Polymeric 6-aminoquinoline, an activated carbamate reagent for derivatization of amines and amino acids by high-performance liquid chromatography

A new polymeric reagent containing the 6-aminoquinoline (6-AQ) tag was developed and applied for the off-line derivatization of amines and amino acids in high-performance liquid chromatography (HPLC). The synthesis and characterization of this polymeric reagent are described. An authentic external standard of a typical amine was synthesized and characterized for the determination of the derivatization efficiency. All amines had a derivatization efficiency higher than 50%; the derivatization of amino acids was performed under optimized phase-transfer catalysis reaction conditions. Derivatized amines and amino acids were separated under conventional reversed-phase conditions and determined by UV and FL detectors. To investigate the practical applications, this polymeric reagent was also used to derivatize protein hydrolysates.     

Facile thiolytic removal of the o-nitrophenylsulphenyl amino-protecting group.

2-Mercaptopyridine was used to effect the selective, mild and efficient cleavage of the o-nitrophenylsulphenyl amino-protecting group from several amino acids and peptides. By utilization of this reagent a stepwise synthesis of the tetrapeptide Thr-Lys-Leu-Arg([Leu3]tuftsin) was successfully achieved. The potential use of 2-mercaptopyridine in mechanized peptide synthesis via the polymeric reagents approach is discussed.     

The amino acid sequence encompassing the active-site histidine residue of lipoamide dehydrogenase from Escherichia coli labelled with a bifunctional arsenoxide

Pyruvate dehydrogenase multienzyme complex (PD complex) in the presence of pyruvate, thiamine pyrophosphate, coenzyme A, and Mg2+ (or NADH) was irreversibly inhibited with the radiolabelled bifunctional aresenoxide p-[(bromoacetyl)amino]phenyl arsenoxide (BrCH2 14CONHPhAsO). The initial reaction of the reagent was with a reduced lipoyl group of the lipoamide acetyltransferase component to form a dithioarsinite complex. Following the normal catalytic reactions, the anchored reagent was delivered into the active site of the lipoamide dehydrogenase (E3) component where an irreversible alkylation ensued via the bromoacetamidyl moiety. Treatment with 2,3-dithiopropanol (to break dithioarsinite bonds) caused the radiolabelled reagent to reside with E3. E3 was isolated from the inhibited PD complex and CNBr cleavage of the inhibited enzyme yielded a single radiolabelled peptide that was purified on a cyanopropyl silica column using high performance liquid chromatography. The radiolabelled amino acid was identified (after acid hydrolysis) as N3-[14C]carboxymethyl histidine in agreement with earlier studies. The radiolabel was located in residue 14 of the peptide for which the sequence was determined as GCDAEDIALTIHAHPTL-EIVGLAAEVFEG. This sequence agrees with the amino acid sequence determined from the gene sequence of E3. The histidine alkylated in the E3 component of the PD complex by BrCH2 14CONHPhAsO is residue-444 and further establishes its active site role.     

Improvement of oxygen-carrying properties of human hemoglobin by chemical modification with a benzene hexacarboxylate-monosubstituted polyoxyethylene

Benzene hexacarboxylate-monosubstituted polyoxyethylene on contact with Hb decreases its oxygen affinity, probably because it specifically interacts with the amino groups of the phosphate-binding site. This site specificity was used to direct the covalent coupling of this polymeric reagent with hemoglobin, in the vicinity of this cleft in order to obtain conjugates with low oxygen affinity and well-defined molecular weight. Such conjugates could thus be regarded as potential candidates for blood substitutes. Covalent fixation of this polymeric site-labeling reagent onto hemoglobin was carried out with the oxy and the deoxy form in the presence of a water soluble carbodiimide. It turns out that the oxygen-binding properties of the resulting hemoglobin derivatives depend on the reaction conditions, yet in all cases the oxygen affinity of the modified protein was lower than that of native hemoglobin and was no longer affected by organic phosphates. These results indicate that phosphate-binding site amines are probably involved in the covalent coupling, although in some conjugates (especially those prepared with high ratios of reagents) other amino groups participate also in the linking to the polymer. Chromatographic analysis and trypic peptide mapping of some conjugates evidenced that the -terminal valine residue was in fact the preferential binding site of hexacarboxylate-monosubstituted polyoxyethylene.     

An accurate colorimetric method for measurement of sulfaminohexose in heparins and heparan sulfates

A modification of a method for hexosamine analysis is presented which adapts it to measurement of sulfaminohexose in heparins and heparan sulfates. Unlike methods of sulfaminohexose analysis based upon coupling with indole, the absorptivity of polymeric and monomeric hexosamines is identical. N-Sulfated hexosamines are specifically deaminated in 33% acetic acid to yield free 2,5-anhydromannose residues which are then coupled to the color reagent 3-methyl-2-benzothiazolinone hydrazone hydrochloride. The sulfaminohexose content of a variety of heparins and heparan sulfates was determined with this methodology and compared with the indole-coupling method. Interferences by amino acids, proteins, and neutral sugar were evaluated in the sulfaminohexose assay and in the originally reported procedure for total hexosamine analysis.     

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.     

Selective N-alkylation of β-alanine facilitates the synthesis of a poly(amino acid)-based theranostic nanoagent

The development of functional amino acid-based polymeric materials is emerging as a platform to create biodegradable and nontoxic nanomaterials for medical and biotechnology applications. In particular, facile synthetic routes for these polymers and their corresponding polymeric nanomaterials would have a positive impact in the development of novel biomaterials and nanoparticles. However, progress has been hampered by the need to use complex protection-deprotection methods and toxic phase transfer catalysts. In this study, we report a facile, single-step approach for the synthesis of an N-alkylated amino acid as an AB-type functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection-deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents for the targeted detection and treatment of cancer.     

Substrate-dependent inactivation of transaldolase activity with tetranitromethane

Transaldolase (Type III) from Candida utilis was found to be inactivated by tetranitromethane only in the presence of the substrates fructose 6-phosphate and sedoheptulose 7-phosphate. This reaction was prevented by the addition of erythrose 4-phosphate or glyceraldehyde 3-phosphate, which are known to accept dihydroxyacetone from the transaldolase-dihydroxyacetone complex, releasing free transaldolase. These results strongly suggest that tetranitromethane does not react with free transaldolase but only with the Schiff-base intermediate. After 1 min of incubation with the reagent at pH 6.0, 4 moles of nitroformate were produced per mole of inactivated enzyme. The modification, probably a nitration or an oxidation of certain amino acid residues of the complex by tetranitromethane, caused a dissociation of the dihydroxyacetone moiety from the complex without any recovery of the enzymatic activity. The fact that the reaction with tetranitromethane takes place only in the presence of substrates indicates that a substrate-mediated change of conformation occurs in transaldolase. Chemical and spectrophotometric evidence is presented showing that tetranitromethane did not modify tyrosine, cysteine, and tryptophan residues in the inactivated enzyme. From amino acid analyses it appears that histidine, serine, proline, methionine, tyrosine, and phenylalanine residues were not altered by this reagent. The possible mechanisms of modification of the transaldolasedihydroxyacetone complex and the chemical nature of the modification by tetranitromethane are discussed.     

Identification of a cross-link in the Escherichia coli ribosomal protein pair S13-S19 at the amino acid level

Escherichia coli 30 S ribosomal subunits and 70 S ribosomes were treated with the bifunctional reagent diepoxybutane, acting as a cross-linker. One major cross-linked protein pair in the 30 S subunit was generated in relatively high yields. This cross-link was shown to consist of ribosomal proteins S13 and S19. Purification of this complex was achieved by a series of conventional and/or high pressure liquid chromatography techniques allowing its isolation in milligram quantities. To reveal the exact position of the two amino acids involved in the cross-link formation, the purified protein pair S13-S19 was subjected to several enzymatic fragmentations, and the resulting peptides were characterized by sequence analysis, amino acid analysis, and fast atom bombardment mass spectrometry. After isolation of the cross-linked peptides, Cys84 in protein S13 and His68 in S19 could be unequivocally identified as the amino acids cross-linked by the bifunctional reagent. This result demonstrates that, despite neutron scattering data which place the centers of mass of S13 and S19 85 A apart, at least these regions of the two proteins are located within a 4-A distance in the ribosomal particle.     

Application of rare-earth elements as labels in studying bilogically active compounds. II. Luminescence spectra and structure of europium complexes for use as shift reagents in NMR studies]

The luminescence spectra and solubility of several europium complexes are investigated. The conditions put upon the complex structure for its successful use as a shift reagent are discussed. Some water-soluble europium complexes with pyridoxaliden amino acids are shown to be fit as possible shift reagents.     

The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.     

On the specificity of the folin and lowry reagents for amine derivatives.

Reactivities of several amine derivatives with the Folin and Lowry reagents were examined. Tertiary amines reacted with the Folin reagent to produce a blue color, and secondary amines having a 2-hydroxyethyl group reacted with the Folin reagent only in the presence of Cu²⁺, i.e., with the Lowry reagent. On the other hand, primary and quarternary amines and amine N-oxides produced no color with either reagent. Reactivities of tertiary amines were greatly influenced by the nature of the N-substituted groups, and the color yield of those forming stable chelate complexes with metals was strongly inhibited by the presence of Cu²⁺, indicating that the formation of a stable complex with Cu²⁺ reduces the reactivity of tertiary amino nitrogen. The requirement of Cu²⁺ for the color development with secondary amines having a 2-hydroxyethyl group may be due to the formation of weak chelate complex with Cu²⁺.     

Chemical modification of spinach ferredoxin: evidence for the involvement of a complex between ferredoxin and ferredoxin:NADP oxidoreductase in NADP photoreduction.

Spinach ferredoxin was modified chemically with trinitrobenzene sulfonic acid (TNBS), a reagent which reacts specifically with amino groups. The trinitrophenylated ferredoxin (TNP-Fd) can accept electrons from Photosystem I as indicated by its full activity in the photoreduction of cytochrome $\text{c}$. The modified protein is inactive, however, in the photoreduction of NADP and cannot form a complex with the flavoprotein, ferredoxin: NADP oxidoreductase. The data presented indicate that the inactivity of the modified protein is the result of modification of a single amino group.     

Effect of cationic cholesterol derivatives on gene transfer and protein kinase C activity.

Four different cationic derivatives of cholesterol were synthesized which contain either a tertiary or a quaternary amino head group, with and without a succinyl spacer-arm. Their ability to inhibit protein kinase C (PKC) activity was measured in a detergent mixed micellar solution. Derivatives containing a quaternary amino head group were effective inhibitors (Ki approx. 12 and 59 microM) of PKC and derivatives containing a tertiary amino head group were approx. 4-20-fold less inhibitory. Liposomes containing an equimolar mixture of dioleoylphosphatidylethanolamine (DOPE) and a cationic cholesterol derivative were tested for the DNA-mediated transfection activity in mouse L929 cells. Highest activity was found with the derivative with low PKC inhibitory activity and with a succinyl spacer-arm. The transfection activity of this tertiary amine derivative, N,N-dimethylethylenediaminyl succinyl cholesterol was dependent on DOPE as a helper lipid; liposomes containing dioleoylphosphatidylcholine and this derivative had little activity. The transfection protocol of this new cationic liposome reagent was optimized with respect to the ratio of liposome/DNA, dose of the complex and time of incubation with cells. Several adherent cell lines could be efficiently transfected with this liposome reagent without any apparent cytotoxicity. However, the transfection activity was strongly inhibited by the presence of serum components.     

Preliminary characterization of novel amino acid based polymeric vesicles as gene and drug delivery agents

The amino acid homopolymers, poly-L-lysine and poly-L-ornithine, have been modified by the covalent attachment of palmitoyl and methoxypoly(ethylene glycol) (mPEG) residues to produce a new class of amphiphilic polymers-PLP and POP, respectively. These amphiphilic amino acid based polymers have been found to assemble into polymeric vesicles in the presence of cholesterol. Representatives of this new class of polymeric vesicles have been evaluated in vitro as nonviral gene delivery systems with a view to finding delivery systems that combine effective gene expression with low toxicity in vivo. In addition, the drug-carrying capacity of these polymeric vesicles was evaluated with the model drug doxorubicin. Chemical characterization of the modified polymers was carried out using (1)H NMR spectroscopy and the trinitrobenzene sulfonic acid (TNBS) assay for amino groups. The amphiphilic polymers were found to have an unreacted amino acid, palmitoyl, mPEG ratio of 11:5:1, and polymeric vesicle formation was confirmed by freeze-fracture electron microscopy and drug encapsulation studies. The resulting polymeric vesicles, by virtue of the mPEG groups, bear a near neutral zeta-potential. In vitro biological testing revealed that POP and PLP vesicle-DNA complexes are about one to 2 orders of magnitude less cytotoxic than the parent polymer-DNA complexes although more haemolytic than the parent polymer-DNA complexes. The polymeric vesicles condense DNA at a polymer:DNA weight ratio of 5:1 or greater and the polymeric vesicle-DNA complexes improved gene transfer to human tumor cell lines in comparison to the parent homopolymers despite the absence of receptor specific ligands and lysosomotropic agents such as chloroquine.     

Marfey’s reagent for chiral amino acid analysis: A review

Summary. The present paper describes characteristics and application of Marfeys reagent (MR) including general protocols for synthesis of the reagent and diastereomers along with advantages, disadvantages and the required precautions. Applications, and comparison with other derivatizing agents, for the resolution of complex mixtures of DL-amino acids, amines and non-proteinogenic amino acids, peptides/amino acids from microorganisms, cysteine residues in peptides, and evaluation of racemizing characteristics have been discussed. Separation mechanisms of resolution of amino acid diastereomers and replacement of Ala–NH₂ by suitable chiral moieties providing structural analogs and different chiral variants and their application as a derivatizing agent to examine the efficiency, and reactivity of the reagent have been focussed. Use of MR for preparing CSPs for direct enantiomeric resolution has also been included.On leave from Department of Chemistry, Indian Institute of Technology Roorkee, Roorkee 247 667, India.     

Structure and Macromolecular Composition of the Jelly Coats of the Urodele Ambystoma mexicanum

Cleavage-stage embryos of the neotenic urodele Ambystoma mexicanum are surrounded by a fertilization envelope and four macroscopic jelly coats termed J₁ (innermost) through J₄ (outermost). In sections prepared for light microscopy, each of the jelly layers stained with protein stains and the periodic acid-Schiff's reagent, but only J₁ stained with alcian blue at pH 2.5. These results suggest that each layer consists of proteins and glycoproteins and that J₁ uniquely contains some sulfate esters. Only J₄ was solubilized with alkaline mercaptan treatment in situ , however, the isolated inner jelly complex (J₁, J₂ and J₃) was easily dissolved in this reagent suggesting that solvent access is impaired in situ . A single alcian blue-staining component plus one protein-staining component were detected on reducing polyacrylamide gel electrophoresis of outer jelly (J₄). In the inner jelly complex (J₁, J₂, J₃), two protein-staining components were detected and no alcian blue-staining components were observed. A predominant polypeptide of 110,000 molecular weight was detected and purified to homogeneity on reducing and denaturing gels of the inner jelly complex. Amino acid analysis of the polypeptide demonstrated a slightly higher fraction of acidic over basic amino acids (Glx+Asx=18.1 mole% vs . Arg + Lys = 11.7 mole%). The N-terminal amino acid was Glu and the sequence of the first eleven amino acids was determined.     

Ion-exchange chromatography in lunar organic analysis

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids.As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10^–12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry.In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.     

Current contributions of peptide synthesis to studies on brain-gut-skin triangle peptides

The usefulness of a strong acid, such as MSA or TFMSA/TFA, as a deprotecting reagent in peptide synthesis was examined. By synthesizing several structurally related brain-gut-skin triangle peptides, a number of advantageous features of the thioanisole-mediated deprotecting procedure were demonstrated. New amino acid derivatives, Arg(Mts), Trp(Mts) and Asp(OChp), were introduced to improve the synthetic methodology of complex peptides and the superior properties of Cys(Ad) were evaluated.     

Selective chemical modification of cytochrome P-450SCC lysine residues. Identification of lysines involved in the interaction with adrenodoxin

Selective chemical modification of cytochrome P-450SCC has been carried out with lysine-modifying reagents. Modification of cytochrome P-450SCC with succinic anhydride was shown to result in loss of its ability to interact with intermediate electron transfer protein - adrenodoxin. To identify amino acid residues involved in charge-ion pairing with complementary carboxyl groups of adrenodoxin, cytochrome P-450SCC complex with adrenodoxin was modified with succinic anhydride. Adrenodoxin was then removed and cytochrome P-450 was additionally modified with isotopically labelled reagent. Subsequent chymotryptic hydrolysis of [14C]succinylated cytochrome P-450SCC and separation of digest obtained by combining various types of HPLC resulted in seven major radioactive peptides. The amino acid sequence of the peptides was determined by microsequencing. The major amino groups modified with radioactive succinic anhydride were found to be at Lys-73, -109, -110, -126, -145, -148 and -154 in the N-terminal sequence of cytochrome P-450SCC molecule and at Lys-267, -270, -338 and -342 in the C-terminal sequence. The role of electrostatic interactions in fixation of cytochrome P-450SCC complex with adrenodoxin is discussed.