Synthesis and evaluation of novel photoreactive α-amino acid analog carrying acidic and cleavable functions

A novel photoreactive α-amino acid bearing an acidic residue and a cleavable diazirine was developed. To mimic common acidic α-amino acids, the residue was designed to be N-acylsulfonamide that possesses an acidic proton and is able to dissociate under the physiological conditions. The inhibition assay of its biotin-tagged derivative with glutamyl endopeptidase from Staphylococcus aureus V8 strain revealed a Ki_app value of 162 μM, which is slightly higher than the K_m value of a common substrate. Upon UV irradiation, this derivative specifically photolabeled glutamyl endopeptidase, l-glutamate dehydrogenase, glutamic oxalacetic transaminase, and l-glutamine synthetase, all the enzymes exhibit high affinity toward acidic α-amino acids. In addition, N-acylsulfonamide group functioned as a cleavable linker in mild basic solution after a brief N-alkylation. Either the multifunctional nature or the simple structure of this acidic α-amino acid surrogate would be useful as versatile photoreactive building block.     

The use of diaminodithiol for labeling small molecules with technetium-99m

To investigate the labeling of small molecules with ^99mTc by the bifunctional chelate approach, we have synthesized both a fatty acid and an estrone derivative containing a chelator of the N₂S₂ type. In the case of the fatty acid, this was a diaminodithiol (DADT) while for the estrone, a diaminodisulfide (DADS) was attached. The estrone derivative (5-(2-methylene estrone 3-methyl ether)-3,3,10,10-tetramethyl-1, 2-dithia-5,8-diazacyclodecane hydrochloride, DADS-E) was prepared by alkylation of DADS while the fatty acid derivative (N-(11-undecanoic acid)-N,N′-bis(2-methyl-2-mercaptopropyl) ethylenediamine hydrochloride, DADT-FA) was synthesized by alkylation of DADS followed by reduction. DADS-E was labeled in ethanol at elevated temperatures while DADT-FA was labeled at room temperature, both by stannous reduction. Paper chromatography showed both to be labeled and reverse-phase HPLC showed multiple peaks for both. Serum stability studies were performed by incubation at 37 °C with aliquots removed at 1 min and 1 day for analysis by size-exclusion HPLC. Initially, little pertechnetate or binding to serum proteins was observed whereas after 1 day the majority of activity in both cases was protein bound with 20 and 38% pertechnetate appearing for DADT-FA and DADS-E respectively. In conclusion, small biologically active molecules may be labeled with ^99mTc through an attached diaminodithiol or diaminodisulfide group.     

Domain-level stability of an antibody monitored by reduction, differential alkylation, and mass spectrometry analysis

Human immunoglobulin G1 (IgG1) contains 12 domains, and each has an intrachain disulfide bond that connects the two layers of antiparallel β-sheets. These intrachain disulfide bonds are shielded from solvents under native conditions. Therefore, accessibility of the disulfide bonds to reduction under conditions that unfold antibody has the potential to be a good indicator of the thermodynamic stability of each domain. The stability of a recombinant monoclonal antibody at the domain level was investigated using a novel method involving reduction of the disulfide bonds in the presence of increasing amounts of guanidine hydrochloride and alkylation with [¹²C]iodoacetic acid, which was followed by reduction of the remaining disulfide bonds and alkylation with [¹³C]iodoacetic acid. The percentage of modification by [¹²C]iodoacetic acid of each cysteine residue was calculated using mass spectra of the cysteine-containing tryptic peptides and used to follow the unfolding of each domain. It demonstrated that the CH2 domain was the least stable domain of the antibody, whereas the CH3 domain was the most stable domain of the antibody. Other domains showed intermediate resistance to the denaturant concentration, similar to the overall unfolding transition monitored by the intrinsic tryptophan fluorescence wavelength shift.     

Enzyme models. The attachment of N-methylhydroxamic acid to cyclohexaamylose. Its reactivity and specificity with phenyl esters

The N-methylacetohydroxamic acid group has been introduced into cyclohexaamylose by the following sequence of reactions: (1) carboxymethylation of cyclohexaamylose by iodoacetic acid, (2) methylation of carboxymethylcyclohexaamylose with diazomethane, and (3) reaction of the carboxymethylcyclohexaamylose methyl ester with N-methylhydroxylamine to form the N-methylacetohydroxamic acid-substituted cyclohexaamylose. By employing purification procedures involving ionexchange chromatography, the synthesis yielded a mono-substituted cyclohexaamylose-N-methylacetohydroxamic acid with selective modification of the C-2, C-3 hydroxyl group side of the cyclohexaamylose ring.p-Nitrophenyl acetate and 2-hydroxy-5-nitro-α-toluenesulfonic acid sultone react 20- and 70-fold faster with cyclohexaamylose-N-methylacetohydroxamic acid than with N-methylmethoxyacetohydroxamic acid. Cyclohexaamylose-N-methylacetohydroxamic acid also displays a marked kinetic stereospecificity for p-nitroover m-nitrophenyl acetate (whereas cyclohexaamylose itself exhibits the reverse stereospecificity). These reactions were shown to be competitively inhibited by cyclohexanol. This evidence indicates that cyclohexaamylose-N-methylacetohydroxamic acid binds the substrate in a reversible complexation step prior to nucleophilic attack and thus is an enzyme model.     

Alkylation of transfer RNA by N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea

The alkylation of a number of purified tRNA preparations by reaction with the carcinogens, N-methyl-N-nitrosourea and N-ethyl-N-nitrosourea was studied in order to investigate the role of nucleic acid structure on the distribution of alkylation products within the nucleotide sequence. The rate of alkylation was greatly increased by increasing the pH over the range 6 to 8 and the degree of alkylation (expressed as moles alkyl groups/mole tRNA) was directly proportional to the concentration of the nitrosamide added and independent of the amount of tRNA present. There was no significant difference in the degree of alkylation of any of the tRNA preparations tested. Reaction with N-ethyl-N-nitrosourea resulted in a degree of alkylation some 13 times less than that produced by reaction with a similar concentration of N-methyl-N-nitrosourea. The major product of the reaction was 7-alkylguanine amounting to about 80% of the total, but 3-methylcytosine, 6-O-methylguanine and 1-methyl-, 3-methyl-, and 7-methyladenine were also identified as products of the reaction of tRNA^fMet with N-methyl-N-nitrosourea.The possibility that preferential alkylation of certain residues within the polynucleotide sequence was produced by reaction with the nitrosamides was examined by degradation of the alkylated tRNA with pancreatic ribonuclease and separation of the oligonucleotide fragments by chromatography on DEAE cellulose. When tRNA^fMet which had been alkylated by reaction with N-methyl-N-nitrosourea or N-ethyl-N-nitrosourea was analysed in this way, the distribution of 7-alkylguanine was, within the limits of experimental error, in agreement with that expected for a random reaction of the alkylating agent with all of the guanosine residues throughout the molecule. A similar result was seen when tRNA^Phe was examined. These results were obtained by alkylation under conditions where the native configuration of the tRNA was maintained and show that the tertiary structure of the nucleic acid does not impart any specificity to the reaction with the nitrosamide producing 7-alkylguanine but the possibility that such specificity does exist for the minor products of alkylation cannot be excluded.     

Discovery of novel N-acylsulfonamide analogs as potent and selective EP3 receptor antagonists

A series of novel N-acylsulfonamide analogs were synthesized and evaluated for their binding affinity and antagonist activity for the EP3 receptor subtype. Representative compounds were also evaluated for their inhibitory effect on PGE₂-induced uterine contraction in pregnant rats. Among those tested, a series of N-acylbenzenesulfonamide analogs were found to be more potent than the corresponding carboxylic acid analogs in both the in vitro and in vivo evaluations. The structure activity relationships (SAR) are also discussed.     

A fluorescent assay for bacterial cell wall lytic enzymes

A sensitive fluorimetric assay is described for the measurement of N-acetylmuramic acid l-alanine amidase as well as lysozyme. The method uses Bacillus subtilis cell walls labeled with fluorescamine on the free amino group of diaminopimelic acid. The method can easily detect the lytic activity of 0.02 μg of pure N-acetylmuramic acid l-alanine amidase in 30 min and of 1 μg of hen egg-white lysozyme in the same period. The method is particularly suitable for measurement of competition between various cell wall preparations for the same enzyme.     

An alkaline proteinase which has inflammatory activity isolated from guinea pig lymphoid cells

A phosphate buffer extract of regional lymph node cells (more than 90% lymphocytes) of guinea pigs immunized with bovine immunoglobulin G contained acid and alkaline proteinase activities. The immunized cells contained approximately twice as much proteinase activities than the normal cells. The alkaline proteinase was purified and two forms could be separated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that one form labeled with [³H]diisopropylfluorophosphate was localized at M_r 28 000 in a single peak in the presence and absence of reducing agent. The enzyme was inhibited by diisopropyl-fluorophosphate, p-chloromercuribenzoate, N-ethylmaleimide and iodoacetic acid, and was also partially inhibited by phenylmethylsulfonyl fluoride. It was relatively heat-stable. Intradermal injection of the purified form into normal guinea pigs induced a mixed neutrophil-mononuclear infiltrate. The immune reaction was reduced in intensity with the diisopropylfluorophosphate-inhibited enzyme. These findings suggests that the proteinase probably participates in the inflammatory response of cellular hypersensitivity.     

Synthesis of betaine lipids: Dipropionylglyceryl(N,N,N-trimethyl)serine

1,2-Dipropionylglyceryl-3-O-3′-(N,N,N-trimethyl)serine was synthesized as a first example of a diglyceride containing an ether-linked hydroxyamino acid with betaine structure. The ether bond was formed by condensation of isopropylideneglycerol (Na alcoholate) and ethyl-2,3-dibromopropionate. Then, the quaternary ammonium group was introduced by replacing the secondary bromine (2-position) with trimethylamine. Finally, the isopropylidene group was split off in acidic medium and the free hydroxyl groups of the glycerol part esterified with propionic acid. The overall yield was 7.5%.     

N-acetylneuraminic acid and sialoglycoconjugate metabolism in fibroblasts from a patient with generalized N-acetylneuraminic acid storage disease

Cultured skin fibroblasts from a patient suffering from generalized N-acetylneuraminic acid storage disease were found to accumulate large amounts (approx. 4.0 μmol/g fresh weight) of free N-acetylneuraminic acid in a lysosome-enriched subcellular fraction. However, there were no detectable deficiencies in lysosomal hydrolase activities (including neuraminidase), and the activities of CMP-N-acetylneuraminic acid synthetase and N-acetylneuraminic acid aldolase were within normal limits. The cellular glycoconjugate composition was normal, and pathologic fibroblasts labeled with either [³H]glucosamine-HCl or $\text{N-[}{}^3\text{H]acetylmannosamine}$ showed a marked accumulation of labeled free N-acetylneuraminic acid, along with elevated incorporation into sialoglycoconjugates. Neither normal nor pathologic fibroblasts secreted labeled free N-acetylneuraminic acid into the culture medium. These results are consistent with an inherited defect in N-acetylneuraminic acid reutilization, resulting in the lysosomal accumulation of the free monosaccharide in generalized N-acetylneuraminic acid storage disease.     

Fluorogenic probe for measuring high-mannose type glycan-specific endo-β-N-acetylglucosaminidase H activity

We synthesized a fluorogenic probe with a high-mannose type heptasaccharide structure to detect the hydrolytic activity of endo-β-N-acetylglucosaminidase from Streptomyces plicatus (Endo-H). The heptasaccharide derivative (1) was labeled with an N-methylanthraniloyl group as a reporter dye at the branching point of the β-mannoside residue and 2,4-dinitrophenyl group as a quencher molecule at the reducing end, which was hydrolyzed by Endo-H, resulting in increased fluorescence intensity. Thus, Endo-H activities could be evaluated easily and quantitatively by measuring the fluorescence signal. Using both this probe (1) and a previously synthesized pentasaccharide probe, the hydrolysis activity of Endo-H and Endo-M were investigated. The results clearly showed a correlation with the substrate specificity of each enzyme.     

Exploration of the use of an acylsulfonamide safety-catch linker for the polymer-supported synthesis of hyaluronic acid oligosaccharides

The synthesis of hyaluronic acid oligosaccharides on polyethylene glycol (PEG) using an acylsulfonamide linker has been explored. Hyaluronic acid is a challenging synthetic target that usually involves the condensation of highly disarmed glucuronic acid building blocks. Amine-ended PEG monomethyl ether was efficiently functionalized with a hydroxyl-terminated acylsulfonamide linker. Suitably protected d-glucosamine (GlcN) and d-glucuronic acid (GlcA) monosaccharide building blocks were coupled to the polymer acceptor using the trichloroacetimidate glycosylation method. The sulfonamide safety-catch linker enables simultaneous cleavage of the monosaccharide from the polymer and orthogonal functionalization for further (bio)-conjugation of the sugar sample. Subsequent glycosylation of PEG-bound glycosyl acceptor to generate hyaluronic acid oligosaccharide chain failed. Model glycosylation experiments in solution and on soluble support using the same unreactive acceptors and donors allows for the synthesis of an orthogonally protected hyaluronic acid disaccharide and suggest that the encountered difficulties could be attributed to the presence of the N-acylsulfonamide.     

Catabolism of polyamines in the rat: Polyamines and their non-α-amino acid metabolites

The metabolic fate of stable isotopically labeled polyamines was investigated after their first and second intraperitoneal injection in rats. Using gas chromatographic and mass fragmentographic analyses of acid-hydrolyzed 24-h urines, some aspects of the polyamine metabolism could be elucidated. After the injections with hexadeutero-1,3-diaminopropane, obly labeled 1,3-diaminopropane was recovered from the urine samples. The rat injected with tetradeuteroputrescine excreted labeled putrescine excreted labeled putrescine, γ-amino-n-butyric acid, 2-hydroxyputrescine and spermidine, while the urine samples of the rat after the injections with tetradeuterocadaverine contained labeled cadaverine and δ-aminovaleric acid. The injections of hexadeuterospermidine led to the appearance of labeled spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, γ-amino-n-butyric acid, 1,3-diaminopropane, β-alanine and spermine. After the injections with octadeuterospermine, labeled spermine, N-(3-aminopropyl)-N′-(2-carboxyethyl)-1,4-diaminobutane, N,N′-bis(2-carboxyethyl)-1,4-diaminobutane, spermidine, isoputreanine, putreanine, N-(2-carboxyethyl)-4-amino-n-butyric acid, putrescine, 1,3-diaminopropane, β-alanine, 2-hydroxyputrescine and possibly γ-amino-n-butyric acid were recovered. Clear differences between the metabolism after the first and second injection were noted for putrescine, spermidine and spermine, which is suggestive for enzyme induction and/or the existence of salvage pathways.     

Nucleotide allosteric regulation of the glutamate dehydrogenases of Teladorsagia circumcincta and Haemonchus contortus

The expression of glutamate dehydrogenase (GDH; EC 1.4.1.3) in L3 of the nematode Haemonchus contortus was confirmed by detecting GDH mRNA, contrary to earlier reports. The enzyme was active in both L3 and adult H. contortus homogenates either with NAD⁺/H or NADP⁺/H as co-factor. Although it was a dual co-factor GDH, activity was greater with NAD⁺/H than with NADP⁺/H. The rate of the aminating reaction (glutamate formation) was approximately three times higher than for the deaminating reaction (glutamate utilisation). GDH provides a pathway for ammonia assimilation, although the affinity for ammonia was low. Allosteric regulation by GTP, ATP and ADP of L3 and adult H. contortus and Teladorsagia circumcincta (Nematoda) GDH depended on the concentration of the regulators and the direction of the reaction. The effects of each nucleotide were qualitatively similar on the mammalian and parasite GDH, although the nematode enzymes were more responsive to activation by ADP and ATP and less inhibited by GTP under optimum assay condition. GTP inhibited deamination and low concentrations of ADP and ATP stimulated weakly. In the reverse direction, GTP was strongly inhibitory and ADP and ATP activated the enzyme.     

Synchrony of Long Duration in Suspension Cultures of Mammalian Cells

THE high-sulphur proteins of α-keratins, which constitute the non-filamentous matrix between the microfibrils, comprise several major groups of proteins, each group consisting of a number of closely related components. They are obtained in a soluble form by reduction of the disulphide bonds of wool and preferential extraction with alkaline thioglycollate at high ionic strength¹. The thiol groups are subsequently stabilized by alkylation with iodoacetic acid.     

Transnitrosation of alicyclic N-nitrosamines containing a sulfur atom

Aromatic and aliphatic nitrosamines are known to transfer a nitrosonium ion to another amine. The transnitrosation of alicyclic N-nitroso compounds generates S-nitrosothiols, which are potential nitric oxide donors in vivo. In this study, certain alicyclic N-nitroso compounds based on non-mutagenic N-nitrosoproline or N-nitrosothioproline were synthesised, and the formation of S-nitrosoglutathione (GSNO) was quantified under acidic conditions. We then investigated the effect of a sulfur atom as the substituent and as a ring component on the GSNO formation. In the presence of thiourea under acidic conditions, GSNO was formed from N-nitrosoproline and glutathione, and an N-nitroso compound containing a sulfur atom and glutathione produced GSNO without thiourea. The quantity of GSNO derived from the reaction of the N-nitrosamines containing a sulfur atom and glutathione was higher than that from the N-nitrosoproline and glutathione plus thiourea. Among the analogues that contained a sulfur atom either in the ring or as a substituent, the thiazolidines produced a slightly higher quantity of GSNO than the analogue with a thioamide group. A compound containing sulfur atoms both in the ring and as a substituent exhibited the highest activity for GSNO formation among the alicyclic N-nitrosamines tested. The results indicate that the intramolecular sulfur atom plays an important role in the transnitrosation via alicyclic N-nitroso compounds to form GSNO.     

Regulation of glutamate dehydrogenase activity in relation to carbon limitation and protein catabolism in carrot cell suspension cultures

Glutamate dehydrogenase (GDH) specific activity and function have been studied in cell suspension cultures of carrot (Daucus carota L. cv Chantenay) in response to carbon and nitrogen supply in the culture medium. The specific activity of GDH was derepressed in sucrose-starved cells concomitant with protein catabolism, ammonium excretion, and the accumulation of metabolically active amino acids. The addition of sucrose led to a rapid decrease in GDH specific activity, an uptake of ammonium from the medium, and a decrease in amino acid levels. The extent of GDH derepression was correlated positively with cellular glutamate concentration. These findings strengthen the view that the function of GDH is the catabolism of glutamate, which under conditions of carbon stress provides carbon skeletons for tricarboxylic acid cycle activity.     

Alkylboronic acids accelerate affinity labelling of acetylcholinesterase with N,N,-dimethyl-2-phenylaziridinium ion

The kinetics of acetylcholinesterase alkylation with N,N-dimethyl-2-phenylaziridinium ion, the anionic-site-directed affinity label, has been investigated in the presence of alkylboronic acids, which are known as the esteratic-site-directed reversible inhibitors of the enzyme. The ternary complex of the enzyme, the aziridinium ion and alkylboronic acid, are formed in this reaction. In the case of propylboronic acid, for which the complete kinetic analysis of the acceleration effect has been carried out, the 85-fold increase in the rate of the enzyme alkylation reaction has been found. This acceleration effect was connected with the alkylation step, whereas the non-covalent binding of the aziridinium ion in the enzyme active centre was even hindered by the alkylboronic acid. The possible mechanism of this kinetic acceleration phenomenon is discussed with special reference to the kinetic data for the spontaneous solvolysis reaction of the aziridinium ion in water and organic solvents.