Fmoc Removal and NCL in the Same Reaction Mixture

In order to evaluate the feasibility of Fmoc removal combined with native chemical ligation in the same reaction mixture, we ligated a model thioester peptide Gly–Cys–Pro–Arg–Ile–Leu–Met–Arg‐^αCOSCH₂CH₂SO₃Na (3) with the model Cys–peptide hydrazide Cys–Leu–Tyr–Arg–Ala–Tyr‐^αCONHNH₂ (2) in standard ligation buffer (200 mm phosphate, 6 m Gu.HCl, 20 mm TCEP), as shown in Figure 2 A. The Cys–peptide hydrazide 2 was obtained by Fmoc removal on peptide 1 using 20 % piperidine in ligation buffer at pH 11.0. After Fmoc removal, the subsequent NCL with the thioester peptide 3 was carried out in the same solution at neutral pH in the presence of exogenous aryl thiol catalyst (20 mm 4‐mercaptophenylacetic acid (MPAA)) and resulted in formation of the desired ligated product 4, without formation of any undesired side products (Figure 2 C). The presence of 20 % piperidine in the reaction mixture did not interfere with the native chemical ligation. Since the pKa of piperidine is 11.1, presumably the piperidine remained protonated at neutral pH, thereby preventing nucleophilic attack on the thioester.

Compatibility of Asn–Gly and Asp–Gly Sequences with Piperidine Treatment

The presence of Asn–Gly or Asp–Gly sequences makes any peptide vulnerable to deamidation or iso‐aspartic acid formation through the intermediacy of aspartimide at high pH, such as the pH 11 used to remove the Fmoc group in our method. ^[9] Therefore, it was imperative to check the compatibility of Asn–Gly or Asp–Gly sequences with the Fmoc removal conditions used in our study. In order to assess the stability of the Asn–Gly or Asp–Gly peptides, we incubated two model peptides containing Asn–Gly or Asp–Gly in their sequence at pH 11 in presence of 20 % piperidine. We found no detectable iso‐aspartyl peptide formation from the Asp–Gly sequence and a negligible amount of deamidated product and iso‐aspartyl peptide formation from the Asn–Gly sequence upon exposure to the optimized Fmoc removal conditions at pH 11, even up to 14 minutes (see Section S4.1 and Figure S7a—c in the Supporting Information).

Multisegment Polypeptide Synthesis

In order to evaluate the efficacy of consecutive one‐pot NCL reactions using Fmoc‐Cys–peptide thioester segments, we compared a typical synthesis of a larger polypeptide through C‐to‐N one‐pot ligations without intermediate purification steps. We selected an 86‐residue polypeptide segment (11, Cys²¹⁷–Cys³⁰²) from Plasmodium falciparum protein Pf‐AMA1 (3D7 strain), which contains a pH‐sensitive Asn–Gly sequence, as a target for synthesis (Figure 3 A).

Figure 3

Multisegment polypeptide synthesis with one‐pot ligation sequences. A) Sequence of the polypeptide segment of P. falciparum protein Pf‐AMA1 (3D7 strain) 11. NCL sites are underlined and highlighted in red and green. B) One‐pot ligation without any intermediate purification step resulted in the final polypeptide in 37 % overall yield. R′=CH₂CH₂SO₃Na. C) Analytical HPLC monitoring of the one‐pot ligation reaction: a) 3 min after the addition of the peptide Cys²⁷⁵–Cys³⁰²‐COOH (5) and Fmoc‐Cys²⁴⁷–Phe²⁷⁴‐^αCOCH₂CH₂SO₃Na (6) in the standard ligation buffer (200 mm PB, 6 m Gu.HCl, 20 mm TCEP) containing 20 mm MPAA. 6′ and 6′′ indicate thiolactone formation from 6; b) The first ligation, within 24 h, resulted in Fmoc‐Cys²⁴⁷–Cys³⁰²‐COOH (8) as the ligated product; c) Fmoc removal from the ligated peptide 8 to give peptide Cys²⁴⁷–Cys³⁰²‐COOH (9); d) 3 min after the addition of peptide Fmoc‐Cys²¹⁷–Lys²⁴⁶‐^αCOCH₂CH₂SO₃Na (7) in the reaction mixture; e) The second ligation was essentially complete within 20 h and furnished polypeptide Fmoc‐Cys²¹⁷–Cys³⁰²‐COOH (10); f) Fmoc removal from the ligated product 10 to give target peptide Cys²¹⁷–Cys³⁰²‐COOH (11). * and ** indicate dibenzofulvene‐TECP adduct and MPAA, respectively. D) Analytical HPLC profile (λ=214 nm) with ESI‐MS showing charge‐state distribution (inset) of the purified polypeptide 11; observed mass 10 123.28±0.06 Da (average of the eight most abundant charge states) and calculated mass 10 123.21 Da (average isotope composition).

For the one‐pot three‐segment C‐to‐N sequential ligation synthesis (Figure 3 B), Fmoc protection of the N‐terminal Cys residue of both the middle segment and the N‐terminal segment was not removed after chain assembly by standard Fmoc‐chemistry SPPS. The corresponding peptide thioesters, Fmoc‐Cys²⁴⁷–Phe²⁷⁴‐^αCOCH₂CH₂SO₃Na (6) and Fmoc‐Cys²¹⁷–Lys²⁴⁶‐^αCOCH₂CH₂SO₃Na (7), respectively, were obtained by activation of the peptide hydrazides by treatment with NaNO₂ followed by thiolysis with sodium 2‐mercaptoethane sulfonate (MESNa). The Fmoc group in both peptides remained intact during the NaNO₂ treatment, and the thioester peptides were obtained in excellent crude purity (see Section S6 in the Supporting Information).

With all three peptide segments in hand, we first ligated the peptide 5 and 6 in ligation buffer (pH 6.9) containing 100 mm MPAA [Figure 3 C, (b)]. The pH of the reaction mixture was then raised to 11 by adding piperidine (20 % (v/v), final concentration) in order to remove the N‐terminal Fmoc group of the ligated product 8, giving quantitative Fmoc deprotection of 8 within 7 min to yield peptide 9 [Figure 3 C, (c)]. To carry out the subsequent NCL reaction with the N‐terminal peptide segment 7 in one‐pot, the pH of the reaction mixture was brought down to 6.9 [Figure 3 C, (d)]. After completion of the ligation reaction [Figure 3 C, (e)], we again raised the pH of the reaction mixture to 11, maintaining the concentration of piperidine to 20 % (v/v), to remove the Fmoc group from the ligated polypeptide 10 [Figure 3 C, (f)]. After the two ligations and two Fmoc‐removal steps in one‐pot, a single purification was carried out to obtain full‐length polypeptide 11 in 37 % yield based on the limiting peptide 5 (Figure 3 D).

Having established an efficient method for Fmoc removal and NCL reactions in the same reaction solution, we wanted to explore the utility of this method for the total chemical synthesis of a typical protein. Human lysozyme contains a polypeptide chain of 130 amino acid residues that includes all twenty of the common proteinogenic amino acids in its sequence (Figure 4 A). It has been employed as a model system in various biochemical and biophysical studies directed towards understanding enzyme catalysis, protein folding, and amyloidogenesis.[ ^10a , ^10b , ^10c ] For the total chemical synthesis, we strategically divided the full‐length lysozyme polypeptide into four peptide segments. ^[5c]

Figure 4

Total chemical synthesis of human lysozyme. A) Amino acid sequence of human lysozyme polypeptide chain [UniProtKB: B2R4C5]. NCL sites are underlined and highlighted in red and green. B) Synthetic strategy for the one‐pot synthesis. C) Synthetic strategy for the convergent synthesis; R′=CH₂CH₂SO₃Na, Ar=C₆H₄CH₂COOH. D) Analytical HPLC chromatogram (left, λ=214 nm) together with ESI‐MS data (right) for: a) purified unfolded full‐length lysozyme polypeptide 20 obtained from the one‐pot synthesis [observed mass 14 700.70±0.04 Da (average isotope composition) and calculated mass 14 700.63 Da (average isotope composition)]; b) purified unfolded full‐length lysozyme polypeptide 20 obtained from the convergent synthesis (observed mass 14 700.72±0.03 Da (average isotope composition) and calculated mass 14 700.63 Da (average isotope composition); c) folded human lysozyme 22 [observed mass 14 692.71±0.07 Da (average of the most abundant isotopologue mass over two observed charge states) and calculated mass 14 692.56 Da (average isotope composition)]. Note the earlier retention time and the narrow distribution of the charge states of the folded lysozyme protein compared with the unfolded polypeptide. E) A cartoon representation of the X‐ray crystal structure of the chemically synthesized human lysozyme. F) Figure showing the alignment of our chemically synthesized human lysozyme structure (this work, PDB ID: 6LFH) with the biosynthetic human lysozyme crystal structure at comparable resolution (1.4 Å resolution, PDB ID: 1JWR), ^[10f] showing an RMSD for C‐α atoms of 0.132 Å.

One‐Pot Synthesis of the Human Lysozyme Polypeptide Chain

The one‐pot synthetic strategy is shown in Figure 4 B. Synthesis of the peptide segments is described in Sections S7.1–S7.4 in the Supporting Information. The first NCL reaction between the Cys–peptide 12 and the peptide thioester 13 at pH 6.7 afforded the ligated product, which was then subjected to 20 % (v/v) piperidine treatment at pH 11 to furnish the deprotected peptide 17 within 7 min (see Section S7.5 and Figure S21 in the Supporting Information). We then reduced the pH of the reaction mixture and carried out the second ligation reaction with the peptide segment 14 a at pH 6.7. The next Fmoc removal cycle in the same reaction mixture, by simply raising the pH to 11.0, maintaining the piperidine concentration to 20 % (v/v), led to complete removal of the Fmoc group from the resultant ligated peptide 18 to afford the deprotected polypeptide 19. The same pH adjustments were repeated for the final ligation reaction with the peptide thioester segment 15 in one pot to deliver the full‐length lysozyme polypeptide 20. After five synthetic steps, a single purification was carried out to give full‐length human lysozyme polypeptide 20 in good yield (36 % based on the starting peptide segment 12). The homogeneity of the synthetic product and its mass were confirmed by LC–MS [Figure 4 D, (a)]. The full‐length polypeptide had an observed mass 14 700.70±0.04 Da (average isotope composition) and calculated mass 14 700.63 Da (average isotope composition).

Convergent Synthesis of the Human Lysozyme Polypeptide Chain

In principle, fully convergent synthesis has advantages over consecutive synthetic reactions or partially convergent synthesis. ^[6] In order to demonstrate the application of Fmoc protection of N‐terminal Cys in peptide thioester segments in a fully convergent synthesis, we prepared the same lysozyme polypeptide as depicted in Figure 4 C.

For the convergent synthesis of the lysozyme polypeptide chain, the N‐terminal half of the full‐length polypeptide was prepared by NCL of the Cys‐terminal peptide hydrazide 14 b and the peptide thioester 15 to afford polypeptide hydrazide 21 (see Figure S23). The C‐terminal half of the full‐length lysozyme polypeptide was obtained, as discussed in the one‐pot strategy, by NCL of Cys–peptide 12 and the Fmoc‐protected peptide thioester 13, followed by piperidine mediated Fmoc removal to give polypeptide 17 (see Figure S24). Both 17 and 21 were purified by reverse‐phase HPLC, then reacted after conversion of 21 to the thioester by treatment with NaNO₂ at reduced temperature followed by addition of MPAA, after which Cys–peptide 17 was added to effect the final NCL reaction at pH 6.7 and to afford the full‐length lysozyme polypeptide 20 (see Figure S25) in 48 % yield (based on the starting peptide segment 17) after purification. The full‐length polypeptide had an observed mass 14 700.72±0.03 Da (average isotope composition) and calculated mass 14 700.63 Da (average isotope composition). The purities of the full‐length lysozyme polypeptide (20) products obtained from convergent synthesis and the one‐pot sequential reactions were similar [Figure 4 D, (a,b)] although the overall yield was slightly reduced.

Folding to Obtain correctly Disulfide‐Linked Human Lysozyme

Finally, the full‐length polypeptide 20 was folded with concomitant formation of disulfide bonds in buffer containing a redox system consisting of 5 mm oxidized glutathione, 2 mm DTT in presence of 0.8 m Gu⋅HCl and 1 mm EDTA in 0.1 m TRIS buffer (pH 8) to furnish folded lysozyme protein 22 after HPLC purification in 38 % yield of isolated product (based on the amount of lysozyme polypeptide 20). ^[5c] The folded lysozyme protein had an observed mass of 14 692.71±0.07 Da (average of the most abundant isotopologue mass over two observed charge states), 8.0 Da less than the mass of the full length polypeptide chain (14 700.7 Da), which is in an excellent agreement with the formation of four disulfide bonds with loss of eight protons [Figure 4 D, (c)]. The earlier retention‐time shift compared with the unfolded lysozyme molecule in reverse‐phase HPLC and the altered charge distribution in the ESI‐MS, characteristics of a folded globular protein, was clearly evident for our chemically synthesized lysozyme molecule. To the best of our knowledge this is only the second successful total synthesis of human lysozyme, ^[5c] after several unsuccessful attempts made in the 1970s from leading laboratories.[ ^10d , ^10e ]

To confirm the correct disulfide bonding pattern and the 3D structure of the folded protein molecule, we crystallized the chemically synthesized lysozyme protein using reported ^[5c] crystallization condition and determined its 3D structure by X‐ray crystallography. The lysozyme crystals were obtained by mixing 2 μL of a solution containing 14 mg mL⁻¹ of folded synthetic human lysozyme 22 in 120 mm LiCl, 2.5 mm HEPES, pH 7.5, and 2 μL of reservoir solution consisting of 30 mm sodium phosphate, 2.5 m NaCl, pH 4.9 using hanging drop vapor diffusion method. We used a crystal that diffracted to 1.46 Å resolution to solve the structure by molecular replacement using the coordinates from PDB ID: 2NWD as the search model for phase determination. The crystallographic model was finally refined using Phenix. ^[11] The refined model had an Rwork/Rfree of 16.2 %/19.6 %. The obtained 3D model (Figure 4 E) revealed correct disulfide formation and had a C‐α root mean square deviation (RMSD) of 0.132 Å (Figure 4 F) compared with the crystal structure reported for recombinant lysozyme protein at a similar resolution. ^[10f]