Labile Linker Research Articles

Strategies in the Design of Solution-Stable, Water-Soluble Prodrugs II: Properties of Micellar Prodrugs of Methylprednisolone

In a previous study, a physical–organic approach to the design of solution-stable, water-soluble prodrugs of the corticosteroid methylprednisolone was outlined, and several 21-esters were synthesized to test the approach. Compounds exhibiting dilute solution stabilities approaching 2 years at 25°C were reported. A complicating factor in more concentrated aqueous solutions of water-soluble prodrugs, however, is the limited extent to which hydrolysis can occur before the solution becomes saturated with respect to the relatively insoluble parent drug. In this study the advantages of micellar prodrugs as water-soluble delivery systems for parenteral administration of relatively insoluble parent drugs are explored. Micellar prodrugs, besides being highly water soluble, have additional advantages in that their micelles (a) solubilize poorly soluble degradation products which may otherwise precipitate and (b) may act as a self-stabilizing influence due to protection of the hydrolytically labile prodrug linkage within the micelle interior. Two 21-esters of methylprednisolone previously identified as having promising dilute solution stability have now been shown to self-associate in aqueous solution at higher concentrations, as determined by solubility, kinetic, and light-scattering measurements. One consequence of self-association is that free methylprednisolone, the product of prodrug hydrolysis, is solubilized in concentrated prodrug formulations. In addition, acid- and base-catalyzed hydrolysis rate constants are altered in the micelles, resulting in further prolongation of shelf life in concentrated solutions. Due to the added benefits of self-micellization, the water-soluble 21-esters investigated exhibit shelf lives exceeding 2 years at 30°C, the upper limit of the controlled room temperature range.

Effect of culture pH on the D-alanine ester content of lipoteichoic acid in Staphylococcus aureus.

The lipoteichoic acid in Staphylococcus aureus growing at high pH values contained very little alanine ester, showing that high overall levels of substitution were not essential for growth. The low alanine content could have resulted from a progressive loss due to base-catalyzed hydrolysis of the labile ester linkages.

PROTEIN INSOLUBILIZATION IN FROZEN GREENLAND HALIBUT (REINHARDTIUS HIPPOGLOSSOIDES)

Frozen storage of minced Greenland halibut (Reinhardtius hippoglossoides) at -10°C resulted in a rapid loss in salt solubility of “my ofibrillar proteins” (approximately 50% in 15 days) and in a gradual loss in water solubility of “sarcoplasmic proteins” (approximately 40% in 120 days). The water and salt inextractable protein from frozen mince (R) was completely soluble in 4% sodium dodecylsulfate (SDS) when a disulfide bond reducing agent such as mercaptoethanol (ME) was present. Other reagents, including urea and Triton X 100, were less effective in solubilizing the protein from mince after frozen storage. Evidence presented supports the thesis that disulfide bond formation contributes to the observed loss in protein extractability during frozen storage of mince. Addition of various thiol reagents to mince prior to freezing was effective in minimizing protein insolubilization. An estimated 50% of the reduced cysteine associated with protein is oxidized in mince after long time storage at -10°C. However, the kinetics of disulfide bond formation do not parallel the time course of protein insolubilization; accordingly, the possibility that disulfide bond formation is a secondary event cannot be excluded. Other lines of evidence indicate that additional covalent or strong bond interactions contribute to the formation of(R). Solubilization and boiling of(R) in SDS/ME does not dissociate high molecular weight aggregates (500–1000 Kdaltons) although these aggregates are disrupted by sodium borohydride reduction. In addition, the degree of hydrolysis of proteins in frozen mince by certain proteolytic enzymes is lower then that of fresh mince despite the complete solubilization of the mince protein by these proteolytic enzymes. The mince protein from frozen fish contains substantially more fluorescence than that of fresh fish. Fluorescence is associated with the high molecular weight peptide fraction after pepsin catalyzed hydrolysis. The amino acid composition of mince proteins after HCl or formic acid/HCl hydrolysis did not change appreciably as a result of frozen storage. The data indicate that covalent bond formation by sulfhydryl residues and other borohydride and acid labile linkages contribute to the loss in solubility of the protein in Greenland halibut mince during frozen storage at -10°C.

Modification of human hemoglobin by covalent association with soluble dextran

Stroma-free Hb solutions present some drawbacks when used as erythrocyte substitutes, mainly because the protein has a short in vivo half-life, due to its small hydrodynamic volume. Covalent coupling of oxyHb with dialdehyde-dextran ( M w ≅ 40 000 ; M n ≅ 25 000 ) leads to adducts whose properties depend upon the pH of the condensations. At pH < 9.6, many labile imine linkages are formed and the conjugates have a high molecular weight at the end of the reaction. In contrast, the final products obtained as pH increases from 9.6 to 10 contain a low-molecular-weight adduct in an increasing ratio; in this case the bond between dextran and Hb is stable and this stability is assumed to result from the rearrangement of a specific imine linkage formed at an NH 2 site of Hb, into a ketoamine group (Amadori rearrangement). Dextran-Hb conjugates have oxygen-binding properties characterized by increased oxygen affinity, and decreased subunit cooperativity and alkaline Bohr effect, relative to unconjugated Hb. These differences become less as the time of condensation reaction decreases and seem to be due to modification of amine groups involved in the salt bridges that stabilize the deoxy form of the protein. Taking into account their oxygen-binding characteristics, the low-molecular-weight conjugates can be regarded as potential erythrocyte substitutes.

Phospholipid substitution of capsular (K) polysaccharide antigens fromEscherichia colicausing extraintestinal infections

The capsular antigens of Escherichia coli causing extraintestinal infections have been analyzed extensively. The K1 antigen which is identical with the capsular antigen of Neisseria meningitidis B [1] has been described by McGuire and Binkley [2] and by Orskov et al. [3]. We have recently reported the structures of the K2, K5, K12, K62 and K82 antigens ([4-7]; Schmidt, M.A. and Jann, K., in preparation). During our study we have found that these K antigens, which are all acidic polysaccharides, tend to aggregate in aqueous solutions. The aggregation could be abolished with Triton X-100, by very mild acid hydrolysis and by treatment with alkali. These results indicated that the polysaccharides contain a lipid moiety in a labile linkage. We report here the characterization of the capsular polysaccharide antigens and the analysis of the lipid moiety of the K I2 and K82 antigens. The lipid was shown to be a phosphatidic acid. During the preparation of this manuscript similar studies were reported by Gotschlich et al. [8] concerning the polyneuraminic acid antigens of Neisseria and also the K92 antigens E. coli. Our results are in agreement with those of Gotschlich et al. and show that termination of these E. coli K antigens with a phospholipid seems to be a general structural feature. 2.1. Bacteria and cultivation

Aspects of the chemistry of donor solvent coal dissolution. The hydrogen-deuterium exchange reactions of tetralin-d 12 with Illinois No. 6 coal, coal products and related compounds

The exchange of hydrogen and deuterium atoms between Illinois No. 6 coal and tetralin-d 12 and naphthalene-d 8 has been studied at 400 °C. These exchange reactions are readily reversible and the evidence suggests that the same kinds of hydrogen atoms exchange with tetralin as with naphthalene. The influence of illinois No. 6 coal, selected coal products and other compounds representative of the structural elements in Illinois No. 6 coal on the rate of the exchange reaction between diphenylmethane and tetralin-d 12 has been studied also. The results indicate that the exchange reaction is initiated by compounds which have labile linkages or reducible structures but not by the weak acids or weak bases. The reactions are initiated by single-bond homolyses and by molecule-induced homolyses.

Formation of alkali labile linkages in DNA by hedamycin and use of hedamycin as a probe of protein-DNA complexes.

Hedamycin forms a stable complex with DNA and introduces alkali labile linkages in the DNA. These labile linkages are located at deoxyguanosine residues and are cleaved by the treatment used for breakage at bases alkylated by dimethyl sulfate. The reaction of hedamycin with all G residues in the chain is not uniform, and certain positions, particularily those in TG tracts, are especially reactive. The reaction of hedamycin with DNA can be inhibited by ethidium bromide, suggesting that intercalation is important in positioning the reactive group of hedamycin near to the base which is modified. The low amount of hedamycin needed to produce observable breakage, its specificity for reaction with DNA and its ability to react with DNA under mild conditions make it suitable for use as a probe of protein-DNA complexes. This was shown by the ability of lac repressor and RNA polymerase to block reaction of hedamycin with the DNA of the lac regulatory region.

Isolation and characterization of a tetrasialoganglioside from mouse brain, containing 9- O-acetyl, N-acetylneuraminic acid

A new ganglioside, containing an alkali-labile linkage, was extracted from mouse brain and purified. It represents 3.6% of total lipid-bound sialic acid in the tissue and was obtained in pure form with a yield of about 35%. It contains sphingosine, glucose, galactose, N-acetylgalactosamine and sialic acid in the molar ratio 1:1:2:1:4 and, upon exhaustive sialidase treatment gives the monosialoganglioside GM1. Partial acid hydrolysis, methylation analysis, gas-liquid chromatography-mass spectrometry and chromium trioxide oxidation studies showed its basic neutral glycosphingolipid core to be ganglio- N-tetraose-ceramide. Three of the four sialic acid residues are N-acetylneuraminic acid and one, as shown by gas-liquid chromatography-mass spectrometry, is 9- O-acetyl, N-acetylneuraminic acid, which contains the alkali labile linkage. 9- O-acetyl, N-acetylneuraminic acid is α-ketosidically linked to position 8 of the N-acetylneuraminic acid residue bound to position 3 of the internal galactose. The other two N-acetylneuraminic acid residues form a disialosyl residue linked to position 3 of external galactose. The complete structure of the studied ganglioside is as follows: NeuAcα2–8NeuAcα2–3Galβ1–3GalNAcβ1–4(9- O-Ac-NeuAcaα2–8NeuAcα2-1′- N-acylsphingosine, and it can be considered as a derivative of the tetrasialoganglioside GQ1b.

Trypanocidal activity of daunorubicin and related compounds.

The anthracycline antibiotic daunorubicin is, in vitro, one of the most potent trypanocidal agents reported1; it permanently abolishes the infectivity of African trypanosomes at less than nanomolar concentrations. In contrast, other anthracyclines such as doxorubicin and nogalamycin must be present in milli-molar concentrations to achieve the same effect (Table 1). Despite its uniquely high activity in vitro, daunorubicin is totally inactive against trypanosomes in infected rodents1. An investigation of this lack of in vivo activity suggested that uptake into trypanosomes is only transient2. We therefore tried to prolong exposure of trypanosomes to the drug by coupling it to a carrier macromolecule. The use of such a carrier should delay removal of the drug from the blood and simultaneously exploit the endocytotic properties of trypanosomes. Ferritin and albumin were chosen as potential carriers as these proteins are endocytosed by trypanosomes3,4. We report here that when the drug was attached by a stable linkage, activity was not maintained, but when attached by a labile covalent linkage the preparation was active both in vitro and in vivo, indicating the desirability of testing other macromolecules, such as dextran5, as carriers for trypanocidal drugs.

Struktur der Zellwandpolysaccharide in der Futtereiweiß‐Hefe Candida spec. H. III. Charakterisierung unterschiedlicher Phosphatbindungen im Mannan‐Protein‐Phosphat‐Komplex

The 31P-NMR spectra of the proteophosphomannan (PPM) and also that of mildly hydrolyzed PPM demonstrated phosphomonoester (in both preparations), acid labile and acid stable phosphodiester linkage, and polyphosphate. Decreasing in size by pronase digestion, separation, purification and characterization of the high and low molecular phosphates by 31P-NMR spectroscopy and chemical analysis revealed the mannan protein is phosphorylated in the N-glycosidically linked carbohydrate parts and in the O-glycosidically linked oligosaccharides. Another phosphate serves as a bridge between the serine of the protein and mannose, mannobioses and mannotrioses and between the threonine and a lipophilic acylglycerid unit. The origin of the polyphosphates has been discussed.

Struktur der Zellwandpolysaccharide in der Futtereiweiß-HefeCandida spec. H. III. Charakterisierung unterschiedlicher Phosphatbindungen im Mannan-Protein-Phosphat-Komplex

The 31P-NMR spectra of the proteophosphomannan (PPM) and also that of mildly hydrolyzed PPM demonstrated phosphomonoester (in both preparations), acid labile and acid stable phosphodiester linkage, and polyphosphate. Decreasing in size by pronase digestion, separation, purification and characterization of the high and low molecular phosphates by 31P-NMR spectroscopy and chemical analysis revealed the mannan protein is phosphorylated in the N-glycosidically linked carbohydrate parts and in the O-glycosidically linked oligosaccharides. Another phosphate serves as a bridge between the serine of the protein and mannose, mannobioses and mannotrioses and between the threonine and a lipophilic acylglycerid unit. The origin of the polyphosphates has been discussed.

Antibodies against sialyloligosaccharides coupled to protein.

The beta-(p-aminophenyl)ethylamine derivatives of sialyloligosaccharides can be coupled to proteins via their phenylisothiocyanate intermediates under conditions that preserve labile sugar linkages. Bovine serum albumin containing 10 to 40 mol of oligosaccharides/mol of protein and keyhole limpet hemocyanin containing 1,100 mol of oligosaccharide/mol of protein have been prepared with the following oligosaccharides: Neu-NAc alpha 2-3Gal beta 1-4Glc, NeuNAc alpha 2-6Gal beta 1-4Glc, Neu-NAc alpha 2-6Gal beta 1-4GlcNAc beta 1-4Glc, Gal beta 1-3[Neu-NAc alpha 2-6]GlcNAc beta 1-4Glc, and NeuNAc alpha 2-3Gal- beta 1-3[NeuNAc alpha 2-6]GlcNAc beta 1-4Glc. Rabbits immunized with these synthetic glycoproteins produce antibodies directed against the oligosaccharides. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides in radioimmunoassay and by double diffusion analysis in agarose gels using oligosaccharide-protein conjugates as precipitating antigens. The antibodies distinguish positional isomers of sialic acid.

Biopolymer synthesis on solid supports

Abstract

Evidence for the association of prolyl hydroxylase from chick embryos with membranes through membrane-bound procollagen

Proline hydroxylase of membrane pellets from tissues of chick embryo was solubilized by several extraction mediums. Three kinds of enzyme linkage with membranes were pointed out: a labile and non specific adsorption, a specific linkage on endogenous procollagen which is linked with the membranes, and a part of membranes themselves. Our results suggest that, in the liver, these three kinds of linkage exist. Whereas, in tibia bones only labile linkages appear by non specific adsorption or through the link with membrane procollagen.

The Cephalosporin Group of Antibiotics

This chapter discusses the cephalosporin group of antibiotics. Two sites in the cephalosporin molecule are: (1) the 7-acyl side chain, leading to the production of 7-acylaminocephalosporanic acids—for example, ring-substituted phenylacetylcephalosporanic acids (analogous to benzylpenicillin) and ring-substituted phenoxyacetylcephalosporanic acids members of both types being active mainly against gram-positive bacteria, and (2) the 3-acetoxymethyl side chain leading to the production of deacetylcephalosporins and deacetoxylcephalosporins. The former have about half the activity of the parent compounds against gram-positive bacteria, such as staphylococci, but much reduced activity against gram-negative bacteria. Removal of the acetoxy group gives 3-hydroxyl derivatives,which are less antibacterial than the parent acetoxy compounds. Cephalosporin C, cephalothin, and cephaloglycin all lose antibacterial activity rapidly when incubated with rat liver homogenate, because of removal of the acetoxy group; however, cephaloridine and cephalexin do not possess these labile ester linkages and are thus, not inactivated.

Gas chromatography and mass spectrometry of some trimethylsilyl derivatives of urinary glucuronides

Guidelines are presented to aid the recognition and interpretation of mass spectra for trimethylsilylated (TMS) glucuronides of aromatic acids and substituted phenols. Masses associated with fragmentation of the derivatized glycon include m e 465, 464, 449, 392, 375, 359, 333, 331, 319, 305, 257, 243, 217, 204, 169, 147, 143, 129, 117, 103, 95, and 93, with m e 375 usually being one of the most abundant masses in the spectrum. Fragmentation of the conjugate after electron impact led to the appearance of a high-abundance fragment of the aglycon having one of the following structures: (a) (TMS) x -Ar-OTMS +, (b) (TMS) x -Ar-OH +, or (e) (TMS) x -AR-O +, where Ar indicates the aromatic ring and (TMS) x indicates polar hydrogens (OH or NH) on the aglycon replaced by TMS groups. Nine of twelve conjugates studied are ethereally linked through a phenolic hydroxyl group; however, the remaining three are ester linked through an aromatic carboxylate group. The latter appcared to be a more labile linkage, since derivatization apparently cleaved the ester bond, forming appreciable quantities of the fully derivatized aglycon and glucopyranurono-(6→1)-lactone. Gas chromatographic retention (methylene units) and mass spectral data are presented for a number of glucuronides isolated from human urine by high-resolution liquid chromatography.

The alanine ester content and magnesium binding capacity of walls of Staphylococcus aureus H grown at different pH values

Recent studies have shown that teichoic acids are functionally involved in the uptake of Mg 2+ and that their ability to bind Mg is reduced by the presence of alanine ester substituents. Walls of Staphylococcus aureaus H grown at pH 5 contain more ester alanine and bind less magnesium than do walls of S. aureaus grown under similar conditions at pH 6 and 7. The differences in alanine content are largely due to its removal by base catalysed hydrolysis of the labile ester linkages during growth in media of pH 6 and 7 and the incorporation of alanine esters into the walls does not appear to be influenced by the pH of the medium in which the bacteria are grown under the conditions described. The decreased magnesium binding capacity of walls of S. aureus grown at pH 5 correlates with their increased alanine ester content but there is no proportional relationship between these two quantities.

Chemical characterization of cytochrome P-450 cam

Electrofocusing experiments indicate isoelectric points at pH 4.55 for cytochrome P-450 cam and 4.67 for P-450 cam-camphor complex. The hemeprotein consists of a single polypeptide chain with asparagine or aspartic acid on the amino terminus and valine on the carboxyl terminus. The amino acid composition based on a molecular weight of 45,000 is reported. A single molecule of ferriprotoporphyrin IX is attached to the protein through acid labile linkages. A small covalently linked carbohydrate moiety was also noted.

A lipid intermediate in the synthesis of a poly-(N-acetylglucosamine 1-phosphate) from the wall of Staphylococcus lactis N.C.T.C. 2102.

1. The enzymic synthesis of the wall polymer poly-(N-acetylglucosamine 1-phosphate) in Staphylococcus lactis N.C.T.C. 2102 was studied by using UDP-[acetyl-(14)C]N-acetylglucosamine and the corresponding nucleotide containing (32)P. 2. Labelled material was extracted from the particulate enzyme preparation with butan-1-ol. Pulse-labelling experiments indicated that this material contained an intermediate in the biosynthesis. 3. The lipid intermediate was partially purified, and chemical and enzymic degradation showed that it was composed of N-acetylglucosamine 1-pyrophosphate in labile ester linkage to an organic-soluble alcohol, possibly a polyisoprenoid alcohol. The methanolysis of sugar 1-pyrophosphate derivatives, including nucleoside diphosphate sugars, is discussed in relation to degradation products obtained from the lipid. 4. The lipids from the particulate enzyme preparation probably contained another compound in which N-acetylglucosamine 1-phosphate is attached to an organic-soluble alcohol; this may participate in the biosynthesis of another polysaccharide. 5. The function of the lipid intermediate in polymer biosynthesis is discussed.

Emergency Treatment of Severe Bacterial Infection

This chapter discusses the cephalosporin group of antibiotics. Two sites in the cephalosporin molecule are: (1) the 7-acyl side chain, leading to the production of 7-acylaminocephalosporanic acids—for example, ring-substituted phenylacetylcephalosporanic acids (analogous to benzylpenicillin) and ring-substituted phenoxyacetylcephalosporanic acids members of both types being active mainly against gram-positive bacteria, and (2) the 3-acetoxymethyl side chain leading to the production of deacetylcephalosporins and deacetoxylcephalosporins. The former have about half the activity of the parent compounds against gram-positive bacteria, such as staphylococci, but much reduced activity against gram-negative bacteria. Removal of the acetoxy group gives 3-hydroxyl derivatives,which are less antibacterial than the parent acetoxy compounds. Cephalosporin C, cephalothin, and cephaloglycin all lose antibacterial activity rapidly when incubated with rat liver homogenate, because of removal of the acetoxy group; however, cephaloridine and cephalexin do not possess these labile ester linkages and are thus, not inactivated.