Os autores graças a divisão de tecnologia de película fina, centro de pesquisa de tecnologia de instrumento (ITRC) e nacional aplicada Research Laboratories, Hsinchu Science Park, Taiwan. Este trabalho foi financiado pelo Conselho Nacional de ciência (conceder n. A maioria dos 105-0210-01-13-01) e Academia Sinica.

<ol>
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Os autores não têm nada para divulgar.

Uma classe de bNAbs de HIV-1, incluindo PG9 e PG16 PGTs 128, 141-145 foram relatados para ser altamente potente para neutralizar a 70-80% de isolados de HIV-1 em circulação. Os epítopos destes bNAbs são altamente conservados entre as variantes de todo o grupo HIV-1 M, portanto eles podem guiar o projeto de immunogen eficaz para uma vacina contra o HIV eliciar neutralizando anticorpos23,24,25 . Como parte dos nossos esforços em curso para identificar os epítopos glicano do HIV-1 amplamente anticorpos neutralizantes, informamos a síntese química e enzimática de um painel de alta manose, híbrido e oligossacarídeos tipo complexo para formar um glicano matriz21, 22. No entanto, as matrizes de glicano comumente usados em lâminas de vidro N- hidroxisuccinimida-revestido (NHS) são incapazes de detectar interações de carboidrato-proteína de baixa afinidade, provavelmente devido à distribuição heterogênea glicano resultante da hidrólise de reactivo N- hydroxysuccimide grupos funcionais em sua superfície. Para superar as limitações dos formatos de matriz convencional, temos desenvolvido uma matriz de glicano um slide de (ACG) de vidro revestido de óxido de alumínio. Realizamos ainda uma comparação de homogeneidade entre a matriz ACG e o NHS comumente usado matriz para provar que a matriz ACG ofereceu uma apresentação glicano mais homogênea na sua superfície22. Nossa análise preliminar de ligação não foi capaz de observar a vinculação de PG9 a qualquer um dos glicanos na matriz de NHS (dados não mostrados). Portanto, para definir a especificidade da ligação do PG9, o glicanos eu-XI estava anexado a uma cauda de ácido fosfônico e impresso no slide ACG com 100 µM dos glicanos individuais (Figura 4). A imobilização de glicano em um slide ACG através da química de fosfonato ofereceu uma distribuição mais estável e homogênea. Cada um dos glicanos foi impresso com cinco repetições e slide imagens foram obtidas de uma varredura de fluorescência após incubação de anticorpo do burro DyLight649-conjugado anti-humano IgG. A análise de ligação de PG9 para os glicanos individuais imprimidos na matriz ACG (Figura 5, painel esquerdo) sugere que PG9 fortemente interagiu com híbrido-tipo glicano (X). As interações foram também detectadas para tipo alta manose Man5 (glicano IV) e o tipo complexo glicano (XI).
As interações de nível moleculares entre PG9 e híbrido-tipo glicano (X) são difíceis de determinar devido à falta de suas informações de estrutura de cristal Co. O glicano híbrido do tipo X é composto de um braço de tipo complexo nos 3-O posição do núcleo e um braço de trimannose ligado ao 6-Ó posição do trissacarídeo central. Ligação do PG9 a híbrido glicano X sugere que PG9 requer tanto manose e ácido siálico resíduos nas proximidades para interações de alta afinidade. Para identificar a combinação de glicano que melhor se encaixa no bolso de vinculação PG9, realizamos um estudo de matriz misturado-glicano. Glicano IV foi misturado com cada glicano de eu-XI na proporção de 1:1 mole e manchado em uma matriz ACG. A análise de ligação de PG9 para cada uma das misturas indicou que uma combinação de homem5 e um SCT (IV + XI) resultou na maior afinidade para PG9 comparado ao IV ou XI sozinho. Além disso, misturas contendo homem5 e aqueles que contêm sialylated antenas, tais como os glicanos VIII e X foram também detectaram22. Pela primeira vez, estes resultados ofereceram uma prova com base em array para o comportamento de vinculação de hetero-os dos glicanos de PG9 que foi sugerido por estudos de estrutura de cristal de PG9 com o HIV-1 gp120 V1/V2 domínio23.
Em conclusão, temos demonstrado uma estratégia eficiente de quimio-enzimático modular para a preparação de altamente diverso N-ligadas a oligossacarídeos que ocorrem em glicoproteínas humanas. Além disso, o desenvolvimento de uma matriz ACG fornece que um meio eficaz determinar interações proteína-carboidrato extremamente fraco e, mais importante, as interações que ocorrem através de hetero-os glicanos.

Os N- glicanos em glicoproteínas estão covalentemente ligados à asparagina (Asn) resíduo de sequon de Asn-Xxx-Ser/Thr o consenso, que afetam vários processos biológicos, tais como reconhecimento de conformação, antigenicidade, solubilidade e lectina de proteína 1 , 2. a síntese química de N-oligossacarídeos vinculados representa um desafio sintético significativo devido à sua enorme heterogeneidade micro estrutural e arquitetura altamente ramificada. A seleção cuidadosa de proteger grupos de sintonizar a reatividade dos blocos de construção, alcançar a seletividade em centros anomérico e uso correto de promotor / activator(s) são elementos-chave na síntese de oligossacarídeos complexos. Para resolver este problema da complexidade, uma grande quantidade de trabalho para avançar N- glicano síntese foi relatada recentemente3,4. Apesar dessas abordagens robustas, encontrar um método eficaz para a preparação de uma vasta gama de N- os glicanos (~ 20.000) permanece um grande desafio.
A taxa de mutação rápida do HIV-1 para atingir a extensa diversidade genética e sua capacidade de escapar de neutralizar a resposta imunitária, está entre os maiores desafios para desenvolver uma vacina segura e profilática contra HIV-15,6 , 7. uma tática eficaz que o HIV usa para evitar a resposta imune do hospedeiro é a glicosilação pós-traducional de gp120 de glicoproteína do envelope com um diverso N-ligados os glicanos derivados do anfitrião glycosylation máquinas8, 9. Um relatório recente sobre a análise precisa de recombinação monomérico HIV-1 gp120 glicosilação de células de rim humano embrionário (HEK) 293T sugere a ocorrência de microheterogeneity estrutural com um padrão característico de célula específico10 , 11 , 12. portanto, a compreensão das especificidades de glicano do HIV-1 bNAbs requer gp120 bem caracterizadas relacionados N- glicano estruturas em quantidade suficiente para análise.
A descoberta da tecnologia microarray de glicano fornecido alta exploração baseada na taxa de transferência de especificidades de uma diversa gama de proteínas carboidratos, adesinas vírus/bactérias, toxinas, anticorpos e lectines13,14 . O arranjo os glicanos sistemática em um formato de matriz baseada no chip poderia determinar interações do proteína-glicano problemático baixa afinidade a apresentação multivalentes15,16,17,18. Este arranjo de chip baseada glicano convenientemente aparece efetivamente imitar interfaces de celular. Para enriquecer a tecnologia e a superar a questão desigual associada com formatos de matriz convencional, nosso grupo desenvolveu recentemente um glicano matriz sobre uma lâmina de vidro revestido de óxido de alumínio (ACG) usando os glicanos de terminou de ácido fosfônico para aumentar a intensidade do sinal, homogeneidade e sensibilidade19,20.
Para melhorar o entendimento atual sobre glicano epitopos de HIV-1 recém isoladas amplamente neutralizando anticorpos (bNAbs), temos desenvolvido uma estratégia modular altamente eficiente para a preparação de um vasto leque de N-ligados os glicanos21 ,22 para serem impressos em um ACG deslize (ver Figura 1). Especificidade de estudos de perfil de bNAbs de HIV-1 em uma matriz ACG ofereceram a detecção incomum de comportamento de vinculação de hetero-glicano de bNAb altamente potente PG9 que foi isolado do HIV infectados indivíduos23,24,25.

<table width="573">
	<tbody>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">64197</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetonitrile</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75058</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Acetic anhydride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108247</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Anhydrous magnesium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7487889</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Boron trifluoride ethyl etherate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">109637</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Bovine serum albumin</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">9048468</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bio-Gel P2 polyacrylamide</td>
			<td style="width:123px;">Bio-Rad</td>
			<td style="width:119px;">1504118</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Bis(cyclopentadienyl)hafnium(IV) dichloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12116664</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">&#946;-1, 4 Galactosyl transferases from bovine milk</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">48279</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="width:168px;height:84px;">BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td>
			<td style="width:123px;">Arrayit</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium molybdate</td>
			<td style="width:123px;">TCI</td>
			<td style="width:119px;">C1794</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Cerium ammonium nitrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">16774213</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Clean glass slide&#160;</td>
			<td style="width:123px;">Schott&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Cytidine-5&#8242;-monophospho-N-acetylneuraminic acid</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">3063716</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Deuterated chloroform</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">865496</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td>
			<td style="width:123px;">Jackson Immuno Research, USA</td>
			<td style="width:119px;">709605098</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dichloromethane</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">75092</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Diethylaminosulfur trifluoride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">38078090</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Dimethylformamide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">68122</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethyl acetate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">141786</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Ethylene glycol</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">107211</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">FAST frame slide incubation chambers</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;"></td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Guanosine 5&#39;-diphospho-b-L-fucose disodium salt&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">15839700</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lab tracer 2.0 software&#160;</td>
			<td style="width:123px;"></td>
			<td style="width:119px;"></td>
			<td style="width:164px;">Section 4 of the Protocol</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GenePix Pro 4300A reader (microarray image analysis)</td>
			<td style="width:123px;">moleculardevices</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">www.moleculardevices.com</td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">GraphPad Prism Software (Image processing )</td>
			<td style="width:123px;">GraphPad Software, Inc</td>
			<td style="width:119px;"></td>
			<td style="width:164px;">http://www.graphpad.com/guides/prism/6/user-guide/</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Lithium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">1310652</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Manganese chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7773015</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Methanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">67561</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">N-butanol</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">71363</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Oxalic acid</td>
			<td style="width:123px;">Acros Organic</td>
			<td style="width:119px;">144627</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Palladium hydroxide</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">12135227</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Phosphate Buffered Saline</td>
			<td style="width:123px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:119px;">10010023</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Pyridine</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">110861</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">P-Toluene sulfonic acid monohydrate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">773476</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Silver triflate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">2923286</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium bicarbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium chloride</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7647145</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="width:168px;height:42px;">Sodium hydrogen carbonate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">144558</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium methoxide&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">124414</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Sodium sulfate</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">7757826</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Toluene&#160;</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">108883</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tris buffer&#160;</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">N/A</td>
			<td style="width:164px;">Ultra-pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="width:168px;height:21px;">Tween-20</td>
			<td style="width:123px;">Amresco</td>
			<td style="width:119px;">9005645</td>
			<td style="width:164px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="width:168px;height:63px;">Uridine diphosphate galactose (UDP-galactose)</td>
			<td style="width:123px;">Sigma Aldrich</td>
			<td style="width:119px;">137868521</td>
			<td style="width:164px;"></td>
		</tr>
	</tbody>
</table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

Apresentamos uma maneira altamente eficiente para a preparação rápida de uma vasta gama de N-ligadas a oligossacarídeos (estima-se que para exceder 20.000 estruturas) que são comumente encontrados em glicoproteínas humanas. Para alcançar a desejada diversidade estrutural, a estratégia começou com a síntese enzimática de quimioterapia de três tipos de módulos de fluoreto de oligosaccharyl, seguidos por sua gradual glycosylations α-seletiva para o 3-Ó e 6-O posiciona do resíduo de manose do trissacarídeo núcleo comum, tendo uma ligação crucial β-mannoside. Ainda mais, nós unimos os N- glicanos à superfície de uma lâmina de vidro revestido de óxido de alumínio (ACG) para criar uma matriz covalente mista para a análise da interação de hetero-ligante com um anticorpo de HIV. Em particular, o comportamento de vinculação de um recém isolados VIH-1 amplamente neutralizante anticorpo (bNAb), PG9, a mistura de espaçadas homem5GlcNAc2 (homem5) e 2,6-di-sialylated bi-antennary complexo tipo N- glicano (SCT ) em uma matriz ACG, abre uma nova avenida para guiar o projeto immunogen eficaz para o desenvolvimento de vacina contra HIV. Além disso, nossa matriz ACG encarna uma poderosa ferramenta para estudar outros anticorpos de HIV para o comportamento de vinculação de hetero-ligante.

Uma estratégia modular de quimio-enzimático para a síntese de um vasto leque de N-os glicanos é apresentada na Figura 1. A estratégia é baseado no fato de que a diversidade pode ser criada no início por quimioterapia-enzimático síntese dos três módulos importantes, seguido pelo mannosylation α-específicos para o 3-O e/ou 6-Ó posição do resíduo manose do comum núcleo trissacarídeo de N- os glicanos. Considerando a diversidade estrutural de bi - tri- e estruturas de tipo complexo tetra-antennary N- glicano, acreditávamos que um conjunto de oligosaccharyl doadores e o aceitador de trissacarídeos núcleo com pré-instalado alquil handlecould ser usado como começar materiais para gerar a diversidade estrutural desejada (Figura 1). A aplicabilidade dos doadores de fluoreto glycosyl na construção de uma biblioteca de complexos glicano provou anteriormente-21.
Para demonstrar a eficácia da nossa estratégia, uma estrutura isomérica bi-antennary (10, Figura 3) foi selecionada para a síntese. O D1 braço antenas 4 e D2 braço antenas 5 de glicano 10 foram gerados em larga escala por métodos enzimáticos quimio. Em particular, o dissacarídeo aceitador 1 foi enzimaticamente galactosylated usando β-1, 4-galactosyltransferase e uridina 5'-diphosphogalactose (UDP-Gal) para formar o trissacarídeo 2. Em seguida, o intermediário 2 foi fucosilado na posição 3 GlcNAc -O na presença de α-1, 3-bandas de Helicobacter pylori (Hpα1, 3 FT) para pagar o desejado Tetrassacarídeo 3. Para ligadura química ao núcleo, blocos 2 e 3 foram peracetylated primeiro, e o redução final p- metoxi fenol grupo foi removido. Finalmente, o flúor foi instalado na presença de DAST para obter o desejado módulos 4 e 5, respectivamente (Figura 2).
Tendo os módulos desejados na mão, em seguida prosseguimos com a stereoselective 3 -O glycosylation de 4 para o núcleo trissacarídeo 6 sob catálise de prata trifluormetanosulfonato e dicloreto de hafnocene para fornecer o respectivo hexasaccharide 7 (Figura 3). A proteção de benzilideno que mascaramento de 4, 6-OH foi removido usando catalítico p-tolueno sulfônico (p- TSA). Aproveitando-se de sua reatividade, 6-OH primário de 8 foi reagiu com flúor módulo 5 sob condições experimentais semelhantes para alcançar a necessária decasaccharide 9. Finalmente, desproteção global foi realizada para obter glicano 10, que caracterizou-se ainda mais usando NMR e espectroscopia de massa (Veja o arquivo de dados suplementares).
Os glicanos contendo uma cauda de amina pentil no final reduzindo foram modificados com os linkers ácidos fosfônico e anexados à superfície ACG através da química de fosfonato (Figura 4). No passado, HIV-1 bNAb PG9 foi exibido por sua especificidade glicano usando matrizes os glicanos homo e hétero para demonstrar para a primeira vez que PG9 interagiu com heteroglycans adjacentes no laço V1/V2 do HIV-1 gp120 superfície (Figura 5).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55855/55855fig1.jpg"> Figura 1: Uma estratégia modular geral para a preparação de N- os glicanos. (A) o número de N- os glicanos gerados por esta estratégia que comumente ocorrem em glicoproteínas humanas é estimado para exceder 20.000. (B) três tipos de módulos preparados por esta abordagem quimioterapia enzimáticos que pode ser usada para glycosylations α-seletive. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig1large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55855/55855fig2.jpg"> Figura 2: síntese de Chemoenzymatic dos módulos. i, UDP-galactose, β 1, 4-GalT, 15 h, 86%; ii, PIB-fucose, α-1, 3-FucT, 15 h, 84%; III, Ac (1)2O, piridina, RT, 12 h; (1) lata, ACN: Tolueno: H2O (3) DAST, Cl2, CH2-30 oC. pode: nitrato de amônio cério; DAST: Trifluoreto Diethylaminosulfur. Nomenclatura do produto: 1, pglucopyranosyl-methoxyphenyl-O-2-acetamido-2-deoxy-β-D--(1→ 2)-α-D-mannopyranoside; 2, p-methoxyphenyl-O-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranoside; 3, p-O-β-D-galactopyranosyl-(1→4)-[α-I-fucopyranosyl-(1→3)-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-α-D-mannopyranoside - metoxifenil; 4, [fluoreto de diacetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-3,4,6-O-triacetyl-α-D-mannopyranosyl 2,3,4,6-O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[3,6-O-; 5, [Fluoreto de fucopyranosyl(1→3)-3,6-O-diacetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-3,4,6-O-triacetyl-α-D-mannopyranosyl 2,3,4,6-O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[2,3,4-O-triacetyl-α-L- <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig2large.jpg" target="_blank">por favor clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55855/55855fig3.jpg"> Figura 3: química glicosilação de módulos de braço D1/D2 para aceitador do núcleo. eu, 4, AgOTf, Cp2HfCl2, tolueno, 4 Å MS, 0 óC a RT, 70%; ii, p- TSA, acetonitrilo, RT, 57%; III, 5, AgOTf, Cp2HfCl2, tolueno, 4 Å MS, 0 óC a RT, 34%; iv, (1) LiOH, 1,4-dioxano: H2O; 90 oC 12 h; (2) Ac2O, piridina, 12 h; (3) Almeida, MeOH, 12 h; (4) Pd(OH)2, MeOH: H2O: HCOOH (5:3:2), H2, 36%. AgOTf: Trifluromethanesulfonate de prata; CP2HfCl2: dicloreto de háfnio Bis (ciclopentadienila), MS: peneiras moleculares, nomenclatura dos produtos: 7, 5-Azidopentyl-O-{[2,3,4,6-O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[3,6-O-diacetyl-2- acetamido-2-Deoxy-β-D-glucopyranosyl]-(1→2)-[3,4,6-O-triacetyl-α-D-mannopyranosyl]}-(1→3)-[2-O-Acetyl-4,6-O-benzylidine-β-D-mannopyranosyl-(1→4)-O-(3,6-di-O-Benzyl-2-Deoxy-2-(2,2,2-trichloroethoxy) carbonylamino-β-D-glucopyranosyl)-(1→4)-O-3,6-di-O-benzyl-2-deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosid. 8, 5-Azidopentyl - O-(2-O-acetil-3,4,6-tri-O-benzyl-α-D-mannopyranosyl-(1→3)-2-O-acetyl-4,6-O-benzylidine-β-D-mannopyranosyl-(1→4)-O-(3,6-di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranosyl)-(1→4)-O-3,6- di-O-benzyl-2-deoxy-2-phthalimido-β-D-glucopyranoside. 9, 5-Azidopentyl-O-{[2,3,4,6-O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[3,6-O-diacetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-[3,4,6-O-triacetyl-α-D-mannopyranosyl]}-(1→3)-{2,3,4,6- O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[2,3,4-O-triacetyl-α-LS160fucopyranosyl-(1→3)-3,6-O-diacetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-3,4,6-O-triacetyl-α-D-mannopyranosyl}-(1→6)-[2-O-acetyl-β-D-mannopyranosyl-( 1→4)-O-(3,6-di-O-Benzyl-2-Deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-D-glucopyranosyl)-(1→4)-O-3,6-di-O-Benzyl-2-Deoxy-2-(2,2,2-trichloroethoxy)carbonylamino-β-Dglucopyranoside 10, 5- Aminopentyl-β-D-galactopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→2)-α-D-mannopyranosyl]-(1→3),-[β-D-galactopyranosyl-(1→4)(α-L-fucopyranosyl-(1→3)-2-2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1→2)- Α-D-mannopyranosyl]-(1→6)-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranosyl-(1→4)-2-acetamido-2-deoxy-β-D-glucopyranoside <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig3large.jpg" target="_blank">clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55855/55855fig4.jpg"> Figura 4: glicano imobilização em uma matriz ACG. (A) modificação química de glicano com amino cauda em um ácido fosfônico cauda para fixação covalente do slide ACG através da química de fosfonato. (B) distribuição dos glicanos sobre uma superfície ACG. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig4large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55855/55855fig5.jpg"> Figura 5: análise de matriz Glyan. (A) estruturas de sintético N- os glicanos que são impressas em uma matriz ACG. (B) vinculação análise de PG9 os glicanos individuais eu-XI imprimido na matriz ACG (painel esquerdo) e misturas de glicano de homem5 misturado com os glicanos eu-XI (painel direito) com concentração de 100 µM. As concentrações molares em µM para PG9 são dadas na legenda. A intensidades de sinal média e desvio-padrão calculado para cinco repetições independentes na matriz são mostrados. As inserções mostram imagens de fluorescência. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig5large.jpg" target="_blank">Clique aqui para ver uma versão maior desta figura.</a>

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Uma abordagem modular para a síntese de N- os glicanos para fixação a um vidro revestido de óxido de alumínio slide (slide ACG) como desenvolveu um microarray glicano e demonstrou-se seu uso para a criação de perfil de um HIV amplamente neutralizando anticorpos.

Quimio-enzimático síntese de N- os glicanos para desenvolvimento de matriz e anticorpos de HIV de criação de perfil

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

chemo-enzymatic-synthesis-n-glycans-for-array-development-hiv

Nanyang Technological University

Research

JoVE Journal

Chemistry

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

8.6K visualizações.  Academia Sinica. Uma abordagem modular para a síntese de N- os glicanos para fixação a um vidro revestido de óxido de alumínio slide (slide ACG) como desenvolveu um microarray glicano e demonstrou-se seu uso para a criação de perfil de um HIV amplamente neutralizando anticorpos.

Vídeo: Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

Semeado Síntese de CdSe / CdS Rod e tetrápodes Nanocristais

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

Química

Preparation of Hydrophobic Metal-Organic Frameworks via Plasma Enhanced Chemical Vapor Deposition of Perfluoroalkanes for the Removal of Ammonia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high surface area microporous materials, such as metal-organic frameworks (MOFs), unique challenges present themselves for PECVD treatments. Herein the protocol for development of a MOF that was previously unstable to humid conditions is presented. The protocol describes the synthesis of Cu-BTC (also known as HKUST-1), the treatment of Cu-BTC with PECVD of perfluoroalkanes, the aging of materials under humid conditions, and the subsequent ammonia microbreakthrough experiments on milligram quantities of microporous materials. Cu-BTC has an extremely high surface area (~1,800 m2/g) when compared to most materials or surfaces that have been previously treated by PECVD methods. Parameters such as chamber pressure and treatment time are extremely important to ensure the perfluoroalkane plasma penetrates to and reacts with the inner MOF surfaces. Furthermore, the protocol for ammonia microbreakthrough experiments set forth here can be utilized for a variety of test gases and microporous materials.

Herein the procedures for plasma enhanced chemical vapor deposition of perfluoroalkanes on microporous materials such as metal-organic frameworks to enhance their stability and hydrophobicity are described. Furthermore, breakthrough testing of milligram quantities of samples is described in detail.

Preparação de hidrofóbicos Metal-Organic Frameworks via Plasma aprimorado Chemical Vapor Deposition de perfluoroalcanos para a remoção de amônia

Plasma enhanced chemical vapor deposition (PECVD) of perfluoroalkanes has long been studied for tuning the wetting properties of surfaces. For high ...

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Síntese e Caracterização de Supramoleculares Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

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 ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Hidroquinona baseada Síntese de ouro Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

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 ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

Sinal de nanossondas rápida realce pela síntese de nanopartículas de ouro em Situ

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Síntese de nanofios de Au substrato ligados através de um mecanismo de crescimento ativo de superfície

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

A Rapid Synthesis Method for Au, Pd, and Pt Aerogels Via Direct Solution-Based Reduction

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various precursor noble metal ions with reducing agents in a 1:1 (v/v) ratio results in the formation of metal gels within seconds to minutes compared to much longer synthesis times for other techniques such as sol-gel. Conducting the reduction step in a microcentrifuge tube or small volume conical tube facilitates a proposed nucleation, growth, densification, fusion, equilibration model for gel formation, with final gel geometry smaller than the initial reaction volume. This method takes advantage of the vigorous hydrogen gas evolution as a by-product of the reduction step, and as a consequence of reagent concentrations. The solvent accessible specific surface area is determined with both electrochemical impedance spectroscopy and cyclic voltammetry. After rinsing and freeze drying, the resulting aerogel structure is examined with scanning electron microscopy, X-ray diffractometry, and nitrogen gas adsorption. The synthesis method and characterization techniques result in a close correspondence of aerogel ligament sizes. This synthesis method for noble metal aerogels demonstrates that high specific surface area monoliths may be achieved with a rapid and direct reduction approach.

A rapid, direct solution-based reduction synthesis method to obtain Au, Pd, and Pt aerogels is presented.

Um método de síntese rápida para Au, Pd e Pt Aerogels através de redução direta baseada em solução

Here, a method to synthesize gold, palladium, and platinum aerogels via a rapid, direct solution-based reduction is presented. The combination of various ...