Experimental and theoretical NMR studies of interaction between phenylalanine derivative and egg yolk lecithin.

Research article Received: 19 November 2013

Revised: 18 January 2014

Accepted: 28 February 2014

Published online in Wiley Online Library

(wileyonlinelibrary.com) DOI 10.1002/mrc.4064

Experimental and theoretical NMR studies of interaction between phenylalanine derivative and egg yolk lecithin Roksana Wałęsa,a Tomasz Ptak,b Dawid Siodłak,a Teobald Kupkaa and Małgorzata A. Brodaa* The interaction of phenylalanine diamide (Ac-Phe-NHMe) with egg yolk lecithin (EYL) in chloroform was studied by 1H and 13C NMR. Six complexes EYL–Ac-Phe-NHMe, stabilized by N–H···O or/and C–H···O hydrogen bonds, were optimized at M06-2X/6-31G(d,p) level. The assignment of EYL and Ac-Phe-NHMe NMR signals was supported using GIAO (gauge including atomic orbital) NMR calculations at VSXC and B3LYP level of theory combined with STO-3Gmag basis set. Results of our study indicate that the interaction of peptides with lecithin occurs mainly in the polar ‘head’ of the lecithin. Additionally, the most probable lecithin site of H-bond interaction with Ac-Phe-NHMe is the negatively charged oxygen in phosphate group that acts as proton acceptor. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: lecithin; peptide; hydrogen bond; NMR; DFT; intermolecular interactions

Introduction Phospholipids are the main components of biological membranes. Lecithin is a very popular and frequently occurring diacylglycerol belonging to phospholipid family. It is formed from choline esterified with a phosphate group and contains glycerol with two fatty acid residues.[1,2] Various combinations of four different fatty acids are found in lecithin molecule. These could be palmitic (16 : 0), stearic (18 : 0), oleic (18 : 1) or linoleic (18 : 2) acids.[3] In Figs 1A and 2A are shown schematic formulas of lecithin substituted with linoleic and stearic acids. The phospholipids exhibit interesting properties because of their amphiphilic structure formed by hydrophilic heads and hydrophobic fatty acid tails. Phospholipids form bilayer liposomes in water.[4] Because of their unique structure and biocompatibility (they are the main components of mammalian cell membranes), liposomes are used as a selective drug delivery system.[5–8] For a long time, the properties and application of liposome-encapsulation drugs in the treatment of leishmaniasis[9] metabolic disorders, fungal diseases[10] and cancer[11] have been studied. The research in liposomal drug delivery has led to commercialization of several anticancer therapeutics.[12] These complex drugs exhibit significant clinical improvement and lower toxicity in comparison with the free compounds. For example, Deol et al.[13] reported that liposome-encapsulated antibiotics have greater efficacies than their free forms. Transport of ions and small molecules through cell membrane is vital for biological functions of living systems. Several molecules are known to promote such transport via membranes. For example, carotenoids are known as membrane plasticizers.[14,15] Peptides could also modify and stabilize liposome membranes.[16] Many natural peptides have been identified as potential therapeutic agents, but the use of peptides as drugs is hindered by their susceptibility to proteolytic degradation and difficulties in penetration

Magn. Reson. Chem. (2014)

through biological membranes. Thus, the main aim of peptide drug design research concentrates on the improvement of the therapeutic index of biologically active substances. It could be achieved by modifications of naturally occurring peptide.[17,18] On the other hand, closure of the peptides in liposomes (encapsulation) is a way to protect them against enzymatic degradation.[19,20] NMR technique as a very powerful analytical method has been applied to study structural and dynamic properties of phospholipid liposomes and interactions between various compounds and lipid membranes.[21–29] For example, numerous 1H NMR investigations on reverse micellar systems aimed at determination of the nature of phospholipid–water interactions were reported.[30–35] These works considered three-component lecithin–benzene–water mixtures by looking at the changes of phosphorus spin–lattice relaxation times (T1). Lecithin and phospholipids are well known and investigated for many years. However, the nature of interaction between peptides and lecithin is still not fully understood. For example, what are the lecithin binding sites and energy of such interactions? In the current study, we address the question of lecithin interaction with a model diamide (abbreviated in the text as Ac-Phe-NHMe) using experimental 1H and 13C NMR spectroscopy supported by theoretical calculations using density functional theory (DFT).

* Correspondence to: Małgorzata A. Broda, Faculty of Chemistry, University of Opole, Oleska St. 48, 45-052 Opole, Poland. E-mail: Małgorzata.Broda@uni. opole.pl a Faculty of Chemistry, University of Opole, Oleska 48 Str., 45-052 Opole, Poland b Department of Basic Medical Sciences, Wroclaw Medical University, Borowska 211 Str., 50-556 Wroclaw, Poland

Copyright © 2014 John Wiley & Sons, Ltd.

R. Wałęsa et al.

1

Figure 1. (A) General formula, proton numbering for the studied compounds. (B) H NMR spectra (400 MHz) of 0.001 M lecithin in 1 : 1 mixture with Ac-Phe-NHMe in CDCl3.

Methods Compounds investigated Diamide Ac-Phe-NHMe was synthesized according to Applewhite and Niemann,[36] crystallized and sublimed. Same analytical data of this compound are given by Broda et al.[37] The method used for isolation and purification of lecithin was a modification of the procedure described by Singelton et al.[38] Ten fresh egg yolks (c.a. 200 g) from commercial eggs were washed with warm water and mixed with 400 ml of acetone using mechanical stirrer for 10 min. The resulting suspension was centrifuged at 2500 rpm for 15 min at 4 °C. The solid was mixed with fresh portion of acetone and centrifuged. The step was repeated six times. The obtained solid was poured into 400 ml of chloroform–methanol mixture (1 : 1), and the solution was left to stand overnight. The suspension was centrifuged at 2500 rpm for 15 min. at 4°C. The solution was decanted and then further filtered through Shott funnel. The solvents were removed on rotary evaporator at 40 °C. The obtained slightly yellow oil was dissolved in hexane (80 ml), and then, acetone (650 ml) was added, and the solution was left to stand overnight. The off-white oily solid was filtered using Shott funnel and then dried in vacuum for 2 h at 40 °C. Meanwhile, the column (30 mm in diameter, 800 mm in effective length, with 500-ml flask, no filter disk but 5 cm of cotton) was filled with previously prepared Al2O3 with activity II. Of Al2O3, 350 g (activity I, 70-230 mesh ASTM, Merck 1.01077, Darmstadt, Germany) was mixed with H2O (8.4 g) for 4 h, then dried for 6 h at 107 °C and cooled; then, 200 ml of eluent (CHCl3 : MeOH, 9 : 1) was added and then decanted to remove the smallest fraction of Al2O3. The last step was repeated four times. The crude product (9.5 g) was dissolved in chloroform so that 5% solution was obtained and purified by column chromatography. The process was monitored using TLC (Silica gel 60, Merck 1.05554, Darmstadt, Germany) using solvents mixture CHCl3 :

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MeOH : H2O, 65 : 25 : 4, and visualized using iodine as well as chlorine–tolidine. The proper fraction (ΔRf ~ 0.25) was gathered and filtered through Shott funnel to remove the Al2O3 particles. The final product was in the form of transparent oily solid (3.3 g). Only a single spot was observed on TLC (thin-layer chromatography) plate for a final product. Analysis calculated for lecithin main component C42H82NO8 (760.10): C, 66.37; H, 10.87; N, 1.84. Found: C, 65.71; H, 10.09; N, 1.88. 13C NMR (400 MHz, CDCl3) δ 172.6, 172.2 (C O), 129.1, 127.1 (C C), 69.5, 65.4, 62.4, 62.1, 58.4 (C–O), 53.5 (C–N), 33.3, 31.0, 28.7–28.4, 26.2, 24.7, 24.0, 21.8, 13.2 (C–C). 1H NMR (400 MHz, CDCl3) δ 5.4–5.2 (4H, m), 5.2 (1H, m), 4.4 (1H, d), 4.3 (2H, s), 4.2–4.1 (1H, m), 4.0–3.9 (2H, m), 3.8 (2H, s), 3.6 (1H, s), 3.4 (9H, s), 2.8 (2H, m), 2.4– 2.3 (4H, m), 2.1–2.0 (4H, m), 1.4–1.2 (42H, m), 0.9 (6H, m). Total integration (82H) excluding water at δ 1.6. NMR measurements The 1H, 13C and 31P NMR measurements were carried out using 400-MHz Bruker Ultrashield NMR spectrometer at room temperature. Bruker TopSpin version 1.3 software was used for data acquisition and processing. All spectra were recorded in CDCl3 and referenced to TMS (tetramethylsilane is a internal standard for 1H and 13C NMR) and to external glufosinate (31P NMR). Very low concentrations were used to avoid Ac-Phe-NHMe association visible from the infrared spectra of similar systems. Thus, the 1H and 31P spectra of 0.001 M solutions of lecithin and lecithin–AcPhe-NHMe complex were studied. However, because of sensitivity problems, the corresponding carbon spectra were observed at significantly higher concentrations (0.02 M). For higher digital resolution, longer acquisition times (4 s) were used for 1H NMR spectra recorded at 400 MHz and spectral width of 4400 Hz. Scans at around 32–512 were collected in case of the proton spectra. 13 C NMR (100 MHz) spectra with 4 k (or more) scans and 3 s



Interaction between Ac-Phe-NHMe and EYL

Figure 2. (A) General formula, carbon numbering for the studied compounds. (B) Ac-Phe-NHMe in CDCl3.

acquisition time duration were recorded. The 31P NMR spectra (162 MHz, acquisition times of 14 s for better digital resolution and spectral width of 16 000 Hz) were also recorded. In addition, T1 relaxation studies of proton and phosphorus signals were performed using inversion-recovery method (IRT1). Besides, several 2D NMR spectra using proton–proton COSY, NOESY and 1H–13C HMQC were recorded to facilitate the assignment of experimental spectra of lecithin with numerous overlapped signals. Theoretical calculations All theoretical calculations in the gas phase were performed with Gaussian 09[39] software at the DFT level using M062X[40,41] density functional and 6-31G* basis set for structure


13

C NMR spectra (400 MHz) of 0.001 M lecithin in 1 : 1 mixture with

optimization of lecithin, Ac-Phe-NHMe and their several potential 1 : 1 complexes. For a large and flexible molecule (lecithin), it is not trivial to find a global minimum structure. In this study, we selected, somehow arbitrary, several possible conformations and chose the one with the lowest energy for further calculations. The selected conformation is reasonable in terms of sterical effects and intramolecular interactions (M06-2X density functional is believed to perform well both for describing H-bonds and weak long range van der Waals interactions). However, no ultimate proofs for the selected lecithin structure as global minimum exist. Regarding Ac-Phe-NHMe, we chose low energy conformers of this compound on the basis of earlier works.[42] The complexes were formed by positioning the C-terminal or N-terminal NH group of the studied diamide toward different carbonyl, ester, phosphate



R. Wałęsa et al. groups and double bond in lecithin (see Fig. 1A). The energy of interaction was evaluated from energy differences between complex and its individual components and was corrected using the counterpoise (CP) method.[43] Next, the vibrational analysis was conducted to assure that the optimized structures correspond to true energy minima (no negative frequencies were observed). Because of the size of the system, a compromise between computational cost and accuracy was chosen. Thus, we selected novel basis sets that are fairly compact while very accurate in predicting nuclear shieldings. Unfortunately, these basis sets were designed for C, H, O and N only (and are not available for P). These basis sets are created by modification of very small STO-3G basis sets and perform very well in predicting carbon shielding. Their excellent performance on providing accurate carbon nuclear shieldings in model dehydropeptides[44] and carbon nanotubes[45] was recently published. Thus, GIAO NMR calculations using VSXC[46] and B3LYP[47,48] density functionals combined with STO-3Gmag[49] basis set for carbon and hydrogen atoms and aug-cc-pVTZ-J basis set designed by Sauer for phosphorus atom were performed.[50–55] The aug-cc-pVTZ-J basis a

Table 1. Observed carbon and proton chemical shifts (in ppm) of 0.001 M lecithin in 1 : 1 mixture with Ac-Phe-NHMe in CDCl3 Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 a

13

C

173.61 173.22 171.44 170.04 136.82 130.00 129.68 129.22 128.06 127.86 126.95 70.42 66.46 63.37 62.97 59.26 54.73 54.48 38.56 34.30 34.12 31.92 31.52 29.44 27.23 27.20 26.15 25.62 24.92 24.83 23.22 22.69 22.58 14.12

Peak no. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

Signal numbering from low to high magnetic field.


1

H

7.220 6.241 5.724 5.340 5.122 4.559 4.404 4.345 4.134 3.972 3.815 3.365 3.043 2.770 2.699 2.288 2.018 1.979 1.582 1.252 0.879

set was also very good in predicting non-hydrogen atom nuclear shieldings[56] and was downloaded from EMSL (Environmental Molecular Sciences Laboratory).[57] The structure of isolated lecithin was assumed from analysis of integral intensities of its proton spectra. Thus, the intensity ratio for several signals indicated the presence of two C18 fatty acids. The first one corresponded to unsaturated linoleic acid (18 : 2) in which the two double bonds are in cis configuration, and the second one was stearic acid with all single C–C bonds. Theoretical chemical shifts were referenced to TMS (tetramethylsilane) calculated at the same level of theory.

Results and Discussion 1

H, 13C and 31P NMR spectra of lecithin in the presence of Ac-Phe-NHMe

Because there are only small changes in proton and carbon NMR spectra of free lecithin upon addition of diamide, we will start the presentation of results by introduction of the corresponding spectra of the mixture. 1H NMR spectrum of the very diluted solution of 1 : 1 mixture of lecithin and Ac-Phe-NHMe in CDCl3 is presented in Fig. 1B. The individual proton signals of lecithin and diamide are numbered starting from the low magnetic field. Obviously, because of magnetic similarity of several fatty acid chain protons, some signals are overlapped and show higher intensity. In Figs 1A and 2A are shown the simplified formulas of both lecithin and diamide together with hydrogen and carbon atom numbering according to the increased magnetic field. In Table 1 are shown the observed proton and carbon signals of lecithin and phenylalanine derivative. The assignment of diamide signals was taken from earlier studies.[58] In the case of lecithin, all proton signals were assigned according to earlier reports.[59–64] Additionally, proton signal assignment was confirmed by 2D NMR spectra, which are shown in the supporting information (Figs S1 and S2). The corresponding carbon spectrum (taken at significantly higher concentration) of lecithin–diamide is shown in Fig. 2B. Some carbon signals, which were missing in the

Figure 3. Sensitivity of lecithin proton signal no. 11 to the presence of diamide (for comparison, the inset shows signal Nos. 9, 10 and 11).



Interaction between Ac-Phe-NHMe and EYL

Figure 4. Hydrogen bonds pattern in M06-2X geometries of lecithin–Ac-Phe-NHMe complexes. Dashed lines represent the intermolecular X–H···O hydrogen bonds.

literature, were assigned from HSQC experiments (see Fig. S3 in the supporting information). There were very small changes of several lecithin signals upon complexation (from 0.001 to 0.003 ppm; see Table S1 in the supporting information). Only two signals, originating from groups within lecithin polar ‘head’ [no. 12 – N(CH3)3 and no. 11 – N–CH2], were perturbated to a higher degree. In Fig. 3 are shown fragments of lecithin 9–11 signals before and after addition of peptide. It was apparent that the most low field signal at about 4.14 ppm, formed by several sharp peaks, was practically unchanged while the most upfield signal at about 3.8 ppm (no. 11 coming from N–CH2) moved to higher field by 0.009 ppm and broadened by about 7% (from 13.51 to 14.46 Hz in the complex). A smaller upfield shift (by 0.005 ppm) was observed for a signal no. 12 at 3.370 ppm (methyl groups attached to nitrogen). Its position in the complex (3.365 ppm) and broadening (from 5.81 to 7.11 Hz, e.g. 22 %) also indicates the formation of complex via diamide H-bonding with a polar


lecithin head. There were also consistent changes (decrease indicating stiffening of the agglomerate structure and mobility) of lecithin proton T1 values upon addition of dipeptide (see Table S2 in the supporting information). A single and fairly broad 31 P NMR peak at 0.021 ppm (ν1/2 = 10.9 Hz) was observed in lecithin solution with diamide (see Fig. S4 in the supporting information). The differences between this spectrum and the spectrum of free lecithin solutions at 0.008 ppm (ν1/2 = 8.3 Hz) are nearly insignificant and will not be discussed in this work. However, here, we only want to stress that the about 25% broadening of 31P NMR signal indicates a formation of higher molecular weight associate(s), which show a decreased mobility in comparison with free lecithin. Broadening of 31P signal upon addition of peptide could be related to decreasing phosphorus mobility, manifested by the lowering of T2 spin–spin relaxation time, directly related to linewidth (ν1/2 = 1/πT2*, where T2* includes magnetic field inhomogeneity). This conclusion is also supported by a small (about 11%) decrease of phosphorous-31 T1 spin–lattice



R. Wałęsa et al. Table 2. The geometrical parameters (in Å and °) of intermolecular hydrogen bonds, torsion angles for the diamide, relative energies (ΔE) and binding energies (in kcal/mol, corrected for BSSE) for the studied lecithin–Ac-Phe-NHMe complexes N(or C)–H···O Complex

H···O

N(C)···O

∠N(or C)–H···O

∠C(or P) O···H

1

1.74 2.47 2.54 2.37 1.82 2.12 2.39 2.31 2.21 2.57 2.53 1.93 2.22 2.25 2.91 2.91 2.67 2.59 1.85 2.69 2.40 2.25 2.69 2.25 1.95 2.76 2.35 2.40 2.35 2.49 2.57 2.60

2.77 3.31 3.06 3.13 2.82 3.13 3.33 3.34 3.18 3.10 3.25 2.94 3.20 3.25 3.40 3.44 3.04 3.19 2.84 3.48 3.35 3.26 3.58 3.25 2.93 3.84 3.41 3.38 3.28 3.37 3.63 3.48

175.9 132.4 (C) 108.1 (C) 137.3 (C) 165.6 152.3 (C) 143.2 (C) 156.0 (C) 146.0 (C) 109.1 (C) 123.1 (C) 176.5 148.0 (C) 151.5 (C) 107.4 (C) 110.4 (C) 99.2 (C) 113.4 (C) 162.9 128.3 (C) 144.1 (C) 153.1 (C) 138.1 (C) 153.5 (C) 161.8 170.7 (C) 161.2 (C) 149.7 (C) 140.8 (C) 135.6 (C) 163.6 (C) 136.1 (C)

148.3 (P) 109.1 109.7 126.2 135.2 (P) 159.0 122.9 135.2 123.7 (P) — — 147.5 111.7 131.5 131.0 — 92.2 (P) 114.3 (P) 137.9 138.3 — 120.7 166.5 112.1 — — 133.7 (P) 162.3 (P) 121.4 109.7 79.8 132.5

2

3

4

5

6

φ

ψ

ΔE

Ebind

87.0

80.2

0.00

31.02

148.7

133.3

0.14

30.88

93.8

60.4

0.16

30.86

163.3

124.1

3.72

27.30

82.6

76.8

14.98

16.04

82.1

83.1

24.54

6.48

relaxation time value of free lecithin (T1 = 0.613 s) upon addition of peptide (T1 = 0.547 s). Besides, comparison of free diamide spectra and their corresponding changes in the presence of lecithin revealed uniform variations of NH chemical shifts (displacements from 6.182 to 6.241 ppm and from 5.593 to 5.724 ppm). These changes are equivalent to low field shift of NH protons taking part in hydrogen bonding. In addition, the formation of lecithin–diamide complex could explain a significant shortening of T1 relaxation time of NH proton at 5.72 ppm (from 1.539 s in free diamide to 0.718 s because of the formation of larger and less mobile aggregate and stronger H-binding). DFT calculations of complexes structures, interaction energies and GIAO NMR parameters The optimized structures of lecithin and Ac-Phe-NHMe calculated in the gas phase at M06-2X/6-31G* level of theory are gathered in Fig. S5 in the supporting information. We obtained 13 optimized 1 : 1 complexes of lecithin with Ac-Phe-NHMe diamide. In this


1

13

Figure 5. Correlation between calculated and experimental H and C NMR chemical shifts for six selected lecithin–Ac-Phe-NHMe complexes.



Interaction between Ac-Phe-NHMe and EYL Table 3. RMS deviation (in ppm) between calculated and observed carbon and proton chemical shifts of 0.001 M lecithin in 1 : 1 mixture with Ac-Phe-NHMe in CDCl3 RMS Complex 1 2 3 4 5 6

VSXC 1

H

1.22 1.13 0.96 1.13 1.13 0.95

RMS B3LYP 1.20 1.10 0.96 1.05 0.84 0.82

VSXC 13

C

work, we present six (1–6) of them with the lowest energy. Their structures, optimized in the gas phase, are shown in Fig. S6. The H-bond patterns, responsible for formation and stabilization of these complexes, are presented in Fig. 4. To simplify this very crowded schemes, in some cases of C–H···O interactions, the protons are not shown on the picture. The selected parameters of the complexes are gathered in Table 2. These include the geometrical parameters of intermolecular hydrogen bonds, torsion angles for the diamide, relative energy for each complex and binding energy (corrected for basis set superposition error BSSE). The complexes in Table 2 are ordered according to the decreasing binding energy. The analysis of interactions, stabilizing the complexes, was performed on the assumption that the X–H···O hydrogen bonds are the main attraction forces (X = N or C). The H-bond criteria described by Steiner and Torshin et al. were used.[65,66] The lowest energy complex 1 is stabilized by very short N–H2···O=P hydrogen bond with H2···O distance equal to 1.74 Å. In addition, four C–H···O interactions occur in this structure. Second in terms of stability, with almost the same energy (ΔE = 0.14 kcal/mol), is complex 2, where the strongest intermolecular interaction is H-bond N–H3···O=P with d(H3···O) = 1.82 Å. As in the previous case, several C–H···O contacts also stabilize the structure 2. Thus, the two lowest energy complexes of lecithin : diamide are stabilized by H-bond between amide N–H and phosphate groups. This interaction is significantly stronger than the N–H···O=C hydrogen bonding because of more negative charges on the oxygen atom of the phosphate group. According to our M06-2X/6-31G* calculations, the Mulliken atomic charges on oxygen atoms in phosphate and carbonyl groups are 0.745 and 0.529, respectively. Next in energy order, with relative energy of only 0.16 kcal/mol, is complex 3 stabilized by N–H2···O=C1 hydrogen bonding and five additional C–H···O contacts. The energies of complexes 1–3 are very similar, so it can be assumed that all of these structures are present in the chloroform solution of lecithin and diamide and that the interaction of the peptide with lecithin in a nonpolar environment occurs mainly in the region of lecithin polar head. The GIAO NMR-calculated results for lecithin protons and carbons, recalculated to the corresponding chemical shifts, were compared with the experimental values, and the individual deviations were gathered in Tables S3 and S4, respectively. The accuracy of theoretical prediction of lecithin proton and carbon chemical shifts is evident from plots correlating theoretically predicted and experimental spectra. Thus, for each complex was created a graph (proton and carbon) showing the results of B3LYP and VSXC density functionals. In each case, a nice linear correlation was observed. As an example, in Fig. 5 are shown


8.79 8.60 8.95 9.04 9.11 9.10

RMS B3LYP 10.36 10.11 10.77 10.45 10.15 10.76

13

1

C+ H

VSXC

B3LYP

6.91 6.80 7.06 7.14 7.18 7.18

8.18 7.98 8.49 8.25 8.00 8.47

the corresponding correlations for complex 1. The individual points were fitted using linear least square method. It is apparent that both density functionals produced slightly different results (slopes), but the scatter of points is similar (very large R2 value of 0.99). The remaining graphs for all complexes are presented in the supporting information in Fig. S7. The overall matching of theoretical chemical shifts of the six complexes with observed spectra is assumed from the total RMS (root mean square) values of deviations between the results calculated with B3LYP and VSXC density functionals and the experiment (Table 3). In the case of proton spectra, the RMS deviations from experiments are between 0.8 and 1.2 ppm, and the B3LYP gives somehow better agreement with experimental chemical shifts. Significantly larger deviations are observed for the predicted carbon chemical shifts (8.6–10.8 ppm), and VSXC produced better agreement with the experiment (RMS of 8.6 ppm for complex 2). When both proton and carbon predictions are considered, the most accurate method is also VSXC for complex 2 (RMS = 6.80 ppm), and B3LYP produced results higher by 1.2–1.7 ppm. Despite small differences, it is interesting to notice that the 1H + 13C VSXC RMS values increase in the same order as the binding energies of the potential complexes (e.g. 1 < 2 < 3 and 4, 5 and 6 are slightly larger). Thus, theoretical prediction of chemical shifts points out to complexes 1–3 as the most probable structures existing in chloroform solution.

Conclusions In this study, we investigated the interactions of model peptide Ac-Phe-NHMe with lecithin molecule in chloroform solution using 1D and 2D NMR spectra and DFT calculations. Based on the obtained results, the following conclusions can be drawn. Both the changes in experimental 1H NMR spectra of Ac-PheNHMe upon addition of lecithin and the theoretical predictions of formation of complexes 1–3, characterized by a fairly large calculated binding energy (from 31.02 to 30.86 kcal/mol, calculated in vacuum), indicate a presence of interactions and formation of complexes in solution of lecithin and diamide in low polarity solvent (CDCl3). According to our molecular modeling studies, the most probable lecithin site of H-bond interaction with Ac-Phe-NHMe is the negatively charged oxygen in phosphate group that acts as proton acceptor. The RMS deviation of theoretically predicted proton and carbon chemical shifts and experimental values is smaller in case of VSXC calculations for the six considered complexes (the RMS of B3LYP results are higher by 1–1.5 ppm).



R. Wałęsa et al. Acknowledgements R. W. is the recipient of a PhD fellowship from a project funded by the European Social Fund. Calculations were carried out in Wrocław Centre for Networking and Supercomputing (http:// www.wcss.wroc.pl) and in the Academic Computer Centre CYFRONET, AGH, Kraków, grant MEiN/SGI3700/UOpolski/063/2006.

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