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. 2024 Mar 4;89(6):4042–4055. doi: 10.1021/acs.joc.3c02907

De Novo Access to BODIPY C-Glycosides as Linker-Free Nonsymmetrical BODIPY-Carbohydrate Conjugates

Clara Uriel $,*, Dylan Grenier $, Florian Herranz $, Natalia Casado §, Jorge Bañuelos §,*, Esther Rebollar , Inmaculada Garcia-Moreno , Ana M Gomez $, J Cristobal López $,*
PMCID: PMC10949249  PMID: 38438277

Abstract

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Recent years have witnessed an increasing interest in the synthesis and study of BODIPY-glycoconjugates. Most of the described synthetic methods toward these derivatives involve postfunctional modifications of the BODIPY core followed by the covalent attachment of the fluorophore and the carbohydrate through a “connector”. Conversely, few de novo synthetic approaches to linker-free carbohydrate-BODIPY hybrids have been described. We have developed a reliable modular, de novo, synthetic strategy to linker-free BODIPY-sugar derivatives using the condensation of pyrrole C-glycosides with a pyrrole-carbaldehyde derivative mediated by POCl3. This methodology allows labeling of carbohydrate biomolecules with fluorescent-enough BODIPYs within the biological window, stable in aqueous media, and able to display singlet oxygen generation.

Introduction

The development of small-molecule fluorophores is a fast-growing research area due to the increasing use of fluorescence imaging methods, in modern research fields, such as biochemistry, molecular biology, and materials science.1 Among these fluorophores, difluoroboron dipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) or BODIPY, i.e., 1 (Figure 1), fluorescent dyes have attracted considerable interest for biological and photophysical studies over the past two decades due to their excellent biocompatibility and tunable photophysical and chemical properties.2 Compared with commonly used fluorophores, the BODIPY dye family exhibits high molar absorption coefficients, insensitivity to the polarity and pH, sharp absorption and emission bands, and high fluorescence quantum yields.3 From a structural standpoint, the BODIPY scaffold consists of two planar pyrrole moieties connected by a methylene bridge and a boron difluoro moiety. From this structural arrangement, two key synthetic precursors to BODIPYs, dipyrromethene (i.e., 2) and dipyrromethane (i.e., 3) (Figure 1), can easily be envisioned.4 Remarkably, the BODIPY scaffold has been compared to a “rigidified” monomethine cyanine dye and a porphyrin’s sibling (“porphyrin’s little sister”).5

Figure 1.

Figure 1

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BODIPY (1), dipyrromethene (2), and dipyrromethane (3) (IUPAC numbering).

In this context, we have been interested in the emergent field of BODIPY-glycoconjugates,6 where carbohydrates, one of the four essential types of biomolecules, are covalently linked to BODIPY fluorophores.7,8 In these derivatives, either component may play a relevant role. For example, the carbohydrate moiety has been shown to internalize,9 solubilize in water,10 target,11 and reduce the cytotoxicity of the fluorophore.12 Conversely, the presence of the fluorophore in BODIPY-carbohydrate conjugates has shown its value in the investigation of carbohydrate-receptor interactions in biological systems with high sensitivity, by way of fluorescently labeled carbohydrates.3,13,14

In general, the synthesis of BODIPY-carbohydrate conjugates benefits from the variety of postfunctional modifications already known in borondipyrromethene derivatives.15 Therefore, the covalent linkage is often achieved by coupling conveniently functionalized BODIPYs with carbohydrates.16 Among these transformations, the copper-mediated azide–alkyne cycloaddition (CuAAC)17 “click-type”18 reactions of either alkyne- or azide-containing BODIPYs play a prominent role. Overall, these approaches lead to the formation of tethered BODIPY-glycoconjugates.

Conversely, to our knowledge, only a small number of approaches to linker-free BODIPY-carbohydrate conjugates have been reported in the literature. Some of these methods have been based on postfunctional BODIPY transformations to incorporate the sugar moiety. For instance, Brothers and co-workers reported on the preparation of sugar-O-BODIPY conjugates in which the fluorophore and the carbohydrate are directly linked through B–O–C bonds.19 We recently described a C-glycosylation approach leading to 2,6-disubstituted BODIPY-carbohydrate derivatives.10b

In addition, protocols based on the de novo synthesis of BODIPY-carbohydrate hybrids have been reported in the literature, albeit scarcely. Recently, two such methods have appeared. First, bis-glucosylated BODIPY dye 6 (Scheme 1a) was obtained by an oxidation/chelation protocol from 5-phenyl dipyrromethane 5, which is readily obtained by condensation of β-C-glucosyl pyrrole 4a with benzaldehyde (Scheme 1a).20 A second method, applied to a wide variety of saccharides, involves the condensation of unprotected reducing sugars (e.g., 7, Scheme 1b) with a pyrrole unit (8) leading to C5-substituted dipyrromethane intermediates 9,21 which can be processed by oxidation/chelation steps, to C8 sugar-substituted BODIPYs, e.g., 10 (Scheme 1b).22 Both methods are based on the well-precedented dipyrromethane → BODIPY transformation, leading to symmetrical BODIPYs 6 and 10 (Scheme 1a,b). In this manuscript, we report a reliable one-pot route to nonsymmetrical BODIPY C-glycosides, i.e., 12 (Scheme 1c). The overall transformation (4 → 12) includes the condensation of glycosyl pyrroles 4 with formyl pyrrole 11, in the presence of POCl3 (Scheme 1c), to afford an intermediate C-glycosyl dipyrromethene 13, which is then complexed in situ by adding triethyl amine and borontrifluoride diethyl etherate to yield BODIPY dyes 12. The process has been performed on a gram scale in one case. Furthermore, postfunctional modifications on carbohydrate-BODIPY hybrids 12 have been implemented to improve the photophysical properties of these BODIPYs.

Scheme 1. (a–c) De Novo Synthesis of Carbohydrate-BODIPY Hybrids.

Scheme 1

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Results and Discussion

We were interested in investigating de novo approaches to linker-free BODIPY-carbohydrate derivatives involving dipyrromethene-based, e.g., 2 (Figure 1), instead of dipyrromethane-based glycosyl intermediates, e.g., 3 (Figure 1). The use of the former would circumvent the need for the oxidation step and allow the BODIPY synthesis to be carried out as a one-pot operation. At the onset of this work, the interest in glycosyl dipyrrane derivatives had been mainly triggered by their use as building blocks in the synthesis of glycoporphyrins and derivatives.23 In this context, 5-glycosyl dipyrromethanes, e.g., 9 (Scheme 1b),24 had been employed as convenient synthetic precursors for meso-glycoporphyrins.25 On the other hand, C-glycosylation26 at C-1 of an unsubstituted dipyrromethane with gluco-, galacto-, and mannopyranosyl trichloroacetimidate glycosyl donors,27 leading to 1-glycosyl dipyrromethanes, has also been reported.28 However, the subsequent boron chelation leading to a glyconjugated BODIPY was not explored.

According to plan, we aimed at exploring well-established synthetic routes to BODIPYs involving glycosyl dipyrromethene intermediates. As starting materials, we selected pyrrole C-glycosides 4 (Scheme 2), readily accessible by C-glycosylation of pyrrole. Accordingly, as glycosyl donors, we selected trichloroacetimidates 14a, 14b, and 14c,27 derived from d-glucose, d-mannose, and d-maltose, respectively, as well as differently protected d-mannose-derived 1,2-methyl orthoesters (MeOEs)29,3015a,b (Scheme 2). The glycosylation of pyrrole with glycosyl trichloroacetimidate donors 14a–c, mediated by BF3·Et2O at −78 °C in CH2Cl2, produced good yields of pyrrole C-glycosides 4a, 4b, and 4c, respectively. On the other hand, the reaction of MeOEs 15a,b with pyrrole, promoted by BF3·Et2O at −30 °C in CH2Cl2, provided good yields of pyrrole C-mannopyranosides 4d and 4e, respectively. All of these glycosylations took place with complete stereoselectivity providing exclusively one of the two possible anomeric isomers.

Scheme 2. Synthesis of C-Glycosyl Pyrroles.

Scheme 2

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With glycopyrroles 4 in hand, we set out to explore several synthetic routes to BODIPYs (routes A to E, Scheme 3). All these routes involved the additional in situ chelation step (not shown in the scheme) of the dipyrromethene intermediates to yield the desired borondipyrromethene derivatives. In our hands, implementation of route A (Scheme 3) consisting of the condensation between pyrrole C-glucoside 4a and its corresponding pyrrole-carbaldehyde 16, easily prepared by the Vilsmeier–Haack formylation of 4a,31 afforded low yield (15%) of bis-glucosyl BODIPY 17, after chelation (Scheme 3 and Scheme S2 in the SI). Alternatively, according to route B (Scheme 3), the reaction of 4a with ethyl orthoformate or ethyl orthobenzoate either gave no BODIPY (17) formation or trace yields of BODIPY 6 (4%), respectively (Scheme S1 in the SI). Along these lines, the application of route C (Scheme 3), involving the one-pot condensation-decarbonylation of pyrrole 2-carbaldehyde 16,32 yielded none of the desired BODIPY 17.

Scheme 3. Synthetic Routes to BODIPY-Carbohydrate Conjugates.

Scheme 3

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Conversely, better results were obtained in the preparation of nonsymmetrical BODIPY-carbohydrate conjugate 12a according to routes D and E (Scheme 3). Such routes involved the condensation of either glycosyl pyrrole 4a with commercially available 2-formyl-3,4-dimethyl pyrrole (11) (route D) or the condensation of 16 with 2,4-dimethyl pyrrole (route E).

Therefore, condensation of 4a with formyl pyrrole 11, mediated by POCl3,33−35 (route D, Scheme 3), followed by chelation with boron trifluoride etherate (Et3N, then BF3·Et2O) at room temperature (r.t.), gave BODIPY 12a in 47% yield (Scheme 3). Likewise, POCl3-mediated condensation of d-glucose-pyrrole-carbaldehyde 16, with 2,4-dimethyl pyrrole, yielded BODIPY 12a, although in a slightly lower yield of 42% (Scheme 3). Based on these results and considering that route D is more convergent than route E since the latter required an additional Vilsmeier reaction on glucoside 4a, we decided to pursue our investigations by optimizing route D.

Interestingly, during the course of these investigations, we found that when the chelation step was carried out at reflux (CH2Cl2), the yield of BODIPY 12a could rise up to 77% (Scheme 4). It is important to note that in this transformation, the formation of variable amounts of symmetrical BODIPY 18 (5–15%) was always observed.36,37

Scheme 4. Access to BODIPY 12a by an Optimized Route D.

Scheme 4

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To validate the route, we next carried out the gram-scale synthesis of 12a (Scheme 5). Thus, chemoselective anomeric deacetylation38 of commercially available 1,2,3,4,6-penta-O-acetyl-d-glucose provided access to 2,3,4,6-tetra-O-acetyl-d-glucopyranose, which upon the reaction with trichloroacetonitrile in the presence of 1,8-diazabicyclo(5.4.0)undec-7-ene (DBU) provided glucosyl donor 14a.27,39C-Glycosylation of pyrrole with glucosyl trichloroacetimidates 14a took place smoothly at −78 °C in the presence of BF3·Et2O, leading to pyrrole C-glucoside 4a. Finally, condensation of 4a with pyrrole-carbaldehyde 11 provided BODIPY 12a in a respectable 73% yield. Formyl pyrrole 11, which is commercially available, could also be uneventfully prepared by Vilsmeier–Haack formylation of 2,4-dimethyl pyrrole.32a

Scheme 5. Gram-Scale Synthesis of 12a from 1,2,3,4,6-Penta-O-acetyl-d-glucose.

Scheme 5

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The scope of this approach was next investigated with the synthesis of additional carbohydrate-BODIPY hybrids 12b–e, from pyrrole C-glycosides 4b–e, respectively (Scheme 6). Thus, the protocol was successfully applied to C-glycosyl derivatives 4b–e, providing good yields of d-mannose- and d-maltose-derived BODIPYs 12b–e.

Scheme 6. Synthesis of Linker-Free Carbohydrate-BODIPY Hybrids 12b–e.

Scheme 6

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Finally, since the chemical and photophysical properties of the BODIPY fluorophores can be modulated by postfunctional modifications on their skeleton, we decided to evaluate a series of transformations on d-glucosyl and d-maltosyl BODIPY derivatives, 12a and 12c, respectively. Thus, we aimed at obtaining water-soluble, red-edge fluorescent derivatives,40 as well as dyes for efficient singlet oxygen production.41

Along these lines, BODIPY 12a was transformed into water-soluble BODIPY 19, by saponification of the sugar acetyl groups under controlled alkaline conditions (Scheme 7).42 On the other hand, iodination at C2 (NIS/BF3·Et2O) was carried out in compounds 19 and 12a (Scheme 7) leading to iodo-BODIPYs 20 and 21, respectively. The latter was transformed into C3-styryl BODIPY 22 by Knoevenagel condensation with benzaldehyde, mediated by piperidinium acetate. Owing to the special properties associated with B(CN)2-BODIPYs,43 we prepared BODIPY 23 from 12a. Thus, starting from compound 23, by way of related synthetic transformations, we were able to access iodinated (NIS) and brominated (NBS), B(CN)2-BODIPYs 24a,b, respectively, and iodo-styryl B(CN)2-BODIPY 25 (from 24a). The corresponding unprotected C-glucosyl BODIPYs 26 and 27 were obtained by removal of the acetyl protecting groups under acidic (HCl/MeOH) rather than alkaline conditions owing to the higher stability of B(CN)2-BODIPYs compared to BF2-BODIPYs,44 under acidic media from derivatives 23 and 24a, respectively.

Scheme 7. Postfunctional Modifications on BODIPY 12a.

Scheme 7

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On the other hand, a related set of reactions carried out on BODIPY-labeled disaccharides 12c (Scheme 8) allowed the preparation of analogous linker-free BODIPY C-maltosides. For instance, saponification of 12c gave water-soluble BODIPY disaccharide 28, whereas iodination produced iodo-BODIPY 29, which could undergo a Knoevenagel condensation resulting in the formation of styryl-iodo-BODIPY disaccharide 30. Again, the transformation of BODIPY 12c into B(CN)2-BODIPY 31 took place efficiently. The latter was then transformed into the iodinated derivative 32. Compound 31 could be saponified under alkaline conditions to protecting-group-free maltoside 33 (Scheme 8). Conversely, attempted acetyl hydrolysis on 31 to 33, under acidic conditions, gave compound 26, where cleavage of the anomeric bond of the terminal glucosyl residue had taken place along with the desired de-O-acetylation. On the other hand, compound 32 could also be efficiently accessed from the corresponding BF2-BODIPY 29 by fluoride to cyanide exchange (TMSCN/BF3·Et2O) (Scheme 8).

Scheme 8. Postfunctional Modifications on BODIPY 12c.

Scheme 8

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The photophysical behavior of the heavy-atom-free glyco-BODIPYs (12a,c and 23, as well as the corresponding unprotected derivatives 19, 28, and 26, respectively) resembled that reported for conventional BODIPYs (such as 1 or 18), supporting the C5 position as suitable for the direct attachment of carbohydrates (Table 1 and Table S1 in the SI). Indeed, albeit the absorption capacity of these glycosyl derivatives decreased (mainly in the unprotected derivatives), all of them displayed strong and sharp emission bands in organic solvents (see methanol in Table 1), with fluorescence efficiencies surpassing 80% and even reaching the 100% for 12c (Table 1). Moreover, the unprotected glycosyl-BODIPYs were also soluble and fluorescent in water (Figure S1 and Table S1 in the SI), especially BODIPY 28 bearing a disaccharidic unit, which also ensured a higher solubility limit (around 0.1 to 1 mM without signs of aggregation-induced quenching, consistent with our previously reported results for related carbohydrate-BODIPY hybrids).10b,42 In this regard, note that the at-boron replacement of the fluorine atoms by cyano moieties is a successful strategy to further enhance the fluorescence efficiency (Table 1).43 In all cases, the B(CN)2-BODIPYs (e.g., 23 and 26) displayed improved fluorescence efficiency with respect to the corresponding BF2-BODIPY (12a and 19). This enhancement of the emission efficiency, attributed to a decrease in nonradiative probability, is strongly correlated with an increased photostability during prolonged irradiation of dyes based on B(CN)2 pattern substitution.43 Photostability in water of B(CN)2-BODIPY 26 and its corresponding BF2-BODIPY 19 was assessed by monitoring the evolution of laser-induced fluorescence under repeated pumping pulses (see the Experimental Section). Consistently, dye 26, which exhibited higher efficiency, also demonstrated greater photostability; its laser-induced emission remained unchanged after 50,000 pump pulses, while its fluorinated counterpart 19 experienced a 25% decrease in emission under identical experimental conditions.

Table 1. Main Photophysical Properties of the Glyco-BODIPYs in Diluted (2 μM) Solutions of Methanola.

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a

Absorption (λab) and fluorescence (λfl) wavelength, molar absorption coefficient at the maximum (εmax), fluorescence quantum yield (Φ), lifetime (τ), and singlet oxygen generation quantum yield (ΦΔ). Full photophysical data in other solvents are listed in Tables S1 and S2 in the SI. The model molecular structures in each case have been added for the sake of clarity.

b

Data in chloroform.

c

Main lifetime (contribution of >90%) of the multiexponential fit (see Table S1 in the SI).

The C2 monobromination of the glycosyl-BODIPY 24b promoted a slight bathochromic shift of the spectral absorption and fluorescence bands but modified strongly the fluorescence signatures (Table 1 and Table S2 in the SI). As it was expected, the bromine heavy atom enhanced the intersystem crossing (ISC), reducing the fluorescence efficiency to less than 10%. Alternatively, the ISC-mediated triplet-state population enabled an effective generation of singlet oxygen45 (approaching the 70%, Table 1). In this regard, the dual response of this monobrominated glycosyl-BODIPY could be suitable for phototheragnostic applications46 since it is able to generate a notable amount of singlet oxygen upon irradiation while retaining enough fluorescence output to visualize the process.

By going from the brominated BODIPY 24b to the monoiodinated glycosyl-BODIPYs 21, 24a, and 29 (as well as the unprotected derivatives 20 and 27), the heavy-atom effect increased the spin–orbit coupling, consequently reducing even more the fluorescence emission that became almost negligible (Table 1 and Table S2 in the SI). Note that in this case, the replacement of fluorine atoms by cyano moieties was not enough to improve the fluorescence response since the iodinated B(CN)2-BODIPYs 24a and 27 exhibited also a faint emission (Table 1). Consequently, these dyes enabled an efficient generation of singlet oxygen, reaching values that surpassed 75% and even 90% (see iodinated BF2-BODIPYs 21 and 29, Table 1). Therefore, these iodinated BODIPYs could act as efficient photosensitizers for photodynamic therapy (PDT). Among the tested compounds, 20 and 27 show promise for their potential use in photodynamic therapy (PDT). While they generate slightly less singlet oxygen than their protected counterparts, compounds 21 and 24a (see Table 1), they have the advantage of being fully soluble in water and remaining stable over time. These qualities make them promising candidates for further preclinical testing as PDT photosensitizers.

Further functionalization of the monoiodinated glycosyl-BODIPY 29, by grafting one styryl group at C3, led to BODIPY 30, which contains a π-extended chromophoric framework. This caused a bathochromic shift of the spectral bands toward the red edge of the visible region (Figure 2). Indeed, the long-wavelength tail of the fluorescence spectrum fell within the biological window (λ > 650 nm). Thus, although the iodine substitution enhanced the triplet-state population, this π-extended derivative retained a remarkable fluorescence efficiency (around 10% at 590 nm, Table 1), together with a singlet oxygen generation of 72%, which was lower than the yield recorded from its iodinated glycosyl-BODIPY analog 29 without a styryl moiety that reached up to 94% (Table 1). Therefore, as detailed for 24b, BODIPY 30 could be a suitable fluorescent photosensitizer for bioimaging-guided PDT, especially once the maltosyl residue is unprotected to ensure solubility in the physiological media.

Figure 2.

Figure 2

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Absorption and normalized fluorescence (darker shading) spectra of glycosyl-BODIPYs 12c and 30, bearing disaccharidic maltosyl residues, in diluted (2 μM) solutions of methanol.

In a first approach, the population of long-lived triplet excited states in these new derivatives was proven by nanosecond-resolved transient absorption (ns-TA) spectroscopy (see the Experimental Section for details). The ns-TA spectra recorded from 21 and 30, as representative examples of the behavior followed by these dyes, showed the characteristic profile previously recorded for iodinated BODIPYs, with two broad absorption bands flanking the ground-state bleaching band centered at 500 nm (Figure S2 in the SI).47 The decay of the positive signals had monoexponential character within the microsecond scale, suggesting that just one long-lived transient triplet state was populated.

To get deeper insights into the long-lived emissions of the new dyes, time-gated spectral analysis induced upon laser excitation at 532 nm in aerated methanolic solutions was performed at room temperature (see the Experimental Section). As it has been reported for other heavy-atom-free single BODIPY fluorophores,48 dye 12a exhibited delayed emission in the 500–600 nm spectral region with a profile similar to its prompt fluorescence (Figure 3 and Figure S3 in the SI). Considering that this emission can be recorded at delay times up to 20 μs, it must unequivocally imply involvement of long-lived triplet excited states harvested through reverse ISC (rISC), giving rise to the detected thermally activated delayed fluorescence (TADF).

Figure 3.

Figure 3

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Delayed emission spectra of BODIPYs 12a, 21, 24a, 24b, and 30 in methanol, recorded at (A) 3 and (B) 10 μs time delay after photoexcitation at 532 nm under ambient conditions. Optically matched solutions were used.

Heavy-atom functionalization of these BODIPYs led to the growing of a dual delayed emission since a new and broad long-wavelength band raised in the 650–900 nm spectral region. When increasing the time delay up to 10 μs, the spectral profile evolved toward a single band since the delayed fluorescence around 560 nm virtually disappeared, while the longer-lasting emission placed at the red edge of the spectrum could be recorded up to 50 μs (Figure 4 and Figure S3 in the SI). On the basis of the T1 energy level detected and computed for other F-BODIPYs as well as boron-substituted related dyes,48 this long-wavelength band can be reliably assigned to the phosphorescence emission.

With respect to the B(CN)2-substituted BODIPY 24a, its corresponding BF2-BODIPY 21 exhibited a faint phosphorescence emission that became undetectable after 10 μs upon photoexcitation (Figure S3 in the SI). In this regard, this effectively quenched phosphorescence emission was in agreement with the highly efficient singlet oxygen generation recorded from 21 that reached a quantum yield up to 93%. An exception to the behavior described above was the delayed emission recorded from the red-emitting dye 30. In this case, the spectral profile, resulting from the delayed fluorescence that peaked at 600 nm and strongly overlapped with its phosphorescence, remained unaltered on increasing the time delay from 3 to 50 μs (Figure 4 and Figure S3 in the SI). The simultaneous detection of delayed fluorescence and phosphorescence emission sustains the balanced fluorescence and singlet oxygen generation recorded from the herein developed dyes advancing their potential as valuable theragnostic agents.49

Conclusions

We have developed a modular, de novo, one-pot, synthetic strategy to linker-free BODIPY-carbohydrate derivatives based on the condensation of readily accessible pyrrole C-glycosides with a pyrrole-carbaldehyde derivative mediated by POCl3 followed by chelation with boron trifluoride etherate (Et3N, then BF3·Et2O). Furthermore, fine adjustment by postfunctional modifications allows ready access to water-soluble, linker-free BODIPY-carbohydrate conjugates, with tailored photophysical properties, which sustain a balanced fluorescence and singlet oxygen generation advancing their potential as valuable phototheragnostic agents. Thus, the overall approach allows the preparation of multifunctional BODIPYs with diverse substitution at C2, C3, C5, and at-boron of the chromophoric core. Such derivatives permit the labeling of carbohydrates with fluorescent-enough BODIPYs amenable to be tracked by bioimaging within the biological window, stable in the physiological media, and displaying singlet oxygen generation, which might prove useful in photodynamic therapy.

Experimental Section

General Information

All solvents and reagents were obtained commercially and used as received unless stated otherwise. Residual water was removed from starting compounds by repeated coevaporation with toluene. All moisture-sensitive reactions were performed in dry flasks fitted with glass stoppers or rubber septa under a positive pressure of argon. In general, reactions were carried out at room temperature (r.t.) unless indicated otherwise. Heating blocks were utilized as heat sources for all reactions requiring elevated temperatures. Anhydrous MgSO4 or Na2SO4 was used to dry organic solutions during workup. Evaporation of the solvents was performed under reduced pressure using a rotary evaporator. Flash column chromatography was performed using 230–400 mesh silica gel. Thin-layer chromatography was conducted on Kieselgel 60 F254. Spots were observed first under UV irradiation (254 nm) then by charring with a solution of 20% aqueous H2SO4 (200 mL) in AcOH (800 mL). All melting points were determined with a Stuart SMP-20 apparatus. Optical rotations were measured on a Jasco P2000 polarimeter with [α]D25 values reported in degrees with concentrations expressed in g/100 mL. 1H, 13C, 19F, and 11B NMR spectra were recorded in CDCl3 or CD3OD at 300, 400, or 500 MHz (1H NMR), 75, 101, or 126 MHz (13C NMR), 376 MHz (19F NMR), and 128 MHz (11B NMR), respectively. Chemical shifts are expressed in parts per million (δ scale) downfield from tetramethylsilane and are referenced to residual protium in the NMR solvent (CHCl3: δ 7.25 ppm, CH3OH: δ 4.87 ppm). Coupling constants (J) are given in Hz. All presented 13C NMR spectra are proton-decoupled. Mass spectra were recorded by direct injection with an accurate mass Q-TOF LC/MS spectrometer equipped with an electrospray ion source in the positive mode. 3,5-Dimethylpyrrole-2-carbaldehyde 11 was prepared according to previously reported Vilsmeier formylation.32a The synthesis of trichloroacetimidate glycosyl donors 14b and 14c was carried out in two steps starting from peracetylated mannose and maltose, respectively, in the same way as described for its d-glucose analog 14a (Gram-Scale Synthesis of BODIPY 12a, Scheme 5); the data of the products thus prepared are in accordance with those described in the bibliography.50 Orthoester 15a(51) was synthesized according to literature procedures, and methyl orthobenzoate 15b was prepared from tetra-O-benzoyl-α-d-mannopyranosyl bromide52 following a published method.53 1,2,3,4,6-Penta-O-acetyl-d-mannopyranose, 1,2,3,6,2′,3′,4′,6′-octa-O-acetyl-d-maltopyranose, and 1,2,3,4,6-penta-O-acetyl-d-glucopyranose were obtained from commercial sources.

General Procedures

General Procedure A: Preparation of C2-Glycosylated Pyrroles

The corresponding glycosyl donor (1 equiv) and pyrrole (5 equiv) were dissolved in anhydrous CH2Cl2 and cooled to an indicated temperature. Then, BF3·Et2O (0.5 equiv) was added. The reaction mixture was stirred until TLC showed a complete disappearance of the glycosyl donor; then, Et3N was added (3.5 equiv); after stirring for 5 min, the crude material was concentrated in vacuo. The residue was purified by silica column chromatography.

General Procedure B: Preparation of C-Glycosyl-BODIPYs

A solution of the corresponding glycosyl pyrrole (1 equiv) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (1.2 equiv) in anhydrous CH2Cl2 (5–20 mL/mmol) was cooled at 0 °C. Then, POCl3 (3 equiv) was added dropwise. The solution was stirred at 0 °C for 15 min and then at room temperature (r.t.) overnight. The reaction mixture was recooled at 0 °C, and triethylamine (10 equiv) and BF3·Et2O (6 equiv) were added dropwise and heated to 40 °C and stirred at that temperature for 3 h. The solution was then diluted with CH2Cl2 (50 mL/mmol), washed with water (2 × 50 mL/mmol) and NaHCO3 (50 mL/mmol), dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica column chromatography.

Reactions and Compounds’ Characterization

2-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-glucopyranosyl)-pyrrole (4a)

The compound 4a was prepared according to the general procedure A starting from 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl-trichloroacetimidate (2.3 g, 4.7 mmol), pyrrole (1.4 mL, 20.3 mmol), and BF3·Et2O (260 μL, 2 mmol) at −78 °C (30 min). After workup (Et3N, 2 mL), the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 8:2 to 6:4) to afford 4a(54,20) (1.5 g, 81%). 1H NMR (CDCl3, 400 MHz): δ 8.42 (s, 1H), 6.77 (td, J = 2.6, 1.7 Hz, 1H), 6.14–6.10 (m, 2H), 5.31 (t, J = 9.35 Hz, 1H), 5.19 (dd, J = 9.2, 5.0 Hz, 1H), 5.15 (dd, J = 9.4, 5.1 Hz, 1H), 4.52 (d, J = 9.9 Hz, 1H), 4.28 (dd, J = 12.4, 4.9 Hz, 1H), 4.17–4.07 (m, 1H), 3.82 (ddd, J = 9.9, 4.9, 2.3 Hz, 1H), 2.07 (s, 3H), 2.04 (s, 3H), 2.01 (s, 3H), 1.90 (s, 3H). HRMS (ESI/Q-TOF) m/z: [M + Na]+ calcd for C18H23NNaO9, 420.1271; found, 420.1276.

2-(2′,3′,4′,6′-Tetra-O-acetyl-α-d-mannopyranosyl)-pyrrole (4b)

Compound 4b was prepared according to the general procedure A starting from 2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl-trichloroacetimidate (2 g, 4.1 mmol), pyrrole (1.4 mL, 20.3 mmmol), and BF3·Et2O (260 μL, 2 mmol) at −78 °C (30 min). After workup (Et3N, 2 mL), the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 8:2 to 6:4) to afford 4b(47) (1.20 g, 74%). 1H NMR (CDCl3, 400 MHz): δ 8.78 (s, 1H), 6.26 (d, J = 1.9 Hz, 1H), 5.47–5.43 (m, 1H), 5.40–5.34 (m, 2H), 4.30–4.06 (m, 4H), 2.17 (s, 3H), 2.06 (s, 3H), 2.04 (s, 3H), 1.98 (s, 3H). HRMS (ESI/Q-TOF) m/z: [M + Na]+ calcd for C18H23NNaO9, 420.1271; found, 420.1266.

2-(2′,3′,6′,2″,3″,4”6″-Hepta-O-acetyl-β-d-maltopyranosyl)-pyrrole (4c)

This compound was prepared according to the general procedure A starting from 2,3,6,2′,3′,4′,6′-hepta-O-acetyl-β-d-maltopyranosyl-trichloroacetimidate (600 mg, 0.77 mmol), pyrrole (0.25 mL, 3.84 mmol), and BF3·Et2O (60 μL, 0.46 mmol) at −78 °C during 2h. After workup (Et3N, 360 μL), the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 8:2 to 6:4) to afford 4c(55) (368.6 mg, 70%). 1H NMR (CDCl3, 400 MHz): δ 8.33 (s, 1H), 6.76 (d, J = 1.7 Hz, 1H), 6.10 (m, 2H), 5.46 (d, J = 4.1 Hz, 1H), 5.42–5.33 (m, 2H), 5.07 (t, J = 9.9 Hz, 1H), 5.02 (t, J = 9.6 Hz, 1H), 4.89 (dd, J = 10.5, 4.0 Hz, 1H), 4.56 (d, J = 9.9 Hz, 1H), 4.48 (dd, J = 12.2, 2.5 Hz, 1H), 4.28–4.23 (m, 2H), 4.11–4.02 (m, 2H), 4.02–3.94 (m, 1H), 3.81 (ddd, J = 9.7, 4.4, 2.4 Hz, 1H), 2.13 (s, 3H), 2.11 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 2.01 (s, 6H), 1.90 (s, 3H).). 13C {1H} NMR (CDCl3, 125 MHz): δ 170.8, 170.7, 170.4, 170.1, 169.8, 169.6, 125.9, 118.8, 108.7, 107.9, 95.8, 77.4, 76.3, 73.9, 73.0, 72.2, 70.1, 69.5, 68.7, 68.1, 63.3, 61.6, 21.1, 21.0, 20.85, 20.7, 20.6. HRMS (ESI/Q-TOF) m/z: [M + Na]+ calcd for C30H39NNaO17, 708.2116; found, 708.2112.

2-(2′-O-Benzoyl-3′,4′,6′-tri-O-benzyl-α-d-mannopyranosyl)-pyrrole (4d)

The compound 4d was prepared according to the general procedure A starting from 3,4,6-tri-O-benzyl mannopyranosyl 1,2-orthobenzoate 15a (250 mg, 0.44 mmol), pyrrole (150 μL, 2.2 mmol), and BF3·Et2O (28 μL, 0.22 mmol) at −30 °C (1 h). After workup (Et3N, 2 mL), the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 8:2) to afford 4d (143 mg, 54%). 1H NMR (CDCl3, 400 MHz): δ 8.47 (bs, 1H), 8.19–8.10 (m, 2H), 7.63–7.54 (m, 1H), 7.49–7.24 (m, 15H), 7.21–7.13 (m, 2H), 6.76–6.72 (m, 1H), 6.12–6.08 (m, 2H), 5.95 (m, 1H), 5.23 (bs, 1H), 4.86 (d, J = 10.8 Hz, 1H), 4.85 (d, J = 11.5 Hz, 1H), 4.69 (d, J = 12.0 Hz, 1H), 4.66 (d, J = 11.4 Hz, 1H), 4.58 (d, J = 12.1 Hz, 1H), 4.53 (d, J = 10.9 Hz, 1H), 4.13 (dd, J = 9.1, 3.2 Hz, 1H), 4.01 (t, J = 9.3 Hz, 1H), 3.84–3.76 (m, 2H), 3.63 (ddd, J = 9.5, 4.6, 2.8 Hz, 1H). 13C {1H} NMR (CDCl3, 125 MHz): δ 165.9, 138.4, 138.3, 138.0, 133.2, 130.2, 130.1, 128.5, 128.4, 128.1, 127.9, 127.8, 127.74, 126.2, 118.7, 108.6, 107.1, 78.6, 75.2, 75.0, 73.9, 73.5, 72.0, 69.8, 69.7. HRMS (ESI/Q-TOF) m/z: [M + Na]+ calcd for C38H37NNaO6, 626.2519; found, 626.2533.

2-(2′-3′,4′,6′-Tetra-O-benzoyl-α-d-mannopyranosyl)-pyrrole (4e)

The compound 4e was prepared according to the general procedure A starting from methyl 3,4,6-tri-O-benzoyl mannopyranosyl 1,2-orthobenzoate 15b (250 mg, 0.4 mmol), pyrrole (60 μL, 0.82 mmol), and BF3·Et2O (30 μL, 0.2 mmol) at −30 °C (2 h). After workup (Et3N, 2 mL), the residue was purified by silica gel column chromatography (hexane/ethyl acetate, 8:2) to afford 4e (200 mg, 77%). 1H NMR (CDCl3, 400 MHz): δ 8.61 (s, 1H), 8.14–8.12 (m, 2H), 8.08–8.03 (m, 2H), 7.97–7.83 (m, 4H), 7.65–7.53 (m, 2H), 7.52–7.24 (m, 10H), 6.93–6.81 (m, 1H), 6.74–6.61 (m, 1H), 6.36 (dd, J = 3.2, 1.8 Hz, 1H), 6.34–6.29 (m, 1H), 6.21 (t, J = 10.0 Hz, 1H), 5.94 (dd, J = 10.0, 3.2 Hz, 1H), 5.40 (bs, 1H), 4.72 (dd, J = 12.2, 2.6 Hz, 1H), 4.49 (dd, J = 12.2, 4.1 Hz, 1H), 4.10 (ddd, J = 10.0, 4.1, 2.6 Hz, 1H). 13C {1H} NMR (CDCl3, 125 MHz): δ 166.4, 166.1, 165.6, 165.5, 133.6, 133.5, 133.4, 133.3, 130.0, 129.9, 129.8, 129.6, 129.1, 129.0, 128.7, 128.6, 128.5, 125.1, 119.5, 109.1, 109.0, 73.2, 71.7, 71.5, 70.1, 67.9, 63.0. HRMS (ESI/Q-TOF) m/z: [M + H]+ calcd for C38H32NO9, 646.2071; found, 646.2067.

1,3-Dimethyl-5-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12a)

According to general procedure B, a solution of C2-glucosylpyrrole 4a (100 mg, 0.25 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (147 mg, 0.3 mmol) in anhydrous CH2Cl2 (5 mL) was reacted with POCl3 (70 mL, 0.75 mmol). The crude was treated with triethylamine (0.35 mL, 2.5 mmol) and BF3·Et2O (0.19 mL, 1.5 mmol). The residue was purified by flash chromatography (hexane/ethyl acetate 6:4) to give derivative 12a as a red solid (106 mg, 77%) along with 1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 18 (1.9 mg, 5%). Data for 12a: [α]D25, +545.8 (c 0.36, CHCl3); Mp 114–116 °C; 1H NMR (CDCl3, 400 MHz): δ 7.11 (s, 1H), 6.84 (d, J = 4.1 Hz, 1H), 6.48 (d, J = 4.1 Hz, 1H), 6.15 (s, 1H), 5.37 (m, 2H), 5.19 (t, J = 9.4 Hz, 1H), 5.01 (d, J = 9.3 Hz, 1H), 4.24 (dd, J = 12.4, 4.7 Hz, 1H), 4.11 (dd, J = 12.3, 2.3 Hz), 3.90 (ddd, J = 10.0, 4.7, 2.2 Hz, 1H), 2.56 (s, 3H), 2.23 (s, 3H), 2.03 (s, 3H), 2.01 (s, 3H), 1.98 (s, 3H), 1.83 (s, 3H). 13C {1H} NMR (CDCl3, 125 MHz): δ 170.8, 170.2, 169.7, 169.6, 164.1, 148.3, 146.3, 136.8, 133.0, 126.6, 125.0, 121.9, 115.4, 74.6, 73.0, 71.0, 68.6, 62.2, 20.8, 20.7, 20.6, 15.3, 11.4. 19F NMR (CDCl3, 376 MHz): δ −142.03 (dq, J = 103.5, 33.4 Hz), −145.48 (dq, J = 103.7, 32.3 Hz). 11B NMR (CDCl3, 128 MHz): δ 0.73 (t, J = 32.7 Hz). HRMS (ESI/Q-TOF) m/z: [M + NH4]+ calcd for C25H33BF2N3O9, 568.2277; found, 568.2293; [M + Na]+ calcd for C25H29BF2N2NaO9, 573.1831; found, 573.1841. Data for 18(56): 1H NMR (CDCl3, 400 MHz): δ 7.01 (s, 1H), 6.02 (s, 2H), 2.51 (s, 6H), 2.22 (s, 6H).

1,3-Dimethyl-5-(2′,3′,4′,6′-tetra-O-acetyl-α-d-mannopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12b)

Compound 12b was prepared according to general procedure B: compound 4b (55.4 mg, 0.14 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (20.6 mg, 0.17 mmol) in anhydrous CH2Cl2 (3 mL) were reacted with POCl3 (39 μL, 0.42 mmol) during 15 min. The crude was treated with triethylamine (77 μL, 0.56 mmol) and BF3·Et2O (106 μL, 0.84 mmol). The residue was purified by flash chromatography (toluene/ethyl acetate 8:2) to give derivative 12b as a red solid (57.7 mg, 75%) along with 1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (18). Data for 12b: [α]D25, +434.2 (c 0.1, CHCl3); Mp 118–120 °C; 1H NMR (CDCl3, 400 MHz): δ 7.16 (s, 1H), 6.89 (d, J = 4.0 Hz, 1H), 6.71 (d, J = 4.0 Hz, 1H), 6.20 (s, 1H), 5.80 (t, J = 3.3 Hz, 1H), 5.46 (dd, J = 8.5, 3.0 Hz, 2H), 5.41 (dd, J = 8.7, 7.6 Hz, 1H), 4.46 (dd, J = 12.2, 4.5 Hz, 1H), 4.25 (dd, J = 12.2, 3.3 Hz, 1H), 3.84 (ddd, J = 7.8, 4.5, 3.3 Hz, 1H), 2.60 (s, 3H), 2.28 (s, 3H), 2.13 (s, 3H), 2.09 (s, 3H), 2.08 (s, 3H), 2.04 (s, 3H). 13C {1H} NMR (CDCl3, 125 MHz): δ 171.2, 170.6, 170.4, 169.7, 165.7, 146.6, 137.4, 133.8, 129.2, 128.4, 125.8, 124.6, 122.6, 116.5, 72.3, 70.8, 70.4, 69.1, 66.9, 62.1, 62.0, 21.1, 21.0, 20.90, 15.6, 11.6. 19F NMR (CDCl3, 376 MHz): δ −140.52 (dq, J = 99.0, 32.0 Hz), −149.48 (dq, J = 96.7, 31.1 Hz). 11B NMR (CDCl3, 128 MHz): δ −0.13 (t, J = 31.7 Hz). HRMS (ESI/Q-TOF) m/z: [M + NH4]+ calcd for C25H33BF2N3O9, 568.2277; found, 568.2290.

1,3-Dimethyl-5-(2′,3′,6′,2″,3″,4″6″-Hepta-O-acetyl-β-d-maltopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12c)

According to general procedure B, a solution of C2-maltosylpyrrole 4c (200 mg, 0.15 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (22 mg, 0.175 mmol) in anhydrous CH2Cl2 (5 mL) was reacted with POCl3 (40 μL, 0.44 mmol). The crude was treated with triethylamine (80 μL, 0.58 mmol) and BF3·Et2O (110 μL, 0.87 mmol). The residue was purified by flash chromatography (hexane/ethyl acetate 3:7) to give derivative 12c as an orange solid (97 mg, 83%). [α]D25, +166.2 (c 0.1, CHCl3); Mp 139–141 °C; 1H NMR (CDCl3, 400 MHz): δ 7.12 (s, 1H), 6.84 (d, J = 4.1 Hz, 1H), 6.43 (d, J = 4.1 Hz, 1H), 6.18 (s, 1H), 5.43–5.39 (m, 2H), 5.37 (dd, J = 10.5, 9.4 Hz, 1H), 5.28 (t, J = 9.4 Hz, 1H), 5.13–5.00 (m, 2H), 4.88 (dd, J = 10.5, 4.1 Hz, 1H), 4.46 (dd, J = 12.3, 2.4 Hz, 1H), 4.27–4.21 (m, 2H), 4.10 (dd, J = 9.8, 8.5 Hz, 1H), 4.04 (dd, J = 12.5, 2.3 Hz, 1H), 3.98 (ddd, J = 10.3, 3.7, 2.3 Hz, 1H), 3.92 (ddd, J = 9.7, 4.5, 2.4 Hz, 1H), 2.59 (s, 3H), 2.26 (s, 3H), 2.10 (s, 3H), 2.09 (s, 3H), 2.07 (s, 3H), 2.02 (s, 3H), 2.01 (s, 3H), 2.00 (s, 3H), 1.84 (s, 3H). 13C {1H} NMR (CDCl3, 125 MHz): δ 170.8, 170.8, 170.7, 170.2, 169.9, 169.6, 164.4, 148.5, 146.3, 137.0, 133.1, 126.5, 124.9, 122.0, 122.0, 115.0, 95.8, 76.4, 73.4, 72.7, 71.7, 70.2, 69.6, 68.6, 68.2, 63.4, 61.6, 21.1, 21.0, 20.8, 20.7, 20.6, 15.4, 11.5. 19F NMR (CDCl3, 376 MHz): δ −142.24 (dq, J = 103.9, 33.3 Hz), −145.48 (dq, J = 103.7, 32.0 Hz). 11B NMR (CDCl3, 128 MHz): δ 0.71 (t, J = 32.7 Hz). HRMS (ESI/Q-TOF) m/z: [M + NH4]+ calcd for C37H49BF2N3O17, 856.3123; found, 856.3116.

1,3-Dimethyl-5-(2′-benzoyl-3′,4′,6′-tri-O-benzyl-α-d-mannopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12d)

Following the general procedure B, a solution of compound 4d (161 mg, 0.27 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (28 mg, 0.225 mmol) in anhydrous CH2Cl2 (7 mL) was treated with POCl3 (63 μL, 0.67 mmol). After stirring for 12 h, triethylamine (0.31 mL, 2.25 mmol) and BF3·Et2O (0.29 mL, 2.25 mmol) were added. The residue was purified by flash chromatography (hexane/ethyl acetate 9:1) to give BODIPY 12d as an orange solid (133 mg, 65%). [α]D25, +1854.2 (c 0.05, CHCl3); Mp 98–100 °C; 1H NMR (CDCl3, 400 MHz): δ 8.01 (m, 2H), 7.53–7.21 (m, 18H), 7.08 (s, 1H), 6.71 (d, J = 4.1 Hz, 1H), 6.45 (d, J = 4.0 Hz, 1H), 6.20 (d, J = 2.9 Hz, 1H), 6.14 (s, 1H), 5.25 (s, 1H), 4.94–4.85 (m, 3H), 4.65–4.58 (m, 3H), 4.16 (t, J = 9.4 Hz, 1H), 4.04 (dd, J = 9.3, 3.0 Hz, 1H), 3.98 (dd, J = 11.4, 3.9 Hz, 1H), 3.87 (dd, J = 11.3, 1.7 Hz, 1H), 3.75 (ddd, J = 9.6, 3.9, 1.7 Hz, 1H), 2.60 (s, 3H), 2.24 (s, 3H). 13C {1H} NMR (CDCl3, 125 MHz): δ 165.2, 162.3, 152.1, 145.1, 138.99, 138.8, 138.0, 136.0, 132.9, 130.3, 130.0, 128.5, 128.5, 128.4, 128.0, 127.7, 127.5, 127.2, 124.9, 121.2, 116.7, 81.7, 79.9, 75.3, 74.2, 74.0, 73.6, 71.3, 69.9, 69.8, 69.5, 15.3, 11.5. 19F NMR (CDCl3, 376 MHz): δ −144.08 (dq, J = 105.0, 32.3 Hz), −146.48 (dq, J = 103.2, 33.7 Hz). 11B NMR (CDCl3, 128 MHz): δ 0.77 (t, J = 33.4 Hz). HRMS (ESI/Q-TOF) m/z: [M + NH4]+ calcd for C45H47BF2N3O6, 774.3528; found, 774.3481. [M + Na]+ calcd for C45H43BF2N2NaO6, 779.3082; found, 779.3075.

1,3-Dimethyl-5-(2′,3′,4′,6′-tetra-O-benzoyl-α-d-mannopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12e)

Following the general procedure B, a solution of compound 4e (210 mg, 0.32 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde 11 (48 mg, 0.39 mmol) in anhydrous CH2Cl2 (15 mL) was treated with POCl3 (100 μL, 0.96 mmol). After stirring for 12 h, triethylamine (0.3 mL, 2 mmol) and BF3·Et2O (0.4 mL, 2.93 mmol) were added. The residue was purified by flash chromatography (hexane/ethyl acetate 8:2) to give BODIPY 12e as a red solid (143 mg, 56%). [α]D25, +1153.4 (c 0.06, CHCl3); Mp 110–112 °C; 1H NMR (CDCl3, 400 MHz): δ 8.11 (m, 4H), 7.92 (m, 4H), 7.61–7.30 (m, 12H), 7.21 (s, 1H), 7.05 (s, J = 4.0 Hz, 1H), 7.00 (d, J = 4.0 Hz, 1H), 6.36 (t, J = 2.9 Hz, 1H), 6.28 (t, J = 9.1 Hz, 1H), 6.21 (s, 1H), 5.99 (dd, J = 9.3, 3.1 Hz, 1H), 5.76 (d, J = 2.8 Hz, 1H), 4.79 (dd, J = 12.1, 3.3 Hz, 1H), 4.63 (dd, J = 12.1, 3.8 Hz, 1H), 4.26 (dt, J = 8.9, 3.5 Hz, 1H), 2.60 (s, 3H), 2.30 (s, 3H). 13C {1H} NMR (CDCl3, 125 MHz): δ 166.5, 166.4, 166.0, 165.6, 165.3, 146.6, 146.4, 137.5, 134.1, 133.5, 133.4, 132.9, 130.4, 130.2, 130.0, 129.9, 129.6, 129.3, 129.1, 128.6, 128.5, 128.3, 125.8, 124.6, 122.8, 117.1, 117.0, 72.2, 72.0, 71.9, 70.2, 67.5, 63.3, 15.6, 11.6. 19F NMR (CDCl3, 376 MHz): δ −141.17 (dq, J = 101.5, 32.2 Hz), −150.98 (dq, J = 101.1, 30.6 Hz). 11B NMR (CDCl3, 128 MHz): δ 0.89 (t, J = 31.5 Hz). HRMS (ESI/Q-TOF) m/z: [M + NH4]+ calcd for C45H41BF2N3O9, 816.2906; found, 816.2885. [M + Na]+ calcd for C45H37BF2N2NaO9, 821.2460; found, 821.2453.

Gram-Scale Synthesis of BODIPY 12a

For 2,3,4,6-tetra-O-acetyl-α,β-d-glucopyranose, 1,2,3,4,6-penta-O-acetyl-α,β-d-glucopyranose (4g, 10.2 mmol) was dissolved in ethyl acetate (150 mL) and DMSO (15 mL); then, aminoethanol (0.62 mL, 10.2 mmol) was added, and the mixture was stirred at r.t. until the reaction was complete (1 h). The crude was diluted with ethyl acetate and washed several times with brine. The organic layer was dried over MgSO4, filtered, and concentrated in vacuo. The residue was purified by column chromatography on silica gel (hexane/ethyl acetate, 1:1) to afford 2,3,4,6-tetra-O-acetyl-α,β-d-glucopyranose47 (2.5 g, 70%).

2,3,4,6-Tetra-O-acetyl-α-d-glucopyranosyl-trichloroacetimidate (14a)

To a stirred solution of 2,3,4,6-tetra-O-acetyl-α,β-d-glucopyranose (2.42 g, 6.95 mmol) in dry CH2Cl2 (25 mL) cooled at 0 °C were added dropwise 1,8-diazabicyclo[5.4.0]undec-7-ene (0.84 mL, 5.6 mmol) and trichloroacetonitrile (2.78 mL, 27.8 mmol). The reaction mixture was stirred at r.t. for 2 h, the solvent was evaporated under reduced pressure, and the crude was subjected to column chromatography (hexane/ethyl acetate, 85:15) to obtain 14a(47) (3.24 g, 95%).

2-(2′,3′,4′,6′-Tetra-O-acetyl-β-d-glucopyranosyl)-pyrrole (4a)

Compound 4a (1.5 g, 81%) was prepared following general procedure A, starting from 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl-trichloroacetimidate 14a (2.3 g, 4.7 mmol), pyrrole (1.4 mL, 20.3 mmol), and BF3·Et2O (260 μL, 2 mmol) at −78 °C (30 min).

1,3-Dimethyl-5-(2′,3′,4′,6′-tetra-O-acetyl-β-d-glucopyranosyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (12a)

According to general procedure B, a solution of C2-glucosylpyrrole 4a (1.18 g, 2.96 mmol) and 3,5-dimethylpyrrole-2-carbaldehyde (437 mg, 3.55 mmol) in anhydrous CH2Cl2 (30 mL) was reacted with POCl3 (0.83 mL, 8.88 mmol). The reaction crude was treated with triethylamine (4.1 mL, 29.6 mmol) and BF3·Et2O (2.25 mL, 17.8 mmol). The residue was purified by flash chromatography (hexane/ethyl acetate 6:4) to give derivative 12a as a red solid (1.19 g, 73%) along with 1,3,5,7-tetramethyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene 18 (31 mg, 7%). The spectroscopic and analytical data of 12a from the gram-scale reaction were consistent with those above for this derivative, obtained in the 0.25 mmol-scale experiment.

Photophysical Properties

The photophysical properties were registered from diluted solutions (around 2 × 10–6 M), prepared by adding the corresponding solvent to the residue from the adequate amount of a concentrated stock solution in methanol, after vacuum evaporation of this solvent. All organic solvents were of spectroscopic grade, and water was of Milli-Q grade. UV–vis absorption and fluorescence spectra were recorded on a Varian model CARY 4E spectrophotometer and an Edinburgh Instruments spectrofluorometer (model FLSP 920), respectively. Fluorescence quantum yields (Φfl) were obtained using commercial BODIPYs (PM546, Φfl = 0.85 in ethanol, for the nonhalogenated dyes, and PM567, Φfl = 0.84 in ethanol, for the halogenated dyes) and cresyl violet (Φfl = 0.54 in methanol, for the π-extended dye 30), as references, from corrected spectra (detector sensibility to the wavelength). The values were corrected by the refractive index of the solvent. Radiative decay curves were registered with the time-correlated single-photon counting technique as implemented in the aforementioned spectrofluorometer. Fluorescence emission was monitored at the maximum emission wavelengths after excitation by means of a Fianium pulsed laser (time resolution of picoseconds) with a tunable wavelength. The fluorescence lifetime (τfl) was obtained after the deconvolution of the instrumental response signal from the recorded decay curves by means of an iterative method. The goodness of the exponential fit was controlled by statistical parameters (chi-square, Durbin–Watson, and the analysis of the residuals).

Nanosecond transient absorption spectra (ns-TAS) were recorded on an LP 980 laser flash photolysis spectrometer (Edinburgh Instruments). Samples were excited by a nanosecond pulsed laser (Nd:YAG laser, Lotis TII 2134) operating at 1 Hz and a pulse width of ≥7 ns, coupled to an OPO, which allows the selection of the excitation wavelength. The transient signals were recorded on a single detector (PMT R928P) oscilloscope for kinetic traces and an ICCD detector DH320T TE cooled (Andor Technology) for time-resolved spectra. Samples were measured aerated and deaerated with nitrogen or oxygen for ca. 15 min before each measurement.

The photoinduced production of singlet oxygen (1O2) was determined by direct measurement of the luminescence at 1276 nm with an NIR detector integrated in the aforementioned spectrofluorometer (InGaAs detector, Hamamatsu G8605-23). The 1O2 signal was registered in a front configuration (front face), 40 and 50° to the excitation and emission beams, respectively, and leaned 30° to the plane formed by the direction of incidence and registration in cells of 1 cm. The signal was filtered by a low cutoff of 850 nm. The 1O2 generation quantum yield (ΦΔ) was determined using the following equation:

graphic file with name jo3c02907_m001.jpg

where ΦΔr is the quantum of 1O2 generation for the used reference (2,6-diiodo-3,5-dimethyl-8-methylthioBODIPY, MeSBDP) being 0.91 in chloroform. Factor α = 1 – 10–Abs corrects the different amounts of photons absorbed by the sample (αPS) and reference (αR). Factor Se is the intensity of the 1O2 phosphorescence signal of the sample (SePS) and the reference (Ser) at 1276 nm. 1O2 quantum yields were averaged from at least five concentrations between 10–6 and 10–5 M.

Photostability

The photostability of the dyes was evaluated from concentrated water solutions (millimolar) contained in 0.1 cm optical-path quartz cells to allow for the minimum solution volume (0.3 mL) to be excited. The liquid solutions were pumped transversely with 5 mJ, 8 ns full width at half-maximum (fwhm) pulses from the third harmonic (355 nm) of a Q-switched Nd:YAG laser (Lotis TII2134) at a 10 Hz repetition rate. The exciting pulses were line-focused onto the cell using a combination of positive and negative cylindrical lenses (f = 15 cm and f = −15 cm, respectively) perpendicularly arranged. The lateral faces of cells were grounded, whereupon no laser oscillation was obtained. Photostability was determined by monitoring the decrease in laser-induced fluorescence intensity after 50,000 pump pulses. The emitted light was collected in a front-face configuration and integrated by a boxcar averager (Stanford Research, model 250) before digitization and computer analysis. The estimated error in photostability measurement was 10%.

Delayed Spectroscopy

Aerated solutions at room temperature of the new dyes contained in 1 cm optical-path rectangular quartz cells were transversally pumped with intense laser pulses from the second harmonic (532 nm) of a Nd:YAG laser (Lotis TII, LS-2147) at a 10 Hz repetition rate. The time-gated emission upon laser photoexcitation, analyzed perpendicularly to the input radiation, was focused onto a spectrograph (Kymera 193i-A, Andor Technologies) coupled to an intensified CCD camera (iStar, Andor Technologies). This camera enables gate widths ranging from nanoseconds up to seconds, and its opening can be delayed in a controlled way with respect to the incoming pump laser pulse. Neither long-pass filters nor band-pass filters were used to remove the excitation laser since we have verified that these filters, especially long-pass ones, under drastic pump conditions, exhibited their own fluorescence and/or phosphorescence emission, which could lead to misunderstanding the experimental results. Each spectrum is the average of at least 200 scans recorded with a gate time of 50 μs. The experiments were usually carried out at an excitation energy fluence of 5 mJ/cm2, which was varied from 1 up to 25 mJ/cm2 to determine the dependence of the delayed fluorescence on the laser fluence. A solution volume of 3 cm3 was used in order to avoid (or at least, to reduce) the risk of photobleaching the sample during the experiments. This experimental setup allowed to carry out the projected measurements even under adverse conditions but avoided to determine properly the efficiency of the delayed emission.

Acknowledgments

This research received financial support from the Spanish Ministerio de Ciencia e Innovación (MCIN)/Agencia Estatal de Investigación (AEI) Grants PID2020-114755GB-C31 and PID2020-114755GB-C33 and PID2021-122504NB-I00 funded by MCIN/AEI/10.13039/501100011033 and by ERDF A way of making Europe. Gobierno Vasco (IT1639-22) is also thanked for financial support. N.C. thanks MINECO (project PID2020-114755GB-C33) for a predoctoral contract.

Data Availability Statement

The data underlying this study are available in the published article and its Supporting Information

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.joc.3c02907.

  • Experimental procedures; copies of 1H, 13C NMR, and photophysical data of all compounds; absorption and steady-state fluorescence spectra, transient absorption spectra, and time-dependent emission spectra of selected representative BODIPY-carbohydrate conjugates (PDF)

The authors declare no competing financial interest.

Supplementary Material

jo3c02907_si_001.pdf (13.6MB, pdf)

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