Darcy C. Burns

Assignment of 1H and 13C spectra and investigation of hindered side‐chain rotation in lupeol derivatives

Complete 1H and 13C spectral assignments are reported for lupeol (1a) and two derivatives where the C‐30 methyl group is replaced by CH2OH (1b) and HC O (1c). Compound 1c shows conformationally dependent substituent effects on 1H chemical shifts. It also shows line broadening of some 13C signals at 25 °C, suggesting hindered rotation of the side‐chain group. This is confirmed by low‐temperature spectra which show splitting of broadened peaks into pairs in a ca 2 : 1 area ratio. The free energy of activation of hindered rotation is estimated as 13.5 kcal mol−1. By contrast, 1a shows no evidence of hindered rotation down to −40 °C although NOE data suggest the presence of two conformers. Spartan molecular mechanics calculations confirm the presence of two stable conformers for 1a and 1c but overestimate the rotational barrier in 1a. The additional barrier in 1c probably reflects loss of conjugative stabilization during rotation. Copyright

Bending Rigid Molecular Rods: Formation of Oligoproline Macrocycles

Bent but not broken: cyclic oligoprolines are accessed in a reaction that effectively bends rigid oligoproline peptides (see scheme; TBDMS=tert-butyldimethylsilyl). The stitching is accomplished during macrocyclization enabled by aziridine aldehydes and isocyanides. Molecular modeling studies suggest that electrostatic attraction between the termini of the linear peptide is pivotal for macrocyclization. The macrocycles were studied by circular dichroism with a polyproline II structure being observed in larger macrocycles.

Getting the Most Out of HSQC and HMBC Spectra

Abstract As the title suggests, this chapter focuses on HSQC and HMBC spectra, two critically important spectra for organic structure determination by two-dimensional NMR. The relative merits of the HSQC sequence and the alternative HMQC sequence are discussed, and the performances of different versions of the HSQC sequence are evaluated, using three test compounds. It is shown that recent modifications of the HSQC sequence have eliminated or minimized earlier weaknesses of this sequence, leading to a more than twofold increase in signal/noise, particularly for edited HSQC spectra. In the case of HMBC, the impact of recent modifications of the sequence is much less than for HSQC. However, the choice of acquisition and processing parameters can have a dramatic effect on the signal/noise of HMBC spectra. A few of the numerous variants of HMBC that have been proposed to compensate for weaknesses of this sequence are considered. While they potentially yield more information, this is at the expense of signal/noise.

Optical Control of Protein–Protein Interactions via Blue Light-Induced Domain Swapping

The design of new optogenetic tools for controlling protein function would be facilitated by the development of protein scaffolds that undergo large, well-defined structural changes upon exposure to light. Domain swapping, a process in which a structural element of a monomeric protein is replaced by the same element of another copy of the same protein, leads to a well-defined change in protein structure. We observe domain swapping in a variant of the blue light photoreceptor photoactive yellow protein in which a surface loop is replaced by a well-characterized protein–protein interaction motif, the E-helix. In the domain-swapped dimer, the E-helix sequence specifically binds a partner K-helix sequence, whereas in the monomeric form of the protein, the E-helix sequence is unable to fold into a binding-competent conformation and no interaction with the K-helix is seen. Blue light irradiation decreases the extent of domain swapping (from Kd = 10 μM to Kd = 300 μM) and dramatically enhances the rate, from weeks to <1 min. Blue light-induced domain swapping thus provides a novel mechanism for controlling of protein–protein interactions in which light alters both the stability and the kinetic accessibility of binding-competent states.

A Circularly Permuted Photoactive Yellow Protein as a Scaffold for Photoswitch Design

Upon blue light irradiation, photoactive yellow protein (PYP) undergoes a conformational change that involves large movements at the N-terminus of the protein. We reasoned that this conformational change might be used to control other protein or peptide sequences if these were introduced as linkers connecting the N- and C-termini of PYP in a circular permutant. For such a design strategy to succeed, the circularly permuted PYP (cPYP) would have to fold normally and undergo a photocycle similar to that of the wild-type protein. We created a test cPYP by connecting the N- and C-termini of wild-type PYP (wtPYP) with a GGSGGSGG linker polypeptide and introducing new N- and C-termini at G115 and S114, respectively. Biophysical analysis indicated that this cPYP adopts a dark-state conformation much like wtPYP and undergoes wtPYP-like photoisomerization driven by blue light. However, thermal recovery of dark-state cPYP is ∼10-fold faster than that of wtPYP, so that very bright light is required to significantly populate the light state. Targeted mutations at M121E (M100 in wtPYP numbering) were found to enhance the light sensitivity substantially by lengthening the lifetime of the light state to ∼10 min. Nuclear magnetic resonance (NMR), circular dichroism, and UV-vis analysis indicated that the M121E-cPYP mutant also adopts a dark-state structure like that of wtPYP, although protonated and deprotonated forms of the chromophore coexist, giving rise to a shoulder near 380 nm in the UV-vis absorption spectrum. Fluorine NMR studies with fluorotryptophan-labeled M121E-cPYP show that blue light drives large changes in conformational dynamics and leads to solvent exposure of Trp7 (Trp119 in wtPYP numbering), consistent with substantial rearrangement of the N-terminal cap structure. M121E-cPYP thus provides a scaffold that may allow a wider range of photoswitchable protein designs via replacement of the linker polypeptide with a target protein or peptide sequence.

CRAPT: an improved version of APT with compensation for variations in JCH

A modified version of the attached proton test (APT) sequence for 13C spectral editing, which we call CRisis‐APT (CRAPT), is developed and tested on representative organic compounds. CRAPT incorporates 13C compensation for refocusing inefficiency with synchronized inversion sweeps (CRISIS) pulses in combination with 1H broadband inversion pulses to give improved compensation for variations in 1JCH along with improved refocusing efficiency. It is shown that CRAPT gives edited 13C spectra with only small losses in sensitivity (between 8% and 15% for strychnine, 1, menthol, 2, cholecalciferol, 3, and isotachysterol, 4), compared with basic 13C spectra obtained on the same compounds. CRAPT also gives significantly better signal/noise than DEPTQ for nonprotonated carbons. Therefore, we conclude that CRAPT is an improvement over APT or DEPTQ or a combination of DEPT135 with a full 13C spectrum for routine 13C spectral editing of organic compounds. Copyright

1H and 13C NMR spectra of (2′S,4′S)-lysergic acid 2,4-dimethylazetidide (LSZ)

First reported (online early view): 6 Jun 2016 in: S.D. Brandt, P.V. Kavanagh, F. Westphal, S.P. Elliott, J. Wallach, T. Colestock, T.E. Burrow, S.J. Chapman, A. Stratford, D.E. Nichols, A.L. Halberstadt. Return of the lysergamides. Part II: Analytical and behavioural characterization of N6-allyl-6-norlysergic acid diethylamide (AL-LAD) and (2′S,4′S)-lysergic acid 2,4-dimethylazetidide (LSZ). Drug Test. Analysis 2017, 9 (1), 38–50 https://doi.org/10.1002/dta.1985