The Investigation Of Cyclodextrin’s Non-covalent Complex By Mass Spectrometry

Non-covalent complexes have been widely used in many fields nowadays so that they attract more and more attention. They can be formed between some small molecule compounds and biological macromolecules, such as protein, polypeptide and oligonucleotide. The investigation of non-covalent complexes will not only push forward the biology and chemistry, but also be beneficial to the development of pharmaceutical industry. Thus a good investigation method is very important, in this thesis, we take the use of electrospray ionization mass spectrometry (ESI-MS), which have experienced great development in the past decades years, as well as other methods as assistant to investigate the non-covalent complexes formed between cyclodextrins and small molecules,such as antidepressant compound, tripeptides, and alkali metal ions.In first part, the non-covalent complexes of α-,β-or γ-cyclodextrin (CD) with antidepressant compound SIPI5358were investigated by electro spray ionization mass spectrometry (ESI-MS), tandem mass spectrometry (MS/MS), UV spectroscopy and fluorescence spectroscopy. The ESI-MS experimental results revealed that SIPI5358can react respectively with a-, P-, or y-CD to form complexes with different coordination numbers. Tandem mass spectra further validated the formation of the non-covalent complexes. The binding of the complexes was also confirmed by UV spectra as well as fluorescence spectra. The formation constant Kf measured by fluorescence spectroscopy experiments was3.45×103mol·L-1for the complex of SIPI5358with (3-cyclodextrinIn second part, to investigate the non-covalent interaction between α-、β-γ-cyclodextrins and peptides, a stoichiometry ofα-、β-、γ-cyclodextrins (CD) with GGG (Gly-Gly-Gly) or GFF (Gly-Phe-Phe) was mixed respectively, and then incubated at room temperature for12h to reach the equilibrium. In positive mode, the electrospray ionization mass spectrometry (ESI-MS) results indicated that α-、β-、γ--CD with GGG or GFF could form non-covalent complexes, respectively. The binding of cyclodextrins with GGG or GFF was further confirmed by collision induced dissociation (CID) in a tandem mass spectrometer. The formation constants of six complexes (GGG+CD and GFF+CD) were determined by mass spectrometric titration. The results showed the formation constants for both GGG’s and GFF’s complexes increased according to the order γ-CD、β-CD、α-CD. The formation constants Kst values for GGG complexes with α-CD、β-CD、or γ-CD are2799.96L-mol"1,2528.73L·mol-1,1697.11L·mol-1,respectively. While the formation constants Kst values for GFF complexes with α-CD、β-CD、or γ-CD are2773.94L·mol-1,2134.03L·mol-1,1330.68L·mol-1respectively. For α-CD、β-CD orγ-CD, the Kst values of GFF complexes containing aromatic group are smaller than those of GGG complexes only containing aliphatic group. The main reason is that in gas phase, with the weaken of hydrophobic force, Van der Waals force plays an important role in the conjugation process of GFF with CD, the coordinating group of GFF is still phenyl group. While in GGG’s complexes, the hydrogen bond dominates in the conjugation process. Our convincing results from the formation constants provides a new evidence, indicating that although the conformations for GFF+CD complexes change slightly when the analysts transfer from solution to gas phase, the phenyl group still takes part in coordinating.In third part, to investigate the influence factors affecting the non-covalent interaction between cyclodextrins and alkali metal ions, a stoichiometry of α,β, γ-CD with alkali metal salts including LiCl, NaCl, KCI, RbCl, CsCl were mixed in a ratio of1:10respectively, and then the solution was left to stand at room temperature for1h to reach the equilibrium. The mass spectrometric results showed that alkali metal ions can conjugate with CDs, and form non-covalent complexes of different coordination ratio. All a-, P-, y-CDs can form mono-valent complexes [CD+M]+(M represents alkali metals) in80%methanol solution. a-CD can only form bivalent complexes [CD+2M]2+with Li+and Na+. β-CD can only form complexes [CD+2M]2+with Li+, Na+and K+. However, y-CD can form [CD+2M]2+with all the five alkali metal ions including Li+, Na+, K+, Rb+and Cs+. Furthermore, some [CD+M+nMCl]+and [CD+2M+nMCl]2+(n=1,2,3...) were also detected, which indicated the higher charge complexes ions containing more alkali metal can hardly form in solution. The binding of a-, P-, and y-CD to alkali metal ions was further confirmed by collision-induced dissociation (CID) in a tandem mass spectrometer. The loss of D-glucose unit series occurred in the fragmentation process of [CD+M]+, indicating all of the complexes underwent the same dissciation reaction mechanism. For the [α-CD+2Li]2+complex, one Li+cation coordinates with four oxygen atoms from primary hydroxyl groups, the other Li+cation coordinates with oxygen atoms from glycosidic bond and primary hydroxyl.