氨基的保护及脱保护

氨基的保护及脱保护策略

编者：彭宪

药明康德新药开发有限公司化学合成部

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目 录

1．

氨基的保护及脱保护概要……………………………………………2 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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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、吡咯、吲哚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。

经典合成反应标准操作—氨基的保护及脱保护
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几种代表性的常用的氨基保护基结构O O X
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, TosOH/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,/dio x/H2O Aloc-Cl/Py
O X O
Boc
X O O
Fmoc
X O O
Alloc
O Cl O TMS
Teoc
伯胺、仲氨、咪唑、 Teoc-Cl/碱/diox/H2O 吡咯、吲哚等
TBAF；TEAF
X O O
Me( or Et)
-
伯胺、仲氨、咪唑、 ROCOCl/NaHCO3,/dio x/H2O 吡咯、吲哚等 邻苯二甲酸酐 /CHCl3/70℃;邻苯二甲 酰亚胺-NCO2Et/aq. Na2CO3 Tos-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
O X N OO X S O
Pht
伯胺
Tos
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
O X CF3
Tfa
X
Trt
MeO OMe X
Dmb
伯胺、仲氨、咪唑、 ArCHO/NaCNBH3/Me OH 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 HCO2H/Pd-C/MeOH; H2/Pd(OH)2/EtOH; TFA; CAN/ CH3CN HCO2H/Pd-C/MeOH; H2/Pd(OH)2/EtOH; CCl3CH2OCOCl/CH3CNPage 3 of 77
OMe X
PMB
X
Bn
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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NaHCO 3 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H 2N(CH 2)nNH 2, 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来中和反应所产生的HCl 。此外，Cbz-ONB （4-O 2NC 6H 4OCOOBn ）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]。

H N R R 2N R R 2Cbz NH 2R 1R 2OOC NHCbz R 1R 2OOC

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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Cbz-Cl 很容易用苯甲醇同光气的反应来制备（见下式），在低温下可以保存半年以上而不发生显著的分解。

CH 2OH 2CH 2OCOCl +HCl

除Cbz-Leu 为油状物外，绝大多数氨基酸的苄氧羰基衍生物都可以得到结晶。有的N -苄氧羰基氨基酸能同它的钠盐按一定比例形成共晶，共晶产物的熔点较高，并难溶于有机溶剂。例如，苯丙氨酸经苄氧羰基化后再加酸析出Cbz-Phe 时往往得到共晶产物（熔点144℃），此共晶产物用乙酸乙酯和1M HCl 一道震摇时可完全转化为Cbz-Phe 而溶于乙酸乙酯中。因此。除Cbz-Gly 以外，一般都是采用酸化后用有机溶剂提取的方法来得到纯的N-苄氧羰基氨基酸。

2.1.1.1 游离氨基酸的Cbz

保护示例

Konda-Yamada, Yaeko; Okada, Chiharu et al., Tetrahedrom ; 2002, 58(39), 7851-7865

Cbz-Cl (18.5 μl, 0.155 mmol) in diethyl ether (0.2 ml) was dropped to a solution of (R)-1 (36.4 mg, 0.129 mmol) in 10% aqueous Na 2CO 3 (1.8 ml) at 0°C, and stirred for 5 h. The reaction mixture was acidified with 10% citric acid, extracted with CHCl 3 (10 mlX3). The organic layer was washed with water, dried over Na 2SO 4, evaporated to give light yellow gels, which were purified by preparative TLC (CHCl 3/MeOH=5:1) to afford (R)-6 (25.7 mg, 47.1%) as yellow amorphous solid. R f = 0.87 (n-BuOH/AcOH/H 2O=4:1:5); [a]D 23 = -27.270 (c = 0.99, CHCl 3);

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2.1.1.2 氨基酸酯的Cbz 保护示例

2.K 2CO 3

12

M. Carrasco, R. J. Jones, S. Kamel et a1., Org. Syn., 70, 29 A 3-L, three-necked, Morton flask equipped with an efficient mechanical stirrer, thermometer, and a dropping funnel is charged with L-methionine methyl ester hydrochloride 1 (117.6 g, 0.56 mol), potassium bicarbonate (282.3 g, 2.82 mol, 5 eq.), water (750 mL), and ether(750 mL), and the solution is cooled to 0°C. Benzyl chloroformate (105 g, 88.6 mL, 0.62 mol, 1.1 eq.) is added dropwise over 1 hr, the cooling bath is removed, and the solution is stirred for 5 hr. Glycine (8.5 g, 0.11 mol, 0.2 eq.) is added (to scavenge excess chloroformate) and the solution is stirred for an additional 18 hr. The organic layer is separated, and the aqueous layer is extracted with ether (2 × 200 mL). The combined organic layers are washed with 0.01 M hydrochloric acid (2 × 500 mL), water (2 × 500 mL), and saturated brine (500 mL), and then dried (Na 2SO 4), filtered, and

evaporated on a rotary evaporator. The resulting oil is further dried in a Kugelrohr oven (50°C, 0.1 mm, 12 hr) to

leave product 2 as a clear oil that solidifies upon cooling: 165–166 g (98–99%), mp 42–43°C.

2.1.1.3 氨基醇的Cbz 保护示例(1)

Cbz-Cl

23THF, H 2O

Clariana, Jaume; Santiago, G. G. et al Tetrahedron: Asymmetry , 2000, 11(22), 4549-4558

Benzyl chloroformate (0.95 ml, 6.7 mmol) was added via syringe into a stirred mixture of aminoalcohol 7 (0.989 g, 5.1 mmol) and sodium carbonate (0.683 g, 6.4 mmol) in the solvent system water (10 ml)–THF (3 ml) maintained at 0°C. The mixture was stirred at room temperature for 18 h (TLC monitoring) and then partitioned between dichloromethane and

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water. The organic phase was dried and evaporated to afford a white solid which was passed through a column of silica gel with hexanes –ethyl acetate (v:v 2:1) to afford the desired product (1.198 g, 72%), mp 125–127°C.

2.1.1.4氨基醇的Cbz 保护示例(2)

Cbz-Cl

23Tol, H 2O H 2O O

Inaba, Takashi; Yamada, Yasuki et al J. Org. Chem ., 2000, 65(6), 1623-1628

To a mixture of toluene (3.85 L), water (3.85 L), and K 2CO 3 (470 g, 3.40 mol) were successively added 1a (770 g, 2.72 mol) and CbzCl (488 g, 2.72 mol) with vigorous stirring at a temperature below 25 °C. After stirring at room temperature for 3 h, triethylamine (27.5 g, 270 mmol) and NaCl (578 g) were successively added, and the mixture was stirred for a further 30 min. The organic layer was separated and concentrated to give the desired product as oil, which was used for the next reaction without purification. The analytical sample was prepared by column chromatography;

2.1.2苄氧羰基的脱去

苄氧羰基的脱除主要有以下几种方法：1). 催化氢解；2). 酸解裂解；3). Na/NH 3（液）还原。 一般而言目前实验室常用简洁的方法就是催化氢解， 但当分子中存在对催化氢解敏感或钝化的基团时，我们就必须采用化学方法如酸解裂解或Na/NH 3（液）还原等。

催化氢解如下式所示。催化氢解的供氢体可以是H 2、环己二烯[1, 2]、1，4-环己二烯

[2]、甲酸铵[3]和甲酸[4-6]等，以后四个为供氢体的反应又叫催化转氢反应，通常这比催化氢化反应更迅速。

N R R 2

CH 3+CO 2+H N R R 2H CH 3+N R COOH R 2

催化剂主要用5-10%的钯-碳、10-20%的氢氧化钯-碳或钯-聚乙烯亚胺，钯-聚乙烯亚

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胺/甲酸对于除去Cbz 要比前两者要好[7]。当HBr/HOAc 脱去Cbz 保护基时，产物往往带又一点颜色，而且分解产生的溴化苄会产生一些副反应并难以除尽，而催化氢解多数能得到无色得产物。由于硫能使催化剂中毒，因此，含有胱氨酸、半胱氨酸等含硫的肽等N-苄氧羰基氨基衍生物一般不用催化氢解法脱除。一般溶剂可以用甲醇，乙醇，乙酸乙酯， 四氢呋喃等，在醇类质子溶剂中反应速度要快的多。

1. G . Briefer, T. T. Nesftrick., Chem. Rew., 1974, 74, 567

2. A. E. Jackson, R. A. Johnstone., Synthesis., 1976, 685; G . M. Anantharamaiah, K. M.

Sivanandaiah., J. Chem. Soc., Perkin Trans. 1, 1977, 490

3. M. Makowski, B. Rzeszotarska, L. Smelka et al., Liebigs Ann. Chem., 1985, 1457

4. D. R. Coleman, G . P. Royer., J. Org. Chem., 1980, 45, 2268

5. B. Eiamin, G . M. Anantharamaiah, G . P. Royer et al., J. Org. Chem., 1979, 44, 3442

6. M, J. O. Anteunis, C. Becu, F. Becu et al., Bull. Soc. Chim. Belg., 1987, 96, 775

7. D. R. Coleman, G . P. Royer., J. Org. Chem., 1980, 45, 2268 D. R. Coleman, G . P. Royer., J.

Org. Chem., 1980, 45, 2268

如果在Boc 2O 存在下用Pd/C 进行氢化，则释放出的胺直接转变成Boc 衍生物[1]。而且这类反应往往要比不加Boc 2O 来的快，其主要由于氢解出来的胺往往会与贵金属有一定的络合，使催化剂的活性降低，和Boc 2O 反应为酰胺后则去除了这一效果。另外有时在氢解时加入适当的酸促进反应也是一样的道理，避免了生成的胺降低反应的活性。

1. M. Sakaitani, K. Hori, Y . Ohfune., Tetrahedron Lett., 1988, 29, 2983

另外当分子中有卤原子(Cl, Br, I)存在时，一般直接用Pd/C 会造成脱卤的发生，一般这种情况下，使用PdCl 2为催化剂，以乙酸乙酯或二氯甲烷为溶剂可较好的避免脱卤的发生。

用MeOH/DMF 为溶剂时，在Cbz-赖氨酸衍生物氢化的过程中会生成N-甲基化的赖氨酸[1]。使用氨为溶剂时，H 2/Pd-C 在-33℃下氢化，肽中的半胱氨酸或蛋氨酸单元不使催化剂毒化，此外，氨还会阻止BnO 醚的还原，所以对Cbz 可得到一些选择性[2-3]。

1. D. R. Coleman, G . P. Royer., J. Org. Chem., 1980, 45, 2268

2. J. P. Mazaleyrat, J. Xie, M. Wakselman., Tetrahedron Lett., 1992, 33, 4301

3. N. L. Benoiton., Int. J. Pept. Petein Res., 1993, 41, 611

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2.1.2.1 5-10%的钯-碳催化氢解示例

H MeOH

C. Jaume; G . G . Santiago et al., Tetrahedron: Asymmetry, 2000, 11(22), 4549-4458 A solution of (R )-8 (0.170 g, 0.52 mmol) in absolute methanol (3 ml) was hydrogenated in the

presence of 15% Pd/C (0.026 g) at room temperature for 12 h. The mixture was filtered (Celite) and

washed with methanol. Then, perchloric acid (0.050 ml, 0.83 mmol) was added and the mixture was stirred for 5 min. The solvent was evaporated to afford (R )-7·HClO 4, mp 233–235°C; [a]D 23=?15.6 (c =0.68, methanol).

2.1.2.2 5-10%的钯-碳催化氢解示例

H 2MeOH

F

NHCbz

OH

H

F

NH 2

OH H

B. Pierfrancesco;

C. silvia et al., Tetrahedron, 1999, 55(10), 3025

A solution of N-Cbz arylglycinol (17) (1.02 mmol) in MeOH (10 mL) was stirred for 15 min in the presence of an excess of Pd(OH)2/C under a dihydrogen atmosphere. The solution was then filtered on a Celite pad and the solvent removed in vaccuo. Purification of the crude afforded the desired free 2-arylglycinols (S)-21 in 87% yield, white solid; [a]D 20=+47.0 (c =0.78, CHCl 3); mp 94-96°C (AcOEt)。

2.1.2.3 Pd/C-甲酸铵催化氢解示例

O

4

10%Pd-C

O

12

Alargov, D. K; Naydenova, Z; Monatsh. Chem., 1997, 128(6-7), 725-732

576.6 mg of compound 1 (1 mmol) was dissolved in 20 ml of methanol. Then 150 mg of ammonium formate (3 mmol) and 75 mg of 10% Pd-C was added and the reaction mixture was stirred at room temperature 10 min and then heated to reflux for 45 min. The mixture was

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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

O O N N H O

O O O 10%Pd-C HCOOH

O

O NH 2

NH 2O O 1

2

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

O

BocHN

N H H /10%Pd-C 2O O O H

N N H

1

2

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 reation 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 PdCl 2催化氢解脱除带卤原子分子上的Cbz 示例

Cl

N

N H N

O

NH

Cl

PdCl 2

Et SiH

Cl

N

N

H N O

N

O O

Cl

1

2

US20030144297

To a solution o compound 1 (900 mg) in methylene chloride (16.5 ml) was addede PdCl 2 (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/CH 2Cl 2 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

2

Pd

4

3

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

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 药明康德内部保密资料 Page 12 of 77

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。

N R Cbz R 2

+H +N R Cbz R 2H +-CH 2Br +CO 2+H.HBr N R 1R 2

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

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 药明康德内部保密资料 Page 13 of 77

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（CH 3SC 2H 5）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]。FSO 3H [2]、CH 3SO 3H [2, 3]、CF 3SO 3H [3, 4]和C 6H 5SCH 3-TFA [5]也是较好的试剂。Me 3SiI 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BBr 3/CH 2Cl 2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CH 2Cl 2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/Me 2S/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.

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

药明康德内部保密资料 Page 14 of 77

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/CHCl 3[1]、HCl/HOAc [2]、HBr/SO 2[3]、液体HBr [4]、TosOH [5]、HI/HOAc [6]、碘化磷[7]、Et 3SiH [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

S N

EtOOC N H O

N 33% HBr AcOH, 91%S N

EtOOC N H O N 2.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

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

药明康德内部保密资料 Page 15 of 77

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

O

H

Br

NH 2

O O

O

Me 3SiI 33O H

Br

NHCbz

O

O O

1

2

US20040204397

Me 3SiI (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. Et 3N (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.

N H N N

N N HN

Cl

2N H N N

N N HN

Cl

Me 3SiI Et 3N, CH 3CN

1

2

US20050203078

2.1 g (4.45 mmol) of compound 1 in 30 ml of CH 2Cl 2 were combined with 1.9 ml (1

3.4 mmol) Me 3SiI 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: CH 2Cl 2/(MeOH/conc. Ammonia 95:5) = 70/30 – 60/40) to yield compound 2 (690 mg, 56%).

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

药明康德内部保密资料 Page 16 of 77

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NaHCO 3 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Boc 2O 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Boc 2O/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O 17标记的氨基酸而不会由于与水交换使O 17丢失[2]。有空间位阻的氨基酸而言，用Boc 2O/Me 4NOH.5H 2O/CH 3CN 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.

Ar NH 2Ar NHBoc

Ar N Boc

Boc O, DMAP 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Boc 2O 生成脲[1]。这个问题可通过该胺NaH 或NaHMDS 反应，然后再与Boc 2O 反应来加以避免[2]。

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

药明康德内部保密资料

Page 17 of 77

Br

H N

Boc 2O DMAP, THF

NH 2

Br

O

O

Br

H N H N Br

+

17%

56%

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。

R NH 2

R NHBoc

+Boc 2O (excess)

BocHN

N

稀酸除去

Boc O

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

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 药明康德内部保密资料 Page 18 of 77

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]D 20 + 25.5° (ethanol c 1.0).

2.2.1.2 氨基酸酯Boc 保护示例

HO COOMe NH 2.HCl

Boc 2O Et 3N HO

NHBoc

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

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 药明康德内部保密资料 Page 19 of 77

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

2.2.1.4 芳胺的单Boc 保护示例

N NH 2

COOH Boc 2O Et 3N, DMF N NHBoc COOH

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 Et 3N (15.2 mL, 108 mmol) was added dropwise at room temperature. To the resulting brown solution was added Boc 2O (11.80 g, 54 mmol). After being stirred for 10 min, the

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

药明康德内部保密资料 Page 20 of 77

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 CH 2Cl 2 (3 X 50 mL). The combined organic phases were then processed in the usual way and chromatographed (13:1 CHCl 3/MeOH) to yield the desired product (4.2 g, 49%).

2.2.1.5 芳胺的双Boc 保护示例

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 CH 2Cl 2 (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/ CH 2Cl 2 1:9, SiO 2): 0.10. Mp: 106-109 °C.

2.2.1.6 酰胺的Boc 保护示例

H

N

HN NH

O

O

DMAP, Et 3N

N

N N O O

Boc Boc

12

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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