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Peptide Synthesis
多肽的合成   

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Abstract

This protocol describes the synthesis of peptides for affinity testing and bioconjugate with solid phase peptide synthesizer at a small scale.

Keywords: Solid phase(固相), Peptide(肽), Fmoc(Fmoc), Scaffold protein(支架蛋白), Microcleavage(microcleavage)

Materials and Reagents

  1. Dichloromethane (DCM)
  2. Dimetylformamide (DMF) (Fisher Science, catalog number: D119-4 )
  3. Trifluoroacetic acid (TFA) (ACRO, catalog number: 13972 )
  4. Thioanisole (TIS) (Sigma-Aldrich,  233781 )
  5. Ethanedithiol (EDT) (TCI, catalog number: E0032 )
  6. 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tertramethyluronumhexafluorophosphate (HBTU) (Anaspec, catalog number: AS-21001 )
  7. N,N-diisopropylethylamine (DIEA) (Santa Cruz Biotech, catalog number: sc-215490 )
  8. Piperidine (biotech. grade, ≥99.5%) (Sigma-Aldrich, catalog number:  571261 )
  9. Dithiothriotol (DTT)
  10. Pyridine
  11. Potassium cyanide
  12. Ninhydrin
  13. Fmoc protected amino acids:
    Fmoc-Ile-OH, Fmoc-Glu (OtBu)-OH, Fmoc-Met-OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)-OH, Fmoc-Arg (Pbf)-OH, Fmoc-Asn (Trt)-OH, Fmoc-Ser (tBu)-OH, Fmoc-Ala-OH, Fmoc-His (Trt)-OH, Fmoc-Gln (Trt)-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH, Fmoc-Pro-OH and Fmoc-Asp (OtBu)-OH, Fmoc-Gly-OH, Fmoc-Cys(Trt)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Trp(Boc)-OH.
  14. Rink Amide Resin (0.66 mmol/g) (AAPPTec, catalog number: RRZ001 or equivalent)
  15. C-18 column (Vydac, catalog number: 218TP510 or equivalent)
    All reagents are ACS grade or synthesis grade
  16. Anhydrous diethyl ether

Equipment

  1. Peptide synthesizer CS-Bio 336x or equivalent
  2. Analytical balance
  3. Swing bucket eppendorf centrifuge or equivalent
  4. High performance liquid chromatograph (HPLC)
  5. Reaction vessel (SKU: PS-G002 )
  6. Liquid chromatograph-mass spectrometer

Procedure

  1. Resin preparation
    1. Weigh 303 mg Rink-Amide resin (0.20 mmol equivalent) into a reaction vessel with filters and connect the up and down outlets to the solvent line and the waste line respectively.
    2. Add 8.0 ml DMF to the resin (washing step), stir gently for 5 min and then remove the solvent via nitrogen pressure (4- 8 psi). Repeat 3 times.

  2. Deprotection/coupling cycle
    1. Add 8.0 ml of 20% piperidine/DMF (vol/vol), stir gently for 1 min (first Fmoc removal step), and then remove the solvent by nitrogen pressure.
    2. Add 8.0 ml of 20% piperidine/DMF (vol/vol), stir gently for 10 min (second Fmoc removal step), and then remove the solvent by nitrogen pressure.
    3. Add 8.0 ml DMF (washing step 1), shake for 30 sec, and then remove the solvent by nitrogen pressure.
    4. Add 8.0 ml DMF (washing step 2), shake for 5 min, and remove the solvent by nitrogen pressure.
    5. Repeat Step 4 four times.
    6. Dissolve 1.0 mmol Fmoc protected amino acid and HBTU (341.37 mg, 0.9 mmol) in 8.0 ml DMF. Mix well by bubbling nitrogen for 30 sec.
    7. Add 2.0 mmol DIEA (1 M, 2 ml) and bubbling nitrogen for 30 sec and wait for 5 min. Add the mixture to resin. Shake for 1 h. and then remove the solvent by nitrogen pressure filtration.
    8. (CRITICAL STEP: Intermittent stirring is preferred over continuous stirring when doing manual synthesis because of the fragility of some resin, in order to minimize the fragmentation of the resin in small-sized particles which can lead to difficulties during filtration. As an alternative, continuous gentle shaking or nitrogen bubbling through the reaction vessel can be applied).
    9. Repeat steps 6 and 7 for double coupling (optional).
    10. Add 8.0 ml DMF (washing step), stir for 30 sec and remove the solvent by nitrogen pressure filtration.
    11. Repeat Step 10 three times.
    12. Repeat the cycle starting from step 3 going to step 12 for each of the subsequent amino acids according to the peptide sequence using the above Fmoc-amino acid derivatives.
      CRITICAL STEP: The progress of the synthesis can be followed at every cycle by using microcleavage test (see below). To speed up the assembly, single couplings can be performed instead of double couplings).
      The requirement for the repetition of the coupling step can be derived from the result of the Kaiser test (see below).
      PAUSE POINT: the synthesis can, in principle, be interrupted at the end of every coupling cycle, when the N-terminal amino group is protected. To avoid undesired removal of the Fmoc group during storage in DMF, wash the peptide-resin five times with DCM and let it dry at room temperature (18-22 °C). Close the syringe with its plunger and cap, and store at 4 °C. Before resuming the synthesis, let the sample reach room temperature, and swell the dry resin as described in step 2.

  3. Removal of the N-terminal Fmoc-group and drying of the peptide resin
    1. Add 8.0 ml of 20% piperidine/DMF (vol/vol), stir gently for 1 min (first Fmoc removal step), and then remove the solvent by nitrogen pressure filtration.
    2. Add 8.0 ml of 20% piperidine/DMF (vol/vol), stir gently for 10 min (second Fmoc removal step), and then remove the solvent by nitrogen pressure filtration.
    3. Add 8.0 ml DMF (washing Step 1), stir for 30 sec, remove the solvent by vacuum filtration.
    4. Add 8.0 ml DMF (washing step 2), stir for 5 min, remove the solvent by nitrogen pressure filtration.
    5. Repeat step 5 four times with DMF.
    6. Wash the resin four times with DCM in a glass filter.
    7. Air-dry the peptide resin.

  4. Peptide cleavage and purification (manually)
    1. Peptide cleavage and deprotection were carried out by a 3 h incubation in a mixture of TFA/TIS/EDT/H2O (94:2:2:2).
    2. The mixture was filtered, and the peptide in solution was precipitated with ice cold anhydrous diethyl ether and centrifuged in eppendorf swing bucket at speed of 1,000 rcf. The resulting peptide was washed four times with ice-cold anhydrous diethyl ether, dried, and dissolved in suitable buffer or solvent (add 1 mM DTT if there is a cysteine).
    3. The peptide was purified by RP-HPLC on a C-18 column. Fractions were collected and lyophilized. The target product was characterized by ESI-MS and was ready for use in the next step in the reaction.

Representative data


Figure 1. Representative HPLC chromatography of a peptide synthesized with this protocol.
Peak at retention time of 11.68 min is the product peak.

Supplement

  1. Kaiser test
    The Kaiser test is a qualitative test for the presence or absence of free primary amino groups, and it can be a useful indication about the completeness of a coupling step. The test is based on the reaction of ninhydrin with primary amines, which gives a characteristic dark blue color. The test requires minimal amounts of analyte and is completed within a few minutes.
    1. Reagents
      1. 0.5 g ninhydrin in 10 ml ethanol (EtOH)
      2. 0.4 ml of 0.001 M potassium cyanide (KCN ) in 20 ml pyridine
    2. Procedure
      1. Transfer a few resin beads to a small glass tube or an Eppendorf tube and wash several times with ethanol.
      2. Add 100 ml of each of the solutions mentioned above (see reagents in this box).
      3. Mix well and place the tube in a preheated oven (115 °C) for 5 min. Light green or no color is negative. Dark blue or purple is positive.
        Critical step to reduce the incidence of false negative results, it is recommended to carry out a parallel positive control. The test is not applicable to N-terminal Pro residues (secondary amine) and N-alkyl amino acids. The test may give false negative results when applied to aggregate sequences.

  2. Microcleavage
    When Fmoc removal data show anomalies, before performing a step that requires the use of particularly expensive materials, or after a critical step, and in general when assembling longer sequences, it may be useful to cleave and analyze a small amount of intermediate product.
    1. Procedure
      1. Transfer a sample containing B1-2 mg dry peptide-resin to a small syringe (2 ml).
      2. Add 300 ml of the cleavage cocktail (Trifluoroacetic acid/H2O/phenol/triisopropylsilane 8.5/0.5/0.5/0.5) to the dried peptide resin, stir gently for 30 s and wait for 3 h (stir gently in-between).
      3. Collect the solution in a small HPLC vial, dilute with 400 ml acetonitrile/water 1/1 and mix.
      4. At this point, the solution can be analyzed in an analytical HPLC system (inject 20 ml) and/or further diluted (1/10) to be injected (2 ml) in liquid chromatograph-mass spectrometer.

Recipes

  1. Amino acid reagent vial
    1.0 mmol Fmoc amino acid per 0.2 mmol equivalent resin
    0.9 mmol HBTU per 0.2 mmol equivalent resin
  2. Peptide cleavage cocktail (5 ml for 200 mg ~300 mg dry peptide-resin)
    4.7 ml TFA
    0.1 ml EDT
    0.1 ml TIS
    0.1 ml water

Acknowledgments

This protocol was adapted or modified from previous work (Coin et al. (2007); Miao et al. (2011); Ren et al. (2010)). This work was supported, in part, by the California Breast Cancer Research Program 14IB-0091 and an SNM Pilot Research Grant (ZC).

References

  1. Coin, I., Beyermann, M. and Bienert, M. (2007). Solid-phase peptide synthesis: from standard procedures to the synthesis of difficult sequences. Nat Protoc 2(12): 3247-3256.
  2. Miao, Z., Ren, G., Jiang, L., Liu, H., Webster, J. M., Zhang, R., Namavari, M., Gambhir, S. S., Syud, F. and Cheng, Z. (2011). A novel 18F-labeled two-helix scaffold protein for PET imaging of HER2-positive tumor. Eur J Nucl Med Mol Imaging 38(11): 1977-1984.
  3. Ren, G., Liu, S., Liu, H., Miao, Z. and Cheng, Z. (2010). Radiofluorinated rhenium cyclized alpha-MSH analogues for PET imaging of melanocortin receptor 1. Bioconjug Chem 21(12): 2355-2360.

简介

该方案描述了用于小规模的用于亲和测试和与固相肽合成仪的生物共轭的肽的合成。

关键字:固相, 肽, Fmoc, 支架蛋白, microcleavage

材料和试剂

  1. 二氯甲烷(DCM)
  2. 二甲基甲酰胺(DMF)(Fisher Science,目录号:D119-4)
  3. 三氟乙酸(TFA)(ACRO,目录号:13972)
  4. 噻苯甲醚(TIS)(Sigma-Aldrich,  233781)
  5. 乙二硫醇(EDT)(TCI,目录号:E0032)
  6. 2-(1H-苯并三唑-1-基)-1,1,3,3-四氢呋喃六氟磷酸盐(HBTU)(Anaspec,目录号:AS-21001)
  7. N,N - 二异丙基乙胺(DIEA)(Santa Cruz Biotech,目录号:sc-215490)
  8. 哌啶(生物技术级,≥99.5%)(Sigma-Aldrich,目录号:571261)
  9. 二硫苏糖醇(DTT)
  10. 吡啶
  11. 氰化钾
  12. 茚三酮
  13. Fmoc保护的氨基酸:
    Fmoc-Ile-OH,Fmoc-Glu(OtBu)-OH,Fmoc-Met-OH,Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH,Fmoc-Asn )-OH,Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH,Fmoc-His(Trt)-OH,Fmoc-Gln(Trt)-OH,Fmoc-Val-OH,Fmoc-Phe-OH,Fmoc -Thr(tBu)-OH,Fmoc-Pro-OH和Fmoc-Asp(OtBu)-OH,Fmoc-Gly-OH,Fmoc-Cys(Trt)-OH,Fmoc-Tyr(tBu)-OH,Fmoc-Trp (Boc)-OH
  14. Rink酰胺树脂(0.66mmol/g)(AAPPTec,目录号:RRZ001或等同物)
  15. C-18柱(Vydac,目录号:218TP510或等价物) 所有试剂均为ACS级或合成级
  16. 无水乙醚

设备

  1. 肽合成仪CS-Bio 336x或等同物
  2. 分析天平
  3. 旋转桶eppendorf离心机或等同物
  4. 高效液相色谱(HPLC)
  5. 反应容器(SKU:PS-G002)
  6. 液相色谱 - 质谱仪

程序

  1. 树脂制备
    1. 将303mg Rink-Amide树脂(0.20mmol当量)与过滤器一起放入反应容器中,并将上下出口分别连接到溶剂管线和废液管。
    2. 向树脂中加入8.0ml DMF(洗涤步骤),轻轻搅拌5分钟,然后通过氮气压力(4-8psi)除去溶剂。 重复3次。

  2. 脱保护/偶联循环
    1. 加入8.0ml 20%哌啶/DMF(体积/体积),轻轻搅拌1分钟(第一次Fmoc除去步骤),然后通过氮气压力除去溶剂。
    2. 加入8.0ml 20%哌啶/DMF(体积/体积),轻轻搅拌10分钟(第二次Fmoc去除步骤),然后通过氮气压力除去溶剂。
    3. 加入8.0ml DMF(洗涤步骤1),摇动30秒,然后通过氮气压力除去溶剂
    4. 加入8.0ml DMF(洗涤步骤2),摇动5分钟,并通过氮气压力除去溶剂
    5. 重复步骤4四次。
    6. 将1.0mmol Fmoc保护的氨基酸和HBTU(341.37mg,0.9mmol)溶于8.0ml DMF中。用氮气鼓泡混合30秒
    7. 加入2.0mmol DIEA(1M,2ml),通入氮气30秒,并等待5分钟。将混合物加入树脂中。摇动1小时。然后通过氮气压滤除去溶剂
    8. (关键步骤:由于一些树脂的脆性,当进行手工合成时,间歇搅拌优于连续搅拌,以便使小粒径树脂中的树脂碎裂最小化,这可导致在过滤期间的困难。作为替代,连续可以施加轻微振荡或通过反应容器的氮气鼓泡)
    9. 重复步骤6和7进行双重耦合(可选)。
    10. 加入8.0ml DMF(洗涤步骤),搅拌30秒,并通过氮气压滤除去溶剂
    11. 重复步骤10三次。
    12. 对于根据肽序列的每个后续氨基酸,使用上述Fmoc-氨基酸衍生物重复从步骤3开始到步骤12的循环。
      关键步骤:可以在每个循环通过使用微切割测试(参见下文)跟踪合成的进展。为了加快装配,可以进行单联接而不是双联接) 重复耦合步骤的要求可以从Kaiser测试的结果(见下文)得出 暂停点:当N末端氨基被保护时,合成原则上可以在每个偶联循环结束时中断。为了避免在DMF中储存期间Fmoc基团的不期望的去除,用DCM洗涤肽树脂5次,并使其在室温(18-22℃)下干燥。关闭注射器及其柱塞和帽,并存储在4°C。在恢复合成之前,让样品达到室温,并按照步骤2所述使干燥的树脂溶胀。

  3. 去除N-末端Fmoc基团并干燥肽树脂
    1. 加入8.0ml 20%哌啶/DMF(体积/体积),轻轻搅拌1分钟(第一次Fmoc除去步骤),然后通过氮气压滤除去溶剂。
    2. 加入8.0ml 20%哌啶/DMF(体积/体积),轻轻搅拌10分钟(第二次Fmoc除去步骤),然后通过氮气压滤除去溶剂。
    3. 加入8.0ml DMF(洗涤步骤1),搅拌30秒,通过真空过滤除去溶剂
    4. 加入8.0ml DMF(洗涤步骤2),搅拌5分钟,通过氮气压滤除去溶剂
    5. 用DMF重复步骤5四次
    6. 在玻璃过滤器中用DCM洗涤树脂四次。
    7. 风干肽树脂。

  4. 肽切割和纯化(手动)
    1. 通过在TFA/TIS/EDT/H 2 O(94:2:2:2)的混合物中孵育3小时进行肽裂解和去保护。
    2. 过滤混合物,溶液中的肽用冰冷的无水乙醚沉淀,并在eppendorf摇摆桶中以1,000rcf的速度离心。 将所得肽用冰冷无水乙醚洗涤四次,干燥,并溶于合适的缓冲液或溶剂中(如果存在半胱氨酸,则加入1mM DTT)。
    3. 在C-18柱上通过RP-HPLC纯化肽。 收集级分并冻干。 通过ESI-MS表征目标产物,并准备用于反应的下一步骤

代表数据


图1.用该方案合成的肽的代表性HPLC色谱。保留时间为11.68分钟的峰是产物峰。

补充

  1. 凯撒测试
    Kaiser试验是游离伯氨基存在或不存在的定性试验,并且它可以是关于偶联步骤的完整性的有用指示。 该测试基于茚三酮与伯胺的反应,其产生特征性深蓝色。 该测试需要最少量的分析物,并在几分钟内完成。
    1. 试剂
      1. 0.5g茚三酮在10ml乙醇(EtOH)中的溶液
      2. 0.4ml 0.001M氰化钾(KCN)在20ml吡啶中的溶液
    2. 程序
      1. 将几个树脂珠转移到小玻璃管或Eppendorf管中,用乙醇洗涤几次
      2. 加入100毫升上述每种溶液(见本框中的试剂)。
      3. 充分混合并将管置于预热的烘箱(115℃)中5分钟。 浅绿色或无颜色为负。 深蓝色或紫色为正。
        关键步骤为减少假阴性结果的发生率,建议进行平行阳性对照。 该测试不适用于N-末端Pro残基(仲胺)和N-烷基氨基酸。 当应用于聚合序列时,该测试可能给出假阴性结果

  2. 微裂纹
    当Fmoc去除数据显示异常时,在执行需要使用特别昂贵的材料的步骤之前,或在关键步骤之后,并且通常在组装较长序列时,切割和分析少量中间产物可能是有用的。
    1. 程序
      1. 将含有B1-2mg干燥肽树脂的样品转移到小注射器(2ml)中
      2. 向干燥的肽树脂中加入300ml裂解混合物(三氟乙酸/H 2 O /苯酚/三异丙基硅烷8.5/0.5/0.5/0.5),缓慢搅拌30秒,并等待3小时 在其间轻轻搅拌)。
      3. 将溶液收集在小的HPLC小瓶中,用400ml乙腈/水1/1稀释并混合
      4. 在这一点上,可以在分析HPLC系统(注射20ml)和/或进一步稀释(1/10)注射(2ml)在液相色谱 - 质谱仪中分析溶液。

食谱

  1. 氨基酸试剂瓶
    1.0mmol Fmoc氨基酸/0.2mmol等价树脂
    0.9mmol HBTU/0.2mmol等量树脂
  2. 肽裂解混合物(对于200mg〜300mg干肽树脂,5ml)
    4.7ml TFA 0.1 ml EDT
    0.1 ml TIS
    0.1 ml水

致谢

该协议是从以前的工作(Coin等人(2007); Miao等人(2011); Ren等人, >(2010))。 这项工作部分地由加利福尼亚乳腺癌研究计划14IB-0091和SNM试验研究授权(ZC)支持。

参考文献

  1. Coin,I.,Beyermann,M。和Bienert,M。(2007)。 固相肽合成:从标准程序到难序列的合成。 em> Nat Protoc 2(12):3247-3256。
  2. M,Z.,Ren,G.,Jiang,L.,Liu,H.,Webster,JM,Zhang,R.,Namavari,M.,Gambhir,SS,Syud,F.and Cheng, 。 用于HER2阳性肿瘤PET成像的新型18F-标记的双螺旋支架蛋白。 a> Eur J Nucl Med Mol Imaging 38(11):1977-1984。
  3. Ren,G.,Liu,S.,Liu,H.,Miao,Z.and Cheng,Z.(2010)。
  • English
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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Miao, Z. (2012). Peptide Synthesis. Bio-protocol 2(14): e233. DOI: 10.21769/BioProtoc.233.
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Hello,
Could you give me the process in details for Fmoc-Ser(tBu) and Fmoc-His(Mmt)?
thanks
S.Ansari
12/28/2012 10:25:13 PM Reply
zheng miao
Department of Radiology, Molecular Imaging Program at Stanford, Stanford University School of Medicine, USA

product #852211 Fmoc-His(Mtt)-OH Novabiochem
product #852019 Fmoc-Ser(tBu)-OH Novabiochem

1/11/2013 3:00:33 PM