Chemical modification of neamine.

The aminocyclitol antibiotic neamine has been modified chemically by removing one or two hydroxyl groups from the 2-deoxystreptamine moiety to give 5- and 6- deoxyneamines (5 and 10), as well as 5,6-dideoxyneamine (15). Their antimicrobial activities were determined against several microorganisms, including kanamycin-resistant strains.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36-100% amide and 0-25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Chemical modification of biocatalysts

Although several powerful methods exist for the redesign of enzyme structure and function these are typically limited to the 20 most abundant proteinogenic amino acids. The use of chemical modification overcomes this limitation to allow virtually unlimited alteration of amino acid sidechain structures. If heterogeneous mixtures of enzyme products are to be avoided, however, the required chemistry should be efficient, selective and compatible with aqueous conditions. Recent advances have been made in the modification of proteinases, aminotransferases and redox enzymes.     

Chemical modification of heparin

Heparin was modified at carboxyl groups by reaction with several pharmacologically important amino-containing compounds in aqueous medium in the presence of 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide. In dependence on the nature of the amine and the ratio of reagents, conjugates containing 36–100% amide and 0–25% isoureidocarbonyl groups were synthesized. Isoureidoarylamide groups are present, along with amide moieties, in the products of heparin modification by hydroxyl-containing aromatic amines. The conjugate of heparin with p-aminobenzoic acid contained oligomeric arylamide.     

Chemical modification of dopamine beta-hydroxylase

Dopamine beta-hydroxylase (3,4- dihydroxyphenylethylamine ,ascorbate:oxygen oxidoreductase (beta-hydroxylating), EC 1.14.17.1) is the terminal enzyme in the biosynthetic pathway of norepinephrine. Chemical modification studies of this enzyme were executed to investigate contributions of specific amino-acid side-chains to catalytic activity. Sulfhydryl reagents were precluded, since no free cysteine residue was detected upon titration of the denatured or native protein with 2-chloromercuri-4-nitrophenol. Incubation of enzyme with diazonium tetrazole caused inactivation of the protein coupled with extensive reaction of lysine and tyrosine residues. Reaction with iodoacetamide resulted in complete loss of enzymatic activity with reaction of approximately three histidine residues; methionine reaction was also observed. Modification of the enzyme using diethylpyrocarbonate resulted in complete inactivation of the enzyme, and analysis of the reacted protein indicated a loss of approx. 1.7 histidine residues per protein monomer with no tyrosine or lysine modification observed. The correlation of activity loss with histidine modification supports the view that this residue participates in the catalytic function of dopamine beta-hydroxylase.     

Chemical modification of aminocyclitol antibiotics

The aminocyclitol antibiotic neamine has been chemically modified at the hydroxyl group on C-6 of the 2-deoxystreptamine moiety. The partially acetylated neamine derivatives, 6,3′,4′-tri-O-acetyl- (3) and 5,3′,4′-tri-O-acetyl-1,3,2′,6′-tetra-N-(ethoxycarbonyl)neamine (4), were prepared by random hydrolysis of the 5,6-O-ethoxyethylidene derivative (2), followed by chromatographic purification. Condensation of 4 and 2,3,5-tri-O-benzoyl-d-ribofuranosyl chloride led to 6-O-(β-d-ribofuranosyl)neamine (7). Analogous condensation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide afforded the corresponding 6-O-(d-hexopyranosyl)neamines.     

Chemical modification of tropomyosin with NBD-chloride

Muscle tropomyosin was modified with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-chloride) at several different pH values. NBD-chloride reacts specifically with SH residue at neutral pH but it reacts with both SH residue and amino residues at alkaline pH. The polymerizability of tropomyosin at low ionic strength and the binding property of tropomyosin to F-actin were not affected by the modification of SH residues but they were lost rapidly by the modification of amino groups, in accordance with the previous report [Johnson, P. & Smillie, L.B. (1977) Biochemistry 16, 2264-2269]. By the addition of heavy meromyosin, labeled tropomyosin which could not bind to F-actin recovered the binding ability to F-actin and it could regulate the superprecipitation of actomyosin in the presence of troponin. Further modification of amino groups (labeling ratios more than 5) led to loss of the regulating ability completely.     

Chemical modification of sarcoplasmic reticulum membranes

The usefulness of chemical cross-linking and ¹²⁵I-labeling techniques in the analysis of protein-protein interactions and membrane polarity was evaluated on sarcoplasmic reticulum membranes. Treatment of fragmented sarcoplasmic reticulum vesicles with glutaraldehyde, dimethylsuberimidate, or copper-phenanthroline leads to the formation of high molecular weight aggregates of the Ca²⁺ transport ATPase; intermediate polymers of functionally and structurally interesting sizes accumulated only occasionally and in amounts of questionable significance. Coupling of membrane proteins with tolylene 2,4-diisocyanate-albumin inhibited tht ATPase activity and caused the appearance of high molecular weight aggregates and a band of about 160 000 dalton which corresponds to the ATPase-albumin complex.Even after the 100 000 dalton band of the Ca²⁺-transport ATPase was severely diminished by cross-linking with copper-phenanthroline or toluene diisocyanate-albumin, the Ca²⁺ binding proteins of sarcoplasmic reticulum remained unreacted. A consistent finding was the presence of dimers of the Ca²⁺ transport ATPase in aged preparations of sarcoplasmic reticulum which were converted upon reduction with β-mercaptoethanol into 100 000 dalton units.Microsomes were labeled with ¹²⁵I in the presence of lactoperoxidase, glucose oxidase, and glucose and the radioactivity oft he various protein components was measured after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The specific activity of calsequestrin was many times greater than that of the Ca⁺ transport ATPase suggesting that it is exposed on the outside surface may be sterically hindered from access by bulky reagents (tolylene diisocyanate-albumin, ferritin-labeled anti-calsequestrin antibodies, proteolytic enzymes, etc.), as calsequestin becomes highly reactive with these agents only after its release from the membrane.     

Chemical modification of rat liver arginase

Chemical modifications were used to search for catalytically important residues of rat liver arginase. The results of carbamoylation, nitration and diazotization suggest that lysyl and tyrosyl residues are not involved in the catalytic function of arginase. The modification of 5--6 tryptophanyl residues by N-bromosuccinimide or 2-hydroxy-5-nitrobenzyl bromide led to about 90% inhibition of the enzyme activity. Photooxidation of 21 histydyl residues also led to considerable inactivation of arginase. The modification of tryptophanyl and histidyl residues did not cause dissociation of the enzyme into subunits.     

Chemical modification study of antisense gapmers

A series of insertion patterns for chemically modified nucleotides [2'-O-methyl (2'-OMe), 2'-fluoro (2'-F), methoxyethyl (MOE), locked nucleic acid (LNA), and G-Clamp] within antisense gapmers is studied in vitro and in vivo in the context of the glucocorticoid receptor. Correlation between lipid transfection and unassisted (gymnotic-using no transfection agent) in vitro assays is seen to be dependent on the chemical modification, with the in vivo results corresponding to the unassisted assay in vitro. While in vitro mRNA knockdown assays are typically reasonable predictors of in vivo results, G-Clamp modified antisense oligonucleotides have poor in vivo mRNA knockdown as compared to transfected cell based assays. For LNA gapmers, knockdown is seen to be highly sensitive to the length of the antisense and number of LNA insertions, with longer 5LNA-10DNA-5LNA compounds giving less activity than 3LNA-10DNA-3LNA derivatives. Additionally, the degree of hepatoxicity for antisense gapmers with identical sequences was seen to vary widely with only subtle changes in the chemical modification pattern. While the optimization of knockdown and hepatic effects remains a sequence specific exercise, general trends emerge around preferred physical properties and modification patterns.     

Chemical modification of Streptococcus flagellar motors

Video techniques were used to record changes in motility of cells of Streptococcus sp. strain V4051 exposed to a variety of protein modification reagents. Starved cells were tethered to glass by a single flagellum, energized metabolically with glucose, or treated with valinomycin and energized artificially via shifts to media containing low concentrations of potassium ion. Experiments were devised that distinguished reagents that lowered the proton motive force from those that blocked the generation of torque (damaged the flagellar motors). Imidazole reagents blocked the generation of torque. Amino, sulfhydryl, dithiol, and disulfide reagents did not. Some of the imidazole, amino, and sulfhydryl reagents had long-term effects on the direction of flagellar rotation.     

Chemical modification of PABA synthase.

p-Aminobenzoic acid (PABA) synthase catalyses the first step in folic acid biosynthesis, the conversion of chorismate to p-aminobenzoate. In general, difficulties in purification have permitted only limited investigation of this enzyme. However, in an attempt to identify possible active site residues, the E. coli enzyme has been incubated with a range of protein modifying agents. Results indicate that cysteine, histidine, arginine and tyrosine residues are important for enzyme activity. Attempts were made to determine the subunits upon which these residues were located.     

Chemical modification of chlorinated microbial polyesters

Chlorination of microbial polyesters poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyoctanoate) (PHO) was carried out by passing chlorine gas through their solutions. The chlorine contents in chlorinated PHB (PHB-Cl) and chlorinated PHO (PHO-Cl) were between 5.45 and 23.81 wt % and 28.09 and 39.09 wt %, respectively. Molecular weights of the chlorinated samples were in the range of between one-half to one-fourth of the original values because of hydrolysis during the chlorination process. Thermal properties of the PHO-Cl were dramatically changed with an increase in its glass transition (T(g) = 2 degrees C) and the melting transition (T(m)). The T(g) of PHB-Cl varied from -20 to 10 degrees C, and its T(m) decreased to 148 degrees C. The chlorinated poly(3-hydroxyalkanoate)s (PHA-Cl) were converted to their corresponding quaternary ammonium salts (PHA-N(+)R(3)), sodium sulfate salts (PHA-S), and phenyl derivatives (PHA-Ph). Cross-linked polymers were also formed by a Friedel-Crafts reaction between benzene and PHA-Cl. The modified PHO derivatives were characterized by (1)H NMR and (13)C NMR spectrometry, Fourier transform infrared spectroscopy, gel permeation chromatography, and differential scanning calorimetry techniques.     

Synthesis and antibacterial activity of novel neamine derivatives

Synthesis and activity of derivatives at the O5 or O6 positions of 1-N-((S)-4-amino-2-hydroxybutyryl)-3′,4′-dideoxyneamine, which is the neamine moiety of arbekacin, were reported. Among these results, the 5-O-aminoethylaminocarbonyl derivative showed effective activity against Staphylococcus aureus expressing a bifunctional aminoglycoside-modifying enzyme AAC(6′)-APH(2″).     

Chemical modification of gastric microsomal potassium-stimulated ATPase

Selective chemical modification was used to examine amino acid residues that might be critical for the operation of the gastric K⁺-stimulated ATPase. Modification of amino groups with the fluorigenic reagent 2-methoxy-2,4-diphenyl-3-dihydrofuranone resulted in selective inhibition of the K⁺-stimulated ATPase and H⁺-transporting activities of the gastric microsomes, while the Mg²⁺-ATPase was not affected. Half-maximal inhibition occurred at about 3 μg 2-methoxy-2,4-diphenyl-3-dihydrofuranone/ml at pH 8.5. ATP provided complete protection against inhibition; the apparent K_m for ATP protection was about 50 μM. Nucleotide selectivity for protection was ATP > ADP > ITP > GTP > CTP > AMP. Sodium dodecyl sulfate gel electrophoresis of the reacted microsomes showed that virtually all the fluorescent label was on the M_r 100 000 peptide band, a very small peptide, and aminolipids. In the presence of ATP there was about 75% reduction in the fluorescent label on the M_r 100 000 peptide, but no change in the labeling of the other components. The arginine specific reagent, butanedione, inhibited Mg²⁺-ATPase and K⁺-ATPase activities, with the former being much less reactive. Similar to 2-methoxy-2,4-diphenyl-3-dihydrofuranone, ATP provided complete protection from butanedione treatment. It is concluded that amino and guanidino groups are critical to the function of the K⁺-ATPase and may be actually at the ATP binding site.     

Chemical modification of insulin in amyloid fibrils

We have investigated the chemical modification of insulin under conditions that promote the conversion of the soluble protein into amyloid fibrils. The modifications that are incorporated into the fibrils include deamidation of Asn A21, Asn B3, and Gln B4. In order to prepare fibrils with minimal deamidation of these residues, the kinetics of aggregation were accelerated by seeding with aliquots of a solution containing preformed fibrils. The resulting fibrils were then reincubated to determine the extent to which chemical modification occurs in the fibril itself. The deamidation of Asn A21 in particular could be followed in detail. Deamidation of this residue in the fibrillar form of insulin was found to occur in only 52 +/- 5% of molecules. This result indicates that there are at least two different packing environments of insulin molecules in the fibrils and suggests that the characterization of chemical modifications may be a useful probe of the environment of polypeptide chains within amyloid fibrils.