[1H] NMR Interactions Probe
Above CMC Methylated CD 9 below CMC
Lollipop, Cup and Ball and then … Leucinolipidyl-Cyclodextrins
Christophe Fajolles1*, Céline Hocquelet1,Véronique Bonnet2, Angelina Angelova3, Florence Djedaïni-Pilard2,Sylviane Lesieur3,Bruno Perly1, and Laurent Mauclaire1
3- Equipe Physico-chimie des Systèmes Polyphasés, UMR CNRS 8612, Université Paris-Sud, F-92296 Châtenay-Malabry Cedex, France
2- Laboratoire des Glucides UMR CNRS 6219, Université Picardie Jules Verne, 33 rue St-Leu, F-80039 Amiens, France
1- CEA, DSM/DRECAM/SCM/LIONS, CEA Saclay, F-91191 Gif Sur Yvette, France
- To study more precisely how these new amphiphilic CDs behave in aqueous solution, NMR was the tool of choice. Relative variation of protons chemicals shifts and changes in dipolar correlation are some manifestations of changes in close environment of these observed nuclei. Inclusion phenomena of cyclodextrins are typical applications. It seemed interesting to study the influence of concentration and temperature on [1H] NMR spectra and on dipolar correlations as obtained by t-ROESY experiments.
Native CD derivative 8 & dilution effect
8 R=H : N-Dodecyl-Nα -(6I-amidosuccinyl-61-deoxy- cyclomaltoheptaose)-L-Leucine (X4)
9 R=CH3 : N-Dodecyl-Nα-(6I-amidosuccinyl-6I-deoxy-2I, 3I- di-O-methyl-hexakis (2II VII, 3II-VII, 6II-VII-tri-O- methyl) cyclomaltoheptaose)-L-Leucine (X2)
- Samples were prepared for 8 and 9 in D2O above CMC (CAC ?) then diluted to get under CMC samples. 9 was 5mM and 1µM. 8 was prepared as a saturated solution at RT then diluted 100 times.
- Solubility and CMC for 8 were so low that 1H spectra were recorded with HDO presaturation. T-ROESY (ref: JACS, 1992,114,3157, TL Huang and AJ Shaka) would have implied a too long spectrometer immobilization and was not recorded under for high dilution under CMC.
- To get comparable spectra all the FID were treated using the same parameters. Calibration was relative to higher field methyl group 5,5’ of the Leucine lateral chain. T-ROESY spectra were treated with symetrisation.
- All the spectra were recorded on a DRX 500 Bruker at 298 K (or otherwise stated) with either BBI 3 axis, or TXI. The solutions were in D2O (Euriso-top). Cyclodextrines are kind gifts of Roquette for β-CD.
Methylated CD derivative 9 & dilution & temperature effect
- Since the 70’s and J.M. Lehn’s « bouquet » cyclodextrin (CD) derivatives, amphiphilic cyclodextrins have attracted much attention. Indeed, a possible approach, as regards pharmaceutical applications, would be to combine the size specificity of CDs with the use of the transport properties of organized structures such as liposomes, vesicles or micelles.
- In most cases, the CDs have been persubstituted at one or more hydroxyl group leading to steric hindrance effects. Under these conditions, the inclusion properties of the cavity could be partially lost. Poor solubility and lack of biocompatibility are some other drawbacks added to long synthesis and purification difficulties.
- Since the early 90’s, monosubstituted derivatives opened a new promising direction. But in order to avoid the autoinclusion observed in the case of the so called “lollipop” molecules, the linear fatty chains had to be substituted by more complex substituants such as cholesteryl or phospholipidyl derivatives.
- This latest class of amphiphilic cyclodextrins displayed self-organisation properties close to phospholipids and further retained the ability of the CD moiety in terms of inclusion of guest molecules. Moreover some of these molecules presented a considerable solubilization power for model membranes (J.incl.Ph., 2002, 44, 317-322)
- To overcome the troubles encountered with phospholipidyl-cyclodextrins, namely high cost, tedious synthesis and rather poor stability, a new class of amphiphilic CDs was designed.When compared to the phospholipidyl-CDs, the peptidolipidyl-CDs differ by the fact that the chiral glycero-phosphate moiety is replaced by an amino-acid providing the following advantages :
- high versatility still retaining the chiral nature at very low cost,
- possibility to play with the length and the number of fatty chains by selecting the amino acid.
9 temperature effect (K)
Native CD 8
- A comparison can be made with the use of quadrupolar split of the same methyl groups when these are deuterated and studied in [2H] NMR. The presence in the same compound of 2 types of methyl groups with different behaviours and close chemical shift can give the same kind of information without any labelling.
- These results can be interpreted as an effect of the aggregative process. Below the CMC, auto-inclusion (dimer or monomer) is preferred. The cyclodextrin cavity is occupied on a greater extend. Above the CMC, the fatty chains prefer the micellar aggregation to inclusion inside the cavity. The aggregation alters the isotropy of the system and then the resolution of the signal as seen on the spectrum
- This result is well illustrated par the effect of temperature on the [1H] spectra of 9 at a millimolar concentration, so well above CMC. Firstly, the quality of the signal decreases strongly at 308K, likely above the cloud point. Then, higher the temperature, higher the field for the signal of the methyl chain. The increase of energy in the system must be found in the micelles. This leads to more auto-inclusion and then a chemical shift.
- At first, auto-inclusion can be observable at all concentrations characterizing the dynamic nature of the phenomena. However above CMC, the interaction with the methyl group of the leucine side chain is more clearly seen associated with discrimination between 5 and 5’. This discrimination is stronger below CMC. The methylation of hydroxyl-6 on 9 leads to a more hindered molecule on the substituted rim of the cyclodextrin. This must enhance the inequivalence of the methyl group 5 and 5’. This effect can also be observed looking at the dipolar interaction of these methyl groups and the alpha proton of L-Leucine.
- These amphiphic cyclodextrins have to be considered as the next step after molecular “lollipop” (ref: J.Mol.struct., 1992, 273, 215-226) and “cup and ball” molecules (ref: J Perkin Trans 2, 1998, 2639-1646). Indeed the lateral chain of leucine can be compared to the BOC group of “cup and ball” molecules. Lollipop had shown very strong autoinclusion of monosubstituted cyclodextrins with linear aliphatic chains over 8 carbon atoms in the backbone. In order to prevent this phenomenon, a BOC group was added at the end of the chain to create “cup and ball” molecules. It was demonstrated that the bulky group did not penetrate deeply inside the cavity. The i-propyl group of the leucine is used as a competitor against autoinclusion of the lauric linear chain. These molecules appears thus as a hybrid concept, a Gemini amphiphilic monosubstituted cyclodextrin with an essentially chiral nature brought by the natural amino-acid and with two strongly different hydrophobic parts, a short bulky one allowing the other part to be involved in aggregation processes. This could bring out the first case of native monosubstituted β-CD with a measurable CMC.
- Studies of the interactions with phospholipids bilayers are underway.