77Se-Enriched Selenoglycoside Enables Significant Enhancement in NMR Spectroscopic Monitoring of Glycan–Protein Interactions

Detailed investigation of ligand–protein interactions is essential for better understanding of biological processes at the molecular level. Among these binding interactions, the recognition of glycans by lectins is of particular importance in several diseases, such as cancer; therefore, inhibition of glycan-lectin/galectin interactions represents a promising perspective towards developing therapeutics controlling cancer development. The recent introduction of 77Se NMR spectroscopy for monitoring the binding of a selenoglycoside to galectins prompted interest to optimize the sensitivity by increasing the 77Se content from the natural 7.63% abundance to 99%. Here, we report a convenient synthesis of 77Se-enriched selenodigalactoside (SeDG), which is a potent ligand of the medically relevant human galectin-3 protein, and proof of the expected sensitivity gain in 2D 1H, 77Se correlation NMR experiments. Our work opens perspectives for adding isotopically enriched selenoglycans for rapid monitoring of lectin-binding of selenated as well as non-selenated ligands and for ligand screening in competition experiments.


Introduction
Mounting awareness in recent decades has highlighted the importance of molecular events in how lectins, a class of carbohydrate-binding proteins, recognize cell surface glycans [1]. Lectin-glycan interactions are involved in cellular processes such as adhesion, intercellular communication, growth and differentiation, cell cycle, and apoptosis. As a result, lectin-mediated processes are implicated in wide range of diseases such as cancer, inflammation and fibrosis, heart disease, and stroke [2][3][4][5][6][7]. Galectins, Gal-1, -3, -7, and -9 in particular, have been implicated in multiple ways in malignant cell proliferation processes [4,5,7,8]. Inhibition of glycan-galectin interactions thus offers a promising perspective towards developing therapeutics influencing these processes. Research on small-molecule-or multivalent glycomimetics as lectin inhibitors is especially worth mentioning in this aspect [9][10][11].
Several analytical techniques (surface plasmon resonance-SPR, isothermal titration calorimetry-ITC, nuclear magnetic resonance-NMR, or X-ray crystallography) are available to investigate these interactions. Among these methods, NMR spectroscopy stands out as it allows studying protein-glycan interactions in solution, i.e., close to physiological conditions at the molecular level [12,13]. Observation of NMR signals of the ligand molecules binding to protein receptors is one of the most powerful approaches to gain insight into structural and dynamic aspects of these interactions [14,15]. NMR spectroscopy is plagued, however, with sensitivity issues, representing a double challenge because of low concentrations and typically weak interactions. Signal overlap is another problem to be dealt with, especially with carbohydrate ligands typically having narrow 1 H chemical shift ranges with consequent serious overlaps in the spectra [16]. Spectral overcrowding quickly becomes unmanageable with oligosaccharide ligands or with carbohydrate mixtures, such as in studies of potential competition with different ligands. 13 C-or 15 N-based NMR approaches are much less affected by spectral overlap, but suffer even more from low sensitivity at natural abundance of these NMR-active isotopes even in sensitivity-enhanced 1 H-detected versions. A continuous methodological challenge in NMR-spectroscopical monitoring is to expand the range of techniques and, here, the introduction of distinct NMR sensors beyond the commonly used 1 H, 13 C, and 15 N isotopes comes into play. Application of 19 F as a reporter nucleus has been proposed as an alternative, combining high sensitivity (0.83% compared with that of 1 H) with superior selectivity owing to a large chemical shift range, as well as specific labeling used to introduce this label just to a few predefined positions of the molecule of interest (typically substituting OH for F) [17][18][19][20]. It is to be noted, however, that fluorinated derivatives are chemically distinct from their natural counterparts; still, very often, they turn out to be good mimics of the parent molecules in terms of structure and binding properties.
The success of the 19 F-approach prompted us to look for a further isotope to be introduced as an NMR reporter into lectin-oligosaccharide interaction analysis. Inspired by the discovery that thiodigalactoside (TDG) is a potent ligand for adhesion/growthregulatory galactose-binding lectins [21][22][23], we have tested selenodigalactoside (SeDG) as a binding partner and its 77 Se isotope as an NMR-spectroscopical sensor. As we have detected rather equal affinities of TDG and SeDG to human galectins-1 and -3 (Gal-1/-3) [24], we initiated 77 Se NMR-based monitoring of SeDG binding to these lectins [25,26]. 77 Se is a spin-1 2 NMR isotope with broad chemical shift range (ca. 3000 ppm), which makes it promising as a selective probe [25,[27][28][29]. Direct 77 Se NMR detection [30,31] is, however, unfavorable owing to the low natural abundance (7.63%) and decreased sensitivity (0.7 of 1 H) of this nucleus. To improve sensitivity, we proposed a 2D 1 H-77 Se HSQMBC (heteronuclear single quantum multiple-bond correlation) experiment via indirect 77 Se detection using CPMG-INEPT (Carr-Purcell-Meiboom-Gill insensitive nuclei enhanced by polarization transfer) out-and-back 1 H→ 77 Se→ 1 H polarization transfer. Theoretically, this approach can yield up to 60-fold ((γ H /γ Se ) 2.5 ) sensitivity enhancement, but, owing to competing relaxation and other transfer processes, a sensitivity gain about 20 can be realized in practice [25,28,32]. A next step toward optimizing 77 Se sensor capacity is to prepare the lectin ligand as an isotopically enriched probe, that is, bringing isotope representation from the natural 7.63% abundance to close to 100%. To exploit this attractive potential, we provide an example by the synthesis of a sample of di(β-D-galactopyranosyl)selenide enriched to 99% in 77 Se isotope ([ 77 Se]DGal). Exploratory experiments reported here with this 77 Seenriched material indeed confirmed our expectations for a spectacular increase in NMR detection sensitivity. We will furthermore demonstrate that combining our advanced 1 H-77 Se HSQMBC NMR method with 77 Se enrichment enables rapid monitoring of lectinbinding of selenated as well as non-selenated ligands and high-throughput screening in competition experiments. An ethanolic solution of NaH 77 Se was prepared following a procedure described by Klayman and Griffin [33]. In a flame-dried flask, isotopically enriched (99%) 77-selenium powder (Laboratory Standards Kft. Budapest, Hungary, lot no. SELM-2445-PK) (50 mg, 0.65 mmol) was stirred in dry ethanol (5 mL) under argon atmosphere (Note). After cooling in an ice bath, sodium borohydride (50 mg, 1.3 mmol) was added and stirring continued until the selenium was dissolved and gas evolution subsided (ca. 5 min). To this clear solution, 1-Br-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose (1, 520 mg, 1.3 mmol) dissolved in dry methyl cyanide (2 mL) was added and stirring continued at room temperature until TLC (hexane/EtOAc 1:1) indicated consumption of 1 (2 h). The reaction mixture was evaporated under reduced pressure, the syrupy residue dissolved in dichloromethane, it extracted with water twice, the organic phase dried (MgSO 4 ), and the solvent was removed in vacuo to yield 2 as a pale-yellow solid at 323 mg (69%). According to its 1 H NMR spectrum, this product was judged to be sufficiently pure, apart from some residual signals from ethanol (1.23/3.70 ppm, see Figure S1, bottom), to be deacetylated in the next step

77 Se-Enriched Di(β-D-galactopyranosyl)selenide (3)
To a solution of 77 Se-enriched di(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)selenide (2) (180 mg) in dry methanol (50 mL), a 25% solution of sodium methoxide in methanol (0.06 mL) was added. After 15 min at room temperature, 0.5 g of Amberlist ® 15H ion exchange resin was added, the resin filtered off after 5 min of stirring, and the filtrate evaporated to dryness under reduced pressure. Recrystallization from methanol furnished 94 mg of white solid (96%). 1 H NMR ( Figure S2) and 13 Figure  S4. It is noteworthy that the protein-ligand concentration ratio in these NMR experiments should always be optimized in accordance with the pertinent binding affinity of the system being studied.  [25]. Simultaneous composite π pulses on the 1 H and 77 Se channels were applied with an equal duration of 90 • pulses (18 µs) achieved with careful adjustment of power levels. The CPMG-INEPT delay ∆ for longrange heteronuclear coupling evolution was adjusted to 45.1 ms. For 77 Se CPD decoupling during FID acquisition (183 ms), the WALTZ16 scheme with a 90 • pulse length of 400 µs was used. 2D 1 H-77 Se CPMG-HSQMBC spectra were recorded for sensitivity comparison on [ 77 Se]DGal and SeDGal samples, with 1024 total data points in the 1 H (t 2 ) dimension and 32 total points in the 77 Se (t 1 ) dimension, using spectral windows of 6.99 ppm (2796 Hz) for 1 H and 6.00 ppm (458 Hz) for 77 Se. On [ 77 Se]DGal sample, 8 scans per t 1 increment were accumulated; in contrast, 360 scans per t 1 increment were used for SeDGal sample. The polarization recovery delay between consecutive scans, d1, was set to 1.7 s. In binding experiments, each 2D 1 H-77 Se CPMG-HSQMBC spectrum was recorded with 1024 total data points in the 1 H (t 2 ) dimension and 2 total points in the 77 Se (t 1 ) dimension, using spectral windows of 6.99 ppm (2796 Hz) for 1 H and 6.00 ppm (458 Hz) for 77 Se. 32 scans per t 1 increment were accumulated and the polarization recovery delay between consecutive scans, d1, was set to 1.7 s.

NMR Experiments
As mentioned in the introduction, we have recently proposed a 2D 1 H-77 Se heteronuclear correlation experiment to enhance the detection sensitivity of 77 Se NMR by replacing direct NMR observation on 77 Se for indirect observation on 1 H nuclei [25,26]. Our 2D 1 H-77 Se CPMG-HSQMBC pulse sequence is based on out-and-back 1 H→ 77 Se→ 1 H polarization transfer via 2,3 J( 1 H, 77 Se) long-range couplings. CPMG-INEPT long-range transfer eliminates signal phase modulation and significantly reduces intensity losses from co-evolving J( 1 H, 1 H) couplings. Furthermore, it can suppress line broadening from chemical exchange, ensuring maximum detection sensitivity and clean signal phases, as was described earlier [25,26,32].
The sensitivity gain using 99% 77 Se-enriched SeDGal ([ 77 Se]DGal) versus the nonenriched specimen (SeDGal) was checked via comparative 2D 1 H-77 Se CPMG-HSQMBC experiments. Figure 1 shows two sets of data corresponding to [ 77 Se]DGal (A) and SeDGal (C), respectively, in terms of signal to noise (S/N) ratios and times of data acquisition (Acq) for samples of equal concentrations. The overall sensitivity improvement calculated from the experimental data in Figure 1

NMR Experiments
As mentioned in the introduction, we have recently proposed a 2D 1 H-77 Se heteronuclear correlation experiment to enhance the detection sensitivity of 77 Se NMR by replacing direct NMR observation on 77 Se for indirect observation on 1 H nuclei [25,26]. Our 2D 1 H-77 Se CPMG-HSQMBC pulse sequence is based on out-and-back 1 H→ 77 Se→ 1 H polarization transfer via 2,3 J( 1 H, 77 Se) long-range couplings. CPMG-INEPT long-range transfer eliminates signal phase modulation and significantly reduces intensity losses from co-evolving J( 1 H, 1 H) couplings. Furthermore, it can suppress line broadening from chemical exchange, ensuring maximum detection sensitivity and clean signal phases, as was described earlier [25,26,32].
The sensitivity gain using 99% 77 Se-enriched SeDGal ([ 77 Se]DGal) versus the nonenriched specimen (SeDGal) was checked via comparative 2D 1 H-77 Se CPMG-HSQMBC experiments. Figure 1 shows two sets of data corresponding to [ 77 Se]DGal (A) and SeDGal (C), respectively, in terms of signal to noise (S/N) ratios and times of data acquisition (Acq) for samples of equal concentrations. The overall sensitivity improvement calculated from the experimental data in Figure 1  To demonstrate the usefulness of 77 Se-enriched ligand in binding experiments, Figu 2A shows the 1D cross section from a 3-minute 2D 1 H-77 Se CPMG-HSQMBC experime of [ 77 Se]DGal (2 mM). This spectrum conveys the reference (100%) signal intensity. It noteworthy that the S/N ratio is more than adequate in this 3-minute spectrum, leavi room to reduce the concentration of the ligand (and, consequently, that of the protein) these experiments. The addition of human galectin-3 (hGal-3, 29 μM) to this sample sulted in a decrease in signal intensity (from 100% to 76%, Figure 2B) by line broadenin which indicated binding of the ligand to the protein [25]. The line broadening in Figu 2B was then partially reversed (from 76% to 86%, Figure 2C) with the introduction of t odigalactoside (TDG, 2 mM) to the sample. This phenomenon is a consequence of reduc relaxation enhancement of [ 77 Se]DGal resonances via competition between the two l ands (TDG and SeDGal) for the same recognition site on hGal-3, as explained earlier [2 It also illustrates that [ 77 Se]DGal can be used via its competitive displacement to indirec monitor the binding of even non-selenated compounds to hGal-3 (or any other target p teins) by our sensitivity-optimized 1 H-77 Se CPMG-HSQMBC method. To demonstrate the usefulness of 77 Se-enriched ligand in binding experiments, Figure 2A shows the 1D cross section from a 3-minute 2D 1 H-77 Se CPMG-HSQMBC experiment of [ 77 Se]DGal (2 mM). This spectrum conveys the reference (100%) signal intensity. It is noteworthy that the S/N ratio is more than adequate in this 3-minute spectrum, leaving room to reduce the concentration of the ligand (and, consequently, that of the protein) in these experiments. The addition of human galectin-3 (hGal-3, 29 µM) to this sample resulted in a decrease in signal intensity (from 100% to 76%, Figure 2B) by line broadening, which indicated binding of the ligand to the protein [25]. The line broadening in Figure 2B was then partially reversed (from 76% to 86%, Figure 2C two ligands (TDG and SeDGal) for the same recognition site on hGal-3, as explained earlier [25]. It also illustrates that [ 77 Se]DGal can be used via its competitive displacement to indirectly monitor the binding of even non-selenated compounds to hGal-3 (or any other target proteins) by our sensitivity-optimized 1 H-77 Se CPMG-HSQMBC method.
room to reduce the concentration of the ligand (and, consequently, that of the protein these experiments. The addition of human galectin-3 (hGal-3, 29 μM) to this sample sulted in a decrease in signal intensity (from 100% to 76%, Figure 2B) by line broadeni which indicated binding of the ligand to the protein [25]. The line broadening in Fig  2B was then partially reversed (from 76% to 86%, Figure 2C) with the introduction of t odigalactoside (TDG, 2 mM) to the sample. This phenomenon is a consequence of reduc relaxation enhancement of [ 77 Se]DGal resonances via competition between the two ands (TDG and SeDGal) for the same recognition site on hGal-3, as explained earlier [2 It also illustrates that [ 77 Se]DGal can be used via its competitive displacement to indirec monitor the binding of even non-selenated compounds to hGal-3 (or any other target p teins) by our sensitivity-optimized 1 H-77 Se CPMG-HSQMBC method. In sum, we have demonstrated that the enhanced detection sensitivity inherent our original 1 H-77 Se CPMG-HSQMBC sequence receives a further significant boost by ing 77 Se-enriched ligands, such as [ 77 Se]DGal, described in this article. The illustra In sum, we have demonstrated that the enhanced detection sensitivity inherent in our original 1 H-77 Se CPMG-HSQMBC sequence receives a further significant boost by using 77 Se-enriched ligands, such as [ 77 Se]DGal, described in this article. The illustrated straightforward introduction of 77 Se into the glycosidic bond opens wide applicability of 77 Se-enriched selenoglycosides for biomedically relevant tissue lectins and beyond. In terms of NMR experimental time, this enhancement allows to obtain data in much less time (a factor of about 170 (12.98 2 ) between solutions of identical concentrations), and this gain is further documented by the results of a ligand competition experiment. The significant sensitivity gain offered by 77 Se-enrichment, such as in [ 77 Se]DGal, enables, furthermore, a fast and specific detection approach for screening the binding of non-selenated ligands as well to hGal-3. The combined sensitivity advantage of this approach will likely aid NMR to become competitive for binding/screening studies at low concentrations nearing physiological/cellular levels with selenated and non-selenated ligands too. Our present study is a first step toward allowing 77 Se-enriched selenoglycosides to become versatile tools in interaction analysis by NMR spectroscopy, and inspires new approaches to combine it with synthetic carbohydrate chemistry, as illustrated herein. The current work opens perspectives for using isotopically enriched selenoglycans alone or in combination of 77 Se with 2 D, 13 C, 15 N, 19 F, and/or 31 P for analyzing the structure and dynamics of ligand binding to proteins by taking advantage of different NMR time scales offered by distinct NMR frequencies of the individual isotopes. Therefore, our method has the potential to support drug development through the rapid and detailed investigation of glycanlectin interactions.