Parallel Homochiral and Anti‐Parallel Heterochiral Hydrogen‐Bonding Interfaces in Multi‐Helical Abiotic Foldamers

Abstract A hydrogen‐bonding interface between helical aromatic oligoamide foldamers has been designed to promote the folding of a helix‐turn‐helix motif with a head‐to‐tail arrangement of two helices of opposite handedness. This design complements an earlier helix‐turn‐helix motif with a head‐to‐head arrangement of two helices of identical handedness interface. The two motifs were shown to have comparable stability and were combined in a unimolecular tetra‐helix fold constituting the largest abiotic tertiary structure to date.


Foldamerresearchhasshownthatsecondarystructures,such
as isolated helices or b-strands,o ccur in ag reat variety of synthetic backbones. [1] In contrast, the design of tertiary folds is ac onsiderable challenge.T his challenge is worth pursuing because tertiary folding is the level at which sophisticated functions emerge in proteins and the same may be expected for foldamers.The way is being paved by impressive progress in protein design [2] and increasing mastery in programming binding interfaces between peptidic structures,i np articular within peptide helix bundles. [2b,c,3] Forinstance,helix bundles have been reported in peptidomimetics,s uch as b-peptides [4] and b-ureas. [5] We have recently introduced the first abiotic tertiary folds,t hat is,f rom backbones that do not relate to peptides or nucleotides. [6] We used the stable helices formed by aromatic oligoamides of 8-amino-quinolinecarboxylic acid [7] (Q in Figure 1) and 6-aminomethylpyridine carboxylic acid [8] as well-defined modules and introduced hydroxy groups at precise positions at their periphery (X and Y in Figure 1) to promote inter-helix hydrogen bonding with amide carbonyl groups (Figure 2a). Va rious types of helix bundling were observed, including parallel trimers and dimers,a nd tilted dimers. [6,9] As opposed to biotic tertiary folds that form in water and are often driven by hydrophobic effects,t hese folds form in organic solvents.A ll these assemblies were homochiral, that is,t hey involved helices that have the same handedness.F urther progress in tertiary structures design will primarily rest on the orchestration of interactions between secondary folds,afar-from-trivial endeavor.H erein we introduce ab inding interface between helices of opposite handedness.U nlike what was recently shown in heterochiral peptide bundling, [10] we demonstrate the equivalence of parallel homochiral and anti-parallel heterochiral abiotic helix association. We also show how the Figure 1. Structures of units Q, Q h , X, Y, T1, T2,a nd foldamer sequences. X and Y are the protected precursors of hydrogen-bonding units X and Y.Sequences are labelled "a"when protected and "b" when deprotected.Sequences end with an 8-nitro group at their Nterminus:this group is noted in the replacementofthe NH group at N-terminal Q units. The T1 unit constitutes an inversion of C!N sequence polarity;sequences that contain T1 thus have two Ntermini. The arrows indicate the hydrogen-bonding patterns between the helices and point towards the hydrogen-bond acceptor.TMSE = 2-trimethylsilylethyl.
two patterns can be combined within the same tertiary fold without having to consider the stereochemistry at each unit, as it would in apeptide.
Tu rn unit T1 ( Figure 1) has been shown to promote homochiral parallel helix bundling between two identical helical segments attached at their C-terminus as,for example in sequence 1 ( Figure 1). [6] Molecular modelling was used to design 1:i ta llowed us to adjust the positions of hydrogenbond donors and led to the replacement of some X units by Y to avoid possible steric repulsions.Adiagram of the helixhelix interface illustrates how the hydrogen-bond donors and acceptors may face each other (Figure 2c,left). This diagram also shows that hydrogen bonding occurs despite the helices having the same handedness:the slope of the main chain (i.e. the angle between its tangent and aplane perpendicular to the axis) should in principle result in some distance between hydrogen-bond donors and acceptors placed on two helices of identical handedness.Y et the large helix diameter and the resulting moderate slope (ca. 158 8)a re such that hydrogen bonding takes place.A nextension of this observation is that an anti-parallel heterochiral helix dimer (Figure 2c,r ight) should not only give rise to as imilar hydrogen-bonding pattern, but in fact lead to ab etter match between the positions of the donors and acceptors because the helical chains have their tangent parallel to each other at the interface,t hat is,t heir slopes have opposite signs (Figure 2c). [11] To test this prediction, we designed turn unit T2 and sequence 2 ( Figure 1). It should be noted that sequence 2 contains the very same nonameric helix segment as 1 but that one of the two is now attached to the turn at its N-terminus.
Thes ynthesis of the Fmoc-protected version of T2 is described in the Supporting Information. In anticipation of the preparation of long oligomers,wedeveloped asolid-phase fragment condensation (SPFC) approach (Figure 3a). Fragments A and B were synthesized using previously reported solid-phase synthesis (SPS) methods. [8c, 12] Fragment A was then cleaved from the resin, purified and coupled to T2terminated fragment B still on the resin. To prepare 2a,t wo identical fragments were condensed. Using mild resin cleavage conditions,o ligomer 4 was obtained as af ree carboxylic acid with its side chains protections,a nd was then converted into the corresponding methyl ester 2a.
The 1 HNMR spectrum of 2a in CDCl 3 shows two sets of signals,s uggesting the coexistence of PM and PP/MM conformers in solution ( Figure S1a in the Supporting Information), as was previously observed for structures containing T1. [6,9] On the contrary,t he deprotected sequence 2b shows one set of sharp NMR signals including for OH resonances (Figure 3c). Thes pectrum is similar to that of 1 (Figure 3b) Double-headed arrows indicate the different distances between the two hydrogen bonds for both X and Y units, as represented in (c). c) Helical net diagramsd epictinghydrogen-bond interfaces between helices. The arrays of six hydrogen-bond donors (yellow) and acceptors (red) belongingt oX (top and bottom) and Y (middle) units are approximated to belong to two planes facing each other.H ydrogen bonds are shown as dotted black lines. Blue and pink rods represent the rims of the helices and are tilted in different directions according to their P or M helix handedness.Ahydrogen-bond array between parallel homochiralh elices (left) can be transformed in an equivalent array between anti-parallel heterochiralh elices (right). In the plane symmetrical central structure, hydrogen-bond donors (reciprocally acceptors) face each other and no hydrogen bond forms:i nverting either helix handedness or sequence orientation makes hydrogen bonding impossible, whereas changing both is aproductive transformation.T he C 2 axis in the diagram at left is best seen in the crystal structure shown in Figure 4a. . Extract of 1 HNMR spectra (500 MHz, CDCl 3 )s howing the NH and OH resonances of 1 [9] (b), 2b (c) and 3b (d). The red dots indicate signals corresponding to OH protons. and indicative of aw ell-folded helix-turn-helix motif.A crystal structure of 2b confirmed the formation of the antiparallel heterochiral helix dimer (Figure 4b). Ther esemblance of the hydrogen-bonding interface in this structure with that of 1 (Figure 4a), is striking.D espite the change of one helix handedness and orientation, the hydrogen-bond donors and acceptors are found at very similar positions ( Figures S2-S3). Thestability of the hydrogen-bonding interfaces was then assessed upon monitoring the effect adding [D 6 ]DMSO into CDCl 3 solutions.B ecause of the rigidity of the aromatic helices,chelate effects are observed between the hydrogen-bonding units that results in an all-or-nothing behavior:t he six hydrogen bonds are disrupted all at once through as mall change of DMSO concentration. Remarkably,t his transition occurred with identical amounts of DMSO (ca. 20 %v ol/vol, Figures S4-S7) for 1 and 2b, showing comparable strengths of the parallel homochiral and anti-parallel heterochiral hydrogen-bonding interfaces.
Differences from peptide helical bundles should be noted: in peptides,b undling is mostly known between a-helices of identical handedness which, to best match at their binding interfaces,g enerally coil around one another. Studies on heterochiral peptide helix bundling [10] and in particular some recent work by Gellman et al., [10a,b] show that homochiral and heterochiral peptide helix interfaces are not equivalent, notably because coil-coiling is not conducive of better complementarity in heterochiral bundles.I nc ontrast, the rigidity of the aromatic helices hampers coiling, at least over short distances,and strictly parallel arrangements form when mediated by turn units,such as T1 and T2.However,aromatic helices may slightly change their local curvature,s oa st o optimize inter-helix interactions:h elix curvature in the structures of 1 and 2b is not rigorously constant and identical to that of relaxed helices not involved in bundling.
In compounds 1 and 2b,h elix handedness control is relative,n ot absolute,a nd guided only by strand orientation as imposed by the turn unit, and by hydrogen-bonding complementarity.T his should in principle allow for the combination of both parallel-homochiral and anti-parallelheterochiral motifs in the same tertiary structure without having to consider the nature of stereogenic centers at each unit as it would in peptides.W ec hallenged this possibility through the design of sequence 3b ( Figure 1). As shown in Figure 4c,d, 3b is expected to fold in as equence of four contiguous helices having either identical or opposite handedness depending on whether they are separated by T1 or T2, with the central YXXQ h Y helical segments each bearing two independent hydrogen-bonding interfaces,o ne homochiral, Figure 4. Crystal structures of compounds 1 [6] (a) and 2b (b). Front view (c) and top view (d) of energy-minimized molecularm odel of 3b. Cartoons indicate helix handedness, Cand N-termini. Side chains of Q, Q h , T1,and T2,included solvent molecules and most hydrogen atoms have been omitted for clarity.F or crystallographic details and the CCDC number see the SupportingI nformation. and one heterochiral. In the design of 3b,w em ade use of helices of different length to avoid creating an extended aromatic surface that might promote aggregation and reduce solubility.S imilarly,w ei ntroduced Q h units with al onger branched alkyl chain inside the sequence to promote solubility ( Figure 1). Oligomer 3a was synthesized combining SPFC (Figure 3a)a nd solution-phase synthesis for the final coupling of T1 with 5.T he 1 HNMR spectrum of protected compound 3a was complex due to the presence of multiple turn units (Figure S1 b) and thus of different conformational stereoisomers (PMMP, PMMM, MMMM, PMPM, PMPP, MMPP and their enantiomers). After removal of the side chain protecting groups,asharp spectrum with only one set of signals was observed for 3b (Figure 3d). Even though an unambiguous structure elucidation could not be achieved in solution or in the solid state,t hese observations altogether suggest that 3b is present in solution in awell-defined folded conformation.
In conclusion, we have introduced an ew well-defined abiotic helix-helix hydrogen-bonding interface and showed that tertiary structures combining different interfaces can be designed, resulting in predictable helix-turn-helix structures composed of helices of different handedness and orientation, ap attern difficult to reach with simple peptides.W ea re currently expanding this work to interfaces between tilted, that is,n on-parallel, helix multimers and will report our progress in due course.
is either in front of or behind the planar helical net diagrams shown. Complementarity also exists with respect to this positioning:w hen one donor lies below the plane shown in the scheme,the acceptor to which it hydrogen bonds lies above the other plane.T he transformationt hat consists in inverting both the handedness and the C!No rientation of ah elix also preservesthe positionning of the acceptors and donors above or below the planes shown in the schemes.