多肽合成工艺流程(英文版).ppt
多肽合成工艺流程
SASRIN (Super Acid Sensitive ResIN) (2-methoxy-4-alkoxybenzyl-alcohol resin)
HO CH2
O CH2
P
CH3O
Peptide is cleavable with 0.5-1.0% TFA in DCM resulted in protected peptide fragments.
✓ Piperidine/DMF (1:4, V/V) is added to the mark;
✓ The mixture is shaken thoroughly and left for 25-30 min;
✓ The resin is filtered off and the absorbance of the filtrate is measured at 301 nm (e = 7800).
The final cleavage results in peptides with COOH group at the C-terminus
The resin is not available for the synthesis of peptides with a sequence on the C-terminal that is sensitive for diketopiperazine formation !
HO CH2 CH3O
O (CH2)3 COOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (Clment of the first amino acid:
多肽药物的生产操作流程
多肽药物的生产操作流程English Answer:Pre-Production.Peptide synthesis design.Raw material procurement.Process optimization and validation.Production.Solid-phase peptide synthesis (SPPS) or liquid-phase peptide synthesis (LPPS)。
Cleavage and deprotection.Purification (HPLC, chromatography)。
Post-Production.Quality control (HPLC, MS)。
Freeze-drying or lyophilization. Formulation and packaging.Quality Assurance.GMP compliance.Regulatory approvals.Stability testing.Additional Considerations.Scale-up and manufacturing.Cost-effectiveness.Market demand.Chinese Answer:生产前。
多肽合成设计。
原料采购。
工艺优化和验证。
生产。
固相多肽合成（SPPS）或液相多肽合成（LPPS）。
切割和脱保护。
纯化（高效液相色谱，色谱法）。
生产后。
质量控制（高效液相色谱，质谱）。
冷冻干燥或冻干。
制剂和包装。
质量保证。
GMP 认证。
监管批准。
稳定性测试。
其他考虑因素。
规模化生产。
成本效益。
市场需求。
y多肽合成路线
Y 多肽合成路线通常包括以下几个步骤：
1. 设计目标多肽序列：根据需求和目标，设计Y 多肽的氨基酸序列。
序列中应包含N-端和C-端氨基酸，以及中间的氨基酸序列。
2. 合成多肽片段：通过固相合成法或液相合成法，逐步合成多肽序列。
合成过程中，通常采用Fmoc（9-氟甲基鸟嘌呤）保护的氨基酸作为合成试剂。
合成多肽片段时，需要注意氨基酸的顺序、肽键的形成以及反应条件。
3. 多肽片段的纯化：合成后的多肽片段可能含有杂质，需要通过柱层析、凝胶过滤或其他分离技术进行纯化。
纯化后的多肽片段应具有较高的纯度和序列正确性。
4. 连接多肽片段：将合成的多肽片段通过适当的连接方式连接成完整的Y 多肽。
这可以通过化学偶联、基因融合或其他方法实现。
5. 去除保护基：在多肽合成过程中，氨基酸残基可能被保护基（如Fmoc）修饰。
在合成完成后，需要将保护基去除，暴露出多肽的天然氨基酸。
6. 多肽折叠与成熟：部分Y 多肽在合成后需要经过折叠才能发挥生物学功能。
因此，对合成的Y 多肽进行折叠和成熟处理，使其具有正确的空间结构和功能。
7. 活性检测与纯度评估：对合成的Y 多肽进行活性检测，以确保其具有预期的生物学功能。
同时，通过多种方法（如HPLC、电泳等）评估多肽的纯度和质量。
8. 储存与应用：将合成的Y 多肽储存于适当条件下，以备后续研究和应用。
Y 多肽在生物医学、药物开发、诊断和治疗等领域具有广泛的应用前景。
多肽合成入门PPT讲稿
N
Y
O
O
R1
R2
+ H2N
N O
N O
Rn N
Rn+1
N
OQ
O
O
R1
R2
XNH
N
N
O
O
R2n+1
R2n+2
N
N
OQ
O
O
• 多肽简介 • 多肽合成原理 • 氨基保护 • 羧基保护 • 侧链功能团的保护 • 肽键生成的方法
氨基保护
• 氨基保护常用的保护基分为烷氧羰基、酰
基，和烷基三大类。因为N烷氧羰基的保护 的氨基酸在接肽时不易发生消旋化，故烷 氧羰基使用最多
氨基保护
• 多肽简介 • 多肽合成原理 • 氨基保护 • 羧基保护 • 侧链功能团的保护 • 肽键生成的方法
羧基保护
• 目前使用的羧基保护大致可以分为三种 • 一种可以用碱皂化脱去，如甲酯、乙酯 • 另一种可以用酸脱酸或碱脱去外，还可以用其
Gln
Q
R基团带负
天冬氨酸
电荷的氨 基酸
(pH=7.0
(aspartic acid) O 谷氨酸(glutamic O
O
时)
acid)
O
N O
H 2
C
O O
N
O
O
O
C H2
Asp Glu
D E
N
R基团带正 赖氨酸(lysine) N
N C
O
H2
Lys
K
电荷的氨 基酸
(pH=7.0
精氨酸
N
(arginine)
N
O O N
CH3 CH
CH 3
多肽固相合成步骤英文描述
多肽固相合成步骤英文描述多肽固相合成是一种合成肽链的方法，它涉及到多个步骤。
以下是多肽固相合成的步骤和英文描述：1. 准备载体：选择适当的树脂，如弱碱性丙烯酰胺树脂(Acrylamide resin)。
Prepare resin: select an appropriate resin, such as weakly basic acrylamide resin.2. 载体预处理：将树脂进行预处理，如使用二氯甲烷和二甲基甲酰胺进行交替洗涤，实现树脂表面的清洁和活化。
Pre-treatment of the resin: pre-treat the resin, such as alternating washing with dichloromethane and dimethylformamide to achieve a clean and activated resin surface.3. 防止侧反应：在肽链合成过程中，需要采取措施防止侧反应的发生，例如使用保护基。
Prevent side reactions: measures need to be taken to prevent side reactions during peptide synthesis, such as using protective groups.4. 合成肽链：通过加入氨基酸单元和活化剂，将肽链逐步合成。
Synthesize the peptide chain: synthesize the peptide chain step by step by adding amino acid units and activators.5. 洗脱肽链：用酸性溶液或氢氟酸将肽链从树脂上洗脱。
Elute the peptide chain: elute the peptide chain from the resin using an acidic solution or hydrogen fluoride.6. 去保护基：使用适当的溶液去除保护基。
多肽偶联的生产工艺流程
多肽偶联的生产工艺流程英文回答：Peptide conjugation is a process used in the production of peptides with attached molecules or functional groups. This process involves the covalent attachment of a specific molecule to a peptide sequence, resulting in a peptide conjugate with enhanced properties or specific functionalities.The production process of peptide conjugates typically involves several steps. Here is a general overview of the process:1. Design and synthesis of the peptide: The first step is to design and synthesize the peptide sequence. This can be done using solid-phase peptide synthesis (SPPS) or recombinant DNA technology, depending on the complexity and length of the peptide.2. Activation of the peptide: Once the peptide is synthesized, it needs to be activated to create a reactive site for conjugation. This can be achieved by introducing a specific functional group, such as a carboxyl group or an amine group, to the peptide.3. Selection and preparation of the molecule to be conjugated: The next step is to select the molecule or functional group that will be attached to the peptide. This molecule can be a drug, a fluorescent dye, a targeting ligand, or any other molecule of interest. The molecule is then prepared for conjugation by introducing a reactive group that can react with the activated peptide.4. Conjugation reaction: The activated peptide and the prepared molecule are mixed together in a suitable reaction buffer or solvent. The reaction conditions, such as temperature, pH, and reaction time, are optimized to ensure efficient conjugation. The reactive groups on the peptide and the molecule react with each other, forming a covalent bond and resulting in the formation of the peptide conjugate.5. Purification and characterization: After the conjugation reaction, the peptide conjugate needs to be purified to remove any unreacted peptide or molecule. Thisis typically done using techniques such as chromatographyor filtration. The purified peptide conjugate is then characterized using analytical techniques, such as mass spectrometry or nuclear magnetic resonance (NMR), toconfirm its structure and purity.6. Evaluation of the conjugate's properties: The final step is to evaluate the properties of the peptide conjugate, such as its stability, solubility, bioactivity, and pharmacokinetics. This can be done through in vitro and in vivo studies to assess the conjugate's potentialapplications in various fields, such as drug delivery, diagnostics, or therapeutics.中文回答：多肽偶联是一种在多肽生产中常用的过程，用于在多肽序列上连接分子或功能基团。
抗多肽制备流程及原理 PPT
R1
R2
H2N
N O
N O
Rn N
Rn+1
N
OH
O
O
3、2 多肽合成原理
化学合成多肽就把氨基酸依照一定的氨基酸排列 顺序和连接方式连接起来。
为了得到具有特定氨基酸顺序的合成多肽,采纳逐
步缩合的定向合成方法,即先将不需要反应的氨基
或羧基用适当的基团保护起来,再进行连接反应,
以保证反应的定向进行。
R1
另一方面,太短的多肽(<10个氨基酸)则会产生识别 特异性特别强的抗体,以至于无法识别整体蛋白,或亲 和性特别低。因此综合考虑制备性抗原地多肽有效长 度一般是10－20个氨基。这种长度的多肽序列会最大 程度的减小生化合成困难,具有一定的水溶性,也会具 有一定程度的二级结构。
(6) 设计合成多肽 一旦抗原序列决定后,接下来就是设计所需合成的
3、6 肽键生成的方法
形成肽键的方法基本能够分为四类:一是羧基 活化法;二是氨基活化法;三是四组份合成法;四是 利用蛋白水解酶的逆反应,亦称酶促合成法。
羧基活化法的基本原理是先将N-保护氨基酸 或肽的a-羧基转变成活化型的RCOX,从而有利于 N来H说2R,取’对代它基进团行X亲的核吸反电应子生性成越R强C,O其N对H羧R’基。的一活般化 能力也越强。
要如此做。
3、 多肽合成原理
3、1多肽简介 肽键是蛋白质分子中氨基酸间的主要连接方式,是
一 胺个键a。lp一ha个-N氨H基2和酸一的个αa-羧lph基a-与CO另O一H脱个水氨缩基合酸而的成α-氨的基酰 之间失去一分子水相互连接而成的化合物称为肽 (peptide),由2 个氨基酸缩合形成的肽叫二肽,由3 个氨 基酸缩合形成的肽叫三肽,少于10 个氨基酸的肽称为 寡肽,由10个以上氨基酸形成的肽叫多肽,因此蛋白质 的结构就是多肽链结构。每个肽在其一端有一自由氨 基,称为氨基端或N-末端,在另一端有一自由羧基,称为 羧基端或C-末端。
多肽合成入门PPT课件
氨基保护
氨基保护常用的保护基分为烷氧羰基、 酰基，和烷基三大类。因为N烷氧羰基的 保护的氨基酸在接肽时不易发生消旋化， 故烷氧羰基使用最多
10
氨基保护
11
多肽简介 多肽合成原理 氨基保护 羧基保护 侧链功能团的保护 肽键生成的方法
12
羧基保护
目前使用的羧基保护大致可以分为三种 一种可以用碱皂化脱去，如甲酯、乙酯 另一种可以用酸脱去，如叔丁酯、对甲
甘氨酸(glycine)
N
N
O
O
N
N
O O
N S
N
N O
N O
N
O
O
C H3
C H3 H
2 C
CH3 CH
CH3 N
H 2
C
N
C H2
H
2
多肽合成简介
1
多肽合成简介
多肽简介 多肽合成原理 氨基保护 羧基保护 侧链功能团的保护 肽键生成的方法
2
多肽简介
蛋白质的基本结构单位 是氨基酸，由20种氨基 酸组成的各种各样的蛋 白质。
每个氨基酸分子（脯氨 酸除外）alpha碳原子上 都结合一个-NH2、一个COOH、一个氢原子，和 一个各不相同的-R
O
O
O
极性不
苏氨酸 (threonine)
N
H3C
HC
O
O
O
Thr
T
带电荷R 基氨基
半胱氨酸 (cysteine)
N
H
2
C
S
O
S
Cys
C
酸
酪氨酸
(tyrosine)
O
N
O
O
H 2
- 1、下载文档前请自行甄别文档内容的完整性，平台不提供额外的编辑、内容补充、找答案等附加服务。
- 2、"仅部分预览"的文档,不可在线预览部分如存在完整性等问题,可反馈申请退款(可完整预览的文档不适用该条件!)。
- 3、如文档侵犯您的权益，请联系客服反馈,我们会尽快为您处理(人工客服工作时间：9:00-18:30)。
90% TFA solution for effective removal.
-SH (Cys)
trityl (Trt)
For selective deprotection
CH2 NH C CH3 O
acetamidomethyl (Acm)
Racemisation during the attachment of Cys derivatives to the resins
C H3 Trt group can be used if on-resin derivatization is required (glycosylation,
phosphorylation). Trt can be cleaved with diluted TFA, while tBu needs
4-Alkoxybenzyl alcohol (Wang) resin:
HO CH2
O CH2
P
Attachment of the first amino acid: Fmoc-Aaa(X)-OH:DIC:DMAP (2:2:0.2 equiv to the resin OH content) in DMF, 1h at RT.
Applied side chain protecting groups in Fmoc-chemistry
Side chain functional group protecting group name (abbreviation)
-OH (Ser, Thr, Tyr)
CH3 H3C C
tert-butyl (tBu)
4-Hydroxymethylphenoxyacetic acid (HMPA) linker:
HO CH2
O CH2 COOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with TFA
4-(4-Hydroxymethyl-3-methoxyphenoxy)butyric acid (HMPB) linker:
Advantages:
excellent pressure stability (continuous flow synthesis) excellent swelling properties (also in water) high diffusion rates available with many types of functional groups low capacity (0.2-0.6 mmol/g), suitable for the synthesis of aggregating peptides, for on resin cyclisation and fragment condansation.
Outline
Resins; Protecting groups; Synthetic protocol; Monitoring; Cleavage technics; Side reactions;
Fmoc/tBu:
Fmoc
tert-butyl
H3C
H3C H3C C CH3
OCH2-P
Rink Amide-AM and Rink Amide-MBHA resins:
OCH3
H3CO Fmoc-HN CH
OCH2-CO-Nle-R
H2N CH2
P
Aminomethyl-PS-DVB
CH3
Peptide cleavage with 90-95% TFA solution.
enantiomerisation!
Determination of loading
(Calculation of the resin capacity) 1.
10-20 mg of dried resin are weighted exactly into a 100 mL measuring flask (for a load of ca. 0.5 meq/g 20 mg is sufficient);
peptides (available for fragment condensation).
ClTrt resin prevents the diketopiperazine formation ! Attachment of Cys and His derivatives to the resin is free from
Similar to Wang resin
4-carboxytrityl linker
O
NovaSyn R TGT alcohol resin
HO
C OH Before use the resin must be converted
to the chloride form by heating with
NH2(mmol/g) = [A301.V(ml)/e301.m(mg)].106
2. ca. 4-6 mg Fmoc-Aaa-resin +400 mL 50% piperidine/DMF 30 min at RT, then filtration dilute with MeOH to 25 mL
Solid phrt II
Application of Fmoc/tBu strategy
Gábor Mező
Research Group of Peptide Chemistry Hungarian Academy of Sciences Eötvös L. University Budapest, Hungary
AcCl or SOCl2 in toluene. Similar to ClTrt resin.
OCH3 H3CO
H2N CH
2,4-dimethoxy-benzhydryl linker NovaSyn R TGR resin
Similar to Rink Amide MBHA resin O
OCH2 C OH
ca. 2 mg Fmoc-Gly-OH +400 mL 50% piperidine/DMF 30 min at RT dilute with MeOH to 25 mL
Capacity of the resin (mmol/g) =
1000.mgly.A3r0es1 in Mgly.mresin.A3g0ly1
1 g ClTrt-resin + 2 mmol Fmoc-Aaa(X)-OH + 8 mmol DIEA in 3-5 mL DCM, for 1.5 h then 0.8 mL MeOH to block the unreacted groups washing with DCM, iPrOH, MeOH, ether
Mgly =297
Rink Amide Resin: synthesis of peptides with CONH2 C-terminus
Cleavage with high concentration of TFA can lead to the break down of
OCH3
the linker
Cl Cl
The final cleavage results in peptides with COOH group at the C-terminus
Cleavage with 90-95% TFA + scavangers results in free peptides Cleavage with AcOH:MeOH(TFE):DCM (1:1:8 or 2:2:6) results in protected
in the presence of DMAP: Fmoc-Cys(Trt)-OH > Fmoc-Cys(Acm)-OH
However, Fmoc-Cys(Acm) at the C-terminal resultes in side reaction:
NH CH C O CH2
O CH2
P
Acm-S CH2 O
SASRIN (Super Acid Sensitive ResIN) (2-methoxy-4-alkoxybenzyl-alcohol resin)
HO CH2
O CH2
P
CH3O
Peptide is cleavable with 0.5-1.0% TFA in DCM resulted in protected peptide fragments.
The basic polymer support is aminomethyl PEG-PS-DVB (NovaSyn R TG) NH2 PEG
HO C H2
O O CH2 C OH
4-hydroxymethylphenoxyacetic acid linker
NovaSyn R TGA resin
There are many different resins and most of them are used for special cases and in individual laboratories. Here only the most widely applied resins will be presented. Resins are based on PS-DVB (1%) copolymer.
HO CH2 CH3O
O (CH2)3 C OOH Attach to aminomethyl PS-DVB resin
Removal of the peptide with diluted TFA
2-Chlorotrityl chloride (ClTrt) resin: