An Improved Strategy for the Chemical Synthesis of 3',5'-Cyclic Diguanylic Acid.

The physiological functions of c-di-GMP and its involvement in many key processes led to its recognition as a major and ubiquitous bacterial second messenger. Aside from being a bacterial signaling molecule, c-di-GMP is also an immunostimulatory molecule capable of inducing innate and adaptive immune responses through maturation of immune mammalian cells. Given the broad biological functions of c-di-GMP and its potential applications as a nucleic-acid-based drug, the chemical synthesis of c-di-GMP has drawn considerable interest. An improved phosphoramidite approach to the synthesis of c-di-GMP is reported herein. The synthetic approach is based on the use of a 5'-O-formyl protecting group, which can be rapidly and chemoselectively cleaved from a key dinucleotide phosphoramidite intermediate to enable a cyclocondensation reaction leading to a fully protected c-di-GMP product in a yield ∼80%. The native c-di-GMP is isolated, after complete deprotection, in an overall yield of 36% based on the commercial ribonucleoside used as starting material. © 2019 by John Wiley & Sons, Inc.

Structure elucidation of the Pribnow box consensus promoter sequence by racemic DNA crystallography.

It has previously been shown that the use of racemic mixtures of naturally chiral macromolecules such as protein and DNA can significantly aid the crystallogenesis process, thereby addressing one of the major bottlenecks to structure determination by X-ray crystallographic methods-that of crystal growth. Although previous studies have provided convincing evidence of the applicability of the racemic crystallization technique to DNA through the study of well-characterized DNA structures, we sought to apply this method to a historically challenging DNA sequence. For this purpose we chose a non-self-complementary DNA duplex containing the biologically-relevant Pribnow box consensus sequence 'TATAAT'. Four racemic crystal structures of this previously un-crystallizable DNA target are reported (with resolutions in the range of 1.65-2.3 Å), with further crystallographic studies and structural analysis providing insight into the racemic crystallization process as well as structural details of this highly pertinent DNA sequence.

Paving the way to personalized medicine: production of some promising theragnostic radionuclides at Brookhaven National Laboratory.

This article reintroduces and reinforces our proposed paradigm that involves specific individual "dual-purpose" radionuclides or radionuclide pairs with emissions suitable for both imaging and therapy and which, when molecularly (selectively) targeted using appropriate carriers, would allow pretherapy low-dose imaging as well as higher-dose therapy in the same patient. We have made an attempt to sort out and organize a number of such theragnostic radionuclides and radionuclide pairs that may thus potentially bring us closer to the age-long dream of personalized medicine for performing tailored low-dose molecular imaging (single-photon emission computed tomography/computed tomography or positron emission tomography/CT) to provide the necessary pretherapy information on biodistribution, dosimetry, the limiting or critical organ or tissue, the maximum tolerated dose, and so forth, followed by performing higher-dose targeted molecular therapy in the same patient with the same radiopharmaceutical. Beginning in the 1980s, our work at Brookhaven National Laboratory with such a "dual-purpose" radionuclide, tin-117m, convinced us that it is arguably one of the most promising theragnostic radionuclides, and we have continued to concentrate on this effort. Our results with this radionuclide are therefore covered in somewhat greater detail in this publication. A major problem that continues to be addressed, but remains yet to be fully resolved, is the lack of availability, in sufficient quantities, of a majority of the best candidate theragnostic radionuclides in a no-carrier-added form. A brief description of the recently developed new or modified methods at Brookhaven National Laboratory for the production of 5 theragnostic radionuclide/radionuclide pair items, whose nuclear, physical, and chemical characteristics seem to show great promise for personalized cancer and other therapies, is provided.

Development of a large scale production of 67Cu from 68Zn at the high energy proton accelerator: closing the 68Zn cycle.

A number of research irradiations of (68)Zn was carried out at Brookhaven Linac Isotope Producer aiming to develop a practical approach to produce the radioisotope (67)Cu through the high energy (68)Zn(p,2p)(67)Cu reaction. Disks of enriched zinc were prepared by electrodeposition of (68)Zn on aluminum or titanium substrate and isolated in the aluminum capsule for irradition. Irradiations were carried out with 128, 105 and 92 MeV protons for at least 24h. After irradiation the disk was chemically processed to measure production yield and specific activity of (67)Cu and to reclaim the target material. The recovered (68)Zn was irradiated and processed again. The chemical procedure comprised BioRad cation exchange, Chelex-100 and anion exchange columns. Reduction of the oxidation degree of copper allowed for more efficient Cu/Co/Zn separation on the anion exchange column. No radionuclides other than copper isotopes were detected in the final product. The chemical yield of (67)Cu reached 92-95% under remote handling conditions in a hot box. Production yield of (67)Cu averaged 29.2 µCi/[µA-h×g (68)Zn] (1.08MBq/[µA-h×g (68)Zn]) in 24h irradiations. The best specific activity achieved was 18.6 mCi/µg (688.2 MBq/µg).

RNA synthesis by reverse direction process: phosphoramidites and high purity RNAs and introduction of ligands, chromophores, and modifications at 3'-end.

We have synthesized and studied the coupling properties of 3'-DMT-5'-CE phosphoramidites. The coupling efficiency per step surpasses 99% in the reverse-direction synthesis methodology, leading to high-purity RNA in a large number of 20- to 21-mers and long-chain oligonucleotides. Our data show that 5'→3' direction synthesis has a distinct advantage compared to the conventional method. As a result, this method of RNA synthesis is expected to be a very useful and practical method of choice for therapeutic-grade RNA. The phosphoramidites, Rev-A-n-bz, Rev-C-n-bz, Rev-C-n-ac, Rev-G-n-ac, and Rev-rU are routinely produced with an HPLC purity of greater than 98% and (31)P NMR purity greater than 99.5%.

RNA Synthesis: phosphoramidites for RNA synthesis in the reverse direction. Highly efficient synthesis and application to convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3'-end.

Defined sequence RNA synthesis by 3'-->5' direction is now well established and currently in use for synthesis and development of vast variety of therapeutic grade RNA and Si RNA etc. A number of such synthetic RNA requires a modification or labeling of 3'- end of an oligonucleotide. The synthesis of 3'- end modified RNA requiring lipophilic, long chain ligands or chromophores, using 3' --> 5' synthesis methodology is challenging, requires corresponding solid support and generally results in low coupling efficiency and lower purity of the final oligonucleotide in general because of large amount of truncated sequences containing desired hydrophobic modification. We have approached this problem by developing reverse RNA monomer phosphoramidites for RNA synthesis in 5' --> 3'- direction. They lead to very clean oligonucleotide synthesis allowing for introduction of various modifications at the 3'- end.

The role of electron-emitting radiopharmaceuticals in the palliative treatment of metastatic bone pain and for radiosynovectomy: applications of conversion electron emitter Tin-117m

A variety of radionuclides continue to be investigated and/or clinically used for different therapeutic applications in nuclear medicine. The choice of a particular radionuclide with regard to appropriate emissions, linear energy transfer (LET), and physical half-life, etc., is dictated to a large extent by the character of the disease (e.g., solid tumor or metastatic disease), and by the carrier to selectively transport the radionuclide to the desired site. An impressive body of information has appeared in the recent literature that addresses many of these considerations. This article summarizes and discusses the role of high-LET electron emitters and their advantage in the treatment of cancer or for other disorders in specific situations. Areas such as bone pain palliation, bone malignancy therapy, and radiation synovectomy are covered in greater detail. Projections are made as to the future directions and progress in these areas. A discussion of the various issues related to the selection criteria that are useful for choosing the appropriate radionuclide for a particular application is included. Use of high-LET electron emitters is discussed in greater detail, with particular emphasis on the use of conversion electron emitter tin-117m for various therapeutic applications.

Uma variedade de radionuclídeos continua a ser investigada e/ou clinicamente utilizada para diferentes aplicações terapêuticas em medicina nuclear. A escolha de um radionuclídeo, considerando-se sua emissão apropriada, transferência linear de energia (LET) e meia-vida física é determinada na maior parte pelo caráter da doença (p.ex., tumor sólido ou doença metastática), e pelo carreador que transporta o radionuclídeo seletivamente para o sítio desejado. Um notável conjunto de informações voltadas para essas considerações tem aparecido na literatura recente. Esse trabalho resume e discute o papel de emissores de elétrons de alta-LET e sua vantagem no tratamento do câncer ou para outras doenças em situações específicas. Abordagens relacionadas com o alívio da dor óssea, a terapia da doença óssea e a sinovectomia por radiação são apresentadas detalhadamente. Projeções para o futuro e os progressos nessas áreas são indicadas. Uma profunda discussão relacionada aos critérios de seleção que são úteis para escolher o radionuclídeo apropriado para que uma aplicação particular seja incluída. O uso de emissores de elétrons de alta-LET é discutido em grande detalhe, com particular ênfase no uso do estanho-117m, um emissor de elétron de conversão, para várias aplicações terapêuticas.

Biodistribution of radioiodinated adenovirus fiber protein knob domain after intravenous injection in mice.

The knob domains from the fiber proteins of adenovirus serotypes 2 and 12 were labeled with radioiodine and then injected into the bloodstreams of mice. Knob proteins with functional binding sites for the coxsackie and adenovirus receptor (CAR) were cleared rapidly from the circulation, with radioactivity appearing predominantly in the stomach, while knob mutants unable to bind to CAR remained in the blood circulation for a prolonged period. The clearance of radiolabeled wild-type knob from the blood was slowed by coinjecting an excess of unlabeled wild-type knob protein. An earlier study showed that (99m)Tc-labeled knob protein with intact CAR-binding activity also cleared rapidly from the blood circulation of mice, with radioactivity accumulating predominantly in the liver (K. R. Zinn et al., Gene Ther. 5:798-808, 1998). Together these results suggest that rapid clearance of knob protein from the blood results from specific binding to CAR in the liver and that the bound knob then enters a degradative pathway. The elevated levels of radioiodine in the stomach observed in our experiments are consistent with deiodination of labeled knob by dehalogenases in hepatocyte microsomes and uptake of the resultant free radioiodine by Na/I symporters in the gastric mucosa. Although CAR has been shown to localize in tight junctions of polarized epithelial cells, where it functions in intercellular adhesion, the results of our study suggest that a subset of CAR molecules in the liver is highly accessible to ligands in the blood and able to rapidly deliver bound ligand to an intracellular degradative compartment.

Bone-seeking therapeutic radiopharmaceuticals

Bone-seeking therapeutic radiopharmaceuticals are utilized on the basis of the radionuclideÆs particulate emissions (primarily low to intermediate beta emission). The requirements therefore are different from those of bone imaging agents that consist mainly of short-lived single photon emitters. Lately, the therapeutic bone seeking radiopharmaceuticals have attained increasing importance due to their potential role in alleviating pain from osseous metastases in cancer patients, for the treatment of joint pain resulting from inflamed synovium (radiosynoviorthesis, or radiosynovectomy), or from various other forms of arthritic disease. There is, however, a paucity of published data on the bio-pharmacokinetics of these agents when used following intravenous administration for bone pain palliation. This paper will briefly review and summarize the presently available chemical and biopharmacokinetic information on the various clinically approved as well as experimental bone-localizing therapeutic radiopharmaceuticals, and make projections on their clinical application for the treatment of primary/metastatic cancer in bone