Definition
Atrial natriuretic peptide (ANP), atrial natriuretic factor (ANF), or atriopeptin, is a hormone secreted by heart muscle cells. It is involved in the homeostatic control of body water, sodium, potassium and fat. It is released by muscle cells in the upper chambers (atria) of the heart (atrial myocytes), in response to high blood pressure. It is closely related to brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP).
Discovery
ANP was discovered in 1981 by a team in Ottawa led by Adolfo J. de Bold after they made the seminal observation that injection of atrial tissue extracts into rats caused copious natriuresis1.
Structural characteristics
ANP is a 28-amino acid peptide with a 17-amino acid ring in the middle of the molecule. The ring is formed by a disulfide bond between two cysteine residues at positions 7 and 23. BNP and CNP also share the same amino acid ring. The ANP receptor occurs as a dimer of a single span transmembrane polypeptide, each containing an extracellular hormone-binding domain and an intracellular domain consisting of a protein kinase-like, ATP-dependent regulatory domain and a GC catalytic domain2.
Mode of action
The activities of ANP are mediated by the ANP receptor or the A-type natriuretic peptide receptor carrying intrinsic guanylate cyclase (GC) catalytic activity. Binding of the hormone to the receptor stimulates GC catalytic activity, thereby elevating intracellular cGMP levels. cGMP in turn, mediates the hormonal actions through cGMP-regulated ion channels, protein kinases and phosphodiesterases.
Functions
ANP stimulates vasodilatation, fluid egress, increases glomerular filtration and salt and water excretion. It also blocks the release and actions of several hormones, including angiotensin II, aldosterone and vasopressin. ANP levels are commonly elevated when there is excessive fluid volume or hypertension, and the hormone may be important in combating these states. It shows promise as an agent to treat heart failure, renal failure and fluid excess states3.
References
1. Mebazaa A and Payen D (1990). Atrial natriuretic factor. Ann Fr Anesth Reanim., 9(2):153-168.
2. Ogawa H, Qiu Y, Ogata CM, Misono KS (2004). Crystal structure of hormone-bound atrial natriuretic peptide receptor extracellular domain: rotation mechanism for transmembrane signal transduction. J Biol Chem., 279(27):28625-28631.
3. Baxter JD, Lewicki JA, Gardner DG (1988). Atrial Natriuretic Peptide. Nature BioTechnology, 6: 529 – 546.
多肽H2N-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-COOH的合成步骤：
1、合成CTC树脂：称取2.45g CTC Resin（如初始取代度约为0.72mmol/g）和2.12mmol Fmoc-Tyr(tBu)-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入5.29mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到 Fmoc-Tyr(tBu)-CTC Resin。结构图如下：
2、脱Fmoc：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-Tyr(tBu)-CTC 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5.29mmol Fmoc-Arg(Pbf)-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10.58mmol DIPEA，5.03mmol 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-Arg(Pbf)-Tyr(tBu)-CTC 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-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
H2N-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin
Fmoc-Cys(Trt)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC 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-Cys(Trt)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-CTC Resin，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H2N-Cys-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-COOH。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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