Anomalous rotational-symmetry breaking in proton arrangement of surface-confined cyclic hydrogen bonds revealed by atomic force spectroscopy.

In hydrogen-bonded materials and biosystems, microscopic proton ordering, which is strongly influenced by the quantum nature of protons, underpins diverse macroscopic phenomena, including phase transitions, chemical reactions, biomolecular processes and collective proton transfer. Yet resolving proton arrangements and characterizing their quantum behavior at the atomic scale remain challenging due to the small size of protons and the lack of effective approaches. Here, we exploit bond-resolved atomic force microscopy and spectroscopy (BR-AFM/AFS) to probe signatures of proton ordering in surface-confined benzimidazole (BI) assemblies. By performing BR-AFS along the apparent H-bond between proton donor and acceptor nitrogen atoms, we extract information consistent with H-bonding directionality and signatures compatible with quantum proton delocalization. We observe anomalous rotational-symmetry breaking in the proton order of the cyclic hexamers, arising from the coexistence of both localized and quantum-delocalized protons. The chirality of a single hexamer can be reversibly switched by altering its adsorption registry combined with collective transfer of six protons. Path-integral molecular dynamics calculations unravel that nuclear quantum effects promote proton delocalization and concomitant reduction of donor-acceptor distance. Our findings enable atomic-level detection of complex proton orders, engineering proton-based quantum states, and elucidation of long-range proton transfer mechanisms in molecular architectures.