Biotage Instrumentos, consumibles y experiencia para excelencia en química medicinal

Sintesis Organica Asistida con Horno de Microondas

Flujo de Trabajo Desarrollo de nuevas Drogas Terapeuticas Sintesis Optimizacion Purificacion Evaporacion Asistida con Microondas Mejora velocidad de reaccion Mayor rendimiento y pureza Permite reacciones “imposibles” Reproducible Amplia biblioteca de aplicaciones Mas de 2,000 unidades instaladas y operando Optimizacion Aditivos de proceso Simples de usar Remocion rapida de productos de reaccion no deseados Purificacion rapida via cromatografia flash Mas purificacion en menos tiempo Detecta cualquier componente Provee informacion sobre pureza de los compuestos Mas de 2,500 unidades instaladss y operando Rapida evaporacion de solventes Solventes Organicos y Agua Formato flexible En modo continuo o por lotes

Introduccion Los reactores de sintesis organica de Biotage utilizan radiacion de microondas como fuente de energia para el lograr alta eficiencia en reaciones quimicas, lo que permite procesos de sintesis mas rapidos y con mayor rendimiento comparado con aquellos en base a calentamiento convencional (conduccion). Bigger Hotter Faster To Inifinity and Beyond 4

Sintesis Organica Tradicional Calentamiento a traves de baño de aceite, o con mecheros bunsen Uso de tecnica de reflujo para trabajar por arriba del punto de ebullicion de los solventes, a falta de viales de reaccion adecuados a alta presion Sistema fragil, complejo y poco reproducible 5

Mecanismo de calentamiento por microondas La radiacion de microondas sobre una muestra determinada, conduce a que sus moleculas vibren y ocurra friccion entre ellas, generando calor desde el interior de la muestra. Conduccion Ionica Oscilacion Dipolar How does microwave energy heat a reaction? There are two major mechanisms by which heating takes place. If two tubes containing water and dioxane are heated at a fixed radiation power for a fixed time, the final temperature will be higher in the water sample. This is due to a mechanism called dipolar polarization. A substance possessing a dipole moment when irradiated with microwaves will generate heat. A dipole is sensitive to external electric fields and will attempt to align itself with the field by rotation. The frequency of the microwave radiation is low enough so that the dipoles have time to respond to the alternating electric field and rotate. The frequency is, however, not high enough for this rotation to precisely follow the field. So as the dipole re-orients to align itself with the electric field, the field is already changing and generates a phase difference between the dipole and the orientation of the field. This results in increased molecular friction and collisions, giving rise to dielectric heating. So dioxane, which does not have the dipole characteristics necessary for microwave dielectric heating, does not heat while water, which has a large dipole moment heats readily. If two samples containing distilled water and tap water are heated, the final temperature of the tube with the tap water will be higher. This is due to the conduction mechanism. A solution containing ions or even a single isolated ion with a hydrogen bonded cluster in the sample will move through the solution under the influence of an electric field, resulting in an increased collision rate. The conduction mechanism is a much stronger interaction than the dipolar mechanism with regards to heat generating capacity. Orientacion Molecular = Friccion Molecular = Calor

Microondas vs. Calentamiento Convencional Calentamiento por Microondas Calentamiento Convencional When heating a sample using microwaves, the sample must be in resonance with the microwaves. Basically the system can consist of single- or multimode cavities: (compartments specially designed for resonance) The sample (load) must be placed in such position in the cavity that resonance will exist i.e. good resonance = lot of energy transfers into the load = rapid heating. Besides resonance, also the chemical properties of the solvent and additives, impacts the heating result 7

Beneficios de calentamiento rapido de las reacciones de sintesis Arrhenius Equation: k = A e–DG‡/RT A partir de la ecuacion de Arrhenius y en relacion a la velocidad de las reacciones, es aceptado el principio de que para una reaccion quimica a temperatura ambiente dada, la velocidad de la reaccion se duplicara por cada incremento de temperatura de 10 grados celsius. Convencional Micro-ondas 23 oC / 12 h = 100 oC / 5 min 23 oC / 16 h = 110 oC / 2 min 70 oC / 16 h = 150 oC / 5 min

Reduccion del tiempo de ciclo de desarrollo de nuevas moleculas Chemistry is an experimental science built up of iterations of hypotheses-experiments-results. Not until the experiment has been conducted and analysed will we know if we can progress in the development process or if we have to get backwards one or more steps trying out a different synthetic route (another hypothesis). What microwave synthesis offers is the possibility to go through many more iterations during the same period of time as compared to conventional techniques leading to quicker results and improved efficiency. Higher yields and cleaner reactions don’t come automatically. They have to be iterated to reach the optimal condition. 9

Ventajas de Sintesis Asistida con horno de Microondas Tiempos de reaccion mas cortos Pruebas rapidas de ideas creativas Incremento en la productividad Mayor rendimiento, pureza Mayor diversidad de reacciones posibles Reacciones “imposibles” ahora son comunes Menor uso de reactivos y solventes Resultados reproducibles Condiciones de sintesis directamente “escalables” para sintesis de alto volumen The short reaction times often enables chemists to think out of the box and rapidly test their creative ideas and increases their productivity. And because the reactions are done at much higher temperatures than possible under conventional heating, sometimes reactions that are very difficult to do conventionally are made possible. The short reaction times usually also result in higher yields and purity. Since we are pushing the reactions with high energy we often find that we need less reagents and solvents and less reactive reagents than conventional synthesis. The results of the reactions are always reproducible, even by a different chemist at a different site. 10

Elementos a considerar con sintesis asistida con horno de microondas De los solventes: Punto de ebullicion Nivel de absorsion de calor Polaridad y variabilidad de polaridad con temperatura Propiedades ionicas Presion de Vapor

Elementos a considerar con sintesis asistida con horno de microondas Interacciones con co-solventes Set temp: 200 °C 4.5 mL Toluene 4.0 Toluene + 0.5 mL DMF

Ejemplos de reacciones de sintesis asistidas con horno de microondas

Reacciones de Reduccion

Hydrogenation Microwave: 4 min, 140oC, 71% Reductions of double bonds using Pd/C and hydrogen in pressurized systems are a very common but tedious reaction to perform. In these uW reactions ammonium formate and cyclhexene have been used as a sourse of hydrogen to eliminate the need for H2 gas in the reaction. Microwave Synthesis supports reactions at extreme temperatures and pressures thus excluding the need for specially designed pressure safety vessels. Biotage Pathfinder data

Reacciones de oxidacion

Pyridinium- dichromate Oxidation No. Oxidizer Solvent Temp (oC) Time (s) Yield % 1 Pyridinium- dichromate DMF 100 300 55 2 Dess Martin DCE 70 200 63 3 NIS/Bu4NI 400 67 4 Ce(SO4)2/NaBrO3 CH3CN/H2O 115 700 25 NIS - N-Iodosuccinimide (1-Iodo-2,5-Pyrrolidinedione; Succiniodimide) Biotage Pathfinder data

Oxidation Microwave: 400 s, 110oC, 100% Microwave: 20 min, 200oC, 65% Biotage Pathfinder data

Oxidation of Alcohols with MP-TsO-TEMPO Method Solvent Temp Time Yield Non-mW CH3CN rt 16 h 95 % mW DCM 100 oC 10 min 100 % 99 % 60 oC 2 h 70 % 5 min 93% 1.5 min 2.5 min Lundin, R. www.biotagepathfinder.com 19

Aminacion Reductiva

Reductive Amination Amines are commonly alkylated via reductive amination using an aldehyde or ketone. Sodium Cyanoborohydride or Sodium Triacetoxyborohydride are the most common reducing agents used. Most reductive aminations are accelerated under acidic conditions. 21

MP-CNBH3 Microwave-assisted Reductive Amination When heating with microwaves, even unreactive ketones reacted smoothly Combination of secondary amines and unreactive ketones gave little product DCM was better than THF in mW reactions Excess amine gave complete conversion however with more acetylated byproduct (Best results with 0.8 eq amine: 1 eq carbonyl) Both THF and DCM were tried as solvents. In most cases DCM worked better. Two methods were tried: (1) Heating all ingredients together at 110 oC/5 min and (2) Premixing the amine and carbonyl at 110 oC/2 min followed by addition of the cyanoborohydride and heating at 110 oC/5 min. No major differences could be seen in the two methods. In some cases the two step procedure gave lower yields. Temperatures higher than 110 oC resulted in amine acetylation. 0.5 mmol amine to 0.6 mmol aldehyde gave best results. Using excess amine gave complete conversion but also resulted in more acetylated amine byproduct. Temp > 110 oC resulted in amine acetylation Pre-mixing amine + Carbonyl made no difference. In some cases this gave lower yields. 22

Reductive Amination with MP-CNBH3 Amine Carbonyl Convn. Yield mW 79 % 98 % 91 % 85 % 39 % 77 % 63 % 69 % 73 % Conventional condition: (1) THF (2) room temp, 16 h Microwave condition: (1) DCM (2) 110 oC, 5-7 min Both THF and DCM were tried as solvents. In most cases DCM worked better. Two methods were tried: (1) Heating all ingredients together at 110 oC/5 min and (2) Premixing the amine and carbonyl at 110 oC/2 min followed by addition of the cyanoborohydride and heating at 110 oC/5 min. No major differences could be seen in the two methods. In some cases the two step procedure gave lower yields. Temperatures higher than 110 oC resulted in amine acetylation. 0.5 mmol amine to 0.6 mmol aldehyde gave best results. Using excess amine gave complete conversion but also resulted in more acetylated amine byproduct. Pannagiotis, P. www. biotagepathfinder.com 23

Amidation

Comparing Procedures Conventional Microwave Temp Room Temp 100 ºC Time 12 h 5 min PS-DCC 2 eq Performing the reaction at 150 C using microwave instead of room temperature, completes the transformation within 5 mins compared to 12h required otherwise. 25

Amide formation Acylation reaction that combines polymer-assisted solution-phase microwave chemistry with solid-phase extraction Both synthesis and purification takes < 15 min 6 different amines were used HOBt scavenged via Si-carbonate SPE Recently, Sauer and colleagues have reported an acylation reaction that combines polymer-assisted solution-phase microwave chemistry with solid-phase extraction (SPE), routinely allowing both synthesis and purification to take place in under 15 min. Conventional conditions require rt/16hrs. The reaction of the acid shown with 6 different amines was accomplished using microwave irradiation for 5 min at 100 C; the coupling agents used were polystyrene-bound carbodiimide and 1-hydroxybenzotriazole (HOBt). After completion, the mixture was run through a plug of Si-carbonate (3-4 equivalents), eluting with methanol, to scavenge the HOBt and any unreacted acid, and the desired product was obtained in high purity. Less reactive amines (dibenzylamine) gave lower conversions after 5 min of microwave heating (37 % vs. 98 %). The authors note in the paper that the “use of a stir-bar facilitates the suspension of PS-carbodiimide resin and mixing of reactions involving insoluble reagents.” And that it does not promote the mechanical degradation of the polymer support. – pg 4724. The authors describe a nice synthetic method using a combination of a microwave-assisted amide coupling using polymer supported carbodiimide with a simple sequestering of HOBt with a silica gel based carbonate. By this combination pure products are available within less than 15 minutes from the start of synthesis. The reaction conditions are amendable to primary, secondary as well as aromatic amines. Noticeable is that amines of low reactivity, such as aniline, provide moderate yields of amides after only five minutes while the overnight reaction at ambient temperature provided less than 10% conversion. D.R. Sauer; D. Kalvin; K.M. Phelan, Org. Lett. 2003, 5, 4721-4724.

NEAT Coupling Microwave: 10 min, 200oC, 82% Biotage Pathfinder data

Reaciones Catalizadas con Metales y Organometicos

Suzuki Coupling 3-Aryl furan-2-ones µW, Solvent Base Temp Yield CH3CN Na2CO3 90 oC decomposition Toluene K2CO3 140 oC 40 % 3-aryl furanones have been reported to have a wide range of activities as antifungal agents. Conventional synthesis of these compounds is generally done over 7 steps. Worthington et al developed a 5 step general method for the synthesis of these compounds with the final step involving a suzuki reaction with various commercially available boronic acids. The solvent in this case played a major role in reaction outcome. The reaction in toluene was successful, even though it was performed at a high temperature than the reaction performed in acetonitrile, underscoring the fact that temperature is not always the culprit during a bad reaction outcome. Optimized reaction conditions found using phenylboronic acid Other Boronic acids yielded 46-63% yield under these conditions Worthington, P. A. et al, Synlett, 2005, 3, 538-540 29

Organometalic Reactions Metal Mediated Reaction Sonogashira Microwave 120 oC, 25 min, 87 % Conventional 100 oC, 24h, 33 % ・ウプサラ大学で実施の、マイクロウェーブ加熱を利用した最初の薗頭反応。 ・種々のハロゲン化アリールとトリメチルシリルアセチレンとの反応を実施 ・MW加熱では5-25分でいずれも80-95%という高収率で目的化合物が得られている ・ブロモベンゼンを用いた場合では、従来法では24時間100℃で33%の収率、MWでは120℃2５分で87% ・イオドベンゼンを用いた場合には５分 Erdeyle, M; Gogoll, A J. Org. Chem. 2001, 66, 4165-4169 30

Organometalic Reactions Domino Ring Closing Metathesis This high temperature ruthenium catalyzed cascade reaction affords bicyclic and tricyclic annulated systems in short times and high yields. Under conventionally heating repetitive addition of catalyst was required due to gradual catalyst decomposition. Using microwave heating the conversions are performed using a single addition of catalyst, using overall 50-80% less catalyst and yet obtaining better yields. Need 50-80% less catalyst compared to conventional Efskind J., Undheim, K. Tetrahedron Lett. 2003, 14, 2837-2839. 31

Organometalic Reactions Preparation of Grignard reagent 89 % Conventionally 0°C, hours Et2O Inert atmosphere Microwaves 60°C, 10min THF Normal atmosphere Grignards can be performed efficiently by heating at 60 oC in the absence of an inert atmosphere. Synthesis: The indicated Grignard reagent was not analyzed or isolated. All yield and characterization data is for the corresponding acid after reacting the Grignard reagent with CO2 (see procedure below). Work-up and purification: Immediately after the heating was completed, a stream of dry CO2 was passed via cannula through the reaction mixture for 3 min at room temperature and then for 2 min at minus 40°C ("dry ice" - acetonitrile bath). The resulting mixture was then subjected to microwave heating at 120 °C for 5 min whereupon it was penetrated by a needle to minimize developed overpressure (caution!). 4N HCl (1.0 mL) was added to the resulting mixture (caution! intense evolution of hydrogen!) and after the gas evolution was over, the reaction mixture was poured into 20 mL of 1N HCl and extracted with MeOtBu (4 x 10 mL). Combined organic extracts were washed with 1N NaOH solution (3 x 10 mL), acidified by careful addition of concentrated HCl to pH~1 and extracted with MeOtBu (3 x 10 mL). Combined organic extracts were washed with brine and dried (Na2SO4). After filtration and solvent evaporation (aspirator) a crystalline residue was dried in vacuo (0.1 Torr) for 1 h affording analytically pure product. Avoids the inconvenience of purging N2 and ice baths. 32

Otras reacciones

Solid-supported TsOH Microwave-assisted Pyrazole Synthesis Entry Acid Method Temp time Yield 1 p-TsOH Non-mW 100 oC 7 h 95 % 2 mW 160 oC 5 min 61 % 3 6 h 84 % 4 With an efficient method to prepare enol ketone 3 in hand, optimization of the next step was undertaken. Reaction of enol ketone 3 with 4-methylphenylhydrazine 4 was carried out under a variety of conditions 60 (Table 2).5,7 Firstly, the use of para-toluenesulfonic acid in ethanol was attempted under non-microwave conditions (100 C), with an excellent yield being obtained in 7 h (entry 1). When these conditions were attempted in the microwave (160 C) a 61% yield of pyrazole 5 was obtained (entry 2). On changing the acid source to silica-supported toluenesulfonic acid,10 excellent yields were obtained in both the thermal and microwave cases (entries 3 and 4). When using the silica-supported reagent no work-up was performed. The crude reaction mixture was evaporated to dryness, and the resulting free flowing solid purified directly by flash column chromatography. The optimum conditions for this reaction (entry 4) yielded pyrazole 5 in 95% yield in 5 min.12 Humphries, P. et al, Tetrahedron Lett, 2006 in press 34

PS-Triphenylphosphine One-pot Wittig olefination Developed & optimized on Emrys™ Liberator Scaled up 30 times in Advancer; 92% yield (95% purity) Here microwaves gives a quick reaction. Solid phase gives a very easy extraction as the normal bi-product tri-phenylphosfin oxide will be bound the the SP and easily filtered away.. The multicomponent set-up a very easy to perform reaction compared to the normally 2-3 steps. And it is also done in large scale. Compare also Jason Tierney´s work on Irori X-cans. 35

Rapid Solvent-free Henry Reaction University of Science and Technology of China, Hefei In this article, the authors have reported on the nitroaldol Henry reaction between nitromethane and various carbonyl compounds. They have studied the reaction with different bases and Lewis acid catalysts, but without the use of any reaction solvent. It was found that good results could be obtained for aldehyde substrates with triethyl amine and without any activation by Lewis acids. In order to make the reaction work with ketones as well, a selection of bases were tried out, and DABCO was found to be the one giving the highest yields in the shortest times. In most cases, Lewis acid catalysis was also needed. Lithium bromide was shown to be most efficient, closely followed by magnesium sulphate. Utilising these optimised conditions, the authors could use a series of aliphatic ketones as substrates, giving products in 63-91% yield. Gan, C.; Chen, X.; Lai, G.; Wang, Z. Synlett 2006, 387-390 36

Niementowski condensation Université de la Rochelle, France The synthesis of tetraaza-pentaphene-5,8-diones described in this article is based on a Niementowski condensation reaction of anthranilic acid derivatives. The anthranilic acid was adsorbed onto graphite, to make use of the microwave-absorbing properties of the solid. The reaction type was also attempted in solution (NMP or DMF), but then it gave much lower yields than with this method. The resulting products were screened for antiproliferative effects on breast cancer cells. Pereira, M. de F.; Picot, L.; Guillon, J.; Leger, J.-M.; Jarry, C.; Thiery, V.; Besson, T. Tetrahedron Letters 2005, 46, 3445-3447 37

One-pot synthesis of 3H-quinazolin-4-ones This reaction does not work with conventional heating! ArQule, Ma, USA and Pfizer, CT, USA Liu et al. have demonstrated the value of microwave-assisted chemistry not only for increasing yields and shortening reaction times but also for expanding the accessible chemical space by generating otherwise unavailable reaction products. The one-pot two step synthesis of 2,3-disubstituted 3H-quinazolin-4-ones from anthranilic acids, carboxylic acids or acyl chlorides and amines, has now been adapted to the synthesis of diverse screening libraries of related quinazolin-4-ones and also the total synthesis of a number of natural products that contain this heterocyclic scaffold. Liu, J.-F.; Lee, J.; Dalton, A. M.; Bi, G.; Yu, L.; Baldino, C. M.; McElory, E.; Brown, M. Tetrahedron Letters, 2005, 46, 1241-1244 38

Microwave Assisted Hydrogenolysis 72 % 64 % Hallberg, A. et al J. Org. Chem. 2005, 70, 938-942

In Situ Carbonylations under air 30-78 % Larhed, M et al. Synlett. 2004, 13, 2335-2338 Larhed, M et al. J. Org. Chem. 2005, 70, 3094-3098 Aryl iodides (0.4 mmol) were allowed to react with hydrazides (3 equiv) in dry THF using palladium acetate (10 mol%) as the precatalyst, DBU (3 equiv) as the base and molybdenum hexacarbonyl as a solid source of carbon monoxide (1 eq). The reactions were performed in sealed vessels under air during 15 mins. Hydrazidocarbonylation were predominantly achieved at a reaction temperature of 110 oC generating the diacylhydrazine products in moderate to good isolated yields (30-78%). Aryl bromides can be used as substrates in amino carbonylation reactions with Herrman’s palladacycle as the precatalyst. The catalyst activity was further improved by addition of Fu’s robust phosphonium salt [(t-Bu)3Ph]BF4 (tetrafluoroborate salt of tri-t-butylphosphine), designed to liberate free t-Bu3P under basic reaction conditions. The lower yields of these reactions are probably a consequence of decomposition of temperature sensitive diacyl hydrazines. Aryl chlorides were found to be inert under the carbonylation conditions. Aromatic acyl sulfonamides have important applications in med chem as carboxylic acid bio-isosteres. Sulfonimides were synthesized from both aryl iodides and bromides by direct carbonylation using Mo(CO)6. The conditions identified in the synthesis of the hydrazidocarbonylations were fully transferrable and were used without the need for re-optimization. Aryl iodides reacted at a lower temperature (110 oC) than aryl bromides (140 oC). 47-88 %

Cyanation Using PS-PPh3 Workup: (1) Filtration (2) Wash resin with Ether (3) Aqueous wash of filtrate (4) Dry and evaporate organic layer A variety of aryl nitriles were prepared in excellent yields from the palladium acetate catalyzed coupling of aryl halides with Zn(CN)2 (Zinc Cyanide) using polymer-supported triphenyl phosphine as the ligand and dimethylformamide as solvent under microwave irradiation conditions. Without the need for any purification, eleven aryl nitriles were synthesized with 84-99% yield and >90% purity by a simple synthesis, filtration, extraction and evaporation sequence. Srivastava, R. R.; Collibee, S. E. Tetrahedron Letters 2004, 45, 8895-8897

Bases de datos de Sintesis Asistidas con horno de microondas Pathfinder 42

Soluciones para Sintesis a traves de Biotage

Estrategias/Formatos de radiacion de microondas MULTI MODE SINGLE MODE Microwave technology was developed during WWII at the radiation laboratory at MIT. In the 1940s virtually all the technology that could impact the microwave oven was developed and well understood. During this period, vacuum tubes called magnetrons were invented capable of generating many kilowatts of electromagnetic power at frequencies that had never been obtained before. These frequencies called microwaves, ranged from 1-30 GHz. Telecommunication and microwave radar equipment occupy many of the band frequencies in these region and in order to avoid interference the wavelength at which many of the domestic and industrial microwaves operate is regulated to 2.45 GHz which is about 12.2 cm. Enfoque sobre cavidad de reaccion - Radiacion dispersa en camara Maxima estabilidad de temperatura - Variabilidad espacial de temperatura sobre el tubo de reaccion dentro de la camara de reaccion

Lider en desarrollo de tecnologia de Sintesis asistida con horno de microondas MAOS (Microwave Assisted Organic Synthesis) Emrys - Primer sistema “single mode” desarrollado para laboratorios de investigacion y desarrollo por Personal Chemistry en 1999 45

Initiator: Single Mode compacto Sistema integrado de bancada Temperatura maxima de proceso 250OC Presion maxima de reaccion 20 Bar 46

Initiator+ Ultima generacion en MAOS

Initiator+ 300OC y 30 bar Pantalla de control de expandida a 10” Opciones de automuestreador de 8 y 60 posiciones 48

Viales de Reaccion Escalables para muestras desde mg hasta gramos Sellados con para permitir reacciones de manera segura aun con reactivos quimicos peligrosos 49

Sintesis en cuatro (4) pasos Secuencia de proceso Sintesis en cuatro (4) pasos Colocar barra de agitacion en vial de reaccion Agregar reactivos y solventes Sellar vial Procesar 50

Seguridad, seguridad, seguridad Triple tier Temperature and pressures are constantly monitored and reaction is controlled, sudden raise or drop in either or will trigger an abortion of the reaction. Must not go beyond 30 bar and/or 300deg. Reaction vessel is maufactured to manage an overpressure beyond 30 bar. The Instrument is designed to control an explosion if one should occur. Magnetron will turn OFF ... …if the lid is not properly closed …if vial temperature > 260ºC vial pressure > 22 bar …if temperature change is  30 ºC/s pressure change is 5 bar/s 0 - 30 bar 40 – 300 ºC Vials: ~ 40 bar > 800 bar 51

Initiator+ Software

Interfaz a usuario Peptide option available when SP Wave module is connected.

Editor de experimento I’m hoping to be able to present regulate on pressure in the front edit screen rather than under advanced edit. This will present the use more easy and accessible. It is a great tool to avoid overshoot in pressure and pre-mature end of run. Set both a target temperature and a target pressure and the system will regulate on which ever is reached first, maximizing the outcome. Anton Paar users have to guess the temperature to avoid aborted runs.

Protocolo de reaccion Highlight the ease of use software with all common parameters on first page. Regulate on pressure and temp will get you the maximum temperature possible but still stay under safety pressure. 55

Resultados – Reportes de Sintesis 56

Resumen Sintesis Organica Asistida con horno de Microondas es una herramienta versatil para acelerar el desarrollo de reaciones de sintesis quimica Desarrollo de reacciones de sintesis mas breves, permiten acelerar el ciclo de descubrimiento y la obtencion de nuevos compuestos mas rapidamente Permite el proceso de reaccciones en condiciones que son imposibles de desarrolar en sintesis convencional de reflujo Metodo de sintesis que puede ser facilmente integrado con resinas de enlace para limpieza de impurezas, y cromatografia Flash para simplificar reproceso y purificacion

Preguntas?

Biotage® Sistemas para Sintesis de Peptidos

Antecedentes La síntesis de péptidos es la producción de péptidos, compuestos orgánicos en los que numerosos aminoácidos se encuentran unidos mediante enlaces peptídicos, llamados también enlaces amida. Como partes integrales de las proteinas, la técnica de síntesis de péptidos ofrece una ventana para el analisis de proteinas. Los péptidos juegan un rol significativos en la biología como hormonas, factores de crecimiento, antibióticos, toxinas y neuropéptidos. Entre las aplicaciones de la síntesis de péptidos se encuentran la de desarrollar péptidos de importancia médica como lo son hormonas y vacunas, también pueden sintetizarse péptidos para producir anticuerpos contra porciones específicas de proteínas o para modificar algunos péptidos naturales con el fin de hacerlos más estables La tendencia en el desarrollo de nuevas terapias apunta a la utilizacion de drogas en base a peptidos, debido a su elevada actividad y especificidad, baja toxicidad, y alto potencial de diversidad quimica. 60

Sintesis de Petidos en Fase Solida La síntesis en fase sólida es la técnica más común para la síntesis de péptidos. Usualmente en este método, los péptidos son sintetizados a partir del extremo del grupo carbonilo (C-terminal) al del grupo amino (N-terminal) de la cadena de aminoácidos En la síntesis de péptidos, un aminoácido protegido en el grupo amino es unido al material de fase sólida (comúnmente, una resina de poliestireno, o polietilenglicol) formando un enlace covalente entre el grupo carbonilo y la resina, generalmente un enlace amido o éster. Luego, el grupo amino es desprotegido y se le hace reaccionar con el grupo carbonilo del siguiente aminoácido protegido en el grupo amino. La fase sólida entonces conduce a un dipéptido. Este ciclo se repite para formar la cadena peptídica deseada. Una vez que la adición de todos los aminoácidos ha sido completada, se separa el péptido sintetizado de la matriz polimérica. 61

Los 20 Aminoacidos basicos 1-Letter Code  3-Letter Code Side chain R Alanine A Ala -CH3 Cysteine C Cys -CH2SH Aspartic acid D Asp -CH2COOH Glutamic acid E Glu -CH2CH2COOH Phenylalanine F Phe -CH2C6H5 Glycine G Gly -H Histidine H His -CH2 (4-imidazoyl) Isoleucine I Ile -CH(CH3)CH2CH3 Lysine K Lys -(CH2)4NH2 Leucine L Leu -CH2CH(CH3)2 Methionine M Met -(CH2)2SCH3 Asparagine N Asn -CH2CONH2 Proline P Pro -CH2CH2CH2- Glutamine Q Gln -(CH2)2CONH2 Arginine R Arg -(CH2)3NH-C(=NH)NH2 Serine S Ser -CH2OH Threonine T Thr -CH(CH3)OH Tryptophan W Trp -CH2 (3-indolyl) Tyrosine Y Tyr -CH2-(C6H4OH) Valine V Val -CH(CH3)2 Proline

Formacion de un enlace peptico

Secuencia en Sintesis de Peptidos en Fase Solida (SPPS)

Desde la perpectiva de procedimiento instrumental Secuencia pre-establecida de pasos que se debe efectuar cuidando una alta precision en los parametros operacionales: tiempo, temperatura, y dispensacion de reactivos Proteccion Deproteccion Acoplamiento Lavado R + P + K + P + Q + Q + F + F + G + L + M + A 65

Sintesis de Peptidos asistida con horno de microondas En la síntesis de péptidos, la radiación de microondas se ha usado para la completar secuencias peptídicas de gran longitud con rendimientos muy elevados y pocos efectos de conversion negativa (racemización) El acoplamiento de los aminoácidos a la cadena peptídica no se cataliza sólo mediante el aumento de la temperatura sino también debido a la radiación electromagnética alterna con la cual se alinea continuamente el esqueleto polar del polipéptido. A causa de este fenómeno, la energía microondas puede evitar la agregación y por tanto incrementar el rendimiento final del péptido. En algunos procesos de sintesis de peptidos, el calentamiento por encima de los 50-55 grados celsius evita también la agregación y acelera el acoplamiento. Las unicas limitaciones detectadas en el uso de microondas, es la racemización que puede producirse durante el acoplamiento de la cisteína y la histidina. La reacción de acoplamiento de estos aminoácidos se realiza a temperaturas más bajas que los otros 18 aminoácidos naturales Uno de los efectos no deseados más importantes es la deshidratación (pérdida de agua) que puede ser muy elevada en ciertos péptidos, como el polipéptido pancreático (PP), efecto secundario que puede también observarse con el simple calentamiento tradicional. 66

Soluciones en Sistemas de Sintesis de Peptidos a traves de Biotage MW RT Biotage is uniquely positioned as the only company which supplies both microwave and traditional room temperature peptide synthesizers The Biotage Syro Wave™ system is a programmable peptide synthesizer that is capable of both conventional room temperature parallel peptide synthesis and microwave assisted peptide synthesis. The system is a fully automated and computer controlled peptide synthesizer, based on a pipetting robot with a single arm. The Biotage SYRO I and SYRO II systems are fully automated and computer controlled peptide synthesizers, based on pipetting robots with one or two arms respectively.

Clasificacion operativa Manual Requiere intervencion del usuario en la ejecucion de todos los pasos requeridos para procesar la sintesis de peptidos Estacion de Trabajo para Petidos con Initiator+ Semi-automatico Solo algunos de los pasos del proceso son efectuados por el equipo (deproteccion Fmoc, y lavado), requiriendo menos intervencion del en el proceso general de sintesis SAP, SAM Automatico Todos la secuencia de sintesis es realizada por el equipo, requiriendo minima intervencion del usuario, una vez iniciada la corrida de la secuencia de sintesis Syro I, Syro 2, Syro Wave, Alstra, SP Wave Biotage is uniquely positioned as the only company which supplies both microwave and traditional peptide synthesizers

Temperatura Ambiente: Biotage SAP & SAM (semi-automaticos) SAP esta diseñado para sintesis de un (1) peptido a la vez SAM diseñado para sintesis simultanea de varios peptidos

Temperatura Ambiente: Biotage Syro I Sistema automatizado para Sintesis de Peptidos Paralela Sistema de un (1) brazo robotico, equipado con 2 bombas de jeringa digitales Para laboratorios con alto volumen de produccion de peptidos (>50 por mes) Poderoso software de programacion y control de la secuencia de sintesis The Biotage SYRO I and SYRO II systems are fully automated and computer controlled peptide synthesizers, based on pipetting robots with one or two arms respectively. The system’s modular construction offers greatest flexibility with respect to the number and size of storage containers for amino acids and reagents as well as reaction vessels. Different reactor blocks with 24, 48 or 96 positions allow for parallel synthesis of peptides in a range from 1 to 300 μmoles. Compact footprint

Temperatura Ambiente: Biotage Syro II Sistema automatizado para Sintesis de Peptidos Paralela Escala de sintesis variable desde 1 a 300umol Sistema de un (2) brazo robotico, equipado con 4 bombas de jeringa digitales Para laboratorios con alto volumen de produccion de peptidos (>50 por mes) Poderoso software de programacion y control de la secuencia de sintesis True automated parallel operation allows for the synthesis of more peptides in shorter times. Flexible reactor formats increase synthesis throughput and result in the highest productivity.

Asistido con Microondas: Biotage Syro Wave™ Sistema de sintesis de Peptido Paralela de alto volumen de produccion asistido con horno de microondas. Collaboration with Knud Jensen and his team to develop new applications for use on the Syro Wave

Biotage Syro Wave™ Sintesis paralela de alto volumen automatizada: alta productividad y eficiencia en costo Disponibilidad de microondas para mejorar eacciones dificiles y reducir tiempo de sintesis de peptidos complejos The Biotage Syro Wave™ system is a programmable peptide synthesizer that is capable of both conventional room temperature parallel peptide synthesis and microwave assisted peptide synthesis. The system is a fully automated and computer controlled peptide synthesizer, based on a pipetting robot with a single arm. Inert Gas and Partial Inert Gas available

Asistido con horno de Microondas: Initiator+ Alstra Biotage Initiaror+ ALSTRA

Initiator+ ALSTRA Especificaciones Sintesis asistida con microondas Totalmente automatizado Tubos de reaccion desechables Para sintesis en escala de 5 µmol - 2 mmol Unidad de control y software de operacion integrado Metodos de sintesis pre-definidos ya disponibles en base de datos de aplicaciones, asi como protocolos abiertos para desarrollo por usuario Agitacion por oscilacion, para mayor eficiencia de acoplamiento Bombas de jeringa digitales Compacto

Asistido con microondas: Initiator+ SP Wave Solucion versatil para sintesis organica y de peptidos Poderosa herramienta de desarrollo para sisntesis de peptidos y petodomimeticos, incluyendo modificaciones complejas, en procedimientos sencillos o de multiples pasos Ideal para sintesis de baja scala (1-10 peptidos por mes, cadenas de menos de 20 aminoacidos por peptido) Operacion semi-automatica reduce el consumo y desecho de reactivos (muchos de alto costo)

Resumiendo…. La sintesis de peptidos en fase solida (SPPS) es una tecnica poderosa para acelerar la sintesis de peptidos y peptomimeticos La utilizacion de radiaccion de microondas contribuye a una reducir el tiempo de sintesis, e incrementa la pureza de las reacciones Calentamiento con microondas mejora la eficiencia de acoplamiento y previene reacciones indirectas. Reduce el costo de sintesis al facilitar el uso de reactivos de acoplamiento estandar Mayor potencial para Quimica Verde a traves del uso de solventes menos agresivos

Productos complementarios para Sintesis de Peptidos tambien disponibles a traves de Biotage Resinas de acoplamiento ChemMatriz 100% polietilenglicol Compatibles con radiacion de microondas Columnas de purificacion de peptidos sinteticos RESOLUX Columnas analiticas, semi-prearativas y preparativas Capacidad para separar peptidos que difieran en tan solo un aminoacido

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