Defense Date


Document Type


Degree Name

Doctor of Philosophy



First Advisor

Sidorov Vladimir


A number of purification techniques such as adsorption, centrifugation, chromatography, extraction, distillation, filtration, precipitation etc. to name a few, are commonly used for the purification. However, due to a constant need of purification of even more complex crude mixtures, be it reaction mixtures, various proteins and DNA from cell extracts or medicinal compounds and fragrances from plant extracts, scientists needed to consistently develop new and improved techniques of purification. One such great advancement in the field of chromatography was the development of affinity chromatography. It revolutionized the field of bioseparations technology, and the dream of single stage purification of complex biological substrates such as proteins, enzymes, co-enzymes, DNA, RNA etc. was realized. Development of various precipitation techniques involving phase tags, acid-base induced precipitation, host-guest interactions, chemoselective precipitation etc. further simplified purification procedures for few substrates. However, only a few examples of chemoselective precipitation are known in literature.1, 2 In 2005, our group synthesized a cyclen-based receptor.9 This receptor showed very strong and very specific affinity for the pyrene based dyes (HPTS, APTS, PTA) under physiological conditions, similar to that of the natural receptors for their ligands. Upon interaction with the dye, this artificial receptor formed a low solubility complex which had micromolar stability. Various structure and activity studies established the essentiality of the macrocyclic structure of the receptor for the dye recognition. We further decided to study the effect of various substituents on the aromatic ring on the binding and quenching affinity of the receptor. A series of receptors bearing electron-withdrawing and electron-donating substituents were synthesized (receptor 4-6). We also synthesized a receptor having aliphatic groups instead of aromatic groups attached to the thiourea group (receptor 7) as well as a receptor lacking thiourea linkage (receptor 8). The results of fluorometric titrations of these receptors were consistent with the previous structure and activity studies, as the receptors having electron withdrawing nitro- and bromo substituents (receptor 4 and 6 respectively) on the aromatic groups (have increased π-stacking) were better receptor than cyclen 1. Interestingly, the receptor 5, bearing an electron-donating methoxy groups on the aromatic ring, has also shown an increase in the affinity towards HPTS. We speculated that this increased affinity could be due to the higher affinity of receptor 5 towards Na+ which was essential for the proper arrangement of the receptor binding arms. The receptor with the aliphatic chain (receptor 7) showed a complicated complexation process and showed variable fluorescence quenching at various receptor concentrations. This behaviour was probably due to the formation of complexes of a various stochiometries. Expectedly, the receptor lacking thiourea group (receptor 8) did not show any interaction.Since this receptor-dye pair formed a low solubility complex of micromolar stability upon interaction, we hypothesized that this receptor-dye pair can be used in the selective separation of substrates by chemoselective precipitation. We reasoned that if the substrate of interest can be attached to the dye, this dye-substrate conjugate then can be selectively precipitated from the reaction mixture upon addition of the receptor. We tried our concept for the separation of lactose from lactose/sucrose mixture. Being a reducing sugar, lactose was labeled with APTS dye, whereas sucrose that lacks the reducing end, remained unmodified. Application of the cyclen receptor to this mixture resulted in precipitation of APTS-lactose conjugate, leaving sucrose as the only component in the solution.10 By coupling APTS dye with lactose via an imine bond, we were able to successfully isolate unmodified lactose from the conjugate by subsequent hydrolysis of the conjugate. Next, we decided to use this dye-receptor pair in affinity chromatography. We hypothesized that if the receptor can be immobilized on some solid support then it can be specifically used as an affinity support for the substrates which are attached to the pyrene-based dyes. A new receptor with a functional group on the cyclen core was needed to immobilize the receptor on the solid support. The receptor 11 having an aromatic primary amine group was synthesized. This receptor was successfully immobilized on the NHS activated agarose resin. A series of fluorometric experiments were performed to determine the specificity and binding ability of the affinity support towards the pyrene dyes. Initial results of these experiments indicated that the affinity support was very effective as indicated by almost 95% fluorescence quench of a solution of 50 nM HPTS and affinity resin. This work has opened several new venues of research, and the applicability of this dye-receptor pair in the fields of biochemistry, pharmacology, cell biology etc. will be explored.


© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

December 2011

Included in

Chemistry Commons