Search

Pradimicin A (PRM-A) and related compounds constitute an exceptional family of natural pigments that show Ca 2+ -dependent recognition of d-mannose (Man). Although these compounds hold great promise as research tools in glycobiology, their practical application has been severely limited by their inherent tendency to form water-insoluble aggregates. Here, we demonstrate that the 2-hydroxyethylamide derivative (PRM-EA) of PRM-A shows little aggregation in neutral aqueous media and retains binding specificity for Man. We also show that PRM-EA stains glycoproteins in dot blot assays, whereas PRM-A fails to do so, owing to severe aggregation. Significantly, PRM-EA is sensitive to glycoproteins carrying high mannose-type and hybrid-type N-linked glycans, but not to those carrying complex-type N-linked glycans. Such staining selectivity has never been observed in conventional dyes, suggesting that PRM-EA could serve as a unique staining agent for the selective detection of glycoproteins with terminal Man residues.

Pradimicins (PRMs) constitute an exceptional class of natural products that show Ca 2+ -dependent recognition of d-mannose (Man). In addition to therapeutic uses as antifungal drugs, the application of PRMs as lectin mimics for glycobiological research has been attracting considerable interest, since the emerging biological roles of Man-containing glycans have been highlighted. However, only a few attempts have been made to use PRMs for glycobiological purposes. The limited use of PRMs is primarily due to the early assumption that the readily modifiable carboxyl group of PRMs is involved in Ca 2+ binding, and thus, not available to prepare research tools. Recently, this assumption has been disproved by structural elucidation of the Ca 2+ complex of PRMs, which paves the way for designing carboxyl group modified derivatives of PRMs for research use. This article outlines studies related to Ca 2+ -mediated Man binding of PRMs and discusses their application for glycobiology., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Naturally occurring pradimicins (PRMs) show specific recognition of d-mannose (d-Man) in aqueous media, which has never been achieved by artificial small molecules. Although the Ca 2+ -mediated dimerization of PRMs is essential for their d-Man binding, the dimeric structure has yet to be elucidated, leaving the question open as to how PRMs recognize d-Man. Thus, we herein report the structural elucidation of the dimer by a combination of X-ray crystallography and solid-state NMR spectroscopy. Coupled with our previous knowledge regarding the d-Man binding geometry of PRMs, elucidation of the dimer allowed reliable estimation of the mode of d-Man binding. Based on the binding model, we further developed an azide-functionalized PRM derivative (PRM-Azide) with d-Man binding specificity. Notably, PRM-Azide stained Candida rugosa cells having mannans on their cell surface through conjugation with the tetramethylrhodamine fluorophore. The present study provides the practical demonstration that PRMs can serve as lectin mimics for use in glycobiological studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Pradimicin A (PRM-A) is a unique natural product that recognizes d-mannopyranoside (Man) in the presence of Ca 2+ ion. Although the Man binding geometry of PRM-A is largely understood, the molecular basis of Man recognition has yet to be established because of the lack of information regarding Ca 2+ binding geometry. In this work, to examine the Ca 2+ binding site of PRM-A, we performed a solid-state nuclear magnetic resonance experiment using 111 Cd 2+ as a surrogate probe for Ca 2+ . Evaluation of 13 C- 111 Cd distances in the [PRM-A/ 111 Cd 2+ ] complexes by rotational-echo double resonance (REDOR) and 111 Cd frequency selective REDOR (FSR) revealed that PRM-A binds 111 Cd 2+ at the anthraquinone moiety, which contradicts the previous hypothesis of the alanine moiety being the Ca 2+ and Cd 2+ binding sites of PRM-A. The distances between Cd 2+ and the carbon atoms at the binding site of PRM-A were found to be 3.5 ± 0.2 Å. Importantly, Man binding was shown not to alter the distances, indicating that [PRM-A/Ca 2+ ] and [PRM-A/Ca 2+ /Man] complexes have similar Ca 2+ binding geometries. This study provides an important clue to understanding the molecular basis of Man recognition of PRM-A.

Pradimicin A (PRM-A) is a unique antibiotic with a lectin-like ability to bind D-mannose (D-Man) in the presence of Ca(2+) ion. Although accumulated evidences suggest that PRM-A recognizes the 2-, 3-, and 4-hydroxyl groups of D-Man, BMY-28864, an artificial PRM-A derivative, was shown not to bind L-fucose (L-Fuc) and L-galactose (lLGal), both of which share the characteristic array of the three hydroxyl groups with D-Man. To obtain a plausible explanation for this inconsistency, we performed co-precipitation experiments of PRM-A with L-Fuc, L-Gal, and their methyl pyranosides (L-Fuc-OMe, L-Gal-OMe) by taking advantage of aggregate-forming propensity of the binary [PRM-A/Ca(2+)] complex. While L-Fuc and L-Gal were hardly incorporated into the aggregate, L-Fuc-OMe and L-Gal-OMe were found to exhibit significant binding to PRM-A. However, increased Ca(2+) concentration abolished this binding, raising the possibility that poor binding of L-Fuc and L-Gal to PRM-A is attributed to their chelation with Ca(2+) ion. This possibility was partly supported by (1)H NMR analysis that detected interaction of L-Fuc and L-Gal with Ca(2+) ion in aqueous solution. These results collectively indicate that PRM-A binds pyranosides of L-Fuc and L-Gal when Ca(2+) concentration is not excessive to trap these sugars by chelation but sufficient to form the [PRM-A/Ca(2+)] complex., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Pradimicins (PRMs) and benanomicins are the only family of non-peptidic natural products with lectin-like properties, that is, they recognize D-mannopyranoside (Man) in the presence of Ca(2+) ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti-HIV activities through binding to Man-containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate-forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM-A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM-A and Man. Evaluation of intermolecular distances by solid-state NMR spectroscopy revealed that the C2-C4 region of Man is in close contact with the primary binding site of PRM-A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co-precipitation experiments using deoxy-Man derivatives, leading to the proposal that PRM-A binds not only to terminal Man residues at the non-reducing end of glycans, but also to internal 6-substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM-A., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Pradimicin A (PRM-A) is a unique antibiotic with a lectin-like ability to recognize d-mannopyranosides (Man) in the presence of Ca(2+) ion. BMY-28864 (1) is a water-soluble analogue of PRM-A, which has been extensively used for studies on the mode of Man recognition and antifungal action of pradimicins. Although it has been assumed that PRM-A and 1 bind Man in a similar fashion, direct experimental evidence has yet to be provided. In this report, we compared Ca(2+) and Man binding of 1 with that of PRM-A through two solid-state NMR experiments. The solid-state (113)Cd NMR analysis using (113)Cd(2+) ion as a surrogate for Ca(2+) ion suggested the similarity in Ca(2+) coordination of PRM-A and 1. The dipolar assisted rotational resonance (DARR) analysis using (13)C-labeled 1 clearly showed that 1 as well as PRM-A binds Man near its carboxyl group. These results collectively indicate that the mode of binding of Ca(2+) ion and Man is nearly identical between PRM-A and 1., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Pradimicin A (PRM-A) is an actinomycete-derived antibiotic with the lectin-like property of being able to recognize D-mannopyranoside (Man) in the presence of Ca(2+) ion. PRM-A and its derivatives have been attracting a great deal of attention as the only family of natural carbohydrate receptors with nonpeptidic skeleton and, more recently, as conceptually novel drug candidates for human immunodeficiency virus (HIV). Despite its scientific interest and potential therapeutic importance, understanding how PRM-A recognizes Man has been severely limited. Conventional interaction analysis of PRM-A with Man in solution has been frustrated by aggregation of PRM-A and the three-component equilibrium consisting of the [PRM-A(2)/Ca(2+)], [PRM-A(2)/Ca(2+)/Man(2)], [PRM-A(2)/Ca(2+)/Man(4)] complexes, and their mixed oligomers. In this Article, we demonstrate the interaction analysis of PRM-A with methyl α-D-mannopyranoside (Man-OMe) in the solid state, which benefits from aggregate-forming propensity of PRM-A and eliminates the problem associated with the complicated equilibrium in solution. Isothermal titration calorimetry (ITC) analysis and coprecipitation experiments revealed that the primary Man binding of PRM-A is markedly tighter than the secondary one, leading to preparation of the solid aggregate solely composed of the [PRM-A(2)/Ca(2+)/Man-OMe(2)] complex. The simple 1:1 complexes of biosynthetically (13)C-enriched PRM-As and [(13)C(6)]Man-OMe facilitated the analysis of the primary Man binding of PRM-A by two-dimensional dipolar-assisted rotational resonance (2D-DARR), which clearly identified that the cavity consisted of D-alanine moiety and ABC rings of PRM-A is the Man binding site. Interestingly, the proposed Man binding site of PRM-A seems to resemble the typical architecture of artificial carbohydrate receptors.