Amplification of chirality in surface-confined supramolecular bilayers

One of the most dramatic effects of supramolecular assembly is the generation of homochirality in near-racemic systems. It is normally infeasible though to flip the absolute chirality of a molecule. Here we rationalize this seemingly contradictory chiral amplification mechanism with a combined scanning tunneling microscopy (STM) and modeling study of surface-grown enantiomerically unbalanced supramolecular bilayers. We identify a chemical equilibrium between opposite but not mirror-image-related twisting molecular geometries of the pure enantiomer, and accordingly two competing aggregation pathways. The nonlinear chiral amplification effect in bilayers of near-racemic mixtures involves the biased adsorption and organization of the majority enantiomer, and the compliance of the minority enantiomer to adopt an energetically less favorable twisting molecular conformation and handed organization. By establishing a direct link between molecular building block architectures and chiral amplification effect, this study provides a general approach to gain insight into cooperative supramolecular assembly in mixed enantiomer systems.

The topic of research and the presentation of the results are very well suited for Nature Communications. In contrast, however, I feel that the authors should take more care to explain their results and the conclusions that can be drawn to the readership of Nature Communications from a broad disciplinary background. The current introductory paragraph is rather long and more historic than solution geared after briefly introducing into the scientific challenge.
N.B. the final lines of the Abstract read very nice, but the language is a bit on the specialists side. What I like here is that it starts with 'generation of homochirality in racemic systems' unlike the first line of the article which spans a bridge between life's homochirality (which is a specific feature of synthesis in biomolecular chemistry) and chiral amplification (which if I am right is more a feature of scientific investigation related to supramolecular assemblies and not to biomolecular synthesis) and is less common in nature.
In my view the 'majority' rules effect is here modified such that the majority forces the minority to change the conformation, to adopt a less favoured conformational state in order to show compliance.
This, I repeat myself, deserves to be explained and evidenced with pride of the authors and should be accepted after mandatory revisions for publication in Nature Communications.
Further comments: Given that the brightness of features in the STM images relates to the topography --the brighter the the protrusion, the larger the apparent height -the distinction in contrast of two isolated strands at nearly …. With the long history of STM studies on longer chain aliphatic derivatives -liquid crystals, thiols and more, the referencing could be improved to evidence the identification of 'chemical' (i.e. HOMO / LUMO) and topographic contrast. I just point at one manuscript here: Y. Qian et al., Langmuir 2003, 19, 6056-6065 P16, line 307: …, while the latTer occurs only IN condition of the coverage exceeding one ML. (or similar).
I agree with the authors that the brightness in the STM images HERE relates to the topography, but this is not generally the case and should be discussed a bit more carefully, possibly with respect to the expected electronic and topographic contrast from the alkane chains and the fluorenyl group. I find the second part of the title not so well justified: " A probe into the complexity of supramolecular copolymerization". It could be that 'supramolecular polymerisation' (It seems like it is a technical term in a significant part of the community, but I admit I was not familiar with it and am not sure if everybody is) is just used as a synonym for 'layer formation' or 'formation/packing of a supramolecular assembly' it may imply that in some cases of polymerization self-organisation will be a prerequisite of polymerization i.e. the formation of a covalent or coordination polymer. In the absence of a bio-catalyst (enzyme) such processes occur at elevated temperatures and in presence of a bio-catalyst it is more the site specifity of the active site. The author's work has enough to offer on its own, this implication is not needed, for sure not in the title while a statement along this line could be added to the conclusion. Thank you once again for your interest in our work and for helping us in improving the manuscript. As you will see, we have made a serious effort to make the manuscript easily accessible to a broad readership.
Modifications to the manuscript can be traced in the "for review only" file indicating via track changes the modifications made.

REVIEWER COMMENTS AND AUTHOR RESPONSE:
Reviewer #1 (Remarks to the Author): 1. The paper describes experiments and supporting theory related to experiments on chirality switching in bilayers of molecules on a graphite surface. This is a comprehensive study and the experimental results and supporting theory appear reliable. However the paper comes across as slightly specialised and as someone with interests in surface assembly but rather peripheral to the sub-field of surface chirality I found it difficult to understand the main points of the paper. Nevertheless I don't question the potential significance of the work and perhaps the authors could overcome some of the issues by revising the paper. In particular I found the 'scene-setting' comments towards the end of page 3, including the diagram Scheme 1 rather difficult to follow.
Author reply: We thank the reviewer for his/her evaluation of our manuscript. 2 We have made substantial changes to both the text and the figures, to clarify the main points, and to make the manuscript more accessible to a wide audience. In particular, Scheme 1 has been revised completely to show how conformational flexibility at the molecular level evolves into diverse self-assembly pathways and eventually leads to the generation of homochirality in near-racemic systems.
2. What is the reference to glycine?
Author reply: The glycine derivative acts as an important reference in this study: 1) Investigations on the glycine derivative are primarily intended to verify the reliability of pathway complexity revealed by theoretical calculations.
2) Considering the similarity between alanine and glycine, investigation on the glycine analogue can act as a good example to show the dramatic effect of methyl side chain on the conformation flexibility of molecular building blocks and therewith the aggregation pathways.
3) For each enantiomer, there are two potential aggregation pathways predicted by the calculation, corresponding to the 1D alignment of two helical conformers. For each pathway, there are two possible supramolecular arrangements, i.e. inclined and near-rectangular. The preferred supramolecular arrangement of the preferred conformer is revealed by STM observations. In contrast, we do not know the assembly behaviour of the less preferred conformer in the absence of experimental observations. However, modelling shows that the energy landscapes of the less preferred conformer of the L-enantiomer (P-type conformer) and D-enantiomer (Mtype conformer) are nearly identical to those of the achiral glycine counterpart. We therefore use the glycine derivative as a reference to predict the preference for spontaneous assembly of the less favoured conformer, if at all it would occur.
3 3. I think in Scheme 1 the authors are trying to get over the idea that a chiral species could include a unit which can undergo conformational changes and pack together either as CW or CCW helix and that both chiral partners could form either CW or CCW helices. I did spend a long time looking at Scheme 1 -a problem is that the helix components don't look very three dimensional so this point is rather confusing. On a related point I didn't understand what was meant by describing one of the pathways as 'silent'.
Author reply: We have changed the scheme. The 'silent' pathway refers to the aggregation pathway which is predicted by theoretical modelling to take place for the energetically less preferred conformer and which in principle could lead to the global minimum state on the energy landscape, but which is not observed by STM for enantiopure systems. We have specified and clarified the meaning of 'silent' pathway -now called "inactive" pathway -in the revised manuscript in the caption of Scheme 1.
4. The discussion of Fig 2 seems clearer -even here there is potential for confusion due to the many acronyms for the different conformations, chiralities and molecules. Also I couldn't follow the arguments at the bottom of p11 -again related to sillent aggregation.
Author reply: As compared to inclined bilayer structures of the pure enantiomers, the achiral analogue FGC18 forms a different monolayer phase, which reflects a near-rectangular alignment and implies that the via theoretical modelling predicted oblique alignment of FGC18 is only a shallow local minimum on the energy landscape. Given the similarity between the energy profiles of FGC18 and the less favoured conformers of FAC18, we envisage that a similar monolayer structure -a phase that is different from the observed bilayer structure of the pure enantiomers -would be obtained upon the spontaneous organization of the less preferred conformers, that is, via a silent (inactive) aggregation pathway. Or looked at it in a different way, the experimental observation of small patches of monolayer δ phase -a near-rectangular network that should in principle only be attained via the 'inactive' pathway -in mixed enantiomer systems is indicative of the presence of the less preferred conformer 4 on the surface. Helical reversal is therefore considered as the most likely reason for the appearance of different phases in the racemic and non-racemic mixtures.
We rewrote the discussion related to the 'silent' ('inactive') pathway. Figure 3 and 4 I can see that there is something interesting going on when both enantiomers are deposited together but can't follow the arguments as to how the origin of the different phases is related to the structures with differing chirality.

On
Author reply: We completely rewrote this section, and developed our arguments in the following way: Experimental observations: • Surface structure of a pure enantiomer shows always the same contrast in a STM image. While L-enantiomer forms exclusively CW bilayers on the surface, only CCW bilayers are observed for the D-enantiomer.
• In contrast to the enantiopure systems, three different types of domains, denoted as λ, ρ and δ, can be identified when a mixture containing both enantiomers is deposited, according to their supramolecular organisation and STM contrast.
• All rows in a ρ domain show the same handedness, which could be CW or CCW.
• A λ domain may contain rows of CW, CCW or both chiralities.
• Small patches of monolayer δ phase are never observed for the pure enantiomers.
Principle of racemate crystallization: • While the chirality/handedness of the supramolecular pattern of an enantiomer is unique (CW or CCW), the self-assembly of a racemate may in principle lead to the formation of two different types of aggregates:

5
• 1) a racemic conglomerate in which a single domain contains only one enantiomer, therewith the organizational chirality of a domain is either CW or CCW, depending on which enantiomer is adsorbed, • 2) a racemic compound, in which both enantiomers are present in the same domain, hence the handedness of the supramolecular pattern in a domain is not determined, and depends on how two enantiomers are organized.
These two typical ways a racemic mixture crystalizes are likely the reason why different phases are observed for the racemate. There is a barrier in between these two organizations on the energy landscapes (see Fig. 2a and 3j).
• Aggregation preference of the less preferred conformer of an enantiomer ("inactive pathway"): the energy profile of the less preferred conformer of an enantiomer is nearly identical to its glycine counterpart. While the near-rectangular organization of the glycine derivative observed by STM is indicative of the fact that the oblique arrangements represent a local minimum which is too shallow to trap the aggregation (see Fig. 2a and 3j), a similar monolayer phase with near-rectangular lattice can be expected from spontaneous aggregation of the less preferred conformer of an enantiomer, which explains the appearance of the δ phase of the racemate. In other words, the presence of the δ phase is indicative of the involvement of the less preferred conformers in aggregation of the racemate.
• CW and CCW bilayers of the racemate: since the preferred molecular conformation and supramolecular organization of two enantiomers are determined to be mirror 6 image related, it is not possible to directly incorporate one enantiomer to the preferred handed lattice of the other one. Therefore, the most feasible way is the compliance of one of both enantiomers to adopt an energetically less favourable twisting molecular conformation and handed organization. As such, CW and CCW bilayers can be obtained with one enantiomer in the bottom layer but the other one in the top layer whereby the L-and D-enantiomers in the two layers possess the same helicity. Theoretical calculations show that a CW (or CCW) supramolecular bilayer of the racemate possesses nearly the same lattice parameters as those of the pure enantiomer, but is ~5 kJ/mol less favoured. Also a small difference in molecular arrangements (Fig. S6) and HOMO (Fig. 4g) is revealed between the supramolecular bilayers of the racemate and the pure enantiomers.
Combining all of the above leads to this conclusion: • λ phase: racemic compound • ρ phase: racemic conglomerate.
6. The conformational freedom available in this molecule certainly leads to interesting effects but the discussion becomes difficult to follow due in part to the many variations and complexity. Perhaps the authors could the revise the paper in a way which makes these arguments in a more accessible manner.
Author reply: We thank the reviewer for the good suggestions. We have made substantial changes to the manuscript, in particular to the scheme and the figures to make sure the paper is easier to read.

Reviewer #3 (Remarks to the Author):
1. Cao and de Feyter present an in-depth study about the self-assembly of a molecule with a complex set of degrees of freedom involving stereo symmetry but also conformational flexibility. This provides a very remarkable milestone in that there is unprecedented insight into the interplay of chirality and conformational changes upon supramolecular organization. This interplay is related to an important molecule and is at least by this reviewer expected to be of general importance in the self organization of molecular material also far beyond the currently investigated system of the chosen Alanine (Amino acid) derivatives. This is a very carefully prepared study and report, both theory and experiment are presented in a very convincing way. I particularly like that the authors chose the achiral analogon of Alanine as the reference material to verify their hypotheses.

Reviewer #2 (Remarks to the Author):
[redacted] 8 Author reply: We thank the reviewer for his/her positive evaluation of our manuscript.
2. The topic of research and the presentation of the results are very well suited for Nature Communications. In contrast, however, I feel that the authors should take more care to explain their results and the conclusions that can be drawn to the readership of Nature Communications from a broad disciplinary background. The current introductory paragraph is rather long and more historic than solution geared after briefly introducing into the scientific challenge.
Author reply: The introductory paragraphs have been modified to emphasize the novelty of this work.
While all the studies thus far focus on the impact of enantiomeric imbalance on the organizing of molecules, in this study we take into account the chemical equilibrium between opposite twisting molecular geometries of the enantiomers and the existence of competing aggregation pathways, and present a molecular level description of the underlying driving forces that lead to the amplification of supramolecular chirality.
3. N.B. the final lines of the Abstract read very nice, but the language is a bit on the specialists side. What I like here is that it starts with 'generation of homochirality in racemic systems' unlike the first line of the article which spans a bridge between life's homochirality (which is a specific feature of synthesis in biomolecular chemistry) and chiral amplification (which if I am right is more a feature of scientific investigation related 9 to supramolecular assemblies and not to biomolecular synthesis) and is less common in nature.
Author reply: We have changed it from 'Such findings highlight the importance of transient molecular conformations, diverse aggregation pathways and competing nucleation and growth processes on the amplification of chirality and supramolecular copolymerization in general.' to 'By establishing a direct link between molecular building block architectures and chiral amplification effect, this study provides a new approach to gain insight into cooperative supramolecular assembly in mixed enantiomer systems.' 4. In my view the 'majority' rules effect is here modified such that the majority forces the minority to change the conformation, to adopt a less favoured conformational state in order to show compliance. This, I repeat myself, deserves to be explained and evidenced with pride of the authors and should be accepted after mandatory revisions for publication in Nature Communications.
Author reply: We have modified Scheme 1 to highlight the compliance of minority conformer/enantiomer in chiral amplification.
Further comments: 5. Given that the brightness of features in the STM images relates to the topography --the brighter the the protrusion, the larger the apparent height -the distinction in contrast of two isolated strands at nearly …. With the long history of STM studies on longer chain aliphatic derivatives -liquid crystals, thiols and more, the referencing could be improved to evidence the identification of 'chemical' (i.e. HOMO / LUMO) and topographic contrast.
I just point at one manuscript here: Y. Qian et al., Langmuir 2003, 19, 6056-6065 Author reply: We have added the reference to support our statement.
6. P16, line 307: …, while the latTer occurs only IN condition of the coverage exceeding one ML. (or similar).
Author reply: The typo has been corrected in the revised version.
7. I agree with the authors that the brightness in the STM images HERE relates to the topography, but this is not generally the case and should be discussed a bit more carefully, possibly with respect to the expected electronic and topographic contrast from the alkane chains and the fluorenyl group.
Author reply: We have compared the supramolecular bilayers of the racemate and the pure enantiomer by means of DFT calculations. Subtle variations in lattice parameters and an energy difference of ~5 kJ/mol were revealed. Since negative bias was applied in this study, therefore isosurface plots of the HOMO to HOMO-3 of the racemate and the pure enantiomers are presented to illustrate the transition in topographic contrast.
8. I find the second part of the title not so well justified: " A probe into the complexity of supramolecular copolymerization". It could be that 'supramolecular polymerisation' (It seems like it is a technical term in a significant part of the community, but I admit I was not familiar with it and am not sure if everybody is) is just used as a synonym for 'layer formation' or 'formation/packing of a supramolecular assembly' it may imply that in some cases of polymerization self-organisation will be a prerequisite of polymerization i.e. the formation of a covalent or coordination polymer. In the absence of a bio-catalyst (enzyme) such processes occur at elevated temperatures and in presence of a bio-catalyst it is more the site specifity of the active site. The author's work has enough to offer on its own, this implication is not needed, for sure not in the title while a statement along this line could be added to the conclusion.
Author reply: The terms 'supermolecular polymerization' and 'supramolecular copolymerization' have both been widely used to describe the formation of non-covalent assemblies in solution, but are less frequently used in surface science. We have changed the second part of the title, from "A probe into the complexity of supramolecular copolymerization" to "A probe into the complexity of cooperative supramolecular assembly" to avoid the misunderstandings. 9. Another suggestion: In the conclusion it may be worth to extrapolate on what molecular building block architectures e.g. other amino acids and beyond, could provide a similar set of properties i.e. an energy for conformational changes that could reduce the 'defect energy' of a racemic crystallization to a pseudo-homochiral one. Also interesting would be a discussion of the energy required for the conformational change as this energy is expected to shift the 'majority rules' guideline to a 'xx% rules' guideline. This could be mentioned together with some statement on how such further insight could be gained in experiment and theory -actually the theory contained in the article here should be able to predict and discuss this issue and I don't recall that I saw it.
Author reply: We thank the reviewer for his/her good suggestions.
We believe that the conformational flexibility of amino acid derivatives is a general fact.
We have compared the opposite twisting forms of a few amino acids (valine, leucine, isoleucine and phenylalanine) analogues with DFT calculations, revealing small energy differences in all these cases.