氨基的保护及脱保护策略-16

氨基的保护及脱保护策略

药明康德内部保密资料

经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

目 录

1． 2．

氨基的保护及脱保护概要……………………………………………2 烷氧羰基类

2-1. 苄氧羰基（Cbz）……………………………………………… 4 2-2. 叔丁氧羰基（Boc）……………………………………………… 16 2-3. 笏甲氧羰基（Fmoc） ………………………………………… 28 2-4. 烯丙氧羰基（Alloc） ………………………………………… 34 2-5. 三甲基硅乙氧羰基（Teoc） …………………………………… 36 2-6. 甲（或乙）氧羰基 …………………………………………… 40

3．

酰基类

3-1. 邻苯二甲酰基（Pht）…………………………………………… 43 3-2. 对甲苯磺酰基（Tos） ………………………………………… 49 3-3. 三氟乙酰基（Tfa） ………………………………………… 53 4．

烷基类

4-1. 三苯甲基（Trt） ……………………………………………… 57 4-2. 2,4-二甲氧基苄基（Dmb） …………………………………… 63 4-3. 对甲氧基苄基（PMB） ……………………………………… 65 4-4. 苄基（Bn） …………………………………………………… 70

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经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

1．氨基的保护及脱保护概要

选择一个氨基保护基时，必须仔细考虑到所有的反应物，反应条件及所设计的反应过程中会涉及的所有官能团。首先，要对所有的反应官能团作出评估，确定哪些在所设定的反应条件下是不稳定并需要加以保护的，并在充分考虑保护基的性质的基础上，选择能和反应条件相匹配的氨基保护基。其次，当几个保护基需要同时被除去时，用相同的保护基来保护不同的官能团是非常有效（如苄基可保护羟基为醚，保护羧酸为酯，保护氨基为氨基甲酸酯）。要选择性去除保护基时，就只能采用不同种类的保护基（如一个Cbz保护的氨基可氢解除去，但对另一个Boc保护的氨基则是稳定的）。此外，还要从电子和立体的因素去考虑对保护的生成和去除速率的影响（如羧酸叔醇酯远比伯醇酯难以生成或除去）。最后，如果难以找到合适的保护基，要么适当调整反应路线使官能团不再需要保护或使原来在反应中会起反应的保护基成为稳定的；要么重新设计路线，看是否有可能应用前体官能团（如硝基，亚胺等）；或者设计出新的不需要保护基的合成路线。

在合成反应中，伯胺、仲氨、咪唑（imidazole）、吡咯（pyrrole ）、吲哚

(又

称：苯并吡咯）和其他芳香氮杂环中的氨基往往是需要进行保护的。已经使用过的氨基保

护基很多，但归纳起来，可以分为烷氧羰基、酰基和烷基三大类。烷氧羰基使用最多，因为N-烷氧羰基保护的氨基酸在接肽时不易发生消旋化。伯胺、仲氨、咪唑、吡咯、吲哚和其他芳香氮氢都可以选择合适的保护基进行保护。下表列举了几种代表性的常用的氨基保护基。

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几种代表性的常用的氨基保护基

结构 OOX缩写 应用 引入条件 脱去条件 H2/Pd-C，供氢体/Pd-C，BBr3/CH2Cl2 or TFA，HBr/HOAc等 3MHCl/EtOAc, HCl/MeOH or diox, TsOH/THF-CH2Cl2, Me3SiI/CHCl3orCH3CN 20%哌啶/DMF，50%哌啶/CH2Cl2等 Ni(CO)4/DMF/H2O; Pd(PPh3)4/Bu3SnH; Cbz 伯胺、仲氨、咪唑、Cbz-Cl/Na2CO3/CHCl3/H2O 吡咯、吲哚等 Boc2O/NaOH/diox/H2伯胺、仲氨、咪唑、O, Boc2O/ /MeOH, Boc2O/Me4NOH/CH3C吡咯、吲哚等 N 伯胺、仲氨等 伯胺、仲氨、咪唑、吡咯、吲哚等 Fmoc-Cl/NaHCO3,/diox/H2O Aloc-Cl/Py OXO Boc XOOFmoc XOOClOTMSO Alloc Teoc 伯胺、仲氨、咪唑、Teoc-Cl/碱/diox/H2O 吡咯、吲哚等 TBAF；TEAF XOOMe( or Et) - 伯胺、仲氨、咪唑、ROCOCl/NaHCO3,/diox/H2O 吡咯、吲哚等 邻苯二甲酸酐/CHCl3/70℃;邻苯二甲酰亚胺-NCO2Et/aq. Na2CO3 Ts-Cl/Et3N TFAA/Py; 苯二甲酰亚胺-NCO2CF3/CH2Cl2 Trt-Cl/Et3N HBr/HOAc; Me3SiI; KOH/H2O/乙二醇 H2NNH2/EtOH，NaBH4/i-PrOH-H2O（6：1） HBr/HOAc, 48%HBr/苯酚（cat） K2CO3/MeOH/H2O; NH3/MeOH; HCl/MeOH HCl/MeOH, H2/Pd/EtOH, TFA/CH2Cl2 HCO2H/Pd-C/MeOH; H2/Pd(OH)2/EtOH; TFA; CAN/ CH3CN HCO2H/Pd-C/MeOH; H2/Pd(OH)2/EtOH; CCl3CH2OCOCl/CH3CN Page 3 of 78

OXNOOXSOPht Ts O伯胺 伯胺、仲氨、咪唑、吡咯、吲哚等 伯胺、仲氨、咪唑、吡咯、吲哚等 伯胺、仲氨、咪唑、吡咯、吲哚等 XCF3 Tfa XTrt MeOOMeX Dmb 伯胺、仲氨、咪唑、ArCHO/NaCNBH3/MeOH 吡咯、吲哚等 PMB-Br/ 伯胺、仲氨、咪唑、K2CO3/CH3CN;PhCH吡咯、吲哚等 O/NaCNBH3/MeOH Bn-Br/Et3N or 伯胺、仲氨、咪唑、K2CO3/CH3CN;PhCH吡咯、吲哚等 O/NaCNBH3/MeOH OMeX PMB X Bn 药明康德内部保密资料

经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

2．烷氧羰基类保护基

烷氧羰基类保护基可用于氨基酸，以在肽合成中减少外消旋化的程度。外消旋化发生在碱催化的N-保护的羧基活化的氨基酸的偶联反应中，也发生在易由N-酰基保护的氨基酸形成的中间体恶唑酮中。

要使外消旋化程度减到最小，需使用非极性溶剂、最弱的碱、低的反应温度，并使用烷氧羰基类保护的氨基酸是有效的。其中常用的有易通过酸性水解去保护的Boc基、由催化氢解去保护的Cbz基、用碱经β-消除去保护的Fmoc基和易由钯催化异构化去保护的Alloc基。

2.1苄氧羰基（Cbz）

苄氧羰基（Cbz）是1932年Bergmann发现的一个很老的氨基保护基，但一直到今天还在应用。其优点在于：试剂的制备和保护基的导入都比较容易；N-苄氧羰基氨基酸和肽易于结晶而且比较稳定；苄氧羰基氨基酸在活化时不易消旋；能用多种温和的方法选择性地脱去。 2.1.1苄氧羰基的导入

苄氧羰基的导入，一般都是用Cbz-Cl。游离氨基在用NaOH 或NaHCO3 控制的碱性条件下可以很容易同Cbz-Cl反应得到N-苄氧羰基氨基化合物。α,β-二胺可用该试剂在pH= 3.5-4.5稍有选择性地被保护，其选择性随碳链地增长而减弱，如H2N(CH2)nNH2, n=2时71%被单保护; n=7时29%被单保护[1]。 氨基酸酯同Cbz-Cl的反应则是在有机溶剂中进行，并用碳酸氢盐或三乙胺（TEA)来中和反应所产生的HCl。此外，Cbz-ONB（4-O2NC6H4OCOOBn）等苄氧羰基活化酯也可用来作为苄氧羰基的导入试剂，该试剂使伯胺比仲胺易被保护，但苯胺由于亲核性不足，与该试剂不反应[2]。

R1HNCbz-ClR2BaseR1R2NCbzNH2R2OOCR1Cbz-ClBaseR2OOCNHCbzR1

1．G. J. Atwell, W. A. Denny., Synthesis, 1984, 1032

2．D. R. Kelly, M. Gingell, Chem. Ind.(London), 1991, 888

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filtered through celite and the filtrate was evaporate to dryness to give 430 mg of compound 2 (98%). This compound was used without further purification in the subsequent step.

2.1.2.4 Pd/C-甲酸催化氢解示例

OOOO1NHNOOOO10%Pd-CHCOOHO2OOONH2NH2

Fyles, T. M.; Zeng, B.; J. Org. Chem., 1998, 63(23), 8337-8345

Compound 1 (0.6 g, 0.8 mmol) was dissolved in 1:1 formic acid/methanol (60 mL) and added to a round-bottom flask (100 mL) containing 1 equiv of palladium catalyst (10% Pd/C, 1.0 g, 0.9 mmol). The mixture was continuously stirred under reflux temperature for 24 h. The catalyst was removed by filtration and washed with an additional 10 mL of methanol. The combined solvents were removed by evaporation under reduced pressure to give Compound 2 (0.34 g, 81%, a white solid, mp 96-98 °C). This compound was used without further purification in the subsequent step.

2.1.2.5 Pd/C催化氢解脱Cbz上Boc示例

OOHNO1NHH2/10%Pd-C(Boc)2OBocHNONH2

WO2004092166

10%Pd-C was addede to a solution of compound 1 (596 mg , 1.77 mmol) and (Boc)2O (773 mg, 3.54 mmol) in etnyl acetate (30 ml). The reaction vessel was evacuated and back-filled with nitrogen (three times), then back-filled with hydrogen (1 atm). After 2 h, the mixture was filtered and concentrated. Purification by silica gel chromatography (30% ethyl acetate/ hexanes - 50% ethyl acetate/ hexanes) gave compound 2 (289 mg, 54%).

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2.1.2.6 PdCl2催化氢解脱除带卤原子分子上的Cbz示例

HNNClCl1OEt3SiHNOOPdCl2ClCl2NNHNONNH

US20030144297

To a solution o compound 1 (900 mg) in methylene chloride (16.5 ml) was addede PdCl2 (30 mg) and triethylamine (0.229 ml). Triethyl silane was added (2 x 0.395 ml) over 2 h. The reaction mixture stirred 1 h and 2 ml of trifluoroacetic acid was added. After 30 min the reaction was basified with 2 N NaOH, extracted with methylene chloride, dried over MgSO4, filtered and concentrated. Chromatography was run on a biotage 40S column with 3-5% MeOH/CH2Cl2 with 0.5% NH4OH to provide compound 2 as a oil (501 mg, 74%).

2.1.2.7 Pd黑催化氢解，用氨为溶剂,半胱氨酸的Cbz脱除示例

OSOHNHCbz3H2Pd4SNH2OOH

Arthur M. Felix, Manuel H. Jimenz et a1., Org. Syn., 59, 159

A dry 1-L three-necked, round-bottomed flask is equipped with a dry ice reflux condenser, a gas-inlet tube, and a magnetic stirring bar as illustrated in the figure. The reaction vessel is

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immersed in an acetone–dry ice bath, and a total of 300 mL of ammonia is passed through a drying tower containing potassium hydroxide pellets and collected in the flask. The bath is removed to permit the reaction to proceed at the boiling point of ammonia (?33℃), and a gentle stream of dry nitrogen is bubbled into the flask. A solution of 0.708 g (0.80250 mole) of N-benzyloxycarbonyl-L-methionine in 10 ml. of N,N-dimethylacetamide 1.02 g (1.40 ml., 0.0101 mole) of triethylamine and 1.25 g of freshly prepared palladium black are added. The nitrogen stream is discontinued and replaced by a stream of hydrogen that has been passed through a concentrated sulfuric acid scrubber. The mixture is stirred under reflux for 5.5 hours to effect hydrogenolysis. The hydrogen stream is discontinued, a flow of nitrogen is resumed, and the dry ice is removed from the reflux condenser, permitting rapid evaporation of ammonia. The flask is attached to a rotary evaporator, and the mixture is evaporated to dryness under reduced pressure. The residue is dissolved in water and filtered through a sintered funnel of medium porosity to remove the catalyst. The filtrate is evaporated to dryness, and the residue (354 mg, 95%) is crystallized from water–ethanol. The white crystalline product, after drying under reduced pressure at 25°, weighs 272–305 mg. (73–82%), m.p. 280–282° (dec.), [α]25D +23.1° (c = 1, aqueous 5 N hydrochloric acid).

酸解脱除 氨基甲酸苄酯在强酸性条件下容易去保护。HBr/HOAc 是酸解脱除苄氧羰基的最常用的试剂[1]。脱除反应主要按下式进行[2]。反应需要消耗2分子的HBr，Cbz的脱除速度随HBr浓度的增大而增大，因此实际上都是采用高浓度的过量HBr/HOAc溶液（1.2M-3.3M）以保证反应的完全。

R2R1NCbz+R2H+R1NCbzH+Br-HBrCH2Br+CO2+R1H.HBrNR2

1. D. Ben-Ishai, A. Berger., J. Org. Chem., 1952, 17, 1564; R. A. Boissonnas, J. Blodinger, A. D. Welcher., J. Am. Chem. Soc., 1952, 74, 5309

2. R. A. Boissonnas, J. Blodinger, A. D. Welcher., J. Am. Chem. Soc., 1952, 74, 5309; J. Meienhofer, E. Schnabel., Z. Naturforsch., 1965, 20b, 661

含有丝氨酸[1]和苏氨酸[2]的肽或其它含羟基的氨基衍生物用HBr/HOAc脱除Cbz时会发生羟基的O-乙酰化反应。虽然O-乙酰基能用碱皂化或氨解脱去，但为了避免这个

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副反应，可以改用HBr/二氧六环或HBr/三氟乙酸来代替HBr/HOAc[3]。由于HBr在三氟乙酸中的溶解度较小，因此不能预先制成HBr/三氟乙酸溶液，而只能将保护的肽或氨基衍生物溶于无水三氟乙酸中，先于0℃下通入干燥的HBr，待Cbz大部分脱除后，再室温通短时间以求完全脱除变化基。Cbz被HBr分解产生的溴化苄能同肽中的某种氨基酸反应，也是需要加以注意的。如，甲硫氨酸的硫原子能同溴化苄反应生成S-苄基甲硫氨酸[4]，防止的办法是加入硫醚（CH3SC2H5）为捕捉剂[5]。色氨酸被HBr/HOAc分解产生有色物质，防止的办法是加入亚磷酸二乙酯。硝基精氨酸会发生硝基的部分脱落，改用液体HBr于-67℃处理可以避免。

1. G. D. Fasman, E. R. Blout., J. Am. Chem. Soc., 1960, 82, 2262

2. S. Fujiwara, S. Moerinaga, K. Narita., Bull. Chem. Soc. Japan., 1962, 35, 438

3. J. Meienhofer, E. Schnabel., Z. Naturforsch., 1965, 20b, 661; 黄惟德等，生物化学与生物物理学报, 1961, 98

4. N. F. Albertson, F. C. Mckay., J. Am. Chem. Soc., 1953, 73, 5323 5. S. Guttmann, R. A. Boissonnas, Helv. Chim. Acta., 1959, 42, 1257

用液体HF在0℃处理10-30分钟即可将Cbz完全脱去[1]。FSO3H[2]、CH3SO3H[2, 3]、CF3SO3H[3, 4]和C6H5SCH3-TFA[5]也是较好的试剂。Me3SiI在氯仿、乙腈中能于几分钟内选择性脱去Cbz和Boc保护基[6]。对于BBr3/CH2Cl2而言，较大分子的肽的Cbz衍生物可在TFA中去除，因为肽在酸中的溶解度比在CH2Cl2中大[7]。从肽中脱去Cbz，可在TFA中添加0.5 M 4-（甲硫基）苯酚[8]或使用HF/Me2S/对甲苯酚[9]（25:65:10,v/v）来抑制Bn+对芳香氨基酸的加成。

1. S. Sakakibara et a1., Bull. Chem. Soc. Japan., 1967, 40, 2164; S. Matsuura, C. H. Niu, J. S. Cohen., J. Chem. Soc. Chem. Commun., 1976, 451

2. H. Yajima, H. Ogawa, H. Sakurai., J. Chem. Soc. Chem. Commun., 1977, 909 3. H. Yajima et a1., J. Chem. Soc. Chem. Commun., 1974, 107 4. H. Yajima et a1., Chem. Pharm. Bull., 1975, 23, 1164

5. Y. Kiso, K. Ukawa, T. Akita., J. Chem. Soc. Chem. Commun., 1980, 101

6. R. S.Lott, V. S. Chauham, C. H. Stammer., J. Chem. Soc. Chem. Commun., 1979, 495 7. J. Pless, W. Bauer., Angew Chem., Int. Ed. Engl., 1973, 12, 147; A. M. Felix., J. Org.

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Chem., 1974, 39, 1427

8. M. Bodanszky, A. Bodanszky., Int. J. Pept. Protein Res., 1984, 23, 287 9. J. P. Tam, W. F. Heath, R. B. Merrifield., J. Am. Chem. Soc., 1983, 105, 6442

此外，已经报道过的还有以下的一些不常用的方法。如HCl/CHCl3[1]、HCl/HOAc[2]、HBr/SO2[3]、液体HBr[4]、TosOH[5]、HI/HOAc[6]、碘化磷[7]、Et3SiH[8]、沸腾的TFA[9]、8M HCl的乙醇液或6 M HCl回流1小时[10]或浓盐酸于25-75℃加热处理1-1.5小时[11]等。

1. G. D. Fasman, M. Idelson, E. R. Blout., J. Am. Chem. Soc., 1961, 83, 709 2. R. B. Merrifield., J. Am. Chem. Soc., 1963, 85, 2149 3. M. Idelson, E. R. Blout., J. Am. Chem. Soc., 1958, 80, 4631 4. M. Brenner, H. C. Curtius., Helv. Chim. Acta., 1963, 46, 2126 5. E. Taschner, B. Liberek, Abstr. Int. Cong. Biochemistry, Vienna 1958 6. E. Waldschmidt-Leitz, K. Kuhn., Chem. Ber., 1951, 84, 381

7. E. Brand, B. F. Erlanger, H. Sachs., J. Am. Chem. Soc., 1952, 74, 1849 8. Birkofer et al., Angew. Chem., Int. Ed., 1965, 4, 417 9. F. Weygand, W. Steglich., Z. Naturforsch., 1959, 14b, 472

10. A.E. Barkdoll, W. F. Ross., J. Am. Chem. Soc., 1944, 66, 567; G. Chelucci, M. Falorni, G. Giacomelli., Synthesis., 1990, 1121 11. J. White., J. Biol. Chem., 1934, 106, 141

2.1.2.8 HBr-AcOH脱除Cbz示例

OEtOOCNSNHNONHCbz33% HBrAcOH, 91%EtOOCNSNHONONH2.HBr

B. Anna; P. Gerald., Heterocycles, 2002, 58, 521

A solution of the amine Cbz compund (208 mg, 0.44 mmol) in 33 % hydrobromic acid in acetic acid (1 mL) and glacial acetic acid (0.6 mL) was stirred at rt for 3 h under an atmosphere of nitrogen. The volatiles were removed in vacuo to leave the free amine

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hydrobromide (168 mg, 91 %) as a brown, highly hygroscopic powder; [α]D =-18.0° (c = 0.4, EtOH);

2.1.2.9 TMSI脱除Cbz示例1

OOONH1BrONHCbzMe3SiIEt3N, CH3CNONH2BrONH2OO

US20040204397

Me3SiI (0.73 ml, 0.73 mmol) was added to a soluton of compound 1 (146 mg, 0.33 mmol) in acetonitrile (10 ml) at room temperature, and the resulting mixture was stirred at room temperature for 2 h. Et3N (0.12 ml) was added and the mixture was stirred at room temperature for 15 min. The solvents were removed in vacuo, and the residue was extracted with ethyl acetate. The combined organics were washed with sodium bicarbonate and brine, dried over sodium sulfate and filtered. Solvents were removed and the residue was used directly in the next step.

HNClNNH1NHCbzNNNMe3SiIEt3N, CH3CNClNNH2NH2HNNNN

US20050203078

2.1 g (4.45 mmol) of compound 1 in 30 ml of CH2Cl2 were combined with 1.9 ml (13.4 mmol) Me3SiI and stirred for 16 h at room temperature. Then 20 ml of MeOH were addede, the mixture was stirred for a further 30 min at room temperature and the reaction mixture was evaporated down completely. The residue was purified by chromatography on silica gel (eluding gradient: CH2Cl2/(MeOH/conc. Ammonia 95:5) = 70/30 – 60/40) to yield compound 2 (690 mg, 56%).

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2.2 叔丁氧羰基（Boc）

除Cbz保护基外，叔丁氧羰基（Boc）也是目前多肽合成中广为采用的氨基保护基，特别是在固相合成中，氨基的保护用Boc而多不用Cbz。Boc具有以下的于的优点：Boc-氨基酸除个别外都能得到结晶；易于酸解除去，但有具有一定的稳定性，Boc-氨基酸能较长期的保存而不分解；酸解时产生的是叔丁基阳离子再分解为异丁烯，它一般不会带来副反应；对碱水解、肼解和许多亲核试剂稳定；Boc对催化氢解稳定，但比Cbz对酸要敏感得多。当Boc和Cbz同时存在时，可以用催化氢解脱去Cbz，Boc保持不变，或用酸解脱去Boc而Cbz不受影响，因而两者能很好地搭配。 2.1.1叔丁氧羰基的导入

游离氨基在用NaOH 或NaHCO3 控制的碱性条件下用二氧六环和水的混合溶剂中很容易同Boc2O反应得到N-叔丁氧羰基氨基化合物[1]。这是引入Boc常用方法之一，它的优点是其副产物无多大干扰并容易除去。有时对一些亲核性较大的胺，一般可在甲醇中和Boc酸酐直接反应即可，无须其他的碱，其处理也方便。

对水较为敏感的氨基衍生物，采用Boc2O/TEA/MeOH or DMF 在40-50℃下进行较好，因为这些无水条件下用于保护O17标记的氨基酸而不会由于与水交换使O17丢失[2]。有空间位阻的氨基酸而言，用Boc2O/Me4NOH.5H2O/CH3CN是十分有利的。

1. D. S. Tarbell, Y. Yamamoto et al., Proc. Natl. Acad. Sci., USA, 1972, 69, 730 2. E. Ponnusamy, U. Fotadar et al., Synthesis., 1986, 48

芳香胺由于其亲核性较弱，一般反应需要加入催化剂，另外对于伯胺，通过DMAP的使用可以上两个Boc.

Boc2O, DMAPBocArNBoc

ArNH2ArNHBoc对于有酚羟基存在的胺，酚羟基上接Boc的速度也是相当快的，因而一般没太大的选择性。 对于有醇羟基存在的，若用DMAP做催化剂，时间长了以后醇羟基也能上Boc， 因此反应尽量不要过夜。

由于氰酸酯的生成，有位阻的胺往往会与Boc2O生成脲[1]。这个问题可通过该胺NaH或NaHMDS反应，然后再与Boc2O反应来加以避免[2]。

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BrBoc2ODMAP, THFBr+O17V%OBrNH2BrHNHNHNO

1. H. J. knolker, T. Braxmeier et al., Angew. Chem., Int. Ed. Engl., 1995, 34, 2497; H. J.

knolker, T. Braxmeier et al., Synlett., 1996, 502; Kessier,A.; Coleman, C. M., et al J. Org. Chem.,

2004, 69(23), 7836-7846

2. T. A. Kelly, D. W. McNeil., Tetrahedron Lett., 1994, 35, 9003

有时在反应中有可能多加了Boc酸酐，当分子中无游离酸碱时很难出去，若一定要除去，一般在体系中加入一些N,N-二甲基乙二胺或N,N-二甲基丙二胺，而后将上了Boc的N,N-二甲基乙二胺或N,N-二甲基丙二胺用稀酸除去。

RNH2Boc2OexcessRNHBoc+Boc2O (excess)H2NNBocHNN稀酸除去

由于Boc对酸敏感，因此在合成过程中用到酸洗或酸溶解等操作时，为了保险起见，尽量不用盐酸而用10%柠檬酸（0.5M）或在低温条件进行。

2.2.1.1 氨基酸Boc保护示例

NH2COOHBoc2ONaOH, t-BuOH, H2ONHBocCOOH Oskar Keller, Walter E. Keller, Gert van Look et al., Org. Syn., 63, 160 A 4-L, four-necked, round-bottomed flask, equipped with an efficient stirrer, a dropping funnel, reflux condenser, and thermometer is charged with a solution of 44 g (1.1 mol) of sodium hydroxide in 1.1 L of water. Stirring is initiated and 165.2 g (1 mol) of L-phenylalanine is added at ambient temperature, and then diluted with 750 mL of tert-butyl alcohol. To the well-stirred, clear solution is added dropwise within 1 hr, 223 g (1 mol) of di-tert-butyl dicarbonate. A white precipitate appears during addition of the di-tert-butyl dicarbonate. After a short induction period, the temperature rises to about 30–35°C. The reaction is brought to completion by further stirring overnight at room temperature. At this 药明康德内部保密资料

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time, the clear solution will have reached a pH of 7.5–8.5. The reaction mixture is extracted two times with 250 mL of pentane, and the organic phase is extracted three times with 100 mL of saturated aqueous sodium bicarbonate solution. The combined aqueous layers are acidified to pH 1–1.5 by careful addition of a solution of 224 g (1.65 mol) of potassium hydrogen sulfate in 1.5 L of water. The acidification is accompanied by copious evolution of carbon dioxide. The turbid reaction mixture is then extracted with four 400-mL portions of ethyl ether. The combined organic layers are washed two times with 200 mL of water, dried over anhydrous sodium sulfate or magnesium sulfate, and filtered. The solvent is removed under reduced pressure using a rotary evaporator at a bath temperature not exceeding 30°C. The yellowish oil that remains is treated with 150 mL of hexane and allowed to stand overnight. Within 1 day the following portions of hexane are added with stirring to the partially crystallized product: 2 × 50 mL, 4 × 100 mL, and 1 × 200 mL. The solution is placed in a refrigerator overnight; the white precipitate is collected on a Büchner funnel and washed with cold pentane. The solid is dried under reduced pressure at ambient temperature to constant weight to give a first crop. The mother liquor is evaporated to dryness leaving a yellowish oil, which is treated in the same manner as described above, giving a second crop. The total yield of pure white N-tert-butoxycarbonyl-L-phenylalanine is 207–230 g (78–87%), mp 86–88°C, [α]D20 + 25.5° (ethanol c 1.0).

2.2.1.2 氨基酸酯Boc保护示例

NH2.HClHOCOOMeBoc2OHOEt3NNHBocCOOMe

Alessandro Dondoni, Daniela Perrone., Org. Syn., 77, 64

A 500-mL, three-necked, round-bottomed flask, is equipped with a magnetic stirring bar, thermometer, reflux condenser protected from moisture by a calcium chloride-filled drying tube, and a pressure-equalizing dropping funnel that is connected to a nitrogen flow line and is charged with a solution of 97% di-tert-butyl dicarbonate (14.3 g, 63.6 mmol) in tetrahydrofuran (100 mL), Methyl serinate hydrochloride (10.0 g, 64.3 mmol) is placed in the flask and suspended in tetrahydrofuran (200 mL) and 99% triethylamine (14.0 g, 138 mmol). The resulting white suspension is cooled with an ice-water bath and the solution of

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di-tert-butyl dicarbonate is added dropwise over a period of 1 hr. After 10 min of additional stirring, the ice-water bath is removed and the suspension is stirred overnight (14 hr) at room temperature, then warmed at 50°C for a further 3 hr. The solvent is removed under reduced pressure and the residue is partitioned between diethyl ether (200 mL) and saturated aqueous bicarbonate solution (250 mL). The aqueous phase is extracted with three 150-mL portions of diethyl ether. The combined organic phases are dried with anhydrous sodium sulfate and concentrated under reduced pressure to give 13.4-14.0 g (95-99% crude yield) of N-Boc-L-serine methyl ester as a colorless oil that is used without further purification. [α] D 23 17.0° (MeOH, c 4.41).

2.2.1.3 Boc酸酐在甲醇中与胺直接反应

COOEtBoc2ONH1MeOHNBoc2COOEt Boc2O (262 g, 1.2 mol) in MeOH (250 ml) was added to a soluton of compound 1 (157.2 g, 1.0 mol) in MeOH (350 ml) at 10°C, and the resulting mixture was stirred at room temperature for 2 h. N1, N1-dimethylethane-1,2-diamine (26 g, 0.3 mol) was added and the mixture was stirred at room temperature for 15 min. The solvent was removed in vacuo, and the residue was dissolved with ethyl acetate (750 ml). The combined organics were washed with 1 N HCl (2 x 250 ml) and brine (2 x 250 ml), dried over sodium sulfate and filtered. The solvent was removed to give compound 2 (250 g, 96%), which was used directly in the next step. 2.2.1.4 芳胺的单Boc保护示例

NH2NCOOHBoc2OEt3N, DMFNNHBocCOOH

Luo, Qun-Li; Liu, Zhi-Ying et al., J. Med. Chem., 2003, 46(13), 2631-2640

3-Aminopyridine-2-carboxylic acid (5.02 g, 36 mmol) was suspended in 60 mL of dry DMF, and Et3N (15.2 mL, 108 mmol) was added dropwise at room temperature. To the resulting brown solution was added Boc2O (11.80 g, 54 mmol). After being stirred for 10 min, the

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mixture was heated at 40-50 °C overnight. The reaction mixture was poured into water and was then extracted with EtOAc (2 X 50 mL). The aqueous phase was acidified to pH 4-5 with 2 M aqueous HCl and then extracted with CH2Cl2 (3 X 50 mL). The combined organic phases were then processed in the usual way and chromatographed (13:1 CHCl3/MeOH) to yield the desired product (4.2 g, 49%).

2.2.1.5 芳胺的双Boc保护示例

BocNH2SSBoc2ONaHMDS, THFSNBocS Macleod, Calim; Mckieman, Gordon J et al., J. Org. Chem., 2003, 68(2), 387-401 A solution of NaHMDS (22.0 mL, 22.0 mmol, 1 M in THF) was added to a solution of the amine (2.11 g, 10.0 mmol) and (Boc)2O (5.46 g, 25.0 mmol) in THF (50 mL) at 0°C under nitrogen. The reaction was allowed to warm to rt and stirred for 16 h. After this time, the reaction was poured into water, extracted into CH2Cl2 (2 X 25 mL), washed with water (2 X 25 mL), dried over Na2SO4, and concentrated to yield a white-yellow solid. Recrystalization from petroleum ether (40-60 °C) gave the imide as needles (3.21 g, 7.80 mmol, 78%). Rf (hexane/ CH2Cl2 1:9, SiO2): 0.10. Mp: 106-109 °C.

2.2.1.6 酰胺的Boc保护示例

HNBoc2OHNO1NHOBocDMAP, Et3NNO2NBocOBocN

Lars G. J. Hammarstr?m, Yanwen Fu et al., Org. Syn., 81, 213

A 2000-mL, three-necked, round-bottomed flask equipped with an argon inlet adapter, glass stopper, and an overhead mechanical stirrer is charged with a suspension of the hydantoin 1 (26.0 g, 154 mmol) in 1000 mL of 1,2-dimethoxyethane. Triethylamine (15.7 g, 154 mmol) is added in one portion, and the resulting white suspension is stirred for 30 min. Di-tert-butyl

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dicarbonate (168.0 g, 770 mmol) is then added by pipette, followed by 4-dimethylaminopyridine (DMAP) (0.2 g, 1.5 mmol). Six additional 0.2 g-portions of DMAP are added at 12 hr intervals during the course of the reaction. The reaction mixture is stirred vigorously for a total of 72 hr, and the resulting light yellow solid is then collected in a Büchner funnel using suction filtration. The filtrate is concentrated to a volume of 60 mL by rotary evaporation, and the resulting solution is cooled to 15°C. The precipitate which appears is collected using suction filtration, added to the first crop, and the combined solids are dissolved in 500 mL of chloroform. This solution is washed with three 200-mL portions of 1.0N HCl, and the combined aqueous phases are extracted with 100 mL of chloroform. The combined organic layers are washed with 100 mL of saturated aq NaHCO3 solution and 100 mL of brine, dried over anhydrous MgSO4, filtered, and concentrated by rotary evaporation. The resulting solid is dried at room temperature at 0.01 mm for 24 hr. The resulting finely ground light yellow solid is suspended in 400 mL of diethyl ether in a 1000-mL, round-bottomed flask equipped with a magnetic stirbar, stirred for 2 hr, and filtered on a Büchner funnel washing with four 50-mL portions of diethyl ether. The product is dried under vacuum (85°C; 0.5 mm) for 24 hr to give 60.0–65.3 g (83-90%) of 2 as a ivory-colored solid.

2.2.1.6 叠氮还原Boc保护示例

OHEtOOCN31COOEtH2/Pd-CBoc2O, EtOAcEtOOCOHCOOEtNHBoc2 Seiki Saito, Kanji Komada, and Toshio Moriwake., Org. Syn., 73, 184 A 500-mL, single-necked, round-bottomed flask, equipped with a Teflon-coated stirring bar, is charged with a suspension of 0.91 g of 10% palladium on carbon catalyst in 100 mL of ethyl acetate. The flask is connected to a normal pressure hydrogenation apparatus and the catalyst is saturated with hydrogen. After removal of the hydrogen, a solution of 18.2 g (0.0785 mol) of 1 and 20.6 g (0.0942 mol) di-tert-butyl dicarbonate in 80 mL of ethyl acetate is added to the suspension of catalyst, a hydrogen atmosphere reestablished, and the suspension is stirred at room temperature under a slight positive pressure of hydrogen for 4–6 hr, The suspension is filtered through a Celite pad, and the pad is rinsed with several portions of ethyl acetate. The combined ethyl acetate solutions are concentrated on a rotary evaporator and finally under high vacuum to give a pale yellow oil that is initially purified by means of a column packed

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with silica gel (100 g) using hexane-ethyl acetate (6:1) as eluent. Fractions containing the product are combined and concentrated on a rotary evaporator to give 23.3 g of crude 2 as a colorless oil. The oily crude 2 is dissolved in 70 mL of hexane-ether (3:1), and the solution is cooled to ?30°C, seeded, and kept overnight at that temperature (freezer) to allow crystallization. The mother liquor is siphoned out while the mixture is kept at ?30°C (dry ice-acetone bath). The crystals are washed with several portions of hexane-ether (3:1) at ?30°C, then dried under high vacuum to provide 12.2–12.7 g of diastereomerically and enantiomerically pure diethyl (2S,3R)-2-(N-tert-butoxycarbonyl)amino-3-hydroxysuccinate (2) as colorless prisms, mp 33–34°C;. The combined mother liquor and the hexane–ether (3:1) washings are concentrated on a rotary evaporator to give a colorless oil, which upon crystallization as above provides an additional 2.7–3.8 g of product 2. The combined yield of crystalline 2 is 15.9–16.5 g (66–73%).

2.2.1.7 吡咯Boc保护示例

Boc2ONHBrDMAPNBocBr Wha Chen, E. Kyle Stephenson et al., Org. Syn., 70, 151 The solution of 2-bromo-1H-pyrrole (9.8 g, 67.2 mmol) in 40 mL of THF is cooled to ?78°C in a dry ice-acetone bath. The flask is equipped with a magnetic stirring bar and a three-way stopcock attached to a balloon filled with nitrogen. To the stirred dark-green solution is added 2.71 g (26.9 mmol) of triethylamine followed immediately by addition of 20.4 g (93.9 mmol) of di-tert-butyl dicarbonate and a catalytic amount (ca. 0.1 g) of 4-dimethylaminopyridine. The flask is evacuated and purged with nitrogen. The mixture is stirred for 8 hr while it is allowed to warm to room temperature. The solvent is removed under reduced pressure at room temperature and 100 mL of hexane is added to the crude product, which is washed with deionized water (3 × 100 mL), dried over sodium sulfate, and concentrated under reduced pressure at room temperature. The crude product is purified by chromatography on amine-treated neutral silica (270 g) using hexane as the eluent. The fractions containing the product are identified by TLC, combined, and concentrated under reduced pressure at room temperature to yield N-tert-Butoxycarbonyl-2-bromopyrrole as a colorless oil (13.5–14.7 g, 82–89%). 药明康德内部保密资料

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2.2.1.8 吲哚Boc保护示例

Boc2OMeONHDMAP, CH3CNMeONBoc G. Tong; P. Ruiyan et al., J. Org. Chem., 1997, 26, 9298 To a solution of 6-methoxy-3-methylindole (5.0 g, 31 mmol) in distilled acetonitrile (150 mL) were added di-tertbutyl dicarbonate (7.44 g, 34.1 mmol) and DMAP (0.195 g, 1.6 mmol). The reaction mixture was stirred at rt for 12 h. The solvent was removed under reduced pressure. The residue was dissolved in CH2Cl2 (100 mL) and washed with an aqueous solution of 1 N HCl (2 x 50 mL). The aqueous layer was extracted with CH2Cl2 (3 x 30 mL). The combined organic ayers were dried (K2CO3). After removal of solvent under reduced pressure, the residue was solidified to afford the product (8.12 g, 99%) as a yellow solid: mp 45-46 °C.

2.2.2 叔丁氧羰基的脱去

Boc比Cbz对酸敏感，酸解产物为异丁烯和CO2（见下式）。在液相肽的合成中，Boc的脱除一般可用TFA或50%TFA（TFA:CH2Cl2 = 1:1,v/v）。而在固相肽合成中，由于TFA会带来一些副反应（如在得到的胺上上一个三氟乙酰基等），因此多采用1-2M HCl/有机溶剂。一般而言用HCl/二氧六环，比较多见。

用甲醇作溶剂，HCl/EtOAc的组合使TBDMS和TBDPS

酯[1]以

及叔丁酯和非酚类酯在Boc脱除时不被断裂，而S-Boc除外[2]。但当同时脱除分子中Boc和叔丁酯, 或分子中有游离羧酸基，千万记住不能用HCl/MeOH,其可将羧酸变为甲酯。同时AcCl/MeOH，则是一个在甲醇中产生无水HCl的便利方法。这些条件也可用来从羧酸制备酯以及形成胺的盐酸盐[3]。

在中性的无水条件下Me3SiI在CHCl3或CH3CN中除了能脱除Boc外，也能断裂氨基甲酸酯、酯、醚和缩酮。通过控制条件可以得到一定的选择性[4]。

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当分子中存在一些官能团其可与副产物叔丁基碳正离子在酸性下反应时，需要添加硫酚(如苯硫酚)来清除叔丁基碳正离子，如此举可防止蛋氨酸和色氨酸的脱Boc时的烷基化[5]。也可使用其它的清除剂，如苯甲醚、苯硫基甲醚、甲苯硫酚、甲苯酚及二甲硫醚[6]。在Boc脱去过程中TBDPS[7]和TBDMS[8]基对CF3COOH是稳定的（在TBS存在，用相对稀一些的10－20 ％TFA）。伯胺衍生物存在下，ZnBr2/CH2Cl2可以选择性的脱除仲胺上的Boc[9]。

1. F. Cavelier, C. Enjabal., Tetrahedron Lett., 1996, 37, 5131

2. F. S. Gibson, S. C. Bergmeier, H. Rapoport., J. Org. Chem., 1994, 59, 3216 3. A.Nudelman, Y. Bechor et al., Synth. Commun., 1998, 28, 471

4. R. S. Lott, V. S. Chauhan et al., J. Chem. Soc. Chem. Commun., 1979, 495; G. A. Olah, S. C. Narang., Tetrahedron., 1982, 38, 2225

5. R. A. T. M. van Benthem, H. Hiemstra et al., J. Org. Chem., 1992, 57, 6083

6. M. Bodanszky, A. Bodanszky., Int. J. Pept. Protein Res., 1984, 23, 565; Y. Masui, N. Chino et al., Bull. Chem. Soc. Jpn., 1980, 53, 464

7. P. A. Jacobi, S. Murphree et al., J. Org. Chem., 1996, 61, 2413 8. J. Deng, Y. Hamada et al., J. Am. Chem. Soc., 1995, 117, 7824 9. S. C. Nigam, A. Mann et al., Synth. Commun., 1989, 19, 3139

2.2.2.1.1 TMSOTf中性条件下脱Boc示例

ONBocHNOHOTBDMSOTfCH2Cl2H2NOTBDMSN

Gilbertson, Scott R; Chang, Cheng-Wei et al., J. Org. Chem., 1998, 63(23), 8424-8431 To a solution containing 2 (1.0 g, 3.9 mmol) in 30 mL of dry CH2Cl2 was slowly added TBDMSOTf (0.9 mL, 4.1 mmol). After stirring the reaction mixture for 6 h, the solvent was evaporated, and the crude product (0.8 g, 75%) was obtaineded, which was used directly in the next step.

2.2.2.1.2 TMSOTf-2,6-lutidine中性条件下脱Boc示例1

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OOOSOOOOSOBocHNTBDMSOTf2,6-LutidineCH2Cl2H2N Kemp, Scott J; Bao, Jiaming et al J. Org. Chem., 1996, 61(20), 7162-7167

12To a stirring solution of compound 1 (800 mg, 2.0 mmol) and 2,6-lutidine (0.4463 ml, 4.0 mmol) in CH2Cl2 (6 mL) was added tert-butyldimethylsilyl triflate (0.690 ml, 3.0 mmol) dropwise over 5 min. After 20 min, saturated NH4Cl (10 mL) was added. The mixture was stirred and separated, and the aqueous layer was extracted with Et2O (3 x 15 mL). The combined organic layers were washed with water (2 x 10 mL) and saturated NaCl (10 mL), dried (MgSO4), and concentrated to give the crude silyl carbamate, which was dissolved in THF (10 mL) and cooled to 0°C. A 1.0 M solution of TBAF in THF (2 mL, 2 mmol) was added over 5 min, and then the solution was stirred at 0°C for 1 h. The solution was concentrated and chromatographed (95:5 CH2Cl2-methanol) through a small plug of silica to give compound 2 (882 mg, 75%) as a clear oil.

2.2.2.1.3 TMSOTf-2,6-lutidine 条件下脱Boc示例2

OHNOO1OOTBDMSOTfO2,6-LutidineCH2Cl2OOHNH2N2OOBocHN

Sakaitani, Masahiro; Ohfune, Yasufumi; J. Org. Chem., 1990, 55(3), 870-876

To a stirred solution of compound l (500 mg, 1.52 mmol) and 2,6-lutidine (0.353 ml, 3.04 mmol) in dry CH2C12 (3.0 mL) at 0 \t-BuMe2SiOTf (0.523 ml, 2.28 mmol). The reaction mixture was stirred at 0°C for 15 min, quenched with saturated aqueous ammonium chloride solution, and extracted with ether several times. The combined organic phase was washed with H2O and then brine, dried (MgSO4), and concentrated in vacuo to give an oily residue, which upon purification by column chromatography on silica gel (elution with 50% ether in hexane) gave O-silyl ester compound 2 (652 mg, 97%): colorless needles; mp 64.0-65.0°C (hexane).

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2.2.2.2 TFA脱Boc示例

TBSONHBocCOOMeTFACH2Cl2TBSONH2COOMeCbzNCbzN

M. Alberto; A. Eduardo et al., J. Org. Chem., 2004, 21, 7004

To a solution of the β-aminoester (0.2 mmol) in CH2Cl2 (3 mL), cooled to 0°C was added TFA (1mL). After the consumption of the starting material (45 min, monitored by TLC), the mixture was evaporated and then saturated aqueous NaHCO3 was added. The aqueous layer was extracted twice with CH2Cl2 (15 mL), and the organic layer washed with brine and dried over anhydrous Na2SO4. The solvent was removered under vacuum, to afford the amine, which were employed without further purification to prepare the Mosher’s diastereoisomeric amides.

2.2.2.3 HCl-Et2O脱Boc示例

ONHBocOOHClEt2OHOOCClH.HNO

C. Mühlemann, P. Hartmann, J. P. Obrecht., Org. Syn., 71, 200

tert-Butyl [1-(tert-butoxycarbonyl)-3-oxo-4-pentenyl]carbamate, 8.73 g (0.0308 mol), is dissolved in 280 mL of an ice-cooled, saturated solution of hydrogen chloride in ether. The solution is kept without stirring at room temperature overnight. The resulting suspension is filtered and the filter cake is immediately washed with dry ether. The washing with ether is repeated four times and, after drying under reduced pressure, 5.48 g (99%) of 4-ketopipecolic acid hydrochloride is obtained as a colorless powder, mp 139–142°C dec.

2.2.2.4 HCl-THF脱Boc示例

OHNHCOOt-BuBocHNH2N HClTHFH2NOHNHCOOHH

J. Wehbe et al., Tetrahedron: Asymmetry, 2004, 15, 851

To the Boc protected amine (0.06 g, 0.17 mmol) dissolved in THF (1mL) was added 2M HCl (1mL, 2 mmol) and the mixture stirred 2 h at room temperature. After evaporation of the solvent, the product was extracted into EtOAc (3. 5mL). The organic layer was dried and

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经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

evaporated under vacuum to afford 17b in 95% yield as a white solid.

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经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

2.2.2.5.1 叔丁酯存在下的脱Boc示例1

OO1Me3SiICHCl3ONH2OOONBoc

US5610144

1.77 ml of Me3SiI are added dropwise at room temperature in the vicinity of 25°C to a soution of 3.8 g of compound 1 in 50 ml of CHCl3. Stirring is contiuned for 30 min, then 20 ml f water are addede. The aqueous phase is separated, then extracted with CHCl3(2 x 20 ml). The organic phases are combined, washed successively with a saturated aqueous Na2CO3(30 ml) and water(2 x 30 ml), then dried over MgSO4 and concentrated to dryness under reduced pressure at 40°C. The mixture of the two diastereoisomers obtained is separated by chromatography on silica (eluent: ethyl acetate/cyclohexane = 1/4). The fractions containing the expected product are combined and concentrated to dryness under reduced pressure at 40°C to give compound 2 (0.5 g), as a yellow-orange oil, used as it is in subsequent syntheses.

2.2.2.5.2 叔丁酯存在下的脱Boc示例1

ONOO1Boc1.1 M HClEtOAcNHOO2O

US2002045623

To asolution of compound 1 (6.3 g, 21.0 mmol) in ethyl acetate (50 ml) was added 1.1 M HCl in ethyl acetate (28.7 ml, 31.5 mmol). The reaction was stirred at room temperature for 1 h, then washed with water, saturated aq. NaHCO3 and brine. The organic phase was dried (MgSO4), filtered and evaporated to afford compound 2 (3.11 g, 74%) as a yellow oil which crystallized upon standing.

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经典合成反应标准操作—氨基的保护及脱保护 药明康德新药开发有限公司

2.2.2.5.3 叔丁酯存在下的脱Boc示例3

NHBocOOOOOTFACH2Cl2OOOONH2O12

WO20040106286

To a solution of compound 1 (149 mg, 0.33 mmol) in CH2Cl2 (2 ml), TFA (1 ml) as added at 0°C and the mixture was stirred for 1 h at 0°C. Saturated aqueous Na2CO3 was added and the mixture was etracted with CHCl3. The etract was purified by silica gel column chromatography to obtained compound 2 (92 mg, 79%).

2.2.2.6 吲哚环上Boc直接加热脱除示例

NHOEtOOCH(neat)MeONBoc1MeO2NHNHHOEtOOCHNHNHH

G. Tong; P. Ruiyan et al., J. Org. Chem., 1997, 26, 9298

Compound 1 (62 mg)was heated (neat) at 160-180 °C for 45 min. The residue was purified by flash chromatography (silica gel, CHCl3/MeOH 95/5) to afford Compound 2 (25 mg) as a solid in 50% yield. 11: [R]27D= - 65.9 (c = 0.97, in CHCl3).

2.3 笏甲氧羰基（Fmoc）

Fmoc保护基的一个主要的优点是它对酸极其稳定，在它的存在下，Boc和苄基可去保护。Fmoc的其他优点是它较易由简单的胺不通过水解来去保护，被保护的胺以游离碱释出[1]。一般而言Fmoc对氢化稳定，但某些情况下，它可用H2/Pd-C在AcOH和MeOH仲脱去[2]。Fmoc保护基可与酸脱去的保护基搭配而用于液相和固相的肽合成[3]。

药明康德内部保密资料

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