Porkodi Kadhirvel Molecular imprints for pharmaceutical drugs of different classes: comparison of sol-gel and acrylic approaches in different formats DEPARTAMENTO DE QUÍMICA E BIOQUÍMICA FACULDADE DE CIÊNCIAS DA UNIVERSIDADE DO PORTO MARCH/2013 FCUP iii Acknowledgments I would like to thank my supervisor, Prof. Dr. António Fernando Silva for accepting me as a doctoral student and giving me the opportunity and his time, which has helped me to come to this point of thesis submission. I offer my profoundest gratitude to my doctoral program co-supervisor, Dr. Manuel Azenha. From the choice of an appropriate topic to the process of experimenting and writing the thesis work, Manel has led me step by step to completion. He has spent his time, effort and co-ordination to my work which has not only helped me to finish the thesis but also has increased the confidence in me. His kind, patient instructions, easily approachable nature and freedom to knock on his doors of Chemistry anytime makes me indebted to him all my life and without whom this work would have never been possible. Obrigadissima Manel! I extend my thanks to Prof. Dr. Börje Sellergren (TU, Dortmund) whose expertise in the subject matter has helped me to submit a fulfilling thesis and also many thanks to him for helping me to squeeze and work with his group. I express my sincerest gratitude to all the colleagues at “University of Porto” and TU- Dortmund” for their invaluable help and support. Financial support gives a great confidence to proceed a work and FCT immensely provided me that support to work in the Universities of Porto, and TU-Dortmund, Germany. Thanks to Dra. Isabel, someone to turn around whenever I needed any help. Thanks to Prof. Paula Gomes who has helped me in technical and scientific discussions. I take time to remember in gratitude Prof. Carlos and Prof. Paulo for all the extra- curricular activities support, which has energized me many times. Thanks to Bala, Abed, Eric, Sudhir, Mahadev, Dianne, João, Isabelle, Luisa and Mariana - my good friends and Chemists who have given a piece of their mind in a right place and time. My special thanks to my friends at Dortmund who helped me to think out of the box and also providing me delicious food with dessert and drinks. iv FCUP It is time to thank my Parents, Grandparents, Sister and Brother-in-law and my dearest nephew Jeeva and niece Neena for giving me their sacrificial support. I would like to thank Nasim, Abol, Fardin and Imran for their care and help. Heartfelt thanks to Sunil Manshukhlal Rajani and Kirit Girdherdas Rajani who have extended their wisdom and knowledge to me in my social network gathering at Porto. Indispensable support from Daisy and Krishna has helped me to run this race and reach a closing phase and making my dream come true. Thank you all once again! Porkodi FCUP v Resumo A impressão molecular tem sido reconhecida como uma das metodologias mais promissoras para a preparação de materiais poliméricos com capacidade de reconhecimento específico para os moldes de impressão, que vão desde pequenas moléculas orgânicas até células inteiras. Essa "memória" molecular pode, entre outros sistemas, ser criado em redes sol-gel (híbrido orgânico-inorgânico) e polímeros acrílicos. Estes últimos teem sido extensivamente investigados enquanto as impressões em sol- gel são menos frequentemente relatadas. No entanto, uma comparação sistemática, realizada no mesmo laboratório,entre as duas abordagens diferentes estava ainda por fazer. Essa comparação, constituiu o principal objetivo do presente trabalho. Materiais de impressão em sol-gel e em acrílico foram preparados em dois formatos diferentes (monólito e esférico) para duas moléculas pertencentes a duas classes diferentes de fármacos, o naproxeno (ácido) e a aminoglutetimida (base), através de uma abordagem de impressão não-covalente. Monómeros funcionais apropriados foram sintetizados, visando a possibilidade de formar fortes complexos monómero-molde (MT), um requisito necessário a priori para a obtenção de matrizes com boa qualidade de impressão. A formação dos complexos não covalentes intermoleculares em sistemas de pré-polimerização foi confirmada por espectroscopia de UV e de RMN. Os materiais de impressão em monólito acrílico foram sintetizados por polimerização radicalar clássica enquanto que o formato esférico foi produzido por um novo processo de enxerto (grafting) e polimerização controlada com agente RAFT. No caso do sol-gel os monólito foram preparados pelo processo típico de hidrólise e condensação na presença de ácido / base como catalisador. Na obtenção de um formato esférico de sol- gel,foi seguida uma técnica de enchimento de poros de sílica esférica. A fim de avaliar o processo de impresão, os correspondentes materiais não-impressos foram sintetizados vi FCUP seguindo o mesmo protocolo, mas omitindo-se o molde. Os materiais foram subsequentemente caracterizados por BET e TGA, a fim de obter os detalhes dos poros e propriedades térmicas, e SEM foi utilizado para investigar a textura da superfície. Uma investigação extensiva foi então realizada por cromatografia líquida, tendo-se para isso empacotado os materiais em pequenas colunas, avaliando-se os parâmetros de desempenho, tais como o factor de impressão e seletividade.Para determinar as propriedades de ligação e parâmetros relacionados com a eficiência cromatográfica, utilizou-se o método rigoroso da análise frontal. Da comparação global sistemática dos diferentes parâmetros estudados, chegou-seà conclusão de que os materiais de impressão acrílica com naproxeno, especialmente no formato de filme fino (MIP-GS-NAP) em partículas esféricas, exibiram características melhores quando comparados com os materiais sol-gel. Para a aminoglutetimida, de novo, as impressões de acrílico demonstraram um desempenho superior para os parâmetros mais importantes, ou seja, capacidade, seletividade e cinética de transferência de massa. Notavelmente, o formato esférico de paredes finas (MIP-GS- AGT), mais uma vez, mostrou vantagens (concretamente maior seletividade e mais rápida transferência de massa) sobre o formato monólito acrílico, e portanto, essa tendência parece ter surgido a partir deste conjunto muito restrito dos dois modelos estudados. FCUP vii Abstract Molecular imprinting has been recognized as one of the most promising methodologies for the preparation of intelligent polymer materials with specific recognition capacities for the template molecules, ranging from small organic molecules to whole cells. Such molecular “memory” can, among other systems, be created in sol-gel (organic-inorganic hybrid) networks and acrylic (organic) polymers. The latter has been extensively investigated while the sol-gel imprints are also frequently reported. Nevertheless, a systematic, within lab, comparison between two different approaches is lacking. That systematic comparison of the two different approaches, constituted the main goal of the present work. Sol-gel and acrylic imprints were prepared in two different formats (bulk and spherical) for two different classes of pharmaceutical drugs-naproxen (acidic) and aminoglutethimide (basic) by non-covalent imprinting approach. Appropriate functional monomers were synthesized, envisaging the possibility of forming strong monomer-template (M-T) complexes, an a priori requisite for obtaining good quality imprinted matrixes. The formation of non-covalent intermolecular M-T complexes in pre-polymerization systems was confirmed by UV and NMR spectroscopy experiments. Acrylic bulk imprints were synthesized by classic radical polymerization whereas the spherical format was produced by novel controlled grafting process using RAFT agent. On the other hand, sol-gel bulk imprints were prepared by typical hydrolysis and condensation process in presence of acidic/basic catalyst. In obtaining a spherical format sol-gel imprints a pore filling technique was followed. In order to evaluate the imprinting behavior, corresponding non-imprinted materials were synthesized following the same protocol but omitting the template. Synthesized imprints/non-imprints were subsequently characterized by BET and TGA in order to obtain pore and thermal properties details, and SEM was used to investigate the surface texture of the materials. Further, an extensive chromatographic investigation was conducted by packing the materials in small columns and assessed by liquid chromatography to obtain the viii FCUP chromatographic performance parameters such as imprinting factor and selectivity. The accurate method of frontal analysis experiment was carried out to get binding properties and column efficiency determining parameters. The results obtained were compared systematically and the overall comparison of the different parameters studied finally gave a conclusion that the acrylic naproxen-imprints, especially in the thin-walled (MIP-G-S-NAP) spherical format, exhibited better features when compared to sol-gel naproxen-imprints. For the template aminoglutethimide, again, the acrylic imprints exhibited superior performance for the most important parameters, namely selectivity, capacity and mass transfer rate. Most noticeably, the thin-walled (MIP-G-S-AGT) spherical format, once more, has shown advantages (specifically higher selectivity and faster mass transfer) over the bulk acrylic format, and therefore a trend appears to have arisen from within this very restricted set of two templates studied. FCUP ix Index Acknowledgments............................................................................................................... iii Resumo ................................................................................................................................. v Abstract .............................................................................................................................. vii Index .................................................................................................................................... ix List of Figures .................................................................................................................. xvii List of Tables ................................................................................................................... xxix List of Equations ............................................................................................................ xxxv List of Abbreviations .................................................................................................... xxxvii Chapter 1 : Introduction ....................................................................................................... 1 Introduction .......................................................................................................................... 1 1.1 Molecular Recognition in Nature ...................................................................... 3 1.2 Artificial Receptors .......................................................................................... 4 1.3 Historic Perspective of Molecular Imprinting .................................................... 5 1.4 Antibody versus MIPs ...................................................................................... 6 1.5 The Basic Strategy of MIPs ............................................................................. 8 1.6 MIP preparative approaches............................................................................ 9 1.6.1 Covalent Imprinting ................................................................................ 11 x FCUP 1.6.2 Non-covalent Imprinting ......................................................................... 11 1.6.3 Semi covalent or Sacrificial Spacer Approach ........................................ 12 1.7 Advantages and Limitations of Covalent and Non-Covalent Approaches ...... 13 1.8 Application of Molecular Imprinting Technology............................................. 15 1.8.1 MIPs in Separation Techniques ............................................................. 16 1.9 Preparative Methods of Molecularly Imprinted Polymers ............................... 18 1.9.1 Organic Polymers .................................................................................. 18 1.9.2 Sol-Gel Organic- Inorganic Hybrids ........................................................ 21 1.10 MIPs in Different Physical Formats................................................................ 22 1.10.1 Limitations of Traditional Bulk Imprinted Polymer Materials ................... 23 1.10.2 Various Ongoing Explorations on Novel Imprinting Strategies................ 24 1.11 Problems and Challenges in Molecularly Imprinted Polymers ....................... 27 1.11.1 Binding Site Heterogeneity and Non-specific Binding ............................. 27 1.11.2 Challenges of Facing Aqueous Environment .......................................... 30 1.12 Organic versus Sol-gel Organic-Inorganic Hybrid .......................................... 31 1.13 Within lab Acrylic Vs. Sol-Gel comparison ..................................................... 33 1.14 Work Plan: Outlook ....................................................................................... 34 1.15 Detailed Work plan ........................................................................................ 35 1.15.1 Templates selected for the project ......................................................... 35 1.15.2 Choice of Functional Monomers ............................................................. 36 1.15.3 Study of Monomer-Template Complex [M-T] Assemblies by Spectroscopic Techniques ..................................................................... 38 1.15.4 Synthesis of Acrylic and Sol-gel Imprinted Matrices ............................... 40 1.15.4.1 Acrylic Bulk Polymers: Classical Approach by Free Radical Mechanism ...................................................................... 40 1.15.4.2 Acrylic Spherical Format: Novel Grafting Technique by Controlled Radical Polymerization ...................................................................... 40 1.15.4.3 Sol-gel Bulk Xerogels: Hydrolysis and Condensation ........... 41
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