Woodward's Reagent Research Articles

Transplantation of Nuclei from Lymphocytes of Adult Frogs into Enucleated Eggs: Special Focus on Technical Parameters

The technique of transplantation of nuclei from adult lymphocytes into enucleated eggs from Xenopus laevis (South African clawed toad) is described. The splenic lymphocytes from the one-nucleolus mutant were bound via their immunoglobulin receptors to nylon fibers, derivatized with the antigen used for immunization. A technique for coupling other cell types with Woodward reagent is also described. The cells were broken by aspiration into a micropipette and injected into enucleated eggs. The egg pronucleus was eliminated by UV treatment followed by surgical removal. The origin of the genome of developing embryos was determined on karyotype preparations by looking for the nucleolar organizer on the chromosome pair No. 12. Participation of egg pronucleus in development was frequent as judged by the incidence of gynogenetic diploid individuals and of tetraploid animals exhibiting characteristics of both recipient egg and somatic cell donor karyotypes. Apparently normal, lymphocyte-dervived tadpoles were obtained, but they died at 12 days. This shows that differentiated cells from adults, such as lymphocytes, can re-express the genes necessary for ontogeny.

Inactivation of Phospholipase C from Bacillus cereus by a Carboxyl Group Modifying Reagent.

Phospholipase C from Bacillus cereus was inactivated by incubation with either of the carboxyl reagents, a water-soluble carbodimide plus a nucleophile or Woodward's reagent K. With the former reagent, the incorporation into the enzyme of the first mol of nucleophile caused a 4-5-fold increase in the Km for dihexanoyllecithin with no significant effect on the Vm. The second mol of nucleophile incorporated caused no further change in Km but destroyed most of the catalytic activity. Modification of the enzyme by carbodiimide plus nucleophile did not alter the relative activity of the enzyme towards micelles and monomolecularly dispersed solutions of diheptanoyllecithin. Furthermore, inactivation by this reagent did not significantly decrease the ability of the enzyme to bind to a substrate-based affinity gel. It was concluded that phospholipase C contains a single carboxyl group that is essential for catalytic activity. The enzyme also contains a total of 4-5 reactive/exposed carboxyl groups.

Grafting of enzymes on collagen films using woodward's reagent “K” and a water-soluble carbodiimide derivative

Two new methods of activation were developed to graft enzymes on collegen films. They involved chemical modifications of surface groups of collagen either by Woodward's reagent "K" or by EDC, a water-soluble derivative of carbodiimide. EDC was a better coupling agent and a detailed study was conducted with this agent. It could be used either in a global method of activation and coupling, or in a two-step procedure of activation of collagen, followed by spontaneous coupling of enzyme. All enzymes tested were successfully bound: malate dehydrogenase, lactate dehydrogenase, aspartate aminotransferase, urease, creatine kinase, hexokinase. The influence on the yield of grafted enzyme, of pretreatment of films, time and temperature of EDC activation, concentration of EDC and enzyme, protecting agents was studied. Stability of enzyme activity on storage was greatly increased after grafting. A co-grafted dual system creatine kinase/heoxkinase, was achieved which exhibited a good efficiency. A striking renaturing process at 0-4degreesC after thermal denaturation, was observed with hexokinase.

Affinity labeling of the primary bilirubin binding site of human serum albumin.

A label for the bilirubin binding sites of human serum albumin was synthesized by reacting 2 mol of Woodward's reagent K (N-ethyl-5-phenylisoxazolium-3'-sulfonate) with 1 mol of bilirubin. This yielded a water-soluble derivative in which both carboxyl groups of bilirubin were converted to reactive enol esters. Covalent labeling was achieved by reacting the label with human serum albumin under nitrogen at pH 9.4 and 20 degrees. Under the same conditions, no covalent binding to the monomers of several proteins could be demonstrated. The number of binding sites for bilirubin and the label were found to be the same, and competition experiments with bilirubin showed inhibition of covalent labeling. The absorption, fluorescence and CD spectra of the label in a complex with human serum albumin were similar to those of the bilirubin human serum albumin complex. However, following covalent attachment to the spectral properties were changed, indicating loss of conformational freedom of the chromophore. Labeling ratios were selected to result in the incorporation of less than 1 mol of label/mol of human serum albumin. Under these conditions, labeling is thought to occur primarily at the high affinity binding site.

The pH-dependence and group modification of beta-lactamase I.

The pH-dependence of the kinetic parameters for the hydrolysis of the beta-lactam ring by beta-lactamase I (penicillinase, EC 3.5.2.6) was studied. Benzylpenicillin and ampicillin (6-[D(-)-alpha-aminophenylacetamido]penicillanic acid) were used. Both kcat. and kcat./Km for both substrates gave bell-shaped plots of parameter versus pH. The pH-dependence of kcat./Km for the two substrates gave the same value (8.6) for the higher apparent pK, and so this value may characterize a group on the free enzyme; the lower apparent pK values were about 5(4.85 for benzylpenicillin, 5.4 for ampicillin). For benzylpenicillin both kcat. and kcat./Km depended on pH in exactly the same way. The value of Km for benzylpenicillin was thus independent of pH, suggesting that ionization of the enzyme's catalytically important groups does not affect binding of this substrate. The pH-dependence of kcat. for ampicillin differed, however, presumably because of the polar group in the side chain. The hypothesis that the pK5 group is a carboxyl group was tested. Three reagents that normally react preferentially with carboxyl groups inactivated the enzyme: the reagents were Woodward's reagent K, a water-soluble carbodi-imide, and triethyloxonium fluoroborate. These findings tend to support the idea that a carboxylate group plays a part in the action of beta-lactamase I.

ChemInform Abstract: TOTAL SYNTHESIS OF FERRICHROME

AbstractZur Synthese der Titelverbindung über 24 Zwischenstufen wurden zwei verschiedene Hexapeptidsequenzen ausgehend von Nδ‐Tosyl‐Nδ‐benzyloxy‐L‐ornithin mittels N‐Hydroxysuccinimide und N,N‐Dicyclohexylcarbodiimid oder nach der Woodward‐Methode dargestellt.

Evidence for an Essential Carboxyl Group for Yeast Phosphoglycerate Kinase: REACTION WITH WOODWARD'S REAGENT K

Abstract An alternative method of purifying 3-phosphoglycerate kinase (EC 2.7.2.3) from yeast is described which employs step-induced pH-gradient elution from phosphocellulose and electrofocusing. The purified enzyme has a specific activity of 1200 to 1400 units per mg when corrected to saturating conditions and 30°. Reaction of this enzyme with Woodward's reagent K. (N-ethyl-5-phenylisoxazolium-3'-sulfonate) leads to rapid inhibition; MgATP affords protection against inactivation. Inhibition obeys pseudo-first order kinetics and there is an enzyme inhibitor complex formed prior to covalent modification. Although up to 5 moles of carboxyl per mole of phosphoglycerate kinase can be modified by Woodward's reagent K, it appears that only a single carboxyl is essential for the enzyme's action. The essential carboxyl appears to be more reactive than the nonessential but modifiable carboxyls. Sedimentation velocity and optical rotatory dispersion studies indicate that there has been no major conformational change concomitant with modification. Possible mechanistic roles for the essential carboxyl are discussed.

Kinetics of Woodward's Reagent K Hydrolysis and Reaction with Staphylococcal Nuclease

The kinetics of the hydrolysis of Woodward's Reagent K has been studied as a function of pH in aqueous solution. Rate constants for the reagent transformation have been combined with rate data for loss of staphylococcal nuclease activity to provide the mechanism for the protein modification. The reactive species for derivatization of the enzyme is the intermediate ketoketenimine. The pH dependence of the rate of the second order reaction between this intermediate and the nuclease yielded a pKapp of 5.22, which is attributed to a carboxyl group or groups of the enzyme.

Total Synthesis of Ferrichrome

Abstract Two different sequence hexapeptides for the synthesis of ferrichrome were prepared using Nδ-tosyl-Nδ-benzyloxy-l-ornithine by means of N-hydroxysuccinimide plus N,N-dicyclohexylcarbodiimide or Woodward reagent K method. The linear hexapeptides (XII·HCl and XIX·HCl) were cyclized by excess N,N-dicyclohexylcarbodiimide to give cyclo-triglycyltri-Nδ-tosyl-Nδ-benzyloxy-l-ornithyl (XX). Detosylation, acetylation and successive reductive debenzylation of XX afforded deferri-ferrichrome (XXIV). Addition of ferric iron to XXIV gave ferrichrome, which had the same crystalline form, superimposable infrared and well-agreeing visible spectra and the same value in optical rotation compared with the data in the literature of natural product.

Modification of carboxyl groups in bovine carboxypeptidase A. I. Inactivation of the enzyme by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K).

Modification of carboxyl groups in bovine carboxypeptidase A. I. Inactivation of the enzyme by N-ethyl-5-phenylisoxazolium-3'-sulfonate (Woodward's reagent K).

Chemical modification of aminoacyl ligases and the effect on formation of aminoacyl-tRNA

Fluorodinitrobenzene (FDNB), Woodward's reagent K, and cyanate were reacted in separate experiments with a crude mixture of Escherichia coli aminoacyl-RNA ligases. Each of these reagents was found to induce selective inhibition of the capacity of certain of these enzymes to esterify 14C-labeled amino acids to transfer RNA's. In particular, on treatment with FDNB, the arginyl-tRNA ligase activity was inhibited 88%, using conditions under which the lysyl-tRNA ligase activity was inhibited 20%. These ligases were amenable to purification, and this same preferential inhibition of the arginyl-tRNA ligase as compared to the lysyl-tRNA ligase was found to occur on treatment of these purified ligases with FDNB. The differential inhibitory effects on individual aminoacyl-RNA ligases varied from one reagent to another.

Structural biochemistry. IV. 3β-Hydroxy-17β-(l-prolyl)amino-androst-5-ene

The synthesis of the title compound III was studied in detail and the following combination of methods was found reliable and convenient. The oxime derivative Ib of ketone la was reduced with sodium–ethanol to 3β-hydroxy-17β-amino-androst-5-ene. The configurational assignment for amine IIa was supported by the results of a comparison with the 17α-epimer and by a proton magnetic resonance study of both isomers. Selective reaction between amine IIa and carbobenzoxy-l-proline was achieved with Woodward's reagent K. Of several procedures explored for removing the carbobenzoxy protecting group from amide IIc, palladium-catalyzed hydrogenolysis proved quite satisfactory. Hydrogenolysis of carbamate IIb to yield prolyl amide III was realized without affecting the Δ5 olefin system. A mass spectral study of amine III and the corresponding 5α-derivative IV confirmed the latter observation. A brief review of procedures for the synthesis of steroidal amines was also presented.

STRUCTURAL BIOCHEMISTRY: II. SYNTHESIS OF 3β-HYDROXY-17β (L-PROLYL-L-PROLYL)AMINO-5α-ANDROSTANE

A practical synthetic route to 3β-hydroxy-17β-amino-5α-androstane (III) was developed and the configuration of the amine substituent established by proton magnetic resonance studies. Condensing amine III with carbobenzoxy-L-proline by means of Woodward's reagent K led to proline derivative IVa. Steroidal peptide Vb was readily prepared by first removing the protecting group from amide IVa by palladium-catalyzed hydrogenolysis and then repeating the peptide-forming and hydrogenation steps. Protection of the 3β-hydroxy group proved to be unnecessary, and the presently reported approach to steroidal peptides proved to be reliable and useful.