Compact, Polyvalent Mannose Quantum Dots as Sensitive, Ratiometric FRET Probes for Multivalent Protein–Ligand Interactions

Abstract A highly efficient cap‐exchange approach for preparing compact, dense polyvalent mannose‐capped quantum dots (QDs) has been developed. The resulting QDs have been successfully used to probe multivalent interactions of HIV/Ebola receptors DC‐SIGN and DC‐SIGNR (collectively termed as DC‐SIGN/R) using a sensitive, ratiometric Förster resonance energy transfer (FRET) assay. The QD probes specifically bind DC‐SIGN, but not its closely related receptor DC‐SIGNR, which is further confirmed by its specific blocking of DC‐SIGN engagement with the Ebola virus glycoprotein. Tuning the QD surface mannose valency reveals that DC‐SIGN binds more efficiently to densely packed mannosides. A FRET‐based thermodynamic study reveals that the binding is enthalpy‐driven. This work establishes QD FRET as a rapid, sensitive technique for probing structure and thermodynamics of multivalent protein–ligand interactions.

Lipoic acid (LA, 3.0 g, ~15 mmol), triethylamine (1.47 g, ~15 mmol) and 30 mL CH2Cl2 were mixed in three-necked bottom flask (250 mL) and then cooled to 0 °C in an ice-bath under N2 under stirring for 30 mins. Methanesulfonyl chloride (1.67 g, ~15 mmol) was the added dropwisely through a syringe (1 mL) and the resulting solution was allowed to slowly warm up to room temperature (RT) and stirred for 5 h. After that, N,N-dimethyl-1,3-propanediamine (1.24 g, ~12 mmol) and triethylamine (0.61 g, ~6 mmol) dissolved in 20 mL CH2Cl2 was slowly added. The resulting solution was stirred at overnight under N2 at RT. The reaction mixture was then transferred to a separation funnel and washed with water (30 mL × 2) and then saturated Na2CO3 solution (100 mL). The organic layer was dried over Na2SO4 and filtered. After evaporation of the solvent, the desired compound (1) was obtained as a yellow oil 1.48 g, yield 34.7%. 1  Step ii) Synthesis of LA-zwitterion (LA-ZW) ligand.
Compound 1 (1.48 g, ~5.2 mmol) was dissolved in 20 mL dry THF and purged with N2 for 30 mins. Then 1,3-propanesultone (1.0 g, ~8 mmol) dissolved in 4 mL dry THF was added through a syringe. The resulting mixture was stirred at RT for 3 days. A turbidity was instantly noticed as 1,3-propanesultone was added, indicating the formation of LA-zwitterion (2) due to its low solubility in THF. After stirring for 3 days, the solvent was evaporated to yield the crude product as a pale yellow solid. The crude product was washed with chloroform (20 mL × 3) and further purified by HPLC to give the pure TA-zwitterion (2) in 23% yield. 1  Scheme S2: Synthetic route to the azido-EG2-mannose derivative.
The mixture was cooled to 0 °C in an ice bath and lipoic acid (2.02 g, 9.8 mmol) in 32 mL of CH2Cl2 was then added dropwise over 30 min under constant stirring. After the addition was complete, the reaction mixture was allowed to warmed up to RT and stirred overnight. The mixture was then filtered off through Celite  and the solid was rinsed with CHCl3. The filtrate was combined and evaporated to dryness, and then added with 100 mL H2O. The resulting solution was washed with diethyl ether (100 mL × 2). The aqueous phase was saturated with NaHCO3 and extracted with CHCl3 (100 mL × 3). The combined organic layers were dried over MgSO4. After evaporation of solvent, the residue was purified by silica gel column chromatography using 20:1 (vol/vol) CHCl3: MeOH as eluting solvent. Each fraction was checked by TLC and the pure product fractions were combined (CHCl3: MeOH =10:1 (vol/vol), Rf (LA-PEG13-N3) = 0.52, Rf (TA) = 0.6, Rf (N3-PEG13-NH2) = 0.04) and evaporated to dryness to give LA-PEG13-N3 (2) as a yellow oil (2. gradually to RT and stirred for 24 h. After filtration and rinse the solid with CHCl3, the filtrate was combined and evaporated to dryness to give an yellow oil. The crude product was purified by silica gel column chtromatgraphy using CHCl2/MeOH (16:1 v/v) as eluting solvent. The desired product (LA-EG3-N3) was obtained as a yellow oil (0.86 g, 80%). Rf (CHCl2/MeOH 10:1 v/v) = 0.64. 1  for 20 mins. The reaction mixture was cooled to 0 °C in an ice bath, and a solution of DCC (0.107 g, 0.52 mmol) in DCM (1 mL) was added dropwise. The reaction mixture was stirred at 0 °C for 1 h before being warmed to RT and stirred for another ~20 h. The reaction mixture (0.50 mL) was transferred to 1.5 mL microcentrifuge tube and 1 mL EtOAc was added and centrifuged at 14 kg for 2 mins. The yellow supernatant 10 was transferred to a round bottom flask. The remaining precipitate was washed by EtOAc (1.5 mL) twice and centrifuged. The supernatants were combined and evaporated to dryness to give a yellow oil. TLC (silica gel, CHCl3/MeOH = 10:1, v/v) Rƒ(Cyclooct -1-yn-3-glycolic acid) = 0.53, Rƒ (LA-PEG600-NH2) = 0.24, Rƒ (DMAP) = 0.42, Rƒ (product) = 0.67. The crude product was purified by silica gel flash column chromatography using CHCl3:MeOH = 20:1 (v/v) as eluting solvent. The Rƒ = 0.67 fractions were collected and combined. After evaporation of solvent, the desired product (4, n = ~13) was obtained as a yellow oil (0.369 g, 75%). 1    The reduced DHLA-PEGn-Man ligands were concentrated and dissolved in chloroform. The concentration of mannose was determined by phenol-sulfuric acid method as described in Section A41 below.

A42) Transmission electron microscopy (TEM) and Dynamic light scattering (DLS)
TEM imaging was performed using a Philips CM200 transmission electron microscope by depositing a drop of the quantum dot solution onto carbon-coated grid as previously described. 2 DLS was performed using a Zetasizer NanoZS (Malvern) using a laser wavelength of 633 nm in disposable polystyrene cuvettes. QD conjugates were in binding buffer (100 mM NaCl, 20 mM HEPES 7.8, 10 mM 14 CaCl2 and 10 g/ml His6-Cys peptide) and measured without filtration. Readings of 10 scans were taken in triplcate and the average values were calculated. 2

A43) Calculation of inter-mannose distance (X) 9
For QD of radius r, each conjugated with N number of ligands, footprint for each ligands: The average deflection angle for each ligand can be calculated by: The inter-mannose distance on the QD surface (X) can be calucated via: where R is hydrodynamic radius of the QD-Man conjugate as measured by DLS (SI Fig. S1

A51: protein production 10,11
Cysteine was introduced into the cDNAs encoding extracellular segment of DC-SIGN to replace residue Q274 or DC-SIGNR to replace R287 for site-specific dye labelling as indicated in the extracellular segment amino acid alignment of DC-SIGN/R below (star indicating Q274 in DC-SIGN and R287 in DC-SIGNR) The mutagenesis was carried out by using synthetic DNA restriction fragments to replace the corresponding wild-type sequences. Standard recombinant DNA techniques were used throughout these experiments. The integrity and successful mutation of the cloned fragments were confirmed by DNA sequencing. 15 Extracellular segments (sequence as above) which were known to form stable homotetramers and faithfully retaining their glycan binding properties 4 were expressed in E.coli and purified by Man-Sepharose affinity column as described previously. 10,11 The monomeric DC-SIGN and DC-SIGNR CRDs were also constructed and purified as described previously. 10,11 The prufity of the recombinant proteins was confirmed by gel electrophresis as shown below: SDS-polyacrylamide gel electrophoresis of DC-SIGN Q274C, DC-SIGNR R287C following purification by Man-Sepharose affinity chromatography. The gel (17.5% polyacrylamide) was stained with coomassie blue.

A52: Protein labelling 12
For labelling DC-SIGNQ274C and DC-SIGNRR287C, proteins were first bound to a mannose-Sepharose  Figure S7. The apparent FRET ratio (I626/I554) was used to calculate the coresponding KD at different temperature (see data analysis below).

A54: Data Analysis: 13,14
For all of the FRET related measurements, the raw fluorescence spectra of labeled protein + QD samples were substracted by the fluorescence spectra of the corresponding dye-labeled protein at same concentration only under identifical conditions to correct the background arising from the acceptor dye direct excitation. improve the fitting by using n as a fitting parameter: n was changed manually in the range of n(0) ± 0.5 in a step size of 0.2. The best fit was evaluated by the resulting R square value (R 2 ) as below (Table S1).

(b) Evaluation of inhibition constant KI. Direct excitation background corrected fluorescence spectra
were used to derive the apparent FRET ratio of I626/I554 as above. It was then plotted against inhibitor (mannose) concnetration. The data were fitted to a simple first order equation as below: Where F is the normalised fraction of dye-labelled-protein bound to QD, (I626/I554)0 is the FRET ratio of dyelabeled protein + QD in the absence of competing mannose, and (I626/I554)m is the apparent FRET ratio in the prescence of mannose. KI is the the concentration of mannose that yields 50% inhibition of QD binding (FRET ratio drops to 50% of the initial level) and [mannose] is the total concentration of mannose.
(c) Evaluation of binding enthalpy and entropy. 14 The apparent FRET ratio (I626/I554) were obtained after correction of dye direct excitation background as above. KD at each temperature was calculated via equation (1) using the Max and n values obtained from the best fit at 25 °C (Table S1) and given in Table S2.

A55) Correlation between FRET ratio and QD bond proteins [1]
For a single QD donor which is in FRET interaction with N identical acceptors (e.g. under idential QD-dye distance r), the FRET efficiency, E, can be given in the following equation: 18 where R0 is the Förster radius of the QD-single dye FRET pair and r is donor-acceptor distance. E can also be measured via the enhanced acceptor emission by the following equation: Where  is a correcting factor for the different dye and QD fluorescence quantum yield. Assuming that the shape of the QD and dye fluorescence spectra are independent of their intensity, then the integrated IQD/IDye ratio should be linearly propotional to the peak intensity ratio, e.g. IQD/IDye =  I554/I626 (where  is a correction factor between the integrated and peak intensity ratio, which is found to be 1.50 here).
The combination of equations (4) and (5) gives the following equation: This yields the following relationship: Hence

I626/I554 = N [ ×× (R0/r) 6 ]
Where   and R0 are all constant values. This equation shows that the apparent FRET ratio I626/I554 should increase linearly with N, the number of acceptors (proteins) bound to each QD if all of the proteins were bound to the QD in the same distance r.

A6) Viral Inhibition Assays 15
Effects of the QD-EG3-Man in inhibiting pseudo-Ebola infection of 293T cells were assessed by using our established procedures. 15