Ferrocene-peptide conjugates represent a hybrid area between organometallic chemistry and biochemistry. In these bioconjugates, the ferrocene (Fc) moiety can serve as molecular scaffold, chromophore, sensitive probe, biological marker, redox active site, etc. Disubstituted Fc systems, in which both cyclopentadienyl rings are substituted, provide influence over the supramolecular structure of the assemblies, and serve as starting materials for the design of electronic biomaterials. Recently, 1'-amino-ferrocene-1-carboxylic acid (Fca) and 1,1'-diaminoferrocene Fc[NH2]2 were recognized as useful tools in bioorganometallic chemistry. This work sketches some novel preparative and structural aspects of Fc-peptide conjugates and explores their applications as biosensors and as polymeric materials. <p>First, I demonstrated that Fca invariably induces a turn-like structure, which is stable in solution and the solid state. The obtained results showed different behaviour for Fca peptides depending on the chirality and position of the attached amino acid. The axial chirality of the Fca is completely dependent on the chirality of the first amino acid attached to the amino terminal of the Fca group.<p>Second, I was able to develop a surface based sensor for the electrochemical detection of papain based on Fc-peptide conjugates. The idea was to place a surface-bound redox probe in close proximity to the electrode surface. In addition, the redox-active Fca label will be part of the recognition site but will not interfere with the recognition process. My sensor provides an attractive alternative for the electrochemical detection of non-labelled non-redox active proteins, which under current detection schemes remains a significant challenge.<p>This work represents a truly important proof of concept for establishing this novel bioorganometallic approach for the electrochemical detection of important biological targets.<p>Last, I was successful in developing a very convenient method to synthesize 1,1'-bis(tert-butoxycarbonylamino)ferrocene as a stable derivative of Fc[NH2]2. This new synthetic approach has circumvented the problems encountered with the explosive diazide usually used as a precursor in the conventional synthons of Fc[NH2]2. Building on this achievement, a series of novel peptide-like oligomeric and polymeric ferrocenyl-amides were synthesized and fully characterized. The electrochemical investigations on these polymers suggested unresolved or no electronic interaction between the ferrocene groups in all systems. These results may reveal the influence of the amide groups on suppressing the electronic interaction between the iron centers in my polymers. <p>Thus, my systematic work on Fc-peptide conjugates lays solid foundations in the fields of structural control, biosensors, and material science.

No description available.

A new technique for conversion of carboxylic acids to carbinols under mild conditions has been developed. This technique requires first formation of enol esters by coupling N-ethyl-5-phenylisoxazolium3' -sulfonate with an appropriate carboxylic acid in acetonitrile, followed by reduction with a 10-fold molar excess of NaBH₄. In addition five carboxyl group "specific" reagents were surveyed for their ability to inactivate the sulfhydryl proteinase papain (EC 3.4.4.10). No inactivation of the enzyme was observed with three of the reagents, diazoacetamide, triethyloxonium tetrafluoroborate, and N-ethyl-5-phenylisoxazolium-3'-sulfonate. The remaining two reagents, N-ethoxycarbony1-2-ethoxy-1,2-dihydroquinoline and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide did indeed inactivate papain. Inactivation by N-ethoxycarbony1-2-ethoxy-1,2-dihydroquinoline appears to be the result of reaction of this reagent with the free thiol group of the enzyme. Treatment of papain with l-ethyl-J-(3-dimethylaminopropyl) carbodiimide resulted in modification of the free thiol group, in 6 to 10 of the nineteen tyrosyl groups, and in six of the fifteen free carboxyl groups. The free thiol group could be essentially completely protected from reaction by converting it to its mercuric derivative with HgCl₂, and the tyrosyl modification was shown to have no effect on enzymatic activity. Reaction of active or mercuripapain with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide in the presence of glycine ethyl ester resulted in incorporation of 6 ethyl glycinate residues per molecule of enzyme, with essentially complete inactivation. In the presence of benzamidoacetonitrile, a competitive inhibitor of papain, only four ethyl glycinate residues were incorporated into active papain with retention of ~80% of enzymatic activity, thus establishing that at least one and perhaps two of the six modified carboxyl groups were located in the active site of the enzyme. The identity of one of these carboxyl groups was postulated to be Asp-158, and thus some evidence was provided for the tentative assignment of a direct mechanistic role in catalysis for this amino acid residue. / Ph. D.

No description available.

No description available.

No description available.

Universiẗat, Veterinärmedizinische Fakultät, Diss., 2005--Leipzig.

Orientador: Jose Luiz Donato / Dissertação (mestrado] - Universidade Estadual de Campinas, Facu.dade de Ciencias Medicas / Made available in DSpace on 2018-08-15T19:17:22Z (GMT). No. of bitstreams: 1 Reque_MariaLucia_M.pdf: 10714154 bytes, checksum: ae7569765a8246a32a670f4b5f2dc205 (MD5) Previous issue date: 2006 / Resumo: A barreira dérmica é representada por uma estrutura lipoprotéica e constitui um ambiente bioquímico altamente complexo. Trata-se de uma barreira muito eficiente ao ingresso de agentes químicos. Não há na literatura nenhum relato do uso de enzimas proteolíticas como agente potencializador da permeação de fármacos presentes em formulações para uso tópico. Neste trabalho desenvolvemos uma formulação de pomada contendo a enzima para potencializar a penetração de diclofenaco dietilamônio na pele, sendo realizado também o estudo da sua estabilidade na formulação através do acompanhamento da sua atividade catalítica, bem como a avaliação in vivo do seu potencial de alergenicidade cutânea. A atividade da papaína como potencializadora da penetração cutânea de diclofenaco dietilamônio em pomada foi avaliada in vitro através de células de difusão de Franz utilizando pele de porco, e in vivo, por meio de testes clínicos com voluntários humanos. O estudo de estabilidade, revelou que a atividade catalítica da papaina permaneceu estável na formulação quando a mesma foi armazenada a temperatura ambiente por pelo menos 24 meses, porém, sob condição acelerada (45°C e 70% U.R.), a enzima perdeu a sua atividade catalítica após seis meses de armazenamento. Não foi observada nos voluntários humanos, nenhuma reação adversa como eritema, pápulas ou vesículas durante o período de avaliação da alergenicidade cutânea da papaína, sendo a enzima aprovada para uso tópico. No teste de permeabilidade in vitro, foi observado um aumento de 50% no acúmulo de diclofenaco após quatro horas de permeação, quando a papaína esteve presente na formulação de diclofenaco dietilamônio em pomada. O teste de permeabilidade in vivo mostrou que a papaína aumentou a penetração de diclofenaco na pele quando a mesma foi tratada anteriormente com pomada contendo papaína e não quando a enzima foi administrada juntamente com o diclofenaco. Foi demonstrado o potencial da papaína como potencializadora da penetração de antiinflamatórios não-esteroidais (AINEs) em formulações de uso tópico, melhorando consideravelmente a eficiência das mesmas / Abstract: The skin barrier is represented by a lipoprotein structure and is a very complex biochemical environmental. It is considered a very efficient barrier to the absorption of many chemical compounds. There is no report about the use of proteolytic enzymes as a penetration enhancer of therapeutic drugs in topic formulations. In this study, an ointment formulation containing papain was developed to increase the diclofenac diethylammonium skin penetration. Stability studies were performed to verify the enzymatic activity after incorporation in the formulation. It was also evaluated the in vivo allergenecity potential of the papain. The penetration enhancing activity of this enzyme was investigated in vitro through Franz-type diffusion cell using porcine skin, and in vivo, through clinical tests with human volunteers. The stability study showed that papain remained stable in the formulation when it was stored at room temperature during 24 months, however, at accelerated condition ( 45°C and 70% of humidity) the enzyme lost its catalytic activity after 6 month of storage. Regarding in vivo allergenicity studies, the human volunteers no observed adverse reactions as eritema, papulas or vesicle during the period of evaluation, being the papain approved to topical use. On in vitro skin permeation study, an increase of about 50% in the diclofenac accumulation was observed after four hours of permeation when the papain was present in the formulation. The in vivo skin permeation study showed that papain increased diclofenac skin penetration when it was treated previously with ointment containing papain and no when the enzyme was administrated together diclofenac. It was demonstrated the papain potential as penetration enhancer in NSAIDs (non steroidal anti-inflammatory drugs) formulation for topical use improving its efficiency / Mestrado / Mestre em Farmacologia

In the first chapter, sodium tetrathionate (TT), a sulfhydryl blocking agent, is assessed for its ability to protect the proteolytic activity (PA) of papaya latex during air, sun or vacuum drying, and of crude papain during storage. By means of Taguchi's L₂₇ (3¹³) fractional factorial design, it was found that the addition of 1% TT significantly increased the retention of PA of papaya latex when it was air dried at a temperature of 55°C. This protection of PA was found to be 23% higher than the one given by the addition of 1% sodium metabisulfite, the compound commonly used in the commercial processing of papaya latex. When drying was carried out either under 27 inches vacuum at 50°C or in the sun, the protective effect of TT on the PA was not significantly different from that of metabisulfite. The PA of crude papain during storage at room temperature was also protected by TT. A loss of 20% of the original PA occurred over a period of 13 wk when crude papain contained 1% TT, compared to a loss of 45% when the crude enzyme preparation contained 1% metabisulfite. In the same chapter five different oxidants for synthesis of TT from thiosulfate are compared, namely: iodine, hydrogen peroxide, ferric chloride, cupric sulfate and sodium vanadate. The results indicated that hydrogen peroxide or sodium vanadate were not only effective in the oxidation but also much less expensive than iodine, which is the most popular oxidant for the synthesis of TT. The results obtained in this chapter warrant the use of TT in the commercial production of commercial papain to prevent the destruction of the enzymes during harvesting, storage, transportation and processing. In the second chapter, chemical modification of pure papain by TT is discussed. Optimization techniques were applied for improving the precision of two methods used in this study: circular dichroism (CD) and proteolytic activity determination. Simplex optimization significantly improved repeatability and signal to noise ratio of the CD scan of papain. A new optimization approach, which was a combination of a central composite rotatable design and simplex optimization, was successfully applied to achieve maximum precision for the proteolytic activity assay of papain using casein as a substrate. This approach may also be applied to other analytical methods to improve the reliability of the experimental data. Influential factors in the inactivation of PA of papain by using TT and reactivation of the inactivated papain by cysteine were carried out using two Taguchi's L₁₆ (2¹⁵) fractional factorial designs. The results indicated that when inactivation was carried out at pH 6.8, with a reaction time of 5 min at 22°C, and a molar ratio of TT to papain of 10, the inactivation reaction was highly reversible upon addition of 20 mM cysteine. Although some interactions of the factors were significant, 70% reactivation was achieved in most cases. Analysis of UV absorbance, near-UV and far-UV CD spectra indicated that there were no major changes in the spectra in papain upon the chemical modification of the enzyme with TT. Secondary structure computed from far-UV CD spectra also demonstrated no significant changes upon this modification. Sulfhydryl data and pH-fluorescence profiles of the modified papain support the hypothesis that reversible blocking by TT results from binding with the single reactive cysteine residue present in papain. Quenching of the intrinsic fluorescence of papain when the modification was carried out using high molar ratios of TT to papain was suggestive of modification of tryptophan residues in the enzyme during the oxidation reaction with TT. Precipitation or insolubilization of pure papain, and of the proteins of papaya latex and commercial papain was observed upon the chemical modification with TT under certain conditions. Addition of β-mercaptoethanol and TT at levels of 100 mM and 50 mM, respectively, precipitated 90% of pure papain. Solubility studies together with electrophoretic analysis of the precipitated papain suggested formation of insoluble aggregates due to the insoluble aggregation as a result of inter-molecular disulfide bonds formation. TT was found to be a competitive inhibitor of both reversible and irreversible inhibition of the enzyme action, when carbobenzoxyglycine p-nitrophenyl ester was used as a substrate. The second order inactivation constant in the absence of substrate was computed to be 16,919 M⁻¹sec⁻¹, indicating that the reaction had a high rate. / Land and Food Systems, Faculty of / Graduate

Muitas estratégias são indicadas a fim de suplantar a baixa permeabilidade de fármacos através da epiderme, umas delas, a de incluir promotores de penetração em formulações farmacêuticas e/ou cosméticas, formulados em sistemas terapêuticos transdérmicos (TTS). Estas substâncias são passíveis de modificar os domínios proteicos da epiderme e removerem provisoriamente a resistência da barreira do estrato córneo, permitindo o acesso dos fármacos aos tecidos viáveis através da circulação sistêmica. A natureza do veículo tópico é conhecida por desempenhar um papel importante na promoção da absorção dentro e através da pele. Os veículos tópicos convencionais, como pomadas, cremes ou géis, exercem predominantemente seus efeitos ao liberar o medicamento na superfície, e as moléculas dos fármacos, então, se difundem através de suas camadas. Os hidrogéis foram obtidos a partir de material polimérico reticulado por processo de radiação ionizante. A cafeína foi escolhida como substância modelo de permeação dérmica, por ser hidrofílica. Entretanto, para que a molécula penetre na barreira cutânea, deve ser associada a promotores de absorção cutânea. A papaína tem sido aplicada na pele íntegra como agente promotor de penetração e absorção cutânea. As enzimas interferem na absorção percutânea de fármacos por duas formas: como um potencializador de penetração e retardador de absorção. Neste trabalho, foram sintetizadas membranas de hidrogéis com poli (N-2- vinil - pirolidona) (PVP), poli (etilenoglicol) (PEG), poli (etilenoglicol diacrilato) (PEG-DA), cafeína e gel de papaína formado de polímero sintético pré-neutralizado para estudo da cinética de permeação cutânea, utilizando ecdise de cobra (Boa Constrictor) como membrana modelo de permeação. As membranas de hidrogel preparadas, foram caracterizadas por análise de termogravimetria (TG), calorimetria exploratória diferencial (DSC), intumescimento, fração gel, microscopia eletrônica de varredura (MEV), e realizados ensaios de permeação dérmica em células de Franz e tomografia de coerência óptica (OCT). O teste de permeação \"in vitro\" desses hidrogéis na ecdise foram satisfatórios, comprovando a eficácia da papaína como promotora de permeação dérmica e contribuindo, assim, para futuros trabalhos que possam explorar ainda mais essa área de entrega de fármacos. / Many strategies are indicated for supplanting the low permeability of pharmaceuticals through the epidermis, one of them to include developers of access in cosmetics formulations, produced in Transdermal Drug Systems. Those substances are capable of modifying the protein dominates of the epidermis and temporarily remove the resistant barrier of the stratum cornea, allowing this way, the access to the pharmaceutical to the viable fabric through the systemic circulation. The nature of the topic vehicle is known for developing an important role of the absorption inside and through the skin. The conventional ways, as ointments, creams or gels, predominated theirs effects by liberating the medication in the superficies, so the pharmaceutical molecules defund beyond their layers. The hydrogels were got from the polymeric reticulate material for ionizing radiation process. The caffeine was chosen as a substance model of the dermis permeation, due to be hydrophilic. Moreover, for the molecule access, it is necessary to the developers of dermal absorption. The papain has been applied in the integra skin as agent promoter of access and dermal absorption. The enzymes interfere in the dermal absorption of the pharmaceutical in two forms: as optimizing, as well as, retarding absorption. This essay we could synthesized hydrogel membranes as Poly (N -2 - vinyl-pirolidon) (PVP), Poly(ethylene glycol) (PEG), Poly (ethylene glycol) diacrylate (PEG-DA), caffeine and papain gel formed by polymeric synthetic pre-neutralized for the studies of kinetic of dermal access for using ecdysis of a snake (Boa constrictor) as a membrane model of access. The membranes of hydrogel prepared for this essay were characterized by thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), swelling, gel fraction, scanning electron microscopy (SEM), and rehearsal of dermal access in Franz cells and optical coherence tomography (OCT). The test of permeation in vitro was satisfactory evidencing the efficacies of papain as a developer of dermal permeation, as well as, contributing for the future work that can explore even more this area of pharmaceutical delivery.