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# The Non-covalent Stereocontrol of Disulfide bonds

Laurent Vial, Julien Leclaire, Elise Dumont, Florent Perret, Adrien Forot, Titouan Chetot

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Laurent Vial, Julien Leclaire, Elise Dumont, Florent Perret, Adrien Forot, et al. The Non-covalent Stereocontrol of Disulfide bonds. 2025. hal-05393306

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# The Non-covalent Stereocontrol of Disulfide bonds

Titouan Chetot,‡ Adrien Forot,‡,† Elise Dumont,|| Florent Perret,‡,† Laurent Vial,*‡,† and Julien Le-claire*‡,†

‡CNRS, Univ. Claude Bernard Lyon 1, ICBMS, UMR5246, F-69622 Villeurbanne, France

†CNRS, ENS de Lyon, Laboratoire de Chimie, UMR 5182, F-69342 Lyon, France

||CNRS, Univ. Côte d'Azur, Institut de Chimie de Nice, UMR7272, F-06108 Nice, France

**ABSTRACT:** Disulfide bonds are pivotal in shaping the structure and function of both natural proteins and synthetic architectures, yet the stereochemical control of their axial chirality has only very recently garnered focused attention. In this perspective article, we have (re)-examined the published structures of natural and synthetic disulfide-containing compounds to uncover the non-covalent interactions that can bias the chirality of the S-S bond toward specific *P* or *M* conformations. Building on these insights, we provide here a conceptual toolbox that could inspire various communities, from molecular biologists exploring the role of disulfide bridges in transmitting stereochemical information within protein structures to organic chemists designing disulfide-based architectures of increased complexity.

## INTRODUCTION

Disulfide bonds play a critical role in biology, as they contribute to fold almost all proteins into well-defined tertiary structures and thereby predetermine their function.¹ Within active sites, they also act as redox mediators during many catalytic processes.² Beyond the biological realm, chemists have extensively exploited the disulfide bond and its dynamic nature across various fields, including systems chemistry (e.g., in dynamic signaling cascades),³ material chemistry (e.g., in self-healing polymers),⁴ and supramolecular chemistry (e.g., in interlocked molecular architectures).⁵ Remarkably, the disulfide bond is a chiral motif, and its handedness may play a non-innocent role in the architectures in which it is embedded. For instance, it was suggested that disulfide bonds in prion proteins may transmit stereochemical information that can influence the manner of folding or refolding into pathogenic forms.⁶ There is therefore a strong incentive to control the stereochemistry of disulfide bonds in both biological and synthetic contexts. Surprisingly, it wasn't until 2022 that supramolecular chemistry began addressing this challenge, showing that intra- and intermolecular non-covalent interactions exerted by chiral neighboring units can affect the stereochemistry of disulfide bonds and vice versa (vide infra).⁷,⁸,⁹

Herein, we decided to decipher the non-covalent interactions that control the stereochemistry of disulfide bonds by examining published structures of natural and synthetic disulfide containing compounds of increasing compositional and structural complexity. When necessary, additional calculations were performed to gain insight into the interactions at work. With this survey, we aim to provide the community with emerging principles on how to encode stereochemical information onto disulfide-containing architectures.

## CHIRALITY OF THE DISULFIDE BOND

**Steroelectronics and conformations.** From a simple bond length perspective, the disulfide linkage seems unlikely to behave as a stereogenic unit, i.e. to fold into stable asymmetric conformations. Within the series of gaseous compounds of generic formula $H_nX-XH_n$ (where X = C, N, O and S and n = 3, 2, 1 and 1 respectively), the central linkage in disulfane $H_2S_2$ is 1.4 times longer than in homologous compounds (Table 1). This should intuitively translate into a rather flat conformational landscape.¹⁰

<table><caption>**Table 1.** Bond length l of gaseous compounds of the $H_nX-XH_n$ generic formula.¹¹</caption><thead><tr><th>H<sub>n</sub>X-XH<sub>n</sub></th><th>C-C</th><th>N-N</th><th>O-O</th><th>S-S</th></tr></thead><tbody><tr><td>l (pm)</td><td>153.5</td><td>144.7</td><td>147.5</td><td>206.0</td></tr></tbody></table>

Regarding the most stable conformation, the central moiety is non-planar in hydrazines $R_2N_2$, peroxides $R_2O_2$ and disulfides $R_2S_2$, contrarily to what is observed in linear alkanes.¹² Yet, the VSEPR theory predicts that the most stable conformation for the R-S-S-R moiety should correspond to a dihedral angle $\chi_1$ of 180° (Figure 1a). (Figure 1a). However, most molecular systems analyzed experimentally and discussed here involve an angle whose value is close to 90°. Calculations confirmed that the bond order is, as expected, close to 1, but that it is almost entirely p in character.¹³ In fact, Pau-ling demonstrated 75 years ago that the two lone pairs borne by a sulfur atom within a disulfide bond are far from being equivalent. One corresponds to a filled 3p atomic orbital while the other is more like a 4s orbital.¹⁴
<figure><img src="image_3.png" /><figcaption>**Figure 1.** Lewis structures of a disulfide bond from (a) VSEPR theory for lone pairs of identical energy levels, (b) molecular orbital analysis involving s-rich and pure p lone pairs, (c) corresponding relative conformational energy diagram as a function of dihedral angle $\chi_1$ in disulfide and dioxide bonds (Gibbs free energies $\Delta_r G^\circ$ in kJ·mol$^{-1}$ for R=H given in brackets), and (d) assignment of *M* and *P* stereodescriptors to chirality axes in disulfide bridges (the green segment highlights the chirality axis). Non-bonding s orbitals were omitted for clarity.</figcaption></figure>

Minimal repulsion between the p-type doublets borne by adjacent sulfur atoms is attained when those orbitals are perpendicular (while repulsion between the 4s spherical orbitals is dihedral angle-independent and remains constant, Figure 1b). Therefore, the most stable geometries of R–S–S–R bridges are skew conformations with dihedral angles $\chi_1$ of + 90° and - 90°, while the *cis*- and *trans*- conformations correspond to local energy maxima (Figure 1c).¹⁵ These mirror conformers also benefits from optimal hyperconjugation between the filled 3p orbital of one sulfur atom and the antibonding $\sigma_{S-R}^*$ orbital of the nearest neighbor.¹⁶

**Assigning stereodescriptors to the disulfide bond.** IUPAC define axial chirality as “[resulting] from the non-planar arrangement of four groups in pairs about a chirality axis”, where a chirality axis is “an axis about which a set of ligands is held so that it results in a spatial arrangement which is not superposable on its mirror image”.¹⁷ According to this definition, disulfides bridges display chirality axes (Figure 1d). Various stereodescriptors exist in the literature to label chirality axes and assign the configuration of disulfide linkages, the most frequently used being *M* and *P*. The Cahn–Ingold–Prelog (CIP) nomenclature considers that the two sulfur atoms define the chirality axis, each of which is bound to groups R₁ and R₂, with R₁ > R₂. The line of sight is then directed along the direction of decreasing R₁ -> R₂ priority. In a Newman representation wherein R₁ is sitting on front top and R₂ positioned on the back right (respectively on the back left), the stereodescriptor assigned to the chirality axis is *P* (respectively *M*, see Figure 1d).

**Dynamics of conformer interconversion.** For both peroxides (R₂O₂) and disulfides (R₂S₂), the *cis* transition state (which involves additional steric repulsion between R groups) is consistently higher in energy than the *trans* transition state. Interconversion between stable mirror conformations therefore systematically goes through the *trans* transition state in linear disulfides (Figure 1c).¹⁸,¹⁹

This activation energy barrier $\Delta_r G^\circ_{trans}$ is nearly five times lower in H₂O₂ (Figure 1c) than in H₂S₂. This difference stems from the reduced lone pair repulsion in peroxides, wherein 2p orbitals of oxygen atoms are more compact than the diffuse 3p orbitals of sulfur atoms in disulfides.²⁰ As a result, the half-life (t₁/₂) at 298 K of each mirror conformer is about 1000 times longer for H₂S₂ (~10⁻¹⁰ s) than for H₂O₂ (~10⁻¹³ s).²¹ This half-life for linear disulfides remains far below the threshold value for atropisomerism, which typically corresponds to t₁/₂ > 1000 s and to energy barrier > 90 kJ·mol⁻¹.²² For comparison, the rotational barriers of ethane (*eclipsed*-staggered and hydrazine (*cis*-skew) are around 11.5 kJ·mol⁻¹ (*eclipsed*-staggered and *cis*-skew, respectively), corresponding to t₁/₂ at 298 K of ~10⁻¹¹ s.²³

Since the lowest-energy pathway corresponds to the *trans* transition state, steric effects of the substituents R have only a moderate impact on the inversion barrier of R₂S₂. Across a series ranging from H₂S₂ to trityl disulfide, the rotational barrier falls within the range of 20-40 kJ·mol⁻¹.²⁴ For the bulkiest species, although the low energy barrier to rotation does not allow the separation of optical an tipodes, their presence can be inferred from low tempera ture NMR data.

## FROM CONFORMERS TO STATIC STEREOISOMERS

As bulkiness of α-substituents is relatively ineffective to increase the energy level of the *trans* transition state, two design options can be envisaged to slow down the interconversion of mirror-image disulfide conformers (Figure 2a). The first consists in forcing this racemization process to occur through the *cis* transition state by integrating the S–S bridge into a ring, wherein *trans* transition state is structurally inaccessible and energetically prohibitive (Figure 2b). To accommodate the structural constraints of small cycles, the dihedral angle of the S–S bridge can be
dramatically reduced (i.e., dihedral angles $χ_1$ of 27° in 1,2-dithiolane and 60° in 1,2-dithiane compared to the typical values near 90° observed in seven- and eight-membered rings).²⁵ In addition, the cis transition state is energetically less favorable than in the linear parent compounds as it corresponds to a flattened portion of the cycle. As a result, inversion barriers in 1,2-dithianes climb up to ~50 kJ·mol⁻¹ (vs ~20 kJ·mol⁻¹ for *n*-propyl disulfide).²⁶ Further rigidification of the backbone can even successfully lead to stable enantiomers, with 5H,8H-dibenzo-[d,f][1,2]-dithiocin standing as the first example of disulfide-centered atropoisomerism.²⁷ On this compound, which was resolved by crystallization, a rotational barrier >100 kJ·mol⁻¹ was measured by dynamic NMR in solution.

<figure><img src="image_4.png" /><figcaption>**Figure 2.** Schematic conformational energy profiles plotted against dihedral angle $χ_1$ for disulfide bonds embedded within: (a) linear, and (b) cyclic frameworks.</figcaption></figure>

To increase the barrier between enantiomorphic disulfide bridges, the second design option consists in further stabilizing the pair of conformers through attractive non-covalent *intra*- or *intermolecular* interactions with neighboring groups. To the best of our knowledge, *intramolecular* interactions (from end groups to the central disulfide unit) alone never completely succeeded so far in freezing disulfide bridges within soluble linear species. As it will be exposed in the rest of this perspective, *intramolecular* interactions (such as hydrogen bond, π-π stacking, steric hindrance or hypervalent interaction) exerted by end groups on the central disulfide moiety had either to be combined with *intermolecular* bonding (mainly with nearest neighbors in the crystal lattice) or with conformational restriction. This situation corresponds to disulfide bridges embedded into a ring bearing substituents, and for which *trans* transition state remains prohibited as previously discussed (Figure 2b). In principle, enantiopure neighboring stereogenic units could be used to attractively or compulsively interact with the disulfide bridge. This could entioselectively lower the energy levels of the stable conformers and raise the energy of the transition states between them. In this specific case, not only is the racemization process slowed down but the equilibrium between the two disulfide enantiomorphs can also be significantly biased. The different flows of stereochemical information from neighboring asymmetric units, and their impact on the population of disulfide-based mirror image conformers will be discussed in this perspective. The four main situations covered by this perspective are conceptually

detailed in the schematic representation Figure 3. In theory, a single static neighboring stereogenic unit can act as an asymmetric sergeant and favor one preferred asymmetric conformation of the disulfide bridge solider (Figure 3a). In practise, it was mainly observed on disulfide containing five-membered rings with a stereogenic center in their vicinity. The second situation corresponds to two static asymmetric sergeants (generally identical units of the same configuration) which cooperatively (Figures 3b) amplify the same asymmetric conformation (or anticooperatively favor opposite conformations, Figure 3c). This is the most frequently reported situation, which corresponds to the crystalline structure of synthetic and biogenic linear dimers as well as disulfide containing rings bearing two stereogenic centers. Case 3d corresponds to disulfide self-assembled from thiol-bearing prochiral units, where both facial and axial chirality emerge during the same process in a concerted manner (and stereochemical information propagates reversibly, between simultaneously formed nearest neighboring stereogenic units).

<figure><img src="image_5.png" /><figcaption>**Figure 3.** Pathways of stereochemical information flow across disulfide-containing molecular frameworks: (a) unidirectional, (b) convergent matching or cooperative, (c) convergent mismatching or anti-cooperative (d) simultaneous and reversible (enabling error correction).</figcaption></figure>

## NEAREST-NEIGHBOUR ASSISTED STEREOCONTROL WITHIN DISULFIDE CONTAINING COMPOUNDS

From the extensive body of published structures of both natural and synthetic disulfide-containing compounds, spanning from small rings to complex biomacromolecules, we have selected only those for which clear evidence could be obtained that neighboring groups exert non-covalent control over the stereochemistry of the disulfide bonds. This feature was generally not examined in the original studies. The studies conducted by Feringa *et al.* and our group are exceptions, as they were specifically conducted to bring the proof of feasibility of such a control (*vide in-fra*).
<figure><img src="image_6.png" /><figcaption>**Figure 4.** (a) Computed diastereomers of (*R*,*R*)-cystine in neutral solution, and (b) pH-dependent protonation states of *L*-cystine, along with the corresponding X-ray crystallographic structures: fully protonated (*R*,*R*)-cystine (red box, CCDC deposit number: 744552), and zwitterionic (*R*,*R*)-cystine (blue box, CCDC deposit number: 1204441). Green dotted lines highlight hydrogen bonds.</figcaption></figure>

**Linear disulfides: from amino acids to proteins.** To determine whether non-covalent interactions exerted by neighboring stereogenic units can bias the proportions of disulfide enantiomorphs, we first examined the smallest motif found in proteins: the oxidized form of the *L*-cysteine (or (*R*,*R*)-cystine, Figure 4a). (*R*,*R*)-cystine contains three stereogenic units: the two asymmetric carbons, which are static stereocenters of the same configuration, surround the conformationally dynamic and chiral disulfide bridge. For decades, the unusually large specific rotation of (*R*,*R*)-cystine relative to other amino acids was ascribed to a contribution of the central bridge to the global optical rotation of the most stable and dominant conformers in solution.²⁸,²⁹ While the rotational barrier of the disulfide bridge of cystine is comparable to those of apolar linear species discussed above (25 kJ·mol⁻¹),³⁴ in-house DFT computational analyses indicate that in neutral solution, the *R*-(*P*-*R*) diastereoisomer is more stable by ~11 kJ·mol⁻¹ than the *R*-*R*-*M* species.³⁰ Thus, it is overwhelmingly predominant (> 99 %), although it remains conformationally labile. Both diastereoisomers form a salt-bridged ten-membered ring, but the former additionally features a favorable intramolecular N-H···S hydrogen bond (Figure 4a).³¹

Depending on the pH of the medium, each diastereoisomeric conformer can be crystallized : the *R*-**M**-*R* is obtained with bis-cationic (*R*,*R*)-cystine from acidic solutions,³² whereas the *R*-**P**-*R* conformer is obtained from neutral solution of zwitterionic (*R*,*R*)-cystine (Figure 4b).³³ At low pH, repulsion between the positively charged ammonium groups is minimized when the disulfide bridge adopts the *M* conformation. This geometry is further stabilized by the intramolecular N-H···S hydrogen bonding between the nitrogen of one Cys monomer and the sulfur atom of the other, resulting in the formation of six-membered rings. Eventually, the sequence of dihedral angles around the disulfide bridge adopts a left-handed helical conformation called +LHSpiral, a secondary structure found in only 1.3% of proteins (Figure 4b, red box).³⁴ In this system, the stereochemical information borne by the asymmetric center of each amino acid is indirectly transferred to the central disulfide bridge through the asymmetric positioning of the ammonium end groups through convergent and matching interactional flows (Figure 3b). This picture is far from anecdotal, as the same secondary

structure induced by the same intramolecular interactions is found in the X-ray structure of the dimethylated analogue, D-penicillamine disulfide (not shown, CCDC number 1230827).³⁵

In the zwitterionic form (neutral pH), the intermolecular C-O···H-N salt bridge become the dominant interaction (Figure 4a, blue box). This intermolecular contact competes is complemented with a secondary five-membered ring N-H···S interaction within the monomeric unit of each dimer. These intra- and intermolecular forces conformationally lock the strands of longitudinally stacked dimers into a right handed supramolecular helix, which exclusively involves all-positive dihedral angles (i.e., +RHSpiral with an incidence of 3.1% in proteins).³⁴ The interpretative picture entails the transmission of stereochemical information from the stereocenter of the amino acid (which positions the donor and acceptor of the salt bridge) into the central disulfide bridge, which thereby becomes the core component of an extended right-handed helix. Overall, the central disulfide bridge is the adjustment variable, which enables optimizing non-covalent interactions in the vicinity of the stereogenic centers (Figure 3b).

Except in specific microorganisms, proteins are isotactic, i.e. made of homochiral amino acids of *L* configuration.³⁶ It is thus legitimate to wonder whether this extended homochiral environment may play the role of an asymmetric lock, i.e. may induce an asymmetric folding of internal disulfide bridges acting as the flexible complementary key.³⁷ In other word, can the asymmetric exclusion volume of the protein tertiary structure have a similar folding effect as the ammonium groups borne by the neighboring chiral center had on the simple *L*-cystine dimer (vide supra). Hogg *et al*. conducted a statistical analysis to answer this question by computing the five dihedral angles (χ₁, χ₂, χ₃, χ₂' and χ₃') surrounding 6874 disulfide bridges of 2776 proteins whose structure was elucidated by X-ray crystallography (Figure 5).³⁴ Surprisingly, this survey delivered a quasi-even distribution of *M* (55.5%) and *P* (44.5%) configurations (corresponding to negative and positive χ₁ angles, respectively). Yet, it also revealed that specific sequences of dihedral angles along the bridges are preferred. Among the 20 possible combinations of positive and negative dihedral angle values, four represent 50.9% of all occurrences (Figure 5). Notably, the sequence consisting entirely of negative dihedral angles (corresponding
to a left-handed helix embedding a L-M-L cysteine moiety) accounts for half of this subset. A simplistic and empirical model, which only considers the amplitude of the dihedral angles (excluding others factors such as *inter-* or *intra*-molecular non-covalent interactions) reveals that this sequence exhibits the lowest dihedral strain energy.

<figure><img src="image_7.png" /><figcaption>**Figure 5.** Dihedral angles (χ) found in L-cystine residues (*left*), and selection of conformers with corresponding signs of dihedral angles, classification and incidence from 6874 disulfide bridges within the X-ray structures of 2776 proteins (*right*).</figcaption></figure>

Deciphering the combination of physical interactions that may promote specific asymmetric conformations around cystine disulfide bridges is the step further, beyond this simplistic model based on molecular mechanics. Protoxin-II, a protein from the tarantula toxin, features the minus left-handed spiral motif (i.e., -LHSpiral, the most prevalent disulfide bond conformation observed in protein structures, *vide supra*) around one of its three S-S bridge involving cysteines 15 and 25.³⁸ As shown in Figure 6a, these two residues are also engaged into a “β-Sheet-like” network of hydrogen bonds, which locks the most remote torsional angles χ₃ and χ₃' (Figure 5). Additionally, the disulfide bridge is buried within the asymmetric protein interior, which likely imposes significant constraints (i.e. acts as an asymmetric lock) on the conformational mobility of the remaining dihedral angles around the bridge (Figure 6b).

<figure><img src="image_8.png" /><figcaption>**Figure 6.** X-ray crystallographic structure of Protoxin-II (zoom) showing: (a) C=O···H-N hydrogen bonds (in green) stabilizing the left-handed helix conformation of a disulfide bridge, and (b) topological constraint brought by the amino acid residues in CPK model around the disulfide bridge in capped sticks preventing rotation around its dihedral angle χ₁ (PDB identifier: 500U).</figcaption></figure>

This specific example illustrates the difficulty faced when trying to decipher the interactions potentially at work to asymmetrical fold disulfide bridges in proteins. Due to the multitude and intricacy of these individual non-covalent interactions within each protein and their diversity from one protein to another, assessing their individual contribution and establishing general principles for stereocontrol remains a significant challenge, that may only be

undertaken by artificial intelligence.³⁹ This also raises the question of whether well-defined, abiotic model systems can provide broader and clearer insights into the phenomena that may govern the asymmetric induced fit of disulfide bonds.

The static stereogenic units, which may cooperatively induce the asymmetric folding of a neighboring disulfide bridge can not only be stereocenters but also supramolecular helices. Conceptually, Gan and co-workers replaced the R-cysteine-based units by two enantiopure et homochiral oligoamide foldamers separated by a central disulfide linkage.⁴⁰ The folding of each aromatic oligoamide is primarily driven by a combination of directional intramolecular hydrogen bonds between amide NH donors and adjacent carbonyl acceptors, as well as π-π stacking interactions between planar aromatic units, which together stabilize the helical conformation and enforce structural rigidity. Examination of the corresponding X-ray crystallographic structure revealed that the axial chirality of the disulfide bond consistently matches the helical screw sense of each foldamer backbone, with the A,A pair cooperatively instructing the bridge into a P configuration only (and the Δ, Δ into a M only, see Figure 7a). This stereochemical congruence indicates that the asymmetric folding of the disulfide bond results from a matching chirality transfer from both bulky helical end groups which schematically act as asymmetric confining units (Figure 3b). This principle can be extended beyond linear systems. In parent macrocyclic architectures, where two enantiopure and homochiral foldamers are joined via disulfide "stackles," a similar convergent stereochemical transfer is observed by Huc and co-workers (Figure 7b).⁴¹ The closed-loop topology imposes additional conformational restriction, resulting in a synchronized chirality between both disulfide bridges and the overall helical twist of the macrocycle. These observations collectively reinforce the view that disulfide bonds, when placed in structurally pre-organized asymmetric nano-environments such as helices (or protein tertiary structures), become stereochemically responsive and can be instructed and store the information transmitted.

<figure><img src="image_9.png" /><figcaption>**Figure 7.** Stereochemical match in disulfide-based foldamers: (a) linear polymer (CCDC deposit number: 2333999), and (b) cyclodimer (CCDC deposit number: 1449708).</figcaption></figure>

Except for cystine, whose high conformational lability prevents the observation of a single well-defined diastereoisomer, stereochemical information transfer from fixed stereocenters to a flexible disulfide bridge has so far been evidenced only on crystallographic structures. In the solid lattice, asymmetric intermolecular interactions with enantiopure nearest neighbors may participate in the
phenomenon, i.e. either synergistically favor the intramolecularly-selected enantiomorph or compete with the intramolecular interactions. With this issue in mind, we deliberately selected intramolecular interactions whose amplitude was so strong that their folding effect is likely to be preserved (albeit attenuated) in solution. This should particularly be the case for disulfide containing macromolecules such as proteins, helical foldamers and macrocycles.

**Cyclic disulfides: towards conformational freezing.** The simplest and most used non-natural chiral disulfide-containing compound is 1,4-dithiothreitol (DTT). More rarely called (2R,3R)-1,4-bis(sulfanyl)butane-2,3-diol, this compound is used to reduce disulfide bonds in proteins in order to denature them and improve their separation during electrophoresis.42 Structurally, the oxidized form (Ox-DDT) involves a pair of adjacent stereocenters of the same configuration and a disulfide linkage, separated by a methylene unit within a six-membered ring (Figure 8a). Formally, it is the cyclized equivalent of homochiral cystine, wherein stereocenters are covalently connected. The crystallographic structure of Ox-DDT only displays disulfide bridges of P conformation, i.e. corresponds to the enantiopure (R, R, P) diastereoisomer. Yet, no intra- or intermolecular non-covalent interactions between substituents borne by the stereocenters and the sulfur atoms can be detected in the solid state.43 The disulfide-containing six-member ring adopts a chair conformation as the parent compound (1R,2R)-trans-1,2-cyclohexanediol does. It is well known that this model diol favors the equatorial positioning of its hydroxy substituents to simultaneously minimize 1,3-diaxial steric repulsion and maximize the intramolecular bond between hydroxyl groups.44 On Ox-DTT, M → P inversion of the disulfide dihedral configuration is accompanied by axial → equatorial chair inversion. In-house density functional theory (DFT) calculations estimate the corresponding Gibbs free energy difference to $ΔG_{eq}^0 = -27.2$ kJ.mol$^{-1}$.30 This translates into a Boltzmann distribution $P/M \sim 10^5$ at 298 K. Assuming that the H-O···H bond energy $ΔG_{HB}^0 = -8.07$ kJ.mol$^{-1}$ is equal in both trans-cyclohexan-1,2-diol and ox-DTT,45 the A value of the hydroxyl group for this disulfide containing chair (wherein the dihedral angle of the SS bridge lies around 60°) is computed to be $A_{OH} = 9.57$ kJ.mol$^{-1}$ using equation 1:

$$ΔG_{eq}^0 = ΔG_{HB}^0 - 2A_{OH} \quad (eq. 1)$$

This indicates that disulfide bridge reinforces the destabilization of the all-axial conformation (as $A_{OH}$ is less than 5 kJ.mol$^{-1}$ in cyclohexane), presumably through additional diaxial interactions between the lone pairs of sulfur atoms and the hydroxyl groups (Figure 7a). This echoes to the kinetic difference observed between linear disulfides and parent alkanes, where ethylene bridges experience comparatively reduced electronic hindrance to rotational freedom (vide supra).46 Inverting one stereogenic center in DTT yields (2R,3S)-1,4-bis(sulfanyl)butane-2,3-diol, commonly known as 1,4-dithioerythritol (DTE). Interestingly, its oxidized form (Ox-DTE) is a meso compound that crystallizes as a mixture of P and M conformers, as both exhibit identical 1,3-diaxial interactions and intramolecular hydrogen bonding (Figure 8b). This demonstrates that when the stereochemistry of the disulfide bridge is governed by two stereogenic centers, they can act as a match pair that

biases the conformational equilibrium (Figure 3b), or as a mismatch pair that eliminates any clear conformational preference (Figure 3c). When both static and dynamic stereogenic units compose the backbone of a ring, inversion of the latter is accompanied by a spatial reorientation of the substituents borne by the latter. This dramatically affects the interactions into which those substituents are engaged (herein attractive such as hydrogen bonds, and repulsive such as 1,3-dixial interactions). Eventually, this results in a substantial energetic bias between enantiomorphic conformations as well as an increase in their interconversion barrier.

<figure><img src="image_10.png" /><figcaption>**Figure 8.** Equilibrium between (a) chair conformations of Ox-DTT, with corresponding X-ray structure of the P conformer (CCDC deposit number: 1145565), (b) chair conformations of Ox-DTE, with corresponding X-ray structure of both enantiomers (CCDC deposit number: 1145564), (c) envelope conformations of (±)-α-lipoic acid, with corresponding X-ray structure of both enantiomers (CCDC deposit number: 1270584), and (d) the most stable stereoisomers of an eight membered cysteinyl-cysteine ring, ox-[Cys-Cys]. Green dotted lines highlight hydrogen bonds.</figcaption></figure>

In the smaller five-membered ring found in (±)-α-lipoic acid, the orientation of substituents are also intimately related to the stability of the conformational enantiomorphs, with the mirror $(R,M)/(S,P)$ enantiomeric pair observed in the solid state (Figure 8c).47 Computational studies have shown that alkyl substituents preferentially adopt pseudo-equatorial positions to minimize steric hindrance in cyclopentanes or heterocyclic analogues.48 Interestingly, an effective flow of stereochemical information between static stereocenters and conformationally labile disulfide bonds can also be observed within larger ring, providing that their backbone include structuring moieties. This is the case of the eight membered cysteinyl-cysteine ring, (noted ox-[Cys-Cys]), which involves two homochiral stereogenic centers (R configuration) separated by a peptide bond on one side, and a CH$_2$-S-S-CH$_2$ sequence (the only conformationally labile portion) on the other side (Figure 8d).49 While the (R,R)-pair instruct the central bridge to quantitatively fold into a P configuration in linear Cystine (the linear homologue, Figure 4), this
configurational sequence can only be obtained if the internal amide linkage adopts the unusual and unfavorable cis configuration in the cyclized system (Figure 8d). This reveals that the amide bond is also eligible for stereochemical instruction and that there is a mismatch between the most stable amide configuration (*trans*) surrounded by a (*R*,*R*) pairs of stereocenters and the most stable disulfide conformation (*P*) surrounded by the same pair of stereocenters. Eventually more than 90% of the population of stereoisomers consists in a *ca.* 70:30 *trans*-R-M-R:cis-R-P-R mixture. Notably, the dominant stereoisomer is also found in the natural nicotinic acetylcholine esterase receptor, a key protein in neurodegenerative disorders.⁵⁰

Remarkably, it was not until 2022 that supramolecular chemistry began to envisage that the conformation of disulfide bonds may be deliberately controlled by non-covalent means, both in the solid state and in solution.⁷ Feringa *et al.* pioneered the field by deciphering the influence of hydrogen bond donors and acceptors on the stereochemistry of neighboring disulfide bridges within dyads made of methyl asparagusic acid (a 1,2-dithiolane derivative) linked to L-alanine methyl ester through a peptide bond (Figure 9). In such architectures, a single static stereocenter (here from alanine) is remotely located on the side chain of the disulfide containing five membered ring. To bias the conformational equilibrium of the latter, the former must preferentially interact with one enantiomorph through intramolecular non-covalent interactions (Figure 3a). The authors showed that in apolar solvents such as cyclohexane, intramolecular S–S···H–N hydrogen bonds can form (Figure 9a). This interaction enables the transfer of asymmetry from the stereogenic center of the amino acid to the disulfide bridge, as observed by circular dichroism (CD) spectroscopy.

<figure><img src="image_11.png" /><figcaption>**Figure 9.** Stereodivergent chirality transfer in a chiral amide assembled from methyl asparagusic acid and L-alanine methyl ester in (a) apolar solvents, (b), polar solvents, and (c) in the solid phase (CCDC deposit number: 2099418). Green dotted lines highlight hydrogen bonds.</figcaption></figure>

Simulation of the CD spectra (using DFT calculations) showed that 65% of the population of disulfide bridges adopts a *M* conformation when the neighboring amino acid is *L*-alanine. Inverting the central chirality of the amino acid resulted in typical mirror-image CD spectra, further supporting the chirality transfer from the stereo-center of the amino acid to the disulfide bridge of the asparagusic unit. The methyl substituent on the 1,2-dithiolane ring is also a crucial structural factor: without it, angle

compression (known as the Thorpe-Ingold effect) is suppressed, and neither S–S···H–N hydrogen bonding nor efficient intramolecular chirality transfer was observed.⁵¹ VT-NMR experiments confirmed that the non-covalent interaction transmitting the stereochemical information from the stereocenter to the bridge was enthalpically favored and entropically unfavored. This entropic penalty is thought to originate from the conformational restriction of the sequence encompassing the 1,2-dithiolane moiety and its amide binding partner. In agreement with Hunter's model of H-bond associations,⁵² the intramolecular transfer of stereochemical information can be disrupted by choosing a strong H-bond accepting solvent. Acetonitrile, a better acceptor than the disulfide moiety, switches the chirality transfer off, by binding intermolecularly with the amide group (Figure 9b). In the solid state, the intramolecular hydrogen bond is replaced by an intermolecular C=O···H-N hydrogen bond yielding helical assemblies, wherein the disulfide bond adopts the *P* configuration albeit with a nearly planar conformation (a dihedral angle of 5°compared to 40° in solution, see Figure 9c). Obviously, the energy penalty associated to this distortion (*vide supra*) is paid by the favorable lattice energy which partially relies on the intermolecular hydrogen bond. By screening various amino acids and dipeptides, the authors later showed that the configuration and dihedral angle value of the disulfide bridge in these helical architectures is high dependent on the structural features of the amino acid side chain (e.g., molecular symmetry, steric hindrance, H-bonding strength, etc.).⁸

A follow-up question, which derives naturally from this analysis, is: can stereochemical information also propagate through disulfide bridges when neighboring stereogenic units are not implemented on the architectures prior to the formation of the bridge(s) but simultaneously, by a self-assembling approach? Disulfide-containing cyclophanes are typically the class of object on which this question can be addressed.⁵³ A subclass of this rather diverse family, based on functionalized 1,4-phenylenedimethanethiol units, was recently reported by Johnson *et al.* (Figure 10).⁵⁴ Although not commented in the original report, the stereochemical self-sorting of these assemblies is remarkable. As in conventional *p*-cyclophanes bearing ethylene spacers,⁵⁵ each phenylene monomer displays facial chirality, once incorporated in the macrocycle and providing that the rotation along the 1–4 axis is hindered.⁵⁶,⁵⁷ Crystallographic analyses reveals that the bis(disulfide) dimer was obtained as a racemic mixture of homochiral species from a prochiral monomeric precursor. Analysis of the configurational-conformational relationship reveals that (*pR–M*)₂ and (*pS–P*)₂ sequences are formed exclusively during the stereo determinant ring closure step (CCDC deposit number: 1906244).⁵⁴ Although the assembling process is kinetic controlled, we further explored *in silico* the three alternative combinations of planar and axial chirality — *M*₂(*pS*)(*pR*), *M*₂(*pS*)₂, and *P*₂(*pS*)₂ — to understand how intramolecular interactions may influence the stability of the macrocyclic diastereoisomers (Figure 10).³⁰ The *P*₂(*pS*)₂ species, was computed to be the most stable, and is indeed observed by X-ray crystallography. Its increased stability with respect to other diastereoisomers originates from optimized π–π interactions, where the aromatic
rings are closely spaced at 3.64 Å, well within the optimal range of 3.5 to 3.9 Å.⁵⁸ In addition, the substantial twist angle of 33° (i.e., rotation around the axis perpendicular to the ring plane) favors head-to-tail interactions between C-Br and C-H dipoles from opposite aromatic rings (Figure 10). The $M_2(pS)_2$ conformer, generated by an inversion of both disulfide bridges, shows intermediate stability. Its structure features a notable slip (i.e., lateral displacement of ring centers) of 1.2 Å and a tilt angle (i.e., deviation from parallel stacking) of 10.6°, which partially disrupt the π-π contact. The energy difference between the two conformers translates into a Boltzmann distribution $P_2(pS)_2/M_2(pS)_2$ (or $M_2(pR)_2/P_2(pR)_2$) of ~10³ at 298 K. Finally, the $M_2(pS)(pR)$ conformer, which involves a bromine-through-the-annulus rotation of one aromatic unit from the previous conformer, lies considerably higher in energy. This conformation has the largest ring separation distance (4.09 Å), likely due to repulsive interactions between bromine atoms, with a twist angle of 9.3°, which strongly weakens the π-π interactions.⁵⁸ Overall, the relative energies of these conformers reflect the effectiveness of their π-π stacking, with greater overlap correlating with enhanced stability. This analysis reveals that chirality planes can be as effective as stereogenic centers to transfer some stereochemical information to disulfide bridges through π-π and dipolar interactions within a conformationally restricted macrocyclic backbone (i.e., prevented bromine-through-the-anulus rotation). This not only supports the feasibility of restraining the conformational liability of disulfide bonds via their incorporation into macrocyclic architectures. It also highlights the diastereoselective self-sorting phenomenon which occurs during macrocyclization, presumably through stereochemical information transfer between nearest neighboring stereogenic units.

<figure><img src="image_12.png" /><figcaption>**Figure 10.** Computed energetic and structural data for three stereoisomers of a cyclophane composed of brominated 1,4-phenylenedimethanethiol units.</figcaption></figure>

Taking molecular complexity a step further, pi-stacking was recently used to direct the self-assembly of 1,3-bis-thiophenol building blocks appended with an amino acid and a nucleobase into well-folded large macrocycles in water (Figure 11).⁵⁹ The 15mer structure obtained and crystallized exhibits a pseudo-$C_5$ axis of symmetry, depicting a pentagon with five groups of three geometrically inequivalent rings. In the crystal phase, stacking occurs between aromatic rings in the i, i+2, and i+4 positions, imposing five disulfide conformational pseudo codons of identical P-M-P sequence. Interestingly, the conformational integrity of the architecture remains intact in solution up to 353K, suggesting that multiplying non-covalent

interactions (here π-π contacts of ca. 3.4 Å) stands as a robust strategy to conformationally lock disulfides bridges in aqueous solution within macrocyclic architectures.

<figure><img src="image_13.png" /><figcaption>**Figure 11.** X-ray crystal structure of a 15mer (CCDC deposit number: 1892919): core part of the foldamer, showing five stacks of three phenyl rings connected by disulfide bonds (a), which favor five repetitions of the same P-M-P conformational codon (b).</figcaption></figure>

The concept of intentional stereocontrol over disulfide bonds by non-covalent means was further explored by some of us, using disulfide-containing cyclophanes. These dyn[n]arenes are self-assembled from functionalized 1,4-bisthiophenol.⁵³a As an illustration of the fundamental principles of dynamic covalent chemistry, exploiting non-covalent interactions enables on demand quantitative assembling of single-size macrocycles. For instance, the octacarboxylate dyn[4]arene is quantitatively produced upon templated self-assembling with spermine, a linear aliphatic tetraamine (Figure 12a).⁶⁰ The template is believed to be the structural initiator during the ring closure.⁹ This ultimate step of the self-assembling process is stereoselectively determinant, as prochiral monomeric faces become chiral, and the restricted conformational mobility dramatically slows down the dihedral inversion process of the disulfide bridges (Figure 3d). A constant regular spacing of the carboxylate groups of the host provides the most stabilizing environment for the polyammonium template homochiral template and corresponds to a $pS_4/pR_4$ facial configuration. This sequence concomitantly instructs the conformation of the disulfide bridges. To accommodate the hindrance of the groups borne in *ortho* position of the phenylene units, the dihedral linkages are forced to adopt a *M*-pR or *P*-pS relative configuration (Figure 12b). Remarkably, the entire stereochemical feature of the octacarboxylate dyn[4]arene is preserved even after the departure of the guest molecule, as observed in both the solid state and in solution.⁹ In the free host, the kinetic barrier for the inversions of the disulfide bridge's dihedral angle was estimated to be around 75 kJ·mol⁻¹, a value that would be expected to cause signal splitting on the NMR timescale if multiple conformers coexisted in solution (which is not observed). These results finally validate the guest-to-host imprinting of stereochemical information which can be stored into the configuration of both disulfide bridges and aromatic units, even after the departure of the instructing template, and in solution.
<figure><img src="image_14.png" /><figcaption>**Figure 12.** (a) X-ray crystallographic structure of an octa-carboxylate dyn[4]arene of *pS*₄P₄ configuration (CCDC deposit number: 1554744), (b) van der Waals contacts between the substituents of the phenyl rings and the sulfur atoms, (c) Non-bonded S···O chalcogen bonds (pink dotted lines), and (d) focus on the deviation from planarity of the aromatic rings in the solid-state structure.</figcaption></figure>

Re-examination of the macrocycle using NCI calculations revealed that the electron-rich oxygen and sulfur atoms are engaged in van der Waals contact, forming five membered rings fused with each aromatic unit (Figures 12c).³⁰ Such non-bonded S···O contacts are chalcogen bonds, and are defined by IUPAC as "net attractive interaction between an electrophilic region associated with a chalcogen atom in a molecular entity and a nucleophilic region in another, or the same, molecular entity".⁶¹ Chalcogen bonding interactions originate primarily from stabilizing n₀ → σ<sub>S-R</sub>* or π<sub>C=O</sub> → σ<sub>S-R</sub>* rather than from electrostatics, solvophobic effects, or dispersion.⁶² Statistically, about 1.3% of methionine and 8.9% of cysteine residues form chalcogen bonds in proteins, contributing to structural stabilization.⁶³ It is worth mentioning that these non-bonded contacts are not limited to oxygen and can involve other heteroatoms like nitrogen.⁶⁴ Within each monomeric unit of the dyn[4]arene, the combined maximization of this attractive interaction and the minimization of the steric hindrance between monomers and linkages results in a slight curvature of the aromatic ring (Figure 12d). This tradeoff clearly illustrates the additional stability which can be brought by hypervalent interactions. Interestingly, no hypervalence (nor consequent disruption of the monomeric unit planarity) was observed from NCI calculations in the parent homochiral octamethoxy dyn[4]arene (not shown, CCDC deposit number: 934188).

A review of the literature revealed other structures where S···O chalcogen bonds act as the primary driving force controlling the stereochemistry of a disulfide bond. In the crystal structure of (*S*,*S*)-α-methylphenylacetyl disulfide, contacts of 3.1 Å (a distance not greater than the sum of the corresponding van der Waals radii) between the oxygen borne by the static stereocenter and the nearest sulfur atom of the bridge instructs the latter into *P* configuration, whereas the (*R*,*R*) enantiomer adopts an *M* twist (Figure 13a).⁶⁵ Here chalcogen bonds synergistically propagates

the chiral information borne by static stereocenters to the central disulfide bridge in simple linear dimers (Figure 3b). They can also act as a stereoselective relay between static stereocenters and conformationally flexible disulfide bond in macrocyclic compounds. In fact, the X-ray crystal structure of a 23-membered cystine-based macrocycle displays an intramolecular S···O interaction with a distance of 3.3 Å (Figure 13b).⁶⁶ Interestingly, the directionality of the interaction corresponds here to a π<sub>C=O</sub> → σ<sub>S-R</sub>* contact, as frequently found in proteins, while organic compounds discussed previously rather involves a n₀ → σ<sub>S-R</sub>* interaction (Figure 13c).⁶³ Notably, this weak hypervalent interaction stabilizes the otherwise strained –LHHook conformation, characteristic of an allosteric disulfide bond (Figure 2).³⁴ Among the 20 possible sequences of dihedral angles along the bridges, only 3 are classified as allosteric disulfides (i.e., -RHStaple, -LHHook, and -/+RHHook). Such bridges differ fundamentally from structural disulfides: rather than providing static stabilization, they act as redox-controlled molecular switches in biological systems.

<figure><img src="image_15.png" /><figcaption>**Figure 13.** X-ray crystallographic structure of: (a) (*R*,*R*,*M*)- and (*S*,*S*,*P*)-α-methylphenylacetyl disulfide (CCDC deposition numbers: 779771 and 779772, respectively), and (b) a 23-membered cystine-based macrocycle (CCDC deposit number: 2415578). Pink and green dotted lines highlight chalcogen and hydrogen bonds, respectively.</figcaption></figure>

Very recently, we demonstrated that diastereoselective self-assembling, wherein facial and axial configuration co-operate, can be achieved on dyn[n]arenes bearing up 32 stereogenic units.⁹ This was achieved without the use of a template, through the rational design of the achiral starting building blocks. The stereoselectivity of the self-assembling was intended to rely on monomeric units bearing bulky groups of different sizes on faces and on the edge (Figure 14a). Exposing the bulkiest group out of the cavity was expected to direct the facial (*In* configuration) while the conformational (*M*-*P*)n sequence of disulfide bridges should minimize clashes between adjacent and opposed units. This design should eventually translate into a cyclopentamer with a (*M*-*In*-*P*-*In*)₂ sequence (Figure 14b). Whereas Feringa's work demonstrated the deliberate encoding of stereochemical information via unidirectional attractive interactions (e.g., hydrogen bonding),⁷,⁸ this study established that such information can also be purposefully encoded in disulfide bonds through non-directional repulsive interactions.
<figure><img src="image_16.png" /><figcaption>**Figure 14.** (a) Rationally designed 1,4-bisthiophenol building block bearing three classes of C-H: axial methine (b, cyan), axial methylene (b, green), equatorial protons (c, grey) protons, and (b) corresponding self-assembled dyn[4]arene of (*M*-In-*P*-In)₂ stereochemistry.</figcaption></figure>

CONCLUSION AND PERSPECTIVE

The selected examples re-examined in this perspective re-
veal that a large variety of attractive (H-bond, salt bridge,
pi-stacking, chalcogen) and repulsive interactions (steric)
can be exploited to asymmetrically fold a disulfide bridge
in the vicinity of a static stereogenic unit. Although it may
be counter-intuitive, steric hindrance is particularly effec-
tive to relay stereochemical information when the disul-
fide bridge is embedded into cyclic backbone (from in
small ring involving 1,3 diaxial interactions to large mac-
rocycles wherein flipping or tilting of monomeric units can
be hindered by substituent effects). This diversity spans to
the instructing stereogenic units asymmetrically interact-
ing with the bridge, which can be stereocenters (central
chirality), helices (helical chirality) or even planes (planar
chirality). Transfer of stereochemical information into di-
sulfide bridges is therefore not restricted to rare molecu-
lar curiosities, and the field of possibility which is rather
vast remains mostly to be explored.

Among the challenges which are open for the synthetic
chemist, one can cite the production of linear species in
solution displaying an asymmetrically folded and kinet-
ically frozen bridge as in natural peptides and proteins.
Our analysis reveals that only a couple of weak interac-
tions are needed to bias the population of enantiomorphs
into 99:1 mixture. Freezing the bridge is more challenging
and may require stronger interactions. Yet and as illus-
trated in this perspective, this can be achieved through
conformational restriction by either embedding the disul-
fide moiety into a cycle or by using bulky asymmetric
neighbors. If the disulfide soldiers can be folded into

enantiopure static units through unidirectional asymmet-
ric instruction from a chiral sergeant, isotactic or syndio-
tactic polydisulfide-based polymers may become syntheti-
cally accessible in one pot. Beyond this picture, which con-
ceptually underpins the field of foldamers, our analysis re-
veals that other paradigms are accessible. First, the amide
bonds, a key structural motif in the protein world which is
generally considered as static stereogenic units can be
similarly instructed cooperatively or anti-cooperatively
with the disulfide bridges. It also raises the question
whether a peptide sergeant may instruct the folding of a
flexible peptide solider via one or several disulfide link-
ages, a scenario which is suspected to be involved in mis-
folding diseases. The second new paradigm concerns the
requirement of a pre-existing chiral enantiopure sergeant,
to induce and propagate the asymmetric folding. As cova-
lent self-assembling of prochiral units with disulfide
bridges can enable the propagation of stereochemical in-
formation between nearest neighbor in a concerted man-
ner, one only needs to identify and design matching dyads,
triads, and beyond. Such a conceptual framework should
afford syndio or isotactic polymers displaying several
classes of codons enabling the chemist to encode stereo-
chemical information into several letter words. It also
opens the perspective of multidimensional propagation of
stereochemical information, i.e. from central sergeants to
peripheral soldiers. Besides the synthetic access to stere-
oregular macromolecular architectures, on demand infor-
mation encoding on synthetic molecular sequence may be-
come accessible by exploiting the toolbox detailed in this
perspective while the reading of the information should
require translocation devices.

AUTHOR INFORMATION

Corresponding Authors

laurent.vial@ens-lyon.fr; julien.leclaire@ens-lyon.fr

Author Contributions

The manuscript was written through contributions of all au-
thors.

AKNOWLEDGEMENTS

Financial support from the French MESR (T.C.) and the
ADEME (A.F.) is gratefully acknowledged. This work was sup-
ported by the LABEX iMUST (ANR-10-LABX-0064) of Univer-
sité de Lyon, within the program “Investissements d’Avenir”
operated by the French National Research Agency (ANR). We
also gratefully acknowledge support from GENCI-IDRIS
(Grand Challenge Jean Zay A0130800609) for the computing
resources.

REFERENCES
¹ Bechtel, T. J.; Weerapana, E. From Structure to Redox: The Diverse Functional Roles of Disulfides and Implications in Disease. *Proteomics* **2017**, *17* (6), 1600391.

² Hogg, P. J. Disulfide Bonds as Switches for Protein Function. *Trends Biochem. Sci.* **2003**, *28* (4), 210–214.

³Laurent, Q.; Martinent, R.; Lim, B.; Pham, A.-T.; Kato, T.; López-Andarias, J.; Sakai, N.; Matile, S. Thiol-Mediated Uptake. *JACS Au* **2021**, *1*(6), 710–728.

⁴ Canadell Ayats, J.; Goossens, J. G. P.; Klumperman, L. Self-Healing Materials Based on Disulfide Links. *Macromolecules* **2011**, *44* (8), 2536–2541.

⁵ Schröder, H. V.; Zhang, Y.; Link, A. J. Dynamic Covalent Self-Assembly of Mechanically Interlocked Molecules Solely Made from Peptides. *Nat. Chem.* **2021**, *13* (9), 850–857.

⁶ Carmack, M. Chirality of the Disulfide in the Prion Proteins. *J. Chem. Inf. Comput. Sci.* **2004**, *44* (1), 286–288.

⁷ Zhang, Q.; Crespi, S.; Toyoda, R.; Costil, R.; Browne, W. R.; Qu, D.-H.; Tian, H.; Feringa, B. L. Stereodivergent Chirality Transfer by Noncovalent Control of Disulfide Bonds, *J. Am. Chem. Soc.* **2022**, *144* (10), 4376–4382.

⁸ Zhang, Q.; Toyoda, R.; Pfeifer, L.; Feringa, B. L. Architecture-Controllable Single-Crystal Helical Self-assembly of Small-Molecule Disulfides with Dynamic Chirality. *J. Am. Chem. Soc.* **2023**, *145*, 6976–6985.

⁹ Zhang, Y.; Ourri, B.; Skowron, P.-T.; Jeamet, E.; Chetot, T.; Duchamp, C.; Belenguer, A. M.; Vanthuyne, N.; Cala, O.; Dumont, E.; Mandal, P. K.; Huc, I.; Perret, F.; Vial, L.; Leclaire, Self-assembly of achiral building blocks into chiral cyclophanes using non-directional interactions. *J. Chem. Sci.* **2023**, *14*, 7126–7135.

¹⁰ Higashi, L. S.; Lundeen, M.; Seff, K. Empirical Relations between Disulfide Bond Lengths, (Nitrogen or Carbon)-Carbon-Sulfur-Sulfur Torsion Angles, and Substituents in Aromatic Disulfides. *Crystal and Molecular Structure of 3,3-Dihydroxydi-2-pyridyl Disulfide*. *J. Am. Chem. Soc.* **1978**, *100*, 8101–8105.

¹¹ Johnson, R. D. III. *NIST 101: Computational Chemistry Comparison and Benchmark Database (CCCBDB)*: National Institute of Standards and Technology: Gaithersburg, MD, 1999. [Online] https://cccbdb.nist.gov (accessed Nov 12, 2025).

¹² Schlegel, H. B.; Skancke, A. Thermochemistry, Energy Comparisons, and Conformational Analysis of Hydrazine, Triazane, and Triaminoammonia. *J. Am. Chem. Soc.* **1993**, *115* (16), 7465–7471.

¹³ Cárdenas-Jirón, G. I.; Cárdenas-Lailhacar, C.; Toro-Labbé, A. Theoretical Analysis of the Internal Rotation, Molecular Structures and Electronic Properties of the XSSX Series of Molecules (X = H, F, Cl). *J. Mol. Struct.: Theochem* **1993**, *282*, 113–122.

¹⁴ Pauling, L. On the Stability of the S₈ Molecule and the Structure of Fibrous Sulfur. *Proc. Natl. Acad. Sci. U.S.A.* **1949**, *35* (9), 495–499.

¹⁵ Orabi, E. A.; Peslherbe, G. H. Computational insight into hydrogen persulfide and a new additive model for chemical and biological simulations. *Phys. Chem. Chem. Phys.* **2019**, *21*, 15988–16004.

¹⁶ Boyd, D. B. Conformational Dependence of the Electronic Energy Levels in Disulfides. *J. Am. Chem. Soc.* **1972**, *94*, 8799–8804.

¹⁷ IUPAC. *Compendium of Chemical Terminology*, 2nd ed. (the "Gold Book"); McNaught, A. D.; Wilkinson, A., Eds.; Blackwell Scientific Publications: Oxford, 1997. Online version (updated 2019), https://doi.org/10.1351/goldbook.A00543 (accessed Nov 12, 2025).

¹⁸ For the conformational energy barriers of H₂O₂, see: Song, L.; Lin, Y.; Wu, W.; Zhang, Q.; Mo, Y. Origins of Rotational Barriers in Hydrogen Peroxide and Hydrazine. *J. Phys. Chem. A* **2005**, *109*, 11097–11104.

¹⁹ For the conformational energy barriers of H₂S₂, see: (a) Lindquist, B. A.; Engdahl, A. L.; Woon, D. E.; Dunning, T. H., Jr. Insights

into the Electronic Structure of Disulfur Tetrafluoride Isomers from Generalized Valence Bond Theory. *J. Phys. Chem. A* **2014**, *118*, 10117–10126; (b) Dixon, D. A.; Zeroka, D. J.; Wendoloski, J. J.; Wasserman, Z. R. The Molecular Structure of Hydrogen Disulfide (H₂S₂) and Barriers to Internal Rotation. *J. Phys. Chem.* **1985**, *89*, 5334–5336.

²⁰ Ryan, P. B., Todd, H. D. Explicit representation of lone-pair orbitals in molecular orbital calculations for NH₃, H₂O, N₂H₄, and H₂O. *J. Chem. Phys.* **1977**, *67* (11), 4787–4793.

²¹ Eyring, H. The Activated Complex in Chemical Reactions. *J. Chem. Phys.* **1935**, *3*, 107–115.

²² Testa, B.; Vistoli, G.; Pedretti, A. Organic stereochemistry. Part 1. Symmetry elements and operations, classification of stereoisomers. *Hely. Chim. Acta* **2013**, *96*, 4–30.

²³ Liu, S.; Govind, N.; Pedersen, L. G. Exploring the Origin of the Internal Rotational Barrier for Molecules with One Rotatable Dihedral Angle. *s*, *129*, 094104.

²⁴ Fraser, R. R.; Boussard, G.; Saunders, J. K.; Lambert, J. B. Barriers to Rotation About the Sulfur–Sulfur Bond in Acyclic Disulfides. *J. Am. Chem. Soc.* **1971**, *93*, 3822–3823.

²⁵ Foss, O. Stereochemistry of Disulfides and Polysulfides. In Organic Sulfur Compounds; Kharasch, N., Ed.; Pergamon Press: Oxford, **1961**; Vol. 1, pp 75–82.

²⁶ Claeson, G.; Androes, G.; Calvin, M. The Activation Energy of Inversion in Substituted 1,2-Dithiane and 1,2-Dioxane Measured by the Nuclear Magnetic Resonance Technique. *J. Am. Chem. Soc.* **1961**, *83*, 4357–4361.

²⁷ Lüttringhaus, A.; Hess, U.; Rosenbaum, H.-J. Conformational Enantiomerism. I. Optically Active 4,5,6,7-Dibenzo-1,2-dithiazocyclooctadiene. *Z. Naturforsch. B* **1967**, *22*, 1296–1300.

²⁸ Field, L. Disulfides and Polysulfides. In Organic Chemistry of Sulfur; Oae, S., Ed.; Plenum Press: New York, **1977**; pp 303–382.

²⁹ Rahman, R.; Safe, S.; Taylor, A. The Stereochemistry of Polysulfides. *Q. Rev. Chem. Soc.* **1970**, *24*, 208–237.

³⁰ The methodology applied for the calculations is as follows: Density functional theory (DFT) calculations were performed at the wB97XD/cc-pVTZ level of theory starting either from available X-ray structures or built manually. Structures were optimized and resulting stationary points were inspected with the VMD software. The Gaussian 16 Rev. C.01 suite of programs was used throughout. When required, non-covalent interaction index (NCI) analyses were performed on optimized structures to visualize non-covalent interaction regions.

³¹ A donor–acceptor distance threshold of 3.1 Å was selected, beyond which hydrogen bonds were considered too weak to contribute significantly to the relative stability of the conformers, see: Espinosa, E.; Molins, E.; Lecomte, C. Hydrogen Bond Strengths Revealed by Topological Analyses of Experimentally Observed Electron Densities. *Chem. Phys. Lett.* **1998**, *285*, 170–173.

³² Anbuchezhiyan, M.; Ponnusamy, S.; Muthamizhchelvan, C. Crystal growth and characterizations of L-cystine dihydrobromide—A semiorganic nonlinear optical material. *Physica B: Condensed Matter* **2010**, *405*, 1119–1124.

³³ Oughton, B. M.; Harrison, P. M. The crystal structure of hexagonal L-cystine. *Acta Crystallogr.* **1959**, *12*, 396–404.

³⁴ Schmidt, B.; Ho, L.; Hogg, P. J. Allosteric disulfide bonds. *Biochemistry* **2006**, *45*, 7429–7433.

³⁵ Rosenfield, R. E., Jr.; Parthasarathy, R. Structure and Conformation of Amino Acids Containing Sulfur. III. The Crystal Structure and Absolute Configuration of 3,3,3',3'-Tetramethyl-D-Cystine (D-Penicillamine Disulfide) Dihydrochloride: An Unusually
Short Intramolecular N-H···S Contact Distance. *Acta Cryst.* **B** *1975*, *31*, 462-468.

³⁶ Mason, S. Biomolecular homochirality. *Chem. Soc. Rev.* **1988**, *17*, 347-359.

³⁷ Wedemeyer, W. J.; Welker, E.; Narayan, M.; Scheraga, H. A. Disulfide bonds and protein folding. *Biochemistry* **2000**, *39*, 4207-4216.

³⁸ Wright, Z. V. F.; McCarthy, S.; Dickman, R.; Reyes, F. E.; Sanchez-Martinez, S.; Cryar, A.; Kilford, I.; Hall, A.; Takle, A. K.; Topf, M.; Gonen, T.; Thalassinos, K.; Tabor, A. B. The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel NaV1.7. *J. Am. Chem. Soc.* **2017**, *139* (37), 13063-13075.

³⁹ For the three major Al-based protein-structure prediction models (a) Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly Accurate Protein Structure Prediction with AlphaFold. *Nature* **2021**, *596*, 583-589; (b) Baek, M.; DiMaio, F.; Anishchenko, I.; Dauparas, J.; Ovchinnikov, S.; Lee, G. R.; Wang, J.; Cong, Q.; Kinch, L. N.; Schaeffer, R. D.; et al. Accurate Prediction of Protein Structures and Interactions Using a Three-Track Neural Network. *Science* **2021**, *373*, 871-876; (c) Lin, Z.; Akin, H.; Rao, R.; Hie, B.; Zhu, Z.; Lu, W.; Smetanin, N.; Verkuil, R.; Kabeli, O.; Shmueli, Y.; et al. Evolutionary-Scale Prediction of Atomic-Level Protein Structure with a Language Model. *Science* **2023**, *379*, 1123-1130.

⁴⁰ Yu, L.; Li, D.; Ma, C.; Kauffmann, B.; Liao, S.; Gan, Q. Redox-Regulated and Guest-Driven Transformations of Aromatic Oligoamide Foldamers in Advanced Structures. *J. Am. Chem. Soc.* **2024**, *146*, 12907-12912.

⁴¹ Tsiamantas, C.; de Hatten, X.; Douat, C.; Kauffmann, B.; Maurizot, V.; Ihara, H.; Takafuji, M.; Metzler-Nolte, N.; Huc, I. Selective Dynamic Assembly of Disulfide Macrocyclic Helical Foldamers with Remote Communication of Handedness. *Angew. Chem. Int. Ed.* **2016**, *55*, 6848-6852.

⁴² Cleland, W. W. Dithiothreitol, a New Protective Reagent for SH Groups. *Biochemistry* **1964**, *3*, 480-482.

⁴³ Capasso, S.; Zagari, A. (4R,5R)-1,2-Dithiane-4,5-diol. *Acta Crystallogr. B* **1981**, *37*, 1437-1439.

⁴⁴ Eliel, E. L.; Wilen, S. H., Eds. Stereochemistry of Organic Compounds, 1st ed.; Wiley Interscience: New York, NY, **1994**.

⁴⁵ C.-Y. Huang; L.A. Cabell; E.V. Anslyn. "Molecular Recognition of Cyclitols by Neutral Polyaza-Receptors: The Strength and Influence of Intramolecular Hydrogen Bonds Between Vicinal Alcohols," *J. Am. Chem. Soc.* **1994**, *116*, 2778-2792.

⁴⁶ Pitzer, R. M.; Lipscomb, W. N. Calculation of the Barrier to Internal Rotation in Ethane. *J. Chem. Phys.* **1963**, *39* (8), 1995-2004.

⁴⁷ Stroud, R. M.; Carlisle, C. H. A single-crystal structure determination of DL-6-thioic acid, C₈H₁₄O₂S₂. *Acta Crystallogr.* **1972**, *B28*, 304-307.

⁴⁸ Stortz, C. A.; Sarotti, A. M. Exhaustive Exploration of the Conformational Landscape of Mono- and Disubstituted Five-Membered Rings by DFT and MP2 Calculations. *RSC Adv.* **2019**, *9*, 24134-24145.

⁴⁹ Creighton, C. J.; Reynolds, C. H.; Lee, D. H.; Leo, G. C.; Reitz, A. B. Conformational Analysis of the Eight-Membered Ring of the Oxidized Cysteinyl–Cysteine Unit Implicated in Nicotinic Acetylcholine Receptor Ligand Recognition. *J. Am. Chem. Soc.* **2001**, *123*, 12664-12669.

⁵⁰ Brejc, K.; van Dijk, W. J.; Klaassen, R. V.; Schuurmans, M.; van der Oost, J.; Smit, A. B.; Sixma, T. K. Crystal Structure of an ACh-

binding Protein Reveals the Ligand-binding Domain of Nicotinic Receptors. *Nature* **2001**, *411*, 269-276.

⁵¹ Beesley, R. M.; Ingold, C. K.; Thorpe, J. F. The formation and stability of spiro-compounds. Part I. spiro-Compounds from cyclohexane. *J. Chem. Soc., Trans.* **1915**, *107*, 1080-1106.

⁵² Hunter, C. A. Quantifying Intermolecular Interactions: Guidelines for the Molecular Recognition Toolbox. *Angew. Chem., Int. Ed.* **2004**, *43*, 5310-5324.

⁵³ For selected recent references, see: (a) Vial, L.; Perret, F.; Leclaire, J. Dyn[n]arenes: Versatile Platforms To Study the Interplay between Covalent and Noncovalent Bonds. *Eur. J. Org. Chem.* **2022**, 2022, e202101274; Otto, S. An Approach to the De Novo Synthesis of Life. *Acc. Chem. Res.* **2022**, *55*, 145-155; (c) Shear, J. B.; Peart, P. A.; Raines, D. J.; Pinkerton, D. M.; Hartley, C. S. Expanding the Scope of Pnictogen-Assisted Cyclophane Self-Assembly. *Eur. J. Org. Chem.* **2022**, 2022, e202200006.

⁵⁴ Shear, T. A.; Lin, F.; Zakharov, L. N.; Johnson, D. W. Design of Experiments" as a Method to Optimize Dynamic Disulfide Assemblies: Cages and Functionalizable Macrocycles. *Angew. Chem. Int. Ed.* **2020**, *59* (4), 1496-1500.

⁵⁵ Hassan, Z.; Spuling, E.; Knoll, D. M.; Lahann, J.; Bräse, S. Planar Chiral [2,2]Paracyclophanes: From Synthetic Curiosity to Applications in Asymmetric Catalysis and Materials. *Chem. Soc. Rev.* **2018**, *47*, 6947-6963.

⁵⁶ Lemiére, G. L.; Alderweireldt, F. C. Proposition for a New Definition of the Chiral Plane and Its Consequences for the Specification of Planar Chirality. *J. Org. Chem.* **1980**, *45*, 4175-4179.

⁵⁷ Assignment of the pS/pR descriptor follows the convention established for planar-chiral pillar[n]arenes, see: Chen, J.-F.; Ding, J.-D.; Wei, T.-B. Pillararenes: Fascinating Planar Chiral Macrocyclic Arenes. *Chem. Commun.* **2021**, *57* (72), 9029-9039.

⁵⁸ Sinnokrot, M. O.; Sherrill, C. D. High-Accuracy Quantum Mechanical Studies of π-π Interactions in Benzene Dimers. *J. Phys. Chem. A* **2006**, *110* (37), 10656-10668.

⁵⁹ Liu, B.; Pappas, C. G.; Zangrando, E.; Demitri, N.; Chmielewski, P. J.; Otto, S. Complex Molecules That Fold Like Proteins Can Emerge Spontaneously. *J. Am. Chem. Soc.* **2019**, *141* (4), 1685-1689.

⁶⁰ Skowron, P.-T.; Dumartin, M.; Jeamet, E.; Perret, F.; Gourlaouen, C.; Baudouin, A.; Fenet, B.; Naubron, J.-V.; Fotiadu, F.; Vial, L.; Leclaire, J. On-Demand Cyclophanes: Substituent-Directed Self-Assembling, Folding, and Binding. *J. Org. Chem.* **2016**, *81* (2), 654-661.

⁶¹ Aakeroy, C. B.; Bryce, D. L.; Desiraju, G. R.; Frontera, A.; Legon, A. C.; Nicotra, F.; Rissanen, K.; Scheiner, S.; Terraneo, G.; Metrangolo, P.; Resnati, G. Definition of the Chalcogen Bond (IUPAC Recommendations 2019). *Pure Appl. Chem.* **2019**, *91*, 1889-1892.

⁶² Pascoe, D. J.; Ling, K. B.; Cockcroft, S. L. The Origin of Chalcogen-Bonding Interactions. *J. Am. Chem. Soc.* **2017**, *139*, 15160-15167.

⁶³ Iwaoka, M.; Takemoto, S.; Tomoda, S. Statistical and Theoretical Investigations on the Directionality of Nonbonded S···O Interactions. Implications for Molecular Design and Protein Engineering. *J. Am. Chem. Soc.* **2002**, *124*, 10613-10620.

⁶⁴ Scheiner, S. On the Properties of X···N Noncovalent Interactions for First-, Second-, and Third-Row X Atoms. *J. Chem. Phys.* **2011**, *134*, 164313.

⁶⁵ Mukhlall, J. A.; Noll, B. C.; Hersh, W. H. Synthesis of Chiral Disulfides: Potential Reagents for Enantioselective Sulfurization. *J. Sulfur Chem.* **2011**, *3*, 199-212.

⁶⁶ Bhardwaj, S. K.; Haridas, V. Hybrid Cyclopeptide-Based Chemical Models for Allosteric Disulfide and Amyloid Self-Assembly. *Chem. Eur. J.* **2025**, *31*, e202500313.