Determination of Controlled Self‐Assembly of a Paracrystalline Material by Homology Modelling with Hybrid NMR and TEM

Abstract Controlling complexity, flexibility, and functionality of synthetic and biomimetic materials requires insight into how molecular functionalities can be exploited for steering their packing. A fused NDI‐salphen (NDI=naphthalene diimide) prototypic artificial photosynthesis material, DATZnS, is shown to be comprised of a phenazine motif, in which the alignment of electric dipole moments in a P2/c supramolecular scaffold can be modulated with bulky substituents. They can also be switched between parallel stacks of dipoles running antiparallel in the DATZnS‐H compared with parallel stacks of dipoles in polar layers running in opposite directions in the DATZnS(3′‐NMe) parent compound. Spatial correlations obtained from HETCOR spectra, collected with a long cross polarization contact time of 2 ms, reveal an antiparallel stacking for the DATZnS‐H homologue. These constraints and limited data from TEM are used to construct a structural model within the P2/c space group determined by the molecular C 2 symmetry. By using homology modelling, a pseudo octahedral coordination of the Zn is shown to follow the packing‐induced chirality with enantiomeric pairs of the Λ and Δ forms alternating along antiparallel stacks. The model helps to understand how the steric hindrance modulates the self‐assembly in this novel class of fused materials by steric hindrance at the molecular level.


Introduction
Spurred by the need for az ero-emission-energy system in the near future, scientists are working to mimic the primary processes of photosynthesis. [1] Even though nature has given the blueprintsf or systems to convert energy from sunlight through photosynthesis,aprincipal challenge lies in finding suitable molecules that mimic the photochemical characteristics of natural photosynthesis for application in durablea rtificial systems by controlled self-assembly. [1g, 2] Recently,astructure-based strategy was presented for the rational de novo design of biomimetic paracrystalline optical materials for application in molecular artificial photosynthesis electrodes. By applying TEM filtering in association with magic angle spinning (MAS)N MR, short-and medium-range order was resolved and used to construct ah igh-resolution packingm odel for paracrystalline fused naphthalene diimide( NDI)-salphen-phenazine (DATZnS(3'-NMe)), prepared by fusing NDI with Zn-salphen, thereby generating ap henazinebridge. [3] This material is ab ioinspired prototypic artificial photosynthetic reaction center.I t exhibits the properties of Zn-salphen to form am etal-organic framework, whereas the NDI and the phenazine can be involvedi ne nhanced p-p stacking. The wavelength at which photonsa re absorbed in the dyad can be adjusted with the functional groups so that it can cover the entire solar spectrum.O pto-electronict unability [4] of the molecule makes this fused materialaversatile building block for chemical engineering purposes. [5] The aliphatict ails enhance the solubility of chemicalp recursors and allow for modification at al ater stage, for example, by using them as al inker to attach self-assembled antenna systemst of unctionalized electrode surfaces. The vacanto rbitals present in the Zn 2 + of the salphen can also accommodate an additional ligand.F or instance,acatalyst can be attached through ac oordinationb ondt ot he scaffold. [6] Compact p-p stacking arising from the phenazine helps to form ar igid scaffold to attach such catalysts. [7] The phenazine aromatic surfaceisflat and electron rich. [8] This createsthe possibilityo fa ttractive van derW aals and charge-transfer interactions similar to supramolecular assemblies built from porphyrins. [9] The properties and applicationso ft heseo rganometallic frameworks can be altered by changing the Zn 2 + with other metals or by introducing af unctionalizedl igand. Considering that the fused salphen-phenazine-NDI motif hasa ne lectric dipolem oment, this provides ah andle for functionalization and chemical engineering of the electronic properties by modulated self-assembly from steric controla tt he molecular level to steer the packing.
To mimic the engineering principles of nature as well as investigatea nd optimize the functional properties using theoretical andc omputational studies ahead of experimental realization, we have to understand how the dimensions and shapes of self-assemblies are determined by steric and electronicpacking factors. [10] The DATZnS-Rm odel system with R = H, 3'-NMe was set up de novo to mimic ac hemically unrelated bacteriochlorophyll light-harvesting system in natural chlorosomes and can be prepared with diverse functional groupst os teer the packing. DATZnS(3'-NMe) forms parallel stacks and antiparallel polar sheetsb ys teric control to modulate the self-assembly.T ransfer of C 2 molecular symmetry into packing symmetry was identified to be predominant in establishing a P2/c packing. Here we resolve aw ell-determined molecular mechanism for controlling the formation of arrays of electric dipolesbyd etermining ah igh-resolution model of ac hemical homologue, DATZnS-H, which belongs to the same class of fused novel chromophore materials ( Figure 1). [11] Computational integration of MAS NMR spectroscopy with Cryo-EM periodograms is opening an ew horizon to high-reso-lution visualization of supramolecular structures with well-defined scaffolding and intrinsic heterogeneity at the molecular level. [10,12] By using TEMa safilter to determiner eflection conditions, along with chemical shift information and distance constraints obtained by cross-polarizationM AS (CP/MAS) NMR 1 H- 13 Ch eteronuclear correlation spectroscopy data, short-and medium-range order can be resolved to determinet he space group and extrapolatedt os imulate ah igh-resolution model for the supramolecular organization of ap aracrystalline material. In this work, we extend the concept with ah omologym odeling step that allows to resolve how the paracrystalline packing adapts to structural variability.W ed etermine the effect of the 3'-NMe functionality at the molecular scale on the shortrange order.I ntermolecular 1 H-13 Cd istance constraints are obtained from frequency-switchedL ee-Goldburg (FSLG) heteronuclear correlation data collected with longer cross polarization contact times from the unlabeled material. [13] The NMR constraints and DFT simulations point to C 2 molecular symmetry of the DATZnS-H, which can be accommodated with aracemic packing of the delta and lambda forms in the P2/c unit cell with the two-fold crystal symmetry axis running along the C 2 molecular symmetry axis that was determined for the parentc ompound. The packing and unit cell parameters were optimized using molecular mechanics, and the 1 H-13 Cc onstraints and ap rincipal reflection in reciprocal space deduced from the electron microscopy were reproducedt ov alidate the packing. The data provide converging evidence that steric control by the bulky 3'-NMe substituent allows for as witch between parallel and antiparallel stacking of the DATZnS motif.

Results
Moderately sized molecules like DATZnS-Rw ith low symmetry and few hydrogens on the extendednetwork of conjugated aromaticr ings, make it feasible to identify selectivei ntermolecular polarization transfer events, to spatially probe the structure. [13] For the DATZnS-H, 13 C1 DCP/MAS NMR resonances are well dispersed, over 170 ppm, due to different functionalities, because there are four carbonyl groups, two phenoxyr ings, and two imide groups present in the fused NDI-zinc-salphenbased chromophore( S1). Narrow NMR lines pointt oawell ordered microstructure.
The 13 Cc hemical shifts s C solid of the DATZnS-H were tentatively assignedb ya nalogy to the NMR response of DATZnS(3'-NMe),a nd the assignment was validated with computational chemicals hifts, which are well in line with the data (Table 1). [1g] Assignments of the primary,s econdary and tertiary carbon atoms can be confirmed with the aid of 1 H-13 Ch eteronuclear dipolar correlation dataacquired with ashort crosspolarization contact time of 0.256 ms, to limit long range intermolecular transfer (S2). For the 13 C, the aliphatic response is shown between 10 ppm and 50 ppm, whereas the aromatic region covers the range from 100 to 170 ppm. The aromatic CH protons are partially resolved in the 1 H-13 Cs pectra ( Figure 2). The terminal methyl group on the alkyl chain has ac haracteristic shift of 13.7 ppm, whereas the alkyl group attached to the electronegative nitrogen resonates with ac hemical shift of 40.7 ppm. The resolved signal around 22.6 ppm is attributed to the 2 7 ,7 7 responses that are essentially indistinguishable,c onfirming at ranslation of C 2 symmetry into packing symmetry with at wofolda xis. The 13, 10 in the phenazine ring are also symmetry relateda nd have characteristic 13 Cc hemical shifts of 100.1 ppm.
In the 1 H-13 Cd ata collectedw ith ac ross polarization contact time of 2ms, there are many long-range correlation signals arising from heteronuclear dipolar transfer between proton and carbon nuclei, which can be intermolecular or between different parts of the DATZnS-Hm olecule ( Figure 2). [12] The protons attached to the tertiary carbon nuclei are well-resolved in the 2D data collected with as hort cross polarization contact time of 256 ms( Figure S2). The differencei si mportant for the selectived etection of long-range correlations at ac ross polarization time of 2ms, to resolve the spatialr earrangement of the DATZnS-H molecules in the P2/c packing relative to the structure of the parent compound.

Discussion
Considering that the two halves of the molecule yield only one set of NMR signals, the data reveal molecular symmetry,e ither achiral s v or chiral C 2 ,w ith the latter being the lowest in energy accordingt ot he DFTc alculations. Hence, both NMR and modeling favor at wo-fold molecular axis, whereas the molecules shouldf orm lamellar packing according to the TEM. To arrive at ah igh-resolution model for the packing, we follow ah omology approach startingf rom the DATZnS(3'-NMe) structure published previously. [1g] The DATZnS-H was positioned in the cell on the two-fold axis. Twos ettings were considered, the originals etting with parallel stacking and as etting, in which the a and c axis were interchanged to obtain as tructure with antiparallel stacking ( Figure S4). Optimizations of unit cell parameters and molecule were performed for both settings and it was found that the antiparallel stacking is 37 kcal mol À1 lower in energy with parameters a = 1.47 nm, b = 1.83 nm, c = 9.6 nm, a = 908, b = 1098 and g = 908 (Figure 4).
The extensive polarization transfer to 4, 5, 3, 6, 3b, and 5b from aromatic protons on the salphen part to the NDI part indicatedi nF igure 2w ith ac ircle validates the antiparallel stacking, which hass hort distances of % 3.5-4 between the salphen protons and NDI carbonscorrespondingw ith the transfer range for heteronuclear correlation signals determined with quantitative simulations of the LGCP signal buildup curve. [1g] The alternative, recognitionb etween the NDI and the salphen part with extended overlap in the parallel stack model is difficult to reconcile with the characteristic heteronuclear transfer in Figure 2. This contrasts with the parallels tacking deduced for the DATZnS(3'-NMe) homologue, in which only correlations were observedb etween the NDI motif and the dimethylamine functionalities, which are proposed to play ad ecisiver ole in steering the packing configurationo ft he latter compound. When parallel stacking is imposed for the DATZnS-H, not only the enthalpy is highert han for antiparallel stacking because of steric hindrance, but also the correlations detected in Figure 2 would requiret ransfer over larger distances beyond5,w hich is unlikely.F inally,t he heteronuclear correlation data for the DATZnS(3'-NMe) homologued on ot exhibit the characteristic correlationsi ndicated with the circle in Figure 2. [1g] Thus, the heteronuclear transfer from 1 Ho ft he salphen to 13 Co ft he NDI can be considered ad ecisive characteristic for structure determinationt od istinguish between parallel and antiparallel stacking.
The distance between two molecules in the direction perpendicular to the plane of the molecule is 4 , [14] indicating strong p-p stacking. The tails are projecting outwards into the space between two stacks ( Figure 4B). The presence of the carbonyl groups and the nitrogen in the NDI with the lone pair induces an extended p-electron delocalization. [15] The electropositive Zn 2 + in the L and D chiral salphen functionalities produce ac avity to which the electronegative bromine of the  .Consideringthat the only difference between the two compounds are the NCH 3 functional groups, chemical control over the dielectric characteristicsa tt he supramolecular level is achieved. Chem. Eur.J.2017, 23,9 346 -9351 www.chemeurj.org 2017 The Authors. Published by Wiley-VCH Verlag GmbH &Co. KGaA, Weinheim next molecule along the b axis is attracted to form the pseudo-octahedral recognition motif, similar to the DATZnS(3'-NMe) homologue (Figure2 and 4B). This electrostatic attraction can stabilize the packing along the axial direction of the molecule. Thep seudo octahedral coordination of the Zn follows the packing induced chirality with enantiomeric pairs of the L and D forms alternating along the antiparallel stack.
In the TEM pattern of Figure 3, the stripes are from alternating regionso fh ighe lectron density containing Zn 2 + with less electron-dense regions of phenazine. Along the antiparallel stack the dipole moments are aligned in opposite directions, which probably constitutes al essr obust scaffold of the packing than for the DATZnS(3'-NMe) parent compound. With the dipolesc ompensated at the level of the antiparallel arrangement within the stack, there is no emerginge lectrostatic componentf or stabilization of planes. This may explain why the DATZnS-H structure appears much less ordered in the TEM, with only the two reflectionso ft he long repeata long the c axis in the Fourier transform. Tightp acking of dyads within the self-assembled stacks providesahigh chromophore density to harvest the solar light efficiently. The pseudo-octahedral recognition motif is ac haracteristic of the supramolecular packing, and is consistentw ith the antiparallel arrangement that transpires from the spatial correlation peaks between salphen and NDI. The structure accommodates the folding of the tails along the phenazine, in line with the 1 H-13 Cd ipolar correlation data collected with the longer cross polarization contact time of 2ms. Steric hindrance from the salphen part restricts parallels tacking. Polymorph analysis shows that the p-p stacking motif remains intact, and it remains as the basic building block. The simulation of the TEM diffraction pattern from the high resolution model generatest he strong pairf rom the lamellar arrangement as observed in the experiment with the distinctr epetition of 1/1.8 nm (data not shown). [16] Conclusions Based on the energy,d ensity,i ntermolecularc orrelations from CP/MAS,r eflection spots in the Fouriert ransform of the TEM image, and homology modelling, we converge upon an antiparallel stacking for DATZnS-Hf orming lamellar sheets in a P2/ c packing arrangement. Pseudo-octahedral coordination of the Zn 2 + and C 2 molecular symmetry produces the twofold axis, whereas the two enantiomeric forms L and D produced by a c-glide symmetry operation lead to ar acemic packing with the alkyl chainsf olded along the phenazine. Thiss tructure is ah omologue of the DATZnS(3'-NMe) structure, and suggests that the NCH 3 functional group can be used to steer the aggregation from ap arallel sheet in DATZnS(3'-NMe) to an antiparallel sheet in DATZnS-H. Hencet he packing of fused NDIsalphen chromophoresf orming ap henazine motif with ad ipole moment in a P2/c supramolecular scaffold can be steeredb yc hemical substituents between antiparallel dipoles and parallel dipolesi nasheet. This concept paves the wayf or the chemical design of supramolecular scaffolds for light harvestinga nd charges eparation, on the way to organic solar fuel cell device concepts that can be programmed to quench the internal field in an antiparallel arrangement, for example, for light harvesting, or exploit the internal field from aligned dipole moments, for example, for the injection of chargei n catalysts for water splitting. 8,0.13 mmol), prepared from 2,3-(p-toluenesulfonamido) -8,9-dibromo-5,12-dihydro-5,12-diazatetracene din-octylimide was dissolved in dry,d egassed DMF (9 mL) under an Ar atmosphere and heated to 80 8Ci nt he dark. [3a] In as eparate flask, salicylaldehyde (40 mg, 0.33 mmol) and zinc acetate dihydrate (265 mg, 1.32 mmol) were dissolved in dry,d egassed DMF (5 mL) and kept under Ar.T his mixture was stirred for 5m inutes and added to the hot DMF solution by means of as yringe. After 6 hours, the reaction mixture was cooled down to room temperature, diluted with 5mLH 2 Oa nd stored overnight at À20 8Ct o induce precipitation. The dark blue precipitate was collected on af ilter and washed with water,e thanol, and dichloromethane to afford (after vacuum drying) 89 mg of ad ark blue solid (59 %y ield from 8,9-dibromo-5,12-dihydro-5,12-diazatetracene di-n-octylimidediamine). M.p. > 300 8C; IR (ATR FTIR): ñ = 2953, 2922,2851,1686,1572,1528,1499,1448,1431,1315,1281,1225,1200,1173,1150,1128,1105,1076,1030,964,914,847, 584 cm À1 .

Experimental Section
The solid-state CP/MAS spectra were recorded with aB ruker AV-750 MHz spectrometer,e quipped with 4mmt riple resonance CP/ MAS probes in dual 1 H-13 Cm ode with as pinning frequency of 13 kHz AE 5Hz. The data were collected at as ample temperature of 298 K. The magic angle was set using the 79 Br resonance from KBr. The pulse sequence for the 2D heteronuclear polarization transfer experiment is shown in Figure S5. The 2D sequence starts with am agic angle preparation pulse on the 1 Hc hannel. After that protons are allowed to evolve for time t 1 with Phase Modulated Lee-Goldburg irradiation to suppress 1 Hh omonuclear dipolar couplings. This is followed by transfer of the magnetization from the protons to carbons by ac ross polarization step. [13,17] The two-pulse phase modulation (TPPM) Scheme was used to decouple proton spins during acquisition while the 13 Cf ree induction decays (FIDs) were recorded in the t 2 domain. [18] For short-range polarization transfer from 1 Hd irectly bonded to 13 C, across-polarization contact time of 0.256 ms was used, and for longer range polarization transfer and the detection of intermolecular correlations, ac ross-polarization contact time of 2msw as employed. The 1 Hc hemical shift was calibrated with a 1 H-13 Cs pectrum of solid tyrosine.HCl [17b] and as cale factor of 0.57 for the FSLG was verified. The data were processed using Bruker To pSpin 3.2 software (Bruker,B illerica, MA). 128 scans were collected for each of the 128 steps in the 1 Hdimension.
Computational chemical shifts were obtained with the Gaussian 03 software package (Gaussian, Inc.,W allingford, CT) using the Becke, Lee, Yang, and Parr (BLYP) exchange-correlation functional with 6-311G basis set. [19] The molecule was geometrically optimized prior to NMR chemical shift calculation.
Biovia Materials Studio Suite (Biovia, San Diego, CA) was used for computational modeling. Am onomer structure of the DATZnS-H core without the aliphatic tails was obtained by optimization with Gaussian 03 (Gaussian, Inc.,W allingford, CT) and placed in the P2/ c unit cell determined for the homologue as described in the results section. Optimization of the cell parameters was performed with the FORCITE module. For geometry optimization the "smart" algorithm setting was used and ac onvergence tolerance of 0.001 kcal mol À1 for energy and 0.5 kcal mol À1 À1 for the force were applied. For the full molecule including the tails, DMol 3 calculations were conducted to estimate the ESP charges. The generalized gradient approximation (GGA) with the Perdew-Burke-Ernzerhof (PBE) functional with double numerical atomic orbital augmented by ap olarization p-function (DNP) basis set was used for DMol 3 calculations. For electron microscopy,samples dissolved in ethanol were applied to ac arbon grid at 83 Kw ith aV itrobot vitrification system (FEI, Hillsboro, OR). Electron microscopy was performed with aT ecnai G2 Polara electron microscope (FEI, Hillsboro, OR) equipped with aG atan energy filter at 115000 magnification (Gatan, Pleasanton, CA). Images were recorded in the zero-loss imaging mode, by using aslit width of 20 eV,with aslow-scan CCD camera at 1micrometer under focus, to have optimal phase contrast transfer at 300 kV.