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Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin.

El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H - Angew. Chem. Int. Ed. Engl. (2010)

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

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Ubiquitin is a highly conserved 76 amino acid protein that can be linked to target proteins through an isopeptide bond between the C-terminal carboxylate of Ub and the ɛ-amine of a lysine residue or N terminus of the target protein... Thiolysine-mediated ligation was also used very recently in segment-based strategies to construct diubiquitins (diUb), while K6- and K29-linked diUbs were obtained by a strategy that relies on a genetic code expanded to the incorporation of N-Boc-lysine (Boc=tert-butoxycarbonyl)... Using a Wang resin and standard coupling conditions (namely, 4 equiv Fmoc-protected amino acid, 4 equiv PyBOP, 8 equiv DIPEA, and single coupling reactions), simultaneous incorporation of all the six selected building blocks led to the synthesis of Ub in high yield (54 % yield of the crude product) and 14 % after refolding (see the Supporting Information) and purification by cation-exchange chromatography (Figure 1)... A control experiment without these dipeptide building blocks did not give a defined product... We first focussed on in situ E1-mediated thioester formation and concomitant native chemical ligation... Initial reactions using 2-mercaptoethanesulfonic sodium salt (MESNa) as a thiol resulted in no detectable ligation... The use of the native chemical ligation catalyst 4-mercaptophenylpropionic acid (MPAA), however, proved effective for the productive formation of diUb, as evidenced by analysis of the crude reaction mixture by sodium dodecasulfate polyacrylamide gel electrophoresis (SDS-PAGE; Figure 3 C)... Production of K27 and K29 linkages proved difficult, most likely because residues K27 and K29 are the least accessible lysine residues in ubiquitin (see the Supporting Information)... After optimization of the ligations under denaturing conditions, we identified the following general conditions as the most efficient for the generation of diUb topoisomers on a preparative scale: UbMESNa and Ub δ-thiolysine mutant are dissolved at 10 mg mL in a 1:1 ratio in the ligation mixture (6 m guanidine hydrochloride (Gdn⋅HCl) pH 8, 50 mm tris(2-carboxyethyl)phosphine (TCEP) and 100 mm MPAA) and incubated overnight at 37 °C; next an additional amount of UbMESNa (0.5 equiv) is added to the ligation mixture to ensure full consumption of all the δ-thiolysine mutant... After preparative HPLC, the anticipated δ-thiolysine-linked diUb conjugates were isolated as MPAA disulfides in all cases (Figure 3 D–G) on a multimilligram scale in yields ranging from 35 to 72 % (See the Supporting Information)... Attempts at radical-mediated desulfurizations at 37 °C by using the radical initiator VA-044 (Figure 3 B), glutathione (40 mm), and TCEP (250 mm) proved unsuccessful... Having routine strategies for the chemical construction of Ub mutants, Ub chains, or specific C-terminal modifications, virtually any Ub derivative is now within practical reach... We believe that the versatility of the methods reported here will accelerate the pace of research into the biology of Ub, thereby opening novel avenues for research and drug discovery.

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A) Synthesis of C-terminally modified Ub. PG=protecting group. a) HFIP/CH2Cl2, 30 min, RT; b) PyBOP, DIPEA, Nu, CH2Cl2, 16 h, RT; c) TFA/iPr3SiH/H2O 3 h, RT. DIPEA=N,N-diisopropylethylamine, HFIP=1,1,1,3,3,3-hexafluoro-2-propanol, PyBOP=benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, TFA=trifluoroacetic acid. B) Turnover of commercial and synthetic UbAMC by USP7 shows identical Michaelis–Menten kinetics.
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fig04: A) Synthesis of C-terminally modified Ub. PG=protecting group. a) HFIP/CH2Cl2, 30 min, RT; b) PyBOP, DIPEA, Nu, CH2Cl2, 16 h, RT; c) TFA/iPr3SiH/H2O 3 h, RT. DIPEA=N,N-diisopropylethylamine, HFIP=1,1,1,3,3,3-hexafluoro-2-propanol, PyBOP=benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, TFA=trifluoroacetic acid. B) Turnover of commercial and synthetic UbAMC by USP7 shows identical Michaelis–Menten kinetics.


Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin.

El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H - Angew. Chem. Int. Ed. Engl. (2010)

A) Synthesis of C-terminally modified Ub. PG=protecting group. a) HFIP/CH2Cl2, 30 min, RT; b) PyBOP, DIPEA, Nu, CH2Cl2, 16 h, RT; c) TFA/iPr3SiH/H2O 3 h, RT. DIPEA=N,N-diisopropylethylamine, HFIP=1,1,1,3,3,3-hexafluoro-2-propanol, PyBOP=benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, TFA=trifluoroacetic acid. B) Turnover of commercial and synthetic UbAMC by USP7 shows identical Michaelis–Menten kinetics.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3021723&req=5

fig04: A) Synthesis of C-terminally modified Ub. PG=protecting group. a) HFIP/CH2Cl2, 30 min, RT; b) PyBOP, DIPEA, Nu, CH2Cl2, 16 h, RT; c) TFA/iPr3SiH/H2O 3 h, RT. DIPEA=N,N-diisopropylethylamine, HFIP=1,1,1,3,3,3-hexafluoro-2-propanol, PyBOP=benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, TFA=trifluoroacetic acid. B) Turnover of commercial and synthetic UbAMC by USP7 shows identical Michaelis–Menten kinetics.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.

AUTOMATICALLY GENERATED EXCERPT
Please rate it.

Ubiquitin is a highly conserved 76 amino acid protein that can be linked to target proteins through an isopeptide bond between the C-terminal carboxylate of Ub and the ɛ-amine of a lysine residue or N terminus of the target protein... Thiolysine-mediated ligation was also used very recently in segment-based strategies to construct diubiquitins (diUb), while K6- and K29-linked diUbs were obtained by a strategy that relies on a genetic code expanded to the incorporation of N-Boc-lysine (Boc=tert-butoxycarbonyl)... Using a Wang resin and standard coupling conditions (namely, 4 equiv Fmoc-protected amino acid, 4 equiv PyBOP, 8 equiv DIPEA, and single coupling reactions), simultaneous incorporation of all the six selected building blocks led to the synthesis of Ub in high yield (54 % yield of the crude product) and 14 % after refolding (see the Supporting Information) and purification by cation-exchange chromatography (Figure 1)... A control experiment without these dipeptide building blocks did not give a defined product... We first focussed on in situ E1-mediated thioester formation and concomitant native chemical ligation... Initial reactions using 2-mercaptoethanesulfonic sodium salt (MESNa) as a thiol resulted in no detectable ligation... The use of the native chemical ligation catalyst 4-mercaptophenylpropionic acid (MPAA), however, proved effective for the productive formation of diUb, as evidenced by analysis of the crude reaction mixture by sodium dodecasulfate polyacrylamide gel electrophoresis (SDS-PAGE; Figure 3 C)... Production of K27 and K29 linkages proved difficult, most likely because residues K27 and K29 are the least accessible lysine residues in ubiquitin (see the Supporting Information)... After optimization of the ligations under denaturing conditions, we identified the following general conditions as the most efficient for the generation of diUb topoisomers on a preparative scale: UbMESNa and Ub δ-thiolysine mutant are dissolved at 10 mg mL in a 1:1 ratio in the ligation mixture (6 m guanidine hydrochloride (Gdn⋅HCl) pH 8, 50 mm tris(2-carboxyethyl)phosphine (TCEP) and 100 mm MPAA) and incubated overnight at 37 °C; next an additional amount of UbMESNa (0.5 equiv) is added to the ligation mixture to ensure full consumption of all the δ-thiolysine mutant... After preparative HPLC, the anticipated δ-thiolysine-linked diUb conjugates were isolated as MPAA disulfides in all cases (Figure 3 D–G) on a multimilligram scale in yields ranging from 35 to 72 % (See the Supporting Information)... Attempts at radical-mediated desulfurizations at 37 °C by using the radical initiator VA-044 (Figure 3 B), glutathione (40 mm), and TCEP (250 mm) proved unsuccessful... Having routine strategies for the chemical construction of Ub mutants, Ub chains, or specific C-terminal modifications, virtually any Ub derivative is now within practical reach... We believe that the versatility of the methods reported here will accelerate the pace of research into the biology of Ub, thereby opening novel avenues for research and drug discovery.

Show MeSH