The synthesis of 8-(6-aminohexyl)-amino-GMP and its applications as a general ligand in affinity chromatography.

The steady-state kinetic behaviors of the five rabbit adrenal norepinephrine N-methyl transferase isozymes have been compared with particular reference to substrate inhibition patterns. Four distinct substrate inhibition patterns were observed. The E-1 isozyme was not subject to inhibition by either substrate, while the E-2 isozyme was inhibited by both substrates. The E-3 and E-4 isozymes were inhibited by norepinephrine only, while E-5 is inhibited only by S-adenosylmethionine. The substrate inhibition constants were sufficiently small in relation to the Michaelis constants to make substrate inhibition an important factor in regulation of activities of the isozymes.     

Purification of malic enzyme by affinity chromatography on immobilized N6-(6-aminohexyl)-adenosine 2',5'-bisphosphate.

Sensitive, rapid, and quantitative methods have been devised for the assay of cellulases and dextranases through the synthesis of two chemically modified carboxymethyl cellulose substrates. One contains a trinitrophenyl group as chromophore. The other contains a fluorescent fluorescamine group. The soluble hydrolytic products in the filtrate released from the substrates by cellulase are thus monitored either spectrophotometrically (for trinitrophenyl group) or spectrofluorometrically (for fluorescamine). The same principle has been applied to the determination of dextranases by utilizing chemically modified Sephadex G-200 containing either group as deseribed above for carboxymethyl cellulose. The methods are sensitive to about 5 μg of enzyme for trinitrophenyl-containing substrates, while the use of fluorescamine-containing substrates is about tenfold more sensitive.     

Preparation of N6-[N-(6-aminohexyl) carbamoyl]-adenine nucleotides and their application to coenzymically active immobilized ADP and ATP, and affinity adsorbents.

Reaction of ADP with hexamethylene diisocyanate in hexamethylphosphoramide followed by treatment in an acidic medium afforded three new adenine nucleotide analogues, N6-[N-(6-aminohexyl)carbamoyl]-ADP, N6-[N-(6-aminohexyl)carbamoyl]-ATP, and N6-[N-(6-aminohexyl)carbamoyl]-AMP in yields of 13%, 12% and 17%, respectively. The occurrence of the ATP analogue may be interpreted in terms of the equilibrium, 2ADP = ATP + AMP. Coenzymic activities of the ADP analogue against acetate kinase and pyruvate kinase were 82% and 20%, respectively, relative to ADP and those of the ATP analogue against hexokinase and glycerokinase were 63% and 87%, respectively, relative to ATP. These analogues were bound to CNBr-activated soluble dextran through their terminal amino group to give an immobilized ADP and an immobilized ATP, each of which was recycled in a system comprising acetate kinase and hexokinase, and when placed in a membrane reactor together with the enzymes, functioned as an immobilized coenzyme continuously yielding glucose 6-phosphate. A series of chemically defined affinity adsorbents were obtained by coupling these analogues to CNBr-activated Sepharose, and were used to separate the enzymes in a mixture of hexokinase, pyruvate kinase, phosphoglycerate kinase, lactate dehydrogenase, and alcohol dehydrogenase.     

Proton magnetic resonance study of 8-(6-aminohexyl)-aminoadenosine 5′-monophosphate

The nucleotide 8-(6-aminohexyl)-amino adenosine 5′-monophosphate (8-AHA-AMP) has been investigated by 220-MHz proton magnetic resonance spectroscopy. The conformation and ionization state of the nucleotide have been determined. The anti-conformation about the glycosyl bond is the preferred form. The interaction between the hexyldiamino chain and the ribose moiety in this conformation gives rise to unusual ribosyl conformation results. The distribution of conformations about the glycosyl bond has little influence on the effectiveness of this nucleotide analog in the purification of dehydrogenases by affinity chromatography. The chemical shift dependence on pH has been carried out on 8-methylaminoadenosine 5′-monophosphate. The 8-aminoadenine ring is protonated at N1 (pK_α 5.0) and at N7 (pK_α 1.5) in acidic solutions. The protonation at N7 is apparently stabilized by a delocalization of charge onto the 8-amino group. The neutrality of the 8-aminoadenine ring at physiological pH is consistent with the effcient binding of the nucleotide by dehydrogenases. An improved method for the preparation of the 8-AHA-AMP is described.     

Affinity chromatography of lactic acid dehydrogenase on N-(6-aminohexyl)oxamate-sepharose

A competitive inhibitor (K_i = 10⁻⁴–10⁻⁵m) for lactic acid dehydrogenase (LDH), N-(6-aminohexyl)oxamate, was synthesized from diethyl oxalate and 1,6-diaminohexane. The affinity column prepared by attachment of this compound to CNBr-activated Sepharose 4B enabled efficient purification of LDH from the marine teleost, Fundulus heteroclitus (Lin.). A similar affinity absorbent prepared by the method of O'Carra and Barry (FEBS Lett. (1972) 21, 281) gave a less satisfactory purification because the LDH was contaminated with phosphorylase a. The reason for this contamination was traced to an incomplete acylation of amino groups on the modified Sepharose beads by the carbodiimide-mediated reaction. Immobilization of the presynthesized affinant, N-(6-aminohexyl)oxamate, assured complete absence of these interfering groups. An alternate synthetic scheme, involving an active ester of ethyl oxalate, was also found to eliminate this problem.     

NMR screening of new carbocyanine dyes as ligands for affinity chromatography

Four new carbocyanines containing symmetric and asymmetric heterocyclic moieties and N‐carboxyalkyl groups have been synthesized and characterized. The binding mechanism established between these cyanines and several proteins was evaluated using saturation transfer difference (STD) NMR. The results obtained for the different dyes revealed a specific interaction to the standard proteins lysozyme, α‐chymotrypsin, ribonuclease (RNase), bovine serum albumin (BSA), and gamma globulin. For instance, the two un‐substituted symmetrical dyes (cyanines 1 and 3) interacted preferentially through its benzopyrrole and dibenzopyrrole units with lysozyme, α‐chymotrypsin, and RNase, whereas the symmetric disulfocyanine dye (cyanine 2) bound BSA and gamma globulin through its carboxyalkyl chains. On the other hand, the asymmetric dye (cyanine 4) interacts with lysozyme and α‐chymotrypsin through benzothiazole moiety and with RNase through dibenzopyrrole unit. Thus, STD‐NMR technique was successfully used to screen cyanine–protein interactions and determine potential binding sites of the cyanines for posterior use as ligands in affinity chromatography. Copyright © 2014 John Wiley & Sons, Ltd.     

Methacryloylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of ferritin

A new metal-chelate adsorbent utilizing 2-methacryloylamidohistidine (MAH) was prepared as a metalchelating ligand. MAH was synthesized using methacryloly chloride and histidine. Monosize nanospheres with an average diameter of 450 nm were produced by emulsion polymerization of 2-hydroxyetylmethacrylate (HEMA) and MAH. Then, Fe³⁺ ions were chelated directly onto the monosize nanospheres. Mon-poly(HEMA-MAH) nanospheres were characterized by Fourier transform infrared spectroscopy, scanning electron microscopy, and elemental analysis. Fe³⁺ chelated monosize nanospheres were used in ferritin adsorption from an aqueous solution. The maximum ferritin adsorption capacity of Fe³⁺-chelated mon-poly(HEMAMAH) nanospheres was 202 mg/g at pH 4.0 in acetate buffer. The non-specific ferritin adsorption on the monpoly( HEMA-MAH) nanospheres was 20 mg/g. The adsorption behavior of ferritin could be modeled using both Langmuir and Freundlich isotherms. The adsorption capacity decreased with increasing ionic strength of the binding buffer. High desorption ratios (> 95% of the adsorbed ferritin) were achieved with 1.0 M NaCl at pH 7.0. Ferritin could be repeatedly adsorbed and desorbed with the Fe³⁺-chelated mon-poly(HEMA-MAH) nanospheres without significant loss of adsorption capacity.     

Design and selection of ligands for affinity chromatography

Affinity chromatography is potentially the most selective method for protein purification. The technique has the purification power to eliminate steps, increase yields and thereby improve process economics. However, it suffers from problems regarding ligand stability and cost. Some of the most recent advances in this area have explored the power of rational and combinatorial approaches for designing highly selective and stable synthetic affinity ligands. Rational molecular design techniques, which are based on the ability to combine knowledge of protein structures with defined chemical synthesis and advanced computational tools, have made rational ligand design feasible and faster. Combinatorial approaches based on peptide and nucleic acid libraries have permitted the rapid synthesis of new synthetic affinity ligands of potential use in affinity chromatography. The versatility of these approaches suggests that, in the near future, they will become the dominant methods for designing and selection of novel affinity ligands with scale-up potential.     

Design and synthesis of affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase

Arachidonic acid (AA) is converted to biologically active metabolites by different pathways, one of the most important of which is initiated by 5-lipoxygenase (5-LO). 5-Hydroxyeicosatetraenoic acid (5-HETE), although possessing only weak biological activity itself, is oxidized to 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a potent chemoattractant for eosinophils and neutrophils. Our main goal is to determine how the biosynthesis of 5-oxo-ETE is regulated and to determine its pathophysiological roles. To achieve this task, we designed and synthesized affinity chromatography ligands for the purification of 5-hydroxyeicosanoid dehydrogenase (5-HEDH), the enzyme responsible for the formation of 5-oxo-ETE.     

Bacitracin and gramicidin S as biospecific ligands for affinity chromatography of human thrombin

It was shown that the cyclic polypeptide antibiotic bacitracin is a competitive inhibitor of fibrinogen clotting by thrombin. Biospecific adsorbents for isolation of thrombin by gramicidin S and bacitracin attachment to silochrome S-80 modified by gamma-glycido-oxypropyl groups were synthesized. The thrombin yield at pH 7.2 and 8.0 was 76.5-96%, purification--6.2-11.6-fold, specific coagulating activity--940-1750 NIH u./mg protein. At pH 6.1 the enzyme does not practically bind to the adsorbents. In all probability, the differences in thrombin binding are due to conformational changes in the enzyme molecule, when pH changes from 6.1 to 7.2. Possible application of the synthesized adsorbents for obtaining laboratory and commercial preparations of thrombin and their perspective use for purification of other blood plasma serine proteinases possessing a narrow specificity are discussed.     

1-(2-Aminoethyl)-3-(arylsulfonyl)-1H-indoles as novel 5-HT6 receptor ligands

Novel 1-(2-aminoethyl)-3-(arylsulfonyl)-1H-indoles were prepared. Binding assays indicated they are 5-HT(6) receptor ligands, among which N,N-dimethyl-N-(2-[3-(1-naphthylsulfonyl)-1H-indol-1-yl]ethyl)amine 8t and N-methyl-N-(2-[3-(1-naphthylsulfonyl)-1H-indol-1-yl]ethyl)amine 8u showed high affinity for 5-HT(6) receptors with K(i)=3.7 and 5.7 nM, respectively.     

N4-(6-aminohexyl)cytidine and -deoxycytidine nucleotides can be used to label DNA

Bisulfite is known to catalyze transamination between cytidine derivatives and amines. Using 1,6-diaminohexane we describe the synthesis and recovery of the 5'-triphosphates of N4-(6-aminohexyl)cytidine and -deoxycytidine (dahCTP). Both may be incorporated into DNA by nick translation with DNA polymerase I of Escherichia coli to provide reactive sites for the attachment of immunological or other labels. Biotinyl dahCTP is actively incorporated into DNA by the same system and can be detected by the binding of streptavidin complexed to an indicator enzyme such as acid phosphatase. Such labeled DNA is a suitable nonradioactive probe for detection of related sequences by hydridization.     

Methacrylamidohistidine in affinity ligands for immobilized metal-ion affinity chromatography of human serum albumin

Different biologands carrying synthetic adsorbents have been reported in the literature for protein separation. We have developed a novel and new approach to obtain high protein adsorption capacity utilizing 2-methacrylamidohistidine (MAH) as a bioligand. MAH was synthesized by reacting methacrylochloride and histidine. Spherical beads with an average size of 150–200 μm were obtained by the radical suspension polymerization of MAH and 2-hydroxyethyl-methacrylate (HEMA) conducted in an aqueous dispersion medium. p(HEMA-co-MAH) beads had a specific surface area of 17.6 m²/g. Synthesized MAH monomer was characterized by NMR. p(HEMA-co-MAH) beads were characterized by swelling test, FTIR and elemental analysis. Then, Cu(II) ions were incorporated onto the beads and Cu(II) loading was found to be 0.96 mmol/g. These affinity beads with a swelling ratio of 65%, and containing 1.6 mmol. MAH/g were used in the adsorption/desorption of human serum albumin (HSA) from both aqueous solutions and human serum. The adsorption of HSA onto p(HEMA-co-MAH) was low (8.8 mg/g). Cu(II) chelation onto the beads significantly increased the HSA adsorption (56.3 mg/g). The maximum HSA adsorption was observed at pH 3.0 Higher HSA adsorption was observed from human plasma (94.6 mg HSA/g). Adsorption of other serum proteins were obtained as 3.7 mg/g for fibrinogen and 8.5 mg/g for γ-globulin. The total protein adsorption was determined as 107.1 mg/g. Desorption of HSA was obtained using 0.1 M Tris/HCl buffer containing 0.5M NaSCN. High desorption ratios (up to 98% of the adsorbed HSA) were observed. It was possible to reuse Cu(II) chelated-p(HEMA-co-MAH) beads without significant decreases in the adsorption capacities.     

Identification of 9-fluoro substituted (-)-cytisine derivatives as ligands with high affinity for nicotinic receptors

(-)-9-Fluorocytisine, (-)-9-methylcytisine and (-)-9-trifluoromethylcytisine were synthesized from the natural product (-)-cytisine. 9-Methyl and 9-trifluoromethyl cytisines display a remarkable affinity at the α(4)β(2) nicotinic receptor subtype (0.2 nM) with a high selectivity versus the α(7) nAChR subtype. Comparison of the affinity values suggests that the size of the substituent at the 9 position of (-)-cytisine seems more important than electronic factors for efficient binding and selectivity at α(4)β(2) nAChRs.