A Concise Synthesis of a BODIPY-Labeled Tetrasaccharide Related to the Antitumor PI-88

A convergent synthetic route to a tetrasaccharide related to PI-88, which allows the incorporation of a fluorescent BODIPY-label at the reducing-end, has been developed. The strategy, which features the use of 1,2-methyl orthoesters (MeOEs) as glycosyl donors, illustrates the usefulness of suitably-designed BODIPY dyes as glycosyl labels in synthetic strategies towards fluorescently-tagged oligosaccharides.

Based on these precedents, we thought it would be of interest to investigate the feasibility of a synthetic approach to BODIPY-labeled PI-88 saccharide components, where the fluorescent dye is incorporated from the beginning of the synthetic sequence. Additionally, we envisioned that the incorporation of the lipophilic BODIPY moiety at the reducing end of a PI-88 saccharide analogue would bring one additional advantage by facilitating the visualization and detection of the synthetic intermediates along the saccharide synthesis [19]. Additionally, in light of some reported literature precedents [20], the incorporation of the lipophilic BODIPY core to the saccharidic ensemble could lead to ameliorated biological activity in the ensuing saccharides. Thus, in this Article, we report a synthetic approach to a PI-88 tetrasaccharide analogue [21] featuring the use of 1,2-methyl orthoester (MeOE) glycosyl donors, i.e., 4 (Figure 1), in which a BODIPY-type fluorescent probe could be attached at the reducing end of the saccharides from the beginning of the synthesis. methyl orthoester (MeOE) glycosyl donors, i.e., 4 (Figure 1), in which a BODIPY-type fluorescent probe could be attached at the reducing end of the saccharides from the beginning of the synthesis.
Finally, glycosyl acceptor 6, containing a mannopyranoside triol unit, was regioselectively mono-glycosylated at O-3'with MeOE disaccharide 9 [44], to yield PI-88 tetrasaccharide precursor analogue 10, in 53% yield (Scheme 4). An alternative glycosylation of 6 with a MeOE monosaccharide, e.g., 4a or 4b, rather than with a disaccharide, i.e., 9, in our hands consistently led to low yields of the trisaccharide analogue. Finally, glycosyl acceptor 6, containing a mannopyranoside triol unit, was regioselectively mono-glycosylated at O-3'with MeOE disaccharide 9 [44], to yield PI-88 tetrasaccharide precursor analogue 10, in 53% yield (Scheme 4). An alternative glycosylation of 6 with a MeOE monosaccharide, e.g., 4a or 4b, rather than with a disaccharide, i.e., 9, in our hands consistently led to low yields of the trisaccharide analogue. To impel the advanced applications of the new glycoprobes, we analyzed the photonic behavior of BODIPY 15 and its saccharide derivatives 16a and 10, under low (photophysical properties) and high (laser properties) irradiation regimes. The replacement of the fluorine atoms at the boron bridge, as in 11 [38], by phenyl groups, as in 15 [45], had low impact on the spectral properties of BODIPY ( Figure 2) but induced both a decrease of the emission efficiency (Table 1) and a biexponential character of the fluorescent lifetime (Table S1), regardless of the environmental properties. The free motion of these phenyl rings chelating the boron atom reduced the planarity of the dipyrrin core (computed bending angles in the dipyrrin core up to 16° in the excited state, Figure 2), increasing the internal conversion processes with a deleterious effect on the fluorescence signal. The labelling of a disaccharide or tetrasaccharide with BODIPY 15, as in compound 16a and 10, respectively, widened the absorption spectrum, while the spectral profile of fluorescence Finally, glycosyl acceptor 6, containing a mannopyranoside triol unit, was regioselectively mono-glycosylated at O-3'with MeOE disaccharide 9 [44], to yield PI-88 tetrasaccharide precursor analogue 10, in 53% yield (Scheme 4). An alternative glycosylation of 6 with a MeOE monosaccharide, e.g., 4a or 4b, rather than with a disaccharide, i.e., 9, in our hands consistently led to low yields of the trisaccharide analogue. To impel the advanced applications of the new glycoprobes, we analyzed the photonic behavior of BODIPY 15 and its saccharide derivatives 16a and 10, under low (photophysical properties) and high (laser properties) irradiation regimes. The replacement of the fluorine atoms at the boron bridge, as in 11 [38], by phenyl groups, as in 15 [45], had low impact on the spectral properties of BODIPY ( Figure 2) but induced both a decrease of the emission efficiency (Table 1) and a biexponential character of the fluorescent lifetime (Table S1), regardless of the environmental properties. The free motion of these phenyl rings chelating the boron atom reduced the planarity of the dipyrrin core (computed bending angles in the dipyrrin core up to 16° in the excited state, Figure 2), increasing the internal conversion processes with a deleterious effect on the fluorescence signal. The labelling of a disaccharide or tetrasaccharide with BODIPY 15, as in compound 16a and 10, respectively, widened the absorption spectrum, while the spectral profile of fluorescence To impel the advanced applications of the new glycoprobes, we analyzed the photonic behavior of BODIPY 15 and its saccharide derivatives 16a and 10, under low (photophysical properties) and high (laser properties) irradiation regimes. The replacement of the fluorine atoms at the boron bridge, as in 11 [38], by phenyl groups, as in 15 [45], had low impact on the spectral properties of BODIPY ( Figure 2) but induced both a decrease of the emission efficiency (Table 1) and a biexponential character of the fluorescent lifetime (Table S1), regardless of the environmental properties. The free motion of these phenyl rings chelating the boron atom reduced the planarity of the dipyrrin core (computed bending angles in the dipyrrin core up to 16 • in the excited state, Figure 2), increasing the internal conversion processes with a deleterious effect on the fluorescence signal. The labelling of a disaccharide or tetrasaccharide with BODIPY 15, as in compound 16a and 10, respectively, widened the absorption spectrum, while the spectral profile of fluorescence matched that of its precursor (Figure 2). It is noteworthy that BODIPYs 10 and 16a displayed a brighter fluorescence with longer lifetimes than their non-glycosylated counterpart 15 in all tested media and regardless of the number of saccharide units appended (Table 1 and Table S1). Thus, glycosylation of the ortho-hydroxymethyl group of the C-8-aryl residue led to a more rigid and compact molecular structure, e.g., 16a and 10, owing to the higher steric hindrance imposed by the bulky disaccharide. In fact, the structural arrangement of the C-8-benzyl residue in 16a was nearly orthogonal (twisting dihedral angle computed in the ground state of 75 • in 15 vs. 85 • in 16a), reducing the internal conversion pathways associated to conformational freedom. Consequently, BODIPY-saccharides 16a and 10 behaved as efficient and stable fluorescent glycoprobes even under laser irradiation conditions, exhibiting a lasing efficiency up to 38% in the green spectral region (540 nm, Table 1) with high photostability, since their laser emission remained at the initial level even after 70,000 pump pulses. This good tolerance to intense and prolonged irradiation is a highly desirable property for fluorescent labels to provide long-lasting bioimages. Therefore, from a photonic point of view, the orthoposition of 8-phenyl BODIPYs is highlighted as a suitable grafting position to tag (oligo)saccharides, even resulting in an amelioration of the photonic performance of the original labeling dye.
matched that of its precursor (Figure 2). It is noteworthy that BODIPYs 10 and 16a displayed a brighter fluorescence with longer lifetimes than their non-glycosylated counterpart 15 in all tested media and regardless of the number of saccharide units appended (Tables 1 and S1). Thus, glycosylation of the ortho-hydroxymethyl group of the C-8-aryl residue led to a more rigid and compact molecular structure, e.g., 16a and 10, owing to the higher steric hindrance imposed by the bulky disaccharide. In fact, the structural arrangement of the C-8-benzyl residue in 16a was nearly orthogonal (twisting dihedral angle computed in the ground state of 75° in 15 vs. 85° in 16a), reducing the internal conversion pathways associated to conformational freedom. Consequently, BODIPY-saccharides 16a and 10 behaved as efficient and stable fluorescent glycoprobes even under laser irradiation conditions, exhibiting a lasing efficiency up to 38% in the green spectral region (540 nm, Table 1) with high photostability, since their laser emission remained at the initial level even after 70,000 pump pulses. This good tolerance to intense and prolonged irradiation is a highly desirable property for fluorescent labels to provide long-lasting bioimages. Therefore, from a photonic point of view, the ortho-position of 8-phenyl BOD-IPYs is highlighted as a suitable grafting position to tag (oligo)saccharides, even resulting in an amelioration of the photonic performance of the original labeling dye.     1 Registered under a soft irradiation regime; dye concentration: 2 µM. Absorption (λ ab ) and fluorescence (λ fl ) wavelength, molar absorption (ε max ) (10 4 M −1 cm −1 ), fluorescence quantum yield (∅), and amplitude-average lifetime (<τ>). 2 Recorded under a hard irradiation regime; dye concentration 2 mM. Peak wavelength for the laser emission (λ la ) and efficiency (Eff (%)) defined as the ratio between the energy of the laser output and the pump energy incident on the cell surface.

Conclusions
In summary, we developed a convergent, efficient, synthetic strategy to BODIPYlabeled PI-88 tetrasaccharide components (10) [46], which serves to illustrate the scope and usefulness of MeOEs as glycosyl donors. The inclusion of the BODIPY-tag from the beginning of the synthesis facilitates the visual recognition (thin-layer chromatography, TLC) of the labeled-saccharide acceptor and the glycosylated products therefrom, among the rest of the non-fluorescent side-products arising from side-reactions of the MeOE glycosyl donor [36]. This feature becomes particularly appealing when excess amounts of glycosyl donors are required to lead the glycosylation to completion. On the other hand, the chemically stable 4, 4'-diphenyl BODIPY derivative (15), used as a tag, displayed good fluorescent properties and photostability under strong and prolonged irradiation, and was also able to withstand all reaction conditions employed in the synthetic sequence leading to 10 [20]. Our results also indicate that the incorporation of carbohydrate subunits at the ortho-hydroxymethyl group of the C-8-aryl substituent has a beneficial effect on the, already, good photophysical features of the BODIPY dye.

General Information
The solvents and reagents used in the transformations included in the manuscript were obtained from commercial sources. In the glycosylation experiments, the adventitious water content was removed by repeated evaporation of the sample with toluene. The temperature at which the reactions were carried out will be mentioned unless room temperature was used. The glycosylation reactions were carried out in dried flasks fitted with rubber septa under an argon atmosphere.
A 5.0 M stock solution of triethyloxonium tetrafluoroborate, employed in the preparation of the BODIPYs, was prepared by dissolving 25 g (0.131 mmol) of the salt in 26.3 mL of anhydrous methylene chloride.
Anhydrous MgSO 4 was used to dry organic solutions during workup. Evaporation of the solvents was performed using a rotary evaporator (Buchi, Flawil, Switzerland). Flash column chromatography was used to purify or separate the samples. Thin-layer chromatography (TLC) was conducted on Kieselgel 60 F254. Spots corresponding to BODIPY-containing molecules were spotted under visible light. TLCs were then inspected under UV irradiation (254 nm) followed by charring with a solution of 20% aqueous H 2 SO 4 (200 mL) in AcOH (800 mL). 1 H and 13 C-NMR spectra were recorded in CDCl 3 at 300, 400, or 500 MHz and 75, 101, or 126 MHz, respectively. Chemical shifts are expressed in parts per million (δ scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent (CHCl 3 : δ 7.25 ppm, CD 3 OD: δ 4.870 ppm). Coupling constants (J) are given in Hz. All presented 13 C-NMR spectra are proton decoupled. Mass spectra were recorded by direct injection with an Accurate Mass Q-TOF LC/MS spectrometer (Agilent Technologies, Santa Clara, CA, USA) equipped with an electrospray ion source in positive mode.

General Procedure for Glycosylation. Procedure A
A previously dried mixture of a glycosyl donor and glycosyl acceptor was dissolved in anhydrous dichloromethane (≈3 mL/0.1 mmol). Previously dried (200 • C, one night) 4Å molecular sieves were added to the mixture. The reaction was cooled to −30 • C, and then BF 3 .OEt 2 (3.0 equiv) was added. After 5-10 min, the reaction mixture was diluted with dichloromethane and the ensuing solution washed with saturated aqueous NaHCO 3 solution. The organic layer was dried over Na 2 SO 4 , filtered, and evaporated under vacuum. The resulting crude mixture was purified by chromatography on silica gel (eluent: hexaneethyl acetate mixtures).

General Procedure for Debenzoylation. Procedure B
The corresponding compound was dissolved in methanol (25 mL/ mmol) and triethylamine (6 mL/ mmol) was added to the resulting solution. The reaction mixture was refluxed overnight, the solvents evaporated, and the ensuing residue concentrated. Purification by flash chromatography was carried out using hexane-ethyl acetate mixtures, as eluent.

General Procedure for Silylation. Procedure C
The corresponding compound was dissolved in dry DMF (20 mL/mmol), and to this solution imidazole (4 equiv.) was added. After stirring for 5-10 min in an ice bath, under argon, terbutyldiphenylsilyl chloride (1.2 equiv.) and a small amount of dimethylaminopiridine DMAP were added. The ice bath was removed, and the reaction mixture was left with stirring at room temperature for 24 h. The reaction mixture was then diluted with ethyl The corresponding polyol was dissolved in pyridine (1 mL/0.1 mmol substrate) and acetic anhydride (0.5 mL/mmol substrate) was then added. The reaction mixture was stirred at room temperature (normally 24 h). After completion of the reaction (t.l.c.), the solvent was evaporated, and the resulting crude mixture was purified by chromatography on silica gel (eluent: hexane-ethyl acetate mixtures).

BF 2 -Bodipy 11
This compound was prepared according to our previously described method [38], from phthalide and pyrrole.

Mannopyranosyl BODIPY 13
This compound was obtained when applying the general procedure for debenzoylation, procedure B, to BODIPY-mannoside 12 (60 mg, 0.087 mmol) followed by flash chromatography (hexane-ethyl acetate; 6:4). Compound 13 (56 mg, 78%): 1 , 0.198 mmol). The mixture, under argon, was refluxed for 24 h. Then, the solvent was evaporated, and the residue dissolved in dichloromethane (10 mL), to which 10 mL of a 1M solution of NaOH were added. The ensuing mixture was kept with stirring for one night. The organic layer was separated and dried over MgSO4, filtered, and evaporated. The residue was purified by for chromatography on silica gel (hexane-ethyl acetate; 8:2) to afford compound 15 (38 mg, 46%). 1