Synthetic Receptor Can Distinguish Between Male and Female Steroid Hormones

[madman]

Synthetic Receptor Can Distinguish Between Male and Female Steroid Hormones

 

[madman]

Abstract
Biological receptors distinguish and bind steroid sex hormones, e.g., androgen-, progestogen-, and estrogen-type hormones, with high selectivity. To date, artificial molecular receptors have been unable to discriminate between these classes of biosubstrates. Here, we report that an artificial polyaromatic receptor preferentially binds a single molecule of androgenic hormones, known as “male” hormones (indicated with m), over progestogens and estrogens, known as “female” hormones (indicated with f), in water. Competitive experiments established the binding selectivity of the synthetic receptor for various sex hormones to be testosterone (m) > androsterone (m) >> progesterone (f) > β-estradiol (f) > pregnenolone (f) > estriol (f). These bindings are driven by the hydrophobic effect, and the observed selectivity arises from multiple CH-π contacts and hydrogen-bonding interactions in the semirigid polyaromatic cavity. Furthermore, micromolar fluorescence detection of androgen was demonstrated using the receptor containing a fluorescent dye in water.








DISCUSSION
We have realized the discrimination between steroid male and female hormones by a synthetic molecular receptor in water. The binding affinity resembles that of natural androgen receptors: Androgenic male hormones are predominantly encapsulated by the receptor even from mixtures including large excess progestogenic and estrogenic female hormones. The key of the present achievement is using a semirigid nanocavity surrounded by polyaromatic frameworks linked by metal ions, which can deform the shape to enhance interactions between the substrates and cavity. The combination of the polyaromatic receptor with high androgen affinity and advanced analytical methods based on single-molecule detection (1–3) would develop novel ultrasensitive analytical devices for steroid sex hormones, one of the most important and complex biosubstrates with high physiological activities.

Fig. 1
Structures of steroid sex hormones, a natural androgen receptor, and a synthetic receptor.

(A) Tetracyclic framework of steroid sex hormones. (B) Crystal structures of a human androgen receptor (6, left) and synthetic receptor 1 (right) shown at the same scale. The binding pocket and cavity are highlighted in yellow. (C) Polyaromatic receptor 1 used here and space-filling representation of the core framework (based on the crystal structure). (D) Representative male hormone, testosterone (2a), and female hormones, progesterone (3a) and β-estradiol (4a).

Fig. 2
Selective binding of testosterone by receptor 1 from mixtures.
(A) Schematic representation of the selective binding of testosterone (2a) by receptor 1 from a mixture of 2a, progesterone (3a), and β-estradiol (4a) (1:1:1 or 1:100:100 ratio) in water. 1H NMR spectra (500 MHz, D2O) of (B) receptor 1 and (C) products obtained from an equimolar mixture of 2a, 3a, and 4a in the presence of 1 at 60°C for 10 min (gray square, 1•3a) and (D) 1•2a. (E) Changes of the 1H NMR chemical shifts (Δδ in ppm) of 2a upon encapsulation by 1. (F) ESI-TOF MS spectrum (H2O, room temperature) of 1•2a.

Fig. 3
X-ray crystal structure of 1’•2a.
(A) Space-filling (for 2a) and cylindrical (for 1’) representation and (B) space-filling representation (the peripheral substituents of 1’ are replaced by hydrogen atoms for clarity). (C) Highlighted positions of 2a inside the polyaromatic shell of 1’ (three different views). (D) Highlighted host-guest interactions of 1’•2a in the cavity (blue, orange, and red dashed lines are CH-π, OH-π, and hydrogen-bonding interactions, respectively)

Fig. 4
Binding affinity of receptor 1 toward male hormones.
(A) Schematic representation of the binding preference of receptor 1 toward male hormones 2a to 2e in water. (B) 1H NMR spectra (500 MHz, D2O, room temperature) of 1•2a (top), 1•2c (middle), and the product (bottom) obtained from an equimolar mixture of 2a and 2c with 1 (blue circle, 1•2a; pale blue square, 1•2c). (C) 1H NMR spectra (500 MHz, D2O, room temperature) of 1•2a (top), 1•2d (middle), and the product (bottom) obtained from an equimolar mixture of 2a and 2d with 1 (blue circle, 1•2a; pale blue triangle, 1•2d). Binding preference of 1 toward (D) 2a and methyltestosterone (5a) and (E) 2d and adrenosterone (5b) in water.

Fig. 5
Binding affinity of receptor 1 toward female hormones.
(A) Schematic representation of the binding preference of receptor 1 toward female hormones 3a to 3c and 4a to 4c in water. 1H NMR spectra (500 MHz, D2O, room temperature) of products obtained from equimolar mixtures of (B) 2e and 3a, (C) 3a and 4a, and (D) 4a and 4c with 1. (E) Binding preference of 1 toward 3b and 5α-androstane (5c) in water.

Fig. 6
Fluorescent detection of testosterone by receptor-dye complex 1•6.
(A) Schematic representation of nanogram-scale fluorescent detection of male hormone 2a using one drop of an aqueous 1•6 solution (8 μM) and their photographs (λex = 356 nm) on a petri dish. (B) Fluorescence spectra (room temperature, λex = 423 nm) of a H2O solution of 1•6 (78 μM, 0.5 ml) before and after addition of 2a (45 nmol) and their photographs (λex = 356 nm).