Walter Korytnyk

Glycosidases in cancer and invasion

SummaryGlycosidases have been demonstrated to be elevated in the interstitial fluid of tumors, sera of animals and patients with tumors, and in some tumor tissue as compared to normal adjacent tissue. Elevations of serum β-N-acetylglucosaminidase and β-glucuronidase most commonly have been found to occur and these enzymes have been shown to be secreted into the extracellular medium by many different tumor cell types in vitro. The mechanism of cellular release of these hydrolytic enzymes probably involves tumor lysosomal exocytosis. Increased tumor glycosidase levels may promote increased tumor cell shedding from primary tumors, local invasion and perhaps be responsible directly, or indirectly for structural changes in tumor cell surface glycoconjugates. These cell surface changes could facilitate tumor cell thrombus formation, secondary site implantation and attachment in the microcirculation to endothelial cells and/or subendothelial basement membrane components.Other studies have demonstrated a correlation between metastatic cell potential and increased endoglycosidase and polysaccharide lyase activity. Generally, metastatic tumor cell variants have been found to be more invasive and capable of degrading proteoglycan basement membrane components, in part due to these increased levels of degradative enzymes. Hence, it is of considerable interest to develop inhibitors against these enzymes. Initial studies with glucuronidase inhibitors in the therapy of bladder tumors have been promising and with the advent of better agents and the use of appropriate in vitro metastatic models it may be possible to design and develop agents which interfere in various metastatic events and limit tumor progression.

ROSFIT: An enzyme kinetics nonlinear regression curve fitting package for a microcomputer

Abstract A nonlinear regression curve fitting package has been specifically developed for enzyme kinetic analyses for use on the Hewlett-Packard HP-85 microcomputer. Data are entered in a conversational manner. Data can be changed, deleted or added, and data sets can be stored and retrieved from a magnetic tape cassette. Data can be fit to any of nine models: the Michaelis-Menten equation, substrate inhibition, random bi bi, ordered bi bi, ping pong bi bi, competitive inhibition, classical noncompetitive inhibition, modern noncompetitive inhibition, or uncompetitive inhibition. The printout for each model consists of several goodness-of-fit statistics, the parameter estimates with 95% confidence intervals, the variance-covariance, and correlation matrices, a residual analysis, and graphs. For example, for competitive inhibition the graphs provided are v vs [S], v vs [I], 1 v vs 1 [S] , and 1 v vs [I] with up to four concentrations of the second compound plotted on each graph. The nonlinear regression algorithm in the package is that of Marquardt. The values determined by ROSFIT are essentially the same as those found using the BMDPAR, BMDP3R, and NONLIN programs on a Univac 90 60 mainframe computer. A typical run for competitive inhibition with 46 data points took a total time of about 18.5 min, not including dat entry time, 14 min for the graph generation, 2 min for the three required iterations, and 2.5 min for miscellaneous operations.

S-, N- and O-glycosyl derivatives of 2-acetamido-2-deoxy-D-glucose with hydrophobic aglycons as potential chemotherapeutic agents and N-acetyl-β-D-glucosaminidase inhibitors

S-, N-, and O-Glycosyl derivatives of 2-acetamido-2-deoxy-D-glucose with hydrophobic aglycons have been obtained as potential, plasma-membrane active agents. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-1-thio-beta-D-glucopyranose (6) was converted into benzyl, diphenylmethyl, triphenylmethyl, and other thioglycosides. Acylation of 6 gave adamantoyl and haloacetyl derivatives. A similar series of N- and O-glycosyl derivatives was obtained from the corresponding NH2-1 and OH-1 analogs of 6, such as O- and N-dinitrophenyl, O- and N-adamantoyl, and N-4-methylbenzylidene derivatives. Several N- and S-glycosyl derivatives were found to inhibit mouse mammary adenocarcinoma (TA3) cells in vitro as well as N-acetyl-beta-D-glucosaminidase from beef liver.

Analogs of cell surface carbohydrates. Synthesis of d-galactose derivatives having an ethynyl, vinyl or epoxy residue at C-5☆

Compounds derived from D-galactose having an ethynyl, vinyl, or epoxide residue at C-5, as well as 7,7-dibromo olefinic, isomeric 7,7-gem-bromofluoro olefinic, and 6,6-gem-difluoro derivatives were obtained from 1,2:3,4-di-O-iso-propylidene-alpha-D-galacto-hexodialdo-1,5- pyranose.

Synthesis and biological activity of some 1-N-substituted 2-acetamido-2-deoxy-β-d-glycopyranosylamine derivatives and related analogs

Several 1-N-substituted derivative [haloacetyl-, glycyl-, (dimethyl)amino-acetyl-, azidoacetyl-, trifluoroacetyl-, and trifluoromethylsulfonyl-] of 2-acetamido-2-deoxy-3,4,6-tri-O-acetyl-beta-D-glucopyranosylamine (1) were synthesized as potential metabolic inhibitors of cellular-membrane glycoconjugates. Several fully acetylated derivatives were found to inhibit growth of mouse mammary adenocarcinoma TA3, leukemia L1210, or leukemia P-288 cells at 1-0.01 mM concentration in vitro. Some of these derivatives were less active after O-deacetylation. Analogs of 1 in which NH2-1 was replaced by OH- or OAc-1 were also active on the same cell systems. The growth-inhibitory activity was correlated with inhibition of the incorporation of 2-amino-deoxy-D-glucose and L-leucine into a macromolecular fraction.

Fluorinated carbohydrates as potential plasma membrane modifiers. Synthesis of 3-deoxy-3-fluoro derivatives of 2-acetamido-2-deoxy-d-hexopyranoses☆

Treatment of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D-allopyranoside with diethylaminosulfur trifluoride or of the 3-O-mesyl derivative with tetrabutylammonium fluoride gave the 2,3-unsaturated compound instead of the expected 3-deoxy-3-fluoro derivative. The latter was obtained when benzyl 2-acetamido-4,6-di-O-benzyl-2-deoxy-3-O-mesyl-alpha-D-allopyran oside was treated with potassium fluoride. Methyl 2-azido-4,6-O-benzylidene-2-deoxy-alpha-D-altropyranoside was converted into the 2-acetamido- and 2-phthalimido-3-O-mesyl derivatives; when treated with fluoride nucleophile, these gave only the 2,3-aziridine derivative. However, treatment of the 2-azido-2-deoxy derivative with diethylaminosulfur trifluoride gave methyl 2-azido-2,3-dideoxy-3-fluoro-alpha-D-mannopyranoside which, after reduction, deprotection, and acetylation, gave the acetylated derivative of methyl 2-acetamido-2,3-dideoxy-3-fluoro-alpha-D-mannopyranoside in excellent yield. These acetylated 3-fluoro derivatives exhibited inhibition of cell growth of murine L1210 leukemia cells in culture at micromolar concentrations.

General methods for modification of sialic acid at C-9. Synthesis of N-acetyl-9-deoxy-9-fluoroneuraminic acid

Methyl 5-acetamido-3,5-dideoxy-2-O-methyl-D-glycero-D-galacto-2-nonulopyrano sate was converted into the 9-O-trityl derivative and the remaining hydroxyl groups were protected as benzyl ethers. Removal of the trityl group, followed by treatment with diethylaminosulfur trifluoride gave the 9-deoxy-9-fluoro derivative, and deprotection N-acetyl-9-deoxy-9-fluoroneuraminic acid (8). In another procedure, coupling of 2-acetamido-2,6-dideoxy-6-fluoro-D-glucopyranose with potassium di(tert-butyl) oxaloacetate, followed by hydrolysis and decarboxylation gave 8. Some of the derivatives were active as inhibitors of growth of mouse mammary adenocarcinoma (TA3) and L1210 cells in culture.

A General Method for the Modification of the 9-Position of N-Acetyl-neuraminic Acid (NANA) and the Synthesis of Its 9-Fluoroanalogue

Abstract Analogs of N-acetylneuraminic acid (1, NANA) are of considerable interest as potential modifiers of cell surface sialic acid. NANA is greatly responsible for the negative charge on cell surface, and plays an important role in cell-to-cell Interactions, immunogenic properties, and metastasis.1 Considerable difficulty has been encountered in the development of approaches for the modification of this molecule.2 The only fluorinated sialic acid which has been reported 1s N-acetyl-3-fluoro-neuraminic acid, a compound which was obtained in only 1% yield.3 A systematic approach to the introduction of protecting groups into NANA, a process which would make specific groups amenable for modification, has not been explored. In this communication we report a successful application of a protection-deprotection approach (Scheme 1) to the synthesis of N-acetyl-9-deoxy-9-fluoroneuraminic acid (7).

Fluorinated carbohydrates as potential plasma membrane modifiers and inhibitors. Synthesis of 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.

Reaction of benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-mesyl-alpha-D-galactopyran oside with cesium floride gave benzyl 2-acetamido-3,6-anhydro-4-O-benzyl-2-deoxy-alpha-D-galactopyranoside instead of the desired 6-fluoro derivative. Acetonation of benzyl 2-acetamido-2-deoxy-6-O-mesyl-alpha-D-galactopyranoside gave the corresponding 3,4-O-isopropylidene derivative. The 6-O-mesyl group was displaced by fluorine with cesium fluoride in boiling 1,2-ethanediol, and hydrolysis and subsequent N-acetylation gave the target compound. In another procedure, treatment of 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-alpha-D-galactose with N-(diethylamino)sulfur trifluoride gave 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-galactose which, on acid hydrolysis followed by N-acetylation, gave 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.

Modifications at C-3 and C-4 of 2-amino-2-deoxy-d-glucose☆

Modifications at C-3 and C-4 of 2-amino-2-deoxy-D-glucose have been developed. A 3,4-double bond was introduced into benzyl 2-acetamido-2-deoxy-3,4-di-O-methylsulfonyl-alpha-D-glucopyranoside by treatment with NaI and Zn. Epoxidation of the double bond with m-chloroperoxybenzoic acid gave an exo-epoxide exclusively. The stereochemistry of the epoxidation product has been confirmed by an alternative synthesis. An analysis of the 1H-n.m.r. spectra indicates that both the 3,4-unsaturated derivatives and the epoxide exist in the OH1 (D) conformation. Nucleophilic reagents (F-, I-) opened the 3,4-epoxide to give 4-substituted derivatives having the D-gulo configuration. Thus, 2-acetamido-1,3,6-tri-O-acetyl-2,4-dideoxy-4-iodo-alpha-D-gulopyranose and 2-acetamido-1,3,6-tri-O-acetyl-3,4-dideoxy-4-fluoro-alpha-D-gulopyranose have been synthesized. Reduction of the double bond in the key intermediate and deprotection gave 2-acetamido-2,3,4-trideoxy-D-glucopyranose. Some of the derivatives were active as inhibitors of growth of mouse, mammary adenocarcinoma cells in culture.