저자는 얇은 영화 기술, 계측 기술 연구 센터 (ITRC)와 국가 적용 연구 실험실, Hsinchu 과학 공원, 대만 감사합니다. 이 작품은 국가 과학 위원회에 의해 지원 되었다 (no를 부여 합니다. 대부분 105-0210-01-13-01) 및 학계 Sinica.

<ol>
	<li>Kim, P. J., Lee, D. Y., Jeong, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Centralized+modularity+of+N-linked+glycosylation+pathways+in+mammalian+cells.">Centralized modularity of N-linked glycosylation pathways in mammalian cells.</a> PloS one. 4, e7317 (2009).</li><li>Townsley, S., Li, Y., Kozyrev, Y., Cleveland, B., Hu, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+Role+of+an+N-Linked+Glycan+on+the+Surface+Antigen+of+Human+Immunodeficiency+Virus+Type+1+Modulating+Virus+Sensitivity+to+Broadly+Neutralizing+Antibodies+Against+the+Receptor+and+Coreceptor+Binding+Sites.">Conserved Role of an N-Linked Glycan on the Surface Antigen of Human Immunodeficiency Virus Type 1 Modulating Virus Sensitivity to Broadly Neutralizing Antibodies Against the Receptor and Coreceptor Binding Sites.</a> J.virol. 90, 829-841 (2015).</li><li>Wang, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+General+Strategy+for+the+Chemoenzymatic+Synthesis+of+Asymmetrically+Branched+N-Glycans.">A General Strategy for the Chemoenzymatic Synthesis of Asymmetrically Branched N-Glycans.</a> Science. 341, 379-383 (2013).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Chemoenzymatic+Synthesis+of+an+N-glycan+Isomer+Library.">Efficient Chemoenzymatic Synthesis of an N-glycan Isomer Library.</a> Chem Sci. 6, 5652-5661 (2015).</li><li>Pritchard, L. K., Harvey, D. J., Bonomelli, C., Crispin, M., Doores, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-+and+Protein-Directed+Glycosylation+of+Native+Cleaved+HIV-1+Envelope.">Cell- and Protein-Directed Glycosylation of Native Cleaved HIV-1 Envelope.</a> J.Virol. 89, 8932-8944 (2015).</li><li>Behrens, A. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Composition+and+Antigenic+Effects+of+Individual+Glycan+Sites+of+a+Trimeric+HIV-1+Envelope+Glycoprotein.">Composition and Antigenic Effects of Individual Glycan Sites of a Trimeric HIV-1 Envelope Glycoprotein.</a> Cell Rep. 14, 2695-2706 (2016).</li><li>Barouch, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenges+in+the+development+of+an+HIV-1+vaccine.">Challenges in the development of an HIV-1 vaccine.</a> Nature. 455, 613-619 (2008).</li><li>Horiya, S., MacPherson, I. S., Krauss, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+Strategies+Targeting+HIV+Glycans+in+Vaccine+Design.">Recent Strategies Targeting HIV Glycans in Vaccine Design.</a> Nat Chem Bio. 10, 990-999 (2014).</li><li>Wang, L. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+carbohydrate+Antigens+for+HIV+Vaccine+Design.">Synthetic carbohydrate Antigens for HIV Vaccine Design.</a> Curr Opin Chem Biol. 17, 997-1005 (2013).</li><li>Tian, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+Glycosylation+on+an+Immunodominant+Region+in+the+V1V2+Variable+Domain+of+the+HIV-1+Envelope+gp120+Protein.">Effect of Glycosylation on an Immunodominant Region in the V1V2 Variable Domain of the HIV-1 Envelope gp120 Protein.</a> PLoS Comput Biol. 12, e1005094 (2016).</li><li>Geyer, H., Holschbach, C., Hunsmann, G., Schneider, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrates+of+human+Immunodeficiency+Virus.+Structures+of+Oligosaccharides+Linked+to+the+Envelope+Glycoprotein+120.">Carbohydrates of human Immunodeficiency Virus. Structures of Oligosaccharides Linked to the Envelope Glycoprotein 120.</a> The J Bio Chem. 263, 11760-11767 (1988).</li><li>Lee, J. H., Ozorowski, G., Ward, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryo-EM+Structure+of+A+Native,+Fully+Glycosylated,+Cleaved+HIV-1+Envelope+Trimer.">Cryo-EM Structure of A Native, Fully Glycosylated, Cleaved HIV-1 Envelope Trimer.</a> Science. 351, 1043-1048 (2016).</li><li>Lonardi, E., Balog, C. I., Deelder, A. M., Wuhrer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Natural+GlycanMicroarrays.">Natural GlycanMicroarrays.</a> Expert Rev Proteomics. 7, 761-774 (2010).</li><li>Paulson, J. C., Blixt, O., Collins, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sweet+Spots+in+Functional+Glycomics.">Sweet Spots in Functional Glycomics.</a> Nat. Chem. Bio. 2, 238-248 (2006).</li><li>Dotsey, E. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+High+Throughput+Protein+Microarray+Approach+to+Classify+HIV+Monoclonal+Antibodies+and+Variant+Antigens.">A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens.</a> PLoS One. 10, e0125581 (2015).</li><li>Wu, C. Y., Liang, P. H., Wong, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Development+of+Glycan+Arrays.">New Development of Glycan Arrays.</a> Org Biomol Chem. 7, 2247-2254 (2009).</li><li>Scurr, D. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+Characterization+of+Carbohydrate+Microarrays.">Surface Characterization of Carbohydrate Microarrays.</a> Langmuir. 26, 17143-17155 (2010).</li><li>Blixt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printed+Covalent+Glycan+Array+for+Ligand+Profiling+of+Diverse+Glycan+Binding+Proteins.">Printed Covalent Glycan Array for Ligand Profiling of Diverse Glycan Binding Proteins.</a> Proc Natl Acad Sci U S A. 101, 17033-17038 (2004).</li><li>Chang, S. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycan+Array+on+Aluminum+Oxide-Coated+Glass+Slides+Through+Phosphonate+Chemistry.">Glycan Array on Aluminum Oxide-Coated Glass Slides Through Phosphonate Chemistry.</a> J. Am. Chem. Soc. 132, 13371-13380 (2010).</li><li>Tseng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1, 773-783 (2016).</li><li>Shivatare, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Convergent+Synthesis+of+Bi-,+Tri-,+and+Tetra-Antennary+Complex+Type+N-Glycans+and+Their+HIV-1+Antigenicity.">Efficient Convergent Synthesis of Bi-, Tri-, and Tetra-Antennary Complex Type N-Glycans and Their HIV-1 Antigenicity.</a> J. Am. Chem. Soc. 135, 15382-15391 (2013).</li><li>Shivatare, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+synthesis+of+N-Glycans+and+Arrays+for+the+Hetero-Ligand+Binding+Analysis+of+HIV+Antibodies.">Modular synthesis of N-Glycans and Arrays for the Hetero-Ligand Binding Analysis of HIV Antibodies.</a> Nat Chem. 8, 338-346 (2016).</li><li>McLellan, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+Hiv-1+gp120+V1/V2+Domain+with+Broadly+Neutralizing+Antibody+PG9.">Structure of Hiv-1 gp120 V1/V2 Domain with Broadly Neutralizing Antibody PG9.</a> Nature. 480, 336-343 (2011).</li><li>Julien, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetric+Recognition+of+the+HIV-1+Trimer+by+Broadly+Neutralizing+Antibody+PG9.">Asymmetric Recognition of the HIV-1 Trimer by Broadly Neutralizing Antibody PG9.</a> Proc Natl Acad Sci U S A. 110, 4351-4356 (2013).</li><li>Willis, J. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long+Antibody+HCDR3s+from+HIV-Native+Donors+Presented+on+a+PG9+Neutralizing+Antibody+Background+Mediate+HIV+Neutralization.">Long Antibody HCDR3s from HIV-Native Donors Presented on a PG9 Neutralizing Antibody Background Mediate HIV Neutralization.</a> Proc Natl Acad Sci U S A. 113, 4446-4451 (2016).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Organic+Chemistry.+Performing+1D+Thin+Layer+Chromatography.">Essentials of Organic Chemistry. Performing 1D Thin Layer Chromatography.</a> JoVE. , (2017).</li><li>de Castro, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+useful+approach+to+the+automation+of+analytical+processes?.">A useful approach to the automation of analytical processes?.</a> J Automat Chem. 12, 267-279 (1990).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Organic+Chemistry.+Nuclear+Magnetic+Resonance+(NMR)+Spectroscopy.">Essentials of Organic Chemistry. Nuclear Magnetic Resonance (NMR) Spectroscopy.</a> JoVE. , (2017).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essentials+of+Analytical+Chemistry.+Introduction+to+Mass+Spectrometry.">Essentials of Analytical Chemistry. Introduction to Mass Spectrometry.</a> JoVE. , (2017).</li><li>Tseng, S. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aluminum+Oxide-Coated+Glass+Slides+for+Glycan+Microarrays.">Preparation of Aluminum Oxide-Coated Glass Slides for Glycan Microarrays.</a> ACS Omega. 1 (5), 773-783 (2016).</li><li>Blixt, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Printed+covalent+glycan+array+for+ligand+profiling+of+diverse+glycan+binding+proteins.">Printed covalent glycan array for ligand profiling of diverse glycan binding proteins.</a> Proc Natl Acad Sci U S A. 101, 17033-17038 (2004).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> GenePix Pro 6.0 Microarray Acquisition and Analysis Software for GenePix Microarray Scanners User’s Guide &amp; Tutorial. , (2017).</li></ol>

저자는 공개 없다.

에이즈-1 bNAbs PG9, PG16, 및 PGTs 128, 141-145를 포함 한 클래스 중화 HIV-1 분리 순환의 70-80%에서 매우 강력한 보고 했다. 이러한 bNAbs epitopes는 높은 전체 HIV-1 그룹 M의 변종 중 보존, 그러므로 그들은 유도 중화 항 체23,24,25 에이즈 백신에 대 한 효과적인 immunogen 디자인을 안내할 수 있습니다. . 에이즈-1 광범위 하 게 항 체를 중화의 glycan epitopes를 식별 하기 위해 지속적인 노력의 일환으로 우리 보고 높은 노 오 스, 하이브리드, 그리고 복합 형식 oligosaccharides glycan 배열21, 형성의 패널의 화학과 효소 합성 22. 그러나, N-hydroxysuccinimide 코팅 (NHS) 유리 슬라이드에 일반적으로 사용 되 glycan 배열 낮은 선호도 탄수화물-단백질 상호 작용, 아마 때문에 이기종 glycan 유통에서 가수분해의 결과 검색할 수 없습니다. 그것의 표면에 반응성 N-hydroxysuccimide 기능 그룹. 기존의 배열 포맷의 한계를 극복 하는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드에 glycan 배열을 개발 했습니다. 우리 더 ACG 배열 하 고 일반적으로 사용 되는 NHS의 동질성 비교 수행 는 ACG 배열22그것의 표면 더 균질 glycan 프레 젠 테이 션을 제공 하는 것을 증명 하기 위해 배열. 우리의 예비 바인딩 분석 PG9 NHS 배열 (데이터 표시 되지 않음)에 glycans의의 바인딩 관찰 하지 못했습니다. 따라서, PG9의 바인딩 특이성을 정의 하려면 glycans 나-사이 phosphonic 산 꼬리에 연결 되었고 개별 glycans (그림 4)의 100 µ M와 ACG 슬라이드에 인쇄. Phosphonate 화학을 통해 ACG 슬라이드에 glycan 동원 정지는 더 안정적이 고 균일 분포를 제공합니다. glycans의 각 5 복제와 인쇄 하 고 슬라이드 이미지 DyLight649 활용 된 당나귀 안티 인간 IgG 항 체 외피 후 형광 검사에서 가져온. ACG 배열 (그림 5, 왼쪽된 패널)에 인쇄 된 개별 glycans 향해 PG9 바인딩 분석 제안 PG9 하이브리드 형 glycan (X)와 함께 강력 하 게 작용 합니다. 상호 작용 높은 노 오 스 유형 Man5에 대 한 감지 했다 (glycan IV) 및 복합 형식 glycan (XI).
하이브리드 유형과 PG9 glycan (X) 간의 분자 수준의 상호 작용은 그들의 공동 결정 구조 정보 부족으로 확인 하기가 어렵습니다. 하이브리드 형 glycan X로 구성 되어 복잡 한 형 팔 3-O 는 코어와 trimannose 팔의 위치에 연결에 6-O 중앙 trisaccharide의 위치. 하이브리드 glycan X PG9 바인딩할 PG9 높은 친 화력 상호 작용의 가까운 부근에만 노 오 스와 sialic acid 잔류물 필요 합니다 제안 합니다. Glycan 조합 PG9 바인딩 주머니에 가장 적합을 확인 하려면 우리는 혼합 glycan 배열 연구를 수행. Glycan IV-11 1:1 몰 비율에서에서 모든 glycan 혼합 되었고 ACG 배열에 발견. 혼합물의 각 PG9 바인딩 분석 남자5 와 SCT (IV + 사이)의 조합을 향해 PG9 IV 또는 사이 혼자에 비해 가장 높은 선호도 결과 표시. 또한, 남자5 및 8 세와 X glycans는 또한과 같은 sialylated 안테나를 포함 하는 혼합물22검색. 처음으로,이 결과 에이즈-1 gp120 V1/V2 도메인23PG9의 크리스탈 구조 연구에 의해 제안 되었다 PG9의 hetero-glycans의 바인딩 동작에 대 한 배열 기반 증거 제공.
결론적으로, 매우 다양 한 N의 준비에 대 한 효율적인 모듈형 화학 효소 전략 우리 증명-oligosaccharides 인간의 glycoproteins에서 발생 하는 연결. 또한, ACG 배열 개발 매우 약한 단백질-탄수화물 상호작용 하 고, 더 중요 한 것은, hetero-glycans를 통해 발생 하는 상호 작용을 결정 하는 효과적인 수단을 제공 합니다.

N-glycans glycoproteins에 covalently에 연결 되는 아스파라긴 (Asn) 잔류물 합의 Asn-Xxx-Ser/Thr sequon의 단백질 구조, antigenicity, 가용성, 및 lectin 인식 등 여러 생물 학적 과정에 영향을 1 , 2. 화학 합성 N-연결 된 oligosaccharides 그들의 거 대 한 구조 마이크로 고 높은 분기 건축 때문에 중요 한 합성 챌린지를 나타냅니다. 보호의 신중한 선택 그룹 빌딩 블록, anomeric 센터, 및 발기인의 적절 한 사용에서 선택 달성의 반응성을 조정/activator(s) 복잡 한 oligosaccharides의 합성에서 핵심 요소입니다. 복잡 한이 문제를 해결 하려면 사전 N-glycan 합성 작품의 좋은 금액으로 알려졌다 최근3,4. 이러한 강력한 접근에도 불구 하 고 찾는 다양 한 N-glycans의 준비에 대 한 효과적인 방법 주요 도전 (~ 20000) 남아 있다.
에이즈-1 광범위 한 유전적 다양성 및 중화 항 체 응답에서 탈출 하는 능력의 급속 한 돌연변이 율 에이즈-15,6 에 대 한 안전 하 고 예방 백신을 개발 하는 가장 큰 과제 중 하나입니다. , 7. 에이즈를 사용 하 여 호스트 면역 반응을 피하기 위해 하나의 효과적인 전술 이다 봉투 당단백질 gp120 다양 한 N의 닢 glycosylation-호스트 glycosylation 기계8에서 파생 된 glycans 연결 9. 인간 미 발달 신장 (HEK) 293T 세포에서 재조합 단위체 HIV-1 gp120 glycosylation의 정확한 분석에 대 한 최근 보고서 제안 특성 셀 전용 패턴10 구조 microheterogeneity의 발생 , 11 , 12. 따라서 이해의 HIV-1 bNAbs glycan 특이성 필요 잘 특징이 gp120 N-glycan 구조 분석에 대 한 충분 한 수량에 관련.
Glycan microarray 기술 발견 제공 다양 한 탄수화물 묶는 단백질, 바이러스/박테리아 adhesins, 독 소, 항 체, 및 lectines13,14의 특이성의 높은 처리량 기반 탐사 . 기준점과 칩 기반 형태로 체계적인 glycans 배열 문제가 낮은 선호도 단백질 glycan 상호 작용 multivalent 프레 젠 테이 션15,16,,1718을 통해 결정 합니다. 이 칩 기반 glycan 배열 셀 인터페이스를 효과적으로 모방을 편리 하 게 나타납니다. 기술을 풍부 관련 된 기존의 배열 형식 고르지 못한 문제를 극복 하 고, 우리의 그룹 최근 개발 phosphonic 산-엔드 glycans를 사용 하 여 신호 강도 향상 하는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드에 glycan 배열 동질성, 그리고 감도19,20.
Glycan epitopes 광범위 중화 항 체 (bNAbs) 새로 격리 된 에이즈-1의에 대 한 현재 이해를 개선 하기 위해, 우리는 N의 광범위의 준비에 대 한 매우 효율적인 모듈형 전략 개발-glycans21 연결 22 는 ACG에 인쇄할 슬라이드 ( 그림 1참조). 매우 강력한 bNAb PG9 HIV 분리 되었던의 이종 glycan 바인딩 동작의 특이 한 검출 감염 개인23,,2425를 제공 하는 프로 파일링 연구 에이즈-1 bNAbs ACG 배열에의 한 특이성

<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

우리 N의 광범위의 빠른 준비를 위한 매우 효율적인 방법 제시-oligosaccharides (초과 20000 구조 추정) 인간 glycoproteins에 일반적으로 발견 되는 연결. 원하는 구조 다양성을 달성 하기 위해 전략 뒤에 3-O 및 6-O 에 그들의 stepwise α 선택적 glycosylations oligosaccharyl 불 소 단위의 세 종류의 화학 효소 합성 시작의 위치는 중요 한 β-mannoside 결합 하는 데 일반적인 코어 trisaccharide의만 노 오 스 잔류물. 우리는 더 HIV 항 체와 이종 ligand 상호 작용의 분석에 대 한 공유 혼합된 배열을 만드는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드의 표면에 N-glycans를 첨부. 특히, 남자 (5)는 새로 고립 된 HIV-1 광범위 중화 항 체 (bNAb), PG9, 밀접 하 게 간격 둔된 남자5GlcNAc2 의 혼합물에의 바인딩 동작 및 2, 6-디-sialylated bi antennary 복잡 한 입력 N-glycan (SCT ) ACG 배열, 에이즈 백신 개발을 위한 효과적인 immunogen 디자인 가이드에 새로운 길을 엽니다. 또한, 우리의 ACG 배열 hetero ligand 바인딩 동작에 대 한 다른 HIV 항 체를 연구 하는 강력한 도구를 구현 합니다.

다양 한 N-glycans의 합성에 대 한 모듈형 화학 효소 전략은 그림 1에 표시 됩니다. 전략은 다양성 α-특정 mannosylation는 3-O 및 6-O 에 다음 세 가지 중요 한 모듈의 화학 효소 합성 하 여 처음에 만들 수 있습니다는 사실에 근거한 일반적인만 노 오 스 잔류물의 위치 trisaccharide Nglycans의 핵심. Bi-트라이-, tetra antennary 복합 형식 N-glycan 구조의 구조 다양성을 고려 하면 우리 oligosaccharyl 기증자와 함께 사전 설치 된 알 킬 handlecould 코어 trisaccharides 수락자로 시작 사용할 수 믿 었 다 원하는 구조 다양성 (그림 1)을 생성 하는 재료. Glycosyl 불 기증자 복잡 한 glycan 도서관 건물에 적용은 입증 되었습니다 이전21.
우리의 전략의 효과 보여, bi antennary 이성질체 구조 (10, 그림 3) 합성에 선정 됐다. D1 팔 안테나 4 와 d 2 팔 안테나 5 glycan 10 의 큰 비늘 화학 효소 방법으로 생성 했다. 특히,이 당 수락자 1 했다 효소 galactosylated trisaccharide 2를 β-1, 4-galactosyltransferase 및 uridine 5'-diphosphogalactose (UDP-Gal)을 사용 하 여. 다음, 중간 2 α-1, 헬리코박터 파일로리 (Hpα1, 3 피트) 원하는 tetrasaccharide 3줄에서 3 fucosyltransferase의 존재에 GlcNAc 3-O 위치에 fucosylated 이었다. 핵심 화학 결 찰, 빌딩 블록 2 와 3 은 첫 번째 peracetylated, 및 줄이는 끝 p-methoxy 페 놀 그룹 제거 되었습니다. 마지막으로, 불 소를 원하는 모듈 4 및 5, 각각 DAST 존재 설치 된 (그림 2).
원하는 모듈을 손에 우리 다음 진행 4 코어 trisaccharide 6 triflate와 각각 hexasaccharide 7 제공 하기 위해 hafnocene dichloride 촉매에서의 stereoselective 3-O glycosylation (그림 3). 그 4, 6-오 마스킹 제거 촉매 p를 사용 하 여 benzylidene 보호-톨루엔 술 폰 산 (p-TSA). 불 소 모듈 5 필요한 decasaccharide 9를 달성 하기 위해 비슷한 실험 조건 하에서 반응 했다 그것의 반응성, 기본 6- 8 의 오의 활용. 마지막으로, 글로벌 deprotection glycan 10, NMR 및 질량 분광학 (보조 데이터 파일 참조)를 사용 하 여 특징 추가 했다를 위해 수행 되었습니다.
줄이는 끝 pentyl 아민 꼬리를 포함 하는 Glycans phosphonic 산 링커 수정 되었고 phosphonate 화학 (그림 4)을 통해 ACG 표면에 부착 된. 마지막으로, HIV-1 bNAb에서 PG9 PG9 에이즈-1 gp120 표면 (그림 5)의 V1/V2 루프에 인접 한 heteroglycans 작용할 처음으로 보여 호모 및 이종 glycans 배열을 사용 하 여 자사의 glycan 특이성에 대 한 상영 되었다.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/55855/55855fig1.jpg"> 그림 1: N-glycans의 준비에 대 한 일반 모듈 전략. (A) N-glycans이이 전략에 의해 생성 된 인간의 glycoproteins에 일반적으로 발생 하는 수가 초과 20, 000으로 추정 된다. (B) α-seletive glycosylations 사용할 수 있는이 화학 효소 접근 방식에 의해 준비 하는 모듈의 3 종류. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig1large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/55855/55855fig2.jpg"> 그림 2: 모듈의 Chemoenzymatic 합성. i, UDP-갈 락 토스, β 1, 4-GalT, 15 h, 86%; ii, GDP-fucose, α 1, 3-FucT, 15 h, 84%; iii, (1) Ac2O, pyridine, RT, 12 h; (1) 수, ACN: 톨루엔: H2O, (3) DAST, 채널2Cl2, C. 수-30 o: 질 산 암모늄 세 륨; DAST: Diethylaminosulfur trifluoride입니다. 제품 명칭: 1, p-methoxyphenyl-O-2-acetamido-2-deoxy-β-D-glucopyranosyl-(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-스캔들-fucopyranosyl-(1→3)-2-acetamido-2-deoxy-β-차원-glucopyranosyl]-(1→2)-α-차원-mannopyranoside-methoxyphenyl; 4, [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 불 소; 5, [2,3,4,6-O-tetraacetyl-β-D-galactopyranosyl]-(1→4)-[2,3,4-O-triacetyl-α-L-fucopyranosyl(1→3)-3,6-O-diacetyl-2-acetamido-2-deoxy-β-D-glucopyranosyl]-(1→2)-3,4,6-O-triacetyl-α-D-mannopyranosyl 불 <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig2large.jpg" target="_blank">하시기 바랍니다 여기를 클릭 보기를 이 그림의 더 큰 버전.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/55855/55855fig3.jpg"> 그림 3: 핵심 수락자 D1/D2 팔 모듈의 화학 glycosylation. 난, 4, AgOTf, Cp2HfCl2, 톨루엔, 4 Å MS 0 oC rt, 70%; ii, p-TSA, 이기, RT, 57%; iii, 5, AgOTf, Cp2HfCl2, 톨루엔, 4 Å MS 0 oC rt, 34%; 4, (1) LiOH, 1, 4-dioxane: H2O; 90 oC, 12 h; (2) Ac2O, pyridine, 12 h; (3) NaOMe, MeOH, 12 h; (4) Pd(OH)2, MeOH: H2O: HCOOH (5:3:2), H2, 36%. AgOTf: 실버 trifluromethanesulfonate; Cp2HfCl2: Bis (cyclopentadienyl) 하프늄 dichloride, MS: 분자 체, 제품 명칭: 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-아 세 틸-3,4,6-트라이-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">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/55855/55855fig4.jpg"> 그림 4: Glycan immobilization ACG 배열에. (A) glycan phosphonic 산으로 아미노 꼬리의 화학 수정 phosphonate 화학을 통해 ACG 슬라이드 공유 첨부 파일에 대 한 꼬리. ACG 표면에 glycans의 (B) 배포. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig4large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/55855/55855fig5.jpg"> 그림 5: Glyan 배열 분석. ACG 배열에 인쇄 된 합성 Nglycans의 (A) 구조. (B) glycans 나-사이 (오른쪽 패널) 100 µ M 농도로 혼합 개별 glycans 나-사이 ACG 배열 (왼쪽된 패널)에 인쇄 하 고 남자5 glycan 혼합물 분석 PG9의 바인딩. PG9 위해 µ M에 어 금 니 농도 전설에 부여 됩니다. 평균 신호 강렬과 표준 오류 배열에 5 개의 독립적인 복제에 대 한 계산 표시 됩니다. 인세트 형광 이미지를 표시합니다. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55855/55855fig5large.jpg" target="_blank">이 그림의 더 큰 버전을 보려면 여기를 클릭 하십시오.</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

Glycan microarray를 개발 하 고 광범위 하 게 항 체를 중화 하는 HIV의 프로 파일링에 대 한 사용 설명 되는 산화 알루미늄 코팅 유리에 첨부 파일에 대 한 N-glycans의 합성에 모듈 접근 (ACG 슬라이드) 슬라이드.

N-glycans 배열 개발 및 HIV 항 체의 항 암 치료 효소 합성 프로 파일링

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 조회수.  Academia Sinica. Glycan microarray를 개발 하 고 광범위 하 게 항 체를 중화 하는 HIV의 프로 파일링에 대 한 사용 설명 되는 산화 알루미늄 코팅 유리에 첨부 파일에 대 한 N-glycans의 합성에 모듈 접근 (ACG 슬라이드) 슬라이드.

비디오: 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.

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

연구

화학

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.

플라즈마 통해 소수성 유기 금속 프레임 워크의 제조 암모니아의 제거에 대한 퍼플 루오로 알칸의 화학 증착을 강화

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.

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.

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.

금 나노 막대의 하이드로 퀴논 기반 합성

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.

수용성 유기 금속 복합 어레이의 합성

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.

에 라 골드 나노 입자 합성에 의해 급속 한 Nanoprobe 신호 향상

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.

활성 표면 성장 메커니즘을 통해 기판 바인딩된 Au 나노 와이어의 합성

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.

Au, Pd, Pt Aerogels 직접 솔루션 기반 감소를 통해 빠른 합성 방법

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