Enantioselective carbohydrate recognition by synthetic lectins in water

These chiral “synthetic lectins” are the first to discriminate between carbohydrate enantiomers, and also show unprecedented affinities for monosaccharide substrates.


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Figure S1. Numbering system for receptors ()-2 and precursors ()-7, as used for the assignment and discussion of NMR spectra. Pyrenyl proton designations (red) are prefixed with "p" (i.e. p4, p1α etc.) while protons from the different spacers (green) are prefixed with an "s" and suffixed by "a,b,c,d" to denote which of the four spacers is involved. Protons from the triamine unit (pink) are prefixed "t" and protons from the dendritic side-chains (blue) are prefixed with "d". Special designations are used for some benzylic hydrogens (e.g. t1αb is the proton on ethyl group 1 closer to spacer b).
The analyte was injected (10 µL of 0.1 mM solution) in acetonitrile/water (1:1) and the eluent was running at 1 mL/min. Detection was achieved using a UV-VIS spectrophotometer. One single peak always eluted either at the front or at several minutes, depending on the eluent.
In another attempt, GlcNAc separopore® 4B agarose resin (Bioworld) 5 was packed into a 1 ml borosilicate FPLC column, and the column was fitted on an AKTA Purifier system (GE Biosciences) at ambient temperature.
Initially the receptor mixture was diluted to 0.1 mg/ml in H2O and 1 ml was loaded onto the column and eluted with an isocratic flow rate of 1 ml/min for 60-180 min. The eluent was monitored at 215 and 280 nm.
As all the material eluted in the void volume of the column, the run was repeated (i) with incubation of ()-2 on the column for 30 minutes before elution, and (ii) using a lower flow rate of 0.1 ml/ml. Neither measure yielded any change. The attempted separation was repeated in 0.1% formic acid/H2O, with isocratic elution at 0.1 ml/min, but again all the material appeared in the void volume. GlcNAc separopore® 4B is used to separate GlcNAc-binding lectins such as Wheat Germ Agglutinin, which binds to GlcNAc more weakly than one enantiomer of 2. It thus seems that the presentation of the GlcNAc units on this column is incompatible with binding to the synthetic lectin. 5 http://www.bio-world.com/productinfo/2_18_162_669/1315/N-Acetyl-glucosamine-GlcNAc-Separopore-Agarose-B-CL.html S18 Figure

1 H NMR titrations
NMR spectra were recorded at 298 K and 600 MHz on a Varian VNMRS 600 equipped with a cryogenically cooled 1 H-observe triple resonance probe. Chemical shifts (δ) are reported in parts per million (p.p.m.). A solution of receptors ()-2 in D2O (99.9% -D, typically 0.15 mM of each receptor) was prepared and typically 400 µL was transferred into a new or thoroughly cleaned and dried NMR tube. This same receptor solution was then used to prepare a stock solution of carbohydrate, ensuring that the concentration of ()-2 did not change during the subsequent titration. In the case of reducing sugars, the stock solution was left overnight so that α and β forms could equilibrate. Aliquots of increasing volume were added from the stock solution into the NMR tube and, after thorough mixing, the 1 H-NMR spectrum was recorded. The first addition typically was 0.5 µL; subsequent additions were made using double the volume of the previous addition (i.e. 1.0, 2.0, 4.0, 8.0 µL, etc.). Signals due to receptor protons were observed to move, as expected for binding with fast exchange on the 1 H NMR timescale, although broadening was also observed in many cases. Where feasible, changes in chemical shift (Δδ) were fitted to a 1:1 binding model using a non-linear least squares curve-fitting programme implemented within Excel. The programme yields binding constants Ka and limiting Δδ as output. An estimated error for Ka could be obtained from individual data points by assuming the determined Ka and δHG. The fits obtained were good in all cases (with r ≥ 0.999), and the thus obtained Ka and Δδ were considered trustworthy. Spectra and analysis curves are shown in Figure S28 - Figure S34. Figure S25. Partial 1 H NMR spectra from the titration of receptors ()-2 (0.15 mM each, 400 μL) with N-acetyl-D-glucosamine 8 (0.90 mM then 501 mM). Peaks marked with empty and red circles were analysed to give binding constants for the diastereomeric complexes (see Figure S26).  Figure S27. Partial 1 H NMR spectra from the titration of receptors ()-2 (0.15 mM each, 400 μL) with D-glucose 9 (1.67 mM then 565 mM). Peaks marked with red circles were analysed to give the binding constant of ~250 M -1 for one of the diastereomeric complexes (see Figure S28). Peaks marked with empty circles are assigned to the second diastereomer. Their early movement during the titration suggests a high binding constant, perhaps larger than that calculated for the first diastereomer.  Figure S29. Partial 1 H NMR spectra from the titration of receptors ()-2 (0.15 mM each, 400 μL) with methyl-β-D-glucoside 10 (0.91 mM then 250 mM). Peaks marked with empty and red circles were analysed to give the binding constant for one of the diastereomeric complexes (see Figure S30).  Figure   S29). Top: Peak marked with red circles in Figure S29, Ka = 250.87 ± 8.04 M -1 (3.21 %); r = 0.99955 ; limiting Δδ = 0.535 p.p.m.. Bottom:

Summary
To assist discussion, it is useful first to label the enantiomers of 2 according to standard practice.
Following Eliel et al., 6 the bicyclic structure of 2 is considered planar chiral, in which chiral plane is that containing the pyrene unit and pendant spacer. To assign a descriptor one first locates the "pilot atom". This is the out-of-plane atom which is closest to the plane and, compared to others at the same distance, is closest to the atom which ranks highest according to CIP rules. In this case the pilot atom is the N attached to p3α, which earns highest priority through its proximity to the CO2 group on the pendant spacer ( Figure S35). The sequence of in-plane atoms attached to the pilot atom is labelled a,b,c, choosing atoms of highest CIP precedence where necessary. In this case c is carbon p3a, rather than p2. When viewed from the pilot atom, the order of a,b,c (clockwise or anticlockwise) determines whether the descriptor should be pR or pS (the prefix p denoting planar chirality). The enantiomer shown in Figure S35 is therefore pS.

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As we could not find an unambiguous NMR assignment of N-acetyl-D-glucosamine 8 in the literature, it was decided to perform this characterisation as part of the present work. Spectra of 8 in D2O at 25 C, and a full assignment of both anomeric forms are given in Figure S39 to To determine which enantiomer bound more strongly to N-acetyl-D-glucosamine 8 (see Figure S25 for the titration experiment) we analysed a mixture of (±)-2 + 8 by means of { 1 H-1 H}-NOESY and -TOCSY NMR spectroscopy at 600 MHz. The concentrations were selected to maximise the number of resonances that could be resolved (particularly the inwards facing s6's), while at the same time having a reasonable concentration of the complex with the stronger binding receptor. We used a 0.42 mM total receptor concentration (so 0.21 mM of each enantiomer) and an N-acetyl-Dglucosamine 8 concentration of 10.6 mM. This was chosen by gradually increasing the concentration of 8; see stacked spectra in Figure S36. Under these conditions the receptor with binding constant Ka = 1280 M -1 will be approximately 93% bound, while the weaker binder (Ka = 80 M -1 ) will be about 46% bound to 8. With the stronger bound complex about twice as populous as the weaker bound complex, it is to be expected that the stronger bound complex could be (partially) assigned with NMR spectroscopy and attributed to receptor pR-2 or pS-2. The resulting spectra are shown in Figure S45 to The entry point into the spin system of the complexes are the s6c protons (7.93 ppm). It is clear that during the titration this resonance is shifted downfield for both enantiomers, but that the limiting chemical shift for one is much larger than for the other; already early on in the 'titration' the visible s6c integrates as one proton, meaning that the other s6c has shifted (to 8.19 ppm, as appeared during the assignment). This is also the case for protons s6b, although the resonance of the major species could not be identified. s6bminor is overlapped with s6dminor around 7.84 ppm. s6d (major) can clearly identified at 7.76 ppm and s6a is essentially unperturbed and appears as a single peak at 8.07 ppm.
It was possible to almost completely assign the resonances for the enantiomer representing the major species present. There were sufficient resolved nuclear Overhauser effect cross peaks (NOEs) between this enantiomer and the carbohydrate. The resonances of the minor species were obviously less abundant and were broadened (e.g. s6bminor, s6cminor and S6dminor resonances) yielding 10.2 S44 only very weak NOEs. Only clearly distinguished NOEs of the major species were used in the analysis to safeguard against possible confounding correlations arising from the minor species, however weak these might be. A full labelling scheme for both enantiomers of 2, as well as the numbering for D-GlcNAc (8), are shown in Figure S37. From the carbohydrate region (3.2 -3.9 ppm, C), it is evident that the α and the β isomers were present, but that the resonances of the β were broadened. These resonances were sharp in a spectrum of pure D-GlcNAc (see Figure S39). None of the α protons display an NOE with any of the receptor resonances whereas there are very clear NOEs with the other hydrogen atoms of D-GlcNAc; β-2, β-5, β-6, β-6', and β-Me. Potential NOEs with the anomeric proton β-1 (should come around 4.62 ppm) were obscured by the overlapping water resonance and indeed no such NOEs could be observed. Protons β-3 (~3.48 ppm) and β-4 (~3.38 ppm) were be identified (although overlapping with α-4). There were no NOEs discernible with β-3 and only two very weak NOEs with β-4 (NB: one of these is crucial to differentiate between pR-2 and pS-2). This means they must be pointing towards the aromatics, furthest away from other H-atoms.
The carbohydrate can be approximately positioned inside the cage by considering the NOEs with the acetyl methyl resonance β-Me (1.976 ppm) and the carbohydrate's methylene resonances β-6 (3.833 ppm) and β-6' (3.679 ppm). NOEs to the β-Me indicate that this unit is emerging from the largest portal, between t1α/β and p2, and in close proximity to s6d, p1α and p10. The S45 carbohydrate's CH2OH is positioned in the smallest portal, between t5α/β and p7, and is also close to s6c (with β-6 pointing towards s6c and β-6'pointing towards t5α/β).
It is therefore concluded that D-GlcNAc 8 in bound as the β-anomer preferentially by the pR enantiomer of 2, in the orientation shown in Figure S38a. A 3D model consistent with the NOE data is shown in Figure S38b. S46 Figure S38. a) The orientation of β-D-GlcNAc 8 in the stronger-binding pR enantiomer of 2, as deduced by 1 H NMR NOE spectroscopy. b) 3D model of the complex after minimisation with Spartan molecular modelling software (Spartan '16 v 2.0.3, molecular mechanics minimisation using the MMFF force field with explicit aqueous solvation).

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NMR spectra and assignments of N-acetyl-D-glucosamine 8 Figure S39. 1 H NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers. Figure S40. 13 C NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers. Figure S41. { 1 H-13 C}-HSQC NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers. Figure S42. { 1 H-13 C}-HMBC NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers. See Figure S43 for a zoom-out. Figure S43. { 1 H-13 C}-HMBC NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers. See Figure S42 for a zoom-in. Figure S44. { 1 H-1 H}-NOESY NMR spectrum of 176 mM N-acetyl-D-glucosamine 8 in D2O including assignments of the alpha (in blue) and beta (in red) anomers.