J. Phys. Chem. B 2009, 113, 15249–15255
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cis- versus trans-Ceramides: Effects of the Double Bond on Conformation and H-Bonding Interactions Shay C. Phillips,† Gemma Triola,‡ Gemma Fabrias,‡ Fe´lix M. Gon˜i,| Donald B. DuPre´,† and M. Cecilia Yappert*,† Department of Chemistry, UniVersity of LouisVille, LouisVille, Kentucky 40292, Research Unit on BioActiVe Molecules (RUBAM), Departament de Quı´mica Biome`dica, Institut de Quı´mica AVanc¸ada de Catalunya (IQAC-CSIC), Barcelona, Spain, and Unidad de Biofı´sica (Centro Mixto CSIC-UPV/EHU) and Departamento de Bioquı´mica, UniVersidad del Paı´s Vasco, Aptdo. 644, 48080 Bilbao, Spain ReceiVed: April 1, 2009; ReVised Manuscript ReceiVed: September 21, 2009
Natural ceramides (Cers) possess a trans double bond between C4 and C5 of the sphingoid chain. This double bond is critical to their cell signaling properties. Both a change from trans to cis and the saturation of this site lead to changes in or loss of biological activity. To explore the conformational impact of the cis double bond, through-bond, and through-space interactions were investigated in hydrated Cers by multidimensional 1H and 13C NMR spectroscopy. Unlike trans-Cer, the cis-isomer exhibited not one but two broad yet resolved resonances for the protons in C1-OH and C3-OH, much like dihydroceramide (DHCer). Temperature-dependent studies and partial isotopic labeling of cis-Cer revealed that relative to trans-Cer, these two OH groups form weaker hydrogen bonds, particularly in the case of C1-OH. Our results also suggest that the cis double bond twists, slightly, the orientation of HO-C1 with respect to HO-C3, thus weakening the hydrogen-bonding network formed between the two OH groups of cis-Cer and bound water molecules. The alteration of the local network of H-bonds may account for the differences observed in the biological activity of the two isomers. Materials and Methods
Introduction Since the roles of ceramides (Cers) as potent lipid second messengers were recognized two decades ago,1-9 these interesting sphingolipids continue to be a theme of exciting research as described in multiple recent reviews.10-18 The cell signaling properties of Cers are critically affected by the double bond between C4 and C5 of the sphingoid chain. For example, Bawab and co-workers found that brain ceramidase decreased its affinity toward its substrate, Cer, when the natural trans configuration was changed to cis or when the double bond was saturated.19 Whereas D-erythro-Cer activated the catalytic subunit of protein phosphatase 2A causing a 3-fold increase in activity, Cers without the trans double bond or with a cis configuration had no detectable effect.20 Our previous studies of trans-Cer in CDCl3 suggested the presence of a tight network of H-bonds connecting the two OH groups in trans-Cer with two water molecules.21 Subsequent studies by Brockman and co-workers supported this possibility.22 When the double bond is absent, this tight, water-assisted H-bonding network is no longer supported. However, comparable biophysical studies have not been pursued yet with the cis isomer. For this reason, the current work extends our previous studies on trans-, dihydro-, and deoxydihydro-Cers to the exploration of conformational changes that may explain the impact of trans-to-cis isomerization on the biological behavior of Cers. * To whom correspondence should be addressed. E-mail: mcyappert@ louisville.edu. † University of Louisville. ‡ Institut de Quı´mica Avanc¸ada de Catalunya (IQAC-CSIC). | Universidad del Paı´s Vasco.
Chemicals. N-Palmitoyl-D-erythro-(cis)-sphingosine (cis-Cer) was synthesized by acylation of the free amine with palmitoyl chloride under Schotten-Baumann conditions, as reported previously.23 N-Palmitoyl-D-erythro-sphingosine (trans-Cer) was obtained from Avanti Polar Lipids, Inc. (Alabaster, AL). Both cis- and trans-Cers were used without further purification. Chloroform, CHCl3, deuterated chloroform, CDCl3, deuterium oxide, D2O, deuterated dimethyl sulfoxide, DMSO-d6, and molecular sieves (1.6-mm pellets) were obtained from SigmaAldrich (St. Louis, MO). Sample Preparation. Samples of both cis- and trans-Cers, in concentrations of 1 and 10 mM, were prepared by dissolving the proper amount of each in CDCl3 or DMSO-d6. Each solution was then heated to 50 °C in a water bath with sonication for 20 min. Samples prepared in this way contained four to five water molecules per Cer. The effect of water on intermolecular and intramolecular hydrogen bond formation was investigated by carrying out a dehydration experiment, as described previously. A portable glovebag filled with nitrogen was used to void the vacuum. To avoid rehydration of the sample with water present in the solvent, 4-5 activated molecular sieves were added to each 1-mL ampule of CDCl3 just prior to addition to the dehydrated sample. Additionally, 2 or 3 activated molecular sieves were added directly into the sample tubes containing the dehydrated sample. Both dehydrated Cer samples were then dissolved in the treated CDCl3. Each sample remained in the glovebag until immediately before analysis. Prior to use, the molecular sieves were activated using a standard microwave by heating at 30 s intervals for approximately 4 min. Deuterated samples were also examined and were obtained by adding 2 µL of D2O directly into the NMR tubes. The final D2O-to-lipid molar ratio, for both isomers, was between 20:1 and 40:1.
10.1021/jp903000m CCC: $40.75 2009 American Chemical Society Published on Web 10/22/2009
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Phillips et al. TABLE 1: Spectral Assignments of (A) 1H and (B) 13C NMR Resonances for 10 mM cis-Cer and trans-Cer in CDCl3 at 25 °Ca chemical shift (ppm)
Figure 1. Schematic representation of the two palmitoylated isomers of Cer used in this study.
One- and Two-Dimensional NMR Studies. NMR experiments were performed with a Varian Inova 500 MHz spectrometer (Palo Alto, CA) equipped with a triple resonance probe. The total number of scans varied to achieve the desired signalto-noise ratio. Temperature-dependent studies were carried out by varying the temperature from 15 to 50 °C for the samples in CDCl3 and from 60 to 80 °C for those in DMSO-d6 to ensure solubilization of the Cers. All one-dimensional spectra were processed using MestReC Version 2.01 (Santiago de Compostela, Spain) on a personal computer. All 2D NMR experiments were carried out with an inverse probe. The 1H-13C heteronuclear multiple quantum coherence (HMQC) spectra were collected for samples of each isomer. For each HMQC experiment, 8 transients of 2048 complex data points/transient were obtained for each of the 512 increments. Sweep widths were 10 and 160 ppm for 1H and 13C, respectively. 1 H-1H nuclear Overhauser effect spectroscopy (NOESY) and 1 H-1H rotating-frame Overhauser effect spectroscopy (ROESY) experiments were conducted with mixing times of 500, 1000, and 1500 ms. NOESY and ROESY experiments were run with 16 transients, 512 increments, and spectral widths of 10 ppm. All 2D experiments were processed using MestReC Version 2.01 (Santiago de Compostela, Spain) on a personal computer. Results and Discussion This study focuses on the conformational preferences of cisCer and compares them with those postulated for trans-Cers in 2002 by Li et al.21 Before presenting the results, it is relevant to mention that Cers are insoluble in water, to the point that, when added to the bulk of an aqueous phase, no Cer molecules orient themselves at the air-water interface.24 This places Cers, at least those with a long N-acyl chain, among the most nonpolar of lipids. When Cers are mixed with phospholipids, their miscibility is exceedingly low, with Cer-rich domains being formed surrounded by almost pure phospholipid.25 In view of these observations in aqueous media and of similar ones summarized in refs 13 and 15, it appears that even in lipid bilayers in aqueous medium an extensive network of H-bonds exists among Cer molecules; thus, the observations in this report are essentially relevant to the physiological situation. The labeling of each carbon is shown in Figure 1 and follows the same rationale described previously in which only the atoms in the acyl chain are denoted with a prime (′) but not those
(A) proton assignment
cis-Cer
trans-Cer
CH3 (Terminal methyls) CH2 (Methylenes) H7 H′3 H6 H′2 HO-C3 HO-C1 H1B H2 H1A H3 H4 H5 NH
0.84 1.24 1.28 1.54 2.04 2.18 2.52 (2.44) 2.62 (2.58) 3.66 3.79 3.93 4.61 5.42 5.54 6.15
0.84 1.23 1.33 1.60 2.08 2.01 2.62 2.62 3.66 3.86 3.92 4.29 5.50 5.74 6.20
(B) carbon-13 assignment
cis-Cer
trans-Cer
CH3 (Terminal methyls) C17 C′3 C6 CH2 (Intermediate methylenes) C16 C′2 C1 C2 C3 C4 C5 O)C-NH
14.35 22.93 26.94 28.11 29.9-29.5 32.16 37.09 62.84 54.99 (54.93) 70.02 128.70 (128.64) 135.01 (135.06) 174.12
14.07 22.64 25.71 32.23 29.0-29.5 31.87 36.81 62.50 54.43 74.69 128.74b 134.29b 173.89
a The values in parentheses correspond to the chemical shifts measured for the 1 mM solution of cis-Cer. All other chemical shifts were identical for both concentrations. b These are the correct chemical shifts for C4 and C5 and were misassigned in our previous report.21
corresponding to the sphingoid base.26,27 Protons were omitted for clarity in Figure 1 but they are labeled with the number of the carbon to which they are attached. Diastereotopic protons at C1 are labeled H1A and H1B. The 1H and 13C NMR results obtained for partially hydrated and dehydrated cis-Cer are first presented and contrasted to those for trans-Cer. From that analysis, possible conformational differences between the two isomers are proposed. Further experimental evidence that supports the proposed arrangements is then presented. 1 H NMR Spectral Assignments. The chemical shifts and assignments of the 1H resonances corresponding to the palmitoylated cis-Cer and trans-Cer are listed in Table 1A. The only chemical shifts that changed as the concentration was increased correspond to the C1-OH and C3-OH proton resonances, which became more deshielded at the higher concentration where more association is expected. This deshielding effect is indicative of the participation of the OH groups in tighter H-bonds as the molecules come closer together. Figure 2 shows the spectra acquired for 10 mM solutions of partially hydrated cis-Cer (Figure 2A) and trans-Cer (Figure 2B) at 25 °C in CDCl3. The molar ratio of water to Cer was estimated from the ratio of the integrated areas corresponding to the water resonance and that of the amide proton NH of Cer. This ratio was determined to be between 4:1 and 5:1. The arrows in Figure 2 point to the major differences between the two spectral traces. These differences are as follows: (i) Whereas in the trans isomer the C1-OH and C3-OH resonances
Conformation and H-Bonds in cis-Ceramide
Figure 2.
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H NMR spectra of 10 mM cis-Cer (A) and 10 mM trans-Cer (B) in CDCl3 at 25 °C.
exhibited the same chemical shift and thus were unresolved, these OH resonances were well resolved in cis-Cer but remained broad. The assignments of C1-OH and C3-OH resonances in the cis isomer were based on their respective bandwidths. The C1-OH resonance is expected to be split by the two neighboring protons (H1A and H1B), whereas that for C3-OH should be affected primarily by only one neighboring proton (H3). The assignments of these OH-related peaks are in agreement with those previously reported for trans-Cer at very low temperature and for dihydro-Cer.21 (ii) Compared to the chemical shifts in trans-Cer, the resonance for H3 in cis-Cer became more deshielded, while those related to H2, H4, and H5 exhibited upfield shifts, with H5 being the most affected. Consequently, the H4 and H5 resonances, which correspond to the protons across the double bond, became significantly closer in chemical shift when changing from trans- to cis-Cer. 13 C NMR Spectral Assignments. The assignments of the 13 C resonances were based on correlations observed in the 1 H-13C HMQC 2D-spectrum of 10 mM cis-Cer at 25 °C in CDCl3. The chemical shift of C5 (δ ) 135.0 ppm) was higher (more downfield) than that of C4 (δ ) 128.7 ppm). Because this trend differs from that reported for trans-Cer with a stearoyl chain,21 the same 1H-13C HMQC experiment was carried out for the palmitoylated trans isomer. Figure 3 shows the H4-C4 and H5-C5 correlations observed in the HMQC spectrum of each isomer and reveals a similar trend for the resonances associated with C5 (δ ) 134.3 ppm) and C4 (δ ) 128.7 ppm) for both the cis and trans isomer. As seen in Figure 3, the C5 resonance appeared more downfield than the resonance due to C4. To further ensure correct labeling of the H4 and H5 resonances, 1H NMR decoupling experiments were carried out in which a specific resonance was saturated and thus could not contribute to the splitting of resonances of neighboring protons. Upon saturation of the H3 resonance, a loss of splitting was observed in the resonance at δ ) 5.42 ppm, confirming its assignment to H4. Additionally, after saturating the H6 reso-
Figure 3. HMQC spectra of 10 mM trans-Cer (A) and 10 mM cisCer (B) in CDCl3 at 25 °C.
nance, the splitting pattern of the resonance at δ ) 5.54 ppm was affected, validating its assignment to proton H5. The mistaken assignments reported for C4 and C5 in our previous report are corrected in Table 1B that lists the 13C chemical shifts and correct assignments for 1 mM (values in parentheses) and 10 mM cis-Cer and 10 mM trans-Cers in CDCl3. Only very slight changes were seen upon increasing
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TABLE 2: Average Values of 1H-1H Coupling Constants Measured in Hz for cis- and trans-Cers in CDCl3 at 25 °Ca protons
10 mM cis-Cer (Hz)
10 mM trans-Cer (Hz)
H1A-H1B H1A-H2 H1B-H2 H2-H3 H3-H4 H2-NH H4-H5
11.5 3.7 2.6 4.0 8.0 7.3 9.9
11.1 3.7 3.0 3.1 6.5 7.3 15.4
a The largest uncertainty in these measurements is 0.1 Hz and is based on the standard deviation of four measurements.
the concentration from 1 to 10 mM cis-Cer. The resonances for C2 and C3 were slightly more shielded at 1 mM but that for C5 was slightly more deshielded for this concentration of cis-Cer; this is an indication of the role of the OH group at C3 in intermolecular associations. Several significant differences were observed when comparing 13C chemical shifts for the cis and trans isomers at 10 mM concentrations. Relative to the trans-Cer, the resonances for C3 and C6 in cis-Cer decreased their chemical shift by 4.7 and 4.1 ppm, respectively. Slight increases in δ (0.3-0.8 ppm) were also observed for C′3, C′2, C2, C1, and C5. The only resonance that exhibited a slight deshielding (downfield shift) with respect to that in trans-Cer was the C5 resonance. Possible reasons for these variations will be discussed later in the section on postulated conformational preferences. Vicinal Proton-Proton Coupling Constants. Table 2 lists the geminal coupling constant between H1A and H1B as well as vicinal 1H-1H coupling constants for both cis- and transCers in CDCl3 (10 mM) at 25 °C. The geminal coupling between H1A and H1B, 2JH1A-H1B, became seemingly larger in absolute value (11.5 Hz) for cis-Cer compared to trans-Cer (11.1 Hz). However, geminal 2JH-H couplings are generally negative because the two protons connected to the same carbon have their spins parallel to each other in the lowest energy state.28 Taking this fact into consideration, the assigned 2JH1A-H1B value for cis-Cer of -11.5 Hz is more negative than the value of -11.1 Hz observed for trans-Cer. The smaller (less positive) geminal coupling in the cis-isomer indicates a decrease in the H1A-C1-H1B bond angle that would result in a decrease in s-character of carbon-centered bond orbitals and, hence, less Fermi contact (FC) along the H1A-C1-H1B coupling pathway.29 As the FC becomes more negative, so does 2JH1A-H1B. It is also possible that 2JH1A-H1B becomes more negative in cisCer because of changes in the strength of H-bonds involving the OH group at C1, as discussed later. Stronger H-bonds involving the OH group as a donor would result in a greater partial negative charge on the O atom. Weaker H-bonds, on the other hand, would reduce this partial negative charge. Consequently, the relative tendency of the OH group to transfer charge into the coupling pathway is greater when the H-bond is stronger. These hyperconjugative interactions (transfer of oxygen electrons to antibonding C-H orbitals, or nO f σ*C-H) yield a positive increase to the FC30 and thus a less negative value for 2JH-H, as observed in the trans isomer. As the strength of the H-bond diminishes, this charge transfer would decrease and the FC would become more negative and result in a more negative value for 2JH-H. Differences were also observed in the vicinal coupling, 3J, between H1B and H2. For the cis isomer, the coupling constant (2.6 Hz) was smaller than that observed in trans-Cer (3.0 Hz), indicating a slight but significant rotation about the bond of
Figure 4. 1H NMR spectra of 10 mM cis-Cer (A) and 10 mM transCer (B) in CDCl3 before and after dehydration at 25 °C.
C1-C2. If the H1A-C1-H1B bond angle had not changed, the decrease in 3JH1B-H2 should have been accompanied by an increase in 3JH1A-H2. Conversely, a reduction of the H1A-C1-H1B bond angle would be expected to reduce both 3JH1A-H2 and 3 JH1B-H2. However, while 3JH1B-H2 decreased, the value for 3 JH1A-H2 remained the same for the two isomers. It is thus proposed that along with a decrease in H1A-C1-H1B bond angle, there must be a rotation about the C1-C2 bond to restore the value of 3JH1A-H2 to 3.7 Hz. The 3JH2-H3 (4.0 Hz) and 3JH3-H4 (8.0 Hz) values for cis-Cer were higher by 0.9 and 1.5 Hz, respectively, than those measured the trans analog (3.1 and 6.5 Hz). These increases indicate rotations about C2-C3 and C3-C4, moving H3 further away from both H2 and H4. Finally, the expected decrease was observed in 3JH4-H5 due to the change in isomerization from trans to cis. Possible explanations for these variations will be addressed later. Temperature Dependencies. Temperature studies were completed to investigate the relative strength of H-bonds involving the C1-OH, C3-OH, and NH moieties in cis- and trans-Cers. Temperature coefficients (TCs) were determined from the slopes of linear regressions of 1H chemical shift values versus temperature plots. Temperature coefficients for amide proton resonances were reported to reflect the participation of the proton in intramolecular or intermolecular H-bonds.31-33 Smaller absolute values of TC are indicative of a greater involvement in intramolecular H-bonds because, in general, intermolecular bonds are weakened with increasing temperature and lead to the shielding (lower chemical shift) of the OH and NH resonances. The temperature coefficients of both isomers, at 1 mM and 10 mM, are listed in Table 3. As the concentration of cis-Cer increased, the absolute values of the three measured TCs increased, particularly for the C1-OH resonance. This suggests that possible intramolecular H-bonds in the monomer are
Conformation and H-Bonds in cis-Ceramide
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TABLE 3: Temperature Coefficients of NH and OH Resonances of cis- and trans-Cers in CDCl3 and DMSO-d6 ∆δ/∆T (ppm/°C) ceramide (a) OH Resonance(s)
1 mM in CDCl3
10 mM in CDCl3
10 mM in DMSO
cis-Cer: C3-OH C1-OH trans-Cer*
-0.0051 ( 0.0002 -0.0044 ( 0.0001 -0.0039 ( 0.0004
-0.0058 ( 0.0003 -0.0061 ( 0.0002 -0.0054 ( 0.0003
-0.0046 ( 0.0002 -0.0044 ( 0.0002 -0.0037(C3-OH) -0.0043(C1-OH)
(b) NH Resonance cis-Cer trans-Cer*
-0.0031 ( 0.0001 -0.0031 ( 0.0002
-0.0035 ( 0.0001 -0.0032 ( 0.0001
-0.0045 ( 0.0003 -0.0049
* Data listed for trans-Cer were previously reported for C18-Cer.21
disrupted and new intermolecular bonds are formed, most likely to water molecules captured within the associated ceramides. Relative to the trans-isomer, the absolute values of the TCs for the C1-OH and C3-OH resonances were larger at both concentrations in the cis isomer. These trends suggest that the OH groups are more involved in intermolecular H-bonds than in the trans isomer. The tight, water-supported internal Hbonding network of C1-OH and C3-OH reported for the trans isomer appears to be disrupted in the cis isomer where the OH groups are likely to be, on average, more bound to water molecules than to each other. The NH resonance for both isomers exhibited similar TCs, indicating that no major changes occurred at this H-bonding site. Temperature coefficients were also measured for 10 mM samples of both isomers in DMSO-d6. Because DMSO is a stronger H-bond acceptor than water, it is expected to disrupt water-mediated H-bonds and form very tight H-bonds with OH and NH groups acting as donors. For the C1-OH and NH resonances, the temperature coefficients were similar. However, the temperature coefficient for the C3-OH resonance in the trans isomer was lower than that for cis-Cer. Due to the bulky nature of the methyl groups of DMSO, it is possible that the cis isomer exhibited a larger temperature coefficient because the DMSO could not get as close to the OH group of C3 due to steric hindrance caused by the proximity of both H4 and H5 on the same side of the C4dC5 double bond. Dehydration and Hydration Effects. Dehydration studies were conducted on samples that were subject to high vacuum for 24 h. Traces A and B in Figure 4 show the 1H NMR spectra of cis- and trans-Cer both before and immediately after dehydration. These studies revealed that immediately after dehydration, the cis isomer retained only one bound water molecule per lipid, while trans-Cer retained two bound water molecules per lipid. The resonances for C1-OH and C3-OH were also no longer visible in cis-Cer, but remained visible in the trans conformer. This trend is important and suggests that the protons C1-OH and C3-OH of cis-Cer are H-bond donors that interact with water molecules that can be removed by vacuum. In the trans isomer, these OH groups are indeed part of a tight hydrogen-bonding network from which the bound water molecules cannot be removed. These results support the previous reported postulate of the ability of the trans double bond of trans-Cer to support or promote a tight internal H-bonding network involving the OH groups on C1 and C3.21 Such an arrangement cannot be formed in the cis isomer as the OH group at C1 does not favor the intramolecular H-bond and interacts with external surrounding water molecules. The cis isomer does, however, retain one water molecule after dehydration and its 1H NMR resonance mimics one of the water resonances held by trans-Cer. We propose that this water
molecule may be held through a tight internal H-bond with the NH group at C2 serving as donor and with the oxygens at OH groups at C1 and C3 acting as acceptors in both isomers and is supported by our results of little change in that region of the molecule. Unlike in the trans-Cer spectrum (not shown) that remained unchanged one hour after dehydration, the spectrum of cis-Cer in Figure 4a shows the presence of another water molecule and as this second water molecule was incorporated, the resonances for C1-OH and C3-OH could also be observed in the spectrum. This is a relevant spectral change that indicates that both of these hydroxyl groups can act as H-bond donors to form a H-bonding network via water bridges but that their ability to participate in intramolecular H-bonds is much less favored than in the trans isomer. Postulated Conformational Preferences Based on the results presented above, we postulate that while the changes in chemical shifts, coupling constants and TCs observed between the cis and trans isomers are subtle, they point to the same conclusion: the OH groups at C1 and C3 of cisCer are no longer locked in the tight H-bond network that was postulated for the trans analog. Unlike in trans-Cer, they sense different electromagnetic environments, as reflected by their different chemical shifts. From the more negative geminal coupling constant for H1A and H1B, we infer that the bond angle H1A-C1-H1B is reduced due to the weakening of the H-bond involving the OH group at C1. As a consequence, the oxygen atom is less negative for the cis-Cer relative to the transCer for which the OH group forms a very tight H-bond. The decrease in bond angle also leads to the lower vicinal coupling constant 3JH1B-H2 for the cis-Cer. This decrease in H-bonding strength is also reflected in the TCs presented in Table 3 for both the C1-OH and C3-OH resonances. The dehydration/hydration experiments provided important details on the role of bound water molecules. Figure 5 shows the postulated arrangements for cis-Cer immediately after dehydration (A) and an hour later (B). Although a single water molecule was retained after dehydration, the OH resonances were not observed. This suggests that the OH groups are not acting as H-bond donors. If they were, the resonances should have been seen. It is possible that, as shown in Figure 5A, the OH group(s) could be H-bond acceptors. Interestingly, upon the trapping of a second water molecule, both the C1-OH and C3-OH resonances appeared. For that reason, Figure 5B shows the two OH groups as donors of H-bonds with a new bound water molecule. The fundamental difference between the two isomers is the inability of forming an intramolecular H-bond between the two
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Figure 6. 1H NMR spectra of 10 mM cis-Cer (A) and 10 mM transCer (B) in CDCl3 before and after partial deuteration at 25 °C.
Figure 5. Proposed conformations of cis-Cer immediately after dehydration (A) and 1 h after dehydration (B).
OH groups in the case of the cis isomer. It is possible that the lone pairs of the oxygen at C3-OH, not being involved in H-bonds with the OH group at C1 could cause the deshielding observed in the resonances for C5 and H5. This difference appears to have profound biological consequences and highlights the relevance of the H-bonding network present in the trans isomer but not in the other analogs studied thus far. The paragraphs below provide further evidence of the proposed conformational differences between the two isomers. Corroboration of the Proposed Arrangements 13
C NMR Isotopic Effects or Secondary-Isotope Multiplets of Partially Labeled Entities (SIMPLE) Experiments. The strength with which OH and NH groups participate in H-bonds
can be directly determined by the degree of splitting of 13C resonances caused by partial deuteration of the H-bonding group(s) attached to a given carbon.34-36 The strength of the H-bond correlates with the magnitude of the two-bond isotope effect, 2∆:37-41 the larger the splitting, the greater the strength of the H-bond. The splitting patterns observed in 13C spectra of both cis- and trans-Cer before (top trace) and after (bottom trace) partial deuteration are shown in panels A and B of Figure 6. The C1 and C3 resonances of cis-Cer were not split after partial deuteration as they were in the trans isomer for which the degree of splitting was comparable for C1 and C3 (∆δ ∼ 0.11-0.12 ppm). These trends validate the proposed participation of the cis C1-OH in H-bonds with surrounding water molecules that can easily exchange and as a result do not lead to the splitting of the C1 resonance. As postulated from the TCs measured for the resonances due to C1-OH and C3-OH in cis-Cer (see Table 3), the broadening of the C3 resonance in cis-Cer corroborates the slightly greater strength of the H-bond formed by this OH group relative to the C1-OH group. However, neither of the two OH groups appeared to be as tightly bound as they are in trans-Cer and even in dihydro-Cer, for which the C3 resonance was split by 0.101 ppm after partial deuteration.21 These differences imply that the cis double bond precludes the formation of H-bonds between the two OH groups. On the other hand, the C2 resonance revealed similar splitting for both isomers reinforcing the proposed absence of significant conformational changes around the amide linkage upon trans-tocis isomerization of C4dC5. ROESY and NOESY Results. 1H-1H ROE and NOE correlations reveal polarization transfer among neighboring protons. These through-space interactions are possible if the protons are within 5 Å of each other. In cis-Cer, interactions of H3-NH and H3-H6 were observed for all mixing times (0.5, 1.0, and 1.5 s). However, at the same mixing times these interactions were not observed in the trans isomer. These
Conformation and H-Bonds in cis-Ceramide
J. Phys. Chem. B, Vol. 113, No. 46, 2009 15255 Acknowledgment. F.M.G. acknowledges financial help from Fundacion Areces and from the Spanish Ministerio de Ciencia e Innovacio´n (Grant No. BFU 2007 62062). References and Notes
Figure 7. 1H NMR spectra of 10 mM cis-Cer in DMSO-d6 (A) and CDCl3 (B).
interactions support the rotation about the C1-C2 bond in cisCer that brings NH closer to H3. The H6-H3 interaction results from the change in isomerization that places H3 in the vicinity of one of the H6 protons in the cis isomer as shown in Figure 5. Additionally, negative cross peaks connecting both C1-OH and C3-OH resonances with the large water resonance were observed in the NOESY spectra for both isomers. The negative volumes of these correlations are due to transfer of polarization via chemical exchange (EXSY). Disruption of H-Bonding Networks Using DMSO. The use of DMSO as solvent induced complete disruption of the watermediated H-bonding network in trans-Cer. This effect was evidenced by significant changes in chemical shifts and TCs.21 As for trans-Cer, the resonances for the OH groups of cis-Cer were shifted downfield (more deshielded) by about 2 ppm (see panel A in Figure 7) when DMSO, rather than CDCl3 (panel B in Figure 7), was the solvent. This is due to the formation of very strong H-bonds with the DMSO. Importantly, the disruption of the H-bonding network resulted in the shielding of the H4 and H5, with H5 being the most affected. This observation confirms that the greater deshielding of H5 may be caused by the presence of water molecules in the vicinity of C3 and of the double bond at C4dC5. In the absence of these water molecules, it is expected that the methyl groups of DMSO would be close to the double bond as the SdO group binds to the OH group at C3. These observations offer credibility to the participation of water molecules in the vicinity of the cis double bond in a H-bonding network as cis-Cer molecules associate with each other. Conclusions These results extend our previous work on the conformational preferences of natural and synthetic Cers and point to the importance of the trans double bond in natural Cers in the formation of a unique connection between C1-OH and C3-OH through a strong internal H-bonding network. Indeed, either the absence of the double bond or a change in isomerization to cis results in the loss of this distinctive motif. Interestingly, little change was observed around the amide linkage upon changing from trans- to cis-Cer, as evidenced by the similarities in spectral features for the NH resonance of both isomers. Taken together, our results suggest that it is most likely that the hydrophilic moiety of the molecule (including the trans double bond) is critical for the relevant biological functions of Cers.
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