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

缩宫素杂质肽[Thr4, Gly7]-Oxytocin

与催产素相比，TGOT的催产素/抗利尿选择性增加了640倍。

TGOT exhibits a 640-fold increase in oxytocic/ antidiuretic selectivity relative to oxytocin.

缩宫素(Oxytocin)的定义

神经垂体激素催产素（OT）是第一个确定其结构的肽类激素，也是第一个以生物活性形式化学合成的肽类激素。它因其子宫收缩活性而引起的“快速出生”而得名。OT也被发现负有责任

The neurohypophysial hormone oxytocin (OT) was the first peptide hormone to have its structure determined and the first to be chemically synthesized in biologically active form. It is named after the “quick birth” which it causes due to its uterotonic activity. OT was also found to be responsible

垂体后叶的泌乳活动【1】。

for the milk-ejecting activity of the posterior pituitary gland 【1】.

缩宫素(Oxytocin)的相关肽

加压素和OT是存在于所有胎盘哺乳动物中的非肽。虽然结构相似，但功能不同。加压素家族的肽在第8位具有共同的碱性氨基酸残基，而OT和相关肽在该位置具有中性氨基酸残基。这种氨基酸残基的化学性质被认为在肽与其各自受体的相互作用中至关重要【2】。

Vasopressin and OT are nonapeptides that are present in all placental mammals. Although similar in structure, they serve different functions. Peptides of the vasopressin family have a basic amino acid residue at position 8 in common, whereas OT and related peptides have a neutral amino acid residue at this position. The chemical nature of this amino acid residue is thought to be of critical importance in the interactions of the peptides with their respective receptors 【2】.

缩宫素(Oxytocin)的类似物

据报道，[2-0-甲基酪氨酸]-催产素（甲基催产素）的抑制特性可以通过氨基甲酰基残基额外取代末端氨基来增强。虽然甲基催产素在某些测定系统上充当OT的抑制剂，并且在其他测定系统上具有类似OT的作用，但其N-氨基甲酰基衍生物在通常用于测定催产素的所有系统上都充当拮抗剂【3】。[Gly7]脱氨基催产素，脱氨基催产素及其各自的retro-D类似物的Corey-Pauling-Koltun模型对于子宫受体的“占领”和“激活”至关重要【5】。[2-O-甲基酪氨酸]-deamino-1-carba-oxytocin，[2-O-甲基酪氨酸]-oxytocin及其二聚体形式在体内对泌乳大鼠乳腺的长期作用显示出生物反应的双相过程，导致假设这些物质表现为激素【5】。用侧链中含有四唑部分的天冬氨酸，谷氨酸和α-氨基己二酸的四唑类似物取代OT中Gln（4）和Asn（5）的酰胺基团，导致类似物的生物活性降低【6】。1-丁酸-2-（O-甲基-L-酪氨酸）-1-碳催产素（卡贝霉素）和/或其他长效催产素类似物用药学上可接受的载体配制，并以足以抑制患者乳腺癌发生或生长的量给药。卡贝霉素和/或其他长效OT类似物也可以用药学上可接受的载体配制，并以足以治疗，预防或缓解患者精神疾病症状的量给药【7,8】。

It has been reported that the inhibitory properties of [2-0-methyltyrosine]-oxytocin (methyloxytocin) can be accentuated by additional substitution of the terminal amino group by a carbamoyl residue. While methyloxytocin acts as an inhibitor of OT on some assay systems and has an OT-like action on others, its N-carbamoyl derivative acts as an antagonist on all the systems which are generally used for the assay of oxytocin 【3】. Corey-Pauling-Koltun models of [Gly7] deaminooxytocin, deaminotocinamide, and their respective retro-D-analogs are essential for "occupation" and "activation" of uterine receptors【4】. The protracted effect of [2-O-methyltyrosine]-deamino-1-carba-oxytocin, [2-O-methyltyrosine]-oxytocin and its dimeric form  studied on the mammary gland of lactating rat in vivo showed a biphasic course of biological response that led to the assumption that the substances behaved like hormonogens【5】. Replacement of the amide groups of Gln(4) and Asn(5) in OT by tetrazole analogues of aspartic, glutamic and alpha-aminoadipic acids containing the tetrazole moiety in the side chains leads to analogues with decreased biological activities【6】. 1-Butanoic acid-2-(O-methyl-L-tyrosine)-1-carbaoxytocin (carbetocin) and/or other long-acting oxytocin analogues are formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to inhibit initiation or growth of breast cancer in the patient. The carbetocin and/or other long-acting OT analogues may also be formulated with a pharmaceutically acceptable carrier and administered in an amount sufficient to treat, prevent or alleviate the symptoms of a psychiatric disorder in the patient【7,8】

缩宫素(Oxytocin)的发现

OT和加压素由Vincent du Vigneaud于1953年分离和合成。1984年阐明了OT基因的结构，1992年报道了OT受体的序列【2】。

OT and vasopressin were isolated and synthesized by Vincent du Vigneaud in 1953. The structure of the OT gene was elucidated in 1984, and the sequence of the OT receptor was reported in 1992【2】.

缩宫素(Oxytocin)的结构特征

所有神经垂体激素都是在Cys残基1和6之间具有二硫键的九肽。这产生了由六个氨基酸的环状部分和COOH末端α-酰胺化的三残基尾部组成的肽。根据第8位的氨基酸，这些肽分为加压素和OT家族：加压素家族含有碱性氨基酸（Lys，Arg），OT家族在此位置含有中性氨基酸。第3位的异亮氨酸对于刺激OT受体和第8位的Arg或Lys作用于加压素受体至关重要。据信这些氨基酸残基极性的差异使加压素和OT肽能够与各自的受体相互作用【1】。

All neurohypophysial hormones are nonapeptides with a disulfide bridge between Cys residues 1 and 6. This results in a peptide constituted of a six-amino acid cyclic part and a COOH-terminal a-amidated three-residue tail. Based on the amino acid at position 8, these peptides are classified into vasopressin and OT families: the vasopressin family contains a basic amino acid (Lys, Arg), and the OT family contains a neutral amino acid at this position. Isoleucine in position 3 is essential for stimulating OT receptors and Arg or Lys in position 8 for acting on vasopressin receptors. The difference in the polarity of these amino acid residues is believed to enable the vasopressin and OT peptides to interact with the respective receptors【1】.

缩宫素(Oxytocin)的作用机制

OT受体是视紫红质型（I类）GPCR家族的典型成员。在GPCR家族成员中，七个跨膜a螺旋是最保守的。GPCR中的保守残基可能参与激活和信号转导至G蛋白的共同机制。OT受体在功能上与Gq/11 a类GTP结合蛋白偶联，该蛋白与Gß一起刺激？磷脂酶C-ß亚型的活性。这导致肌醇三磷酸和1,2-二酰基甘油的产生。肌醇三磷酸触发细胞内储存的Ca2+释放，而二酰基甘油刺激蛋白激酶C，其磷酸化未鉴定的靶蛋白。最后，为了响应细胞内[Ca2+]的增加，引发了各种细胞事件。

The OT receptor is a typical member of the rhodopsin-type (class I) GPCR family. The seven transmembrane a-helices are most highly conserved among the GPCR family members. Conserved residues among the GPCRs may be involved in a common mechanism for activation and signal transduction to the G protein. OT receptors are functionally coupled to Gq/11 a class GTP binding proteins that stimulate together with Gß? the activity of phospholipase C-ß isoforms. This leads to the generation of inositol trisphosphate and 1, 2-diacylglycerol. Inositol trisphosphate triggers Ca2+ release from intracellular stores, whereas diacylglycerol stimulates protein kinase C, which phosphorylates unidentified target proteins. Finally, in response to an increase of intracellular [Ca2+], a variety of cellular events are initiated.

缩宫素(Oxytocin)的功能

刺激泌乳（泌乳）：乳腺肺泡被平滑肌（肌上皮）细胞包围，平滑肌（肌上皮）细胞是OT的主要靶细胞。OT刺激肌上皮细胞收缩，导致牛奶被喷射到导管和水箱中。

Stimulation of milk ejection (milk letdown): Mammary alveoli are surrounded by smooth muscle (myoepithelial) cells which are a prominent target cell for OT. OT stimulates contraction of myoepithelial cells, causing milk to be ejected into the ducts and cisterns.

出生时刺激子宫平滑肌收缩：在妊娠后期，子宫平滑肌细胞上OT受体的丰度增加，这与子宫（有时还有母亲）的“易怒性”增加有关。当胎儿刺激子宫颈和阴道时，OT在分娩过程中释放，它增强子宫平滑肌的收缩以促进分娩或分娩。

Stimulation of uterine smooth muscle contraction at birth: During the later stages of gestation, there is an increase in abundance of OT receptors on uterine smooth muscle cells, which is associated with increased "irritability" of the uterus (and sometimes the mother as well). OT is released during labor when the fetus stimulates the cervix and vagina, and it enhances contraction of uterine smooth muscle to facilitate parturition or birth.

如果子宫收缩不足以完成分娩，医生和兽医有时会服用OT（催产素）以进一步刺激子宫收缩。

In cases where uterine contractions are not sufficient to complete delivery, physicians and veterinarians sometimes administer OT (pitocin) to further stimulate uterine contractions.

母亲行为的建立：在分娩期间，脑脊液中OT的浓度增加，OT在大脑中的作用在建立母亲行为中起主要作用【1】。

Establishment of maternal behavior: During parturition, there is an increase in concentration of OT in cerebrospinal fluid, and OT acting within the brain plays a major role in establishing maternal behavior【1】.

缩宫素(Oxytocin)的相关文献

1. Gimpl  G,  Fahrenholz F (2001). The Oxytocin Receptor System:Structure, Function, and Regulation. Physiol Rev., 81(2):629-683.

2. Van Kesteren RE, Smit AB, De Lange RP, Kits KS, Van Golen FA, Van Der Schors RC, De With ND, Burke JF, Geraerts WP (1995). Structural and Functional Evolution of the Vasopressin/Oxytocin Superfamily: Vasopressin-Related Conopressin Is the Only Member Present in Lymnaea, and Is Involved in the Control of Sexual Behavior. J  Neurosci., 15(9): 5989-5998.

3. Bisset GW, Clark BJ,  Krejci I, Polacek I,  Rudinger J (1970). Some pharmacological properties of a synthetic oxytocin analogue 11-N-carbamoyl-hemicystine-2-0-methyltyrosinel-oxytocin (carbamoylmethyloxytocin), an antagonist to the neurohypophysial hormones. Br. J. Plarinac., 40: 342-360.

4. Hechter O, Kato T, Nakagawa SH, Yang F, Flouret G (1975). Contribution of the Peptide Backbone to the Action of Oxytocin Analogs (stereoisomers/hormone action). PNAS., 72(2):563-566.

5. Barth T, Flegel M, Jost K (1976. Protracted milk-ejecting effect of some oxytocin analogues in rats. Endocrinol Exp., 10(1):65-71.

6. Manturewicz M, Grzonka Z, Borovicková L, and Slaninová J (2007). Oxytocin analogues with amide groups substituted by tetrazole groups in position 4, 5 or 9. Acta biochimica Polonica, 54(4):805.

7. Cassoni P, Sapino A, Papotti M, Bussolati G(1996). “Oxytocin and Oxytocin-Analogue F314 Inhibit cell Proliferation and Tumor Growth of Rat and Mouse mammary carcinomas,” Int. J. Cancer., 66(6):817-820.

8. Cassoni P, Sapino A, Fortunati N, Munaron L, Chini B, Bussolati G (1997). “Oxytocin Inhibits the Proliferation of MDA-MB231 Human Breast-Cancer Cells Via Cyclic Adenosine Monophosphate and Protein Kinase A,” Int. J. Cancer., 72:340-344.

二硫键广泛存在与蛋白结构中，对稳定蛋白结构具有非常重要的意义，二硫键一般是通过序列中的2个Cys的巯基，经氧化形成。
 

形成二硫键的方法很多：空气氧化法，DMSO氧化法，过氧化氢氧化法等。
 

二硫键的合成过程，  可以通过Ellman检测以及HPLC检测方法对其反应进程进行监测。  
   

如果多肽中只含有1对Cys，那二硫键的形成是简单的。多肽经固相或液相合成，然后在pH8-9的溶液中进行氧化。      
 

当需要形成2对或2对以上的二硫键时，合成过程则相对复杂。尽管二硫键的形成通常是在合成方案的最后阶段完成，但有时引入预先形成的二硫化物是有利于连合或延长肽链的。通常采用的巯基保护基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。我们分别列出两种以2-Cl树脂和Rink树脂为载体合成的多肽上多对二硫键形成路线：
 

二硫键反应条件选择    
 

 二硫键即为蛋白质或多肽分子中两个不同位点Cys的巯基（-SH）被氧化形成的S-S共价键。 一条肽链上不同位置的氨基酸之间形成的二硫键，可以将肽链折叠成特定的空间结构。多肽分 子通常分子量较大，空间结构复杂，结构中形成二硫键时要求两个半胱氨酸在空间距离上接近。 此外，多肽结构中还原态的巯基化学性质活泼，容易发生其他的副反应，而且肽链上其他侧链 也可能会发生一系列修饰，因此，肽链进行修饰所选取的氧化剂和氧化条件是反应的关键因素， 反应机理也比较复杂，既可能是自由基反应，也可能是离子反应。      

反应条件有多种选择，比如空气氧化，DMSO氧化等温和的氧化过程，也可以采用H2O2，I2， 汞盐等激烈的反应条件。
 

空气氧化法： 空气氧化法形成二硫键是多肽合成中最经典的方法，通常是将巯基处于还原态的多肽溶于水中，在近中性或弱碱性条件下（PH值6.5-10），反应24小时以上。为了降低分子之间二硫键形成的可能，该方法通常需要在低浓度条件下进行。
 

碘氧化法：将多肽溶于25%的甲醇水溶液或30%的醋酸水溶液中，逐滴滴加10-15mol/L的碘进行氧化，反应15-40min。当肽链中含有对碘比较敏感的Tyr、Trp、Met和His的残基时，氧化条件要控制的更精确，氧化完后，立即加入维生素C或硫代硫酸钠除去过量的碘。 当序列中有两对或多对二硫键需要成环时，通常有两种情况：
 

自然随机成环:       序列中的Cys之间随机成环，与一对二硫键成环条件相似；
 

定点成环：       定点成环即序列中的Cys按照设计要求形成二硫键，反应过程相对复杂。在 固相合成多肽之前，需要提前设计几对二硫键形成的顺序和方法路线，选择不同的侧链 巯基保护基，利用其性质差异，分步氧化形成两对或多对二硫键。       通常采用的巯基保护 基有trt, Acm, Mmt, tBu, Bzl, Mob, Tmob等多种基团。