Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

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Abstract

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E2) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD–E2 conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer–E2 conjugates, CD–E2 was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.

Introduction

β-Cyclodextrins (CDs) are torus-shaped saccharides consisting of a shallow interior cavity, a primary face of 7 hydroxyls and a secondary face of 14 hydroxyl groups. CDs and modified CDs have many interesting biological properties due to their unique structure, which can be altered most readily by chemical modification of the primary face.1, 2 The hydrophobicity of the CD cavity has dominated thinking in terms of CD host–guest chemistry via formation of non-covalent inclusion complexes and native and commercially available CD derivatives, the latter generally heterogeneous compounds, have been used as efficient drug carriers.3 Alternatively, the CD torus can be used as a scaffold to attach drug moieties in order to enhance their effectiveness by facilitating drug delivery or specificity; for example, CD–epoxysuccinyl peptide conjugates were reported as inhibitors of membrane-associated cathepsin B, known to be secreted in several tumor cell lines.4 The synthesized CD–peptide conjugates were full inhibitors of extranuclear cysteine proteases and were reported not to be membrane-permeant. Other examples of CD conjugates as specific ligands of cell-surface receptors include opioid peptides,5 gastrin peptides,6 and mono- and oligosaccharides.7 These approaches rely in part on the assumed membrane impermeability of CD conjugates, based upon the natural hydrophilicity of CD conjugates and a molecular weight (>1500) above the perceived limits of facile, passive membrane transport.

The development of chemically and biologically stable estrogen–macromolecule conjugates has been a challenge of increasing importance given the interest in differentiating non-classical ligand-activated estrogen receptor (ER) actions versus the classical ER function as a ligand-activated nuclear receptor. Estrogens are able to elicit rapid responses that are thought to be mediated by extranuclear receptors that may be the classic ER, splice variants, or G-protein coupled receptors.8, 9, 10 The non-classical, extranuclear action of E2 elicit activity via cytosolic kinase cascades, which may have genomic effects via epigenetic actions or transcription factors other than ER.11, 12 The classical, genomic actions of ER as a nuclear receptor have been differentiated from membrane-associated ER activity by use of commercially available protein conjugates such as E2–BSA12, 13, 14 and E2–peroxidase.15 These conjugates have been widely used, but as remarked by Katzenellenbogen, they have critical limitations such as leakage of free E2 either non-covalently bound or from biological degradation of the conjugate in cell cultures.8, 13, 16 In order to overcome these problems and develop efficient E2–macromolecule conjugates, Katzenellenbogen and co-workers utilized a polyamidoamine (PAMAM) dendrimer as core architecture instead of a protein, leading to an E2-conjugate selective for extranuclear ER.

Click chemistry is commonly used today as a synonym for the Huisgen cycloaddition, although originally defined by Sharpless as encompassing simple synthetic steps giving quantitative conversions.17 Quantitative procedures for formation of CD-conjugates avoid the complex chromatography required to remove by-products of incomplete derivatization. Herein, novel estradiol–CD conjugates were prepared using click chemistry and the estrogenic activity was assayed in order to test the ability of CD derivatives to provide selective probes of estrogenic activity.

Section snippets

Synthesis of ACD conjugates

A CD-conjugate persubstituted at the C6 position of the primary face was the initial target. Aminocyclodextrins (ACDs) are a specific subset of persubstituted CDs, which may be readily prepared by direct amination of per-6-bromo-per-6-deoxy-CD or per-6-iodo-per-6-deoxy-CD ACDs possess an annulus of amino groups in place of the ether oxygens of native CD and a corona of pendant side chains; at neutral pH the presence of the positively charged annulus diminishes the ability of the ACD cavity to

Conclusions

The classical mechanism of estrogen action is defined by: (i) binding of an estrogenic ligand to ER in the cytoplasm; (ii) ER dimerization and translocation to the nucleus; (iii) complexation with the ERE domain of DNA; (iv) recruitment of coregulator proteins; (v) transcription and expression of E2-responsive gene products. Non-classical mechanisms utilize extranuclear receptors that are activated by estrogenic ligands, often via rapid signaling cascades, however these may also induce

Materials and general methods

All chemicals and solvents were obtained from Sigma–Aldrich and Fisher unless otherwise stated and purified prior to use according to known procedures when necessary: 17β-ethynylestradiol (Steraloids, Newport, RI), NHS–rhodamine (PIERCE, Rockford, IL), and all other reagents were used as received. DMF and Et3N were distilled from CaH2. β-Cyclodextrin and all CD derivatives were dried under vacuum in a drying pistol over P2O5 with refluxing acetone prior to use. Moisture sensitive reactions were

Acknowledgments

This research was supported by the National Institutes of Health (CA 102590) and DOD contract W81XWH-07-1-0445. Dr. Zhiqiang Wang and Dr. Kuanqiang Gao are acknowledged for technical assistance.

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