Synthesis of phenylazonaphtol-β-D-O-glycosides, evaluation as substrates for beta-glycosidase activity and molecular studies

Background Phenylazonaphtol-β-D-O-glycosides are alternative substrates for the detection of enzymatic activity of β-glycosidases which are involved in various important processes. These azoic compounds are currently exploited as prodrugs for colonic disease due the presence of β-glycosidase activity in the gut flora and therefore allowing the release of the drug at the specific site. Results Phenylazonaphtol-β-D-O-glucoside 3a and galactoside 3b were prepared via diazonium salt conditions under weak acidic conditions which do not compromise the O-glycosidic bond stability, by coupling reaction between 2-naphtol sodium salt with aminoglycosides 1a and 1b. The resulting phenylazonaphtol glycosides 2a and 2b were deprotected affording the phenylazonaphtol glycosides 3a and 3b in quantitative yield. The galactoside glycoside 3b was assayed as substrate for in vitro β-galactosidase enzymatic activity showing strong absorbance after releasing of the azoic chromophore. Also, docking studies were performed to determine the best pose as well as the interactions between the ligand and the residues located at the active site. Conclusions The methodology developed for synthesizing the phenylazonaphtol glycosides described proved to be convenient for generating azoic functionalities in the presence of glycosidic bonds and the glycosides suitable as alternative substrates and potentially useful prodrugs in the treatment of colonic diseases.

Background β-glycosidases are hydrolytic enzymes involved in a number of important processes such as defense mechanism, growth regulation [1], gene markers in transgenic plants [2], and prodrugs [3]. The most commonly used substrates for the histochemical localization of β-glycosidases contain the 5-bromo-4-chloro-3-indolyl chromophore attached to most of the known monosaccharides through an O-glycosidic bond [4]. After enzymatic hydrolysis, the water soluble indoxyl intermediate must undergo an oxidative dimerization to produce a blue precipitate. However, in some cases before dimerization occurs to produce the indigo dye, some diffusion takes place, producing false positives at some regions in cells lacking enzymatic activity [5]. In addition, phenylazonaphtol glucosides have been synthetically prepared and tested in transgenic plants containing the β-glucuronidase activity which is a widely used gene marker. A comparative test between the commercially available glycosidic indoxyl substrates with the alternative phenylazonaphtol glucuronides resulted in partial diffusion for the indigo dye and no detectable diffusion for the phenylazonaphtol chromophore [6,7].
On the other hand, glycosides when attached to pharmacologically active substances can be used as prodrugs for improving availability, stability, and particularly for specific delivery of the active compound at the target organ. For instance, steroids, antitumor, and anti-inflammatory compounds have been attached to glycosides and evaluated as specific delivery prodrugs [8]. Also, azoic β-glycosides provided to be useful as prodrugs particularly against colitis, Crohn's disease, and colorectal cancer since the delivery at colon level might be achieved by the action of azoreductase or glycosidase present at the colonic microflora [9].
Due the potential usefulness of phenylazonaphtol glycosides in processes mentioned above, we developed a methodology for preparing phenylazonaphtol glycosides under weak acidic conditions and the resulting azoic glycosides evaluated as β-galactosidase substrates with potential application as prodrugs for colon diseases.

Methods
The preparation of the phenylazonaphtol glycosides 3a and 3b was achieved according to Scheme 1 consisting in the coupling reaction between 2-naphtol in the sodium salt form with 4-aminophenyl tetracetyl glycopyranoside [10][11][12] 1a and 1b under modified diazonium salt condition, which is mainly the use of acetic acid instead of stronger acids, with sodium nitrite at 0°C to produce the diazonium salts which was condensed in situ with 2naphtol sodium salt providing the corresponding phenylazonaphtol tetracetyl glucopyranoside 2a and 2b. Final removal of the acetate protecting groups under Zemplen conditions provided the target phenylazonaphtol glycosides 3a and 3b as a reddish solid in 60% yield.

Results and discussion
The deprotected glycoside 3b was tested as substrate for the detection of glycosidase activity using commercial Escherichia coli β-galactosidase. Thus, the hydrolytic activity resulted in the cleavage of the glycosidic linkage, releasing the phenylazonaphtol chromophore which is observed as an intense reddish color with absorption at 410 and 455 nm, as it is observed in the absorption graphic ( Figure 1) and as observed in the vials ( Figure 2).

Docking studies
The active site architecture showing the interactions between the phenylazonaphtol galactoside 3b and E. coli βgalactosidase pocket was performed using AutoDock 4.2 [14] and Chimera [15] software packages as visualization system. The protein assigned for the docking analysis was E. coli β-galactosidase (PDB IDs: 1JYN), and the residues chosen were Asn102, Asp201, Asn460, His418, Glu461, Glu537, His540, Phe 601, and Trp999 which have been described to participate in the interactions with its natural substrate lactose [16]. β-galactosidase is a retaining glycosidase with a double displacement mechanism, and O its interactions with the ligand proceed with participation of water and the ions Mg 2+ and Na 1+ within the catalytic cavity [17]. The docking analysis was performed to find the minimized conformations according to the docking protocols which use the Lamarckian Genetic Algorithm for searching the most favorable ligand-protein complex interactions. Thus, as a result of the docking analysis, we found the minimized conformer with the galactose moiety positioned toward the active site having a binding energy of −11.62, showing hydrogen bond interactions of C-3 and C-4 hydroxyl groups of galactoside moiety with Glu461 residue, in agreement with those reported for the natural substrate lactose [16] (Figure 3). The ribbon representation was also analyzed by using the chimera visualizing system to show the region of the cavity and the residues involved in the interaction with the substrate 3b (Figure 4).
In addition, the surface representation shows that the galactopyranoside moiety is embedded into the pocket interacting with the residues while the azoic chromophore

1-[(4-tetra-O-acetyl-β-D-glucopyranosyloxy phenyl)azo]-2naphtol (2a)
Compound 1a (0.543 g, 1.23 mmol) was dissolved in 8 mL of THF, and the flask was cooled to 0°C. After the addition of sodium nitrite (85 mg, 1.23 mmol) and 0.5 mL of acetic acid/H 2 O (1:1, v/v), the reaction was kept under ice-bath temperature with stirring for 15 min. 2-naphtol sodium salt dissolved in 5 mL of THF/H 2 O (1:1, v/v) was added dropwise in another flask, and the reaction was kept at 0°C with stirring for 30 min, allowing the reaction to reach room temperature and stirred for another 60 min. The solvent was removed under vacuo, diluted with CH 2 Cl 2 (30 mL) and washed with cold 2 N NaOH solution (3 × 15 mL) and water (20 mL). The organic phase was dried over anhydrous sodium sulfate and evaporated on