Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations

Abstract

Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid-phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C-terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield-sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi-segment templated NCL approach permits the one-pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of the full-length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one-pot approach removes the need for intermediate purification. We report the synthesis of two E. coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

Keywords: chemical protein synthesis; native chemical ligation; peptide conjugation; peptide-thioester precursor; templated peptide ligation.

Figure 1.

Multi-segment templated peptide ligations. (A) Simultaneous templated ligation of three peptide segments. Peptides functionalized with traceless tethers are conjugated via orthogonal templating pairs (e.g., short complementary DNA strands or clickable moieties). This templated approach improves reaction efficiency, but because of simultaneous ligations at both junctions, unwanted cross-ligation and internal peptide cyclization occur (red dotted arrows) competing with the desired reactions (green arrows). (B) Controlled Activation of Peptides for Templated NCL (CAPTN). Peptide 1 and 2 are tethered together via maleimide/thiol conjugation and selective peptide-Nbz activation favors ligation between peptide 1 and 2 (green arrow) while preventing cyclization of peptide 2 (red dotted arrow). Clicking of peptide 3 then enables templated ligation to peptide 2 via peptide-NHNH₂ activation (green arrow) without cross-ligations (red dotted arrow). This one-pot approach is compatible with our Lys and Glu “helping hand” traceless linkers. Peptide modifications are indicated by symbols, with corresponding chemical groups detailed in the figure legend.

Figure 2.

Maleimide/thiol-mediated CAN. Peptide 1a-NHNH2 was functionalized with a thiol-containing HH linker and peptide 2a with a maleimide-HH linker. Peptides 1a and 2a were conjugated to yield 3a (2.0 mM 1a and 2a, 6 M GnHCl, pH 6.8, r.t. 10 min). CAN was initiated with the conversion to 4a with subsequent ligation resulting in 5a (0.5 mM 3a, 15 eq NaNO₂, 100 mM MPAA and TCEP, 6 M GnHCl, pH 6.8, r.t. 1 h). HH linker removal (0.25 mM 5a, 1 M NH₂OH, 6 M GnHCl, pH 6.8, r.t. 2 h) yielded the desired peptide 6a demonstrating the utility of this second conjugation reaction needed for a three-segment system. The (*) indicates the cleaved HH linkers. Underlined and bolded Lys (K) indicate placements of HH linkers. Peptide modifications are indicated by symbols, with corresponding chemical groups detailed in the figure legend.

Figure 3.

Three-segment templated NCL. (A) Three-segment templated NCL with simultaneous peptide-NHNH₂ activation. Peptide 1-NHNH₂ was conjugated to peptide 2-NHNH₂ by maleimide/thiol-functionalized HH linkers to generate conjugate 4 (1.2 mM 1 and 2, 6 M GnHCl, pH 6.8, r.t. 10 min). Peptide 3 was functionalized with a DBCO-containing HH linker and clicked to 4 via the azide-functionalized HH linker on peptide 2 (0.85 mM 3 and 4, 6 M GnHCl, pH 6.8, r.t. 2 h). The resulting conjugate 5 underwent simultaneous templated NCL by peptide-NHNH₂ 1 and 2 activation (0.43 mM 5, 15 eq NaNO₂, 100 mM MPAA and TCEP, 6 M GnHCl, pH 6.8, r.t. 3 h). Following removal of HH linkers (0.22 mM ligated 5, 1 M NH₂OH, 6 M GnHCl, pH 6.8, r.t. o/n), the desired product was obtained (6, green, 70% RP-HPLC yield), but cross-ligation between peptide 1 and 3 as well as cyclization of peptide 2 were observed (7, red, 6.5% and 8, red, 18% RP-HPLC yield respectively). (B) Controlled Activation of Peptides for Templated NCL (CAPTN). Peptide 1’-Nbz was functionalized with a maleimide-containing HH linker and conjugated to peptide 2-NHNH₂ via its thiol HH linker (1.3 mM 1’ and 2, 6 M GnHCl, pH 6.8, r.t. 10 min). Peptide-Nbz templated NCL was performed to yield 4’ by selectively activating peptide 1’ (1.0 mM conjugate, 100 mM MPAA, 6 M GnHCl, pH 6.8, 37°C, 3 h). Following MPAA extraction by Et₂O, conjugate 5’ was obtained by clicking peptide 3 to 4’ via the azide-functionalized HH linker on peptide 2 (0.85 mM 3 and 4’, 6 M GnHCl, pH 3, r.t. 2 h). Peptide-NHNH₂ templated NCL was performed by peptide 2 activation (0.43 mM 5’, 15 eq NaNO₂, 100 mM MPAA and TCEP, 6 M GnHCl, pH 6.8, r.t. 3 h). HH linker removal (0.22 mM ligated 5’, 1 M NH₂OH, 6 M GnHCl, pH 6.8, r.t. o/n) yielded the desired product (6, green, 87% RP-HPLC yield) with less peptide 2 cyclization (8, red, 6.6% RP-HPLC yield) and negligible cross-ligation between peptide 1’ and 3. For (A) and (B), peptides 1 and 1’ have the same primary sequence but differ in their respective crypto-thioesters. Peptide 2 contains all canonical amino acids except for Met (substituted with Nle). Underlined and bolded Lys (K) indicate placements of HH linkers. Peptide modifications are indicated by symbols, with corresponding chemical groups detailed in the figure legend.

Figure 4.

AlHx-mediated CAN of S16. (A) Intermolecular peptide-NHNH₂ NCL of 9 with 10 (1.0 mM 9, 1.5 mM 10, 15 eq NaNO₂, 100 mM MPAA, 20 mM TCEP, 6 M GnHCl, pH 6.8, r.t. 48 h). The ligation at the sluggish Ile thioester performed poorly, resulting in significant thioester hydrolysis 9b (red) and unreacted 10 (red) as well as minimal product formation 11 (green, 20% RP-HPLC yield). (B) AlHx-mediated CAN of S16. Peptides 12 and 13 were clicked together via the azide-functionalized AlHx linker on 12 and the DBCO-functionalized AlHx linker on 13 (1.8 mM 12 and 13, 6 M GnHCl, pH 6.8, r.t. 1 h). Two peaks are expected due to the formation of regioisomers.^[20e] Peptide-NHNH₂ templated NCL was then accomplished by in situ thioester activation (14a) yielding ligated conjugate 14 (1.0 mM conjugate, 15 eq NaNO₂, 100 mM MPAA, 100 mM TCEP, 6 M GnHCl, pH 6.8, r.t. 2.5 h). Importantly, minimal thioester hydrolysis was observed. Desulfurization to 15 was done post-dialysis and completed in 5.5 h (0.33 mM 14, 60 mM VA-044, 120 mM GSH, 150 mM TCEP, 6 M GnHCl, pH 6.5, r.t. 5.5 h). Traceless cleavage of the AlHx linkers (0.2 mM 15, 25 mM [Pd(allyl)Cl]₂, 25 mM GSH, 6 M GnHCl, pH 8, 37°C, 45 min) followed by DTT quenching of the Pd (40 mM DTT, 6 M GnHCl, pH 7, r.t., 10 min) revealed full-length linear S16 (16, green, 81% RP-HPLC yield) in much cleaner and efficient one-pot manner, outperforming the intermolecular approach. Met in 9 and 12 was substituted with Nle. Underlined and bolded Glu (E) indicate placements of HH linkers. Peptide modifications are indicated by symbols, with corresponding chemical groups detailed in the figure legend.

Figure 5.

CAPTN-mediated synthesis of S17. (A) Intermolecular peptide-Nbz NCL of 17 with 18 (1.0 mM 17, 1.2 mM 18, 100 mM MPAA, 20 mM TCEP, 6 M GnHCl, pH 6.8, 37°C, 24 h). The ligation at the sluggish Val thioester resulted in thioester hydrolysis 17b (red) and unreacted 18 (red) with 50% product formation 19 (green, RP-HPLC yield). (B) CAPTN-mediated synthesis of S17. Peptide 20-Nbz was functionalized with a maleimide-containing HH linker and conjugated to peptide 21-NHNH₂ via its thiol HH linker (2.0 mM 20 and 21, 6 M GnHCl, pH 6.8, r.t. 10 min). Peptide-Nbz templated NCL was performed on conjugate 23 by selective peptide-Nbz activation (1.7 mM 23, 100 mM MPAA, 6 M GnHCl, pH 6.8, 37°C, 3 h) and yielding the desired ligated conjugate 24 (green). Following MPAA extraction by Et₂O, 22 was clicked to 24 via the azide-functionalized HH linker on 21 (1.0 mM 22 and 24, 6 M GnHCl, pH 3, r.t. 2 h). Peptide-NHNH₂ templated NCL was performed resulting in full-length ligated S17 conjugate (0.65 mM conjugate, 15 eq NaNO₂, 100 mM MPAA and TCEP, 6 M GnHCl, pH 6.8, r.t. 4 h). Desulfurization was conducted in one pot by first extracting MPAA with Et₂O, then initiating with VA-044 (0.11 mM conjugate, 60 mM VA-044, 120 mM GSH, 300 mM TCEP, 6 M GnHCl, pH 6.5, 60°C, 4 h). The reaction was then purified by RP-HPLC to isolate the desulfurized S17 conjugate (25) with 15% isolated yield over five reactions done in one pot. AlHx removal was achieved in 4 h (0.38 mM 25, 20 mM Pd/TPPTS, 10 mM GSH, 6 M GnHCl, pH 8, 37°C, 4 h) followed by Ddap removal in 2 h (0.19 mM conjugate, 1 M NH₂OH, 6 M GnHCl, pH 6.8, r.t. 2 h). Dialysis and PdCl₂ treatment (5.0 mM PdCl₂, 6 M GnHCl, pH 7, r.t., 30 min) with subsequent DTT quenching (250 mM DTT, 6 M GnHCl, pH 7, r.t., 10 min) removed remaining Acm yielding linear S17 (26) that was purified by RP-HPLC with 27% isolated yield over the last three reactions. Met in 17 and 20 was substituted with Nle. Underlined and bolded Lys (K) and Glu (E) indicate placements of HH linkers. Peptide modifications are indicated by symbols, with corresponding chemical groups detailed in the figure legend.