KG fue apoyado por la subvención P-1760 Fundación RA Welch, TCU Instituto Andrews de Matemáticas y Educación la Ciencia (a KG), TCU Investigación y Creatividad Actividad Grant (a KG) y TCU SERC Grant (a JHS).

1. Síntesis de biotina-Fc-cisteína (1) <ol><li> Métodos en fase sólida para producir unidos a la resina 1. <ol><li> Coloque resina cargada biotina (250 mg, 0,145 mmol) en una jeringa de vidrio fritado y se hinchan la resina mediante la elaboración de dimetilformamida (5 ml) y agitando la jeringa en un agitador de laboratorio durante 20 min. Expulsar a la solución y repita dimetilformamida hinchazón una vez más. </li><li> Eliminar el grupo protector Fmoc mediante la adición de 4-6 ml de 20% de piperidina en dimetilformamida a la jeringa seguido de 10-15 min de agitación. Repita el proceso de desprotección con otro 4-6 ml de piperidina. Lavar la resina con una secuencia de 3x dimetilformamida, 3x dimetilformamida: metanol (1: 1), metanol 3x: diclorometano (1: 1), 3x diclorometano, ~ 5 ml cada uno. Hacer una prueba de la ninhidrina (+) en una pequeña muestra (~ 10) de las perlas para confirmar la desprotección con éxito por la presencia de azul después del calentamiento. </li><li> Mezcle una solución que contiene ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico(203,3 mg, 0,4350 mmol), 1-hidroxibenzotriazol hidrato (58,8 mg, 0,413 mmol), diisopropil carbodiimida (0,0673 ml, 0,435 mmol), diisopropiletilamina (0,0757 ml, 0,435 mmol), y una mezcla 4: 1 de diclorometano y dimetilformamida. Dibuje esta en la jeringa poroso y agite suavemente en un agitador de laboratorio durante 6 horas. Entonces expulsar la solución de la jeringa y lavar como se describió anteriormente. </li><li> Realizar la prueba de la ninhidrina (-) como se describe anteriormente para confirmar acoplamiento. La prueba de la ninhidrina todavía puede ser útil para confirmar acoplamiento a pesar del color anaranjado de la perla derivado de la unión del resto que contiene hierro. </li><li> A continuación, eliminar el grupo Fmoc mediante la adición de 20% de piperidina en dimetilformamida y se lavó como se describe anteriormente. El ensayo de ninhidrina (+) se debe utilizar para confirmar la eliminación de Fmoc. </li><li> Preparar una solución compuesta de Fmoc-Cys (Trt) -OH (254,8 mg, 0,4350 mmol), 1-hidroxibenzotriazol hidrato (58,8 mg, 0,4125 mmol), diisopropil carbodiimida (0,0673 ml, 0,4350mmol), diisopropiletilamina (0,0757 ml, 0,4350 mmol), y una mezcla 4: 1 de diclorometano y dimetilformamida. Añadir este cóctel acoplamiento cisteína la jeringa poroso y agite suavemente durante 6 horas. Lave usando el protocolo descrito anteriormente. </li><li> Confirmar el acoplamiento mediante la prueba de la ninhidrina (-), seguido de la eliminación del componente de Fmoc con piperidina al 20% y lavado. Verifique el terminal libre amina mediante la prueba de la ninhidrina (+). </li></ol></li><li> La escisión de 1 de la resina. <ol><li> Prepare una solución de TFA (9,45 ml), agua (0,25 ml), 1,2-etanoditiol (0,25 ml), y silano triisopropilo (0,1 ml), añadirlo a la jeringa y agite suavemente durante 4 horas. </li><li> Recoger la solución rojo-marrón resultante en un tubo Eppendorf y se evapora el TFA lentamente usando una corriente de aire. </li><li> Añadir éter dietílico frío (~ 15 ml) al tubo Eppendorf para precipitar 1, que formará agitación suave. Aislar el producto a través de centrifugación (1 g, 5 min). Trepetición de gallina ciclos de lavados de éter de dietilo (~ 60 ml total) y centrifugar para obtener 1 como un sólido rojo / marrón. </li></ol></li></ol> 2. Caracterización y Análisis de 1 <ol><li> Confirmar que la identidad coincide con la conectividad y la composición que se muestra en la Figura 2 usando 1 H (16 scans) y 13 C NMR (512 exploraciones) en metanol deuterado (300 l) y el análisis ESI-MS. Cuente con los siguientes resultados: Espectro de RMN 1H (CD3OD) δ / ppm: 1,407-1,684 (m, 6H), 2,245 (t, 2H), 2,665-3,150 (m, 12H), 4,015 (t, 1H), 4,104 (d, 2H ), 4,274 (q, 1H), 4,426 (d, 2H), 4,479 (q, 1H), 4,595 (t, 1H) y 13 C espectro de RMN (CD3OD) δ / ppm: 24,644 (CH 2), 25.472 (CH 2), 28,051 (CH 2), 28,300 (CH 2), 35,474 (CH 2), 38,698 (CH 2), 39,241 (CH 2), 39,717 (CH 2), 55,340 / 55,538 (anillo Cp), 60,286 (CH), 61.964 (CH), 62.521 / 62.821 (Cp-ring), 66.038 / 66.170 (Cp-ring), 69.153 / 69.328 (Cp-ring), 71.468 / 71.593 (anillo Cp), 76.466 (CH), 171.770 (C = O), 175,361 (C = O). ESI-MS (m / z): Encontrado: 639.00 [1 + Na] +, teórico: 639,1 [1 + Na] + y HR-MS (m / z): Encontrado: 617,2049 [1 + H] +, teórico: 617.1622 [1 + H] +. </li><li> Realizar HPLC, análisis elemental para confirmar la composición de aislado 1. Llevar a cabo cromatogramas de HPLC usando una columna de fase inversa C8 con 100% de MeOH a un caudal de 0,5 ml / min. Nota: los tiempos de retención de HPLC fueron: 3,198 a 4,674 min. </li></ol> 3. Inmovilización de 1 sobre una superficie de oro <ol><li> Polímero Cut respaldado diapositivas de oro en cuadrados de ~ 0,25 en 2. </li><li> Llenar un vaso de precipitados de 50 ml con una solución de agua DI de 1 (~ 1 mM). </li><li> Añadir la diapositiva de oro al vaso de precipitados y cubrir con un vidrio de reloj. Todosow el portaobjetos de vidrio para incubar O / N a temperatura ambiente sin agitación. </li><li> Retire la diapositiva oro de la solución y permitir que se seque al aire. </li><li> Obtener la digitalización de imágenes de microscopía electrónica utilizando un microscopio electrónico de barrido (o equivalente) para observar inmovilizado 1. </li></ol>

The authors have nothing to disclose.

La síntesis de derivados del ferroceno asimétricas es un reto en solución. Por ejemplo, los intentos para producir 1 en solución resultó en bajos rendimientos del producto deseado (menos de 20%). Asimismo, las reacciones que utilizan el ácido carboxílico 1&#39;-amino-ferroceno (sans Fmoc) y resina de biotina unido resultaron en producto insoluble consistente con el producto polimerizado reportado por Baristic et al. y el producto mínimo 47. Esto se complica aún más...

Los biosensores son dispositivos pequeños que emplean la tecnología de reconocimiento biomolecular como la plataforma para el análisis selectivo y se utilizan por su especificidad, velocidad y bajo costo. Biosensores electroquímicos para la detección de biomoléculas están a la vanguardia de este campo debido a su simplicidad, eficacia de costes, y alta sensibilidad. 1,3 La anatomía general de estos sensores es un electrodo equipado con una molécula de reconocimiento específica para el marcador biológico de interés . La unión del biomarcador por la molécula de reconocimiento se traduce en un cambio local de potencial o corriente que puede ser detectado por medición simple. Hasta la fecha, el fragmento de reconocimiento puede variar de enzimas, anticuerpos 4-8, 9-12 células enteras, 13-16, 17-20 receptores péptidos 21-23 y 24 de ADN y se han centrado en gran medida de mayor tamaño, las moléculas biológicas. 25-28 Investigación los esfuerzos en este campo se han concentrado principalmente en inmunosensores where una inmunoglobulina es inmovilizado con un núcleo activo redox (tal como ferroceno) y se utiliza para detectar un anticuerpo de interés. Estos estudios han sido excluidos de las aplicaciones clínicas debido a la escasa precisión y tiempo de consumo derivados de las complicaciones derivadas de la utilización de antígenos / anticuerpos. 1,3 creciente atención se ha centrado en la detección de moléculas pequeñas (menos de 1 kg / mol) de biomédica , la comida y el interés ambiental, además de la seguridad nacional. 29 Los ejemplos más conocidos de dispositivos biosensores son monitores de glucosa en la auto-prueba, que tienen serigrafiadas electrodos enzimáticos acoplados a un metro amperométrica bolsillo. Estos sistemas utilizan típicamente un método coulométrica donde se mide la cantidad total de carga generada por la reacción de oxidación de la glucosa durante un período de tiempo. Dispositivos negociables deben ser portátil, robusto y de mano para hacer un uso fácil para la población en general. Etiquetas redox tales como ferroceno son necessary para proporcionar la detección electroquímica de biomarcadores o pequeñas moléculas en solución como la mayoría de los biomarcadores no son intrínsecamente electroquímicamente activo. 30-38 ferroceno es una molécula organometálica que es un estándar de oro para la electroquímica, lo que hace que sea una excelente opción para la integración en biosensores electroquímicos. Especies activas redox a base de ferroceno ya han ganado considerable atención debido a su pequeño tamaño, buena estabilidad, el acceso sintético conveniente, modificación química fácil, lipofilicidad relativa, y la facilidad de sintonización redox. 3,30-42 moléculas pequeñas basado en el núcleo ferroceno tiene han utilizado ampliamente como detectores de iones metálicos y moléculas pequeñas. 32-38,43 sistemas de focalización especies más grandes, tales como biomoléculas han utilizado la fijación de grandes anticuerpos o inmunoglobulinas a derivados del ferroceno que han sido incrustados en una superficie electroquímica. 1,3,39 , 44 En cada caso, la intensi potencial y actualty de la pareja redox Fe III / Fe II se alteró tras el acoplamiento molecular, produciendo de este modo un nuevo mango espectroscópica que indica la presencia de la molécula de analito. Este cambio se debe a la amplia superposición que se produce entre el sistema pi de los anillos de ciclopentadienilo y las de hierro orbitales d. Si el sistema pi se modifica, es decir, derivado o se hace reaccionar, a continuación, la interacción orbital, a su vez, el cambio. Esto afectará el núcleo Fe y puede ser observada como un cambio en el potencial del par Fe III / Fe II. 40,45,46 Estas propiedades hacen que un sistema de este tipo atractivo para su uso como un agente de cuantificación en un inmunoensayo electroquímico o biosensor. Con el fin de producir sistemas de ferroceno que contiene específicas para capacidades de biosensores es óptimo para modificar un anillo de Cp con el específico para una molécula diana bio-receptor y utilizar el otro anillo Cp como una correa de sujeción molecular para la lectura electroquímica o elecelectrodo (Figura 1). La síntesis de estos derivados de ferroceno asimétricas es desafiada por reacciones secundarias y la formación de especies diméricas y poliméricas formadas sobre la reticulación intermolecular. 47 Sin embargo, la química de acoplamiento de la producción de un enlace amida es la ruta más directa para proporcionar derivados simples de ferroceno que implican componentes biológicos tales como péptidos y sus metabolitos. Por lo tanto, las técnicas de fase sólida desarrollados por primera vez en la década de 1950 por Merrifield de síntesis de péptidos se pueden aplicar a los compuestos organometálicos que contienen ferroceno. Mediante el uso de la molécula de ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico ortogonalmente sustituido, un sistema de ferroceno que puede contener un resto receptor (biotina), lectura electroquímica (ferroceno), y el componente de inmovilización-enlazador (cisteína) tiene ha construido y se detalla en este documento. La síntesis de este bio-conjugado se discute, así como pruebas para la inmovilización en una superficie de oro. Este represen trabajots la primera presentación de un sistema compuesto de biotina, ferroceno y un aminoácido para la inmovilización en una superficie de oro.

<table><tbody><tr><td>Biotin Novatag Resin</td><td>NovaBiochem</td><td>8550510001</td><td></td></tr><tr><td>TORVIQ 10 ml Luer Lock Fritted Syringe</td><td>Fisher</td><td>NC9299151</td><td></td></tr><tr><td>piperidine</td><td>Acros</td><td>P/3520/PB05</td><td></td></tr><tr><td>ninhydrin test</td><td>Sigma-Aldrich</td><td>60017-1ea</td><td></td></tr><tr><td>1&amp;rsquo;-Fmoc-amino-ferrocene-1-carboxylic acid</td><td>Omm Scientific</td><td>Special Order</td><td></td></tr><tr><td>&lt;em&gt;N,N&amp;prime;&lt;/em&gt;-Diisopropylcarbodiimide</td><td>Sigma-Aldrich</td><td>D125407-5G</td><td></td></tr><tr><td>Fmoc-Cys(Trt)-OH</td><td>Novabiochem</td><td>8520080025</td><td></td></tr><tr><td>trifluoroacetic acid</td><td>Sigma-Aldrich</td><td>T5408</td><td></td></tr><tr><td>1,2-ethanedithiol</td><td>Sigma-Aldrich</td><td>2930</td><td></td></tr><tr><td>triisopropyl silane</td><td>Sigma-Aldrich</td><td>233781</td><td></td></tr><tr><td>Eppendorf tubes (20 ml)</td><td>any source</td><td></td><td></td></tr><tr><td>methanol</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dichloromethane</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>dimethylformamide</td><td>any source</td><td></td><td>dry with molecular sieves prior to use &amp;amp; store in 100 ml media bottle for easy usage</td></tr><tr><td>centrifuge</td><td>any source</td><td></td><td></td></tr></tbody></table>

synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology

La detección temprana es la clave para el éxito del tratamiento de la mayoría de las enfermedades, y es particularmente imprescindible para el diagnóstico y el tratamiento de muchos tipos de cáncer. Las técnicas más comunes utilizados son Imaging modalidades tales como imágenes por resonancia magnética (MRI), tomografía por emisión de Topografía (PET), y computarizada Topografía (TC) y son óptimas para la comprensión de la estructura física de la enfermedad, pero sólo se pueden realizar una vez cada cuatro a seis semanas debido a la utilización de agentes de formación de imágenes y el coste total. Con esto en mente, el desarrollo de &quot;punto de atención&quot; técnicas, como biosensores, que evalúan el estado de la enfermedad y / o la eficacia del tratamiento en el consultorio del médico y lo hacen de una manera oportuna, revolucionaría protocolos de tratamiento. 1 Como un medio para explorar biosensores basados ​​ferroceno para la detección de moléculas biológicamente relevantes 2, se han desarrollado métodos para producir bio-conjugados ferroceno-biotina descritos en este documento. Este informe se centrará en un sistema biotina-ferroceno-cisteína que puede ser inmovilizada sobre una superficie de oro.

La forma unida a resina de 1 se muestra en la Figura 2. La unión covalente del componente de ferroceno da lugar a una coloración naranja a las perlas de resina que es persistente con lavado continuo e indicativo de una plancha inmovilizada que contiene el complejo en comparación con la absorción de hierro por el componente PEG de la perla de resina. La forma libre de resina de 1 es idéntico en color a las perlas de resina. Después de la eliminación del compuesto ...

Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Metodología sintética para asimétrica Ferroceno derivados Sistemas de Bio-conjugadas vía sólida metodología a base de resina de fase

Early detection is a key to successful treatment of most diseases, and is particularly imperative for the diagnosis and treatment of many types of cancer. ...

synthetic-methodology-for-asymmetric-ferrocene-derived-bio-conjugate

Research

JoVE Journal

Chemistry

9.6K vistas.  Texas Christian University. The synthesis of asymmetric species of ferrocene is challenging using solution techniques. This report focuses on the methods carried out to produce a ferrocene-biotin bioconjugate using facile and clean reactions accomplished via solid-phase synthesis. Incorporation of a thiolate moiety is shown to impart the ability for immobilization on gold surfaces.

Video: Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

Síntesis de Proteínas bioconjugados<em&gt; vía</em&gt; Química cisteína-maleimida

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Investigación

Química

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

Síntesis de un conjunto complejo de metal-orgánico soluble en agua

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Constructing Thioether/Vinyl Sulfide-tethered Helical Peptides Via Photo-induced Thiol-ene/yne Hydrothiolation

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene hydrothiolation. In addition, this protocol describes the preparation of vinyl-sulfide-tethered peptides using in-solution intramolecular thiol-yne hydrothiolation between amino acids that possess alkene/alkyne side chains and cysteine residues at i, i+4 positions. Linear peptides were synthesized using a standard Fmoc-based solid-phase peptide synthesis (SPPS). Thiol-ene hydrothiolation is carried out using either an intramolecular thio-ene reaction or an intermolecular thio-ene reaction, depending on the peptide length. In this research, an intramolecular thio-ene reaction is carried out in the case of shorter peptides using on-resin deprotection of the trityl groups of cysteine residues following the complete synthesis of the linear peptide. The resin is then set to UV irradiation using photoinitiator 4-methoxyacetophenone (MAP) and 2-hydroxy-1-[4-(2-hydroxyethoxy)-phenyl]-2-methyl-1-propanone (MMP). The intermolecular thiol-ene reaction is carried out by dissolving Fmoc-Cys-OH in an N,N-dimethylformamide (DMF) solvent. This is then reacted with the peptide using the alkene-bearing residue on resin. After that, the macrolactamization is carried out using benzotriazole-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop), 1-hydroxybenzotriazole (HoBt), and 4-Methylmorpholine (NMM) as activation reagents on the resin. Following the macrolactamization, the peptide synthesis is continued using standard SPPS. In the case of the thio-yne hydrothiolation, the linear peptide is cleaved from the resin, dried, and subsequently dissolved in degassed DMF. This is then irradiated using UV light with photoinitiator 2,2-dimethoxy-2-phenylacetophenone (DMPA). Following the reaction, DMF is evaporated and the crude residue is precipitated and purified using high-performance liquid chromatography (HPLC). These methods could function to simplify the generation of thioether-tethered cyclic peptides due to the use of the thio-ene/yne click chemistry that possesses superior functional group tolerance and good yield. The introduction of thioether bonds into peptides takes advantage of the nucleophilic nature of cysteine residues and is redox-inert relative to disulfide bonds.

We present a protocol for the construction of thioether/vinyl sulfide-tethered helical peptides using photo-induced thiol-ene/thiol-yne hydrothiolation.

Construcción de Thioether/sulfuro de vinilo-atado Hydrothiolation de péptidos helicoidales mediante foto-inducida tiol-eno/Ino

Here, we describe a detailed protocol for the preparation of thioether-tethered peptides using on-resin intramolecular/intermolecular thiol-ene ...

Solid-phase Synthesis of [4.4] Spirocyclic Oximes

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of solid-phase synthesis, regenerating Michael (REM) linker strategies, and 1,3-dipolar cycloaddition, a library of structurally similar heterocycles, both with and without a spirocyclic center, can be constructed. The main advantages of the solid-support synthesis are as follows: first, each reaction step can be driven to completion using a large excess of reagents resulting in high yields; next, the use of commercially available starting materials and reagents keep the costs low; finally, the reaction steps are easy to purify via simple filtration. The REM linker strategy is attractive because of its recyclability and traceless nature. Once a reaction scheme is completed, the linker can be reused multiple times. In a typical solid-phase synthesis, the product contains either a part of or the whole linker, which can prove undesirable. The REM linker is &#34;traceless&#34; and the point of attachment between the product and the polymer is indistinguishable. The high diastereoselectivity of the intramolecular 1,3-dipolar cycloaddition is well documented. Limited by the insolubility of the solid support, the reaction progression can only be monitored by a change in the functional groups (if any) via infrared (IR) spectroscopy. Thus, the structural identification of intermediates cannot be characterized by conventional nuclear magnetic resonance (NMR) spectroscopy. Other limitations to this method stem from the compatibilities of the polymer/linker to the desired chemical reaction scheme. Herein we report a protocol that allows for the convenient production of spirocyclic heterocycles that, with simple modifications, can be automated with high-throughput techniques.

Here we present a protocol to demonstrate an efficient method for the synthesis of spirocyclic heterocycles. The five-step process utilizes solid-phase synthesis and regenerating Michael linker strategies. Generally difficult to synthesize, we present a customizable method for the synthesis of spirocyclic molecules otherwise inaccessible to other modern approaches.

Síntesis en fase sólida de oximas espirocícliclos [4,4]

A convenient synthetic route for spirocyclic heterocycles is well sought after due to the molecule&#39;s potential use in biological systems. By means of ...

Synthesis and Bioconjugation of Thiol-Reactive Reagents for the Creation of Site-Selectively Modified Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. Yet maleimide-based conjugates display limited stability in vivo because the succinimidyl thioether linkage can undergo a retro-Michael reaction. This, of course, can lead to the release of the radioactive payload or its exchange with thiol-bearing biomolecules in circulation. Both of these processes can produce elevated activity concentrations in healthy organs as well as decreased activity concentrations in target tissues, resulting in reduced imaging contrast and lower therapeutic ratios. In 2018, we reported the creation of a modular, stable, and easily accessible phenyloxadiazolyl methyl sulfone reagent &#8212; dubbed &#8216;PODS&#8217; &#8212; as a platform for thiol-based bioconjugations. We have clearly demonstrated that PODS-based site-selective bioconjugations reproducibly and robustly create homogenous, well-defined, highly immunoreactive, and highly stable radioimmunoconjugates. Furthermore, preclinical experiments in murine models of colorectal cancer have shown that these site-selectively labeled radioimmunoconjugates exhibit far superior in vivo performance compared to radiolabeled antibodies synthesized via maleimide-based conjugations. In this protocol, we will describe the four-step synthesis of PODS, the creation of a bifunctional PODS-bearing variant of the ubiquitous chelator DOTA (PODS-DOTA), and the conjugation of PODS-DOTA to the HER2-targeting antibody trastuzumab.

In this protocol, we will describe the synthesis of PODS, a phenyoxadiazolyl methyl sulfone-based reagent for the site-selective attachment of cargos to the thiols of biomolecules, particularly antibodies. In addition, we will describe the synthesis and characterization of a PODS-bearing bifunctional chelator and its conjugation to a model antibody.

Síntesis y Bioconjugation de tioles reactivos reactivos para la creación de sitio selectivamente modificados Immunoconjugates

Maleimide-bearing bifunctional probes have been employed for decades for the site-selective modification of thiols in biomolecules, especially antibodies. ...

Preparation of Site-Specific Cytotoxic Protein Conjugates via Maleimide-thiol Chemistry and Sortase A-Mediated Ligation

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as growth factor receptors on their surface. This feature enables development of highly selective therapeutics, the protein bioconjugates, composed of targeting proteins (antibodies or receptor ligands) connected to highly cytotoxic drugs by a specific linker. Due to very high affinity and selectivity of targeting proteins the bioconjugates recognize marker proteins on the cancer cells surface and utilize receptor-mediated endocytosis to reach the cell interior. Intracellular vesicular transport system ultimately delivers the bioconjugates to the lysosomes, where proteolysis separates free cytotoxic drugs from the proteinaceous core of the bioconjugates, triggering drug-dependent cancer cell death. Currently, there are several protein bioconjugates approved for cancer treatment and large number is under development or clinical trials.
One of the main challenges in the generation of the bioconjugates is a site-specific attachment of the cytotoxic drug to the targeting protein. Recent years have brought a tremendous progress in the development of chemical and enzymatic strategies for protein modification with cytotoxic drugs. Here we present the detailed protocols for the site-specific incorporation of cytotoxic warheads into targeting proteins using a chemical method employing maleimide-thiol chemistry and an enzymatic approach that relies on sortase A-mediated ligation. We use engineered variant of fibroblast growth factor 2 and fragment crystallizable region of human immunoglobulin G as an exemplary targeting proteins and monomethyl auristatin E and methotrexate as model cytotoxic drugs. All the described strategies allow for highly efficient generation of biologically active cytotoxic conjugates of defined molecular architecture with potential for selective treatment of diverse cancers.

Here we provide detailed protocols for a site-specific labeling of proteins with cytotoxic drugs using maleimide-thiol reaction and sortase A-mediated ligation.

Cancer is currently the second most common cause of death worldwide. The hallmark of cancer cells is the presence of specific marker proteins such as ...

Bioquímica

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Construcción de péptidos cíclicos penetrantes de células para mejorar la penetración de barreras biológicas

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

Construcción de péptidos cíclicos usando un centro de sulfonio en la correa

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Solid Phase Synthesis of a Functionalized Bis-Peptide Using "Safety Catch" Methodology

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This technique quickly proved advantageous over its solution-phase predecessor in both time and labor. Improvements concerning the nature of solid support, the protecting groups employed and the coupling methods employed over the last five decades have only increased the usefulness of Merrifield's original system. Today, use of a Boc-based protection and base/nucleophile cleavable resin strategy or Fmoc-based protection and acidic cleavable resin strategy, pioneered by R.C. Sheppard, are most commonly used for the synthesis of peptides1.
Inspired by Merrifield's solid supported strategy, we have developed a Boc/tert-butyl solid-phase synthesis strategy for the assembly of functionalized bis-peptides2, which is described herein. The use of solid-phase synthesis compared to solution-phase methodology is not only advantageous in both time and labor as described by Merrifield1, but also allows greater ease in the synthesis of bis-peptide libraries. The synthesis that we demonstrate here incorporates a final cleavage stage that uses a two-step "safety catch" mechanism to release the functionalized bis-peptide from the resin by diketopiperazine formation.
Bis-peptides are rigid, spiro-ladder oligomers of bis-amino acids that are able to position functionality in a predictable and designable way, controlled by the type and stereochemistry of the monomeric units and the connectivity between each monomer. Each bis-amino acid is a stereochemically pure, cyclic scaffold that contains two amino acids (a carboxylic acid with an &alpha;-amine)3,4. Our laboratory is currently investigating the potential of functional bis-peptides across a wide variety of fields including catalysis, protein-protein interactions and nanomaterials.

The efficient solid-phase peptide synthesis of a functionalized bis-peptide trimer utilizing a "safety catch" cleavage procedure from HMBA resin is described.

Síntesis en fase sólida de un péptido funcionalizado Bis-El uso de &quot;Cierre de seguridad&quot; Metodología

In 1962, R.B. Merrifield published the first procedure using solid-phase peptide synthesis as a novel route to efficiently synthesize peptides. This ...

Biología

Solid Phase Synthesis

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize winning invention where a reactant molecule is bound on a solid support and undergoes successive chemical reactions to form a desired compound. When the molecules are bound to a solid support, excess reagents and byproducts can be removed by washing away the impurities, while the target compound remains bound to the resin. Specifically, we will showcase an example of solid-phase peptide synthesis (SPPS) to demonstrate this concept.

Solid Phase Synthesis: Principles, Peptide Synthesis

Síntesis en fase sólida

Source: Vy M. Dong and Diane Le, Department of Chemistry, University of California, Irvine, CA
Merrifield&#39;s solid-phase synthesis is a Nobel Prize ...

Metodología sintética para asimétrica Ferroceno derivados Sistemas de Bio-conjugadas vía sólida metodología a base de resina de fase

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