Part I of this thesis describes a method for the isolation of a specific family of aminoacyl-tRNAs. Reaction of the aminoacyl-tRNAs with the N-hydroxysuccinimide ester of 2-naphthoxyacetic acid produces N-2-naphthoxyacetylaminoacy1-tRNAs which possess sufficient affinity for BD-cellulose to allow their separation from unmodified tRNA by simple chromatographic steps. The N-2-naphthoxyacetyl amino acid is removed by mild alkaline hydrolysis and the iso-acceptor tRNAs are separated by chromatography on BD-cellulose. tRNA₁Gly and tRNA₂Gly have been purified from brewer's yeast (Saccharomyces cerevisiae) by this method and a simplified procedure for the large scale isolation of these tRNAs was developed. The object of Part II of this thesis was to develop a new method of nucleotide sequence analysis based on the following steps: (i) specific introduction of hydrophobic groups onto the 3'- or 5'-terminals of polynucleotides, (ii) subsequent cleavage of the appropriately derivatized polymer under conditions which favor rupture of only one bond per molecule of polymer, (iii) separation of fragments bearing the derivative from those not bearing the derivative, (iv) resolution of derivatized fragments according to chain length, (v) complete degradation of the purified fragments from step (iv) with the reagent used to generate these fragments followed by identification of the components so liberated from each fragment, and finally, (vi) assembly of the data obtained from step (v) to order the composite fragments from 5' and 3' ends to derive an unambiguous primary sequence. It was reasoned that N-2-naphthoxyacetylglycyl-tRNA₁Gly and tRNA₂Gly could be used as a model polymer bearing an appropriate 3’-derivative, that BD-cel1u1ose chromatography could effect step (iii), that DEAE-cel1ulose-7 M urea column chromatography could effect step (iv) and that either DEAE-cellulose chromatography or combined chromatographic-electrophoretic techniques could effect step (v). A method for placing a hydrophobic derivative onto the 5’-terminus of polynucleotides needed to be developed. Further, it was necessary to discover if available endonuc1eases could degrade the model polymer in a manner approaching random, single-hit kinetics. To ascertain the feasibility of the proposed method, two studies were undertaken. First, the 3'-terminal fragments released by exhaustive digestion of N-2-naphthoxyacetylglycyl-tRNA₁Gly and tRNA₂Gly with RNase T₁ were isolated by the procedure to verify the efficiency of a number of the required steps. The fragments (from the results of the nucleostide-analysis and on the assumption that the 3'-sequence cytidy1y1(3’-5’)cytidy1y1 (3’5’adenosine is common to all functional tRNAs) are (Cp, Ap)CpCpA and (Up, Ap, Cp, Cp) CpCpA. The relative yields of the two fragments suggested they were derived from tRNA₁Gly and tRNA₂Gly, respectively. Second, a study was undertaken of the degradation products liberated from crude and purified glycyl-tRNAs as well as from crude and purified N-2-naphthoxyacety1g1ycy1-tRNAs by RNase T₁ (and, to a lesser extent, by pancreatic RNase). RNase T₁ was found to degrade crude glycyl-tRNA with a product distribution suitable for testing the feasibility of the method. It was not possible, however, to label specifically the glycyl-oligonucleotides with a hydrophobic group in the presence of other degradation products. Fragmentation patterns suitable for execution of the proposed procedure were not obtained with crude or purified N-2-naphthoxyacetylglycyl-tRNAs with either RNase T₁ or with pancreatic RNase. Studies on the pH-dependent activity of RNase T₁ degradation of tRNA showed, in addition to the expected optimum at pH 7-5, a second pH.optimum at pH 4.5. The latter activity was dramatically augmented by the presence of 7 M. urea. Studies on the specificities of several RNase T₁ preparations under various conditions showed the presence of substantial levels of adenosine as an end-group of tRNA degradation products. Preliminary results of a search for reagents capable of adding specifically an aromatic residue onto the 5'-terminus of polynucleotides demonstrated that reacting either 2-naphthyl-phosphoromorpholidate or 2, 4-dinitrof1uorobenzene with tRNA resulted in increased binding between the modified tRNAs and BD-cel1ulose. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

The syntheses of glycos-3-yl amino acids, 2-deoxy glycos-3-yl amino acids,and deoxy amino uronic acid derivatives (homologous of the sugar moiety of the polyoxins) are reported. 5,3-Spiro-pyrrolidone carbohydrate derivatives, formed by intramolecular cyclization from doubly branched-chain a, y-diami.no acids, are also described. The ketose, 1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ulose (8)'', was condensed with 2-phenyl-5 (4)-oxazolinone (89), in the presence of lead (II) acetate as catalyst, to yield (E)-and (Z)-2-phenyl-4- (1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)-5(4) oxazolone (143) and (144), as a 1:1 mixture,in a 75% yield. The stereo-specific synthesis of 143 was achieved by changing the reaction solvent from dimethoxyethane to tetrahydrofuran. Methanolysis of a mixture of 143 and 144 afforded a mixture of (E)- and (Z)-methyl-N-benzamido-a-(1,2:5,6-di-O-isopropylidene-a-D-ribo-hexofuranos-3-ylidene)glycinate (145) and (146) in a 90% yield. Catalytic hydrogenation of the mixture of 145 and 146 afforded methyl-D-2 (and L-2) -3-deoxy-1,2 :5 ,6-di-0_-isopropylidene-a-D-allofuranos-3-yl)-N-benzoylglycinate (147) and (148) which were separated by silica gel chromatography. Methyl 4,6-0-benzylidene-2-deoxy-q-D-erythro-hexopyranoside-3-ulose (153), was allowed to react with 2-phenyl-5(4)-oxazolone (89) to yield (E)- and (Z)-2-phenyl~4-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)-5(4)-oxazolone (154) and (155) in 13% and 27% yields, respectively. Methanolysis of 154 and 155_ afforded (Eland (Z)-methyl-N-benzamido-a-(methyl-4,6-0-benzylidene-2,3-dideoxy-a-D-erythro-hexopyranos-3-ylidene)glycinate (156) and (157) in quantitative yields. Compound 156 was catalytically"hydrogenated to afford methyl-D-2-(methyl-4,6-0-cyclohexylmethylidene-2,3-dideoxy-q-g-arabino-hexopyranos-3-yl)-N-cyclohexylcarboxylglycinate (167) in a 71% yield. Similarly, _15_7 afforded methyl-D-2- (methyl-4 ,6-0-cyclohexylmethylidene-2 , 3-dideoxy-q-D-ribo-hexopyranos- 5-yl) -N-cyclohexylcarboxylglycinate (170) in a 68% yield. 2-Phenyl-5(4)-oxazolone (89) was condensed with 3-0-benzyl-l,2-O-isopropylidene-q-g-xylo-pentodialdo-1,4-furanose (173), prepared by known procedures, to afford a mixture of (E)- and (Z)-4-(3-0_-benzyl-5-deoxy-1,2-0-isopropylidene-q-D-xylofuranos-S- ylidene)-2-phenyl-5(4)-oxazolone (174) and (175) in a 45% yield. Methanolysis of the mixture of 174 and 175 afforded (E)- and (Z)-methyl-6-N-benzamido-3-0-benzyl-5,6-dideoxy-l,2-0-isopropylidene-q-D-xylo-heptofur-5-enuronate (176) and (177) in an 85% yield. Hydrogenation followed by hydrogenolysis of the mixture of 176 and 177 gave a mixture of methyl-6-N-benzamido-5,6-dideoxy - 1,2-0-isopropy'lidene-a-D-gluco (and g-L-ido) -heptofuranuronat (180) and (181) . Transesterification of 180 and 181 afforded the ethyl esters which were separated into pure ethyl-6-N-benzamido-5,6-dideoxy-1,2-0-isopropylidene-a-D-gluco (and g-L-ido)-heptofuranuronate (182) and (183) by fractional crystallization. Compound 183 was hydrolyzed in hot aqueous ethanolic barium hydroxide solution to afford 6-amino-5,6-dideoxy-1,2-0-isopropylidene-g-L-ido-heptofuranuronic acid (184) in a 77% yield. The 1,3-dipolar addition of diazomethane to (Z)-3-deoxy-1,2:5,6-di-0-isopropylidene-3-C-methoxycarbonylmethylene-a-g-ribo-hexofuranose (13) , prepared from 8_ by application of the Wittig reaction, afforded a mixture of spiro-AA- and A^-pyrazolines, which were hydrogenated at high pressure with Raney nickel, to afford spiro-3,4'-S_- (3,3-dideoxy-1,2:5,6-di-O-isopropylidene-q-p-ribo-hexofuranose)-3'-R-amino-2'-pyrrolidone (192) , spiro-3,4'-S~(3,3-dideoxy-l,2:5,6~di-0-isop ropy lidene-a-D-ribo--hexo furanose)-3'-S_-amino-2'pyrrolidone (195) , spiro- 3,4' - R- (3, 3-dideoxy-l ,2 :5 ,6-di-^-isopi'opylidene-a-D-ri_bo_-hexofuranose)-3'-R-amino-2'-pyrrolidone (194) , and spiro-3,4*-R-(3, 3-dideoxy-1,2:5,6-di-0-isopropylidene-a-p-ribo-hexofuranose)- 3'-S-amino-2'-pyrrolidone (195) in yields of 16, 32, 14, and 18%^respectively. Compounds 192, 195, 194, and 195 were acetylated to yield the crystalline derivatives 196, 197.. 198, and 199, Selective de-0-i sop ropy l.i den at ion of compound 196 followed by oxidative cleavage with sodium meta-periodate gave an aminal 200 which was acetylated to afford spiro-3,4' -S_- (3, 3-dideoxy- 1,2-0-isopropylidene-a-D-erythro-pentodia.ldo-1,4-furanose)-3'-R-acetamido-2'-pyrrolidone 3',5 - R-aminal-5,1'-diacetate (202). Compound 19 7 was^selectively de-O-isopropylidenated and oxidized with periodate to yield spiro-3 ,4 '-S-(3,3-dideoxy--1,2-0-isopropylidene-a-D-erythro-pentodialdo-1,4-furanose) -3' -S_- acetamido-2 ' -pyrrolidone (201) . Complete de-0_-isopropylidenation of 197 afforded spiro- 3,4'-S_~ (3,3-dideoxy-g-D-ribo-hexopyranose) -3' -S_-acetamido-2 ' -pyrrolidone (203) in a 30% yield. An optically active amino acid Wittig reagent was prepared as follows. L-Cystine diethyl ester dihydrochloride (205) was chlorinated to yield 3-chloro-L-alanine ethyl ester hydrochloride (206) which was converted to N-acetyl-g-chloro-L-alanine ethyl ester (207). Compound 207 was reacted with tr:i phenyl nhosplunc and sodium iodide to afford cthyl-N-acetyl-3- (triphenyJphosphoniuiniodo)-a-L-a] an ate (204) in a 51% yield. / Science, Faculty of / Chemistry, Department of / Graduate

Ribonucleic acids are concentrated in certain parts of the cell. Separation of these parts usually precedes isolation of the ribonucleic acids they contain. This separation is accomplished by a process of cell fractionation (Mathias, 1966).

Modern concepts of the structure, function and metabolism of proteins have evolved painstakingly through the centuries. In September, 1772, Rutherford discovered the gas which was later named nitrogen by Chaptal in 1790 . The fundamental importance of nitrogen in the body was only appreciated after Gay-Lussac and Thenard had pioneered chemical procedures for organic analysis. Magendie applied this knowledge to dietary constituents and concluded that nitrogen was an essential component of the diet. His early insight into metabolism was depicted in his view that body constituents are continuously being replaced, the rate being dependent on the tissue. In 1838 the term"protein" was coined by Gerard Mulder for substances containing a basic nitrogenous component.

The current renaissance in biological research has evoked widespread interest in the field of lipid metabolism. While extensive studies during the past decade have greatly expanded our knowledge of the subject, these researches have been directed primarily to the serum lipids. By far the greatest accumulation of lipid in the mammalian organism is found in the depot fat. This tissue had, until fairly recently, been generally regarded as a semi-static food store, expanding and shrinking in response to a relative excess or deficit of dietary calories. This simple concept is no longer tenable. The pioneering work of a few workers in the field during the past few decades has more recently interested many others and the accumulated evidence has established the fat depot among the more active organs in the mamnalian organism.

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published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy