Nonclassical Oddâeven Alternation in Mixed-Chain

Article pubs.acs.org/crystal

Nonclassical Odd−even Alternation in Mixed-Chain Diacylethanolamines: Implications of Polymorphism Pradip K. Tarafdar, S. Thirupathi Reddy, and Musti J. Swamy* School of Chemistry, University of Hyderabad, Hyderabad 500 046, India S Supporting Information *

ABSTRACT: N,O-Diacylethanolamines (DAEs), derived by the O-acylation of bioactive N-acylethanolamines (NAEs), are most likely present in biological membranes. In the present study, a homologous series of mixed chain DAEs with an N-palmitoyl chain and varying O-acyl chains (n = 6−16) have been synthesized and characterized. Differential scanning calorimetry (DSC) studies revealed a nonclassical type of odd−even alternation in the enthalpy and entropy of melting, with the odd−even effect becoming more prominent with increase in the O-acyl chain length. This was rationalized by analyzing the 3-dimensional structures of several DAEs. In most cases, two types of packing polymorphs (α and β) were observed; while the α polymorph is characterized by a mixed type of chain packing, in the β polymorph the chain packing is symmetric. Analysis of the crystal structures revealed that the odd−even effect is manifested through differences in the packing of acyl chains, with the packing being closer when both chains are even. A possible role of polymorphism in the odd−even effect has also been considered, and it was suggested that the nonclassical behavior observed in the alternation could be attributed to the presence of different fractions of the α and β forms in the bulk mixture. Similar nonclassical odd−even effects and polymorphism may exist in other mixed chain systems, which adopt an “L” shape.

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INTRODUCTION Lipids, the amphiphilic molecules, not only form the structural basis of biomembranes but also play functionally significant roles as signaling molecules in a variety of cellular processes.1−4 Investigation of their structures is therefore crucial for understanding the roles played by them in nature. However, due to the presence of long and flexible alkyl (or acyl) chains in them, it has generally been difficult to obtain high quality single crystals of lipids and, hence, very few of them could be crystallized and investigated with respect to 3-dimensional structure.5 Interestingly, lipids exhibit polymorphism in the hydrated state, leading to the formation of various types of phases, such as gel, liquid-crystalline, inverse hexagonal, and cubic phases, which are well characterized.6−8 However, there are very few investigations reporting the characterization of polymorphism in the solid (crystalline) state of lipids. Studies reporting crystal structures of lipids, especially those dealing with polymorphism, are of great interest, not only from an academic perspective but also in view of the wide use of lipids in the formulation of cosmetics and pharmaceuticals and in the food industry, where an understanding of the molecular structure and intermolecular interactions will help in their better use.9,10 Although the LIPID MAPS (LIPID metabolites and pathways strategy) Structure Database (LMSD) contains more than 30,000 unique lipid structures,11 very few of them © 2012 American Chemical Society

have been characterized by single crystal X-ray diffraction analysis. N-Acylethanolamines (NAEs) and their precursors N-acylphosphatidylethanolamines have been studied for several decades due to their putative role in combating stress and in view of their interesting biological and medicinal properties.12−15 O-Acylation of NAEs yields a new class of lipids, namely N,O-diacylethanolamines (DAEs). Since O-acylation of NAEs was shown to be catalyzed by rat heart cell free preparations or by lipases, it appears very likely that DAEs would be present as minor constituents in biological membranes and play specific role(s) in the parent organism/ tissue.12,14,15 To understand the possible role played by DAEs, it is essential to investigate the properties of these molecules in a systematic manner. Since the N- and O-acyl chains of DAEs can be identical or different, it would be important to investigate the structure and phase behavior of DAEs with matched acyl chains as well as with mixed acyl chains. In a recent study, we have investigated the thermotropic phase transitions of DAEs with matched chains.15 In another report we have shown that DAEs with mixed acyl chains adopt an “L” shape in the solid state and exhibit packing polymorphism.16 Received: June 2, 2011 Revised: December 31, 2011 Published: February 24, 2012 1132

dx.doi.org/10.1021/cg200698t | Cryst. Growth Des. 2012, 12, 1132−1140

Crystal Growth & Design

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Table 1. Crystallographic Data for N,O-Diacylethanolamines of Even N- and O-Chains at 298 K formula formula wt. crystal system space group a (Å) b (Å) c (Å) α β γ Z V (Å3) Dcalc (g cm−3) R1 wR2 data completeness (%) GooF

NPOL α form (NPOLα)

NPOL β form (NPOLβ)

NPOM α form (NPOMα)

NPOM β form (NPOMβ)

C30H59NO3 481.78 orthorhombic Pca21 9.046(2) 4.8948(12) 69.909(17) 90.00 90.00 90.00 4 3095.4(13) 1.034 0.0597 0.1455 97.8 0.959

C30H59NO3 481.78 monoclinic P21/c 68.592(4) 4.8795(3) 8.7780(6) 90.00 92.2620(10) 90.00 4 2935.7(3) 1.090 0.0627 0. 1690 98.7 1.061

C32H63NO3 509.83 orthorhombic Pca21 9.087(5) 4.925(3) 74.87(4) 90.00 90.00 90.00 4 3350(3) 1.011 0.1085 0.2630 99.5 1.032

C32H63NO3 509.83 monoclinic P21/c 74.26(3) 4.913(2) 9.069(4) 90.00 92.577(7) 90.00 4 3306(2) 1.024 0.1464 0.2994 94.1 1.138

a trace of ethanol yielded crystals of a second polymorph of NPOL. Thin colorless crystals of N-palmitoyl-O-nonanoylethanolamine (NPON) and N-palmitoyl-O-undecanoylethanolamine (NPOU) were also grown at room temperature from dichloromethane having a trace of methanol. Measurements were carried out at room temperature (ca. 298 K) and 100 K with a Bruker SMART APEX CCD area detector system using a graphite monochromator and Mo Kα (λ = 0.71073 Å) radiation obtained from a fine-focus sealed tube. Data reduction was done using the Bruker SAINTPLUS program. Absorption correction was applied using the SADABS program, and refinement was done using the SHELXTL program.19,20 The crystal parameters of all the crystals studied by X-ray diffraction are presented in Tables 1 and 2.

In the present study, a homologous series of DAEs of fixed N-acyl chain (n = 16) and varying O-acyl chains (n = 6−16) have been synthesized and their thermotropic phase transitions have been characterized by differential scanning calorimetry (DSC). The analysis of DSC data revealed a distinct pattern in the odd−even alternation, which is qualitatively different from that observed for other lipids including fatty acids, NAEs, and DAEs with matched chains. This led to the question as to why the odd−even alternation phenomenon of DAEs with mixed acyl chains is different from that observed in other lipids. We have tried to understand this interesting aspect by analyzing the crystal structures of several mixed-chain DAEs. The results obtained suggest a definite role for polymorphism in the odd− even alternation.

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Table 2. Crystallographic Data for N,O-Diacylethanolamines of Even N-Acyl and Odd O-Acyl Chains at 298 K

EXPERIMENTAL SECTION

Materials. Long chain fatty acids, CH3-(CH2)n-COOH, of even and odd chain lengths (n = 4−14) were purchased from Aldrich (Milwaukee, WI, USA). Oxalyl chloride was obtained from Merck (Germany). Ethanolamine and other solvents were purchased locally. Synthesis of N,O-Diacylethanolamines. N-Palmitoylethanolamine (NPEA) was prepared by the reaction of palmitoyl chloride with ethanolamine as reported earlier.17 N-Palmitoyl, O-acyl (C6 to C16) ethanolamines were synthesized by the dropwise addition of 1.2 mol equiv of the acid chloride in dichloromethane to a solution containing 1 mol equiv of NPEA in the same solvent, under constant stirring.16 The crude products obtained were purified by silica-gel column chromatography using increasing concentration of ethyl acetate in n-hexane as the eluent and recrystallized from a mixture of dichloromethane and acetone. Overall yields ranged around 55− 60%. The products were further characterized by FTIR and 1H NMR spectroscopy, and representative FTIR and 1H NMR spectra corresponding to N-palmitoyl-O-lauroylethanolamine (NPOL) are given in Figures S1 and S2, respectively. Details of spectral assignment are given in the Supporting Information. Crystallization, X-ray Diffraction, and Structure Solution. Thin colorless crystals of N-palmitoyl-O-lauroylethanolamine (NPOL) and N-palmitoyl-O-myristoylethanolamine (NPOM) were grown at room temperature from dichloromethane containing a trace of methanol. Indexing of a number of crystals for each sample indicated that only one crystal form was obtained for NPOL, whereas NPOM yielded two different crystal forms, orthorhombic and monoclinic, suggesting the presence of two different polymorphs which were further analyzed by single-crystal X-ray diffraction. Crystallization experiments using a mixture of dichloromethane and toluene (1: 1 v/v) containing

formula formula wt crystal system space group a (Å) b (Å) c (Å) α β γ Z V (Å3) Dcalc (g cm−3) R1 wR2 data completeness (%) GooF

NPON

NPOU

C27H53NO3 439.70 monoclinic P21/c 64.921(7) 4.8832(5) 9.0299(9) 90.00 90.679(2) 90.00 4 2862.5(5) 1.020 0.0883 0.3016 99.9 1.029

C29H57NO3 467.76 monoclinic P21/c 69.079(11) 4.8901(8) 8.9962(14) 90.00 91.818(3) 90.00 4 3037.4(8) 1.023 0.1143 0.3465 97.9 1.038

Differential Scanning Calorimetry. DSC studies were carried out on a Perkin-Elmer PYRIS diamond differential scanning calorimeter. Samples of dry DAEs (1−3 mg) were weighed accurately into aluminum sample pans, covered with an aluminum lid, and sealed by crimping. Reference pans were prepared similarly, but without any sample in them. Heating and cooling scans were performed from room temperature (ca. 25 °C) to about 100 °C at a scan rate of 1.0°/min or 2.0°/min, and each sample was subjected to two heating scans and one cooling scan. Transition enthalpies were determined by integrating the 1133



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peak area under the transition curve. In all cases, only the first heating scan was considered for further analysis. Transition entropies were determined from the transition enthalpies, assuming a first order transition according to the expression21

ΔHt = T ΔSt

Table 3. Chain-Melting Phase Transition Temperatures (Tt), Enthalpies (ΔHt), and Entropies (ΔSt) of DAEsa O-acyl chain length, n

Tt (°C)

ΔHt (kcal·mol−1)

ΔSt (cal·mol‑1·K−1)

C6 C7 C8 C9 C10 C11 C12 C13 C14 C15 C16b

74.7 75.7 77.7 78.1 81.1 82.4 87.5 87.7 87.1 87.6 90.1

15.43 16.22 17.65 18.53 20.5 20.77 21.85 22.1 24.52 23.86 26.38

44.36 46.49 50.3 52.75 57.87 58.41 60.58 61.24 68.06 66.13 72.62

(1)

where T is the transition temperature and ΔHt values are taken at this temperature in order to calculate the corresponding ΔSt values.

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RESULTS AND DISCUSSION Differential Scanning Calorimetry. Heating thermograms corresponding to dry DAEs (recrystallized from dichloromethane and acetone mixture) of even and odd, mixed acyl chains are shown in Figure 1. This figure shows that each a

Data obtained from the DSC thermograms of dry samples of DAEs with fixed N-palmitoyl and varying O-acyl chain lengths (n = 6−16) are shown. bFor C16 the values obtained are close to the previously reported values (Tt = 89.9, ΔHt = 26.4, ΔSt = 72.7, ref 15).

varying O-acyl (C6−C16) chains are given in parts A and B, respectively, of Figure 2. In both cases it is observed that the even and odd series independently exhibit linear dependence of the calorimetric parameters on the acyl chain length. This results in a zigzag pattern, when the data obtained with the even and odd chain length series are viewed together with the values of enthalpy and entropy for the odd chain length series being slightly lower than those of the even chain length series. Similar trends were observed in the phase transition temperatures and thermodynamic parameters characterizing the thermotropic phase transitions of long-chain hydrocarbons, fatty acids, N-acylethanolamines, and N,O-diacylethanolamines with matched chain lengths,22−24 and the phenomenon is wellknown as “odd−even alternation”. However, in the case of DAEs with mixed chains, the two linear fits corresponding to the even and odd series intersect at lower chain lengths (Figure 2), whereas, in the case of other lipids (e.g. hydrocarbons, fatty acids, NAEs, DAEs with matched chains), it was observed that the linear fits for the two series are nearly parallel to each other.15,22−25 In other words, for DAEs with mixed acyl chains the differences in the thermodynamic parameters between the even and odd series are smaller when the O-acyl chains are shorter and increase with increase in the length of O-acyl chains. This nonclassical trend in odd−even alternation in DAEs with mixed chains will be discussed in more detail below. The enthalpy and entropy data for the mixed-chain DAEs with a fixed N-acyl (palmitoyl) chain and varying O-acyl chains of even and odd chain lengths could be independently fit to expressions 2 and 3 given below,22 as observed earlier with NAEs with even and odd acyl chains:23,25

Figure 1. DSC heating thermograms of DAEs with fixed N-palmitoyl chains and different O-acyl chains (n = 6−16). (A) Thermograms of DAEs with an even number of C-atoms in the O-acyl chains. (B) Thermograms of DAEs with an odd number of C-atoms in the O-acyl chains. The number of C-atoms in the O-acyl chain is indicated against each thermogram.

DAE exhibits a major transition, which was found to correspond to the capillary melting point of the compound. Some of the DAEs show minor transitions at lower temperatures, and in some cases the thermogram consists of two sharp endotherms that are closely spaced. It is unlikely that the two peaks arise due to the presence of a significant impurity, because in such a case the impurity will lead to broadening of the endotherm rather than yielding a second sharp peak in the thermogram. These observations indicate the possibility of polymorphism in DAEs with mixed chains. When the samples were subjected to a second heating scan, the additional transitions disappeared in some cases, and small decreases have been noted in the transition enthalpies. This suggests that during the cooling scan, some of the DAEs do not come back to the structure of the initial starting form. Therefore, in all cases the first heating scan was considered for further analysis and the total area under the major and minor transitions was integrated to get the transition enthalpies. The transition temperatures (Tt), transition enthalpies (ΔHt), and transition entropies (ΔSt) obtained are presented in Table 3. Chain Length Dependence of Transition Enthalpy and Transition Entropy. The chain length dependences of transition enthalpy and transition entropy for the chain-melting phase transitions of DAEs of fixed N-acyl (palmitoyl) and

ΔHt = ΔHo + (n − 2)ΔHinc

(2)

ΔSt = ΔSo + (n − 2)ΔSinc

(3)

where ΔHo and ΔSo are the end contributions to ΔHt and ΔSt, respectively, arising from the fixed N-palmitoyl chain, the central polar region, and the methyl group of the O-acyl chain. ΔHinc and ΔSinc are the incremental values of ΔHt and ΔSt contributed by each CH2 group of the O-acyl chain. Linear least-squares analysis of the chain length-dependent values of ΔHt and ΔSt for the DAEs with even and odd O-acyl chains yielded the incremental values (ΔHinc and ΔSinc) and end contributions (ΔHo and ΔSo). These values are listed in Table 4. 1134



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Figure 2. Chain length dependence of transition enthalpies (ΔHt) and transition entropies (ΔSt). Transition enthalpies (A) and transition entropies (B) obtained for the thermotropic phase transitions of N,O-diacylethanolamines of mixed acyl chains are plotted against the number of methylene units (n − 2, where n is the number of C-atoms in the O-acyl chains) in each acyl chain. Data obtained from the heating scans are shown.

Table 4. Incremental Values (ΔHinc, ΔSinc) of Chain Length Dependence and End Contributions (ΔHo, ΔSo) to Phase Transition Enthalpy and Entropy of Dry N,O-Diacylethanolamines with Mixed Acyl Chainsa

a

lipid

ΔHinc

ΔHo

ΔSinc

ΔSo

DAEs (even chain length) DAEs (odd chain length) NAEs* (even-chain length)

1.09 ± 0.04 0.94 ± 0.05 0.82 ± 0.02

11.19 ± 0.4 11.81 ± 0.53 −0.10 ± 0.26

2.81 ± 0.13 2.38 ± 0.17 2.01 ± 0.06

33.60 ± 1.32 35.50 ± 1.64 2.12 ± 0.71

Values for N-acylethanolamines (NAEs) with even acyl chain lengths, taken from ref 25 are also given for comparison.

The end contributions of enthalpy and entropy for DAEs (both even O-acyl chains and odd O-acyl chains) are comparable to the phase transition enthalpy and entropy observed for NPEA.25 This is consistent with the fact that the N-acyl chains in the mixed chain DAEs are packed similar to the N-acyl chains of NPEA26 (discussed below) and, hence, their contribution to the phase transition enthalpy is comparable to that of NPEA.25 On the other hand, both ΔHinc and ΔSinc are higher for DAEs with even O-acyl chains as compared to those with odd O-acyl chains (Table 4). This suggests that while the N-acyl chains are packed equally well for all the DAEs investigated here, the O-acyl chain packing is tighter for the DAEs with even O-acyl chains as compared to those with odd O-acyl chains. This aspect is considered further below in conjunction with the chain packing details derived from the crystal structures. Description of the Structures. Initial cell indexing (at 298 K) of several crystals of N-palmitoyl-O-myristoylethanolamine (NPOM) indicated the presence of two types of crystals from the same batch, suggesting polymorphism. Both types of crystals were further characterized by single crystal Xray diffraction, and the crystal structures were solved. The crystal parameters obtained are given in Table 1, and the molecular structure of the α polymorph (NPOMα) is shown in Figure 3A. The atomic coordinates and equivalent isotropic displacement parameters for all non-hydrogen atoms are given in Table S3. The bond distances and bond angles involving all the non-hydrogen atoms are given in Table S4 whereas the corresponding torsion angles are given in Table S5. It is seen from Figure 3A that the hydrocarbon portions of the two acyl chains (C14−C1, corresponding to the O-acyl chain and C19−C32, corresponding to the N-acyl chain) of the molecule

Figure 3. ORTEP plots of the DAEs: (A) α polymorph of NPOM; (B) α polymorph of NPOL; (C) NPON.

are in the all-trans conformation. The amide carbonyl and N−H groups are also in the trans geometry. The torsion angles observed for the two acyl chain regionsexcepting the C17− C18−C19−C20 angle, which is −69.0°are all close to 180° and are fully in agreement with the above observation. The gauche conformation at the C18−C19 bond results in a distinct change in the chain direction leading to a bending of the molecule, thus giving it a structure akin to the letter “L”. The conformation of the N-palmitoyl chain and the central ethanolamine part of the molecule are essentially identical to those observed in the structure of NAEs such as NMEA and NPEA.17,26 1135



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Figure 4. Packing diagrams of NPOM: (A) view along the b-axis of the α polymorph; (B) view along the b-axis of the β polymorph. The length of the hydrophobic region is also indicated. The crystal lattice of the β form has two different types of hydrophobic regions, one shorter in length and the other longer, whereas in the α form only one type of hydrophobic region is present due to mixed type chain packing.

N-Palmitoyl-O-myristoylethanolamine (NPOM) adopts an essentially similar structure in the β polymorph (NPOMβ) (Figure S3A). The atomic coordinates and equivalent isotropic displacement parameters of selected atoms, bond angles, bond lengths, and torsion angles corresponding to this structure are presented in Tables S6−S8. The C17−C18−C19−C20 torsion angle in NPOMβ is found to be 68.2°, which is slightly less than the value observed in NPOMα. This gauche conformation at the C18−C19 bond bends the NPOMβ molecule and gives it an “L” shape, with both acyl chains coming off the two ends of the ethanolamine moiety. The molecular structure of the α polymorph of N-palmitoylO-lauroylethanolamine (NPOLα), shown in Figure 3B, is isostructural with NPOMα. The other form of N-palmitoylO-lauroylethanolamine, namely NPOLβ, is isostructural with NPOMβ (see Figure S3B). The atomic coordinates and equivalent isotropic displacement parameters of selected atoms, bond angles, bond lengths, and torsion angles of these two crystals are presented in Tables S9−S14. Both the polymorphs of NPOL also adopt an “L” shape in the solid state due to the gauche conformation at the C16−C17 bond. The molecular structures of N-palmitoyl-O-nonanoylethanolamine (NPON) and N-palmitoyl-O-undecanoylethanolamine (NPOU) are similar to the β polymorph of NPOL and NPOM. The ORTEP of NPON is given in Figure 3C, and it is seen from the figure that this DAE with an odd O-acyl chain also adopts an L-shape in the solid state. An ORTEP of NPOU is given in Figure S3C. The atomic coordinates and equivalent isotropic displacement parameters of selected atoms, bond angles, bond lengths, and torsion angles of these two molecules are given in Tables S15−S20. Molecular Packing. A packing diagram of NPOMα along the b-axis is given in Figure 4A. The NPOM molecules in the α polymorph are packed in layers that are stacked in such a way

that the methyl groups of the O-acyl chains from one layer face the methyl groups of the N-acyl chains of the adjacent layer, resulting in a mixed packing arrangement.16 The methyl ends of the stacked layers are in van der Waals contact with the closest methyl−methyl distance between opposite layers and the same layer being 3.968 Å and 4.925 Å, respectively. The layer thickness (C1−C32 distance) in the structure of NPOMα is 34.69 Å, and the all-trans N- and O-acyl chains of the molecule are tilted by 34.6° and 32.5°, respectively, with respect to the normal to the respective methyl end planes. The packing diagram of the β polymorph of NPOM (NPOMβ) along the b-axis is given in Figure 4B. The packing of NPOM molecules in the β polymorph is different from that found in the α polymorph. In NPOMβ the molecules are packed in layers in such a way that the methyl groups of the O-acyl chains from one layer face the methyl groups of the O-acyl chains of the next layer, instead of the methyl groups of the N-acyl chain of the next layer as seen in the packing of NPOMα. Similarly, the N-acyl chains from one layer face the N-acyl chains of the next layer, resulting a symmetric packing arrangement.16 The layer thickness (C1−C32 distance) in NPOMβ is 34.40 Å, and the all-trans N- and O-acyl chains of the molecule are tilted by 34.5° and 34.0°, respectively, with respect to the normal to the respective methyl end planes. Packing diagrams of NPOMα along the a-axis and of NPOMβ along the c-axis are given in Figures S4A and S4B, respectively. The α polymorph of NPOL (NPOLα) is isostructural with NPOMα and has a similar packing arrangement (Figure S5). The packing is of the mixed type, where the methyl groups of the O-lauroyl chains from one layer face the methyl groups of the N-palmitoyl chains, with the closest methyl−methyl distance between opposite layers and the same layer being 3.919 Å and 4.895 Å, respectively. The layer thickness (C1−C30 distance) in NPOLα is 32.19 Å, and the all-trans N- and O-acyl 1136



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Figure 5. View along the long axis, illustrating packing of DAEs. (A) α-polymorph of NPOL (packing of both N-acyl and O-acyl chains); (B) packing of O-acyl chains in the β-polymorph of NPOL; packing of (C) N-acyl and (D) O-acyl chains in NPOU (β-polymorph). Since the chain packing is of the mixed type in the α-polymorph, both N-acyl and O-acyl chains face each other and pack similarly along the long axis. The N-acyl chain packing in the β-polymorph of NPOL is very similar to the chain packing observed in the α-polymorph (not shown). On the other hand, in NPOU (which is similar to the β-polymorph of NPOL), the N-acyl chain packing (panel C) is similar to that in NPOL, but the O-acyl chain packing (panel D) is less compact.

basis of packing of the hydrocarbon chains.22 To investigate the odd−even alternation in the thermodynamic properties of DAEs in detail, we determined the crystal structures of odd and even chain DAEs and compared the molecular packing and intermolecular interactions in them. A close view of the packing along the long axis shows that, for DAEs with even O-acyl chains (NPOL), both the polymorphs have similar packing arrangement (Figure 5A and B). In DAEs with odd O-acyl chains, the packing interactions between N-acyl chains (C16 = even) are similar to those in NPOL, whereas the packing interactions between the O-acyl chains are distinctly different from the interactions between the even N-acyl chains (Figure 5C and D). The distance between the closest methyl groups in opposite layers is shorter in DAEs with even O-acyl chains as compared to DAEs with odd O-acyl chains (Table 5 and Figure 5). This

chains of the molecule are tilted by 34.7° and 33.7°, respectively, with respect to the normal to the respective methyl end planes. Similarly, the β polymorph of NPOL (NPOLβ) is isostructural with NPOMβ and has a similar packing arrangement as observed in the crystal lattice of NPOMβ (see Figures 4B and S6). The packing is of the symmetric type, where the methyl groups of the O-lauroyl chains from one layer face the methyl groups of the O-lauroyl chains of the adjacent layer and N-palmitoyl chains from one layer face the methyl groups of the N-palmitoyl chains of the next layer. The layer thickness (C1−C30 distance) in NPOLβ is 31.64 Å and the all-trans N- and O-acyl chains of the molecule are tilted by 35.6° and 35.7°, respectively, with respect to the normal to the respective methyl end planes. The packing of NPON and NPOU is similar to that in the β polymorphs of NPOL and NPOM (see Figures S7 and S8). The acyl chains are packed in a symmetric manner, where the methyl groups of N-palmitoyl chains of one layer face the methyl groups of N-palmitoyl chains of the adjacent layer and methyl groups of odd O-acyl chains face the methyl groups of odd O-acyl chains of the next layer. The structures of NPOLα and NPOMα were also solved at low temperature (100 K), and their packing and intermolecular interactions were found to be similar to those observed in the structures determined at 298 K (see Supporting Information, Table S21, Figure S12). Therefore, it may be expected that the low temperature structures of the other DAEs investigated here will also be similar to those determined at 298 K. Even−Odd Alternation in Calorimetric Parameters. Previously, alternation in long chain compounds such as n-alkanes, alcohols, and fatty acids has been explained on the

Table 5. Distance between the Terminal C-Atoms between the Layers in N,O-Diacylethanolamines with Even and Odd O-Acyl Chains

compd

O-methyl to O-methyl distance (opposite layer) (Å)

O-methyl to O-methyl distance (same layer) (Å)

N-methyl to N-methyl distance (opposite layer) (Å)

N-methyl to N-methyl distance (same layer) (Å)

NPOO NPON NPOD NPOU

3.875 4.070 3.902 4.092

4.866 4.883 4.883 4.890

3.918 3.917 3.906 3.904

4.866 4.883 4.883 4.890

packing difficulty with odd chains (longer interbilayer distance) can be explained in terms of a geometric model, proposed 1137



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Figure 6. Schematic representation of the packing of (A) a diacylethanolamine with even N- and O-acyl chains and (B) diacylethanolamine with an even N-acyl chain and odd O-acyl chain (symmetric type chain packing). (C) Predicted packing of diacylethanolamine with an odd O-acyl chain with mixed type chain packing, which leads to less compact packing than that of the symmetric one (see text for details).

earlier to rationalize the melting point alternation in n-alkanes.27 A similar rationale suggests that the shape of N-palmitoyl-O-octanoylethanolamine (even N- and O-acyl chains) can be approximated to a rectangle, whereas the shape of the homologous N-palmitoyl-O-nonanoylethanolamine (even N-acyl and odd O-acyl chains) can be approximated to be a trapezoid in two dimensions (Figure 6). It is shown schematically in Figure 6 that the rectangle can be packed more tightly than a trapezoid, where similar packing will create some void space (marked in black). Therefore, the distance between the closest methyl groups in opposite layers (C1−C1) will be more in the case of DAEs with odd O-acyl chains as compared to the DAEs in which both N- and O-acyl chains are even. This is consistent with the results from the crystal structures (Figure 5 and Table 5). Thus, DAEs with even N- and O-acyl chains have close intermolecular contact at both ends, whereas DAEs with an odd O-acyl chain and an even N-acyl chain possess close intermolecular interaction on one side and at the other end the distances between terminal methyl groups are longer. This leads to a less dense packing in DAEs with an odd O-acyl chain, which is also reflected in the packing coefficients of the corresponding crystals. The packing coefficient for NPOO is 64.9%, which is higher than the values observed for NPON (63.2%) [the β polymorph of NPOO is considered, since its packing is similar to the packing observed in NPON]. Similarly, the packing coefficient of NPOD is 64.8%, which is also higher than that of NPOU (63.6%). These results suggest that the acyl chains in DAEs with both even chains are packed more tightly than those with odd O-acyl chains. Polymorphism in Mixed Chain DAEs. Generally, different packing arrangement with similar lattice energy can increase the possibility of polymorphism.28 Recently, on the basis of packing considerations and the structures of two DAEs with mixed chains, we predicted that at least two polymorphic forms should be possible for an L-shaped lipid.16 In the present studies the crystal structures of NPOL and NPOM in two different polymorphic forms have been solved by single-crystal X-ray diffraction, wherein the DAE molecules are packed in different possible arrangements. The acyl chain packing is of the mixed type in the α polymorph and the symmetric type in the β polymorph. The atomic resolution structures of two polymorphic forms in DAEs, which are “L” shaped, lend further support to the prediction that at least two polymorphic forms will be present for a lipid which adopts an “L” or “I” shape in the solid state.16

Although we were successful in solving the crystal structures of two polymorphic forms of DAEs with even acyl chains, in the case of DAEs with odd acyl chains, crystals of the α form could not be obtained despite several crystallization attempts. Our geometric model discussed above provides a possible explanation for the difficulty to obtain crystals of the α form. It is obvious from the model that the α form with odd O-acyl chain (mixed type chain packing) will experience less tight packing than the β form (Figure 6C). The mixed type chain packing with odd acyl chains will result in void space on both sides and the C−C distance will be more, resulting in a less compact acyl chain packing. Therefore, the α form is expected to be less stable than the β form in DAEs with odd O-acyl chains and this could be the reason why crystals of the α form could not be obtained for these lipids. Polymorphism and Odd−Even Alternation. The above studies suggest that in DAEs with odd acyl chains the β form is likely to be more stable than the α form and therefore the β form is expected to be predominant after crystallization. Now what about DAEs with mixed even acyl chains? Crystallization of DAEs with even, mixed acyl chains yielded two polymorphic forms. The percentage of a particular form in the mixture can give an idea of the kinetically controlled form, since in many cases it is regarded that the nucleation and growth of a crystal are under kinetic, rather than thermodynamic, control.29,30 Although it is difficult to estimate the relative content of the two forms in a mixture, random cell indexing indicated that for NPOM, NPOO and NPOD the mixture of crystals obtained from dichloromethane and methanol contained more crystals of the α form than the β form for each lipid. For NPOL we have been able to generate the β form only once and successive crystallization attempts always led to the α form. These observations indicate that for DAEs with mixed, even acyl chains, the α-form is the predominant one. Additional supporting evidence in this regard comes from crystal structure analysis which also suggests that the percentage of α form is likely to be more than that of β form. It has been suggested recently that a general correlation can be derived between approximate domain ratio and the expected crystallographic R-values.31 High R factor of polymorph II of aspirin, an analgesic, has been suggested to be due to an intergrowth of two “polymorphic” domains.31 A comparison of the R factors of the α and β polymorphic forms of DAEs showed that the R factor of α form is lower than that of the β form for all DAEs (Table 1 and Table S30). Therefore, it is likely that crystals of the β form contain a small fraction of the 1138



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mixed chain systems, which adopt an “L” shape in the solid state.

α form. This suggests that the percentage of α form is likely to be more in the mixture and the α form is kinetically controlled. While the α form is kinetically controlled the β form appears to be thermodynamically more stable. The distance between the closest methyl groups in opposite layers (C−C distance) in the β form is slightly lower than that in the α form for all the four DAEs investigated so far (Table 6), although this slight

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CONCLUSIONS A homologous series of N,O-diacylethanolamines with mixed acyl chains, which are biologically relevant lipids, have been synthesized, and their thermotropic phase transitions have been characterized by DSC. Analysis of DSC studies on DAEs with mixed chains yielded the thermodynamic parameters associated with the phase transition and revealed a qualitatively distinct pattern in the odd−even alternation. The odd−even effect becomes prominent with increase in the O-acyl chain lengths, which was not observed in fatty acids, NAEs, and other lipid systems. To explain the interesting odd−even effect, 3dimensional structures of four DAEs, two with odd O-acyl chains (NPOU, NPON) and two with even acyl chains (NPOL, NPOM), have been solved by single crystal X-ray diffraction. The structures demonstrate that the conformation of N-acyl chain in DAEs is very similar to that found in different NAEs, and all the DAEs adopt an “L” shape in the solid state. In addition, the structures of DAEs with mixed, even acyl chains have been solved in two different polymorphic forms at atomic resolution. In polymorph α the chain packing is mixed, whereas in the β form it is symmetric. The odd−even effect was explained by analyzing the molecular packing from crystal structures, which suggests that the DAEs with odd acyl chains pack with voids, whereas those with even N- and O-acyl chains pack more tightly. The role of polymorphism has been discussed, which may be responsible for the deviation of odd−even behavior from classical to nonclassical. A similar nonclassical odd−even effect and polymorphism may exist in other mixed chain systems, which adopt an “L” shape in the solid state.

Table 6. Distance between the Terminal C-Atoms between the Layers in the α and β Polymorphic Forms of N,ODiacylethanolaminesa

a

compd

methyl−methyl distance (1st type, opposite layer) (Å)

methyl−methyl distance (2nd type, opposite layer) (Å)

methyl−methyl distance (same layer) (Å)

NPOOα NPOOβ NPODα NPODβ NPOLα NPOLβ NPOMα NPOMβ

3.943 3.918 3.916 3.906 3.919 3.879 3.968 3.952

3.943 3.875 3.916 3.902 3.919 3.884 3.968 3.946

4.882 4.866 4.882 4.883 4.895 4.880 4.925 4.913

For NPOO and NPOD, the data were taken from ref 16.

difference in the layer distances are in the error range of structure determination. However, this may result in a more favorable van der Waals’ interaction, thus making the β form thermodynamically more stable. The DSC thermogram and temperature dependent powder X-ray diffraction (PXRD) data of NPOL show a phase transition ∼80−82 °C before the main transition (Figure 1 and Figure S13). The PXRD pattern of NPOL at 25 °C is close to the simulated PXRD pattern of NPOLα and PXRD pattern at 80 and 85 °C is close to that of NPOLβ (Figure S13 and S14). This suggests a probable phase transition from NPOLα to NPOLβ with temperature. The above analysis derived from crystal structures of DAEs with mixed acyl chains suggests that the odd−even alternation is most likely due to differences in the packing of acyl chains and that the nonclassical behavior observed in the alternation could be attributed to the presence of different fractions of the α and β forms in the bulk mixture. Now, why is a similar pattern in odd−even alternation not observed in DAEs with matched acyl chains? The difference in the thermodynamic stability between the α and β polymorphs may arise from the symmetry (the α form is asymmetric, whereas the β form is symmetric) and also from van der Waal’s interaction. In the α form only one type of hydrophobic region (brick) is present, whereas the β form consists of two types of hydrophobic regions (bricks): one longer and another shorter (see Figures 4 and S15; see also ref 16). Such a difference in the size of the hydrophobic segments, which we refer to as hydrophobic bricks, is expected to lead to differences in the van der Waal’s interaction in the two polymorphic forms. In DAEs with matched acyl chains, the length of hydrophobic bricks will be the same in both the polymorphs and the two polymorphs may not differ significantly in their thermodynamic stability. This may be the reason why the pattern of odd−even alternation observed earlier in matched chain DAEs15 is different from that observed in mixed chain DAEs in the present study. In this regard, we predict the possibility of a similar anomalous odd−even effect in other

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ASSOCIATED CONTENT

S Supporting Information *

Parts of the Experimental Section and Results and Discussion as well as crystallographic cif files for all the structures determined; Tables S1−S31, containing IR and NMR spectral data for all the DAEs reported, fractional atomic coordinates and isotropic displacement parameters for all the crystal structures presented as well as selected bond distances, bond angles, and torsional angles; and Figures S1−S15, giving representative IR and NMR spectra and presenting packing diagrams and hydrogen bonding patterns of selected examples as well as a schematic diagram. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. Telephone: +91-40-23134807. Fax: +91-40-23012460. Web site: http://chemistry.uohyd.ernet.in/∼mjs/.

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ACKNOWLEDGMENTS This work was supported by the Centre for Nanotechnology supported by the Department of Science and Technology (India) at the University of Hyderabad, in which M.J.S. is a coinvestigator. P.K.T. and S.T.R. were supported by Senior and Junior Research Fellowships, respectively, from CSIR (India). Use of the National Single Crystal Diffractometer Facility (SMART APEX CCD single crystal X-ray diffractometer) at 1139



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(30) Dunitz, J. D. Chem. Commun. 2003, 7, 545−548. (31) Bond, A. D.; Boese, R.; Desiraju, G. R. Angew. Chem., Int. Ed. 2007, 46, 618−622.

the School of Chemistry, University of Hyderabad, funded by the Department of Science and Technology (India), is gratefully acknowledged. We thank Dr. P. Raghavaiah for help with X-ray diffraction data collection and Prof. S. Pal for advise on the crystal structure analysis. The School of Chemistry is a Centre for Advanced Study of the University Grants Commission (India).

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