Synthesis and characterisation of an iodinated bile-salt derivative for photoaffinity labelling

The synthesis and characterisation of a novel iodinated bile salt derivative, 125I-labelled 3 beta-azidocholylhistamine, is described. The derivative is handled by rat liver in a similar manner to taurocholate and binding to bovine serum albumin, a well-characterised bile acid-binding protein, is demonstrated. The suitability of the derivative for photoaffinity labelling is assessed.     

Synthesis and analysis of a methyl ether derivative of tetracycline which inhibits growth of Escherichia coli

Tetracycline is a widely used broad spectrum antibiotic. A derivative of tetracycline was synthesized by methylation (-CH3) of the phenolic hydroxyl group, with the use of diazomethane (CH2N2). A methyl ether group is then formed from the reaction with diazomethane, which replaces the hydroxyl group. The newly formed derivative has reduced hydrogen bonding capability relative to the unmodified tetracycline. An infrared spectra shows the appearance of the ether group on the derivative and the Log P calculations indicate that the derivative has increased lipophilic tendency. The Lipophilic Substituent Constant calculated for the tetracycline derivative is 0.46, indicating a lipophilic substituent. The tetracycline derivative was soluble in aqueous solvents and was stable for more than five weeks when stored at < or = 0 degrees C. The derivative was placed in tissue culture utilizing Luria-Bertani (LB) media, at a concentration of 12.0 microg/mL and inhibited the growth of E. coli (XL-1 blue) from 15% to 20% within the initial sixteen hours.     

Determination of tryptophan, tyrosine, and phenylalanine by second derivative spectrophotometry

Second derivative spectrophotometry has been useful for the determination of aromatic amino acids. However, published methods produce erroneous results, because those methods measure second derivative values by the vertical distance between peak and trough which is subject to variation according to the aromatic amino acid composition of proteins. This paper presents a method of second derivative spectrophotometry which measures second derivative absorbance values by means of the vertical distance from baseline to the derivative curve at a wavelength specifically assigned to each aromatic amino acid, and makes corrections for the interference from other amino acids at the same wavelength. The Appendix describes a computational method for obtaining absolute values of second derivative absorbances directly from normal absorbance values without using the spectrophotometer's derivative mode, because most commercial instruments produce completely arbitrary second derivative values which make comparison of data obtained on two different instruments impossible.     

Preparation and some properties of a derivative of wheat germ agglutinin with altered biological activity

An inactive derivative of wheat germ agglutinin, which is a strong activator of blood platelets, was prepared by selective chemical modification of the lectin with cyanogen bromide at acid pH. The derivative was then used as a probe to learn about the initial events in platelet stimulation by physiological agents. Amino acid analysis of the modified lectin confirmed specific cleavage of a methionine residue. Gel filtration studies indicated a molecular weight for the lectin derivative similar to the unmodified lectin. In gel electrophoresis in the presence of sodium dodecyl sulfate, reduced samples of the derivative showed two bands and the main component migrated slightly faster than the native lectin. The derivative retained the capacity to precipitate an antibody to the lectin although at least one of the antigenic sites was lost due to chemical modification. The derivative did not compete with the unmodified lectin for binding to platelets. Unlike the parent lectin, the derivative did not aggregate platelets even at a ten fold higher concentration. Under similar conditions, there were about 1.0 X 10(5) binding sites/platelet for the lectin derivative with an apparent dissociation constant of 1.7 microM compared to 5 X 10(5) sites/cell and a dissociation constant of 0.4 microM for the native lectin. Overnight incubation of platelets or red cells with the derivative in microtiter plates showed about 2-5% agglutinating activity for the derivative compared to the unmodified lectin. Incubation of platelets with the lectin derivative inhibited platelet aggregation by thrombin while aggregation induced by a number of other agents was not significantly affected. This inhibitory effect of the lectin derivative on thrombin-induced platelet aggregation could be readily reversed with GlcNAc. The lectin derivative may be a useful tool to explore the structure-function relationship of cell surface components.     

A crystalline A14-(2-nitro-4-trimethylammoniophenyl) derivative of bovine insulin

[O-(2-Nitro-4-trimethylammoniophenyl)-TyrA 14]insulin (bovine) is a product formed on reaction of bovine insulin with the hydrophilic reagent 1-fluoro-2-nitro-4-trimethyl-ammoniobenzene iodide (TAN-F) in an aqueous buffer at pH 8.00. The derivative was isolated and its purity established by standard procedures. The identity of the derivative was determined by degrative studies with alpha-chymotrypsin. The addition of zinc to the above reaction decreases the yield of the title derivative, but increases the yield of the [N alpha-TAN-GlyA1] derivative. [N alpha-Boc-GlyA1]insulin was also reacted with the above mentioned reagent in an attempt to improve the yield of the A14-tyrosine derivative. The biological activity of this microcrystalline derivative was found to be 12.4 units/mg as measured by the mouse convulsion assay.     

1H-n.m.r. studies on the existence of substrate binding sites in bovine pancreatic ribonuclease A

The reaction of ribonuclease A with either 6-chloropurine riboside 5'-monophosphate or the corresponding nucleoside yields one derivative, with the reagent covalently bound to the alpha-amino group of Lys-1, called derivative II and derivative E, respectively. We studied by means of 1H-n.m.r. at 270 MHz the interaction of these derivatives with different purine ligands. The pK values of His-12- and -119 were obtained and compared with those resulting from the interaction with ribonuclease A. The results showed that the interaction of derivative E with 3'AMP is similar to that described for RNase A as the pK2 of His-12 is increased while that of His-119 remains unaltered. However, derivative II presents some differences as it was found an enhancement of the pK2 values of both His-12 and His-119. Interaction of derivative II and derivative E with dApdA increases the pK2 of His-119, whereas a decrease is found when it interacts with ribonuclease A. These results suggest that the phosphate group and the nucleoside of both derivatives are located in regions of the enzyme where natural substrate analogues have secondary interactions and they can be interpreted as additional binding sites.     

A stable bis-allyloxycarbonyl biotin aldehyde derivative for biotinylation via reductive alkylation: application to the synthesis of a biotinylated doxorubicin derivative

A novel, stable, biotin aldehyde derivative is reported in which the biotin moiety is N1,N3-protected by the allyloxycarbonyl group. The derivative is stable to sodium cyanoborohydride mediated reductive alkylation and is cleaved under mild Pd [0] catalysis. This novel biotin aldehyde should have wide application in avidin- and streptavidin-based detection systems and bioassays. The derivative is utilized in the synthesis of a biotinylated doxorubicin analogue that retains topoisomerase activity.     

Sperm ultrastructure of the honey bee (Apis mellifera) (L) (Hymenoptera, Apidae) with emphasis on the nucleus-flagellum transition region

The flagellum of Apis mellifera (Hymenoptera, Apidae) consists of two mitochondrial derivatives, an axoneme and two accessory bodies. The mitochondrial derivatives are of unequal size and lie parallel to the axoneme. In the larger derivative four regions can be distinguished while in the smaller, only three. The region occurring only in the larger derivative consists of paracystalline material. The smaller mitochondrial derivative terminates anterior to the larger one. An extremely long centriolar adjunct is observed between the nucleus and the smaller mitochondrial derivative. This adjunct is compact, very electron dense and gradually tapers from base toward apex, finishing at the anterior extremity of the axonemal microtubules. In this flagellar region, there is only one accessory body present between the larger mitochondrial derivative and the axoneme. Anteriorly, the tips of the axonemal microtubules are inserted in a well developed mass of granular appearance. This material surrounds the nuclear base, separating it from the anterior end of the larger mitochondrial derivative. We believe that the structure identified here as a centriolar adjunct is homologous to that observed in Formicidae, Ichneumonoidea and Symphyta. Therefore, very probably, it is common to most Hymenoptera.     

Syntheses of fused heterocyclic compounds and their inhibitory activities for squalene synthase.

A variety of fused heterocyclic compounds (2-11) were synthesized as a modification of the lead compound 1a and evaluated for their inhibition of squalene synthase. 4,1-Benzothiazepine derivative 2, 1,4-benzodiazepine derivative 6, 1,3-benzodiazepine derivative 7, 1-benzazepine derivative 9, and 4,1-benzoxazocine derivative 10 potently inhibited squalene synthase activity, whereas the 4,1-benzoxazepine derivatives 1 was the most potent inhibitor. 4,1-Benzothiazepine S-oxide derivative 4, 1,4-benzodiazepine derivative 5, 1,3,4-benzotriazepine derivative 8, and 1,2,3,4-tetrahydroquinoline derivative 11 were found to be weakly active. Comparison of the X-ray structures of these compounds (1a, 2, 4, 5, 7 and 10) suggests that orientation of the 5- (or 6)-phenyl group is important for activity.     

Lysine biosynthesis in Saccharomyces. Conversion of α-aminoadipate into α-aminoadipic δ-semialdehyde

The reduction of alpha-aminoadipate to alpha-aminoadipic delta-semialdehyde by a cell-free extract of Saccharomyces is shown to be a three-step process. First the amino acid reacts with ATP to form an adenylyl derivative. Then the adenylyl derivative of alpha-aminoadipate is reduced in the presence of NADPH. In the third step the reduced adenylyl derivative of the amino acid is cleaved to form alpha-aminoadipic delta-semialdehyde. The presence of Mg(2+) is necessary for the first and second steps. The third step does not need any cofactors. The product of the first step was isolated by chromatography after incubating the cell-free extract of Saccharomyces with alpha-aminoadipate, ATP and Mg(2+). The isolated product was identified as an adenylyl derivative of alpha-aminoadipate and could be converted into alpha-aminoadipic delta-semialdehyde under the stated experimental conditions. The product of the second step was too unstable to be identified.     

Use of site-directed mutagenesis to obtain isomorphous heavy-atom derivatives for protein crystallography: cysteine-containing mutants of phage T4 lysozyme

Five different cysteine-containing mutants of the lysozyme from bacteriophage T4 were used to explore the feasibility of using site-directed mutagenesis to generate isomorphous heavy-atom derivatives for protein crystallography. Cysteines 54 and 97, present in wild-type lysozyme, can be readily reacted with mercuric ion to produce an excellent isomorphous heavy-atom derivative. Mutants with an additional cysteine at position 86, 146, 153 or 157, or with Cys 97 replaced by Val, were engineered by site-directed mutagenesis. The mutant lysozyme Thr 157----Cys reacts with mercuric chloride to give an excellent new derivative although Cys 157 is only approximately 60% substituted with the heavy atom. The cysteine at position 146 is largely buried but reacts readily with mercuric chloride. In this case the isomorphism is poor and the resultant derivative is of marginal quality. Cys 153 reacts rapidly with mercuric ion but the derivative crystals do not diffract. The mutant Pro 86----Cys does not yield a particularly good heavy-atom derivative. This is due in part to a loss of isomorphism associated with the mutation. In addition, Cys 86 shows very little reactivity towards mercurials even though it is fully exposed to solvent. The mutation Cys 97----Val was used to explore the possibility of creating an independent derivative by deleting a heavy-atom site already present in wild-type lysozyme. In all cases that were tested, the quality of the heavy-atom derivative was improved by using as an isomorphous pair mercury-substituted mutant versus non-substituted mutant rather than mercury-substituted mutant versus (non-substituted) wild-type lysozyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new derivative of vitamin C and its application to the synthesis of labelled ascorbic acid

1. The preparation of a mono-O-cyclohexylidene derivative of l-ascorbic acid is described. 2. The new compound is shielded by the cyclohexanone group at C-5 and C-6 of the ascorbic acid molecule, while the double bond between C-2 and C-3 is kept intact. 3. The double bond of the new derivative is more resistant to oxidation than its parent compound. 4. Ascorbic acid is easily regenerated by mild acid hydrolysis. 5. The new derivative facilitates the synthesis of (14)C-labelled vitamin C.     

Conformational isomerizations of the poly(dA-dT) and poly(amino2dA-dT) duplexes involving the unusual X-DNA double helix: a fourth derivative spectrophotometric study

Fourth derivative spectrophotometry has been applied to monitor conformational isomerizations of polynucleotides for the first time. The transitions studied have been the B-A and A-X isomerizations of poly(dA-dT) and the B-X one of poly(amino2dA-dT). Parameters obtained from the fourth derivative spectra have been used to follow these conformational changes. The A form of poly(dA-dT) has been characterized by a new fourth derivative peak at 293.0 nm which can be associated to interstrand adenine-adenine interactions. Furthermore, some of the fourth derivative peaks in the long wavelength region (270-310 nm) can be related to stacking interactions present in the polynucleotide double helices. The tentative assignment of these peaks, particularly that at 299.0 nm in the derivative spectra of poly(amino2dA-dT), to n----pi electronic transitions is discussed.     

2'',3''=Carbonates in the synthesis of uridine 5''-deoxy and 2'',5''-dideoxy derivatives.

Detritylation of 2',3'-O-carbonyl-5'-O-trityluridine (Ia) with ethereal hydrogen chloride affords 2',3'-O-carbonyluridine (Ib; 83%) which is converted by mesylation to the 5'-mesylcarbonate Ic (75%). Reaction of compound, Ic with tetrabutylammonium bromide in DMF affords the 5'-bromo carbonate Id (77%) which is reduced with tributyltin hydride to the 5'-deoxyuridine 2',3'-cyclic carbonate Ie (70%). When heated with imidazole, compound Ie affords the 2,2'-anhydro derivative IIa (76%) which is converted to the 2'-chloro derivative IIIa (88%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIa gives 2',5'-dideoxyuridine (IIIb; 68%). When heated with NaHCO3 in DMF, the 5'-bromo carbonate Id affords the anhydro bromo derivative IIb (50%) which is converted to the 2',5'-dichloro derivative IIIc (86%) on heating with HC1/DMF. The tributyltin hydride reduction of compound IIIc affords the 2',5'-dideoxy derivative IIIb (59%). Alkaline hydrolysis of the 2,2'-anhydro derivative IIa affords the arabinosyl derivative IVa which is converted to the diacetyl derivative IVb (34%) by acetylation. When refluxed in water, the 2',3'-cyclic carbonates Ib, Id, and Ie are hydrolysed to the parent nucleosides, namely, uridine (Va; 81%), 5'-bromo-5'-deoxyuridine (Vb; 78%), and 5'-deoxyuridine (Vc; 83%). Hydrolysis of carbonates Ib and Ie is accompanied by the formation of the 2,2'-anhydro derivatives IIc (10%) and IIa (5%) as by-products.     

The interaction of mitochondrial transhydrogenase with derivatives of coenzyme A

The 2,4-dinitrophenyl derivative of dephospho-CoA and the 7-nitrobenzofurazan-4-yl derivative of CoA are competitive inhibitors (Ki 3 microM and 2.6 microM respectively) of mitochondrial transhydrogenase with regard to NAD+ and NADPH respectively. The 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA is a competitive inhibitor with regard to both transhydrogenase substrates with the same Ki equal to 0.3 microM. The pattern of transhydrogenase inhibition with the 7-nitrobenzofurazan-4-yl derivative of dephospho-CoA indicates that one molecule of the inhibitor binds simultaneously to both the NADP(H) and the NAD(H) binding sites of the enzyme. This result is evidence of the short distance between the NADP(H) and the NAD(H) binding sites.     

Second derivative fluorescence spectroscopy of tryptophan in proteins

The second derivatives of N-acetyl- -tryptophan amide (AcTrpNH₂) fluorescence spectra were characterised in order to describe changes in the tryptophan environments of proteins. This tryptophan model compound was studied in several media with different degrees of hydrophobicity. The effect of tyrosines on the derivative spectra was also determined in situations in which both tyrosine and tryptophan were excited. An analysis of fluorescence second derivative spectra suggests that AcTrpNH₂ fluorescence emission is composed of two main bands. Increasing solvent polarity resulted in a red-shift by both bands and a relative increase in the emission efficiency of the shortest wavelength band. The applicability of fluorescence second derivative is shown through several examples. Turbidity observed in whole membrane extracts, for example, is eliminated by using second derivative spectra. Melittin, human and bovine serum albumins and the carboxypeptidase–PCI complex were studied as examples of the use of fluorescence second derivative spectroscopy to monitor changes in structural characteristics when these proteins were subjected to various transitions.     

The individual tyrosines of proteins: their spectra may or may not differ from those in water or other solvents

The overall derivative spectrum of a protein is the sum of the individual derivative spectra just as the overall ultraviolet spectrum of a protein is the sum of its component parts. The RNase and DNA binding protein Sso7d has two tyrosines and one tryptophan. We used two mutant forms of the protein to show that the individual aromatics contribute derivative spectra that can be explained on the basis of their environments. We used mutant forms of iso-1-cytochrome c to estimate the contributions of the single tryptophan and three of the five tyrosines to the overall derivative spectrum. The tryptophan spectrum is not exceptional. The comparable tyrosine spectra are more complex. The derivative spectrum of individual tyrosines does not correspond to that expected on the basis of concentration. This is a reflection of two factors: (1) the extent to which mutations are sensed distally through the introduction and compression of packing defects; and (2) the extent to which electronic transitions of tyrosine are influenced by nearby atoms. This influence could take the form of tyrosine residing in an area where the dielectric coefficient is not uniform; it could also result from tyrosine bumping into neighboring atoms with lower frequency than it does in solution.     

An electron spin resonance study of high spin forms of cobalt(II) bovine carbonic anhydrase

The ESR spectra of bovine Co(II) carbonic anhydrase at 7 K at low and high pH and of the iodide derivative have been analyzed. The spectrum of the low pH form shows axial symmetry whilst that at high pH is rhombically distorted. This anisotropy is still more accentuated in the iodide derivative. The high pH (hydroxyl) form and the iodide derivative are thought to have a tetracoordinate trigonal pyramidal structure, with a fifth more distant axial ligand. The low pH form is consistent with a pseudotetrahedral geometry previously postulated.     

DAPI derivative: a fluorescent DNA dye that can be covalently attached to biomolecules

The preparation of a DAPI (4′,6-diamidino-2-phenylindole) derivative is described. The resulting derivative retains the fluorogenic property upon binding to double-stranded DNA. Its ability for bioconjugation through amide linkage is demonstrated.