Synthesis of carbohydrate–BODIPY hybrids

n-Pentenyl 1,2-orthoesters in regioselective glycosylations

Some years ago, in collaboration with Prof. Fraser-Reid’s group, we addressed the issue of regioselectivity in the glycosyl coupling of monosaccharide polyols with n-pentenyl 1,2-orthoester glycosyl donors (NPOE), e.g., 4 (Fig. 1) [4], aiming at simplifying the synthetic protocols toward oligosaccharides by reducing the number of protection/deprotection steps. In this context, we found that NPOEs selectively glycosylated primary alcohols in the presence of secondary hydroxyl groups in several primary/secondary diol acceptors [5].

Fig. 1:

NPOE glycosyl donor 4 and methyl mannopyranoside triol acceptors 5, 6, and 7. The more reactive hydroxyl groups in the glycosylation reactions with NPOE 4 are indicated with an arrow.

We observed a similar selectivity trend in the glycosylation of three different mannopyranoside-triol acceptors, i.e., 5, 6, and 7 (Fig. 1) [6].

Thus, the reaction of 3,4,6-triols, e.g., 5, and 2,4,6-triols, e.g., 6 (Fig. 1), with NPOE 4, resulted in the selective glycosylation of their primary hydroxyl group. We also observed a remarkable selectivity in the glycosylation of 2,3,4 mannopyranoside triols, e.g., 7, containing three secondary hydroxyl groups, with glycosyl donor 4 (Fig. 1) [6].

Regioselective synthesis of PI-88 analogs

More recently, to validate MeOEs as useful glycosyl donors in oligosaccharide synthesis, we turned our attention to PI-88 (aka muparfostat) [10], the first heparanase inhibitor [11] to progress to the clinic [12]. Thus, PI-88 reached phase III clinical trials for post-resection hepatocellular carcinoma, however, it failed to be approved for use [13].

PI-88 consists of a complex mixture of monophosphorylated polysulfated mannose saccharides, recently depicted as 9 and 12 (Fig. 2), by Ferro and coworkers [14, 15]. Among them, the major components are the α-(1 → 3)/α-(1 → 2)-linked penta-10 (∼60 %) and tetrasaccharide 11 (30 %) derivatives (Fig. 2) [16, 17]. All α-(1 → 3) linked mannoses, i.e. 12 (Fig. 2), were also present although in minor amounts [14, 15].

We explored several synthetic routes to the penta- and tetrasaccharide analog components of PI-88 based on regioselective glycosylations with MeOEs 8 [18]. In all the routes investigated, we employed n-pentenyl glycoside (NPG) 13 [19] as the reducing-end component. In the ensuing derivatives, the terminal double bond in the anomeric pentenyl group could be used in additional conjugation events. [20] In addition, MeOEs could be selectively activated in the presence of NPG glycosides.

One of these routes is depicted in Scheme 2. Thus, chemoselective glycosylation of NPG 13 with MeOE 8a yielded a tri-O-benzoyl disaccharide intermediate (67 % yield), which after de-O-benzoylation and selective mono-silylation – at the primary hydroxyl group – furnished triol disaccharide 14. Chemo- and regioselective glycosylation of 14, at its 3′OH group, with MeOE disaccharide 15 allowed access to tetrasaccharide 16 (63 % yield). The latter was then de-O-benzoylated to heptaol tetramannoside 17, which could be regioselectively glycosylated at its 3′′′OH with MeOE 8b to yield n-pentenyl pentamannoside 18.

Scheme 2:

Synthetic route to tetra- and pentasaccharide PI-88 analogs 16 and 18, respectively, by regioselective glycosylations of mannose polyols with MeOEs 8.

Regioselective synthesis of BODIPY glycosides as PI-88 analogs

The use of small organic fluorescent molecules has significantly contributed to recent developments in chemical biology and biomedicine when used in combination with fluorescence spectroscopy, or microscopy, detection techniques [21, 22]. In this context, using BODIPY fluorophores in combination with carbohydrates has proven to be especially useful in bio-applications [23], [24], [25]. Along this line, we decided to evaluate the possibility of incorporating BODIPY aglycon 19a (Scheme 3a, rather than n-pentenyl alcohol) into the synthesis of PI-88 analogs. BODIPY 19a had been shown to display remarkable photophysical properties [26, 27]. Our synthetic route to 19a involved a one-pot transformation using phthalide and pyrrole as the starting materials, followed by in situ coordination with BF₃.Et₂O (Scheme 3a) [28]. A slight variation of the aforementioned protocol, replacing BF₃.Et₂O by B(Ph)₃ in the coordination step allowed us access to B-Ph₂ BODIPY 19b (Scheme 3b). Indeed, the chemical substitution of the fluorine atoms by phenyl groups at boron had been reported to increase the chemical stability of the ensuing BODIPYs [29], which proved to be useful in our synthesis of the PI-88-BODIPY analogs, vide infra.

Scheme 3:

Synthesis of BODIPYs 19a and 19b (a, b). Attempted glycosylation/deprotection of BODIPY 19a (c). Synthetic route to BODIPY containing tetrasaccharide PI-88 analog 24 from BODIPY 19b (d, e).

Thus, even though glycosylation of 19a with MeOE 8a yielded the sought BODIPY glycoside 20, its saponification under different reaction conditions failed to yield the desired 2-OH mannopyranoside. Instead, depending on the reaction conditions employed, compounds 21a or 21b, where the fluorine atoms at boron have been replaced by methoxy groups, were obtained (Scheme 3c) [30]. Fortunately, glycosylation of BODIPY 19b with MeOE 8a followed by de-O-benzoylation, led to the desired 2-OH mannopyranoside 22 that underwent a subsequent glycosylation reaction with MeOE 8a to furnish BODIPY disaccharide 23a in moderate yield (Scheme 3d).

Protecting group manipulations in disaccharide 23a, involving saponification and monosilylation of the primary hydroxyl group, paved the way to 2′,3′,4′-triol 23c. The latter was regioselectively glycosylated at O-3′ with MeOE disaccharide 15, leading to PI-88 BODIPY-tetrasaccharide analog 24 in a fair 53 % yield (Scheme 3e).

BODIPY as aglycons in saccharide synthesis: proof of concept

To demonstrate the usefulness of BODIPY 25 as a fluorescent aglycon in the synthesis of saccharides, we tackled the preparation of branched and linear trisaccharides 29 and 31, respectively (Scheme 4a and b).

Scheme 4:

Synthesis of branched BODIPY-containing trisaccharide 29 (a), and linear BODIPY-containing trisaccharide 31 (b), by sequential glycosylation of BODIPY 25 with several glycosyl donors.

Accordingly, access to trisaccharide 29 was carried out by glycosylation of 25 with mannopyranosyl trichloroacetimidate 26, followed by de-O-benzoylation of the ensuing tetrabenzoyl mannopyranoside to a tetraol intermediate, which was regioselectively glycosylated (at O-6) with phenyl thioglycoside 27, and with NPOE 28 (at O-3) (Scheme 4a). Final saponification of the benzoyl protecting groups yielded trisaccharide 29 in fairly good yield (26 % yield, five steps).

Alternatively (Scheme 4b), BODIPY 25 was glycosylated with phenyl thioglycoside 30, followed by de-O-silylation of the ensuing monosaccharide to unveil the primary hydroxyl group. Iteration of the process (glycosylation with 30, de-O-silylation) allowed the preparation of a disaccharide intermediate that was finally glycosylated with phenyl thioglycoside 27 to yield a trisaccharide intermediate that upon saponification produced linear saccharide 31 (Scheme 4b).

Trisaccharides 29 and 31, displayed high water solubility and remarkable fluorescence efficiency in water, reaching quantum yield values up to 81 % [38]. In contrast to their excellent luminescence in organic solvents, many BODIPYs become weakly or non-emissive in aqueous media owing to their tendency to associate forming H-type aggregates, by assembly of the dyes in a head-to-tail manner. H-aggregates are characterized by hypsochromic shifted absorption bands and no or weak fluorescence emission. In this context, branched derivative 29, which was soluble in water at concentrations as high as 2 mM, displayed a slight trend toward molecular (H-)aggregation upon increasing the dye concentration in water. This phenomenon was observed at concentrations higher than 0.1 mM, as revealed by the increased absorbance observed at the short-wavelength vibronic shoulder (Fig. 4). Nevertheless, it is important to mention that – highly fluorescent – BODIPYs at concentrations up to 0.1 mM in water, already fulfilled the requirements normally demanded in biological microscopy studies.

Fig. 4:

Normalized absorption spectra of trisaccharide 29 at different concentrations in H₂O. The growing absorption corresponding to the H-aggregate is highlighted.

Access to linker-free glyco-BODIPYs by Ferrier-type C-glycosylation

Among the existing synthetic methods to access carbohydrate–BODIPY hybrids, scarce literature examples are dealing with glycosylation reactions. During our studies on the glycosylation of BODIPYs containing hydroxyl groups (e.g., Scheme 3a and b), we had not observed the formation of any compound arising from the competing C-glycosylation reaction of the dipyrromethene moiety. Indeed, attempted C-glycosylation of BODIPY 32a (Scheme 5) with some mannopyranosyl trichloroacetimidate donors failed to give any coupling product. Conversely, the reaction 32a with tri-O-acetyl D-glucal 33 as the donor, under Ferrier rearrangement [49, 50] conditions, successfully led to bis-β-BODIPY-C-glycoside 34a as the sole regio- and stereoisomer in 68 % yield (Scheme 5) [51]. Likewise, Ferrier-type glycosylation of azidomethyl BODIPY 32b with 33 produced bis-monosaccharidic BODIPY 34b in 70 % yield, also in a completely stereocontrolled manner (Scheme 5). On the other hand, the reaction of hydroxymethyl BODIPY 32c with D-glucal 33 produced BODIPY 35a containing three sugar units, again as the sole isomer. Saponification of 35a (Et₃N/MeOH 1:4) led to hydrophilic BODIPY 35b. The latter was soluble in water up to the millimolar range, retaining a remarkable fluorescence efficiency (77 %, H₂O). Noticeably, this derivative showed no tendency to intermolecular aggregation in water, a well-known deactivation pathway in BODIPYs, and the increment of dye concentration in water altered neither the absorption nor the fluorescence profiles.

Scheme 5:

Synthesis of linker-free bis-monosaccharidic BODIPYs 34a, b by Ferrier-type C-glycosylation of BODIPYs 32a, b with commercially available tri-O-acetyl D-glucal 33. Access to BODIPY 35a, containing three sugar units, by Ferrier-type glycosylation of 32c with D-glucal 33.

Access to glyco-BODIPYs by CuAAC reaction of bis-propargyl BODIPYs with azido-sugars

In an unrelated approach to carbohydrate–BODIPY hybrids, we recently described the synthesis and click copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) reactions [52, 53], of 2,6-dipropargyl BODIPY derivatives, e.g., 37 (Scheme 6), with azido-containing carbohydrates to yield bis-triazolyl derivatives 38 [54].

Scheme 6:

Synthesis of 2,6-di-propargyl BODIPYs 37, and their CuAAC reactions with azido-sugars to gain access to carbohydrate–BODIPY hybrids 38.

The reported method involved the initial preparation of bis-propargyl BODIPYs 37, by a Nicholas propargylation/decobaltation protocol [55], of a series of 8-substituted 1,3,5,7-tetramethyl BODIPYs (36), followed by CuAAC click-type reaction with azido-containing sugars.

Accordingly, the CuAAC reaction of BODIPY 39 with the carbohydrate derivatives 40, and 41, and cholesteryl derivative 42, gave rise to glyco-BODIPYs 43a,b, and 44 (Fig. 5). Thus, protected or unprotected sugar derivatives, e.g., 40 and 41, respectively, could be used (CuSO₄, sodium ascorbate, 3.0 equiv azido compound, THF/H₂O, pressure tube, 65 °C) to access either hydroxyl-protected or hydroxyl-free glyco-BODIPYs, e.g., 43a or 43b, respectively. Furthermore, using limited amounts of the first azido component, the CuAAC reaction could be performed sequentially with two azido-containing molecules to obtain differently substituted BODIPYs. For instance, the CuAAC reaction of 39 with cholesteryl azide 42 (0.9 equiv) allowed the synthesis of a monosubstituted BODIPY-cholesteryl derivative intermediate (40 % yield) that could undergo a second CuAAC reaction with 40 to furnish compound 44 (78 % yield), where installation of one carbohydrate and one cholesteryl derivative at the BODIPY core had taken place (Fig. 5).

Fig. 5:

Bis-triazolyl BODIPY–carbohydrate hybrids 43a, b and 44, obtained by CuAAC reaction of BODIPY 39 with azido-derivatives 40–42.