The pyrrolidine ring structure is prevalent in numerous natural alkaloids and is an important feature of the proline and hydroxyproline residues that pervade biochemistry in peptides and proteins. The chemical and biological properties of substituted pyrrolidine derivatives, along with many other compounds, hinge on the relative stereochemistry. It is well established that the presence of fluorine in an organic molecule can significantly influence the stereochemical behavior. Consequently, various molecular properties, such as polarity, viscosity, and intra- and intermolecular interactions, are impacted by the C‒F bond. These features underlie the important role of selective fluorination in pharmaceuticals and agrochemicals development .

For instance, substituted 3-fluoropyrrolidines, particularly in the form of 2-carboxy derivatives (fluoroprolines), have been extensively explored. These compounds represent valuable nonnatural amino acids, and depending on the regio- and stereochemistry of fluorine substitution, fluoroproline substututions can enhance the conformational stability of proline-rich proteins such as collagen . Therefore, pyrrolidine derivatives are particularly susceptible to conformational control induced by a fluorine substituent.

Figure 1: a) Pseudoequatorial and pseudoaxial conformations of pyrrolidine. b) Cis- and trans-isomers of 3-fluoropyrrolidine.

Protonation of 3-fluoropyrrolidine generates the 3-fluoropyrrolidinium cation, and this typically results in a highly favored conformation in both the gas phase and solution where the fluorine and nitrogen atoms are cis, mirroring the behavior observed in analagous 4- and 6-membered ring systems . This conformational preference is attributed to an electrostatic gauche effect, where an attractive NH2+∙∙∙Fδ− interaction reinforces the well-known hyperconjugative gauche effect. Additionally, NH∙∙∙F hydrogen bonding has been proposed to play a role in stabilizing conformers of certain 3-fluoroalkylamines and the respective cations .

Intramolecular hydrogen bonds involving either the carboxy or hydroxy group of 4R- and 4S-hydroxyproline have been identified as key factors in stabilizing the favored conformations in the gas phase. Therein, the contribution of a gauche effect due to electron delocalization is considered to be secondary . However, stabilization via intramolecular hydrogen bonding does not seem to significantly impact the conformational stability of 3-fluoropiperidine. In this context, the cis-conformer, with the axial fluorine atom facing the N-hydrogen atom, is either equally or only slightly more stable than the other three conformers in both the gas phase and implicit water . Hence, it appears that strong intramolecular interactions, and not only hydrogen bonding, govern the orientation of the fluorine atom in the F‒C‒C‒N fragment, favoring the cis-isomer of 3-fluoropyrrolidine.

Figure 2: Flat representations of 2,3-, 3,4-, and 2,4-difluoropyrrolidines. The potential effects resulting from the addition of a second 1,2- or 1,3-fluorine atom (nN→σ*CF anomeric and σCH→σ*CF fluorine gauche effects) on the conformation of different stereoisomers is explored.

Achieving conformational control upon the introduction of a second fluorine atom in fluoropyrrolidine presents challenges. Unlike the chair-like conformation of six-membered rings, five-membered rings lack the geometric arrangement necessary to most effectively accommodate anomeric and gauche effects . It is noteworthy that vicinal difluorination has previously been demonstrated to minimize conformational bias in fluoroprolines , offering some insight into the potential outcomes of this study.

In this study, initially, the conformational equilibrium of 3-fluoropyrrolidine and the corresponding cation were analyzed to establish a benchmark for selecting an appropriate theory level for subsequent calculations. Then, the different isomers of the 1,2- or 1,3-difluorinated pyrrolidines were each be subjected to quantum-chemical analysis.

In order to achieve the most accurate conformational depiction of the difluorinated pyrrolidines through density functional theory (DFT), a benchmark study was conducted. This study compares the crystallographic geometry of a pyrrolidinium salt with the CCSD/DGTZVP geometry of the pyrrolidinium cation (with an exclusively axial C–F bond) and the energy of 3-fluoropyrrolidine conformations. Various combinations of DFT methods (B3LYP-D3BJ, ωB97XD, and PBEPBE) and basis sets (DGTZVP and 6-311++G**) were evaluated. The selection of these functionals was based on whether or not they included dispersion terms that could influence the absolute energy. These functionals are widely used and have demonstrated strong performance in numerous evaluations and validations across the literature. Similarly, for the basis sets, a comparison was made between a triple-zeta valence plus polarization basis set with diffuse and tight d-functions (DGTZVP) and a Pople-based large basis set with polarizability and diffuse functions, often referred to as high standard.

Figure 3: MAE comparing the geometry parameters (bond length, bond angle, and dihedral angle) obtained from DFT calculations at the CCSD/DGTZVP level. The MAE for the crystal structure compared to the CCSD structure is 20.3316. “a” denotes DGTZVP and “b” denotes 6-311++G**.

Table 1: Conformational energy (ΔG0, kcal⋅mol−1) and population (%) of 3-fluoropyrrolidine according to different theoretical levels for the gas phase and implicit DMSO (in parentheses), along with the MAE compared to the CCSD/DGTZVP level.

aa = DGTZVP. bb = 6-311++G**.

Figure 4: Exhaustive illustration of all conformational, configurational, and constitutional isomers of difluorinated pyrrolidine, illustrating the relative orientation of the C–F and N–H bonds used to build the input geometry for the calculations. The pairs 10/11 and 14/15 were degenerate. Consequently, only one structure of each pair was computed.

From the pool of 24 potential conformational, configurational, and constitutional isomers of the difluorinated pyrrolidines (i.e., 1–24), two pairs were degenerate (i.e., 10/11 and 14/15), reducing the number of distinct structures to 22. However, due to the considerable flexibility of the five-membered ring and the low barrier for pyramidal interconversion of the N–H moiety, some inputs converged to the same isomer post-geometry optimization. Consequently, a total of 10 different structures was identified in the gas phase, with two additional structures observed in implicit DMSO (using solvation model density, SMD), bringing the total to 12.

Figure 5: Stable difluorinated pyrrolidines derived from gas-phase calculations performed at the B3LYP-D3BJ/6-311++G** level. Conformers: 1/4, 2/3, 9/12, and 18/19. Stereoisomers: 1 /4, 9/12, and 17/19.

Table 2: Relative Gibbs free energy (kcal⋅mol−1) for the geometry-optimized difluorinated pyrrolidines.

Table 3: Relative electronic energy (ΔEfull = ΔELewis + ΔEnon-Lewis) in isodesmic relationships and significant electron delocalization derived from NBO calculations (kcal⋅mol−1) for the difluorinated pyrrolidines in the gas phase.

aSum of antiperiplanar σCH→σ*CF interactions.

Figure 6: σCH→σ*CF fluorine gauche interaction, which also occurred in 19, and anomeric interaction in isomer 19. The nN→σ*CF electron delocalization stabilized 19 by 22.43 kcal⋅mol−1.

Quantum-chemical calculations were conducted at the B3LYP-GD3BJ/6-311++G(d,p) level to assess isodesmic relationships for the most stable conformations of all isomers of 1,2- and 1,3- difluorinated pyrrolidines. This level of theory was chosen for its superior performance compared to other methodologies, as demonstrated through comparisons of the method with structural data on the 3-fluoropyrrolidinium cation and 3-fluoropyrrolidine. The conformational space of 2,3-, 2,4-, and 3,4-difluoropyrrolidines is notably dictated, both in the gas phase and implicit polar solution, by the N to C–F bond anomeric effect. Additionally, albeit less significant, electron delocalization from the C–H bonding orbital to the C–F antibonding orbital plays a crucial role in lowering the energy of isomers 17 and 19 relative to 1, 2, and 3. These insights deepen our understanding of the energetic principles governing molecular structures and provide valuable guidance in designing selectively fluorinated stereoisomers to influence preferred conformations when designing functional molecules.

The benchmark study was conducted for 3-fluoropyrrolidine and 3-fluoropyrrolidinium cation to determine the optimal theoretical level for further investigations involving 2,3-, 2,4-, and 3,4-difluoropyrrolidines, compared to the CCSD/DGTZVP level . The evaluated levels encompassed the B3LYP-GD3BJ , ωB97XD , and PBEPBE functionals, along with the 6-311++G(d,p) and DGTZVP basis sets. Following the identification of B3LYP-GD3BJ/6-311++G(d,p) as the most reliable method, it was employed to optimize the geometry and compute frequencies (to derive Gibbs free energy) for the difluorinated pyrrolidines. These computations were conducted in both the gas phase and employing an implicit DMSO solvent using SMD . Subsequently, a NBO analysis was performed for the gas-phase-optimized geometry at the same level of theory, utilizing the DEL (NOSTAR) keyword to discern Lewis- and non-Lewis-type contributions to the total electronic energy, alongside individual electronic delocalization interactions. The calculations were all performed using the Gaussian 16 package of software .

The authors are grateful to Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, funding code 001), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 306830/2021-3), and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) for the financial support of this research.

Ana Flávia Candida Silva: formal analysis; investigation. Francisco A. Martins: formal analysis; investigation; supervision; writing – review & editing. Matheus P. Freitas: conceptualization; formal analysis; supervision; writing – original draft; writing – review & editing.

All data that supports the findings of this study is available in the published article and/or the supporting information to this article.