Structure and Thermotropic Phase Behavior of a Homologous Series

Article pubs.acs.org/JPCB

Structure and Thermotropic Phase Behavior of a Homologous Series of Bioactive N‑Acyldopamines S. Thirupathi Reddy,† Pradip K. Tarafdar,† Ravi Kanth Kamlekar, and Musti J. Swamy* School of Chemistry, University of Hyderabad, Hyderabad 500 046, India S Supporting Information *

ABSTRACT: N-Acyldopamines (NADAs), which are present in mammalian nervous tissues, exhibit interesting biological and pharmacological properties. In the present study, a homologous series of NADAs with varying acyl chains (n = 12−20) have been synthesized and characterized. Differential scanning calorimetric studies show that in the dry state the transition temperatures, enthalpies, and entropies of NADAs exhibit odd−even alternation with the values corresponding to the even chain length series being slightly higher. Both even and odd chain length NADAs display a linear dependence of the transition enthalpies and entropies on the chain length. However, odd−even alternation was not observed in the calorimetric properties upon hydration, although the transition enthalpies and entropies exhibit linear dependence. Linear least-squares analyses yielded incremental values contributed by each methylene group to the transition enthalpy and entropy and the corresponding end contributions. N-Lauroyldopamine (NLDA) crystallized in the monoclinic space group C2/c with eight symmetry-related molecules in the unit cell. Single-crystal X-ray diffraction studies show that NLDA molecules are organized in the bilayer form, with a head-to-head (and tail-to-tail) arrangement of the molecules. Water-mediated hydrogen bonds between the hydroxyl groups of the dopamine moieties of opposing layers and N−H···O hydrogen bonds between the amide groups of adjacent molecules in the same layer stabilize the crystal packing. These results provide a thermodynamic and structural basis for investigating the interaction of NADAs with other membrane lipids, which are expected to provide clues to understand how they function in vivo, e.g., as signaling molecules in the modulation of pain.

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immune system.6 Another important class of lipid molecules are N-acylethanolamines (NAEs), which have been studied for several decades due to their putative role in combating stress and interesting biological and medicinal properties.7−9 Among the NAANs, amides of long chain fatty acids with dopamineN-acyldopamines (NADAs)are a growing group of neuroactive lipids (neurolipins). Recent studies have revealed that NADAs with different fatty acyl chains serve as signaling molecules and play a major role in the modulation of pain. For example, N-arachidonyldopamine (NArDA) activates both human and rat vanilloid receptor subtype1 (TRPV1) with an efficiency equivalent to that of capsaicin.10 It also acts as a cannabinoid receptor1 (CB1) agonist and induces hypothermia, hypolocomotion, catalepsy, and analgesia in mice.11,12 Apart from the agonist activity toward CB1 and TRPV1 receptors, NArDA also activates a redox-sensitive p38 MAPK pathway that stabilizes COX-2 mRNA, resulting in the accumulation of the COX-2 protein.13 Analysis of the bovine striatal extract indicated the existence of other NADAs, such as N-palmitoyldopamine (NPDA), N-stearoyldopamine (NSDA), and N-oleoyldopamine (OLDA) besides NArDA, which was

INTRODUCTION Lipids are simple amphiphilic molecules which generally do not possess complex structures like proteins and nucleic acids, although in the presence of excess water they form a variety of supramolecular assemblies depending on their structure and other factors such as ionic strength, pH, and temperature. Among the various aggregated structures adopted by hydrated lipid dispersions, the bilayer assembly is found in the membranes of cells and organelles. However, for long lipids have been considered to be dull molecules compared to other biomolecules such as proteins that catalyze biochemical reactions and nucleic acids that carry genetic information necessary for producing proteins. This traditional view of lipids has changed significantly over the past two decades or so, and their role as second messengers in cell signaling, cell function, and health has been emphasized.1,2 Recently, lipid metabolites and pathways strategy (LIPID MAPS) initiative in lipidomics has been taken up to identify and quantitate the major and minor lipid species in mammalian cells.3−5 The goal of this is to quantitate the changes in various lipid species in response to perturbation and to characterize their biochemical and biophysical roles. Among the various classes of lipids, N-acyl conjugates of amino acids and neurotransmitters (NAANs) have attracted considerable attention in recent years because of their potential roles in the nervous system, vasculature, and the © 2013 American Chemical Society

Received: March 20, 2013 Revised: June 20, 2013 Published: June 24, 2013 8747

dx.doi.org/10.1021/jp402750m | J. Phys. Chem. B 2013, 117, 8747−8757

The Journal of Physical Chemistry B

Article

Scheme 1. Synthesis of N-Acyldopamines

Synthesis and Characterization of N-Acyldopamines. N-Acyldopamines (NADAs) were synthesized by a slight modification of a previous procedure11 involving a simple condensation between fatty acid and dopamine hydrochloride. Briefly, to a solution of fatty acid (1.2 equiv) in acetonitrile kept at 4 °C, triethylamine (1.5 equiv) and isobutyl chloroformate (1.2 equiv) were added. The mixture was stirred for an hour under nitrogen atmosphere and diluted with water, and the mixed anhydride was extracted with ethyl acetate. The mixed anhydride was allowed to react with a solution of dopamine hydrochloride (1.3 equiv) in dimethylformamide (DMF) containing triethylamine (1.5 equiv) overnight at 0 °C under inert atmosphere. After completion of the reaction, dichloromethane was added to the solution followed by water, and the sample was mixed vigorously to remove DMF. The organic layer was washed 5−6 times with distilled water to completely remove DMF, dried over anhydrous sodium sulfate, and concentrated. The crude product obtained was purified on a silica gel column by using increasing concentrations of ethyl acetate in hexane and recrystallized from a mixture of acetonitrile and acetone at −20 °C. Overall yields ranged between 60 and 65%. The NADAs obtained were characterized by TLC, melting point, FTIR, 1H NMR and 13C NMR spectroscopy, and high-resolution mass spectrometry. IR spectra (KBr pellet) were recorded on a Jasco FTIR 5300 spectrometer. 1H NMR spectra were obtained at room temperature in CDCl3 (solvent) on a Bruker Avance NMR spectrometer operating at 400 MHz, whereas 13C NMR spectra were recorded at room temperature in CD3OD at 100 MHz on the same spectrometer. Mass spectra of NADAs were obtained in the positive ion mode on a Bruker Maxis high-resolution mass spectrometer, equipped with an electrospray ionization system. Capillary melting points of the compounds were recorded on a Superfit (Mumbai, India) melting point apparatus. Briefly, solid NADA samples were packed in glass capillaries with one end sealed, and the melting of the compound was monitored visually by means of a magnifying lens which was built into the apparatus. Temperature was measured to ±0.1 degree accuracy with a calibrated thermometer. Differential Scanning Calorimetry. DSC studies on solid NADAs were carried out on a PerkinElmer PYRIS Diamond differential scanning calorimeter. About 1−2 mg of dry NADAsobtained by recrystallization as described above was weighed accurately into aluminum sample pans, covered with an aluminum lid, and sealed by crimping. Reference pans

isolated earlier from the same tissue.14 OLDA is a capsaicin-like lipid with agonist activity for TRPV1 but devoid of affinity to CB1 receptors. OLDA is also involved in cellular Ca2+ trafficking and behavioral alternations.14 The naturally occurring saturated N-acyldopamines such as NPDA and NSDA did not activate TRPV1, although they significantly enhanced the TRPV1-mediated effects of NArDA, called the entourage effect.15 However, recent findings on the agonist activity of N-octanoyldopamine suggest that TRPV1 activation might be a functional property of the whole class of N-acyldopamine derivatives.16 Interestingly, NPDA, OLDA, and NArDA were found to inhibit IgE-mediated allergic response in RBL-2H3 cells,17 suggesting potentially therapeutic/preventive application for these compounds. In addition, OLDA was found to cross the blood−brain barrier (BBB) and therefore may serve as a prodrug of dopamine.18 The above observations suggest that NADAs may represent a new source of biochemically and pharmacologically important compounds. Although the structure−activity relationships of NADAs have not been evaluated in detail, there are possibilities that NADAs may disrupt lipid packing at the target protein− lipid interface.6 While the dopamine moiety in the headgroup may provide specificity, the lipid tail can insert into cell membranes and alter the membrane dynamics, which could affect the functional properties of membrane proteins (e.g., CB1, TRPV1). In order to understand how NADAs interact with other membrane lipids and perturb membrane structure and dynamics, it is important to investigate the physical properties, phase behavior, and interaction of these compounds with other membrane components such as phospholipids. As the first step in this direction, we have performed differential scanning calorimetric studies on a homologous series of NADAs with saturated acyl chains in order to characterize the phase transitions of dry and hydrated forms of NADAs, and the results obtained are reported here. In addition, we have determined the 3-dimensional structure of NLDA, which will be helpful in understanding the physical properties of NADAs and to establish structure−activity relationship for them.

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MATERIALS AND METHODS Materials. Fatty acids and dopamine hydrochloride were obtained from Sigma (St. Louis, MO), and triethylamine was obtained from Merck, Germany. Isobutyl chloroformate was purchased from Lancaster (Eastgate, England). All solvents were purchased from Sisco Research Laboratory (Mumbai, India) and were distilled and dried prior to use. 8748



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(amide-I) and 1556−1548 cm−1 (amide-II). The amide N−H stretching band was seen at 3308-3296 cm−1, and the O−H stretching of the catechol moiety was observed around 3470− 3445 cm−1, whereas the C−N stretching band was seen at 1118−1111 cm−1. The stretching, bending, and rocking modes of the polymythelene portion of the hydrophobic acyl chains were seen at 2921−2849, 1466−1456, and 719 cm−1, respectively. The aromatic C−H stretching bands were seen at 3089−3077 cm−1, and bending vibrations (from ortho and meta protons) were found around 866−862 and 819−815 cm−1. The FTIR data for all the NADAs are summarized in Table S1. The 1H NMR spectra of NADAs showed resonances at 2.17−2.19 δ (2H, t) and 1.58−1.61 δ (2H, m) for the methylene groups at α and β positions to the carbonyl moiety, at 5.56−5.82 δ (1H, bs) for the amide N−H, at 3.50−3.57 δ (2H, q) for the methylene group adjacent to the amide N−H, and at 2.72−2.74 δ (2H, t) for the methylene group connected to the aromatic ring. The two hydroxy protons gave broad singlets at 7.4−8.0 δ and 5.61−6.32 δ, whereas the aromatic protons gave resonances between 6.58 and 6.84δ. Resonance of the terminal methyl group was seen at 0.88−0.92 δ (3H, t), whereas the resonance of the polymethylene group was observed at 1.24−1.27 δ (16−32H, m). These data are summarized in Table S2. A representative 13C NMR spectrum of N-lauroyldopamine is given in Figure S3. The spectrum shows a resonance at 13.04 δ corresponding to the terminal methyl group of the acyl chain. Resonances corresponding to the methylene groups are seen at 22.33, 25.68, 29.33, 31.67, 34.57, 35.78, and 40.81 δ as well as between 28.87 and 29.34 δ (five peaks with the peak at 29.34 being more intense than the others) and six resonances between 114.92 and 144.86 (corresponding to the aromatic carbons) and a peak at 174.87 δ (carbonyl carbon). These values are consistent with the structure of N-lauroyldopamine. All other NADAs gave similar spectra, except that the intensity of the peak at ∼29.34 δ increased with increase in the acyl chain length. The 13C spectral data for all the NADAs are given in Table S3. A high-resolution ESI mass spectrum of NLDA is shown in Figure S4. The most intense peak seen at 336.2536 matches well with the molecular ion of the compound ([M + H]+, calcd mass 336.2538). In addition, prominent peaks are also seen at 671.4982 ([M2 + H]+, calcd mass 671.4998) and 1006.7397 ([M3 + H]+, calcd mass 1006.7458), which correspond to proton adducts of dimer and trimer of NLDA. In addition, sodium ion adducts of M, M2, and M3 are also seen in the spectrum of NLDA. For both proton adducts and sodium ion adducts, the intensity of the peaks decreases with increasing size of the adduct. In each case, the proton adduct is more intense as compared to the sodium ion adduct. Other NADAs also yielded essentially similar mass spectra. These data are given in Table S4. The above results from FTIR, 1H and 13C NMR spectroscopy, and ESI mass spectrometry are fully consistent with the structures of N-acyldopamines and indicate that they are of high purity. Differential Scanning Calorimetry. Heating thermograms corresponding to dry NADAs of even acyl chain lengths are presented in Figure 1A, and those corresponding to the odd chain length series are shown in Figure 1B. The thermograms show that each NADA exhibits a major endothermic transition, which corresponds to the capillary melting point of the compound. In some cases, particularly for NADAs with shorter

were prepared similarly, but without any sample in them. Heating and cooling scans were performed from room temperature (ca. 25 °C) to about 120 °C at a scan rate of 1.5°/min, and each sample was subjected to three heating scans and two cooling scans. Transition enthalpies were determined by integrating the area under the transition curve, and transition entropies were calculated from the transition enthalpies assuming a first-order transition according to the equation19,20 ΔHt = TtΔSt

(1)

where Tt is the transition temperature and ΔHt values taken at this temperature were used to calculate the corresponding ΔSt values. DSC studies of hydrated NADAs were carried out on a VPDSC microcalorimeter from MicroCal (Northampton, MA). Samples of NADAs (3−4 mg) were weighed accurately in aluminum pans and transferred into clean, dried glass test tubes. Each sample was dissolved in dichloromethane/ methanol (2:1; v/v) mixture, and a thin film of the lipid was obtained by blowing a gentle stream of dry nitrogen gas over the solution. Final traces of the solvent were removed by vacuum desiccation for at least 3 h. Then 20 mM sodium phosphate buffer, pH 7.4 was added to the thin film, and the sample was subjected to several cycles of freeze−thawing with intermittent vortexing in order to obtain a homogeneous suspension, which was used for DSC studies. Each sample was subjected to three heating scans and two cooling scans between ca. 10 and 110 °C at a scan rate of 1°/min. Crystallization, X-ray Diffraction, and Structure Solution. Thin plate type, colorless crystals of N-lauroyldopamine (NLDA) were grown at room temperature from acetonitrile containing a trace of acetone. X-ray measurements were carried out at room temperature (ca. 25 °C) on a Bruker SMART APEX CCD area detector system using a graphite monochromator and Mo Kα (λ = 0.717 073 Å) radiation obtained from a fine-focus sealed tube. The minimum resolution of X-ray diffraction measurements was 0.84 Å. Data reduction was done using Bruker SAINTPLUS program. Absorption correction was applied using SADABS program, and refinement was done using SHEXTL program.21 Structure refinement was carried out using 1172 [>2σ(Fo)] observed reflections and converged into a final R1 = 0.1777 and wR2 = 0.4383, with goodness of fit = 1.597. Crystal Parameters of N-Lauroyldopamine. Molecular formula, C20H35NO4; molecular weight, 353.49; crystal system, monoclinic; space group (Sg), C2/c; unit cell dimensions (with standard deviations in parentheses): a = 77.42(6) Å, b = 4.900(2) Å, c = 10.919(6) Å, α = 90°, β = 90.966(11)°, γ = 90°; volume of the unit cell, V = 4142(4) Å3; number of molecules in the unit cell, Z = 8.

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RESULTS Synthesis and Characterization of N-Acyldopamines. A homologous series of N-acyldopamines (NADAs) with saturated acyl chains containing 12−20 C atoms have been synthesized in the present study by a simple condensation of the fatty acids with dopamine hydrochloride. The structure and purity of NADAs were characterized by TLC and FTIR, 1H NMR and 13C NMR spectroscopy, and ESI mass spectrometry. Representative FTIR and 1H NMR spectra of N-palmitoyldopamine are given in Figure S1 and Figure S2, respectively. FTIR spectra (KBr pellet) of recrystallized NADAs showed absorption bands due to amide linkage at 1641−1637 cm−1 8749



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Figure 1. DSC heating thermograms of dry N-acyldopamines. (A) Thermograms of NADAs with even number of C atoms in the acyl chains. (B) Thermograms of NADAs with odd number of C atoms in the acyl chains. The number of carbon atoms in the acyl chain is indicated against each thermogram.

Figure 2. DSC heating thermograms of hydrated NADAs. (A) Thermograms of NADAs with even number of C atoms in the acyl chains. (B) Thermograms of NADAs with odd number of C atoms in the acyl chains. The number of carbon atoms in the acyl chain is indicated against each thermogram.

entropies, ΔSt, were calculated from transition enthalpies using eq 1. The values obtained are listed in Table 1. Chain Length Dependence of Transition Enthalpy and Transition Entropy. Chain length dependences of transition enthalpy and transition entropy for the chain-melting phase transitions of dry NADAs are given in Figures 3A and 3B, respectively. In both cases, NADAs with even and odd acyl chains independently exhibit linear dependence of ΔHt and ΔSt on the chain length. However, when the data obtained with even and odd acyl chain lengths are viewed together, a zigzag pattern is seen with the values of enthalpy and entropy for the odd acyl chain lengths being slightly lower than those of even acyl chain lengths. That is, the calorimetric data exhibit odd− even alternation, and this aspect will be discussed in more detail below. The transition enthalpies and transition entropies of dry NADAs for even and odd acyl chain lengths could be fit well to expressions 2 and 3:22

acyl chains, an additional minor transition was observed. These minor transitions suggest the possibility of polymorphism in NADAs. When the samples were subjected to a second heating scan, in most cases the thermograms became more complex, involving multiple transitions (Figure S5), possibly due to partial degradation of the samples when subjected to heating to high temperatures. Therefore, in all the cases only the first heating scan was considered for further analysis, i.e., to estimate transition temperatures, enthalpies, and entropies, and the values obtained are presented in Table 1. DSC studies were also carried out on NADAs upon hydration with 20 mM phosphate buffer (pH = 7.4). Each NADA gave a single, sharp phase transition in the hydrated state and the transitions were reversible, although enthalpy of the transition decreased slightly when the sample was subjected to several cycles of heating and cooling. Thermograms from the first heating scans for NADAs with even and odd acyl chains are shown in Figures 2A and 2B, respectively. The phase transition temperatures, Tt, were estimated from the peak positions of the DSC endotherms, whereas the transition enthalpies, ΔHt, were obtained by integrating the area under the curve after buffer subtraction, normalization, and baseline correction. Transition

ΔHt = ΔH0 + (n − 2)ΔHinc

(2)

ΔSt = ΔS0 + (n − 2)ΔSinc

(3)

where n is the number of C atoms in the acyl chain and ΔH0 and ΔS0 are the end contributions to the ΔHt and ΔSt,

Table 1. Transition Temperatures (Tt), Transition Enthalpies (ΔHt), and Transition Entropies (ΔSt) of N-Acyldopamines in Dry and Hydrated States (Average Values of Three Measurements with Standard Deviations Are Given) dry NADAs acyl chain length 12 13 14 15 16 17 18 19 20

Tt (°C) 78.0 78.6 87.0 88.1 94.5 94.0 99.2 97.8 100.9

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1 0.1

ΔHt (kcal/mol) 10.27 9.10 12.70 12.50 14.49 14.03 16.01 15.31 16.50

± ± ± ± ± ± ± ± ±

0.03 0.10 0.01 0.22 0.31 0.02 0.17 0.01 0.19

hydrated NADAs ΔSt (cal/mol/K) 29.3 28.4 35.3 34.6 39.4 38.2 43.0 41.3 44.1

± ± ± ± ± ± ± ± ±

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0.1 0.3 0.1 0.6 0.8 0.1 0.5 0.1 0.5

Tt (°C) 73.9 78.7 82.5 85.8 88.7 91.1 93.3 94.8 96.1

± ± ± ± ± ± ± ± ±

0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.1 0.1

ΔHt (kcal/mol) 3.60 5.35 6.40 7.35 9.40 10.65 12.60 13.65 14.75

± ± ± ± ± ± ± ± ±

0.14 0.21 0.28 0.63 0.91 0.07 0.85 0.92 0.35

ΔSt (cal/mol/K) 10.4 15.2 18.0 20.5 26.0 29.3 34.4 37.1 40.0

± ± ± ± ± ± ± ± ±

0.4 0.6 0.8 1.8 2.5 0.2 2.3 2.5 1.0



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Figure 4. Chain length dependence of transition enthalpies (A) and transition entropies (B) of hydrated NADAs. Values of ΔHt and ΔSt were plotted against the number of methylene (CH2) units (n − 2) in the acyl chains. Solid line represents linear least-squares fit of the data.

Figure 3. Chain length dependence of transition enthalpies (A) and transition entropies (B) of dry NADAs. Values of ΔHt and ΔSt are plotted against the number of methylene (CH2) units (n − 2) in the acyl chains. Solid and dotted lines represent linear fits of the data for even- and odd-chain length series, respectively.

values and end contributions of transition enthalpy and entropy were obtained. These values are also listed in Table 2. Chain Length Dependence of Transition Temperatures. The transition temperatures of both dry and hydrated NADAs increase with increasing chain length (Figure 5). However, in both cases magnitude of the change decreases with increase in the chain length. Additionally, for the dry NADAs an alternation is seen between the odd-chain length and evenchain length series, with the even chain length series exhibiting slightly higher transition temperatures than odd chain length

respectively, arising from the terminal methyl group and polar region of N-acyldopamine molecule. ΔHinc and ΔSinc are the average incremental values contributed by each CH2 group to ΔHt and ΔSt, respectively, and the values of these parameters obtained from linear least-squares analysis are listed in Table 2. Table 2. Incremental Values (ΔHinc, ΔSinc) of Chain Length Dependence and End Contributions (ΔH0, ΔS0) to Phase Transition Enthalpy and Entropy of N-Acyldopaminesa dry NADAs thermodynamic parameters ΔHinc (kcal/mol) ΔH0 (kcal/mol) ΔSinc (cal/mol/K) ΔS0 (cal/mol/K)

even chain length 0.95 1.01 2.27 7.22

(±0.07) (±0.97) (±0.2) (±2.62)

odd chain length 0.87 0.73 2.11 6.10

(±0.1) (±1.42) (±0.26) (±3.64)

hydrated NADAs 1.42 −10.55 3.76 −26.99

(±0.04) (±0.57) (±0.11) (±1.55)

a

Average values of transition enthalpy and transition entropy given in Table 1 have been used for the linear fitting of the data. Errors shown in parentheses are fitting errors obtained from the linear least-squares analysis.

Linear chain length dependence of transition enthalpy and transition entropy observed here indicates that structures of the NADAs of different even chain lengths are very similar in the solid state. Similarly, the data suggest that structures of all oddchain NADAs are also likely to be very similar. This suggests that the molecular packing and intermolecular interactions in all the even chain length NADAs are likely to be very similar, and determination of the 3-dimentional structure of any one of them is likely to give a good idea of the molecular packing and intermolecular interactions present in the crystalline state for the entire homologous series. Chain length dependences of transition enthalpy and transition entropy for the thermotropic phase transitions of hydrated NADAs are shown in Figures 4A and 4B, respectively. Interestingly, for the hydrated samples no clear-cut even−odd alternation is seen, and the values of ΔHt and ΔSt for the entire homologous series could be fitted satisfactorily to a straight line. From the linear least-squares analysis, the incremental

Figure 5. Chain length dependence of phase transition temperatures of NADAs in the dry (A) and hydrated (B) conditions. Solid and dashed lines in (A) correspond to nonlinear least-squares fits of the transition temperatures of even-chain length and odd-chain length series to eq 7. Solid line in (B) corresponds to nonlinear least-squares fit of the Tt values of all hydrated NADAs to eq 7. 8751



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additional water molecule is also seen, which is found in the same asymmetric unit. The atomic coordinates and equivalent isotropic displacement parameters for all non-hydrogen atoms are given in Table S5. The bond distances and bond angles involving the non-hydrogen atoms are presented in Table S6, whereas the torsion angles are given in Table S7. These data show that the hydrocarbon portion (C10−C20) of NLDA molecule is in an all-trans conformation as all the torsion angles corresponding to the acyl chain region are in the neighborhood of 180°. The dihedral angle data presented in Table S5 also shows that all the carbon atoms of the phenyl ring, the two oxygen atoms belonging to the catechol moiety, and the C7 carbon atom are in the same plane, which is expected due to the sp2 hybridization of the ring carbons. The torsion angles of C1−C7−C8−N1, C7−C8−N1−C9, and C8−N1−C9−-C10 are 171.66°, 129.5°, and 174.9°, respectively. This shows that geometry of the NLDA molecule is essentially linear until the C8 atom. The structure of dopamine moiety is similar to that seen in the crystal structure of dopamine hydrochloride as well as the MD simulated structure of dopamine bound to its receptor (dopamine D2 receptor).26,27 Therefore, acylation does not seem to alter the conformation adopted by the dopamine moiety in the crystal. Molecular Packing and Molecular Area. Packing diagrams of NLDA viewed down the c-axis and a-axis are shown in Figures 7A and 7B, respectively. The NLDA molecules are packed head-to-head (and tail-to-tail) in stacked bilayers. The O−H···O hydrogen bonds between the head groups of opposite leaflets mediated by water molecules stabilize this arrangement (see Figure 8A). In addition, the amide groups of adjacent molecules are also involved in hydrogen bonds. The carbonyl oxygen atoms of adjacent

compounds (Figure 5A), whereas such alternation was not observed with the hydrated samples (Figure 5B). With the dry samples, both for even series and odd series, the Tt values increase in a smooth progression but with decreasing increments as the chain length is increased. As the acyl chain length increases, the total contribution from the polymethylene portion toward the total enthalpy and entropy of the phase transition will be sufficiently large that the end contributions are negligible in comparison. At infinite acyl chain length, eqs 2 and 3 can be reduced to22 ΔHt = (n − 2)ΔHinc

(4)

ΔSt = (n − 2)ΔSinc

(5)

Then the transition temperature for infinite chain length, Tt∞, will be given by Tt∞ = ΔHinc/ΔSinc

(6)

T∞ t

From the data presented in Table 2, the values for NADAs of even and odd acyl chain lengths (in the dry state) have been estimated using eq 6 as 418.5 and 412.3 K, respectively. Similarly, the T∞ t value for the hydrated NADAs was estimated as 377.7 K. For a number of diacyl lipids as well as single chain amphiphiles, which exhibit linear dependence of transition enthalpy and transition entropy on chain length, it has been shown that the data can be fit to the equation20,23−25 Tt = ΔHt /ΔSt = Tt∞[1 − (n0 − n′0 )/(n − n′0 )]

(7)

where n0 (= −ΔH0/ΔHinc) and n′0 (= −ΔS0/ΔSinc) are the values of n at which the transition enthalpy and transition entropy extrapolate to zero. It can be seen from Figure 5A that the transition temperatures of NADAs with even number of C atoms as well as odd-number of C atoms in the acyl chains independently fit rather well with eq 7. With the hydrated NADAs the transition temperatures for the entire homologous series could be satisfactorily fit by nonlinear least-squares method to eq 7, as seen clearly in Figure 5B. Additionally, the fitting parameters also yielded the T∞ t values for even and odd NADAs in the dry state as 395.1 ± 7.6 and 390.8 ± 0.9 K and as 393.7 ± 1.5 K for the hydrated NADAs. These values are in reasonable agreement with the T∞ t values estimated using eq 6. Description of the Structure. An ORTEP of the crystal structure of N-lauroyldopamine is shown in Figure 6 along with the atom numbering for all non-hydrogen atoms. In this plot an

Figure 7. Packing diagrams of NLDA. (A) A view down the c-axis. (B) A view down the a-axis. Color code: carbon, gray; nitrogen, blue; oxygen, red.

Figure 6. ORTEP showing the molecular structure of N-lauroyldopamine (NLDA). 8752



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Figure 8. (A) Hydrogen-bonding pattern in the crystal lattice of NLDA. A close-up view of the bilayer is shown displaying N−H···O and O−H···O type hydrogen bonding down the c-axis. The acyl chains are truncated to show an enlarged view of the headgroup region with hydrogen bonds shown in dashed lines. (B) Packing of the catechol moieties in the crystal lattice. The aromatic rings are laterally displaced, precluding any possibility of π-stacking interactions. Carbon atoms are shown as open circles, nitrogen atoms are shown in blue, and oxygen atoms are in red.

is 26.75 Å2, which is higher than the molecular area of different NAEs (21.95−22.03 Å2)28−30 as well as in diacylethanolamines with matched chains (21.42−22.13 Å2) whose crystal structures have been determined earlier.20,31,32 The larger molecular area and higher tilt angle in NLDA suggest that the acyl chains are apart from each other. Hydrogen-Bonding and Intermolecular Interactions. In order to understand the intermolecular hydrogen-bonding interactions in the crystal structure of NLDA, the molecular packing in the crystal was examined from various angles. Figure 8A shows the hydrogen-bonding interactions between NLDA molecules in the crystal lattice. These include intermolecular hydrogen bonds between the O−H groups of the catechol moieties from opposite leaflets mediated via bridging water molecules as well as H-bonds between amide N−H and carbonyl oxygen atoms of adjacent NLDA molecules in the

molecules point in opposite direction, providing appropriate juxtaposition of the amide carbonyl and N−H moieties of adjacent molecules to form N−H···OC type hydrogen bonds. The terminal methyl groups of the acyl chains in stacked bilayers are in van der Waals contact, with the closest methyl−methyl distance between opposite layers and same layer being 3.705 and 4.9 Å, respectively. The bilayer thickness (C20−C20 distance) for NLDA is 47.96 Å. The layer thickness (C20−O2 distance) in the crystal structure of NLDA is 22.78 Å, and the all-trans N-acyl chains of the molecule are tilted by 46.16° with respect to the normal to the methyl end plane. This value is considerably higher than the tilt angles observed in the crystal structures of several N-acylethanolamines (35°− 37°).9,28−30 The higher tilt angle may arise from the larger area of the headgroup in NADAs, containing the catechol moiety. The area per each NLDA molecule in the bilayer plane 8753



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inside the membrane interior, thus making the amide bond less accessible to the enzyme. However, there are no reports regarding the physicochemical properties of NADAs, which can help to understand the above interesting properties. In the present study we have investigated the phase behavior of a homologous series of NADAs bearing saturated fatty acyl chains and compared their behavior with NAEs, which are also single-chain amphiphiles that exhibit interesting biological properties. In addition, we have determined the structure of NLDA at atomic resolution and analyzed the intermolecular and packing interactions in the crystal lattice, which will be helpful in understanding the physical properties of NADAs and for establishing structure−activity relationships. Incremental Enthalpy and Entropy. Linear least-squares analysis of transition enthalpies and entropies of dry as well as hydrated NADAs yielded ΔH0 and ΔS0 (the end contributions to transition enthalpy and entropy, respectively, arising from the terminal methyl group and polar region of N-acyldopamine molecule) as well as ΔHinc and ΔSinc (the average incremental contribution of each CH2 group to the transition enthalpy and entropy). Theoretically, ΔHinc gives a measure of cohesiveness of the acyl chain packing. It can be seen from Table 2 that ΔHinc of NADAs in the dry state (for both even and odd acyl chain lengths) is lower than the ΔHinc in the hydrated state. In the case of NAEs it was found that ΔHinc of the dry state is higher than that of the hydrated state.24,25,34 This difference in the ΔHinc of NADAs and NAEs possibly arises from the hydrophobic effect and π-stacking of the aromatic rings of the catechol moiety. Analysis of the packing interactions in the crystal structure of NLDA shows that the catechol moieties in adjacent layers are laterally displaced, resulting in a lack of πstacking interaction of the aromatic rings in the solid state (Figure 8B). The end contribution of entropy, ΔS0, is also more negative for hydrated samples as compared to the dry state. The negative ΔS0 is also indicative of the hydrophobic effect playing a role in the packing of NLDA, which could arise due to the ordering of water molecules around the catechol moiety of the headgroup.35 Together, these observations suggest that the hydrophobic effect and possibly π-stacking in hydrated form bring the NLDA molecules closer, which results in a more compact packing of the acyl chains. On the other hand, the acyl chains in NAEswhich do not have any nonpolar moieties in the headgroup region that can be involved in hydrophobic/π-stacking interactionsare more tightly packed in the dry state.28−30 In the solid state of NLDA, water-mediated hydrogen bonds between two catechol moieties help them to pack. Each hydrogen bond likely provides an enthalpic stabilization of 5−6 kcal/mol.36 The enthalpic stabilization by H-bonding likely puts a geometric constraint such that π-stacking between catechol moieties would be difficult to achieve. Also, the acyl chains in the NLDA crystal are far apart and less tightly packed in the solid state. The closest and longest methyl−methyl distances in the same layer of NLDA are 4.9 and 6.2 Å, whereas in NAEs, the closest and longest methyl−methyl distances in the same layer were 4.88 and 5.4 Å, respectively. Together, these observations further confirm that the acyl chains are less tightly packed in solid NLDA. The decrease in the melting transition temperature upon hydration in NAEs was >15 deg, whereas in NADAs the decrease is in the neighborhood of ∼5 deg. Altogether, our data strongly suggest that upon hydration the acyl chains become more tightly packed in NADAs, and the most likely factors responsible for this are hydrophobic effect and π-stacking.

same side of the bilayer. Each hydroxyl group of the dopamine moiety forms a water-mediated H-bond with a hydroxyl group of the dopamine moiety of an NLDA molecule from the opposing layer. Thus, each NLDA molecule from one leaflet is connected to an NLDA molecule from the opposite leaflet by two water-mediated H-bonds. Each water-mediated H-bond connects the OH group on the C-1 atom of an NLDA molecule to the OH group on the C-2 atom of an NLDA molecule in the opposite layer (Figure 8A). While the OH group on the C-1 acts as a hydrogen bond donor, the OH group on the C-2 acts as a hydrogen bond acceptor, with a water molecule mediating these interactions. All the O−H···O hydrogen bonds in NLDA have two types of O···H distances: the O2···H distance is 2.108 Å, whereas the O1···H distance is 2.316 Å. The corresponding distances between the donor and acceptor oxygen atoms are 2.802 and 2.855 Å, respectively. These hydrogen bonds are nonlinear with the angle between covalent bond and hydrogen bond being 142.27° and 121.17°, respectively. In addition to the hydrogen bonds between the hydroxyl groups, there are also strong hydrogen bonds between the amide N−H and carbonyl oxygen atoms of adjacent NLDA molecules (Figure 8A). The distance between the hydrogenbonded carbonyl oxygen and amide hydrogen atom is 2.116 Å, and the N···O distance is 2.972 Å. The angle between the covalent and hydrogen bond (N−H···O angle) is 173.23°. The O−H···O hydrogen bonds form extended networks, which stabilize the bilayer arrangement in the crystal lattice. The N− H···O hydrogen bonds connect adjacent NLDA molecules in the same plane. However, while the N−H group of each NLDA molecule forms a hydrogen bond with an adjacent molecule in another unit cell on one side, the carbonyl oxygen atom of the same NLDA molecule forms a hydrogen bond with the N−H group of an adjacent NLDA molecule in another unit cell on the other side.

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DISCUSSION N-Acylated derivatives of amino acids, neurotransmitters, and ethanolamine constitute an important class of bioactive lipids. Among these, considerable attention has been focused on Nacylethanolamines (NAEs) for nearly 5 decades in view of their putative stress combating role.7−9,25,33 Over the past 15 years our laboratory has been investigating the structure, phase behavior, and membrane interactions of NAEs with the objective of establishing structure−activity relationships for them.9,25 Recently, N-acyl amino acids and neurotransmitters (NAANs) have attracted much attention in view of their biological properties.6 However, structure−activity relationships for these compounds are still largely unexplored. NAcyldopamines (NADAs), which are produced by the condensation of fatty acids with dopamine, possess interesting biological activity in vivo and in vitro. NADAs with Narachidonyl and N-oleoyl chains activate TRPV1 channel whereas NADAs bearing stearoyl and palmitoyl chains were found to increase the activity of N-arachidonyl dopamine,15 an effect termed as entourage ef fect. It is believed that these molecules act by direct binding to the receptor or by changing the physical properties of the membrane.6 The entourage ef fect shown by NSDA and NPDA suggests that NADAs alter the membrane structure. In addition, OLDA has been reported to cross the BBB with an efficiency that is many times greater than that of dopamine18 and that it resists hydrolysis by FAAH.11 This retardation in the hydrolysis could be attributed to its interaction with biomembranes, with the acyl chain embedded 8754



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Odd−Even Chain Alternation in Thermodynamic Parameters. The data presented in Figures 3 and 5 show that the phase transition temperatures and thermodynamic parameters (ΔHt and ΔSt) for dry NADAs exhibit an odd− even alternation. Similar trends were observed in the transition temperatures and thermodynamic properties of long-chain hydrocarbons, fatty acids, N-acylethanolamines and N-, Odiacyl ethanolamines with matched as well as mixed chains in the solid state.20,22,25,34 This alternation has been explained on the basis of packing of hydrocarbon chains. In long-chain fatty acids, for example, it was shown that differences in the packing properties between the terminal methyl groups between the even and odd acyl chain lengths can explain the differences in the physical properties.22 Such differences do not arise in the methyl group packing if the chains are perpendicular to the methyl group plane. However, if the hydrocarbon chains are tilted with respect to the plane of the methyl groups, their packing modes can differ, leading to alternation in the physical properties.22 The hydrocarbon chain tilt seen in the crystal structure of NLDA is consistent with the alternation in the thermodynamic parameters between the homologous series of NADAs containing even and odd acyl chain lengths. Phase Behavior and Biological Implications. The phase behavior of amphiphiles is guided by the packing parameter, P (= v/lca0, where v and lc are the volume and chain length of the hydrophobic portion, respectively, and a0 is the optimal surface area occupied by the molecule at the hydrocarbon interface).37 Amphiphiles for which P ≈ 1 favor the formation of a lamellar phase, with amphiphilic bilayers separated by water layers. The lamellar phase is the common structure seen in biological membranes. If P > 1, i.e., amphiphiles with a large effective chain volume and/or a small headgroup possess a reverse wedge shape and tend to promote formation of inverse mesophases. N-Acylethanolamines (NAEs) with small headgroup form lamellar crystalline phase at low temperature, whereas at high temperature they form lamellar liquid crystalline phase and at least two isotropic phases.24,38 At high temperatures, the crystalline chains becomes fluid, resulting in an effective increased hydrocarbon volume. This provides a wedge-shaped conformation to small headgroup NAEs necessary for inverse phases (inverse micelles, inverse cubic) formation. Addition of an ethanol moiety to NAEs forms diethanolamide with a relatively large headgroup, which also has been shown to favor lyotropic lamellar phase formation.39 However, for shorter chain diethanolamides micellar phase is observed at low temperature also and in excess water.39 The headgroup area of NADAs is not expected to be too large because of π-stacking, and they most likely form lamellar crystalline phase at low temperature and lamellar fluid phase above transition temperature. However, we do not rule out the possibility of other nonlamellar phases at high temperatures and particularly at low hydration. Another popular surfactant, monoacylglycerides, form a variety of phases in water ranging from lamellar, inverted hexagonal, bicontinuous cubic phases, etc.40 Apart from packing parameter, the H-bond plays an important role in determining the existence of various phases. For example, N-(2-hydroxydodecyl)-L-alanine, an N-alkyl amino acid, forms lamellar phase with intermolecular H-bond interactions,41 whereas in N-alkyl ureas H-bonding results in high curvature, which favors the formation of inverse micellar (L2) phase at high temperature.39,42 The π-stacking as well as H-bonding ability of NADAs makes them an attractive choice for detailed analysis of binary phase behavior in water.

A knowledge of thermodynamic parameters associated with the phase transitions of a homologous series of lipids, especially when they exhibit linear dependence on chain length, allows an estimation of the end contribution of enthalpy and entropy. The end contributions give a measure of the enthalpic and entropic contributions arising from the headgroup moiety to the overall phase transition enthalpy and entropy.23,32 This was tested in our earlier report on N,O-diacylethanolamines (DAEs) of fixed N-acyl chain with different O-acyl chains, and it was found that the end contributions could be attributed to N-acyl part of the molecule, with the incremental contribution coming from polymethylene part of the O-acyl chain. In the present study, the end contribution of enthalpy, ΔH0, mainly arises from the dopamine (DA) moiety. It was observed that upon hydration dopamine moiety gains a substantial enthalpic stabilization (−10.55 kcal/mol) with the expense of ordering of water structure (reduction in entropy). The enthalpic stabilization could arise from hydrophobic, πstacking and hydrogen-bonding interactions and provides a possible explanation as to how dopamine accumulates in a dense core carotid body.43 This enthalpic stabilization in a hydrophobic environment also provides further clues on the interaction of DA with its receptors. It was found that the receptor binding site of DA comprises of aromatic amino acid residues, which may help to gain hydrophobic as well as πstacking interactions. Apart from the enthalpic stabilization of dopamine moiety, NADAs were found tightly packed in aqueous medium, which may explain their ability to cross the blood−brain barrier and remain sufficiently stable under hydrolyzing condition. It is possible that such tightly packed chains in NADAs may be localized in lipid raf ts in biomembranes and may play a role in modulating the activity of membrane proteins. The entourage effect produced by NPDA and NSDA20 may arise from the interaction of tightly packed NADAs with other constituents of biomembranes. Miscibility studies between NADAs with other lipids, such as phosphatidylcholine and phosphatidylethanolamine, would be required to further investigate their possible role in lipid raft formation. Such studies are currently underway in our laboratory.

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CONCLUSIONS

In this study, a homologous series of N-acyldopamineswhich are biologically relevant and neuroactive lipidshave been synthesized, and their thermotropic phase transitions were characterized by differential scanning calorimetry. A linear dependence was observed in the thermodynamic parameters, ΔHt and ΔSt, associated with the chain-melting phase transitions. The 3-dimensional structure of N-lauroyldopamine, solved by single crystal X-ray diffraction, revealed that the molecules of this lipid adopt an extended, essentially linear geometry, with the hydrophobic acyl chains facing each other, i.e., a head-to-head (and tail-to-tail) arrangement, akin to a bilayer structure. The acyl chains are tilted considerably (by ∼46°) with respect to the bilayer normal. Studies aimed at understanding the interaction of these molecules with other membrane lipids/drugs would be expected to provide clues to understand their role in biological processes and pharmaceutical applications. 8755



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(8) Chapman, K. D. Occurrence, Metabolism, and Prospective Functions of N-Acylethanolamines in Plants. Prog. Lipid Res. 2004, 43, 302−327. (9) Swamy, M. J.; Tarafdar, P. K.; Kamlekar, R. K. Structure, Phase Behaviour and Membrane Interactions of N-Acylethanolamines and NAcylphosphatidylethanolamines. Chem. Phys. Lipids 2010, 163, 266− 279. (10) Huang, S. M.; Bisogno, T.; Trevisani, M.; Al-Hayani, A.; Petrocellis, L. D.; Fezza, F.; Tognetto, M.; Petros, T. J.; Krey, J. F.; Chu, C. J.; et al. An Endogenous Capsaicin-Like Substance with High Potency at Recombinant and Native Vanilloid VR1 Receptors. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 8400−8405. (11) Bisogno, T.; Melck, D.; Bobrov, M. Y.; Gretskaya, N. M.; Bezuglov, V. V. N-Acyl-Dopamines: Novel Synthetic CB1 Cannabinoid-Receptor Ligands and Inhibitors of Anandamide Inactivation with Cannabimimetic Activity in vitro and in vivo. Biochem. J. 2000, 351, 817−824. (12) Bezuglov, V.; Bobrov, M.; Gretskaya, N.; Gonchar, A.; Zinchenko, G.; Melck, D.; Bisogno, T.; Di Marzo, V.; Kuklev, D.; Rossi, J. C.; et al. Synthesis and Biological Evaluation of Novel Amides of Polyunsaturated Fatty Acids with Dopamine. Bioorg. Med. Chem. Lett. 2001, 11, 447−449. (13) Navarrete, C. M.; Perez, M.; Vinuesa, A. G.; Collado, J. A.; Fiebich, B. L.; Calzado, M. A.; Munnoz, E. Endogenous N-AcylDopamines Induce COX-2 Expression in Brain Endothelial Cells by Stabilizing mRNA Through a p38 Dependent Pathway. Biochem. Pharmacol. 2010, 79, 1805−1814. (14) Chu, C. J.; Huang, S. M.; Petrocellis, L. D.; Bisogno, T.; Ewing, S. A.; Miller, J. D.; Zipkin, R. E.; Daddario, N.; Appendino, G.; Di Marzo, V.; et al. N-Oleoyldopamine, a Novel Endogenous CapsaicinLike Lipid That Produces Hyperalgesia. J. Biol. Chem. 2003, 278, 13633−13639. (15) De Petrocellis, L.; Chu, C. J.; Moriello, A. S.; Kellner, J. C.; Walker, J. M.; Marzo, V. D. Actions of two Naturally Occurring Saturated N-Acyldopamines on Transient Receptor Potential Vanilloid 1 (TRPV1) Channels. Br. J. Pharmacol. 2004, 143, 251−256. (16) Tsagogiorgas, C.; Wedel, J.; Hottenrott, M.; Schneider, M. O.; Binzen, U.; Greffrath, W.; Treede, R. D.; Theisinger, B.; Theisinger, S.; Waldherr, R.; et al. N-Octanoyl-Dopamine is an Agonist at the Capsaicin Receptor TRPV1 and Mitigates Ischemia-Induced Acute Kidney Injury in Rat. PLoS One 2012, 7, e43525. (17) Yoo, J.-M.; Park, E. S.; Kim, M. R.; Sok, D.-E. Inhibitory Effect of N-Acyl Dopamines on IgE-Mediated Allergic Response in RBL-2H3 Cells. Lipids 2013, 48, 383393. (18) Przegaliński, E.; Filip, M.; Zajac, D.; Pokorski, M. N-OleoylDopamine Increases Locomotor Activity in The Rat. Int. J. Immunopathol. Pharmacol. 2006, 19, 897−904. (19) Marsh, D. Handbook of Lipid Bilayers; CRC Press: Boca Raton, FL, 1990. (20) Kamlekar, R. K.; Tarafdar, P. K.; Swamy, M. J. Synthesis, Calorimetric Studies and Crystal Structures of Diacylethanolamines with Matched Chains. J. Lipid Res. 2010, 51, 42−52. (21) Sheldrick, G. M. A Short History of SHELX. Acta Crystallogr. 2008, A64, 112−122. (22) Larsson, K. Physical Properties−Structural and Physical Characteristics. In The Lipid Handbook; Gunstone, F. D., Harwood, J. L., Padley, F.B., Eds.; Chapman and Hall: London, 1986; pp 321− 384. (23) Marsh, D. Biomembranes. In Supramolecular Structure and Function; Pifat, G., Herak, J. N., Eds.; Plenum Press: New York, 1982; pp 127−178. (24) Ramakrishnan, M.; Sheeba, V.; Komath, S. S.; Swamy, M. J. Differential Scanning Calorimetric Studies on the Thermotropic Phase Transitions of Dry and Hydrated Forms of N-Acylethanolamines of Even Chainlengths. Biochim. Biophys. Acta 1997, 1329, 302−310. (25) Marsh, D.; Swamy, M. J. Derivatized Lipids in Membranes: Physico-Chemical Aspects of N-Biotinyl Phosphatidylethanolamines, N-Acyl Phosphatidylethanolamines and N-Acylethanolamines. Chem. Phys. Lipids 2000, 105, 43−69.

ASSOCIATED CONTENT

S Supporting Information *

Data from FTIR, 1H NMR, and 13C NMR spectroscopy as well as ESI MS spectrometry. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel +91-40-2313-4807; Fax +91-40-2301-2460/0145; e-mail [email protected], [email protected] (M.J.S.). Author Contributions †

S.T.R. and P.K.T. contributed equally to this study.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a research grant from the Department of Science and Technology (India) to M.J.S. S.T.R., P.K.T., and R.K.K. were supported by Senior Research Fellowships from the Council of Scientific and Industrial Research (India). Use of the National Single Crystal Diffractometer Facility (SMART APEX CCD single crystal Xray diffractometer) at 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 his help in the solution of the crystal structure of NLDA. The University Grants Commission (India) is acknowledged for for their support through the UPE and CAS programs to the University of Hyderabad and School of Chemistry, respectively.

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ABBREVIATIONS USED NADA, N-acyldopamine; NArDA, N-arachidonyldopamine; NLDA, N-lauroyldopamine; NPDA, N-palmitoyldopamine; NSDA, N-stearoyldopamine; DA, dopamine; DSC, differential scanning calorimetry; MAPK, mitogen-activated protein kinase; MCF-7, michigan cancer foundation-7; COX-2, cyclooxygenase-2; mRNA, messenger RNA; TRPV1, transient receptor potential vanilloid-1; Tt, transition temperature; ΔHt, transition enthalpy; ΔSt, transition entropy; ΔHinc, incremental enthalpy; ΔSinc, incremental entropy; ΔH0, end contribution of enthalpy; ΔS0, end contribution of entropy.

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REFERENCES

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Structure and Thermotropic Phase Behavior of a Homologous Series

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