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. 2023 Jul 28;86(7):1862-1869.
doi: 10.1021/acs.jnatprod.3c00421. Epub 2023 Jul 11.

Unmasking the True Identity of Rapamycin's Minor Conformer

Emily B Crull  1 , Ajay N Jain  2 , Paul C D Hawkins  3 , Ann E Cleves  2 , Edmund I Graziani  4 , R Thomas Williamson  1
Affiliations

Unmasking the True Identity of Rapamycin's Minor Conformer

Emily B Crull et al. J Nat Prod. .

Abstract

Rapamycin, a well-known macrocyclic natural product with myriad biological activities, has been the subject of intense study since its first isolation and characterization over five decades ago. Rapamycin has been found to adopt a single conformation in the solid state (both when protein bound and uncomplexed) and exists as a mixture of two conformations in solution. Early work established that the major conformer in solution is the trans amide isomer but left the minor conformer mostly uncharacterized. Since that time, it has been widely accepted that the minor conformer of rapamycin is the cis amide, based solely on analogy to FK-506, another potent immunosuppressive compound with some shared key structural elements. To address this long-standing and unresolved question, the solution structure of the minor conformer of rapamycin was investigated using a combination of NMR techniques and computational methods and determined to be a trans amide species with rotation about the ester linkage.

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Conflict of interest statement

The authors declare the following competing financial interest(s): This work was financially supported in part by Apertor Pharmaceuticals Inc.

Figures

Figure 1
Figure 1
Structures of rapamycin (1) and FK-506 (2) with the FKBP binding region of rapamycin highlighted.
Figure 2
Figure 2
Overlaid crystal structures of rapamycin from the CSD (RAPMCN11, purple) and in a binary complex with FKBP-12 (5HKG, gold) with insets showing the trans orientation of the amide bond within the FKBP-binding region (left) and the pseudocyclohexyl conformation of C25–C29 (right)., Amide torsion: RAPMCN11 = −167.6°, 5HKG = 177.7°. Heavy-atom RMSD = 0.67 Å.
Figure 3
Figure 3
Chair cyclohexane geometry in the C24–C30 region of rapamycin (PDB code 5HKG, gold) compared to a 2-hydroxycyclohexyl group from a diterpene structure from the CSD (CSD code ODICAJ, purple). RMSD for CC(O)CC(=O): 0.14 Å.
Figure 4
Figure 4
Overlaid crystal structures of the pipecolate–ketoamide regions of FK-506 from the CSD (FINWEE, purple, amide is cis) and in a binary complex with FKBP-12 (5HUA, green, amide is trans).
Figure 5
Figure 5
(R)-N-Boc-pipecolic acid (3).
Figure 6
Figure 6
15N HMBC spectra of (A) (R)-N-Boc-pipecolic acid (3), (B) FK-506 (2), and (C) rapamycin (1) in DMSO-d6.
Figure 7
Figure 7
Conformational ensembles derived from initial literature NMR restraints. (A) Crystal structure conformation of rapamycin bound to FKBP (green, PDB Code 5HKG) with the lowest energy major and minor conformers (cyan and magenta, respectively). (B) Low-energy major and minor conformer ensembles (cyan and magenta, respectively).
Figure 8
Figure 8
A series of HSQC spectra showing how some resonances, such as those for H2-3, H2-4, H2-5, and H2-6, barely migrated while others, like those for H2, H28, H27, H25, and H31, moved more drastically between the major (green) and the minor (purple) conformers.
Figure 9
Figure 9
Key ROESY correlations that support two conformational alternatives for the cyclohexyl side chain to the rest of the molecule for the major (green) and minor (purple) conformational forms.
Figure 10
Figure 10
Final conformational ensembles (with the FKBP-bound reference conformer shown in green). (A) Low-energy major conformer ensemble (orange). (B) Low-energy minor conformer ensembles (slate). (C) Comparison of the final minor conformer ensemble with the closest matching conformers from the initial minor ensemble (magenta).
Figure 11
Figure 11
Torsions exhibiting more than a 30° difference between the major and the minor conformation.
Figure 12
Figure 12
Distribution of differences in torsion angle between the major and the minor conformations of rapamycin (1). A similar chart for FK-506 (2) can be found in the Supporting Information.
Figure 13
Figure 13
Potential energy surface plot of the amide and ester torsions for (A) FK-506 (2) and (B) rapamycin (1).
See this image and copyright information in PMC

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