Team:Tuebingen/Notebook/Protocols/SyntheticPeptides

From 2014.igem.org

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<h3>Synthetic Peptides</h3>
<h3>Synthetic Peptides</h3>
<p>Peptides were obtained by solid-phase synthesis using the Fmoc strategy.
<p>Peptides were obtained by solid-phase synthesis using the Fmoc strategy.
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Cleavable TentaGel&reg; R RAM from <i>Rapp Polymers</i> was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel&reg;-Resin was performed with 20 % piperidine in dimethylformamide (2x15 min). Cysteine was activated with HBTU (0.97 eq.) and added twice in 5-fold excess. The product was washed 5 times in DMF and 2 times in dichloromethane (DCM). Deprotection of the Fmoc-protected amino group of the coupled cysteine was performed like the deprotection of the TentaGel&reg;-Resin. HBTU (0.97 eq.) activated lysine (Fmoc-Lys(Mtt)-OH) was coupled twice in 3-fold excess, with the second coupling taking place overnight. The 4-methyltrityl (Mtt) protection group was removed by washing the resin with 1 % trifluoroacetic acid (TFA) in DCM with gravity flow. Carboxyfluorescein was activated with HBTU (0.97 eq.) and coupled twice to the free &epsilon;-amino group of the lysine, whereas the second coupling occurred overnight. After extensive washing and deprotection of the &alpha;-amino group, the synthesis of the tag-specific region was performed automatically using an Invatis AG ResPep System. Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Ahx-OH were used for this purpose. <i>N</i>-methyl-2-pyrrolidone (NMP) was added to the solvents increasing the solubility of the amino acids and prevented precipitation in the machine.</p>
+
Cleavable TentaGel&reg; R RAM from <i>Rapp Polymers</i> was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel&reg;-Resin was performed with 20 % piperidine in dimethylformamide (2x15 min).</p>
-
+
<img src="https://static.igem.org/mediawiki/2014/2/29/Tuebingen14_Fmoc.jpg"style=" width: 400px;">
 +
<p id="picTextCenter">Figure 1: Mechanism for the deprotection of Fmoc-protected amines</p>
 +
<p>Cysteine was activated with HBTU (0.97 eq.) and added twice in 5-fold excess. The product was washed 5 times in DMF and 2 times in dichloromethane (DCM). Deprotection of the Fmoc-protected amino group of the coupled cysteine was performed like the deprotection of the TentaGel&reg;-Resin. HBTU (0.97 eq.) activated lysine (Fmoc-Lys(Mtt)-OH) was coupled twice in 3-fold excess, with the second coupling taking place overnight. The 4-methyltrityl (Mtt) protection group was removed by washing the resin with 1 % trifluoroacetic acid (TFA) in DCM with gravity flow. Carboxyfluorescein was activated with HBTU (0.97 eq.) and coupled twice to the free &epsilon;-amino group of the lysine, whereas the second coupling occurred overnight. After extensive washing and deprotection of the &alpha;-amino group, the synthesis of the tag-specific region was performed automatically using an Invatis AG ResPep System. Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Ahx-OH were used for this purpose. <i>N</i>-methyl-2-pyrrolidone (NMP) was added to the solvents increasing the solubility of the amino acids and prevented precipitation in the machine.</p>
 +
<img src="https://static.igem.org/mediawiki/2014/b/bf/Tuebingen14-HBTU.jpg"style=" width: 400px;">
 +
<p id="picTextCenter">Figure 1: Mechanism of the coupling reaction</p>
<p>The processed product was cleaved from the resin and unprotected from their side chain protection groups using a cleavage solution consisting of 670 mg of phenol, 450 &mu;l of water, 450 &mu;l of triisopropylsilyl (TIPS) and 9 ml of TFA. The resulting peptides were precipitated in cold diethyl ether and lyophilized overnight. Purification was performed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electrospray ionization (ESI).</p>
<p>The processed product was cleaved from the resin and unprotected from their side chain protection groups using a cleavage solution consisting of 670 mg of phenol, 450 &mu;l of water, 450 &mu;l of triisopropylsilyl (TIPS) and 9 ml of TFA. The resulting peptides were precipitated in cold diethyl ether and lyophilized overnight. Purification was performed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electrospray ionization (ESI).</p>

Revision as of 00:00, 18 October 2014


Protocols

Synthetic Peptides

Peptides were obtained by solid-phase synthesis using the Fmoc strategy. Cleavable TentaGel® R RAM from Rapp Polymers was used for synthesis. Deprotection of the Fmoc-protected amino group on the surface of the TentaGel®-Resin was performed with 20 % piperidine in dimethylformamide (2x15 min).

Figure 1: Mechanism for the deprotection of Fmoc-protected amines

Cysteine was activated with HBTU (0.97 eq.) and added twice in 5-fold excess. The product was washed 5 times in DMF and 2 times in dichloromethane (DCM). Deprotection of the Fmoc-protected amino group of the coupled cysteine was performed like the deprotection of the TentaGel®-Resin. HBTU (0.97 eq.) activated lysine (Fmoc-Lys(Mtt)-OH) was coupled twice in 3-fold excess, with the second coupling taking place overnight. The 4-methyltrityl (Mtt) protection group was removed by washing the resin with 1 % trifluoroacetic acid (TFA) in DCM with gravity flow. Carboxyfluorescein was activated with HBTU (0.97 eq.) and coupled twice to the free ε-amino group of the lysine, whereas the second coupling occurred overnight. After extensive washing and deprotection of the α-amino group, the synthesis of the tag-specific region was performed automatically using an Invatis AG ResPep System. Fmoc-Ala-OH, Fmoc-Asn(Trt)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Gly-OH, Fmoc-His(Trt)-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-Met-OH, Fmoc-Phe-OH, Fmoc-Pro-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH and Fmoc-Ahx-OH were used for this purpose. N-methyl-2-pyrrolidone (NMP) was added to the solvents increasing the solubility of the amino acids and prevented precipitation in the machine.

Figure 1: Mechanism of the coupling reaction

The processed product was cleaved from the resin and unprotected from their side chain protection groups using a cleavage solution consisting of 670 mg of phenol, 450 μl of water, 450 μl of triisopropylsilyl (TIPS) and 9 ml of TFA. The resulting peptides were precipitated in cold diethyl ether and lyophilized overnight. Purification was performed by high-performance liquid chromatography (HPLC) and the resulting product was checked by mass spectroscopy using electrospray ionization (ESI).