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國立清華大學
碩士論文
多價性半乳糖衍生物的合成與
其針對凝集素之生醫應用
Synthesis of Multivalent Galactoside Derivatives toward
Biomedical Application for Lectins
系別：化學系
學號：103023570
研究生：賴育聰 (Lai, Yu-Tsung)
指導教授：王聖凱博士 (Dr. Wang, Sheng-Kai)
中華民國一 O 五年九月
中文摘要
凝集素與醣體間的交互作用力廣泛存在於多種重要生理反應以及生物機制中。
但因為單一的凝集素與醣體之間的交互作用往往非常微弱，為凝集素的研究帶來困
難。而解決作用力的不足除了藉由多價性效應之外，開發具有更高親和力的醣體也
是解決方法之一。
在本研究的第一部分中，我們針對綠膿桿菌生物膜開發出抑制劑。在抗生素廣
泛使用的現代，綠膿桿菌對於多數的抗生素產生抗藥性，而生物膜的形成更加局限
治療方法。綠膿桿菌感染以及形成生物膜主要藉由其凝集素 LecA 針對細胞表面半
乳糖殘基結合。近年發現帶有芳香環的半乳糖可以提高 LecA 的結合能力。我們開
發出多種不同的芳香性半乳醣苷來做為配基以探討作用力強弱與結構之關係。同時
我們開發出脯胺酸多肽作為多價性骨架，藉由其 PPII 構形來維持有效距離來針對
LecA 之相鄰之結合位置結合，並以此骨架合成多價醣肽材料做為抑制劑，此多價性
材料設計在微陣列篩檢中有不錯的效果。
此外，由於肝細胞表面存在大量特有的凝集素去唾液酸糖蛋白受體(ASGPR)，
藉由此受體與半乳糖殘基結合所產生的胞吞作用可以將藥物載體送入細胞內以達到
基因藥物作用之目的。所以我們嘗試將開發出的芳香性半乳醣苷修飾於奈米粒子表
面上並乘載螢光 siRNA，藉由 Hep3B 肝癌細胞攝取實驗，我們在共聚焦顯微鏡下發
現芳香性半乳醣苷比一般未修飾之奈米載體高出十倍，此結果有助於未來發展相關
結構之配基來修飾於肝疾病藥物載體。
I
Abstract
Carbohydrate-lectin interaction involved in many biological process and mechanism.
However, the weak binding affinity of monovalent carbohydrate ligand and lectin is the
difficulty to exploit carbohydrate for lectin research. In addition to exploiting multivalent
interaction, developing high affinity monovalent carbohydrate derivatives is also a
solution to overcome the difficulty.
In past decades, antibiotics were commonly used and cause P. aeruginosa rapidly
develop resistance during the course of treating an infection. In addition to the resistance
of P. aeruginosa, biofilm formation is a more severe issue for human health. The ability
of infection and biofilm formation of P. aeruginosa were mainly caused by LecA lectin to
specifically bind to galactose residue of cell surface. Recent years, it was reported that the
aromatic galactoside have higher affinity to LecA than galactose. In this part of thesis, we
synthesized kinds of aromatic galactoside as ligand to understand the relationship
between the aromatic structure and the affinity. Apart from this, we also develop
multivalent scaffold by using polyproline for its PPII structure to maintain the effective
distance. We designed and synthesized aromatic galactoside conjugated polyproline
material as multivalent ligand binding to neighboring binding site of LecA. After
microarray scanning, we found that the multivalent materials were effective for LecA
binding test.
Furthermore, asiaglycoprotein receptor (ASGPR) is an endocytotic cell surface lectin
expressed by hepatocyte, which recognized galactoside residue of asiaglycoprotein. So
we tried to use the aromatic galctoside as ligand and modified on nanoparticle carrier to
test the uptake ability. After modification of the surface of nanoparticle carrier, the
fluorescent siRNA were used as cargo to test the uptake ability of the human hepatoma
Hep3B cell line. In Hep3B cell uptake assay, we found that modified nanoparticle was
II
elevated the ability of uptaken by Hep3B about 10 times higher than unmodified under
confocal microscopy. These result may be helpful for designing more valid ligand
structure to modified drug carrier.
III
目錄
中文摘要 ...........................................................................................................................................I
Abstract ........................................................................................................................................... II
目錄 ................................................................................................................................................ IV
圖目錄 ............................................................................................................................................ VI
表目錄 ......................................................................................................................................... VIII
式目錄 ............................................................................................................................................ IX
流程目錄 ......................................................................................................................................... X
縮寫表 ............................................................................................................................................ XI
第一章、緒論 .................................................................................................................................. 1
1.1.前言 ........................................................................................................................................ 1
1.2.醣類與凝集素之交互作用 .................................................................................................... 2
1.3.多價性效應 ............................................................................................................................ 3
1.3.1.多價性效應原理 ............................................................................................................. 3
1.3.2.多價性效應對於凝集素的研究 ..................................................................................... 7
1.4.脯胺酸多肽的特性及應用 .................................................................................................... 8
1.4.1.脯胺酸多肽結構與特性 ................................................................................................. 8
1.4.2.脯胺酸多肽在生物研究上的應用 ................................................................................. 9
1.5.綠膿桿菌凝集素抑制劑 ...................................................................................................... 10
1.5.1.綠膿桿菌對於人類健康的危害 ................................................................................... 10
1.5.2.綠膿桿菌生物膜的形成及抗藥性機制 ....................................................................... 11
1.5.3.綠膿桿菌可溶性凝集素 I(PA-IL)的結構與特性 ........................................................ 12
1.5.4.綠膿桿菌凝集素 LecA 抑制劑作為抗綠膿桿菌材料之研究..................................... 14
1.5.5. 多價性綠膿桿菌生物膜抑制劑 ................................................................................. 16
1.6.醣脂質對藥物載體奈米粒子的選擇性控制研究 .............................................................. 20
1.6.1. RNAi 藥物以及肝相關疾病研究 ................................................................................ 20
1.6.2. RNAi 藥物輸送奈米粒子載體種類與性質 ................................................................ 21
1.6.3.藥物輸送奈米粒子材料對於肝病的研究 ................................................................... 23
IV
1.6.4. ASGPR 結構以及胞吞機制 ........................................................................................ 25
1.6.5.醣分子配體對於 ASGPR 親和力研究 ........................................................................ 27
1.7. 研究動機 ............................................................................................................................ 28
第二章、結果與討論 .................................................................................................................... 31
2.1.單價 LecA 抑制劑合成 ....................................................................................................... 31
2.1.1. β-芳基半乳醣苷之合成 ............................................................................................... 31
2.1.2. Gb3 醣體之合成 .......................................................................................................... 40
2.1.3. 以微陣列(Microarray)篩檢 LecA 與抑制劑之結合能力 .......................................... 45
2.1.4.微陣列試驗結果與討論 ............................................................................................... 46
2.2.特定間距之二價醣肽 LecA 抑制劑合成 ........................................................................... 48
2.2.1.醣肽材料合成之策略 ................................................................................................... 48
2.2.2.多肽固相合成脯胺酸多肽骨架 ................................................................................... 50
2.2.3.具疊氮基之芳香性半乳醣苷合成 ............................................................................... 53
2.2.4.疊氮-炔類的[3+2]環化加成 ......................................................................................... 55
2.2.5.圓二色光譜結構分析 ................................................................................................... 57
2.2.6. 微陣列試驗之結果與討論 ......................................................................................... 60
2.3.半乳糖衍生物修飾奈米藥物載體表面對於肝細胞之研究 .............................................. 64
2.3.1.醣修飾脂質材料合成之策略 ....................................................................................... 64
2.3.2.具丙炔基之半乳糖衍生物之合成 ............................................................................... 65
2.3.3.半乳糖衍生物接合脂質化合物之合成 ....................................................................... 68
2.3.4. 肝細胞對於奈米粒子攝入實驗 ................................................................................. 69
2.3.5. NPs 攝入結果與討論 .................................................................................................. 71
第三章、實驗方法與材料 ............................................................................................................ 73
3.1. Material and methods .......................................................................................................... 73
3.2. Synthetic procedures and characterization .......................................................................... 74
第四章、參考文獻 ...................................................................................................................... 129
V
圖目錄
圖一、Seeberger 研究團隊設計符合 ASGPR 構形的半乳糖配體來對 ASGPR 結合 2 ............. 2
圖二、(1)單價交互作用示意圖、(2)多價交互作用示意圖 7 ....................................................... 3
圖三、多價性交互作用在生物反應中所涉及的四種反應機制 8 ................................................ 5
圖四、抗體利用多價結合來引發免疫反應 7。 ............................................................................ 6
圖五、不同種類的多價性醣綴合物 8 ............................................................................................ 8
圖六、PPI、PPII 之立體結構示意圖 18 ......................................................................................... 9
圖七、Maecke 和 Wennemers 團隊合成的腫瘤細胞探針示意圖 19 .......................................... 10
圖八、生物膜形成以抵抗外在環境示意圖 21 ............................................................................. 11
圖九、LecA 結構示意圖 25 ........................................................................................................... 13
圖十、Gb3Cer/CD77 抗原結構圖 ................................................................................................ 13
圖十一、合成四價抑制劑抑制綠膿桿附著感染示意圖 25 ......................................................... 14
圖十二、β-芳基半乳醣苷對於 LecA 胺基酸殘基提供額外作用力 31....................................... 15
圖十三、對於 LecA 具有高親和力的 2-萘基 β-D-半乳醣苷 8 .................................................. 16
圖十四、 Chevolot 與 Morvan 教授團隊合成八價樹突狀分子 34 ............................................ 17
圖十五、(a) Pieters 教授團隊以聚糖作為 spacer 材料設計符合結合位置的二價抑制劑。(b) 利

用理論計算模擬與 LecA 結合之情況，其骨架確實可以維持結合間距 38。 .......................... 18
圖十六、Winssinger 研究團隊同時調控距離與半乳糖(galactose)配體來對 Lec A 結合 39 .... 19
圖十七、各種 RNAi 修飾及奈米載體提升在體內穩定性 46 ..................................................... 23
圖十八、Bi, Tri, Tetra-antennary 聚醣結構示意圖 54................................................................. 25
圖十九、ASGPR 於肝細胞表面結構組成示意圖 58 ................................................................... 26
圖二十、arabinogalactan 之聚醣結構 62。 .................................................................................. 28
圖二十一、化合物 38 之 1H-1H COSY 光譜展開圖，化學位移在 4.00 ppm 之 4’-H 與 2.39 ppm

之羥基的耦合現象判斷羥基位於半乳糖四號位置 .................................................................... 43
圖二十二、選擇性還原開環之反應機構 69 ................................................................................. 43
圖二十三、市售之塗布玻片修飾方法示意圖 72。 ..................................................................... 45
圖二十四、不同單價抑制劑利用微陣列所篩檢結果，以相對螢光強度為縱軸作圖。......... 48
圖二十五、Brimble 教授團隊利用 CuAAC 合成 MUC1 片段醣肽衍生物 73。....................... 49
VI
圖二十六、脯胺酸多肽骨架(41-44)之 CD 光譜結果 ................................................................. 58
圖二十七、醣肽化合物(60-64)與同長度之脯胺酸多肽骨架 42 之 CD 比較圖。 ................... 59
圖二十八、醣肽化合物 65 與相同長度骨架 43 CD 光譜 .......................................................... 59
圖二十九、不同抑制劑利用微陣列所篩檢結果，以相對螢光強度為縱軸作圖 .................... 61
圖三十、醣肽化合物 65-70 在不同濃度下所求得之滴定曲線 ................................................. 62
圖三十一、市售之疊氮基磷脂質結構(化合物 76)..................................................................... 65
圖三十二、共聚焦顯微鏡拍攝圖之結果 .................................................................................... 70
圖三十三、Hep3B 對於不同修飾之 NPs 的相對攝取量長條圖。 ........................................... 71
VII
表目錄
表一、以 PTC 反應建構乙醯化 β-芳基半乳醣苷之 aglycone 結構與產率 .............................. 33
表二、去乙醯化之 β-芳基半乳醣苷結構與產率 ........................................................................ 38
表三、化合物 55-75 之產率以及結構 ......................................................................................... 56
表四、微陣列篩檢之化合物結構與名稱對照表 ........................................................................ 61
表五、65-70 在微陣列測試中所測得之 IC50 數值 .................................................................... 63
VIII
式目錄
式一、式二、芐基化保護芳香環上羧酸基以利 PTC 反應進行。 ........................................... 32
式三、化合物 18 之合成 .............................................................................................................. 37
式四、氫化去除化合物 5 及 8 之芐基保護 ................................................................................ 37
式五、脯胺酸多肽骨架與 LecA 配體進行疊氮-炔類的[3+2]環化加成環化加成 ................... 55
式六、以 CuAAC 合成半乳糖衍生物接合脂質化合物 91-96 ................................................... 68
IX
流程目錄
流程一、合成乙醯化 β-芳基半乳醣苷反應路徑 ........................................................................ 33
流程二、化合物 15 合成路徑圖 .................................................................................................. 35
流程三、化合物 17 之合成路徑 .................................................................................................. 36
流程四、醣予體 34 之合成 .......................................................................................................... 41
流程五、醣受體 38 之合成 .......................................................................................................... 42
流程七、兩個丙炔基(propargyl group)修飾之特定距離脯胺酸多肽合成 ................................ 51
流程八、具疊氮基之芳香性半乳醣苷化合物 50-54 合成 ......................................................... 54
流程九、化合物 82-86 之合成 ..................................................................................................... 66
流程十、化合物 90 之合成 .......................................................................................................... 67
X
縮寫表
Ac Acetyl
Ac2O Acetic anhydride
ASGPR Asialoglycoprotein receptor
Bn Benzyl
BnBr Benzyl bromide
MeCN Acetonitrile
Cbz n-Butyl Carbobenzyloxy
CuI Copper(I) iodide
CuSO4 Copper(II) sulfate
Cy3 Cyanine 3
DAPI 4',6-Diamidino-2-phenylindole
DCM Dichloromethane
DIEA N,N-Diisopropylethylamine
DMF N,N-Dimethylformamide
DMAP 4-Dimethylaminopyridine
DMSO Dimethyl sulfone
DOPA 3,4-Dihydroxy-L-phenylalanine
DOTAP N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride
DSPE 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine
EtOAc Ethyl acetate
EtOH Ethanol
FAM Carboxyfluorescein
Fmoc 9-Fluorenylmethyloxycarbonyl
HATU 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide
XI
hexafluorophosphate
HBTU N,N,N’,N’-Tetramethyl-O-(1H-benzotriazol-1-yl)uranium
hexafluorophosphate
HOBT Hydroxybenzotriazole
Hyp Hydroxyproline
HRMS High resolution mass spectrometry
HBr Hydrobromic acid
Me Methyl
MeOH Methanol
M.S. Molecular sieve
NHS N-Hydroxysuccinimide
NIS N-Iodosuccinimide
NPs Nano particles
PBS Phosphate-buffered saline
PEG Poly ethylene glycol
Ph Phenyl
PPII Polyproline II
Pyr. Pyridine
r.t. Room temperature
TBAI Tetrabutylammonium iodide
Tf Trifluoromethanesulfonic
TFA Trifluoroacetic acid
TLC Thin layer chromatography
THF Tetrahydrofuran
TMS Trimethylsilyl
XII
第一章、緒論
1.1.前言
在生物體內，許多生物過程藉由蛋白質辨識細胞表面或是其他蛋白質上的醣體
才得以進行，使得醣體在生物體內扮演重要的角色。除此之外，這些醣體亦是一些
毒素，細菌的辨識目標，且通常都具有相當的專一性。例如: 泌尿道致病性大腸桿
菌(Uropathogenic E. coli)，是常見的泌尿道感染病原之一，其細胞膜表面的粘附素
FimH 對尿道上皮細胞表面的甘露糖具有辨識能力，進而黏著造成感染。細菌通常具
有對於細胞表面的醣體辨認、結合的蛋白質，藉此聚集達到感染宿主的目的。故醣
分子可以成為一種醫藥上可以應用的工具，可以抑制許多細菌的致病因子，像是:
聯苯基-α-D-甘露醣苷(biphenyl-α-D-mannosides) 與 FimH 有相當高的親和性，可以做
為 FimH 的抑制劑1。但是通常單一醣體對於特定蛋白質的作用力微弱，難以應用來
做為有效的生醫工具。所以目前，許多研究團隊不只著重於人工合成對於目標蛋白
質有親和性的醣體，同時也著重於研究蛋白質構形來設計多價性(multivalent)材料，
來達到提升親和力的作用。例如: Seeberger 研究團隊利用不同間距骨架搭配半乳糖
(galactose)來篩選出最符合去唾液酸醣蛋白受體(asialoglycoprotein receptor，ASGPR)
多聚體構形的合成分子以增加其親和力(圖一)2。正因為蛋白質與醣體作用十分複雜，
所以如何增加醣分子作用力來對於相關蛋白質的應用為相當重要的研究目標。
1
圖一、Seeberger 研究團隊設計符合 ASGPR 構形的半乳糖配體來對 ASGPR 結合
2
。
1.2.醣類與凝集素之交互作用
凝集素(Lectins)是一種對醣蛋白上的醣體具有高度特異性(specificity)的結合
蛋白，對於特定醣體具有專一性的非免疫來源(non-immune origin)蛋白質。不同
種類的凝集素廣泛存在於細菌、植物以及動物體中，不過通常並不歸類於酵素、
抗體的一種。植物凝集素是第一種被確定的凝集素，其主要功能大多涉及防衛機
制來對抗寄生物、蕈類以及捕食者，但其功能依舊十分複雜並尚未被完整了解。
另外一方面，病原體如細菌、病毒所具有的凝集素，被確認為一種黏著分子，促
進病原體附著在宿主細胞表面以及形成生物膜(biofilm)。甚至一些細菌凝集素為
致病因子，例如毒素(toxins)，在感染的過程中做為主要的致病因素3。典型的凝
集素至少有兩個或兩個以上的醣結合位置(carbohydrate-binding sites)4，同時會與
不同紅血球細胞表面的醣交互作用，隨後交叉鏈接(cross-linking)產生沉澱，這種
凝集素造成現象被稱為細胞凝集(agglutination)，是早期判斷凝集素的現象之一5。
2
不過雖然醣類分子與凝集素相互結合產生作用的例子在自然界中比比皆是，但是
其非共價(non-covalently)作用力再加上可逆的結合方式造成單醣分子與凝集素結
合能力不高，造成利用醣分子來研究凝集素的困難6。
1.3.多價性效應
1.3.1.多價性效應原理
相較於一般單價交互作用(monovalent interaction)為單一醣分子與受體的結
合，多價性交互作用(multivalent interaction)發生在具有多個結合位置的多價受體
(multivalent receptors)，如凝集素(Lectins)，與帶有多個配基(ligands)的多價配體
(multivalent ligands)，如細胞表面的醣蛋白，如示意圖(圖二)7。
圖二、(1)單價交互作用示意圖、(2)多價交互作用示意圖 7。
當兩個以上的配基通過特定間距鏈接的配體，對於適當結構且具有兩個以上
3
結合位置的目標蛋白質而言，其結合能力可以顯著的提升。以熱力學解釋，大部
分多價交互作用比起多個單價配體連接到受體上的焓(enthalpy)雖然相差不多，但
多價配體的熵(entropy)因為配基鏈接起來的緣故，在構形(conformation)適當的情
況下，其移動與轉動所具有的熵可以約略視為單分子運動進而降低因結合所減少
的熵，導致自由能(例如 Gibbs free energy)減少更多，造成多價作用大多結合力較
高 7。此外，雖然單價配體與受體的交互作用是十分直截了當地，但多價交互作
用並非如此，其交互作用十分複雜且難有理論模型可以完全詮釋。主要原因是因
為交互作用總體的結合解離平衡反應是由多個單一的配基與受體的平衡反應所
組成，通常平衡反應之間又存在動力學競爭與不同的交互作用機制。自然界中複
雜的多價交互作用以增強作用力的機制大致上可以分為四種，如圖三8所示，以
下敘述之:
1) Chelate effect:
具有多個結合位置的受體同時和帶有多個配基的分子結合，提升親和力，
如同多價的凝集素同時和帶有多醣基的分子直接產生螯合。
2) Receptor cluster:
單價受體與配基先結合，剩餘的多價受體或配基再通過群聚行為(cluster
behavior)來符合彼此最適當的空間與結合位向，加強親和力。通常發生
在細胞膜上，受體可以通過在雙層磷脂質擴散來達到和多價配體最佳的
結合位置與位向。
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3) Subsite binding:
配體同時帶有對主結合位置以及副結合位置(secondary binding site)具有
親和性的配基來形成異二價(heterodivalent)結合，提升專一性以及親和
力。
4) Statistically effect:
透過高密度配基來提升和受體結合的機率，使受體不易脫離與配體的結
合。例如多價醣基化結構針對單價凝集素的結合9。
圖三、多價性交互作用在生物反應中所涉及的四種反應機制 8。
雖然多價性交互作用(Multivalent interaction)的理論並不具有可以完全解釋
的理論模型，但多價性交互作用的概念與發展卻能解決許多生物上的研究問題以
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及合理詮釋許多重要的生物體內反應，例如免疫系統的活化。不同種類的抗體通
常都具有多個等價數的受體：兩個(IgD, IgE, IgG, IgA)，四個(IgA)，六個(IgA)，
甚至多至十個(IgM)。多個等價數的受體會與抗原表面的多個配體所結合，來引
發巨噬細胞清除病原體以及其他免疫反應。位於抗體尾端可結晶區(Fc portion)的
甘露糖(Mannose)殘基會與巨噬細胞表面的甘露糖受體結合，當只有一個可結晶
區(Fc portion)與受體結合，其作用力是非常弱的，並不足以誘導巨噬細胞的免疫
反應。不過當多個抗體同時辨認到同一個病原體導致高密度的抗體群聚起來而形
成多價性配體，透過高密度配基來提升和受體結合的機率，這些抗體的可結晶區
(Fc portion)對於巨噬細胞表面的多個受體所產生的作用力則會強大到足以引發
巨噬細胞的免疫反應(圖四)7。
圖四、抗體利用多價結合來引發免疫反應 7。
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1.3.2.多價性效應對於凝集素的研究
凝集素與醣類分子在生物體中的作用扮演相當重要的角色，同時醣類分子也
是研究凝集素最適合的工具之一。雖然凝集素對於醣類分子具有專一性，但是由
於非共價性(non-covalently)鍵結，可逆性的(reversible)結合模式，以及相對微弱
的結合(解離常數(dissociation constant, Kd)為毫莫爾濃度範圍)10，造成研究上的困
難。近年來許多研究團隊設計一連串的多價性醣綴合物(glycoconjugate) (圖五)8，
利用多價性效應來解決單價醣類研究上的困難。例如：醣簇分子(glycoclusters)
為化學合成具有特定數量結合表位(binding epitopes)的分子；醣樹枝狀分子
(glycodendrimers)是具有多支鏈所組成的球形結構，具有特定的價數的同時仍然
保有部分單一分子的特性；多醣體聚合物(glycopolymers)為具有更高價數的巨分
子，但其配體數量難以控制11，且在價數提高的同時，其聚合物骨架也帶來毒性
的問題；醣奈米粒子(glyconanoparticles)提供大量的配基(主要為 50-150 個醣修飾
殘基)分布於奈米粒子表面。近年來奈米材料對於生物應用上的發展越來越快速，
醣奈米粒子對於抵抗病原體感染的研究在生物體內以及動物模型上都有亮眼的
成果12、13；載體蛋白(carrier protein)在生物體內的作用是非常靈敏的，所以在蛋白
質的特定位置上修飾醣類表位(carbohydrate epitopes)來作為凝集素研究的材料是
近年來許多團隊努力開發的對象，近年內的相關結果則對於設計抗感染藥物有很
重要的貢獻14、15、16。
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圖五、不同種類的多價性醣綴合物 8。
多價性醣綴合物(glycoconjugate)不僅在近年來使我們得以一窺生物體中凝
集素的作用，利用多價性交互作用提高凝集素與醣修飾配體的親和力，我們更得
以解決許多生物上的困難。例如透過合成多價性配體來阻絕許多病原體病原體凝
集素，利用具有高親合性的配體來多堵塞住凝集素的結合位置，阻絕病原體在細
胞組織上的附著。多價性交互作用為凝集素研究提供更有效的策略。
1.4.脯胺酸多肽的特性及應用
1.4.1.脯胺酸多肽結構與特性
脯胺酸多肽螺旋 II(polyproline helix II, PPII)是常見的二級結構，在許多天然
蛋白質以及生理過程中扮演重要的角色。寡聚脯胺酸序列通常在水溶液下為左旋
的 PPII 結構，每三個脯胺酸序列會形成 360 度的螺旋，造成相隔三個醯胺鍵的
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脯胺酸會重合，彼此之間的軸向距離為 9.4 Å ，而在低極性溶液下則會形成右旋
的脯胺酸多肽螺旋 I(polyproline helix I, PPI)，每 3.3 個脯胺酸會使結構旋轉一圈，
其軸向距離為 5.4 Å 17 (圖六)18。因為水溶液下的 PPII 結構特性已經被明確定義且
證實，使脯胺酸多肽具有作為位置選擇性(site-specific)生物研究工具的可行性。
圖六、PPI、PPII 之立體結構示意圖 18。
1.4.2.脯胺酸多肽在生物研究上的應用
由於脯胺酸多肽結構特性非常適合用來調控距離，同時因為結構較許多天然
胺基酸序列結構更具剛性 (rigidity) 。對於多價性骨架而言，其
結構越具剛性與越適合受體位向，其與受體的結合可以降低更多的 entropic
penalty，所以對於研究多個受體之間距而言是理想的工具，例如: Maecke 和
Wennemers 團隊將對於前列腺腫瘤(prostate adenocarcinoma)細胞過度表現的
GRP-R (gastrin-releasing peptide receptor)有專一性的兩種多肽分子連接在脯胺酸
多肽的側鏈，藉 PPII 結構的特性合成間距不同的放射性探針，來開發對於腫瘤
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細胞表面受體間距相符合的材料，得到最為符合間距的探針，藉此得以在生物體
內標示前列腺腫瘤細胞位置(圖七)19。
圖七、Maecke 和 Wennemers 團隊合成的腫瘤細胞探針示意圖 19。
1.5.綠膿桿菌凝集素抑制劑
1.5.1.綠膿桿菌對於人類健康的危害
一般而言，病原體通過辨識宿主細胞表面的表位(epitopes)來黏著與穿透宿主
細胞進而造成感染。伺機性病原體綠膿桿菌(pseudomonas aeruginosa)是一種好氧
的葛蘭氏陰性(Gram-negative)細菌，廣泛存在於生活周遭，甚至在通風系統中也
是無處不在，是醫院內主要造成免疫力低下的病人產生併發性感染的主因之一，
其併發症包括敗血症(septicaemia)、泌尿道感染、胰腺炎(pancreatitis)、皮膚炎
(dermatitis)，尤其是對於囊腫纖維化(cyctic fibrosis)病人的肺部感染產生的呼吸道
劣化，是最為嚴重且往往導致死亡的症狀。綠膿桿菌主要透過兩種位於外膜(outer
membrane)上可溶性凝集素，LecA (PA-IL)及 LecB (PA-IIL)來附著在細胞組織上以
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及形成生物膜(biofilms)。LecA (PA-IL)及 LecB (PA-IIL)分別辨識細胞表面上的半
乳糖殘基(galactose residue)以及岩澡糖(fucose)來做結合20。透過兩種凝集素結合
醣蛋白與醣鞘脂質(glycosphingolipid, GSLs)的醣類表面，細菌得以在組織表面大
量黏著感染，形成生物膜。
1.5.2.綠膿桿菌生物膜的形成及抗藥性機制
生物膜是在黏附表面的細菌群聚形成的組織，包覆在生成的細胞外基質
(extracellular matrix)21內。生物膜的構成有助於細菌在不利於生長的環境下拓殖，
甚至在不同薄膜區塊的細菌會產生不同的基因表現，來對抗外在環境22。形成生
物膜最重要的影響在於細菌會形成具保護功能的細胞外基質，可以阻絕生物體內
的免疫機制以及抗生素療程(圖八)21。
圖八、生物膜形成以抵抗外在環境示意圖 21。
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生物膜對抗外在抗生素以及免疫系統可能的機制十分多樣且複雜，像是抗生
素或是免疫物質對於細胞外基質的穿透性低落、或是薄膜內營養環境的不同造成
部分細胞處於生長緩慢的狀態而對於抗生素有很好的抵抗性，亦或生物膜內的細
菌表型(phenotypes)改變來保護薄膜等因素。同時生物膜會釋放出游離的細菌細胞
來持續感染其他上皮細胞表面，造成持續感染以及擴增新的薄膜 21。綠膿桿菌對
於大部份抗生素有抗藥性，能快速地產生抗藥性突變，而生物膜的抗藥機制讓現
今發展對於綠膿桿菌有效的抗生素療程更加侷限。
1.5.3.綠膿桿菌可溶性凝集素 I(PA-IL)的結構與特性
綠膿桿菌可溶性凝集素 I(PA-IL; gene LecA)，LecA 蛋白，是第一個被單離出
來的綠膿桿菌凝集素，其由 121 個胺基酸組成，為同源四聚體(homotetramers)結
構23。兩個碳水化合物結合位置的最短距離約略為 26 Å 。由 LecA 與半乳糖的錯
合物結晶結構可得知四聚體由四個 β-sandwich 摺疊單體結構組成，而 LecA 與半
乳糖的交互作用需要鈣離子的輔助，每一個和半乳糖結合的結合位置皆需橋接一
個鈣離子24，如圖九25所示。
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圖九、LecA 結構示意圖 25。
LecA 蛋白主要由綠膿桿菌群體感應(quorum sensing)系統控制下表現在細菌
外膜上，辨認醣鞘脂質以及群聚彼此來表達致病因子。LecA 對於治療上的挑戰
除了附著組織以及引發生物膜形成之外，Römer 教授團隊在 2014 年亦發現 LecA
對於上皮細胞表面 globotriaosylceramide(或稱 Gb3/CD77 抗原，亦為 Pk 血型抗
原)(圖十)有高度結合能力26，其結合會促進非吞噬型(nonphagocytic)細胞產生內化
(internalization)綠膿桿菌的現象，使綠膿桿菌侵入正常細胞27，對肺及膽的上皮細
胞造成損害。
圖十、Gb3Cer/CD77 抗原結構圖
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1.5.4.綠膿桿菌凝集素 LecA 抑制劑作為抗綠膿桿菌材料之研究
在抗生素廣泛使用的現代，綠膿桿菌對於越來越多的抗生素產生抗藥性，而
生物膜的形成更加局限治療的方法28。因為抗生素研究與開發不易，抗綠膿桿菌
化合物更是如此，所以開發新的抗綠膿桿菌方法是目前迫切所需的。細菌凝集素
對於醣基化目標的結合可以由單醣的競爭來抑制，如半乳糖、甘露糖、岩藻糖皆
被測試來限制細菌感染。在小鼠肺炎(murine pneumonia)細胞模型中利用半乳糖與
岩藻糖配合抗生素使用，可以有效的限制綠膿桿菌的擴散29。在綠膿桿菌附著人
類呼吸道上皮細胞實驗中，利用人類母乳寡醣可以顯著的抑制其附著30。因此藉
由合成新的抑制劑來競爭凝集素與醣基化目標的策略成為一種可以有效對抗綠
膿桿菌的方法，例如 Vidal 團隊在 2014 年發表的四價抑制劑，通過與 LecA 的結
合來抑制細菌凝集、生物膜形成以及黏著上皮細胞(圖十一)25。
圖十一、合成四價抑制劑抑制綠膿桿附著感染示意圖 25。
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LecA 除了對於天然的 globotriaosylceramide(Gb3/CD77 抗原 ) 以及
αGal(1,4)-Gal 雙醣苷的相關結構有良好的親和力之外，研究發現芳香性醣苷配基
(aromatic aglycones)在半乳糖與凝集素結合位置結合之餘，可以額外提供與 LecA
之 His50 殘基產生的 CH-π非共價作用力，同時特定芳香性醣苷配基結構會降低
更多 entropy penalty ，造成 LecA 對於化學合成的 β- 芳基半乳醣苷 (β-aryl
galactoside)有更好的結合能力(圖十二)31。
圖十二、β-芳基半乳醣苷對於 LecA 胺基酸殘基提供額外作用力 31。
抑制 LecA 來抵抗綠膿桿菌感染成為了一種可行且有效的方法，近年來越來越
多研究團隊開始研究對於結合 LecA 能力更好的抑制劑，且除了簡單的苯環結構
醣苷配基可以有效提高對於 LecA 的親和力之外，Reymond 教授團隊發現 2-萘基
(naphthyl)作為醣苷配基的 2-萘基 β-D-半乳醣苷(2-Naphthyl β-D-galactopyranoside)
(圖十三)8，其親和力相較具苯基(phenyl)醣苷配基半乳糖化合物提高一倍，相較
半乳糖提高 21 倍。這些對於 LecA 具有高結合能力的單價抑制劑研究，也同時增
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加多價性抑制劑的發展空間。
圖十三、對於 LecA 具有高親和力的 2-萘基 β-D-半乳醣苷 8。
1.5.5. 多價性綠膿桿菌生物膜抑制劑
LecA 為同源四聚體結構，具有四個結合位置，為了增強抑制劑與細胞表面
半乳糖殘基的競爭，近年來許多研究團隊針對四聚體短邊的兩個結合位置開發出
多價性結構的 LecA 抑制劑來抑制綠膿桿菌的附著以及生物膜形成。醣規則樹枝
狀分子(glycodendrimers)是近年來許多團隊採用的多價性材料之一，例如
Reymond 教授團隊在 2011 年第一個發表出四價 LecA 配體且成功抑制綠膿桿菌
生物膜，其多價性骨架(multivalent scaffold)即是採用樹枝狀(dendrimeric)胜肽骨架，
對於 LecA 的解離常數可以達到 100 nM，是一般半乳糖的 875 倍32。此後越來越
多種多價性骨架應勢發展，例如:Vidal 教授在 2013 年發表以環狀四聚胺基葡萄糖
(cyclic tetra-glucosamine)為核心所發展出來的半乳醣苷配體可以達到 79 nM 的解
離常數33。在 2014 年，Chevolot 與 Morvan 教授團隊以甘露糖為核心藉由磷雙酯
(phosphodiester)建構八價半乳醣苷配體(圖十四)34，在微陣列逆向酵素聯結親合分
析法實驗中(Microarray reverse enzyme-linked lectin assay)中，其與 LecA 之親和力
可達一般半乳糖的 1361 倍 34。醣規則樹枝狀分子因為透過高密度配基來提升和
16
受體結合的機率，且甚至可以進一步調控其幾何構形，近年來許多團隊都以規則
樹枝狀分子來作為多價性骨架，其與 LecA 結合的解離常數約為 50-100 nM8。
圖十四、 Chevolot 與 Morvan 教授團隊合成八價樹突狀分子 34。
醣規則樹枝狀分子大量被開發成為 LecA 的抑制劑，因其具有高價數，甚至可
以配合幾何位向設計以及距離調控。但大多數的醣規則樹枝狀分子其調控間距長
度的材料大多具伸縮性，缺乏剛性，在水溶液下容易摺疊，難以在兩個以上的碳
水化合物結合位置間維持長度35，造成大多醣規則樹枝狀分子鮮少能應用螯合作
用(Chelate effect)機制，大多是以其多數配基造成統計學上機率提升(Statistically
effect)機制，導致與 LecA 結合程度無法有效降低其自由能。如同多醣體聚合物
(glycopolymers)及醣奈米粒子(glyconanoparticles)，醣規則樹枝狀分子因其高密度
的配基，具有容易促使凝集素凝集(aggregation)的現象，降低治療上的表現36。相
反的，針對相鄰的結合位置所設計來符合蛋白質間距的寡架數小分子抑制劑對於
寡聚凝集素的抑制並不具有上述缺點，且可以達到足夠的親和力，像是 Bundle
17
教授在 2000 年發表的志賀毒素(Shiga toxin)抑制劑為針對相鄰的結合位置所設計
的小分子十價抑制劑，對於志賀毒素的解離常數可以達到低 nanomolar 範圍，為
最成功的例子之一37 。在 LecA 的小分子寡價(oligovalent)抑制劑研究中，像是
Pieters 教授團隊在 2012 年合成的兩價抑制劑 35，採用具有剛性結構的聚醣(glycan)
作為調控距離的間隔(spacer)材料，以一般的 β-半乳糖為配基，其抑制效果可以
達到 28 nM，為二價抑制劑目前所達到最好的效果。在理論計算的結晶模型中，
其骨架確實可以維持間距來和四聚體短邊的兩個結合位置結合，顯著提升半乳糖
的結合能力38(見圖十五)，為目前對於 LecA 的最佳結合力之二價抑制劑。
圖十五、(a) Pieters 教授團隊以聚糖作為 spacer 材料設計符合結合位置的二價抑
制劑。(b) 利用理論計算模擬與 LecA 結合之情況，其骨架確實可以維持結合間
距 38。
在 2014 年，Römer 教授與 Winssinger 教授研究團隊系統性地開發兩價 LecA
抑制劑，對於幾何構形以及間距(spacer)、配基種類來做具有全面性的檢測(圖十
18
六)39，其骨架採用肽核酸(peptide nucleic acid, 簡稱 PNA)，在微陣列(microarray)
實驗結果發現當 β-苯硫基半乳醣苷(β-thiophenyl galactoside)為配基且骨架符合
LecA 構形時，其解離常數可以達到 82 nM。為了有效利用配基與受體之結合來
增強多價性作用力，以符合蛋白質構形去設計可以固定構形之骨架以及配基為為
合理且有效的策略。
圖十六、Winssinger 研究團隊同時調控距離與半乳糖(galactose)配體來對 Lec A 結
合 39。
總結上述文獻研究，除了開發對於凝集素更具親和力的配基之外，利用具有
剛性結構材料作為骨架對於多價性研究而言不僅可以達到螯合作用 (Chelate
effect)機制，同時越具剛性的骨架或間距材料除了可以維持在生物體中的有效長
度外，更能有效率的利用配基結合。開發具剛性骨架的寡價數抑制劑為凝集素相
關研究提供非常具有潛力的契機。
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1.6.醣脂質對藥物載體奈米粒子的選擇性控制研究
1.6.1. RNAi 藥物以及肝相關疾病研究
近年來，許多慢性肝臟疾病盛行，成為全球性的醫療議題之一，像是 B 型肝
炎(hepatitis B virus)病毒感染，在全球有超過三億五千萬的帶原者40，造成每年超
過一百萬的死亡案例，而原因在於晚期階段的肝臟疾病通常伴隨嚴重併發症且缺
乏有效的治療手段。肝臟有超過 80%的細胞為實質肝細胞(hepatocyte)，其表面有
大量的運輸蛋白以及胞吞受體，多數肝臟疾病例如:A、B、C 型肝炎(hepatitis A, B
or C)以及非酒精性脂肪肝炎(alcohol-induced steatphepatitis)等疾病皆和肝細胞
(hepatocyte)的感染與病變有關，因此許多治療策略皆以肝細胞作為目標設計藉由
內化機制來吸收藥物41。
干擾性核醣核酸(RNA Interference; RNAi)為一種基因療法，藉由小分子 RNA
直接針對序列配對引發後轉錄修飾基因靜默(gene silencing)作為治療方法，對於
跳躍基因(transposons)以及病毒等侵入性遺傳因子，RNAi 可以有效的靜默(silence)
病變細胞中的特殊片段基因，直接或間接地影響病變細胞表現。近年來 RNAi 發
展迅速，針對肝臟部分的疾病例如:肝癌42、病毒感染43，特別是遺傳性疾病方面
的治療研究皆有相當良好的突破44，為肝病提供一個具有潛力的治療方法。不過
雖然 RNAi 療法在體外實驗研究上非常成功，但對於臨床療法與體內實驗而言尚
有許多障礙，其中包括:對於生物體內酵素的穩定性差、在血液循環半生期短，
無法針對目標組織或細胞，以及缺乏進入細胞的機制45，所以發展安全以及更有
20
效率的 RNAi 藥物輸送方法是目前 RNAi 藥物面臨的主要挑戰。
1.6.2. RNAi 藥物輸送奈米粒子載體種類與性質
鑒於 RNAi 在體內所遇到的問題，近年內發展出許多化學修飾方法或藥物傳
遞材料來增加 RNAi 藥物在體內輸送的穩定性以及在血液循環中的抗降解能力，
以及增加對於特定細胞或組織的選擇性。常見的化學修飾與材料來做為體內輸送
載體如圖十七46所示，例如:(1)對於易水解的 2’-羥基修飾為甲基等取代基增加在
體內的穩定性，或利用鎖核酸(Locked nucleic acid, LNA)作為 RNAi 材料，但通常
對於目標細胞的吸收效率低落。如圖十七(A)。 (2) 以病毒作為載體的方式輸送，
慢病毒屬(lentiviruses)以及腺病毒（Adenoviruses）等為常見 RNA 載體，可以藉
由基因操作修飾對於目標細胞受體具專一性的病毒衣殼(capsid)蛋白質以增加專
一性，如圖十七(B)。但因病毒較具免疫原性(immunogenic)易引發免疫反應，且
乘載的 RNAi 藥物相比非病毒載體較有限，侷限其發展。 (3)無機奈米粒子載體，
大多利用金、碳、矽材料組成載體，適合發展作為具生物可容性高且非免疫原性、
非毒性載體材料，其表面修飾具選擇性生物分子，例如癌症抗體，可以有效針對
癌細胞做結合以輸送 RNAi 藥物，如圖十七(C)。但其發展限制在載體缺乏核內體
脫離（endosome escape）機制，對 RNAi 藥物保護不足。 (4)聚合物載體，聚合
物材料例如聚乙烯亞胺 (polyethylenimine, PEI) 、聚乳酸聚甘醇酸
(poly(lactic-co-glycolic acid), PLGA)，此類型聚合物經多年研究大多具有生物相容
21
性、生物可降解以及安全等性質，以 PLGA 作為材料的聚合物為例，其奈米粒子
遵循核內體脫離（endosome escape）機制，再加上細胞內化粒子之後表面的相反
電荷的特性，使 RNAi 藥物得以釋放於細胞質47。此外，載體表面修飾聚乙二醇
（Polyethylene glycol, PEG）可以有效提高奈米粒子在血液循環中的時間以及滯
留在體內腫瘤細胞位置48，十分適合作為 RNAi 藥物載體，如圖十七(D)。 (5)脂
質體材料，利用形成雙層脂質，利用親水端結合 RNAi 藥物或是化療藥物。脂質
載體除了可以提升藥物在血液循環中的穩定性之外，陽離子脂質體的形成更能透
過細胞膜表面的副電荷增加細胞攝取能力，在對於肺癌組織細胞實驗有很好的抑
制效果49，如圖十七(E)。但雖然正電荷脂質體作為藥物輸送材料有助於細胞攝取，
但在體內常造成誘導 I 型及 II 型干擾素(interferon)產生免疫反應以及對於肝臟產
生毒性，限制脂質體載體的發展50，故近年來許多研究利用中性的脂質取代陽離
子型態脂質來降低其毒性，不過大多轉染(transfection)效率低下。Huang 教授團
隊在 2010 年發展出一種固態奈米脂質(Solid lipid nanoparticles, SLNs)利用聚合玻
尿酸 (Hyaluronic acid) 插入 DSPE
(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine)-PEG 作為陽離子脂質材料，可
以降低其毒性，如圖十七(F)。此外，在脂質體表面修飾可以針對癌症抗原的抗體
可以在體內實驗中有效傳遞 RNAi 藥物到小鼠肺部病變細胞，增加此類材料的發
展潛力51。
22
圖十七、各種 RNAi 修飾及奈米載體提升在體內穩定性 46。
Huang 教授團隊在 2012 年發展出新型材料作為固態奈米脂質內核，採用磷
酸鈣作為固態核心，磷酸鈣核心溶於酸性環境下，預期在胞吞後以增加核內體滲
透壓方式脫離核內體，釋放其封裝的 RNAi 藥物，而外層磷脂質採用 DSPE-PEG，
利用 PEG 的長鏈可以增加脂質體周圍的立體障礙，防止血液中的抗體辨識脂質
體，增加脂質體在體內的循環時間，同時藉由修飾具專一性配體於脂質體表面可
以增加對於目標細胞的選擇性，在生物體內實驗對於癌症細胞有良好的藥物傳遞
特性52。
1.6.3.藥物輸送奈米粒子材料對於肝病的研究
對於許多肝臟疾病，肝細胞(hepatocyte)不外乎是最為理想的基因療法目標，
23
主要由於藥物可以系統性地進入以及具有高度胞吞特性。不過除了肝細胞之外，
人體有百分之八十的巨噬細胞分布於肝臟，容易攝取外來的病原體以及大分子量
的治療藥物，導致許多藥物輸送系統喪失能力。而近十年來，肝臟疾病案例急遽
增加，主要因為缺乏有效治療藥物53。而 RNAi 藥物以及奈米載體的發展有助於
肝臟疾病的治療。雖然奈米載體的發展能夠提高 RNAi 藥物在血液循環的半生期
之外，未能有效針對特定細胞，尤其是肝臟做為藥物傳遞目標。所以提升肝細胞
對於載體的攝取是必須要面對的問題。
李遠川教授研究發現，哺乳類動物肝細胞表面凝集素，去唾液酸醣蛋白受體
(asialoglycoprotein receptor，ASGPR)，其具有碳水化合物結合位置之次單元
(subunit)彼此之間存在叢聚效應(cluster effect)形成多聚體，對於半乳糖或乙醯半
乳糖胺(N-Acetylgalactosamine, GalNAc)的 triantennary 及 tetraantennary 寡醣(示意
圖如圖十八所示54)具有高親和性，進而調控胞吞作用(endocytosis)55，而 ASGPR
為哺乳類動物肝細胞特有的受體，幾乎只表現在肝細胞表面，其他組織細胞缺乏，
故 ASGPR 對於藥物傳遞策略上為很好的目標。
24
圖十八、Bi, Tri, Tetra-antennary 聚醣結構示意圖 54。
為了提升肝細胞對於載體的攝取，許多團隊利用表面接合半乳糖56或是乙醯
半乳糖胺57配體的脂質體奈米載體成為有效的策略。
1.6.4. ASGPR 結構以及胞吞機制
ASGPR 為一種鈣離子補助(Ca2+-dependent)碳水化合物結合蛋白，主要分布
於肝細胞表面，主要功能為透過辨識、結合、胞吞以清除去唾液酸醣蛋白
(asialoglycoprotein)、凋亡細胞和脂蛋白等，維持血漿中醣蛋白的動態平衡。而
ASGPR 由兩種 homologous 次單元組成，分別為分子量 46 KDa 的 H1 單元及分
子量 50 KDa 的 H2 單元。兩種次單元在人類肝細胞表面以非共價作用力組成
ASGPR，組成比例大多為 H1:H2=2-5:1，較常為三聚體形式，比例為 H1:H2=2:1，
如圖十九58所示。
25
圖十九、ASGPR 於肝細胞表面結構組成示意圖 58。
雖然關於 ASGPR 的研究很多，其功能大多已被研究發現，但其結構除了 H1
蛋白質結構域(protein domain)已經透過結晶結構繞射得知外59，H2 次單元尚未被
完整了解，以至於缺乏整個寡聚凝集素三維結構資訊。對於想透過 ASGPR 作為
對象以傳遞藥物到肝細胞的策略而言是主要的障礙。
ASGPR 引導胞吞作用主要利用受體與配體先行結合，引發細胞膜出芽形成格
形蛋白(clathrin) 被膜小窩(coated pit)包覆 ASGPR 與配體所形成的複合物，藉此
發生內化作用。ASGPR 與半乳糖或乙醯半乳糖胺配體在 pH 6 環境下透過鈣離子
輔助結合，當進入細胞之後格形蛋白所形成的囊會被脫去，複合物則會再次被包
覆，形成核內體(endosome)，之後核內體與溶酶體(lysosome)融合，在 pH 為 5.4
的溶酶體環境下，鈣離子與 ASGPR 親和力下降，進而釋放配體蛋白與溶酶體作
26
用降解，而 ASGPR 則會經由一系列再循環核內體(recycling endosome)回到細胞
表面60。此機制有助於設計 pH 敏感奈米載體，例如 Huang 教授團隊設計的磷酸
鈣作為固態核心，在此胞吞機制下能夠正確地釋放藥物，對於肝病治療研究而言
為適合的材料 52。
1.6.5.醣分子配體對於 ASGPR 親和力研究
ASGPR 對於末端為半乳糖或乙醯半乳糖胺的去唾液酸糖蛋白
(asialoglycoprotein)的強結合能力，其主要原因為配體的多價性效應。與單一半乳
糖的親和能力約在 millimolar 範圍，但當配體價數提高，像是 biantennary、
triantennary、tetraantennary 去唾液酸醣蛋白，解離常數分別可以達到 10-6、5×10-9、
10-9 mM61。除了利用天然的 multiantennary 寡醣例如 arabinogalactan (結構如圖二
十所示62)之外，許多研究利用合成多價性半乳糖及乙醯半乳糖胺提高肝細胞的攝
取能力，例如 Ernst 教授團隊在 2008 年利用三價半乳糖及乙醯半乳糖胺配體提高
具 ASGPR 的肝腫瘤細胞 HepG2 細胞的攝取量 58。Seeberger 教授團隊在 2015 年
利用 α-螺旋(α-helix)coiled-coil 胜肽作為距離調控骨架以及利用不同長度的 PEG
做鏈接，設計符合人類 ASGPR 構形的三價半乳糖材料，可以更有效的讓 HepG2
細胞攝取 2。雖然大多研究對於提高親和力的方法為提高價數以及設計多價性骨
架以符合 ASGPR 結構，卻少有研究對於直接開發有效的單價配體。同時因為乙
醯半乳糖胺對於 ASGPR 親和力相比半乳糖高出 10-50 倍63，故單價配體研究多以
27
乙醯半乳糖胺作為材料開發新的配體，例如 Finn 與 Mascitti 教授團隊在 2012 年
合成一系列的乙醯半乳糖胺衍生物，在乙醯半乳糖胺不同的位置修飾官能基或保
護基。其最佳的解離常數可以達到 0.69 μM，對做為藥物載體修飾材料為很好的
選擇64。
圖二十、arabinogalactan 之聚醣結構 62。
開發高親和性的醣分子配體可以應用在修飾奈米載體表面以達到增加藥物
輸送至肝臟的能力之外，也能夠利用多價性骨架開發出更高親和性的材料，對於
ASGPR 結構及構形有進一步的了解的可能。
1.7. 研究動機
凝集素與醣體的作用與許多生理反應以及生物體間交互作用息息相關，例如
細菌凝集素對於宿主細胞表面的醣體辨認以及感染。半乳糖與許多重要的凝集素
具有一定程度的親和力，不過天然單價半乳糖對於凝集素作用力不高，對於凝集
素研究以及應用是最大的問題之一。而多價性交互作用與開發更具親和力之醣分
28
子皆為凝集素研究提供了解決的方法。因此本論文希望合成一系列的半乳醣苷配
基，配合多價性骨架針對特定凝集素作結合，期望解決凝集素在生物上之相關問
題。
第一部分希望設計並合成多種 LecA 抑制劑。綠膿桿菌在醫院內一直是嚴重
的併發感染原因之一，其本身對於大多數抗生素具有抗藥性。綠膿桿菌凝聚在體
內形成生物膜為更嚴重的衛生健康相關議題，其對於抗生素以及免疫系統的抵抗
性是目前臨床治療挑戰。藉由合成綠膿桿菌凝集素 LecA 抑制劑來防止綠膿桿菌
附著感染以及抑制生物膜形成在許多文獻中皆具有佐證，為相當合理的策略。針
對相鄰的結合位置所設計的寡價數骨架伴隨距離調控可以達到低 nanomolar 解離
常數，而脯胺酸多肽分子由於在水溶液中偏好以相對一般多肽更具剛性之 PPII
結構為主，為相當合適的距離調控材料。芳香性醣苷配基被證實可以有效提高對
於 LecA 親合能力，故本論文希望能開發結合能力更佳之芳香性半乳醣苷以及芳
香性半乳醣苷脯胺酸多肽化合物，期望結合並抑制 LecA，以及進一步測試芳香
性半乳醣苷脯胺酸多肽接合材料的生物膜抑制能力。
第二部分希望以合成半乳糖衍生物接合脂質化合物來增加固態奈米脂質藥
物載體對於肝臟輸送能力之研究。ASGPR 為肝細胞上特有的凝集素，對於半乳
糖殘基具結合能力，進而產生胞吞作用，可以做為載體選擇性結合之目標。近年
來許多研究將半乳糖直接修飾於脂質末端合成醣奈米粒子作為藥物載體，但半乳
糖親和力往往不足以明顯提升對於肝臟輸送能力。除了藉由合成多價半乳糖接合
29
以提升肝細胞選擇性之外，開發其他更具親和力半乳糖衍生物作為配體為一種更
有效率的方法。大多研究著重於乙醯半乳糖胺的修飾，尚未有研究以半乳糖為材
料進行修飾，雖然天然乙醯半乳糖胺對於 ASGPR 的結合能力較佳，但其合成以
及價格相對半乳糖而言較難以取得，且 ASGPR 結構尚未被完全解析，半乳糖依
然具有發展的潛力。故這部分實驗嘗試將具芳香性 aglycone 修飾於半乳糖
anomeric center，期望在結合位置之外產生新的非共價作用力或是降低結合產生
的 entropy penalty。最後將半乳糖衍生物接合至脂質材料上，修飾於 Huang 教授
52
團隊在 2012 年發展出的固態奈米脂質材料表面，期望可以更進一步提升載體
的藥物傳遞能力，增加肝細胞的攝取。而除了增加選擇性之外，可以藉由肝細胞
攝取實驗比較半乳糖衍生物之親和力，有益於日後多價性材料研究發展。
30
第二章、結果與討論
2.1.單價 LecA 抑制劑合成
天然的 globotriaosylceramide(Gb3Cer)為 LecA 在生物體中辨識的主要目標之
一，Gb3 對於 LecA 抑制劑研究而言是良好的材料，不過因為其化學合成方法繁
複，尤其是 1,2-cis 醣苷鍵建構不易，對於形成 Galα1,4-Lac 之醣基化(glycosylation)
反應選擇性低下，造成總體產率低下為最主要的問題。
近年來因許多研究發現 β-芳基半乳醣苷相對一般半乳糖對於 LecA 有更好的
結合能力，甚至有些芳香性半乳醣苷與 LecA 之結合能力 31 比報導之 Gb3 衍生物
的結合能力 20 更好。此外 β-芳基半乳醣苷材料合成方法較 Gb3 為簡單，非常適
合作為 LecA 抑制劑材料。
本部分實驗想開發以及探討作為 aglycone 之芳香環取代基結構的延長或修
改結構是否對於 LecA 之結合能力有所增加，期望芳香環共軛結構可以固定並符
合結合 LecA 之結構構形，以減少結合時所產生之 entropic penalty，最後和 Gb3
衍生物做比較。
2.1.1. β-芳基半乳醣苷之合成
鑒於 β-芳基半乳醣苷與 LecA 之高親和性，開發更有效的半乳醣苷為本論文
的目標之一。Roy 教授於 1994 年利用相轉催化(phase transfer catalyzed, PTC)反應
建構許多 β-O-aryl 及 β-S-aryl 醣苷65。PTC 反應之優點在於可以完全轉換 α-glycosyl
31
halides 之 anomeric 位向合成 β-醣苷建，可以有效率的合成多種 β-O-aryl 及 β-S-aryl
醣苷。本論文大部分 O-aryl 半乳醣苷主要利用 Roy 教授之 PTC 反應方法以及條
件，合成 β-芳基半乳醣苷來做為 LecA 抑制劑。
芳香性 aglycone 部分，其中具有酸基的芳香環化合物為了能夠進行 PTC 反
應，需先以芐基化(benzylation)保護酸基。芐基化 aglycone 酸基部分使用等當量
弱鹼拔去酸基的氫離子來活化酸基的親和能力進行芐基化保護，可以選擇性的將
芐基保護在酸基上(式一、二)，達到不影響 PTC 反應進行之目的。
(式一)
(式二)
式一、式二、芐基化保護芳香環上羧酸基以利 PTC 反應進行。
合成 β-芳基半乳醣苷部分，以天然半乳糖為起始物，以吡啶(pyridine)與醋酸
酐(acetic anhydride, Ac2O)進行全乙醯化反應，反應十二小時後以萃取移除吡啶與
醋酸酐，經管柱層析得到全乙醯保護之化合物 3。化合物 3 於冰浴下加入氫溴酸
反應一小時回到室溫再反應一小時，將一號位置置換為溴，因溴化後產物易水解，
故在萃取移除氫溴酸後，不經管柱層析直接置於真空乾燥後進行醣基化反應。將
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上述溴化產物作為予體，加入市售 O-aryl 化合物作為受體，硫酸氫四丁基銨
(Tetrabutylammonium hydrogen sulfate, TBAHS)作為相轉催化劑，將上述材料溶於
二氯甲烷(dichloromethane, CH2Cl2)後，加入 1 N 氫氧化納水溶液催化醣基化反應
進行，由 TLC 判定反應是否完成，當 glycosyl halides 消耗完後，經萃取及管柱
層析即可得到各種乙醯化之 β-芳基半乳醣苷，如流程一與表一所示。
流程一、合成乙醯化 β-芳基半乳醣苷反應路徑
表一、以 PTC 反應建構乙醯化 β-芳基半乳醣苷之 aglycone 結構與產率。
Yield
entry O-aryl acceptor Product
(for 2 steps)
1 49%
2 38%
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3 50%
4 43%
5 42%
6 31%
7 24%
8 37%
9 49%
10 45%
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除了表一所涵蓋的芳香環 aglycone 之外，期望能合成具更多共軛芳香環結構
以探討與 LecA 之結合能力影響，故將部分芳基半乳醣苷化合物利用耦合的方式
延長其共軛系統。將化合物 4 經過氫氣及 10% Pd/C 將硝基轉換為胺基之化合物
14 後，參考文獻作法66，於 140 °C 下與 2-氯-4 甲基喹啉(2-chloro-4-methylquinoline)
在乙醇溶液下迴流，經管柱層析後得到化合物 15，如流程二所示。
流程二、化合物 15 合成路徑圖
而具有碘苯基之化合物 6，可以利用耦合反應增加其共軛芳香環結構。利用
碘化銅以及 L-脯胺酸之螯合物作為催化劑將化合物 7 與 5-硝基吲哚(5-Nitroindole)
在二甲基亞碸(Dimethyl sulfoxide, DMSO)於 100°C 鹼性條件下進行 Ullmann
coupling。但由於在碳酸鉀之鹼性條件下進行，部分的半乳糖乙醯基保護會在此
條件下被去除，需要再經過一次乙醯化反應，得到化合物 16。隨後為了提升化合
物 16 去乙醯化(deacetylation)之後的水溶性能力以及應用性，將吲哚之硝基藉由
35
氫化反應得到化合物 17，如流程三所示。
流程三、化合物 17 之合成路徑
S-aryl 半乳醣苷在文獻上大多和 O-aryl 半乳醣苷與 LecA 之結合能力相似，
不過文獻上大多 aglycone 採用 2-萘硫酚(2-naphthalenethiol)及苯硫酚(thiophenol)，
鮮少採用雜原子芳香環。為了更進一步比較多種 β-芳基半乳醣苷之間的關係，將
2-巰基吡啶(2-Mercaptopyridine)作為 aglycone 合成 β-芳基半乳醣苷。其作法採用
文獻之合成方法67，將化合物 3 依照前述方法置換一號位置為溴，接著加入 2-巰
基吡啶溶於丙酮以及甲苯之溶液，利用碳酸鉀非勻相催化醣基化反應進行，隨後
經減壓濃縮及萃取，管柱層析後即得到化合物 18，如式三所示。
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式三、化合物 18 之合成
其中為了增加化合物 5 與化合物 8 之水溶性以及後續應用，須將芐基化保護
移除。利用氫化反應來移除芐基保護，得到化合物 19、20，如式四所示。
式四、氫化去除化合物 5 及 8 之芐基保護。
在合成及修飾完乙醯化 β-芳基半乳醣苷 7、9-15、17-20 後，利用甲醇鈉的甲
醇溶液進行乙醯基水解，得到化合物 21-32，如表二所示。
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表二、去乙醯化之 β-芳基半乳醣苷結構與產率。
Acetylated
entry Product Yield
glycan
Quant.
1
yield
2 83%
3 82%
4 64%
5 81%
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6 91%
7 98%
8 92%
9 68%
Quant.
10
yield
11 59%
Quant.
12
yield
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完成乙醯基水解之後，將所合成之 β-芳基半乳醣苷(化合物 21-32)以 10%
DMSO 水溶液配製為 1mM 濃度，儲存於-20°C 以便日後與 LecA 之相關生物試驗
使用。
2.1.2. Gb3 醣體之合成
為了探討 β-芳基半乳醣苷與天然配體 Gb3 之差異，實驗部分想利用 Gb3 作
為對照，比較 β-芳基半乳醣苷之效果。而 Gb3 之衍生物價格昂貴，為了能在後
續實驗以及生物測試使用，需要合成 Gb3 以供日後使用。
本部分實驗將 Gb3 之合成分為非還原端(non-reducing end)之半乳糖為醣予體
合成，以及還原端(reducing end)之乳糖(lactose)醣受體部分，藉由醣基化反應建
構 αGal(1,4)-Lactose 之醣苷鍵。半乳糖醣予體合成部分，為了提高醣基化之活性
以及建構 1,2-cis 之醣苷鍵，採用芐基保護較酯類保護適合，離去基方面則採用甲
硫酚(thiocresol)作為離去基，具高穩定性以及高反應性之優點，為廣泛使用之離
去基。首先將前述之化合物 3 與甲硫酚以三氟化硼-乙醚錯合物(Boron trifluoride
diethyl etherate, BF3·OEt2)於無水之二氯甲烷進行 Helferich 醣基化反應，合成 β 位
向之化合物 33，隨後以甲醇鈉之甲醇溶液，於無水條件下進行乙醯基去保護，經
過中和與乾燥後，利用氫化鈉(sodium hydride)以及溴化芐(benzyl bromide)於二甲
基甲醯胺(dimethylformamide)溶液加入四丁基碘化銨(Tetrabutylammonium iodide,
TBAI)催化下進行全芐基化保護，經萃取與管柱層析後得到化合物 34，如流程四
40
所示。
流程四、醣予體 34 之合成
醣受體合成部分，如流程五所示，以市售之乳糖為起始物，以前述全乙醯化
反應之方法利用吡啶與醋酸酐進行全乙醯化，得到全乙醯保護之化合物 35，隨後
利用氫溴酸於冰浴下反應一小時，將一號為置置換為溴，因溴化後產物溶液水解，
故僅萃取移除氫溴酸之後，不經管柱層析即進行醣基化反應，加入芐基-2-羥基乙
胺甲酸酯(benzyl 2-hydroxyethyl carbamate)作為受體，上述溴化產物為予體，氰化
汞(Mercury(II) Cyanide, Hg(CN)2)，作為 promotor，於無水乙腈(acetonitrile, MeCN)
在 70°C 反應兩小時，之後在室溫下反應八小時，生成化合物 36，於氫核磁共振
光譜之結果，於化學為移 4.40 ppm 為新生成之 C-1 位置之氫，其耦合常數為 7.9
Hz，確認為 β 位向。將上述產物利用甲醇鈉之甲醇溶液水解乙醯基保護之後，於
非還原端半乳糖之四號及六號位置的羥基利用苯甲醛二甲醇縮醛(benzaldehyde
dimethyl acetal)經樟腦磺酸(Camphorsulfonic acid, CSA)催化在乙腈溶液下形成
benzylidene acetal 保護，隨後不經管柱層析，直接中和與抽乾，和溴化芐於二甲
基甲醯胺進行芐基化保護，得到化合物 37。而後為了得到非還原端半乳糖之四號
未保護之羥基來進行醣基化反應，採用酸催化還原之方式來進行選擇性開環反應。
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依照文獻之方法68，利用三乙基矽烷(triethylsilane)在三氟醋酸(trifluoroacetic acid)
酸性條件下，於冰浴下反應三小時，萃取及管柱層析後即可得到化合物 38，可借
助 1H-1H COSY 光譜驗證，由化學位移在 4.00 ppm 之 4’-H 與 2.39 ppm 之羥基的
耦合現象判斷(圖二十一)。
流程五、醣受體 38 之合成。
42
圖二十一、化合物 38 之 1H-1H COSY 光譜展開圖，化學位移在 4.00 ppm 之
4’-H 與 2.39 ppm 之羥基的耦合現象判斷羥基位於半乳糖四號位置。
酸催化還原開環反應為常見之選擇性還原 benzylidene acetal 方法之一，其機
構如圖二十二69所示，因為 6 號所受環上拉電子能力影響較弱，造成 6 號位形成
oxonium ion 之中間物較 4 號形成 oxonium ion 之中間物穩定，造成此中間體被矽
烷還原之後於 C-6 位置形成芐基，得到在 C-4 位置選擇性開環之產物。
圖二十二、選擇性還原開環之反應機構 69。
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合成 Gb3 衍生物如流程六所示，醣基化反應參考文獻之反應條件70將醣予體
34 及醣受體 38 以及經過活化之 AW-300 分子篩先於無水二氯甲烷溶液中反應 30
分鐘後，置於-45°C 下反應十分鐘，依序加入 N-碘代丁二酰亞胺(N-iodosuccinimide,
NIS)做為活化劑及三氟甲磺酸三甲基矽酯(Trimethylsilyl trifluoromethanesulfonate,
TMSOTf)提供酸性條件以進行醣基化反應，反應三小時後經萃取及管柱層析，其
α-位向產物單離之產率為 67.4%。隨後利用 10% Pd/C 以及氫氣進行氫化反應去
除芐基保護以及苄氧羰基(Carboxybenzyl)保護，此部分反應須由氫核磁共振光譜
來判定是否有將苯環區的訊號去除。最後藉由重氮轉換反應(Diazotransfer)71將胺
基轉換為疊氮基，在 Zn2+催化下與 TfN3 反應後經由逆相 C-18 矽膠管柱純化獲得
化合物 40。
流程六、Gb3 衍生物化合物 40 之合成
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2.1.3. 以微陣列(Microarray)篩檢 LecA 與抑制劑之結合能力
為了進行快速篩檢單價配體之效果，本部分實驗採用微陣列(Microarray)作為
分析工具。微陣列為一種將反應物附著於矽晶片等載體上的技術，其優點在於所
需反應試劑的量極少化，且能夠同時快速篩檢大量化合物。
在微陣列的應用中，若要將反應的主體連接至玻璃等載體上，其表面須經過
官能基化的處理，並在欲連接至微陣列上的主體做相對應的官能基修飾，以市面
上有在販售的塗布玻片為例：利用以 N-Hydroxysuccinimide (NHS)基團活化之酯
類與-NH2 反應形成醯胺鍵而連接；或將表面表現為環氧乙烷(Epoxide)，並與親
合性基團行開環加成反應，如圖二十三72。
圖二十三、市售之塗布玻片修飾方法示意圖 72。
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微陣列(Microarray)實驗由實驗室開發之實驗系統所測試，由實驗室相關人員
林則學同學操作。操作上，首先先將市售之 LecA 蛋白溶於以 1 mg/ mL 之濃度
溶於 PBS 後，以 Lumiprobe 公司所建議之標準方法，將 Cy3-NHS 對 LecA 進行
標記。完成反應與經過透析後，我們利用超微量分光光度計(nanodrop)測量每個
蛋白質所被標記 Cy3 之程度，經過計算平均為 2 Cy3/ LecA。隨後我們在塗布 NHS
的玻片上(Schott Nexterion slide H)以 50 μM 濃度之單價半乳糖分子以及三價半乳
糖樹狀分子 Gal3-dendrimer 利用微陣列點陣平台列印於玻片上，將單價半乳糖分
子以及三價半乳糖樹狀分子修飾於玻片表面來與單價配體抑制劑競爭對於 LecA
的結合，求得抑制劑與 LecA 之相對結合能力。在醯胺鍵耦合反應完成後，我們
將玻片經過乙醇胺去活化未反應之 NHS 基團以及清洗，最後在玻片上分割出不
同化合物之反應區塊，將預混合之不同濃度抑制劑以及 Cy3 標記之 LecA 溶液置
於玻片表面作用一小時後來與半乳糖樹狀分子競爭結合(LecA 濃度為 17 μg/ mL)。
隨後經過清洗以及陰乾，經過螢光掃描後，我們藉由被玻片上競爭結合之 LecA
的螢光強度可以得知抑制劑的相對結合能力。
2.1.4.微陣列試驗結果與討論
文獻上過去已合成出許多對於 LecA 有良好結合能力的芳香基半乳醣苷來作
為單價抑制劑 31，在許多試驗中相較於天然半乳糖皆有較好的 LecA 結合能力。
在 microarray 篩檢試驗中，經過嘗試多種 microarray 條件的最佳化之後，我們以
46
250 μM 之芳基半乳醣苷濃度進行微陣列篩檢，結果如圖二十四所示，圖中螢光
強度越高表示被玻片上的半乳糖樹狀分子與單價抑制劑競爭結合 LecA 越多，表
示抑制劑能力越差。結果顯示雖然大部分所合成的芳香基半乳醣苷不如預期有比
文獻報導過之化合物 21 來對於 LecA 有更好的結合能力，但像我們仍然可以發現
許多趨勢存在。例如我們將對位取代之化合物 21 改為間位羧酸基取代，其結合
能力可以再次提升，但將甲基多取代於苯環上會影響其原本作用力導致親和力下
降，在 microarray 篩檢中對於 LecA 並沒有結合的現象產生，其螢光強度與未加
入任何半乳糖衍生物分子之控制組相似。而將文獻已知有良好作用力的 2-萘酚改
為 1-萘酚結構會導致親和力下降，而將兩個苯環以兩個原子分隔，我們發現對位
取代的芐基對提升親和力並沒有幫助，反而造成結合能力得下降，此外雜原子芳
香環取代之化合物 29 其與 LecA 之結合能力相對於相似苯環結構化合物 26 有良
好的結合能力，此部分有助於進一步開發 LecA 抑制劑。而更多共軛環結構之化
合物 30-32 很可惜的並沒有符合原本之設計目的，藉由增加芳香環結構來減少結
合時產生的熵變化來做為提升親和力之手段，此部分結果需要更多相關結構化合
物才能定論。總體而言，在這部分實驗中合成的部分芳香基半乳醣苷對於 LecA
之結合能力具有不錯的結果，此外雖然某些化合物對於 LecA 結合能力並不理想，
但在後續實驗部份我們依然可以利用這些結構合成寡價數抑制材料來測試其在
多價性骨架上之表現。
47
圖二十四、不同單價抑制劑利用微陣列所篩檢結果，以相對螢光強度為縱軸作
圖。
2.2.特定間距之二價醣肽 LecA 抑制劑合成
由於許多凝集素包括 LecA 在生物體中皆以多聚體方式存在，而針對多聚體
相鄰的結合位置所設計的寡價數骨架伴隨距離調控可以達到低 nanomolar 解離常
數，所以這部分實驗設計採用脯胺酸多肽作為骨架，藉由其在水溶液中之結構特
性，合成特定距離來同時對於 LecA 四聚體短邊兩個結合位置結合之二價抑制劑，
而配基部分則採用 β-芳基半乳醣苷作為材料，期望能開發出更有效之綠膿桿菌抑
制劑。
2.2.1.醣肽材料合成之策略
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為了達到將脯胺酸多肽做為控制醣配基距離之骨架，必須將醣配基與多肽或
是胺基酸進行連接，此部分需要準確且方便的方法。透過一價銅催化之疊氮與炔
類的[3+2]環化加成(Azide-alkyne Huisgen cycloaddition)，亦稱 CuAAC，為許多研
究上廣泛應用的方法，其可以讓分別擁有疊氮以及炔基之兩分子進行環化加成，
形成三唑環(triazole ring)，例如: Brimble 教授團隊除了利用 CuAAC 建構 N-乙醯
葡糖糖胺(N-Acetylglucosamine)為核心的多價醣體之外，更分別以疊氮以及末端
缺修飾之醣分子與 MUC1 胜肽序列配對，合成癌症細胞表面過度表現之 MUC1
片段的醣肽衍生物，可以應用於癌症疫苗的研究(圖二十五)73。
圖二十五、Brimble 教授團隊利用 CuAAC 合成 MUC1 片段醣肽衍生物 73。
本部分實驗設計想利用疊氮-炔類[3+2]環化加成反應(CuAAC)來作為醣肽材
料之合成，將脯胺酸多肽分子的特定位置修飾上末端炔官能基，以便和具有疊氮
基(azido group)之醣分子配基接合。
49
2.2.2.多肽固相合成脯胺酸多肽骨架
LecA 四聚體結構之相鄰結合位置距離約為 26 Å ，而脯胺酸多肽分子若以
PPII 構形作為骨架，相隔每三個醯胺鍵結構會旋轉一圈，回到相同位置，軸向距
離為 9.4Å，設計相隔三個脯胺酸距離倍數之骨架來符合凝集素結合位置之間距以
及探討親和力強弱差別為本論文想探究的目標之一。多肽骨架採用固相多肽合成
法(Solid phase peptide synthesis, SPPS)合成特定位置具末端炔之脯胺酸多肽。構築
單元除了利用市售之 Fmoc-L-proline 外，本實驗利用
Fmoc-trans-4-propargyloxy-L-proline 在胜肽序列中的特定位置來控制醣基化位置，
以合成具特定間距之抑制劑。
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流程七、兩個丙炔基(propargyl group)修飾之特定距離脯胺酸多肽合成
固相合成步驟可分為四部分:
1. 去保護(deprotection):利用 20%哌啶於 DMF 溶液，將市售樹脂上之 Fmoc 保護
基或是胺基酸構築單元之 Fmoc 保護基去除，得到末端為具親核性的胺基狀態。
2. 耦合(coupling):以 HBTU, HOBt, DIPEA 作為耦合試劑，以 DMF 為溶劑，將胺

51
基酸構築單元作為反應物，依欲合成之多肽序列依照順序採用不同胺基酸構築單
元進行耦合。
3. 去活化(capping): 在耦合反應中，雖然試劑及構築單元皆以高當量進行反應，
但依然具有未反應之胺基存在的可能性。未反應之胺基在後續耦合反應中仍然會
繼續反應，延續其胺基酸序列。為了避免其繼續反應造成後續分離問題，對於在
耦合反應中未反應完全的胺基則利用 50%乙酐於吡啶溶液進行乙醯化來避免其
繼續反應造成後續純化問題。
4. 切除(cleavage):當胜肽鏈合成結束後，必須將其與樹脂分開以獲得目標產物。
依照使用樹脂不同所使用的切除方法也不盡相同，在此部分實驗，我們利用三氟
醋酸(trifluoroacetic acid, TFA)以及三異丙基矽烷(Triisopropylsilane, TIS) 作為還
原劑以及水，以 TFA:TIS:H2O=38:1:1，來切除樹脂上的胜肽鏈，並將切除之溶液
以減壓蒸餾移除過多的酸，之後以高效能液相層析儀(High Performance Liquid
Chromatography, HPLC)分離。
依實驗設計需求，在此部分實驗依序合成間隔 3、6、9、12 醯胺鍵之丙炔基
(propargyl group)修飾之脯胺酸多肽 41、42、43、44，如流程七所示。
52
2.2.3.具疊氮基之芳香性半乳醣苷合成
為了將第一部分合成之半乳醣苷利用 CuAAC 連接在具末端炔之脯胺酸多肽，
需要將芳香性半乳醣苷上修飾疊氮基。為了快速得到多種具疊氮基之芳香性半乳
糖衍生物，此部分實驗採用進行醯胺鍵形成(amide bond formation)的方式來進行
具疊氮基之芳香性半乳醣苷合成，以化合物 9、14、17、19、20 為適合修飾之目
標，因其芳香環上皆有酸基或是胺基取代，適合利用化學修飾來增加其後續應
用。
化合物 9、14、17、19、20 分別與 6-azidohexanoic acid 或
2-(2-azidoethoxy)ethanamine 耦合形成醯胺鍵，經過萃取以及純化得到化合物
45-49，最後利用甲醇鈉的甲醇溶液去除乙醯保護，得到化合物 50-54，如流程八
所示，最後為了方便後續實驗進行，將化合物 50-54 配製為 100 mM 之 DMSO 溶
液，於-20°C 保存。
53
流程八、具疊氮基之芳香性半乳醣苷化合物 50-54 合成。
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2.2.4.疊氮-炔類的[3+2]環化加成
為了利用具末端炔修飾之多肽骨架 41-44 及具疊氮基醣分子化合物 40、50-54
進行銅催化疊氮-炔類的[3+2]環化加成來合成目標醣肽化合物。將化合物 40、
50-54 分別以特定當量數加入多肽骨架 41-44 配成之水溶液，再加入一價銅試劑
催化進行 CuAAC 反應，得到化合物 55-75，詳見表三，反應式如式五所示，另
外因為 Gb3 合成步驟繁複，故僅以最理想距離之多肽骨架 43 來合成 Gb3 衍生醣
肽化物來與其餘醣肽化合物比較。
式五、脯胺酸多肽骨架與 LecA 配體進行疊氮-炔類的[3+2]環化加成環化加成
55
Azidoglycan:
Propargylpeptide:
表三、化合物 55-75 之產率以及結構
Azidoglycan 40 50 51 52 53 54
propargylpeptide
55 56 57 58 59
41 ND[a] (86%) (89%) (87%) (83%) (70%)
60 61 62 63 64
[a]
42 ND (89%) (85%) (91%) (64%) (61%)
65 66 67 68 69 70
43 (98%) (82%) (90%) (96%) (71%) (64%)
71 72 73 74 75
[a]
44 ND (91%) (92%) (88%) (79%) (68%)
[a]:因實驗設計未需要此種組合之醣肽材料，以 ND (not determined)代表未進
行此組合之實驗。
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2.2.5.圓二色光譜結構分析
為了確認骨架之 PPII 結構以及探討芳基半乳醣苷連接上骨架之前後是否有
PPII 結構上的改變。將所有合成的胜肽以 Far-UV 範圍內的波長測量圓二色
(Circular dichroism, CD)光譜。圓二色光譜因為容易使用且靈敏度高，在蛋白質以
及 DNA 分子結構鑑定上為常使用的研究工具，特別是在二級結構變化上為合適
的測量工具。原二色光譜屬於吸收光譜，藉由光譜儀產生之平面偏極光通過蛋白
質、胜肽等具有光學活性之結構物質時，會因為光學物質與偏極光之電磁場作用
產生穿透率不同，造成偏極光出入射光之角度偏轉，藉由此特性可以來測量具有
光學活性的分子結構。
為了比較 CuAAC 前後結構之差異，所以我們分別測量脯胺酸多肽骨架(41-44)
與進行 CuAAC 後的醣肽分子(55-75)以進行比較。首先脯胺酸多肽骨架圓二色光
譜結果如圖二十六所示，所有的脯胺酸多肽皆在水溶液下以 100 μM 濃度，攝氏
25 度下測量。結果發現在 228 nm 波長範圍附近皆會有一最大正值的訊號，同時
在 200-210 nm 範圍皆有一最小負值訊號，與 PPII 結構的特徵訊號相符，表示測
定的胜肽在水溶液中皆為 PPII 結構。
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圖二十六、脯胺酸多肽骨架(41-44)之 CD 光譜結果
而醣肽化合物(55-75)之 CD 光譜部分，我們採用相隔兩個脯胺酸循環間距之
骨架之醣肽化合物(60-64)來比較之間差異，如圖二十七所示。同樣發現在 228 nm
波長範圍附近皆會有一最大正值的訊號，同時在 200-210 nm 範圍皆有一最小負
值訊號，與 PPII 結構的特徵訊號大致相符，來確認醣肽分子(55-75)依然維持在
水溶液中的 PPII 結構。
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圖二十七、醣肽化合物(60-64)與同長度之脯胺酸多肽骨架 42 之 CD 比較圖。註[a]:
因在純水中溶解度不佳，故此光譜實驗於 90 %甲醇水溶液下測量。
除此之外，以 Gb3 衍生物為配基之二價醣肽化合物 65 同樣為了確認 Gb3 衍
生物與脯胺酸多肽骨架 43 的連接並沒有改變 PPII 結構，我們同樣將 65 進行 CD
光譜之結構鑑定，其結果如圖二十八所示，依然和前述結果相符，仍然維持 PPII
構形。
圖二十八、醣肽化合物 65 與相同長度骨架 43 CD 光譜。
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2.2.6. 微陣列試驗之結果與討論
為了得知合成出來之二價醣肽抑制劑與 LecA 的結合能力比較，利用微陣列
篩檢來得到其相對強度。以 25 μM 濃度之抑制劑採用前述方法進行微陣列篩檢實
驗。我們將結果分別以配基結構分類，骨架由短至常依序排列，結果如圖二十九
所示。結果可以發現，當骨架長度為三個脯胺酸多肽循環時，每種芳香性半乳醣
苷衍生物皆會出現最好的 LecA 結合能力，與當初實驗設計的結果相符合。此外，
除了以 26 做為配基的醣肽化合物 58、63、69、74 之外，在三個脯胺酸多肽循環
距離骨架下，其餘配基對於 LecA 結合能力皆高於 Gb3 衍生物之醣肽化合物 65，
特別是化合物 68，在掃瞄結果下幾乎沒有殘留螢光於玻片上，有相當好的結合效
果。另外我們發現在單價抑制劑篩檢實驗中，結合能力不明顯的化合物 32，在經
過骨架上後修飾後，也能達到好的結合效果，顯示化合物 32 在脯胺酸多肽骨架
上可以更有效的與 LecA 結合。以上結果顯示脯胺酸多肽骨架對於 LecA 而言為
適合之骨架材料，在間距控制為適當距離時可以達到最佳化之多價性交互作用
力。
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圖二十九、不同抑制劑利用微陣列所篩檢結果，以相對螢光強度為縱軸作圖。
表四、微陣列篩檢之化合物結構與名稱對照表。註[a]:未進行此組合之實驗
Scaffold (turns) 41 (1) 42 (2) 43 (3) 44 (4) Monovalent
Ligand
55 60 66 71
56 61 67 72
57 62 68 73
58 63 69 74
59 64 70 75
- [a] - [a] 65 - [a]
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初步篩檢結果顯示醣肽化合物在三個脯胺酸多肽循環間距下有最好之親和
力結果，為了得到在此間距骨架中各種芳香性半乳醣苷配基對於 LecA 之相對的
結合能力指標，我們將此距離骨架所合成之化合物 65-70 分別以多種濃度於微陣
列試驗中篩檢，將螢光強度與濃度作圖，得到滴定曲線(titration curve)來求得 IC50
之數值(圖三十，表五)。在 IC50 比較下，66、67、68、70 在此微陣列試驗下數值
皆十分相近，皆在 1 μM 附近，與上述之結果相符合。而效果最好的為以化合物
32 做為配基的化合物 70，為本實驗中所設計與合成之最佳二價抑制劑，雖然在
單價配體比較中並無發現其對於 LecA 之作用力有明顯之提升，但在多價性骨架
上卻可以達到最佳結合能力，此方面原因可能需要與蛋白質結合之相關結構分析
來進一步驗證需才能定論。除此之外，在微陣列篩檢中結合能力明顯較弱的化合
物 69，其 IC50 數值皆高於 66、67、68、70 許多，此部分也與前述篩檢結果相符
合。而化合物 65 可能因為 IC50 遠高於其他化合物，以致無法在此濃度區間求得
IC50。
圖三十、醣肽化合物 65-70 在不同濃度下所求得之滴定曲線。
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表五、65-70 在微陣列測試中所測得之 IC50 數值。
Bivalent Inhibitor 65 66 67 68 69 70
IC50 value (μM) ND* 1.261 1.124 1.427 153.5 1.033
註*:因為在此微陣列篩檢中無法得到其滴定曲線，故無法得知其 IC50 數值。
綜合以上所得到之結果，雖然在單價抑制劑篩檢中單價配基的效果並不如預
期般有明顯之提升，但藉由開發出針對 LecA 之相鄰結合位置的二價抑制劑以及
距離調控骨架，以骨架之 PPII 構形來維持在生物試驗中所達到的有效長度以增
加多價性效應，在顯示結果中有大幅度之提升，特別是醣肽化合物 70 為本實驗
中所得到最佳之二價抑制劑，相對於其配基而言有最為明顯之提升。除此之外，
因為微陣列試驗篩檢只能快速得到抑制劑結合能力之強弱差異，尚且無法得知其
對於抗生物膜之能力，缺乏應用方面數據，此部分實驗日後會加以測試以了解其
應用性。
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2.3.半乳糖衍生物修飾奈米藥物載體表面對於肝細胞之研究
因為目前文獻中少有利用芳香環化合物作為 aglycone 之半乳醣苷作為
ASGPR 之配體，而開發具更高親和力之配體來進行肝臟相關之研究非常具有潛
力。本部分實驗藉由合成半乳糖衍生物之脂質材料修飾於奈米粒子表面以期待利
用與 ASGPR 結合所產生之胞吞作用增進奈米藥物載體被肝細胞攝取之能力。
2.3.1.醣修飾脂質材料合成之策略
為了使用芳基半乳醣苷來做為配體增加奈米粒子之攝取能力，將第一節所合
成出的多種芳基半乳醣苷中挑選出多種可以進行耦合反應來增加應用性之化合
物 9、14、17、19、20，利用 CuAAC 來將芳基半乳醣苷接合至奈米粒子表面脂
質材料以達到修飾奈米粒子表面之目的。同時為了探討芳基半乳醣苷是否較一般
半乳糖配體之結合能力好，可以藉由合成半乳糖接合脂質化合物所修飾奈米粒子
來作為對照。
本實驗與清華大學生物醫學工程研究所陳韻晶教授實驗室合作，以市售之疊
氮基磷脂質化合物 76(DSPE-PEG(2000) Azide)(圖三十一)來作為修飾目標，欲藉
由合成具炔丙基(propargyl)之半乳醣衍生物以 CuAAC 反應接合。
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圖三十一、市售之疊氮基磷脂質結構(化合物 76)
2.3.2.具丙炔基之半乳糖衍生物之合成
分別將化合物 9、14、17、19、20 與 4-戊炔酸(pentynoic acid)或丙炔胺進耦
合反應形成醯胺鍵，經過萃取以及純化得到化合物 77-81，最後利用甲醇鈉的甲
醇溶液去除乙醯保護，得到化合物 82-86，如流程九所示。
65
流程九、化合物 82-86 之合成
66
同時為了比較芳基半乳醣苷與一般半乳醣苷之差異，需藉由合成具丙炔基之
半乳糖化合物來做為對照組。以化合物 3 為起始物，先試以三氟化硼-乙醚錯合
物(Boron trifluoride diethyl etherate, BF3·OEt2)為醣活化劑(activator)與炔丙醇
(propargyl alcohol)進行醣基化反應，但結果除了 α、β 選擇性不佳之外，反應性低
落與反應結果混亂為最大的問題，故改採用反應性更佳之三氯亞醯胺酯基
(trichloroacetimidate)官能基作為離去基，以酸催化醣基化反應之方式作為策略。
首先，先將化合物 3 以苯甲胺(benzylamine)去除一號位之乙醯保護，經管柱層析
後可以得到一號位置為保護之化合物 87，再以 1,1,1-三氯乙腈
(1,1,1-Trichloroacetonitrile)以及有機鹼 DBU 催化形成三氯亞醯胺酯基，經快速管
柱層析之後得到化合物 88，因三氯亞醯胺酯基之高度反應性，並不穩定，故經管
柱層析後隨即減壓去除溶劑後直接進行醣基化反應，以炔丙醇做為予體，在無水
環境下利用三氟甲磺酸三甲基矽酯進行反應，可得單一 β 位向之化合物 89。最後
利用甲醇鈉的甲醇溶液去除乙醯保護，得到目標產物 90，如流程十。
流程十、化合物 90 之合成。
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2.3.3.半乳糖衍生物接合脂質化合物之合成
將所合成之具末端炔化合物 90、82-86 與市售之疊氮基脂質化合物 76 以
CuAAC 進行加成反應可以得到目標之半乳糖衍生物接合脂質化合物 91-95，如式
六所示，之後經由逆相 C-8 固相萃取管柱(solid phase extraction column)將產物純
化分離，經過凍乾(lyophilization)，並保存於-20°C 以供後續實驗使用。
式六、以 CuAAC 合成半乳糖衍生物接合脂質化合物 91-96。
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除了藉由 MALDI-TOF 來確認是否成功在脂質材料 76 利用 CuAAC 來連接末
端炔化合物 90、82-86，我們可以於氫核磁共振光譜之結果加以確認，以化合物
92 為例，雖然大部分半乳糖的特徵訊號被聚乙二醇的訊號掩蓋，不過苯環區之對
苯酚訊號之化學位移以及耦合常數皆與起始化合物 82 位置大致符合，而 triazole
ring 之訊號於化學位移 7.60 之單重峰，積分為一個氫。
2.3.4. 肝細胞對於奈米粒子攝入實驗
ASGPR 為肝細胞表面已知和胞吞作用有關之特有受體。因此為了探討不同
β-芳基半乳醣苷所修飾之脂質材料是否能增加奈米藥物載體被肝細胞所攝取之
能力，我們藉由以不同半乳糖衍生物修飾奈米載體粒子表面，並利用奈米載體粒
子乘載具螢光標記之 siRNA，混入培養液培養後，經共聚焦顯微鏡可以藉由細胞
內螢光強度判定攝取能力差別。
實驗採用人類肝癌細胞株Hep3B作為目標細胞，Hep3B為已知具有較多
ASGPR表現之細胞之一74。此細胞實驗以及奈米粒子載體由清華大學生物醫學工
程研究所陳韻晶教授實驗室陳冠臻同學所製備，主要以文獻中的製備方式加以修
改所製備。奈米粒子載體(NPs)內層由DOPA、NaHPO4 、CaCl2 以及螢光標記之
siRNA所組成，外層為DOTPA、DSPE-PEG2000與cholesterol 以及半乳糖衍生物
接合脂質化合物91-96（莫耳比1:0.99:2:0.01）混合形成的脂質層，合成半乳糖衍
生物修飾NPs以進行後續細胞實驗。且為了加以比較未修飾之NPs所造成之差異，
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我們藉由合成不具有修飾之NPs來作為對照組。
操作上，先將 Hep3B 細胞 (105 / 1 mL / well) 培養在12-well plates 上培養，
並將內含有5’-FAM螢光標定之siRNA並且表面修飾不同半乳糖衍生物之NPs
(0.15 nmole)加入以含有1 mL serum 培養液培養的細胞中，於37°C培養4小時後，
以1× PBS清洗後以4% PFA (in 1× PBS)固定細胞，經由複染上DAPI 後將利用共
聚焦顯微鏡進行拍攝與統計計算。共聚焦顯微鏡之拍攝圖(圖三十二)，其中藉由
DAPI對於細胞核染色之藍色螢光來得知細胞數量，藉由siRNA所標記之綠光螢光
相對強度，我們可以知道NPs攝入的程度。
圖三十二、共聚焦顯微鏡拍攝圖之結果。
為了進一步比較每一組 NPs 所造成的差異，以圖像處理軟體統計強度以及計
算分析，將每一組共聚焦顯微鏡拍攝圖中的 FAM 之螢光強度(綠光)除以 DAPI
所造成之螢光強度(藍光)，即可得到相對 NPs 被攝入之情形。最後以未修飾任何
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醣體之 NP(PEG)數據為基準，以長條圖方式作圖(圖三十三)，得到相對於表面未
修飾醣體之差異程度變化。
圖三十三、Hep3B 對於不同修飾之 NPs 的相對攝取量長條圖。
2.3.5. NPs 攝入結果與討論
對於增加 RNAi 藥物進入肝細胞能力的提升，目前文獻大多採用乙醯胺基半
乳糖或是一般半乳糖來作為配基，藉由與 ASGPR 結合進而促進胞吞作用，少有
以直接在半乳糖一號位接上 O-aryl 形式的官能基做為策略來開發對於 ASGPR 更
具親和力的配體。本次實驗將各種合成之配體 91-96 修飾 NPs 表面以進行 Hep3B
細胞攝入實驗，比較未修飾或經一般半乳糖修飾之 NPs 之結果。結果顯示，在一
號位置接上對位取代之苯酚結構，如 92 及 96，其被 Hep3B 所攝入量為沒有半乳
糖修飾之 NPs 的將近十倍。除此之外，我們發現若將苯酚結構以間位(meta-)官能
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基取代，如 93 是以間位取代酸基以銜接脂質材料 DSPE-PEG(2000)，會開始減弱
NP 之被攝取能力，推測因為 ASGPR 對於半乳糖配體之結合能力下降造成降低胞
吞作用。此外，若將苯酚之芳香結構改為萘酚作為 aglycone 會發現 NPs 被攝取
之能力大幅下降，甚至低於一般半乳糖修飾 NPs 數值，其數值與位經任何修飾之
NPs 相似，推測過於龐大的芳香環結構，特別是稠合芳香環(fused aromatic ring)，
不僅沒有增加額外對於 ASGPR 的親合能力，甚至會進一步影響半乳糖與碳水化
合物結合位置的結合能力。
總結以上結果，合成特定芳基半乳醣苷修飾在 NPs 表面對於含有 ASGPR 之
肝癌細胞攝取 NPs 之能力可以有效提升。此外，雖然乙醯胺基半乳醣因為對於
ASGPR 本身就有比較好的結合能力(為半乳糖之 50 倍)，本身非常適合來做為增
加載體粒子的修飾，但因為由半乳糖為起始物之合成步驟繁瑣，而乙醯胺基半乳
醣市售價格昂貴，開發芳基半乳醣苷相對而言為較好的選擇之一，以結果而言，
雖然並沒有大幅度提升 NPs 被細胞攝取之能力，但一號位經過對位苯酚取代之半
乳糖相對於大部分文獻所採用的半乳糖修飾已經增加許多肝細胞攝取之能力，當
取代基位置改變或增加環數量皆可能會降低 ASGPR 之親和力，但保留對位苯酚
結構並在對位取代吲哚並沒有太大影響。就發現結果而言，單一對位苯環取代的
結構對於肝細胞攝取能力是有所提升的，具有發展相關結構化合物作為配基之潛
力。
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第三章、實驗方法與材料
3.1. Material and methods

General: All chemicals were purchased as reagent grade and without further
purification. Ethyl acetate (EtOAc) and hexane were distilled before column
chromatography. Dichloromethane (CH2Cl2) and acetonitrile (CH3CN) were distilled
over calcium hydride. Tetrahydrofuran (THF) was extra-dry grade stored with
molecular sieve purchase from Acros. Anhydrous N,N-dimethylformamide(DMF) was
purchase from JT Baker. Molecular sieve for glycosylation were AW-300(Aldrich) and
activated by flame and microwave. Reaction were monitored with analytical thin layer
chromatography(TLC) on E. Merck silica gel 60 F254 plate(0.25 mm) and visualized
under UV(254 nm) and staining with p-anisaldehyde, ceric ammonium molybdate,
ninhydrin or potassium permanganate. Column chromatography was carried out on
Silica Gel 60 (E. Merck). High resolution ESI-TOF mass spectra were measured on a
Bruker–Impact HD, EVOQ. Matrix-assisted laser desorption ionization-time of flight.
(MALDI-TOF) mass spectra were measured on a Bruker Daltonics, Autoflex III
smartbeam LRF200-CID. Reverse-phase HPLC was performed on Agilent
technologies 1260 Infinity. Peptides were purified on a VyDac C18 (25 cm x 10 mm),
column operating at a flow rate of 3 mL min-1 using a mobile phase of 0.1% TFA in
H2O and acetonitrile. All reactions were carried out in oven-dried glassware under
atmosphere of argon. NMR spectra were recorded on a Mercury 400 or Bruker AV-400
and these are recorded in parts per million (δ ppm) units. For 1H NMR spectra,
chemical shifts are expressed in ppm from CDCl3 (7.24 ppm) or CD3OD (3.31 ppm)
or D2O (4.80 ppm) or (CD3)2CO (2.04 ppm) or (CD3)2SO (2.50 ppm). Coupling
constant (J) are reported in hertz (Hz). s, singlet; d, doublet; t, triplet; q, quartet; m,
73
multiplet.1H NMR spectra were reported in this order: chemical shifts; number of
proton; multiplicity; coupling constant(s). Mass spectra were obtained ESI mode.
3.2. Synthetic procedures and characterization

General procedure for the glycosyl bromide synthesis and PTC (phase transfer
catalyzed) glycosylation (protocol A): The acetylated glycoside(compound 4 )
dissolved in 33% HBr / acetic acid solution at 0 °C in iced bath stirred for 30 minute.
Then removed iced bath, stirred for 1.5 hours. Upon completion of reaction, the
reaction were quenched by iced water and diluted with EtOAc, the resulting solution
was washed with saturated NaHCO3(aq) (three times), brine, dried over Na2SO4,
filtered, evaporated to dryness, then evaporated in vacuum for 1 hour obtained
glycosyl bromide as colorless oil.
After one hour in vacuum, the resulting glycosyl bromide, 1.1 eq.
tetrabutylammoniumhydrogensulfate(TBAHS) and 2 eq. aromatic aglycones were
dissolved in CH2Cl2, then 1M NaOH(aq) added and stirred vigorously for few hours.
When completion of reaction, the mixture were diluted with EtOAc, then washed with
1 M NaOH(aq) 3 times, H2O ,brine, dried over Na2SO4. The residue was purified
using silica gel chromatography (EtOAc/Hexane system) to yield desired product.
General procedure for hydrogenation (protocol B): starting material were dissolved
or suspended in methanol with 10% v/v formic acid, then 10% Pd/C was added to.
The mixture were evaporated in vacuum few seconds and back-filled with hydrogen
for 3-5 times. Then stirred under atmosphere of hydrogen. After few hours, removed
the hydrogen balloon and dilute with methanol, solid were filtered through a pad of
Celite. The residue was purified using flash column.
General procedure for deacetylation (protocol C): Acetylated starting material were
74
dissolved in methanol, then added 30 % NaOMe in Methanol solution, stirred at 0 °C.
Upon completion of reaction, the reaction were neutralized with Dowex 50x resin,
after filtration removal of resin and removal of solvent by rotary evaporator to give
deacetylated product.
General procedure for linker coupling to glycan (protocol D): The compounds
with amino or carboxylic acid group (9、14、17、19、20) and azido or terminal alkynyl
linker were dissolved in CH2Cl2 stirred at 0 °C, then DIEA
(N,N-Diisopropylethylamine) and HATU was added sequentially and stirred overnight.
Upon completion of reaction, the mixture was diluted with CH2Cl2 and washed with
saturated NaHCO3(aq), water, and brine. After dried over Na2SO4, filtered, then
purified by silica gel chromatography to furnish conjugated compound.
3-Benzyloxycarbonylphenol (1)75. 3-Carboxyphenol (1.00 g, 77.24 mmol) and
Na2CO3 (0.78 g, 7.24 mmol) were dissolved in 10 mL anhydrous DMF and stirred for
5 minutes, then benzyl bromide (0.86 mL, 7.24 mmol) was added at 0 °C stirred for
another 5 minutes and allowed to brought to r.t. Upon the completion of reaction, iced
H2O was added to the mixture and diluted with ether, then the organic layer was
extracted with H2O 5 times, brine, then dried over Na2SO4, filtered, concentrated and
purified by silica gel chromatography (EtOAc/hexane = 1:6 to 1:3) to furnish 1. (1.76
g, 82%). 1H NMR (400 MHz, CDCl3): δ =7.76 (s, 1H), 7.67 (s, 1H), 7.60 (dd, J = 7.8,
0.9 Hz, 1H), 7.35-7.24 (m, 5H), 7.20 (t, J = 7.9 Hz, 1H), 7.09-7.06 (m, 1H), 5.28 (s,
2H).
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Benzyl-6-hydroxy-2-naphthoate (2)34. 6-Hydroxy-2-naphthoic acid (0.20 g,
1.06 mmol) in 2 mL aqueous methanol 90%, Cs2CO3 (0.17 g, 0.53 mmol) was added.
The solution was stirred at room temperature for 30 min, then remove solvent at
reduced pressure and co-evaporated with toluene. The resulting cesium salt was
suspended in 1 mL anhydrous DMF, cooled to 0 °C and benzyl bromide (0.13 mL,
1.06 mmol) was added. After 1 h stirring, the solution was warm up to room
temperature and stirring for further 10 h before the solvent was removed under
reduced pressure. The residue was taken up into water and then extracted with EtOAc
and the combined organic layers were dried over Na2SO4 and the solvent was removed
under reduced pressure. Purified by silica gel chromatography (EtOAc/hexane = 1:10
to 1:3) to furnish 2. (0.25 g, 84%). 1H NMR (400 MHz, CDCl3): δ =8.56 (s, 1H), 8.02
(d, J = 8.6, 1H), 7.81 (d, J = 8.5, 1H), 7.64 (dd, J = 8.6, 1.6 Hz, 1H), 7.49-7.48 (m,
2H), 7.42-7.33 (m, 3H), 7.18-8.14 (m, 2H), 5.43(s, 2H).
Penta-O-acetyl-D-galactopyranose (3)76.D-Galactose (19.60 g, 108.80 mmol)
was dissolved in 150 mL pyridine, and then acetic anhydride 150 mL was added in ice
bath, after 10 minutes ice bath was removed. The resulting mixture were stirred under
a argon atmosphere at r.t. for 15h. The reaction was quenched by MeOH in ice bath.
The solvent were removed under reduced pressure. The residue was diluted with
EtOAc (150 mL) and washed with saturated NaHCO3(aq), water, and brine. Dried
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over Na2SO4, filtered, then purified by silica gel chromatography (EtOAc/hexane =
1:4 to 1:1) to yield 3 (43.92 g, 89%). 1H NMR (400 MHz, CDCl3): δ =6.31 (d, J = 3.7
Hz, 1H, H-1), 5.46 (app. t, J = 9.9 Hz, 1H, H-4), 5.15-5.06 (m, 2H, H-2, H-3), 4.25
(dd, J = 12.9, 4.3 Hz, 1H, H-6a), 4.11-4.06 (m, 2H, H-5, H-6b), 2.16, 2.08, 2.01, 2.00
(5s, 3H×5, Ac).
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O-(4-Nitrophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (4) .
According protocol A. Compound 3 (2.00 g, 5.12 mmol) was dissolved in 4 mL 33%
HBr / acetic acid for bromination. After bromination procedure, the glycosyl bromide
were dissolved in 25 mL CH2Cl2 with TBAHS (1.74 g, 5.12 mmol) and nitrophenol
(0.926 g, 6.66 mmol), and then 8 mL 1M NaOH(aq) added. After PTC glycosylation
procedure and extraction in protocol A, the crude were purified by silica gel
chromatography (EtOAc/hexane/CH2Cl2 = 1:5:0.6 to 1:2:0.3) to furnish compound 4
(1.17 g, 49%). [𝛼]30

D -7.9 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 8.20-8.17
1
(m, 2H), 7.07-7.05 (m, 2H), 5.50 (dd, 1H, H-2, J = 10.4, 7.9 Hz), 5.45 (app. d, 1H,
H-4, J = 3.3 Hz), 5.15 (d, 1H, H-1, J = 7.9 Hz), 5.12 (dd, 1H, H-3, J = 10.4, 3.3 Hz),
4.22-4.09 (m, 3H, H-6a, H-6b, H-5), 2.17,2.05,2.00 (3s, 3H×4, Ac); HRMS (ESI): calc.
for C20H23NO12Na [M + Na] +, 492.1112, found 492.1112.
O-(3-Benzyloxycarbonylphenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranosid
77
e (5). According protocol A. Compound 3 (1.72 g, 4.41 mmol) was dissolved in 4 mL
33% HBr / acetic acid for bromination. After bromination procedure, the glycosyl
bromide were dissolved in 20 mL CH2Cl2 with TBAHS (2.50 g, 4.41 mmol) and
compound 1 (1.36 g, 5.96 mmol), and then 7 mL 1M NaOH(aq) added. After PTC
glycosylation procedure and extraction in protocol A, the crude were purified by silica
gel chromatography (EtOAc/hexane/CH2Cl2 = 1:4:0.5 to 1:2:0.3) to furnish compound
5 (0.94 g, 38%). [𝛼]31

D -45.0 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ=
1
7.78-7.76 (m, 1H), 7.68-7.67 (m, 1H), 7.45-7.30 (m, 6H), 7.18 (ddd, 1H, J = 8.2 Hz,
2.6 Hz, 0.9 Hz), 5.48 (dd, 1H, J = 10.2, 7.9 Hz, H-2), 5.44 (app. d, 1H, J = 3.4 Hz,
H-4), 5.34 (d, 2H, J = 1.2 Hz), 5.10 (dd, 1H, J = 10.3, 3.4 Hz, H-3), 5.08 (d, 1H, J =
7.9 Hz, H-1), 4.15 (d, 2H, J = 6.5 Hz, H-6), 4.06 (app. t, J = 6.4 Hz, H-5), 2.16, 2.04,
2.02, 1.99 (4s, 3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.4, 170.2, 170.1, 169.3,
165.7, 156.8, 135.8, 131.7, 129.6, 128.6, 128.3, 128.2, 124.5, 122.0, 117.6, 99.6, 77.2,
71.2, 70.8, 68.5, 66.9, 61.4, 20.7, 20.6(2C), 20.54; HRMS (ESI): calc. for
C28H30O12Na [M + Na] +, 581.1629, found 581.1638.
O-(4-Iodophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (6)78. According
protocol A. Compound 3 (1.00 g, 2.56 mmol) was dissolved in 2 mL 33% HBr /
acetic acid for bromination. After bromination procedure, the glycosyl bromide were
dissolved in 12 mL CH2Cl2 with TBAHS (0.87 g, 2.56 mmol) and iodophenol (0.85 g,
3.84 mmol), and then 4.5 mL 1M NaOH(aq) was added. After PTC glycosylation
procedure and extraction in protocol A, the crude were purified by silica gel

78
(0.70 g, 50%). [𝛼]26
D +7.4 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 7.58-7.55
1
(m, 2H), 6.77-6.74 (m, 2H), 5.45 (dd, 1H, J = 10.4, 7.9 Hz, H-2), 5.42 (d, 1H, J = 3.0
Hz, H-4), 5.07 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.97 (d, 1H, J = 7.9 Hz, H-1),
4.21-4.06 (m, 2H, H-6a, H-6b), 4.03 (app. t, 1H, J = 6.6 Hz, H-5), 2.15, 2.04, 1.98 (3s,
3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1, 169.3, 156.7, 138.4,
119.1, 99.4, 86.1, 71.0, 70.7, 68.4, 66.7, 61.3, 20.7, 20.6, 20.6, 20.6; HRMS (ESI):
calc. for C20H23O10INa [M + Na] +, 573.0228, found 573.0232.
O-(2,3,6-Trimethylphenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (7).
According protocol A. Compound 3 (135 mg, 0.334 mmol) was dissolved in 0.5 mL
bromide were dissolved in 1.5 mL CH2Cl2 with TBAHS (117 mg, 0.345 mmol) and
2,3,6-trimethylphenol (88 mg, 0.629 mmol), and then 0.5 mL 1M NaOH(aq) added.
After PTC glycosylation procedure and extraction in protocol A, the crude was
purified by silica gel chromatography (EtOAc/hexane = 1:4 to 1:2) to furnish
compound 7 (67.2 mg, 43%). [𝛼]30 1

D -11.5 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
δ= 6.89-6.84 (m, 2H), 5.50 (dd, 1H, J = 10.5, 8.0 Hz, H-2), 5.37 (d, 1H, J = 3.3 Hz,
H-4), 5.06 (dd, 1H, J = 10.5, 3.5 Hz, H-3), 4.74 (d, 1H, J = 8.0 Hz, H-1), 4.10-4.02 (m,
2H, H-6a, H-6b), 3.75 (app. t, 1H, J = 6.9 Hz, H-5), 2.22, 2.20, 2.15 (3s, 3H×3), 2.19,
2.11, 1.99, 1.93 (4s, 3H×4, Ac); HRMS (ESI): calc. for C23H30O10Na [M + Na] +,
489.1731, found 489.1746.
79
Benzyl-4’-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthoate
(8)34. According protocol A. Compound 3 (1.30 g, 3.34 mmol) was dissolved in 3 mL
bromide were dissolved in 17 mL CH2Cl2 with TBAHS (1.25 g, 3.67 mmol) and
compound 2 (1.30 g, 4.69 mmol), and then 6 mL 1M NaOH(aq) added. After PTC
glycosylation procedure and extraction in protocol A, the crude was purified by silica
gel chromatography (EtOAc/hexane = 1:4 to 1:2) to furnish compound 8 (0.85 g,
42.0%). [𝛼]31
D +11.2 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 8.56 (s, 1H),
1
8.05 (dd, 1H, J = 8.6, 1.5 Hz), 7,85 (d, 1H, J = 9.0 Hz), 7.73 (d, 1H, J = 8.6 Hz), 7.46
(d, 1H, J = 7.0 Hz), 7.39-7.31 (m, 4H), 7.22 (dd, 1H, J = 9.0, 2.4 Hz), 5.54 (dd, 1H, J
= 7.9, 10.4 Hz, H-2), 5.48 (d, 1H, J = 3.3 Hz, H-4), 5.38 (s, 2H), 5.23 (d, 1H, J = 7.9
Hz, H-1), 5.15 (dd, 1H, J = 10.4, 3.3 Hz, H-3), 4.24-4.14 (m, 3H, H-5, H-6a, H-6b),
2.17, 2.05, 2.04, 2.00 (3H×4, Ac); 13

C NMR (100 MHz, CDCl3): δ= 170.2, 170.1,
167.0, 169.3, 166.4, 156.3, 136.5, 136.0, 131.2, 130.9, 128,9, 128.5, 128.2, 128.2,
127.1, 126.2, 126.1, 119.6, 110.8, 99.1, 71.2, 70.7, 68.5, 66.8, 66.7, 61.5, 20.6, 20.6,
20.5, 20.5; HRMS (ESI): calc. for C32H32O12Na [M + Na] +, 631.1786, found
631.1789.
O-(5-Aminonaphthyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (9).
80
According protocol A. Compound 3 (2.00 g, 5.13 mmol) was dissolved in 4 mL 33%
HBr / acetic acid for bromination. After bromination procedure, the glycosyl bromide
were dissolved in 25 mL CH2Cl2 with TBAHS (1.25 g, 3.67 mmol) and
5-amino-1-naphthol (1.23 g, 7.70 mmol), and then 8 mL 1M NaOH(aq) added. After
PTC glycosylation procedure and extraction in protocol A, the crude was purified by
silica gel chromatography (EtOAc/hexane/CH2Cl2 = 1:4:0.5 to 1:1:0) to furnish
compound 9 (0.77 g, 31%). [𝛼]25 1

δ= 7.51 (app. t, 2H, J = 8.7 Hz), 7.29 (t, 1H, J = 8.0 Hz), 7.24 (t, 1H, J = 8.0 Hz), 7.02
(d, 1H, J = 7.6 Hz), 6.75 (d, 1H, J = 7.2 Hz), 5.66 (dd, 1H, J = 10.4, 8.0 Hz), 5.47 (d,
1H, J = 3.1 Hz), 5.14 (dd, 1H, J = 10.5, 3.4 Hz), 4.26-4.06 (m, 4H, H-6a, H-5), 2.15,
2.04, 2.01, 2.00 (3H×4, Ac); 13

C NMR (100 MHz, CDCl3): δ= 170.2, 170.2, 170.0,
169.6, 153.2, 141.9, 126.5, 126.3, 124.5, 124.2, 115.8, 112.1, 110.3, 109.0, 99.9, 70.9,
70.6, 68.4, 66.8, 61.3, 20.7, 20.5(3C); HRMS (ESI): calc. for C24H28NO10 [M + H] +,
490.1708, found 490.1711.
79
O-(4-Biphenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (10) .
According protocol A. Compound 3 (242 mg, 0.621 mmol) was dissolved in 0.5 mL
bromide were dissolved in 3 mL CH2Cl2 with TBAHS (232 mg, 0.683 mmol) and
4-Phenylphenol (159 mg, 0.931 mmol), and then 1 mL 1M NaOH(aq) added. After
PTC glycosylation procedure and extraction in protocol A, the crude was purified by
silica gel chromatography (EtOAc/hexane = 1:4 to 1:2) to furnish compound 10 (75
81
mg, 24.2%). [𝛼]30
D +12.7 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 7.53-7.48
1
(m, 4H), 7.40 (app. t, 2H, J = 7.5 Hz), 7.33-7.29 (m, 1H), 7.07-7.04 (m, 2H), 5.50 (dd,
1H, J = 10.4, 8.0 Hz, H-2), 5.45 (d, 1H, J = 3.6 Hz, H-4), 5.11 (dd, 1H, J = 10.4, 3.4
Hz, H-3), 5.07 (d, 1H, J = 8.0 Hz), 4.26-4.21 (m, 1H, H-6a), 4.18-4.12 (m, 1H, H-6b),
13
4.10-4.05 (m, 1H, H-5), 2.17, 2.07, 2.05, 2.00 (4s, 3H×4, Ac); C NMR (100 MHz,
CDCl3): δ= 170.3, 170.2, 170.1, 169.4, 156.4, 140.4, 136.4, 128.7, 128.2, 127.0, 126.8,
117.1, 99.6, 71.0, 70.8, 68.6, 66.9, 61.3, 20.7, 20.6(2C), 20.53; HRMS (ESI): calc. for
C26H28O10Na [M + Na] +, 523.1575, found 523.1565.
O-(4-(Benzyloxy)phenoxy)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside
(11)79. According protocol A. Compound 3 (121 mg, 0.310 mmol) was dissolved in
0.5 mL 33% HBr / acetic acid for bromination. After bromination procedure, the
glycosyl bromide were dissolved in 1.5 mL CH2Cl2 with TBAHS (108 mg, 0.318
mmol) and 4-Benzyloxyphenol (115 mg, 0.579 mmol), and then 0.5 mL 1M NaOH(aq)
added. After PTC glycosylation procedure and extraction in protocol A, the crude was
compound 11 (62 mg, 37%). [𝛼]30 1

D +5.4 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3):
δ= 7.41-7.28 (m, 5H), 6.95-6.84 (m, 4H), 5.43 (dd, 1H, J = 10.4, 8.0 Hz, H-2), 5.42
(dd, 1H, J = 3.8, 0.7 Hz, H-4), 5.06 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 5.01 (s, 2H), 4.90
(d, 1H, J = 8.0 Hz, H-1), 4.23-4.11 (m, 2H, H-6a, H-6b), 3.98 (td, 1H, J = 6.7, 1.0 Hz,
H-5), 2.16, 2.06, 2.02, 1.99 (4s, 3H×4, Ac); HRMS (ESI): calc. for C27H30O11Na [M +
Na] +, 553.1680, found 553.1693.
82
O-(8-Quinolinyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (12).
According protocol A. Compound 3 (267 mg, 0.684 mmol) was dissolved in 1 mL
8-quinolinol (144 mg, 1.030 mmol), and then 1 mL 1M NaOH(aq) added. After PTC
glycosylation procedure and extraction in protocol A, the crude was purified by silica
gel chromatography (EtOAc/hexane = 1:4 to 1:1) to furnish compound 12 (158 mg,
49%). [𝛼]30
D -49.5 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 8.89 (dd, 1H, J =
1
4.2, 1.7 Hz), 8.12 (dd, 1H, J = 8.3, 1.7 Hz), 7.55 (dd, 1H, J = 6.9, 2.6 Hz), 7.45-7.38
(m, 3H), 5.69 (dd, 1H, J = 10.5, 8.0 H, H-2), 5.45 (dd, 1H, J = 3.4, 1.0 Hz, H-4), 5.34
(d, 1H, J = 8.0 Hz, H-1), 5.15 (dd, 1H, J = 10.1, 3.4 Hz, H-3), 4.25-4.14 (m, 2H, H-6a,
13
H-6b), 4.02 (td, 1H, J = 6.7, 0.9 Hz, H-5), 2.17, 2.08, 2.01, 2.00 (4s, 3H×4, Ac); C
NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.2, 169.7, 152.7, 149.8, 141.0, 135.7,
129.5, 126.2, 123.3, 121.5, 116.7, 101.2, 71.0, 70.8, 68.8, 66.9, 61.3, 20.9, 20.6,
20.6(2C); HRMS (ESI): calc. for C23H25NO10Na [M + Na] +, 498.1371, found
498.1378.
O-(2-(6-Benzoyl)naphthalenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside
83
(13). According protocol A. Compound 3 (218 mg, 0.560 mmol) was dissolved in 1
mL 33% HBr / acetic acid for bromination. After bromination procedure, the glycosyl
6-Benzoyl-2-naphthol (201 mg, 0.810 mmol), and then 1 mL 1M NaOH(aq) added .
After PTC glycosylation procedure and extraction in protocol A, the crude was
compound 13 (144 mg, 45%). [𝛼]31 1

δ= 8.20 (s, 1H), 7.93 (dd, 1H, J = 8.5, 1.6 Hz), 7,85-7.79 (m, 4H), 7.59 (tt, 1H, J = 7.4,
1.9 Hz), 7.51-7.79 (m, 2H), 7,38 (d, 1H, J = 2.3 Hz), 7.24 (dd, 1H, J = 8.9, 2.4 Hz),
5.55 (dd, 1H, J = 10.4, 7.9 Hz, H-2), 5.48 (d, 1H, J = 3.0 Hz, H-4), 5.24 (d, 1H, J =
7.9 Hz, H-1), 5.15 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.28-4.12 (m, 3H, H-6a, H-6b,
H-5), 2.18, 2.06, 2.05, 2.01 (4s, 3H×4, Ac); HRMS (ESI): calc. for C31H30O11Na [M +
Na] +, 601.1680, found 601.1687.
80
O-(4-Aminophenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (14) .
According protocol B. Compound 4 (1.17 g, 2.50 mmol) was dissolved in 20 mL
methano, then 100 mg 10% Pd/C was added for hydrogenation. After hydrogenation
procedure in protocol B, the crude was purified by silica gel chromatography
(EtOAc/hexane = 1:3 to 1:1) to furnish compound 14 (0.96 g, 87%). [𝛼]30

D +5.7 (c 1.0,
CHCl3); 1H NMR (400 MHz, CDCl3): δ= 6.8-6.77 (m, 2H), 6.57-6.54 (m, 2H),
5.41-5.36 (m, 2H, H-2, H-4), 5.03 (dd, 1H, J = 7.1, 3.1 Hz, H-3), 4.82 (d, 1H, J = 8 Hz,
H-1), 4.2-4.04 (m, 2H, H-6a, H-6b), 3.94 (app. t, 1H, J = 6.7 Hz, H-5), 2.12, 2.04,
84
2.00, 1.96 (4s, 3H×4, Ac); HRMS (ESI): calc. for C20H25NO10Na [M + Na] +, 462.1371,
found 462.1375.
O-(4-((4-Methylquinolin-2-yl)amino)phenoxy)-2,3,4,6-tetra-O-acetyl-β-D-gal
actopyranoside (15). Compound 14 (200 mg, 0.293 mmol) and
2-chloro-4-methylquinoline (102 mg, 0.570 mmol) were dissolved in 5 mL EtOH,
refluxed under argon for 12h. Upon completion of reaction, the mixture was
evaporated, then purified by silica gel chromatography (EtOAc/hexane = 1:4 to 1:1) to
furnish compound 15 (248 mg, 89%). [𝛼]30 1

D +8.7 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ= 7.74 (dd, 2H, J = 15.5, 8.2 Hz), 7.55-7.51 (m, 1H), 7.46 (d, 2H, J = 8.8
Hz), 7.29-7.24 (m, 1H), 6.97 (d, 2H, J = 8.9 Hz), 6.71 (s, 1H), 5.46 (dd, 1H, J = 10.4,
8.0 Hz, H-2), 5.42 (d, 1H, J = 3.2 Hz, H-4), 5.09 (dd, 1H, J = 10.5, 3.4 Hz, H-3), 4.95
(d, 1H, J = 7.9 Hz, H-1), 4.23-4.10 ( 2H, H-6a, H-6b), 3.99 (app. t, 1H, J = 6.5 Hz,
H-5), 2.53 (s, 3H), 2.15, 2.07, 2.02, 1.99 (4s, 3H×4, Ac). 13C NMR (100 MHz, CDCl3):
170.3, 170.2, 170.1, 169.3, 154.3, 152.9, 146.8, 146.0, 135.7, 129.6, 126.4, 124.1,
123.6, 122.8, 122.5, 118.0, 111.5, 100.2, 70.9, 70.8, 68.6, 66,8, 61.2, 20.7, 20.6(2C),
20.5, 18.9; HRMS (ESI): calc. for C30H33N2O10Na [M + Na] +, 581.2130, found
581.2143.
85
O-(4-(5-Nitro-1H-indol-1-yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyran
oside (16). Compound 6 (440 mg, 0.80 mmol), 5-nitroindole (156 mg, 0.96 mmol),
CuI (46 mg, 0.24 mmol), K2CO3 (276 mg, 2.00 mmol) and L-proline (55 mg, 0.48
mmol) were premixed in vacuum for 1h. Then degassed DMSO (4 mL) was added
under argon stream and heated to 100 °C stirred for 8hr. the reaction mixture was
cooled to r.t., diluted with EtOAc (20 mL), and filtered through a plug of Celite. The
filtrate was concentrated in vacuum. The residue was dissolved in 3 mL pyridine, then
2 mL acetic anhydride was added with catalytic amount DMAP
(4-Dimethylaminopyridine), stirred for 2 h. The reaction was quenched by MeOH in
ice bath. After concentrated in reduced pressure, the residue was purified by silica gel
chromatography (EtOAc/hexane = 1:4 to 1:1) to give compound 16 (331 mg,
71%). [𝛼]28
D +4.7 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 8.61 (d, 1H, J =
1
2.2 Hz), 8.08 (dd, 1H, J = 9.2, 2.2 Hz), 7.42-7.37 (m, 4H), 7.19-7.15 (m, 2H), 6.82 (dd,
1H, J = 3.4, 0.5 Hz), 5.52 (dd, 1H, J = 10.4, 7.9 Hz, H-2), 5.47 (dd, 1H, J = 3.4, 0.6
Hz, H-4), 5.14 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 5.11 (d, 1H, J = 7.9 Hz, H-1),
4.27-4.23 (m, 1H, H-6a), 4.18-4.14 (m, 1H, H-6b), 4.11-4.08 (m, 1H, H-5), 2.19, 2.09,
2.05, 2.01 (4s, 3H×4, Ac). 13C NMR (100 MHz, CDCl3): δ= 170.2, 170.1, 170.0, 169.3,
155.9, 141.9, 138.7, 133.6, 131.3, 128.1, 126.0, 118.1, 118.0, 117.7, 110.1, 105.3, 99.3,
C28H28N2O12Na [M + Na] +, 607.1534, found 607.1535.
86
O-(4-(5-Amino-1H-indol-1-yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyra
noside (17). According protocol B. Compound 16 (235 mg, 0.402 mmol) was
dissolved in 4 mL methanol, then 23 mg 10% Pd/C was added for hydrogenation.
After hydrogenation procedure in protocol B, the crude was purified by silica gel
chromatography (EtOAc/hexane = 1:2 to 1:1 with 1%TEA) to furnish compound 17
(170 mg, 76%). [𝛼]25

D +3.1 (c 1.0, CHCl3); H NMR(400 MHz, CDCl3): δ= 7.38-7.36
1
(m, 2H), 7.27 (d, 1H, J = 8.8 Hz), 7.17 (d, 1H, J = 3.2 Hz), 7.12-7.09 (m, 2H), 6.94 (d,
1H, J = 2.2 Hz), 6.65 (dd, 1H, J = 8.7, 2.2 Hz), 6.46 (d, 1H, J = 3.2 Hz), 5.50 (dd, 1H,
J = 10.4, 7.9 Hz, H-2), 5.46 (d, 1H, J = 2.9 Hz, H-4), 5.12 (dd, 1H, J = 10.4, 3.4 Hz,
H-3), 5.06 (d, 1H, J = 7.9 Hz, H-1), 4.26-4.22 (m, 1H, H-6a), 4.18-4.13 (m, 1H, H-6b),
13
4.08-4.02 (m, 1H, H-5), 2.18, 2.09, 2.04, 2.01 (4s, 3H×4, Ac). C NMR (100 MHz,
CDCl3): δ= 170.3, 170.2, 170.1, 169.3, 154.9, 140.1, 135.5, 130.9, 130.1, 128.2, 125.3,
118.0, 113.0, 110.8, 105.7, 102.3, 99.8, 71.1, 70.8, 68.6, 66.8, 61.3, 20.7, 20.6 (2C),
20.5; HRMS (ESI): calc. for C28H31N2O10 [M + H] +, 555.1980, found 555.1980.
2-(2’,3’,4’,6’-tetra-O-acetyl-β-D-galactopyranosylmercapto)pyridine (18) 81 .
Compound 3 (500 mg, 1.280 mmol) was dissolved in 2 mL 33% HBr / acetic acid for
bromination procedure and extraction in protocol A. After bromination procedure, the
87
glycosyl bromide and K2CO3 (212 mg, 1.536 mmol) were dissolved in 2.5 mL dry
acetone and 1.5 mL dry toluene. After glycosylation, the reaction mixture was
evaporated to dryness, then diluted with CH2Cl2 and H2O. The organic layer was
washed with saturated NaHCO3(aq). After driing with Na2SO4 and concentrated, the
crude was purified by silica gel chromatography (EtOAc/hexane = 1:4 to 1:2) to

D +17.4 (c 1.0, CHCl3); H NMR (400
MHz, CDCl3): δ= 8.38 (d, 1H, J = 4.7 Hz), 7.47 (t, 1H, J = 7.7 Hz), 7.17 (d, 1H, J =
7.9 Hz), 7.02-6.99 (m, 1H), 5.73 (d, 1H, J = 10.4 Hz, H-1), 5.41 (d, 1H, J = 3.3 Hz,
H-4), 5.34 (app. t, 1H, J = 9.9, 3.2 Hz, H-3), 4.06-4.01 (m, 3H, H-6a, H-6b, H-5), 2.09,
1.94, 1.92, 1.91(4s, 3H×4, Ac); HRMS (ESI): calc. for C19H23NO9SNa [M + Na] +,
464.0986, found 464.0992.
4-(tetra-O-acetyl-β-D-galactopyranosyloxy)benzoic acid (19). According
protocol B. Compound 5 (491 mg, 0.879 mmol) was dissolved in 5 mL CH2Cl2 with
10% v/v (0.5 mL) formic acid, then 50 mg 10% Pd/C was added for hydrogenation.
After hydrogenation procedure in protocol B, the crude was purified by silica gel
chromatography (EtOAc/hexane = 1:2 to 1:1) to furnish compound 19 (356 mg, 86%).
[𝛼]31
D -2.5 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3): δ= 7.81 (d, 1H, J = 7.7 Hz),
1
7.71 (s, 1H), 7.38 (app. t, 1H, J = 8 Hz), 7.23 (d, 1H, J = 7.8 Hz), 5.49 (dd, 1H, H-2, J
= 10.4, 7.9 Hz), 5.45 (d, 1H, H-4, J = 3.2 Hz), 5.12 (dd, 1H, H-3, J = 10.0 Hz, 3.4 Hz),
5.11 (d, 1H, H-1, J = 7.0 Hz), 4.18 (d, 2H, H-6, J = 6.2 Hz), 4.11 (app. t, 1H, H-5, J =
6.2 Hz), 2.17, 2.06, 2.05, 2.00 (4s, 3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.9;
170.5; 170.2; 170.1; 169.4; 156.7; 130.8; 129.6; 124.9; 122.7; 117.5; 99.2; 71.2; 70.7;
88
68.5; 66.9; 61.6; 20.6; 20.5; 20.5(2C); HRMS (ESI): calc. for C21H24O12Na [M + Na] +,
491.1160, found 491.1151.
6-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyloxy)-2-naphthoic acid (20)34.
According protocol B. Compound 8 (313 mg, 0.515 mmol) was dissolved in 5 mL
CH2Cl2 with 10% v/v (0.5 mL) formic acid, then 30 mg 10% Pd/C was added for
hydrogenation. After hydrogenation procedure in protocol B, the crude was purified
by silica gel chromatography (EtOAc/hexane = 1:1) to furnish compound 20 (217 mg,
81%). [𝛼]31
D +11.6 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 8.63 (s,1H), 8.09
1
(d, 1H, J = 8.5 Hz), 7.89 (d, 1H, J = 9.0 Hz), 7.87 (d, 1H, J = 8.6 Hz), 7.36 (d, 1H, J =
2.2 Hz), 7.24 (dd, 1H, J = 8.8, 2.1 Hz), 5.55 (dd, 1H, J = 10.4, 8.0 Hz, H-2), 5.49 (d,
1H, J = 3.2 Hz, H-4), 5.24 (d, 1H, J = 7.9 Hz, H-1), 5.16 (dd, 1H, J = 10.4, 3.3 Hz,
H-3), 4.28-4.14 (m, 3H, H-5, H-6a, H-6b), 2.18, 2.06, 2.05, 2.01 (s, 3H×4, Ac); 13C
NMR (100 MHz, CDCl3): δ= 171.6, 170.4, 170.2, 170.1, 169.4, 156.7, 137.0, 131.9,
131.4, 129.0, 127.4, 126.3, 125.5, 119.6, 110.9, 99.2, 71.3, 70.8, 68.6, 66.9, 61.5, 20.7,
20.7(2C), 20.6; HRMS (ESI): calc. for C25H26O12Na [M + Na] +, 541.1316, found
541.1309.
O-(4-aminophenyl)-β-D-galactopyranoside (21)82. According protocol C.
Compound 14 (50 mg, 0.114 mmol) was dissolved in 1.2 mL MeOH and stirred at 0
89
°C, then 10.7 μL 30% NaOMe in methanol solution was added. After deacetylation
procedure, the residue were evaporated in vacuum without further purification to
furnish compound 21 (36 mg, quantitative yield). [𝛼]31 1

D -38.2 (c 0.5, MeOH); H
NMR (400 MHz, CD3OD): δ= 6.95-6.91 (m, 2H), 6.70-6.66 (m, 2H), 4.68 (d, 1H, J =
7.8 Hz, H-1), 3.88 (d, 1H, J = 2.7 Hz, H-3), 3.77-3.71 (m, 3H, H-6a, H-6b, H-2), 3.61
(m, 1H, H-5), 3.54 (dd, 1H, J = 9.7, 3.4 Hz); 13C NMR (100 MHz, CD3OD): δ= 152.4,
143.3, 119.3, 117.8, 104.3, 71.8, 74.9, 72.4, 70.2, 62.4.
4-(β-D-galactopyranosyloxy)benzoic acid (22). According protocol C.
Compound 19 (57 mg, 0.122 mmol) was dissolved in 1 mL MeOH and stirred at 0 °C,
then 23 μL 30% NaOMe in methanol solution was added. After deacetylation

D -52.1 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 7.73 (s, 1H), 7.69-6.67 (m, 1H), 7.41-7.33 (m, 2H), 4.92 (d, 1H, J = 7.8
Hz, H-1), 3.92 (d, 1H, J = 3.3 Hz, H-4), 3.82 (dd, 1H, J = 9.7, 7.8 Hz, H-2), 3.78-3.70
13
(m, 3H, H-6a, H-6b, H-5), 3.60 (dd, 1H, J = 9.7, 3.3 Hz, H-3); C NMR (100 MHz,
CD3OD): δ= 169.5, 159.1, 133.2, 130.5, 124.6, 122.5, 118.9, 102.9, 76.9, 74.8, 72.2,
70.1, 62.3; HRMS (ESI): calc. for C13H17O8 [M + H] +, 301.0918, found 301.1408.
O-(2,3,6-Trimethylphenyl)-β-D-galactopyranose (23). According protocol C.
90
then 13.2 μL 30% NaOMe in methanol solution was added. After deacetylation

D -2.8 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 6.86-6.80 (m, 2H), 4.57 (d, 1H, J = 7.8 Hz, H-1), 3.87 (d, 1H, J = 3.5 Hz,
H-4), 3.84 (dd, 1H, J = 9.8, 7.7 Hz, H-2), 3.74-3.58 (m, 1H, H-6a), 3.62-3.58 (m, 1H,
H-6b), 3.54 (dd, 1H, J = 9.8, 3.4 Hz, H-3), 3.35 (td, 1H, J = 6.2, 1.0 Hz, H-5), 2.30,
2.26, 2.20 (3s, 3H×3); 13C NMR (100 MHz, CD3OD): δ= 154.7, 136.6, 131.4, 130.3,
128.8, 126.7, 106.2, 76.4, 74.9, 73.2, 70.0, 62.0, 20.1, 17.6, 13.7; HRMS (ESI): calc.
for C15H22O6Na [M + Na] +, 321.1309, found 321.1305.
2-(β-D-galactopyranosylmercapto)pyridine (24) 83 . According protocol C.

D -54.1 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 8.37 (d, 1H, J = 4.9 Hz), 7.73-7.68 (m, 1H), 7.54-7.51 (m, 1H), 7.19-7.16
(m, 1H), 5.12 (d, 1H, J = 9.8 Hz, H-1), 3.94 (d, 1H, J = 3.4 Hz, H-4), 3.78-3.67 (m,
3H, H-2, H-5, H-6a, H-6b), 3.57 (dd, 1H, J = 9.2, 3.3 Hz, H-3); 13C NMR (100 MHz,
CD3OD): δ= 159.6, 149.9, 138.8, 124.5, 121.9, 86.8, 80.8, 76.3, 70.8, 70.5, 62.6;
HRMS (ESI): calc. for C11H15NO5SNa [M + Na] +, 296.0563, found 296.0562.
91
6-(β-D-galactopyranosyloxy)-2-naphthoic acid (25). According protocol C.

D -69.4 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 8.54 (s, 1H), 7.99 (dd, 1H, J = 8.6, 1.7 Hz), 7.94 (d, 1H, J = 9.0 Hz), 7.84
1H, J = 7.7 Hz, H-1), 3.95 (d, 1H, J = 3.4 Hz, H-4), 3.88 (dd, 1H, J = 9.7, 7.8 Hz, H-2),
13
3.84-3.77 (m, 3H, H-6a, H-6b, H-5), 3.64 (dd, 1H, J = 9.7, 3.4 Hz, H-3); C NMR
(100 MHz, CD3OD): δ= 158.9, 138.4, 131.9, 131.9, 130.1, 128.4, 127.5, 126.9, 126.9,
121.0, 111.7, 102.7, 77.2, 74.9, 72.5, 70.3, 62.5; HRMS (ESI): calc. for C17H17O8 [M -
H] -, 349.0929, found 349.0942.
O-(5-Aminonaphthyl)-β-D-galactopyranoside (26). According protocol C.
Compound 9 (97 mg, 0.197 mmol) was dissolved in 1.5 mL MeOH and stirred at 0 °C,

D -28.6 (c 1.0, DMSO); H NMR (400 MHz,
CD3OD): δ= 7.81 (dt, 1H, J = 8.5, 0.8 Hz), 7.64 (dt, 1H, J = 8.5, 0.8 Hz), 7.33 (dd, 1H,
J = 8.4, 7,8 Hz), 7.25-7.19 (m, 2H), 5.04 (d, 1H, J = 7.8 Hz, H-1), 3.97 (dd, 1H, J =
92
9.7, 7.8 Hz, H-2), 3.94 (d, 1H, J = 3.2 Hz, H-4), 3.81-3.77 (m, 2H, H-6a, H-6b),
13
3.75-3.71 (m, 1H, H-5), 3.63 (dd, 1H, J = 9.7, 3.4 Hz, H-3); C NMR (100 MHz,
(CD3)2SO): δ= 153.2, 144.3, 126.5, 125.9, 123.7, 123.5, 115.8, 109.9, 108.7, 108.1,
101.6, 75.6, 73.4, 70.5, 68.2, 60.5; HRMS (ESI): calc. for C16H19NO6Na [M + Na] +,
344.1105, found 344.1093.
84
O-(4-Biphenyl)-β-D-galactopyranoside (27) . According protocol C.

D -15.5 (c 1.0, DMSO); H NMR (400 MHz,
(CD3)2SO): δ= 7.63-7.58 (m, 4H), 7.43 (app. t, 2H, J = 7.5 Hz), 7.33-7.29 (m, 1H),
7.13-7.11 (m, 2H), 5.20 (d, 1H, J = 5.0 Hz), 4.87 (d, 1H, J = 7.8 Hz, H-1), 4.89 (d, 1H,
J = 6.8 Hz), 4.68 (m, 1H), 4.54 (d, 1H, J = 4.4 Hz), 3.72 (app. t, 1H, J = 3.4 Hz, H-4),
3.62-3.48 (m, 4H, H-6a, H-6b, H-2, H-5), 3.44-3.42 (m, 1H, H-3); 13C NMR (100
MHz, (CD3)2SO): δ= 157.2, 139.8, 133.7, 128.9, 127.6, 126.8, 126.3, 116.7, 101.1,
75.5, 73.3, 70.3, 68.1, 60.4; HRMS (ESI): calc. for C18H20O6Na [M + Na] +, 355.1152,
found 355.1150.
93
O-(4-(Benzyloxy)phenoxy)-β-D-galactopyranoside (28). According protocol C.

D -37.3 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 7.43-7.29 (m, 5H), 7.08-7.04 (m, 2H), 6.92-6.89 (m, 2H), 5.03 (s, 2H),
4.73 (d, 1H, J = 7.8 Hz, H-1), 3.89 (d, 1H, J = 3 Hz, H-4), 3.80-3.72 (m, 3H, H-6a,
13
H-6b, H-2), 3.63 (m, 1H, H-5), 3.55 (dd, 1H, J = 9.7, 3.4 Hz); C NMR (100 MHz,
CD3OD): δ= 155.6, 153.5, 138.89, 129.5, 128.8, 128.6, 119.2, 116.7, 104.0, 76.9, 74.9,
72.4, 71.5, 70.2, 62.4; HRMS (ESI): calc. for C19H22O7Na [M + Na] +, 385.1258,
found 385.1249.
85
O-(8-Quinolinyl)-β-D-galactopyranoside (29) . According protocol C.
°C, then 19 μL 30% NaOMe in methanol solution was added. After deacetylation

D -66.6 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 8.84 (dd, 1H, J = 4.3, 1.6 Hz), 8.38 (dd, 1H, J = 8.3, 1.6 Hz), 7.67-7.53
(m, 4H), 5.02 (d, 1H, J = 7.8 Hz, H-1), 4.07 (dd, 1H, J = 9.9, 7.7 Hz, H-2), 3.96 (d, 1H,
J = 3.4 Hz, H-4), 3.89-3.78 (m, 3H, H-6a, H-6b, H-5), 3.68 (dd, 1H, J = 9.8, 3.4 Hz,
3-H); 13
C NMR (100 MHz, CD3OD): δ= 153.8, 150.1, 140.1, 138.6, 131.0, 128.5,
123.0, 122.5, 114.6, 103.9, 77.4, 74.2, 72.1, 70.2, 62.6; HRMS (ESI): calc. for
94
C15H18NO6 [M + H] +, 308.1129, found 308.1117.
O-(2-(6-Benzoyl)naphthalenyl)-β-D-galactopyranoside (30). According
protocol C. Compound 13 (56 mg, 0.096 mmol) was dissolved in 1 mL MeOH and
stirred at 0 °C, then 9 μL 30% NaOMe in methanol solution was added. After
deacetylation procedure, the residue were evaporated in vacuum without further
purification to furnish compound 30 (41 mg, 89%). [𝛼]30 1

D -53.0 (c 0.5, MeOH); H
NMR (400 MHz, CD3OD): δ= 8.20 (s, 1H), 7.92-7.84 (m, 3H), 7.81-7.79 (m, 2H),
7.66 (tt, 1H, J = 7.4, 1.3 Hz), 7.59-7.53 (m, 3H), 7.39 (dd, 1H, J = 8.9, 2.4 Hz), 5.09 (d,
1H, J = 7.8 H, H-1), 3.95 (d, 1H, J = 3.4 Hz, H-4), 3.89 (dd, 1H, J = 9.7, 7.8 Hz, H-2),
13
3.86-3.78 (m, 2H, H-6a, H-6b, H-5), 3.65 (dd, 1H, J = 9.7, 3.4 Hz, H-3); C NMR
(100 MHz, CD3OD): δ= 198.6, 159.2, 139.3, 138.3, 134.3, 133.6, 133.1, 132.2, 130.9,
129.7, 129.5, 128.7, 127.0, 121.2, 111.8, 102.7, 77.2, 74.9, 72.3, 70.3, 62.5; HRMS
(ESI): calc. for C23H22O7Na [M + Na] +, 433.1258433.1253, found 433.1253.
O-( 4-((4-Methylquinolin-2-yl)amino)phenoxy)-β-D-galactopyranoside (31).
According protocol C. Compound 15 (87 mg, 0.150 mmol) was dissolved in 1.5 mL
MeOH and stirred at 0 °C, then 14 μL 30% NaOMe in methanol solution was added.
After deacetylation procedure, the residue were evaporated in vacuum without further
95
D -10.1 (c 1.0, DMSO); H
NMR (400 MHz, (CD3)2SO): δ= 9.17 (s, 1H), 7.85 (d, 2H, J = 8.8 Hz), 7.82 (d, 1H, J
= 8.3 Hz), 7.64 (d, 1H, J = 8.3 Hz), 7.54 (dd, 1H, J = 8.2, 7.0 Hz), 7.27 (dd, 1H, J =
8.0, 7.0 Hz), 7.02 (d, 2H, J = 8.8 Hz), 6.85 (s, 1H), 5.15 (d, 1H, J = 5.0 Hz), 4.85 (d,
1H, J = 5.5 Hz), 4.76 (d, 1H, J = 7.6 Hz), 4.67-4.66 (m, 1H), 4.50 (d, 1H, J = 4.4 Hz),
3.72 (app. t, 1H, J = 3.5 Hz, H-4), 3.60-3.51 (m, 4H, H-6a, H-6b, H-2, H-5), 3.44-3.39
13
(m, 1H, H-3), 2.55 (s, 3H); C NMR (100 MHz, (CD3)2SO): 154.1, 152.1, 147.1,
144.0, 135.8, 129.1, 126.6, 123.7(2C), 122.2, 119.7, 116.6, 113.5, 101.8, 75.4, 73.4,
70.4, 68.2, 60.4, 18.3; HRMS (ESI): calc. for C22H25N2O6 [M + H] +, 413.1707, found
413.1703.
O-(4-(5-Amino-1H-indol-1-yl)phenoxy)-β-D-galactopyranoside (32).
According protocol C. Compound 17 (45 mg, 0.080 mmol) was dissolved in 1 mL
MeOH and stirred at 0 °C, then 7.5 μL 30% NaOMe in methanol solution was added.

D -35.8 (c 0.5, MeOH); H
2.0 Hz), 6.72 (dd, 1H, J = 8.7, 2.1 Hz), 6.42 (dd, 1H, J = 3.1, 0.5 Hz), 4.89 (d, 1H, J =
7.8 Hz, H-1), 3.92 (d, 1H, J = 2.8 Hz, H-4), 3.84 (dd, 1H, J = 9.8, 7.8 Hz, H-2),
3.81-3.76 (m, 2H, H-6a, H-6b), 3.71-3.68 (m, 1H, H-5), 3.61 (dd, 1H, J = 9.7, 3.4 Hz);
C NMR (100 MHz, CD3OD): δ= 157.4, 140.4, 135.9, 132.8, 131.5, 129.4, 126.1,
13
118.9, 114.8, 111.7, 108.1, 103.2, 103.1, 77.0, 74.8, 72.3, 70.2, 62.4; HRMS (ESI):
96
calc. for C20H23N2O6 [M + H] +, 387.1551, found 387.1552.
4-methylphenyl-2,3,4,6-tetra-O-acetyl-1-thio-β-D-galactopyranoside (33) 86 .
Compound 3 (3.16 g, 8.10 mmol) and thiocresol (2.76 g, 21.70 mmol) was dissolved
in 25 mL dry CH2Cl2, then boron trifluoride diethyl etherate was added at 0 °C. After
40 h stirring, the reaction was diluted with 25 mL CH2Cl2, and then washed with
NaHCO3(aq) 3 times, then brine, dried over Na2SO4. After concentrated and silica gel
(2.66 g, 72%). 1H NMR (400 MHz, CDCl3): δ= 7.39 (d, 2H, J = 8.1 Hz), 7.10 (d, 2H,
J = 8.0 Hz), 5.38 (dd, 1H, J = 3.4, 0.7 Hz, H-4), 5.20 (app. t, 1H, J = 10.0 Hz, H-2),
5.01 (dd, 1H, J = 9.9, 3.3 Hz, H-3), 4.62 (d, 1H, J = 10.0 Hz, H-1), 4.19-4.06 (m, 2H,
H-6a, H-6b), 3.90-3.87 (m, 1H, H-5), 2.32 (s, 3H), 2.09, 2.08, 2.02, 1.95 (4s, 3H×4,
Ac).
4-methylphenyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside (34) 87 .
According protocol C. Compound 33 (7.80 g, 17.16 mmol) was dissolved in 60 mL
MeOH and 30 mL CH2Cl2 stirred at 0 °C, then 0.97 mL 30% NaOMe in methanol
solution was added. After deacetylation procedure, the residue were evaporated in
vacuum without purification, portion of the residue (100 mg) and
Tetrabutylammonium iodide (7.7 mg, 0.021 mmol) were dissolved in 5 mL anhydrous

97
DMF, cooled to 0 °C, stirred under argon. After BnBr (0.34 mL, 2.80 mmol) added,
60% NaH (100 mg, 2.50 mmol) was added to, and stirred overnight at room
temperature. After completion of benzylation, the mixture were diluted with EtOAc
(25 mL) and washed with saturated NH4Cl (15 mL), dried over Na2SO4, filtered, and
concentrated for silica gel chromatography (EtOAc/hexane = 1:5 to 1:2) to afford
thioglactoside 34. (271 mg, quantitative yield). [𝛼]25 1

D -0.5 (c 1.0, CHCl3); H
NMR(400 MHz, CDCl3): δ= 7.53-7.51 (d, 2H, J = 8.2 Hz), 7.46-7.43 (m, 3H),
7.38-7.21 (m, 17H), 7.03 (d, 2H, J = 7.9 Hz), 5.01 (d, 1H, J = 11.5 H, H-1), 4.86-4.73
(m, 4H), 4.66 (d, 1H, J = 3.2 Hz), 4.63 (d, 1H, J = 1.1 Hz), 4.48 (m, 1H), 4.02 (d, 1H,
J = 2.5 Hz, H-4), 3.95 (app. t, 1H, J = 9.4 Hz, H-2), 3.73-3.62 (m, 4H, H-3, H-5, H-6a,
H-6b), 2.32 (s, 3H); HRMS (ESI): calc. for C41H42O5SNa [M + Na] +, 669.2645, found
669.2657.
D-Lactose octaacetate (35)88. D-Lactose (10.0 g, 27.75 mmol) was dissolved in
100 mL pyridine, and then acetic anhydride 78.6 mL was added in ice bath, then ice
bath was removed after 10 minutes. The resulting mixture were stirred under an argon
atmosphere at r.t. for 36h. The reaction was quenched by MeOH in ice bath. The
solvent were removed under reduced pressure. The residue was diluted with EtOAc
(150 mL) and washed with saturated NaHCO3(aq), water, and brine. Dried over
Na2SO4, then purified by silica gel chromatography (EtOAc/hexane = 1:2 to 1:1) to
furnish 35 (α-major). (19.1 g, quantitative yield). 1H NMR (400 MHz, CDCl3): δ=
6,23 (d, 1H, J = 3.8 Hz, H-1), 5.44 (dd, 1H, J = 10.2, 9.3 Hz, H-3), 5.33 (dd, 1H, J =
98
3.5, 1.0 Hz, H-4’), 5.10 (dd, 1H, J = 10.4, 7.9 Hz, H-2’), 4.98 (dd, 1H, J = 10.3, 3.7
Hz, H-2), 4.93 (dd, 1H, J = 10.4, 3.5 Hz, H-3’), 4.46 (d, 1H, J = 7.9 Hz, H-1’), 4.42
(dd, 1H, J = 12.2, 2.0 Hz, H-6a), 4.15-4.04 (m, 3H, H-6’a, H-6’b, H-6b), 3.98 (ddd,
1H, J = 10.1, 4.1, 2.0 Hz, H-5’), 3.86 (m, 1H, H-4), 3.79 (app. t, 1H, J = 7.4 Hz, H-5),
2.16, 2.15, 2.13, 2.04, 2.04, 2.03, 1.99, 1.94 (8s, 3H×8, Ac)
2-(Benzyloxycarbonyl)aminoethyl-2,3,4,6-tetra-O-acetyl-β-D-galactopyranos
yl-(1 → 4)-2,3,6-tri-O-acetyl-β-D-glucopyranoside (36)88. According protocol A.
Compound 35 (1.0 g, 1.474 mmol) was dissolved in 2 mL 33% HBr / acetic acid for
bromination. After bromination procedure, the glycosyl bromide, benzyl
2-hydroxyethyl carbamate (0.58g, 2.948 mmol) and Hg(CN)2 (0.41 g, 1.621 mmol)
were dissolved in 10 mL absolute CH3CN and stirred at 70 °C for 2 h, then keep
reacted in r.t. Upon the reaction completion, the mixture was evaporated to dryness
and diluted with CH2Cl2, then washed with H2O, saturated NaHCO3(aq), brine, after
concentrated and purified by silica gel chromatography (EtOAc/hexane = 1:1 to 2:1)
to give compound 36 (0.548 g, 46%). [𝛼]25 1

D +5.7 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ= 7.32-7.27 (m, 5H), 5.31 (d, 1H, J = 3.2 Hz, H-4’), 5.16 (app. t, 1H, J = 9.2
Hz, H-3), 5.09-5.05 (m, 3H, H-2’, CH2Ph), 4.91 (dd, 1H, J = 10.4, 3.4 Hz, H-3’), 4.84
(dd, 1H, J = 9.3, 8.2 Hz, H-2), 4.47-4.40 (m, 3H, H-6a, H-1’, H-1), 4.12-4.01 (m, 3H,
H-6’a, H-6’b, H-6b), 3.83 (app. t, 1H, J = 6.9 Hz, H-5’), 3.79-3.71 (m, 2H, H-4,
CHCHCH2NH), 3.69-3.63 (m, 1H, CHCHCH2NH), 3.54 (ddd, 1H, J = 9.6, 4.9, 1.9 Hz,
H-5), 3.35-3.33 (m, 2H, CH2CH2NH), 2.11,2.04, 2.02, 1.96, 1.93 (5s, 3H×5, Ac), 2.00
(s, 6H, 3H×2, Ac); HRMS (ESI): calc. for C36H47NO20Na [M + Na] +, 836.2584, found
99
836.2590.
2-(Benzyl((benzyloxy)carbonyl)amino)ethyl-2,3-di-O-benzyl-4,6-O-benzylide
ne-β-D-galactopyranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D-glucopyranoside (37).
According protocol C. Compound 36 (0.515 g, 0.633 mmol) was dissolved in 10 mL
After deacetylation procedure, the residue were evaporated in vacuum. The crude
residue was dissolved in 2.5 mL absolute CH3CN, then benzaldehyde dimethyl acetal
(0.137 mL, 0.912 mmol) was added and stirred at r.t. under argon, followed by
addition of camphorsulfonic acid (14.2 mg, 0.061 mmol) and the temperature was
raised to 45 °C. Upon the reaction completion, the mixture was brought to r.t., and the
reaction mixture was neutralized with Et3N, concentrated and evaporated in vacuum
for few hours. The resulting residue from previous reaction was dissolved in a solution
of BnBr (0.601 mL, 5.061 mmol) and 5 mL anhydrous DMF at 0 °C under argon, to
which 60 % NaH (0.228 g, 5.693 mmol) was added, followed by rigorous stirring.
After completion of benzylation, the mixture was diluted with EtOAc, and the
resulting solution was washed with saturated NH4Cl(aq),dried over Na2SO4, filtered,
to give compound 38 (0.546 g, 75%). [𝛼]29 1

D +9.4 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ= 7.53-7.50 (m, 5H), 7.49-7.09 (m, 35H), 5.47 (s, 1H), 5.19 (d, 1H, J = 10.6
Hz), 5.15 (d, 2H, J = 17.6 Hz), 4.86-4.84 (m, 2H), 4.80-4.69 (m, 5H), 4.62-4.51 (m,
100
3H), 4.45 (d, 1H, J = 7.9 Hz, H-1), 4.29 (m, 2H, H-1’, CH2Ph), 4.21 (d, 1H, J = 12.1
Hz, H-6’a), 4.03-3.74 (m, 7H, H-4’, CH2NBnZ, H-6’b, H-4, H-2, H-6a), 3.67-3.26 (m,
13
8H, H-6b, CH2CH2NBnZ, H-2’, H-3, H-5, H-3’), 2.93 (s, 1H, H-5’); C NMR (100
MHz, CDCl3): δ= 156.3, 156.1, 128.8, 138.4, 138.3, 138.0, 137.7, 137.6, 136.5, 136.4,
128.7, 128.4, 128.2, 128.1, 128.7, 128.0, 127.9, 127.7, 127.5, 127.5, 127.3, 127.3,
127.1, 127.0, 126.4, 103.5, 102.6, 101.1, 82.8, 81.7, 79.5, 78.7, 77.2, 75.6, 75.1, 74.9,
73.4, 72.8, 71.4, 68.8, 68.0, 67.8, 67.1, 66.2, 51.3, 47.0, 45.9; HRMS (ESI): calc. for
C71H73NO13Na [M + Na] +, 1170.4974, found 1170.4988.
2-(Benzyl((benzyloxy)carbonyl)amino)ethyl-2,3,6-tri-O-benzyl-β-D-galactop
yranosyl-(1→4)-2,3,6-tri-O-benzyl-β-D- glucopyranoside (38). Compound 37
(0.661 g, 0.576 mmol) was dissolved in a solution of Et3SiH (1.014 mL, 6.91 mmol)
and 5 mL dry CH2Cl2 ,then actvated AW-300 molecular sieve 1.2 g was added to
stirred for 1 at 0 °C under argon. (0.530 mL, 6.91 mmol) TFA was added to the
resulting solution at 0 °C, stirred for 3 h from 0 °C to r.t. Upon the reaction completion,
the residue was diluted with CH2Cl2, and washed with saturated NaHCO3(aq), water,
and brine. Dried over Na2SO4, then purified by silica gel chromatography
(EtOAc/hexane = 1:5 to 1:3) to furnish compound 38. (0.471 g, 71%). [𝛼]29

D +16.5 (c
1.0, CHCl3); 1H NMR (400 MHz, CDCl3): δ= 7.37-7.35 (m, 2H), 7.32-7.20 (m, 2H),
7.23-7.20 (m, 15H), 7.10-7.08 (m, 1H), 5.13 (d, 2H, J =14.9 Hz), 4.97 (dd, 1H, J =
10.7, 1.9 Hz), 4.83-4.24 (m, 15H), 4.01-3.93 (m, 3H), 3.97-3.75 (m, 1H), 3.66-3.25 (m,
15H), 2.39 (s, 1H); 13C NMR (100 MHz, CDCl3): δ= 156.4, 156.2, 139.0, 138.6, 138.1,
137.9, 136.6, 136.5, 128.4, 128.4, 128.2, 128.0, 128.0, 127.8, 127.7, 127.6, 127.5,
101
127.2, 103.6, 102.4, 82.8, 81.7, 81.0, 79.3, 77.2, 76.3, 75.3, 75.2, 75.0, 73.4, 73.0, 72.7,
71.9, 68.4, 68,1, 67.9, 67.2, 66.1, 51.4, 47.1, 45.9; HRMS (ESI): calc. for
C71H75NO13Na [M + Na] +, 1172.5131, found 1172.5144.
2-(Benzyl((benzyloxy)carbonyl)amino)ethyl-2,3,4,6-tetra-O-benzyl-α-D-galac
topyranosyl-(1→4)-O-(2,3,6-tri-O-benzyl-β-D-galactopyranosyl)-(1→4)-2,3,6-tri-
O-benzyl-β-D-glucopyranoside (39). Compound 38 (0.131 g, 114 mmol) and 34
(0.110 g, 0.170 mmol) and 0.3 g molecular sieve were suspended in 3 mL dry CH2Cl2
stirred at r.t. for 1h. Then cooled to -45 °C stirred under dry argon. NIS (0.031 g, 0.182
mmol) and TMSOTf (2 μL, 0.011 mmol) was added sequentially, stirred for 2 h and
brought the temperature to -15 °C. The reaction was quenched by 10% Na2SO3(aq)
and Et3N. The residue was diluted with CH2Cl2, and washed with saturated
NaHCO3(aq), water, and brine. Dried over Na2SO4, then purified by silica gel
chromatography (EtOAc/hexane = 1:5 to 1:3) to furnish compound 39. (0.128 g,
67%). [𝛼]31
D +27.7 (c 1.0, CHCl3); H NMR (400 MHz, CDCl3): δ= 7.40-7.12 (m,
1
60H), 5.13 (d, 2H, J = 15.1 Hz), 5.08-5.05 (m, 2H), 4.86 (d, 2H, J = 11.3 Hz),
4.81-4.41 (m, 16H), 4.37-4.22 (m, 5H), 4.16 (t, 1H, J = 8.9 Hz), 4.12-3.92 (m, 7H),
13
3.89-3.77 (m, 2H), 3.65-3.24 (m, 11H), 3.18-3.15 (m, 1H); C NMR (100 MHz,
CDCl3): δ= 156.4, 139.0, 138.9, 138.7, 138.6, 128.5, 138.4, 138.3, 138.2, 137.9, 137.8,
137.7, 136.5, 128.5, 128.4, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.1, 128.0,
127.9, 127.8, 127.6, 127.5, 127.5, 127.5, 127.4, 127.4, 127.3, 127.2, 127.1, 127.1,
102
103.6, 102.7, 100.7, 82.4, 81.6, 81.6, 79.4, 76.9, 76.5, 75.1, 74.9, 74.9, 74.8, 74.7, 73.7,
73.2, 73.1, 73.0, 73.0, 72.4, 72.0, 51.4, 47.1, 45.9; HRMS (ESI): calc. for
C105H109NO18Na [M + Na] +, 1694.7537, found 1694.7456.
2-Azidoethyl-α-D-galactopyranosyl-(1→4)-β-D-galactopyranosyl-(1→4)-β-D
-glucopyranoside (40). According protocol B. Compound 39 (69 mg, 0.040 mmol)
was dissolved in 0.6 mL CH2Cl2 and 1.5 mL CH2Cl2 with 10% v/v (0.5 mL) formic
acid (0.3 mL), then 10mg 10% Pd/C was added for hydrogenation. After
hydrogenation procedure in protocol B, the crude was concentrated and evaporated in
vacuum for few hours. The resulting residue was dissolvedin the solution of 0.4 mL
MeOH and 0.2 mL H2O and K2CO3 (7.3 mg, 0.053 mmol), ZnCl2 (0.2 mg, 0.0017
mmol), and 0.2 mL TfN3 in CH2Cl2 solution were added sequentially, stirred at 0 °C
under argon for 5 h the brought to r.t.. TFN3 in CH2Cl2 solution was made by reported
method71 : (92 mg, 1.416 mmol) NaN3 dissolved in 0.25 mL H2O and 0.4 mL CH2Cl2
stirred at 0 °C, then (0.1 mL, 0.355 mmol) Tf2O was added to the solution carefully
and stirred for 2h at 0 °C. After 2 h, the aqueous layer was extracted with 0.2 mL
CH2Cl2 twise. The organic combined layer was reacted with amino aubstrate straightly.
Upon completion of reaction, the mixture was evaporated to dryness, and diluted with
H2O, purified by semi-preparative C18 HPLC column with 5 % MeCN in 0.1%
TFA(aq) to furnish 40. (20 mg, 87%). [𝛼]24 1

D +36.9 (c 1.0, H2O); H NMR (400 MHz,
D2O): δ= 4.93 (d, 1H, J = 3.7 Hz, H-1’’), 4.52 (d, 1H, J = 8.2 Hz, H-1’), 4.50 (d, 1H, J
13
= 8.2 Hz, H-1), 4.06-3.54 (m, 20H), 3.32 (t, 1H, J = 8.4 Hz); C NMR (100 MHz,
103
D2O): δ= 103.3, 102.1, 100.3, 78.6, 77.4, 75,4, 74.9, 74.4, 72.9, 72.2, 70.9, 70.8, 69.1,
69.0, 68.6, 68.5, 60.5, 60.4, 60.0, 50.5; HRMS (ESI): calc. for C20H35N3O16Na [M +
Na]+ 596.1910, found 596.1904.
O-(4-(6-Azidohexanamido)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyrano
side (45). According protocol D. Compound 14 (150 mg, 0.341 mmol) and
6-azidohexanoic acid (73 μL, 0.512 mmol) was dissolved in 1.5 mL DMF at 0 °C, and
DIEA (92 μL, 0.512 mmol), HATU (195 mg, 0.512 mmol) was added sequentially.
After coupling procedure and extraction in protocol D, the residue was concentrated
and purified by silica gel chromatography (EtOAc/hexane = 1:3 to 1:1) to give
compound 45 (150 mg, 76 %). [𝛼]31 1

D +4.0 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ= 7.42-7.38 (m, 2H), 6.96-6.92 (m, 2H), 5.44 (dd, 1H, J =10.4, 8.0 Hz, H-2),
5.42 (app. d, 1H, J = 3.4 Hz, H-4), 5.08 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.95 (d, 1H, J
= 8.0 Hz, H-1), 4.22-4.11 (m, 2H, H-6a, H-6b), 4.01 (app. t, 1H, J = 6.8 Hz, H-5), 3.26
(t, 2H, J = 6.8 Hz), 2.33 (t, 2H, J = 7.4 Hz), 2.15,2.05,2.03,1.99 (4s, 3H×4, Ac),
13
1.77-1.70 (m, 2H), 1.65-1.58 (m, 2H), 1.47-1.40 (m, 2H); C NMR (100 MHz,
CDCl3): δ= 170.9, 170.3, 170.2, 170.0, 169.4, 153.4, 133.4, 121.4, 117.5, 100.1, 71.0,
70.7, 68.6, 66.8, 61.3, 51.1, 37.2, 28.5, 26.3, 24.9, 20.7, 20.6(2C), 20.5; HRMS (ESI):
calc. for C26H34N4O11Na [M + Na] +, 601.2116, found 601.2129.
104
O-(3-((2-(2-Azidoethoxy)ethyl)carbamoyl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-
galactopyranoside (46). According protocol D. Compound 19 (227 mg, 0.485 mmol)
and 2-(2-azidoethoxy)ethanamine (0.2 mL, 0.970 mmol) was dissolved in 2.5 mL
CH2Cl2 at 0 °C, and DIEA (130 μL, 0.730 mmol), HATU (277 mg, 0.730 mmol) was
added sequentially. After coupling procedure and extraction in protocol D, the residue
was concentrated and purified by silica gel chromatography (EtOAc/hexane = 1:3 to
1:1) to give compound 46 (205 mg, 73%). [𝛼]31 1

D -3.2 (c 1.0, CHCl3); H NMR (400
MHz, CDCl3): δ= 7.45 (t, 1H, J = 2.3 Hz), 7.41 (dt, 1H, J = 7.2 Hz, 1.1 Hz), 7.31 (app.
t, 1H, J = 8.0 Hz), 7.09 (ddd, 1H, J = 8.2, 2.6, 1.0 Hz), 5.46 (dd, 1H, J = 10.4, 8.0 Hz,
H-2), 5.43 (dd, 1H, J = 3.4, 0.8 Hz, H-4), 5.09 (d, 1H, J = 8 Hz, H-1), 5.07 (dd, 1H, J
= 10.4, 3.4 Hz, H-3), 4.20-4.04 (m, 3H, H-6a, H-6b, H-5), 3.68-3.61 (dd, 2H, J = 5.4
Hz, 4.7 Hz), 2.14, 2.03, 2.02, 1.98 (4s, 4×3H, Ac); 13C NMR (100 MHz, CDCl3): δ=
170.2, 170.0, 169.8, 169.2, 166.5, 156.7, 135.9, 129.4, 121.2, 120.0, 115.4, 99.0, 70.9,
70.6, 69.9, 69.5, 68.3, 66.7, 61.2, 50.4, 39.5, 20.5, 20.4(2C), 20.3; HRMS (ESI): calc.
for C25H32N4O12Na [M + Na] +, 603.1909, found 603.1922.
O-(6-((2-(2-Azidoethoxy)ethyl)carbamoyl)naphthalen-2-yl)-2,3,4,6-tetra-O-a
cetyl-β-D-galactopyranoside (47). According protocol D. Compound 20 (157 mg,

105
0.304 mmol) and 2-(2-azidoethoxy)ethanamine (0.15 mL, 0.75 mmol) was dissolved
in 1.5 mL DMF at 0 °C, and DIEA (81 μL, 0.455 mmol), HATU (173 mg, 0.455 mmol)
was added sequentially. After coupling procedure and extraction in protocol D, the
residue was concentrated and purified by silica gel chromatography (EtOAc/hexane =
1:3 to 1:1) to give compound 47 (179 mg, 94%). [𝛼]31 1

D +11.8 (c 1.0, CHCl3); H NMR
(400 MHz, CDCl3): δ= 8.26 (s, 1H), 7.84 (dd, 1H, J = 8.6, 1.8 Hz), 7.83 (d, 1H, J =
9.0 Hz), 7.33 (d, 1H, J = 2.4 Hz), 7.21(dd, 1H, J = 8.9, 2.4 Hz), 5.54 (dd, 1H, J = 10.5,
7.9 Hz, H-2), 5.47 (d, 1H, J = 3.4 Hz, H-4), 5.20 (d, 1H, J = 8.0 Hz, H-1), 5.14 (dd,
1H, J = 10.5, 3.4 Hz, H-3), 4.27-4.22 (m,1H, H-6a), 4.19-4.12 (m, 2H, H-5, H-6b),
3.75-3.68 (m, 6H, CH2×3), 3.41-3.39 (m, 2H, CH2), 2.18, 2.06, 2.05, 2.01 (3H×4, Ac);
C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1, 169.4, 167.3, 155.9, 135.7,
13
130.8, 130.4, 129.2, 127.4, 127.3, 124.5, 119.6, 110.9, 99.3, 71.2, 70.8, 70.2, 69.9,
C29H34N4O12Na [M + Na] +, 653.2065, found 653.2088.
O-(5-(6-Azidohexanamido)naphthalenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactop
yranoside (48). According protocol D. Compound 9 (150 mg, 0.306 mmol) and
6-azidohexanoic acid (65 μL, 0.460 mmol) was dissolved in 1.5 mL DMF at 0 °C, and
compound 48 (141 mg, 73%). [𝛼]26 1

D -48.0 (c 1.0, CHCl3); H NMR (400 MHz,
CDCl3): δ= 7.97 (d, 1H, J = 8.4 Hz), 7,87 (d, 1H, J = 7.4 Hz), 7.53-7.38 (m, 4H), 7.07
106
(d, 1H, J = 7.6 Hz), 5.69 (dd, 1H, J = 10.5, 8.0 Hz, H-2), 5.49 (d, 1H, J = 3.2 Hz, H-4),
5.17 (d, 1H, J = 8.0 Hz, H-1), 5.15 (dd, 1H, J = 10.1, 3.2 Hz, H-3), 4.29-4.24 (m, 1H,
H-6a), 4.19-4.12 (m, 2H, H-6b, H-5), 3.29 (t, 2H, J = 6.3 Hz), 2.51 (t, 2H, J = 7.6 Hz),
13
1.86-1.79 (m, 2H), 1.68-1.6 3(m, 2H), 1.55-1.51 (m, 2H); C NMR (100 MHz,
CDCl3): δ= 171.5, 170.3, 170.2, 170.1, 169.7, 153.4, 132.0, 128.6, 126.4, 125.9, 125.8,
122.3, 119.6, 115.8, 108.9, 100.0, 71.1, 70.7, 68.5, 66.8, 61.3, 51.2, 37.1, 28.6, 26.4,
25.2, 20.8, 20.6(2C), 20.6; HRMS (ESI): calc. for C30H36N4O11Na [M + Na] +,
651.2273, found 651.2295.
O-(4-(5-(6-Azidohexanamido)-1H-indol-1yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-
D-galactopyranoside (49). According protocol D. Compound 9 (87 mg, 0.157 mmol)
and 6-azidohexanoic acid (34 μL, 0.236 mmol) was dissolved in 1 mL DMF at 0 °C,
and DIEA (42 μL, 0.236 mmol), HATU (90 mg, 0.236 mmol) was added sequentially.
compound 49 (75 mg, 69%). [𝛼]26 1

δ=7.87 (d, 1H, J = 1.8 Hz), 7.38-7.35 (m, 3H), 7.30 (bs, 1H), 7.24 (d, 1H, J = 3.0 Hz),
7.20 (dd, 1H, J = 8.9, 2.0 Hz), 7.13-7.09 (m, 2H), 6.59 (d, 1H, J = 3.1 Hz), 5.50 (d, 1H,
J = 10.4, 8.0 Hz, H-2), 5.46 (d, 1H, J = 3.0 Hz, H-4), 5.12 (dd, 1H, J = 10.4, 3.4 Hz,
H-3), 5.07 (d, 1H, J = 8.0 Hz, H-1), 4.26-4.21 (m, 1H, H-6a), 4.17-4.11 (m, 1H, H-6b),
4.08-4.05 (m, 1H, H-5), 3.26 (t, 2H, J = 6.9 Hz), 2.37 (t, 2H, J = 7.4 Hz) , 2.18, 2.08,
2.04, 2.01(4s, 3H×4, Ac), 1.80-1.73 (m, 2H), 1.66-1.57 (m, 2H), 1.50-1.40 (m, 2H);
107
C NMR (100 MHz, CDCl3): δ= 171.1, 170.3, 170.2, 170.1, 169.3, 155.1, 134.9,
13
133.3, 131.0, 129.1, 128.7, 125.4, 117.9, 116.5, 112.9, 110.3, 103.4, 99.6, 71.0, 70.7,
68.5, 66.8, 61.3, 51.1, 37.2, 28.5, 26.3, 25.1, 20.7, 20.6(2C), 20.5; HRMS (ESI): calc.
for C34H39N5O11Na [M + Na] +, 716.2538, found 716.2544.
O-(4-(6-Azidohexanamido)phenyl)-β-D-galactopyranoside (50). According
protocol C. Compound 45 (34 mg, 0.059 mmol) was dissolved in 0.5 mL MeOH and

D -32.0 (c 0.5, MeOH); H
7.7 Hz, H-1), 3.90 (app. d, 1H, J = 3.3 Hz, H-4), 3.81-3.72 (m, 3H, H-6a, H-6b, H-2),
3.68-3.65 (m, 1H, H-5), 3.57 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 3.33-3.29 (m), 2.36 (t,
13
2H), 1.76-1.68 (m, 2H), 1.68-1.60 (m, 2H), 1.50-1.42 (m, 2H); C NMR (100 MHz,
CD3OD): δ= 174.2, 155.8, 134.4, 122.7, 118.1, 103.3, 77.0, 74.9, 72.3, 70.2, 62.4,
52.3, 37.6, 29.7, 27.4, 26.5; HRMS (ESI): calc. for C18H26N4O7Na [M + Na] +,
433.1694, found 433.1684.
O-(3-((2-(2-Azidoethoxy)ethyl)carbamoyl)phenyl)-β-D-galactopyranoside
(51). According protocol C. Compound 46 (41 mg, 0.072 mmol) was dissolved in 0.5
108
mL MeOH and stirred at 0 °C, then 7 μL 30% NaOMe in methanol solution was added.

D -35.3 (c 1.0, MeOH); H
NMR (400 MHz, CD3OD): δ= 7.55 (s, 1H), 7.47 (dd, 1H, J = 7.6, 0.5 Hz), 7.37 (app. t,
1H, J = 7.8 Hz), 7.27 (m, 1H), 4.92 (d, 1H, J = 7.8 Hz, H-1), 3.91 (d, 1H, J = 3.2 Hz,
H-4), 3.84-3.66 (m, 8H, H-6a, H-6b, H-2, H-5, CH2×2), 3.61-3.56 (m, 3H, CH2, H-3),
3.39 (app. t, 2H, J = 4.7 Hz). 13C NMR (100 MHz, CD3OD): δ= 170.0, 159.2, 137.0,
130.6, 122.0, 121.0, 116.6, 102.8, 77.0, 74.8, 72.2, 70.9, 70.3, 70.2, 62.4, 51.7, 40.9;
HRMS (ESI): calc. for C17H24N4O8Na [M + Na] +, 435.1486, found 435.1479.
O-(6-((2-(2-Azidoethoxy)ethyl)carbamoyl)naphthalen-2-yl)-β-D-galactopyra
noside (52). According protocol C. Compound 47 (41 mg, 0.072 mmol) was
dissolved in 0.5 mL MeOH and stirred at 0 °C, then 8 μL 30% NaOMe in methanol
solution was added. After deacetylation procedure, the residue were evaporated in
vacuum without further purification to furnish compound 52 (37mg, 95%). [𝛼]31

D
–47.4 (c 1.0, MeOH); 1H NMR (400 MHz, CD3OD): δ= 8.32 (s, 1H), 7.89 (dd, 1H, J
= 9.0, 2.1 Hz), 7.87-7.82 (m, 2H), 7.55 (s, 1H), 7.39-7.36 (m, 1H), 5.06 (d, 1H, J = 7.7
Hz, H-1), 3.94 (d, 1H, J = 3.4 Hz, H-4), 3.87 (dd, 1H, J = 9.6, 7.8 Hz, H-2), 3.86-3.77
(m, 3H, H-6a, H-6b, H-5), 3.73-3.69 (m, 4H), 3.65-3.62 (m, 3H, H-3, CH2), 3.41-3.39
(m, 2H); 13C NMR (100 MHz, CD3OD): δ= 170.4, 158.4, 137.5, 131.5, 131.1, 130.1,
128.6, 128.5, 125.4, 121.0, 111.7, 102.7, 77.2, 74.8, 72.3, 71.0, 70.5, 70.3, 62.5, 51.8,
41.0; HRMS (ESI): calc. for C21H26N4O8Na [M + Na] +, 485.1643, found 485.1636.
109
O-(5-(6-Azidohexanamido)naphthalenyl)-β-D-galactopyranoside (53).

D -22.2 (c 1.0, MeOH); H
NMR (400 MHz, CD3OD): δ= 8.35 (d, 1H, J = 8.4 Hz), 7.64 (d, 1H, J = 8.5 Hz), 7.58
(d, 1H, J = 7.5 Hz), 7.49-7.43 (m, 2H), 7.28 (d, 2H, J = 7.6 Hz), 5.08 (d, 1H, J = 7.8
Hz, H-1), 3.99 (dd, 1H, J = 9.7, 7.8 Hz, H-2), 3.95 (d, 1H, J = 3.4 Hz, H-4), 3.83-3.73
(m, 3H, H-6a, H-6b, H-5), 3.64 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 3.35 (t, 2H, J = 6.7 Hz),
2.56 (t, 2H, J = 7.3 Hz), 1.83 (quintet, 2H, J = 7.6 Hz), 1.73-1.66 (m, 2H), 1.60-1.52
(m, 2H); 13C NMR (100 MHz, CD3OD): δ= 175.6, 155.0, 133.8, 131.3, 128.1, 127.3,
125.8, 124.8, 122.1, 117.3, 110.7, 103.2, 77.1, 75.0, 72.3, 70.3, 62.4, 52.3, 37.2, 29.7,
27.5, 26.6; HRMS (ESI): calc. for C22H28N4O7Na [M + Na] +, 483.1850, found
483.1838.
O-(4-(5-(6-Azidohexanamido)-1H-indol-1yl)phenyl)-β-D-galactopyranoside
(54). According protocol C. Compound 48 (45 mg, 0.065 mmol) was dissolved in 0.5
110
purification to furnish compound 54 (37 mg, quantitative yield). [𝛼]28
D -25.6 (c 1.0,
MeOH); 1H NMR (400 MHz, CD3OD): δ= 7.86 (d, 1H, J = 1.9 Hz), 7.43-7.36 (m, 4H),
7.28 (d, 2H, J = 8.6 Hz), 7.23 (dd, 1H, J = 8.8, 1.5 Hz), 6.59 (d, 1H, J = 3.0 Hz), 4.93
(d, 1H, J = 7.7 Hz, H-1), 3.93 (d, 1H, J = 3.3 Hz, H-4), 3.89 (dd, 1H, J = 9.6, 7.9 Hz,
H-2), 3.82-3.78 (m, 3H, H-6a, H-6b, H-5), 3.61 (dd, 1H, J = 9.6, 3.4 Hz, H-3),
3.33-3.31 (m, 2H), 2.40 (t, 2H, J = 7.4 Hz), 1.79-1.72 (m, 2H), 1.69-1.62 (m, 2H),
1.52-1.47 (m, 2H); 13C NMR (100 MHz, CD3OD): δ= 174.3, 157.7, 135.5, 134.8,
132.5, 130.6, 130.0, 126.4, 118.9, 117.8, 114.2, 111.2, 104.1, 103.1, 77.1, 74.8, 72.3,
70.2, 62.5, 52.3, 37.7, 29.7, 27.5, 26.6; HRMS (ESI): calc. for C26H31N5O7Na [M +
Na] +, 548.2116, found 548.2110.
O-(4-(Pent-4-ynamido)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside
(77). According protocol D. Compound 14 (32 mg, 0.077 mmol) and 4-pentynoic acid
(14 mg, 0.116 mmol) was dissolved in 0.5 mL CH2Cl2 at 0 °C, and DIEA (20 μL,
0.116 mmol), HATU (44 mg, 0.116 mmol) was added sequentially. After coupling
procedure and extraction in protocol D, the residue was concentrated and purified by
silica gel chromatography (EtOAc/hexane = 1:3 to 1:1) to give compound 77 (28 mg,
70%). [𝛼]27
D +3.9 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3): δ= 7.42-7.40 (m, 2H),
1
6.96-6.93 (m, 2H), 5.44 (dd, 1H, J = 10.4, 8.0 Hz, H-2), 5.42 (d, 1H, J = 3.5 Hz, H-4),
5.08 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.96 (, 1H, J = 8.0 Hz, H-1), 4.16 (m, 2H, H-6a,
H-6b), 4.02 (t, 1H, J = 6.6 Hz, H-5), 2.62-2.55 (m, 5H), 2.16, 2.05, 2.03, 1.99 (4s, 3H×
4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1, 169.4, 169.2, 153.5,
111
133.3, 121.5, 117.5, 100.0, 82.7, 71.0, 70.7, 69.6, 68.6, 66.9, 61.3, 36.0, 20.7,
20.6(2C), 20.5, 14.7; HRMS (ESI): calc. for C25H29NO11Na [M + Na] +, 542.1633,
found 542.1630.
O-(3-(Prop-2-yn-1-ylcarbamoyl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopy
ranoside (78). According protocol D. Compound 19 (79 mg, 0.168 mmol) and
propargyl amine (17 μL, 0.252 mmol) was dissolved in 1 mL CH2Cl2 at 0 °C, and
compound 78 (66 mg, 77%). [𝛼]28 1

δ= 7.46-7.42 (m, 2H), 7.34 (t, 1H, J = 7.9 Hz), 7.12 (ddd, 1H, J = 8.1, 2.5, 0.9 Hz),
5.47 (dd, 1H, J = 10.4, 7.9 Hz, H-2), 5.44 (d, 1H, J = 3.5 Hz, H-4), 5.09 (d, 1H, J = 8
Hz, H-1), 5.09 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.23-4.12 (m, 4H, CH2, H-6a, H-6b),
4.06 (td, 1H, J = 6.1, 0.8 Hz, H-5), 2.26 (t, 1H, J = 2.5 Hz), 2.16, 2.05, 2.04, 1.99 (4s,
3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ=170.5, 170.2, 170.0, 169.4, 166.3, 156.8,
135.3, 129.7, 121.4, 120.5, 115.6, 99.1, 79.4, 71.8, 71.2, 70.7, 68.4, 66.9, 61.4, 29.6,
20.7, 20.6(2C), 20.5; HRMS (ESI): calc. for C24H27NO11Na [M + Na] +, 528.1476,
found 528.1483.
112
O-(6-(Prop-2-yn-1-ylcarbamoyl)naphthalen-2-yl)-2,3,4,6-tetra-O-acetyl-β-D-
galactopyranoside (79). According protocol D. Compound 20 (44 mg, 0.085 mmol)
and propargyl amine (8 μL, 0.128 mmol) was dissolved in 0.5 mL CH2Cl2 at 0 °C, and
compound 79 (46 mg, 97 %). [𝛼]27 1

D +10.9 (c 0.5, CHCl3); H NMR (400 MHz,
CDCl3): δ= 8.25 (s, 1H), 7.85 (d, 1H, J = 9.0 Hz), 7.82 (dd, 1H, J = 8.6, 1.6 Hz), 7.77
(d, 1H, J = 8.6 Hz), 7.34 (d, 1H, J = 2.2 Hz), 7.23 (dd, 1H, J = 8.9, 2.4 Hz), 6.37 (t, 1H,
J = 4.8 Hz), 5.54 (dd, 1H, J = 10.4, 7.9 Hz, H-2), 5.48 (d, 1H, J = 3.4 Hz, H-4), 5.21
(d, 1H, J = 7.9 Hz, H-1), 5.14 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 4.30 (dd, 1H, J = 5.1,
2.6 Hz), 4.27-4.13 (m, 3H, H-6a, H-6b, H-5), 2.29 (t, 1H, J = 2.5 Hz), 2.18, 2.06, 2.05,
2.01 (4s, 3H×4, Ac); 13

C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1, 169.4,
116.9, 156.1, 135.8, 130.8, 129.8, 129.1, 127.6, 127.5, 124.4, 119.7, 110.9, 99.2, 79.5,
72.0, 71.2, 70.8, 68.6, 66.9, 61.5, 29.9, 20.7, 20.6(2C), 20.5; HRMS (ESI): calc. for
C28H29NO11Na [M + Na] +, 578.1633, found 578.1629.
O-(5-(Pent-4-ynamido)naphthalenyl)-2,3,4,6-tetra-O-acetyl-β-D-galactopyra
noside (80). According protocol D. Compound 9 (81 mg, 0.166 mmol) and
4-pentynoic acid (29 mg, 0.249 mmol) was dissolved in 1 mL CH2Cl2 at 0 °C, and
113
compound 80 (84 mg, 89%). [𝛼]27 1

δ= 7.97 (d, 1H, J = 8.5 Hz), 7.86 (d, 1H, J = 7.1 Hz), 7.77 (s, 1H), 7.62 (d, 1H, J = 8.4
Hz), 7.45 (t, 1H, J = 7.8 Hz), 7.39 (t, 1H, J = 7.8 Hz), 7.06 (d, 1H, J = 7.6 Hz), 5.68
(dd, 1H, J = 10.4, 8.0 Hz, H-2), 5.49 (d, 1H, J = 3.2 Hz, H-4), 5.18-5.13 (m, 2H, H-1,
H-3), 4.28-4.24 (m, 1H, H-6a), 4.19-4.11 (m, 2H, H-6b, H-5), 2.72-2.67 (m, 4H), 2.19,
2.06, 2.02, 2.01 (4s, 3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1,
170.1, 169.7, 153.2, 132.0, 128.7, 126.3, 125.8, 125.7, 122.4, 119.7, 116.1, 108.9, 99.9,
83.0, 71.1, 70.6, 69.9, 68.4, 66.8, 61.3, 36.0, 20.8, 20.7, 20.6, 20.6, 15.0; HRMS (ESI):
calc. for C29H31NO11Na [M + Na] +, 592.1789, found 592.1805.
O-(4-(5-(Pent-4-ynamido)-1H-indol-1-yl)phenyl)-2,3,4,6-tetra-O-acetyl-β-D-g
alactopyranoside (81). According protocol D. Compound 17 (55 mg, 0.099 mmol)
and 4-pentynoic acid (18 mg, 0.148 mmol) was dissolved in 0.5 mL CH2Cl2 at 0 °C,
and DIEA (27 μL, 0. 0.148 mmol), HATU (56 mg, 0.148 mmol) was added
sequentially. After coupling procedure and extraction in protocol D, the residue was
to give compound 81 (44 mg, 69%). [𝛼]28 1

D +6.5 (c 0.5, CHCl3); H NMR(400 MHz,
CDCl3): δ= 7.88 (d, 1H, J = 1.8 Hz), 7.44 (bs, 1H), 7.38-7.35 (m, 2H), 7.25-7.20 (m,
2H), 7.12 (d, 1H, J = 8.8 Hz), 6.60 (d, 1H, J = 3.1 Hz), 5.51 (dd, 1H, J = 10.4, 8.0 Hz,
114
H-2), 5.46 (d, 1H, J = 3.1 Hz, H-4), 5.12 (dd, 1H, J = 10.4, 3.4 Hz, H-3), 5.07 (d, 1H,
J = 7.9 Hz, H-1), 4.26-4.11 (m, 2H, H-6a, H-6b), 4.07 (app. t, 1H, J = 6.7 Hz, H-5),
2.65-2.58 (m, 4H), 2.18, 2.09, 2.04, 2.01 (4s, 3H×4, Ac); 13C NMR (100 MHz, CDCl3):
δ= 170.3, 170.2, 170.1, 169.4, 169.2, 155.2, 135.0, 133.4, 130.8, 129.2, 128.8, 125.5,
118.0, 116.6, 113.0, 110.4, 103.5, 99.7, 83.0, 71.1, 70.7, 69.5, 68.6, 66.8, 61.3, 36.1,
20.7, 20.6(2C), 20.5, 14.9; HRMS (ESI): calc. for C33H35N2O11 [M + H] +, 635.2235,
found 635.2251.
O-(4-(Pent-4-ynamido)phenyl)-β-D-galactopyranoside (82). According
protocol C. Compound 77 (18 mg, 0.034 mmol) was dissolved in 1 mL MeOH and
purification to furnish compound 82 (11 mg, 88%). [𝛼]34

D –45.2 (c 0.4, MeOH); H
1
7.7 Hz, H-1), 3.90 (d, 1H, J = 3.0 Hz, H-4), 3.81-3.72 (m, 3H, H-6a, H-2, H-6b),
3.68-3.64 (m, 1H, H-5), 3.59-3.56 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 2,57-2.53 (m, 4H),
2.28 (t, 1H, J = 2.3 Hz); 13C NMR (100 MHz, CD3OD): δ= 172.1, 155.9, 134.3, 122.7,
118.1, 103.3, 83.5, 77.0, 74.9, 72.3, 70.3, 70.2, 62.4, 36.7, 15.6; HRMS (ESI): calc.
for C17H21NO7Na [M + Na] +, 374.1210, found 374.1202.
115
O-(3-(Prop-2-yn-1-ylcarbamoyl)phenyl)-β-D-galactopyranoside (83).

D -50.0 (c 0.5, MeOH); H
NMR (400 MHz, CD3OD): δ= 7.55 (t, 1H, J = 1.9 Hz), 7.48-7.46 (m, 1H), 7.39 (t, 1H,
J = 7.9 Hz), 7.29 (ddd, 1H, J = 8.3, 2.5, 0.8 Hz), 4.92 (d, 1H, J = 7.7 Hz, H-1), 4.14 (d,
2H, J = 2.6 Hz), 3.91 (d, 1H, J = 2.2 Hz, H-4), 3.82 (dd, 1H, J = 9.6, 7.6 Hz, H-2),
3.81-3.71 (m, 2H, H-6a, H-6b), 3.59 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 2.60 (t, 1H, J = 2.5
Hz); 13C NMR (100 MHz, CD3OD): δ= 169.4, 159.3, 136.6, 130.7, 122.1, 121.4,
116.7, 102.9, 80.7, 77.0, 74.8, 72.2, 72.1, 70.2, 62.5, 30.0; HRMS (ESI): calc. for
C16H18NO7 [M - H] -, 336.1089, found 336.1104.
O-(6-(Prop-2-yn-1-ylcarbamoyl)naphthalen-2-yl)-β-D-galactopyranoside (84).
According protocol C. Compound 79 (23 mg, 0.041 mmol) was dissolved in 1 mL
purification to furnish compound 84 (20 mg, quantitative yield). [𝛼]34

D -61.5 (c 1.0,
MeOH); 1H NMR (400 MHz, CD3OD): δ= 8.32 (s, 1H), 7.91 (d, 1H, J = 9.0 Hz), 7.85
116
1H, J = 7.8 Hz, H-1), 4.20 (d, 2H, J = 2.4 Hz), 3.94 (d, 1H, J = 3.4 Hz, H-4), 3.88 (dd,
1H, J = 9.6, 7.8 Hz, H-2), 3.84-3.77 (m, 3H, H-6a, H-6b, H-5), 3.64 (dd, 1H, J = 9.6,
3.4 Hz, H-3), 2.62 (t, 1H, J = 3.5 Hz); 13C NMR (100 MHz, CD3OD): δ= 169.8, 158.5,
137.6, 131.6, 130.7, 130.1, 128.8, 128.6, 125.3, 121.0, 111.7, 102.8, 80.9, 77.2, 74.9,
72.3, 72.0, 70.3, 62.5, 30.0; HRMS (ESI): calc. for C20H22NO7 [M + H]+, 388.1391,
found 388.1384.
O-(5-(Pent-4-ynamido)naphthalenyl)-β-D-galactopyranoside (85). According
protocol C. Compound 80 (41 mg, 0.072 mmol) was dissolved in 0.5 mL MeOH and

D -55.9 (c 0.5, MeOH); H
NMR (400 MHz, CD3OD): δ= 8.36 (d, 1H, J = 8.4 Hz), 7.72 (d, 1H, J = 8.5 Hz), 7.59
(d, 1H, J = 6.6 Hz), 7.49-7.42 (m, 2H), 7.28(d, 1H, J = 7.6 Hz), 5.07 (d, 1H, J = 7.8
Hz, H-1), 3.99 (dd, 1H, J = 9.7, 7,8 Hz, H-2), 3.95 (d, 1H, J = 3.4 Hz, H-4), 3.81-3.73
(m, 3H, H-6a, H-6b, H-5), 3.64 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 2.76-2.73 (m, 2H),
2.65-2.61 (m, 2H), 2.39 (t, 1H, J = 2.6 Hz); 13C NMR (100 MHz, CD3OD): δ= 173.6,
154.9, 133.8, 131.3, 128.2, 127.3, 125.8, 124.8, 122.2, 117.6, 110.7, 103.2, 83.7, 77.1,
75.0, 72.4, 70.6, 70.3, 62.4, 36.4, 15.8; HRMS (ESI): calc. for C21H23NO7Na [M +
Na]+, 424.1367, found 424.1350.
117
O-(4-(5-(Pent-4-ynamido)-1H-indol-1-yl)phenyl)-β-D-galactopyranoside
(86).According protocol C. Compound 81 (36 mg, 0.056 mmol) was dissolved in 0.5
purification to furnish compound 86 (20 mg, 74.1%). [𝛼]30 1

D -30.0 (c 0.5, MeOH); H
NMR(400 MHz, CD3OD): δ= 7.86 (d, 1H, J = 1.8 Hz), 7.43-7.36 (m, 4H), 7.30-7.26
(m, 2H), 7.24 (dd, 1H, J = 8.9, 2.0 Hz), 6.59 (dd, 1H, J = 3.5, 0.6 Hz), 4.93 (d, 1H, J =
7.7 Hz, H-1), 3.93 (d, 1H, J = 3.3 Hz, H-4), 3.84 (dd, 1H, J = 9.7, 7.8 Hz, H-2),
3.83-3.71 (m, H-6a, H-6b, H-5), 3.61 (dd, 1H, J = 9.7, 3.4 Hz, H-3), 2.63-2.54 (m, 4H),
2.30 (t, 1H, J = 2.4 Hz); 13C NMR (100 MHz, CD3OD): δ= 172.3, 157.7, 135.5, 134.9,
132.4, 130.6, 130.1, 126.5, 119.0, 117.7, 114.2, 111.2, 104.2, 103.1, 83.6, 77.1, 74.9,
72.3, 70.3, 70.2, 62.5, 36.8, 15.7; HRMS (ESI): calc. for C25H26N2O7Na [M + Na]+,
489.1632, found 489.1626.
2,3,4,6-tetra-O-acetyl-β-D-galactopyranose (87) 89 . Benzyl amine (0.35 mL,
3.20 mmol) was added to a solution of compound 3 (1.00 g, 2.56 mmol) in 13 mL THF.
The reaction mixture was stirred at room temperature for 17 h. After removal of
solvents, CH2Cl2 was added to dissolve the residue and this solution was washed three
times with 1.0 M HCl, followed by saturated NaHCO3(aq) and brine, and dried over
118
anhydrous sodium sulfate. After concentration, the crude product was purified by flash
column chromatography (EtOAc/hexane = 2:1), giving product 87 (0.68 g, 76%). 1H
NMR (400 MHz, CDCl3): δ= 5.46 (d, 1H, J = 3.6 Hz, H-1), 5.42 (d, 1H, J = 2.4 Hz,
H-4), 5.36 (dd, 1H, J = 10.6, 3.4 Hz, H-3), 5.09 (dd, 1H, J = 10.8, 3.6 Hz, H-2), 4.42 (t,
1H, J = 6.6 Hz, H-5), 4.05 (dd, 1H, J = 6.7, 2.1 Hz, H-6a, H-6b), 2.10, 2.05, 2.00, 1.95
(4s, 3H×4, Ac).
2-propynyl 2,3,4,6-tetra-O-acetyl-β-D-galactopyranoside (89) 90 . Compound
87 (0.68 g, 1.95 mmol) was dissolved in 10 mL of anhydrous CH2Cl2 and treated with
trichloroacetonitrile (1.96 mL, 19.5 mmol). This solution was cooled in an ice bath for
10 min and treated with DBU (0.55 mL, 3.9 mmol). The reaction was stirred at 0 oC
for 2 h and the solvent was removed. The crude product was purified by flash column
chromatography (EtOAc/hexane = 1:2), then concentrated in vacuum for 3 h, giving
trichloroacetimidate residue. The trichloroacetimidate, propargyl alcohol (0.774 mL,
13.3 mmol) and freshly dried 4 Å molecular sieves (0.8 g) in dry CH2Cl2 (7 mL) was
stirred at room temperature for 1 h under argon. The mixture was cooled to -40 oC.
TMSOTf (59 μL, 0.266 mmol) was added to the reaction mixture. After 3 h, the
reaction was quenched by addition of Et3N. The resulting solution warmed up to room
temperature, and filtred through celite. The filtrate was washed with satured
NaHCO3(aq), brine and water. After concentration, the crude product was purified by
flash column chromatography. (EtOAc/hexane = 1:2), giving product 89 (0.40 g, 53%
for two steps). [𝛼]28

D +10.5 (c 0.5, CHCl3); H NMR (400 MHz, CDCl3): δ= 5.37 (d,
1
1H, J = 3.5, 0.9 Hz, H-4), 5.19 (dd, 1H, J = 10.4, 8.0 Hz, H-2), 5.03 (dd, 1H, J = 10.4,
119
3.4 Hz, H-3), 4.71 (d, 1H, J = 8.0 Hz, H-1), 4.35 (d, 1H, J = 2.4 Hz), 4.18-4.06 (m, 2H,
H-6a, H-6b), 3.91 (td, 1H, J = 6.8, 0.8 H, H-5), 2.44 (, 1H, J = 2.4 Hz), 2.12, 2.04,
2.02, 1.96 (4s, 3H×4, Ac); 13C NMR (100 MHz, CDCl3): δ= 170.3, 170.2, 170.1, 169.5,
98.6, 78.2, 75.3, 70.8, 70.7, 68.5, 66.9, 61.2, 55.9, 20.7, 20.6(2C), 20.5.
1-propynyl-β-D-galactopyranoside (90)91. According protocol C. Compound
89 (91 mg, 0.236 mmol) was dissolved in 1 mL MeOH and stirred at 0 °C, then 22 μL
30% NaOMe in methanol solution was added. After deacetylation procedure, the
residue were evaporated in vacuum without further purification to furnish compound
90 (53 mg, quantitative yield). [𝛼]29 1

D -40.2 (c 0.5, MeOH); H NMR (400 MHz,
CD3OD): δ= 4.43-4.41 (m, 3H, H-1, CH2), 3.83 (dd, 1H, J = 3.3, 0.9 Hz, H-4),
3.79-3.69 (m, 2H, H-6a, H-6b), 3.55-3.4 (m, 3H, H-5, H-2, H-3), 2.85 (t, 1H, J = 2.5
Hz). HRMS (ESI): calc. for C9H14O6Na [M + Na] +, 241.0683, found 241.0679.
120
General procedures for solid-phase peptide synthesis. Rink amide resin
(50-100 mg, 0.55-0.7 mmol/g dertermined by loading test) was swollen in DCM for 30
min then washed with DCM (3 × 3 mL) and DMF (3 × 3 mL). The resin was treated with
piperidine (20 vol % in DMF, 2 × 3 mL) for 15 min then washed with DMF (3 × 3 mL),
DCM (3 × 3 mL) and DMF (3 × 3 mL). A preactived solution of Fmoc-protected amino
acids (4 equiv.), HOBT (4 equiv.), HBTU (4 equiv.) and DIEA (4 equiv.) in DMF (final
concentration 0.2 M) was added and resin shaken at 40 oC for 1 h. The resin was washed
with DMF (3 × 3 mL), DCM (3 × 3 mL) and DMF (3 × 3 mL). The resin was treated with
Ac2O (10 vol % in pyr. 3 mL) for 10 min. The resin was washed with DMF (3 × 3 mL),
DCM (3 × 3 mL) and DMF (3 × 3 mL). This procedure was repeated until all amino acids
were coupled to the resin.
Cleavage of peptide from Resin and Purification. The resin was treated with a
mixture of TFA, triisopropylsilane and water (95:2.5:2.5 v/v/v, 2 × 2 mL), the reaction
mixture was shaken at r.t. for 1 h and then filtered. The resin was washed with DCM (3 ×
3 mL), and the washes combined with the filtrate. The combined filtrates were
concentrated. The residue was dissolved in acetonitrile and purified by reverse phase
HPLC using a linear gradient of a solvent system consisting of solvent A (water/0.1%
TFA) and solvent B (acetonitrile). All peptides were characterized by MALDI-TOF mass
spectrometry.
121
Rink amide resin (100 mg, 0.064 mmol) as starting material to SPPS. MALDI-TOF:
calc. for C38H51N7 O9Na [M + Na]+ 772.36, found 772.97; HPLC method (10 to 15%
solvent B over 5 min, then 15 to 25% solvent B over 15 min ), Rt = 19.3 min, 76% yield.
calc. for C53H72N10O12Na [M + Na]+ 1063.52, found 1063.89; HPLC method (10 to 15%
122
solvent B over 25 min), Rt = 25.1min, 56% yield.
General procedures for copper(I)-catalyzed alkyne-azide cycloaddition of
glycopeptide synthesis:
Compound 55-75. To a solution of azido glycoside (2.5 equiv., 80-120 mM) in
DMSO and alkynyl peptide (1 equiv, 10 mM) in H2O were premixed, then CuSO4•5H2O,
sodium ascorbate and ligand were added to reach 1 mM Cu(I) as final concentration. The
reaction was stirred at r.t. for 1 h. The reaction mixture was purified by reverse phase
HPLC using a linear gradient of a solvent system consisting of solvent A (acetonitrile)
and solvent B (water/0.1% TFA). The product was characterized by MALDI-TOF mass
spectrometry.
Data for 55: MALDI-TOF: calc. for C74H103N15O23Na [M + Na]+ 1592.72, found 1592.86;
123
HPLC method (10 to 95% solvent B over 45 min ), Rt = 13.7 min, 86% yield.
HPLC method (10 to 15% solvent B over 5 min, then 15 to 27% solvent B over 25 min ),
Rt = 13.9 min, 89% yield.
Rt = 18.9min, 87% yield.
HPLC method (10 to 35% solvent B over 15 min ), Rt = 16.2min, 83% yield.
HPLC method (10 to 95% solvent B over 45 min ), Rt = 19min, 70% yield.

124
Data for 65: MALDI-TOF: calc. for C108H163N19O47Na [M + Na]+ 2501.08, found
2500.83; HPLC method (10 to 95% solvent B over 45 min ), Rt = 11.2min, 98% yield.
Data for 69: MALDI-TOF: calc. for C112H150N21O29 [M + H]+ 2253.08, found 2253.96;
Data for 70: MALDI-TOF: calc. for C120H156N23O29 [M + H]+ 2383.15, found 2384.14;
125
General procedures for copper(I)-catalyzed alkyne-azide cycloaddition of lipid
conjugation:
Compound 91-96. Azido lipid (1 equiv, 10 mM) was dissolved in the solution of
alkynyl glycoside 82-86、90 (2 equiv., 40 mM) in MeOH, then CuSO4•5H2O, sodium
ascorbate and ligand were added to reach 1 mM Cu(I) as final concentration. The reaction
was stirred at r.t. for 1 h. The reaction mixture was purified by C8 SPE (solid extraction
column) using using a 10% stepwise gradient from 0-100% MeOH in water. Fractions
with the desired compound was combined, reduced in vacuum and lyophilized from a
mixture of water and methanol to give the desired compound as a white powder. The
product was characterized by MALDI-TOF mass spectrometry.
126
Data for commercial azido lipid 76 : MALDI-TOF : 2949.91± n×44.0
Data for 91: MALDI-TOF: found 3192.06 ± n×44.0, Apart from 76 for 242.15, calc. for
C9H14O6Na [M + Na]+ 241.09. Quantitative yield.
C17H21NO7Na [M + Na]+ 374.14. Quantitative yield.
C16H19NO7Na [M + Na]+ 360.13. 99% yield.
C20H21NO7Na [M + Na]+ 410.14. 97% yield.
Data for 95: MALDI-TOF: found 3353.69± n×44.0, Apart from 76 for 403.78, calc. for
C21H23NO7 [M +H]+ 402.15. Quantitative yield.
Data for 96: MALDI-TOF: found 3440.83± n×44.0, Apart from 76 for 490.92, calc. for
C25H26N2O7Na [M +Na]+ 489.18. Quantitative yield.
127
128
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附錄
附錄目錄
化合物 1 之 1H 核磁共振光譜 ..................................................................................... 1
化合物 2 之 1H 核磁共振光譜 ..................................................................................... 2
化合物 3 之 1H 核磁共振光譜 ..................................................................................... 3
化合物 4 之 1H 核磁共振光譜 ..................................................................................... 4
化合物 5 之 1H 核磁共振光譜 ..................................................................................... 5
化合物 5 之 13C 核磁共振光譜 .................................................................................... 6
化合物 6 之 1H 核磁共振光譜 ..................................................................................... 7
化合物 6 之 13C 核磁共振光譜 .................................................................................... 8
化合物 7 之 1H 核磁共振光譜 ..................................................................................... 9
化合物 8 之 1H 核磁共振光譜 ................................................................................... 10
化合物 8 之 13C 核磁共振光譜 ...................................................................................11
化合物 9 之 1H 核磁共振光譜 ................................................................................... 12
化合物 9 之 13C 核磁共振光譜 .................................................................................. 13
化合物 10 之 1H 核磁共振光譜 ................................................................................. 14
化合物 10 之 13C 核磁共振光譜 ................................................................................ 15
化合物 11 之 1H 核磁共振光譜.................................................................................. 16
化合物 12 之 1H 核磁共振光譜 ................................................................................. 17
化合物 12 之 13C 核磁共振光譜 ................................................................................ 18
化合物 13 之 1H 核磁共振光譜 ................................................................................. 19
化合物 14 之 1H 核磁共振光譜 ................................................................................. 20
化合物 15 之 1H 核磁共振光譜 ................................................................................. 21
化合物 15 之 13C 核磁共振光譜 ................................................................................ 22
化合物 16 之 1H 核磁共振光譜 ................................................................................. 23
化合物 16 之 13C 核磁共振光譜 ................................................................................ 24
化合物 17 之 1H 核磁共振光譜 ................................................................................. 25
化合物 17 之 13C 核磁共振光譜 ................................................................................ 26
化合物 18 之 1H 核磁共振光譜 ................................................................................. 27
化合物 19 之 1H 核磁共振光譜 ................................................................................. 28
化合物 19 之 13C 核磁共振光譜 ................................................................................ 29
化合物 20 之 1H 核磁共振光譜 ................................................................................. 30
化合物 20 之 13C 核磁共振光譜 ................................................................................ 31
化合物 21 之 1H 核磁共振光譜 ................................................................................. 32
化合物 21 之 13C 核磁共振光譜 ................................................................................ 33
化合物 22 之 1H 核磁共振光譜 ................................................................................. 34
化合物 22 之 13C 核磁共振光譜 ................................................................................ 35
化合物 23 之 1H 核磁共振光譜 ................................................................................. 36
化合物 23 之 13C 核磁共振光譜 ................................................................................ 37
化合物 24 之 1H 核磁共振光譜 ................................................................................. 38
化合物 24 之 13C 核磁共振光譜 ................................................................................ 39
化合物 25 之 1H 核磁共振光譜 ................................................................................. 40
化合物 25 之 13C 核磁共振光譜 ................................................................................ 41
化合物 26 之 1H 核磁共振光譜 ................................................................................. 42
化合物 26 之 13C 核磁共振光譜 ................................................................................ 43
化合物 27 之 1H 核磁共振光譜 ................................................................................. 44
化合物 27 之 13C 核磁共振光譜 ................................................................................ 45
化合物 28 之 1H 核磁共振光譜 ................................................................................. 46
化合物 28 之 13C 核磁共振光譜 ................................................................................ 47
化合物 29 之 1H 核磁共振光譜 ................................................................................. 48
化合物 29 之 13C 核磁共振光譜 ................................................................................ 49
化合物 30 之 1H 核磁共振光譜 ................................................................................. 50
化合物 30 之 13C 核磁共振光譜 ................................................................................ 51
化合物 31 之 1H 核磁共振光譜 ................................................................................. 52
化合物 31 之 13C 核磁共振光譜 ................................................................................ 53
化合物 32 之 1H 核磁共振光譜 ................................................................................. 54
化合物 32 之 13C 核磁共振光譜 ................................................................................ 55
化合物 33 之 1H 核磁共振光譜 ................................................................................. 56
化合物 34 之 1H 核磁共振光譜 ................................................................................. 57
化合物 35 之 1H 核磁共振光譜 ................................................................................. 58
化合物 36 之 1H 核磁共振光譜 ................................................................................. 59
化合物 37 之 1H 核磁共振光譜 ................................................................................. 60
化合物 37 之 13C 核磁共振光譜 ................................................................................ 61
化合物 38 之 1H 核磁共振光譜 ................................................................................. 62
化合物 38 之 13C 核磁共振光譜 ................................................................................ 63
化合物 39 之 1H 核磁共振光譜 ................................................................................. 64
化合物 39 之 13C 核磁共振光譜 ................................................................................ 65
化合物 40 之 1H 核磁共振光譜 ................................................................................. 66
化合物 40 之 13C 核磁共振光譜 ................................................................................ 67
化合物 45 之 1H 核磁共振光譜 ................................................................................. 68
化合物 45 之 13C 核磁共振光譜 ................................................................................ 69
化合物 46 之 1H 核磁共振光譜 ................................................................................. 70
化合物 46 之 13C 核磁共振光譜 ................................................................................ 71
化合物 47 之 1H 核磁共振光譜 ................................................................................. 72
化合物 47 之 13C 核磁共振光譜 ................................................................................ 73
化合物 48 之 1H 核磁共振光譜 ................................................................................. 74
化合物 48 之 13C 核磁共振光譜 ................................................................................ 75
化合物 49 之 1H 核磁共振光譜 ................................................................................. 76
化合物 49 之 13C 核磁共振光譜 ................................................................................ 77
化合物 50 之 1H 核磁共振光譜 ................................................................................. 78
化合物 50 之 13C 核磁共振光譜 ................................................................................ 79
化合物 51 之 1H 核磁共振光譜 ................................................................................. 80
化合物 51 之 13C 核磁共振光譜 ................................................................................ 81
化合物 52 之 1H 核磁共振光譜 ................................................................................. 82
化合物 52 之 13C 核磁共振光譜 ................................................................................ 83
化合物 53 之 1H 核磁共振光譜 ................................................................................. 84
化合物 53 之 13C 核磁共振光譜 ................................................................................ 85
化合物 54 之 1H 核磁共振光譜 ................................................................................. 86
化合物 54 之 13C 核磁共振光譜 ................................................................................ 87
化合物 77 之 1H 核磁共振光譜 ................................................................................. 88
化合物 77 之 13C 核磁共振光譜 ................................................................................ 89
化合物 78 之 1H 核磁共振光譜 ................................................................................. 90
化合物 78 之 13C 核磁共振光譜 ................................................................................ 91
化合物 79 之 1H 核磁共振光譜 ................................................................................. 92
化合物 79 之 13C 核磁共振光譜 ................................................................................ 93
化合物 80 之 1H 核磁共振光譜 ................................................................................. 94
化合物 80 之 13C 核磁共振光譜 ................................................................................ 95
化合物 81 之 1H 核磁共振光譜 ................................................................................. 96
化合物 81 之 13C 核磁共振光譜 ................................................................................ 97
化合物 82 之 1H 核磁共振光譜 ................................................................................. 98
化合物 82 之 13C 核磁共振光譜 ................................................................................ 99
化合物 83 之 1H 核磁共振光譜 ............................................................................... 100
化合物 83 之 13C 核磁共振光譜 .............................................................................. 101
化合物 84 之 1H 核磁共振光譜 ............................................................................... 102
化合物 84 之 13C 核磁共振光譜 .............................................................................. 103
化合物 85 之 1H 核磁共振光譜 ............................................................................... 104
化合物 85 之 13C 核磁共振光譜 .............................................................................. 105
化合物 86 之 1H 核磁共振光譜 ............................................................................... 106
化合物 86 之 13C 核磁共振光譜 .............................................................................. 107
化合物 87 之 1H 核磁共振光譜 ............................................................................... 108
化合物 89 之 13C 核磁共振光譜 ...............................................................................110
化合物 90 之 1H 核磁共振光譜 ................................................................................ 111
化合物 92 之 MALDI-TOF 質譜圖 ..........................................................................112
9.0
7.760
7.673
7.606
8.5
7.589
7.588
7.352
7.334
8.0
1.00 7.303
1.05 7.300
1.02 7.293
7.5
2.08 7.284
3.04 7.264
7.244
1.09
7.217
7.0
1.03 7.198
7.178
7.089
6.5
7.086
7.083
7.069
7.066
6.0
7.063
5.280
7.609
5.5
2.02
5.0
1
1
4.5
4.0
化合物 1 之 H 核磁共振光譜
化合物 1 之 1H 核磁共振光譜
3.5
3.0
2.5
2.0
1.5
1.0
0.5
ppm
8.557
9.0
8.035
8.013
7.820
1.00 7.799
8.5
7.651
7.634
1.04 7.630
8.0
7.495
1.12 7.476
1.13 7.419
1.95 7.402
7.5
2.73 7.383
2.14 7.367
7.350
7.0
7.332
7.240
7.183
7.165
6.5
7.161
7.157
7.151
6.0
7.145
譜
5.5
2.19 5.434
5.0
2
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.007
-0.001
ppm
9.0
8.5
8.0
7.5
7.240
7.0
6.317
6.5
6.308
1.00 5.705
5.684
5.478
6.0
5.454
5.428
5.149
5.5
1.04 5.124
5.098
2.27 5.089
5.072
5.0
5.065
1
5.063
4.272
3
4.5
4.261
1.13 4.240
2.19 4.230
4.114
4.0
4.104
4.091
4.089
化合物 3 之 H 核磁共振光譜
3.5
4.062
4.056
3.0
2.5
2.95 2.163
2.98 2.076
3.14 2.024
2.0
3.32 2.009
2.93 1.998
1.5
1.0
0.5
ppm
9.0
8.5
8.203
1.91 8.180
8.0
7.5
7.240
7.074
2.00 7.069
7.056
7.0
7.051
6.5
5.523
5.503
5.497
6.0
5.477
5.463
0.90 5.455
5.5
0.98 5.161
5.142
1.04 5.133
0.99 5.124
5.0
5.107
5.098
4.223
4
4.5
4.206
4.196
3.34 4.180
4.161
4.0
4.146
4.136
4.120
3.5
4.105
4.087
3.0
2.5
3.00 2.169
2.051
6.04
2.021
2.0
3.06 2.001
1.5
1.0
0.5
-0.029
ppm
7.674
7.425
7.421
9.0
7.403
7.385
7.381
7.376
8.5
7.364
7.349
7.343
8.0
7.335
0.96 7.331
0.92 7.323
7.318
7.5
6.13 7.309
1.02 7.300
7.240
7.0
7.194
7.191
7.187
7.185
6.5
7.173
7.171
7.167
6.0
7.165
5.502
5.482
1.07 5.476
5.5
0.96 5.456
1.89 5.445
2.00 5.436
5.0
5.335
5.332
5.113
5
5.105
4.5
5.092
1.97 5.088
5.079
1.00
4.0
5.072
4.162
4.146
4.079
3.5
4.063
4.046
2.160
3.0
2.038
2.018
1.993
-0.024
2.5
7.784
2.79 7.759
2.98 7.680
2.89
2.0
3.14
1.5
1.0
0.5
ppm
210
200
190
180
170.42
170.18
170.05
170
169.33
165.72
160
156.75
150
135.84
140
131.71
129.58
128.60
130
128.31
128.15
124.54
120
122.01
117.57
6
110
99.37
100
90
化合物 5 之 13C 核磁共振光譜

77.32
80 77.00
76.68
71.22
70.77
70
68.54
66.88
61.38
60
50
20.68
40
20.62
20.54
77.20
30
20
ppm
9.0
8.5
8.0
7.576
7.569
7.564
1.92 7.551
7.5
7.547
7.240
6.767
7.0
6.760
2.00 6.756
6.742
6.738
6.5
5.465
5.457
5.446
6.0
5.439
5.427
5.419
5.088
5.5
1.97
5.080
5.062
1.01 5.054
5.0
0.97 4.982
4.963
4.210
7
4.192
4.5
4.182
2.45 4.164
1.01 4.142
4.0
4.127
4.114
4.098
3.5 4.080
4.063
4.042
4.026
3.0
4.009
2.5
3.03 2.154
6.45 2.035
2.0
2.87 1.985
1.5
1.0
0.5
ppm
210
200
190
180
170.319
170.185
170.087
170
169.325
160
156.662
150
138.417
140
130
119.115
120
8
110
99.398
100
90

86.100
77.317
80 77.000
76.682
71.043
70.667
70
68.435
66.722
61.289
60
50
40
30
20.697
20.646
20
20.636
20.556
ppm
9.0
8.5
8.0
7.5
7.240
6.893
7.0
6.874
2.07 6.864
6.845
6.5
5.528
5.508
6.0
5.502
5.482
5.370
0.93 5.362
5.5
0.98 5.077
5.068
0.77 5.050
5.0
5.042
1.00 4.752
4.732
9
4.104
4.5
4.087
4.076
1.98 4.064
4.0
4.060
0.81 4.047
4.037
3.5 4.019
3.763
3.746
3.729
3.0
0.40 2.223
2.5
2.80 2.202
3.02 2.186
1.89 2.154
3.13 2.106
2.0
3.02 1.995
2.88 1.929
1.5
1.0
0.5
ppm
8.5580
8.0691
8.0654
9.0
8.0476
8.0438
7.8629
0.96 7.8405
8.5
7.7433
7.7217
0.97 7.4665
7.4490
8.0
0.98 7.3947
0.98 7.3772
7.3589
2.00 7.3450
7.5
4.08 7.3411
0.95 7.3304
7.3239
7.3166
7.0
7.3058
7.2400
7.2313
6.5
7.2254
7.2089
7.2030
5.5680
6.0
5.5482
5.5420
1.04 5.5222
1.00 5.4827
5.5
5.4745
1.99 5.3827
0.99 5.2370
1.01 5.2172
5.0
5.1700
5.1618
5.1441
5.1357
10
4.5
4.2448
4.2353
3.10 4.2274
4.2127
4.0
4.1896
4.1756
4.1577
3.5 4.1503
4.1365
3.0
2.5
2.77 2.1681
2.92 2.0524
2.76 2.0393
2.0
2.0053
3.14
1.5
1.0
0.5
ppm
210
200
190
180
170.2172
170.1137
169.9805
170
169.2778
166.3548
160
156.3413
150
136.5358
135.9698
131.1635
140
130.8970
128.9197
128.5258
130
128.1667
127.1408
126.1697
120
126.1066
119.4486
110.7639
11
110
99.0562
100
90

77.3181
80 77.0000
76.6818
71.1661
70.6962
70
68.4931
66.8268
66.7152
60
61.4520
50
20.6281
20.5590
20.4761
40
20.5375
128.1911
30
20
ppm
9.0
8.5
7.5334
8.0
7.5116
7.4892
7.3119
2.08 7.2920
7.5
1.13 7.2716
1.09 7.2624
7.2440
0.97 7.2228
7.0
7.0255
1.03 7.0066
6.7581
6.5
6.7401
5.6878
5.6679
6.0
5.6618
5.6418
1.06 5.4701
1.00 5.4623
5.5
5.1596
5.1510
2.14 5.1350
5.1247
5.0
5.1154
4.2569
4.2393
4.2289
12
4.5
4.2114
3.24 4.1614
1.24 4.1462
4.1336
4.0
4.1183
4.1045
4.0872
3.5 4.0711
4.0550
3.0
2.5
3.10 2.1528
3.28 2.0381
2.0081
2.0
6.31 2.0040
1.5
1.0
0.5
-0.0301
ppm
210
200
190
180
170.2400
170.1869
170.0182
170
169.5686
160
153.1728
150
141.9449
140
126.4945
130
126.3239
124.4660
124.1633
120
115.8176
112.0664
110.3028
13
110
108.9635
99.8722
100
90

77.3185
80 77.0000
76.6815
70.9007
70.6429
70
68.4149
66.8250
61.2929
60
50
40
30
20.6745
20
20.4919
ppm
7.529
8.5
7.527
7.522
7.512
7.490
8.0
7.423
7.405
4.29 7.386
7.327
7.5
2.33
7.325
1.08 7.307
2.18 7.288
7.0
7.240
7.066
7.061
7.045
6.5
7.037
5.523
5.503
5.497
6.0
5.477
5.458
1.16 5.449
5.5
1.00 5.130
5.121
1.20 5.104
1.00 5.095
5.0
5.083
5.063
4.256
4.239
4.5
4.228
14
3.82 4.210
4.178
4.0
4.163
4.150
4.135
4.122
3.5 4.105
4.087
4.070
4.054
3.0
2.5
3.00 2.172
2.066
6.43 2.046
2.0
2.86 2.004
1.5
1.0
0.5
-0.022
ppm
210
200
190
180
170.30
170.21
170.08
170
169.35
160
156.37
150
140.35
140
136.43
128.73
128.21
130
127.04
126.83
120
117.14
15
110
99.62
100
90

77.32
80 77.00
76.68
71.00
70.80
70
68.63
66.85
61.33
60
50
40
30
20.68
20.60
20
20.53
ppm
7.371
7.359
7.355
7.340
9.0
7.321
7.317
7.313
8.5
7.307
7.300
7.293
7.286
8.0
7.283
7.279
7.240
7.5
5.00 6.947
6.939
6.933
6.922
7.0
4.39 6.916
6.908
6.886
6.5
6.879
6.872
6.862
6.856
6.0
6.847
5.457
5.437
5.5
2.13 5.431
5.423
1.19 5.422
2.17 5.412
5.0
1.14 5.081
5.072
5.055
16
4.5
5.046
5.005
2.31 4.907
1.14 4.887
4.0
4.233
4.215
4.205
3.5
4.187
4.152
4.136
4.124
3.0
4.108
4.002
3.985
2.5
3.968
3.12 2.161
3.30 2.067
3.56 2.060
2.0
3.25 2.024
1.990
-0.022
1.5
3.999
3.983
3.966
1.0
0.5
ppm
8.891
8.884
8.880
9.0
0.94 8.128
8.124
8.107
8.103
8.5
7.562
7.556
0.96 7.545
8.0
7.538
7.454
0.96 7.435
7.423
7.5
3.22 7.412
7.402
7.391
7.0
7.381
7.240
5.716
5.696
6.5
5.690
5.670
5.455
6.0
5.453
5.447
0.97 5.444
0.94 5.351
5.5
1.00 5.331
0.99 5.275
5.170
5.0
5.162
5.144
5.136
17
4.252
4.5
4.235
2.14 4.224
1.02 4.207
4.0
4.180
4.163
4.152
4.135
3.5
4.036
4.034
4.020
3.0
4.017
4.003
4.000
2.5
3.10 2.170
3.09 2.076
2.010
2.0
5.83 2.007
1.5
1.0
0.5
-0.026
ppm
210
200
190
180
170.28
170.24
170.16
170
169.70
160
152.67
149.76
150
140.97
140
135.72
129.47
130
126.23
123.27
121.54
120
116.72
18
110
101.20
100
90

77.32
80 77.00
76.68
70.98
70.83
70
68.78
66.94
61.26
60
50
40
30
20.86
20.57
20
20.61
ppm
7.823
7.813
7.808
9.0
7.803
7.799
7.792
7.614
8.5
7.612
0.94 7.608
7.598
1.00
8.0
7.593
3.99 7.588
1.05 7.577
1.92 7.574
7.5
0.93 7.571
1.26 7.507
7.488
7.0
7.470
7.381
7.375
7.248
6.5
7.240
7.226
7.220
6.0
5.575
5.555
1.11 5.548
5.529
5.5
0.98
0.93 5.487
5.480
0.94 5.249
5.0
5.230
5.169
5.160
19
5.143
4.5
5.135
3.24 4.282
4.259
4.0
4.249
4.227
4.216
4.197
3.5
4.183
4.171
4.169
3.0
4.165
4.150
4.116
2.5
3.34 2.179
2.062
5.64
2.057
2.0
2.88 2.009
1.5
1.0
0.5
ppm
9.0
8.5
8.0
7.240
6.806
6.798
6.792
7.5
6.781
6.776
6.767
7.0
6.574
1.93 6.565
6.560
1.96
6.548
6.5
6.543
6.535
5.408
6.0
5.387
5.383
5.378
5.362
5.5
2.04 5.050
5.041
1.02 5.024
5.0
5.015
1.00 4.830
4.810
20
4.200
4.5
4.183
2.28 4.172
4.155
4.0
1.03 4.122
4.106
4.094
3.5 4.077
4.059
4.042
3.961
3.0
3.944
3.928
2.5
3.00 2.124
3.06 2.036
2.000
2.0
2.91
3.05 1.958
1.5
1.0
0.5
ppm
9.0
7.771
7.750
7.732
7.712
8.5
7.551
7.533
7.515
8.0
7.513
2.08 7.466
1.30 7.444
7.290
7.5
2.31
7.272
1.08
7.254
7.252
2.02
7.0
7.240
1.00 6.985
6.963
6.706
6.5
5.480
5.460
5.454
6.0
5.434
5.426
5.418
5.255
5.5
2.07
5.103
0.40 5.095
1.03 5.077
5.0
0.99 5.069
4.958
4.955
21
4.938
4.5
4.936
2.22 4.231
1.01 4.213
4.0
4.202
4.185
4.149
4.133
3.5
4.120
4.104
4.007
3.0
3.990
3.974
3.14 2.530
2.5
3.24 2.151
3.23 2.066
3.25 2.024
2.0
2.98 1.992
1.5
1.0
0.5
-0.033
ppm
210
200
190
180
170.2661
170.1807
170
170.0540
169.3446
160
154.2890
152.9381
150
146.7974
146.0426
140
135.6504
129.6326
126.3499
130
124.1266
123.5635
122.8362
120
122.4649
117.9544
111.5021
22
110
100.2362
100
90

77.3180
80 77.0000
76.6819
70.8832
70.7804
70
68.6354
66.8275
61.2440
60
50
40
30
20.6817
20.5708
20
20.5119
18.8515
ppm
8.093
8.087
8.070
8.065
9.0
7.420
7.413
1.00 7.405
8.5
7.399
7.396
1.03 7.387
7.383
8.0
7.375
7.240
7.186
7.5
4.05 7.178
7.172
2.17 7.161
7.156
7.0
1.01 7.147
6.825
6.823
6.5
6.816
6.815
5.543
5.523
6.0
5.516
5.497
1.15 5.476
5.5
1.01 5.475
5.468
2.09 5.466
5.151
5.0
5.143
5.124
5.117
23
4.5
5.104
1.07 4.275
1.12 4.258
1.16 4.247
4.0
4.230
4.182
4.166
3.5
4.154
4.138
4.120
4.113
3.0
4.103
4.096
4.085
2.5
4.080
2.77 2.186
2.98 2.091
3.19 2.046
2.0
3.22 2.014
1.5
1.0
0.5
ppm
190
180
170.18
170.06
170
169.95
169.26
160
155.94
150
141.89
138.74
140
133.57
131.31
130
128.09
126.01
118.09
120
117.98
117.70
110.13
110
105.32
24
99.32
100
90
77.32
80

77.00
76.68
71.02
70.58
70
68.40
66.69
61.19
60
50
40
30
20.61
20.50
20
20.43
ppm
7.382
9.0
7.377
7.365
7.360
7.279
8.5
7.257
7.240
7.177
8.0
7.169
7.118
0.98 7.110
7.104
7.5
0.98
1.09 7.093
0.86 7.088
6.948
2.00
7.0
6.942
0.93 6.666
0.95 6.660
6.644
6.5
0.90
6.638
6.462
6.454
6.0
5.526
5.506
1.08 5.500
5.480
5.5
0.92 5.461
1.07 5.454
1.00 5.135
5.0
5.126
5.108
5.100
25
5.070
4.5
1.11 5.050
1.07 4.264
1.01 4.247
4.0
4.236
4.218
4.179
4.163
3.5
4.150
4.135
4.081
3.0
4.063
4.046
4.018
2.5
3.19 2.182
3.13 2.086
3.05 2.041
2.0
3.04 2.011
1.5
1.0
0.5
ppm
210
200
190
180
170.28
170.19
170.08
170
169.34
160
154.89
150
140.09
140
135.48
130.87
130.09
130
128.19
125.25
120
117.94
113.00
110.84
26
110
105.67
102.32
99.77
100
90

77.32
80 77.00
76.68
71.05
70.77
70
68.59
66.82
61.31
60
50
40
30
20.71
20.61
20
20.54
ppm
9.0
8.5
8.382
1.00 8.370
8.0
7.489
7.470
7.451
0.96 7.240
7.5
7.177
0.90 7.157
0.91 7.019
7.0
7.006
7.004
7.000
6.988
6.5
5.741
6.0
5.715
1.02 5.415
5.407
5.366
5.5
0.91
0.89 5.341
5.315
0.96 5.133
5.0
5.125
5.108
5.100
27
4.5
3.49 4.039
4.0
3.5
3.0
2.5
2.77 2.088
3.20 1.940
2.0
1.919
5.78 1.908
1.5
1.0
0.5
ppm
9.0
8.5
7.818
8.0
1.12 7.799
1.08 7.711
7.405
7.5
1.07 7.385
7.365
0.93
7.240
7.218
7.0
6.5
5.519
5.500
6.0
5.493
5.473
5.460
1.17 5.452
5.5
1.00 5.141
5.133
2.10 5.116
5.0
5.107
5.099
4.188
4.172
28
4.5
4.130
2.22 4.114
1.24 4.098
4.0
7.236
3.5
3.0
2.5
3.13 2.167
2.063
6.02 2.055
2.0
3.06 1.998
1.5
1.0
0.5
ppm
210
200
190
180
170.90
170.54
170.20
170
170.06
169.36
160
156.70
150
140
130.83
129.64
130
124.94
122.74
120
117.45
29
110
99.15
100
90

77.32
80 77.00
76.68
71.19
70.68
70
68.46
66.94
61.57
60
50
40
30
20.61
20.53
20
20.46
ppm
9.0
1.02 8.629
8.5
8.095
8.074
1.08 7.902
8.0
1.06 7.880
1.01 7.779
7.757
7.254
7.5
1.05
0.54 7.249
7.240
0.39
7.232
7.0
7.227
5.575
5.555
6.5
5.549
5.529
5.490
6.0
5.482
5.249
1.00 5.229
7.362
5.5
0.98 7.357
0.94 5.179
0.97 5.170
5.0
5.153
5.144
4.280
4.258
30
4.5
4.248
3.14 4.226
4.195
4.0
4.181
4.168
4.163
4.149
3.5
3.0
2.5
3.04 2.181
2.064
6.22
2.056
2.0
3.24 2.013
1.5
1.0
0.5
ppm
210
200
190
180
171.64
170.38
170.23
170
170.13
169.41
160
156.67
150
137.02
140
131.85
131.43
128.97
130
127.35
126.31
125.49
120
119.62
110.86
31
110
99.15
100
90

77.32
80 77.00
76.68
71.26
70.78
70
68.56
66.87
61.51
60
50
40
30
20.72
20.66
20
20.57
ppm
9.0
8.5
8.0
6.952
6.943
7.5
6.938
6.927
6.921
7.0
2.03 6.912
2.02 6.704
6.695
6.690
6.5
6.679
6.673
6.665
6.0
4.687
5.5
4.667
3.883
3.876
3.767
5.0
3.758
1.00 3.750
3.744
32
4.5
3.738
3.733
3.714
3.621
4.0
0.98
2.90 3.619
1.03 3.608
3.605
1.04 3.5
3.591
3.589
3.558
3.549
3.0
3.533
3.525
3.349
2.5
3.318
3.314
3.310
3.306
2.0
3.302
1.5
1.0
0.5
ppm
210
200
190
180
170
160
152.41
150
143.26
140
130
119.30
120
117.75
33
110
104.31
100
90

80 76.80
74.89
72.40
70
70.21
62.39
58.71
60
49.64
49.43
49.21
50
49.00
48.79
48.57
40
48.36
30
20
ppm
9.0
8.5
7.733
7.690
7.687
7.671
8.0
1.00 7.668
7.413
0.88
7.393
7.5
1.96 7.374
7.358
7.355
7.352
7.0
7.340
7.338
7.335
6.5
6.0
4.927
4.908
5.5
3.928
3.920
3.842
5.0
1.38 3.822
3.818
3.798
34
3.784
4.5
3.776
3.765
0.91 3.732
4.0
1.46 3.719
3.702
2.86 3.615
0.82 3.607
3.5
3.591
3.583
7.693
3.0
7.675
2.5
2.0
1.5
1.0
0.5
ppm
210
200
190
180
169.51
170
159.12
160
150
140
133.20
130.51
130
124.61
122.47
118.86
120
35
110
102.89
100
90

80 76.90
74.77
72.21
70
70.11
62.27
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20
ppm
9.0
8.5
8.0
7.5
6.864
7.0
6.845
2.24 6.816
6.797
6.5
6.0
4.581
4.561
3.876
5.5
3.867
3.845
3.839
5.0
3.820
3.739
3.723
0.99
3.711
36
4.5
3.695
3.624
3.609
4.0
1.98 3.597
1.06 3.582
1.12 3.558
3.549
3.5
0.98
3.533
1.20
3.525
3.368
3.0
3.366
3.350
3.337
3.335
2.5
3.09 3.318
2.89 3.314
2.84 3.310
2.0
3.306
3.302
2.299
2.255
1.5
2.201
3.353
1.0
0.5
ppm
210
200
190
180
170
160
154.72
150
140
136.57
131.36
130.27
130
128.75
126.66
120
37
110
106.23
100
90

80 76.42
74.88
73.18
70
70.03
62.03
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20.05
20
17.58
13.73
ppm
9.0
8.377
8.364
7.725
8.5
1.00 7.722
7.706
7.704
8.0
7.702
7.686
1.06 7.683
1.07 7.537
7.5
7.535
1.04 7.516
7.515
7.0
7.189
7.176
7.173
7.170
6.5
7.158
6.0
5.5
5.137
1.13 5.112
3.943
5.0
3.937
3.775
3.760
38
4.5
3.744
3.737
3.725
3.713
4.0
1.02
3.702
4.15 3.686
1.04 3.669
3.5
3.584
3.575
3.561
3.553
3.0
3.318
3.314
3.310
2.5
3.306
3.302
2.0
1.5
1.0
0.5
ppm
210
200
190
180
170
159.63
160
149.93
150
138.78
140
130
124.51
121.91
120
39
110
100
90
86.77

80.82
80
76.34
70.84
70
70.45
62.60
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20
ppm
9.0
8.544
8.004
0.94 8.000
8.5
7.982
7.978
0.99 7.946
8.0
0.91 7.924
0.92 7.848
7.827
0.97
7.565
7.5
1.01 7.559
7.396
7.390
7.0
7.373
7.367
6.5
6.0
5.087
5.068
5.5
3.950
3.941
1.05 3.901
3.882
5.0
3.877
3.869
3.857
40
4.5
3.851
3.842
0.97 3.828
3.816
4.0
1.01
3.811
2.77 3.801
1.00 3.790
3.5
3.776
3.770
3.659
3.651
3.0
3.635
3.626
3.318
2.5
3.314
3.310
3.306
2.0
3.302
1.5
1.0
0.5
ppm
210
200
190
180
170
158.8831
160
150
138.3786
140
131.9227
131.8810
130.0744
130
128.3965
127.5252
126.8987
120.9460
120
111.7061
41
110
102.7108
100
90
77.2120
80 74.8672
72.2496
70.2760
62.5036
70
49.6379
49.4251
49.2122
60
49.0000
48.8563
48.7874
48.5740
50
48.3612
126.9192
40
30
20
ppm
7.826
7.824
7.822
9.0
7.805
7.803
7.651
8.5
7.632
7.630
7.628
7.347
8.0
1.19 7.328
1.20 7.326
7.307
7.5
1.35 7.247
2.25 7.228
7.226
7.207
7.0
1.00 7.188
6.849
6.846
6.5
6.830
6.827
5.053
5.034
6.0
4.976
3.991
3.972
5.5
3.967
3.948
1.42 3.943
3.936
5.0
3.812
3.801
3.784
42
4.5
3.774
3.745
3.740
2.38 3.728
4.0
2.07 3.726
1.12 3.723
1.13 3.710
3.5
3.642
3.633
3.617
3.609
3.0
3.318
3.314
3.310
2.5
3.306
3.302
7.800
7.653
2.0
7.649
1.5
1.0
0.5
ppm
210
200
190
180
170
160
153.23
150
144.31
140
126.53
130
125.88
123.72
123.51
120
115.83
109.85
43
108.69
110
108.06
101.60
100
90

80
75.58
73.36
70.53
70
68.24
60.47
60
50
40.14
39.93
39.72
39.51
40
39.30
39.09
38.88
30
20
ppm
9.0
8.5
7.627
7.605
7.582
8.0
7.450
4.20 7.431
7.413
7.5
2.09 7.332
1.00 7.329
2.00 7.312
7.0
7.295
7.293
7.129
7.108
6.5
5.203
5.190
6.0
4.898
4.881
4.862
4.693
5.5
4.680
0.95 4.542
4.531
0.94
5.0
3.721
1.10 3.712
1.02 3.616
0.95 3.603
44
4.5
3.593
3.573
3.561
4.0
3.546
1.14 3.521
3.507
4.38 3.497
3.5
1.19 3.481
3.443
3.421
3.0
3.354
2.509
2.504
2.493
2.5
2.500
2.498
3.731
2.0
6.351
5.620
4.668
1.5
1.0
0.5
ppm
210
200
190
180
170
160
157.16
150
139.76
140
133.67
128.86
127.61
130
126.83
126.25
120
116.71
45
110
101.07
100
90

80
75.54
73.30
70.29
70
68.14
60.38
60
50
40.14
39.93
39.72
39.51
40
39.30
39.09
38.88
30
20
ppm
9.0
7.426
7.423
8.5
7.405
7.374
7.371
7.366
8.0
7.354
7.350
7.335
7.5
5.00 7.310
7.306
2.28 7.289
7.075
7.0
1.99 7.066
7.060
7.049
6.5
7.043
6.923
6.914
6.908
6.0
6.897
6.891
5.5
5.026
4.741
2.23 4.722
5.0
3.891
1.22 3.883
3.802
46
4.5
3.781
3.773
3.761
3.756
4.0
1.20
3.748
3.08 3.737
1.22 3.719
1.14 3.5
3.647
3.634
3.631
3.617
3.0
3.571
3.563
3.547
2.5
3.538
2.0
1.5
1.0
0.5
ppm
210
200
190
180
170
160
154.421
152.360
150
140
137.713
128.279
130
127.622
127.373
120
118.012
115.477
47
110
102.826
100
90

78.962
80 75.710
75.644
73.677
71.161
70
70.314
69.029
61.229
60
50
40
30
20
ppm
9.0
8.848
1.00 8.842
8.5
8.350
1.01
8.329
8.0
4.32
7.5
3.319
7.0
3.314
3.306
3.301
7.574
6.5
7.554
7.540
7.535
6.0
7.515
7.496
5.027
5.008
5.5
4.099
4.079
1.41 4.075
5.0
4.056
3.966
3.959
3.893
48
4.5
3.871
3.863
1.02 3.839
0.93
4.0
3.831
3.12 3.818
0.90 3.796
3.713
3.5
3.705
3.688
3.680
3.0
3.310
8.853
8.838
2.5
2.0
1.5
1.0
0.5
ppm
190
180
170
160
153.82
150.11
150
140.12
140
138.61
130.99
130
128.49
122.99
122.54
120
114.64
110
103.87
49
100
90
80

77.41
74.17
70 72.11
70.23
62.56
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20
ppm
7.870
7.867
7.849
9.0
7.845
7.807
7.794
7.790
8.5
7.786
1.00 7.679
7.676
3.12
8.0
7.673
2.03 7.662
1.17 7.657
3.09 7.653
7.5
1.19 7.642
7.639
7.636
7.0
7.593
7.587
7.569
7.549
6.5
7.535
7.531
7.402
6.0
7.396
7.380
7.374
5.103
5.5
5.084
3.957
1.22 3.949
5.0
3.914
3.895
3.890
50
3.871
4.5
3.861
3.851
1.03 3.836
4.0
1.29 3.825
3.28 3.819
1.01 3.810
3.798
3.5
3.784
3.779
3.671
3.0
3.662
3.646
3.638
3.318
2.5
3.314
3.310
3.306
2.0
3.302
1.5
1.0
0.5
ppm
210
198.58
200
190
180
170
159.15
160
139.33
150
138.30
134.28
133.57
140
133.11
132.15
130.93
130
129.70
129.53
128.70
120
127.02
121.17
111.81
51
110
102.70
100
90

80 77.21
74.87
72.25
70
70.27
62.51
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20
ppm
1.00 9.168
9.0
7.865
8.5
7.843
7.834
7.813
7.651
8.0
1.88
1.04 7.630
0.96 7.560
0.94 7.542
7.5
7.522
0.91 7.293
7.275
1.99
7.273
7.0
0.94 7.255
7.036
7.014
6.5
6.853
2.508
6.0
2.496
2.491
5.159
5.5
5.147
4.857
0.99 4.843
4.768
5.0
0.98 4.749
0.97 4.667
0.96 4.655
52
4.5
0.96 4.502
4.491
3.724
3.716
4.0
3.707
1.19 3.598
4.41 3.563
3.5
1.27 3.544
3.524
3.513
3.438
3.0
3.427
3.416
2.85 3.406
2.5
3.394
3.360
2.546
2.503
2.0
2.500
7.539
1.5
1.0
0.5
ppm
210
200
190
180
170
160
154.10
152.06
150
147.14
144.03
140
135.84
129.10
130
126.59
123.70
122.16
119.67
120
116.64
113.53
53
110
101.79
100
90

80
75.44
73.35
70.37
70
68.17
60.43
60
50
40.14
39.93
39.72
39.51
40
39.30
39.09
38.88
30
20
18.29
ppm
9.0
7.361
8.5
7.355
7.344
7.338
7.250
8.0
7.247
7.242
7.237
7.5
7.229
2.00 7.225
2.94 7.019
0.86 7.014
7.0
6.735
0.82 6.730
6.713
6.5
6.708
0.74
6.427
6.426
6.419
6.0
6.418
4.908
4.888
5.5
3.923
3.916
3.863
0.66 3.844
5.0
0.60 3.839
3.828
3.820
54
4.5
3.810
3.793
3.789
0.92 3.781
4.0
3.00 3.776
0.90 3.760
3.747
1.02
3.5
3.723
3.708
3.695
3.693
3.0
3.678
3.676
3.622
2.5
3.614
3.598
3.590
3.318
2.0
3.314
3.310
3.306
1.5
3.302
1.0
0.5
ppm
210
200
190
180
170
160
157.35
150
140.36
140
135.88
132.81
131.54
130
129.36
126.11
118.88
120
114.81
111.65
55
110
108.14
103.18
103.14
100
90

80 77.00
74.83
72.27
70
70.21
62.44
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30
20
ppm
9.0
8.5
8.0
7.399
7.5
2.03 7.379
7.240
2.05 7.114
7.094
7.0
6.5
5.389
5.387
5.381
6.0
5.379
5.221
5.196
5.171
5.5
1.00 5.029
1.00 5.021
1.02 5.004
5.0
4.996
4.636
1.01 4.611
4.187
56
4.5
4.170
4.159
2.12 4.141
4.0
1.03 4.108
4.093
4.080
4.064
3.5
3.904
3.902
3.886
3.0
3.871
3.869
2.5
3.07 2.324
2.83 2.095
3.27 2.079
2.022
2.0
3.15
3.05 1.951
1.5
1.0
0.5
ppm
7.532
7.512
7.458
9.0
7.454
7.437
7.429
7.383
8.5
7.377
7.373
7.368
8.0
7.360
7.352
2.37 7.350
7.341
7.5
2.92
17.98 7.340
7.337
1.93 7.326
7.0
7.322
7.306
7.240
7.042
6.5
7.022
5.025
4.996
6.0
4.859
4.834
4.795
4.766
5.5
4.759
1.19 4.730
1.33 4.663
5.0
4.655
2.82
4.634
0.88 4.631
57
0.79 4.523
4.5
2.03 4.494
4.470
0.92 4.441
4.0
1.00 4.024
1.87 4.018
1.80 3.978
3.5
3.954
3.931
3.732
3.709
3.0
3.706
3.692
3.667
2.5
3.658
2.82 3.650
3.634
3.628
2.0
3.617
2.324
2.189
1.5
2.078
1.690
1.313
1.295
1.0
1.278
0.5
ppm
6.232
6.223
5.461
9.0
5.438
5.435
5.412
8.5
5.338
5.335
5.329
5.327
8.0
5.122
5.102
5.096
7.5
5.076
4.999
4.989
4.973
7.0
4.964
4.953
4.944
6.5
4.927
1.00 4.919
4.469
4.449
6.0
4.441
0.10
4.437
1.02 4.411
5.5
1.08 4.406
0.11 4.152
1.08 4.136
0.17 4.124
5.0
1.06 4.120
1.03 4.113
4.108
58
1.16
4.5
4.102
1.08 4.085
3.36 4.072
1.05 4.067
4.0
1.19 4.058
1.12 4.039
3.994
3.5
3.974
3.969
3.963
3.874
3.0
3.872
3.856
3.840
2.99
2.5
3.837
2.84 3.815
2.89 3.791
8.72 3.767
2.0
2.83 2.156
3.18 2.146
2.136
1.5
2.128
2.104
2.094
2.072
1.0
2.040
2.036
2.031
0.5
2.026
2.018
2.005
1.986
1.943
1.939
ppm
7.318
9.0
7.310
7.298
7.240
8.5
5.312
5.304
5.162
5.140
8.0
5.116
5.091
5.071
7.5
5.064
5.00 5.057
5.047
4.933
7.0
4.925
4.907
4.899
6.5
4.857
4.837
4.834
4.813
6.0
4.470
4.447
4.428
5.5
1.17 4.417
1.12 4.397
4.116
3.23 4.100
5.0
1.20 4.089
1.16 4.072
2.09 4.059
59
4.5
1.08 4.048
4.040
2.93 4.018
4.013
4.0
1.22
2.31 4.006
3.850
1.21 3.833
3.5
1.18 3.815
2.56 3.794
3.782
3.769
3.0
3.753
3.728
3.705
2.5
3.01 3.690
2.63 3.682
3.63 3.666
3.657
2.0
6.02
3.641
2.70
3.631
3.12 3.565
1.5
3.560
3.552
3.548
3.541
1.0
3.536
3.528
3.523
0.5
3.351
3.339
2.114
2.041
2.022
2.003
ppm
7.526
7.521
7.517
9.0
7.511
7.508
7.473
8.5
7.463
7.454
7.449
7.395
8.0
7.390
7.375
7.372
5.08
7.5
7.352
24.99 7.332
11.16 7.325
7.312
7.0
7.308
7.305
7.299
6.5
7.296
7.288
7.284
7.266
6.0
7.247
7.240
7.228
1.00
5.5
7.217
1.05 7.208
1.93 7.203
7.190
5.0
2.05
7.182
5.74 7.173
3.11 5.465
60
4.5
1.04 5.205
2.20 5.178
1.02 5.168
5.124
4.0
7.33 4.835
4.797
4.785
8.35
3.5
4.770
4.758
4.742
4.726
3.0
1.01
4.574
4.541
4.510
2.5
4.459
4.440
4.305
4.275
2.0
4.229
4.199
4.029
1.5
4.020
3.863
3.859
3.832
1.0
3.828
3.785
3.765
0.5
3.761
3.741
3.641
3.628
3.387
2.930
ppm
210
200
190
156.33
156.06
180
138.75
138.40
138.27
170
137.96
137.73
137.62
160
136.54
136.40
128.68
150
128.48
128.36
128.20
128.12
140
128.07
127.95
127.92
130
127.67
127.53
127.45
120
127.31
127.26
127.13
61
110
127.04
126.39
103.52
100
102.62
101.14
82.80
90
81.71

79.46
78.65
80 77.32
77.21
77.00
76.68
70
75.60
75.13
74.88
60
73.44
72.82
71.40
50
68.76
68.01
67.78
40
67.09
66.18
51.29
30
46.99
45.86
20
ppm
7.370
7.362
7.357
9.0
7.353
7.318
7.297
8.5
7.296
7.293
7.267
7.243
8.0
7.240
7.233
7.216
2.29
7.5
7.207
22.88 7.202
15.34 7.101
1.42 5.150
7.0
5.113
4.986
4.981
6.5
4.959
4.954
4.755
4.718
6.0
4.693
4.689
4.663
5.5
4.659
4.634
2.04 4.629
1.00 4.569
5.0
4.537
4.520
15.78 4.490
62
4.5
4.423
4.415
4.409
4.395
4.0
3.25
4.365
1.06 4.346
4.341
3.5
12.46 4.316
4.311
4.005
3.975
3.0
3.950
3.767
3.658
2.5
1.00 3.640
3.634
3.621
3.593
2.0
3.588
3.569
3.545
1.5
3.517
3.476
3.463
3.458
1.0
3.452
3.445
3.439
0.5
3.353
3.330
3.301
2.386
7.091
7.084
ppm
210
200
190
180
170
156.43
156.16
138.98
138.56
160
138.14
137.85
136.63
150
136.48
128.44
128.39
140
128.28
128.20
128.04
130
127.97
127.77
127.68
120
127.56
127.46
127.17
63
103.62
110
102.41
82.77
81.74
100
81.02
79.31
77.32
90
77.20

77.00
80 76.68
76.31
75.27
75.16
70
74.97
73.42
73.04
60
72.73
71.93
68.36
50
68.06
67.92
67.21
40
66.06
51.37
47.06
45.94
30
20
ppm
7.382
7.335
7.317
9.0
7.303
7.287
7.271
8.5
7.267
7.253
7.238
7.227
8.0
7.201
7.197
7.191
7.5
7.187
7.178
59.99
7.159
7.135
7.0
7.130
7.121
5.149
6.5
5.111
5.076
5.048
4.877
6.0
4.849
4.785
4.771
5.5
4.751
4.742
2.10 4.733
2.01 4.563
5.0
2.04 4.547
15.87 4.525
2.94 4.515
64
4.5
2.12 4.496
1.20 4.488
4.472
2.20
4.467
4.0
5.24 4.457
2.41 4.447
2.35 4.438
3.5
5.27 4.408
4.16 4.347
1.09 4.336
4.317
3.0
4.287
4.262
4.247
2.5
4.163
4.085
4.066
4.058
2.0
4.034
4.016
3.987
1.5
3.982
3.962
3.956
3.655
1.0
3.631
3.610
3.529
0.5
3.513
3.491
3.472
3.319
3.297
3.267
ppm
138.849
138.685
138.597
210
138.526
138.435
138.339
200
138.225
137.941
137.834
190
137.709
128.463
128.346
180
128.302
128.215
128.186
170
128.163
128.135
128.090
128.063
160
128.044
127.926
127.773
150
127.619
127.544
127.524
140
127.499
127.421
127.365
130
127.292
127.157
127.100
120
127.077
103.581
102.720
65
110
100.652
82.430
81.622
81.545
100
79.362
77.318
90
77.202
77.000

76.917
80 76.683
76.514
75.139
74.939
70
74.897
74.814
74.712
60
73.673
73.189
73.117
50
72.967
72.950
72.369
40
71.996
69.367
68.070
67.958
30
67.828
67.756
67.617
20
67.238
67.180
51.380
47.055
45.941
ppm
7.5
7.0
4.939
4.930
4.800
4.535
6.5
4.514
4.511
4.490
4.363
4.347
6.0
4.331
4.069
4.057
4.026
5.5
4.017
4.006
3.975
3.946
5.0
0.95 3.926
3.913
3.898
3.885
2.00 3.878
4.5
0.94 3.867
3.851
3.841
3.830
66
4.0
3.812
20.40 3.800
3.790
3.779
3.5
3.771
0.95 3.759
3.752
3.744
3.726
3.0
3.718
3.701
3.688
3.683
2.5
3.662
3.653
3.646
3.638
2.0
3.632
3.610
3.589
3.569
3.561
1.5
3.547
3.536
3.345
3.324
1.0
3.303
0.5
ppm
210
200
190
180
170
160
150
140
130
120
67
110
103.27
102.13
100.31
100
78.62
77.36
90
75.43

74.85
74.39
80 72.86
72.17
70.92
70
70.83
69.13
68.95
68.52
60
60.52
60.37
60.02
50
50.53
68.56
40
30
20
ppm
9.0
8.5
8.0
7.5
2.01
7.2400
5.4639
5.4440
7.0
2.10
5.4378
5.4263
5.4179
5.0898
6.5
5.0812
5.0636
5.0550
4.9618
6.0
4.9418
4.2182
4.2007
5.5
2.03 4.1900
4.1726
4.1513
1.04 4.1356
5.0
1.00 4.1230
4.1074
4.0309
4.0139
68
4.5
3.9974
3.2806
2.13 3.2637
1.00 3.2467
4.0
2.3525
2.3341
2.3154
2.1540
3.5
2.0483
2.01 2.0329
1.9859
6.9584
3.0
6.9508
6.9450
6.9331
2.04 6.9282
2.5
3.07 6.9192
3.20 1.7709
1.7522
2.99 1.7332
2.0
2.87 1.7139
2.14 1.6951
1.6530
2.27
1.5
1.6354
2.10 1.6174
1.6049
1.5987
1.0
1.5812
1.4738
1.4612
1.4541
0.5
1.4451
1.4359
1.4231
1.4146
1.3987
ppm
210
200
190
180
170.8674
170.3203
170.1817
170
170.0431
169.3653
160
153.4341
150
140
133.4121
130
121.3474
120
117.5419
69
110
100.0554
100
90

77.3176
80 77.0000
76.6821
70.9592
70.7307
70
68.5986
66.8274
61.2893
60
51.1409
50
40
37.1468
28.5417
30
26.2710
24.9156
20.6728
20
20.5920
20.5124
ppm
7.418
7.405
7.402
9.0
7.399
7.329
7.309
8.5
7.289
7.240
7.109
7.106
8.0
7.102
7.100
1.07 7.088
7.5
1.07 7.086
1.05 7.082
7.080
1.04 6.625
7.0
5.482
5.463
5.456
6.5
5.436
5.431
5.429
5.422
6.0
5.420
5.097
1.11 5.091
5.5
5.083
0.95
5.077
5.065
2.07
5.056
5.0
4.194
4.176
4.166
70
4.5
4.149
4.136
3.27 4.122
4.108
4.0
4.103
6.30 4.085
4.073
3.5 4.070
2.14 4.067
4.058
4.055
3.0
4.040
4.038
3.684
2.5
3.672
3.06 3.669
2.96 3.659
3.652
2.0
3.13
3.00 3.642
3.638
3.631
1.5
3.611
3.370
3.358
3.356
1.0
3.346
2.144
2.033
0.5
2.025
1.976
ppm
190
180
170.23
169.98
169.80
170
169.15
166.51
160
156.68
150
140
135.88
129.44
130
121.17
119.97
120
115.38
110
71
98.96
100
90
77.32
77.00
80

76.68
70.92
70 70.55
69.89
69.52
68.32
66.73
60
61.19
50.35
50
39.53
40
30
20.47
20.38
20
20.34
ppm
9.0
8.2565
7.8578
8.5
7.8533
1.09 7.8444
7.8363
7.8319
8.0
2.01 7.8219
0.97 7.7645
7.7430
7.3396
7.5
1.00 7.3335
1.05 7.2395
7.2307
7.2246
7.0
7.2084
0.91 7.2023
5.5632
5.5434
6.5
5.5370
5.5173
5.4792
5.4708
6.0
5.2159
5.1960
1.05 5.1536
5.5
0.94 5.1450
5.1274
1.02 5.1188
0.98 4.2513
5.0
4.2414
4.2276
4.2160
4.1901
72
4.5
4.1757
1.17 4.1532
2.12 4.1418
4.1400
4.0
3.7472
3.7340
6.30 3.7283
3.7243
3.5
2.02 3.7166
3.7140
3.7046
3.6960
3.0
3.6836
3.4096
3.3974
3.3858
2.5
2.1787
3.09 2.0599
6.01 2.0520
2.0084
2.0
2.89 -0.0261
4.2654
4.1278
4.1248
1.5
1.0
0.5
ppm
210
200
190
180
170.31
170.19
170.07
170
169.36
167.30
160
155.91
150
140
135.65
130.79
130.41
129.15
130
127.42
127.31
124.51
119.56
120
110.87
73
110
99.25
100
90

77.32
80 77.00
76.68
71.19
70.76
70.17
70
69.93
68.55
66.86
61.47
60
50.63
50
39.73
40
30
20.69
20.62
20
20.53
ppm
9.0
7.9853
8.5
7.9643
7.8826
7.8641
7.5319
8.0
1.93
7.5108
7.4856
7.4651
7.5
4.04 7.4460
7.4223
1.12 7.4025
7.3825
7.0
7.0800
7.0611
5.7128
6.5
5.6930
5.6866
5.6667
5.4963
6.0
5.4881
1.00 5.1822
0.98 5.1666
5.5
5.1610
5.1413
2.09 5.1333
4.2863
5.0
4.2701
4.2595
4.2436
74
4.5
4.1932
1.03 4.1776
2.10 4.1659
4.1506
4.0
4.1314
4.1156
5.1622
3.5
3.3081
1.95 3.2924
3.2762
2.5240
3.0
2.5051
2.4879
1.67 2.1936
2.5
2.0642
3.01 2.0231
3.46 2.0168
6.32 1.8443
2.0
2.07 1.8277
2.13 1.8108
1.6788
2.19
1.5
1.6610
1.6427
1.6258
1.5823
1.0
1.5517
1.5292
1.5127
0.5
1.8588
1.7885
ppm
210
200
190
180
171.53
170.32
170.21
170
170.08
169.66
160
153.35
150
140
132.03
128.57
126.41
130
125.93
125.77
122.28
120
119.58
115.77
75
108.92
110
100.00
100
90

77.32
80 77.00
76.68
71.14
70.66
70
68.47
66.83
61.33
60
51.19
50
40
37.13
28.58
30
26.37
25.16
20.77
20
20.63
20.56
ppm
7.867
7.376
7.369
9.0
7.354
7.346
7.300
8.5
7.240
7.232
7.217
7.212
8.0
0.93 7.195
7.190
2.87 7.120
7.5
1.09 7.098
0.65 6.591
0.97 6.583
5.525
7.0
1.90 5.505
5.499
0.94 5.479
6.5
5.459
5.452
5.134
5.125
6.0
5.108
5.099
1.11 5.077
5.5
1.00 5.058
4.258
1.17 4.241
0.99 4.230
5.0
4.213
4.173
4.158
76
4.5
4.145
1.13 4.130
1.15 4.080
1.07 4.063
4.0
4.047
3.281
3.264
3.5
3.247
2.01 2.386
2.368
2.349
3.0
2.177
2.084
1.98 2.038
2.5
2.008
3.28
1.785
3.18 1.766
3.34 1.747
2.0
3.19 1.728
2.33 1.664
2.47 1.660
1.5
2.30 1.647
1.628
1.610
1.592
1.0
1.496
1.476
1.467
0.5
1.458
1.445
1.438
7.128
7.115
7.103
ppm
210
200
190
180
171.07
170.26
170.15
170
170.05
169.32
160
155.13
150
140
134.93
133.28
130.95
129.15
130
128.71
125.43
117.92
120
116.52
112.85
77
110.27
110
103.40
99.59
100
90

77.32
80 77.00
76.68
71.00
70.69
70
68.52
66.77
61.25
60
51.13
50
40
37.16
28.52
30
26.27
25.07
20.65
20
20.55
20.49
ppm
9.0
8.5
7.468
8.0
7.459
7.454
7.442
7.437
7.5
1.94
7.428
7.091
1.96 7.083
7.0
7.077
7.066
7.060
7.052
6.5
4.821
4.802
6.0
3.900
3.892
3.807
3.800
5.5
3.789
3.779
3.761
5.0
3.756
1.00 3.750
3.735
78
3.722
4.5
3.683
3.670
3.668
0.98
4.0
3.653
3.05 3.587
1.01 3.578
1.02 3.562
3.5
3.554
3.318
3.314
3.0
3.310
3.303
3.306
2.382
2.5
2.06 2.363
2.344
1.760
2.0
1.741
2.14 1.722
2.06 1.703
1.684
1.5
2.07
1.675
1.658
1.651
1.0
1.640
1.621
1.603
1.498
0.5
1.496
1.484
1.477
1.468
1.459
1.446
ppm
210
200
190
180
174.178
170
160
155.809
150
140
134.429
130
122.650
120
118.106
79
110
103.339
100
90

80 76.963
74.854
72.284
70
70.219
62.422
52.295
60
49.638
49.425
49.212
50
49.000
48.787
48.574
40
48.362
37.626
29.668
30
27.427
26.450
20
ppm
9.0
8.5
8.423
7.548
7.475
8.0
7.457
7.456
1.00 7.393
0.98
7.5
7.373
1.01 7.354
0.98 7.285
7.283
7.0
7.267
7.265
7.262
6.5
4.931
4.912
4.882
6.0
3.919
3.911
3.843
3.823
5.5
3.819
3.810
3.800
5.0
1.23 3.784
3.775
3.762
80
3.738
4.5
3.724
3.711
3.697
4.0
1.00 3.686
8.21 3.675
3.02 3.662
3.613
3.5
2.02 3.604
3.599
3.588
3.0
3.579
3.575
3.562
3.401
2.5
3.390
3.378
3.314
2.0
3.310
3.306
3.318
3.302
1.5
7.477
1.0
0.5
ppm
210
200
190
180
170.01
170
159.15
160
150
140
136.97
130.60
130
122.04
121.04
120
116.62
81
110
102.82
100
90

77.00
80 74.77
72.19
70.88
70.32
70
70.17
62.42
51.70
60
49.64
49.43
49.21
50
49.00
48.79
48.57
40
48.36
40.88
30
20
ppm
9.0
8.5
0.88 8.319
7.907
7.885
8.0
1.14
1.93 7.865
7.845
0.95 7.550
7.5
0.87 7.385
7.363
5.073
7.0
5.054
3.945
3.937
6.5
3.898
3.878
3.873
3.854
6.0
3.840
3.834
3.828
5.5
3.810
3.804
1.00 3.796
3.786
5.0
3.773
3.731
3.718
82
4.5
3.705
3.698
0.97 3.694
1.09 3.655
4.0
3.23 3.647
4.18 3.635
3.23 3.622
3.5
3.414
1.92 3.403
3.391
3.318
3.0
3.314
3.310
3.306
2.5
3.302
7.913
7.890
7.824
2.0
7.391
7.368
3.864
1.5
1.0
0.5
ppm
210
200
190
180
170.42
170
158.39
160
150
137.49
140
131.51
131.07
130.09
130
128.62
128.50
125.35
120
120.95
111.70
83
110
102.73
100
90

80 77.16
74.84
72.25
70.95
70
70.45
70.26
62.50
60
51.76
50
40.95
40
30
20
ppm
9.0
8.5
8.366
1.10 8.345
7.652
7.631
8.0
7.589
1.34 7.571
1.21 7.489
7.5
2.32 7.470
1.07 7.449
7.429
7.289
7.0
7.270
5.085
5.066
6.5
4.877
4.015
3.995
6.0
3.990
3.971
3.953
3.944
5.5
3.831
3.814
1.05 3.805
5.0
3.790
3.784
3.779
3.760
84
4.5
3.754
3.744
1.22 3.733
4.0
1.13 3.655
3.84 3.647
1.20 3.631
3.623
3.5
2.30 3.368
3.352
3.334
3.0
3.318
3.314
3.310
2.34 3.306
2.5
3.302
2.578
2.560
2.0
2.541
2.57 1.865
2.48 1.846
2.51 1.828
1.5
1.809
1.790
1.734
1.0
1.717
1.699
1.680
1.663
0.5
1.597
1.576
1.559
1.546
1.538
ppm
190
180
175.5491
170
160
154.9781
150
140
133.7997
131.3209
128.1351
130
127.3442
125.8062
124.8165
122.0764
120
117.3120
110.6491
110
103.2003
85
100
90
80

77.0994
74.9829
72.3543
70 70.2613
62.4271
52.3523
60
49.6390
49.4253
49.2122
49.1939
50
49.0000
48.7879
48.7777
40
48.5748
48.3608
37.2254
29.7049
30
27.5196
26.5881
20
ppm
9.0
8.5
7.860
7.856
8.0
7.427
0.87 7.406
7.386
4.30 7.364
7.5
2.22 7.294
7.272
1.02 7.246
7.0
7.243
7.224
7.221
0.92
6.591
6.5
6.583
4.940
6.0
4.920
3.930
3.921
5.5
3.864
3.844
3.840
3.825
5.0
0.96
3.821
3.815
3.797
86
4.5
3.781
3.765
3.752
0.98 3.736
4.0
1.38 3.722
3.11 3.706
1.04 3.629
3.5
3.620
2.10 3.605
3.596
3.330
3.0
3.314
3.310
3.306
2.5
2.14 3.302
2.422
2.403
2.384
2.0
2.43 1.792
1.774
2.52
1.755
2.25
1.5
1.736
1.718
1.688
1.671
1.0
1.653
1.634
1.617
0.5
1.523
1.503
1.485
1.466
ppm
210
200
190
180
174.29
170
160
157.69
150
135.48
140
134.82
132.49
130.62
130
130.04
126.41
118.93
120
117.76
114.15
111.20
87
110
104.14
103.12
100
90

80 77.05
74.84
72.27
70
70.23
62.46
52.30
60
49.64
49.43
49.21
50
49.00
48.79
48.57
40
48.36
37.70
29.67
30
27.45
26.56
20
ppm
9.0
8.5
8.0
7.423
7.400
7.5
1.94 7.384
7.240
6.955
7.0
2.00 6.950
6.938
6.933
6.5
5.465
5.446
5.439
6.0
5.428
5.420
5.093
5.5
2.05 5.084
5.066
5.058
1.07
4.967
5.0
0.99 4.947
4.219
4.201
88
4.5
4.191
4.173
2.36 4.154
1.10 4.138
4.0
4.126
4.110
4.033
3.5
4.016
4.000
2.709
2.618
3.0
2.611
2.603
4.73 2.594
2.5
2.587
3.41 2.579
2.94 2.574
2.82 2.562
2.0
2.549
3.16
2.157
2.050
1.5
2.034
1.988
1.0
0.5
ppm
v
210
200
190
180
170.34
170.20
170.05
170
169.39
169.21
160
153.53
150
140
133.27
130
121.51
120
117.54
89
110
100.03
100
90
82.74

77.32
80 77.00
76.68
70.97
70.74
70
69.63
68.62
66.85
60
61.30
50
40
36.00
30
20.67
20.59
20
20.51
14.73
ppm
7.460
7.456
7.450
9.0
7.439
7.437
7.420
8.5
7.418
7.358
7.338
7.319
8.0
7.240
7.135
2.12 7.133
7.5
7.129
1.03 7.126
1.00 7.114
7.112
7.0
7.108
7.106
6.439
6.5
6.427
5.492
5.473
6.0
5.466
5.446
5.436
2.02 5.104
5.5
5.100
5.096
2.07 5.080
5.0
5.070
4.225
4.223
4.219
90
4.5
4.217
4.32 4.212
1.08 4.210
4.0
4.206
4.203
4.192
4.174
3.5
4.159
4.145
4.131
3.0
4.117
4.077
4.075
4.060
2.5
0.84 4.045
3.16 4.043
3.43 2.265
2.0
2.93 2.259
3.02 2.253
2.159
2.051
1.5
2.041
1.990
1.0
0.5
ppm
210
200
190
180
170.541
170.161
170.000
170
169.366
166.311
160
156.767
150
140
135.337
129.703
130
121.441
120.506
120
115.626
91
110
99.096
100
90
79.362

77.318
80 77.000
76.682
71.756
71.162
70
70.666
68.435
66.896
60
61.413
50
40
29.632
30
20.658
20.554
20
20.480
ppm
9.0
8.248
7.861
7.838
7.832
8.5
7.828
1.00 7.810
7.806
0.90
8.0
7.779
1.04 7.758
0.97 7.348
7.342
7.5
0.99
7.249
0.62 7.239
0.62 7.226
7.0
7.221
6.381
6.369
6.357
6.5
0.95 5.567
5.547
5.541
6.0
5.521
5.483
1.10 5.475
5.221
5.5
0.98
5.202
1.03 5.157
1.05 5.149
5.0
5.131
5.123
4.307
92
4.300
4.5
2.06 4.294
1.19 4.288
2.10 4.271
4.0
4.258
4.248
4.236
4.223
3.5
4.191
4.176
4.154
3.0
4.143
4.128
2.301
2.295
2.5
1.25
3.49 2.288
3.66 2.181
2.062
2.0
3.12 2.053
2.99 2.012
1.5
1.0
0.5
ppm
210
200
190
180
170.32
170.19
170.09
170
169.37
166.93
160
156.05
150
135.83
140
130.82
129.82
129.11
130
127.59
127.51
124.36
120
119.74
110.91
93
110
99.23
100
90
79.50

77.32
80 77.00
76.68
71.95
71.22
70
70.77
68.57
66.86
60
61.46
50
40
29.87
30
20.71
20.64
20
20.55
ppm
9.0
7.986
7.965
8.5
7.874
7.856
1.00 7.771
8.0
0.94 7.616
0.87 7.595
1.07 7.476
7.457
7.5
1.48
1.13 7.437
7.407
1.12 7.387
7.0
7.367
7.240
7.072
7.053
6.5
5.710
5.690
5.684
6.0
5.664
1.13 5.494
5.485
1.14
5.5
5.175
5.166
2.34 5.157
5.140
5.0
5.132
4.283
4.267
94
4.5
4.256
1.31 4.240
2.28 4.190
4.174
4.0
4.162
4.147
4.127
3.5
4.112
2.707
2.695
3.0
2.221
2.191
3.95 2.107
2.061
2.5
2.021
3.21 2.014
3.93 2.721
2.0
6.01 2.687
2.684
2.668
1.5
1.0
0.5
ppm
210
200
190
180
170.32
170.19
170.09
170
169.37
166.93
160
156.05
150
135.83
140
130.82
129.82
129.11
130
127.59
127.51
124.36
120
119.74
110.91
95
110
99.23
100
90
79.50

77.32
80 77.00
76.68
71.95
71.22
70
70.77
68.57
66.86
60
61.46
50
40
29.87
30
20.71
20.64
20
20.55
ppm
9.0
7.878
7.874
7.442
8.5
7.383
7.377
7.361
8.0
1.00 7.353
1.16 7.247
7.240
1.11 7.229
7.5
1.25 7.224
0.81 7.207
1.23 7.202
7.0
2.20 7.126
7.104
0.98 6.601
6.593
6.5
5.530
5.510
5.504
6.0
5.484
5.464
1.17 5.456
5.138
5.5
1.02
5.129
1.22 5.111
1.06 5.103
5.0
5.085
5.065
4.264
96
4.247
4.5
1.18 4.236
1.24 4.218
1.21 4.179
4.0
4.163
4.151
4.135
3.5
4.106
4.089
4.072
4.056
3.0
2.651
2.635
4.01 2.628
2.5
2.620
3.20 2.616
3.14 2.605
4.17 2.592
2.0
3.17 2.576
2.182
2.087
1.5
2.042
2.012
1.0
0.5
ppm
210
200
190
180
170.29
170.18
170.08
170
169.35
169.20
160
155.19
150
140
134.98
133.43
130.77
129.19
130
128.79
125.53
117.96
120
116.55
112.98
110.35
97
110
103.47
99.65
100
90
82.98

77.32
80 77.00
76.68
71.06
70.73
70
69.51
68.56
66.80
60
61.29
50
40
36.13
30
20.71
20.60
20
20.54
14.88
ppm
9.0
8.5
7.473
8.0
7.465
7.460
7.448
7.443
7.5
2.08
7.435
7.091
2.11 7.083
7.0
7.078
7.066
7.061
7.052
6.5
6.0
4.822
4.803
3.900
5.5
3.893
3.807
3.801
3.789
5.0
1.22 3.778
3.762
3.757
98
4.5
3.751
3.735
3.722
1.00 3.683
4.0
3.08 3.670
3.667
0.96 3.652
0.95
3.5
3.638
3.588
3.580
3.564
3.0
3.555
3.318
4.00 3.314
2.5
3.310
0.94 3.306
3.302
2.572
2.0
2.556
2.548
2.543
1.5
2.534
2.290
2.284
2.279
1.0
0.5
ppm
210
200
190
180
172.12
170
160
155.85
150
140
134.33
130
122.67
120
118.13
99
110
103.34
100
90

83.51
80 76.96
74.87
72.30
70.30
70
70.23
62.43
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
36.72
30
20
15.59
ppm
9.0
7.555
8.5
7.550
7.545
7.481
8.0
7.478
7.462
1.07 7.459
1.08 7.407
7.5
1.08 7.387
1.05 7.367
7.303
7.0
7.299
7.297
7.282
7.278
6.5
7.276
4.935
4.916
6.0
4.146
4.140
3.919
5.5
3.910
3.839
3.820
3.815
5.0
1.35
3.807
3.796
3.781
100
4.5
3.774
3.762
2.22 3.740
3.736
4.0
1.20
5.00 3.725
3.713
1.07 3.611
3.5
3.603
3.587
3.578
3.318
3.0
3.314
3.310
0.92
3.306
2.5
3.302
2.607
2.601
2.595
2.0
7.305
7.285
1.5
1.0
0.5
ppm
210
200
190
180
169.44
170
159.26
160
150
140
136.55
130.68
130
122.13
121.33
120
116.73
101
110
102.90
100
90

80.70
80 77.04
74.82
72.23
72.06
70
70.21
62.45
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
29.96
30
20
ppm
9.0
8.5
1.04 8.323
7.924
7.901
8.0
1.09 7.851
1.97 7.848
7.556
1.11 7.551
7.5
1.00 7.394
7.388
7.372
7.366
7.0
6.5
6.0
5.076
5.057
5.5
4.206
4.200
3.946
1.04 3.938
5.0
3.898
3.879
102
3.874
3.855
4.5
3.840
2.12 3.835
1.14 3.826
4.0
1.21 3.810
3.797
3.03 3.787
1.29 3.774
3.5
3.768
3.656
3.648
3.632
3.0
3.623
3.314
0.90 3.310
2.5
3.306
2.657
2.629
2.623
2.0
2.616
1.289
3.318
3.302
1.5
1.0
ppm
210
200
190
180
169.84
170
158.53
160
150
137.64
140
131.58
130.65
130.12
130
128.80
128.59
125.30
120
121.04
111.73
103
110
102.76
100
90

80.87
80 77.19
74.88
72.27
72.02
70
70.29
62.52
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
30.00
30
20
ppm
9.0
8.5
8.370
0.87 8.348
7.726
7.704
8.0
7.596
0.94 7.579
0.95 7.487
7.5
2.05 7.465
1.00 7.443
7.422
7.286
7.0
7.267
6.5
5.083
6.0
5.063
4.014
3.995
5.5
3.990
3.971
1.17 3.952
3.943
5.0
3.813
3.804
3.788
104
4.5
3.778
3.758
3.753
1.94 3.742
4.0
3.18 3.731
3.655
1.06 3.646
3.5
3.631
3.622
3.318
3.314
3.0
3.310
2.15 3.306
2.10 3.302
2.5
0.70 2.764
2.746
2.729
2.653
2.0
2.646
2.636
2.630
1.5
2.616
2.613
2.394
2.387
1.0
2.381
0.5
ppm
190
180
173.62
170
160
154.94
150
140
133.78
131.33
128.15
130
127.30
125.78
124.80
122.16
120
117.61
110.71
110
103.24
105
100
90
83.69
80
77.10

75.00
72.38
70.57
70
70.27
62.44
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
36.41
30
20
15.81
ppm
7.866
10
7.861
7.433
7.425
7.420
7.408
7.402
9
7.394
7.383
7.368
7.361
7.300
7.292
8
7.287
1.00
7.275
7.270
4.45 7.262
2.09 7.254
0.98 7.249
7
7.232
7.227
6.592
0.94 6.591
6.584
4.940
4.921
6
3.930
3.922
3.865
3.846
3.841
3.825
106
1.16 3.822
3.815
3.797
3.794
3.781
3.765
1.07 4 3.753
3.737
4.49
3.736
1.02 3.725
3.723
3.720
3.718
3
3.707
3.706
4.45 3.631
3.622
0.94 3.607
3.598
2
3.310
2.627
2.616
2.600
2.590
2.582
1
2.577
2.570
2.560
2.553
2.543
2.310
2.303
2.297
6.582
ppm
210
200
190
180
172.25
170
160
157.73
150
135.51
140
134.89
132.41
130.63
130
130.08
126.45
118.96
120
117.74
114.15
111.20
107
110
104.15
103.14
100
90

83.61
80 77.08
74.86
72.28
70.33
70
70.24
62.47
60
49.64
49.43
49.21
50
49.00
48.79
48.57
48.36
40
36.80
30
20
15.74
ppm
9.0
8.5
8.0
7.240
5.462
7.5
5.453
5.423
5.417
5.378
7.0
5.370
5.351
5.343
6.5
5.258
5.111
5.103
5.085
6.0
5.075
5.066
0.98 5.040
5.5
0.99 5.023
1.25 5.015
4.998
0.89
4.675
5.0
4.657
4.441
4.425
108
4.5
0.98 4.408
4.138
1.05 4.126
2.01 4.108
4.0
4.092
4.085
4.076
3.5
4.068
4.057
4.051
4.040
3.0
4.035
4.023
4.006
2.5
3.992
2.73 3.985
3.38 3.973
3.937
2.0
3.67
3.921
3.00 3.905
2.100
1.5
2.050
2.004
1.946
1.0
0.5
ppm
9.0
8.5
8.0
7.5
7.240
7.0
5.372
5.366
5.211
5.191
6.5
5.185
5.165
5.042
6.0
5.034
5.016
5.008
4.715
5.5
1.00 4.695
0.99 4.354
0.97 4.348
5.0
4.305
4.178
0.96
4.162
4.150
109
4.5
1.94 4.133
4.119
2.27 4.112
4.0
1.00 4.102
4.091
4.074
3.5 4.058
3.925
3.923
3.908
3.0
3.906
3.891
5.374
5.363
2.5
0.91
3.11 2.446
2.440
3.02 2.434
2.0
2.88 3.889
2.90 2.119
2.041
2.022
1.5
1.956
1.0
0.5
ppm
210
200
190
180
170.34
170.18
170.08
170
169.51
160
150
140
130
120
110
110
98.61
100
90

78.16
77.32
80 77.00
76.68
75.33
70
70.81
68.45
66.94
61.16
60
55.86
50
40
20.74
20.62
20.53
30
70.78
20
ppm
9.0
8.5
8.0
7.5
7.0
4.468
6.5
4.462
4.429
4.423
6.0
4.415
4.406
4.382
4.376
5.5
3.836
3.829
3.790
5.0
3.772
3.761
3.744
3.735
111
4.5
3.07
3.722
3.706
3.693
4.0
1.00 3.552
3.534
2.27
3.528
3.25 3.520
3.5
3.517
3.514
3.510
3.0
1.03 3.501
3.496
3.488
3.472
2.5
3.464
3.314
3.310
2.0
3.306
2.860
2.854
2.848
1.5
3.318
3.302
3.838
1.0
3.827
0.5
ppm
Intens. [a.u.]
Intens. [a.u.]
3192.060 3369.647
3104.163
3324.068 3281.238
300
3457.047
3060.766 3368.065 3193.118 3501.333
300
3038.112
250
3412.053
3149.341
3545.486
200
2994.088
3456.339 3588.935
200 2972.322
3105.341
150
3500.402 3633.097
2949.867 3061.511
2928.279
3543.890 100
3677.119
100 2906.017 3039.028
50
0 0
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
m/z m/z
化合物 91 之 MALDI-TOF 質譜圖化合物 92 之 MALDI-TOF 質譜圖

Intens. [a.u.]
Intens. [a.u.]
3355.440 3361.286
3223.369
3317.296
3443.415
3493.379
3179.308 500
3487.313
3537.280
600 3229.456
3157.400 3531.411 3185.251

400
3580.993
3575.307 3141.142
3625.137
400 3091.569
300
3619.242
3069.678
3669.459
3120.490
200
3663.561 3713.073
3025.632
200
3003.376 3076.617
3706.949 3757.216
2981.496
100
3751.353
0 0
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
m/z m/z

112
Intens. [a.u.]
Intens. [a.u.]
3331.814 3440.831
3463.523
3243.327 3353.046 3572.932
500 3551.386 800
3308.979 3616.816
3660.865
3595.661
3199.412 3264.922
400
600
3704.683
3639.594
3220.880
3155.488
300
3683.731 3749.107
3110.886 400
3177.014
3792.845
200 3727.329
3132.631
3066.870 3836.593
3771.416
200
3880.125
100
3815.485 3088.540
2690.125
2513.460
0 0
2000 2200 2400 2600 2800 3000 3200 3400 3600 3800 2000 2200 2400 2600 2800 3000 3200 3400 3600 3800
m/z m/z
113
114

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國 立 清 華 大 學

Biomedical Application for Lectins

研究生：賴育聰 (Lai, Yu-Tsung)

指導教授：王聖凱 博士 (Dr. Wang, Sheng-Kai)

治療方法。綠膿桿菌感染以及形成生物膜主要藉由其凝集素 LecA 針對細胞表面半

乳糖殘基結合。近年發現帶有芳香環的半乳糖可以提高 LecA 的結合能力。我們開

我們開發出脯胺酸多肽作為多價性骨架，藉由其 PPII 構形來維持有效距離來針對

面上並乘載螢光 siRNA，藉由 Hep3B 肝癌細胞攝取實驗，我們在共聚焦顯微鏡下發

difficulty to exploit carbohydrate for lectin research. In addition to exploiting multivalent

interaction, developing high affinity monovalent carbohydrate derivatives is also a

solution to overcome the difficulty.

synthesized kinds of aromatic galactoside as ligand to understand the relationship

distance. We designed and synthesized aromatic galactoside conjugated polyproline

material as multivalent ligand binding to neighboring binding site of LecA. After

Furthermore, asiaglycoprotein receptor (ASGPR) is an endocytotic cell surface lectin

expressed by hepatocyte, which recognized galactoside residue of asiaglycoprotein. So

structure to modified drug carrier.

表目錄 ......................................................................................................................................... VIII

1.5.3.綠膿桿菌可溶性凝集素 I(PA-IL)的結構與特性 ........................................................ 12

1.5.4.綠膿桿菌凝集素 LecA 抑制劑作為抗綠膿桿菌材料之研究..................................... 14

1.5.5. 多價性綠膿桿菌生物膜抑制劑 ................................................................................. 16

1.6.1. RNAi 藥物以及肝相關疾病研究 ................................................................................ 20

1.6.2. RNAi 藥物輸送奈米粒子載體種類與性質 ................................................................ 21

1.6.5.醣分子配體對於 ASGPR 親和力研究 ........................................................................ 27

1.7. 研究動機 ............................................................................................................................ 28

2.1.單價 LecA 抑制劑合成 ....................................................................................................... 31

2.1.1. β-芳基半乳醣苷之合成 ............................................................................................... 31

2.1.2. Gb3 醣體之合成 .......................................................................................................... 40

2.1.3. 以微陣列(Microarray)篩檢 LecA 與抑制劑之結合能力 .......................................... 45

2.2.特定間距之二價醣肽 LecA 抑制劑合成 ........................................................................... 48

2.2.6. 微陣列試驗之結果與討論 ......................................................................................... 60

2.3.4. 肝細胞對於奈米粒子攝入實驗 ................................................................................. 69

2.3.5. NPs 攝入結果與討論 .................................................................................................. 71

3.1. Material and methods .......................................................................................................... 73

3.2. Synthetic procedures and characterization .......................................................................... 74

第四章、參考文獻 ...................................................................................................................... 129

圖六、PPI、PPII 之立體結構示意圖 18 ......................................................................................... 9

圖七、Maecke 和 Wennemers 團隊合成的腫瘤細胞探針示意圖 19 .......................................... 10

圖九、LecA 結構示意圖 25 ........................................................................................................... 13

圖十、Gb3Cer/CD77 抗原結構圖 ................................................................................................ 13

圖十二、β-芳基半乳醣苷對於 LecA 胺基酸殘基提供額外作用力 31....................................... 15

圖十三、對於 LecA 具有高親和力的 2-萘基 β-D-半乳醣苷 8 .................................................. 16

圖十四、 Chevolot 與 Morvan 教授團隊合成八價樹突狀分子 34 ............................................ 17

圖十五、(a) Pieters 教授團隊以聚糖作為 spacer 材料設計符合結合位置的二價抑制劑。(b) 利

圖十六、Winssinger 研究團隊同時調控距離與半乳糖(galactose)配體來對 Lec A 結合 39 .... 19

圖十七、各種 RNAi 修飾及奈米載體提升在體內穩定性 46 ..................................................... 23

圖十八、Bi, Tri, Tetra-antennary 聚醣結構示意圖 54................................................................. 25

圖十九、ASGPR 於肝細胞表面結構組成示意圖 58 ................................................................... 26

圖二十、arabinogalactan 之聚醣結構 62。 .................................................................................. 28

圖二十一、化合物 38 之 1H-1H COSY 光譜展開圖，化學位移在 4.00 ppm 之 4’-H 與 2.39 ppm

圖二十三、市售之塗布玻片修飾方法示意圖 72。 ..................................................................... 45

圖二十五、Brimble 教授團隊利用 CuAAC 合成 MUC1 片段醣肽衍生物 73。....................... 49

圖二十七、醣肽化合物(60-64)與同長度之脯胺酸多肽骨架 42 之 CD 比較圖。 ................... 59

圖二十八、醣肽化合物 65 與相同長度骨架 43 CD 光譜 .......................................................... 59

圖三十、醣肽化合物 65-70 在不同濃度下所求得之滴定曲線 ................................................. 62

圖三十三、Hep3B 對於不同修飾之 NPs 的相對攝取量長條圖。 ........................................... 71

表二、去乙醯化之 β-芳基半乳醣苷結構與產率 ........................................................................ 38

表三、化合物 55-75 之產率以及結構 ......................................................................................... 56

表五、65-70 在微陣列測試中所測得之 IC50 數值 .................................................................... 63

國立清華大學

指導教授：王聖凱博士 (Dr. Wang, Sheng-Kai)

更多 entropy penalty ，造成 LecA 對於化學合成的 β- 芳基半乳醣苷 (β-aryl