Synthesis and solvodynamic diameter measurements of closely related mannodendrimers for the study of multivalent carbohydrate–protein interactions

Summary This paper describes the synthesis of three closely related families of mannopyranoside-containing dendrimers for the purpose of studying subtle structural parameters involved in the measurements of multivalent carbohydrate–protein binding interactions. Toward this goal, two trimers 5 and 9, three 9-mers 12, 17, 21, and one 27-mer 23, varying by the number of atoms separating the anomeric and the core carbons, were synthesized using azide–alkyne cycloaddition (CuAAc). Compound 23 was prepared by an efficient convergent strategy. The sugar precursors consisted of either a 2-azidoethyl (3) or a prop-2-ynyl α-D-mannopyranoside (7) derivative. The solvodynamic diameters of 9-mer 12, 17, and 21 were determined by pulsed-field-gradient-stimulated echo (PFG-STE) NMR experiments and were found to be 3.0, 2.5, and 3.4 nm, respectively.


Synthesis and solvodynamic diameter measurements of closely related mannodendrimers for the study of multivalent carbohydrate-protein interactions
Introduction Multivalent carbohydrate-protein interactions are at the forefront of a wide range of biological events which have triggered a plethora of versatile synthetic methods for the design of potent inhibitors and glycomimetics [1][2][3][4]. Among the diverse strategies leading to efficient ligands, glycopolymers [1, [5][6][7], glycodendrimers [7][8][9][10][11][12][13][14], and sugar rods [15,16] have retained most attention. An additional approach that has gained keen interest resides in the modifications of both the aglycon [17][18][19] and substituent residues [20][21][22] of the targeted sugar moieties through extensive studies of quantitative structure-activity relationships (QSARs). In most of the studies related to aglycon modifications, it was concluded that aromatic glycosides possessed improved binding properties due to the ubiquitous presence of aromatic amino acids in the cognate binding sites Scheme 1: Synthesis of mannosylated trimers 5 and 9 using trimesic acid core transformed into propargylated (2) and azidopropylated (6) scaffolds and then coupled by "click chemistry" with either 2-azidoethyl (3) or propargyl (7) mannopyranosides. [23][24][25]. This is also supported by the recent findings that the sugar backbones themselves also possess a hydrophobic side that orients the sugars in appropriate aromatic amino acid rich pockets [26][27][28].
Unfortunately, due to the inherent complexity of studying multivalent binding interactions, researchers have used experimental conditions that often biased the intrinsic phenomena under investigations [29]. For instance, when evaluating thermodynamic parameters by isothermal calorimetry (ITC), scientists used either truncated versions of for instance, tetrameric lectins such as ConA, or diluted conditions to avoid precipitation of the complexes [30,31]. Alternatively, the application of surface plasmon resonance (SPR) also creates artificial situations not sufficiently related to the natural cellular events, thus requiring complex mathematical algorithms [32]. Most solidphase immunoassays (ELLA, ELISA) also fall under the same criticism by providing unusually high (or too close) sugar densities. Also important and in spite of the two decades of glycodendrimer chemistry [7], there is still no general rule to allow predicting which structural parameters would be optimal for the binding interactions.
In order to gain more insight into this direction, we designed herein three families of closely related mannopyranoside clusters (glycodendrimers) aimed at evaluating their relative binding abilities against the hometetrameric leguminous lectin ConA from Canavalia ensiformis by inhibition of haemagglutination and by turbidimetry. The latter would allow us to measure relative kinetic factors involved in the cross-linking lattice formation using soluble partners.

Results and Discussion
In order to critically evaluate the subtle structural parameters imparted by glycodendrimers in deciphering their relative thermodynamic and kinetic abilities towards multivalent lectins, we designed three families of closely related mannopyranoside dendrimers. Scheme 1 describes the preparation of trimers 5 and 9 built around benzene-1,3,5-tricarboxamide (BTA or trimesamide core) having respectively nine and ten atoms between the anomeric and the benzene carbon, hence differing by a distance of only ~1.5 Å. Schemes 2-4 illustrate the syntheses of 9-mers 12 and 21 using the same trimesic acid core, together with a phloroglucinol template to initiate the synthesis of homologue 17, but incorporating 2-amino-2-hydroxymethylpropane-1,3diol as a branching unit (TRIS) at the G(1) level. Thus, compounds 12, 17, and 21 differ by having nine atoms between the anomeric carbon and the focal quaternary carbon of TRIS followed by two, four, and nine atoms to reach the benzene carbon, respectively (~4, 6, and 12 Å). Finally, the synthesis of a 27-mer mannosylated dendrimer 23 is shown in Scheme 5.
The syntheses of 9-mers 12, 17 and 21 are illustrated in Schemes 2-5 and follow a conceptually identical strategy to the one described above for trimers 5 and 9. Toward this goal, propargylated 9-mer scaffold 10 [17] was treated under the same CuAAc conditions with azide 3 to provide peracetylated 11 in 83% yield which upon Zemplén de-O-acetylation gave 12 in essentially quantitative yield (Scheme 2). Complete spectral characterization ( 1 H, 13 C NMR and HRMS) concluded for the Scheme 4: Convergent synthesis of further "extended" 9-mer 21 using mannosylated bromoacyl dendron 18 transformed into azide 19 followed by CuAAc coupling to tripropargylated core 2.
aforementioned structure having twelve atoms in the linking arm (see Supporting Information File 1).
Analogously, the extended 9-mer glycodendrimer 17, possessing fourteen atoms between the anomeric carbon and the benzene carbon, was prepared according to Scheme 3. Thus, phloroglucinol (13) was carefully O-alkylated with the previously synthesized bromoacetylated TRIS derivative 14 [36] using K 2 CO 3 in DMF to provide 15 in 43% yield. Again, the structural integrity of 15 was fully assessed by the simplicity of its 1 H NMR symmetrical patterns that showed the characteristic singlets for the three amide protons at δ 6.85 ppm, relative to the three benzene protons (δ 6.17 ppm) and the six O-acyl protons at δ 4.36 ppm (core) compared with the peripheral acetylenic methylenes (18H), inner methylene of TRIS (18H), and the terminal alkyne protons (9H) at δ 4.16, 3.87, and 2.48 ppm, respectively.
Toward the last and further extended 9-mer 21, a convergent strategy was rather adopted (Scheme 4). This strategy has the clear advantages of providing an easier purification process from partially substituted end-products together with a better assessment of complete surface group modifications. Hence, known 14 [36] was first cycloadded to mannosylazide 3 under the above CuAAc conditions. The "click reaction" proceeded exceptionally efficiently and provided bromoacylated dendron precursor 18 in 94% yield. Substitution of the bromide by azide also proceeded uneventfully (NaN 3  Finally, a 27-mer mannosylated G(1)-dendrimer 23 was similarly prepared using an accelerated convergent strategy (Scheme 5). This time, the nonapropargylated scaffold 10 was "clicked" under CuAAc with trimeric azidodendron 19 to give 22 in an acceptable yield of 63% after silica gel column chro- matography, corresponding to an excellent 95% yield per individual dendron's incorporation. The complete disappearance of propargylic signals in the 1 H NMR spectrum supported complete conversion. Note that working with peracetylated sugar precursors allows less tedious purification practices as opposed to working with unprotected sugars which often necessitate purification by cumbersome dialysis followed by HPLC treatment. Here again, the complete structural integrity of the final product can be readily confirmed from its characteristic spectral identification. Ultimately, dendrimer 23 was deprotected under the usual Zemplén conditions in 82% yield. Once again, all the relative integrations for each proton presented on the surface were in perfect agreement with those of the middle and internal regions. Interestingly, high resolution mass spectrometry ( + TOF technique) resulted in the formation of multicharged adducts that matched the expected theoretical patterns, especially the one corresponding to [M + 7H] 7+ , as illustrated in Scheme 5 (insert).

NMR diffusion studies
To accurately estimate the various structural factors involved in the intricate binding interactions between our synthetic multi-meric mannosides and ConA, we determined their relative diffusivity measurements by NMR spectroscopy. In fact, diffusion NMR spectroscopy has recently become a method of choice to access information about sizes and shapes of macromolecular species by measuring their diffusion coefficients in a given solvent [17,37]. The size of nonavalent compounds 12, 17, and 21, and more particularly their solvodynamic radii, was thus estimated with the help of pulsed-field-gradient stimulated echo (PFG-STE) NMR experiments using bipolar pulse pairslongitudinal-eddy-current delay (BPP-LED) in D 2 O at 25 °C. Stimulated echoes were used since they avoid signal attenuation due to transverse relaxation while bipolar gradient pulses reduce gradient artefacts [38]. The diffusion rates (D) were calculated from the decay of the signal intensity of the common H-5 proton (δ = 2.98 ppm) located on each epitope with increasing field gradient strength (Figure 1a). In all cases, monoexponential behavior was observed (Figure 1b), which was manifested as a linear decay of the logarithm of the signal intensity as a function of the gradient strength. This behavior is consistent with a spherical and unimolecular character of the glycodendrimers, confirming the absence of aggregation phenomena in aqueous solution under the working concentra-   (Table 1).
As expected, nonavalent conjugates 12, 17, and 21 presented solvodynamic diameters in the range of roughly 3 nm when considering the decay of distinctive and common H-5 signals. These values remained consistent with similar congeners described earlier and harboring different epitopes [17]. The variation of the complexity of anchoring functionalities in the middle region with the incorporation of amide functions and triazole groups is responsible for a diameter enhancement for 21 when compared with 12, as expected. On the other hand, rather counter-intuitive tendencies were observed since the apparently slightly extended structure 17 was measured as the smallest molecule of the family in water. A specific spatial arrangement of the dendrons that emanate from 1,3,5-O-alkylations on the aromatic core in 17, compared to the one generated in BTAscentered structures 12 and 21, could explain this observation. Also, these discrepancies might result from the general amphiphilic behavior of this kind of macromolecules [39]. In fact, these glycoclusters shared common structural factors with hydrophilic peripheral moieties and an aromatic central core but the introduction of distinct functionalized linkers may change the overall hydrophobic/hydrophilic balances of the structures. As such, they could engage supplementary intramolecular hydrogen bonding or hydrophobic interactions that could mediate their three-dimensional arrangement in aqueous media. Moreover, it is also reported that the relative spatial distribution of the branches around the C=O-centered BTAs strongly depends on the nature of the substituents [40]. This hypothesis can partly explain the discrepancy observed for the calculated diameter of 21 (Table 1,

Conclusion
The syntheses of three related families of mannosylated glycoclusters and glycodendrimers were efficiently accomplished around a benzene core and using the CuAAc methods now routinely used in this field [9,41,42]. The targeted compounds were based on trimesic acid scaffold which is known to properly expose the surface sugar groups to tetrameric lectins such as ConA [43] and the LecA lectin from Pseudomonas aeruginosa [17]. With these closely related families of mannosylated dendrimers in hand, together with their known relative size in solution, we are now well positioned to evaluate their binding behavior against their cognate proteins and this work will be published in due course [44].
The study of subtle structural variations and the nature of anchoring functions, as observed in diffusivity experiments, could represent a first step towards rational interpretation to explain the differential kinetic behavior within a closely related family of glycoclusters.

Experimental General remarks
All reactions in organic medium were performed in standard oven-dried glassware under an inert atmosphere of nitrogen using freshly distilled solvents. CH 2 Cl 2 was distilled from CaH 2 and DMF from ninhydrin, and kept over molecular sieves.  [38]. The diffusion experiment employed a bipolar pulse-field gradients stimulated echo sequence as proposed by Wu et al [45]. The gradient pulse duration δ was 4 ms and the diffusion times (Δ) were 40 to 50 ms to ensure that the echo intensities were attenuated by at least 80%. A complete attenuation curve was obtained by measuring 30 gradient strengths, which were linearly incremented between 1. 8  , δ = gradient duration, Δ = diffusion delay, and τ = pulse length for bipolar pulses. All diffusion spectra were processed in Mat NMR [47].

Glycodendrimer synthesis
Procedure A: multiple CuAAc couplings on polypropargylated cores To a solution of polypropargylated core (1.00 equiv) and complementary azido synthon (1.25 equiv/propargyl) in a THF/ H 2 O mixture (1:1) were added sodium ascorbate (0.30 equiv/ propargyl) and CuSO 4 ·5H 2 O (0.30 equiv/propargyl). The reaction mixture was stirred at 50 °C for 3 h then at room temperature for an additional 16 h period. Ethyl acetate (10 mL) was added and the resulting solution was poured in a separatory funnel containing 25 mL of EtOAc and 30 mL of a saturated aqueous solution of NH 4 Cl. Organics were washed with (2 × 25 mL) of saturated NH 4 Cl aq , water (2 × 20 mL) and brine (1 × 10 mL). The organic phase was then dried over MgSO 4 and concentrated under reduced pressure. Column chromatography on silica (DCM/MeOH 100:0 to 90:10) afforded the desired glycocluster.

Procedure B: Zemplén de-O-acetylation procedure for insoluble hydroxylated derivatives
The acetylated compound was dissolved in anhydrous MeOH and a solution of sodium methoxide (1 M in MeOH, 5 µL every 20 min until precipitation) was added. An additional 100 µL was then injected and the heterogeneous reaction mixture was stirred at room temperature for 24 h. The solvent was then removed with a Pasteur pipette and a mixture of anhydrous MeOH/DCM (4:1, 5 mL) was added to the residual white foam. A vigorous agitation is maintained for an additional 15 min period. After removal of the solvents with a Pasteur pipette, the residue was dissolved in H 2 O (3 mL), and the pH was adjusted to 7 by the addition of ion-exchange resin (Amberlite IR 120 H + ). After filtration, the solvent was removed under vacuum with a rotary evaporator and lyophilized to yield the fully deprotected glycocluster.