CONH Groups Research Articles

Engineering the weak N–H⋯π hydrogen bond in 4-tritylbenzamide host and controlling the interaction through guest selection

The title amide host 1 crystallizes in the wheel-and-axle framework via amide N–H⋯O dimer and includes several aromatic and aliphatic guest molecules in cavities of 40 Å2 size between supramolecular axles. Bulky triphenylmethyl groups make it impossible for the second NH donor to engage in strong hydrogen bonding and this promotes a weak intermolecular N–H⋯π interaction in inclusion adducts of aromatic and hydrophobic guests (structure type 1, guest = xylenes, chloro/bromo-toluene). On the other hand, the N–H⋯π interaction is absent for guests with CO groups because of stronger N–H⋯Oguest hydrogen bonding (type 2, guest = EtOAc, MeNO2). Crystal structures of both types are virtually identical except for the rotation of CONH2 group that transforms the N–H⋯π interaction to the N–H⋯O hydrogen bond. In addition to controlling the occurrence of the weak N–H⋯π hydrogen bond through host⋯guest recognition, a third structure type with N–H⋯Ohost and N–H⋯π hydrogen bonds is present in the anisole adduct. Amide group conformations, strong and weak hydrogen bonds, and close packing of aromatic residues determine the three structure types of composition 1·(guest)0.5 in space group P. The CH2Cl2 solvate, 1·(CH2Cl2)1.5, has a different crystal packing in space group C2/c with guest molecules included in channels between amide dimers and also between Ph3C groups. The design and control of the weak N–H⋯π hydrogen bond are shown for the first time in a family of isomorphous crystal structures. Infrared spectroscopy and variable temperature X-ray diffraction are consistent with the hydrogen bond nature of the N–H⋯π interaction. Differential scanning calorimetry and thermal gravimetric analysis confirm the functional behavior of inclusion host 1 and show differences in the release of CH2Cl2 molecules from the two types of channels. Crystal latttice energies follow the order structure type 3 < type 2 < type 1 in the range of −90 to −76 kcal mol−1 per host molecule.

A theoretical and spectroscopic study of carbamazepine polymorphs

AbstractThe drug carbamazepine has been modeled, using ab initio density functional theory calculations [B3LYP/6–31G(d)], both as a single molecule and as a dimer. The predicted geometry of the single molecule calculation is compared with the crystallographic data on each of the polymorphs (carbamazepine forms I and III). From the predicted geometry it is possible to calculate the IR and Raman spectra; these predictions compare favorably with the observed spectra of both polymorphs of carbamazepine for most of the bands. The spectral differences between the polymorphs are more striking in the IR than the Raman spectra, with strong IR bands at 1688 and 1396 cm−1 in form I shifting to 1678 and 1388 cm−1 in form III. Analysis of the potential energy distributions for the calculated normal modes reveals that the vibrations are localized across different ring systems of the carbamazepine structure. Most notably, the polymorph‐sensitive modes in the IR spectra are localized to the pendant CONH2 group; it is these modes that show the greatest disparity from the calculated spectra, and it is this group that is perturbed in the polymorph crystal structures. The calculated dimer structure is similar to that of the single molecule, but the polymorph‐sensitive IR modes are significantly better predicted by the dimer calculation. Copyright © 2004 John Wiley &amp; Sons, Ltd.

Negative ion mass spectra of Cys-containing peptides. The characteristic Cys gamma backbone cleavage: a joint experimental and theoretical study.

The Cys residue initiates characteristic backbone cleavages of [M-H](-) anions of Cys-containing peptides. A combination of experiment and theory suggests that these processes are initiated by molecular recognition between the C-terminal CONH(-) group (in this study all peptides have C-terminal CONH(2) groups) and the SH in the Cys side chain to form an S-H...O=C hydrogen bond. This process is exothermic by 60 kJ mol(-1) (calculations at the HF/6-31G(d)//AM1 level of theory). The structure of this reactive intermediate has the NH(-) of the amide group and the central CH of the Cys residue locked into position such that these groups effect an S(N)2 process to form an intermediate which can either (i) dissociate to give an RNH(-) species [the delta ion (process endothermic by 37 kJ mol(-1) with a barrier of 132 kJ mol(-1))], or (ii) effect deprotonation within the intermediate to eliminate RNH(2) to give the gamma backbone cleavage anion in a reaction exothermic by 40 kJ mol(-1) with a barrier of 132 kJ mol(-1). Collision-induced mass spectra of the [M-H](-) anions of five selected Cys-containing peptides all contain gamma and (gamma-H(2)S) anions. Three of these spectra also show the less favoured delta cleavage anions.

Formation of hydrogen bonding in ionized poly(N‐isopropylacrylamide) gels by continuous water exchange

AbstractThe effects of continuous water exchange on the swelling behavior of poly(N‐isopropylacrylamide‐co‐sodium acrylate) gel were studied. The diameter of gels and the Na+ concentration in the solvent were measured at several constant intervals after the solvent (the distilled deionized water) was exchanged. The diameter decreased at room temperature as the solvent was exchanged with water, and it continued to decrease even after more than about 97% of the initial Na+ diffused into the water. Thus, the final swelling ratio of the gel was only slightly larger than that of the neutral poly(N‐isopropylacrylamide) gel. To reveal the structural change in molecular level, solid‐state 13C cross‐polarization/magic‐angle spinning, solid‐state 1H combined rotation and multiple‐pulse spectroscopy, and swollen‐state 13C dipolar decoupled/magic‐angle spinning NMR experiments were carried out for several dried and swollen samples by varying the times of water exchange. As a result, the intensity and position of the carboxyl peak changed, and the relative intensity of the nonionized carboxyl groups of gels increased with an increasing number of water exchanges. These results indicated that hydrogen bonding was formed between the two, nonionized carboxyl groups (COOH) and/or between the COOH and CONH groups. The macroscopic polymer network shrinkage is discussed in terms of the replacement of counterions Na+ by H+ and of the formation of intermolecular hydrogen bonding. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 1090–1098, 2004

Substituent Effects in Discotic Liquid Crystals

A series of novel mesogens have been prepared by a five-fold Sonogashira reaction of terminal acetylenes with a functionalized pentabromophenol. The corresponding side chain polymers were prepared by a polymer analogous substitution reaction. The mesogens differ in the nature of the substituents, linking five hexyl tails to the aromatic core, i.e. CH2, O, S, SO2 and CONH groups. A wide range of mesophases and corresponding transition temperatures has been detected, ranging from low melting nematic phases to highly stable columnar phases. The widely variable phase behavior is described in terms of specific intermolecular interactions.

Nowy sposob syntezy hydrofobowo modyfikowanego poli(kwasu akrylowego)

A new method of the synthesis of hydrofobically modified poly(acrylic acid) (HMPAA) (Scheme A) is based on a dissolution of initial poly(acrylic acid) (PAA) in thionyl chloride, which at temp. 60 °C reacts with COOH groups so the chloroyl groups COCI create. Chloroyl groups were subjected to partial amidation with dodecylamine and then the unreacted COCl groups were hydrolized to the carboxyl groups. HMPAA obtained this way contained 2.55 % of nitrogen and in aqueous solutions (0.1 M NaCl) showed, in viscometric measurements, the behavior being characteristic for amphiphylic polymers. It means the dependence η s p /c = f(c) (Fig. 2) significantly deviates from a straight line in contrast to the initial PAA (Fig. 1). Further confirmation of an amphiphylic character of HMPAA is the decrease in surface tension of water from 72.7 mN/m (just water) to 42 mN/m (1 % solution of HMPAA), at temp. 25 °C. 1 3 C NMR spectra of PAA and HMPAA (Fig. 4 and 5) were interpreted. In HMPAA spectrum there are additional signals coming from carbon atom of CONH group and from C atoms of aliphatic chain from dodecylamine. An explanation of the differences of the courses of curves of potentiometric titration of PAA and HMPAA (Fig. 6 and 7) is also given.

A SERS spectroelectrochemical investigation of the interaction of butylethoxycarbonylthiourea with copper surfaces

Abstract The interaction of the sulfide mineral flotation collector, n -butylethoxycarbonylthiourea (BECTU), with copper surfaces at pH 9.2 has been investigated by voltammetry and Raman scattering spectroscopy, NMR, Raman and FTIR spectroscopies have been applied to characterise BECTU and its copper(I) compound to provide a basis for interpreting the spectroelectrochemical data. In the copper compound, the organic molecule is bonded to metal atoms through both its sulfur atom and the nitrogen of the NHCO group of BECTU with the release of the proton. Voltammetry showed that the copper compound is formed above approximately −0.4 V and its presence on the surface inhibits oxide formation. Surface enhanced Raman scattering (SERS) spectra from copper electrodes in the presence of BECTU showed that the major surface species was the copper compound at potentials more than or equal to –0.4 V. The SERS spectrum at approximately −0.5 V was significantly different to that observed from for the bulk compound. This spectrum is interpreted in terms of charge transfer chemisorption of BECTU onto copper atoms in the metal surface.

Trans-3,4-Dimethyl-4-(3-carboxamidophenyl)piperidines: A novel class of μ-Selective opioid antagonists

trans-3,4-Dimethyl-4-(3-carboxamidophenyl)piperidines constitute a novel class of μ opioid receptor antagonists. The CONH2 group was found to be an effective isostere of the phenolic OH moiety. Structure–activity relationships at the piperidine nitrogen position led to the identification of several ligands displaying high affinity toward the cloned human μ opioid receptors, good selectivity μ/δ, μ/κ, and potent in vitro antagonist activity.

Peptide helices with pendant cycloalkane rings. Characterization of conformations of 1-aminocyclooctane-1-carboxylic acid (Ac8c) residues in peptides.

A pentapeptide, Boc-Leu-Ac8c-Ala-Leu-Ac8c-OMe 1, an octapeptide, Boc-Leu-Ac8c-Ala-Leu-Ac8c-Ala-Leu-Ac8c-OMe 2 and a tripeptide, Boc-Aib-Ac8c-Aib-OMe 3 containing the 1-aminocyclooctane-1-carboxylic acid residue (Ac8c) were synthesized and conformationally characterized by x-ray diffraction studies in the crystal state. Peptides 1 and 2 were also studied by NMR in CDC13 solution. Peptide 1 adopts a purely 3(10)-helical conformation in crystals, stabilized by three intramolecular 1 <-- 4 hydrogen bonds. Peptide 2 in crystals is largely 3(10)-helical with distortion in the backbone at the N-terminus by the insertion of a water molecule between Ac8c (2) CO and Ala (6) NH groups. Peptide 3 forms a C10-ring structure, i.e. a type III (III') beta- turn conformation stabilized by an intramolecular 1 <-- 4 hydrogen bond. Five cyclooctane rings assume boat-chair conformations, whereas the sixth [Ac8c(8) in 2] is appreciably distorted, resembling a chiral intermediate in the pseudorotational pathway from the boat-chair to the twisted boat-chair conformation. Internal bond angles of the cyclooctane rings are appreciably distorted from the tetrahedral value, a characteristic feature of the cyclooctane ring. Peptide 1 crystallized in the space group P212121 with a = 11.900(4) A, b = 18.728(6) A, c = 20.471(3) A and Z = 4. The final R1 and wR2 values are 0.0753 and 0.2107, respectively, for 3901 observed reflections [Fo > or = 3 sigma (Fo)]. Peptide 2 crystallized in space group P21 with a = 12.961(5) A, b = 17.710(10) A, c = 15.101(7) A, beta = 108.45(4) degrees and Z = 2. The final R1 and wR2 values are 0.0906 and 0.1832, respectively, for 2743 observed reflections [Fo > or = 3sigma (Fo)]. 1H-NMR studies on both the peptides strongly suggest the persistence of 3(10)-helical conformations in solution. Peptide 3 crystallized in the space group P21/n, with a = 10.018(1) A, b = 20.725(1) A, c = 12.915(1) A and Z = 4. The final R1 and wR2 values are 0.0411 and 0.1105, respectively, for 3634 observed reflections [Fo > or = 4sigma (Fo)].

Phenyl-Perfluorophenyl Synthon Mediated Cocrystallization of Carboxylic Acids and Amides

The significance of face-to-face π−π stacking of phenyl and perfluorophenyl rings (Ph-PhF), a robust supramolecular synthon in aromatic and perfluoroaromatic crystal structures, is studied in the cocrystallization of simple aromatic carboxylic acids and amides. X-ray crystal structures of C6H5COOH·C6F5COOH 1, C6H5CONH2·C6F5CONH2 2, and C6H5CONH2·C6F5COOH 3 are analyzed to understand the role of Ph-PhF synthon in directing self-assembly and hydrogen bonding in these cocrystals. The strong hydrogen bond donor acidity of C6F5COOH and C6F5CONH2 together with mixed stacks of phenyl and perfluorophenyl rings steer acid···acid and amide···amide hydrogen bonding in cocrystals 1 and 2. Acid···amide hydrogen bonding is sufficiently strengthened by donor acidity and acceptor basicity in 3 that the role of the Ph-PhF synthon is weaker because the aromatic rings stack with lateral offset. The complex C6H5COOH·C6F5CONH2 4 could not be obtained under similar crystallization conditions. The crystal structure of C6F5CONH2 is determined to compare molecular conformation and hydrogen bonding with motifs in the cocrystals. This study shows the viability of the Ph-PhF synthon in the presence of strong hydrogen bonding COOH and CONH2 groups for crystal engineering.

Two-dimensional infrared spectroscopy and principal component analysis studies on a new azobenzene derivative supramolecular system based on hydrogen bonds.

Infrared (IR) spectra of a supramolecular assembly with an azobenzene derivative and intermolecular hydrogen bonds have been measured in the temperature range from 30 to 200 degrees C to investigate heat-induced structural changes and thermal stability. Principal component analysis (PCA) and two kinds of two-dimensional (2D) correlation spectroscopy, variable-variable (VV) 2D and sample-sample (SS) 2D spectroscopy, have been employed to analyze the observed temperature-dependent spectral variations. The PCA and SS 2D correlation analyses have demonstrated that the complete decoupling of hydrogen bonds in the supramolecular assembly occurs between 110 and 115 degrees C, which is in good agreement with the results of a differential scanning calorimetry (DSC) study for the heating process. The PCA of the IR spectra in the region of 3600-3100 cm(-1) has illustrated that there are at least four principal components for the different NH2 and CONH species in the present supramolecular system. The VV 2D correlation spectroscopy study has provided information about the structure and strength of hydrogen bonds of NH2 and CONH groups and their temperature-dependent variations. The different species of hydrogen-bonded NH2 and CONH groups in the supramolecular system can be clarified by the VV 2D correlation analysis. The VV 2D correlation analysis has also revealed the specific order of the temperature-induced changes in the hydrogen bonds of NH2 and CONH groups.

Discriminating chiral molecules of (R)-PPA and (S)-PPA in aqueous solution by ECSTM.

Pricking the surface of chirality: The chirality of (R)- and (S)-2-phenylpropionamide molecules has been directly observed on Cu(111) in solution by using electrochemical scanning tunneling microscopy (see images). Around the chiral center, the methyl and CONH2 groups maintain the chiral configuration, although the R and S molecules adsorb onto Cu(111) in the same (4×4) symmetry array.

The Proton Affinity and Entropy of Protonation of Lysinamide. The Effects of Intramolecular Proton Solvation

The equilibrium constants for the gas-phase proton transfer from protonated tri-n-propylamine to lysinamide at several temperatures have been measured using Fourier transform ion cyclotron resonance mass spectrometric techniques. The thermodynamic values obtained from a van't Hoff plot are ΔH = −4.36 ± 0.85 kcal mol-1 and ΔS = −12.25 ± 2.34 cal mol-1 K-1. These values lead to derived values of the proton affinity (PA) and entropy of protonation (ΔSp) of lysinamide of 241.4 ± 0.9 kcal mol-1 and 10.9 ± 2.2 cal mol-1 K-1, respectively. A sophisticated calculation in the literature suggests that the former is identical with the PA of lysine as would be expected. The PA of lysinamide exceeds that of 1,5-diaminopentane by 2.5 kcal mol-1, which is consistent with an expected favorable interaction between the carbonyl oxygen of the CONH2 group and the strong hydrogen bond in protonated lysinamide. The ΔSp value is in good agreement with a kinetic-method determination of the entropy of protonation of lysine. It is...

Theoretical and experimental examination of -caprolactam dimer stability

The cyclic dimer of ε-caprolactam (C2) is a very stable compound as indicated by its high melting point (347 °C), low solubility in most solvents, and relatively low reactivity toward hydrolysis. FTIR measurements in solid state confirmed the existence of hydrogen bonds between NH protons and CO groups. Upon heating to 200 °C these bonds are not broken, but are merely weakened. This is shown by shifts of the NH stretching band toward higher wave numbers as well as by shifts of other characteristic bands. Temperature controlled NMR measurements on 0.3 and 30% C2 solutions in formic acid gave temperature coefficients of NH proton signals of −0.006 and −0.009 ppm/K, respectively, strongly indicating intermolecular hydrogen bonding. In the same concentration range signals for NH protons and CO carbons were shifted toward higher fields with higher C2 concentration. Using density functional theory (DFT) calculations it was not possible to successfully perform geometry optimization on any C2 structure with intramolecular hydrogen bonds. The trans form of C2 was found to be 7.0 kcal/mol lower in energy than the cis form. Symmetrical structures of C2 based on published trans X-ray structures were found to have comparable energies. Hydrogen bonds between trans C2 and discrete molecules of formic acid, water and N-methylacetamide, as model for solvents and a neighboring C2 molecule, were found to be comparable in energy. Full geometry optimization of a dimer of trans C2 gave energy stabilization of 8.25 kcal/mol per H-bond. These results show C2-C2 polar interactions to be comparable with polar interactions of C2 with solvents. The stability of C2, however, cannot be explained only by hydrogen bonding. It is likely that nonpolar interactions between C2 molecules complement the stabilization by hydrogen bonding. Optimized dimer of two trans cyclic-ε-caprolactam-dimer molecules (Etot = −1 461.0682582 H).

Conformational Changes and Orientation of Humicola lanuginosa Lipase on a Solid Hydrophobic Surface: An in Situ Interface Fourier Transform Infrared-Attenuated Total Reflection Study

This study was done to better understand how lipases are activated at an interface. We investigated the conformational and solvation changes occurring during the adsorption of Humicola lanuginosa lipase (HLL) onto a hydrophobic surface using Fourier transform infrared-attenuated total reflection spectroscopy. The hydrophobic surfaces were obtained by coating silicon attenuated total reflection crystal with octadecyltrichlorosilane. Analysis of vibrational spectra was used to compare the conformation of HLL adsorbed at the aqueous-solid interface with its conformation in solution. X-ray crystallography has shown that HLL exists in two conformations, the closed and open forms. The conformational changes in HLL caused by adsorption onto the surface were compared with those occurring in three reference proteins, bovine serum albumin, lysozyme, and α-chymotrypsin. Adsorbed protein layers were prepared using proteins solutions of 0.005 to 0.5 mg/mL. The adsorptions of bovine serum albumin, lysozyme, and α-chymotrypsin to the hydrophobic support were accompanied by large unfoldings of ordered structures. In contrast, HLL underwent no secondary structure changes at first stage of adsorption, but there was a slight folding of β-structures as the lipase monolayer became complete. Solvation studies using deuterated buffer showed an unusual hydrogen/deuterium exchange of the peptide CONH groups of the adsorbed HLL molecules. This exchange is consistent with the lipase being in the native open conformation at the water/hydrophobic interface.

Backbone cleavages of [M - H](-) anions of peptides. Cyclisation of citropin 1 peptides involving reactions between the C-terminal [CONH](-) residue and backbone amide carbonyl groups. A new type of beta cleavage: a joint experimental and theoretical study.

This paper reports the study of backbone cleavages in the collision-induced negative-ion mass spectra of the [M - H](-) anions of some synthetic modifications of the bioactive amphibian peptide citropin 1 (GLFDVIKKVASVIGGL-NH(2)). The peptides chosen for study contain no amino acid residues which could effect facile side-chain cleavage, i.e. Ser (-CH(2)O, side-chain cleavage) and Asp (-H(2)O) are replaced by Ala or Lys. We expected that such peptides should exhibit standard and pronounced peaks due to alpha cleavage ions (and to a lesser extent beta cleavage ions) in their collision-induced negative-ion spectra. This expectation was realised, but the spectra also contained peaks formed by a new series of cleavage anions. These are produced following cyclisation of the C-terminal CONH(-) moiety at carbonyl functions of amide groups along the peptide backbone; effectively transferring the NH of the C-terminal CONH(-) group to other amino acid residues. We have called the product anions of these processes beta' ions, in order to distinguish them from standard beta ions. Some beta' ions also fragment directly to some other beta' ions of smaller mass. The reaction coordinates of alpha,beta and beta' backbone processes have been calculated at the HF/6-31G*//AM1 level theory for simple model systems. The initial cyclisation step of the beta' sequence is barrierless and exothermic. Subsequent steps have a maximum barrier of +40 kcal mol(-1), with the overall reaction being endothermic by some 30 kcal mol(-1) at the level of theory used. These calculations take no account of the complexity of the conformationally flexible peptide system, and it is surprising that each of the two reacting centres can 'find' each other in such a large system.

An analysis of the hydration thermodynamics of the CONH group

The hydration thermodynamics of the CONH group play a fundamental role for the stability of the native conformation of globular proteins, but cannot be measured in a direct manner. The values of the thermodynamic functions have to be extracted from experimental measurements on model compounds using group additivity approaches. The estimates determined by Makhatadze and Privalov in the temperature range 5–100°C are used in the present study in view of their qualitative reliability. They are analyzed by means of a suitable approach that couples scaled particle theory calculations with the application of the modified Muller's model. It results that the negative entropy change is caused by the excluded volume effect for cavity creation, exaggerated in liquid water by the small size of water molecules themselves; the negative enthalpy change is determined by the H-bond energetics, formation of CONH–water H-bonds, and reorganization of water–water H-bonds. The negative heat capacity change, a striking feature of CONH hydration thermodynamics, is because the H-bonds in the hydration shell of the CONH group are less broken than those in bulk water in the temperature range examined.Key words: peptide group, hydration, excluded volume effect, H-bonds, two-state model, negative heat capacity change.

Stereoselective Electron-Transfer Reactions of Myoglobin and Cytochrome c with Chiral Viologen-Radical Cations

Abstract Electron-transfer (ET) reactions of metmyoglobin (metMb) and the oxidized form of cytochrome c (cyt c(III)) with chiral viologen-radical cations containing (1-phenyl- and 1-cyclohexylethyl)carbamoylmethyl groups were examined at pH 7.0 (10 mM phosphate buffer), ionic strengths (I) of 0.040–0.50 M, and temperatures of 10–35 °C. In the metMb system, the pseudo first-order rate constant of the decay of the viologen-radical cation was saturated with increasing concentrations of metMb, indicating that metMb interacts with the viologen-radical cation to form a complex followed by an intramolecular ET reaction. The stereoselectivity was observed in both the complex formation and the intramolecular ET processes for the (S,S)-isomers of chiral viologens. The ratios of the association constants and the intramolecular ET rate constants of the (S,S)-isomer with those of the (R,R)-isomer were 1.1–1.3 and 1.1–1.5, respectively. The saturation kinetics was not observed for the ET reaction between metMb and the achiral benzylviologen-radical cation, which has no CONH bond, suggesting that the hydrogen bonding of the CONH group of the chiral viologen with the side chain of the polypeptide in metMb plays an important role in the stereoselectivity. No appreciable complex formation was observed in the ET reaction between cyt c(III) and viologen-radical cations, although the stereoselectivity was observed. The ratios of the second-order rate constants of the (S,S)-isomer with those of the (R,R)-isomer were 1.1–1.4. The stereoselectivity is discussed based on the activation parameters.

An analysis of the hydration thermodynamics of the CONH group

An analysis of the hydration thermodynamics of the CONH group

PREPARATION AND PHYSICAL PROPERTIES OF BLEND FILMS FROM SODIUM ALGINATE AND POLYACRYLAMIDE SOLUTIONS

Blend films of sodium alginate and polyacrylamide (PAAm) were prepared by mixing the aqueous solution of both samples at a different ratio. All blend films obtained are optically clear to the naked eye. The structure and physical properties of the films were studied by FT-infrared (FT-IR), wide angle X-ray diffraction (WAXD), differential thermal analysis (DTA), thermogravimetic analysis (TGA), scanning electron microscopy (SEM), and tensile strength test. The results showed that the occurrence of interactions between -COO−, -OH groups of sodium alginate and -CONH2 groups of PAAm in the blends through hydrogen formation. The blend films exhibited the higher thermal stability and improved mechanical properties in dry states. These properties had the maximum value around 20 wt% PAAm content in the blend film. The morphological transition of the blend films from sodium alginate-like to PAAm-like was observed by scanning electron microscopy.