The steroid nucleus, sterane, is composed of three cyclohexane rings and one cyclopentane ring. The six carbon atoms of a cyclohexane ring are not fixed rig idly in space, but are capable of interchanging through turning and twisting between several structural arrangements in space called conformations.
The two principal conformations of a cyclohexane ring are the chair and boat forms (see Figure 1).
Fig1. Principal conformational representations of cyclohexane. The left conformer of cylohexane is a chair, while the right conformer is a boat. They interchange about a million times per second.
Each of the two substituent groups on the six carbon atoms of the cyclohexane ring may exist in either the general plane of the ring and are designated as equatorial (e) or a plane perpendicular to the ring plane and are designated as axial (a). For the equatorial bonds, it is possible to superimpose on the equatorial notation an indication of whether they are below (α) or above (β) the general plane of the ring. Cyclohexane is highly conformationally mobile, interchanging between the boat and chair forms many thousands of times per second. The most stable form of the cyclohexane ring is the chair form; in this conformer there is a greater interatomic distance between the equatorial and axial hydrogens than in the boat form. Figure 2 illustrates the nature of all of the equatorial (e) and axial (a) hydrogens on the cholestane and coprostane ring structures.
Fig2. Comparison of structural relationships resulting from cis or trans A:B ring fusion in 5α-cholestane versus 5β-cholestane (top row) and cholestanol versus coprostanol (second row). In 5α-cholestane and cholestanol (left side of rows 1 and 2) the A:B ring fusion is trans (C-19-methyl-C-5H) while in 5β-cholestane and coprostanol (right side of rows 1 and 2) the A:B ring fusion is cis (C-19-methyl-C-5H). The orientation of substituents around carbon-5 for the cis and trans circumstances are illustrated in the bottom row (•–• indicates carbon–carbon bonds). Finally, with respect to the hydroxyl on C-3, its beta orientation is maintained for both cholestanol and coprostanol in spite of their different A/B ring fusions.
As indicated, the B and C rings of both cholestane and coprostane are locked into chair conformations (see Figure 2). Although, in principle, the A ring of both these steroids is free to interchange between the boat and chair representations, the chair form is believed to be much more favored. In the case of vita min D steroids, which do not have an intact B ring due to the breakage of the carbon-9-carbon-10 bond (thus they are termed seco steroids), the A ring is much more conformationally mobile than that of the usual cholesterol-derived steroids.
An important point for the reader to consider is that the usual structural representation given for steroids provides no clear designation of either the three-dimensional geometry or the space-filling aspects of the electron orbitals associated with each atom involved in the formation of the requisite bonds required for the full molecular structure. A comparison for cholestanol is given in Figure 3 of the planar representation (A), the planar conformational model (B), a Dreiding three- dimensional model emphasizing bond lengths and angles (C), and a Corey-Pauling three-dimensional space-filling model (D). Certainly the space-filling molecular representation most closely approximates the reality of the three-dimensional shape of the steroid and thus provides an insight into the overall molecular shape required to produce a biological response.
Fig3. Four structural representations of cholestanol. (A) A typical two-dimensional structure of cholestanol drawn on a page. (B) A planar conformational model, also drawn on a page. (C) A Dreiding three-dimensional “stick” model of cholestanol emphasizing bond angles and interatomic distances. (D) A Corey-Pauling three-dimensional space-filling model of cholestanol. The C and D models can either be manually assembled from a kit of all possible parts or can be modeled in three dimensions on a computer using a highly sophisticated program.
The approach of steroid conformational analysis has been of great value to the organic chemist as a tool to predict or understand the course of synthetic organic chemical reactions. It is also known that conformational considerations play an increasingly useful role in the understanding of steroid hormone-receptor interactions. Steroid receptors are known to have very precise ligand specificities; see for example, Table 1, which illustrates the structural preferences of the nuclear receptor for the steroid hormone 1α,25(OH)2-vitamin D3. It would be surprising, therefore, if the intimate local structure of the receptor’s ligand-binding site did not have the capability of distinguishing and discriminating between the presence or absence of a key hydroxyl or the various conformational forms of the same steroid hormone.
Table1. Ligand Specificity of the Nuclear 1α,25(OH)2D3 Receptor
Figure 4 presents a schematic view of the vita min D receptor (VDR) as it interacts with the three key hydroxyls on carbons 1α, 3β, and 25 of the steroid hormone 1α,25(OH)2D3 to create three anchoring hydrogen bonds (X, Y, & Z) which collectively create a functional ligand-receptor complex (see Figure 4A). The importance of these three hydrogen bonds has been deduced via evaluation of the three-dimensional structure of the VDR as determined via X-ray crystallographic analysis with its bound ligand the 1α,25(OH)2D3 steroid hormone. As shown in panel B of Figure 4, a modified ligand [25(OH)D3] which is missing the key 1α-hydroxyl group on the A-ring, can only bind to the VDR 0.15% as well as the natural hormone, 1α,25(OH)2D3 (see Table 1); thus it is unable to form a stable ligand-receptor complex and therefore it is not able to produce significant biological responses. Comparable structure-functions studies for all the steroid hormones have been carried out by many pharmaceutical companies to identify analogs of the natural steroid hormone(s) which are able to produce useful selective biological responses.
Fig4. Absence of the 25-hydroxyl of the steroid hormone 1α,25(OH)2D3 reduces the affinity of 1α (OH)D3 to bind to the vitamin D receptor (VDR) by ~660 fold. The schematic model of the VDR illustrates how the receptor for 1α,25(OH)2D3 first “captures” and then forms a stable hydrogen bond (with X) with the 25-hydroxyl group on the end of the conformationally mobile 8-carbon side chain. This is followed by the conformationally mobile A ring’s two hydroxyls of 1α,25(OH)2D3 (the 1β and the 3β) to form a stable (using Y and Z) receptor-ligand complex. The X, Y, and Z labels indicate known binding domains on the receptor’s interior which each form a stabilizing hydrogen bond with the three hydroxyls of 1α,25(OH)2D3 when the ligand is docked inside the VDR. Panel A illustrates the proposed steering effects of the 25-hydroxyl group docking with X which then permits capture of the conformationally active A ring’s two hydroxyls by Y and Z. Panel B illustrates the consequences of the absence of the side chain 25-hydroxyl group on the poor ability of 1α (OH)D3’s 1α- and 3β-hydoxyls to be captured by the VDR. This is reflected by the fact that 1α (OH)D3 binds to the VDR only 0.15% as well as 1α,25(OH)2D3 (100%; see Table 1 and its legend). Thus the following calculation can be made: [RCI for 1α,25(OH)2D3] / [RCI for 1α (OH)D3] = [100] / [0.15] = 666-fold difference in relative binding in favor of 1α,25(OH)2D3.
