The binding of micellar lipids to chitosan.

The lipid binding capacity of chitosan (partially deacetylated chitin) was determinined with respect to micellar solutions of bile salts, dodecyl sulfate, natural ox bile and an artificial mixed microemulsion. The stoichiometry was determined following the separation of the solid phase by filtration or centrifugation. The major variables in the extent of binding were the pH and ionic strength, suggesting that the interactions are mainly of ionic nature. It is noteworthy that under optimal conditions chitosan could bind, i.e., coprecipitate, with 4-5 times of its weight with all the lipid aggregates tested. These results have a bearing on the nutritional and pharmacological applications of chitosan. The analyses of the components from the precipitates with microemulsion and ox bile show a significant selectivily of binding caused by hydrophobic interactions.

Exploratory studies of lipid-pectin interactions.

This study was undertaken to elucidate the mechanism responsible for the hypolipidemic action of pectin. The experiments reported here were designed to test if direct molecular interactions exist between pectin and lipids. Equilibrium dialysis of pectin and taurocholate showed binding only at high nonphysiological ionic strength. When lipid microemulsions and micelles of low charge density were used, unambiguous proof of binding to pectin were obtained by NMR spectroscopy and gel exclusion chromatography. The results suggest that the interaction in mainly by hydrogen bonds involving the pectin carboxylic moieties. The quantitation of lipid binding by pectin could be established only in presence of polyvalent cations using a membrane filtration technique. Under optimum conditions, pectin can bind four times its weight in lipids. Although the techniques presented here are physical-chemical, the conclusions are highly relevant to bioavailability. These results represent the first successful demonstration of direct lipid-polysaccharide interactions in biochemistry, and they have an obvious bearing on the physiological absorption process. The intestinal binding of dietary and biliary lipids by pectin may be a major mechanism of action of this hypolipidemic polysaccharide. This paper also cells attention to techniques which could be beneficial for the in vitro evaluation of plant fibers.

The enzymatic hydrolysis of the phosphate ester bond in some thionucleotides.

We prepared the 5'- and 3'-O-phosphorothioate esters of the antitumor agent O2 : 2'-anhydro-1-beta-D-arabinosylcytosine. We also included in this study esters of 2'-thio-2'-deoxycytidine, namely, 2'-S-dCyd-2' : 3'-P, 2'-S-dCyd-2'-P, and 2'-S-dCyd-3'-P, along with natural nucleotides. These compounds were subjected to the action of Escherichia coli alkaline phosphatase, potato acid phosphatase, and bovine pancreatic ribonuclease A. The data were analyzed by Lineweaver-Burk plots to obtain Km and KI values. Only 2'-S-dCyd-2'-P was a substrate for alkaline phosphatase; the anhydro-araCyt phosphorothioates were good competitive inhibitors, while 2'-S-dCyd-3'-P did not associate with the enzyme. Acid phosphatase hydrolyzed all four monoesters investigated, including the S-phosphorothioate. The cyclic phosphorothioate, 2'-S-dCyd-2' : 3'-P was neither hydrolyzed by, nor associated with, ribonuclease A. ORD spectroscopy was also used in an attempt to relate the structural features of analogs to the peculiarity of their hydrolysis.

Mechanistic possibilities in prebiotic thiophosphate chemistry.

Two types of reactivities of thiophosphates have been demonstrated: one being nucleophilic displacement by the P-S moiety of nucleoside phosphorothioates and the other, phosphorylation via P-S cleavage as the driving force. We have designed a system where both displacement on carbon and P-S cleavage are possible. Adenosine derivatives have been synthesized with 5'-deoxy-5'-chloro and 5'-O-tosyl substitutions as leaving groups utilizing the 3'-O-phosphorothioate as the biphilic center. The main products of cyclization were 5'-O-tosyl and 5'-chloroadenosine 2':3'-cyclic phosphate. Formation of 3':5'-S-phosphorothioate was slow even using an excellent leaving group. This is possibly due to hydrogen bonding between the 2'-OH and the neighboring P-O.--KOH hydrolysis of the cyclic phosphorothioate yielded 2'(3') phosphorothioates in a 1:1 ratio. The 2' and 3' isomers were separated and used to study the relative rates of cyclization. The cyclization via P-S cleavage of 2'(3')-O-phosphorothioates showed that the 2' isomer was more reactive. This is the first report of superior reactivity of the 3'-OH of a ribonucleoside.

Isolation of pyrimidine isostichs from chick erythrocyte DNA.

The isolation of pyrimidine oligonucleotides (isostichs) from chick erytyrocyte DNA was accomplished on a preparative scale with the goal of providing starting material for further chemical modifications. The DNA was degraded by the method of Burton and Petersen and the isostichs were obtained via paper chromatography and ion-exchange chromatography. The various isostichs were further fractionated into base compositional isomers, the frequency of which was determined as mole percent of total pyrimidines. Although this work was not intended to be an analytical study, the large quantities of DNA used allowed the measurement of most compositional isomers with an accuracy of 5% of the listed values. The data for long oligothymidylates are less reliable. The results of the present study revealed the anticipated bias in the distribution of pyrimidine isostichs in favor of longer chain lengths as compared to that expected for a random distribution. Isostichs above chain length 10 occur in amounts approximately 7 times greater than calculated.

Novel prebiotic systems: nucleotide oligomerization in surfactant entrapped water pools.

Oligomerization of 5' -TMP in water pools entrapped by dodecyl-ammonium chloride surfactant aggregates in benzene: hexane in the presence of dicyanodiimide at temperatures ranging from 21 degree -72 degree resulted in the formation of linear and cyclic oligonucleotides containing up to pentamers. Effects of temperature, time and surfactants have been examined. Rate constants for the formation of oligomers have been determined at five different temperatures. These data afforded values of (formula: see text). Prebiotic significance of these results are discussed.

Preparation of 2'-thio-2'-deoxycytidine 2':3'-phosphorothioate.

The synthesis of a novel ribonucleotide analog 2'-thio-2' deoxycytidine 2':3'-O,S-phosphorothioate is described. In the first step, 2,2'-anhydro 1-beta-D-arabinosylcytosine was thiophosphorylated by the action of dithiophosphate, a process which gave predominantly the 3'-O-phosphorothioate isomer. An intramolecular displacement reaction led to the formation of the title compound. Structure and reactivity of this thioanalog differ substantially from 2':3'-CMP.

Compartmentalization of amino acids in surfactant aggregates. Partitioning between water and aqueous micellar sodium deodecanoate and between hexane and dodecylammonium propionate trapped water in hexane.

Cationic amino acids, arginine and lysine partition differentially from water into aqueous micellar sodium dodecanoate. Conversely, partitioning of serine, glycine, aspartic acid, glutamic acid, threonine, alanine, proline, valine, leucine, phenylalanine and isoleucine do not vary appreciably. Partitioning from neat hexane into dodecylammonium propionate trapped water in hexane is, however, dependent upon both electrostatic and hydrophobic interactions. These results imply that the interior of dedecylammonium propionate aggregates is negatively charged and is capable of hydrogen bonding in addition to providing a hydrophobic enviroment. The solubilities of amino acids in neat hexane substantiate the previously derived amino acid hydrophobicity scale. Relevance of partitioning in these systems to the postulated selective amino acid compartmentalization is discussed.

Tumor-inhibiting properties of the neutral P-O-ethyl ester of adenosine 3':5'-monophosphate in correlation with its crystal and molecular structure.

The P-O-ethyl ester of cAMP has been synthesized, its inhibition of solid and ascites tumors studied, and its pattern of urinary excretion followed. Et-cAMP is more effective than cAMP against solid sarcoma 180 in mice and against Ehrlich ascites carcinoma cells in tissue culture. The urinary excretion pattern of injected E-t-cAMP suggests that about two-thirds of the injected dose (13 mumol per animal) is retained in the rat rather than being promptly excreted. Liver slice studies of the effect on glycogenolysis suggest that the Et-cAMP is converted to cAMP intracellularly. The compound crystallizes in space group P21 with one molecule per asymmetric unit. The base ring has the anti conformation. The ethyl group is endo to the base ring and is axial in the flattened chair-conformer six-membered ring formed by the 3'-5' O-P-O cyclization. In most other respects the structure of the compound is closely similar to the known structures of other cyclic nucleotides.

The use of s-2-cyanoethyl phosphorothioate in the preparation of oligo 5'-deoxy-5'-thiothymidylates.

An improvement of our strategy for the stepwise synthesis of oligo 5'-deoxy-5'-thiodeoxyribonucleotides [Chladek and Nagyvary (1972) J. Amer. Chem. Soc. 94, 2079] involves the use of 5'-O-tosylthymidine 3'-S-2-cyanoethyl phosphorothioate. The displacement of the tosylate by thymidine 3'-phosphorothioate and subsequent alkaline deblocking afforded the dinucleotide (Tps)2. The process of displacement and deblocking was repeated three more times at an average yield of 30 percent per step. The corresponding bifunctional derivative of deoxyadenosine was found much less reactive and practically unsuitable for repeated chain elongation. The ORD and CD spectra of the analogs are similar to those of the natural oligonucleotides.

Synthesis and properties of some cyclic AMP alkyl phosphotriesters.

Cyclic AMP was converted to its phosphotriesters according to the classical approach of phosphate activation with a sulfonyl chloride, followed by esterification with an alcohol. The methyl, ethyl, propyl, butyl and cetyl triesters were prepared, and some of their physical-chemical properties determined. Alkaline hydrolysis of these alkyl phosphotriesters resulted predominantly in ring opening. On the other hand, nucleophilic attack by thiourea led to the formation of cAMP as the main product. The conclusion can be drawn from these results that cAMP phosphotriesters could serve as suitable storage forms of cAMP, and cyclic triesters may be the best vehicle of transporting nucleotides through biological membranes.