The 5-O-beta-D-galactofuranosyl-containing peptidophosphogalactomannan of Penicillium charlesii. Characterization of the mannan by 13C NMR spectroscopy

Penicillium charlesii secretes a galactofuranosyl and phosphodiester-containing peptidophosphogalactomannan (pPGM). A linear mannan was prepared from pPGM by treatment with 48% aqueous HF which selectively cleaves galactofuranosyl and phosphodiesters; treatment with alkaline borohydride releases the mannan from the polypeptide. Mannan from P. charlesii cultured in D-[1,2-13C2]glucose contained mannopyranosyl residues which were enriched in 13C at both C-1 and C-2 and, to a lesser extent, at C-5 and C-6. The mannan was examined with a combination of 13C NMR INADEQUATE pulse sequence and selective 13C saturation to assign the resonance frequency of anomeric carbons directly coupled to specific C-2 signals. Three species of mannosyl residues, each substituted with a glycosidic linkage at C-2, and a fourth species substituted at C-6 and not substituted at C-2 were observed. Mannan obtained from P. charlesii cultured in D-[6-13C]glucose contained mannopyranosyl residues which were enriched in 13C primarily in C-6. The mannan was examined by DEPT 13C NMR to determine the number of species which were substituted at C-6. Mannan, treated as described above, contained a 1----6-linked mannopyranosyl species. pPGM contains minor quantities of at least four other substances attached to hydroxymethyl groups of the hexosyl residues.     

Probing the active site of reconstituted human placental aromatase [Poster 02]

To gain some insight into the factors that determine active site-ligand binding and to delineate which sites of the androstene nucleus are critical for recognition by the aromatase enzyme, we compared the effect of various substituents located at the biologically important C-2, 6, 11, 16, 17, and 19 positions. Based on two independent measurements, namely optical difference spectra and inhibition of enzyme activity, we report: (a) oxygen functions at positions C-11 do not appear to induce difference spectra although they cause weak inhibition; (b) 2ß- and 16-hydroxyandrostenes induce type-I spectra and weak inhibition; (c) C-6 and C-19-substituted androgens are type-I spectrum inducers and strong inhibitors; (d) the presence of nonspecific binding sites on the aromatase enzyme cannot be ruled out.     

Steroidal carbonitriles as potential aromatase inhibitors

Estrogens, responsible for the growth of hormone-dependant breast cancer are biosynthesized from androgens involving aromatase enzyme in the last rate limiting step. Inhibition of aromatase is an efficient approach for the prevention and treatment of breast cancer. Novel 4-phenylthia derivatives (2, 3 and 7) have been synthesized as aromatase inhibitors. The synthesized compounds (2, 3 and 7) exhibited noticeable enzyme inhibiting activity. Kinetics study of these compounds (2, 3, and 7) showed negligible inhibition of the enzyme under conditions conducive for irreversible inhibition of the enzyme. Introduction of unsaturation at C-4, C-1 & 4 or C-4 & 6 (compounds 5, 9 and 11) was observed to not be an effective strategy for entrancing aromatase inhibiting activity in 17-oxo-16β-carbonitrile derivatives. The D-seco derivatives (13-15 and 17) having unsaturation at C-4, C-1 & 4 or C-4 & 6 along with carbonitrile function in ring-D showed complete loss of aromatase inhibiting activity.     

Hydroxylation of [3H]5 alpha-androstane-3 beta,17 beta-diol by whole tissue, epithelial cells and fibroblasts from the same hyperplastic human prostate

Hydroxylations of 3 beta-hydroxy 5 alpha-dihydro C19-steroids are terminal reactions by which male accessory sex organs dispose of intracellular androgens. Cellular androgen egress is of particular interest in benign prostatic hyperplasia (BPH) where the elevated nuclear 5 alpha-dihydrotestosterone-receptor content may be implicated in the etiology of the disease. We here report substitution of hydroxyl groups at C-6 alpha, C-7 beta and predominantly at C-7 alpha of [3H]5 alpha-androstane-3 beta,17 beta-diol on incubation of 3 and 8.5 nM substrate concentrations with minced and explanted human BPH tissue. Fibroblasts isolated from the same prostatectomy specimen hydroxylated 3 nM radiosubstrate mainly at C-6 alpha, with extensive metabolism to 17-oxosteroids. Epithelial cells from the same tissue source substituted to the same extent at the three positions. Competing 3 beta-hydroxysteroid dehydrogenase exceeded hydroxylase activity only in epithelial-cell cultures. Our findings support previous evidence that prostatic epithelial and stromal cells make different contributions to androgen disposition by the 3 beta-hydroxysteroid pathway.     

Preparation and some properties of rat skeletal-muscle polyribosomes

1. A series of d-galactose derivatives substituted at C-1 and C-6 were tested for active accumulation by everted segments of hamster and rat intestine. 2. d-Galactose and 6-deoxy-6-fluoro-d-galactose were accumulated far more rapidly than 6-deoxy- and 6-chloro-6-deoxy-d-galactose, and this is interpreted as due to hydrogen-bonding at C-6 during the transport process. 3. 6-Bromo-6-deoxy- and 6-deoxy-6-iodo-d-galactose were not actively transported, indicating that the allowed size of substituent at C-6 lies between that of chlorine and bromine atoms. 4. Similar results were obtained at C-1. Both methyl alpha-d-galactopyranoside and methyl beta-d-galactopyranoside were well transported, but methyl beta-d-thiogalactopyranoside and 1-deoxy-d-galactose were not transported; d-galactopyranosyl fluoride was transported, but only poorly. Again hydrogen-bonding is suggested. 5. It is proposed that d-glucose is the ideal structure for active transport and that binding occurs at C-1, C-2, C-3, C-4 and C-6. Loss of two or more of these bonds usually causes loss of active transport. 6. By plotting Lineweaver-Burk plots of the rates of transport of the galactose derivatives, the apparent V and K(m) values were obtained. With hamster intestine both these values were very reproducible. Contrary to expectation, V varied for different sugars. 7. The K(i) of some of the analogues modified at C-1 and C-6 was determined with methyl alpha-d-glucoside as substrate. 8. An attempt to alkylate the carrier by using methyl 3,4-anhydro-alpha-d-galactoside was unsuccessful. There was no evidence that this compound was bound to the carrier.     

Synthesis of 11 alpha-hydroxyprogesterone haptens

C-4 and C-6 bridged haptens of 11 alpha-hydroxyprogesterone, an anti-androgen, were respectively synthesized via 4, 5 and 5 alpha, 6 alpha epoxide ring openings using 3-mercaptopropanoic acid. Substitution of 6-bromo derivative of progesterone using the same reagent unexpectedly afforded a C-4 substituted product instead of the normal C-6 substituted product previously reported in the literature.     

Design, synthesis, and docking studies of novel benzopyrone derivatives as H(1)-antihistaminic agents

Two new series of 2H-1-benzopyran-2-one derivatives substituted at C-6 and/or C-7 with propanolamines, and/or piperazine propanol derivatives have been synthesized and assayed for the H(1)-histamine antagonist. Twelve of the 20 newly synthesized 4- substituted benzopyrones have shown potent antihistaminic H(1) activity. In addition, molecular modeling and docking of the tested compounds into high affinity histamine binding protein (HBP) and histamine N-methyltranseferase (HNMT) active site in complex with its bound inhibitor (diphenhydramine) was performed in order to predict the affinity and orientation of these compounds at the active sites. The ICM score values show good agreement with predicted binding affinities obtained by molecular docking studies as verified by pharmacological screening. The results showed similar orientation of the target compounds at HBP, and HNMT active sites compared with reported histamine H(1) antagonist. Also, it was concluded that in order for the compounds to be active, they must bind with both active sites of HNMT enzyme (two pockets) to inhibit it. Compounds 8c, 8i, 11g, 11i, and 11k; observe the maximum activities.     

Chemical structure of the lipid A component of Plesiomonas shigelloides and its taxonomical significance

Abstract The chemical structure of the lipid A moiety of the lipopolysaccharide of the type strain of Plesiomonas shigelloides was elucidated. It consists of a β-(1 → 6)-linked glucosamine disaccharide carrying phosphate groups at C-1 of the reducing and at C-4' of the non-reducing glucosamine. It contains a total of 6 residues of fatty acids, 2 amide-linked and 4 ester-linked. The amino groups of the backbone disaccharide are N -acylated by substituted 3-hydroxyacyl residues: at the reducing glucosamine by 3-O-(14:0)14:0; and at the non-reducing glucosamine by 3-O-(12:0)14:0.
Two residues of 3-hydroxytetradecanoic acid are linked to C-3 and C-3' of the glucosamine residues; the hydroxy groups of these ester-linked 3-hydroxytetradecanoic acids are unsubstituted. In free lipid A, the hydroxyl groups at C-4 and C-6' are unsubstituted, indicating that the 2-keto-3-deoxyoctonic acid (KDO) is linked to C-6' of the non-reducing glucosamine, as was shown with enterobacterial lipid A. The taxonomical significance of these structural details is discussed.     

TEMPO-mediated selective oxidation of substituted polysaccharides--an efficient approach for the determination of the degree of substitution at C-6

2,2,6,6-Tetramethyl-1-piperidinyloxy radical (TEMPO)-mediated oxidations of substituted polysaccharides were studied at pH 10.2 and at a temperature of 0 °C with NaOCl as the oxidant. The reaction is highly selective, and it was shown that the oxidation can proceed to a yield of nearly 100%. The oxidation process was investigated for several substituted polysaccharides, especially for a series of hydroxypropyl guar gums with different molar degrees of substitution. It was shown that this oxidation can be used for the determination of the degree of substitution at C-6 of the polysaccharide by comparing the difference in oxidation yield between substituted and natural polysaccharides. Studies on several hydroxypropyl guar gums showed that the degrees of substitution at C-6—for MS of 0.08, 0.34, 0.62, and 1.08—are 0.06, 0.24, 0.40, and 0.44, respectively. The results were extended to other polysaccharides such as carboxymethyl cellulose, cationic guar gum, carboxymethyl pullulan, and methyl cellulose. It can be concluded that the TEMPO-mediated oxidation is a useful method for the determination of the DS at the substituted C-6 position for different kinds of modified polysaccharides.     

Three iridoid glycosides from Viburnum furcatum

Three new bitter iridoid glycosides having an 8,10,11-oxygen substituted iridoid skeleton with an isovaleryl moiety at C-1, have been isolated from the ether and ethyl acetate soluble fractions of the leaves of Viburnum furcatum. Two of them had a glucose moiety at C-11 of the iridoid skeleton and a p-coumaroyl group linked to C-6 of the sugar, and they were found to be geometrical isomers about the double bond of the p-coumaroyl2 moiety. The third one was characterized as a alloside of the same aglycone.     

Differences in steroid specificity for rat androgen binding protein and the cytoplasmic receptor.

Two proteins in the rat, androgen binding protein (ABP) and the cytoplasmic receptor (CR), have high affinity and limited capacity for binding androgens. To determine the structural requirements for binding with high affinity, each protein was partially purified and the ability of over 100 steroids to compete with [3H]dihydrotestosterone (17 beta-hydroxy-5 alpha-androstan-3-one) for binding sites was assessed. The results indicate marked differences in the steroid specificities of the two proteins. Some alterations of dihydrotestosterone at C-2 or C-2 and C-3 increase binding to ABP two to four-fold. Similarly, the affinity of 17 beta-hydroxy-7 alpha-methyl-4-estren-3-one for ABP increases two-fold when a double bond is created at C-14. Addition of a methyl group in the alpha position at C-7 or C-17, or an ethinyl group at C-17 cause little change in affinity; however, modifications at C-11 and C-17 beta, and deletion of the methyl group at C-10 significantly impair binding to ABP. Binding to the CR is maintained or increased by deletion of the methyl group at C-10. Binding is lessened by modifications at C-3 and C-17 beta. Most alterations at C-2, C-7, C-11, and C-17 alpha have only minor effects on binding to the CR. These studies should provide a molecular basis for predicting the effects of specific structural modifications. When some modifications at C-2 or C-2 and C-3 are combined with changes at C-17 beta, the resulting steroids retain very high affinity for ABP and very limited binding to the CR. Such steroids may provide a means for assessing the function of ABP.     

Structure-activity relationships of 2-, 4-, or 6-substituted estrogens as aromatase inhibitors

Aromatase, which is responsible for the conversion of androgens to estrogens, is a potential therapeutic target for the selective lowering of estrogen levels in patients with estrogen-dependent breast cancer. To develop a novel class of aromatase inhibitors, we tested series of 2- and 4-substituted (halogeno, methyl, formyl, methoxy, nitro, and amino) estrones (7 and 9), as well as series of 6alpha- and 6beta-substituted (alkyl, phenalkyl, and alkoxy) estrones (13 and 14), and their estradiol analogs (8, 10, 11, and 12) as aromatase inhibitors. All of the inhibitors examined blocked the androstenedione aromatization in a competitive manner. Introduction of halogeno and methyl functions at C-2 of estrone as well as that of a phenalkyl or methyl function at the C-6alpha or C-6beta position markedly increased affinity to aromatase (apparent K(i) value=0.10-0.66 microM for the inhibitors versus 2.5 microM for estrone). 6alpha-Phenylestrone (13c) was the most powerful inhibitor among the estrogens studied, and its affinity was comparable to that of the androgen substrate androstenedione. Estradiol analogs were much weaker inhibitors than the corresponding estrone compounds in each series, indicating that the 17-carbonyl group plays a critical role in the formation of a thermodynamically stable enzyme-inhibitor complex.     

The structure of unit B-type glycopeptides from porcine thyroglobulin

The structure of Unit B-type glycopeptides (monosialo-type and disialo-type) was investigated by Smith degradation, methyllation, and mass spectral analysis. These glycopeptides contain three peripheral sugar chains. Two are composed of D-galactose residues linked at C-6 and 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and the other is composed of a D-galactose residues linked at C-6, a 2-acetamido-2-deoxy-D-glucose residues linked at C-4, and a D-mannose residue linked at C-2. Most of these peripheral sugar chains are linked to two inner D-mannose residues which are substituted at C-3 and C-6, and constitute branching points. L-Fucose and N-acetyl-neuraminic acid residues are nonreducing terminal groups, and a di-N-acetylchitobiose moiety is linked to an asparagine residue in the peptide moiety. By methylation analysis of the oligosaccharide obtained by hydrazinolysis of the disialoglycopeptide, the L-fucose residues was found to be linked to C-6 of the 2-acetamido-2-deoxy-D-glucose residue linked to the asparagine residue. From these results, and from the previously reported data on the sugar sequence and the anomeric configurations of the linkages between sugar residues, structures for these glycopeptides are proposed.     

Steroid structural requirements for high affinity binding to human sex steroid binding protein (SBP)

The sex steroid binding protein (SBP) which binds androgens circulating in the blood of man has been examined to determine the structural requirements for high affinity binding. SBP was purified partially and the ability of each of more than 150 steroids to compete with [³H]dihydrotestosterone (17β-hydroxy-5α-androstan-3-one) for binding to SBP was assessed.Binding was enhanced by reduction of the Δ⁴ double bond to 5α-dihydro, addition of a methyl group at C-4 and in one case unsaturation at C-14, 15. Affinity was always reduced by modifications of the C-17β hydroxy. Binding was also severely decreased by deletion of the keto moiety at C-3; however, relatively high affinity was retained by an alcohol or an unsubstituted pyrazole group at C-3. Certain alpha surface substitutions such as 17α-ethinyl had limited effects on binding; whereas, other modifications such as 7α-methyl or 17α-methyl caused significant reduction in binding. Most modifications at C-2, 6, 9 or 11 also impaired affinity, and the 5β steroids had reduced affinity.     

Purification and mode of action of two different arabinoxylan arabinofuranohydrolases from Bifidobacterium adolescentis DSM 20083

Two novel arabinofuranohydrolases (AXH-d3 and AXH-m23) were purified from Bifidobacterium adolescentis DSM 20083. Both enzymes were induced upon growth of Bi. adolescentis on xylose and arabinoxylan-derived oligosaccharides. They were only active with arabinoxylans and therefore denoted as arabinoxylan arabinofuranohydrolases. Their optimal activity was at pH 6 and 30–40 °C. They were very specific in their mode of action and were clearly different from AXH-m from Aspergillus awamori. AXH-m23 released only arabinosyl groups, which were linked to the C-2 or C-3 position of singly substituted xylose residues in arabinoxylan oligomers. AXH-d3 hydrolysed C-3-linked arabinofuranosyl residues of doubly substituted xylopyranosyl residues of arabinoxylans and arab- inoxylan-derived oligosaccharides. No activity was observed with C-2-linked arabinofuranosyl residues of these doubly substituted xylopyranosyl residues, or against C-2- and C-3-linked arabinofuranosyl residues of singly substituted xylopyranosyl residues. The combination of AXH-d3 and AXH-m showed low debranching activity with highly substituted glucurono-arabinoxylans. However, arabinoxylan from wheat flour was debranched almost completely. Received: 12 January 1999 / Accepted: 17 January 1999     

Pim kinase inhibitory and antiproliferative activity of a novel series of meridianin C derivatives

A novel series of meridianin C derivatives substituted at C-5 position were prepared. These derivatives were tested for their kinase inhibitory potencies against all three family members of the pim kinases (Pim-1, Pim-2 and Pim-3). In addition, their antiproliferative activity towards three human leukemia cell lines as MV4-11, Jurkat clone E6-1 and K562 has been evaluated. Structure activity relationships at C-3 and C-5 positions of indole were performed to better understand the mechanism behind the enhanced potency. Compound 7f, the most active compound of the series showed a single-digit nanomolar IC₅₀ with selectivity towards Pim-1 kinase.     

Inhibition of Src kinase activity by 7-[(2,4-dichloro-5-methoxyphenyl)amino]-2-heteroaryl-thieno[3,2-b]pyridine-6-carbonitriles

7-[(2,4-Dichloro-5-methoxyphenyl)amino]thieno[3,2-b]pyridine-6-carbonitriles with various heteroaryl groups at C-2 are inhibitors of Src kinase activity. Of these new analogs, compounds substituted at C-2 by a 3,5-furan or a 2,5-pyridine had the best activity in the Src enzyme and cell assays.     

Structural requirements for binding to the sugar-transport system of the human erythrocyte

The structural requirements for binding to the glucose/sorbose-transport system in the human erythrocyte were explored by measuring the inhibition constants, K(i), for specifically substituted analogues of d-glucose when l-sorbose was the penetrating sugar. Derivatives in which a hydroxyl group in the d-gluco configuration was inverted, or replaced by a hydrogen atom, at C-1, C-2, C-3, C-4 or C-6 of the d-glucose molecule, all bound to the carrier, confirming that no single hydroxyl group is essential for binding to the carrier. The binding and transport of 1-deoxy-d-glucose confirmed that the sugars bind in the pyranose form. The relative inhibition constants of d-glucose and its deoxy, epimeric and fluorinated analogues are consistent with the combination of beta-d-glucopyranose with the carrier by hydrogen bonds at C-1, C-3, probably C-4, and possibly C-6 of the sugar. Both polar and non-polar substituents at C-6 enhance the affinity of d-glucose derivatives relative to d-xylose, and d-galactose derivatives relative to l-arabinose, and it is suggested that the carrier region around C-6 of the sugar may contain both hydrophobic and polar binding groups. The spatial requirements at C-1, C-2, C-3, C-4 and C-6 were explored by comparing the relative binding of d-glucose and its halogeno and O-alkyl substituents. The carrier protein closely approaches the sugar except at C-3 in the d-gluco configuration, C-4 and C-6. d-Glucal was a good inhibitor, showing that a strict chair form is not essential for binding. 3-O-(2',3'-Epoxypropyl)-d-glucose, a potential substrate-directed alkylating agent, bound to the carrier, but did not inactivate it.     

7-(aryl/heteroaryl-2-ylethynyl)-4-phenylamino-3-quinolinecarbonitriles as new Src kinase inhibitors: addition of water solubilizing groups

New 4-phenylamino-3-quinolinecarbonitriles with a 7-ethynyl group substituted by a pyridine, phenyl or thiophene ring containing basic water solubilizing groups were prepared and evaluated as Src kinase inhibitors. Of these new analogs, potent activity was observed with compounds having a (2,4-dichloro-5-methoxyphenyl)amino group at C-4, a methoxy or ethoxy group at C-6, and a pyridyl group bearing a dimethylamine or N-methylpiperazine on the ethynyl group at C-7.     

Structures of the major oligosaccharides from a human rectal adenocarcinoma glycoprotein

Four oligosaccharides in the reduced form were isolated from RMG (a mucin-type glycoprotein from a human rectal adenocarcinoma). They were 1) Sia alpha s2 leads to 6GalNAc-ol; 2) Sia alpha 2 leads to 6(Gal beta 1 leads to 3)GalNAc-ol; 3) Sia alpha 2 leads to 6(GlcNAc beta 1 leads to 3)GalNAc-ol; and 4) Sia alpha 2 leads to 6(GalNAc alpha 1 leads to 3)GalNAc-ol. The amounts of oligosaccharides 1, 2, 3, and 4 corresponded to 27, 5, 11, and 8% of the total N-acetylgalactosaminitol produced on alkaline borohydride treatment of RMG. To determine the structures of oligosaccharides 2, 3, and 4, a mixture of the three was subjected to methylation analysis which revealed that the N-acetylgalactosaminitol was substituted at both C-3 and C-6 and other sugars at the nonreducing ends. Desialized oligosaccharides were prepared, and the structures were deduced by analysis of the permethylated sugars on gas-liquid chromatography/mass spectrometry. Anomeric configurations were determined by exoglycosidase digestions except for galactose which was analyzed by chromium trioxide oxidation.