A Hybrid Solid‐State NMR and Electron Microscopy Structure‐Determination Protocol for Engineering Advanced para‐Crystalline Optical Materials

Abstract Hybrid magic‐angle spinning (MAS) NMR spectroscopy and TEM were demonstrated for de novo structure determination of para‐crystalline materials with a bioinspired fused naphthalene diimide (NDI)–salphen–phenazine prototype light‐harvesting compound. Starting from chiral building blocks with C 2 molecular symmetry, the asymmetric unit was determined by MAS NMR spectroscopy, index low‐resolution TEM diffraction data, and resolve reflection conditions, and for the first time the ability to determine the space group from reciprocal space data using this hybrid approach was shown. Transfer of molecular C 2 symmetry into P2/c packing symmetry provided a connection across length scales to overcome both lack of long‐range order and missing diffraction‐phase information. Refinement with heteronuclear distance constraints confirmed the racemic P2/c packing that was scaffolded by molecular recognition of salphen zinc in a pseudo‐octahedral environment with bromide and with alkyl chains folding along the phenazine. The NDI light‐harvesting stacks ran orthogonal to the intermolecular electric dipole moment present in the solid. Finally, the orientation of flexible lamellae on an electrode surface was determined.

Abstract: Hybrid magic-angle spinning (MAS)N MR spectroscopy and TEM were demonstrated ford en ovo structure determination of para-crystalline materials with ab ioinspiredf used naphthalened iimide( NDI)-salphen-phenazine prototype light-harvesting compound. Startingf rom chiral buildingb locks with C 2 molecular symmetry,t he asymmetric unit wasd etermined by MAS NMR spectroscopy,i ndex low-resolutionT EM diffraction data,a nd resolve reflection conditions, and for the first time the ability to determine the space group from reciprocal space data using this hybrid approach was shown. Transfer of molecular C 2 symmetry into P2/c packing symmetryp rovided ac onnection across length scales to overcome both lack of long-range order and missingdiffraction-phase information. Refinement with heteronuclear distance constraints confirmed the racemic P2/c packing that was scaffolded by molecular recognition of salphen zinc in ap seudo-octahedral environment with bromide and with alkyl chains folding along the phenazine.T he NDI light-harvesting stacks ran orthogonal to the intermolecular electric dipole moment present in the solid. Finally,t he orientation of flexible lamellaeo na ne lectrode surface was determined.
Chemical self-assembly to bridget he gap from dead to living matter is ac hallenging field. Controlling complexity,f lexibility, and functionalityo fs ynthetic and biomimetic materialr equires [1] engineering soft para-crystalline compoundst hat lack crystalline long-range order in at least one dimension. However,r esolving their microscopic and mesoscopic order at atomic resolution from the anisotropic background heterogeneity has proven to be quite difficult. Only recently has the hybridb ottom-up magic-angle spinning (MAS) NMR spectroscopy and top-downe lectron microscopy (EM) structure-determinationmethodology shownp romise to overcome the limitations of either technique. [2] MAS NMR spectroscopy is intrinsically am icroscopic method [3] that cannot solve as tructure de novo because packing order is determined by minimizing steric hindrance with screw axes or glide planes at higher levels in the structural hierarchy.F or small organic molecules forming microcrystals, modeling protocols with ac onjectured rathert han determined space group are used, making MAS NMR spectroscopy ac hemical shift filter for selectiona nd validation.
Earlier,w eshowed fort he largest biological (protein-free and grossly heterogeneous) light antennae that the limitations of MAS NMR spectroscopy can be diminished by using a2 D TEM periodogram as ab and-pass filter to resolve sparse regions of nonzero intensity in reciprocal space. [4] Here we present the important next step by showing that genuines pacegroupi nformationc an be extracted from TEM data in reciprocal space.
To demonstrate this, we use ap rototypical engineered biomimeticc hromophore light-harvesting material that consistso f fused Br-substituted naphthalene diimide (NDI), phenazine, and Zn-salphen buildingb locks ( Figure 1). [5] This system, denoted as DATZnS(3'-NMe), modelst he chlorosome that is built from parallel stacks of bacteriochlorophyll (BChl) connected by metalc oordination in ar ecognitionm otif. The BChl stacks form polarizable curveds heetsa nd tubes that are thought to yield ad ielectric response upon excitation with crossingo f energyl evels and coherent mixing of excitons tates for energy transport. Although the model is chemically unrelated to BChl, we show that it self-assembles into stacks that form extended polar curveds heets like the naturalp aradigm. The NDI of the model is capable of overlap,t he phenazine carries the electric dipole,and the Zn-salphen provides ar ecognition motif for coordination. [6] The molecular symmetry and asymmetricu nit were determined from MAS NMR shifts collectedw ith 13 Ca tn atural abundance. We indexed the TEM diffraction and determined both the unit-cellparameters and agenuine space group by analysis of systematic absences in as parse reciprocal space-intensity pattern with the help of the molecular-symmetry information obtainedb yM AS NMR spectroscopy.T he technologiesw ere then merged at the molecular level and unit cell. Because indexing of an unknown structure is not possible by TEM alone, the diffraction-phase problem was implicitly overcome by connectinga cross length scales through transfer of molecular symmetry to packing symmetry.T his provided as tructural underpinning for engineering supramolecular material in the desired sheets with parallela ligned dipoles. Fourier-transform filtering in reciprocal space averaged static heterogeneity in real space and enabled extrapolatingt he short-and medium-range ordering in a para-crystalline lattice to establish afull 3D lattice model,w hich we refinedw ith MAS NMR heteronuclear 1 H-13 C correlation data. Finally,w ed etermined the orientation of DATZnS(3'-NMe) on as urfacet or elate to the geometry of biomimeticmaterialine lectrode applications.
The symmetric NMR response of the DATZnS(3'-NMe) provided conclusive evidence that the asymmetric unit was half of the molecule ( Figure S1, Table S1 in the Supporting Information). Of the two possible configurations, the syn form with am irror plane running along the centero ft he phenazine motif was approximately 10 kcal mol À1 less stable than the anti form that had atwofold axis. [7] This was the first step in our approach.
High-resolution TEM of the system on acarbon grid revealed curved lamellae (Figure 2A). The Fourier transform showed strong centrosymmetric reflectionsa t1 /1.685 nm À1 and perpendicularly as eries of spots at 1/0.547 nm À1 with as ystematic absence indicating h0l (l = 2n)r eflections ( Figure 2B). Both strong features were attributed to first-order reflectionsa nd pointedt ot he molecule along the 1.685 nm direction with its C2a xis representing P2p acking symmetry.T he systematic absence revealed ana dditional translation with as crew axis or glide plane.Ascrew in ad ifferent direction would imply an orthorhombic cell, in contrast with EM images that revealed am onoclinic cell with intensity at 1/1.24 nm À1 that was attributed to second-order reflectionsf or ar ealistic density of 1.67 gcm À2 ( Figure S5 in the Supporting Information). This left ag lide plane that explained both the systematic absence at 1/ 0.547 nm À1 in Figure 2a nd the absence of first-order reflections in Figure S5 in the Supporting Information. The mirror operation produced ar acemic packing, with the wings of the salphen forming enantiomeric chiral L and D pairs. [10] This led to a P2/c space group with four inequivalent sites, two from the twofold axis in the DATZnS(3'-NMe) and two from the enantiomeric pair.T his represents the second step of our approach,i nw hich we overcame the diffraction-phase problem and indexedt he TEM data to resolve ag enuine space group. Considering the weakness of the reflection spots in Figure S5 in the SupportingI nformation, there could be other polymorphsa sw ell, but these did not pass the TEM diffraction filter.
The P2/c structure was modeled with unit-cell dimensions a = 0.547 nm, b = 1.685 nm, c = 2.517 nm, and b = 1028,d etermined with TEM to an energy of 170.5 kcal mol À1 (see S10 in the Supporting Informationf or details). To validate the structure, we performed ano ptimization without constraining the cell, which led to virtually the same result. Next, long-range transfer signals were identified in ah eteronuclear 1 H-13 Cd ataset recorded with al ong contact time of 4msb yc omparing with data collected for as hort mixing time ( Figure S3 in the Supporting Information) and observed between the 3' 1 ,3 ' 2 , 3'' 1 ,a nd 3'' 2 protons of salphen and the 4, 5 13 Cn uclei in the NDI motif ( Figure 3). Transfer involving 3'-NMe and 4, 5 13 Cprovided strong NMR evidence for molecular recognition between the NDI parto ft he molecule and the salphen motif of an adjacent molecule. The transfer of polarization,that is observed between protons on the alkyl chain and the 11,1 2, 3b,5 b,1 3 a, 9a,1 4 b,8 a,1 0, and 13 13 Cn uclei on the phenazine backbone, positions the alkyl chain in the packing.T he buildupo fL ee-Goldburg cross-polaization (LGCP) signals for the 4, 5a nd 13a, and 9a 13 Cn uclei from Figure S4 in the Supporting Information was in line with as imulation of transfer over approximately 4 . ( Figure 4). [11] The 3b,5 b 13 Cn uclei in the central part of the NDI motif correlated with the 2 1 and 7 1 CH 2 protons. This revealed the formation of slipped J-aggregatesf or the NDI, with the alkyl tail above the plane of an eighbouring molecule. It implied that the correlationsa nd buildupf rom protons at the alkyl chain to 13a,9 a 13 Cn uclei were also intermolecular.S imi- larly,b uildup of CP intensity from the alkyl CH 2 to the quaternary 13 Co nt he phenazine core could be considered intermolecular from the abundantc loud of protons on the alkyl chain.
Strong p-p stacking interactions and aligned electric dipoles explained why the materialh ad ah igh density,alow energy, and wasi nsoluble. The aliphatic tails were oriented in the same direction as the salphen wings andw ere situated in voids between the phenazine moieties. Tight packing with the alkyl chains folded along the phenazine bridge of an eighboring molecule explained the observation of strong heteronu-  [8] (D) simulated diffraction pattern obtained with the NMR-derived geometry in the P2/c space group; [9] (E) orientation of DATZnS(3'-NMe) on the carbon grid (orange color); (F) viewed along the b axis;a nd (G) along the a axis.  (Figure 3). The molecular recognition and distortedo ctahedral surrounding of the Zn 2 + ion put the 3'-NMe of salphen at 0.45 nm from the 4, 5 13 Cn uclei in NDI, which was in quantitative agreement with the LGCP buildup kinetics and its simulation. This refinement represents the third step in our approach.
In our final and fourth step we determined the orientation of the material on the surfacebysimulating the diffraction pattern, thereby validating the indexing and space group. Av iew along the 0.69, 0, 0.69 lattice vector yielded the best matchf or the density and the diffractionp attern ( Figure 2C,D ). [9] The analysisv alidated the systematic absence of reflections from the c-glide plane in the P2/c space group and showed that À101 and 10À1w ere quenched ( Figure 2B). Thes trong 010 and 0 À10 originated from lamellar spacing and alternating regions of Zn-salphen and NDI. Thep henazine dipoles were aligneda long the surfacea nd were perpendicular to the NDI stacks that ran parallelt ot he surfacewith the plane of the NDI rings at an angle of 458 (Figures2E, F). Figure 5s hows how molecular recognitionl eads to at ransfer of molecular symmetry for scaffolding. Whereas steric hindrance favors screw axes or glide planes( symmetry operations with at ranslational component) to allow for interpenetration of symmetry-related molecules, the screw axis is apparently suppressed in DATZnS(3'-NMe) in favour of at wofold axis to accommodate intramolecular C 2 symmetry ( Figure 5A). This is possible because of the rich structural variability introduced with the nonplanar metal salphen. It allows forp acking in an achiral P2/c space group with ar acemic mixture of the two enantiomeric species, thereby circumventing the need for as crew axis in favour of a cglide plane with inversion symmetry in the structure.
With C 2 molecular symmetryp reserved, DATZnS(3'-NMe) selfassembles into polar planesw ithout inversion symmetry elements, thereby mimicking the parallel stacking in the chlorosome antenna ( Figure 5B), for which the selectivity induced by chirality emerges at the salphen motif. The electric dipoles align and form extendeda rrays with ap ositive and an egative side to support charge separation following light absorption in the NDI columns running perpendicular to the electric-field direction( Figure 5). DATZnS(3'-NMe) forms extended chiral layers in the proposed 3D model, arisingf rom planar arrangements of individual C 2 motifs (known as organizational chirality), in which the net dipole moment is canceled owing to antiparallel layers.
In conclusion, we have demonstrated hybrid MAS NMR spectroscopy and TEM for de novo structure determinationofabioinspired para-crystalline material. The concept can be further developed with, for example, pattern recognitiona cross TEM and NMR datasets to facilitate the applicability and broaden the scope. This paves the wayf or structure determination of advanced organics upramolecular materials that bridge the gap from dead to living matter and are inaccessible to highresolution diffraction methods. LGCP buildup curves of the polarizationt ransfer to 4/5 (red) and 13a/9a (green) carbon nuclei compared with asimulatedbuildup for aheteronuclear 1 H-13 Cs pin pair separated by approximately4 (blue). The buildup curves representing 3 (black solid lines) and 5 (black dotted lines) are also shown.