200 μm. F, phospho-FRS2 positive blood vessels were counted in a tumor section derived from each tumor. Nine tumors were examined in each treatment group. Percentage of phospho-FRS2 positive blood vessels relative to total CD31-positive vessels is expressed as the mean ± SEM (n = 9). ‡, <i>P</i> < 0.05.</p

Synthesis and Scalable Conversion of l‑Iduronamides to Heparin-Related Di- and Tetrasaccharides

A diastereomerically pure cyanohydrin, preparable on kilogram scale, is efficiently converted in one step into a novel l-iduronamide. A new regioselective acylation of this iduronamide and a new mild amide hydrolysis method mediated by amyl nitrite enables short, scalable syntheses of an l-iduronate diacetate C-4 acceptor, and also l-iduronate C-4 acceptor thioglycosides. Efficient conversions of these to a range of heparin-related gluco-ido disaccharide building blocks (various C-4 protection options) including efficient multigram access to key heparin-building block ido-thioglycoside donors are described. A 1-OAc disaccharide is converted into a heparin-related tetrasaccharide, via divergence to both acceptor and donor disaccharides. X-ray and NMR data of the 1,2-diacetyl iduronate methyl ester and the analogous iduronamide show that while both adopt ¹<i>C</i>₄ conformations in solution, the iduronate ester adopts the ⁴<i>C</i>₁ conformation in solid state. An X-ray structure is also reported for the novel, ⁴<i>C</i>₁-conformationally locked bicyclic 1,6-anhydro iduronate lactone along with an X-ray structures of a novel distorted ⁴<i>C</i>₁ iduronate 4,6-lactone. Deuterium labeling also provides mechanistic insight into the formation of lactone products during the novel amyl nitrite-mediated hydrolysis of iduronamide into the parent iduronic acid functionality