Phospholipid conjugates: formation of the intramolecular π-cation complex

Abstract

Phospholipid conjugates consist of functionally different classes of molecules: phospholipid drug conjugates, fluorescent lipid probes and lipid molecular motors. All these conjugates are molecules that bear a functional group– a drug, a fluorophore or a molecular motor attached to the phospholipid. The conjugation is needed to incorporate a functional group into the lipid bilayer of liposome or lipid nanoparticle and thus, either modulate the effect of the drug or bring a new function to the liposome. Here, using NMR spectroscopy and quantum chemistry calculations, we show that phospholipid conjugates can form intramolecular π-cation complexes between quaternary ammonium group of the phosphatidylcholine and aromatic ring of the conjugated moiety. We also report on how to avoid the π-cation complex formation. If the linker between the aromatic moiety and the choline group is long enough the formation of π-cation complex is not observed.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13065-025-01526-x.

Introduction

Phosphatidylcholine (PC) is a class of phospholipids that incorporate choline as a headgroup. It is a major component of biological membranes. Because of natural abundance and nontoxicity, PC is used in various medicine/biology related applications such as liposomes and lipid nanoparticles. One of the lines of PC centered R&D is the construction of phosphatidylcholine conjugates (PhC). The aim is to incorporate a functional group into the lipid bilayer of liposome or lipid nanoparticle and thus either modulate the effect of the drug or bring a new function to the liposome (Fig. 1A). The functional group can be a drug, a fluorophore or a molecular motor. Thus, the PhC super class consists of functionally different classes of molecules: phospholipid drug conjugates (PDC), fluorescent lipid probes (FLP) and lipid molecular motors (LMM)

Fig. 1.

(A) Schematic representation of a liposome and bilayer loaded with PhC. (B) Synthetic lipids and PhCs used in the study. DPPC– dipalmitoylphosphatidylcholine. tA-PC– type-A azo-indane phosphatidylcholine, tB-PC– type-B azo-indane phosphatidylcholine, az-PC– azobenzene-phosphatidylcholine. С. Annotations of atoms and groups of atoms used in the study

The PDC is probably the most popular class of phospholipid conjugates. Recent reviews on the topic [1, 2] discusses several PDCs, with the paclitaxel PC conjugates being amongst the first mentioned examples. Due to the paclitaxel’s bulky hydrophobic structure, its incorporation inside the lipid bilayer is very challenging. However, its conjugation with phosphatidylcholine addresses this issue [3, 4]. The same is true for porphyrins [5, 6]. For less bulky molecules like mitomycin C [7], chlorambucil [8], cisplatin [9], colchicinoids [10, 11], camptothecin [12], doxorubicin [13] and small molecule MYC inhibitor [14], the conjugation with lipids does improve drug properties such as drug loading efficiency, stability, systemic toxicity, and controlled drug release [15].

The FLP pretend to be the first synthesized lipid derivatives. Being known since 60s, these have been used in membrane studies and cell imaging (see recent review [16] and examples [17, 18]).

LMM is the least populated class of PhC. Being introduced in the late 90s [19] they a have narrow field of application– the control of membrane structure and dynamics [20–22]. For example, raft or domain organization in the membrane could be controlled by LMM using light [20, 22].

Despite of differences in function, the most of PhC have the same structural pattern. Phosphatidylcholine has a special part called a “functional group” attached to the “sn-2 position” of the lipid glycerol backbone through a “hydrocarbon linker”. (Fig. 1). The popularity of the sn-2 position is based on two key factors. The first thing is the starting compound, which is a lipid with a free hydroxy group in the sn-2 position. This can be made synthetically [23] or purchased commercially. Commercial availability is based on the availability of corresponding enzyme– phospholipase A2, not phospholipase A1. The second factor is phospolipases A2 in mammals that are targets for PhC as they are involved into inflammation processes. Thus, many PhC are developed to either sense PLA2 activity [24] or utilize PLA2 activity to release a drug [10, 25].

In line with our ongoing work on different PhC structures [10, 17, 26, 27], we developed a new type of LMM, structure shown on Fig. 1B, in which we identified molecular interactions within PhC molecules. Those interactions were clearly seen by distortions in the NMR spectra of PhC. It appeared that these were originated from a π-cation complex formation inside the PhC molecule. Using experimental and in-silico methods, below we describe the phenomenon, investigate its molecular reasons, and find out the rule on how to avoid it.

Experimental

Synthesis

The starting compounds (1) were obtained previously [28]. The general procedure was as follows: The starting compound (1) was alkylated with hexyl bromide to give the phenyl ether (2). The reaction was carried out in dry boiling DMF (dimethylformamide) with potassium carbonate as the base. The reaction was completed in four hours. The reaction mixture was then mixed with water and extracted with ethyl acetate. Product (2) was isolated via chromatography on silica using a mixture of petroleum ether and ethyl acetate (10:1) as the eluent. Yields were about 80–92%. The hydrolysis of (2) with finely powdered NaOH in a CH₂Cl₂:MeOH (9:1) followed by treatment with 1 N HCl gave the acid (3), which was used further without purification. The acid (3) was introduced into the Stiglich esterification reaction with lyso-PC using DIC as condensing agent, DMAP as base and dry CH₂Cl₂ as a solvent. The reaction was kept overnight. The reaction mixture was evaporated, dissolved in CHCl₃:MeOH 8:2 and applied to silica column. Eluent composition was CHCl₃:MeOH 8:2 followed by CHCl₃:MeOH: H₂O 65:25:4. Final purification over alumina using pure CHCl₃ give tA-PC, tB-PC or az-PC. Isolated yields were about 56–64%.

tA-PC

¹H-NMR, 700 MHz, CDCl₃:MeOH 1:1: 7.86 (dd; J₁ = 9.00, J₂ = 2.31; 2 H, I = 2), 7.70 (dd, J₁ = 8.02, J₂ = 2.94, 2 H, I = 2), 7.33 (d, J = 8.33, 1H, I = 1), 7.12 (d, J = 8.26, 1H, I = 0.05), 7.00 (dd, J₁ = 9.065, J₂ = 2.66, 2 H, I = 2), 6.90 (dd, J₁ = 9.11, J₂ = 0.91, 2 H, I = 0.10), 6.85 (wide s, 1H, I = 0.05), 6.77 (dd, J₁ = 9.03, J₂ = 1.89, 2 H, I = 0.1), 6.57 (dd, J = 9.03, 2 H, I = 0.1), 5.33 (m, 4 H + 1H, I = 4.08), 4.45 (dt, J₁ = 3.64, J₂ = 12.18, 1H, I = 0.99), 4.25 (m, 2 H, I = 2), 4.20 (dd, J₁ = 6.93, J₂ = 11.97, 1H, I = 1.06), 4.04 (m, 2 H + 2 H, I = 3.85), 3.63 (m, 1H, I = 1.16), 3.58 (m, 2 H, I = 2.08), 3.19 (d, J = 7, 9 H, I = 9), 3.00 (ddd, 1H, I = 1), 2.92 (m, 2 H, I = 2), 2.74 (q, J = 6.16, 1H, I = 1.15), 2.56 (dd, J₁ = 16.03, J₂ = 8.82, 1H, I = 1), 2.46 (m, 1H, I = 1.1), 2.28 (dt, ), 2.01 (m, I = 3.22), 1.80 (m, 2 H, I = 2.16), 1.56 (m, 2 H, I = 2.42), 1.48 (dt, 2 H, I = 2.21), 1.30 (I = 26), 0.92 (m, 3 H, I = 3.14), 0.87 (dt, 3 H, I = 3.66).

tB-PC

¹H-NMR, 700 MHz, CDCl₃:MeOH 1:1: 7.87 (d, J = 8.75, 2 H, I = 2.00, E-), 7.53 (m, 1H, I = 1, E-), 7.29 (m, 1H + 1H, I = 1.93, E-), 7.14 (m, 1H, I = 0.29, Z-), 7.07 (d, J = 7.56, 1H, I = 0.26, Z-), 7.01 (d, J = 8.82, 2 H, I = 2.04, E-), 6.96 (d, J = 8.89, 2 H, I = 0.52, Z-), 6.76 (d, J = 9.03, 2 H, I = 0.45, Z-), 6.62 (d, J = 7.63, 1H, I = 0.22, Z-), 5.30 (m, 4 H + 1H, I = 3.65), 4.45 (dd, J1 = 12.4, J2 = 3.22, 1H, I = 1.06), 4.25 (m, 2 H, I = 2.68), 4.19 (m, 1H, I = 1.23), 4.05 (m, 2 H + 2 H, I = 4.6), 3.94 (t, J = 6.51, 2 H, I = 0.45, Z-), 3.66 (m, 1H, I = 1.12), 3.59 (m, 2 H, I = 2.65), 3.21 (d, J = 5.53, 9 H, I = 10.5, ratio 1:5), 2.86 (m, 1H, I = 1.06), 2.76 (m, 1H, I = 1), 2.58 (dd, J1 = 8.54, J2 = 6.93, I = 1), 2.47 (m, 1H, I = 1), 2.30 (m, 2 H, I = 2.93), 2.02 (m, 4 H, I = 2.74), 1.79 (m, 2 H, I = 2.3, E-), 1.75 (m, 2 H, I = 0.51, Z-), 1.59 (m, 2 H, I = 3.39), 1.49 (m, 2 H, I = 2.45), 1.30 (wide m, I = 35), 0.92 (t, 3 H, I = 3.37), 0.88 (m, 3 H, I = 4.5).

az-PC

¹H-NMR, 700 MHz, CDCl₃:MeOH 1:1: 7.87 (ddd, J₁ = 8.96, J₂ = 2.1, J₃ = 3.15, 2 H, I = 2), 7.79 (ddd, J1 = 8.33, J₂ = 2.17, J₃ = 2.52, 2 H, I = 1.95), 7.35 (dd, J₁ = 8.4, J₂ = 1.96, 2 H, I = 1.83), 7.16 (dd, J₁ = 6.72, J₂ = 1.96, 2 H, I = 0.1, Z-), 7.00 (ddd, J₁ = 8.96, J₂ = 2.1, J₃ = 3.15, 2 H, I = 2), 6.89 (dd, J₁ = 6.37, J₂ = 1.96, 2 H, I = 0.1, Z-), 6.81 (dd, J₁ = 6.72, J₂ = 1.96, 2 H, I = 0.1, Z-), 6.76 (dd, J₁ = 6.37, J₂ = 1.96, 2 H, I = 0.1, Z-), 5.32 (m, 6 H, I = 3.99), 5.24 (m, 1H, I = 1.2), 4.39 (dd, J₁ = 3.15, J₂ = 12.18, 1H, I = 0.93), 4.22 (m, 2 H, I = 2.08), 4.15 (dd, J₁ = 11.98, J₂ = 7, 2 H, I = 1.11), 4.00 (m, 2 H, I = 2.32), 3.61 (m, 1H, I = 0.99), 3.56 (t, J = 4.5, 2 H, I = 2.03), 3.18 (d, J = 7.21, ratio 1:5.5, 9 H, I = 9), 3.02 (m, 2 H, I = 2), 2.74 (m, 4 H, I = 3), 2.29 (m, 1H, I = 1), 2.23 (t, J = 7.1, 2 H, I = 1.75), 2.02 (m, 4 H, I = 4.1), 1.81 (m, 2 H, I = 2.19), 1.59 (d, J = 9.0 Hz, 1H), 1.54 (dd, J = 8.8, 5.8 Hz, 2 H), 1.48 (ddd, J = 14.8, 7.4, 4.6 Hz, 2 H), 1.36 (dt, J = 7.3, 3.8 Hz, 4 H), 1.36–1.29 (m, 1H), 1.31–1.27 (m, 1H), 1.26 (s, 7 H), 1.29–1.20 (m, 5 H), 0.99–0.84 (m, 7 H).

NMR-spectroscopy

The spectra were recorded on a Bruker Avance III 700 MHz NMR spectrometer equipped with a room-temperature triple resonance TXI probe. NMR was used to confirm the structures of the synthesized compounds and to measure the diffusion coefficients. The structures were confirmed through a standard set of 1D ¹H, ¹³C and 2D homonuclear DQF-COSY and heteronuclear ¹-¹³ C HSQC with corrected multiplicity, ¹-¹³ C and ¹-¹⁵ N HMBC, ¹H-¹H NOESY and ROESY NMR.

The diffusion coefficient D was measured using the pulsed gradient stimulated echo sequence with water suppression and convection compensation (PGSTE-watergate [29]). Spectra were acquired using 16 scans and 32 linear gradient g₁ steps from 13.93 to 52.95 Gs/cm. The diffusion delay Δ was set to 400 ms.

After acquisition, it turned out that the distance between the Ch_Me groups’ signals of interest at 3.15–3.25 ppm was comparable to the peak widths (Fig. 4, A). To avoid the effects of peak overlapping, the group of signals was deconvoluted as a sum of three Lorentzian shapes along the ¹H chemical shift axis, and simultaneously the decay of each peak’s intensity E along the g1 gradient axis was approximated with the following equation [29]:

Fig. 4.

A, B. DOSY NMR experiments. (A) Singlets from Ch_Me group of lyso-PC (left peak, reference) and tA-PC in open (no π-cation complex) and closed (internal π-cation complex) forms. Signal is fitted with the sum of Lorentzian curves. (B) Dependence of signal intensity on the field gradient

1

Here E₀ is the amplitude at zero gradient, γ is the proton gyromagnetic ratio, δ=2.0 ms, δ₂=1.12 ms, g = g₁-12.82 Gs/cm. The error of D was determined as the standard deviation of this approximation.

Quantum chemical calculations

Geometry optimization was performed as follows: three-dimensional structures generated in ChemSketch were preliminarily optimized at BP86 level with def2-SVP basis set, using COSMO solvent model for EtOH solvent including D4 dispersion correction. The resulting structures were further optimized with the B3LYP/def2-TZVP using the following keywords as solvent model and correction as well: COSMO EtOH, D4, VERYTIGHTOPT. The B3LYP functional with def2-TZVP basis set was used because it is very standard and frequently found in literature, thus allowing us to compare data from different sources. All calculations were carried out using ORCA 4.2.1 software [30].

Monolayer experiments

The monolayer properties of the phospholipid conjugates mixed with matrix lipid, were characterized using a Langmuir-type film balance designed to measure the surface pressure (π) as a function of cross-sectional molecular area (A), MicroTrough XS (Kibron inc, Finland).

Lipid monolayers were formed by spreading (15 µl aliquots) mixtures made from stock solutions dissolved in chloroform-methanol (5:1). Total lipid concentration was 0.5 mg/ml. The total volume of the buffer was 75 ml and the initial area of the monolayer before compression was 110 cm². After spreading on the subphase surface, lipid films were compressed at a rate of ⩽4 Å2/molecule/min after a delay period of 30 min. Subphase was produced using water previously purified by two sequential distillations. The film balance was housed in an isolated box. The PhCs under study were azo-derivatives. They could perform trans to cis isomerisation upon excitation with UV-light. To keep PhC solely in trans form the monolayer was illuminated with blue light (480 nm). All experiments were performed in triplicate.

Analyses of monolayer isotherms

Monolayer compressibilities were obtained numerically from π − A isotherms through the following equation:

2

where A is the area per molecule at the indicated surface pressure (π). We expressed our data as the reciprocal of CS, defined as the surface compressional modulus (CS^− 1). High CS^− 1 values correspond to low lateral elasticity among packed lipids forming the monolayer. CS^− 1 values are known to be especially sensitive to phosphatidylcholine acyl structure.

The total free energy of mixing lipids in molar fractions X1 and X2 were determined as follows:

3

where.

4

and.

5

We calculated ∆G^mix (J/mol) at π = 20 mN/m.

Results and discussion

PhC used in the study

PhC used in the study are depicted in Fig. 1B. These are phosphatidylcholines which bear an azo-benzene derivative incorporated inside hydrophobic tail in the sn-2 position. tA-PC and tB-PC are derivatives of indane hydrocarbon. They differ in the respective orientation of the indane ring and the azo-group: tA-PC bears 6-(4-hydroxyphenyl) azo indane, tB-PC bears 4-(4-hydroxyphenyl) azo indane. The az-PC bears 4-hydroxyazobenzene. The synthesis and properties of azo-indanes in comparison with hydroxyazobenzene was reported in [28]. The synthesis pathway of lipid derivatives tA-PC, tB-PC and az-PC is shown in Fig. 2.

Fig. 2.

Synthesis of the compounds. Top. Molecular motors used as starting compounds: indane derivative type A (tA, left), indane derivative type B (tB, center) and azo-benzene (az, right). Bottom. General scheme of PhC synthesis

Detailed structures of fragments used in this study are depicted in Fig. 1C. These assignments were used in the NMR and quantum chemical calculations. The choline moiety has Ch_Me, Ch_α and Ch_β groups. The Ch_Me is a set of three CH₃ groups attached to the N atom. Ch_α and Ch_β are CH₂ groups located between N and O atoms of the choline. Aromatic moiety has four Ar groups, Ar₁ - Ar₄, two nitrogen groups N₁ and N₂ and two aromatic tail groups T_o and T_α. Aliphatic tail group T has sequential groups T_α, T_β, Tγ and terminal T_Me group.

Partial distortions observed in the NMR spectra

As depicted in Fig. 3, the ¹H NMR spectra of PhCs reveal unusual distortions of several signals. The distortions are observed in the forms of splitting and broadening. To be more specific, signals from phenolic ring of conjugated moiety Ar₂ and Ar₃, and choline protons Ch_α and Ch_Me are distorted (Fig. 3). Signals from other groups are not distorted. The character and power of the distortion depends on the structure of conjugated moiety. The most pronounced distortion is observed in the tA-PC spectra which shows splitting of signals of both aromatic and cholinic protons. The Ch_Me signal is divided into two peaks with almost equal intensity. On the other hand, the tB-PC shows mostly broadening of Ch_α signal, a slight broadening of Ar₂ and Ar₃ signals and splitting of Ch_Me signal into two signals of unequal intensity can also be clearly noticed. Notably, the az-PC has a spectrum with almost no distortions, except for Ch_Me group which is splitted into two signals of unequal intensity (Fig. 3).

Fig. 3.

Top. 1H NMR Spectra of PC-conjugate tA-PC. Distorted regions are highlighted. Arrows point to zoomed regions and their annotations. Bottom. Zoomed regions of ¹H NMR spectra of tA-PC and corresponding regions of tB-PC and az-PC. Ar₂, Ar₃, Ch_α, Ch_Me signals show splitting, which could not be predicted from molecular structure. Others signals do not show additional splitting, see for example T_γ, T_Me signals

Splitting of Ch_Me signal in the NMR spectra and failure of the monomer/dimer assumption

The distortions on NMR spectra are believed to be related to the molecular structure of the compounds. This let us to a deeper investigation of the signals of the Ch_Me group which are observed around 3.2 ppm region. As a consequence of the free rotation of the group, all nine protons are equivalent, resulting in a singlet signal with an integral value of nine. Concurrently, the ¹H spectra of PC-conjugates (Fig. 3) exhibit the splitting of the singlet into two signals, 7.2, 5.5, 7 Hz apart, for az-PC, tB-PC, and tA-PC, respectively (Fig. 3, inset Ch_Me). The peak ratios are 1:5.06, 1:2.73, and 1.05:1 for 4-az-PC, tB-PC, and tA-PC, respectively.

First, we had put forth the hypothesis that lipids were capable of forming dimers and thus, could exist in solution as a combination of dimeric and monomeric molecular forms. Thus, the observed splitting in Ch_Me signal was believed to be caused by the coexistence of monomers and dimers. To test this assumption, the diffusion rates of the signals were measured (see Fig. 4A, B). We employed lyso-phosphatidylcholine (lyso-PC) as a reference molecule. This compound possesses a single hydrophobic tail and no aromatic conjugated part, and it is known to be monomeric. Our findings revealed that the singlets corresponded to molecular forms exhibiting a similar diffusion coefficient (Fig. 4B). Namely, molecular form with Ch_Me peak at 3.195 ppm had diffusion coefficient of (4.41 ± 0.01)*10^− 6 cm²/sec; molecular form with Ch_Me peak at 3.205 had a diffusion coefficient of (4.49 ± 0.01)*10^− 6 cm²/sec and lyso-PC had diffusion coefficient of (4.75 ± 0.04)*10^− 6 cm²/sec. Monomer and dimer should have had very different diffusion coefficients but the measured diffusion coefficients were very close to each other. According to the Einstein-Stokes equation, the diffusion coefficient D is inversely proportional to the hydrodynamic radius of the diffusing particle. Even if we had approximated the monomer and dimer as spheres of the same density, which is the most compact approximation, the diffusion coefficient should have decreased at least by . However, the alteration of D value between different peaks did not exceed 9% and was likely the result of imperfect shape of NMR signals or overlapping with some minor signal. Thus, we concluded that the molecules in solution should exist in the form of monomers. To reveal whether NMR distortions could have an origin in the intramolecular interactions, we performed NOESY experiments.

NOESY experiments reveal internal complex formation

The NOESY spectra of the tA-PC is depicted in Fig. 5. The NOESY spectra of tB-PC is presented in supplementary materials. In the NOESY spectra of tA-PC and tB-PC, a cross-correlation of the signals at 3.21 ppm (CH_Me) and 7.88 ppm (Ar₃) is evident, which suggests the spatial proximity of the methyl groups of choline and the ortho-protons of the alkoxyphenol ring. This phenomenon may be attributed to the intramolecular π-cation interaction. The ring currents of the aromatic system result in a shift of the signals of the protons in close proximity to it towards a strong magnetic field.

Fig. 5.

(A) NOESY NMR spectra of tA-PC. (B) The inset shows the cross-peak for choline Ch_Me group and phenolic Ar₃ group

Thus, we refer the ratios of Ch_Me signal intensities below as π/N ratios.

The π/N ratios (Fig. 3) indicate that the interaction is most strongly manifested for tA-PC and weakest for az-PC. To investigate the structure of complexes we performed quantum chemical calculations.

3D structures of complexes by quantum chemical optimization

First, we obtained structures of tA-PC, tB-PC and az-PC with and without internal π-cation complexes formed. These are shown in Fig. 6B and A, respectively. The initial geometry for quantum chemical optimization was obtained by manually rotating the molecular groups to place the Ch_Me group close to the Ar₃ group. This was done because conformation generation using openBabel software does not yield a conformer with Ch_Me and Ar₃ groups close to each other.

Fig. 6.

Geometries of tA-PC, tB-PC and az-PC without (A) and with (B) π-cation complexes formed. The structures were obtained by quantum chemistry calculations at B3LYP level of theory. Choline and aromatic groups are highlighted by stick representation. Structures of tA-PC, tB-PC and az-PC in xyz format and z-matrices for the optimized geometries are presented in supplementary materials

The optimized geometries of tA-PC, tB-PC and az-PC were predicted to be different in the relative orientation of the Ch_Me and Ar₃ groups. In the case of tA-PC, those groups were very close to each other. tB-PC had Ch_Me groups shifted towards the Ar₂ group. The Ch_Me of the az-PC was located near the azo N₁ and N₂ atoms rather than near the Ar₂ or Ar₃ groups.

The geometry data support NMR data qualitatively. The proximity of Ch_Me and Ar₃ groups inside the π-cation complex corresponds to the π/N ratios of the Ch_Me NMR signal. The closer Ch_Me and Ar₃ groups are to each other the higher the peak ratio is.

π-cation complex formation originates from electrostatic interactions between a cation and p-orbitals. We also calculated Mulliken charges for atoms involved in the interaction and tried to figure out a relationship between the charges and the π/N ratios of the Ch_Me NMR signal. The result is depicted in Fig. 7. It is clear that Mulliken charges are either close to each other or are within an acceptable error range for all compounds. π/N ratio does not depend on Mulliken charges in the non-complexed conformations of tA-PC, tB-PC and az-PC. The same is true for complexed conformations with an exception of Ch_Me protons (Fig. 7B). These have slightly increased charges in the case of az-PC. None the less, we cannot pronounce any hypothesis which connects charges on atoms with π/N ratio. The supplementary material contains the HOMO and HOMO-1 orbitals of the compounds. Again, there is no obvious relationship between the shape of the π orbitals and the π/N ratio.

Fig. 7.

Calculated charges of ChMe and Ar3 groups vs. experimental π/N ratio. (A) Charges were calculated for structures without internal π-cation complex. (B) Charges were calculated for structures with internal π-cation complex. Mean values and standard deviations are presented for all atoms in each group

With electrostatic interactions being sorted out, we propose the following explanation of the difference in π-complex formation in tA-PC, tB-PC and az-PC: The probability of π-cation complex formation depends on the conformational freedom of the linker between the lipid backbone and the aromatic moiety. Moreover, predefined orientation of sp-2 and sp-3 atoms in the indane ring affects the probability. tA-PC and tB-PC have a linker more rotationally restricted than az-PC due to their structures. The az-PC has 2 CH₂ groups while tA-PC and tB-PC have only one. tA-PC and tB-PC differ from each other by orientation of the linker with respect to Ar₃ and Ch_Me groups.

To test the above hypothesis, we synthesized PhCs with long linkers. In this case, the linkers were 11 carbon atoms long. Since chain elongation leads to the increase in the number of possible conformers [31], we were suspecting that the probability of the π-cation complex formation would be reduced for all compounds. In fact, we observed no splitting or other distortions in the NMR spectra of the compounds (see supplementary materials) (see Fig. 8).

Fig. 8.

Structures of long tail derivatives. Synthesis is to be published elsewhere

PhC effect on lipid packing

The monolayer properties of the phospholipid conjugates mixed with lipid were characterized using a Langmuir-type film balance. The data in Fig. 9 are presented as reciprocal of CS– the surface compressional modulus (CS^− 1) versus pressure. High CS^− 1 values correspond to low lateral elasticity among packed lipids forming the monolayer.

Fig. 9.

The compressivity plots. The compressivity was calculated from the raw experimental data. The experimental error was calculated as the standard deviation over 100 sequential data points. The plots show the compressivity smoothed by the moving average with a window size of 100 points. (A) Experimental error in compressivity determination for two independent runs on tB-PC. (B) Compressivity (black - DOPC, green - tB-PC, orange − 30% tB-PC in DOPC). (C) Compressivity (black - DOPC, green - tA-PC, orange − 30% tA-PC in DOPC). (D) Compressivity (black - DOPC, green - az-PC, orange − 30% az-PC in DOPC)

Figure 9 shows compressivity plots of the lipid derivatives tA-PC, tB-PC and az-PC and their mixtures with DOPC (30/70). Pure lipid derivatives and DOPC lipid have different compressivity plots. This corresponds to different molecular structures of the derivatives. At the same time, the compressivity plots of pure DOPC and DOPC/lipid derivative mixtures do not differ significantly from each other. The latter may be due to a relatively good mixing of the compounds. To better assess the interactions between the derivatives and DOPC, we calculated the total free energy of mixing (∆G_mix). This parameter is often used to get a better idea of the nature of the interactions between lipids. ∆G_mix<0 indicates good miscibility while ∆G_mix>0 indicates repulsive interactions between two compounds. (see [32–34]). The ∆G_mix for lipid derivatives are presented in Table 1.

Table 1.

∆G_mix of the lipid derivatives

tA-PC (J/mol)	tB-PC (J/mol)	az-PC (J/mol)
-520.5	-220.7	-475.5

For all derivatives, ∆G_mix is negative, indicating good lipid miscibility. More interestingly, different derivatives have different π/N ratios, but the compressivity plots of mixed monolayers look almost the same and do not differ significantly from the pure DOPC plot. We conclude that the formation of intramolecular π-cation complexes does not affect the compressibility plots.

Biological relevance

Interactions between aromatic rings and positively charged groups (π-cation interactions) are important in structural biology. These interactions are found in many protein structures and thus, play a big role in how proteins fold and how they recognize other molecules [35]. In the study of lipid membranes, these interactions affect how the membranes interact with proteins. Integrated membrane proteins have aromatic amino acids, such as tryptophan, located at the polar head region of the lipid bilayer [36]. Interfacial membrane proteins, such as phospholipases C, can bind more easily to membrane lipids through tyrosines, which form stable interactions with choline groups of lipid molecules [37]. At the same time, π-cation interactions are much less studied than hydrogen bonds and π–stacking. In the current study, we have shown that phospholipid conjugates bearing aromatic moieties are also capable of forming π-cation complexes with positively charged choline groups. The formation of such complexes does not significantly alter lipid packing in monolayers. We suggest that other types of lipid aggregates, such as liposomes and micelles, will behave in the same way.

Conclusion

Herein, we report the synthesis of three new phospholipid conjugates. Using NMR spectroscopy and quantum chemical calculations which were found to be in well agreement with each other, we have shown that phospholipid conjugates can form intramolecular π-cation complexes between quaternary ammonium group of the phosphatidylcholine and aromatic ring of the conjugated moiety. The approach to avoid the π-cation complex formation is to place relatively long linkers between the lipid and the conjugated moiety.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

IAB - conception and design of the work, interpretation of data, writing the manuscript. VPS, EFK, KIM - the acquisition, analysis, and interpretation of data, VPS - chemical synthesis, EFK - NMR experiments, KIM -monolayer experiments. AE - interpretation of data, revision of the manuscript.

Funding

This work was supported by the Ministry of Science and Higher Education of the Russian Federation (theme no. 122011300058-3 (beforee 01.01.2025), 125012000470-0 (number is changed from 01.01.2025).

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations

Ethical approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.