**1. Introduction**

The recognition of the crucial role of heparanase enzyme, an endo-β-D-glucuronidase able to degrade heparan sulfate (HS) in the extracellular matrix and basement membranes, in a number of pathological processes, such as metastasis and angiogenesis, has triggered the development of heparanase inhibitors [1–6]. Among these inhibitors PI-88, a mixture of functionalized mannose oligosaccharides, i.e., **1, 2**, (Figure 1), that potently inhibited heparanase and in vitro angiogenesis, has been considered as a promising candidate and has received considerable attention [7]. On the other hand, fluorescent labeling of biomolecules has been recognized as a research topic of great significance, since such labeling facilitates the investigation of glycoconjugates and their interaction in biological systems at high sensitivity [8–12]. Among the types of fluorescent dyes commonly employed as tags, borondipyrromethene (BODIPY, 4, 4-difluoro-4-bora-3a, 4a-diaza-s-indacene) dyes, e.g., **3** (Figure 1) have excelled. Several reasons can be cited for this preference: their high fluorescent quantum yields (∅), excellent photochemical and chemical stabilities, and, arguably, the relatively facile modulation of their photophysical and/or chemical properties by means of synthetic postfunctionalization of their indacene core [13–18].

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.

**Citation:** Ventura, J.; Uriel, C.; Gomez, A.M.; Avellanal-Zaballa, E.; Bañuelos, J.; García-Moreno, I.; Lopez, J.C. A Concise Synthesis of a BODIPY-Labeled Tetrasaccharide Related to the Antitumor PI-88. *Molecules* **2021**, *26*, 2909. https:// doi.org/10.3390/molecules26102909

Academic Editor: Ugo Caruso

Received: 14 April 2021 Accepted: 12 May 2021 Published: 14 May 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Figure 1.** PI-88 (**1**,**2**), BODIPY (**3**), IUPAC numbering) and 1,2-methyl orthoester glycosyl donors (**4**) used in this study.

#### **2. Results**

TBDPSO BzO BzO

P

Bn = benzyl; Bz = benzoyl;

TBDPS = tert-butyldiphenyl silyl

P

Several synthetic approaches to analogues of tetra- and pentasaccharides **1** have already been reported [22–28]. In those approaches, different types glycosyl donors have been employed. We have been interested in the use of 1,2-methyl orthoesters (MeOEs) [29–33] as an inexpensive alternative to Fraser–Reid's *n*-pentenyl orthoester glycosyl donors (NPOEs) [34–36]. The former derivatives were shown to display good regioselectivity, similar to that of NPOEs [31]. Accordingly, our convergent 1 + 1 + 2 approach to BODIPYtagged tetramannan, **10** (Scheme 1) was based on our previous studies on the selective mono-glycosylation at position *O*-3 in mannopyranose substrates possessing a 2,3,4 triol moiety, i.e., **6**, **7** (Scheme 1), with MeOE glycosyl donors, **4a**, **4b**, and **9 [31]**. Thus, our strategy involved two glycosidic disconnections **A** and **B** (Scheme 1). Disconnection **A** (Scheme 1), leading to BODIPY-disaccharide **6**, was envisioned by glycosylation of a benzylated mannopyranoside **5**, exposing only one hydroxyl group (*O*-2) for the glycosyl coupling, with MeOE donor **4a**. On the other hand, disconnection **B** (Scheme 1) was imagined by regioselective mono-glycosylation of triol **6**, at position *O*-3', according to precedents from our research group [31], by a disaccharide MeOE donor, **9**.

O

Ph

O

OMe

= Bz;

5 7 SPh **Scheme 1.** Retrosynthesis of PI-88 tetrasaccharide analogue **10**, involving the sole use of 1,2-methyl orthoester glycosyl donors, **4** and **9**.

**BODIPY**

The synthetic route started with the glycosylation of BODIPY **11 [37]**, readily available through a one-pot transformation using phthalide and pyrrole as the starting materials, followed by in situ coordination with BF3.OEt<sup>2</sup> [38], with methyl orthoester **4c**, to yield BODIPY glycoside **12** (80% yield, Scheme 2a). However, attempted saponification of the

3 2

2-*O*-Bz substituent in **12**, by treatment with Et3N or NaOMe in methanol, resulted in the production of the undesired B(OMe)2-BODIPY derivatives **13** and **14**, where the fluorine atoms were replaced by methoxy groups (Scheme 2a). These derivatives, although also fluorescent [39], proved to be labile under the acidic conditions required in the next glycosylation events. We then turned our attention to borondiphenyl BODIPY **15**, a more chemically robust yet fluorescent BODIPY analogue [40]. Access to **15** was also affected by slightly modifying our one-pot procedure [38], from phthalide and pyrrole, by simply replacing BF3.OEt<sup>2</sup> by B(Ph)<sup>3</sup> in the borondipyrromethene ring closing reaction (Scheme 2b) [41].

**Scheme 2.** (**a**) Glycosylation of BODIPY **11** with MeOE **4c** and saponification attempts; (**b**) one-pot synthesis of 4,4-diphenyl BODIPY **15**.

The synthetic routes to the two fragments in the convergent approach to PI-88 tetrasaccharide analogue **10**, glycosyl acceptor **6** and glycosyl donor **9**, are depicted in Scheme 3a,b. Thus, glycosylation of hydroxymethyl BODIPY **15** with benzylated MeOE **4c**, followed by saponification (NaOMe/MeOH), led to BODIPY-mannopyranoside **5** (Scheme 3a). The latter was then glycosylated with tri-*O*-benzoyl MeOE **4a**, to yield BODIPY disaccharide **16a** in moderate yield (52%). Next, protecting group manipulations in compound **16a**, including de-*O*-benzoylation leading to tetraol **16b**, and selective monosilylation at the primary hydroxyl group at O-6' in the latter [terbutyldiphenylsilyl chloride, 4-dimethylaminopyridine (DMAP), in dimethyl formamide (DMF)] produced 2',3',4'-triol **6**, (Scheme 3a).

On the other hand, the route to MeOE-disaccharide donor **9** started from phenyl thiomannopyranoside **7** (Scheme 3b). Accordingly, regioselective mono-glycosylation at *O*-3 of 2,3,4-triol **7** with MeOE **4b,** based on precedents from our research group [31,42], yielded phenyl 1-thiomannopyranoside **8** in good yield (Scheme 3b). To conclude, a threestep sequence from **8**, including perbenzoylation, anomeric bromination, and orthoester formation from an intermediate glycosyl bromide according to Wei et al. [43], allowed its conversion to MeOE disaccharide donor **9** (Scheme 3b).

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.

**Scheme 3.** (**a**) Synthesis of BODIPY disaccharide acceptor **6**; (**b**) access to MeOE-disaccharide donor **9**, from thioglycoside **7**, by regioselective glycosylation with MeOE **4b** and subsequent orthoester formation on the ensuing disaccharidic thioglycoside intermediate **8**.

9 O O BnO **Scheme 4.** Regioselective glycosylation of triol **6** with MeOE-disaccharide **9** leading to tetrasaccharide **10**.

O

6

BF<sup>3</sup> .OEt<sup>2</sup> - 30 oC, CH2Cl<sup>2</sup> 53% 10 N B N Ph Ph N B N Ph Ph 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 *ortho*- position 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.

**Figure 2.** Absorption (in blue) and normalized fluorescence (in red) spectra of BODIPY **15** and its glycosylated derivative **16a** in diluted solution of ethyl acetate. The spectral profiles of **10** fully resemble those of **16a**. The excited state optimized geometry of **15** in two different views is also enclosed with key dihedral angles to show the bending of the chromophore.

**Table 1.** Photophysical <sup>1</sup> and laser <sup>2</sup> properties of BODIPY **15**, and glycosylated derivatives **16a** and **10**, in ethyl acetate. For the sake of comparison, the corresponding photophysical data of the F-BODIPY counterpart (in ethanol) have been added. For additional photophysical data, see Table S1.


μ λ λ ε <sup>−</sup> <sup>−</sup> ∅ τ <sup>1</sup> Registered under a soft irradiation regime; dye concentration: 2 µM. Absorption (λab) and fluorescence (λfl) wavelength, molar absorption (εmax) (10<sup>4</sup> M−<sup>1</sup> cm−<sup>1</sup> ), fluorescence quantum yield (∅), and amplitude-average lifetime (<τ>). <sup>2</sup> Recorded under a hard irradiation regime; dye concentration 2 mM. Peak wavelength for the laser emission (λla) and efficiency (Eff (%)) defined as the ratio between the energy of the laser output and the pump energy incident on the cell surface.

#### **3. 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

λ

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.

#### **4. Materials and Methods**

#### *4.1. 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<sup>4</sup> 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 H2SO<sup>4</sup> (200 mL) in AcOH (800 mL). <sup>1</sup>H and <sup>13</sup>C-NMR spectra were recorded in CDCl<sup>3</sup> 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 (CHCl3: δ 7.25 ppm, CD3OD: δ 4.870 ppm). Coupling constants (*J*) are given in Hz. All presented <sup>13</sup>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.

#### *4.2. General Procedures*

#### 4.2.1. 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 BF3.OEt<sup>2</sup> (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<sup>3</sup> solution. The organic layer was dried over Na2SO4, filtered, and evaporated under vacuum. The resulting crude mixture was purified by chromatography on silica gel (eluent: hexaneethyl acetate mixtures).

#### 4.2.2. 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.

#### 4.2.3. 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

acetate and extracted with a saturated aqueous NaHCO<sup>3</sup> solution and brine. The combined organic solutions were dried over anhydrous MgSO<sup>4</sup> and evaporated under vacuum. The resulting crude mixture was purified by chromatography on silica gel (eluent: hexane-ethyl acetate mixtures).

## 4.2.4. General Procedure for Characterization of Polyol Derivatives as Peracetates. Procedure D

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).
