烷氧羰基類保護基可用于氨基酸，以在肽合成中減少外消旋化的程度。外消旋化發(fā) 生在堿催化的 N-保護的羧基活化的氨基酸的偶聯(lián)反應(yīng)中，也發(fā)生在易由 N-?；Ｗo的 氨基酸形成的中間體惡唑酮中。
要使外消旋化程度減到小，需使用非極性溶劑、弱的堿、低的反應(yīng)溫度，并使 用烷氧羰基類保護的氨基酸是有效的。其中常用的有易通過酸性水解去保護的 Boc 基、 由催化氫解去保護的 Cbz 基、用堿經(jīng)β-消除去保護的 Fmoc 基和易由鈀催化異構(gòu)化去保 護的 Alloc 基。
1、芐氧羰基（Cbz）
芐氧羰基（Cbz）是 1932 年 Bergmann 發(fā)現(xiàn)的一個很老的氨基保護基，但一直到今 天還在應(yīng)用。其優(yōu)點在于：試劑的制備和保護基的導(dǎo)入都比較容易；N-芐氧羰基氨基酸和肽易于結(jié)晶而且比較穩(wěn)定；芐氧羰基氨基酸在活化時不易消旋；能用多種溫和的方法 選擇性地脫去。
1.1 芐氧羰基的導(dǎo)入
芐氧羰基的導(dǎo)入，一般都是用 Cbz-Cl。游離氨基在用 NaOH 或 NaHCO3 控制的堿 性條件下可以很容易同 Cbz-Cl 反應(yīng)得到 N-芐氧羰基氨基化合物。α,β-二胺可用該試劑 在 pH= 3.5-4.5 稍有選擇性地被保護，其選擇性隨碳鏈地增長而減弱，如 H2N(CH2)nNH2, n=2 時 71%被單保護; n=7 時 29%被單保護[1]。氨基酸酯同 Cbz-Cl 的反應(yīng)則是在有機溶 劑中進行，并用碳酸氫鹽或三乙胺來中和反應(yīng)所產(chǎn)生的 HCl。此外，Cbz-ONB （4-O2NC6H4OCOOBn）等芐氧羰基活化酯也可用來作為芐氧羰基的導(dǎo)入試劑，該試劑 使伯胺比仲胺易被保護，但苯胺由于親核性不足，與該試劑不反應(yīng).
Cbz-Cl 很容易用苯甲醇同光氣的反應(yīng)來制備（見下式），在低溫下可以保存半年以 上而不發(fā)生顯著的分解。
除 Cbz-Leu 為油狀物外，絕大多數(shù)氨基酸的芐氧羰基衍生物都可以得到結(jié)晶。有的 N-芐氧羰基氨基酸能同它的鈉鹽按一定比例形成共晶，共晶產(chǎn)物的熔點較高，并難溶于 有機溶劑。例如，苯丙氨酸經(jīng)芐氧羰基化后再加酸析出 Cbz-Phe 時往往得到共晶產(chǎn)物（熔 點 144℃），此共晶產(chǎn)物用乙酸乙酯和 1M HCl 一道震搖時可完全轉(zhuǎn)化為 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機溶劑提取的方法來得 到純的 N-芐氧羰基氨基酸。
游離氨基酸的 Cbz 保護示例
氨基酸酯的 Cbz 保護示例
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 (Na2SO4), 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.
氨基醇的 Cbz 保護示例(1)
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 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.
氨基醇的 Cbz 保護示例(2)
To a mixture of toluene (3.85 L), water (3.85 L), and K2CO3 (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;
1.2、芐氧羰基的脫去
芐氧羰基的脫除主要有以下幾種方法：1). 催化氫解；2). 酸解裂解；3). Na/NH3（液） 還原。一般而言目前實驗室常用簡潔的方法就是催化氫解， 但當(dāng)分子中存在對催化氫 解敏感或鈍化的基團時，我們就必須采用化學(xué)方法如酸解裂解或 Na/NH3（液）還原等。
催化氫解如下式所示。催化氫解的供氫體可以是 H2、環(huán)己二烯、1，4-環(huán)己二烯 [2]、甲酸銨和甲酸等，以后四個為供氫體的反應(yīng)又叫催化轉(zhuǎn)氫反應(yīng)，通常這比催化 氫化反應(yīng)更迅速。
催化劑主要用 5-10%的鈀-碳、10-20%的氫氧化鈀-碳或鈀-聚乙烯亞胺，鈀-聚乙烯亞胺/甲酸對于除去 Cbz 要比前兩者要好。當(dāng) HBr/HOAc 脫去 Cbz 保護基時，產(chǎn)物往往帶又一點顏色，而且分解產(chǎn)生的溴化芐會產(chǎn)生一些副反應(yīng)并難以除盡，而催化氫解多數(shù)能得到無色得產(chǎn)物。由于硫能使催化劑中毒，因此，含有胱氨酸、半胱氨酸等含硫的肽 等 N-芐氧羰基氨基衍生物一般不用催化氫解法脫除。一般溶劑可以用甲醇，乙醇，乙 酸乙酯， 四氫呋喃等，在醇類質(zhì)子溶劑中反應(yīng)速度要快的多。
如果在 Boc2O 存在下用 Pd/C 進行氫化，則釋放出的胺直接轉(zhuǎn)變成 Boc 衍生物[1]。而且這類反應(yīng)往往要比不加 Boc2O 來的快，其主要由于氫解出來的胺往往會與貴金屬有 一定的絡(luò)合，使催化劑的活性降低，和 Boc2O 反應(yīng)為酰胺后則去除了這一效果。另外有時在氫解時加入適當(dāng)?shù)乃岽龠M反應(yīng)也是一樣的道理，避免了生成的胺降低反應(yīng)的活性。
另外當(dāng)分子中有鹵原子(Cl, Br, I)存在時，一般直接用 Pd/C 會造成脫鹵的發(fā)生，一般 這種情況下，使用 PdCl2為催化劑，以乙酸乙酯或二氯甲烷為溶劑可較好的避免脫鹵的 發(fā)生。
用 MeOH/DMF 為溶劑時，在 Cbz-賴氨酸衍生物氫化的過程中會生成 N-甲基化的賴 氨酸。使用氨為溶劑時，H2/Pd-C 在-33℃下氫化，肽中的半胱氨酸或蛋氨酸單元不使 催化劑毒化，此外，氨還會阻止 BnO 醚的還原，所以對 Cbz 可得到一些選擇性。
5-10%的鈀-碳催化氫解示例
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·HClO4, mp 233–235°C; [a]D23=?15.6 (c=0.68, methanol).
5-10%的鈀-碳催化氫解示例
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]D20=+47.0 (c=0.78, CHCl3); mp 94-96°C (AcOEt)。
Pd/C-甲酸銨催化氫解示例
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 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.
Pd/C-甲酸催化氫解示例
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.
Pd/C 催化氫解脫 Cbz 上 Boc 示例
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).
酸解脫除
氨基甲酸芐酯在強酸性條件下容易去保護。HBr/HOAc 是酸解脫除芐氧 羰基的常用的試劑[1]。脫除反應(yīng)主要按下式進行[2]。反應(yīng)需要消耗 2 分子的 HBr，Cbz 的脫除速度隨 HBr 濃度的增大而增大，因此實際上都是采用高濃度的過量 HBr/HOAc 溶液（1.2M-3.3M）以保證反應(yīng)的完全。
含有絲氨酸和蘇氨酸的肽或其它含羥基的氨基衍生物用 HBr/HOAc 脫除 Cbz 時 會發(fā)生羥基的 O-乙酰化反應(yīng)。雖然 O-乙酰基能用堿皂化或氨解脫去，但為了避免這個副反應(yīng)，可以改用 HBr/二氧六環(huán)或 HBr/三氟乙酸來代替 HBr/HOAc。由于 HBr 在三 氟乙酸中的溶解度較小，因此不能預(yù)先制成 HBr/三氟乙酸溶液，而只能將保護的肽或氨 基衍生物溶于無水三氟乙酸中，先于 0℃下通入干燥的 HBr，待 Cbz 大部分脫除后，再 室溫通短時間以求完全脫除變化基。Cbz 被 HBr 分解產(chǎn)生的溴化芐能同肽中的某種氨基 酸反應(yīng)，也是需要加以注意的。如，甲硫氨酸的硫原子能同溴化芐反應(yīng)生成 S-芐基甲硫 氨酸，防止的辦法是加入硫醚（CH3SC2H5）為捕捉劑[5。色氨酸被 HBr/HOAc 分解產(chǎn)生有色物質(zhì)，防止的辦法是加入亞磷酸二乙酯。硝基精氨酸會發(fā)生硝基的部分脫落，改 用液體 HBr 于-67℃處理可以避免。
用液體 HF 在 0℃處理 10-30 分鐘即可將 Cbz 完全脫去。FSO3H、CH3SO3H、 CF3SO3H和 C6H5SCH3-TFA也是較好的試劑。Me3SiI 在氯仿、乙腈中能于幾分鐘內(nèi) 選擇性脫去 Cbz 和 Boc 保護基。對于 BBr3/CH2Cl2而言，較大分子的肽的 Cbz 衍生物 可在 TFA 中去除，因為肽在酸中的溶解度比在 CH2Cl2 中大。從肽中脫去 Cbz，可在 TFA 中添加 0.5 M 4-（甲硫基）苯酚或使用 HF/Me2S/對甲苯酚（25:65:10,v/v）來抑 制 Bn+對芳香氨基酸的加成。
此外，已經(jīng)報道過的還有以下的一些不常用的方法。如 HCl/CHCl3、HCl/HOAc、 HBr/SO2、液體 HBr、TosOH、HI/HOAc、碘化磷、Et3SiH、沸騰的 TFA、 8M HCl 的乙醇液或 6 M HCl 回流 1 小時或濃鹽酸于 25-75℃加熱處理 1-1.5 小時 等。
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