•Enantiospecific interactions are probed using a functionalized AFM cantilever•A force difference of 70 pN between homo- and heterochiral pairs is obtained•The force is directional resulting from transient spin exchange interactions•Toy model calculations reveal the short range and directionality of the force Most biological molecules are chiral. Interestingly, in life, they appear in one chirality. This is not trivial as most of non-biological chemistry is achiral. Indeed, the earliest evidence for amino acids on earth suggests that both enantiomers were present. One of the driving forces to preserve homochirality in life may be enantiomer-specific forces. Here, the interaction between chiral peptides is probed using a chiral molecule functionalized atomic force microscope. The results show that the same-handed (chirality) polypeptides generate large attraction potentials (∼10 Kcal/mol), in agreement with simulations. This force, related to the chiral-induced spin selectivity (CISS) effect, is short ranged and directional and is therefore especially relevant to crowded biological systems. This force places symmetry constraints on protein folding, it may provide a reason for preserving chirality in life. For applications, these results may promote simulations enabling to improve chiral synthesis. Enantiospecific biorecognition interactions are key to many biological events. Commonly, bio-affinity values, measured in these processes, are higher than those calculated by available methods. We report here the first direct measurement of the interaction force between two chiral peptides (right- and left-handed helical polyalanine peptides) and the quantification of difference in the interaction force between homochiral and heterochiral pairs of molecules using atomic force microscope (AFM), together with supportive calculations based on a simple theoretical model. A force difference of 70 pN between same and opposite enantiomer interactions is measured. Additional measurements show spin dependency and fast decay of the interaction term, consistent with spin exchange interactions. This short range enantiospecific interaction term is especially relevant in crowded biological systems. The results shed light on the importance of spin and exchange interactions in biological processes. Enantiospecific biorecognition interactions are key to many biological events. Commonly, bio-affinity values, measured in these processes, are higher than those calculated by available methods. We report here the first direct measurement of the interaction force between two chiral peptides (right- and left-handed helical polyalanine peptides) and the quantification of difference in the interaction force between homochiral and heterochiral pairs of molecules using atomic force microscope (AFM), together with supportive calculations based on a simple theoretical model. A force difference of 70 pN between same and opposite enantiomer interactions is measured. Additional measurements show spin dependency and fast decay of the interaction term, consistent with spin exchange interactions. This short range enantiospecific interaction term is especially relevant in crowded biological systems. The results shed light on the importance of spin and exchange interactions in biological processes. Nature is based on chiral molecules, namely molecules that appear in two forms, enantiomers, which are mirror images of each other. Interestingly, chiral biomolecules, such as proteins and sugars, appear in nature mainly as one enantiomer. The origin of “homo chirality” in nature was—and is—discussed very intensively in the literature.1Blackmond D.G. The origin of biological homochirality.Cold Spring Harb. Perspect. Biol. 2019; 11: a032540Crossref PubMed Scopus (42) Google Scholar However, the focus of this work is related to a more fundamental question, i.e., why did nature preserve chirality so persistently over the many millions of years of evolution? In other words, does chirality per se, independent on the specific handedness, provide properties that serve an important role in life? The ability of biological molecules to interact selectively with each other is at the heart of all biological processes and the basis of many pharmaceutical concepts. Two important properties, related to chirality, characterize interactions in nature, i.e., very high enantioselectivity and the relatively fast rates of very complex processes, e.g., the rate of protein folding2Ivankov D.N. Finkelstein A.V. Prediction of protein folding rates from the amino acid sequence-predicted secondary structure.Proc. Natl. Acad. Sci. USA. 2004; 101: 8942-8944Crossref PubMed Scopus (164) Google Scholar, 3Qiu L. Pabit S.A. 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Chirality-induced spin polarization places symmetry constraints on biomolecular interactions.Proc. Natl. Acad. Sci. USA. 2017; 114: 2474-2478Crossref PubMed Scopus (108) Google Scholar However, such spin-related interaction term has never been measured directly before. Herein, we use atomic force spectroscopy, to directly measure the enantioselective interaction between oligopeptides of different handedness. Simple modeling of the spin-related enantiospecific interaction energies shows that the spin constraint also imposes directionality in the interaction. The enantioselectivity measured here does not correspond to any of the established biorecognition mechanisms related to structural properties, such as the “lock and key” model,7Fischer E. Einfluss der configuration auf die Wirkung der enzyme.Ber. Dtsch. Chem. Ges. 1894; 27: 2985-2993Crossref Scopus (1171) Google Scholar and induced fit and allosteric interactions,8Schneider H.J. Limitations and extensions of the lock-and-key principle: differences between gas state, solution and solid state structures.Int. J. Mol. Sci. 2015; 16: 6694-6717Crossref PubMed Scopus (29) Google Scholar and neither can it be explained through differential, enantiomer-specific, long-range electrostatic interactions.9Ramanan R. Dubey K.D. Wang B. Mandal D. Shaik S. Emergence of function in P450-proteins: a combined quantum mechanical/molecular mechanical and Molecular Dynamics study of the reactive species in the H2O2-dependent cytochrome P450SPα and its Regio- and enantioselective hydroxylation of fatty acids.J. Am. Chem. Soc. 2016; 138: 6786-6797Crossref PubMed Scopus (41) Google Scholar, 10Wang Z. Danovich D. Ramanan R. Shaik S. Oriented-external electric fields create absolute enantioselectivity in Diels–alder reactions: importance of the molecular dipole moment.J. Am. Chem. Soc. 2018; 140: 13350-13359Crossref PubMed Scopus (78) Google Scholar, 11Dubey K.D. Shaik S. 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Although additional research will be needed to delineate the exact scope and pervasiveness of this effect, it can be expected to alleviate some of the notable discrepancies between observed versus computed enantiospecificity, interaction energies, and reaction rates. Furthermore, the directionality emerging from our model may help to explain the high efficiency of many complex bioprocesses, such as protein folding and enzymatic reactions, since it significantly reduces the phase space the systems have to explore. When considering bio-related chemical processes, the distribution of charges in the reacting species is of major importance. Obviously, these distributions are a direct result of the spatial positions of the charged electrons and nuclei in the molecular systems. However, next to charge, electrons also have another property, their spin, which is their angular momentum and can have two orientation values. In organic molecules, the spin is typically not coupled significantly to the molecular frame; therefore, the orientation of the spin relative to this frame is not defined. Consequently, in such a case, the electron’s spin direction does not affect the interaction between molecules, i.e., the exchange term of the dispersion interaction is spin independent.18Danovich D. Shaik S. Neese F. Echeverría J. Aullón G. Alvarez S. Understanding the nature of the CH···HC interactions in alkanes.J. Chem. Theory Comput. 2013; 9: 1977-1991Crossref PubMed Scopus (90) Google Scholar For chiral molecules, however, this is not the case. In the last 2 decades, it was established that when electrons are displaced in a chiral system, the rate of this displacement depends on their spin. This property was termed the chiral-induced spin selectivity (CISS).19Naaman R. Paltiel Y. Waldeck D.H. Chiral molecules and the electron spin.Nat. Rev. Chem. 2019; 3: 250-260Crossref Scopus (224) Google Scholar In chiral systems, the spin that results from the charge displacement is strongly coupled to the molecular frame, such that spins of one type are displaced faster than the other, depending on the handedness of the molecule and the direction of motion. Charge displacement occurs whenever two chiral molecules approach each other, resulting in the formation of induced electric dipoles (see Figure 1A). Consequently, the emergence of these dipoles implies that at each electric pole, there is at least a fraction of an unpaired electron, i.e., spin polarization is intrinsically associated with the electric dipole formation in chiral molecules.20Abendroth J.M. Nakatsuka N. Ye M. Kim D. Fullerton E.E. Andrews A.M. Weiss P.S. Analyzing spin selectivity in DNA-mediated charge transfer via fluorescence microscopy.ACS Nano. 2017; 11: 7516-7526Crossref PubMed Scopus (58) Google Scholar The concept of charge polarization accompanied by spin polarization was verified in experiments in which the interaction of chiral molecules with ferromagnetic substrates was probed.21Banerjee-Ghosh K. Ben Dor O.B. Tassinari F. Capua E. Yochelis S. Capua A. Yang S.H. Parkin S.S.P. Sarkar S. Kronik L. et al.Separation of enantiomers by their enantiospecific interaction with achiral magnetic substrates.Science. 2018; 360: 1331-1334Crossref PubMed Scopus (173) Google Scholar,22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar Hence, when two chiral molecules interact, a spin-dependent interaction term emerges,23Happer W. Tam A.C. Effect of rapid spin exchange on the magnetic-resonance spectrum of alkali vapors.Phys. Rev. A. 1977; 16: 1877-1891Crossref Scopus (159) Google Scholar which is dependent upon the relative handedness of the molecules. Here, we present experiments in which the force between chiral oligomer attached to the tip of an atomic force microscope (AFM) and a monolayer made from oligomers that possess either the same or opposite handedness, or oligomers that are achiral, are monitored. The energies that associate with the enantiospecific interactions are larger by more than a factor five compared with the thermal energies at room temperature. These experimental results are accompanied by model calculations that show the role of the spin exchange interaction and its effect on the interaction energies and their angle-dependent distribution. Previous work22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar has shown that exchange interactions can be probed using modified atomic force spectroscopy (AFS). This past study probed exchange interactions between ferromagnetic substrates and helical peptides. AFS is widely used to examine biological interactions and functions24Bizzarri A.R. Cannistraro S. The application of atomic force spectroscopy to the study of biological complexes undergoing a biorecognition process.Chem. Soc. Rev. 2010; 39: 734-749Crossref PubMed Google Scholar and is used for the study of binding and unbinding of proteins.25Hinterdorfer P. Baumgartner W. Gruber H.J. Schilcher K. Schindler H. Detection and localization of individual antibody-antigen recognition events by atomic force microscopy.Proc. Natl. Acad. Sci. USA. 1996; 93: 3477-3481Crossref PubMed Scopus (1021) Google Scholar, 26Hugel T. Seitz M. The study of molecular interactions by AFM force spectroscopy.Macromol. Rapid Commun. 2001; 22: 989-1016Crossref Scopus (323) Google Scholar, 27Florin E.L. Moy V.T. Gaub H.E. Adhesion forces between individual ligand-receptor pairs.Science. 1994; 264: 415-417Crossref PubMed Scopus (1715) Google Scholar This study utilizes the earlier mentioned method to probe spin exchange interactions between helical (hence chiral) peptides and demonstrate the relation between enantiomer selectivity and spin. A chemically modified standard gold-coated AFM cantilever was used. The functionalized cantilever is applied to generate a distance-dependent force curve based on short-ranged spin exchange interaction. The gold AFM tip was functionalized with polyethylene glycol (PEG), 60 nm long, bound to a helical peptide, L-AHPA, where AHPA is alpha helix polyalanine (AHPA) [HS-PEG-NH-AAAAKAAAAKAAAAKAAAAKAAAAKAAAAKAAAAK-COOH] (see Figures 1C and 1D). The AHPA was chosen due to its strong spin separation rates.28Ghosh S. Mishra S. Avigad E. Bloom B.P. Baczewski L.T. Yochelis S. Paltiel Y. Naaman R. Waldeck D.H. Effect of chiral molecules on the electron’s spin wavefunction at interfaces.J. Phys. Chem. Lett. 2020; 11: 1550-1557Crossref PubMed Scopus (30) Google Scholar The AFM tip was functionalized in such a way that the peptide’s carboxyl group is facing the substrate (see experimental procedures section for details). The PEG acts as a spacer to reduce nonspecific interactions and the whole system is immersed in ethanol to eliminate capillary forces as done in Ziv et al.22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar The measured samples consist of self-assembled monolayers of the same helical polypeptide adsorbed on a gold substrate. The adsorption process ensures a peptide alignment such that the carboxylic group is facing up, so that there are no covalent bonds possible between the oligopeptide on the AFM tip and the one adsorbed on the substrate. Over a thousand curves of force versus distance were measured, which were subsequently examined manually. Following previous studies,22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar,29Das P. Duanias-Assaf T. Reches M. Insights into the interactions of amino acids and peptides with inorganic materials using single-molecule force spectroscopy.J. Vis. Exp. 2017; 121e54975Google Scholar,30Razvag Y. Gutkin V. Reches M. Probing the interaction of individual amino acids with inorganic surfaces using atomic force spectroscopy.Langmuir. 2013; 29: 10102-10109Crossref PubMed Scopus (41) Google Scholar only curves that showed a clear pulling event (see in Figure S1) were further analyzed as single-molecule rapture events. A worm-like chain (WLC) model was then fitted on the specific interaction’s pulling event, and the pulling force was retrieved. The mean pulling force (MPF) was calculated by averaging over the selected pulling forces. The forces were averaged in order to achieve simple unbiased analysis. The interaction is expected to be transient; however, the time dependence of the tip’s interaction with the surface is not included in this study and is addressed is previous work.22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar The timescale of this transient effect is relatively long since it is controlled by the adsorption and desorption kinetics as discussed in previous works.21Banerjee-Ghosh K. Ben Dor O.B. Tassinari F. Capua E. Yochelis S. Capua A. Yang S.H. Parkin S.S.P. Sarkar S. Kronik L. et al.Separation of enantiomers by their enantiospecific interaction with achiral magnetic substrates.Science. 2018; 360: 1331-1334Crossref PubMed Scopus (173) Google Scholar,22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar The force between the same and different enantiomers is presented in Figure 2. Right- or left-handed helical oligopeptides (L-AHPA or D-AHPA, respectively) were measured when adsorbed on the gold substrate. As control experiments, we measured a monolayer of an achiral 12-mercaptododecanoic acid with a comparable length and an equivalent carboxylic head group facing up. The MPF of the molecules is shown in Figure 2A. The relatively strong binding forces are attributed to the exchange interaction due to the CISS effect. A force difference of 70 ± 10 pN between the interaction of the homochiral pair of oligopeptides (L-AHPA monolayer [L-AHPA]) and the interaction of the heterochiral pair (D-AHPA monolayer—L-AHPA adsorbed AFM tip) is obtained. The interaction’s energy is retrieved by integrating the force distance curve over the pulling distance. An average energy difference of about 0.3 eV is measured (Figure S2). The force measured here and the interaction energy agree with the splitting between singlet and triplet states (∼1 eV). We attribute the low forces to nonspecific interactions that are the same for all samples (i.e., Coulomb and dispersive forces). The higher forces are attributed to the spin-dependent exchange interaction and is different for homo and hetero chiral interactions, i.e., this force is stronger between same enantiomers (L-L) than opposite enantiomers (L-D). The differentiation between high and low forces is discussed in the supplemental experimental procedures (Figure S8; Table S2). As a control experiment, we checked achiral molecules as well. The MPF between the L-AHPA adsorbed on the tip and achiral control was lower than the force for both enantiomers but without statistical significance. It is interesting to note that the MPF of the plain gold sample is lower than the MPF of the chiral L-L interaction. At first sight, this may be surprising since the interaction between a gold substrate and a carboxylic group has a coordinated character, but it can be explained by the gold being coated with organic contamination.31Paik W. Han S. Shin W. Kim Y. 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Effect of chiral molecules on the electron’s spin wavefunction at interfaces.J. Phys. Chem. Lett. 2020; 11: 1550-1557Crossref PubMed Scopus (30) Google Scholar As a result, spin-dependent charge is moving from the substrate through the adsorbed molecule. This charge and its spin can affect the interaction between the molecules. The same functionalized tip, as described earlier, was used and a constant applied magnetic field perpendicular to the surface was applied during the measurement (see Figure 3A1). The results are presented in Figures 3A2 and 3A3. The MPF difference between up and down magnetizations is 28 ± 9 pN (Figure 3A2). These results support the notion that the interactions strength is spin dependent. A clear difference is also seen in the force distributions histograms (Figure 3A3). The effect of the substrate magnetization on the force measured, in the case of chiral molecules, can also be a result of more efficient charge penetration from the substrate into the chiral molecule, when the injected charge has the preferred spin for the given handedness.28Ghosh S. Mishra S. Avigad E. Bloom B.P. Baczewski L.T. Yochelis S. Paltiel Y. Naaman R. Waldeck D.H. Effect of chiral molecules on the electron’s spin wavefunction at interfaces.J. Phys. Chem. Lett. 2020; 11: 1550-1557Crossref PubMed Scopus (30) Google Scholar Here, the charge redistribution is caused by the adsorption37Ben Dor O. Yochelis S. Radko A. Vankayala K. Capua E. Capua A. Yang S.H. Baczewski L.T. Parkin S.S.P. Naaman R. Paltiel Y. Magnetization switching in ferromagnets by adsorbed chiral molecules without current or external magnetic field.Nat. Commun. 2017; 8: 14567Crossref PubMed Scopus (86) Google Scholar and the pulling of the two molecules.22Ziv A. Saha A. Alpern H. Sukenik N. Baczewski L.T. Yochelis S. Reches M. Paltiel Y. AFM-based spin-exchange microscopy using chiral molecules.Adv. Mater. 2019; 31e1904206Crossref PubMed Scopus (26) Google Scholar These processes are known to change the polarization of the molecule and, therefore, result in an exchange of charge with the substrate. This charge transport from the substrate increases the spin density at the interaction region between the molecule attached to the tip and the adsorbed molecule. To evaluate this effect, we investigated the interaction between a tip coated with achiral molecule and oligopeptide adsorbed on magnetized substrates (Figure S3). In this case, the difference in the force measured for the two directions of magnetization is within the noise range of the system. Thus, the main difference in force, under opposite magnetic field, is a result of spin-dependent exchange interactions. We have shown so far that spin is affecting the biorecognition pulling force. To relate the results to exchange interactions only, and to differentiate them from mechanical and structural-related forces, we probed the interaction range. The spin exchange interaction is characterized by overlap of wave functions and is, therefore, short ranged. In a previous study, the decay length of the spin-dependent interaction was determined to be about 0.7 nm.38Metzger T.S. Mishra S. Bloom B.P. Goren N. Neubauer A. Shmul G. Wei J. Yochelis S. Tassinari F. Fontanesi C. et al.The electron spin as a chiral reagent.Angew. Chem. Int. Ed. Engl. 2020; 59: 1653-1658Crossref PubMed Scopus (39) Google Scholar To probe the range of the effect observed in this study, a layer of achiral amino-acid (glycine), 0.4 nm long, was added to the respective helical peptide monolayers (L/D-AHPA), which are 5.4 nm high. The adsorption of the glycine was done following a protocol reported in the literature,39Buttafoco L. Engbers-Buijtenhuijs P. Poot A.A. Dijkstra P.J. Daamen W.F. van Kuppevelt T.H. Vermes I. Feijen J. First steps towards tissue engineering of small-diameter blood vessels: preparation of flat scaffolds of collagen and elastin by means of freeze drying.J. Biomed. Mater. Res. B Appl. Biomater. 2006; 77: 357-368Crossref PubMed Scopus (72) Google Scholar and the experimental layout and of the monolayer with glycine is presented in Figure 3B1. The samples were measured with the same functionalized AFM cantilever as before. The results are presented in Figures 3B2 and 3B3. A difference in the MPF of 25 ± 4 pN was measured, suggesting that the effect is still apparent but weaker than without the achiral separation. An additional verification of the range of the force was performed with samples having a thicker bi-layer of glycine (0.8 nm) (see Figure S4), and the results are presented in the inset of Figure 3B3. Note the higher MPF for the longer bi-layer due to possible intertwining of the molecules. In the case of a sample in which the chiral molecules are separated by 0.8 nm, the difference in the force between the enantiomers has completely disappeared, suggesting a length dependence of the force as expected in the case of spin-exchange-related interactions.38Metzger T.S. Mishra S. Bloom B.P. Goren N. Neubauer A. Shmul G. Wei J. Yochelis S. Tassinari F. Fontanesi C. et al.The electron spin as a chiral reagent.Angew. Chem. Int. Ed. Engl. 2020; 59: 1653-1658Crossref PubMed Scopus (39) Google Scholar It is important to note that these distances are relevant to the crowded and high-pressure in vivo environments. These results also suggest that the penetration of the helical peptide into the monolayer is also short ranged, otherwise the structural differences would become apparent in the interaction. It is important to appreciate that when a chiral molecule interacts with achiral one, the spin exchange interaction term vanishes, since the spin