Glucuronidation with Allyl quillaic ester

第六章附圖

Scheme 22. Glucuronidation with Allyl quillaic ester

Entry Compound Promotor Temperature Product 1 25 TMSOTf 0 ℃ messy^a, 98 < 5%

2 25 B(PhF5)3 rt messy^a, 98：17%; orthoester：20%

3 31 TMSOTf 0 ℃ messy^a, 99：26%

4 31 B(PhF₅)₃ 0 ℃ 99：32%; orthoester：none 5 31 B(PhF5)3 rt 99：38%; orthoester：none 6 32 TMSOTf 0 ℃ 66：60%; orthoester：12%

7 32 B(PhF₅)₃ 0 ℃ 66：49%; orthoester：none 8 32 B(PhF₅)₃ rt 66：45%; orthoester：none 9 79 TMSOTf 0 ℃ messy^a, 100：55%; α：10%

10 79 B(PhF₅)₃ 0 ℃ 100：41%; α：12%

11 79 B(PhF₅)₃ rt 100：59%; α < 5%

12 90 B(PhF5)3 rt 101：50%; orthoester：none

a messy: side reactions of quillaic acid transacylation 產物：

2.3 玻尿酸分解酶抑制劑活性

我們以兩種方法檢測玻尿酸分解酶抑制劑的活性：混濁度試驗和 Morgan-Elson 反應法，分別以完整玻尿酸含量和玻尿酸降解產物含量兩個角度來評估玻尿酸分解酶的活性。

混濁度試驗是利用玻尿酸會和具有長碳鏈的 CTAB（cetyltrimethylammonium bromide）反應產生不溶性複合物，因此，可依據混濁度檢測玻尿酸含量，若玻尿酸分解酶活性高，玻尿酸被分解，反應後加入 CTAB 產生的不溶性複合物少，混

濁度低；反之，混濁度高，則表玻尿酸分解酶活性被抑制⁷⁰。

圖十顯示了不同濃度的 92 反應後的混濁度，可以發現即使將濃度提升至 1 mM，

其混濁度和完全不加抑制劑的混濁度無明顯差異，表 92 並無顯著抑制活性。

圖十、不同濃度之化合物 92 抑制玻尿酸水解酶之能力

Morgan-Elson 反應法是利用玻尿酸被降解後會產生 free form 的 N-terminal，會

第三章結論

我們針對三萜類進行葡萄醣醛酸化反應做優化研究，探討 6 種不同的葡萄醣醛酸基給予者和兩種作為醣接受者的三萜類化合物在不同活化劑、溫度、溶劑中

的反應結果。（圖十二）

我們發現，當使用 B(PhF5)3為活化劑時，具有較好的β 選擇性，值得一提的是，使用此活化劑時，反應溫度為一個重要的因素，冰浴下反應時容易有 orthoester 的副產物生成，但如果將反應升溫至室溫，orthoester 副產物消失。

醯類保護基方面，Ac 保護基因為立障較小，反應時容易有副反應的發生。Bz 和 Piv 保護基兩者沒有太大的差別，端看葡萄醣醛酸基接受者的不同而有不同的產率，接受者為齊墩果酸時，Bz 保護產率較高；皂皮酸為接受者時，Piv 保護的產率較高。但在選擇性方面，Piv 保護基因為立障效應，較不會原酯的副產物生成。

使用 TBS 為保護基反應活性最好，和兩個三萜類化合物的反應產率都高，我們認為其具有發展的可能性，未來要進行三萜類化合物的葡萄醣醛酸化反應時，

TBS 保護基是一個很好的選擇。

圖十二、（A）醣給予者；（B）醣接受者

（A）（B）

在以本研究中所獲得的葡萄醣醛酸衍生物進行玻尿酸分解酶抑制劑之活性探討實驗中，發現在 1 mM 下仍無明顯抑制活性。這些化合物的活性將再繼續進行探討。

本研究提供了優化的葡萄醣醛酸化反應條件，未來有助於進一步合成更多葡萄醣醛酸衍生物。

第四章實驗部分

saccharic acid 1,4-lactone (β-glucuronidase inhibitor), hyaluronic acid (HA)

Sigma-Aldrich:

Hyaluronidase (HAse) from bovine testes, Type I-S, lyophilized powder: Sigma H3506, Lot. SLBV1921, 587 units/mg Solid

6. DMAB 溶液(10X)：100 mg DMAB 完全溶解於 125 μL 10 N HCl 後加入 875 μL 2.5% hexadecyltrimethylammonium bromide (CTAB)之 2% NaOH 終止反應，以 SpectraMax^® Paradigm^® Multi-Mode Microplate Reader (Molecular Devices, p-dimethylaminobenzaldehyde 溶於 125 L 10 N HCl 後加入 9.875 mL 冰醋酸），於 37 ℃下反應 20 分鐘後，以 13,200 r.p.m.於 4 ℃下離心 15 分鐘，取 200 mL 至 96 孔盤中，以 SpectraMax^® Paradigm^® Multi-Mode Microplate Reader (Molecular Devices, Sunnyvale, CA, USA)偵測吸收值（λ = 585 nm），計算抑制率後，以 GraphPad Prism 7 軟體繪圖。

4.2 一般實驗方法

Acetic acid, acetonitrile (ACN), acetone, Amberlyst IR120 (H⁺), boron trifluoride diethyl etherate (BF3·OEt2), N-bromosuccinimide (NBS), 1,8-Diazabicyclo[5.4.

0]undec-7-ene (DBU), 4-dimethylamino pyridine (DMAP), dimethylformamide (DMF), N,N-Diisopropylethylamine (DIPEA), Na2S2O3, NH4Cl, pyridine, tetrahydrofuran (THF), p-thiocresol, trifluoromethanesulfonate (TMSOTf), triethylamine, D-glucose, Hydrogen bromide solution 33 wt. % in acetic acid (33% HBr/AcOH),

tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf), tetrabutylammonium

iodide (TBAI), benzoyl chloride (BzCl), tris(pentafluorophenyl)borane (B(PhF₅)₃), pivaloyl chloride (PivCl), dimethoxymethyl benzene, iodobenzene diacetate (TBAI), 2,2,6,6-Tetramethyl-1-piperidinyloxy (TEMPO)

Alfa Aesar:

Trichloroacetonitrile (Cl3CCN), p-toluenethiol (TolSH)

Fisher Scientific:

2,2-dimethoxypropane, NaHCO3, K2CO3,NaOH, KOH, Pd/C

RDH:

Molecular sieves 4 Å , Celite^®

Merck KGaA:

CDCl3, Kieselgrl 60 silica gel 40-63 μm (230-400 mesh)

Sigma-Aldrich:

Ac2O, CD3OD, borane tetrahydrofuran (BH3．THF)

友和貿易股份有限公司:

ACS-CHCl3, methanol, EtOAc, hexane

景明貿易股份有限公司:

Dichloromethane (CH2Cl2), dimethylformamide (DMF)

4.3.2 實驗儀器

4.3.2.1 核磁共振（NMR）

使用 Bruker AMX-400（400 MHz）、Bruker DPX-200（200 MHz）或 AV III-600

（600 MHz），以溶劑 CDCl3或 CD3OD 配置樣品，以 ppm 為化學位移之單位，校正標準為 CDCl₃（1H: 7.26 ppm, 13C: 77.2 ppm）、CD₃OD（1H: 3.31 ppm, 13C: 49.0 ppm）。1H-NMR 分裂形式定義如下：s, 單峰（singlet）; d, 雙分裂（doublet）; t, 三分裂（triplet）; q, 四分裂（quartet）; m, 多分裂（multiplet）。耦合常數（coupling constant）以 J 表示，單位為 Hz。

4.3.2.2 薄層層析（Thin layer chromatography, TLC）

使用 Merk Kieselgrl 60 F254 之矽膠（silica gel）薄層層析片，利用流動相展開後，以紫外燈（254 nm）照射觀察，也可以顯色劑 anisaldehyde 或 cerium 染片後加熱觀察。

4.3.2.3 管柱層析

Silicycle 60 矽膠 40-63 μm（230-400 mesh, Merck）

4.3.2.4 質譜

Bruker BioTOF II^TM ESI-TOF

4.3.2.5 高壓液相層析（HPLC）

SHIMADZU (system controller: CBM-20A, photodiode array detector:

SPD-M20A, pump: LC-20AT, autosampler: SIL-20AHT). 純化使用 HPLC Ascentis-RP18（5 μm, 250 mm × 10 mm）管柱進行分離，化合物 92 流動相分離條 件如表三所示，流速為 2.0 mL/min，以 210 nm 波長偵測。化合物 92 純度分析使 用 HPLC Mightysil-RP18（5μm，250 mm × 4.6 mm），流動相為 isocratic H₂O + 0.1%

TFA：30%，CAN：70%，流速為 1.0 mL/min，以 210 nm 波長偵測。

4.4 合成步驟及數據

 General procedure for imidate formation (Method A) Step 1.

To a stirred solution of thio compound (1 equiv.) in acetone/H2O, 9/1 (0.1~0.2 M) was added NBS (4 or 6 equiv.) at rt. After being stirred for 2 h, the reaction was quenched by NaHCO3(aq.), Na2S2O3(aq.) and then removed acetone under reduced pressure. The mixture was diluted with CH2Cl2, washed by NaHCO3(aq.), Na2S2O3(aq.)

brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography to give hemiacetal product.

Step 2.

To the hemiacetal product (1 equiv.) dissolved in anhydrous CH2Cl2 (0.05~0.2 M) was added Cl3CCN (6 equiv.), DBU (0.2 equiv.) at rt under N2 atmosphere. The reaction mixture was stirred for 2 h. The resulting mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give imidate glucuronide.

 General procedure for glucuronidation (Method B)

To the suspension of glucuronyl imidate (1.2 equiv.), benzyl oleanolate or allyl quillaic ester (1 equiv.) and active 4 Å molecular sieve powder in anhydrous CH2Cl2

(40~60 mM) was added B(PhF₅)₃ or TMSOTf (0.1 eq) at rt under N₂ atmosphere. After being stirred for 30 min, the reaction was quenched by Et3N and then filtered. The resulting mixture was concentrated under reduced pressure and purified by column chromatography.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-acetyl-α-D-glucopyranosid)uronate (25)⁷²

Compound 25 (150 mg, 95%) as white foam: R_f 0.28 (EtOAc/Hexanes, 1/3) was synthesized according to method A by using compound 70 (250 mg, 0.57 mmol) and purified by flash chromatography (silica gel, EtOAc/Hexanes, 1/3). 25: ¹H NMR (200 MHz, CDCl3) δ 8.72 (s, 1H, NH), 6.63 (d, J = 3.6 Hz, 1H, H-1), 5.62 (dd, J = 10.2, 9.4 Hz, 1H, H-4), 5.26 (dd, J = 10.1, 9.4 Hz, 1H, H-3), 5.14 (dd, J = 10.1, 3.6 Hz, 1H, H-2), 4.49 (d, J = 10.2 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.01 (s, 9H, OCOCH3*3) ppm.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-benzoyl-α-D-glucopyranosid)uronate (31)

Compound 31 (207 mg, 56%) as white foam: R_f 0.49 (EtOAc/Hexanes, 1/3) was synthesized according to method A by using 71 (348 mg, 0.55 mmol) and purified by flash chromatography (silica gel, EtOAc /Hexanes, 1/4). 31: ¹H NMR (200 MHz, CDCl3) δ 8.68 (s, 1H, NH), 7.99-7.86 (m, 6H, Ar-H), 7.55-7.31 (m, 9H, Ar-H), 6.91 (d, J = 3.5 Hz, 1H, H-1), 6.29 (dd, J = 10.2, 9.9 Hz, 1H, H-3), 5.75 (dd, J = 10.1, 9.9 Hz,

1H, H-4), 5.63 (dd, J = 10.2, 3.5 Hz, 1H, H-2), 4.76 (d, J = 10.1 Hz, 1H, H-5), 3.69 (s, 3H, OCH3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 167.4, 165.6, 165.4, 160.4, 133.8, 133.6, 130.0, 129.9, 128.6, 92.9 (C-1), 90.6, 71.1, 70.3, 69.7, 69.4, 53.2 ppm.

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-pivaloyl-α-D-glucopyranosid)uronate (32)

Compound 32 (0.7 g, 56%) as white foam: R_f 0.41 (EtOAc/Hexanes, 1/10) was synthesized according to method A by using compound 72 (1.5 g, 2.65 mmol) and purified by flash chromatography (silica gel, EtOAc /Hexanes, 1/15). 32: ¹H NMR (400 MHz, CDCl3) δ 8.71 (s, 1H, NH), 6.65 (d, J = 3.3 Hz, 1H, H-1), 5.69 (dd, J = 10.1, 9.8 Hz, 1H, H-3), 5.30 (dd, J = 10.2, 9.8 Hz, 1H, H-4), 5.19 (dd, J = 10.1, 3.3 Hz, 1H, H-2), 4.48 (d, J = 10.2 Hz, 1H, H-5), 3.71 (s, 3H, OCH3), 1.15-1.10 (m, 27H, OCOC(CH3)3*3) ppm; BBD¹³C NMR (100 MHz, CDCl3) δ 177.2 (OCOC(CH3)3), 176.9 (OCOC(CH3)3), 176.8 (OCOC(CH3)3), 167.4 (C-6), 160.5 (C=NH), 92.6 (C-1), 90.7 (CCl3), 70.8 (C-5), 69.5 (C-3), 68.8 (C-2), 68.7 (C-4), 53.1 (OCH3), 38.9 (OCOC(CH3)3), 38.8 (OCOC(CH3)3), 27.2 (OCOC(CH3)3), 27.1 (OCOC(CH3)3) ppm.

28-O-allyl quillate (61)

To a solution of quillaic acid (803 mg, 1.65 mmol) in THF/H₂O (10/1 v/v, 8 mL) was treated with allyl bromide (286 μL, 3.30 mmol), potassium carbonate (455 mg, 3.30 mmol) and TBAI (30 mg, 0.08 mmol) under rt. The mixture was heated at reflux for 3.5 h. The reaction mixture was concentrated under reduced pressure. The residue was then diluted with CH2Cl2, washed by H2O, dried over MgSO4 and concentrated to afford a crude residue which was purified by column chromatography (silica gel;

EtOAc/Hexanes, 1/3) to give compound 61 (797 mg, 92%) as white foam: Rf = 0.43 (EtOAc/Hexanes, 1/2); ¹H NMR (400 MHz, CDCl3) δ 9.35 (s, 1H. H-23), 5.87-5.78 (m, 1H, OCH2CHCH2), 5.36 (t, J = 3.4 Hz, 1H, H-12), 5.27 (d, J = 17.1 Hz, 1H, OCH2CHCH2), 5.18 (d, J = 10.4 Hz, 1H, OCH2CHCH2), 4.57-4.39 (m, 3H, H-16, OCH2CHCH2), 3.75 (dd, J = 11.3, 4.1 Hz, 1H, H-3), 3.03 (dd, J = 14.2, 3.5 Hz, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 2.08-1.82 (m, 5H), 1.81-1.56 (m, 7H), 1.55-1.40 (m, 2H), 1.38-1.28 (m, 4H), 1.28-1.20 (m, 2H), 1.17 (d, J = 8.7 Hz, 1H), 1.13-0.98 (m, 5H), 0.94-0.93 (m, 7H), 0.88 (s, 3H), 0.70 (s, 3H); BBD¹³C NMR (100 MHz, CDCl3) δ 207.3, 176.4, 142.8, 132.0, 122.3, 118.0, 74.7, 71.7, 65.1, 55.2, 48.6, 48.0, 46.5, 46.3, 41.3, 40.5, 39.7, 38.0, 35.8, 35.3, 35.3, 32.7, 32.2, 30.7, 30.3, 26.7, 25.9, 24.5, 23.2, 20.6, 16.9, 15.6, 8.8 ppm; HRMS⁺ (ESI-TOF) calcd for C33H51O5 [M+H]⁺ 527.3731, found 527.3733.

28-O-Allyl-3-O-(methyl 2,3,4-tri-O-pivaloyl-β-D-glucopyranosiduronate)quillate (66)

Compound 66 (16.5 mg, 60%) as a white solid: R_f 0.40 (EtOAc/Hexanes, 1/4) was synthesized according to method B by using 32 (20 mg, 0.033 mmol), 61 (15 mg, 0.028 mmol) and TMSOTf (0.5 μL, 0.0028 mmol); ¹H NMR (600 MHz, CDCl3) δ 9.40 (s, 1H, H-23), 5.88-5.82 (m, 1H, OCH2CHCH2), 5.38 (t, J = 3.4 Hz, 1H, H-12), 5.31-5.16 (m, 4H, OCH2CHCH2, H-3′, H-4′), 5.10 (t, J = 8.8, 8.2 Hz, 1H, H-2′), 4.52-4.47 (m, 4H, H-1′, OCH2CHCH2, H-16), 3.99 (d, J = 10.0, 1H, H-5′), 3.84 (dd, J = 11.7, 4.62 Hz, 1H, H-3), 3.72 (s, 3H, OCH3), 3.06 (dd, J = 14.3, 4.1 Hz, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 1.89-1.87 (m, 4H), 1.79-1.73 (m, 5H), 1.67-1.62 (m, 4H), 1.48-1.39 (m, 2H), 1.33-1.29 (m, 4H), 1.16-1.08 (m, 30H), 1.04-0.99 (m, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.90 (s, 3H), 0.86 (m, 1H), 0.70 (s, 3H) ppm; BBD¹³C NMR (150 MHz, CDCl3) δ 207.5 (C-23), 177.1, 176.6, 176.5, 167.5 (C-6′), 142.9, 132.3 (OCH2CHCH2), 122.7 (C-12), 118.3 (OCH2CHCH2), 100.8 (C-1′), 81.3 (C-3), 75.0 (C-16), 72.5 (C-5′), 72.1 (C-3′), 71.4 (C-2′), 69.7 (C-4′), 65.3 (allylic CH2), 54.6, 52.9, 49.2, 48.9, 46.7, 46.5, 41.5, 40.8, 39.9, 38.9, 38.8, 38.2, 36.1, 35.6, 35.6, 32.9, 32.4, 30.9, 30.5, 29.8, 27.3, 27.3, 27.2, 27.1, 24.9, 24.7, 23.4, 20.2, 17.2, 15.8, 10.4 ppm; HRMS⁺ (ESI-TOF) calcd for C55H85O14 [M+H]⁺ 969.5934, found 969.5904.

28-O-benzyl oleanolate(67)⁷³

To a stirred solution of oleanolic acid (10.0 g, 21.9 mmol), K₂CO₃ (4.55 g, 32.9 mmol) and TBAI (81.0 mg, 0.219 mmol) in aqueous THF (100 mL) was added benzyl chloride (3.8 mL, 32.9 mmol) at rt. The reaction was heated at reflux and stirred 12 h.

The resulting mixture was concentrated under reduced pressure, the residue was diluted with CH2Cl2, washed by water and brine, dried over MgSO4 and then concentrated. The residue was recrystallized for methanol to obtain 67 (10.16 g, 84%) as a white crystal;

1H NMR (200 MHz, CDCl3) δ 7.40-7.29 (m, 5H, Ar-H), 5.28 (t, J = 3.4 Hz, 1H, H-12), 5.08-5.05 (m, 2H, CO2CH2Ph), 3.28-3.12 (m, 1H, H-3), 2.88 (dd, 1H, H-18), 2.02-1.80 (m, 3H), 1.75-1.57 (m, 6H), 1.54-1.45 (m, 3H), 1.45-1.15 (m, 8H), 1.12 (s, 3H), 1.05-0.98 (m, 2H), 0.97 (s, 3H), 0.91 (s, 3H), 0.89 (s, 3H), 0.87 (s, 3H), 0.77 (s, 3H), 0.73 (s, 1H, H-5), 0.60 (s, 3H) ppm; BBD ¹³C NMR (50 MHz, CDCl3) δ 177.6, 143.8, 136.5, 128.5, 128.1, 122.6, 79.16 (C-3), 66.1, 55.3, 47.7, 46.8, 46.0, 41.8, 41.5, 39.4, 38.8, 38.6, 34.0, 33.3, 32.9, 32.5, 30.8, 27.7, 27.3, 26.0, 23.7, 23.5, 23.2, 18.5, 17.0, 15.7, 15.4 ppm; HRMS⁺ (ESI-TOF) calcd for C37H55O3 [M+H]⁺ 547.4146, found 547.4140.

Methyl 1,2,3,4-tetra-O-acetyl-α-D-glucopyranosiduronate (69)⁷⁴

Glucuronolactone (11 g, 62.4 mmol) in methanol (70 mL) was added NaOH (0.25 g, 6.24 mmol) and the reaction mixture was stirred for 5 h. The resulting mixture was concentrated by rotavapor to give a residue which was added pyridine (80 mL) and then slowly added AcOH (50 mL) at 0 ℃. The reaction mixture was stirred for 12 h, and then was concentrated, purified by column chromatography (silica gel, EtOAc/Hexanes, 1/2 to 1/1) to give compound 69 (19 g, 81%) as yellow foam: R_f 0.53 (EtOAc/Hexanes, 1/1); α-anomer: ¹H NMR (200 MHz, CDCl3) δ 6.39 (d, J = 3.6 Hz, 1H, H-1), 5.50 (dd, J

= 10.2, 9.4 Hz, 1H, H-4), 5.21 (dd, J = 10.1, 9.4 Hz, 1H, H-3), 5.11 (dd, J = 10.1, 3.6 Hz, 1H, H-2), 4.41 (d, J = 10.2 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.18 (s, 3H, OCOCH3), 2.03 (s, 6H, OCOCH3), 2.01 (s, 3H, OCOCH3*2) ppm.

Methyl p-tolyl 2,3,4-tri-O-acetyl-1-thio-β-D-glucopyranosiduronate (70)⁷⁵

To a stirred solution of compound 69 (13.8 g, 36.6 mmol) in anhydrous CH2Cl2 (40 mL) was added 33% HBr/HOAc (40 mL) in ice bath under N2 atmosphere. Upon completion of the reaction after 6 h, the reaction was quenched by ice water and then it was diluted with CH2Cl2, washed by ice water, H2O, brine, dried over MgSO4 and then concentrated to afford crude glucuronyl bromide. To a stirred solution of the glucuronyl bromide (14.6 g, 36.8 mmol) in 1.0 M Na2CO3(aq.) (40 mL)/EA (40 mL) was added HSTol (9.1 g, 73.5 mmol) then TBAI (2.72 g, 7.35 mmol) at rt. After being stirred for 12 h, the reaction was quenched by saturated NaHCO3 and then was diluted with EA, washed by saturated NaHCO3, brine, dried over MgSO4 and then concentrated. The residue was purified by column chromatography (silica gel, EtOAc/Hexanes, 1/5 to 1/2) to give compound 70 (12.1 g, 75%) as a white solid: Rf 0.38 (EtOAc/Hexanes, 1/3); ¹H NMR (200 MHz, CDCl3) δ 7.37 (d, J = 8.0 Hz, 2H, Ar-H), 7.11 (d, J = 8.0 Hz, 2H, Ar-H), 5.23 (dd, J = 9.5, 8.6 Hz, 1H, H-3), 5.11 (dd, J = 9.6, 9.5 Hz, 1H, H-4), 4.89 (dd, J = 8.6, 10.0 Hz, 1H, H-2), 4.64 (d, J = 10.0 Hz, 1H, H-1), 3.99 (d, J = 9.6 Hz, 1H, H-5), 3.73 (s, 3H, OCH3), 2.32 (s, 3H, SPhCH3), 2.06 (s, 3H, OCOCH3), 1.97 (s, 6H, OCOCH₃*2) ppm.

Methyl (p-tolyl 2,3,4-tri-O-benzoyl-1-thio-β-D-glucopyranosid)uronate (71)

To a stirred suspension of compound 70 (177 mg, 0.404 mmol) in MeOH (5 mL) was added Na (1 mg, 0.0404 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by amberlite H+. The reaction mixture was filtered and concentrated. The residue was added anhydrous CH2Cl2 and then added BzCl (0.281 mL, 2.42 mmol), TEA (0.563 mL, 4.04 mmol) and DMAP (4.94 mg, 0.0404 mmol) in ice bath under N2 atmosphere. After being stirred for 6 h, the reaction was quenched by saturated NH4Cl and then it was diluted with CH2Cl2, washed by H2O, saturated brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/Hexanes, 1/5) to afford compound 71 (220 mg, 87%) as white solid : R_f 0.33 (EtOAc/Hexanes, 1/4); ¹H NMR (400 MHz, CDCl3) δ 7.97-7.80 (m, 6H, Ar-H), 7.55-7.13 (m, 13H, Ar-H), 5.89 (dd, J = 9.6, 9.3 Hz, 1H, H-3), 5.60 (dd, J = 9.7, 9.6 Hz, 1H, H-4), 5.45 (dd, J = 9.6, 9.3 Hz, 1H, H-2), 4.98 (d, J = 9.8 Hz, 1H, H-1), 4.33 (d, J = 9.7 Hz, 1H, H-5), 3.71 (s, 3H, OCH3), 2.36 (s, 3H, SPhCH3) ppm; BBD¹³C NMR (100 MHz, CDCl3) δ 167.2, 165.3, 139.2, 134.3, 133.5, 130.0, 129.9, 128.6, 128.4, 86.7 (C-1), 76.6, 73.6, 70.2, 53.1, 21.4 ppm;

HRMS⁺ (ESI-TOF) calcd for C₃₅H₃₁O₉S [M+H]⁺ 627.1683, found 627.1688.

Methyl (p-tolyl 2,3,4-tri-O-pivaloyl-1-thio-β-D-glucopyranosid)uronate (72)

To a stirred suspension of 70 (4.57 g, 10.3 mmol) in MeOH (15 mL) was added Na (23.6 mg, 1.03 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by Amberlyst IR120 (H⁺). The reaction mixture was filtered and concentrated.

The residue (1.0 g, 3.2 mmol) was added anhydrous CH2Cl2 (10 mL) and then added PivCl (2.4 mL, 19.1 mmol), TEA (4.4 mL, 32 mmol) and DMAP (39.1 mg, 0.32 mmol) in ice bath under N2 atmosphere. After being stirred for 6 h, the reaction was quenched by NH4Cl and then it was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/Hexanes, 1/8) to afford 72 (1.5 g, 83%) as white solid: Rf 0.72 (EtOAc/Hexanes, 1/2);¹H NMR (200 MHz, CDCl3) δ 7.37 (d, 2H, Ar-H), 7.13 (d, 2H, Ar-H), 5.36 (dd, J = 9.3, 9.1 Hz, 1H, H-3), 5.19 (dd, J = 9.8, 9.3 Hz, 1H, H-4), 5.02 (dd, J = 10.1, 9.1 Hz, 1H, H-2), 4.67 (d, J = 10.1 Hz, 1H, H-1), 4.05 (d, J

= 9.8 Hz, 1H, H-5), 3.74 (s, 3H, OCH3), 2.35 (s, 3H, SPhCH3), 1.03-1.00 (m, 27H, OCOC(CH3)3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 176.8 (OCOC(CH3)3), 176.3 (OCOC(CH3)3), 176.1 (OCOC(CH3)3), 166.9 (C-6), 138.7 (Ar-C), 133.6 (Ar-C), 129.7 (Ar-C), 127.6 (Ar-C), 86.7 (C-1), 76.2 (C-5), 72.6 (C-3), 69.0 (C-2), 68.9 (C-4), 52.6 (OCH3), 38.6 (OCOC(CH3)3), 27.0 (OCOC(CH3)3), 21.1 (SPhCH3) ppm; HRMS⁺ (ESI-TOF) calcd for C₂₉H₄₃O₉S [M+H]⁺ 567.2622, found 567.2609.

Methyl

(p-tolyl 2,3,4-tri-O-tert-butyldimethylsilyl-1-thio-β-D-glucopyranosid)uronate (74) To a stirred suspension of 70 (4.57 g, 10.3 mmol) in MeOH (15 mL) was added Na (23.6 mg, 1.03 mmol) at rt. Upon completion of the reaction after 3 h, the reaction was quenched by amberlite H+. The reaction mixture was filtered and concentrated. The residue (1 g, 3.18 mmol) was added anhydrous DMF (5 mL) and then added TBSCl (2.88 g, 19.1 mmol), imidazole (0.78 g, 11.5 mmol) at rt under N2 atmosphere. The mixture was heated to 80℃. After being stirred for 12 h, the reaction was quenched by H2O, concentrated and then it was diluted with CH2Cl2, washed by H2O, brine, dried over MgSO4 and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel; EtOAc/Hexanes, 1/20) to afford 74 (0.7 g, 32%) as a colorless syrup: Rf 0.72 (EtOAc/Hexanes, 1/6);¹H NMR (200 MHz, CDCl3) δ 7.45 (d,

Methyl

(trichloroacetimidoyl-2,3,4-tri-O-tert-butyldimethylsilyl-α-D-glucopyranosid)urona te (79)

Compound 79 (25 mg, 54%) as a colorless syrup: Rf 0.59 (EtOAc/Hexanes, 1/10) was synthesized according to method A by using compound 74 (43.8 mg, 0.067 mmol) and purified by flash chromatography (silica gel, EtOAc/Hexanes, 1/30 to 1/20). 79: ¹H NMR (200 MHz, CDCl₃) δ 7.26 (s, 1H, NH), 5.56 (s, 1H, H-1), 4.47 (s, 1H), 4.10 (s, 1H), 3.98 (s, 1H), 3.73 (s, 3H, CO2CH3), 3.39 (s, 1H), 0.95 (m, 3H), 0.94 (s, 6H), 0.92 (s, 3H), 0.91 (s, 6H), 0.86 (s, 3H), 0.85 (s, 6H), 0.21 (s, 3H), 0.20 (s, 3H), 0.14 (s, 3H), 0.13 (s, 3H), 0.06 (s, 3H), 0.05 (s, 3H) ppm; ¹³C NMR (50 MHz, CDCl3) δ 170.0 (CO₂CH₃), 169.0 (C=NH), 90.3 (C-1), 77.4, 76.9, 71.7, 71.2, 52.1 (CO₂CH₃), 26.1 (Si(CH3)2C(CH3)3), 25.9 (Si(CH3)2C(CH3)3), 25.7 (Si(CH3)2C(CH3)3), 18.4 (Si(CH₃)₂C(CH₃)₃), 18.2 (Si(CH₃)₂C(CH₃)₃), 18.0 (Si(CH₃)₂C(CH₃)₃), -4.0 (Si(CH3)2C(CH3)3), -4.8 (Si(CH3)2C(CH3)3), -5.1 (Si(CH3)2C(CH3)3) ppm.

p-tolyl 4,6-O-benzylidene-3-O-benzyl-1-thio-β-D-glucopyranoside (84) ⁷⁶

To a stirred solution of 83 (7.03 g, 18.7 mmol) in anhydrous ACN (20 mL) was added dimethoxy-methylbenzene (5.46 mL, 37.3 mmol), TsOH (0.362 mg, 1.87 mmol) at rt under N2 atmosphere. Upon completion of the reaction after 1 h, the reaction was quenched by TEA. The reaction mixture was concentrated under reduced pressure and recrystallized by ether to give 84 (6.85 g, 79%) as a white solid: Rf 0.38 (EtOAc /Hexanes, 1/4); ¹H NMR (200 MHz, CDCl3) δ 7.48-7.28 (m, 12H, Ar-H), 7.16-7.10 (m, 3H, Ar-H), 5.56 (s, 1H, CHPh), 4.85 (dd, J = 32.8, 21.3 Hz, 2H, OCH2Ph), 4.56 (d, J = 9.5 Hz, 1H, H-1), 4.38 (dd, J = 10.4, 4.7 Hz, 1H, H-4), 3.78 (dd, J = 8.6, 10.4 Hz, 1H, H-3), 3.58-3.43 (m, 4H, H-2, H-5), 3.50 (dt, J = 6.6, 4.6, 2.2 Hz, 2H, H-6), 2.55 (d, J = 2.2 Hz, 1H, OH), 2.35 (s, 3H, SPhCH3) ppm.

p-tolyl 2-O-benzoyl-4,6-O-benzylidene-3-O-benzyl-1-thio-β-D-glucopyranoside (85)⁷⁶

To a stirred solution of 84 (7.4 g, 15.9 mmol) in anhydrous CH₂Cl₂ (30 mL) was added BzCl (2.8 mL, 23.9 mmol), TEA (3.3 mL, 23.9 mmol), DMAP (0.39 g, 3.18 mmol) at 0 ℃ under N2 atmosphere. After being stirred for 8 h, the reaction was quenched by NH4Cl and then diluted with CH2Cl2, washed by H2O, brine, dried by MgSO4. Collected organic layer and then concentrated under reduced pressure. The residue was recrystallized by ether to give 85 (7.8 g, 86%) as a white solid: Rf 0.56 (EtOAc/Hexanes, 1/4); ¹H NMR (200 MHz, CDCl3) δ 8.02 (d, J = 1.2 Hz, 2H, Ar-H), 7.65-7.31 (m, 10H, Ar-H), 7.12-7.08 (m, 6H, Ar-H), 5.60 (s, 1H, CHPh), 5.25 (dd, J = 10.0, 8.4 Hz, 1H, H-2), 4.76 (d, J = 10.0 Hz, 1H, H-1), 4.74 (dd, J = 32.8, 21.3 Hz, 2H, OCH2Ph), 4.42 (dd, J = 10.4, 4.8 Hz, 1H, H-4), 3.92-3.75 (m, 3H, H-3, H-6), 3.54 (dt, J

= 10.2, 4.8 Hz, 1H, H-5), 2.35 (s, 3H, SPhCH3) ppm.

2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-D-glucopyranoside (86)⁷⁷

To a stirred solution of 85 (1 g, 1.76 mmol) in anhydrous BH3‧THF (8.8 mL, 8.79 mmol), CH2Cl2 (5 mL) was added TMSOTf (32 μL, 0.176 mmol) at 0 ℃ under N2

atmosphere. After being stirred for 5 h, the reaction was quenched by TEA, MeOH and then concentrated under reduced pressure. The residue was recrystallized (silica gel, EtOAc/Hexanes, 1/6) by ether to give 86 (0.93 g, 92%) as a white solid: Rf 0.23 (EtOAc/Hexanes, 1/4); ¹H NMR (200 MHz, CDCl3) δ 8.06 (d, J = 1.2 Hz, 2H, Ar-H), 7.63-7.29 (m, 10H, Ar-H), 7.16-7.07 (m, 7H, Ar-H), 5.23 (dd, J = 10.0, 9.0 Hz, 1H, H-2), 4.87-4.61 (m, 5H, OCH2Ph*2, H-1), 4.00-3.46 (m, 2H, H-6), 3.85 (dd, J = 9.0, 8.9 Hz, 1H, H-3), 3.45 (dd, J = 9.5, 8.9 Hz, 1H, H-4), 3.47 (ddd, J = 9.5, 4.6, 2.6 Hz, 1H, H-5), 2.32 (s, 3H, SPhCH3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 165.3, 138.6, 137.8, 137.7, 133.4, 130.0, 129.9, 128.7, 128.6, 128.4, 128.2, 127.8, 86.4 (C-1), 84.1, 79.7, 75.5, 75.3, 72.6, 62.2, 21.3 ppm; HRMS⁺ (ESI-TOF) calcd for C34H35O6S [M+H]⁺ 571.2149, found 571.2136.

2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-D-glucopyranosyluronate (87)

To a stirred solution of compound 86 (54.2 mg, 0.095 mmol) in CH2Cl2/H2O, 2/1 (1.5 mL) was added TEMPO (1.48 mg, 0.0095 mmol), BAIB (45.9 mg, 0.14 mmol) at rt.

After being stirred for 12 h, the reaction was quenched by Na2S2O3 and then diluted with CH2Cl2, washed by NaHCO3, Na2S2O3, brine, removed H2O by MgSO4. Collected organic layer and then concentrated under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/CH₂Cl₂/Hexanes, 1/1/3) to give 87 (46 mg, 83%) as a white solid: Rf 0.13 (EtOAc/Hexanes, 1/1); ¹H NMR (200 MHz, CDCl3) δ 7.99 (d, J = 1.2 Hz, 2H, Ar-H), 7.65-7.31 (m, 10H, Ar-H), 7.13-7.10 (m, 7H, Ar-H), 5.27 (dd, J = 9.0, 7.1 Hz, 1H, H-2), 4.90 (d, J = 9.0 Hz, 1H, H-1), 4.66 (dd, 2H, OCH2Ph), 4.17 (d, J = 7.5 Hz, 1H, H-5), 3.93 (dd, J = 7.8, 7.5 Hz, 1H, H-4), 3.82 (d, J = 7.1 Hz, 1H, H-3), 2.30 (s, 3H, SPhCH3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 170.5, 165.2, 138.8, 137.4, 133.6, 130.0, 128.6, 128.4, 128.3, 128.1, 127.9, 86.3 (C-1), 81.8, 78.7, 74.8, 71.8, 21.3 ppm; HRMS⁺ (ESI-TOF) calcd for C34H32O7SNa [M+Na]⁺ 607.1761, found 607.1762.

Methyl (2-O-benzoyl-3,4-di-O-benzyl-1-thio-β-D-glucopyranosid)uronate (88) To a stirred solution of 87 (34 mg, 0.058 mmol) in DMF (1 mL) was added K2CO3 (4 mg, 0.029 mmol), MeI (6 μL, 0.087 mmol) at rt. The reaction was heated to 80 ℃, after being stirred for 12 h, the reaction was quenched by NH4Cl and then diluted with CH2Cl2, washed by H2O, brine, dried by MgSO4. Collect organic layer and then concentrate under reduced pressure. The residue was purified by column chromatography (silica gel, EtOAc/Hexanes, 1/4) to give 88 (28 mg, 81%) as a white solid: Rf 0.58 (EtOAc/Hexanes, 1/2); ¹H NMR (200 MHz, CDCl3) δ 8.00 (d, J = 1.2 Hz, 2H, Ar-H), 7.65-7.31 (m, 10H, Ar-H), 7.14-7.06 (m, 7H, Ar-H), 5.23 (dd, J = 10.0, 8.6 Hz, 1H, H-2), 4.74 (d, J = 10.0 Hz, 1H, H-1), 4.70 (dd, 4H, OCH2Ph), 4.03-3.82 (m, 3H, H-3, H-4, H-5), 3.76 (s, 3H, OCH3), 2.30 (s, 3H, SPhCH3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 168.5, 165.1, 138.6, 137.7, 137.5, 133.6, 133.4, 130.0, 129.8, 128.6, 128.4, 128.2, 127.9, 87.4 (C-1), 83.4, 79.3, 78.3, 75.4, 75.3, 52.7, 21.3 ppm; HRMS⁺ (ESI-TOF) calcd for C35H34O7SNa [M+Na]⁺ 621.1917, found 621.1926.

Methyl

(trichloroacetimidoyl-2-O-benzoyl-3,4-O-benzyl-α-D-glucopyranosid)uronate (90) Compound 90 (27 mg, 60%) as a white foam: R_f 0.45 (EtOAc/Hexanes, 1/3) was synthesized according to method A by using compound 88 (27 mg, 0.055 mmol), Cl3CCN (34 μL, 0.33 mmol), DBU (1.5 μL, 0.011 mmol) and was purified by column chromatography (silica gel, EtOAc/Hexanes, 1/5). 90: ¹H NMR (200 MHz, CDCl3) δ 8.56 (s, 1H, NH), 7.94 (d, J = 1.2 Hz, 2H, Ar-H), 7.58-7.27 (m, 7H, Ar-H), 7.18 (s, 6H, Ar-H), 6.64 (d, J = 3.5 Hz, 1H, H-1), 5.39 (dd, J = 9.9, 3.5 Hz, 1H, H-2), 4.86-4.63 (m, 4H, OCH2Ph*2), 4.48 (d, J = 10.4 Hz, 1H, H-5), 4.27 (dd, J = 10.4, 9.3 Hz, 1H, H-4), 4.01 (dd, J = 9.9, 9.3 Hz, 1H, H-3), 3.74 (s, 3H, OCH3) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 168.8, 165.5, 160.5, 137.5, 133.6, 129.9, 129.2, 128.7, 128.6, 128.4, 128.2, 127.9, 93.7 (C-1), 79.2, 78.8, 77.4, 75.7, 72.8, 72.2, 52.9 ppm.

28-O-benzyl-3-O-(methyl 2,3,4-tri-O-benzoyl–β–D-glucopyranosyluronate) oleanolate (91)

Compound 91 (30 mg, 52%) as a white solid: R_f 0.56 (EtOAc/CH₂Cl₂/Hexanes, 1/1/4) was synthesized according to method B by using 31 (46 mg, 0.082 mmol), 67 (30 mg, 0.055 mmol) and B(PhF5)3 (3 mg, 0.0055 mmol) and purified by flash chromatography (silica gel, EtOAc/CH2Cl2/Hexanes, 1/1/15 to 1/1/10). 91:¹H NMR (200 MHz, CDCl3) δ 7.95-7.82 (m, 6H, Ar-H), 7.52-7.29 (m, 14H, Ar-H), 5.90 (dd, J = 9.8, 9.6 Hz, 1H, H-3′), 5.69-5.54 (m, 2H, H-2′, H-4′), 5.27 (t, J = 3.4 Hz, 1H, H-12), 5.07-5.04 (m, 2H, CO2CH2Ph), 4.88 (d, J = 7.7 Hz, 1H, H-1′), 4.30 (d, J = 9.7 Hz, 1H, H-5′), 3.69 (s, 3H, OCH3), 3.13-3.10 (m, 1H, H-3), 2.92-2.83 (m, 1H, H-18), 2.00-1.75 (m, 5H), 1.73-1.62 (m, 4H), 1.54-1.45 (m, 2H), 1.34-1.13 (m, 8H), 1.08 (s, 3H), 1.03-1.00 (m, 1H), 0.98-0.93 (m, 1H), 0.91 (s, 3H), 0.89 (s, 3H), 0.84 (s, 3H), 0.69 (s, 3H), 0.66 (s, 1H, H-5), 0.61 (s, 3H), 0.55 (s, 3H) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 177.6, 167.5, 165.9, 165.3, 165.0, 143.8, 136.5, 133.5, 133.3, 129.9, 128.5, 128.4, 128.1, 122.7, 103.4 (C-1′), 90.9 (C-3), 72.9 (C-5′), 72.5 (C-2′), 71.9 (C-4′), 70.6 (C-3′), 66.1, 55.5, 53.0, 47.7, 46.8, 45.9, 41.8, 41.5, 39.4, 38.9, 38.5, 36.8, 33.9, 33.2, 32.8, 32.5, 30.8, 27.8, 25.9, 23.8, 23.5, 23.1, 18.2, 16.9, 16.3, 15.3 ppm; HRMS⁺ (ESI-TOF) calcd for C65H77O12 [M+H]⁺ 1049.5410, found 1049.5411.

3-O-β-D-glucopyranosyloxyuronate quillaic acid (92)

To a stirred solution of 91 (30 mg, 0.029 mmol) in MeOH/THF, 2/3 (3 mL) was added Pd/C (3 mg,), TsOH (0.362 mg, 10% w/w) at rt under H2 atmosphere (balloon).

Upon completion of the reaction after 5 h, the reaction mixture was filtered, concentrated under reduced pressure and purified by column chromatography. The resulting compound then dissolved in KOH/THF, 1/4 (5 mL) at rt. The reaction was heated to 66 ℃ reflux. After being stirred for 12 h, the reaction was quenched by Amberlyst IR120 (H⁺), concentrated under reduced pressure and purified by HPLC to give 92 (10 mg, 52%) as a white solid: R_f 0. 20 (CH₂Cl₂/MeOH, 5/1). The HPLC column used in the purification was Ascentis-RP18 (5 μm, 250 mm × 10 mm). Solution A: ddH₂O. Solution B: ACN. Mobile phase: see Table 3. The flow rate was 2 mL/min.

Peaks were detected at 210 nm.; ¹H NMR (600 MHz, CD3OD) δ 5.24 (t, J = 3.4 Hz, 1H,

28-O-benzyl-3-O-(methyl 2,3,4-tri-O-acetyl–β–D-glucopyranosyluronate) oleanolate (94)

Compound 94 (30 mg, 52%) as a white solid: R_f 0.56 (EtOAc/CH₂Cl₂/Hexanes, 1/1/4) was synthesized according to method B by using compound 25 (46 mg, 0.082 mmol), 67 (30 mg, 0.055 mmol) and B(PhF₅)₃ (3 mg, 0.0055 mmol) and purified by flash chromatography (silica gel, EtOAc/CH2Cl2/Hexanes, 1/1/10 to 1/1/6). 94: ¹H NMR (200 MHz, CDCl3) δ 7.40-7.29 (m, 5H, Ar-H), 5.27 (t, J = 3.4 Hz, 1H, H-12), 5.25-5.08 (m, 3H, H-2′, H-3′, H-4′), 5.07-5.04 (m, 2H, CO2CH2Ph), 4.58 (d, J = 7.8 Hz, 1H, H-1′), 4.00 (d, J = 9.5 Hz, 1H, H-5′), 3.74 (s, 3H, OCH3), 3.13-3.05 (m, 1H, H-3), 2.95-2.86 (m, 1H, H-18), 2.02-2.00 (m, 9H, OCOCH3*3), 2.00-1.75 (m, 5H), 1.73-1.62 (m, 2H), 1.54-1.45 (m, 3H), 1.43-1.30 (m, 4H), 1.29-1.13 (m, 5H), 1.08 (s, 3H), 0.98-0.93 (m, 2H), 0.91 (s, 6H), 0.90 (s, 3H), 0.86 (s, 3H), 0.72 (s, 3H),0.66 (s, 1H, H-5), 0.58 (s, 3H) ppm; BBD¹³C NMR (50 MHz, CDCl3) δ 177.6, 170.4, 169.6, 169.3, 167.4, 143.8, 136.5, 128.5, 128.1, 122.6, 103.1 (C-1′), 90.8, 72.6, 72.3, 71.6, 69.7, 66.1, 55.6, 53.0, 47.7, 46.8, 46.0, 41.8, 41.5, 39.4, 39.0, 38.5, 36.8, 33.9, 33.2, 32.8, 32.5, 30.8, 27.8, 25.9, 25.8, 23.8, 23.5, 23.2, 20.8, 18.3, 16.9, 16.5 ppm; HRMS⁺ (ESI-TOF) calcd for C₅₀H₇₁O₁₂ [M+H]⁺ 863.4940, found 863.4931.

28-O-benzyl-3-O-(methyl 2,3,4-tri-O-pivaloyl–β–D-glucopyranosiduronate) oleanolate (95)

Compound 95 (13 mg, 48%) as white solid: R_f 0.77 (EtOAc/CH₂Cl₂/Hexanes, 1/1/4) was synthesized according to method B by using compound 32 (20 mg, 0.033 mmol), 67 (15 mg, 0.028 mmol) and B(PhF₅)₃ (1.5 mg, 0.0028 mmol) and purified by flash chromatography (silica gel, EtOAc/CH2Cl2/Hexanes, 1/1/15 to 1/1/10). 95:¹H NMR (400 MHz, CDCl3) δ 7.35-7.29 (m, 5H, Ar-H), 5.35 (dd, J = 9.4, 9.0 Hz, 1H, H-3′), 5.27 (t, J = 3.4 Hz, 1H, H-12), 5.22 (dd, J = 10.0, 9.4 Hz, 1H, H-4′), 5.14-5.01 (m, 3H, H-2′, OCH2Ph), 4.64 (d, 1H, H-1′), 4.02 (d, J = 10.0 Hz, 1H, H-5′), 3.71 (s, 3H, OCH3), 3.11-3.07 (m, 1H, H-3), 2.91-2.87 (m, 1H, H-18), 2.00-1.93 (m, 1H), 1.83-1.74 (m, 3H), 1.70-1.62 (m, 4H), 1.54-1.48 (m, 2H), 1.47-1.20 (m, 8H), 1.18-1.10 (m, 33H), 1.03-1.00 (m, 1H), 0.91 (s, 3H), 0.90 (s, 3H), 0.89 (s, 3H), 0.85 (s, 3H), 0.72 (s, 3H), 0.68 (s, 1H, H-5), 0.58 (s, 3H) ppm; BBD¹³C NMR (100 MHz, CDCl3) δ 177.5, 177.2, 176.7, 176.4, 167.6, 143.7, 136.5, 128.5, 128.1, 122.6, 102.5 (C-1′), 89.4, 72.7 (C-5′), 72.3 (C-3′), 71.8 (C-2′), 69.9 (C-4′), 66.1, 55.8, 52.8, 47.7, 46.9, 46.0, 41.8, 41.5, 39.4, 39.1, 38.9, 38.6, 36.8, 34.0, 33.3, 32.8, 32.5, 30.8, 28.4, 27.7, 27.6, 27.3, 27.2, 26.0, 25.8, 23.8, 23.5, 23.2, 18.3, 16.9, 16.4, 15.4 ppm; HRMS⁺ (ESI-TOF) calcd for C59H89O12 [M+H]⁺ 989.6349, found 989.6352.

28-O-benzyl-3-O-(methyl

2,3,4-tri-O-tert-butyldimethylsilyl-β-D-glucopyranosiduronate) oleanolate (96) Compound 96 (16 mg, 63%) was synthesized according to method B by using compound 79 (20 mg, 0.029 mmol), 67 (13 mg, 0.024 mmol) and B(PhF5)3 (1.5 mg, 0.0029 mmol). The purification of compound 96 was under flash column chromatography (silica gel, EtOAc/CH2Cl2/Hexanes, 1/1/15 to 1/1/10). 96: ¹H NMR (200 MHz, CDCl3) δ 7.40-7.29 (m, 5H, Ar-H), 5.28 (t, J = 3.4 Hz, 1H, H-12), 5.13-4.99 (m, 2H, CO2CH2Ph), 4.84 (d, J = 5.76 Hz, 1H, H-1′), 4.25 (m, 2H), 3.75-3.68 (m, 5H, OCH3), 3.11-3.02 (m, 1H, H-3), 2.92-2.85 (d, 1H, H-18), 2.02-1.80 (m, 5H), 1.75-1.57 (m, 4H), 1.54-1.45 (m, 3H), 1.45-1.15 (m, 9H), 1.11 (s, 3H), 0.94 (s, 3H), 0.92 (s, 3H), 0.91 (s, 3H), 0.89 (s, 9H), 0.88 (s, 9H), 0.87 (s, 9H), 0.85 (s, 3H), 0.80 (s, 3H), 0.73 (s, 1H, H-5), 0.60 (s, 3H), 0.10 (s, 6H), 0.09 (s, 6H), 0.07 (s, 3H), 0.05 (s, 3H) ppm; BBD

13C NMR (50 MHz, CDCl3) δ 177.6, 170.8, 143.7, 136.6, 128.5, 128.1, 122.8, 103.9 (C-1′), 88.7, 79.0, 73.0, 71.7, 66.1, 55.9, 52.1, 47.8, 46.9, 46.0, 41.8, 41.5, 39.5, 39.2, 38.9, 36.9, 34.0, 33.3, 32.9, 32.5, 30.9, 28.2, 27.8, 26.0, 23.8, 23.5, 23.2, 18.3, 18.0, 17.0, 16.7, 15.5, -3.9, -4.1, -4.8 ppm; HRMS⁺ (ESI-TOF) calcd for C62H107O9Si3

[M+H]⁺ 1079.7217, found 1079.7223.

28-O-benzyl-3-O-(methyl-2-O-benzoyl-3,4-di-O-benzyl-β-D-glucopyranosyluronate) oleanolate (97)

Compound 97 (9.5 mg, 50%) as a white solid: R_f 0.55 (EtOAc/Hexanes, 1/3) was synthesized according to method B by using compound 90 (14 mg, 0.022 mmol), 67 (10 mg, 0.018 mmol) and B(PhF5)3 (1.0 mg, 0.0022 mmol). The purification of compound 97 was under flash column chromatography (silica gel, EtOAc/Hexanes, 1/15 to 1/10).

97:¹H NMR (400 MHz, CDCl₃) δ 7.98 (d, J = 1.2 Hz, 2H, Ar-H), 7,55 (t,1H, Ar-H), 7.41 (m, 3H, Ar-H), 7.35-7.29 (m, 7H, Ar-H), 7.15-7.09 (m, 6H, Ar-H), 5.30 (dd, J = 10.0, 8.4 Hz, 1H, H-2), 5.26 (t, J = 3.4 Hz, 1H, H-12), 5.10-5.02 (m, 2H, CO2CH2Ph), 4.80-4.58 (m, 5H, H-1′, H-5′, H-4′, OCH2Ph), 3.96-3.81 (m, 2H, OCH2Ph), 3.81 (m, 1H, H-3′), 3.74 (s, 3H, OCH3), 3.07-3.00 (m, 1H, H-3), 2.90-2.85 (m, 1H, H-18), 2.00-1.93 (m, 1H), 1.83-1.74 (m, 2H), 1.70-1.62 (m, 3H), 1.54-1.48 (m, 2H), 1.47-1.20 (m, 8H), 1.20-1.12 (m, 3H), 1.07 (s, 3H), 1.00-0.94 (m, 2H), 0.90 (s, 3H), 0.88 (s, 3H),0.84-0.82 (m, 1H), 0.80 (s, 3H), 0.65 (s, 3H), 0.60 (s, 1H, H-5), 0.56 (s, 3H), 0.53 (s, 3H) ppm;

BBD¹³C NMR (100 MHz, CDCl3) δ 177.5, 168.9, 165.1, 143.8, 137.8, 137.7, 133.2, 130.0, 129.9, 128.6, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.8, 122.7, 103.7 (C-1′), 90.5, 82.0, 79.5, 75.2, 75.1, 74.6, 73.7, 66.1, 55.6, 52.7, 47.7, 46.8, 46.0, 41.7, 41.5, 39.4, 38.9, 38.5, 36.7, 34.0, 33.2, 32.7, 32.5, 30.8, 29.8, 27.9, 27.7, 26.0, 25.8, 23.8, 23.5, 23.2, 18.2, 16.9, 16.3, 15.3 ppm; HRMS⁺ (ESI-TOF) calcd for C₆₅H₈₀O₁₀Na [M+Na]⁺ 1043.5644, found 1043.5638.

28-O-Allyl-3-O-(methyl 2,3,4-tri-O-acetyl-β-D-glucopyranosyluronate)quillate (98) Compound 98 (15 mg, 17%) as a white solid: Rf 0.19 (EtOAc/Hexanes, 1/3) was synthesized according to method B by using 25 (50 mg, 0.104 mmol), 61 (46 mg, 0.0867 mmol) and B(PhF5)3 (5.3 mg, 0.0104 mmol) and purified by flash chromatography (silica gel, EtOAc/Hexanes, 1/4). 98: ¹H NMR (400 MHz, CDCl₃) δ 9.35 (s, 1H, H-23), 5.87-5.78 (m, 1H, OCH2CHCH2), 5.38 (t, J = 3.4 Hz, 1H, H-12), 5.29 (d, 1H, J = 17.08 Hz, OCH2CHCH2), 5.21-5.13 (m, 3H, H-3′, H-4′, OCH2CHCH2), 4.91 (dd, J = 8.2, 7.9 Hz, 1H, H-2′), 4.53-4.42 (m, 3H, H-16, OCH2CHCH2), 4.43 (d, J

= 7.9 Hz, 1H, H-1′), 3.97 (d, J = 9.4 Hz, 1H, H-5′), 3.78-3.73 (m, 4H, H-3, OCH3), 3.11-3.05 (m, 1H, H-18), 2.14 (t, J = 13.2 Hz, 1H, H-19), 2.07-2.04 (m, 4H), 2.00 (s, 3H), 1.99 (s, 3H), 1.81-1.68 (m, 4H), 1.79-1.65 (m, 6H), 1.50-1.38 (m, 2H), 1.34 (s, 3H), 1.30-1.20 (m, 3H), 1.17-1.05 (m, 3H), 1.00 (s, 3H), 0.96 (s, 3H), 0.94 (s, 3H), 0.91-0.89 (m, 4H), 0.70 (s, 3H) ppm; BBD¹³C NMR (100 MHz, CDCl3) δ 206.4, 176.4, 170.2, 167.2, 132.3, 122.6, 118.3, 102.1 (C-1′), 83.4, 75.0, 72.5, 72.0, 71.0, 69.5, 65.3, 55.1, 53.1, 48.9, 48.8, 46.6, 46.5, 41.5, 40.7, 39.9, 38.2, 36.0, 32.9, 32.9, 30.9, 30.6, 27.1, 24.7, 20.7, 20.6, 17.1, 15.8, 10.1 ppm; HRMS⁺ (ESI-TOF) calcd for C46H67O14 [M+H]⁺ 843.4525, found 843.4525.

28-O-Allyl-3-O-(methyl 2,3,4-tri-O-benzoyl-β-D-glucopyranosyluronate)quillate (99)

Compound 99 (12 mg, 38%) as a white solid: R_f 0.23 (EtOAc/Hexanes, 1/4) was synthesized according to method B by using 31 (20 mg, 0.036 mmol), 61 (16 mg, 0.030 mmol) and B(PhF5)3 (1.5 mg, 0.0036 mmol) and purified by flash chromatography (silica gel, EtOAc/Hexanes, 1/5). 99: ¹H NMR (400 MHz, CDCl3) δ 9.19 (s, 1H, H-23), 7.95-7.81 (m, 6H, Ar-H), 7.54-7.26 (m, 9H, Ar-H), 5.86-5.82 (m, 2H, H-3′, OCH2CHCH2), 5.59 (dd, J = 9.6, 9.4 Hz, 1H, H-4′), 5.45 (dd, J = 9.7, 7.8 Hz, 1H, H-2′), 5.38 (t, J = 3.4 Hz, 1H, H-12), 5.28 (dd, J = 17.1, 1.4 Hz, 2H, OCH2CHCH2), 4.74 (d, J = 7.8 Hz, 1H, H-1′), 4.51-4.46 (m, 3H, H-16, OCH2CHCH2), 4.27 (d, J = 9.4 Hz, 1H, H-5′), 3.89-3.82 (m, 1H, H-3), 3.68 (s, 3H, OCH3), 3.05 (dd, J = 14.2, 3.5 Hz, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 2.02-1.83 (m, 4H), 1.81-1.68 (m, 5H), 1.50-1.38 (m, 4H), 1.31 (s, 3H), 1.30–1.20 (m, 3H), 1.17-1.05 (m, 3H), 1.01 (s, 3H), 0.96 (s, 3H), 0.92 (s, 3H), 0.89 (s, 3H), 0.85-0.82 (m, 1H), 0.68 (s, 3H) ppm; BBD¹³C NMR (100 MHz, CDCl3) δ 208.3, 176.4, 167.4, 165.7, 165.4, 164.9, 142.9, 133.6, 133.4, 132.3, 130.0, 129.9, 129.2, 128.9, 128.8, 128.6, 128.5, 122.7, 118.3, 102.1 (C-1′), 83.4, 75.1, 72.8, 72.2, 71.5, 70.4, 65.3, 54.4, 53.0, 49.8, 48.8, 46.6, 46.5, 41.5, 40.7, 39.9, 38.2, 36.2, 35.5, 32.9, 32.3, 30.9, 30.5, 27.1, 25.0, 24.8, 23.4, 20.2, 17.1, 15.8, 10.7 ppm;

HRMS⁺ (ESI-TOF) calcd for C₆₁H₇₃O₁₄ [M+H]⁺ 1029.4995, found 1029.4995.

28-O-Allyl-3-O-(methyl

2,3,4-tri-O-tert-butyldimethylsilyl-β-D-glucopyranosiduronate) quillate (100)

Compound 100 (22.5 mg, 59%) as a colorless syrup: R_f 0.51 (EtOAc/Hexanes, 1/4) was synthesized according to method B by using compound 79 (30 mg, 0.043 mmol), 61 (19 mg, 0.036 mmol), B(PhF₅)₃ (2 mg, 0.0043 mmol) and was purified by column chromatography (silica gel, EtOAc/Hexanes, 1/15 to 1/8). 100: ¹H NMR (400 MHz, CDCl3) δ 9.39 (s, 1H, H-23), 5.90-5.82 (m, 1H, OCH2CHCH2), 5.38 (t, J = 3.4 Hz, 1H, H-12), 5.38-5.18 (m, 2H, OCH2CHCH2), 4.63 (d, J = 5.0 Hz, 1H, H-1′), 4.52-4.47 (m, 3H, OCH2CHCH2, H-16), 4.21 (d, 2H), 3.97-3.93 (m, 1H, H-3), 3.73 (s, 3H, OCH3), 3.66 (s, 1H), 3.56 (d, 1H), 3.09-3.02 (m, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 1.99-1.86 (m, 4H), 1.84-1.70 (m, 5H), 1.67-1.65 (m, 2H), 1.54-1.52 (m, 1H), 1.45-1.40 (m, 2H), 1.33 (s, 3H), 1.25-1.20 (m, 2H), 1.14-1.12 (m, 2H), 1.08 (s, 3H), 1.05-1.00 (m, 2H), 0.96 (s, 3H), 0.95 (s, 3H), 0.90 (s, 12H), 0.86 (s, 9H), 0.85 (m, 9H), 0.71 (s, 3H), 0.09 (s, 3H), 0.08 (s, 3H), 0.07 (s, 3H), 0.05 (s, 3H), 0.04 (s, 3H), 0.02 (s, 3H) ppm;

BBD¹³C NMR (100 MHz, CDCl3) δ 206.9 (CHO), 176.5, 170.5, 142.9, 132.3 (OCH2CHCH2), 122.8 (C-12), 118.2 (OCH2CHCH2), 100.0 (C-1′), 78.8, 78.5, 75.1, 72.9, 65.3 (allylic CH₂), 54.9, 52.2, 48.9, 48.0, 46.8, 46.5, 41.5, 40.8, 39.9, 38.3, 36.0, 35.6, 35.6, 32.9, 32.4, 30.9, 30.5, 27.1, 26.0, 25.9, 24.8, 24.6, 23.4, 20.2, 18.0, 17.2, 15.7, 10.3, -4.1, -4.2, -4.3, -4.3 ppm; HRMS⁺ (ESI-TOF) calcd for C₅₈H₁₀₃O₁₁Si₃ [M+H]⁺ 1059.6803, found 1059.6815.

28-O-Allyl-3-O-(methyl

2-O-benzoyl-3,4-O-benzyl-β-D-glucopyranosyluronate)quillate (101)

Compound 101 (7.5 mg, 42%) as a white solid: R_f 0.31 (EtOAc/Hexanes, 1/3) was synthesized according to method B by using compound 90 (14 mg, 0.022 mmol), 61 (9.5 mg, 0.018 mmol) and B(PhF5)3 (1.0 mg, 0.0022 mmol). The purification of compound 101 was under flash column chromatography (silica gel, EtOAc/Hexanes, 1/10 to 1/6). 101:¹H NMR (400 MHz, CDCl₃) δ 9.14 (s, 1H, H-23), 8.00 (d, J = 1.2 Hz, 2H, Ar-H), 7.59-7.28 (m, 6H, Ar-H), 7.25-7.09 (m, 7H, Ar-H), 5.87-5.78 (m, 1H, OCH2CHCH2), 5.36 (t, J = 3.4 Hz, 1H, H-12), 5.29-5.16 (m, 3H, OCH2CHCH2, H-2′), 4.78-4.58 (m, 4H, OCH2Ph, H-1′, H-5′), 4.50-4.42 (m, 4H, H-4′, H-16, OCH2CHCH2), 3.92 (dd, 2H, OCH2Ph), 3.75-3.70 (m, 5H, H-3′, H-3, OCH3), 3.08-3.01 (m, 1H, H-18), 2.14 (t, J = 13.6 Hz, 1H, H-19), 1.86-1.83 (m, 4H), 1.76-1.74 (m, 4H), 1.52-1.47 (m, 2H), 1.42-1.35 (m, 2H), 1.30 (s, 3H), 1.25 (s, 3H), 1.19-1.08 (m, 4H), 0.96 (s, 3H), 0.95 (s, 3H), 0.89 (s, 3H), 0.88 (s, 3H), 0.85-0.84 (m, 1H), 0.66 (s, 3H) ppm;BBD¹³C NMR (100 MHz, CDCl3) δ 208.6, 176.5, 168.8, 165.1, 142.8, 137.7, 137.6, 133.3, 132.3, 129.9, 129.8, 128.6, 128.5, 128.4, 128.2, 128.1, 127.9, 122.7, 118.2, 102.5 (C-1), 83.2, 81.8, 79.4, 75.2, 75.0, 74.5, 73.3, 65.3, 54.5, 52.7, 49.7, 48.9, 46.6, 46.5, 41.5, 40.7, 39.9, 38.1, 36.2, 35.6, 32.9, 32.2, 30.8, 30.5, 29.8, 27.1, 25.0, 24.8, 23.4, 20.2, 17.1, 15.7, 10.7 ppm; HRMS⁺ (ESI-TOF) calcd for C₆₁H₇₆O₁₂Na [M+Na]⁺ 1023.5229, found 1023.5224.

第五章參考文獻

1. Wong, C. H.; Krasnova, L., Highlights in chemical glycobiology. Isr. J. Chem. 2019, 59, 18-22.

2. Huang, G.; Chen, X.; Huang, H., Chemical modifications and biological activities of polysaccharides. Curr. Drug Targets 2016, 17, 1799-1803.

3. Krasnova, L.; Wong, C. H., Understanding the chemistry and biology of glycosylation with glycan synthesis. Annu. Rev. Biochem. 2016, 85, 599-630.

4. Cao, H.; Hwang, J.; Chen, X., 14. Carbohydrate-containing natural products in medicinal chemistry. Research Signpost Publications 2011, 411-431.

5. Petrella, R. J., Hyaluronic acid for the treatment of knee osteoarthritis: long-term

Aesthetic Plast. Surg. 2004, 28, 295-300.

8. Alekseeva, L. I.; Benevolenskaia, L. I.; Nasonov, E. L.; Chichasova, N. V.; Kariakin, A. N., [Structum (chondroitin sulfate)--a new agent for the treatment of osteoarthrosis]. Ter. Arkh. 1999, 71, 51-53.

9. Oduah, E. I.; Linhardt, R. J.; Sharfstein, S. T., Heparin: Past, Present, and Future.

Pharmaceuticals (Basel) 2016, 9, 38.

10. Hou, N.; Cai, B.; Ou, C. W.; Zhang, Z. H.; Liu, X. W.; Yuan, M.; Zhao, G. J.; Liu, S.

M.; Xiong, L. G.; Luo, J. D.; Luo, C. F.; Chen, M. S., Puerarin-7-O-glucuronide, a water-soluble puerarin metabolite, prevents angiotensin II-induced cardiomyocyte hypertrophy by reducing oxidative stress. Naunyn Schmiedebergs Arch. Pharmacol.

在文檔中藉由保護基策略探討三萜之 3-O-葡萄糖醛酸化反應的優化研究 (頁 49-135)

Glucuronidation with Allyl quillaic ester

第六章 附圖

Scheme 22. Glucuronidation with Allyl quillaic ester

2.3 玻尿酸分解酶抑制劑活性

第三章 結論

第四章 實驗部分

4.2 一般實驗方法

4.3.2 實驗儀器

4.4 合成步驟及數據

第五章 參考文獻

第六章附圖

第三章結論

第四章實驗部分

第五章參考文獻