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Langmuir 1997, 13, 1952-1956
Liposomes from Phosphatidyl Nucleosides: An NMR Investigation Silvio Bonaccio,† Donatella Capitani,‡ Anna Laura Segre,*,‡ Peter Walde,† and Pier Luigi Luisi† Istituto di Strutturistica Chimica, G. Giacomello, CNR, Area della Ricerca di Roma, C. P. 10, I-00016 Monterotondo Stazione, Rome, Italy, and Institut fu¨ r Polymere, ETH-Zu¨ rich, CH-8092 Zu¨ rich, Switzerland Received May 13, 1996. In Final Form: January 17, 1997X
We describe an NMR analysis of liposomes obtained from two phosphatidyl nucleosides, namely 5′(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine (1) and 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine (2). This analysis is mostly based on 1D- and 2D-1H-NMR at high field and is aimed at investigating intramolecular interactions between groups within the same lipid molecule and intermolecular interactions between neighboring lipid molecules in the liposome aggregate. Particular care was taken to choose mixing times which reduce the possibility of spin diffusion effects. The presence of particular cross peaks in 2D-1H-NOESY experiments permits us to assess the presence of syn- and anti-conformations of the aromatic head groups both in the monomeric nucleotides and in the liposomes. The lack of any particular ring effect in liposomes suggests that no stacking interactions of the nucleobases are present. On the other hand, the presence of other distinct cross peaks indicates short interatomic distances between a given aromatic base and the sugar moiety of an adjacent phosphatidyl nucleoside headgroup. These data permit one to draw a structural model of the liposome surface which accounts for the poor binding characteristics of phosphatidyl nucleoside liposomes toward complementary oligonucleotide strands.
Introduction In a previous paper1a we have reported on the behavior of liposomes obtained from phosphatidyl nucleosides, in particular 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)cytidine (1) and 5′-(1-palmitoyl-2-oleoyl-sn-glycero(3)phospho)inosine (2), which were obtained enzymatically from the corresponding phosphatidylcholines and nucleosides. The interest in this type of supramolecular lipid aggregates is twofold: On the one hand, they permit one to investigate whether and to what extent the chemical and binding properties of nucleic acids can be expressed also on the spherical structure of the liposomesswhether, for example, complementary base-pairing recognition also works at this novel structural level. On the other hand, phosphatidyl nucleosides and the corresponding liposomes appear to be interesting from the biomedical point of view.2,3 In order to display the information and recognition properties typical of nucleic acids, the bases of the phosphatidyl nucleosides in the liposomes must be available for binding. Since cytidine or inosine is part of the polar headgroup, one may expect that it is exposed to water. On the other hand, the bases of these nucleosides also possess a strong hydrophobic character, and it may therefore be that they are reclined back into the bilayer and not available for surface binding. In fact, preliminary studies1b showed no significant binding availability of liposomes made of 1 or 2. * To whom to address correspondence. † Institut fu ¨ r Polymere, ETH-Zu¨rich. ‡ Istituto di Strutturistica Chimica, CNR, Rome. X Abstract published in Advance ACS Abstracts, March 1, 1997. (1) (a) Bonaccio, S.; Walde, P.; Luisi, P. L. J. Phys. Chem. 1994, 98, 6661-6663; 10376. (b) Bonaccio, S. Liposomen aus Phosphatidylnukleosiden. Dissertation Nr. 11232, ETH-Zu¨rich, 1995. (2) Hostetler, K. Y.; Stuhmiller, L. M.; Lenting, H. B. M.; van den Bosch, H.; Richman, D. D. J. Biol. Chem. 1990, 265, 6112-6117. (3) Shuto, S.; Itoh, H.; Sakai, A.; Nakagami, K.; Imamura, S.; Matsuda, A. Bioorg. Med. Chem. 1995, 3, 235-243.
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The present NMR investigation was aimed at clarifying this point and the more general one of the conformation of the phosphatidyl nucleosides in the corresponding liposomes, focusing on the nucleoside headgroup. In particular, the following, three questions will be addressed in this work: (i) whether the base has an “in-” or an “out-” orientation with respect to the bilayer of the liposomes; (ii) whether the base assumes a syn- or an anti-conformation (see Figure 1); and (iii) whether the bases are engaged in intra- or intermolecular interactions. The present study will be restricted to the two phosphatidyl nucleosides indicated above as 1 and 2, including their mixture. In the meantime, we have prepared also several other phosphatidyl nucleosides, bearing for example adenine and uracil bases. The corresponding liposomes and other supramolecular aggregates formed thereof are presently under investigation, and the results will be reported at a later time. We will utilize here mostly 1D- and 2D-1H-NMR, i.e. NOESY techniques at high field, in order to investigate intramolecular interactions between groups within the same lipid molecule and intermolecular interactions between neighboring lipid molecules. One technical problem must be pointed out from the beginning: all our measurements of the liposome samples are carried out at 47 °C, as at lower temperature the resolution is not sufficient for a quantitative analysis (the signals are too broad). © 1997 American Chemical Society
Liposomes from Phosphatidyl Nucleosides
Figure 1. Schematic representation of the two most relevant conformations of cytidine and inosine. The two bases cytosine and hypoxanthine have either a syn- or an anti-conformation with respect to the sugar moiety.
Materials and Methods Reagents and Preparation of Liposomes. 1 and 2 were synthesized from 1-palmitoyl-2-oleoyl-sn-glycero(3)phosphocholine (POPC), and the corresponding nucleosides by the help of phospholipase D as described before.1a In this reference details on the preparation of the liposomes (extrusion of multilamellar liposomes through polycarbonate membranes and final extrusion through membranes with 50 nm pores) are also given. Note that pD ) pH (meter reading) + 0.4.4 1H-NMR Measurements. 1H-NMR measurements were performed at 600.13 MHz on a Bruker AMX spectrometer. 2DNOESY experiments5a were performed in the phase sensitive mode (TPPI),6 typically using 1K of memory for 512 increments. 1D-NOESY buildup was performed using soft pulses.5b The data were processed with AURELIA software.7
Results and Discussion Case of Cytidine Monophosphate and Inosine Monophosphate. Since we are dealing with problems of the relative geometry of atoms within the polar headgroups, or between adjacent headgroups, it is apparent that the methods of choice are high-field 1D-1HNMR spectral analysis followed by 2D-1H-NOESY experiments. Before embarking into the study of liposomes made of phosphatidyl nucleosides, it is necessary to analyze cytidine monophosphate and inosine monophosphate in order to have suitable references. The chemical shifts (given in ppm) and relative assignments for cytidine monophosphate are as follows: H-6 ) 8.10 d; H-5 ) 6.13 d; H-1′ ) 6.00 d; H-2′ ≈ H-3′ ≈ 4.34 m; H-4′ ) 4.24 m; H-5′ ) 4.05 m; H-5′′ ) 3.97 m. The chemical shifts (given in ppm) and relative assignments for inosine monophosphate are as follows: H-8 ) 8.56 s; H-2 ) 8.21 s; H-1′ ) 6.13 d; H-2′ ) 4.79 m; H-3′ ) 4.52 m; H-4′ ) 4.36 m; H-5′ ≈ H-5′′ ) 4.01 m. Since the molecules are rather small, NOESY experiments were performed with a mixing time of 500 ms. (4) Wu¨thrich, K. NMR in Biological Research: Peptides and Proteins; North-Holland Publishing Company: Amsterdam, 1976. (5) (a) Jeener, J.; Meier, B. H.; Bachmann, P.; Ernst, R. R. J. Chem. Phys. 1979, 71, 4546-4553. (b) Kessler, H.; Oschkinat, H.; Griesinger, C.; Bermel, W. J. Magn. Reson. 1986, 70, 106-133. (6) Marion, D.; Wu¨thrich, K. Biochem. Biophys. Res. Commun. 1983, 113, 967-974. (7) Bruker Analytische Messtechnik GMBH, AURELIA Software, 1994.
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Figure 2. 1D-NOE buildup data for cytidine monophosphate (13.9 mM in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD 8): T ) 27 °C, mixing times between 100 and 800 ms. The irradiated resonance at 8.10 ppm is due to H-6.
In cytidine monophosphate (spectra not shown) the strongest cross peak is the one between proton H-6 (8.10 ppm) and the peak at 4.34 ppm, which is due to both H-2′ and H-3′. Due to this ambiguity, and due to the strong coupling condition between these protons, this cross peak will be completely disregarded.8 One cross peak is present between proton H-6 (8.10 ppm) of the base and the anomeric proton H-1′ (6.00 ppm) of the sugar moiety, pointing to a syn-conformation. Moreover, two other cross peaks can be observed between H-6 and H-5′ (4.05 ppm) as well as between H-6 and H-5′′ (3.97 ppm), both pointing to the anti-conformation. A 1D-NOE buildup experiment was performed by irradiating H-6, see Figure 2. From this experiment and distance geometry calculations performed with the Hyperchem program, it is clear that both isomers must be present, with the syn-isomer slightly more populated: for the syn-isomer, the distance between H-6 and H-1′ is 2.3 Å; for the anti-isomer, H-6-H-1′ ) 3.6 Å, and the distance between H-6 and H-5′ and/or H-5′′ is 2.7 Å (only distances shorter than 5 Å between H-6 and the ribose are given). Thus, cytidine monophosphate in D2O solution at 27 °C is present in both the synconformation and the anti-conformation. A strong peak is observed between the adjacent protons H-5 and H-6, which are also J-coupled. This cross peak may be assumed as an internal standard in the 2D-1HNOESY experiments, since the distance between these two nuclei (2.45 Å, as estimated from bond lengths and bond angles)9 is well-known. It’s also worth noticing that H-5 does not show any other cross peak. The 2D-1H-NOESY experiment (not shown) of inosine monophosphate suggests again the existence of both conformations (syn-as well as anti): H-8 shows a cross peak with H-1′, pointing to a syn-conformation (∼2.6 Å in the syn-conformer and 3.8 Å in the anti-conformer), and H-8 also shows a very strong cross peak with H-2′, pointing to an anti-conformation (∼3.6 Å the shortest distance in the syn-conformer and ∼3.1 Å in the anti-conformer) while a contact of H-8 with H-3′ can only be due to the antiisomer (since a distance shorter than 5 Å is present only (8) Ernst, R. R.; Bodenhausen, G.; Wokaun, A. Principles of Magnetic Resonance in One and Two Dimensions; Claredon Press: Oxford, 1987; Chapter 7, p 374. (9) Landolt-Bo¨rnstein. Numerical Data and Functional Relationships in Science and Technology; New Series, Group VII, Vol. 1, “Nucleic Acids”; Springer Verlag: Berlin, Heidelberg, 1989; Subvol. a, pp 1821.
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Figure 3. 1D-NOE buildup data for inosine monophosphate (11.9 mM in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD 8): T ) 27 °C, mixing times between 100 and 800 ms. The irradiated resonance at 8.56 ppm is due to H-8.
in the anti-3-endo isomer). A 1D-NOE buildup by irradiating H-8 is shown in Figure 3. Interatomic distances were calculated with the Hyperchem program. Again a strong peak between H-8 and H-5′ and H-5′′ was not considered due to the strong coupling present between these protons. All these data point to the presence of both syn- and anti-isomers, with the anti-isomer more populated than the syn-isomer. In summary, the bases of cytidine monophosphate and inosine monophosphate in aqueous solution at 27 °C exist in both conformations, syn and anti, exchanging in a fast equilibrium; in cytidine the syn-isomer is slightly favored, while in inosine the anti-isomer is preferred. Case of the Phosphatidyl Nucleosides. It is known that the polar headgroup of phosphatidylcholines and other phospholipids can assume different orientations with respect to the bilayer plane.10 The two limit cases are encountered when the polar headgroup is either roughly parallel or roughly perpendicular to the bilayer plane. On the other hand, as we have discussed above, the aromatic base can basically assume either the syn- or the anticonformation with respect to the sugar moiety. Thus, there are a variety of basic possibilities for the orientation of the nucleoside headgroup on the outer and inner surface of phosphatidyl nucleoside liposomes. Keeping all the possibilities in mind, and having clarified the assignment of the reference compounds, we can now proceed to examine the NMR properties of liposomes made of phosphatidyl nucleosides. One important methodological aspect must be discussed first: the mixing time chosen for performing 2D-1HNOESY experiments. As pointed out previously,11 too long mixing times produce strong spin diffusion cross peaks, which in turn may lead to wrong interpretations; in fact, in micelles and other supramolecular lipidic systems, significant spin diffusion may be present even at relatively short mixing times (data not shown). (10) (a) Browning, J. L. In Liposomes: From Physical Structure to Therapeutic Applications; Knight, C. G., Ed.; Elsevier/North-Holland Biomedical Press: Amsterdam, 1981; pp 189-242. (b) Hauser, H.; Pascher, I.; Pearson, R. H.; Sundell, S. Biochim. Biophys. Acta 1981, 650, 21-51. (c) Hauser, H.; Pascher, I.; Sundell, S. Biochemistry 1988, 27, 9166-9174. (d) Hu¨bner, W.; Mantsch, H. H.; Paltauf, F.; Hauser, H. Biochemistry 1994, 33, 320-326. (11) (a) Keepers, J. W.; James, T. L. J. Magn. Reson. 1984, 57, 404426. (b) Olejniczak, E. T.; Gampe, R. T., Jr.; Fesik, S. W. J. Magn. Reson. 1986, 67, 28-41.
Figure 4. 2D-1H-NOESY spectrum of liposomes from 1 (11.1 mM in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD 8): T ) 47 °C, mixing time 25 ms. (A, top) full spectrum; (B, bottom) details of the spectrum showing the relevant cross peaks.
All this is paramount to say that, in order to properly study the 2D-1H-NOESY properties of liposomes, a contact time must be chosen which rules out the possibility of spin diffusion effects. To this purpose a series of measurements was performed with different contact times: 150, 100, 50, and 25 ms, respectively. The criterion for assessing whether or not a spin diffusion effect was present was the absence of any residual cross peak between the terminal methyl group of the fatty acid chains at 0.9 ppm and both the methylene protons of the glyceride moiety near 4.5 ppm and the fatty acid olefinic protons at 5.35 ppm (see Figure 4A). Only with a mixing time as short as 25 ms were no spurious cross peaks found; thus only the corresponding data were used here (the other data are not shown). It must be clear that, due to this very short mixing time,
Liposomes from Phosphatidyl Nucleosides
only strong cross peaks, corresponding to rather short distances, can be observed, while interatomic distances of the order of 4 Å or more cannot be detected. The chemical shifts of the aromatic protons observed in the case of liposomes of 1 are quite similar to those found for cytidine monophosphate. This seems to rule out a significant stacking between the aromatic bases within the liposomes. In fact, in the case of stacking, the magnetic anisotropy of an aromatic ring would cause a strong upfield shift on the neighbor one and vice versa.12 A similar conclusion had been reached on the basis of ultraviolet spectroscopy, which showed the lack of any significant perturbation of the aromatic chromophors.1 The comparison of the 1H-NMR spectrum of liposomes from 1 with the 1H-NMR spectrum from POPC13,14 does not indicate an interaction between the pyrimidine base (cytosine) and the glycerol moiety. In fact, the resonances of the glycerol backbone protons are practically unaffected by the presence of the nucleoside headgroup. It is also apparent from the 2D-1H-NOESY experiments that no cross peaks are present between the aromatic protons and either the glycerol moiety or the fatty acid chains (e.g. no cross peaks between H-5 or H-6 and CH-O-CO at 5.5 ppm). Thus, the polar head as a whole is located away from both the alkyl chains and the glycerol part; and furthermore adjacent aromatic bases do not interact with each other. It is also interesting to notice from Figure 4B that the anomeric proton H-1′ yields observable cross peaks with both aromatic protons (H-5 and H-6), suggesting that the sugar moiety of one unit is interacting with, i.e. must be very near to, both aromatic protons. Only two possible conformations may give rise to the observed cross peaks. The first is a conformer A in which contacts both between H-1′ and H-5 and between H-1′ and H-6 are intermolecular, as sketched in Figure 5A. Note that locally this is an anti-conformation. The second possible conformer (B) has an intramolecular contact between H-1′ and H-6 and an intermolecular contact between H-1′ and H-5. Note that locally this is a synconformation (Figure 5B). The situation in the case of liposomes containing 2 is rather similar, although these liposomes of 2 per se are not stable and tend to aggregate.1 In order to avoid aggregation, liposomes from a mixture of POPC and 2 (molar ratio 1:4) have been prepared. The full 2D-1HNOESY spectrum, recorded at 47 °C, is shown in Figure 6A, and details of the spectrum, showing the cross peaks with the ring H-8, are given in Figure 6B. Again, cross peaks between the anomeric H-1′ and the aromatic protons H-2 and H-8 could be detected. This indicates that also in this case the ribose part of the phosphatidyl nucleoside must be in close contact with the base, most likely through intermolecular interactions between two neighboring phospholipid molecules.15 Let us consider finally the liposomes obtained from the 1:1 mixture of the two lipids 1 and 2. We obtain the same cross peaks as those previously observed for the neat samples (see Figure 7). The lack of new cross peaks suggests that no particular interactions between the (12) Waugh, J. S.; Fessenden, R. W. J. Am. Chem. Soc. 1957, 79, 846-849. (13) Finer, E. G.; Flook, A. G.; Hauser, H. Biochim. Biophys. Acta 1972, 260, 49-58. (14) Dorovska-Taran, V.; Wick, R.; Walde, P. Anal. Biochem. 1996, 240, 37-47. (15) As evident from Figure 6, the NMR signals of POPC and 2 are split, indicating either that in addition to the liposomes other supramolecular structures are present or that possibly domains rich in one of the two lipids are formed in the liposome bilayer. We did not further study this effect.
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Figure 5. (A, top) Schematic representation of conformer A in liposomes made of 1 (with anti-conformation of the bases), showing the intermolecular interaction between H-1′ and H-5 and between H-1′ and H-6. (B, bottom) Schematic representation of conformer B in liposomes made of 1 (with syn-conformation of the bases), showing the intramolecular contact between H-1′ and H-6 and an intermolecular contact between H-1′ and H-5. The distance a between H-5 and H-6 is 2.45 Å, and the distance b between H-1′ and H-5 or H-6 is 2.8 Å.
purine and the pyrimidine bases are present, which in turn suggests that the two bases are distinctly compartmentalized, possibly building patches of the same surfactant type within a given liposome. Concluding Remarks The NMR analysis described here permits one to gain an insight into the geometry of the headgroup of phosphatidyl nucleosides in the liposomes. The fact that the aromatic bases have the same resonances in liposomes and in the model compounds in water indicates their exposure to water; on the other hand, the interaction of one given base with the sugar moiety of a neighboring base may explain why the bases are not easily available for complementary base pairing. From the NMR analysis of liposomes made of 1, one can propose for the orientation of the headgroup of this phosphatidyl nucleoside molecule the schematic picture shown in Figure 5. This also indicates how an intermolecular interaction between a base and a sugar of neighboring units can come about. However, it is clear that only energy minimum calculations, possibly coupled with molecular dynamics studies, can give a more precise evaluation of the structure and in particular of the packing interactions. One may now go back to the goals and expectations set for the liposomes made of phosphatidyl nucleosides, aimed at combining the supramolecular properties of liposomes with the recognition chemistry of nucleic acid bases.
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Figure 6. 2D-1H-NOESY spectrum of liposomes from a mixture of POPC and 2 in the molar ratio POPC:2 ) 1:4 (total concentration, 13.8 mM; concentration of 2, 11.1 mM; in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD 8). T ) 47 °C; mixing time 25 ms. (A, top) full spectrum; (B, bottom) details of the spectrum showing the relevant cross peaks.
Clearly the structural features of the cytidine and inosine bases in phosphatidyl nucleoside liposomes as shown here are not very suitable for complementary recognition and binding. Can one utilize these NMR data to propose a further development of the work with potentially succesful binding properties? The first idea that comes to mind is to use spacer groups between the phosphate part and the nucleoside so as to increase the distance of the aromatic base from the sugar moiety of the neighboring units and to push it at the same
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Figure 7. 2D-1H-NOESY spectrum of liposomes from an equimolar mixture of 1 and 2 (total concentration 21.6 mM in 20 mM deuterated sodium phosphate buffer, 20 mM KCl, 0.2 mM EDTA, pD 8). T ) 47 °C; mixing time 25 ms. (A, top) full spectrum; (B, bottom) details of the spectrum showing the relevant cross peaks. The subscripts i and c refer to inosine and cytidine, respectively.
time more into the aqueous external medium. One elegant way to do so would be to couple dinucleotides to the phosphoglyceride moiety or to have two sugar units separating the base from the glycerol moiety. Studies in this direction are presently in progress in our group. Acknowledgment. The work is supported by a COST action D7 program (molecular recognition). The authors thank J. Bella for assistance in the setup of NOE buildup experiments. LA9604745
