Beatrice Wagner

Saturation Transfer Difference NMR Spectroscopy for Identifying Ligand Epitopes and Binding Specificities

We have studied the potential of saturation transfer nuclear magnetic resonance (NMR) experiments in different mode to screen compound mixtures for binding activity and to characterize binding epitopes on the ligand. We have developed a protocol based on the transfer of saturation from the protein to bound ligands which by dissociation is moved into solution where it is detected (Fig. 1; Mayer and Meyer 1999; Peters and Meyer). By subtracting a spectrum, where the protein is saturated from one without protein saturation, a spectrum is produced where only signals of the ligand(s) remain in the difference spectrum. The irradiation frequency is set to a value where only protein resonances and no resonances of free ligands are located. Usually, irradiation frequencies around 1.5 ppm are practical because no ligand resonances are found in this spectral region, whereas the significant line width of protein signals still allows selective saturation. If the ligands show no resonances in the aromatic spectral region, the saturation frequency may also be placed there.

Synthetic Potential of Fucosyltransferase III for the Synthesis of Fluorescent‐labeled Milk Oligosaccharides

Various fundamental biologic roles of milk oligosaccharides have been recognized; however, their structure‐affinity relationship is still not fully revealed. Herein, we describe the synthesis of the fluorescent‐labeled milk oligosaccharides 3‐(5‐dimethylaminonaphthalene‐1‐sulfonylamino)propyl β‐D‐galactopyranosyl‐(1→3)‐[α‐L‐fucopyranosyl‐(1→4)]‐2‐acetamido‐2‐deoxy‐β‐D‐glucopyranoside (1) and 3‐(5‐dimethylamino‐naphthalene‐1‐sulfonylamino)propyl β‐D‐galactopyranosyl‐(1→3)‐[α‐L‐fucopyranosyl‐(1→4)]‐β‐D-glucopyranoside (2) as useful tools for synthetic, analytic, and biologic applications. For the fucosylation of lactose and lacto‐N‐biose, the chemical and the enzymatic syntheses using fucosyltransferase III were compared.

Cutaneous iontophoretic delivery of CGP69669A, a sialyl Lewis(x) mimetic, in vitro.

Abstract:  The aim was to investigate the feasibility of using iontophoresis for the cutaneous delivery of the E‐selectin antagonist CGP69669A, a sialyl Lewisx‐glycomimetic with potential activity against inflammatory skin diseases. The effects of current density and formulation on iontophoretic transport were evaluated in porcine and human skin in vitro. Cumulative permeation of CGP69669A increased with current density (69.73 ± 9.51, 113.97 ± 26.80 and 160.44 ± 13.79 μg/cm2 at 0.1, 0.3 and 0.5 mA/cm2, respectively) and drug concentration (37.42 ± 13.13, 78.96 ± 23.13 and 160.44 ± 13.79 μg/cm2, at 1, 3 and 5 mg/ml, respectively). In contrast, passive delivery was negligible. Although permeation from a 2% hydroxyethyl cellulose gel was lower than that from aqueous solution, skin deposition – more relevant for the local treatment of dermatological conditions – was 3‐fold higher. The results demonstrated that although CGP69669A cannot be delivered passively into the skin it is an excellent candidate for transdermal iontophoresis, a technique that is ideally suited to the delivery of glycomimetics.

Development and validation of a HPAE-PAD method for the quantification of CGP69669A, a sialyl Lewisx mimetic, in skin permeation studies

A simple, rapid, precise and specific isocratic HPAE-PAD method for quantification of CGP69669A was developed and validated. CGP69669A is a glycomimetic of sialyl Lewis(x) and an antagonist of E-selectin with potential application in the treatment of inflammatory skin disease. Quantification was performed using a Dionex CarboPac(TM) PA-200 anion-exchange column (3 × 250 mm) with 100 mm NaOH solution as mobile phase, a flow rate of 0.50 mL/min and an injection volume of 10 μL. A quadruple potential waveform was used to detect the carbohydrate (+0.1 V from 0.00 to 0.40 s, -2.0 V from 0.41 to 0.42 s, +0.6 V at 0.43 s and -0.1 V from 0.44 to 0.50 s with current integrated between 0.20 and 0.40 s for detection) and rafinose was employed as an internal standard. The optimized conditions enabled rapid elution of CGP69669A (at 3.0 min) without interference from solvent peaks or substances present in the skin. The method showed good intra- and inter-day precision and accuracy and the response was linear from 1.0 to 25 µg/mL. This is the first validated direct method for the quantification of CGP69669A. It will now be employed in studies investigating the topical and transdermal delivery of CGP69669A in vitro and in vivo and it should also be of use for other applications of this molecule.

Iontophoretic Delivery of Glycomimetics-A New Approach for the Treatment of Inflammatory Skin Diseases

A key event in the inflammatory response is the recruitment of leukocytes from the microcirculation to the inflamed tissue through endothelial-dependent mechanisms that include leukocyte tethering and rolling, activation, firm adhesion and diapedesis to the interstitium. Leukocyte tethering and rolling along the endothelial surface is mediated by a family of carbohydrate-binding proteins (E-, P- and L-selectins). While L-selectin is constitutively expressed on the surface of leukocytes, expression of E- and P-selectin is upregulated on the endothelial surface during inflammation.