Boronate affinity gels

A polyacrylamide-boronate affinity gel, Affi-gel 601 , has been used for the separation of cyclic adenosine monophosphate, cyclic guanosine monophosphate, and cyclic cytidine monophosphate from their corresponding 5-nucleotides and nucleosides. A simple direct assay of 3 5 -cyclic nucleotide phosphodiesterase activity has been developed, based on the use of the gel. 

We also found that boronate affinity gels failed to retain nicotinamide-photolabeled fragment A or its peptides. In contrast, radiolabeled peptides from fragment A irradiated in the presence of phosphate- or adenine-labeled NAD bound quantitatively to such resins and could be eluted at pH 5.0. Since boronate gels bind compounds containing unblocked vicinal hydroxyls, we concluded that neither of the ribosyl moieties of NAD was present (at least in unmodified form) in the nicotinamide-labeled photoproduct. 

E. H. Pfadenhauer, S. D. Tong. Determination of inosine and adenosine in human plasma using high performance liquid chromatography and boronate affinity gel. J. Chromatogr. 

To reduce the binding pH, Scouten and co-workers first synthesized a novel boronate affinity ligand, catechol [2-(diethylamino)carbonyl-4-bromomethyljphenylboronate, which contains intramolecular B-O coordination. Then, this ligand was coupled to sulfhydryl cellulose. The novel boronate affinity gel bound the glycoprotein horseradish peroxidase (HRP) at neutral condition (pH 7.0), at which the immobilized enzyme retain 90.12% of its original activity. 

X. C. Liu and W. H. Scouten, Studies on oriented and reversible immobilization of glycoprotein using novel boronate affinity gel,/. Mol Recognita 1996, 9(5-6), 462-467. 

The use of thiopropyl-Sepharose and boronic acid-agarose is an example of covalent chromatography, since relatively strong but reversible covalent bonds are formed between the affinity gel and specific macromolecules. 

Fig. 1 Immunoblots of acid extracts of nuclear preparations from mouse epidermal cells JB6 (clone 41) which had been treated with active oxygen produced by xanthine/xanthine oxidase (50 pg/ml xanthine plus 5 Xg/ml xanthine oxidase) for 30 min or 5 pg/ml MNNG for 20 min. The extracts were purified on a boronate affinity column and the proteins separated on 15% polyacrylamide gels (34). A polyclonal rabbit antibody against histone H3 (37) was used for the immunoblot and the blot was reacted with [i Sjj-iabeled donkey anti-rabbit IgG. Densitometer scannings are shown at the right side of the autoradiogram and were obtained with a Zeineh "soft laser" scanning densitometer.

These gels are based on immobilised alkyl boronic acids. They have a selective affinity for 1,2- or 1,3-diol groupings such as those found in catechol-containing molecules such as dopamine and in sugars or glycosides. 

Lyon and Phelps [57] have evaluated various glycan-substituted AH-Sepharose gels to be used for affinity chromatography. Srivastava and Farooqui reported the elution of hyaluronidase from heparin-Sepharose columns using heparin or hyaluronan solutions as eluent [58]. Alternatively, affinity chromatography of hyaluronidase has been performed using concavalin A-Sepharose [58] or Matrex gel phenyl boronate [59] columns. 

Novel tetrahedral boronate complexes have been prepared as potential ligands for affinity chromatography either from the amination (LiN(TMS)2) of a-haloalkylboronates followed by A-acetyl-ation (i.e., (132)) or, alternatively, from the a-iodo derivatives and acetamidine (i.e., (133)) (Scheme 17). The 1,3,2-dioxaborinanes proved to be less stable than the pinanediol derivatives to silica gel. "B NMR as well as other data confirmed their chelated nature (e.g., (132) d 6.8) <92JOM(43i)255>. Spiroborates (e.g. (134)) have also been prepared from boric acid, diols, and enolizable 1,3-dicarbonyl compounds <86JPR755>. Vinylzirconocenes (135) are converted to vinylboronates (136) with chloroboranes <910M3777>. The hydrozirconation of 5-vinylboronates (137) produces the mixed... 

Synthesis and purification of 2 dNAD+. We used p-NMN+ adenyl transferase to synthesize [adenylate- P]2 dNAD+ from [a-3 ]2 dATP. Residual 2 dATP was removed by affinity chromatography on a boronate gel as described elsewhere (14). The elimination of contaminating P-NMN+ (Fig. lA), which also boimd to the boronate gel, was facilitated by its conversion to nicotinamide ribose by treatment with bacterial alkaline phosphatase (Fig. IB). Figure 1C shows that following preparative HPLC, a single peak corresponding to pure 2 dNAD was detected. It is important to note that all of the 32p radiolabel co-eluated with this peak. 

Kanekiyo explored linear polymer imprinting (1-D), the formation of a 1 1 dimer between anionic polymers and cationic polymers was utilised. The polyanion fragment contains boronic acid units to complex with AMP (Scheme 49). On removal of AMP from the precipitated polyion complex a cleft that has the memory for the AMP template is created." It was demonstrated that this cleft shows high affinity with AMP and the precipitate (gel) displays reversible swelling-shrinking in response to the binding of AMP. When this gel is deposited on a QCM resonator, it responds to small changes in the concentration of AMP." ... 

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