Solid-phase synthesis of terminal oligonucleotide-phosphoramidate conjugates

A novel phosphoramidite, N,N-diisopropylamino-2-cyanoethyl-9-anthracenemethyl phosphoramidite 1, was prepared and coupled with the terminal 5'-hydroxyl of support-bound T10 and the putative phosphite triester intermediate was subsequently reacted with iodine in the presence of either water or a series of primary and secondary amines. The reactivity of 1 compared to a previously reported benzyl phosphoramidite 2 was also investigated: oxidation of the product of coupling 2 with CPG-T10-5'OH under aqueous conditions resulted in greater than 30% of the benzyl moiety being retained. In contrast, essentially complete loss of the 9-anthracenemethyl group was observed using 1 under the same conditions. Oligonucleotides modified with a terminal phosphate monoester, lipophilic, fluorescent or cationic groups were thus prepared.

Novel methodology for the preparation and purification of oligonucleotides incorporating phosphorothiolate termini

A novel phosphoramidite; N,N-diisopropylamino-2-cyanoethyl-ortho-methylbenzylphosphoramidite 1, was prepared. The reaction of 1 with DMTrT and subsequent derivatisation of the phosphite triester product under solution-phase, Michaelis–Arbuzov conditions was investigated. Coupling of 1 with the terminal hydroxyl groups of support-bound oligodeoxyribonucleotides and subsequent reaction with an activated disulfide yielded oligonucleotides bearing a terminal, phosphorothiolate-linked, lipophilic moiety. The oligomers were readily purified using RP-HPLC. Silver(I)-mediated cleavage of the phosphorothiolate linkage and desalting of the oligonucleotides were performed readily in one step to yield cleanly the corresponding phosphate monester-terminated oligomers.

Reactions of polycarbonate with cyclohexene oxide and phosphites: A density functional study

Epoxides and phosphites are often used as additives to stabilize the properties of polymers, including bisphenol A polycarbonate (BPA-PC). We describe density functional (DF) calculations of the reactions of cyclohexene oxide (CHO, cyclohexane epoxide) and phosphites with chain segments of BPA-PC, with the aim of identifying possible reaction paths and energy barriers. The reactions of CHO with the OH-terminated PC chains and with the carbonate group are exothermic, although there is an energy barrier in each case of more than 10 kcal/mol. A comparison of results for different CHO isomers demonstrates the importance of steric effects. The reactions between the same groups of the PC chain and the phosphites 2-[2,4-bis(tert-butyl)phenoxy]-5,5-dimethyl-1,3,2-dioxaphosphorinane] (BPDD) and trimethyl phosphite (TMP), and their phosphonate isomers are characterized by large energy barriers.

A novel structural class of photoswitchable oligonucleotide

Directed Michaelis–Arbuzov reactions of support-bound internucleotide O-benzyl- or O-methyl-phosphite triesters with meta-phenylazobenzylamine or alkane-/glycol-linked a,x-diamines were effected in the presence of iodine. The corresponding tritylated phosphoramidate-linked 11-mers were fully deprotected and released from the support under standard conditions and the fast- and slow-diastereoisomers of both the E- and the Z-meta-phenylazobenzyl-appended oligomers were readily resolved by RP-HPLC. The primary amine-functionalised oligonucleotides were either purified, detritylated and then finally treated with Nhydroxysuccinimidyl carboxylic acid ester derivatives of photoswitchable moieties (Route A) or first derivatised and then subsequently purified and detritylated (Route B). This latter route enabled resolution of fast- and slow-isomers of the trityl-on oligomers bearing novel photoswitchable azopyridine or 9-alkoxyanthracene moieties using RP-HPLC, following which the pure diastereoisomers were detritylated and characterised by MALDI-MS.

Studies on the biodegradation of fosfomycin: Growth of Rhizobium huakuii PMY1 on possible intermediates synthesised chemically

The first step of the mineralisation of fosfomycin by R. huakuii PMY1 is hydrolytic ring opening with the formation of (1R, 2R)-1,2-dihydroxypropylphosphonic acid. This phosphonic acid and its three stereoisomers were synthesised by chemical means and tested as their ammonium salts for mineralisation as evidenced by release of P-i. Only the (1R, 2R)-isomer was degraded. A number of salts of phosphonic acids such as (+/-)-1,2-epoxybutyl-, (+/-)-1,2-dihydroxyethyl-, 2-oxopropyl-, (+/-)-2-hydroxypropyl-, (+/-)-1-hydroxypropyl- and (+/-)-1-hydroxy-2-oxopropylphosphonic acid were synthesised chemically, but none supported growth. In vitro C-P bond cleavage activity was however detected with the last phosphonic acid. A mechanism involving phosphite had to be discarded as it could not be used as a phosphorus source. R. huakuii PMY1 grew well on (R)- and ( S)- lactic acid and hydroxyacetone, but less well on propionic acid and not on acetone or (R)- and (+/-)-1,2-propanediol. The Pi released from (1R, 2R)-1,2-dihydroxypropylphosphonic acid labelled with one oxygen-18 in the PO3H2 group did not stay long enough in the cells to allow complete exchange of O-18 for O-16 by enzymic turnover.

Application Of the Michaelis-Arbuzov Reaction to the Synthesis Of Internucleoside 3'-S-Phosphorothiolate Linkages

The 5'-O-monomethoxytrityl-3'-S-(aryldisulfanyl)-3'-deoxythymidines 7 and 8 have been prepared by the reaction of 5'-O-monomethoxytrityl-3'-thiothymidine with the appropriate arenesulfenyl chloride. These disulfides undergo a Michaelis–Arbusov reaction with simple trialkyl phosphites to yield 5'-O-monomethoxytrityl-3'-thiothymidin-3'-yl O,O-dialkyl phosphorothiolates. More interestingly, 3'-deoxy-3'-S-(2, 4-dinitrophenylsulfanyl)-5'-O-monomethoxytritylthymidine 8 reacts with a variety of thymidin-5'-yl dialkyl phosphites to give dithymidine phosphorothiolate triesters with the phosphorothiolate group protected with either a methyl or a 2-cyanoethyl group. 3'-O-(tert-Butyldimethylsilyl)thymidin-5'-yl triethylammoniumphosphonate 17 is converted into the corresponding bis-(O-trimethylsilyl) phosphite by treatment with bis(trimethylsilyl)trifluoroacetamide. in situ Reaction of this phosphate with disulfide 8 gives, after work-up, the dithymidine phosphorothiolate diester directly. Methylation of compound 17 with methyl chloromethanoate, followed by silylation and subsequent reaction with disulfide 8, gives the methyl-protected dithymidine phosphorothiolate triester.

Synthesis of novel pyrophosphorothiolate-linked dinucleoside cap analogues in a ball mill

Michaelis–Arbuzov reactions of S-aryl disulfide derivatives of 3′-thiothymidine or 5′-thioadenosine with tris(trimethylsilyl) phosphite proceeded in high yields to the corresponding phosphorothiolate monoesters. Subsequent hydrolytic desilylation and phosphate coupling were effected in one-pot using liquid-assisted grinding in a vibration ball mill. Novel 3′,5′- and 5′,5′-pyrophosphorothiolate-linked dinucleoside cap analogues were thereby prepared.

Photopyroelectric study of thermal properties and phase transitions in selected solids

This thesis presents in detail. the theoretical developments and calculations which are used for the simultaneous determination of thermal parameters, namely thermal diffusivity (a). thermal effusivity (e), thermal conductivity (K) and heat capacity (cr ) employing photopyroelectric technique. In our calculations. we have assumed that the pyroelectric detector is supported on a copper backing. so that there will be sufficient heat exchange between the heated pyroelectric detector and the backing so that the signal fluctuations are reduced to a minimum. Since the PPE signal depends on the properties of the detector that are also temperature dependent. a careful temperature calibration of the system need to be carried out. APPE cell has been fabricated for the measurements that can be used to measure the thermal properties of solid samples from ~ 90 K to ~ 350 K. The cell has been calibrated using standard samples and the accuracy of the technique is found to be of the order of± 1%.In this thesis, we have taken up work n photopyroelectric investigation of thermal parameters of ferroelectric crystals such as Glycine phosphite (NH3CH2COOH3P03), Triglycine sulfate and Thiourea as well as mixed valence perovskites samples such as Lead doped Lanthanum Manganate (Lal_xPb~Mn03) Calcium doped (Lal_xCaxMnOJ) and Nickel doped Lanthanum Stroncium Cobaltate (Lao~Sro5Ni,Col_x03).The three ferroelectric crystals are prepared by the slow evaporation technique and the mixed valence perovskites by solid state reaction technique.Mixed valence perovskites, with the general formula RI_xA~Mn03 (R = La. Nd or Pr and A = Ba, Ca, Sr or Pb) have been materials of intense experimental and theoretical studies over the past few years. These materials show . colossal magneloresis/ance' (CMR) in samples with 0.2 < x < 0.5 in such a doping region, resistivity exhibits a peak at T = T p' the metal - insulator transition temperature. The system exhibits metallic characteristics with d %T > Oabove Tp (wherep is the resistivity) and insulating characteristics with d % T < 0 above T p. Despite intensive investigations on the CMR phenomena and associated electrical properties. not much work has been done on the variation of thermal properties of these samples. We have been quite successful in finding out the nature of anomaly associated with thermal properties when the sample undergoes M-I transition.The ferroelectric crystal showing para-ferroelectric phase transitions - Glycine phosphite. Thiourea and Triglycine sulfate - are studied in detail in order to see how well the PPE technique enables one to measure the thermal parameters during phase transitions. It is seen that the phase transition gets clearly reflected in the variation of thermal parameters. The anisotropy in thermal transport along different crystallographic directions are explained in terms of the elastic anisotropy and lattice contribution to the thermal conductivity. Interesting new results have been obtained on the above samples and are presented in three different chapters of the thesis.In summary. we have carried investigations of the variations of the thermal parameters during phase transitions employing photopyroelectric technique. The results obtained on different systems are important not only in understanding the physics behind the transitions but also in establishing the potentiality of the PPE tool. The full potential of PPE technique for the investigation of optical and thermal properties of materials still remains to be taken advantage of by workers in this field.

Field evaluation of systemic inducing resistance chemicals at different growth stages for the control of apple (Venturia inaequalis) and pear (Venturia pirina) scab

Two field trials were conducted using established apple (Malus cv. Golden Delicious) and pear (Pyrus communis 'Williams' Bon Chretien') to assess the efficacy of three commercially available systemic inducing resistance (SIR) products, Messenger (a.i. Harpin protein), Phoenix (a.i. Potassium phosphite) and Rigel (a.i. Salicylic acid derivative) applied at four different growth stages of tree development (bud break, green cluster, 90% petal fall, early fruitlet) against the foliar pathogens Venturia inaequalis and Venturia pirina which cause apple and pear scab respectively. A conventional synthetic fungicide (penconazole) used within the UK for apple and pear scab control was included for comparison. Little efficacy as scab protectants was demonstrated when each SIR product and penconazole was applied at only two growth stages (bud break, green cluster). However when the above compounds were applied at three or more growth stages efficacy as scab protectants was confirmed. The synthetic fungicide penconazole provided greatest protection against apple and pear scab in both the 2006 and 2007 field trials. There was little difference in the magnitude of scab protection conferred by each SIR agent. Results suggest application of at least three sprays during bud break to early fruitlet formation with an appropriate SIR agent may provide a useful addition to existing methods of apple and pear scab management under field conditions. (C) 2009 Published by Elsevier Ltd.

Experimental Chemotherapy against Trypanosoma cruzi Infection Using Ruthenium Nitric Oxide Donors

The ruthenium NO donors of the group trans-[Ru(NO)(NH(3))(4)L](n+), where the ligand (L) is N-heterocyclic H(2)O, SO(3)(2 -), or triethyl phosphite, are able to lyse Trypanosoma cruzi in vitro and in vivo. Using half-maximal (50%) inhibitory concentrations against bloodstream trypomastigotes (IC(50)(try)) and cytotoxicity data on mammalian V-79 cells (IC(50)(V79)), the in vitro therapeutic indices (TIs) (IC(50)(V79)/IC(50)(try)) for these compounds were calculated. Compounds that exhibited an in vitro TI of >= 10 and trypanocidal activity against both epimastigotes and trypomastigotes with an IC(50)(try/epi) of <= 100 mu M were assayed in a mouse model for acute Chagas` disease, using two different routes (intraperitoneal and oral) for drug administration. A dose-effect relationship was observed, and from that, the ideal dose of 400 nmol/kg of body weight for both trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) (isn, isonicotinamide) and trans-[Ru(NO)(NH3) 4imN](BF4) 3 (imN, imidazole) and median (50%) effective doses (ED50) of 86 and 190 nmol/kg, respectively, were then calculated. Since the 50% lethal doses (LD(50)) for both compounds are higher than 125 mu mol/kg, the in vivo TIs (LD(50)/ED(50)) of the compounds are 1,453 for trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and 658 for trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3). Although these compounds exhibit a marked trypanocidal activity and are able to react with cysteine, they exhibit very low activity in T. cruzi -glycosomal glyceraldehyde-3-phosphate dehydrogenase tests, suggesting that this enzyme is not their target. The trans-[Ru(NO)(NH(3))(4)isn](BF(4))(3) and trans-[Ru(NO)(NH(3))(4)imN](BF(4))(3) compounds are able to eliminate amastigote nests in myocardium tissue at 400-nmol/kg doses and ensure the survival of all infected mice, thus opening a novel set of therapies to try against trypanosomatids.

Compatibilidade entre acaricidas e fertilizantes foliares em função de diferentes águas no controle do ácaro da leprose dos citros Brevipalpus phoenicis

A adição de fertilizantes foliares à calda acaricida é frequentemente empregada na citricultura com o intuito de reduzir os custos das aplicações. Todavia, as implicações desta prática, na maioria dos casos, são desconhecidas. O objetivo do trabalho foi avaliar o efeito de caldas acaricidas em mistura com fertilizantes foliares e preparadas com diferentes águas no controle do ácaro B. phoenicis. Foram realizados dois experimentos em laboratório, nos anos de 2009 e 2010, utilizando-se de frutos de laranja para conter ácaros Brevipalpus phoenicis. Um dos experimentos constou de três bioensaios, nos quais se procurou verificar o efeito das misturas entre fertilizantes foliares e os acaricidas cyhexatin, propargite e acrinatrhrin sobre B. phoenicis. No outro experimento, além de verificar o efeito das misturas de fertilizantes com os acaricidas propargite e acrinatrhrin, buscou-se também avaliar o efeito de águas coletadas em diferentes fontes utilizadas no preparo das caldas sobre B. phoenicis. Os resultados evidenciaram que a aplicação dos fertilizantes foliares cloreto de zinco, cloreto de manganês, ureia e a mistura de fosfito de potássio + ureia + cloreto de zinco não afetaram a ação dos acaricidas cyhexatin, propargite e acrinathrin sobre o controle de B. phoenicis. As misturas dos cloretos de zinco e de manganês com o sulfato de magnésio e a adição de fosfito de potássio diminuíram a eficiência dos acaricidas propargite e acrinathrin, não devendo, a princípio, ser adicionadas numa mesma aplicação. Águas provenientes dos municípios paulistas de Itápolis, Pirangi e Pirassununga interferiram na ação dos acaricidas propargite e acrinathin sobre B. phoenicis, sendo que a água coletada em Itápolis apresentou resultados superiores em termos de eficiência. Verificaram-se alterações dos valores de pH e da condutividade elétrica após a adição de alguns dos fertilizantes à calda acaricida.

Método titrimétrico para determinar fosfito em amostras agroindustriais

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Eletrodo modificado em filme de paládio para a determinação voltamétrica de fosfito

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Evaluation of different foliar fertilizers on the crop production of sugarcane

Influence of different foliar fertilizers (phosphite, micronutrients, biostimulant, phosphite + micronutrients, phosphite + biostimulant, micronutrients + biostimulant and phosphite + micronutrients + biostimulant) on yield of sugarcane was evaluated after fertilization at 30, 90 and 150 days after harvesting two-year-old sugarcane. The experiment was carried out in a commercial crop employing a randomized block design in four replicates. Higher stalk masses were observed for fertilization at 30 days after harvest, and the higher content of sucrose, total recoverable sugar and Brix degrees were observed for sugarcane fertilized after 150 days. Statistical analysis (Duncan's test) revealed no significant variation (P & 0.05) in Brix degree, sucrose content and total recoverable sugar. For total recoverable sugar x stalk weight (the main payment type for sugarcane producers), the following sequence (time treatment, fertilizer composition) 30-days, micronutrient + biostimulant; 150-days, biostimulant; and 90-days, biostimulant increased 11%, 17%, and 21% the yield of sugarcane. © 2013 Copyright Taylor and Francis Group, LLC.

Efeito de diferentes águas e fertilizantes foliares em caldas acaricidas no controle do ácaro Brevipalpus phoenicis (Geijskes, 1939) (Acari: Tenuipalpidae) em citros

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)