Methylphosphonate (MP) oligodeoxynucleotides (MPOs) are metabolically stable analogs of conventional DNA containing a methyl group in place of one of the non-bonding phosphoryl oxygens. All 16 possible chiral R(P) MP dinucleotides were synthesized and derivatized for automated oligonucleotide synthesis. These dimer synthons can be used to prepare (i) all-MP linked oligonucleotides having defined R(P) chirality at every other position (R(P) chirally enriched MPOs) or (ii) alternating R(P) MP/phosphodiester backbone oligonucleotides, depending on the composition of the 3'-coupling group. Chirally pure dimer synthons were also prepared with 2'-O-methyl sugar modifications. Oligonucleotides prepared with these R(P) chiral methylphosphonate linkage synthons bind RNA with significantly higher affinity than racemic MPOs.

Re-examination of the structure of pyridine coenzymes in solution by use of the 220-MHz high-frequency nuclear magnetic resonance spectrometer indicates that there is primarily one folded structure that is in rapid equilibrium with an open form. Reduced DPN + and reduced analogs of DPN + exist predominantly with the B side of the dihydropyridine ring folded against the adenine moiety. (The oxidized coenzymes appear to exist in the same folded structure.) Furthermore, the ribose protons undergo very little conformational change upon reduction of the pyridine ring; this observation strongly suggests a considerable similarity between the folded forms of the oxidized and reduced coenzymes. A model of the folded structure is presented.

ABSIRACT We have found that poly(L-lysine) can be a very effective agent in preventing the growth of Ehrlich ascites tumors in mice. When given optimal doses of poly(L-lysine) (Mr 60 X 103) intraperitoneally for 5 consecutive days, beginning on day 1 after inoculation with Ehrlich ascites cells, White Swiss mice show nearly a 100% remission from subsequent tumor growth. Rechallenge of "cured" animals with tumor cells, however, shows no long-term immunological protection. In tissue culture, poly(L-lysine) shows a related potent cytotoxicity with HeLa cells; interestingly, the D isomer has properties strikingly different from those of the L isomer. In addition, there is a strong molecular weight dependence in that the small polylysine (Mr 3 X 103) possess less than 1/20th the cytotoxicity of large polymers (Mr 70 X 103) on a weight basis in both cell culture and animal studies. At the same time, none of these lysine polymers gives any significant increase in life span to BDF1 mice infected with L1210 murine leukemia cells. We have also further explored the mechanism by which the polylysines express their cytotoxicity. These data indicate that lysine polymers show cell specificity in their action and in some cases they may be beneficial as potent antineoplastic agents, particularly when molecular weight is taken into consideration. For more than 25 years, poly(L-lysine) has been known to have unusual biological properties. Early studies showed that it decreased the infectivity of tobacco mosaic virus (1), disturbed thrombin formation in rats (2), blocked the development of bacteriophage (3, 4), protected chicken embryos from animal viruses (5, 6), and possessed antibacterial activity (7). There was also an early report that indicated that polylysine had some activity against murine tumors (8). More recently, polylysine has been found to exhibit a large number of unique membrane properties. These include the ability to enhance the cellular uptake of macromolecules (9), to inhibit iodide uptake by thyroid slices (10), to produce pathogenesis of the glomerular epithelium (11), to act as an anticholinesterase (12), to specifically agglutinate the lymphocytes from cancer patients (13), and to either increase or decrease the transport of specific radioisotopes into cells (14). These effects are probably associated with the polycationic character of polylysine and are probably due to specific interactions on the cell membrane.Recently, we have been examining the ability of polylysine to serve as an efficient drug carrier (15). In doing the controls for these studies, we found that poly(L-lysitie)s of a certain Mr, (60 X 103) could induce 100% remissions in mice inoculated with Ehrlich ascites when the polymer was administered at increased doses. This paper presents results on the antineoplastic activity and toxicity as functions of polymer molecular weight and concentration. Also, we will provide further information into the mechanism accounting for the antineoplastic and cytotoxic activities of polylysine. MATERIALS AND ...

Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-06-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the 06 position of N2-ibu-06-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 j.mole scales.These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.

We describe the development of several hybridization assay formats involving acridinium-ester-labeled DNA probes. The simplest of these is a homogeneous assay procedure that requires only three steps to complete, including a 5-s detection step. Using this format, we have detected target sequences in the 10(-16) to 10(-17) mol range; when rRNA is the target, this translates to 3000 to 300 bacterial organisms. The entire assay can be carried out in less than 30 min. This is the first homogeneous DNA probe assay to be of practical use in the clinical laboratory, and it represents a major simplification of hybridization formats. We also demonstrate the use of this homogeneous assay format to discriminate single-base differences between two closely related target sequences and to detect DNA as well as RNA target molecules. By combining homogeneous hybrid discrimination with solid-phase separation, we have been able to decrease background readings from unhybridized probe to only a few parts per million. This enhances assay sensitivity about 10-fold, to a range of 10(-17) to 10(-18) mol of target. We are in the process of further improving the performance of these assays.