Preparations And Studies Of Artificial Ion Channels With High Selectivity Based On Helical Structures

Natural ion channel plays an extremely important role in the normal physiological activities of life,and participates in a lot of basic physiological activities.Highly specific selectivity is an attractive feature of natural ion channels.Natural channels are not easy to purify and liable to denature in vitro,which makes it rather difficult to study the selectivity of protein ion channels directly.However,biomimetic ion channels have many advantages,such as good stability,relatively simple structure and so on.The study of artificial ion channels can help us better understand the natural protein ion channels.Artificial ion channels mainly divide into cation channels and anion channels.Most of the artificial anion channels are Cl~-channels,while the research of artificial cation channels concentrates mainly on Na~+and K~+channels with high selectivity.The research contents of this thesis focus on biomimetic cation channels.Nowadays,the research of biomimetic cation channel is not only limited to the design and synthesis of a new channel structure,but also committed to the pursuit of selectivity of biomimetic ion channel.Although the K~+/Na~+selectivity ratio of artificial K~+channels can reach about 20 at present,there is still a big gap between this selective level and natural K~+channel proteins.Therefore,how to further obtain artificial ion channels with higher selectivity has become a problem that restricts the development of the field.With the attitude of learning from nature,we carefully studied the selective filter of natural K~+channel,and found that the internal diameter of the selective filter is only 2.8? ,which is only slightly larger than the diameter of K~+.Therefore,we decided to explore the influence of the inner diameter of the channel on the ion selectivity,hoping to obtain a bionic ion channel with higher selectivity and provide a new idea for the design of highly selective ion channels.The detailed work are as follows:1.To construct highly efficient and selective supramolecular potassium channels through pore size adaptationAromatic foldamers have the advantage of easy prediction of molecular conformation.Our group has constructed a class of typical hollow helix molecules by electrostatic repulsion interaction.The pyridine pentamer reported previously is a highly selective K~+channel with pyridine oxadiazole as the repeating unit and an inner diameter of 3.8? .Consequently,in this part of the work,we replace some units of pyridine pentamer(1)with o-phenanthroline with smaller radian.Through simulation calculation,the inner diameter of the newly designed synthetic channel 2 is 2.7angstroms defined by van der Waals surfaces,which is very close to the size of K~+(2.76? ).Through the ion transport experiments and the planar lipid bilayer experiments,it is found that the K~+selective transportation capacity of 2 is higher than that of the previously published channel 1.The K~+/Na~+permeability ratio of 2 measured by the asymmetric planar lipid bilayer experiments can reach 18.2,which is the highest value measured in the field of artificial K~+channels.In addition to the ultra-high K~+selectivity,channel 2 also has a transport activity similar to that of the natural channel Gramicidin A(g A).The construction of K~+channels with high selectivity and high transport activity in this part of the work inspired us to further explore the influence of bionic ion channel aperture on selectivity.2.Construction of artificial sodium ion channel by using the restricted aperture design of the helical channelMany of the cation artificial ion channels are related to K~+channels,while the selective transport of Na~+is rarely reported.From the previous part of work,we found that the appropriate aperture will create an ultra-high selective transportation capacity.On the basis of the previous work,we continue to reduce the diameter of the spiral channel.So,in this part,we synthesized a helical polymer(HP1)with only o-phenanthroline and oxadiazole,and compared it with a pyridine-oxadiazole helical polymer(HP2)transporting K~+.The simulation calculation results show that the inner diameter of the HP1 is only 2.3? defined by van der Waals surfaces,which is just larger than the diameter of Na~+(2.04? )and at the same time smaller than that of the K~+(2.76? ).It is exciting to find that HP1 is a selective Na~+channel through ion transportation experiments,and the selective transport ratio of Na~+/K~+is 1.3.The asymmetric planar lipid bilayer experiment can also conclude that HP1 is a Na~+channel,and the Na~+/K~+selective permeability ratio is 1.9.This part of the work fully demonstrates that the aperture of 2.3? can limit the transportation of K~+.In this part,although we have obtained a channel for selective transportation of Na~+,the problem is that its transmission capacity for Na~+is only slightly better than that for K~+.3.A highly selective sodium ion channel screened from foldamers using o-phenanthroline-oxadiazole as basic elementsFew Na~+channels have been reported in the field of cationic artificial ion channels, and even fewer are highly selective ones.From the above work,we know that HP1with an inner diameter of 2.3? can selectively transport Na~+,but its maximum Na~+/K~+selectively transport ratio is only 1.3.We really want to improve its Na~+selectivity.Therefore,in this part of the work,we use the same repeating units as HP1 to design and synthesize three oligomers:o-2mer,o-3mer and o-4mer.O-2mer is an open C-shaped molecule due to its short skeleton.The skeletons of o-3mer and o-4mer are sufficient to rotate into a helical structure,so the two molecules are helical oligomers with an inner diameter of 2.3? .The single crystal analytical data of o-3mer gives sufficient molecular structure information for the three oligomers.Through ion transport experiments,we found that o-3mer and o-4mer are selective transport channels for Na~+,but to our surprise,o-2mer is a selective transport channel for Na~+.Most importantly,through ion transport experiments,it was found that the selective transport ratio of Na~+/K~+of o-3mer could reach up to 5.2.This indicates that the Na~+channel with the highest selectivity has been successfully screened out in this part of the work.4.Potassium ion transport properties of helical polymers based on o-phenanthroline-pyridine alternationIn the first part of the work we know that the aperture of 2.7? allows the helical channel to transport K~+in a highly selective manner.Based on this,we wondered whether helical polymers with the same structure would have better K~+selectivity.Therefore,in this part of the work,we synthesized helical polymer(1)with o-phenanthroline-pyridine as alternating repeating units.Disappointingly,the K~+/Na~+selective transport ratio of 1 was found to be only 1.7 by ion transport experiments.However,fluorescence titration experiments showed that 1 had almost no response to Na~+in the low concentration single helix state.Since 1 is thought to form a supramolecular channel to transport ions in the membrane,it leads us to suspect that the reason for the poor selectivity of the polymer is the insufficient length of 1.This part of the work led us to realize that the selectivity of oligomers and polymers with the same structure is also very different.The exact reason is unknown and further detailed research is needed.5.Construction and characterization of a new type of helix channel with a larger apertureThe helical molecules reported in our previous work are all folded due to electrostatic repulsion interactions.Due to the limitation of conditions,there are relatively few kinds of elements used to construct the aromatic helical foldamers.In this part of the work,we applied hydrogen bonding interactions to our helix construction system to further enrich the types of helical channels.In this part,we introduced pyridine-amide into the pyridine-oxadiazole helical polymer,constructed a new helical polymer through the synergy of electrostatic repulsion and intramolecular hydrogen bonding interactions,and studied its channel properties.The circular dichroism data shows that the new polymer can be folded into a helical conformation.Ion transport experiments and planar lipid bilayer experiments have proved that this polymer can be used as an ion channel,but unfortunately,the pore size of the channel is too large to show any selectivity.This part of the work expands the types of our helical channel structures and shows that it is feasible to introduce other interactions to construct helix channels.This strategy lays the foundation for the construction of a variety of helical ion channels,and has a profound impact on the construction of new types of helial channels.