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Bis-benzyl dianions were prepared from the three symmetrical dimethylbiphenyls in yields of 79-86%, and shown to react with a variety of electrophiles on the benzylic carbons. Electrophiles included dimethyl and diethyl sulfate, isopropyl bromide, t-butyl iodide, allyl chloride, trimethylsilyl chloride, trimethylgermyl bromide, and trimethyltin chloride. Reaction of the dianions with (alpha),(omega)-dihalides provides an excellent route to n.0 - and n.0.n.0 cyclophanes. An X-ray study on 14.0 paracyclophane showed it to have a conformation different from that observed in solution by NMR; MM2 calculations were used in determining what the solution conformation is likely to be. Oxidation of the dianions produced the monomer dihydrophenanthrene from the ortho isomer, dimer 2.0.2.0 metacyclophane from the meta isomer, and polymer from the para isomer. An X-ray study on 2.0.2.0 metacyclophane showed it to have the same conformation in the crystal as is observed in solution by NMR. An NMR method for determining the angle of twist in a biphenyl based on the chemical shift of the ortho hydrogens was developed. Many oxidizing agents were evaluated for the oxidation of the dianion from 2,3-dimethyl-1,3-butadiene to 1,2,5,6-tetramethylenecyclooctane; cupric bromide was found to be the best.

The aim of this research was to study the mechanism involved in the synthesis of deoxyhypusine, the intermediate step in the synthesis of the amino acid hypusine. Oeoxyhypusine is derived from the butylamine moiety of a spermidine molecule which is added to the famino group of one lysine in the eukaryotic initiation factor 40 (eIF-4D). Initially, a hepatoma tissue cell (HTC) lysate with a pH of 9.5 in glycine buffer and with a depleted spermidine pool supported deoxyhypusine synthesis in protein. Since CHES buffer was as efficient as glycine buffer, the synthesis of deoxyhypusine was pH dependent (optimum ∼9.2) and not buffer dependent. Next, several inhibitors were used in the cell-free system to block deoxyhypusine synthesis. Only guazatine, a plant amine oxidase inhibitor, completely inhibited deoxyhypusine synthesis. This suggested that an oxidase was involved in deoxyhypusine synthesis. In addition factors were investigated as possible allosteric stimulators of deoxyhypusine formation. NAD⁺, NADH, FAD⁺, FMN⁺, and as nicotinamide were tested for effects on deoxyhypusine formation. NAD⁺ was the most efficient stimulator, but NAOH and nicotinamide also stimulated deoxyhypusine formation. Although these factors increased the synthesis of deoxyhypusine, these assays were done in buffer with low concentrations of spermidine. When the spermidine pool was replenished, these effects were diminished. Thus, it appeared that NAD⁺ may lower the apparent K(m) for spermidine without affecting the V(max) of deoxyhypusine synthesis. The inhibition of deoxyhypusine synthesis by guazatine implied the involvement of a polyamine oxidase. Therefore, the effect of oxygen depletion on deoxyhypusine formation was investigated. The depletion of oxygen reduced the level of deoxyhypusine synthesis to 12% of the control. This activity could be restored to 85% by reoxygenation of the lysate. Thus in support of the suggestion made by the guazatine data, a spermidine oxidase in involved in deoxyhypusine formation. The most significant contribution of this work was the development of a cell free system to study deoxyhypusine. This synthesis required an unusually high pH in vitro and required polyamine depletion (Chapter 2). In addition, synthesis requires a unique spermidine oxidase that is blocked by a guazatine and is conditionally stimulated by NAD⁺ (Chapter 3).

The experimental results of the current work have two parts. First the synthesis, characterization, and spontaneous copolymerization of two highly electrophilic imines, tricyanomethanimine and methyl 3-aza-α, β-dicyanoacrylate. 1,1-Dichloro-2,2-dicyanoethylene or methyl 3,3-dichloro-2-cyanoacrylate reacted with excess sodium azide to give the corresponding diazides, which smoothly underwent thermolysis in solution to give a solution of the novel tricyanomethanimine or methyl 3-aza-α, β-dicyano-acrylate. Attempted isolation gave only oligomers, but reactions using solution of these imines succeeded. Cyclopentadiene and 2,3-dimethyl-1,3-butadiene afforded hetero Diels-Alder reactions. N,N-Dimethylaniline with tricyanomethanimine gave a p-substituted derivative, but with methyl 3-aza-α, β-dicyanoacrylate only a charge transfer complex was formed. Imines - p-methoxystyrene copolymers were obtained. These imines are as reactive as TCNE, but in contrast can also polymerize. Second, a new unsymmetrically substituted quinodimethane was synthesized, characterized, and copolymerized with electron donating monomers. Oxidation of the 1-cyano-1-phenylmethylene-4-cyano-4-ethoxycarbonylmethylene with MnO₂ gave mostly poly-7,8-dicyano-7-ethoxycarbonyl-8-phenylquinodimethane (DCEPQ), but depolymerization by sublimation yielded 45% of DCEPQ. This compound was a mixture of cis and trans isomers. It was homopolymerized by anionic initiation. High molecular weight copolymers of DCEPQ-styrene, DCEPQ-p-methylstyrene and DCEPQ-p-methoxystyrene(p-MeOSt) were formed spontaneously in 1,2-dichloroethane. All polymerizations occurred by a radical mechanism. High molecular weight polymers formed at low conversion. DCEPQ - p-MeOSt copolymerizations yielded alternating copolymers. From spontaneous polymerization of DCEPQ with NVC no copolymer was obtained. All of these polymerizations begin with a bond forming mechanism and propagate by polyaddition.

In this study, synthetic routes to certain short acyl chain phospholipids were developed. Their inhibitory or substrate properties for phospholipase A₂ were then examined. An improved method for the acylation of glycerophosphocholine is described using a mixed fatty acid - trifluoroacetic acid anhydride. This partial synthetic route is particularly suitable for obtaining short acyl chain phosphatidylcholines. A new pathway for constructing the unnatural sn-1-phosphatidylcholines is also described, starting from L-arabinose. This is converted first to a key intermediate, 2,3-O-isopropylidene-sn-glycerol, which is then phosphorylated and transformed into sn-glycero-1-phosphocholine. This can be acylated as above to give sn-1-phosphatidylcholines. Routes to the short chain phosphatidylethanolamines were investigated and discussed. A procedure, using phospholipase D, was used to convert L-diC₆ PC into L-diC₆ PE in a transphosphatidylation reaction. Failed attempts to obtain shorter chain homologs by this and other methods are also detailed. The kinetics of inhibition of the phospholipase A₂ hydrolysis of L-diC₆ PC by the D-isomer are also reported for the monomeric concentration range. It was found that the D- enantiomer did not behave as a pure competitive inhibitor, and that an enzyme-substrate-inhibitor complex can exist. The implications of these results with regard to PLA₂ hydrolysis of mixed micelles is discussed. The PLA₂ substrate properties of both the anionic and zwitterionic diC₆ PE's were also studied. It was established that the anionic diC₆ PE is either a very poor substrate relative to the zwitterionic diC₆ PE, or acts as a competitive inhibitor towards its hydrolysis. Similarly, the rates of base-catalyzed acyl ester hydrolysis are about 18 times greater for the zwitterionic than for the anionic diC₆ PE. The importance of a protonated amino group in both these hydrolyses studies is noted. In addition, certain physical properties of diC₆ PE, such as its critical micelle concentration and carbon-13 NMR spectrum, are also given.

We have chosen the murine immunoglobulin M (IgM) as system to study glycoprotein biosynthesis and carbohydrate processing. Secreted IgM heavy chain (m) has five glycosylation sites which location and structures have been determined. m chain variable region (VH) is involved in antigen binding, while the constant region (CH) is responsible for the effector functions in which the carbohydrate plays an important role. We have determined the carbohydrate structures present at each glycosylation site of IgM produced by a hybridoma cell line (PC 700) and its derived mutants and compared them to IgM from myeloma cell MOPC 104E. PC 700 mutants secrete altered IgM. The alterations include: deletion of one or more constant domains (mutants: 128, 313, and 562) and m chain hyperglycosylation (mutants 21 and 38). Gene analysis indicated that deletions can arise from two different mechanisms. One of these involve a major gene change (mutant 128), while others come from base point mutations (mutants 313 and 562). Cells 21 and 38 did not appear to have m gene insertions. Determination of purified single glycosylation site structures show that PC 700 m chain is processed only to biantennary. Heavy chain protein fragmentation and carbohydrate studies indicate that mutants 21 and 38 alterations are due to an increase in oligosaccharide processing and reduction of unprocessed structures. There is a trend of processing going from PC 700 < 21 < 38. In addition, our results show how growth cell conditions can affect the carbohydrate processing without altering the determinants of m chain oligosaccharide structures. Studies on the IgM molecule illustrate the need for precisely define structure-function relationships. This would allow the selection of the best antibodies for studies such as those involved in immunotherapy.

A number of α-melanotropin analogues have been prepared. Insight towards the development and understanding of the functional roles of each of the amino acid residues important for high melanotropic potency in a number of biological systems was studied. The melanotropin analogue Ac- α-MSH₄₋₁₂-NH₂ contains all of the structural requirements necessary for obtaining full biological potency on the lizard (Anolis carolinensis) melanophore and S-91 mouse melanoma. On the frog (Rana pipiens) melanophore, the melanotropin analogue Ac-[Nle⁴]-α-MSH₄₋₁₃-NH₂ possesses full melanotropic potency relative to the native hormone. The smallest melanotropin analogue capable of eliciting any biological response was Ac-α-MSH₆₋₉-NH₂ (Ac-His-Phe-Arg-Trp-NH₂) on all biological systems studied. The low biological activity found in previous studies for smaller melanotropin analogues may have been due to a trace contamination of a potent melanotropin. The importance of each of the thirteen amino acid residues of α-melanotropin in contributing to the melanotropic actions in a number of biological systems is discussed. We were unable to confirm the reported presence of a second independent active sequence in α-melanotropin. The structural requirements for prolongation of biological activity (following removal of exogenous hormone from the assay medium) and enzyme stability have been studied. Analysis suggests species-dependent differences in the structural relationships of the amino acid residues in the 4, 7 and 11 positions for prolonged melanotropic response. Analogues prepared with D-phenylalanine in place of its L-enantiomer in the seventh positions of α-melanotropin analogues generally results in an increased resistance to enzymatic degradation towards rat brain homogenate, rat serum, and to the purified enzymes, trypsin and chymotrypsin. Some of these analogues have been shown to be useful probes for the understanding of melanotropic actions in a number of biological systems. Two α-melanotropin analogues have been prepared which possess partial agonism on the mouse melanoma adenylate cyclase assay. A number of these analogues should prove useful in future studies directed towards a more detailed study of melanotropin receptor systems in a large variety of biological systems. The first known synthesis of melanin concentrating hormone (MCH) is reported. The overall synthetic yield of this cyclic heptadecapeptide was 14%. The chemical, physical and biological properties of synthetic MCH and naturally occurring telost MCH were in agreement. MCH was found to be a full agonist in stimulating melanosome dispersion in both the frog and lizard bioassay. Therefore MCH can stimulate melanosome dispersion or contraction depending upon the bioassay studied. Preliminary studies were done towards understanding the mechanisms of MCH action on telost fish.

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No description available.

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