Platform Synthetic Lectins for Divalent Carbohydrate Recognition in Water

Biomimetic carbohydrate receptors (“synthetic lectins”) have potential as agents for biological research and medicine. However, although effective strategies are available for “all‐equatorial” carbohydrates (glucose, etc.), the recognition of other types of saccharide under natural (aqueous) conditions is less well developed. Herein we report a new approach based on a pyrene platform with polar arches extending from aryl substituents. The receptors are compatible with axially substituted carbohydrates, and also feature two identical binding sites, thus mimicking the multivalency observed for natural lectins. A variant with negative charges forms 1:2 host/guest complexes with aminosugars, with K₁>3000 m ^?1 for axially substituted mannosamine, whereas a positively charged version binds the important α‐sialyl unit with K₁≈1300 m ^?1.     

N‐Alkyl Ammonium Resorcinarene Salts as High‐Affinity Tetravalent Chloride Receptors

N‐Alkyl ammonium resorcinarene salts (NARYs, Y=triflate, picrate, nitrate, trifluoroacetates and NARBr) as tetravalent receptors, are shown to have a strong affinity for chlorides. The high affinity for chlorides was confirmed from a multitude of exchange experiments in solution (NMR and UV/Vis), gas phase (mass spectrometry), and solid‐state (X‐ray crystallography). A new tetra‐iodide resorcinarene salt (NARI) was isolated and fully characterized from exchange experiments in the solid‐state. Competition experiments with a known monovalent bis‐urea receptor ( 5 ) with strong affinity for chloride, reveals these receptors to have a much higher affinity for the first two chlorides, a similar affinity as 5 for the third chloride, and lower affinity for the fourth chloride. The receptors affinity toward chloride follows the trend K₁?K₂?K₃≈ 5 >K₄, with K_a=5011 m ^?1 for 5 in 9:1 CDCl₃/[D₆]DMSO.     

Sugar‐Decorated Sugar Vesicles: Lectin–Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self‐assembles when unilamellar bilayer vesicles of amphiphilic β‐cyclodextrins are decorated with maltose and lactose by host–guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate–adamantane conjugates strongly bind to β‐cyclodextrin (K_a≈4×10⁴ M ^?1). The maltose‐decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose‐decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D ‐glucose or β‐cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.     

1H‐NMR relaxometric studies of interaction between apoptosis specific MRI paramagnetic contrast agents and micellar models of apoptotic cells

¹H‐NMR was previously used to analyze the interaction between peptides (E3 and R826) selected by phage display to target apoptotic cells and phospholipidic models of these cells. In order to avoid the use of apoptotic cells and to obtain a fast evaluation of the efficiency of the potential MRI contrast agents obtained by grafting these peptides and their scramble analogs on a paramagnetic gadolinium complex, their proton relaxometric behavior was investigated in the presence of micelles mimicking healthy and apoptotic cells. Their preferential interaction with 1,2‐dipalmitoyl‐sn‐glycero‐3‐phospho‐l ‐serine micelles mimicking apoptotic cells as compared with 1,2‐dipalmitoyl‐sn‐glycero‐3‐phosphocholine micelles modeling healthy cells was shown by nuclear magnetic relaxation dispersion profiles and the enhancement of the transverse proton relaxation rates at 60 MHz. The association constant values confirm the stronger interaction of the selected conjugated peptides (K_a Gd‐PMN‐E3(gadolinium 2,2′,2′′,2′′′‐[((4‐carboxy)pyridine‐2,6‐diyl)bis(methylenenitrilo)]‐tetrakis acetate) grafted with E3 peptide): 2.43 10⁴ m ^?1; K_a Gd‐DTPA‐R826(gadolinium ((1‐p‐isothiocyanatobenzyl)‐diethylenetriaminepentaacetate) grafted with R826 peptide): 2.91 10⁴ m ^?1) as compared with their conjugated scrambles (K_a Gd‐PMN‐E3sc(gadolinium 2,2′,2′′,2′′′‐[((4‐carboxy)pyridine‐2,6‐diyl)bis(methylenenitrilo)]‐tetrakis acetate) grafted with E3 scramble peptide): 0.18 10⁴ m ^?1; K_a Gd‐DTPA‐R826sc(gadolinium ((1‐p‐isothiocyanatobenzyl)‐diethylenetriaminepentaacetate) grafted with R826 scramble peptide): 0.32 10⁴ m ^?1) even if the conjugation of E3 and R826 seems to decrease their interaction. Copyright © 2015 John Wiley & Sons, Ltd.     

A Macrocycle Based on a Heptagon‐Containing Hexa‐peri‐hexabenzocoronene

A cyclophane is reported incorporating two units of a heptagon‐containing extended polycyclic aromatic hydrocarbon (PAH) analogue of the hexa‐peri‐hexabenzocoronene (HBC) moiety (hept‐HBC). This cyclophane represents a new class of macrocyclic structures that incorporate for the first time seven‐membered rings within extended PAH frameworks. The saddle curvature of the hept‐HBC macrocycle units induced by the presence of the nonhexagonal ring along with the flexible alkyl linkers generate a cavity with shape complementarity and appropriate size to enable π interactions with fullerenes. Therefore, the cyclophane forms host–guest complexes with C₆₀ and C₇₀ with estimated binding constants of K_a=420±2 m ^?1 and K_a=(6.49±0.23)×10³ m ^?1, respectively. As a result, the macrocycle can selectively bind C₇₀ in the presence of an excess of a mixture of C₆₀ and C₇₀.     

Enantioselective Alkynylation of Trifluoromethyl Ketones Catalyzed by Cation‐Binding Salen Nickel Complexes

Cation‐binding salen nickel catalysts were developed for the enantioselective alkynylation of trifluoromethyl ketones in high yield (up to 99 %) and high enantioselectivity (up to 97 % ee). The reaction proceeds with substoichiometric quantities of base (10–20 mol % KOt‐Bu) and open to air. In the case of trifluoromethyl vinyl ketones, excellent chemo‐selectivity was observed, generating 1,2‐addition products exclusively over 1,4‐addition products. UV‐vis analysis revealed the pendant oligo‐ether group of the catalyst strongly binds to the potassium cation (K⁺) with 1:1 binding stoichiometry (K_a=6.6×10⁵ m ^?1).     

1H NMR studies of binary and ternary dapsone supramolecular complexes with different drug carriers: EPC liposome,SBE‐β‐CD and β‐CD

Binary and ternary systems composed of dapsone, sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD), β‐CD and egg phosphatidylcholine (EPC) were evaluated using 1D ROESY, saturation transfer difference NMR and diffusion experiments (DOSY) revealing the binary complexes Dap/β‐CD (K_a 1396 l mol^?1), Dap/SBE‐β‐CD (K_a 246 l mol^?1), Dap/EPC (K_a 84 l mol^?1) and the ternary complex Dap/β‐CD/EPC (K_a 18 l mol^?1) in which dapsone is more soluble. Copyright © 2014 John Wiley & Sons, Ltd.     

Negatively Charged Yellow‐Emitting 1‐Aminopyrene Dyes for Reductive Amination and Fluorescence Detection of Glycans

1‐Aminopyrenes with three ω‐hydroxylated N‐alkylsulfonamido or alkylsulfonyl residues in positions 3, 6, and 8 were prepared, O‐phosphorylated, and applied for reductive amination of oligosaccharides. The dyes (?≈20 000 m ^?1 cm^?1) with six negative charges (pH≥8) and low m/z ratios enable labeling and fluorescence detection of reducing sugars (glycans) related to the most structurally and functionally diverse class of natural products. Under excitation with a 488 nm laser, the new glycoconjugates emit yellow light of about 560 nm, outperforming (with respect to brightness and faster electrophoretic mobilities) the corresponding APTS derivatives (benchmark dye with green emission in conjugates).     

Modulation of Excited‐State Proton Transfer of 2‐(2′‐Hydroxyphenyl)benzimidazole in a Macrocyclic Cucurbit[7]uril Host Cavity: Dual Emission Behavior and pKa Shift

The effect of the macrocyclic host, cucurbit[7]uril (CB7), on the photophysical properties of the 2‐(2′‐hydroxyphenyl)benzimidazole (HPBI) dye have been investigated in aqueous solution by using ground‐state absorption and steady‐state and time‐resolved fluorescence measurements. All three prototropic forms of the dye (cationic, neutral, and anionic) form inclusion complexes with CB7, with the largest binding constant found for the cationic form (K≈2.4×10⁶ M ^?1). At pH≈4, the appearance of a blue emission band upon excitation of the HPBI cation in the presence of CB7 indicates that encapsulation into the CB7 cavity retards the deprotonation process of the excited cation, and hence reduces its subsequent conversion to the keto form. Excitation of the neutral form (pH≈8.5), however, leads to an increase in the keto form fluorescence, indicating an enhanced excited‐state intramolecular proton‐transfer process for the encapsulated dye. In both the ground and excited states, the two pK_a values of the HPBI dye show upward shifts in the presence of CB7. The prototropic equilibrium of the CB7‐complexed dye is represented by a six‐state model, and the pH‐dependent changes in the binding constants have been analyzed accordingly. It has been observed that the calculated pK_a values using this six‐state model match well with the values obtained experimentally. The changes in the pK_a values in the presence of CB7 have been corroborated with the modulation of the proton‐transfer process of the dye within the host cavity.     

Insight into How Telomeric G‐Quadruplexes Enhance the Peroxidase Activity of Cellular Hemin

The toxic oxidative damage of G‐quadruplexes (G4), linked to neurodegenerative diseases, may arise from their ability to bind and oxidatively activate cellular hemin. However, there have been no precise studies on how telomeric G4 enhances the low intrinsic peroxidase activity of hemin. Herein, a label‐free and nanopore‐based strategy was developed to explore the enhancement mechanism of peroxidase activity of hemin induced by telomeric G4 (d(TTAGGG)_n). The nanopore‐based strategy demonstrated that there were simultaneously two different binding modes of telomere G4 to hemin. At the single‐molecule level, it was found that the hybrid structural telomeric G4 directly binds to hemin (the affinity constant (K_a)≈10⁶ m ^?1) to form a tight complex, and some of them underwent a topological change to a parallel structure with an enhancement of K_a to approximately 10⁷ m ^?1. Through detailed analysis of the topology and peroxidase activity and molecular modeling investigations, the parallel telomere G4/hemin DNAzyme structure was proven to be preferable for high peroxidase activity. Upon strong π–π stacking, the parallel structural telomere G4 supplied a key axial ligand to the hemin iron, which accelerated the intermediate compound formation with H₂O₂ in the catalytic cycle. Our studies developed a label‐free and single‐molecule strategy to fundamentally understand the catalytic activity and mechanism of telomeric DNAzyme, which provides some support for utilizing the toxic, oxidative‐damage property in cellular oxidative disease and anticancer therapeutics.     

Encapsulation of AGE‐Breaker Alagebrium by Cucurbit[7]uril Improved the Stability of Both Its Carbonyl α‐Hydrogen and Thiazolium C2‐Hydrogen

As determined by both ¹H NMR and UV/Vis spectroscopic titration, ESI‐MS, isothermal titration calorimetry, and DFT molecular modeling, advanced glycation end products (AGE) breaker alagebrium (ALA) formed 1:1 guest–host inclusion complexes with cucurbit[7]uril (CB[7]), with a binding affinity, K_a, in the order of magnitude of 10⁵ m ^?1, thermodynamically driven by both enthalpy (ΔH=?6.79 kcal mol^?1) and entropy (TΔS=1.21 kcal mol^?1). For the first time, a dramatic inhibition of keto–enol tautomerism of the carbonyl α‐hydrogen of ALA has been observed, as evidenced by over an order of magnitude decrease of both the first step rate constant, k₁, and the second step rate constant, k₂, during hydrogen/deuterium exchange in D₂O. Meanwhile, as expected, the reactivity of C2‐hydrogen was also inhibited significantly, with an upshift of 2.09 pK_a units. This discovery will not only provide an emerging host molecule to modulate keto–enol tautomerism, but also potentially lead to a novel supramolecular formulation of AGE‐breaker ALA for improved stability and therapeutic efficacy.     

Glycoluril‐Derived Molecular Clips are Potent and Selective Receptors for Cationic Dyes in Water

Molecular clip 1 remains monomeric in water and engages in host–guest recognition processes with suitable guests. We report the K_a values for 32 1? guest complexes measured by ¹H NMR, UV/Vis, and fluorescence titrations. The cavity of 1 is shaped by aromatic surfaces of negative electrostatic potential and therefore displays high affinity and selectivity for planar and cationic aromatic guests that distinguishes it from CB[n] receptors that prefer aliphatic over aromatic guests. Electrostatic effects play a dominant role in the recognition process whereby ion–dipole interactions may occur between ammonium ions and the C=O groups of 1 , between the SO₃^? groups of 1 and pendant cationic groups on the guest, and within the cavity of 1 by cation–π interactions. Host 1 displays a high affinity toward dicationic guests with large planar aromatic surfaces (e.g. naphthalene diimide NDI+ and perylene diimide PDI+) and cationic dyes derived from acridine (e.g. methylene blue and azure A). The critical importance of cation–π interactions was ascertained by a comparison of analogous neutral and cationic guests (e.g. methylene violet vs. methylene blue; quinoline vs. N‐methylquinolinium; acridine vs. N‐methylacridinium; neutral red vs. neutral red H⁺) the affinities of which differ by up to 380‐fold. We demonstrate that the high affinity of 1 toward methylene blue (K_a=3.92×10⁷ m ^?1; K_d=25 nm ) allows for the selective sequestration and destaining of U87 cells stained with methylene blue.     

Cucurbit[7]uril: A High‐Affinity Host for Encapsulation of Amino Saccharides and Supramolecular Stabilization of Their α‐Anomers in Water

Cucurbit[7]uril (CB[7]), an uncharged and water‐soluble macrocyclic host, binds protonated amino saccharides (D ‐glucosamine, D ‐galactosamine, D ‐mannosamine and 6‐amino‐6‐deoxy‐D ‐glucose) with excellent affinity (K_a=10³ to 10⁴ M ^?1). The host–guest complexation was confirmed by NMR spectroscopy, isothermal titration calorimetry (ITC), and MALDI‐TOF mass spectral analyses. NMR analyses revealed that the amino saccharides, except D ‐mannosamine, are bound as α‐anomers within the CB[7] cavity. ITC analyses reveal that CB[7] has excellent affinity for binding amino saccharides in water. The maximum affinity was observed for D ‐galactosamine hydrochloride (K_a=1.6×10⁴ M ^?1). Such a strong affinity for any saccharide in water using a synthetic receptor is unprecedented, as is the supramolecular stabilization of an α‐anomer by the host.     

Solid‐contact Acetate‐selective Electrode Based on a 1,3‐bis(carbazolyl)urea‐ionophore

The construction and study of solid‐contact acetate‐selective electrodes is described using a 1,3‐bis(carbazolyl)urea derivative as a neutral hydrogen‐bonding ionophore and poly(3,4‐ethylenedioxythiophene) as the solid contact. It was shown recently that this ionophore binds acetate (logK_ass=4.98) that is used as primary ion in this study. The electrodes show linearity over the activity range of 10^?4.50–10^?1.10 with a sub‐Nernstian slope of ?51.3 mV per decade and a detection limit of 10^?5.00. The anion‐selectivity pattern of these electrodes deviates markedly from the Hofmeister pattern. When adding ionophore to the membrane the logarithm of the selectivity coefficient (logK) for SCN^? decreased from 6.5 to 1.2, logK for I^? decreased from 5.7 to 0.9, logK for NO₃^? decreased from 4.3 to 0.6 and logK for Br^? decreased from 3.3 to 0.1. The selectivity coefficients of hydrophilic anions such as Cl^?, F^?, HPO₄^2?, and SO₄^2? are significantly lower than in case of the ionophore‐free membrane. It was discovered that the constructed electrodes are also relatively selective to bicarbonate. This work is an important step towards the further development of solid‐contact anion‐selective electrodes.     

Halide‐Anion Binding by Singly and Doubly N‐Confused Porphyrins

The halide‐binding properties of N‐confused porphyrin (NCP, 1 ) and doubly N‐confused porphyrins (trans‐N₂CP ( 2 ), cis‐N₂CP ( 3 )) were examined in CH₂Cl₂. In the free‐base forms, cis‐N₂CP ( 3 ) showed the highest affinity to each anion (Cl^?, Br^?, I^?) with association constants K_a=7.8×10³, 1.9×10³, and 5.8×10² M ^?1, respectively. As metal complexes, on the other hand, trans‐N₂CP 2–Cu exhibited the highest affinity to Cl^?, Br^?, and I^? with K_a=9.0×10⁴, 2.7×10⁴, and 1.9×10³ M ^?1, respectively. The corresponding K_a values for cis‐N₂CP 3–Cu and NCP 1–Cu were about 1/10 and 1/2, respectively, of those of 2–Cu . With the help of density functional theory (DFT) calculations and complementary affinity measurements of a series of trisubstituted N‐confused porphyrins, the efficient anion binding of NCPs was attributed to strong hydrogen bonding at the highly polarized NH moieties owing to the electron‐deficient C₆F₅ groups at meso positions as well as the ideally oriented dipole moments and large molecular polarizability. The orientation and magnitude of the dipole moments in NCPs were suggested to be important factors in the differentiation of the affinity for anions.     

多吡啶锌配合物键合与切割DNA研究

The complex of Zn[(phen)(dione)Cl]ClO_4·H_2O(where phen is 1,10-phenanthroline and dione is 1,10-phenan-throline-5,6-dione)has been synthesized and characterized.The interaction of the complex with DNA was investi-gated using UV absorption,fluorescence spectroscopy and electrophoresis measurements.The results show that thecomplex mainly binds to the double helix of DNA with intercalation mode and the binding constant K is 2.4×10~4mol~(-1)·L.Moreover,the complex can efficiently cleave plasmid DNA at physiological pH and temperature.Thecleavage occurs via a hydrolysis mechanism,which is showed by adding radical scavengers,rigorously anaerobicexperiments,analysis for malondialdehyde-like products,and the hydrolysis experiment of BDNPP with a rate con-stant k_(obs)of 5.3×10~(-6)s~(-1).     

The salt–cocrystal spectrum in salicylic acid–adenine: the influence of crystal structure on proton‐transfer balance

At one extreme of the proton‐transfer spectrum in cocrystals, proton transfer is absent, whilst at the opposite extreme, in salts, the proton‐transfer process is complete. However, for acid–base pairs with a small ΔpK_a (pK_a of base ? pK_a of acid), prediction of the extent of proton transfer is not possible as there is a continuum between the salt and cocrystal ends. In this context, we attempt to illustrate that in these systems, in addition to ΔpK_a, the crystalline environment could change the extent of proton transfer. To this end, two compounds of salicylic acid (SaH) and adenine (Ad) have been prepared. Despite the same small ΔpK_a value (≈1.2), different ionization states are found. Both crystals, namely adeninium salicylate monohydrate, C₅H₆N₅⁺·C₇H₅O₃^?·H₂O, I , and adeninium salicylate–adenine–salicylic acid–water (1/2/1/2), C₅H₆N₅⁺·C₇H₅O₃^?·2C₅H₅N₅·C₇H₆O₃·2H₂O, II , have been characterized by single‐crystal X‐ray diffraction, IR spectroscopy and elemental analysis (C, H and N) techniques. In addition, the intermolecular hydrogen‐bonding interactions of compounds I and II have been investigated and quantified in detail on the basis of Hirshfeld surface analysis and fingerprint plots. Throughout the study, we use crystal engineering, which is based on modifications of the intermolecular interactions, thus offering a more comprehensive screening of the salt–cocrystal continuum in comparison with pure pK_a analysis.     

β‐Turn Analogues in Model αβ‐Hybrid Peptides: Structural Characterization of Peptides Containing β2,2Ac6c and β3,3Ac6c Residues

The effect of gem‐dialkyl substituents on the backbone conformations of β‐amino acid residues in peptides has been investigated by using four model peptides: Boc‐Xxx‐β^2,2Ac₆c(1‐aminomethylcyclohexanecarboxylic acid)‐NHMe (Xxx=Leu ( 1 ), Phe ( 2 ); Boc=tert‐butyloxycarbonyl) and Boc‐Xxx‐β^3,3Ac₆c(1‐aminocyclohexaneacetic acid)‐NHMe (Xxx=Leu ( 3 ), Phe ( 4 )). Tetrasubstituted carbon atoms restrict the ranges of stereochemically allowed conformations about flanking single bonds. The crystal structure of Boc‐Leu‐β^2,2Ac₆c‐NHMe ( 1 ) established a C₁₁ hydrogen‐bonded turn in the αβ‐hybrid sequence. The observed torsion angles (α(?≈?60°, ψ≈?30°), β(?≈?90°, θ≈60°, ψ≈?90°)) corresponded to a C₁₁ helical turn, which was a backbone‐expanded analogue of the type III β turn in αα sequences. The crystal structure of the peptide Boc‐Phe‐β^3,3Ac₆c‐NHMe ( 4 ) established a C₁₁ hydrogen‐bonded turn with distinctly different backbone torsion angles (α(?≈?60°, ψ≈120°), β(?≈60°, θ≈60°, ψ≈?60°)), which corresponded to a backbone‐expanded analogue of the type II β turn observed in αα sequences. In peptide 4 , the two molecules in the asymmetric unit adopted backbone torsion angles of opposite signs. In one of the molecules, the Phe residue adopted an unfavorable backbone conformation, with the energetic penalty being offset by a favorable aromatic interaction between proximal molecules in the crystal. NMR spectroscopy studies provided evidence for the maintenance of folded structures in solution in these αβ‐hybrid sequences.     

Evidence for a Rapid Degenerate Hetero‐Cope‐Type Rearrangement in [Cp*W(S)2S‐CH2‐CHCH2]

Treatment of the salt [PPh₄]⁺[Cp*W(S)₃]^? ( 6 ) with allyl bromide gave the neutral complex [Cp*W(S)₂S‐CH₂‐CH?CH₂] ( 7 ). The product 7 was characterized by an X‐ray crystal structure analysis. Complex 7 features dynamic NMR spectra that indicate a rapid allyl automerization process. From the analysis of the temperature‐dependent NMR spectra a Gibbs activation energy of ΔG^≠ (278 K)≈13.7±0.1 kcal mol^?1 was obtained [ΔH^≠≈10.4±0.1 kcal mol^?1; ΔS^≠≈?11.4 cal mol^?1 K^?1]. The DFT calculation identified an energetically unfavorable four‐membered transition state of the “forbidden” reaction and a favorable six‐membered transition state of the “Cope‐type” allyl rearrangement process at this transition‐metal complex core.     

β,β‐(1,4‐Dithiino)subporphyrin Dimers Capturing Fullerenes with Large Association Constants

β,β‐(1,4‐Dithiino)subporphyrin dimers 7‐syn and 7‐anti were synthesized by the nucleophilic aromatic substitution reaction of 2‐bromo‐3‐(4‐methoxyphenylsulfonyl)subporphyrin 4 with 2,3‐dimercaptosubporphyrin 5 under basic conditions followed by axial arylation. Additions of C₆₀ or C₇₀ to a dilute solution of 7‐anti (ca. 10^?6 m ) in toluene did not cause appreciable UV/Vis spectral changes, while similar additions to a concentrated solution (ca. 10^?3 m ) resulted in precipitation of complexes. In contrast, dimer 7‐syn captured C₆₀ and C₇₀ in different complexation stoichiometries in toluene; a 1:1 manner and a 2:1 manner, respectively, with large association constants; K_a=(1.9±0.2)×10⁶ m ^?1 for C₆₀@ 7‐syn , and K₁=(1.6±0.5)×10⁶ and K₂=(1.8±0.9)×10⁵ m ^?1 for C₇₀@( 7‐syn )₂. These association constants are the largest for fullerenes‐capture by bowl‐shaped molecules reported so far. The structures of C₆₀@ 7‐anti , C₇₀@ 7‐anti , C₆₀@ 7‐syn , and C₇₀@ 7‐syn have been determined by single‐crystal X‐ray diffraction analysis.