Synthesis of Tetravalent Thio- and Selenogalactoside-Presenting Galactoclusters and Their Interactions with Bacterial Lectin PA-IL from Pseudomonas aeruginosa

Synthesis of tetravalent thio- and selenogalactopyranoside-containing glycoclusters using azide-alkyne click strategy is presented. Prepared compounds are potential ligands of Pseudomonas aeruginosa lectin PA-IL. P. aeruginosa is an opportunistic human pathogen associated with cystic fibrosis, and PA-IL is one of its virulence factors. The interactions of PA-IL and tetravalent glycoconjugates were investigated using hemagglutination inhibition assay and compared with mono- and divalent galactosides (propargyl 1-thio- and 1-seleno-β-d-galactopyranoside, digalactosyl diselenide and digalactosyl disulfide). The lectin-carbohydrate interactions were also studied by saturation transfer difference NMR technique. Both thio- and seleno-tetravalent glycoconjugates were able to inhibit PA-IL significantly better than simple d-galactose or their intermediate compounds from the synthesis.


Introduction
Lectins from pathogenic organisms could be important virulence factors. These specific carbohydrate-binding proteins could be involved in the recognition and adhesion processes in host-pathogens interactions [1]. Consequently, carbohydrate-based inhibitors of lectins are promising potential therapeutics [2]. Lectins are usually multivalent oligomeric proteins, frequently displaying an avidity effect and increased affinity to complex glycosylated surfaces. Therefore, the multivalent inhibitors containing several carbohydrate residues are suitable for disrupting lectins' binding to host cells or tissues [3]. The opportunistic pathogen Pseudomonas aeruginosa is a Gram-negative bacterium, causing chronic and potentially lethal lungs infections in immunocompromised humans, mainly patients suffering from cystic fibrosis (CF). It is the most widespread pulmonary pathogen associated with CF and significantly influences morbidity and mortality [4,5]. P. aeruginosa associated with CF and significantly influences morbidity and mortality [4,5]. P. aerugin produces a tetrameric D-galactose-specific lectin named PA-IL (LecA) which is conside to be involved in adhesion, biofilm formation, cellular invasion and cytotoxicity [6][7][8][9][10] Our previous works presented several potential inhibitors of various lectins of b terial and fungal origin [11]. Recently, a tetravalent lead compound I for anti-adhes therapy of Pseudomonas aeruginosa infections was developed ( Figure 1) [12]. Our current aim is to synthesize the thio-and selenoglycoside analogues of the te valent lead-structure as well as their intermediate compounds to investigate their bind properties towards lectin PA-IL and the effect of sulfur and selenium on lectin bindi Several selenium-containing carbohydrates are known from the literature. They were s thesized for various purposes [13], exploiting the inherent potential of the selenium cleus [14]. Selenoglycosides were used as glycosyl donors [15], for protein glycoconju tion in site-selective glycosylation by Se-S-mediated ligation [16], as enzyme inhibitors GlcNAcase [17] and as novel glycosidase inhibitors [18]). Mono-and divalent seleno lactosides and diselenide digalactosides proved to be potential ligands to biomedic relevant galactophilic lectins [19]; non-glycosidically linked Se-containing pseudodis charides were also synthesized as BanLec and ConA lectin ligands [20]. Selenium-link neoglycoconjugates, pseudodisaccharides [21], selenenylsulfide-linked glycopepti and glycoproteins [22] were also prepared. Moreover, the presence of a selenium nucl provides an excellent opportunity for structural analysis of biomolecules by NMR and ray spectroscopy [14] and to study the lectin-carbohydrate interactions using sophi cated 77 Se-NMR methods [23]. Selenium could be also potentially used as a trace for selective detection of compounds in the biofluids [24]. A further advantage of the Se terglycosidic linkage is its higher stability towards hydrolases [17,18].

Synthesis
PA-IL is known to recognize and interact with D-galactose-containing ligands spe ically. In order to develop potential enzymatically stable ligands for this lectin, we h synthesized thio-and selenogalactoside-containing glycoconjugates based on the l structure ( Figure 1).
Methyl α-D-galactopyranoside 1 was used as standard, as a natural ligand of the tin. For the synthesis of an oligovalent ligand by click-strategy [25], propargyl 2,3,4,6-te O-acetyl-1-thio-β-D-galactopyranoside [26] (2a) and propargyl 2,3,4,6-tetra-O-acetyl-1 leno-β-D-galactopyranoside (2b) were synthesized starting from peracetylated galacto ranoside bromide via thio-or selenouronium salt and propargylation ( Figure 2). T deacetylated propargyl S-galactoside 3a [27] and propargyl Se-galactoside 3b were a suitable for investigations of their potential binding properties to the lectin PA-IL. Di lactosyl diselenide 4a and digalactosyl disulfide 4b [19] are known from the literature a were also suitable and available for binding studies as divalent galactoside ligands. T Our current aim is to synthesize the thio-and selenoglycoside analogues of the tetravalent lead-structure as well as their intermediate compounds to investigate their binding properties towards lectin PA-IL and the effect of sulfur and selenium on lectin binding. Several selenium-containing carbohydrates are known from the literature. They were synthesized for various purposes [13], exploiting the inherent potential of the selenium nucleus [14]. Selenoglycosides were used as glycosyl donors [15], for protein glycoconjugation in site-selective glycosylation by Se-S-mediated ligation [16], as enzyme inhibitors (O-GlcNAcase [17] and as novel glycosidase inhibitors [18]). Mono-and divalent selenogalactosides and diselenide digalactosides proved to be potential ligands to biomedically relevant galactophilic lectins [19]; non-glycosidically linked Se-containing pseudodisaccharides were also synthesized as BanLec and ConA lectin ligands [20]. Selenium-linked neoglycoconjugates, pseudodisaccharides [21], selenenylsulfide-linked glycopeptides and glycoproteins [22] were also prepared. Moreover, the presence of a selenium nucleus provides an excellent opportunity for structural analysis of biomolecules by NMR and X-ray spectroscopy [14] and to study the lectin-carbohydrate interactions using sophisticated 77 Se-NMR methods [23]. Selenium could be also potentially used as a trace for the selective detection of compounds in the biofluids [24]. A further advantage of the Se-interglycosidic linkage is its higher stability towards hydrolases [17,18].

Synthesis
PA-IL is known to recognize and interact with D-galactose-containing ligands specifically. In order to develop potential enzymatically stable ligands for this lectin, we have synthesized thio-and selenogalactoside-containing glycoconjugates based on the lead structure ( Figure 1).

Inhibition of PA-IL with Thio-and Selenogalactosides
The inhibitory potential of tetravalent thio-and selenogalactosides as well as their intermediate compounds was investigated by hemagglutination inhibition assay with microscope detection [28]. The minimal inhibitory concentrations (MIC) of compounds were determined and their inhibitory potencies were calculated and semi-quantitatively evaluated by the comparison with simple monosaccharide D-galactose (standard). All tested compounds were able to inhibit hemagglutination caused by the lectin PA-IL (see Table 1 and Figure S3). The monovalent intermediates 3a and 3b (propargyl 1-thio-and 1-selenoß-D-galactopyranoside) showed eight times better inhibitory potency than D-galactose, possibly due to the additional interactions via their sidechains. Compounds 4a and 4b (digalactosyl disulfide and digalactosyl diselenide) displayed inhibitory potency 16 and 8, respectively. Although these compounds are theoretically divalent, they have no spacer and are not supposed to bind to the two binding sites simultaneously; therefore, the potencies comparable with monovalent compounds were expected. Both tetravalent compounds 7a and 7b were 256 times better inhibitors than D-galactose; taking into account the effect of several galactose units in the single compound (parameter β), they were 64 times better than D-galactose. These results are the same as the potency obtained for the lead structure I. The substitution of oxygen in the glycosidic linkage did not affect the inhibitory effect on lectin PA-IL.

Inhibition of PA-IL with Thio-and Selenogalactosides
The inhibitory potential of tetravalent thio-and selenogalactosides as well as their intermediate compounds was investigated by hemagglutination inhibition assay with microscope detection [28]. The minimal inhibitory concentrations (MIC) of compounds were determined and their inhibitory potencies were calculated and semi-quantitatively evaluated by the comparison with simple monosaccharide D-galactose (standard). All tested compounds were able to inhibit hemagglutination caused by the lectin PA-IL (see Table 1 and Figure S3). The monovalent intermediates 3a and 3b (propargyl 1-thio-and 1-selenoß-D-galactopyranoside) showed eight times better inhibitory potency than D-galactose, possibly due to the additional interactions via their sidechains. Compounds 4a and 4b (digalactosyl disulfide and digalactosyl diselenide) displayed inhibitory potency 16 and 8, respectively. Although these compounds are theoretically divalent, they have no spacer and are not supposed to bind to the two binding sites simultaneously; therefore, the potencies comparable with monovalent compounds were expected. Both tetravalent compounds 7a and 7b were 256 times better inhibitors than D-galactose; taking into account the effect of several galactose units in the single compound (parameter β), they were 64 times better than D-galactose. These results are the same as the potency obtained for the lead structure I. The substitution of oxygen in the glycosidic linkage did not affect the inhibitory effect on lectin PA-IL.

H STD and Competition NMR Experiments
Ligand-based STD NMR experiments [29][30][31] were performed to support and further characterize the binding of β-D-galactoside-containing ligands to PA-IL. This technique is able to disclose the structural regions of the ligands that are involved in the binding. Moreover, further information on the interaction-such as binding site and relative affinitycan be obtained in competition experiments when suitable reference (natural) ligand is available.
In the present study, Me α-D-Gal (1) served as a reference ligand and its binding to PA-IL was unambiguously confirmed with the STD NMR spectra shown in Figure 3B. All galactosyl ring protons and also the methyl protons of OCH 3 group show similar STD effects (for resonance assignment see the 1 H NMR spectrum in Figure 3A), suggesting that Me α-D-Gal being a small ligand may fit completely in the binding pocket of PA-IL.
In the succeeding competition experiments STD NMR spectra were recorded on samples containing both the natural (1) and one of the mono-or tetravalent ligands in a one-to-one molar ratio. The STD signals belonging to the well-resolved resonances (i.e., the ones separated from the resonances of 1 of tested compounds-marked by blue arrows in the STD spectra of Figure 3D,F, and also in Figure S2) confirm that all investigated mono-and tetravalent ligands bind to PA-IL. Moreover, the STD effects observed on the CH 2 protons-marked by dotted blue arrows in Figure 3D,F-suggest that binding of compounds 7a and 7b to PA-IL involves certain hydrophobic contacts of the spacers in the formation of the complex.
Moreover, the STD signal attenuation of the reference ligand 1-monitored on wellseparated (non-overlapping) resonances of 1 and marked by filled red circles in Figure 3B,D,F-confirms that the particular ligands compete for the same (or partially overlapped) binding site of PA-IL. Considering the 1:1 ratio of the competing ligands in the sample, the substantial drop observed in the STD signal intensities suggest that the tetravalent ligands 7a and 7b show significantly higher affinity towards PA-IL than the natural ligand 1 used in the competition assay.
The STD NMR spectra of the monovalent ligands (3a and 3b) show similar signal patterns (see Figure S2), confirming their binding to PA-IL. The attenuation of STD signals of 1 observed in the competition experiments, however, was significantly weaker, indicating lower affinity of the monovalent ligands towards PA-IL. It should be noted that the relative affinity order of the ligands assessed (qualitatively) in the competition NMR experiments is in accordance with the inhibition data given in Table 1.
In summary, tetravalent Sand Se-galactoclusters synthesized by click-chemistry were found to be suitable ligands of the lectin PA-IL in vitro, with significant, about 64 times better inhibitory activity than simple D-galactose. We can also conclude that enzymatically stable S- [32] and Se-interglycosidic linkages [17,18] do not influence the potency of ligands compared with the appropriate O-glycosides [12]. We could prove using STD-NMR techniques that the multivalent ligands compete with the natural ligand for the binding sites of the protein. In the future, multivalent selenoglycosides will provide a great opportunity to investigate the lectin-carbohydrate interactions in biologically relevant environments by highly sensitive and selective advanced 77 Se-NMR methods. As D-galactose and L-fucose were applied for treatment of CF in an open clinical trial [33], novel Sand Se-galactoconjugates can be possible candidates in anti-adhesion therapy of cystic fibrosis as inhalational drugs.

General Methods
Optical rotations were measured at room temperature with a Perkin-Elmer 241 automatic polarimeter. TLC analysis was performed on Kieselgel 60 F 254 (Merck) silica gel plates with visualization by immersing in a sulfuric-acid solution (5% in EtOH) followed by heating. Column chromatography was performed on silica gel 60 (Merck 0.063-0.200 mm) and flash column chromatography was performed on silica gel 60 (Merck 0.040-0.063 mm). Organic solutions were dried over MgSO 4 and concentrated under vacuum. The 1 H (500 MHz) and 13 C NMR (125.76 MHz) spectra were recorded with Bruker Avance II 500 spectrometer (Bruker, Billerica, MA, USA). Chemical shifts are referenced to Me 4 Si or DSS (0.00 ppm for 1 H) and to solvent signals (CDCl 3 : 77.00 ppm, CD 3 OD: 49.15 ppm for 13 C). ESI-QTOF MS measurements were carried out on a maXis II UHR ESI-QTOF MS instrument (Bruker, Billerica, MA, USA), in positive ionization mode. The following parameters were applied for the electrospray ion source: capillary voltage: 3.6 kV; end plate offset: 500 V; nebulizer pressure: 0.5 bar; dry gas temperature: 200 • C and dry gas flow rate: 4.0 L/min. Constant background correction was applied for each spectrum, the background was recorded before each sample by injecting the blank sample matrix (solvent). Na-formate calibrant was injected after each sample, which enabled internal calibration during data evaluation. Mass spectra were recorded by otofControl

Compound 3b
A catalytic amount of NaOMe (pH~9) was added to a stirred solution of ester 2b (150 mg, 0.33 mmol) in dry MeOH (5 mL) and stirred overnight at room temperature. The reaction mixture was neutralized with Amberlite IR-120 H + ion-exchange resin, filtered and evaporated; then, the crude product was purified by flash column chromatography

Compound 7a
A catalytic amount of NaOMe (pH~9) was added to a stirred solution of ester 6a (115 mg, 0.4 mmol) in dry MeOH (5 mL) and stirred overnight at room temperature. The reaction mixture was neutralized with Amberlite IR-120 H + ion-exchange resin, filtered and evaporated; then, the crude product was purified by flash column chromatography

Compound 7b
A catalytic amount of NaOMe (pH~9) was added to a stirred solution of ester 6b (100 mg, 0.26 mmol) in dry MeOH (5 mL) and stirred overnight at room temperature. The reaction mixture was neutralized with Amberlite IR-120 H + ion-exchange resin, filtered and evaporated; then, the crude product was purified by flash column chromatography

Hemagglutination Inhibition Assay (HIA)
PA-IL was produced and purified as previously described [7]. The lectin was dissolved in the suitable buffer (20 mM Tris/HCl, 150 mM NaCl, 5 mM CaCl 2 , pH 7.5) to a concentration of 0.25 mg·mL −1 . The lectin was mixed with synthetized galactosides and serially diluted in the buffer in a 5 µL:5 µL ratio. The final (working) concentration of the lectin was therefore 0.125 mg·mL −1 . Then, a total volume of 10 µL of 20% papain-treated, azid-stabilized red blood cells B − in the buffer was added, after which the mixture was thoroughly mixed and incubated for 5 min at room temperature. After incubation, the mixture was again mixed, transferred to a microscope slide and examined. The examination was conducted using the Levenhuk D2L NG Digital Microscope (Levenhuk, Tampa, FL, USA). Images were obtained with a Levenhuk D2L digital camera (Levenhuk, Tampa, FL, USA) using the software ToupView for Windows (Levenhuk, Tampa, FL, USA). The positive (experiment without an inhibitor) and negative control (experiment without the lectin) were prepared and processed in the same way using the appropriate volume of dissolving buffer instead of the omitted components. The minimal inhibitory concentration (MIC) of the inhibitor able to inhibit hemagglutination was determined and compared with the standard (D-galactose), and the potency of the inhibitor was calculated (MIC of the standard/MIC of the inhibitor).

1 H STD NMR Experiment
All NMR measurements were performed on a Bruker Avance II 500 spectrometer (Bruker, Billerica, MA, USA) operating at 500.13 MHz for 1 H and equipped with 5-mm triple-resonance (txi) probe-head with z-axis gradients. 1 H STD NMR spectra were recorded on samples dissolved in D 2 O (1M Tris-d 11 , 0.5 mM CaCl 2 , pH 7.5, T = 303 K) with the molar ratio of the ligand to PA-IL of about 100:1. The concentration of PA-IL tetramer was kept as low as ca. 10 µM to avoid aggregation upon addition of the ligand(s). For selective saturation of protein resonances, a train of bandselective E-BURP-1 (90 • ) shaped pulses of 50 ms each with a maximum B1 field strength of 75 Hz was employed yielding a total irradiation time of 3 s. For irradiation at aliphatic (CH 3 ) region of PA-IL the E-BURP-1 pulses were applied at −0.3 ppm, while for recording the reference (off-resonance) spectrum, the irradiation frequency was set at −27 ppm. Offand on-resonance data were recorded at alternate scans and the corresponding FIDs were collected in separate memories for subsequent processing and for the generation of STD spectra. Competition STD NMR experiments were performed following the experimental protocol as given above on samples containing two ligands (natural ligand and one of the mono-or multivalent ligands) and PA-IL lectin in 100:100:1 molar ratio. STD spectra were typically recorded with 2000-2400 transients to obtain a suitable signal-to-noise ratio for the analysis.
Supplementary Materials: The following are available. Figure S1: NMR spectra of the new compounds. Figure S2: The STD NMR spectra of the monovalent ligand 1, 3a and 3b in the presence of PA-IL. Figure S3: Influence of D-galactose, compounds 3a, 3b, 4a, 4b, 7a and 7b on hemagglutination caused by lectin PA-IL.