沉浸式翻译Delivery Summary
交货摘要
A request was made by IBI on Apr_29_2024 to synthesize 10.0 mg of DRA6~ DR10-10 with a delivery date on Jul_02_2024.
IBI 于 Apr_29_2024 日提出合成 10.0 mg DRA6~ DR10-10 的请求，交货日期为 Jul_02_2024。
Compound ID | Amount Delivered(mg) | Date Delivered | # of Steps | Notebook Page |
Project Summary
项目概要
The synthetic route is based on a procedure supplied by IBI
合成路线基于 IBI 提供的程序.
Experimental for largest scale run:
最大规模运行的实验：
General procedure for preparation of DRA6 - Notebook Page: MH00475-1
DRA6 的一般制备步骤 - 笔记本 页码： MH00475-1
Peptide Synthesis:
多肽合成：
The peptide was synthesized using standard Fmoc chemistry.
该肽是使用标准 Fmoc 化学方法合成的。
Resin preparation: To a solution of Rink AMIDE MBHA resin (0.1 mmol, 1.00 eq, Sub: 0.31 mmol/g), the resin was added DMF (30 mL) and agitated with N2 at 25 °C for 2 h. Then the mixture was filtered to get the resin. The resin was washed with DMF (30.0 mL * 5)
树脂制备：向 Rink AMIDE MBHA 树脂（0.1 mmol，1.00 eq，Sub：0.31 mmol/g）溶液中加入树脂 DMF （30 mL），并在 25 °C 下用 N2 搅拌 2 小时。然后过滤混合物得到树脂。用 DMF （30.0 mL * 5） 洗涤树脂.
Deprotection: 20% piperidine in DMF (25.0 mL) was added and agitated the resin with N2 for 10 min. The resin was washed with DMF (30.0 mL * 5) and filtered to get the resin.
脱保护：加入 20% 哌啶的 DMF 溶液 （25.0 mL），并用 N2 搅拌树脂 10 分钟。 用 DMF （30.0 mL * 5） 并过滤得到树脂。
Coupling: A solution of Fmoc-Pro-OH (0.1 mmol, 3.00 eq), DIEA (0.2 mmol, 6.0 eq) and HBTU (0.095 mmol, 2.85 eq) in DMF (10.0 mL) was added to the resin and agitated with N2 for 30 min at 25°C. The resin was then washed with DMF (30.0 mL * 5)
偶联：将 Fmoc-Pro-OH（0.1 mmol，3.00 eq）、DIYA（0.2 mmol，6.0 eq）和 HBTU（0.095 mmol，2.85 eq）在 DMF （10.0 mL） 中的溶液加入树脂中，并在 25°C 下用 N2 搅拌 30 分钟。 然后用 DMF （30.0 mL * 5） 洗涤树脂.
Repeat above step 2 to 3 for the coupling of following amino acids: (2-68)
重复上述步骤 2 至 3 以偶联以下氨基酸：（2-68）.
Note:
注意：
# | Materials | Coupling reagents |
1 | Fmoc-Pro-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
2 | Fmoc-Ser(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
3 | Fmoc-Gly -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
4 | Fmoc- Ser(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
5 | Fmoc- Gly -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
6 | Fmoc- Thr(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
7 | Fmoc-Glu(OtBu) -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
8 | Fmoc- Thr(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
9 | Fmoc-Arg(Pbf) -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
10 | Fmoc- Pro -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
11 | Fmoc- Leu -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
12 | Fmoc- Thr(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
13 | Fmoc-Ala-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
14 | Fmoc-Leu-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
15 | Fmoc- Glu(OtBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
16 | Fmoc-His(Trt) -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
17 | Fmoc- Leu -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
18 | Fmoc- Glu(OtBu) -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
19 | Fmoc- Ala -OH(3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
20 | Fmoc- Ser(tBu)-OH(3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
21 | Fmoc- Leu -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
22 | Fmoc- Arg(Pbf)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
23 | Fmoc-Gly -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
24 | Fmoc-Leu-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
25 | Fmoc-Ala -OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
26 | Fmoc-Ala -OH(3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
27 | Fmoc- Thr(tBu)-OH (3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
28 | Fmoc- Ser(tBu)-OH(3.00 eq) | HBTU (2.85 eq) and DIEA (6.00 eq) |
29 | Fmoc-Leu-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
30 | Fmoc- Glu(OtBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
31 | Fmoc- Ser(tBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
32 | Fmoc-Ala -OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
33 | Fmoc- Glu(OtBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
34 | Fmoc-Gly-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
35 | Fmoc-Gly-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
36 | Fmoc-Gly -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
37 | Fmoc-Gly-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
38 | Fmoc-Lys(Dde)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
39 | Fmoc-Gly-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
40 | Fmoc-Gly-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
41 | Fmoc- Glu(OtBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
42 | Fmoc- Leu-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
43 | Fmoc- Leu-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
44 | Fmoc- Trp(Boc)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
45 | Fmoc- Glu(OtBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
46 | Fmoc- Val-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
47 | Fmoc- Phe-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
48 | Fmoc- Glu(OtBu) -OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
49 | Fmoc- Lys(Boc)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
50 | Fmoc-Ala -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
51 | Fmoc- Lys(Boc)-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
52 | Fmoc- Lys(Boc) -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
53 | Fmoc- Glu(OtBu) -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
54 | Fmoc-Asp(OtBu) -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
55 | Fmoc-Leu-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
56 | Fmoc- Tyr(tBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
57 | Fmoc- Lys(Boc)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
58 | Fmoc- Ser(tBu) -OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
59 | Fmoc- Tyr(tBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
60 | Fmoc- Asp(OtBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
61 | Fmoc- Ser(tBu)-OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
62 | Fmoc- Thr(tBu) -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
63 | Fmoc-Phe -OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
64 | Fmoc- Thr(tBu)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
65 | Fmoc- Gly-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
66 | Fmoc-Gln(Trt) -OH(3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
67 | Fmoc- Aib-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
68 | Boc-His(Trt)-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
The Dde deprotection: After the coupling of Boc-His(Trt)-OH, add 3% H2N·NH2/DMF (20.0 mL) and react on 30 min and then repeat it for one more time. Drain and wash with DMF (20.0 mL) for 5 times.
Dde脱保护：Boc-His（Trt）-OH偶联后，加入3% H2N·NH2/DMF （20.0 mL） 反应 30 分钟，然后再次重复一次。沥干并用 DMF （20.0 mL） 洗涤 5 次。
Repeat above step 3 for the coupling of the following amino acid: (69-72)
重复上述步骤 3 以偶联以下氨基酸：（69-72）
Note:
注意：
69 | Fmoc-AEEA-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
70 | Fmoc-AEEA-OH (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
71 | Fmoc- Glu-OtBu (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
72 | C20Diacid(tBu) (3.00 eq) | HATU (2.85 eq) and DIEA (6.00 eq) |
Peptide Cleavage and Purification:
肽切割和纯化：
Add cleavage solution (20.0 mL, 92.5% TFA/2.50% TIS/2.50% H2O/2.50% 3-Mercaptopropionic acid) to the flask containing resin at room temperature and stirred for 1.5 h.
在室温下将裂解液（20.0 mL，92.5% TFA/2.50% TIS/2.50% H2O/2.50% 3-巯基丙酸）添加到含有树脂的培养瓶中，并红搅拌 1.5 小时。
Precipitated the peptide with cold isopropyl ether (300 mL). Filter and collect the filter cake. The filter cake was washed with isopropyl ether (150 mL * 2). Dry the crude peptide under vacuum 2 h to get the crude peptide (1.50 g)
用冷异丙醚 （300 mL） 沉淀肽。过滤并收集滤饼。滤饼用异丙醚 （150 mL） * 2 洗涤。在真空下干燥粗肽 2 小时，得到粗肽 （1.50 g）.
The crude peptide was purified by prep-HPLC (TFA condition: A: 0.075 % TFA in H2O, B: ACN) to give the final product DRA6 (12.5 mg, 96.10% purity, TFA) as a white solid. The DRA6 was confirmed via LCMS (MH00475-1-P1-P1C, Rt= 1.162 min) and HPLC (MH00475-1-P1-P1B, Rt= 9.979 min).
通过制备 HPLC 纯化粗肽（TFA 条件：A：0.075 % TFA 的 H2O，B：ACN），得到最终产物 DRA6（12.5 mg，96.10% 纯度，TFA）为白色固体。 通过 LCMS （MH00475-1-P1-P1C，R t= 1.162 min） 和 HPLC （MH00475-1-P1-P1B，R t= 9.979 min） 确认 DRA6。
Purification conditions:
净化条件：
Separation condition | |
Dissolution condition | Dissolve in 10%ACN+H2O |
Instrument | Gilson GX-281 |
Mobile Phase | A: H2O (0.075% TFA in H2O) |
B: CH3CN | |
Gradient | 30-50-50min, Retention time: 25min |
Column | luna ,10um, c18,100A,25mm+Gemin,5um, c18,110A |
Flow Rate | 20 mL/min |
Wavelength | 220/254 nm |
Oven Tem | 30oC |
Spectrum:
光谱：
LCMS: MH00475-1-P1-P1C, Rt= 1.162 min, MS cal.: 7925.04, MS observed: [M+4H]4+ = 1982.27
LCMS：MH00475-1-P1-P1C，R t= 1.162 分钟，MS 校准：7925。04， MS 观察到：[M+4H]4+ = 1982.27
HPLC: MH00475-1-P1-P1B, Rt= 9.979 min, purity: 96.10%
高效液相色谱：MH00475-1-P1-P1B，R t= 9.979 min，纯度：96.10%