一、概述
二硒代-硒酸酯连接两个蛋白或者肽段的方法简称为DSL方法，英文全称为Diselenide–Selenoester Ligation。该方法可以在无其它辅助反应物的条件下，快速连接两个肽段，形成新的酰胺键，脱硒后便可产生新的天然肽或者蛋白质。
二、步骤
1，通过Fmoc-固相肽合成（SPPS）方案合成适当功能化的肽二硒化物和肽硒酸酯片段。
（此方法是目前快速合成肽段的首选方法，成功率高且可以进行各种结构设计）
2，通过DSL融合这些片段，得到新的更长的片段。
（通常进行连接1至10分钟内完成）
3，对它们进行化学选择性脱硒，新片段变成天然合成蛋白。
（用于在β-硒天冬氨酸（（β-Se）-Asp）和γ-硒代谷氨酸（（γ-Se）-Glu）残基，脱硒反应通常完成在1-10分钟内，而对于硒衍生的Leu，Phe和Pro以及Sec，这种转化通常进行6-8小时）
三、应用
通过DSL-脱硒化作用合成脂联素翻译后修饰的胶原结构域硒代半胱氨酸（硒代半胱氨酸的氧化形式）和两种抑制Tick的凝血酶。
Synthesis of adiponectin (19–40) selenoester, Part 1: loading to Cl-TCP(Cl) ProTide Resin
1) Weigh 0.5 g (240 μmol) of Cl-TCP(Cl) ProTide Resin (0.48 mmol/g) into a 10-mL fritteddisposable reaction vessel and allow to swell for 40 min in dry DCM (5 mL) while shaking at roomtemperature using a mechanical shaker.
2) Expel the liquid from the reaction vessel and wash the resin with 10 × 5 mL of DCM.
3) Dissolve 268 mg of Fmoc-Met-OH (0.72 mmol, 3 equiv.) in 3 mL of DCM in a 21-mL glass vial,using sonication to aid dissolution, if necessary.
4) Add 615 μL of DIPEA (3.6 mmol, 15 equiv.) using a micro-syringe, and agitate the vial for 30 s atroom temperature.
5) Draw up the reaction solution into the fritted syringe bearing the resin and allow the syringe toshake on a mechanical shaker at room temperature for 16 h.
6) Expel the reaction solution from the syringe and wash the resin with 5 × 5 mL of DCM, 5 × 5 mL ofDMF and 5 × 5 mL of DCM.
7) Add 5 mL of CTC resin capping solution (17:2:1 (vol/vol/vol) DCM/MeOH/DIPEA) to the resinand allow the syringe to shake at room temperature for 40 min.
8) Expel the solution from the syringe and wash the resin with 5 × 5 mL of DMF, 5 × 5 mL of DCMand 5 × 5 mL of DMF.
Synthesis of adiponectin (19–40) selenoester, Part 2: determination of resin-loading
9) Treat the resin with 3 mL of Fmoc-deprotection solution (20 vol% piperidine in DMF) for 3 min.Expel the solution from the resin and retain for subsequent measurement. Repeat the treatmenttwice more, again retaining the deprotection solution. Wash the resin with a further 1 mL ofdeprotection solution and combine with the previously retained solutions.
10) Wash the resin with 5 × 5 mL of DMF, 5 × 5 mL of DCM and 5 × 5 mL of DMF and set the resinaside until the resin loading has been determined.
11) Dilute 100 μL of the retained Fmoc-deprotection solution in a further 10 mL of fresh Fmocdeprotection solution, using a 10-mL volumetric flflask.
12) Add 1 mL of fresh Fmoc-deprotection solution to a quartz cuvette and blank the spectrophotometerat λ = 301 nm.
13) Add 1 mL of diluted Fmoc-deprotection to the paired cuvette and measure the absorbance atλ = 301 nm.
14) Calculate the resin loading using the following formula:Resin loading (μmol) = A301nm × dilution volume (mL) × dilution factor × 1,000/7,800 M/cmWhich for the above volumes simplififies to the following:Resin loading (μmol) = A301nm × 10,000/78For loading of 2-chlorotrityl chloride resins, we typically achieve loadings of ~70% of that statedby the manufacturer.? TROUBLESHOOTING
Synthesis of adiponectin (19–40) selenoester, Part 3: fragment extension via automatedSPPS
15) Place 100 μmol of the resin prepared in Step 10 into an appropriate synthesizer vessel.
16) Run the fragment sequence (TTTQGPGVLLPLPKGACTGW) on an automated peptide synthesizerwith heating, using appropriate coupling cycles. Note that the cysteine residue (Cys, C (boldface))should be incorporated bearing side-chain Acm protection to prevent intramolecular transesterifification of the selenoester by the thiol of the unprotected cysteine side chain, leading to theformation of the corresponding thiolactone. The thiolactone intermediate would be competent in asubsequent DSL reaction under additive conditions but at a substantially reduced rate.
17) Upon completion of the automated SPPS, transfer the resin to a new fritted reaction syringe andwash the resin with 5 × 5 mL of DMF, 5 × 5 mL of DCM and 5 × 5 mL of DMF.
18) Repeat Steps 9 and 10 to remove the N-terminal Fmoc-protecting group. It is not necessary toretain the deprotection solutions this time.
19) Dissolve 243 mg of Boc-Glu(OtBu)-OH (800 μmol, 8 equiv.) and 114 mg of Oxyma (800 μmol, 8equiv.) in 1 mL of DMF in a 21-mL glass vial, using sonication to aid the dissolution, if necessary.
20) Add 82 μL of DIC (800 μmol, 8 equiv.) to the vial using a micro-syringe and agitate for 30 s toensure thorough mixing before adding the coupling solution to the resin from Step 18. Note thatthe N-terminal residue of any selenoester-functionalized fragment is coupled as a Boc-protectedamino acid so that the N terminus can remain protected during the selenoesterifification step andthen be deprotected during the subsequent acidic global deprotection step.
21) Allow the resin to shake at room temperature for 1 h, and then expel the coupling solution. Treatthe resin with a fresh batch of coupling solution (prepared as described in Steps 19 and 20) for afurther hour at room temperature with shaking.
22) Expel the solution from the syringe and wash the resin with 5 × 5 mL of DMF and 5 × 5 mL ofDCM and dry the resin.
Synthesis of adiponectin (19–40) selenoester, Part 4: solution-phase selenoesterifification
23) Split the resin to perform selenoesterifification at a 50-μmol scale.
24) Wash the resin with 10 × 5 mL of DCM.
25) Treat the resin with 5 mL of HFIP cleavage cocktail for 40 min at room temperature with shaking.
