Molecular recognition of surface-immobilized carbohydrates by a synthetic lectin

Summary The molecular recognition of carbohydrates and proteins mediates a wide range of physiological processes and the development of synthetic carbohydrate receptors (“synthetic lectins”) constitutes a key advance in biomedical technology. In this article we report a synthetic lectin that selectively binds to carbohydrates immobilized in a molecular monolayer. Inspired by our previous work, we prepared a fluorescently labeled synthetic lectin consisting of a cyclic dimer of the tripeptide Cys-His-Cys, which forms spontaneously by air oxidation of the monomer. Amine-tethered derivatives of N-acetylneuraminic acid (NANA), β-D-galactose, β-D-glucose and α-D-mannose were microcontact printed on epoxide-terminated self-assembled monolayers. Successive prints resulted in simple microarrays of two carbohydrates. The selectivity of the synthetic lectin was investigated by incubation on the immobilized carbohydrates. Selective binding of the synthetic lectin to immobilized NANA and β-D-galactose was observed by fluorescence microscopy. The selectivity and affinity of the synthetic lectin was screened in competition experiments. In addition, the carbohydrate binding of the synthetic lectin was compared with the carbohydrate binding of the lectins concanavalin A and peanut agglutinin. It was found that the printed carbohydrates retain their characteristic selectivity towards the synthetic and natural lectins and that the recognition of synthetic and natural lectins is strictly orthogonal.


2-[2-(2-Aminoethoxy)ethoxy]ethanol (2)
The synthesis was done according to literature under argon. [1] The crude educt 1 (6.00 g, 34.4 mmol) was dissolved in THF (50 mL) and triphenylphosphine (11.7 g, 44.7 mmol) was added in two portions. After stirring for 48 h at room temperature, water (1.13 mL) was added and stirring was continued for another 48 h. The solvent was removed under reduced pressure and the residue is afterwards dried under high vacuum at 45 °C overnight. Water (119 mL) was added and the formed precipitate is removed by filtration. After washing the solid with water (200 mL), the solvent was evaporated under reduced pressure and the product was dried in high vacuum at 70 °C overnight. S6

2-[2-(2-Aminoethoxy)ethoxy]ethyl β-D-glucopyranoside (7)
The synthesis was performed as described in literature under an atmosphere of argon. [5] To a solution of 6 (327 mg, 533 mol) in dry MeOH (10 mL) was added a solution of sodium methoxide (cat. amounts) in dry MeOH (3 mL). After stirring for 2 h at room temperature, DOWEX HCR-W2 (H + , washed with MeOH) was added until a neural pH was reached. The suspension was filtered and washed with MeOH (10 mL). To this, palladium on carcoal (32 mg, 10 wt %) was added and the suspension was hydrogenated for 24 h at room temperature. After filtration over Celite ® to remove the catalyst and washing with MeOH (100 mL), the solvent was evaporated and the residue was dried in high vacuum to give the desired product.  S9

2-[2-(2-Aminoethoxy)ethoxy]ethyl β-D-galactopyranoside (10)
The synthesis was performed as described in literature under an atmosphere of argon. [9] To a solution of 9 (191 mg, 312 mol) in dry MeOH (5 mL) was added a solution of sodium methoxide (cat. amounts) in dry MeOH (1 mL). After stirring for 2 h at room temperature, DOWEX HCR-W2 (H + , washed with MeOH) was added until a neural pH was reached. The suspension was filtered and washed with MeOH (10 mL). To this, palladium on carcoal (32 mg, 10 wt %) was added and the suspension was hydrogenated for 24 h at room temperature. After filtration over Celite ® to remove the catalyst and washing with MeOH (100 mL), the solvent was evaporated and the residue was dried in high vacuum to give the desired product.

1,2,3,4,6-Penta-O-acetyl β-D-mannopyranoside (11)
The synthesis was performed as described in literature. [7] A suspension of anhydrous sodium acetate (5.00 g, 60.0 mmol) and acetic anhydride (60.0 mL, 640 mmol) was refluxed for 5 minutes. After addition of D-(+)-mannose (10.0 g, 60.0 mmol) refluxing was continued for further 30 min. The hot solution was poured on ice water (300 mL) and the mixture was extracted with DCM (3  50 mL). Drying with MgSO 4 , filtering, removing the solvent under reduced pressure gave the crude product which was dissolved in DCM (100 mL) and washed S11 with sat. NaHCO 3 (3  50 mL). After drying over MgSO 4 , filtration, evaporation of the solvent under reduced pressure and drying in high vacuum, the product was obtained.

2,3,4,6-Tetra-O-acetyl-α/β-D-mannopyranosyl trichloroacetimidate (12)
The synthesis was performed as described in literature under an atmosphere of argon. [6] A solution of 11 (3.00 g, 7.68 mmol) and hydrazine acetate (850 mg, 9.22 mmol) in dry DMF (15 mL) was stirred at 60 °C for 1 h. The mixture was diluted with EtOAc (50 mL), washed with water (3  30 mL) and brine (1  30 mL), dried over MgSO 4 and evaporated to dryness under reduced pressure to yield a yellow oil. After dissolving the oil in dry DCM (10 mL), trichloroacetimidate (7.70 mL, 76.8 mmol) were added and the solution was cooled to 0 °C. The solution was treated with 1,8-diazabicycloundec-7-ene (DBU) (0.152 mL, 768 mol) and stirred at 0 °C for 1 h. After warming to room temperature, stirring was continued for another hour and then the solvents were removed under reduced pressure. The brown oil was purified by silica gel column chromatography using cyclohexane:EtOAc = 1:1 as eluent.

1,8-Diamino-3,6-dioxaoctane-D-neuraminic acid (16)
The synthesis was performed under an atmosphere of hydrogen. 15 (170 mg, 296 mol) was dissolved in dry MeOH (5 mL). After the addition of palladium on carcoal (17 mg, 10 wt %), the suspension was hydrogenated for 24 h at room temperature. After filtration over Celite ® to remove the catalyst and washing with MeOH (100 mL), the solvent was evaporated and the residue was dried in high vacuum to give the desired product.

N-tert-Butoxycarbonyl ethylendiamine (17)
The synthesis was performed as described in literature under an atmosphere of argon. [ 8 ] Ethylendiamine (12.2 mL, 183 mmol) was dissolved in dry DCM (160 mL) and the solution was cooled to 0 °C. After drop-wise addition of a solution of di-tert-butyl dicarbonate (Boc 2 O) (4.00 g, 18.3 mmol) in dry DCM (100 mL) over 3 h at 0 °C, the solution was stirred overnight at room temperature. The reaction mixture was washed with water (4  100 mL), dried over MgSO 4 , filtered and evaporated under reduced pressure. After drying in high vacuum the product was obtained.

Solid phase peptide synthesis (SPPS)
Standard operation procedure loading of the resin (SOP 1): Loading of the resin was performed according to literature under an atmosphere of argon. [10] The according amino acid (2 equiv relative to resin loading) was dissolved in dry DCM and a small amount of DMF (SPPS grade) and is added to 2-chlorotrityl resin (loading: 1.50 mmol/g). After addition of DIPEA (2 equiv relative to resin loading) the mixture is agitated by a slow stream of argon for 5 min. Following another addition of DIPEA (3 equiv relative to resin loading), agitation is continued for 1 h. The remaining reactive groups of the resin are quenched with MeOH p.a.
Determination of the beads´ loading [11] was done by suspending 6.00 mg dry beads in 20% piperidine in DMF (1 mL) and stirring for 20 min at room temperature. 100 µL of the supernatant were diluted with DMF p.a. (10 mL) and the absorbance from 290 to 310 nm is measured against DMF p.a. Loading can be calculated by using the following formulas: Standard operation procedure for step-wise chain elongation via SPPS (SOP 2): SPPS was performed according to literature. [1,2] Dry beads were swelled in DMF p.a. for 45 min while shaking the reaction vessel. After sucking of the DMF p.a., the Fmoc-group is cleaved with 20% piperidine in DMF for 20 min. The resin is washed with DMF p.a. (4 times) and alternately with DCM p.a. (3 times) and isopropanole p.a. (3 times) to fully remove the piperidine. Success of the cleavage was controlled by the Kaiser test. Equal amounts of a 5% ninhydrin solution in EtOH, 80% phenol in EtOH and 0.001 M KCN in pyridine were mixed with a few washed beads and heated for 1 min to 100 °C. A blue color of the beads indicated free amine functions on the resin. The coupling step was performed by suspending the resin in a solution of the according Fmoc-protected amino acid (3 equiv) and Oxyma pure ® (3.6 equiv) in DMF (SPPS grade). After addition of DIPCDI (3.3 equiv), the suspension was shaken for 2 h and then the solvent was sucked off. Washing of the beads with DMF (SPPS grade, 4 times) was followed by the Kaiser test which should not result in a color change if the coupling was successful. The deprotection and coupling steps were continued until the desired peptide sequence was obtained.
Standard operation procedure for cleavage of resin and removal of permanent protection groups (SOP 3): Cleavage of the resin and removal of permanent protection groups was performed according to literature. [1,12] Prior to splitting of the peptide from the resin, the Fmoc-group was removed by treatment with 20% piperidine in DMF for 20 min. After washing with DMF p.a. (4 times) and alternately with DCM (2 times) and isopropanole p.a. (2 times), the beads were suspended in a solution of TFA:H 2 O:EDT:TIS = 94:2.5:2.5:1 and stirred for 5 h at rt. After filtration, the beads were washed with TFA (5 times) and the filtrate was concentrated until the peptide started to precipitate. Addition of cold Et 2 O resulted in complete precipitation and the suspension was keep overnight in the freezer. The peptide was collected by filtration, washed with Et 2 O and dried in high vacuum to yield the peptides as white, hygroscopic solids which were stored under argon. S18

FITC-HisHis (22)
The appropriate amount of labeled tripeptide 21 was dissolved in phosphate buffer (100 mM, pH 7.4) and stirred in an open vial until the Ellman-test was negative. The resulting dimer was used without further purification for ITC and fluorescence measurements.

Isothermal titration calorimetry of FITC-HisHis with NANA
ITC measurements were performed using a NanoITC system (Calorimetry Sciences Cooperation, USA) and ITC Run software. Sample solutions were prepared by dissolving the appropriate amount of FITC-HisHis and NANA in phosphate buffer (100 mM, pH 7.4). Before filling the cell, the solution was degassed for 30 min. A solution of 2 mM FITC-HisHis was titrated with a degassed solution (30 min) of 40 mM NANA. All measurements were performed at 23 °C using a stirring rate of 300 rpm and a 400 s interval between each injection. To determine the heat of dilution, a NANA solution was titrated into phosphate buffer (100 mM, pH 7.4). The heat of dilution was subtracted from the raw heat data. The data were fitted to a 1:2 model using a spread sheet method. [14] Figure S1: A) Raw data of ITC titration of 2 mM FITC-HisHis with 40 mM NANA in phosphate buffer (100 mM, pH 7.4); B) Fit obtained from the raw data shown in A) (first data point is not used for the fit).

X-ray photoelectron spectroscopy
X-ray photoelectron spectroscopy (XPS) of Si-wafers functionalized with an epoxideterminated SAM and printed with NANA ink using a PEG coated flat stamp are shown in Figure S3 and S4. While nearly no N(1s) signal was detected in the epoxy SAM, a clear N(1s) signal at 400 eV can be observed when the NANA ink is printed. Two nitrogen peaks are identified: a major peak which arises from the amide bonds in the carbohydrate ink and a minor one that is attributed to traces of absorbed triethylammonium salt that could not be removed by rinsing or sonification and which originates from the triethylamine added as an additive in the ink solution. The C(1s) peak shows a splitting into the C-C (285 eV), C-O (287 eV), CC, CO and residual epoxide C-O signals (289 eV) as expected for the NANAterminated SAM.

Surface plasmon resonance (SPR)
In order to confirm the interaction between HisHis and NANA with SPR a commercially available polycarboxylate hydrogel sensor surface was employed. Both the spectrometer and the hydrogel chips were provided by XanTec bioanalytics GmbH, Düsseldorf, Germany. The functionalization of the polycarboxylate hydrogel with amine terminated NANA was performed by N-hydroxysuccinimide (NHS) activation and subsequent peptide coupling. All SPR experiments were carried out in NH 4 CO 3 buffer (100 mM, pH 7.8) with a flow rate of 10 µL/min at 25 °C. HisHis (0.5-2.0 mM in NH 4 CO 3 buffer) was applied to the NANA functionalized surface for 10 min. A small but significant SPR signal increase was observed upon the addition of HisHis. The initial rate and extent of surface binding correlated with the concentration of HisHis (0.5-2.0 mM) applied to the sensor. However, it was not possible to obtain sufficiently reproducible data to perform a quantitative analysis of the peptidecarbohydrate interaction. The poor quality of the SPR signal is certainly due to the low molecular weight of HisHis which limits any further SPR investigations.