专肽生物_定制多肽合成服务_多肽合成定制服务_多肽合成厂家_多肽合成公司

专肽生物合成用于蛋白质-蛋白质相互作用研究的生物素化肽。尽管生物素可以在 N 端或 C 端引入（通过赖氨酸残基），但我们建议使用 N 端修饰，因为它成本低、成功率高、周转时间短且易于操作。因为多肽合成是从 C 端到 N 端合成的，因此，N 端修饰是 SPPS步骤的最后一步，不需要额外的特定缩合步骤。相比之下，C 端修饰需要额外的步骤，并且通常更复杂。当然，原则上生物素可以定位在任何地方。

生物素可以通过多种不同的接头或间隔物与肽分离。尽管如此，还是建议包含一个灵活的间隔物，例如 Ahx（一个 6 碳接头），以使生物素标签更加稳定或灵活。

专肽生物在 N 端或 C 端提供生物素化：生物素-N 端、赖氨酸-生物素-肽中间和赖氨酸-生物素-C 端。
专肽生物还可以使用 Ahx 接头或长碳 (LC) 接头提供生物素化：生物素-Ahx-N 末端、Lys-Ahx-生物素-肽中间、Lys-Ahx-生物素-C-末端。

（生物素结构）

示例：
GRGDS在N端和C端标记生物素的结构展示。

1、GRGDS在N端标记生物素，不增加Ahx 接头

2、GRGDS在N端标记生物素，增加一个Ahx 接头

3、GRGDS在C端标记生物素，不增加Ahx 接头

4、GRGDS在C端标记生物素，增加一个Ahx 接头。

Definition
Corticotropin-releasing factor (CRF) represents an early chemical signal of the stress response and activates the hypothalamus–pituitary–adrenal axis in response to a stressful stimulus.

Discovery
The existence of CRF was first proposed in 1955 by Guillemin and Rosenberg and Saffran and Schally. Initially isolated from ovine hypothalamus and characterized as a 41 amino acidpeptide in 1981, CRF was subsequently characterizedfrom rat hypothalami, and the identical structure was deduced for human CRF on the basis of the cDNA sequence of the human CRF precursor gene 1,2. Eckart K et al., in 2001 developed strategies permitting synthesis of CRF analogs for CRFBP or CRFR without cross-reaction. They found that one residue of the ARAE motif served as a switch enhancing or preventing ligand binding to CRFBP. This knowledge was applied to the development of peptidic CRF agonists and antagonists 3.

Structural Characteristics
CRF is a 41- amino acid peptide, was originally isolated from the hypothalamus. The stretch of amino acid residues 22–25, Ala-Arg-Ala-Glu, of human/rat CRF, representing the ARAE motif, was found to be responsible for the high affinity of hyrCRF to CRFBP, in contrast to the low affinity of ovine CRF containing the sequence Thr-Lys-Ala-Asp instead. Interestingly, each residue of the ARAE sequence contributed to the high affinity of hyrCRF, as was demonstrated by single residue exchanges. Although sauvagine (Svg), another peptide of the CRF peptide family, binds to CRFBP with only low affinity, the Svg analog containing the ARAE sequence, instead of Glu21-Lys22-Gln23-Glu24, binds with high affinity. However, it has not yet been investigated whether all residues of the ARAE motif are important  4,5.  Specific modifications and substitutions of CRF that led to the discovery of antagonists with extended duration of action as compared to that of astressin {cyclo(30−33)[dPhe12,Nle21,Glu30,Lys33,Nle38]hCRF(12-41)}. These additional modifications included elongation of the peptide chain by three residues at the N-terminus, its acetylation, and the [CαMeLeu27] substitution to yield cyclo(30−33)[dPhe12, Nle21,CαMeLeu27,Glu30,Lys33,Nle38]Ac-hCRF(9-41), which was found to be longer acting than astressin which further increases the efficiency (potency, duration of action, and bioavailability) of this family of antagonists 6. 

Mode of Action
CRF exhibits its biologic actions through G rotein-dependent receptors. To date, mainly two subtypes of CRF receptors (CRFRs), CRFR1 and CRFR2, have been identified. The central actions of CRF-like peptides are also modulated by the water-soluble CRF binding protein (CRFBP). Consistent with the results of experiments on CRFBP deficient mice, the physiological role of endogenous CRFBP in the central nervous system limit the availability of free ligand for CRFR-mediated actions in brain regions where CRFBP, CRFR, and CRF are colocalized. In addition, central injection of CRF antagonists such as a-helical CRF9–41 (a-hel-CRF9–41), which are bound by CRFBP with high affinity, release endogenous CRF by displacement from CRFBP. The CRF antagonist anti-Svg-30 (aSvg-30) is selective for CRFR2 and useful in in vivo experiments because of its high solubility in cerebrospinal fluid (CSF), the antagonists a-hel-CRF9–41 and Ast are not selective, and bind with different affinities to CRFBP, CRFR1, and CRFR2 7,8.

Functions

Homeostasis, CRF plays a major role in the maintenance or restoration of homeostasis by stimulating the activity of the hypothalamic-pituitary-adrenal (HPA) axis.

Biological function, it also acts within the brain to control immune, reproductive and cardiovascular functions as well as catecholamine release, drug withdrawal, behavior, mood, and anxiety thereby implicating CRF not only as central mediator of stress responses, but also in a variety of stress srelated or -unrelated peripheral functions 9.

On central nervous system, CRF mediates the hypothalamic-pituitary-adrenocortical stress axis. CRF was later found to be widely distributed also outside the hypothalamus throughout the central nervous system 8.

Neurotransmitter, CRF functions as a neurotransmitter or neuromodulator eliciting a wide spectrum of autonomic, electrophysiological, and behavioral effects.

Peripheral sites,  in addition to pituitary and central nervous system effects, some effects of CRF in vitro and in vivo have been found at various peripheral sites, where specific binding sites for CRF or messenger RNA (mRNA) for CRF receptors have been localized as well 1.

References

1.     Guillemin R, Rosenberg B (1955). Humoral hypothalamic control of anterior pituitary: a study with combined tissue cultures. Endocrinology, 57:599-607.

2.     Shibahara S, Morimoto Y, Furutani Y, Notake M, Takahashi H, Shimizu S, Horikawa S, Numa S (1983). Isolation and sequence analysis of the human corticotropin-releasing factor precursor gene. Embo. J., 2(5):775-779.

3.     Eckart K, Jahn O, Radulovic J, Tezval H, van Werven L, Spiess J (2001). A single amino acid serves as an affinity switch between the receptor and the binding protein of corticotropin-releasing factor: Implications for the design of agonists and antagonists. PNAS., 98(20):11142-11147.

4.     Sutton SW, Behan DP, Lahrichi SL, Kaiser R, Corrigan A, Lowry P, Potter E, Perrin MH, Rivier J, Vale WW (1995). Ligand requirements of the human corticotropin-releasing factor-binding protein. Endocrinology, 136(3):1097-1102.

5.     Jahn O, Eckart K, Sydow S, Hofmann BA, Spiess J (2001). Pharmacological characterization of recombinant rat corticotropin releasing factor binding protein using different sauvagine analogs.  Peptides, 22(1):47–56.

6.     Rivier JE, Kirby DA, Lahrichi SL, Corrigan A, Vale WW, Rivier CL (1999). Constrained corticotropin releasing factor antagonists (astressin analogues) with long duration of action in the rat. J. Med. Chem., 42(16):3175–3182.

7.     Spiess J, Dautzenberg FM, Sydow S, Hauger RL, Rühmann A, Blank T, Radulovic J  (1998). Molecular Properties of the CRF Receptor. Trends Endocrinol Metab., 9(4):140–145.

8.     Brauns O, Liepold T, Radulovic J, Spiess J (2001). Pharmacological and chemical properties of astressin, antisauvagine-30 and alpha-helCRF: significance for behavioral experiments.  Neuropharmacology, 41:507–516.
9.     Gulyas J, Rivier C, Perrin M, Koerber SC, Sutton S, Corrigan A, Lahrichi SL, Craig AG, Vale W, Rivier J  (1995). Potent, structurally constrained agonists and competitive antagonists of corticotropin-releasing factor. PNAS,. 92(23):10575-10579.

CRF (corticotropin-releasing factor) is a 41-peptide produced mainly in the hypothalamus. The peptide hormone stimulates ACTH release from the anterior lobe of the pituitary gland. CRH plays an important role in the endocrine, behavioral, and immune response to stress and probably as well in the regulation of energy balance. The human sequence EEPPISLDLTFHLLREVLEMARAEQLAQQAHSNRKLMEII amide also corresponds to the sequence of canine, feline, murine, and porcine CRH.

多肽Biotin-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2的合成步骤：

1、合成MBHA树脂：取若干克MBHA树脂（如初始取代度为0.5mmol/g）和1倍树脂摩尔量的Fmoc-Linker-OH加入到反应器中，加入DMF，搅拌使氨基酸完全溶解。再加入树脂2倍量的DIEPA，搅拌混合均匀。再加入树脂0.95倍量的HBTU，搅拌混合均匀。反应3-4小时后，用DMF洗涤3次。用2倍树脂体积的10%乙酸酐/DMF 进行封端30分钟。然后再用DMF洗涤3次，甲醇洗涤2次，DCM洗涤2次，再用甲醇洗涤2次。真空干燥12小时以上，得到干燥的树脂{Fmoc-Linker-MHBA Resin}，测定取代度。这里测得取代度为 0.3mmol/g。结构如下图：

2、脱Fmoc：取1.51g的上述树脂，用DCM或DMF溶胀20分钟。用DMF洗涤2遍。加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Linker-MBHA Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取1.36mmol Fmoc-Ile-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入2.72mmol DIPEA，1.29mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Ile-Linker-MBHA Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Ile-Linker-MBHA Resin

Fmoc-Ile-Ile-Linker-MBHA Resin

H2N-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Glu(OtBu)-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

H2N-Glu(OtBu)-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

Fmoc-Ser(tBu)-Glu(OtBu)-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Ser(tBu)-Glu(OtBu)-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHA Resin，结构如下：

5、生物素反应连接：在上述树脂中，加入适当DMF后，再加入1.36mmol 生物素到树脂中，再加入2.72mmol DIPEA、1.29mmol HBTU，鼓氮气反应30分钟。用2倍树脂体积的DMF 洗涤3次树脂（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。 得到Biotin-Ser(tBu)-Glu(OtBu)-Glu(OtBu)-Pro-Pro-Ile-Ser(tBu)-Leu-Asp(OtBu)-Leu-Thr(tBu)-Phe-His(Trt)-Leu-Leu-Arg(Pbf)-Glu(OtBu)-Val-Leu-Glu(OtBu)-Met-Ala-Arg(Pbf)-Ala-Glu(OtBu)-Gln(Trt)-Leu-Ala-Gln(Trt)-Gln(Trt)-Ala-His(Trt)-Ser(tBu)-Asn(Trt)-Arg(Pbf)-Lys(Boc)-Leu-Met-Glu(OtBu)-Ile-Ile-Linker-MBHAResin。 结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品Biotin-Ser-Glu-Glu-Pro-Pro-Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn-Arg-Lys-Leu-Met-Glu-Ile-Ile-NH2。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽等。

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