Name: synthetic methodology for asymmetric ferrocene derived bio-conjugate systems via solid phase resin-based methodology
Uploaded: 2015-03-12T14:00:00.000+00:00
Duration: 7 min 7 s
Description: Watch this Scientific Journal Video about Synthetic Methodology for Asymmetric Ferrocene Derived Bio-conjugate Systems via Solid Phase Resin-based Methodology at JoVE.com

KG foi apoiada pela concessão P-1760 RA Fundação Welch, TCU Andrews Instituto de Matemática e Ciências da Educação (para KG), TCU pesquisa e criatividade Atividade Grant (a KG) e TCU SERC Grant (a JHS).

1. Síntese de biotina-Fc-cisteína (1) <ol><li> Métodos em fase sólida para produzir resina-1 ligado. <ol><li> Coloque biotina resina carregada (250 mg, 0,145 mmol) para uma seringa de vidro sinterizado e inchar a resina através da elaboração de dimetilformamida (5 mL) e agitando a seringa num agitador de laboratório durante 20 min. Expelir a solução e repetir dimetilformamida inchaço mais uma vez. </li><li> Remover o grupo protector Fmoc por adição de 4-6 ml de 20% de piperidina em dimetilformamida para a seringa, seguido por 10-15 min de agitação. Repetir o processo de desprotecção com mais de 4-6 ml de piperidina. Lava-se a resina com uma sequência de 3x dimetilformamida, 3x dimetilformamida: metanol (1: 1), metanol 3x: diclorometano (1: 1), diclorometano 3x, ~ 5 ml cada. Fazer um teste de ninidrina (+) sobre uma pequena amostra (~ 10) dos grânulos para confirmar a desprotecção bem sucedida pela presença de azul após aquecimento. </li><li> Misturar uma solução contendo ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico(203,3 mg, 0,4350 mmol), hidrato de 1-hidroxibenzotriazole (58,8 mg, 0,413 mmol), carbodiimida de diisopropilo (0,0673 ml, 0,435 mmol), diisopropil etil amina (0,0757 ml, 0,435 mmol), e uma mistura 4: 1 de diclorometano e dimetilformamida. Desenhe esta dentro da seringa de frita e agitar suavemente num agitador de laboratório durante 6 horas. Em seguida expelir a solução a partir da seringa e lavagem conforme descrito anteriormente. </li><li> Realizar o teste da ninidrina (-), tal como descrito acima para confirmar acoplamento. O teste de ninidrina ainda podem ser úteis na confirmação de acoplamento apesar de a cor laranja do cordão de derivados de a fixação da porção contendo ferro. </li><li> Em seguida, remover o grupo Fmoc através da adição de 20% de piperidina em dimetilformamida e lavado como descrito acima. O teste de ninidrina (+) deve ser utilizado para confirmar a remoção do Fmoc. </li><li> Prepara-se uma solução composta por Fmoc-Cys (Trt) -OH (254,8 mg, 0,4350 mmol), hidrato de 1-hidroxibenzotriazole (58,8 mg, 0,4125 mmol), carbodiimida de diisopropilo (0,0673 ml, 0,4350mmol), diisopropiletilamina (0,0757 mL, 0,4350 mmol), e uma mistura 4: 1 de diclorometano e dimetilformamida. Adicione este cocktail acoplamento cisteína a seringa frita e agitar suavemente durante 6 horas. Lavar utilizando o protocolo descrito anteriormente. </li><li> Confirmar acoplamento utilizando o teste de ninidrina (-), seguida por remoção do componente de Fmoc com 20% de piperidina e lavagem. Verifique se o terminal de acesso gratuito amina usando o teste de ninidrina (+). </li></ol></li><li> A clivagem de um a partir da resina. <ol><li> Adicione uma solução de TFA (9,45 ml), água (0,25 ml), 1,2-etanoditiol (0,25 ml), e silano de triisopropilo (0,1 ml), adicioná-lo à seringa e agitar suavemente durante 4 h. </li><li> Recolher a solução vermelha-castanha resultante num tubo de Eppendorf e evapora-se o TFA lentamente com uma corrente de ar. </li><li> Adicionar éter dietílico frio (~ 15 ml) para o tubo de Eppendorf para precipitar 1, que irá formar uma ligeira agitação. Isolar o produto por meio de centrifugação (1 g, 5 min). Then ciclos repetidos de lavagens de éter etílico (~ 60 ml total) e centrífuga para obter 1 como um sólido vermelho / marrom. </li></ol></li></ol> 2. Caracterização e Análise de 1 <ol><li> Confirmar que a identidade corresponder a conectividade e composição mostrada na Figura 2, usando 1 H (16 varrimentos) e 13 C RMN (512 scans) em metanol deuterado (300 ul) e análise de ESI-MS. Espere os seguintes resultados: 1 H RMN Espectro (CD 3 OD) 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) e espectro de 13 C RMN (CD 3 OD) δ / ppm: 24,644 (CH2), 25,472 (CH 2), 28,051 (CH2), 28,300 (CH2), 35,474 (CH2), 38,698 (CH2), 39,241 (CH2), 39,717 (CH2), 55,340 / 55,538 (Cp-anel), 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 (Cp-ring), 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] + e HR-MS (m / z): Encontrado: 617,2049 [1 + H] +, Teórico: 617.1622 [1 + H] +. </li><li> Realizar HPLC, análise elementar para confirmar a composição de um isolado. Realizar cromatogramas de HPLC utilizando uma coluna de fase reversa C8 com MeOH a 100% a uma taxa de fluxo de 0,5 ml / min. Nota: tempos de retenção de HPLC foram: 3,198-4,674 min. </li></ol> 3. Imobilização de 1 sobre uma superfície de ouro <ol><li> Cut polímero apoiado lâminas de ouro em quadrados de ~ 0,25 em 2. </li><li> Encher um copo de 50 ml com uma solução de água Dl de 1 (~ 1 mM). </li><li> Adicionar a lâmina de ouro para o copo e cubra com um vidro de relógio. Tudoow da lâmina de vidro a incubar O / N à temperatura ambiente sem agitação. </li><li> Remover o slide de ouro a partir da solução e permitir que ela seca ao ar. </li><li> Obter imagens de microscopia eletrônica de varredura, utilizando um microscópio eletrônico de varredura (ou equivalente) para observar imobilizada 1. </li></ol>

The authors have nothing to disclose.

A síntese de derivados de ferroceno assimétricas é um desafio em solução. Por exemplo, as tentativas para produzir uma solução em resultou em baixos rendimentos do produto desejado (menos de 20%). Do mesmo modo, reacções que utilizam ácido carboxílico 1&#39;-amino-ferroceno (sans Fmoc) e biotina ligada resina resultou em um produto insolúvel consistente com o produto polimerizado relatado por Baristic et al. e produto mínima. 47 Isto é ainda mais complicada pel...

Biossensores são pequenos dispositivos que utilizam a tecnologia de reconhecimento biomolecular como a plataforma para análise seletiva e são utilizados para a sua especificidade, velocidade e baixo custo. Os biossensores eletroquímicos para a detecção de biomoléculas estão na vanguarda deste campo, devido à sua simplicidade, custo-benefício, e alta sensibilidade. 1,3 A anatomia geral destes sensores é um eletrodo equipado com uma molécula de reconhecimento específico para o marcador biológico de interesse . A ligação do marcador por molécula de reconhecimento resulta de uma alteração local do potencial ou da corrente que pode ser detectada por uma simples medição. Até à data, a porção reconhecimento pode variar de enzimas, anticorpos, 4-8 9-12 células inteiras, 13-16, 17-20 receptores peptídeos 21-23 e 24 e de DNA em grande parte com foco em moléculas maiores, biológicos. 25-28 Research esforços nessa arena têm se concentrado principalmente na imunossensores where uma imunoglobulina é imobilizada com um núcleo activo redox (tais como ferroceno) e utilizados para detectar um anticorpo de interesse. Estes estudos foram excluídos aplicações clínicas devido à baixa precisão e tempo de consumo decorrentes das complicações decorrentes do uso de antígeno / anticorpos. 1,3 crescente atenção centrou-se na detecção de moléculas pequenas (menos de 1 kg / mol) de biomédica , alimentos e interesse ambiental, além de segurança nacional. 29 Os exemplos mais conhecidos de dispositivos biossensores são monitores de glicose de auto-teste, que têm tela impressa eletrodos enzimáticos acoplados a um metro amperometric bolso. Estes sistemas utilizam tipicamente um método coulométrico onde a quantidade total de carga gerada pela reacção de oxidação da glicose é medida ao longo de um período de tempo. Dispositivos mobiliários deve ser portátil, robusto e de mão de fazer uso fácil para a população em geral. Tag Redox como ferrocene são necessry para proporcionar a detecção eletroquímica de biomarcadores ou pequenas moléculas em solução como a maioria dos biomarcadores não são intrinsecamente eletroquimicamente ativa. 30-38 Ferroceno é uma molécula organometallic que é um padrão-ouro para eletroquímica, o que o torna uma excelente opção para a integração em biossensores eletroquímicos. Espécies ativas redox à base Ferroceno já acumulou considerável atenção devido ao seu pequeno tamanho, boa estabilidade, acesso sintético conveniente, modificação química fácil, lipofilicidade relativa, e facilidade de ajuste redox. 3,30-42 Pequenas moléculas baseado no núcleo ferrocene tem sido amplamente utilizado como detectores de íons metálicos e pequenas moléculas. 32-38,43 Sistemas dirigidas a espécies maiores, como biomoléculas têm utilizado a ligação de anticorpos grandes ou imunoglobulinas para derivados do ferroceno que foram incorporados em uma superfície eletroquímica. 1,3,39 , 44 Em cada caso, o potencial de corrente e intensity do par redox Fe III / Fe II foi alterado por acoplamento molecular, produzindo, assim, um novo identificador espectroscópica indicando a presença da molécula de analito. Esta mudança surge a partir da sobreposição extensiva que ocorre entre o sistema-pi dos anéis ciclopentadienilo e os de ferro orbitais d. Se o sistema pi é modificado, ou seja, derivado ou reagir, em seguida, a interacção orbital, por sua vez, a mudança. Isto irá afectar o núcleo Fe e pode ser observada como uma mudança no potencial do par Fe III / Fe II. 40,45,46 Estas propriedades tornam um tal sistema atractivo para uso como um agente de quantificação em um imunoensaio electroquímico ou biossensor. De modo a produzir sistemas de ferroceno contendo capacidades específicas para biossensores é óptima para modificar um anel de Cp com a bio-receptor específico para uma molécula alvo e utilizar o outro anel de Cp como uma amarra molecular para a leitura electroquímica ou electrode (Figura 1). Síntese destes derivados do ferroceno assimétricos é desafiado por reações colaterais e a formação de espécies diméricas e poliméricas formadas sobre intermolecular cross-linking. 47 No entanto, o acoplamento química produzindo uma ligação amida é o caminho mais direto para fornecer derivados simples de ferrocene envolvendo componentes biológicos tais como péptidos e seus metabolitos. Portanto, as técnicas de fase sólida primeiro desenvolvidos na década de 1950 por Merrifield de síntese de péptidos podem ser aplicados a compostos organometálicos contendo ferroceno. Através da utilização da molécula de ácido 1&#39;-Fmoc-amino-ferroceno-1-carboxílico ortogonalmente substituído, um sistema de ferroceno, que pode incluir uma porção do receptor (biotina), leitura electroquímica (ferroceno), e o componente de imobilização-ligante (cisteína) possui Foram construídas aqui detalhado. A síntese deste bio-conjugado é discutido bem como prova para a imobilização sobre uma superfície de ouro. Este trabalho represenst a primeira apresentação de um sistema composto de biotina, ferroceno e um aminoácido para a imobilização sobre uma superfície de ouro.

<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

A detecção precoce é uma chave para o sucesso do tratamento da maioria das doenças, e é especialmente indispensável para o diagnóstico e tratamento de vários tipos de cancro. As técnicas mais comuns utilizados são métodos de imagem, como a ressonância magnética (MRI), Positron Emission Topografia (PET), e Topografia Computadorizada (CT) e são ideais para a compreensão da estrutura física da doença, mas só pode ser realizada uma vez a cada quatro a seis semanas, devido à utilização de agentes de imagiologia e de custo global. Com isto em mente, o desenvolvimento de &quot;point of care&quot; técnicas, como biossensores, que avaliam o estágio da doença e / ou a eficácia do tratamento no consultório do médico e fazê-lo em tempo hábil, iria revolucionar os protocolos de tratamento. 1 Como um meio para explorar biossensores ferroceno base para a detecção de moléculas biologicamente relevantes 2, foram desenvolvidos métodos para produzir conjugados de bio-ferroceno-biotina aqui descritos. Este relatório vai se concentrar em um sistema-biotina-ferrocene cisteína que pode ser imobilizada em uma superfície de ouro.

A forma ligada resina de 1 é mostrada na Figura 2. A ligação covalente do componente ferroceno dá origem a uma coloração laranja para as pérolas de resina que é persistente com lavagem contínua e indicativo de um complexo de ferro contendo imobilizada em oposição a absorção de ferro pela PEG componente da esfera de resina. A forma livre da resina-1 é idêntica em cor para as pérolas de resina. A seguir à remoção do composto a partir da resina-esferas, ...

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.

Metodologia sintética para Asymmetric Ferroceno Derivados Sistemas Bio-conjugadas via Metodologia base de resina em Fase Sólida

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

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Chemistry

9.6K visualizações.  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.

Vídeo: 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.

Síntese de Bioconjugados de proteínas<em&gt; via</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 ...

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.

Síntese de uma matriz de complexo de metal-orgânicos solúveis em água

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.

Construindo Tioéter/sulfeto de vinil-amarrados induzida por peptídeos helicoidais Via foto Thiol-ene/ino Hydrothiolation

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.

Fase sólida síntese de [4,4] Spirocyclic oximas

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.

Síntese e Bioconjugation dos reagentes Thiol-reativa para a criação de Site-seletivamente modificado Immunoconjugates

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

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.

Construção de Peptídeos Cíclicos Penetrantes em Células para Maior Penetração de Barreiras 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.

Construindo peptídeos cíclicos usando um centro de sulfônio on-tether

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.

Síntese em Fase Sólida de um funcionalizada Bis Peptide-Usando o &quot;Safety Catch&quot; Metodologia

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

Biologia

Functionalized Spirocyclic Heterocycle Synthesis and Cytotoxicity Assay

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring systems is challenging. An efficient methodology to synthesize these compounds was recently published that described the solid phase synthesis in four steps rather than the previously reported five steps. The advantage of this shorter synthesis is the elimination of the use of toxic reagents. Low-loading Regenerating Michael (REM) linker-based resin was found to be crucial in the synthesis as high-loading versions prevented the addition of reagents containing bulky phenyl and aromatic side chains. The colorimetric 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) assay was used to examine the cytotoxicity of micromolar concentrations of these novel spirocyclic molecules in vitro. MTT is readily available commercially and produces relatively fast, reliable results, making this assay ideal for these spirocyclic heterocycles. Orthogonal ring structures as well as furfurylamine (a precursor in the synthesis method containing a similar 5-member ring motif) were tested.

Here, we describe a bioassay using 3-(4&#8242;,5&#8242;-dimethylthiazol-2&#8242;-yl)-2,5- diphenyltetrazolium bromide (MTT) to test previously synthesized spirocyclic oximes.

Síntese Espirocíclica Funcionalizada de Heterociclos e Ensaio de Citotoxicidade

Spirocyclic heterocycles have recently been reported in literature to be potential drugs for cancer therapy. The synthesis of these novel orthogonal ring ...

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

Síntese de 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 ...

Metodologia sintética para Asymmetric Ferroceno Derivados Sistemas Bio-conjugadas via Metodologia base de resina em Fase Sólida

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