Protein structure determination is largely reliant on crystallography (X-ray, neutron or electron), electron cryo-microscopy (Cryo-EM) or NMR experiments. Refinement is the final step in obtaining accurate three-dimensional atomic model based on experimental data. Since the quality of the data (e.g., resolution) is rarely sufficient to utilize these data alone, this step has traditionally relied on parameterized libraries that describe stereochemistry of the molecules in question. The libraries used in major refinement packages do not describe unusual local arrangements of protein residues in Ramachandran space, novel ligands, or non-covalent interactions such as π stacking, halogen, hydrogen or salt bridges. The methods we are developing in the Q | R project [1-4], which is our next generation open-source software package (http://github.com/qrefine), combine experimental data with chemical restraints derived from quantum-chemical methods. These procedures allow at present quantum refinement of proteins based on both X-ray crystallography or Cryo-EM experiments. Quantum refinement has shown to significantly improve model geometry, considering both the overall aspects of model and model-to-data fit statistics, as well as specific detailed structural features, in particular the hydrogen bonding. [1] M. Zheng, J. R. Reimers, M. P. Waller, P. V. Afonine, Acta Cryst. D 73, 45 (2017) [2] M. Zheng, N. W. Moriarty, Y. Xu, J. R. Reimers, P. V. Afonine, M. P. Waller, Acta Cryst. D 73, 1020 (2017) [3] M. Zheng, M. Biczysko, Y. Xu, N. W. Moriarty, H. Kruse, A. Urzhumtsev, M. P. Waller, P. V. Afonine, Acta Cryst. D 76, 41-50 (2020) [4] L. Wang, H. Kruse, O. V. Sobolev, N. W. Moriarty, M. P. Waller, P. V. Afonine, M. Biczysko, Acta Cryst. D 76, 1184–1191 (2020)

In this work, we present a comprehensive structural study of cytisine, a potent nicotinic agonist, for which we aim to clarify its bioactivity using high-resolution rotational spectroscopy. In a first step, we used our chirped-pulse spectrometer to characterize two different conformers presenting axial and equatorial arrangements of the piperidine NH group. In sight of the crucial role of the environment of the heteroatoms in cytisine molecule for docking the nicotinic receptor, we used a cavity-based technique to resolve the ¹⁴N quadrupole hyperfine structure in a second step. It has allowed us to obtain a detailed structural description of the molecule, clarifying the disposition of the piperidine NH group and further revealing an exotic intramolecular NH···N interaction. This intramolecularity justifies the over-stabilization of the axial conformer over the equatorial form and demonstrates the positive action of this alkaloid on the nicotinic receptor.

In ferrocene (Fe(C₅H₅)₂), the first discovered metallocene, iron is sandwiched between two cyclopentadienyl rings in an eclipsed configuration. Ferrocene is an orange solid that sublimates easily and is stable at high temperatures. Due to its symmetry (D_5h) this compound does not have a dipole moment, so it is not active in the microwave region and consequently, its gas phase structure is not accessible through microwave spectroscopy. However, as it has been shown for triacetone triperoxide [1], its complexation with water makes it possible. In this work, we have done a combined theoretical and experimental work to observe and analyze the microwave spectrum of ferrocene – H₂O. The theoretical computations predict two possible low-energy structures of the complex. In one form, water lies in the σ_h plane of ferrocene. In the other form, water is close to the C₅ axis of ferrocene on top of one of the cyclopentadienyl rings. Both forms have been observed. The most intense spectrum is that of a symmetric top with satellite patterns consistent with the effects of the free rotation of water. The rotational constant B determined for this spectrum is close to that predicted for the second axial form so that we can conclude that water is located along the C₅ axis and freely rotating around it. This motion averages the ferrocene – H₂O structure to that of a symmetric top. Different isotopic species have been detected, including ⁵⁴Fe and ¹³C in their natural abundances, which have made it possible to determine the structure of the heavy atom skeleton of ferrocene and the axial location of water. A second weaker rotamer with an asymmetric top spectrum has rotational constants very close to those predicted for the other ferrocene-water conformer. Experimental and theoretical work is still in progress. 1. Blanco, S.; Macario, A.; Garcia-Calvo, J.; Revilla-Cuesta, A.; Torroba, T.; Lopez, J.C.; Microwave Detection of Wet Triacetone Triperoxide (TATP: Non-Covalent Forces and Water Dynamics. Chem. Eur. J. 2021, 27, 1680–1687.

Solid samples of progesterone (m.p. 126^°C), one of the essential hormones, have been vaporized by laser ablation and probed in a supersonic expansion using a broadband Fourier transform microwave spectroscopy. The analysis of around 150 rotational transitions revealed the existence of a single conformation. Like for the related testosteroneI. León, E. R. Alonso, S. Mata, and J. L. Alonso, J. Phys. Chem. Lett. 2021, 12, 6983-6987. progesterone adopts an extended configuration which is the most stable form predicted by quantum-chemical calculations. Due to the methyl group internal rotation, A-E splittings have been observed and allowed for the precise determination of the barrier height. The considerable molecular size of progesterone, one of the largest ever attempted solid, illustrates the potential of the LA-CP-FTMWE. R. Alonso, I. León, J. L. Alonso, Intra- and Intermolecular Interactions between non-covalently Bonded Species. Elsevier, 2021, 93-141.echnique in structural chemistry.
Acknowledgments: The authors thank the financial fundings from Ministerio de Ciencia e Innovacion ( PID2019-111396GB-I00) and Junta de Castilla y León (VA077U16 and VA244P20).

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Footnotes:

I. León, E. R. Alonso, S. Mata, and J. L. Alonso, J. Phys. Chem. Lett. 2021, 12, 6983-6987., E. R. Alonso, I. León, J. L. Alonso, Intra- and Intermolecular Interactions between non-covalently Bonded Species. Elsevier, 2021, 93-141.t

Allose, an aldohexose sugar, is a rare monosaccharide. It differs from the archetypal glucose in the hydroxyl group at the C3 position. However, this slight variation seems to be decisive in its natural abundance, as well as its biological role. Because of the structure-property relationship and to shed light on the effects of epimerization, we have brought β-D-allose into the gas phase using laser ablation techniques, and its conformational panorama has been characterized using chirped-pulse Fourier transform microwave (LA-CP-FTMW) spectroscopy. Three conformers have been unequivocally identified based on the spectroscopic rotational parameters. All the detected conformers exhibit a counter-clockwise arrangement (cc) network formed by an intramolecular hydrogen bond similar to what is observed in β-D-glucose. In opposition, we found that the intramolecular hydrogen bonds in β-D-allose are stronger than in β-D-glucose, which could have drastic biological implications.

The rotational spectra of over 30 isotopologues of chlorobenzene (C₆H₅Cl, C_2v) have been collected over portions of the 2 – 360 GHz frequency region. The transitions of these isotopologues were least-squares fit to complete sextic Hamiltonians with the support of computationally predicted spectroscopic constants. The resultant rotational constants of all available isotopologues, alongside high-level computational corrections for vibration-rotation interaction and electron-mass distribution, were used to determine a highly precise semi-experimental equilibrium (r_e^\textSE) structure of chlorobenzene. Finally, advanced quantum mechanical calculations were performed at the CCSD(T)/cc-pCV5Z level to compare to the experimental results. Analysis of the chlorobenzene r_e^\textSE structure will provide insight into the limitations of molecular structure determination when some atoms lie close to (or directly on) principal axes, a difficulty observed in previous molecular structure determinations.

Molecular structures determine spectroscopic parameters that allow molecular identification and reveal qualitative information about bonding and energetics. Over the years, a number of distinct operational definitions of molecular structure (bond lengths and bond angles) have emerged and some confusion often exists when theoreticians and experimentalists debate “a bond length”. Here I briefly review the practical value of accurate molecular structures and survey existing experimental and theoretical methods for determining them. I place particular emphasis on the Kraitchman r_s substitution structure, the computationally obtained r_e structure and the semi-experimental r_e^SE structure. Ultimately, the most satisfactory method for determining very high-accuracy structures today is a mixed experimental-theoretical approach that uses data from microwave spectroscopy and quantum-chemical calculations. After discussing that, I talk in detail about a different approach to obtaining Kraitchman substitution structures that we have recently employed.

In this reflective presentation, we will discuss a fruitful, decade long collaboration between the speaker and Prof. Gamil Guirgis at the College of CharlestonN. A. Seifert; G. A. Guirgis; B. H. Pate, J. Mol. Struct 2012, 1023, 222.nd Gamil's now distinct contributions to organosilicon chemistry. In particular, we will focus on Gamil’s facilitation of undergraduate students and young scientists in their study of the chemical and spectroscopic properties of these molecules, as well as his use of microwave and infrared spectroscopy (and spectroscopists!) as essential methods for chemical analysis. Our discussion will primarily focus on recent microwave studies, such as silylcyclohex-2-ene, 1,1-difluorosilylcyclohex-2-ene and cyclopentylsilane, whose spectra were recently acquired in the Grubbs lab at Missouri S&T. However, given the timely nature of celebrating a decade of collaboration between Gamil and the speaker, we will highlight the history and past results of Gamil’s collaborations with microwave spectroscopists in the past decade, which stretches across multiple laboratories and research groups, many of which have been featured at ISMS in past years.Examples include: T. M. C. McFadden; N. Moon; F. E. Marshall, et al., Phys. Chem. Chem. Phys 2022, 24, 2454; G. A. Guirgis; J. S. Overby; M. H. Palmer, et al., J. Phys. Chem. A 2012, 116, 7822; G. A. Guirgis; R. E. Sonstrom; A. J. Clark, et al., J. Phys. Chem. A 2019, 123, 4389.inally, we use this story as motivation to discuss the collaborative interface between the “spectroscopist” with the “chemist”. As microwave spectroscopy continues to climb up the formidable but traversable mountain towards mainstream chemical applicability, Gamil’s work with microwave spectroscopists offers a unique and compelling example of how microwave spectroscopy and spectroscopists can provide (and have provided) essential services for those interested in chemical synthesis.

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Footnotes:

N. A. Seifert; G. A. Guirgis; B. H. Pate, J. Mol. Struct 2012, 1023, 222.a Examples include: T. M. C. McFadden; N. Moon; F. E. Marshall, et al., Phys. Chem. Chem. Phys 2022, 24, 2454; G. A. Guirgis; J. S. Overby; M. H. Palmer, et al., J. Phys. Chem. A 2012, 116, 7822; G. A. Guirgis; R. E. Sonstrom; A. J. Clark, et al., J. Phys. Chem. A 2019, 123, 4389.F

There has been continued interest in the structure of substituted cyclobutanes. In this work, we measured the rotational spectrum of cyclobutanecarboxylicacid (CBCA) for the first time using a chirp-pulse and a cavity-based Fourier transform microwave spectrometers. To aid in our analysis of the spectrum, we performed potential energy surface scans at B3LYP/ aug-cc-pVTZ level in the ring–COOH dihedral angle of both equatorially- and axially-substituted CBCA. These scans revealed a unique local minimum and a shallow, symmetrical double-well at the global minimum, implying the existence of four distinct stable (yet facilely interconvertible) conformers. We re-optimized these conformers using both density functional theory and second-order Møller–Plesset perturbation theory with the aug-cc-pVTZ basis set and computed their relative energies, dipole moments, and rotational constants. We will present and discuss the corresponding assignments of features in the rotational spectrum.

6-Aminopenicillanic acid (6-APA) is one of the essential intermediates in synthesizing semisynthetic and naturally occurring penicillins. We have transferred 6-APA into the gas phase using laser ablation techniques and characterized its conformational panorama using chirped-pulse Fourier transform microwave (LA-CP-FTMW) spectroscopy. The spectroscopic parameters derived from the spectrum analysis conclusively identify the existence of four conformers of 6-APA. The 14N nuclear quadrupole coupling constants have been analyzed, allowing an accurate structural determination. The observed structures correlate nicely with the biological function of 6-APA.